all

Humphrey, Harry B.

Studies in the physiology and morphol-
ogy of some California Hepaticae. 1908

7

ie

PROCHEDINGS

OF THE

WASHINGTON ACADEMY OF SCIENCES

VoL. X, PP. I-50. Puates J-II. JANUARY 18, 1908.

STUDIES IN THE PHYSIOLOGY AND MORPHOL-
OGY OF SOME CALIFORNIA HEPATIC.

By Harry B. Humpurey, B.S.
AcTING INSTRUCTOR IN BOTANY, LELAND STANFORD JUNIOR UNIVERSITY.

THE morphology and physiology of the Hepatice have been
treated by many authors, but their work has been confined mainly
to the group as a whole. The intimate relations of the liver-
worts to their environments, have however, received only inci-
dental treatment. Ordinarily we are inclined to associate with
the Hepaticze an environment characterized by moisture and
shade. To a certain extent we are justified in doing so, for
the majority of the known species occur in just such a habitat,
many of the larger and more striking ones, such as Monoclea,
Dumortiera and some species of Aneura, being common in the
more humid regions of the tropics. On the other hand many
species are known to occur normally in parts of the world where
climatic conditions are not so evenly balanced as in the tropics ;
many thrive in extreme northern and southern regions where
they are subject to great variations in temperature, while those
growing in regions like the west coast of the United States
must adapt themselves to prolonged periods of drought alter-
nating with six or seven months of rainy weather.

It was with a view of ascertaining the nature and influence
of these various conditions common to certain California hepat-
icz that the present study was undertaken.

Proc. Wash. Acad. Sci., January, 1908. I

2 HUMPHREY

The author wishes to acknowledge his obligations to Pro-
fessor Douglas H. Campbell and Associate Professor George
J. Peirce under whose direction the work was pursued. Thanks
are also due Professor Alexander W. Evans, Dr. Marshall A.
Howe and Professor Roland Thaxter for assistance in the
determination of material and for the use of certain hepatice
sent to the writer.

PARASITISM AND SAPROPHYTISM IN HEPATICZ.

The association of certain fungi with hepatice was first
described in detail by Leitgeb,’ who, in his studies on Pézlidium
ciliare, observed the infection of young sporogonia. He found
that all such sporogonia were more or less abnormal in their
mode of segmentation and inferred from this that the infected
organs were structurally effected by the action of the fungus.

Following Leitgeb, a number of writers have observed fungus
infection in other hepatice. As early as 1879, Kny’ discovered
sterile fungal hyphe in the rhizoids of Lunularza and Mar-
chantia. ‘These, he states, were found to be present in rhizoids
undergoing a process of regeneration, which process may have
been stimulated by the hyphz. Cavers*® has observed that
when Marchantza and Lunularia grow in ordinary soil free of
humus the rhizoids are penetrated by hyphe which grow up-
ward as delicate filaments showing cross-walls at rather long
intervals. These hyphe occasionally become branched but
never, so far as he has observed, reach the tissue of the thallus.
On the other hand, he has found that when these plants grow
on humus soil the hyphz extend into the compact tissue of the
thallus, to which in fact, they are largely confined.

Golenkin * observed the presence of endotrophic mycorrhiza in
Marchantia palmata, M. paleacea, Preissia commutata, Tar-

1Leitgeb: Untersuchungen iiber die Lebermoose, Heft 2, p. 58; Tafel 3,
Fig. 26.

*Kny and Bottger, 1879: Ueber eigenthiimliche Durchwachsungen an den
Wurzelhaaren zweier Marchantiaceen. Verhandl. d. bot. Vereins d. Prov.
Brandenburg, p. 2 of Separate.

3 Cavers, 1903: Saprophytism and Mycorrhiza in Hepatice. The New Phy-
tologist, Vol. II, No. 2, pp. 32-33.

4Golenkin, 1902: Die Mycorrhiza ahnlichen Bildungen der Marchanteen.
Flora, Band 90, p. 209.

if

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 3

gionia hypophylla, Plagiochasma elongatum and fegatella
conica. In all these the hyphe are confined to the more com-
pact tissue of the thallus.

Jeffrey,’ in his paper on the Gametophyte of Botrychium vir-
ginianum, describes an endophytic fungus associated with this
fern. His observations show that conidia develop singly within
the host cells usually at the end of a hypha. These peculiar
conidia germinate zz sztu, generally producing a germ-tube
which forces its way into the neighboring cells of the host.
Within certain cells of the host he observed the development of
vesicular structures along the enclosed hyphz. Similar struc-
tures have been observed by Bruchmann? in his recent studies
on Ophioglossum vulgatum. In his Fig. 42 he has indicated
fungal structures similar to those common to the endophytic
hyphe found by the author in cells of Aneura multifida major.

The same writer® in his work upon Botrychium lunaria
describes an endophytic fungus infecting the cells of the pro-
thallium. He states that the hyphz usually find entrance to the
prothallial tissue through the rhizoids though in some instances
they are found penetrating the outer, cuticularized surface of
the prothallium. Once within the cells of the host, the hyphe
fill the cells with their sclerotia-like structures and veszcular
enlargements. All starch of the infected cells disappears, but
he finds oil and albumen within the hyphe which he regards as
reserve products to be utilized later by the growing embryo.
Thus he recognizes a symbiotic relationship between fungus and
host, and in another part of the same paper he advances the
theory that the endophyte is a probable means in assisting the
prothallium to withstand the rigors of hot and cold weather.

1Jeffrey, 1898: The Gametophyte of Botrychtum Rae University of
Toronto Studies, No. 1, p. 12.

* Bruchmann, 1904: Ueber das Prothallium und die Keimpflanze von Ophzo-
Zlossum vulgatum L. Botanische Zeitung, Heft XII, Taf. VIII, Fig. 42 und
Fig. 42a.

? Bruchmann, 1906: Ueber das Prothallium und die Sporenpflanze von Bofry-
chium lunaria Sw. Flora, Band 96, Heft 1, pp. 210-211.

4 HUMPHREY

THE PARASITIC FUNGUS ASSOCIATED WITH FOSSOMBRONIA
LONGISETA AUST.

In the writer’s recent studies on the Development of /ossom-
bronia longiseta* no reference was made to the structure of the
infecting fungus; in fact, the material then in hand was too
scanty to make anything like a satisfactory study. Since the
publication of the above paper, however, excellent material has
been found in various stages of development, and the relation of
fungus to host has been clearly worked out.

So far as could be ascertained, the host is invaded from with-
out by hyphe that make their entrance through the rhizoids or
directly through the cells of the host stem. The infection of
the rhizoids is very similar to that described by Cavers’ for
Lophozia, Cephalozia and other hepatice. As a rule the
hyphez not only invade the rhizoids but extend throughout the
tissues of the host, regardless of the nature of the substratum,
thus differing somewhat from the nature of infection reported
by Cavers where he has observed that the degree of infec-
tion varies with the amount of humus in the soil. In F/os-
sombronia, as a rule, the hyphe infecting rhizoids alone were
confined to the inner wall of the organ, though in some instances
not a few rhizoids were found in which hyphez had penetrated
through the wall to the exterior as shown in Fig. 3. The host
thus affected was growing in soil containing little humus and
in no case were these external hyphe observed to grow longer
than those figured. The hyphez confined to the rhizoids are
very seldom branched and are rather remotely septate. In cer-
tain parts, however, usually at the end of a branch, short seg-
ments not unlike conidia develop and are abstricted from the
hypha. The subsequent behavior of these has not been
observed. It is, however, quite probable that these structures
furnish one means of transfer of infection. I did not find in
Frossombronia anything comparable with the conidia described
by Jeffrey * as occurring in the endophyte of Botrychium vir-
ginianum.

1Humphrey, 1906: The Development of Possombronia longiseta Aust. Annals
of Botany, Vol. XX, No. LXXVII.

A oc: cit.

2 170G; (Cit.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 5

In a few instances infected rhizoids were found to have under-
gone considerable modification of form as seen in Fig. 5. On
the other hand very similar modifications were repeatedly ob-
served in uninfected rhizoids. ‘These were very likely due to
the stimulus of contact with compact soil. Whether the fungus
is instrumental in the development of such malformations as the
one shown in Fig. 5 cannot be determined at this writing. It
is, however, hardly probable, for the writer has observed that
when infected plants are transferred to Knop’s solution the new
rhizoids though seriously infected maintain a uniform direction
of growth and are apparently unaffected by the hyphe.

In a previous paper’ mention was made of the fact that /os-
sombronza plants in many instances developed a tuberous growth
which, on careful examination, was found to contain a more or
less complex growth of fungus hyphe. Microchemical tests
demonstrated the presence of a large quantity of starch, oil and
nitrogenous food products within the cells of this tuberous
growth and this, no doubt, in a measure accounts for the greater
development of the fungus in this region. So far as the author
has observed, none of these tuberous growths is free from infec-
tion. It is hardly probable, however, that this structure is
directly due to the activity of the fungus for it is a structure
common to a number of other hepatice such as Geothallus
tuberosus Campb., Lezccza cancellata Tayl., Possombronta tuber-
tfera Goebl., and a number of others in which no fungus infec-
tion has been observed. Specimens of Geothallus tuberosus
have been carefully examined by the writer with a view to
ascertaining the presence of infection, but in all material ex-
amined the tubers were uninfected and filled with reserve food
products as described by Campbell,’ ’95. No doubt the tuber
in the case of Fossombronia longiseta serves the same purpose
as in Geothallus tuberosus or in Fossombronia tuberifera, 7. é.,
as a special structure to carry the plant through a more or less
prolonged dry season. Howe®* has shown that in specimens of

EV oe, cit:

Campbell, 1896: A New California Liverwort. Botanical Gazette, Vol. 21,
Wo. kp i.

? Howe, 1899: The Hepatice and Anthocerotes of California. Memoirs of
the Torrey Botanical Club, Vol. VII, p. 8o.

6 HUMPHREY

Fossombronia longiseta collected in southern California where
the dry season is longer, this tuberous growth is considerably
more marked than in the case of plants in the northern half of
the state. Owing to the earthquake of April 18, 1906, a break
occurred in a water-pipe not far from Stanford University. As
a consequence a considerable tract of ground was well irrigated
throughout the past, very prolonged dry summer. Upon this
thoroughly moistened soil the writer found, growing vigorously,
a considerable number of plants of Hossombronza longiseta,
Anthoceros pearsont, Targionia hypophylia, and some species
of the more common mosses. Here were growing a number
of plants accustomed to summer desiccation and it occurred to
the writer that under these conditions of increased humidity
certain structural changes might result both in the development
of the gametophyte and sporophyte.

Careful examination of a large number of these plants re-
vealed no evidence of anything in the nature of a tuberous
growth in any part of the thallus though all plants examined
showed fungus infection. It would seem therefore that this
instance would lend some support to the inference that, so far
as our species of Hossombronza is concerned, these small tuber-
like structures are purely adaptive and their development
depends largely upon certain external factors. Whether growth
under similar conditions would result in the reduction or dis-
appearance of the tubers in such plants as Geothallus tubero-
sus or Anthoceros phymatodes is a matter that has not yet been
tested. In these forms the tuber is a well-marked and doubtless
long-established modification of the thallus and if reduced at
all would probably require a considerable period of time during
which conditions of constant moisture are allowed to act.
Peirce,’ ’06, has demonstrated that such a well-marked char-
acter as the dorsiventrality of certain liverwort gametophytes is
not a hereditary character as commonly supposed but is pri-
marily due to the formative influence of light.

As elsewhere stated, some plants of Hossombronza longiseta
were studied in which the rhizoids were apparently free from

1 Peirce, 1906: Studies of Irritability in Plants. Annals of Botany, Vol. XX,
No. LXXX, p. 459.

"
a Ni me hg ete alee gti ithe gt Spe sh idaho ager ~

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 7

hyphe, though the more compact tissue of the thallus was in
many such instances thoroughly infected. Thus it would seem
that infection need not necessarily take place through the rhi-
zoids. Material carefully fixed in chromic acid (1 per cent.)
and stained with the Fleming’s triple combination seemed to
give very satisfactory results, though it was found necessary to
allow more time to the action of the stain than was customary
in staining organs or structures of the host. Sections through
the stem of infected plants almost always showed a well-de-
fined zone of cells more or less given over to hyphe. This is
not unlike the condition observed by Cavers'* (03) in Fegatella
and by Golenkin’ (02) in a number of forms cited above.
Golenkin states that the cells of this zone, though still retaining
their protoplasm and nuclei, are void of chlorophyll and starch.
Careful examination of infected cells of Hossombronza, except
where infection was so far advanced that the cells were occu-
pied by developing sclerotia, revealed the presence of varying
amounts of chlorophyll and starch, depending upon the number
and vigor of invading hyphe.

Unlike the forms described by Golenkin, this fungus is not
necessarily confined to any given zone, for any portion of the
thallus may be invaded by hyphz extending out from the more
compact parts. ‘Though several hundred fruiting plants have
been studied, no evidence of infection of either antheridium or
archegonium has yet been seen, and the sporophyte is appar-
ently free from the attack by fungus.

In material fixed in January, 1904, about three months after
the beginning of the rainy season, almost the entire tissue of the
stem was packed with hyphe, many branches extending through
the outer layer of cells of the host into the substratum.

Although the fungus was present in virtually the whole of
the stem-tissue, strictly speaking, it was found that at this stage
of its development the hyphe were more or less confined to the
innermost cells of the stem while those in proximity to the
growing point as well as the leaves were apparently free from
infection. The epidermal cells and those immediately beneath

? Loe. cit. 02 33:
* LOE; Cit... ps 200:

8 HUMPHREY

showed evidences of comparatively few hyphez which evidently
served to connect the more functionally active interior system
with those in the rhizoids and with the substratum. Thus far
the habit of growth of this fungus agrees quite consistently with
those described by Cavers.

At about this period in its development one may observe, scat-
tered here and there throughout the infected zone, vesicles of
considerable size and presenting a variety of form. ‘These ap-
pear as great enlargements of the hyphe and are full of rather
coarsely granular protoplasm (Fig. 6). This protoplasm has
within it minute nuclei, at first rather few in number but as the
vesicles become older and larger increasing in number until
they quite fill the interior of the vesicle. It was further found
that in some instances at least these vesicles are connected with
the exterior by hyphe (Fig. 8) and along the course of these
hyphz may be seen certain of these nuclear bodies. Whether
or not they make their escape to the exterior as zodspores could
not be demonstrated, but their development and subsequent be-
havior is suggestive. In his recent work upon the development of
Ophioglossum pendulum and other members of the genus,
Campbell” has demonstrated the presence of thin-walled vesi-
cles in the endophyte. In these the behavior of the nuclei
resembles that occurring in the vesicles of the endophytic fun-
gus found in the Fossombronza. In Fossombronia these vesicles
completely fill the host cells in which they occur, in some cases
causing a considerable distention of the cell.

As stated above, the amount of chlorophyll or of starch and
other products of metabolism varies with the degree of infection.
Certain cells of the host seem packed with hyphe, and a micro-
chemical test fails to reveal any of the products of the normal
cell, and while a large part of the stem appears to be dying,
the leaves and the uninfected stem tissue seem to be vigorous
and unaffected by the presence of the fungus. Comparison of
infected plants with those entirely or nearly free from fungus in-
vasion seems to demonstrate that up to the vesicle stage of devel-
opment the growth of the host is not abnormally stimulated, in

tC; Cit., p.32—33.
2 Campbell, 1907.

far daneres Moreen ek Meee ferry ae Yew ments en ae

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 9

fact an extensive examination of plants of both sorts leads one
to conclude that the presence of the fungus is of no advantage
to the host and in many instances is an evident detriment. In
March, 1906, while making some field studies upon /ossom-
bronia, the writer observed that certain plants appeared to be
dying. The affected plants were in some instances almost
white, apparently from loss of chlorophyll. Closer examina-
tion with a hand-lens revealed the presence of a great number
of minute black bodies along the length of the stem and in the
leaves. These were especially abundant near the base of the
leaf. On further examination with the microscope it was found
that these black bodies were sclerotia within the cells of the host
and the development of these resulted ultimately in the death of
the host. It was at first thought probable that this fungus was
distinct from the one found invading the rhizoids and stem tissue,
but a careful study seems to show that the two are identical. At
least so far as concerns the host the development of these scler-
otia marks the period of greatest activity of the fungus, and its
truly parasitic nature is manifested. Up to the appearance of
the sclerotia the life of the host is not seriously affected but as
soon as the hyphe begin to extend and to form the structures
that later develop sclerotia a very evident drain upon the vitality
of the host sets in and increases with the further development
of these structures.

It was at first thought that these might be perithecia of some
ascomycete but microtome sections demonstrated their true
nature. Wherever they occur the cells containing them become
considerably distended and completely filled. Their presence
causes no abnormal development of the cell as regards thickness
of wall or form, though the cell-contents become much modified
to the extent that all vestige of any starch, oil, cytoplasm, etc.,
has disappeared, even the nucleus is absorbed and the entire
cell cavity is filled by the sclerotium.

The behavior of the hyphz with reference to the nucleus and
chromatophores as described by Cavers' for the fungus infect-
ing Monoclea does not occur in the case of the fungus under

‘Cavers, 1904: Contributions to the Biology of the Hepatice. Part I.
Targionia, Reboulia, Preissta, Monoclea, p. 39.

IO HUMPHREY

consideration. In Afonoclea the fungus is confined to a sharply
defined mycorrhizal zone three or four cell layers in thickness.
Many of the cells in this zone are filled with branched hyphe,
tufts of which seem to envelop the nucleus. In some cases
even the chromatophores, like the nucleus, become surrounded
by similar tufts of branching hyphe in a manner quite sug-
gesting the formation of a lichen.

In Fossonbronza the hyphe preceding the development of a
sclerotium are but sparsely ramified. The first indication of the
sclerotium is the increase in diameter of the hyphz within a cell
followed later by the profuse development of short, thick
anastomosing branches between which may at first be seen
spaces of varying size which ultimately disappear as the
sclerotium increases in extent (Fig. 10). The nucleus and
chromatophores at first visible finally disappear, doubtless being
taken up by the actively growing fungus. As yet the writer
has failed to observe how these structures are consumed.

According to Czapek' the tissues of MWarchantia, Fegatella,
Lunularia and other hepatice contain an antiseptic principle
which he calls ‘‘sphagnol” because of its abundance in the
peat-mosses. He has shown that this substance exists in com-
bination with the cellulose of the cell-walls and exerts an
inhibitive influence upon the development of moulds and
bacteria. This, Cavers’ thinks, suggests the view that in the
case of certain Fusarium-like fungi the ‘*‘ sphagnol ” may serve
to regulate the growth of the fungus and prevent symbiosis
from passing into parasitism. In the case of Hossombronza it
cannot be shown that at any time during a period of infection the
invading fungus maintains a symbiotic relation with reference
to the host, for an examination of infected cells shows that the
presence of hyphz sets up a disturbance of the cell-metabolism,
the cell becoming impaired to such an extent as ultimately to
cause its death. The degree of this impairment varies with
the activity and extent of the parasite. To be sure, many
infected plants appear to be quite as healthy and fertile as

1Czapek, 1889: Zur Chemie der Zell membranen bei den Laub. und Leber-
moosen. Flora, Band 86, p. 361.
JOG: Cit:, p.'33-

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICH II

others yet free from the fungus, but it is owing to the fact that
the degree of infection is but slight and the vigor of the host
is sufficient to throw off for a time the harmful effects of the
parasite just as the leaves of cultivated lettuce maintain apparent
full vigor during early stages of the infection by Bvremza
lactuca.'

Experiments designed to ascertain the effect of the fungus
upon very young plants of either Hossombronza or Fimbriaria
have thus far failed, and it cannot be decisively stated here that
Fimbrvaria is at all susceptible to infection by this fungus. The
examination of a large number of plants of all stages of develop-
ment has shown our common species, /. californica, compara-
tively free from infection by any fungus. Recently, however,
the author has found a few plants of this species attacked by a
fungus which seems, in material thus far examined, to be con-
fined entirely to the cells of the first four layers on the ventral
side of the thallus. Hyphe in considerable numbers were ob-
served in both the smooth and tuberculate rhizoids. These
showed but slight tendency toward branching, in many instances
extending the entire length of a rhizoid without producing a
branch. The hyphe confined to the rhizoids, compared with
those within the mycorrhizal zone of the thallus, are very much
more delicate; but upon extending into the cells of the thallus
they branch profusely and develop relatively thick and tough
walls. The fungus resembles the one described above for /os-
somoronza, in that it is filamentous and septate. Structures com-
parable to vesicles, conidia or sclerotia have not yet been
observed. The presence of the fungus seems in no wise to re-
tard the growth or affect the vigor of the thallus and no plants
have yet been found in which the hyphe have penetrated the
chlorophyll-bearing tissue.

THE PARASITIC FUNGUS ASSOCIATED WITH ANEURA
MULTIFIDA MAJOR.

While in the field collecting Aneura the writer observed that
a considerable number of plants had taken on a rusty brown

‘An illuminating discussion of symbiosis and parasitism is to be found in
Plant Physiology. Peirce. Pp. 85-92 inclusive.

I2 HUMPHREY

color. Upon careful examination it was found that they were
in a dying condition owing to the presence of a fungus. A
large quantity of material consisting of plants in a normal con-
dition as well as those visibly affected by the fungus were
brought into the laboratory in September, 1906, where the rela-
tion of fungus to host could be more carefully studied. This
species of Aneura is found growing on moist surfaces of rocks
along streams, on decaying logs and moist banks of soil in
which there is considerable clay. Material collected from all
three of these sources contained a large number of infected
plants so it 1s hardly probable that the fungus is one confined
to plants growing on decaying wood.

Aneura muliiida major, as well as other species of this genus,
under certain conditions produce two-celled gemme in great
numbers. It was found that many of these after a short time
had germinated and some were infected. Figure 15 shows a
young plant resulting from the germination of a gemma, into the
older cells of which a fungus had penetrated by well-marked
haustoria. ‘The diseased cells contained less chlorophyll and
showed unmistakable evidences of the harmful effect of the
fungus. Young plants of varying age and size were found to
be infected. It was first thought likely that infection took place
directly while the gemma was still within its mother cell, but a
very careful examination of gemme failed to support any such
view. It seems that young plants of less than four cells are
rarely infected. The fungus develops conidia freely and it is
probable that infection is brought about by their germination.
A small number of young plants developing from germinating
spores were observed, but as yet none of these has shown the
presence of a fungus.

In the older plants, hyphe from within the cells of the thallus
grow downward extending into the rhizoids, though this is by
no means so common as in the case of Fossombronza or Fim-
briarta. Many rhizoids whether infected or not present a
strong tendency to branch as shown in Fig. 16, aand 6. This
is undoubtedly due to the influence of contact stimulus; in fact,
rhizoids not in contact with the substratum are all quite simple
and more delicate.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 13

In his studies upon Lophocolea bidentata, Cavers* found that
the tissues of the gametophyte are entirely free from hyphe,
but the rhizoids which grow out in tufts from the bases of the
amphigastria penetrate the substratum of rotten wood and there
become profusely branched like the haustoria of many fungi.
This, he considers, enables the liverwort to assume a more or
less saprophytic existence. He has not however shown that the
plant actually does adapt itself to this mode of life, nor does it
appear that we can assume the branching of the rhizoids as due
to anything other than contact stimulus. Peirce and Randolph”
have demonstrated that in the case of certain attached fresh-
water alge and in many marine forms the development of a
holdfast is directly and wholly the result of contact stimulus.
The complexity and extent of the holdfast were found to vary
with the degree of roughness of the surface of the substratum ;
young plants grown on ground glass developing much more
elaborate holdfasts than similar plants grown on smooth glass
while those grown in dust-free water developed no holdfasts at all.

In Knop’s solution the writer now has plants of /ossombronza,
Cryptomitrzum and other liverworts growing that were removed
from a normal soil substratum some months ago. Still attached
to these plants are some of the old rhizoids which are all more
or less gnarled and modified, some exhibiting short lateral proc-
esses. Since placing the plants in Knop’s solution a great
number of rhizoids have developed and in every,*7stance they
are perfectly straight and unmodified and much more delicate
in structure than those that had grown while the plants were in
their normal habitat. We have here exactly the same behavior
exhibited by the secondary roots and root-hairs of higher plants.
If, as some botanists maintain, the rhizoids of bryophytes are
simply organs of attachment is it not probable that, as in the
case of certain algz, these branches are due to the stimulus
afforded by contact?

Aside from Aneura and Fossombronza the writer has observed
branching rhizoids in Cephalozta bicuspidata, due probably to

BICOC.“Gite,(p.. 32: .
*Peirce and Randolph, 1905: Studies of Irritability in Algz. Botanical
Gazette, 40° pp. 321-350.

14 HUMPHREY

the same stimulus, as they were not infected by hyphe. The
fungus associated with Azeura differs from that attacking Fos-
sombronza both as to development and habit of growth. In the
case of the /ossombronza fungus we find it to be more truly
endophytic, once infection has been brought about, while the
fungus associated with Azeura seems to be epiphytic in habit,
developing haustoria-like branches which in turn develop other
branches penetrating and drawing sustenance from the host
cells. The hyphe are septate and profusely branched, especially
within the tissues of the host. The hyphe within the rhizoids
are in nearly every instance unbranched and in no case were
they seen to grow through the wall of the rhizoid communicating
with the exterior as in Hossombronza.

The physiological effect of the fungus upon the host is quite
as marked as any we have yet studied. The cell walls of
Aneura are very much thicker than those of most of our liver-
worts, and the plants are vigorous in habit. Each cell contains
usually one large oil body and numerous chromatophores.
Fungal hyphz penetrate the cell walls without any resulting
modification and in some instances a single branch may pass
through several cells without producing haustoria or branches
of any sort. Ordinarily, however, after entrance to the host
tissues is effected, the fungus becomes quite extensive, as seen
in Fig. 13, where three affected cells are represented. In these
it will be observed that the large oil body has already disap-
peared and the number of chromatophores is somewhat below
the normal (compare Fig. 12 with Fig. 14). Ultimately the cells
become in some instances packed with hyphe and at this stage
the death of the cell rapidly ensues. Such cells when micro-
chemically tested are void of starch, no nucleus or cytoplasm
- can be made out, and the chromatophores have undergone
complete disorganization ; in fact, little remains but the cell wall.
In certain plants where the fungus had reached an advanced stage
the hyphe within certain cells had developed into knot-like
structures suggesting the beginning of sclerotia; and in a few
instances these had, when sufficiently nourished, developed into
blackish thick-walled sclerotia completely filling and distending
the cell cavity. These, in appearance and structure, very much

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC: 15

resemble the sclerotia formed in the cells of Fossombronza,
though they do not develop in such great numbers. This is
doubtless due to the fact that the comparatively early decline
and death of the host tissues impairs the vigor and health
of the parasite before these structures can develop in great
numbers.

As soon as the host cells begin to show the effects of para-

sitism there develop upon the surface of the host at indefinite
points along the surface-hyphe, perithecia-like structures (Fig.
11) the real function of which, however, has not been demon-
strated. Professor Roland Thaxter kindly attempted to iden-
tify the fungus for me but being unable to determine the true
nature of these structures was forced to wait for more advanced
stages. The writer has prepared a number of slides of micro-
tome sections which show these bodies to be hollow.
_ Beyond a wall no other structures have yet been observed in
connection with the interior of these somewhat globular bodies.
They seem to develop from short processes that appear here and
there along the external hyphe. ‘They assume very early the
spherical habit and develop at various points on their surface
short spine-like processes at first pointing in different directions
but ultimately either disappearing or lying all in one plane at
the base of the structure bearing them. On certain host plants
the tissues of which have already begun to turn brown, these
perithecia-like bodies develop in such numbers as to be almost
in contact, and the complex of hyphz on the surface and within
the host is very elaborate. Two-celled conidia were found on
the surface of the host but it could not be proved that these
spores belonged to the fungus infecting Azeura, although no.
other fungus was observed in the material.

It is the intention of the writer to work out the development
and life history of this fungus and the one associated with Fos-
sombronza more fully in a later paper. At the present moment
the main object is to discuss the relation of parasite and host.
In the case of this fungus there is less evidence of anything
comparable to a symbiotic relation than in the relation of the
Fossombronza parasite to its host. In fact, as soon as infection
is brought about, the host seems to show signs of resulting

16 HUMPHREY

injury. ‘The infection spreads from plant to plant quite rapidly.
In a dinner-plate full of material, at first the majority of plants
examined were unaccompanied by fungus, but within three
weeks, on one side of the plate all the plants were turning brown;
on examining these they were found to be infected. The fun-
gus seems usually to make its attack through the ventral side
of the thallus, though occasionally a plant would be found with
hyphez on both sides. Microtome sections failed to reveal any
vesicular structures within the host.

Aneura, being naturally less compact and vigorous than such
forms as Fegatalla or Lunularza, furnishes a poorer field for a
parasitic fungus and seems less able to cope with the fungus.
This, however, may be partly due to greater vigor of the par-
asite. It must also be noted that infected plants behave dif-
ferently under varying conditions. <Aneura multifida major
as we find it here is semi-aquatic as to habitat and when grow-
ing under perfectly normal conditions may be considerably in-
fected without showing any effect other than becoming yellow-
ish green in color. If, however, these same plants are brought
into the laboratory, even though well supplied with moisture,
under a bell-jar they become brown in color finally dying,
while uninfected plants seem to thrive quite as well as when
growing out-of-doors under normal conditions. Plants grow-
ing along streams, though receiving little less than the required
amount of water, if infected, seem less able to throw off the
effects of the fungus than plants growing under perfectly
normal conditions of moisture, light, etc.

Aside from the fungi found in association with Fossombronza,
Fimbriaria and Aneura, the writer has had occasion to study
infected material of Anthoceros pearsoni and Porella bolanderz.
In habit of growth the fungus associated with Axthoceros is
very much like that infecting Azeura though none of the
material examined showed any structures suggestive of fruiting
organs other than conidia which were being formed in consid-
erable numbers by abstriction along the external hyphe. They
branch freely and are seen to anastomose not at single points
here and there but continuously for some length, both hyphe
being rather minutely septate along the length of contact. The

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 17

hyphe produce short lateral processes which grow in length
until they come in contact with a similar branch of a neighbor-
ing hypha effecting conjugation with it; or in case it fails to
meet with such a branch it is seen to anastomose with a near-
by hypha and in this manner a complicated weft of hyphe may
develop over the surface of the host. The same seems to be
true with regard to the hyphe within the host cells, though to
less extent. Within certain cells (Fig. 19) dark colored scle-
rotia-like structures develop, though in the material studied
these were by no means abundant. With a limited amount of
infected material nothing definite can be said as to the relation
of the fungus to the host. All plants examined however seemed
practically unaffected by the fungus. Infected cells exhibited
the single chromatophore in every case except where sclerotia
had formed. Just what would have been the effect had the
fungus been as far advanced as in the case of Hossombronza or
Aneura is mere conjecture. Our species of Anthoceros have,
growing within the thallus, colonies of a species of /Voséoc, but
the writer saw no evidence of fungal hyphe invading these.

Porella bolanderz is infected by a fungus of somewhat dif-
ferent habit, though its ultimate effect upon the host is not
greatly different from what has been described for Hossombronza
and Aneura.

The principal hyphal trunks appear for the most part to be
intercellular, thus differing in this respect from the others
described above. A single hypha finding entrance to a leaf for
example, will ramify to some extent, and these various primary
branches grow along between the cells of the host, thus forming
an intercellular complex that may effect nearly every cell.
From these primary branches, haustoria find entrance to the
host cells and here they become more or less branched, with the
result that the cells lose all their contents except the nucleus;
this seems to persist. After the disorganization and disappear-
ance of the chromatophores, the fungus structures are easily
visible and in many of these cells may be seen exceedingly
thick-walled chlamydospores, in some instances quite filling the
enclosing wall of the host cell (Fig. 17).

The fungus is not confined wholly to the interior of the tissue.

Proc. Wash. Acad. Sci., January, 1908.

18 HUMPHREY

In many cases a complex of hyphe over-runs the surface of a
leaf and it is not uncommon to find chlamydospores in connection
with these external hyphz. One-celled conidia are abstricted
in large numbers from branches of the external hyphe. Sclerotia
have not yet been found, though occasionally a cell may be seen
well filled with knots of hyphez, too loose in structure, however,
to be regarded as sclerotia. Cells in which these knots occur
are always dead and apparently empty.

The fungus behaves toward the host as a parasite. Its hyphe
produce haustoria in great numbers which in the course of time
invade all the cells of a leaf, thus impairing greatly its ability to
elaborate food materials. The younger leaves of the host are
apparently uninfected, and even though the older parts of the
thallus may be dead, growth continues at the growing point and

the plant may grow indefinitely, though very evidently diseased. ©

The writer has not thus far observed a fungus associated with
our species of Spherocarpus or with Targionia hypophylla,
although Golenkin’ reports the existence of an endotrophic
mycorrhiza for the latter, and it may be that further investigation
will demonstrate the presence of a fungus in Zargzonza as we
find it here. Asa matter of fact it is not improbable that all
the hepatice of this region may on further study prove to be
- infected, the fungus being either a symbiont or, what is more
likely, a parasite. The writer has seen no evidence in favor of
the view that the fungus stimulates the growth or in any way
benefits the host, though the presence of hyphe may lead up to
the development of abnormal structures such as modified root-
hairs, enlarged cells, etc.

Bruchmann,’ in his studies on Botrychium lunarza, concludes
as follows: ‘* Der physiologische Nutzen des Zusammenlebens
von Prothallium und Pilz scheint mir auch hier nicht darin zu
bestehen, dass die wenigen und unscheinbaren Pilzfaden,
welche aus dem Substrat des Gamophyten in ihn emmiinden,
belanglose Humusstoffe ihm zufiithren, Sondern nur darin,
dass die von den functionsfahigen Rhizoiden der wachsenden
Schutelpartie aufgelésten und herbeigeftihrten Baustoffe mit-

THSOE. Cit, 4p. 209s
aoc. iCit.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC I9

telst des Stoffwechsels des Endophyten in haltbare Reservestoffe
umgewandelt und aufgespeichert werden, von denen namentlich
die auffallend grosse Oelmenge dem Prothallium die Fahigkeit
verleiht, in dem sandigen Boden auch wahrend der Sommerhitze
und der Winterkalte von Austrocknung bewahrt zu bleiben.”

As already stated, the author has found, in the case of all
infected forms thus far investigated, oil and of necessity
albumen, in the living cells of the gametophyte. In Fossom-
bronza and Aneura at a certain stage in the development of the
endophyte there is a minimum quantity of these products within
the host cells containing hyphe and a maximum amount of
these same products within the cells of the fungus. The result
is the death of the host or, at least, a very serious check to its
development, resulting in small sickly plants. In such a case
we cannot possibly regard the food products derived from the
gametophyte as reserve materials later to be given up by the
fungus as nourishment to further growth of the host.

The only forms examined not showing harmful effects from
association with a fungus were /7mdbriarza, Hegatella and
Anthoceros. Of the first and the last, only a very limited
amount of infected material was found, consequently little light
can be thrown upon actual relation of host to endophyte. Howe’
has reported an endophytic fungus associated with Anthoceros
olneyz Aust., the septate hyphze of which produce at the ends
of lateral branches, globular clusters within which are produced
numerous dark spore-like cells bearing some resemblance to
those of the Tilletiaceze. He has found the same parasite in
A. ravenellit Ala. (Mohr.) and the same or one very similar in
A.hallit Aus. He regards this fungus as a parasite, though no
detailed account is given relative to the character of the asso-
ciation of the two. |

DEVELOPMENT OF FRUITING ORGANS OF FEGATELLA CONICA
IN CALIFORNIA.

fegatella conica (Conocephalum conicum) is described by

Howe’ as growing in moist, deeply shaded places, especially

1 Howe, 1898: The Anthocerotacee of North America, Bulletin of the Torrey
Botanical Club, Vol. 25, No. 1.
2 Loe, €it., ps 59:

20 HUMPHREY

on stones and rocks beside streams. It is not common in Cali-
fornia and was first collected by Professor Underwood at Fel-
ton, Santa Cruz County, in 1888. In July, 1906, it was found
in fruiting condition by the author on a trip up San José Cajfion
in the Santa Lucia Mountains in Monterey County. So far as
is known, plants bearing receptacles had not been found in Cali-
fornia prior to the above named date, though fruiting plants
have been collected by C. V. Piper near Seattle, Wash. Con-
ditions during the rainy season there and in the parts of Cali-
fornia where Fegatelia thrives are similar. The dry season near
Puget Sound is occasionally interrupted by summer showers
which may in combination with other factors influence the
development of fruiting organs.

Fegatella may be found in many of our near-by cajions; in
fact, it has been observed to occur along the banks of creeks
well down upon the plain of the Santa Clara valley, but almost
always it will be found in particularly well watered and densely
shaded localities, a combination of conditions very essential to
the normal development of the plant in this region of long, dry
summers.

Antheridial plants bearing receptacles have been collected
from several localities, though these plants are by no means
common. Normally, they are of such vigorous, robust habit as
to spread over a considerable area by mere vegetative growth,
and it is not uncommon to find several square feet entirely cov-
ered by Fegatella to the exclusion of everything else.

On finding female plants in fertile condition a field study was
made relative to their habitat, and conditions affecting growth
and development of fruiting organs. San José Cajfion is a deep,
narrow gorge, the floor of which is well watered by a never-
failing stream. The north wall of the cafion consists very
largely of precipitous rock ledges that in many places come
abruptly to the stream’s edge and are only sparsely clothed with
Artemesita, Adenostoma and a few other plants of xerophytic
habit. The south wall rises less abruptly into hills averag-
ing seven to eight hundred feet in height. \Theseyhavews
covering of rich humus and the vegetation at the base along
the creek and well up on the south slope consists of Seguoza

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICHZ 21

sempervirens, Alnus rhombtfolia, Salix lastolepis and other
trees which afford abundant shelter for a variety of shade-loving
plants. Here also thrive several species of mosses and liver-
worts that in less favored localities would either die or pass into
a dormant, air-dry condition.

Fegatella occurs throughout the length of the cafion on both
banks of the creek, though most abundantly on the south side
where there is a minimum of direct sunlight and evaporation.
Five trips were made during the past season for at least a dis-
tance of three miles toward the head of the cafion and each time
a diligent search was made for fruiting plants which were found
in but few, very limited areas, where the exposure was north or
northeast. It was observed that no fruiting plants were to be
found in places void of sunlight, and the same was true when
the plants grew within six or eight inches of the water’s surface
in light of the same intensity as fell upon nearby fruiting plants.
Plants bearing receptacles in greatest number were found ona
flat elevation about eighteen inches above the water in a break
between trees through which fell a flood of strong, diffused
light and at midday direct sunlight. This level bed of Fega-
tella was bathed constantly by seepage of spring-water from a
moss and fern grown cliff forming the background, upon which
were found a few fruiting plants of /egatella and Aneura pin-
guts. Wherever fruiting plants occurred, it was noted that the
combined conditions of light and moisture were relatively about
the same. In places where there was strong light but insuffi-
cient moisture the plants were observed to wither, and when
these conditions were reversed there was always a strikingly
vigorous vegetative development, some individual plants meas-
uring ten or more inches in length.

The time of fertilization of our California plants is not defi-
nitely known. The antherozoids are discharged during March
and April and receptacles still containing intact antheridia have
been found in some of our cafions as late as August.

In England, according’ to Cavers,’ the receptacles begin to
develop in early spring but do not mature until about the end of
June, while the earliest stages in the development of the sporo-

WLOC:. Citas, Pe Qos

22 HUMPHREY

gonium are observed about the middle of July. Owing to the
very slow development of the sporogonium the spores are not
discharged until the following spring. Here in California our
plants behave differently. After fertilization the female recep-
tacles grow to about two to two and one half millimeters in diam-
eter. Though the plants may be amply supplied with moisture
from below throughout the summer, proper humidity and tem-
perature conditions do not prevail until the beginning of the
winter or rainy season. ‘Throughout the summer the growth of
the receptacle is so slight as to be almost imperceptible. San
José Creek empties into Carmel Bay and in late summer and
autumn, the cafion, being near the sea, is quite commonly filled
with heavy fogs for a part of the day, leaving all vegetation
dripping wet. While these fogs are not equivalent to the
drenching rains of winter, they do play a great role in the revi-
val of certain mosses, liverworts and lichens and doubtless stimu-
late their growth to a certain extent. At any rate, up to the
latter part of August the archegonial receptacle of /egatella
increases but slightly in size, though the increase is sufficient to
enable one to distinguish fertilized from unfertilized receptacles.
By the first of October (before any rain had fallen) the sporo-
gonium had advanced tothe spore tetrad stage and remained in
about this condition until the opening of the rainy season. Fol-
lowing this, rapid development ensues and the spores are dis-
charged in January.

This liverwort might be considered as viviparous, since the
spores germinate within the capsule. Cavers states that ger-
mination occurs within the capsule before the spores are dis-
charged, but does not say how long a time elapses between
the first evidences of germination, and the ultimate elongation
of the peduncle and dehiscence of the capsule. With our
plants germination-stages were found at least a month preced-
ing the liberation of the spores. How much earlier than this
germination actually begins cannot be said, as material for
examination was not available at that time. Germination does
not take place simultaneously in all the spores of a single cap-
sule. One may find within a capsule every stage from
the ungerminated spore to the six-cell stage, and it is to be

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 23

noted that a certain percentage of spores do not germinate at
all, but may be seen in a collapsed and shrunken condition
within the capsule.

Most of the plants examined were infected by a mycorrhizal
fungus that agrees closely in structure and habit with the one
described by Cavers. Our observations confirm this as to the
presence of starch within infected cells which is contrary to the
view of Golenkin,’ who investigated a number of infected
hepatice, including Fegatella, and states that infected cells
contain no starch or chloroplasts.

Our material was found growing on sandy soil containing
considerable humus and it was found that but few rhizoids
contained hyphze. No plants were observed in which the
ventral tissue of the midrib was infected to any extent as
shown by Cavers in plants collected near the sewer-like drains
of tanneries and similar works. All infected plants were in
vigorous, healthy, growing condition and so far as could be
ascertained, the presence of a fungus entails no such disastrous
effects as one may observe in more delicate forms such as
Fossombronia or Aneura.

ADAPTATION OF CERTAIN LIVERWORTS TO THE DRY SEASON.

It is commonly assumed that bryophytes are, in general, mois-
ture-loving plants. While this is to a great extent true it may
easily be shown that there are many important exceptions.
Among the mosses we have only to note such forms as Grzm-
mia, Hedwegia, Andreea and many others common to different
parts of the world growing on exposed rock surfaces, with abil-
ity to revive promptly and resume growth after the first rain.
Likewise many foliose Jungermanniacez, common in the east-
ern part of the United States, are capable of withstanding
drought periods of considerable length, lying practically dor-
mant throughout the rainless interval. ‘To what extent eastern
thallose Jungermanniacee are capable of withstanding drought
is a question, that so far as the writer is aware, has not yet been
investigated.

In this section of California, and more particularly the low-

'Loc. cit., pp. 209-220.

24 HUMPHREY

lying country about San Francisco Bay there is an interesting
hepatic flora consisting of types of most of the principal groups.
All of these forms fruit luxuriantly and are seldom found sterile
during any one growing season.

Among the more common hepatice of this region, and espe-
cially in the vicinity of Stanford University, are certain species
of /eccza, including ft. glauca and Lt. trichocarpa, both of
which grow vigorously in localities of extreme exposure; and
especially is this true of the latter, a liverwort structurally
adapted to such a habitat. Aside from several species of Atzccza
two species of /zmbriaria (Asterella), F. californica and F.
violacea are met with, the former being quite common. In cer-
tain localities, especially along the high banks of arroyos, Cryf-
tomitrium tenerum is found to be abundant. Along with it and
in places of severer exposure, occur formations consisting almost
exclusively of the highly resistant Zargzonza hypophylla.

Along the banks of our coast-range streams, even where
these come down into the Santa Clara valley, occur such widely
distributed forms as Fegaiella conica and Marchantia poly-
morpha. These however always grow in close proximity to
the water where throughout the year they may never be entirely
deprived of a supply of moisture. It is doubtful if these forms
would be capable of withstanding the effects of a dry season.
Three years ago the writer transferred several plants of /ega-
tella to a locality in the valley where conditions of light and
substratum were restored as nearly as could be done, at the same
time modifying and finally cutting off altogether the supply of
moisture. By lessening for a whole summer season the supply
of moisture it was found that growth of the thallus was greatly
checked and the resulting branches were much shorter. Plants
that were deprived of water dried up and died, all ability to
recover being lost as soon as the tissue had given up a certain
amount of the water of constitution. It cannot here be said to
what extent Marchantia polymorpha is capable of withstanding
drought, but since it is almost never found except where there
is a fairly constant supply of moisture it would probably endure
desiccation little better than Fegatella contca. Other moisture-
loving liverworts found in moist shaded cafions of the outer

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 25

coast ranges are two species of Aneura (fticcardia), A. multifida
major and A. pinguzs, the latter being far more sensitive to
change of environment than the former, but neither is capable
of withstanding much loss of water. They are both found
growing at their best when, if not actually in running water, they
are at least so situated as to receive a constant supply. The
finest, most vigorous specimens I have ever collected have been
found growing in such a position as to receive constant dripping
of fresh water from above, which so far as has been observed
does not materially influence the development of fruiting organs
as is the case when Fegatella is too abundantly supplied.

Several specimens of /tzcczocarpus natans found growing on
soil about a small lake in the foot-hills were brought into the
laboratory along with enough of the substratum to prevent any
shock resulting from transference. These along with some
Aneura multifida major were placed in a shallow earthern
saucer and exposed to the dry air of the laboratory until in a
normal air-dry condition. In this condition they were gradually
moistened but no sign of recovery was apparent in any part of
the tissue of either plant, and after several days of normal mois-
ture supply they showed no signs of life. It is apparent from
this and several other experiments upon hygrophilous forms
that they lack the degree of adaptability to changed climatic
conditions exhibited by such forms as /tzccza, Targzonia or
Porella.

Aside from several genera of Ricciaceze and Marchantiacee,
two species of Spherocarpus and one of Fossombronia, F..
longiseta, represent the thallose Jungermanniaceze for the
region about San Francisco Bay, as well as higher up into the
hills and mountains. So far as habit is concerned Sphero-
carpus is hardly to be compared to /ossombronza for the one is
an annual while the other remains alive throughout the entire
summer. We have here a relatively small representation of
leafy liverworts, the most common being Porella bolanderz,
and Frullania bolanderi, while in certain isolated localities one
may find ftadula complanata growing on tree trunks and
Lophozta ventricosa forming reddish-brown patches on exposed
hillsides that receive abundant moisture during the rainy season.

26 HUMPHREY

The Anthocerotacee are represented by Anthoceros fust-
formis, A. pearsont and A. phymatodes the first two being
very common, while the third, though not so widely distributed,
is very abundant in certain localities near Stanford University.
All three species are found growing luxuriantly in places of
severe exposure, though thriving best in slightly shaded locali-
ties. At one time it was supposed that A. fuszformis and A.
pearsont were annuals but they were afterwards shown by
Campbell’ to be perennial in habit. So far as the writer has
observed Axthoceros phymatodes survives through the agency
of its conspicuous tubers. In fact, it seems that virtually all of
the thallus dies during the dry season except these structures
which are packed with food-materials and serve as very effec-
tive water-storage organs. ‘The other two species, while surviv-
ing the dry season, resume development with but a compara-
tively small amount of the old thallus persisting, though both
species are highly mucilaginous and especially adapted to
extreme conditions as described by Campbell.” Careful exami-
nation of a large number of recently revived plants failed to
demonstrate the presence of sex organs even in their early
stages and it is probable that these develop after the new
growth of thallus is well under way. With the exception of the
two species of Spherocarpus it has been shown that all of our
xerophytic forms are perennial, persisting from year to year
unless artificially interfered with. Just how extended a period
of desiccation they are capable of withstanding has never, so
far as I know, been determined. In the autumn of 1903 the
writer attempted to germinate spores of Hossombronza longiseta
that had been collected in 1896. Some of the old plants along
with the spores were placed in petri dishes containing distilled
water, while others were sown upon moist earth. None of the
spores germinated nor did the plants revive. The material was
sent me from an eastern herbarium and I do not know the con-
ditions affecting the material after collection. The failure of
the plants to revive and of the spores to germinate can throw
little light upon the question.

1Campbell, 1904: Resistance of Drought by Liverworts. Torreya, Vol. 4,

No. 6, p. 84.
*Campbell, 1895: 1st ed., Mosses and Ferns, p. 117; 2d ed., 1905, p. 123.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 27

So long as the water of constitution is not disturbed the de-
gree of desiccation seems to make no perceptible change in the
reviving powers of all our common xerophytic forms. In the
preparation of a previous paper’ quantitative experiments were
made upon Fossombronza longiseta to ascertain the actual water
content of air-dried plants and to test the vitality of plants and
spores that had been for several weeks subjected to the action
of such a powerful drying reagent as glacial phosphoric acid.
The results obtained showed that the plants of this species in
the normal air-dry condition contain an appreciative amount of
water which may be removed without the slightest injury to the
vitality of the plant, while spores subjected to the same treat-
ment germinate quite as readily as those under perfectly normal
conditions. ‘To extend these results the same experiment was
carried out with Zargzonza hypophylla, Kimbriaria californica,
feiccia glauca, Porella bolandert, Cryptomitrium tenerum and
again with Fossombronza longiseta. All material was collected
early in September before the first rains, was brought into the
laboratory where, with a fine stiff brush and other instruments,
it was carefully freed from any soil particles that might be
clinging to the rhizoids. ‘Then to correct an error that might
creep in from the absorption of moisture by the plants from the
fingers or breath of the operator the plants were again subjected
to normal air-dry conditions a number of days.

Following this the five lots of each plant, were carefully
weighed on a chemical balance and then placed at once in the
recelving chamber of a desiccator over a quantity of glacial
phosphoric acid. Here the material was left from September
21, 1906, until February 9, 1907, when it was again weighed
and the respective weight losses recorded. ‘The weights before
and after artificial drying are given below:

Normal Air-dry Loss of Weight After Four
Fimbriaria. Weight. Months Desiccation.
lot I 0.05 g. 0.0025 g.
lot 2 0.05 g. 0.0007 g.
lot 3 thrown out as defective.
lot 4 0.072 g. O.009I g.
lot 5 0.105 g. 0.0047 g.

4Toc. cit.

28 HUMPHREY

Normal Air-dry Loss of Weight After Two
Targionia. Weight. Mouths Desiccation.
lot I 0.055 g- 0.0035 g-
lot 2 0.070 g. 0.0150 g.
lot 3 0.052 g. 0,0066 g.
lot 4 0.075 g. 0.0046 g.
lot 5 0.055 g. 0.0017 g.
Normal Air-dry Loss of Weight After Four
Cryptomityium. Weight. Months Desiccation.
lot I 0.050 g. 0.00250 g.
lot 2 0.040 g. 0.00310 g.
lot 3 0.040 g. 0.00325 g.
lot 4 0.060 ¢g. 0.00230 ¢g.
lot 5 thrown out as defective.
Normal Air-dry Loss of Weight After Two
Riccia glauca. Weight. Months Desiccation.
lot I 0.030 g. 0.00170 g.
lot 2 ; 0.035 &. 0.00200 g.
lot 3 0.030 g. 0.06260 g.
lot 4 0.030 g. 0.00310 g.
lot 5 0.025 g. 0.00330 g.
Normal Air-dry Loss of Weight After Four
Fossombronia. Weight. Months Desiccation.
lot I 0.025 g. 0.0020 g.
lot 2 O1O208ocen 0.0020 g.
lot 3 0.040 g. 0.0020 g.
lot 4 0.030 g. 0.0026 g.
lot 5 0.025 g. 0.002I g.
Normal Air-dry Loss of Weight After Four
Porella. Weight. Months Desiccation.
lot I 0.420 g. 0.0215 g.
lot 2 0.320 g. 0.0180 g.
lot 0.350 g. 0.0150 g.
lot 4 0.402 g. 0.0170 g.
lot 5 0.402 g. O.OI51 g.

The wide discrepancies existing between the weights of dif-
ferent lots of Fimbrzarza and of Zargzonza are probably due to
the fact that certain lots were largely made up of thick, bulky
plants capable of retaining more moisture than the smaller
thinner plants composing other lots. Then too, some allowance
must be made for small particles of soil clinging to the rhizoids,
though care was exercised in the removal of all foreign matter
from the plants employed in the experiment. It will be noticed
that in the case of Cryptomitrium, Lossombronia and the others
where the plants were of more uniform size the discrepancies
are slight.

a
b=

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 29

Of the above forms /vmbriarza, Targionia, and Porella are
the most sensitively hygroscopic; at least when moistened they
are the first to become turgid and resume activity.

By dividing the total loss in weight due to artificial desicca-
tion by the air-dry weight we obtain in percentages the actual
fraction of the air-dry weight that is lost in drying. For exam-
ple it was found that Fzmbrzarza, lost 6.06 per cent. of its air-
dry weight while losses for the other five were: Zargztonza,
i2.3\per cent.; Cry plomirim, 5.80 per cent. ; 7i2cc7a, o.47
per cent. ; ossombronza, 8.23 per cent. ; Porella, 4.57 per cent.

From these figures it will be seen that in the normal air dry
state, 7Zargzonza contains the greatest amount of the water of
constitution, while Fossombronza and Aticcza contain about two
thirds as much. Fore/la, the only leafy liverwort used in the
experiment, had given up all but a very small percentage of its
air-dry weight followed closely by Crypftomztrium, one of the
Marchantiacee.

As a rule our summers are characterized by heavy morning
fogs which drift in over the mountains from the ocean. These
hang over the valley for three or four hours after sunrise greatly
reducing the temperature and leaving behind, each morning of
their occurrence, considerable precipitation, enough sometimes
to result in dripping from eaves. The past summer (1906), how-
ever, was unusually free from these fogs, even late in the season
when ordinarily they are heaviest and most frequent. Fog-
less mornings are characterized here by slight or even no pre-
cipitation of dew and are forerunners of hot, extremely dry
days. During such a season as the one just described it is
probable that our liverworts remained in an absolutely dormant
condition. It has many times been demonstrated that all our
perennial forms on being moistened revive in from half an hour
to twelve hours, resuming growth very promptly if the supply
of moisture is constant. Campbell’ has shown in a recent paper
that this peculiar adaptation is a property not only of certain of
the hepaticz but belongs also to Gymnogramme triangularts, a
fern common to this region, which dries up in summer, remain-
ing dormant throughout the season, without the resulting death

PVC. Cites Pp. O5-

30 HUMPHREY

of the leaves as occurs in the majority of ferns. Not infre-
quently one finds in the field quite fully developed prothallia of
this species early in the autumn before the winter rains have
actually set in. It is hardly possible for these to have devel-
oped from spores that had germinated at any other time than in
spring or early summer. In fact, it has been experimentally
shown by Professor Peirce’ that the prothallia of this species
may endure prolonged desiccation without evidence of the
slightest injury, for on being moistened they very promptly re-
vive and young sporophytes develop normally from these gameto-
phytes of the preceding season. Perennial prothallia develop-
ing tubers have been reported by Goebel? for the allied fern
Anogramme cherophylla, common in southern Europe. Such
structures belong to some of our liverworts but have not yet
been shown to occur in the prothallia of any of our ferns. Aside
from the common fern Gymnogramme, other pteridophytes native
to California, ¢. 9., Selaginella biglovet and S. lepidophylla,
the latter the well known ‘resurrection plant,” are reported by
Campbell in the paper above referred to as remaining in a dry
and dormant condition throughout the greater portion of the
year, resuming active growth during the rainy season, at first
absorbing considerable water through their leaves as do the
leaves of Gymnogramme.

To test the vitality of those liverworts that had been naturally
and then artificially desiccated, a certain number of plants from
each lot were placed on moist earth and others in Knop’s
nutrient solution. In both cases the plants revived promptly
and are now growing vigorously, producing fruiting organs in
great number. A surprisingly small portion of the thallus of
Targionta, Riccia, Fimbriarza and Porella was actually dead ;
though in Fossombronia, Cryptomitrium and Anthoceros phy-
matodes only the apical end including a small portion of the
thallus appeared to be alive. Within five hours revived plants
from each species were fixed with chromic one per cent. fixing
solution and run up through the alcohols and bergamot oil to

?Campbell, 1904: Resistance of Drought by Liverworts. Torreya, Vol. 4,
No. 6, p. 85.
3 Goebel, 1898: Organography of Plants. Part II, Vol. 1, p. 426.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 31

parafin. The same method of fixation, dehydration, clearing
and infiltration were employed here as detailed in a previous
paper on the development of Fossombronia longiseta, barring
slight modifications. It was found that the best results could
be obtained by following the method of dehydration described
by Chamberlain." The use of diffusion shells and the ‘‘ con-
stant drip” process have proved somewhat unreliable, not
always giving satisfactory results for such tissues as prevail
among the more delicate hepatice. Instead of transferring
from absolute alcohol directly to a 50 per cent. solution of
bergamot oil and then to pure bergamot, a more gradual trans-
fer was employed and a similar plan was followed in running up
through the paraffins.

In the autumn of 1903 the writer revived some material con-
sisting of Hossombronza and Fimbriarza and after a few hours
placed them in a fixing solution. On studying sections of each
it was observed that in the case of /ossombronzia, not only was
a considerable portion of the thallus alive but it was found that
there were nearly mature antheridia and archegonia. Sections
of /zmbriarza through the median sulcus proved the presence
of well-advanced antheridia. This suggested the possibility of
a similar early development of sex organs in other forms than
the two mentioned above. A number of slides of Azccza, Tar-
gionia, and Cryptomitrium as well as of FHossombronza and
Fimbriarva were prepared and in each instance, at least, anther-
idia have been found. Only in Fossombronza and Porella have
we found archegonia. In /zmbriarza and Cryptomitrium
these organs occur on special receptacles none of which showed
any sign of development. In Azccza and Crytomztrium only
early stages of the antheridium were present which was rather
contrary to anticipation. A large number of plants of each
genus were sectioned and the great majority of them were
sterile. Quite early in the rainy season we have been in the
custom of collecting quantities of /tzccza in which no difficulty
is experienced in obtaining virtually all stages in the develop-
ment of the sex organs. For this reason the writer feels that a
more exhaustive examination of this species ought to reveal

1 Chamberlain, 1905: Methods in Plant Histology, p. 22.

_ a

32 HUMPHREY

fruiting organs quite as advanced as those of Zargionia or
Fimbriaria. As for Cryptomitrzum, it is not surprising to find
so few antheridia, for even as late as the first week of February
sections of Crypfiomitrium revealed the presence of mature
antheridia while in the case of zmbrizarza they may be found
almost ripe before the beginning of the wet season. Abrams!’
in his studies of Crypftomztrium found that the antheridia
matured earlier than the archegonia and informs me that he
found fairly well advanced stages of the antheridium as early
as November. In FPored/a not only all stages in the develop-
ment of sex organs are to be found but well-developed sporo-
phytes as well. This accounts for the very sudden appearance
of mature sporophytes shortly following a few days of wet
weather. Growing as it ordinarily does on the exposed sur-
faces of rocks and tree trunks, it is liable to severe exposure
and may be left quite dry in a few hours of wind or sunshine.
This fact may have some influence upon its reproductive habits
and, in a measure, account for the advanced sporogonia found in
revived material. At any rate Forella is scarcely to be com-
pared with such thallose forms as /tzccza or Targzonia. ‘The dif-
ferences in habit and structure might well beget differences rela-
tive to the appearance and time of development of the sex organs.

In another part of this paper the writer mentions having
found Fossombronia longiseta so situated out of doors as to re-
ceive a constant supply of moisture from a leak in a water
pipe. Many of these plants were examined on November I
very near the close of the dry season and were found bearing
embryos in various stages of development and in some instances
well advanced sporogonia as shown in Fig. 1. By the first of
January the spores and elaters had matured and some capsules
had already dehisced.

Not having observed these plants at the time of fertilization
it cannot be definitely said just when the antheridia and arche-
gonia matured. However, the approximate date may be ascer-
tained by observing the rate of development of the sporogonium
and of the ripening of the spores. In 1904 the first rain of

1Abrams, 1899: Structure and Development of Cryftomttrium tenerum.
Botanical Gazette, Vol. 28, pp. II0-I21.

“et
ul SG ee

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 33

any consequence fell on the twenty-first of August, but liver-
worts barely revived by it were overtaken by a period of dry,
hot weather which lasted till September 23, when the rainy
season began. It might justly be assumed that the resumption
of growth, as regards Fossoméronza at least, began at this time,
More than the usual amount of rain was recorded for October
and November and growing conditions were very favorable.
Material collected in early February showed sporongia in which
the spore-mother cells were preparing to pass into the tetraspore
condition, and six weeks later material was collected bearing
ripe spores and elaters. From this it will be seen that, having
a favorable rainy season, the time required to produce ripe
spores is approximately six months after the first rains of any
consequence. Judging from the advanced development in which
one finds antheridia and archegonia at the close of the summer
season, fertilization must occur within a few weeks after
resumption of growth is well established.

With regard to the plants that had grown unchecked through-
out the summer, it must be noted that temperature and humidity
conditions are widely different from those prevailing in winter.
Though the plants were well irrigated, the atmospheric condi-
tions were such as to influence to some extent the growth and
behavior of the thallus and fruiting organs. Assuming, how-
ever, the same rate of development of the sporogonia for these
plants as for those grown in winter, fertilization must have
occurred during July and later. Growing in a paraflin-coated
glass dish containing Knop’s nutrient solution, the writer has
under observation several plants of Fossombronza, placed there
a month ago in air-dry condition as nearly free from soil parti-
cles as they could be rendered by the use of a fine, stiff brush.
Antheridia and archegonia then in the initial stages of develop-
ment are now about as far advanced as were the oldest of those
observed as soon as the plants had revived —in the course of
twelve hours. These oldest ones are now maturing. It will
be shown elsewhere, however, that Knop’s solution affords a
very active stimulus to development of the thallus and fruiting
organs, probably shortening the time of their normal develop-
ment considerably.

Proc. Wash. Acad. Sci., January, 1908.

34 HUMPHREY

At any rate, it is evident that under conditions prevailing in
this part of California a second crop of antheridia appears before
the close of a single growing season, and in the case of Fossom-
bronza at least, when the supply of moisture is maintained, ferti-
lization and ultimate development of the sporophyte proceed
regularly, the spores ripening three or four months earlier than
those of the winter crop. It is probable that the second growth
of antheridia and archegonia begin to develop at least a month
before the close of the rainy season, and this fact is of particu-
lar importance to the plant as it insures the maturity of the
sporophytic generation well within the rainy season.

Certain of our liverworts show a tendency to develop tubers
or other structures enabling them to resist drought. For ex-
ample, in the southern part of the State, Geothallus tuberosus is
a form bearing well-marked tubers, while in our own locality
Frossombronza develops tuber-like thickenings of the stem, and
well-defined tubers are to be found in the case of Anthoceros
phymaiodes. All the plants employed in this study showed the
presence of scales or hairs which in some instances secrete
mucilage; these along with the mucilage cells within the thallus
are structures undoubtedly serving as water-storage organs.

According to Howe,’ aside from Anthoceros phymatodes, tuber-
ous structures are not uncommon among plants of A. pearsonz,
both species growing ordinarily in exposed places. In other
parts of the world, hepatica bearing tubers have been found.
For example Goebel’ describes two species of Authoceros, A.
argentinus and A. dichotomus and a species of Hossombronza,
F. tuberifera, common in certain parts of Chili. He regards
the organ in the last named species a true tuber not unlike the
same structure developed by our Axthoceros phymatodes, but
considers the tubers of A. argentinus and A. dichotomus as trans-
formed branches of the thallus, the ends of which, have become
swollen and filled with reserve food products, very similar to
what prevails in our common species A. pearsonz. Goebel *
also reports certain species of /tzccza as developing tubers,

MCoc. cit. p. 164.
4120; \cit., (Pp. 293:
2NOc. Cit... p. ZO.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC: 35

notably an Italian form in which he observed entire portions of
the thallus developed into tuber-like structures. Stephani’ has
also described similar structures for /tzccza bulbtfera. None of
our Californian Riccias has yet been described as bearing
tubers or tuber-like growths, though /t¢ccca trichocarpa, men-
tioned elsewhere in this paper, is well adapted to withstand
severe exposure, the thallus being densely clothed with tawny
sete .3 to .65 mm. long, with those toward the apex in as many
as eight to twelve series, affording a very effective protection
against excessive drying. ‘Though these tuber-like structures
wherever they have been examined are always found to con-
tain large quantities of reserve food, there is little doubt that
they are instrumental as water-storage organs.

STUDIES IN THE GERMINATION OF SPORES.

In a previous paper the writer has described and figured the
germination of the spores of Mossombronza longiseta. ‘The
results of experiments then carried out showed the early devel-
opment of this species to agree closely with certain forms, not-
ably Spherocarpus californicus, investigated by Campbell.’

Most of the hepaticee of this region produce spores character-
ized by a heavy, more or less sculptured exosporium which
Leitgeb * found to consist of two parts or layers of which the
inner belongs to the spore itself and is the exine, while the
perinium or outer layer is formed later and consists of parts of
the sporocyte. Leitgeb suggests the probability of the perinium
as a protection against drought but in the light of more recent
studies of certain forms producing thick-walled spores, e. ¢.,
Corsinia, Pretssia, Anthoceros and Spherocar pus, it may be held
that the perinium can certainly have nothimg to do with a rest-
ing period, for spores of these forms germinate readily within a
few days after being sown. As to the thick exosporium fur-
nishing a protection against excessive dryness there can be little
doubt, although such forms as Corsznza and the aquatic Ric-

1 Stephani, 1898: Species Hepaticarum. Bull. de l’Herb. Boiss., Vol. VI, p.
333

2 Campbell, 1896: Notes on Spherocarpus. Erythea, Vol. IV, No. 5, Pl. 2.

* Leitgeb, 1884, Ueber Bau und Entwicklung der Sporenhaute, Graz.

36 HUMPHREY

cieze produce spores of similar character. Goebel,' however,
suggests that localities favorable to Corsznza may not always be
moist, and in the case of the aquatic Ricciez the perinium very
effectively serves as a protection against invasion of the spore by
fungal hyphe.

Although the great majority of our xerophilous liverworts
produce thick-walled spores there are such exceptions as Por-
ella bolanderi and Fradula complanata whose spores are protected
by a thin, frail exosporium and are incapable of withstanding
prolonged drying. These liverworts grow on tree trunks or
exposed surfaces of rocks where they are subject to sudden

drying out and we should naturally expect their spores to be
better protected.

The character of the spore-wall, however, might suggest the
possibility of these forms being naturally more hygrophilous in
character, while their adaptation to a dry habitat is merely one
phase of the extreme adaptability common to both forms.

In order to get at the relationship of the exospore to the spore
a study of the life conditions of the several species is necessary
and some light will be thrown upon the subject when we ascer-
tain at what time, in nature, the spores of a large variety of
forms germinate. With a large number of forms it has been
repeatedly shown that spores sown on soil under conditions as
nearly normal as possible germinate only after a long period of
rest.

In June, 1904, the writer sowed on sterilized earth, a quantity
of spores of Hossombronza that had been gathered a few days
before sowing. ‘The culture was made in an earthen saucer
and placed within another close-fitting earthen saucer, in which
was maintained a supply of water so that the dirt was kept con-
stantly moist from below. ‘The culture was placed on a table
near a window where it could receive a nearly normal supply
of light. During the latter part of August a few spores had
germinated but not until October did they germinate in any
great number. The spore of Hossombronza, while perhaps not
as well protected as that of Avmbrzarza or Targionza, nevertheless
is furnished with a resistant exosporium and is well adapted to

1 Loc. cit., 107.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 37

the xerophilous conditions under which the plant grows. The
spores are positively known to maintain their vitality for at least
two years but for how much longer, cannot be said. The thin-
walled spores of such forms as Aneura, Fegatella, Radula and
Porella withstand but little drying, losing all germinating power
within a few months at most, which fact would seem to be evi-
dence in favor of the statement that the thick exosporium is a
protective structure.

Spores of Anthoceros fustformis one year and eight months
old, when sown on sterilized soil germinated copiously within
two weeks. Here we have an instance of spores that are known
not to require a resting period before germinating, yet, unlike
Aneura, the spores of Anthoceros are known to endure at least
two years desiccation. Spores of /zmbriarza californica and
Targionia hypophylla two years of age were sown on sterilized
earth and kept well watered. These germinated within twelve
and fifty-six days respectively. This wide discrepancy is diffi-
cult to account for. The soil was kept as free from fungi as
possible: however, only a few of the Zargzonza spores ger-
minated and this fact may have been due to reduced vitality from
prolonged drying. Further experiments with spores of this
genus are necessary ; in fact, germination experiments conducted
out of doors are the ones of greatest value. Such a series of
culture experiments was started more than a year ago in the
Stanford Arboretum but before results of any value were ob-
tained the cultures were accidentally destroyed by a workman.
It was then too late in the season to start another series, it being
very near the close of the rainy season. A fresh collection of
spores was made from the 1906 crop and several artificial cul-
tures were started indoors, using both sterilized and non-steril-
ized earth and Knop’s nutrient solution. The spores used in
the various cultures were of the following forms: /tzccza glauca,
Fimbriaria violacea, F. californica, Targionia hypophylla,
Fossombronia longiseta, Cryptomitrium tenerum and Aneura
multiida major.

No spores of the first mentioned plant have yet germinated,
though sown six months ago after a resting period of five
months. Spores of both species of Azmbrzarza failed to ger-

38 HUMPHREY

minate, though sown at the same time as those in the case of
Piccta. A very few of the spores of either Hossombronia or
Cryptomitrzum germinated. ‘Though he did not duplicate in
every case the cultures of the writer, Professor Peirce had a
similar experience with all spores of the same (1906) crop.
This apparent loss of vitality is difficult to account for, unless
due to the unusual character of the weather’ during the spring
months. Ordinarily our last rains occur in April, by which time
the spores of the above-named hepatice have either been dis-
charged or are maturing. An examination of the daily record
of precipitation from April 1 to the close of the season may be
instructive. Up to April 1 from January 1 the total rainfall
amounted to 10.04 inches. From April 1 to June 15 the record
was as follows:

Amount of Amount of

Date. Precipitation. Date. Precipitation.
JENDIFUL “UW AdoasaaeboonecncicanoaD! 0.06 May Walt is snk sc cesteneseeeen 0.02
77 \” IC) .ceseaoanagonag secaaoone 0.01 OF ANI snes Godinain“soocSaqcec: 0.34
SEMI Qieecetescles.setes calayiaacis O.11 sO ieZOhaecantseaaeae 6 ee neeee 0.04
PC ik pepedbes sogenecaboousos 0.03 Be) Aifscdconaecagsoncchas oo" 0.51
COTE DY 7 ae eee oeelsis cata noise te 0.29 SS UDO auch rage ante teresa Gain
AMA ZOR Aamo taenesesscwsiles trace {ta WN 2 Orcicaette cetera etme O.11
IMEI 216) soe conestoooeSapantads 0.01 | UAE YanacecconcceBorsomoucnde 0.02
ri Th ib snpsoddsonoAdonsaSAI005 0.02 i ent ocob Acc ondads a aca 0.73
nie | MEYsondricoa seoescon nd ops ee 0.24

OE 2 OT casement eelae nee trace

From the above record it may readily be seen that from
April 1 to April 23 the total precipitation amounted to but .07
of an inch — scarcely more than a trace. Between the twenty-
third and twenty-seventh enough rain fell to revive the already
dried mosses and liverworts to a condition of perfectly normal
turgidity and they remained so at least a week after the last rain-
fall. Now during April the spores of a number of our liver-
worts are only beginning to mature, and it is barely possible
that the prolonged dry period during that month interfered with
the proper ripening of the spores. On the other hand, spores
of certain forms, for example, Hossombronza, which had fully
matured during March but were not gathered till after the
rainy season, failed to germinate ; and this suggests the possibility
of the vitality of the spores having been impaired by successive

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 39

rains and rapid desiccation following one upon the other. What-
ever be the cause of impairment the fact remains that almost
no spores germinated even under most painstaking precautions
as to culture conditions. To get at the root of the matter, how-
ever, a series of experiments is necessary in which the student
should duplicate as nearly as possible the conditions prevailing
from April 1 to June 15 of 1906.

It is an unsettled question as to whether the spores of many
liverworts actually require a prolonged resting-period, and there
is still need of investigation along this hne. We are familiar
with the fact that the spores of most xerophilous forms do lie
dormant several weeks before germinating but it has not been
shown in very many instances that this resting-period is inti-
mately connected with the preparation of the spore for germina-
tion. The writer has demonstrated beyond doubt that the spores
of Hossombronia longiseta, under certain conditions, do not re-
quire a resting-period. Cultures were prepared using different
media with a view to testing, if possible, the relative influences
of the various media upon germination. One lot of spores col-
lected on January 10, 1906, were sown January 12 on pulver-
ized earth that had been thoroughly steam-sterilized and well
watered with distilled water. Care was exercised to select soil
on which Fossombronza grows vigorously out of doors; this
was placed in a crystallization dish and covered with one half
of a petri dish to keep the culture as free from dust as possible.
A number of crystallization dishes were thoroughly cleaned
and then covered on the inside with a thin coating of paraffin
to prevent the water used from taking up any impurities that
might exist in the glass. In three of these, Knop’s nutrient
solution of the following proportions was placed:

AO) ores oiht Yeia. closes cscs cisce sista waists isle eilaisjelaleitice 500 c.c
SCM WILT ALC ccene deen deenine aelsdenealacaine eee 2.00 grams.
MAS HeStUM SUL Mates. ce saccecascsseseoeroe 0.5 oe
Potassium nitrate...... he cpocon pes ocwnoo00G8s 0.5 ie
Porassiuim phosphates. vaccesecsscenascoasess 0.5 es

Other solutions of varying strength were employed as follows :

BOE steerer a dese vei dwihnseneivameees svceas estedans 500 c.c
CAleMIMA METALS... < eee. calSonccpes nsec senases 2.00 grams.
Masnesinin Sul phiatess.iscsscsncccucvecneses. 0.5 es
POPaSSH IM MIEKALC .vocs,\cecccstubceses tess oslean Os “

Potassium phosphate omitted.

40 HUMPHREY

Other stock solutions were made up in which the amounts of
the various salts used in a normal solution were reduced in
quantity one fourth and one eighth, though the quantity of dis-
tilled water was kept the same, 500 c.c., in all the solutions.
It will be noticed that from one solution the acid salt, potassium
phosphate, was left out with a view to testing its value as a
stimulus to germination. Spores of the same lot were sown in
the various solutions the same day they were collected; the cul-
ture dishes were carefully covered and placed on a north window-
sill where a uniform amount of diffused light fell upon each
culture. In another dish that had been paraffin-coated, water
that had been twice distilled was placed and on the same date
spores of the same lot were sown. ‘These germinated in con-
siderable numbers within twelve days. Some of the water used
in this culture was taken from the supply bottle and evaporated
on a slide. On examination, a precipitate of organic particles
was found, showing that certain compounds had passed over
with the water in distilling, and it was thought probable that
these acted as a stimulus, for the spores in this culture germi-
nated before those in the culture containing none of the acid
salt. In fact these latter seemed to show no indications of any
preparation for germination, such as change in color of exospor-
ium from very dark brown to a lighter more translucent shade.
The distilled water used in the preparation of the various cul-
ture solutions was from the same stock bottle as that used in the
distilled-water culture. Why spores collected at the same time
and sown at the same time on these two media should have
germinated earlier in distilled water containing organic impuri-
ties than in a culture solution void of one of its constituent salts
is difficult to account for unless in the latter we have a physio-
logically unbalanced solution which has been shown by Loeb?
to exercise a certain toxic effect upon certain marine and fresh-
water animals and by Osterhout’ in his experiments upon alge.
This, however, needs far more extensive investigation than has
yet been given to it.

1Loeb, 1905: Pfliiger’s Archiv, 107: 252.

2 Osterhout, 1906: On the Importance of Physiologically Balanced Solutions

for Plants. University of California Publications. Botany, Vol. 2, No. 10, pp.
229-230.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 4I

The spores in the unbalanced culture solution germinated
within six days after those in the normal Knop’s solution, which
would indicate the existence of some stimulating factor in that
solution. Another lot of distilled water was then prepared by
allowing the first fourth or fifth part to distil as waste water, thus
getting rid of the larger part of the volatile substances. Only
the middle three fifths was saved and this was redistilled in the
same manner. The best portion of this was again distilled and
only the middle portion of the product was collected ; in this way
water of a high degree of purity was obtained. This was used
in the preparation of another distilled water culture and spores
of Fossombronza sown eighteen days ago show no evidence of
germinating. Spores sown on Knop’s solution, normal, germi-
nated within fourteen days producing vigorous germ tubes with
rather more than the usual amount of chlorophyll. These young
plants have grown more rapidly and vigorously than those in
distilled water that germinated approximately at the same time.
Spores sown in the normal solution germinated two days later
than those in either of the reduced solutions but in the former
there was a far greater number of young plants two days after
the first observation of germination.

Spores of /imbriarza californica collected in March, 1905,
were sown in Knop’s solution (normal) and germinated promptly
within twelve days. Young plants in this culture that are now
sixteen to twenty days old are characterized by the unusual
length of the germ-tube, in some instances measuring more than
one and a half millimeters from the basal end to the first cell
cut off at the distal end of the germ-tube. In some instances
two germ-tubes were seen to emerge from a single spore as
figured by Campbell’ for Zargionia hypophylla. Spores of
the same species were sown in an earthen saucer filled more
than half-full of finely pulverized non-sterilized soil. These
germinated promptly and in great numbers. Both cultures
were so placed as to receive light from a north window: the
Knop’s solution culture received somewhat the greater amount
of light being in a glass dish, coated part way up with paraffin.
None of the young plants of the soil culture produced germ-

"Eo. cit., Ist €dz,) p..65% 2d ed-, p. 66.

42 HUMPHREY

tubes of more than usual length while some showed the forma-
tion of cross-walls very early. The young plants in the nutri-
ent solution have grown rapidly, producing in some instances
as advanced a development as is seen in Fig. 21, within eigh-
teen days. A very few young plants in which there was only
a slight germ-tube development had already begun to assume
the dorsi-ventral form common to this genus. Desiccated
plants of Azmbrzaria, Reiccia and Fossombronia when placed
in Knop’s solution revived quickly producing within a few
days a greatly increased number of rhizoids and in the case of
Fossombronta, many more than the usual number of antheridia,
closely covering the dorsal surface of the young growing shoot.
From these and other results it seems highly probable that
Knop’s nutrient solution exerts a stimulative influence upon the
development of the thallus, rhizoids, and reproductive organs
as well as the germination of the spores. Further investigation,
however, with solutions of varying strength and composition is
needed in order definitely to determine the nature and chemical
composition of the salt or combination of salts that act as a
. stimulus to growth.

According to Goebel,’ germination in the Marchantiez when
compared with other thallose liverworts presents certain char-
acteristic differences. He separates the product of germina-
tion of a spore of Prezssza for example, into three structures,
z. €., the germ-tube, the ‘‘ pro-embryo” and the young plant.
He finds that the ‘* pro-embryo” is positively heliotropic and
develops at its end a flattened cell mass which he terms the
germ disk. This forms at right angles to the direction of the
light-rays and the young plant arises from one quadrant of the
germ disk as shown in his Fig. 95.

On the same page he states that a similar germination is
common in the Ricciez contrary to the description and figures
of Campbell? who studied the germination of /tzccza trichocarpa
and a number of other forms. He refers to Campbell’s Fig. 9,
p. 38, as failing to bear out his statement that the axis of growth
in the young plant is continuous with that of the germ-tube.

AL OG. Ctl, Pak It.
CICOENGIt. TStrede, (p..39% 2died., 9. 8o.

v bet

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICA! 4

An examination of the figure referred to, however, will show
that although an angle was present in the young gametophyte
it developed in the germ-tube itself, the upper portion of which
lies in the direct axis of growth of the young plant, no such
structure as a pro-embryo being present. In his study of /7m-
briaria and Targionza his figures show an early development
of the young plant not unlike that in the case of /tzccza tricho-
carpa. ‘The writer’s investigations relative to the germination
of both Fossombronia and /imbriarta agree in detail with those
of Campbell in the forms studied by him (Figs. 22 and 23).
MacDougal’ has shown that, given the necessary intensity of
light, its direction will vitally influence the form and habit of a
living plant as well as the disposition of various organs incident
to its development. This being true, we might, @ przor:,
assume that Goebel’s Predssca and ARiccéa spores on germinat-
ing take on the angular form shown in his Fig. 95 because of
certain external factors and, most of all, light, instead of trying
to account for it on grounds of heredity or of certain inherent
factors. In fact, Peirce,” Rosenvinge,* and others have experi-
mentally shown that in the case of certain alge, germinating
spores are profoundly influenced by the direction and intensity
of light. Eliminating so far as possible all other external factors
it was found that the young plants always grew towards the
light, while the rhizoid or the holdfast was negatively helio-
tropic. Peirce has demonstrated that in addition to this, light
exerts a directive influence upon the formation of cross-walls
in sporelings of alge, liverworts and certain ferns, these walls
always forming at right angles to the direction of the light rays.*
With ferns and liverworts he has further shown that when the
long axis of the germ-tube lies in the direction of the light rays,
the continued growth of the young plant will be in the same
direction. More than this, he has demonstrated that when the
germ-tube ruptures the spore wall on the side away from the |

1 MacDougal, 1903: The Influence of Light and Darkness upon Growth and
Development. Memoirs of the New York Botanical Garden, Vol. II.

MOE: City, P.330-

3 Rosenvinge, 1889: Influence d’agents exterieurs sur lVorganisation polaire
et dorsiventral des;plantes. Revue Generale de Botanique, 1.

* Peirce, 1906: Studies of Irritability in Plants, p. 453.

44 HUMPHREY

light there is a strong tendency to turn toward the light, and if
this is not accomplished by the germ-tube, the further develop-
ment of the young plant will at once or gradually assume the
direction of positive heliotropism.

DURATION OF VITALITY OF CERTAIN GREEN OR THIN-
WALLED SPORES.

A failure to germinate the spores of Aneura multifida major
after they had been allowed to become several weeks old,
induced the writer to prepare a series of cultures to test the life
duration of the spores of Axeura, Feadula, Porella and
Fegatella.

The first spore cultures of all these forms were started on the
same day of collection in order to determine whether the spores
were ready for germination under normal conditions. Practi-
cally all spores germinated in cultures consisting of sterilized
earth, Knop’s solution and distilled water. I next allowed
spores of Fegatella to dry for three days, by placing them on a
sheet of paper on the laboratory table. These were then sown
in the various culture media. Many of the spores had already
germinated within the capsule but these along with all spores
still ungerminated failed to show the slightest sign of any
recovery after days of constant moisture and favorable light.
Later, Professor Peirce demonstrated that one hour’s drying
under normal conditions was sufficient to kill all spores, and
further experiments with the spores of this species were re-
garded unnecessary. ‘The spore wall in the case of Hegatella
is very thin, allowing rapid loss of the water of constitution,
resulting in a general contraction of spore contents and collapse
of the spore wall.

On January 12 cultures were started, sowing spores of Ahadula
complanata collected on the ninth of the same month. Prac-
tically all of these germinated within a few days, so another
trial was not made till the twenty-eighth of January. Approxi-
mately ten per cent. of the spores of this sowing failed to
recover, the cytoplasm and other spore contents having con-
tracted towards the center forming a brownish mass.

On February 11 a third culture was started, the spores now

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATIC 45

having dried thirty-two days. Between 20 and 25 per cent.
of these spores failed to recover. The last lot of spores were
sown on March 3 —fifty-two days after collecting. Approxi-
mately forty per cent. of these were desiccated beyond recovery.
Time does not permit at the present further testing of the spores
of this species but judging from the results of the above cultures
very few if any spores could endure longer than a few months
at the most.

Porella bolanderz was then experimented with, cultures being
started at various intervals using the same media as in preced-
ing cultures. The results obtained were not greatly different
from those in the case of /radu/a though in my last culture
which was made on March 5 not more than half the spores have
recovered and fewer still have germinated after eleven days.
The spores in this culture had been allowed to dry thirty-eight
days, or fourteen days less than was allowed spores used in the
last ftadula culture. Making due allowance for the possibility
of tuxic effects due to the activity of bacteria or fungi that might
have been in the various cultures it is highly improbable that
the failure of constantly increasing numbers of spores to revive
could have been entirely due to any such cause and we are jus-
tified in the conclusion that the spores of these forms and prob-
ably some others not yet investigated are incapable of enduring
more than a comparatively slight amount of desiccation.

SUMMARY.

1. Plants of Fossombronia longiseta, as it occurs here, are
seldom free from infection by a fungus, which, in its relation
to the host, acts as a true parasite. In early stages the fungus
is confined to the rhizoids and compact tissue of the stem, ulti-
mately extending throughout all vegetative tissues of the gameto-
phyte. Large, black sclerotia develop within the cells of the
leaves and peripheral cells of the stem, resulting in the death
of the host.

No instance of infection of sex organs or sporogonium has
been observed.

2. Kimbriaria californica has been found to be infected by
a fungus, the hyphz being confined to the outermost three or

46 HUMPHREY

four layers of ventral cells and to both sorts of rhizoids. Its
relation to the host is apparently symbiotic.

3. A fungus, evidently epiphytic in habit, was found associ-
ated with Aneura multifida major. Its relation to the host is
that of a parasite.

4. Fungi were also found associated with Azthoceros pearsont
and Porella bolanderz. In both cases the fungus is epiphytic,
sending hausteria-like branches into the tissues of the host.

5. Fertilization in Fegatella conica in California occurs in
early spring. At the close of the dry season the spores are in
the tetrad stage. With the commencement of the rainy season,
the growth of the female receptacle is resumed and the spores
mature early in January.

6. When hygrophilous forms such as Aneura pinguis or
Fegatella conica are transferred to a dry habitat and allowed
to become desiccated they are unable to recover.

7. All of our xerophilous forms such as /imbrzarza cali-
fornica, Targionia hypoyhylla, several species of /ticcza, Fos-
sombronza, and others resume growth promptly after months of
extreme drought. When artificially desiccated over glacial
phosphoric acid until further loss of weight is imperceptible, on
being moistened they revive quite as promptly as when nor-
mally air-dried.

8. Artificial drying does not impair germination of spores of
xerophilous forms.

9g. In Porella bolandert material examined at the close of
the dry season exhibits holdover antheridia and archegonia and
well-advanced sporogonia. Other xerophilous forms of peren-
nial habit show sex organs in various stages of development.

10. Fossombronia growing in a habitat of constant moisture
matured spores in March and December to February.

11. Mucilage secreting structures are common to many forms
and especially to our species of Anthoceros. A. pearsond and
A. phymatodes are rendered resistant to drought by the presence
of tubers. Similar structures, though less developed, are com-
mon to Hossombronza longiseta in dry regions.

12. Spores of xerophilous forms are known to retain their
vitality at least two years and probably longer. In nature they
pass through a resting period of several weeks.

PHYSIOLOGY AND MORPHOLOGY OF CALIFORNIA HEPATICZ 47

13. Spores of Hossombronza longiseta sown in Knop’s solution
and in impure distilled water germinated in the former within
fourteen days and in the latter within twelve days after removal
from the capsule. Spores sown on sterilized soil show no evi-
dence of germination after sixty-two days.

14. The spores of Fegatella conica, Aneura pinguis, Porella
bolanderz’ and ftadula complanata were found incapable of
withstanding prolonged desiccation. Germination occurs nor-
mally within the capsule or very soon after dispersal.

BIBLIOGRAPHY.
Abrams.

1899 Structure and Development of Cryftomitrium tenerum (Hook.) Aust.
Botanical Gazette, Vol. 38.

Bruchmann.
1904 Ueber das Prothallium und die Keimpflanze von Ophioglossum vulgatum
L. Botanische Zeitung, Heft XII, Tafel VIII.
1906 Ueber das Prothallium und Sporenpflanze von Botrychium lunarza Sw.
Flora oder Allgemeine Botanische Zeitung. Band 96, Heft 1.

Campbell.

1896 The Development of Geothallus tuberosus Camp. Annals of Botany,
Vol. IX.

1896 Notes on Spherocarpus. Erythea, Vol. IV, No. 5, Pl. 2.

1895 Mosses and Ferns, Ist ed. 1905, 2d ed.

1904 Resistance of Drought by Liverworts. Torreya, Vol. 4, No. 6.

1907 Studies on the Ophioglossacee. Annales du Jardin Botanique, Vol.
XXiI.

Cavers.
1903 On Saprophytism and Mycorrhiza in Hepatice. The New Phytologist,
Molly No. 2:
1904 On the Structure and Biology of Fegatella contca Corda. Annals of
Botany, Vol. XVIIT, No. LXIX, Plate VI.
1904 Contributions to the Biology of the Hepatice. Part 1, Zargionza, Re-
boulia, Pretssta, Monoclea.

Chamberlain.
1905 Methods in Plant Histology.

Czapek.
1889 Zur Chemie der Zell-membranen bei den Laub und Lebermoosen.
Flora, Band 86.

Goebel.
1898 Organography of Plants, Part II, Vol. 1.

Golenkin.
1902 Die Mychorrhiza ahnlichen Bildungen der Marchantiaceen, Flora, Band

go.

48 HUMPHREY

Howe.
1898 The Anthocerotacee of North America. Bulletin of the Torrey Botan-
ical Club, Vol. 25, No. 1.
1899 The Hepatice and Anthocerotes of California. Memoirs of the Torrey
Botanical Club, Vol. VII.
Humphrey.
1906 The Development of Yossombronia longiseta Aust. Annals of Botany,
Vol. XX, No. LXXVII.
Jeffrey.
1898 The Gametophyte of Botrychium virginianum. University of Toronto
Studies, No. I.
Kny and Bottger.
1879 Ueber eigenthiimliche Durchwachsungen an den Wurzelharren zweier
Marchantiaceen. Verhandl. d. bot. Vereins d. Prov. Brandenburg.
Leitgeb.
Untersuchungen iiber die Lebermoose, Heft 2, Tafel 3.
1884 Ueber Bau und Entwicklung der Sporenhaute. Graz.

Loeb.
1905 Pfliiger’s Archiv, 107.
MacDougal.
1908 The Influence of Light and Darkness upon Growth and Development.
Memoirs of the New York Botanical Garden, Vol. II.
Osterhout. ‘
1906 Onthe Importance of Physiologically Balanced Solutions for Plants.
University of California Publications. Botany, Vol. 2, No. Io.
Peirce.
1906 Studies of Irritability in Plants. Annals of Botany, Vol. XX, No.
LXxX.
Peirce and Randolph.
1905 Studies of Irritability in Alge. Botanical Gazette, No. 40.
Rosenvinge.
1889 Influence d’agents exterieurs sur l’organisation polaire et dorsiventrale
des plantes. Revue generale de Botanique. I.

Stephani.
1898 Species Hepaticarum. Bulletin de l’Herbier Boissier, Vol. VI.

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EXPLANATION OF FIGURES IN PLATES 1 AND II.

PrGea IT.

Nun W

COs

12.

15.

Diagram of a young Sporogonium of Fossombronia longiseta collected
November 1, 1906, near close of the dry season. Found in a locality of
constant moisture.

. Mature plant of Fossombronta longiseta. Wad grown allsummer. Co)-

lected January 6, drawn January 7.

. Living rhizoid of F. longiseta perforated by hyphe. >< 560.

. Hyphe passing from cells of thallus into rhizoid. > 560.

. Abnormal rhizoid of /. longiseta containing hyphe. >< 560.

. Thin-walled vesicle of the fungus associated with /. longiseta, within

stem tissue of the host. > 560.

. Hypha complex within cells of the host, #. longiseta. > 560.
. Vesicle and hyphe apparently communicating with the exterior.

560.

. (a) and (6). Formation of conidia by abstriction of hvpha of the fun-

gus infecting F. longiseta. >< 560.

. Development of sclerotia in cells of a leaf of F. longrseta. > 560.
. Perithecia-like structures developing from hyphe on the surface of

Aneura multifida major.
Uninfected cells of Aneura multifida major showing oil-bodies, o. b., and
chromatophores. XX 335.

. Infected cells of Aneura multifida major. Note the absence of oil-bodies

and small number of chromatophores. > 335.

. Three cells of Aneura multifida major showing knots of hyphe. Chlo-

rophyll and other cell contents wanting; celis, dead. >< 335.
Infection of a young plant of Aveura multifida major that has developed
from agemma. 560.

16a. Branching rhizoids of Aneura multifida major. X 80.
166. Branching rhizoids of Lepidozza attenuata. < So.

17.

18.

23

Longitudinal section of young sporogonium of Porella bolander? near
close of the dry-season. > about 50.
Portion of an infected leaf of Porella bolanderi showing chlamydospore

(c). X 560.

. Developing sclerotia within leaf cells of Porella. > 560.
. Infection of tissue of Antheceros pearsoni, showing sclerotium. 560.
- Portion of plant of VFegatella contca showing receptacle. Collected

December 28, 1906; drawn January 2, 1907. Ad. nat.
Apical end of a young Fimbriarza plant. > 560.
Young plant of Fossombronia longiseta. X 560.

All drawings except Fig. 1, 16 and 23 were made with the aid of an Abbé

camera lucida.

(50)

Ee ; ‘i ae:
ah ae) tp a
a ne r Ne my : :

PLATE II.

Was#. AcaDd. Sci., VOL. X

Proc,

EY DEL.

HUMPHR