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THE

PROCEEDINGS

OF THE

LINNEAN SOCIETY

OF

New SoutH WALES

FOR THE YEAR

1950

VOL. LXXV.

WITH TWELVE PLATES.
442 Text-figures.

SYDNEY:
PRINTED AND PUBLISHED FOR THE SOCIETY BY

AUSTRALASIAN MEDICAL PUBLISHING CO. LTD.
Seamer Street, Glebe, Sydney,

and
SOLD BY THE SOCIETY.
1950.

ii

CONTENTS OF PROCEEDINGS, 1950.

PARTS 1-2 (Nos. 347-348).

(Issued 6th June, 1950.)

Pages.
Presidential Address: The Last Haunts of Demons: A Comparative Study of
secretion and Accumulation. By KR: eNe Robertson] 2 Seen cee ie ar i-xx
Council Elections Shs We GPR Godel, Li sowamlifes Game Wa" ecelN oicegeyog OA Negte MOY tte aero aT at XX
Balance Sheets for the Year 1949-50 iy AP ee LAE ee he PTE ON en ohn O b= O.aN il
Abstract of “Proceedings i tl ecco ee cdo ute earn eho clay can ain aie ie eee ar XXiv
Australian Ephemeroptera. Part I. Taxonomy of New South Wales Species
and Evaluation of Taxonomic Characters. By Janet EH. Harker. (One
hundred and one Text-figures.) ahead ikrlbsaetac eae te colton! GS outs Se RMR a oan ear 1-34
A Study of the Alkaline Phosphatase Reaction in Tissue Sections. Part I.
The Possibility of its Quantitaive Use. By K. W. Cleland. (Nine Text-
figures. ) : 35-53
A Study of the Alkaline Phosphatase Reaction in Tissue Sections. Part II.
The Historical and Cytological Validity. By K. W. Cleland. (Plates
i and ii) a Oo Ta eer enn Netra Ne nies Meare Se A gad aie 54-69
Dilation of the Foot in Uber (Polinices) strangei (Mollusca, Class Gastro-
poda). By Muriel C. Morris, Linnean Macleay Fellow in Zoology. (Three
Text-figures. ) age coi A Nee gk bh Se 70-80
Reduvioidea from New South Wales. Notes and Descriptions (Hemiptera).
By Petr Wygodzinsky. (Communicated by J. W. T. Armstrong.) (Thirty-
four Text-figures. ) ef ce 9 ate TERRE OG 0 ais ir ee ae 81-88
The Hair-Tracts in Marsupials. Part IV. Direction Characteristics of Whorls
and Meristic Repetition of Radial Fields. By W. Boardman. (One Text-
figure.) SPREE: Mee STON CMe MAI EOI e Gd) aco) OSM GS! Bic ac 89-95

A Hybrid Hucalyptus. By Daeryor..) (Plates: isang ive) ae eee 96-98

CONTENTS.

PARTS 3-4 (Nos. 349-350).
(Issued 22nd September, 1950.)

Cytological Studies in the Myrtaceae. III. Cytology and Phylogeny in the
Chamaelaucoideae. By S. Smith-White. (Plates v and vi; one hundred
and one Text-figures. )

A Revision of the Australian Species of the Genus Lagenophora Cass. By
Gwenda L. Davis. (Plate vii; thirteen Text-figures. )

A Review of and Contribution to Knowledge of Phylloglossum Drummondii
Kunze. By Frances M. V. Hackney, D.Sc. (Twenty Text-figures. )

Notes on the Aphediinae of Australia (Coleoptera, Scarabaeidae). The
Aphodius tasmaniae, howitti, yorkensis, andersoni complex. By B. B.
Given. (Ten Text-figures.) (Communicated by P. B. Carne.)

The Morphology of the Immature Stages of Aphodius howitti Hope (Coleoptera,
Scarabaeidae, Aphodiinae). By P. B. Carne. (Plate viii; thirteen Text-
figures. )

Notes on Australasian Simuliidae (Diptera) II. By M. J. and I. M. Mackerras.
(Fifteen Text-figures.)

Revision of the Genus Solenogyne Cass. By Gwenda L. Davis. (Fourteen
Text-figures. )

The Classification of Bacteria, with Special Reference to Non-pathogenic
Hubacteria. By H. J. Ferguson Wood .. .. ..,~..

Australian Polyporaceae in Herbaria of Royal Botanic Gardens, Kew, and
British Museum of Natural History. By G. H. Cunningham ..

> (Dud ew
: "62 ry

lil

Pages.

99-121
122-132

133-152

153-157

158-166
167-187
188-194
195-213

214-249

iv CONTENTS.

PARTS 5-6 (Nos. 351-352).

(Issued 21st December, 1950.)

Page.
Further Notes on Experimental Crossing within the Aédes scutellaris Group of
Species (Diptera, Culicidae). By A. R. Woodhill 251-253
/
The Hair Tracts in Marsupials. Part V. A Contribution on Causation. By

W. Boardman 254-266
The Hair Tracts in Marsupials. Part VI. Evolution and Genetics of Tract

Pattern. By W. Boardman 267-272
The Hair Tracts in Marsupials. Part VII. A System of Nomenclature. By

W. Boardman. (Two Text-figures.) 273-278
A Note on the Non-reciprocal Fertility in Matings between Sub-species of

Mosquitoes. By S. Smith-White 279-281
Respiration and Cell Division of Developing Oyster Eggs. By K. W. Cleland:

(Five Text-figures. ) 282-295
The Intermediary Metabolism of Unfertilized Oyster Eggs. By K. W. Cleland.

(Three Text-figures.) «.. 296-319
A Further Contribution on the Life-History of Pherosphaera. By C. G. Elliott.

(Communicated by P. Brough.) (Plates ix—xii; twenty-two Text-figures.) 320-333
A New Genus and Four New Species of Phloecharinae (Coleoptera, Staphy-

linidae) from the Australian Region. By W. O. Steel. (Communicated by

J. W. T. Armstrong.) (Twenty-nine Text-figures. ) 334-344
Notes on the Morphology and Biology of Anabarrynchus fasciatus Macq., and

other Australian Therevidae (Diptera, Therevidae). By Kathleen M. I.

English. (Forty-seven Text-figures. ) 2. ¢ 345=959
Abstract of Proceedings XXV—-XXxXi
Taist Of) MeEmbDers4 (7s NOUN es (Tet Mi Tae CS RE) tea ert ir te in .. XXXiI-xXxXxXvi
List of Genera and Species XXXVil
List of Plates XXXVIi

Index ee AMET EMOIT SOELEO AR, CRI (Aad, tie: Ga old Mais MBS Cae XXXVili-xl

PRESIDENTIAL ADDRESS.

ANNUAL GENERAL MEETING. Ne
WEDNESDAY, 29th Marcu, 1950.

The Seventy-fifth Annual General Meeting was held in the Society’s Rooms, Science
House, Gloucester Street, Sydney, on Wednesday, 29th March, 1950.

Dr. R. N. Robertson, President, occupied the Chair.

The Minutes of the Annual General Meeting held on 30th March, 1949, were read
and confirmed.

PRESIDENTIAL ADDRESS.

It is my privilege to deliver the 75th Presidential Address to this Society, instituted
in 1874 “for the cultivation and study of the Science of Natural History in all its
branches”. Those of us who have derived social and intellectual pleasure and profit
from the activities of the Society, owe a considerable debt to the wisdom and generosity
of our predecessors. From the time of its inauguration, when it began with 124
members, the Society has gone on from strength to strength, fostering the study of
Natural History. Not only have most local scientists interested in Natural History
been active members, but, thanks to the generous benefaction of Sir William Macleay,
the Society has been able to stimulate a considerable amount of research. It has survived
difficult times—two economic depressions, two world conflicts, to say nothing of the
disastrous Garden Palace fire of 1882 when it lost all its possessions and records.
Despite these vicissitudes, the Society has established high standards of scientific work
and its publications enjoy a high reputation.

It is pleasing to be able to report that the past session, despite some problems
which have given the Council cause for concern, seems to have maintained the high
standards of previous years. Our greatest problem is to meet the rising costs,
particularly of publication, with the reduced income from the Society’s investments,
due to the decline in interest rates in recent years. We have already found it necessary
to reduce the size of the ProcEEDINGS, and now that research in natural history has
recovered from its temporary setback during the war, we are receiving more papers
than we are able to publish. An approach to the State Government for an increase in
the annual grant was not successful and, consequently, at recent special meetings, it
has been decided that the subscription, which has been one guinea since 1893, should be
increased to two guineas from 1st March, 1950. é

The Society held only seven General Meetings during the year, as two meetings had
to be cancelled because of lighting restrictions. The attendance at meetings averaged
35. If the interest shown by members is continued, rearrangement of the existing
meeting room may need consideration, as the present maximum seating capacity is 50.
It has been the Council’s policy to arrange meetings to provide, as far as possible, for
varied interests. Authors of papers have co-operated by presenting their papers in an
interesting manner and a number of notes and exhibits have been arranged. The last
meeting of the session took the form of a symposium, “The Surface of the Bacterial
Cell”, jointly with the New South Wales Society for Experimental Biology. The
speakers were Dr. R. J. Swaby, Miss P. M. Rountree, and Mr. W. J. Scott, and a lively
discussion followed. At the September meeting an interesting talk of considerable
historical interest to the Society was given by Dr. N. W. G. Macintosh on “Crania in
the Macleay Museum’. Some of the crania described were collected by the late Baron
Nicolai Nicolaevitch Miklouho—Maclay on his expeditions to northern Papua and the
Philippines; others were from the collection of Sir William Macleay. Dr. Macintosh’s
paper is noteworthy as being the first anthropological paper presented to the Society for

A

ii PRESIDENTIAL ADDRESS.

many years. Our thanks are due to Mrs. P. Messmer, Mr. A. Musgrave, Mr. Ian Fraser
and the Linnean Macleay Fellows for notes and exhibits at various meetings.

Exchanges received from scientific societies and institutions totalled approximately
1,480. A number of requests for exchanges was received and new exchanges were
entered into with: Ecole Polytechnique de Jassy, Roumania; Anales del Jardin Botanico
de Madrid; Matematisk—Naturvetenskapliga Biblioteket, Stockholms Hogskola, Sweden;
Gotesborgs Kungl. Vetenskaps— och Vitterhetssamhalle, Sweden. The steady accumula-
tion of periodicals received has caused the Library to become congested, and additional
steel shelving has been ordered. Duplicate periodical parts have been disposed of to
libraries in Australia and New Zealand, and lists of duplicate books for sale will be
available shortly. The Library will require planning to allow for expansion in the
future. Loans from the Library, particularly interstate inter-library loans, have
increased.

Volume 74 of the Society’s Procrrpines, reduced in size because of increased costs,
was all published during 1949; it consists of 236 + xxiv pages, 4 plates and 207 text-
gures. This is approximately half the size of Volume 73. With the exception of a
grant towards one paper, the total cost, £495, was borne by the Society. The
PROCEEDINGS have now been placed on a fixed subscription basis for overseas and
local non-member subscribers, and this is proving a most satisfactory arrangement.
There has been an increased demand from abroad for reprints and parts of the
PROCEEDINGS.

The nett return from Science House for the year was £547.

The numerical strength of the Society at 28th February, 1950, was: Ordinary
Members, 218; Honorary Member, 1; Life Members, 15; Corresponding Members, 4;
Total, 238. During the year 22 new members were elected, one member was lost by
death and the names of four members were removed under Rule VII.

In May, the State Government called on organisations interested in fauna protection
to nominate three members to serve on the Fauna Protection Panel, under the Fauna
Protection Act (1948). The Society asked permission to nominate a member, and later
Mr. E. Le G. Troughton was appointed. Other members of this Society who are on the
Panel are Mr. J. R. Kinghorn and Professor P. D. F. Murray. The Society was invited
by Unesco to send a representative to the International Technical Conference on the
Protection of Nature, at Lake Success, in August, 1949. Mr. Troughton, who was abroad
at that time, was able to attend this conference, as well as the meetings of the Uno
Scientific Conference on the Conservation and Utilization of Resources embracing water,
soil, minerals, flora and fauna, and fire control, which was held at the same time. Later
in the year the Society was invited to advise the Fauna Protection Panel on the reserva-
tion of natural areas.

The Meteorological Bureau asked the Society to advise it on the compilation of lists
of plants and animals suitable to use for making phenological observations. This
request arose originally in 1947 from a conference of Directors, of Meteorological
Services. A committee consisting of Messrs. Musgrave, Zeck, Millett, Dr. H. S. McKee,
Miss Morris and the Secretary, was formed. This committee has completed a list of
plants, and has submitted it to the Meteorological Bureau; a list of insects is not yet
finalized. The committee has offered to check the lists for northern Victoria and
southern Queensland, and also to advise inland weather observers concerning the
collection of insects.

Linnean Macleay Fellowships.

In 1948 the Council appointed four Fellows for 1949, viz., Miss M. Hindmarsh
(Botany), Miss A. Millerd (Biochemistry), Miss M. Morris (Zoology) and Miss J.
Balmain (Biochemistry). The first three Fellows made satisfactory progress in their
investigations during the year. Miss Balmain resigned in June to go to England where
she is now working at Dairy Research Laboratories, Reading. She was at the time of her
resignation investigating inhibition of microbial dehydrogenase activity by a series of

PRESIDENTIAL ADDRESS. iii

compounds systematically related to phenol and benzoic acid. Good progress was made
but no final results were achieved in the six months during which she held a Fellowship.

Miss Morris continued her survey of the seasonal variation of the plankton of the
Hawkesbury Estuary for the first six months of the year. The collection of material
was discontinued in May as a full twenty-four months’ series of samples had then been
collected. The material has been studied qualitatively and the quantitative estimations
of the catches are now being carried out. It is hoped that this section of the work
will be completed and prepared for publication early this year. A twelve months’ series
of samples collected by the Fisheries Division of C.S.I.R.O., from various stations from
the mouth of the Hawkesbury Hstuary to Windsor, has been studied, with the intention
of determining to what extent the plankton population varies with changes in salinity.
The qualitative and quantitative studies of these catches are now completed and the
work is being prepared for publication. An investigation of the mode of action of the
foot of the sandsnail, Uber (Polinices) strangei, was completed in March, 1949, and the
paper accepted for publication by the Society.

Miss Hindmarsh carried out investigations of the processes concerned with mitosis
in plant cells, by studying the relationship of certain compounds to the various stages
of cell division. Work on the effects of sulphanilamide and p-aminobenzoic acid on
meristematic cells has been continued. Sulphanilamide is known to inhibit cell division
by preventing the initiation of prophase and the formation of the mitotic spindle.
Earlier work showed that roots from onion bulbs had a very variable number of dividing
cells. Some time was spent in improving techniques for growing onion roots which
would give more consistent results. To produce aseptic and less variable material, onion
seedlings were grown under modified culture conditions. Although untreated onion
seedling roots have a fairly uniform number of dividing cells, roots treated with either
sulphanilamide or p-aminobenzoic acid solutions are not so uniform. This variability
is increased in roots treated with a mixture of the two substances. The conditions
under which a typical sulphanilamide inhibition of cell division is produced were found
to be different in bulb roots and seedling roots.

Miss Millerd was able to isolate the succinoxidase system from potato tuber
(Solanum tuberosum L.). The system has been studied aerobically and anaerobically
by the standard Warburg and Thunberg techniques. The effects of pH, redox dyes
(2:6 dichlorphenolindophenol, thionine and methylene blue) and inhibitors (e.g.,
cyanide, phenyl-mercuric acetate), on succinic dehydrogenase and on the complete
system, have been investigated. The enzymic properties of the system have been
compared with those of the corresponding system from animal tissue. The results of
these investigations are being prepared for publication.

The Council reappointed three Fellows for 1950: Miss Muriel Morris to a Fellowship
in Zoology, Miss Mary Hindmarsh to a Fellowship in Botany, and Miss Adele Millerd
to a Fellowship in Biochemistry. We wish them success in their investigations.

Macleay Bacteriologist.

The position of Macleay Bacteriologist was advertised in Great Britain, France,
Denmark, Holland, the United States, and Australia, and five applications were received.
The Council, on the recommendation of the Bacteriology Committee, considered that
the application of Dr. Y. T. Tchan was outstanding and offered him the appointment
for a five-year period. I am happy to be able to announce that Dr. Tchan has accepted
this appointment and we hope that he will be able to commence his work in a few months.
Dr. Tchan, who is of Chinese nationality, has had a distinguished career as a bacteri-
ologist and is at present in charge of the laboratory work and demonstrations in soil
microbiology, Institut Pasteur, and of the soils section of the French Colonial Ministry,
Paris. Dr. Tehan has specialized in soil bacteriology and has a number of publications
to his credit. Arrangements will be made for Dr. Tchan to continue this type of work
at the University of Sydney. I am sure that I am speaking for all members of the
Society when {i say that we are looking forward to a happy and profitable association
with Dr. Tchan.

iv PRESIDENTIAL ADDRESS.

Notes to Members.

We offer our congratulations to Mr. W. H. Maze, who has recently been appointed
Registrar at the University of Sydney, to Dr. J. L. Still, who was appointed to the Chair
of Biochemistry in the same University, and to Dr. E. V. Mercer, recently awarded the
degree of Doctor of Philosophy at Cambridge.

Mr. J. M. Vincent and Dr. N. C. W. Beadle were on study leave in the United States
and Great Britain during the year.

Obituary.

It is recorded with regret that Walter John Enright died on 27th September, 1949,
aged 75, after having been a member of the Society since 1914. He was born in Maitland
and was considered to be one of the finest criminal lawyers in the State, and a noted
geologist. For nine years, including two years as Mayor, he served on the West Maitland
Municipal Council. Mr. Enright graduated in 1893 as a Bachelor of Arts of the University
of Sydney, where he was also one of the first students to graduate under Professor Sir
Edgeworth David. Throughout his life he took a keen interest in geology and accom-
panied Professor David on his survey of the Maitland Coalfields. He was also an
expert on aboriginal lore and spent some time living with the remnants of the Kuttung
tribe at Port Stephens, and later, at Sawyer’s Point on the Karuah River, was admitted
as a member of the tribe. In recent years he accompanied Professor A. P. Elkin to the
north coast of New South Wales to gain further knowledge. Mr. Enright had been a
President of the New South Wales Anthropological Society, Fellow of the Anthropological
Institute of Great Britain and Ireland, Fellow of the Royal Geographical Society of
Great Britain, member of the Geographical Society of New South Wales, Member of
the Royal Society of New South Wales, Member of the Royal Australian Historical
Society, and of the Ornithologists’ Union. *

Tue Last Haunts or DEMoNS: A COMPARATIVE STUDY OF SECRETION AND ACCUMULATION.
PROLOGUE.

In addressing you on this occasion, I am conscious of the changes in biological
thought which the Society has witnessed during the 75 years of its existence. Two
extracts from the first two Presidential Addresses by Mr. William, later Sir William,
Macleay, will be sufficient to remind you of the state of biological knowledge 75 years
ago. Of the theory of evolution, that topic of intense controversy, he said, “It is
evident, I think, from the general tenor of scientific literature for some years past.
that the evolution theory, long so unpopular, and which under Lamarck’s teaching gained
so few proselytes, has, under the superior fascinations of Darwin’s admirable work,
‘The Origin of Species’, become the fashionable faith. But what may be generally
believed is not necessarily true or worthy of belief’. And a year later, after discussing
the same topic, he goes on, “But the settlement of such a question as that of spontaneous
generation is a matter of much more importance to the world at large than the most
ingenious speculations upon subjects of which we know nothing, and’are never likely
to Know much. The chief scientific discovery of the year is the entire disproval of the
doctrine” (of spontaneous generation).

Subsequently the Society witnessed the aceeptance of the theory of evolution and
the gradual enlightening of biological thought by the glow of this brilliantly
co-ordinated material. After the acceptance of the theory of evolution, perhaps the most
marked changes in biological thought during the Society’s existence were due to the
continued success of explanations of living mechanisms in terms of the properties of
matter. During the period there has been a receding of those troubled waters which
have characterized the science of biology for generations, when biology was subjected
alternately to the vitalistic flood and the mechanistic tide. We have gradually settled
to something less turbulent. The biologist is prepared to continue his investigations
without the unshakable convictions and furious polemics which characterized this age-
old issue. I do not propose to address you on the vitalist-mechanist controversy but
rather, in attempting an address sufficiently wide to interest more than one section of

PRESIDENTIAL ADDRESS. Vv

our members and sufficiently specialized to be of some scientific value, to describe a
physiological mechanism which shows similar principles in two widely separated
organisms. Our Society started when comparative morphology was the fashion in
biology: our acceptance of the theory of evolution has led us to look for similar
principles underlying physiological mechanisms in different organisms. I wish to
discuss this one particularly because it has only recently yielded to the physico-chemical
approach.

One of my most distinguished predecessors in this Presidential Chair, Professor
J. T. Wilson, devoted some portion of his Presidential Address of 1898 to the problem of
how far mechanism, i.e., physico-chemical theories are capable of being utilized in the
explanation of the phenomena of living activity. And in his own words a year later,
“ventured to state the conviction that, in so far as a strictly scientific or natural-
historical representation of these phenomena is the object aimed at, this can only be
given in terms of physical cause, or mechanism”. This early and clear warning as to
the logically dangerous position of the vitalistic exponents is still worth reading today.
In the subsequent year, Professor Wilson, who was still President, found it necessary
to return to this topic, for meantime there was published, in the ‘Nineteenth Century”,
a paper by the great physiologist, J. S. Haldane, who stated the neovitalist position. To
Haldane, the physiologist, it became unbelievable that the complexity of the processes
undertaken by certain organs of animals could be explicable in terms of physics and
chemistry. “To any physiologist’, he writes, “who candidly reviews the progress of the
last 50 years, it must be perfectly evident that, so far from having advanced towards a
physico-chemical explanation of life, we are in appearance very much further from one
than we were 50 years ago.’ In particular, Haldane found it difficult to believe that the
explanations of secretion, growth, development, nutrition, heredity and excitability
should be purely mechanical. You will be familiar with the progress that has been
made in the physico-chemical explanations of some of these ‘phenomena, but it is
particularly with that of secretion that I wish to deal.

The Search for Demons.—Let me explain why secretory processes should present
very special difficulty to a physico-chemical explanation. We know that the “no perpetual
motion” law, the second law of thermodynamics, however this concept is stated, is
obeyed by all the physical universe of which we have experience. This law, which
means that energy cannot flow from a low level to a higher one, is obeyed in all its
forms. Thus a warm body cannot grow warmer at the expense of a cooler body. A
low source of electrical potential does not charge a cell with a higher electrical potential.
Molecules do not diffuse from where they are in low concentration to where they are in
high concentration, so secretion and accumulation, defined as movement of ions in
biological systems against concentration gradients, would appear, at first sight, to
negate the second law of thermodynamics. It is, perhaps, no wonder that some began
to look for something special in the way of a force, not familiar to physics. We have
the suggestion from Johnstone (1914) and from Lewis and Randall (1923) that there
may be phases within a living cell in which the second law of thermodynamics is not
obeyed. As late as 1940, in a discussion of secretion, R. W. Gerard wrote, “Can living
things, then, transcend the second law? Is this a basis for ‘vital’ action? Some have
thought so, and possibly the time has not yet come to consider the question closed.
None the less, the probabilities are strongly against it’.

How can the plant or animal cell bring about the movement of substances in such
a direction as to reverse the spontaneous movement we should expect on statistical
physical laws? Let me confess that I have always been fascinated, as have many others,
with the idea of the demon of Clerk-Maxwell. He suggested that a minute being, able
to see individual molecules, operating a frictionless door in a wall separating two
compartments of a vessel filled with gas would be able to take advantage of the natural
movement of the molecules to separate them into a compartment of fast moving
molecules and a compartment of slow moving molecules. As every fast moving molecule
approaches from the left the demon opens the door but closes it again to every molecule
approaching from the right, so more fast moving molecules are accumulated on one side

AA

vi PRESIDENTIAL ADDRESS.

than on the other. Thus, such a demon residing in the living cell would be able to
open the surface doors to the desirable molecules, to shut them fast to those that were
undesirable, and soon all required would be inside. The suggestion that the living cell
in secretion or accumulation might operate by a mechanism which is unknown in
physics is tantamount to postulating the existence of such a demon. It will be my
purpose in this address to show that no such demon is necessary. Let me provide a
clue at the outset. The Maxwellian demon could counteract the physical law only so
long as he himself did not draw on any energy for his existence or for his work in the
opening and shutting of doors. The living cell is not short of energy and our problem
is one of how the energy is applied to secretion or accumulation.

Defining the Problem.—This property of living cells of transferring substances from
a solution in which the concentration is low to a solution in which the concentration
is high seems to be fairly general. It is useful to speak of the mechanism by which
this is brought about as the mechanism of active transport, a term which will be used
through this address to distinguish the movement of the substances into or out of
living cells by a process other than diffusion or ionic exchange. It is useful further
to distinguish those instances in which a substance is transferred from the exterior to
the interior of the cell against a concentration gradient, referred to here as accumulation,
from those in which the substance is transferred to a high concentration outside a cell
which will be referred to as secretion.

There are many examples of. secretion and accumulation of a wide variety of
compounds, and no attempt is made here to list either the types of secretion or of
secretory organs. It is of interest to members of the Society to note that the Presidential
Address of Professor W. J. Dakin in 1935 on the subject “The Aquatic Animal and its
Environment” was largely concerned with the consequences of the secretory mechanisms
responsible for the maintenance of the steady state difference between the body fluids
and the external environment. It is interesting, too, that even at that date Professor
Dakin acknowledges our almost complete lack of knowledge of the mechanism whereby
these differences are maintained. Other familiar examples are to be seen in the kidneys,
in the intestine of animals, in the gastric mucosa, in frog skin and in plant cells.
Substances undergoing active transport seem to range from water and individual ions
to organic molecules such as sugar. The problems relating to this wide variety of
fields have been reviewed in books such as those by Hoagland (“Lectures on the
Inorganic Nutrition of Plants’, 1944) and Hodber (‘Physical Chemistry of Cells and
Tissues’, 1946). The mechanisms have been the subject of review articles such as those
by Krogh (1946) and Ussing (1949).

In this address no attempt will be made to cover all these fields. It is proposed to
review the work on two tissues, widely separated in evolution—the parenchyma cells
of plant roots and the oxyntic cells of gastric mucosa in frogs. These two organs are
chosen partly because of the detailed work which has been done, and partly because, in
their .physical similarities, despite evolutionary separation, they illustrate what may be
an underlying principle of general application to all secretory and accumulatory organs.

HISTORICAL.

Ton Accumulation in Plants.—Apparently it was a long time after the discovery by
de Saussure (1804) of the uptake of different substances in varied proportions that it
was realized that some complicated mechanism of accumulation was operating in the
plant cell. At first it was thought that the uptake of these various substances was
dependent entirely upon their power of diffusion into the cells by virtue of being
dissolved in water and that the differences in proportion were due to the utilization
or destruction of the compounds after absorption. Thus Sachs, in his textbook, states
(using A. W. Bennett’s translation, 1875): “. . . will be clear that the accumulation
of certain substances in the interior of plants depends in the first place on whether the
compound of that which is present in the surrounding water is decomposed by the
plant; that, moreover, the constituents of the different compounds must accumulate
in the plant in definite quantities according to the extent in which they are needed;

PRESIDENTIAL ADDRESS. Vil

and that finally the quantitative composition of the substances in question within the
plant usually bears no resemblance to that of the surrounding water.” Leunis (1883)
quotes examples of selective uptake, such as those of De Saussure, but states that the
process is based on the laws of osmosis. Nathansohn (1901) showed that the chloride
content of a seaweed Codium tomentosum, transferred from sea water to an isotonic
solution free of chloride, did not approach physiological equilibrium as long as the
material remained turgid. He further showed, in 1903, that there was an unequal
absorption of the two ions of any salt. In the early years of this century, it came to be
realized that there was something other than diffusion and utilization of ions involved
in the absorption by plant cells. From this time on there was a number of examples
which showed, first, that ions entered plant cells independently of other ions present
in the solution, in quantities which differed widely from the proportion in which the
ions were present in the external solution, and, second, that the entry of ions was not
governed by the concentration differences between the internal. and external. medium.
Spectacular examples of this kind of ionic movement into cells were obtained. For
instance, it was shown that marine organisms frequently had very high concentrations
of potassium compared with the concentration in sea water, and the concept of
differential permeability was established. It was, however, still thought that the proto-
plasmic lining of the plant cell constituted a semipermeable membrane through which
the dissolved substances diffused to the interior of the cell, but which exercised a certain
degree of selective permeability so that some ions were barred where others could enter.
During the first twenty years of the century, due largely to the work of Osterhout, who
published various papers on this topic, of Pantanelli (1915), of Stiles and Kidd (1919),
and of Redfern (1922), it became clear that entry by exchange accounted for the unequal
absorption of ions from the external solutions. In exchange phenomena, ions entered in
exchange for mobile ions already present in the tissue; these mobile ions passed to the
external solution. At the same time, however, it was shown that absorption of both
anions and cations from a dilute solution often resulted in much higher concentrations
in the cell than in the external solution and that no corresponding ions appeared in the
external solution. Hence the phenomenon of accumulation of ions, which could not be
accounted for by simple exchange, was clearly established. By 1930, it was possible
to distinguish three modes of entry: (1) by diffusion, (2) by exchange, and (3) by the
active transport or accumulation mechanism which resulted in relatively high internal
concentrations.

During the next few years, two additional discoveries were made. The first was
that the accumulation process was connected in some way with the metabolism of the
cell and especially with aerobic respiration. It is in connection with this work that the
name of Steward is particularly associated. In a series of papers on cut potato tissue
beginning in 1932, Steward (vide Steward, 1937) showed the very close dependence of
the accumulatory mechanism upon the uptake of oxygen by the tissue. This work was
confirmed for other tissues, particularly for accumulation by barley roots, by Hoagland
and Broyer (1936). At the same time in a series of researches commencing in 1933, on
the relation between the respiration rate and the uptake of salt by wheat roots,
Lundegardh and Burstrom showed that an increase in the salt accumulation by roots
was accompanied by an increase in the oxygen uptake. At that stage LundegArdh and
Burstrom (1933) described this increase in respiration rate as the anion respiration, a
discovery which was to lead subsequently to a useful hypothesis of the mechanism of
accumulation.

Up to 1930, in spite of the knowledge of accumulation, there had been few attempts
to formulate a possible mechanism. Evidence from the work on large cells like those
of Valonia and Nitella, where the vacuolar sap can be obtained with a minimum of
injury to the cytoplasm, suggested very strongly that the accumulation occurred with
both ions free in solution in the vacuolar sap. Briggs and Petrie (1928) showed that
the observed accumulation could not be accounted for by ionic exchange or Donnan
equilibria, unless it were assumed that the cell consisted of two separate phases with

Vili PRESIDENTIAL ADDRESS.

the cation accumulating in one and the anion accumulating in the other; this seemed
to be an unlikely explanation of the phenomenon.

S. C. Brooks (1929) suggested that accumulation could result from exchange of
cations for hydrogen ions and of anions for bicarbonate ions formed from the respiratory
carbon dioxide, the nett result being the entry of both ions of a salt from the external
solution. Brooks considered it would be necessary to postulate that the cell maintained
a mosaic of cation permeable and anion permeable areas through which the cations and
anions respectively might exchange. Briggs (1930) criticized this hypothesis of Brooks
and pointed out that a mosaic would be inadequate to account for accumulation by
exchange for two ions, such as bicarbonate and hydrogen passing to the exterior of the
cell. The same criticism applies to the modified hypothesis by Brooks (1937). Briggs
pointed out that it would be possible for an exchange mechanism to bring about accumu-
lation only if there were a phase of cationic permeability alternating in time with a
phase of anionic permeability. While this system would be satisfactory on theoretical,
thermodynamic grounds, it could not be tested experimentally.

So, by 1935, the dependence of the mechanism on respiration had been clearly
illustrated, but a satisfactory hypothesis of the connection between respiration as the
energy providing process, and accumulation as a mechanism, nad not been formulated.

Hydrochloric Acid Secretion in Animals.—The realization that hydrochloric acid is
responsible for the acidity of the stomach of animals was arrived at early by at least
some investigators, for in 1824 Prout advanced the view that the acid in the stomach
was “free or, at least, unsaturated muriatic acid”. In spite of this there seems to have
been a long period of uncertainty as to whether the acid so apparent was, in fact,
hydrochloric acid, and a good deal of uncertainty as to its origin. Claude Bernard*
(1859), for instance, reached the conclusion that the gastric secretion did not acquire
its acid properties until it reached the superficial region of the mucosa and had mixed
with foods of the stomach, a belief which amounts to the suggestion that the acid is
formed externally to the glands and possibly by a fermentation of the mucus. Briicke
(1859), on the other hand, reached the opinion that the acid was formed in the glands.
Other investigators, after repeated attempts, were unable to associate the acid formation
with the glands themselves. It was not until this century that further work, with
modified micro-chemical techniques, by Fitzgerald (1910), finally established that the
hydrochloric acid was free in the secretion which appears in the canaliculi, but left
open the question of whether the acid or its hydrogen ions occurred in the cytoplasm
of the cells. It was pointed out that the cytoplasm must constitute a membrane
permeable to H+ and Cl ions, but not to Nat, K*, PO,--, or HCO-,, and here Fitzgerald
makes the suggestion that “if we suppose that the cytoplasm is so constituted as to
maintain the ionisation of the hydrogen and the chlorine in the presence of alkalies, we
attribute a property to it that could also be responsible for the formation of acid in
the first place in the cytoplasm”’.

As far back as 1845, Bence Jones had suggested the hypothesis that the formation
of acid frees alkali, and in 1859 Briicke assumed that if free acid were’liberated in the
gastric tubules, an alkali of corresponding strength must pass into the blood and lymph.
Further extensive work showed that the alkali liberated to the blood by the stomach
was equivalent to the hydrochloric acid secreted.

It is not proposed to give a complete historical account of the work in relation to
the secretion of HCl. This work has been well summarized by Hollander (1943), and
by Babkin (1944). By 1940, it was clear that hydrochloric acid passed from the gastric
mucosa into the stomach and simultaneously an approximately equivalent amount of
alkali was liberated into the blood. The importance of carbon dioxide in relation to
this alkali had been suspected, but the relation was elucidated only by the discovery
(Davenport, 1939) of a high concentration of carbonic anhydrase in the parietal or
oxyntic cells of the mucosa. Some investigators, notably Ni and Lim (1928), Hanke
(1937) and Teorell (1933), had compared the rates of secretion and of respiration
without formulating any comprehensive hypothesis as to the mechanism.

* Quoted from Fitzgerald (1910).

PRESIDENTIAL ADDRESS. 1X

RECENT EXPERIMENTAL EVIDENCE.

Accumulation in Plant Cells.—Entry of ions by the phenomena of diffusion and
exchange may be important under certain circumstances, but, generally speaking, these
lend themselves to a comparatively straightforward physical explanation. Diffusion to
equality of concentration inside and outside the cells does not seem to be a very common
phenomenon. Whereas cations diffuse and exchange .readily between the cell wall,
cytoplasm and external solution, such movement by anions is restricted because of the
large number of indiffusible anions (e.g., proteins) which cannot escape from the cell,
particularly in the cytoplasm. This has been illustrated, for instance, in discs of carrot
tissue by the electrical diffusion potential which is established when salt is allowed to
diffuse along a concentration gradient through the tissue (Briggs and Robertson, 1948).
This restriction of entry of anions from the external solution, due to the presence of a
high percentage of indiffusible anions in the cytoplasm, may well account for much of
the high resistance of the cytoplasm to the entry of salt (vide Frey-Wyssling, 1948).
Local concentrations of lipoids may also contribute to this resistance, but the importance
of the occurrence of lipoid areas in plant cells has not been fully assessed.

Because of the high percentage of indiffusible anions, many of which are accompanied
by diffusible cations in the cell, most of the exchange which occurs is cationic. The
nature and quantity of the ions leaving the cell depend both on the previous history of
the tissue and on the composition of the external solution. As the ions from the interior
of the cell are replaced by ions exchanging from the external solution, the subsequent
changes in ionic balance may affect the level of metabolic intermediates, some of which,
such as the organic acids, are themselves ions. This has been well illustrated by the
work of Ulrich (1941) and of Burstrom (1945). It can be shown, for instance, that when
entry of cations exceeds that of anions, as when potassium bicarbonate is applied, in the
external solution, the excess cations are balanced by an increase in organic acid anions
and ‘an increase in the total organic acid in the cell. When, as from solutions with a
divalent cation, the uptake of anions exceeds that of cations, the organic acid anions
and the total organic .acid in the cell decrease. These changes in organic acids are
related to changes in respiration rate, which are consistent with the acids being inter-
mediates of the respiration process.

Despite the interesting connection between exchange phenomena of this type and
the entry of ions, this mechanism alone is not adquate to explain the active transport
process which brings about the accumulation of both cations and anions in the cell
without the appearance of an equivalent quantity of ions in the external solution. It
is necessary here to explain some of the characteristics of the process of salt accumula-
tion before an attempt is made to relate it to the respiration with a view to formulating
a hypothetical mechanism.

The ions which are absorbed seem to exist in free solution in the cells and are not
absorbed in high concentration because they are destroyed or combined after entry;
two kinds of evidence suggest that the ions are free in solution. The ions are liberated
readily on rupture of the cells indicating that they are not adsorbed on the cell
constituents. In the large cells of Nitella, it has been possible to withdraw solution
from the vacuole with very little injury to the cytoplasm and to find the accumulated
jons free in this solution. Further, the accumulated ions exert the expected osmotic
pressure.

The accumulatory power of the cells is considerable. Many figures have been
published which illustrate the high ratio of the concentration of a particular salt in the
cell to the concentration of the same salt in the external medium. It is common, for
instance, to find that tissue is capable of reducing the external concentration almost to
zero and simultaneously of building up an internal concentration which is hundreds of
times more concentrated than the external solution. The rate of accumulation of salt is
of the order of 0-4 x 10° gram molecules per gram of tissue per hour. This rate
of accumulation does not remain constant with time, being highest immediately after
the salt comes into contact with the surface of each cell, and gradually falling as the

x PRESIDENTIAL ADDRESS.

internal concentration rises. During this period it is necessary to distinguish the entry
of the salt by the accumulation process, from the entry of the individual ions by
exchange; these two processes have been examined in detail by Stiles and Skelding
(1940, a, b) and Stiles and Dent (1946). The accumulation rate proper falls, however,
as the internal concentration of salt increases. At no stage is there any suggestion that
the entry of both ions of the salt along a concentration gradient is important, for the
rate of accumulation does not seem to be altered sharply after the internal concentration
becomes equal to that of the external solution.

When the accumulation rates are compared by taking initial rates at different
concentrations, it is found that the concentration of the external solution limits the
rate at low concentrations, but that the rate approaches a maximum asymptotically
as concentration is increased. This shows very clearly that the accumulatory mechanism
is basically dependent upon some internal factor in the cells and, except at very low
concentrations, is not a function of external concentration. Temperature increases the
vate of accumulation and the calculated energy of activation suggests that the active
transport is dependent not on a diffusion process, but possibly on a chemical process.
The salt that is accumulated is retained by the cell and is not lost to the external
solution even when the concentration in the external solution is reduced to a low value
by the absorption of salt, or by transferring the tissue back to water. The process of
accumulation can be influenced by various inhibitors; the evidence from inhibitor
experiments will be discussed later when accumulation and respiration are being
considered.

Since the accumulation of salt cannot be brought about (unless one admits that it
defies the second law of thermodynamics) without the expenditure of energy on the
part of the cell, it is natural that investigations should be designed to determine the
relationship between the rate of respiration and salt accumulation. Lundegardh and
Burstrom (1933) showed that increase in salt accumulation was accompanied by an
increase in respiration rate, which they termed the anion respiration. This respiration,
which is also known as the salt respiration, was subsequently shown to occur in various
tisSues (Steward, 1937, Robertson, 1941). It is unfortunate that an early controversy
about the validity of this concept of anion or salt respiration obscured the vision of a
number of investigators and prevented the ready recognition of the value of this
discovery as a basis for a hypothetical mechanism. It can, however, be shown in a
variety of tissues that, if cells have been respiring in water, the addition of salt brings
about an increase in the rate of respiration which may, under some circumstances, be
as great as 100%. This increased rate of respiration is achieved in a very short time
(almost as quickly as the salt reaches the surfaces of all the cells), and subsequently
continues as long as the salt concentration at the surface of the cell is maintained,
despite the change in the rate of accumulation. Salt respiration, as this increase will
be termed here, increases with external concentration of salt at low concentrations, but
becomes independent at higher concentrations. It is increased by increasing temperature
and has a high energy of activation (Robertson, 1944). Transfers of tissue from salt
solution to water are followed by a slow decline in the rate of salt respiration (Robertson
and Thorn, 1945).

It soon became clear that, at least in those tissues examined in detail, the accumula-
tion mechanism was bound up with the extra respiration which results from the
stimulus of the salt applied to the external surface of the cells. The accumulatory
mechanism is not dependent merely upon the respiratory level but appears to depend
upon some specific stages in the respiration. For instance, most tissues will cease to
accumulate and may even lose salt already accumulated if changed from aerobic to
anaerobic conditions. Further, the addition of methylene blue, which actually increases
the oxygen uptake of respiring tissue, results in a cessation of the accumulatory process
(Hoagland and Broyer, 1942). It was, however, the evidence from experiments with
inhibitors which established firmly the dependence of the accumulation on the salt
respiration. It was found that the salt respiration was inhibited by cyanide, azide and
carbon monoxide; these inhibitors also stopped accumulation. These same inhibitors

PRESIDENTIAL ADDRESS. xi

were either without effect on the basal respiration of tissue respiring in water, or
inhibited it only partially (vide Robertson and Turner, 1945). Since it was generally
considered that sensitivity of these inhibitors indicated a cytochrome (iron-porphyrin)
system as the carrier of hydrogen ions and electrons to the oxygen involved in respira-
tion, this suggested that accumulation was probably linked in some way to the operation
of a cytochrome system. This suggestion was first made by Lundegardh.

The first attempt to obtain a quantitative relation between the amount of salt
accumulated and the salt or anion respiration were made by Lundegardh and Burstrom
(1933). They showed the factors which increase salt respiration also increase the salt
accumulation and, taking this together with the inhibitor evidence, Lundegardh developed
a hypothesis for the relation of the accumulatory processes to the cytochrome system.
This hypothesis, which was first stated briefly in 1939, has been modified from time to
time and is here given in the form which was restated by Robertson and Wilkins
(1948). Lundegardh’s hypothesis depends on the fact that when hydrogen atoms are
released from respiratory intermediates by the dehydrogenases, the electron of each
hydrogen atom passes to the cytochrome carrier, and the hydrogen ion is liberated into
the medium. Subsequently, by the intervention of cytochrome oxidase, the electron
passes to an atom of oxygen, the hydrogen ion is picked up from the medium and, on
the transfer of two electrons and two hydrogen ions to one oxygen atom, a molecule
of water is formed.

Lundegardh suggested that a substance such as cytochrome which carries electrons
in one direction because of the change in valency in the iron atom could carry anions
in the opposite direction. Simultaneously, the hydrogen ions liberated as the result of
the dehydrogenase activity pass to the exterior of the cell, and it was assumed that the
eations from the exterior would enter the cell by exchange with those hydrogen ions.
Thus the driving force for the accumulation against the concentration gradient would
be the continuous production of positive and negative charges which traverse the
cell boundary in exchange for the ions of the external solution. Since the hydrogen ions
and electrons are ultimately united with oxygen to form water, ions disappear from the
external solution.

Lundegardh’s hypothesis suggested the possibility of putting the relation between
ion accumulation and salt respiration on a quantitative basis. If the Lundeg&rdh
hypothesis were correct, the maximum possible rate of accumulation would occur when
one anion enters as one electron is transferred by the carrier system, and when, simul-
taneously, one cation enters in exchange for the corresponding hydrogen ion. Since
salt respiration has a respiratory quotient of unity and may therefore be considered as
a normal carbohydrate respiration, the overall quantitative relation between oxygen
uptake and water formation may be represented in the following equation:

C, Hy O, + 60, + 6H.0 = 6CO. + 12H.0

This allows for the fact that all the oxygen which enters into the respiratory process
makes its appearance as water, and each oxygen molecule taken up in respiration is
therefore balanced by four hydrogen atoms. This means that for each oxygen molecule,
four hydrogen ions are required at the cytochrome oxidase and four electrons have
traversed the cytochrome carrier system. Thus the maximum theoretical accumulation
rate on the Lundegardh hypothesis should be four times as much monovalent salt
accumulated as oxygen taken up in the salt respiration process when both are expressed
in gram molecules. To test this hypothesis quantitatively, we carried out a number
of experiments on carrot tissue (Robertson and Wilkins, 1948). These experiments,
designed to compare the rate of accumulation and the rate of salt respiration when
neither was limited by the external concentration of salt, showed that the ratio of
equivalents of salt accumulated to gram molecules of oxygen consumed in salt
respiration approached the hypothetical value of four. Thus these quantitative
observations which are not in themselves proof of Lundegardh’s hypothesis, are
consistent with the possibility that the production of hydrogen ions and electrons in
respiration is the driving mechanism for the accumulation of salt in the cells.

xii PRESIDENTIAL ADDRESS.

Many other factors must be taken into account before this hypothesis can be
regarded as complete. Some of these factors have been considered by Lundegardh
(1940, 45, 49). It is necessary to postulate either that there is a specific carrier
mechanism for the anions and a separate carrier mechanism for the cations or that, as
Briggs has suggested, there is a short period during which the cytoplasm is permeable
to anions followed by a period in which the cytoplasm is permeable to cations. If this
occurred with a cell in a solution of potassium chloride, for instance, hydrochloric acid
would be formed in the cell during the anion permeable phase, with the anion from
the external solution balanced by the hydrogen ion liberated from respiration. If then
the cell became permeable to cations and temporarily impermeable to anions, the
hydrogen ions could be exchanged to the exterior for the potassium ions of the external
solution. The significance of this will be discussed subsequently in consideration of
the theory of secretory and accumulatory processes.

Whatever the mechanism of active transport of ions across the cell to the vacuole,
it is necessary to postulate some degree of resistance to the back diffusion of ions which
have been accumulated in the cell in a concentration greater than that of the external
solution. It is not yet clear where this resistance to back diffusion occurs. It seems,
however, likely that the resistance to ionic movement by free diffusion is quite high
since all impedance measurements of plant cells (cf. Cole and Curtis, 1938) indicate
a high resistance. Further, it seems unlikely that the mechanism of active transport
would be adequate to cope with back diffusion of ions if the resistance of the cytoplasm
were as low as that of water. This can be seen in experiments in which inhibitors
such as cyanide are used to block the active transport mechanism, when only low rates
of leakage from the cells are observed. Lundegardh considered that most of this
resistance to back diffusion is due to a surface layer of lipoid, similar in structure to
the Langmuir monolayers. This view is supported by Holm-Jensen, Krogh and
Wartiovaara (1944) from their work on Nitella and Tolypellopsis. The evidence for
most of the resistance of the cytoplasm being at or near the tonoplast has been discussed
by Arisz (1945). The position of the high resistance zones of the cytoplasm requires
further investigation.

Any hypothesis of the mechanism of salt accumulation must also make allowance
for the specificity of absorption of different ions. On any simple electrochemical theory,
no marked difference would be expected between, for instance, the absorption of sodium
and that of potassium, but in many biological systems the cells may absorb one of these
ions in much greater quantity than the other. It seems therefore necessary, as pointed
out by Ussing (1949), to postulate that an entry of ions by the active transport
mechanism occurs as the result of temporary combination with cell substances whose
combining power with different ions varies considerably. That such substances exist
in biological systems and differ in their combining power, even between similar ions
such as sodium and potassium, is a very reasonable possibility (vide Franck and Mayer,
1947), but so far no such substance has been identified as a carrier in ‘active transport
mechanisms.

Recent work has suggested the possibility that the accumulatory mechanism may, to
some extent, depend upon the phosphate transfer system of the living cell. Since this
system is the best understood energy-transfer mechanism linked to the respiratory
process, it is not surprising that there have been suggestions for some time that the
accumulation mechanism might depend on it (Hoagland, 1944: Nance, 1949). Some
evidence for this suggestion has recently been obtained with work on the effect of 2,
4-dinitrophenol which has been shown to inhibit the transfer of energy-rich phosphate
from respiration (Loomis and Lipmann, 1948). Robertson and Wilkins (unpublished
data) have found that this compound inhibits accumulation without depressing
respiration. It is not yet clear whether the phosphorylations are directly concerned
in the active transport mechanism or whether this inhibition of accumulation is due to
some disorganization of the cytoplasmic structure consequent upon the inhibition of
phosphate transfers.

PRESIDENTIAL ADDRESS. Xlil

Lundegardh has tried to correlate the accumulatory mechanism of roots with the
electrical potential difference obtained across the root surface and influenced in various
ways by the presence of ions in the external solution. These potential differences are
difficult to interpret because of the complexity of the system across which the difference
is measured. Lundegdardh himself believes that this potential difference and modifications
obtained changing from one solution to another, are associated primarily with the outer
surfaee of the epidermal cells of the root, and bases this belief upon the rapid change in
potential consequent on a change in the external solution (Lundegardh, 1941). Ions
absorbed by the cut root are concentrated in the sap bleeding from the cut end, which
is at a potential different from that of the external solution. The maintenance of this
potential difference may assist in the movement of one of the ions of a salt, but can
hardly, on our present knowledge, be related directly to the active transport of both
ions. Lundegardh’s results are important, however, and suggest a causal relation
between the magnitude of this potential difference and the respiration rate.

So far, therefore, evidence suggests that there is a relation of the accumulatory
mechanism to the later stages in the respiratory process and that the continued
production of positive and negative charges, resulting from the cytochrome system,
might provide the energy necessary for the accumulation of ions from the external
solution. Any such hypothesis, however, requires to be adequately co-ordinated with
observations on the specificity of ion accumulation and on the effect of 2, 4-dinitrophenol
which may be assumed to be preventing phosphate transfer. —

Hydrochloric Acid Secretion.

The process of secretion by the gastric mucosa results in the production of hydrogen
ions in concentrations exceeding (by about three million times) the concentration in
the blood. Simultaneously, chloride ions are secreted in approximately equivalent
concentrations to the hydrogen ions and in higher concentrations (by about 1-6 times)
than the chloride of the blood. Secretion can continue over long periods, working at the
rate of 10,000 calories per gram molecule HCl secreted, and is dependent upon the
respiration. Following the work of Davenport, it was suggested by Davenport and
Fisher (1940) that carbon dioxide, which is changed to carbonic acid by the action of
carbonic anhydrase, is associated with the formation of hydrochloric acid and perhaps
directly concerned in its production. Subsequently, however, following very careful
study of the secretion of hydrochloric acid in isolated pieces of frogs’ gastric mucosa,
Davies has shown that the activity of carbonic anhydrase is associated with, but is not
the direct cause of the acid production. This work by Davies and his collaborators, like
that of investigators of accumulation in-plant tissue, has attempted to establish the
quantitative relationship between the stimulated respiration, which occurs when the
mucosa is acted on by histamine to commence secretion, and the hydrochloric acid
production. The stimulated respiration is sensitive to cyanide as an inhibitor and
secretion ceases when this respiration is inhibited. Anaerobic conditions also bring
about a cessation of secretion. Respiration but not secretion is stimulated by brilliant
cresyl blue and fluoride, whilst respiration is increased but secretion is inhibited by
azide and 2, 4-dinitrophenol. The similarity of the secretory mechanism and the stimu-
lated respiration to accumulation and salt respiration in plant cells, except in behaviour
to azide, might therefore suggest an underlying principle common to both mechanisms.

In recent years, Davies and his co-workers have examined the quantitative relation
between the amount of oxygen uptake in the stimulated respiration and the hydrochloric
acid secreted by the oxyntic cells of the mucosa. Harlier workers had not produced
adequate evidence of this quantitative relation, though it can be calculated from the
data of some workers that the amount of HCl secreted was in considerable excess of the
amount of oxygen absorbed. In 1948 Crane and Davies showed that 11 gram molecules
of hydrochloric acid were secreted for each gramme molecule of oxygen taken up.
Consequently the simple relationship of ionic secretion to the hydrogen ion production
resulting from and electron transport through the cytochrome system, must be modified
to apply to the secretion of hydrochloric acid.

X1V PRESIDENTIAL ADDRESS.

The gastric mucosa presents excellent material for study of phenomena of this
kind because it is possible to determine the electrical potential between the secretory
and non-secretory sides of the mucosa, and even to impose external electrical potentials
across the secretory system. In the resting condition the secretory side of the isolated
mucosa is about 30 mV negative in the external circuit to the non-secretory side, when
both are in a biological saline medium. Experiments with silver chloride and glass
electrodes show that the mucosa in the resting condition is apparently impermeable
to chloride ions and to hydrogen ions in the range from pH5 to pH8. If connected
electrically, the two sides of the mucosa can provide electric power continuously. When
secretion begins, the potential difference across the mucosa falls and the resistance rises.
When the natural potential difference is enhanced by passing a current through secreting
mucosa, the rate of secretion is increased and when the natural potential difference is
opposed, the rate of acid secretion is decreased or stopped. Clearly, the secretion of the
hydrochloric acid is associated with the activity of an electro-chemical system which
succeeds in separating hydrogen ions, not only those from the dehydrogenases of respira-
tion, but also from some other source, from their corresponding hydroxyl ions. The
hydroxyl ions have a very short existence because under the action of carbon dioxide
and carbonic anhydrase, they are combined to give bicarbonate ions which then pass
back from the oxyntic cells into the blood stream in the “alkaline tide” which has been
recognized for some time. An exactly equivalent amount of acid and alkali are
produced, the acid being secreted. to the lumen of the stomach and the bicarbonate
passing to the blood stream. Following the early theory enunciated by Crane, Davies
and Longmuir (1948), Davies and Ogston (1949, 1950*) have extended the theory to
make specific proposals for a way in which metabolic energy is produced and applied
to the secretion of this quantity of hydrochloric acid. As this theory is of considerable
importance in its possible general application to secretory and accumulatory processes,
it will be discussed in some detail.

Davies and Ogston distinguished two mechanisms by which hydrogen ions may be
generated, either from the substrates of respiration or from water. The first mechanism
is essentially the same as that discussed already, based on Lundergardh’s hypothesis
for the accumulation of salts in plant cells. By this mechanism the maximum production
of hydrogen ions is four per molecule of oxygen. The second mechanism proposed uses
the energy of the unstable phosphate esters to transport the hydrogen ions derived from
water. It is suggested that a carrier substance reacts with hydrogen ions and a reduced
iron-porphyrin to give reduced carrier and oxidized iron-porphyrin. This reduced carrier
then becomes phosphorylated by creatine phosphate and diffuses through a region of the
cell which is impermeable to free hydrogen ions. It then liberates hydrogen ions as the
result of losing inorganic phosphate and undergoing oxidation by the iron-porphyrin
enzyme. The ferrous-ferric system returns to its initial state by transferring electrons,
the carrier and the phosphate diffuse back, chlorides diffuse to the hydrogen ions to
maintain electrical neutrality and the nett result is the hydrolysis of unstable phosphate
and a transport of hydrochloric acid. This mechanism can transfer two hydrogen ions
per phosphate, so that if the phosphate/oxygen ratio is two, eight hydrogen ions may be
transported with one oxygen molecule taken up.

If the two mechanisms are working together, it is possible for a maximum secretion
of twelve hydrogen ions per oxygen molecule absorbed. Thermodynamic considerations
showed that the free energy of the hydrolysis of creatine phosphate would be sufficient
to account for the secretion of two hydrogen ions per phosphate.

Simultaneously, Davies and Ogston make some suggestions as to the observed
potential difference. It is suggested that the outer surface of the resting oxyntic cell is
impermeable to H* ions and to Cl ions so that the surface acts, relative to the interior,
as a reversible sodium electrode. Inside the cell, hydrogen ions and sodium ions are
maintained at different concentrations by the secretory mechanism. At the onset of
secretion the permeability of the outer surface of the cell is changed so that it becomes

*T am indebted to Dr. Davies and Dr. Ogston for permission to see this manuscript before
publication.

PRESIDENTIAL ADDRESS. XV

permeable to hydrogen and to chloride. This tends to cause the observed drop in the
potential difference across the cell. The effect of an external source of current on the
rate of secretion of active mucosa may be simply electrolytic. This electrolytic effect
is not observed in the resting mucosa, probably because of the low permeability to
hydrogen ions.

The Davies and Ogston theory, therefore, proposes two mechanisms whereby the
high ratio of hydrochloric acid secreted to oxygen absorbed can be explained and the
relationship of the mechanism to the phosphate transfers is suggested. These two
mechanisms working together in parallel would explain a high ratio of secretion to
oxygen uptake. If the two mechanisms weré working in series, a low ratio would be
obtained, but the system would still depend upon phosphate transport. The possible
general significance of these theoretical considerations will be discussed in the next
section.

PRINCIPLES OF ACCUMULATION AND SECRETION OF IONS.

We are now in a position to review the principles involved in the ability of living
cells to secrete or accumulate ionic substances. Various hypotheses have been made from
time to time, but most of these are based either on insufficient information about the
chemical entities of the cell or on erroneous assumptions about the physical constitution
of the cytoplasm. The most valuable discussion of the theoretical mechanisms involved
is that given by Franck and Mayer (1947) in which they discuss, without specific
relation to particular secretory processes, the principles involved in the expenditure of
chemical energy and its application to performing useful osmotic work. The value of
observations on salt accumulation in plants and hydrochloric acid secretion in animals
is that they permit the introduction of hypothetical mechanisms dependent upon known
biochemicai activities of the cell.

The underlying principles of secretion have been dealt with very clearly by Davies
and Ogston (1950). Where secretion or accumulation implies nett movement of
electrolyte to a higher chemical potential, these authors point out that constraint must
be placed on at least one ion. Since a single ion cannot be secreted alone, because of
the requirement of maintenance of electrostatic neutrality, a second ion follows that
upon which the constraint has been placed. It is useful to distinguish these two ionic
behaviours in Davies and Ogston’s term of “primary” secretion for the ion undergoing
constraint and “secondary” secretion for that which follows to maintain the electro-
static neutrality. Initially the metabolism of the cell must be applied in such a way as
to produce and separate electric charges and it is interesting to note in this connection
that one of the most important cell mechanisms bringing this about is the cytochrome
system in which an electron is removed from a hydrogen ion. Similarly it is possible
for the constraint on an ion to be brought about by a carrier which combines so
effectively with a “primary” ion that it can remove it from its partner and transfer it
elsewhere in the cell. If it were possible for a carrier simultaneously to combine with
both ions of a salt and to lose those two ions elsewhere in the cell an accumulation of
salt would occur. As Franck and Mayer pointed out, this is possibly an over-simplified
picture.

Whatever the mechanism, the primary cause of secretion is the production and
separation of positive and negative charges and in many respects the first mechanism
suggested above for the secretion of hydrochloric acid, where the hydrogen ion is
liberated when an electron is passed through the cytochrome system, represents the
simplest expression of the mechanism. After the primary secretion of these hydrogen
ions, chloride ions can be regarded as moving toward the hydrogen as the result of the
‘establishment of a potential gradient. Such a mechanism alone, however, could not
explain the secretion of both ions of a salt, and now it is necessary to introduce the
concept referred to by Davies and Ogston as “tertiary” secretion. Supposing that the
hydrogen ion formed initially were transported to the non-secretory surface in exchange
for the cation which accompanied the chloride, then salt would have been secreted and
the cation is said to have entered by “tertiary” secretion.

Xvi PRESIDENTIAL ADDRESS.

A further necessary consideration is that there must be some selective permeability
to the ions on which the process operates. Thus, if hydrochloric acid, for instance, is
being secreted, the cell must be permeable to the chloride ion, but relatively impermeable
to the hydrogen which is the ion of primary secretion. If tertiary secretion is to occur
so that the cation accompanying the chloride is to enter, the cell must become permeable
to the primary ion (hydrogen) and to the tertiary cation. For the reasons pointed out
by Briggs (1930), the secondary entry of chloride following the primary secretion of
hydrogen and the tertiary exchange of hydrogen for the cation could not occur simul-
taneously with ions free in solution. Such a mechanism could operate only if the phase
of permeability to chloride were followed, in time, by a phase of permeability to cation.
It is not possible to have both ions of a salt moving in solution in the same direction
against a concentration gradient, because the potential gradient can favour the move-
ment of only one of the ions. If, however, the ions were not free in solution, but each
ion were combined firmly to an appropriate carrier, the secretory mechanism could
operate in the manner outlined. The anions would be passed to the secretory surface
as a carrier passes anions formed by the cell to the non-secretory surface. Another
carrier would pass hydrogen ions formed by the cell to the non-secretory surface and
carry cations back to the secretory surface.

As long as the quantity of ions secreted does not exceed that which corresponds to
hydrogen ions and electrons coming from the substrates of respiration at the point of
action of the cytochrome system, this system will account for the drive to secrete or
accumulate. We have seen, however, that the hydrochloric acid secretion, as examined
by Davies and his collaborators, appears to exceed that which would be possible on the
basis of the operation of the cytochrome system alone. The hypothesis, suggested by
Davies and Ogston, accounts for the additional hydrogen ions as being formed in a
phosphorylating cycle. It is interesting to note that the passage of electrons through the
cytochrome system is still involved in this second mechanism. Schematically these two
mechanisms are summarized in Text-fig. 1.

These two mechanisms working in parallel would be adequate to explain the
secretion of hydrochloric acid. Perhaps in the accumulation by plants, the two systems
operate in series so that the amount accumulated never exceeds that which is possible
from the liberation of charges by hydrogen from substrate molecules. If this were so,
it would account for the inhibitory effect of a substance like 2, 4-dinitrophenol which can
prevent phosphate transfers.

Davies and Ogston have discussed the thermodynamic relationships of the mechanism
of secretion and have reached the conclusion that the energy liberation in the two
mechanisms suggested would be adequate to bring about the concentration of hydrochloric
acid observed on the secretory side. A detailed thermodynamic examination of the
suggested mechanism applied to plant cell secretion has not yet been carried out.
It has previously been pointed out (Robertson, 1941) that the energy necessary to
bring about accumulation represents a very small fraction of the total energy liberated
simultaneously in the process of salt respiration.

The mechanisms suggested by Davies and Ogston are shown to be compatible with
the potential differences observed in the resting and in the secreting mucosae. The
application of similar hypotheses to plant cytoplasm in the accumulating state and
further consideration of Lundegardh’s work, may lead to a satisfactory explanation of
the potential differences observed, and reveal the connection between these potentials
and respiration on the one hand and salt accumulation on the other. Davies and
Ogston conclude with some suggestions as to how variations of these two mechanisms
could be applicable to other types of secretion, such as secretion of acids by the tubules
of the kidney, absorption of bicarbonate from the tubular fluid, secretion of bicarbonate
in the pancreas and secretion of neutral salts. Thus we have the opportunity of formu-
lating hypotheses which can be tested by suitable experimental observations on some
of these tissues. We have reached a stage in the investigation of secretion and accumula-
tion when reasonable explanations, using biochemical entities of the cell, can be

PRESIDENTIAL ADDRESS. Xvii

suggested for the general principles of these two processes. There is still much to do,
much we do not know, and there are many gaps to be filled.

Perhaps the largest gap in our knowledge is that which arises from our ignorance
of the submicroscopic organization of the cytoplasm and of the distribution of the active
constituents of the cell. It is perhaps necessary here to warn against hypotheses which

FIRST MECHANISM

SECRETORY SIDE NON-SECRETORY SIDE
es + 4H (FROM SUBSTRATE) 4HCO3-
|, ( +4CO,
2S hn) A heya A Ee BAG ER

V

2S + (4H* + 4Fett

ELECTRON TRANSFER
| (IRON-PORPHYRIN i,
SYSTEM)
++
Aga ar OL, te ATO
SECRETION

4cl~ 4cl™

IMPERMEABLE
TO Ht, OH” AND HCO;

SECOND MECHANISM

NON-SECRETORY SIDE

SECRETORY SIDE

CHP CHP Ee

HYDROLYSIS CH, + CrP

CH, +P P+Cr

Cy Para vue 2Fet+*+ CH,
\} ELECTRON TRANSFER
aaa a
Co (atlpata bess APO a C tp QM 4p QYIGO=
2H,0 + 2CO,

ACT

IMPERMEABLE
TO H+, OH- AND HCO3-

Text-fig. 1.
The two mechanisms suggested by Davies and Ogston (1950) for the secretion of
hydrochlorie acid. Symbols: §S, a carrier (e.g., flavoprotein); C, a carrier;
Cr, creatine; P, phosphate. It is assumed that the separating structure in the
second mechanism is less permeable to CH, than to CH,P and C or that CH,P
is decomposed only on the secretory side.

suggest the orientation of the functional constituents in relation to hypothetical cell
membranes whose existence is difficult to demonstrate and whose positional relation to
the active constituents of the cell is practically unknown. That there is an external
membrane consisting of a few layers of oriented molecules in a lipo-protein complex,
seems a very likely hypothesis for some cells. There is, however, no evidence that such
an external membrane is of universal occurrence and it may be that the active
constituents of the cytoplasm operate in some mesh not protected from an external
solution by a neatly arranged lipoid membrane. The lipoid-like properties which

Xviii PRESIDENTIAL ADDRESS.

explain so many observations on permeability of the cytoplasm need not necessarily
be on the surface, but may be at some depth in the cytoplasm. Possibly it is here
that the specific substances which act as carriers of ions, pick up the ions, and
differentiate in their powers of combination between different ionic substances. These
carriers themselves may depend for their existence upon the high degree of organization
of the cytoplasm and might, if they depended upon a lipo-protein complex, for instance,
lose their identity with the destruction of the cytoplasm in any way. This could explain
why it is difficult to find ions associated with any particular substances when secretory
or accumulatory cells are broken up for analysis. In this connection, it is interesting
to note the very marked differences obtained by Jacobson and Overstreet (1947), using
radio-active ions, between the absorptive capacity of living and killed plant cells.
Furthermore, if active transport is intimately concerned with the late stages of respira-
tion and perhaps with the phosphorylations which accompany them, we shall have to
examine the possibility of this mechanism being included in the organized regions of the
cells in which these respiratory actions occur. Recent work points to the particulate
nature of the cyclophorase system and the probable association of the dehydrogenases
and phosphorylases with the cytochrome enzymes in the mitochondria. The possible
significance of these observations in relation to the suggested secretory mechanisms has
still to be explored.

HIPILOGUE.

We have reached a partial explanation of the accumulatory and secretory mechanisms.
However incomplete and however much detail remains to be filled in, we can see that
there are reasonable possibilities, depending only on the kind of biochemical changes
which we know to be going on continuously in the cell, for explaining the once
mysterious mechanisms of secretion and accumulation. We see that the living cell
does not disobey the second law of thermodynamics and that the demons have been
exoreized.

The living cell has the characteristic property of harnessing the energy flux to
perform useful work. Here, there is no incompatability with physical law, but, as
Schrodinger (1948) points out, the living cell ‘feeds on negative entropy” or, unlike
the physical universe, “produces order from order’. The secretory mechanism is just
a special example of the living organism’s ability to use energy derived in abundance,
fundamentally from carbohydrate, itself formed in accordance with physical laws, to
do useful work. In doing so it is using matter which is obeying the laws of matter so
that to return to the words of J. T. Wilson with which I began, “in so far as a strictly
scientific representation of this phenomenon is the object, this can only be given in
terms of physical cause or mechanism’. On this, perhaps, the modern biologist has
resolved the old controversy between the vitalists and the mechanists. The organism
exhibits properties which though not apparent from a study of non-living matter, are
consistent with all the facts derived from such a study; such properties do not merit
the terms “vital force’. The demons of the vitalists were never more than the flight
of fancy which Clerk-Maxwell intended his demon to be.

I wish to thank Professor C. W. Emmens, Dr. H. S. McKee and Miss M. J. Wilkins
for their helpful criticism of the manuscript on which this address is based.

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PRESIDENTIAL ADDRESS. bebe

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, 1899.—Presidential Address. Proc. LINN. Soc. N.S.W., 24: 1-29.

The Honorary Treasurer, Dr. A. B. Walkom, presented the Balance Sheets for the
year ended 28th February, 1950, duly signed by the Auditor, Mr. S. J. Rayment, F.C.A.
(Aust.), and he moved that they be received and adopted, which was carried unanimously.
A vote of appreciation for Dr. Walkom’s care of the Society’s finances was passed.

No nominations of other candidates having been received, the President declared
the following elections for the ensuing year to be duly made:

President: D. J. Lee, B.Sc.

Members of Council: R. H. Anderson, B.Sc.Agr.; W. R. Browne, D.Sc.; N. A.
Burges, M.Sc., Ph.D.; A. N. Colefax, B.Sc.; S. J. Copland, M.Se.; D. J. Lee,
B.Sce.; J. M. Vincent, B.Sc.Agr

Auditor: S. J. Rayment, F.C.A. (Aust.).

A cordial vote of thanks to the retiring President was carried by acclamation.

Xxi

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XXiv

ABSTRACT OF PROCEEDINGS.

ORDINARY MONTHLY MEETING.
29th Marcu, 1950.

Mr. D. J. Lee, President, in the Chair.

Library accessions amounting to 56 volumes, 250 parts or numbers, 18 bulletins,
16 reports, and 1 pamphlet, total 341, had been received since the last meeting.

PAPERS READ (by title only).
1. Reduvioidea from New South Wales. Notes and Descriptions (Hemiptera). By
Petr Wygodzinsky. (Communicated by J. W. T. Armstrong.) :
2. Australian Ephemeroptera. Part I. Taxonomy of New South Wales Species and
Evaluation of Taxonomic Characters. By Janet E. Harker.

ORDINARY MONTHLY MEETING.
26th Apri, 1950.
Mr. D. J. Lee, President, occupied the Chair.

The President announced that the Council had elected Dr. A. B. Walkom to be
Honorary Treasurer and Dr. Lilian Fraser, Dr. G. D. Osborne, Dr. R. N. Robertson and
Mr. A. R. Woodhill to be Vice-Presidents for the 1950-51 session.

The President congratulated Dr. I. M. Mackerras on being awarded the Clarke
Memorial Medal of the Royal Society of New South Wales for 1950 for his work on
Diptera, and Dr. T. B. Kiely for obtaining the degree of D.Sc.Agric., and the award of
the Royal Society’s Medal for his work on the Black Spot of Citrus. Miss Muriel Morris,
Linnean Macleay Fellow in Zoology, was congratulated on obtaining the M.Sc. degree of
the University of Sydney.

The President referred to the death on 2nd April, 1950, of Professor W. J. Dakin,
who had held the Chair of Zoology in the University of Sydney from 1929 to 1948 and
who had been a member of the Society since 1929. He was a member of Council from
1930 to 1943 and President during the 1934-35 session. He was a noted authority on
marine biology. ‘

Messrs. K. G. Brown, Randwick; L. D. Crawford, B.Se., Newtown; Dr. K. V.
Krishnamurthy, M.A., Sydney University; and Mr. A. K. O’Gower, B.Sc., Harbord, were
elected Ordinary Members of the Society.

Library accessions amounting to 7 volumes, 47 parts or numbers, 2 bulletins, and
4 reports, total 60, had been received since the last meeting.

PAPERS READ.

1. A Study of the Alkaline Phosphatase Reaction in Tissue Sections. Parts I and II.
By K. W. Cleland.

2. Dilation of the Foot in Uber (Polinices) strangei (Mollusea, Class Gastropoda).
By Muriel C. Morris, Linnean Macleay Fellow in Zoology.

3. The Hair Tracts in Marsupials. Part IV. Direction Characteristics of Whorls
and Meristic Repetition of Radial Fields. By W. Boardman. :

4. A Hybrid Eucalyptus. By L. D. Pryor.

AUSTRALIAN EPHEMEROPTHRA.

PART I. TAXONOMY OF NEW SOUTH WALES SPECIES AND EVALUATION OF
TAXONOMIC CHARACTERS.

By JANET H. Harker, B.Sc., Department of Biology, New England
University College, Armidale, N.S.W.

(One hundred and one Text-figures.)

[Read 29th March, 1950.]

Contents. Page.

Introduction 1
Taxonomy 2
Taxonomic Gingimnctene 2
Types °
Method of Daseaiwvion 6
Key to the Hphemeroptera of New Sorin Ww nies 6
Description of Species 8

(o°e)

SUperhamiullyarsaActOld Camere eis nn ore eens ome BR Oe TRL ino S eg has bee ta She do MEO Mee
Ea hyaeueptOpMLebiidale mens in or tletdalen t,he /S Recbhy Pn Gee MRD SS Bates ee PTS pean cane 8

Genus Atalophlebia Se Ae <p We = Esti ba Ere ae bE Sh ea de BE A BeaNcs MU CEE Ge 2 a ee FOE eo 8
Genus Leptophlebia RRL? SR I eel ed eA a Ur ie A AUr rai nie Aim Re roonael Reieh > eect 7
CLETUS CLE CLIC AU IN, Oi ac een SOME RRS eae) Re, See ou seein any Spiers Pa aoa eeepee aNre ch
Family Baétidae 5 ieeeny alin alert hoe ee ra ean Sida ac ain Dein hace aon Mac i nmin a de RO RE 22
Genus Baétis ee CC Aig Tecan ists MEN Te cea Las Men STU ee os mesh hc BERN s Anes oy ee GMOS meas ewes Roe b
Genus Cloéon UPS EMI 2 Gel dar oye Nea Re PCat Os, Aare ERS Man ee eR we ae eer) aan SETAE

Family Caénidae

Genus Caénis

Superfamily entasonoides

Genus Atopopus
Check-List of Australian Sneccen
Techniques :
Appendix I. Genus Wreioneiia 2 :
Appendix II. Description of a Specimen Seon ERG
Acknowledgements
References

bo bo bo
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Ol a ae, )

INTRODUCTION.

The Ephemeroptera or mayflies have attracted the attention of entomologists in
Europe and America far more than in Australia, perhaps the main‘reason being the
lack in Australia of the large swarms which have compelled notice elsewhere.

Linné first divided the Ephemeroptera into two groups within the genus Hphemera,
basing the division on the presence or absence of the appendix dorsalis. Further
subdivision was based on the presence or absence of hindwings (Baétis and Cloéon
Leach, 1815) and then on the combination of this and the character used by Linné
(Brachycercus Curtis and Caénis Stephens). Later entomologists divided the order on
the basis of cross veins (Burmeister, 1839) and the number of joints in the tarsi, and
in 1843 Pictet based a scheme on venation and the condition of the eyes.

Eaton published the first monograph in 1871, upon which all later classification
has been based; it is in this monograph that the first Australian species are described
apart from two isolated descriptions—Atalophlebia costalis Burm. and Atalophlebia
australasica Pict.

In his second monograph, “Monograph of Recent Ephemeridae” (1883-1888), Haton
described seven Australian species. Seventeen species are mentioned by Ulmer (1916)
in his results of Dr. Mjoberg’s Scientific Expedition, which collected mainly in
Queensland.

B

2 AUSTRALIAN EPHEMEROPTERA,

No further work was published until Tillyard (1933) described the mayflies of the
Mount Kosciusko region, which he followed with a redescription of the genotype of
Atalophlebia and later listed thirteen Australian species from Tasmania (1935).

Phillips (1930) in describing the New Zealand Ephemeroptera has clarified the
position of several genera which are represented in both Australia and New Zealand.

TAXONOMY.
EVALUATION OF TAXONOMIC CHARACTERS.

Many of the characters on which the classification of Australian Ephemeroptera
has been based seem unsatisfactory. An evaluation of these characters is therefore
made, being based on long series of specimens. Definition of terms used is given
where it is thought necessary, otherwise all terminology is taken from Snodgrass (1935).

Head. The head is of the usual insect form, but the extraordinary reduction of
the mouth parts and their accompanying musculature leaves a shelf-like projection
above the space where the mouth parts would normally be. The nasal carina (a
longitudinal ridge on the shelf in front of the middle ocellus) varies in size, as does
the shelf itself, but it varies to a certain extent within a species.

The antennae are reduced, with an almost vestigial flagellum; the scape is usually
short and stout, and the pedicel varies in length and form, being reduced in Atalophlebia
and very stout in Caénis.

The compound eyes show a great range in size and form, those of the male being
larger than the female in all described Australian species. The males of some species
show subdivision of the eyes, the upper region being larger and lighter in colour
than the lower. The extreme development is seen in Baétidae, where “turban eyes”
are present, the upper division being pedunculate on the lower. This development may
have evolved from the habit of mating in the air, and the consequent necessity for
the male to see the female above (Needham, Traver and Hsu, 1935).

Three ocelli are present, varying in size with sex to a certain extent. The middle
ocellus tends to atrophy, while the spatial relations of all three vary generically. For
example, they lie far apart in Caénis, close together in Baétis, and become ascalophoid
in Atalophlebia.

Thorax. The mesothorax is the largest division of the thorax, being itself sub-
divided into the antecosta, prescutum, scutum, scutellum and postscutellum (Needham,
Traver and Hsu, 1935).

The morphology of the thorax has been little used as a taxonomic character, except
for occasional reference to location of colour markings. But, although the colour and
marking of the thorax are a useful guide in most cases, a good deal of variation
occurs and the colour marking for any species described must not be followed as a
rigid limit.

_ Abdomen. This consists of ten segments with paired genital openings and specialized
genitalia pertaining to the ninth segment of the male. In the male the sternum bears
the articulated styliger plate (forceps base), which in turn bears the forceps or
appendicular clasping organs, each forcep consisting of two parts, the distal stylus
(usually this alone is known as the ftorcep), and the basal coxopodite. There is
evidence that the styliger plate is formed by the union of the median part of the
sternum with the coxopodite, and the separation of this from the rest of the sternum
(Snodgrass, 1935).

The styli themselves are commonly jointed, usually into three segments, but
some show two or four, and those of Caénis are unjointed.

The basal part of the stylus can be easily confused with the coxopodite in some
cases—the only way of determining the stylus is to trace the origin of the stylus
muscles, which always arise in the coxopodite (Snodgrass, 1935). Many authors have
not taken this into account, but it is urged that where the segmentation of the
forceps is taken as a taxonomic character the correct evaluation of the styli should
be recognized.

BY JANET FE. HARKER. a

The penes are primitively two tubular processes, but usually are found to be
united basally to some degree, and on the penes appendages are common and are
chitinous and usually backwardly directed.

The genitalia are probably one of the most constant of characters, and therefore
one of the most important; owing to the large number of descriptions from either
pinned specimens or specimens in which the genitalia could not be adequately examined
there has been insufficient use of the genitalia as a taxonomic character.

In the female the sternum of the ninth segment is extended backward, and
although it varies in shape considerably it is fairly constant within a species.

The Caudal Filaments. These are two, or three, in number. The two lateral
ecerci are always present, but the appendix dorsalis may be present and equal in
length to the cerci or be in various stages of abortion to complete absence. The
presence or absence of the appendix dorsalis has been deemed more important than
is warranted, as there is one case at least where it is absent in some specimens and
present in others (Atalophlebia australis), yet it is a useful guide to species as long
as the fact that it is only a guide is kept in mind.

Text-figures 1-2. Atalophlebia parva.
1, right and left forewings superimposed, cross-veins which appear in only one wing are
dotted; 2, wing base, Ag axillary region, AxC axillary cord, D intermediary plate, HP humeral
plate.

The lateral margins of the abdomen may show remarkable lateral extensions
which are usually directed posteriorly; this is of most constant occurrence in
Siphlonuridae, but the shape of the lateral margins varies considerably in the
Leptophlebiidae, and is important taxonomically.

Legs. All the legs are weak and in many genera are completely useless for
walking, but they are nevertheless highly differentiated. The forelegs are most
specialized, differing from the middle and hind pair, and in the male they are usually
considerably lengthened and hold the female in copulation—the tarsi being turned
backward for this purpose as the male approaches the female from underneath.

The relative proportions of the tibia and tarsus have been used by some authors
and seem to be a constant character if kept to a reasonable comparison, that is a
relative length comparison; this has been used as a generic characteristic by Tillyard
in the Siphlonuridae.

4 AUSTRALIAN EPHEMEROPTERA,

The tendency for fusion of the first tarsal joint with the tibia is of importance
taxonomically, the fusion taking place most frequently in the hind leg, so that it is
usually the hind leg which is used in comparisons. The relative proportions of the
tarsal joints is another character used by Tillyard, but the variation found within a
series of specimens was such that it is not used in this paper. The claws of the pair
on each tarsus may be similar in form, both being hooked at the tip, or one may be
blunt and flattened, or even both may take this latter form.

Specimens have been captured, and also bred in the laboratory, in which legs
have been regenerated in the late nymphal stages and show varying degrees of
reduction in the imago. When both legs have been damaged there is no indication
that this has occurred. Thus, as damage to the nymphal appendages is not infrequent,
discretion should be used in the comparison of measurements.

Wings. The terms applied to venation are those interpreted by Tillyard (1932).
Table 1 shows systems used by other authors in specific descriptions.

TABLE I.

TILLYARD 1932
AND NEEDHAM AND

EATON 1883

EATON 1883

COMSTOCK AND
TILLYARD 1923 MORGAN 19/2 NEEDHAM [9/2

THIS PAPER TRAVER 1935 TILLYARD 1926 (NUMBERS) (NAMES)
Pie fe ee c+ c c c G c t COSTA
Rp Akh e eaeen Se- Se Se Se Sc Se 2 SUBCOSTA
at an ae Ry + R Ry Ry Ry Ry 3 RADNUS
Ro- Ro Ro IR3a My R5 =
IR3t+ = IR 2R RS = - BRANCHES OF
2 a
R39 R3a ae = = + SECTOR
IR30+ IR3b 4R3a - - =
R3b- R3b SRaa RS Ra =
IR3d + IR3b Rab INTERPOLATED Ra 4 J
VEIN 1.
Rigs R3 Rays R3 Mo Rs 3 CUBITUS
Tote MA, + Ry May Rye M3 M G PRAEBRACHIAL
IMA~ or IMA Rab = Ma =
MA3+ Rs MA- Rs Mg M3 G POBRACHIAL
MP) — My MP, My Cuy Cur 7

Ma INTERPOLATED
VEIN 2

ICuA - 1Cuy Cuyb 1A
Cu Ast Cua Cuyb Cuye 1A
CuP - 1A 2A
AXILLARIES
A)- 1A
1A+
Aat
A3Agt =
(AXILLARIES )
wile —|
AUTHORS FOLLOWING THIS SYSTEM

The cross vein system has frequently, and incorrectly, been referred to as constant.
Text-fig. 1 shows the right and left wings of one specimen superimposed, those cross
veins which appear on only one wing being dotted; this reveals such a variation even
in the same specimen that the use of cross veins as a diagnostic character is rejected.

Marginal veinlets between the longitudinal veins may be present, and the number
of these is a distinct generic character. The wing base (Text-fig. 2) is unusual, the
Ephemeroptera holding the wings vertical at rest and not flexing them as do most
insects. At the base of each wing is a humeral plate between the costal vein and the
tergite, the posterior part of the wing base is continuous with the posterior margin
of the tergum, but the weak sclerites in the axillary region seem to have some
homology with those of other insects.

BY JANET E. HARKER. 5

The shading of the wing and its coloration are of value and are usually considered
constant, at least in the subimago; however, the effect of physiological variation within
the animal, particularly in the larva, alters the relative amount of pigmentation of the
cuticle, and this may result in an effect on the wing shading.

The hindwing is of more importance in some respects than the forewing, par-
ticularly in reference to the shape of the anterior margin, which appears to be
constant. The presence or absence of the hindwing is in some families used for
generic segregations.

NYMPH.

The nymphal characters can be used with possible advantage over those of the
adult, as it is probably in the former stage that specific divergence occurs.

Head. The shape of the head varies considerably, and in its extremes is used to
characterize families. 'The shape of the head is associated with the type of habitat
and the habits of the larva. The position of the eyes varies with the shape of the head,
being dorsal in Atalophlebia, which have flattened heads, and lateral in Siphlonuridae,
with hypognathous heads. The colour of the eyes is fairly constant and has at times
a taxonomic significance; in the male the eyes become divided in the late instars.
The antennae vary in diameter and in length, but they are so easily broken that little
significance should be attached to them.

Mouth Parts. The labrum is present in all Australian forms so far described;
it takes the form of a transverse, more or less rectangular plate with spines and hairs
variously disposed over the surface, and the anterior edge varies in shape considerably
and is often of specific importance.

The mandibles are divided into two distinct areas: the canine and molar areas.
The canine area in particular has often been credited with taxonomic significance,
but the variation in a series is extreme, and the development also varies considerably
with the stage of development of the nymph. Between the canine and molar areas
arises the prostheca (otherwise known as lacinia mobilis, mandibular palp, or man-
dibular endopodite); this varies between a large structure bearing a brush of hairs to
a minute hairless spine.

The maxillae have a separate cardo and stipes, but the galea and lacinia are
fused lengthwise and their fused “plate” often ends in a tooth or hook, or more often
in a thick brush of hairs below which is a row of pectinate “rakes”. The maxillary
palp is modified in various ways; it is probable that the primitive form is three-
segmented and covered with hairs, but these segments may be reduced to two or may
be greater than three (fifteen in Ameletopsis), and any of the segments may lose their
sensory hairs.

The labium is of primitive form and the palpal segments may differ in relative
length and the form of the paraglossa is often a specific variant.

Thorax. The variation in the thorax appears to depend largely on the habitat;
probably the variation is a function of the muscular development. The colour and
markings are useful characters, but the same precautions must be taken as with the
use of colour in the adult.

Legs. The legs vary greatly with habit. In forms which live in swift-flowing
streams the front femora are often greatly thickened, and in other forms which
flatten themselves against rocks the legs are flattened with the femora broad and the
tibiae slender. All the legs bear a one-jointed tarsus with a single claw, which varies
greatly in form.

Gills. Abdominal gills occur only on segments one to seven, being present on
the dorsal side of the lateral margin, and their number and disposition are of
considerable importance. The two principal types are lamelliform or plate-like and
filiform or threadlike; one pair of gills may become enlarged to cover the others—
this is a modification found in forms frequenting silty waters and creeks which partially
dry up in summer.

6 AUSTRALIAN EPHEMEROPTERA,

From this discussion of the various parts of the insect it appears that very few
characters are stable enough to be used as primary taxonomic characters; however, it
is not by single characters that species are separated, but rather by combinations
which only become recognizable when a long series of specimens is examined.

TYPES.
All type specimens have been deposited in the Australian Museum, Sydney, and
paratypes are being deposited in the British Museum. Wherever possible, for each

species a holotype (the male imago), allotype (female imago), morphotype (male sub-
imago), allomorphotype (female sub-imago), and a nymphal morphotype have been
selected. The terminology used is that defined by Davis and Lee (1944).

Tillyard has described several species for which the type specimens have not been
able to be traced in any Australian Museum, the Commonwealth Scientific and
Industrial Research Organization Entomological Branch, or the British Museum. It
has not been found possible to recognize these species from the descriptions.

METHOD OF DESCRIPTION.

As text-figures are a clearer guide than verbal descriptions, the latter have been
abbreviated. :

Measurements. All measurements have been made from a series of a minimum of
twenty specimens, unless otherwise stated. The measurement given is the average
of these figures, and the range of the measurements is given in brackets.

General Colour. This has been ascertained by naked eye, being the general colour
impression given by the mayfly.

Wings. Where the cross venation varies the text-figure shows the maximum number.

Life Cycle. Wherever possibie, the imagines, subimagines and nymphs have been
connected by breeding them from the nymphal stage; as a further check, it having
been found that on no occasion has the egg differed in the late nymphal stage from
that of the imago or subimago, the relationship of these stages has been shown by
the eggs.

Specimens Examined. These have all been collected by the author unless otherwise
stated.

Text-figures. These have all been drawn with the aid of a camera lucida.

Females, Subimagines. Where the male imago has been described in detail the

following descriptions of other forms only give details in which they differ from the
male.

KrY TO THE EHPHEMEROPTERA OF NEW SOUTH WALES.
Families suspected or known to be present, but not actually described, are included in
this key.
IMAGINES.

Superfamilies.
1. In forewing veins MP and CuA strongly divergent at base. MP, strongly bent towards
CuA basally. Hind tarsi with four movable joints or less; if a fifth is present it is

immovably gunited! toy D1. pete. cisysccscy ciemen cuege eae Oewene © uckshode SEO Reese Fie S.F. EPHEMEROIDEA.

In forewing veins MP and CuA parallel at base or weakly divergent. Fork of MP nearly
SVIMMEEVICAL! re layeipsrctrc wis aire, ei create atalrayisiette Paw e font Vet sUc eae ney cialis heueTeuueie nomen ene xovektt Cnc eRe Reel licteae n= nete Neuen 2.

2. Hind tarsi with four movable joints, if a fifth is present it is immovably united to
151 0): a Cs Ta aete Conn Peeters octets heise boii oie ao ccna oo OOOO mo CO AO Cac os S.F. BAETOIDEA.

re ths oie Oreo ao Od. oo Gat dom S.F. HEPTAGENOIDEA.
[Atopopus spadix, sp. nov.)

Superfamily BA®mTOIDBA.
1. Forewing with tornus from two-fifths to half the length of the wing from the base, CuA
nearly straight, with a descending series of pectinate branches. CuP curved concavely

[open O70 Ue eee Giercho 0G One Ona a SRO Re OosE Toma Uoodac OCs CO OOnOO DS SIPHLONURIDAE.
Forewing with tornus at not more than one-quarter of wing length from base. CuP
SIFMOIGL LY: NCUnVER PE Yaiiee sree Serotec. Hone meh ctelelie rete [ten eretayeoreNoveR RoR y meray neha cram Mener ster sens 2.

2: ebMfthe) Lorewin'e WVvAY clearly; (forked!) Gwe. Week. cpeneh eye ele de lodch manele Retekelsi cede tcPeleieromey elec ifthe clroferene 3.

In the forewing MA not forked, although behind it are two free veins which are not
attached at base. Usually few cross veins. Hindwing small and narrow, sometimes
absent, swith at Osten (Orns LON SI LIGIN al Vell S ae ieee nee eienicnelsten enero n nanan ana BAETIDAE.

BY JANET E. HARKER. "7

Wings milky or infuscated, ciliate on hind margin. Hind wings absent, but may be
occasionally present in subimago. No unattached intercalaries, frequently only few

CROSSMAVC IIS pamrran sree mene nee cwr tera piace castro since honed Secret Re R evap dees racer er erik bat leVieea cb aacloeecen ek el esceyerers CAENIDAE.
Wings hyaline, hind wings nearly always present, wings with numerous cross veins .... 4.
In forewing CuP usually widely separated at base from CuA, but lying close to A, No

unattached intercalated veins between media and cubitus ............ LEPTOPHLEBIIDAE.

CuP in forewing approximating at base to CuA, but widely separated from A,. Several,
usually two, unattached intercalated veins between media and cubitus, and also in
front of the posterior branch of the media ......................... EPHEMERELLIDAE.

Family BAETIDAE.
Hind wing present, forewing small with marginal veinlets in sets of two or more ....Baétis.
Hind wing absent, forewing with marginal veinlets single .......................... Cloéon.
[Cloéon fluviatile Ulm.]

Family CAENIDAE (BRACHYCEROIDAE Lestage).
Wings not exceptionally broad, ratio of length to breadth approximately 3:1
ee EMA MER Da veN ech a TRE SA SRE A a eR cot Maeva ai ce IS tei ahd AUG ante ake: anabehehete odavenctecte Shs Tasmanocoenis.
Wings exceptionally broad near base, ratio of length to breadth 2:1 or less ........ Caenis.
[Caenis scotti Till.]

Genus Baetis.

iL,

Oo

Costal angulation of hindwing acute .........5:.2......-.-:te-e-- B. baddamsae, sp. nov.
Costal angulation of hindwing not acute ..................:..0+002- B. confluens, sp. nov.

Family LEPTOPHLEBIIDAE.

Warcsall Glass enll imekerony acl waveboenie 9) 5 deodooousodoloooucok obo oouboaoous db ooooo de 6 Pa5
Of every pair of tarsal claws one broad and obtuse, the other narrow and uncinate .... 3.
in GiwANnewmMOLremor less sobulselya Sub Owael sangecior-) sen ciel rele eles) crae Siaaie Atalophlebia.
Hindwing oblong, oblique; its marginal area abbreviated and relatively very broad
0 0 00 6 0.6 WTOIREABS:Oin Be Ort r ose bee ciliaris oObrony sce aye iris ar olicuitan elle Eee eeinacaracn an eR enn aca Adenophlebia.
Hindwing obtusely ovate or oval; its marginal area narrow throughout and extended .. 4.
Hindwing strongly angulated in front; its marginal area narrow throughout and far
OK S ING C CUMMME ats its Tos errata oe trom teow tesiyes erea even civcass tom serearecrehioy sale deren tet ston awe nahin emenetrose aenittic es ceria Thraulus.
Appendix dorsalis much shorter than caudal filaments ...................... Blasturus.
Appendix dorsalis equal to the caudal filaments .............0.. eee eee ee eee 5.
Penes separated almost to base; a long flap-like appendage, narrowed distally, is attached
near apex of each and extends inwards between lobes of penes ........... Leptophlebia.
[L. crassa, sp. nov.]
ReEnesmwithoutenap=lkemappenGdacey mrs cus sain silicic) ens siies vile cist s)cbcieioien acl een i eee eae Deleatidium.

Genus ATALOPHLEBIA.

IMAGINES.

Wore wing ess Giam Boel) sren, sha Meme odosaconoonconb00 neon dbccbonodobdueeoDbDUAD OD 2.
MORAINE MNO Wawa. So) ime, ihe Wem ca Sdrocaucscoodnoaccdo Uubenoou0GunCo UDO bOOUDUO 3.
No cross veins present in basal region of C—Sc area .................. A. parva, sp. nov.
Cross veins present in basal region of C—Sc ....................... A. marowana, sp. nov.
Sculpturing on the egg reticulate without any other marking being present ..............

© outa “ith: dh CHOLOAOEO eee DaE RATER RECITES TRRr SUn TE SP SEU for Rea MIR OU ie Se nn RP ORE oA A. albiterminata Till.
INO. BS BUSVON GY taco oid elaral cea mecieliatote okaey cad Saal yee ean iat Ista ee PerIAEAsacd TntoLb..ata Rano eG ban ale) Sea rE RS AML Qttatiey tte tea 4,
Sculpturing on egg reticulate with circular markings at the angles of the “cellular lines”

B06 GHOkG O 1O°S-0l BLO CC CEuE RO ERR GUST RE ROSE ONIAZ OMS LenS RG: DRT DE CIaG ase Hoa emaAiMaracic sn itern caer A. longicaudata, sp. nov.
Sculpturing reticulate with raised circular areas also present ........ A. incerta, sp. nov.

Female imago A. maculosa unknown.

The male imagines cannot be satisfactorily differentiated except on the genitalia (Text-

figs. 19-27).
SUBIMAGINES.
PEELOT CWI Stet ODES CH aiweactepiecce a eee hens, oss, 6) sooh ene Tevisi cue eves, syiet epee uctten tes ne, emer stiectarlec ete) aharsliocula: Gu Syreueyheye ego's Be
LNOTEWMINE TAO SIRE I NG poop edo Gatloe odo one GUD bo eno oo MoGUU Oo Odoe.ADee Bind obo UuICoD 6 2.
2. Cross veins absent in basal region C-Se area ...........+-.-....+e20-- A. parva, sp. nov.
Cross veins present in basal region C—Se area .................+--- A. marowana, sp. nov.
Pale amMpdammMarkwalmost «completers wea-iaiwscie ca ciecus ceetercieeucleia ch sicke caciene icc A. incerta, sp. nov.
Ibpenlnoley Sook uAse cilayooucen oeeen” eopeawaim kes oto_o-cmoloeeschc nose eto b'p Oasia eo ip.olG oma o iGo pido min aidihrmoiooo clog onto 4,
4. Forewing darkly blotched, whole wing darkly shaded .......... A. longicaudata, sp. nov.
Forewing not so heavily shaded, little shading of cellS ............ A. albiterminata Till.

Subimago of A. maculosa unknown.

1.

Family SIPHLONURIDAE.
MP, and IMP attached basally to CuA. Tarsal claws alike. Costal angulation of hindwing
MEAP [ORI BNE aocbos poo me ob ddd eo eum oo od On D Oo OUD OOO mo ue a Koen Ameletoides Till.
MP, and IMP normal in forewing of every pair of tarsal claws, one blunt and one
ALC ULLC Mam tes eemen su cerctis nae ah esveetaprensWayeh aiciajie chiar nylateetctiaiassisah epstiaclc awe: cifeires cise) elon sive etishcah obistiaieh a oh ol @Rettete lov af abahe ehopateuse ens 2.

8 AUSTRALIAN EPHEMEROPTERA,

20 TRarsi) four SELMEMCCA (iF Meee cteneuetAd vote ch ste i-leeet ol etc hehe cuaere ete eUaneronee tier eat Ns Tasmanophlebia Till.
Warsi’ five Sseemented® |» s.6- Fite, tous orca otre rey ped cayenay oo CoN Tawa le the). <ird Palen ete Coloburiscus Eat.
NYMPHS.
Superfamilies.

1. Mandible with an external tusk projecting forward ................... S.F. EPHEMEROIDBA.
Mandibles without such’ a. tusk! %s.i2 S<.. hee eee oe «A I ie or ee etic cn sles ees ie 2:

2. Head strongly depressed. Eyes dorsal. Upper member of each gill pair plate-like ......
PU DE Rela ahaa aleve databe hal atateladetatele va leuale Hitene Ne (ar eeere re ome aetetns Re. ces ote enema eae Nts S.F. HEPTAGENOIDEA.

Head not? strongly wdepressed)) chives) lateral. -yarserwerierse ited oeionet ieiel eran notes S.F. BAETOIDEBA.

Superfamily BAETOIDEA.

1. ‘Caudal filaments’ frineéd on! Doth VSides oy. cisrae uererehes coc aew ere esi oe eral Ree note relied tel ce eee eens 2.
Caudal flamentseiringed on) inners bord emnonlivaecmnciasneieecccreieieraieneiaianel noises iene ne ieen eee oe

2. Seven pairs of gills inserted laterally at sides of abdomen, sometimes all filamentous or
first) pain sreduced and others leat-likelsea acm cei eee eee LEPTOPHLEBIIDAE.

Five or six pairs of gills, first pair very small, second enlarged, covering the following
pairs, which... bear: ia Monge Girin ge: eeigetiensis ceed cute evecare sueueitel- (xeuovenciio ath sled teceemetens CAENIDAE.

3. Body cylindrical, head bent downwards, hind corners of abdominal segments not produced
EER RCE AL acre Cia OIGic LD tig Oo RO EEO OOO oo e aalolad Slain Ob Ome Gon oosaUoU aos BAETIDAE.

Body more or less flattened, head held horizontally or nearly so, hind corners of abdominal
sezments produced sbackwanrdsim saci doen ke Ornelas ciently eR ree erent SIPHLONURIDAE.

Family BAETIDAE.

1. Gills—lamellae double on abdominal segments 1-6 ............. 22s eee e eee e eevee Cloéon.
2. Gills—lamellae single on all abdominal segments ...............-02.2 cee eeeeeeeeee Baétis.
Family LEPTOPHLEBIIDAE.
nae Git O Siiets) bie aaa ert racic cack eaEnercraly Mncictsy chu scictod Old Grulla dala dic, Giaiemio.c 6 HU Boom O Oa eso Deliatidiwm.

COS UD (0 Kopel o) Kaas ep eit cache ota cio a CROPS cicktr Ofc e ERED OO Oa ERC tG Go DOctOIG Oo ola DiS Co OOo oc oe 2.

2. Gills on abdominal segment 1 deeply forked with slender linear divisions, on other segments

oval, lamelliform and fringed around the entire margin .................... Thraulus.

INOt AIS! BDOVE | a cicieetel Sa eye rersve: seicueprelteuayicti vole lords yahous S609, dy/spaweylove, ss esi ets enele Ne foc teneme Ls heweraie neuen aisiear eter By

3. Gills lanceolate with narrowed tail-like tip. Lateral spines on abdominal segments 8 and 9

SHO PERE, Cte HUSPE GALT ES Chat Center CRON Ge CHICO ONCE cao 30 hcl Eoin Dio, ta ciGia 0.0.0 eo mncid Sl oia Leptophlebia.

Gills either lanceolate or digitate, spines usually on abdominal segments 2 and 9 .....

areal ithe, Petree eancite gla, ai rctals sak Sadie aU 128 Ue oh obra Sata nl yall sts eak UE RUL UPbee Ran aus se cua Bemets TPA ny Sica trae Atalophlebia.
Genus Atalophlebia. ,

Le MGAWS (GISItAte rose sos acai We eice ev Suelo eee ara revel eae enone shail ade GS c terere or ele eee ree anole eno HORE tema 2:

Gills Wan Ceolates, , Fasyccacsreb eum tovl cee abs We apecrea cee Slits ere rer enamel One ohne let oeiboternelrans ich peewee A. parva, sp. nov.

ry, (CIN seo olloioheateywes Yalby salle yeeVSMS 6 ou dato ooo coe mb gonnoaGndbouDSe A. albiterminata Till.

CesT ESERR rhb or i amy MeetonC Ne eo ta etowo ictorasiecterore EaGin cioalcic dete. aclotaacicn yOcareroicr olor A. incerta, sp. nov.

Family SIPHLONURIDAE.

1. Thorax humped, very broad. Gills deeply bifid ...:................... Coiaburiscus Fat.
Thoraxnotehumped. iGillsslamellate:, sc se Hie he Bid catalina ei oles geese s aR cacked ener 2:

2. Dorso-ventrally flattened. Gills carried dorsally on abdominal segments ...............
AGO Dirk OD OI OOO Ce rd ao ooo ope UE ato oe oop moor Od Tasmanophlebia Till.
Nymphs slightly laterally flattened. Gills carried laterally ............ Ameletoides Till.

DESCRIPTIONS OF SPECIES.
Genus ATALOPHLEBIA Haton.

Synonymy: Atalonella* Needham and Murphy, Bull. Lloyd Libr., 24, 4, 1924.
Genotype: A. australis Walk.

Imago.

“Hindwing in front somewhat arched, the summit of the arch obtusely sub-angular,
situated usually before the middle of the curve; sub-costa strongly arched, meeting
the margin very obliquely; radius usually nearly straight, constituting as it were the
chord of the are described jointly by the sub-costa and the portion of the margin
included between its extremity and the radius; hence while the narrow marginal area
is broadest at its base and acuminate at its termination, the sub-marginal area is
broadest at the middle or a little before the middle, and tapers gradually to its oblique
apex. Cross veins abundant in the forewing, those in the marginal area before the bulla
well defined. At the terminal margin the longitudinal nervures are provided with
curved simple branchlets and Bone are no isolated veinlets. The two intercalar nerves

* See “Appendix Us ja), 8h):

BY JANET E. HARKER. 9

of anal-axillar interspace of the forewing have simple branchlets, and usually the hinder
one, close to its proximal extremity, curves forward to unite with the other, which

simply curves forwards to join the anal nervure .... Tarsal ungues all nearly alike,
small, narrow and hooked at the tip .... Forcep limbs of male 3 jointed.” (Eaton,
1881.)

Nymph.

Body flattened dorso-ventrally.

Head: The eyes are lateral and antennae long.

Mouth Parts: Both the maxillary and labial palps are three-segmented. The labium
is notched medio-anteriorly and the maxillae bear a broad brush of terminal hairs.

Legs: The claws are all toothed.

Caudal Filaments: These bear short hairs at the intersegmental regions.

Gills: Paired and lanceolate or lamelliform gills are borne on the first seven
abdominal segments.

ATALOPHLEBIA LONGICAUDATA, Sp. Nov.

Holotype o&, Tenterfield, 2,831’, 10: 1948.

Allotype 9, Pine Forest, Armidale, 3,300’, 9: 1948.

Morphotype Subimago, Marowan Cr., Glen Innes, 3,520’, 10: 1948.

Paratype, Alloparatype, Morphoparatype, Marowan Cr., Glen Innes, 3,520’, 10: 1948.

DESCRIPTION.
Male Imago.

Measurements: Body length, 17 mm. (15-18 mm.). Cerci, 30 mm. (28-31 mm.).
Appendix dorsalis, 20 mm. (16-32 mm.). Forewing, 16 mm. (14-17 mm.). Hindwing,
3:5 mm. (3:0-4:0 mm.).

General Colour: Black with lighter markings and with a distinct white line running
beside the prescutoscutal suture.

Head: The head is black with ill-defined lighter markings, and bears antennae with
black pedicels and dark brown fiagellae. The lower lobes of the eyes are slightly darker
than the dull orange-brown upper lobes and the ocelli are white with black bases.

Thorax: This is black with light markings and white outlines to the prescutoscutal
sutures.

Abdomen: The abdomen is brown-yellow with dark brown markings, and in some
specimens there is a white posterior edge to the last three segments.

Wings: In the forewing the pterostigma is shaded brown, all the veins are black,
and the cross veins in the C and Sc area are shaded (Text-fig. 39). In the hindwing
all the veins are opaque and colourless except those in the C—Sec region, which are brown.

Legs: The legs are yellow-brown with dark brown markings. The fore-femora
bear one or two longitudinal brown markings, but the hind-femora darkens only at
its proximal end, as do all the tibiae. The tarsal claws are all narrow and uncinate.

Genitalia (Text-fig. 20): The forceps are brown with lighter distal joints and the
oenes are uniformly brown.

Caudal Filaments: These are black, or in some specimens lighter towards the
distal end, the tip ranging from white to a colour not appreciably different from that
of the base. The cerci are longer and stouter than the appendix dorsalis.

Female Imago.

Measurements: Body length, 15 mm. (11:0-18:0 mm.). Cerci, 20 mm. (18-22 mm.).
Appendix dorsalis, 13 mm. (11:0-15:0 mm.). Forewing, 14 mm. (11:0-17-°0 mm.). Hind-
wing, 3°38 mm. (3:0—5:0 mm.).

Egg (Text-fig. 36): These shows a sculpturing of pentagonal figures with circular
markings at the angles of each pentagon.

Male Subimago.
Measurements: Body length, 15-5 mm. (10°5-18:5 mm.). Cerci, 19:0 mm. (17:0—21-0
mm.). Appendix dorsalis, 13-0 mm. (12:0-18:0 mm.). Forewing, 15:0 mm. (11-:0-18-0
mm.). Hindwing, 4:0 mm. (3:5-5:0 mm.).

10 AUSTRALIAN EPHEMEROPTERA,

General Colour: Yellow-brown with dark brown markings.

Head: The head is yellow-brown with dark brown markings, and bears yellow-
brown antennae which are slightly lighter at the tips. The upper lobes of the eyes
are yellow-brown and the lower dark brown to black. The ocelli are yellowish, and the
lateral ones in particular are very prominent.

Thorax: The thorax is dark brown with light brown markings and a white or
purplish tinge at the wing base.

Abdomen: This is yellow-brown with dark markings on either side of the median
line, and the posterior edge of each segment is outlined with brown. The lateral
margins of each segment come to a point at about two-thirds of their length, which
gives the appearance of a serrated edge to the whole abdomen.

Wings (Text-fig. 38): The wings are creamy with dull brown irregular shadings
and shading on all the cross veins, although the longitudinal veins are opaque and
nearly colourless.

Female subimago (Text-fig. 7).
Nymph unknown.
Specimens Hxaamined.

Imago. Serpentine R., Point Lookout, 4,000’, 10: 1948; Dumaresque Cr., Armidale, 3,000’,
11:1948, 9:1948, 10:1948; Pine Forest Cr., Armidale, 3,000’, 9:1948; Marowan Cr., Glen
Innes, 3,520’, 10:1948; Tenterfield, 2,831’, 10:1948; Badga, 3,000’, 12:1939, H. M. Stephens;
Solitary Cr., Tarana, 4,:1941, E. Garret; Mienna, Tas., 3,300’, 12: 1928; Shoalhaven R., 2,500’,
12:1929, H. M. Stephens; Bolaro, 3,400’, 12: 1928, H. M. Stephens.

Biology.

This species has only been found in the vicinity of flowing streams, over which it
flies in a characteristic manner. This flight covers about two hundred yards and
consists of a gliding motion in the downstream direction and a rapid dart upstream.
In the downstream flight both males and females trail their caudal filaments in the
water and are made conspicuous by the habit of holding the filaments at right angles
to the body.

ATALOPHLEBIA ALBITERMINATA Till.

Proc. Roy. Soc. Tasmania, 1935.

Holotype 3%, Lake Echo, Tasmania, 2:1933, Tillyard.

Allotype 9°, Morphotypes Subimagines and Nymphs, Lake Echo, Tasmania, 2: 19338, Tillyard.

Note.—It has not been possible to trace any of these types, so a neotype has been
selected. The neotype is not, however, topotypical, as no further specimens have been collected
from Lake Echo to my knowledge.

Neotype 9, Marowan Cr., Glen Innes, 3,520’, 9: 1947.

DESCRIPTION.
To the original description by Tillyard the following features are added.

Male Imago.

No male imagines have been collected by the author, but pinned specimens are
present in the Australian Museum collection. As these pinned specimens are useless
for an adequate description only measurements are given.

Measurements: Body length, 16-5 mm. Cerci, 39 mm. Appendix dorsalis, absent.
Forewing, 15 mm. Hindwing, 3 mm. Foreleg, 3:0, 3:6, 6:0 mm. MHindleg, 3-6, 7:5,
3-0 mm.

Female Imago (Text-fig. 3).

Measurements: Body length, 15 mm. Cerci, 11 mm. (10-15 mm.). Appendix dorsalis,
11 mm. (10-15 mm.). Forewing, 15 mm. (13-17 mm.). Hindwing, 4:0 mm. (3-5—4-5
mm.). Foreleg, 2-5, 2-4, 2:0 mm. Hindleg, 3-0, 2:0, 2:0 mm.

General Colour: Dark brown.

Head: Dark brown with yellow markings, and grey-brown eyes and grey ocelli.

Thoraz: This is yellow-brown with dark brown markings. i

Abdomen: The abdomen is yellow-brown with very distinct dark brown markings.

BY JANET E. HARKER. 11

Wings (Text-fig. 40): The wings are hyaline except in the costal and subcostal
area of the forewing, which is brown with darkly shaded cross veins; all the venation
is black.

Legs: The hindlegs are lighter than the dark brown forelegs, and their tarsi are
almost white.

Cerci: These are dark coloured, but in some specimens may end in a white tip.

Egg: The eggs show a hexagonal sculpturing and are laid in a gelatinous substance
which dissolves as it passes through water.

Female Subimago.

General Colour: Yellow with distinct brown markings.

Head: The head is yellow with brown markings, black eyes, and white ocelli borne
on black bases.

Thorax: This is yellow with dark brown to black markings.

Wings (Text-fig. 41): The wings are yellowish with black veins and have all cross
veins shaded dull grey. In the hindwings Sc reaches almost to the apex, Rs is forked
and arises about half-way along MA, and MP is forked and an intercalary is present.

Caudal Filaments: These are dark brown with black annulations and a broad black
band at the end of each fourth segment.

Nymph.

Measurements: Body length, 10-16 mm.

General Colour: This varies considerably with the environment, more so than in
any other nymph known to the author; generally it is dark brown, grey, greenish
brown or pale cream.

Head: This is cream or light brown with brown markings, and the antennae are
approximately one and a half times as long as the head and thorax, with the basal
segment light brown, and the following segments transparent. The eyes are brown
or black.

Mouth Parts (Text-figs. 64-66): The labrum has a median incision on its anterior
border which bears five to seven flat, dentate processes; recurving spines are also
present on this border and bend in towards the central line; posterior to these is
another row without recurving tips. On the lateral angles spines are present and
directed at an angle following the curve of the labrum. In the mandibles the outer
canines have three or fourth teeth and the inner two, the prosthecas are sigmoid in
shape with a brush of well-developed, inwardly directed hairs. The molar surfaces
are well developed with 10-13 parallel serrated ridges, and a few scattered hairs are
present on the outer surface of the mandible. The maxillae bear three-segmented palps,
of which the middle segments bear spines on the inner surface and the apical segments
are covered with hairs on the outer surface. A row of pectinate “rakes” is present
below the brush of terminal hairs on the plate. The labium bears three-segmented
yellow palps of which the first segments carry hairs, but the second are practically
bare. The paraglossae have hairs present on the anterior edge and the glossae, which
are rectangular with rounded corners, bear a dark patch lengthwise in a central
position and very few hairs, those which are present being mainly on the basal surface.
A single row of spines is present close to the distal end of the glossae.

Legs: Creamish-yellow, and on the inner surface of the tibia there are rows of
branched hairs.

Gills: These are double and present on the first seven abdominal segments,
decreasing in size posteriorly.

Biology.

The nymphs live among debris and clinging to the bottom of rocks in running
streams, frequently in the more sluggish parts. The early instar nymphs often lie
_ partially buried among the sand or debris of the bottom, while the older nymphs run
backwards and sideways with great agility seeking shelter in crevices rather than
swimming.

12 AUSTRALIAN EPHEMEROPTERA,

Specimens Examined.

Imagines. Marowan Cr., Glen Innes, 3,520’, 9: 1947.

Nymphs. Dumaresque Cr., Armidale, 3,300’, 9: 1948, every month 1947; Pine Forest Cr.,
Armidale, 1947, Commissioners Waters, Armidale.

ATALOPHLEBIA MAROWANA, Sp. NOV.

Holotype Subimago ¢&, Marowan Cr., Glen Innes, 3,520’, 10: 1948.
Allotype Subimago 9, as above.
Paratypes Serpentine R., Point Lookout, 4,000’, 10: 1948.

Text-figures 3-18. Imagines.

3, Atalophlebia albiterminata, female; 4, Atalophlebia incerta, male; 5, Atalophlebia macu-
losd, male; 6, Atalophlebia parva, male; 7, Atalophlebia longicaudata, female subimago; &8,
Atalophlebia marowana, female subimago; 9, Leptophlebia crassa, male subimago; 10,
Deleatidium annulatum, male imago; 11, species described in Appendix II, female imago; 12,
Baétis baddamsae, female subimago; 13, Caénis scotti, female subimago; 14, Baétis confluens,
male imago; 15, Baétis baddamsae, male imago. Nymphs. 16, Leptophlebia crassa; 17, species
described in Appendix II; 18, Caénis scotti. (Various magnifications. )

DESCRIPTION.
Male Subimago.

Measurements: Body length, 9-2 mm. (7:0-10-0 mm.). Cerci, 15-0 mm. Appendix
dorsalis, 15-0 mm. Forewing, 6:8 mm. (4:0-8:0 mm.). Hindwing, 1:3 (1:0-1:8 mm.).
Foreleg, 2-0, 3:0, 1:0 mm. Hindleg, 2:0, 2-0, 1:0 mm.

General Colour: This is yellow-brown with dark brown markings and almost black
annulations on the abdomen.

Head: The head is yellow-brown with black antennae, and eyes of which the upper
lobes are grey-brown and the lower dark brown. The ocelli are large, bulging, and
dark brown.

BY JANET E. HARKER. 13

Thorax: This is yellow-brown with dark brown markings.

Wings (Text-figs. 51, 61): The wings are grey with clear longitudinal veins and
brown or black cross veins.

Legs: The legs are yellow-brown with dark brown areas surrounding the femoro-
tibial articulation. The tarsi are four-jointed and the tarsal claws all narrow and
uncinate.

Caudal Filaments: These are yellow-brown with dark annulations on the first
proximal segment.

Female Subimago (Text-fig. 8).
Head: The eyes are black and the ocelli are white on a black base and widely
separated.
Eggs: The egg is spindle-shaped and sculptured with irregular star-shaped markings
with a central pit (Text-fig. 30).

Imago, nymph unknown.
Specimens Hxamined.
Subimagines. Tillbuster Cr., Armidale, 3,300’, 11:1947; Serpentine R., Point Lookout, 4,000’,
10:1948; Marowan Cr., Glen Innes, 3,520’, 10:1948; The Creel, Thredbo R., 3,000’, 11: 1928,
H. M. Stephens.

ATALOPHLEBIA MACULOSA, Sp. NOV.
Holotype 3%, Serpentine R., Point Lookout, 4,000’, 10: 1948.
Allotype 2, Paratypes, Serpentine R., Point Lokout, 4,000’, 10: 1948.

DESCRIPTION.
Male Imago (Text-fig. 5).

Measurements: Body length, 11:5 mm. (10-0-13:0 mm.). Cerci, 24:0 mm. Appendix
dorsalis, 24:0 mm. Forewing, 11-4 mm. (10-0-15-:0 mm.). Hindleg, 3-0, 3-0, 1:0 mm.

General Colour: This is black with yellow or yellow-red stripes running transversely
on the abdomen.

Head: The head is black with eyes of which the upper lobes are orange and the
lower black, and the ocelli are white on black bases.

Thorax: The thorax is black with white bands beside the prescutoscutal suture.

Abdomen: This is dark brown with a yellow or yellowish-red band on the posterior
margin of each segment, except the last three, which often bear a white margin. The
lateral margins are smooth and rounded.

Wings (Text-fig. 43): The wings are clear with very dark venation, and about half-
way along Sc, usually in the region of the bulla, a striking black spot occurs which
covers two or three cells.

Legs: The forelegs are dark brown, and some specimens show a slightly lighter
line marking the tarsal joints. The mid- and hindlegs are yellow to orange, with one
black mark about half-way along the femur and another on the femoro-tibial joint. In
all the legs the tarsi are darker than the other segments.

Genitalia (Text-fig. 22): The forceps are light yellow, and the penes, which are
dark brown, are large and distinctive.

Caudal Filaments: These are uniformly black, and the appendix dorsalis may be
similar to the cerci or may be slightly thinner.

Female Imago.
The body is often stouter than that of the male.

Subimago and nymph unknown.
Specimens Hxamined.

Imagines. Serpentine R., Point Lookout, 4,000’, 10:1948; Marowan Cr., Glen Innes,
3,520’, 10:1948.

ATALOPHLEBIA INCERTA, SP. NOV.
Holotype &%, Gara R., Armidale, 3,330’, 4: 1947.
Allotype 2°, Morphotype subimago, Paratypes, Gara R., Armidale, 4: 1947.

14 AUSTRALIAN EPHEMEROPTERA,

DESCRIPTION.
Male Imago (Text-fig. 4).

Measurements: Body length, 10:0 mm. (7:5-12:0 mm.). Cerci, 30 mm. (17-45 mm.).
Appendix dorsalis, 25 mm. (17-30 mm.). Forewing, 10 mm. (8:0-12°5 mm.). Hindwing,
2-5 mm. Foreleg, 8-0 mm. (6:5-10-°0 mm.). Hindleg, 5-5 mm. (4:5-6-0 mm.).

General Colour: This is dark brown.

Head: The eyes are black and the ocelli white.

Thorax: This is dark brown with black markings and a white outline to the
prescutoscutal suture.

Abdomen: The abdomen is of slightly lighter colour than the thorax and has black
markings.

Legs: The legs are yellow-brown with dark brown markings on the femora, four-
segmented tarsi, and tarsal claws which are all narrow and uncinate.

Wings: These have brown venation and are clear except in the C—Se region of the
forewing, which is opaque, particularly towards the apex, in which the cross veins are
all shaded.

Genitalia: The genitalia are similar to A. australis, but the penes are more separated
than in this species.

Caudal Filaments: These are dark brown.

Female Imago.
Egg (Text-fig. 29): On the egg are hexagonal markings separated by circular
areas.

Male Subimago.

Wings: The forewing is heavily shaded with dull brown, giving in some cases the
complete lambda pattern described for this genus by Tillyard (1933), but in others the
shading is much less noticeable so that it gives a superficially complete brown colour
to the wing.

Nymph.

Measurements: Body length, 9:0 mm.

General Colour: This is light brown to greenish-brown.

Mouth Parts (Text-figs. 68-71): The labrum bears a median concavity with five
dentate incisions on the anterior border, and sparse hairs on the outer anterior angles.
The mandibles show a variable number of teeth in both the right and left canines,
the minimum being five in the outer canine; and the molar area of the left mandible
is larger than that of other Atalophlebia. The labium bears a two-segmented palp and
the paraglossae are large, with two distinct rows of spines, while the glossae are
small and ovate with three encircling rows of spines.

Abdomen: In the abdomen the lateral margins are produced into striking recurving
spines.

Gilis (Text-fig. 67): The gills are trifurcate, the three finger-like processes on each
gill of a pair arising from a broad lamellate process. ,

Specimens Examined.

Imagines. Lake Leake, Tas., 2,000’, 1:1929, H. M. Stephens; Dumaresque Cr., Armidale,
3,300’, 2:1947; Gara R., Armidale, 3,000’, 3: 1948.

Nymphs. Dumaresque Cr., Armidale, 3,300’, 4: 1947.

ATALOPHLEBIA PARVA, SP. NOV.

Holotype 30, Gara R., Armidale, 3,333’, 3: 1948.

Allotype 9, and Morphotype, Gara R., Armidale, 3,333’, 3:1948.

Paratypes, Dumaresque Cr., Armidale, 3:1948.

DESCRIPTION.
Male Imago (Text-fig. 6).

Measurements: Body length, 7-0 mm. (6:5-8-5 mm.). Cerci, 70 mm. Appendix
dorsalis, absent. Forewing, 7:0 mm. (6:5-8-0 mm.). Hindwing, 1:2 mm. Foreleg, 1:5
2°3, 25 mm. (Tarsi, 0-8, 0-09, 0:09, 0:15 mm.). MHindleg, 1:4, 2:0, 0-5 mm. (Tarsi,
0-09, 0-09, 0-09, 0-15 mm.).

BY JANET BE. HARKER. 15

25a

Text-figures 19-27. Penes. (‘“a” after a number indicates lateral view.)
19, Atalophlebia marogwana; 20, Atalophlebia longicaudata; 21, Atalophlebia parva; 22,
Atalophlebia maculosa; 23, Atopopus spadixz ; 24, Deleatidium annulatum ; 25, Leptophlebia crassa;
26, Baétis confluens; 27, Baétis baddamsae. (All figures x 25.)

16 AUSTRALIAN EPHEMEROPTERA,

General Colour: This is a yellowish-brown, except for the thoracic region, which
is black.

Head: The head is black with short colourless antennae and white ocelli. The
eyes are divided into an upper yellow to orange-brown region and a lower dove-grey
or black.

Thorax: This is dark brown with black markings and a distinct white line which
runs down beside the prescutoscutal suture.

Abdomen: The abdomen is dull brown with lighter brown markings and lighter
intersegmental areas. ;

Wings: These are transparent with creamy venation, except in the costal area of
the forewing, which is opaque, particularly in the pterostigmatic region (Text-figs.
Ab OE

Legs: The forelegs have reddish-brown femora with dark brown markings and
yellow-brown tibiae and tarsi. The mid- and hindlegs are yellow-brown with dark
brown markings. The tarsal claws are all narrow and uncinate.

Genitalia (Text-fig. 21): The forceps are three-jointed; the basal joint is dull
brown changing to white distally, and the two distal joints are white.

Caudal Filaments: The appendix dorsalis is absent. The cerci are yellow-brown
with dark brown annulations.

Female Imago.

Measurements: Body length, 6-5 mm. (5:5-9-0 mm.). Cerci, 7:00 mm. Appendix
dorsalis, absent. Forewing, 6:2 (5:5-6-5 mm.). Foreleg, 1:0, 1:0, 0-5 mm.

General Colour: This is dark reddish-brown with the thorax showing distinctly
darker brown.

Head: The eyes are greyish-black.

Abdomen: This is more uniform in colour than that of the male imago, being
dark reddish-brown with a lighter coloured mid-dorsal line.

Eggs: The eggs are very distinctive. They bear two rows of appendages at either
pole and the immature eggs show a sculpturing of hexagonal figures each with a
central pit, while the mature eggs show radiating lines from the pit to the angles
of the hexagon (Text-fig. 35).

Male Subimago.
The presence of uniformly grey wings distinguishes this stage from the imago.

Female Subimago.

In the majority of female subimagines the appendix dorsalis is present. Measure-
ment, 7°0 mm. (6-5-8-5 mm.).

Nymph.

Measurements: Body length, 8:0 mm.

General Colour: This is chocolate-brown. ;

Head: The head is rectangular in shape when viewed from the dorsal surface.
The eyes are black and the ocelli black with white bases.

Mouth Parts: The labrum (Text-fig. 75) is slightly concave on its anterior margin,
the concavity being dentate. The mandibles (Text-figs. 73, 74) bear outer and inner
canines with a variable number of teeth. The molar surface of the left mandible tapers
to a sharp point on the inner edge and a chitinized angular projection is present on
the inside edge below the molar surface. The prostheca is present in both mandibles,
but is both more acuminate and more heavily chitinized apically in the right. The
maxillae (Text-fig. 77) bear three-segmented palps, of which the basal two segments
are about equal in size while the distal segment is much shorter. The anterior edge
of each maxilla is fringed with a thick brush of brown hairs, below which is a row
of about twenty parallel pectinate “rakes”. The labium (Text-fig. 76) bears two
three-segmented palps, the distal two segments being much narrower than the proximal
segment. Spines occur on the basal segment, particularly on the outer margin, and

BY JANET E. HARKER. 17

also on the distal portion of the middle segment. The paraglossae bear a group of
spines on the anterior margin, while the glossae are densely covered with hairs.

Thorax: This is yellow-brown with curving, dark-brown markings.

Abdomen: The abdomen is dull brown with yellowish-brown markings.

Legs: The femur and tarsus each bear one dark band, and the claws are prominent
and hooked at the tips.

Caudal Filaments: These are yellow-brown with dark annulations.

Gills (Text-fig. 72): These are paired and lanceolate in shape. The outer member
of each pair narrows at about two-thirds of its length and then expands to a
lamelliform tip.

Biology.
The subimago of this species is very sluggish and apparently attracted to light
colours, as it will settle on the clothing of the collector and shows no tendency to
fly away.

Specimens Hxamined.

Imago. Queanbeyan R., 1,901’, 2:1948; Gara R., Armidale, 3,300’, 4:1948, G. Davis;
Dumaresque Cr., Armidale, 3,000’, 3: 1948; Commissioners Waters, Armidale, 3,000’, 4: 1948;
Serpentine R., Point Lookout, 4,000’, 10:1948; Mienna, Tasmania, 3,000’, 12:1928, H. M.
Stephens.

Nymphs. Queanbeyan R., 1,901’, 2:1948; Gara R., Armidale, 3,000’, 4:1948, G. Davis;
Dumaresque Cr., Armidale, 3,000’, 3: 1948.

Genus LEPTOPHLEBIA Westwood.

Intro. Mod. Classif. Ins., 2: 31, 1840.

Synonymy: Huphyurus Bengtsson, Ent. Tidskr., 38:177, 1914.
Genotype: L. marginata Linn.

Male Imago.

Wings: In the forewing the posterior branch of Rs sags posteriorly; in the hind-
wing the costal margin is very flatly arcuate with a shallow depression in the middle
region. Cross veins are abundant in both wings.

Genitalia: The forceps are three-segmented, but a fourth may be present. The
penes usually each bear an acuminate spine at the tip, and a reflex spur reaching down
to the base of the central notch.

Legs: One of each pair of tarsal claws on a tarsi is blunt and the other hooked.

Nymph.
The gills are lanceolate and double, and there are well-developed lateral spines on
the abdominal segments.

LEPTOPHLEBIA CRASSA, Sp. NOV.

Holotype Subimago ¢, Dumaresque Cr., Armidale, 3,300’, 3: 1947.

Allotype Subimago 2, Dumaresque Cr., Armidale, 3,300’, 8: 1947.

Morphotype Nymph, Barrington Tops, 4,750’, 3: 1948, B. McMillan.

Paratypes, as above.

DESCRIPTION.
Male Subimago (Text-fig. 9).

Measurements: Body length, 11-5 mm. Cerci, 20:0 mm. Appendix dorsalis, 19-0 mm.
Forewing, 13:0 mm. Hindwing, 3:0 mm. Foreleg, 3-0, 2-5, 1:5 mm. Hindleg, 3-5, 2-0,
0-8 mim.

General Colour: This is yellow with brown markings.

Head: The head is yellow with chocolate-brown markings and brown antennae.
Both regions of the eyes are dark brown and the ocelli are white.

Thorax: The thorax is yellow with definite brown markings.

Abdomen: This is yellow with dark brown to black markings; on the lateral
margin, which is slightly acute, there is a distinct black spot.

Wings (Text-fig. 60): The wings are grey with slightly darker veins, and in the
hindwing the costal margin shows a slight concavity near the middle.

Cc

18 AUSTRALIAN EPHEMEROPTERA,

Legs: These are yellow with brown markings, the tarsi being four-segmented and
bearing tarsal claws, of which one of each pair is narrow and acuminate and the
other blunt.

Genitalia: The torceps are three-segmented, and from the tip of each penis projects
a weak acuminate spine.

Caudal Filaments: These are yellow with a single brown band at the distal end
ot the basal segment. The appendix dorsalis is slightly shorter than the cerci.

Text-figures 28-37. Eggs.

28, Atalophlebia albiterminata; 29, Atalophlebia incerta; 30, Atalophlebia marowana; 31,
species referred to in Appendix II; 32, Deleatidium annulatum; 33, Clo€on fluviatile; 34,
Leptophlebia crassa; 35, Atalophlebia parva; 36, Atalophlebia longicaudata; 37, Caénis scotti.
(All figures x 60.)

Female Subimago.

Measurements: Body length, 12-5 mm. (9-0-14:0 mm.). Cerci, 20°0 mm. Appendix
dorsalis, 19°70 mm. Forewing, 11:0 mm. (9:0-12:0 mm.). Hindwing, 2-5 mm. (2-0-4:0
mm.). Foreleg, 3:0, 2-0, 0‘°8 mm. Hindleg, 3-0, 2:5, 0:8 mm.

Head: The head is darker than that of the male. :

Egg: The egg is almost spherical and bears raised knobs, each one of which is
surrounded by a circle of minute knobs.

Nymph.

Measurements: Body length, 9:0 mm. Cerci, 9:0 mm. Appendix dorsalis, 10:0 mm.

General Colour: Yellow with dark markings on the abdomen.

Head: The head is dark brown to black with white ocelli and short antennae.

Mouth Parts (Text-figs. 79-83): The labrum has no median concavity but bears
a row of minute hairs along the anterior border; the lateral margins are extended
and incurved. The left mandible has both the outer and inner canines bearing only
one tooth, which is sharply pointed apically, and a prostheca which is acuminate and
chitinized apically; the right mandible has serrated canines and no chitinized process
in the prostheca. The maxillae bear three-segmented palps, of which the two distal
segments bear hairs. The “plate” region is flattened and disc-shaped, and bears a tuft
of brown hairs intermingled with which are recurving pectinate “rakes”. The labium

BY JANET E. HARKER. 19

bears three-segmented palps, the segments of which decrease in size distally. The
paraglossae are egg-shaped, with the narrow end facing inwards towards the glossae,
and bear spines along the anterior borders and the latero-anterior angles. The glossae
are narrow, with almost parallel sides, and a bear a few hairs at the tips.

Thorax: The thorax is light brown with darker markings, and the wing buds are
generally black.

Abdomen: The lateral margins of the abdomen are produced into spines, of which
those of the ninth segment are the longest.

Legs: The legs are almost colourless, but may be tinged with brown along the
ventral portion of the femora and tarsi. The tarsal claws bear large denticles.

Gills: The gills are paired, lanceolate, and seven in number. The gills on the
third abdominal segment are largest and the following pairs decrease in size with
order (Text-fig. 78).

Specimens Hxaamined.

Subimagines. Serpentine R., Point Lookout, 4,000’, 10: 1948; Marowan Cr., Glen Innes,
3,520’, 10:1948; Mienna, Tas., 3,300’, 12:1928, H. M. Stephens.

Nymphs. Dumaresque Cr., Armidale, 3,300’, 7:1948; Tumut R., Talbigo, 1,200’ (under
rocks), 3:1948, B. McMillan; Barrington Tops, 4,750’, 3: 1948, B. McMillan.

Genus DELEATIDIUM Haton.

Trans. Ent. Soc. London, 1899.
Genotype: D. lilli Eat.

Imago.

“Distinguished as a genus from Leptophlebia by the male imago having genitalia
conformable in pattern to those of an Atalophlebia” (Haton).

Nymph.
Body flattened dorso-ventrally.
Head: Square in shape, with eyes borne laterally. Mouth parts similar to
Atalophlebia.
Gills: Single.

DELEATIDIUM ANNULATUM, Sp. nov.

Holotype ¢%, Serpentine R., Point Lookout, 4,000’, 10: 1948.
Allotype 2, Morphotype Subimago, Paratypes, Talbigo, 1,200’, “at dusk’, 12:1947, B.
MeMillan.

DESCRIPTION.
Male Imago (Text-fig. 10).

Measurements: Body length, 10:0 mm. Cerci, 11:0 mm. Appendix dorsalis, 11:0 mm.
Forewing, 9:0 mm. Hindwing, 15 mm. Foreleg, 1:5, 3:0, 0-5 mm. Hindleg, 2-0, 1-0,
0-5 mm.

General Colour: This is tan.

Head: The head is orange with brown markings, while the upper region of the
eyes is pale reddish-brown, and the lower and the ocelli are grey.

Thorax: Creamish-orange with light brown markings.

Abdomen: The abdomen is creamish-orange with definite markings which change
in the posterior segments from dark brown to red-brown.

Wings: The forewing is faintly opalescent in the C-Se region, in which the cross
veins are unevenly shaded; elsewhere the wings are clear with brown venation.

Legs: The legs are cream with two red-brown bands on the femora, and on each
tarsus one claw of the pair is narrow and acuminate and the other is blunt.

Genitalia: The forceps are yellow and the penes red-brown.

Caudal Filaments: These are cream with definite brown annulations.

Subimago.
Wings: The wings are grey-brown in colour.
cc

EPHEMEROPTERA,

AUSTRALIAN

20

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BY JANET E. HARKER. 21

Specimens Hxamined.
Imagines. Juanama Cr., Talbigo, 1,200’ (at dusk), 12: 1947, B. McMillan; Tumut-Talbigo,
900’-1,200’ (in rain), 12:1947, B. McMillan; Marowan Cr., Glen Innes, 3,500’, 10:1948;

Serpentine R., 4,000’, 10: 1948.
Family BAETIDAE.
Imago.

Head: In the male the development of the compound eye is outstanding and is
known as a “turban eye”; the upper part is raised on a broad pedestal and is lighter
in colour than the lower, normally rounded, portion.

Thorax: The pronotum of the female is narrow, closely connected to the mesonotum
and receding behind.

Wings: In the forewing the cross veins are usually greatly reduced, and along the
margin is developed a series of short marginal veinlets between the branches of the
main veins. The middle branches of all the treads are disconnected, except occasionally
IR,b and ICuA, and the anal area is greatly enlarged, with a reduction of anal venation.
The hindwings are greatly reduced or absent.

Caudal Filaments: The appendix dorsalis is aborted.

Nymph.
Mouth Parts: The maxillary palps are three-segmented.
Gills: Seven pairs are present, all being exposed and lamelliform.

Genus BAETIS Leach.

Brewst. Edinb. Encycl., 9: 137, 1815.

Synonymy: Brachyphlebia Westwood; Introd. Mod. Classif. Ins., 2:25, 1840. Cloé Pictet;
Hist. Nat. 2 Ephem. Neurop., 1843 Acentrella Bengtsson; Hnt. Tidskr., 1912.

Genotype: B. binoculatus Linn.

Imago.

Wings: The forewing is 2-9 mm. and has marginal intercalaries occurring in pairs
and basal costal cross veins are entirely wanting. In the hindwing there are only
two or three longitudinal veins.

Genitalia: The forceps are four-segmented, the basal segment being the stcutest
but contracting at its distal end, and the third segment being the longest.

Nymph.

Head: The head is hypognathous.

Mouth Parts: The labrum bears a notch in a median position on its anterior
margin. The mandibular canines are large with blunt teeth. The maxillary palp may
be three- or two-segmented.

Gills: Single, obtusely ovate or obovate gills are present on abdominal segments 1-7.

BAETIS BADDAMSAE, Sp. Nov.

Holotype o, Guyra, 4,300’, 9: 1948, G. Baddams.

Allotype 2, Guyra, 9:1948.

Morphotypes Subimagines, nymphs, Billy’s Cr., Grafton-Armidale Rd., 3: 1948.
Paratypes, Marowan Cr., Glen Innes, 3,520’, 10: 1948.

DESCRIPTION.
Male Imago (Text-fig. 15).

Measurements: Body length, 6:5 (6-0-7-0 mm.) or 11:6 mm. (11:0-12:0 mm.). Cerci,
10:2 mm. or 16:0 mm. Appendix dorsalis, absent. Forewing, 6:3 mm. (5:5-7-0 mm.) or
9-9 mm. (9-0-11-0 mm.). Hindwing, 2:0 mm. or 3:0 mm. Foreleg, 1:5, 2:0, 2-5 mm.
Hindleg, 4:0, 4:0, 2-0 mm.

Text-figures 38-51. Forewings.
38, Atalophlebia longicaudata, subimago; 39, costal area imago, Atalophlebia longicaudata ;
40, Atalophlebia albiterminata, costal area imago; 41, Atalophlebia albiterminata subimago; 42,
Baétis confluens; 43, Atalophlebia maculosa; 44, Atalophlebia incerta; 45, Atalophlebia parva;
46, species referred to in Appendix II; 47, Caénis scotti; 48, Deleatidium annulatum; 49, Baétis
baddamsae ; 50, Atopopus spadix; 51, Atalophlebia marowana. (All figures x 4.)

AUSTRALIAN EPHEMEROPTERA,

bo
bo

General Colour: Black with conspicuous orange eyes.

Head: This is black with very short antennae and eyes of which the upper lobes
are bright orange and the lower dark brown or black.

Thorax: The thorax is black with very narrow yellow markings.

Abdomen: In the abdomen the anterior two segments are black with yellow
markings and the following segments are brown with slightly darker markings in
small areas; a distinct white line runs along the lateral margins.

Wings: These are colourless with yellow-brown venation, and in the hindwing the
anterior margin rises to an acute peak close to the base.

Legs: The legs are grey-brown and the fore-tarsi have a distinctly crenulate
outline. The tarsal claws of each pair are dissimilar, one being narrow and acuminate
and one blunt and ovate.

62

Text-figures 52-63. Hindwings.

52, Atalophlebia albiterminata, subimago; 53, Atalophlebia maculosa; 54, Atopopus spadiz ;
55, species referred to in Appendix II; 56, Deleatidium annulatum; 57, Atalophlebia parva; 58,
Baétis confluens; 59, Atalophlebia incerta; 60, Leptophlebia crassa; 61, Atalophlebia marowana ;
62, Baétis baddamsae ; 63, Atalophlebia longicaudata. (All figures x 16.)

BY JANET E. HARKER. 23

Genitalia: The forceps are four-jointed.
Caudal Filaments: Basally these are brown with lighter annulations, but distally
they are cream with dark annulations.

Female Imago.

General Colour: This is brown with a black head and thorax.

Head: The eyes are grey.

Thorax: This is dark brown with a distinctive yellow pattern.

Eggs: The eggs are of a distinctly greenish colour when first laid, but darken
after the first day; there is no sculpturing on the egg at all. The eggs are laid in
masses which stick together by means of a gelatinous substance which adheres closely
to the substratum.

Subimago (Text-fig. 12).
General Colour: Creamy-yellow with darker thorax.
Wings: The wings are faintly grey with yellow venation.
Legs: These are yellow with segments outlined with dark brown.

Nymph.

Measurements: Body length, 10-0 mm. or 6:0 mm.

General Colour: Grey-brown.

Head: The head is cream with dull brown markings, black eyes and grey ocelli.

Mouth Parts (Text-figs. 84-88): The labium is deeply notched in the middle region
of the anterior margin, which also bears a row of spines.

The mandibles bear blunt canines, the inner one bearing three teeth and the outer
not being toothed, and a prostheca being present only in the right mandible. The
maxillae bear two-segmented palps, of which the distal segments are the longest. The
galea-lacinia narrows anteriorly, the galea region terminating in an acuminate point
while the lacinia region is much broader and ends in a row of spines. On the labium
the palps are two-segmented, the basal segments being the longest and broadest and
the distal segments bearing spines on the outer surface; the distal segment also appears
to have an inner lobe, which is deeply incised from the outer lobe. The paraglossae
and glossae are of similar dimensions and are both pointed, the glossae bearing a row
of spines on the inner surface.

Biology.

These Baétids are seldom found flying over water; they appear to emerge and fly
almost perpendicularly upwards out of sight. The nymphs are present among algae,
where they can be seen gently raising and lowering their caudal filaments as they feed.

There appear to be two distinct physiological races present in this species. From
a large series which was collected from one location in a stream all the imagines,
subimagines and nymphs could be divided into two distinct groups on size, the groups
not overlapping.

Specimens Haamined.

Imagines. Gara R., Armidale, 3,300’, 12:1947; Guyra, 4,300’, 9:1948, G. Baddams;
Tillbuster Cr., Armidale, 3,300’, 9:1948; Marowan Cr., Glen Innes, 3,520’, 10:1948; Mienna
Cr., Tasmania, 3,300’, 12: 1928, H. M. Stephens.

Nymphs as above and Billy’s Cr., Armidale-Grafton Rd., 1,800’, 3: 1948.

. BAETIS CONFLUENS, Sp. NOV.
Holotype %, Dumaresque Cr., Armidale, 3,300’, 9: 1947.
Allotype 2°, Paratypes, Dumaresque Cr., Armidale, 3: 1948.

DESCRIPTION.
Male Imago (Text-fig. 14).
Measurements: Body length, 45 mm. Cerci, 9:0 mm. Forewing, 4:0 mm. Hindwing,
05 mm. Foreleg, 1:1:2 (2:9 mm.). Hindleg, 3:2:2 (1:5 mm.).

General Colour: Light brown to orange-brown.

24 AUSTRALIAN EPHEMEROPTERA,

Head: The head is light brown with “turban” eyes, of which the surfaces are orange
and the pedestal yellow, and the ocelli are slightly stalked.

Thoraz: This is light brown with darker brown and yellow markings.

Abdomen: This is light brown with sparse markings and the lateral margins are
slightly concave, especially in the posterior segments.

Wings (Text-fig. 42): In the forewing the costal and apical area of the subcostal
region is slightly milky, Rs forks close to the base and IR,b commences close to the
fork, MA is unfolded. In the hindwing very few cross veins are present and the
anterior margin is convex.

Legs: The legs are light brown and the tarsal claws of each pair on a tarsus are
dissimilar, one being narrow and acuminate and one blunt and ovate.

Subimago and nymph unknown.

Specimens Examined.
Dumaresque Cr., Armidale, 3,300’ (rain), 9: 1947.

CLOEON FLUVIATILE Ulm.

Archiv. fiir Naturgeschichte, 11, 1919.

To the original description all that need be added is a description of the egg.

Egg: Slightly ovate, cream in colour. Sculpturing reticulate; the markings surround
almost circular smooth areas (Text-fig. 33).

Specimens Examined.
Armidale, 3,300’, 9: 1948.
Family CAENIDAE.
(Brachycercidae Lestage.)

The type genus of this family, genus Caénis, was suppressed by Lestage as a
synonym of Brachycercus Curtis, which changed the family name to Brachycercidae.
Since then no other authors have followed this alteration, and there seems some doubt
as to the true synonymy of these two genera. Therefore until further evidence can be
put forward it is proposed to use the name in general usage, Caénis.

Imago.

Head: The compound eyes are button-shaped and widely spaced in both sexes,
the antennal second joint is at least twice as long as the first, usually longer, and the
posterior margin of the head is nearly straight.

Wings: The anal area is well rounded and usually has only one anal vein. The
cross vein system is greatly reduced and no free marginal veinlets are present. The
forewing is 2-5 mm. in length and the hindwing is nearly always absent.

Nympn.
The gills of the second abdominal segment are enlarged and form a gill cover
for the succeeding gills.
The only genus recorded from Australia is the Type genus.

Genus CAENIS Stephen.

Ill. Brit. Ent., 6:61, 1835.

Synonymy: Ordella Campion, Ann. Mag. Nat. Hist., 11, s. 9, 64:515. Ordella Lestage,
Ann. Soe. Ent. Belg., 65: 61.

Genotype: C. halterata Fab.

Imago.

Wings: Exceptionally broad near the base (a feature which distinguishes Oaénis
from the Tasmanian genus Tasmanocaénis Lestage). The cross veins usually occur
singly in each intercalary region. t

Antennae: The second joint of the antennae is not more than twice the length
of the basal joint.

Appendix dorsalis: Always present and may be slightly longer than the caudal
filaments.

BY JANET E. HARKER. 25

Nymph.

The nymph is important in distinguishing this genus from the closely related
Brachycercus and Tricorythodes, so that although these two genera have not been
found in Australia they are mentioned in comparison with Caénis, as it is very likely
both of the former genera are present in Australia.

Mouth Parts: These are similar in all three genera, but the labrum is less broad
in Caénis than Brachycercus and its apical margin is slightly concave. The glossae
and paraglossae are fused in Tricorythodes but differentiated in the other genera.

Gills: The gills on segments 3-6 are single in Caénis and Brachycercus, but double
in Tricorythodes.

The postero-lateral spines of the abdominal segments are upcurved in Brachycercus
but not in the other genera.

CAENIS scorTi1 Till.

Proc. Roy. Soc. Tasmania, 1935 (published 1936).

Holotype Imago, Esk River, Clarendon, Tasmania, 3: 19338.

Tillyard has only described the male imago of this species. The imago has not
been found at all in Australia, and the five specimens taken were unfortunately placed
in spirit and not allowed to undergo their final ecdysis.

Text-figures 64-77.
Atalophlebia albiterminata nymphal mouth parts. 64, right mandible; 65, left mandible;
66, labrum. Atalophlebia incerta nymph. 67, gill; 68, right mandible; 69, left mandible; 70,
labrum; 71, labium. Atalophlebia parva nymph. 72, gill; 73, right mandible; 74, left mandible;
75, labrum; 76, labium; 77, maxilla. (All figures x 4.)

Female Imago.
Fog: (Text-fig. 37.)

Female Subimago (Text-fig. 13).

Five specimens only in the series.

Measurements: Body length, 6-4 mm. Cerci, 2-5 mm. Appendix dorsalis, 3-5 mm.
Forewing, 5:0 mm. Hindwing, absent.

General Colour: Cream with dark brown markings, grey-brown antennae and dark
brown eyes and ocelli.

Thorax: This is cream with dark brown markings and a dull brown mesonotum.

Abdomen: The abdomen is cream with brown stippled markings, a light cream
venter with black markings, and the lateral margins are gently curved.

26 AUSTRALIAN EPHEMEROPTERA,

Wings (Text-fig. 47): The wings are opaque with dark brown venation. The
hindwings are absent.

Legs: In the foreleg the femur is white with a brown outline and the tibia and
tarsus light grey; the mid- and hindlegs are white with black markings. The tarsal
joints are four-segmented, but the divisions are very indistinct. Of each pair of tarsal
claws one is blunt and rounded and the other curved and pointed.

Nymph (Text-fig. 18).

Measurements: Body length, 5-5 mm.

General Colour: Cream with dull brown markings giving an impression of light
dull brown.

Head: This is cream with brown markings, black eyes and short antennae.

Mouth Parts (Text-figs. 92-96): The labium is slightly concave on its anterior
margin, the lateral margins of the concavity being slightly dentate. The mandibles
bear two groups of canines with a varying number of teeth in each group; the molar
surface is strongly chitinized and a prostheca is present. In each maxilla the plate
(fused galea and lacinea) is almost pointed, being strongly convex on its inner surface
and coneave on its outer, with just the distal portion convex; a few spines are
present at the distal extremity and the distal end of the outer surface is hairy. The
palp is three-jointed, the proximal segment being the longest, and spines are present
on the inner edge of the distal segment. The labium bears three-jointed palps, the
basal joints of which are stout and short and the third joints are very short; all three
joints bear stout spines. Both glossae and paraglossae are almost oblong and also
bear stout spines.

Thorax: This is cream with brown markings, there being two large semi-circular
markings on the pronotum and scattered markings on the mesothorax; the pronotum
is narrow and collar-like.

Abdomen: The abdomen is cream with brown markings, the venter is creamy
white with a dark line following the alimentary canal, and the lateral. margins are
convex with slightly incurving tips.

Legs: The legs bear black markings and rows of spines are present around the
tibio-tarsal joint. The tarsal claws bear fine denticles.

Gills: There are only six gills present. The first (Text-fig. 89) is extremely small
and of remarkable form, being apparently jointed into three segments, or sometimes
four, and bearing hairs. The second gill (Text-fig. 90) is modified to form a gill cover
for the remaining pairs and is heavily chitinized all over. The last four pairs are
filamentous and very flexible (Text-fig. 91).

Biology.

‘The nymph is distributed widely, occurring in every district from which mayflies
have been collected. It has been found below the surface of gravel bottoms of the
running streams, but more often is found in the muddy banks or the silted edges of
a stream. It is often present in streams which are drying up, usually either in the
mud bottom or under and amongst any algae which may be present. In the laboratory
it has been kept alive under a small clump of Spirogyra, which retained some moisture,
for a period of five weeks; it has also been found under the caked surface of the
muddy bottom of dishes which have dried out.

The adult appears not to be as prevalent as the abundance of larvae would lead
one to expect, but this may be a result of collecting at the wrong times; however, it
is the only nymph which has not been able to be bred through in the laboratory.

Specimens Hxamined.
Imago. Dumaresque Cr., Armidale, 3,000’, 8: 1947.

Nymphs. Umberumberka Dam, Silverton Shire, 983’, 8:1947, A. Stokes; Gara R.,
Armidale, 3,000’, 2:1948; Queanbeyan R., 1,901’, 2:1948: Pine Forest, Armidale, 3,000’,
10: 1947.

BY JANET E. HARKER. 27

Genus ATOPOPUS Eaton.
Ent. Mo. Mag., 17-18:191-197, 1881.
Genotype: A. tarsalis Ent.

Imago Male.
“Foreleg about as long as body; tarsus about 1:4 as long as tibia, and this nearly
1:2 as long as femur. The tarsal joints in order of shortening rank 1, 2, 3, 4, 5, and
the first about 1:3 as long as the second, and nearly half as long as the tibia. Hind-
tarsus as long as second, and nearly 0:6 as long as femur. The first joint about 3-4
as long as the second, and upward of 1:1 as long as the tibia. Ungues unlike each
other in every tarsus. Forceps limbs 3 jointed... .”

Text-figures 78-101.

Leptophlebia crassa nymph. 78, gill; 79, right mandible; 80, left mandible; 81, labrum;
82, maxilla; 83, labium. Baétis baddamsae nymph. 84, labium; 85, maxilla; 86, labrum; 87,
right mandible; 88, left mandible. Caénis scotti nymph. 89, gill from ist abdominal segment ;
90, gill from 2nd abdominal segment; 91, gill from 3rd abdominal segment; 92, right mandible;
93, left mandible; 94, maxilla; 95, labrum; 96, labium. Species referred to in Appendix II,
nymph. 97, frontal process of head; 98, labium; 99, maxilla; 100, right mandible; 101, left
mandible. (Various magnifications.)

ATOPOPUS SPADIX, Sp. NOV.

Holotype ¢, Armidale, 3,300’, 10: 1948.
Allotype 2, Paratypes, Badja, 3,000’, 11:1929, H. M. Stephens.

DESCRIPTION.
Male Imago.
Measurements: Body length, 9-5 mm. Cerci, 12:0 mm. Appendix dorsalis, absent.
Forewing, 9:0 mm. Hindwing, 2:0 mm. Foreleg, 1:8, 3:2, 4:0 mm. MHindleg, 2:0, 1-0
2-5 mm.

28 AUSTRALIAN EPHEMEROPTERA,

General Colour: Red-brown with clear black markings.

Head: This is red-brown with dark brown markings, eyes which are dark brown
and undivided, and white ocelli.

Thorax: This is dark brown with black margins to the sutures except that of the
prescutoscutal, which is white.

Abdomen: The abdomen is yellowish-red to red-brown with clear-cut brown marking.

Wings: The wings are clear with yellow venation, and the cross veins in the C-Sce
area of the forewing are tinged with reddish-brown.

Legs: The legs are yellow with two dark markings on the femora. The hind- and
midlegs, as is usual in this genus, are remarkable for their proportions, and the tarsal
claws on each tarsus are dissimilar.

Genitalia: The forceps are three-segmented and yellow in colour.

Female Imago.
As only pinned specimens were examined no eggs could be described.

Nymph unknown.

Specimens Examined.

Armidale, 3,300’, 10:1948; Badja, 3,000’, 11:1929, H. M. Stephens.

Although the practice of describing species from pinned specimens is not followed in
general, in the case of this species, where a sufficient number of specimens in spirit were
not obtainable for a good series, the series has been completed with pinned specimens from
the H. M. Stephens collection, which are in very good condition.

CHECK LIST OF AUSTRALIAN SPECIES.
(Including Tasmania.)

Superfamily BAETOIDEA.
Family LEPTOPHLEBIIDAE.

ATALOPHLEBIA HEitn., 1881; Tillyard, 1933. Type A. australis Walk.
albiterminata Till., 19385. Proc. Roy. Soc. Tasmania. Type location unknown.
australasica Pict., 1843, in Baétis; Leptophlebia Eaton, 1871; Eaton, 1888; Ulmer,

1917. Synonym: A. costalis Burm. Type location: British Museum.
australis Walk., 1853, in Hphemera; Leptophlebia Etn., 1871; Etn., 1888; Till.,
1933. Type location: British Museum.
brunnea Till., 1935. Type location: British Museum.

*costalis Burm., 1893, in Baétis; Potomanthus Pict., 1853; Leptophlebia Htn., 1871;
Etn., 1888; Hutton, 1898, Trans. N.Z. Inst., Vol. 31; McLachlan, 1894, Ent. Mo.
Mag.; Ulmer, 1920. Synonym of A. australasica Pict.

delicatula Till., 1935. Type location unknown.

*furcifera Etn., 1871, in Leptophlebia; Etn., 1888. Synonym of Deleatidium furcifera.
Type location: National Museum of Victoria.

fuscula Till., 19385. Type location unknown.

hudsoni Till., 1935. Type location unknown.

ida Till., 1935. Type location: British Museum. ,

inconspicua Etn., 1871, in Leptophlebia; Etn., 1888. Type location: Hope Museum,
Oxford.

lucida Ulm., 1917. Type location: Stockholm Museum.

sexfasciata Ulm., 1917. Type location: Stockholm Museum.

simillima Ulm., 1919. Type location: Stockholm Museum.

superba Till., 1935. Type location: British Museum. Var. pallida Till., 1935.

*strigata Etn., 1871, in Leptophlebia; Etn., 1888; Ulmer, 1920, in Delatidium.

uncinata Ulm., 1917. Type location: Stockholm Museum.

LEPTOPHLEBIA Westwood, 1840. Genotype: L. marginata Linn., in Ephemera.
*australis Etn.,'1871; Etn., 1888, in Atalophlebia.
*furcifera Etn., 1871; Etn., 1888, in Atalophlebia.
*inconspicua Etn., 1871; HEtn., 1888, in Atalophlebia.

*strigata Etn., 1871; Etn., 1888, in Atalophlebia.

BY JANET E. HARKER.

DELEATIDIUM Htn., 1899. Genotype: D. lilli Htn.
strigatum Htn., 1899. Synonym: Huphyurus bicornis Ulm.
furcifera Etn., 1871, in Leptophlebia; Htn., 1888, in Atalophlebia.

Family SIPHLONURIDAE.
TASMANOPHLEBIA Till., 1921. Genotype: 7. lacustris Till.
lacustris Till., 1921. Type location: British Museum.
lacus-coerulei Till., 1983. Type location unknown.
nigriscens Till., 1933. Type location unknown.
AMELETOIDES Till., 1933. Genotype: A. lacus-albinae Till., 19338.

29

CoLopuriscus EHat., 1864, Hnt. Mo. Mag. Synonym: Coloburis. Genotype: C. humeralis

Walk., in Hphemera.
giganticus Till., 1933. Type location: C.S.I.R.O., Canberra.
haleuticus Eat., 1871. Type location: National Museum of Victoria.
munionga Till., 1933. Type location: C.S.I.R.O., Canberra.
tonnoiri Lest., 1935. Type location unknown.

Family BAETIDAE.

Bakris Leach., 1815, Brewst. Edinb. Encycl. Genotype: B. binoculatis Linn.
*australasica Pict. Synonym of Atalophlebia costalis Burm.
frater Till., 1935. Type location: British Museum.

Croton Leach, 1815. Genotype: C. dipterum Linn., in Ephemera. Synonyms:

Pictet, 1843; Cloéopsis Vayssiére, 1882.
fluviatile Ulm., 1919. Type location: Stockholm Museum.
tasmaniae Till., 1935. Type location: British Museum.
virens Klap., 1905. Synonym: C. viridis Till., 1935; Ulm., 1916.

Family CAENIDAE.

CAENIS Steph., 1935, Ill. Brit. Hnt. Genotype: C. halterata Fab. (in Ephemera).

scotti Till., 1936. Type location: British Museum.

NEW SPECIES DESCRIBED IN THIS PAPER.

ATALOPHLEBIA.
incerta.
longicaudata.
maculosa.
marowana.
parva.

ATOPOPUS.
spadix.

BARETIS.
baddamsae.
confluens.

DELEATIDIUM.
annulatum.

LEPTOPHLEBIA.
crassa.

GEOGRAPHICAL DISTRIBUTION OF GENERA OCCURRING IN AUSTRALIA.

Cloé

Atalophlebia: New Zealand, Chile, Ceylon, Cape of Good Hope, Japan, South Africa.

Leptophiebia: Europe, North America, Chile.
Deleatidium: New Zealand, Chile.
Tasmanophlebia:

Ameletoides:

Coloburiscus: New Zealand, North America.

* Denotes synonymy.

30 AUSTRALIAN EPHEMEROPTERA,

Baétis: Burope, Canada, Greenland, Egypt, North America, Central and South America,
Indo-Malayan Region.

Cloéon: Europe, Indo-Malayan region, Japan, Chile, China, North America.

Caénis: Europe, Egypt, Morocco, Cape Colony, Ceylon, North America.

Atopopus: Borneo.

TECHNIQUES.
Preservation.

All stages are fixed in Carnoy’s fluid and preserved in 70% alcohol. Pinned
specimens are extremely unsatisfactory, as the colour darkens and body markings
usually become indistinguishable, due to the shrivelling of the specimen. By fixing the
specimen it is ensured that if new taxonomic techniques are evolved entailing histo-
logical examination the paratype will be in a suitable condition.

Preparation.

Genitalia. The terminal segments of the male are dissected away and the forceps
and penis dissected apart prior to any preparation—this was found to be the surest
way of preventing the parting of the two halves of the penis. The parts are then
boiled in KOH until the flesh dissolves, rinsed in water, allowed to stand for a minute
in glacial acetic acid, and washed again in water. If they are not perfectly cleared
the process can be repeated. It was found that if slides were prepared of the genitalia
some detail was lost in that only one view of the penis could be observed; therefore
the parts were mounted on a glass slip in glycerine jelly and placed in the same jar
as the insect from which they were taken. The advantage of this method is that
the preparation can be moved into any position with a warm needle, and there can be
no confusion as to which insect they refer.

Wings. When necessary wings were mounted by removing and placing in a drop
of alcohol on a glass slide, the alcohol allowed to evaporate and a cover slip sealed on.
Care must be taken to prevent the mounting medium from covering the wing itself,
as some of the veins become invisible.

Mouth Parts. These were prepared in a similar fashion to the genitalia, but
mounted in Canada balsam.

Eggs. These were dissected out and stained with neutral red or Orsein, and
examined with an oil-immeision lens.

APPENDIX I.
Genus ATALONELLA Needham and Murphy.
Bull. Lloyd Libr. 24, Ent. Ser., 4:1-79, 1924.
The erection of this genus has been criticized by Lestage (1931); to his arguments
the following points are added and cases cited.

Forewing.

1. Costal cross veins in basal half of Sc space supposed numerous in Atalophlebia
and wanting in Atalonella. It has been found in several series of forewings that the
cross veins in some species may be present or absent in this area, e.g., Atalophlebia
fuscula Till.

2. Costal veins of stigmal region erect in Atalophlebia, aslant in Atalonella; this
varies within a series, but not very noticeably. Intermediate forms occur and it would
be difficult to differentiate between erect and slanting.

3. Bisector of MA, MP fork at its proximal end nearer MP, in Atalophlebia, in the
middle of the fork in Atalonella. This has been found to vary so much that the two
types have even been found on the right and left wings respectively of the same
specimen.

4. CuA, straight in its apical third in Atalophlebia, curved in Atalonella. This,
too, has been found to vary in a series.

BY JANET E. HARKER. al

Hindwing.

1. Tip of subcostal vein at nine-tenths of wing length in Atalophlebia, three-quarters
in Atalonella. This is a constant character in all the specimens examined.

2. Upper lobe of median vein normal in Atalophlebia, disconnected at base in
Atalonella. This again has been found to occur in both forms, one in each wing of a
single specimen. However, the apparently missing piece can be distinguished if the
wing is stained so that care is needed in the use of such a character.

3. Cross veins between the anal veins present in Atalophlebia, absent in Atalonella.
These may vary within a series.

Table 2 gives species showing combinations of Atalonella and Atalophlebia characters.

TABLE 2.
Species. Atalonella Characters. Atalophlebia Characters.
Atalophiebia brunnea Number cross veins in costal | Bisector MA, MP closer to
Till. area. Cross veins in stig- MP,. Upper fork of median
mal region aslant. CuA, vein normal.

straight apically. Tip Se
at three-quarters wing
length. Bisector lower fork
MP absent. Cross veins
between anal veins absent.

Atalophlebia delicatula Cross veins in stigmal area | Cross veins in basal half of
Till. aslant. Bisector MA, MP Se-C area present. CuA,
in middle. Tip Se at three- vein straight at tip.
quarters wing length. Upper
fork median vein detached.
Bisector of lower fork MP

absent.
Atalophlebia longi- Bisector of MA, MP fork | Cross veins in the basal half
caudata, sp. nov. medianly placed. CuA, Se region numerous. Stig-
curved all the way to the mal cross veins erect. Tip
tip. Se at nine-tenths wing

length.

Nymph.

In distinguishing between the nymph of Atalophlebia and Atalonella Needham and
Murphy apply characters which not only appear in combination within each genera,
but these characters are also applied in two conflicting descriptions of Atalophlebia
characters.

To Atalophlebia nymphs are assigned the character ‘Posterior lateral angles of
rear abdominal segments not tipped with thin flat lateral spines’ (page 11), whereas
on page 36, in comparing Atalophlebia and Atalonella, lateral spines are said to be
present on abdominal segments 5-9.

Again on page 11, “Femora not dilated’; page 36, “Femora dilated, Atalonella
femora slender’; and in reference to Atalonella, page 11, ““Femora dilated’’.

APPENDIX II.

A specimen has been collected from Dumaresque Cr., Armidale, with unusual
characters. As only one adult has been bred out the author cannot describe a new
species, but it seems likely that this specimen cannot be included in any of the
known genera. %

DESCRIPTION.
Female Imago.
Measurements: Body length, 13:0 mm. Cerci, broken. Forewing, 13:0 mm. Hindwing,
3:0 mm. Foreleg, 3:0, 3:0, 1:5 mm. MHindleg, 3-0, 2:5, 1:0 mm.
General Colour: Cream with red-brown marks on the abdomen.

32 AUSTRALIAN EPHEMEROPTERA,

Head: The head is cream with brown markings, black eyes, short antennae
(0-3 mm.), and cream ocelli with brown bases.

Thorax: The thorax is cream with very faint light brown outlines to the thoracic
sclerites.

Abdomen: This is cream with red-brown markings, and the lateral margins are
rounded.

Wings: The wings have dark brown or red-brown veins which in the C-Se region
of the forewing are slightly shadowed, the wing itself in this region being opaque.

Legs: These are cream or white without any markings, and the claws of each pair
on a tarsus are dissimilar, one being hooked and the other blunt.

Nymph.

Measurements: Body length, 14 mm.

General Colour: Yellow with darker markings.

Head (Text-fig. 97): This is triangular with two small projections arising from
the head on either side of the labrum (not from the mandibles) 1:4 mm. long and
each bearing hairs at the tip. The head is light golden-brown with chocolate-brown
markings. The eyes are brown and the antennae are quite stout and bear whorls of
longish hairs.

Mouth Parts (Text-figs. 98-101): The labrum bears a small convex protuberance
in the middle region of the anterior margin, and hairs and small spines are present
on the antero-lateral angles together with a row of stout spines which slant inwards
towards the mid-line. The mandibles are stout with long hairs on the outer margin.
The outer canines bear two teeth, and the inner, one; a prostheca is present and the
molar region is very broad. The maxillae bear two-segmented palps, of which the
distal joints are almost rectangular with broad anterior edges bearing hairs; only a
few hairs are present at the distal end of the plates, below which are single rows of
pectinate spines. The labium bears two-segmented palps, of which the distal segments
are short and conical, and each bears a row of stout spines on the inner surface.
The paraglossae are broad and bear a clump of stout spines in the centre of the
ventral surface, the glossae are slightly kidney-shaped with a mid-longitudinal line of
spines. :

Thorax: The prothorax is oblong and golden-brown with chocolate-brown markings.

Abdomen: This is similar to the thorax, but with black markings; the venter is
white with a single median black line following the alimentary canal.

Legs: The legs bear long hairs.

Gills: These are double and lanceolate with a long filamentous distal end, and
hairs all over the surface of the gill. They are white with black tracheae.

Caudal Filaments: These are colourless with whorls of hair at the end of each
segment. The appendix dorsalis is slightly longer than the cerci.

Biology.
The nymph lives under the surface of the stream bed and can ‘only be found by
dredging.

ACKNOWLEDGMENTS.

To all those who have collected specimens throughout the course of this work I am
indebted, and in particular to Mr. B. McMillan.

The advice and criticism given by Mr. D. J. Lee, of the School of Public Health and
Tropical Medicine, University of Sydney, has been stimulating and of great assistance. To
Mr. D. Davies, of the same school, I am indebted for the redrawing of Table I.

To all the members of the Biology Department of New England University College,
Armidale, I am indebted for their assistance and co-operation. In particular I should like
to acknowledge the continual encouragement, advice and criticism given by Mrs. G. L. Davis,
without which the revision would not have been undertaken.

References.
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, 1980.—Kritische Bemerkungen ueber einige nordische Ephemeropteren, nebst
beschreibung nueur Larven. Lunds Univ. Arssk., N.F., Avd. 2, Bd. 26: 1-27.

BY JANET E. HARKER. 33

BrADLEY, J. C., 1931.—A laboratory guide to the study of insect wings. New York.

BrueEs and MELANDER, 1932.—Mus. of Comp. Zool, 73.

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Huxuey, J., 1932.—Problems of Relative Growth.

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LEAcH, 1815.—Brewst. Edinb. Encycl., 9: 137.

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Ent. France, xcviii.

—- , 1930.—Notes sur le premiere Brachycercidien découvert dans la faune Australienne
Tasmanocoenis tonnoiri sp. nov. et remarques sur la famille des Brachycercidae Lest. Mém.
Soc. Hnt. Belg., xxiii: 49-60.

MorGan, ANN, 1912.—Homologies in the wing veins of mayflies. Ann. Ent. Soc. America,
5: 89-106.

, 1913.—Contribution to the biology of mayflies. Ann. Ent. Soc. Amer., 6: 371-413.
, and GRIERSON, MARGARET, 1932.—The functions of the gills in burrowing mayflies.
Physiol. Zool., 5: 230-245.
MurpHy, H., 1922.—Notes on the biology of Baétis. Lloyd Libr. Bull., 22, Ent. Ser., No. 2: 1-46.
NEEDHAM, J. G., 1918.—Aquatic insects, Ward and Whipple. Fresh Water Biology, pp. 867-946.
, and Luoyp, J. T., 1916.—Life of inland waters. Ithaca, N.Y.
, and MurpHy, H., 1934.—Neotropical mayflies. Bull. Lloyd Libr., 24, Ent. Ser., 4:1-79.
, and NEEDHAM, P. R., 1927.—A guide to the study of freshwater biology. New York
and Albany. Pp. 10-14.
, TRAVER, J. R., and Hsu, Y., 1935.—The biology of mayflies. Ithaca, N.Y.

PHILLIPS, J. S., 1930.—A revision of the New Zealand Ephemeroptera. Trans. N.Z. Inst.,
61: 271-390.

, 1931.—Studies of New Zealand mayfly nymphs. Trans. Ent. Soc. London, 79.

Picrert, J. F., 1843.—Histoire Naturelle, générale et particuliére des Insect Neuroptéres.
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SCHENK, H. J., and MCMAsTERS, J. H., 1935.—Procedure in Taxonomy.

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SPEITH, H. T., 1933.— The phylogeny of some mayfly genera. Jour. New York Ent. Soc.,
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TILLYARD, R. J., 1917.—The biology of dragonflies. Cambridge, 1-396.

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, 1923.—Descriptions of two new species of mayfly from New Zealand. Trans. N.Z.

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, 1923.—The wing venation of the order Plectoptera. Journ. Linn. Soc. London,
35: 143-162.

————, 1924.—Plectoptera. Australian Encyel., 309-310.
, 1926.—Insects of Australia and New Zealand. Angus and Robertson, Sydney, 1-5
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23297-1384, 237-272.

———, 1936.—The Trout-Food Insects of Tasmania. Part ii. Monograph of the Mayflies
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60
2.
c

i.

34 AUSTRALIAN EPUEMEROPTERA,

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35

A STUDY OF THE ALKALINE PHOSPHATASH REACTION IN
TISSUE SECTIONS.

PART I. THE POSSIBILITY OF ITS QUANTITATIVE USE.

By K. W. CLELAND.
(Department of Histology and Embryology, University of Sydney.)

(Nine Text-figures.)

[Read 26th April, 1950.]

Table of Contents. Page

Introduction 35
Methods-—

(a) Photometric 36

(b) Histological 37

(¢) Chemical : 37
Accuracy of the HO LOHACRS erate 38
Inorganie basis of the reaction—

(a) Demonstration of calcium phosphate a ARMAS Em UAV P Tp eee EERE ie Ryne

(b) Validity of demonstration method HE VUNS= sere. nope eenee te tee abe 2 Ey age eS 9

(c) Formation of calcium phosphate aes ae 7 a ah ee Sie Suh a 6 ey eel)
The behaviour of the enzyme in sections—

(a) Comparison of fixatives eke Bech hake ene ease Ae ¢ Gres SCREEN en IRAy ae Meu ERS CoN (ort came Ze

(b) Stability of the enzyme in Beetions ah om Hen Sripe eae TAN AR EG a Rein td ayRe eb tun he:
Conditions of optimal enzyme activity—

(a) Effect of substrate concentrations Scans ae ke cee ee te isk ae UL ear a Ra EMT) B 2 3

(b) Effect of pH SuSE: 3 ul ye eS A (Gti eH ene ae SD VM cra ae eMC tt |e be, UMM ee Rien et SMe Fb

(c) Effect of buffer eoncentrtions Ce Aileen | OR am A OE pe a! Were CM a ey ead

(d) Effect of activators PS DAS ete aN GAPS ORR he Do OO Bee WM ee aS Te LAR RAS SO LEAN R RATA

(e) Effect of inhibitors Ree PE: Ry A Mae ete WI Lhe ees ete TU pio Mato eis 2a eet)

(if) THES Oi? CAlEtoNIN GCOMOSMIENON carves s5 -'56 “poe ocoflon »eo wo ues ne €o 4
Significance of the photometric estimate—

(a) Time/density relation ae LEANER Rte, ee arc 8 i cas ee RIAN

(b) Relation between density Arnal phosphate Content one ection! ROT RARE AO cE Sa AS SEIS PU STIAS
DISCUSSION Ime Dine RELY ciara Ure Soe SEL e seat, oheeet SESE gy pate Wekt Py ocieees econ MI MESE 0 LECUEML ih be ee thee bora 140
Summary oO. ese Fp IBS eS EE eerie. Samechens errr emer ant yee ae Seto ewe eM sere cae MEIN oa «eye 5k
Acknowledgement Ste AC sch SUS Ul than. Nealcleh, ATENEO coy UP TEOE BALAN en AE MEE BA, PMG S (n CER te Rem sy MRO)
References 52

INTRODUCTION.

Despite the enormous body of data comprising formal genetics, the mechanism of
gene reproduction and the method by which the genes exert their effect in the
cytoplasm are still only matters of speculation.

Before the problem can be rationally approached at the biochemical level much
further data on the chemical composition, and especially the enzyme constitution, of
the nucleus will be necessary. :

As a contribution to this problem a study of the variations in amount of a number
of phosphatases in the meiotic and spermateleotic cycles of the testis cells of guinea-pigs
was projected, making use of the reaction in tissue sections.

The first histochemical demonstration of phosphatase appears to have been made
by Robison and Soames (1930), who incubated thin slices of limb bones in solutions
of pH 7:4 containing calcium, inorganic phosphate and hexosephosphate. After pro-
longed incubation the calcium phosphate in the slices was demonstrated by the
von Kossa method. Rosenheim and Robison (1934) applied the same methods to
kidney, aorta and lung. The pictures obtained by these methods have little resemblance
to those resulting in the modern phosphatase technique, because in the former the pH
was sub-optimal and wandering of calcium phosphate and enzyme occurred in the unfixed

36 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

tissue. Although Martland and Robison (1929) had observed that bone phosphatase
survived treatment with alcohol, this fact was not applied to the histochemical study
of the enzyme for another ten years.

In 1939 Gomori and Takamatsu greatly improved the method by introducing
fixation, replacing freehand sections with microtome sections, and by using an optimal
pH and a greater substrate concentration. Gomori (1941) replaced the generally
unsatisfactory von Kossa method of demonstrating the calcium phosphate formed by
enzyme action with the current cobalt conversion method. Kabat and Furth (1941)
suggested the routine addition of magnesium to the incubating solution.

Despite the enormous body of literature which has accumulated on the distribution
of and physiological changes undergone by the enzyme in various tissues and cells,
there have been few attempts to examine the reaction critically. The most important
study of the validity of the reactions has been made by Danielli (1946), who concluded
that the reaction was capable of giving a valid intracellular localization of the enzyme.
Lison (1948) in a review of the reaction is of the opinion that diffusion and adsorption
of the enzyme may, when the tissue is poorly fixed, result in false intracellular
localizations. Various aspects of the method have been examined by Emmel (1946),
Stafford and Atkinson (1948), and Doyle (1948).

Histochemical results are usually expressed in subjectively determined grades of
colour. While the human eye is well able to compare the depths of two colours and
to arrange a series of colours in order of their depth, it cannot determine the
quantitative relationships in such series.

The first serious attempt to give a quantitative expression to cytochemical and
histochemical results appears to be that of Marza and Chiosa (1935). By means of a
comparison ocular the object in the microscope field was compared with a series of
colours produced by known concentrations of the substance being studied. The method
was applied by Marza et al. (1937) in a study of the histochemistry of the ovary of
Fundulus.

Apart from the methods of Caspersson (1936, 1941), which are specialized and
require apparatus beyond the reach of most laboratories, the first use of photoelectric
methods in histochemistry appears to have been made by Stowell (1942) in a study
of the variations in intensity of the Feulgen reaction during experimental carcino-
genesis. Stowell did not, however, demonstrate that the intensity of the Feulgen
reaction was a valid estimate of the desoxyribonucleic acid content of tissue sections.
This fact has been very elegantly established by Di Stefano (1948).

The difficulty of applying ordinary photoelectric methods to cytochemical problems
is the difficulty in getting enough output to work the usual type of measuring
instrument. This problem has been resolved in two ways. Pollister and Ris (1947) and
Pollister and Moses (1949) have made use of electron multiplier photocells which give
a large output and a low noise signal ratio, while Ely and Ross (1948) and Stowell
(1948) have used densitometry of photomicrographs.

"In the present investigation use has been made of an ineecumen a purchased locally
for use as a photomicrographic exposure meter. The instrument is rather crude but
is adequate tor the present study, since all the points at issue may be decided by
photometry of microscope fields rather than of cells and parts of cells.

The aim of this investigation has been to establish the optimal conditions for
demonstrating alkaline phosphatase in tissue sections and to decide whether it is
possible to obtain a valid estimate of the enzyme in tissues and cells. Such an
investigation forms a necessary preliminary to the projected study noted above.
Because it has a number of technical advantages over other tissues, renal cortex has
been the main tissue studied. The temperature of incubation has been 40°C. throughout.

Merrnops.
(a) Photometric.
The photometric unit consisted of a vacuum photocell with a caesium cathode, and
a two-stage D.C. amplifier, the output of which was read on a microampmeter.

lad

BY Kk. W. CLELAND. 3%

The photocell was mounted in a light-tight cylinder. Opposite the cathode, and of
the same diameter, was an aperture which could be closed by a sleeve inside the cell
mounting. From this aperture projected a cylinder one inch long.

Coupling with the microscope was effected by means of a Reichert demonstration
ocular. A fitting was made which was placed over the end of the horizontal arm of
the demonstration ocular, and into it the spout of the photocell mounting was
inserted. All junctions were light-tight. This method of coupling allows simultaneous
visualization of the field and measurement of its light absorption.

Illumination of the microscope field was by critical illumination of the Kohler
type, the light source being a low-voltage concentrated filament lamp, run on heavy-duty
accumulators. The lamp input was regulated by a variable resistance.

The response of the apparatus over the light intensities used was tested and
slight deviations from logarithmic response noted. These were shown to be due to
the amplifier, not the photocell.

The mean percentage transmission readings of the experimental sections were
expressed as a percentage of the transmission of the control sections incubated in the
absence of substrate. The resulting transmission was expressed in “density units’ by
reference to a standard semi-logarithmic graph, the ordinate being log.% transmission
and the abscissa being so divided that 100 density units occur between 100 and 30%
transmission, the curve being taken from the standard response calibration. One
density unit thus corresponds to an optical density (2-log.G) of 0:00523.

(b) Histological.

The organs, removed from male guinea-pigs, killed by a blow on the head, were
cut into fairly large pieces, about 7 mm. thick. The use of these large pieces of
tissue is justified later. After fixation at 2°C. for 18-24 hours, dehydration was
completed by cold absolute alcohol (8-24 hours). Clearing was in two changes of
benzol (6-24 hours) and embedding in paraffin was accomplished in the shortest
feasible time (2-5 hours). The acetone, alcohol and benzol volumes were at least
50 times the volume of the tissue. Sections were cut at 8 microns, the microtome
being adjusted to minimize paraffin compression (Dempster, 1943).

The sections were flattened in a beaker of distilled water immersed in a thermo-
statically controlled water bath at 39-40°C. The time needed for complete flattening
was determined and a time about half as much again chosen (usually 20-30 seconds).
All sections in the series were flattened for exactly this time and were mounted
serially in groups of two on numbered slides.

Aiter drying in the incubator for minimal times, the slides were stored in specimen
jars with ground-glass greased lids. in tie bottom of the jar was a layer of silica
gel (with indicator), and the jar was stored in a refrigerator.

Under these conditions of storage no detectable change in the phosphatase content
of sections could be demonstrated photometrically over periods of up to four months
after cutting of the sections. Sections which had remained in sealed jars over spent
silica gel at room temperature for a period of six months still gave a typical reaction
with little or no diminution in intensity.

All authors who have studied the stability of the enzyme in sections—e.g., Danielli
(1946), Lison (1948), Doyle (1948)—seem agreed that much of the enzyme is lost in
a period of about two weeks after cutting. The discrepancy between these reports and
the present experience is very difficult to understand.

Tissues embedded in paraffin blocks were also found to be stable for periods of at
least nine months. This is again at variance with the reports of Lison and Doyle.

Surface areas were determined by planimetry of microprojection drawings; in
kidney sections only the cortical area was measured.

(c) Chemical.

All inorganic chemicals used in this study were of analytical grade. The glycero-
phosphate and phenylphosphate were B.D.H. laboratory reagents, the former being a
mixture of a and B@ ester.

D

38 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

Calcium phosphate was extracted from tissue sections by means of N/10 HCl and
the inorganic phosphorus determined by the molybdenum blue method, using stannous
chloride as the reducing agent. The absorption was measured with the same photo-
metric unit as was used on the microscope, the range being 1-l0ug. P. The precision
(2 x C.V.) was 3% at the 4yg. level and wherever possible aliquots containing
approximately 4ug. were taken.

All pH measurements were made with a Jones pH electrometer and measured to
the nearest 0-1 unit.

The phosphate solubilities were determined by addition of an excess of inorganic
phosphate to the solution under test, centrifuging the precipitate and analysis of the
supernatant fluid.

ACCURACY OF THE PHOTOMETRIC ESTIMATE.

The following are the main sources of variability:

(a) Errors in setting zero and full-scale deflection. These seldom reach half a
unit and can be ignored, since they are frequently checked.

(bv) Histological variability in different fields. The coefficient of variation of fields
of renal cortex in any one section was 5%. Since 10 readings are made on each section
the coefficient of variation of the section mean due to this source is 1:5%.

(c) Variation in section thickness. This source of error is difficult to assess
accurately. By using sections as thick as possible without obscuring the cytological
picture it can be minimized, and the use of section pairs eliminates the effects of any
tendency of the microtome, due to mechanical instability, to cut alternate thick and
thin sections. Richards (1944), using an interference method of estimating section
thickness, quotes a standard deviation of 0-3u for sections cut with a microtome setting
of 10u. This gives a coefficient of variation at the 5% level of significance of 6%. The
method of flattening (if any) is not stated. Linderstrom-Lang, Holter and Ohlsen
(1934) have reported an error of 3% (in weight) in paraffin sections, due mainly to
variations in the microtome.

(d) Variation due to section flattening. This appears to be the largest potential
source of variability, and for this reason stringently controlled flattening technique was
used. In four groups of ten serial sections, flattened in the way described, the coefficient
of variation of the surface area was found to be 1:5-2%.

(e) Variability due to the histochemical procedures was presumably the largest
source in the present case; an idea of its magnitude can be obtained from a study of
the total variability.

The total variability of density was studied in four groups of ten serial sections
which had been subjected to the phosphatase reactions and the C.V. found to vary
between 3% and 6%. By the use of four sections in any density determination the
standard error is reduced to about 24%, and the 5% probability limits are thus about
5% of the mean. In all the experiments of the present paper four sections were used
in the computation of the density units quoted.

Tue INorRGANIC BASIS OF THE REACTION AND HISTOCHEMICAL TECHNIQUE.

Since in the present case it was intended to use the histochemical method in a
quantitative manner, some knowledge of the inorganic basis of the technique seemed
desirable, both as a general background and as a means of devising suitable methods.

(a) The Demonstration of Calcium Phosphate.

(1) Washing atter incubation should remove any particles of calcium phosphate
adhering’ to the section and neutralize the buffer used for incubation, since alkaline
solutions can cause an increased blank reaction by interaction with cobalt. These
aims were met in the present work by using M/20 CaCl. dissolved in M/50 veronal
buffer of pH 7:5. The solution is saturated with inorganic phosphate before use.
Addition of phenyl mercuric nitrate prevents bacterial growth. Four dips in the
solution is sufficient.

BY K. W. CLELAND. 39

(ii) Conversion to cobalt phosphate. Some conditions affecting the solubility of
cobalt phosphate are shown in Table 1.

TABLE 1.
Solubility of Cobalt Phosphate at Room Temperature.
Precipitant
(at M/20). | Vehicle. pH. | wg. P/10 ml.
|
Co(NO,), .. .. | Distilled water. | 6-2 154
y> +CaCle 7 eal F . a 138
CaCl, os Kits ae Pe Fh 6:7 5
Co(NOs;)2 o8 .. | Veronal (M/50). 7-2 8
oe O° 06 op aa 7-4 4
55 CAO a, 5 ss D 9 8-5
CaCl, ES na oe 55 aa 7-4 115
i

It will be seen that cobalt phosphate has a considerable solubility when precipitated
in distilled water, and that a great reduction in solubility results when veronal buffer
is used as a vehicle for the cobalt. When the cobalt phosphates prepared from distilled
water and veronal buffer were washed twice with distilled water and re-suspended in
distilled water considerable differences in solubility were noted. After leaving overnight
the former was soluble to an extent of 5mg. P/10 ml., while the latter showed a solubility
of 140ug. P/10 ml.

Large quantities of cobalt phosphate may be dissolved in glycine buffers.

For these reasons the conversion of calcium to cobalt phosphate was made by the
use of cobalt nitrate (M/20) dissolved in veronal buffer of pH 7:3; in tissue sections
the reaction was complete in 10 minutes. The solution is saturated with inorganic
phosphate before use.

(iii) Washing. Danielli (1946) has indicated that this step is a critical one. By
the use of the veronal-cobalt solution its latitude is increased, as indicated above. In
this investigation, washing after the cobalt solution was performed in three jars of
distilled water, the slide being dipped four times in each jar.

(iv) Conversion to cobalt sulphide. Because of the variability of yellow ammonium
sulphide both between batches and in the same batch with time from manufacture, it is
very difficult to standardize this step.

Since the importance of this factor was not appreciated until late in this investiga-
tion, the densities quoted in different experiments are not strictly comparable.

With the stock ammonium sulphide used in the present study no difference in
maximum depth of colour produced was detected in dilutions down to 1 in 200, but
below this the colour developed was reduced and the cytological picture was changed.

In the work here reported a dilution of 1 in 100 was used; the reaction was
complete in five minutes.

(v) Danielli has indicated that the dehydration in alcohol is a eritical step. In
order to standardize this step better the slides were dipped six times in two lots of
tap-water, blotted gently, dipped four times in two lots of absolute alcohol and then in
two lots of xylene. The mountant used was Gurr’s Euparal. The alcohols were
frequently changed.

(0) Validity of Demonstration Method.

As a test of the validity of the histochemical technique for visualization of the
inorganic phosphate, and to obtain some idea of the relation of the density to the
absolute amounts of inorganic phosphate, experiments with known amounts of phosphate
incorporated in cigarette papers were performed.

Pieces of rice cigarette paper were impregnated with known amounts of sodium
phosphate and dried. A control piece received only distilled water.

40 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

When these papers are placed in an M/50 CaCl, solution of pH 9-5 considerable
losses of phosphorus occur, presumably because of non-absorption of the resulting
calcium phosphate. When placed directly in buffered cobalt nitrate, put through
ammonium sulphide in the usual way, blotted, dried for minimal times in the incubator
and mounted, a linear relation between the density and the amount of phosphorus
present was found over a wide range, as shown in Text-fig. 1.

This linearity being established, it was then necessary to show that no loss of
phosphorus, like that found in cigarette papers, occurs in sections incubated in buffer
substrate. As indicated by Table 2, no significant loss of phosphate from tissue sections
occurs in the course of the histochemical incubation.

TABLE 2.
Loss of Phosphate from Kidney Sections During Incubation.

Time | 1 hr. 15 hr. | 21 hr. 40 hr.
Increase in phosphorus calcium in fluid, | |

Ug. Ne oA is ate ao 1 5 8 12
Phosphorus present in sections, wg. .. | 40 120 130 150

Gomori (1949) has suggested that one of the complicating factors of the histo-
chemical reaction is the balance between formation of calcium phosphate and its
solution or diffusion from the section. The present results do not favour such an
interpretation, provided the solution is saturated with calcium phosphate before
incubation.

It may then be concluded that the techniques used for the demonstration of the
calcium phosphate formed by enzyme action are suitable for quantitative use.

(c) The Formation of Calcium Phosphate.

(i) Choice of Buffer. Of the three common buffers available in the pH range of
alkaline phosphase borate has the greatest acid-buffering capacity.

In the histochemical reaction borate buffer was found to give higher final densities
than veronal. (Cf. Table 7.)

In fresh tissue homogenates or tissue sections studied non-histochemically (Ca
absent from incubating fluid) it was found that the enzyme activity in borate and
veronal buffers was the same in testis but less with borate in kidney.

Borate buffers, usually with glycine added (p. 43) were mainly used in this
study. Since buffer capacity is probably important (p. 44), equimolar mixtures of
borate and veronal, which give linear buffering over this range, were used in the study
of the pH optimum.

(ii) The Solubility of Calcium Phosphate. The solubility of calcium phosphate
was unaffected by calcium concentration, buffer composition or concentration, but was
very dependent on pH, as shown in Table 3. :

TABLE 3.
Effect of pH on Solubility of Calcium Phosphate.
ug. P/10 ml.
pH. | Solution.
10-0 1
9°5 2
9-0 4
8-5 10
8-0 30
5 105

M/50 borate buffer, M/100 calcium ciloride. Temperature 40° ©.

BY K. W. CLELAND. 41

That this increasing solubility is important in the histochemical method is shown
in Table 4, where the density produced in the presence and absence of excess inorganic
phosphate at several pH values is shown.

TABLE 4.

Effects of Excess Phosphate on Density Resulting after Incubation
at Various pH Vatues.

‘ |
Excess |
pH. Phosphorus. Density.
i
|
9-5 Present. 39
9-5 | Absent. 40
8-7 _Present. 35
8-7 Absent. 34
8-2 Present. 24
8-2 Absent. 15
7:7 Present. D
O°U Absent. 0
7-0 Present. 1
7:0 Absent. 0

Incubation times vary for the different pH values. M/100
glycerophosphate, M/100 mg., M/50 borate buffer pH 9-4.
Kidney sections.

At temperatures below 40°C. an increase in the solubility of calcium phosphate
was observed.

(iii) Choice of Calcium Compound and its Concentration. Calcium chloride and
nitrate were compared for their effect on enzyme activity at several concentrations
over the range M/1000 to M/10 and no differences found. Since the former was
available in A.R. grade it was preferred, the concentration used being M/100.

THE BEHAVIOUR OF THE HINZYME IN SECTIONS.
(a) Comparison of Fixatives.

The preservation of enzyme is difficult to compare in differently treated sections
because of a number of histological variables, namely:

(i) Shrinkage of material. A comparison of the shrinkage produced by different
fixatives may be based on the cube of the mean proximal tubule radius.

(ii) The degree of flattening. Flattening not only corrects the paraffin compression
caused by the microtome but may also cause an expansion of the section by increasing
the intertubular area. This latter factor will depend on the degree of hardening of the
intertubular connective tissue by the fixative. The time taken for flattening of the
sections forms a basis for comparing different fixatives.

TABLE 5.
Apparent Enzyme Activity after Various Fixatives.

Time for Shrinkage Enzyme Blank
Fixative. | Flattening. Factor. Density. Density.

Absolute acetone ae ee = 1 min. | 100 31 85
80% alcohol Ey ih a uae 2 min. 105 29 10
25% pyridine in 80% alcohol .. ae 5 min. 90 35 12
20% pyridine |
70% alcohol i : aie oy: More than 80 16 24
10% formalin ) 10 min.

M/50 borate buffer pH 9:4, M/100 Mg and substrate. Incubation 30 mins. Kidney sections. The values
quoted are the means of four separate experiments.

42 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

A further factor to be considered in the choice of fixative is the intensity of the
blank reaction, which was found to vary with different fixatives.

Table 5 shows the effect of different fixatives on kidney enzyme. It will be seen
that when the histological factors are considered there is little difference in enzyme
preservation between the first three fixatives; the fourth causes much enzyme destruction
and is associated with a high blank density. There was little difference in the quality
of fixation with the first three fixatives and acetone was preferred in the present study.

Slight apparent increases in the enzyme activity of blocks fixed at room temperature
or of blocks smaller than usual were shown to be due to differences in shrinkage.

In only two blocks of about 80 used in the present study was there any evidence
of a gradient of enzyme from surface to interior; these two cases were evidently due
to incomplete clearing in benzol.

ite)

90

x
ie}

DENSITY
uw
{eo}
DENSITY

30}

0.05 O10 Ols
\ ug P PER MM?

10 5
3 HOURS OF EXTRACTION

ae
pee
Le

am
oe

DENSITY
LOG DENSITY

5 I = 2k
2 HOURS OF EXTRACTION 4 LOG INCUBATION TIME (MINS)

,

Text-figures 1-4.

Figure 1.—The relation between known concentrations of inorganic phosphate incorporated
in cigarette papers and the density (in units defined on p. ue resulting from the histochemical
procedure adopted.

Figure 2.—Loss of enzyme in kidney sections incubated for varying times in buffer with
Ca and Mg ions (M/100) but without substrate. The density is that resulting after a subsequent
incubation with substrate. Curve 1 is typical of the majority of kidney sections tested.
Curves 2 and 8 are from sections showing much loss of activity, curve 2 demonstrating the
protective action of glycine (0:006M). ‘The stimulation of enzyme activity after short periods
of incubation is due to the presence of magnesium ions.

Figure 3.—The effect of the presence of calcium phosphate in the section on the loss of
enzyme activity. Curve 1 is from sections incubated with substrate, the calcium phosphate
extracted and the section reincubated with substrate. Curve 2 is from slides in which the
primary incubation was conducted in the absence of substrate, and curve 3 from slides where
Ca was omitted in the primary incubation. All sections are of kidney.

Figure 4.—The time course of the phosphatase reaction in kidney sections at various
substrate concentrations.

BY K. W. CLELAND. 43

(bo) Stability of the Enzyme in Sections.

In a study of the enzyme surviving after various times of incubation in a number
of different substrate free solutions the following points were noted:

(i) In the majority of kidney blocks studied the loss of activity after 24 hours’
incubation in borate buffer of pH 9-5 was less than 15%; some blocks, especially testis,
may show almost complete loss. This rapid inactivation could not be related to any
known factor in their past history.

(ii) The loss of activity was relatively uninfluenced by the presence of magnesium
in the incubating solution, suggesting that the loss is not due to loss of coenzyme.

(iii) Veronal, borate and glycine buffers (M/50) gave essentially the same inactiva-
tion rates when tested at pH 9-4.

(iv) The inactivation was not influenced by borate buffer concentration over the
range M/50-M/10. i

(v) The losses were greater the higher the pH, but the differences are not marked.

(vi) A decision between inactivation or solution of enzyme could not be made.

The stability of a considerable number of enzyme solutions is known to be increased
by the presence of amino-acids (cf. Weil-Malherbe, 1948). Alkaline phosphatase solu-
tions are stabilized (or activated) by the presence of glycine at 0:006M concentrations
(Bodansky, 1936).

Text-fig. 2 indicates that the addition of glycine stabilizes the enzyme in sections
also; the effect was maximal at 0:006M concentration in tissue sections.

The enzyme changes occurring under the conditions of the histochemical reaction
were investigated by removing with acetate buffer (M/10, pH 5) the calcium phosphate
in sections incubated for various periods, reincubating the sections with substrate for
30 minutes, and determining the resulting density. In one control series the first
incubation is performed without substrate and in another with substrate but without Ca.
The latter series permits a study of the effect of substrate per se.

The results, which are shown in Text-fig. 3, indicated that the enzyme was
considerably stabilized by the presence of calcium phosphate in the sections.

The experiments reported above indicate that there will be little loss of enzyme
during incubation in the histochemical reaction in sections with initially high enzyme
activity. In sections with low initial activity considerable losses of activity would be
possible.

CONDITIONS OF OPTIMAL ENZYME ACTIVITY.
(a) Effect of Substrate Concentration.
With glycerophosphate as substrate the section density increased with increasing
substrate concentration up to the highest concentration tested (M/10), the effect being
the same for both testis and kidney, as shown in Table 6.

TABLE 6.
Effect of Substrate Concentration: on Enzyme Activity.

Substrate
Concentration. M/10. | M/50. M/100. M/1000.
Kidney ab a3 70 | 51 32 20
Testis ve ae 81 63 46 21

M/50 borate glycine buffer pH 9-4, M/100 Mg and Ca. Incubation 30 mins. for
kidney and 60 mins. for testis. The figures quoted are density units.

Phenylphosphate and glycerophosphate at M/100 gave similar densitities, but little
change in enzyme activity occurred with the former over the range M/10-M/1000. The
same difference in substrate enzyme affinities has been noted in test-tube experiments
by Schmidt and Thannhauser (1943). The time course of the reaction with M/100

44 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

eglycero- and phenylphosphate are the same over the range of incubation times—fifteen
minutes to four hours. This indicated that diffusion of the substrate through the
calcium phosphate shell around the enzyme sites was unlikely to be a limiting factor
at this glycerophosphate concentration.

When the time course of the reaction at three glycerophosphate concentrations is
followed it is found that the slopes of the three lines are different, as shown in
Text-fig. 4. The causes are threefold: (a) the normal relation between enzyme activity
and resulting density, (b) the changing relation between calcium phosphate content of
the section and density, (c) movement of calcium phosphate in the section. These
three factors will be discussed later.

When sections are incubated to a given density at different substrate concentrations
by varying the incubation time, important changes in apparent cytological localization
result, a phenomenon which will be discussed in Part II of this paper.

(b) Effect of pH.

Text-fig. 6 indicates that the pH optimum of kidney and testis for short-term
experiments was 9:4, a value similar to that found for the test-tube enzyme.

An apparent change of pH optimum with time was noted with kidney sections, as
may be inferred from Text-fig. 6. When sections are incubated to a given density at
different pH values by varying the incubation time, differences in apparent cytological
localization occur. The main difference is a greater density in the nuclei at the lower
pH values due to factors which will be discussed in another paper. It is to these
factors that the apparent change in pH optimum with time is due. The mechanism is
outlined on page 46.

(c) Effect of Buffer Concentration.

An unexpected considerable increase in histochemical density was found with high
borate buffer concentrations, as shown by Table 7, veronal buffer showing little change
with different concentrations.

TABLE 7.
Effect of Buffer Concentration.
Concentration. | M/10. | M/20. M/50. M/200
| |
Kidney : i
Verona] .. 5.0] 62 61 538 =
Borate es 89 72 64 62
Testis : |
Veronal anal 34 — 32 =
Borate Pe | 52 = | 38 —
|

pH 9-4. M/40 glycerophosphate, M/100 Mg and Ca. Incubated 45 mins. for kidney
and 60 mins. for testis. The figures quoted are density units. ,

The activity at the high borate concentrations appears to be increased at all
intracellular sites. The interpretation of the phenomenon is difficult, the following
seeming the most likely.

At the sites of enzyme activity localized shifts to a more acid pH occur, due to
phosphate both free and in combination with calcium, and to the local excess cation
caused by removal of calcium. High buffer concentration is more able to neutralize
this acid shift.

The apparent stimulating effect of high borate concentration on enzyme activity is
not demonstrable in homogenates or in tissue sections incubated in the absence of
calcium.

(d) Effect of Activators.
Test-tube experiments have implicated the following metal ions as possible coenzymes
for phosphatases: Mg, Mn, Co, Zn.

BY K. W. CLELAND. 45

When tested at pH 8-2, in the presence of an excess of inorganic phosphate, and
an ion concentration of M/100, Mg gave a greater stimulation than Mn, while Co gave
about twice the density of Mg. By placing the section incubated in the cobalt solution
directly into ammonium sulphide it could be demonstrated that the primary precipitate
was all cobalt phosphate. The main cytological difference in the Co incubated sections
was that the brush border reaction was very wide.

At pH 9-4, at which pH calcium phosphate was the least soluble phosphate, Mg was
found to be the best activator, as indicated by Table 8.

TABLE 8.
Effect of Ions at pH 9-4.

|
| Concentration Section
Buffer. Ton. (M). Density.

Borate 4: Bee Ses 2, Co 1074 23
Borate glycine aa a, AS Co I@=8 24
Borate Ege ae Se Beco Mn 10-4 22,
Borate & 5s ti apt) Mg 1073 25
Borate glycine is Pr: eee || Mg 10-3 25
Borate NG ie 26 | Mg Ope 29
Borate glycine | — — | 21
Borate | — = | 21-5

M/100 glycerophosphate and Ca. Incubation 30 mins. Kidney sections.

By the following experiment it was possible to infer that the apparent activation by
Co at the lower pH was due to differences in the properties of calcium and cobalt
phosphates. Cobalt can be dissolved in M/50 glycine buffer to M/100 concentration
at pH 9:0, at which pH cobalt phosphate is about five times more soluble than calcium
phosphate. Three series of slides were incubated for the same time in the following
solutions: (a@) M/100 Co in glycine buffer with added substrate; (0) solution (a@) with
M/100 Ca added; (c) glycine buffer of the same pH as the previous with substrate and
Ca added. Control sections were incubated in these three solutions in the absence of
substrate.

The resulting densities of the sections were 65, 34 and 32 units respectively.

It is demonstrated in the next section that the presence of calcium phosphate at
the enzyme site causes an inhibition of the enzyme. The greater width of the brush
border reaction in sections incubated with cobalt in the absence of calcium suggests
that cobalt phosphate causes less enzyme inhibition than calcium phosphate.

It is unfortunate that these cobalt solutions cause such a high blank impregnation
of the tissue, a factor which makes visual interpretation difficult, for the relations in
enzyme content at the various cellular sites are more truly represented than by the
classical technique. :

The enzyme activation by Mg was found to be maximal at a Mg concentration
of M/100.

Zine ion was found to have an apparent inhibitory action on the section density
produced, varying from 70% to 5% at M/600 and M/60,000 Zn concentration respectively.
This was apparently due to the great insolubility of Zn phosphate; some of the
primary precipitate is in this form and is not converted to cobalt phosphate, thus
appearing in the section as colourless zinc sulphide. Moog (1943) has reported an
inhibition of the histochemical reaction in the chick embryo by zine ions.

(e) Effect of Inhibitors.

The effect on the histochemical density of a number of substances known to inhibit
the enzyme in solution is shown in Table 9.

46 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

TABLE 9.
Effect of Inhibitors on the Activity of Phosphatase in Sections.

Inhibitor. KCN. Taurocholate. Hydrosulphite. | Na.s. | Nil.

|
|
Concentration (M.) scl}; dle Om Ome Lome | 107° 10-5 |) 102 1073 —_
Density units ae ae 6 14 |} -ilfe}ob) Wer | 22 21-2 0 17/75) 20

No Mg. M/100 glycerophosphate M/50 borate buffer. pH of solutions adjusted to 9-4 after addition of inhibitor.
Incubation 30 mins. Kidney sections.

Bodansky (1936) has shown that kidney phosphatase is inhibited about 50% by
taurocholate. There is little or no effect on the enzyme in sections. Moog (1943) has
found an inhibition of the enzyme in chick embryo sections by taurocholate.

Sizer (1941) demonstrated that ox kidney phosphatase is inhibited strongly by
hydrosulphite, but in guinea-pig kidney sections a slight activating effect is seen.
Sizer was unable to demonstrate much inhibition by cyanide or sulphide, but Schmidt
and Thannhauser (1943) found a 90% inhibition of purified intestinal phosphatase by
M/60 cyanide. Fell and Danielli (1943) found ammonium sulphide a powerful inhibitor
ot skin phosphatase in both test tube and sections.

Emmel (1946) has described a reversible inhibition of rat kidney phosphatase in
sections and noted an apparent activation of the enzyme after thorough removal of the
cyanide. This could not be demonstrated in guinea-pig kidney, only about 75% of the
original enzyme density being recovered after cyanide treatment.

(f) Effect of Calcium Concentration.

The effect of calcium concentration on the density resulting after the histo-
chemical reaction is shown in Table 10; in both organs the density is less at M/10
than at M/100, and at concentrations less than M/100 there is an increased density
with kidney sections but a decreased density with testis sections.

TABLE 10.
Effect of Ca Concentration on Apparent Enzyme Activity in Sections.

| |
Ca Concentration. | M/10. | —-M/100. M/1000. M/10,000.
Kidney Radice 12 34 42 36
Testis = RUE 32 55 | 35 1

Values quoted are density units. M/50 borate glycine buffer pH 9-4. M/100 Mg.
M/40 substrate. Incubation 30 mins. kidney and 45 mins. testis. Testis sections 10.

The interpretation of these phenomena is as follows. At calcium concentrations
less than M/100 some phosphate diffuses from the enzyme site before it is trapped as
calcium phosphate. Some of this calcium phosphate is secondarily adsorbed by the
tissue section, and some escapes to the incubating solution (shown experimentally).
In kidney sections the main site of enzyme activity is at the brush borders and the
enzyme at this site is normally rapidly inhibited by the accumulated calcium phosphate
(cf. Table 11). Owing to this fact loss of phosphate has little effect on the final density
at this site and the slight loss of density is more than counterbalanced by the phosphate
absorbed by other parts of the section. The overall density thus shows an increase
at low calcium concentrations. In testis incubated for short periods the enzyme is very
little inhibited by calcium phosphate at the enzyme sites, which are fairly evenly
distributed in the sections (cf. Table 11). Practically all the phosphorus that the
enzyme is potentially capable of splitting thus appears in the section: when any is lost

BY K. W. CLELAND. 47

to the incubating solution, as happens with low calcium concentrations, there is an
overall loss of density.

The lower density at Ca concentrations greater than M/100 is due to depression
of enzyme activity; the cytological picture is identical with that found when the
enzyme activity is lowered by other means (cf. Part II of this paper).

SIGNIFICANCE OF THE PHOTOMETRIC HSTIMATE.

(a) Time Density Relation.

It is evident from previous figures that when the logarithm of density is plotted
against the logarithm of incubation time a rectilinear relation holds for kidney.

That the same relation holds in tissues with a different intracellular partition of the
enzyme is shown in Text-fig. 7. The log./log. relation is evidently characteristic of the
kinetics of the histochemical reaction and almost certainly describes the reaction rate
in the various cell components which contribute to the overall density of the micro-
scope field. Text-fig. 8 indicates that the relation is not due to any peculiarity of the
histochemical visualization procedure or the photometric method.

A strong inhibition of the enzyme activity, increasing with incubation time, is
implied by these figures. An indication of the degree and time course of the inhibition
in testis and kidney sections may be obtained from Table 11.

TABLE 11.
Comparing the Enzyme Activity of Tissue Sections when the Phosphate is Trapped as Calcium
Phosphate as in the Histochemical Reaction (+Ca) and when it is Free to Diffuse into the
Incubating Fluid (—Ca).

Ca Increase of Phosphorus (uwg.) in Sections (+Ca ) and
Fluid (—Ca).

Organ. " ms at
|
1 Hour. | 2 Hours. | 4 Hours.
| |
Testis oo 75 | 117 | 185
— 88 191 | 364
Kidney + 111 | 145 178
_ 485 | 950 | 1780

M/50 borate buffer pH 9-4. M/100 Mg. Ca, when present, M/100. Substrate
M/20. Approximate total section area, 11 cm.? for each organ.

There are two possible explanations of this inhibition: lowering of substrate
concentration at the enzyme surface by a diffusion barrier due to the calcium phosphate
or actual inhibition of the enzyme by the high concentrations of inorganic phosphate
which is present at the enzyme site as calcium phosphate (cf. Schmidt and Thannhauser,
1943).

The only satisfactory way of deciding between these two alternatives has been by
the use of substrates showing different affinities for the enzyme; this type of evidence
favours the latter alternative (p. 43).

In Text-fig. 7 it will be noted that the kidney line has a lower slope than the
adrenal or testis lines. This is due to the fact that the brush borders are very active,
giving a high density at low incubation times, but, because of the inhibition by split
products, the density contribution by this source increases only slowly with time of
incubation. The slope of the kidney line is thus mainly determined by the reaction in
the other parts of the cells of the proximal tubules, which occupy a relatively small
area in the section. In the adrenal cortex high densities are reached at an early
stage of incubation because of the high enzyme content of the cortical cytoplasm of
the cells, which occupies a larger relative area than do the brush borders in the case
of kidney. The slope of the adrenal line is higher than that of kidney, because the
chief factor determining it, the reaction in the rest of the adrenal cortical cell. is

LOG DENSITY

48 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. TI,

spread over a much larger relative area than was the case in kidney. In testis the
density is low at small incubation times because the enzyme is not present at any
site at concentrations comparable with that in kidney brush border or the cortical
cytoplasm of the adrenal cortex cells, and the slope of the line is high because the
enzyme is distributed over a wide area in the microscope field.

(b) Relation between Photometric Density and Inorganic Phosphate Content of Sections.

In a study of the time course of the histochemical reaction in sections of different
thickness it was found that the slope of the time density plots varied with section
thickness. The causes to be considered were variation of the adequacy of diffusion

TESTIS 2

DENSITY
at:
LOG DENSITY

10.5 10 95 9 8 75 - jie g ate 3 3
5 PH 7 LOG INCUBATION TIME (MINS)
: aN
= ;
“ x A
a We 3
J
. =
nw WW .
a 4
waa Z
i =)
oa g
5 = a,
=
wn 3 VA
>
a .
fo)
ac
a
rs d
Tr: erage oe
* A
2 g = 2% (0) 20 40 6 80
LOG INCUBATION TIME (MINS) 8 LOG INCUBATION TIME (MINS) 9 DENSITY

Text-figures 5-9.

Figure 5.—The effect of hydrogen ion concentration on the phosphatase activity of testis
and kidney sections, as measured by the histochemical technique. Incubation conditions: M/50
borate veronal buffer. M/100 Ca and Mg. M/100 substrate for kidney, M/40 for testis;
incubation time 30 mins. kidney and 120 mins. testis. An excess of inorganic phosphate was
present at all pH values.

Figure 6.—The time course of the histochemical reaction at different hydrogen ion
concentrations. Note the apparent change in pH optimum between pH 8:7 and 9-2, due mainly
to wandering of calcium phosphate from the enzyme site, as will be demonstrated in
another paper.

Figure 7.—The time course of the histochemical reaction in sections of three different
organs. Note the difference in slope, due mainly to differences in the cytological distribution
of the enzyme, as explained in the text. The conditions of incubation are the same for the
three organs.

Figure 8.—The time course of the phosphatase reaction in kidney and testis sections as
measured by the amount of phosphorus extractable from the section after varying incubation
times.

Figure 9.—The relation between phosphorus extractable from tissue sections and the
photometric density of the sections. While the density is a valid estimate of phosphate
content of model systems (fig. 1), this figure indicates considerable deviation from Beer’s

law in tissue sections, owing to the uneven distribution of the very intensely light-absorbing
cobalt sulphide.

100

BY K. W. CLELAND. 49

of substrate or visualizing reagents with section thickness or inadequacy of the density
as a measurement of inorganic phosphate in sections. The latter was the simplest to
test; in the experiment shown in Table 12 it will be seen that the amount of inorganic
phosphate extractable from the section is proportional to the section thickness but the
inorganic phosphate/density ratio is different in sections of different thickness.

TABLE 12.

Relation between Section Thickness, Inorganic Phosphate Content and Density in Kidney
Sections of Different Thickness.

} Phosphorus. Cortex Area. g. P/20
Thickness. Density. | (ug.) (Mim.) units/mm.?
|
5 63°5 9-3 91 0-082
8u 78 14 95-5 0-0379
lw 91 19-2 97 0-0435

M/50 borate glycine buffer pH 9-4. M/100 Mg, Ca. Substrate M/20. Incubation
time, 2 hours.

In order to systematize this finding, experiments were conducted in which the
density and inorganic phosphate content of sections incubated for various times was
determined.

When the phosphorus content, per unit density and area, was plotted against
density the curves shown in Text-fig. 9 resulted. It will be seen that in kidney
sections a change of relation occurs even at low densities and is fairly constant in rate.
Testis sections show a constant relation up to medium densities and then change
rather rapidly. It is this change of relation which causes the deviations in testis
sections, of the plot of incubation time/density in Text-fig. 7. A similar effect is
probably responsible for the deviation in adrenal sections at the longer incubation times.

When the figures derived from this experiment are used to calculate the amounts
of inorganic phosphate present in the sections of different thickness mentioned above,
it was found that the calculated phosphate content was proportional to section thickness
at all incubation times.

The most likely interpretation of these findings is as follows. In kidney the brush
borders react very strongly at small incubation times. Very active parts of the brush
border reach at an early stage a density such that all of the incident light is absorbed.
As the incubation time is increased, more brush border areas become completely
absorbing. Although completely absorbing the light in the finished preparation, these
areas still produce phosphorus by substrate splitting, this extra phosphate not con-
tributing to the total photometric density. The observed density/phosphorus ration
implies that the rate of reaching this 100% light-absorbing state is fairly constant
for kidney. In testis the situation is different. The apical ends of the Sertoli cells
are the most active constituent but react at a much slower rate than the brush
border of the kidney. Furthermore, they cover a larger relative area than the brush
borders. For this reason the density-phosphorus relation is constant up to medium
densities. After this, Sertoli cell areas become 100% absorbing and reach this stage
much more synchronously than the brush borders, resulting in a rapid change in the
above relation.

DISCUSSION.

To establish the possibility of a histochemical estimation of the enzyme content,
either relative or absolute, of tissue sections is a difficult task requiring a thorough
investigation of all the points involved.

The first requirement is a method of estimating the reaction products of the
enzyme as they occur at the enzyme site. It has been shown in this paper that the
cobalt conversion method of Gomori (1941), somewhat modified, is capable of giving

50 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

a reasonably accurate estimate of inorganic phosphate incorporated in cigarette papers.
It is desirable to check conclusions derived from such model experiments in two
respects—to show that Beer’s law is obeyed not only in model systems where the
substance is homogeneously distributed, but also, if possible, in tissue sections where
the distribution is rarely homogeneous, and to show that any losses of the substance
which occur during the colorimetric procedures are similar in both model system and
tissue section. Both these points were shown to be important in the present study:
Beer’s law was not obeyed in tissue sections and losses of phosphate occurred in
cigarette papers when they were subjected to exactly the same treatment as tissue
sections. The fact that no significant loss of phosphate occurs in tissue sections during
incubation implies that the calcium phosphate arising by enzyme action is adsorbed by
the section. This adsorption, while it makes possible a clear demonstration of the
enzyme in the section, suggests caution in interpreting the picture so obtained as a
valid intracellular localization of the enzyme: it is quite possible that different cell
components have different powers of adsorbing calcium phosphate.

The second requirement is that no loss of enzyme should occur during the
incubation, or if it does, that no difference in the percentage lost occurs in different
histological or cytological elements of the section. It is evident from the experiments
detailed in this paper that little loss of enzyme need be expected at least over relatively
short incubation times, and especially where, owing to high enzyme content, the enzyme
is rapidly protected by a calcium phosphate shell. The enzyme at the brush borders,
at first sight, appears to be more resistant to inactivation and less inhibited by the
inhibitors used than the enzyme in other cell sites. This, however, is no evidence of
differential loss or inhibition of the enzyme: because of the autoinhibitory nature of
the phosophatase reaction, the enzyme at the brush borders may be strongly reduced
in amount or activity without showing a comparable change in the density visible at
this site in histochemical preparations. This same factor complicates the interpretation
of Emmel’s (19460) results with cyanide inhibition.

In estimations of enzymes it is desirable, although not absolutely necessary, to
determine the conditions of optimal activity of the enzyme. The conditions prescribed
by Gomori (1941) call for a veronal buffer of approximately M/50 concentration and a
substrate concentration have been found to shew considerable decreases in pH over a
period of days when in constant use, and the present study has shown that borate
buffers of higher molarity are preferable. When the pH falls below the optimum, as
can happen quite readily with veronal buffers, considerable changes in the apparent
cytological partition of the enzyme occur. An inspection of published figures for
various organs suggests that this factor has been operating. It has also been shown
in this study that the substrate concentration recommended by Gomori is insufficient
to saturate the enzyme. The saturating concentration of glycerophosphate appears to
be in excess of M/10, a concentration considerably higher than that found necessary
to saturate the fresh enzyme in test-tube experiments. It seems possible that, in the
process of fixation, some barrier is formed around the enzyme site which obstructs the
free diffusion of substrate molecules to the active groups of the enzyme. A difference
in this barrier might explain the differences recently noted by Wang and Grossman
(1949) between the enzyme activity of alcohol fixed and frozen dried sections of
various organs.

A linear relation between incubation time and amount of substrate split makes for
a convenient and accurate estimate of an enzyme. Non-linearity may be caused by
enzyme inactivation, a fall in substrate concentration, or an inhibition of the enzyme
by substrate split products. Schmidt and Thannhauser (1943) have found that the
organic part of the phosphate ester causes little inhibition of alkaline phosphatase
while inorganic phosphate has a considerable inhibiting effect. They have interpreted
this to mean that it is the phosphate moiety of the substrate which combines with
the enzyme, and have suggested that this is the reason for the relative non-specificity
ot the enzyme. In the alkaline phosphatase reaction in tissue sections the enzyme is
strongly inhibited by the inorganic phosphate liberated from the substrate, which,

BY K. W. CLELAND. 51

instead of diffusing away and thus becoming diluted, is retained at the enzyme site as
calcium phosphate. This fact greatly complicates attempts to estimate the enzyme
content of tissues or cells by the histochemical method, especially since the study of
the time/density relation has indicated that the inhibition is somewhat anomalous.
This inhibition can give rise to quite false visual estmates of the relative activity of
different cell components: for example, it would not be suspected from inspection of a
routine histochemical preparation that the brush borders of kidney tubules have an
activity which must be some hundredfold that of any other component of the tubule
cell. Estimates of the enzyme activity based on the alcohol moiety (Menten et al., 1944;
Manheimer and Seligman, 1948; Loveless and Danielli, 1949) would theoretically be
preferable to estimates based on the phosphate, since the autoinhibition would be
less. These methods would be difficult to standardize, however, and would need much
more critical examination than they have yet received.

The section on the time/density relation seems to indicate that the most satisfactory
method of comparing the enzyme activity of two organs by histochemical means would
be based on the density produced in incubation times sufficiently short to obviate the
split product inhibition. Thus the density found in testis and -kidney sections in
Text-fig. 7 is, by extrapolation to 1 minute incubation, 2-5 and 10 units respectively,
suggesting that testis has a relative activity about 23% that of kidney. It will be
shown in another paper that this estimate is in accord with test-tube assays of the
enzyme in fresh tissue homogenates.

An approximate calculation of the absolute enzyme activity of a fresh tissue
equivalent of the kidney sections in Text-fig. 7 indicates that the calculated activity is
of the same order as found in fresh tissue homogenates, and fails to indicate that much
destruction of the enzyme can be ascribed to the preparation of the histological sections.

Reasons have already been given for thinking that the methods for estimating the
enzyme content of tissue sections are also applicable to estimates of the intracellular
partition of the enzyme.

Thus reasonably accurate estimates of the cytological distribution of alkaline
phosphatase seem technically feasible. Whether such estimates would indicate a
physiological reality depends on two points which have not been investigated in the
present paper. These are that no movement of the substances used in localization of
the intracellular enzyme occurs, and that there is no loss or no differential loss of
enzyme between the time the cell is removed from the living animal and the time
when the histochemical incubation begins. These two factors will be considered in
Part II of this paper.

SUMMARY.

1. A photometric apparatus suitable for the determination of the light absorption
of microscope fields is described and its method of use indicated.

2. Loss of enzyme with time in paraffin blocks or in tissue sections on slides is
very small.

3. The sources of variability in the photometric estimate are considered and the
procedures adopted shown to give precise results.

4. In order to standardize the histochemical demonstration of calcium phosphate
a number of modifications have been made to the technique, and the technique adopted
is shown to give a valid estimate of the phosphate content of model systems.

5. The effect of pH on the solubility of calcium phosphate is stressed and the
importance of this factor in the histochemical technique shown.

6. A number of: fixatives have been compared for their ability to preserve enzyme,
and acetone or 80% alcohol found to be the most suitable.

7. Factors influencing the stability of the enzyme during incubation of tissue
sections are studied and the addition of 0:006M glycine found to be desirable. The
presence of calcium phosphate in sections is found to have a stabilizing effect.

8. The enzyme activity increases with glycerophosphate concentration, the enzyme
still being unsaturated at M/10 substrate.

ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. I,

vl
bo

9. The pH optimum of the enzyme in sections is the same as of the enzyme in
solution, but owing to peculiarities of the histochemical reaction an apparent change
of pH optimum occurs in kidney sections.

10. Borate buffers of high concentration are in general shown to be preferable to
the classical veronal buffer.

11. Magnesium ion at a concentration of M/100 is the most effective enzyme
activator.

12. The time/density relation in tissue sections is best expressed by a double
logarithmic plot, different organs showing different slopes in such plots. This is shown
to be due to differences in the cytological distribution of the enzyme.

13. There is a strong inhibition of the enzyme by split products retained at the
enzyme site. The degree of inhibition is different in different organs, because of
differences of cytological distribution of the enzyme.

14. The light absorption of tissue sections is often not a valid estimate of the
phosphate extractable from the sections. The invalidity is different for different organs.

15. Difficulties in comparison of the enzyme activity of organs differing much in
the cytological distribution of the enzyme are indicated. “A valid comparison of the
enzyme activity of different cell sites would be possible under certain conditions.

ACKNOWLEDGEMENT.

The author is indebted to Professor C. W. Stump and Mr. D. J. Lee for criticism of the
manuscript.

References.

BopDANSKyY, O., 1936.—The accelerant effect of amino acids on the activity of bone phosphatase.

J. Biol. Chem. 114: 273.
, 1936.—Are the phosphatases of bone, kidney, intestine and serum identical? J. Biol.

Chem., 118: 341.

CASPERSSON, T., 1936.—Uber den chemischen Aufbau der Strukturen des Zellkernes. Skand.
Arch. Physiol., 73 (supp.).

—, 1940.—Methods for the determination of absorption spectra of cell structures. J. Roy.
Micros. Soc., 60:8.

DANTIELLI, J. F., 1946.—A critical study of techniques for determining the’ cytological position
of alkaline phosphatase. J. Hap. Biol., 22:110.

DeMpstTER, W. T., 1943.—Paraffin compression due to the rotary microtome. Stain Tech., 18:13.

Di STerano, H. S., 1948.—A cytochemical study of the Feulgen nucleal reaction. Proc. Nat.
Acad. Sci., 34:75.

DoyLe, W. L., 1948.—Effect of dehydrating agents on phosphatases of mouse appendix. Proc.
Soc. Exp. Biol. Med., 69: 43.

Evy, J. O., and Ross, M. H., 1948.—Nucleic acid content in intestines of rats after X radiation.
Cancer Research, 8: 285.

EMMEL, V. M., 1946a.—The intracellular distribution of alkaline phosphatase after various
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Gomori, G., 1939.—Microtechnical demonstration of phosphatase in tissue sections. Proc. Soc.
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ORM

KapatT, E. A., and FurtH, J., 1941.—A histochemical study of the distribution of alkaline
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BY K. W. CLELAND. 53

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A STUDY OF THE ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS.
PART Il. THE HISTOLOGICAL AND CYTOLOGICAL VALIDITY.

By K. W. CLELAND,
Department of Histology and Embryology, University of Sydney.

(Plates I and II.)

{Read 26th April, 1950.]

Table of Contents. Page.

Introduction o4
Methods Be tet nie Re cel ie axe 54
Validity of the cytological localizaticns 55
(a) Visualization of cobalt phosphate .. 5d

(b3 Movement of cobalt phosphate 55

(c) Movement of calcium phosphate 55

(i) Effect of calcium concentration 56

(ii) Effect of reducing enzyme activity 56

Gii) Effect of pH 57

(iv) Discussion 5 57

(d@) Movement of enzyme 58

(i) General considerations 58

(ii) Positive evidence ; : 59

(iii) Negative evidence 3 Baer: trey Re Cans tee : i PEE e : Sy KEY

(iv) Discussion .. Lay age eae ie tie Be A Si a 5 ee PEGI ee tie 78 « So KN)

The histochemical validity of the reaction .. oe Me Mn Dame eK Me Fite ee a5 Pld teed PO!
(i) Phosphatase-activity in different organs .. ate aoe Are, ar ae eS ae a Go, (pil

(GO)) SONNE Aine! Thosolleloy~e CrowAycaKsy whol GChhaxsresme Oesens 5.5 so oc on ca oc a0 os WP

(iii) Iiffect of fixation on amcunt and distribution of enzyme RN ee fh oy | OE
DISCUSSION Me ik hes. 2 CR ee RE Re ea en se ee a
Summary Sit, yee) 1 bom) ee hee CURE EIS sie LOA et Re IS 2S AEE Pe ee GC
Acknowledgment ae a ioe Bei ae abe fe Shs bh Seo ne Lee aes Lite ete oie se 67
References .. Bas Phe an AS) oH ska ae a SEA cist? SANS RING Rie ee Ra) ba Se eS Ot
iwddendum™® i.) Ses. a Se BS er Be ED ee 2
Explanation of plates nl ae aie ie Tene a OE RE a ies enor 2 SEE Sy eee mele eR os 2 SAN TRE MRG'S

INTRODUCTION.

In a previous paper (Cleland, 1950) an investigation of the possibility of quanti-
tative use of the histochemical reaction in tissue sections was made. ;

_ It was shown that a reasonable estimate of the enzyme content of tissues and cells
was possible, provided that: (a) no movement of the localizing substances or enzyme
occurred, and (vb) no differential loss of enzyme occurred in different cell components or
organs.

The present communication records a study of these two points.

Mernops.

Unless otherwise stated materials and methods are the same as described in the
previous paper.

Phosphatase activity was determined in distilled water homogenates prepared by
means of a Waring blendor fitted with a microcup. With the following conditions:
veronal buffer, M/20, pH 9-4; M/20 glycerophosphate; M/100 Mg.; temperature 40°C.;
the activity was expressed as the amount of phosphorus split from the substrate (in ug.)
by 100 mg. fresh weight in 30 minutes.

Homogenates for the centrifugal studies were prepared in normal saline. The
activity remaining in the supernatant after the following centrifugal treatment: 150 x g.

BY K. W. CLELAND. 55

for 3 minutes in an International SBI centrifuge; 10,000 x g. for 10 minutes in a Servall
SSIa centrifuge and 20,000 x g. for 30 minutes in the same instrument: was expressed as
a percentage of the activity in the unfractionated homogenate.

The absolute activity of the enzyme in tissue sections was determined with the same
conditions used for determination of the homogenate activity except that Ca was
rigorously excluded from the solution.

In the enzyme determinations phosphorus analyses were made by the method of
Fiske and Subbarow.

VALIDITY OF THE CYTOLOGICAL LOCALIZATIONS.

In a multistep technique such as this, false localizations can arise at any step and,
to prove its validity, it is necessary to investigate each step individually, starting from
the last and progressing to the first.

(a) Visualization of Cobalt Phosphate.

As the dilution of ammonium sulphide was decreased below 1/260 or the pH of
sodium sulphide below 7:5, important changes in the physical state of the cobalt sulphide
resulted. Instead of the usual homogeneous reaction in the ground cytoplasm in kidney
sections the reaction was in the form of granules having the distribution and shape of
mitochondria, the total density being slightly reduced. This could be interpreted in two
ways: either that at higher ammonium sulphide concentrations a cobaltammine complex
is formed which diffuses from the granules in which phosphatase is known to be present
(see later); or that the slow conversion to sulphide favours solution of the cobalt
phosphate which is then precipitated from solution in the tissue in the form of granules
of cobalt suiphide. A decision between these two alternatives may be made with ordinary
ammonium sulphide concentrations by increasing the ammonium concentration by
addition of ammonium chloride, and by increasing the solubility of cobalt phosphate by
dissolving the ammonium sulphide in glycine buffer of the same pH as the ammonium
sulphide solution itself. The first procedure has no effect, but the second gives an
accentuation of the picture found with low ammonium sulphide concentrations. The
correct picture is, then, the homogeneous distribution normally found.

It is of interest to note that the nuclei do not adsorb the cobalt phosphate when it
is made more soluble by the above method. As will be shown later, much of the cellular
alkaline phosphatase is bound to the mitochondria, and these survive fixation. The
fact that the phosphatase cannot be shown on these granules in tissue sections suggests
that the method may be invalid for cellular localizations.

(b) Movement of Cobalt Phosphate.

This may move either during its formation from calcium phosphate or during the
washing of the section after the conversion. In view of the fact that the cytological
distribution is the same with the present technique, which greatly reduces the solubility
of the cobalt phosphate in both steps, as with the classical technique, means that these
steps are unlikely to be associated with any significant migration of the compounds.
This conclusion is strengthened by the fact that an identical cytological distribution is
found when the calcium phosphate is visualized directly with alizarin added to the
incubating solution.

(c) Movement of Calcium Phosphate.

Danielli (1946) has reported experiments indicating that nuclei can adsorb calcium
phosphate from supersaturated solution or can serve as condensation points under these
conditions. It was not possible to reproduce the experimental conditions used by
Danielli, but the observation has been confirmed by a number of different methods.
Danielli reported that this calcium phosphate adsorbing property of the nuclei does not
invalidate the cytological localization of the enzyme. The experiments reported here
do not support the latter contention, which appears to be almost correct for kidney
under optimal conditions of enzyme activity and short incubation times, but is not

a6 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. II,

correct for organs with lower activity than kidney, or for kidney at longer incubation
times.
The experiments on which these views are based are detailed below.

(i) Effect of Calcium Concentration.

When sections of kidney and testis are incubated in substrate buffer solutions
containing M/100 Ca and M/1000 Ca, for the time required to give equal density in the
two series, it is found that the apparent cytological distribution of the enzyme is different.
At M/100 Ca concentration the nuclear reaction in the proximal tubules is quite weak,
the cytoplasmic reaction moderate and the brush bordér reaction intense. At the lower
Ca concentration the nuclear reaction is much stronger in the proximal tubules and is
increased in other cortical tubules, the cytoplasmic reaction is increased in intensity
and the brush border reaction slightly diminished. In testis, the main difference between
sections incubated at the two calcium concentrations is that the nuclei have a higher
density at the lower calcium concentration, the effect being less noticeable than in
kidney. These alterations in relative density of various cell sites become more marked
as the calcium concentration is lowered even further (Figs. 1 and 2, Pl. i).

With low calcium concentrations the diffusion of calcium ions will be insufficient
to trap the phosphate liberated by the enzymatic hydrolysis immediately it is formed.
The effect of low calcium concentration will then be to favour trapping more uniformly
in the section and the above observations thus provide independent proof of the ability
of nuclei to adsorb calcium phosphate liberated in their vicinity. Nuclei do not adsorb
calcium phosphate (in a form demonstrable by the histochemical reactions) from a
substrate free incubating solution saturated with this compound.

This does not, in itself, prove that the calcium concentration usual in the technique
(approx. M/100) is insufficient to cause immediate trapping of the phosphate at the
enzyme site.

As has already been stated (Cleland, 1950), high concentrations of calcium cause
a reduction in the final density of sections as compared with those incubated in solutions
of normal calcium content. When sections incubated to the same final density in the
presence of M/10 and M/100 Ca are compared microscopically, it is found that the
differences are indistinguishable from those which occur in cases where the enzyme
activity is depressed to a comparable degree by lowering the substrate concentration,
or by the addition of KCN (Figs. 3 and 4, Plate i). This suggests that Ca concentrations
of the order of M/100 are adequate to cause immediate trapping of the enzymatically
liberated phosphate.

(ii) Effect of Reducing Enzyme Activity.

As the enzyme activity of kidney sections is reduced by various means, and the
resulting cytological picture compared with that in sections of normal activity, both
series being incubated for the times required to give the same final density, it is found
that, in the sections in which the activity is reduced, the nuclei have a much greater
density. Furthermore, the nuclear density increases with increasing enzyme inhibition.

Thus when slides incubated in M/100 substrate are compared with those incubated
to the same histological density in M/1000 substrate, it is found that, in the latter, the
nuclei are much more heavily impregnated and the brush border reaction is noticeably
weaker (Figs. 5 and 6, Plate ii). With substrate concentrations of the order of 10-° to
10-° M the brush border reaction is very weak and the nuclei heavily impregnated. No
increase in density occurs in sections incubated without substrate in buffer calcium
solutions saturated with inorganic phosphate.

When the activity of the kidney sections is reduced by other means—e.g., by incuba-
tion in solutions containing _ KCN—exactly the same phenomena occur as were observed
when the activity was reduced by lowering the substrate concentration. It seems very
unlikely that the nuclear enzyme would be so different from that in the rest of the cell
that it would simultaneously have so different a Michaelis constant and be activated by
cyanide and high Ca concentration instead of inhibited.

BY K. W. CLELAND. 57

These observations on kidney tissue sections are much less marked, but still demon-
strable, in testis sections.

(iii) Effect of pH.

In kidney sections, reduction of the rate of enzyme action by lowering the pH results
in a much more marked nuclear impregnation than that shown when other methods of
reducing enzyme action were used (Fig. 7, Plate ii). Four hypotheses could account for
this: (a) that the nuclear enzyme is more readily inactivated at the higher pH; (b) that
the nuclear enzyme is activated at the lower pH; (c) that the nuclear enzyme has a
lower pH optimum than the rest of the cell; (d) that the greater solubility of calcium
phosphate at the lower pH (Cleland, 1950) favours more movement of this substance
in the section during incubation, with subsequent adsorption of it by the nuclei. An
experiment designed to test the first two alternatives is shown in Table 1. These two
hypotheses are excluded. In some experiments, differences between slides 2, 4 and 6
suggested that there was some loss of final density in the nucleus caused by pretreatment
of the slides at the higher pH; thus under certain conditions hypothesis (a) accounts for
a small part of the difference.

TABLE 1.
Analysis of Cause of Higher Nuclear Reaction at Lower pH.

Pretreatment Incubation with
Slide. without Substrate. Substrate. Result.
1 1 hr. at pH 9-5 1 hr. at pH 9:5 Same as 3.
2 do. 1 hr. at pH 8:3 Same as 6.
3 nil 1 hr. at pH 9°5 Poor nuclear reaction.
4 1 hr. at pH 8:3 1 hr. at pH 8:3 Same as 6.
5 do. 1 hr. at pH 9:5 Same as 3.
6 nil 1 hr. at pH 8°3 Good nuclear reaction.
@ 2 hr. at pH 9-5 nil Usual diffuse blank reaction.
8 2 hr. at pH 8-3 nil do.

All solutions saturated with inorganic phosphate. M/50 borate veronal buffer, M/100
subtrate and Mg. Kidney sections.

The third hypothesis is made unlikely by the observation that the relative nuclear
reaction increases down to the lowest pH tested (7:7) and a pH optimum difference of this
order seems very unlikely. A decision between the third and fourth hypotheses can,
however, be made by a study of nuclei separated from fixed or fresh tissue. This would
exclude the possibility of absorption by the nuclei of calcium phosphate. formed at other
cell sites, and make possible a study of the pH optimum of the nuclear enzyme itself.

Nuclei were separated from fresh guinea pig kidney by methods described later,
smeared on slides, dried and fixed in acetone. The enzyme present in these separated
nuclei has the same pH optimum as that found for whole tissue sections. This, then,
means that the higher nuclear activity at the lower pH is most probably due to the fact
that calcium phosphate has a higher solubility at the lower pH.

Testis sections show this pH effect to a lesser degree than kidney while adrenal
cortex shows it in greater degree.

(iv) Discussion.

It can be shown by the following experiment that calcium phosphate once precipitated
in the section does not redistribute itself. Kidney sections were incubated for one hour
in the presence of substrate, thoroughly washed in three changes of buffer calcium, and
then placed in buffer calcium at pH 9:5 and 8:0 (both saturated with phosphate) for
periods up to 24 hours. An unincubated slide was placed with each of these in order to
determine whether any substrate had been carried over with the slides. No redistribu-

ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. II,

ol
C

tion of density could be demonstrated at either pH, indicating that any calcium phosphate
movement must occur during its formation.

The following hypothesis is capable of integrating all the observations. The calcium
phosphate is formed in the molecular state and is freely diffusible in this state. When
the local concentration is such that supersaturation occurs, the calcium phosphate
precipitates in the colloidal form, a state in which no diffusion is possible. Certain
cell constituents because of their electrostatic properties, are capable of attracting the
molecular form to give a supersaturation in their vicinity with subsequent precipitation
of the colloidal form.

This hypothesis explains the effects observed on reducing the rate of enzyme action
in kidney, since with lower activity, less of the molecular form will reach a concentra-
tion sufficient to give precipitation in the colloidal form, with consequent diffusion of
the molecular form and its adsorption by the nuclei. When not only the rate of
formation is reduced but also the precipitation threshold of the calcium phosphate is
raised, as it is by reducing the pH, the effect is much more marked. In testis where
the activity is normally considerably lower than kidney, diffusion of calcium phosphate
plays a considerable part in the picture obtained under normal conditions of incubation,
and the above effects observed in kidney are thus less evident.

That the molecular form of calcium phosphate is strongly adsorbed to the tissue
section, and does not diffuse into the medium is shown by the following experiment.
Sections of kidney and testis were incubated for four hours at two substrate concen-
trations. Analyses of the increase of phosphorus in the incubating fluid and the sections
were made with the results shown in Table 2. The results obtained are best explained by
diffusion of inorganic phosphate (untrapped by the calcium ions) from the section.

TABLE 2.
Loss of Phosphorus from Sections of Different Enzyme Activity.

Substrate P in Sections. P Increase in

Organ. | Concentration. (ug.) Fluid. (ug.)
Testis .. ae 3 Evebdl M/100 155 8
M/1000 102 5
Kidney et Ss foul M/100 180 13
M/1000 145 4

Active tissue area approximately 11 cm* M/50 borate buffer M/100 Mg and Ca.
Incubation 4 hours.

Figures quoted for increase of phosphorus in the incubating fluid are probably slight
overestimates ; bacterial substrate splitting is probably greater in the fluid containing
sections than in the control fluid without sections.

This strong adsorption of the molecular form of calcium phosphate implies that this
substance is probably capable of wide migration in tissue sections.

Since this work was done the paper of Jacoby and Martin (1949)* has been seen.
The present work confirms their findings and defines the conditions under which the
migration of the calcium phosphate is likely to give serious errors of cytological
localization of the enzyme.

(d) Movement of Enzyme.

(i) General Considerations.

That it is desirable to entertain the possibility of adsorption of enzyme, at least as
far as the nucleus is concerned, is shown by the following considerations.

(a) As will be shown later, a significant fraction of the alkaline phosphatase is
present in a soluble form. The purified enzyme is not precipitated by acetone concen-
trations less than 50% (Schmidt and Thannhauser, 1943). Since the normal perme-

* See also J. Anat., 1949, 83: 351.

BY K. W. CLELAND. 59

ability properties of the nucleus (Callan, 1948) are likely to be changed during the
process of fixation, there is a possibility of diffusion of the enzyme into the nucleus during
fixation. In floating out the sections after cutting, this possibility would be even greater.

(bo) The isoelectric point of the nuclear substance, at least when fixed or partly
fixed, is quite low. In at least some groups of enzymes, a rough positive relation is found
between the pH optimum and the isoelectric point. The possibility of complex formation
between chromatin and alkaline phosphatase is therefore indicated.

(c) The adsorption of protein under certain conditions by tobacco mosaic virus has
been shown to occur by Kleczkowski (1946) and by Lauffer (1948). The general aspects
of the problem have been discussed by Pirie (1948). The adsorption of enzymes (lipase,
catalase and phosphatase) from dilute solution by vaccinia virus, which contains similar
quantities of the same type of nucleic acid as the cell nucleus, has been shown to occur
by Hoagland et al. (1942).

(d) Dounce and Beyer (1948) have shown that nuclei can take up arginase from
solution. Since they worked with whole nuclei, separated by relatively gentle means,
this suggests that the permeability properties of mammalian nuclei may be different
from those of the amphibian cocyte, Callan (1948), or else the properties are changed by
relatively gentle means.

(e) Lison (1948) has considered the possibility of enzyme adsorption in the histo-
chemical technique and thinks that it plays a considerable role. However, he presents
no experimental evidence to support the view.

(ii) Positive Evidence in Tissue Sections.

The evidence in favour of an adsorption of enzyme by cell nuclei in a histochemically
demonstrable form may be summarized thus:

(a) In almost all organs showing a positive cytoplasmic reaction for alkaline phos-
phatase the nuclei also show a positive reaction. In a few cases the nuclear reaction is
greater than the cytoplasmic reaction.

In all these cases it is not possible to be sure that the observed picture is not due
to adsorption of calcium phosphate by the nuclei.

(b) In guinea-pig kidney the nuclear reaction is usually confined to the cells of
the proximal tubules, the other cortical and medullary tubules showing little or no
reaction, except with very long incubation times. In the latter case this reaction is
probably not due to enzyme action.

If the reaction in the nuclei of the proximal tubules is due to adsorption of enzyme
from the cytoplasm of these tubules, the reaction in nuclei separated from whole kidney
would be expected to differ from that found in tissue sections since, during preparation,
all nuclei would be exposed to similar concentrations of soluble enzyme.

Whole guinea-pig kidney was homogenized in a Waring blendor microcup in 10 vols.
of saline. The homogenation time was 45 secs. and the blendor was run at half voltage
since full voltage resulted in complete fragmentation of the nuclei. The nuclei were
separated from the homogenate by centrifugation with fields between 70 and 100 g., the
separation being microscopically controlled for each run. The principal final contaminant
was red cells, no attempt being made to remove these. The concentrated nuclear
suspension was smeared on slides in the same manner as used for blood films, fixed for
varying times in acetone, and subjected to the histochemical reaction. Good nuclear
suspensions could also be prepared from fixed tissues, the blendor then being run at
full voltage.

All nuclei so prepared show a slight positive reaction, which increased very little
with increasing incubation time. While individual nuclei differ somewhat in their
density, there was no evidence of the presence of the two populations of nuclei expected
from the results in tissue sections.

These results are consistent with the hypothesis that enzyme may be taken up from
solution by nuclei, and that this enzyme, under favourable conditions, may give rise to
a slight reaction in the histochemical procedure.

The histochemical reaction is usually found to be denser in smears of nuclei
separated from distilled water homogenates.

60 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. II,

(iii) Negative Evidence.
The evidence against movement of enzyme being an important invalidating faetor
in the histochemical technique, may be summarized as follows:

(a) In cells with a very intense cytoplasmic reaction, in which the adsorption of
calcium phosphate by the nuclei should be minimal, as indicated in the previous section,
the nucleus should give no reaction if it contains no intrinsic enzyme and does not
adsorb any from the cytoplasm during fixation. Such a situation occurs in the poly-
morphs of guinea-pig blood films fixed in acetone, where a jet black cytoplasmic reaction
surrounds the colourless lobes of the nucleus. This only applies to short incubation
time; longer incubation times result in a nuclear impregnation which is probably caused
by calcium phosphate adsorption.

(b) If adsorption of enzyme occurred only during fixation, some difference in the
nuclear reaction would be expected between blocks of different size. In very small blocks
of tissue (1 mm. thick) fixation by acetone would be almost instantaneous, while in large
blocks (8 mm. thick) the fixation would be slow and some time would elapse before the
enzyme precipitating concentration of acetone was reached. In experiments of this
type no differences in the nuclear reaction could be found.

The possibility of enzyme redistribution during the floating out process cannot,
however, be excluded, since it will be demonstrated later that enzyme soluble before
fixation does not become insoluble after fixation.

(c) If nuclei are capable of adsorbing enzyme in a form demonstrable by histo-
chemical means, it should be possible to demonstrate a positive reaction in nuclei
separated from a source which is completely phosphatase negative but which have been
subsequently exposed to a solution of phosphatase. Avian red cells provide a convenient
source of nuclei showing no trace of a reaction under normal conditions.

A solution of phosphatase was prepared from ox kidney in the following way.
Minced ox kidney was suspended in 1% vol. of 95% alcohol, stirred for 30 mins. and
filtered. This process was repeated three times and the resulting tissue preparation
stored in the refrigerator in closed containers. The preparation was found to be quite
stable. The soluble enzyme was extracted from this by blending in four vols. of water
and the resulting suspension filtered. Ammonium sulphate was added to 50% concen-
tration and the precipitate collected. After dialysis against 1% NaCl solution over-
night the solution was centrifuged at 20,000 g. for 15 mins. and diluted to an activity of
1200ug.P split/ml. enzyme/30 mins. under the conditions described in the next section.
This enzyme activity is approximately double that of the soluble enzyme in the proximal
tubules of guinea-pig kidney.

Pigeon red cells were washed in four lots of 10 vols. of 1% NaCl. They were
suspended in four vols. of 1% NaCl and added to an equal volume of the above enzyme
solution. The mixture was blended until about 90% of the red cells had been disrupted.
The cell membrane was removed by this method, in contrast to, other methods of
cytolysis, where the cell membrane is found to surround the nucleus.

The nuclei were centrifuged down, the supernatant removed and replaced by 50%
acetone. After 30 mins. this was replaced by absolute acetone which was allowed to act
overnight in the refrigerator. After pouring off the acetone the preparation was taken
up with sufficient distilled water to give a thick suspension which was then smeared
on slides. These were subjected, either immediately, or after further acetone fixation, to
the histochemical reaction. In neither case could any reaction be demonstrated.

(iv) Discussion.

Previous workers have approached this problem in other ways. Danielli (1946) has
reported that the reaction is the same in rat kidney after a wide variety of fixatives,
and Emmel (1946) has compared the reaction found in frozen dried material with that
found after normal methods of fixation. While these results are suggestive, they do not
eliminate the possibility that movement of soluble enzyme occurred during the floating-
cut process.

BY K. W. CLELAND. 61

The evidence on the possibility of enzyme movement giving rise to false nuclear
localizations in the histochemical reaction is thus mainly negative. The slight positive
reaction found in all isolated kidney nuclei as against the reaction in tissue sections
could be interpreted as due to inactivation of the enzyme in all the nuclei of the latter
due to the paraffin embedding, the positive nuclear reaction found in the proximal tubule
nuclei in tissue sections being due to adsorption of calcium phosphate.

THE HISTOCHEMICAL VALIDITY OF THE REACTION.

(i) Phosphatase Activity of Different Organs.

It has been demonstrated in the previous section that the phosphatase reaction has
a number of grave defects making its use in cytological localization of the enzyme unsure.
It remained to see what value the method has as a histochemical technique. Little
objection can be raised to a qualitative localization of the enzyme to a given histological
element, but the quantitative aspects, and, more particularly, the validity of negative
results, has not yet been investigated satisfactorily.

Attempts at test tube assay of phosphatases in different animals and in different
organs from the same animal appear to have been made rather infrequently. Some of
the relevant data are collected in Table 3.

TABLE 3.
| | H
Animal. Kidney. | Intestinal Liver. Lung. | Spleen. | Pancreas. |; Testis. | Adrenal. | Author.
i+ Mucosa.
}
| | |
ep OD By OR aS orgs 1
: 100 | 0:27 heck | | 2
100 8 45 24 3
Mouse hed 100 260 | 0-3 3°3 1:6 2
100 202 4:5 9 hei | Aes | | 3 3
100 4-5 | 9
Rabbit .. | 100 435 ate Oa ! | 4
100 292 Sylois BD} Bo} | | 5
100 267 47-5 43 | 44 | 2 | 19 3
Cat ad 100 212 8 54 6-5 5 | 5
100 650 4 24 6 4 12 3
— | |
Dog co 100 360 5-6 10 26-5 | 6
100 630 Hemel p12 22 27 7-5 3
Man ey. 100 240 16-5 | 27 aes
Chicken .. 100 | 360 29 7
Guinea-pig 100 650 6-5 31 | 108 3
100 11-7 15-6 26 | 8
| i = |- -: S ee
Number of
animals 12 LH | 6 yaa |
Range .. 9-13 14-18 19-34 |

Literature key: 1. Truhlar et al. (1939). 2. Greenstein (1941). 3. Macfarlane et al. (1934). 4. Hoffmeyer
et al. (1946). 5. Kay (1928). 6. Gad (1946). 7. Moog (1946). 8. Present investigation. 9. Kochachian and
Fox (1944).

The methods used for the data quoted in Table 3 are very diverse, a factor which
probably explains some of the discrepancies in the estimation of the activity of the same
organ in the same animal by different authors. The only data for guinea-pig which
could be found were those of Macfarlane et al. (1934), the data being obtained by a
method using very suboptimal pH and very long incubation time, and not including
analyses for testis and spleen, organs very suitable for comparison by histochemical
methods.

62 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. II,

The results obtained in the present investigation are shown in Table 3 as percentages
of the value of homologous kidney cortex. They may be converted to absolute values by
veference to the mean value for kidney which was 1127ug.P/30 mins./100 mg. fresh
weight, with a range of 950-1400.

A rough histochemical estimate of the enzyme in tissue sections was made for these
four organs in three complete experiments, in which the histological procedures were
the same for the organs in each experiment. The sections were incubated for 45 mins.
under conditions identical with the test tube enzyme determinations. The results were
as tollows (expressed as density units defined in the previous paper): Kidney 51 (range
45-55), Testis 42 (range 38-45), Spleen 20-5 (range 18-23). In liver no consistent
increase over the blank density could be detected at this incubation time, or after four
hours’ incubation.

The difficulties of making histochemical comparisons of the enzyme activity in
sections of organs in which the distribution of the enzyme is different, has already been
stated (Cleland, 1950), but it will be seen that the values given by kidney, testis and
spleen are plausible comparisons of their relative enzyme content, while that given by
liver is very much lower than would be expected from the test tube experiments.

(ii) Soluble and Insoluble Enzyme in Different Organs.

It is known that phosphatases occur in bound and free form, and it is of considerable
importance for interpretation of the histochemical reaction to know whether the enzyme
in liver is less tightly bound to the cell particulates, and, if so, to what extent it became
insoluble after the fixation used in the histochemical reaction.

The first point was investigated by centrifugal fractionation of homogenates.

The results of two experiments are shown in Table 4, results from two other
experiments, performed with different centrifugal fields, being completely in accord with
those quoted. The value quoted is a percentage of the value in the whole homogenate.

TABLE 4.
Phosphatase Partitions in Various Centrifugal Fractions in Different Organs.

Organ. Supernatant 1. Supernatant 2. Supernatant 3.
% % %
Testis 75 27 22-5
82 30 25-5
Kidney .. a As 40 | 19 14
42-5 20 14
Liver pa a4 Pact 97 85 87
97 100 91
Spleen .. ee ae 76 29 21-5
81 36°5 PISO)

The composition of the fractions removed was as follows:—Testis: (1) sperm heads,
tails, middle pieces; some unbroken nuclei, small granules, developing acrosomes; (2) few
sperm tails, many small granules; (3) very small granules. Kidney: (1) fibre, few
nuclei, granules; (2) small granules; (3) microscopically unidentifiable. Liver: (1)
occasional nucleus, granules; (2) small granules; (3) very small granules. Spleen:
(1) fibre, some whole leucocytes, leucocyte nuclei, polymorph lobes; (2) few fibres,
granules; (3) microscopically unidentifiable. Fraction I in all tissue contained a few
unbroken erythrocytes.

It will be seen that, with the exception of liver, the major part the alkaline phos-
phatase is bound to the particulate matter of the cytoplasm, mainly to the large granule
fraction. Fragmented brush borders of kidney are almost certainly included in this
fraction as granules, since it was impossible to trace them as morphological entities.
Chromosomes from nuclei occur in fractions I and II.

BY K. W. CLELAND. 63

It seems unlikely that the low percentage of bound enzyme obtained for liver is due
to autolytic freeing of the phosphatase from the granules, since available assays for
cathepsin in different organs (rat) indicate that liver has a lower activity than spleen
or kidney (Maver, Dunn and Greco, 1948).

(iii) Hffect of Fixation on Amount and Distribution of the Enzyme.

Having indicated that guinea-pig liver phosphatase is present mainly in soluble form,
it remained to be seen whether any binding of the soluble enzyme occurred during
fixation. Pieces of the above four organs were removed from guinea-pigs, weighed,
and placed in 20 vols. of cold acetone in a refrigerator. After 24 hours the acetone was
replaced by cold absolute alcohol, this being allowed to act for periods of three days and
14 days in two experiments each containing organs from two individual guinea-pigs.
After fixation, the organs were placed in 10 vols. of saline and homogenized in the
blendor for two minutes. Two drops of octyl alcohol were added after blending to break
the foam. The homogenate was removed, 1/10 vol. of M/5 veronal buffer pH 9-4 added,
and allowed to remain at room temperature for one hour. After mixing, a sample of
the whole homogenate was taken for assay. The remainder was centrifuged at 20,000 x g.
for 15 minutes, and a sample of the supernatant, which was clear, taken for phosphatase
assay. The largest component present in the homogenate in appreciable quantity was the
cell nucleus, the majority of which appeared to survive disintegration. Granules retained
their integrity.

The phosphatase activities of the whole homogenates were within the normal activity
range for the organ concerned, indicating that little or no loss of activity is caused by
fixation, a fact noted by other authors (Danielli, 1946, Stafford and Atkinson, 1948, Doyle,
1948), and no difference could be found between the tissues fixed for three and 14 days.
No change in the solubility of the enzyme in kidney or spleen could be demonstrated, an
increase of 5% in the solubility of the testis enzyme was noted, while the percentage
of soluble enzyme in liver had declined to 60%.

It will be seen that fixation, while decreasing the solubility of the liver enzyme some-
what, cannot be considered to preserve soluble enzyme in a form likely to be demon-
strated in the histochemical reaction, and, therefore, that the reaction demonstrates only
the bound enzyme of the cell. This possibility has been considered by Lison (1948), but
no evidence was adduced.

The enzyme solubility factor is still insufficient to account for the low values found
for the activity of guinea-pig liver sections in the histochemical technique. In order to
study this matter further, to see what degree of inactivation was produced by imbedding
in paraffin and the preparation of tissue sections in a form suitable for histochemical
examination, and whether this was different for different organs, the absolute activities .
of the phosphatase in tissue sections of the above four organs was determined. The
results are shown in Table 5. :

TABLE 5.
Phosphatase Activity of Tissue Sections for Various Organs.

g. P Liberated per 100 sq. mm. of Surface Area.
Percentage
Organ. of Kidney.
1 Hour. 2 Hours. 4 Hours.
Testis 10-1 ZS 43 21
Kidney 50-2 106 204 100
Liver 1:91 3:25 4-6 2°3
Spleen 7:0 i550 29-4 14°5

Three points emerged from this experiment:
during the incubation and the activity is sufficiently low under the conditions of the

(1) Enzyme in the liver is inactivated

64 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. II,

histochemical technique to account for the low values found by that technique. (2) Other
organs show little inactivation over the times used and their activity in comparison with
kidney is within the normal limits found for homogenates. (3) Assuming a shrinkage
in volume of 50%, a not unlikely value in view of the experiences of Stowell (1941) and
Tarkham (1931), a caleulation of the enzyme activity of fresh tissue equivalent to these
sections fails to indicate that any loss of enzyme activity can be ascribed to the histo-
logical procedures involved in the preparation of spleen, kidney or testis sections. This
is at variance with the findings of Stafford and Atkinson (1948) and Doyle (1948).

DISCUSSION.

The demonstration that cobalt sulphide can precipitate in the section in granular
form under certain conditions, implies that the interpretation of such structures as the
perinuclear granules described by Deane and Dempsey (1945) as Golgi apparatus in
kidney sections, is rather difficult; they do not appear to be sufficiently constant for
the possibility of artefact to be excluded. No structure which could be unequivocally
interpreted as Golgi apparatus has been found in the guinea-pig kidney sections studied
here. The blackening in the Golgi apparatus in the epithelial cells of small intestine
is constant enough to be regarded as valid, but whether it is due to enzyme is another
matter. A blackening in what appears to be the Golgi apparatus of adrenal cortex cells
(Bennett, 1940) has been noted in the present study.

The demonstration of blackening in the chromosomes by Willmer (1942), Krugelis
(1942, 1946), Danielli and Catcheside (1946) and others, and the reports of nuclear
reactions by a large body of histochemists, cannot with any certainty be related to the
presence of phosphatase, because of the demonstration of adsorption of calcium phosphate
by chromatin. This is especially so because in those cells in which the chromosome
reaction is described, the cytoplasmic reaction is usually slight, a state of affairs which
is shown by the present study to give the most troublesome calcium phosphate wandering.
This fact also removes much of the grounds for the interesting speculation of Brachet
and Jeener (1948).

In view of the property possessed by nuclei of adsorbing calcium phosphate from
supersaturated solution in the incubating fluid (Danielli, 1946), the use of unusual
substrates, such as nucleic acids, often at a relatively low pH, does not seem likely to
give valid results. At low pH and in the presence of a source of nitrogen bacterial
growth is quite rapid; splitting of the complex substrate by the bacterial enzymes
would yield calcium phosphate in the vicinity of the section, with probable adsorption
of it by the nuclei. To complicate matters further, these low pH solutions are not
usually presaturated with calcium phosphate, which, in a previous paper (Cleland, 1950),
has been shown to be desirable. The resulting picture in these cases is a balance between
a number of factors: loss of calcium phosphate from enzyme sites to the solution before
the latter becomes saturated due to bacterial splitting of substrate, wandering of calcium
phosphate from the enzyme site after saturation of the solution, adsorption of calcium
phosphate from the solution which has become supersaturated due fo bacterial action,
and destruction of the enzyme owing to the long incubation times used. The findings of
yomori (1949) are interesting in this connection.

These remarks are not meant to imply that there is no alkaline phosphatase in cell
nuclei: merely that it is not possible to obtain unequivocal evidence of its presence by
means of the histochemical reaction.

The evidence for the presence of phosphatase in the nucleus is, however, very slight.
Apart from a statement by Mirsky and Ris (1946) not accompanied by any experimental
details, and a footnote report in a paper by Potter (1947) to the effect that large
quantities of apyrase occur in liver cell nuclei, the only report of which the author is
aware is that of Dounce (1943), who has shown that isolated liver nuclei contain about
twice as much phosphatase on a weight basis as whole liver cells. No other nuclei_
appear to have been examined for their phosphatase content, and if the nucleur arginase
distribution is generally applicable (Dounce and Beyer, 1948), considerable variation
between nuclei from different sources can be expected.

BY K. W. CLELAND. 65

In view of the reports reviewed in the section on adsorption of enzyme, this obesrva-
tion of phosphatase in liver nuclei must be regarded with some reserve until suitable
adsorption controls on isolated nuclei have been performed. The evidence on non-
adsorption of enzyme adduced in this paper will not necessarily be applicable to studies
of isolated nuclei assayed in the test tube; in the histochemical reaction the greater
proportion of the enzyme not firmly affixed to the protoplasmic structure will be lost to
the incubating fluid, while in the test tube assay-all the enzyme both fixed and adsorbed
will be measured.

Three methods have been used to study the enzyme constitution of the nucleus;
reactions in tissue sections with microscopic localization of a coloured product produced
at the enzyme site (literature in Glick, 1949); test tube assays of enzyme in nuclei
separated from fresh tissues (Dounce, 1943; Lan, 1943; Dounce and Beyer, 1948); test
tube assays of enzyme in cell fragments produced by centrifugal stratification and
breaking of whole cells, in which fragments without nucleus are usually compared
with fragments containing the nucleus and a little cytoplasm. Shapiro (1935), Holter
(1936), Holter and Kopac (1937), Ballentine (1940) and others.

The first of these methods seems unlikely, in view of the present experience with
the alkaline phosphatase reaction, which has a primary localizing product of very low
solubility, to yield a valid localization of an enzyme and proof of the validity or invalidity
of such methods may be difficult or even impossible. The fact that some enzymes have
to be studied in unfixed sections introduces serious possibilities of extraction of enzyme
and tissue substance during the procedures. Quantitation of such methods presents
considerable difficulty and the interpretation of negative results is unsure.

The second method has two drawbacks: the possibility of adsorption of enzymes,
and, in view of the work of Pollister and Leuchtenberger (1948), the strong likelihood of
extraction of enzymes.

Inasmuch as the cell is very little injured by the third method (sea urchin eggs so
broken are capable of development, Harvey, 1932, et seq.), and the nuclear membrane,
being still surrounded by some cytoplasm, can be expected to preserve its normal
permeability, this method appears to be the most satisfactory of the three.

Phosphatase belongs to a group of hydrolytic enzymes whose physiological role is,
in general, obscure. Members of this group, on present knowledge, appear to be a
liability to the cell rather than an asset, inasmuch as they cause degradation of key
substances in the energy forming mechanisms of the cell. The fact that histochemists
have related phosphatase to, inter alia, glycogen synthesis, nucleic acid synthesis and
breakdown and protein synthesis, is, perhaps, a measure of our ignorance of the physio-
logical role of the enzyme rather than of any property of the enzyme itself. A very
suggestive approach to the physiological role of one of this group of apparently inimical
enzymes (one causing cozymase destruction) has been made by Mellwain (1949). It
seems possible that phosphatases may play a similar role in the regulation of energy
formation in the cell.

The finding that alkaline phosphatase is, except in liver, associated with the large
granules of the cytoplasm of the guinea-pig organs studied, is of interest in view of the
current intensive enzymological study of this cell fraction. It appears that, with the
exception of the glycolytic system, most of the energy-yielding reactions of the cell are
present on this granule fraction (Cross, Taggart et al., 1949).* In an unpublished study
of the cytoplasmic distribution of a number of the Krebs cycle enzymes in oyster eggs,
it has been founa that the enzymes are associated with the cytoplasmic granules, probably
on that fraction identified cytologically as yolk (Cleland, 1947). Mitochondria evidently
retain their enzyme integrity even on undergoing a cytological differentiation; Du Buy
et al. (1949) have shown that the enzyme composition of melanoma granules is very
similar to that of amelanotic granules, both resembling the enzyme spectrum reported
by other authors of large granules from normal tissues. It would appear likely that this

* Since cytologically validated by Kennedy and Lehninger, J. Biol. Chem., 1949, 179: 957.

66 ALKALINE PHOSPHATASE REACTION IN TISSUE SECTIONS. MI,

segregation of the energy-yielding reactions of the cell on the large granule fraction
is one of the prime laws of the cytoplasmic structure of the animal cell.

The extraordinary resistance of the kidney enzyme to extraction from tissue sections
is reminiscent of the firm binding of such enzymes as cytochrome oxidase and succinic
dehydrogenase to the tissue particulates: the latter enzymes have not been obtained in
a truly soluble form. As further evidence of the strong binding of the alkaline
phosphatase the methods used for the preparation of the purified enzyme may be cited.
These involve digestion of the tissue with trypsin (Schmidt and Thannhauser, 1943;
Ehrensvard, 1933). It would appear that enzymes such as these are built into the
structure of the granule; others, such as malice and lactic dehydrogenases, which may
be extracted in a soluble form from acetone powders, are evidently retained by forces
of a more secondary nature.

Moog and Steinbach (1946) have reported that significant quantities of the alkaline
phosphatase of the chick embryo are bound to granules. When their fraction 2, which
must contain most of the large granules, is included in the estimate of the particulate
phosphatase, the percentage of soluble enzyme, up to 10 days’ incubation, is between
25% and 30%, a figure similar to that found for guinea-pig kidney, spleen and testis in
the present study. Between 10 and 12 days’ incubation the percentage of soluble enzyme
aoubled in the chick embryo. Moog and Steinbach (1945) have reported that much of
the apyvase activity of the chick embryo is also associated with the particulate matter
ot the cytoplasm.

The demonstration that the percentage of bound phosphatase may change during
development (Moog and Steinbach, 1946)* and the demonstration in the present study
that lyoenzyme is not made insoluble by histological fixation, when taken together,
indicate that caution is required in interpreting reports of changes in alkaline phos-
phatase activity during development (Brachet, 1947, a, b; Krugelis, 1947, a, b: Moog,
1943: Zorzoli, 1947). Thus while Moog (1943) records decreases in histochemical
alkaline phosphatase activity of most organs of the chick embryo after eight days’
incubation, estimates of total alkaline phosphatase in the test tube show a continuous
increase over this period (Moog, 1946). The discrepancy is evidently due to change
in the proportion of bound enzyme.

In a previous study of oogenesis in the oyster (Cleland, 1947) no alkaline phos-
phatase reaction could be demonstrated. Later test tube studies have indicated that the
mature oocytes have about 15% of the activity of guinea-pig spleen. This is sufficient
to be demonstrable histochemically if the enzyme were bound. Since it is all present in
soluble form, no histochemical reaction is demonstrable.

Similar difficulties of interpretation arise where a change in the intensity of the
histochemical reaction occurs in a pathological condition or is associated with some
physiological change.

SUMMARY.

(a) Low ammonium sulphide concentrations can give rise to spurious granular
precipitates of cobalt sulphide.

(b) Wandering of calcium phosphate and its adsorption by nuclei is a factor which
makes interpretation of the cytochemical reaction virtually impossible. This factor
becomes more serious when the enzyme activity is low, and is very serious when the
incubation of the sections is conducted at a low pH.

(c) The weight of evidence is against any false localization of the enzyme being due
to movement of the enzyme during or after fixation.

(d@) The alkaline phosphatase activities of guinea-pig kidney, testis, liver and spleen,
in test tube experiments, is compatible with the activity displayed in the histochemical
reaction for all organs except liver.

(e) The enzyme is associated with the large granule fraction in testis, kidney and
spleen, but the greater part of the liver enzyme is not bound to granules.

*Mazia (1949) has recently shown that desoxyribonuclease is progressively bound to
tissue particulates during Arbacia development.

BY K. W. CLELAND. 67

(f) Histological fixation causes virtually no change in the solubility of unbound
enzyme, and results in little or no enzyme inactivation.

(g) The enzyme activity in tissue sections is almost entirely due to enzyme which
was bound before fixation. No loss of enzyme could be ascribed to the embedding process.

ACKNOWLEDGEMENT.

This work has been assisted by a grant to the Department from the University of Sydney
Cancer Research Fund.

The author is indebted to Professor C. W. Stump and Mr. D. J. Lee for eriticism of
the manuscript.

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ADDENDUM.

,

Since this paper was written the paper of Ruyter and Neumann (1949)* has been
seen. While their presentation is a little difficult to follow, their main point appears to
be that the reaction, as usually practised, is likely to give invalid results because of
extiaction and wandering of enzyme in the lower concentrations of alcohol. With the
tissue sections used in the present study we have been unable to find any evidence of
extraction of enzyme by 30% alcohol even after 30 hours in this reagent.

EXPLANATION OF PLATES I AND II.

All photomicrographs are of guinea-pig kidney sections at a final magnification of 130
diameters. Orthochromatie plates were used throughout and the exposures were chosen to
give maximum contrast for the nuclei, this causing considerable loss of contrast in the brush
borders and cytoplasm of the proximal tubules. Exposure and development times were
identical for all figures.

*Ruyter, J. H. C., and Neumann, H., 1949.—A eritical examination of the histochemical
demonstration of the alkaline phosphomonoesterase. Biochim. Biophys. Acta, 3: 125.

BY K. W. CLELAND. 69

PLATE I.

Figs. 1 and 2. Comparing the distribution of density in sections incubated to the same
visual density in the presence of different concentrations of Ca. Borate buffer (M/50) pH 9-4,
M/50 substrate, M/100 Mg. Ca concentration M/100 for Fig. 1 with incubation time 30 minutes;
M/100,000 for Fig. 2 with incubation time 4 hours.

Note the increased cytoplasmic and especially nuclear density at the lower calcium
concentration. This indicates that nuclei are able to adsorb calcium phosphate generated in
their vicinity by enzyme action.

Figs. 3 and 4. Comparing the distribution of density in slides incubated to the same
final density, with different rates of enzyme action. Borate buffer (M/50) pH 9:4, M/50
substrate, M/100 Mg. Ca concentration M/100 for Fig. 38, with incubation time 90 minutes,
and M/10 for Fig. 4, with incubation time 4 hours.

When the rate of formation of calcium phosphate is slowed, this results in a greater
migration of calcium phosphate to the nuclei.

PLATE II.

Figs. 5 and 6. Comparing the distribution of density in slides incubated to approximately
the same final density, with different rates of enzyme action. Borate buffer (M/50) pH 9-4,
M/100 Ca and Mg. Substrate concentration M/10 for Fig. 5, with incubation time 30 minutes,
and M/1000 for Fig. 6, with incubation time 4 hours.

Even though. the final density of the slide incubated at the lower substrate concentration
is less than that of the control, the nuclear density is both absolutely and relatively greater.
Similar results to those shown in Figs. 3 to 6 were also obtained when the rate of enzyme
activity was depressed with potassium cyanide.

Fig. 7. Showing the distribution of density in slides incubated at pH 8:5 (veronal borate
buffer). Other conditions same as in previous figures. When not only the rate of enzyme
action is depressed but the precipitation threshold of calcium phosphate is raised by lowering
the pH, adsorption of calcium phosphate by the nuclei is greatly increased. With comparable
overall density, the nuclear impregnation decreases with increasing pH, as also does the
solubility of calcium phosphate.

Fig. 8 Showing a typical control section, after four hours’ incubation under the same
conditions as Figs. 1, 3 and 5, but in the absence of substrate. The nuclear impregnation
shown here is more than in the control sections corresponding to the other figures because this
was prepared on the same day as it was photographed, while the others were over a month old
and some fading had occurred in these. The dark bodies in the glomeruli and between the
tubules are red cells.

DILATION OF THE FOOT IN UBER (POLINICHS) STRANGEI
(MOLLUSCA, CLASS GASTROPODA).

By Murtet C. Morris, Linnean Macleay Fellow in Zoology.
(Three Text-figures. )

{Read 26th April, 1950.]

INTRODUCTION.

For the past forty to fifty years it has been universally accepted that the
phenomenon of turgescence and extension of certain parts of the body, in particular
the foot, in most Molluscs is due to the forcing of large quantities of blood into the
organs concerned. Thus Pelseneer writes in regard to the blood in Molluses (Pelseneer,
1906): “The volume of blood in some groups, particularly in the Lamellibranchs and
Gastropods, is so great that it plays a very important part in the turgescence of various
parts of the integument, by filling the tegumentary sinuses during the relaxation of
their muscles.”

However, various workers of the early and middle nineteenth century held that
water from the surrounding medium, taken up either directly into the blood vessels
or into a set of “aquiferous” tubes completely separate from the blood system, was
responsible for this turgescence. This theory was applied almost exclusively to the
phenomenon of turgescence of the Molluscan foot. Many different theories accounting
for the uptake and output of this water and its transmission through the foot were
put forward—some of them backed with experimental evidence and some of them
purely theoretical. The story of the birth, life and death of this “Wasseraufnahme”’
theory, as it was called, is a particularly interesting one, and Justus Carriere has left
a very good and comparatively detailed account of its development from its birth in
the work of Delle Chiaje (Descrizione di un nuovo Apparato di canali acquosi scoperto
negli animali invertibrati marini delle due Sicilie, in Memorie sulla storia e anatomia
degli animali senza vertebre del regno di Napoli 1823-29. Tom. 11, p. 259 ff., 1826) to
its death at the hands of some six or seven workers during the 1880’s and 1890’s
(Carriére, 1882). These workers of the latter part of the nineteenth century proved
conclusively for certain species of Lamellibranch and Gastropod Molluses that the
expansion of the foot and other parts could be adequately explained by the forcing of
blood into the parts concerned. Their assumption that a similar state of affairs existed
in all other Lamellibranch and Gastropod Molluses is understandable when one considers
the large range of specimens investigated and the development of the study of
comparative anatomy.

‘However, in 1884, just as the old ‘‘Wasseraufnahme” theory was beginning to lose
popularity, Schiemenz published a paper in which he claimed to have demonstrated the
existence of an “‘aquiferous” system in a family of Prosobranch Molluscs—the Naticidae
(Schiemenz, 1884). Up to this time he had strongly opposed the ‘“Wasseraufnahme”
theory and had decided to carry out a series of experiments to prove his point, using
Natica josephina, because of its large and extensible foot. To his amazement, his
results seemed to show that water from the external medium was being used by the
animal for expansion of the foot. He later (Schiemenz, 1887) carried out an investi-
gation of the mode of uptake, transmission and expulsion of this water and claimed
to have demonstrated the existence of pores on the edge of the foot opening into a
series of water cavities completely separate from the blood vessels.

BY MURIEL C. MORRIS. tal

When Schiemenz’s papers first appeared, they were quite readily accepted by a
number of workers, for the old “Wasseraufnahme” theory still had a small following,
but when this theory finally lost its hold it must have been obvious that here was a
startling and isolated exception to what was by then an accepted theory, namely that
the expansion of the foot in Lamellibranchs and Gastropods was due to the forced flow
of blood. However, a survey of the literature of the past sixty years reveals that
Schiemenz’s theories regarding water-uptake in the Naticidae are still accepted by some
writers, even though no workers have touched seriously on this problem of expansion
of the Naticid foot since that time.

The common occurrence of a Naticid, Uber (Polinices) in the neighbourhood of
Sydney drew the attention of the late Professor W. J. Dakin, of the Zoology Department,
Sydney University, to this anomalous situation, and particularly to the strange hesitancy
in the literature to give a full explanation of the phenomenon of foot expansion in the
Naticidae. For instance, in several standard text-books the authors lead the reader
to understand that the uptake of sea-water may be accepted as a factor in the extension
of the Naticid foot, without giving any details or correlating the phenomenon with the
entirely different means of extension in all other Molluscs in which expansion of the
foot has been investigated. It seemed clear that here was a strange gap in regard to
our knowledge of the physiology of the Molluscs, the existence of an isolated phenomenon
which was not merely a problem of the Naticidae, but one which should be linked up
with the fundamental physiology of the phylum. When it was realized that no check
had been made on Schiemenz’s work and that practically nothing original had appeared
on the subject of water systems in the Molluscs during the past forty to fifty years,
the advisability of an inquiry became obvious.

On Professor W. J. Dakin’s suggestion it was decided that an investigation of the
conditions in the local species of Naticids should be undertaken. It was felt that if
the existence of an “aquiferous’” system in the Naticidae was refuted, the one real
stumbling block to the theory that turgescence in all Molluscs is due to the forcing
of blood into the parts concerned would be removed. If, on the other hand, such a
system was proved to be present, a very desirable confirmation of at least part of
Schiemenz’s work would be achieved, and possibly an interesting field for further
research would be reopened.

GENERAL NOTES ON THE NATICIDAE.

The Naticidae are Prosobranch Gastropod Molluses belonging to the order Meso-
gastropoda, which, in the modern classification of the Gastropod Molluscs, includes all
the members of the old tribe Taenioglossa of the Monotocardia.

As in all Prosobranchs, the foot is divided by an oblique furrow into an anterior
propodium (PR., Text-fig. 1) and a posterior metapodium (METP., Text-fig. 1). It has
become highly modified for burrowing in the mud and sand, the propodium developing
a “shell-fold” (PR.SH.FL., Text-fig. 2) shaped like a ploughshare, which can completely
cover the head and anterior half of the shell; whilst the metapodium has also developed
a “shell-fold” (METP.SH.FL., Text-fig. 2), which, when expanded, is present as a collar
round the back edge of the shell. Whilst the “shell-fold” of the metapodium goes
straight over into the metapodial tissue proper, the edge of the propodial ‘‘shell-fold”
is separate from the edge of the propodium proper except for a region X—X’ (Text-
fig. 2) at the anterior edge of the propodium. The actual point of junction, X, of this
“Shell-fold” with the propodium proper is shown more clearly in Text-fig. 3.

In Uber the propodium and the propodial “shell-fold” are darkly pigmented except
at the edges. The edge at the anterior tip and back along the sides to just behind
the junction of the propodium and the propodial ‘‘shell-fold” is a creamy yellow to
orange, whilst the edges of the rest of the ‘‘shell-fold” and of the propodium proper are
light grey (Text-fig. 2). The metapodium and metapodial ‘‘shell-fold” are usually not
as darkly pigmented as the propodial tissue, the edges occasionally being more lightly
pigmented than the bulk of metapodial tissue (Text-fig. 2).

FF

72 DILATION OF FOOT OF UBER (POLINICES) STRANGEI,

It is probably correct to say that in no other Gastropod is there such a remarkable
extension of the body from the shell as that observed in the family Naticidae, and the
volume change is certainly such as to arouse interest and inquiry. When fully expanded
the foot appears as a frill of oedematous flesh completely surrounding the shell, whilst

METP

PR.SH.FL.

CEPH. TENT. 2

MT.
PR. SH.FL.

METP. SH.FL. METP. EX.AP.

3

Text-figures 1-3.

Fig. 1, under-surface of anterior half of fully expanded foot of Uber strangei (x2). Fig. 2,
fully expanded Uber strangei (x 1-5). Fig. 3, postero-lateral view of Uber strangei from right
side (x2). CEPH.TENT., cephalic tentacles; COL.MUS., columella muscle; EX.AP., exhalant
aperture; IN.AP., inhalant aperture; MN., mantle; MT., mouth; METP., metapodium;
METP.SH.FL., metapodial shell-fold; OP., operculum; PR., propodium; PR.SH.FL., propodial
shell-fold; SH., shell; X., point of separation of propodial shell-fold from propodium, right
side; X.’, point of separation of propodial shell-fold from propodium, left side; Y., space
enclosed by operculum, metapodial shell-fold and shell where water is sometimes trapped.

BY MURIEL C. MORRIS. 73

the shell is almost covered with the “‘shell-folds’” (Text-fig. 2). In some of the biggest
specimens observed, in which the shell measured five cm. from anterior to posterior
edges when placed with the operculum down, the foot has been found to measure as
much as sixteen cm. from anterior tip to posterior tip. In specimens of average size,
with a shell size of three cm., the’ foot in the normal expanded condition usually
measures about ten cm.

When the animal contracts, this large mass of foot and “shell-folds” is withdrawn
into the space of the comparatively small shell, which is closed by the operculum
attached to the upper side of the metapodium (Text-fig. 3).

Three species of Naticids occur commonly in the Sydney area—Uber (Polinices)
strangei (Reeve), Uber (Polinices) conicus (Lamarck). and Uber (Polinices) aulaco-
glossa (Pilsb. and Van.), whilst very occasionally Mamilla (Polinices) melanostoma
(Swainson), which occurs commonly in Queensland but does not extend much beyond
the Queensland—New South Wales border, makes its appearance. Generally Uber
strangei is confined to the estuarine flats, whilst Uber conicus and Uber aulacoglossa
occur most commonly on the sandy beaches. They occur at the low-water level of
spring tides, and consequently are seldom found on anything but very wet sand, usually
preferring to be at least half covered with water. While collecting specimens it has
been observed that they make their appearance about an hour before low tide, and
then as the tide begins to rise again they disappear very quickly. Those species
found on the estuarine flats seem to prefer the pools hollowed out in the sandy areas
to the more muddy Zostera covered areas, and it is in these pools that the largest
specimens are to be found.

The large ploughshare-shaped propodium and the propodial “shell-fold” are obviously
adaptations of the foot for progression through the sand and mud. The propodium
forces its way through the sand half an inch to an inch below the surface, so that all
of the propodium and the propodial “shell-fold” and part of the shell are covered with
mud. The only part of the animal which is visible is the metapodium and posterior
part of the shell.

The tracks left in the sand by the animal are very characteristic. They consist of
a smooth depression, one-half to two inches wide, depending on the size of the animal,
with raised edges formed by the large bulk of propodial tissue burrowing under the
sand, which, when it comes in contact with the shell, falls away on either side, building
up a raised edge to the depression made by the propodial tissue. The metapodium
following along behind simply smooths over the depression. At one end of the track
there is usually a mound which, on closer investigation, proves to be the animal itself
burrowing along beneath the sand. If disturbed in any way they will immediately
burrow well down below the surface. It is not certain whether .they continue to
creep along once they are well down below the surface of the sand, or whether they
remain stationary and then burrow up to the surface later on to continue their
progression. The fact that a large number of the specimens found burrowing well
beneath the surface at the end of a track are in a contracted or semi-contracted state
seems to indicate the latter course.

The estuarine flats are criss-crossed with literally hundreds of these tracks at low
tide, each of them pursuing a devious course and forming an interlacing network with
other tracks. Pyrazus ebeninus leaves a very similar track, but in this case it is
formed mainly by the shell dragging along behind the animal and not so much by
the foot itself.

MATERIAL.

Uber strangei was used almost exclusively for the experiments presented in this
paper. The specimens were collected from Gunnamatta Bay on Port Hacking and from
Sailor’s Bay on Middle Harbour, Port Jackson, and the actual work was carried out
mainly at the Zoology Department, Sydney University. On two occasions, 17/1/49
to 25/1/49 and 14/3/49 to 17/3/49, when there was a need for good aquarium conditions

74 DILATION OF FOOT OF UBER (POLINICES) STRANGET,

and a constant supply of specimens, the work was carried out at the Fisheries Division
of the Commonwealth Scientific and Industrial Research Organization at Cronulla.

EXPERIMENTAL RESULTS.

When an expanded specimen of Uber is disturbed so that the foot contracts and
the soft parts are withdrawn into the shell, the effect is rather striking. The extended
parts of the animal disappear from view quickly and at the same time a large volume
of fluid escapes from the “mouth” of the shell. It will be seen later that Schiemenz
(1884) claimed that the greater part of this fluid had come from the “aquiferous” spaces
in the foot, where it had played a part in the expansion of that organ.

The propodium is withdrawn first with no loss of fluid, and it is only when the
metapodium, bearing the operculum, is withdrawn that fluid is seen to escape from the
“mouth” of the shell. This is very noticeable when observing an animal in the field,
where it is continuously encountering obstacles in its path. On closer observation,
however, a slight increase in the size of the metapodium as the propodium contracts,
or a corresponding decrease in the size of the metapodium as the propodium is stretched
out, is noticeable—pointing to the fact that a fluid is being alternately compressed into
or withdrawn from the metapodium as the propodium is withdrawn or stretched out.

The amount of fluid given off naturally varies with the size of the specimen, but
it is somewhere in the order of 4—7 ml. for an average-sized specimen when fully
expanded (Table I). In this connection it should be pointed out that Schiemenz’s
figures for the total fluid given off when Natica josephina contracted (Schiemenz, 1884,
table, p. 535) are amazingly large when compared with those given in Table 1 for

TABLE 1.

Fluid Expelled when Fully Expanded Specimens of Uber strangei
of Various Sizes Contracted.

Ml. Fluid

Expelled. Size of Specimen.
9-0 Large specimen.
8-0 5 3
728) “6 3
06) Af a
7:0 if 5
7-0 a ‘a
6-5 A 5
6-0 | ws 53
6-0 | Ms A
5:8 Medium sized specimen.
5-525 ap » »
Bob) es 6) oH
5-0 op » »
5-0 a 5 ;
4-0 ns) _ 3
3°0 Small specimen.
Paps) Pe aA
2-0 | ” ”

Uber strangei. The figures for shell volume which he gives (Schiemenz, 1884, table,
p. 535) show that most of the specimens he was using were larger than the specimens
of Uber strangei used here; but even so the figure which he gives for the total fluid
volume given off by a Natica of a certain size is much greater than that observed for
an Uber of corresponding size. It seems very likely that he did not remove as much
of the surface moisture and mucus as possible before measuring the amount of fluid
given off when the specimens contracted.

BY MURIEL C. MORRIS. US

When measuring the amount of fluid given off, care must be taken to make sure
that the animal is fully expanded before inducing it to contract, because the amount
of fluid varies with the degree of expansion. This fact would at first seem to indicate
clearly that at least some of the fluid given off has played a part in the expansion of
the foot; but there is another factor, the “free-space” of the shell, which has to be
considered.

It was soon realized that when the animal expands, water from the outside will
be sucked into the space, termed here the ‘free-space’, between the animal and its
shell—a space which, when the animal is in a contracted state, is filled with foot
tissue but, when the foot is expanded, forms a partial vacuum and so sucks water in
from outside. This water, of course, is pushed out when the animal tater contracts
and the space is again filled with foot tissue. The size of the ‘free-space’ and
consequently the amount of water taken up into it and given off from it will vary,
for the one specimen, with the degree of expansion. Thus the fact that more fluid,
i.e. total fluid, is given off by a fully expanded specimen than by a semi-expanded
specimen could be attributed to the variation in the size of the “free-space” under these
conditions, and so need not necessarily point to the fact that the fluid given off has
played a part in the expansion of the foot.

The existence of this “free-space” can be quite easily demonstrated—for instance,
if a hole is drilled in the shell and the animal induced to expand and then contract
again, fluid which normally would have escaped via the “mouth” of the shell will be
seen to escape through this hole.

Although it did not seem likely that this “free-space” could account for all the fluid
given off when the animal contracts, it was deemed wise to investigate the matter
by repeating an experiment carried out by Schiemenz, who had also realized the
Significance of this ‘free-space’ (Schiemenz, 1884). The experiment had actually been
devised by Agassiz (Agassiz and Siebold, 1855). The volume of fluid given off by a
fully expanded specimen was measured and compared with the volume of the shell,
the latter being determined by immersion of the empty shell in a graduated cylinder
of water. It was realized that if the volume of the fluid was greater than the volume
of the shell it would mean that some of the fluid must have been given off by the
animal itself, because the “free-space” obviously could not accommodate it all. The
figures for the volume of the shell give a liberal estimation of the size of the “free-
space” because a lot of the space inside is normally occupied by the visceral hump, so
the “free-space” will always be less than the volume of the shell. Schiemenz’s figures
for this experiment (Schiemenz, 1884, table, p. 535) show that the fluid given off was
far in excess of the volume of the shell—as much as two to three times as great in
many cases. Agassiz (Agassiz and Siebold, 1855) himself states that the volume of
fluid given off when Natica heros contracted was two to three times as great as the
shell volume, but he gives no figures to support this. The figures obtained when this
experiment was repeated are given in Table 2.

In most cases the volume of fluid expelled is slightly greater than the volume of
the shell, but it is especially to be noted that in no case is the difference as great as
that observed by Schiemenz, and in some cases it is less than the volume of the shell.

At first it was assumed that the fluid given off was sea-water, either from the
“free-space” of the shell or possibly from ‘“aquiferous” spaces in the foot—if such
spaces existed. However, when the fluid given off by four specimens was examined
it was found to contain blood corpuscles. This pointed to the fact that some of the
fluid given off was blood, which, although it might not be a normal component of the
fluid expelled, but only present when the animal contracts under stress (which will
always be the case when handling an Uber, no matter how much care is taken), must
be taken into account in all volume measurements. Schiemenz had observed the
presence of blood corpuscles in the fluid (Schiemenz, 1884) and had assumed that they
had gained entry into the water cavities (and so to the outside when the animal
contracted) by bursting of blood vessels during rapid contraction. However, he did

76 DILATION OF FOOT OF UBER (POLINICES) STRANGEI,

TABLE 2.
Comparison of Volume of Fluid Expelled by Fully Expanded Specimens
of Uber strangei with the Volume of their Shells.

Presence (p) or
M1. Fluid Volume of Shell. Absence (a) of

Expelled. Mt. Blood Corpuscles.
9-0 8-0 ?
Coe 6-0 ?
7:5 5-0 2
7:0 6°75 2
6:5 4-0 ?
6-0 4-0 p
5-8 3°25 ?
5-525 5-0 ?
5-5 6-0 ?
5-0 6-0 p
3:0 3-0 p
2-5 2-0 p
2-0 20 ?

not mention the possibility that this blood component of the fluid given off, together
with the water from the “free-space” of the shell, and any moisture and mucus adhering
to the animal, could make up the whole of the fluid given off and that possibly no
water was being expelled from the foot itself. This possibility must not be overlooked,
especially as in no case did the present set of observations show that the amount of
fluid expelled was much in excess of the volume of the shell. As regards the surface
moisture and mucus, Schiemenz makes no mention of having wiped the outside of the
animal to remove as much as possible before measuring the fluid given off. (The
surface fluid, especially that lying in the space behind the shell (Text-fig. 3), the floor
of which is formed by the operculum, the roof by the shell itself and the walls by the
metapodial “shell-fold’”, is a very important factor and care was always taken to
remove as much as possible.)

As it seemed at this stage that the fluid given off consisted of three, and possibly
four, parts—(a) “free-space” water, (0) surface water and mucus, (c) blood, (@) water
from ‘“‘aquiferous’” system, if present—it was decided to try removing the shell so
that the ‘free-space’ component (a) of the fluid could be eliminated and the surface
water and mucus (vb) component greatly decreased, as removal of the shell facilitates
removal of surface moisture and mucus. It was realized that if a large quantity of
fluid was still given off, parts (a) and (0) of the total fluid given off when the shell
was still on couid be regarded as unimportant, in which case the relative importance
of parts (c) and (d) would have to be determined. If, on the other hand, no fluid
was given off after removal of the shell, it could be assumed that parts (a@) and (bd)
formed all of the fluid given off when the intact animal contracted. If a little was
still given off it would mean that, whilst (a) and (0) made up most of the fluid, (c)
and (d) also played a part, and their relative impcrtance would have to be ascertained.

The shell was removed by carefully cutting it away with bone forceps until only
the region where the columella muscle is attached remained. If care is taken and the
animals kept for two to three days under good conditions, they recover from this
operation quite successfully. Once the animals had recovered and expanded again,
they were removed in an expanded condition from the aquarium, as much as possible
ot the surface moisture and mucus removed, and the fluid given off collected. In one
case the fluid was examined for blood corpuscles. The results are given in Table 3.

These results show that even when component (a) and the major part of com-
ponent (b) are removed, quite a large quantity of fluid is still given off. In the case
of the one specimen in which the fluid was examined for blood corpuscles, the latter

BY MURIEL C. MORRIS. 77

TABLE 3.

Fluid Expelled by Fully Expanded Specimens of Uber
strangei after Removal of their Sheils.

Presence (p) or

MI. Fluid Absence (a) of

Expelled. Blood Corpuscles.
5-0 | p
4-0 ?
3:0 | ?
1:0 | ?

were found to be present in considerable numbers, which meant that the five ml. of
fluid given off consisted of two, and possibly three, components: (0) surface moisture
and mucus, almost negligible, (c) blood, (d@) water from “aquiferous” system, if present.

The question was whether components (0b) and (c) could together account for all
of the fluid given off when the shell was removed, or whether part of this fluid could
come from a set of “aquiferous” cavities in the foot.

The results of another experiment, carried out on a specimen with shell removed,
are interesting in this connection. The animal was weighed in a contracted state and
then in an expanded state. It was realized that if any increase in weight occurred
during expansion it would be due to, firstly, extra moisture adhering to the enlarged
surface (as the surface area of the foot would naturally increase on expansion) and,
secondly, to water taken up into the foot for expansion (if such a process existed).
The results are given in Table 4.

TABLE 4.
Differences in Weight between Contracted and Fully Expanded Specimens of Uber strangei
after Removal of their Shells.

Ml. Sea-water of

Weight when Weight when MI. Fluid | Density 1-02
Contracted. Expanded. Later Given | Necessary to
Gm. Gm. | Off. Account for

Weight Increase.

6-530 8-930 | 1-0-2-0 | 2-35
6-230 15-280 5-0 | 8-87
6-230 10-270 ? 3-99
5-475 9-445 | ? 3-89
3-646 5-455 | 1-0 eee

These results show that there is a definite increase in weight in each case. The last
column in Table 4 gives the amount of sea-water which would have to adhere to the
surface of the animal if the extra surface water adhering to the expanded foot alone
were to account for the weight increase. It is quite obvious, in the case of the first
four examples, that such a volume of sea-water could not have been adhering to the
surface at the time of weighing, especially as great care was taken to remove as much
as possible before weighing. Part, and probably most, of the weight increase must
have been due to something that was taken up by the animal during. expansion. It is
Significant that the increase in weight in each case is roughly proportional to the
amount of fluid given .off when the animal later contracted.

Although this last experiment indicated that water was taken up by the foot
during expansion, it was realized that obviousiy the animal must be able to expand
to quite a considerable degree without taking up water in order that the foot can
be brought into contact with the water.

~]
io 8

DILATION OF FOOT OF UBER (POLINICES) STRANGEI,

It was thus decided to see if the animal would expand, or at least protrude the
toot through the “mouth” of the shell, on a dry or slightly moist surface. After many
unsuccessful attempts, one animal placed on perfectly dry linoleum did expand its foot
to assume the normal creeping position and to move about. However, the foot tissue,
especially the metapodium, had a flat, thin appearance instead of the rounded oedematous
appearance of a normally expanded foot. The propodium was heavily creased, while the
edges of the metapodium were curled up. When the animal later contracted, it did
so without loss of fluid. Several other specimens behaved similarly when placed on
damp blotting paper, damp sand or slightly moist glass. It might be.argued that the
animals did not expand fully because of the unusual environments of dry linoleum,
damp blotting paper, etc., and not because there was no water present to be taken up
into the “aquiferous” spaces. However, an animal which has expanded in sea-water
and has been placed on either dry linoleum or damp blotting paper will crawl about
and react quite normally.

It would thus appear that if water is used in the expansion of the foot it is
needed only in the final stages of this process.

DISCUSSION.

The experiments described on the measurement of the fluid given off when the
specimens contracted, both with shell intact and with shell removed, together with
the observations on shell volume and those on weight change during expansion,
indicate that water, in this case sea-water, is taken up during the expansion of the foot,
whilst the observations on the expansion of specimens in dry or slightly moist locations
show that this water is used only during the final stages of expansion.

However, in the author’s opinion it is not possible to be as certain about the
matter as Schiemenz was because of the comparatively small volume and weight
differences observed in the first experiments described in this paper compared with
the large volume differences that he observed.- The experiments merely indicate that
Schiemenz’s statement that water is taken up into the foot during expansion is probably
correct and that this uptake occurs to a lesser degree than Schiemenz believed.

It is quite obvious that before a completely accurate picture of the mechanism of
expansion can be obtained a full study of the blood system and the mode of circulation
will have to be undertaken, in order that the relationship between blood system and
“aquiferous” spaces can be fully understood. Schiemenz (1887) did inject the blood
system of several specimens of Natica josephina, and as a result claimed the existence
ot a rather special type of blood system. This consisted of a closed system of arteries,
capillaries and veins in the foot, completely separated from the water cavities and
with none of the sinuses that are usually present in the Molluscan foot. All the
histological elements, such as muscles, glands and nerves, were completely surrounded
by sheaths protecting them from the sea-water taken in during expansion of the foot.
(The water cavities were merely the spaces between the muscles and other tissue
elements of the foot. The sheaths surrounding the muscles and glands enclosed a
blood sinus (Gewebe sinus) which bathed these elements and received its blood from
the capillaries via a sub-epithelial sinus. Because of the comparatively crude histological
techniques in use at the time, his injection pictures, and consequently his whole
theory of the blood system, must be regarded with doubt.

It will also be necessary to demonstrate definitely the existence of pores opening
from the outside into the foot. Although a large series of sections of the foot were
made, the author has not been able to demonstrate the existence of any pores opening
from the outside into the foot. Obviously pores must be present if water is going
to be used in the expansion of the foot. The uptake of water osmotically would be
much too slow a process to account for the rapid expansion of the foot of Uber strangei.
Schiemenz (1884, 1887) claimed to have demonstrated the presence of pores at the
junction of the propodium and the propodial “shell-fold” in Natica josephina, but his
description of the pores was not very convincing—possibly because he found it necessary

BY MURIEL C. MORRIS. 79

to go to great lengths to prove that they were pores and not merely rents in the
epithelium.

Schiemenz admitted (Schiemenz, 1887) that his theory of the actual mechanism of
water-uptake through these pores was weak. It would appear that he propounded his
theory because he was unable to demonstrate the existence of an effective pump system
or to show that the beating of cilia on the ciliated surface of the epithelium of the
foot was sufficient to cause a current of sea-water to pass through the pores into the
“aquiferous” cavities. Obviously there must be some better means of ensuring the
uptake of water into these “aquiferous” cavities than that described by Schiemenz,
but as yet it has not been demonstrated.

As regards the jets of fluid which Schiemenz (1884) described as spraying out from
the foot at the point of junction of the propodium and the propodial ‘‘shell-fold’’,
similar jets have been observed only once or twice during the course of this work,
and Schiemenz himself admitted only having seen them on rare occasions. He was
quite sure that these jets were due to water coming out through the pores rather
forcibly, but each time the author observed them the animal was contracting rather
vigorously, and it seems very probable that they were caused by water or the body
fluids escaping through rents formed in the epithelium during violent contraction. It
is understandable, of course, that if pores are present along the edge of the propodium,
rents are more likely to occur near or around these pores than elsewhere during
violent contraction.

There is one aspect of the question which should be stressed. This is in reference
to the misleading statements on the subject of turgescence in the Molluses which
appeared in the standard text-books at the beginning of the century, and which are
still appearing in almost unaltered form in the modern text-books.

The standard text-books (Lang, 1896; Pelseneer, 1906) side-stepped the issue when
dealing with the phenomenon of turgescence in the Molluscs. The fact that these
early authors were willing to accept the work of others, even on a controversial subject,
is understandable. It was inevitable that, when dealing with such a wide field as the
phylum Mollusca, they would strike new and still controversial subjects which, because
of lack of time, they themselves could not investigate. It is unfortunate, however,
that they did not state the case as it really stood at that time. It perhaps would
have been wiser if they had pointed out that, while the forcing of blood into the
parts concerned could almost certainly account for the phenomenon of turgescence in
the Lamellibranchs, this theory could not be applied unreservedly in the case of the
Gastropods because the situation was complicated by the fact that turgescence in the
Naticidae had recently been shown to be due to the uptake of water into a series of
“aquiferous” cavities.

A survey of recent editions of standard text-books (Borradaile, Potts, Hastham,
Saunders, 1935; Kukenthal, 1925; Parker and Haswell, 1940; Thomson, 1944) reveals
practically the same attitude to the phenomenon of turgescence in the Molluscs as
that held by the authors of text-books at the beginning of the century. Most of them
definitely state that turgescence in Lamellibranchs is due to the forcing of blood into
the parts concerned, but none of them give an explanation of the phenomenon of
turgescence in the Gastropods. A few refer to the “‘aquiferous” system in the Naticidae—
giving no details and without correlating the phenomenon with the state of affairs in
other Molluscs—but even this startling exception is mostly ignored.

As a result of this failure to state the case as it really stands, there has grown
up among all except specialists in the field the idea that turgescence in all Molluscs
has been proved to be due to the forcing of blood into the parts concerned. The state
of affairs in the Naticidae has consequently either been overlooked by research workers
or simply accepted as an exception which does not warrant investigation.

SUMMARY.
(1) The explanation of turgescence and expansion of parts in the Molluscs is briefly
considered. It is pointed out that in one small group of Prosobranch Molluscs, the

80 DILATION OF FOOT OF UBER (POLINICES) STRANGEI,

Naticidae, expansion of the foot is still considered to be due to the uptake of water
into a set of ‘aquiferous” cavities in the foot, whilst in all other Molluses it is
universally accepted nowadays that the forcing of blood into the parts concerned is
the chief factor in’ expansion.

(2) Some general notes on the Naticidae are given..

(3) Observations on the amount of fluid expelled during contraction by specimens,
both with shell intact and with shell removed, together with observations on shell
volume and on weight change during expansion, point to the fact that water is taken
up during expansion.

(4) Observations on the partial expansion of specimens on dry linoleum, moist
sand, ete., show that this uptake only occurs during the latter stages of expansion
and to a lesser degree than was formerly thought.

(5) Two factors leave a doubt in the author’s mind. Firstly, the volume of fluid
expelled by an Uber of a certain size was much less than that observed by Schiemenz
for a Natica of similar size; secondly, the differences between shell volume and volume
of fluid expelled were smaller than those observed by Schiemenz for Natica. Possible
explanations for these variations are given.

(6) Lines for further research are indicated.

(7) The discussion of the phenomenon of turgescence in text-books dealing solely
with the Mollusca, and also in text-books of invertebrate zoology, is considered.

ACKNOWLEDGEMENTS.

I would like to express my sincere thanks to the late Professor W. J. Dakin, formerly of the
Zoology Department, University of Sydney, for his assistance and guidance both in the
carrying out of the work and in its preparation for publication. My thanks also to Professor
P. D. F. Murray, Mr. A. N. Colefax and Mr. R. Endean, of the Zoology Department, and to
Mr. G. Humphrey, of the Biochemistry Department, University of Sydney, for their many
helpful suggestions and criticisms; to the staff of the Fisheries Division of the Commonwealth.
Scientific and Industrial Research Organization at Cronulla for making available their aquarium
facilities.

References.

AGASsiIz, L., StpBoutpD, C. TH., 1855.—Ueber das Wassergefasssystem der Mollusken. JZ. wiss.
Zool., Band 7: 176-180.

BORRADAILE, L., Potts, F., EASTHAM, L., SAUNDERS, J., 1935.—The Invertebrata. Second [dition.
Cambridge University Press, London.

CARRIERE, JUSTUS, 1882.—Die Fussdritisen der Prosobranchier und das Wassergefass-System der
Lamellibranchier und Gastropoden. Archiv. mikr. Anat., Band 21: 387-46.

KUKENTHAL, W., 1925.—Handbuch der Zoologie. Band 5, Erste Leiferung, Seites 44, 45, 87.
Walter de Gruyter und Co., Berlin und Leipsig.

LANG, A., 1896.—Text-book of Comparative Anatomy. Translated by Bernard and Bernard.
Macmillan and Co., London.

PARKER, T. J.. HASWELL, W. A., 1940.—A Text-book of Zoology. Sixth Edition, revised by Otto
Lowenstein. Macmillan and Co., London.

PBLSENEER, P., 1906.—Treatise on Zoology, Pt. 5, Mollusca. Edited by E. Ray Lankester.
Adam and Charles Black, London. ,

SCHIEMENZ, P., 1884.—Uber die Wasseraufnahme bei Lamellibranchiaten und Gastropoden
(einschliesslich der Pteropoden). Mitt. zool. Sta. Neapel, Band 5, Drittes und Viertes
Heft: 509-543.

, 1887.—Uber die Wasseraufnahme bei Lamellibranchiaten und Gastropoden (einschlies-
slich der Pteropoden). Mitt. zool. Sta. Neapel, Band 7, Drittes Heft: 423-472.

THOMSON, J. A., 1944.—Thomson’s Outlines of Zoology. Ninth Edition, revised by James

Ritchie, Oxford University Press, London.

81

REDUVIOIDEA FROM NEW SOUTH WALHS.
NOTES AND DESCRIPTIONS (HEMIPTERA).

By PETR WycopzInsky, Instituto de Medicina Regional, University of
Tucuman, Argentine.
(Communicated by J. W. T. Armstrong.)

(Thirty-four Text-figures. )

[Read 29th March, 1950.]

INTRODUCTION.

It is to be supposed that Australia is one of the regions of the Harth least known
as to its fauna of the Reduvioidea. There is no doubt that the continent possesses
many highly interesting elements, the study of which might shed new light on many
questions of phylogeny and zoogeography.

It was therefore with great pleasure that we accepted for determination the
specimens sent by Mr. J. W. T. Armstrong, of Callubri, Nyngan, New South Wales.
All of these have been collected in New South Wales and several species and even
genera have proved to be new to science. Our sincerest thanks are due to Mr. Armstrong,
whose excellent field work is of great value to Australian entomology.

The present paper, being the second of a series (the first being Wygodzinsky, 1949),
contains locality records and notes for some known species, as well as the description
of two very interesting new genera, one belonging to the Hnicocephalidae, the other to
the reduviid subfamily Emesinae. We are obliged to Professor Dr. R. L. Usinger, of
Berkeley, California, for information regarding the enicocephalid.

Following Mr. Armstrong’s wishes, the types of the new species and certain other
specimens are being deposited in the Australian Museum, Sydney. We thank Mr.
Armstrong for the kind permission to retain some specimens for our own collection.

Dr. I. Mackenzie Lamb very kindly assisted us in linguistic matters, for which
our thanks are due to him.

Family HE NICOCEPHALIDAER.
USINGERIELLA, gen. nov.

Enicocephalinae. Medium-sized, slender species; body surface shining, with sparse
delicate bristles, and small setiferous tubercles at certain regions of the body.

Head elongate, constriction behind eyes not very pronounced, the posterior lobe
subrectangular, somewhat broader posteriorly than in front. Eyes of moderate size.
Ocelli situated laterally at anterior border of posterior lobe of head. Rostrum short
and stout, first joint short, second about twice as long, third short again, delicate in
lateral view. Antennae not quite as long as head and pronotum together, the first joint
shortest, the other three subequal, the last one very faintly spindle-shaped.

Pronotum unarmed, strongly flattened on disc above. Anterior lobe transversely
rectangular, with a fovea on disc on either side, anterior and posterior lobes very
short, the former subtrapezoidal, the latter transversely band-shaped, shallowly but
distinctly emarginate on hind border. Anterior and median lobe with a median
longitudinal impression. Pronotum laterally with 1 + 1 carinae beset with numerous
small setiferous tubercles. Scutellum broad at base, subtriangular, somewhat pointed
posteriorly, its disc flattened, smooth.

Forewings entirely membranous, all veins with slender bristles, which are more
humerous at margins of wing. Venation characterized by its closed discal cell, basal
cell wanting; stigma not perceptible.

G

82 REDUVIOIDEA FROM NEW SOUTH WALES,

Anterior coxal cavities closed behind. Front femora strongly incrassate, with a
small process ventrally near its base. Fore-tibiae strongly dilated. Anterior tarsi
one-segmented, with two strong subequal claws. Mid-legs slender and short; hind-
femora strongly thickened. Mid- and hind-tarsi two-jointed.

Abdomen without special features. Male genital characters unknown.

Genotype: Usingeriella boganensis, sp. n.

We have great pleasure in dedicating this new genus to Professor R. L. Usinger.

It is somewhat difficult to place Usingeriella in one of the tribes of the Enico-
cephalinae adopted by Jeannel (1941), owing chiefly to the impossibility of examining
the genitalia of the male; most probably it belongs to the Systelloderini. By its wing
venation, Usingeriella approaches Nesenicocephalus Usinger, 1938, from the Pacific
region, a genus which was not known to Jeannel (1941). Ours differs from
Nesenicocephalus by numerous characters, such as the much longer head, the very
differently shaped pronotum, the strongly thickened front legs, the posteriorly closed
anterior coxal cavities and the presence of small setiferous tubercles on various body
regions. We are informed by Dr. Usinger (in litt.) that our new genus also seems
to have affinities with the species described by Bergroth (1907) as Gamostolus tonnoiri,
from New Zealand.

Wyegodzinsky del.
Text-figs. 1-11. Usingeriella boganensis, gen. nov., sp. n., female, holotype.

Fig. 1, general aspect. Fig. 2, head, lateral view. Fig. 3, pronotum and scutellum, as
seen in microscopical preparation. Fig. 4, thorax, seen from below. Fig. 5, setiferous tubercles
of pronotum (high magnification). Fig. 6, foreleg. Fig. 7, process at base of fore-femur.
Fig. 8, group of spines at apex of fore-tibia. Fig. 9, group of spines at ventral surface of
fore-tarsus. Fig. 10, hind leg. Fig. 11, veins of forewing.

USINGERIELLA BOGANENSIS, Sp. Nl.
Female.
Length to apex of abdomen 4:5; maximum width of pronotum 0-75 mm.
Colour of head to base of antenniferous tubercles, as well as pronotum, fusco-
testaceous; anterior portion of head, rostrum, antennae, legs, scutellum, abdomen and

BY PETR WYGODZINSKY. 83

membrane testaceous;: fore-tarsi and claws ferrugineous; setiferous tubercles dark
brown. Eyes and ocelli reddish.

Body surface slightly shining, beset with sparse bristles of moderate length.
Setiferous tubercles in small number, present on lateral and ventral surface of posterior
lobe of head, ventral surface of fore-trochanter and femur, prosternum (rather
numerous), lateral regions of pronotum, anterior acetabula, meso- and metapleura,
sides of carina of scutellum and thoracic sterna.

General shape as in Text-fig. 1. Forewings somewhat shorter than extended
abdomen.

Head as in Text-figs. 1-2. Eyes relatively small; their distance dorsally about
three times their width, seen from above. In lateral view the eye does not attain the
dorsal and ventral surface of head. Postocular region of head slightly rounded,
about as wide as long. Rostrum as in Text-fig. 2, curved backwards in life. Length of
first joint of antennae 0-2 mm.; relative length of joints I-IV = 1:2:7:2-5:2-3. All joints
with slender bristles in small number, rather inconspicuous. Last joint slightly
spindle-shaped.

Pronotum as in generic description; shape and relative size of the three lobes
as in Text-figs. 1 and 3. Suture between mid- and hind-lobe with small shallow
excavations. 4+4 apodemes visible in microscopical preparation (Text-fig. 3).

Legs as in generic description and Text-figs. 6-10. Process at base of fore-femur
as in Text-fig. 7. Spines at inner apical angle of fore-tibia as in Text-fig. 8, five rather
slender and two very short ones, one of the latter widened apically; spines of inner
surface of anterior tarsus as in Text-fig. 9.

Venation of forewings as in generic description and Text-figs. 1 and 11.

Type: One female, holotype, in the Australian Museum, Sydney.

Locality: Bogan River, N.S.W.* Armstrong coll.

It is to be hoped that the comparison with additional species of the genus will
allow us later to establish the truly specific characters of the insect in hand.

Family REDUVIIDAE.
Subfamily EMESINAE.
EMPICORIS RUBROMACULATUS (Blackburn, 1889).

Localities: Bogan River, N.S.W., J. W. T. Armstrong coll. (one male, author’s collection) ;
Acacia Plateau, N.S.W., J. W. T. Armstrong coll. (one male, in coll. Armstrong, one female,
Australian Museum).

This is a cosmopolitan species.

ARMSTRONGULA, gen. Nov.

Rather small, delicate species. Body surface smooth, opaque, not hairy. Adults
winged or apterous.

Head very short, rounded behind eyes; ante- and postocular region of about equal
size. Ocelli wanting; transversal furrow between eyes present. Ventral surface of
head with 1+1 rows of numerous long and delicate spines. Rostrum slender, not
elbowed, its joints of subequal length; upper (morphologically ventral) surface of
first joint with several long spine-like bristles.

Antennae inserted dorsally and laterally at anterior third of anteocular portion of
head; antenniferous tubercles rather prominent, stout. -Antennae long and very slender,
second and third joints shorter than first one, the fourth very short.

Pronotum (in winged and apterous form) constricted near hind margin; not
pedunculate. Anterior lobe subovate, convex dorsally, posterior lobe not overlapping
mesonotum except at anterior extremity. Superior lobe of anterior acetabula with a
forwardly and downwardly directed process. Mesonotum subrectangular. Scutellum
simple, subtriangular. Metanotum distinct in winged form, knobbed apically. Hind
border of prosternum slightly emarginate.

* Specimens of this insect were taken singly during the spring on the underside of logs,
mostly on wet, Swampy ground; though recently (November) one was taken in a much
drier situation under a board on ‘red’ ground. J. W. T. Armstrong.

84 REDUVIOIDEA FROM NEW SOUTH WALES,

Fore-coxa slender, dorsal surface basally and ventral surface apically with some
spine-like erect bristles. Femur rather slender, slightly curved at base. Trochanter
and femur with regularly arranged spines of various size; those on femur arranged in
four longitudinal series, all of which commence near base of joint: one antero-ventral
and one postero-ventral series, the former interrupted near base; the postero-ventral
series composed of large spiniferous processes, the antero-ventral series composed of
small spiniferous tubercles and slender spines. Between these two series there are on
the ventral surface two more complete series, composed of smaller spines only. Fore-
tibia over half as long as fore-femur and about as long as coxa, straight, rather stout,
somewhat widened apically, ventrally with two series of rather long spine-like bristles.
Tarsus much shorter than tibia, together with the latter as long as trochanter and
femur together; three-jointed, rather heavily chitinized, ventrally with short simple
setae, some of them spine-like, practically bare above. Two short simple claws present.
Mid- and hind-legs simple, very delicate, the hind-femora surpassing considerably the
apex of the abdomen. Mid- and hind-tarsi three-jointed, the joints of about equal
size, not strongly chitinized, hairy on all surfaces; claws simple.

Venation of forewing of the Ploiaria type, viz., with one large elongate discal cell,
at its base laterally a small elongate submarginal cell. Stigma carried almost to apex
of forewing. :

Abdomen slender, elongate, subfusiform, somewhat widened near middle. Sternites
convex, without median carina.

Genotype: Armstrongula tillyardi, sp. n.

This very pretty new genus, which we have pleasure in naming for its collector,
is nearly related to the African Tinna, specimens of which are before us. Armstrongula
differs from Tinna chiefly by the considerable number of spines on the underside of
the head, the slender spines on the upper surface of the first rostral joint (absent in
Tinna) and the four distinct rows of spines on the ventral surface of the fore-femora.

ARMSTRONGULA TILLYARDI, sp. Nn.
Winged Male.

Length to apex of forewings 7:0-7:5, maximum width of thorax 0-7-0-8 mm.
General colour stramineous. The following regions piceous: head dorsally and laterally
(Text-figs. 12-13), rostrum with all of the first, apical two-thirds of the second and
basal two-thirds of the third segment laterally; all joints of antennae, with exception
of one small whitish annulus at base and apex of first and apex of second joint;
pro- and meso-thorax dorsally and laterally as in Text-figs. 12-13; metanotum (post-
scutellum) with exception of the whitish apex; two large annuli of fore-coxa which
join ventrally; ventral half of fore-trochanter; three large annuli of fore-femur, one
sub-basal, one submedian and one subapical, these much wider than the intervening
whitish spaces; two large annuli of fore-tibia, one sub-basal and one apical; second and
third joint of fore-tarsus; meso- and meta-pleura; lower half of median and posterior
acetabula; a streak on lower half of median and posterior coxa; median and hind-
femur and tibia; ventral surface of abdomen. Ground colour of forewings greyish
stramineous, mottled with brownish (Text-fig. 23); veins of anterior half of wing creamy
white, on posterior half, piceous. The dark pigment sometimes fading to reddish, the
extension of the reddish pigment obviously variable individually.

Surface of head and pronotum minutely wrinkled, of mesonotum very minutely
granulous; at low magnification both appear dull. Pubescence absent. Surface of
abdomen ventrally as on mesonotum, beset sparsely with very short bristles that
become somewhat more numerous towards apex.

Head as in Text-figs. 12-14; ante- and post-ocular region of about equal length.
Transverse constriction between eyes only faintly curved. Eyes large, dorsally about
as wide as interocular space, ventrally somewhat more approximated; in lateral view
the eyes attain ventral and dorsal surface of head. Antenniferous tubercles stout.
Ventral surface of head laterally with 1+1 rows of 7-8 ventrally directed slender spines
inserted at regular intervals (Text-fig. 13, and as in Text-fig. 15). Rostrum shining,

BY PETR WYGODZINSKY. 85

Wygodzinsky del.
Text-figs. 12-34. Armstrongula tillyardi, gen. nov., sp. n.

Fig. 12, head and thorax of male, seen from above. Fig. 12a, scutellum and metanotum
of same. Fig. 13, same, lateral aspect. Fig. 14, same, seen from below. Fig. 15, head of
female, lateral view. Fig. 16, head and thorax of female, seen from above. Fig. 17, foreleg
of male, antero-lateral view.. Fig. 18, detail of fore-femur. Fig. 19, detail of fore-tarsus, one
claw shown only. Fig. 20, detail of hind-femur. Fig. 21, detail of apical portion of hind-tibia.
Fig. 22, hind-pretarsus with one claw shown. Fig. 23, forewing of male. Fig. 24, genital region
of male, seen from above. Fig. 25, same, lateral view. Fig. 26, posterior process of male
hypopygium, with its bristles, high magnification. Fig. 27, clasper. Fig. 28, aedeagus, lateral
view. Fig. 29, genital region of female, seen from above. Fig. 30, same, lateral aspect.
Fig. 31, posterior tergites. Fig. 32, seventh sternite, with lobe of eighth sternite and anterior

gonapophysis. Fig. 33, posterior gonapophysis (high magnification). Fig. 34, tergite V of
female.

86 REDUVIOIDEA FROM NEW SOUTH WALES,

slender, only very faintly curved at base, its joints of about equal length (Text-fig. 13) ;
first joint on upper surface with two rows of four upwardly directed spines each.
Antennae very slender, without distinct pilosity; length of first joint 6-0 mm.; relative
length of joints = 1:0:58:0-47:0-17.

Pronotum distinctly shorter than head, about as wide as long, its sides rounded,
its hind margin emarginate; antero-lateral processes short, truncate apically; slightly
convex above, its posterior two-thirds with a delicate median longitudinal furrow.
Lateral aspect as in Text-fig. 138. Ventral aspect of prothorax as in Text-fig. 16;
apparent posterior border emarginate. Mesonotum dorsally as in Text-fig. 12, longer
than pronotum, its sides subparallel; slightly convex above, its median longitudinal
furrow extending for its whole length; hind margin almost straight, sharply excavate
at centre. Mesonotum laterally with two subparallel carinae. Scutellum and post-
seutellum as in Text-fig. 12a; the former semicircular, its centre elevated longitudinally,
the elevation bifid at anterior margin. Disc of metanotum flattened, its apex tuberculi-
form, somewhat elevated.

Forelegs as in generic description and Text-figs. 17-19. Coxa dorsally at base with
2-3 spines, its posterior half on antero-ventral surface with a series of about eight
spiniform bristles. Trochanter with 2-3 spines, the largest one strongly inclined,
inserted upon a stout ventral process. Femur more or less parallel-sided; postero-ventral
series beginning at base of joint, composed of five stout setiferous processes on which
there are inserted strong spines, the latter being distinctly longer than the diameter of
the joint. Antero-ventral series composed of one basal spine, inserted near base of
joint, and 8-9 additional ones, which are much shorter and more delicate than those
of the other series. The two intermediate series composed each of about 25-30 strong
bristles of variable size, several of them being distinctly lateral of the principal row.
Tibia and tarsus as in generic description and Text-figs. 17 and 19. Median and
posterior legs simple, slender, posterior femur surpassing considerably apex of abdomen;
bristles of tibia and femur stout and short (Text-fig. 20), those of apical portion of
tibia slender and much longer (Text-fig. 21). Length of median femur 6, posterior
femur 8-8, median tibia 9-1 and posterior tibia 10-2 mm. Median and hind-tarsi and
claws as in generic description; pretarsus and claws as in Text-fig. 22.

Forewing attaining apex of abdomen, its venation and pattern as in Text-fig. 23;
large discal cell a little longer than apical vein.

Abdomen elongate, slender, widest at middle. General aspect of genitalia as in
Text-figs. 24-25. Hypopygium beset with rather numerous short bristles, apically with
a very short rounded process (Text-fig. 26). Clasper slightly curved, not strongly
chitinized apically, beset with numerous bristles of moderate length (Text-fig. 27).
Aedeagus elongate, with a rather complex internal structure (Text-fig. 28).

Apterous Female.

Rather similar to male, which makes a detailed description unnecessary.

Length 7:0 mm. Pattern much as in male, dark pigment less extensive; abdominal
sternites stramineous to whitish, darker near sides and hind border. Tergites
stramineous, with an elongate median longitudinal stripe of subtriangular shape that
does not attain the anterior margin of the segment (Text-fig. 34).

Eyes much smaller than in male (Text-fig. 15), their distance dorsally corres-
ponding to a little less than twice their width. In lateral aspect the eyes do not attain
dorsal or ventral surface of head. Meso- and meta-notum as in Text-fig. 76; the former
with a median longitudinal furrow behind, the latter with a median longitudinal
carina which is somewhat widened behind. Wing pads of fore- and hind-wings distinct,
but very small (Text-fig. 16).

Abdominal tergites somewhat longer than wide, slightly emarginate behind, at hind
margin in centre with a small but distinct tubercle which is not included in the dark
stripe mentioned above (Text-fig. 31). General aspect of genital region (difficult to
make out) as in Text-figs. 29-30. Seventh sternite (Text-fig. 32) with 141 sub-
rectangular antero-lateral processes; posterior border indented in centre. Lobes of

BY PETR WYGODZINSKY. 87

eighth sternite very short, rounded. Anterior gonapophyses reduced to small chitinized
rods (Text-fig. 32, A). Median gonapophyses (not figured) very delicate, of the usual
fan-shaped type. Posterior gonapophyses (Text-fig. 33) well developed, subtriangular,
their dise anteriorly with several short and stout bristles, their hind margin with some
large and heavily chitinized spines.

Types: One male, holotype, one female, allotype, Australian Museum; one female, paratype,
in coll. Armstrong; one male, one female, paratypes, author’s collection.
Locality: Bogan River, N.S.W., J W. T. Armstrong coll.

The species is dedicated to the memory of that great Australian entomologist
R. J. Tillyard.

Subfamily HOoLopriLiInak.
PTILOCNEMUS LEMUR Westwood, 1840.

Localities: Bogan River, N.S.W., J. W. T. Armstrong Coll. (one male, author’s collection) ;
Inverell, N.S.W., Armstrong coll. (one female, author’s collection; one male, one female in
coll. Armstrong; one female, Australian Museum).

This pretty species is rather common in Australia. Its biology is not well known
and it would be worth further study.

Subfamily STENOPODINAE.
SASTRAPADA AUSTRALICA Stal, 1874.

Locality: Acacia Plateau, N.S.W., Armstrong coll. (one male, author’s collection).
This specimen corresponds well to Stal’s description, differing only in its slightly

larger size (12:5 mm.).

Subfamily REDUVIINAE.
ARCHILESTIDIUM ORNATULUM Breddin, 1900.

Locality: Acacia Plateau, N.S.W., J. W. T. Armstrong coll. (one male, one female, author’s
collection ; two males in coll. Armstrong; one male, Australian Museum).

This is the genotype; only one additional species has been described (cinnabaricum
China, 1925, from Victoria).

Subfamily HARPACTORINAE.
ENDOCHUS (PNIRSUS) CINCTIPES Stal.
Locality: Inverell, N.S.W., Armstrong coll. (one female, author’s collection).
The species was described from “Australia borealis”. Though we have not seen the
type, we are fairly sure of our determination, owing to the complete agreement of the
specimen before us with the original description.

NYLLIUS Stal, 1859.

Nyllius Stal, 1859, Oefv. K. Vet.-Akad. Foerh., 16 (8): 365.
Nyllius Stal, 1868, Kongl. Sv. Vet.-Akad. Handl., 7 (11): 98.
Nyllius Stal, 1874, Kongl. Sv. Vet.-Akad. Handl., 12 (1): 42.
Orgetorixa China, 1925, Ann. Mag. Nat. Hist., 9 (11): 486.

There is nothing in China’s description that would differentiate his Orgetorixa from
Stal’s Nyllius. It is possible that China’s only species, australicus, is different from
Nyllius asperatus Stal, considering its much larger size. We therefore maintain China’s
species for the time being.

NYLLIUS AUSTRALICUS (China, 1925).

Locality: Acacia Plateau, N.S.W., J. W. T. Armstrong coll. (one female, author’s col-
lection, one female in coll. Armstrong).

SUMMARY.

The author gives distributional and synonymical notes on some Reduvioidea of
New South Wales. In the Enicocephalidae there is described Usingeriella boganensis,
gen. nov., sp. n., apparently related to Nesenicocephalus Usinger, 1939, from the Pacific

REDUVIOIDEA FROM NEW SOUTH WALES,

io.)
~

(Hawaii and the Philippines). In the Reduviidae there is described Armstrongula
tillyardi, gen. nov., sp. n., an emesine related to the African Orthunga Stal. The
harpactorine Orgetoriza China, 1925, is considered a synonym of Nyllius Stal, 1859.
Distributional notes on some other reduviids are given.

References.

JEANNEL, R., 1941.—Les Hénicocephalides. Monographie d’un groupe d’Hémiptéres hémato-
phages. Ann. Soc. Ent. France, 110: 273-368.

UsINGpR, R. L., 1939.—A new genus of Pacific Island Enicocephalidae with new species from
the Hawaiian and Philippine Islands (Hemiptera). Proc. Haw. Ent. Soc., 10 (2) : 267-270.

WyGopzINsky, P., 1949.—Reduvioidea of New South Wales. Description of a remarkable new

genus and species of Emesinae (Reduviidae, Hemiptera). Rev. Brasil. Biol., Rio de Janeiro,
SY) (CD)p & 1995

89

THE HAIR TRACTS IN MARSUPIALS.

PART IV. DIRECTION CHARACTERISTICS OF WHORLS AND MERISTIC REPETITION
OF RADIAL FIELDS.

By W. BoArDMAN.
(Department of Zoology, University of Melbourne.)

(One Text-figure.)

[Read 26th April, 1950.]

INTRODUCTION.

An analysis of tract pattern as exhibited by the collection of marsupial pouch
young on which this investigation is based throws into relief the importance of the
radial field (convergent and divergent) and its whorled variant as a factor in pattern
formation and pattern evolution. The results of this analysis will be discussed
subsequently. In the present contribution a group of generalizations is submitted
relative to some attributes of these fields.

DIRECTION CHARACTERISTICS OF WHORLS.

Both divergent and convergent radial fields often appear as the familiar divergent
(centrifugal) and convergent (centripetal) whorls. The direction of whorling may be
either clockwise or counter-clockwise, using the terms as Schwalbe (1910, 1911) defined
them. Whorling of a radial field may occur invariably at a particular site as with the
genal convergence found in the genus Wallabia, or only infrequently as in the case of
the commonly present inter-ramal median convergence. Median divergent radial fields
in the marsupial groin, but not elsewhere, appear always to be free of whorling;
bilateral pairs of radial fields in the same situation are constantly present in whorled
form. Sometimes whorled and unwhorled conditions may alternate in the species, as
with the occipital divergent whorl of Trichosurus vulpecula. f

The direction of whorling is found to conform to definite rules having applicability
to the species in the case of median unpaired structures but to the order Marsupialia
as a whole for bilateral pairs.

MEDIAN DIVERGENT WHORLS.

Median divergent whorls occur both on the dorsal and ventral surfaces. Dorsal
whorls, mostly situated in the cranial half of the body, are characteristic of the
Macropodinae and Phascolarctidae, and are also found in some of the Phalangerinae.
A ventral whorl at the root of the throat is present in Vombatus hirsutus. The strong,
nearly median whorl in the groin of Onychogalea fraenata (Boardman, 1946, Fig. 39) is
difficult to interpret considering the certainly abnormal arrangement of the surrounding
fields: it is probably morphologically equivalent to the whorl found in the Macropodinae
(except Dendrolagus) on each side behind the knee. '

The Dorsal Surface. A survey of the material indicates that the directional
character, clockwise or counter-clockwise, of dorsal median whorls is constant within
the limits of the species. The series of Trichosurus vulpecula vulpecula and of Phasco-
larctos cinereus, in which the median dorsal whorled system is clockwise and counter-
clockwise respectively, may be quoted as examples. The material available does not
present any exceptional cases, though their occurrence might be anticipated in view of
the statistical studies carried out on the crown whorl of man (Schwarzburg, 1927),

ace

90 HAIR TRACTS IN MARSUPIALS,

where a clockwise condition has been shown to be genetically dominant to the less
frequently occurring counter-clockwise form. The fact that nearly related species of
the same genus (Trichosurus vulpecula and fuliginosus, and Wallabia agilis and bicolor,
for instance) may show opposite directions of rotation supports this possibility.

The Ventral Surface. There is insufficient evidence to contend that direction of
rotation is similarly constant for the species in the case of median whorls on the
ventral surface, since only a single specimen of Vombatidae, Vombatus hirsutus hirsutus,
has been recorded as possessing the medially placed divergent whorled system.
Considering, however, the clear rules that exist for paired whorled systems on both
the dorsal and ventral surfaces (v. infra) it seems unlikely that species constancy for
direction is not applicable equally to both the dorsal and ventral surfaces when the
whorl is single and medial.

Median Longitudinal Pairs of Whorils. Occasionally a median whorl may appear
doubled in a longitudinal direction. The phenomenon has not been observed in
marsupials other than in Trichosurus vulpecula and T. fuliginosus.* In two of the
vulpecula series an additional whorl occurs on the vertex in front of the normal whorl
between the ears; its direction in both specimens is counter-clockwise, that is, opposite
to its partner. In one of the two examples of fuliginosus a median longitudinal pair
is substituted for the single whorl; the additional whorl, in this case apparently the
hinder one, has the same direction of rotation as its partner (see’ Text-figure 1). No
explanation can be offered for this anomaly.

MEDIAN CONVERGENT WHORLS.

Median convergent radial fields frequently show whorling in three situations—on
the crown (as in the Macropodinae), in the inter-ramal position (as in Trichosurus
vulpecula), and at or in the vicinity of the umbilicus (as in Sarcophilus harrisii).
Where two or more specimens of a species show whorling of a given convergent field
only similarity in direction characteristics has been recorded. In Sarcophilus harrisii,
for example, the umbilical convergence is, in a litter of four females, clockwise. While
the evidence is not as ample as in the case of median divergent whorls, a like rule—
species constancy of direction of rotation—would seem to hold.

BILATERALLY PAIRED DIVERGENT WHORLS.

Rules for direction are applicable to bilateral pairs of whorls wherever they occur
on the body. In the Marsupialia separate rules are determined for the dorsal and
ventral surfaces.

The Dorsal Surface. There are only three locations. on the dorsal surface where
bilaterally paired divergent whorled systems develop. Considering these from before
backwards, the first will be the small whorls immediately behind the caudal margin
of the rhinarium that give rise to the well-known rhinal reversal present in some
phalangers and in all except one (Macropus major) of the Macropodidae before me.
The second site is the oeciput and neck region, as in some Macropodinae, where,
however, it is commoner to find the whorled system single and medial (the relationship
between single and double whorled systems in the same location is discussed below).
Finally, paired whorls may be present in the sacral region; Phascolarctos is the only
marsupial recorded as having them. Divergent radial systems are frequent at the
median angle of the eye but they appear not to be subject to whorling. In such
bilateral pairs of whorls the two members have the relationship of mirror images the
one to the other and the whorl on the left side is counter-clockwise. No exceptions to
this rule were observed.

The Ventral Surface. As with dorsal paired systems, bilaterally paired whorls on
the ventral surface have the relationship of mirror images. However, in contra-
distinction to the direction of rotation on the dorsal surface, the left member of a
ventral pair is clockwise when viewed from the ventral aspect. Here also there are

* Compare, however, repetition (which may be simultaneously longitudinal and lateral)
of the dorsal whorl in Phascolarctos cinereus (v. infra).

BY W. BOARDMAN. 91

only three situations for the occurrence of the whorls—on the throat or cranial part
of the thorax (Peramelidae, Tarsipedinae, Vombatidae, Potoroinae), in the axilla or on
the upper arm adjacent (Phalangerinae, Phascolarctidae, Macropodinae), and in the
groin (Thylacininae, Peramelidae, Macropodinae). A single exception has been noted in
the axillary whorls of Onychogalea fraenata, which have the opposite direction of
whorling.

BILATERALLY PAIRED CONVERGENT WHORLS.

Convergent whorls having the relationship of bilateral pairs (mirror images)
are found only infrequently in marsupials. On the dorsal surface they do not occur
elsewhere than in the vicinity of the lateral canthus (some Phalangerinae, some
Macropodinae). On the ventral surface a pair is present one on each flank of
Thylacinus and Thylogale. The present material would seem to point to the same
directional rules holding for both convergent and divergent systems, that is, on the
dorsal surface the left member of a pair is counter-clockwise, on the ventral surface
it is the right member that is counter-clockwise. The only exception is in the whorl
between the eye and ear in Pseudocheirus; on the left side it is clockwise and not
counter-clockwise, as might be expected from a position so near to the mid-dorsal line.

The genal convergent radial field of some Macropodinae appears invariably to be
whorled, the direction of rotation being in accord with the rule for the dorsal surface.
A single specimen of the Trichosurus vulpecula vulpecula series (Boardman, 1946, Fig. 19)
shows this whorl as an abnormality and conforming to the rule for the ventral, not
the dorsal, surface.

THE CASE OF SUPERNUMERARY (ABNORMAL) WHORLS.

Isolated single whorls additional to the normal complement of the species may
occur in the marsupial skin. Such whorls may be convergent or divergent; occasionally
both types are found in a single individual (see, for example, Perameles gunnii (Board-
man, 1946, Fig. 11). Certain individuals seem particularly subject to development of these
supernumerary structures, the majority of examples being recorded from two specimens:
the bandicoot Perameles gunnii already mentioned, and a wallaby, Onychogalea fraenata
(Boardman, 1946, Fig. 39). The direction characteristics of supernumerary whorls
situated to the right or left of the middle line support the validity of the rules enumerated
above, that is, they most frequently conform to the rules for the position occupied as if
they were members of bilateral pairs. Of eleven such whorls only one is exceptional in
this respect.

LIMB WHORLS.

In Phascolarctos cinereus and in two macropods, Thylogale sp. and Onychogalea
fraenata, whorls develop on the hind-limbs. Phascolarctos has only a single whorl
(clockwise on the left, counter-clockwise on the right limb) situated medially just
above the ankle. The two macropods, each represented by only a single specimen,
display a pair of whorls distally on the shank, one placed on its lateral and one on its
medial aspect. Thylogale sp. has the left lateral whorl clockwise, the right counter:
clockwise, the left medial whorl counter-clockwise, the right clockwise. For each
corresponding whorl site in Onychogalea fraenata the opposite direction of whorling
holds. The directional constancy of the whorls in the Phascolarctos series indicates
that direction of rotation of limb whorls is probably species constant. The data provided
by the three species taken together suggest that the direction of whorling on the hind-
limbs is independent of that of whorled structures of the trunk.

Whorls occur medially on the forelimbs in Thylogale sp. and Macropus, major.
Thylogale has the whorl on the forearm, but in Macropus major it lies proximal of
the elbow. In both the direction of whorling conforms to the rule for the ventral
surface of the body—clockwise on the left, counter-clockwise on the right. This is
not coincidence and might in fact be anticipated by a consideration of the homologies
of these particular whorls, concerning which there seems little doubt that in both

HH

92 HAIR TRACTS IN MARSUPIALS. IV,

cases they are to be classified as laterally displaced axillary whorls. Axillary whorls
occur in the Macropodinae except in the genus Dendrolagus and in the two species
having forelimb whorls.

THE RULES ESTABLISHED FOR OTHER MAMMALIAN ORDERS.

Schwalbe (1911) has summarized his findings in the Cercopithecidae and Simiidae.
In these two families of Anthropoidea it appears that on the vertex, belly and nape
whorls are clockwise on the right side of the body, counter-clockwise on the left side;
on the side of the head, the breast, side of the abdomen and on the elbow the opposite
holds. The rules cover both centrifugal and centripetal types of whorls. Voigt (1857)
has enunciated a similar rule for man.

In man attention has further been concentrated on the crown or so-called occipital
whorl, and several studies of its distribution both in the single and bilaterally double
condition have appeared. Gates (1946) has summarized the work in this field. The
contribution of Schwarzburg (1927) on school children in Germany may be taken as
typical of the results obtained. Predominantly the whorl is medial and clockwise;
cases in which it was counter-clockwise were only a quarter as frequently encountered.
Of all cases examined, 7% had the whorled system bilaterally double, the majority
having the left member clockwise and the right counter-clockwise.

Merristic REPETITION OF RADIAL FIELDS.

Bateson (1894) has classified an immense number of cases of serial duplication of
parts which he differentiated from other fluctuations in structure under the term
meristic variation. The term is applicable to a group of cases in the marsupial
material before me in which radial fields are, in a minority of specimens of some
species, duplicated either laterally or longitudinally. The duplication, when it occurs,
is independent of whether the radial field involved is or is not whorled in the single
condition.

DUPLICATION OF DORSAL DIVERGENT FIELDS IN THE DIPROTODONTIA,

A median dorsal whorl is characteristic of the Phascolarctidae and Macropodinae,
and is also found in some of the Phalangerinae. The whorled system is most usually
situated on the nape or occiput, less frequently between the shoulders, and occasionally,
as in the genus Dendrolagus (Rothschild and Dollman, 1936), at the middle of the
back or near the root of the tail. When the whori occurs between or cranial of the
shoulders it is subject to repetition which may be transverse or longitudinal or, more
rarely, in both directions in one and the same specimen.

The case of the koala, Phascolarctos cinereus, illustrates the phenomenon in its
simplest and also in its most complex expression. The hair tracts of ten specimens have
to date been recorded in the literature. Of these, seven agree with the original account
of Wood Jones (1923) in that there is present a single counter-clockwise whorl in the
mid-dorsal line between the attachments of the forelimbs; one specimen (Boardman,
19436) has the dorsal whorled system duplicated so that a bilateral symmetrical pair is
substituted; two specimens (Boardman, 1948a) have instead of the single median whorl
a series of three bilateral pairs, of which the middle pair would appear to correspond
to the single median structure.

A bilaterally doubled condition of the same whorl is apparently common in the
Macropodinae (Osphranter robustus and Wallabia dorsalis, for example). As only a
single specimen is available of the species showing the double condition it cannot
be said whether or no the single and double forms of the whorled system are alternatives
for the species, as in Phascolarctos cinereus. No case can be cited of a macropod with
longitudinal doubling of the whorl. In Macropus major, where two dorsal divergent
centres do in fact occur in the middle line of the back, there seems little doubt, in
view of the distance separating the centres of the two fields, that the hinder of the two
is a new development in the genus.

BY W. BOARDMAN. 93

In the Phalangerinae the dorsal whorl appears not to be subject to lateral twinning
but in two species longitudinal duplication occurs. Thus two of the series of Trichosurus
vulpecula and one of the two examples of 7. fuliginosus have the occipital whorl longi-
tudinally doubled. In TZ. vulpecula the additional whorl has a rotation direction
opposite to that of the partner, but in 7. fuliginosus the two whorls are similarly
orientated (Text-figure 1).

Text-figure 1. Longitudinal doubling of the median dorsal divergent whorl in
Trichosurus fuliginosus. Both whorls have the same direction characteristics.

Except in the case of Trichosurus vulpecula duplication, whether lateral or longi-
tudinal or both simultaneously, produces pairs of whorls which conform to the rules
for direction of rotation on the dorsal aspect of the body (v. supra).

DUPLICATION OF THE GULAR DIVERGENT FIELD IN Vombatus hirsutus.

A mid-ventral whorled field occurs at the root of the neck of Vombatus hirsutus
hirsutus and has the same capacity for longitudinal duplication described above for
dorsal systems in the Phalangerinae. The case for longitudinal doubling rests on a
single specimen of the subspecies (Boardman, 1948a). No comment can be made on
the directional relationship of ventral longitudinal pairs, since in this single specimen
displaying them they are in the form of unwhorled divergent fields.

Bilaterally paired whorls, presumably homologous with this gular whorl, occur in
both subspecies of Vombatus ursinus, but as the ventral surface is known from only
a single specimen in each case it is not possible to state whether or no the doubling
is a variable feature of the species.

INGUINAL DIVERGENT FIELDS IN Thylacinus cynocephalus.

The male of Thylacinus cynocephalus shows a well-developed unwhorled divergent
radial field having the scrotal stalk at its centre. In the female a bilateral pair of
whorled fields occurs (Boardman, 1945); the left member is clockwise, the right
counter-clockwise. This difference can be interpreted in one of two ways: it either
represents an example of meristic repetition or of sex-dimorphism. Sex-dimorphism of
hair tracts is a phenomenon of extremely restricted occurrence and is not Known to be
concerned with whorl duplication. It seems most likely, then, that the condition in the
female Thylacinus is a further case of meristic variation.

EVOLUTIONARY SIGNIFICANCE OF DUPLICATION OF RADIAL FIELDS.
Most of the radial fields, both diverging and converging, and their whorled variants
described for marsupials in this work occur either as a single median system or
duplicated. The alternative conditions may, in the case of certain fields (v. supra), be

94 HAIR TRACTS IN MARSUPIALS. IV,

present among the members of one and the same species. Meristic repetition of this
kind is probably commoner than the cases cited would indicate. In the Macropodinae,
for example, both the single and duplex conditions of the nuchal whorl occur in the
related species of the genus Wallabia, but the material includes only one example of
each species. It is likely that in such related groups examination of series would
provide further cases. It must not be assumed, however, that alternative forms of
radial fields within the species limits are at all general. A survey of the material
points to there being no grounds for doubting that these fields, whether in the
single or doubled condition, are constant in character in the majority of species.

Two facts emerge when the gross distribution within a group (say family or
subfamily to take an arbitrary unit) of single and double versions of the same radial
system is considered. Firstly, for any particular field presenting these two alternatives
there are more species with the single than with the double condition. The inguinal
radial field system in the Peramelidae and the nuchal whorl in the Macropodinae may
be cited as illustrating this point. Further, in the cases of meristic repetition so far
recorded the duplex condition affects only a minority of the members of a species.
Secondly, there is a marked tendency within a group where both conditions exist for
the single condition to have a greater frequency of occurrence in the lower members.
Thus Perameéles gunnii alone among the peramelids examined has regularly a duplex
condition of the inguinal whorled system. Bensley (1903) has stated that this species
is ‘undoubtedly a derivative of nasuta’; it must, then, represent a relatively advanced
stage of evolution within the Peramelidae. The large wallabies (Wallabia spp.)
represent a later phase of evolution than the smaller Macropodinae (Bensley, 1903).
It is in these larger species that the nuchal whorl may appear as a double system.
It is significant, too, that meristic repetition is most commonly and probably only
found at higher evolutionary levels. Thylacinus, Trichosurus and Phascolarctos, for
example (v. supra), all represent terminations of evolutionary trends in modern
marsupials.

Taking these facts together, it would seem highly probable that of the two
expressions of a radial field, single and doubled, the single condition is the more
primitive, and that generally, doubling, whether it be a constant or occasional feature
of a species, coincides with a later stage of marsupial evolution.

SUMMARY.

1. The direction characteristics (clockwise or counter-clockwise) of median divergent
whorls on the dorsal surface are constant for the species. This is probably true also
of median divergent whorls on the ventral surface and of median convergent whorls
on both dorsal and ventral surfaces.

2. Bilaterally paired whorls have the relationship of mirror images the one to
the other. On the dorsal surface the left whorl is counter-clockwise; on the ventral
surface it is clockwise. This appears to be true for both convergent and divergent
systems.

3. Supernumerary whorls conform to the direction rules for their situation as though
they were members of bilateral pairs.

4. The direction of rotation of whorls on he limbs is probably constant for the
species, but is independent of that of whorled structures on the trunk.

5. The phenomenon of meristic repetition of the radial field, frequent in marsupial
material, is discussed. Where duplication occurs the pairs of whorls conform to the
directional rules for the dorsal and ventral surfaces respectively.

6. It is considered on the available evidence that, in general, the single median
radial field represents the primitive condition, the equivalent duplex system being a
later development in pattern evolution.

References.
BATESON, W., 1894.—Materials for the Study of Variation. London.
Benstey, B. A., 1903.—On the Evolution of the Australian Marsupialia. Trans. Linn. Soc.
Lond., 9 (ser, 2) :'83-217.

BY W. BOARDMAN. 95

BOARDMAN, W., 1943a.—On the External Characters of the Pouch Young of Some Australian
Marsupials. Awst. Zool., 10: 138-160.
, 1943b.—The Hair Tracts in Marsupials. Part i. Description of Species. Proc. LINN.
Soc. N.S.W., 68: 95-113.
————, 1945.—-Some Points in the External Morphology of the Pouch Young of the Marsupial
Thylacinus cynocephalus Harris. Ibid., 70: 1-8.
, 1946.—The Hair Tracts in Marsupials. Part ii. Description of Species (continued).
Ibid., 70: 179-202.
GATES, R. R., 1946.—Human Genetics. New York.
JONES, F. Woop, 1923.—The External Characters of Pouch Embryos of Marsupials. No. 5.
Phascolarctus cinereus. Trans. Roy Soc. S. Aust., 47: 129-135.
ROTHSCHILD, LORD, and DOLLMAN, G., 1936.—The Genus Dendrolagus. Trans. Zool. Soc. Lond.,
21: 477-548.
SCHWALBE, G., 1910.—Uber die Richtung der Haare bei den Halbaffen. In Voeltzkow. Reise
in Ostafrika in den Jahren 1903-1905, Bd. iv: 207-265.
, 1911.—Uber die Richtung der Haare bei den Affenembryonen. In Selenka, Studien
liber Entwickelungsgeschichte der Tiere, Bd. v: 205 pp.
SCHWARZBURG, W., 1927.—Statistische Untersuchungen itiber den menschlichen Scheitelwirbel
und seine Vererbung. Zeits. Morph. Anthrop., 26: 195-224.
VoietT, C. A., 1857.—Abhandlung tiber die Richtung der Haare am menschlichen K6rper. Denks.
math.-naturw. Klasse Kaiserl. Akad. Wiss., Wien, 13:50 pp.

A HYBRID EUCALYPTUS.
By L. D. Pryor, M.Sc., Dip. For.
(Plates III and IV.)
[Read 26th April, 1950.]

A good deal of interest has been created from time to time in the possibility of the
occurrence of natural hybrids of Eucalyptus. Ewart (1930) states: “the occurrence of
hybrids between certain species is well established”. Mueller (1882) says: ‘Natural
cross-fertilization of flowers only exceptional and then partial, artificial hybridization
easy.” Further, Mueller (1884) says: ‘“Hybridism does not seem to explain the origin
of these aberrant forms in a genus where cross-fertilization is guarded by a calycine lid.”

Baker and Smith (1902) support this view in the following statements:

“.. . it appears difficult to understand how natural hybridization pertains in the
origin of Eucalyptus species, the essential organs being protected by an operculum, and in
almost every instance pollen grains are found adhering to stigmas before the operculum
falls off.” . . . “Cross-fertilisation in the case of eucalypts is, in our opinion, quite
exceptional. .. .”

In the same connection Maiden (1924) says: “I have tested all the following points
as regards #. vitrea ...and its characters are possessed in about equal proportions by
E. coriacea and E. amygdalina. I think I have produced sufficient evidence to show that
my suggestion as to the hybrid character of H. vitrea is a very reasonable one. That
hybridization occurs in the genus and that there is much evidence of it, I consider to be
absolutely proved.”

The general difficulties associated with the classification of the genus Eucalyptus
have been mentioned in several places by various writers. This has been partly respon-
sible for the suggestion that hybrids are of frequent occurrence on the one hand, and for
the relatively small amount of reliable information which has been published concerning
their existence or otherwise, on the other hand. In making an examination of any
individual plani, it is always necessary to reject the tendency to describe an individual
as of hybrid origin simply because it has characters which are intermediate between those
of other known groups. It is true this is one of the characters which many hybrids may
be expected to possess, but in itself it is only one of the factors to be considered in
establishing the hybrid origin of a given individual. The determination of a specimen
as.a hybrid between two established species may be supported by evidence in the
following way: ,

1. From morphological characters, as described above, its position may be
indicated as intermediate in some degree between the putative parents.

2. Evidence relating to its field occurrence.

3. Evidence of its genetic constitution deduced by breeding or examining progeny
from the individual concerned.

4. The synthesis of a similar type by controlled hybridizing.

There are few, if any, cases for which information on all of these aspects is avail-
able. From Maiden’s comments on #. vitrea, and subsequent examination, it is known
that individuals occur in several areas which present considerable variation and which
have, with some reserve, been referred to EH. vitrea R. T. Baker. There are several
forms which are thus described occurring on the Southern Tablelands, and one of them
has been examined in some detail. This tree has morphological characters which are
intermediate between #. dives Schauer and EF. pauciflora Sieber. Firstly the bark, while
characteristically of the persistent Peppermint kind at the butt, does not extend far into
the larger limbs which become smooth as #. pauciflora. The fruits are intermediate in

BY L. D. PRYOR. 97

size between H. dives and EH. pauciflora and the leaves have venation which is somewhat
more longitudinal than H. dives but less so than #H. pauciflora, while again the texture
of the leaves is somewhat coarser than that of H. dives but not so coriaceous as that of
EH. pauciflora. The peduncle also is shorter than H. pauciflora but longer than H. dives
and the wood, macroscopically, shows the same intermediate position, being lighter in
colour than #. dives but darker than EH. pauciflora, apart from other intermediate
characters. In short, in the major botanical features, specimens are found which are
intermediate in several important morphological characters between these well-established
species.

Trees of this particular kind are found fairly widely spread in many parts of the
Southern Tablelands south of Goulburn and Canberra. Trees are frequently youthful,
possibly around 40 to 50 years old, and they are invariably found in a site which is the
junction between H. pauciflora and H. dives. This distribution is fairly frequent in some
parts of the Tablelands where stony ridges adjoin cold plains. The tree is never found
as a distinct population, but always as an odd specimen among either E. dives or
H. paucifiora. From a specimen such as this, seed was collected. This was open-pollinated
and presumably, as it was from an isolated tree, was either selfed or back-crossed with
one or other of the putative parents, viz., H. dives Schauer and H. pauciflora Sieber.
Progeny secured from this seed has shown considerable segregation of characters and
has resulted, after three years, in the production of young plants (only 16 were
preserved) ranging from forms very close to H. pauciflora to forms close to H. dives.
As the juvenile foliage of H. dives carries broadly elliptical or ovate leaves for an
indefinite number of pairs, and that of H. pauciflora changes after a few pairs (3-5)
to the alternate condition, a very good character is available for estimating degree of
segregation. Characters of venation, stem colour, leaf texture, also correspond with
the other characters apparent in the segregates.

The synthesis of such a hybrid has not yet been achieved experimentally, but the
evidence available suggests strongly that this form described as H. vitrea is, in fact, a
naturally occurring hybrid between H. dives and EH. paucifiora. It may be added that
there is no general difficulty in supposing that such a hybrid could be formed as firstly
the two species are relatively closely related in their morphological characters, the cross
is not a wide one within the genus, they occur naturally side by side in the field, and
they also flower together in early spring. :

The occurrence of isolated specimens only and the fact that many of the trees
are approximately the same age, suggest perhaps the extensive regeneration which
followed heavy land clearing and ringbarking some 50 or 60 years ago, may have
released many seedlings and caused a mass regeneration which would permit the
survival here and there of occasional hybrids, as has been suggested by Brett (1949).
Assuming this hybrid is of the F, generation, the absence of extensive natural popula-
tions of segregates between the apparent parents is simply explained by the fact that
up to the present date there has been practically no regeneration under sheep grazing
conditions since the one which initially followed the first heavy clearing.

The figures (Plates iii and iv) show firstly the intermediate characteristics in leaf
structure and fruits and buds in a naturally occurring tree, compared with the putative
parents, and also the type of variation obtained in the leaves of progeny from one of
these trees.

SUMMARY.

Evidence from field occurrence, morphological affinity and open-pollinated progeny
tests is produced which strongly indicates the natural occurrence of a hybrid between
Eucalyptus paucifiora Sieber and Hucalyptus dives Schauer, in the Australian Capital
Territory and Southern Tablelands of New South Wales. The hybrids have been
previously referred to Hucalyptus vitrea R. T. Baker.

References.

Baker, R. T., and SmirH, H. G., 1902.—A Research on the Eucalypts especially in regard to
their Essential Oils. Government Printer, N.S.W.: 15.

98 A HYBRID EUCALYPTUS,

Brett, R. G., 1949.—Private communication.

Ewart, A. J., 1930.—Flora of Victoria. Government Printer, Victoria: 797.

MUELLER, F. v., 1822, 1884.—Eucalyptographia. Government Printer, Victoria, 8 (1882), 10
(1884).

MAIDEN, J. H., 1924.—A Critical Revision of the genus Eucalyptus. Government Printer, N.S.W.,
6: 167.

EXPLANATION OF PLATES.
PLATE ITI.
Upper specimens show for comparison, mature foliage, fruits, and buds of Hucalyptus
pauciflora Sieber, Eucalyptus vitrea R. T. Baker, Hucalyptus dives Schauer, with characteristic
juvenile foliage of Hucalyptus dives and Hucalyptus pauciflora.

PLATH IV.

The range of juvenile foliage obtained from seedlings raised from a single tree of Eucalyptus
vitrea, arranged from a type approaching Hucalyptus dives, upper left, to a type approaching
Bucalyptus paucifiora, bottom right. Note the change from approximately opposite to markedly
alternate leaves, the very short petiole or practically sessile leaves to markedly petiolate leaves,
the thin stems of the Hucalyptus dives type, to the thick, almost fleshy stems of the type tending
to Hucalyptus pauciflora.

m=

Proc. Linn. Soc. N.S.W., 1950. PLATE I.

Alkaline phosphatase reactions in tissue sections.

Proc. Linn. Soc. N.S.W., 1950. PUATH IT:

aA gg

ees:
WSS Pe

Alkaline phosphatase reactions in tissue sections,

Pie NGUd} 1006,

Proc. Linn. Soc. N.S.W., 1950.

“BISPA-T WOLy PI9S OL) PISCE
BLY P/ITED FT PUE SIA J S22M7IQ SIZEOPIO2S JO SAf/UIAN?

calyptus,

id Ku

A hybr

Proc. Linn. Soc. N.S.W., 1950. AE hve

E. dives, Schauer _ -Evitrea, RI Baker E pauctHora, Sieber

£ dives, Schauer juvetmles — £ paucilor2, Sieber juveniles

A hybrid Eucalyptus.

39

CYTOLOGICAL STUDIES IN THE MYRTACEAE.
III. OYTOLOGY AND PHYLOGENY IN THE CHAMAELAUCOIDEAE.

By S. SmirH-WHITE,
Department of Botany, University of Sydney.

(Plates v and vi; One hundred and one Text-figures. )
[Read 31st May, 1950.]

INTRODUCTION.

It has been shown that the basic chromosome number in the larger subdivisions
of the Myrtaceae—the Leptospermoideae and the Myrtoideae—is eleven (Atchison 1947;
Smith-White 1948), and this number is remarkably constant. The occurrence of
seeondary association, and other conditions, suggested that this basic number is itself
a derived one, probably from an original set of six. The present study was undertaken
with this hypothesis in mind, and in the hope that it might yield evidence with a
bearing on the phylogenetic relationships between the three tribes of the family.

Cytology has been used in a number of cases to indicate the phyletic relationships
within and between groups, and to show the nature of the processes that have controlled
speciation (e.g., Babcock 1942, 1947; Anderson 1937). In a natural group evolutionary
trends may remain discernible, and depending on the nature of.the processes involved,
cytological data may be of considerable value in the interpretation of phylogeny.

TAXONOMY AND DISTRIBUTION OF THE CHAMAELAUCOIDEAE.

The Chamaelaucoideae has usually been associated systematically with the Lepto-
spermoideae, and the two tribes have much in common both in morphology and in
geographical distribution. Both are essentially Australian, but the Chamaelaucoideae
is much more restricted both in its range and in the number and diversity of its
species. Whereas the Leptospermoideae extends to New Zealand, Oceania, and through
Indonesia to South-East Asia, the Chamaelaucoideae is entirely restricted to Australia,
and shows its maximum diversity only in the south-western corner of the continent.

The Chamaelaucoideae is, however, of considerable interest in connection with the
origin of the Australian myrtacecus flora. Andrews (1913) considered the group to be
derived, through the Leptospermoideae, from some ‘generalized’ Hugenia-like or
Myrtus-like tropical mesophytic ancestor, the development of xerophytic characters
being a response to the gradually increasing aridity of the Australian climate. Bews
(1926) supported this opinion, maintaining that the xerophytic habit in angiosperms is
derived from a primitive mesophytic one. On other grounds, including floral morphology
and geographical distribution, the tribe has sometimes been considered the more
primitive. Woolls (1884) expressed the opinion that the typically Australian genera
of the Myrtaceae originated in south-west Western Australia, and quoted Hooker in
support of that opinion. Woolls to some extent foreshadowed the theory put forward
by Vavilov (1926, 1939) that the primary regions of plant groups are characterized by
great botanical diversity. Since the Myrtoideae is absent from Western Australia, and
is centred in tropical America, the distribution of the three tribes would suggest at
least a dual phylogenetic origin of the family from primitive types of the Myrtiflorae,
with the Chamaelaucoideae either giving rise to or derived from the Leptospermoideae
through connecting genera such as Scholtzia.

The number of genera and approximate number of species dn the Chamaelaucoideae
and their distribution as between Hastern and Western Fes are given in Table 1.
This table has been compiled from the Flora Australiensis (Bentham 1866), from the
Floras of the various States by Bailey (1900, 1909), Black (1926), Ewart (1930) and

I

100 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

Maiden (1916), and from various papers by Black, Cheel, Gardner, and White. No
claim is made for any finality or completness in the figures.

MATERIAL AND METHODS.

This paper deals with the cytology of 17 species and taxonomic varieties of the
Chamaelaucoideae, representing seven genera and all three subtribes of the tribe.
A summary of the data presented is given in Table 2, in which the arrangment of
the species is essentially that of Bentham (1866). Most of the species are native to the

TABLE 1.
Distribution of the Genera and Species of the Chamaelaucoideae.

|
Number of Species. |
Genus. Total.
Eastern.* Western.+ Cosmopolitan.

Euchamaelauceae.

Actinodium .. ae Bee — 1 | —_— 1

Darwinia... rf ae 10 30 | — | 40

Homoranthus ae ae 3 = = 3

Rylstonea oe se be 1 = — 1

Verticordia .. 26 26 1(?) 46 = | 47

Pileanthus .. Le ie = 3 | = | 3

Chamaelaucium ys ve — 12 oo 12
Calythriceae.

Calythriz ae soe Ks 12 34 2 48

Lhotzkya vy. af aa 3 7 = 10
Thryptomeneae.

Homalocalyx | 2t — — r 2

Thryptomene 4 26 2t 32

Micromyrtus 5 12 1 18
Total Chamaelaucoideae ot 41 171 5 217

* Includes the northern areas of the Northern Territory.
+ The south-west part of the Continent.
t= Species with a strictly northern distribution.

environs of Sydney, but Chamaelaucium uncinatum has been studied from cultivated
material, and other material of Western Australian species has been forwarded by
Miss Baird and Mr. G. Smith of the University of Western Australia, and by Miss
Eardley of the University of Adelaide. The identification of this material is due to the
collaborators mentioned.

Most of the work recorded was concerned with meiosis in pollén mother cells,
and the observations were made chiefly from temporary acetocarmine crushes. Aqueous
fixation, followed by Feulgen staining gave satisfactory preparations with the smaller
anthers of Darwinia but was less satisfactory with Chamaelaucium. The introduction
of a treatment with 4% ammonium oxalate, as suggested by Hillary (1940) was found
to be an advantage. More detailed observations of meiosis in Darwinia fascicularis and
Chamaelaucium uncinatum were also made on material fixed in Randolph’s CRAF
modification of Navashin’s fluid, embedded, and cut at 14 and 20 microns. The
Feulgen-Light Green method of Semmens and Badhuri (1939) proved useful in the
study of chromosome-nucleolus relationships, although such preparations were found to
fade rapidly. 3

Pollen fertility was determined in the dextrin-sorbitol stain mountant previously
deseribed (Smith-White 1948).

BY S. SMITH-WHITE. 101

Observations were made with a 2 mm. apochromatic objective, an achromatic
condenser of 1:3 n.a., and a 20x compensating eyepiece. Drawings were made with an
Abbe camera lucida, giving a magnification at bench level of x 3900, and have been
reduced to x 2600 in reproduction. Photographs are at the magnifications indicated.

MEIOSIS: GENERAL OUTLINE.
For the present purpose the species Darwinia fascicularis and Chamaelaucium
uncinatum are considered as representative of the 6- and 11-chromosome types within
the tribe.

(i) Darwinia fascicularis Rudge. n = 6.

At the time of meiosis the anthers are minute and the pollen mother cell mass
is usually only 3—4 cells across and little more in length. The cells‘are tightly packed

TABLE 2.
Summary of Cytological Data.
Tribe Chamaelaucoideae.

Seasonal
Dis- No. of Haploid Occurrence
Species. tribution. Localities. Plants Chromosome| of Meiotic
Examined. No. Stages.
Euchamaelauceae.
Actinodium Cunninghamit .. | W. Aus. King George’s 1 (?) 6 Sept.
Sound.
Darwinia citriodora aia =. |) We -Atus: Perth. 1(?) 6 Sept.—Oct.
D. taxifolia ue Be ae |e Atus? Blackheath. 10 6 May-July.
D. biflora a ae so || JR AEE Pymble. 10
Epping. 10 6 May—July.
Ryde. 10
D. grandiflora .. ae oo || dak UIs Berowra. 10
St. Ives. 3 6 June-Aug.
D. intermedia ae ae oa || J3_ ANGE. St. Ives. 10
Loftus. 10 6 Aug.—Dec.
National Park. 5
D. taxifolia v. grandiflora .. | E. Aus. Bulli. 10
Helensburgh. 10 6 April-June.
D. tazifolia f. tetraflora EK. Aus. King’s Tableland. 10 6 July.
D. fascicularis EK. Aus. Pymble. 10 |
St. Ives. 10
National Park. 10 6 Aug.—Dec..
Helensburgh. 10
| Kuring-gai. 10
Verticordia sp... W. Aus. Perth. 1(?) ‘8 Aug.
V. plumosa W. Aus. Perth. 1(?) 8 Aug.
V. farnesiana.. a W. Aus. Adelaide (Cult.). il ia
Chamaelaucium uncinatum W. Aus. Sydney (Cult.). 8 11 May-July..
Calythriceae.
Calythrix tetragona EK. Aus. Pymble. 5
National Park. 10 11 Aug.
C. Fraseri We AMIE Perth. u@) | ifalt Oct.—Nov..
Thryptomeneae.
Thryptomene Mitchelliana .. | E. Aus. Melbourne (Cult.). | 11 Aug.
Micromyrtus microphylla -- | HK. Aus. Wahroonga. 5 | 11 Aug.
|

and do not separate easily (Text-fig. 1). The nuclei measure only 8-10 microns in
diameter (Text-fig. 2), and are of the vesicular type reported by McAulay and
Cruickshank (1937) in Hucalyptus, and described by Manton (1935). There is present
in each only a single nucleolus, 3-4 microns in diameter, and the nucleoplasm is clear
except for a number of minute Feulgen-positive bodies, probably heterochromatic
in nature.

102 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

The early stages of prophase are normal. There is the usual increase of nuclear
size, to a maximum of 17-20 microns at pachytene. At zygotene, pairing of the
heterochromatic granules, which may represent the resting stage prochromosomes, is
often apparent. At pachytene, the paired threads may be distributed uniformly near
the surface of the nucleus (Text-fig. 3), or they may form a more or less compact
mass which may or may not include the nucleolus (Text-figs. 4, 5). It is generally
assumed that synezesis is artefactual, but the impression is gained from the present
material that it may represent some real substage of pachytene. Whatever its signifi-
cance, it provides a useful condition for determining the attachment of chromosome
threads to the nucleolus, as illustrated in Text-fig. 4. Never more than one attached
chromosome (bivalent) thread was observed in D. fascicularis or any other species
of the genus. At pachytene, the nucleolar-associated chromatin body is of relatively
large size (cf. Plate v, fig. 1), and it is usually closely applied to the nucleolus.

Contraction of the bivalents during diplotene-diakinesis is often irregular (Text-
fig. 6). Individual bivalents may possess one, two or occasionally three chiasmata,
but any complete analysis of chiasmata in diplotene nuclei has not been accomplished.
At diakinesis and at metaphase, interstitial chiasmata may occur (Text-fig. 7).

TABLE 3.
Chromosome Arrangement in Darwinia.
Configurations.
| Irregular. Total.
5 (1). 6 (0). |
|
Ze
D. fascicularis— |
1-M 3 | 5 -- 8
| 5 | ) — 5
1-A tie ic ae n| 12 1 a= | 13
2-M Ae prac OR 3 1 | 4
2-A at ab Sat 23 7 — | 30
D. grandiflora— | |
1M by ite SU 53 | 9 2 64
2-—M ee 25 Mia 35 | 5 1 | 41
Total ts a fi 88 | 14 3 | 105
| |
| | |
|
Grand total ii cy 111 21 | 3 135

| | / |
|

Following diakinesis there is a short condition of prometaphase (Text-fig. 8), when the
bivalents lie in a close group in the centre of a clear region of the cytoplasm. It is
considered that this stage provides an opportunity for the development of secondary
association if the necessary chromosome relationships exist. :

At first metaphase (Text-figs. 9, 10, Plate v, fig. 3), pairing is complete. Univalents
and multivalent associations are not found. The bivalents are of approximately uniform
size, although some other species have one bivalent distinctly larger than the others
(vide infra). Both at first and second metaphases secondary association is notably
absent, in contrast with the condition found in the 11l-chromosome genera studied. In
the absence of secondary association the bivalents at first metaphase and the chromo-
somes at second metaphase are evenly spaced in the configurations shown to be stable
by Kuwada (1929) and Maeda and Kato (1929). The most frequent arrangement
consists of five chromosomes in a ring, with one centrally placed (Text-fig. 9), and
a much less frequent arrangement is of all six chromosomes in an evenly spaced ring
(Text-fig. 10, Plate v, fig. 4). The frequency of these arrangements is indicated by
the data in Table 3.

At early first anaphase the chromosomes may be associated by one or two terminal
chiasmata or by interstitial chiasmata (Text-figs. 11-14). In the latter case the

BY S. SMITH-WHITE. 103

chromosomes'may show late disjunction, and this behaviour is apparently responsible
for the lagging and false “bridges” illustrated in Text-figs. 15, 16 and 17. Rarely it
offers the possibility of non-disjunction and unequal partition. The stretching of the
region of a chromosome between the centromere and an interstitial chiasma is often
considerable and the submedian position of the centromere becomes quite evident in
such cases.

Late anaphase (Text-fig. 18) is normal, and telophase is notable only for the
marked clumping of the chromosome groups before the development of the interphase
nuclei (Text-fig. 19). In the interphase nucleus the dispersion of the chromosomes
is not complete and heterochromatic granules remain distinctly larger than in fully
resting nuclei. The nuclei are considerably flattened (Text-fig. 22) and the chromo-
somes tend to lie on the inner surfaces of the nuclei relative to the cell as a whole,
giving the “saucer” stage described by McAulay and Cruickshank in Hucalyptus. With
the smaller number of chromosomes in Darwinia this appearance is less conspicuous.
Never more than one nucleolus per nucleus has been observed at interphase, and this
is usually eccentrically placed and is associated with only one chromosome or
prochromosome.

Interphase passes without conspicuous change into second prophase. At second
metaphase (Text-figs. 23, 24, Plate v, fig. 5) and anaphase (Text-figs. 25, 26) the spindles
may lie in the same plane or at right angles to one another, giving either a quartite or
tetrahedral arrangement of the microspores. These stages again illustrate the absence
of secondary association. Second telophase shows also a clumping of the chromosomes
before the microspore nuclei open out (Text-figs. 27-29). In each microspore nucleus
soon after formation only a single nucleolus is developed, in association with one of
the chromosomes. The nuclei are now similar to the original pollen mother cell nuclei,
but are smaller and have fewer heterochromatic granules (Text-fig. 30).

Cytokinesis does not commence until after the full development of the microspore
nuclei. It is then accomplished by furrowing, a method apparently normal to many
dicotyledons, first described by Farr (1916, 1918).

(ii) Chamaelaucium uncinatum Schau. n = 11.

Except for the difference in chromosome number, and for details dependent on
this, meiosis in Ch. uncinatum follows a similar pattern as in Darwinia. Resting
premeiotic nuclei, ca. 10 microns in diameter, have a larger number of heterochromatic
granules, and four of these may be associated with a single large nucleolus, although
this association has not been consistently evident.

Prophase development is again similar, but at pachytene two bivalent threads may
be attached to the nucleolus (Text-figs. 32, 33; Plate v, fig. 11), one by a larger hetero-
chromatic mass than the other. This attachment is again most readily determined at
synezesis. At diakinesis (Text-fig. 34) two bivalents still show nueleolar association.

Chiasma conditions are similar to those found in Darwinia. At diakinesis and at
first metaphase (Text-figs. 35, 36) there may be one or two terminal chiasmata, or
occasionally interstitial chiasmata in each bivalent. At metaphase, 11 bivalents are
regularly formed, and these are arranged in a compact equatorial plate on the spindle
(Text-figs. 37-42, Plate v, fig. 12). The bivalents are of uniform size, but are distinctly
smaller than in Darwinia. The arrangement of the chromosomes on the plate is
considerably affected by the occurrence of secondary association. In the absence of
secondary attraction, the stable arrangement of 11 chromosomes would be as a ring
of eight, with three centrally placed, as demonstrated by Kuwada (1929), Muto (1929),
Ogawa (1929), and Alam (1936). Alam (1936) and Raghaven (1938) have demonstrated
how the occurrence of secondary association can upset such an arrangement. In
Ch. uncinatum the arrangement 8 (3) is distinct only in those cases showing a minimum
of secondary association (Text-fig. 37). This grouping reaches a maximum of five
pairs (Text-fig. 41). Lesser degrees of association are more common, and sometimes
associations of threes may occur (Text-fig. 42). Table 4 indicates the frequency of
secondary association in Ch. uncinatum. Following Alam and Raghaven, secondary

104 CYTOLOGICAL STUDIES IN THE MYRTACEAE. ITI,

24

Text-figures 1-26. Darwinia fascicularis Rudge. 1. L. sect. of an anther just before
meiosis (x 1300). 2. Pollen mother cell in premeiotic resting stage. Two heterochromatic
bodies are shown associated with the nucleolus. 3. Pachytene. 4 and 5. Pachytene showing
the synezetic knot, when the nucleolar associated chromatin is distinct. 6. Late diplotene,
with irregular condensation of the bivalents. 7. Diakinesis. There is one nucleolus-associated
bivalent. 8. Prometaphase. 9 and 10. 1-M plates showing different configuration. 11-14. Early
anaphase stages. In 12 the chromosomes have been separated in drawing to show the chiasma

BY S. SMITH-WHITE. 105

pairs have been recorded as representing one association each, and triple groups as two
associations, for the purposes of the table.

First anaphase and telophase (Text-fig. 43) closely resemble the corresponding stages
in Darwinia, but interphase shows an important difference. Frequently two nucleoli
may be formed in one or both nuclei of a pollen mother cell, and these may be of
similar or of widely different size (Text-fig. 44, Plate v, fig. 13). The occurrence of
the two nucleoli becomes very conspicuous after the use of Semmens’ and Badhuri’s
Feulgen-Light Green technique, which also demonstrates the association with each
nucleolus of a single heterochromatic granule. Single nuceoli, presumably originating

TABLE 4.
Secondary Association in Chamaelaucium uncinatum.
No. of Secondary
Associations. 0 ib 2 3 4 5 Total.
1-M as ata ae fe 1 6 9 14 11 7 49
2-M a6 Ae eh ue 2 1 2 4 2 0 11
Total .. ae ee 3 7 11 18 13 a 60

by fusion, possess two heterochromatic granules. Two nucleoli also occur in the micro-
spore nuclei following the second devision (Text-fig. 51, Plate v, fig. 15). The high
frequency of only single nucleoli would suggest that fusion occurs soon after the
formation of the nucleoli.

Second prophase (Text-figs. 45, 46), metaphase (Text-figs. 47-49, Plate v, fig. 14),
anaphase and telophase resemble the same stages in Darwinia, except for the occurrence
of secondary association. At second anaphase (Text-fig. 50), occasional laggards may
be seen. Cytokinesis also occurs by furrowing.

CYTOLOGY OF THE GENERA AND SPECIES.
I. Subtribe HUCHAMAELAUCEAE.

Of the seven genera of this subtribe, four, containing over 60 species, are confined
to Western Australia, and two, with five species, are entirely eastern. Darwinia is
the only genus extending throughout the continent, but it is also predominantly
western.

1. Actinodium. n = 6.

A monotypic western genus. Material of A. Cunninghamii Schau. was forwarded by
Miss Baird and was obtained from the King George’s Sound district.

In premeiotic mitoses in sporogenous tissue a diploid number of 12 was determined
(Text-fig. 52). The chromosomes are of uniform size and show no conspicuous features
of morphology apart from submedian centromeres. Failure to observe satellites or
secondary constrictions in the material cannot be accepted as critical.

In meiosis in the pollen mother cells the similarity with Darwinia is very apparent.
The nucleolar-associated heterochromatin is particularly conspicuous at pachytene
(Plate v, fig. 1). From first metaphase to second metaphase (Text-figs. 53, 54, Plate v,
fig. 2) the chromosomes show the same type or arrangement and give no evidence of
secondary association.

relationships. 15-17. Anaphase stages showing late disjunction of a bivalent, giving the appear-
ance of false bridges. 18. 1-A in polar view. 19. 1-T, showing the clumping of the two chromo-
some groups. 20. Interphase. 21 and 22. Second prophase. 22 shows the development of the
“saucer” stage. 23. 2-M, showing the typical chromosome configuration. 24-26. Second anaphase.
All text-figures are at x 2600 unless otherwise stated.

106 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

HZ

558)

“celrth a,
36

BZ

Text-figures 27-51. 27-30. D. fascicularis, showing a series of developmental stages from
second telophase to cytokinesis. 31-36. Chamaelaucium wuncinatum. 31. Pollen mother cell
nucleus prior to meiosis. 32. Pachytene. There are two pairs of nucleolar organizers.
33. Another pachytene nucleus. 34. Diakinesis. Two bivalents are associated with the
nucleolus. 35. 1-M, in polar view. 36. 1-M or early 1-A in side view to show normal chiasma
relationships. Chamaelaucium uncinatum. 37-42. Different 1-M plates showing different degrees
of secondary association. 43. First telophase. 44. Interphase, showing two nucleoli per
nucleus, prophase, showing the “saucer’’ stage. 47-49. 2-M plates showing different degrees

BY S. SMITH-WHITE. 107

The close relationship assumed between Actinodium and Darwinia on morphological
grounds (Cheel 1922) is supported by the limited cytological evidence. Undoubtedly one
is derived from the other, but Actinodium may be a relic type, restricted by reason of
genetic homogeneity (Stebbins 1942) or a new form of recent origin.

2. Darwinia. n = 6.

A genus of about thirty-seven species, divided into two sections. The section
Genetyllis is probably the more primitive and is represented in both eastern and western
floras. All the species dealt with in this paper belong to this section. The section
Schaumannia, entirely western, connects the genus with Verticordia and with the
eastern genus Homoranthus.

D. citriodora Benth. n = 6.

Vicinity of Perth; Miss A. M. Baird, Mr. G. Smith. Cytological conditions are
similar to those described for D. fascicularis (Text-figs. 55-59). There is no evidence
of secondary association, and meiosis is quite regular. At first metaphase the chromo-
somes are of rather larger size, and one bivalent is distinctly larger than the others
of the complement. This large bivalent may be late in disjunction, but no other
abnormalities were seen.

Darwinia taxifolia sensu lata.

D. taxifolia was first described by A. Cunningham, but Bentham (1866) included
as synonymous with it another species, D. intermedia, also described by Cunningham.
Cheel (1922) has shown the two-species to be distinct in both morphological character
and geographical range, and at least deserving of varietal rank. In the author’s
experience the species of D. taxifolia as understood by Bentham is a complex group and
contains a number of forms of more or less doubtful specific rank, viz.:

D. taxifolia A. Cunn., sensu strictu.

D. taxifolia var. biflora Cheel.

D. grandiflora Baker and Smith.

D: taxifolia var. grandiflora Bentham.

D. intermedia A. Cunn. (= D. taxifolia var. intermedia Cheel).
D. taxifolia f. tetraflora.

All these forms appear to possess (1) district geographical ranges and perhaps
also ecological requirements, and (2) constant morphological differences. No natural
hybridization between them can occur and they would appear to constitute separate
genetic systems and to possess the properties of species as accepted by Huxley (1939,
1942), Darlington (1940), Timofeeff-Ressovsky (1940), Muller (1940), Dobzhansky
(1941) and Mayr (1942, 1948).

(a) Darwinia taxifolia A. Cunn., sensu strictu.

According to Cheel (1922) this form has a wide distribution from the Blue Mountains
to Jervis Bay. The material studied, from an isolated population at Blackheath, agreed
essentially with the description given by Cheel, except for a rather higher proportion
of flowers in pairs than in fours. The character of the floral bracts distinguishes the
form sharply from D. biflora.

Except that the chromosomes are slightly smaller, cytological conditions in the form
are normal and typical of the genus. Meiosis did not appear irregular, but in view of
the pollen condition found, a more careful examination is necessary. In ten plants
selected at random from the population, so little pollen was produced that satisfactory
counts could not be made, but the pollen grains seen appeared small and deficient.
It is not possible to decide at the present stage whether this lack of fertility is
cytologically, genetically or physiologically determined. The isolation of the population,

of secondary association. 50. 2-A, showing laggard chromosomes in each spindle. It is not
clear whether these are due to misdivision at first anaphase. 51. Newly formed microspore
nuclei in a pollen mother cell. Some show two nucleoli, but in two these have already fused.
All text-figures are at x 2600 unless otherwise Stated.

108 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

allowing no opportunity for hybridization, did not give any expectation of the pollen
sterility found.
(b) D. taxifolia var. biflora Cheel. n = 6.

Populations of this form from localities extending from Ryde to Pymble were
examined. The species was found in association with D. fascicularis, but not with
any other forms of the D. taxifolia group.

The meiotic divisions show six bivalents of small but even size (Text-fig. 60), and
cytological behaviour appears typical of the genus. In some plants univalents may
occur with a frequency of from 1% to 5% of the pollen mother cells, and microcytes
may be formed with a similar frequency at the tetrad stage. In the population examined
for pollen fertility a high figure was obtained (Table 7), but it is probable that other
populations are more variable.

(c) D. grandiflora Baker and Smith. n= 6.

A tall shrubby species quite distinct from the var. grandiflora of Bentham, and
with a distinct range, from St. Ives northwards to Hawkesbury River. Material from
St. Ives and Berowra was examined.

Meiosis in the species is normal (Text-figs. 61-64, Plate v, figs. 6, 7). Harly
separation of “rod” bivalents with single terminal chiasmata may give a false
impression of univalents, but true univalents are at least rare. The type of chromosome
arrangement at metaphase, as in D. fascicularis, suggests a lack of any secondary
attractions (Table 3).

Pollen fertility in the Berowra material was found to be high (Table 7), except for
one plant showing complete sterility suggestive of physiological rather than meiotic
breakdown (Plate vi, figs. 4, 5).

(d) D. taxifolia var. grandiflora Bentham. n = 6.

A prostrate stoloniferous form most closely related to D. intermedia, but readily
recognized by its unusual habit and the larger size of its leaves, floral parts and
pollen. It occurs at moderate altitudes from Helensburgh southwards to Buili and

TABLE 5.
Tetrad Formation in Darwinia taxifolia var. grandiflora.
|
No. of Microspores |
per Tetrad. | 2; 3 4 5 6 Total.

Plant No. 1 Aes =| 1 2 420 27 8 458

Plant No. 2 Be SN eA) 1 196 5 1 203
|
|

Total 3 Re east 3 616 32 9 | 661

West Dapto. Two populations, from Helensburgh and Bulli, have been studied. In
the former locality considerable variation in pollen fertility was found (Table 7, Plate vi,
fig. 7) and this variation may be correlated with the fact that the species not only
co-exists with D. fascicularis, but also contacts, but does not overlap, the distribution of
its close relative D. intermedia. In the Bulli locality pollen fertility is higher and
meiosis is regular (Text-figs. 65-67). Table 5 shows the degree of regularity in tetrad
formation in two plants from this locality, and the extent of pollen fertility is given in
Table 7. In several plants “giant” pollen grains (Plate vi, fig. 6) occur with a frequency
of as much as 1%, suggesting the occasional failure of meiosis.

(e) D. intermedia A. Cunn. n = 6.

A prostrate but not stoloniferous form with a fairly wide distribution extending
from Helensburgh northwards to Kuringai. Material has been studied from several
localities, chiefly at Loftus and at St. Ives. Material from Loftus showed reasonably

BY S. SMITH-WHITE. 109

regular meiosis and a consequent high pollen fertility (Table 7), but the St. Ives
population showed a very considerable variability in pollen. fertility and an unusual
frequency of abnormalities at meiosis. In this latter material the somatic chromosome
number is 12 (Text-fig. 68), but owing to the considerable frequency of univalents at
1-—M, and of divided univalents at 1-A and 2-M (Text-figs. 69-74) decisive meiotic
counts were not easy to obtain.

In some plants of this material up to eight univalents per nucleus are found at
1—M, and in one case the mean frequency per nucleus was 5:4. Such univalents show
the usual failure of congression to the metaphase spindle (Text-figs. 72, 73) and
misdivision in the equatorial region of the spindle at 1—A (Text-fig. 74, Plate v, fig. 8).
Apart from their abnormal behaviour, the univalents are not easily distinguished
from bivalents, seen in polar view.

Neither bridges, multiple association nor other evidences of structural chromosomal
changes were observed in any plants of this population, and although their presence
cannot be excluded, it is possible that the partial failure of pairing is due to a
reduction in chiasma frequency caused either by structural or genotypic factors. The
formation of restitution nuclei has not been observed, but its occurrence is suggested
by the presence of occasional “giant” pollen grains in some pollen samples.

TABLE 6.
Tetrad Formation in Darwinia intermedia. A plant showing 48-9% bad pollen.

No. of Microspores Unequal Equal
per Tetrad. 2 3 4 4 5 6 eae Total.
| |
es | —3
No. of tetrads observed .. ft 0 0 21 91 28 8 1 149
Total No. of microspores included 0 0 84 364 140 48 7 643

Bad pollen found in pollen counts be ah Ste Se -. 48:9%
Bad pollen expected from tetrad data (see text) a ee -- 43°-4%
V=1 23 with 1 D.F. 5% level of x7=3-54.

At second metaphase unequal plates are frequent, and these may contain half-
chromosomes and whole chromosomes. Text-figure 75 shows a late second prophase cell
in which one half-chromosome has been lost in the cytoplasm. In the left-hand nucleus
there are four whole chromosomes and five half-chromosomes, and in the nucleus on
the right there are three whole chromosomes and four half-chromosomes. This con-
dition could arise from a first metaphase with three bivalents and six univalents,
followed by 1—A in which one univalent has passed without division to the left and
the other five have divided at late 1—A, with one of the half-chromosomes failing to
reach the right-hand group. Chromosomes lost in the cytoplasm may degenerate or
may be organized into microcytes at the end of the first or second divisions.

Thus telophasic groups are formed which are grossly deficient or unbalanced. As
a result of these irregularities, the number of microspores per tetrad is variable and
the microspores are of variable size. In Table 6 the frequency of tetrads with different
numbers of microspores is given for a plant showing 48:9% of sterile pollen. On the
assumption that viable pollen will be produced only from tetrads with four visually
equal microspores, a ratio of 279 non-viable to 364 viable pollen grains would be
expected. Analysis shows this proportion to be in satisfactory agreement with the
pollen viability actually found. Although it is obvious that some microspores from
unequal tetrads may give viable pollen grains, the agreement is considered sufficiently
close to indicate that meiotic irregularities could account for the pollen sterility found.

The considerable differences in pollen fertility found, not only between plants
but also between populations, in this and other species of Darwinia and also within
species of Callistemon and other genera of the Myrtaceae (Smith-White 1948) show that
the inferences drawn by Lawson (1930) must be treated with reserve.

110 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

bee
se
@
(Hin

Text-figures 52-78. 52-54. Actinodiwm Cunninghamii. Mitotic metaphase in young anther

tissue. 53. 1-A. 54. 2-M. Configurations are similar to those in Darwinia. 55-59. Darwinia
citriodora. 55. 1-M. 56

and 57. The same division, in 57 the chromosomes being separated in

drawing. 58. 1-A. The larger bivalent is late in disjunction. 59. 2-M.

In all cases the larger
size of one chromosome is evident and secondary association is absent. 60. D. biflora, 1-M.

The bivalents are slightly smaller than in other species. 61-64. D. grandiflora B. & S. 61. 1-M.
62 and 63, early 1-A stages in side view. Early disjunction of some bivalents, as in 63, may
give a false impression of the presence of univalents. 64. 2-M. 65-67. D. taxifolia var.
grandiflora Benth. 65. 1-M. 66 and 67. Early and late 1-A stages. 68-75. D. intermedia.

BY S. SMITH-WHITE. 111

(f) D. taxifolia f. tetraflora. n = 6.

This distinct form, or perhaps ectotype, is found in a windswept situation on
King’s Tableland, at the extreme western limit and greatest altitude for the species.
It is a prostrate shrub which possesses foliage resembling D. fascicularis, but in some
floral characters it approaches D. intermedia. Its characters are suggestive of a perhaps
remote hybrid origin. The meiotic divisions, however, are quite regular, and pollen
tertility is high (Text-fig. 77; Plate vi, fig. 2; Table 7).

Darwinia fascicularis Rudge. n = 6.

This species, which is the dominant one on the sandstone soils of the central coast
of New South Wales, is relatively constant in morphological characters, but shows
variability in characters such as pigmentation of the leaves and young shoots, in the
essential oil in the leaves and to some extent in habit. Haviland (1884) and Brewster
(1915) have shown that the species is dependent on small birds for pollination and
that self-pollination is almost entirely precluded. This latter statement probably also
applies to the other members of the genus studied and might account for the normally
low seed fertility of all species even when pollen fertility is high.

Plants from a number of localities have been examined for meiotic irregularities
and for the presence of pollen sterility (Table 7). Considerable variability in pollen
fertility between plants and between populations occurs, and in some cases the character
of the non-viable pollen suggests that it is caused by meiotic irregularities, whilst
in others it appears to be physiologically or genetically controlled. In no cases,
however, have meiotic irregularities comparable with those in D. intermedia been
found.

The pollen grain mitosis has generally proved very difficult to find in the Myrtaceae.
Text-figure 78 shows a metaphase of this division. The chromosomes are of similar
length, ca. 1:5—2:0 microns, and have clearly marked median or submedian centromeres.
One chromosome carries an indistinct secondary constriction which may represent
the nucleolar organizer.

Pollen Fertility in Darwinia.

The data presented in Table 7 indicate that most, if not all, species of Darwinia
examined show very considerable variability in pollen fertility. Since the work of
Rosenberg on Drosera (1927) it has been usual to accept the occurrence of pollen sterility
as indicative of interspecific hybridization (Allen 1929), and Lawson (1930) has made
inferences on this basis concerning the importance of hybridization in the origin of
the Australian flora. The present data show that in the Myrtaceae such inferences must
be treated with reserve, at least until more is known of the breeding systems of the
species concerned.

The distributions of the eastern Australian species of Darwinia show a number of
peculiarities. All species tend to be divided into comparatively small communities,
perhaps on the basis of ecological conditions. The degree of isolation between such
populations seems to be of all degrees, from partial and slight to what might
reasonably be considered absolute, and between the “species” of the D. taxifolia group
this isolation has a geographical and probably a genetic basis. It would appear from
casual inspection that the size of interbreeding populations is frequently small,
sometimes very small, comprising in some cases the plants over an area of a few
square chains, and that gene-migration between such populations may be very slow.

68. Mitotic metaphase in young anther tissue. 69 and 70. 1-M plates in polar view. Univalents
are in outline. 71 and 72. Marly 1-A stages in*side view. The univalents congress to the
plate either erratically or not at all. 73. 1-A, showing eight univalents, the maximum number
seen. 74. 1-A. Univalents left in the equator of the spindle divide into their chromatids.
75. Second prophase. Hach nucleus contains a different number of whole and half-chromosomes,
and one half-chromosome (stippled) has been lost in the cytoplasm. See text for further
explanation. 76. D. tavifolia Cunn. 1-M. 77. D. tawifolia f. tetraflora. 1-M. 78. D. fascicularis.
Mitotic metaphase of the pollen grain division. One chromosome carries a poorly defined
secondary constriction. All text-figures are at x 2600 unless otherwise stated.

112 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

Wright (1940) has shown that such a condition within a species may lead to
diversity by the operation of random selection, and even to the fixation of dis-
advantageous characters. It has been proved that extensive species usually contain
many gene mutations and chromosomal changes, which may attain different frequency
concentrations in different parts of the species range, and Huxley (1942) has termed
such changes in gene concentration “clines”. The existence of clines and of discontinuous
changes in gene frequencies has been adequately established in Drosophila (Spencer
1947).

In the species of Darwinia it is suggested that the dissection of the total species
population into small more or less isolated breeding groups has permitted the establish-
ment of discontinuous changes in the frequency of structural changes involving small
chromosome segments, and of different gene mutations, some of which might affect
chiasma frequencies, pollen fertility, and similar characters. Perhaps gene migration
between breeding groups is sufficient to prevent the final attainment of equilibrium.
Under such conditions pollen sterility cannot be accepted as due to any recent or
extensive interspecific hybridization.

TABLE 7.
Pollen Fertility in Darwinia spp.
Mean Percentage Fertile Pollen for Plants and Populations. Means of Four Determinations per Plant.

| | | | | |
| | | Popu-
Species. Population. | 1 2 3 4 5 6 u Sarin | 10 (lation; F IP
| | Means
al |
| |
| |
D. biflora .. .. | Epping. 99-5/98-2|96-8)98-5/97-7|98-5/98-11/98-3 96-7/95-8 98-0 | 1-38 DS.
| | laa =
grandifloraB.&S. | Berowra. | — 196-5/97-7/97-1)97-7/97-8 92-3)94-9 95-2/93- 4! 96-0 | 2-41 |<0-05
| | ls
—-— a
Vv. grandiflora | | | |
Benth... .. | Bulli. 86-0,94-9197-9/94-3)/96-3)92-8 93-9|96-7/98-3/68-3) 93-3 |15-06 |<0-001
| | |
| |
IDGY ce .. | Helensburgh. |49-1)78-5)/26-5)93-7|/86-6)45-0)98-1 91-7 88°1/75-5) 75-4 | 9-88 |<0-001
pee tees beeen) | | |
intermedia | St. Ives. 84:0/48-2/55-2/80-0/82-4/33-5)44-2/53-8 67-9/50 9 59-7 |12-93 |<0-001
| eee |
| |
Dorey .. | Loftus. 86-0)95-7/91-4)92-1)93-8|83-6 88-1/82-8 91-7/96-0, 90°6 | 1-13 ILS.
| | | |
| i | | | | | |
taxifolia tetraflora | King’s Table- | | |
land. 197-6/98-5|78-0|81-9|73-9)/65-2/92-2'98-6|/95-8/99-0,; 91-0 | 3-26 | <0-01
| | | | | a
| | |
fascicularis .. | Kuring-gai. '84-2/97-5]95-6|76-6/94-4/84-3/74-9/52-0/92-6 —| 85-7 | 8-37 |<0-001
| | | | | |
Do: Gs: .. | Helensburgh. | —1|91-4|63-5|98-3/99-8 96-8 —* |99-6/99-6 —1} 95-6 |79-72 |<0-001
! : | | | ee ee
Dove .. | National Park.|83-7/99-9)93-3)65-2/53-1)63-2)17-2)/73-5)91-0) —*| 74-9 |71-44 |<0-001
| | :

1 Data for plants eliminated owing to their very low fertility, close to 0%, of a different type and not suggesting
meiotic breakdown.

2 All means and the analysis of variance have been obtained in terms of the angular transformation of the data.

3 Test for variance between populations. F'y,.5,=15:74. P<0-001.

3. Verticordia.

The two sections of this genus are entirely western in distribution. All the
material examined belongs to the section Huverticordia, which connects closely with
Darwinia.

Verticordia sp. Collected and forwarded by Miss Baird. n = 8.

At 1—M there are regularly eight bivalents on the plate (Text-figs. 82, 83, Plate v,
fig. 9). First anaphase and second metaphase are usually normal (Text-figs. 84, 85),
but rarely true chromosome bridges are formed (Text-fig. 86), indicating that the
plant examined was heterozygous for a small inversion. Tetrad formation is regular.

BY S. SMITH-WHITE. 113

Verticordia plumosa Druce. n = 8.

Material identified and forwarded by Miss A. M. Baird and Mr. G. Smith, Perth.
Very few meiotic stages were obtained. The haploid number determined at 1-—M
(Text-fig. 87) and 1—A was in agreement with the previous material.

Verticordia farnesiana (?). n=11.

Material forwarded by Miss Hardley, of Adelaide, from cultivated plants. The
specific name is given as Synonymous with V. plumosa. Meiosis in the material was
regular, with a haploid number of 11, determined at 1-M and 2-M. At both stages
one chromosome or bivalent is of relatively large size (Text-fig. 88).

The disagreement in chromosome number found in the material available suggests
that Verticordia may occupy a very interesting evolutionary position in the
Chamaelaucoideae, but verification and detailed study of the genus are required before
this position can be established.

4. Chamaelaucium.
Ch. uncinatum Schau. n = 11.

Details of meiosis in this species have already been described. Numerous cultivated
plants and some material of a red-flowered variety forwarded by Miss Baird have been
examined without detection of either meiotic irregularities or of any considerable
pollen sterility. One young seedling purchased from a Gordon nurseryman, however,
showed very considerable pollen sterility (Plate vi, fig. 9). Unfortunately this plant
failed to survive and its meiotic behaviour was not studied.

II. Subtribe CALYTHRICEAE.

The two genera in this subtribe are chiefly western. In Calythriz, the larger
~f the genera, the widespread C. tetragona is polymorphic and might well be divided
into several species.

Calythria tetragona Labill. n = 11.

Material examined from several localities was found to be uniform in cytological
constitution and behaviour. At diakinesis (Text-fig. 89) eleven bivalents are formed,
and one at least is associated with the nucleolus. At 1—M, 1—A and 2-M (Text-figs.
90-94, Plate v, figs. 16, 17) one of the bivalents is of relatively large size, and this large
bivalent may be late in disjunction at anaphase. When first observed, this large bivalent
was tentatively assumed to indicate fusion, but the presence of large bivalents in
Darwinia citriodora and in Verticordia removes its significance in this respect.

Secondary association in the species is very marked (Text-figs. 90, 94), reaching a
maximum of five secondary pairs at both 1-M and 2—M. In side view of 1—M, the large
bivalent is seen to remain unassociated. Rare abnormalities occur in this, as in most
other species of the Chamaelaucoideae. Text-figure 95 illustrates a first telophase with
a persistent bridge and two dividing univalents in the mid-region of the spindle. The
frequency of such conditions is too low to cause any serious pollen sterility. Plate v,
figures 18 and 19, illustrates the failure of unpaired chromosomes to congress to the
1-M plate, and the misdivision of univalents at 1—A.

Calythriz Fraseri A. Cunn. n = 11.
Material from Miss Baird, Perth.

The count at 1-M (Text-fig. 96) confirms that made in C. tetragona. The size
difference of one bivalent of the complement seems less marked.

Ill. Subtribe THRYPTOMENEAE.
A subtribe of phylogenetic interest, since it contains genera such as Scholtzia
which connect the Chamaelaucoideae with the Leptospermoideae.

Thryptomene.
Only one species has been examined.

114 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

Text-figures 79-101. 79-81. D. intermedia. 79. Tetrads with abnormal numbers of micro-
spores (x 650). 80 and 81. Good and bad pollen grains (x ca. 650). 82-86. Verticordia sp.
82. 1-M. 88. 1-M or early 1-A in side view. The bivalents have been separated in drawing.
84. 1-A. 85. 2-M. 86. 2-A, showing an inversion bridge. The fragment could not be recognized.
87. V. plumosa. 1-M. 88. V. farnesiana. 2-M. One of the 11 chromosomes is distinctly larger
than the others. 89-95. Calythrix tetragona. 89. Diakinesis. 90. 1-M, polar view. 91. 1-M
in side view. 92. 1-A. In figures 90-92 the large bivalent is easily recognized and is often

BY S. SMITH-WHITE. 115

Th. Mitchelliana F.v.M. n = 11.

The 1—M plate consists of eleven bivalents (Text-fig. 97). Secondary association
is marked and reaches a maximum of five groups in approximately 10% of the pollen
mother cells.

Micromyrtus.
This genus is closely allied to the preceding and is the only one represented in
eastern New South Wales.

M. microphylla Benth. n = 11.

In the pollen mother cells of this species the meiotic process is closely similar to
that in Chamaelaucium. A particularly good example of the association of two bivalent
chromosomes with the nucleolus at zygotene-pachytene was found (Text-fig. 98), in
which zygotene association of the chromosomes had not been completed, so that one
bivalent appeared attached by two separate organizers. At 1—M and 2—-M (Text-figs. 99,
101, Plate v, fig. 20) eleven bivalents are formed and show considerable secondary
association. In side views of 1—M (Text-fig. 100) the bivalents are seen to be associated
chiefly by terminal chiasmata and the secondary association of similar bivalents is
also apparent.

DISCUSSION.
(a) Chromosome Arrangement and Secondary Association.

Kuwada (1929) and his associates have discussed in detail the characteristics of
chromosome arrangement on the metaphase plate at meiosis and have shown that stable
configurations conform with those to be expected on the assumption that the determining
factor is the repulsion between the chromosomes, acting in the limited space of the
spindle. With equal repulsions the stable configuration will provide equal and maximum
spacing.

Although secondary association was first observed by Kuwada in 1910, its signifi-
cance was not appreciated until much later. Darlington (1928), Darlington and Moffett
(1930), and Moffett (1931) drew attention to its significance as indicating ancestral
homologies between the chromosomes concerned and made use of its occurrence to
demonstrate the secondarily balanced nature of the Pomoideae. Lawrence (1931a,
19316) has demonstrated its significance in Dahlia, and Catcheside (1934) has shown
by a consideration of secondary association that secondarily balanced polyploidy occurs
in Brassica. Since that time the occurrence of such secondary association has been
used by many investigators for the inference of polyploidy or secondary polyploidy.

Secondary association is a variable phenomenon, and the maximum rather than
the minimum degree occurring is of significance. It is likely to be most pronounced
when the chromosomes are small and when, as in Darwinia, there is a marked prometa-
phase prior to the development of the spindle. Under such conditions the absence of
secondary grouping of the chromosomes, and close conformity with the theoretically
stable types of configuration may be accepted as evidence of lack of ancestral homology
between the chromosomes. In all the species of Darwinia and in the related Actinodium,
where the haploid number is 6, the frequency of the theoretically stable configura-
tion 5 (1) (ef. Table 3) provides this evidence. In the 1l-chromosome general
Chamaelaucium, Calythrix, Thryptomene and Micromyrtus the theoretically stable
configuration 8 (3) is rare, and maximum secondary association is given with the
formation of five pairs, leaving six independent groups of one or two chromosomes. A
similar condition has’ been reported in the Leptospermoideae (Smith-White 1948) and

late in disjunction. 93 and 94. 2-M, showing the large chromosome and different degrees of
secondary association. 95. 1-A, showing a persistent chromosome bridge and laggard chromo-
somes which are dividing on the spindle. 96. Calythrix Frasert. 1-M. 97. Thryptomene
Mitchelliana. 1-M. 98-101. Micromyrtus microphylla. 98. Zygotene-pachytene, showing two
nucleolar-associated bivalent chromosomes. 99. 1-M. 100. 1-M in side view. Chiasmata are
terminalized. 101. 2-M. Secondary association is evident. All text-figures are at x 2600 unless
otherwise stated. fi

J

116 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

there can be little doubt that the basic chromosome set for both tribes is six, the
number now found in Darwinia and its allied genera.

(b) Nucleoli and Attached Chromosomes.

The occurrence of several nucleoli per nucleus is not infrequent, and DeMol in
1926 was perhaps the first to suggest a possible relation between nucleolar number
and polyploidy. Since the discovery of satellited chromosomes by Navashin in 1912
their function as nucleolar organizers has also been established. Thus there is a
close correspondence between the number of nucleolar chromosomes and the number
of nucloli formed at telophase. Gates (1942) has reviewed this relationship between
nucleoli and nucleolar chromosomes and their bearing on the inference of polyploidy.
Although the number of nucleolar chromosomes and nucleoli may be increased either
by polysomy or by translocation and reduplication of chromosome segments, Gates is
of the opinion that truly basic diploids have only one pair of nucleolar chromosomes
and that when the evidence of the number of nucleolar-associated chromosomes at
prophase, and of the number of nucleoli formed at telophase supports the evidence of
secondary association, the inference of secondary polyploidy is justified.

In the six-chromosome genera Darwinia and Actinodium only a single nucleolus
has been observed in premeiotic pollen mother cell nuclei, and this has been presumably
formed by the fusion of two initiated in the telophase of the previous diploid mitosis.
At prophase of meiosis there is a single large and conspicuous heterochromatic body
attached to the nucleolus, and at diakinesis a single bivalent is associated with the
nucleolus. In interphase and recently formed microspore nuclei never more than a
single nucleolus per nucleus has been observed. This evidence points to the inference
that Darwinia has only a single nucleolar organizing chromosome in its haploid
complement.

In Chamaelaucium and in Micromyrtus the presence of two separate nucleolar
organizers associated with the nucleolus is frequently evident at pachytene of meiosis.
At interphase and in recently formed microspore nuclei two nucleoli per nucleus are
often found, but seem liable to early fusion. The evidence of nucleolar number and
of the number of nucleolar associated chromosomes in the Chamaelaucoideae suggests
that the number found in Darwinia is basic and the more frequent 11-chromosome
genom is a derived one. The evidence fully supports the inference drawn from the
study of secondary association.

(c) The Basic Chromosome Number and the Phylogeny of the Myrtaceae.

If the hypothesis that the basic number in the Myrtaceae is six and that the
predominant number 11 has been derived from it by polyploidy and structural change
is accepted on the basis of the evidence presented, several important questions need
to be discussed.

(1) The actual method by which the 1l-chromosome genom has originated is
searcely susceptible to experimental test, but polyploidy, followed by unequal trans-
locations, permitting the loss of a centromere (Darlington 1937, Tobgy 1943) would
seem to be the most probable method. The total loss of a pair of chromosomes, however,
might not be impossible soon after or immediately consequent upon the occurrence
of the tetraploid condition, particularly if the original tetraploid was subject to some
meiotic irregularity.

(2) The implications of the hypothesis in regard to phylogenetic relationships in
the family. The hypothesis put forward is incompatible with Andrew’s view (1913)
that the Myrtoideae is primitive, that the Leptospermoideae have been derived from
the Myrtoideae, and that the Chamaelaucoideae represent an extreme specialization. The
Chamaelaucoideae, and particularly the Euchamaelauceae, are shown to be primitive
in respect to chromosome number, although they are certainly not so in some morpho-
logical features. It is possible, if features of geographical distribution are also
considered, that the Myrtoideae in America and the Leptospermoideae-Chamaelaucoideae
in Australia have evolved separately from primitive Myrtiflorae stock. Such an assump-
tion would necessarily infer that the primitive stock possessed a haploid chromosome set

BY S. SMITH-WHITE. 117

of six and that the derivation of a secondary number of 11 has occurred independently
in the two groups. In view of the frequency of the number 11 in many families this
independent origin is not unlikely. This assumption also means that the family as at
present constituted is not a monophyletic one.

Data on chromosome number in the other families of the Myrtifloreae would be
of value as indicating the basic number for the primitive stock of the order. These
families are almost entirely tropical, and the Melastomaceae is the only family
mentioned in Tischler’s lists (1927, 1931, 1936, 1937, 1938) and in Darlington and
Janaki (1944). The number of species in this family for which such data is recorded
is small (Table 8). The haploid number of 28 reported by Suguira is too high to be
basic, but the haploid number 12 reported for Bertolonia and three other genera is of
interest, since it could be easily derived by polyploidy from a basic set of six. At
least this evidence does not conflict with the present hypothesis.

TABLE 8.
Chromosome Numbers in the Melastomaceae.
(From Tischler’s Lists.)

Species. n 2n Authority.

Centradenia floribunda .. ae — 24-26 Heitz, 1926.

Bertolonia marmorata es se 12 — Ruys, 1925.

B. aenea .. ae Ae ata = 28-32 Heitz, 1926.

Melastoma candidum a och 28 — Sugiura, 1936.

M. sanguineum .. ots he — 56 Matsuura and Suto, 1935.
Miconia racemosa .. nia dd 12 — Ruys, 1925.

Mouriria anomala ae ft — 24 Ruys, 1925.

Memecylon floribundum .. Ae — 24 Ruys, 1925.

Triuranthera Winkleri .. iA 24 — Ruys, 1925.

(3) The significance of the occurrence of secondary polyploidy in the evolution of
the Myrtaceae. At the present time the 11-chromosome genom is almost universal
throughout the Myrtaceae. The six-chromosome genom is found only in two genera of
a single subtribe and must be regarded as a remnant. Obviously the 11-chromosome
genom has proved more successful in the course of the evolutionary development of
the group.

The importance of polyploidy in plant evolution and speciation has been discussed
by many authors and has been reviewed by Muntzing (1936) and by Stebbins (1940,
1947). Babcock (1942, 1947) includes polyploidy as one of the important, but secondary,
evolutionary processes, of lesser importance than gene mutation or chromosomal altera-
tions, and Manton (1932) and Stebbins (1940) consider that polyploidy may lead to
diversity of form and speciation, but that it is not of importance in the origin of
major taxonomic groups. Stebbins points out that polyploidy leads to a reduction in
the visible mutation rate and in the phenotypic expression of recessives, and would
consequently lower the effect of selection; but Crane (1939) states that polyploids
have a wider range of variation than diploids. It has been suggested by Aase (1935)
that by the increase in the number of genes in the genom a greater total mutation
rate is possible and that the presence of multiple sets of chromosomes provides pro-
tection against the immediate deleterious effect of most gene mutations. Polyploidy
may thus allow greater polymorphism, which, as pointed out by Stebbins (1942),
provides greater adaptability. Stebbins has more recently (1947) inclined to the view
that the woody types of Angiosperms, in which chromosome numbers of 11 to 14 are
common, have been derived from more ancient types with haploid numbers of 5, 6 or 7,
a view supported by the data for the Leguminoseae (Senn 1938, 1942) and the Anonaceae
(Bowden 1945), as well as by the present paper. The evolutionary dominance of
higher chromosome numbers at the present time suggests that it has conferred some
evolutionary advantage, either directly, as suggested by Muntzing (1936), or due to

118 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

the consequences of hybridization. Stebbins (1947) puts forward the view that not
only is polyploidy a frequent consequence of hybridization, but that it may enable
species previously inter-sterile to produce hybrids and so permit of an increase in the
range of gene exchange.

Darlington (1937) points out that secondary polyploidy, by creating a new balance
of genetic factors, is likely to cause more profound phenotypic changes than primary
polyploidy. The frequency of secondary association in many plant groups with prime
numbers of chromosomes in their haploid sets (e.g. 11, 13, 17) suggests that secondary
polyploidy may be of considerable importance in the evolution of the Angiosperms.

Whilst gene mutation, small chromosomal alterations and segregation following
hybridization have undoubtedly been responsible for nearly all speciation in the
Australian Myrtaceae, the most important step in the origin of the group is considered
to be the origin of the secondary haploid genom of 11 chromosomes. This step must
rank as of equal importance with that concerned in the origin of the Pomoideae; the
consequent increased variability has allowed an “explosive” speciation whilst the six-
chromosome forms have been forced into a geographical corner, where they have
suffered morphological specialization and specific restriction.

(4) The taxonomic and cytological sequence in the Huchamaelauceae. Within the
Euchamaelauceae it is possible to arrange the genera in a more or less linear sequence
on morphological grounds (Bentham 1866, Cheel 1922) with connecting species between
the genera and sections of the larger genera. This sequence is illustrated diagram-
matically below:

Actinodium
6 NG
Darwinis
Homoranthus Bae (Genetyllis)
4 we i

Rylstonea Darwinia
2 (Schaumannia)
?

Verticordia
(Euverticordia)
8,11

’

|

Verticordia

(Catocalyptra) BN
?

Pileanthus
?

Chamae laucium
11

The cytological data presented in the present paper cover too few of the genera
to support or deny this arrangement, but they are at least suggestive. A more detailed
survey of the subtribe might well have important phylogenetic and taxonomic
consequences. i

SUMMARY.

1. Seventeen species and varieties, representing seven genera and all three sub-
tribes of the Chamaelaucoideae have been cytologically examined. Haploid numbers
of 6, 8 and 11 have been found, the lower numbers being new for the Myrtaceae.

2. Evidence is produced that the haploid genom of six is primitive and basic for
the tribe and family. This evidence concerns the occurrence of secondary association
of chromosomes and the chromosome-nucleolar relationships.

3. The evolutionary and phylogenetic consequences of this hypothesis are considered.
The origin of the 11l-chromosome genom is considered to have begun an epoch in the
evolution of the family.

4. The theory that the Chamaelaucoideae have been derived through the Lepto-
spermoideae from the Myrtoideae is denied. The Chamaelaucoideae may be primitive to

BY S. SMITH-WHITE. 119

'

the Leptospermoideae, but the Myrtoideae are more likely to have had a separate origin
from primitive Myrtiflorae. The family is in this sense polyphyletic.

5. The data presented on pollen fertility in Darwinia reduce the significance of
the inferences drawn by Lawson on the importance of interspecific hybridization as a
cause of speciation in the family. It is suggested that variation in pollen fertility is
a direct consequence of the dissection of the total species populations into small isolated
breeding groups. ;

References.

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ALAM, Z., 1936.—Cytological Studies in Some Indian Oleiferous Cruciferae. Ann. Bot., 50: 88-102.
ALLEN, H. H., 1940.—Natural Hybridisation in Relation to Taxonomy. The New Systematics.
Ed. J. Huxley, London.
ANDREWS, H. C., 1913.—The Development of the Natural Order Myrtaceae. Proc. LINN. Soc.
N.S.W., 38: 529-568.
ANDERSON, H., 1937.—Cytology in Relation to Taxonomy. Bot. Rev., 3: 335-350.
ATCHISON, H., 1947.—Chromosome Numbers in the Myrtaceae. Amer. J. Bot., 34: 159-164.
Bascock, EH. B., 1942.—Systematics, Cytogenetics and Evolution in Crepis. Bot. Rev., 8: 139-190.
, 1947a.—Cytogenetics and Speciation in Crepis. Advances in Genetics. Vol. i. New
York. |
, 1947b.—The Genus Crepis. Part I. Univ. Calif. Publ. Bot. Vol. xxi.
Batitey, F. M., 1900.—The Flora of Queensland. Brisbane.
, 1909.—Comprehensive Catalogue of Queensland Plants. Govt. Printer, Brisbane.
BENTHAM, G., 1866.—Flora Australiensis. Vol. iii. Lovell Reeve and Co., London.
Brews, J. W., 1926.—Studies in the Hcological Evolution of the Angiosperms. I. New Phyt.,
26: 1-21.
Buack, J. M., 1926.—Flora of South Australia. Vol. iii. Govt. Printer, Adelaide.
Brewster, A. A., 1915.—Observations on the Pollination of Darwinia fascicularis Rudge.
Proc. LINN. Soc. N.S.W., 40: 753-58.
BowpENn. W. M., 1945.—Chromosome Numbers in the Higher Plants. 2 Acanthaceae to
Myrtaceae. Amer. J. Bot., 32: 81-92.
CATCHESIDE, D. G., 1934.—The Chromosomal Relationships in the Swede and Turnip Groups
of Brassica. Ann. Bot., 48: 601-633.
CHEEL, E., 1922.—Notes on the Genera Darwinia, Homoranthus and Rylstonea in New South
Wales, Queensland and South Australia. J. Roy. Soc. N.S.W., 56: 62-77.
CRANE, M. B., 1939.—The Origin and Behaviour of Cultivated Plants. The New Systematics.
Ed. J. Huxley, London.
DARLINGTON, C. D., 1928.—Studies in Prunus. i and ii. J. Genet., 19: 213-256.
, 1937.—Recent Advances in Cytology. Second Edition. London.
, 1940.—Taxonomic Species and Genetic Systems. The New Systematics. Ed. J. Huxley,

London.
DARLINGTON, C. D., and JANAKI AMMAL, 1944.—A Chromosome Atlas of Cultivated Plants.
London.
, and Morrett, A. A., 1930.—Primary and Secondary Balance in Pyrus. J. Genet.,
22)2 L29-15 1.

DOBZHANSKY, TH., 1941.—Genetics and the Origin of Species. Revised ed. New York.
Ewart, A. J., 1930.—Flora of Victoria. Govt. Printer, Melbourne.
Farr, G. H., 1916.—Cytokinesis in the Pollen Mother Cells of Certain Dicotyledons. Mem.
N. Yk. Bot. Gard., 6: 253-317.
, 1918.—Cell Division by Furrowing in Magnolia. Amer. J. Bot., 5: 379-395.
GATES, R. R., 1942.—Nucleoli and Related Structures. Bot. Rev., 8: 337-410.
HAVILAND, E., 1884.—Occasional Notes on Plants Indigenous to the Immediate Neighbourhood
of Sydney. Proc. LINN. Soc. N.S.W., 9: 67-71.
HILLARY, B. B., 1940.—Uses of the Feulgen Reaction in Cytology, ii. Bot. Gaz., 102: 225-235.
Hux.Ley, J. H., 1940.—The New Systematics. London.
, 1942.—Evolution, The Modern Synthesis. London.

Kuwapa, Y., 1929.—Chromosome Arrangement i. Model Experiments with Floating Magnets
and Some Theoretical Considerations. Mem. Coll. Sci. Kyoto Imp. Univ., B, 4: 200-264.
LAWRENCE, W. J. C., 1931a.—The Genetics and Cytology of Dahlia variabilis. J. Genet., 24:

257-306.
, 1931b.—The Secondary Association of Chromosomes. Cytologia, 2: 352-394.
Lawson, A. A., 1930.—The Origin of Endemism in the Angiosperm Flora of Australia. PRoc.
LINN. Soc. N.S.W., 55: 371-380.
McAu.tay, A. L., and CRUICKSHANK, F. D., 1937.—The Male Meiotic Cycle in Eucalyptus.
Pap. and Proc. Roy. Soc. Tas., 1937: 41-44.
MaAgEpA, T., and Kato, K., 1929.—Chromosome Arrangement. vii. The Pollen Mothers Cells
of Spinacia oleracea and Vicia Faba. Mem. Coll. Sci. Kyoto Imp. Univ., B, 4: 327-345.
MAIDEN, J. H., 1916.—A Census of the Plants of N.S.W. Govt. Printer, Sydney.

120 CYTOLOGICAL STUDIES IN THE MYRTACEAE. III,

MANTON, I. 1932.—Introduction to the General Cytology of the Cruciferae. Ann. Bot., 46:
509-569.

, 1935.—Some New Evidence on the Physical Nature of Plant Nuclei from Interspecific
Polyploids. Proc. Roy. Soc., B, 118: 522-547.

Mayr, E., 1942.—Systematics and the Origin of Species. New York.

, 1948.—The Bearing of the New Systematics on Genetical Problems. The Nature of
Species. Advances in Genetics. Vol. ii. Ed: M. Demerec. New York.

Morrett, A. A., 1931.—The Chromosome Constitution of the Pomoideae. Proc. Roy. Soc., B,
108 : 423-446.

Mutter, H. J., 1940.—Bearings of the Drosophila Work on Systematics. The New Systematics.
Ed. J. Huxley. London.

Muto, A., 1929.—Chromosome Arrangement ii. The Meiotic Divisions in the Pollen Mother
Cells of Phaseolus Chrysanthos and Cassia occidentalis. Mem. Coll. Sci. Kyoto Imp. Univ.
B, 4: 265-271.

MUNTZING, A., 1936.—The Evolutionary Significance of Autopolyploidy. Hereditas, 21: 263-378.

Oaawa, K., 1929.—Chromosome Arrangement v. Pollen Mother Cells in Torilis Anthriscus and
Pucedanum japonicum. Mem. Coll. Sci. Kyoto Imp. Univ., B, 4: 309-322.

RAGHAVEN, T. S., 1988.—Morphological and Cytological Studies in the Capparidaceae. ii. Ann.
Bot., N.S., 2: 75-96.

ROSENBERG, O., 1927.—Hereditas, 8: 305-338. Cited from Alam, 1936.

SEMMENS, C. 8., and BapHurti, P. N., 1939.—A New Technique for the Differential Staining of
the Nucleoli and Chromosomes. Stain Techn., 14: 1-5.

SENN, H. A., 1938.—Cytological Evidence of the Status of the Genus Chamaechrista Moench.
J. Arn. Arb., 19: 153-157.

, 1942.—Relation of Anatomy and Cytology to the Classification of the Leguminoseae.
Chron. Bot., 7: 306-308.

SMITH-WHITE, S., 1948.—Cytological Studies in the Myrtaceae. ii. Chromosome Numbers in
the Leptospermoideae and Myrtoideae. Proc. LINN. Soc. N.S.W., 73: 16-36.

STEBBINS, G. L., 1940.—The Significance of Polyploidy in Plant Evolution. Amer. Nat., 74:
54-66.

, 1942.—The Concept of Genetic Homogeneity as an Explanation of the Existence of
Rare and Endemic Species. Chron. Bot., 7: 252.

, 1947.—Types of Polyploids: Their Classification and Significance. Advances in
Genetics. Vol. i. Ed. M. Demerec. New York.

TIMOFEEFF-RESOVSKY, N., 1940.—Mutations and Geographical Variation. The New Systematics.
Ed. J. Huxley. London.

TISCHLER, G., 1927.—Pflanzliche Chromosomenzahlen. i. Tab. Biol., 4: 1-83.

, 1931.—Ibid. ii. Tab. Biol., 7: 109-226.

, 1935.—Ibid., iii. Tab. Biol., 11: 281-304.

, 1936.—Ibid., iii. Tiel ii. Tab. Biol., 12: 1-115.
, 1938.—Ibid., iv. Tab. Biol., 16: 162-218.

Toney, H. A., 1943.—A Cytological Study of Crepis fuliginosa and C. neglecta and their Fi
Hybrid, and its Bearing on the Mechanism of Phylogenetic Reduction in Chromosome
Number. J. Genet., 45: 67-111.

Vavitov, N. I., 1926.—Studies in the Origin of Cultivated Plants. Bull. Appl. Bot. Select., 16:
1-248.

, 1940.—The New Systematics of Cultivated Plants. The New Systematics. Ed.
J. Huxley. London.
WooLis, W., 1884.—On the Myrtaceae of Australia. Proc. LINN. Soc. N.S.W., 9: 641-648.
WRIGHT, S., 1940.—The Statistical Consequences of Mendelian Heredity in Relation to Speciation.
_ The New Systematics. Ed. J. Huxley. London.

DESCRIPTION OF PLATES V ANID VI.

PLATE V.
(All photographs x 2000.)

Fig. 1. Pollen mother cells of Actinodiwm Cunninghamii in prophase. The heterochromatic
nucleolar organizer is large and conspicuous. Acetocarmine.

Fig. Actinodium Cunninghamii. 1-A in polar view. Acetocarmine.

Fig. 3. Darwinia fascicularis. 1-M, showing the 5 (1) configuration. Feulgen.

Fig. 4. D. fascicularis. 1-M. The 6 (0) configuration. Feulgen.

Fig. 5. D. fascicularis. 2-M. Feulgen.

'

to

Fig. 6. D. grandiflora B. & S. 1-M. Feulgen.

Fig. 7. D. grandiflora B. & S. 2-M. Feulgen.

Fig. 8 D. intermedia. 1-A, showing a dividing univalent. St. Ives population. Aceto-
carmine.

©

Fig. Verticordia sp. 1-M. Acetocarmine.

BY S. SMITH-WHITE. 1a

Figs. 10-15. Chamaelaucium uncinatum. Feulgen-Light Green. Fig. 10. Resting pollen
mother cell nucleus showing the heterochromatic nucleolar-associated chromatin. 11. The two
nucleolar-associated bodies are shown attached to the single nucleus in a prophase nucleus.
12. 1-M. 18. Interphase, showing two nucleoli in each nucleus. 14. 2-M. 15. A pollen mother
cell with microspore nuclei, one showing two nucleoli.

Figs. 16-19.—Calythrix tetragona. Acetocarmine. Fig. 16. 1-M. The large bivalent is
conspicuous. 17. 1-M, side view. 18. 1-M, side view with two univalents near the poles.
19. 1-A, side view of the spindle showing misdivision of laggard chromosomes.

Fig. 20. Micromyrtus microphylla. 2-M. Acetocarmine.

PLATE VI.

(All photographs x ca. 150.)

Fig. 1. Darwinia fascicularis. Kuringai. Plant No. 2.

Fig. 2. D. taxifolia f. tetraflora. WKing’s Tableland No. 9.
Fig. 3. D. intermedia. St. Ives No. 6.

Fig. 4. D. grandiflora. Berowra No. 7.

Fig. 5. D. grandiflora. Berowra No. 1.

Fig. 6. D. taxifolia v. grandifiora. Bulli No. 10.

Fig. 7. D. taxifolia v. grandiflora. Helensburgh.

Fig. 8. Chamaelaucium uncinatum. Cult. National Park.
Fig. 9. Chamaelaucium wneinatum. Gordon seedling.

A REVISION OF THE AUSTRALIAN SPECIES OF THE GENUS
LAGENOPHORA CASS.

By Gwenpa L. Davis, Department of Biology, New England University College,
Armidale, N.S.W.

(Plate vii; thirteen Text-figures. )

[Read 31st May, 1950.]

INTRODUCTION.

Cassini (1816) erected the genus Lagenifera to accommodate specimens collected in
Australia by Labillardiére, and described by that author as a new species of Bellis
(B. stipitata). In 1818 Cassini wrote “there are two corrections to make in my first
number in the Bulletin of December, 1816: ... and the name of the genus Lagenifera has
been changed to that of Lagenophora’. Although the earlier synonym has priority, it
was never accepted in general usage, and in the opinion of the present writer, the later
name should be retained.

The genus Jxauchenus was described by Cassini (1828) from a single specimen
collected at Port Jackson by d’Urville, and which he considered was intermediate
between Lagenophora and Bellis. This genus was listed as synonymous with Lagenophora
by Bentham (1866).

A. Richard (1832) erected the genus Microcalia for Forster’s Calendula pumila from
New Zealand, but although he stated reasons for excluding this specimen from Calendula,
he made no comparison with Lagenophora. De Candolle (1836) redescribed Richard’s
Microcalia australis as Lagenophora Forsteri.

Bentham, since he had at his disposal plants collected from the early days of
Australian exploration, handled comparatively long series of specimens, where earlier
workers had had to be content with one or two. As a result, he was able to note
variations within species, and relegated many former names to synonymy. In the last
fifty years collections in Australian herbaria have grown considerably so that as genera
come under revision earlier workers’ concepts of component species must sometimes be
further modified when populations thought by them to be discrete, are shown to
represent merely extremes in a continuously variable series.

The present writer is of the opinion that the species included by Cassini in his genus
Solenogyne are generically distinct and that Lagenophora, in its revised sense, is
represented in Australia by only two species.

Distribution of the Genus.

Lagenophora is widely distributed throughout the New World from Liukiu Island
to the Hast Indies and Ceylon, throughout Australia, New Zealand and Subantarctie
Islands, New Caledonia and Fiji, while seven species are recorded ‘in Index Kewensis
from South America. Since the original descriptions of the three species from the
Sandwich Islands (L. Maviensis H. Mann, L. Hrici C. N. Forbes and L. Helena C. N.
Forbes) are devoted to vegetative characters, it cannot be determined whether they
belong to this genus in its revised sense.

The original descriptions of the twenty-seven valid species of this genus are devoted
almost exclusively to sich vegetative character as would be most likely influenced by the
habit, and variation within a species is not recorded. Such characters, unsupported by
differences of a stable nature such as may be expected in the fruits, are insufficient for
specific status and it is suggested that further work will show that a large proportion
of these species are ecological variations.

Categories.
The general principles put forward in a previous paper (Davis, 1948) are applied also
in this revision. On the material available the use of infraspecific categories is not

BY GWENDA L. DAVIS. 123

justified, and the very considerable vegetative variation shown by one species is seen
to be continuous. The factors governing this variation have not yet been determined,
but it is thought that they are ecological.

Evaluation of Taxonomic Characters.

A taxonomic character is one which is inherited as such and whose form is not
influenced by the environment. It therefore remains constant under whatever conditions
a particular plant may be growing, and is referred to as a primary taxonomic character.
Habit, texture, shape and number of leaves are the result of the interaction between
genetic factors and the environment and consequently should not in general be used as
primary taxonomic characters. Secondary taxonomic characters are purely vegetative
and are frequently, though not invariably, linked with primary ones. These are
frequently of importance in identifying immature specimens in which the primary
taxonomic characters are not yet developed.

In this revision a large number of specimens were critically examined and an
attempt was made to evaluate the taxonomic importance of all characters whether
previously used or not. The results of this comparison are set out below under
appropriate headings.

Habit: Although L. stipitata shows very considerable variation in mode of growth
it can be readily distinguished from L. Hwegelvi, which is usually a more robust plant
with large bracts on the scapes. The two species can be separated on this character in
the absence of fruits. The variation within L. stipitata was found to be continuous so
no infraspecific categories were recognized.

Leaves show wide limits of continuous variation and are of no taxonomic value.

Indumentum of a septate-hairy nature is always present in some degree and conse-
quently is to be regarded as a generic character.

Capitula are usually largest in L. Huegelii, but the lower limits of measurement in
this species are overlapped by the upper limits of L. stipitata.

Involucral bracts are of little or no value in specific determination, their size
depending on that of the capitulum as a whole.

Ray and disc florets are similar in both species, though they tend to be most
numerous and smallest in L. stipitata.

Anthers bear a terminal appendage to the connective in both species and no
variation was seen.

Stylar branches are dimorphic, and identical in similar florets in both species.

Receptacle varies in size in proportion to the size of the capitulum, consequently is
usually largest in L. Huegelii.

Fruits are of primary taxonomic importance, though the difference between those
of the two species is one of dimensions rather than character.

Specimens Examined.

Through the courtesy and co-operation of the Directors of the various Australian
herbaria and several private collectors, a long series of specimens has been critically
examined. These are all listed under the appropriate species, the source in each case
being indicated as follows:

National Herbarium, Sydney (NSW).
National Herbarium, Melbourne (MEL).
Brisbane Herbarium (BRI).
University of Adelaide (AD).

State Herbarium, Perth (PERTH).
University of Tasmania (HO).
University of Florence (FLO).

J. M. Black (JMB).

J. B. Cleland (JBC).

F. A. Rodway (FAR).

N. A. Wakefield (NAW).

IK

124 AUSTRALIAN SPECIES OF LAGENOPHORA CASS,

Specific Descriptions.

Both species have been redescribed from a longer series of specimens than was
available to the original authors, and the specific limits have been extended to cover all
variations. Marked departures from the usual condition are discussed separately in the
text. The various measurements given are to be regarded more as a general indication
ot size than absolute criteria, since in all structures more or less considerable size
variation is found on the same plant. All figures are camera lucida drawings.

TAXONOMY.
COMPOSITAR, tribe ASTEROIDEA.

Lagenophora Cass., Bull. Soc. Philom. Paris (1818), 34.

Synonymy: Lagenifera Cass., Bull. Soc. Philom. Paris (1816), 199; Iawauchenus Cass.,
Dict. Sci. Nat., LVI (1826), 176; Microcalia A. Rich., Fl. Nova Zel., t. 30 (1832), 230.

Erect or ascending stoloniferous perennials from 2-8-39 cm. high, with a septate-
hairy indumentum on all vegetative parts. Leaves up to 15 cm. long, 3:2 em. broad,
oblanceolate to elliptical in gross outline, acute to obtuse, with serrate, crenate or
undulating margins, petiolate, basally clustered in erect plants, but commonly cauline
as well when the plant has an ascending mode of growth. Jnflorescences 1-15, 4-11 mm.
diameter excluding the rays, borne on scapes or axillary peduncles which are provided
with 3-11 linear to narrow elliptical bracts. IJnvolucral bracts 35-54 in 3-4 rows, the
innermost 2:2-5 mm. long, 0:5-1-2 mm. broad, linear to linear-lanceolate or obovate,
obtuse or acuminate, with torn-ciliate margins and septate hairs on outer surface.
Bracts become reflexed when fruits are shed. Ray florets 60-100, the rays 2-5-5 mm.
long, 0:4-0-°9 mm. broad in 3-4 rows, white, pale pink or bluish, rounded distally.
Disc florets 12-20, staminate, sterile, corolla about 1 mm. long, tubular, 4-5 lobed
distally. Stamens 4-5, present only in disc florets. Anthers united laterally forming
a tube around the style, connective prolonged beyond the level of the pollen sacs to
form a terminal appendage. Stylar branches dimorphic, those of the ray florets smooth,
those of the disc papillose on outer surfaces. Receptacle 1:1—3-8 mm. broad, 0:5-1:9 mm.
high, truncate hemispherical, bearing small elevations on the sides, corresponding to the
points of attachment of the ray florets. The sterile disc florets are borne on the
flattened summit of the receptacle. Fruit an inferior achene, 2:7-4:3 mm. long,
0-8—2-1 mm. broad, straw-coloured to dark brown, more or less ecrescentic to obovate,
flattened, bearing a narrow beak curved away from the centre of the receptacle and
surmounted by a paler collar.

Type species: Lagenophora stipitata (Labill.) Druce.

Key to the Species.

Erect or ascending plants with 3-6 linear or filiform bracts on scapes or peduncles. Fruits
rather crescentic, tapering distally into a narrow curved microscopically’ glandular beak
RE ha SOE one a TORR on ard Gaiam Mar stolord aciokd Oro ordi, Bea aoa a Hoenn eeo oo 1. L. stipitata

*Hrect plants whose scapes bear 5-11 narrow-elliptical sessile leaves. Fruits obovate, flattened,
microscopically glandular distally passing abruptly into a curved neck ...... 2. L. Huegelii

1. LAGENOPHORA STIPITATA (Labill.) Druce.
Rep. Bot. Exch. Cl. Brit. Isles, 1916 (1917), 630.
(Text-figs. 1-10; Plate vii.)

Synonymy: Bellis stipitata Labill., Pl. Nov. Holl. II (1806), 55; Lagenophora Billardieri
Cass., Dict. Sci. Nat., XXV (1822), 111; L. Billardieri Cass. var. a pusilla DC., Prod. v (1836) ;
307; L. Billardieri Cass. var. B media DC., l.c.; L. Billardieri Cass var. y glabrata DC., l.c.;
Trauchenus sublyratus Cass., Dict. Sci. Nat., LVI (1828), 176; Lagenophora gracilis Steetz in

Lehmann, Pl. Preiss, 1 (1845), 428; L. latifolia W. J. Hooker, Lond. Journ. Bot., VI (1847),

113; L. montana W. J. Hooker var. a major W. J. Hooker, l.c.; L. montana W. J. Hooker var.
B minor W. J. Hooker, l.c.

Syntype series: Eleven specimens. “New Holland. Herbarium Webbianum ex herb.
Labillardiére” (FLO).

Erect or ascending stoloniferous perennials 2-8-39 em. high, septate-hairy all over.
Leaves usually petiolate, up to 15 cm. long, 2-2 em. broad, oblanceolate to elliptical in
gross outline, serrate, crenate or undulating margins, acute to obtuse, clustered basally,

Pa

AUSTRALIAN SPECIES OF LAGENOPHORA CASS, 125

cauline leaves present or absent. Inflorescences 1-15, 4-10 mm. diameter, borne on
seapes or axillary unbranched peduncles bearing 3-6 linear or filiform acuminate bracts
up to5 mm. long. Jnvolucral bracts 35-50, 2-2-4 mm. long, 0-5-1 mm. broad, in 3—4 rows,
linear to linear-lanceolate, usually acuminate, torn-ciliate, septate-hairy on outer surface,
reflexed when fruit are shed. Ray florets 60-100, 2-5-4 mm. long, 0-4—0-7 mm. broad, in
3-4 rows, female, white, pale pink or bluish. Disc florets 12-20, staminate, sterile, the
corolla tube 4-5 lobed. Receptacle 1:1-2 mm. broad, 0:5-1:9 mm. high, truncate-
hemispherical, with small elevations corresponding to the points of attachment of the
florets. Fruit 2:7—4:3 mm. long, 0-8 mm. broad, straw-coloured to dark brown, more or
less crescentic with a narrow curved microscopically glandular beak surmounted by a
paler collar. The curve of the beak is directed away from the centre of the receptacle.

Habitat: Grassland and open forest.
Range: Coast and tablelands of eastern and southern Australia as far west as Spencer’s
Gulf, throughout Tasmania and islands of Bass Strait. Also recorded from tropical Asia.

Specimens examined: Queensland: Yarrabah, 7.1918, N. Michael, No. 414 (BRI); Atherton,
1.1919, C. T. White (BRI); Bellenden Ker Range, 1881, Karton (MEL); Bellenden Ker Range,
1891, S. Johnson (MEL); Herberton, 7.1.1912, F. H. Kenny (BRI); Mackay, A. Dietrich
(MEL); Rockhampton, 10.2.1868, P. O’Shanesy (MEL); Rosedale, L. G. Dovey (BRI);
Glasshouse Mts., sandy ground, 5.1910 (BRI); Virginia, near Brisbane, 16.9.1916, C. T. White
and F. N. Evans (BRI); Pine River, I. W. Slatter (BRI); Ithaca Creek, F. M. Bailey (BRI) ;
Helidon, J. Shirley (BRI); Moreton’s Bay, Leichhardt (MEL); hills near Mt. Gravatt, “ray
florets white’, 14.3.1931, C. T. White, No. 7406 (BRI); Wellington Point, 11.1891 (BRI);
Jimboomba, 5.1921, H. Cheel (NSW); Lockyer, 1875, Hartmann (MEL); Cunningham’s Gap,
6.1892, BE. M. Bailey (NSW); Inglewood, 3.1911, J. L. Boorman (NSW); McPherson Range,
Haster, 1909, C. T. White (BRI).

New South Wales: Upper Barwan, 11.1882, B. Wilson (MEL); Tenterfield, 3.11.1886, E.
Betche (NSW, MEL); Timbarra, New England, C. Stuart, No. 444 and 781 (MEL); Richmond
River, 1877, C. Fawcett (MEL) ; Clarence River, 11.1875, Wilcox (MEL); Perretts (Tyringham)
and Bald Hills station (Grafton-Armidale road), 12.1893, J. H. Maiden (NSW); Moona River,
Walecha, 2.1884, A. R. Crawford (MEL); Port Macquarie, 2.1898, J. L. Boorman (NSW) ;
Cundletown, Manning River, 12.1899, EH. Cheel (NSW); Barrington Tops, in Swamp, 1.1934,
L. Kraser and J. Vickery (NSW); Barrington Tops, in grassland, 7.1.1934, L. Fraser and
J. Vickery (NSW); William’s River, near Salisbury, 11.1.1934, L. Fraser and J. Vickery
(NSW); Sandgate, Newcastle, 8.11.1902, “white flowers’, R. H. Cambage (NSW); Christie’s
aully, Mooney Mooney Creek, 9.1926, W. F. Blakely, G. P. Darnell-Smith and D. W. C. Shiress
(NSW); Mt. Wilson, 4.1896, J. H. Maiden (NSW) ; Mt. Victoria, 1.1915, A. A. Hamilton (NSW) ;
Jamieson Valley, 10.1894, J. J. Fletcher (NSW) ; Jenolan Caves, 11.1899, W. F. Blakely (NSW) ;
Hawkesbury Agricultural College, Richmond, 23.10.1906 (NSW); Hornsby, chiefly in forest
land, 4.1914, W. F. Blakely (NSW); Port Jackson, 7.1855, EF. Mueller (MEL); Botany Bay,
7.1855, EF. Mueller (MEL); Como, 6.3.1887, R. Collie (NSW); Mt. Kembla, 7.11.1891, J. J.
Fletcher (NSW); Austinmer, 16.7.1933, 22.4.1934, G. Rodway (FAR); Seven Mile Beach, Berry,
“in sand’, 17.2.1930, EF. A. Rodway (FAR); Kangaroo Valley, 30.4.1931, F. A. Rodway (FAR) ;
Comerong Island, Shoalhaven River, 18.9.19282-F. A. Rodway (FAR); Roseby Park, Shoalhaven
River, 10.1916, 30.3.1931, F. A. Rodway (FAR); Crookhaven Heads, 1.1.1941, FE. A. Rodway
(FAR) ; Huskisson, paddock, 29.3.1931, 2.4.1939, F. A. Rodway (FAR); Jerrawangala, 4 miles
south of Wandandian, 11.3.1934, F. A. Rodway (FAR); Ulladulla, 1883, W. Bauerlen (MEL) ;
Bateman’s Bay district, 10.1890, W. Bauerlen (MEL), Sassafras, 26 miles s.w. of Nowra, about
2,000 ft., moorland, 14.6.1942, F. A. Rodway (FAR); Braidwood district, 3,000 ft., 10.1886,
W. Bauerlen (MEL); Bimberi Peak, Queanbeyan, 5.1.1912, R. H. Cambage (NSW) ; the Peaks,
Yarrangobilly, 24.1.1933, W. A. W. de Barzeville (NSW); Nimitybelle to Tantawonglo Mts.,
12.1896, J. H. Maiden (NSW); Mt. Kosciusko, 5,500 ft., 11.1.1930, T. Harris (NSW).

Victoria: Hume River, 1886, S. Jepheott (MEL); Victorian Alps, 5,000 ft., 12.1921, A. J.
Tadgell (MEL); around bogs at the Scout Hut, Middle Creek, Bogong High Plains, 5,400 ft.,
usually growing in the shade of Snow Gums, 4.2.1949, J. H. Willis (MEL); ranges near Omeo,
metamorphic schist, 3,000 ft., Stirling (MEL); Snowy River, 1.1854, F. Mueller (MEL); near
Mt. Ellery, 1886, HE. Merrall (MEL); Nungatta, Genoa, Weatherhead (MEL); Genoa district,
2.1885, W. Bauerlen (MEL); moist sandy flats behind ““Marshmead’”’ homestead, N.E. portion
of Mallacoota Inlet toward the Howe Range, growing with Selaginella wliginosa, Caladenia
carnea var. pygmaea and other swamp vegetation, 24.10.1948, J. H. Willis (MEL) ; Cape Conran,
N. A. Wakefield (NAW); Southern Croajingolong, N. A. Wakefield (NAW); Orbost, 3.1937,
EF. Robbins (MEL) ; Newmerella, near Orbost, 28.5.1902, Grove (MEL); Avon River, Gippsland,
1882, Howitt (MEL); Mt. Wellington, 3.1861, F. Mueller (MEL); McAllister River, F. Mueller
(MEL); Mt. Baw Baw, 4-5,000 ft., 12.1860, FE. Mueller (MEL); Red Jacket Creek, Victorian
Alps, 18738, Gargurevich (MEL); Warburton rly. line between Killara and Woori-Yallock,
$8.4.1904, P. R. H. St. John (MEL); Dandenong Range, 1.1853, F. Mueller (MEL); Dandenong
Creek, 1.1853, FEF. Mueller (MEL); Anderson’s Creek, between Doncaster and Warrandyte,

126 AUSTRALIAN SPECIES OF LAGENOPHORA CASS,

14.3.1902, P. R. H. St. John (MEL); Brighton, 10.1852, F. Mueller (MEL); Skye River, near
Frankston, “in damp heath land scrub’, 5.12.1904, P. R. H. St. John (MEL); Plenty Ranges,
11.1898, C. Walter (MEL, NSW); Upper Yarra, 10.1892, C. Walter (MEL); Maryborough, 1874,
Cc. Maplestone (MEL); Bolmarra, near Ballarat, H. Wooster (MEL); near Ballarat, 1.1853,
F. Mueller (MEL); Meredith, 1884, S. Johnson (MEL); near Geelong, 1885, J. B. Wilson
(MEL); Geelong, H. B. Williamson (MEL); Curdie’s River, 12.1873 (MEL); Hawkesdale,
11.1900, 11.1901, H. B. Williamson (MEL) ; Portland (MEL); Glenelg River, F. Mueller (MEL) ;
Grampians, ‘‘wet flats’, 11.1880, D. Sullivan (MEL).

Bass Strait: King Island, 1882, E. Spong (MEL); King Island, A. Neate (MEL); Flinders
Island, 6.11.1844, Milligan, n.607 (MEL); Flinder’s Island, 6.11.1844, R. C. Gunn, n.67 (HO).

Tasmania: Circular Head, 6.11.1837, Gunn, n.67 (NSW, HO); Woolworth, 30.3.1837, Gunn,
n.833 (NSW); Emu Bay, 7.11.1841, Milligan, n.607 (MEL); Penquite, 10.12.1844, Gunn, n.67
(NSW, HO); Emu Bay and Hampshire Hills, 15.11.1841, Milligan (MEL); Hampshire Hills,
Milligan, n.1024 (MEL); Surrey Hills and Macquarie Hbr., Milligan (MEL); Mt. Bischoff,
1884, F. Kayser (MEL); Waratah, 12.1924, A. H. S. Lucas (NSW); Mt. Barrow and foothills,
Ce. Dorset, 1.1922, H. M. R. Rupp (NSW); Launceston, S. Hannaford (MEL); moist ground
near Glendher, Launceston, 1.1862 (MEL); Wilmot, 12.1915, F. A. Rodway (FAR); South
Esk River, 1886, S. Oakden (MEL); St. Marys, 11.1925, E. Rees (HO); Swanport, Storey
(MEL); Somerset Co., “light bush’, 14.2.1948, W. M. Curtis (HO); Arthur’s Lakes, 18.2.1843,
Gunn, n.833 (NSW); Interlaken, 3.1948, Liphott (HO); St. Peter’s Pass, 24.1.1931, O. Rodway
(HO); Mt. Dundas, 12.1893 (NSW); Margin of Lake St. Clair, 13.2.1845, Gunn, n. 1148
(NSW); National Park, about 200 ft., 27.2.1943, W. M. Curtis (HO); Russell Falls, 3.1910,
EH. Cheel (NSW); Mt. Dromedary, 2.1894, L. Rodway (HO); Mount Direction, 6.1922, EF. A.
Rodway (FAR); Hast Risdon, 13.11.1937 (HO); Bellerive, 2.1893, L. Rodway (NSW); Neck,
marsh, 11.1.1837, Gunn, n.832 (NSW); Port Arthur, 1892, J. Bufton (MEL); near landslip,
Glenorchy, 3.1.1881, Simpson (MEL); near Hobart, 12.1906, H.H.D.G. (JMB); Hobart, 11.1898,
F. A. Rodway (FAR); Domain, Hobart, 1.1894, L. Rodway (HO); Huon Road, beyond Fern-
tree, dry heath, 1,000 ft., 2.2.1931, E. Atkinson (HO); Mt. Wellington, 1.1839, Gunn, n.1148
(NSW); Mt. Nelson, 11.1913, L. Rodway (HO); gully beside Mt. Nelson Road, 11.2.1937,
Consett Davis (FAR); ‘‘Melaleuca’’, Blackman’s Bay, 11.1935, F. A. Rodway (FAR); South-
port, 1.1856, ?F. Mueller (MEL); Hartz Mts., 1.1901, A. H. S. Lucas (NSW); D’Entrecasteaux
Rivulet, Recherche, 2.1852 (MEL); Van Diemen’s Land, R. Brown (MEL).

South Australia: Mt. Remarkable. Campbell’s Creek, ‘‘ray pink’ (JMB); Lake Bonney,
1882, 1887, C. Wehl (MEL) ; Hindmarsh, 3.1.1924, J. B. Cleland (JBC); National Park, “burnt
area, ligules bluish”, 30.12.1939, J..B. Cleland (JBC); Mt. Lofty Ranges, 1.1894, O. E. Menzel
(AD, NSW); Mt. Lofty, 1.4.1918, E. H. Ising (JMB); Aldgate, 12.1896, O. E. Menzel (NSW) ;
Scott’s Creek, 31.10.1906, H.H.D.G. (JMB); Coromandel Valley, 1850, Blondowsky (MEL) ;
Clarendon, 12.1883, R. Tate (AD); Mt. Compass, 11.1930, J. B. Cleland (JBC); Square Water-
nole, “pink”, 22.2.1928 (JMB); Myponga, 26.1.1908, H.H.D.G. (JMB); Back Creek, Inman
Valley, “pale pink’, 5.11.1934, J. B. Cleland (JMB); Back Valley, Encounter Bay, 17.11.1930,
28.10.1934, J. B. Cleland (JBC, JMB); Hills between Inman River and Hindmarsh Tiers, 1.1926,
J. B. Cleland (JBC); Hindmarsh Tiers, 22.5.1928, 13.1.1929, J. B. Cleland (JBC); Port Victor,
Francis (AD); Encounter Bay, near Hall’s Creek, 11.1.1934, J. B. Cleland (JBC); Davenport
and Rivoli Bay, 10.1848, F. Mueller (MEL); Mt. Gambier, 1867, Wehl (MEL); Mt. Gambier,
Wilhelmi (MEL) ; Port Macdonnell, 1.11.1941, J. B. Cleland (JBC).

Kangaroo Island: Rocky River, ray blue, 18.11.1924, J. B. Cleland (JBC, JMB); Middle
River, 10.1905, E. Ashby (NSW); Peaty flats, 28.10.1924, T. G B. Osborn (JMB); Rav. de
Casvars, 3.2.1948, J. B. Cleland (JBC); Karatta, bare temporarily submerged flat near river,
flowered purple, 9.11.1886 (MEL).

Through the courtesy of Professor Pichi-Sermolli, a photograph (Plate vii) was
obtained of a sheet of eleven specimens in the herbarium of the University of Florence
labelled ‘““Herbarium Webbianum—ex herb. Labillardiére—Nova Hollandia’. The sheet
bears the determination “Bellis stipitata’, and is extensively annotated. Professor Pichi-
Sermolli, in a personal communication, states: “all Labillardiére’s type specimens in our
herbarium have extensive notes, like that of Bellis stipitata. In these notes the original
description, or part of it, is quoted. Together with it, very often observations on the
affinity of the species, further characters (not quoted in original description) of flowers
and fruits and suggestions on systematical value of genera and species are added.
Frequently in these notes some words are deleted and replaced with other ones, which
are quoted in the original description of the book by Labillardiére.’ Professor Pichi-

Text-figures 1-10, L. stipitata.
1-2, showing extremes of variation in habit, x 4; 3, capitulum, x 6; 4, receptacle, x 13;
5, ray floret, x 10; 6, disc floret, x 10; 7, stigma of dise floret, x 20; 8, anthers, x 10; 9, fruit
from one of Labillardiére’s specimens at the University of Florence, x 10; 10, distribution.

’

BY GWENDA IL. DAVIS.

128 AUSTRALIAN SPECIES OF LAGENOPHORA CASS,

Sermolli later wrote that he had sent a specimen of the handwriting in question to the
British Museum, where it was identified as that of Labillardiére. In habit, florets and
fruits (Text-fig. 9) these specimens agree with the original description and accom-
panying figures, so have been nominated the syntype series, from which one specimen
may be selected as lectotype.

It is of interest that in the National Herbarium, Melbourne, there are four mounted
specimens from Steetz’s collection labelled by Steetz “In insula Van Diemen leg. cl.
Labillardiere ipse, mecumque communicavit amicus cl. Sonder, 1843”. The date, as in
Steetz’s other specimens, refers to the date of receipt by him and not that of collection.
These specimens are very similar to those of the type series, of which they may be
duplicates, and have been used as a basis of comparison in this work.

Cassini (1822) redescribed Labillardiére’s specimens as Lagenophora Billardiéri,
with the note ‘‘we have made this description from dry specimens collected by M.
Labillardiére at Cape Van-Diemen’’, consequently the specimens at Florence are also
the type series of the later name. Cassini subsequently examined further material in
both Labillardiére’s and Sieber’s collections, and in 1826 erected three varieties based
on small vegetative differences. His var. media corresponds with var. typica of modern
usage, but the other two (var. pusilla and var. glabrata) are to be regarded as relatively
small vegetative variations and were never accepted in general usage.

Trauchenus sublyratus, a monotypic genus, was described by Cassini (1828) to
accommodate a single dry specimen collected at Port Jackson by d’Urville, and which
he distinguished from Lagenophora ‘by its dise composed of numerous flowers, perhaps
hermaphrodite, or at the very least provided with a conspicuous false ovary, by its
crown composed of numerous flowers arranged in two rows, by the involucral scales
being homogeneous from one end to the other, and by the ovaries having a thick and
sticky neck”. Since these are characters of Lagenophora and not points of difference,
Trauchenus sublyratus was relegated to synonymy by later workers.

Bentham (1866), quoting Steetz (1845), listed Brachycome pumila in his synonymy
of L. Billardiéri, but, in view of the fact that in the original description Walpers (1843)
stated “pappus shortly coronate”’, the present writer has omitted it from the synonymy,
pending Walper’s specimen (“Van Diemen’s Land’’) being traced.

Lagenophora gracilis was reduced to synonymy by Bentham (1866). The type
specimen, according to Steetz, was collected by Roé and deposited in the Court
Herbarium of Vienna. In the National Herbarium, Melbourne, is a specimen (coll.
“Thotsky, New Holland’) labelled “ZL. gracilis” in Steetz’s writing, and accompanied
by an envelope containing three fruits. This envelope is inscribed by Steetz “mature
achenes of Lagenophora gracilis nobis, from the herbarium of the Prince of Vienna’,
and suggests that these fruits were removed from the type specimen and retained by
Steetz in his own collection. They are identical with those of L. stipitata.

Hooker’s two species L. latifolia (“Mt. Wellington, Gunn”) and L. montana are
merely “hanes given to vegetative variations and, as pointed out by Bentham (1866),
pass gradually into the normal form. Ll. montana is divided by Hooker into two
varieties, var. a major (“Marlborough and Woolnorth’”) and var. B minor (“Mt.
Wellington, Gunn’). In the National Herbarium, Sydney, are specimens in Gunn’s
collection from Marlborough and Woolnorth which have been nominated haptotypes of
the first variety, since they are almost certainly duplicates of those sent to Hooker.
There is also a specimen collected by Gunn from Mt. Wellington, which is the type
locality of both L. latifolia and L. montana var. 6 minor. Since this specimen agrees
equally well with both descriptions, it is uncertain of which it is the haptotype.

Bentham (1866) drew attention to the wide variation in size of the inflorescences
and rays of L. Billardiéri, and on this established two varieties, microcephala with small
inflorescences and short rays, the commonest form in tropical and subtropical regions,
and normalis, with larger heads and rays, which is commonest in southern districts. He
also pointed out that Labillardiére’s own specimens were almost intermediate between
these two varieties. The present writer’s observations have supported Bentham’s state-

BY GWENDA L. DAVIS. 129

ments to the extent that the average diameter of the Queensland specimens examined
was 5-5 mm. (excluding the rays), that of specimens from New South Wales 7-4 mm.,
while that of Victorian specimens was 8-6 mm. These measurements being taken from
dried specimens are, probably, not in themselves accurate, but they do indicate a progres-
Sive increase in size from north to south of eastern Australia.

Variation in the habit of the plant (Text-figures 1, 2) as well as shape and arrange-
ment of the leaves is considerable and the peaks of variation are in some instances far
removed, but are connected by intermediate forms. As far as can be judged with only
dried specimens, young plants possess a basal rosette of radical and clustered lower
cauline leaves. As growth proceeds, and especially if dense herbage is present, a main
stem bearing axillary peduncles and cauline leaves develops and the plant may assume
an ascending type of growth. Unfortunately ecological notes are seldom available and
how far vegetative features are influenced by environmental factors and age of the
plant is a matter for speculation until some experiments being undertaken at the present
time are completed. It is of interest that the single specimen examined from Lord Howe
Island (28.8.1843, L. Leichhardt, MEL) was identical with mainland ones.

Variation in the fruits of L. stipitata is slight and confined to small size differences,
the general shape and proportions remaining the same. The slight variation in the
curvature of the neck noticed in some specimens did not appear constantly and it was
thought to be influenced by drying condition. Bentham (1866) recorded ‘‘sometimes
[the neck is] as long as the breadth of the achene, sometimes very short’. By tracing
the development of the fruit it has been found that the neck lengthens as the fruit
matures, and in the young fruit is hardly apparent.

2. LAGENOPHORA HUEGELIT Benth., Enum. Pl. Hueg. (1837), 59.
(Text-figures 11-13.)

Synonymy: Lagenophora Guniiana Steetz in Lehmann. Pl. Preiss (1845), 431.

Perennial herbs 7—31:-5 cm. high, the septate-hairy indumentum on scapes and
leaves being visible macroscopically. Leaves radical, up to 14:3 em. long, 3-2 cm. broad,
oblanceolate, dentate to serrate, tapering proximally into a short or long petiole. Scapes
2-6, robust, bearing 5-11 narrow-elliptical, sessile, acuminate leaves up to 1-6 cm. long,
4 mm. broad, entire or distally and acutely toothed. These leaves are frequently confined
to the lower half of the scape, but in some specimens are borne along the whole length,
becoming smaller and finally filiform below the capitulum. Inflorescences 2—4, 8-11 mm.
~diameter excluding the rays. IJnvolucral bracts 42—54, in several rows, the inner ones
up to 5 mm. long, 1:2 mm. broad, narrow-obovate, usually obtuse to subacute, the
margins torn-ciliate. Outer bracts narrower and shorter. Ray florets about 70, the rays
1—5 mm. long, 0-3-1 mm. broad, in about 4 rows, ‘white’, ‘pink’. Disc florets up to
3 mm. long, 1 mm. broad. Receptacle: 2-4 mm. broad, 1-1:8 mm. high, truncate hemi-
spherical. Fruits 3:9-4 mm. long, 1:2-2-1 mm. broad, dark brown, obovate, flattened,
microscopically glandular distally and usually bearing a few white straight hairs on
each flat surface. Neck microscopically glandular.

Habitat: Forest land.

Range: Gippsland, Yarra River and Western Victoria, Hobart and Tasman Peninsula, in
Tasmania, Yorke Peninsula, Kangaroo Island and south-east of South Australia, and south-
west division of Western Australia.

Specimens examined :

Victoria: Gippsland (MEL); Yarra R., 10.1852, F. Mueller (MEL); Little R., Fullager
(MEL); Wimmera, 1890, J. P. Hckert (MEL); West Wimmera, Walter (MEL); Shire of
Dimboola, 2.10.1897, F. M. Reader (MEL); in dampish red gum forest about one mile east of
Cherry Pool, towards the Black Range, upper Glenelg River area, 4.11.1948, J. H. Willis
(MEL); Grampians, 10.1907, H. B. Williamson (MEL); Moyston, 10.1872, D. Sullivan (MEL):
Wando R., forest, 2.10.1842, J. G. Robertson (NSW).

Tasmania: Hobart, 11.1898, F. A. Rodway (FAR): Hobart, 12.1923, A. H. S. Lueas
(NSW); Domain, Hobart, 23.9.1943, W. M. Curtis (HO); Sandy Bay, 11.1894, L. Rodway
(HO); Port Arthur, 1892, J. Bufton (MEL).

South Australia: Wirrabara forest, 10.1889, W. Gill (MEL); Yorke Peninsula, 1888,
Beythieu (MEL); Tanunda, 9.1847, ?F. Mueller (MEL); South Para R., under trees,

130 AUSTRALIAN SPECIES OF LAGENOPHORA CASS,

21.10.1887 (MEL) ; Lyndoch, 8.10.1927, J. B. Cleland (JBC); Forest Range, “ligule very short”,
14.10.1934, J. B. Cleland (JBC); National Park, Belair, 5.11.1905 (JBC); Lofty Ranges, F.
Mueller (MEL); Mt. Lofty Range (JMB); Kuitpo, 23.9.1928, J. B. Cleland (JBC); Mt.
Compass, ‘‘rays apparently very short”, 14.10.1934, J. B. Cleland (JBC); Port Elliot, 9.1896,
O. E. Menzel (NSW, AD); Hall’s Creek, Encounter Bay, 23.5.1932, J. B. Cleland (JBC);
Encounter Bay, 29.10.1934, J. B. Cleland (JBC); Mulgundawa, Lake Alexandrina, 3.10.1906

(JBC).

Text-figures 11-13, L. Huegelii.
11, habit, x 4; 12, fruit, x 10; 13; distribution.

Kangaroo Island: Waratta, “Grassy ground, riverside, flowers pink’, 9.11.1886, O. Tepper

(MEL).

Western Australia: South Hutt, Oldfield (MEL); Darling Ranges, ‘rays very short, white’’,
9.1907, M. Koch (MEL, PERTH); close to Perth, Preiss No. 118, “emi” 1843 (MEL); near
Perth, 7.1911, F. Stoward (NSW); South Perth, 15.7.1923, W. M. Carne (PERTH); Midland
Junction, 19.7.1897, R. Helms (NSW); Claremont, ‘‘in sandy soil, ray white’, 6.1901, W. V.
Fitzgerald (NSW); Cottesloe, 8.1908, J. B. Cleland (NSW); Swan R., Oldfield (MEL); North
Fremantle, 10.7.1897, R. Helms (NSW); Armadale, 10.1911, F. Stoward (NSW); between Swan
River and Geographe Bay, 1881, J. Forrest (MEL); Busselton, 1870, A. E. Pries (MEL) ; Black-
wood River, 1875, Hester (MEL); Vasse R., Oldfield (MEL); Yallingup, 10.1909, J. H. Maiden
(NSW); Lowdon, 8.1909, M. Koch (NSW); Big Brook, Warren District, M. Koch, No. 2584

BY GWENDA L. DAVIS. 131

(BRI); Gordon R., “wet places”, Oldfield (PERTH); Tenterden, 23.9.1902, A. Morrison
(PERTH); Stirling Range (MEL); Kalgan R., “grassy places’, Oldfield (MEL); Mouliup,
Albany, 3.10.1930, E. Elder (FAR); King George’s Sound, 1889, Clarke (MEL).

The original specimens handled by Bentham were collected by Hiigel from the Swan
River and King George’s Sound, but unfortunately are not available in Australia, and
type selection has not been possible. In a redescription of this species (1866) Bentham
lists a longer series of specimens, including ‘“‘Wando R., J. G. Robertson’, and three
specimens from Robertson’s collection bearing this locality label with the date
(2.10.1842) are in the National Herbarium, Sydney. In the absence of the type, these
specimens have been used as a basis of comparison.

Among specimens from Steetz’s collection in the National Herbarium, Melbourne,
is one bearing the type data of Lagenophora Gunniana (‘In insula Van Diemen leg. cl.
Gunn (Herb. Gunnian, No. 510)’’), which has been nominated lectotype of this species,
though the name was reduced to synonymy by Bentham (1866).

The relationship between L. Huegeliit and L. stipitata is close and specimens of each
species have been examined which tended towards the other. Typical plants of
L. Huegelui are robust and rather coarse, with relatively large leaves on the scapes, but
more slender specimens are sometimes seen which approach L. stipitata in appearance.
In all cases, however, the broad scape leaves are diagnostic of L. Huegelii in the
vegetative state. Certain specimens from the lower south-west of Western Australia
(Big Brook, Gordon River, Yallingup, Lowdon) were much more slender than usual,
and consequently were not unlike L. stipitata. In each case, however, well-developed
bracts were present on the scapes and the fruits, though not mature, were more robust
than in L. stipitata.

A number of specimens of L. Huegelii were examined in which the rays were
extremely short, not exceeding 1 mm. in length. Although a discontinuity in this
character appears to be present, breeding experiments and a much larger series of
specimens are necessary before this variation can be more than recorded.

In view of their distribution, these two species might be regarded as subspecies, but
the present writer is of the opinion that although both populations undoubtedly arose
from a common ancestor comparatively recently, they have now diverged beyond
subspecific status and are best regarded as distinct but closely related species. Text-
figures 5-8, although drawn from L. stipitata, are equally applicable to L. Huegelii.

ry

ACKNOWLEDGEMENTS.

In carrying out this work I am indebted to the Directors of various herbaria and private
collectors who generously placed collections at my disposal. I would like particularly to
acknowledge the co-operation in this respect of Mr. R. H. Anderson of the National Herbarium,
Sydney, Mr. A. W. Jessep and Mr. J. H. Willis of the National Herbarium, Melbourne, Mr.
C. T. White of Brisbane Herbarium, Professor J. G. Wood, Professor J. B. Cleland and Mr.
J. M. Black of Adelaide University, Mr. C. A. Gardner of Perth Herbarium, Miss W. Curtis of
the University of Tasmania, Dr. F. A. Rodway of Nowra, and Mr. N. A. Wakefield of Cann
River, Victoria. ‘

Finally, I am indebted to Professor R. Pichi-Sermolli of the University of Florence for

sending photographs of Labillardiére’s specimens in that herbarium, one of which is reproduced
in this paper, and carrying out examinations of these specimens on my behalf.

References.

Baitey, F. M., 1900.—Queensland Flora, iii: 793-794, Brisbane.

BENTHAM, G., 1837.—Enumeratio Plantarum quas in Novae Hollandiae ora austro-occidentali ad
Fluvium Cygnorum et in sinu Regis Georgii collegit Carolus Liber Baro de Hiigel, p. 59,
Vienna.

, 1866.—Flora Australiensis, iii: 506-508, London.

Buack, J. M., 1929.—Flora of South Australia, iv: 580-581, Adelaide.

Cassint, H., 1816.—Bulletin des Sciences de la Société philomatique de Paris, p. 199.

, 1818.—Ibid., p. 34.

, 1822.—Dictionaire des Sciences naturelles, xxv: 111.

, 1828.—Ibid., Ivi: 176.

Davis, G. L., 1948.—Revision of the genus Brachycome Cass., Part I. Australian species. Proc.
Linn. Soc. N.S.W., Ixxiii: 142-241.

L =

132 AUSTRALIAN SPECIES OF LAGENOPHORA CASS.

Davis, H. F. C., 1941.—Taxonomic Categories. Aust. Jour. Sc., iv, No. 2: 49-52.

—————., and Len, D. J., 1944.—The Type Concept in Taxonomy. Ibid., No. 1: 16-19.

Dp CANDOLLE, A. P., 1836.—Prodromus Systematis naturalis regni vegetabilis, v: 307.

Druce, —, 1916.—Report of the Botanical Exchange Club of the British Isles, p. 630.

Ewart, A. J., 1930.—Flora of Victoria, pp. 1090-1091, Melbourne.

Hooxer, W. J., 1847.—London Journal of Botany, vi: 113.

LABILLARDIERE, J. J. DB, 1806.—Novae Hollandae plantorum specimen, ii: 55, Paris.

LEHMANN, C. (Ed.), 1845.—Plantae Preissianae, i: 428, 431.

Maipen, J. H., 1916.—Census of New South Wales Plants, p. 196, Sydney.

Moore, C., and BretrcHe, E., 1893.—Handbook of the New South Wales Flora, pp. 261-262,
Sydney.

Ropway, L., 1903.—Tasmanian Flora, pp. 77-78, Hobart.

WALPERS, W. G., 1843.—Repertorium botanices systematicae, ii: 584.

DESCRIPTION OF PLATE VII.
Photograph of sheet of specimens in the herbarium of the University of Florence collected
in Australia by Labillardiére and named by him Bellis stipitata.

CORRIGENDUM TO REVISION OF THE GENUS BRACHYCOME CASS. PART III.
(Proc. Linn. Soc. N.S.W., xxiv (1949), p. 151.)

The record of Brachycome Mulleroides from the Snowy River is erroneous. These specimens
have since been shown to be B. ptychocarpa.

133

A REVIEW OF AND CONTRIBUTION TO KNOWLEDGE OF PHYLLOGLOSSUM
DRUMMONDII KUNZE.*

By FrRAnNcES M. V. HAcKNEy, D.Sc.
(Twenty Text-figures. )
[Read 31st May, 1950.1]

INTRODUCTION.

The genus Phylloglossum (Lycopodiaceae) is monotypic, being represented by the
species Phylloglossum Drummondii. This species is limited in its distribution to parts
of Western and South Australia, Tasmania and New Zealand. The sporophyte
generation was first described by Kunze in 1843, and has received much attention from
Bower, Jeffrey, Osborn, Sampson and several other investigators. The gametophyte
generation is imperfectly known, the only original descriptions being those of Thomas,
Sampson and Holloway.

However, despite the fact that so much has been done to elucidate the morphology
and anatomy of the sporophyte, the literature on the subject is scattered and somewhat
fragmentary. There is need of a comprehensive and connected account of Phylloglossum
in its various aspects. Furthermore, the investigations carried out by the writer throw
a little more light on certain controversial aspects of the morphology and anatomy.
It was for these reasons that the present paper was prepared.

The sporophyte generation will be described and discussed first. The literature
dealing with the gametophyte generation and the embryo sporophyte will then be
reviewed.

The final section of the paper will be devoted to a discussion of the phylogenetic
status and affinities of Phylloglossum.

MATERIALS AND METHODS. '

Most of the material was obtained from Adelaide, South Australia. Some of this
material was germinated at Sydney University by Dr. P. Brough, who prepared from
it slides showing the development of the “dropper” and young tuber (see later). A
few of the plants examined were collected from Swampy ground near Cannington, a
suburb of Perth, Western Australia, by Miss A. M. Baird, University of Western
Australia. The plants from South Australia were all examined in the preserved
condition, but some specimens from Western Australia were still living while under
examination.

Method of Preparation for Microscopic Study.

The material was fixed in 1% chromoacetic acid, washed thoroughly in water and
subsequently passed through successive concentrations of alcohol from 10% to 100%.
It was then passed through mixtures of alcohol and chloroform, and thence to pure
chloroform; it was finally embedded in pure paraffin wax (melting point 51°-52°C.)
and serial microtome sections were prepared.

THE SPOROPHYTE GENERATION.
GROSS MORPHOLOGY OF THE PLANT.

Phylloglossum Drummondii is a small tuberous perennial which attains a total
length of 1:5 to 5-0 cm. during the growing season (autumn and early spring, i.e., May
to October, approximately). The general features of a typical well-developed fertile
plant are shown in Text-figure la. Above ground level appears a crown of simple
quill-like leaves, generally not more than 3 cm. in length. Poorly developed plants

* The investigations described in this paper were carried out while the writer was a fourth-
year honours student in Botany at the University of Sydney.

LL

134 PHYLLOGLOSSUM DRUMMONDII,

bear only one or two leaves, but well-developed plants may have as many as twenty.
A young plant in its first year produces only one leaf, and the number is usually
increased in the second year. However, the number of leaves produced does not depend
entirely on the age of the plant, since some young plants complete their first three
years of growth without producing more than one leaf in each growing season, while
others produce several leaves in their second year.

In the mature fertile plant (Text-figure la) the axis terminates in a comparatively
long peduncle, bearing a small cone. Measurements taken from various plants show
that the length of the cone varies from 0-4 cm. to 1:0 cm., approximately, according to
the size of the plant. The peduncle is nearly always unbranched, but rare specimens
have been reported in which the peduncle bifurcates into equally well-developed portions.
Thomas (1901) states that “the branching always takes place above the lowest
sporophyll, sometimes quite at the base of the spike, near the lowest leaf (sporophyll),
sometimes further up, or even close to the apex of the strobilus’. However, Bower
(1908, p. 297) presents a figure in which the peduncle is branched below the lowest
sporophyll, the two branches being unequally developed.

The cone bears short, rather fleshy sporophylls, each of which gives rise to a
single reniform, homosporous sporangium on its adaxial surface. On dehiscence the
valves gape, allowing the yellow spores to be shed gradually.

From the region of the stem at the base of the crown of leaves arise from one to
three relatively long, thick, unbranched roots, which run horizontally just below the
surface of the ground. These roots are covered throughout their length with root hairs.
Also embedded in the soil are two stalked tuberous bodies; one of these, referred to
as the “current tuber’, has given rise to the present year’s growth; the other is the
“young tuber’, by means of which the plant is enabled to perennate. The stalk,
or “dropper”, of the young tuber arises from the stem of the plant which has been
produced by the growth of the current tuber.

All vegetative parts of the plant except the young tuber die at the end of the
growing season. The young tuber lies dormant in the soil until the following autumn,
when growth is initiated. Germination of tubers to produce either sterile or fertile
plants has been described by Bower (1885) and will be referred to later in this paper.

The Relation of External Morphology to Ecological Conditions.

Osborn (1919) has made some observations on the ecology of Phylloglossum
growing near Adelaide, South Australia. He points out that there are two sets of
climatie conditions which tend to alter the ground level in the area where Phylloglossum
has been studied by him. During the dry summer months the surface of the soil may
become desiccated and wind erosion may take place. During the wet winter months,
or even following occasional heavy summer rains, there may be a flow of water over
the surface of the soil. This results in erosion of soil from some areas and subsequent
deposition of soil over other areas. Alterations in ground level due to these climatic
factors would not amount to more than a few millimetres rise or fall, but these
variations could influence the growth of a plant as small as Phylloglossum.

Osborn has paid special attention to the behaviour of the tuber as an organ of
perennation. The developing “dropper” or young tuber stalk usually attains such a
length as to cause the young tuber and the current tuber to lie side by side in the
soil. However, the young tuber is sometimes formed several millimetres above or
below the current tuber. Osborn considers that “the depth at which the new tuber is
formed in the soil in relation to ground level is not a matter of chance, but is the
result of definite growth of the plant producing it. If for any reason the current tuber
is buried by deposition of more soil above it, the burial is compensated for by the
development of a short stalk bearing the new tuber. Conversely, if, by removal of the
surface soil, the current tuber is brought near the surface, the new tuber is sunken
to a greater depth.” Statistical analysis of data showed that over 80% of the plants
examined formed their new tubers at depths of 9 to 12 mm.

BY FRANCES M. V. HACKNEY. 135

The existence of Phylloglossum in some areas is very precarious, being threatened
alternately by burial and desiccation, as a result of the ecological conditions under
which it grows. In plants where considerable adjustment for depth of tuber has to
be made, or where the early onset of the dry season halts growth, the new tuber may
be smaller than that produced in the previous growing season. Thus the plant does
not always develop steadily from season to season towards its maximum size.

From the number of old tuber coats in the immediate vicinity of the plant it is
sometimes possible to assess its age. Osborn gives figures showing a single-leaved
plant entering on its fourth year, a two-leaved plant in its fifth year, and a three-leaved
plant entering on at least the sixth year of its existence.

Vegetative Multiplication in Phylloglossum.

The tuber of Phylloglosswm is generally regarded as an organ of perennation rather
than of vegetative multiplication. As a rule, only one tuber is formed by each plant
at the end of the growing season. Occasionally, however, two tubers are formed.
Thomas (1901) was the first to observe double tuber formation in Phylloglossum. He
recorded that this phenomenon occurred most frequently in larger plants, but sometimes
occurred in smaller plants. One plant possessing only a single leaf was observed by
Thomas to be forming two new tubers. The two tubers may arise on the same side
or on opposite sides of the plant, simultaneously or in succession. The present writer
observed only one small plant (three leaves) having two new tubers. In this specimen
the presence of two tubers appeared to have been due to the dichotomous branching
of a single dropper (see Text-figure 10).

The infrequency with which Phylloglossum forms more than one new tuber
indicates that double tuber formation is not of much significance as a method of
vegetative multiplication. Sampson (1916) mentions five plants, out of a collection of
one hundred, which possessed two new tubers apiece. The present writer, after
examining over one hundred plants, has found only one possessing two new tubers.

Osborn (1919) has recorded and described the regeneration of Phylloglossum plants
from detached leaves. This method of vegetative reproduction may be compared with
that of Lycopodium ramulosum, in which gemmae are produced from old roots and
detached leaves (Holloway, 1916). Osborn first observed vegetative reproduction of
Phyloglossum in the field and later succeeded in promoting it experimentally in the
laboratory. Detached leaves kept on moist soil under favourable conditions may retain
turgidity and greenness for two or three months. Curvature of the leaves in a vertical
plane is a frequent phenomenon preliminary to the initiation of vegetative propagation.
The first step in vegetative propagation is the development of one or more irregular,
greenish-yellow cell masses at or near the proximal end of the leaf. From a growing
point differentiated on the lower surface of the cell mass a dropper and tuber develop
which resemble those of a mature plant. Occasionally more than one dropper and
tuber develop from one cell mass, due to the initiation of more than one meristematig
zone. After the development of the tuber one or more minute “leaflets” may be
developed from the cell mass. The young plant, consisting of dropper, tuber and
“leaflets”, is then complete and capable of carrying on an independent existence.
Text-figure 2 shows diagrams indicating stages in this method of reproduction (drawn
by the writer from Osborn’s description).

Osborn was unable to give any estimate of the frequency with which new plants
were formed from detached leaves under field conditions. When plants of Phylloglossum
are turgid the leaves are very brittle. In Osborn’s opinion, a blow from a falling
twig or a wind-swept leaf might be sufficient to break them. Under suitable conditions
production of new plants from detached leaves might be of considerable importance
in the field.

DETAILED MorPHOLOGY AND ANATOMY OF THE SPOROPHYTE GENERATION.
THE ROOT.
It is unusual for a root to be formed during the first year or two of the life of
the plant. The function of absorption during this time is presumably carried on directly

136 PHYLLOGLOSSUM DRUMMONDII,

Text-figures 1, 2, 4, 5.*

Figure 1, a and b. Two mature plants of Phylloglossum. Figure 1a represents'a typical
well-developed fertile plant, while Figure 1b represents a small sterile plant bearing two new
tubers (abnormal). Figure la, x 33; Figure 1b, x 5%. n.t. = new tuber; o.t. = older tuber; 1 =
leaf; r = root; e = cone; horizontal line represents soil level.

Figure 2, Diagrams A to E. Vegetative reproduction of Phylloglossum from detached
leaves (see text), x 3%. cm. = cell mass; d = dropper or stalk of tuber; m = meristematic zone;
lm = “leaflet’’? meristem; 1 = “leaflet”; p = invaginated growing point; t ='tuber. Drawn from
description by Osborn (1919).

Figure 4, Diagrams A to F. Diagrams illustrating Bower’s description of germination of
the tuber at the onset of the growing season (See text), x 4%. A = one-leaved stage; B = three-
leaved stage; C = five-leaved stage; D = apical growing point depressed; E = root and dropper

of tuber developing; F = almost fully developed sterile plant; a = apical growing point;
1 = leaf; ti: = tuber; ad = depressed apical growing point; r = root; d = dropper of new
tuber; te = new tuber.

Figure 5, a and b. Development of the young tuber in a fertile plant (x 9) (from a slide
belonging to Dr. P. Brough, University of Sydney). Figure 5a@ shows the dropper beginning to
develop, with the meristematic apex directed downwards. Figure 5b shows the young tuber
at a later stage in longitudinal section, with the meristematic apex directed upwards and
enveloped in the invaginating tissue. See text for details. ect = current tuber; os = remains of
dropper of ct; m = meristematic apex of new tuber; d = dropper; r = root; p = peduncle;
] = leaf; yt = young tuber; it = invaginating tissue.

* For Text-figure 3 see page 139.

BY FRANCES M. V. HACKNEY. 137

through the tuber wall, which bears many rhizoid-like outgrowths. Specimens past the
second growing season usually bear one to three long, straight, unbranched roots,
covered with numerous root hairs. Bower (1885), describing the germination of the
tuber at the onset of the growing season, showed that the root is of exogenous origin.
It originates as an excrescence on the surface of the main axis. Furthermore, the
superficial cells of the very young root undergo both periclinal and anticlinal divisions,
so that there is no clearly marked dermatogen. As development proceeds, periclinal
divisions become more and more frequent at the growing tip; this results in the
formation of a root cap.

The internal structure of the mature root is very simple (see Text-figure 3). The
epidermal cells give rise to unicellular root hairs and enclose a broad cortex with
large intercellular spaces. In the material examined by the writer the outer cortex
had a distinctly lacunar appearance in both longitudinal and transverse section. This
feature was noted by Wernham (1910), but other writers do not mention it. It is
too definite to be an artifact due to the method of fixation of the tissues. It has
probably been developed in response to the high moisture content of the habitat and
consequently will vary in degree in material from different localities. The presence
of lacunae in the lower (subterranean) regions of the stem (see later) and of large
air spaces in the aerial parts of the plant may also be due to the same cause.

The cortex is bounded internally by the endodermis. In Phylloglossum the root
is the only organ in which an endodermis has been recognized. The stele consists
of xylem elements surrounded by small, closely packed parenchymatous cells. In the
root figured above (Text-figure 3) the xylem was diarch, but in other roots examined
it was monarch. Bower refers to the small parenchymatous cells surrounding the
xylem as phloem. They differ from the cortical cells in having smaller dimensions
and lacking intercellular spaces, so probably represent a rudimentary or reduced type
of phloem. In longitudinal section they were observed to be elongated, with oblique
end walls. They were similar in all respects to the “phloem” cells shown in Text-
figures 15, a and b, occurring in the stem and leaf.

THE TUBER.

For many years the morphology of the tuber was a puzzle to botanists. Bower
(1885) observed and described in detail the germination of a number of tubers of
Phylloglossum. A brief résumé of his observations will be of interest at this stage.
At the time of Bower’s work the structure of the tuber was not understood. The
tuber was misinterpreted as being homologous with the protocorm of Lycopodium (see
later section). Consequently, parts of Bower’s original description of its germination
are presented in terms which are now known to be inapplicable. However, the actual
observations recorded, are accurate, and, in more up-to-date terms, can be summarized
as follows. At the onset of germination the growing point sunken in the apex of the
tuber elongates to give rise to the new axis of the plant. The first leaf then makes its
appearance as a lateral outgrowth upon the axis. Other leaves are then produced to
form a crown. According to Bower, these later leaves are usually produced in pairs
“with some irregularities of arrangement’. However, later and more detailed work
indicates that the arrangement of the leaves is more generally a much-compressed
spiral (see later section). Bower records that “in plants which do not produce
cones, the apex of the plant becomes depressed after the production of the leaves and
the young tuber begins to develop from a growing point in the centre of this depression.
As a result of specially localized intercalary growth, the growing point of the tuber
gradually becomes inverted; the tuber stalk elongates and enters the soil. Sub-
sequently the meristematic apex of the tuber is turned once again into the vertical
position as a result of increased growth on one side. During this period of growth
the first root arises from the side of the plant opposite to that on which the new
tuber is being formed.”

Text-figure 4 shows a series of diagrams drawn by the writer from Bower’s
description of germination as above.

138 PHYLLOGLOSSUM DRUMMONDII,

In those plants which produce cones the apex does not become depressed, but
srows up in an erect position and produces the sporophylls. In such plants the young
tuber is recorded by Bower as developing from a growing point in a depression near
the base of the peduncle. ;

Bower described the mode of germination of Phylloglossum as “‘strikingly similar
to the embryonic stages of Lycopodium’. Believing the tuber of Phylloglossum to be
homologous with the protocorm of Lycopodium cernuum, he was of the opinion that “we
may regard Phylloglossum as a form which retains, and repeats in its sporophore
generation, the more prominent characteristics of the embryo as seen in Lycopodium
cernuum”’. He concluded that Phylloglosum was “a permanently embryonic form of
Lycopod”, regarding it as the most primitive in structure of all living Pteridophytes.
However, detailed anatomical studies reveal striking differences between the tuber of
Phylloglossum and the protocorm of Lycopodium. The resemblances between the two
structures are superficial. The phylogenetic status and affinities of Phylloglossum will
be discussed in detail later in this paper.

The tuber, whether that of a sterile or a fertile plant, develops exogenously.
Text-figure 5 shows the young tuber developing in a fertile plant. During development
the orientation of the growing point is gradually inverted, owing to increased
meristematie activity on one side of the distal region of the dropper. A continuation
of this growth gradually invaginates the organic apex, which consequently is no
longer visible externally. This development finally leaves the growing point at the
base of a long narrow canal.

The mature tuber consists of a stalk or dropper and a swollen basal storage
region (the tuber proper). ‘Text-figure 6 shows the young tuber in longitudinal
section. The bulk of the tuber proper consists of thin-walled storage parenchyma.
This tissue is composed of rather large isodiametric cells with very few and very
small intercellular spaces. Hach cell has a prominent nucleus and is packed with
minute starch grains. The density of the cell contents here is in marked contrast
with those of the corresponding cells of the current tuber, which are almost exhausted
of carbohydrate at the end of the growing season. The storage tissue is surrounded
by several layers of elongated, transparent cells with scarcely any contents. There
is a well-defined epidermis, consisting of cells which are elongate in longitudinal
section and have pits and peculiar thickenings on their radial walls (cf. Bower, 1885).
There is a nucleus in each epidermal cell. Some of the epidermal cells give rise
te prolonged root-hairlike outgrowths which may be cut off from the parent cells
by walls.

The meristematic apex appears as a cone of tissue above the storage region.
The cells have large nuclei, each with one nucleolus, and dense cytoplasmic contents.

Above and around the meristematic apex is the narrow canal formed by the
growth of the invaginating tissue. This canal has been traced from the region
immediately above the organic apex to the top of the mature dropper.

Unlike those of the tuber proper, the epidermal cells of the dropper are not
thickened. The main tissue of the dropper is composed of narrow, elongated paren-
chymatous cells with pointed ends. The vascular strand is not in the centre of the
dropper, but on one side of the central canal, from which it is separated by some
parenchymatous cells. It is composed of weakly lignified scalariform tracheids,
similar in shape to the parenchymatous cells. In the region encircling and enclosing
the meristematic apex of the tuber is an inverted funnel of tracheidal tissue. This
has been described by both Wernham and Osborn as an expansion of the vascular
strand of the tuber stalk. Such an interpretation may be true, but, on the other
hand, the tissue in question may have been formed by the convergence of several

small tracheidal strands developed independently of one another in the invaginating
tissue.

BY FRANCES M. V. HACKNEY. 139

There is apparently no highly developed phloem in the tuber stalk. Around the
tracheids of the tuber strand are a number of elongated parenchymatous cells which
may represent a rudimentary type of phloem (cf. those already described in the root).

The anatomy of the tuber has been further studied from transverse sections cut
at various levels. The storage tissue appears much the same in transverse section
as in longitudinal section (see Text-figure 7a). It is surrounded by parenchymatous
cells of the inner wall, with no sign of vascular tissue. The epidermal cells are oblong
in transverse section and the thickenings on their radial walls are plainly seen. These
thickenings are well developed and markedly striated. Bower (1885) reported that the

12'S) DP

—_s
TD ep
ie ae

—-

eae sy
ane

Text-figures 3, 6, 7.*

Figure 3. Transverse section of root, x 100. px = protoxylem; mx = metaxylem; 1] = lacuna:
rh = root hair.

Figure 6. Longitudinal section of young tuber, x 40. d= dropper; c = canal; t = tracheids;
ma = meristematic apex; st = storage tissue; e = epidermis.

Figure 7, ad, b and c. Figure 7a: epidermis and storage tissue as they appear in transverse
section of the new tuber, x 80. Figure 7b: tissues seen in transverse section of young tuber
at level of meristematic apex; m = meristematic tissue; x = canal; t = tracheidal cell.
Figure 7c: tissues seen in transverse section of dropper ; px = protoxylem ; mx = metaxylem.

thickenings gave a mauve colour with Schulze’s solution and were not stained with
aniline sulphate; he concluded that they were not lignified. In some of the sections
cut by the writer the thickenings stained red with safranin, suggesting lignification,
but in other sections they remained unstained. When sections from mature tubers
were treated with sulphuric acid and iodine the thickenings remained colourless,
indicating that they did not consist of cellulose.

* Wor Text-figures 4 and 5 see page 136.

140 PHYLLOGLOSSUM DRUMMONDII,

In a transverse section of the tuber at the level of the apical growing point the
latter appears as a central circular mass of meristematic cells, separated from the
surrounding tissue by the canal (see Text-figure 7b). The parenchymatous cells of
the invaginating tissue are roughly isodiametric in transverse section and their
contents are scanty. There is a small nucleus in each cell. The epidermal cells are
thickened as described above. In transverse section the extent of the vascular
tissue in the invaginating tissue is noteworthy. In some specimens it is represented
by a few degenerate tracheids grouped on one side of the canal. This type of vascular
system appears to be confined to the smaller tubers. In the larger ones a more
interesting development is seen, inasmuch as a more or less complete ring of degenerate
tracheids appears in the invaginating tissue. This ring corresponds to the rim of
the funnel of tracheids seen in longitudinal sections of a large tuber (Text-figure 6).
In transverse sections of the tuber at the level of the meristematic apex these tracheids
are seen in oblique section. The thickenings on their walls are not pronounced and
do not stain with safranin. Those in hand-cut sections of a mature tuber gave the
cellulose reaction with sulphuric acid and iodine. Evidently lignification does not
take place here. Text-figure 7c shows the dropper of the tuber in transverse section.
The mesarch nature of the vascular strand is clearly evident.

The diagrams shown in Text-figure 8 represent the vascular tissue seen in a
series of transverse sections at various levels of a large tuber and its dropper (from
a fertile plant). Text-figure 8 A shows the groups of degenerate tracheids arranged
in a ring in the invaginating tissue around the meristematic apex. The circular
arrangement does not persist for a great distance up the dropper; in a section cut
at a level slightly higher than 8 A it has given place to a horseshoe-shaped arrangement,
8B. Text-figure 8C fat a higher level than B) shows the tracheidal strands arranged
in a semicircle. In the succeeding diagrams the vascular strand of the tuber stalk
appears as a broken arc of xylem with its concave side directed away from the main
body of the plant. The shape and orientation of similar bundles have been noted
by Sampson (1916), who pointed out that in several well-developed plants examined
the form of the stele changes as it passes down the dropper. When it is first.
separated from the stele of the main stem the tuber stele is convex inwards; by the
time it enters the dropper the curvature is in the opposite direction. In well-developed
plants the tuber stele sometimes makes a sharp upward bend after passing out from
the main stele and before going down the dropper. Sampson’s observations of stelar
behaviour in well-developed plants have been confirmed by the present writer.

In the droppers of small tubers examined by the writer the vascular strand is
not horseshoe-shaped, but small and compact in transverse section (see Text-figure
8D). -~ The protoxylem of all vascular strands is mesarch and there is no definite
bundle sheath.

THE STEM.

'The vascular system of the stem of Phylloglossum is extremely variable. A number
of stems of fertile plants of all sizes were examined by means of serial transverse
sections. The extent of development of vascular tissue is greater in larger plants
than in smaller ones.

Below the departure of the root traces there is no vascular tissue in the stem.
The root traces are continuous with the stele of the main axis before they pass out
into the roots. The series of diagrams in Text-figure 9 demonstrate this in a plant
bearing a single root. The young tuber and its vascular strand are also included in
these diagrams. Text-figure 9A represents a transverse section of the axis of the
plant cut below the point of origin ‘of the root. The dotted regions are lacunae. The
dropper of the young tuber is seen in section alongside the main axis. In 9B the
root trace is seen in oblique section, moving out from the main axis. Note the
lacunar tissue in the root. Text-figure 9C represents a transverse section cut at a
higher level than 9B. The stele of the main axis is now recognizable as such, the
root trace having lost its identity. The dropper of the young tuber is now shown

BY FRANCES M. V. HACKNEY. 141

joined to the main axis; the tuber stele is still seen in transverse section. In 9D the
main stele, with perforations, is again plainly shown. The tuber stele is seen in
oblique section, moving through the cortex of the main axis.

The diagrams shown in Text-figure 10 were made from sections of a plant bearing
two roots. “ In this specimen two root traces are continuous with the stele of the
main axis.

Behaviour of the Stele of the Stem in Fertile Plants.

(A) Medium-Sized Fertile Plants.

Text-figure 11 shows the distribution of vascular tissue in portion of the stem of
a typical medium-sized plant. Text-figure 11 A represents a cross section cut below
the departure of the tuber strand, the young tuber being included in the diagram.
The stem stele in the base of this specimen was a solid protostele of irregular shape.
In some specimens the protostele is medullated and the xylem is broken up into a few
irregularly anastomosing strands. Text-figure 11 B represents a section at a slightly
higher level than 11 A, at the junction of the dropper and the main axis. The stem
_ stele is as before, but the tuber trace is seen in oblique section as it moves across
to pass into the dropper. Text-figure 11 C shows a section a little higher up the stem.
The stele appears here to be a medullated protostele with two arcs of xylem, the
smaller of which is the tuber trace. The next diagram (11D) shows the stele just
above the departure of the tuber trace. There is a distinct gap in the xylem of the
main stele, apparently due to the departure of the tuber trace. This gap is a constant
feature of the stele in this region, and in larger plants is even more pronounced than
here. In the present specimen the first leaf trace has already separated from the
right-hand side of the main stele in 11D. In 118# the leaf trace has moved into a
position directly opposite the centre of the main stele. At first sight and without a
knowledge of 11D, the leaf trace would appear to be responsible for the gap in the
main stele, but this, of course, is not so. The shift in position of the leaf trace was
observed in several specimens. Jeffrey (1908) observed similar instances of leaf
traces occupying misleading positions opposite stelar gaps for which they were not
responsible. In Text-figure 11 F another leaf trace has been given off, leaving no gap.
In 11G the third leaf trace has been given off, and a second gap has appeared in
the central stele, opposite the second leaf trace. Since it makes its appearance after
the leaf trace has been given off, this gap is merely a perforation and not a leaf gap.
In 11H two leaves have separated off and the base of the third is shown still
attached to the stem. At this level the leaf trace is not in the centre of the leaf, as
it is higher up. The stem stele has now passed into the base of the peduncle. Its
irregular behaviour is obvious from 11G and H. It is a medullated protostele in
which perforations appear which form no particular pattern.

(B) Stems of Large Fertile Plants.

The behaviour of the stele in a fertile plant much larger than the above is
shown in Text-figure 12. The section represented by the first diagram (12 A) was
cut at a level comparable with that at which that shown in Text-figure 11D was cut.
The stele in this region is a comparatively large, horseshoe-shaped protostele, from
which four leaf traces have already been given off. The gap where the tuber stele
has been given off is large and distinct.

Text-figure 12 B represents a section cut a short distance above that shown in
the preceding diagram. The stele is now clearly a medullated protostele with
irregular perforations in the xylem. In many specimens the tuber gap persists as far
as the base of the peduncle, but in this specimen it has been bridged by tracheids
a short distance above the departure of the tuber strand.

Vestigeal Leaf on Tuber Stalk.

The diagrams in Text-figure 13 show the arrangement of the vascular tissue
in another large fertile plant. This plant is of special interest because it exemplifies
the occurrence of what is probably a vestigeal leaf arising from the tuber stalk.

142 PHYLLOGLOSSUM DRUMMONDII,

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c: 15D

Text-figures 8-13.

Figure 8, Diagrams A to G. Diagrammatic representation of arrangement of vascular
tissue in transverse sections of a large tuber and its dropper. Vascular tissue is indicated
by black masses; m = apical meristem of tuber; ec = canal. See text for details.

Figure 9, Diagrams A to D. Diagrammatic representation of arrangement of vascular
tissue (black) in a series of transverse sections of the base of the stem in a plant bearing
one root (x 12). 1 = lacuna; T = vascular strand of tuber; R = vascular strand of root;
S = vascular strand of stem. See text for details.

Figure 10, Diagrams A to D. Diagrams showing the distribution of vascular tissue
(black) in a series of transverse sections of the base of the stem in a plant bearing two
roots (x 12). Lettering as in Figure 9. See text for details.

BY FRANCES M. V. HACKNEY. 143

Mettenius (1867) observed a small ligulate piece of tissue, frequently found above the
new tuber. He regarded this as an atrophied leaf, since he observed a series of
transitional forms between it and the normal leaves of the plant. Bertrand observed
a similar body and named it the “organe de Mettenius”. His material showed no
transitional leaves. Bower (1886) noticed that in several specimens examined by
him a leaf placed above the new tuber was smaller than the other leaves. He referred
to this as the “supernumerary leaf”’.

In several large specimens, Sampson (1916) observed a small hump of tissue
borne in the angle between the new tuber and the peduncle of the cone. In some
specimens the hump of tissue was visible only in a microscopic examination; in others
it was visible to the naked eye. Occasionally a small but otherwise normal leaf was
found in its place. Sampson observed a complete series of transitional forms between
the microscopic outgrowths of tissue and the small normal leaves.

Text-figure 13 A represents a section just above the point of attachment of the
young tuber to the base of a large fertile plant examined by the writer. Three root
traces and the vascular strand of the young tuber appear at this level, in oblique
section, entering the stem. In Text-figure 13B the root traces have converged to
form an irregular ring of tracheids in the centre of the stem. The young tuber
strand is moving through the cortex. The next development is of interest (13C). The
stem stele becomes a complete ring of xylem, distinctly medullated, from which seven
leaf traces are given off almost simultaneously; meanwhile the young tuber trace (T)
occupies a position very close to the central stele. In 13D perforations have appeared
in the xylem cylinder; the tuber trace is still distinctly visible (in oblique section at
this level). Text-figure 13 HE shows the tuber trace connected with the main stele.
Of particular interest is the presence of a tiny group of tracheids, running from the
tuber trace into the small hump of tissue just opposite the tuber trace. This hump
probably represents a vestigeal leaf and corresponds to those described by Sampson
(1916). The group of tracheids may be interpreted as the vascular trace of this
vestigeal leaf.

Just above the region where the young tuber strand joins the main stele, a gap
similar to that previously described for another plant (Text-figure 11) becomes apparent
(Text-figure 13F). As in the specimen previously described, this gap seems to be
definitely associated with the exit of the tuber trace. In ascending the main stem
further, the stele, with its associated leaf traces, repeats the type of behaviour shown
in Text-figure 11. It is worth noting that, in some large fertile plants, Sampson
observed that the tuber stele made a sharp upward bend as it passed out through the
cortex of the main stem towards the dropper. A similar bend was observed in one
plant by the present writer.

Behaviour of the Stele in Sterile Plants.

In some plants the roots and the characteristic crown of leaves are present and
a new tuber is formed, but no cone is produced. Text-figure 14 presents a diagrammatic
reconstruction of the vascular system of a typical sterile plant, built up from a
series of longitudinal sections. In this specimen a single root trace enters the base
of the short stem to join the main stele. Four leaf traces are given off from the
main stele. The latter assumes what can only be described as a broken-up, diffuse
appearance just below the point at which the leaf bases separate from the very short

Figure 11, Diagrams A to H. Distribution of vascular tissue (black) in portion of the
stem of a typical medium-sized plant (x 12). See text for details. S = vascular strand of
stem; T = vascular strand of tuber; L = leaf trace.

Figure 12, Diagrams A and B. Behaviour of the stele in a large fertile plant (x 12).
See text. S = stele of stem; L = leaf trace; P = typical perforation in xylem; = gap
in stele. ‘

Figure 13, Diagrams A to F. Arrangement of vascular tissue (black) in a large fertile
plant (x 12). See text for details. T = tuber strand; R = vascular strand of root; S = stele
of stem; L = leaf trace; vlt = probable vascular strand of vestigeal leaf.

144 PHYLLOGLOSSUM DRUMMONDII,

stem. In transverse sections of sterile plants the stele appears in oblique section in
this region. Except for the leaf traces there is a complete absence of vascular tissue
from the upper part of the stem. There is no sign of vascular tissue in the stalk of
the current tuber. It must be remembered that this is not the original dropper of
the current tuber, corresponding to that seen in connection with the new tuber, but
a new structure produced during germination of the tuber by growth of its apical

Text-figures 14, 18, 20.*

Figure 14. Diagrammatic representation of the vascular system of a typical sterile plant,
built up from a series of longitudinal sections (x 6). 1 = leaf; lt = leaf trace; ss = stele of
main stem; ts = tuber strand; yt = young tuber; ot = old tuber. See text for details.

Figure 18. Longitudinal section of a mature cone (x 30). S = sporophyll; sp = spor-
angium; st = sporophyll trace. :

Figure 20, A,.B, € and D. Diagrams illustrating the development of the embryo as
described by Thomas. See text for full details. G = gametophyte, or prothallus; F = foot;
L = leaf; S = stem apex. The dotted line represents soil level.

meristem. Traces of vascular tissue may be seen in the remains of the original
dropper, which is now represented by some torn tissue around the top of the current
tuber.

There is usually no medulla in the stele of a sterile plant. The most striking
feature of the anatomy of sterile plants is the apparent disappearance of the stele
from the upper part of the stem. Sampson (1916) has also commented on this feature
and offers the explanation that it may be due to a sharp bend in the axis (cf. the bend
already mentioned as occurring in the tuber steles of some fertile plants). The
stele of the main axis may have bent over to pass down the dropper of the young
tuber. If this be so, the plant would consist of an unbranched axis bending over to

* For Text-figures 15, 16, 17, 19, see page 146.

BY FRANCES M. V. HACKNEY. 145

form the annual storage tuber. This explanation is all the more feasible in view
of the fact, already mentioned, that, in sterile plants, Bower (1885) has identified
the growing point of the new tuber with the stem apex.

Although in most of the sterile plants examined by the present writer the young
tuber is probably derived from the main stem apex, this is not true of all sterile
plants. Sampson describes one sterile plant (the largest examined) in which the
tuber was apparently a lateral structure, as in fertile plants. The stele of the stem
in this plant was medullated and showed the tuber gap characteristic of large
fertile plants.

It seems probable that there are two types of sterile plants; one type consists of
a simple unbranched axis bending over to form the annual storage tuber, the other
consisting of a branched axis, one branch of which forms the tuber while the other
is arrested in development before the formation of the peduncle.

Tissues Present in Stem and Leaf.

Text-figure 15a shows a transverse section of the stem of a large fertile plant above
the departure of the leaf traces. Text-figure 16a depicts a longitudinal section of the
same region. The stele is a medullated protostele in which the ring of xylem tracheids
is interrupted by perforations and surrounded by small closely packed parenchymatous
cells. One of the most striking features of the xylem is the presence of cavities in
the centres of the groups of tracheids. The writer observed a few small mesarch
protoxylem elements at the sides of these cavities (cf. Wernham, 1910). In the
transverse sections cut by the writer no tissue was discernible which could be
definitely identified as phloem. In longitudinal sections, however, certain large,
elongated cells were observed outside the xylem. These probably represent a rudi-
mentary or reduced type of phloem (cf. those seen in the root and tuber). Each of
these cells contains a prominent nucleus and some cytoplasm. No sieve areas are seen
on their walls, and they resemble the surrounding cells of the cortex in many respects,
including their staining properties. Bower (1885) mentions similar cells, pointing out
that they would probably serve the purpose of phloem, but both Wernham and
Sampson report failure to find any sign of phloem in the stem. No definite bundle
sheath can be identified in either stem or leaf.

Nothing unusual has been observed about the epidermis and cortex, except that
the intercellular spaces in the cortex are often very large. The large intercellular
spaces are best seen in Text-figure 16a.

Text-figure 150 shows a transverse section of a leaf; Text-figure 16b shows a
longitudinal section of the same tissues. There is a single leaf strand, usually with
a cavity in the centre. The cavities seen in Phylloglossum may aptly be compared
with the protoxylem cavities of such a type as EHquisetum. Where cavities are absent
their positions are occupied by protoxylem elements. The probable rudimentary or
reduced phloem, the cortex and the epidermis of the leaf are similar to those already
described for the stem.

THE PEDUNCLE AND THE CONE.

The peduncle is comparatively long and bare of leaves except for the sporophylls
arranged in a compact cone at the top. It is usually unbranched (see section on gross
morphology of the whole plant). No transitional forms occur between the vegetative |
leaves and the sporophylls. The number of sporophylls varies from four to twelve or
more, according to the size of the cone, the average number in a well-developed cone
being eight.

At the base of the peduncle the stele consists of a number’ of irregular, anas-
tomosing, mesarch strands, arranged in the central regicn. Hames (1936) refers to
this as a dictyostele. He also refers to the medullated protostele of the lower part
of the stem as a siphonostele. The use of these terms in this connection is very con-
fusing, as they do not apply in their generally accepted meanings. Strictly speaking,
a siphonostele is one in which the xylem forms a ring around the medulla and is

146 PHYLLOGLOSSUM DRUMMONDI,

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PI ITD
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Text-figures 15, 16, 17, 19.*
Figure 15, a and b. Figure 15a shows a transverse section of the stem of a large fertile
plant above the departure of the leaf traces (x 40). Figure 15b shows a transverse section
of a leaf (x 40). See text. mx = metaxylem; px = protoxylem; p = perforation in xylem;

It = leaf trace; s = stamate; pxe = protoxylem cavity.
Figure 16, a and Db. Figure 16a@ shows a longitudinal section of the stem of a large

fertile plant above the departure of the leaf traces (x60). Figure 16b shows a longitudinal
section of a leaf (x60). See text. Lettering as in Figure 15. ph? = probable rudimentary
or reduced phloem.

Figure 17, a, b and c. Figure 17a represents the mature cone in transverse section (x 40).
sp = sporangium; st = sporophyll trace; mvs = main vascular strand of peduncle. Figure 170
shows details of spores viewed from three angles. Figure 17¢ shows the thickened epidermis

* For Text-figure 18 see page 144.

BY FRANCES M. V. HACKNEY. 147

broken at intervals by leaf gaps which do not overlap; a dictyostele is a further
development of the siphonostele in which the leaf gaps occur closely enough for two
or more to appear in the same cross-section. As there are no leaf gaps in Phylloglossum
there can be neither siphonostele nor dictyostele in the true sense of these terms.
Hames also mentions an amphiphloic siphonostele in the peduncle. In view of the facts
that the extent of the phloem is difficult to determine and that there is no reference
to possible rudimentary or reduced phloem cells having been found anywhere but
outside the xylem, the stele cannot be regarded as amphiphloic.

Throughout the region between the base of the peduncle and the base of the cone
the xylem cylinder is usually medullated and perforated by gaps, but at the base of
the cone it becomes a solid mesarch protostele. In many cone-bearing plants the cone
axis appears to be a conservative region, retaining ancestral features (cf. the reten-
tion of centripetal metaxylem in the cone axes of certain cycads). In Phylloglossum
the sporophyll traces are given off from the main stele close together in spiral
succession. In most cones a few cauline tracheids are left at the top of the cone
axis after the departure of the last sporophyll trace.

Text-figure 17a shows the mature cone in transverse section. The mesarch proto-
stelic structure of the axial vascular strand is clearly seen. The lower portions of
three sporophylls and the sporangia borne by them are included in the section. The
section was cut above the departure of the first sporophyil trace; the second sporophyll
trace is shown in oblique section as it passes into the base of its sporophyll. The
third sporophyll trace is visible in transverse section, the material having been cut
below the point of attachment of the third sporophyll.

The sporangia are reniform in transverse section and contain numerous uniform
jetrahedral spores with pitted sculpturings on their curved bases. Text-figure 170
Shows details of spores viewed from three angles. No developmental stages were
available, but formation of the sporangium presumably occurs in the same manner as
in Lycopodium. In the sporangia examined only the outer thickened wall cells were
intact, so that tapetum and inner wall cells were not observed.

The epidermal cells are thickened on the radial and inner tangential walls
(Text-figure 17c). They have peculiar wavy outlines when seen in surface view
(Text-figure 17d).

In a longitudinal section of the cone (Text-figure 18) the sporangia are seen to
possess short stalks and to be foliar in origin. Each sporangium is protected by the
downwardly directed dorsal (abaxial) lobes of the sporophylls above it and by the
upturned tip of its own sporophyll. There are no ligules in Phylloglossum.

THE GAMETOPHYTE GENERATION.

The germination of the spores and the early stages of the development of the
gametophyte have never been observed. The only descriptions of the gametophyte are
those of Thomas (1901), Sampson (19160) and Holloway (1935). Of these writers
Thomas alone had access to more than one specimen. Thomas, collecting in the
ficinity of Auckland, New Zealand (see Holloway, 1935), obtained prothalli of
rnylloglossum growing naturally among parent sporophytes. He mentionéd the fact
that they were only discoverable in three of the areas searched, although sporophyte
plants were abundant in all these areas. Sampson recorded failure of all attempts
to raise prothalli from spores, and found only one badly preserved specimen in some

of the sporangium in section, with the stomium consisting of thin-walled cells. Figure 17d
represents a surface view of the epidermis of the sporangium, the cellS having peculiar wavy
outlines.

Figure 19, A, B and C. Figure 19 A: diagrammatic representation of shapes of prothalli.
The dotted line indicates soil level. Figure 19 B: an archegonium in which the egg is ready
for fertilization. Figure 19 C: two antheridia. The one on the right-hand side contains sperm
mother cells; the one on the left-hand side is almost empty, the sperms having matured and
the lid having been lost, allowing their escape. Drawings made from descriptions by Thomas
and by Holloway. 19 A, x 7; 19B and 19C, x 272.

148 PHYLLOGLOSSUM DRUMMONDII,

Australian material to which she had access. Holloway was also unsuccessful in his
attempts to produce prothalli from spores, and was able to obtain only one specimen
in the field, near Auckland.

Apparently very special conditions are necessary for the germination of the spores.
These are probably not of regular annual occurrence, even where sporophytes are
numerous. Thomas suggests that perhaps the most important of these conditions is
the presence of a fungus with which the prothallus may live symbiotically (cf. the
symbiotic fungi found in all species of Lycopodium whose developmental stages are
known). Thomas adds that it is not impossible that the prothallus may begin life
as a saprophyte, dependent upon an endophytic fungus. Among his specimens he
found one young prothallus which was quite colourless except for a faint yellow tinge
at the upper end, as well as two others, without sexual organs, showing only very few
chloroplasts. When the young prothallus is prevented from forming chlorophyll by
being buried in the soil it will doubtless be colourless.

All fully developed (sexually mature) prothalli recorded had green tissue above
the soil.

The prothalli examined by Thomas varied greatly in external form. The variations
are apparently not entirely due to differences in age, but may be due in part to
obstacles encountered in the soil during growth, or to differences in depths at which
development of the prothalli commenced in the soil. The length of the prothalli
varied between 6 mm. and less than 2 mm.

General Features of the Prothallus.

The general features of the prothallus as described by Thomas may be summarized
as follows. The subterranean parts of the body consist of an oval ‘tubercle’ and a
eylindrical shaft of varying length and thickness. The “tubercle” is probably the
first part to be formed on germination of the spore. The length of the shaft is
probably related to the depth at which germination occurs below the surface of the
soil. At the level of the soil surface there is a slight expansion of the top of the
shaft to form a crown, on which the sexual organs are developed. With the exception
of the archegonial necks, the whole of the upper part of the prothallus is green.
Chloroplasts are most abundant in the part just below the crown. The upper cells of
the shaft may be green, but the colour fades out as the shaft passes into the soil.
In some specimens there is a region of conspicuous meristematic activity at one side
of the shaft, just below the crown.

There is little internal differentiation of the tissues. The cells of the “tubercle”
of the mature gametophyte are of rounded polygonal form with scanty protoplasmic
contents; those of the shaft are elongated, rectangular at the surface, longer and more
pointed in the centre. Starch is often abundant in these cells. Thomas observed very
fine hyphae of an endophytic fungus in the cells of the lower half of the prothallus.
These hyphae pass in through the rhizoids and often form a dense mass on the
surface of the “tubercle’’.

Male and female sex organs develop on the same prothallus and the archegonial
necks are plainly noticeable on the crown. On each young prothallus Thomas found
only two or three archegonia, but on older prothalli there may be ten or twenty.
They appear to be formed in basipetal succession. The necks project from the surface
of the prothallus, usually in two tiers of four cells each. The venter, with the large
egg cell, lies at a short depth below the surface. The antheridia lie sunken in the
crown, covered by a single layer of cells. There are no multicellular paraphyses among
the sexual organs, but on some parts of the crown the surface cells are slightly
papillose.

Thomas’ description of the prothallus was made from a number of specimens
but no text-figures were published with it. Thomas obviously intended to follow his
first paper with a second, which was never published. Holloway (1935) gives a
description of a single specimen, together with a set of text-figures. No archegonia
are present in this specimen, but antheridia occur in considerable numbers, sunken

BY FRANCES M. V. HACKNEY. 149

in the tissue of the crown. So closely are the antheridia packed that usually only a
single layer of cells separates each from its neighbour. They are covered by a single
layer of cells, in which a definite cap cell is present. Most of the antheridia in the
specimen are empty, but a few are still packed with sperms.

The drawings of gametophyte organs shown in Text-figure 19 were made by the
writer to illustrate the descriptions by Thomas and by Holloway.

An endophytic fungus was present in Holloway’s specimen (cf. Thomas’ observa-
tion). Sampson (19160) also observed fungal hyphae in and between the cells of
the prothallus, but her specimen was too poorly preserved to show other details.

THE HMBRYO SPOROPHYTE.

Information concerning the embryogeny of Phyllogiossum is scanty. Thomas (1901)
gives a description of the development of the embryo. This description has since been
rendered somewhat less valuable by the fact that Thomas, like Bower, believed the
tuber of Phylloglossum to be homologus with the protocorm of Lycopodium. In some
places where Thomas speaks of the “protocorm” he was evidently referring to the
young tuber. The single prothallus examined by Sampson (19160) carried a young
sporeling which was forming a storage tuber at an early stage of its life (see later).
Thomas also refers to the first leaf as a protophyll, but this interpretation is clearly
erroneous. The organ in question is not a protophyll but a true leaf, because a
clearly defined vascular strand is present when development is complete.

Thomas’ description of the development of the embryo emphasizes its similarity
to that of the embryo of Lycopodium cernuum. However, the comparison appears to
depend for its aptness on the interpretation of the tuber as the protocorm. If
there be no protocorm the development of the embryo of Phylloglossum will resemble
Xhat seen in Lycopodium clavatum rather than that seen in Lycopodium cernuum.

Text-figure 20 shows a series of diagrams drawn by the writer to illustrate the
facts recorded in Thomas’ description.

Early in development the embryo grows obliquely downwards and outwards; the
lowest part forms the foot, while the stem apex and the first leaf are formed at the
opposite end. The tip of the leaf is the first part to burst through the prothallus
and its emergence causes a split to occur down the side of the prothallus; at this
stage the embryo “is a short, cylindrical body, bluntly tapering at either end and
connected with the prothallus by the foot, which now appears to be lateral in
position; the ends of the embryo represent the stem apex and the first leaf respectively”’.
No root formation has occurred in any of Thomas’ specimens, but a root is shown
in Sampson’s specimen (see later). According to Thomas, “the first leaf grows up
and attains a height of 2-3 cm. above the ground. It becomes green even before it
emerges from the prothallus. Soon after its emergence stomata are formed and a
thin strand of tracheids appears in the centre. The first leaf has the same structure

as any other small leaf formed in later years.” Thomas states that the stem apex
erows downwards into the soil and ‘forms .a protocorm, apparently in the same
manner that the adult plant forms its annual storage tuber”. From his description of

this ‘protocorm” and from later work by Sampson (19160) it is clearly not a protocorm
but a tuber.

Sampson describes a single specimen of a sporeling at a fairly advanced stage
of development. It consisted of a downwardly directed stem apex, the first leaf, a
single root and an “embryonic swelling” partly embedded in the prothallus. Sampson
debates whether the swelling is similar to the foot found in an embryo of the
Lycopodium clavatum type or to the protocorm of Lycopodium cernuum. Her main
reasons for considering the second possibility were: (1) the description given by
Thomas, discussed above, and (2) the fact that the swelling was partly extra-
prothallial. However, Sampson remarks that the extra-prothallial appearance may
have been merely due to shrinkage of the poorly preserved gametophyte tissue. As
Thomas’ belief in the existence of a protocorm is based on a misunderstanding of the
nature of the tuber, and as he clearly observed the presence of a foot, there seems to

150 PHYLLOGLOSSUM DRUMMONDII,

be no reason why the embryonic swelling observed by Sampson should not be interpreted
as a foot.

The specimen examined by Sampson demonstrates that, although root formation
is not common in the first year of growth (see Thomas, 1901), it does occur in some
instances and probably arises in the embryonic region between the points of origin of
the leaf and the foot.

GENERAL DISCUSSION OF THE PHYLOGENETIC STATUS AND AFFINITIES
Or PHYLLOGLOSSUM.

The simplicity of the plant body and the superficial similarity of the tuber to the
protocorm (the presence of which in some Lycopodian embryos was once regarded as
a primitive feature) have misled many of the early morphologists (e.g., Bower, 1885,
Thomas, 1901). Subsequent research has shown that Phylloglossum must no longer
be regarded as “the most primitive living Pteridophyte”, but as a highly specialized
plant adapted to a geophilous habit. Its tuber is not homologous with the protocorm.
The available data is against the view that Phylloglossum forms a protocorm at any
stage of its life.

THE MORPHOLOGY OF THE TUBER.

The morphology of the tuber was not understood for many years after the first
descriptions of the plant were published. Bower (1885) believed that the tuber was
homologus with the protocorm of Lycopodium cernuum, and this belief was shared by
several of his successors.

In 1908 Jeffrey suggested that the gap frequently observed in the vascular tissue
at the base of the stem might be due to the passing out of the tuber strand, but he
did not discuss the morphology of the tuber. Wernham (1910) was of the opinion
that Phylloglossum was an extremely specialized type. He considered that the gap
in the stele of the stem was ‘an ancestral character, and one of considerable
importance”. He was of the opinion that it might represent the leaf gap of a
megaphyllous leaf which had been suppressed by specialization. Phylloglossum,
according to Wernham, might resemble J’mesipteris, which he described as ‘‘micro-
phyllous” in its lower portion and “megaphyllous” in its upper portion. This opinion
was never widely accepted. However, Wernham’s paper is one of the earliest published
attempts to prove that Phylloglossum, “far from being a primitive form, is highly
specialized’’.

Sampson (1916a) considers that the occurrence of the stelar gap is a strong
piece of evidence in favour of the interpretation of the tuber as a modified branch.
The full evidence for this view, presented first by Sampson and later verified by the
present writer (with the exception of observation 2), may be summarized as follows:

(1) In large fertile plants a gap is left in the medullated protostele of the main
axis by the exit of the vascular strand of the tuber.

(2) Where two tubers are produced on opposite sides of the plant two gaps are
present in the main stele. é

(3) The stele of the tuber is frequently concave inwards as it passes out through
the cortex of the main axis; i.e., it shows a gap corresponding to that left in the
main stele.

(4) The dropper of the tuber sometimes bears leaves, usually reduced.

(5) In some specimens there is evidence, from the bend in the vascular strand of
the ‘tuber, that the direction of growth of the tuber was not originally downwards.

, (6) In sterile plants the tuber is usually derived from the apical growing point.

An additional piece of evidence is contained in the study of the embryology (see
above). Early in the life of the sporophyte a storage tuber is formed from the stem
region of the embryo.

In view of these facts there can be little doubt: that the tuber of Phylloglossum,
far from being a primitive organ, as was once generally believed, is in reality a

~

BY FRANCES M. V. HACKNEY. 151

relatively recent structure developed to meet the peculiar climatic (edaphic) GONE ORE
to which Phylloglossum became exposed.

Evidences of Reduction in Phylloglossum.
Evidences that Phylloglossum is a highly specialized form which has undergone
reduction, rather than an example of extreme primitiveness, are as follows:

(1) The highly developed cone and tuber are advanced features. They are more
advanced, for instance, than the reproductive structures seen in the more primitive
species of Lycopodium and Isoétes.

(2) The presence of a medulla in the stele is not generally regarded as a primitive
feature.

(3) The presence of vestigeal leaves (on the tuber stalk) indicates reduction.

(4) The protoxylem cavities and the perforations indicate reduction in the xylem;
reduction may also account for the occurrence of a solid protostele in small specimens.

(5) The rudimentary nature of the phloem may be a secondary development due
to reduction. This point is, of course, open to argument.

(6) The root is absent during the early life of most plants, in spite of the fact
that roots are produced in some sporelings.

(7) In most plants branching occurs only once a year, that is, when the new tuber
is initiated. Occasionally, however, two new tubers may be formed, or branching
may occur in the cone axis. It is at present impossible to tell whether this branching
is due to the retention of an ancestral character (palingenetic) or the manifestation
of a new feature (cenogenetic). As very primitive Pteridophytes (e.g., the Psilo-
phytales) branch freely, the former alternative is the more probable.

The conception of Phylloglossum as an extremely primitive form is of historical
interest rather than of phylogenetic significance.

AFFINITIES OF PHYLLOGLOSSUM.

The Lycopodian affinities of Phylloglossum are indicated by many morphological
and anatomical features.

(1) The roots are exogenous and adventitious in origin. Their internal structure
recalls that of Stigmarian rootlets and the roots of Isoétes.

(2) The tuber appears to be homologus with the method of perennation seen
in Lycopodium inundatum, where the whole plant dies down except the tip of the
rhizome, which persists as a resting bud (see Holloway, 1916).

(3) The anatomy of the stem shows several features characteristic of the
Lycopodiales. The stele is a mesarch protostele; in large specimens medullation and
perforation of the xylem cylinder frequently occur, but true leaf gaps are never
formed.

(4) Dichotomous branching sometimes occurs.

(5) The leaves are very simple in structure, with no definite palisade tissue, and
each is supplied by a single mesarch strand which dies out before reaching the tip.

(6) The cone, with its spirally arranged sporophylls, is very like that of
Lycopodium. Hach sporophyll bears a single sporangium which resembles that of
Lycopodium in structure, position and mode of dehiscence. There are no ligules.

(7) The spores are of one type only.

(8) The gametophyte appears to be similar to that of Lycopodium cernuum.

(9) From the available evidence the development of the embryo appears to be:
similar to that of L. clavatum. The occurrence of a protocorm has not been.
demonstrated.

SUMMARY.

The external morphology of the sporophyte is described briefly and its relation to
ecological conditions is discussed, and the methods of vegetative reproduction are
considered.

The morphology and anatomy of the sporophyte are detailed under the following
héadings: Root; Tuber; Stem (including leaves) ; and Pedunele and the Cone.

Mu

152 PHYLLOGLOSSUM DRUMMONDII.

The literature describing the gametophyte and the embryo is critically reviewed.
The phylogenetic status and affinities of Phylloglossum are discussed. It is shown that,
far from being an extremely primitive form, Phylloglossum is probably a type in which
apparent simplicity has been achieved as a result of modification and reduction through
specialization.

ACKNOWLEDGEMENTS.

The writer expresses her gratitude to Dr. P. Brough, Botany Department, University of
Sydney, for his guidance in this work, for the use of his collection of material, and for his
helpful criticism of the manuscript. She also wishes to express her thanks to Professor
E. Ashby, who was Professor of Botany at Sydney University at the time when this work
was carried out, for the use of his laboratories and facilities.

References.
Bower, F. O., 1885.—On the Development and Morphology of Phylloglossum Drummondii.
Phil. Trans. Roy. Soc., 176: 665-678.
, 1908.—The Origin of a Land Flora. Macmillan and Co., London.
Eames, A. J., 1936.—Morphology of Vascular Plants. Lower Groups. McGraw-Hill Book Co.
Inc., London and New York.
Houuoway, J. E., 1916.—Studies in the New Zealand Species of Lycopodium. II. Methods of
Vegetative Reproduction. R. Brit. Trans. and Proc. N.Z. Inst., xlvii: 513-520.
, 1935.—The Gametophyte of Phylloglossum Drummondii. Ann. Bot., xlix: 513-520.
JEFFREY, E. C., 1908.—Are There Foliar Gaps in the Lycopsida? Bot. Gaz., xlvi, 4: 241-258.
OSBORN, T. G. B., 1919.—Some Observations on the Tuber of Phylloglossum. Ann. Bot.,

33: 485-516.
Sampson, Kk., 1916a@—The Morphology of Phylloglossum Drummondii Kunze. Ann. Bot.,
30: 315-331.
, 1916b.—Note on a Sporeling of Phylloglossum attached to a Prothallus. Ibid.,
30: 605-607.

THomAS, A. P. W., 1901.—Preliminary Account of the Prothallus of Phylloglossum. Proc.
R. Soe. Lond., 69: 285-291.

WERNHAM, H. F., 1910.—The Morphology of Phylloglossum Drummondii. Ann. Bot., xxiv:
335-347.

153

NOTES ON THE APHODIINAE OF AUSTRALIA (COLEOPTERA, SCARABAEIDAE).
THE APHODIUS TASMANIAE, HOWITTI, YORKENSIS, ANDERSONI COMPLEX.

By B. B. Given, Entomologist, Department of Scientific and Industrial Research,
New Zealand.

(Communicated by P. B. Carne.)
(Ten Text-figures.)
[Read 28th June, 1950.]

INTRODUCTION.

The species tasmaniae Hope and howitti Hope of the genus Aphodius, have long
been the cause of considerable controversy. The species andersoni Blackburn and
yorkensis Blackburn have not previously been carefully considered with the first two
species, as they are (if valid species) relatively rare and of no known economic
importance. What we have long accepted as A. howitti is a major pasture pest in
Victoria, South Australia, and parts of New South Wales, and it is this fact which now
makes it necessary that the systematic status of this and other closely allied species
should be carefully examined.

This, the first of what is hoped to be a series of papers on the Aphodiinae, is mainly
a collection of extracts from official and personal files, and published descriptions and
opinions. Little personal research has been done by the writer, and the only really
original contribution is a series of line drawings.

APHODIUS HOWITTI Hope.
Proc. Ent. Soc. Lond., 1846, p. 147.

tasmaniae Hope, Proc. Ent. Soc. Lond., 1846, p. 147.

australasiae Blanchard, Voy. Pole Sud 4, 1853, p. 101.

andersoni Blackburn, Proc. Roy. Soc. Vic., xvii, Pt. 1, 1904, p. 154.

longitarsis Redtenbacher, Reise der ... Fregatte Novara um die Erde, 1867, p. 58.

This is a rather variable species as regards structure, colour, and size. Its distribu-
tion is wide (from Eyre Peninsula through south-eastern South Australia and Victoria
to south-eastern New South Wales), and it is often extremely common.

The following description is greatly condensed, as detailed descriptions have
frequently been published in the past.

Colour normally black, but sometimes brownish or, rarely, reddish. Ventral surface
and coxae brownish. Lateral and basal borders of pronotum usually reddish brown,
anterior angles testaceous. Entire surface nitid.

Head broad and finely punctured, clypeal margin narrowly reflexed, anterior clypeal
margin evenly curved, or straight and somewhat angled laterally. Medially on the frons,
behind the clypeal suture, is a distinct but small tubercle, while laterally, in front of the
eyes and on the suture, are raised areas. Eyes black, not prominent. Antennae rather
small, and not highly distinctive.

Pronotum glabrous except for elongate marginal bristles; evenly convex, narrowly
margined, finely punctured, and with distinct though not prominent anterior angles.
Posterior angles almost completely rounded. The pronotum of the male is much larger
and somewhat more convex than in the female, although in both sexes (particularly
males) size is very variable.

Hlytra strongly convex, elongate, and with nine sharply impressed, narrow striae
on each elytron; inter-strial spaces strongly convex, with a rather irregular row of
punctures on either side, each puncture bearing a minute hair.

154 APHODIINAE OF AUSTRALIA,

Fore-tibiae strongly toothed, with the teeth more slender in the female. Spaces
between teeth crenulate.
Length, 8:5-12 mm.; breadth, 3-8—5:3 mm.

Explanation of Synonymy, etc.

Harold (Berl. Zeit., 1859) appears to have been the first to doubt the validity of
Hope’s species howitti, and at that time reported the two species tasmaniae and howittt
to be identical. In 1861 (loc. cit.) he reversed his opinion as a result of an examination
of further material. An account of this is to be found in Blackburn’s paper (Proc. Roy.
Soc. Vic., 1904, pp. 1538-4). However, in this paper Blackburn makes an error in saying
that tasmaniae was described a year later than howitti. Actually, howitti was described
on the same page as tasmaniae, but below it. Blackburn considers that Harold’s reversal
of opinion was due to the fact that in the first case he was examining specimens of one
sex, and in the second, specimens of opposite sex.

Junk (Cat. Coleopt., 1910) does not consider howitti and tasmaniae as being
synonymous, but places australasiae Blanch. as a synonym of howiétti, and longitarsis
Redt. as a synonym of tasmaniae. Andersoni Blackb. is left valid, and a second species,
australasiae (Boheman, Res. Hugen., 1858), is also left. This last species should be
renamed (Blanchard’s species had priority), but whether or not it is synonymous within
this group is not herein determined. However, Blackburn apparently considers it to be
close to A. frenchi Blackb., which. relationship would exclude it from the group under
discussion.

Schmidt (Genera Insectorum) considers the two species howitti and tasmaniae to
be both valid.

Resulting from correspondence with Miss W. Kent Hughes (Division of Entomology,
C.S.1.R.O., Canberra) in 1931, Mr. G. J. Arrow, of the British Museum, examined types of
andersoni, howitti and tasmaniae, and concluded that the last two species were the same,
and that a specimen sent by Miss Hughes, named howitti by Mr. A. Lea, agreed with the
type of andersoni. Miss Hughes, in her reply, writes:

“You mention in your letter that A. andersoni Blackb. had smooth elytral intervals
and more punctured head and thorax. Some of my specimens agree with A. andersoni
Blackburn, as regards the puncturation of the head and thorax, but seem to have the
elytral structure of A. howitti Hope. Would it be possible for these all to belong to one
variable species, or for one or other of them to be a variety or sub-species? When
looking through the series one seems to get a gradual change from one to the other.”

In 1939, correspondence was conducted between Mr. A. L. Tonnoir, Division of
Economic Entomology, Canberra, and Mr. H. J. Carter on the subject. In his first letter
Mr. Tonnoir mentions the tubercles on the head of howitti, stating that his information
was obtained from J. W. Evans, who obtained it from Renaud Paulian of Paris. Carter’s
final opinion was that the three species howitti, tasmaniae, and andersoni, were the
same, and that all should be called A. tasmaniae Hope.

In the Tasmanian Journal of Agriculture (Feb. 1), 1941, p. 29, J. W. Evans places
on record his findings resultant on correspondence with M. Paulian of the Paris Natural
History Museum. The presence of the three tubercles on the head (vid. sup.) are
mentioned and illustrated, and Evans also considers the anterior outline of the head
(clypeus) to be distinctive. He also makes an interesting and very important statement
as follows:

“With regard to their distribution, A. tasmaniae occurs in southern Tasmania and
has been bred only from larvae collected at Gretna and Huon Island. A. howitti has
been reared from larvae collected at several places on the north-west coast and Flinders
Island, and is the injurious species in Victoria and South Australia, and probably New
South Wales.” (Note.—The present writer has not yet seen specimens of the typical
mainland form from Tasmania.)

Finally, type material was again examined at the request of Mr. P. B. Carne of the
Division of Entomology, C.S.I.R.O., Canberra. This examination was carried out by Mr.

BY B. B. GIVEN. 155

E. B. Britton of the British Museum, who makes the following observations on the Hope
material and Blackburn’s type of andersoni, in a letter dated 5th June, 1948:

“I am quite certain that they belong to the same species. The only difference is
that the type of tasmaniae is 10 mm. long while that of howittii is only 8 mm. Hope’s
descriptions differ only in the matter of size and colour.

“The type of A. andersoni is in our collections here. The length is 9-5 mm. and
colour yellowish brown. The outline of the clypeus differs slightly from that of
tasmaniae and the tubercles of the frons are less prominent, but I do not hesitate to
consider this also synonymous with howitti.

“The series of specimens labelled tasmaniae in our collections is mixed. The majority
of the specimens are the true howitti but there are five examples of a species with hairy
elytra apparently restricted to Tasmania. This is, I suppose, the species which Blackburn
‘and others have identified as tasmaniae.

“There are two other species, australasiae Blanchard and longitarsis Redt., recorded
by Schmidt as synonyms of howitti and tasmaniae respectively. The descriptions of
these species are fairly detailed and yet make no mention of hairs on the elytral intervals.
It seems certain that these are howitti and not the ‘tasmaniae’ of Blackburn. This latter
species is, therefore, left in need of a name.” ;

Britton also gives differences of the hind tarsi, sides of pronotum, and posterior
pronotal angles, as separating characters for the species tasmaniae (= howitti, =
andersoni, etc.) and “tasmaniae”’ as erroneously accepted in Australia.

On Hope’s type of A. howitti, the label reads “howettii. However, as “howitti’” was
employed in Hope’s published description, it being stated that the species was named
after its collector, a Mr. Howitt, it is safe to assume that the specimen label is incorrect.

In a later letter, dated 6th July, 1948, Britton replies to queries regarding
A. yorkensis, and gives the results of a comparison between types and specimens of
“howitti” and “tasmaniae”’ (howitti and pseudotasmaniae respectively in the nomencla-
ture introduced in this paper). He also examined the genitalia of the two species and
declares them to be quite distinct in this regard. All points mentioned in this
comparison bear out Britton’s earlier observations and conclusions; that is, that the
so-called species andersoni, howitti and tasmaniae are actually all howitti and that what
had previously been considered to be tasmaniae iS an undescribed species (herein
described as pseudotasmaniae, n. sp.).

From the above extracts and summaries, it is obvious that Hope’s species tasmaniae
and Blackburn’s andersoni must be reduced to synonymy under howitti Hope. The two
species actually occurring, howitti and pseudotasmaniae, have been carefully examined,
and certain characters found to be useful in separating them. Seven males and fifteen
females from Tasmania, and eighteen males and sixteen females from the mainland
were studied, and the following characters were found to occur:

Tubercles on head.—All specimens from the mainland with the exception of one
female from Canberra had well-developed, or at least definite, tubercles. In two
Tasmanian males, head tubercles (median in particular) were faintly developed.

Clypeal outline.—This character was found to be extremely variable, and diagnos-
tically useless.

Hairiness of elytra—Distinctly hairy elytra were present on all males from
Tasmania, but on no other specimens. )

Genitalia. —From three males from Tasmania and three from the mainland, genitalia
were dissected and examined. Constant differences were again found and these are
illustrated in Text-figs. 9 and 10. These figures are somewhat idealized to illustrate
points of difference, but no actual distortion of proportion occurs. Male genital characters
are not suitable for general use on account of the extreme fragility of some parts of
the structure, and examination must be made before mounting in any clearing mountant
as certain important parts are liable to become practically invisible.

156 APHODIINAE OF AUSTRALIA,

APHODIUS YORKENSIS Blackburn.

Trans. Roy. Soc. S.A., 1892, p. 209.

In characters of colour (ruto-ferrugineous), coarse puncturing of head, truncate
clypeus, puncturing of elytra, and proportions of the male pronotum, the pair before me
agree very well with Blackburn’s description, and the only characters mentioned below
are those not mentioned by Blackburn, but which appear to be useful for separation
from howitti and pseudotasmaniae.

Re

la 2a

| 9 | 9a 10 10a

Text-figures 1-10.

Figs. 1, la.—Pronotum, dorsal and anterior, A. yorkensis.

Figs. 2, 2a, 3, 3a.—Pronotum showing range for howitti or pseudotasmaniae.
Figs. 4, 4a.—Head, yorkensis.

Figs. 5, 5a.—Head, howitti.

Figs. 6, 6a.—Head, pseudotasmaniae.

Figs. 7, 7a.—Fore-tibiae, male and female, yorkensis.

Figs. 8, 8a.—Fore-tibiae, male and female, howitti.

Figs. 9, 9a—Male genitalia, howitti.

Figs. 10, 10a.—Male genitalia, pseuwdotasmaniae.

Head very broad, clypeus strongly reflected anterolaterally, clypeal suture of male
(in specimen examined) apparently deeply arcuate in its median part. Lateral raised
areas on suture as well-developed as in howitti.

BY B. B. GIVEN. a lyr

Pronotum of male very large, and so strongly convex as to obscure portion of lateral
margins when viewed from above. (Text-fig. 1.)

Fore tibia somewhat curved, and with the margins between the teeth smooth. Fore
tarsus with apical segment shorter than preceding two together (Text-figs. 7, 7a; cf.
Text-figs. 8, 8a).

Ventral surface more densely haired than in howitti. The sizes of the specimens
before me are as follows:
Male.—Length, 10-8 mm.; breadth, 4:8 mm.
Female.—Length 8-9 mm., breadth 3-8 mm.

Note.—Mr. E. B. Britton, of the British Museum, has kindly examined the type of
this species. He describes the colour as being a uniform cherry red, and confirms the
smoothness of the fore-tibial outline between the teeth, the fore-tibial curvature or
anterior concavity, and the male pronotal characters. Mr. Britton’s letter is dated
6th July, 1948.

APHODIUS PSEUDOTASMANIAE, Nn. Sp.

This species has been long known as A. tasmaniae in error. An analysis of the
confusion which has given rise to this error appears earlier in this paper, under
A. howitti.

Very close to A. howitti, but differing in being devoid of tubercles on the head,
having conspicuous rows of hairs on the elytra of the male, and in details of the male
genital terminalia.

Material examined:

Holotype male, collected by Key, Kerr and Carne, 11 m. S.E. of Waddamana, Tasmania,
January, 1948. Allotype female, collected by Mr. J. R. Cunningham, Kingston,
Tasmania, February, 1948; C.S.I.R.O. Collection, Canberra.

Two paratype males, three paratype females, collected by J. R. Cunningham, Kingston,
Tasmania, February, 1948; pair in collection of Entomology Division, Dept. S. & I.R.,
New Zealand; male and two females in collection of Dept. of Agriculture, Hobart,
Tasmania.

Paratype male, paratype female collected by H. Nye, Glenora, Tasmania, February, 1939;
Collection F. HE. Wilson, Melbourne.

Paratype male, paratype female, collected by J. W. Evans, Glenelg, Gretna, Tasmania,
emerged 2.2.39 (reared?) ; collection of Dept. of Agriculture, Hobart.

Two paratype females, collected by J. W. Evans, Glenelg, Gretna, Tasmania, emerged
2.2.39 (reared?), one paratype male, five paratype females, collected by Messrs. Key,
Kerr and Carne at Broadmarsh, Bothwell, Scamander, and Waddamana in Tasmania,
January, 1948; C.S.I.R.O. Collection, Canberra.

SUMMARY.

On evidence submitted by Mr. E. B. Britton of the British Museum and others, the
species tasmaniae Hope is herein reduced to synonymy under the species howitti Hope.
Material from Tasmania, previously considered to be tasmaniae, is described as a new
species, pseudotasmaniae. The species andersoni Blackburn is also reduced to synonymy
under howitti Hope, and yorkensis Blackburn retains its status.

Acknowledgments.

I wish to thank Mr. P. B. Carne for material and information from the files and collections
of the Division of Entomology, Commonwealth Scientific and Industrial: Research Organization,
Canberra, A.C.T. I am also indebted to Mr. H. B. Britton, of the British Museum, for making
comparisons with type material, and to Mr. F. EH. Wilson of Melbourne for the loan of his
collection of the Aphodiinae, for the loan of literature, and for his valued personal opinions on
the group. To the South Australian Museum, I owe thanks for the loan of a pair of Aphodius
yorkensis Blackburn. Finally, I am indebted to the Tasmanian Department of Agriculture for
the loan of specimens of Tasmanian forms.

158

THE MORPHOLOGY OF THE IMMATURE STAGES OF APHODIUS HOWITTI HOPE
(COLEOPTERA, SCARABAEIDAE, APHODIINAE).

By P. B. Carne, B.Agr.Sc.*
(Plate viii, thirteen Text-figures.)
[Read 28th June, 1950.]

INTRODUCTION.

Aphodius howitti Hope in the larval stage is a pest of pastures, occurring in economic
densities in the south-eastern portion of Australia, including Tasmania.

This paper describes the external morphology of the immature stages, no detailed
account of which has been published previously. The author had the opportunity to
consult an unpublished thesis prepared by Miss D. M. Cumpston in 1939, in which the
raster and epipharynx of the larva are described briefly, the terminology introduced by
Hayes (1929) being employed. However, the more recent terminology originated by
Boéving (1942) appears preferable, although primarily designed for use in the description
of larval Phyllophaga (Melolonthinae). Recently, this terminology was extended for use
in the description of larval Coprinae (Ritcher, 1945). It is possible to describe Aphodiine
larvae almost completely in the terms used in these publications, as the morphology of
the Aphodiinae is intermediate between that of the Coprinae and that of the
Melolonthinae.

The method of description used is patterned to some extent on that employed by
Ritcher in describing the species Anomala innuba Fab. (Ritcher, 1943), the writer being
of the opinion that the excellent descriptions published by this author (Ritcher, 1943,
1945a, 1945b and 1945c) provide models which can profitably be paralleled in the
description of Australian larval Scarabaeidae.

The larvae were collected in the vicinity of Canberra and Weetangera (A.C.T.);
Queanbeyan, Burra, Crookwell, Tumbarumba (N.S.W.); Ballarat, Hamilton, Melbourne
(Victoria); and Gretna (Tasmania).

The majority of the figures are composite: the slide preparations of the mouth-
parts employed in making the figures, together with the series of larvae from the
above localities, have been deposited in the Museum of the Division of Entomology,
C.S.:LR-0., Canberra, A.C.T:

Larvae were fixed for ten minutes in Carls’ fixative maintained at approximately
70° C., and preserved in 70 per cent. alcohol. Slide preparations were made in polyvinyl
alcohol mounting medium.

The larva of Aphodius howitti Hope is distinctive among other pasture scarabaeids
in that it has a brownish-black head capsule, raster pattern as illustrated (Text-fig. 12),
and a mode of life which involves the making of a vertical burrow in the soil from
which it emerges at night to feed on dung or vegetation.

The only larvae with which it might be confused are those of Aphodius pseudo-
tasmaniae Given and A. yorkensis Blackb. In neither of these species is the larva
known, but in view of the similarity of the three species in the adult stage, it seems
probable that their larvae would exhibit little macroscopic differentiation. Given
(1950) observes that A. pseudotasmaniae is restricted to Tasmania, and A. yorkensis
to the Yorke Peninsula of South Australia.

DESCRIPTION OF THE LATE (PREPUPAL) THIRD INSTAR LARVA.
The body is cylindrical and typically scarabaeiform (Text-fig. 1); when at rest,
the mouthparts approximate to the anus. The length of twenty prepupae ranged from
13 mm. to 18 mm. with a mean length of 15:0 mm.

* An officer of the Division of Entomology, C.S.1I.R.O.

BY P. B. CARNE. 159

Head (Text-fig. 2).

The cranium* is equal in width to the prothorax, surface shining, dark brown to
black. The mean maximum width of cranium of 100 prepupae taken from seven sites
was 2:97 mm., with a S.D. (Standard Deviation) of 0:11 mm. The clypeofrontal suture
(CS) is distinct, and is bounded laterally by the precoilae (PCL). The frontal sutures
(FS) are fine, and meet well in front of the hind margin of the head, forming an angle
of approximately 60°. The epicranial suture (HS) is about one-half the length of one
of the frontal sutures. The frons (F) bears a pair of prominent anterior frontal setae
(AFS), between which lie a pair of paramedian pigmented depressions. Each anterior
frontal angle (AA) carries a single seta.

Epicranium (Text-fig. 2, E): The epicranium possesses four pairs of dorso-
epicranial setae (DES). Of each group of four setae, three lie in a transverse row,
while the fourth lies slightly cephalad of the junction of the frontal sutures. Five
setae occur on each side of the epicranium, and a sixth lies caudad of each anterior
frontal angle.

Antenna (Text-figs. 2, A, and 3): The antennae are approximately equa! in length to
the cranium, 4-segmented and borne on a basal piece fused to the epicranium. The first
segment is longer than the second, and the second longer than the third. The ultimate
segment is reduced to a small conical structure terminated by four or five senscry pegs
(SP) and a long terminal hair (TH). A very similar structure is found in the larvae
of the Coprinae (Ritcher, 1945c). The second segment carries a single microsensillum
(MIS) dorsally, the third bears a ring of four or five short setae distad, and an oval
convex sensorial spot (SS). The fourth segment possesses a small, apparently concave,
sensorial spot.

Clypeus (Text-fig. 2, CY): The clypeus is trapezoidal and is not divided into post-
and preclypeus. A pair of short paramedian setae are present, laterad of which are
two pairs of exterior clypeal setae (ECS), the more median pair being the longer.
These setae all arise from an approximate mid-transverse line.

Labrum (Text-fig. 2, L): The labrum is asymmetric, trilobed, in length approxim-
ately equal to the clypeus, and in width to the clypeus at its distal margin. Two pairs
of setae lie across the mid-transverse line, a paramedian pair, and a pair near the
lateral edges of the labrum. The lateral lobes bear three setae set close together, the
central seta of each triad being the longest. The apical lobe carries a pair of short setae.

Epipharynxe (Text-fig. 4): The epipharynx is broader than long and weakly trilobed.
The acanthopariae each bear six or seven setae (ACP), the posterior four or five being
snort and curved slightly cephalad; the anterior two setae, which are about twice as
long, are set in slightly from the edge. Plegmata are absent.

The acroparia carries two rows, each of four setae (ACR), one row along the edge
of the epipharynx, the other set in from it. Between the acroparia and the acanthopariae
lie the clithra (CA). The haptomerum (H) is a hump bearing two large macrosensilla
(MAS). The pedium (PE), which is raised above the general level of the epipharynx, is
bare except for a row of fine non-articulated processes which runs obliquely across the
right side. The pedium is bounded cephalad by the protophoba (PPH), which consists
of a series of small appressed processes, at the bases of which are about 19 sensilla.
Laterad, the pedium is bounded by the dexiophoba (DPH) and the laeophoba (LPH), and
caudad by the mesophoba (MPH), all of which consist of non-articulated processes, those
of the laeophoba being finer than those of the dexiophoba. Caudad, the latter is modified
to form a plate-like structure which is fringed with processes similar to those of the
dexiophoba proper, and which is borne perpendicular to the plane of the epipharynx as
a whole. Between the lateral phobae and the acanthopariae lie extensive gymnopariae
(GP). Chaetopariae are absent.

A pair of stout setae are present near the lateral margins of the epipharynx,
adjacent to the clypeolabral condyles. A dexiotorma and laeotorma (DX, LT) are
present, fused mesad and produced cephalad to form a long anterior epitorma (ETA).

* “Cranium” is here used to denote that part of the head capsule consisting of the
epicranium and the frons, i.e., the immovable part of the head.

N

160

IMMATURE STAGES OF

APHODIUS HOWITTI.

BY P. B. CARNE. 161

Caudad of the mesophoba is a variable, faintly chitinized structure, subtriangular in
shape, adjacent to which lie four to six sensilla. The affinities of this structure are
doubtful. Somewhat similar structures occurring in Coprine larvae are termed crepides
by Ritcher (1945c).

Mandibles (Text-figs. 2, M, and 5-6): These are asymmetric, the scissorial area
(SA) reddish-black and stout, cutting edge notched, with a tooth proximad of the notch.
The left mandible is longer than the right and overlaps the latter (viewed dorsally).

Dorsad (Text-fig. 5), each mandible bears three setae near its exterolateral margin,
and a fourth smaller seta, often obscured, adjacent to the preartis (PA). A brustia
(BR) is present on each mandible, while an acia (AC) is present on the left mandible
only.

Ventrad (Text-fig. 6), on each mandible, is borne the postartis (PTA) and the
ventral process (VP). The right mandible bears two or three small groups of bristles.
Of these, only one group is paired on the left mandible. The central portion of each
mandible is a pale yellow in colour, the molar structures, and the edges generally,
grading red to black. No oval stridulating area is present, as it is in the Melolonthinae.
The surface bears a large number of minute asperities, not shown in the figures. These
are particularly noticeable about the postartis.

Mavilla (Text-figs. 7 and 8): ventrad (Fig. 7), the maxilla is composed of cardo
(CAR), stipes (ST), galea (G), and lacinia (LAC). The cardo is longer than wide, also
the stipes. The lacinia and galea are entirely separate. The cardo bears a variable
number. of setae, usually four. The stipes bears two setae: one long proximal seta and
a somewhat shorter distal seta. The ‘galea terminates in a simple uncus (U) from
which extends caudad a row of 10 to 12 short setae. A number of larger setae are
grouped about the uncus, only one arising from the ventral surface of the galea. The
lacinia ends in a notched uncus, proximad of which is a very stout seta. The lacinia
carries a single seta proximad.

Dorsad (Text-fig. 8), the stipes bears two setae, proximad of which lies a row of
11 to 15 stridulating teeth (SD), the apices of which are directed cephalad. The galea
possesses distad a group of five or six stout setae. The lacinia carries a row of 10 to 11
setae, longer than those in the row on the ventral surface of the galea. The maxillary
palpus (MP) is 4segmented, and arises from a palpifer (PF) which bears a singlet seta
on its ventral surface. The third segment bears a pair of setae; the fourth ends in a
group of sensory pegs. ;

Labium. (Text-figs. 7 and 8): Ventrad (Text-fig. 7), the labium consists of a large
subtrapezoidal postmentum (PMP) and a terminal prementum (PRM), which carries a
pair of labial palpi (LP). The postmentum is bare except for a pair of setae, one in each
proximolateral corner. Adjacent to each seta is a single sensillum. The prementum
bears proximad a pair of rather long setae, laterad of which are one or two pairs of very
small setae; and distad, four pairs of setae. he latter consist of a paramedian pair, a
pair of very long setae, each situated directly proximad of a labial palp, and, laterad of
these, a third pair of shorter setae close to the edge of the prementum. The fourth pair

Text-figures 1-4.

Fig. 1.—Third instar larva, left view. PRSC, prescutum; PS, prothoracic shield; SCL,
scutelium; SCU, scutum; V, venter.

Fig. 2.—Third instar larva, cranium, front view. A, antenna; AA, anterior frontal angle;
AFS, anterior frontal setae; CS, clypeal suture; CY, clypeus; DES, dorsoepicranial setae; BH,
epicranium; ECS, exterior clypeal setae; ES, epicranial suture; F, frons; FS, frontal suture;
L, labrum; M, mandible; PCL, precoila.

Fig. 3.—Third instar larva, right antenna. MIS, microsensillum; SS, sensorial spot; SP.
sensory pegs; TH, terminal hair.

Fig. 4.—Third instar larva, epipharynx. ACP, acanthoparia; ACR, acroparia; CA, clithrum;
DPH, dexiophoba; DX, dexiotorma; ETA, epitorma; GP, gymnoparia; H, haptomerum; LPH,
laeophoba; LT, laeotorma; MAS, macrosensillum; MIS, microsensillum; MPH, mesophoba ;
PE, pedium; PPH, protophoba.

162 IMMATURE STAGES OF APHODIUS HOWITTI.

Text-figures 5-11.
Fig. 5.—Third instar larva, mandibles, dorsal aspect. AC, acia; BR, brustia; M, molar
lobe; PA, preartis; SA, scissorial area.
Fig. 6.—Third instar larva, mandibles, ventral aspect. PTA, postartis; VP, ventral process.

Fig. 7.—Third instar larva, maxillae and labium, ventral aspect. CAR, cardo; G, galea;
LAC, lacinia; MP, maxillary palpus; PF, palpifer; PMP, postmentum; PRM, prementum,
U, uncus,

BY P. B. CARNE. 163

are very small and lie proximad of the labial palps, caudad of the very long setae.
Adjacent to each is a sensillum.

Dorsad (Text-fig. 8), is located the glossa (GL), which bears a complex of sclerotized
masses, the oncyli (O). A longitudinal row of short setae lies proximad of each labial
palp, the row curving laterad near the base of the palps; proximad of this row of setae,
and running obliquely, is a row of non-articulated spines, which, towards the sides of the
glossa, are much reduced and triangular in shape. A transverse row of very small,
closely appressed processes occurs on the glossa distad of the oncyli. At the base of
each process (which is directed caudad) is a microsensillum. The glossa bears two
pairs of setae near its distal edge; between the more proximal pair is a group of four
sensilla.

Thorac.

The thorax consists of pro-, meso-, and metathoracic segments. The prothoracic
segment bears a pair of sub-oval heavily chitinized areas, the prothoracic shields
(Text-fig. 1, PS). Ventrad of these are the prothoracic spiracles. The meso- and meta-
thoracic segments are divided dorsally into three annulets, the prescutum (PRSC), the
scutum (SCU), and the seutellum (SCL). Hach annulet bears a transverse row of long
setae.

Spiracles (Text-figs. 1, 9): Only the prothoracic segment bears spiracles. Each
consists of a respiratory plate (RSP) which measures 0:26 mm. along its major axis.
There is a spiracular slit (SST) and a bulla (BU). The concavity of each spiracle faces
caudad.

Legs (Text-figs. 1, 10, 11): These are well developed, the larva being capable of far
more rapid movement than most scarab larvae. The prothoracic legs are slightly
shorter than the mesothoracic, and these slightly shorter than the metathoracic pair.

Each leg consists of a long cylindrical coxa (COX), a short trochanter (TRO), a ~
markedly clavate femur (FEM), and a fused tibiotarsus (TTS), which ends in a single
claw (CL). Setation is sparse, there being about seven setae on the coxa, and about six
on the trochanter (which bears ventrally the longest setae on the legs). The tibiotarsus
is more plentifully supplied with setae. The claws of the prothoracic and mesothoracic
legs are slightly longer than those of the metathoracic legs (ratio 2:0: 1:9: 1-6). The
distal portion of each claw (Text-fig. 11) is a reddish-brown; the proximal portion is a
pale yellow and bears two minute setae.

Abdomen.

The abdomen consists of ten segments, of which only the first eight bear spiracles.
The first six segments are dorsally divided into prescutum, scutum and scutellum, as in
the meso- and metathoracic segments. Rows of short setae are present on all the
annulets of the abdominal segments from the scutellum of the first to the scutellum of
the fifth segment. Long setae are present on the prescutum of the first segment, and are
interspersed among the short setae caudad. Setae are fewer on the scutum of the sixth
segment, while the scutellum, and subsequent segments caudad, which are undivided
dorsally, carry only sparse long setae. The tenth segment bears, dorsad, the venter (V),
which has a transverse anal slit.

Hach of the abdominal spiracle-bearing sclerites carries two long setae. The sternum —
of each segment bears a transverse row of six to eight mixed long and short setae. The
venter is bare. Ventrad, the tenth segment bears the raster.

Spiracles: There are eight abdominal spiracles, the concavities of which face
cephalad. Measurements made along the major axis of the respiratory plate show that

Fig. 8.—Third instar larva, maxillae and labium, dorsal aspect. CAR, cardo; G, galea;
GL, glossa; O, onecylus; PF, palpifer; SD, stridulating teeth.

Fig. 9.—Third instar larva, left prothoracie spiracle. BU, bulla; RSP, respiratory plate;
SS, spiracular slit. :

Fig. 10.—Third instar larva, left mesothoracic leg. CL, claw; COX, coxa; FEM, femur ;
TRO, trochanter; TTS, tibiotarsus.

Fig. 11.—Third instar larva, left mesothoracic leg, distal portion. Cl, claw; TTS, tibiotarsus.

164 IMMATURE STAGES OF APHODIUS HOWITTI,

all the abdominal spiracles are smaller than the prothoracie pair, with the exception of
the pair on the sixth abdominal segment. Those of the eighth abdominal segment are
the smallest. The following are measurements made on a single specimen: Prothoracic
spiracle 0:26 mm. Abdominal spiracles 1-8: 0-14, 0-20, 0:22, 0-22, 0-24, 0-26, 0-18, 0:12 mm.

Raster (Text-fig. 12): The raster is composed of a septula, a pair of palidia, a pair
of tegillae, and a campus. The septula (S) is ovoid and extends from close to the lower
anal lip to a point somewhat short of the middle of the tenth abdominal segment. Each
palidium (PLA) is composed of from five to seven blunt translucent pali (P). Each

Text-figures 12, 13.
Fig. 12.—Third instar larva, raster. B, barbula; C, campus; P, palus; PLA, palidium;
S, septula; T, tegilla.
Fig. 13.—Pupa, left view.

palus is about twice as long as wide and has a very short constricted stem. At its
point of articulation there is a chitinized structure embedded in the tissues laterad.
Boving describes four distinct classes of pali (Boving, 1942), but the pali of Aphodius
are unlike any described by him.

The tegillae (T) extend from the barbulae (B) to the palidia, and are not united
anterior to the septula. The setae composing the tegillae are hamate and curved caudad.
The barbulae consist each of a group of usually five prominent setae. The campus (C)
is narrow and bare although sometimes possessing one or two very small non-hamate
setae mesad. Two very small hamate setae are borne caudad of the septula.

Range of Variation.

Epipharyns: The acanthopariae consist each of five to seven setae; about 50 per
cent. of the larvae studied had seven setae on each side. Frequently the number was
reduced to six on the right side, less frequently on the left. The haptomeral macrosen-
silla in rare cases numbered three instead of two.

Mazillae: The number of setae in the row upon the lacinia varied from 10 to 11
with an average of 10-6 (16 specimens examined). The number of setae’in the row on

BY P. B. CARNE. 165

the galea varied from 10 to 12 with an average of 11:5 (15 specimens). The number
of stridulating teeth on the stipes varied from 11 to 15 with an average of 12:3 (13
specimens).

Raster: The palidia consist of five to seven pali, the most frequent arrangement being
five on one side and six on the other, with a tendency for the lower number to be
found on the left side. A variable number of non-hamate setae (rarely more than four)
were found either in the campus or among the setae of the tegillae.

In the course of examination of larval mouthparts, a series of second instar larvae
from Crookwell, New South Wales, was found to contain a considerable proportion of
individuals in which the epipharynx and raster exhibited a large number of super-
numerary setae. It was at first thought that these setae were those of the third instar,
showing through the second instar tissues just prior to ecdysis. However, this
phenomenon was not observed in specimens from any other locality, and moreover, there
existed a complete gradient from normal to “fully aberrant” in the Crookwell series, this
latter fact removing any question of a second species of Aphodius being involved.

A microphotograph of the epipharynx of an aberrant specimen is reproduced in
Plate VIII, which should be compared with Text-figure 4. The maxillae are similarly
possessed of many additional setae, while in the raster (but not in every case) each
seta is paired with another rather less pigmented seta. The larvae were active and of
normal size.

DISTINGUISHING FEATURES OF INSTARS.

The instar of a specimen may be determined with ease from a number of points of
difference:

1. Cranium: The maximum width of cranium is useful as an indicator of instar.
The following figures (Table 1) are derived from a series of measurements of over 1,000
specimens, all of which were collected at Dickson Experiment Station, A.C.T., during
the winters of 1946 and 1947.

It will be noted that there is neither contact nor overlap between the ranges
characteristic of each instar.

TABLE 1.

Widths of crania of Aphodius howitti larvae from Dickson Eaperiment
Station, Canberra, A.C.T.

Number of Mean Width of -

Instar. Specimens. Cranium (mm.).| Range (mm.). S.D.
ist 40 1:05 0-81 —1-14 0-0625
2nd 97 1:82 | 1:67 —1-95 0-0619
3rd 1,031 2°83 2°29 — 3-29 0-1407

2. Epipharynx: The clithra are absent in the first instar, and present in a reduced
state in the second instar.

3. Raster: The first instar raster is characterized by barbulae, the setae of which
are relatively extremely long, and by the absence of palidia. In the second instar, the
barbulae are relatively much shorter. Palidia are not present, although the setae towards
the inner edge of each tegilla possess pigmented supporting structures.

PUPA.

The exarate pupa is a light yellow in colour and specimens collected at Dickson
Station measured 1:0 to 1:2 cm. in length. It possesses a tough semi-transparent skin,
a large mounded tubercle on the head, and in pupae from which male beetles will emerge,
a finger-like projection caudad (Text-fig. 13).

The sheaths enveloping the legs terminate in pairs of fine hairs. In older pupae,
when the adult structures are well developed and clearly visible as in Text-fig. 13, it may
be seen that the tibiae of the meso- and metathoracic legs each carry two sharp spines, and
that the abdominal spiracles are borne on the pleural membrane, dorsad of which, on the

166 IMMATURE STAGES OF APHODIUS HOWITTI.

pupal skin, are irregularly shaped areas of thickening. The antennae may be seen to
be 9-segmented. These observations enable the pupa to be identified immediately as one
ot the larger Aphodiinae. The relatively large size of the pupa would prevent confusion
with that of any other species of the subfamily known in Australia, again with the
exception of the two species mentioned above, i.e., A. pseudotasmaniae Given and
A. yorkensis Blackb.

EGG.

The eggs are found in the soil in batches of 20 to 50, the average being about 35.

Eggs collected just prior to hatching are pale yellow, ovoid, and measure approximately
1 mm. along their major axis. The membrane is smooth and devoid of ornamentation.

SUMMARY.

Series of third instar larvae collected from a wide range of sites in south-eastern
Australia have been examined: the present paper describes in detail the external
morphology of the larva and the variations observed. Emphasis has been laid on those
structures of taxonomic importance in other sub-families of the Scarabaeidae.

Larvae of the two earlier instars have been studied and the distinguishing features of
the three larval instars are listed. The pupa and egg are described briefly.

ACKNOWLEDGEMENTS.

The writer wishes to thank Dr. K. H. L. Key of the Division of Entomology, C.S.1I.R.O.,
for his criticism of the draft of this paper, Mrs. M. Spencer for reference to her thesis, and the
many persons who have supplied him with larval material. Miss B. M. Rothery provided technical
assistance, and Mr. T. Pickard the microphotograph for Plate viii.

The work described in this paper was carried out as part of the research programme of the
Division of Entomology, C.S.1.R.O.

References.

Bovine, A. G., 1942.—A classification of larvae and adults of the genus Phyllophaga. Mem. Ent.
Soc. Washington, No. 2, 72. pp.
GIVEN, B. B., 1950.—Notes on the Aphodiinae of Australia. Proc. LINN. Soc. N.S.W., Ixxv:

153-157. .
Hayegs, W. P., 1929.—Morphology, taxonomy and biology of larval Scarabaeidae. Jil. Biol. Mono.,
Le ial)

RITcHER, P. O., 1945.—The Anomalini of eastern North America with descriptions of the larvae
and a key to species (Coleoptera: Scarabaeidae). Kentucky Agric. Exp. Sta. Bull., 442,
23 pp.

———, 1945a.—Rutelinae of eastern North America with descriptions of the larva of
Strigodermella pygmaea (Fab.) and three species of the tribe Rutelini (Coleoptera:
Scarabaeidae). Kentucky Agric. Exp. Sta. Bull., 471, 15 pp.

————, 1945b.—North American Cetoniinae, with descriptions of larvae and keys to genera
and species (Coleoptera: Scarabaeidae). Kentucky Agric. Eup. Sta. Bull., 476, 33 pp.

== --= , 1945¢e.—Coprinae of eastern North America, with descriptions of larvae and keys to
genera and species (Coleoptera: Scarabaeidae). Kentucky Agric. Exp. Sta. Bull., 477, 17 pp.

EXPLANATION OF PLATE VIII.

Microphotograph of the epipharynx of a second instar larva of Aphcdius howitti Hope, taken
at Crookwell, New South Wales. See explanation in text.

167

NOTES ON AUSTRALASIAN SIMULIIDAEH (DIPTERA). II.

By M. J. Mackerras and I. M. Mackerrras,
Queensland Institute of Medical Research, Brisbane.

(Fifteen Text-figures.)
[Read 28th June, 1950.]

INTRODUCTION.

Since our previous paper (1949), we have accumulated a considerable quantity of
new material, much of which was obtained during a tour of Cape York and north-eastern
Queensland. The present notes include observations on the aurantiacum group of
Cnephia, descriptions of five new species (one Cnephia, three Simulium, one Austro-
simulium), a new subspecies of Cnephia tonnoiri Drum., the early stages and male of
Simulium faheyi Tayl., and records extending the known distribution of previously
described species. The Queensland fauna has been increased from nine to seventeen
species.

References listed in our earlier papers are not repeated here.

*

|-Omm.

Text-fig. 1.-—Lateral view of larvae of (a) Cnephia tonnoiri orientalis, and
(b) Simulium ornatipes, showing form of abdomen.

The Genus CnepHis End.

We were previously unable to give characters separating the larvae from Simuliwm.
We find that they may be recognized by the truncated posterior end of the abdomen, the
maximum width being at the seventh segment, as compared with the more fusiform
abdomen of Simulium (Text-fig. 1). They are also more waxy and opaque in appear-
ance, and the ventral incisure of the head capsule is shallow, contrasting with the deep
incisure of all known Australasian species of Simuliuwm (Text-fig. 2). The incisure is
shallow in several species of Austrosimulium also, but that genus is distinguished by
the anal sclerite, and the body is fusiform like Simulium.

Although its early stages are still unknown, we now consider that C. wmbratorum
(Tonn.) should be placed in the aurantiacum group.

ce)

4

68 NOTES ON AUSTRALASIAN SIMULIIDAE,

Keys to species of aurantiacum group.

Adults.

Orange to reddish brown flies; Cu, gently curved; claws of 2? with powerful basal tooth.

Wing with dark marking at Lorie Ob AR ik ycvereiecretchous otestolene lo ous. creielakolelicteieys anche teioiatoie bere ieienenehened: 2
Wines without darkemarkin oie reteietciokerteielalerefoteicineicnedeteieistat-isleishcnelsvcielehat-ienteloneieien ete iatetateie iets 3
Scutum brown scaled, with median and dorsocentral vittae of golden scales .. strenua n. sp.
Scutum uniformly covered with golden scales .............-c2ccseceecce tonnoiri (Drum.) *
Large orange species; propleural hairs present ............-++--..0-- aurantiacum (Tonn.)
Small to medium reddish brown species; propleural hairs absent ...... umbratorum (Tonn.)

* All subspecies.

0:5mm,

S. clathrinum

A, fulvicorne

Text-fig. 2.—Head capsules of larvae, showing ventral incisure.

Pupae.
Abdomen with strong terminal hooks, chaetotaxy as in Text-fig. 4; gill filaments many-
branched, arborescent.
Pleural membrane of abdominal segments 5-7 without chitinous plates ....................
Be ee peat Soh eral cove ere teria erates toe rats New oes tee torotete te tegen eaeret tee aurantiacum (Tonn.) strenua n. sp.

Pleural membrane of abdominal segments 5-7 with rounded chitinous plates .......... 2

Gilliiilamentsvantler-like ys aib out 9210-4 Oe rewenciedeeeieneieielelieieieieneieieneionsrone tonnoiri tonnoiri (Drum.)

Gill filaments slender, in sweeping curves, about 50-70 ......... tonnoiri orientalis n. subsp.

Gillgilamentsestout-pa boutwlh=2 Ome ee Rie ncoriciiene iene enone tonnoiri fuscoflava M. & M.
Larvae.

Abdomen truncate, widest at 7th segment; cuticle waxy, opaque; antennae short (Text-
fig. 11); ventral incisure of head capsule shallow (Text-fig. 2); anal sclerite without
backwardly directed strut; rectal gills simple; ventral papillae absent.

Proleg with prominent palp-like processes at either side of apical segment; arms of anal

sclenite reduplicateds pricicycuscs ches eC elieie ior sia chiens Fonehekcnoue rien Relea Menon IIR strenua n. sp.
Proleg without such prominent processes; arms of anal sclerite Single .................. 2
Posterior circlet broad, rows closely placed, teeth light brown, small and numerous, difficult
tomsce individually mateo OMOLAMeETERSiMiericesreaeicieictonelspelcteren casters ienananaens aurantiacum (Tonn.)
Circlet narrower, rows not so closely placed, teeth dark, larger, and can be seen individually
at less *tham 50M AMEeCETS Keg xe) wyataioten ce sate titel cabo vavlelis eohke kets Talla lfcirahecieveustsyshertel iene tenekonowe omen menete 3
Circlet with 18-24 teeth per row .... tonnoiri tonnoiri (Drum.) tonnoiri orientalis n. subsp.
Ginrcletawithsaboutelomteethien Crarowwaremee ein rine neice iene tonnoiri fuscoflava M. & M.

O:|mm.

CG. tonnoiri orientalis C. aurantiacum C. strenua

Text-fig. 3—Hypopygia of males of Cnephia spp.

CNEPHIA AURANTIACUM (Tonn.).
The characters of adults and larvae previously reported have been confirmed. In

addition, the upper facets of the male eyes are smaller and more numerous than in other

BY.M. J. AND I. M. MACKERRAS. 169

species of the genus, being about 0:04 mm. in diameter and in more than 20 rows
vertically and across. The abdomen, in specimens we have seen, is less hairy than in
C. tonnoiri. The hypopygium (Text-fig. 3) has the distal end of the anterior part of
the phallosome distinctly concave and the distal end of the apodemes more heavily
armed than in C. tonnoiri.

The posterior part of the cocoon is of more definitely “wall-pocket” shape than in
the other species, but is loosely constructed anteriorly, so that the head and thorax of
the pupa often hang freely from the opening.

Pupae are best separated from C. tonnoiri by the absence of pleural plates on
_ abdominal segments 5-7. The gill filaments number about 30 to 40, and are stouter
than in C. tonnoiri orientalis, with broadly angled branches, and a stiff, antler-like form;
they cannot be distinguished readily from those of C. strenua and C. tonnoiri tonnoiri.

The posterior circlet of the larva has a more distinctive appearance than can be
indicated in words, and may be seen quite easily in unmounted specimens at the
magnification indicated. The median notch in the anal sclerite is closed posteriorly, so
that the body of the sclerite appears to have a hole in it (Text-fig. 5).

New distribution.—Queensland: South coast district. Little Nerang Creek, elevation 300 ft.,
August. Harly stages on reeds in fairly fast, cold, clear, turbulent water; adults bred from the

pupae.

Omm.

05mm.

=
€ tonnoin_ orientalis CAonnorri fuscoflaya Cstrenya | \

Text-figz. 4—Pupae of Cnephia spp. Top, abdominal chaetotaxy ;
below, respiratory horns.

CNEPHIA TONNOIRI (Drum.).

All subspecies are to be distinguished from C. aurantiacum: as adults by the dark
spot on the wing, the almost invariable absence of propleural hairs, the more extensively
darkened mid and hind legs, the larger upper facets of the ¢ eyes (about 0-06 mm. in
diameter and in about 15 rows vertically, 12 across), and by the ¢ hypopygium (Text-
fig. 3), the distal end of the anterior part of the phallosome being gently sinuous and the
apodemes relatively weak; as pupae by possessing well defined, rounded chitinous plates
on the pleural membrane of abdominal segments 5 to 7 (Text-fig. 4); and as larvae by
the coarser posterior circlet and open median notch in the anal sclerite (Text-fig. 5).

The three subspecies now recognized are distinguished from each other primarily
on the pupae.

Cnephia tonnoiri tonnoiri (Drum.).

The respiratory filaments of the pupa number about 20 to 40, the main branches
are a little stouter than in the eastern race, relatively stiff, with wide angle of branching

170 NOTES ON AUSTRALASIAN SIMULIIDAE,

and antler-like appearance similar to C. strenua in Text-fig. 4 and also very like the
filaments of C. aurantiacum.
Distribution.—Western Australia (localities given in previous paper).

Cnephia tonnoiri orientalis, n. subsp.
Types: Holotype 9, allotype ¢, morphotype pupa and larva, from Little Nerang Creek,
south coastal Queensland, September, in the Division of Entomology, C.S.I.R.O., Canberra, A.C.T.

Adults.

Indistinguishable from C. tonnoiri fuscoflava as originally described (Mackerras and
Mackerras, 1948, p. 238). They may be darker than the typical subspecies, but the only
specimens we have seen from Western Australia are rather old, and we did not
previously give sufficient weight to the possibility that they may have faded. We have
searched for propleural hairs on a considerable series of specimens, and found two or
three weak hairs near the lower margin of the sclerite on one side only in two females.
As these hairs form a well-defined group on the surface of the sclerite in C. aurantiacum,
this character remains substantially reliable. Hypopygium of male as in Text-fig. 3.

Cocoon.
A shapeless bag, as in other subspecies.

Pupa.

Chaetotaxy and terminal spines as in other subspecies. Gill filaments about 50 to
70 in number, rather slender, branching mostly close to the base with narrow fork, more
flexible than in the typical subspecies and sweeping forward in even curves (Text-fig. 4).
Specimens from the type and southern localities have about 50 filaments, those from
Springbrook 506 to 70, but are otherwise indistinguishable.

Larva.

As typical subspecies. The posterior circlet is composed of well-spaced rows of
eighteen to twenty-four medium-sized teeth.

Distribution.—Tasmania: Rheban (Griffiths R., Sandspit R.), January. A.C.T.: Canberra,
November (Tonnoir) ; Coree Creek, November, January (Tonnoir) ; Cotter R. and Paddy’s R.,
November (Mackerras). Queensland: Little Nerang Creek (300 ft.), August, September;
Purling Brook (Springbrook area), 2,000 ft., December.

Biology.

The early stages occur in clear, moderate to fast, turbulent streams, generally
adjacent to, rather than in, the line of fastest flow. They are nearly always attached
to vegetation, rarely to stones. Adults have not been collected in the field.

Cnephia tonnoiri fuscoflava M. & M.
The pupal gill filaments are stouter and fewer than in the other subspecies (Text-
fig. 4), and there are fewer teeth in the posterior circlet of the larva. Adults are
indistinguishable from C. tonnoiri orientalis.

Distribution.—Queensland: Still only known from the type locality on Stradbroke Is., our
(1949) record of it from Little Nerang Creek being in error.

,

CNEPHIA STRENUA DN. Sp. :
Types.—Holotype 92, allotype @, morphotype pupa,and larva, from the Cascades, Freshwater
Creek, Cairns district, north Queensland, September, in the Division of Entomology, C.S.I.R.O.,
Canberra, A.C.T.

Distinctive features.

A large species. Adults resemble C. tonnoiri, but are distinguished by the dark
antennae, scutal pattern, black hairs at sides of scutellum, differently marked legs, and
less hairy abdomen. Pupae similar to C. aurantiacum. Larvae gigantic when full
grown, with very dark head and very broad posterior circlet of very fine teeth; on each
side of the distal segment of the proleg there is a conspicuous, palp-like process, which
has not been seen in any other species we have examined.

Female.
Length: Body 3-5 to 4 mm., wing about the same.

BY M. J. AND I. M. MACKERRAS. 171

Head.—F rons about one-eleventh of head width, dark greyish brown, with fine
goiden hairs. Antennae with basal three segments light brown, remainder dark brown.
Face and palpi brown.

Thorax.—Scutum rich brown, covered with brown scales showing golden reflections
anteriorly and posteriorly in certain lights, and with narrow but distinct median and
dorsocentral vittae of golden scales. Scutellum with conspicuous long black hairs.
laterally and short golden ones centrally. Pleurae yellowish brown, darker on lower
part of sternopleuron. A few propleural hairs are present in all specimens.

Legs.—Coxae and femora yellowish, but with dark brown tips to femora. Basal
quarter of tibiae yellowish, remainder dark brown. Hind metatarsus similar; other
tarsal segments brown, with narrow yellow zone at base. Calcipala large, of typical
form of the group; pedisulcus shallow, corrugated. Claws with strong basal tooth, as in
other species of the group.

C.tonnoiri_orrentalis C. aurantiacum C.strenua

Text-fig. 5—Submenta (above) and anal sclerites (below) of larvae of Cnephia spp.

Wings.—Hyaline, with small but distinct dark marks at base and at fork of R as in
C. tonnoiri. Halteres large; stem creamy yellow, knob dark brown.

Abdomen.—Dark greyish brown dorsally, with a fringe of golden Hairs near distal
edge of each segment. Spermatheca nearly smooth; external genitalia and genital
fork as in C. tonnoiri.

Male.

The upper facets of the eyes are about 0:06 mm. in diameter and in about 15 rows
vertically, 12 across. The antennae are pale on only the first and basal half of the
second segment; the scutal vittae are less well defined, and there is a rather indefinite

‘patch of golden scales above and in front of wing roots; the legs are less darkened;
otherwise as in female.

Hypopygium (Text-fig. 3) with the distal end of the anterior part of the phallosome
lightly chitinized and produced into a prominent process, which at first sight resembles
a median piece, but is seen on close examination to be continuous with the distal edge.
There is a rather prominent, hairy lobe on the inner side of the coxite.

Cocoon.

Length 3-4 mm. Like C. tonnoiri, soft, simple bag-like; dark, and usually including
foreign material. Attached to substrate all along ventral wall, generally in groups of
two or more. The head and thorax of the pupa often project from the cocoon.

Pupa.
Length 3-455 mm. The respiratory organ has a short, wide stem, which divides
at once into several wide branches, the posterior being the longest. Each main trunk

172 NOTES ON AUSTRALASIAN SIMULIIDAE,

gives off a number of spreading, antler-like branches. Many of these divide again,
forming a complex group of about 50 to 55 filaments arranged as in Text-fig. 4, in which,
however, only about two-thirds of the filaments are shown.

The pupa cannot be distinguished on these and other easily seen characters from
Cc. aurantiacum, but does appear to differ, in that the third abdominal sternite is more
strongly chitinized and bears three instead of two hooks laterally on each side, the
tergites are finely tuberculate on their posterior portions only (tuberculate all over in
aurantiacum), and there are usually more than five stiff hairs on each side of the ninth
tergite (five in aurantiacum). The first of these characters may be variable, and the
others can only be seen in cleared and mounted material.

Larva.

Length of gill-spot larvae 8-11 mm. Very large, thick, brownish larvae, with paler
integument ventrally at posterior end. Head capsule and its appendages very heavily
pigmented, dark brown to black, obscuring pattern on dorsum of head. Submental plate
characteristic, with prominent central tooth and eight or nine pairs of stout, blade-like
spines on each side (Text-fig. 5). Gill-spot as in Text-fig. 14. Thoracic proleg robust
and armed with a conspicuous circlet of dark hooks, which are more closely set than
in other known species; the lateral cuticular plates are well developed and bear
numerous long spines, and there are two pointed palp-like processes at the base of the
apical narrower portion of the proleg (Text-fig. 14).

Anal sclerite with median notch closed posteriorly to form a “hole”: its posterior
limbs double. There is a conspicuous row of 10-12 tubercles bearing short spines on each
side between the posterior limb of the selerite and the upper edge of the circlet, and also
a patch of fine, short hairs on each side above the posterior limbs. Posterior circlet
exceptionally well developed, the rows of hooks being so closely packed and the hooks
so numerous as to render counting them impracticable. Individual hooks are rather
smaller than in C. tonnoiri, and are also smaller than the hooks on the proleg.

Distribution.—Known only from the type locality in North Queensland.

Biology.

This is a remarkable species. The larvae are enormous, so large that we scarcely
believed that they were Simuliid larvae when we first saw them; yet their bulk must be
made up of muscle and essentially larval organs, for the pupae and adults are little if

S.tnornatum S. peregrinum

Text-fig. 6.—Heads of females of Simuliwm spp.

any bigger than their near relatives. Most larvae were found in cascades, at points
where a powerful jet of water was concentrated between narrow walls of rock and
shooting over 4 sharp ledge. The depth of water was about twelve inches, and the
flow so strong that an arm or leg could only be held against it with the greatest
difficulty. The larvae were clustered thickly on the bottom ledge, where the flow was
strongest. It is from their adaptation to withstand such a battering that the specific

BY M. J. AND I. M. MACKERRAS. 173

name is derived. By contrast, a few were also found in company with Simulium
aureonigrum in a small tributary, on a nearly vertical face of rock over which the water
poured in a thin, fast layer. When removed from the water, the larvae adhered to our
hands in a way we had not seen previously, and were quite difficult to remove.

Pupae were in groups on the rocks, in rougher, more sheltered places below the
brink, where the water spread out fanwise into a fast, but much thinner and less
forceful layer. They were quite difficult to collect intact. No adults were seen in the
field.

The Genus SimuLtium Latr.
So many species have been added that our previous keys have become obsolete.

Keys to Species.

Females.
ti, IEYE@-@UIETO BE RSEY OURS) “6 6: 8p pnoeokS ERIE DL GsG cad) CEE SCRC He RR EeeRE GES Sree CL IG Ren Eee ERE LE GIG aiCr Ol nee e cnee eer ir 2
Pre-lar ame, Wyld COM OICHIONIS WII KEES Goocoucccodncndc0dcodbdcD oD bdUdOobDoDGuUGUUS 3
2. Medium-sized species; legs conspicuously marked with black and yellow ...... ornatipes Sk.

Small species; legs predominantly dark (basal two-thirds of hind metatarsus pale) ........
PR Me snes ice acral a) ero Wala peise) Soy cis Toa she ame vouey alaive auccisive ieilerenalisye aus jo euscel ailelia: oman bkepieis peregrinum n. sp.
3. Minute, pale species; antennae entirely yellowish fawn; femora and tibiae predominantly

Grea, WOUlOWys ONOeuaeen UNARAUKOIA)) GoouunccouodcnuuvodomoUUuUuUDddOOS Simulium sp. B.
Larger, darker species; antennae with at most the basal segments pale; femora and tibiae
OSS CL OTIMUET rately aes Ol AT Ko esis yee eee alot pales etatee teiepreoec tthe races a lePol aivsliosecovel cy cetan aya vrortay aniaj.e / snencniahel opleitetaner Scwlouente 4

4. Scutum with golden median and dorsocentral lines; abdomen with tergites 2-4 black scaled;
Dom AaLe mere yi She black rather Shimin' Sam cpeierereiciycesicne cieiersiersiene sions ene clathrinum M. & M.
Seutum without discrete golden lines; abdomen with pale scales on some tergites, including
Fes MTR ree aN ref cReINS otaNe SSCS ev ey eos Swe SCS eS O AN Daa Seles) a) ariaiteteligitelansabee Gralla eins) eva eos abene tole Aun weariane crane 5

5. Basal segments of antennae brown; very dark specieS ............... cece eee eee eee wees 6
Basal segments of antennae orange to brownish yellow; not such dark species .......... "

6. Scutum and second abdominal tergite adorned with rich golden scales; tergites 3-5 entirely
dark, 6-8 sprinkled with creamy gold scales ...................... aureonigrum n. sp.

Scutum and second abdominal tergite with dull golden to silvery scales; tergites 3-7 with
incomplete golden or creamy fringe and few or no pale scales on the disc, 8 with a few

SCABUCLCOMUATERSCATOS Tr etch ce ctsmemcnene seme iee same eMeee exich eleven on leveotel aie eet ame beak tierien inornatum n. sp.
Scutum and second abdominal tergite with creamy gold to silvery scales; tergites 3-5 with

a few apical silvery scales, 6-8 speckled with silvery scales .............. melatum Wh.

7. Brownish black and golden species; second abdominal tergite with at most an incomplete
golden fringe; claws with small, sub-basal tooth ........................ faheyi Tayl.
Brownish black and creamy gold species; second abdominal tergite quite densely covered
with creamy gold scales; claws with small, sub-basal tooth ............ papuense Wh.

Black and silvery species; second abdominal tergite with median patch or apical band of
Shp, SEUSS Clays ViwhsdoOme Weel SoaccanducucdndouuduogcdbuuguGS nicholsoni M. & M.

Notes.—(1) We have omitted two New Guinea species, S. oculatum End. and
S. wilhelmlandae Smart, because it is uncertain to which groups they belong; the
former may bé close to S. clathrinum, the latter to S. peregrinum. (2) The claw
eharacters are useful, but can only be seen properly in cleared and mounted prepara-
tions. Females of S. ornatipes and S. peregrinum have very large basal teeth on the
claws, S. clathrinum has medium-sized teeth, often detectable in pinned specimens,
S. aureonigrum, S. inornatum, S. faheyi and S. papuense have small sub-basal teeth,
S. melatum has minute teeth deeply set in the concavity of the claw, and S. nicholsoni
fias none (Text-fig. 7).

Males

“Tl, Piresenlleire: Bivona, RST WIA ae eaters ene Peres Pere OR RRP sa erred penpals, SU Rete es) Oa RE ert. 2
Pr@e=fllaie BREE, WH COMOCUIOMS TEN SOAIIES so coacooccocds eco cocb dood OdooKUOouOOKGUUOS 3

2. Medium-sized species; upper facets of eye less than 0:04 mm. in diameter, in about 16 rows;
legs conspicuously marked with black and yellow ...................... ornatipes Sk.

Small species; upper facets of eye more than 0:05 mm. in diameter, in 10 rows; legs
MiReConNAINU CENA Sooo co oeaoat dacconadgaacacapoodoaueacuoloodouo peregrinum n. sp.

3. Upper facets of eye more than 0:04 mm. in diameter, in about 12 rows .................. 4
Upper facets of eye less than 0:04 mm. in diameter, in about 16 rows .................. 6

4. Abdominal tergites 2 and 5-8 with golden scales arranged much asin @ ...... papuense Wh.
Abdominal tersites) 2=8) velvety black, ttomemtose! 47.5... sees acs sedacceuueceses nee 5

5. Seutum with three golden lines usually discernible .................... clathrinum M. & M.

Scutum without indication of median and dorsocentral golden lines .... aureonigrum n. sp.

174 NOTES ON AUSTRALASIAN SIMULIIDAE,

co |

Anterior part of phallosome with straight or gently sinuous distal edge

A i eR EES SES co HG SU Bho Ai RE EIGI Gn Gis Broo are nicholsoni M. & M. faheyi Tayl.
Anterior part of phallosome with markedly concave distal edge ....................... 7
Anterior part of phallosome forming an open bay distally ................... melatum Wh.
Anterior part of phallosome with bay almost enclosed distally ............ inornatum Nn. sp.

Note.—The abdominal adornment of S. papuense is most unusual. Our specimen

may be an intersex, but the eyes, legs and genitalia are normal, and Wharton’s descrip-
tion of the allotype agrees.

bo

~)

clathrinum papuense

aureonigrum inomatun melatum faheyi nicholsoni

Text-fig. 7.—Claws of females of Simulium spp.

Pupae

Cullgnlamentsinnimerousscrb ORESCEn tA tte eee enc eine ee creer: papuense Wh.
Gill filaments 8 honweach Side: Ser. osycio acc ciakecsore cisee eRe Monel de hE oe ueetoaeret peregrinum Nn. sp.
Gilly filaments: 6 tom Veach: ‘Side: wenccaicse costae bie ete CEs cue eosin der PRON heVeTie (a STS La etusns NCI ete RRR 2
Gill filaments 4 vor Gach Sid eso sei wy Sucks sia cca auctsccouetdehsi-sise dl ey oparalte ere see oy Rov oWr Fe sek aces CSRS RRR CR enero 3
Respiratory organ with well-marked stem; filaments directed forward close together ......

Bre antes te Pes lotrn iced sity tens ic bexayshio ever ta adis aa eerie Sy al ictke Hose Me sLOMeHst ohana eelereusRelsyoksicnce ais sen eeowenekens nicholsoni M. & M.
Respiratory organ with short stem; filaments spreading more widely .......... faheyi Tayl.
Cocoon with deep collar; filaments narrow, subeaual, directed forward close together ......

Ci osciovundid cho lola ol Olomio Gaia DIOllad 6 CO Dime Deco moO Gedo e come o 4.ao.0 ble 'od Goon clathrinum M. & M.
Cocoon) with narrow: Collar Or NOME! .i ss cesserclena o. overensuete ne aden clseentrerel erence none eesiene a ieee tote +

Filaments very thick, pale, relatively smooth, subequal, spreading widely .... ornatipes Sk.
Filaments of moderate thickness, darker, irregularly mammillated, subequal, directed forward
(6) Kofs(e\int Of 2-4 cl 015) aaah none ene EAI nae REINS, cy CCS Te REPRE RON icy ci omS GIG ol citer ctaro aici Moin Gaol alc melatum Wh.
Filaments of intermediate thickness, dark, finely patterned, of unequal length, spreading
WIGEliy aU aie eanc inl a teleb kale tole hater es cueunaoas on aesclin s-amapavalsNe ya ovens Ren ohoele sus eaenuauten slags imornatum Nn. sp.

Filaments narrow, dark, finely patterned, subequal, spreading .......... aureonigrum n. sp.
Larvae
Rectalesilis) Simple ses ee Ae er Ms tree a ace racers ee ee eee eerste LESS Cane usec sucPan ck Sere Lael ureWene ems 2
Rectal: cillsy compound (ce ines fae ereretenc teats © chote Pale ev evcetereueikets ohone onenel oilei tiered hol hakelicneraioncReltensenehens 5
A broken line of brown scales ventro-laterally on each side anterior to the circlet ...... 3
INO PLOWNnYScalesmin sthis .pOSLtLOMGperccueak a ieleus keke Cee Cro nc Ton Peden NUN Orn etc ekicticn RemcnaCRcn one a:
Posterior part of head rather uniformly pigmented; ventral papillae usually indefinite or
£21] Of<1=) 08 Reso MORS halo taCuCe TG ONGHELOIOL econo on ciceclulencuGucl Gaeceee GID DIDS 0 OO) OGIO COG Ooo cob. melatum Wh.
Posterior part of head with a W-shaped pattern; ventral papillae well defined ............
arieiatcaiien eles orteiceinlcet stfsue fel eintist atyaycslic Lome ialetre mer eaoust Gita u ons solic! le eve de ORM RCa CM mea Leimotnene aeRetten tebe te Reate inornatum Mn. sp.
Smaller, more yellowish larvae; head pattern negative type ............ nicholsoni M. & M.
Larger, darker larvae; head pattern positive type .......................--- ornatipes Sk.
Small larvae; antennae conspicuous, nearly as long aS head ............. peregrinum Nn. sp.
Larger larvae; antennae inconspicuous, about half as long as head ..................6. 6
A broken line of brown scales ventro-laterally on each side anterior to the circlet ..........
Ra SII CE Rae CRE RERCIC TE, Eee OMAR GlS GG. Oi clad o'O-4'0 FO oo Od Dt o aureonigrum n. sp.
No brown 'sealesiin, this! PoOsitiomy sie wich lere eee a etoelc rioieuen Guswoemee ice aEsl eesiey eile (e)aircy ells eee ewe teneneneie 7
Head pattern indefinite or partly negative in type; ventral incisure extending about half-
way, to) base of SuUbmemtun Tye Wa. citar ieee iver tene tote easiseeetencneeael oil ole arenes faheyi Tayl.
Head pattern positive type; ventral incisure extending more than half-way to base of
sSubmientumy by 26s Sh eG, Ge Ue EEE ERROR Perera eta. nh ne) age eee ete 8
Ventral incisure reaching base of submentum; head pattern conspicuous, bullet-shaped
Cae IPRS Cid CHEE RRO OSE TOP ELECT RCL HOD Ol O CICA OTRAS OG OO ON OO Dio Decne. a papuense Wh.

Ventral incisure not reaching base of submentum; head pattern cruciate .. clathrinum M. & M.

BY M. J. AND I. M. MACKERRAS. 175

Notes—(1) The ventro-lateral scales at the posterior end of the abdomen are some-
times inconspicuous or absent in young larvae of S. melatum. (2) The ventral papillae
do not provide such clear-cut distinctions as in Austrosimulium. They are large in
peregrinum, medium to small in ornatipes, nicholsoni, faheyi, aureonigrum and
imornatum, indefinite or ventro-lateral in melatum and papuense, and absent (though
ventro-lateral swellings are present) in clathrinum.

SIMULIUM ORNATIPES Sk.
New distribution.—Queensland: Springbrook, 2,000 ft. (south coast district), December;
various coastal streams between Nambour and Gympie, February, April, May; Babinda, N.Q.,
September (the most northerly record so far on the mainland). :

Wu|.0

|

“Wi}.0

f

Text-fig. 8.—S. peregrinum. @: a, antenna; b, palp; c, fore leg; d, mid leg;
e, hind leg; f, genitalia. of: g, hypopygium. :

SIMULIUM PEREGRINUM DN. Sp.
Types: Holotype 2, allotype ¢, morphotype pupa and larva, from Black Camp Creek, Cape
York, August, in the Division of Entomology, C.S.I.R.O., Canberra, A.C.T.

Distinctive features.

A very smali species, lacking pre-alar scales, but possessing lower sternopleural
(“mesosternal’) hairs and largely pale hind metatarsus like the clathrinum group.
Adults with base of abdomen creamy to yellowish in both sexes; head of male wider
than thorax, upper facets of eye greatly enlarged. Pupae with respiratory apparatus
longer than the body, dichotomously branched, forming eight slender filaments. Larvae
with very prominent ventral papillae, compound anal gills, and antennae which are
nearly as long as the head.

This is the first species of Edwards’ sub-group C to be found on the mainland of
Australia. It is typical of the sub-group in all respects, and quite distinct from the

176 NOTES ON AUSTRALASIAN SIMULIIDAE,

clathrinum group, though it shows features which suggest that the latter may have
evolved from the former in the isolation of their Australasian extension. Similarly,
S. ornatipes, which lacks lower sternopleural hairs, would appear to represent the sub-
group D in a somewhat modified form in this region.

S. peregrinum is separable from the species of sub-group C described by Edwards
from Java most conspicuously on pupal characters, and from S. wilhelmlandae Smart.
(= Morops pygmaea End.) by the colouration of the antennae and abdomen. Its name
is derived from the fact that it is, in a sense, a wanderer from the home of its relatives.

, Female.

Length: Body 1:5 mm.; wing 1-4 mm.

Head wide and rounded. Frons about one-sixth of head width (Text-fig. 6).
brownish black, shining. Antennae eleven-segmented; basal three or four segments
brownish yellow, remainder dark brown. Proboscis very short, so that the palpi appear
to be long; both dark brown, with the terminal segment of the palp paler.

Thorax.—Scutum and scutellum uniformly greyish black, rather shining; dise
thinly and evenly covered with short, fine, golden hairs, which are rather denser and
stronger at the sides and on the scutellum; apical edge of scutellum with a row of long
black hairs. Pleurae uniformly dark greyish brown. Pre-alar area bare; a tuft of
white propleural hairs present; lower sternopleural hairs fine, pale, and rather scattered.

Wings clear, veins brownish yellow, hairs black. There is the usual row of spinules
on the costa and a few on the distal part of R,; upper surface of R with a single row
of black hairs. Halteres with stem light brown, knob pale lemon yellow.

Legs (Text-fig. 8)—Fore tarsi larger and more powerful than mid, distinctly
thickened but not flattened; the enlargement is more obvious than in the clathrinum
group. Hind femora and tibiae swollen, about twice as thick as fore or mid, the tibiae
angulated beyond the middle posteriorly. Calcipala and pedisulcus well developed. All
claws with a powerful basal tooth.

Fore and mid coxae cream; femora brown, covered with golden scales on most of
anterior surface; tibiae brown, with creamy knees and golden scales on about proximal
third; tarsi deep brown, almost black. Hind coxae brown, cream apically; femora
brown, with rather scattered pale golden scales; tibiae brown, with creamy knees and
a covering of pale golden scales on basal half anteriorly; metatarsi cream and covered
with creamy golden scales on basal four-fifths, brown on distal fifth; remaining tarsal
segments dark brown, but with a ring of pale scales at bases of second and third
segments.

Abdomen.—First tergite brown, creamy in centre and with a creamy fringe; second
largely cream, but more or less narrowly brown along apical edge; 3 to 5 brownish
black, tomentose, with dark hairs; 6 to 8 black and shining. Venter cream basally,
light brown apically. Terminalia, genital fork and spermatheca as in Text-fig. 8.

The female from Smoko Creek is a little larger; tergites 1 and 2 of its abdomen
are more completely yellow; only the basal three-fourths of the hind metatarsi are
pale; but otherwise it is similar to the Cape York specimens. It came from an exactly
similar pupa.

Male.

Head large, rounded, wider than thorax. Upper facets of eye about 0:05 mm. in
diameter,* in ten rows vertically and transversely. Antennae creamy yellow, except for
the distal 2 to 4 segments, which are darkened. Scutum fairly densely covered with
creamy gold scales, which are larger and more conspicuous than in the female. Pleurae,
wings and legs as in female; claws simple, of typical male form. Abdomen with first
tergite dark brown, with long brown fringe; second mostly yellow, with apical brown
zone widening at sides, the pale part covered with rather shining tomentum; remaining
segments velvety black, except for conspicuous, silvery, tomentose sublateral patches on
5 to 7; venter as in female.

* Hye facet measurements given in this paper were made on cleared preparations mounted
in Canada balsam.

BY M. J. AND I. M. MACKERRAS. 177

Hypopygium (Text-fig. 8) with style shorter than coxite, ending in a single spine.
Anterior part of phallosome strongly convex and longitudinally striate ventrally;
posterior part with a pair of heavily chitinized, deeply pigmented, somewhat irregular
plates, possibly corresponding to the chitinized bars in Cnephia. Apodemes large and
powerful, terminating in a beak-like structure with three divisions. No median piece
detected.

Cocoon.
Wall-pocket type; smooth, very thin and delicate; no collar and no central dorsal
projection.

Pupa. :
Length: Body 2-1 mm., filaments 2:2 mm.

There is a group of four long hairs on each side of the scutum just anterior to its
highest point; abdominal armature weak, inconspicuous, of normal distribution, except
for the presence of patches of minute spines on the ventral surface of segments 4 to 6.
Respiratory apparatus (Text-fig. 10) with a distinct stem, branching dichotomously to
form eight very long, slender filaments; the lower and outer pairs are longer than the
upper and inner. The tips of these delicate filaments break off easily, so that discrep-
ancies in their relative lengths are seen in different specimens.

Larva.
Small; length of gill-spot larva about 3:7 mm.

Head pattern (Text-fig. 11) of positive type, rather indefinite; generally of broadly
rectangular form, with a somewhat darker median zone merging into a darkened, trans-
verse, posterior triangle. Antennae very large, more than three-fourths the length of
the head; basal segment longer than apical, and without apparent subdivision into two
parts. Submentum as in Text-fig. 12.

Gill-spot brown, characteristic, the long stem and posteriorly coiled filaments being
easily seen (Text-fig. 14).

Abdomen.—Ventral papillae triangular, very large and conspicuous. Rectal gills
compound, each with about four lobes. Posterior circlet narrow, rows well spaced, each
composed of 12 rather large teeth. Anal sclerite as in Text-fig. 12. There are no ventro-
lateral dark scales in front of the circlet.

Distribution.—North Queensland: Cape York (Black Camp Creek, Black Gin Creek),

August, September; Russell River, near Babinda, and Smoko Creek, near Bramston Beach,
September.

Biology.

In the type locality larvae were abundant on reeds, sticks, and dead leaves in the
swifter parts of small, clear, shaded creeks running through fairly dense bush. Pupae
were present in the same situations, but only a few could be found, although gill-spot
larvae were not uncommon. Conditions in Smoko Creek were similar to those on Cape
York, but the Russell River was a swifter, more open stream. Adults were not seen in
the field.

SIMULIUM CLATHRINUM M. & M.

Adults, pupae and larvae from North Queensland resemble those from the south,
but the cocoons (Text-fig. 15) differ in that, although they have the same lattice-like
construction and well-developed collar, the mouth has a clearly defined, often thickened
edge, which gives it something the appearance of an A. bancrofti cocoon under a hand
lens. Many pupae in the Babinda district had the filaments broken off short, and none
of these gave rise to adults.

New distribution.—Queensland: Currumbin Creek, near N.S.W. border, January; swifter
coastal streams between Nambour and Gympie, February, April, May (southern race). Cape
York, Black Gin Creek, August; Babinda district, abundant in larger, swifter streams and also

in a small jungle creek near Bramston Beach, September; Innisfail district, Berner Creek,
September (all these being northern race).

178 NOTES ON AUSTRALASIAN SIMULIIDAE,

SIMULIUM AUREONIGRUM N. sp.

Types: Holotype 9, allotype ¢, morphotype pupa and larva, from a small tributary near the
Cascades, Freshwater Creek, Cairns district, October, in the Division of Entomology, C.S.I.R.0.,
Canberra, A.C.T.

Distinctive features.

Five Australian species of Simulium are now known to have pupal respiratory organs
with four filaments. S. ornatipes stands apart on group and general characters, and
S. clathrinum is well differentiated in all stages, but the other three (aureonigrum,
melatum, inornatum) are closely related. They are dark species, with more rounded
heads than 8S. nicholsoni and S. faheyi (Text-fig. 6), and with larvae having ventro-
lateral patches of brown scales just in front of the posterior circlet, suggesting at first
sight a rudimentary form of the ventral chitinous ring found in some species of
Austrosimulium.

S._sureonigrum S. mlatum S. inornatum S._faheyi

Text-fig. 9—¢ hypopygium of S. auwreonigrum; anterior part of phallosome
of other species.

S. aureonigrum is distinguished from its near relatives: in the female by the rich
golden scales on the jet black scutum and by the arrangement of the golden scales on the
abdomen; in the male by the large upper facets of the eyes and gently sinuous distal
margin of the anterior part of the phallosome, in both of which respects it is nearer to
S. clathrinum; in the cocoon by its close weave and lack of a central dorsal projection;
in the pupa by the form and arrangement of the gill-filaments (Text-fig. 15); and in the
larva by the compound rectal gills.

Female.

Length: Body 2:3 mm., wing 2:3 mm.

Head.—F rons about one-fifth of head width, tapering towards antennae; grey, with
scattered golden scales. Face grey, with some pale scales. There is a rim of golden
scales on the occiput behind the eyes. Antennae with first two segments light brown,
remainder brownish black. Proboscis dark brown, palpi brownish black.

Thorax.—Scutum black, fairly densely covered with rich golden scales, mixed on the
dise with some patchily distributed black ones. The black scales also form fairly
definite, narrow dorsocentral lines, which converge slightly from behind forward and
then diverge for the anterior fourth of their length as in S. nicholsoni. Pleurae uniformly
dark grey, with paler grey reflections in certain lights. Pre-alar scales rich to creamy
gold; pronotal, propleural and lower sternopleural scales pale gold.

Legs—Fore and mid coxae covered with golden scales, hind coxae black. Femora
black, with rather irregularly distributed golden scales. Tibiae black, with paler gold
(silvery in certain lights) scales on basal half, extending more distally on outer surface.
Fore and mid tarsi black. Hind metatarsus with creamy zone on basal two-thirds;
remaining segments completely dark. Calcipala and pedisuleus as in SNS. clathrinum.
Claws with a small sub-basal tooth.

Wings hyaline, veins dark. Halteres with brown stem and creamy yellow knob.

Abdomen.—First segment dark brown laterally, paler medially, with a deep golden
fringe. Second deep brown, blackish at apex, and bearing a conspicuous median patch of
rich golden scales, which extend laterally as an apical golden line to join the golden
patches on the side of the abdomen. Third to fifth entirely black dorsally. Sixth with

BY M. J. AND I. M. MACKERRAS. 179

a smooth, somewhat tomentose area in centre, seventh and eighth entirely smooth, not
as shining as S. clathrinum; all three bearing scattered golden scales, especially near
their apical edges. Side of abdomen with broad patches of rich golden scales on
segments 2 to 5, a few on 6, and only two or three on 7. Venter dull yellowish brown.
External genitalia and genital fork as in other species of the group.

Male.

Upper facets of eyes as in S. clathrinum, averaging 0-042 mm. in diameter and in
about 12 rows vertically and across. ‘The antennae are entirely dark. The scutum is jet
black, fairly densely covered with rich golden scales, which are somewhat irregularly
mixed with black ones in the central area, though with no indication of definite lines.
The legs are similar to the female, except that the zone of pale scales on the hind tibia
is more sharply limited distally, and the pale area on the hind metatarsus is rather
vague and indefinite. ;

Abdomen dark brown to black, with apex of first segment lighter brown; second to
fourth covered with velvety black tomentum, remainder with velvety black tomentum in
centre, somewhat shining at sides, where there are silvery reflections in certain lights.
Venter with basal two or three segments dull yellowish brown, remainder dark.
Hypopygium similar to that of S. clathrinum.

Cocoon.
Length about 2:7 mm. along base. An open wall-pocket type, smooth and fairly
closely woven; with a well-defined, darkened, rolled edge, but without collar or central
dorsal projection.

Pupa.

Length 2-5 mm. Cephalic and thoracic hairs slender. Head and thorax densely
covered with minute, irregularly arranged tubercles, which become more triangular and
spiny on the under side of the head, legs and wing covers. Respiratory organ (Text-
fig. 15) with a very short stem, which is covered with minute triangular spines. The
four filaments come off almost simultaneously; the first sweeps upwards and directly
forwards; the second upwards, inwards and forwards; the third downwards, inwards,
forwards and upwards; and the fourth downwards, outwards, forwards and finally curves
upwards and inwards. Abdominal chaetotaxy normal.

Larva.

Length 5 to 6 mm. when full grown. Body white to slaty grey in colour, with the
usual darker mottling. Antennae about half the length of the head, projecting a little
beyond basal segment of mouth brushes. Head pattern positive type, usually forming a
well-defined “W” as in Text-fig. 11, but sometimes rather diffuse. Ventral incisure
deeper than wide, extending about half-way to base of submentum, and forming a
rounded triangle anteriorly. Submentum as in Text-fig. 12; five pairs of hairs on
submental plate. ;

Gill-spot (Text-fig. 14) L-shaped, with three main trunks showing in the anterior,
upright limb, which is shorter than the horizontal limb.

Ventral papillae present, small and triangular, rather better defined than in related
species. Rectal gills compound; accessory lobes variable, usually three on each main
lobe, but sometimes fewer and occasionally none on one or two lobes. Anal sclerite as
in Text-fig. 12. Posterior circlet composed of about 90 rows of hooklets, with about 12
hooklets per row. There are two patches of flat, brownish scales ventro-laterally on
each side just anterior to the circlet.

Distribution.—Known only from the type locality in north Queensland.

Biology.

Larvae and pupae were found adhering to rock and dead leaves, in company with a
few Onephia strenua, in a thin layer of clear water coursing rapidly over an almost
vertical face of rock. The stream was quite small and well shaded, in contrast to the
powerful, turbulent flow in the adjacent, larger, open Freshwater Creek, where C. strenuwa
was abundant but no Simuliwm larvae were found. Adults were not seen in the field.

180 NOTES ON AUSTRALASIAN SIMULIIDAE,

Many of the larvae were parasitized by Mermethid worms, which sometimes almost
filled the posterior swollen part of the abdomen of the host. In one larva a Micro-
sporidian occupied much of the hinder part of the abdomen.

SIMULIUM MELATUM Wh.

Wharton, 1949, p. 406; from Blue Mountains and Sydney district, N.S.W. (type locality
Lett River, Hartley).

Our material agrees substantially with Wharton’s description and with specimens
he kindly presented to the Institute, though with minor differences.

A. fulvicorne

S. nicholsoni 05mm

ee ee

Text-fig. 10.—Respiratory organs of pupae.

Female.
Black and creamy gold to silvery, as compared with the black and rich gold of
S. aureonigrum. The dorsum of the abdomen shows a silvery fringe to first segment,
2 with a conspicuous patch of silvery scales, 3 to 5 black, with an occasional apical
silvery scale, 6 to 8 smooth, slightly shining, and speckled with silvery scales. The
pale area on the hind metatarsus is better defined than in Wharton’s description. Claws
with a very small tooth, which is deeply set in the concavity (Text-fig. 7).

Male.

The upper facets of the eyes, as Wharton points out, are relatively small, resembling
those of S. nicholsoni rather than S. clathrinum; they average 0:033 mm. in diameter
and are in about 16 rows. The abdomen is covered with velvety black tomentum, with
the usual ashy reflections from the shiny lateral patches on segments 5 to 7. The
hypopygium is distinguished by the deep but widely open bay in the distal end of the
anterior part of the phallosome (Text-fig. 9). This is a true indentation, and its
appearance is different from that of the arched anterior part when seen more or less
end on.

Cocoon and Pupa.

As described by Wharton; the cocoon has a well-marked central dorsal projection
and is coarsely woven.

BY M. J. AND I. M. MACKERRAS. 181

Larva.

Difficult to separate from S. aureonigrum and S. inornatum. The head pattern is
generally more diffuse, and the antennae and bases of the mouth-brushes are shorter
relative to the length of the head. The gill-spot is bigger in all dimensions (Text-
fig. 14). Ventral papillae variable, usually none, but sometimes as well developed as in
S. inornatum. The rectal gills are simple. A single group of flat brown scales is present
‘ventro-laterally on each side in front of the circlet in grown larvae, but sometimes
cannot be seen in young specimens.

New distribution.—New South Wales: Gara River, Armidale district (about 3,000 ft.), May,
A, F. O’Farrell. Queensland: Purling Brook (2,000 ft.), Springbrook area, December, larvae
and pupae at the edge of a fast, clear stream in company with Cnephia tonnoiri orientalis,
S. ornatipes, Austrosimulium mirabile, A. furiosum and A. victoriae; tributary of Freshwater
Creek, Cairns district, October, in company with S. aureonigrum (two pupae, from one of which
a 2 emerged).

S. peregrinum

C.strenua

S.aureonigrum S. melatum

Text-fig. 11.—Heads of larvae.

SIMULIUM INORNATUM D0. Sp.

Types: Holotype 2, allotype ¢, morphotype pupa and larva, from a small unnamed creek
at 1,300 ft. on the Springbrook Road, south Queensland, December, in the Division of Entomology,
C.S.1.R.O., Canberra, A.C.T.

An undistinguished species, intermediate in many respects between S. aureonigrum
wnd S. melatum, but sufficiently distinct on characters of the male and pupa to be
regarded as a separate species.

Female.

Head, thorax, legs, wings and halteres generally as in VN. melatum, but with more of
‘a tendency to a dull gold coloration. Scutum with broad though rather indefinite black
dorsocentral stripes, which widen posteriorly almost to reach the median line and lateral
margins. Claws with a small sub-basal tooth. First segment of abdomen with a
conspicuous creamy fringe; second with a patch of golden scales on tergite: third to
fifth with brownish black scales on dise and irregularly scattered creamy golden ones
along the apical edge; sixth rather shiny in centre, seventh and eighth rather shiny over
whole dorsum, all three bearing some dull golden scales which are mainly apical in
position; the lateral patches on segments 2 to 5 are silvery.

182 NOTES ON AUSTRALASIAN SIMULIIDAE,

Male.

Velvety black and golden, as in the other species, and with the usual lateral ashy
patches on the abdomen. Resembles S. melatum in the upper facets of the eyes, being
only moderately enlarged and in about 16 rows. Distinguished from all Australian
species of the genus by the hypopygium, the anterior part of the phallosome being deeply
excavated distally to form a nearly circular bay, which is almost closed by inwardly
projecting arms (Text-fig. 9).

Cocoon.

Wall-pocket type, rather finely woven, with no collar, a rolled anterior edge, and a

well-marked central dorsal projection.

Pupa.

Length about 3 mm. Head and thorax covered with minute tubercles. Chaetotaxy
normal. Respiratory organ (Text-fig. 15) with short dark stem which gives rise simul-
taneously to four diverging filaments of unequal length, the ventral and ventro-lateral
branches being the longest. The filaments are stiff, and curve forward and upward; the
tips may be produced into a very delicate flexible extension which is readily broken off.

Larva.

Resembles S. awreonigrum in all respects, except that the rectal gills are simple, and
the gill-spot is larger, though not as broad as that of 8. melatum. From the latter it is
distinguished by the more definitely W-shaped head pattern, somewhat longer antennae,
and well-defined ventral papillae. ,

Distribution.—Only known from the type locality in south Queensland.

Biology. ;

Larvae and pupae were present in moderate numbers on rock, dead leaves, and the
fine roots of a semi-aquatic plant in a small, steep, shady creek. The water was clear
and moderately fast, but only an inch or so deep and a foot or so wide. The stream
appeared to be drying out fairly rapidly. In the previous year we had taken a few
larvae and pupae (which failed to emerge) about two miles lower down the same road
at an elevation of 900 ft. They were in a similar creek, but on an edge of rock where
the water poured over in a miniature fall.

SIMULIUM NICHOLSONI M. & M.

New distribution.—Queensland: Several coastal streams between Nambour and Gympie. The
most northerly limits known for S. nicholsoni are the Mackenzie River inland and just south of
Fraser Island on the coast.

SIMULIUM FAHEY! Tayl.

Types: Taylor’s type @ is in the School of Public Health and Tropical Medicine, Sydney.
We have designated an allotype ~ from Lennon’s Creek, near Babinda, N.Q., and morphotype
pupa and larva from Berner Creek, and: lodged them in the same collection. Additional specimens
(2, d%, pupa, larva) are in the Division of Entomology, C.S.I.R.O., Canberra, A.C.T.

The status of this species is established on the basis of fresh material from Taylor’s

type locality, and a re-examination of the holotype, which was,kindly lent us by Mr.
D. J. Lee of the School of Public Health and Tropical Medicine, Sydney, who also
permitted us to mount a leg. It is close to S. nicholsoni, but the differences in the
female, pupa and larva are sufficient to warrant specific separation.

Female.

S. faheyi differs from S. nicholsoni in possessing a small sub-basal tooth on the claw
(Text-fig. 7), so doubtful specimens can be identified by clearing and mounting a leg.
Its general coloration is brownish black (darker in fresh material than in the type,
which is thirty years old) and golden, as compared with the black and silvery coloration
of S. nicholsoni, On the abdomen, the fringe of the first segment is golden; the dorsum
of the second is usually entirely brown, but sometimes with a more or less incomplete
golden fringe; third and fourth entirely brown; sixth to eighth tomentose and sprinkled
with brown and pale golden scales, which are not so numerous and conspicuous (nor as
pale) as in §. nicholsoni or S. papuense. Other differences are listed in the key. It is

BY M. J. AND I. M. MACKERRAS. 183

interesting that S. papuwense, which has very different pupae and larvae, resembles
S. faheyi so closely in the female.

Male.
Similar to S. nicholsoni in all respects, including the hypopygium.

Cocoon.
Wall-pocket type, similar to S. nicholsoni, though usually more coarsely woven.

Pupa.
Northern specimens have the head and thorax fairly densely but not quite uniformly
covered with small, blunt tubercles, like S. nicholsoni, and the thoracic and abdominal
chaetotaxy also resembles 8S. nicholsoni. The chief differences are in the respiratory

Ly

ee

\\

S. peregrinum

ok

05mm.
Olmm, Olmm.

Text-fig. 12—From left to right: antenna, submentum, lateral and dorsal views
of tip of abdomen of larvae of Simuliwm spp.

apparatus (Text-fig. 10). Stem very short, 0:07 mm. (0°35 in nicholsoni), dark and
spiny (paler and with smaller, paler spines in nicholsoni). The stem divides into four
branches (immediately into six in nicholsoni), the medial and lateral of which again
divide into two. The six filaments diverge more than in S. nicholsoni, but continue
forward, and the tips curve inward towards each other. The filaments are about 2 mm.
long, but their distal portions are delicate and readily broken off, perfect specimens
being difficult to find.

In southern specimens the tubercles on the head and thorax are sparse and some-
times restricted to a narrow row on each side of the mid line. The gill filaments also
tend to be shorter. In pupae from Tin Can Bay they are about 1-5 mm. long, and in
those from Fraser Island they are remarkably short (about 1 mm.), and usually more
robust, or at any rate the tips are usually intact. This is reflected also in the gill spot
of the larva, the filaments being only long enough to complete one circle and the tips
sometimes projecting out from the top of the spot.

YP

184 NOTES ON AUSTRALASIAN SIMULIIDAR,

Larva.

S. faheyi can be distinguished immediately from S. nicholsoni by its possessing
compound rectal gills, each main digitation usually having three accessory lobes, though
sometimes fewer. Other differences are minor. The head pattern of nicholsoni is
negative, the median longitudinal stripe and two oval areas at the base on each side
being pale; in faheyi the pattern (Text-fig. 11) is of similar form but indeterminate type,
the median stripe often being pigmented, but the oval areas at base usually pale. The
antennae are longer (about 0°35 mm.) in nicholsoni than faheyi (0°30 mm.). The ventral
incisure extends about half-way to the base of the submentum and is rather square ended
in nicholsoni; it is deeper and usually U- or V-shaped in faheyi (Text-fig. 2). In the
gill-spit, the long, pale stem shows conspicuously on the anterior edge in nicholsoni,
whereas in faheyi this edge is also pale, but the dividing filaments can be made out
(Text-fig. 14). The ventral papillae are more conspicuous in faheyi than in nicholsoni.

Distribution.—Queensland: Innisfail district, Berner Creek (type locality), September,
numerous larvae, pupae and bred adults; Babinda district: Lennon’s Creek, small creeks on
Babinda-Boulders Rd., jungle creek near Bramston’s Beach, Russell River, Fishery Creek, all
September. Southern Queensland: Figtree Creek, Fraser Island, April; creek near Tin Can Bay,
April; Kin Kin Creek, near Lake Cootharabah, April; Six-Mile Creek, Cooroy, May—last three
in company with S. nicholsoni; Blunder Creek, Oxley, April, May. The Lawn Hill specimen
previously recorded appears to be correctly placed, but more material is needed to check the
record from the Northern Territory.

Biology.

S. faheyi seems to be as abundant and widespread in the north as S. nicholsoni is in
the south, and the early stages were found in broadly similar situations, having a
preference for moderately fast, smoothly flowing, open streams, and especially for
attachment to reeds and grass beneath the water. It was found alone or in company
with 8S. clathrinum (which was equally abundant) in the north, and in company with
one or more of S. ornatipes, S. nicholsoni, A. furiosum and A. bancrofti in the various
southern streams. A female was taken at Bramston Beach attempting to bite man; in
view of the abundance of the early stages, this habit would appear to be as occasional
as in 8. nicholsoni.

The Genus AUSTROSIMULIUM Tonn.

AUSTROSIMULIUM MIRABILE M. & M.

New distribution.—Queensland: Babinda district, Smoko Creek, small jungle creek near
‘Bramston’s Beach, and small creek on Babinda-Boulders Road, all September. Springbrook
area: unnamed creek at 1,300 ft., in company with S. inornatwm, November; Purling Brook
(2,000 ft.), December. (Previously only known from the type locality at Dawson’s Creek on
the slopes of Mt. Glorious, 8.Q.) :

AUSTROSIMULIUM FULVICORNE 0. Sp.

Types: Holotype ¢%, mounted on a slide, morphotype pupal skin and larva, in spirit, from
Yanky Jack Creek, Fraser Island, February, D. Mackerras, in the Division of Entomology,
C.S.1.R.O., Canberra, A.C.T. <

,

Distinctive features.

Belongs to the mirabile group and most nearly related to that species, but so well
differentiated that we feel justified in describing it on less material than we would
ordinarily require. The male is distinguished from A. cornutum by the shape of the
antennae and entirely yellow segments 4 to 10, and from A. mirabile by antennal
coloration and absence of dark spots on the wing; the pupa from A. mirabile by the
rounded, club-shaped end of the spiny respiratory horn; and the larva from A. mirabile
by the longer, darkened basal segment of the antenna and by the head pattern.

Male (in spirit).
Length: Body (abdomen over-extended) 4 mm.; wing 1:8 mm. (the true size is
about the same as A. mirabile).

Head.—Wider than thorax; upper facets of eyes moderately enlarged, in 15-16 rows.
Antennae (Text-fig. 13) distinctly shorter than in A. mirabile but of same general form,

BY M. J. AND I. M. MACKERRAS. 185

with second segment much longer than others; the ninth and tenth are incompletely
separated (cf. A. bancrofti); first and second segments dark brown, third a brighter
brown, fourth to tenth yellow. Face dark brown, with silvery hairs; mouth parts and
palpi brown. There appear to be some golden hairs around the occiput.

Thorax.—Scutum dark brown, and appears to be covered with silvery scales.
Pleurae brown.

Legs.—Not so uniformly dark brown as in a spirit specimen of A. mirabile. The
fore and mid femora and tibiae are pale brown, almost yellowish, and the hind femur and.
tibia are also pale, but margined with dark pigmentation, especially on the tibia. All
knees are yellowish. All tarsi (including hind metatarsus) are dark brown. Calcipala
and pedisulcus as in Text-fié. 13. The claws are of normal male form.

——+

O:lmm.
O:'lmm.

>——_——S— +
O-Imm.

Text-fig. 13.—A. fulvicorne. ¢: a, antenna; b, palp; ce, hind tarsus. Larva: d, head
pattern ; e, antenna; f, submentum; g and h, lateral and dorsal views of tip of abdomen.

Wings.—Veins dark, and hairs on veins strong, with a few outstanding black hairs
on basal section of R as in A. mirabile; but without the dark pigmented spots, except a
trace at the fork of R, and completely without the groups of long dark bristles which are
associated with these spots in A. mirabile. Halteres with stem brownish and knob
creamy.

Abdomen.—Chitinous parts of tergites dark brown, membrane pale; hairs appear to
be black. No trace could be seen of the silvery, tomentose patches, which are charac-
teristic of A. mirabile and clearly visible on a spirit specimen as well as on pinned
material. The hypopygium resembles that of A. mirabile, and has a similar acute setulose
ventral swelling on the anterior part of the ffhallosome. The style, however, has only
two spines, as in A. cornutum and A. crassipes. The spines are variable in A. mirabile;
one specimen now before us has three on each side, and another four on one side and
two and a small one on the other.

Note—tThe colour description is to be taken as general rather than precise, and may
require amendment when fresh material is available. The comparison with the male of
A. mirabile was, however, made entirely on spirit specimens.

186 NOTES ON AUSTRALASIAN SIMULIIDAE,

Cocoon.
Length: 1:8 mm. Simple, finely woven wall-pocket type, with central dorsal
projection, like that of A. mirabile.

Pupa.

Thoracic integument with exceedingly minute, irregularly distributed tubercles, and
a large, stout, curved pair of posterior thoracic hairs as in A. mirabile. The abdominal
chaetotaxy is apparently normal, but the specimen is somewhat damaged. The respira-
tory horn (Text-fig. 10) is flattened, blade-like, with rounded end, and covered with
numerous strong, sharp, black spines. The filaments are not very numerous, arise
mainly from the sides and internal surface and but few from the lateral surface, are
rather longer than the horn, and of the usual beaded appearance.

Larva.
Length: 5 mm. Creamy white, with greyish brown mottling. Head with broad,
dark pattern (Text-fig. 13), which is distinctly wider than in A. mirabile. Antennae
similar to A. cornutum, with brownish basal segment, shorter than the paler, slender

wus

\

. aureonigrum

C. strenua S.inornatum S. melatum

Text-fig. 14.—Lateral view of full-grown larvae, showing gill-spots; note size of
C. strenua. (This figure is intended for use in conjunction with our earlier
figure—1949, Text-fig. 19, p. 403.)

distal segment (Text-fig. 13). Ventral incisure shallow, wider than deep (Text-fig. 2).
Submentum as in Text-fig. 13, with five strong hairs on each side.
Gill-spot (Text-fig. 14) pear-shaped, dark, with the spiny horp distinctly visible.
Rectal gills simple. Large ventral papillae present. Anal sclerite with the usual
backwardly directed struts and the incomplete ventral chitinous ring characteristic of
the group, the upper end being swollen as in A. mirabile (Text-fig. 13). Cirelet similar
to other species.
Distribution.—Queensland: Only known from the type series from Fraser Island, comprising
about 20 larvae and one adult male with its pupal shell and cocoon.

Biology.

The larvae and pupa were on grass in fairly swift, evenly flowing clear water in a
small creek, deeply cut in the sand and shaded by vegetation growing over from the
banks.

AUSTROSIMULIUM CRASSIPES (Tonn.).
New distribution.—Queensland: Small tributary of Cave Creek (upper Numinbah Valley),
March. (Previously known only from Victoria and the Blue Mountains, N.S.W.)

BY M. J. AND I. M. MACKERRAS. 187

AUSTROSIMULIUM BANCROFTI (Tayl.).
New distribution.—Queensland: Coastal creeks between Nambour and Gympie, February,
April.
AUSTROSIMULIUM PESTILENS, M. & M.
New distribution.—New South Wales: Nyngan, March, August, J. Armstrong. Not pre-

viously recorded outside Queensland; specimens kindly submitted by Mr. D. J. Lee, School of
Public Health and Tropical Medicine, Sydney.

AUSTROSIMULIUM FURIOSUM (SK.).

New distribution.—Queensland: Fig-tree Creek, Fraser Island, April; several small coastal
streams between Nambour and Gympie, April, May; Purling Brook, Springbrook area (2,000 ft.),
December.

AUSTROSIMULIUM VICTORIAE (Tonn.).
Queensland specimens differ from southern ones in that the long dorsal projections

on the cocoon are completely missing (Text-fig. 15). Adults, pupae and larvae are,
however, not to be separated from Canberra specimens, and we hesitate at present to use

S. clathrinum S. peregrinum
northern race

S. aureonigrum

S.inornatum A.victoriae
TEaPALinG Queensland race

Text-fig. 15.—Cocoons and pupae (for use in conjunction with our earlier figure—
1949, Text-fig. 20, p. 404, but it is to be noted that the respiratory organ of one
side only is shown here).

the cocoons alone to differentiate species 01 subspecies. Nevertheless, differences in the
shape of the cocoon in different parts of the geographical range have been observed in
other species also (S. ornatipes, S. clathrinum), and may indicate that genetic differences
are developing. Much more material would be needed to attack this problem.

New distribution.—Queensland: Little Nerang Creek (300 ft.), September; Purling Brook
and a small creek in the rain forest, Springbrook area (2,000 ft.), December. (Not recorded
previously north of Wentworth Falls, N.S.W.)

References.

Macxkerras, M. J., and Mackmrras, I. M., 1948.—Simuliidae (Diptera) from Queensland. Aust.
I SG, IRA (03), ile wel,

Mackerrras, I. M., and MAckerras, M. J., 1949.—Revisional notes on Australasian Simuliidae
(Diptera). Proc. LINN. Soc. N.S.W., lxxiii: 372-405.

WHARTON, R. H., 1949.—New species of Simuliidae (Diptera, Nematocera) from New South
Wales. Proc. Linn. Soc. N.S.W., lxxiii: 406-412.

Wee

188

REVISION OF THE GENUS SOLENOGYNE CASS.

By Gwenpa L. Davis, Department of Biology, New England University
College, Armidale.

(Fourteen Text-figures. )

[Read 26th July, 1950.]

INTRODUCTION.

Cassini (1828) described the genus Solenogyne from a dry specimen in Mérat’s
herbarium, which had been collected in the Port Jackson district. He wrote: “this genus
is distinguished from Lagenophora by its non-radiate and discoidal inflorescence, the
crown composed of tubular flowers in many rows, by the ovaries lacking a collar and
by the involucral bracts being applied and foliaceous all over.”

The genus Emphysopus was erected by J. D. Hooker in 1847 to accommodate certain
specimens collected in Tasmania by Gunn, but no comparison was published between this
new genus and either Lagenophora or Solenogyne.

In 1860 Hooker incorporated Emphysopus in Lagenophora with the comment “at
one time, as I thought, worthy of being kept generically separate, but upon reconsidera-
tion I am induced to unite it with Lagenophora’.

Mueller (1866) listed Hooker’s genus in the synonymy of a new species of Solenogyne,
but Bentham (1867) sank both Solenogyne and Emphysopus in Lagenophora.

Although Moore and Betche (1893) once more separated Solenogyne (in which they
included Hmphysopus) from Lagenophora, most later workers have followed Bentham,
whose treatment was retained by Maiden (1916).

The investigations of the present writer have supported Moore and Betche, and the
genus Solenogyne is now reinstated in generic rank. Lagenophora and Solenogyne agree
in that the capitulum is made up of two types of florets, the outermost of which are
arranged in three or four rows and are pistillate. The central florets in both genera
are staminate and although the stylar branches are densely papillose and relatively
large, they function only in brushing the pollen out of the anther tube, and the ovaries
are abortive. These two genera are undoubtedly closely allied but the tubular or lipped
outer florets of Solenogyne and the marked difference in fruit are characters which
merit generic recognition.

Affinities.

In the absence of cytological evidence the affinities of Solenogyne and Lagenophora
‘can only be discussed in a general way. Nevertheless Babcock (1947) states: “the
phylogenetic significance of chromosome number, size and shape can be interpreted only
in relation to or with the aid of other criteria’, and although “the evidence from
comparative morphology, cytology and genetics has been combined in the determination
of interspecific relationships in Crepis”, he asserts that differences in chromosomes are
always reflected in morphology. In view of this a discussion of some evolutionary trends
shown by these genera, though based on comparative morphology alone, may not be
entirely worthless.

In Brachycome it has already been noted (Davis, 1948) that the innermost dise
florets seldom develop fruit, and it was suggested that this might be due to the earlier
maturing outer florets commandeering the vascular supply of the receptacle. When the
ray and outer disc florets of B. marginata Benth. were removed those at the centre still
failed to produce normal fruits. Although these experiments were not sufficiently
extensive to be of great value in themselves, they did suggest another explanation, that
is, that the inner dise florets might be partially or wholly sterile, and further work is
being carried out along these lines. If in Brachycome the species can be arranged in

BY GWENDA L. DAVIS. 189
a series from normal fertility of the inner disc florets to their partial and finally
complete sterility, then the evolution is indicated of the condition in Lagenophora and
Solenogyne where all the disc florets are sterile. The strap-like ray florets of
Lagenophora are identical with those of Brachycome, and if the relationship with
Solenogyne is as close as comparative morphology indicates, then the question must be
decided whether tubular ray florets are primitive or derived. It is suggested that had
this tubular condition been the retention of a primitive character, little or no variation
would be shown. The fact that all conditions, from completely tubular to 2-lipped
and finally 1-lipped with minute ray are found, indicates that Solenogyne originated from
a Lagenophora-like ancestor, and that genetic stability has not yet been attained. The
opinion that Solenogyne is of more recent origin than Lagenophora is supported by its
more restricted distribution.

Distribution of the Genus.

Solenogyne is confined to eastern and southern Australia from south-eastern Queens-
land through New South Wales and Victoria to south-eastern South Australia and is
widely distributed’: in Tasmania. A single record exists for ‘North Queensland’, but
unfortunately no exact locality accompanies the specimen.

According to Cheeseman (1925) one variety has become naturalized in New Zealand
on Banks’ Peninsula and near Wellington. Specimens have been examined from the
latter locality and were identical with those from New South Wales.

Nomenclature.
Some changes in nomenclature have been necessary on priority grounds and are
discussed in the groups concerned.
Type terminology is that of Davis and Lee (1944).

Categories.

This genus consists of two populations which, although vegetatively distinct, bear
identical fruits. As in Brachycome (Davis, 1948) and Lagenophora (Davis, 1950), the
principle has been followed that only morphological differences in the fruits justify
specific status, and that constant and discontinuous differences in vegetative characters
are varietal in nature. It therefore follows that Solenogyne is a monotypic genus of two
varieties.

Evaluation of Taxonomic Characters.

All morphological characters were examined and limits of variation noted. Although
few of these characters were found to be continuously variable, and therefore of no
value in distinguishing between populations, the results of these examinations are
discussed below. Habit: scapigerous, but considerable variation in size was noted, which
could probably be largely correlated with habitat. Unfortunately collector’s notes seldom
supply details of habitat. Indumentum of a septate-hairy nature was ‘always present,
but the degree of development was variable. Leaves showed a slight amount of variation
in the dissection of their margins and in one variety the specimens fell into three
geographic groups on this and other vegetative characters. Léaves alone, however, have
little diagnostic value. Scapes supply the primary taxonomic character in distinguishing
between the two varieties, and although some variation was observed both in breadth
and length, intermediate forms were not found. Capitula, florets and fruits were identical
in both varieties.

Specimens Hxamined.

All specimens examined are listed under the appropriate category, together with
collector’s notes. The source of each specimen is indicated in brackets as in a previous
paper. (Davis, 1950.)

Descriptions.
It is only when a genus is made up of two or more species that generic characters

can be recognized apart from specific. Since Solenogyne is a monotypic genus no generic
diagnosis is given.

190 REVISION OF SOLENOGYNE CASS,

Specific and varietal descriptions are based on available material, consequently as
further specimens are collected these descriptions may require modification. Limits of
variation are indicated for all characters, but size measurements are to be regarded
merely as a guide.

Type Species: SOLENOGYNE BELLIOIDES Cass.

SOLENOGYNE BELLIOIDES Cass.
Dict. Sci. Nat., lvi (1828), 174.

Perennial herbs, 1:5-18 em. high, more or less septate-hairy on leaves and scapes.
Leaves up to 11 ecm. long, 2:1 cm. broad, radical, oblanceolate, dentate or coarsely
toothed, rarely entire. Scapes filiform or robust, provided with 1—4 broad-linear bracts,
the lowest of which are up to 1:3 cm. long, and the uppermost scale-like. Capitula 2-26
on each plant, 5-7 mm. diameter. IJnvolucral bracts 22-30 in 3-4 rows, 1:5-2:2 mm. long,
0-5-1:3 mm. broad, glabrous or microscopically glandular, linear’ to narrow-obovate,
obtuse, and the margins torn ciliate. Outer florets about 55, in 3-4 rows, tubular at the
base with.distal half to third split to form a short entire or toothed ray, 0-7 mm. long,
female. Central florets about 10, 5-toothed, tubular, regular, 2-2 mm. long, staminate,
ovary abortive. Stylar branches of outer florets linear, smooth and diverging at
maturity, those of the inner florets unequal in length, densely glandular hairy and
appressed. Stamens 4-5, present only in the central florets. Anthers united into a tube,
and the connective is prolonged distally to form a terminal appendage. Receptacle
1-1-3 mm. broad, 1 mm. high, convex, shallowly pitted. Fruit an inferior achene,
2:2-2-6 mm. long, 0:7-1:-2 mm. broad, light to dark brown, narrow-elliptical to narrow-
obovate, flattened, smooth. Pappus absent.

Key to the Varieties.

Scapes filiform, usually exceeding the leaves, rarely less .................... var. a bellioides
SYMONS HOMIE, WIGUEYI hy ane s.ceeohinee We MEMES Sodaheoododsconsdconoasonbnensone var. B Gunnii

Solenogyne bellioides Cass. var. a bellioides comb. et stat. nov. (Text-figures 1-11.)

Synonymy: Solenogyne bellioides Cass., Dict. Sci. Nat., lvi (1828), 174; Solenogyne brachy-
comoides F. Muell., Fragm., v (1866), 62; Lagenophora Solenogyne F. Muell., ex Benth, FI.
Aust., iii (1866), 506.

Herbs 5:5-18 em. high in which the indumentum of septate hairs is not dense but can
be seen macroscopically. Leaves up to 6:5 em. long, 2:1 em. broad. Scapes filiform, in
thickness one-seventh to one-tenth the breadth of the inflorescence, and usually at least

twice as long as the leaves, rarely less. Jnvolucral bracts turn downwards when fruit
are mature.

Habitat: Grassland and open forest.

Range: South-eastern Queensland, northern tablelands and middle western districts of New
South Wales, and central coast south to Hill Top.

Specimens examined.—Queensland: Ridges about Brisbane, F. M. Bailey (BRI); Brisbane
River (MEL); Botanic Gardens, Brisbane, 2.1915, C. T. White (BRI); Goodna, forest country,
edge of scrub, 17.3.1$17, C: T. White (BRI); Laidley, 3.1920, C. T. White (BRI); Gatton,
10.1906, J. F. Bailey (BRI); Gatton Agriculture College, moderately common in mixed native
pasture, poor soil, badly drained subsoil, 19.2.1930, C. T. White, No. 6637 (BRI); Toowoomba,
4.1916, C. T. White (BRI).

New South Wales: Tenterfield, 10.1886, H. Betche (NSW); Tenterfield, C. Stuart, No. 939
(MInL) ; Timbarra, C. Stuart, No. 99 (MEL); Clarence River, Beckler (MEL, NSW) ; Warialda,
3.1906, H. M. R. Rupp (NSW); Glen Innes, 29.10.1886, E. Betche (NSW); Clifton, C. Stuart,
No. 41 (MEL); Tingha, 6.1917, J. L. Boorman (NSW); Armidale, 31.1.1941, G. L. Davis
(FAR) ; University College, pasture land, under trees, 28.11.1949, G. L. Davis (FAR) ; Bundarra-
Uralla Road “pasture land’, 13.11.1949, G. L. Davis (NSW); Namoi River, 1890, Musson
(MEL); Coonamble, 4.1913, W. H. Potts (NSW); Murrurundi, 3.1895, F. Fraser (NSW) ;
Moonan Brook, near Scone, 1883, H. Carter (MEL); Gulgong, 4.1901, J. H. Maiden and
J. L. Boorman (NSW); Barrangan, beyond Mudgee (MEL); Hill End, 4.1885, J. Lauterer
(MEL); Hornsby, 4.1914, W. F. Blakely (NSW); Botanic Gardens, Sydney, 4.1914, W. F.
Blakely (NSW); Hurstville, 3.5.1899, I. Cheel (NSW); Penshurst, 10.1909, EB. Cheel (NSW) ;
Liverpool, ‘‘amongst grasses on hill in Hucalypt forest”, 15.4.1931, C. E. Hubbard and E. Cheel,
No. 8486 (BRI); Hill Top, J. Shirley (BRI).

BY GWENDA L. DAVIS. 1197

The first record of this population was in 1828 when Cassini erected the monotypic
genus Solenogyne (S. bellioides) to accommodate “a dry specimen which appears to
have been collected in the neighbourhood of Port Jackson, and which is to be found
among the unnamed Synantherée in the herbarium of M. Mérat” (original description).
Although this specimen has not yet been traced, Cassini’s thorough description leaves
no doubt as to its identity.

This same population was later described by Mueller (1866) as S. brachycomoides,
and Cassini’s name was listed in synonymy. In the original description and immediately
following the name of the new species, Mueller inserted the words “Lagenophora
Solenogyne F.M. coll.’, apparently referring to specimens distributed by him with that
identification. When Bentham (1866) incorporated Solenogyne in Lagenophora he used
Mueller’s manuscript name which he attributed to him. Since Bentham’s description
is the first to which the name Lagenophora Solenogyne is formally applied, the epithet
itself should date from that time and not, as is generally quoted, from Mueller’s earlier
mention in synonymy.

The type locality of S. brachycomoides is “near the town of Clifton, New England,
C. Stuart”, while Bentham, in connection with L. Solenogyne, quoted specimens from
three localities, one of which was ‘New Hngland, C. Stuart”. In the National Herbarium,
Melbourne, is a folder of specimens labelled by Mueller “Solenogyne brachycomoides,
Clifton, New England, C. Stuart’, and marked as having been examined by Bentham. One
of these specimens (Text-figure 1) has therefore been selected as lectotype of both
Solenogyne brachycomoides and Lagenophora Solenogyne.

Since the genus Solenogyne is reinstated in the present paper, Cassini’s original
epithet is retained and the source of the slightly different spelling in Index Kewensis
(S. bellidioides) has not been traced.

Variation within this variety is confined to the size of the plants, other characters
remaining constant. One specimen was examined in which the indumentum was so
dense that the plant had a woolly appearance, but owing to the uncertainty of the
locality from which it was collected, it has not been listed above. A field label is
attached to the specimen, and bears, in Leichhardt’s writing, the data ‘the wool leaved
daisy, near water, 9th March, 1843”.

Solenogyne bellioides Cass. var. 8 Gunni (Hook. f.), n. comb.
(Text-figures 12-14.)

Synonymy: Hmphysopus Gunnii Hook. f., Lond. Journ. Bot., vi (1847), 114; Solenogyne
vellioides Sond. in Linnaea, xxv (1852), 480; Lagenophora Emphysopus Hook. f., Fl. Tasm., 1
(1860), 187; L. Gunnii (Hook. f.) J. M. Black, Fl. S.A., Pt. 4 (1929), 580.

Haptotype: Tasmania, 1835, Gunn, No. 512 (NSW).

Septate-hairy herbs, 1:5-11:3 cm. high. Leaves up to 11 cm. long, 1:4 cm. broad.
Seapes robust, in thickness one-fifth to one-sixth the breadth of the inflorescence.
Involucral bracts do not turn downwards at maturity of fruits.

Habitat: Grassland and open forest.

Range: Tablelands, middle west and central coast of New South Wales, throughout
Victoria and south-eastern South Australia; east coast of Tasmania, and a single record from
Queensland. Naturalized on Banks’ Peninsula and near Wellington in New Zealand (Cheeseman,
1925).

Specimens examined.—Queensland: North Queensland, J. H. Tenison-Woods (MEL).

New South Wales: Clarence River, Beckler (MEL); Glen Innes, 6.1917, J. L. Boorman
(NSW); Ebor Falls, 31.1.1941, G. lL. Davis (FAR); University College, Armidale, 28.11.1949,
G. L. Davis (FAR): Moona Plains, Walcha, 1884, A. R. Crawford (MEL); Warrambungle
Ranges, 10.1899, W. Forsyth (NSW); Barrington Tops, on granite, 7.1.1934, L. Fraser and
J. Vickery (NSW); Swashfield, via Oberon, 31.1.1941, F. Johnson (NSW); west of Blue
Mountains, 7.1893, G. King (MEL); Putting Green, Blackheath, 19.1.1937, N. Popel (NSW) ;
Jenolan Caves, 3.1900, W. F. Blakely (NSW); Ryde Bowling Green, 10.1944, Cruth (NSW) ;
near Tempe, 10.1897, W. Forsyth (NSW); Hurstville, 18.5.1899, E. Cheel (NSW); Moss Vale,
28.10.1910, E. Cheel (NSW); Lake George, 4.1898, EH. Betche (NSW); Queanbeyan, 1.1888,
EK. Betche (NSW); Nelligen-Braidwood Road, Top of Clyde Mt., in grass at roadside, 25.4.1932,
EF. A. Rodway (FAR); Wagga Wagga, 3.1885, R. Thom (MEL); Upper Murray River, 1887,
C. French, Jr. (MEL) ; Edward’s River, 10.1875, F. Mueller (MEL).

eo
bo

REVISLON OF SOLENOGYNE CASS,

Text-figures 1-14.

BY GWENDA L. DAVIS. 193

Victoria: Snowy plains on the Limestone River (MEL) ; Genoa district, 3.1885, W. Bauerlen
(MEL); in meadows above the Snowy River, F. Mueller (MEL); Kilmore, 1883, R. Thom
(MEL) ; Laverton, 4.1911, J. Starr (MEL) ; near Station Peak (MEL) ; sources of the Campaspe
River, 6.1887, J. Dickinson (MEL) ; Moyston, 4.1872, D. Sullivan (MEL) ; Wando Vale, 21.6.1842,
J. G. Robertson (NSW) ; Wimmera, 10.1899, D’Alton (MEL) ; Wimmera, 1890, H. Davis (MEL) ;
Wimmera, swampy places, 16.4.1898, F. M. Reader (MEL).

Tasmania: East coast, south of Elephant Pass, on a grazed short-grass roadside bank near
sea-level, 18.11.1942, H. D. Gordon (HO); Mt. Rumney, Mountain Top, 1,236 ft., 25.4.1931,
EF. H. Long (HO); New Town, Spicer (MEL); Domain, Hobart, 5.1892, L. Rodway (HO) ;
Mt. Nelson Range, 1.1893, L. Rodway (NSW); Tasmania, 1835, Gunn, No. 512 (NSW).

South Australia. Open pastures on State hills, Belair, 26.5.1882, R. Tate (AD); entrance to
Long Gully, North Park, 22.4.1910, H.H.D.G. (JMB); Port Elliot, in paddock near Cliff House,
9.5.1918, H. W. Andrews (JMB); Clarendon, 5.1882 (MEL); the Bluff, Encounter Bay, on
eropped grassy hills, 1.1924, J. B. Cleland (JBC, JMB); Encounter Bay, 12.5.1928, J. B.
Cleland (JBC) ; Hall’s Creek, Encounter Bay, 23.5.1932, J. B. Cleland (JBC).

J. D. Hooker (1847) described Hmphysopus Gunnii from a specimen collected in
Tasmania by Gunn, but later (1860) he included this species in Lagenophora as
L. Emphysopus. In Gunn’s collection in the National Herbarium, Sydney, is a specimen
with the label “512.1835”, which has been nominated haptotype (Text-figure 12).

Solenogyne bellioides Sond. is listed in the above synonymy on Bentham’s authority
and the fact that the original description agrees with this variety, but no syntype
specimens are available in Australia.

The specimens examined fall into three groups on vegetative characters:

1. Small plants from 1:7—6-3 cm. high with 2-8 inflorescences. The indumentum of
septate hairs is very sparse, so that the plants are almost glabrous. Leaves are up to
3:4 em. long, 1:3 cm. broad, closely and regularly serrate. This form has only been seen
from New South Wales localities.

2. Larger plants from 4:2-11 cm. high bearing up to 17 inflorescences. Septate hairs
are more abundant and visible macroscopically on stems and leaves, but are not
sufficiently numerous to appear woolly. Leaves up to 9:8 cm. long, 1:6 cm. broad, the
teeth further apart and the margin not closely serrate. Only seen from southern New
South Wales, western Victoria, Belair and Mt. Graham in South Australia.

3. Densely septate-hairy plants of a rather woolly appearance, 2:5-8-8 cm. high
with 3-9 inflorescences. Leaves up to 7:6 cm. broad, the teeth of which are similar to
the preceding form. This is the most widely distributed form, occurring from the
Clarence River, through the highlands of New South Wales, Victoria and Tasmania.

It is considered that the series of specimens available is not sufficiently large to
justify these three apparently distinct forms being recognized under separate status,
since more intensive collecting may yield specimens which bridge the apparent
discontinuities.

No other variation was seen in this variety except an abnormal condition in a
specimen from the Snowy River, in which two of the scapes bifurcated distally and each
branch was terminated by an inflorescence.

ACKNOWLEDGEMENTS.

The specimens on which this revision was based were lent for that purpose by the Directors
of the various public herbaria, and I would like to acknowledge the co-operation in this respect
of Mr. R. H. Anderson, National Herbarium, Sydney; Mr. A. W. Jessep and Mr. J. H. Willis,
National Herbarium, Melbourne; Mr. C. T. White, Brisbane Herbarium; Miss W. Curtis,
University of Tasmania; as well as the following private collectors: Professor J. B. Cleland
and Mr. J. M. Black of University of Adelaide, and Dr. F. A. Rodway of Nowra.

Text-figures 1-11, S. bellioides var. bellioides.—1, habit. lectotype of S. brachycomoides and
Lagenophora Solenogyne, x %.: 2, capitulum, x 5. 3. receptacle after shedding of fruits, x 5.
4 and 5, ray florets and young fruits showing extremes of variation, x 20. 6, stylar branches of
ray florets, x 20. 7, fruit, x 10. 8, dise florets, x 10. 9, anthers, x 20. 10, stylar branches of
dise floret, x 20. 11, distribution.

Text-figures 12-14.—S. bellioides var. Gunnii.—12, habit. Haptotype of Bmphysopus Gunnii,
x 4. 13, receptacle after shedding of fruits, x 5. 14, distribution.

194 REVISION OF SOLENOGYNE CASS.

References.

Bascock, [. B., 1947.—The Genus Crepis. Pt. 1. Berkeley and Los Angeles.
BENTHAM, G., 1866.—Flora Australiensis, iii: 506-508. London.
Buack, J. M., 1929.—Fiora of South Australia, iv: 580-581. Adelaide.
CASSINI, H., 1828.—Dictionaire des Sciences Naturelles, lvi: 174.
Davis, G. L., 1948.—Revision of the genus Brachycome Cass., Pt. 1. Australian species. Proc.
LINN. Soc. N.S.W., lxxiii, pts. 3-4: 142-241.
, 1950.—Revision of the Australian species of the genus Lagenophora Cass. Proc. LINN.
Soc. N.S.W., Ixxv: 122.
Davis, H. F. C., and Ler, D. J., 1944.—The Type Concept in Taxonomy. Aust. Jowrn. Sci., vii,
No. 1: 16-19.
Druceg, 1916.—Report of the Botanical xchange Club of the British Isles, p. 630.
Hooxer, J. D., 1847.—London Journal of Botany, vi: 113-114.
, 1860.—Flora Tasmaniae, i: 189. London.
MAIDEN, J. H., 1916.—Census of New South Wales Plants: 196. Sydney.
Moore, C., and BETcHE, I., 1893.—Handbook of the New South Wales Flora: 261-262. Sydney.
MuveE.urr, F., 1866.—Fragmenta Phytographiae Australiae, v: 62. Melbourne.

195

THE CLASSIFICATION OF BACTERIA.
WITH SPECIAL REFERENCE TO NON-PATHOGENIC EUBACTERIA.*

By H. J. Ferguson Woop, M.Sc., B.A., A.A.C.I.,
Division of Fisheries, C.S.I.R.O., Cronulla, N.S.W., Australia,

[Read 26th July, 1950.]

INTRODUCTION.

An urgent need in bacteriology to-day is a revision of the classification of bacteria.
At the present time, two determinative systems are in common use, that of Lehmann
and Neumann (1930), which is used on the Continent of Europe and to a large extent
in Britain, and that given in Bergey’s Manual of Determinative Bacteriology (5th Ed.,
1943), which is used in America and by workers in general bacteriology in Australia.
In Britain, Topley and Wilson’s textbook on the Principles of Bacteriology and Immunity
deals with classification using a slight modification of the classification of Buchanan
(1917) and its later development by Bergey et al. But the section on non-pathogenic
forms is weak.

In my work on bacteria of marine origin I have found that a revision of the systems
now in use is essential before any studies on the relationships of any flora can be
profitably undertaken. In a previous study (Wood, 1940) I found that a number of
types isolated did not fit any species described by Bergey, whereas Lehmann and
Neumann’s classification proved too general. It was felt at the time that many of the
strains did not deserve specific rank and I begged the question by using a decimal
system to describe them. It may well be that, with some genera, such a system may be
the best method of describing the strains until further work has shown what can be
justly regarded as differentiating characters.

A growing amount of literature deals with bacterial classification, while earlier
work has been ably summarized by a number of authors. None of the classifications is
free from serious faults, the conclusion being that bacterial classification is a subject of
considerable difficulty, owing to the apparently simple nature of the organisms and
more especially to their extreme variability.

The best starting point appears to be a critical study of some of the more prominent
suggestions which have been made, There seems, however, to be a limit to this sort
of work; at a certain stage one can only go back to the bacterial groups with which one
is familiar to see how they fit into the suggested scheme. This suggestion has already
been made by Bruce White (1937). Once one knows the range of variation of his
organisms and can show how to arrange them into groups and sub-groups, so that other
workers can compare different floras, one has a working classification of genera and
species. If this working system fits one of the determinative classifications, the latter
may be claimed to be satisfactory for the purpose. I feel sure that such a combination
of the practical with the speculative will give a much clearer picture than can be seen
at present. Lately van Niel with the non-sulphur purple bacteria and Stanier with the
Cytophagas have successfully used the method.

In any classification there are bound to be difficulties, for, to the taxonomist,
“natura non facit saltem”. Frequently species and often genera can only be determined
by making arbitrary divisions at convenient points—e.g., in the case of Pseudomonas
and Vibrio. The student should realize that these divisions are arbitrary and that they

*The work described in this paper was carried out as part of the programme of the
Division of Fisheries, Commonwealth Scientific and Industrial Research Organization,
Australia.

196 THE CLASSIFICATION OF BACTERIA,

will only rarely mark real breaks in continuity. Observed breaks are often due to lack
of knowledge of intermediate forms. Therefore, in recording his results, a writer should
state where in each group his divisions occur and why he has drawn them. Then if
he or another writer wishes to alter the divisions it can be done without invalidating
the records of other workers or throwing the whole system out of gear. The debated
question (Zobell and Upham, 1944, Zobell and Feltham, 1934, Stanier, 1941) of whether
a distinctive marine flora should be recognized cannot be resolved until it is agreed by
bacteriologists whether failure to grow on fresh water media is sufficient grounds for
creating new species. On this distinction depends whether or not a marine flora can
be regarded as homologous with a land flora. This question of the possibility of
continued interchange of sea and soil bacteria is highly important in marine biology.
Hence my own enforced excursion from marine bacteriology into the taxonomic field and
my insistence that taxonomy must be pragmatic.

DIFFICULTIES IN THE WAY OF ADEQUATE CLASSIFICATION.
A. BACTERIAL VARIATION.

Recent literature abounds with reports of variation in bacteria which some authors
claim is mutational in character and therefore bound up with the genetics of the
organisms—e.g., Severens and Tanner (1945). If mutants occur, as now seems certain,
we should have to regard each culture as containing potentially several mutant strains.
By selective culture we can increase the numbers of the strain most suited to our
chosen conditions till we get a pure culture which may or may not retain the tendency
to mutate. On the other hand, we may, for example, by growing a culture in 3%
sodium chloride, induce a salt-tolerant strain, or the whole culture may become salt
tolerant and have to be trained again to grow on fresh water media. Such strains
would be more labile than the mutant strains.

We have, in the literature, records of variation in morphology, serology, biochemical
reactions, nutrition requirements, pigmentation, enzymic activity, phage resistance,
resistance to bactericides and loss of function. A study of the papers given or referred
to in the Cold Spring Harbour Symposium of Quantitative Biology II, 1946, will show
this.

This brings us to the question of type cultures. Lehmann and Neumann (1930)
give numerous examples of type cultures which in their laboratory behaved very
differently from the reactions given for them by the original recorder. I have found
that my own cultures, when kept for a year or more, showed considerable variation from
the original, and that the confirmation of a reaction of such cultures for comparison
with a given strain is often impossible. Type cultures of these organisms may there-
fore be quite misleading and, although they are a valuable aid to diagnosis, they must
be used with caution. In other words, a worker must know the probable expected
variation before he decides whether his organism can be classed with the type culture
of the species.

Another point 1s that reactions which will serve to differentiate one species may be

useless in differentiating another. I took four pure cultures, two each of Staphylococcus*
and Corynebacterium, plated them and selected ten colonies of each. Each of the forty
colonies was then tested as a separate organism. Table 1 shows the reactions of these
forty cultures and the originals on the usual diagnostic media.

It will be seen that Staphylococcus was reasonably constant in its fermentation of
certain sugars—e.g., glucose, maltose, mannitol, salicin, etc.—and inconstant in xylose,
raffinose, etc. Corynebacterium, on the other hand, was inconstant throughout so that
it is doubtful whether sugars can be used to determine species of this genus.

Lehmann and Neumann, while admitting that there appears to be a reason for
separating Pseudomonas and Vibrio, have pointed out that the separation is difficult at
times, as one frequently finds cultures which show only a proportion of curved or
straight rods. In such cases, one must rely on the “scientific tact” of the observer until

*T have adopted Cowan’s suggestion (private communication) that Cohn’s description of
Micrococcus is inadequate and therefore Staphylococcus must be used for these forms.

197

BY E. J. FERGUSON WOOD.

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198 THE CLASSIFICATION OF BACTERIA,

a definite differentiation can be made. A similar problem confronts us when we meet
a spore-forming aerobe with polar flagella. Is it to be classified as a spore-forming
Pseudomonas or an aberrant Bacillus? As Stanier and van Neil (1941) point out, we
can only determine such points by studying the physiological characteristics of the
organism for, since we have in Sporosarcina a spore-forming coccus, it is logical to
accept a spore-forming Pseudomonas. The same applies to fluorescent cocci, spore-
formers, ete. Until recently I regarded Proteus as a distinctive genus on account of its
inability to produce gas from lactose and its possession of a urease; but lately a strain
which gave only a small quantity of gas in glucose and saccharose after ten days’
incubation was isolated in this laboratory; this organism obviously forms a link between
Proteus and the non-gas-producing Bacteria. Rustigan and Stuart (1943) give as
characteristics of Proteus that it gives a small quantity of gas in 48 hours and attacks
urea rapidly. The extreme morphological variation in Corynebacterium intergrading
with the Coccaceae is another case in point. One culture isolated here showed only
coccoid forms and was classified as Staphylococcus until the diffusible red pigment
associated with C. erythrogenes appeared. Slide cultures showed rod forms with snap-
ping division after 8 to 16 hours, but at 48 hours all forms were coccoid. Van Niel
(1946) records similar changes in morphology in Corynebacterium, while Burke,
Schwartz and Klise (1943) record a Staphylococcus aureus strain which regularly
showed rod forms in cultures less than 12 hours old. This discounts Rahn’s theory that
“morphological changes are usually decided changes”. Topping (1937) records motile
Corynebacteria and her findings seem to be accepted. I have also isolated these forms,
motile with polar flagella but otherwise identical with Corynebacterium species.
Pigment production is another unreliable character. Lehmann and Neumann (1930)
point out that the Coccaceae often show single colonies with pigmented and non-
pigmented sectors and I have frequently found such colonies in the Coccaceae, These
authors go on to show the relationship between some of the non-pigmented and
pigmented forms, and bearing this in mind, use colour variations as specific characters
only. Pigment variation is well known in the Serratia group where different shades
of red and non-pigmented strains occur. Bergey also accepts this idea for the Coccaceae,
though he does not point out the relationships, but uses pigmentation to separate
Achromobacter from Flavobacterium while he is not consistent in retaining this character
in the two genera.

In addition, van Niel (1944) has pointed out that the non-sulphur purple bacteria
show characters putting sections of them in the Coccaceae, Pseudomonadaceae, ete. He
places them in a photosynthetic group parallel with the Eubacteria. He also points out
that loss of photosynthetic activity would transfer these bacteria to the heterotrophic
bacteria and a similar loss of photosynthetic activity among the primitive algae would
make these also indistinguishable from heterotrophic bacteria. The marine micro-
flagellates are particularly difficult to separate as some become heterotrophic in absence
of light.

The argument as to whether heterotrophs preceded autotrophs or vice versa is
probably unimportant and it may well be that development in both directions occurred.
Luria (1947) points out that in its present stage, bacterial genetics cannot be expected
to “bring under control the hornet’s nest of bacterial taxonomy but may suggest some
useful precautions in approaching it”. He shows that ‘‘species, genera and even tribes
are often separated on the basis of character differences that may be brought about
by a single mutational step, for example the tribes Eschericheae and Proteae, the genera
Salmonella and Eberthella, the species Staphylococcus aureus and Staphylococcus albus
in the classification of Bergey’s Manual. Genera (for example Phytomonas) are
separated from closely related groups (Pseudomonas) on the basis of plant patho-
genicity, a character that may well arise or disappear by mutation. It is obvious that
such mutable properties can be used only in practical determinative keys without claim
to any taxonomic significance. Even then the greatest caution should be observed, since
variable characters may prove too elusive to “permit recognition of organisms of
practical importance. In many cases, description of a variability pattern might prove

BY E. J. FERGUSON Woop. 199

a better taxonomic criterion than description of any one or more of the variable pheno-
typic traits themselves” (Luria, 1947). I had already reached the same conclusions from
my study of the Corynebacteria and Coccaceae, basing them on purely practical deter-
minative considerations.

With the motile strains of Corynebacterium, all, on prolonged culture, lost their
motility and then became indistinguishable from the rest of the Corynebacterium strains
I was investigating. I was unable to induce motility on any medium. As I intended
to keep these cultures as type cultures and submit them to the National Collection of
Type Cultures at the end of the war, I was concerned at the variation, which is just
another example of the danger of placing too much reliance on type cultures.

This means that we have to rely on differentiation mainly on the original description
of the species, and, with changing methods over the years, correlation becomes increas-
ingly difficult.

B. THE COMPOSITION OF MEDIA.

It is well known that slight variations in the composition of media cause changes
in the observed reactions of bacteria. This becomes important. as soon as we begin to use
physiological characters for differentiation. For example, if British, American and
German peptones give different results it becomes very difficult to assess the reactions
in peptone media, while many organisms do not grow well in the absence of peptone.
During the war we could not, at this laboratory, get Lab-Lemco meat extract and were
forced to use whatever peptone we could buy—we had always used Witte peptone for
carbohydrate media—and we had to change from Japanese agar to Australian agar
made from Gracilaria confervoides, which has a higher setting point and other peculiar
properties. We could get no uniformity in the production of green water-soluble pigment
from Pseudomonas strains, or in the red diffusible pigment of Corynebacterium
erythrogenes. Even the use of Georgia and Poe’s and of Gessard’s media was not wholly
satisfactory in establishing fluorescence or pyocyanin production in Pseudomonas, the
trouble apparently lying in the agar. The production of hydrogen sulphide appears to
depend on the meat or yeast extract used, Australian “Marmite” giving different results
from “Yeastrel”. The nitrate reduction test, normally unsatisfactory, becomes more so
with chemicals from different sources. In Australia skimmed powdered milk is no longer
manufactured and this has given another source of error.

C. STAINING REACTIONS.

Here, too, one finds difficulties. Many Coccaceae and Corynebacteria are Gram
positive no matter which of the recognized methods are used, but others are positive by
some methods, negative by others. Age of culture and composition of media also inter-
fere and must result in anomalies, unless the worker is familiar with the particular
group under study. As an example, Corynebacterium helvolum is described by Bergey
(5th Edition) as Flavobacterium helvolum, and is recorded by him as Gram negative,
whereas I have found it Gram positive, as does Jensen (1934).

When one considers the difficulties enunciated above, one sees the futility of the
system of specific description and at times of generic description given in Bergey’s
manual and finds the necessity of weighing Bergey’s species in the light of one’s
knowledge of variation. I have recently used a punched card system for sorting my
organisms and have applied it also to descriptions of species by Bergey and his followers,
checking back against the full description given. It is surprising to find how many of
these species are indistinguishable from one another and for this reason alone cannot
stand.

PREVIOUS SCHEMES OF CLASSIFICATION.

Bacteriological literature is full of papers on the classification of the bacteria and
this literature has been ably reviewed by a number of authors. “Natural” systems have
been proposed based on morphology (including the Gram stain), physiology and function,
and these characters frequently have been combined in an arbitrary and at times
confused manner with unfortunate results. The morphologists include the earlier

Q

200 THE CLASSIFICATION OF BACTERTA,

bacteriologists; the physiologists really began with Orla-Jensen; while function,
especially pathogenicity, is stressed by Bergey and the Society of American Bacteriolo-
gists in such genera as Phytomonas, Erwinia, Xanthomonas, Corynebacterium, etc.

A very sound discussion of previous systems is given in Kluyver and van Niel’s
paper (1936). These authors show the importance of morphology in defining the primary
groups and the necessity for careful evaluation of physiological characters in subdivision.
They point out that one should not push morphological divisions too far. They do not
agree with Prévot (1940) that physiological characters must be confined to the delimita-
tion of species. They discriminate between ‘‘incidental” and “genuine” immotility, a
distinction which is fundamentally important. For example, the non-motile Gram
negative rods morphologically and physiologically resembling the genus Pseudomonas
should be regarded as ‘incidentally’ non-motile, whereas the Corynebacteria should be
regarded as “genuinely” immotile. However, Topping and I have found motile Coryne-
bacteria—which in this case would be classed as “incidentally motile”. This shows a
relationship between Corynebacterium and Pseudomonas which is strengthened by the
frequency of pleomorphism in both genera and the occasional occurrence of snapping
division in the latter genus, which I have observed in slide cultures.

The system of Migula (1895) given in Engler and Prantl follows Cohn (1872) in
applying a binomial classification. This system was based on morphology and stressed
the flagellation of motile organisms which is nowadays accepted as a highly useful
character. Buchanan (1917) developed a system on which was founded the classification
now used by the Society of American Bacteriologists in Bergey’s Manual. This was a
distinct advance, being put forward as a contribution to an expanding science. This
system was also primarily morphological though physiological characters were also used
extensively. Orla-Jensen (1909) put forward a physiological classification, neglecting
morphological characters except flagellation, and assumed that physiological characters
were phylogenetic, putting Streptococcus, etc., with the Peritrichineae, and Corynemonas,
Mycomonas and Actinomyces among the Cephalotrichineae, while Mycobacterium and
Corynebacterium were removed to another family. Kluyver and van Niel (1936) give
further criticism of Orla-Jensen’s suggestions which is sufficient to show that physio-
logical criteria alone do not provide a satisfactory classification. They point out, how-
ever, that these suggestions do emphasize the importance of physiology, an importance
which most subsequent writers have been ready to concede. We can follow the line of
thought from Migula through Chester (1901), Orla-Jensen and Buchanan to the present
Bergey’s Manual, but unfortunately the later developments appear to most other writers
and to me as unfortunate.

Lehmann and Neumann in Europe produced a textbook of determinative bacteriology,
which, while reasonably sound as far as it goes, is too conservative for our present
knowledge. Despite this, British and European bacteriologists still prefer Lehmann and
Neumann to Bergey. I have found myself forced to abandon Bergey and think it
desirable to set out my views in this paper.

PREVIOUS SCHEMES OF CLASSIFICATION.
A. LEHMANN AND NEUMANN CLASSIFICATION.

Lehmann and Neumann’s classification (Bacteriologische Diagnostik, Sth ed.,
Breed’s translation, 1930) gives a classification of the Schizomytes which appears to be
more reliable than that of Bergey in that they take into full consideration the variability
of bacteria. On this account, they are perhaps rather conservative in the number of
genera which they accept. Some of these, for example the genus Bacterium, become
rather unwieldy in size, at the same time including organisms with fairly wide range
of characteristics. The number of species in each genus is much less than in Bergey
but it is probable that they are more well founded. Jensen has used Lehmann and
Neumann’s classification in his work on the soil organisms of the Actinomycetales
(Jensen, H. L., 1931, 1932, 1934). Lehmann and Neumann’s classification is based
primarily on morphological characters, thus following the classification of the higher
orders of plants and animals. Actually they carry this morphological differentiation

BY E. J. FERGUSON WOOD. 201

right down to genera, and it is at this point that the system begins to show signs of
strain.

In this paper I am primarily concerned with Lehmann and Neumann’s two orders,
Schizomycetales and Actinomycetales. Kluyver and van Niel point out that the biggest
contribution of these two authors was their recognition of the relationship between
Corynebacterium, Mycobacterium and Actinomyces, although for physiological reasons
Kluyver and van Niel find it difficult to include C. diphtheriae with Corynebacterium.
This is due to lack of Knowledge of the variability of the genus in its ability to ferment
carbohydrates, and once this variability is recognized the difficulty disappears. One
wonders why the editors of Bergey’s Manual have been so slow to accept Lehmann and
Neumann’s suggestion.

Apart from Desmobacteriaceae and Spirochaetaceae with which I am not familiar
and which have been dealt with by Kluyver and van Niel, I would, in the main, accept
Lehmann and Neumann’s families, although I would certainly rearrange some of the
genera and subgenera. It is when one reaches a consideration of the genera of this
system that the difficulties are realized. Hven if we accept their subgenera as genera
the classification is still inadequate in some respects. I believe, however, that if this
were done the result would be a better classification than Bergey’s.

The following quotation shows the appreciation by Lehmann and Neumann of the
difficulties caused by variation:

“If this classification is reviewed then it cannot be denied that the families and
the genera are frequently connected by intermediate forms. The following may be
recalled: The border-line between the Coccaceae and Bacteriaceae is made indistinct by
oval and lancet-shaped cocci and by species which sometimes form cocci, then rods (see
the later description of Streptococcus acidi and Bacterium melitense). It is frequently
difficult ‘to “distinguish between Streptococcus (‘Diplococcus’) and Micrococcus and-
Sarcina. Twisted forms occur in the life cycle of many rods; flagella and endospores
occur in quite different species so that it would lead to an entirely artificial grouping if
a classification were based exclusively upon flagella or upon endospores.

“Various only relatively acid-fast species form transitions between Corynebacterium
and Mycobacterium. The border between Mycobacterium and Actinomyces is made very
indefinite because of the fragility and the relative acid-fastness of many Actinomycetes.
Similar transitions have been found in other parts of the plant and animal system.
They cause difficulties in regard to names, but only because the nomenclature of Linné
that is still used is founded on hypotheses which prevailed 100 years before Darwin.”
(Breed’s translation.)

B. BERGEY’S MANUAL OF DETERMINATIVE BACTERIOLOGY.

Bergey’s Manual of Determinative Bacteriology set out to be a comprehensive list
of described species of bacteria and to present these forms in something resembling a
natural system. Based on Migula and Chester, it incorporated some of Orla-Jensen’s
then revolutionary ideas but has since degenerated into a “splitters” paradise of genera
and species. This is no doubt the result of trying to reconcile divergent views on
classification by a number of eminent bacteriologists who have assisted in editing the
various editions. The work has come in for a great deal of criticism in recent years.
Rahn (1937), Kluyver and van Niel (1936) and Stanier and van Niel (1941) have been
particularly searching in their criticisms.

The arbitrary use of characters in the system is most striking, producing as it does
such unfortunate results, as is also “‘the utter disregard for the significance of mutual
relationships between natural groups” (Kluyver and van Niel). Another bad feature is
the uncritical recording of species described elsewhere without taking any note of their
variation or of their distinguishing features. My main criticism of this part of the
Manual is that American bacteriologists as a whole do not realize the variability of
bacteria, and have accepted the Manual as a precedent for creating further species on
flimsy evidence. I shall reserve my detailed criticism of the Manual till I come to
discuss the genera and species. It is obvious that this work will have very limited

202 THE CLASSIFICATION OF BACTERIA,

usefulness until it is completely revised in the light of the recent criticism and until
all genera and species are pruned to a bare minimum. It is usual in classification for
schools of “splitters” to be followed by schools of ‘“lumpers’’, and there are signs that
this is about to take place. The papers of Taylor (1938), Topping (1937), Jensen
(1943), van Niel (1944), Stanier (1942) are examples of this tendency, and I feel
constrained to follow their example by suggesting places where the pruning knife may
well be wielded. Each writer has of course his own ideas of pruning, and these ideas
are governed by his own experience with regard to his special branch. At the moment,
in Great Britain there is a strong move in the direction of “lumping”.

C. KLUYVER AND VAN NIEL’S CLASSIFICATION.

Kluyver and van Niel, in their paper of 1936, suggest a classification based on
morphological and katabolic grounds, but it would appear to the writer that, in certain
cases, they have gone too tar in this respect; for example, they cannot fit Coryne-
bacterium diphtheriae within the saphrophytic Corynebacteria because of its fermenta-
tive capacity. The writer has found that quite a number of Corynebacteria from marine
sources are fermentative and there appears to be a continuous gradation between non-
fermentative and fermentative strains. As a variation in fermentative capacity can be
shown to exist in Corynebacterium, it is to be presumed that other genera are likely to
show this property. So the use of katabolic activity as a major generic criterion seems
to be fraught with some danger. In certain cases, such as the use of photosynthesis,
there would seem to be less difficulty, and there are good grounds for basing some
genera, e.g., Clostridiwm, on anaerobic growth. In the case of Streptococcus, on the
other hand, the writer isolated numerous strains of anaerobic streptococci from teeth.
In all cases, although the organisms were obligate anaerobes on first isolation, they
later grew quite well in aerobic culture. The writer feels that the solution to this
problem is, as suggested by Bruce White, along the lines of a study of a group of strains
building up from the strain to the variety and from the variety to the species, and
thence to the genus, keeping in mind, however, the criteria used in existing classifica-
tions. This would entail a great deal of study by bacteriologists working with any
given material, but should go a long way to confirming or refuting the work of the
determinative school of bacteriologists. The writer does not hold, however, with Bruce
White’s suggestion of using serology as a panacea for* all ills, since although the
Salmonella group fit well into a serological scheme this applies to very few other
groups. One may note the failure of Rustigan and Stuart to obtain serological differen-
tiation among the closely related Proteus group. Most bacteriologists have at one time
or another tried to solve their classification problems by serological methods, but few
of them have been able to get results consistent enough to warrant publication. Thus,
Bruce White’s implied suggestion that non-medical bacteriologists have ignored the
serological method has more apparent than real foundation. As an example of the
' jnapplicability of this method, the genus Rhizobium shows serological relationships
which cut straight across the host relationships. In such a case; the host relationships
must stand and the serological relationships must be discarded in classification. In
genera such as Achromobacter, where no pathogenic organisms are known, it is difficult
to decide just where to start serological investigation. On the other hand, serological
tests on the coliform bacteria, Hrivinia and Serratia, suggest a close relationship
between these genera which, confirmed by other characters, seems to show that they do
not merit generic rank. Kluyver and van Niel have made some very pertinent observa-
tions on the desiderata of a natural classification of bacteria. They state “We are of
the opinion that Prévot rightly emphasized the priority of morphological over physio-
logical characters, yet his restriction that the application of the latter should be confined
to the limitation of species is of quite arbitrary nature. In accepting a taxonomic value
for physiological characters, it cannot be understood why it could not also be applied
for the demarkation of higher systematic units.” Hence it follows in an attempt to
subdivide the organisms belonging to one ‘natural’ group of bacteria into species
one shall have to create as many species as there are organisms which differ in

Co

BY BE. J. FERGUSON WOOD. 20

“sufficiently fundamental” characters, regardless of the possible existence of inter-
mediate types. It depends entirely upon the “scientific tact” of the investigator which
characters will be deemed worthy of the designation “sufficiently fundamental’.

D. RAHN’S SYSTEM.

Rahn’s system of classification divides the EHubacteriales primarily on spore forma-
tion, then on the Gram reaction, relations to oxygen, and lastly flagellation and form.
It does not seem sound to me to make two primary divisions so unequal as that based
on spore formation. Then the complete separation of Lactobacillus from the Actinomy-
cetales, which this system implies, seems unnatural, although it still retains them in the
same group as the Streptococci. The relationship to oxygen is a character of doubtful
value when used for a major division, as there are so many facultative organisms. For
these reasons I do not see my way clear to accept Rahn’s system.

EH. PRINGSHEIM’S SYSTEM.

The system of Pringsheim (1923) is a development of Lehmann and Neumann’s
system. He separates the purple bacteria as Rhodobacteriales with two families—
Rhodobacterinae and Thiorhodinae—and places the much disputed Beggiatoaceae with
the Chlamydobacteriaceae in the Chlamydobacteriales. He keeps the order Actinomy-
cetales in the sense of Lehmann and Neumann put alters the name to Mycobacteriales.

In Hubacteriales he has three families, Coccaceae with three genera—Streptococcus,
Micrococcus (retaining the Staphylococcus group and the Gonococcus group although the
Staphylococcus group corresponds to the wider sense of Micrococcus without Neisseria),
the Sarcina: Bacteriaceae with two genera—Bacterium and Bacillus, the former including
a Gram negative group with three sub-groups—dysentery group, non-liquefying coli
group and the liquefying Proteus group, and a Gram positive group—the long lactic
acid bacteria (obviously out of place here), containing an aerobic and an anaerobic
group, and lastly Spirillaceae with three genera, Pseudomonas, Vibrio and Spirilliaim.

F. STANIER AND VAN NIEL’S CLASSIFICATION.

The most adequate system as far as it goes is that of Stanier and van Niel (1941).
The system proposed by Kluyver and van Niel, in which morphology is used for dividing
bacteria into tribes, and mode of nutrition for genera, is abandoned and Haeckel’s
kingdom Monera is divided into two: Myxophyta which represent the photo-synthetic
chlorophyll-bearing representatives producing oxygen, and their colorless non-
photosynthetic counterparts, and Schizomycetae which are non-photosynthetic or photo-
synthetic without oxygen production. The Schizomycetae are divided into three classes:
Eubacteriae with rigid cell walls, flagellar motility (when motile), reproduction by trans-
verse fission and spores as endospores, cysts or conidia; Myxobacteriae without rigid
cell walls, with creeping motility and spores as microcysts or fruiting bodies; and
Spirochaetae without a rigid cell wall and with motility by means of an axial filament
or modified fibrillar membrane.

The Hubacteriae are divided into three orders:

1. Rhodobacteriales—photosynthetic but not producing oxygen.

2. Hubacteriales—non-photosynthetie unicellular, no conidia.

3. Actinomycetales—non-photosynthetic usually mycelial, with conidia (Jensen’s
emendation accepted by the authors).

The Myxobacteriae have one order, Myxobacteriales, and the Spirochaetae one order,
Spirochaetales.

This scheme has the advantage of having the Rhodobacteriales, Eubacteriales and
Actinomycetales (including Mycobacterium and Corynebacterium) as parallel units,
suggesting their probable parallel development. The authors show the probable deriva-
tion of the Rhodobacteriales and.the Actinomycetales from the Hubacteriales and suggest
that the Beggiatoaceae and Myxobacteriae are also derived from the Eubacteriales
through the Chroococceales.

204 THE CLASSIFICATION OF BACTERIA,

G. VAN NIEL’S SUGGESTIONS.

An interesting discussion on the possibility of an adequate bacterial classification is
that of van Niel (1946). He now considers that a natural classification is impossible at
the present time and suggests that bacteriologists content themselves with working out
a series of keys based on different properties, each organism being diagnosed by cross
reference to the keys. This seems to me ideal rather than practical, requiring a new
approach that will leave the identity of a given organism in doubt until adequate keys
are produced. I do not think we can afford to break with the past in the manner
suggested, although van Niel’s work is too penetrating and founded on too long an
experience in bacteriology to be lightly dismissed. I feel that we should compromise by
studying existing classifications and abandoning any genera that are not adequately
differentiated, while at the same time revising our concept of genus and species.

THE GENUS-SPECIES CONCEPT.

Van Niel shows clearly that Cohn, while creating the idea of a binomial classification
of the bacteria, did not necessarily mean to give bacterial genera and species the Linnean
significance. The Binomial system was merely a convenience, and as such it appeals
to me. The mistake some bacteriologists have made is in tacitly accepting a Linnean
significance and in insisting that bacterial genera and species must fall into orders and
families and thence assuming that we should aim at a phylogenetic classification. More-
over, some have assumed that a determinative classification is a phylogenetic one. Most
of the anomalies we have encountered since have been due to such conceptions. If, as
van Niel suggests, we realize this, I think we can find a way out of the dilemma. Our
genera and other groupings then are convenient, not absolute, and we can draw our
dividing lines at the most convenient point. As for species, these must be still more
flexible and must allow for the wide range of variation which has been shown to be
possible. Later we may find that some of our genera have phylogenetic significance
while others have not. This inequality of status will not be a serious obstacle in deter-
minative work. We thus provide ourselves with a working tool until we improve our
knowledge of bacterial genetics to the point where a strictly phylogenetic classification
becomes possible.

THE BASES OF CLASSIFICATION.

As van Niel points out, there has in the past been a great deal of discussion on the
relative merits of morphological and physiological features for bacterial classification, but
it has mainly centred on their taxonomic rather than their determinative importance.
The question of their pragmatic importance has not been so carefully considered. It is
this that I would stress. Stanier and van Niel use both morphological and physio-
logical characters in their grouping of the bacteria, but they have carefully chosen the
characters used as being the most constant characters available. It is that fact which
commends their classification to me. They have also kept in view the apparent phylo-
genetic relationships. Despite van Niel’s criticism of the use of the terms “order”,
“family”, “senus” and “species”, these are convenient and it jis necessary from a
practical point of view to make at first broad and then ever-narrowing distinctions. If
we regard the groupings as pigeon holes we may find that in dividing our pigeon holes
into smaller ones we have a few things left over. We must remember that in every
filing system we have our “miscellaneous” file. Van Niel would avoid this by a series
of cross indices, but we must always have some sort of label on our files and the genus-
species concept does give us a label which is easy to understand: and we must have a
system for attaching our labels. We have, then, to choose characters which are constant
enough to use as labels. We find a few characters, some morphological and some physio-
logical, which we can use. Ability to photosynthesize under a given set of conditions
is a sound character, and so is a tendency towards mycelium formation or branching.
These probably have phylogenetic value and are an obvious starting point. Broadly
speaking, the Gram stain is allied with resistance to certain bactericides, e.g., the sulpha
drugs, and therefore this stain is a useful physiological character. Shape, presence and
location of flagella, presence or absence and form of spores are also useful and may in

BY E. J. FERGUSON WOOD. : 205

some cases represent phylogenetic trends. Even range of variation, as with the Actino-
mycetales (if we retain this order), is a useful character. I have used these in the
decisions regarding the validity of genera, but have disregarded others, such as pigmenta-
tion, which experience has shown are not so constant. The usual biochemical tests
have not, in the organisms I have investigated, stood up to the test. They seem to vary
too much within a given strain, and cannot be correlated with one another or worked
out on a statistical basis. If used at all, they must, I submit, be confined to specific
distinctions. Colour also belongs to this group.

In addition, these characters are, as Luria points out, alterable by a single mutation
and such mutations have been shown to occur. The change from an obligate autotrophic
or obligate anaerobic existence to a heterotrophic or aerobic existence requires a change
in enzyme systems, and is therefore not alterable by a single mutation.

Once we have put a label on our organisms there is no reason why we should not
cross-index our organisms on a basis of, say, red pigment or cellulose digestion as van
Niel suggests, and I agree with him that there is no reason for keys to follow the
classification. I also agree with him that the more keys the better, provided the
characters used are reasonably stable. Agar digestion, for example, would be inadmis-
sible, as the property is easily lost on subculture, while cellulose digestion, being rela-
tively stable, is admissible. 2

THE SUGGESTED CLASSIFICATION OF THE BACTERIA.

Even with all these well-considered systems to guide us it does not seem possible
to provide a system which will be acceptable to all. The following classification is
set out as the one seeming to suit my own conceptions and to fulfil my own needs. It
develops the Huropean system in the light of more recent work and deliberately rejects
many of the genera set up by the Society of American Bacteriologists as being either
ill-founded (those based on pathogenicity or pigment production) or unnecessary. The
main requirement of any proposed system at the moment is simplicity, reducing the
number of genera and species, with the desideratum that even the genera and species
retained be further reduced, if it can be shown that this can be conveniently done.
It is felt that a few well-marked genera and species are preferable to a number of
vaguely defined ones. It is also strongly urged that new genera and species should
be created only when ample evidence for their creation is available and when it is
definitely of advantage to the science that they should be created. Frequently, perhaps,
we shall have to rely on the “scientific tact” of the bacteriologist in this respect.
Every author should, however, when creating new species or genera, define his creations
as clearly as possible, including in his definitions the characters which differentiate
them from the nearest allied genera or species.

The divisions as far as orders follow Stanier and van Niel (1941), except for the
deletion of the order Actinomycetales.

Kingdom Monerra Haeckel.
Micro-organisms which do not appear to possess true nuclei or plastids and for
which sexual reproduction cannot be readily demonstrated.*

Sub-kingdom MYXxOPHYTA.

Unicellular or multicellular organisms which, if motile, show creeping motility.
The predominant type of metabolism is photosynthetic with oxygen production. The
photosynthetic pigments are chlorophylls a and b, accompanied by phycocyanin and
sometimes phycoerythrin. Non-photosynthetic colorless organisms which are clearly
recognizable as counterparts of photosynthetic genera are also included.

Sub-kingdom SCHIZOMYCETAE.
Unicellular or mycelial organisms, which, if motile, may creep or may move by
means of flagella or an elastic axial filament or fibrillar membrane. Metabolism is

* The possession of true nuclei by bacteria is claimed by some writers, as is a form of
Sexual reproduction. Some of these claims appear well founded but cannot yet be regarded
as indisputable. It is, however, thought necessary to modify Stanier and van Niel’s description.

206 THE CLASSIFICATION OF BACTERIA,

predominantly non-photosynthetic, but, if photosynthetic, is without oxygen production.
The photosynthetic members of this division never contain chlorophylls a or 0
phycocyanin or phycoerythrin.

Class EUBACTERIAE.

Unicellular or mycelial organisms. If unicellular, they may be spherical, rod-
shaped or spiral. Motility when present always by flagella. Multiplication by transverse
fission. Resting stages, if present, may be endospores, cysts or conidia. Two orders,
Rhodobacteriales, Eubacteriales,

Class MYXOBACTERTAE.

Unicellular rod-shaped organisms. Always show creeping motility (never flagella).
Multiplication by transverse fission. Resting stages, if present, may be microcysts,
sometimes contained within larger cysts. The individual microcysts or the larger cysts
may be borne on or in fruiting bodies of various shapes. One order, Myxobacteriales.

Class SPIROCHAETAE.

Unicellular spiral organisms. Always motile by means either of an elastic axial
filament or of a modified fibrillar membrane. Multiplication by transverse fission. No
resting stages known. One order, Spirochaetales.

Order RHODOBACTERIALES. Stanier and van Niel (Pringsheim).
Unicellular organisms. No resting stages known. Photosynthetic, not producing
oxygen.
Order EUBACTERIALES. Stanier and van Niel (Buchanan).
Unicellular organisms. Resting stages, if present, may be either endospores, cysts
or conidia. Non-photosynthetic.

This paper deals with only one order, EHubacteriales.

The Order EUBACTERIALES.

1. Family Coccaceae—Pribram.

Spores absent or rare, cells approximately spherical, usually Gram-positive.

This classification is in accord with Pringsheim and Lehmann and Neumann, except
for Neisseria, which, on account of its usual Gram-negative staining, parasitism and
difficulty of culture except on serum media, seems to warrant generic rank. While
parasitism itself should not in the writer’s opinion be used to characterize genera, it
may in certain cases be used in conjunction with other considerations. That is why
parasitism is accepted here and rejected for Corynebacterium.

The genus Staphylococcus Rosenbach cannot be adequately separated from the genus
Micrococcus Cohn, as it differs from the latter genus in being “usually parasitic’ and
possibly in degree of tolerance to a high pH. As some of the Micrococci are able to be
facultative parasites, Bergey’s distinction has no foundation. The same applies to the
genus Gaffkya. Prévot’s family Neisseriaceae is suppressed, while Veillonella appears
to be Micrococcus or Staphylococcus, the two described species differing markedly in
their carbohydrate reactions.

N.B.—It would be interesting to test non-parasitic members of this family to the
sulpha drugs, acridines, penicillin and other drugs, sensitivity to which is known to
be related to the Gram-staining properties, especially as these drugs attack the Gram-
negative as well as the Gram-positive pathogenic forms. One of these drugs might
well have determinative significance.

The differentiating character used is the number of planes of division, not because
it is definitive in itself, but because it separates Streptococcus and Neisseria, two
usually parasitic genera, from the sometimes parasitic Staphylococcus and the non-
parasitic Sarcina.

A. Genus Streptococcus Billroth. Division in one plane. Chains frequent. No
flagella. Gram-positive. Aerobes or facultative anaerobes. Closely related to Neisseria,
Lactobacillus and to Corynebacterium.

BY E. J. FERGUSON WOOD. 207

B. Genus Neisseria Trevisan. Division in one plane forming diplocoecci. No flagella.
Gram-negative. Aerobes or facultative anaerobes. All parasitic.

Oo. Genus Staphylococcus Rosenbach. Division in two planes. Flagella rare. Gram-
positive or Gram-variable. Aerobic or aerophilic. Related to Sarcina and possibly to
Corynebacterium. Staphylococcus is preferable to Micrococcus, as Cohn’s description
is hardly sufficient.

D. Genus Sarcina Goodsir. Division in three planes on suitable media. Not always
distinguishable from Staphylococcus. Often in packets. Some species motile. Gram-
positive. Usually aerobic or partly anaerobic. Related to Corynebacterium.

#H. Genus Sporsarcina Orla-Jensen. Division in three planes. Motile, peritrichous.
Endospores formed.

2. Family Bacteriaceae—Cohn.

Gram-negative (one genus Gram-positive). Rod-shaped organisms. Flagella, when
present, peritrichous. Usually fermentative, tendency towards gas production. No
endospores.

The families Bacteriaceae and Pseudomonadaceae are differentiated primarily on
flagellation, which in itself would be dubious procedure. However, it normally agrees
with power to ferment carbohydrates, pigment production and other characters which
seem to most bacteriologists to justify the separation of these two families.

The Bacteriaceae are probably the most difficult group to classify adequately.
Bergey gives a number of families which I consider should be reunited. ;

The Parvobacteriaceae of Rahn appears to form a doubtful family based as,it is on
size and growth in body fluids. Most authors are dubious about size as a characteristic,
but the three tribes of organisms appear to have some common characters. Pasteurella
and Malleomyces (Pfeifferella) are differentiated on reaction on milk and should
probably constitute only one genus, Pasteurella. The next tribe has one genus, Brucella,
and the next tribe, Haemophileae, three genera, Haemophilus, Noguchia and Dialister,
of which Rahn, I feel correctly, recognizes only Haemophilus. We have thus three
genera, Pasteurella, Brucella and Haemophilus, which I consider fit well into the
Bacteriaceae and are characterized by their small size and a tendency to bipolar
Staining.

Rahn (1937) follows Orla-Jensen in creating a family, Lactobacteriaceae, to include
the Streptococci, while most morphologists include these with the Coccaceae. If we
follow Orla-Jensen’s classification we are faced with the desirability of including also
Corynebacterium, which seems to be closely related to Lactobacillus but shows affinities
also with Sarcina and possibly Streptococcus and Staphylococcus. It seems best, there-
fore, for the time being to follow the morphologists by including Streptococcus with the
Coccaceae, though Cowan and Gibson (private communications) would include this genus
with Lactobacillus, with which it has a great deal in common. The correct position of
this genus is admittedly difficult. This leaves us with the genera Lactobacillus and
Propionibacterium, both of which are Gram-positive, pleomorphic, non-motile rods with a
tendency towards metachromatic staining. For this reason they do not fit into the
Bacteriaceae.

The family, as I envisage it, includes Rahn’s Enterobacteriaceae and some of
Cohn’s Bacteriaceae. Listerella and Microbacterium are not included in the organisms
of this group. Klebsiella should be suppressed as being insufficiently differentiated from
Hscherischia and Aerobacter, while these two genera show so many intergrading forms
that they can hardly be sustained as separate genera. Erwinia is not admitted, as it
is based only on pathogenicity for plants, while Serratia can only with difficulty be
sustained on pigment production, especially as this is not constant. The last two are
shown by various authors to be similar in reactions to the coli-aerogenes group. I
would include all these under the name Hnterobacter Rahn, emend.

Of Cohn’s Bacteriaceae as given by Bergey, Cellulomonas, as being obviously a
hotch-potch of organisms with one common factor, decomposition of cellulose, must be
Suppressed. It would be as logical to form a genus on the basis of agar or chitin

208 THE CLASSIFICATION OF BACTERTA,

digestion or the decomposition of mucin. Achromobacter and Flavobacterium separated
on pigmentation must be combined—I suggest as the genus Bacterium Migula emend.
Species of the genus Actinobacillus, being non-motile and pleomorphic and occurring in
lesions resembling actinomycosis, may be Gram-negative Proactinomyces or Actinomyces
or may be better classed as a genus of the Pseudomonadacede. Their acid fastness is
not recorded. Bacteroides is regarded by Rahn as a mixed genus, and I would follow
him in suppressing it. Musobacterium appears to be closely related to Corynebacterium
as a Gram-negative anaerobic branch and has actually been assigned to this genus
by Lehmann and Neumann. This genus and Actinobacillus obviously do not belong
here. Alcaligenes, incorporated by Bergey with Rhizobium in the Rhizobiaceae, is a
mixture of Corynebacterium and Bacterium (Achromobacter and Flavobacterium),
although one type culture is stated by Conn (1942) to have polar flagella and is therefore
Pseudomonas.

We are thus left with the following genera:

A. Genus Pasteurella Trevisan. Small Gram-negative rods showing bipolar staining,
microaerophilic, haemophilic. Usually grow on ordinary media.

B. Genus Brucella Meyer & Shaw. Small Gram-negative rods, no marked bipolar
staining, microaerophilic. Grow on ordinary media.

C. Genus Haemophilus Winslow et al. Small Gram-negative rods, often showing
bipolar staining. Microaerophilic to anaerobic. Haemophilic. Do not grow on ordinary
media.

~ Note—It appears rather difficult to retain these three genera and perhaps they
should be united. There is some indication of serological affinity between them.

D. Genus Enterobacter Rahn emend. Gram-negative rods and filaments, motile
with peritrichous flagella or non-motile. Acid and gas produced from glucose and
lactose. Members appear to be antigenically related.

E. Genus Proteus Hauser. Gram-negative pleomorphic rods and filaments, motile
with peritrichous flagella. Acid and gas produced from glucose rapidly but in small
quantities. No gas from lactose. Usually characteristic spreading growth. Decompose
urea rapidly. Serological groups difficult to separate.

F. Genus Salmonella Ligniéres. Gram-negative rods, motile with peritrichous
flagella ov non-motile. Acid and often gas produced from carbohydrates. Gas not
normally produced from lactose or saccharose. An antigenically related group.

G. Genus Shigella Castellani Chalmers. Gram-negative non-motile rods producing
acid but no gas from carbohydrates. Do not liquefy gelatin. Living in intestinal tract
ot warm-blooded animals. Often pathogenic.

H. Genus Kurthia Trevisan. Gram-positive rods. Peritrichous flagella. No acid
from carbohydrates.

I. Genus Bacterium Migula emend. Gram-negative rods, motile with peritrichous
flagella or non-motile. Gas not formed from carbohydrates.

3. Family Pseudomonodaceae—Winslow et al.

Gram-negative rods, straight or curved. Motile with polar flagella or non-motile.
May show snapping division. Poor fermentation of carbohydrates is usual; no gas
formed. No endospores.

The differentiation of non-motile Bacterium from non-motile Pseudomonas is
difficult and may at times be impossible. In such cases yellow to orange cell pigment
suggests Bacterium; greenish diffusible pigment, Pseudomonas; fermentation of glucose
and no other carbohydrates, production of ammonia from urea, asparagin and peptone
suggest Pseudomonas.

This group seems related to the motile Staphylococci, some of which are feebly
Gram-positive and through the Gram-positive motile Corynebacterium forms described
by Topping and myself to the Actinomycetaceae. The non-sulphur purple bacteria are
a photosynthetic offshoot of this group and the autotrophic Nitrosomonas forms are
another branch. Hydrogenomonas and Methanomonas are probably normal Pseudo-

BY E. J. FERGUSON WOOD. 209

monadaceae, as few of these organisms have been tested under the conditions under
which the two last-mentioned genera were isolated. :

Of the genera listed in Bergey’s Manual, the two differentiated on the basis of
cellulose digestion, Cellvibrio and Cellfaicicula, should be suppressed, as suggested by
Stanier (1941). Cellfalcicula is obviously synonymous with Pseudomonas, though,
owing to its metachromatic granules and pleomorphism, it has some affinities with
Corynebacterium (cf. Corynebacterium fimi and the motile polar flagellate Corynebacteria
isolated by Topping and myself). Phytomonas distinguished by pathogenicity for
plants should be suppressed, as the genus Pseudomonas as a whole shows a tendency
towards pathogenicity under certain circumstances. Instances are the records of
occasional pathogenicity for fish of Pseudomonas strains normally inhabiting water.
Protaminobacter appears to be merely a group of Pseudomonas strains with a rather
specialized enzyme structure and has close affinities with the Gram-negative, pleomorphic
organisms which I have isolated from sea-water and which, apart from their Gram
reaction, closely resemble Corynebacterium. Jensen (1931) points out the relationship
between Mycoplana and the Actinomycetaecae. It is possibly best to retain the genus
Mycoplana, dropping the physiological characterization of Gray and Thornton and using
it to denote all Gram-negative highly pleomorphic forms which appear to bridge the
gap between Pseudomonas and the Actinomycetaecae. Both motile and non-motile
forms would be included in this genus. Actinobacillus appears to be related to these
organisms, while Fusobacterium may be the anaerobic representative, although
Sanarelli (1927) considers it to be related to the Spirochaetes rather than to the
Actinomycetaceae.

We are left with the following genera:

A. Genus Vibrio Miiller. Gram-negative non-sporing curved rods with only a
single curve. Polar flagella.

B. Genus Spirillum Ehrenberg. Gram-negative non-sporing spiral forms with polar
flagella. ;

O. Genus Pseudomonas Migula. Gram-negative non-sporing straight rods. Non-
motile or motile with polar flagella. Usually produce acid in glucose broth. May produce
a green fluorescent pigment. j

D. Genus Mycoplana Gray and Thornton emend. Gram-negative highly pleomorphic
rods with granules. Non-motile or motile by polar flagella. Carbohydrates not readily
fermented.

E. Genus Actinobacillus Brumpt. Gram-negative pleomorphic non-motile rods.
Carbohydrates usually fermented.

F. Genus Fusobacterium Knorr. Gram-negative rods, fusiform, non-motile.
Anaerobic. (The place of this genus is considered doubtful. Cowan (verb. com.) places
it with the Mycobacteriaceae. )

4. Family Bacillaceae—Fischer.

Rods producing endospores: usually Gram-positive. Flagella, when present, generally
peritrichous.

Bergey’s Manual gives two genera:
A. Genus Bacillus—aerobic forms.
B. Genus Olostridium—obligate anaerobic forms.

Imsenecki (1945) has shown that these two genera show cytological differences
and for this reason they appear to be soundly based. Bergey’s separation of the genus
Bacillus into species groups is also sound. I have found that certain physiological
characters are more constant for one group, e.g., sugar fermentation in Staphylococci
than for another, e.g., the same reactions in Corynebacterium. This means that there
is justification for using different physiological characters to separate different groups.

The spore-forming genus Bacillus contains types (Aerobacillus group) which in
other respects resemble Hnterobacter in the production of acid and gas from carbo-
hydrates, Pseudomonas (B. serusitidis with polar flagella and only weakly Gram-positive

210 THE CLASSIFICATION OF BACTERIA,

or Gram-variable), Bacterium (B. subtilis with peritrichous flagella thus resembling
Bacterium), so that we may regard spore formation as a possible phylogenetic end
point of development for each group. Some recent work has shown that asporogenous
strains of spore formers do exist. The genus Aerobacillus may also be soundly based.
This idea is strengthened by the existence of a sporing coccus with peritrichous
flagella—Sporosarcina ureae. As well as the Gram-positive forms there exist Gram-
negative species with often a tendency towards pleomorphism—the Bacillus fusiformis
group which suggests a relationship with the Fusobacterium—Actinobacillus group. I
have recently isolated from marine muds spore-forming pleomorphic bacillus strains
which suggest spore-forming types of Corynebacterium, Mycobacterium or Actinomyces.

This leaves two genera—Acetobacter and Azotobacter—which Bergey raises to the
status of monogeneric families. Both these genera seem to have relationships with the
Pseudomonas—Mycoplana—Corynebacterium group. They should, I think, be left as
genera incertae sedis ov placed in a family with the other Gram-negative pleomorphic
organisms.

These Gram-negative pleomorphic forms—Mycenlana, Actinobacillus, Fusobacterium,
Azotobacter and Acetobacter, should probably be separated from the Pseudomonadaceae
as a separate family, the Mycoplanaceae, but I would not stress the point at this stage.

5. Family Actinomycetaceae—Buchanan.

I do not think that future bacteriologists will be able to accept the order Actino-
mycetales as separate from the Eubacteriales owing to the obvious links between the
Coccaceae, Lactobacillus and Corynebacterium on the one hand and Pseudomonas, the
Gram negative pleomorphic bacteria and Corynebacterium on the other. I have included
Lactobacillus and Propionibacterium with the Actinomycetaceae. If this is done,
Streptococcus seems out of place here. H. L. Jensen (1931-1934) has shown the
transition between the non-acid-fast Corynebacteria and Proactinomyces and the acid-
fast Mycobacterium, Proactinomyces and Actinomyces. It is possibly a question whether
the line should be drawn separating the group, as is done in this paper, on the basis of
the Gram stain, regarding the non-sporing Gram negative bacteria as Pseudomonadaceae
and the non-sporing Gram positive bacteria as Actinomycetaceae or whether Lactobacillus
and Propionibacterium should be separated from Corynebacterium and included with
Streptococcus in a separate family Lactobacillaceae. One could justify including
Corynebacterium with the Lactobacilli on account of the polar flagellate members of
this genus found by Topping and myself, and one could also justify the inclusion in the
Actinomycetaceae of the pleomorphic granular Gram-negative forms. Transitional
forms among the bacteria are so frequent that it seems to me that no indisputable line
can be drawn at all.

Cohn and Dimmick (1947) seem in error when they state that Mycobacterium has
been extended to include “all acid-fast forms whether or not branching occurs” or ‘‘many
branching forms whether or not they are acid-fast”. Jensen has created the genus
Proactinomyces to include the branched forms, one group of which are acid-fast, the
other non-acid-fast.

The occurrence of snapping cell division appears to me as to Jensen a valid
character for differentiating Corynebacterium as it can easily be demonstrated by using
the agar slide culture described by Topping. In such cases there is no inference,
although the presence of V. and N. forms, if they predominate, can safely be regarded
as strong evidence. In my study of marine Corynebacteria I have encountered all types
from those showing branching to forms normally coccoid, and have found from agar
culture that they are in the young stages rods with true snapping division. They range
from forms morphologically identical with mitis strains of C. diphtheriae to diphtheroid
forms. The sugar reactions cannot be regarded as diagnostic, as although I agree with
Cohn and Dimmick that they are characteristically poor fermenters, variation in the
direction of strong fermentation does occur. I cannot therefore accept their genus
Arthrobacter.

BY E. J. FERGUSON WOOD. 211

6. Family Actinomycetaceae—Lehmann and Neumann (1927).

Gram positive rods, frequently pleomorphic. Non-motile or rarely motile by means
of polar flagella. No endospores. Conidia may be formed.

A. Genus Leuconostoc. Gram positive, non-motile, coccoid forms, rods may occur.
Ferments carbohydrates producing lactic acid.

B. Genus Lactobacillus Cohn. Gram positive, non-motile rods frequently forming
chains, tendency towards pleomorphism and irregular staining. Aerobic. Ferments
carbohydrates producing lactic acid. Not acid-fast. Normal division.

C. Genus Propionibacterium. Gram positive, non-motile rods. Pleomorphic. Meta-
chromatic granules usual. Microaerophilic. Carbohydrates actively fermented producing
propionic and acetic acids and carbon dioxide. Not acid-fast. Normal division, or at
least no record of snapping division.

D. Genus Corynebacterium Lehmann and Neumann emend Jensen. Gram positive
rods, usually non-motile; when motile, flagella polar. Pleomorphic, with club-shaped
and coccoid forms frequent, some branching. Cystites common. Metachromatic granules
or irregular staining characteristic. Carbohydrate fermentation usually poor. Acid
formation normally weak. Not acid-fast. Snapping cell division, giving V, N and
palisade arrangement.

H. Genus Mycobacterium Lehmann and Neumann emend Jensen. Rods, staining
with difficulty. Acid-fast. Pleomorphism including branched forms frequent. Slipping
cell division giving characteristic bundles.

F. Genus Proactinomyces Jensen. Gram positive rods and mycelial forms.
Frequently acid-fast. Pleomorphic, conidio-spores. No spores on aerial mycelium.

G. Genus Actinomyces Harz. Mycelial forms. Conidial spores in aerial mycelium.

H. Genus Micromonospora QMrskov. Mycelial forms. Spores single, terminal or on
branches of vegetative mycelium.

The group is characterized, as Jensen points out, by a wide range of variation and
the genera are in practice difficult to separate. The variation probably shows their
phylogenetic relationships. Jensen (1931-32) points out that some species show charac-
teristics of both Corynebacterium and Mycobacterium and others of Mycobacterium and
Proactinomyces, and that it is therefore impossible to draw a hard and fast line between
the genera. Other species, however, are not so variable and show the validity of the
genera. The anomalous species may best be regarded as bridging forms.

I have found in Corynebacterium an almost continuous gradation of types from those
fermenting no carbohydrates to those fermenting many. The fermentation is as a rule
slow and a high degree of acidity is not reached (M.R. negative). Experiments have
shown that fermenting strains may appear in non-fermenting cultures, so that sugar
fermentations cannot be relied upon for specific diagnosis in this genus. There seem,
however, to be good grounds for separating Corynebacterium from Lactobacillus and
Propionibacterium on the basis of carbohydrate fermentation and snapping cell division.
I am not certain, however, whether Propionibacterium should be retained as a separate
genus.

DISCUSSION.

This paper is intended to outline a classification of the Bacteria which removes a
good many of the objections raised by other authors of the two classifications most
generally in use, that of Lehmann and Neumann, and Bergey et al., and which also
smooths out’ some of the difficulties I have found as a bacteriologist in classifying
organisms that I have encountéred during twenty years of bacteriological research,
first in plant pathology, then in medical and water bacteriology and latterly in marine
' bacteriology.

It brings out some relationships which seem apparent to me and which I do not
think other writers have recorded.

Finally, it is intended to provoke thought among bacteriologists, especially among
those bacteriologists who have in the past been prone to ignore the problems of classi-
fication. I wish to stress the point that adequate studies of the relationships of bacterial

212 THE CLASSIFICATION OF BACTERTA,

assemblages cannot be made until taxonomy has become more orderly than it is at
present.

In presenting this paper I realize that I am laying myself open to criticism. I am
doing so deliberately in order to provoke discussion, hoping that science will gain by
such criticism. I also hope that some of the suggestions I have made will be accepted,
for example the strong connection between Pseudomonas, the Gram-negative pleomorphic
organisms and the Actinomycetaceae.

SUMMARY.

An attempt is made in this paper to provide a determinative classification of the
Bacteria which will be of greater practical use than those of Lehmann and Neumann and
of Bergey et al. It follows Stanier and van Niel and then defines genera mainly on
morphological grounds. The classification is not phylogenetic, as the author realizes
that we do not know enough about the genetical relationships of bacteria to create a
natural classification. For this reason it must be clearly understood that the divisions
are not used in Linnean sense but are convenient groupings for determination.

The bias of the paper is admittedly towards general rather than medical bacteriology
because the writer feels that the field of general bacteriology has been sadly neglected.

It is impossible for any determinative classification to accommodate satisfactorily all
the organisms that a research worker may encounter, so that there will be times when
intensive study of a particular group of organisms will be necessary in order to allot
a generic name to a given organism.

This paper does not go beyond genera, because although the writer has at one time
or another encountered most of the genera, he has studied only a few of them in
sufficient detail to discuss specific relationships.

References.

Bereny, D. H., et al., 1943.—Bergey’s manual of determinative bacteriology, 5th ed. Williams.
and Wilkins.

————,, 1948.—Ibid., 6th Ed.

BucHANAN, R. E., 1917.—Studies in the nomenclature and classification of the bacteria.
Ue 1ex0KGiin, 725 ibys):

BurRKE, V., SCHWARTZ, H., and Kuiser, K. 8., 1943.—Morphological lifecycle of a Staphylococcus-
like organism and modification of the cycle. J. Bact., 45: 415.

CHESTER, F. D., 1901.—A manual of determinative bacteriology. Macmillan Co., N.Y.

CoHN, F., 1872.—Untersuchungen tiber Bakterien. Beitr. Biol. Pfl., 1: 127.

Conn, H. J., 1942.—Validity of the genus Alkaligenes. J. Bact., 44: 353.

, and DimmickK, J., 1947.—Soil bacteria similar in morphology to Mycobacterium and

Corynebacterium. J. Bact., 54: 291.

IMSENECKI, A., 1945.—On the structure of anaerobic bacteria. J. Bact., 49:1.

JENSEN, H. L., 1931.—Contributions to our knowledge of the Actinomycetales. II. Proc. LINN.
Soc. N.S.W., lvi: 345.

1932.—Contributions to our knowledge of the Actinomycetales. IV. Ibid., lvii: 364.

, 1934.—Studies on saprophytic Mycobacteria and Corynebacteria. Ibid., lix: 19.

Kiuyver, A. J., and vAN Nigeu, C. B., 1936.—Prospect for a natural classification of bacteria.
Centr. f. Bakt., II, 94: 369. :

LEHMANN, K. B., and NEUMANN, R. O., 1930.—Bacteriologische Diagnostik. 8 Aufl. Munchen.
Trans. R. S. Breed.

Luria, S. E., 1947.—Recent advances in bacterial genetics. Bact. Rev., 11:1.

MAXWELL, M. L., 1945.—Fellmongering investigations. IV and V. C.S.I.R. Bull, 184:57, 89.

Micuna, E., 1895.—Schizophyta, in Engler und Prantl. Die Naturlichen Pflantzenfamilien I,

MEV e ls
ORLA-JENSEN, S., 1909.—Die Hauptlinien des naturlichen Bakteriensystems. Centr> fa Bakr,
TUES 97724 8 BAS).

Prevotr, A. R., 1940.—Manual de Classification et de Determination des Bacteries Anaerobies.
Monograph. de l’Inst. Pas. Massen et Cie, Paris.

PRINGSHEIM, HE. G., 1923.—Zur Kritik der Bakterien-systematik. Lotos, 71: 357.

RAHN, O., 1937.—New principles for the classification of bacteria. Centr. f. Bakt., II, 78:1-2;
96: 273. :

2RUSTIGAN, R., and Sruartr, C. A., 1943.—Taxonomic relationships in the genus Proteus. Proc.
Soc. Haper. Biol. Med., 58: 241.

SANARELLI, G., 1927.—Origine commune des spirochetes et des bacilles fusiformes. Compt. rend.
de Soc. Biol., 96: 1136.

BY E. J. FERGUSON WOOD. 213

SEVERENS, J. M., and TANNER, J. W., 1945.—The inheritance of environmentally induced
characters in bacteria. J. Bact., 49: 383.
STANIER, R. Y., 1941.—Studies on marine agar digesting bacteria. J. Bact., 42: 526.
, 1942.—The Cytophaga group. A contribution to the morphology of Myxobacteria.
Bact. Rev., 6: 143.
, and VAN NIEL, C. B., 1941.—The main outlines of bacterial classification. J. Bact.,
42: 437.
Taytor, C. B., 1938.—Further studies of B. globiforme and the incidence of this type of
organism in Canadian soils. Soil Sci., 46: 307.
Toppine, L. E., 1937.—The predominant micro-organisms in soils, I Description and classifica-
tion of the organisms. Centr. f. Bakt., Il, 97: 289.
VAN NIEL, C. B., 1944.—The culture, general physiology, morphology and classification of the
non-sulphur purple and brown bacteria. Bact. Rev., 8:1.
, 1946.—The classification and natural relationships of bacteria. Cold Spring Harb.
Symp. of Quant. Biol., I1: 285.
WHITE, P. BRucE, 1937.—Remarks on bacterial taxonomy. Centr. f. Bakt., II, 95:145.
Woop, EF. J. F., 1940.—Studies on the marketing of fresh fish in eastern Australia. II. The
bacteriology of spoilage of marine fish. C.S.I.R. Pam. 100.
ZOBELL, C. EH., and FELTHAM, C. B., 1934.—Preliminary studies on the distribution and
characteristics of marine bacteria. Bull. Scripps Inst. Oceanog. Tech. Ser., 3: 279-296.
, and UPHAM, H. C., 1944.—A list of marine bacteria including descriptions of sixty
new species. Ibid., 5, (2): 239.

214

AUSTRALIAN POLYPORACEAE IN HERBARIA OF ROYAL BOTANIC GARDENS,

KEW, AND BRITISH MUSEUM OF NATURAL HISTORY.
By G. H. CUNNINGHAM, Plant Diseases Division, Scientific and
Industrial Research Department, Auckland, New Zealand.

Amauroderma pullatus

rudis
TUG OSUS

Coltricia acupunctata

brunneo-leuca
fruticum
haskarlii

laeta
oblectans
placodes
schweinitzeii
weberiana

Coriolus albo-vestidus

ambiguus
aZureus
beckleri
blumei
bowmani
carneo-niger
cingulatus
corrugatus
elongatus
epitephrus
flabelliformis
hirsutus
lactineus
leoninus
meyenii
occidentalis
paleaceus
pargamenus
peradeniae
pinsitus
sanguineus
sinulosus
sprucei
subcaperatus
velutinus
versicolor
xanthopus
zonatus

Daedalea abietina

latissima
saepiaria
subferruginea
trabea

Fomes applanatus

australis
caryophyllaceus
clelandii

(Communicated by N. A. Burges.)
[Read 26th July, 1950.]

Contents.
Page.
2238 Fomes conchatus
239 durus
240 endapalus
216 gilvus
220 lividus
see 226 lloydii
228 mastoporus
230 pectinatus
233 pyrrhocreas
236 riIMOSUS
241 scaber
247 SCTUPOSUS
216 Senex
216 setulosus
218 spadiceus
218 Strigatus
219 zealandicus
219 Fomitopsis annosa
PAPAL concava
221 connata
a 222 hemitephra
224 ochroleuca
225 scutellata
226 tasmanica shi
228 Fuscoporia ferruginosa
229 laevigata
23 punctata
5 232 Ganoderma lucidum
233 Hexagona alveolaris
234 dorcadidea
235 gunnii
236 polygramma
236 similis sae ae
240 tenwis
242 thiaitesii
243 vespacea
243 wightii
246 Inonotus setiporus
246 tabacinus
247 Lenzites aspera
248 beckleri
216 betulina
230 glabrescens
240 palisoti
243 scalaris
245 tenuis
Palit unicolor ‘
218 Merulius binominatus
221 corium
221 pallens

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BY G. H. CUNNINGHAM. 215

Contents—continued.

Page. Page.
Polyporus adustus .. ..... s6) ido BLO - JLFOMPOrUS WOWIS 65° (4c “co oc Bie na) aed lis)
CUUDULUWSh mace ak tte eke kate SIR tod 16 venustus 246
arcularius eae ee wire mete ete edit vernicifluus Rh Seni Betis CHS
atro-maculus an ae we p He eee versatilis : : 246
australiensis Spa yaa af eis Snake conser alse CICHOTIGMSUS oo oo 06 66 HRN RUB
berkeleyi Sey RTA EERO es Stony yepenner dow) VIiNOSUS ME ELEN Sy eA peed aie
borealis So etond dicts eee je ecroea reed Ue) virgatus VAR. HAAN OED) hs Re Dee oA
campylus SS atic cusan Aas Fane wera aa) xerophyllus cb gol 6c Biche Eee ees
CULE LUSHMRIR , POe ee be GUL. are Msg peer Oi zonalis Bidah ase Ca ose een LAS
colensoi 221 Poria atro-vinosa DPE evi ses Seles weer te)
dichrous 223 calcicola 220
dictyopus 223 carneo-lutea 221
dryadeus 224 dictyopora 223
elegans 224 eupora 225
fusco-lineatus 226 hyalina 228
grammocephalus 227 hyposclera 229
hartmannii 228 leucoplaca 230
hypomelanus 229 livida ‘ Aoi
infernalis 229 medulla-panis 232
lacteus 229 mucida 232
liebmannii 230 radula 239
melanopus Es) subvinecta 243
merulius 9392 tarda 244
mylittae 933 undata 245
pelliculosus 236 vaporaria 246
pes-caprae 236 versipora 246
porphyrites 237 victoriae 247
portentosus .. cme woke els 237 vulgaris 247
MROUAYOOUIS 355 oo oo oO 238 wakefieldiae 247
pusillus 238 Trametes cristata 223
rhinocephalus 239 devexa 223
rhipidium 239 drummondii 224
rugiceps 240 fee 225
TUusSsiceps ; 240 ferrea 225
SCOMULOSIUS Hamer ey ny tate 241 floccosa 226
spiculifer 243 feodata 226
sulphureus 243 lilacino-gilva 231
superpositus .. 244 protea 237
tephroleucus 244 proteiforma 238
tephronotus 244 pyurrhocreas 239
thelephoroides 245 serpens 240
tumulosus 245
INTRODUCTION.

Several months were spent in England in 1948 studying collections of Polyporaceae
from Australia, Tasmania and New Guinea in the herbarium of the Royal Botanic
Gardens, Kew; and the Berkeley and Broome types in the herbarium of the British
Museum of Natural History, South Kensington. This paper provides a complete list
of species and their synonyms, together with collections from this region available
for study in these herbaria.

I am grateful to Sir Edward Salisbury, F.R.S., Director of the Royal Botanic
Gardens, and Miss E. M. Wakefield, Deputy Keeper of the herbarium, for allowing me
to examine the Kew collections; to Dr. J. Ramsbottom, Keeper of the herbarium of
the British Museum of Natural History, and Mrs. Balfour Brown, who kindly made
available for study specimens in the herbarium of that institution.

Valid species are set in capitals, synonyms in italics and incidental species, misdeter-
minations, etc., in capitals and lower case. Literature references were checked in the
library at Kew, so that these are accurate both for types and synonyms. I have added
the Australian States to locality references, since these were seldom inserted on the
sheets at Kew.

R

216 AUSTRALIAN POLYPORACEAE,

1. ABIETINA, DAEDALEA Fr., Syst. Myc., Vol. 1, p. 334, 1821.
abietina, Lenzites Fr., Epicrisis, p. 407, 1838.

The following collections from Australia are under the cover of Lenzites abietina
at Kew: “Rockhampton, Q., Mrs. Thozet’”, “Port Denison, Q., Shann”’”, and “Australia,
S.12, S.65”. . '

acervatus, Polyporus Lloyd = Polyporus grammocephalus.

2. ACUPUNCTATA, COLTRICIA (Berk.), nov. comb.

latus, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 325, 1839, type ex Van Diemen’s
Land, Gunn.; non Fries, Syst., p. 384, 1821.

acupunctata, Trametes Berk., Jour. Linn. Soc., Vol. 13, p. 164, 1873, type ex Lord
Howe Island, J. P. Fallagher.

aratus, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 53, 1878, type was based on the
same collection as 7. acupunctata.

Other collections at Kew are: “Tasmania”, “Endeavour River, Q., Persietz’’,
“Daintree River, Q., Pentzcke, No. 143”, all being filed under P. latus; “Brisbane, Q.,
Bailey”, which is under the cover of P. aratus.

acuta, Trametes Cke. = Trametes floccosa.

adami, Polystictus Cke. = ? Polyporus obovatus.

adusta, Lenzites Lloyd = Daedalea subferruginea.

38. ADUSTUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 363, 1821. ,

demissus, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 52, 1845, type ex Swan River,
W. Aus., Drummond, No. 50.

ochraceo-stuppeus, Polystictus Lloyd, Letter 63, D. 11, 1916, type ex Petersham,
N.S.W., E. Cheel.

Additional collections at Kew are: ‘Narranoom, Vic., Stranger’, filed under P.
adustus; “Daintree River, Q., Pentzcke”’, placed by Cooke under Polyporus pallescens Fr.

aequus, Polystictus Lloyd = Coriolus versicolor.

albertinii, Polyporus Lioyd = Coltricia schweinitzi.

albida, Hexagona Berk. = Hexagona vespacea.

4. ALBIDUS, POLYPORUS (Trog.) Fr., Epicrisis, p. 475, 1838.

Under the cover of Polyporus fragilis is a specimen ex “Richmond River, N.S.W.,
Camara” which on the sheet was referred by Cooke to P..destructor. It is of P. albidus
in the sense that this species is now interpreted in northern Europe.

albo-fuscus, Polyporus Lloyd = Polyporus portentosus.

albo-niger, Polyporus Lioyd = Polyporus atro-maculus.

5. ALBO-VESTIDUS, coRIOLUS (Lloyd), nov. comb.

albo-vestidus, Polystictus Lloyd, Myc. Notes, No. 69, p. 1192, 19238, type ex Adelaide,
S. Aus., J. B. Cleland, No. 752.

6. ALVEOLARIS, HEXAGONA (Fr.) Murr., Bull. Torrey Bot. Club, Vol. 31, p. 327, 1904.
alveolaris, Cantharellus Fr., Syst. Myc., Vol. 1, p. 322, 1821.
ohiensis, Favolus Berk. & Mont., in Mont. Syll., p. 171, 1856, type ex Ohio, U.S.A.
hispidulus, Favolus Berk. & Curt., Jour. Linn. Soc., Vol. 10, p. 321, 1865, type ex
Cuba.

Under Favolus hispidulus is filed one specimen ex “‘Australia’’ which matches North
American collections of H. alveolaris; a second, under the same cover, ex “‘N.Z., Colenso’’,
is a fragment of Polyporus colensoi. Under Favolus ohiensis is filed one collection ex
“Hndeavour River, Q., Persieh, No. 202”.

alveolaris, Polyporus (Bosc) Fr. Cooke referred P. collybioides, type ex ‘Richmond

River, N.S.W.’”, to this species. It does not agree with other collections in the cover »

at Kew, but is a poorly preserved specimen of Polyporus arcularius.
7. AMBIGUUS, CORIOLUS (Berk.), nov. comb.
ambigua, Daedalea Berk., Lond. Jour. Bot., Vol. 4, p. 305, 1845, type ex Ohio.
lactea, Trametes Fr., Nov. Symb., p. 96, 1851, type ex Hurope.
aesculi, Daedalea (Fr.) Murr., N. Am. Fl, Vol. 9, p. 126, 1908.
Specimens at Kew are “Brisbane, Q.”’, labelled Daedalea aesculi by Lloyd; and
“Clarence River, N.S.W., Wilcox’. Both are filed under Trametes ambigua Fr., which
is a different species.

amboinensis, Polyporus Fr. Two specimens so named from Australia are of
Ganoderma lucidum.

BY G. H. CUNNINGHAM. 217

angustus, Polyporus Berk. = Polyporus tephronotus.
8. ANNOSA, FOMITOPSIS (Fr.) Karst., Rev. Myc., Vol. 3, p. 18, 1886.
annosus, Polyporus Fr., Syst. Mye., Vol. 1, p. 373, 1821.

A specimen ex “Richmond River, N.S.W.” so referred by Cooke is resupinate and
cannot be identified with certainty. It possesses the same microstructure as F. annosa.

anthracophilus, Polyporus Cke. = Polyporus campylus.

applanata, Daedalea Kl. = Lenzites palisoti.

9. APPLANATUS, FOMES (Pers. ex Wallr.) Gill., Champ., p. 686, 1878.

applanatus, Polyporus (Pers.) Wallr., Fl. Krypt. Germ., Vol. 4, p. 591, 1833.

incrassatus, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 41, 1878, type ex Cape
York Peninsula, Q., Challenger Expedition.

scansilis, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 58, 1878, type ex Juan
Fernandez Islands, Challenger Expedition.

Other collections at Kew are: “Upper Hunter River, N.S.W., Miss Carter” and
“Endeavour River, Q., Persieh”’, both filed under Fomes marginatus. The types of
Polyporus incrassatus and P. scansilis possess the same type of cuticle, context and
spores, so are obviously synonyms. One other collection, filed under P. incrassatus,
is ex “New Guinea, Capt. Armit”’.

aprica, Polyporus Berk., Fl. Tas., Vol. 2, p. 254, 1860. No specimen is at Kew, so
that the species cannot now be identified.

arata, Hexagona Berk. = Inonotus setiporus.

aratus, Polyporus Berk. = Coltricia acupunctata.

archeri, Polyporus Berk. = Polyporus merulius.

10. ARCULARIUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 342, 1821.

similis, Polyporus Berk., Lond. Jour. Bot., Vol. 2, p. 635, 1843, type ex Brazil.

sSquamiger, Favolus Berk., Jour. Linn. Soc, Vol. 13, p. 166, 1873, type ex New
England, N.S.W.

armitii, Polyporus Muell. & Kalch., ex CkKe., Grev., Vol. 10, p. 94, 1882, type ex
Dunrobin, Q. a

collybioides, Polyporus Kalch., ex Cke., Grev., Vol. 10, p. 94, 1882, type ex Richmond.
River, N.S.W.

The following Australian collections are at Kew: “Brisbane, Q., F. M. Bailey, No.
708”, “Botanic Gardens, Sydney, N.S.W., H. Cheel’, and “Illawarra, N.S.W., Kirton,
No. 145”. Filed under the cover of P. lentus are “Guntawang, N.S.W., Hamilton, No. 6”
and “Govt. Domain, near Melbourne, F.v.M.”’; under P. similis “Endeavour River, Q..,,
Persieh” and “Goode Island, Torres Strait, Powell’; and under Favolus squamiger are:
“Australia” and “New England, N.S.W.”.

argentatus, Polyporus Cke. = Coriolus paleaceus.

armitii, Polyporus Muell. & Kalch. = Polyporus arcularius.

ascoboloides, Polyporus Berk., Jour. Linn. Soc., Vol. 13, p. 162, 1873. The type ex
“Australia” is an unidentifiable mycelial fragment growing upon debris from the forest
floor. :

11. ASPERA, LENZITES (KI.) Fr., Hpicrisis, p. 405, 1838.
aspera, Daedalea Kl., Linnaea, Vol. 8, p. 480, 1838.
nivea, Lenzites Cke., Grev., Vol. 15, p. 94, 1887, type ex Russell River, Q., Sayer,
No. 50. } ;

The following collections at Kew agree with the type ex Mauritius—“Brisbane, F. M.
Bailey, No. 165”, “Daintree River, Q., Pentzcke’’, “Dunk Island, Q., W. Cottrell-Dormer,,
No. 31”, “Richmond River, N.S.W., Camara, White’, and “New Guinea, Jola River,
W. H. Armit”; filed under L. nivea is “Bellenden Ker, Q., No. 835”; and placed by Massee
under Daedalea subsulcata is “Sunday Island, Kermadecs, W. R. B. Oliver’.

atro-hispidus, Polyporus Lloyd = Polyporus pelliculosus.

12. ATRO-MACULUS, PoLyPORUS Lloyd, Myc. Notes, No. 67, p. 1162, 1922 (name only);
Stevenson and Cash, Bull. Lloyd Library, No. 35, p. 98, 1936.
albo-niger, Polyporus Lloyd, in herb. Kew.
Part of the type ex “L. Rodway, Tasmania” is at Kew; as is also a second specimen
labelled P. albo-niger Lloyd ex “Hobart, Tasmania, L. Rodway”. In Mycological Notes,
p. 1162, Lloyd recorded the species but did not publish a description. He filed one

218 AUSTRALIAN POLYPORACEAR,

with the specimens in his herbarium and after Lloyd’s death this was published by
Stevenson and Cash.

13. ATRO-VINOSA, PORIA CkKe., Grev., Vol. 14, p. 110, 1886.

atro-vinosus, Polyporus Cke., Grevy., Vol. 10, p. 131, 1882.

The type ex “Clarence River, N.S.W., Wilcox” and a second collection ex “Mt.
Napier, Vic.’”’ are the only specimens at Kew.

atypus, Polyporus Lev. Two specimens ex “Strickland River, New Guinea, Armit’,
filed under this cover by Cooke, were referred by Lloyd to Polyporus brunneolus Berk.
They are of Coriolus paleaceus.

aulacophylla, Daedalea Berk. = Lenzites tenwis.

aureus, Merulius Fr. Cooke (Hdbk., p. 168, 1892) recorded the species from
Queensland. No specimens are at Kew from this region.

14. AUSTRALIENSIS, POLYPORUS Waket., Kew Bull. Mise. Inf., p. 157, 1914.

The following collections from Australia are at Kew: Type ex “Coomera River, Q.,
C. T. White’; “Victoria, F. Campbell’, named P. retiporus by Cooke; “Geographe Bay,
W. Aus.’’, referred to P. stypticus by Cooke; “Toowoomba, Q.’ and “Grampian Mts.,
Vic., Sullivan’’, placed under P. portentosus by Cooke; “Johnstone River, Q.”’, filed by
Cooke under P. nidulans Fr.

15. AUSTRALIS, FOMES (Fr.) Cke., Grev., Vol. 14, p. 18, 1885.

australis, Polyporus Fr., Elench., p. 108, 1828.
brownii, Hifvingia Murr., Western Polypores, p. 39, 1915.

The type, which was described from a specimen collected on a tree trunk on a
Pacific Ocean island, no longer exists. At Kew, however, is a plant labelled P. australis
in Fries’ handwriting, which may be regarded as the neotype. It is imperfect in that
the cuticle is wanting; but in microstructure, size of spores and pores, etc., matches
numerous collections from this region. As the spores are the largest known of the
ganoderma type, the species may be identified readily by this feature alone. A co-type
specimen of Elfvingia brownii at Kew, ex “California, Brown’, is of the same species.

awhitu, Fomes G. H. Cunn. = Fomes endapalus.

16. AZUREUS, coRIOLUS (Fr.) G. H. Cunn., Plant Diseases Division Bull, 75. p. 7, 1948.
azureus, Polystictus Fr., Nov. Symb., p. 93, 1851, type ex Mirador, Mexico, Dr.
Liebman.

The species is common in New Zealand, collections from this region matching part
of the type at Kew. One collection from Australia is at Kew, ex “Zoological Gardens,
Perth, W. Aus., W. N. Cheesman’’, filed under Polystictus versicolor.

badio-lutescens, Polyporus Kalch. = Coriolus occidentalis.

baileyi, Merulius Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 62, 1883.

The type, ex “Brisbane, Q., F. M. Bailey, No. 171’, is an agaric, which Bresadola,
on the type sheet, noted as being “Paxilli sp—Pax. pannoidi Fr. valde affinis’. On the
sheet the specific name is spelled “baileie’, and in Saccardo’s Sylloge Fungorum, Vol. 6,
p. 414, 1888, was published as “baylei”.

' 17. BECKLERI, CoRIOLUS (Berk.), nov. comb.
beckleri, Polyporus Berk., Jour. Linn. Soc., Vol. 13, p. 162, 1873.

Only the type collection is at Kew, ex “Clarence River, N.S.W., Dr. Beckler, No. 16”.
On the type sheet it had also been labelled P. scobinaceus Berk.

berkeleyi, Lenzites Lev. = Lenzites betulina.

18. BECKLERI, LENZITES Berk., Jour. Linn. Soc., Vol. 13, p. 161, 1873.

torrida, Lenzites Kalch., ex Cke., Grev., Vol. 8, p. 154, 1880, type ex Richmond
River, N.S.W.

guilfoylei, Lenzites Berk., ex CkKe., Grev., Vol. 10, p. 64, 1881, type ex Tweed River,
N.S.W., Guilfoyle.

Other Australian collections at Kew are: the type ex “Clarence River, N.S.W., Dr.
Beckler, No. 10” and “Trinity Bay, Q., M. Sayer, No. 36”; under L. faventina Cald.
Cooke filed “Brisbane, Q., No. 559” and “Brisbane, Q., No. 202”; “Goode Island, Torres
Strait” was misnamed Daedalea subsulcata B. & Br. by Cooke. No specimen of Lenzites
torrida is at Kew; but the illustration in Grevillea, Vol. 10, Pl. 144, f. 21, shows it to
have been based on L. beckleri.

BY G. H. CUNNINGHAM. 219

19. BERKELEYI, POLYPORUS Fr., Nov. Symb., p. 56, 1851.
retiporus, Polyporus Cke., Grev., Vol. 12, p. 15, 1883, type ex Daintree River, Q.,
T. Pentzcke.
zelandicus, Polyporus Cke., Grev., Vol. 16, p. 113, 1888, type ex New Zealand,
T. Kirk, No. 309.
One other specimen is at Kew, ex “Illawarra, N.S.W., Camara”, which was by

Cooke referred to P. retiporus.

20. BETULINA, LENZITES Fr., Epicrisis, p. 405, 1838.
betulina, Daedalea Fr., Syst. Myc., Vol. 1, p. 333, 1821.
flaccida, Lenzites Fr., Epicrisis, p. 406, 1838.
berkeleyi, Lenzites Lev., Ann. Sci. Nat., Ser. III, Vol. 5, p. 122, 1846.

Collections at Kew are “Western Point, Vic.”’, “Richmond River, N.S.W., Camara,
No. 89” and “Sugar Loaf Mt., N.S.W., F.v.M.’, the two last being filed under L. flaccida,
which is merely a thin form of the species. In Handbook, p. 101, 1892, Cooke recorded
the species also from Queensland, but there are no specimens at Kew from that State.

betulinus, Polyporus Fr. A specimen so named by Cooke, ex “Toowoomba, Q.’, is
of Polyporus portentosus.

bicolor, Hexagona McAlp. = Hexagona gunnii.

biennis, Polyporus Fr. Cooke (Hdbk., p. 114, 1892) recorded the species from
Queensland. There is no specimen at Kew from this region.

bifasciata, Lenzites Cke. & Mass. = Daedalea subferruginea.

biformis, Polyporus Fr. A specimen so labelled by Cooke, ex ‘Near Melbourne, Vic.,
F. Reader’, is of P. elongatus.

21. BINOMINATUS, MERULIUS Mass., Kew Bull. Mise. Inf., p. 104, 1913. The only
specimen at Kew is the type ex “Brisbane, Q., F. M. Bailey, Nov., 1912”.

bireflexus, Polyporus Berk. & Br. = Coriolus zonatus.

biretum, Polyporus Kalch. = Coltricia fruticum.

bistratosus, Polyporus Berk. & Cke. The species, a Fuscoporia, type ex ‘“Paricatuba”’,-
is not in this region, Australian records being based on such species as Poria vincta
(= P. radula), Poria medulla-panis and Poria calcicola.

blepharistoma, Poria (Berk. & Br.) Cke. The only Australian collection under this
cover at Kew, ex “Toowoomba, Q.’’, is an immature specimen of Poria mucida.
22. BLUMEI, CORIOLUS (Lev.), nov. comb. \

blumei, Polyporus Lev., Ann. Sci. Nat., Ser. III, Vol. 2, p. 185, 1844, type ex Java.

gallopavonis, Polyporus Berk. & Br., Trans. Linn. Soec., Ser. II, Vol. 2, p. 59, 1883,
type ex Brisbane, Q., Bailey.

sub-zonalis, Polyporus Cke., Grev., Vol. 19, p. 44, 1890, type ex Daintree River,
Q., Pentzcke. —

Other collections at Kew are “Christmas Island, Kermadecs, H. W. Rietz’, named
P. sub-zonalis by Cooke; “Endeavour River, Q., Persietz’’, filed under Polyporus versicutus
Berk. & Curt. There is also a specimen ex “Samoa, C. G. Lloyd” which is filed under
P. gallopavonis.

23. BOREALIS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 366, 1821.

Two collections are at Kew from Australia: “Loddon River, Vic., Wallace’ and
“Melbourne, Vic., Reader’.

24. BOWMANI, CORIOLUS (Berk.), nov. comb.

bowmani, Daedalea Berk., Jour. Linn. Soc., Vol. 13, p. 166, 1878.

The only specimen at Kew is the type ex “Herbert’s Creek, Q., E. M. Bowman”.
It is white, almost resupinate, but not daedaloid. Cooke (Hdbk., p. 163, 1892) mis-
spelled the name D. bowmanni. : ; -

breviporus, Polyporus Cke. = Fomes scruposus.

brisbanensis, Polyporus Berk. & Br. A herbarium name applied to specimens ex
“Brisbane, Q., Nos. 136, 138”, filed under Polyporus havannensis Berk. & Curt. They
are of Fomitopsis ochroleuca.

brown, Hlfvingia Murr. = Fomes australis.

broomei, Polyporus Rabh. = Poria undata.

220 AUSTRALIAN POLYPORACEAE,

brumalis, Polyporus Fr. Though recorded by Cooke (Hdbk., p. 112, 1892) from
Queensland, no specimens from this region are at Kew. Obviously the record was based
on specimens of P. arcularius.

brunneo-albus, Polyporus Fr. Cooke (Hdbdk., p. 148, 1892) referred P. brunneo-leucus
as a synonym of this species. The former name cannot be used, since it was erected
by Fries (Nov. Symbd., p. 94, 1851) without a diagnosis or reference to a type, and was
published after P. brunneo-leucus.

25. BRUNNEO-LEUCA, COLTRICIA (Berk.), nov. comb.
brunneo-leucus, Polyporus Berk., Lond. Jour. Bot., Vol. 5, p. 4, 1846.
corrivalis, Polyporus Berk., Jour. Linn. Soc., Vol. 13, p. 162, 1873, type ex Adelaide,
S. Aus., Dr. Schomburgk, No. 47. f
illudens, Daedalea Cke. & Mass., Grev., Vol. 21, p. 37, 1892, type ex Kurrumburra,
Vic., Mrs. Martin, Nos. 1066, 1026, 1037.

Types of the synonyms listed match the type of P. brunneo-leucus ex “Van Diemen’s
Land, R. Gunn, Esq.”. No other specimens are at Kew from this region.

brunneolus, Polyporus Berk. One specimen so referred by Cooke, ex “Daintree
River, Q., T. Pentzcke’’, is too imperfect for identification, being partly destroyed by
insects. No other collection from this region is at Kew.

buibipes, Polyporus Fr. Specimens of the type of P. cladonia (= Coltricia oblectans)
were referred by Cooke to P. bulbipes, but do not resemble others under that cover.

byrsinus, Polyporus Mont. Cooke (Hdbk., p. 150, 1892) recorded the species from
Queensland, but there are no specimens at Kew from this region. His record was
probably based on a specimen of C. occidentalis.

26. CALCICOLA, PORTIA Sacc. & Syd., Syll. Fung., Vol. 14, p. 192, 1899.
calceus, Polyporus Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 55, 1875, non Schw., 1834.
Though several collections from Australia are at Kew, none is filed under this

cover. A specimen ex “New Guinea, Strickland River, Bauerlen” is filed under Fomes
bistratosus; “Richmond River, N.S.W.” is under Poria calcea; “Brisbane, Q., ex herb.
Broome” is under Poria medulla-panis; ‘“‘Daintree River, Q., Pentzcke”’ is in the cover
of Poria fatiscens; “Milsom Island, Hawkesbury River, N.S.W., J. B. Cleland, No. 17”
is under Poria selecta; “Clarence River, N.S.W., Wilcox” and “Toowoomba, Q.,
Hartmann” are filed under Poria victoriae.

callosa, Poria (Fr.) Cke. A collection ex “Endeavour River, Q., Persiech” was filed
under this species by Cooke but does not agree with other specimens in this cover
at Kew.

27. CAMPYLUS, POLYPORUS Berk., Fl. Tas., Vol. 2, p. 252, 1860.

gunnii, Polyporus Berk., Fl. Tas., Vol. 2, p. 253, 1860, type ex Back River Gully,
Tasmania.

anthracophilus, Polyporus Cke., Grev., Vol. 12, p. 16, 1883, type ex Rockhampton,
Q., Mrs. Thozet.

laurencii, Polyporus Berk., in herb. Kew.

rosettus, Polyporus Lloyd, Myc. Notes, No. 43, p. 601, 1916, type ex Australia,
W. A. Scarfe.

wilsonianus, Polyporus Lloyd, Mye. Notes, No. 55, p. 787, 1918, type ex Victoria,
Jas. Wilson.

Types of the synonyms match the type of P. campylus, ex “Tasmania, Archer”.
Other collections at Kew are “North Gippsland, Vic., Dr. Tisdale’, referred to P. gunnii;
“Port Phillip, Vic.’’, ‘““Daylesford, Wallace”, ‘Gippsland, Vic., Murray’, “Melbourne, Vic.,
F. Reader”, “S.W. Australia, T. Muir” and ‘Australia’, filed under P. anthracophilus;
under P. tephronotus is “Pennant Hills, Parramatta River, N.S.W., Challenger Expedi-
tion”; and under P. pelliculosus is “Gippsland, Vic., Webb”.

caperatus, Polyporus Berk. Two specimens so referred by Cooke, ex “Richmond
River, N.S.W., Camara’, are of Coriolus corrugatus. Under the cover of P. lanatus is
a plant ex “Port Denison, Q., Shann’” which Lars Romell on the sheet referred to
P. caperatus. It is a specimen of OC. occidentalis.

carneo-fulvus, Fomes (Berk.) Cke. Two collections placed under this cover by
Cooke, ex “Tropical Queensland, Bailey’ and “Bellenden Ker, Q.” are of Fomes conchatus
or F. gilvus.

BY G. H. CUNNINGHAM. 221

28. CARNEO-LUTEA, PORIA Rodw. & Clel., Papers and Proc. Roy. Soc. Tas. for 1929,
p. 18, 1929.

Represented at Kew by a co-type specimen ex “Bullahdelah, N.S.W., J. B. Cleland,
No. 10”.

29. CARNEO-NIGER, CORIOLUS (Berk.), nov. comb.

carneo-niger, Polyporus Berk. ex CKe., Grev., Vol. 12, p. 15, 1883.

Three specimens are mounted on the type sheet at Kew, ex “Daintree River, Q.,
Pentzcke”. One is typical and may be regarded as the holotype.

carneus, Polyporus Nees. Australian collections at Kew filed under this cover are
“Melbourne, Vic., G. LeFevre’, ‘“Narranoom, Vic., Stranger’, and “Brisbane, Q.”. These
are collections of Trametes lilacino-gilva. A specimen ex “Hndeavour River, Q., Persieh”
filed under this cover = Coriolus paleaceus; and a collection ex “Bot. Gardens, Sydney,
N.S.W., A. Grant” is of Fomes gilvus.

carteri, Trametes Berk., ex Sacc. & Trott., Syll. Fung., Vol. 9, p. 196, 1891. The
type ex “Bombay, H. J. Carter” and a collection ex “National Park, Adelaide, S. Aus.,
W. N. Cheesman, No. 3” are of Trametes protea.

cartilagineus, Polyporus Berk. & Br. = Fomes durus.

30. CARYOPHYLLACEUS, FOMES (Berk. & Curt.) Cke., Grev., Vol. 15, p. 22, 1886.
caryophyllaceus, Polystictus Berk. & Curt., ex CKe., Grev., Vol. 14, p. 78, 1886.
One collection from Australia, ex “Dunk Island, Q., W. Cottrell-Dormer, No. 38”

matches the type, ex Venezuela.

chilensis, Polyporus Fr. Recorded by Cooke, as Fomes chilensis (Hdbk., p. 130,
1892) from Queensland. The only specimen from Australia in this cover at Kew,
ex “Australia, F.v.M.’’, is of Fomes mastoporus.

chioneus, Polyporus Fr. Though not uncommon in New Zealand, there are no
collections at Kew from Australia. A collection filed by Cooke under this cover, ex
“Richmond River, N.S.W., Camara” = Polyporus lacteus; those ex ‘Melbourne, Vic.,
Reader” and “Trinity Bay, Q., Sayer, No. 7’ = Fomitopsis ochroleuca.

chlorina, Poria Mass. = Poria versipora.

chrysoleucus, Polyporus Kalch. = Trametes proteda.

cichoraceus, Polyporus Berk. = Inonotus setiporus.

cinereo-fuscus, Fomes Currey. Though recorded by Cooke (Hdbdk., p. 136, 1892)
from Queensland, there is no specimen at Kew from this region.

31. CINGULATUS, CORIOLUS (Fr.), nov. comb.
cingulatus, Polyporus Fr., Linnaea, Vol. 5, p. 518, 1830.

The following collections are at Kew: “Lower Ardur River, Gulf of Carpentaria,
Q.”, “Dunk Island, Q., W. Cottrell-Dormer, No. 2’, “Daintree River, Q., Pentzcke”’ and
“Endeavour River, Q., Persieh”’. The last two were filed by Cooke under P. cubensis.
cinnabarinus, Polyporus Fr. = Coriolus sanguineus.

cinnamomeo-squamulosus, Polyporus P. Henn. = Polyporus russiceps,

32. CITREUS, POLYPORUS Berk., Jour. Linn. Soc., Vol. 13, p. 162, 1873.

Only the type is at Kew, ex “Clarence River, N.S.W., Dr. Beckler, No. 3”.

cladonia, Polyporus Berk. = Coltricia oblectans.

33. CLELANDII, FOMES Lloyd, Letter 60, p. 11, 1915; Myc. Notes, No. 40, p. 550, 1916.

The species is represented at Kew by part of the type ex “Tupperak, N.S.W., J. B.
Cleland, No. 52”.

clelandii, Lenzites Lloyd = Daedalea trabea.

cognatus, Polyporus Kalch. in herb. Kew. Referred by Cooke (Hdbk., p. 140, 1892)
as a synonym of Polystictus stereinus, which is, in turn, a synonym of Polyporus
liebmannit.

34. COLENSOI, POLYPORUS Berk., Fl. N.Z., Vol. 2, p. 178, 1855.

multiplex, Polyporus Berk., in herb. Kew.

Australian collections at Kew are: “Victoria” and “Australia, Mueller teste Massee”
(a note on the sheet to this effect by Lloyd), the latter being labelled P. multiplex
Berk. It is merely a pallid form with identical microstructure.

bo
bo
bo

AUSTRALIAN POLYPORACEAE,

colliculosa, Trametes Berk. A specimen from Australia under this cover at Kew ex
“Port Denison, Q., Shann” is not the same as other collections included therein, the
pores being much too small. Unfortunately I was unable to identify the plant.

collybioides, Polyporus Kalch. = Polyporus arcularius.

compressus, Polyporus Berk. = Fomitopsis ochroleuca.

35. CONCAVA, FOMITOPSIS (Cke.), nov. comb.

concavus, Fomes Cke., Grev., Vol. 19, p. 44, 1890.

Two collections are at Kew, the type ex “Johnstone River, Q., Berthoud” and
“Brisbane, Q., F. M. Bailey’. On the type sheet Bresadola referred specimens to
P. ferreus Berk. and P. dochmius Berk. The latter is a synonym of Trametes ferrea,
which is distinct from Fomitopsis concawa.

36. CONCHATUS, FOMES (Pers. ex Fr.) CkKe., Grev., Vol. 14, p. 18, 1885.
conchatus, Polyporus (Pers.) Fr., Syst. Myc., Vol. 1, p. 376, 1821.

One collection ex “Endeavour River, Q., Persieh” is filed under this cover at Kew.
A second, probably of this species, ex ‘“Broger’s Creek, N.S.W., Bauerlen’’, was filed by
Cooke under P. murinus.

conchoides, Polyporus (Mont.) Lloyd = Polyporus thelephoroides.

confluens, Polyporus Fr. One collection referred to this species by Cooke, ex “Lord
Howe Island, Camara’ I was unable to identify; a second ex “Port Denison, Q., Shann”
is of Polyporus fusco-lineatus.

37. CONNATA, FOMITOPSIS (Fr.), nov. comb.
connatus, Polyporus Fr., Elench., p. 92, 1828.
One Australian collection at Kew, ex “Moreton Bay, Q., F. M. Bailey’, agrees with

European specimens in the herbarium.

contigua, Fuscoporia (Pers. ex Fr.) G. H. Cunn. Under the cover of Poria contigua
is part of a specimen ex “Swan River, W. Aus., Drummond” labelled on the sheet
Poria ferrugineus; the other portion is filed under Poria ferruginosa. The plant is
Fuscoporia punctata.

contrarius, Fomes (Berk. & Curt.) Cke. The species was recorded from Queensland
by Cooke (Hdbk., p. 132, 1892), but there are no specimens from this region at Kew.

cookei, Hexagona Sacc. = Hexagona vespacea.

cookei, Trametes Sacc. = Trametes floccosa.

38. CORIUM, MERULIUS Fr., Hlench., p. 58, 1828
pelliculosus, Merulius Cke., Grev., Vol. 19, p. 109, 1881, type ex Victoria, Mrs.
Martin, No. 762.
eriophorus, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 60, 1883,
type ex Brisbane, Q., F. M. Bailey, No. 419.
Other Australian collections at Kew are ‘Adelaide, S. Aus., W. N. Cheesman”,
“Tasmania” (seven specimens named by Berkeley) and “Swan River, W. Aus., Nos.
249, 253%.

corrivalis, Polyporus Berk. = Coltricia brunneo-leuca.

39. CORRUGATUS, CORIOLUS (Pers.), nov. comb. ,
corrugatus, Polyporus Pers., in Gaud. Voy. Freye. Bot., p. 172, 1826.
sanguinea, Daedalea K1., Linnaea, Vol. 8, p. 481, 18338.
persoonii, Polystictus Cke., Grev., Vol. 14, p. 85, 1886.

The following collections are at Kew filed under the cover of Polystictus persoonii:
“Goode Island’, “Upper Daintree River, Q., Harris, No. 35”, “Tropical Queensland,
Bailey”, “Trinity Bay, Q., Sayer’, “Brisbane, Q., ex herb. Broome”, ‘““New Guinea, Armit”’,
“Tweed River, N.S.W., Guilfoyle’ and “Gippsland, Victoria”. A ninth collection ex
“Richmond River, N.S.W., Camara” is filed under P. caperatus. In the cover some
sheets are labelled Daedalea sanguinea, others Polyporus corrugatus, both synonyms
of C. corrugatus.

corticola, Poria (Fr.) Cke. Under this cover are two collections. One, ex ‘Australia’,
is a resupinate fragment of Trametes protea; the other, ex “N.Z., Colenso, No. 923”, is
of Polyporus merulius.

cretaceus, Polyporus Lloyd = Polyporus portentosus.

BY G. H. CUNNINGHAM. 223

crinigera, Hexagona Fr. Though recorded by Cooke (Hdbk., p. 164, 1892) from
Queensland, there are no specimens from this region at Kew or the British Museum of
Natural History.

40. CRISTATA, TRAMETES CkKe., Grev., Vol. 10, p. 182, 1882.

Australian collections at Kew are the type ex “Port Denison, Q., Shann, No. 26’,
“Russell River, Q@., W. A. Sayer, No. 47’, “Brisbane, Q., ex herb. Broome, No. 190” and
“Clarence River, N.S.W., Wilcox’. The last two were filed by Cooke under Trametes
curreyt.

eryptarum, Fomes (Fr.) Cke. The two collections from Australia under this cover
are of different species. “Brisbane, Q., No. 978” is a contorted specimen of Fomitopsis
scutellata; “Victoria, No. 1014” is a Poria, probably P. vaporaria.

cubensis, Polyporus Mont. Two collections so named by Cooke, ex “Daintree River,
Q., Pentzcke” and “Endeavour River, Q., Persieh” are of Coriolus cingulatus.

cupreo-nitens, Polyporus Kalch. = Coriolus xanthopus.

cupreo-roseus, Polyporus Berk. One specimen from Australia filed under this cover
= Trametes lilacino-gilwa.

cupuliformis, Polyporus Berk. & Curt. = Polyporus pusillus.

curreyi, Polyporus Berk. = Fomes strigatus.

curreyi, Trametes Cke. Of the Australian collections filed under this cover two,
“Clarence River, N.S.W., Wilcox” and “Brisbane, Q., ex herb. Broome, No. 190’, are
of Trametes cristata; one, ex “Upper Hunter River, Miss Carter”, is of T. drummondii;
and one, ex “Adelaide, W. N. Cheesman”, is either 7. drummondii or T. protea, probably
the latter.

cyclodes, Polyporus Fr. A specimen ex “Australia”, labelled by Berkeley Trametes
occidentalis (to which species it was correctly referred) was filed by Cooke under
P. cyclodes.

daedalioides, Trametes Berk., Ann. Nat. Hist., Vol. 3, p. 325, 1839.

tasmanica, Daedalea Sacc., Syll. Fung., Vol. 6, p. 384, 1886.

The type, ex “Van Diemen’s Land, Messrs. Gunn and Laurence, ex herb. Hooker’,
is a resupinate specimen of some 7’rametes. Saccardo arbitrarily changed the name to
D. tasmanica on Cooke’s statement (Grev., Vol. 15, p. 54, 1886) that the species was a
Daedalea. It is poroid and a Trametes as the genus is now defined.

decipiens, Hexagona Berk. = Trametes drummondait.

demissus, Polyporus Berk. = Polyporus adustus.

deplanata, Daedalea Fr. = Lenzites palisoti.

destructor, Polyporus Fr. A specimen ex “Richmond River, N.S.W., Camara”’, filed
under the cover of P. fragilis, was on the sheet referred by Cooke to P. destructor. It
is the plant which is now known in northern Hurope as P. albidus.

41. DEVEXA, TRAMETES Berk., Jour. Linn. Soc., Vol. 13, p. 165, 1873.

On the sheet of the type ex “Tweed River, N.S.W., Dr. Guilfoyle” Bresadola had
written: “= Polystictus occidentalis K1., forma obesa, juvenilis, hymenio nondum
evoluto”. It is a distinct species, nevertheless. Other Australian collections at Kew
are: “Pennant Hills, Parramatta River, N.S.W., Challenger Expedition’, “Toowoomba,
Q., Hartmann” and “North Gippsland, Vic., H. Tisdall’”. The last is filed under Trametes
heteromalla, and on the sheet Lars Romell had noted ‘= T. devexa Berk.”’.

42. DICHROUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 364, 1821.

Australian specimens at Kew are: “Enoggera, near Brisbane, Q., W. N. Cheesman”
and “Macedon, Vic., Mrs. Martin, No. 435”. Others from this region under the cover
of P. dichrous are of P. thelephoroides.

43. DICTYOPORA, PORTA Cke., Grev., Vol. 14, p. 114, 1886.

dictyoporus, Polyporus Cke., Grev., Vol. 12, p. 17, 18838.

The type ex “Toowoomba, Q., Hartmann” is the only authentic collection under

this cover at Kew.

44. picTyopus, PoLyPporuS Mont., Ann. Sci. Nat., Ser. II, Vol. 3, p. 349, 1835.
Though the species is abundant in New Zealand there are no collections at Kew

224 AUSTRALIAN POLYPORACEAE,

from Australia. A specimen so named by Berkeley, filed under this cover, ex
“Parramatta, Challenger Expedition”, is of P. melanopus.

dielsii, Polyporus P. Henn. = Polyporus portentosus.

diminutus, Polyporus Mass. = Polyporus rhipidium.

discolor, Hexagona Fr., Nov. Symb., p. 102, 1851. Named by Fries from Western
Australia (as Favolus discolor Fr., Pl. Preiss, Vol. 2, p. 136, 1847). No type specimen is
known, and as the description is inadequate the name should be deleted.

dispar, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 101, 1882. The type was from
“Victoria”. No specimens are at Kew or the British Museum of Natural History.

dochmius, Polyporus Berk. & Br. = Trametes ferred.

45. DORCADIDEA, HEXAGONA (Berk. & Br.), nov. comb.
dorcadideus, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 57, 1883.

The type, ex “Enoggera Creek, near Brisbane, Q., No. 374, herb. Broome, No. 155”,
is in the British Museum of Natural History. No specimens are at Kew.

46. DRUMMONDI, TRAMETES, Nov. nom.

decipiens, Hexagona Berk., Lond. Jour. Bot., Vol. 4, p. 57, 1845.

Type is ex “Swan River, W. Aus., Drummond, Nos. 151, 152”. Other collections at
Kew are “Lower Ardur River, N.Q.’, “Moruya, N.S.W., W. N. Cheesman”, ‘Snowy River,
Bauerlen, No. 230’, “Melbourne, LeFevre, Nos. 202, 204, 209”, “Dimboola, Vic., No. 65”,
“Murray River, Vic., C. French” and “Upper Hunter River, N.S.W., Miss Carter”. The
last was filed by Cooke under Trametes curreyi.

Though described as a Hexagona, the species is a Trametes. As the combination
T. decipiens is preoccupied (Bres., Ann. Myc., Vol. 18, p. 40, 1920) I have renamed the
species, which is not the same as that described by Bresadola, after the collector of the
type, James Drummond, who sent to Berkeley many collections from Western Australia.

47. DRYADEUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 374, 1821.
At Kew there is one collection ex “Beenak, Vic., J. H. Willis”.
durissima, Hexagona Berk. & Br. Cooke (Hdbk., p. 164, 1892) recorded the species
from Victoria. As there are no specimens at Kew from this region, it is probable he
based the record on a collection of Hexagona gunnii, which the species resembles
closely.
48. DURUS, FOMES (Jungh.), nov. comb.
durus, Polyporus Jungh., Ann. Sci. Nat., Ser. II, Vol. 16, p. 315, 1841.
cartilagineus, Polyporus Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 49, 1875, type ex
Dolosbagey, Ceylon.
testudo, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 59, 1883, type
ex Brisbane, Q., F. M. Bailey, No. 323, in British Museum of Natural History.
The following collections are at Kew: “Fishery Falls, Q., G. W. Priest’, “Dunk
Island, Q., W. Cottrell-Dormer, No. 29”, the latter being filed under Polyporus durus, —
“Trinity Bay, Q., Sayer’, “Daintree River, Q., Pentzcke”’ and “Johnstone River, Q., -
Berthoud” Cooke filed under P. cartilagineus.

49. ELEGANS, POLYPORUS Fr., Epicrisis, p. 440, 1838.
guilfoylei, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. ’II, Vol. 2, p. 58, 1888,
type ex Tweed River, N.S.W., Dr. Guilfoyle.

Collections at Kew are: “Endeavour River, Q., Persieh’; “Brisbane, Q., No. 168,
herb. Broome”, filed under P. picipes; “Tumbulgum, R. T. Burke’, filed under
P. melanopus; and under P. gwilfoylei, which is merely a spathulate form, are “Aus-
tralia”, “Samoa, C. G. Lloyd” and ‘“Baridi, Papua, C. E. Carr’. On the type sheet of
this synonym Bresadola had noted: ‘Ne pas specifiquement distincte de Polyporus
elegans.”

50. ELONGATUS, CoRIOLUS (Berk.) Pat., Essai tax., p. 94, 1900.
elongatus, Polyporus Berk., Lond. Jour. Bot., Vol. 1, p. 149, 1842.
hodgkinsoniae, Polyporus Kalch., ex Cke., Grev., Vol. 10, p. 96, 1882, type ex
Richmond River, N.S.W., Maria Hodgkinson.
Australian collections at Kew are: “Tropical Queensland, Bailey”, “Trinity Bay,
Q., Sayer”, “Bloomfield River, Miss Bauer”, “Shoalhaven, N.S.W., Bauerlen”, “Sugar
Loat Mt., N.S.W.”, “New England, N.S.W.”, “Port Phillip, Vic.’, ‘““New Guinea, Armit”

BY G. H. CUNNINGHAM. 225

and “Van Diemen’s Land, Gunn”, this last being filed under Polyporus friesii. Under
P. biformis is placed a specimen ex “Near Melbourne, Vic., F. Reader’’.
emerici, Polyporus Berk. = Polyporus fusco-lineatus.
51. ENDAPALUS, FOMES (Berk.) Cke., Grev., Vol. 14, p. 20, 1885.
endapalus, Polyporus Berk., Jour. Linn. Soc., Vol. 138, p. 163, 1873.
fusco-dresdensis, Polyporus Lloyd, Myc. Notes, No. 66, p. 1112, 1922, type ex
Tasmania, L. Rodway.
awhitu, Fomes G. H. Cunn., Plant Diseases Divn. Bull. 79, p. 16, 1948, type ex
Awhitu Peninsula, Auckland, N.Z.
Types of P. fusco-dresdensis and F. awhitu match the type of F. cngapalns ex
“Tweed River, N.S.W., Dr. Guilfoyle’’.
epileucus, Polyporus Fr. A specimen so named by Cooke, ex “Australia, Reader’’,
is of Polyporus tephroleucus.
52. EPITEPHRUS, CORIOLUS (Berk.), nov. comb.
epitephra, Trametes Berk., Jour. Linn. Soec., Vol. 13, p. 165, 1873.
The type is ex ‘“‘Adelaide,.S. Aus., Schomburgk, No. 26”. Other collections at Kew are

“St. Vincent Gulf, N.S.W.”, labelled Trametes dealbata Kalch.; and “Moruya, N.S.W.,
W. N. Cheesman, No. 43’. A specimen under this cover, ex “N.Z., Colenso, No. 525”,
is a somewhat distorted plant of Trametes protea.

eriophorus, Polyporus Berk. & Br. = Merulius corium.

eucalypti, Polyporus Kalch. = Coriolus paleaceus.

eucalyptorum, Polyporus Fr. = Polyporus portentosus.

53. EUPORA, PORIA (Karst.) Cke., Grev., Vol. 14, p. 110, 1886.

euporus, Polyporus Karst., Not. Sallsk. Faun. Fl. Fenn., Vol. 9, p. 360, 1868.

One collection is at Kew, ex “Moruya, N.S.W., W. N. Cheesman, No. 111”.

exotephrus, Fomes (Berk.) Cke. Under this cover is filed a small specimen, ex
“Near Trinity Bay, Q., W. A. Sayer’, which is of Fomitopsis hemitephra.

extensus, Polystictus Berk. = Coriolus occidentalis.

fasciatus, Fomes (Fr.) Oke. A specimen filed under this cover, ex “Barron River,
Q.”, is of Fomitopsis hemitephra.

fatiscens, Poria (Berk. & Rav.) Cke. The plant so named by Cooke, ex “Daintree
River, Q., Pentzcke’’, is of Poria calcicola.

faventina, Lenzites Cald. Two collections filed under this cover by Cooke, ex
“Brisbane, Q., F. M. Bailey, Nos. 202, 559”, are thin specimens of Lenzites beckleri.

favoloides, Hexagona Cke. = Hexagona vespaced.

54. FEEI, TRAMETES (Fr.) Lloyd, Syn. gen. Fomes, p. 226, 1915.

feei, Polyporus Fr., Linnaea, Vol. 5, p. 518, 1830.

The following collections agree with specimens at Kew from Brazil (type locality)
and Cayenne, ex herb. Paris: “Trinity Bay, Q., Sayer’, “Queensland”, “Mosman River,
Barnard” and “Astrolabe, New Guinea, Armit’’.

55. FERREA, TRAMETES (Berk.), nov. comb.

ferreus, Polyporus Berk., Lond. Jour. Bot., Vol. 6, p. 502, 1847.
dochmius, Polyporus Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 50, 1875, type ex

Ceylon.
seriatus, Polyporus Kalch., ex Cke., Grev., Vol. 10, p. 102, 1882, type ex Victoria.

Australian collections at Kew which match the type ex “Hautane Range, Ceylon”
are filed under Fomes ferreus (Berk.) Cke. They are “Port Denison, Q., Shann’,
“Daintree River, Q.’, “Johnstone River, Q., Berthoud’, “Richmond River, N.S.W.,
Camara’, “Tweed River, N.S.W., Camara” and “New Guinea, Armit’”. Under Fomes
oblinitus (Berk.) Cke. is an additional collection ex “Clarence River, N.S.W., Moreton”’.

ferrugineus, Poria (Berk.) Cke. Under the cover of Poria contigua is part of a
specimen ex “Swan River, W. Aus., Drummond” which Berkeley had named as above.
The other part is filed under Poria ferruginosa. The plant is Fuscoporia punctata.

56. FERRUGINOSA, FUSCOPORIA (Schrad. ex Fr.) Murr., N. Am. Fl., Vol. 9, p. 5, 1907.

ferruginosus, Polyporus (Schrad.) Fr., Syst. Myc., Vol. 1, p. 378, 1821.
obliquus, Polyporus Fr., Syst. Mye., Vol. 1, p. 378, 1821.
One specimen, ex “Pennant Hills, Parramatta River, N.S.W., Challenger Expedition”,

is filed under Fomes obliquus. Other Australian collections under the cover of F. obliquus

226 AUSTRALIAN POLYPORACEAE,

are of several species. “Clarence River, N.S.W., Wilcox” = Fuscoporia laevigata; “Swan
River, W. Aus., Drummond” = Fuscoporia punctata. One ex “Melbourne, F. Reader,
No. 24” is now merely a fragment of brown mycelium.

57. FLABELLIFORMIS, CORIOLUS (KIl.), nov. comb.
flabelliformis, Polyporus W1l., Linnaea, Vol. 8, p. 483, 1833.
murinus, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 72, 1875.
glirinus, Polystictus Walch. ex Cke., Grev., Vol. 14, p. 83, 1886, type ex South

Africa, McOwan.
rufo-rigidus, Polystictus Lloyd, Myc. Notes, No. 73, p. 1330, 1924, type ex Melbourne,
Cc. C. Brittlebank.

Collections at Kew are: ‘“‘Port Denison, Q., Shann’’, “Johnstone River, Q., Berthoud”,
“Dunk , Island, Q., W. Cottrell-Dormer”, “Brisbane, Q., Bailey’, “Tweed River, N.S.W.,
Guilfoyle’, “Clarence River, N.S.W., Camara, Thorneton’’, “Clarence River, N.S.W., Dr.
Beckler” (the last filed under Polystictus versicolor), “New Guinea, Armit’, “Strickland
River, New Guinea” and “S.H. New Guinea, Capt. Armit’” (the last is under P. pictilis),
“Suva” (under Polystictus comptus), “South Australia, J. B. Cleland’ (one of three
specimens filed under Polystictus versicolor).

flaccida, Lenzites Fr. = Lenzites betulina.

58. FLOCCOSA, TRAMETES (Jungh.), nov. comb.

floccosus, Polyporus Jungh., Ann. Sci. Nat., Ser. II, Vol. 16, p. 3138, 1841.

acuta, Trametes Cke., Grev., Vol. 10, p. 132, 1882, type ex Richmond River, N.S.W.,
Camara, No. 733.

cookei, Trametes Sacc., Syll. Fung., Vol. 6, p. 342, 1888.

One collection is under this cover at Kew, ex “Moruya, N.S.W., W. N. Cheesman”’.
floridanus, Polyporus Berk. Cooke (Hdbk., p. 146, 1892) recorded the species from
Queensland, but there are no specimens from this region at Kew.

59. FOEDATA, TRAMETES (Berk.), nov. comb.
foedatus, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 41, 1878.

The type ex “Cape York, Q., Challenger Expedition” consists of two plants in
good condition.

fomentarius, Fomes (Fr.) Kickx. The species is not present in this region. Collec-
tions filed under this cover at Kew are of several species. ‘‘Port Jackson, N.S.W.,
Hewland” and ‘‘Twofold Bay, N.S.W., White’ = Fomes setulosus; ‘““King George Sound,
W. Aus., Harris” = Fomes scruposus; “N.Z., Ohaiwai, Dr. Berggren” = Fomes mastoporus.

fragilis, Polyporus Fr. One specimen filed under this cover, ex “Richmond River,
N.S.W., Camara” = Polyporus albidus; a second, ex “Gippsland, Vic., Webb’, may be
the same but is too damaged for accurate identification.

friesii, Polyporus Kl. Two plants so named by Berkeley, ex “Van Diemen’s Land” =
Coriolus elongatus.

frondosus, Polyporus Fr. Cooke (Hdbk., p. 118, 1892) recorded the species from
Tasmania, but there are no specimens at Kew from this region.

60. FRUTICUM, COoLTRICIA (Berk. & Curt.), nov. comb.
fruticum, Polyporus Berk. & Curt., Jour. Linn. Soc., Vol. L0, p. 310, 1869.
biretum, Polyporus Kalch., Hedw., Vol. 15, p. 114, 1876, type ex Clarence River,
N.S. W.

Two Australian collections match the type ex Cuba. One, “Mt. Williams, Bellingen
River, N.S.W.”, was filed under Fomes inflexibilis; the second, ex “Endeavour River, Q.,
Persieh”’, was placed by Cooke under Polyporus pubescens Fr.

fulvus, Fomes (Fr.) Cke. Australian collections placed under this cover at Kew
are of two species. “King George Sound, W. Aus., Harris” and “Port Denison, Q.,
Shann” = Fomes setulosus; “Toowoomba, Q., Hartmann” = Fomitopsis ochroleuca.

funalis, Polyporus Fr. = Coriolus leoninus.

fusco-dresdensis, Polyporus Lloyd = Fomes endapalus.

61. FUSCO-LINEATUS, POLYPORUS Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 1,
p. 401, 1879.

platotis, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 1, p. 401, 1879, type
ex Brisbane, Q., C. E. Broome.

BY G. H. CUNNINGHAM. 227

emerici, Polyporus Berk. ex Cke., Grev., Vol. 10, p. 96, 1882, type ex Brisbane, Q.,
KF. M. Bailey, No. 204.
macroter, Polyporus Berk. & Br., in herb. Kew.
magnoporus, Polyporus Lloyd, Myc. Notes, No. 66, p. 1111, 1922, type ex Australia,
Jas. Wilson, No. 6.
The type, ex “Brisbane, F. M. Bailey, Nos. 80, 204’, consisting of two specimens, is
in the herbarium of the British Museum of Natural History. One specimen is a
duplicate of the type of P. emerici at Kew. Other collections at Kew are “Hndeavour
River, Q., Persieh’’, filed under Polyporus nilgheriensis, and “Port Denison, Q., Shann”,
placed by Cooke under P. confluens.

fusco-maculatus, Polyporus Bres. & Pat. = Polyporus udus.

galactinus, Polyporus Berk. A specimen so referred by Cooke, ex ‘Endeavour
River, Q., Pentzcke” = Polyporus tephroleucus.

gallopavonis, Polyporus Berk. & Br. = Coriolus blumei.

gausapatus, Polyporus Kalch. = Trametes protea.

gilvo-rigidus, Polyporus Lioyd = Inonotus tabacinus.

62. GILVUS, FomMES (Fr.) Lloyd, Letter 42, p. 6, 1912.
gilvus, Polyporus Fr., Elench., p. 104, 1828.
rubiginosus, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 324, 1839, type ex Mauritius.
laurencii, Polyporus Berk., Fl. Tas., Vol. 2, p. 254, 1860, type ex Van Diemen’s

Land, Lawrence.
scabrosus, Polyporus Berk., in herb. Kew.

Australian collections at Kew are: “Pennant Hills, Parramatta River, N.S.W.,
Challenger Expedition” (four specimens, two being of F. gilvws, one F. scruposus
and one the form known as F. hookeri); “Clarence River, N.S.W., Wilcox’, filed under
P. laurencii; “Brisbane, Q.”’, under Polyporus plebius; and “New Guinea’, named
P. holosclerus.

glabra, Lenzites Lloyd = Lenzites glabrescens.

glabratus, Polyporus Kalch. The type collection was from “Clarence River, N.S.W.”.
No specimens are at Kew from this region.

63. GLABRESCENS, LENZITES (Berk.), nov. comb.
glabrescens, Daedalea Berk., Jour. Linn. Soc., Vol. 16, p. 39, 1878.

glabra, Lenzites Lloyd, Myc. Notes, No. 56, p. 811, 1918, type ex Russell Islands,
Solomons, E. Cheel.

Only the type collection is at Kew, ex “Parramatta River, N.S.W., Challenger
Expedition”. On the sheet Bresadola had written: “Tout a fait identique au Lenzites
platyphylla Lev.” Specimens of the latter are not at Kew, so I have been unable to
compare the two species.

glabro-tabacinus, Polystictus Lloyd = Inonotus tabacinus.

glirinus, Polystictus Kalch. = Coriolus flabelliformis

gourliaei, Polyporus Berk. = Trametes protea.

64. GRAMMOCEPHALUS, POLYPORUS Berk., Lond. Jour. Bot., Vol. 1, p. 148, 1842.

muelleri, Polyporus WKalch. ex Cke., Grev., Vol. 10, p. 97, 1882, type ex Richmond
River, N.S.W.
acervatus, Polyporus Lloyd, Myc. Notes, No. 64, p. 1006, 1920, type ex Singapore.

The following Australian collections match the type ex Philippine Islands: “Daintree
River, Q., Pentzcke”’, “Endeavour River, Q., Persieh’, “Port Denison, Q., Shann’’,
“Brisbane, Q., Bailey, No. 808”, “Cape Direction, Q., D. Thomson”, “Richmond River,
N.S.W., Camara” and “Lake Barine, H. Flecker” are under the cover of P. grammo-
cephalus. “Brisbane, Q., C. H. Btoome” is labelled P. macroter, a herbarium name for
P. fusco-lineatus; and under Favolus spathulatus is filed a branched specimen ex “Dunk
Island, Q., W. Cottrell-Dormer, No. 41”.

gryphaeformis, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 54, 1845. The type was
from “Swan River, W. Aus., Drummond, No. 149”. No cover is at Kew or British
Museum of Natural History.
2 guilfoylei, Lenzites Berk. = Lenzites beckleri.

guilfoylei, Polyporus Berk. & Br. = Polyporus elegans.

228 AUSTRALIAN POLYPORACEAE,

65. GUNNII, HEXAGONA Fr., Ann. Nat. Hist., Vol. 7, p. 452, 1841.

vesparius, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 323, 1839.

bicolor, Hexagona McAlp., Proce. Linn. Soc. N.S.W., p. 123, 1904.

olivacea, Hexagona Lloyd, Letter 53, p. 11, 1914, type ex Victoria, Jas. Wilson.

praetervisa, Trametes Bres., Ann. Myc., Vol. 18, p. 39, 1920, type ex Seven Hills,
Australia, Torrend.

The name was changed by Fries, who held that P. vesparius was too close to
P. vespaceus Pers. Four collections from Australia are at Kew: the type ex “Van
Diemen’s Land, Gunn’, “Van Diemen’s Land, Lawrence”, “Grampian Mts., Vic.,
Sullivan” and “Melbourne, Vic., LeFevre, No. 201’.

gunnii, Polyporus Berk. = Polyporus campylus.

66. HARTMANNII, POLYPORUS CkKe., Grev., Vol. I}, TO), ales, alfexes3,

The type, which is excellently preserved, was from “Toowoomba, Q., Hartmann,
No. 10”. Other collections from the region are “Gladesville, Parramatta River, N.S.W.,
Miss Margaret Flocton” and “Brisbane, Q., Bailey”.

67. HASKARLII, COLTRICIA (Lev.), nov. comb.
haskarlii, Polyporus Lev., Ann. Sci. Nat., Ser. III, Vol. 2, p. 190, 1844.

The following collections are at Kew: “Upper Daintree River, Q., Harris’, ““Bellenden
Ker, Q., Bailey”, ‘Toowoomba, Q., Hartmann”, “Clarence River, N.S.W., Camara, No. 70”
and “Strickland River, New Guinea, Bauerlen”’.

hemileucus, Fomes (Berk. & Curt.) Cke. Cooke (Hdbk., p. 132, 1892) recorded the
species from Queensland. As there are no specimens from this region at Kew it is
probable that the record was based on a specimen of Fomitopsis hemitephra.

68. HEMITEPHRA, FOMITOPSIS (Berk.) G. H. Cunn., Plant Diseases Division Bull. 76,
p. 2, 1948.
hemitephrus, Polyporus Berk., Fl. N.Z., Vol. 2, p. 179, 1855.

hypopolius, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 99, 1882, type ex Rock-
hampton, @., Mrs. Thozet.

Australian collections which match the type ex “N.Z., Colenso” are: ‘Brisbane, Q.,
W. N. Cheesman, No. 47”, “Trinity Bay, Q., W. A. Sayer” (the latter is filed under
Polyporus exotephrus); “Barron River, Q.’’, placed by Cooke under Fomes fasciatus;
“Moruya, Blue Mountains, N.S.W., W. N. Cheesman’”’; and “Sydney, N.S.W., W. Froggatt’,
misnamed Fomes martius by Lloyd.

heteromalla, Trametes Cke. = Coriolus occidentdlis.

69. HIRSUTUS, CORIOLUS (Fr.) Quel., Fl. Myc. Fr., p. 389, 1888.
hirsutus, Polyporus Fr., Syst. Myec., Vol. 1, p. 367, 1821.
pisiformis, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 98, 1882, type ex Richmond
River, N.S.W., Camara.

Australian collections at Kew are: “Endeavour River, Q., Persietz’, ‘Rockhampton,
Q., Mrs. Thozet”, “Toowoomba, Q., Hartmann”, “Trinity Bay, Q., Karsten’, “Sunnybrook,
Q., C. G. Greenham”’’, “Toowoomba, Q., Hartmann” (this last filed under P. glirinus),
_“Tilawarra, N.S.W., Karsten”, “Nat. Park, Sydney, N.S.W., W. N. Cheesman”, “Clarence
River, N.S.W., Beckler’, “North Gippsland, Vic., H. Tisdale’; and “Tasmania, Dr.
Milligan”’.

hispidulus, Favolus Berk. & Curt. = Hexagona alveolaris.

hispidus, Polyporus Fr. Cooke (Hdbk., p. 123, 1892) recorded the species from
Queensland, but there are no specimens from the region at Kew.

hodgkinsoniae, Polyporus Kalch. = Coriolus elongatus.

hololeuca, Trametes Kalch. = Polyporus portentosus.

holosclerus, Fomes (Berk.) Cke. One specimen filed under this cover, ex “New
Guinea, Capt. Armit” = Fomes gilvus; a second, ex “Endeavour River, Q., Persietz” =
F. scruposus.

70. WYALINA, PORIA (Berk.) Cke., Grev., Vol. 14, p. 109, 1886.
hyalinus, Polyporus Berk., Fl. Tas., Vol. 2, p. 255, 1860.

The type was from “Tasmania, Archer, growing on naked wood of an eucalypt’. -

A second collection at Kew is from “Orange, N.S.W., J. B. Cleland, No. 18”.

BY G. H. CUNNINGHAM. 229

71. HYPOMELANUS, POLYPORUS Berk. ex Cke., Grev., Vol. 15, p. 51, 1886.

Under the cover of Polyporus melanopus are two Australian collections which match
the type ex ‘“N.Z., Grey River”. They are ex “Toowoomba, Q., Hartmann” and
“Melbourne, Vic., Dr. Berggren’’.

hypopolius, Polyporus Kalch. = Fomitopsis hemitephra.

72. HYPOSCLERA, PORIA (Berk.) Cke., Grev., Vol. 14, p. 110, 1886.
hyposclerus, Polyporus Berk. ex Cke., Grev., Vol. 10, p. 103, 1882.

The following Australian collections at Kew match the type from Tahiti: ‘Brisbane,
Q., C. E. Broome’, “Trinity Bay, Q., Sayer, No. 9’, “Kin Kin, Q., No. 3850’, “Katoomba,
Blue Mts., N.S.W., W. N. Cheesman” and “Clarence River, N.S.W., Wilcox”. One
collection, filed under the cover by Cooke, ex ‘‘Daintree River, Q., Pentzcke’” = Poria
undata; a second ex “Dunk Island, Q., W. Cottrell-Dormer, No. 14” = Poria radula.

hypothejus, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 102, 1882. The type was
from Richmond River, N.S.W. No specimens are at Kew from this region.

hystriculus, Polyporus Cke. = Polyporus pelliculosus.

igniarius, Fomes (Fr.) Kickx. The species has not been found in this region.
Australian collections at Kew, filed under this cover, are of the following species:
“Daintree River, Q., T. Pentzcke” = Fomes senex; “Port Denison, Q., Shann” = Fomes
setulosus; “N.Z., Ohaiwai, Dr. Berggren” = F. australis.

illotus, Polyporus Kalch. = Trametes protea.

illudens, Daedalea Cke. & Mass. = Coltricia brunneo-leuca.

imbricatus, Polystictus Lloyd, Myc. Notes, No. 55, p. 791, 1918. The co-type specimen
at Kew, ex “Sydney, N.S.W., J. H. Maiden” is too fragmentary to name. Lloyd’s figure
(f. 1191) and description suggest that the species may have been based on P. berkeleyi.

incompta, Daedalea Berk., Trans. Linn. Soc., Ser. II, Vol. 2, p. 61, 1883, type ex
Port Douglas, Q., J. HE. T. Woods.

No specimens are at Kew or the British Museum of Natural History.

inconcinna, Daedalea Berk. = Hexagona vespacea.

incrassatus, Polyporus Berk. = Fomes applanatus.

73. INFERNALIS, POLYPORUS Berk., Lond. Jour. Bot., Vol. 2, p. 637, 1843.

Specimens which match the type, ex “Arrial das Merces, South America” are:
“Gippsland, Vic., Webb’, ‘“Moonan Brook, Vic., Miss Carter’, “New Caledonia, Dr. F.
Sarasin” (the last filed under Polyporus blanchetianus Mont.) and “New Guinea, Fly
River, Bauerlen”, which is under the cover of P. russiceps.

inflexibilis, Fomes (Berk.) Cke. The Australian record is based on two collections
ex “Mt. Williams, Bellingen River, N.S.W.”. They are of two species, Coltricia fruticum
and Fomes scruposus.

intermedia, Daedalea Berk. = Hexagona vespacea.

intonsus, Polyporus Berk., Fl. Tas., Vol. 2, p. 252, 1860, type ex Tasmania, Archer.
No cover is at Kew, and the description is too incomplete to allow of identification.

intybaceus, Polyporus Berk. = Inonotus setiporus.

laceratus, Polyporus Berk. = Coriolus pargamenus.

Jactea, Trametes Fr. = Coriolus ambiguus.

74. LACTEUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 359, 1821. One collection, ex
“Richmond River, N.S.W., Camara” is at Kew, filed by Cooke under Polyporus chioneus.

75. LACTINEUS, CORIOLUS (Berk.), nov. comb.
lactineus, Polyporus Berk., Ann. Nat. Hist., Vol. 10, p. 3738, 1842.
levis, Trametes Berk., Lond. Jour. Bot., Vol. 6, p. 507, 1847, type ex Ceylon.
Under this cover at Kew are the following collections: “Dunk Island, Q., W. Cottrell-
Dormer, No. 5” (intermediate between this species and C. sprucei), “Katoomba, Blue
Mts., N.S.W., W. N. Cheesman”, “Lilyvale, N.S.W., A. A. Hamilton’, “Mt. Dandenong,
Vic., comm. H. McLennan” and “Strickland River, New Guinea, Bauerlen’. A specimen
ex “Australia” was by Cooke placed under Trametes hololeuca. Under Trametes levis
are filed: ‘Port Denison, Q., Shann’”, “Toowoomba, Q., Hartmann’, “Richmond River,
N.S.W., Camara” and “Parramatta River, N.S.W., L. Weber”.

AUSTRALIAN POLYPORACEAR,

bo
oo
oO

76. LAETA, COLTRICIA (CkKe.) G. H. Cunn., Plant Diseases Division Bull. 77, p. 8, 1948.
laetus, Polyporus Cke., Grev., Vol. 12, p. 16, 1883.
lateritius, Polyporus Lioyd, Letter 67, p. 12, 1918.
Collections at Kew from Australia agree with the type ex “Upper Owens River,
Vic., Mrs. McCann”. They are “Guntawang, N.S.W., Hamilton’, ‘Victoria, Dr. Winter”
and “Gippsland, Vic., Mrs. Murray”’.

77. LAEVIGATA, FUSCOPORIA (Fr.) G. H. Cunn., Plant Diseases Division Bull. 73, p. 9,
1948.
laevigatus, Polyporus Fr., Hym. Eur., p. 571, 1874.
Under the cover of Poria ferruginosa is a collection ex “Clarence River, N.S.W.,
Wilcox’; a second, ex “Australia”, is filed under Poria rufitincta (Berk. & Curt.) Cke.
lanatus, Polyporus Fr. A specimen ex “‘Port Denison, Q., Shann’’ filed under this
cover by Cooke is of Coriolus occidentalis.

78. LATISSIMA, DAEDALEA Fr., Syst. Myc., Vol. 1, p. 340, 1821.

Under Daedalea microsinulosa Kl. (= D. sinulosa Fr.) are filed two Australian
collections, ex ““Endeavour River, Q., Persietz’ and “Port Denison, Q., Shann’’. A speci-
men ex ‘Daintree River, Q., Pentzcke’’, placed under this cover by Cooke, is of Poria
medulla-panis.

lateritius, Polyporus Lloyd = Coltricia laeta.

latus, Polyporus Berk. = Coltricia acupunctata.

laurencti, Polyporus Berk. = Fomes gilvus, and also a herbarium name for P.
campylus.

lenis, Polyporus Lev. Under the cover of Polystictus trizonatus is a specimen ex
“Upper Yarra, Vic., Lucas’’ to which Bresadola had attached a note ‘= Polyporus lenis
Lev. = form of P. occidentalis Kl1., old, naked”. The plant is a slightly discoloured
specimen of Coriolus zonatus.

lentus, Polyporus Berk. Two collections from Australia, placed by Cooke under
this cover, are of Polyporus arcularius. They were from the following localities:
“Guntawang, N.S.W., Hamilton, No. 6” and “Govt. Domain, Vic.’.

79. LEONINUS, CORIOLUS (KI.), nov. comb.
leoninus, Polyporus K1l., Linnaea, Vol. 8, p. 486, 1833.
funalis, Polyporus Fr., Epicrisis, p. 459, 18388.
mons-veneris, Polyporus Jungh., Batay. Genootsch. Verhand., Vol. 17, p. 61, 1839.
One specimen from Australia, ex “Lower Ardur River, Gulf of Carpentaria, Q.”’, is
under the cover of Polyporus leoninus. A second, ex “Endeavour River, Q., Persieh’’, is
filed under P. funalis.
leonotis, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 73, 1875. The type was ex
“Australia”. No specimens are at Kew or British Museum of Natural History.
leucocreas, Polyporus Cke. = Polyporus portentosus.

80. LEUCOPLACA, PORTIA (Berk.) CKe., Grev., Vol. 14, p. 113, 1886.
leucoplacus, Polyporus Berk., Fl. N.Z., Vol. 2, p. 180, 1855.
macrospora, Poria Rodw. & Clel., Papers and Proc. Roy. Soc. Tas. for 1929, p. 81,
1929. i
The type is from New Zealand, ex “N.Z., Colenso’’, and is matched by the type of
P. macrospora ex “National Park, S. Aus.”. An Australian specimen ex “Kurrumburra,
Vic., No. 1044” filed under the cover at Kew is not the same species.
levis, Trametes Berk. = Coriolus lactineus.
libum, Polyporus Berk., Jour. Linn. Soc., Vol. 13, p. 163, 1873. The type ex ‘“‘Tweed
River, N.S.W., Guilfoyle’” is merely a fragment which has been so damaged by insects
as to be unrecognizable.

81. LIEBMANNII, PoLYporus Fr., Nov. Symb., p. 59, 1851.
mutabilis, Polyporus Berk. & Curt., Ann. Mag. Nat. Hist., Ser. II, Vol. 12, p. 438,
1853. F
sterinus, Polyporus Berk. & Curt., Jour. Linn. Soc., Vol. 10, p. 308, 1869.
rigescens, Polyporus Cke., Grev., Vol. 14, p. 12, 1885.
stereoides, Polystictus Berk. & Curt. ex Cke., Grev., Vol. 14, p. 78, 1885.
cognatus, Polyporus Kalch., in herb. Kew.

‘BY G. H. CUNNINGHAM. 231

Specimens from this region at Kew are “Clarence River, N.S.W.”’, filed under
P. luteus, and on the sheet referred by Lloyd to P. mutabilis; “Aloha, Papua, C. EH.
Carr”; and “Samoa, C. G. Lloyd’, which was identified by Bresadola as P. liebmannii.

82. LILACINO-GILVA, TRAMETES (Berk.) Lloyd, Syn. gen. Fomes, p. 226, 1915.
lilacino-gilvus, Polyporus Berk., Ann. Nat. Hist., Ser. I, Vol. 3, p. 324, 1839.
rosea, Trametes Lloyd, Letter 59, p. 5, 1915, type ex Australia, J. B. Cleland.
stowardii, Trametes Lloyd, Myc. Notes, No. 48, p. 6838, 1917, type ex W. Australia,
Dr. F. Stoward.
Australian collections at Kew are: “Hnoggera, Brisbane, Q., W. N. Cheesman,

No. 77’, “Daintree River, Q., Pentzcke, No. 219’, “Pennant Hills, Parramatta River,
N.S.W., Challenger Expedition”, “Tilba Tilba, N.S.W., Miss Bates’, “Gippsland, Vic.,
Murray, Webb’, “Loddon River, Vic., Wallace’, “Ballarat, Vic., C. French’, “S.W. Aus.,
T. Muir’, “Van Diemen’s Land, Gunn” and “Australia”, this last being filed under
Polyporus cupreo-roseus Berk. Under the cover of Polyporus carneus are “Brisbane, Q.’,
“Melbourne, Vic., G. LeFevre, No. 212” and “Narranoom, Vic., Stranger”’.
limbatus, Polyporus Fr. Cooke (Hdbk., p. 148, 1892) recorded the species from
Victoria, but there are no specimens from this region at Kew.
lineato-scaber, Fomes Berk. & Br. = Fomes strigatus.
83. LIVIDUS, FOMES (Kalch.) Sacc., Syll. Fung., Vol. 6, p. 206, 1888.
lividus, Polyporus Kalch., ex CkKe., Grev., Vol. 10, p. 103, 1882.
luridus, Fomes (Kalch.) Cke., Grev., Vol. 14, p. 21, 1885.
Collections from Australia at Kew are the type ex “Richmond River, N.S.W.”,

“Clarence River, N.S.W., Thorneton”, “Imbil State Forest, Q., J. B. Cleland, No. 14’,
“Hawkesbury River, N.S.W., J. B. Cleland” and “Lismore, N.S.W., J. B. Cleland”.

84. LIVIDA, PoRIA Cke., Grev., Vol. 10, p. 131, 1882.

The type ex “Clarence River, N.S.W., Wilcox” is probably a resupinate form of
Fomes lividus.

85. LLOYDII, FOMES Cleland, Toadstools ... of S. Aus., p. 200, 1935.
The type was from “National Park, S. Aus., J. B. Cleland’. No specimens are
at Kew.

86. LUCIDUM, GANODERMA (Ley. ex Fr.) Karst., Rev. Myc., Vol. 3, p. 17, 1881.
lucidus, Polyporus (Leyss.) Fr., Syst. Mye., Vol. 1, p. 353, 1821.

Under the cover of Ganoderma lucidum are filed two collections ex “Stradbroke
Island, Q., C. E. Hubbard” and “Queensland, Bailey’. Under Fomes lucidus (Fr.) Cke.
are “Lower Ardur River, Gulf of Carpentaria, Q.”’, “Dunk Island, Q., W. Cottrell-Dormer,
No. 11”, “Brisbane, F. M. Bailey’, “Lord Howe Island, Camara’, “Port Denison, Q.,
Fitzalan, Shann’, “Endeavour River, Q., Persieh”’, “Trinity Bay, Q., Karsten’. Filed
- under Polyporus amboinensis are “Trinity Bay, Q.” and “Johnstone River, Q.”.

luridus, Fomes (Kalch.) Cke. = Fomes lividus.

luteo-nitidus, Polyporus Berk. One specimen so referred, ex “Brisbane, Q.”, does
not agree with the type, but is a form of Polyporus zgonalis with a brief lateral stem.

luteo-olivaceus, Polyporus Berk. & Br. = Coltricia placodes.

luteus, Polyporus Nees. A specimen ex “Clarence River, N.S.W., Wilcox” referred
here by Cooke does not resemble collections under the cover named by Fries. Lloyd
referred it—correctly, I believe—to P. mutabilis (= P. liebmannit).

macrospora, Poria Rodw. & Clel. = Poria leucoplaca.

macroter, Polyporus Berk. & Br. = Polyporus fusco-lineatus.

maculatissimus, Polyporus Lloyd = Polyporus portentosus.

magnoporus, Polyporus Lloyd = Polyporus fusco-lineatus.

marginatus, Fomes (Fr.) Gill. The species has not been found in this region,
collections at Kew from Australia being of the following species: “North Queensland”,
“Endeavour River, Q., Persieh’ and “Mt. Macdonald” = Fomitopsis ochroleuca;
“Hndeavour River, Q., Persieh” and “Upper Hunter River, N.S.W., Miss Carter” = Fomes
applanatus; “N.Z., Hokianga, Dr. Berggren” = Fomitopsis hemitephra.

martius, Fomes (Berk.) Cke. Lloyd so named a specimen of Fomitopsis hemitephra
ex “Sydney, N.S.W., W. Froggatt’’.

Ss

932 AUSTRALIAN POLYPORACEAE,

87. MASTOPORUS, FOMES (Lev.) CkKe., Grev., Vol. 13, p. 118, 1885.
mastoporus, Polyporus Ley., Ann. Sci. Nat., Ser. III, Vol. 2, p. 182, 1844.
ponderosus, Polyporus Kalch. ex Cke., Grey., Vol. 10, p. 99, 1882.
Two specimens are at Kew from this region. One, ex ‘Australia, F.v.M.’, is filed
under F. chilensis; the other, ex “N.Z., Ohaiwai, Dr. Berggren’, had been placed under
F. fomentarius.

88. MEDULLA-PANIS, PORTIA (Fr.) Cke., Grev., Vol. 14, p. 109, 1886.
medulla-panis, Polyporus Fr., Syst. Myec., Vol. 1, p. 380, 1821.

Collections at Kew are: “Dunk Island, Q., W. Cottrell-Dormer, No. 18’, “Daintree
River, Q., Pentzcke”’ (the latter labelled Daedalea latissima), “Mt. Lofty, S. Aus.,
J. B. Cleland’, “Sydney, N.S.W., J. B. Cleland’, “Hawkesbury River, N.S.W., J. B.
Cleland’, “Richmond River, N.S.W., Mrs. Hodgkinson”, “Melbourne, Vic., F. M. Reader,
No. 384” (the last filed by Cooke under Poria tarda) and “Strickland River, New Guinea,
Bauerlen, No. 48” (filed under Fomes bistratosus).

megaloporus, Polyporus Lloyd = Polyporus russiceps.

megaloporus, Polyporus Mont. A collection ex “Coomera River, Q., C. T. White”
filed under this cover appears to be of Polyporus russiceps.

89. MELANOPUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 347, 1821.

Only one authentic specimen is at Kew from this region; this, ex ‘‘Parramatta
River, N.S.W., Challenger Expedition”, was filed by Cooke under Polyporus dictyopus.
Collections under P. melanopus ex ‘Toowoomba, Q., Hartmann” and “Melbourne, Vic.,
Dr. Berggren” are of P. hypomelanus; that ex “Tumbulgum, R. T. Burke” = P. elegans.

membranicinctus, Polyporus Berk. = Polyporus merulius.

menziesii, Trametes Berk. = Trametes protea.

90. MERULIUS, POLYPORUS Berk., Fl. Tas., Vol. 2, p. 254, 1860.

archeri, Polyporus Berk., Fl. Tas., Vol. 2, p. 255, 1860.
membranicinctus, Polyporus Berk. ex Cke., Grev., Vol. 15, p. 26, 1886.
tasmaniae, Polyporus Berk., in herb. Kew.

Three collections from Tasmania are at Kew, the type ex “Tasmania, Archer’, type
of P. archeri ex “Tasmania, Archer” and type of P. membranicinctus ex ‘Tasmania,
No. 1379”. Two collections from. New Zealand are also at Kew, “N.Z., Colenso, No. 136”,
filed under Poria fusco-carnea Pers., and “N.Z. Colenso, No. 923” placed under the cover
of Poria corticola.

91. MEYENII, CORIOLUS (KI.), nov. comb.
meyenti, Polyporus K1., Nova Acta Acad. Leop.-Carol., suppl. 19, p. 236, 1843.
obstinatus, Trametes Cke., Grev., Vol. 12, p. 17, 1883.

Collections at Kew are: “Tropical Queensland, Bailey’, “Endeavour River, Q.,
Persieh” (type of JT. obstinatus), ‘““Baridi and Yodda River, Papua’, “Strickland River,
New Guinea, Bauerlen” and ‘‘Samoa, C. G. Lloyd”.

mirabilis, Hexagona Lloyd = Hexagona thwaitesii.

mollis, Trametes Fr. Cooke (Hdbk., p. 160, 1892) recorded the species from New
South Wales, but no specimens from Australia are at Kew.

mollusea, Poria (Fr.) Cke. One specimen from Australia, ex “Endeavour River,
Q., Persieh’’, is filed under this cover at Kew. It is not P. mollusca, but a much-decayed
pileate species, too damaged to identify.

mons-veneris, Polyporus Jungh. = Coriolus leoninus.

92. MUCIDA, PORTIA (Pers. ex Fr.) Cke., Grev., Vol. 14, p. 109, 1886.
mucidus, Polyporus (Pers.) Fr., Syst. Mye., Vol. 1, p. 382, 1821.

A specimen ex “Toowoomba, Q.’, filed by Cooke under Poria blepharistoma, is an
immature plant of Poria mucida.

muelleri, Daedalea Berk. = Lenzites palisoti.

muelleri, Hexagona Berk. = Hexagona tenuis.

muelleri, Polyporus Kalch. = Polyporus grammocephalus.

muelleri, Trametes Berk. = Coriolus paleaceus.

multilobus, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 95, 1882. The type was from
“Richmond River, N.S.W.”. No specimens are at Kew.

BY G. H. CUNNINGHAM. 233

multiplex, Polyporus Berk. = Polyporus colensoi.

multisetosus, Polyporus Lloyd = Fomes setulosus.

murinus, Polyporus Kalch. = Coriolus flabelliformis.

murinus, Polyporus Lev. The collection from “Broger’s Creek, N.S.W., Bauerlen”
does not resemble other specimens under this cover at Kew. It is probably of Fomes
pectinatus, but of this I am not certain, as spores were not found.

mutabilis, Polyporus Berk. & Curt. = Polyporus liebmannii.

myelodes, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 73, 1875. No specimens are
at Kew or British Museum of Natural History.

93. MYLITTAE, POLYPOoRUS Cke. & Mass., Grev:, Vol. 21, p. 37, 1892.
australis, Mylitta Berk., Ann. Mag. Nat. Hist., Vol. 3, p. 326, 1839.

Only sclerotia are at Kew, ex “Beechworth, Vic., J. W. Howard’, which were named
Mylitta australis by Berkeley. Cooke (Hdbk., p. 249, 1892) placed M. australis under
the Ascomycetes.

nanus, Polyporus Mass. = Polyporus rhipidium.

nidulans, Polyporus Fr. The specimen on which this record was based, ex
“Johnstone River, Q.’, is of P. australiensis.

nigripes, Fomes (Fr.) Cke. Cooke (Hdbk., p. 127, 1892) recorded the species from
New South Wales, but there are no specimens from this region at Kew.

niphodes, Poria (Berk. & Br.) Cke. Cooke (Hdbk., p. 154, 1892) recorded the species
from New South Wales. No specimens from this region are at Kew, the record
probably being based on a specimen of Poria calcicola.

nivea, Lenzites Cke. = Lenzites aspera.

novo-guineensis, Polyporus P. Henn. = Polyporus virgatus.

94. OBLECTANS, COLTRICIA (Berk.) G. H. Cunn., Plant Diseases Division Bull. 77,
p. 3, 1948.
oblectans, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 51, 1845.
cladonia, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 51, 1845.
perdurans, Polyporus Kalch. ex Cke., Grev., Vol. 9, p. 1, 1880.

The following Australian collections match the type at Kew, ex “Swan River,
W. Aus., Drummond, No. 157”: “Swan River, W. Aust., Drummond, No. 220”, the type
ot P. cladonia which is filed under the cover of P. bulbipes; “‘Gaisford, Q., E. Bowden’’,
“Pennant Hills, Parramatta River, N.S.W., Challenger Expedition”, “Sydney, North |
Shore, N.S.W., Whitelegge, No. 7’, “Oweo, J. Sterling’, “Melbourne, Vic., F. M. Reader’”’,
“Tasmania, W. Archer’, “Van Diemen’s Land, Mr. Gunn” and “S.W. Aus., T. Muir,
Nos. 110, 122” (No. 122 is filed under P. parvulus).

oblinitus, Fomes (Berk.) Cke. A specimen ex “Clarence River, N.S.W., Moreton”
so referred by Cooke = Trametes ferrea.

obliquus, Polyporus Fr. = Fuscoporia ferruginosa.

obovatus, Polyporus Jungh., Ann. Sci. Nat., Ser. II, Vol. 16, p. 316, 1841. Cooke
(Hdbk., p. 139, 1892) recorded the species from Queensland and New South Wales under
the name Polystictus adami. No collections from these localities are at Kew, the only
specimen being from New Guinea, also filed under P. addami. Bresadola referred this
plant to P. obovatus.

obstinatus, Trametes Cke. = Coriolus meyenii.

95. OCCIDENTALIS, CORIOLUS (KI.), nov. comb.
occidentalis,. Polyporus K1., Linnaea, Vol. 8, p. 486, 1833.
occidentalis, Trametes (K1.) Fr., Hpicrisis, p. 491, 1838.
heteromalla, Trametes Cke., Grev., Vol. 10, p. 182, 1882, type ex Mt. Dromedary,
N.S.W., Miss Bates.
extensus, Polystictus Berk. ex CkKe., Grev., Vol. 14, p. 82, 1886, type ex Daintree
River, Q., Persietz.
badio-lutescens, Polyporus Kalch., in herb. Kew.
subcongener, Daedalea Berk. ex Cke., Grev., Vol. 19, p. 93, 1892, type ex Australia.
Additional Australian collections at Kew are: “Trinity Bay, Q., Karsten”,
“Hndeavour River, Q., Persietz’, “Daintree River, Q., Pentzcke’’, “Port Denison, Q.,

Shann” (the last filed by Cooke under P. lanatus and by Lars Romell referred to

SS

234 AUSTRALIAN POLYPORACEAE,

P. caperatus), “Australia” (referred by Cooke to P. cyclodes), “Richmond River, N.S.W.,
Camara’, “Tarwin, Gippsland, Vic.’, “Moe Swamp, Gippsland, Vic.”, “North Gippsland,
Vic., H. Tisdall”. The last three are filed under 7. heteromalla, and the specimen from
North Gippsland was by Lars Romell referred to 7. devexa.

ochraceo-stuppeus, Polystictus Lloyd = Polyporus adustus.

96. OCHROLEUCA, FOMITOPSIS (Berk.) G. H. Cunn., Plant Diseases Division, Bull 76,
p. 5, 1948.

ochroleucus, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 53, 1845.

compressus, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 53, 1845, type ex Swan
River, W. Aus., Drummond, No. 141.

brisbanensis, Polyporus Berk. & Br., in herb. Kew.

ungulata, Trametes Berk., Jour. Linn. Soc., Vol. 13, p. 165, 1873, type ex Adelaide,
S. Aus., Schomburgk, No. 2.

scrobiculata, Trametes Berk., Grev., Vol. 6, p. 70, 1877, type ex Melbourne, Vic.,
LeFevre, No. 212.

varia, Trametes Lloyd, Myc. Notes, No. 66, p. 1114, 1922, type ex Tasmania,
L. Rodway.

The type is from “Swan River, W. Aus., Drummond, Nos. 248, 285”. Additional
collections from Australia are: “Quiedong, Q., No. 454” (filed under P. ostraeformis),
“Brisbane, Q., Nos. 136, 138” (filed under P. havannensis and on the sheet labelled
P. brisbanensis), ““Trinity Bay, Q., Sayer, No. 7’ (placed under the cover of P. chioneus).
“Endeavour River, Q., Persieh” and “Mt. Macdonald” (filed under Fomes marginatus),
“Toowoomba, Q., Hartmann” (under cover of Fomes fulvus), “Nambour, Blackball
Range, Q., W. N. Cheesman, No. 59”, “Pennant Hills, Parramatta River, N.S.W.,
Challenger Expedition”, “Gippsland, Vic., Mrs. Campbell’, “Melbourne, Vic., F. M.
Reader” and “W. Aus.”.

ochro-flava, Trametes Cke. Cooke (Hdbk., p. 159, 1892) recorded the species from
Queensland, but there are no specimens from this region at Kew.

odontoporus, Polyporus Kalch. in herb. Kew. A specimen labelled Poria odontoporia
by Cooke, ex “Guntawang, N.S.W., Hamilton, No. 4’, is of Poria versipora growing
over the surface of an irregular piece of wood.

ohiensis, Favolus Berk. & Mont. = Hexagona alveolaris.

olivacea, Hexagona Lioyd = Hexagona gunnii.

orbicularis, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 324, 1839. The type, ex
“Van Diemen’s Land, Gunn, No. 16, on living bark’’, is, as Bresadola had noted on the
type sheet, a species of Septobasidium.

orbiformis, Fomes (Fr.) Cke. Cooke (Hdbk., p. 130, 1892) recorded the species from
Victoria, but there are no specimens from this region at Kew.

ornithorhynchi, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 96, 1882. The type was
from ‘Richmond River, N.S.W., Maria Hodgkinson”. No cover for this species is at
Kew.

ostraeformis, Polyporus Berk. One specimen ex “Quiedong, Q., No. 454” is filed
under this cover at Kew. As Bresadola had noted on the sheet, it is of Fomitopsis
ochroleuca. \ ,

ovinus, Polyporus Fr. A specimen so named by Cooke, ex “Queensland, Campbell,
No. 96” = Amauroderma rudis.

97. PALEACEUS, CORIOLUS (Fr.), nov. comb.
paleacea, Trametes Fr., Nov. Symb., p. 97, 1851.
muelleri, Trametes Berk., Jour. Linn. Soc., Vol. 10, p. 320, 1869, type ex Victoria
River, Q., F.v.M. ;
eucalypti, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 738, 1875, type ex Rock-
hampton, Q., Mrs. Thozet.
picta, Trametes Berk., Trans. Linn. Soc., Ser. II, Vol. 2, p. 61, 1883, type ex
Moreton Bay, Q., F. M. Bailey, Nos. 11, 14. ,
argentatus, Polyporus Cke., Grev., Vol. 15, p. 20, 1886, type ex Narranoom, Vic.,
Stranger.
Additional collections at Kew are: “North Queensland”, “Brisbane” and “Dunk
Island, Q., W. Cottrell-Dormer, No. 28”, which are filed under Polyporus rudis Berk.;

“Bellenden Ker, Q., Bailey, Nos. 820, 821” and “Victoria, Mrs. Martin, No. 634’, placed

BY G. H. CUNNINGHAM. 235

under Polyporus eucalypti; “Endeavour River, Q., Persietz’, “Daintree River, Pentzcke”
and “New Guinea, Armit”, filed under Trametes muelleri; one specimen ex “Hndeavour
River, Q., Persietz’’ is filed under Trametes picta, a second from the same source under
Polyporus carneus; “Yerrigong, Nowra, N.S.W., F. A. Rodway” and “Dunk Island, Q.,
W. Cottrell-Dormer, No. 13” are filed under Trametes lilacino-gilva; “Trinity Bay, Q.,
Karsten, No. 2”, ‘Loddon River, Vic., No. 6’, “Toowoomba, Q., Hartmann” and
“Narranoom, Vic., Stranger’ are under Polyporus pallisiert.

98. PALISOTI, LENZITES Fr., Hpicrisis, p. 404, 1838.
palisoti, Daedalea Fr., Syst. Myc., Vol. 1, p. 335, 1821.
laevis, Daedalea Hook., in Kuenth., Syn., p. 9, 1822.
.deplanata, Daedalea Fr., Linnaea, Vol. 5, p. 518, 1830.
polita, Daedalea Fr., Linnaea, Vol. 5, p. 514, 1830.
applanata, Daedalea K1., Linnaea, Vol. 8, p. 481, 1833.
repanda, Lenzites (Mont.) Fr., Epicrisis, p. 404, 1838.
pallida, Lenzites Berk., Lond. Jour. Bot., Vol. 1, p. 146, 1842.
muelleri, Daedalea Berk. ex Cke., Grev., Vol. 19, p. 93, 1891.
Collections of species listed above, and other combinations, though varying somewhat
in size and colour, possess the same microstructure and cannot be separated on any
constant feature. Australian collections at Kew are: “Stannary Hills, Q., Dr. T. L.

Bancroft, No. 2039’, filed under Lenzites deplanata; “‘Toowoomba, Q., Hartmann’’, under

the cover of Lenzites repanda; ‘‘Toowoomba, Q.”, type of Daedalea muelleri. On the
sheet of this last Bresadola had noted “= Tr. ludificans Ces. = forma abortiva L.
applanata’’.

99. PALLENS, MERULIUS Berk., Outlines, p. 256, 1860.

Collections from Australia at Kew filed under this cover are: ‘Victoria, No. 430’,
“Tasmania” (three collections) and “Tarwin, No. 21”’.

pallescens, Polyporus Fr. Specimens ex “Daintree River, Q., Pentzcke” are thick
plants of Polyporus adustus.

pallida, Lenzites Berk. = Lenzites palisott.

pallisieri, Polyporus Berk. ex Cke., Grev., Vol. 10, p. 98, 1882. The type, ex
Saskatchewan, Canada, was referred to Fomes subroseus (Weir) Ov., in Rhodora, Vol.
25, 1923. It is not the same as Flomes pallisieri (Berk.) Cke., Grev., Vol. 14, p. 21, 1885,
which is based on the species later renamed by Cooke Polyporus argentatus, a synonym
of Coriolus paleaceus. Collections from Australia at Kew so referred by Cooke are of
Coriolus paleaceus.

100. PARGAMENUS, CORIOLUS (Fr.), nov. comb.

pargamenus, Polyporus Fr., Hpicrisis, p. 480, 1838.
laceratus, Polyporus Berk., Ann. Nat. Hist., Vol. 2, p. 392, 1839.

Collections at Kew from Australia are filed under P. laceratus. They are: “Brisbane,
Q., Bailey” and “Illawarra, N.S.W., Karsten, No. 43”. Another specimen ex ‘Adelaide,
S. Aus., W. N. Cheesman” may be the same but is too imperfect for accurate diagnosis.

parilis, Polyporus Fr., Pl. Preiss., Vol. 2, p. 136, 1847. No cover is at Kew. The
species was described from material collected in Australia, but the description is too
. incomplete to enable it to be recognized.

parvulus, Polyporus Kl. A specimen from Australia, ex “S.W. Aus., T. Muir, No.

110”, filed under this cover = Coltricia oblectans. Bresadola noted on the sheet that it
is a synonym of 0. cinnamomea.

101. PECTINATUS, FOMES (KI.) Cke., Grev., Vol. 10, p. 20, 1885.
pectinatus, Polyporus K1l., Linnaea, Vol. 8, p. 485, 1833.

The following collections from this region are at Kew: “Campbell’s Creek, Q.,
H. Fletcher”, “Condamine River, Q., Hartmann, No. 513” (the latter filed under Fomes
spadiceus and referred by Cooke to P. substygius Berk., by Bresadola to P. nilgheriensis),
“New Guinea, Capt. Armit, No. 18” and ‘“N.Z., Bay of Islands, No. 334”.

pelles, Polyporus Lioyd = Polyporus pelliculosus.

pelliculosus, Merulius Cke. = Merulius corium.

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AUSTRALIAN POLYPORACEAE,

102. PELLICULOSUS, POLYPORUS Berk., Lond. Jour. Bot., Vol. 7, p. 575, 1848.

hystriculus, Polyporus Cke., Grev., Vol. 15, p. 16, 1886, type ex Melbourne, Vic.,
F. M. Reader, No. 13.

pelles, Polyporus Lloyd, Syn. sect. Apus Polyporus, p. 327, 1915, type ex Queensland,
EK. Jarvis.

atro-hispidus, Polyporus Lioyd, Myce. Notes, No. 57, p. 823, 1919, type ex Victoria,
Jas. Wilson.

strigoso-albus, Polyporus Lloyd, Myc. Notes, No. 73, p. 1329, 1924, type ex Australia,
J. B. Cleland.

The type ex Penguite is not at Kew; but two specimens, ex “Tasmania, Archer”
so labelled by Berkeley may be regarded as neotypes. The following collections agree
with these. “Upper Yarra, Vic., Lucas” and “Western Port, Vic., Musgrave”. One
specimen so referred by Cooke, ex “Gippsland, Vic., Webb” = Polyporus campylus.

pendula, Daedalea Berk. = Lenzites wnicolor.

pentzckei, Polyporus Kalch., Proc. Linn. Soc. N.S.W., Vol. 8, p. 175, 1884. The type
was from “Daintree River, Q., Pentzcke”’. No specimens are at Kew.

103. PERADENIAR, CORIOLUS (Berk. & Br.), nov. comb.

peradeniae, Polyporus Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 51, 1875.

Bresadola referred the species to P. cervino-gilvus Jungh., Crypt. Java, p. 45, 1838;
Petch in Lloyd’s Mycological Notes, No. 67, p. 1163, 1922, to P. zeylandicus Berk. with
as synonyms P. diversiporus and P. personatus Berk. Both were published on later
pages of the same periodical in which Polyporus peradeniae was described. Filed under
P. peradeniae are the following collections from Australia: “Dunk Island, Q., W. Cottrell-
Dormer, No. 44’, ‘Nambour, Blackball Range, Q., W. N. Cheesman”’, “‘Daintree River,
Q., Pentzcke’’, ‘‘Clarence River, N.S.W., Thorneton’’, “Richmond River, N.S.W., Camara’,
and “Creswick, near Ballarat, Vic., W. N. Cheesman”’.

perdurans, Polyporus Kalch. = Coltricia oblectans.

peroxydatus, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 39, 1878. The type was
ex “Pennant Hills, Parramatta River, N.S.W., Challenger Expedition”. No specimens are
at Kew or British Museum of Natural History.

persooni, Poiystictus Cke. = Coriolus corrugatus.

104. PES-CAPRAE, POLYPORUS (Pers:) Fr., Syst. Myc., Vol. 1, p. 354, 1821.

tasmanicus, Polyporus Mass., Kew Bull. Misc. Inf., p. 179, 1899.

Both collections, ex “Australia” and “Tasmania, L. Rodway’, type of P. tasmanicus,
resemble European specimens of P. pes-caprae. Massee’s name cannot be used, since
it is preoccupied by P. tasmanicus Berk., 1860.

phellina, Trametes Berk., Jour. Linn. Soc., Vol. 13, p. 164, 1873. The type was from
“New England, Australia”. No cover is at Kew.

picipes, Polyporus Fr. Portion of a specimen ex “Brisbane, Q., No. 168, ex herb.
Currey” is under this cover at Kew. The specimen is of P. elegans.

picta, Trametes Berk. = Coriolus paleaceus.

pictilis, Polyporus Berk. A specimen at Kew, so labelled by Cooke, ex “S.H. New
’ Guinea, Capt. Armit” = Coriolus flabelliformis.

105. PINSITUS, coRIOLUS (Fr.) Pat., Tax. hymen., p. 94, 1900.

pinsitus, Polyporus Fr., lench., p. 95, 1828.

Collections at Kew are “Port Denison, Q., Shann’’, “Dunk Island, Q., W. Cottrell-
Dormer, No. 39”, ‘Endeavour River, Q., Persieh’, “Daintree River, Q., Pentzcke” (the
last filed by Cooke under P. vellereus), “Clarence River, N.S.W., Wilcox” (placed under
P. rufescens). A specimen so named by Massee, ex “N.Z., Colenso, No. 1427” = Coriolus
hirsutus.

pisiformis, Polyporus Kalch. = Coriolus hirsutus. Type specimens ex “Richmond
River, N.S.W.” are immature and about the size of a grain of wheat. Their micro-
structure is that of C. hirsutus.

106. PLACODES, COLTRICIA (Kalch.), nov. comb.

placodes, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 73, 1875.
luteo-olivaceus, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 1, p. 402, 1879.
The type of P. luteo-olivaceus ex “Brisbane, Q., Bailey” matches that of P. placodes

ex “Rockhampton, Q., Mrs. Thozet”, the prior name. Other collections at Kew, filed

BY G. H. CUNNINGHAM. 237

under P. luteo-olivaceus are: ‘‘Port Denison, Q., Shann, No. 19”, “Endeavour River,
Persieh”, “Toowoomba, Q., Hartmann’, “Hnoggera, Q., C. T. White’, “Toowoomba, Q.”,
“Clarence River, N.S.W., Wilcox’, “Richmond River, N.S.W., Camara’ and ‘“Moonan
Brook, Vic., Miss Carter”.

platotis, Polyporus Berk. & Br. = Polyporus fusco-lineatus.

plebius, Polyporus Berk. A specimen ex “Brisbane, Q., herb. Broome” so referred
is ot Fomes gilvus. No others are at Kew from this region.

pocula, Polyporus (Schw.) Berk. & Curt. = Polyporus pusillus.

polita, Daedalea Fr. = Lenzites palisoti.

107. POLYGRAMMA, HEXAGONA (Mont.) Fr., Epicrisis, p. 497, 1838.
polygrammus, Polyporus Mont., Ann. Sci. Nat., Ser. II, Vol. 8, p. 365, 1837.

rigida, Hexagona Berk., Jour. Linn. Soc., Vol. 16, p. 54, 1878, type ex Lord Howe
Island, J. P. Fallagher.

subtenuis, Hexagona Berk. ex Cke., Grev., Vol. 19, p. 103, 1891, type ex Rockhampton,
Q., Mrs. Thozet.

Additional collections at Kew are: “Australia, 8.15, 8.19”, “Queensland, Walter’,
“Brisbane, Q., ex herb. Broome’, “Goode island, Torres Strait, Powell’, ‘Toowoomba, Q.,
Hartmann”, “Tweed River, N.S.W., Camara” and “Illawarra, N.S.W., Johnson”. Under
Hexagona rigida are filed “Lord Howe Island, C. French, Jr.”, “Kin Kin, Q., W. D.
Francis’, “Dunk Island, Q., W. Cottrell-Dormer, No. 35”, and “New Guinea, Capt. W.
Armit”. Under the cover of H. nitida is one collection ex “Samoa, W. Powell” which
Lloyd on the sheet referred to H. tenwis. Under H. subtenuis are “Queensland, Bailey”’,
“Port Denison, Q., Shann” and “Botanic Gardens, Sydney, N.S.W., A. Grant’.

polymorphus, Polyporus Rostk. Under this cover Cooke placed four fragments ex
“Endeavour River, Q., Persieh’’. They are too fragmentary to name, but do not
resemble Huropean specimens in the cover.

ponderosus, Polyporus Kalch. = Fomes mastoporus.

108. PORPHYRITES, POLYPORUS Berk., Hook. Jour. Bot., Vol. 8, p. 196, 1856.

Specimens under this cover at Kew are: “Tropical Queensland, Bailey” and
“Johnstone River, Q., Berthoud”.

109. PORTENTOSUS, POLYPORUS Berk., Lond. Jour. Bot., Vol. 3, p. 188, 1844.
eucalyptorum, Polyporus Fr., Pl. Preiss., Vol. 2, p. 135, 1847, type ex Australia.
hololeuca, Trametes Kalch., Hedw., Vol. 15, p. 114, 1876, type ex Victoria.

leucocreas, Polyporus Cke.,- Grev., Vol. 8, p. 55, 1879, type ex N.Z., Coromandel,
No. 311.

dielsii, Polyporus P. Henn., Hedw., Vol. 42, p. 75, 1903, type ex Western Australia.

cretaceus, Polyporus Lloyd, Syn. sect. Apus Polyporus, p. 302, 1915, type ex Tasmania,
L. Rodway.

maculatissimus, Polyporus Lioyd, Myce. Notes, No. 66, p. 1113, 1922, type ex Tasmania,
L. Rodway, No. 1037.

albo-fuscus, Polyporus Lioyd, Mye. Notes, No. 73, p. 1318, 1924, type ex South
Australia, J. B. Cleland, No. 873. :

Though the type ex “Swan River, W. Aus., Drummond, No. 125” is now somewhat
fragmentary, the microstructure is so characteristic that identification is possible on
this feature alone. Other collections at Kew are: “Toowoomba, Q., No. 11’, ““Toowoomba”’
(the latter filed under P. betulinus), “North Queensland”, “Parramatta River, N.S.W.,
Challenger Expedition’, “Melbourne, Vic., F. M. Reader’, “Tasmania”, “New Guinea,
Capt. Armit” and “N.Z., York Bay, G.H.C.”’, the last identified by Lloyd as
P. eucalyptorum.

praetervisa, Trametes Bres. = Hexagona gunnii.

110. PROTEA, TRAMETES (Berk.), nov. comb.

proteus, Polyporus Berk., Lond. Jour. Bot., Vol. 2, p. 514, 1848.

rigida, Trametes Berk. & Mont., Ann. Sci. Nat., Ser. III, Vol. 11, p. 240, 1849, type
ex Bahia, Brazil.

gourliaei, Polyporus Berk., Fl. Tas., Vol. 2, p. 2538, 1860, type ex Van Diemen’s
Land, ex Wm. Gourlie.

menziesii, Trametes Berk., in herb. Kew.

chrysoleucus, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 72, 1875, type ex
Rockhampton, Q., Thozet, No. 768.

238 AUSTRALIAN POLYPORACEAE,

semidigitaliformis, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 39, 1878, type ex
Pennant Hills, Parramatta River, N.S.W.

gausapatus, Polyporus Walch. ex Cke., Grev., Vol. 10, p. 102, 1882, type ex Richmond
River, N.S.W.

illotus, Polyporus JIkalch. ex Cke., Grev., Vol. 10, p. 102, 1882, type ex Richmond
River, N.S.W.

carteri, Trametes Berk., ex Sacc., Syll. Fung., Vol. 9, p. 196, 1891.

Additional collections at Kew from this region are: ‘“‘Toowoomba, Q., Hartmann”,
“Moreton Bay, Q., Bailey”, ‘Pennant Hills, Parramatta River, N.S.W., Challenger
Expedition” and “Tweed River, N.S.W., Guilfoyle’, filed under 7. protea. Under
Polystictus versicolor is a collection ex ‘South Australia, J. B. Cleland”; under Trametes
carteri is filed “Adelaide, National Park, S. Aus., W. N. Cheesman, No. 3”. A resupinate
specimen ex “Clarence River, N.S.W., Wilcox” is filed under Poria corium Kze.; a
second, ex “Australia”, is under Poria calcicola. Under T. gausapata, labelled on the
sheet T. menziesii, is placed “Kurrumburra, Vic., No. 1039”; a second collection under
this cover is “Endeavour River, Q., Persietz”. Under 7. versiformis Cooke placed
“Rockhampton, Q., Mrs. Thozet, No. 131”; and “New Caledonia, F. J. Roberts” is filed
under 7. gibbosa.

111. PROTEIFORMA, TRAMETES (Cke.), nov. comb.

proteus, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 102, 1882, non Berk., 1843.
proteiformis, Polystictus Cke., Grev., Vol. 14, p. 81, 1886.

The only specimen at Kew is the type ex ‘Melbourne, Vic., F. v. Mueller’. The
name was changed by Cooke since Polyporus proteus was preoccupied.

112. PROTEIPORUS, POLYPORUS CkKe., Grev., Vol. 12, p. 15, 1883.

The type ex “Toowoomba, Q., Hartmann” and “Gippsland, Vic.” are the only
collections at Kew. Lloyd (Syn. stip. Polyp., p. 162, 1912) referred the species as a
synonym of P. rufescens; but it is doubtful if the latter occurs in the region.

proteus, Polyporus Kalch. = Trametes proteiforma.

protracta, Lenzites Fr. = Daedalea trabea.

pubescens, Polyporus Fr. A collection from Australia, ex “Endeavour River Q.,
Persieh”, so referred by Cooke, consists of two plants of Coltricia fruticum.

pullagerii, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 54, 1878. No specimens are
at Kew or British Museum of Natural History.

113. PULLATUS, AMAURODERMA (Berk.), nov. comb.

pullatus, Polyporus Berk. ex Cke., Grev., Vol. 13, p. 117, 1885, nomen nudum.
pullatus, Fomes Berk. ex Cke., Grev., Vol. 15, p. 21,-1886.

Specimens from Australia, ex ‘‘Brisbane, Q., F. M. Bailey, Nos. 730, 764, 766” and
“Trinity Bay, Q., Karsten”, match the type at Kew ex Hong Kong.

pullus, Fomes (Berk. & Mont.) Cke. Though recorded from Queensland by Cooke
(Hdbk., p. 133, 1892) there are no specimens from this region at Kew.

114. puNcrATA, FUSCOPORIA (Fr.) G. H. Cunn., Plant Diseases Division, Bull. 73,
p. 11, 1948.

punctatus, Polyporus Fr., Hym. Eur., p. 572, 1874.

One specimen of the species, ex “Swan River, W. Aus., Drummond”, is filed under
Poria contigua and on the sheet labelled Poria ferrugineus. Part of the same specimen
is also under the cover of Poria ferruginosa.

115. PUSILLUS, POLYPORUS (Fr.), nov. comb.

pusillus, Favolus Fr., Linnaea, Vol. 5, p. 511, 1830. :
cupuliformis, Polyporus Berk. & Curt., Hook. Jour. Bot., Vol. 1, p. 1038, 1849.
pocula, Polyporus (Schw.) Berk. & Curt., Proc. Am. Acad. Arts and Sci., Vol. 4,
p. 122, 1858.
Specimens from this region, ex- ‘Melbourne, Vic., Mrs. Martin, No. 606” (filed

under P. cupuliformis) and “N.Z., Bay of Islands, No. 236” (filed under Favolus
rhipidium), match part of the type ex Brazil. A collection at Kew named Favolus
pusillus, ex “Van Diemen’s Land, Gunn” = Polyporus rhipidium.

116. PYRRHOCREAS, FOoMES Cke., Grev., Vol. 14, p. 11, 1885.

Represented at Kew by the type collection, ex “New Guinea, Capt. Armit’”. On the
sheet Bresadola had noted: ‘= Polyporus albo-marginatus Ley. = laeticolor Berk. =
kermes Berk.” Lloyd, on the type sheet, referred the species to P. croceus Pers.

BY G. H. CUNNINGHAM. 239

117. PYRRHOCREAS, TRAMETES Berk., Jour. Linn. Soc., Vol. 13, p. 164, 1873.

The type consists of two specimens ex “Herbert*s Creek, Q.”. Other collections
which match the type are “Brisbane, Q., F. M. Bailey, No. 337’, ‘Daintree River, Q.,
Pentzcke”, ‘“Guntawang, N.S.W., Hamilton, No. 26’, “Clarence River, N.S.W., Wilcox’’
and “Samoa, C. G. Lloyd”, the last being filed under P. proteus Berk.

quadrans, Polyporus Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 1, p. 400, 1879.
No specimens are at Kew or British Museum of Natural History.

quercina, Daedalea Fr. A specimen so referred, ex “Bot. Gardens, Adelaide, S. Aus.,
W. N. Cheesman”, is a weathered plant of Lenzites aspera.

radiato-rugosis, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 323, 1839. No specimens
are at Kew or British Museum of Natural History.

118. RADULA, PORTIA (Pers. ex Fr.) CkKe., Grev., Vol. 14, p. 111, 1886.

radulus, Polyporus (Pers.) Fr., Syst. Myc., Vol. 1, p. 383, 1821.
vinctus, Polyporus Berk., Ann. Mag. Nat. Hist., Ser. II, Vol. 9, p. 196, 1852.

Specimens ex ‘Daintree River, Q., Pentzcke” are filed under Poria vincta; ‘Dunk
Island, Q., W. Cottrell-Dormer, No. 14” is under Poria hyposclera; and “Bellenden Ker,
Q., Karsten” Cooke placed under Fomes bistratosus.

rasipes, Polyporus Berk. = Polyporus vernicifluus.

repanda, Lenzites (Mont.) Fr. = Lenzites palisoti.

retiporus, Polyporus Cke. = Polyporus berkeleyi.

119. RHINOCEPHALUS, POLYPORUS Berk., Fl. Tas., Vol. 2, p. 253, 1860.

The type, ex “Tasmania, Archer’, is the only collection at Kew.

120. RHIPIDIUM, POLYPORUS Berk., Lond. Jour. Bot., Vol. 6, p. 319, 1847.

rhipidium, Favolus (Berk.) CKe., Grev., Vol. 15, p. 58, 1886.
diminutus, Polyporus Mass., Jour. Bot., Vol. 34, p. 153, 1896.
nanus, Polyporus Mass., in herb. Kew.

Filed under Favolus rhipidium are the following collections: “Sunday Island,
Kermadecs, W. R. B. Oliver’, “Raoul Island, Kermadecs, McGillivray, voyage of H.M.S.
Herald”, “Illawarra, N.S.W., Kirton, No. 44”, “Pennant Hills, Parramatta River, N.S.W.,
Challenger Expedition’, “Victoria, Dr. Winter’ and “Melbourne, Vic., F. M. Reader”.
Under P. nanus is a collection ex “Port Phillip, Vic., F. M. Reader, No. 31”; and
under Favolus pusillus is filed one ex “Van Diemen’s: Land, Gunn’’.

rigescens, Polyporus Cke. = Polyporus liebmannii.

rigida, Hexagona Berk. = Hexagona polygramma.

rigida, Trametes Berk. & Mont. = Trametes protea.

rigidus, Polyporus Lloyd = Inonotus tabacinus.

121. RIMOSUS, FOMES (Berk.) Cke., Grev., Vol. 14, p. 18, 1885.

Under this cover at Kew the type collection is given as “Van Diemen’s Land,
Lawrence”; but in the published description it was stated to be “Swan River, W. Aus.,
Drummond, No. 144”. One other specimen under the cover, ex “Daintree River, Q.,
Pentzcke” = Fomes setulosus.

rosea, Trametes Lioyd = Trametes lilacino-gilva.

rosettus, Polyporus Lloyd = Polyporus campylus.

rubidus, Polyporus Berk. Three specimens so referred, ex “North Queensland’,
“Brisbane, Q.” and “Dunk Island, Q@., W. Cottrell-Dormer, No. 28”, are of Coriolus
paleaceus.

rubiginosus, Polyporus Berk. = Fomes gilvus.

122. RUDIS, AMAURODERMA (Berk.), nov. comb.

rudis, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 323, 1839.

Specimens at Kew match the type ex “Tasmania, herb. Sir W. Hooker”. These
are: “Queensland, F. M. Bailey’, “Queensland, Campbell, No. 96” (the latter filed under
Polyporus ovinus), ““Melbourne, Vic., F. Reader, No. 40” and “New Caledonia, J. F.
Roberts”. One specimen so labelled, ex “N.Z., Colenso”’, = P. xerophyllus.

rufa, Poria (Fr.) Cke. Though recorded from Victoria by Cooke (Hdbk., p. 155,
1892), there are no specimens from this region at Kew.

rufescens, Polyporus Fr. Collections from Australia do not resemble those from
India on the same sheet. One, “Clarence River, N.S.W., Wilcox” = Coriolus pinsitus;

240 AUSTRALIAN POLYPORACEAE,

a second, ex “New Guinea’, was on the sheet referred by Boedijn to 7. corrugata;
two others, ex “Pennant Hills, Parramatta River, N.S.W.” and “Lake Muir, S.W. Aus.,
T. Muir’, are unrecognizable.

rufi-tincta, Poria (Berk. & Curt.) Cke. A specimen so referred by Cooke, ex
“Australia, herb. Kalchbrenner” = Fuscoporia laevigata.

rufo-lateritius, Polyporus Kalch. ex Cke., Grev., Vol. 10, p. 104, 1882. No specimens
are at Kew. The description of the type ex “Richmond River, N.S.W.” suggests the
species was based on Polyporus merulius.

rufo-rigidus, Polystictus Lloyd = Coriolus flabelliformis.

rufo-rugosus, Polyporus Lloyd = Coriolus zonatus.

123. RUGICEPS, POLYPORUS Lloyd, Myc. Notes, No. 73, p. 1329, 1924.

Part of the type is at Kew, ex “Victoria, Jas. Wilson”.

124. RUGOSUS, AMAURODERMA (Fr.), nov. comb.

rugosus, Polyporus Fr., Elench., p. 74, 1828, non Nees, 1826.

The following collections from this region are at Kew: “Pennant Hills, Parramatta
River, N.S.W., Challenger Expedition”, “S.E. New Guinea, Capt. Armit” and “Mt.
Bedford, New Guinea, Capt. Armit”.

125. RUSSICEPS, POLYPORUS Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 48, 1875.
cinnamomeo-squamulosus, Polyporus P. Henn., Engl. Bot. Jahrb., Vol. 30, p. 43, 1901.
megaloporus, Polyporus Lloyd, Myce. Notes, No. 48, p. 684, 1917.

The only authentic collection at Kew from this region is ex “Coomera River, Q.,

C. T. White’, filed under Favolus megaloporus (Mont.) Bres. One collection, ex “New
Guinea, Fly River, Bauerlen” = Polyporus infernalis; a second, ex ‘New Guinea,
Strickland River, Bauerlen” = P. xerophyllus.

126. SAEPIARIA, DAEDALEA Fr., Syst. Myc., Vol. 1, p. 333, 1821.
saepiaria, Lenzites Fr., Epicrisis, p. 407, 1838.

A collection ex “National Park, Adelaide, S. Aus., W. N. Cheesman, No. 35’, filed
under Lenzites saepiaria, agrees with specimens from Upsala, so named by Fries.

salicinus, Fomes (Fr.) Cke. The specimen so referred, ex ‘“‘Trinity Bay, Q., Sayer,
No. 40” = Coltricia weberiana.

sanguinarius, Polyporus Kl. ~Bresadola referred to this species, on the sheet, a
specimen ex “Clarence River, N.S.W., Moreton” which Cooke had filed under P. oblinitus
Berk. It is Trametes ferrea.

sanguinea, Daedalea Kl. = Coriolus corrugatus.

127. SANGUINEUS, CORIOLUS (Fr.), nov. comb.
sanguineus, Polyporus Fr., Syst. Mye., Vol. 1, p. 371, 1821.
cinnabarinus, Polyporus Fr., Syst. Mye., Vol. 1, p. 371, 1821.
coccineus, Polyporus Fr., Nov. Symb., p. 67, 1851.
puniceus, Polyporus Kalech., Rev. Mye., Vol. 4, p. 95, 1882.
semi-sanguineus, Polystictus Lloyd, Letter 39, p. 8, 1912. f
In Kew herbarium collections are placed under Polystictus sanguineus and
_P. cinnabarinus. In some instances the collection has been divided and placed under
both. There is no constant feature upon which two species. may be maintained,
consequently collections should be merged under C. sanguineus, since this precedes
P. cinnabarinus on the same page of Systema.

Australian collections at Kew are: “Trinity Bay, Q., W. A. Sayer’, “Endeavour
River, Q., Persieh’”’ (under both covers), “Daintree River, Q., Pentzcke”, ““Condamine
River, Q.’, “Port Denison, Q., Shann”, “Herberton, Q.”, “Twofold Bay, Q., White”,
“Britie Island, Q., C. E. Hubbard”, “Stadbroke Island, Q., C. E. Hubbard’, “Moreton
Bay, Q., F. M. Bailey’, “Pennant Hills, Parramatta River, N.S.W., Challenger Expedition”
(in both covers), “Sydney, N.S.W., J. B. Cleland’, “Mt. Dromedary, N.S.W., Miss
Bates”, Richmond River, N.S.W., Camara”, “Narranoom, Vic., Stranger’, “Melbourne,
Vic., G. LeFevre”, ‘North Gippsland, Vic., H. Tisdall’, “W. Aus.”, ‘King George Sound,
Harris”, “Tasmania, L. Rodway, No. 1379”.

128. SCABER, FoMES (Berk.) Lloyd, Syn. genus Fomes, p. 249, 1915.

igniarius var. scaber, Polyporus Berk., Ann. Nat. Hist., Vol. 3, p. 324, 1840.
tepperi, Fomes Lioyd, Syn. genus Fomes, p. 256, 1915.

BY G. H. CUNNINGHAM. 241

Lloyd erected the species upon a specimen ex “Tasmania’’ which at Kew was filed
under Fomes rimosus, though published as Polyporus igniarius var. scaber. A second
specimen is at Kew ex “Victoria, ex herb. Lloyd’. Filed under Fomes tepperi is part
of the type ex “Norwood, S. Aus., J. G. O. Tepper” and in addition ‘Sydney, N.S.W.,
J. B. Cleland, No. 291”. These two collections match the type of F. scaber.

scabriusculus, Polyporus Berk., Jour. Linn. Soc., Vol. 18, p. 384, 1881. No specimens
are at Kew or British Museum of Natural History.

scabrosus, Polyporus Berk. = Fomes gilvus, pro parte.

129. SCALARIS, LENZITES (Berk. & Br.), nov. comb.

scalaris, Daedalea Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 61, 1883.

The type, ex “Brisbane, Q., Bailey, No. 429” is in the British Museum of Natural
History.

scansilis, Polyporus Berk. = Fomes applanatus.

schomburgkii, Daedalea Berk. = Lenzites tenuis.

130. SCHWEINITZII, COLTRICIA (Fr.) G. H. Cunn., Plant Diseases Division, Bull. 7

p. 7, 1948.
schweiniteii, Polyporus Fr., Syst. Myc., Vol. 1, p. 351, 1821.
tabulaeformis, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 302, 1845.
albertinii, Polyporus Lloyd, Syn. stip. Poly., p. 160, 1912.

Two specimens are at Kew from Australia, “Brisbane, Q., F. M. Bailey, No. 763”,
filed under P. tabulaeformis; and “Kndeavour River, Q., Persieh’’, which Lloyd erected
as P. albertinii because the spores were slightly coloured, a condition not uncommon in
Dld specimens.

~

131. SCOPULOSUS, POLYPORUS Berk., Hook. Jour. Bot., Vol. 6, p. 143, 1854.

A specimen ex “Daintree River, Q., Pentzcke” resembles the type, ex Sikkim, India.

scorteus, Polyporus Fr. Recorded by Cooke (Hdbk., p. 149, 1892) from New South
Wales. No specimens are at Kew from this region. The species is in any event a
synonym of Coriolus occidentalis.

scrobiculata, Trametes Berk. = Fomitopsis ochroleuca.

132. SCRUPOSUS, FOMES (FY.) G.'H. Cunn., Plant Diseases Division, Bull. 79, p. 11,
1948.

sSeruposus, Polyporus Fr., Epicrisis, p. 473, 1838.
breviporus, Polyporus Cke., Grev., Vol. 12, p. 17, 1883.

Australian collections at Kew are filed under several species. In the cover of
Fomes gilvus are “Enoggera, Q., F. M. Bailey’, ““Toowoomba, Q., Hartmann”, “Pennant
Hills, Parramatta River, N.S.W., Challenger Expedition’ and “‘Port Albert, Vic.”. Under
Fomes fomentarius is “King George Sound, W. Aus., Harris’. “Bellingen River, N.S.W.”
is filed under Polyporus fruticum. Under-Foemes holosclereus is “Endeavour River, Q.,
Persietz”’. Filed under Fomes inflexibilis is “Mt. Williams, Bellingen River, N.S.W.”.
Under Polyporus laurencii is “Gippsland, Vic., Miss Camppell’”’. The type of P. breviporus
is ex “Endeavour River, Q., Persietz’.

133. SCUTELLATA, FOMITOPSIS (Schw.) G. H. Cunn., Plant Diseases Division, Bull. 76,
p. 6, 1948.

scutellatus, Polyporus Schw., Trans. Am. Phil. Soc., Ser. II, Vol. 4, p. 157, 1832.
atro-albus, Fomes P. Henn., Monsunia, Vol. 1, p. 144, 1899.
yoshinagae, Polyporus Lloyd, Letter 54, p. 5, 1915.

\ Collections from this region, especially those from New Zealand, match part of the
type at Kew, “Bethlehem, U.S.A., ex herb. Schweinitz’. Only one specimen from
Australia is at Kew, ex “Brisbane, Q., No. 978’, filed under Fomes cryptarum.

selecta, Poria Karst. A specimen so filed, ex ““Milsom Island, Hawkesbury River,
N.S.W., J. B. Cleland, No. 17” = Poria calcicola.

semi-digitaliformis, Polyporus Berk. = Trametes protea.

134. SENEX, FOMES (Nees & Mont.) CkKe., Grev., Vol. 13, p. 118, 1885.

senex, Polyporus Nees & Mont., Ann. Sci. Nat., Ser. II, Vol. 5, p. 70, 1836.

Two collections are at Kew: “Daintree River, Q., T. Pentzcke”’ (filed under Fomes
igniarius) and “Dunk Island, Q., W. Cottrell-Dormer, No. 8”.

seriatus, Polyporus Kalch. = Trametes ferrea.

242 AUSTRALIAN POLYPORACEAE,

sericea, Hexagona Fr. Though recorded by Cooke (Hdbk., p. 164, 1892) from
Queensland, there are no specimens at Kew from Australia.
serpens, Merulius Fr. Cooke (Hdbk., p. 169, 1892) recorded the species from
Queensland, but there are no specimens from the region at Kew.
135. SERPENS, TRAMETES Fr., Hym. Hur., p. 586, 1874.
serpens, Daedalea Fr., Syst. Myc., Vol. 1, p. 340, 1821.

stephensii, Polyporus Fr. ex Berk., Outlines, p. 252, 1860.

Two collections are at Kew from Australia, “Queensland, C. Lumholtz”’ and “Pennant
Hills, Parramatta River, N.S.W., Challenger Expedition’.

136. SETIPORUS, INONOTUS (Berk.), nov. comb.

intybaceus, Polyporus Berk., Lond. Jour. Bot., Vol. 1, p. 149, 1842.
setiporus, Polyporus Berk., Lond. Jour. Bot., Vol. 6, p. 505, 1847.
cichoraceus, Polyporus Berk. ex Fr., Nov. Symb., p. 92, 1851.
tabacina, Hexagona Levy., in Zoll. Syst. Ind. Arch., p. 15, 1854.
arata, Hexagona Berk., Jour. Linn. Soc., Vol. 16, p. 438, 1877.

The species was first named P. intybaceus; but as this was preoccupied, Fries
changed the name to P. cichoraceus Berk. Meanwhile Berkeley had again named it
P. setiporus, so this becomes the valid specific name. The following collections are at
Kew, all filed under P. cichoraceus: “Trinity Bay, Q., Sayer, No. 2”, “North Queensland”,
“Brisbane, Moreton Bay, Q., Bailey’, ‘“Morunga, N.S.W., W. N. Cheesman’, “Sugarloaf
Mt., N.S.W., F.v.M.”, “Tweed River, N.S.W., Camara, No. 86”, “Clarence River, N.S.W.,
Wilcox’, ‘“‘Box Hill, Vic., Mrs. Martin, No. 446” and “Sealer’s Cove, No. 143”. The type
ot P. setiporus was from Ceylon, of P. intybaceus from Philippines, and of H. arata
from Aru Island.

137. SETULOSUS, FOMES Lloyd, Syn. gen. Fomes, p. 248, 1915.

multisetosus, Polyporus Lloyd, Myce. Notes, No. 63, p. 976, 1920, type ex Ararat,
Vic., E. J. Semmens, No. 5.
Collections from this region match part of the type at Kew ex Ceylon. They have

been filed under several species: “‘Twofold Bay, Q., White’, “Port Jackson, N.S.W.,
Hewland” and “N.Z., Colenso, No. 25” are under the cover of Fomes fomentarius; “Port
Denison, Q., Shann” and “King George Sound, W. Aus., Harris’ are under Fomes fulvus;
“Port Denison, Q., Shann” is also under Fomes igniarius; “Daintree River, Q., Pentzcke’”’
is filed under Fomes rimosus.

138. SIMILIS, HExAGONA Berk., Lond. Jour. Bot., Vol. 5, p. 4, 1846.

Two collections are filed under this cover at Kew, the type ex “‘Australia—gathered
by one of the officers of the Beagle” and ‘“Australia’’.

similis, Polyporus Berk. = Polyporus arcularius.

139. SINULOSUS, coRIOLUS (KI.), nov. comb.

sinulosa, Daedalea K1., Linnaea, Vol. 8, p. 482, 1833.
microsinulosa, Daedalea Fr., E/picrisis, p. 495, 1838.

Collections at Kew from Australia are: “Endeavour River, Q., Persietz’’ and ‘Port
Denison, Q., Shann”’.

sinuosa, Poria (Fr.) Cke. Cooke (Hdbk., p. 156, 1892) recorded the species from
New South Wales, but there are no specimens from Australia at Kéw.

sordidus, Polyporus Berk. Two collections from Australia are filed under this cover
at Kew. One, ex “Govt. Domain, Melbourne, Vic.”, is fragmentary but probably Coriolus
paleaceus; the second, ex “Queensland”, is, as Bresadola had referred it on the sheet,
a specimen of Fomes strigatus.

140. SPADICEUS, FOMES (Berk.) Cke., Grev., Vol. 14, p. 20, 1885.

spadiceus, Polyporus Berk., Ann. Nat. Hist., Vol. 18, p. 388, 1839.

The two collections at Kew agree with the type ex India. They are ex “Tropical
Queensland, Bailey” and “Strickland River, New Guinea, Bauerlen, Nos. 50, 54”. A
third collection, filed under this cover, ex “Condamine River, Q., Hartmann, No. 513”,
Cooke referred to P. substygius Berk., Bresadola to P. nilgheriensis Mont. It is of
Fomes pectinatus.

spathulatus, Favolus (Jungh.) Bres. <A collection so referred, ex “Dunk Island,
Q., W. Cottrell-Dormer, No. 41”, is a branched specimen of Polyporus grammocephalus.

BY G. H. CUNNINGHAM. 243

141. SPICULIFER, POLYPORUS CkKe., Grev., Vol. 15, p. 20, 1886.

Two collections are at Kew, both labelled type: “North Gippsland, Vic., Webb” and
“North Gippsland, Vic., H. Tisdall, No. 23”. A third, ex “N.Z., Waitaki, No. 348’, filed
under this cover, is a weathered specimen of Coriolus hirsutus.

142. SPRUCEI, CORIOLUS (Berk.), nov. comb.

sprucei, Trametes Berk., Hook. Jour. Bot., Vol. 8, p. 236, 1856.
sprucei, Daedalea Berk., Trans. Linn. Soc., Ser. II, Vol. 1, p. 402, 1879.

Four Australian collections at Kew agree with the type, ex Panure, namely, ‘‘Cape
York, Q.”’, “Endeavour River, Q., Persieh’, “Rockhampton, Q., Mrs. Thozet, No. 136”
and “Port Jackson, N.S.W., Dr. Wolls’”. The species is closely related to C. lactineus.

squamiger, Favolus Berk. = Polyporus arcularius.

sterinus, Polyporus Berk. & Curt. = Polyporus liebmannii.

stereoides, Polystictus Berk. & Curt. = Polyporus liebmannii.

stowardiu, Trametes Lloyd. = Trametes lilacino-gilwva.

strangeri, Polyporus Kalch., Proc. Linn. Soc. N.S.W., Vol. 7, p. 106, 1883. Type ex
“Riverina, N.S.W.”. No specimens are at Kew or British Museum of Natural History.
The description suggests the species was based on a specimen of P. hypomelanus or
P. infernalis.

striata, Lenzites Fr. = Daedalea trabea.

143. STRIGATUS, FOMES (Berk.) CkKe., Grev., Vol. 14, p. 20, 1885.

strigata, Trametes Berk., Lond. Jour. Bot., Vol. 6, p. 502, 1847.

cerophyllaceus, Polyporus Currey, Trans. Linn. Soc., Ser. II, Vol. 1, p. 120, 1882.
sneato-scaber, Fomes Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 59, 1883.
curreyi, Polyporus Berk. ex Cke., Grev., Vol. 15, p. 21, 1886.

The following collections from this region agree with the type ex Hautane, Ceylon,
at Kew: “North Queensland, J. E. T. Wood, No. 357”, type of F. lineato-scaber (in
British Museum of Natural History), “Trinity Bay, Q., No. 527”, “Clarence River,
N.S.W., Wilcox” and “Yambi Bay, New Caledonia, Dr. Sarasin, No. 37”.

strigoso-albus, Polyporus Lloyd = Polyporus pelliculosus.

strumosus, Polyporus Fr. Cooke (Hdbk., p. 125, 1892) recorded the species from
Victoria, but there are no specimens at Kew from this region.

stypticus, Polyporus Fr. A specimen so referred by Cooke, ex “Geographe Bay,
W. Aus.”, was filed by Miss Wakefield under Polyporus australiensis.

suaderis, Polyporus Lioyd = Fomitopsis tasmanica.

144. SUBCAPERATUS, cCORIOLUS (Lloyd), nov. comb.

subcaperatus, Polystictus Lloyd, Myc. Notes, No. 64, p. 996, 1920.

Part of the type is at Kew, ex “Sydney, N.S.W., J. B. Cleland, No. 560”.

subcongener, Daedalea Berk. = Coriolus occidentalis.

145. SUBFERRUGINEA, DAEDALEA (Berk.), nov. comb.

subferruginea, Lenzites Berk., Hook. Lond. Jour. Bot., Vol. 6, p. 134, 1854.

bifasciata, Lenzites Cke. & Mass., Grev., Vol. 21, p. 37, 1892, type ex Victoria, Mrs.
Martin, No. 995. :

adusta, Lenzites Lloyd, Myc. Notes, No. 65, p. 1072, 1921, type ex Australia, J. B.
Cleland.

The type of L. bifasciata is the only Australian collection at Kew. It matches the
type of D. subferruginea from Khasia Mts., India.

subsuleata, Daedalea Berk. & Br. Specimens ex “Goode Island, Torres Strait” =
Lenzites beckleri; those from “Sunday Island, Kermadecs, W. R. B. Oliver” = Lenzites
aspera. Both were filed under the cover of D. subsulcata.

subtenuis, Hexagona Berk. = Hexagona polygramma.

subvaporaria, Poria Cke. = Poria versipora.

146. SUBVINCTA, PORTA (Berk. & Br.) CkKe., Grev., Vol. 14, p. 109, 1886.

subvinctus, Polyporus Berk. & Br., Jour. Linn. Soc., Vol. 14, p. 54, 1875.
The type ex “Tasmania” is the only collection at Kew.

subzonalis, Polyporus Cke. = Coriolus blumei.

147. SULPHUREUS, PoLypoRUS Fr., Syst. Myc., Vol. 1, p. 357, 1821.

Three collections are at Kew from Australia: “Endeavour River, Q., Persietz,
No. 26”, “Brisbane, Q., F. M. Bailey, No. 647” and “Lake Cootharata, Q., F. M. Bailey”.

244 AUSTRALIAN POLYPORACEAE,

148. SUPERPOSITUS, POLYPORUS Berk., Jour. Linn. Soc., Vol. 13, p. 161, 1873.

The type, from “New England, Vic., Australia’, is matched by a second collection
at Kew ex “Strickland River, New Guinea”.

tabacina, Hexagona Ley. = Inonotus setiporus.

149. TABACINUS, INONOTUS (Mont.) Karst., Rev. Myc., Vol. 3, p. 19, 1881.

tabacinus, Polyporus Mont., Ann. Sci. Nat., Ser. III, Vol. 2, p. 349, 1835.

xverampelinus, Polyporus Kalch. ex Thuem., Grev., Vol. 4, p. 72, 1875, type ex
Rockhampton, Q., A. Thozet.

glabro-tabacinus, Polystictus Lloyd, Myc. Notes, No. 67, p. 1152, 1922, type ex
Hobart, Tasmania, L. Rodway, No. 1152.

rigidus, Polyporus Lloyd, Myc. Notes, No. 738, p. 1319, 1924, nomen nudum.

gilvo-rigidus, Polyporus Lloyd, Myc. Notes, No. 74, p. 1334, 1925, type ex N.Z.,
Dunedin, H. Kk. Dalrymple.

Additional collections at Kew from this region are: ‘National Park, Adelaide,
S. Aus., W. N. Cheesman, No. 97” and “New Guinea, Rev. Chalmers’, the latter being
filed under P. leprieurii Mont.

tabulaeformis, Polyporus Berk. = Coltricia schweinitzii.

150. TARDA, PORTA (Berk.) Cke., Grev., Vol. 14, p. 109, 1886.

tardus, Polyporus Berk., Lond. Jour. Bot., Vol. 4, p. 56, 1845.

The type ex “Swan River, W. Aus., Drummond, No. 130” is the only collection at
Kew from this region. One other specimen filed under this cover, ex “Melbourne, Vic.,
Reader” = Poria medulla-panis.

tasmaniae, Polyporus Berk. On the type sheet of Poria membranicincta is a
specimen labelled Polyporus tasmaniae Berk. As Bresadola had noted on the sheet,
both are synonyms of Polyporus merulius.

tasmanica, Daedalea Sace. = Trametes proted.

151. TASMANICA, FOMITOPSIS (Berk.), nov. comb.

tasmanicus, Polyporus Berk., Fl. Tas., Vol. 2, p. 254, 1860.

cuneatus, Fomes Lloyd, Syn. gen. Fomes, p. 217, 1915, type ex N.Z., Colenso, No. 369.

suaderis, Polyporus Lloyd, Myc. Notes, No. 59, p. 859, 1919, type ex Tasmania,
L. Rodway.

The type, ex “Tasmania, Archer’, is matched by the types of the synonyms
F. cuneatus and P. suaderis. These are the only collections at Kew.

tasmanicus, Polyporus Mass. = Polyporus pes-caprae.

152. TENUIS, HEXAGONA (Hook.) Fr., Epicrisis, p. 498, 1838.

tenuis, Boletws Hook., in Kuenth. Syn. Vol. 1, p. 10, 1822.
muelleri, Hexagona Berk., Jour. Linn. Soec., Vol. 13, p. 166, 1873.
Collections ex ‘Cape York, Q., Challenger Expedition” and “New England, Vic.,

Australia” (type of H. muelleri) are at Kew.

153. TENUIS, LENZITES (Berk.), nov. comb.

tenuis, Daedalea Berk., Lond. Jour. Bot., Vol. 1, p. 151, 1842.
aulacophylla, Daedalea Berk., Jour. Linn. Soc., Vol. 13, p. 166, 18738, type ex
Australia, Schomburgk, No. 58.
schomburgkii, Daedalea Berk. ex Cke., in Muell. Fragm. Phyto. Aus., Suppl. to
Vol. 11, p. 86, 1880, type ex Australia. A
The following collections are at Kew, filed under D. aulacophylla: ‘Rockhampton,

Q., Mrs. Thozet’’, “Cape York, Q.’, “Goode Island, Powell’, “Thursday Island, Bauerlen,
No. 55”, “Raoul Island, Kermadecs, H. M. Herald” and ‘Clarence River, N.S.W., Wilcox’’.
They agree with the type ex Philippines.

tenuissimus, Merulius Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 2, p. 62, 1883.
The type ex “Brisbane, Q., Bailey, No. 173” now consists of some mycelium upon a
fragment of rotting wood. It is unrecognizable.

154. TEPHROLEUCUS, POLYPORUS Fr., Syst. Myc., Vol. 1, p. 360, 1821.

Two collections are at Kew from this region: “Australia, Reader’ was filed by
Cooke under P. epileucus; “Endeavour River, Q., Pentzcke’” was placed by Cooke under
P. galactinus. A specimen ex “Tasmania, Mt. Wellington, Eaton’ placed here by
Cooke = P. tephronotus.

155. TEPHRONOTUS, POLYPORUS Berk., Fl. Tas., Vol. 2, p. 252, 1860.

angustus, Polyporus Berk., Fl. Tas., Vol. 2, p. 253, 1860.

BY G. H. CUNNINGHAM. 245

Collections at Kew are the type ex “Tasmania, Archer’, the type of P. angustus,
ex “Tasmania, Archer”, “Tasmania, Mt. Wellington, Haton”’ filed under P. tephroleucus.
One specimen ex “Clarence River, N.S.W., Wilcox” filed under P. tephronotus by
Cooke = Poria radula; a second, ex “Pennant Hills, N.S.W., Challenger Expedition” =
Polyporus campylus.

tepperi, Fomes Lloyd = Fomes scaber.

testudo, Polyporus Berk. & Br. = Fomes durus.

156. THELEPHOROIDES, POLYPORUS (Hook.) Fr., Epicrisis, p. 473, 1838.
thelephoroides, Boletus Hook., in Kuenth. Syn., Vol. 1, p. 10, 1822.
conchoides, Gloeoporus Mont., Hist. Cuba, p. 385, 1842.

The type, ex “Loxa, Peru, Humboldt, No. 222”, is excellently preserved at Kew. It
is matched by specimens of Gloeoporus conchoides later described by Montagne. Only
one collection is at Kew from Australia, ex “Moruya, N.S.W., W. N. Cheesman’’, though
there are several from New Zealand, where the species is abundant.

157. THWAITESII, HEXAGONA Berk., Am. Acad. Arts & Sci., Vol. 4, p. 122, 1860.
mirabilis, Hexagona Lloyd, Syn. gen. Hexagona, p. 37, 1910, type ex Samoa, C. G.
Lloyd.
Specimens from Australia at Kew are “Port Denison, Q., Fitzalan, Shann’,

“Toowoomba, Q@., Hartmann, No. 52’, “Soane River, Behen’, and “Bloomfield River,
Bauer”. The last was referred on the sheet by C. J. Humphrey to H. capillacea Pat.
tomentosus, Polyporus Fr. Recorded by Cooke (HdbkK., p. 137, 1892) from Victoria
and Queensland, but no specimens are at Kew from this region. ;
torrida, Lenzites Kalch. = Lenzites beckleri.

158. TRABEA, DAEDALEA (Pers.) Fr., Syst. Myc., Vol. 1, p. 335, 1821.
trabea, Lenzites Fr., Epicrisis, p. 406, 1838.
striata, Lenzites Fr., Epicrisis, p. 406, 1838.
protracta, Lenzites Er., Vet. Akad. Forh., p. 52, 1851.
clelandu, Lenzites Lloyd, Myce. Notes, No. 61, p. 887, 1919.
Collections at Kew from this region are ex “Brisbane, Q., F. M. Bailey, No. 629”,
_filed under L. striata; “Brisbane, Q., C. E. Broome”, under cover of 7. rigida Berk.
& Mont.; “Angaston, S. Aus., W. N. Cheesman, No. 4” and “N.Z., Waitaki, No. 254’, the
latter being filed under L. abietina.
tricholoma, Polyporus Mont. One collection so referred by Cooke, ex “Endeavour
River, Q., Persieh”, is a small-pored form of P. arcularius.
trizonatus, Polyporus Cke. = Coriolus zonatus. The type, ex “Upper Yarra, Vic.,
Lucas’, is a discoloured specimen of C. zonatus. On the type sheet Bresadola had
noted “Polyporus lenis Lev., a form of Polystictus occidentalis Kl., old, naked’. A
second specimen of C. zonatus on the same sheet was noted by Bresadola as “Probably
P. velutinus”’.

159. TUMULOSUS, POLYPORUS Cke. & Mass. ex Cke., Grev., Vol. 17, p..55, 1889.
The type was from “Brisbane, Q., F. M. Bailey”. Only a sclerotium is now at Kew.

160. UDUS, POLYPORUS Jungh., Ann. Sci. Nat., Ser. II, Vol. 16, p. 320, 1841.
fusco-maculatus, Polyporus Bres. & Pat., in Lloyd’s Myce. Notes, No. 6, p. 49, 1901.
Under the cover of P. rasipes are two collections of P. wdus at Kew from Australia,
ex “Daintree River, Q., Pentzcke’ and “Richmond River, N.S.W., Camara”. P. fusco-
maculatus, ex Samoa, was based on the same species.
umbrinella, Hexagona Fr. Cooke (Hdbk., p. 165, 1892) recorded the species from
Queensland, but no specimens from the region are at Kew.

161. UNDATA, PORTIA (Pers.) Bres., Ann. Myc., Vol. 1, p. 78, 1903.
undatus, Polyporus Pers., Myc. Hur., Vol. 2, p. 90, 1825.
broomei, Polyporus Rabh. ex Berk. & Br., Trans. Linn. Soc., Ser. II, Vol. 1, p. 402,
1879, type ex Brisbane, Q., No. 398, in British Museum of Natural History.
Two collections at Kew from Australia are “Daintree River, Q., Pentzcke’, filed
under Poria hyposclera; and ‘Clarence River, N.S.W., Wilcox’, which is under Poria
radula.
ungulata, Trametes Berk. = Fomitopsis ochroleuca.

246 AUSTRALIAN POLYPORACEAE,

162. UNICOLOR, LENZITES (Fr.), nov. comb.
unicolor, Daedalea Fr., Syst. Myc., Vol. 1, p. 336, 1821.
pendula, Daedalea Berk., Fl. N.Z., Vol. 2, p. 180, 1855.

Cooke (Hdbk., p. 162, 1892) recorded the species from Queensland, but there are
no specimens from Australia at Kew. Daedalea pendula, type ex “N.Z., Colenso”, is a
pendulous form.

163. VAPORARIA, PORIA (Fr.) Cke., Grev., Vol. 14, p. 111, 1886.

vaporarius, Polyporus Fr., Syst. Myc., Vol. 1, p. 382, 1821.

Collections from Australia which resemble European specimens are “Brisbane, Q.,
Bailey”, “Swan River, W. Aus., Drummond”, “Melbourne, Vic., No. 1014” (the last filed
under Fomes cryptarum), and “Tasmania, Archer’. Two collections so referred by
Cooke, ex “Moe Swamp, Gippsland, Vic.” and “Clarendon, Vic.”’, are of Poria versipora,
as are all New Zealand specimens under this cover.

varia, Trametes Lloyd = Fomitopsis ochroleuca.

varius, Polyporus Fr. Cooke (Hdbdk., p. 116, 1892) recorded the species from
Queensland, Western Australia and Tasmania. No specimens are at Kew from Australia.

vellereus, Polyporus Berk. Under this cover at Kew are two collections from
Australia, ex “Endeavour River, Q., Persieh’”’ and “Daintree River, Q., Pentzcke”’. Both
are of Coriolus pinsitus.

164. VELUTINUS, CoRIOLUS (Fr.) Quel., Ench., p. 175, 1886.
velutinus, Polyporus (Pers.) Fr., Syst. Myc., Vol. 1, p. 368, 1821.
One specimen of three from “South Australia”, filed under Polystictus versicolor,
is of this species.

165. VENUSTUS, POLYPORUS Berk., Lond. Jour. Bot., Vol. 4, p. 55, 1845.
Two collections are at Kew from Australia, the type ex “Swan River, W. Aus.,
Drummond, No. 135” and “Brisbane, Q., No. 101’.

166. VERNICIFLUUS, POLYPORUS Berk., Fl. Tas., Vol. 2, p. 254, 1860.
rasipes, Polyporus Berk., Jour. Linn. Soc., Vol. 16, p. 49, 1878.

The type is ex “Tasmania, Archer’. The type of P. rasipes ex ‘‘Admiralty Islands,
Challenger Expedition” matches this. Other collections, filed under P. rdasipes, are ex
“Trinity Bay, Q., Sayer’ and “New Guinea, Capt. Armit”. The former Bresadola
doubtfully referred to P. brasiliensis.

167. VERSATILIS, POLYPOoRUS Berk., Lond. Jour. Bot., Vol. 5, p. 150, 1846.

One collection is under this cover at Kew, ex “Nambour, Blackball Range, Q.,
W. N. Cheesman”. Two others, filed under this cover, ex “Rockhampton, Q., Mrs.
Thozet” and “Clarence River, N.S.W., Camara, No. 67”, are probably specimens of
Irpex flavus.

versicolor, Fomes P. Henn. = Fomes rimosus.

168. VERSICOLOR, CORIOLUS (Fr.) Quel., Ench., p. 175, 1886.
versicolor, Polyporus (l.) Fr., Syst. Myc., Vol. 1, p. 368, 1821.
aequus, Polystictus Lloyd, Myce. Notes, No. 62, p. 933, 1920, type ex Tasmania,
L. Rodway. “

The only specimens at Kew are “Mt. Lofty, Adelaide, S. Aus., W. N. Cheesman”,
“Tasmania” and the type of P. aequus. Others filed under this cover are of C. zonatus.
_ versicutus, Polyporus Berk. & Curt. Specimens so referred by Cooke, ex “Endeavour
River, Q., Persietz’” and ‘Daintree River, Q., Pentzcke, No. 143”, are of Coltricia
acupunctata. A third, ex “Endeavour River, Q., Persietz’”’ = Coriolus blumei.
versiformis, Trametes Berk. & Br. An Australian collection ex “Rockhampton,
Q., Mrs. Thozet, No. 131”, so referred by Cooke = Trametes protea.

169. VERSIPORA, PORIA (Pers.) Rom. Svensk Bot. Tidskr., Vol. 20, p. 15, 1926.
versiporus, Polyporus Pers., Myc. Eur., Vol. 2, p. 105, 1825.
chlorina, Poria Mass., Kew Bull. Mise. Inf., p. 95, 1906, type ex Christmas Island.
subvaporaria, Poria Cke., in herb. Kew.

The following collections from Australia are at Kew: ‘‘Toowoomba, Q., Hartmann”,
filed under Poria vulgaris; “Guntawang, N.S.W., Hamilton, No. 4’, under Poria odonto-
poria; “Moe Swamp, Gippsland, Vic.” and “Clarendon, Vic.’’, under Poria vaporaria.

BY G. H. CUNNINGHAM. 247

170. VESPACEA, HEXAGONA (Pers.) Fr., Epicrisis, p. 497, 1838.
vespaceus, Polyporus Pers., Gaud. Voy. Freye., p. 169, 1827.
inconcinna, Daedalea Berk., Lond. Jour. Bot., Vol. 1, p. 151, 1842.
albida, Hexagona Berk., Jour. Linn. Soc., Vol. 16, p. 47, 1878.
intermedia, Daedalea Berk., Jour. Linn. Soc., Vol. 18, p. 385, 1881.
favoloides, Hexagona Cke., Grev., Vol. 14, p. 118, 1886.
cookei, Hexagona Sace., Syll. Fung., Vol. 6, p. 363, 1888.

Collections at Kew from this region are: “Malamon Island, Challenger Expedition’,
type of H. albida; “New Guinea, Hollrung”’’, filed under H. albida; “Strickland River,
New Guinea, Bauerlen, No. 20”, type of H. favoloides which Saccardo changed to
H. cookei because the name was preoccupied; and “Australia”, type of Daedalea
intermedia.

vesparius, Polyporus Berk. = Hexagona gunnii.

171. VICTORIAE, PORIA (Berk.) CkKe., Grev., Vol. 14, p. 111, 1886.

victoriae, Polyporus Berk. ex Cke., Grev., Vol. 10, p. 103, 1882.

Three collections are at Kew, the type ex “Victoria, F.v.M.’’, “Daintree River, Q.,
Pentzcke” and “Hawkesbury River, N.S.W., J. B. Cleland’. Others under this cover
from Australia are of Poria calcicola.

172. VICTORIENSIS, POLYPORUS Lloyd, Myc. Notes, No. 65, p. 1095, 1921.
Part of the type is at Kew, ex “South Australia, J. B. Cleland’’.
vinctus, Polyporus Berk. = Poria radula.

173. vVINOSUS, POLYPORUS Berk., Ann. Mag. Nat. Hist., Ser. IX, Vol. 2, p. 195, 1852.

A collection ex “Strickland River, New Guinea, Capt. Armit” agrees with the type
ex St. Domingo.

violacea, Poria (Fr.) Cke. A specimen so labelled by Cooke, ex “Victoria, No.
1029” = Poria spissa.

174. vVIRGATUS, POLYPORUS Berk. & Curt., Jour. Linn. Soc., Vol. 10, p. 304, 1869.

novo-guineensis, Polyporus P. Henn., in Schum. & Holir., Flora von Kais. Wilh. Land,
p. 6, 1889.
The type of P. novo-guineensis matches specimens of P. virgatus at Kew from the

type locality, Cuba.

5s) VULGARIS, PORTA (HY) Gray, Nat. Arr. Brit. Pl, Vol. 2; p. 639, 1821. One
specimen ex “Richmond River, N.S.W.” is probably of this species, though I was not
able to verify this by examination under the microscope. A collection ex ““Toowoomba,
Q., Hartmann” so referred by Cooke = Poria versipora.

176. WAKEFIELDIAE, PORIA Rodw. & Clel., Papers & Proc. Roy. Soc. Tas. for 1929,
p. 85, 1929.
Part of the type is at Kew, ex “Sydney,N.S.W., J. B. Cleland, No. 20”.

177. WEBERIANA, COLTRICIA (Bres. & P. Henn.), nov. comb.
weberianus, Polyporus Bres. & P. Henn., in litt.
weberianus, Fomes Sace., Syll. Fung., Vol. 9, p. 174, 1891.
A specimen so named by Bresadola, ex “Brisbane, Q., C. E. Broome’, is under the
cover of P. fruticum at Kew. A second, ex ‘Trinity Bay, Q., Sayer, No. 40”, is filed
nnder Fomes salicinus.

178. WIGHTII, HExAGoNA (KI1.) Fr., Nov. Symb., p. 100, 1851.

wightii, Polyporus K1., Linnaea, Vol. 7, p. 200, 1832. -
wrightii, Hexagona (Kl.) Fr., Epicrisis, p. 496, 1838.

The following Australian collections are at Kew: “Port Denison, Q., Fitzalan,
Shann”, “Toowoomba, Q., Hartmann, No. 52’, “Blomfield River, Bauer” and “Soane
River, Behen”’’.

wilsonianus, Polyporus Lloyd = Polyporus campylus.

179. XANTHOPUS, CORIOLUS (FY.), nov. comb.

xzanthopus, Polyporus Fr., Syst. Myc., Vol. 1, p. 350, 1821.
cupreo-nitens, Polyporus Kalch. ex Thuem., Myce. Univ., No. 1702, 1881.

Collections at Kew from this region are: ‘Goode Island, Q., W. Powell’, ‘‘Cape
York, Q., Challenger Expedition”, “Brisbane, Q.”’, ‘“Rockhampton, Q., Mrs. Thozet’,
“Dunk Island, Q., W. Cottrell-Dormer’’, “North Queensland”, “Endeavour River, Q..

248 AUSTRALIAN POLYPORACEAE,

Persietz’, “Trinity Bay, Q., Karsten’, “Upper Daintree River, Q., Harris, Pentzcke’,
“Port Denison, Q., Fitzalan”, “Clarence River, N.S.W., Dr. Beckler’, “Tweed River,
N.S.W., Camara’, “Mossman River, Barnard’, “Melbourne, Vic.” (type of P. cupreo-
nitens), “New Guinea, Capt. Armit, No. 2’, “Solomon Islands, Dr. Guppy”, “Fly River,
New Guinea, Everett’s Expedition, Bauerlen”’.

rerampelinus, Polyporus Kalch. = Inonotus tabacinus.
verophyllaceus, Polyporus Currey = Fomes strigatus.

180. xEROPHYLLUS, POLYPORUS Berk., Fl. N.Z., Vol. 2, p. 178, 1855.

Specimens at Kew which match the type, ex “N.Z., Hooker herb.”’, are “N.Z.,
Colenso, No. 532”, filed under P. rudis; and “New Guinea, Strickland River, Bauerlen’”’,
which is under the cover of P. russiceps.

181. ZEALANDICUS, FOMES Cke., Grev., Vol. 14, p. 18, 1885.
zealandicus, Polyporus Cke., Grev., Vol. 8, p. 55, 1879.
hamatus, Fomes (Corner) Imaz., Jour. Jap. Bot., Vol. 16, p. 586, 1940.

The type, consisting of three specimens, “N.Z., Coromandel, Dr. Berggren, Nos. 309,
310” and an additional collection ex “Sunday Island, Kermadecs, W. R. B. Oliver”,
are at Kew.

zelandicus, Polyporus Cke. = Polyporus berkeleyi.
182. ZONALIS, POLYPORUS Berk., Ann. Nat. Hist., Vol. 10, p. 375, 1842.

Collections at Kew from this Ree lon are: “Near Trinity Bay, Q., W. A. Sayer’,
“Buderim Mts., Q., C. T. White’’, “Brisbane, Q.” (the last is filed under P. luteo-nitidus),
“Strickland River, New Guinea’ and “New Guinea, Armit’’.

183. ZONATUS, CORIOLUS (Fr.) Quel., Ench., p. 175, 1886.
zonatus, Polyporus Fr., Syst. Myc., Vol. 1, p. 368, 1821.
bireflexus, Polyporus Berk. & Br. ex Cke., Grev., Vol. 10, p. 101, 1882.
rufo-rugosus, Polyporus Lloyd, Myc. Notes, No. 78, p. 1331, 1924.
orbiculata, Polyporus Col., in herb. Kew.
trizonatus, Polyporus CkKe., Grev., Vol. 12, p. 17, 1883.

The following collections from this region are at Kew: “Brisbane, Q., No. 172”
(type of P. bireflexus), “Illawarra, N.S.W., Camara” (under P. elongatus), ‘““Nymitiballe,
Bauerlen, No. 246” and “Upper Yarra, Vic., Lucas” (both under P. trizonatus), “Tas-
mania, L. Rodway” (type ot P. rufo-rugosus), ““N.Z., Colenso, No. 1052” (filed under
Polystictus versicolor and in the herbarium labelled P. orbiculata), ‘‘N.Z., Colenso,
Nos. 837, 838” and “N.Z., T. Kirk, No. 88” (filed under P. versicolor).

References.
BerKELEY, M. J., 1855.—Fungi, in J. D. Hooker’s The Botany of the Antarctic Voyage. II.
Flora Novae-Zealandiae, part ii: 172-210.
, 1860a.—Fungi, in J. D. Hooker’s The Botany of the Antarctic Voyage. III. Flora
Tasmaniae, vol. ii: 241-282.
, 1860b.—Outlines of British Fungology.
' Bourpot, H., and GAuzIn, A., 1928.—Hymenomycetes de France.
CLELAND, J. B., 1935.—Toadstools and Mushrooms and other Larger Fungi of South Australia,
Part Il. Adelaide: Government Printer.
Cooke, M. C., 1892.—Handbook of Australian Fungi.
CUNNINGHAM, G. H., 1947.—New Zealand Polyporaceae. 1. The genus Poria. Dept. Sci. Ind.
Res. Plant Diseases Bull. 72, as cited in text.

, 1948a.—New Zealand Polyporaceae. 2. The genus Fuscoporia. Ibid., 73.

, 1948b.—New Zealand Polyporaceae. 3. The genus Polyporus. Ibid., 7
————, 1948c.—New Zealand Polyporaceae. 4. The genus Corioluws. Ibid., 75.
———., 1948d.— New Zealand Polyporaceae. 5. The genus Fomitopsis. Ibid., 76.

1948e.—New Zealand Polyporaceae. 6. The genus Coltricia. Ibid., 7
—————, 1948f.—New Zealand Polyporaceae. 7. The genus Jnonotus. Ibid., 7
19489.—New Zealand Polyporaceae. 8. The genus Fomes. Ibid., 79.

, 1948h.—New Zealand Polyporaceae. 9. Trametes, Lenzites and Daedalea. Ibid., 80.
Frins, E. M., 1821.—Systema Mycologicum, vol. i.

, 1828.—Elenchus fungorum, vols. i and ii.

, 1838.—Epicrisis systematis mycologici seu synopsis Hymenomycetum.
—————,, 1847.—-Fungi, in Plantae Preissianae, vol. ii.

BY G. H. CUNNINGHAM. 249

Fries, ©. M., 1851.—Novae Symbolae Mycologicae, in Nova Acta Soc. Sci. Upsala, Ser. III,
vol. i: 17-136.
, 1874.—Hymenomycetes europaei.
GILLET, C. C., 1878.—Champignons qui croissent en France.
Luoyp, C. G., 1910.—Synopsis of the Genus Hexagona. The Mycological Writings of C. G.
Lloyd, vol. 3 (Hex.).
, 1915.—Synopsis of the Genus Fomes. Ibid., vol. 4 (Fom.) : 209-288.
, 1915.—Synopsis of the Section Apws of the Genus Polyporus. Ibid., vol. 4 (Ap.):
289-392.
MONTAIGNE, J. P., 1842.—Histoire physique, politique et naturelle de l’ile de Cuba.
Murriuu, W. A., 1907-08.—Polyporaceae, in North American Flora, vol. 9, parts 1-2:1-132.
PATOUILLARD, N., 1900.—EHssai taxonomique sur les familles et les genres des Hymenomycetes.
Persoon, C. H., 1825.—Mycologia europaea, vol. ii.
QUELET, L., 1886.—Enchiridion fungorum.
, 1888.—Flore mycologique de la France.

ron

a

Proc. Linn. Soc. N.S.W., 1950. PLATE y.

Cytological Studies in the Myrtaceae.

ee ey)
fees Ah

Proc. Linn. Soc. N.S.W., 1950. PLATE VI.

‘Al ° eatin
ok “Ss
en)
a ~
oes Se
ne oe

FES nee

i
i

Cytological Studies in the Myrtaceae. III.

a

Proc. Linn. Soc. N.S.W., 1950.

PLATE VII.

Australian Species

of Lagenophora.

Proc. Linn. Soc. N.S.W., 1950. PLATE VIII.

Immature Stages of Aphodius howitti Hope.

251.

FURTHER NOTES ON EXPERIMENTAL CROSSING WITHIN THE AEDES
SCUTELLARIS GROUP OF SPEHECIHS (DIPTERA, CULICIDAB).

By A. R. WooDHILL,
Pd Department of Zoology, University of Sydney.

[Read 27th September, 1950.]

INTRODUCTION.

In a previous paper (Woodhill, 1949) an account was given of experimental crossing
between Aédes scutellaris scutellaris Walker and Aédes scutellaris katherinensis
Woodhill, and in this it was shown that the cross was fertile using kKatherinensis males
and scutellaris females, but that the reciprocal cross was sterile. In the present paper
a description is given of back crosses with the original parents and also of crosses
between other species of the scutellaris group. The distribution of the species used is
given in the map accompanying the previous paper, and the breeding and crossing
procedure is similar to that previously described.

CROSSING EXPERIMENTS.

TABLE 1.
Results of Crossing F, Hybrids (produced from Male Katherinensis x Female Scutellaris) with Both Parents.
|
Crosses, Numbers and Sexes. Approximate
Number Period of Egg Percentage
of Eggs Production. Eggs
Hybrid. scutellaris. | katherinensis. Produced. Hatched.
|
|
1376S 10799 1220 5/12/49-12/12/49 0
10699 11839 | 480 | 4 a 100
135¢3 9729 | 1150 21/12/49-27/12/49 100
9722 135¢5 650 Fy 3 100

It will be seen that the only sterile cross was that of hybrid males crossed with
katherinensis females and that in each-case where hybrid males were used the egg
production was considerably greater than where males of the original parents were
used. In all the above crosses copulation was frequent and samples of the females
showed living spermatozoa in the spermathecae. Larvae resulting from the fertile eges
were bred through to the adult stage and appeared quite normal. No further information
has been obtained as to a possible explanation of the sterility occurring in these crosses..

In all the crosses shown in Table 2 copulation was frequently observed and living
spermatozoa were present in the spermathecae of the females, but nevertheless all the
crosses were completely sterile.

DISCUSSION.

In the back crossing experiments the only sterile cross occurred when katherinensis
Was used as the female parent, and it will be remembered that in the original crosses
between scutellaris and katherinensis (Woodhill, 1949) the Katherinensis females gave
sterile eggs. There is obviously some factor associated with katherinensis females
which renders them sterile unless crossed with males of their own subspecies. Two
Similar cases occurring in mosquito crosses are now known, i.e. Downs and Baker
(1949), who crossed Aédes aegypti with Aédes albopictus, and Perry (1949), who crossed
Aédes scutellaris scutellaris (= A. hebrideus) with Aédes pernotatus. Both these authors
found that one cross was fertile, whereas the reciprocal cross gave sterile eggs, and

ay

252 EXPERIMENTAL CROSSING WITHIN AEDES SCUTELLARIS GROUP,

it would appear that this phenomenon is peculiar to closely related forms of mosquitoes.
Partial sterility of this type is recorded in other groups of animals, but in these cases
the sterility occurs in the F, adult hybrids, whereas in the experiments mentioned above
no development of the embryo occurs in the F, eggs. With reference to Table 2, it is
clear that Aédes pseudoscutellaris Theobald and Aédes scutellaris scutellaris Walker are
completely isolated genetically, and this confirms the opinion of Farner and Bohart
(1945), who accorded specific status to these forms on morphological and geographic
evidence. As was to be expected, Aédes scutellaris katherinensis Woodhill was com-
pletely sterile when crossed with A. pseudoscutellaris. Perry (see above) concludes as a

TABLE 2.
Results of Crossing A. pseudoscutellaris Theobald with A. scutellaris scutellaris Walker and A. scutellaris katherinensis
; Woodhill.
|
Crosses, Numbers and Sexes. |
Approximate | | Percentage
| Number of | Period of Egg Eggs
pseudo- | Eggs Production. Hatched.
scutellaris. scutellaris. | katherinensis. Produced.
|
158g. 10529 2400 11/7/49-25/ 7/49 0
9235 4999 260 | 3/6/49-20/ 6/49 0
11399 14499 1090 11/7/49-25/ 7/49 0
6329 | 995d 120 3/6/49--20/ 6/49 0
164g 17029 4700 2/9/49-16/ 9/49 } 0
2000S 13529 2700 30/9/49-14/10/49 0
9999 | 635d 790 2/9/49-16/ 9/49 0
9329, 125d'¢ 1100 30/9/49-14/10/49 0

result of his work that A. scutellaris scutellaris and A. pernotatus should be regarded
as distinct species owing to “théir ability to hybridize only with extreme difficulty under
laboratory conditions”. It should, however, be pointed out that only small numbers of
individuals, up to 16, were used in his experiments. The author found the same
difficulty with small numbers of scutellaris and katherinensis, but when 60 to 100
individuals of both sexes were used, large numbers of hybrids were readily obtained.
It could be argued, therefore, that the question as to whether pernotatus should be
regarded as a subspecies of scutellaris or as a distinct species (on the grounds of
genetical isolation) should be left open until more comprehensive crossing experiments
are carried out. On the other hand, as Perry points out, the two forms differ markedly
in their feeding habits, 4nd no intermediate forms have been taken in the field in areas
where the two forms occur together.
SUMMARY.

(1) Back crosses of F, hybrids (produced from male katherinensis x female
scutellaris ) were made: with both parents. All the crosses were fertile with the
exception of hybrid males crossed with katherinensis females. In this cross all egg
were completely sterile.

(2) A. pseudoscutellaris Theobald was crossed with A. scutellaris scutellaris Walker
and with A. scutellaris katherinensis Woodhill, and in all cases the eggs were completely
sterile.

(3) The bearing of these experiments on problems of speciation in the Aédes
scutellaris group of species is discussed.

ACKNOWLEDGEMENTS.

The author wishes to acknowledge with gratitude the assistance given by Miss M. Besly,
Miss M. Morris and Mr. J. R. Henry, who acted as blood donors and submitted to considerable
irritation on many occasions.

BY A. R. WOODHILL. 253

References.

Downs, W. G., and BAksrR, R. H., 1949.—Experiments in Crossing Aédes (Stegomyia) aegypti
Linnaeus and Aédes (Stegomyia) albopictus Skuse. Science, 109, No. 2826, 200-201.
FarNeR, D. S., and BoHart, R. M., 1945.—A Preliminary Revision of the Scutellaris Group.

U.S. Naval Medical Bull., 44 (1) : 21-37.

Prrry, W. J., 1950.—Biological and Crossbreeding Studies on Aédes hebridews and Aédes
pernotatus. Ann. Hunt. Soc. Amer., 43 (1) :1238-136.

STONE, A., and FARNER, D. S., 1945.—Further Notes on the Aédes scutellaris Group. Proc. Biol.
Soc. Washington, 58: 155-162.

WoOoDHILL, A. R., 1949.—A Note on Experimental Crossing of Aédes (Stegomyia) scutellaris
seutellaris Walker and Aédes (Stegomyia) scutellaris katherinensis Woodhill (Diptera,
Culicidae). Proc. LINN. Soc. N.S.W., 74: 224-226.

THE HAIR TRACTS IN MARSUPIALS.
PART V. A CONTRIBUTION ON CAUSATION.
By W. BoarpMAN,

Department of Zoology, University of Melbourne.

[Read 27th September, 1950.]

Contents.

Page
Statement of the Problem oH Beas Sea Seed wpaete.) aude eve Manet Ce da ee ane oy
Species Constancy of Definitive Teer ations 3 Bid eoino se a)
Species Constancy of Pattern Development A csc Sa ee eee ets CMa rene th bs Pa

The Concept of Primary and Secondary Hair Conneinatinn
The Evidence for Primary Status of Radial Field Centres: 255
(a) Genetics of Radial Fields 256
(b) Meristic Repetition of Centres 256
The Secondary Status of Intervals 25%
The Orientation of the Follicle : es 257
The Divergent Centre Considered as a Gaoutin Genre he 259
Relationship of Divergent and Convergent Centres to the BrOcess of. een Gmanin 260
These eltsConsidered: asa MosaicroreLolarized SElain Held Sii-ye cite cies eee rst mE Gy)
Interchangeability of Divergent and Convergent Centres Pe ies ME rele WN ore Deito. cic. Rea
IDYRCOUGIOM 506 s6 60°) da “os og ‘si (G6 G5) geod ABSes vibe (oe wba, 95 do 152 BO
Summary Se pagel Ve RE ae Se Ba oa NE et cece ie cave) UA) Pte rece ws ew
Acknowledgements aN CME Cae ee eae ee RN eer Vn Ree aN d Mtn charts ou GG oo CARE
IRCLCrENICES® bea) eke hse Age SAS NDR ag We LE Seger ses ce TOin Le MaRS], ACE iMeagisy, Werstrci mt eam a AR

In Part IV (Proc. Linn. Soc., 1950, 75:89-95) of this work variations in expression
ot the centre of the radial field were considered. Convergent and divergent centres
are now diseussed in relationship to the development of definitive tract pattern and
through them an approach is made to the problem of causation.

STATEMENT OF THE PROBLEM.

Authors are in general agreement on what constitutes the primitive hair arrange-
ment in the mammalian skin. The primitive condition may be defined as “craniocaudal
in the dorsal half, more or less, of head, neck and trunk, and for the rest, caudoventral
in varying degree; on the limbs postaxially and distally” (Boardman, 19460). None of
the marsupials examined in the course of this work shows an unmodified primitive
picture, though some of the phascogales and lower phalangers make a close approach
to it. Regional alteration of major or minor extent in the direction of the hair shaft
as found in the primitive condition gives rise to hair tracts such as have been described
for many marsupials and other mammals.

No evidence has been forthcoming to justify doubt that tract pattern is species
constant except for abnormalities of expression, and, once laid down, persists unchanged
throughout life (wv. infra). Constancy of pattern form implies that there is regularity
in the direction of flow of hair within the tracts and that this regularity must be the
outcome of a precise placing of the individual hair shaft in ontogeny. All attempts at
explanation of regional differentiation in the direction taken by the growing hair have
been and must continue to be a search for forces within the body which act to confer
this orientation on the follicle. Any theory of causation must suggest explanation for
the primitive condition and the derivation from it of the: most complicated pattern,
together with the varying degrees of pattern complexity which examination of this
marsupial material has shown to exist between these extremes. The problem is essentially
concerned with conditions in the skin at the time of the laying down of the follicle
anlagen, which, from their earliest appearance exhibit a definitive orientation (Landauer,
1925a, has summarized the literature bearing on this point).

BY W. BOARDMAN. 255

SPECIES CONSTANCY OF DEFINITIVE TRACT PATTERN.

As early as 1837 Eschricht* expressed the belief that tract pattern was constant
for the species, that different species may have different patterns, and that the pattern
peculiar to a species was laid down in embryonic development and remained unaltered
throughout life. Evidence of the soundness of these conclusions has steadily accumulated,
particularly through the work of Schwalbe (1910, 1911) on the Anthropoidea, de Beaux
(1917, 1918, 1924a, 1924b, 1927), on Anthropoidea, Carnivora and other groups, and of
Niedoba (1917) on an extensive series of domestic animals. Further confirmation is
provided by the large collection of marsupial pouch young examined in the course of
the present work. Of a total of about one hundred and sixty known species of
marsupials the hair tracts of fifty-three have been recorded, thirty-seven being described
from series of two or more specimens. No grounds exist anywhere in this collection
for doubting Eschricht’s views on pattern constancy nor can any evidence be adduced
_that pattern changes with age over the range of size presented by the collection.

SPECIES CONSTANCY OF PATTERN DEVELOPMENT.

A considerable literature exists relative to the ontogeny of the mammalian coat.
To Voigt (1857) and Ludwig (1921) we owe details for man, Schwalbe (1910, 1911)
for anthropoids other than man, Dry (1926) for the mouse Mus musculus, Fraser (1928)
for the white rat, Colin (1943) for the guinea-pig, Toldt (1907)+ for Vulpes vulpes,
Gibbs (1938) for Trichosurus vulpecula, and Landauer (1925a) for Didelphys virginiana.
Schonherr (1937) has provided a detailed account of hair succession in the region of a
divergent whorl. It is not necessary to review this work in detail. Wherever develop-
ment of the mammalian coat has been investigated the sequence of events leading to
its formation has been found to be constant for the species and always involves the
development of hair in small localized areas which gradually increase in extent until
the primary coat is laid down. In the absence of negative findings the body of evidence
available seems sufficiently substantial to accept the species constancy of pattern
development as a generalization for the mammalia.

THE CONCEPT OF PRIMARY AND SECONDARY HAIR CONFIGURATIONS.

Voigt (1857) charted the hair tracts of man as a series of fields each of which has
hairs radiating from a divergent centre. He considered that the slope of the hair and
the direction in which it pointed were the outcome of regional differences between the
rate of growth of the epidermis relative to the dermis. The divergent centres situated
within the constituent fields of the pelage were held to be focal points of the growth
forces concerned, and these centres he regarded as primary. All other hair configura-
tions were classified as secondary in the sense that their occurrence at the margin of
the fields indicated that they were brought into existence by forces operating within
the fields. |

Any approach to the question of causation must be preceded by an evaluation of
the status of the peculiar configurations of hairs (centres, intervals, ridges and partings)
associated with tract formation. What are the primary features of the pelt concerned
with tract formation and what are the consequential effects? Voigt’s classification arose
from his theory that differential tensions in the skin layers were causal in tract
formation. It is profitable to approach this question in the first instance independently
of notions of causal factors. If a group of hair configurations is in fact dependent for
expression on the presence of another group, then the latter will display attributes—
Mendelian inheritance and meristic repetition, for example—not found in the former.
In what follows evidence is presented to demonstrate that divergent and convergent
centres together constitute a primary group and that all other hair configurations are
secondary.

* Information from Voigt (1857).
y+ Information from Schwalbe (1911).

256 HAIR TRACTS IN MARSUPIALS. V,

THE EVIDENCE FOR PRIMARY STATUS OF RADIAL FIELD CENTRES.
(a) Genetics of Radial Fields.

The genetics of radial fields in mammals, including man, has received considerable
attention, but no work of this kind has been done for marsupials. The particular
section of the literature relevant to the present purpose is that in which the alternative
conditions of presence or absence of radial fields has been determined as due to gene
substitution.

Craft and Warner (1934) report that in swine whorls which occur over the loin
and rump are hereditary. Their data “support the hypothesis that the swirl is due to
the interaction of two dominant complementary genes which may be present in either
a homozygous or heterozygous combination to produce the swirl’. Gates (1946) in
resuming this work comments that a single factor with low penetrance would equally
well explain the results.

The most complete analysis is provided by the work ,on the genetics of rosette
pattern in the guinea-pig. The divergent centres (rosettes) characteristic of the rough
coat are the outcome of a single gene substitution which, alone, produces reversal of
hair direction on the hind toes; the extent of rosette formation is progressively increased
by the presence of modifiers (Castle, 1925; Sewall Wright, 1935).

The cases given above could be added to considerably (see, for instance, Landauer,
1926, 1929).

No direct evidence from breeding experiments can be brought forward to support
the contention that convergent centres are genetically determined in the same way
as divergent centres. It is of interest that while geneticists working on the guinea-pig’s
coat have been preoccupied with divergent centres examination of a rough coat shows
that both divergent and convergent centres may be involved in production of the
roughness. That convergent centres are primary characters is suggested by the alterna-
tives of presence or absence observed for some of them. Thus, within the genus
Perameles all species examined, with the exception of P. gunnii, possess a convergence
on the elbow. Further, in a series of Perameles sp. from Western Australia (Part III)
containing three specimens, one member has the normally occurring elbow whorl
absent. A case in the same category is provided in the occurrence of a convergent
whorl on the cheek in one of the series of Trichosurus vulpecula (Part II, Fig. 19,
Boardman, 1946a). The sporadic occurrence of the mid-dorsal convergent centre in, man
may also be cited (Wood Jones, 1927, 1934).

(b) Meristic Repetition of Centres.

Within the limits of the species alternative conditions are found for the focus of
some radial fields; the centre may be single or double or, sometimes, multiple. The
point may be illustrated by an extreme case, the mid-dorsal whorl of Phascolarctos,
which, while most commonly single and median, is often bilaterally doubled and
occasionally subdivided into as many as six separate centres collectively having the
same general effect as the single median whorl. It is difficult to visualize these
alternative conditions as other than an innate capacity of the field’s centre to undergo
duplication. Such a property could only be consonant with classification as a primary
constituent of the pelt. Part IV of this series (Boardman, 1950a) deals at length with
meristic repetition of the divergent centre in the Marsupialia, including a detailed
account of the phenomenon in Phascolarctos cited above. The type of variation is shown
to be one of common occurrence.

In the case of the convergent centre evidence of duplication as an alternative
condition for the species is not so ample, being restricted to two cases—the chin
convergence of Phascolarctos (Part II, Fig. 26) and the mid-ventral (umbilical) con-
vergence in Trichosurus vulpecula hypoleucus (Part III, p. 194).

Supernumerary whorls, both divergent and convergent, have been recorded in
marsupials. Reference to them is made in Part IV (Boardman, 1950a). The phenomenon
is readily separable from that of duplication of the centres of radial fields discussed above,
where additional whorls always lead to a condition that is symmetrical with respect

BY W. BOARDMAN. 257

to the longitudinal median line of the dorsal or ventral surface and are demonstrably
derived from single median structures. Supernumerary whorls, on the other hand, are
sporadically distributed in the pelt and show, no relationship to existing normally
occurring centres. ;

THE SECONDARY STATUS OF INTERVALS.

Convergent and divergent intervals, usually referred to as “‘kreuze” in the German
literature, constitute the principal hair configuration classified as secondary. The
conditions for their formation are provided by currents from two divergent centres
meeting and diverging towards two oppositely situated convergent centres or their
equivalent. By equivalent is meant the extremities of the ears, limbs, and tail which
act in the skin as though physiologically similar to the more precisely defined convergent ~
centre. Examples of the method of formation are seen, for example, in Dasyurinus
geoffrou, Sarcophilus harristi, Perameles gunnii, and Trichosurus vulpecula (Part II,
Figs. 5, 8, 13 and 20 respectively, Boardman, 1946a). The terms ‘convergent’ and
“divergent” as applied to the interval have no significance except as a convenience
in description.

It is necessary to evaluate these intervals by the same criteria as have been used
above to determine the status of the radial field. There is no genetic evidence that
they are other than consequential effects of the presence of divergent and convergent
centres. The facies of an interval is predictable by a consideration of the conditions in
the surrounding fields. Intervals are not known to present alternative single or duplex
expressions and the occurrence of an abnormal interval cropping up in the manner of
a supernumerary whorl is unknown. The intervals satisfy the requirements of a
feature of secondary status.

THE ORIENTATION OF THE FOLLICLE.

It is a well-established fact that the slope of the developing hair shaft with
reference to the skin surface is apparent from the earliest stages of the hair anlagen
(see Landauer, 1925a, for references). Additionally, it will be self-evident from a
consideration of the regularity in direction of the hair streams composing a pattern
of tracts that the individual hair will also emerge in a determined plane lying at right
angles to the skin surface. In other words, the direction taken by the growing hair
in any specified part of the body is a constant for the species.

Voigt (1857), working principally on human material, analysed the relationships
of hairs in the divergent radial field and commented on their arrangement in lines
radiating from the centre. Ludwig (1921), working on human material and a series
of domestic animals, confirmed this finding and extended it by the statement that the
lines always ran between a divergent and a convergent centre. That placing of hairs
in rows is general in the Mammalia is implicit in the well-known work of De Meijere
(1894) on hair arrangement. The truth of the generalization that the flow of hair is
from divergent centre to convergent centre (or its equivalent) certainly holds for the
Marsupialia, as evidenced by examination of the extensive collection on which this
work rests. A search for exceptions in published accounts of the hair tracts of
members of other orders of the Mammalia has yielded negative results.

The neglect of Ludwig’s important generalization is probably attributable to the
manner in which the arrangement of follicles in rows is distorted in some animals
(the sheep, for example) by the simultaneous development of several generations of
follicles with consequent crowding so that the lines are difficult if not impossible to see.
In the phalanger, Trichosurus vulpecula, the primary hairs cover the whole body before
the secondary and tertiary hairs break through (Gibbs, 1938). The primary coat is
therefore presented in its simplest form. The species, moreover, has a moderately
complicated tract pattern which, combined with the peculiarities of the hair succession,
makes it particularly valuable for study in this context. The hair tracts of Trichosurus
vulpecula are well known (Wood Jones, 1920; Boardman, 1946a, 1949). Gibbs (1938)
has given an account of hair succession in the building up of the coat. The pattern
of the tracts is determined by the disposition of a series of divergent centres in

258 HAIR TRACTS IN MARSUPIALS. V,

association with a group of convergent centres or their equivalent. The divergent
centres are situated on each side at the hind margin of the rhinarium, medially on
the crown, at the medial canthus, on the chin, and in the axilla; the preauricular
reversal presumably originates in a centre within the external ear, but its position
could not be defined. Convergent centres occur on the mid-ventral line caudal of the
interramal papilla, at about the middle of the abdomen (the umbilical convergence),
at the cloacal hillock, and in the male associated with the base of the scrotum; a
poorly defined convergent point is present just below and in front of the angle of the
mouth. Occasionally a convergent centre is developed on the cheek (Part II, Fig. 19,
Boardman, 1946a). The extremities of the limbs, tail and ears have, as explained
above, the equivalence of convergent points in that hairs are directed towards them,

The account that follows is based on a series of pouch young not previously
recorded. Only four specimens of the series are relevant and these are presented in
order of extent of hair development. Th crown-rump length is given and the damp
weight, the latter appearing to be a much more accurate basis for size comparison.

Specimen I. A male (crown-rump length 35 mm., weight 2:9 gm.), Canberra, Australian
Capital Territory; coll. R. N. Wardle, May, 1943.

The specimen is at an early stage of coat development, not all of the body surface
being yet clothed with the first generation hairs. Only a few vibrissae at the caudal
margin of the mystacial zone have come through. Delimitation of the fields can be
made accurately, as the hairs in general are arranged in rows which can be traced
from divergent centre to convergent centre or equivalent. The region surrounding
the central point of the occipital divergent radial field is still hairless, but an inner
zone of shorter shafts, as shown by comparison -with the next member of the series,
indicates that the wave of growing hairs is progressing towards the centre. This
phenomenon of initiation of hair growth on a circle surrounding the centre from which
growth then proceeds both away from and towards the centre is apparently general
in Trichosurus, as all divergent centres show it in some degree. The bilaterally paired
whorls at the margin of the rhinarium, to the presence of which the rhinal reversal
is due (Part II, Fig. 19, Boardman, 1946a) appear not to be active at this stage, so that
their territory on the dorsal and lateral aspects of the snout is naked. The field
associated with the canthal divergent centre which meets that of the rhinal centre in a
divergent interval is formed anterior to the canthus as though the rhinal field were
there. Apparently the skin is organized to conform with the definitive pattern before
the development of the hairs.

The front of developing hairs from the occipital and axillary whorls which enters
into the composition of the mid-dorsal (umbilical) convergence has not quite reached
its central point. There are no indications that the centre is other than passive at this
stage of coat formation. The same condition obtains for the interramal convergent
point into whose formation enter hairs from all the divergent centres of the head,
including the occipital whorl. i

A mid-ventral longitudinal strip of skin apparently coincident with the site of the
sternal gland and the medial and lateral surfaces of the ear are naked.

Specimen II. A headless female; Canberra, Australian Capital Territory; coll. W.
Boardman, 25th November, 1943.

The second specimen is at about the same stage of hair development as the first
and shows no differences from it.

Specimen III. A male (crown-rump length 40 mm., weight 3-2 gm.); Canberra,
Australian Capital Territory; coll. W. Eldridge, 5th June, 1944.

This example, somewhat larger than the first, shows the development of hairs at
a later stage. The mystacial vibrissae are well developed. The rhinal centres are

BY W. BOARDMAN. 259

active, but a small zone on the rhinal side of the future divergent interval in front
of the eye is still naked or at most the hairs are just breaking the skin surface. The
growth wave from the dorsal and axillary whorls has reached the mid-ventral convergent
point. Only the scrotum is without hair at the hinder end of the body. The medial
naked patch on the ventral thorax presumed related to the sternal gland is still
present and the ear both laterally and medially remains naked.

Specimen IV. A female (crown-rump length 45 mm., weight 10-7 gm.); Canberra,
Australian Capital Territory; coll. W. Boardman, 16th October, 1944.

Hair development shows a considerable advance on the previous members of the
series. The medial naked patch on the ventral thorax remains and the ear is hairless
both laterally and medially.

The arrangement of hair around the interramal convergent centre of this specimen
shows very clearly the manner in which hair streams from separate sources entering
into the composition of the field surrounding such a centre retain, at this stage of
development, their identity. The zone around this particular point receives hairs
traceable back to divergent centres on the chin, medial canthus, margin of rhinarium,
and occiput; each of these fields has its own peculiarities of hair length, hair density
and so on.

THE DIVERGENT CENTRE CONSIDERED AS A GROWTH CENTRE.

In 1934 Sewall Wright expressed the view that the rosettes of the rough guinea-
pig’s coat were “physiologically isolated growth centres” modifying the antero-posterior
gradient. Sewall Wright does not appear to have developed his ideas beyond their
formal statement. That this interpretation is correct for the guinea-pig is readily
confirmed by examining embryos of rough forms at the earliest stage of hair develop-
ment, where the positions of the rosette centres are marked by the precocious
development of follicle clusters. Ludwig (1921), paying particular attention to the
occipital whorl in man, has advanced the same idea of follicle activity spreading from
a centre. In the marsupials, when the development of the pelt as shown, for example,
in Trichosurus (see above and Gibbs, 1938) is considered, the conclusion is inescapable
that the divergent centre, whatever its position in the skin, is a centre of activity from
which a follicle growth wave is initiated. A crucial example is provided by a specimen
of Phascolarctos in the lumbar region of which follicle activity has, judged by the
differential density of the follicle population, originated in the sacral whorls, and on the
distal hind-limb from the whorl] on the ankle. The same phenomenon is readily observed
in connection with the gular centres of the family \Peramelidae.

The question arises: Can this demonstrated activity of the centre of the divergent
field be regarded as general for mammals? The uniformity in structure of the skin
and its appendages in the class suggests uniformity of underlying processes which,
with the facts available, point to the generalization being tenable. Much of the literature
dealing with coat development is not usable in this context, as the hair tracts of the
forms considered are not known.

Coincidence of the initial centres of follicle growth with the position of divergent
centres may be obscured. As indicated above in discussing T'richosurus vulpecula, the
first development of follicles is on the circumference of a circle of considerable radius
about the centre. Follicle differentiation then proceeds from this ring towards the
centre and, beyond it, centrifugally. An extreme example of the phenomenon is seen
in the development of the mid-back divergent centre of Macropus major. The condition
as presented by well-furred pouch young is figured in Part II, Fig. 42, Boardman,
1946a. The centre is seen to lie over the lumbar region and from it there extends
forwards to the occiput a feathering which terminates in a divergent interval.
Examination of younger specimens shows that the centre of the system initially
includes the feathering also. In the early stages of coat development on the dorsal trunk
the hair is seen to radiate from the margin of a fusiform naked area extending along
the mid-back from the occiput to the definitive centre.

260 HAIR TRACTS IN MARSUPIALS. V,

RELATIONSHIP OF DIVERGENT AND CONVERGENT CENTRES TO THE PROCESS OF
Harr GROWTH.

Evidence is presented above to support the status of divergent and convergent centres
as primary characteristics of the coat genetically controlled for position and incidence. A
fundamental similarity between divergent and convergent centres is suggested by several
considerations. Both kinds appear in an identical way as abnormalities (v. supra) and
both may be at the focal point of whorled systems. Further, there are grounds for
believing that occasionally a divergent system may be replaced at the same locus by a
convergent system (v. infra). In the elaboration of the primary coat, however, these two
structures are not of equal value. The divergent centre is apparently a focal point of
activity (v. supra). Follicles first make their appearance at the site of divergent centres
and from them the follicle growth wave extends centrifugally. It has been pointed out how
the follicles are developed along lines and how each line is as it were drawn from a
divergent to a convergent centre. There is no evidence in this marsupial material that
the convergent centre is in any way concerned with the initiation of follicle growth
as is the case with the divergent centre. Lines of successively developed follicles grow
towards the convergent centre and finally enter into spatial relationships around it.
The follicles around a convergent centre may be traceable through their developmental
lines back to two or more divergent centres. It is readily observed and significant
that for some time after a convergent centre has become defined by follicle growth the
characteristics of the fields (hair density, hair character, etc.) associated with the
several contributory divergent centres retain their identity (see the account of
Trichosurus vulpecula above).

The tip of the tail, limb extremities, and tips of the ears are regarded as
physiologically equivalent to convergent centres in that they enter into similar relation-
ships with the divergent centres where the laying down of hair rows is concerned and
appear not to be the site of initiation of hair growth unless, as in the case of the
hind-limb of Phascolarctos, a divergent centre occurs distally.

The inactivity of the convergent centre with respect to hair production is probably
due to its later appearance in the skin or later attainment of a threshold for some
requirement necessary in the laying down of follicle anlagen.* Subsequent activity at
the convergent centre is shown by such features of the mature pelt as regional
difference in. the length attained by the hairs. Thus many marsupials, particularly the
macropods, show an early lengthening of the hairs of the cloacal hillock, which is the
site of a convergent centre. The same phenomenon is observed in the mid-ventral trunk
convergence of ungulates, and on man and other animals in the formation of the
beard, which appears to be associated with the interramal convergence.

THE PELT CONSIDERED AS A MOSAIC OF POLARIZED HAIR FIELDS.

Establishment of the generalization that hairs develop in rows and that the rows,
however tortuous their course might be, always run between divergent and convergent
centres, justifies the viewpoint that the skin in its expression of hair tract pattern may
be viewed as a mosaic of hair fields. Ideally the hair field will be spindle-shaped,
with a divergent centre at one pole and a convergent centre at the other. All the hairs
within a field will point towards the convergent centre. In general, both convergent
and divergent centres will act as the poles of two or more of these fields. The hairs
entering into the formation of convergent centres will not all be from the same source

“The system of divergent centres associated with a given pattern does not necessarily
become active in follicle development for all its members simultaneously. In the account given
above of the ontogeny of the coat in Trichosurus vulpecula it was noted that the medial canthal
divergence emerges as a centre of follicle production before the more cranially situated rhinal
centre, so that for some time the divergent interval between them is formed only on the
side adjacent to the eye. If the postulate is sound that divergent and convergent centres are
fundamentally similar in their physiological significance, then the existence of differences in

timing of attainment of threshold levels as between divergent and convergent centres related
through hair rows would be anticipated.

BY W. BOARDMAN. 261

(divergent centres), and these differences, as shown by examination of the interramal
convergence of Trichosurus vulpecula (v. supra), can often be seen as differences in the
character and arrangement of the hairs.

INTERCHANGEABILITY OF DIVERGENT AND CONVERGENT CENTRES.

It has been suggested above that the divergent and convergent centres have the
same fundamental function in relationship to the physiology of tract development. If
there is this similarity between the two types of centre one might reasonably look
for cases, either in ontogeny or phylogeny, where the one has become substituted for
the other. No evidence is forthcoming from this marsupial material that alternative
conditions of divergence or convergence may obtain at a given situation on the skin in
the same species. From the.phylogenetic aspect, however, a case from the Peramelidae
seems relevant. In this family hair tracts of the groin may be determined by the
presence of a single or double divergent radial system or of a medial convergent
system. The two alternatives have been figured in Part I (Boardman, 19430) for
Tsoodon obesulus (Fig. 9) and Perameles myosura notina (Fig. 10). Both alternatives
occur within the genus Perameles. The probability is present, then, that substitution
of one type of field for the other may take place.

DISCUSSION.

Explanation has been the objective of nearly all workers on hair tracts. The
theories* that have been advanced fall into two groups representing fundamentally
different approaches to the subject.

In the first group the early primordium of the follicle is regarded as subject to
tension and pressure through the influence of which the definitive slope and direction
of the developing hair is determined. The literature of these mechanical theories has
been reviewed by a number of authors, notably Schwalbe (1910, 1911), Landauer (1925a,
1925bd) and Biedermann (1928). The most recent work advocating mechanical
explanation is that of Landauer (1925b).

The second and smaller group of theories was inaugurated with Ludwig’s paper
(1921), using human material and some domestic animals. Colin (1943) followed with
a study based on the embryogenesis of hair follicles in the guinea-pig. Both of these
workers propounded theories to explain differential hair direction, and in each case
the theory rests on a gradient-field concept. Ludwig advanced the idea that growth in
area of the skin is “quantitatively pluricentral’, each surface element increasing
through reproduction of its own special cells. Further, these growing surface elements
grow faster in some directions than others. From this postulated pluricentral expansion
and differential growth rates Ludwig concludes that in the skin there exist growth
lines running between points of the highest and lowest growth rate. These lines
determine the pattern of follicles. This ingenious theory is primarily concerned with
explanation of deviations from the arrangement of the hair in the primitive condition.
The nature of the work makes a critical examination difficult in that histological or
experimental evidence having bearing on it is at present not available. Colin (1943)
accounted for hair direction by suggesting “that the more actively growing regions give
off some kind of growth-promoting influence, possibly a substance or substances which
diffuse to other regions. As a consequence, a physiological gradient with its high
at the posterior end and its low somewhere on the head becomes established; similar
gradients develop in the limbs and ears with highs at the distal ends. The resulting
differential growth within the follicles directs the latter away from the more active
regions .... The formed hairs accordingly point in general toward regions of active
growth as measured at the time of formation of their primordia.’ Colin’s hypothesis
is open to a number of objections, both in itself and arising out of its application to
explain the data derived from this study of marsupial hair tracts. The theory depends

* Hair tracts have been advanced: by Kidd (1903, 1920) as a case of the inheritance of
acquired characters. This viewpoint has been strongly supported by Wood Jones (1924, 1925,
1943). Consideration of the question is deferred to a later contribution.

262 HAIR TRACTS IN MARSUPIALS. V,

on the reality of “differential growth of the opposite sides of the early primordium
itself’. Beyond an expression of opinion by the author arising from examination of
his material, there seems to be no histological evidence presented in the paper that
the slope of the early primordium is traceable to or correlated with differential growth.
The theory would identify divergent centres and convergent centres as focal points of
low and high metabolic activity respectively. The role of the divergent centres as the
point from which the follicle growth waves take their origin and the passivity, at least
in the early stages of coat formation, of the convergent centre, make it highly probable
that the opposite is the case (v. supra). The idea of a diffusible substance being in
any way connected with hair slope seems to be negatived by the absence of any
evidence of graded effect from the centre of diffusion outwards. Also there is a
complete absence (v. supra) of any kind of mingling or.resultant of forces at tract
margins as would almost certainly occur in the presence of such a substance diffusing
from adjacent centres. Colin takes the view that the points of high growth rate of the
developing body are coincident with convergent centres as they occur in the formed
coat. The general growth in area of the skin is, of course, closely correlated with the
changing contours of the body as growth proceeds. But the placing of divergent
and convergent centres appears to have no relationship to probable regions of low and
high growth rate for the body generally. The point is made clear by consideration of
the variable position of the median dorsal divergent centre in the members of the
macropod genus Dendrolagus (Rothschild and Dollman, 1936). This centre may be
present at the level of the shoulders, the mid-back, or near the root of the tail. Super-
imposed upon the general growth contours of the skin there is apparently a second
and separate system of centres of activity concerned with the elaboration of the hair
covering with its extensive implications in skin organization.

The regional differential orientation of the hair that gives rise to tract formation
presents a number of problems. It is necessary to seek explanation for:

1. The presence of divergent and convergent centres.

2. The slope of the hair, that is, the angle it makes with the skin surface.

3. The direction in which the sloping hair is pointed, that is, on a line joining a
divergent to a convergent centre.

4. The centrifugal extension of the follicular growth wave from a centre and the
arrangement of the developing follicles on lines joined to the centre.

5. The whorling of divergent and convergent centres.

Sufficient evidence has now accumulated (v. supra) to make tenable the view that
divergent and convergent centres are centres of activity which, by their number and
arrangement, determine the pattern of tracts. The follicle growth wave has been shown
(in marsupials) to take its origin from or in the vicinity of a divergent centre. The
subdivision of the growth wave to produce tract units is conditioned by the presence
of the so-called convergent centres upon which a given segment of the growth wave
converges. Further, evidence has been adduced that fundamentally the divergent and
convergent centres are similar in nature. The manner in which the lines of follicles
converge towards a point (centre) shows that while activity at the point is not such as
to permit follicle initiation it is not quiescent, but is in some way concerned in the
course taken by the follicles. As has been expressed earlier in this paper, it seems
permissible to view any given pattern of tracts as a mosaic of separate fields each
polarized by the presence of a divergent and convergent centre. The nature of the
activity at the centres seems to be responsible for the initiation of the follicle growth
wave.

Landauer (1925b) has emphasized the necessity for recognizing that the definitive
orientation gf the hair shaft involves two components—the angle that it makes with
the skin surface, and the direction in which it is pointing. His theory of causation,
however, advances a common cause (skin tension) as responsible for the two components.
Within a given tract the slope of the hair tends to remain constant, but the direction
in which it is pointing is subject to wide variation in relationship to the tract poles.
Illustrative of the extent of fluctuation in direction, the course of hairs from the occipital

BY W. BOARDMAN. 263

whorl in Trichosurus over the shoulder and then caudalwards on the ventral surface
to the mid-belly convergence may be cited. There seems no reason why a common
cause should be sought for the two components of hair direction. The tip of the
developing hair is always directed away from the centre in association with which the
follicle growth wave is initiated. This rule has no exceptions in the present marsupial
material. There is, however, nothing to indicate that the primary slope as distinct
from the direction of pointing is the outcome of activity at the centre. It is proposed
here to advance the view that slope is an intrinsic quality of the follicle. It will be
apparent that much more is involved than mere inclination of the hair shaft with
reference to the surface of the skin. There are probably constant relationships of the
sebaceous gland, the arrector pili muscle and the epithelial bed to the inclined follicle,
though confirmation of this has not been included in the present research. Support
for the viewpoint comes from two sources. Firstly, slope is correlated with features
both in the structure of the hair itself and in its environment. Landauer (1925a) has
gathered together the relevant literature. Thus Stoss reports correlation between skin
thickness and angle of inclination of hair in the horse and cattle; Landauer, utilizing
data from Frédéric, showed that the same was true for man, and later (Upham and
Landauer, 1935) this was confirmed on more extensive material; Lehmann’s data for
sheep establish a correlation between thickness of hair and slope. Secondly, the experi-
mentally demonstrated individuality of the feather with reference to orientation seems
admissible as supporting evidence. Thus Lillie (1942) reports: “If a papilla dissected
completely free from all attachments is rotated through 90° or 180° around its own
axis and re-implanted, the feather which develops from it is similarly disorientated, in
the latter case upside down with the dorsal surface next the skin.’ It should be
borne in mind that feathers, like hairs, are arranged in tracts within which feathers
develop in an orderly sequence from localized areas of proliferation (Holmes, 1935).
There is, then, a probability that slope per se is an innate quality of the follicle
correlated with certain physical features, particularly thickness of the skin and
thickness of the hair shait.

Evidence has been presented (v. supra) that the follicle growth wave is made up
of lines of follicles which extend from a divergent centre towards a convergent centre.
Two points here call for explanation—the fact that the hair shaft primarily grows
away from the centre of initiation of follicle growth, and that the so-directed shaft.
lies on a line connecting the divergent centre with a convergent centre. No suggestion
is forthcoming from this materiai as to how the growth centre from which the growth
wave originates brings about this orientation of the follicle. The second point, the
geometrical pattern of lines along which the follicles grow, finds a possible explanation
by applying the principle of ultrastructural organization as put forward by Weiss in
connection with his experimental work on the growth of nerve fibres. The advancing
lines of follicles that constitute the growth wave, each line drawn along a definite route
from a divergent centre to a convergent centre, produce an impression that there
must be directive tracks of some kind in the skin. The phenomenon of follicle succession
and line direction has been discussed above for Trichosurus vulpecula. Ludwig (1921)
held the opinion that the growth lines existed at the microscopic level but was unable
to detect them. Weiss (1934) writes:

“The formation of connecting fiber tracts is strikingly demonstrated when two
spinal ganglia are cultivated in a thin plasma membrane at some distance from each
other: A narrow streak of cells and nerve fibers grows then in a perfectly straight
direction along the line connecting the two cultures. The assumption that the
phenomenon is due to ‘chemotactic’ attraction could be decisively disproved. The real
mechanism is as follows: The proliferative activity of the cultures entails dehydration
of the surrounding medium. The dehydration causes contraction. Contraction proceeding
from two centers establishes tensions which, in the space between the two centers,
reach a maximum of intensity and are oriented in the direction of the connecting line.
These oriented tensions evoke a correspondingly oriented pattern of ultrastructures in
the medium, an ultrastructural ‘bridge’ leading from one center to the other. The

264 HAIR TRACTS IN MARSUPIALS. V,

outgrowing nerve fibers simply follow the ‘bridge’. On the basis of these results the
fact is explained that in embryogenesis intracentral fiber tracts develop between centers
of accelerated growth (Coghill) and that peripheral nerves are ‘attracted’ by growing
organs (Detwiler).”

The existence of the system of centres of activity in the mammalian skin may be
interpreted as an example in vivo of Weiss’s tissue culture experiment, from which it
might be concluded that the activity of the centres has produced between them a series
of micellar lines related causally to the precise tracks followed by the advancing
follicles.

The whorling of the hair often observed about the divergent and convergent centre
finds no explanation in the present work. The phenomenon would seem to imply the
existence of a separate problem. In Trichosurus vulpecula two centres usually strongly
whorled in well-furred material, the occipital divergent centre and the mid-ventral
(umbilical) convergent centre, appear not to be whorled at the time that the primary
coat is laid down. This would suggest that the whorling is a later development,
perhaps conditioned in its appearance by differential growth about the centre. The
separateness of this phase of the hair tract problem is shown by Schwarzburg’s (1927)
work on the occipital whorl in man, where it was demonstrated that clockwise rotation
is dominant to counter-clockwise rotation.

SUMMARY.
1. The hair tracts problem is stated.
2. Evidence is submitted that tract pattern:
(a) is constant for the species,
(b) does not vary with age,
(c) is constant in its developmental stages for the species.

3. Divergent and convergent centres are shown to be primary configurations of the
pelt in that their presence is dependent on the genotype. Divergent and convergent
intervals are regarded as secondary characteristics the formation of which is conditioned
by the grouping of centres.

4. The primary hairs are shown to be disposed in lines running between divergent
and convergent centres.

5. The divergent centre is identified as a growth centre from which the growth
wave of follicles takes origin.

6. Evidence is tendered that divergent and convergent centres, while not equally
concerned in the development of the pelt, are fundamentally similar in nature. :

7. There are grounds for believing that the divergent and convergent centres may
occasionally be interchangeable. ;

8. The skin, with reference to its hairy eovering, is considered to be a mosaic of
fields each unit of which is polarized by a divergent and convergent centre.

9. Current theories of causation involving gradient-field concepts are critically
examined. , ,

10. Two components are recognized in the definitive orientation of the hair shaft—
the angle made by it with the skin surface (its slope) and the direction in which
it is pointing. Slope is considered to be an intrinsic quality of the follicle.

11. The geometrical pattern of follicle lines running between divergent and con-
vergent centres is considered as possibly due to activity at the centres having produced
an ultrastructural organization of micellar (ultramicronic) lines along which the
follicles grow.

ACKNOWLEDGEMENTS.

I am greatly indebted to Professor W. E. Agar, of the University of Melbourne, and to
Professor P. D. F. Murray, of the University of Sydney, for helpful discussion and critical
reading of the manuscript.

References. ?
Geaux, O. DE, 1917.—Studi sui neonati dei mammiferi (Forma esterna). Parte prima. Primati
e carnivori fissipedi. Cap 1-4. Arch. Ital. Anat. Embriol., 15: 467-541.

BY W. BOARDMAN. 265

BEAUX, O. DE, 1918.—Id., Cap 5-9. Ibid., 17: 144-215.

, 1924a.—Studien tiber neugeborene Saugetiere (aussere Form). Carnivora fissipedia.
Kapitel X-XI. Zool. Jahrb. Syst., 47: 331-378.

, 1924b.—Beitrag zur Kenntnis der Gatting Potamochoerus Gray. Ibid., 47: 379-504.

, 1927.—Studien tiber neugeborene Sdugetiere (&ussere Form). Carnivora fissipedia.
Kapitel XII-XIIJ. Ibid., 54: 1-38.

BIEDERMANN, W., 1928.—Vergleichende Physiologie des Integuments der Wirbeltiere. Hrgebnisse

1

der Biologie, 4: 360-680. 3

BOARDMAN, W., 19438.—The Hair Tracts in Marsupials. Part i. Description of species. Proc.
LINN. Soc. N.S.W., 68: 95-113. :
, 1946a.—The- Hair Tracts in Marsupials. Part ii. Description of species, continued.
Ibid., 70: 179-202.
, 19466.—The Hair Tracts in Monotremes; their Relationship to the Mammalian
Primitive Condition. Univ. Qld. Pap., Dept. Biol., II, 7: 7 pp.
, 1949.—The Hair Tracts in Marsupials. Part iii. Description of species, concluded.
Proc. LINN. Soc. N.S.W., 74: 192-195.
, 1950.—The Hair Tracts-in Marsupials. Part iv. Direction characteristics of whorls
and meristic repetition of radial fields. Ibid., 75: 89-95.
CASTLE, W. E., 1925.—Heredity in rabbits and guinea-pigs. Bibliographia Genetica, 1: 419-458.
ConiIn, E. C., 1945.—Hair direction in mammals: embryogenesis of hair follicles in the guinea-
pig. J. Morph., 72: 191-223.
Crarr, W. A., and WARNER, H. A., 1934.—Observations on different types of hair whorls in
mammals, andthe inheritance of hair whorls in swine. Proc, Oklahoma Acad. Sci., 14: 23-27.
Dry, F. W., 1926.—The coat of the mouse (Mus musculus). J. Genetics, 16: 287-340.

FRASER, Doris A., 1928.—The development of the skin of the back of the albino rat until the
eruption of the first hairs. Anat. Rec., 38: 203-224.

Gates, R. R., 1946.—Human Genetics. New York.

GIBBS, HELENA, 1938.—A study of the development of the skin and hair of the Australian
Opossum, Trichosurus vulpecula. Proc. Zool. Soc. Lond., Ser. B, 108: 611-648.

HoumMeEs, ANNE, 1935.—The pattern and symmetry of adult plumage units in relation to the
order and locus of origin of the embryonic feather papillae. Am. J. Anat., 56: 513-536.
JoNES, F. Woop, 1920.—The external characters of pouch embryos of marsupials, No. 1.

Trichosurus vulpecula var. typicus. Trans. Roy. Soc. 8S. Aust., 44: 360-373.
, 1924.—On the causation of certain hair tracts. J. Anat. Lond., 59: 72-79.
, 1925.—The hair pattern of a’ kangaroo; a study of cause and effect. J. Mammal,
G3) Wea,
, 1927.—The middorsal hair whorl of man. Am. J. Phys. Anthrop., 5: 89-95.
, 1934.—The dorsal hair tracts of the Australian aborigine. J. Anat. Lond., 69: 91-92.
, 1943.—Habit and Heritage. London.
Kipp, W., 1903.—The Direction of Hair in Animals and Man. London.
, 1920.—Initiative in Evolution: London.
LANDAUER, W.,: 1925a.—On the Hair Direction in Mammals. J. Mammal., 6: 217-232.
, 1925b.—Bemerkungen zu Ludwigs Hypothese der Morphogenese des Haarstrichs.
Zool. Anz., 64: 235-244.
, 1926.—Die Vererbung von Haar- und Hautmerkmalen, ausschliesslich Farbung und
Zeichnung, mit Beriicksichtigung von Rassedifferenzierung und Deszendenz. Zeitschrift f.
induktive Abstammungs- wu. Vererbungslehre, 42: 113-226.

, 1929.—Die Vererbung von Haar- und Hautmerkmalen, ausschliesslich Farbung und
Zeichnung. Ii. Ibid., 50: 356-415. 2

Linuig, F. R., 1942.—On the development of feathers. Biol. Rev., 17: 247-266.

Lupwie, E., 1921.—Morphologie und Morphogenese des Haarstrichs. Zeit. Anat. Entwickl., 62:
59-152.

MEIJERE, J. C. H. pr, 1894.—Utber die Haare der Saugethiere, besonders liber ihre Anordnung.
Morph. Jahrb., 21: 312-424.

NIEDOBA, T., 1917.—Untersuchungen tber die Haarrichtung der Haussdiugetiere. Anat. Anz.,
50: 178-192 and 204-216.

ROTHSCHILD, LorD, and DOLLMAN, G., 1936.—The Genus Dendrolagus. Trans. Zool. Soc. Lond.,
21: 477-548.

ScHONHmERR, H., 1937.—Uber die Wachstumsbewegung der Haaranlagen am Scheitelwirbel des
Menschen. Anat. Anz., 85: 193-208.

SCHWALBE, G., 1910.—Uber die Richtung der Haare bei den Halbaffen. In Voeltzkow, Reise in
Ostafrika in den Jahren, 1903-1905, 4: 207-265.

266 HAIR TRACTS IN MARSUPIALS. V.

SCHWALBE, G., 1911.—Uber die Richtung der Haare bei den Affenembryonen. In Selenka, Studien
liber Entwickelungsgeschichte der Tiere, 5: 205 pp.

SCcHWARZBURG, W., 1927.—Statistische Untersuchungen tiber den Menschlichen Scheitelwirbel
und seine Vererbung. Zeit. Morph. Anthrop., 26: 195-224.

Toutpr, K., 1907.—Studien tiber das Haarkleid von Vulpes vulpes. Annalen des k.k. naturhis-
torischen Hofmuseums Wien, 22: 210-313.

UPHAM, ELIZABETH, and LANDAUER, W., 1935.—The relation of thickness of cutis and subcutis
to hair slope in human skin. Anat. Rec., 61: 359-366.

Voiet, C. A., 1857.—Abhandlung tiber die Richtung der Haare am menschlichen K6rper. Denks.
math.-nattirw. Klasse Kaiserl. Akad. Wiss., Wien, 13: 50 pp.

Wetss, P., 1934.—In vitro experiments on the factors determining the course of the outgrowing
nerve fiber. J. Hap. Zool., 68: 393-448.

WRIGHT, S., 1934.—Genetics of abnormal growth in the guinea pig. Cold Spring Harbor
Symposia on Quantitative Biology, 2: 137-147.

, 1935.—On the genetics of rosette pattern in guinea pigs. Genetica, 17: 547-560.

THE HAIR TRACTS IN MARSUPIALS.
PART VI. EVOLUTION AND GENETICS OF TRACT PATTERN.
: By W. BoarpMAN,
Department of Zoology, University of Melbourne.
[Read 27th September, 1950.]

Contents.

Page.
The Primitive Condition Considered in Relationship to Growth Centres .. ts PSG eG Sule et AOE
Stages in Tract Development and their Evolutionary Significance SW rane Nea sano tam Se yet anh eT AG)
Genetics of Hair Tracts .. . NS, ears eT Pein Sehg hated aE ON EY oP ENO etal a tae are O.
Sex-Dimorphism with ye deones io Biiiona Bie Naat setae ca itetedy RoR GN ONO:
The Gular Whorls of the Peramelidae—A ne orenetic ‘Dhoemation SES BUSHY he SOMA) crue reo one GU)
Summary oy He ang os Be A Ph Bete ee RAR A ey Hd em rg Aiba > Ben mabed ee aa a beats 6th Bish Oty base 2 iil!
TNCKCHON CCS MERE Raty ese tect: Ra ae N em rcp RET, aden y REb Me Tate at FAP Cd Fy yates vc dvad valve Br Ne ohh e

Evidence has been submitted (Part V) that any given tract pattern may be regarded

as a mosaic of fields the number and form of which is determined by the skin’s
. complement of divergent and convergent centres. The centres are shown to be primary
characteristics of the skin in the sense that their presence or absence is conditioned by
the genotype. It follows that the phylogeny of pattern is most likely to be expressible
in terms of change in the number and distribution of these centres. Support for this
view is provided by a consideration of tract sequence in the polyprotodont family
Dasyuridae and the diprotodont subfamily Phalangerinae. The material representing
other families is not sufficiently ample for formal presentation in this context.

Tur Prowirive Conprrion CoNSIDERED IN RELATIONSHIP TO GRrowTH CENTRES.

In the primitive condition (Boardman, 1946b), the simplest of tract patterns,
divergent (growth) centres are situated at the extremity of the snout and on the chin
so that growth of hair is initiated at the cranial end of the body and spreads as a
growth wave caudally. The course of the lines of follicles is determined by the influence
of the caudal end of the body and the distal extremities of the ears and limbs which are
regarded as physiological equivalents of convergent centres. The relationship of the
follicle growth wave to divergent and coivergent centres has been discussed in Part V.
Universaliy in marsupials the cloacal hillock is the site of a convergence with, in
consequence, a convergent interval between it and the root of the tail. A litter of
seven Antechinus flavipes (M.4793) in which the primary coat is not completely laid
down illustrates these points. The dorsal half of the body is haired from the cranial
end almost to the level of the hind limbs and it is apparent from the direction taken by
the lines of follicles and the gradient in hair size from the edge of the advancing wave
eranially that hair growth was initiated at the extremity of the snout. The fore-limb
is haired laterally but not the hind-limb. The gular region is haired and there is weak
hair growth on the ventral thorax. Origin of the growth wave on the ventral aspect is
similarly traceable back to the divergent centre on the chin. Confirmatory evidence from
the Marsupialia is provided by juveniles of Myrmecobius fasciatus (Part III, Boardman,
1949), which in its tracts differs very little from the primitive condition.

STAGES IN TRACT DEVELOPMENT AND THEIR HVOLUTIONARY SIGNIFICANCE.

‘The comparative approach to pattern made possible by the wide selection of
marsupials available for this study early showed that pattern could be classified using
as criteria for separation the number and situation of the centres of radial fields.
Differences between one pattern and another are essentially differences involving
divergent and convergent centres. This is best shown on the present material by analysing

U

268 HAIR TRACTS IN MARSUPIALS. VI,

pattern form in the polyprotodont family Dasyuridae and the diprotodont subfamily
Phalangerinae. It is seen that pattern within these two groups falls readily into stages
of ascending order of complexity from the most primitive to the most highly developed
genera. The authority of Bensley (1903) is cited for what are primitive and what are
highly developed genera in the groups discussed.

The facility with which the tracts can be classified as indicated leaves no doubt that
pattern evolution has been through successive additions of divergent and convergent
centres on a primitive ground-plan. While primitive genera and highly developed
genera can be nominated with reasonable certainty the relationship of genera between
the two extremes is less clear so that no attempt has been made to elaborate a phylo-
genetic arrangement. The pattern grades are arranged below in an arbitrary linear
series. It is not at present known what constitutes a unit of increment in this
evolutionary sense for divergent and convergent centres so that the presentation does
not necessarily portray in detail the actual sequence of events leading to the formation
of any particular pattern.

Family DASYURIDAE.

Of the five pattern grades observable in the family the first and secon belong to the
Phascogalinae, the third, fourth and fifth to the Dasyurinae.

Subfamily PHASCOGALINAE.

Stage 1.—The simplest pattern amongst the polyprotodonts is that of the Sminthopsis-
group (Sminthopsis, Antechinus and Planigale) of this subfamily. The coat presents a
close approach to the hypothetical primitive condition. Except in the scrotal region of the
males (v. infra) there are no added divergent or convergent centres so that the hairs
generally flow from the nasal region caudally or caudally and ventrally. The cloacal
hillock is the site of a convergence common to all marsupials.

Stage 2.—The second stage is represented by the Dasycercus-group (Dasycercus,
Dasyuroides and Phascogale). It is derivable from the Sminthopsis-group by the
addition of the elbow and interramal convergences. The interramal convergence is not
always fully expressed (see Dasycercus cristicauda, Boardman 1946a, Figs. 1 and 2).

Subfamily DASYURINAE.
Stage 1—The first stage, represented in this material only by the single genus
Sarcophilus, differs from the Smithopsis-group of the Phascogalinae by the addition of
the mid-ventral (umbilical) convergence.

Stage 2.—A re-examination of the specimens of Dasyuwrus quoll leads to the conclu-
sion that the somewhat vague disturbances in the gular hair are caused by a bilateral
pair of whorls associated with a distorted interramal convergent point. If this be so
the stage would be derivable from the Dasycercus-group of the Phascogalinae.

Stage 3—Stage 3 follows Stage 2 by the addition of an umbilical convergence. It
includes the Dasyurinus-group (Dasyurinus and Dasyurops). The derivation of Stage 3
from Stage 2 implies homology of the blurred gular whorls of Dasyurus and the axillary
whorls of Dasyurinus and Dasyurops. This homology is in accord with the facts of
variation in whorl] pattern (v. infra).

Subfamily PHALANGERINAE.
Four stages in tract elaboration may be defined between the primitive Acrobates and
the highly developed Trichosurus.

Stage 1—In Acrobates pygmaeus the! simple condition shown in the Sminthopsis-
group of the Phascogalinae is repeated. The primitive nature of the tracts is not altered
by the presence of the flying membrane.

Stage 2.—The second stage is represented by Cercartetus, in which appear an inter-
ramal convergent point and a preauricular stream (presumably from a divergence
associated with the external ear). These two features together give rise to a divergent
interval on the face.

BY W. BOARDMAN. 269

Stage 3.—The third stage is reached in Petaurus and Schoinobates by the addition
of a convergent point in the vicinity of the lateral canthus (in Petaurus papuanus this
point has not been recorded).

Stage 4—Pseudocheirus differs from Stage 3 by the addition of an umbilical conver-
gent point. The conspicuous convergent whorl between the eye and the ear is regarded
as homologous with the point more usually situated nearer to the eye.

Stage 5.—In Dactylopsila a dorsal whorl] is found superimposed on the conditions
characteristic of Stage 4.

Stage 6.—Trichosurus represents a considerable advance on the preceding stage in
that both axillary and medial canthal divergent centres are present. The centre at the
lateral canthus is not developed.

The polyprotodont and diprotodont stocks of the Marsupialia represented above by
the Dasyuridae and Phalangerinae respectively are distinct lines of descent that appear
to have separated very early in the history of the group. In polyprotodonts tract
patterns have, except for the medial canthal divergence of the Peramelidae, evolved
beyond the primitive state only on the ventral aspect of the body. In diprotodonts, on
the other hand, dorsal and ventral surfaces have been affected. Comparing the ventral
surface of polyprotcdonts and diprotodonts with reference to tract evolution it is readily
seen that the end result is in both cases the same. That is to say, for each divergent or
convergent centre in the Polyprotodontia there is an equivalent structure occupying a
similar situation in the Diprotodontia. This fact is illustrated by reference to the
analysis of tract pattern in the Dasyuridae and Phalangerinae. Inguinal radiating fields,
not represented in either dasyures or phalangers are, when they occur, the same in both
polyprotodonts and diprotodonts. An apparent exception to this generalization is
provided by the ventral thoracic convergence in Thylacinus which is not found else-
where in marsupials. It might be anticipated that one or both of the two principal
marsupial stocks would, in their more highly developed forms, produce centres not
represented in the other. Hlsewhere than in this particular centre Thylacinus conforms
to the generalization. A consideration of the tracts of the dorsal surface as presented
by the constituent families of the Diprotodontia leads to a similar conclusion, namely,
that the most highly developed group, the Macropodinae, includes all features present
in other groups.

A given centre may show considerable variation in position. Consider the paired
divergent whorls, most commonly situated in the axilla, which are present in all
Macropodidae examined except Dendrolagus. The whorl may be displaced laterally onto
the upper arm (Macropus major) and the forearm (Thylogale sp.), or cranially onto
the upper thorax (Bettongia penicillata) and even as high as the root of the neck
(Potorous tridactylus apicalis). The occurrence of this series of positions about the
more frequent axillary locus for the whorl, the relationship of the genera cited, and the
absence of other whorled systems with which it might be confused, make tenable the
assumption that in each case it is the same whorl! that is being considered. The material
presents no evidence of overlap of the territory that might be occupied by adjacent
centres.

Comparison of the hair tracts of the Polyprotodontia and Diprotodontia, bearing in
mind the restriction on the variation in position of any particular centre, raises the
point that there must exist for the marsupial skin a maximal complement of divergent
and convergent centres. The evaluation of this maximal figure (to be discussed in a
subsequent contribution) can be satisfactorily made only through comparative studies
which enable what might be called initial centres to be distinguished from derived
centres produced by subdivision of the initial units. The subdivision (meristic
repetition) of centres is discussed at length in Part IV.

With reference to tract pattern, then, the situation in the marsupial skin is such
that evolution proceeds by successive increments of centres towards the attainment of
a saturation density, each centre, divergent or convergent, having its own territory of
occurrence. A similar end-result is attained irrespective of whether development is
along the distinct lines presented by the Polyprotodontia or the Diprotodontia.

270 HAIR TRACTS IN MARSUPIALS. VI;

GENETICS OF HAIR TRACTS.

The genic mechanism may have evolved in two ways. There may have been
successive mutations leading to the production of centres in particular areas. With this
interpretation the relatively precise zonation of centres and the relationships of diver-
gent and convergent types, affecting similarly polyprotodont and diprotodont lines, is
difficult to explain except in terms of orthogenetic trends. On the other hand, it may
be postulated that a mutation or mutations occurred in the ancestral stock permitting
the potential development of centres up to the saturation density (v. supra) for the
marsupial skin and that the extent to which they appear is conditioned by the effects
of a system of modifying genes. This viewpoint would explain the similar steps by
which pattern has evolved in polyprotodonts and diprotodonts and the attainment in
both of these major groups of a similar end-point for pattern form. While no evidence
from marsupial breeding is available, support for the second alternative is given by the
work of Sewall Wright (1934, 1935) on the genetics of rosette pattern in guinea-pigs
which is interpreted by him as implying the existence of such a genic background to the
pattern grades that may be defined for these rodents. Variation of rosette pattern in
guinea-pigs is similar to pattern variation in the Marsupialia as a whole.

SEx-DIMORPHISM WITH REFERENCE TO PATTERN.

Only one aspect of pattern following sex is found in the Marsupialia and that is
what has been referred to in the descriptive portion of this work as the prescrotal
reversal or prescrotal reversed triangle. The condition has been figured for Dasycercus
cristicauda (Part II, Fig. 3, Boardman, 1946a@). It consists of the development of a
divergent centre at the base of the scrotal stalk with a convergent centre in the middle
line a short distance in front. The distinctness of the triangular field of reversed
hairs is attested by the fact that often the field remains naked or very sparsely haired
after the development of dense hair in the surrounding areas. In species having
prescrotal reversal of this type the female shows no variation in the primitive caudo-
ventral trend of the inguinal region except in Tarsipes spenserae where a similar but
indistinctly developed area of reversal is found in a female also.

Prescrotal reversal is probably universal in the Phascogalinae but there is at present
no evidence that the peculiarity is found in the Dasyurinae. It occurs in Myrmecobius
fasciatus. As mentioned above, it is recorded for Tarsipes spenserae and is also found
in some of the less highly developed members of the Phalangerinae (Acrobates pygmaeus
and Cercartetus nanus). It is doubtful if the reversal as found in the Phascogalinae
occurs in the higher phalangers or in the macropods, but the material is insufficient to
provide data on which to formulate a rule. ;

The simplest explanation is that the dimorphism is a secondary sexual character.
The tact that the female of Tarsipes spenserae has the configuration weakly developed
is against interpretation as a case of sex-limited inheritance.

Tur GuLar WHORLS OF THE PERAMELIDAE—A PHENOGENETIC EXPLANATION.

The family Peramelidae is characterized by the presence of a whorled system on the
throat. What might be called the typical condition is that of a bilateral pair of whorls,
mirror images of each other and conforming to the marsupial directional rule for the
ventral surface, that is, the left member is always clockwise (Part IV, Boardman, 1950a).
The paired condition has been figured for Perameles nasuta (Boardman, 1943a).
Variation consists in the suppression of one whorl independently of the other which
maintains unchanged both its lateral position and direction of flow. This unilateral
suppression is indiscriminately on the right or left side within the species and is
independent ot sex (see Hchymipera cockerelli, Part I, Fig. 11, Boardman, 1943b; Isoodon
obesulus obesulus, Part II, Fig. 9, Boardman, 1946a). The chances of one or other of
the three conditions of the gular whorled system (paired whorls, a single right whorl,
or a single left whorl) occurring are approximately equal if the family is considered as
a whole. Of a total of nineteen specimens showing gular whorls, seven (three males
and four females) have the double system, six (one male and five females) have a single
whorl clockwise and on the left, six (again one male and five females) have a single

BY W. BOARDMAN. ale

whorl counter-clockwise and on the right. Three of the ten examples with a double-
whorled system have one member of the pair more strongly developed than the other.
Specimen R.13042, a female of the Perameles gunnii series, Shows this point very well in
having the right member very small and practically overwhelmed so far as effect on hair
disturbance is concerned by the whorl on the left side.

The male of the series of Macrotis minor minor (Part II, Boardman, 1946a) has no
gular whorls at all. The remainder of the series consists of two females having each a
single whorl. At first sight this seems to be an extreme case of the tendency to suppress
whorls, but the fact that the male is devoid of elbow and inguinal whorls as well
suggests that a more profound genic disturbance is involved which is not in this context
comparable with the phenomenon of unilateral suppression.

Right-left asymmetry, the essential peculiarity of the variability of these gular
‘whorls, has a parallel in certain teeth in fossil skulls. Simpson (1944) has recorded
of the functionless P? in the Oligocene viverrid Hoplophoneus that it ‘‘may vary from
a well-developed state to complete absence, not only within one race but also within one
individual (i.e., the left side may differ from the right side)”. A further case is provided
by the degenerating P*® of the Paleocene Multituberculate Ptilodus montanus. Simpson
does not deal specifically with the probable genetic background of these tooth vagaries
but it would appear from the general viewpoint expressed that he visualizes the accumu-
lation of small degenerative mutations leading gradually, in the absence of selection-
pressure, to the suppression of the teeth and during suppression bringing about
disequilibrium in the factors determining the range of variability.

Goldschmidt (1946) has commented at length on the examples of tooth variation
given by Simpson. Goldschmidt has also submitted an alternative phenogenetic
explanation which rests on known genetic facts. Briefly, this explanation takes
cognisance of the peculiarities of some of the homoeotic mutants in Drosophila. Amongst
these mutants are genes of low penetrance, highly variable expression, and with a high
degree of right-left asymmetry. The right-left asymmetry has been shown by statistical
analysis of tetraptera, tetraltera and podoptera to be the outcome of the absence of
right-left correlation, each wing reacting independently in the mutant. Goldschmidt
demonstrates that, by altering such features as time of determination and threshold
levels ‘“‘one single mutant with a type of action as discussed, causing any rudimentation,
can produce all the conditions of variability, etc., described by Simpson”. Goldschmidt
has developed his thesis at length in several works (see, for example, 1938 and 1940).

The genetics of the peramelid gular whorls may be viewed from a similar angle—
a mutation having arisen to alter the developmental timing characteristic of the whorl
pair relative to each other thus introducing right-left asymmetry. The situation is, then,
comparable to the cases of Simpson’s relative to degenerating teeth and to the homoeotic
mutants (though no homoeosis is involved in such a situation). Z

In discussing the high variability of degenerating teeth Simpson stresses that “It
is so commonly true that degenerating structures are highly variable that this may be
advanced as an empirical evolutionary generalization”. There is no evidence that this
high variability with right-left asymmetry is associated with degeneration in the case
of peramelid gular whorls. The variability of the whorls probably differs from the
expression of variability in Simpson’s skulls in that only one member of the whorl pair
is involved. Whether the whorl is absent or at any stage of development short of
complete expression the remaining whorl is invariably fully developed and produces hair
reversal on the throat little if at all less than the pair together would produce. The
question of degeneration does not seem to arise with reference to these whorls.

SUMMARY.
1. The primitive condition of hair tracts is expressed in terms of growth centres.

2. Successive levels of tract complexity within the Dasyuridae (Polyprotodontia)
and Phalangerinae (Diprotodontia) are presented as evidence that tract evolution has
proceeded by the continued addition of divergent and convergent centres on a primitive
ground-plan.

V

bo
be |
bo

HAIR TRACTS IN MARSUPIALS. VI,

2d

3. Evolution of the gene complex with reference to tract pattern is considered.
The phylogeny ot tract pattern suggests that there is operative a principal gene or
genes the expression of which is dependent on a system of modifiers.

4. The known cases of sex-dimorphism of tracts are recorded. It is concluded that
the phenomenon is interpretable as a secondary sexual character.

5. The vagaries of phenotypic expression of the gular whorls in the Peramelidae
are stated. The right-left asymmetry is attributed to a mutation having altered develop-
mental timing of the whorl pair relative to each other. It is concluded that the high
variability shown by the whorls is not associated with degeneration.

References.

BeENSLEY, A., 19038.—On the Evolution of the Australian Marsupialia: with Remarks on the
Relationships of the Marsupials in General. Trans. Linn. Soc. Lond., Zool., 9 (Ser. 2):
83-217.

BOARDMAN, W., 19438a.—On the External Characters of the Pouch Young of Some Australian
Marsupials. Aust. Zool., 10% 138-160.

, 1943b.—The Hair Tracts in Marsupials. Part i. Description of Species. .Proc. LINN.
Soc. N.S.W., 68:.95-113.

, 1946a.—The Hair Tracts in Marsupials. Part ii. Description of Species, continued.
Ibid., 70: 179-202.

, 1946b.—The Hair Tracts in Monotremes; their Relationship to the Mammalian
Primitive Condition. Univ. Qld. Pap., Dept. Biol., Il, 7: 7 pp.

, 1949.—The Hair Tracts in Marsupials. Part iii. Description of Species, concluded.
Proc. LINN. Soc. N.S.W., 74: 192-195.

, 1950a.—The Hair Tracts in Marsupials. Pant live Direction Characteristics of
Whorls and Meristiec Repetition of Radial Fields. Ibid., 75: 89-95.

, 1950b.—The- Hair Tracts in Marsupials. Part v. A Contribution on Causation.
Ibid. (in press).

GOLDSCHMIDT, R., 1938.—Physiological Genetics. New York.
, 1940.—The Material Basis of Evolution. New Haven, London and Oxford.

, 1946.—‘An Empirical Evolutionary Generalization”, Viewed from the Standpoint of
Phenogenetics. Am. Nat., 80: 305-317.

Simpson, G. G., 1944.—Tempo and Mode in Evolution. New York.
WricGHT, S., 1934.—Genetics of Abnormal Growth in the Guinea Pig.. Cold Spring Harbor
Symposia on Quantitative Biology, 2: 137-147.
, 1935.—On the Genetics of Rosette Pattern in Guinea Pigs. Genetica, 17: 547-560.

THE HAIR TRACTS IN MARSUPIALS.
PART VII. A SYSTEM OF NOMENCLATURE.
By W. BoarDMAN,
Department of Zoology, University of Melbourne.

(Two Text-figures. )
[Read 27th September, 1950.]

Divergent and convergent centres the distribution and relationships of which
condition hair tract formation (Part V) are not disposed haphazardly in the skin but
are found only in particular situations. Hach centre has a territory of occurrence
and the territories of neighbouring centres seem not to overlap (Part VI, Boardman,
1950c). ‘There is apparently a definable maximal concentration of centres which is an
intrinsic property of the group. Comparison of polyprotodont and diprotodont tract
evolution and consideration of the probable genetic mechanism (Part VI, Boardman,
1950c) leaves little doubt that centres of the same type occupying the same situation
are homologous structures.

With these generalizations it becomes possible to formulate provisionally a system
of nomenclature using symbols applicable to the Marsupialia and almost certainly
(v. infra) to the Mammalia as a whole. In the proposed terminology divergent and
convergent centres are recorded relative to the dorsal and ventral surfaces. The
centres, divergent and convergent respectively, are numbered from the cranial end
caudally except as mentioned below. In drawing up such a nomenclature several points
must be taken into consideration. Evidence has been presented (Part IV, Boardman,
1950a) that some bilateral pairs of centres are derived from a single medial structure.
The derivation of bilateral pairs from medial units may be general. Thus, the divergent
centre on the forearm in Thylogale has been shown to be equivalent to the axillary
centres of macropods generally (Part VI, Boardman, 1950c) and must be presumed
equivalent to the single medial whorl as exhibited by Vombatus hirsutus (Parts II
and IV, Boardman, 1946a, 1950a). However, in cases such as the divergence at the
medial canthus, the elbow convergence and the genal convergence where the specimens
give no evidence that affinity is with a known medial point, the centre is referred to
by abbreviation and is not included in the numerical system used for other centres.
The material suggests but does not provide further grounds for believing that duplica-
tion may have occurred longitudinally and that subsequent bilateral duplication has
taken place in one only of the daughter centres. Longitudinal duplication is a well-
established phenomenon as a meristic variant and simultaneous longitudinal and
transverse duplication is known to occur (Part IV, Boardman, 1950a). This viewpoint
would explain the triangular arrangement of divergent centres in the groin of
Perameles gunnii (Part II, Figs. 12 and 13, Boardman, 1946a@), and of the interramal
convergence and the convergent centre occasionally seen in the vicinity of the angle
of the mouth, as in Trichosurus vulpecula (Part II, Fig. 19, Boardman, 1946a). The
nearness of the centres in the cases cited is suggestive of relationship through duplica-
tion. The unique convergence on the mid-ventral thorax of Thylacinus (Part I,
Fig. 8, Boardman, 1943) is omitted from consideration at this stage pending further
information relevant to its status; it may be related to the elbow convergence in the
same manner as has-been suggested above for the convergence at the angle of the mouth
and the interramal convergence.

The following abbreviations are used: a = ankle, C = convergent, D = divergent,
d = dorsal, e = elbow, g = genal, h = heel, k = canthal, 1 = lateral, m = medial, p = ear
(pinna).

274 HAIR TRACTS IN MARSUPIALS. VII, <

DIVERGENT CENTRES OF HEAD, NECK AND TRUNK.

Dorsal Surface.—There appear to be four divergent centres in the dorsal series
additional to those associated with the eye and ear (v. infra). The first (Ddl1) is placed
at the end of the snout and is present in all marsupials. The centre of this system is
usually diffuse and apparently may be single or bilaterally doubled. When doubled
and whorled, as in Jrichosurus vulpecula and Onychogalea fraenata (Part II, Figs. 19
and 38, Boardman, 1946a), the result is the well-known rhinal reversal. The second
(Dd2), restricted to diprotodonts, occupies the region from the crown back to about the
level of the inferior angle of the scapula. The third (Dd3) is fairly constant in
position on the mid-back; it is represented in the collection only by Macropus major
(Part II, Fig. 42, Boardman, 1946a@). The fourth (Dd4) occurs over the sacrum back
to the root of the tail; Phascolarctos has this centre doubled over the sacrum; in
Dendrolagus dorianus it is situated at the root of the tail (Miklouho-Maclay, 1885).

Ventral Surface.—Three divergent centres occur on the ventral surface. The first
(Dv1), of universal occurrence in marsupials, is situated on the chin. The second
(Dv2) is most commonly found in a bilaterally paired condition associated with the
axilla, but may be located on the gular region, ventral thorax in front of the caudal
limits of the axilla, or on the fore-limb (Part VI, Boardman, 1950c). Dv3, in the
inguinal region, may be single and median as in Jsoodon obesulus (Part I, Fig. 9,
Boardman, 1943), bilaterally doubled as in Thylacinus (Boardman, 1945, Fig. 1) or
arranged as three centres at the angles of a triangle as in Perameles gunnii (Part II,
Figs. 12 and 13, Boardman, 1946a). In Macropus major the lateral members of the
Dv3 pair have again subdivided to give a transversely placed pair on each side of the
middle line (Part II, Fig. 43, Boardman, 1946a).

CONVERGENT CENTRES OF THE HEAD, NECK AND TRUNK.

Dorsal Surface—Other than that associated with the lateral canthus (v. infra)
only one convergent centre (Cdl) is found on the dorsal surface. It occurs on the
crown in the Macropodinae (see, for example, Macropus major, Part II, Fig. 42,
Boardman, 1946a) and occasionally in Phascolarctos.

Ventral Surface.—Four sites of convergence are found on the ventral aspect of the
body. The cranialmost (Cvl) is that at or in the vicinity of the interramal papilla.
Most marsupials possess it. The second (Cv2) is the familiar umbilical convergence,
though its coincidence with the umbilical scar is exceptional rather than otherwise.
Cv2 is bilaterally doubled in Thylacinus (Part I, Fig. 6) and Thylogale (Part I, Fig. 24,
Boardman, 1943). The third (Cv3) occurs in front of the cloacal hillock and may be
present within the pouch area or near the root of the scrotum. The caudalmost member
of the series, Cv4, is the convergence at the cloacal hillock; it is universal in marsupials.
All of these convergent centres are shown by Trichosurus vulpecula (Part II, Fig. 20,
Boardman, 1946a).

A genal convergence (Cg) is present in some marsupials.

CENTRES ASSOCIATED WITH THE Hyr, EAR AND LIMBS.

Except for the divergent centre on the upper arm of Macropus major and on the
forearm of Thylogale (Part VI, Boardman, 1950c) none of the centres found associated
with the eye, ear and limbs can be demonstrated as derived from medial loci of the
head, neck or trunk, although, as mentioned above, it is possible that this is so.
Consequently, no attempt is made at this stage to give them a place in the numbered
series defined above and they are here referred to by non-committal abbreviation.

The eye.—Very often in marsupials a divergent centre (Dmk) is associated with
the medial canthus (for example, Onychogalea fraenata, Part II, Figs. 37 and 38,
Boardman, 1946a). A further centre, apparently always convergent (Clk), is similarly
associated with the lateral canthus. Sometimes the centre at the lateral canthus may
be at some distance from it as in Pseudocheirus (Part Il, Fig. 17, Boardman, 1946a).

.

BY W. BOARDMAN. 275

The ear.—The frequent presence of a preauricular reversal would suggest that a
divergent centre may be situated in the cavity of the external ear but its focal point is
difficult to define in this marsupial material. It is provisionally designated Dp.

The fore-limb.—The elbow convergence (Ce) is often present in marsupials; it is
always single.

The hind-limb.—Except in the Phascogalinae, the heel seems always to be the site
of a convergence (Ch) which produces reversal to a greater or lesser extent on the
plantar surface. It is very strongly developed in peramelids.

Divergent systems on the hind-limb occur rarely. Phascolarctos (Part II, Fig. 27,
Boardman, 1946a) possesses a distinct whorl on the ankle (Da), and what is probably a
duplicated version of this has been recorded for two macropods (Thylogale sp. and
Onychogalea fraenata).

Text-figures 1 and 2.

(

Schematie representation of the relationship of divergent and convergent centres in the
marsupial skin (Fig, 1, dorsal aspect; Fig. 2, ventral aspect). See text for explanation of
symbols.

APPLICABILITY OF THE SYSTEM OF NOMENCLATURE TO THE MAMMALIA AS A WHOLE.

The symbolized terminology submitted above is based on the collection of marsupial
young, a systematic account of the hair tracts of which has been recorded in Parts I-III
(Boardman, 1943, 1946a, 1949) of this work. The collection is representative of the
several groups and includes nearly a third of known marsupials. A summary of the
hair tracts of these species arranged according to the terminology is presented in Table 1.

276 HAIR TRACTS IN MARSUPIALS. VI,

TABLE 1.

Résumé of Marsupial Tract Pattern Arranged in Accordance with the Proposed Nomenclature. Centres of Universal
Occurrence in the Order are Omitted. (For explanation of symbols, see text.)

| }

Name. oi fed | <tlai les | 3] lai les | Reference.
ODIDIDIFPIFPISISIS( &F| lS) al sicis
AIAIAIAIAISOlO/OlO/AlOlOl/OlO/A

PHASCOGALINAE—
Sminthopsis crassicaudata c. 1949*, p. 192
Sminthopsis crassicaudata macrura 1943, p. 97
Sminthopsis crassicaudata centralis 1946a, p: 181
Antechinus flavipes flavipes 1943, p. 96
Antechinus maculatus — 1943, p. 96
Planigale ingrami = — 1943, p. 97
Dasycereus cristicauda + a5 1946a, p. 179
Dasyuroides byrnei = ar 1946a, p. 181
Phascogale sp. ar oo 1949, p. 192
DASYURINAE—
Sarcophilus harrisii — = —|- 1943, p. 98; 1946a, p. 182
Dasyurus quoll oa = +} -+ 1943, p. 97
Dasyurinus geoffroii g. ar + aF\lae 1946a, p. 181
Dasyurinus geoffroii fortis + +} -F +} 1946a, p. 181
Dasyurops maculatus ae ee PS] Ht =) = =] K+] FP] 19460, p. 182
THYLACININAE—
Thylacinus cynocephalus .. 5 Sse Sala S| S| SB sey ss ios. then;
MYRMECOBIIDAE—
Myrmecobius fasciatus = .. |—|—}/—-}/—-}—-}—-|—-}-|-— —|—|+]/—|—}| 1949, p. 192
PERAMELIDAE—
Isoodon obesulus obesulus . . 2. |=} —)=l-4] +] =] —)— | —1 +) —] —| +] 4] —| 1946a, p. 182
Isoodon obesulus fusciventer eS SE el iti See 1943p. 100
Tsoodon macrourus . . He ee Sp ae SH Se] = 1948, pe LOW
Isoodon torosus is a ee ffm p yy FP =] =] =) HP 4] —) =] +) 4+) —| 1948, p. 101
Perameles nasuta .. 5 le ele ate ete ee ed ee SE 9435 pe, 102.
Perameles gunnii.. ne 2. Jf} —} +) +) =} +] —|—] +}-] =] —]4+]—]} 1946a, p. 1838
Perameles myosura myosura ee Jo} —P—l ya] y 4] =) —) 4+] —] —] +] +) —] 1948, p. 101
Perameles myosura notina ee fol Sia cig ap || ar 1943, p. 101
Perameles sp. ae + + sir lar 1949, p. 194
Macrotis minor minor an + 4 + +/+ 1946a, p. 184
Echymipera cockerelli be 22 fp] y Ey 4) =} +] —] =] +] —] —] —] +} —] 1948, p. 102
TARSIPEDINAE— “
Tarsipes spenserae .. Te .. |—f—} =} +) —] =} +) —] —] +] =| —] —] +] —| 1948, p. 102; 1949, p. 194
PHALANGERINAE—
Acrobates pygmaeus AG .. |—}—}—}—|—]-}—|—|-—| —} —| -| —| +] —] 1948, p. 103; 1946a, p. 185
Cercartetus concinnus me ee f=] slap Ht HP] =] =] =] =] =) - | 4) 1949, p. 194
Cercartetus nanus nanus .. .. |=}—}—}—}—|—}+}/—|-—]-|—-| -|-| +]—] 1946a, p. 185 ‘
Cercartetus nanus unicolor .. |—|—|—}—}—}—} +) —}—}-| —| —| —| +) —} 1946a, p. 185
Petaurus breviceps ah .. |—|—}—}—}—}—}+)—}—} +) +) -—| —| +|—| 1946a, p. 186
Petaurus australis .. a oe | Sf Hl HH Fl HK) I |=] +] =| 19460, pv. 186
Petaurus norfolcensis as .. f—f—}—}—-}—-}—y 4+ —|+)/+)—|—|+]—] 1946a, p. 186
Petaurus papuanus. . im .. |—}|—}—}—}—}—}+)—-|—| +) -—| —| —| +} —| 1946a, p. 187
Schoinobates volans. . 44)/ =) | 4E)/ 2 ee 1948, p. 104; 1946a, p. 188
Pseudocheirus laniginosus aR IPae Fl 5 3F 1946a, p. 187
Pseudocheirus convolutor ec. = -E} +)+ “+ 1946a, p. 187
Dactylopsila picata + — +/+ +/+ + 1948, p. 104
Trichosurus vulpecula v. so fy} SE) I Hf SEI EN) Nf SE lf | eS) aa, fo, TSS
Trichosurus vulpecula hypoleucus +} —|—|-+/—|—]}+/+}—|+]—]—]—]+]—] 1946a, p. 190; 1949, p. 194
Trichosurus fuliginosus ee |e) —lHl +} —) —) +) +) —] +) —!) —] —| +] —| 1946a, p. 190
PHASCOLARCTIDAE—
Phascolarctos cinereus +} —} +} +) —|—} +] +]—|+]—|] —| —| +/+] 1948, p. 106; 1946a, p. 192
VOMBATIDAE—
Vombatus ursinus u. We .. |—|/—}—}+}/—}—-}]—| —] —| +] —| —|—| +} —} 1946a, p. 192
Vombatus ursinus tasmaniensis .. |—|—|—|+|—|—|+]—|/—|+]—]—]—|+]—] 1946a, p. 194
Vombatus hirsutus h. ae ». }—}—}—} +) —)| —|—) —| —1| +) —-] —| —} +] —| 1946a, p. 195
POTOROINAE— ;
Bettongia penicillata ae ee |=] —]—} +) —}—] +] 4+} +] +] —| —| +] +] —| 1948, p. 106
Potorous tridactylus apicalis ee) |p] ) Pf] 4) +] +] —]—| —| +] —]} 1946a@, p. 195
Bettongia lesueur graii 5 ee }—}—}—) +) —)—} +) +) +)4+]—) —| +/4+]—] 1949, p. 195
Caloprymnus campestris .. -» |—}—|—| +] —| —] +] —| —] +) -—-] —| —| +] —] 1946a, p. 196

BY W. BOARDMAN. | 27

=~]

TABLE 1.—Continued.
Resumé of Marsupial Tract Pattern Arranged in Accordance with the Proposed Nomenclature. Centres of Universal
Occurrence in the Order are Omitted. (For explanation of symbols, see text.) i

ate :
|
Name. oi od | Hl oi] od] 3] Sled les|* ieee ata] Reference.
S\SIS PIF Slee |S) Els) wo] S].c} s|
RIAIAIAIASO/O/O/O/A/OlO;O/O/A
|
Sea ay eas male iI—| J—|— | |— |
MACROPODINAE— |
Dendrolagus inustus Je ee ee tet tet ee a AS pe ON
. Onychogalea fraenata | hy lf ef | ef ae er +|—|+]+| 1946a, p. 197
Thylogale sp. Sel |h-lhar ll arikar atallecta Rate +|]+) 1948, p. 108
Wallabia agilis Stele elt ictal icfalllctallictell ctelpctlliate lector OLGA. steel O9
Wallabia bicolor | | SPARSE ++ ais + + 1943, p. 111; 1946a, p. 199
Wallabia dorsalis +)/—)]—}+)+)+ + sr/—=|\Sr + 1946a, p. 200
Wallabia sp.’ ey) i] ff | a + 1946a, p. 200
Setonix brachyurus aD ; sr) Sar Sel Sey arse ate ar 1949, p. 195 '
Osphranter robustus AG bo =|—]-E} + + —|+ 1943, p. 112; 1946a, p. 200
Macropus major .. ne 2. {PAY —] 4) 4) 4+) 4) +) 4) 4+ = +}—| 1946a, p. 201

* Dates refer to papers by W. Boardman, see list of references. + See also Boardman (1945).

The scattered literature on hair tracts of mammals other than marsupials is, in the
aggregate, extensive and appears to be sufficient to serve as a basis for consideration of
the question of general applicability of the nomenclatural system that has been proposed.

For groups other than Primates the extensive work of Kidd (1900, 1902, 1903a,
19030, 1904, 1920) is the principal source although the usefulness of the descriptions is
restricted since only adult material was examined. To this should be added the papers
of De Beaux (1924a, 1924b, 1927, 1931) on fissiped carnivores, Potamochoerus and
Bradypus, of Wood Jones (1940) on Pedetes, of Niedoba (1917) on domestic animals
(rabbit, guinea-pig, sheep, goat, pig, cat, dog, cow, buffalo, horse, ass, mule), and of
Boardman (19460) on monotremes.

The Primates have attracted more attention than other eutherian groups. In

addition to the classical accounts of the hair tracts of man accurate information is avail-
able on a wide range of genera. Schwalbe (1910, 1911) has written on Galago, Lemur,
Propithecus, Indris, Tarsius, Macacus, Semnopithecus, Nasalis, Simia, Gorilla and
Anthropopithecus. De Beaux (1917, 1918) has written on Hylobates, Guereza, Cerco-
pithecus, Maimon, Choiropithecus, Papio, Hamadryas, Hapale, and Galago. Brandt (1940)
has discussed hair arrangement in Chiromys, Loris, Tarsius, Cebus, Pithecia, Macacus,
Cercopithecus, Hylobates, Anthropopithecus, and Gorilla, and .Osman Hill (1947) in
Arctocebus.
; A critical examination of this literature, particularly of that concerning the Primates,
leads to the conclusion that the principle of classification of tract pattern by reference
to divergent and convergent centres is applicable generally in mammals. Further, the
scheme drawn up for marsupials would serve equally well for other mammalian orders
as far as their hair tracts are at present known. There appears to be throughout the
class a unity of plan for the distribution of divergent and convergent centres. It would
seem that the spatial relationships of this system of centres (Part V) is within limits,
constant from order to order and therefore is to be regarded as a class attribute. As
Kidd (1904) has written, “No mammalian Order has been found possessing characteristic
whorls, featherings and crests’.

SUMMARY.
1. A symbolized nomenclature is proposed of divergent and convergent centres as
they are found in the marsupial skin.
2. The principle of classification is held to be applicable to the Mammalia generally.
3. It is suggested that the spatial relationships of the system of centres (divergent
and convergent) is, within definable limits of variation, constant for the Mammalia as a
whole.

bo
-~]
oo

HAIR TRACTS IN MARSUPIALS. VII.

References.

BeEAvux, O. DE, 1917.—Studi sui neonati dei mammiferi (Forma esterna). Parte prima. Primati
e carnivori fissipedi. Cap. 1-4. Arch. Ital. Anat. Embriol., 15: 467-541.
, 1918.—Id., Cap. 5-9. Ibid., 17: 144-215.
, 1924a.—Studien tiber neugeborene Sdéugetiere (A4ussere Form). Carnivora fissipedia.
Kapitel X-XI. Zool. Jahrb. Syst., 47: 331-378.
——, 1924b.—Beitrag zur Kenntnis der Gattung Potamochoerus Gray. Ibid., 47: 379-504.
, 1927.—Studien tiber neugeborene Sdugetiere (aussere Form). Carnivora fissipedia.
Kapitel XII-XIIl. Ibid., 54: 1-38.
, 1931.—Studi sui neonati dei mammiferi. 11 neonato di Bradypus tridactylus L.
Arch. Zool. Torino, 15: 35-82.
BoaRDMAN, W., 1943.—The hair tracts in marsupials, Part i. Description of species. Proc.
LINN. Soc. N.S.W., 68: 95-113. ;
————, 1945.—Some points in the external morphology of the pouch young of the marsupial,
Thylacinus cynocephalus Harris. Ibid., 70: 1-8.
, 1946a.—The hair tracts in marsupials. Part ii. Description of species, continued.
Ibid., 70: 179-202. :
, 1946b.—The hair tracts in monotremes; their relationship to the mammalian primitive
condition. Um. Qld. Pap. Dept. Biol., ii, 7: 7 pp. F
, 1949.—The hair tracts in marsupials. Part iii. Description of species, concluded.
Proc. LINN. Soc. N.S.W., 74: 192-195.
, 1950a.—The hair tracts in marsupials. Part iv. Direction characteristics of whorls
and meristic repetition of radial fields. Ibid., 75: 89-95.
, 1950b.—The hair tracts in marsupials. Part v. A contribution on causation. Ibid.,
75 (in press).
, 1950e—The hair tracts in marsupials. Part vi. Evolution and genetics of tract
pattern. Ibid., 75 (in press).
BRANDT, W., 1940.—The prenatal development of the hair tracts in primates. Human Biol.,
12: 203-231.
Hiuut, W. C. OSMAN, 1947.—The lorisoid genus Arctocebus: observations based on the type
material. Proc. Roy. Soc. Edin. (section B), 62: 248-265.
JONES, F. Woop, 1940.—The external characters of a neonatal Pedetes. Proc. Zool. Soc. Lond.,
110B: 199-206.
Kipp, W., 1900.—The significance of the hair-slope in certain mammals. Proc. Zool. Soe. Lond.,
1900: 676-686.
, 1902.—Certain habits of animals traced in the arrangement of their hair. Ibid.,
1902, ii: 145-158.
, 1903a.—Notes on the hair-slope for four typical mammals. Ibid., 1903, i: 79-83.
, 1903b.—The Direction of Hair in Animals and Man. London.
, 1904.—On proposed additions to the accepted systematic characters of certain
mammals. Proc. Zool. Soc. Lond., 1904, i: 142-150. ° :
MiIkLOUHO-MaAc.Lay, N. DE, 1885.—Notes on the direction of the hair on the back of some
kangaroos. Proc. LINN. Soc. N.S.W., 9: 1151-1158.
NIEDOBA, T., 1917.—Untersuchungen tiber die Haarrichtung der Haussaugetiere. Anat. Anz.,
Jena, 50: 178-192 and 204-216.
SCHWALBE, G., 1910.—Uber die Richtung der Haare bei den Halbaffen. In Voeltzkow, Reise in
Ostafrika in den Jahren 1903-1905, Bd. iv: 207-265.
, 1911.—Uber die Richtung der Haare bei den Affenembryonen. In Selenka, Studien
uber Entwickelunysgeschichte der Tiere, Bd. v: 205 pp.

bo
-]
vo)

A NOTE ON NON-RECIPROCAL FERTILITY IN MATINGS BETWEEN
SUBSPECIES OF MOSQUITOES.

By S. SMITH-WHITE,
Botany Department, University of Sydney.

[Read 27th September, 1950.]

The non-reciprocal fertility of matings between Aédes scutellaris scutellaris and
A. scutellaris katherinensis reported by Woodhill (1949a, 1949b, 1950) is of very con-
siderable interest in relation to the problem of speciation in mosquitoes. Matings
between the two subspecies prove to be fully effective and give fertile hybrids which
show characteristic Mendelian segregation in the F, when the subspecies scutellaris is
used as the female parent, but when katherinensis is used as female parent the mating
is ineffective and the eggs laid are completely inviable. The occurrence of cryptic and
sibling species in Anopheles and other genera has been well established (Mayr, 1942),
but in such cass sterility barriers and sexual isolation between the forms is complete.
In the present case, Woodhill has pointed out that the two forms would presumably
interbreed should they occur in the same territory, and in the absence of evidence to
the contrary they must be ranked as geographical subspecies. Comparable cases of
non-reciprocal isolation between “races” of other species of Aédes have been described
by Perry (1949) and by Downs and Baker (1949), so that the case is not exceptional.
It may even be of frequent occurrence in the Culicidae.

The origin and nature of non-reciprocal fertility is also of genetical interest because
it may involve an interaction between the nucleus and the cytoplasm. The existence
of undefined cytoplasmic incompatibility as an isolating mechanism between species
is probably of frequent occurrénce, but, where it involves complete and absolute isolation,
it is not susceptible to analysis. It is important that the present case should be actively
investigated by any methods which may become available.

Woodhill’s data demonstrate that in the sterile mating A. s. katherinensis x A. s.
scutellaris* effective copulation and insemination occur, live spermatozoa being found
in the spermathecae of the mated females. Two possibilities must be considered.

Firstly, the scutellaris sperm may fail to enter the katherinensis eggs, either because
of the serological conditions in the host females or for mechanical or other reasons.
There is abundant evidence that insemination, or even the mere act of copulation,
may serve as a stimulation to ovulation in insects, and the high egg production obtained
from the mating is of little significance. However, the low egg production of females
of Aédes albopictus when mated with A. aegypti, as reported by Downs and Baker (l.c.)
may indicate that copulation alone provides insufficient stimulus. It must be emphasized
that unless partial development of the hybrid embryos or actual fusion of gamete nuclei
can be demonstrated, it will be difficult to exclude this first possibility, but it would seem
to be improbable in view of the backcross data obtained by Woodhill.

The second possibility is that the sperm enter the eggs but that either they fail to
effect fertilization or the hybrid embryos die at a very early stage of development.
This possibility has implications of far-reaching significance, involving an incom-
patibility interaction between the cytoplasm of the katherinensis egg and the scutellaris
nuclear genom in the haploid sperm or the hybrid embryo. An explanation, tenable in
mammals, that the hybrid embryo is serologically incompatible with the body fluids of
the mother, is untenable in the Culicidae, where the entry of the sperm into the egg
occurs immediately prior to oviposition and where the actual nuclear fusion may occur

*In conformity with the usual genetical convention, throughout this paper the female
parent is written to the left and the male parent to the right of the x sign in denoting a mating.

WwW

280 NON-RECIPROCAL FERTILITY IN MOSQUITOES,

after this act, as it does, for example, in Anopheles (Nicholson, 1921). In the reciprocal
mating scutellaris x katherinensis the cytoplasm of the scutellaris egg must be fully
compatible with the katherinensis genom both in the haploid sperm and the hybrid
embryo.

The following formal scheme, although at present little more than speculation,
could explain such behaviour and leads to predictions capable of experimental verification
or disproof. It is dependent on the assumption that the insect sperm contributes
little or no cytoplasm to the zygote. There is evidence trom the inheritance of
CO.-sensitivity in Drosophila (L’Heritier, 1948) that the sperm of Diptera may contribute
a little cytoplasm to the zygote, but this would be small in comparison with the quantity
of cytoplasm contributed by the egg and would not be likely to invalidate the hypothesis.

If the cytoplasm of katherinensis contains a factor or factors incompatible with the
nuclear genom of scutellaris, the nuclear-cytoplasmic constitution of katherinensis may
be written as KKx, where K represents one haploid katherinensis genom, and x represents
the cytoplasmic factor. Similarly, scutellaris may be written SS;, where , may represent
a different cytoplasmic factor, characteristic of scutellaris cytoplasm, but not incom-
patible with the katherinensis genom, or merely the absence of x. It is not at present
suggested that x is necessarily particulate in nature.

The mating SS; x KKx would involve S; eggs and K sperms and would give the
hybrid combination SK;, which would be viable, but the mating KKx x SSs, involving
Kx eggs and S sperm, would fail. Further, it would be expected that, on backcrossing
the fertile hybrid SK, to both parents reciprocally the following matings would be
successful and would give progeny of the constitutions indicated.

Gametes Involved. Constitution of
Mating. fe) 3 Backeross Progeny.*

pets M. SKs SK

SKs x SSs — DS) — Ss
2 2
Sk SK

SSs x SKs Ss — Ss
2 2

pa Z Sks zs Sk _

SKs x KKk 5 K — Ks

SK
On the other hand, the mating KKx x SK;, which would involve Kx eggs and 2

sperms, might give either partial or complete sterility, according to the number of genes
in the S genom involved in the incompatibility interaction. In the unlikely event
that only one such “S” gene were involved, 50% of compatible sperm would be produced,
but with a number, n, of “S” genes, inherited independently, the expected proportion
of compatible sperm would be only 1/2". With numerous “S” genes randomly distributed
on the chromosomes and inherited in three linkage groups (the chromosome complement
of Aédes consists of three pairs of chromosomes), sperm from F, males lacking all
portions of the S genom, and consequently pure for the K genom, would be rare, if
they cecurred at all, and the backcross to KKx females would be completely unsuccessful.
It may be noted that meiosis in F, males has been found to be normal, with a mean
chiasma frequency above 2 per bivalent and not obviously lower than that in the
parental subspecies.

So far prediction is in agreement with the experimental evidence, on the assumption
that many “S” genes are involved. It is desirable that the hypothesis should be

Sk
* The formula 9 is used to designate the product of reduction division in the hybrid to
indicate that as the result of crossing over it will contain a haploid genom made up in part
of units of the S genom and in part of the K genom. The actual result in any particular
gamete will depend on the positions of chiasmata and the accidents of chromosome assortment.

‘

BY S. SMITH-WHITE. 281

tested further, and this could be done by a series of backcrosses of the types
KKiQ? x (SKs99 x KKiJd'S') bd
KKx9? x [(SKs99 x KKid'g) 92 x KKxdd1dS
ete.
It would be expected that the S genom would be gradually broken down, at a
rate dependent on the number of “S” genes involved and on their chromosomal
distribution, leading to partially fertile matings in the later backcross generations.

If this thesis of incompatibility between nuclear genes and cytoplasmic factors
can be established, it would be theoretically possible to analyse the nature of the
cytoplasmic factors involved and, so far as the author is aware, no comparable cases of
interspectific or inter-racial cytoplasmic sterility barriers have been analysed in plants
or animals, with the possible exception of the mating groups and the “killer” (Kappa)
substance in Paramoecium (Sonneborn, 1947). In the present case it should be
possible to determine the nature of the cytoplasmic factors, whether particulate and
plasmagenic or diffuse and perhaps hormonal, and also to determine the degree of
dependence of the x factor on the KK nucleus. Such an analysis would seem to require
the use of a third subspecies, compatible with katherinensis when the latter is used as
female, and compatible also with scutellaris. Since many subspecies of Aédes scutellaris
are already known (Woodhill, 1949a), and probably many more remain to be discovered,
these requirements should not be unattainable. Crosses of katherinensis with this third
species, and back crosses to the third species, always using the latter as the male, would,
when outcrossed to scutellaris, indicate whether the , cytoplasmic factor was dependent
on the KK nucleus or independent and permanent.

References.

Downs, W. G., and Baker, R. H., 1949.—Experiments in Crossing Aédes (Stegomyia) aegypti L.
and Aédes (Stegomyia) albopictus Skuse. Science, 109: 200-201.

L/HERITIER, PH., 1948.—Sensitivity to COz in Drosophila—A Review. Heredity, 2: 325-348.

NicHouson, A. J., 1921.—The Development of the Ovary and Ovarian Egg of a Mosquito,
Anopheles maculipennis Mieg. Quart. Jowr. Micr. Sci., 65: 395-448.-

Mayr, E., 1942.—Systematics and the Origin of Species. Columbia Univ. Press, N. York.

PreRRY, W. J., 1950.—Biological and Crossbreeding Studies on Aédes hebrideus and Aédes
pernotatus. Ann. Ent. Soc. Amer., 43 (1) :1238-136.

SONNEBORN, T. M., 1947.—Recent Advances in the Genetics of Paramecium and Huplotes.
Advances in Genetics. Ed. M. Demerec. Vol. 1. Academic Press Inc., N. York.

WoopHiLtt, A. R., 1949a.—A New Subspecies of Aédes (Stegomyia) scutellaris (Diptera,
Culicidae) from Northern Australia. Proc. LINN. Soc. N.S.W., 74:140-144.

———, 1949b.—A Note on Experimental Crossing of Aédes (Stegomyia) scutellaris scutellaris
Walker and Aédes (Stegomyia) scutellaris katherinensis Woodhill. Proc. LINN. Soc.
N.S.W., 74: 224-226. 2

, 1950.—Further Notes on Experimental Crossing within the Aédes scutellaris Group
of Species (Diptera, Culicidae). Proc. Linn. Soc N.S.W., 75; 251.

282

RESPIRATION AND CELL DIVISION IN DEVELOPING OYSTER EGGS.
By K.-W. CLELAND.
Department of Histology and Embryology, University of Sydney.
(Five Text-figures. )
[Read 25th October, 1950.]

INTRODUCTION.

Since Hertwig made the first observation of fertilization in the Naples sea urchins
in 1875, work on invertebrate eggs has played an important part in the history of
cytology and cell physiology. Warburg in 1908 initiated the study of the relations which
exist between the metabolism of developing eggs and the morphological changes which
they undergo; since this time many workers have contributed to the study of these
relations, which today are still far from elucidated.

The advantages of invertebrate, more particularly marine invertebrate, eggs are
not far to seek: they are readily removed from the parent in the form of cells and
washed free of other ovarian contaminants; they are usually in physiologically uniform
state and, being separated in a fluid to which they would normally be voided, are
remarkably stable in vitro; their availability in large numbers, physiologically uniform,
is an advantage not possessed by tissue cultures, and the rapid, continued synchronized
division in developing eggs makes them unique as material for physiological studies
on cell division.

Most of this type of work has been done on the small, relatively alecithal eggs of
sea urchins. Since they keep poorly under laboratory conditions, their study implies the
existence of a well equipped marine laboratory in reasonable proximity to extensive sea
urchin beds, facilities which are not locally available.

On the other hand, the rock oyster (Ostrea commercidalis) which is cultivated in
the Sydney area, will remain viable for up to six weeks out of water and is readily
maintained under laboratory conditions in small amounts of aerated sea water. Its
eggs, which are small, although made rather opaque by the presence of large numbers
of small yolk granules (Cleland, 1947), are available for a considerable period in a
fertilizable condition, have a satisfactory respiratory rate, and develop very rapidly.

The present work, which, with the exception of the colchicine experiments, was
performed in the 1948 season, was undertaken in order to define the gross aspects of
the respiratory metabolism of unfertilized and developing oyster eggs. In succeeding
papers the metabolism will be analysed in greater detail and such knowledge applied
to physiological studies of the mitotic process.

Apart from the work of Ballentine (1940) on O. virginica, there have been no
previous observations made on respiration in developing oyster eggs; Lucke and Ricca
(1941) have made a study of the permeability of the eggs of O. virginica.

MATERIALS AND METrHODs.

The source and maintenance of the adults and the method of preparing crude egg
suspensions are the same as described in a previous paper (Cleland, 1947).

Crude egg suspensions were strained and freed of cytolysed eggs, vesicular tissue
contaminants, etc., by three cycles of suspension in 50 volumes of sea water and centrifu-
gation at 35 x g for 60-75 seconds. The number of viable eggs separated from one oyster
varied between 5 and 30 x 10°.

Sperm was separated from male oysters in about 5 ml. of sea water and, after
straining, the resulting suspension centrifuged at 125 x g for 3 minutes to remove
spermatocytes, spermatids and non-motile sperm. Such sperm suspensions will retain
their fertilizing capacity tor up to four days when stored in a refrigerator at about 3°C.

BY K. W. CLELAND. 283

Fertilization was performed by suspending the washed eggs in 50-100 volumes of
sea water, adding about 1/200 volume sperm suspension and leaving for 15 minutes.
After this time eggs were washed once, as above, to remove sperm, suspended in about
10 volumes of sea water and placed either into Warburg vessels for immediate use or
into 150 ml. Erlenmeyer flasks shaken on the Warburg bath. In all the experiments
quoted in this paper the fertilization and development percentage was in excess of 90%
unless otherwise stated.

Oxygen consumption was determined by the direct method of Warburg in vessels of
15-20 ml. capacity. R.Q. was determined by the direct method of Warburg and by the
first method of Dickens and Simer. All procedures are described in Dixon (1943) and
Umbreit et al. (1945). In the tables and figures the respiratory rate quoted for. any ~
given time is the rate found for the previous time period; e.g., a value quoted for
8 hours after fertilization refers to the rate over the period 24-3 hours.

Egg numbers were determined on aliquots made to a final volume of 5 ml. This
was placed in an optically plane-bottomed dish of 4 cm. diameter, the suspension agitated
and, after settling, the number of eggs in twenty-four microscope fields counted. The
mean number per field was multiplied by the factor relating the surface area of the
microscope field to that of the dish. The coefficient of variation of the mean number
of eggs per field was 3:9%, which was a close approach to the theoretical value derived
from the Poisson distribution.

The volume of the eggs used was determined on aliquots by the centrifuge method
of Shapiro (1935). Because of the absence of jelly layer the eggs pack more quickly
and compactly than sea urchin eggs; a centrifugal field of 700 x g acting for 5 minutes
gave a valid estimate of volume.

Total nitrogen was determined by combustion of aliquots in H.SO, and H.O., steam
distillation of the digest in the Parnas Wagner apparatus, and Nesslerization of the
distillate.

After the completion of an experiment on developing eggs, development was followed
in aliquots diluted 1/200 for a further 18 hours. In all cases at least 50% of the eggs
were alive and had developed into typical trochophores. The low percentage development
is associated with, and was presumed due, to bacterial growth in the cultures.

Determinations of the stage of development in the closed Warburg flasks were made
by reference to a parallel shaken Hrlenmeyer flask culture. This flask was stoppered
and had provision for CO. absorption.

All experiments were conducted in a Warburg bath maintained at 26°C. with a
shaking rate of 90 cycles/min. and an amplitude of 6 cm.

RESULTS.

In twenty-nine determinations the number of eggs in 1 ml. of packed eggs was
found to vary from 13-17 million, with an average of 14-5. One ml. of eggs contained
an average of 28 mg. of total nitrogen with a range of 24-32 (25 determinations). One
ml. of eggs had a dry weight of 240 mg. with a range of 230-260 (six determinations).

Effect of Continued Shaking.

It has been known for a long time that the rate and amplitude of shaking is an
important factor in studies of invertebrate egg respiration by the Warburg method
(e.g. Whitaker, 1933). In the present instrument the amplitude was fixed and the rate
could not be reduced below 90 cycles per minute. The influence of time of shaking was
investigated by removing aliquots from a large, gently aerated, stationary culture of
fertilized eggs at hourly intervals, recovering the eggs by centrifugation, and trans-
ferring them to a Warburg vessel, which was then shaken and read until the end of the
experiment. The results, which are shown in Table 1, indicate that eggs may safely be
shaken for periods of seven hours without altering their respiratory rate. Loss of eggs
by cytolysis, as determined by successive estimates of volume, was almost invariably
found in both stationary and shaken cultures, up to a maximum of 10% of the initial
volume of eggs.

284 RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

Respiration of Unfertilized Eggs.

The majority of unfertilized egg batches could be shaken constantly in Warburg
flasks for 7-8 hours without change in respiratory rate (Text-fig. 1, curves 1 and 2). In
about 20% of cases eggs shaken for 5-6 hours showed a considerable increase in
respiratory rate as shown in Text-figure 1 (curves 3 and 4). Examination of such eggs
revealed signs of cytolysis, the eggs appearing perfectly spherical and more light-

04
ae
= y
< e
GE WA :
a 74 -~~e~ (ee ~~9- fee” Bla gle
O eg de ge visell ae
= Sails ad Thee ju wr ee ee reeeneene ~~
<< ~e
=
ae
Y)
ud
(ae

SHAKING BEGUN —?

fe) 2 3 4 6
HOURS AFTER REMOVAL

Text-figure 1.

Respiratory rate of unfertilized eggs during prolonged shaking. Curves (1) and (2) were
typical of the majority of batches studied, curves (3) and (4) from batches showing evidence
of cytolysis at the end of the experiment. The respiratory rate is expressed as wl. O2/5 x 10°
eges/% hour.

TABLE 1.
Effect of Continued Shaking on the Respiratory Rate of Developing Eggs.

Hours After Fertilization.

Flask ese atte eR oe ; Mi. : ‘i hy
No. | | l | -
De wiles Dead Woks sual oR | 4 | 43 5 Bye 6 64 7
|
— |
| |
I 30 35 Abe. |v Seta, spe ath aeGe 68 65 85, | 95 103
2 = ~ £3 ee DOUen ear o0 menemaao 63 | 62 Pe eee) 94
3 a — = = oy 49) | 55 GBao l= BD) 75 86 95
4 _ - | | | | 66 63 83 90 100
5 | ile 78 85 95
a b

Respiratory rate is expressed asl. O, per flask per half hour. The slight variations between flasks are probably
due to varying losses of eggs during the centrifugation and resuspension of the samples. The times of somatoblast
division and hatching are shown at @ and b. é

BY K. W. CLELAND. 285

absorbing or having an appearance corresponding to abnormality I in egg suspensions
described in a previous paper (Cleland, 1947). It is not known whether this increased
respiratory rate occurred in stationary cultures also.

The respiratory rate of 40 batches of unfertilized eggs, some of them from individual
oysters, some from pooled eggs, is shown in Text-figure 2, where observations of the
state of the germinal vesicle are also recorded.

In a previous paper (Cleland, 1947) it was stated that the germinal vesicle of the
oyster does not break down until fertilization occurs. More intensive study of the
material has shown that the eggs fall into three classes in their behaviour to fertilization:
(a) those which show no germinal vesicle breakdown and no activation even though
sperm penetration has occurred; (0) those which show ho breakdown or incomplete
breakdown in sea water but which show activation on fertilization; (c) those which pass

NUMBER

te emesis ia
2 RESPIRATORY RATE (ul/ml/hr)

Text-figure 2.
Frequency histogram of the respiratory rate of unfertilized eggs. The black areas are from
batches showing more than 75% germinal vesicle breakdown, the dotted areas from batches
with 10-75% breakdown, and the white areas from batches showing less than 10% breakdown.

to the first maturation metaphase in sea water and remain at this stage in the absence
of fertilization. Eggs taken at the height of the breeding season almost invariably
belong to the latter class; the second class was only rarely found.

The respiratory rate of class (c) eggs has tended in later seasons to be lower than
that found characteristic for eggs in the 1948 season.

Effect of Fertilization on Respiratory Rate.

The effect of fertilization on the respiratory rate was investigated on six batches
of eggs (each from one oyster only), fertilization being effected by tipping in 0:1 ml.
of sperm suspension from the sidearm of the Warburg flask. The respiration of this
quantity of sperm was negligible.

In Text-figure 3 the mean and two of the experimental curves are shown. Im all
curves no increase in respiratory rate was found until the onset of the first cleavage

286 - RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

division. After this the curve may show a smooth rise, as in curve 3, or show
differences in respiratory rate with different stages of the division cycle, as in curve 2
or curve 1, which is the mean curve of the six experiments.

The data indicate that the maximal respiratory rate is found in the prophase,
metaphase and possibly anaphase, while the minimal rate is found in telophase and the
resting stage. The differences between different experiments were evidently due to
differences in the degree of synchronization of division; in the poorly synchronized eggs
the curve was smooth, while in the well-synchronized it showed the mitotic correlation.
Synchronization as good as that found in Arbacia eggs (Fry, 1936) has never been
found in the present material.

Respiratory Rate during Development.

The respiratory rate of 20 batches of eggs was followed for periods of up to eight
hours after fertilization. At the peak of the season, when synchronization is good and
more than 95% of the eggs are fertilized curves typical of those shown in Text-figure 4

e)
@
23
ia
< a
fy 2 a eo |
Ve) a & i
x ‘ig Ve ]
e q Ve
= Hi Fg
: ; Ve
a Lf
at S ’ VA ee e
tJ —__ *__,_ —° ——e —e sie Weg
(ae Ay ia e mals WA
ites. oo lize
O BODIES ; CLEAVAGES
N o ; : 4

HOURS AFTER FERTILISATION

Text-figure 3.
Respiratory rate (expressed as ul. O2/5 x 10° eggs/4 hour) following fertilization.

were found. The first two plateaus may be somewhat masked, as in Text-figure 5, when
the middle of the critical periods falls near a point of reading.

The early development of 0. commercialis, which does not appear to differ signifi-
cantly from the development of O. gigas (Fujita, 1929) or O. virginica (Brooks, 1880)
may be divided into the following stages: (a) stage of meiotic divisions lasting for
about 60 minutes after fertilization; (b) early cleavage stage up to 3-4 hours after
‘fertilization; (c) stage of disappearance of the macromere, due to division of the
“somatoblast” (Fujita) and occurring at about three and a half hours after fertilization;
this is associated with depressed cleavage rate; (d) later cleavage stage terminating
at 4-5 hours after fertilization; (e) the first signs of hatching are seen in a non-
translational rotatory movement of the larvae which becomes progressively more rapid
and appears to be associated with a depressed mitotic activity; (f/f) stage of increasing
swimming intensity which is associated with increasing rate of translational movement

BY K. W. CLELAND. 287

and occurs between hatching and about six and a half hours after fertilization; (g) stage
of maximum swimming intensity is reached about seven hours after fertilization. At
this stage the larvae in stationary cultures arrange themselves in vertical columns as
described by Roughley (1933).

It will be noted in Text-figure 4 that three plateaus in the respiratory rate are
present: one at somatoblast division, one at hatching and one when maximal swimming
rate is reached. The first two are evidently due to depression of the mitotic rate and
the third to attainment of maximal ciliary activity, the latter probably accounting for
most of the rise after hatching.

Effect of Blocking Cell Division on Respiratory Rate.

In order to see what relation the rise in respiration had to cell division, experiments
were performed in which colchicine was added from the side arm at different times
after fertilization. The results of such an experiment (one of three) are shown in
Text-figure 5. Similar results were obtained with a number of other mitotic inhibitors
and will be considered in a later paper of this series. Almost identical results were
obtained over a hundredfold colchicine concentration range (M/100—M/10,000), sug-
gesting that penetration of the alkaloid or primary inhibition of respiratory enzymes
was not the explanation of the results. Below the minimum biologically effective
concentration (approximately 10-*M) colchicine was without effect on respiration.

Effect of Suspending Medium.
Table 2 records one of the three experiments performed showing the influence of
suspending medium and pH on the respiration of developing eggs. Glycine has been

x Zs TABLE 2.
Effect of Suspending Medium and pH on Respiration of Developing Eggs.
Hours After Fertilization. 4 |
Initial ih i _| Final
Medium. ; pH. | | > Wiel
13 2 24 3 3h 4 44 5 53 6a
Sea water .. 2s 7-85 70 82 108 144 148 1/9 L8H) 225 280 275 | 7-4
M |
Sea water + ——
; 100
KHL Oe a as 5:8 65 74 | 100 130 130 150 150 184 220 230 | Bo
Sea water +7" glycine 8) 70 84 117 150 160 190 190 250 315 325 7:6
5 |
M
Sea water + — phos-
a 100" | |
phate buffer 6-3 67 75 106 135 137 165 165 207 245 250 | 6:3
Sea water + HCl 6-0 65 78 104 135 | 138 | 162 | 164 195 235, 240 | 6-0
a b |

The respiratory rate is expressed as Ll. O, per half hour per 5 x 10° eggs. a and b show the times of somatoblast
division and hatching. No difference in the amount of egg breakdown in the various media could be detected.

recommended as a sea water buffer by Robbie (1948). The increased respiratory
rate with glycine, especially after hatching, was presumed due to utilization of the
glycine; glycine added from the side arm to larvae in sea water causes a rise in
‘respiratory rate to the level of larvae which have developed in glycine sea water. The
more marked depression of respiratory rate after hatching at low pH was associated
with, and presumed due to, depressed ciliary activity.. Similar results were obtained
with unfertilized eggs. /

Respiratory Quotient during Development.

Since development proceeds normally at a pH where CO. retention can be satis-
factorily corrected for by the method of Johnson (Umbreit et al., 1945) periodic
respiratory quotient determinations on one egg sample are possible by the direct

'

288 RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

method of Warburg. The validity of the KCO. correction for pH was established by
estimates of the increase in the CO, which could be liberated by H.SO,. Continuous R.Q.
determinations were made on fifteen batches (from individual females) of developing
eggs suspended in M/100 KH.PO, and sea water (pH 5-8) and, in about half of these,
values typical of those shown in Nos. 1-3 of Table 3 were obtained where little or no
change during development was found. In the other half (Nos. 4-6) the R.Q. appeared to
rise, especially after hatching. No pH change was detectable in any of these experiments.
Since this rise occurred in cases where the amount of eggs per flask was higher it was
suspected that some metabolic product was causing a depression of oxygen uptake in the
flask without KOH. Since this was abolished by replacing the gas atmosphere by fresh
air it appeared that the relatively high CO. tension in the gas space was responsible.

It will be seen from Table 3 that some variation in respiratory quotient is found
in different egg batches and that little or no change occurs on fertilization.

TABLE 3.
Respiratory Quotient during Development.
|
| Hours After Fertilization.
Expt. | Unfer- é
No. tilized. | | l |
14 2 Oe alleen 3h | 4 ae 5 Bee a6
| | |
|
1 0-82 0-84 | 0-82 0-82 0-83 0-81 | 0°86 0-83 | 0-85 0-84 —
2 0-80 0-78 | 0-79 0-78 0:78 0-76 0-81 0-77 0-82 0-83 | 0-82
3 0-78 0-81 0-738 0-83 0-85 0-81 0-84 | 0-80 —_ — |
4 0-78 0-79 | 0-79 0-79 0-77 0-81 0-82 0-81 0-84 0-89 | =
5 0-84 0-84 0:86 | 0-91 0:87 0-94 0-84 | 0-90 0-96 0:94 | 0-97
6 0-76 | 0-78 | 0°80 0-82 0-84 0-83 0-86 0-88 0:94 0:99 | 1-03
1 | | | |

In eight experiments parallel determinations of R.Q were made with the direct
method of Warburg and the first method of Dickens and Simer. These determinations,
some of which are shown in Table 4, indicated that the results of the two methods
were in fair agreement. The R.Q. of eggs in sea water was the same as that of eggs
in KH.PO, buffered sea water in determinations by the Dickens and Simer method.

TABLE 4.
Comparison of the Respiratory Quotient by the Direct Warburg and Dickens and Simer Methods.
Hours After Fertilization. >
Experiment
Number. Method.

14-3 3-4 4-5 5-6

il Direct Warburg ae Hi ee ee 0:79 0-80 0-82 0-384
Dickens and Simer or Pic le 0:78 0:78 0-380 0-81 -

|
ase al

2 ; Direct Warburg Br co a iy 0-79 | 0-78 0-82 0-383

Dickens and Simer oe oH ro 0-82 ‘| 0-80 0-84 0°85

3 | Direct Warburg... Hs Ss 3 0-80 | 0+82 0-82 0-85

Dickens and Simer ap We a 0-80 0-80 0-79 | 0-82

DISCUSSION.

In Cartesian diver determinations of respiratory rate Holter and Zeuthen (1944),
Zeuthen (1944) and Borei (1948) have found that there is a decline of respiration of
unfertilized eggs of Ciona, Rana, Psammechinus and Asterias, with time after removal
from the ovary, a constant rate being reached 2-3 hours after removal. Such a

BY K. W. CLELAND. 289

declining respiration appears never to have been observed in Warburg experiments and
was not found in the present case even when the first reading was made twenty
minutes after removal of eggs from the ovary.

(e)
(e)
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——— (eS)
FIRST CLEAVAGE SECOND CLEA- INCREASING MAXIMUM
STAGES VAGE STAGES SWIMMING SWIMMING
RATE RATE

I 2 3 4 5 6 7 8
4 HOURS AFTER FERTILISATION
—
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I 2 3 4 5 6
5 HOURS AFTER FERTILISATION

Text-figures 4 and 5.

Fig. 4.—Respiratory rate (expressed as pl. O2/5 x 10° eges/i hour) and the recognizable
morphological stages during development. ‘

Fig. 5.—Effect of the addition of colchicine on the respiratory rate of dev eloping eggs
Curve 1 shows the respiratory rate of colchicine-treated unfertilized eggs, and curve 4 is the
control developing eggs. In curves 2 and 3 colchicine was tipped from the sidearm at the
points indicated; the development ceased in these eggs at the following metaphase. Colchicine
concentration 10“M; respiratory rate is expressed as ul. O2/flask/% hour.

290 RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

The rather sudden marked increase in respiratory rate which is capable of
occurring in unfertilized eggs (Text-figure 1) suggests that the respiratory mechanism
is not normally functioning maximally but is presumably limited by such factors as rate
of substrate mobilization (cf. Ballentine, 1940).

The effect of fertilization of an egg on its respiratory rate has been determined
in a large number of animals. These observations have been collected in Table 5. An
attempt was made to make this table comprehensive in respect of genera that have
been studied, but not in respect of authors studying the same form. References not
quoted in the present paper may be found in Ballentine (1940).

The only previous attempt to explain the difference in respiratory change at
fertilization in different animal species appears to have been. that of Whitaker (1933).
He suggested that there was a normal range of respiratory rate which was necessary

for development in any egg; unfertilized eggs with rates lower than this showed an
TABLE 5.
Changes in Respiratory Rate on Fertilization in Eggs of Different Animals.
Nuclear Ratio | Nuclear | Ratio
Type. Genus. F./UF. Author. Type. Genus. F./UF. Author.
1 Nereis. 1-4 Whitaker (1931). 3 Rana. Gis il | Brachet (1934a).
‘3 1:25 | Barron (19382). 3 ca, Zeuthen (1944).
| Mactra. 1:8 Ballentine (1940). Bufo. 1 | Stefanelli (1938).
2 Ciona. 1:7 Tyler and Humason Fundulus. 16:7 Boyd (1928).
(1937). x 45 1(?) | Philips (1940).
3 ca. i Holter and Zeuthen 4 Dendraster. 2-6 Tylerand Humason
(1944), | (1937).
Cumingia. 0-45 | Whitaker (1931). Fucus. | 1:85 | Whitaker (1931).
| Ostrea. 1-4 Ballentine (1940). | Strongylo- Sie Tyler and Humason
| ns | 1:0 Cleland (1950). || centrotus. (1937).
Chaetopterus.| 0-81 | Ballentine (1940). | Psamme- 1-9 Laser and Roths-
PY | 0-54 | Whitaker (1931). | chinus. child (1939).
| Asterias.* | 0-98 | Tang (1981). | | * 6 | Shearer (1922).
nS ca. 1 Borei (1948). | | EA) | 3-6 | Borei (1948).
| Sabellaria. 1-12) Faure: Hremiet * || | Paracen- 6 | Warburg (1915).
| (1922). trotus.
Urechis 1-15 Tyler and Humason = 4-7 | Brock et al. (1938).
| (1937). | Arbacia. 5 | Tang (1931).
| act sen 2-6 | Shapiro (1936).
| 3 4:5 | Ballentine (1940).

*Proceeds to complete maturation in the absence of fertilization.
References not quoted in this paper may be found in Ballentine (1940).

increase, while eggs with higher rates showed a decline on fertilization. He noted a
number of exceptions to this rule, and the criticism of Gerard and Rubenstein (1934)
have cast some doubt on the whole hypothesis. Indeed calculation of the respiratory
rate per unit volume of eggs in the animals shown in Table 5 indicates that the
fertilized egg values vary over a sevenfold range. Four types may be distinguished in
the nuclear state of eggs on spawning (Just, 1938; Wilson, 1925): (a) eggs in which
the germinal vesicle remains intact on spawning and breaks down only after fertilization;
(b) eggs which proceed to the first maturation metaphase after spawning; (c) eggs
which proceed to the second maturation metaphase after spawning; (d) eggs with
haploid resting nucleus on spawning.

The data recorded in Table 5 have been arranged in these four groups. The
assigning of animals to any group has been difficult, requiring a reference to the
embryological literature, where available or by analogy with related forms.

The following points emerge from this comparison: (a) in the fourth group, com-
prising mostly the sea urchins, a rise in respiratory rate of at least twofold occurs on

wat

BY K. W. CLELAND. 291

fertilization; (6) the third and second groups are characterized by either no change
or a depression; the first group shows a moderate or marked rise.

The apparent exceptions to this generalization are: (1) Ciona, where Tyler and
Humason find a considerable increase on fertilization; this rise was not found in Ciona
eges by Holter and. Zeuthen. (2) Ostrea, where Ballentine found a rise at some
unspecified time after fertilization, while the present work has failed to find any rise
associated with fertilization per se. (3) Fundulus, where Boyd found a marked increase
which disappeared when the first cleavage division occurred. This phenomenon could
not be confirmed by Philips (1940) and the rise on fertilization is therefore questionable.

It will be seen that these exceptions are all capable of other explanation and cannot
be considered as critical evidence against the generalization. There is thus a fair
correlation between no rise in respiratory rate on fertilization and a kinetic state of
the nucleus and a moderate or marked rise with a non-kinetic nucleus. This suggests
that respiration is increased in eggs either by previous entry into the meiotic divisions
or by sperm penetration.

That this postulated rise in respiratory rate on entry into the meiotic divisions is
not due solely to the increased respiratory rate found in cells during the division
cycle is indicated by those cases in the second class where the rate shows a considerable
decrease on fertilization. This suggests that the germinal vesicle breakdown itself is
the main cause of the rise in the second class, the higher rate being subject to further
modification by fertilization in forms such as Chaetopterus and Cumingia.

The rise in respiratory rate on germinal vesicle. breakdown may well be due
primarily to physical changes in the cytoplasm and is perhaps analogous to the rise
found in some of the batches of unfertilized eggs shown in Text-figure 1. It is not
difficult to imagine how liberation of the very aqueous germinal vesicle contents
(Cleland, 1947; Harris, 1939) could cause such physical change and, indeed, a consider-
able difference in cytoplasmic viscosity has been found (centrifuge method) between
eggs with intact and broken germinal vesicles in the present material.

Generalizations such as this, derived from a comparative study of respiratory
change on fertilization, cannot be regarded as any more than suggestive. There are,
however, two more direct lines of evidence suggesting its validity. In’ the present
material there is a difference in respiratory rate between unfertilized eggs with and
without germinal vesicle breakdown (Text-figure 2). At first sight these two groups
seem to overlap somewhat in respiratory rate. However, when the percentage of
germinal vesicle breakdown is considered it will be seen that the overlapping could be
quite satisfactorily explained on that basis, the probable mean respiratory rate for
eggs showing more than 90% germinal vesicle breakdown, being about 425 wul./ml./hr.
and that of eggs showing less than 10% breakdown being about 200 wl./ml./hr. A
considerable number of attempts were made to follow the respiratory rate of eggs
during the germinal vesicle breakdown. No increases could be demonstrated, but by
the time readings could be begun breakdown was already well advanced and the
experiments are thus not conclusive. In a previous experiment (Cleland, 1947) the
respiratory rate of unfertilized eggs was found to be considerably lower than that
found in the present series. This difference may be due to the fact that the germinal
vesicles did not break down until after fertilization in the 1947 experiment.

Borei (1948) has been able to follow the respiration of Asterias eggs during germinal
vesicle breakdown and the meiotic divisions. He was able to show that there was a
marked increase in respiratory rate when these processes occurred and that the activa-
tion persisted after the meiotic divisions were completed.

Studies on the respiratory rate during mitosis are of considerable importance in
assessing the metabolic cost of the various stages. The available evidence has been
considered by Zeuthen (1944, 1947). The present work confirms that of Zeuthen, but
owing to the incomplete synchronization, accurate quantitative estimates of the
respiratory rate at various stages were not possible. It would appear, however, that the

Main energy requirement occurs during prophase when chromosome synthesis is

bo
©
bo

RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

occurring.* The absence of any demonstrable change in the maturation divisions
where no chromosome synthesis occurs is in agreement with this.

Changes in respiratory rate and development with pH have been studied by a
number of authors. In general there is a depression of respiration with increasing
hydrogen ion concentration (e.g., Warburg, 1910; Root, 1930) and. the present material
is no exception. The effect of pH on development is different in different forms, even
among genera of sea urchins. For example, Warburg (1910) found that Strongylo-
centrotus eggs were unable to develop at pH 6, while Arbacia eggs can develop over a
wide pH range (Smith and Clowes, 1924). In the present form a slight delay in
development of ten minutes in four and one-half hours is found at pH 5-8.

In a series of papers (1933-1938, reviewed by Tyler, 1942) Tyler has presented
strong evidence that the amount of energy (as measured by respiration) required to
perform a given amount of morphogenetic work is remarkably constant under a variety
of experimental conditions. He was unable satisfactorily to “dissociate” (Needham, 1936,
1942) the total respiration from the final morphogenetic result. It would appear that a
partial dissociation has been accomplished in the present material by low pH; up to
the hatching stage the same amount of morphogenetic work is accomplished with the
consumption of significantly less oxygen.

A number of authors have attempted to study the causes and significance of the
rise in respiration during development by experimental interference with normal
development. Lindahl (1936), Lindahl and Ohman (1938) found that when normal
development of sea urchin eggs was modified by lithium treatment, which itself had
no effect on initial respiratory rate, the normal rise in respiration was inhibited.
Brachet (1938) found that Chaetopterus eggs, when activated by KCl to a differentiation
without cleavage in which the nucleus undergoes anomalous division cycles and the
cytoplasm differentiates to give a unicellular trochophore-like body, show a slower rise
in respiration than in normally devéloping eggs. Tyler and Horowitz (1938) have
reported a similar situation in Urechis eggs, and Andresen et al. (1944) have demon-
strated the very anomalous “development” of Ciona syncytia is associated with con-
siderable rise in respiratory rate. Horowitz (1940) demonstrated that when gastrulation
of sea urchin eggs is prevented by thiocyanate, the respiration of the “‘dauerblastulae”
does not rise above that typical of the normal blastula. Andresen et al. (1944) have
expressed the opinion that the reason for the rise of respiratory rate during development
lies in the chemical potentialities of the unfertilized egg and that once the egg is
activated there follows a more or less predetermined series of chemical events which
are largely independent of associated morphological changes.

The experiments reported in this paper do not support this opinion; when cleavage
is prevented by colchicine there is no increase in respiratory rate and the normal rise
in respiration is evidently a function of both the increase in cell number and the
continued sequence of cell divisions, during which the respiration is raised (ctf.
Text-fig. 3).

The facts emerging from the colchicine experiment are as follows: (i) Unfertilized
eges show a slight decrease (10%) in respiratory rate after treatment with colchicine.
This decrease is greater than that found in unfertilizable eggs (other experiments),
suggesting that at least part of the depression is due to interference with the meiotic
spindle. (ii) The application of colchicine at the two-cell stage causes a greater
depression of respiratory rate than that found in unfertilized eggs. (iii) The application
at later stages leads to an even greater inhibition, but the constant level is
higher than that found in the earlier stages of development. (iv) In all cases except
unfertilized eggs there is a time lag between application of colchicine and full respira-
tory depression. The absence of a concentration effect implies that this is not due
to delay in penetration of the eggs by the alkaloid. ‘

* The recent experiments of Bullough (Nature, 165: 493, 1950) are in agreement with this.
Also Bap. Cell. Res., 1: 410.

BY K. W. CLELAND. 293

The interpretation placed on these observations is as follows. Colchicine, by inter-
ference with the spindle, abolishes part of the activity of the cell and, this activity being
abolished, that fraction of the total respiration concerned in producing the energy
required for it is also abolished. The lag in effect is due to the phase specificity of the
colchicine and to the absence of complete synchronization of the divisions both in
single eggs and in different eggs of the batch. The colchicine stable residual respiration,
which increases with development, is regarded as a maintenance respiration, i.e. the
respiration concerned in the production of the energy normally used in the maintenance
of the cells as living systems. The observations indicate that this fraction of the total
respiration increases with increase in cell number (cf. Robbie, 1948), although not
proportionately to the increase in the activity metabolism associated with the increase
in the number of dividing cells.

This interpretation is supported by the study of the respiratory rate during
development. When the mitotic rate is physiologically depressed, as it is at the
somatoblast division and hatching stages, the respiratory rate either remains constant
or shows a decrease, the former presumably when synchronization of development and
the choice oi observation times is not good, the latter when it is more perfect.

The above concept of maintenance metabolism has no relation to that proposed by
Fisher and Stern (1942), Fisher and Henry (1944), Ormsbee and Fisher (1944),
Moog (1944). In the latter concept the maintenance metabolism is regarded as
qualitatively different from the activity metabolism, while in the former the distinction
is purely quantitative. : -

The observation that CO, causes an inhibition of respiration -in oyster eggs is in
qualitative agreement with the work of Root (1930) on Arbacia eggs. Invertebrate
eges evidently differ in this respect from mammalian tissues, which are often stimulated
or stabilized by the presence of CO, or bicarbonate (Laser, 1935, 1942).

Needham (1930) has adduced considerable evidence, mostly from respiratory
quotient studies, to support a hypothesis of a succession of energy sources during
development (carbohydrate — protein — fat). Later (Needham, 1942) this was
modified to exclude the cleavage stages and the succession considered to begin at
gastrulation. During the cleavage stages the R.Q. differs widely in different animals:
1:0-0-8 for Urechis (Horowitz, 1940); 0-6—-0-7 for the frog (Brachet, 1934); 0-75—-0-85
for Paracentrotus (Ohman, 1940). Assuming that these determinations are valid, there
thus appears to be ho substrate preferentially oxidized during the cleavage stages.

In the developing oyster egg the R.Q. suggests combustion of either protein or
a mixture of carbohydrate and fat. Analyses of ammonia production of eggs indicated
that the combustion of protein was insufficient to account for more than a part of the
normal respiration; thus the second alternative seems the more likely.

; SUMMARY.

1. Methods of handling the eggs of the oyster are described.

2. Some batches (about 20%) ot unfertilized ‘eggs may show sudden marked
increases in respiratory, rate about four hours after removal from the ovary; this is
usually correlated with morphological signs of cytolysis.

3. A considerable difference in respiratory rate was found between unfertilized eggs
in which the germinal vesicle remains intact and those in which it breaks down. It is
suggested that a causal relation exists between these two factors.

4. No increase in respiratory rate occurs on fertilization until the onset of the
first cleavage division.

5. Slight periodicity in the respiratory rate during the early cleavages suggests
that the respiratory rate is higher in the pre-anaphase stages of division.

6. The respiratory rate rises considerably during development and shows three
plateaus: at somatoblast division, at hatching and at attainment of maximum swimming
intensity. The first two plateaus are associated with depression of the mitotic rate.

7. Eggs are capable of unretarded normal cleavage at pH 5-9. At this pH significantly
less oxygen is consumed in reaching the hatching stage than in sea water at a pH of 7-5.

294 RESPIRATION AND CELL DIVISION IN OYSTER EGGS,

8. Colehicine abolishes the rise in respiratory rate during development. A respira-
tory depression is found after colchicine treatment, and it appears after a lag period.
The depression is slight in unfertilized eggs but becomes greater with increasing
development of the eggs. A lower constant level is reached which is higher with
increasing development of the egg. These facts are interpreted in terms of activity and
maintenance fractions of the total respiratory metabolism.

9. The respiratory quotient has a value of about 0-80. This is apparently due to
combustion of a mixture of carbohydrate and lipide. No change in R.Q. takes place
during the first six hours of development.

10. The reported respiratory change on fertilization in a range of animals is
examined and a hypothesis suggested which accounts for most of the facts.

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BY IX. W. CLELAND. 295

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THE INTERMEDIARY METABOLISM OF UNFERTILIZED OYSTER EGGS.
By K. W. CLELAND,
Department of Histology and Embryology, University of Sydney.
(Three Text-figures. )
[Read 25th October, 1950.]

Contents.
Page
Introduction a ain een hae Psy Raa ied ie GM Wier aN en re A enc eARON Seep WL) utah ay Ue
Mateuialskanaunictnods hss gh Bee ys KO aneet psesth eae Se rats RA ee an eee CR 5 ch yy pe
297

Analyses of Eggs ay Phe aS ats tea Mis iG ha At) Yn tical set SRT. Bee a HEI he eee ote ie
Experiments on Whole ase
(a) Effect of inhibitors 2
(b) Effect of methylene blue Anal pose eubete Aes ne We ASV EMEA) Wheto er) ieee (ONRA A eS OY ye
(c) Glyecolysis 2
(d) High energy phospuate AeTREKSOR. 3
Experiments on Homogenates—

(a) General as Reena ey aARS el SES Ci Ta ee I ee Seber Oi a, ey ie eee Ere eR OR eM C(O
(b) Effect of dopyeteannts IE Eee cs NEY eb esi ROB e oe a eh coin oo? SIR eae owe oe ~ SADE
(c) Effect of lipides ottate >. 503
(d) Effect of amino acids 303

(e) Effect of tricarboxylic acid cycle sintenmediates. Se Sack et APOE, ete eb EO aT ek eae gr ee hl a pera (pee
Specific Enzymes in Homogenates— :

(a) Cytochrome oxidase 30¢
(b) Succinoxidase 306
(c) Malic oxidase ae 307
(d) Miscellaneous Reeirais tel enzymes 308
Complex Enzyme Systems in Homogenates—
(a) Glycolysis F ate ~ 308
(b) Tricarboxylic aera ay 311
(ce) High energy phosphate PHOTO. 312
Discussion 312
Summary 317
er omiede nents 318
References 318

INTRODUCTION.

In a previous paper (Cleland, 1950a@) a study was made of the respiratory rate and.
respiratory quotient of oyster eggs during early development.

If work such as this is to be elaborated and related to a lower biochemical level,
some knowledge of the enzyme constitution of the egg, the pathways of its metabolism
and the method of biological energy production is essential.

‘Since the final aim of the present work is to study the mitotic process, the above
knowledge is required before any results obtained with the present material can be
related to other biological material of which metabolic properties are known. This
knowledge is also required as a background for the study of the effect of inhibitors on
the mitotic process.

The aim of the present paper is, then, to outline the intermediary metabolism of the
oyster egg. Since only an outline is necessary at this stage, no detailed study of the
properties of the enzymes involved has been attempted. * Wherever possible whole eggs
have been studied or, when these were inapplicable, unfractionated homogenates.

The work was performed in the 1948—50 seasons.

MATERIALS AND METHODS.
The preparation of egg suspensions has already been deseribed (Cleland, 1950a). In
the present case no attention was paid to the fertilizability of the eggs, but most reactions
have been studied on both fertilizable and unfertilizable batches.

BY K. W. CLELAND. 297

Standard manometric methods (Umbreit et al., 1945) were employed, the flasks
having 15-20 ml. capacity. Some experiments were performed with flasks of 5-6 ml.
capacity. Differences of 5% in respiratory rate are considered significant. The tempera-
ture of incubation has been 26°C. throughout.

Cozymase was prepared by the method of Williamson and Green (1939); ATP by
the method of Le Page (1945); adenylic acid by the alkaline hydrolysis of ATP
(Lohman, 1932) with subsequent separation in the Ba soluble alcohol precipitable fraction
of the hydrolysate; cytochrome C by the method of Kielin and Hartree (1937); aceto-
acetic acid by the method of Schaffer (1921); crystalline sodium pyruvate from freshly
vacuum distilled pyruvic acid by the method of Robertson (1942). All other chemicals
were from commercial sources (usually B.D.H.).

All substrates and inhibitors were used as neutral solutions (glass electrode) of the
sodium salt.

Unless otherwise stated, preparations for analysis were deproteinized with trichlor-
acetic acid in a final concentration of 10%.

Lactic acid was determined by the method of Barker and Summerson (1941);
pyruvic acid by the method of Friedman and Haugen (1941); phosphorus by the method
of Fiske and Subbarow (1925); amino acids in tungstic acid filtrates by the method
of Folin (1922) with glycine as standard; tyrosine by the method of Folin and
Ciocalteu (1929).

Glycogen was separated by the method of Lindberg (1945), hydrolysed for 30
minutes with 5N H.SO,, and the reducing sugar determined by the method of Folin and
Malmros (1929). The latter method was also used for the determination of the reducing
substance in tungstic acid filtrates.

The barium salt fractionation and analysis was performed according to Le Page
and Umbreit (1945).

Nitrogen was determined by direct Nesslerization of H.SO,-HCI1O, digests. Protein N
was taken as that precipitable by 10% trichloracetic acid, polypeptide N as the N
precipitable by tungstic acid treatment of trichloracetic filtrates, and non-protein N that
remaining in the filtrate after tungstic acid precipitation.

All colour determinations were made with a Spekker photoelectric absorptiometer.

The cyanide studies were made according to the instructions of Robbie (1946) for
balanced KOH/KCN centre well mixtures.

The nitrogen and nitrogen CO, mixtures were from commercial cylinders used
without further purification. The oxygen uptakes were usually nil and always less than
2% of the control in air.

The preparation of homogenates is described later.

ANALYSES OF HGGS.

Glycogen, monosaccharide, lactic acid and amino acids are present, as shown in
Table 1, where the distribution of nitrogen is also shown. Rough analytical values for
fat and phospholipide have already been given (Cleland, 1947). The fairly high amino
acid content (4% of dry weight) is still considerably less than the amounts found in.
Urechis eggs (Horowitz, 1939) or Strongylocentrotus eggs (Orstrom, 1942). Fairly high
amino acid contents are found in rapidly growing normal mammalian material
(Christensen and Streicher, 1948; Christensen et al., 1948). Although elevated in
hyperplastic epidermis, the amount is much reduced when neoplastic change supervenes
(Roberts and Tishoff, 1949). .The large proportion of the total nitrogen in granules is
expected from the high P granule content of the eggs (Cleland, 1947).

The glycogen and total carbohydrate content of the eggs is considerably less than
that found in sea urchins (Hutchens et al., 1942; Orstrom and Lindberg, 1940).

A number of the phosphorylated intermediates found in mammalian material are
also present in oyster eggs, as shown in Table 2.

The figures for inorganic phosphate are very much higher than those quoted by
Chambers and White (1949) for Arbacia eggs, but are of the same order as the figures

298 METABOLISM OF UNFERTILIZED OYSTER EGGS,

of Crane (1947) for Arbacia. The seven-minute hydrolysable P values are somewhat
lower than those found in Arbacia by the above authors.

Of the total seven-minute hydrolysable P approximately 65% was present in the Ba
insoluble fraction and is thus considered to be from ATP and ADP (hexose diphosphate

TABLE 1.
Presence of Substrates and Distribution of Nitrogen.

| Number of Content,
Substance. | Analyses. Mg./ml. Eggs.
| |
|
Glycogen... a wa ze: || 9 3:1 -6-2
Reducing substance Be te ar 5 1:0 -2:3
Lactic acid ii aH at | 18 1 0-02-0-05
Pyruvie acid if ne st Beal 15 | 0 -0-01
Free amino acids .. a2 56) won 6 7-11
é 5 tyrosine As aA af: at 9 i= 10s)
= |
Granular* N 4 | 40-55
Protein N 3 | 74-80 -% Total
Polypeptide N 3 6-15 N
Non-protein N 3 6-10 J

|

* Precipitable by a force of 20,000 xg. acting for 30 minutes.

contributes less than 5% of the total). A further 15% can be accounted for as glucose-1-
phosphate in the Ba soluble fraction. The remainder was not accounted for.

EXPERIMENTS ON WHOLE EGGs.
(a) Effect of Inhibitors.

The effect of a number of inhibitors on the respiration of whole eggs is shown in
Table 3. 7

TABLE 2.
Presence of Phosphorylated Compounds.

Number of

Fraction. Material. Analyses. Content or Method of Identification.
|
Inorganic P an .. | Whole eggs. 7 250-530 g./ml. eggs.
7 min. hydrolysable P .. 7 5 100-320 wg./ml. eggs.
JMve Chl ANID 5 .. | Ba-insoluble frac- 5 P,, pentose, N, 2650 A absorption.
Hexose diphosphate YA tion. 5 Fructose. s
Phosphoglycerie acid on 2 Resistant P.
|
Fructose 6 phosphate .. | Ba-soluble, alcohol | 5 Fructose.
Cozymase .. nes .. | precipitable frac- 4 Absorption at 3400 A.
Adenylie acid a Be tion. 4 Ratio absorption at 2650 and 3400 A.

Spectrophotometrie studies were made with a Beckman spectrophotometer.

These inhibitor studies suggest: (a) that most of the oxygen consumption is
mediated by a cytochrome oxidase system (cyanide and azide and possibly fluoride
inhibition); (0b) that respiration is accompanied by phosphorylation and that the
phosphorylation partly controls the respiratory rate (dinitrophenol effect cf. Loomis and
Lipmann, 1946); (c) that enzymes whose activity depends on —SH groups, either
oxidative or glycolytic or both, are concerned in the respiration (iodoacetic acid and
phenylmercurie nitrate effects).

Ee oe

BY K. W. CLELAND. 299

(bo) Effect of Methylene Blue and Possible Substrates.

Methylene blue at concentration M/300 was found to stimulate the respiration some
200%-400%, a fact usually interpreted to mean that the cytochrome-cytochrome oxidase
step is limiting. Hydroquinone at a concentration of M/10 gave, after correction for
auto-oxidation by the method of Schneider and Potter (1943) stimulations varying from

~0-30% (pH 7:0), while the addition of succinate, malate, malonate, glucose or pyruvate
(M/20) had no demonstrable effect on respiration.

TABLE 3.
Effect of Inhibitors on Respiration.
Concentration (M.).
Inhibitor. | } pH.
Ome 10° Ome 10-4 10-> Om |
a |
| | }
Cyanide crits OR need ey eel oe eS 7 15} 0. |G G5 95 i 7-5
Azide x ae a3 na 50 | 64 85 100 6-5
Fluoride .. ar ae Be 2) 94 100 — | a —- | es
Todoacetate ote is At = | 33 | 64 92 | 100 | — | uh
Phenylmercuric nitrate Ne — | — 20* 90 100 -— | 015)
Dinitrophenol; Ne ae — | — | 120 140 | 175 100 | a
}
H | J

The figures quoted are percentages of the control respiration, the absolute value of which varied from
100-300 21. in the experimental period of 60-120 mins.

* Variations of 10-25% were encountered.

7 Both the position and height of the maximum stimulation have varied considerably in different egg batches
(cf. Table 7).

In order to determine whether the absence of stimulation by the latter substances
might be due to their non-penetration, experiments of the following type were performed.

To an approximately 50% suspension of eggs in sea-water, pyruvate was added and
the suspension shaken for 45 minutes. Samples of the whole suspension and the cell-free
supernatant were treated with trichloracetic acid and analysed for pyruvate. The
non-solvent volume was determined by the osmotic method (cf. Brooks and Brooks,
1940).

The results of a typical experiment are shown in Table 4. No pyruvate was used
by the eggs during the period of the experiment.

TABLE 4
Penetration of Pyruvate into Eggs.
Volume of suspension (ml.) 3-4 Calculated pyruvate in supernatant
$5 > eggs ae At iL 9835) on an hypothesis of :
Pyruvate in suspension (ug./ml.) | 240 non-penetration 397 g./ml.
as ; supernatant ,, 388 complete ,, 300 ,,
Non-solvent volume per cent. .. | 42

The data in Table 4 indicate that, within the limits of experimental error, no
penetration of pyruvate occurs. Since it appeared likely that the eggs would be
impermeable to most substrates, all studies of intermediary metabolism were conducted
on homogenates.

(ce) Glycolysis.

The experiments shown in Text-fig. 1 and Table 5 indicate (a) that considerable
quantities of CO, are evolved by eggs suspended in bicarbonate—CO:z buffers, and that the
COs arises from bicarbonate, (b) that the amounts of lactic and pyruvic acids accumu-
lating are far less than expected from the manometric acid production, (c) that although
similar quantities of lactic and pyruvic acid accumulate under aerobic conditions no
manometric acid production is demonstrable.

300 METABOLISM OF UNFERTILIZED OYSTER EGGS,
|

TABLE 5.

| |
| Change in

Experiment Gas Initial . | CO. Bicarb. oj Paks a!
Number. Phase. | Bicarb. Evolved. | Recovered. | Total. |
| (ul.) (ul.) ba) ee na) Lactic. | Pyruvie.
| | | (g.) | (ue.)
| | |
1 N./CO, 310 aaa 265 emesis lage eit © pena)
0,/CO, 315 — | 307 | — +12 4®
2 N./CO. 316 59 | 250 309 +10 +12
Oy COM 320 = | Soap = =| 2
- | | j —
3 | Ne COL en| 360 | 35°5 315 350-5 +7 +8
en On COsay a 340 — | 334 | — +7 +3
| |
| | | |

All figures are expressed as ul. CO,. Approx. 0°3 ml. egg/flask. Experiment duration 60 mins. Gas phase
5% CO, in Nz or Os.
Egg breakdown was slight and identical in amount under aerobic and anaerobic conditions.

Glycolytic inhibitors cause a depression of acid production, as shown in Table 6.
It will be seen that the inhibition by iodoacetate and phenylmercuric nitrate increases
with time, a phenomenon probably due to the relative impermeability of the eggs to
these substances, while the fluoride inhibition is constant. Since fluoride seems less
likely to affect other acid-producing mechanisms (e.g., ATP splitting) than the other
two inhibitors, and is later shown to inhibit glycolysis in extracts, a conclusion that at
most only 25% of the acid production is due to glycolysis seems warranted.

TABLE 6.
Effect of Glycolytic Inhibitors on Acid Production.
Phenyl Mercuric
Time Period. NaF. lodoacetate. Nitrate.
(10? M.) (10-? M.) (10°? M.)
0-45" wy ate ass 75 93 | 83
45-90 .. ie Be 77 72 52
!

The figures quoted are percentages of the control which evolved 331. CO, in the 90
minutes. The flasks were of 5 ml. capacity and contained 0-15 ml. eggs, 0-02 M.
bicarbonate (final conc.) and 5% CO, in Nz in the gas phase.

; Large amounts (about 800 wl. CO./ml. eggs) of acid are released on cytolysis of
oyster eggs by saponin or taurocholate under both aerobic and ,anaerobie conditions.
This acid production is proportional to the amount of eggs taken after retention
corrections have been applied; this is difficult to reconcile with even a one-step enzyme
system let alone a complex system like the glycolytic system. The cytolytic acid
production is very insensitive to inhibitors (iodoacetate, phenylmercuric nitrate, fluoride,
phloridzin) and to the addition of glycogen, and it was therefore concluded that it
was non-metabolic. The most likely explanation is that the egg’s interior is normally
maintained at a more acid pH than the surrounding environment.

Injury of eggs under anaerobic conditions, with subsequent increased permeability
and a slow neutralization of intracellular acid groups, is probably responsible for a
portion of the acid production of whole eggs; eggs recovered from these anaerobic
experiments frequently fail to fertilize, while those from the aerobic experiments are still
capable of fertilization.

A turther fraction of the acid production can be ascribed to splitting of ATP (cf.
Table 7) and phosphomonoesterase and lipase activity (cf. Table 16).

BY K. W. CLELAND. 301

No acid’ production could be demonstrated on fertilization of oyster eggs.

If the conclusions derived from the inhibitor experiments are correct, the glycolytic
acid production is equivalent to approximately 15 wl. of CO./hr./0:3 ml. eggs, or to 60 ug.
of lactic acid and/or pyruvic acid. This latter value is considerably less than that
found in actual analyses (Table 5). This implies either that the conclusion is invalid
or that some mechanism of anaerobic removal of pyruvate or lactate exists. That the
latter alternative is correct is indicated by the homogenate experiments on pyruvate
utilization (Table 19), where it is shown that the rate of anaerobic removal is sufficient
to account for the non-accumulation of these substances.

In a previous study (Cleland, 1950a) it was inferred from the respiratory quotient
-values that carbohydrate and lipide were being oxidized. If aerobic and anaerobic
glycolytic rates are equal, as appears likely from the analyses, the inferred glycolytic

‘rates are sufficient to account for the carbohydrate moiety of the respiration of these
eggs (intact germinal vesicles), provided the pyruvate is completely oxidized. That the
latter occurs is indicated by the homogenate experiments (Table 21).

(d) High Energy Phosphate Formation.

: High energy phosphate bonds, which are regarded as the ultimate source of usable
biological energy, are formed in mammalian tissues both in breakdown of carbohydrate
to lactic acid and-in the oxidization of pyruvate through the tricarboxylic acid cycle
(Lipmann, 1941; Potter, 1944; Cross et al., 1948).

The production of high energy phosphate bonds is strongly inhibited by dinitrophenol
(Loomis and Lipmann, 1946; Cross et al., 1948), the stimulating effect of this compound
on respiration being ascribed to its obviating the need for inorganic phosphate in the
aerobic process (Loomis and Lipmann, 1946), or to its ability to cause or promote break-
down of some labile phosphate ester and thus to’ make available more inorganic
phosphate (Tepley, 1949).

In the present material the presence of ATP and/or ADP and adenylic acid has
been shown (Table 2). The latter compound causes a stimulation of glycolysis
(Table 15), which implies that high energy phosphate bonds arise in this reaction.
The stimulating effect of dinitrophenol on respiration has been shown in Table 3.

The effect of varying experimental conditions on the high energy phosphate content
of whole eggs is shown in Table 7, where one of the five experiments performed is
described. It will be seen that a graded decrease in the content occurs when the

TABLE 7.
High Energy Phosphate Changes under Various Conditions.

: iy P,. Pees
Inhibitor. Conc. Gas Phase. ul. Or. (ug.) (Ug.) (Ug.)

= = Air. 128 87 99 186

= — Reduced O.. 69 | 108 84 192

= —_— No. 3 113 75 188

Cyanide Re ae 1073 M. Air. ial | 115 73 188
Dinitrophenol ... .. 10-3 M. is | 346 | © 3128} 69 191
& nA La 107M. 5 485 120 69 189

i ae tye 10~° M. Fe 194 96 | 90 186

|

Each flask contained approximately 0:3 ml. of eggs with broken germinal vesicles. Experiment duration
90 mins.
P, refers to inorganic phosphate and P, to 7 min. hydrolysable P.

!

respiration is decreased either by reduction of oxygen tension or by the action of
cyanide. This indicates that the bonds are formed during oxidative metabolism, the
decrease presumably being due to utilization and/or autolysis by apyrase action. It
will furthermore be seen that dinitrophenol in appropriate concentration causes a
greater decrease than anaerobic conditions, although causing a marked increase in

302 METABOLISM OF UNFERTILIZED OYSTER EGGS,

oxygen consumption; this is possibly due to interference with high energy phosphate
formation not only in respiration but also in glycolysis.

By analysis of barium salt fractions it was shown that most of the decrease occurred
in the Ba precipitable fraction. This implies that the greatest loss occurs in the ATP
and ADP fractions.

Barth and Jaeger (1947) have studied the effect of anaerobic conditions on the high
energy phosphate content of developing frogs’ eggs. A much slower decrease was
found than in the present material. This is presumably due to the fact that the
glycolytic system is quite active in the frog’s egg and is capable of providing sufficient
energy for the early cleavages to occur. In the oyster egg anaerobic conditions cause
arrest of development. =

Robbie (1947) has described in developing eggs three types of response to anaerobic
conditions: (a@) those which are capable of maintenance and development, e.g. Fundulus;
(b) those capable of maintenance only, e.g. Arbacia; (c) those incapable of either
maintenance or development under anaerobic conditions, e.g. Echinarachnius (Robbie,
1948). These differences are possibly due to variations in the energy supply available
from the glycolytic system.

EXPERIMENTS ON HOMOGENATES.
(a) General. ‘

Homogenates were prepared in an ice-jacketed ground-glass apparatus of the Potter
type. Depending on the type of reaction under study either distilled water or a phosphate
saline mixture (0-2M KCl, 0-1M MgCl, 0-01M—phosphate buffer pH 7-6) was used as an
homogenizing medium.

TABLE 8.
Effect of Additions on Homogenate Respiration.
|
| Respiration.
Addition. Concentration. Percentage
(M.) | Control.

|

|
Cytochrome C ee A oe DeelOme | 195
Methylene blue ae Be aa 33K Om ' 145
Succinate * 58 Ae i 10° 185
Malonate & Be eh ie 107+ 65
Cyanide Be Ste ae is 10° 8-5
Iodoacetate .. bi 3 z% 10° 57
Arsenite a Se a3 ae Om? 82
Phenyl mercuric nitrate a 0 10° 50

Each flask contains 1 ml. 50% homogenate (phosphate saline) in a final
volume of 2 ml. Experiment duration, 90 mins. Control respiration, 114 wl.

The respiration of homogenates was relatively independent of time of homogenation.
In all cases the time selected was that required to cause complete cytolysis of 95%
of the eggs (with the present apparatus about two minutes). Packed eggs were usually
homogenized in an equal volume of homogenizing fluid.

While the pH optimum of homogenate respiration is approximately 8-0 it is
difficult to buffer at this pH. For this reason phosphate buffers (final concentration
M/25) of pH 7:5 were used in the homogenate experiments. Cytochrome, when added,
was used at a final concentration of 2 x 10M.

Typical homogenate respiration curves are shown in Text-figures 2 and 3. The
decline in the respiratory rate of uncomplemented homogenates is evidently due to at
least two factors: depletion of substrate in those cases where linear respiration occurs
in the presence of added substrate, and changes in the enzyme system itself where
this linearity is not found. :

' The metabolic properties of whole eggs and homogenates are compared in Table 23.

The effects of a number of additions on homogenate respiration are shown in Table 8.

~

BY K. W. CLELAND. 303

The stimulation found with cytochrome C is expected from previous deductions on
whole eggs, the stimulation found with methylene blue is less than that found with
whole eggs, and, in contrast to its lack of effect on whole eggs, which it evidently did
not penetrate, succinate gives a marked stimulation of homogenate respiration.

In contrast to its marked effect on the respiration of whole eggs, dinitrophenol has
only a small effect on homogenate respiration (cf. Table 22). This effect is reduced
still further when the phosphate concentration is increased.

Malonate and arsenite powerfully inhibit certain specific enzymes of the tricarboxylic
acid cycle in vertebrate tissues (Krebs, 1943). The results quoted above imply either
that none or only part of the homogenate respiration proceeds through the cycle or that
the enzymes of the oyster cycle have different properties from the classical material.

The addition of phosphate is without effect on the respiration of homogenates.
This is possibly due to the fact that sufficient is already present; Cross et al. (1949)
have found that the vastly more active cyclophorase preparation from kidney requires
only 1 w»M/ml. for maximal activity.

(0) Effect of Carbohydrates.

The effect of some carbohydrates on homogenate respiration is shown in Table 9.
It will be seen that significant stimulation of respiration is found with glycogen and
fructose. When ATP is also added, glucose as well as fructose gives increases in the
respiratory rate (cf. Text-figure 2). ATP alone has little or no effect.

TABLE 9.
Effect of Carbohydrates on Homogenate Respiration.
Con- Number of Percentage
Substance. centration. Experiments. Control.
Glycogen .. =e = ae 0-6% 4 115-130
M
=— 92-103
Glucose / 100 4
5 +ATP ra Sep poses ue 1 130
Fructose a % 4 107-120 :
» +ATP a3 1 150
Arabinose whe Br 35 4 100-105
Galactose .. ae Se om _ | 4 102-106
Xylose tn 4 98-104

Control respiration, 90-220ul. Duration, 1-2 hrs. Cytochrome added in two
experiments. ATP, when added, 1 mg./flask.

(c) Effect of Lipides.

The effect of some lipides and lipide derivatives on homogenate respiration is shown
in Table 10. H will be seen that some of the lower fatty acids give significant increases
im some preparations and that lecithin gives a considerably greater and more consistent
stimulation. A considerable portion of the lipide store of the eggs is phospholipide
(Cleland, 1947). Hydrolytic products of fats and phospholipides, glycerol and glycero-
phosphate, stimulate respiration, but higher fatty acids are consistently inhibitory.
This inhibition has been described in rat liver preparations, but in these the addition
of ATP enables utilization of the fatty acid to occur (Lehninger, 1945). This is not the
case in egg homogenates. The non-oxidation of higher fatty acids was not affected by
the addition of tricarboxylic acid cycle intermediates (cf. Lehninger, 1945; Graffin and
Green, 1948).

(ad) Effect of Amino Acids.
The following amino acids (M/100 final concentration) failed to cause a significant
increase or decrease in homogenate respiration (3 expts.): glycine, dl-alanine l-leucine,

s

304 METABOLISM OF UNFERTILIZED OYSTER EGGS,

l-tyrosine. Dl-aspartate gave stimulations of 5-20% (5 expts.) and lglutamate caused
stimulations of 50-100% (12 expts.). The stimulation by glutamate was increased
about 20% by addition of cozymase and the oxidation of glutamate was accompanied
by ammonia production.

TABLE 10.
Effect of Lipides and Lipide Derivatives on Homogenate Respiration.

|
| Concentration. Number of Percentage
Substance. (M.) Experiments. Control.
|
estate uiand) . ste howe ete arocu: 10-2 6 | 90-98
Propionate . . a a ral . 4 96-103
Butyrate .. nv ah alt a 4 105-144
Valerate i, am ak pel i 4 |i}: 95-130
Caproate .. Ae te ae ll 5 4 | 103-133
Caprylate .. si 5 sei | i 4 | 67-78
oe as a6 ie ns Om 2 } 92-97
. +ATP .. a x) 5 | 2 | 90-100
Palmitate .. cy ae ‘ed 3 2 91-95
re SA TP) 2 Be ey e 2 | 92-97
Lecithin .. As By ake 0:5% 4 | 115-160
Glycerol .. aie As; i Om 3 120-128
Glycerophosphate . . AIG Bis A Deva: 120-130
Acetoacetate 12 oie Ae 45 4 92-103
|

The control respiration varied from 90-170 ul. in different experiments of duration
1-2 hrs. Cytochrome added in some experiments ; ATP when added 2 mg./flask.

(e) Hffect of Tricarboxylic Acid Intermediates.

The effect of a number of tricarboxylic cycle intermediates on the respiration of
homogenates is shown in Table 11. It will be seen that all caused a marked stimulation
of homogenate respiration whether the homogenate was made in distilled water or
phosphate saline and in the presence or absence of added carrier (methylene blue or
cytochrome). Typical curves are shown in Text-figure 2.

TABLE 11.
Effect of Tricarboxylic Acid Cycle Intermediates on Homogenate Respiration.
Substrate M Number of Percentage
100 Experiments. Control. e

Citrate oe yg om Ps 8 130-160
a keto glutarate oe 55 th 5 135-175
Succinate te nh a ats 40 | 150-350
Fumarate ad os as 30. | if 140-190
Malate ae 5 as Sid 20 150-200
Pyruvate ie he Ae “fs 12 | 130-185
Lactate a. oad ws Sal 6 122-140

Some experiments were performed without added oxygen carrier, some
with methylene blue and some with cytochrome. The final homogenate con-
centration has varied between 12% and 25%.

SPECIFIC ENZYMES IN HOMOGENATES.
(a) Cytochrome Ovidase.

The presence of cytochrome oxidase in the egg homogenate is indicated by the
typical experiment shown in Table 12.

160

) 120

we

80

OXYGEN UPTAKE

40

BY K. W. CLELAND. 30D

80

-4
ie) et rr
ra) ——— -2

ay ee
= ee
(a)
uJ
<
iQ peal
el Me i
ray
n“
e)
O
fe)
nu
ce) 30 60 90 120

! TIME (MINS)

40 60 80

OXYGEN UPTAKE (UL)

20

30 60 90 120 ° 15 30 45 60

2 TIME (MINS) 3 TIME (MINS)

a : Text-figures 1-3.

Fig. 1. Typical anaerobic acid production curves for whole eggs. The eggs were suspended
in sea-water to which was added 0:1 volume of M/4 bicarbonate. The‘ flasks were gassed
with 5% CO, in N» for 5-10 minutes, and equilibrated for 10 minutes in the bath before the
first reading was taken. The volume of packed eggs per flask was as follows: 0:15 ml. in (1),
0-38 ml. in (2), 0:29 ml. in (3); 0-32 ml. in (4).

Fig. 2. Typical respiration curves for homogenates, and the effect of substrate additions.

Curve 1: Flask of 5 ml. capacity containing 0-5 ml. of 25% homogenate, 0:1 ml. cytochrome
and 0-2 ml. phosphate buffer in a final volume of 1 ml. Curve 1A: Same with M/100 pyruvate
added. Curve 2: Different homogenate but flask contents same as in (1). Curve 2A: Same with
fructose (M/100) and ATP (1 mg.) added. Curve 3: Flask of 15 ml. capacity containing
1 ml. of 50% homogenate and 0:4 ml. of phosphate buffer in a final volume of 2 ml. Curve 4:
Different homogenate but flask contents similar to (3) except for the addition of 0-2 ml. of
eytochrome. Phosphate buffer: M/5, pH 7:5. Cytochrome: 2 x 10-4M. It will be seen that
addition of substrate may prevent the decline in respiratory rate of uncomplemented homo-
genate (2 and 2A) or have little effect on the decline (1 and 1A). This suggests that two
factors are concerned in the decline: exhaustion of substrate and injury to the enzyme system.
The endogenous respiration of the homogenates in curves (3) and (4) is more nearly linear
Owing to the smaller dilution of the homogenate. The addition of cytochrome did not
consistently affect the linearity.

[Legend continued on page 306.]

75

306 METABOLISM OF UNFERTILIZED OYSTER EGGS,

The enzyme is saturated with a cytochrome concentration of approximately
4x10°M. Bands indistinguishable from those of cytochrome C may be seen in egg
suspensions with a comparison microspectroscope.

TABLE 12.
Cyutochtome Oxidase in Egg Homogenates.

System. ul. O2.
|
1 Homogenate ane ye oe 14
2 (1) +hydroquinone.. oe 88
3 (1) +eytochrome ae Eu 22,
4 (2) +eytochrome ae a 217
5 (4) +eyanide ae a id 60
6 (4) —homogenate an Bi 46
|

Each flask contained 1 ml. of 10% distilled water homogenate,
M z
0-5 mil. = phosphate buffer (pH 7-5) in a final volume of 3 ml.

Hydroquinone final concentration 2:5x10-?M, cytochrome
4x10-° M, cyanide 10°-*M. Experiment duration, 60 minutes.

(b) Succinoxidase.

In Table 13 the results of a typical experiment indicating the presence of succin-
oxidase are shown.

The complete system, (4) in Table 13, is saturated by approximately 1:5 x 10°M
cytochrome, and is slightly stimulated by the presence of 5 x 10*M Ca++ and Al*+*+, as
noted by Schneider and Potter (1943) for the mammalian enzyme. The pH optimum
was approximately 8-2.

TABLE 13.
Succinoxidase in Egg Homogenates.

System. : [l. Oo.
| |
1 | Homogenate .. 33 aS 19
2 | (1)+eytochrome be ee 26
3 | (1)+succinate .. as ae 31
4 | (3)+cytochrome ak Ae 75 ‘
5 (4) + cyanide oe <a seal 3
6 (3)+methylene blue .. ae 48
7 (5)+methylene blue .. 4% 39

Each flask contained 1 ml. of 10% distilled water homogenate,
M i
0-5 ml. z phosphate buffer (pH 7-5) in a final volume of 3 ml.

Succinate final concentration 2-5 x10~* M, cytochrome 2 x 107° M,
cyanide 10-°M, methylene blue 3:3x10°°M. Experiment
duration, 1 hr.

The effect of some inhibitors on the succinoxidase system is shown in Table 14.
The effects of these inhibitors are in general similar to those described by Humphrey
(1947) for adductor muscle. As in the latter, the relatively small inhibition by malonate

Fig. 3. Effect of tricarboxylic acid cycle intermediates on homogenate respiration. Each
flask, of 5 ml. capacity, contained 0:5 ml. of 40% homogenate, 0:2 ml. phosphate buffer
(M/5, pH 7-5), and 0-1 ml. cytochrome (2 x 10-4M) in a final volume of 0-9 ml. Curve 1 is the
control, curve 2 has pyruvate, curve 3 a-ketoglutarate, curve 4 malate, and curve 5 succinate,
added in final concentration M/100. Apart from the control the greatest deviation from
linearity is found in the pyruvate curve. This is consistent with the operation of the tri-
carboxylic acid cycle, since pyruvate is not oxidized as such, but only after a labile condensation
reaction, while the other substrates are oxidized directly.

=~]

BY K. W. CLELAND. 30

is noteworthy. Indeed the effect with changing succinate concentration does not give
much support to the idea that malonate is acting competitively with succinate in the
present material.

TABLE 14,
Effect of Inhibitors on Succinoxidase.

Concentration. Succinate. O, Uptake
Inhibitor. (M.) (M.) Percentage
| Control.

Malonate MT: = Ete 1077 Spc Om2 835)
“5 ae = ot i 10-2 é 36
= Bi ave ae 3 Zeal Oms 30
Ef a fs se Ome Ome 65
Pe oe S su 10-3 10-2 90
Iodoacetate ae tes ot Om? 25a Ome 59
3 oie a aie 10-3 13 88
Phenyl mercuric nitrate Bey 10-3 aa 3
35 53 Ae Ome ie 65
5 3 Ble Ome a3 110
Arsenite .. >... ae me 10-2 107? 54
re Ss as he § 10-8 is 85

Flask contents similar to those indicated in Table 13. Data are from several
experiments of duration 1-2 hrs. The inhibitions are -calculated from the ratio of
(System (4)+inhibitor minus system (2)+inhibitor) and (system (4) minus system (2)),
the code numbers being those shown in Table 13.

(ce) Malic Oxidase.

The oyster egg homogenate contains a system capable of oxidizing malic acid,
which is similar to that described in mammalian tissues by Potter (1944). The results
of a typical experiment are shown in Table 15.

TABLE 15.
Malice Oxidase in Egg Homogenates.
System. ul. Os.
1 | Homogenate ae ae 5 10
2 | (1) -+malate iy re Ey 21
3H (1) + glutamate ae ys sh 18
Aan (1)+eytochrome .. aa ae 16
5 | (4) + malate OE Se _ 37-5
6 (4) +glutamate ies re ae 29-5
7 | ()+malate hare e RI 107
Oma (7) +cozymase aes ae ees 122

Each flask contains 1 ml. of 12-5% distilled water homogenate

M :
0-5 ml. = phosphate buffer (pH 7-5) in a final volume of 3 ml.

Malate final concentration 2107? M, glutamate 2x 10-2 M,
cytochrome 2x10-°M, cozymase 1 mg./flask. Experiment
duration, 1 hr.

The system is saturated by a cytochrome concentration of about 1-5 x 10°M. The
addition of nicotinamide to the system rarely had any effect, suggesting that cozymase
degrading enzymes are absent or of very low activity. The marked stimulation of the
oxidation of malate by glutamate implies the presence of an active glutamic oxalacetic

308 METABOLISM OF UNFERTILIZED OYSTER EGGS,

aminopherase. The relatively small effect of cozymase suggests that the enzyme is
normally fairly saturated with the coenzyme and is in accord with the postulated
absence of cozymase destroying enzymes.

(ad) Miscellaneous Hydrolytic Enzymes.

The presence in egg homogenates of a number of hydrolytic enzymes is indicated in
Table 16. It will be seen that the activity of acid and alkaline phosphatase, proteinase
and lipase is tairly low. The activity of apyrase is rather high for invertebrate tissue,
being of the same order as that found in the adductor muscle of the present species
(Humphrey, 1949) and higher than that found in Arbacia eggs (Connors and Scheer,
1947).

The amylase activity is unexpectedly high and is not accompanied by any phosphate
uptake. It is very improbable that the activity is due to contamination of the egg
suspensions with small digestive diverticula fragments, since the latter have never

TABLE 16.
Hydrolytic Enzymes in Egg Homogenates. .
Typical
5 Concen- Activity
Enzyme. Substrate. tration. Buffer. pH. Activator. Method. (per 0-1 ml.
Eggs/4 hr.).
M M ;
Alk. phos- | Glycerophosphate. — Veronal. 9-0 Mes ) A Pinorg. 25g.
phatase. 20> : 100
Acid phos- = | e Acetate. | 4°8 — ; an Pe
phatase. | M
Apyrase .. | ATP. TG Veronal. 7:5 — rf 140 wg.
Lipase .. | Tween 80. , 5% Bicarb.-CO,. A —_ CO, displaced. 1-8uM.
Proteinase | Haemoglobin. | 2G Acetate. 3395) —- Anson (1988). 35 ug.
Amylase | Glycogen. | 0-4% Phosphate. Views) NaCl (1%). | AGlycogen. 4-5 mg.

a Correction for changes in the blank (without substrate) was applied in all cases.

been recognized in suspensions, and the activity is unchanged on further washing of
the eggs;. the enzyme is soluble and not bound to cell granules (Cleland, 1950b).
Furthermore, although the alkaline phosphatase activity of the digestive diverticula is
high (Cleland, 1947) that of egg homogenates is low.

The question of the histochemical detection of the alkaline phosphatase in the
ege has been considered elsewhere (Cleland, 1950c).

; COMPLEX ENZYME SYSTEMS IN HOMOGENATES.
(a) Glycolysis. ;

The experiments on whole eggs indicated that lactic and pyruvic acids were produced
under aerobic. and anaerobic conditions. In order to study the system further, recon-
struction experiments were carried out with homogenates. As in the case of whole
eggs, non-metabolic acid production vitiated manometric estimates of glycolysis. The
three typical experiments incorporated in Table 17 indicate the following: (a) that in
both whole homogenate and in homogenate supernatant, pyruvic acid is the main acid
formed; (0b) that both adenylic acid (or ATP) and cozymase are coenzymes for the
glycolysis; (c) that when the granular components of the homogenate are eliminated by
high-speed centrifugation a considerable apparent stimulation of glycolysis occurs in
the reconstructed system, a fact suggesting that some mechanism of anaerobic pyruvate
removal is present. Little pyruvate accumulates in the complete system under aerobic
conditions in homogenates, although, owing to removal of the aerobic pyruvate utilizing
system, considerable amounts may accumulate in extracts incubated aerobically.

BY K. W. CLELAND. ~ 309

The effect of the classical glycolytic inhibitors (cf. Stotz, 1945) on the reconstructed
system is shown in Table 18. It will be seen that the oyster egg system behaves
similarly to the mammalian system. ;

TABLE 17.
Anaerobic Glycolysis in Egg Homogenates and Extracts.
= Whole Homogenate. Supernatant.*
System.

ug. Pyruvic. ug. Lactic. ug. Pyruvic. ug. Lactic.
il Enzyme preparation shy as 8 4-1 0 0
2 (1) + glycogen a ae ats 1183 4:3 11 1:6
3 (2) +cozymase xe ae igh 18 6:0 13-5 | 1-2
4 (2)+ATP a aM 355 as 32 6:3 26 2-3
5 (2)+adenylic acid .. ae ne 27 5:8 22 23
6 (4) +cozymase os ae ahs 52 9-2 36°5 Bom

= = |

(6) Homologous enzyme me aN 26-5 6-1 83 3-4

* After centrifugation for 5 mins. at 10,000 xg.
Three experiments are delimited by the thick lines. The final volume was 1 ml. in each experiment which con-

M.
tained phosphate buffer (pH 7-5) of final concentration a5 The additions were as follows: glycogen 6 mg.,

cozymase, ATP, and adenylic acid 1 mg. The enzyme preparation in the whole homogenate experiment was 0-5 ml.
of 40% homogenate, in the supernatant experiment 0-5 ml. of 20% homogenate supernatant and in the homologous
enzyme experiment 0-5 ml. of 30% homogenate or the supernatant therefrom. The duration of all experiments was
2 hours and the flasks were gassed with nitrogen.

In order to analyse the anaerobic pyruvate degrading mechanism further, the
removal of added pyruvate by homogenates and extracts was studied. The experiment
detailed in Table 19 indicates the following: (a) that anaerobic pyruvate removal is
quite high, being only slightly less than that found under aerobic conditions; (0) that
the removal is stimulated by the presence of carbon dioxide and/or bicarbonate; and

TABLE 18.
Effect of Inhibitors on Glycolysis in Extracts.
ug. Acid Formed.
Concentration.
Inhibitor. (M.)
Pyruvic. Lactic.
—— ~— ; 65 Bow
Iodoaeetate ae ane aA 10° 8-5 io}
Phenyl mercuric nitrate ete Om 0 0
Fluoride ae he Sed 10°? 0 0:8
Phloridzin e ae iy Ome 5 0

M
Each flask contained 0:5 ml. of 40% homogenate supernatant and 0-2 ml. 5

phosphate buffer (pH 7-5) in a final volume of 1 ml. The additions were identical to
those in system (6) of Table 17. The experiment duration was 2 hours, the flasks being
gassed with nitrogen.

(c) that the removal does not involve a mere conversion to lactate; (d) that the system
is present on the granular component of the homogenate.

The system, or part of it, is evidently labile, since the CO, and bicarbonate stimula-
tions were usually less than shown in Table 19, as also were the rates of removal of

}

310 METABOLISM OF UNFERTILIZED OYSTER EGGS,

pyruvate. The experiments, however, suggest that CO, fixation plays at least some role
in pyruvate removal. Experiments were therefore conducted in which changes in the
bicarbonate and CO, content were followed. The results of the two extreme experiments
are shown in Table 20. Owing presumably to the lability of the CO. fixing mechanism,
the results, while not really satisfactory, do indicate that in some preparations (Expt. 1)
some CO, fixation occurs, while in others the removal is due entirely to decarboxylation
(Expt. 2).

TABLE 19.
Removal of Added Pyruvate by Homogenates.
| Bicarbonate | Pyruvate Lactate
Enzyme. Gas Phase. | (Final Removed. Formed.
Concentration). (ug.) (ug.)
| |
|
|
Homogenate .. | Air. -—— | 107 0
3 oH Bic. No — 67 | Berl
bs a =; . 0-005 M 85 1-4
3 am 50 || BY COsiiv is | 0-025 M 91-5 2-1
Supernatant* el N, | — SE TAKO) 28

* Centrifuged for 5 minutes at 10,000 xg.
Each flask contained 0-5 ml. of 30% phosphate saline homogenate or the supernatant therefrom

M
and 0:2 ml. of a phosphate buffer (pH 7-5) in a final volume of 1 ml. At the beginning of the

experimental period, which was 60 mins., 110g. of pyruvate was added from the siderarm.

The experiments detailed in Table 5 on the acid production of whole eggs are not
sufficiently precise to decide whether CO. fixation or decarboxylation is responsible for
the lack of accumulation of pyruvate in whole eggs. Unfortunately, owing to failure
of the supply of eggs, it has not yet been possible to perform precise experiments to
decide this issue. The lability of the homogenate system, however, suggests that CO,
fixation may play a considerable role in whole eggs.

TABLE 20.
The Mechanism of the Anaerobic Pyruvated Utilization.

| |
|

Experiment I. | Experiment II.
| |
Control. Experimental. | Control. Experimental.
Pyruvate added (ug.) in ae 223 | oe 226
‘< recovered ({4g.) . . — 185 | = | 163
x used (ug.) .. as = 38 | = | 63
| | |
CO, evolved (1.) ore ate 8-9 7-25 24 | 31-5
Bicarbonate recovered (t11.) .. 56 | 53-6 42-5 47
Ao bell (RIS) Wet 1 i yee eae 64-9 | 60-75 66-5 | 78-5
Difference (21.) A Aid —4-15 +12
Pyruvate used/CO, produced | —9-1 +5:-25

|
\

Hach flask, of 5 ml. capacity, contained 0-5 ml. of phosphate saline homogenate (20% in

M
Experiment I and 32% in Experiment II) and 0-1 ml. ‘G bicarbonate in a final volume of 0-7 ml.

The side arm contained 0-1 ml. of 100% trichloracetic acid which was tipped at the end of the
experimental period (90 minutes in Experiment I and 120 minutes in Experiment II) in order
to recover the bicarbonate. The flasks were gassed with a mixture of 1% CO.in Ny». The pyruvate

M
(0-1 ml. of 50 into the main chamber } and bicarbonate were added from opsonic pipettes.
J

BY K. W. CLELAND. 311

(0) Tricarborylic Acid Cycle.

The tricarboxylic acid cycle was formulated on the following three basic observa-
tions (Krebs, 1943): (1) ability of the posulated intermediates to cause a marked
stimulation of respiration; (2) a synthesis of citrate from added oxalacetate; (3)
oxidative formation of succinate from oxalacetate or fumarate in malonate poisoned
systems. The last of these is regarded by Krebs as the most important.

The first of these conditions appears to be fulfilled in the present material (Table 11)
in those intermediates which were tested. Glutamate and, to a much less extent,
aspartate also cause increases in the oxygen consumption of homogenates.

In view of the considerably lower respiratory rate and the low. efficiency of
malonate as an inhibitor of succinoxidase in the present material as compared with
pigeon breast muscle, it seemed unlikely that the second and third conditions could be
fulfilled.

The tricarboxylic acid cycle appears to be the only mechanism known in animal
tissues whereby pyruvate is completely oxidized. If it could be shown that pyruvate
oxidation approached completion in the present material the evidence in favour of the
operation of the cycle would therefore be strengthened.

When pyruvate was added to phosphate saline homogenates (4 experiments) the
extra oxygen consumed varied from 28% to 70% of the theoretical requirement for
complete combustion.

It has been shown that the enzymes of the cycle are present on the large granule
fraction of mammalian liver and kidney (Kennedy and Lehninger, 1949, Green et al.,
1948) and in this material the oxidation of pyruvate is complete. In the oyster egg
the tricarboxylic acid cycle enzymes have been shown to be present on the egg granules
(Cleland, 19500). Because of the low activity of the respiration in eggs it seemed
possible that the low efficiency of oxidation of pyruvate in egg homogenates might be
due to loss of cycle intermediates from the granules to the medium during the oxidation
of the pyruvate.

If this were so, some improvement in the efficiency of utilization of pyruvate might
be expected if pyruvate and some other cycle intermediate were oxidized together.
This was found to be the case in three experiments, one of which is shown in Table 12.

TABLE 21.
Oxidation of Pyruvate under Different Conditions.
| Pyruvate [ ete att
System. | tl. Os. Excess Used. |
| Op. | (Ug.) | ae
|
1 Homogenate .. ot Ss | 132 — — —
2 (1) +pyruvate BS ne est 151 | 19 110 38%
3 (1) +succinate a8 ae a | 272 — | — —
4 (3) +pyruvate mi ms sea" th 305 | 33 74 I, 90%
5 (1)+citrate .. wt ss a 193 | — | — | =
6 (5) +pyruvate eh e al 217 21 48 98%
7 (1)+malate .. &: a He 224 | == | = 2
8 (7) +pyruvate Se ue 50 | 247 | 23 | 70 80%

i :
Each flask contained 1 ml. of 35% phosphate saline homogenate, 0:4 ml. = phosphate buffer (pH 7-5) and 0-2 ml.
M.

2x107-4M cytochrome C in a final volume of 2 ml. Succinate, citrate and malate concentrations were Aaa

Pyruvate (110g./flask) was added from the side bulb at the beginning of the experimental period which was 82
minutes. '

When either citrate, succinate or malate is present during the oxidation of the pyruvate
the rate of pyruvate utilization is reduced but the efficiency of oxidation approaches
100%. Citrate causes the most marked depression of pyruvate utilization and gives the
most complete combustion, as would be expected on the above hypothesis. Kennedy and

2g

312 METABOLISM OF UNFERTILIZED OYSTER EGGS,

Lehninger (1949) have described an inhibition of pyruvate utilization by malate in the
rat liver granule system.

(c) High Energy Phosphate Formation.

That some high energy phosphate formation occurs during the respiration of
homogenates is indicated in the experiment shown in Table 22. Under anaerobic
conditions or in dinitrophenol treated homogenates a considerable decrease in the seven-
minute hydrolysable P occurs. The endogenous respiration of homogenates is almost
capable of maintaining the P; and when tricarboxylic acid cycle intermediates are being
oxidized, the P, content shows a slight increase.

TABLE 22.
in Homogenates under Different Conditions.

High Energy Phosphate Changes

)

|

| Oxygen

System. Gas Used. | 1Dip IPs. Portis

Phase. (ul.) (Ug.) | (U.g.) | (ug.)

1 Homogenate (at beginning) .. | — | — | 87 fete 226) . 109-5
2 (1) at end & oe Neto ete ALD 18:5 | 88 | 22 110
3 (2)+10-* M dinitrophenol Air | 22 99 15 | 114
4 | (2)+suecinate and pyruvate .. | Air | 42 82 “sil 26 108
5 | (2) Ay uh 4 oy N2 0:5 95 | 9 | 104

Each flask, of 5 ml. volume, contained 0:5 ml. of 50% phosphate saline homogenate and 0-1 ml. of 210-4 M
M.-
cytochrome in a final volume of 0:8 ml. Succinate and pyruvate were added in a final concentration of Tan The

experiment duration was 30 minutes. P, refers to inorganic P, P; to 7 minute hydrolysable P.

Keltch et al. (1949) have shown that a particulate enzyme system from Arbacia
eggs is capable of oxidative phosphorylation when using a number of tricarboxylic acid
cycle intermediates.

DISCUSSION. ;

The experiments on the penetration of substrates (Table 4) indicated that inverte-
brate cells do not necessarily have the same permeability properties as mammalian cells.
The impermeability may be confined to free living marine invertebrate cells, since
various preparations from earthworms appear to be freely permeable to a variety of
substrates (O’Brien, personal communication).

The vertebrate cell or tissue is normally in a spatially limited, substrate containing
environment and even when leaving the parent organism (e.g. sperm) the environment
does not change greatly. In contrast, the spawned pelagic invertebrate egg is voided into
an enormous substrate-free environment where intracellular substrate loss would be
irrevocable.

The slight or absent permeability of the present material to natural substrates
would, therefore, appear to have a definite survival value. Considerably larger molecules
(respiratory and mitotic inhibitors) are able to exert their typical biological effect in
the present material, which suggests that the impermeability is rather selective.

Impermeability to natural substrates has necessitated the use of acellular homo-
genates. This has considerably complicated the analysis of intermediary metabolism
because of uncertainty about the relation of the homogenate respiratory mechanisms to
those of the whole egg.

In Table 9 it was shown that glycogen caused a significant stimulation of homogenate
respiration. Since glycolysis from glycogen occurs in homogenates (Table 17) and the
end products of glycolysis are readily oxidized (Table 11) it may be concluded that
the respiratory stimulation caused by glycogen proceeds over these pathways. Hexo-
kinase is evidently present in the eggs, since added ATP enables a considerably
increased respiration to be maintained by fructose and glucose (Table 9; Text-fig. 2).

BY K. W. CLELAND. 31

is)

It may therefore be concluded that whole eggs are capable of using in their oxidative

metabolism both the polysaccharide and monosaccharide which they contain (Table 1).
The greater effect of fructose on uncomplemented homogenates may be due to

different affinities of hexokinase for fructose and glucose (cf. Meyerhof, 1947).

In a previous paper (Cleland, 1950a) the respiratory quotient -of whole eggs was
shown to be approximately 0:80, a value which appeared to result from oxidation of
carbohydrate and lipide. As already indicated, the maximal glycolytic rates found in
extracts would yield sufficient pyruvate to maintain this quotient; the glycogen and
monosaccharide content found is sufficient to allow the quotient to be maintained for
about eight hours.

Some of the lower fatty acids are able to stimulate the oxygen consumption of
homogenates (Table 10). The mechanism was not defined, but the variability of the
results suggests that the reactions involved may be complex. As in the uncomplemented
mammalian preparations, higher fatty acids at higher concentrations caused inhibitions
of homogenate respiration, and even with added ATP and a low fatty acid concentration,
under which conditions mammalian liver preparations actively oxidize the fatty acid
(Lehninger, 1945, 1946), no increase in respiration could be demonstrated. In view of
the presence of large amounts of fat in the present material (Cleland, 1947) and of
a respiratory quatient suggestive of considerable lipide oxidation (cf. Table 23) the
failure of homogenates to oxidize higher fatty acids may probably be ascribed to
lability of the system (cf. Lehninger, 1945, 1946) or to a saturating concentration of
fatty acid being already present in the uncomplemented homogenate. Acetoacetic acid
is a product of fatty acid oxidation in the mammalian liver system, and either it or
some precursor or product is believed to condense with oxalacetate and thus to enter

the tricarboxylic acid cycle (cf. Breusch, 1948, Grafflin and Green, 1948). In contrast to
pyruvate, acetoacetate is without effect on egg homogenate respiration. It cannot be
decided whether this is due to a real qualitative difference between the oyster and
mammalian material or whether it is due to lability of the condensation mechanism. —

The experiments with amino acid additions indicate that no d-amino acid oxidase
is present in the eggs. Apart from an active glutamic enzyme and an aspartic
metabolizing system of low activity, amino acids do not appear to be normal respiratory
substrates for homogenates. Whole eggs, however, show a slightly increased respiratory
rate in the presence of glycine (Cleland, 1950a), which suggests that some of the
homogenate inactivity may be due to the lability of the enzymes concerned. In view
of the low ammonia production of the whole eggs it is not improbable that the rather
- large amounts of free amino acids present (Table 1) are used in synthetic reactions
(e.g. chromosome synthesis) rather than serving as energy sources. |

A superficially typical cytochrome oxidase of relatively high activity has been
found in the present material (Table 12) and cyanide causes inhibition of the respira-
tion of whole eggs comparable in magnitude with that found in mammalian material.
It is fairly certain, therefore, that the link with oxygen in whole eggs is through the
cytochrome system. There do not appear to be many reports of cytochrome oxidase
in invertebrates. In cyanide inhibition studies, Robbie (1949) has indicated that the
respiration of a wide range of invertebrate tissues is inhibited by cyanide in greater
or lesser degree. Vanadium-containing invertebrates are evidently an exception. The
presence of cytochrome oxidase in sea urchin eggs is well established not only by
substrate and inhibitor studies on whole eggs but also by isolation of an enzyme
system (Krahl eft al., 1941).

Succinoxidase (Table 13) has been described in few invertebrates. The literature
has been considered by Humphrey (1917). It is also present in several tissues of the
earthworm (O’Brien, personal communication) and is probably present also in the sea
urchin egg (Crane and Keltch, 1949; Keltch et al., 1949). As in the case of the
adductor muscle of the present material and in the earthworm preparations, malonate
causes relatively small inhibitions of the enzyme (Table 14). This fact indicates that
considerable caution must be exercised in the interpretation of inhibitor experiments
on unknown material.

314 METABOLISM OF UNFERTILIZED OYSTER EGGS,

Malic oxidase (Table 15) does not appear to have been described previously in
invertebrate material, although malate has been shown to stimulate the respiration of
an enzyme preparation from sea urchin eggs (Keltch et al., 1949). The inferred
presence of a glutamic-oxalacetic transanimase is interesting inasmuch as this system
is very widespread and active in mammalian material (Herbst, 1944).

The activity of the hydrolytic enzymes, proteinase, amylase and lipase (Table 16)
is high enough to yield considerably more breakdown products than could be removed
by the oxidative metabolism of the intact egg. Increases in the activity of these
enzymes, which is evidently controlled in the intact egg, would have to be considered
among possible factors responsible for the increases in respiratory rate of eggs during
development (Cleland, 1950a). Increases in their intracellular activity may also
contribute to the increased respiration found in injured eggs.

It is not difficult to envisage a physiological role for the proteinase and lipase in
the eggs, but the significance of the high amylase activity is difficult to assess. One
possibility would be an insoluble and inaccessible glycogen-amylase complex capable of
yielding substrate to the glycolytic system, but no evidence of this possibility has
been found in studies of the intracytoplasmic distribution of amylase or glycogen
(Cleland, 19500).

Owing to false leads arising from unexpected features of the system, analysis of
the glycolytic mechanism proved difficult. The facts emerging from the analysis are
as follows: (a) Whole eggs and homogenates produce considerable amounts of acid, of
which only a very small part can be ascribed to the lactic and pyruvic acids accumulating
(Table 5). (0b) Experiments on cytolytic acid production, some of which are summarized
in the present paper, suggested beyond reasonable doubt that most of the acid
production was non-metabolic. A considerable fraction of uncytolysed whole-egg acid
production and the acid production of homogenates was viewed in the same light.
(c) The effect of glycolytic inhibitors on whole-egg acid production (Table 6) suggested
that the true:glycolytic acid production is still greater-than can be accounted for by
the lactic-pyruvic acid analyses. (d) In the homogenate studies (Table 19) it was
shown that pyruvate, the main glycolytic acid produced, disappears in sufficiently large
amounts to account for the discrepancy noted in (Cc).

Unfertilized and recently fertilized sea urchin eggs produce considerable amounts
of acid under anaerobic conditions, and, at least on fertilization, under aerobic con-
ditions (e.g. Laser and Rothschild, 1939). Large amounts of acid are produced on
injury or actual cytolysis of the egg (e.g. Runnstrom, 1936; Rothschild, 1939). Apart -
from the absence of acid production on fertilization in the oyster, there are points of
similarity to the situation in the sea urchin egg. Small amounts of lactic acid also
oecur in the sea urchin egg (Perlzweig and Barron, 1926; Hutchens et al., 1942).

Carbohydrate metabolism in the sea urchin egg is still unsatisfactorily known.
Lindberg (1948, 1946) has presented evidence that carbohydrate is broken down, not
through the usual glycolytic cycle but through phosphogluconiec acid and pentoses. The
more recent investigations of Lindberg and Ernster (1948) are not inconsistent with ,
this view.

Some of the conclusions about sea urchin carbohydrate metabolism have been based
on the lack of effect of iodoacetate. In view of the work of Barron and Tahmisian
(1948), Harting (1947) and Humphrey (1949) this lack of effect must be interpreted
with caution.

The glycolytic system in the present material is similar to mammalian material in
its coenzyme needs and reaction to inhibitors. It differs quite markedly, however, in
the lactate pyruvate equilibrium: pyruvic acid far exceeds the lactic acid accumulation.
Accumulation of both acids is probably not uncommon, in invertebrates (cf. Humphrey,
1949). In earthworm muscle both acids accumulate (O’Brien, personal communication).
The oyster egg appears to be the most extreme case of pyruvate accumulation yet
encountered.

BY K. W. CLELAND. 315

The possibility that the pyruvate accumulation is an artefact must be considered.
The phenomenon would be possible if some active system capable of oxidizing reduced
cozymase were present. The latter possibility is considered unlikely for the following
reasons: (a) the pyruvate accumulation is unaffected by rigorous exclusicn of the final
traces of oxygen from the nitrogen (passage over copper at 360°C.); (0b) the cozymase
oxidizing systems are evidently present on the granular fractions, since malate and
glutamate oxidation is found to be associated with the granules (Cleland, 19500) while
the glycolytic system is found to be most active in homogenate supernatants where it
is qualitatively similar to the system in whole homogenates (Table 17); (c) competition
for reduced coenzyme by malic dehydrogenase in a CO, fixing mechanism would seem
to be ruled out by the fact that qualitatively similar results are obtained in whole
homogenates and in supernatants therefrom, while the mechanism is known to be
present on the granules (Table 19). Since lactic dehydrogenase appears to be present
(Table 11), it seems reasonable to infer that the pyruvate accumulation is due to the
equilibrium properties of this enzyme.

Meyerhof and Geliazkowa (1947) have shown that the differences found in the
glycolytic mechanism of mammalian homogenates and homologous extracts is due to the
activity of the apyrase which is present mainly on the cell granules. This factor is
unlikely to play any part in the difference between whole homogenate and the super-
natant therefrom in the present material for the following reasons: (a) Glycogen is
used as substrate rather than glucose. (0b) Adenylic acid is almost as active as ATP
(Table 17). (c) The apyrase activity is relatively low and is evenly distributed
between granules and ground cytoplasm (Cleland, 19500). (qd) The alternative explana-
tion proposed is quite adequate to account for the facts.

Hvidence suggestive of CO, fixation has been presented, (Tables 19 and 20). The
fact that the malic, fumaric and succinic utilizing systems, like the system using
pyruvate anaerobically, are on the granules (Cleland, 19500) is consistent with this
view, as also is the fact that these substances would all be capable of rapid oxidation
when aerobic conditions returned.

If the CO. fixing mechanism operates in whole eggs, as seems likely on general
grounds, it would. provide a mechanism of oxidizing the reduced cozymase produced
during glycolysis and would also yield considerably more energy than mere breakdown
tO pyruvate (Ochoa, 1947).

The data shown in Table 21 have indicated that homogenates are capable of
oxidizing pyruvate completely under certain conditions, and the conclusion was drawn
that this oxidation took place via the_tricarboxylic acid. Owing to the relative
‘insensitivity of the methods available and the low metabolic rate of the present material
the experiments considered critical by Krebs (1943) did not seem feasible. However,
the evidence adduced for the cycle (utilization of cycle intermediates, complete oxida-
tion of pyruvate), together with the fact that the enzymes concerned are bound to cell
granules and behave as part of a complex in the centrifugal field (Cleland, 19500), as
does the cyclophorase complex in mammalian material (Green et al., 1948), seems
adequate. ;

The respiratory metabolism of whole eggs and homogenate is compared in Table 23.

It will be seen that the respiratory rate of homogenates may exceed that of
homologous whole eggs. The impression has been formed that this is found in eggs
with low initial respiratory rate (i.e. eggs with unbroken germinal vesicles; cf.
Cleland, 1950a@) and is, further, more direct evidence that the respiratory rate of whole
eges is far from maximal.*

The respiratory quotient has been found to be slightly lower in homogenates than
in whole eggs. This fact and the insignificance of the difference between the cyanide
inhibitions indicates that no new oxygen transport mechanisms or oxygen-consuming
systems (e.g. lipide auto-oxidation) arise on cytolysis.

* A phenomenon first observed by Warburg (Arch. ges. Physiol., 1914, 158: 189).

\

316 METABOLISM OF UNFERTILIZED OYSTER EGGS,

The difference in methylene blue stimulation is probably due to the fact that in
whole eggs there is usually insufficient penetration to cause visible staining of the egg
interior (Cleland, 1947) while in homogenates strong staining of the granules occurs,
a situation not unlikely to cause some disruption of the enzyme complex contained
thereon. The fact that any stimulation occurs with methylene blue may be interpreted
in either of two ways: (a) that the cytochrome-cytochrome oxidase system is limiting;
the high activity of the cytochrome oxidase (Table 12) suggests that it is the cytochrome
which is deficient; or (0b) that some of the dehydrogenases are present in soluble form
in the ground cytoplasm while all of the normal oxygen transport mechanisms are
present on granules; methylene blue enables the soluble dehydrogenases to function
maximally and eliminates the need for electron transport from enzyme to granule.

The strong succinoxidase and glycolytic inhibitor phenylmercuric nitrate (Tables
14 and 18) causes a marked inhibition of whole-egg respiration. That the main
inhibition in whole eggs is due to inhibition of succinoxidase acting in the tricarboxylic
cycle is suggested by: (a) the magnitude of the inhibition (pyruvate catabolism accounts

; TABLE 23.
Comparison of the Respiration of Whole Eggs and Homologous Homogenate.

Characteristic. Whole Eggs. Homogenate. Remarks.
|
Respiratory rate 25 9 of = Increase or decrease. —
a quotient a a8 id 0-80 0:76

Oxidative phosphorylation aa ath Marked. Slight. Tables 7 and 22.

Respiration in the presence of (as per- }
centage control) :
HCN (107° M) pe an 5:4 8:

Ba 5 | No cytochrome added.
Methylene blue (3 x 107% M) aM 256 165 55 Fr
Dinitrophenol (10~*° M) ae ue 180 118 No P added.
Phenyl mercuric nitrate (10~° M) = 18 47 Data from single egg batch.
Todoacetate (10-* M) ad He ES 35 61 wa bs Ms
Malonate (107! M).. be ne a No penetration 65 - a AS
Arsenite (10~? M) i a ae 67 76

for only a portion of the total respiration); (0) the smaller inhibition in homo-
genates does not indicate that lipide oxidation is much affected by this inhibitor;
(c) iodoacetate and arsenite which are less effective inhibitors of succinoxidase
than phenylmercuric nitrate give less inhibition of egg respiration; (d) fluoride, —
a strong inhibitor of glycolysis at M/100 (Table 18) causes much less inhibition of
respiration than the above two compounds. The phenylmercuric nitrate residual
respiration would thus appear to give a crude estimate of the respiration not mediated
by the tricarboxylic acid cycle. If this were so, other succinoxidase inhibitors would
be expected to give inhibition of thé phenylmercuric nitrate sensitive respiration of
whole eggs approximately proportional to their effect on succinoxidase (Table 14).
This appears to be so for arsenite but not for iodoacetate.

In homogenates the phenylmercuric nitrate inhibition is considerably less than
in whole eggs, suggesting that only about 50% of the total homogenate respiration
proceeds through the tricarboxylic acid cycle. The inhibitions caused by arsenite and
malonate in whole homogenates are consistent with this hypothesis, but the inhibition
by iodoacetate, as in the case of whole eggs, is greater than would be expected.

That the phenylmercuric nitrate residual respiration is due to oxidation of lipides
to fragments small enough to enter the tricarboxylic acid cycle seems worth considering.
The greater residual respiration together with the lower respiratory quotient in
homogenates as compared with whole eggs is consistent with this.

Dinitrophenol causes a much smaller stimulation of homogenate than of whole-egg
respiration. This may be due either to injury to the phosphorylating mechanisms in the

BY K. W. CLELAND. 317

former, making these less obligatory in the oxidation, or to differences in the availability
of inorganic phosphate at the enzyme sites in homogenates and whole eggs. The
differences in high energy phosphate formation in homogenates and whole eggs (Tables
7 and 22) suggest that the former factor may be more important; however, until more
is known about the intracellular distribution of inorganic phosphate, and its rate: of
diffusion in cytoplasm, the latter factor cannot be excluded.

The magnitude of the respiratory stimulation caused by dinitrophenol (Tables 3
and 23) strongly suggests that the availability of inorganic phosphate normally plays
a role in limiting the respiratory rate of whole eggs. During the development of the
ege the rising respiratory rate may thus be due to a greater rate of hydrolysis of the
ATP arising during respiration. This might be caused by more rapid utilization in the
endergonic cellular syntheses involved (e.g. chromosome, cell and nuclear membrane
syntheses) and/or increases. in apyrase activity. The effects of colchicine on the
respiration of developing eggs (Cleland, 1950a) could also be interpreted on the former
hypothesis. Owing to the arrest of the cellular syntheses by colchicine, phosphate
turnover is diminished and the respiratory rate is depressed to a lower constant level.

SUMMARY.

In order to provide a background for analysis of the metabolic changes during
development and for the use of inhibitors in studies of the mitotic process, attempts
were ‘made to outline the intermediary metabolism of oyster eggs. The following points
were elicited.

Whole Eggs:

(1) Glycogen, monosaccharide, phospholipide and neutral fat are present; free
amino acids are present in considerable amount but not do appear to be metabolically
active.

(2) Fructose phosphates, phosphoglyceric acid, cozymase, adenylic acid and ATP are
present. Small quantities of lactic and pyruvic acid occur.

(3) Some 90-95% of the respiration is mediated by a cytochrome-cytochrome oxidase
system. Phenylmercuric nitrate causes an 80% inhibition of respiration, a fact inter-
preted in terms of its inhibition of the tricarboxylic acid cycle. ©

(4) Hggs are impermeable to natural substrates, a fact making the use of homo-
genates obligatory for study of intermediary reactions.

(5) Anaerobic acid production far exceeded that expected on the grounds of lactic
and pyruvic acid analyses. Experiments on eggs during cytolysis strongly suggested
that much of this acid production was non-metabolic. A rough estimate of true
glycolytic rate was made by the use of glycolytic inhibitors.

(6) The high energy phosphate content of eggs is depleted by cyanide inhibition,
anaerobic conditions and dinitrophenol, indicating that phosphate pond energy is
generated during respiration.

Homogenates.

(7) Glycogen and fructose stimulate the respiration; with ATP added, glucose
does also.

(8) Some of the lower fatty acids and phospholipide stimulate the respiration;
none was found with higher fatty acids. .

(9) Of six amino acids tested only aspartate and glutamate caused respiratory
stimulation.

(10) Citrate, a-ketoglutarate, succinate, fumarate, malate, pyruvate and lactate
cause marked respiratory stimulation.

(11) Typical cytochrome oxidase, malic oxidase and succinoxidase systems have
been demonstrated. The latter shows a relatively small malonate inhibition.

(12) Lipase, proteinase, amylase, acid and alkaline phosphatase, and apyrase have
been demonstrated. The amylase is very active.

318 METABOLISM OF UNFERTILIZED OYSTER EGGS,

(13) The glycolytic system requires addition of cozymase and adenylic acid or
ATP for maximal activity. Pyruvie acid is the main end product and greater
accumulation is found when the granules are centrifuged from the homogenate.

(14) The latter fact is due to an anaerobic pyruvate removing mechanism on the
granules. At least part of the removal seems due to a CO. fixing mechanism.

(15) Evidence for the operation of a tricarboxylic acid cycle is presented. The
most important evidence is the complete oxidation of pyruvate.

(16) Some phosphate bond energy is formed during the respiration of homogenates.

ACKNOWLEDGEMENTS.

The collaboration of Mr. B. R. A. O’Brien, of this Department, in some of the early
experiments is gratefully acknowledged. Miss P. Smith has given valuable technical assistance.
Thanks are due to the Division of Fisheries, C.8.1.R.O., Cronulla, for the gift of oysters.

The Department of Biochemistry kindly made available some of the commercial fine
chemicals used, and the work has been aided by a grant from the University of Sydney
Cancer Research Fund.

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\

BY K. W. CLELAND. 319

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320

A FURTHER CONTRIBUTION ON THE LIFE HISTORY OF PHEROSPHAERA.

By CHARLES G. Exxiorr, M.Se., Botany School, University of Melbourne.
(Communicated by Dr. Patrick Brough.) <

(Plates ix—xii and Twenty-two Text-figures. )

[Read 29th November, 1950.]

This paper describes the development of the gametes and the proembryo of the
Tasmanian conifer Pherosphaera hookeriana Archer, which features were not adequately
dealt with by Lawson (19230). The development of the embryo from the stage at which
the prosuspensor begins to elongate has been the subject of a previous communication
(Elliott, 1948).

FERTILITY AND POLLINATION.

The material of Pherosphaera hookeriana used in the present investigation was
collected in the Mt. Field National Park, Tasmania, in February, 1948. In my previous
paper figures were given for the fertility of the material containing the youngest
embryos collected on 22nd March, 1947. Similar figures have been worked out for some
of the material collected from the same plants on 13th February, 1948. The site of these
two collections is near tree line at 4,000 feet on Mt. Mawson, on a shelf just below the
top of the range, which affords a slight but not very great amount of protection from
the prevailing weather. Another collection was made on 15th February at Eagle Tarn,
a small lake nestling at the foot of Mt. Mawson in a sheltered position (altitude 3,390
feet). The figures for all three lots of material are presented in Table 1.

The winter of 1946 was unusually severe, and as Table 1 shows, the fertility of the
Mt. Mawson material in the following summer, 1947, was less than in 1948; the winter
of 1947 was not severe.

=

TABLE 1. wae
Numbers of Ovules, their Fertility, and Stage of Abortion of Unpollinated Ovules in Pherosphaera
hookeriana.
Percentage
Number Total Percentage Unpollinated
Material. of Cones Number of Fertile. Ovules Aborting
Examined. Ovules. Early.
Mt. Mawson, 22 Mar., 1947 62 213 25-0 86-25
Mt. Mawson, 13 Feb., 1948 40 180 36:7 79:8
Eagle Tarn, 15 Feb., 1948 70 315 : 33°3 12-9

For the purpose of these tables, a fertile ovule is simply one that contains a well
developed prothallus. As a rule unpollinated ovules abort, and do not develop large
prothalli. Only very occasionally is a well developed prothallus found when there is no
evidence of any pollen tube in the nucellus. The nucellus and embryo sac, and more
particularly the integument, in unpollinated ovules may, however, continue to enlarge
for a time before ceasing to grow. Some abortive ovules were very small. Others were
nearly full sized, but both contained a mere shell of a nucellus or a very degenerate
prothallus. A distinction could be drawn at about half the normal size of the fertile
ovules. The stage at which the abortion took place is also indicated in Table 1. It
will be seen that in the exposed situation a much higher proportion of ovules aborts at
an early stage than at the more sheltered Hagle Tarn.

No attempt has been made to determine the fertility of ovules at different stages of
development. It is to be expected that even in the presence of pollen tubes fertilization

l -

BY C. G. ELLIOTT. 321

might not take place, and hence the difference in “fertility” recorded between the two
lots of Mt. Mawson material and between the two 1948 collections may be too great
as the Mt. Mawson 1948 material was mostly in pre-fertilization stages.

In Table 2 the distribution of fertile ovules among cones is shown. In both lots of
material from the exposed situation the actual distribution does not differ significantly
from a Poisson series, that is to say, ovules are pollinated at random. However, this is
not true of the sheltered situation, where significant excesses above expected numbers
are found in the class 0 fertile ovules per cone, and also in the classes 3, 4 and 5 fertile
ovules per cone. The average total number of ovules per cone is 4-5. Apparently pollen
blows in a cloud in the sheltered situation, and some cones are in a more favourable
position than others in respect to the passing pollen grains.

TECHNIQUE.
Material was fixed in the field in formalin-acetic-alcohol. For examination, the
ovules were removed from the cones and their integuments dissected off. The nucellus
-and the prothallus within it were then embedded using butyl alcohol as dehydrating

TABLE 2.
Distribution of Fertile Ovules among Cones on Pherosphaera hookeriana, Compared with that Expected on Random
Distribution. ;
Number of Cones with » Fertile Ovules.
Material. P
n=0 1 2 3 hast 5 6
Mt. Mawson, 22 Mar., | Found a 22 28 10 2 0 0
1947 bie. a 0:16
Expected .. 26-0 22-6 9-8 2-9 0:6 0-1
Mt. Mawson, 13 Feb., | Found Wes 5 12 16 6 1 0 0
1948 Mf oy 0:12
Expected .. 1:7 12-7 10-4 5-8/ 2-4 0:8 0-2
agle Tarn, 15 Feb., | Found Bie} 21° 19 12 12 4 2} 0
1948 a ant 0-04
Expected .. 15-6 23-4 17-6 8:8 3-3 Wes 0-2

P=probability that the numbers found constitute a Poisson series.

agent and solvent for paraffin. Microtome sections were stained with iron haematoxylin
and counterstained with Bismarck brown. Squash preparations were also made, these
being stained with carmine or by the Feulgen technique. :

=

STAGES OF DEVELOPMENT IN THE MATERIAL.

All the collecting in 1948 was done within a few days, but in different situations
different stages were found, and each collection contained a wide range of stages. In
these natural populations pollination apparently continues for a long time, and hence a
tremendous number of ovules would have to be examined to study any one stage very
intensively. In the present investigation some 200 fertile ovules have been examined.
Most abundant in the material from Mt. Mawson (13th February) were archegonia prior
to the ventral canal division and pollen tubes with body cells. The first division of the
zygote illustrated in Pl. xi, fig. 22, came from this material, but such an advanced
stage was exceptional. The cones collected at Hagle Tarn (15th February) yielded
ovules in which developing and mature gametes, fertilization, and all proembryo stages
were observed, together with embryo systems in which the prosuspensors. were elonga-
ting and embryos beginning to grow. Similar and later stages were found in cones
collected near Boronia Moor on 13th February (not referred to in the section on
pollination and fertility).

22 LIFE-HISTORY OF PHEROSPHAERA,

oo

DEVELOPMENT OF THE MALE GAMETES.

According to Lawson (19230) the pollen grains of Pherosphaera at the time of
shedding contain two nuclei—the tube nucleus and the generative nucleus. The pollen
tubes growing through the nucellus contain three nuclei—the tube nucleus, and the
products of the division of the generative nucleus: the body cell and the stalk or
sterile nucleus. The body cell has a densely staining sheath and contains a large
nucleus, generally somewhat excentrically placed, with a conspicuous nucleolus, the
centre of which does not stain so heavily (Text-fig. 1). The other two nuclei are
generally found close to the body cell. In Pl. ix, fig. 4, the tube nucleus is unusually
far from the body cell. The stalk nucleus may be found either ahead of, or behind, or
sometimes to the side of, the body cell. Sterling (1948) reports that in Taxus the stalk
nucleus moves round the side of the body cell, so the same is probably the case in
Pherosphaera. It is generally considerably flattened against the body cell (PI. ix, fig. 2).
In this respect it resembles that of Saxegothea (Looby and Doyle, 1939, p. 111 and
Pl. 3, fig. 22). It also has the appearance of the nuclei figured by Sinnott (1913) in
Podocarpus dacrydioides (his Pl. 9, fig. 50) and P. ferrugineus (Pl. 5, fig. 2), although
these were considered to be non-functional male gametes. However, the gametes in
Saxegothea (Looby and Doyle) and Pherosphaera (as will be shown below) are very
different from the stalk nucleus, and Sinnott’s interpretation is in error. In fact the
adherence of the stalk nucleus to the body cell in this fashion seems to be a feature
of most podocarps. i

The division of the body cell nucleus is shown in PI. ix, fig. 3. The division results
in two nuclei of equal size and each with a large nucleolus (Text-fig. 2). They resemble
the body cell nucleus except in size. One of them is generally much closer to the edge
of the body cell than the other. The more centrally placed nucleus enlarges considerably,
while the other nucleus does not enlarge so much. At maturity the male cells are
generally unequal, but the degree of inequality is variable. Text-figure 3 shows a case of
extreme inequality in the two nuclei, although this is a young stage. Text-figures
5 and 7 and PI. ix, fig. 7, may be regarded as typical, but the two nuclei may be more
equal in size. The larger nucleus is surrounded by a densely staining sheath which
becomes less homogeneous as development proceeds (Pl. ix, figs. 7, 8; Text-figs. 5, 6).
Text-figure 9 shows part of the sheath of the male complex illustrated in Pl. ix, fig. 7.
There are dark staining vesicular portions in protoplasm which does not stain so
heavily. The smaller male nucleus is pushed well to the side of the protoplast, so
that its outer side is quite naked, being covered by no cytoplasm at all as far as can
be seen. In Pl. ix, fig. 6, part of the sheath has come off, but this exceptional male
complex is interesting, as it shows the rest of the cytoplasm clearly, and also demon-
strates that in Pherosphaera two male cells are not formed—there is no membrane of
any sort dividing the protoplast, such as occurs in Saxegothea (Looby and Doyle,
1939) and Phyllocladus (Young, 1910).

As development proceeds the male nuclei stain more and more intensely. In the
very young male nuclei small chromomeres only are visible. At a later stage, represented
in Text-figure 6, heavily staining chromosomes are seen, and they are thrown into coils
of large and varying diameter (Pl. ix, fig. 5 and Text-fig. 8). The full length of the
chromosome is not so intensely stained at this stage, but the less heavily stained portions
are also coiled (Text-fig. 8).

Up to this time the nucleolus still stains intensely, but as the chromosomes become
intensely staining throughout their length the nucleolus loses its’ black colour with
hematoxylin and appears reddish. Its fragmentation is not the reason for the dark
staining of the male nuclei. The nucleolus in Text-figure 5 is red and the chromosomes
are more closely coiled, apparently staining heavily throughout their length.

In the mature gametes (Pl. ix, fig. 8) the chromosomes are more regularly coiled,
and the diameters of the gyres considerably less than in the earlier stage described.
The appearance of the mature gamete, which has been described by Haupt (1941) and

BY C. G. ELLIOTT. 323

Sterling (1948) as coarsely granular, is due to the coiled chromatids lying close
- together.

The chromosomes of the extruded nucleus are always less heavily stained than those
of the nucleus near the centre. The difference in appearance between the two male
nuclei is shown in Text-figures 5, 6 and 7 and PI. ix, figs. 6 and 7.

‘Although they stain very intensely with haematoxylin, the gametes contain little
or no desoxyribosenucleic acid. Even in material kept in alcohol for two years,
metaphase and anaphase chromosomes in the prothallial tissue and proembryo are well!
stained in the Feulgen technique following 10 minutes’ hydrolysis at 60°.

The coiling seen in the young gametes presumably represents a relic coiling, but
the mature gametes seem to show a narrower, and therefore newly developing, spiral.
The cytology of the male gametes of conifers deserves further study.

The male gametes of Pherosphaera are thus rather different from those of most
podocarps hitherto described. The two male nuclei are unequal, both in size and the
degree to which their chromosomes are coiled (or uncoiled ?) and capable of staining
with haematoxylin. The male nuclei are equal in size in Saxegothea (Looby and Doyle,
1939) and in Microcachrys (Lawson, 1923a); and where they are unequal, as in
Podocarpus andinus (Looby and Doyle, 1944a) and Phyllocladus (Young, 1910), the
smaller nucleus is not extruded from the body cell protoplast. In each of these cases
also the male nuclei are separated by a membrane into two cells. Stiles (1911) claimed
the gametes of Dacrydium were unequal, but one cannot say whether his drawing
represents a gamete or a stalk nucleus. There is, however, considerable resemblance
between the gametes of Pherosphaera and the Hupodocarpus species described by
Coker (1902).

THE OVULE AND DEVELOPMENT OF THE ARCHEGONIA.

The structure of the ovule may be seen from PI ix, fig. 1. The peculiar lips at
the micropylar end are noteworthy. These appear to be present from early stages
(Lawson, 19230, Text-figs. 7-9). Lawson did not suggest they had any function in
pollination. The nucellus is free from the integument right to the base. Occasionally
the nucellus is found to contain two prothalli placed side by side, as in Text-figure 14,
but this phenomenon is very rare—frequency less than 1%. The female prothallus has
no “tent-pole’’.

One of the most significant features of the female gametophyte of Pherosphaera
hookeriana is that the mature archegonia are deeply buried by the growth of the
prothallus. This was overlooked by Lawson, although his Text-figures 26 and 27 show
buried necks. , Lawson said that “each archegonium has its own small and shallow
depression over the neck” (pp. 511-2). This burial of archegonia is an important
resemblance between Pherosphaera and other podocarps, in all of which, this occurs.

Lawson records ‘three or occasionally four’ as the most frequent number of
archegonia. I have found a wider range. In 100 prothalli the number of archegonia
ranged from 2 to 6; the mean was 3:56 + 0:09.

Text-figure 10 shows the neck of an archegonium still in a superficial position, but
the cells of the prothallus are just beginning to encroach over it. This stage resembles
that in Sazegothea in Looby and Doyle’s (1939) Pl. 2, fig. 10. The nucleus in the
young archegonium has a characteristic structure, which is shown in Text-figure 10
and Pl. ix, fig. 9. It has a comparatively large and conspicuous nucleolus, and chromo-
somes can barely be seen in it. The nucleus is situated near the neck (Text-fig. 10
and Pl. ix, fig. 9), a position which it retains until the ventral canal division, which
takes place close to the neck. The very young archegonium has a large central vacuole
and very little cytoplasm, but the amount of cytoplasm rapidly increases, and in
Pl. ix, fig. 9, there are numerous small vacuoles as well as what remains of the large
central one. At this stage the neck is already deeply buried. The buried neck is also
well shown in Pl. ix, fig. 10. The neck generally consists of four cells, but eight are
sometimes found. In one prothallus a six-celled neck was observed.

324 LIFE-HISTORY OF PHEROSPHAERA,

The ventral canal nucleus is seen in Pl. ix, fig. 10. It is found near the neck,
while the egg nucleus takes up a position in the centre of the archegonium. As the
ventral canal nucleus was not often seen it is concluded that it degenerates quickly,
although in several archegonia which had not been entered by a pollen tube, three or
even four nuclei could be seen. The amount of cytoplasm continues to increase and
appears to radiate in strands from the egg nucleus, but apart from this asteroids are
not evident. Finally, the archegonium is nearly full of cytoplasm, and the appearance
of the stained egg nucleus is somewhat similar (Pl. ix, fig. 11; Pl. x, fig. 12). In
favourable cases chromosomes can be seen in the egg nucleus showing relic (?) coiling,
as illustrated in Looby and Doyle’s (1944a) photographs of Podocarpus andinus (their
Pl. 11, figs. 4, 5 and 8). A nucleolus surrounded by chromocentres is generally seen
in the egg nucleus (PI. ix, fig. 11); the structure is especially well shown in carmine
stained preparations.

Text-figures 1-7. Pherosphaera hookeriana.

1. Body cell. with stalk and tube nuclei. 2. Young male gametes shortly after division of
body cell nucleus. 3. Male gametes in early stage of development but showing marked
inequality of size. Stalk and tube nuclei together in front of male complex. 4. Early stage
of development of intense staining of chromosomes of gametes. 5. Well-advanced stage in
development of gametes. 6. Gametes in mid-stage of development, similar to those shown in
Text-fig. 8 and Pl. ix, fig. 5. 7. Typical male gametes. Magnifications, all figures, x 500.

The egg nucleus is not surrounded by a broad sheath of differentiated cytoplasm.
In this respect Pherosphaera resembles Saxegothea (Looby and Doyle, 1939) and pre-
sumably also Dacrydium (Sinnott, 1913, Pl. 5, fig. 3), and differs from Podocarpus
andinus (Looby and Doyle, 1944@) and P. spicatus and P. dacrydioides (Sinnott).

The cells of the prothallus surrounding the archegonium are usually differentiated
as a jacket layer (PI. ix, fig. 10; Pl. x, figs. 13, 16, 17). Archegonia are usually
separated from one another by at least one layer of cells, but occasionally two are in
direct contact (Pl. ix, fig. 11). In older archegonia, especially unfertilized ones, the
outline of the egg nucleus becomes highly irregular.

RELATION OF POLLEN TUBE TO ARCHEGONIUM, AND FERTILIZATION.

The pollen tubes grow through the nucellus towards the apex of the prothallus
(Pl. ix, fig. 4) and spread out over it, passing to the sides. They then grow between

BY C. G. ELLIOTT. ; 325

the nucellus and prothallus to approximately the level of the archegonia. Lawson
(19236) mentions their being outside the megaspore membrane, but since this is very
thin, hardly any thicker than many cell walls in the surrounding tissue, and is often
much buckled, it is very difficult to be certain which side of the membrane the pollen
tube is on. However, the archegonia are buried at an early stage, and the pollen tube
must therefore penetrate some prothallial tissue to reach them. A pollen tube growing
in prothallial tissue is shown in PI. ix, fig. 1. In Text-fig. 13 the pollen tube has grown
between the nucellus and prothallus and then inwards to reach the archegonia.
Lawson considered that the pollen tubes “have their full growth before the
archegonia are organized” and that their course is “well established before the archegonia

\2 13 14

Text-figures 8-14. Pherosphaera hookeriana.

8. Some of the chromosomes in male gametes showing coiling and intense staining (x 1,200).
9. Detail of part-of sheath of male gamete shown in Pl. ix, fig. 7 (x 1,200). 10. Archegonium
prior to ventral canal division, with prothallial tissue just beginning to grow over the neck
(x 350). 11. Archegonium with pollen tube above resulting in ‘“‘suspension’’ of neck cells.
Details from several sections included (x 350). 12. Section of archegonium showing male
gamete fusing with egg nucleus, and the second male nucleus just above it (x 350). 13. Course
of pollen tube between nucellus and prothallus, thence inwards to neck (ruptured) of

archegonium. Top of prosuspensor shown (x 200). 14. Two prothalli side by side in same
ovule (x50).

326 LIFE-HISTORY OF PHEROSPHAERA,

appear’. But there is no evidence of any correlation between pollen tube growth and
archegonium initiation, since the differences between the mean number of pollen tubes
per ovule for different numbers of archegonia (Table 3) are not significant. When the
number of pollen tubes is in excess of the archegonia, all body cells may form gametes,
and in one instance as many as three male complexes were found outside the same
archegonium; or some pollen tubes may grow very little and remain some distance from
any archegonia, and the body cell nucleus does not then divide. But in several cases,
where the number of pollen tubes was equal to or less than the number of archegonia,
a small body cell was still found high up in the nucellus.

The pollen tubes generally reach the archegonia about the time of the ventral
canal division. The pollen tube presses down on the neck, resulting in the “suspension”
of the neck cells (Text-fig. 11), but owing to the small size of the archegonia, about
two-fifths the diameter of those of Podocarpus andinus, this phenomenon is not so
striking as in other podocarps. The occurrence of the ventral canal division is not
related to the presence of pollen tubes, however, as the structure of the nucleus of old
unfertilized archegonia is that of an egg nucleus. The body cell nucleus may divide
before or after the ventral canal division, since in one case in which the body cell
Was dividing, the nuclei of the nearby archegonia had “egg nucleus” structure, while
in two others the archegonium nucleus had a large nucleolus and little else visible in it.

TABLE 3.
Numbers of Pollen Tubes in Relation to Number of Archegonia in Siaty-five Ovules of Pherosphaera hookeriana.
Pollen |
Tubes. 0 1 2 3 4 5 6 7 8 9 | 10 11 Mean.
Archegonia
2 1 2
3 1 5 5 3 | 1 1 1 3:176
4 1 2 vi 10 @ | 638 1 1 3-161
5 2 6 2, 1 1 1 1 3:143
|
i |

The entry of the male nuclei into the archegonium is always through the neck.
The rupture of the neck probably takes place from within. Pl. x, fig. 12, shows
cytoplasm protruding from an archegonium with male gametes outside. It does not
protrude as far as in Podocarpus andinus (Looby and Doyle, 19440). In Pl. x, fig. 13,
there is no sign of a pollen tube outside, but the neck is ruptured all the same. In
Pl. x, fig. 15, the male nuclei are about to enter the archegonium, and necks through
which male cells have entered may be seen in Text-figs. 15 and Pl. x, figs. 14, 17 and 18.

When the functional male nucleus comes in contact with the egg nucleus its chromo-
somes still stain intensely (Pl. x, fig. 16). Before the membrane separating the two
fusing nuclei disappears, however, the male nucleus assumes the appearance of the
female nucleus (PI. x, fig. 17). (One of the male nuclei in PI. x, fig. 14, is seen to be in
contact with the egg nucleus in another section.) Similar phenomena have been
described by Haupt (1941) and Allen (1946).

The number of nuclei of the pollen tube which enter the archegonium seems to be
variable in such cases where it was possible to identify them with certainty. The
second male nucleus generally enters (Text-fig. 12 and Pl. x, fig. 14).° It appears to
have remained outside in Text-fig. 15. Possibly one of the stalk and tube nuclei has
entered the archegonium in Pl. x, fig. 17, but neither has in Pl. x, fig. 14. The latter
would appear to be the general rule. The sheath of the male gametes can generally
be distinguished at this stage.

DEVELOPMENT OF THE PROEMBRYO.
In its general features the development of the proembryo of Pherosphaera resembles
that of other podocarps. The first division of the zygote takes place in the centre of

BY C. G. ELLIOTT. 327

the archegonium, but later free nuclear divisions take place in the lower part, while
the cytoplasm in the upper part of the archegonium becomes rather diffuse (PI. xi,
figs. 23-7). Four divisions normally take place, giving sixteen free nuclei. The cyto-
plasm cleaves into uninucleate protoplasts beginning from below, and this distinguishes
what I shall call the primary embryo units from a tier above (Text-fig. 18), which
divides (Text-figs. 20, 19) to give the prosuspensors and the open cell tier (Text-figs.
21, 22). The nuclei of the primary embryo units divide to give binucleate embryo units
(Text-fig. 21). This feature is characteristic of podocarps, but in Pherosphaera the
binucleate cells, instead of remaining binucleate, immediately proceed to form two-celled
units. The binucleate cell phase is of very short duration, and if binucleate embryo
units persist, as reported previously (Hlliott, 1948), it can only be regarded as highly
exceptional.

_ In the third and fourth nuclear divisions, at metaphase the spindle is surrounded
by a clear area (Pl. xi, figs. 25, 27), which is still evident at anaphase on occasions
(Pl. xi, fig. 26). By late anaphase (PI. xii, fig. 28) all appearance of an intranuclear
nature has been lost. However, in the first two divisions an intranuclear nature may

TABLE 4.

Numbers of Nuclei (Resting, and up to Prometaphase) in Proembryos of Pherosphaera
hookeriana up to 8-nucleate Stage.

Large Nuclei in Extra Small Nuclei | Extra Small Nuclei Number of

Proembryonic within Pro- Extruded from Observations.
Cytoplasmic Mass. embryonic Cyto- Proembryo.
plasmic Mass.

eo to ||
|

|

DID MOOR Rh RR im bo
| |
‘ l roe | |
FPRENPDNEP RP OHPEP HEH pL

Simmer |

* Nuclei even sized, but not very large.
+ Uncertain.

not be apparent even at metaphase (PI. xi, fig. 22). There is no question here of the
great disparity between size of spindle and fusion nucleus visible at the first division in
Podocarpus andinus. The situation is similar to that in Sciadopitys (Tahara, 1937),
where the first division is not intranuclear, while in later divisions (his Fig. 9) the
chromosomes seem to. be surrounded by a clear zone.
: The base of the archegonium is not very long and pointed, hence tiering of nuclei
is not marked. At the 16-free nuclear stage there are two layers of nuclei well defined,
but the lower group may itself be two-tiered. Text-figure 17 is typical. As a result.
the number of prosuspensors and primary embryo units, and arrangement of the latter,
is variable. Sometimes only two primary embryo units are formed; the numbers three
and four are most frequent, but only where there are four or more embryo units are
they in two tiers. There is no evidence of a “phragmoplast” during wall formation;
cell formation is entirely by cleavage of the protoplast, beginning from below.

The division to form the prosuspensor tier takes place before that in the primary
embryo units. In Text-figure 20 the upper tier is near metaphase while the nuclei in
the embryo units are in prophase. The division in the primary embryo units is less

wo
iw)
co

LIFE-HISTORY OF PHEROSPHAERRA,

advanced in Text-figure 19, where the upper tier division is in telophase, and in Pl. xii,
fig. 29, the open cell tier has been formed and the nucleus in the embryo units is in
prophase. Where there is more than one tier of primary embryo units the division of
nuclei in the cells nearer the prosuspensors takes place ahead of that below. In Text-

|

; Text-figures 15-22. Pherosphaera hookeriana.

15. Archegonium with pierced neck and proembryo. The second male nucleus is outside
‘the neck. Proembryo contains four large nuclei (two in this section) and three small (two
visible) being constricted off. 16. Four of eight free nuclei in prophase of fourth free nuclear
division. 17. Sixteen-free nuclear stage, showing a “rosette’’ above. 18. Proembryo in which
primary embryo units are delimited, but division to give prosuspensor and open cell tiers
has not yet taken place. 19. Primary embryo unit nucleus in early prophase and upper tier
division in telophase. Two-celled ‘rosette’ present. 20. Mid-prophase of division of primary
embryo units, and prometaphase of upper tier division. Four-nucleate rosette above. Detail
from two sections. 21. Prosuspensor and open cell tiers formed, with stages in formation of
binucleate units from primary embryo units. Drawing made up from several adjacent sections.
22. Binucleate embryo unit, prosuspensor and open cell tier. Magnifications: Text-fig. 15, x 350;
all others, x 500.

BY C. G. ELLIOTT. 329

figure 21 the upper cells show a binucleate cell and late anaphase of division, while the
terminal cell. shows metaphase.

Unfortunately in no case were the two nuclei of a binucleate cell sufficiently in the
same plane of focus to make a good photograph possible. Pl. xii, fig. 30, is the best
example, though this is a stage in formation of a two-celled unit, as a cleavage plane
is being formed across the centre of the cell. In Pl. xii, fig. 32, two two-celled units
may be seen. j

Although Tahara’s (1937) account of the proembryo of Sciadopitys is not as clear
as could be desired, there are some features which seem to be similar to those in
Pherosphaera. After the fifth division, wall formation is confined to the lower parts
(cf. Pherosphaera, Text-fig. 18) with relict nuclei above; and the sixth division consists
of simultaneous formation of prosuspensor and open cell tier above and two-celled
units below. In Pherosphaera these two processes are not so closely synchronized.
Tahara’s Figure 11 shows prophase in several primary embryo units, in the tier which
gives rise to prosuspensor and open cell tier, and in several relict nuclei above.

THE ORIGIN OF “ROSETTE” HMBRYOS.

“Rosette” embryos are of general occurrence in Pherosphaera (Hlliott, 1948). It
is unfortunate that this term has to be used, for the embryos here referred to are in
no way comparable with those of certain Pinaceae. However, a better name has yet to
be thought of.

In the free nuclear stages it would be expected that the numbers of nuclei found
would be 2, 4, 8 and 16. In Pherosphaera other numbers, such as 38, 6, 7 and 9, also occur.

In Text-figure 15 seven nuclei, of which four are seen in the section drawn, are
found in the denser protoplasm at the base of the archegonium—four of them large,
and three much smaller. The smaller ones appear in the process of being constricted
off. Plate xi, figs. 23 and 24, are two sections through the same proembryo in which
there are four large nuclei (three, visible in fig. 23 and the fourth in fig. 24) and a
small one (fig. 24) already cut off from the main embryonic mass of cytoplasm. The
difference in size is significant. The observations of proembryo nuclei up to the
eight-nucleate stage are set out in Table 4.

Supernumerary spindles have been found in several archegonia (Table 5). A series
of three spindles is shown in PI. x, figs. 19, 20, 21. In all such cases two have been

TABLE 5.
Spindles (Metaphase and Anaphase) Seen in Proembryonic Cytoplasmic Mass in
Pherosphaera_ hookeriana.

Number of Observations.
Division Number,
and Number of
Spindles Expected. Expected
Number of One Extra Two Extra
Spindles Only. Spindle. Spindles.
1 (1) 1
2a(2) 1 3
3 (4) 1 1 1
4 (8) 4 —_ =

found to be much larger than the other. It has not been possible to count exactly the
number of chromosomes on each, but the evidence is very suggestive that two spindles
are diploid (Pl. x, figs. 19, 20) and one haploid (fig. 21). On this presumption, the
small nuclei in Text-figure 15 and Pl. xi, fig. 24 may be considered haploid. Their
origin is apparently from the non-functional male gamete, or one of the other male
nuclei, or possibly from one of the cells of the female gametophyte which happen to

330 LIFE-HISTORY OF PHEROSPHAERA,

enter the archegonium: the cells of the jacket layer about the time of the first division
of the zygote appear indistinct from the archegonium, and their nuclei divide
synchronously with the first two free nuclear divisions. However, in no case did the
supernumerary male nuclei appear to be about to divide. Few studies have been made
of the fate of the supernumerary male nuclei, but Allen (1946) found in Pseudotsuga
that, though they sometimes may divide, they remain in the upper part of the
archegonium. Their identification is always difficult, since at this time numerous bodies
of nucleus-like appearance (as stained with haematoxylin) are found in the archegonium
(cf. Looby and Doyle, 1939, p. 112).

Thus in the proembryo of Pherosphaera the nuclei derived from the zygote are
frequently accompanied by smaller nuclei, which are probably haploid and therefore
of gametophytie origin, although this has not been definitely ascertained. These small
nuclei divide synchronously with those of the embryo while they are in a common
eytoplasmic mass, but later they are separated off from the proembryo and lead an
independent existence in the upper part of the archegonium, and their divisions
generally cease to be synchronous.

Relict nuclei (diploid nuclei from the zygote which do not contribute to the cellular
proembryo) occur very rarely, if at all, in Pherosphaera. Where it is possible to count
the number of nuclei with certainty, as it is up to the eight-nucleate stage, it appears
that no nucleus from the zygote (so determined by its large size, especially towards
prophase of the next division) is constricted off from the cytoplasm containing the
proembryo. Hight spindles only occur in the dense cytoplasm in every case where this
fourth division was observed, and though in the 16-nucleate stage it is generally too
difficult to count the nuclei to be quite sure about the presence of relicts, counts, when
certain, of the numbers of prosuspensors and primary embryo units indicate that all
16 nuclei had taken part in the formation of these structures. In two cases, however,
only seven prosuspensors and one embryo unit could be found, and since rosettes were
absent in these proembryos, only three instead of four free nuclear divisions could have
taken place. In Text-figure 17 the nucleus by itself looks very like those in the
protoplast below it, and it may be a relict.

It is unlikely that any rosette embryos come from the open cell tier, as these
nuclei appear to ‘degenerate very quickly. On the other hand, nuclei resembling the
male nuclei may be found in the archegonium at a late stage (e.g., Pl. xii, fig. 31, cf.
PL oe, whe 1055).

SoME FEATURES OF THE “HARLY”’ EMBRYO.

The main features of the development of the embryo have already been described
(Elliott, 1948). The prosuspensor elongates and pushes the two-celled units some
distance into the prothallial tissue before any further development takes place. Food
reserves are built up in the prothallial cells as soon as the prosuspensor begins to
elongate. Starch, however, is confined to the central zone of tissue, as in Saxegothea
(Doyle and Looby, 1939, p. 132). Hach two-celled unit develops.as an independent
embryo which produces embryonal tubes. The development is a good example of
determinate cleavage polyembryony.

It was recorded that developing embryos regularly contain binucleate cells, quite
apart from the binucleate embryo unit phase. In Pl. xii, fig. 35, three embryos are
seen. The nuclear division in the cell marked with an arrow is at telophase, but there
is no trace of a wall being formed across the equator of the spindle, which is itself
scarcely evident. Pl. xii, figs. 33 and 34 (two photographs of the same section in
different planes of focus) show two embryos. Each of the two cells in the right-hand
embryo is binucleate. In the right-hand cell of this embryo, however, a furrow is
being formed on the outside of the protoplast, but has not yet reached the centre. In
fig. 33 the furrow appears as a definite cleavage plane right across the centre of the
cell. In fig. 34, in the plane of focus of one of the nuclei, the furrow is not nearly so
evident, and does not extend between the nuclei. The fixative used would tend to
enlarge, but hardly to create, such furrows, and I regard them as significant and

BY C. G. ELLIOTT. 331

tangible evidence of cleavage plane formation. Moreover, the evidence goes to show
that similar cleavage planes develop in the binucleate cells leading to two-celled units,
just as in Podocarpus andinus (Looby and Doyle, 19440) leading to four-celled units
from a quadrinucleate protoplast.

It is of interest to record that during the elongation of the prosuspensor the
two-celled units pass through a stage during which they are difficult to’fix and stain
well. They emerge from this condition before further development takes place in the
embryo units, the lower embryo units emerging before those nearer the prosuspensor.
A similar phenomenon has been described for the binucleate embryo units in Podocarpus
andinus (Looby and Doyle, 19440, p. 261).

DISCUSSION.

In some features of its life history described in this paper Pherosphaera hookeriana
shows close similarities to other well-known podocarps, as well as considerable
differences. The structure of the body cell, the burial of the neck of the archegonium at
an early stage, and the general features of embryogeny up to the binuclate stage are
features shared with other podocarps. On the other hand, the form of the male gametes
“and the presence of two-celled embryo units, as well as the absence of male prothallial
cells (Lawson, 1923b) are important differences. Pherosphaera also has unique
epidermal structures (Florin, 1931).

I previously suggested that in respect of cone morphology Pherosphaera has close
affinities with Dacrydium; and I would now stress what I observed before, that we can
hardly estimate the significance of what we now know about Pherosphaera until more
is known about the life history of Dacrydium. Little enough is known of D. cupressinum,
and in leaf form it differs widely from the group including D. bidwillii and D. franklinii,
which in external appearance at least bear a closer resemblance to Pherosphaera
hookeriana. The present paper, though by no means an exhaustive account, can provide
some more substantial basis for comparison when the life history of Dacrydium is
better understood. :

SUMMARY.

Comparisons of the numbers of fertile ovules, stage of abortion of unpollinated
ovules, and randomness of distribution of fertile ovules in three collections of
Pherosphaera hokeriana suggest that severity of season affects fertility, while in
sheltered situations, as contrasted with exposed, unpollinated ovules grow large and
ovules are pollinated at random.

The male nuclei are usually unequal in size and the smaller one is extruded from
the protoplast that contained them, but is not delimited therefrom by a membrane.
During development of the male gamete relic coiling of the chromosomes is apparent.
The chromosomes increase greatly in their intensity of staining with haematoxylin, the
length of chromosome so staining developing progressively. :

The archegonia are buried at an early stage before pollen tubes reach them. In such
features aS the number of archegonia, and the nuclear divisions in them, the female
gametophyte is independent of the stage of development of the male gametophyte.

The development of the proembryo up to the binucleate stage resembles that in
other podocarps, but the binucleate cells immediately form two-celled units.

Rosette embryos probably arise from haploid nuclei which occur in the protoplast
with diploid nuclei, but from which they are constricted off. Diploid relict nuclei have
not been observed.

References.

ALLEN, G. S., 1946.—HEmbryogeny and Development of the Apical Meristems of Pseudotsuga.
I. Fertilization and Harly Hmbryogeny. Amer. J. Bot., 33: 666-677.

Cokur, W. C., 1902.—Notes on the Gametophytes and Embryo of Podocarpus. Bot (Gaz:
37: 89-107.

Doyue, J.. and Loopy, W. J., 1939.—HEmbryogeny in Sawegotheq and its relation to other
Podocarps. Set. Proc. R. Dwblin Soc., 22: 127-147. i

EvuiotTr, C. G., 1948.—The Embryogeny of Pherosphaera hookeriana. Proc. LINN. Soc. N.S.W.,
73 :120-9.

332 LIFE-HISTORY OF PHEROSPH AERA,

FLorRIN, R., 1931.—Untersuchungen zur Stammesgeschichte der Coniferales und Cordaitales.
I. Morphologie und Epidermisstruktur der Assimilationsorgane bei den rezenten Koniferen.
K. Svenska Vetensk. Akad. Handl., 3 Ser., 10 (1).
Haupt, A. W., 1941.—Oogenesis and Fertilization in Pinus lambertiana and P. monophylla. Bot.
Gaz., 102: 482-498.
LAWSON, A. A., 1928a.—The Life History of Microcachrys tetragona. Proc. LINN. Soc. N.S.W.,
48:177-1938.
, 1923b.—The Life History of Pherosphaera. Proc. LINN. Soc. N.S.W., 48: 499-516.
Loopy, W. J., and Doy.Le, J., 1939.—The Ovule, Gametophytes, and Proembryo in Saxegothea.
Sci. Proc. R. Dublin Soc., 22: 95-117.
, 1944a.—The Gametophytes of Podocarpus andinus. Sci. Proc. R. Dublin Soc.,
23 : 222-237.
, 1944b.—Fertilization and Early Embryogeny in Podocarpus andinus. Sci. Proc. R.
Dublin Soc., 23: 257-270.
Sinnott, E. W., 1913.—The Morphology of the Reproductive Structures in the Podocarpineae.
Ann. Bot., Lond., 27: 39-82.
STERLING, .C., 1948.—Gametophyte Development in Taxus cuspidata. Bult. Torrey tot. Cv,
75: 147-165.
STILES, W., 1911.—A Note on the Gametophytes of Dacrydiwm. New Phytol., 10: 342-7.
TAHARA, M., 1937.—Contributions to the Morphology of Sciadopitys verticillata. Cytologia,
Fujii jub. vol.: 14-9.
Younc, M. S., 1910.—The Morphology of the Podocarpineae. Bot. Gaz., 50: 81-100.

EXPLANATION OF PLATES.

PLATE Ix.

1. Section of ovule showing integuments, micropyle, nucellus, and prothallus with
archegonium and pollen tube. x 50.

2. Body cell with stalk nucleus. x 400.

3. Division of body cell nucleus. x 400.

4. Pollen tube growing through nucellus, showing body cell and tube nucleus. x 300.

5. Coiled and heavily staining chromosomes in young male gamete. Section adjacent to
that shown in Text-figure 8. x 1,000.

6. Male complex with peculiar sheath. x 400.

7. Typical male gametes in mid-stage of development. x 400.

8. Mature male gamete. x 400.

9. Young archegonium with buried neck. Small arrows (top and bottom left) indicate
boundary between prothallus and nucellus. x 300.

10. Archegonium with ventral canal nucleus, showing buried neck cells. x 300.

11. Two archegonia in direct contact, Showing characteristic appearance of mature egg
nucleus. x 300. :

re

IPGATE xs

12. Archegonium with cytoplasm protruding through neck. Male gametes above, the
smaller nucleus in focus. x 300.

13. Archegonium showing egg nucleus and jacket cells, with cytoplasm protruding from
neck, but no male complex outside. x 300.

14. Two male nuclei inside archegonium (one of them in contact with egg nucleus in
another section), ruptured neck through which they entered, and stalk and tube nuclei outside.
x 300.

15. Male gametes about to enter archegonium through neck; functional gamete out of
focus, nucleolus of second male nucleus visible. x 300.

16. Male nucleus just in contact with egg nucleus. x 300.

17. Later stage of fusion of nuclei. Two other male nuclei visible in archegonium above.
x 300.

18. Archegonium in which second proembryo division is taking place. Shows neck through
which male gametes entered. x 300.

19, 20, 21. Three spindles visible in single proembryo. Those in 19 and 20 presumed diploid,
the smaller in 21, haploid. x 1,000.

PLATE XI.

2. First division of fusion nucleus. x 1,000.
, 24. Four free nuclei; three of them in 23 and the fourth in 24, which also shows small
nucleus forming rosette. x 400.

25. Metaphase of second free nuclear division. x 400.

26. Anaphase of third free nuclear division. x 400.

27. Late prophase of fourth free nuclear division, with a ‘rosette’ in which two nuclei
are dividing. x 400.

BY C. G. ELLIOTT. 333

PLATE XII.
28. Late anaphase of fourth free nuclear division. x 400.
29. Primary embryo unit (nucleus in prophase), prosuspensors and open cell tier, with
division of nucleus in a “‘rosette”’ above. x 400.
30. Proembryo showing binucleate unit in process of conversion into two-celled unit. x 500.
31. Supernumerary nuclei in archegonium with prosuspensors below. x 400.
32. Proembryo with two-celled units. x 400.

33, 34. Two views of embryo in different planes of focus, showing formation of cleavage
plane in binucleate cell. x 850.

35. Hmbryos at tip of elongating prosuspensor. That marked by arrow shows absence of
cell plate formation, leading to binucleate cell. x 485.

334

A NEW GENUS AND FOUR NEW SPECIES OF PHLOECHARINAE (COLEOPTERA,
STAPHYLINIDAE) FROM THE AUSTRALIAN REGION.

By W. O. STEEL.
(Communicated by J. W. T. Armstrong.)

(Twenty-nine Text-figures. )

[Read 25th October, 1950.J

INTRODUCTION.

Up to the present three species of Phloecharinae have been described from the
Australian region, all of them being placed in the genus Phloecharis; these are
P. antipodum Fauvel from Australia, P. maori Cameron from New Zealand, and
P. australis Fauvel from New Caledonia. The first two of these, and presumably also
the third (see below), differ, particularly in the form of the maxillary palpi, from true
Phloecharis and cannot be retained therein. A new genus is necessary for these
species and the description of this, together with those of four new species, is given
below.

No specimens of P. australis have been available for study, but there seems to be
no reason why this should not be congeneric with the Australian species. Bernhauer
(Col. Cat. 129, 1933, 1024) records this species as occurring in Queensland, though no
other reference to this has been found. There are, however, tour specimens in the
British Museum of Natural History from Queensland which bear Bernhauer’s label
“Phloecharis australis Fauv.”, and these would appear to be the insects on which the
Australian record is based. These specimens are referable to P. maori Cam., with
‘which, from Fauvel’s description (Rey. d’Ent., 21, 1902, 257), australis could well be
conspecific. Against this contention, however, there is the fact that Fauvel knew the
New Zealand species, to which he gave the manuscript name maori, besides implying
(l.e.p. 257) that it was different from qautstralis. It appears advisable, therefore, not
to synonymize australis and maori until such time as authentic australis can be examined
and also, until then, to delete this species from the Australian list.

P. maori, though known to Fauvel from New Zealand and described by Cameron
from that country, appears to be very rare there. It is shown here, however, to be
widely distributed and apparently common in Australia, and it would appear, therefore,
that the species is an Australian one which has, by some means or other, spread to
New Zealand.

The figures illustrating this paper have all been drawn with the aid of a camera
lucida. Those of the mouth-parts and aedeagi are from Euparal mounts and the rest
from dried specimens. The aedeagi and mouth-parts are figured as transparencies, that
is to say, internal chitinizations, etc., are shown, but in the case of the former, as
the unextruded internal sac shows no features of taxonomic significance, this is omitted
in all cases. The figures illustrating surface sculpture were made using a vertical
illuminator. a

I am greatly indebted to Mr. H. M. Hale and. Mr. H. Womersley, of the South
Australian Museum, tor making available to me a large amount of material, together
with Mr. A. M. Lea’s notes on the specimens. Thanks are also due to Mr. J. Balfour-
Browne, of the British Museum of Natural History, for facilities for using the collections
there, and to Messrs. J. W. T. Armstrong and K. M. Guichard.

Genus PSEUDOPHLOECHARIS, gen. nov.
In general facies, very similar to Phloecharis Mann.
Head narrower than the pronotum, about as long as broad (excluding eyes), the
post-ocular portion (about posterior third) normally retracted into the thorax. The

BY W. 0. STEEL. 305

eyes about one-third the total length of the head, rounded and rather prominent, so
that the visible portion of the head appears more or less triangular (Text-figures 12-18).
Posterior head suture* distinct on dorsal surface, almost absent on the ventral. Ventral
head sutures well separated, converging from base to about the level of the middle
of the eyes, then diverging towards front; distinct throughout their whole length. .

Antennae with all the segments sparsely setose, the seventh to eleventh also with
short pubescence. \

Labrum (Text-figure 3) transverse, the front margin very lightly rounded, the
anterior angles broadly rounded; the surface with a few punctures bearing long setae.
Mandibles (Text-figures 2, 4) rather short and stout, pointed apically, the right with
a small rounded tooth at about middle internally, the left internally with two very
fine tubercles near apex and a third often absent, presumably abraded at about middle.
Both just behind middle internally with a membranous area which bears a fringe of
very fine, short setae; below this, at base, with a small file-like molar area. Inner lobe
of maxillae (Text-figure 1) broader than the outer, at apex with numerous rather stout
setae and, externally, a curved pointed spine. The outer with a brush of fine setae
apically. Maxillary palpi with the first segment very small, a little longer than broad,
the second much longer and broader than the first, widened apically, the third widened
apically, shorter than the second, the fourth distinctly longer than and as broad as the
third, elongate pyriform. All the segments with numerous setae. Mentum distinctly
transverse, trapezoidal, narrowed in front. Glossae not distinguishable from the
paraglossae, the ligula consisting of two lobes which are fused except in front and
which bear one or two fine setae. Lobes of the hypopharynx well developed, densely
setose internally. Labial palpi with the first segment distinctly longer than broad,
the second much shorter and a little narrower, slightly transverse, the third narrower
than and distinctly longer than the second.

Pronotum transverse, variable in shape. - Prosternal process narrow and pointed,
extending backwards between the anterior coxae for about one-third of their length.
Mesosternal process narrow, rounded apically, extending backwards between the inter-
mediate coxae for about three-quarters of their length and slightly overlapping the
short, bluntly pointed metasternal process. Metasternum variable in length, without a
process posteriorly. Elytra variable in length, the wings sometimes atrophied.

Third to sixth, first to third visible abdominal segments about equal in length,
the seventh and eighth distinctly longer. Sternite of the third segment with a median
longitudinal keel on basal half. Tergites of segments four and five on each side of
middle, besides the normal puncturation and pubescence, with a diagonal row of fine,
strongly curved setae. Posterior margin of the tergite of the seventh segment with a
fine, whitish membranous fringe (even in the brachypterous species).

Posterior coxae transverse, strongly expanded laterally above dorsal to the femora,
also with a small expansion below ventral to the trochanter. Tibiae finely and rather
closely setose, with a few stouter setae on each side at apex. Tarsi about two-thirds as
long as their respective tibiae, the second segment about twice as long as broad, inserted
almost at the base of the first, which, from above, appears very short. It, however,
extends below the second and is nearly as long as this (as in Phioecharis). The third
segment distinctly shorter than the second, the fourth a little shorter than the third,
the fifth much longer, about as long as the four preceding together.

Genotype: Phloecharis maori Cameron.

Range: Australia, New Zealand and (presumably ) New Caledonia.

Little is known as to the habits of the species of this genus, but they would appear
to be similar to those of Phloccharis spp. The larvae are as yet unknown.

This genus differs from Phloecharis Mann. mainly in the structure of the maxillary
palpi and ligula. In this latter genus the second segment of the maxillary palpi is
longer and distinctly narrower, the third is a little longer than the second and much
wider, while the fourth is much shorter than the third and less than half as wide,

*See Steel, Hnt. Mon. Mag., 85, 1949, 282.
A-1

336 NOTES ON AUSTRALIAN PHLOECHARINAE,

SS
5)

ems mise, peace:
scarce :
Rien
Whoter
Sy ode

ay, =
~ a*
— 7
bt

Text-figures 1-11.
1. Pseudophloecharis maori (Cam.), maxillae and labium (scale = 0-2 mm.). 2. Pseudo-
phloecharis maori (Cam.), left mandible (scale = 0:1 mm.). 3. Pseudophloecharis maori (Cam.),
labrum (scale as 1). 4. Pseudophloecharis maori (Cam.), right mandible (scale aS 2).

ive)
iw)
~]

BY W. 0. STEEL.

bluntly pointed apically, with the sides almost straight. The ligula has the glossae
and paraglossae distinct, the former extending forwards some distance beyond the latter.

Besides the above, there are also some more obscure differences, at least from
Phloecharis subtilissima Mann. (the only species of Phloecharis which has been available
for dissection). In this species the scale-like setae on the ventral surface of the labrum
are rather numerous and all small, whereas in Pseudophloecharis spp. these are
distinctly fewer in number and the posterior two are about twice as large as the rest.
These setae are shown by dotted lines in Text-figure 3. The left mandible of
P. subtilissima has a small bifid tooth at about middle which is absent in
Pseudophloecharis.

The ground sculpture of the head, pronotum and elytra varies somewhat in
Pseudophloecharis, but that of the abdomen is more or less constant for all the species
dealt with here. The tergites are finely and rather closely punctured and pubescent,
the ground sculpture of those of the third to sixth (first to fourth visible) segments is
more or less reticulate (Text-figure 10, though not always as regular as this), whereas
on those of the seventh and eighth it is distinctly closer and alutaceous (Text-figure 11).
The sternites are sculptured as the tergites.

KEY TO THE KNOWN SPECIES OF PSEUDOPHLOECHARIS,

1. Sutural length of elytra shorter than the pronotum ................... brachyptera, n. sp.
Sutural length of elytra distinctly longer than the pronotum ...............0....0..0005 2

2. Sides of the pronotum sinuate before the posterior angles ................0.00.0000. 8
Sides of the pronotum not sinuate before the posterior angles .................0.00euee 4

3. Sinuation of the pronotal sides very marked (Text-fig. 13), elytra shorter in relation to the
pronotum (pronotum: sutural length about 1:1:2) ............... 2. sinuatum, n. sp.
Sinuation of the pronotal sides less marked (Text-fig. 14), elytra longer in relation to the
_pronotum (pronotum: sutural length about 1:1:4) ............... 3. antipodum (F vl, )

4, Head, elytra and abdomen black or blackish, the pronotum lighter. Dise of pronotum
AEE Tap A teLeMe Cog cucnownsiee ih Perera (aerot sc easel aorere reece = yamaha flanaya are ienstanane) ite tamas 4. fuscum, n. sp.’
Colour yellowish-brown to brown, pronotum convex ............ Dele hee aoe pS i at aN te 5

5. Punecturation of pronotum very close, as fine as that of the abdominal tergites. Pronotum
distinctly more narrowed in front than behind (Text-fig. 18). Aedeagus as Text-

fel HUNTS SPT eee ia are Marre RS ose Lg Sere ooeey ote mE Se eee teiichanveutenn Ieee el eapereuctest 6. maori (Cam.)

Puncturation of pronotum less close, coarser than that of the abdominal tergites. Pronotum
(Text-figs. 16, 17) rather variable in shape, but less narrowed in front than in the
preceding species. Aedeagus as Text-figures 25, 26 ........... 5. occidentalis, n. sp.

1. PSEUDOPHLOECHARIS BRACHYPTERA, h. Sp. (Text-figures 5, 8, 12, 19-20).

Rather flattened. Dorsal surface of head yellowish red to pitchy red, pronotum and
abdominal tergites yellowish red, elytra yellowish red with about the basal fourth and a
narrow area along the suture black. Mandibles, palpi and legs yellowish red, antennae
with the first three segments yellowish ‘red, the rest pitchy. Ventral surface wholly
yellowish red. Pubescence yellowish. . sit

Length: 2:25-2-5 mm.

Tergites of abdomen moderately shining, the head, pronotum and elytra less so,
greasy lustrous. Head distinctly, but not very closely punctured and pubescent, the
punctures coarser than those on the abdomen; between the punctures with a well marked
ground sculpture similar to that on the pronotum. Antennae with the third segment
about as long as the second, the fourth and fifth subequal in length, longer than broad,
the sixth to tenth about equal in length, but increasing gradually in breadth, the sixth
a little transverse, the tenth rather strongly so, about twice as broad as long, the
eleventh distinctly longer, as broad as and about twice as long as the tenth, bluntly
pointed apically.

©

5. P. brachyptera, n. sp., ground sculpture of pronotum (x 225). 6. P. sinuatum, n. sp., ground
sculpture of pronotum (x 225). 7. P. maori (Cam.), ground sculpture of pronotum (x 225).
8. P. brachyptera, n. sp., ground sculpture of elytra (x 225). 9. P. maori (Cam,), ground
sculpture of elytra (x 225). 10. P. antipodwm (Fvl.), ground sculpture of tergite of fourth
abdominal segment (x 225). 11. P. antipodwm (Fyl.),. ground sculpture of tergite of seventh
abdominal segment (x 225), :

338 NOTES ON AUSTRALIAN PHLOECHARINAE,

Pronotum about one and one-half times as broad as long, broadest at about middle,
the sides rounded in front, distinctly sinuate behind, the anterior angles rounded, the
posterior scarcely rounded, subrectangular, the anterior margin lightly emarginate, the
posterior lightly sinuate. Puncturation and pubescence similar to that on head, but
distinctly closer; between the punctures with a well-marked, more or less regular
coriaceous ground sculpture (Text-fig. 5). On each side, at base, with a shallow
impression.

Elytra very short, about one and one-quarter times as broad as long, the sutural
length a little shorter than the pronotum. Puncturation and pubescence as close as that
on the pronotum, but the punctures a little finer and somewhat raised. Ground sculpture
well marked and more or less regular (Text-fig. 8).

[ eee
é Uae >
7 n¢

™
\

12
Text-figure 12. P. brachyptera, n. sp., head, thorax and elytra (scale = 1 mm.).
Tergites of abdomen with puncturation and pubescence a little closer than that of
the pronotum, the punctures somewhat finer. Ground sculpture as noted above (see
generic description).

Aedeagus as Text-figures 19-20.

Victoria: Dividing Range (ex coll. Blackburn).

Holotype (c¢) and allotype in the South Australian Museum; no further specimens seen.
This species may be easily distinguished from all the others dealt with here by

the short elytra. In these it is similar to Phloecharis spp. of the subgenus Scoiodytes
Saulcy, but, apart from the generic characters, differs in the normally developed eyes.

2. PSEUDOPHLOECHARIS SINUATUM, Nn. sp. (Text-figures 6, 138, 21-22).

Rather flattened. Dorsal surface of head blackish red to black, pronotum red, elytra
red, with a black, triangular, basal area, tergites of abdomen blackish except apically,
where they are reddish. Antennae, mandibles, palpi and legs yellowish red. Ventral
surface of head and prosternum red, meso- and metasternum blackish, abdominal
sternites reddish. Pubescence yellowish.

Length: 2-5-3 mm.

Greasy lustrous, the abdominal tergites a little more shining. Head distinctly, but
not closely punctured and pubescent, the punctures coarser than those on the abdomen;
between the punctures with a ground sculpture similar to that on the pronotum, but a
little less marked. Antennae with the third segment about as long as the second, the
fourth and fifth about equal in length, longer than broad, the sixth to tenth about equal
in length, but gradually becoming broader, the sixth hardly transverse, the tenth trans-

BY W. 0. STEEL. 339

verse, less than twice as broad as long, the eleventh distinctly longer,-nearly twice as
long as the tenth, blunily pointed at apex.

Pronotum about one and one-half times as broad as long, broadest at about middle,
the sides rounded in front, distinctly sinuate behind, the anterior angles rounded, the
posterior subrectangular, hardly rounded, the anterior margin lightly emarginate, the
posterior lightly sinuate. Puncturation and pubescence similar to that on head, but
distinetly closer; between the punctures with a well-marked, rather irregular, coriaceous
ground sculpture (Text-fig. 6). On each side, at base, with a shallow impression.

Elytra about as broad as long, the sutural length about one and one-quarter times
as long as the pronotum. Puncturation and pubescence as close as that on the pronotum,
but the punctures somewhat finer. Ground sculpture well marked and more or less
regular (as brachyptera, Text-fig. 8).

16 17

Text-figures 13-18.
Head, pronotum and elytra of: 13. P. sinwatwm, n. sp. 14. P. antipodum (Fvl.). 15. P.
fuscum, n. sp. 16-17. P. occidentalis, n. sp. 18. P. maori (Cam.). The scale, in each
case, = 1 mm,

340 NOTES ON AUSTRALIAN PHLOECHARINAE,

Tergites of abdomen with puncturation and pubescence a little closer than that of
the pronotum, the punctures finer. Ground sculpture as noted above (see generic
description).

Aedeagus as Text-figures 21-22.

Victoria (Blackburn): Holotype.

Tasmania: Hobart, allotype; Mt. Wellington (Lea).

Holotype (@) and one paratype (¢) in the South Australian Museum, allotype in the
British Museum (Nat. Hist.).

This species is in some ways very close to brachyptera, the pronotum being prac-
tically identical in form and the puncturation very similar. It may, however, be easily
distinguished from this by the longer elytra and the form of the aedeagus. From the
other species of the genus it is easily separated by the form of the pronotum.

3. PSEUDOPHLOECHARIS ANTIPODUM (Fauvel) (Text-figures 10-11, 14, 23-24).
Phloecharis antipodum Fauvel, Ann. Mus. Civ. Gen., 18, 1878, 483. Blackburn, Trans. Roy.

Soc. S. Aust., 1888, 191.

Rather flattened. Dorsal surface of head reddish to black, pronotum and abdomen
red, the latter sometimes darker, elytra red, with a basal triangular mark blackish.
Mandibles, antennae, palpi and legs yellowish red. Ventral surface red. Pubescence
yellowish.

Length: 2-2-5 mm.

Greasy lustrous, the abdominal tergites slightly more shining. Head distinctly, but
not very closely, punctured and pubescent, the punctures coarser than those on the
abdomen; ground sculpture similar to that on the pronotum, but a little less marked.
Antennae similar to those of sinuatum, q.v.

- Pronotum a little more than one and one-half times as broad as long, broadest at
about middle, the sides rounded, slightly sinuate before the posterior angles, which are
subrectangular and not rounded, the anterior angles rounded, the anterior margin lightly
emarginate, the posterior lightly sinuate. Puncturation and pubescence similar to
that on the head, but distinctly closer; the ground sculpture well marked, very similar
to that of brachyptera (Text-fig. 5). On each side, at base, with a shallow impression.

Elytra very slightly broader than long, the sutural length a little more than one and
one-third times as long as the pronotum. Puncturation and pubescence as close as
that on the pronotum, but the punctures somewhat finer. Ground sculpture distinct and
more or less regular (as brachyptera, Text-fig. 8).

Tergites of abdomen with puncturation and pubescence a little closer than that on
the pronotum, the punctures finer. 'Ground sculpture as noted above (see generic
description).

Aedeagus as Text-figures 23-24.

Type Locality: Western Australia. Type in the British Museum (Nat. Hist.).

Specimens examined: 5, the type in the British Museum collection and four
specimens from the South Australian Museum.

Records:

Western Australia.
South Australia: Lucindale with Rhytidoponera forelli Craw. (Lea); Adelaide

(Blackburn).
New South Wales: Forest Reefs (Lea); Glen Innes (Lea).

In the shape of the pronotum this species is intermediate between sinuatum and
fuscum, The aedeagus is, however, quite different from either of these, being longer
in relation to its breadth than in any other known species of the genus.

4. PSEUDOPHLOECHARIS FUSCUM, 0. sp. (Text-figures 15, 29).

Rather flattened. Dorsal surface of head black, pronotum blackish red to black,
sometimes lighter (? immature), elytra black, sometimes reddish at apex, abdominal
tergites black with the apices reddish. Mandibles brown, palpi and legs yellowish
brown. Antennae brown with the basal segments yellowish brown. Ventral surface
black with the apices of the abdominal sternites reddish, Pubescence yellowish.

Length: 2 mm,

BY W. 0. STEEL. 341

Head and pronotum greasy lustrous, the elytra and abdomen a little more shining.
Head distinctly, but not very closely, punctured and pubescent, the punctures coarser
than those on the abdomen; ground sculpture similar to that on the pronotum. Antennae
very similar to those of sinwatum, q.v.

Pronotum nearly one and two-thirds times as broad as long, broadest a little behind
middle, not sinuate before the posterior angles, the sides rounded, the anterior angles
rounded, the posterior angles scarcely rounded, obtuse, the anterior margin lightly
emarginate, the posterior lightly sinuate. Puncturation and pubescence similar. to
that on the head, but closer; the ground sculpture well marked, more or less regular,
similar to that of brachyptera (Text-figure 5). On each side, at base, with a shallow
impression.

Elytra very slightly broader than long, the sutural length a little more than one and
one-third times as long as the pronotum. Puncturation and pubescence as close as that
on the pronotum, the punctures a little finer. Ground sculpture distinct, similar to
that of maori (Text-fig. 9).

Tergites of abdomen with puncturation and pubescence a little closer than that on -
the pronotum, the punctures finer. Ground sculpture as noted above (see generic
description).

Aedeagus as Text-figure 29.

South Australia: Port Lincoln (Blackburn), holotype; Myoponga (A. H. Elston).

Victoria (Blackburn): Allotype; Glenferrie (K. M. Guichard).

Holotype (¢), allotype and one paratype in the South Australian Museum, one paratype
in the British Museum (Nat. Hist.) and one paratype in my collection.

In puncturation and general facies this species is very close to antipodum (Fvl.).
It differs, however, in the colour, non-sinuate pronotum, the ground sculpture of the
elytra and the aedeagus. The ground sculpture of the elytra of fuscum and maori is

distinct from that of all the other species in that it is reticulate instead of alutaceous
(cf. Text-figs. 8 and 9).

5. PSEUDOPHLOECHARIS OCCIDENTALIS, nh. sp. (Text-figures 16-17, 25-26).

Rather convex, the disc of the pronotum scarcely depressed. Dorsal surface lighter
or darker brown, the head and/or the elytra sometimes darker. Mandibles, palpi and
legs yellowish-brown, antennae with the basal segments yellowish brown, the remainder
darker. Ventral surface lighter or darker brown. Pubescence yellowish.

Length: 2—2:5 mm.

Tergites of abdomen moderately shining, the remainder less so, greasy lustrous.
Head distinctly, though not very closely, punctured and pubescent, the punctures coarser
than those on the abdomen; between the punctures with a ground sculpture similar
to that on the pronotum. Antennae very similar to those of sinuatum, but with the
penultimate segments a little less transverse.

Pronotum rather variable in shape, from one and one-half to one and five-eighths
times as broad as long, broadest at or a little behind middle, the sides rounded, the
anterior: angles rounded, the posterior scarcely so, obtuse, the anterior margin lightly
emarginate, the posterior lightly sinuate. Puncturation and pubescence similar to that
on the head, but distinctly closer; ground sculpture more or less regular, similar to
that of brachyptera (Text-fig. 5). On each side, at base, with a shallow impression,
which is very indistinct and sometimes almost absent. ~ ;

Elytra very slightly broader than long, the sutural length about one and one-third
times as long as the pronotum. Puncturation and pubescence as close as that on the
pronotum, the punctures a little finer. Ground sculpture more or less regular (as
brachyptera, Text-fig. 8).

Tergites of abdomen with puncturation and pubescence slightly closer than that of
the pronotum, the punctures a little finer. Ground sculpture as noted above (see generic
description).

Aedeagus as Text-figures 25-26.

BY W. 0. STEEL. 343

Western Australia: Donnybrook (Lea) (holotype and allotype); Swan River (Lea) ;
Newcastle (now known only as Toodyay) (Lea); Darling Ranges, on Xanthorrhoea (Lea) ;
Yanchep, 13-23.xi.1935 (R. E. Turner).

Holotype (¢), allotype and four paratypes in the South Australian Museum, one paratype
in the British Museum (Nat. Hist.), and one paratype in my collection. One further specimen
in the South Australian Museum.

Occidentalis differs from the species previously described in the more convex form,
particularly of the pronotum. The puncturation of the head and pronotum is also
rather finer. In some ways (convex pronotum and colour) it is close to maori, but
can be easily distinguished from this by the shape of and the coarser and less close
puncturation of the pronotum, the ground sculpture of the elytra and the form of the
aedeagus. d

The pronotum (as noted above) varies somewhat in shape and it was at first thought
that more-than one species was represented in the material before me. Examination
of the aedeagi, however, disproved this. In the material examined, the specimens with
the less transverse pronotum have this broadest at about the middle of its length
(Text-fig. 17), whereas those with this more transverse have it broadest behind the
middle and a little more narrowed in front than behind (Text-fig. 16). Whether this is
generally true for the species can only be shown by the examination of a much larger
number of specimens.

The species appears to be more localized in its distribution than the others, being
apparently restricted to Western Australia.

6. PSEUDOPHLOECHARIS MAORI (Cameron) (Text-figures 1-4, 7, 9, 18, 27-28).

Phloecharis maori Cameron, Ann. Mag. Nat. Hist., 11, 1944, 779.

? Phloecharis australis Fauvel, Rev. d’Ent., Caen, 22, 1903, 257.

Moderately convex, the disc of the pronotum not depressed. Dorsal surface lighter
or darker brown, the head often a little darker. Mandibles, palpi and legs yellowish
brown, antennae brown, slightly lighter basally. Ventral surface lighter or darker
brown. Pubescence yellowish.

Length: 1:75-2:25 mm.

Head, pronotum and elytra rather dull, the abdomen somewhat more shining. Head
moderately closely punctured and pubescent, the punctures as fine as those of the
abdomen; ground sculpture similar to that of the pronotum. The eyes somewhat larger
than in the other species. Antennae with the third segment a little shorter than the
second, the fourth shorter than the third, slightly longer than broad, the fifth a little
longer and wider than the fourth, longer than broad, the sixth to tenth about equal
in length, but increasing gradually in breadth, the sixth very slightly transverse, the
tenth about one and one-half times as broad as long, the eleventh nearly twice as long
as the tenth, rounded apically.

Pronotum a little variable in shape, slightly more than one and one-half times as
broad as long, broadest just in front of base, distinctly more narrowed in front than
behind, the sides rounded; the anterior angles rounded, the posterior rather sharply
rounded, obtuse, the anterior margin lightly emarginate, the posterior lightly sinuate.
.Puncturation and pubescence close, the punctures as fine and close as those on the
abdomen; ground sculpture as Text-figure 7. On each side, at base, with an indistinct,
shallew impression.

Text-figures 19-29.
19. P. brachyptera, n. sp., aedeagus, ventral view. 20. P. brachyptera, n. sp., aedeagus,
lateral view. 21. P. sinuatwm, n. sp., aedeagus, ventral view. 22. P. sinwatwm, n. sp., aedeagus,

lateral view. 23. P. antipodum (Fvl.), aedeagus, ventral view. 24. P. antipodum (Fvl.),
aedeagus, lateral view. 25. P. occidentalis, n. sp., aedeagus, ventral view. 26. P. occidentalis,
n. sp., aedeagus, lateral view. 27. P. maori (Cam.), aedeagus, ventral view. 28. P. maori

(Cam.), aedeagus, lateral view. 29. P. fuscwm, n. sp., aedeagus, lateral view. (This figure is
more or less a reconstruction, as the only male specimen of this species available for study
had the aedeagus partially extruded and somewhat damaged. ) The scale, in each case,
= 0-4 mm.

B-1

344 NOTES ON AUSTRALIAN PHLOECHARINAE,

Elytra somewhat impressed on each side of the suture, a little broader than long,
the sutural length about one and one-third times as long as the pronotum. Puncturation
and pubescence similar to that on the pronotum, ground sculpture as Text-figure 9.

Tergites of abdomen closely and finely punctured and pubescent, ground sculpture
as noted above (see generic description).

Aedeagus as Text-figures 27-28.

Type Locality: Glen Hope, New Zealand. Type in the Broun collection, British Museum
(Nat. Hist.).

Specimens examined: 54, the type and six other specimens in the British Museum (Nat.
Hist.), 46 specimens from the South Australian Museum, and one specimen from the collection
of Mr. J. W. T. Armstrong.

Records:

Western Australia: Swan River, with Iridomyrmex nitidus Mayr. (J. S. Clark);
Donnybrook on Xanthorrhoea (Lea); Darling Ranges (Lea).

South Australia: Lucindale (Feuerheerdt).

New South Wales: Galston (Lea); Muswellbrook (Lea); Sydney; Wollongong (Lea) ;
Springwood (Mrs. L. Smith).

Queensland: Mt. Tambourine (Lea).

Tasmania: Hobart (Lea); Launceston (Lea); Mole Creek (Lea); Ulverstone (Lea) ;
Waratah (Lea and Carter) ; Huon River (Lea); King Is. (Lea).

New Zealand: Glen Hope (Broun); Tairua (Broun); Ngaruaroahia (Broun).

? New Caledonia: Boulari (Saves).

This species is very easily recognized by the close and fine puncturation and
pubescence of the head and pronotum, the punctures being as fine as those on the
abdomen. The shape of the pronotum, though showing a slight variation, is also
characteristic, this being always broadest a little in front of the base.

There is a greater variation in size than has been noted in any of the previous
species, but this is no doubt partially due to the much larger number of specimens
examined.

Maori appears to be the commonest and most widely distributed species of the
genus. As previously noted, it agrees very well with the description of Phloecharis
australis Fvl., and an examination of the New Caledonian species will probably show
the two to be identical.

NOTHS ON THE MORPHOLOGY AND BIOLOGY OF ANABARRHYNCHUS
FASCIATUS MACQ. AND OTHER AUSTRALIAN THEREVIDAE
(DIPTERA, THEREVIDAE).

By KATHLEEN M. I. ENGLISH,
Department of Zoology, University of Sydney.
(Forty-seven Text-figures. )

[Read 29th November, 1950.]

INTRODUCTION.

The family Therevidae is of world-wide distribution and it is well represented im
Australia. Tillyard (1926) gave the number of species as 56. Later, Mann (1928-1933)
described new species and sank some names as synonyms, and he recognized 75 species
in 14 genera; but he states: “It must be stressed that these are ‘Revisional Notes’
only, as insufficient material is available to allow a complete revision of the family.”

No description has been found of the immature stages of any Australian species,.
and very little detailed information on the immature stages is available anywhere.

The earliest records I have seen of Therevidae larvae are by Bouché (1834) and. _
Westwood (1840). The latter gives two other earlier references. Descriptions of larvae
and pupae have been given by Brauer (1883), Williston (1896), Lundbeck (1908),.
Collinge (1909), Felt (1912), Malloch (1915), de Meijere (1916), Issac (1925), and
Bhatia (1934).

In all these papers the descriptions of the larvae have family characteristics only,
there are no details of the head or mouth-parts, and the text-figures by de Meijere, of
mouth-parts, are the only ones which may have some generic characters.

In this paper records are given of the occurrence of A. fasciatus Macq., and the
larva and pupa are described in some detail. Records are given also of the occurrence
of eight other species, and the larva and pupa of each is described briefly, it being
considered necessary to describe only the features that distinguish the species.

ANABARRHYNCHUS FASCIATUS Macquart. Text-figures 1-13.

A. fasciatus was described by Macquart (1848), but the decription is more accessible
in Australia in Mann’s Revisional Notes, pt. 1, -p. 151 (1928). In 1947, when specimens
were submitted to him for identification, Mr. Mann states: “It is a species that I know
from Sydney only.”

Occurrence.

Many larvae were found in the sandy soil of a small garden at Rose Bay, Sydney,
N.S.W. They were particularly numerous in two small plots where fruit fell from the
overhanging branches of a peach tree. The peaches were infected with fruit fly, and
in January, 1947, the garden was left untended for some weeks, the fallen fruit
‘accumulated and fruit fly and other larvae developed unhindered. In February, when
this decaying fruit was being picked up, one large Therevid larva was seen on the
soil under the remains of one peach; and in March, when the soil was being dug over
where the fallen fruit had lain, nine Therevid larvae were turned up in a few minutes.
In April, during a week-end of gardening, thirty larvae of various sizes were found,
but only ten of the larger ones were kept. From March to October, 1947, 91 Therevid
larvae were collected in various parts of the small garden and many more were seen
but not taken. Many were found near the surface and were uncovered by just scratching
the top soil; others were found deeper down when digging, but it was not possible
to determine at what depth exactly they were in the soil. In colour most of them
were white or cream, but some of those found in October had a distinct pink tinge;

346 NOTES ON AUSTRALIAN THEREVIDAE,

Ve bea
Anterior \\. Antenna.
Dorsal Hour:
sh
Abdominal
Spiracle.
/2. aS
4 es Aplerior
Fae Ly MOTD, Paps. Se oF
Be Labrutn
Labial Pape
Posterior
/’ Sorrack.

\Posfer/or
Pseudopods. [22

Text-figures 1-12. Anabarrhynchus fasciatus.

Larva, lateral view, x 73.

Head of larva, lateral view, x 120.

Antenna of larva, x 500.

Head of larva, ventral view, x 120.

Vertical section of larval head, showing labrum and labium, x 120.
6. Mandible and maxilla of larva, x 185.

ot wm Ww bo

BY KATHLEEN M. I. ENGLISH. 347

others appeared to have alternating dark and light bands; this was evidently due to
some dark-coloured foodstuff showing through the thin-walled part of the segments.

The larvae are carnivorous and it was necessary to keep each larva in a jar or tube
by itself, for if several were left together overnight there would be only one surviving in
the morning. No real attempt was made to supply the larvae with food. In the soil
under the peach trees were many larvae and pupae of fruit fly and another Acalyptrate
fly. Some of these were given to a few of the Therevid larvae and were apparently
acceptable, as the larvae were consumed and some of the pupae were pierced and the
contents extracted.

When found many of the larvae had small mites attached to them; and some had
brown spots on the skin, evidence of some diseased condition, for these did not survive
long; others, after having been kept in captivity for a time, developed this condition
and later died.

Of the larvae collected at Rose Bay in 1946 and 1947, 48 pupated, and from these
there emerged 31 of A. fasciatus and four of other species. Seven pupae were parasitized
by a Bombyliid, of which four adults emerged and three were dissected out in the
pupal stage. One adult of A. fasciatus was obtained from a larva collected in 1946 in
the Hornsby valley, and seven adults were obtained from larvae collected at Woolwich
in 1949.

The pupal period varied from 26-28 days in early September to 14-15 days in the
latter part of October, and 10 days for one specimen in February.

Some adults were collected in the garden at Rose Bay, but not as many were
seen as might have been expected from the number of larvae in the soil. No eggs were
found nor was egg laying observed.

Larva. Text-figures 1-9 and 13.

- The larva (Text-fig. 1) is about 25 mm. long and about 1 mm. in width. It has a
dark brown chitinized non-retractile head and a long slender body consisting of three
thoracic and ten abdominal segments. The thoracic segments each bear a pair of
latero-ventral hairs, the first segment tapers anteriorly to the small head, and near its
posterior border is situated the anterior spiracle. The first six abdominal segments are
each divided by a constriction into two parts, and the first seven segments have, laterally,
a pattern of depressed lines. The eighth segment bears the posterior spiracles. The
tenth segment is divided by a constriction into two parts: the proximal part bears
three pairs of hairs and the distal part bears the anus on the ventral surface and two
pseudopods at the apex.

The Head.—The head (Text-fig. 2) is-about_3 mm. long, it is broad posteriorly and
tapers to a point anteriorly, it is rounded on the dorsal surface and is almost flat on the
ventral surface. The epicranium is a shining brown, the posterior edge is much darker,
and laterally there is a white or colourless area of an irregular shape. ‘On each side
of the epicranium are ten or more papillae, two of which occur, one above the other,
in the posterior end of the white area. At the anterior edge of this area is the antenna
(Text-fig. 3), a curious structure of two very dissimilar parts, the larger part shaped
like a pointed dome on a short thick stalk, the smaller part shaped like a candle,
with a pointed palp at the top, and the whole encircled by a ridge. On the posterior
portion of the epicranium is a large forwardly directed bristle or hair.

On the ventral surface of the epicranium (Text-fig. 4) there is a large chitinized
ventral plate, and on each side of it are two long bristles and one papilla.

Internally the box-like “pharynx support” through which runs the esophagus (Text-
fig. 5) is very similar in arrangement to that described for Apiocera maritima Hardy
(These PROCEEDINGS, 1xxi, p. 298, 1947).

Posterior end of larval exuvia with pseudopods, x 120.
8. Anterior spiracle of larva, surface view, x 185.
9. Posterior spiracle of larva, surface view, x 185.

* 10. Pupa, lateral view, x 73.
11. Pupa, anterior end, ventral view, x 7}.
12. Pupa, posterior end, ventral view, x 7.

348 NOTES ON AUSTRALIAN THEREVIDAE,

The anterior portion of the head contains the mouth-parts and bears bristles and
palps externally. On each side near the lateral posterior edge are three bristles
(Text-fig. 2). The one nearest the median line I have called the anterior dorsal hair;
it is much longer than the one immediately below; near the ventral edge is another
short bristle. In front of these is a large two-jointed palpus. On the ventral surface
(Text-fig. 4) is a centrally situated fleshy lobe armed with six spines, called by
de Meijere (1916) the prementum. Further forward are a pair of slender labial palps,
and near the anterior edge are two pairs of lobes or palps, which I have called the
anterior maxillary palps.

Mouth-parts. The labrum (Text-fig. 5) curves downwards and the anterior part is
hidden by the mandibles when the head is viewed from the side. In the anterior part
of the labrum there is a pair of small spines set in a depression; nearer the apex
the edge forms an upturned peak, and below this the chitin is armed with spines and
hairs. Below the labrum is the hypopharynx, articulated posteriorly to the ventral rods,
and from it the salivary duct runs back below the ventral rods. Below the hypopharynx
is the labium, a laterally compressed structure composed of a chitinous rod above with
fleshy lobe below covered with fine hairs or spines. The arrangement and shape of the
hypopharynx and labium is very similar to that described for Apiocera maritima Hardy.
On each side of the labrum are the strongly chitinized pointed mandibles (Text-fig. 6)
with a fine saw-tooth lower edge and some small barbs on the upper edge. The maxillae
are set outside the mandibles, they are not heavily chitinized, and, as well as the
small anterior palps, they are provided with a beard, or thick tuft, of long hairs.

Behind the head is the capsule rod (Text-fig. 13). It articulates with the posterior
dorsal peak of the epicranium and extends through the prothorax into the anterior part
of the mesothorax and can be seen through the skin; in the live larvae it can be seen
moving actively with the movements of the head. The rod is about 1 mm. in length,
it is strongly chitinized and further strengthened by very heavy chitin in the shaft
and in the distal portion of the wider part.

The posterior pseudopods are used continuously for locomotion when they are turned
downwards at right angles to the body; they are about half the length of the distal
part of the last segment. In the larva itself no structure is visible in the pseudopods,
but in the larval exuvia a very definite chitinized structure is visible (Text-fig. 7); this
probably enables the larva to grip surfaces by suction.

The spiracles (Text-figs. 8 and 9). The anterior pair are situated on the posterior
part of the prothorax, and the posterior pair are on the eighth abdominal segment.
Both pairs are strongly chitinized and show up on the live larva as almost black spots.

Pupa. Text-figures 10, 11 and 12.

Pupal exuviae vary in length from 8-9 mm. The head bears a pair of slender
antennal processes, each ending in a slender spine. The sheath of the labrum is very
‘short and does not extend to the edge of the sheath of the proboscis. On the thorax
there is a strong alar spine on each side at the base of the wing sheath; and each
spiracle is placed on a small high tubercle. The first abdominal segment bears, on
each side, one long spine laterally near the spiracle, and three small dorso-lateral
spines. The second and succeeding abdominal segments, except the last, each have a
single incomplete row of fine bristles near the posterior edge; the bristles are mostly
short on the second segment and they increase in length on each succeeding segment.
On the dorsal surface the bristles are numerous; on the ventral surface there are six
or seven on the second segment and they increase on each succeeding segment to twelve
or thirteen in the last row. lLaterally there are four or five bristles below the
spiracle on each lateral prominence. The last segment bears on each side two very
small bristles, and it ends in two tubercles, each bearing a long spine. The abdominal
spiracles are on small high tubercles.

No difference could be found between the male and female pupae of this species,
though there are very slight differences noted in the other species described in this paper.

BY KATHLEEN M. i. ENGLISH. 349

ter/or

Art er/or 4.
axillary Fates.

~ Dorsal Horr.
SS

NS
S
~

Labial Papp.

Mulerior, -~
Dorsal Harr

Popillae ta
White Area. ee e
Pepe in Anterior Dorsal Harr.
/ : While Area
Meniary lr 7 - ~~ oe
or Beara. ae
Labial Pie Sipe

Dorsal Harr

Atierior a Anitecior Dersal Hatr.
20. - Mailary Paps. Af. Pople \ 22
a ; Whe Area.
Latta Pap. <
r Antena-- ‘
‘ ieee
4 Papihae 1

Whe Area.

Text-figures 13-22.

13. Anabarrhynchus fasciatus, capsule rod of larva, x 60.

14. Acupalpa semiflava, capsule rod of larva, x 60.

15. Platycarenum quinquevittata, head of larva, lateral view, x 120.
16. Anabarrhynchus maritimus, head of larva, lateral view, x 120.

17. Ectinorrhynchus variabilis, head of larva, lateral view, x 120.
18. Taenogera superbus, head of larva, lateral view, x 120.

19. Acraspisa trifasciata, head of larva, lateral view, x 150.

20. Acuplpa semiflava, head of larva, lateral view, x 120.

21. Agapophytus albobasalis, head of larva, lateral view, x 120.

22. Agapophytus aterrimus, head of larva, lateral view, x 120.

iv)
Oo
a

NOTES ON AUSTRALIAN THEREVIDAE,

PLATYCARENUM QUINQUEVITTATA Macquart. Text-figures 15, 23-26.
Recorded by Mann from N.S.W. and Tasmania.

Occurrence. j

Adults were collected at Narooma in January, 1937 and 1938, on beach sand; and
at Cronulla, on 5th October, 1947, among grasses on ‘the sand-dunes behind the beach,
where they were very numerous, and there were many mating couples; three pairs
were caught.

Larvae were collected at Narooma, Cronulla and Yarra Bay (Botany Bay). The
larvae are found in sand, either in the beach above high-water mark or in the dunes
and sand-hills behind the beach. They travel at times just below the surface of the
sand and leave behind a fine line, or track, of raised sand in a very minute “rick-rack”
pattern. These fine tracks, about one or two mm. in width, sometimes extend for long
distances and are often very numerous. There was no opportunity to observe whether
these tracks occur at any time of the year or only at certain seasons; nor was it
possible to determine for how long they would remain on the sand if not levelled by
rain or blown away by wind.

Narooma: In November, 1938, two large larvae were found in surface sand on the
beach.

‘Cronulla: In May, 1947, six larvae were collected in the sand-hills behind the beach;
they were found by scraping away the top sand at the ends of the “rick-rack” tracks.

Yarra Bay: In July, 1948, one larva was found among the roots of the spinfex
growing in the sand at the back of the beach. It had its mouth-parts sunk into a small
beetle pupa, and it continued to. feed even after being uncovered. In October, 1948,
five larvae were found among spinifex roots in the sand. Many Lepidoptera and some
Coleoptera larvae were present also in the sand among the grass roots. Four adults
were obtained from these larvae—two males and two females—and the pupal period
was obtained for two. One emerged in early December (pupal period 18 days); one
emerged in early February (pupal period 16 days).

Larva. Text-figure 15.

The larvae. are large, up to 30 mm. in length and 1:5 mm. in width. On the
epicranium the two papillae in the white area are placed close together and one above
the other. The anterior dorsal hair is short, the anterior maxillary palps large, and
maxillary tuft long and strong. The labrum has a small indentation, but it lacks the
distinct upturned peak of A. fasciatus. :

The capsule rod is heavily chitinized and shaped as in A. fasciatus (Text-fig. 13).
The posterior pseudopods are minute, less than one-fifth of the length of the distal part
of the last segment. The spiracles are not very heavily chitinized.

The dark capsule rod shows very conspicuously through the skin of the thorax;
this and the minute ‘posterior pseudopods make it possible to determine the species of
the live larva if the habitat is known. ‘

Pupa. Text-figures 23-26.

Pupal exuviae vary in length from 8 to 9 mm. The head bears slender antennal
processes, each terminating in a long spine. The sheath of the labrum is short. The
thorax bears a long alar spine on each side; the spiracles are each on a small high
tubercle.

The first abdominal segment bears on each side one long spine laterally, near the
spiracle, and three long dorso-lateral spines. The second and succeeding abdominal
segments, except the last, bear fine bristles near the posterior edge. On the dorsal
surface the bristles are in two rows: an anterior row of shorter bristles which become
shorter on each succeeding segment, and a posterior row of longer bristles which
become longer on, each succeeding segment. On the ventral surface the bristles are
long and short, irregularly placed; they become longer on each succeeding segment.
On the lateral prominences there are short and long bristles; the number and arrange-

BY KATHLEEN M. I. ENGLISH. 351

ment is irregular. The last segment is divided by a constriction into two parts. The
proximal part bears laterally ten or twelve bristles, short and long; the distal part
bears at the apex two small thin bristles. The abdominal spiracles are borne on very
small tubercles.

In the male pupa (Text-fig. 25), on the last segment, in addition to the lateral
bristles, there are nine or ten short bristles on the ventral surface.

ANABARRHYNCHUS MARITIMUS Hardy. Text-figures 16 and 27-29.
Recorded by Mann from Tasmania, N.S.W. and Queensland.

Occurrence.

Adults were collected at Narooma in January, 1937, 1938, 1939, and in November,
1938. More were collected at Cronulla on 5th October, 1947, where they were numerous
on the tall grasses on the sand-dunes behind the beach. Some mating couples were
seen and one pair was caught.

One larva was found at Yarra Bay, 10th July, 1948, among the roots of spinfex
growing in sand at the back of the beach. On 13th September it had pupated and on
9th October a male emerged before 8 a.m. This was the only specimen obtained from
a larva.

Larva. Text-figure 16.

No measurement was made of the larva before it pupated. On the epicranium the
two papillae in the white area are placed close together,-one above and a little behind
the other. The anterior dorsal hair is short, anterior maxillary palps large, and
maxillary tuft long and strong. The labrum has a very small prominence near the
apex, but it has not the distinct peak of A. fasciatus. The capsule rod is heavily
chitinized and shaped as ‘in A. fasciatus (Text-fig. 13). In the larval exuvia the
posterior pseudopods are about the same length as in A. fasciatus and they have the
same structure at the extremity (Text-fig. 7), though not quite so heavily chitinized.
The spiracles are fairly heavily chitinized.

Pupa. Text-figures 27, 28 and 29.

The pupal exuvia is 9 mm. long. The head bears slender antennal processes, each
ending in a slender spine. The sheath of the labrum is short. The thorax bears a
long alar spine on each side; the spiracles are each on a small tubercle.

The first abdominal segment bears, on each side, a long bifid spine near the spiracle,
and two long dorso-lateral spines. The second and succeeding abdominal segments,
except the last, bear a single incomplete row-of fine bristles near the posterior edge,
and most of these become longer on each succéeding segment. On the dorsal surface
the bristles are numerous on the second segment, but the number is reduced a little
on succeeding segments. On the ventral surface there are seven to nine bristles on
each side of a central space. On the lateral prominences there are seven to nine
bristles on segments two to six, and five on the seventh. The last segment is divided
by a constriction, the proximal part bears laterally two short bristles, and on the
ventral. surface four short bristles. The distal part bears, at the apex, two very small
spines. The abdominal spiracles are borne on very small tubercles.

No female pupa was obtained for comparison.

ECTINORRHYNCHUS VARIABILIS Macquart. Text-figures 17 and 30-32.

Recorded by Mann from Tasmania, N.S.W., Queensland and Western Australia.

Occurrence. u

No adults were collected.

Among the larvae of A. fasciatus collected in garden soil at Rose Bay in April, 1947,
was one from which a male of this species emerged in September; and two males .
emerged from larvae collected in garden soil at Woolwich, one in September, 1949
(pupal period 33 days), and one in August, 1950. These were the only specimens
obtained.

352 NOTES ON AUSTRALIAN THEREVIDAE,

_--- Thoracic §, yoiracle.— ~— —_ 33.

Were!
S, yiracle

Z
Ab dora!
sera
/

Pee?

26. Sheath of Labrurn
29

Thoracie--F
_ Spiracle.

Sheath of
Proboscls.~

Text-figures 23-35. Pupae, showing lateral view of whole pupa and ventral views of
anterior and posterior ends of pupae. \

23, 24, 25, 26. Platycarenuwm quinquevittatds x Th.
27, 28, 29. Anabarrhynchus maritimus, x Th.

30, 31, 32. EHctinorrhynchus variabilis, x Tk.

33, 34, 35. Taenogera superbus, x Th.

BY KATHLEEN M. I. ENGLISH. 353

Larva. Text-figure 17.

No measurements were made of these larvae before they pupated. On the epicranium
the two papillae in the white area are placed well apart, one behind and slightly above
the other. The anterior dorsal hair is short. The anterior maxillary palps are smaller
than in the previous species described, but they are as long as the basal segment of
the labial palp. In the maxillary tuft the hairs are sparse. The labrum has no peak
near the apex.

The capsule rod is shaped es in A. fasciatus (Text-fig. 13), but not quite so heavily
chitinized. In the larval exuvia the posterior pseudopods appear to be nearly as long
as in A. fasciatus, and they have the same heavily chitinized structure (Text-fig. 7).
The spiracles are fairly heavily chitinized.

Pupa. Text-figures 30, 31 and 32.

The pupal exuviae are 10-11 mm. in length. The head bears short thick antennal
processes, each bearing a short strong spine with a very short spine at its base.

The sheath of the labrum is short. There are no alar spines on the thorax; the
spiracles are each on a long slender tubercle.

The first abdominal segment bears on each side one very small thorn-like spine
laterally near the spiracle, and one very small dorso-lateral spine, but in one specimen
there were no spines on this segment. The second and succeeding segments, except
the last, are armed with spines near the posterior edge. On the dorsal surface, on
segments two to five, there are about twelve spines, long and short; segment six may be
like segment five or more like segment seven; on segment seven there are six long
spines. On the ventral surface on segments two to seven are two spines placed well
apart; they increase in length on each succeeding segment. On the lateral prominences,
on segments two to six, are two spines, one long and one short; on segment seven they
are both long. The last segment bears, on the anterior portion, one short spine
laterally and two short spines ventrally; the distal portion bears at the apex two long
spines. The abdominal spiracles are borne on long slender tubercles, and on segments
three to seven these bear a small peak on the anterior edge.

No female pupae were obtained for comparison.

TAENOGERA SUPERBUS Schiner. Text-figures 18 and 33-35.
Recorded by Mann from Queensland and N.S.W.

Occurrence.
Two adults, a mating couple, were collected at Yass, N.S.W., in December, 11928.
Three larvae were found in soil at Yass in August, 1930, and from these emerged
two females and one male in November. The pupal period was not obtained.

Larva. Text-figure 18.

The largest larva was 24 mm. in length. On the epicranium the two papillae in
the white area are placed well apart, one behind and slightly above the other. The
anterior dorsal hair is long. The largest of the anterior maxillary palps are as long
as the basal segment of the labial palp. This species appears to have an extra pair of
maxillary palps covered with hairs instead of the maxillary tuft of the other species
in this paper. The labrum has a very small peak near the extremity. The capsule rod
is shaped as in Acupalpa semiflava (Text-fig. 14), but it is not so heavily chitinized.
In the larval exuvia there is no chitinized structure at the extremity of the posterior
pseudopods. The spiracles are not heavily chitinized.

Pupa. Text-figures 33, 34 and 35.

The longest pupal exuvia was 12 mm. in length. The head bears short thick
antennal processes, each terminating in .a strong spine with a very small spine at its
base.

The sheath of the labrum is short. On the thorax there are no alar spines; the
spiracles are each on a long slender tubercle.

vo
oO
roe

NOTES ON AUSTRALIAN THEREVIDAE,

There are no spines on the first abdominal segment. The second and succeeding
abdominal segments, except the last, are armed with strong spines near the posterior
edge. On the dorsal surface, on segments two to five, there are ten to fourteen spines,
long and short; on segments six and seven there are six long spines. On the ventral
surface, on segments two to seven, there are two spines placed apart; on the seventh
segment the spines are longer and placed closer. On the lateral prominences, on
segments two to seven, there are two spines, one long and one short. The last segment
is divided by a constriction into two parts; on the proximal part, on each side, is one
small dorso-lateral spine. The distal part bears at the apex two long spines. The
abdominal spiracles are borne on long slender tubercles, and on segments three to
seven these bear a small peak on the anterior edge.

The male pupa bears, in addition, on the last segment two small spines on the
ventral surface.

ACRASPISA TRIFASCIATA Krober. Text-figures 19 and 36-38.
Recorded by Mann from N.S.W. and Northern Territory of Australia.

Occurrence.
Adults were collected at Yass, N.S.W., where they were numerous in October, 1932.

Two larvae were found in soil at Yass in August and October, 1930; from these
emerged a male and a female in November. The pupal period was not obtained.

Larva. 'Text-figure 19.

The larvae were about 14 mm. in length. On the epicranium the two papillae in
the white area are placed close together, one behind and a little above the other. The
anterior dorsal hair is short. The anterior maxillary palps minute, maxillary tuft scant
or absent. The shape of the labrum could not be determined. The capsule rod is shaped
as in Acupalpa semiflava (Text-fig. 14), but is not so heavily chitinized. In the larval
exuvia the posterior pseudopods have some structure at the extremity but it is not
chitinized and its formation could not be determined. The spiracles are not heavily
chitinized.

Pupa. Text-figures 36, 37, 38.
The pupal exuviae are 6 mm. in length. The head bears short thick antennal
processes, each terminating in a slender spine which may have a minute spine at its
base.

The sheath of the labrum,is short. On the thorax there are no alar spines; the
spiracles are each on a long slender tubercle.

The first abdominal segment bears, on each side, one strong bristle laterally near
the spiracle, and three very slender dorso-lateral bristles. The second and succeeding
segments, except the last, are armed with long bristles and short spines near the posterior
edge. On the dorsal surface, on segments two to six there are six slender bristles
and eight short spines; on the seventh segment are six bristles but only two or three |
very small spines. On the ventral surface, on segments two to seven there are four
long bristles and one or two short bristles also on segments six and seven. On the
lateral prominences, on segments two to seven are two long bristles. The last segment
bears, on each side, four or five long and short dorso-lateral bristles, and on the ventral
surface in the male pupa are three strong bristles. Apically the last segment ends in
two tubercles, each ending in a spine. Abdominal spiracles are borne on tall slender
tubercles.

In the female pupa the short spines are more numerous on the dorsal surface of all
abdominal segments. The last segment bears two or three long dorso-lateral bristles
on each side, and there are no bristles on the ventral surface. ;

ACUPALPA SEMIFLAVA Mann. Text-figures 14, 20, 39-41.
Recorded by Mann from Queensland.

BY KATHLEEN M. I. ENGLISH. 355

/ SU
a] Abdonind
Splracle.
7
[
I
I /
Abdominal Spirdele.
47,
Shear of, LabruiiN. £4. Sheath of Ee

JE. G XX F/,
(Sheath of
PIObOSCIS. «

Shealh of~~

Thoratic---{
-Prabosc/s.

Sorracle.

Text-figures 36-47. Pupae, showing lateral view of whole pupa and ventral views of
anterior and posterior ends of pupae.

Bes Bl, BE Acraspisa trifasciata, x12.

39, 40, 41. Acupalpa semiflava, x 7s.

42, 43, 44. Agapophytus albobasalis, x 73.

Abe 46. 47. Agapophytus aterrimus, x 7d.

356 NOTES ON AUSTRALIAN THEREVIDAE,

‘Occurrence.
One adult was collected at Yass, N.S.W., in October, 1932. Two larvae were found
in soil at Yass in September, 1932, and from these two females emerged in. November
and December. The pupal period was not obtained.

Larva. Text-figures 14, 20.

No measurements were made of the larvae before they pupated. On the epicranium
the two papillae in the white area are placed close together, one behind the other. The
anterior dorsal hair is short, the anterior maxillary palps minute, maxillary tuft scant
or absent. The curved labrum has no peak or depression. The capsule rod (Text-fig. 14)
is shorter than in A. fasciatus, the shaft is not so heavily chitinized, the wide part is
longer and narrower and has no heavily chitinized portion at the posterior end. In
the larval exuvia the posterior pseudopods are very short and the extremity is not
chitinized. The spiracles are not heavily chitinized.

Pupa. Text-figures 39, 40, 41.

Length of pupal exuviae § mm. The head bears short thick antennal processes, each
bearing a slender spine, with a small basal spine. Sheath of the labrum is long; it
reaches beyond the sheath of the proboscis. On the thorax there are no alar spines;
the spiracles are each on a high slender tubercle on a rounded base.

The first abdominal segment bears, on each side, one slender bristle (sometimes
bifid) near the spiracle, and one dorso-lateral bristle. The second and succeeding
segments, except the last, bear bristles and spines near the posterior edge. On the
dorsal surface, on segments two to seven, are long bristles and short spines, irregularly
placed. On the ventral surface, on segments two to seven, are long and short bristles,
irregularly placed. On the lateral prominences, on segments two to seven, are two long
bristles, and in some cases some short bristles also. The last segment bears, on the
proximal part, three small dorso-lateral bristles on each side; the distal part divides
at the apex into two small tubercles, each ending in a spine. The abdominal spiracles
are borne on long slender tubercles and, on segments two to seven, these terminate in
a small spine. There is no male pupa for comparison.

AGAPOPHYTUS ALBOBASALIS Mann. Text-figures 21, 42—44.
Recorded by Mann from Queensland, N.S.W. and South Australia.

/

Occurrence.
Two adults, a mating pair, were collected at Yass, N.S.W., in December, 1929.
Larvae were found in 1930 at the same locality, and from these seven adults
emerged, four females and three males. Pupal periods obtained were 22—23 days for two
females which emerged on December 6, and 17 days for a male which emerged on
December 10.

Larva. . Text-figure 21. r
Larvae varied in length from 20 to 23 mm. On the epicranium the two papillae in
the white area are placed well apart, one behind the other. Anterior dorsal hair is
long, anterior maxillary palps minute. The maxillary tuft scant or absent. The
labrum has no peak or depression. The capsule rod is shaped as in Acupalpa semiflava
(Text-fig. 14). In the larval exuvia the posterior pseudopods are very short and they
have no chitinized structure at the extremity. The spiracles are not heavily chitinized.

Pupa. Text-figures 42, 438, 44.

Length of pupal exuviae 9 mm. The head bears thick antennal. processes, each
terminating in a strong spine which may have a minute spine at its base. The sheath of
the labrum is long. On the thorax there are no alar spines; the spiracles are each on
a long slender tubercle on a round base.

The first abdominal segment bears, on each side, a long strong bristle near the
spiracle and two strong dorso-lateral bristles. The second and succeeding segments,

BY KATHLEEN M. I. ENGLISH. / SOI

except the last, bear short spines and long bristles near the posterior edge. On the
dorsai surface, on segments two to five, are short spines and long bristles, irregularly
placed; on segments six and seven are six long bristles. On the ventral surface, on
segment two are two long bristles; on segment three are two long and two short; and
on segments four to seven are four long bristles. On the lateral prominences, on
segments two to seven are two long bristles and in some cases a short one also. The
last segment bears, in the female pupa, two small dorso-lateral bristles on each side;
the distal part divides into two tubercles, each bearing a long spine. The spiracles are
borne on long slender tubercles and, on segments two to seven, these each end in a very
small spine. The male pupa bears on the proximal part of the last segment two very
small spines on the ventral surface.

AGAPOPHYTUS ATERRIMUS Mann. Text-figures 22, 45-47. -
Recorded by Mann from Queensland, N.S.W. and Victoria.

g Occurrence.
No adults were collected.
Larvae were found at Yass, N.S.W., in 1930, mostly in the soil at the base of hollow

rotting tree stumps, and from these three females and one male emerged in December.

Pupal periods obtained were: 25 days for one male, which emerged on 5th December,

and 18 days for one female, which emerged on 13th December.

Larva. Text-figure 22.

The largest larvae were 27 mm. in length. On the epicranium the two papillae in
the white area are placed well apart, one behind the other. The anterior dorsal hair
is long, anterior maxillary palps minute, maxillary tuft short and thick. The labrum
has no peak or depression. The capsule rod is shaped much as in Acupalpa semiflava
(Text-fig. 14), but in some specimens it is more heavily chitinized at the posterior end.
In the larval exuvia the posterior pseudopods are short and there is no chitinized
structure at the extremity. The spiracles are fairly heavily chitinized.

: Pupa. Text-figures 45, 46, 47.

Length of pupal exuviae 9-11 mm. The head bears thick antennal processes, each
ending in a thin spine with a minute spine beside it. The sheath of the labrum is
long. On the thorax there are no alar spines; the spiracles are each borne on a long
slender tubercle. ~

The first abdominal segment bears, on each side, one long strong bristle near the
spiracle and one short slender dorso-lateral bristle. On the dorsal surface, on segments
two to four, near the posterior edge, are numerous short strong spines; on segments five
to seven the spines are short, slender and few in number. On the ventral surface
segments two to five may bear two; minute spines or none; segments six and seven
bear two long strong spines. The lateral prominences each bear two long strong spines.
The last segment bears, on each side, one very small dorso-lateral spine in the female
pupa; the distal part ends in two tubercles, each bearing a spine. The spiracles are
borne on long slender tubercles.

The male pupa bears on the proximal part of the last segment two small strong
Spines on the ventral surface, and there may be an additional very small latero-dorsal
spine on each side.

NOTES ON HABITS.

In the prepupal stage the larvae of the Therevidae assume a very characteristic
position. The larva lies in the soil in a curved position somewhat like the letter U,
‘or almost in a circle; if it is uncovered the larva straightens itself and slowly makes
its way below the surface of the soil, then re-assumes its curved position. The hard
skin of the larva does not allow it to contract very much, the thoracic segments are
Slightly shortened and become bead-like in appearance, and the abdominal segments
contract a little.

358 NOTES ON AUSTRALIAN THEREVIDAE,

Adult Therevidae are fairly numerous, but not as many are to be seen as might
be expected from the prevalence of the larvae; they fly lazily, and some species fly
with the legs hanging downwards, in the manner of certain wasps.

TENTATIVE KEYS FOR THE GENERA DESCRIBED IN THIS PAPER.

Larvae. f

1. Anterior maxillary palps as long as, or longer than, the basal segment of the labial palp,
asa ins, Rextsfieure 26h ohicvencsileil td eoicees Gol epee eiel Seoeee ad eons ean taue: eben eee tet tl =, Sonete ee em eaaes 2
Anterior measillanyvapalpsamilnute sashimi Nhext=11 een Omeeteiense teleiene leanne i nena nen ae ean ree 5

2. Papillae in lateral white area of head placed one above the other, or nearly so, as in
Mext=TSULES pel Sy AME, UGH Ls cecie) eiearres aes vetieitelcohe tere ehicged ceded iremericic Nerietierts tot nohe Rewehomonenee Reena deena 3
Papillae in lateral white area’ of head placed one well behind the other, as in Text-

SOE =41 D0 cee Wil LA Cte and Bice ioncloiS ong nef Coote kore avaud is a eaatarmen oie. d a olo.cl bo micidmle held br alo ool aabadced 6 4

3.. Posterior pseudopods minute, iess than one-fifth of the length of the distal part of the
LASER SCLIN EMUE oe cis Coons ty eobed sacha) enous ba iors: toler on cs eater Remo eH Re nebo cic na eae ees aeons OMe nal’ercoens Platycarenum
Posterior pseudopods long, about half the length of the distal part of the last segment
GRextahi sll) bee eer ee Miers. cekicey aed Sue chstobnlets: eisie sUstanetetace letra tetemetneyepol state Anabarrhynchus

ab Aico Corse InEhie Sino (Mbexqmles I). godoncooosnbodcodvcoobdenUODOGDBS Ectinorrhynchus
PNaeeroye COmseyl ineyne Moray (Caberqeaaey Alke})) og sb0osaucccaoboddo yet Nee ha rokstineletePAn eeort Taenogera

by UNimigrevone Clopasenl lobo aoe, ys) tool Wberqakenoreey IG) bog oGacadvegedosnudieoegoooD sc ouesedon 6
Anterior dorsal hair long (Text-figs. 21 and 22)" 22.5.2. see eee ee we apevekses -Agapophytus

6. Papillae in lateral white area of head about as far apart as papillae below antenna (Text-
PT PLSD Yb 5 Ay eee acwcteaes radiator stu eocticenieunn ciasararcde tate sells febiel ie nace oinalle PausWtcR ais Leqiseas key Ouchie rel ie lente lnokrstoes Acraspisa

Papillae in lateral white area of head about twice as far apart as papillae below antenna

(ASSES AON! Seccooon dos dsb ogooe bods oo eo do Cao os ObOR Ado OTO OOD DOCS JDO o0 Acupalpa
Pupae.

ie NhHoOraxewiAbhalarespinewals) se Mext=hl Sir is2 Oued me sucdeteteyenaisietene men shokeielc werent heme omens iela team een 2
PhHOLAR WACHOUE MATAR USDIN Ci icais dan sie fs-cke sieve Di ee tebe tact rc te HOHE, ah eeticlle Tener ARNE eeu ATR seer ar ema 3
2. Dorsal abdominal bristles in two rows on segments two to seven (Text-fig. 23) .........
Se Ee INA Se SIC ROR TEMEM El Bt GN Peace Gado Oct sco deen TeE OIE Cia is ororoers Crore bro Td TiasoFG Le TororaG Platycarenum
Dorsal abdominal bristles in one row on segments two to seven (Text-figs. 10 and 27)
ee Pe oc RON ates ore yn rete eon ree Bol camer bret Oe SEL LAU a aan ae Sprite, aemea oi! tr or Wet awe Mire fae rch seve Anabarrhynchus
5. Sheath of labrum short, not reaching to edge of sheath of proboscis, as in Text-figures
Y EnaCl BB “GacooscoobccandgiodoadoaogocdosapeaqocooapogaebeogdsvooUd GROUNDS DOS 4
Sheath of labrum long, reaching beyond edge of sheath of proboscis, as in Text-figure 41
Bee PO ee EE oo PIS aL SN Ua od ch Dactate ai eaE coe Teme O Tes aot Or ececone iomianeG o chasd OSTOGO Dido 0 5
4. First abdominal segment with very small spines or without spines; on { Hetinorrhynchus
segments 2-7 abdominal spines strong (Text-figs. 30 and 33) ...... ] Taenogera
First abdominal segment with long spines; on segments 2-7 abdominal spines slender (Text-
1g OD) ha EEN aout ecicd Cau ete Or riniet anaes (Mi ater ata se Nec eae LAC CLS ex ree e cs 3 GBP OTC CHRSEONS.O Acraspisa
os ANGE Coins Serene Gherxuansss 22) ehael 25))) Geascéoacdcccdcsodonosbocdos Agapophytus
AN ool ouoanbaeyl Siophaversy Skesovoksie> (CAME rine) BES) g sias clolscoooadGgonrdccouoSceosuooono donde Acupalpa

CONCLUSIONS.

Therevid larvae are tound frequently in gardens and bushland soil, but they are
apparently of no economic importance, and the adult flies are inoffensive; these facts
probably explain why there is so little published information on their life history.

The keys given for the larvae and pupae described in this paper are tentative only,
for one cannot say whether the characters selected will be of use for other genera.
For the larvae it was necessary to find very small differences to distinguish them, and
it would be difficult, if not impossible, to identify the living larvae. The pupae, on
the whole, are more readily distinguished, but it was impossible to separate satisfactorily
the genus Hctinorrhynchus trom the genus Taenogera. I hesitated to use the small
differences in the abdominal spines because these vary considerably in different
individuals of the same species and they even vary on the two sides of the same
individual.

The specimens used in the preparation of this paper, e.g. the adult flies with pupal
exuviae and slide mounts of the larval exuviae, have been deposited in the Macleay
Museum at the University of Sydney.

ACKNOWLEDGEMENTS.
The writer is indebted to Professor P. W. F. Murray and Mr. A. R. Woodhill, Department
of Zoology, University of "Sydney, who made available laboratory accommodation at the

Department; and to Mr. J. S. Mann, of the Biological Section, Department of Public Lands,
Queensland, who identified the adult flies.

BY KATHLEEN M. I. ENGLISH. 359

References.

BuaTiA, H. L., 1934.—A Note on the Life-history of Psilocephala sequa Walker (Fam.
Therevidae: Diptera). Ind. Jowr. Sci., Delhi, iv, pp. 203-204, 1 pl.

BRAUER, F., 1883.—Densk. K. Akad. Wiss. Wien, Math. Nat., xlvii, p. 29.
BoucHk, P. F., 1834.—Naturgeschiche der Insecten, pp. 45-46, Taf. 4, f. 16-20.

COLLINGE, W. E., 1909.—Observations on the Life-history and Habits of Thereva nobilitata Fabr.
‘and other Species. Jowr. Econ. Biol. iv, pp. 14-17, 1 pl.

FELT, EH. P., 1912.—N.Y. State Mus. Bull., 155, Ann. Report, p. 121.

Issac, P. V., 1925.—Some Observations on the Life History and Habits of Phycus brunneus
Wied. (Thereva). Mem. Dept. Agric. India, Calcutta, Hut. Ser. ix, pp. 29-30, 1 pl.

LUNDBECK, W., 1908.—Diptera Danica, pt. 2, pp. 136-138.

Macquart, J., 1841-1850.—Diptéres exotiques nouveaux ou peu connus. Suppl. 1-4. (Extrait
des Mémoires Société Royale des Sciences de VAgriculture et des Arts, de Lille, 1844.)
(A. fasciatus, 1848, Suppl. 3, p. 32, and 1850, Suppl. 4, p. 102.)

MAuuocH, J. R., 1915.—Some Additional Records of Chironomidae from Illinois and Notes on
other Illinois Diptera. Bull. Ill. St. Lab. Nat. Hist., xi, pp. 334-336.

MANN, J. S., 1928-1933.—Revisional Notes on Australian Therevidae. Pt. 1, Awst. Zool., v, 1928,
pp. 151-194. Pt. 2, Awst. Zool., vi, 1929, pp. 17-49. Pt. 3, Awst. Zool., vii, 1933, pp. 325-344.

MEIJBRE, J. C. H. bdE, 1916.—Beitrage zur Kenntnis der Dipteren Larven und Puppen. Zool.
Jahr. Abt. System., xl, pp. 210-214.

TILLYARD, R. J., 1926.—Insects of Australia and New Zealand, p. 362.
WESTWoopD, J. O., 1840.—An Introduction to the Modern Classification of Insects, II, p. 550.
WILLISTON, S. W., 1896.—Manual of the Families and Genera of North American Diptera, p. 68.

c-1

XXV

ABSTRACT OF PROCEEDINGS.

ORDINARY MONTHLY MERTING.

3ist May, 1950.

Dr. Lilian Fraser, Vice-President, in the Chair.

Dr. Fraser announced that the Council had elected Professor P. D. F. Murray to fil
a vacancy on the Council.

Dr. Fraser congratulated Miss Muriel Morris, Linnean Macleay Fellow in Zoology
on the award of an International Federation of University Women Fellowship for study
overseas. Miss Morris had arranged to continue her studies on plankton at Oxford
University.

Mr. F. K. Rickwood, B.Sec., Sydney University, was elected an Ordinary Member of
the Society.

Library accessions amounting to 39 volumes, 182 parts or numbers, 22 bulletins, 3
reports and 6 pamphlets, total 252, had been received since the last meeting.

PAPERS READ.
1. Cytological Studies in the Myrtaceae. III. Cytology and Phylogeny in the
Chamaelaucoideae. By S. Smith-White.

2. A Review of and Contribution to Knowledge of Phylloglossum Drummondti. By
Frances M. V. Hackney.

3. A Revision of the Australian Species of the Genus Lagenophora Cass. By Gwenda
L. Davis.

ORDINARY MONTHLY MEBRTING.

28th JuNE, 1950.
Mr. D. J. Lee, President, in the Chair.
Mr. A. A. Day, Bexley, was elected an Ordinary Member of the Society.
Library accessions amounting to 9 volumes, 119 parts or numbers, 3 bulletins, 7
reports and 3 pamphlets, total 141, had been received since the last meeting.

PAPERS READ.
1. Notes on the Aphodiinae of Australia (Coleoptera, Scarabaeidae, Aphodiinae).
By B. B. Given. (Communicated by P. B. Carne.)

2. The Morphology cf the Immature Stages of Aphodius howitti Hope (Coleoptera,
Scarabaeidae, Aphodiinae). By P. B. Carne.

3. Notes on Australasian Simuliidae (Diptera). II. By M. J. Mackerras and I. M.
Mackerras.

NOTES AND EXHIBITS.
Coral Genera of Heron Island (Barrier Reef). By K. E. W. Salter and M. A. Besley.
Situated practically on the Tropic of Capricorn (23° 27’ S.), Heron Island is close
to the southern limit of the Great Barrier Reef. A careful study of the coral fauna of
the reef immediately north of this island shows that there are at least fifty-one species
ot Madreporaria. Preparatory to completion of all identifications, the following twenty-
five genera are placed on record as occurring in this region;

XXVi ABSTRACT OF PROCEEDINGS.

PERFORATA. IMPERFORATA.
Acropora (seven species) Pocillopora (one species) *
Turbinaria (one species) Stylophora (one species)
Astraeopora (one species) Seriatopora (one species)
Montipora (three species) Acrhelia (one species)
Porites (two species) * Galaxea (one species)
Goniopora (one species) Cyphastrea (one species)
Dendrophyllia (one species) Echinopora (one species)

Favia (seven species)

FUNGIDA. Favites (four species)
Fungia (two species) + Coeloria (four species)
Psammocora (two species) Platygyra (one species)
Coscinaria (one species) Merulina (one species)

Hydnophora (three species)
Lobophyllia (two species)
Symphyllia (one species)

Coral growth is inhibited by certain factors such as the covering of their polyps by
sand and by the active movements of the sea. Once identification is established,
statistical data can be compiled and comparative counts made to show the relative
abundance of particular species in relation to the above factors in the environment.
The fauna and its growth as it occurs under such inhibitors contrasts with its distribu-
tion in deep pools where both sanding and wave action are reduced to a minimum.

ORDINARY MONTHLY MEETING.
26th JuLy, 1950.

Mr. D. J. Lee, President, in the chair.

Miss Elise E. Sellgren, B.Sc., Coogee, was elected an Ordinary Member of the
Society.

The President announced that following a request from the Information Service,
C.S.I.R.O., which had resulted from one of the recommendations of the Royal Society
Scientific Information Conference (July, 1948), the Council had decided that a synopsis
of each paper published in the PROCEEDINGS would appear below the title of the paper.
Such a synopsis will not interfere in any way with the arrangement of the paper
but will facilitate the reading of the articles in the PRocEEDINGS. The synopses will be
used as abstracts by Australian and overseas abstractors.

The President drew members’ attention to the fact that Dr. Yao-tseng Tchan,
Macleay Bacteriologist to the Society, would be arriving in Sydney shortly.

The President wished Miss Morris bon voyage on the eve of her departure to
England.

Library accessions amounting to 6 volumes, 131 parts or numbers, 35 bulletins, 4
reports, total 176, had been received since the last meeting.

PAPERS READ.
1. A Classification of the Bacteria, with Special Reference to Non-pathogenic
Eubacteria. By E. J. Ferguson Wood. ‘
2. Australian Polyporaceae in Herbaria of Royal Botanic Gardens, Kew, and British
Museum of Natural History. By G. H. Cunningham. (Communicated by N. A. Burges.)
3. A Revision of the Genus Solenogyne Cass. By Gwenda L. Davis.

NOTES AND EXHIBITS.

Mitotic Poisons. By Miss M. Hindmarsh.

A large number of very different chemical substances are known to inhibit mitosis
in plant cells, but the inhibition is not always the same. Many act like colchicine and
prevent spindle formation. However, they are usually not as efficient as colchicine in
producing polyploids, since they also inhibit the entry into prophase and so gradually

* Additional species are thought to occur.
7; Only two species were found at Heron Island; but a third was recorded from the
Capricorns at One Tree Island.

ABSTRACT OF PROCEEDINGS. XXvil

suppress mitosis. Other substances produce effects similar to X-ray induced abnormalities
which result in a permanent alteration to the chromosomes of the cell.

Mr. A. Musgrave exhibited specimens of Stephanitis queenslandensis Hacker, 1927,
a member of the family Tingidae (Lace Bugs). The insect was first described by
Hacker in the Mem. Q’land Mus., ix, p. 28, from specimens from Tambourine Mountain
and Brisbane, 8.Q., on Stephania hernandifolia Walp., and in the following year he
recorded it in the same publication from the Cairns district and Magnetic Island, N.Q.
Specimens are in the Australian Museum collection from Lane Cove, Sydney, 28th April,
1946, collected by Mr. N. W. Rodd, on Azalea leaves, and also from Lindfield, 13th
January, 1941, on Rhododendrons. Specimens have also been collected at Canberra,
A.C.T., by Dr. A. J. Nicholson, February, 1944, and identified by Mr. Hacker as his
species. This insect is well known to growers of Rhododendrons and Azaleas in the
Sydney district, and references to the Lace Bug appear in salesmen’s catalogues.

The genus Stephanitis is known from the Palaearctic and Oriental regions, and
at least three species are known to attack Azaleas and Rhododendrons in Europe,
United States of America, and South Africa, the best known being Stephanitis
(Leptobyrsa) rhododendri Horvath, 1905, first recorded from Holland upon cultivated
Rhododendrons. The Australian insect appears to be very closely related to this
widespread Huropean insect, which has been the subject of many papers and articles
in scientific journals abroad. Nothing appears to have been written about the economic
aspects of our Australian species, though it is understood that the N.S.W. Department of
Agriculture has been carrying out experiments for its control.

ORDINARY MONTHLY MEETING.

30th AUGUST, 1950.

Mr. D. J. Lee, President, in the chair.

Miss Joy G. Garden, B.Sc.Agr., Sydney, and Mr. R. A. Oxenford, B.Sc., Lane Cove,
were elected Ordinary Members of the Society.

The President ccngratulated. Miss Adele Millerd, Linnean Macleay Fellow in
Biochemistry, on the award of a research fellowship which would enable her to study
at the California Institute of Technology.

Library accessions amounting to 14 volumes, 120 parts or numbers, 6 bulletins, 2
reports, 11 pamphlets, total 153, had been received since the last meeting.

The meeting then took the form of a number of short talks on various aspects of
research in the New South Wales Department of Agriculture.

Dr. C. J. MAGEE introduced the work of the Department.

Mr. GRAHAME Epe@ar, Director of Veterinary Research, spoke on various aspects of
the Division of Animal Industry. Investigations on problems of the animal industry of
this State are centred in the Division of Animal Industry whose’ research centre is
located at the Veterinary Research Station, Glenfield. The problems fall into three main
eategories, namely, animal health, nutriticn and genetics. Whilst it is possible to make
these bread differentiaticns, all three overlap and it is impossible to establish each as a
separate entity. The importance of the work during the last ten years has been empha-
sized by the urgent necessity of increasing food production, especially of animal origin.
‘Extraordinary inflated values for wool and leather have also stressed the significance of
the animal industries in the national economy. The appraised price of wool during the
war years was fixed at 1s. 3d. per lb., whereas the Australian Wool Clip during the
1949/50 selling season realized an overall price of nearly 5s. per lb. Animal health
research will always remain paramount as maximum production from our flocks and
herds can never be achieved unless a satisfactory standard of health is maintained.
Disease in an’mals and birds is the greatest source of economic loss. Whilst Australia
is happily situated in being free of many of the major livestock epizootics, the presence
of Rinderpest, Foot and Mouth Disease, Rabies and many other animal plagues in the
countries immediately north of Australia presents an ever present threat. The aims
and objects of animal health research lie in the fields of preventive medicine,

f

XXViil ABSTRACT OF PROCEEDINGS.

The Veterinary Research Station, Glenfield, was established in 1923, and during the
27 years of its existence has developed and extended enormously in its field of activities.
Since its inception over 400 scientific articles and papers have been published by the
professional officers on the staff. The work of the Station falls into two categories,
diagnosis and research. The diagnostic material received ranges from 20,000 to 30,000
specimens per annum, from animals and birds in various parts of New South Wales.

Research investigations on specific projects are located also at other centres in
different parts of the State. For instance, at the Shannon Vale Nutrition Station, Glen
Innes, a comprehensive series of experiments has been in progress for ten years whose
aim is the investigation of the nutrition and breeding of Merino sheep on the granitic
soils of New England. At Trangie Agricultural Experiment Station, blowfly investiga-
tions and progeny testing of Merinos are being intensively pursued and a new laboratory
was opened early last year for studies and research on wool fibre. The Barooga Field
Station located on the Murray River near Tocumwal is devoted solely to the investiga-
tion of the problem of Toxaemic Jaundice in sheep. This work is a co-operative effort
with the Division of Animal Health and Production of C.S.I.R.O. Genetic and nutritional
investigations of poultry are centred at the Seven Hills Poultry Experiment Farm.

Mr. Edgar then briefly mentioned investigations on tuberculosis and mastitis in
cattle, contagious pustular dermatitis and blowfly strike in sheep, swine fever in pigs,
fowl-tick fever in poultry. He also mentioned the new Nutrition Laboratory at Glenfield
and experiments in the drought-feeding of merino sheep, the effects of diet on butterfat
production, and under genetics, investigations on sheep and poultry.

Dr. S. L. Macinpor, Principal Research Agronomist, gave a brief account of the
organization, progress and results of plant research conducted primarily on the ten
Experiment Farms and two Agricultural Colleges in New South Wales. An active
programme of plant introduction has been pursued as the basis of plant breeding
investigations. The introduction of the rust resistant variety Walsh has enabled the
linseed acreage in the State to increase from 500 acres in 1947-48 to an estimated 20,000
acres in 1950. Breeding for flag smut resistance in wheat has been more successful than
breeding for resistance to stem rust. In 1942 and again in 1948 highly rust-resistant
wheat varieties became susceptible to new races of stem rust. Varieties of better baking
quality released by the University and the Department have resulted in one-third of the
State’s wheat acreage being sown to wheats of medium to strong baking quality. Three
new potato varieties bred by the Department and two recently introduced are expected
to be worth £150,000 annually in increased production. Hight new hybrid maize strains
are expected to raise yields at least 20%. Considerable use has been made of genetic
theory in the development of hybrid maize programmes. Experiments with crops and
pastures are designed so as to determine the best varieties, fertilizers and methods of
establishment in each region. Spectacular results have recently been obtained using
molybdenum on cauliflowers, an expenditure of 3s. to 4s. per acre preventing losses of
’ £100 to £300 per acre from Whiptail disease. Considerable success is being obtained in
the use of selective weedicides on blackberries, and also on water hyacinth, Noogoora
burr and Bathurst burr. In a New England rotation experiment the incorporation of
red clover in an oats-maize cropping sequence has resulted in a greater total yield of
oats and maize being obtained from four crops where Red Clover is included than from
six where the red clover is omitted. Additional rotation experiments are being
commenced to determine the best type of pasture, the length of pasture and the desirable
cropping sequence in the arable phase. A rate of stocking experiment with deferred
grazing is being conducted at Trangie in co-operation with the C.S.I.R.O.

Dr. F. T. Bowman, Special Fruit Research Officer, discussed research in Horticulture.

The research work of the Division of Horticulture deals with the problems of fruit
production, handling and processing, and in organization it corresponds broadly with
that of the Division of Plant Industry. The Division has a number of experiment
orchards and other facilities for the carrying out of research, and often conducts its
work on growers’ properties, in packing houses, ete., where the problems occur,

ABSTRACT OF PROCEEDINGS. xxix

The Division deals with perennial plants, and thus the problems of horticulture
range from the propagation of plants, setting out the orchard, early care and training
of the plants, to care of the mature and ageing plants. Other problems arise from the
annual product, the fruit, from the stage of blossom bud initiation, through development
and harvesting, to preparation for market or some form of storage or processing, as the
case may be. ;

They were often concerned with cumulative effects, e.g., in irrigation practice, as well
as with the effects of different seasonal influences, e.g., spray injuries. Hence, the
results of research on fruit must usually be based on many years’ work.

Some typical examples of research in the different sections of the subject are:
Propagation research (introduction of rootstock for fruit trees), soil management of
orchards (contour planting; suitable covercrops; and reduced tillage), irrigated soils,
pruning, cropping (pollination; chemical thinning; stop drop sprays), fruit handling
and processing.

Me. S. L. Attman, Senior Entomologist, described the Entomological Branch which,
as part of the Department of Agriculture, was formed in 1890, and for the past 60 years
has been concerned with insect pest problems. Some of the earlier members specialized
in systematic work, but in recent years the emphasis has been increasingly on the
applied side. At the present time the staff consists of 14 entomologists, three of whom
are permanently located in country districts. Most phases of pest control are under-
taken, and this calls for co-operation with other Divisions of the Department of Agri-
culture, and also outside bodies, including the Department of Health.

Pests of fruit, vegetables and field crops, medical and veterinary pests, stored
products and grain pests, industrial pests and quarantine safeguards against pest intro-
ductions have all received careful attention.

New insecticides and new equipment now tend to dominate the pest control field and
present-day investigations are mainly concerned with reviewing pest control schedules
from this viewpoint.

Dr. C. J. MAcer, Chief Biologist, discussed research in the Biological Branch. The
research work of the Biological Branch is directed mainly at preventing losses from plant
diseases. In addition, some attention is given to dairy and food bacteriology and general
biology.

The nature of the plant disease investigations varies but follows a conventional
plan of determining the cause, exploring the conditioning factors and endeavouring to
develop control measures. It usually happens that the control phase of the investigation
is the most prolonged, consisting of a prescribed programme based on the researches and
a series of modifications of the programme until a thoroughly satisfactory one that suits
the disease, the grower and everybody else (i.e., the consumer and Health authorities)
is evolved.

Examples of the nature of the work undertaken are indicated by the following
investigations: Bunt, Black Spot of Apples, Whiptail of Cauliflower, Bean Scald,
Hxanthema (copper deficiency of Citrus), Phytophthora Rot of Citrus, Scaly Butt of
Citrus, Stem-pitting of Grapefruit, Black Spot of Citrus, Bunchy Top of Bananas, and
Foliar Nematode of Chrysanthemums.

ORDINARY MONTHLY MEETING.
27th SEPTEMBER, 1950.
Mr. D. J. Lee, President, in the chair.

Protessor H. N. Barber, M.A., Ph.D., Hobart, Tasmania; Mr. J. M. Hallinan, Bexley;
Dr. Y. T. Tehan, Sydney, and Mr. J. L. Willis, B.Sc., Artarmon, were elected Ordinary
Members of the Society.

The President referred to the death of Dr. G. A. Waterhouse, who had been a member
of the Society since 1897 and was elected a Corresponding Member in 1943.

XXX ABSTRACT OF PROCEEDINGS.

The President announced that the Council is prepared to receive applications for
Linnean Macleay Fellowships tenable for one year from list January, 1951, from qualified
candidates. Applications should be lodged with the Secretary not later than Wednesday,
1st November, 1950.

Library accessions amounting to 3 volumes, 33 parts or numbers, 3 bulletins, total
39, had been received since the! last meeting.

PAPERS READ.

1. The Hair Tracts in Marsupials. V. A Contribution on Causation. By W.
Boardman. (By title only.)

2. The Hair Tracts in Marsupials. VI. Evolution and Genetics of Tract Pattern.
By W. Boardman. (By title only.)

3. The Hair Tracts in Marsupials. VII. A System of Nomenclature. By W.
Boardman. (By title only.)

4. Further Notes on Experimental Crossing within the Aédes scutellaris Group of
Species (Diptera, Culicidae). By A. R. Woodhill.

5. A Note on Non-reciprocal Fertility in Matings between Sub-species of Mosquitoes.
By S. Smith-White.

Professor N. A. Burcres and Dr. N. C. W. BEADLE gave talks on Recent Changes in the
Vegetation of north-western New South Wales.

Dr. N. C. W. BEADLE showed a number of kodaslides illustrating the striking contrast
in the condition of the saltbush-bluebush country before and after the good rains of
1949 and 1950. The luxuriance of the regenerating communities, with their relatively
high frequency of recently established perennials suggests that complete recovery of the
_ communities will occur if stocking rates are judiciously controlled.

PROFESSOR BurRGES gave a brief account of the changes which have occurred in the
Mulga country in the neighbourhood of Tibooburra since the abnormal rains of 1949.
Kodachrome slides and a short length of coloured cinema film were used to illustrate the
general appearance of the Mulga dune country in February and August, 1950.

ORDINARY MONTHLY MEETING.
25th OctroBer, 1950.
Mr. D. J. Lee, President, in the Chair.
Mr. K. R. Sharp, Cooma, N.S.W., was elected an Ordinary Member of the Society.

The President referred to the death of Dr. B. L. Middleton, of Murrurundi, who had
been a member since 1937. :

The President announced that the Council is prepared to receive applications for
Linnean Macleay Fellowships tenable for one year from ist January, 1951, from qualified

‘candidates. Applications should be lodged with the Secretary not later than Wednesday,
Ist November, 1950. :

Library accessions amounting to 29 volumes, 60 parts or numbers, 47 bulletins, 1
report and 3 pamphlets, total 140, had been received since the last meeting.

PAPERS READ.

1. Respiration and Cell Division in Developing Oyster Eggs. By K. W. Cleland.

2. The Intermediary Metabolism of Unfertilized Oyster Eggs. By K. W. Cleland.

3. A New Genus and Four New Species of Phloecharinae (Coleoptera, Staphylinidae)
from the Australian Region. By W. O. Steel. (Communicated by J. W. T. Armstrong.)

Professor P. D. F. Murray gave a short talk on The Fusion of Long Bones.

In the normal guinea-pig the tibia and fibula are not fused, but in guinea-pigs whose
fibulae have been experimentally fractured one of the two halves comes into contact with
the tibia and presumably rubs against it. In the region of contact the periostea fuse

ABSTRACT OF PROCEEDINGS. Xxxi

and transform themselves into a pad of cartilage between the two large bones. Then
blood vessels attack the cartilage from each of the two bones and resorb it, while bone
is deposited in its place, as in ordinary “endochondral ossification”’.

Fusion between pairs of long bones occurs normally in many animals, and it
seemed of interest to find out whether the fusion occurred by the same process where
it was normal as in the guinea-pig, where it is abnormal. In the rat, the normal fusion
of the tibia and fibula occurs just as in the guinea-pig, but in the metatarsals of the
young chick, the tibia and fibula, and radius and ulna of the frog, the fusion occurs
without the formation of cartilage.

Mr. B. R. A. O’Brien gave a short talk on Some Aspects of Regeneration in Harth-
worms.

ORDINARY MONTHLY MEETING.
29th NovemMBER, 1950.

Mr. D. J. Lee, President, in the Chair.

The President announced that Miss Mary Hindmarsh had been reappointed to a
Linnean Macleay Fellowship in Botany for 1951, and that Mr. N. C. Stevens and Mr.
T. G. Vallance had been appointed Linnean Macleay Fellows in Geology for 1951.

Library accessions amounting to 7 volumes, 80 parts or numbers, 3 bulletins, 6
reports and 16 pamphlets, total 112, had been received since the last meeting.

PAPERS READ.

1. A Further Contribution on the Life History of Pherosphaera. By Charles G.
Elliott. (Communicated by P. Brough.) (By title only.)

2. Notes on the Morphology and Biology of Anabarrhynchus fasciatus Macq., and
other Australian Therevidae (Diptera, Therevidae). By Kathleen M. I. English.

TaLkKS on northern Australia were given by PROFESSOR J. MAcDONALD HoumgEs, “The
Geographical Background to the Problems of North Australia”; Dr. W. KIRKLAND,
“Medical Problems of North Australia’; and Dr. N. W. G. Macintosu, “Comments on
the Physical Types of the Aborigines of Arnhem Land”. A talk prepared by Mr. W.
PoGGENDORF on “Problems of Agriculture” was given by Mr. EH. B. Furpy.

SPECIAL GENERAL MEETINGS.

Two Special General Meetings were held on 8th and 22nd February, 1950, respec-
tively, to alter Rule VI to read as follows:

Line 1: “The Annual Subscription shall be two guineas... .
Lines 10-11: “. ..., the sum of twenty guineas in lieu of further Annual’
Subscriptions.”

Two Special General Meetings were held on 25th October and 29th November, 1950,
respectively, to make an addition to Rule VI, as follows: “Any Ordinary Member who
has paid the Annual Subscription for forty years shall be exempt from further
payments.”

”

1899
1947

1932
1946
1901

1942
1931

LIST OF MEMBERS.
(15th December, 1950.)

ORDINARY MEMBERS.

Abbie, Professor Andrew Arthur, M.D., B.S., B.Se., Ph.D., c.o. University of Adelaide,
Adelaide, South Australia.
*Albert, Michel Francois, ‘“‘Boomerang”’’, 42 Billyard Avenue, Blizabeth Bay, Sydney.
‘*Allman, Stuart Leo, B.Sc.Agr., M.Sc., Entomological Branch, Department of Agriculture,
Farrer Place, Sydney.
Anderson, Miss Beverley I., 19 Kareela Road, Chatswood, N.S.W.

Anderson, Robert Henry, B.Sc.Agr., Botanic Gardens, Sydney.

*Armstrong, Jack Walter Trench, “Callubri’’, Nyngan, N.S.W.

Ashby, Professor Hric, D.Sc., D.I.C., F.L.S., Vice-Chancellor’s Lodge, Lennoxvale, Belfast,
N. Ireland.

Aurousseau, Marcel, B.Sc., c.o. Mr. G. H. Aurousseau, 16 Woodland Street, Balgowlah,
N.S. W.

Baddams, Miss Greta, B.A., B.Sc., New England University College, Armidale, N.S.W.

Balmain, Miss Judith Hope, M.Sc., Shinfield Dairy Research Institute, Reading, Eng.

*Barber, Professor H. N., M.A., Ph.D., University of Tasmania, Hobart, Tasmania.

*Beadle, Noel Charles William, D.Sc., Botany School, Sydney University.

Bearup, Arthur Joseph, 66 Pacific Avenue, Penshurst, N.S.W.

Beattie, Mrs. Joan Marion, M.Sc. (née Crockford), Bradley Street, Cobar, N.S.W.

Benson, Professor William Noel, B.A., D.Sc., F.G.S., University of Otago, Dunedin, New
Zealand.

Besly, Miss Mary Ann Catherine, B.A., 7 Myra Street, Wahroonga, N.S.W.

Birch, Louis Charles, B.Ag.Se., M.Se., Department of Zoology, Sydney University.

Blake, Stanley Thatcher, M.Sec., Botanic Gardens, Brisbane, Queensland.

Boardman, William, M.Sc., Department of Zoology, University of Melbourne.

Bradhurst, Miss Peggy Joan, B.Sc., 25 Belgium Avenue, Roseville, N.S.W.

Brett, Robert Gordon Lindsay, B.Sc., 7 Petty Street, West Hobart, Tasmania.

Brough, Patrick, M.A., D.Se., B.Se.Agr., Botany School, Sydney University.

Brown, Kenneth George, 6 Dolphin Street, Randwick, N.S.W.

Browne, Mrs. Ida Alison, D.Sc., Department of Geology, Sydney University.

Browne, Lindsay Blakeston Barton, 34 Kent Road, Rose Bay, N.S.W.

Browne, William Rowan, .D.Sc., Department of Geology, Sydney University.

Browning, T. O., Waite Agricultural Research Institute, Adelaide, South Australia.

Bryan, Clement, B.A., Intermediate High School, Corowa, N.S.W.

Burden, John Henry, 1 Havilah Street, Chatswood, N.S.W.

*Burges, Professor Norman Alan, M.Sc., Ph.D., Botany School, Sydney University.

Burkitt, Professor Arthur Neville St. George Handcock, M.B., B.Se., Medical School,
Sydney University.

Campbell, Mrs. Emily Mary, 22 Madeline Street, Hunter’s Hill, N.S.W.

Campbell, Thomas Graham, Council for Scientific and Industrial Research, Box 109,
Canberra, A.C.T.

*Carey, Professor Samuel Warren, D.Sc., Geology Department, University of Tasmania,
Hobart, Tasmania.

Carne, Phillip Broughton, B.Agr.Se. (Melb.), Division of Entomology, C.S.I.R.O., Box 109,
Canberra, A.C.T. ;

Carne, Walter Mervyn, c.o. Department of Commerce and Agriculture, Reliance House,
Flinders Lane, Melbourne, Victoria.

Carroll, Miss Dorothy, B.A., B.Se., Ph.D., D.1I.C., Science House, 157 Gloucester Street,
Sydney.

*Chadwick, Clarence Earl, B.Sc., Entomological Branch, Department of Agriculture, Farrer
Place, Sydney.

Cheel, Edwin, 40 Queen Street, Ashfield, N.S.W.

Christian, Stanley Hinton, Malaria Survey, Banz, Central Highlands, via Lae, New
Guinea.

*Churchward, John Gordon, B.Sc.Agr., Ph.D., 1 Hunter Street, Woolwich, N.S.W.

Clark, Laurance Ross, M.Sc., C.S.I.R.O., Box 109, Canberra, A.C.T.

Cleland, Professor John Burton, M.D., Ch.M., 1 Dashwood Road, Beaumont, Adelaide,
South Australia.

Cleland, Kenneth Wollaston, M.B., Department of Anatomy, Sydney University.

Colefax, Allen Neville, B.Sce., Department of Zoology, Sydney University.

* Life Member.

1946
1942
1947
1908
1928
1950

1945
1948
1950
1936
1934
1925
1937
1927

1948

1937
1926
1946

1948
1941

1949
1947
1930

1947
1948
1948
1930
1939

1950
1935
1944

1946
1936
1939

1950
1228
1917
1947

1932
1947
1930
1938

1943
1930

1943

1932
1942

1917
1938

1947
1945

LIST OF MEMBERS. XXXL

]
Colless, Donald Henry, Borneo Malaria Research, Labuan, British North Borneo.
Copland, Stephen John,“B.Se., Chilton Parade, Warrawee, N.S.W.
Costin, Alec Baillie, 12 Barambah Road, Roseville, N.S.W.
Cotton, Professor Leo Arthur, M.A., D.Sc., Department of Geology, Sydney University.
Craft, Frank Alfred, B.Sc., 10 Bank Street, Wellington, N.S.W.
Crawford, Lindsay Dinham, B.Sc., 4 Dalton Avenue, West Hobart, Tasmania.

Davis, Mrs. Gwenda Louise, B.Sc., New England University College, Armidale, N.S.W.

Davison, Miss Daphne Claire, M.Sc., ‘“Carinya’’, Waratah Street, Palm Beach, N.S.W.

Day, Alan Arthur, 13 Besborough Avenue, Bexley, N.S.W.

Day, Maxwell Frank, Ph.D., B.Sc., C.S.I.R.O., Box 109, Canberra, A.C.T.

Day, William Hric, 23 Gelling Avenue, Strathfield, N.S.W.

de Beuzeville, Wilfred Alexander Watt, J.P., ““Melamere”’, Welham Street, Beecroft, N.S.W.-.

Deuquet, Camille, B.Com., 126 Hurstville Road, Oatley, N.S.W.

*Dixson, Sir William, “‘Merridong’’, 586 Gordon Road, Killara, N.S.W.

Drover, Donald P., Institute of Agriculture, University of Western Australia, Nedlands,,.
W.A. i

Dulhunty, John Allan, D.Sc., Department of Geology, Sydney University.

Dumigan, Edward Jarrett, 10 High Street, Toowoomba, Queensland.

Durie, Peter Harold, B.Sc., C.S.I.R.O., Veterinary Parasitology Laboratory, Yeerongpilly,
Brisbane, Queensland.

ry

Ealey, Eric H. M., 18 Ray Road, Epping, N.S.W.

Edwards, Hric Thomas, Ph.D., M.Sc.Agr., National Press Pty. Ltd., 126-130 Phillip Street,.
Sydney.

Elliott, John Henry, 8 Shellcove Road, Neutral Bay, N.S.W.

Endean, Robert, M.Sce., 15 Milton Avenue, EHastwood, N.S.W.

English, Miss Kathleen Mary Isabel, B.Se., 1 Mt. Morris Street, Woolwich, N.S.W.

Fenton, Miss Enid Grace, 45 Cecil Street, Gordon, N.S.W.

Fraser, Ian McLennan, 131 Fox Valley Road, Wahroonga, N.S.W.

Fraser, Miss Judith A., 14 Milray Avenue, Wollstonecraft, N.S.W.

Fraser, Miss Lilian Ross, D.Sc., ‘Hopetoun’, 25 Bellamy Street, Pennant Hills, N.S.W.
Frohlich, Mrs. Frances Marie Veda, D.Sc., No. 5, 176 Ebley Street, Bondi Junction.

Garden, Miss Joy Gardiner, B.Se.Agr., Botanic Gardens, Sydney.

*Garretty, Michael Duhan, M.Se., ‘Surry Lodge’, Mitcham Road, Mitcham, Vic.

Greenwood, William Frederick Neville, c.o. Colonial Sugar Refining Co. Ltd., Lautoka,
Fiji.

Griffiths, Mrs. J. F. G., B.Se. (née Crust), 81 Muston Street, Mosman, N.S.W.

Griffiths, Mervyn Edward, M.Sc., Australian Institute of Anatomy, Canberra, A.C.T.

*Gunther, Carl Ernest Mitchelmore, M.B., B.S., D.T.M., Bulolo, New Guinea.

Hallinan, John Michael, “Quirindi’’, 65 Dunmore Street, Bexley, N.S.W.

Hamilton, Edgar Alexander, 16 Hercules Street, Chatswood, N.S.W.

Hardy, George Huddleston Hurlstone, ‘‘The Gardens’, Letitia Street, Katoomba, N.S.W.

Harker, Miss Janet Elspeth, B.Sc., Department of Zoology, University of Manchester,
Manchester, Eng.

Harris, Miss Thistle Yolette, B.Sc., 14 Pacific Street, Watson’s Bay, N.S.W.

Henderson, David Leonard Wylie, L.S.M.1.S., “Berida’, R.M.B. 420, Bourke, N.S.W.

Heydon, George Aloysius Makinson, M.B., Ch.M., Flat 5, 79 O'Sullivan Road, Rose Bay..
N.S.W.

Hill, Miss Dorothy, M.Se., Ph.D., Department of Geology, University of Queensland,
Brisbane, Queensland.

Hindmarsh, Miss Mary Maclean, B.Se., Botany School, University of Sydney, N.S.W.

Holmes, Professor James Macdonald, Ph,D., B.Se., F.R.G.S., F.R.S.G.S., Department of
Geography, Sydney University. :

Horowitz, Benzoin, Hngr.Agr.S., Dr.Agr.Se. (Cracow, Poland), Waite Institute, Private
Mail Bag, Adelaide, S.A. —

Hossfeld, Paul Samuel, M.Se., 132 Fisher Street, Fullarton, South Australia.

Humphrey, George Frederick, M.Sc., Department of Biochemistry, Sydney University.

Jacobs, Ernest Godfried, “‘Cambria’’, 106 Bland Street, Ashfield, N.S.W.
Jacobs, Maxwell Ralph, D.Ing., M.Sc., Dip.For., Commonwealth Forestry Bureau, Canberra,
A.C.T.

Johnson, Lawrence Alexander Sidney, 178 Beecroft Road, Cheltenham, N.S.W.
Johnston, Arthur Nelson, B.Sc.Agr., Hawkesbury Agricultural College, Richmond, N.S.W.

* Life Member.

XXX1V LIST OF MEMBERS.

1907 Johnston, Professor Thomas Harvey, M.A., D.Sc., F.L.S., University of Adelaide, Adelaide,
South Australia.

1948 Joklik, Wolfgang, M.Se., 74 New South Head Road, Vaucluse, N.S.W.

i987 Jones, Mrs. Valerie Margaret Beresford, M.Se. (née May), Moolooiabel Esplanade, |
Narrabeen, N.S.W.

1980 Joplin, Miss Germaine Anne, B.Sce., Ph.D., ‘Huyton’, 18 Wentworth Street, Eastwood,
N.S.W.

1948 Jopling, Alan Victor, B.Sc., B.E., 11 Waverton Road, Waverton, N.S.W.

1933 Judge, Leslie Arthur, 87 Eastern Road, Turramurra, N.S.W.

1949 Keast, James Allen, 313 West Botany Street, Rockdale, N.S.W.

1937 Kesteven, Geoffrey Leighton, B.Se, c.o. F.A.O., United Nations, Milawa Mansions,
Para-atit Road, Bankok, Siam.

1938 Kesteven, Hereward Leighton, D.Se., M.D., The Hospital, Cooktown, Queensland.

1948 Kiely, Temple Baylis, Caroline Street, East Gosford, N.S.W.

1938 Kinghorn, James Roy, C.M.Z.S., Australian Museum, College Street, Sydney.

1949 § Kooptzoff, Miss Olga, 322 Moore Park Road, Paddington, N.S.W. ;

1950 Krishnamurthy, Kolipakkam Venkatesa, M.A., Ph.D., 1/3B D’silva Road, Mylapore,
Madras, 4, S. India.

1939 Langford-Smith, Trevor, M.Se., Ministry of Post-War Reconstruction, Canberra, A.C.T.

1946 Larcombe, Miss Pauline Gladys, B.Sc., 17 Ethel Street, Burwood, N.S.W.

1944 Lascelles, Miss June, M.Sc., Department of Biochemistry, University of Oxford, Oxford,
England.

1946 Lawrence, James Joscelyn, B.Sc., 91 Boundary Street, Clovelly, N.S.W.

1932 Lawson, Albert Augustus, 9 Wilmot Street, Sydney.

1940 Lazer, Mrs. Ruth, M.Se. (née Wolf), 25 Sir Thomas Mitchell Road, Bondi, N.S.W.

1934 Lee, Mrs. Alma Theodora, M.Se. (née Melvaine), P.O., Hornsby.

19386 Lee, David Joseph, B.Sc., School of Public Health and Tropical Medicine, Sydney
University.

1945 Liddell, Miss Jean Joyce, Janet Clarke Hall, Trinity College, Carlton, N3, Vic.

i943 Lothian, Thomas Robert Noel, Botanic Gardens, Adelaide, South Australia.

1949 Lower, Harold Farnham, 7 Avenue Road, Highgate, Adelaide, South Australia.

1948 Macintosh, Neil William George, M.B., B.S., Department of Anatomy, University of
Sydney, N.S.W.

1945 Mackerras, David.

1922 Mackerras, Ian Murray, M.B., Ch.M., B.Sc., Queensland Institute of Medical Research,
Herston Road, Valley, Brisbane, Queensland.

1931 *Mair, Herbert Knowles Charles, B.Se., 5 Collaroy Street, Collaroy Beach, N.S.W.

1948 Manefield, Tom, Jr., B.Sc., 14 Maida Road, Epping, N.S.W.

1948 Marks, Miss Elizabeth Nesta, M.Sc., Biology Department, University of Queensland,
Brisbane, Queensland.

1949 Marshall, Professor Charles Edward, B.Sc., Ph.D., D.Se., F.G.S., M.I.Min.E., Department
of Geology, University of Sydney, N.S.W.

1932 Martin, Donald, B.Se., Stawell Avenue, Hobart, Tasmania.

1905 Mawson, Sir Douglas, D.Se., B.E., F.R.S., University of Adelaide, Adelaide, South
Australia.

1933 Maze, Wilson Harold, M.Se., University of Sydney.

1932 McCulloch, Robert Nicholson, B.Sc.Agr., B.Sc., Roseworthy Agricultural College, Rose-
worthy, South Australia.

1948 McKee, Hugh Shaw, B.A., D.Phil. (Oxon.), Council for Scientific and Industrial Research,
Private Bag, P.O., Homebush, N.S.W.

1947 McMillan, Bruce, 171 Lawson Street, Hamilton, Newcastle, N.S.W.

1949 McPhail, Miss Isabel Jean, B.Se., New England University College, Armidale, N.S.W.

1944 Mercer, Frank Verdun, B.Se., Department of Botany, University of Sydney.

1947 Messmer, Mrs. Pearl Ray, 64 Treatts Road, Lindfield, N.S.W.

1949 *Miller, Allen Horace, «1281 Canterbury Road, Punchbowl, N.S.W.

1938 Miller, David, Ph.D., M.Sce., F.R.S.N.Z., F.R.E.S., Cawthron Institute, Nelson, New Zealand.

1948 Millerd, Miss Alison Adele, M.Sc., 51 Park Avenue, Roseville.

1947 Millett, Mervyn Richard Oke, B.A., 19 Avoca Street, South Yarra, Vic.

1946 Millington, Richard James, New England University College, Armidale, N.S.W.

1949 Minter, Philip Clayton, 49 Cotswold Road, Strathfield, N.S.W.

1947 Morris, Miss Muriel Catherine, B.Sc., S.M.H.E.A., 6 First Avenue, Cooma, N.S.W.

1944 Moye, Daniel George, B.Sc., Dip. Ed., S.M.H.E.A., 6 First Avenue, Cooma, N.S.W.

1939 Moye, Mrs. Joan, B.Sc. (née Johnston), S.M.H.E.A., 6 First Avenue, Cooma, N.S.W.

1926 Mungomery, Reginald William, c.o. Bureau of Sugar Experiment Stations, Department
of Agriculture and Stock, Brisbane, B.7, Queensland.

* Life Member.

* Life Member.

LIST OF MEMBERS. XXXV

Murray, Professor Patrick Desmond Fitzgerald, M.A., D.Se., Department of Zoology,
University of Sydney, N.S.W.

Musgrave, Anthony, F.R.E.S., Australian Museum, College Street, Sydney.

Myers, Kenneth, 3 Longworth Avenue, New Lambton, N.S.W.

Newman, Professor Ivor Vickery, M.Sc., Ph.D., F.R.M.S., F.L.S., Department of Botany,
University of Ceylon, Colombo 3, Ceylon.

Nicholson, Alexander John, D.Sc., F.R.E.S., C.S.I.R.O., Box 109, Canberra, A.C.T.

*Noble, Norman Scott, D.Se.Agr., M.Se., D.I.C., C.S.1.R.O., 314 Albert Street, Hast
Melbourne, C.1, Victoria.

Noble, Robert Jackson, B.Se.Agr., Ph.D., 324 Middle Harbour Road, Lindfield, N.S.W.

North, David Sutherland, 42 Chelmsford Avenue, Lindfield, N.S.W.

O’Brien, Brian Robert Alexander, Department of Anatomy, Sydney University.

O’Farrell, Antony Frederick Louis, A.R.C.Sec., B.Se., F.R.E.S., New England University
College, Armidale, N.S.W.

O’Gower, Alan Kenneth, B.Sc., 59 Brighton Street, Harbord, N.S.W.

Oke, Charles George, 34 Bourke Street, Melbourne, C.1, Victoria.

Osborn, Professor Theodore George Bentley, D.Sc., F.L.S., Professor of Botany, University
of Oxford, Oxford, England.

Osborne, George Davenport, D.Sc., Ph.D., Department of Geology, Sydney University.

Oxenford, Reginald Augustus, B.Se., 227 Burns Bay Road, Lane Cove, N.S.W.

*Pasfield, Gordon, B.Sc.Agr., 20 Cooper Street, Strathfield, N.S.W.

Perkins, Frederick Athol, B.Sc.Agr., Biology Department, University of Queensland,
Brisbane, Queensland. ’

Phillips, Miss Marie Elizabeth, M.Sc., 4 Morella Road, Clifton Gardens, N.S.W.

Plomley, Kenneth Francis, 50 Domain Street, South Yarra, Melbourne, Victoria.

Pollak, Mrs. Audrey, 33 Arundel Street, Glebe, N.S.W.

Pollak, John Kurt, 33 Arundel Street, Glebe, N.S.W.

Pope, Miss Elizabeth Carington, M.Se., Australian Museum, College Street, Sydney.

Priestley, Professor Henry, M.D., Ch.M., B.Sc., 54 Fuller’s Bridge Road, Chatswood, N.S.W.

Pryor, Lindsay Dixon, M.Sc., Dip.For., Parks and Gardens Section, Department of the ~
Interior, Canberra, A.C.T.

Purchase, Miss Hilary Frances, B.Sc.Agr., School of Agriculture, University of Sydney,
N.S. W. :

e

Raggatt, Harold George, D.Sc., 485 Bourke Street, Melbourne, Victoria.

Rickwood, Frank Kenneth, B.Sc., Department of Geology, Sydney University.

Riek, Edgar Frederick, B.Sc., C.S.I.R.O., Box 109, Canberra, A.C.T.

Roberts, Noel Lee, 43 Hannah Street, Beecroft, N.S.W.

Robertson, Rutherford Ness, B.Sc., Ph.D., Food Preservation Research Laboratory,
C.S.1.R.O., Private Mail Bag, Homebush, N.S.W.

Ross, Donald Ford, c.o. Ross Bros. Pty. Litd., 545-547 Kent Street, Sydney.

Roughley, Theodore Cleveland, B.Sc., F.R.Z.S., Chief Secretary’s Department, Box 304,
G.P.O., Sydney.

Salter, Keith Eric Wellesley, B.Sc., ‘“Hawthorn’’, 48 Abbotsford Road, Homebush, N.S.W.

«Scammell, George Vance, B.Sc., 7 David Street, Clifton Gardens, N.S.W. ~

Scott, Miss Beryl, B.Sc., Department of Geology, University of Tasmania, Hobart, Tas.

Selby, Miss Doris Adeline, M.Sc., M.B., 11 Locksley Street, Killara, N.S.W.

Sellgren, Miss Elise Evelyn, B.Sce., 76 Bream Street, Coogee, N.S.W.

Sharp, Kenneth Raeburn, B.Sc., Eng. Geology, S.M.H.H.A., Cooma, 4S, N.S.W.

Shaw, Miss Dorothy Edith, B.Sc.Agr., 119 Bellevue Parade, Hurstville, N.S.W.

Sherrard, Mrs. Kathleen Margaret, M.Sc., 43 Robertson Road, Centennial Park, Sydney.

Shipp, Erik, 59 William Edward Street, Longueville, N.S.W.

Smith, Robert, B.Sc. (Agric.), Institute of Agriculture, University of Western Australia,
Nedlands, Western Australia.

Smith, Miss Vera Irwin, B.Sc., F.L.S., ““hoana’’, Mt. Morris Street, Woolwich, N.S.W.

Smith-White, Spencer, B.Sc.Agr., 7 Merriwa Street, Gordon,_N.S.W. :

Southcott, Ronald Vernon, M.B., B.S., 12 Avenue Road, Unley Park, Adelaide, South
Australia.

Spencer, Mrs. Dora Margaret, M.Sc. (née Cumpston), 16 Attunga Street, Woollahra,
N.S. W.

Stanley, George Arthur Vickers, B.Sc., c.o. Messrs. Robison, Maxwell and Allen, 19 Bligh
Street, Sydney.

Stead, David G., ‘“Boongarre’’, 14 Pacific Street, Watson’s Bay, N.S.W.

Stevens, Neville Cecil, B.Se., 12 Salisbury Street, Hurstville, N.S.W.

XXXVi LIST OF MEMBERS.

1948
1911

1940

1950
1949

1944
1943
1946
1921
1949

1923

1902
1949

1942

Still, Professor Jack Leslie, B.Sc., Ph.D., Department of Biochemistry, Sydney University,
N.S. W.

Stokes, Miss Anne, B.Sc., 45 Walkerville Terrace, Gilberton, S.A.

*Sulman, Miss Florence, ““Burrangong’’, McMahon’s Point, N.S.W.

Taylor, Keith Lind, B.Sc.Agr., Forestry Commission, Division of Wood Technology, 96
Harrington Street, Sydney.

Tchan, Yao-tseng, Dr., és Sciences (Paris), Botany School, Sydney University.

Thorp, Mrs. Dorothy Aubourne, B.Se. (Lond.), ‘“Carinya’’, Tara Street, Kangaroo Point,
Sylvania, N.S.W.

Thorpe, Ellis William Ray, B.Se., New England University College, Armidale, N.S.W.

Tindale, Miss Mary Douglas, M.Sc., 60 Spruson Street, Neutral Bay, N.S.W.

Tipper, John Duncan, A.M.I.E.Aust., Box 2770, G.P.O., Sydney.

*Troughton, Ellis Le Geyt, C.M.Z.S., F.R.Z.S., Australian Museum, College Street, Sydney.

Turnock, Miss Betty May Blanche, B.Se., New England University College, Armidale,
N.S. W.

Vallance, Thomas George, B.Se., 57 Auburn Street, Sutherland, N.S.W.

Veitch, Robert, B.Se., F.R.E.S., Department of Agriculture and Stock, William Street,
Brisbane, Queensland.

Vickery, Miss Joyce Winifred, M.Se., Botanic Gardens, Sydney.

Vincent, James Matthew, B.Se.Agr., Dip.Bact., Faculty of Agriculture, Sydney University.

*Voisey, Alan Heywood, D.Sec., New England University College, Armidale, N.S.W.

Walkom, Arthur Bache, D.Se., Australian Museum, College Street, Sydney.

Wallace, Murray McCadam Hay, B.Sc., P.O. Box 127, Katanning, Western Australia.

Ward, Mrs. G. W., B.Se., c.o. Agent-General for Tasmania, 457, Strand, London, W.C.2.

Ward, Melbourne, Gallery of Natural History and Native Art, Medlow Bath, N.S.W.

Wardlaw, Henry Sloane Halero, D.Se., F.A.C.I., Department of Physiology, Sydney
University.

Waterhouse, Douglas Frew, M.Sc., C.S.1.R.O., Box 109, Canberra, A.C.T.

Waterhouse, John Teast, B.Se., “Invermay”’’, Collarenebri, N.S.W.

Waterhouse, Lionel Lawry, B.E., ‘“‘Rarotonga’’, 42 Archer Street, Chatswood, N.S.W.

Waterhouse, Professor Walter Lawry, D.Sc.Agr., M.C., D.I.C., Faculty of Agriculture,
Sydney University.

Watson, Irvine Armstrong, Ph.D., B.Se.Agr., Faculty of Agriculture, Sydney University.

Wearne, Walter Loutit, c.o. James Gilberg & Co., 143 Elizabeth Street, Sydney.

Whaite, Mrs. Joy Lilian, c.o. Mrs. D. Bailey, Lower Street, Panania, N.S.W.

Wharton, Ronald Harry, B.Sc., Institute of Medical Research, Kuala Lumpur, Malaya.

*Whitley, Gilbert Percy, Australian Museum, College Street, Sydney.

Wilkins, Miss Marjorie Jessie, B.Sc., 33 Muston Street, Mosman, N.S.W.

Willings, Mrs. Lucy May, B.A., C.S.I.R.O., Division of Fisheries, P.O. Box 21, Cronulla,

- N.S.W.

Willis, Jack Lehane, B.Sc., 15 Muttama Road, Artarmon, N.S.W.

Wilson, Ralph Dudingston, M.Se., M.Se.Agr., Biological Branch, Department of Agricul-
ture, Box 36A, G.P.O., Sydney.

Winkworth, Robert Ernest, Botany Department, University of Melbourne, Carlton, N.3,
Victoria.

Womersley, Herbert, F.R.E.S., A.L.S., South Australian Museum, Adelaide, South
Australia.

Wood, Edward James Ferguson, C.S.I.R.O., Marine Biological Laboratory, P.O. Box 21,
Cronulla, N.S.W. : 4

Woodhill, Anthony Reeve, B.Sc.Agr., Department of Zoology, Sydney University.

Zeck, Mrs. Anne, 694 Victoria Road, Ryde, N.S.W.
Zeck, Emil Herman, 694 Victoria Road, Ryde, N.S.W.

HONORARY MEMBER.

Hill, Professor James Peter, D.Se., F.R.S.Z., F.Z.S., F.R.S., ‘“Kanimbla’’, Dollis Avenue,
Finchley, London, N.3, England.

CORRESPONDING MEMBERS.

Broom, Robert, M.D., D.Se., F.R.S., Transvaal Museum, Pretoria, Transvaal, South Africa.

Jensen, Hans Laurits, D.Se.Agr. (Copenhagen), State Laboratory of Plant Culture,
Department of Bacteriology, Lyngby, Denmark. :

Rupp, Rev. Herman Montague Rucker, B.A., 24 Kameruka Road, Northbridge, N.S.W.

* Life Member.

LIST OF NEW GENERA AND SPECIES.

Vou. 75.
Genera.
Page
Armstrongula (Reduviidae) .. .. .. 88
Pseudophloecharis (Staphylinidae) .. 304
Usingeriella (Hnicocephalidae) .. .. 81
Species.
Page.
annulatum (Deleatidium) EL maculosa (Atalophlebia)
aureonigrum (Simulium) 178 marowana (Atalophlebia)
baddamsae (Baetis) 21 occidentalis (Pseudophloecharis)
boganensis (Usingeriella) 82 orientalis (Cnephia tonnoiri)
brachyptera (Pseudophloecharis) Ol parva (Atalophlebia)
eonfluens (Baetis) Be ee 3 peregrinum (Simulium)
crassa (Leptophlebia) ily pseudotasmaniae (Aphodius)
fulvicorne (Austrosimulium) 184 sinuatum (Eseudopiigech aris)
fuscum (Pseudophloecharis) 340 spadix (Atopopus)
incerta (Atalophlebia) 13 strenua (Cnephia)
inornatium (Simulium) ! 181 tillyardi (Armstrongula)
longicaudata (Atalophlebia) 9

. i-ii.—Alkaline Phosphatase Reactions in Tissue Sections.

lii-iv.—A Hybrid Eucalyptus.

LIST OF PLATES.
PROCEEDINGS, 1950. =

v—vi.—Cytological Studies in the Myrtaceae. III.

vii—dAustralian Species of Lagenophora.

vilii—Immature Stages of Aphodius howitti Hope.

ix—xli.—Pherosphaera hookeriana.

XXXVii

Page.

tS
epi:
.. d41
. 170
abe A
So I)
Soe llayrs
Go Bax
Beall
> UO)

84

XXXV1ii

INDEX. ;

(1950.)
Page. Page.
Abstract of Proceedings ........ XX1V—XXXi Boardman, Ww.—
Aédes scutellaris Group, Further Notes The Hair-Tracts_in Marsupials. Part
on Experimental Crossing within VE FGF O,010:0 0:50 0 Foie O14 0 FOO ONG O03 89
Lhe? athe ORAM On eee 251 The Hair-Tracts in Marsupials. Part
A Further Contribution on the Life- V. A Contribution on Causation .. 254
History of Pherosphaera ......... 320 The Hair-Tracts in Marsupials. Part
Av Hybrid (mucalyptus ). 02moe ae ceo 96 Mae ; eee: and Genetics of ae
‘ : : rac atlernn Gasket eee
Bc AE BIOS LEI Se PSE Tia The Hair-Tracts in Marsupials. Part
WIBSTS RIQGEORE,, EEN sooe doo: daneso VII. A System of Nomenclature .. 273
Alkaline Phosphatase Reaction in Bowman, F. T., Talk by Sreuia
TSS D SECUIO RS PEUNE Nb poop 39 Be Brown, K. G., elected a Member ...... XXiv
yNlhemehons Se iby, MMW he Gb aac coca ono XxXix :
: g . Burges, Professor N.’A., Talk by ..... SRONGN:
INGEN OW IRN WAS Baio gonodomooooc TSO
Anabarrynchus fasciatus Macq., Notes Carne, P. B., The Morphology of the
on the Morphology and Biology of, Immature Stages of ‘Aphodius
and other Australian Therevidae howitti Hope (Coleoptera, Scara-
(DugUeNE, WS ENTIERE) on sie0 poet een baeidae, Aphodiinae) ............ 158
A New Genus and Four New Species of Classification of Bacteria, with Special
Phloecharinae (Coleoptera, Staphy- Reference to Non-pathogenic Eubac-
linidae) from the Australian COTA on ans eae ay cee kan A es Dene ee 195
ReSiIon * Wasieeecsec oe Aone 334 Cleland, K. W.—
A Note on the Non-reciprocal Fertility A Study of the Alkaline Phosphatase
in Matings between Sub-species of Reaction in Tissue Sections. Pt. I 35
Mosquitoes ...-..--- sess eee reese 279 Bt SL 430. ee eae | SAN ee ae 54
Aphodiinae of Australia (Coleoptera, Respiration and Cell Division in
Scarabaeidae), Notes on the ...... 153 Developing Oyster Eggs ......... 289
Aphodius howitti Hope (Coleoptera, The Intermediary Metabolism of Un-
Scarabaeidae), The Morphology of fertilized Oyster Eggs ............ 296
the Immature Stages of .......... 158 Coral Genera of Heron Island (Barrier
A Review of and Contribution to Reef) Gixmipit) seo XXV-XXVi
Knowledge of Phylloglossum Drum- Councils Electionsaeene eee XxX
mondit IEHOWARY So daoo8 IOI DOO Crom ID ic 133 Crawford, 1D, IDS, elected a Member ... xxiv
A Revision of the Australian Species of Cunningham, G. H., Australian Poly-
the Genus Lagenophora Cass. see 122 poraceae in MHerbaria of Royal
A Study of the Alkaline Phosphatase Botanic Gardens, Kew, and British
Reaction in Tissue Seetions. Pt. I. Museum of Natural History ...... 214
The Possibility of its Quantitative Cytological Studies in the Myrtaceae.
WSC ey crepe eee Neeley gh Nee 35 lel Cytology and Phylogeny in the
A Study of the Alkaline Phosphatase Chamaclaucoideae Hans eee 99
Reaction in Tissue Sections. Pt. II.
The Histological and Cytological Dakin, Professor W. J., reference to
Validity Baneaeeen, cra taNmas gue at Pe pine Sika pee 54 Geath Ol. 2 i a ae ei one XXiv
Australasian Simuliidae (Diptera) II, DES. Gaeade, Ib
Ik ores i he EN A eee Pe one f T 167 A Revision of the Australian Species
ve a ran ae Be Nee Sey a 1 ; of the Genus Lavyenophora Cass. .. 122
aor ery ae ae outh Be Revision of the Genus Solenogyne
SIMS incl JSWeIEnnein Cole UN Cass.) , BG Sr ae ee 188
mea Cinema ieineies See sia Serie eg bf 1 Day, A. A., elected a Member ......... XXV
Australian Polyporaceae oi eee das Dilation of the Foot in Uber (Polinices)
Royal Botanic Gardens, up and strangei (Mollusca, Class Gastro-
British Museum of Natural History 214 Doda) ere ee ee 70
Bacteria, Classification of, with Special ID(okeehe, (Cnenehaers, AMIE Joe Goaoosusones XxXvii
Reference to Non-pathogenie Eubae: ‘d&An, Grahame, Talk by «0. 0+ 0+ a
WOE 9 29h pec buad 8 dn6. BONS); -- 195 Pyliott, C. G., -A Further Contribution
Balance Sheets for 1949-50 ...... Xxi-xxili oni ithe uilnte © Gistory) wobl Phere
Barber, Professor H. N., elected a ~ Oy) O11, ROR RG oe Nae ore 5 e - 320
p IMCD CIAS.. HI eybieve ccs eerere hk Siclegeee XxX1X English, Kathleen M. I., Notes on the
Beadle, sNmiG@smwee bal ka Dy > ac eniaaewen XXX Morphology and Biology of Anabar-
Besly, Mary A., and Salter, K. E. W., rynchus fasciatus Macq., and other
Coral Genera from Heron Island Australian Therevidae (Diptera,
(Barrier Reef) (Exhibit) .... xxv—xxvi THELEVICRE) hae Sa eerie eal crete wore: 345

‘

Page.
Ephemeroptera, Australian ........... 1
Hucalbypurs: -Ae Ebyprids soso 2). so 96
Exhibits. Lace Bugs (Stephanitis

queenslandensis), Mitotic Poisons xxvii
Exhibits at meetings .... xxv, xxvi, xxxvii

Fauna Protection Panel .............. li

Fertility in Matings between Sub-
species of Mosquitoes. A Note on
INOMEGSCIPTO CAG ceeishcnecoushetals bosheneeeav oe 279

Fraser, Lilian, elected a Vice-President xxiv

Further Notes on Experimental Cross-
ing within the Aédes_ scutellaris
Group of Species (Diptera, Culi-

GUGIEKS) i 1 G SIA RIESE EEE CIDE Oo eee oer Zou
Garden, Joy G., elected a Member .... xxvii
Given, B. B., Notes on the Aphodiinae

of Australia (Coleoptera, Scara-

[DEVON EVE) Beate NON eerie ge drt ea 153
Hackney, Frances M. V., A Review of

and Contribution to Knowledge of

Phylloglossum Drummondii Kunze 133
Hair-Tracts in Marsupials—

IPE NY ho: 3 Seo a a Eels Ge eae 89

Pt. V. A Contribution on Causation 254
Pt. VI.- Evolution and Genetics of

MI AG Cure UUE TEMG arcs. cle Setrecees, sian boeiovele 267
Pt. VII.- A System of Nomenclature .. 273
Hallinan, J. M., elected a Member .... xxix

Harker, Janet E., Australian HEphemer-
OOS IE iE ale ea tear ea 1
Hindmarsh, Mary M.—

Linnean Macleay Fellow in Botany,

TUCO EE CONE: 275 Rn Nel aD NT ea iii
Mitotic Poisons (Exhibit) ......... XXVi
Reappointed to Fellowship ........ Xxxi

Kiely, T. B., award of Royal Society’s

MICOS Bo BES poe EN iee enee reCrnere a XXIV
GK ANG MV ened KD Yin eisai one ae XXXi
Krishnamurthy, K. V., elected a Member xxiv
Lagenophora Cass., A Revision of the

Australian Species of the Genus .. 122
Lee, D. J:, elected President .......... xXx
irae NOLES\AOIM) cclens i/c ui Sc esele ciavece oor li
Linnean Macleay Fellowships ........ ii

Appointments for 1950 ............. ili
Appomtuments tor 1951525. .55....-.- XXXi
Macdonald Holmes, Professor J., Talk

IDA et ea year eye ep stNc eile © eetshace =e diene XXxi
MacindoesSiia:. Lali Dyce ..... 6.2. XXVili
Macintosh, N. G. W., Talk by ........ XXx1
Mackerras, I. M., award of Clarke

Wilerinioraienl IMIGCEN See oodetosuoeode XXIV
Mackerras, M. J. and I. M., Notes on

Australasian Simuliidae (Diptera)

TUES cag cae seahe or eB ies Cr Cle beero) Seana oe ne ace 167
Macleay Bacteriologist, appointment of iii
Mite en Cider Mall kee Dye tys.5 Aye ihe tellers os eee XXix
Marsupials, The Hair-Tracts in—

1tig UWS Sati SIO GREED ERE CRS ae 89
EUCHRE Seger aoa wrth eM er Sh eintte ie ia, ails da veyed 254
Teena VIN tatty eas Pe ee ya GZ 267
1, WAHL ale

XXX1X

Members, Notes of Interest of ........ iv
Middleton, B. L., reference to death of xxx
Millerd, Adele—

Award of Fellowship to .......... XXVil
Linnean Macleay Fellow in _ Bio-
Chiemistryn swionkeiOle eens wala lili
Morris, Muriel C.—
Award of Overseas Fellowship ..... XXV
Dilation of the Foot in Uber
(Polinices) strangei (Mollusca,
Class Gastropoda) see 70
Linnean Macleay Fellow in Zoology,
WOT nO dete saben css cis toe eel aye gues cota aeolian ili
Mosquitoes, A Note on Non-reciprocal

Fertility in Matings between Sub-

SPECIES) Ol eter ecial Mera A alee ee ee 279
Murray, Professor P. D. F.—
MlectedmtomCounciliee ee see ee XXV
REN Ken yarns oc carne ae esuene re drags ayer eae as SRONOXE
Musgrave, A., Lace Bugs (Stephanitis
queenslandensis) (Exhibit) XXVii
Myrtaceae, Cytological Studies in ..... 99
New Genera and Species, List of ... xxxvii
Northern Australia, Talks on ........ XXxi
Notes on Australasian Simuliidae
@Dipleras) eae eye es aera tee 167
Notes on the Aphodiinae of Australia
(Coleoptera, Scarabaeidae).' The
Aphodius tasmaniae, howitti, york-
ensis, andersoni Complex ........ 153
Notes on the Morphology and Biology of
Anabarrynchus fasciatus Macq., and
other Australian Therevidae (Dip-
tera es Mnerevidae) cso ese yes hoe 345
Obituary—W. J. Hnright ............. iv

OPIBEIe I. | 1B Je VA Ne lone Lod bob oo coc SROXOXGT
O’Gower, A. K., elected a Member .... xxiv
Osborne, G. D., elected a Vice-President xxiv
Oxenford, R. A., elected a Member .... xxvii
Oyster Hggs—

Respiration and Cell Division in
DS VETOP UM eases |S eE eke ee Coates 282
The Intermediary Metabolism of
Wantertilized. = Vasc hens ote salons ae 296
Phenological Observations, : Assistance
tc Meteorological Bureau .......... ii
Pherosphaera, A Further Contribution
on the Life History of ........... 320
Phloecharinae (Coleoptera, Staphy-
linidae). A New Genus and Four
New Species of, from the Australian
EUG STON Sc etervnarty kennel arcucticceyercnel ateite 334
Phylloglossum Drummondii Kunze. A
Review of and Contribution to
Konto wlediees OR) sass cone Siskeceuatteces eects 133
IPlatvesHMaIStwOls iret ls suc stekecd sv eishe au. XXXVIi
letopeeinoloret, Wo, EWI ly cooaacodgonwdc XXxi

Polyporaceae, Australian, in Herbaria
of Royal Botanic Gardens, Kew, and
British Museum of Natural History 214
Presidential Address i
Pryor, L. D., A Hybrid Eucalyptus .... 96

e) (ajlefiejlefelisiiel «ele uireitellaji™) 6

Reduvioidea from N.S.W., Notes and
Descriptions (Hemiptera)

xl INDEX.

Respiration and Cell Division in
Developing Oyster Eggs .......... 282
Revision of the Genus Solenogyne Cass. 188

‘’ Rickwood, F. K., elected a Member ... xxv
Robertson, R. N.—
Elected a Vice-President ........... XXiV

The Last Haunts of Demons: A Com-
parative Study of Secretion and
Accumulation (Presidential Address) i

Salter, K. HE. W., and Besly, Mary A.,

Coral Genera of Heron Island
(Barrier Reef) (Exhibit) .... xxv—xxvi
Sellgren, Elise E., elected a Member .. xxvi
Sharp, K. R., elected a Member ....... XOXONG
Simuliidae (Diptera) II, Notes on Aus-
ENAVASTAM Nie eee ec cnche elec oe eae 167
Smith-White, S.—
A Note on the Non-reciprocal Fer-
tility of Matings between Sub-

species of Mosquitoes ............ 279
Cytological Studies in the Myrtaceae,

1 EIU) Ear hne aes irate anes sue east ae CoC EMEMS IEE GUNES or truer 99
Solenogyne Cass., Revision of the Genus 188
Special General Meetings ............ XXXi
Steel, W. O., A New Genus and Four

New Species of Phloecharinae
(Coleoptera, Staphylinidae) from
une PAUIStralianeeeslOnimen eee nese 334
Stevens, N. C., appointed a Linnean
MacleayaHellows soe see eee DOO
Synopsis,. to appear below title of
DADETS eye ee Sey Ue te OL Oa TE AN Xxvi
Tchan, Yao-tseng—
Appointed Macleay Bacteriologist ... iii
ANTEIDVENL tha SGN Gokesgeoaccesoobe XXvi
Mlectedsa Member sacri rire siaoe elcr. Xxix

Page

The Hair-Tracts in Marsupials—
Parte UW yinc Giclees eee bee Oe ee oe 89
Parties aad Sti2 Stee eraion bis Ce 254
Partials oi 6G bee eee ee ate 267
Part} Vile isis boda eee ee ee ee 273

The Intermediary Metabolism of Unfer-
tilized Oyster Hegegs

The Last Haunts of Demons: A Com-
parative Study of Secretion and
Accumulation (Presidential Address) i

The Morphology of the Immature Stages
of Aphodius howitti Hope (Coleop-
tera, Scarabaeidae, Aphodiinae) .. 158

Uber (Polinices) strangei, Dilation of
the, Hooteine 6s cee ei asee a eee 70

Vallance, T. G., appointed a Linnean

Macleay Hellowatasec. saeco XXXi
Walkom, A. B., elected Honorary

PEASUNET | fish Se auefelcst svlayette euctoenene XXiV
Waterhouse, Dr. G. A., reference to

death Ob Ge Cee as Sane XXix
Willis, J. L., elected a Member ....... Xxix

Wood, E. J. Ferguson, The Classifica-

tion of Bacteria, with Special
Reference to Non-pathogenic Eubac-
tenia’. oo Eee ae eee 195
Woodhill, A. R.—
Elected a Vice-President ........... XX1V
Further Notes on Experimental Cross-
ing within the Aédes scutellaris
Group of Species (Diptera, Culi-
CIDAE)) mee ene ee, SRE ore 251
Wygodzinsky, Petr, Reduvioidea from
New South Wales, Notes and
Descriptions = iien. secre een ne 81

Proc. Linn. Soc. N.S.W., 1950. PLATE IX.

Pherosphaera hookeriana.

Proc. Linn. Soc. N.S.W., 1950. PLATE X.

Pherosphaera hookeriana.,

Proc. Linn. Soc. N.S.W., 1950. PLATE XI.

Pherosphaera hookeriana,

a tS a
he reat

yy. w
Atea

Proc. Linn. Soc. N.S.W., 1950. PLATE XII.

Pherosphaera hookeriana,.

: (Issued 6th June, 1950.) > f 6 LtA
: =
Vol. LXXV. | _ Nos. 347-348. !
Parts 1-2.
THE
PROCEEDINGS
|| LINNERN SOCIETY
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WOODS HOLE, Mass.

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LIST OF OFFICERS AND COUNCIL, 1950-51.

President:
D. J. Lee, B.Se.
Vice-Presidents:
A. R. Woodhill, B.Sc.Agr. Lilian Fraser, D.Sc.
G. D. Osborne, D.Sc., Ph.D. R. N. Robertson, B.Sc., Ph.D.
Hon. Treasurer: A. B. Walkom, D.Sc.

Secretary: Dorothy Carroll, B.A., B.Sc., Ph.D., D.C.

Council:

R. H. Anderson, B.Sce.Agr. G. D. Osborne, D.Sce., Ph.D.
W. R. Browne, D.Sc. R. N. Robertson, B.Se., Ph.D.
Professor N. A. Burges, M.Se., Ph.D. T. C. Roughley, B.Sce., F.R.Z.S.
Dorothy Carroll, B.A., B.Sc.; Ph.D., D.1I.C. E. Le G. Troughton, C.M.Z.S., F.R.Z.S.
A. N. Colefax, B.Se. _ J. M. Vincent, B.Sc.Agr., Dip.Bact.
S. J. Copland, B.Se. A. B. Walkom, D.Sc.
Lilian Fraser, D.Sc. H. S. H. Wardlaw, D.Sc., F.A.C.I.
Professor J. Macdonald Holmes, B.Sc., Professor W. L. Waterhouse, M.C.,

Ph.D., F.R.G.S., F.R.S.G.S. D.Se.Agr., D.1.C.
D. J. Lee, B.Sc. A. R. Woodhill, B.Se.Agr.

Auditor: S. J. Rayment, F.C.A. (Aust.).
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\

PROCEEDINGS, LXXV, PARTS 1-2, 1950.

CONTENTS.
Pages.
Presidential Address: The Last Haunts of Demons: A Comparative Study of
Seeretion and) Accumulation: By, “Rav N. ARODertSON ase) atte chet j-xx -
Council Elections Mee: Se Me ori cA Nae Me,” TAG GR Ree igen ea fathr . OS 2.0.
Balance ‘Sheets’ for the. Year 1949-50 co rds ea Se in is eo ee Le ee eee Xe
Abstract of Proceedings La kan oh A abies ties 2k Re Ao isle ea ce ae xxiv
Australian Ephemeroptera. Part I. Taxonomy of New South Wales Species
and Evaluation of Taxonomic Characters. By Janet EH. Harker. (One ‘ is
hindred) atid one \Rext-firtires) 0. 21 o. aaua ee he a ee ee ee 1-34

A Study of the Alkaline Phosphatase Reaction in Tissue Sections. Part I.
The Possibility of its Quantitative Use. By K. W. Cleland. (Nine Text-
PRS URES Taare ae le iy eR OLN car TEL OR Wear es i cae ann Dg ato 35-53

A Study of the Alkaline Phosophatase Reaction in Tissue Sections. Part II.
The Histological and Cytological Validity. By K. W. Cleland. (Plates is
i and ii) BY sebaatates ntgte cont era oped! NN SS (Pants Ce Sat alr an 54-69

Dilation of the Foot in Uber (Polinices) strangei (Mollusca, Class Gastro-
poda). By Muriel C. Morris, Linnean Macleay Fellow in Zoology. (Three =
Text-figures) SLUG Wate ARAM GL DY (GS, “eRe 15. See te aie Sata ie ae 70-80

Reduvioidea from New South Wales. Notes and Descriptions (Hemiptera).
By Petr Wygodzinsky. (Communicated by J. W. T. Armstrong.) (Thirty- ;
four Text-figures) De Dt a ae NED I taint de ay Ca ie ce a ga 81-88

The Hair-Tracts in Marsupials. Part IV. Direction Characteristics of Whorls
and Meristic Repetition of Radial Fields. By W. Boardman. (One Text-
figure) esta ete

A Hybrid Bucalyptus. By L. D. Pryor. (Plates iiiand iv) .. .. .. .. 96-98 a

(issued 22nd September, 1950.)

Vol. LXXV. Nos. 349-350.
Parts 3-4.

THE

Pech BOIINGS

OF THE

MIANEAN SOCIETY

OF

NEw SOUT Eat Bitten |

LIBRARY

MAR 2 6 195)

FOR THE ae

1950.

WOOBS HOLE, MASS

|

Parts 3-4 (99-249).
CONTAINING PAPERS READ IN MAY-AUGUST.

With four plates.
[Plates v-viii.]

g SYDNEY:

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Registered at the General Post Office, Sydney, for transmission
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Agent in Hurope:
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The Linnean Society of New South Wales

LIST OF OFFICERS AND COUNCIL, 1950-51.

President:
D. J. Lee, B.Se.
Vice-Presidents:
A. R. Woodhill, B.Sc.Agr. Lilian Fraser, D.Sc.
G. D. Osborne, D.Sc., Ph.D. R. N. Robertson, B.Sc., Ph.D.
Hon. Treasurer: A. B. Walkom, D.Sc.

Secretary: Dorothy Carroll, B.A., B.Sc., Ph.D., D.I.C.

Council:
R. H. Anderson, B.Sc.Agr. Professor P. D. F. Murray, M.A., D.Sc.
W. R. Browne, D.Sc. G. D. Osborne, D.Se., Ph.D.
Professor N. A. Burges, M.Sc., Ph.D. R. N. Robertson, B.Se., Ph.D.
Dorothy Carroll, B.A., B.Sc., Ph.D., D.LC. T. C. Roughley, B.Sc., F.R.Z.S.

EB. Le G. Troughton, C.M.Z.S., F.R.Z.S.

A. N. Colefax, B.Sc. : 2 2

me ee ae baat es J. M. Vincent, B.Sc.Agr., Dip.Bact.
pperseses Jifeehh A. B. Walkom, D.Sc.

Titan Braser, 2. Sc: H. S. H. Wardlaw, D.Sc., F.A.C.1L
Professor J. Macdonald Holmes, B.Sc., Professor W. L. Waterhouse, M.C.,

Ph.D., F.R.G.S., F.R.S.G.S. D.SeAgr., D.I.C.
D. J. Lee, B.Sc. A. R. Woodhill, B.Se.Agr.
Auditor: S. J. Rayment, F.C.A. (Aust.).

NOTICE.

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BuLLOuGH, W. S., 1939.—A Study of the Reproductive Cycle of the Minnow in Relation
to the Environment. Proc. Zoot. Soc. Lond., 109, A, Pt. 1: 79-108.

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PROCEEDINGS, LXXV, PARTS 3-4, 1950.

CONTENTS.

Cytological Studies in the Myrtaceae. III. Cytology and Phylogeny in the
Chamaelaucoideae. By S. Smith-White. (Plates v and vi; one hundred
and one Text-figures. )

A Revision of the Australian Species of the Genus Lagenophora Cass. By
Gwenda L. Davis. (Plate vii; thirteen Text-figures.)

A Review of and Contribution to Knowledge of Phylloglossum Drummondii
Kunze. By Frances M. V. Hackney, D.Sc. (Twenty Text-figures.)

Notes on the Aphodiinae of Australia (Coleoptera, Scarabaeidae). The
Aphodius tasmaniae, howitti, yorkensis, andersoni complex. By B. B.
Given. (Ten Text-figures.) (Communicated by P. B. Carne.)

The Morphology of the Immature Stages of Aphodius howitti Hope (Coleoptera,
Scarabaeidae, Aphodiinae). By P. B. Carne. (Plate viii; thirteen Text-
figures. )

Notes on Australasian Simuliidae (Diptera) II]. By M. J. and I. M. Mackerras.
(Fifteen Text-figures. )

Revision of the Genus Solenogyne Cass. By Gwenda L. Davis. (Fourteen
Text-figures. ) a

The Classification of Bacteria, with Special Reference to Non-pathogenic
Hubacteria. By EH. J. Ferguson Wood ..

Australian Polyporaceae in Herbaria of Royal Botanic Gardens, Kew, and
British Museum of Natural History. By G. H. Cunningham ..

Pages.

99-121

122-132

133-152

153-157

158-166

167-187

188-194

195-213

214-249

:
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Nos. 351-352.

THE

PROCEEDINGS

OF THE

LINNEAN SOCIETY

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Marine Biological Laboratory}
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CONTAINING PAPERS READ IN SEPTEMBER-NOVEMBER,
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FOR THE YEAR

1950.

Parts 5-6 (250-859; xxv

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SyDNEr:

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LIST OF OFFICERS AND COUNCIL, 1950-51. ae
President:
D. J. Lee, B.Sc.
: Vice-Presidents: ‘
A. R. Woodhill, B.Se.Agr. Lilian Fraser, D.Sc. NY <

G. D. Osborne, D.Sc., Ph.D. R. N. Robertson, B.Se., Ph.D. :
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Professor N. A. Burges, M.Sc., Ph.D. T. C. Roughley, B.Se., F.R.Z.S.
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A. N. Colefax, B.Sc. J. M. Vincent, B.Sc.Agr., Dip.Bact.
Ss. J. Copland, B.Sc. A. B. Walkom, D.Sc. — a
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D. J. Lee, B.Se. A. R. Woodhill, B.Se.Agr. — : i

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‘ Notice To AUTHORS.

Renate: are requested to assist in facilitating the Society’s publishing work by observing
the following recommendations when preparing papers for submission to the Council. Perusal
of the PROCEEDINGS will show the general style to be adopted.

Manuscript.—Manuscripts should be double-space typed with a margin of at least one inch on
the left-hand side, top and bottom of each page. The title should be spaced clearly above
the text on the first page. The original should be submitted and a copy retained by the
author for checking proofs. A Table of Contents, on a separate sheet, should accompany
each manuscript; this is not necessarily for publication, but will serve to show the proper
relation of the headings. A manuscript when submitted to the Council should be complete
in every detail, both with regard to text, references and illustrations, for any extensive
alterations: or corrections made on the proofs, if allowed, will be at the author’s expense.
No words except generic and specific names, and those to be printed in italics, should be
underlined. An Abstract of the paper should accompany the manuscript.

Perberonces: Reterénces should be carefully checked by the author, who is alone responsible
for their accuracy. They should be listed alphabetically at the end of the manuscript, and
should be cited in the text by the author’s name, e.g., Bullough (1939) or (Bullough, 1939) ;
and in the list thus:

BuLutoueH, W. S., 1939.—A Study of the Reproductive Cycle of the Minnow in Relation
‘ to the Environment. Proc. Zeol. Soc. Lond., 109, A, Pt. 1; 79-108.

_ Abbreviations. —Standard abbreviations should be used in tabulations and after numerals in the
' ‘text. The abbreviations of names of periodicals should conform to those in the World List
of Scientific Periodicals.

by number. -Hach table, provided with a heading descriptive of the contents, should be
submitted on a separate sheet.

rt I lustrations..

_ Plates.—The size should not pr eeed 7% x 5 in. except if the subject will bear reduction. A
number of small photographs should ie arranged to make one plate. Photographs should
Show good contrast and be printed on glossy paper.

_ Line drawings will, as far_as possible, be printed in the text. Drawings should be made on
white board or Stiff white paper with Indian ink. It is advisable to arrange a number
of text-figures, all to bear the same reduction, on a single large sheet, which can be
reduced to one-third, one-half or one-quarter the original size, giving a block 8 x 5 in. or
4 x 5 in. without loss of essential detail. Hach separate figure should be clearly numbered
and all lettering should be plain and large enough to be clearly readable when reduced.
-If co-ordinate paper is used for graphs it should be blue-lined. The explanation of Text-
figures should be supplied on a separate sheet; but the explanation of Plates should be
given at the end of the manuscript. Text-figures and Plates should be numbered consecu-
tively and referred to in the text by number. Indicate in the manuscript where each
Text-figureé or group of figures is to be inserted.

Eee ropts should be. corrected and returned as soon as possible tceether with an order for. any
reprints required in addition to the 50 supplied gratis by the Society.

Tabulations.—Tables should be numbered consecutively and referred to specifically in the text.

ras

PROCEEDINGS, LXXV, PARTS 5-6, 1950.

CONTENTS.

_ Further Notes on Experimental Crossing within the Aédes scutellaris Group of
Species (Diptera, Culicidae). By A. R. Woodhill

The Hair Tracts in Marsupials. Part V. A Contribution on Causation. By
W. Boardman

The Hair Tracts in Marsupials. Part VI. Evolution and Genetics of Tract
Pattern. By W. Boardman

The Hair Tracts in Marsupials. Part VII. A System of Nomenclature. By
W. Boardman. (Two Text-figures.)

A Note on the Non-reciprocal Fertility in Matings between Sub-species of
Mosquitoes. By S. Smith-White

Wha» Ae
Respiration and Cell Division of Developing Oyster Eggs. By K. W. Cleland.

Cirve: DextAeUres yy aya ess ciated ins cates ate ek ares

The Intermediary Metabolism of Unfertilized Oyster Eggs. By K. W. Cleland.
(Three Text-figures.)

A Further Contribution on the Life-History of Pherosphaera. By C. G. Elliott.
(Communicated by P. Brough.) (Plates ix—xii; twenty-two Text-figures.)

A New Genus and Four New Species of Phloecharinae (Coleoptera, Staphy-
linidae) from the Australian Region. By W. O. Steel. (Communicated by
J. W. T. Armstrong.) (Twenty-nine Text-figures.) .. tees To

Notes on the Morphology and Biology of Anabarrynchus fasciatus Macq., and
other Australian Therevidae (Diptera, Therevidae). By Kathleen M. I.
English. (Forty-seven Text-figures.)

Abstract of Proceedings

Page.

251-253

254-266

267-272

273-278
279-281
282-295
296-319 s i

320-333

Dist of Members. 2250 9. 509 Se a A ea

List of Genera and Species Bee ENS

List of Plates eee ela Pcie Whe Ont eR Lie re ORC Te reW ICTS SMIET k o haptabe tata hig: Cen ae a

Index Soe a TS ge bo eae vi Saree isha! Sanpete ham REAM Bee Xxxviii-xl

~ EA