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OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

No. 134, 175 pages, frontis., 91 figures, 26 tables December 22, 1979

The Biology and Physiology of the Living Coelacanth

Edited By

John E. McCosker and Michael D. Lagios

California Academy of Sciences,
San Francisco, California 94118

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PUBLISHED BY THE ACADEMY

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The Biology and Physiology of the Living Coelacanth

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Drawing by Robt. Day: © 1966 The New Yorker Magazine, Inc.

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

No. 134, 175 pages, frontis., 91 figures, 26 tables December 22, 1979

The Biology and Physiology of the Living Coelacanth

Edited By
John E. McCosker and Michael D. Lagios
California Academy of Sciences,
San Francisco, California 94118
CADEMy.
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E563
5
co oO
O GP
ONpED

SAN FRANCISCO

PUBLISHED BY THE ACADEMY

COMMITTEE ON PUBLICATIONS

Laurence C. Binford, Chairman
Tomio Iwamoto, Editor
Frank Almeda, Jr.
William N. Eschmeyer
George E. Lindsay

(US ISSN 0068-5461)

The California Academy of Sciences
Golden Gate Park
San Francisco, California 94118

PRINTED IN THE UNITED STATES OF AMERICA
BY ALLEN PRESS, INC., LAWRENCE, KANSAS

CONTENTS

Introduction. By MICHAEL D. LAGIOS ‘and JOHN E. McCOSKBR .........4..46-

NUMBER

Ihe

2

“-.

My story of the first Coelacanthh By M. COURTENAY-LATIMER ................

The influence of the Coelacanth on African ichthyology.
Biya Vin G ATE MESS MID sh ook G/acoks va 805 se airs a ee eee

. Inferred natural history of the living Coelacanth.

By ORINVE SMCCOSIGERS 2:..sy.0 fos8s Sees soa he 6 he wee Hote ae nite See

. The Coelacanth and the Chondrichthyes as sister groups: A review of shared apo-

morph characters and a cladistic analysis and reinterpretation.

ByaVilGHAre-D. DAGIOS SOR «tse ae ee ee Ss eee, er rae eee
Coelacanths: Shark relatives or bony fishes? By LEONARD J. V. COMPAGNO,
Wt MPAene Duy OY Mile EVAR ID ICAGIOS a... 0s © oo rs eaten rice aio ene Ee ee ee
Ventral gill arch muscles and the phylogenetic relationships of Latimeria.

JB Bek OR VA UBS BS eater ae toe kl eae Ogre Ie Mr RE RE SPREE ROAR dear GY AA Gar ob 018.60

Observations on the structure of mineralized tissues of the Coelacanth, including
the:scalessand, their associated odontodes,” By W= A. MILLER ~.525.2225- 202-2 eee
Mechanisms of osmoregulation in the Coelacanth: Evolutionary implications.

ByAROBERT We GRIPPITH andsPETERK: TiePANG®: 22555 s220.62 bee een nee
Some biochemical parameters in the Coelacanth: Ventricular and notochordal fluids.
Bye Ost RAS MIUSSE'NE infos oe oe a Sale a Dis Oho cree he ee ee eee
Chordate cytogenetic studies: An analysis of their phylogenetic implications with
particular reference to fishes and the living Coelacanth. By GUIDO DINGERKUS...

. Immunochemical and biological studies with growth hormone in extracts of pituitaries

imoMlexistine primitive fishes. By TETSUO HAYASHIDA. .o. oe oe eee

. Evolution of the creatine kinase isozyme system in the primitive vertebrates.

By SUZANNE JE. EISHER andiGREGORY S. WEUIAM, «i: 2.5 235 scr atone eee

. Amino acids and taurine in intracellular osmoregulation in marine animals.

By J. B. LOMBARDINI, PETER K. T. PANG and ROBERT W. GRIFFITH ........

. Recapitulation of the discussion at the close of the 15 June 1977 symposium on the

relationships of primitive fishes, with particular reference to the Coelacanth.
ranscribediby SUSAN BROWN and'GEORGE BROWN ..%. - 23.9332 e502 2s oes

Pages

DEDICATION

This volume is dedicated to the memory of Faith Atkins Breeden, a patient and persistent
supporter of scientific endeavors.

ACKNOWLEDGMENT

Publication of this volume was made possible by a grant
from the Charline Breeden Foundation.

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 5 pages

December 22, 1979

INTRODUCTION

By

Michael D. Lagios and John E. McCosker

California Academy of Sciences,
San Francisco, California 94118

Forty-one years ago today a most remarkable
event occurred off the coast of South Africa.
The discovery by a young woman scientist of a
steely-grey, heavily-scaled, curious living fossil
fish thought extinct for 70,000,000 years was the
stuff of dreams. It was immediately heralded as
the biological find of the century by adventurer
and ichthyologist James Leonard Brierly Smith.
Perhaps no single living organism has so dra-
matically affected the public consciousness and
scientific imagination as Latimeria chalumnae.
For scientists it provided a unique opportunity
to gaze backward at evolution, through its living
tissues, at a lineage dating to the Early Devo-
nian. For the rest of humankind, the celebration
of the coelacanth in irreverent prose by Ogden
Nash, in song by Charles Rand, through the vi-
sual arts by New Yorker cartoonist Robert Day,
and cinematographically through the *‘Monster
From the Black Lagoon,’ bears witness to the
appeal of this unicorn reborn.

In addition to the excitement generated by the
presumed antiquity of the living coelacanth was
the immediate disposition of the scientific com-
munity and the lay press to consider Latimeria
the nexus to tetrapod evolution. In 1954 Science
Newsletter wrote that “‘the coelacanth is be-
lieved to have given rise to amphibians and to
be the ‘missing link’ in man’s eventual evolution

from sea creatures.”’ It is fitting that two score
years since its celebrated discovery the coel-
acanth undergo reappraisal. The present collec-
tion of papers, the majority of which were pre-
sented in an AAAS symposium in June 1977,
provides an interpretive spectrum which will
tantalize, though not completely satisfy, devout
coelacanthophiles. Some of these works will re-
affirm the established systematic position of the
coelacanth while others are very divergent from
the prevailing interpretation, suggesting a rela-
tionship with a hypothetical paleozoic pre-
chondrichthyean group. A wide spectrum of re-
search methods and materials was used by the
various contributors in their evaluation of the
coelacanth. These range from novel re-evalua-
tions of more conventional hard calcified tissues
such as gill arches and teeth to newer ap-
proaches using soft tissue morphology, compar-
isons of the antigenic structure of peptide hor-
mones and the definition of creatine kinase
isoenzymes.

This volume is based on tissues and data col-
lected by the Expedition of the Royal Society
(1969) to Aldabra and Cosmoledo and the
French/British/American (1972) and the Califor-
nia Academy of Sciences (1975) expeditions to
Grande Comore. Popular accounts of the 1972
Expedition by Griffith (1973), Thomson (1973)

nN

OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGure |. Fishermen’s pirogues at rest in Iconi, Grande Comore.

5°
hn
a = J

;
7 i)

*
tee +s

afta

; ’ eke ‘ = <¢_
_ alte a inte DOR WE es ees.
FIGURE ; John E. McCosker and Michael D. Lagios dissecting a preserved coelacanth at the Veterinary Laboratory,
Moroni, Grande Comore. Photo courtesy of Al Giddings.

LAGIOS & McCOSKER: INTRODUCTION 3

eer

wen

FiGure 3. Members of the 1975 California Academy of Sciences Expedition (Daniel Robineau was not present). Top row:
John McCosker, David Powell, Chuck Nicklin, Sandy McCosker, and Lester Gunther. Bottom row: Al Giddings, Sylvia Earle,
John Breeden, and Michael Lagios.

=

COM RES

é * , Pe .
Ce. (OTS EXPRDITHONCDELACANTE

FiGURE 4. Postage stamp issued in honor of the 1975 Expedition.

4 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE 5.
examine the juvenile 1974 specimen and the eggs of the 1972 ovigerous female specimen. Photo courtesy of Al Giddings.

and Anthony (1976) and of the 1975 Expedition
by McCosker and McCosker (1976) provide in-
sights into the difficulties encountered by each
group such that the elusive goal of capturing,
studying and returning with a living coelacanth
remains unsatisfied. The California Academy of
Sciences Expedition, supported primarily by the
Charline Breeden Foundation, was successful in
returning with two specimens which had been
frozen soon after capture. Tissues from those
specimens were distributed to thirty laboratories
with the assistance of the Society for the Pro-
tection Of Old Fishes. Given the present politi-
cal situation in the Comores, it seems unlikely
that such research materials will soon again be-
come available, and for that reason we are even
more grateful for the support provided by gen-
erous benefactors and agencies.

{t would be asking too much from such a di-
verse field of approaches and interpretations to

Daniel Robineau and Jean Anthony of the Paris Museum and Sylvia Earle of the California Academy of Sciences

reach a consensus regarding the systematic po-
sition of the coelacanth. What we have attempt-
ed to provide for the readers is a unique oppor-
tunity to field the question for themselves. We
also feel that this volume will contribute signif-
icantly through its historical articles by Mmes.
Courtenay-Latimer and Smith and through the
comprehensive listing of direct and indirect lit-
erature citations which deal with coelacanths.

In summation, we take pride in the knowledge
that two score years thence, a more lucid view
of Latimeria is surfacing. ‘‘Coelacanth’’ has
found its place in public parlance, as fondly cited
by the late Professor JLB Smith in telling the
story of ‘‘a prominent member of the British
Parliament who, in attacking an opponent,
called him a ‘Coelacanth’ on the grounds that
from his long silence in that august assembly it
was a surprise to find him still alive,’ or as uti-
lized by Time Magazine in describing Richard

LAGIOS & McCOSKER: INTRODUCTION

M. Nixon as a “‘Coelacanth of American anti-
Communism.’ Yet the most astute observer of
the enigmatic Latimeria was the late Ogden
Nash. We are pleased to introduce this volume
with his quatrain:

It jeers at fish unfossilized

As intellectual snobs elite;
Old Coelacanth, so unrevised,
It doesn't know it’s obsolete.

LITERATURE CITED

ANTHONY, J. 1976. Opération coelacanthe. Vivre et revivre
Paventure 7. Collection ed. by A. and J. Arthaud. Librarie
Arthaud, Paris. 201 pp.

GRIFFITH, R. W. 1973. A live coelacanth in the Comoro Is-
lands. Discovery 9:27-33.

McCosker, S., AND J. E. McCosker. 1976. To the islands
of the moon. Pac. Discovery 29:19-32.

THOMSON, K. S. 1973. Secrets of the coelacanth. Nat. Hist.
82:58-65.

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 5 pages

December 22, 1979

MY STORY OF THE FIRST COELACANTH

By

M. Courtenay-Latimer

Tsitzikama, South Africa

Most ichthyologists believe that the modern
coelacanth story began in 1938 with the discov-
ery of a curious mauvy blue fish on the deck of
Captain Goosen’s boat. Yet for me, the coel-
acanth story actually started as far back as 1911,
and were it not for an island with a strange at-
traction and several men I met along the way,
the coelacanth story might not have happened.
At the age of four years I went to visit my moth-
ers maternal home on the Addo heights from
where at a certain time of the evenings the rays
of the Bird Island Lighthouse could be seen re-
flecting on the upstairs bedroom windows. Being
nervous of the rays of light, my mother drew the
curtains, and said “‘It is only the Lighthouse
rays guiding the sailors and all people sailing on
the dark seas.’’ This story of the light so im-
pressed me, that the wonder grew.

As I grew older I again visited the farm for
school vacations and always my interest in Bird
Island and the lighthouse stood out as a most
intriguing feature, more especially as the young
folk made special evening visits to a point to get
a clearer view of the island and lighthouse. But
children had to be seen and not heard and it was
not until the age of 16 that I was allowed the
privilege of joining the young people and really
watching the lighthouse rays. Now an urge

sprung up within me to visit the island. I felt I
must get to the island. But why? No lady went
to an isolated place like that!

Six years later I became the appointed curator
of the East London Museum at the princely sum
of £2 per month. I was in my seventh heaven.
I had always dreamed of working in a museum
but here too, ladies were not accepted. However
the Museum had been built for East London and
I was appointed and expected to do the best for
the official opening by the Cape Provincial
Administration on 24 September 1930.

The Museum collection was very sad; all
kinds of odd specimens and six birds sadly eaten
up by dermestid beetles. However I sorted and
cleaned and then from home brought in the Lati-
mer collections (for as a family we had amassed
some good treasures). Now as a young and very
enthusiastic worker my life was thrown into
building and collecting for the Museum. Week-
ends, public holidays and personal vacations
were put to the benefit of the Museum. I inter-
viewed fishing clubs and the manager of the fish-
ing trawlers to save material for me, and in no
time beautiful marine material was brought in.
At this stage I first met Dr. JLB Smith. He vis-
ited the Museum and was most intrigued with
the mounts I had made of the small fish which
I could handle. He as always was most encour-
aging and gave me greater inspiration to do my
very best in this work.

During the following five years I worked un-
der the guidance of Sir Henry Meir, Mr. D.
Boyce, and Dr. Ernest Gill, learning about var-

COURTENAY-LATIMER: FIRST COELACANTH

ious aspects of museum work. My desire to visit
Bird Island remained undaunted, until 1935
when I met a Mr. Patterson, Director in Chief
of Islands off South Africa. No words could ex-
press my excitement. But he was adamant and
could not give me permission to go alone to Bird
Island, BUT PROMISED if I got one other older
lady to go with me he would grant me permission
to visit Bird Island for at least a six month stay!

On my return to East London I lost no time
in persuading my mother to accompany me. But
my father refused to hear my plea. After my
perpetual begging, he finally but very reluctantly
gave his consent.

I wired Mr. Patterson and on the Ist Novem-
ber 1936 my mother and I were ready to sail on
the packet boat at 5 a.m. Then a wire arrived to
say my father was joining us. The panic that
followed was unbelievable but in the end Port
Captain Tarpey had pity on me and organized
that my father, mother and I would set sail on
the packet boat for Bird Island. No one could
believe the joy—AT LAST!

After one and a half hours sailing the Captain
of the packet boat said he was afraid the surf
was too rough and we may not be able to land.
After heaving to for an hour or two the wind
dropped, and we finally landed on the west side
of the island.

A dream come true. To mom and dad proba-
bly a very bleak outlook. I was enthralled the
gannets were nesting and their constant calls
carra! carra! carra! were almost deafening. I
was enthralled by the smell of the guano and the
joy of the colonies of Jackass Penguins that lined
the route to our residence for our six weeks stay.

Once we settled in, no time was lost in inves-
tigating the life on the island, the reefs, and the
fishing off the shores of the island. Many after-
noons found me way out at sea fishing and col-
lecting sea birds and thus it was we met Captain
Goosen of the trawler Nerine. He was interested
in my collections and finally came ashore to see
what I had collected. Thereafter I used to go out
to the trawler and collect material from their
nets.

The time dawned for my return to East Lon-
don. I had now collected 15 huge packing cases
of sea birds and marine material but how to get
them back? I appealed to the trawler Captain
who was only too happy to help me. Thus the
contact was started and after my return to East
London Capt. Goosen and a Mr. Peacock be-

came firm Museum friends, and continued col-
lecting and bringing in material which I could
cope with in my small Museum though not too
many fish were handled during this time as our
space was very limited and I had no way of
keeping the specimens fresh until I had time to
mount them.

22 December 1938 dawned a hot, shimmering
summers day. At 10:30 my newly installed
phone rang to say the trawler Nerine had docked
and had a number of specimens for me. I was
busy completing the creating of a fossil reptile
in a case, and at first thought “‘what shall I do
with fish now? So near Christmas?’ Then I con-
sidered I should go down and wish the men on
the trawler a ‘‘Happy Christmas.’’ So I rang for
a taxi and went down to the fishing wharf. It was
now 11:45 and all the men had left leaving an
old Scotsman who said *‘Lass they have all gone
but I will show you the specimens set aside for
you by Capt. Goosen.”’ I went onto the deck of
the trawler Nerine and there I found a pile of
small sharks, spiny dogfish, rays, starfish and
rat tail fish. I said to the old gentleman ‘‘They
all look much the same, perhaps I won't bother
with these today’’; then, as I moved them, I saw
a blue fin and pushing off the fish, the most beau-
tiful fish I had ever seen was revealed. It was 5
feet long and a pale mauvy blue with irridescent
silver markings. *‘What is this?’’ I asked the old
gentleman. “‘Well lass,’ said he, ‘‘this fish
snapped at the Captain’s fingers as he looked at
it in the trawler net. It was trawled with a ton
and a half of fish plus all these dogfish and oth-
ers.’ ‘Oh’ I said, “‘this I will definitely take to
the Museum, but I shall not worry with the
rest.”’ I called Enoch, my native boy, and with
a bag we had brought down he and I placed it
in the bag and carried it to the taxi. Here, to
my amazement the taxi man said ‘‘No stinking
fish in my taxi!’ I said ‘‘Well you can go, the
fish is not stinking—I will call another taxi.”
With that, he allowed us to put it into the boot
of the taxi.

On arrival at the East London Museum I
dumped it onto the table vacated by the fossil
I had just assembled. And then began a library
search of our meagre literature on fish. I
searched the library. At the back of my mind I
had written lines whilst at school on a ganoid
fish—this kept ticking in my mind. Was it? No
it could not be! Was it a lungfish gone balmy?
No it could not be.

8 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE |.

Then I decided the only person who would
help me without a laugh would be Dr. JLB Smith
of Rhodes University, Grahamstown. But to
preserve it? What must I do. Now 3 p.m., the
blue had faded, becoming a dirty grey. I phoned
one of my Trustees, Mr. W. Sargent, who said
he would lend me a hand truck. I sent Enoch to
fetch it and we then took it through the streets
of East London to 19 Nahoon View Road, where
a friend, Mr. R. Center, practised taxidermy. He
was not good at fish mounting, but I reckoned
he would be able to advise and help me for prior
to this I had asked my chairman Dr. Bruce Bays
to place it in the mortuary at the hospital. I ap-
proached the cold storage. But no one was anx-
ious to have a stinking fish. It was NOT stinking;
it was fresh and beautiful, but it was a blazing
hot day so I had to work fast. Mr. Center was
kindness itself. We got it onto a table and placed
bands of cloth soaked in formalin and securely
wrapped newspaper over it.

I wrote Dr. Smith and sent a sketch, hoping
to hear from him in a day or two. But no news.
24th still no news. Christmas day and no reply.
At 5 p.m. on the 26th, I visited Mr. Center. In
great fear, we opened the casing and found the
formalin had not penetrated through the heavy
scales. We sat and considered what should be

‘Almost armour like”’ scales of the coelacanth, described by Miss Courtenay-Latimer in her letter of 23 December
1938 to JLB Smith. Photo courtesy of Al Giddings.

done. Mr. Center said the outer casing was hard.
He considered he could skin it and commenced
straight away. The flesh was bluish green and
very oily. He cut the tongue away and said I
best keep it until he got a form made. I returned
to my home, very disappointed and worried, for
I felt the fish was an important discovery and
needed proper care.

On 3 January 1939, at 10 a.m. a wire from Dr.
Smith arrived: ““MOST IMPORTANT PRE-
SERVE SKELETON AND GILLS = FISH
DESCRIBED,”’ I trotted off to Mr. Center but
alas the inners had been dumped. We went
through the refuse dumps but no luck. Mr. Cen-
ter had saved what he could of the fish which
was the entire outer casing and the hard boney
tongue.

Letters and telephone messages went back
and forth. A Mr. Kirsten who did a great deal
of photography for the Museum had taken pho-
tographs on the morning of the 22 December for
me; but when I contacted him he said in taking
the spool from his camera it slipped and the en-
tire spool was spoilt. My Board of Trustees were
not interested.

It was 16 February 1939. We had been having
terrific rains, and at 9 a.m. Dr. JLB Smith
phoned the day before he came to the East Lon-

COURTENAY-LATIMER: FIRST COELACANTH

FIGURE 2.

don Museum. As he walked into my small office
Where I had the now mounted fish he said, “I
always KNEW somewhere, or somehow a prim-
itive fish of this nature would appear.”

A reporter from the Daily Dispatch came in
with a photographer. At first he, Dr. Smith gave
them no satisfaction. Then he sat down at my
desk and told them about the wonder of this
find. But said he: “‘No photographs.”? ‘‘Oh”’
pleaded I, ‘‘all the photographs taken after being

The author and Professor Smith viewing the unpacking of the second coelacanth at Grahamstown, South Africa,
31 December 1952. Photo courtesy of JLB Smith Institute of Ichthyology.

brought to the Museum on 22 December have
been lost, please could they take one shot.”
Reluctantly he agreed, and how wise he had
been, for that photographer sold those photo-
graphs to every newspaper in the world plus the
Illustrated News, and when we, the East Lon-
don Museum, wanted a copy, he made us pay
Gon

The news of the discovery was now world
wide and phone calls were received from all over

10 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, NO. 134

the world. One museum phoned to state that I
was to admit that the fish had been dragged up
from a muddy grave! When I refused to admit
to this they said ‘‘What proof have you?”’ I stat-
ed ‘‘When I took the fish off the trawler it was
mauvish blue in colour but by 3 p.m. had faded
to a dirty grey, which proved the pigments had
died.”

A public viewing for one day was arranged
and people flew from all over in a single day and
evening we had over 20,000 visitors to see this
wonderful discovery. My Board of Trustees now
became extremely interested and were anxious
to get a sale for the specimen. This shocked me.
So once again I appealed to my friend and sup-
porter Dr. JLB Smith who now took the entire
burden of the fish and its discovery onto his
shoulders. The specimen was packed and sent
to his special care at his private residence,
where we felt that he should be in sole charge
and have the honour to be able to work on this
discovery.

The public of East London, now aroused,
plied the Board of Trustees with many whys and
wherefores and stirred up great irritation. On the

return of the specimen the coelacanth was
placed on an all-day view, where the public
streamed to view it. Thereafter it was taken to
the South African Museum to be properly
mounted by Mr. Drury, the taxidermist at the
South African Museum, Cape Town.

It was not until January 1940 when the re-
mounted specimen together with a coloured cast
returned to the East London Museum, where it
remains today as one of the Museum’s chief ex-
hibits.

This story is one of the most astounding rec-
ords of a woman’s intuition, for:

had I never gone to Bird Island;

had I never met Dr. JLB Smith who, of all the
scientists I met as a young girl struggling
with meagre funds in a small Museum, al-
ways gave encouragement and never criti-
cism; and

had I not gone to the wharf to wish the men
a Happy Christmas,

there never would have been a coelacanth dis-
covery in South Africa, on 22 December 1938.

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 6 pages

December 22, 1979

THE INFLUENCE OF THE COELACANTH ON
AFRICAN ICHTHYOLOGY

By

Margaret M. Smith

J.L. B. Smith Institute of Ichthyology,
Grahamstown, South Africa

When the Coelacanth was pulled up onto the
deck of a trawler on December 22nd 1938, not
only did it rock the zoological world but it also
had a profound effect on South African ichthy-
ology, mainly through the man who was pre-
pared to make this staggering announcement
when all his senses reeled at the impossible
come true.

Before the appearance of the living coel-
acanth, the study of fishes in South Africa had
begun with Andrew Smith during the first half
of the nineteenth century. At the beginning of
the twentieth century that remarkable and in-
defatigable zoologist J. D. Gilchrist described
numerous deepsea forms, and together with W.
W. Thompson published a number of excellent
checklists of South African fishes with extensive
synonymy and bibliographic references.

A fateful faux pas then changed the direction
of ichthyological research when Gilchrist forgot
a dinner engagement with the then Director of
the South African Museum. The latter was so
incensed that he brought Keppel H. Barnard out
from England and directed him to work on the
South African marine fishes! To this end he
bought every book available on sea fish so that
the South African Museum obtained almost
every important treatise ever produced on ma-

rine fish. When Barnard completed his mono-
graph in 1927 he was, to quote his own words,
‘sick of fishes.’’ He then turned his fine intellect
to elucidate the problems of the South African
crustacea and mollusca, leaving the icuinyuiog-
ical field wide open.

Into this, timidly publishing his first fish paper
in 1931, came a chemist and dedicated research
worker, James Leonard Brierly Smith. Living in
Grahamstown, he soon came to serve the four
nearby museums: Port Elizabeth, Grahams-
town, Kingwilliamstown and East London (as
honorary curator of fishes), and by 1938 had es-
tablished himself overseas as an up-and-coming
ichthyologist.

When, therefore, JLB announced the capture
of a living coelacanthid fish, a fish believed ex-
tinct for over 50 million years, it was not sur-
prising that while his statement was immediately
accepted overseas, at home it was met with
complete disbelief!

I remember so well our return to Grahams-
town. The announcement with the photograph
had already appeared in the East London Daily
Dispatch. We went straight to the Albany Mu-
seum to lay this incredible news before the man
who encouraged JLB to work on fishes. We
were met with a stoney face—how could JLB

12 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGuReE 1.

have made such a terrible mistake. ‘‘Have you
seen its picture?’” asked my husband. ‘Yes, of
course’’ was the reply—‘‘that’s just a Kob (Ar-
gyrosomus hololepidotus, family Sciaenidae)
with a regenerated caudal.’’ JLB assured him
that it was no such thing. He’d caught and han-
died thousands of Kob during his lifetime and
this fish was far removed from being a Kob!
Nothing would convince him and we left.

The next day JLB met Dr. Liebenberg, a bot-
anist who worked at the Albany Museum and an
old friend. He was most upset, placed his hands
on my husband’s shoulders and said ‘‘What on
earth made you do this dreadful thing?’’ ‘‘What
dreadful thing, Lieb?’ ‘‘This coelacanth non-
sense’ was the reply. ‘‘You’ll never again be
able to hold up your head in any scientific com-
munity.’ “‘But it is a Coelacanth!’’ he insisted
“No, man, it can’t be. Old H— says it isn’t, and
if he says it isn’t then it can’t possibly be one.”’
He was inconsolable, and it wasn’t JLB who
convinced him it was a Coelacanth.

However, in America, the ichthyologists were

JLB Smith holding a venomous stonefish during field collections at Pinda.

a lot less skeptical. “If JLB Smith says it is a
Coelacanth, then it must be. I know his work
well and he'd never give a wrong diagnosis on
a matter like this.”’

One of the immediate results of the capture
was a flood of correspondence. The most fas-
cinating came from palaeontologists from all
over the world. Not only did letters from ich-
thyologists pour in, but they also sent their re-
prints with queries or nomenclatorial advice.
The palaeontological literature we received at
that time still forms an important part of the In-
stitute’s library and as such is available to
workers all over South Africa.

JLB decided to follow no particular school as
far as the nomenclature of the bones was con-
cerned. He considered it right and proper that
a non-palaeontologist, and therefore someone
completely outside the “‘battle of the names,”
should describe the fish. So he gave the bones
numbers, enabling subsequent workers to de-
cide exactly what names they should bear.

Many years later when the Danish coelacanth

SMITH: INFLUENCE ON AFRICAN ICHTHYOLOGY

PREMIO £100

RECOMPENSE

Examine este peixe com cuidado, Talvez lhe dé sorte. Repare nos dois rabos que possui e nas suas
estranhas barbatanas. O unico exemplar que a ciéncia encontrou tinha, de comprimento, 160 centimetros. Mas ja
houve quem visse outros, Se tiver a sorte de apanhar ou encontrar algum NAO O CORTE NEM O LIMPE DE
QUALQUER MODO — conduza-o imediatamente, inteiro, a um frigorifico ou peca a pessoa competente que
dele se ocupe. Solicite, ao mesmo tempo, a essa pessoa, que avise imediatamente, por meio de telgrama, o profes-
sor J. L. B, Smith, da Rhodes University, Grahamstown, Uniao Sul-Africana.

Os dois primeiros especimes serao pagos a razao de 10.000$, cada, sendo o pagamenty garantido
pela Rhodes University e pelo South African Council for Scientific and Industrial Research. Se conseguir obter
mais de dois, conserve-os todos, visto terem grande valor, para fins cientificos, « as suas canseiras serao bem
recompensadas.

REWARD

COELACANTH

Look carefuliy at this fish. It may bring you good fortune. Note tk
and the fins. The only one ever saved for science was 5 ft (160 cm.) long. Others
you have the good fortune to catch or find one DO NOT CUT OR CLEAN IT ANY WA* : get 1t whole at
once to a cold storage or to some responsible official who can care for it, and ask bk o notify Professor
J. L. B. Smith of Rhodes University Grahamstown, Union of S. A., immediately by tel aph. For the first
2 specimens £100 (10.000 Esc.) each will be paid, guaranteed by Rhodes University ar hy the South Afri-
ean Council for Scientific and Industrial Research. If vou get more than 2, sav: the._ cil, as every one is
valuable for scientific purposes and you will be well paid.

uliar double tail,
> been seen. Tf

Veuillez remarquer avec attention ce poisson. I] pourra vous apporter bonne chance, peut étre.
Regardez les deux queuex qu'il posséde et ses étranges nageoires. Le seu] exemplaire que la science a trouvé
avait, de longueur, 160 centimétres. Cependant d’autrés ont trouvés quelques exemplaires en plus.

Si jamais vous avez la chance d’en trouver un NE LE DECOUPEZ PAS NI NE LE NETTOYEZ
D'AUCUNE FACON, conduisez-le immediatement, tout entier, a un frigorifique ou glaciére en Gemandui a une
personne competante de s’en occuper. Simultanement veuillez prier a cette personne de faire part telegraphi-
quement a Mr. le Professeus J. L. B. Smith, de la Rhodes University, Grahamstown, Union Sud-Africaine,

Le deux premiers exemplaires seront payés a la raison de £100 chaque dont le payment est ga-
ranti par la Rhodes University et par le South African Council for Scientific and Industrial Research.

Si, jamais il vous est possible d’en obtenir plus de deux, nous vous serions trés grés de les con-
server vu qu’ils sont d’une trés grande valeur pour fins scientifiques, et, neanmoins les fatigues pour obtan-
tion seront bien recompensées.

FiGure 2. Reward poster circulated along the east African coast which resulted in the identification of the 1952 coelacanth

by Capt. E. E. Hunt.

palaeontologist, Jgrgen Nielsen came to South
Africa in 1953 to see the second coelacanth, we
suggested he should visit East London, just over
100 miles away to see the first coelacanth.
‘‘Quite unnecessary” he said, “‘the descriptions
and photographs of the various structures in
your monograph on the first coelacanth are more
than adequate. I have no need to see them for
myself.”

South Africa had been rocked by the discov-
ery of Latimeria. Anglers and trawlers alike
were looking out for strange sea creatures.
Specimens flowed into museums. Everyone was
aware of Smith of Grahamstown. One of the best
material rewards was his being made an Hon-
orary Foreign Member of the American Society
of Ichthyologists and Herpetologists, and as
such received the journal Copeia. American ich-

14 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE 3.

thyologists not only sent him rare books but
helped fill in the earlier parts of Copeia. The re-
sult was that for many years we had the only set
of Copeia in Africa. So herpetology also bene-
fitted!

This, the true turning point in his career, was
obscured by the second world war. For the next
eight years, until the war ended, he continued
to teach chemistry, do research work in chem-
istry and produced two chemistry text books
during that time.

In 1946 the South African Council for Scien-
tific and Industrial Research came into being,
and JLB Smith was made one of its first three
senior bursars. The coelacanth had done its
work well, and the University released Smith to
concentrate on his ichthyological work, to pro-
duce THE SEA FISHES OF SOUTHERN AF-
RICA and to hunt for the home of the living
coelacanths. It is of interest to note that Lie-
benberg, the botanist who was so upset about
JLB’s announcement of the capture of the first
coelacanth was an important link in the chain

us

Captain E. E. Hunt, the discoverer of the second coelacanth, aboard his vessel at Dzaoudzi, Isle Mayotte. Two
weeks later this craft was destroyed by a cyclone.

~
&

ey

that eventually invited JLB to undertake the
writing of THE SEA FISHES OF SOUTHERN
AFRICA. He knew JLB had at one time started
writing a book to aid anglers to identify the fish-
es they caught. I wonder how much the East
London coelacanth added to this.

By the end of the war there was little doubt
that the East London coelacanth was a stray.
Most of the “‘wise’’ scientists concluded that it
had come from the great depths, the Danes even
mounted a ‘‘deep sea’? round the world expe-
dition to look for coelacanths. They amassed a
wealth of wonderful material, but no coel-
acanths. Even though they passed close to the
Comoro Islands, they probably fished in waters
too deep for them! Smith, however, maintained
that the coelacanths lived among rocks, at
depths able to be reached by hook and line and
in areas remote from biologists. As an angler he
was even able to predict how a coelacanth would
play when once hooked. He deduced that the
East London specimen had drifted down on the

SMITH:

INFLUENCE ON AFRICAN ICHTHYOLOGY

wt —_/ im a* a‘

Meare

FiGure 4. JLB Smith and ‘‘Malania anjouanae,” the second coelacanth specimen, 29 December 1952. Captain Hunt is at
JLB’s right. Monsieur P. Coudert, Governor of the Comores, is at his left.

southward flowing Mozambique—Agulhas Cur-
rent. He proposed that it must have come from
somewhere south of Cape Delgado where the
great south equatorial current divides to wash
the coast of east Africa, where one branch
swings northwards up the Kenya coast and the
other southwards to South Africa.

When the mammoth work THE SEA FISHES
OF SOUTHERN AFRICA was completed in
1949, JLB and I set off on a series of expeditions
up the east coast of Africa to seek the home of
the coelacanths. We also were interested in
studying the tropical fishes, for the major re-
cruitment of the South African fauna to this day
comes down the Mozambique—Agulhas Current.
These expeditions were wonderful training times
for me. In 1952 I started using diving techniques
to collect in subtidal areas. One who has never
dived cannot appreciate the magic of tropical
reefs; the coral with all its attendant animals

bathed in clear warm blue water is a marvelous
sight.

By the time we tracked the coelacanth home
to the Comoro Islands, I knew most of the shal-
low-water fishes that flitted in and out of the
coral gardens. We collected, photographed,
painted and preserved fish specimens as we
slowly explored the virgin reefs north of Beira
to Kenya.

This resulted in a steady stream of publica-
tions from JLB’s pen. Soon ichthyologists the
world over began to realise the wealth of marine
life along the east coast of Africa. Small feeble
fishes from as far away as the Philippines were
found in east African waters. Closely related
species to those from Japan were discovered in
the Mozambique channel. Our publications
eventually caused an American ichthyologist to
write that it would seem the great Indo-Pacific
was just one little puddle after all!

16 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGure 5. The author scrubbing the second coelacanth outside of the Department of Ichthyology, Rhodes University.

The eventual discovery of the Comoro Islands
as the home of the coelacanths did not imme-
diately stop all expeditions. By now the need to
collect tropical marine species even further
afield spurred us on to work at the islands north
of Madagascar—from the Seychelles down to
the Aldabras—and twice again along the Mo-
zambique coast before JLB Smith called a halt
to devote the remainder of his life to publishing
monographs and smaller papers on the material.

In 1968 at the age of 70, ‘‘coelacanth’’ Smith
died. During his lifetime the coelacanth had riv-
etted the attention of the zoological world on
South Africa. It had aided the establishment of
the Department of Ichthyology at Rhodes Uni-
versity so that JLB Smith could devote all his
time to his beloved fishes. Ichthyological and
palaeontological books poured into his library
now safely incorporated in the Institute’s li-
brary. The search for the coelacanth brought
both of us collecting among reefs never before

visited by any scientists, and a knowledge of
western Indian Ocean fishes that will stand me
in good stead for the rest of my life.

The finding of the home of the coelacanths
caught the imagination of the world and added
considerably to Smith’s reputation, so firmly es-
tablished by the identification of the East Lon-
don coelacanth. The foyer of the new building
that houses the JLB Smith Institute of Ichthy-
ology has been specially designed to house an
extensive and well documented permanent ex-
hibition of the story of the coelacanth. It starts
400 million years ago, and proceeds first to a full
scale model of the East London coelacanth and
finally to a special glass tank displaying the
““second’’ coelacanth. It is fitting that so many
visitors to the quiet little city of Grahamstown
find their way to pay homage to, and to stand
gazing in awe at, the story of the fish that has
done so much for South African ichthyology.

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 8 pages

December 22, 1979

INFERRED NATURAL HISTORY OF THE
LIVING COELACANTH

By

John E. McCosker

California Academy of Sciences,
San Francisco, California 94118

INTRODUCTION

It is patently presumptuous to describe the
natural history and behavior of a living fish
which, though from a lineage traceable to the
Upper Devonian, has never been observed in a
healthy living condition. To date more than 88
specimens of the living coelacanth, Latimeria
chalumnae Smith, have been captured, but with
the exception of brief observations of dying
specimens made by Millot (1955), Locket and
Griffith (1972), and a British Broadcasting Com-
pany photographer (D. Attenborough, pers.
comm.), most of the knowledge concerning the
behavior of this species is based upon inferential
evidence. To that body of knowledge I will
herein contribute additional data gathered dur-
ing the 1975 California Academy of Sciences
Coelacanth Expedition and the subsequent
study of recent and older museum specimens of
Latimeria. Other data collected by researchers
working at Grande Comore and previously un-
published are included here and gratefully ac-
knowledged.

With the exception of the first known speci-
men of Latimeria, all subsequent captures have
been made off the islands of Grande Comore and
Anjouan in the Comoran archipelago. The Com-
oro islands have undergone dramatic political

upheaval since our 1975 expedition. The over-
throws of the French colonial government, the
Abdallah government, the Soilih government,
and the Denard government have closed the
country to western expeditionary groups. Dur-
ing the Soilih government all official records
were destroyed (Anon. 1978) including, as I
have ascertained, all recent coelacanth catch
data. I am therefore including catch record data
which were provided me by the Department of
Production in Moroni in 1975, through inter-
views with Comoran fishermen and their chief
Mohammed Ali Chabane, and through subse-
quent correspondence with Said Bacar Moussa
of Mitsamouli, Grande Comore. These data
(Appendix 1), supplement the coelacanth catch
records tabulated by Millot et al. (1972).

OCEANOGRAPHIC SETTING OF
COELACANTH CAPTURES

The restriction of coelacanth captures since
1938 is probably a result of oceanographic and
sociological conditions unique to Grande Co-
more and Anjouan. Grande Comore and An-
jouan are high volcanic islands which lack a fish-
able fringing reef and inner lagoon; Grande
Comore and Anjouan are rare environments in
that the submarine topography of most other

18 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES. No. 134

Itsandra

. Maogueni

_N'Gouni Volcano

M' Bachili

*_i:Moindzaza
Volcano

} Mitzoudje

FiGuRE 1.
acanth capture cites identified. Soundings are from Forster
(1974).

Bathymetric map of Grande Comore with coel-

nearby volcanic coastlines has been smoothed
by sedimentation and/or coral growth along their
slopes (Forster 1974). Whereas the shallow la-
goons of other Indian Ocean islands are pre-
ferred fishing grounds, their absence at Grande
Comore and Anjouan has necessitated that fish-
ing be done from the deep reef edge at depths
of 20-200 m. Another contributory factor is the
Comoran fishery for the deep-reef associated
oilfish, Ruvettus pretiosus, a fish of special sig-
nificance to Islamic culture because of its pur-
ported antimalarial properties when used as a
salve. The submarine topography of Grande Co-
more is particularly sheer along the southwest
coast except for that along the village of Iconi
where a small fan spreads seaward from the
N’Gouni volcanic rim (Fig. 1). This has been the
primary site for coelacanth capture (16 of 86
specimens), probably a result of the proximity
to Iconi, the major fishing village of Grande Co-
more, as well as the favorable oceanographic
conditions which exist there.

A popular misconception exists concerning

the depth of capture of Latimeria. Coelacanths
are not bathypelagic as is often suggested, and
have been captured between depths of 70 and

20,
15}
Latimeria
caught 10:
5
O
100 200 300 400 500 600
depth (m)
FiGURE 2. Reported depth of capture of 65 coelacanths.

600 m. It is quite likely that Latimeria prefers
a habitat of 200 m or more but occasionally
makes feeding forays into shallower water, par-
ticularly during and about the dark of the new
moon phase. It is my impression that the depth
of capture recorded by Millot et al. (1972) for
many coelacanths is in error and exaggerated
toward greater depths. Supporting evidence is
based on interviews made by the author and
Sandra McCosker (McCosker and McCosker
1976) of Comoran fishermen and of M. Francis
Debuissy, then Chief Veterinarian of the De-
partment of Production of the Comores. Native
fishermen employ a hand-line fishing method
which, due to currents, suspends the line
obliquely in the water column. The actual length
of line is by necessity considerably greater than
the actual depth of capture. This was verified by
1975 Expedition members by accompanying
Iconi coelacanth fishermen onto the fishing
grounds and comparing their opinion of depth
with the actual depth as measured by scuba div-
ing. Francis Debuissy informed us that Comoran
fishermen often erroneously report their dis-
tance from shore as the depth of capture, such
that a fisherman fishing one-half kilometer from
shore will report that as a depth of 500 m.

Presented in Figure 2 are combined data from
Millot et al. (1972: fig. 4) and 15 subsequent cap-
tures. The majority of fishes are purportedly
captured between 150-300 m, which probably
reflects the apparent depth of the oilfish-catching
effort. The 100-300 m bathymetric horizon
forms a narrow band | km or less in width
around Grande Comore.

TEMPERATURE AT DEPTH OF CAPTURE

It has been suggested that Latimeria inhabits
submarine caves wherein freshwater aquifers

McCOSKER: COELACANTH NATURAL HISTORY

FIGURE 3.

seep (J. Anthony, pers. comm.; Forster 1974).
Submarine recesses were observed by expedi-
tion members who, while scuba diving, discov-
ered cool, freshwater aquifers between depths
of 30 and 70 m. Forster (1974), citing the geol-
ogist B. G. J. Upton, described the geologic con-
dition responsible for this phenomenon:

Although the basaltic rock is mainly hard
and impervious there are porous clinkery
horizons and longitudinal holes within the
lava flows, both of these systems would
make good aquifers. It is therefore likely
that the considerable rainfall on the high
ground of M’Kartala, which has no per-
manent surface streams, could provide a
supply with sufficient pressure to flow out
even at 200 m depth.

It is important to note that the coelacanth catch
is increased during January, February, and
March (see Fig. 5) in spite of the reduced fishing
effort due to the monsoon rains. A fairly good

Coelacanth fisherman with handline.

temporal correlation exists between the rainfall
at Maoueni, Grande Comore, and the incidence
of coelacanth capture. Maoueni, is located on a
broad lava fan which flows onto the SE coast.
This correlation is in agreement with the drought
of 1975/1976 and the unfortunate lack of coel-
acanth captures during January—March 1975, a
period of normal monsoon rainfall when the fish-
ing effort was increased several fold on behalf
of our expedition (McCosker and McCosker
1976). Fewer coelacanths than normal were cap-
tured throughout Comores during the drought
condition which existed across the SE African
subcontinent in 1975-1976.

The temperature at the depth of coelacanth
capture appears to be slightly below surface
temperatures. The majority of coelacanth cap-
tures have been near or above the deep ther-
mocline which flanks the steep island slope.
Anjouan data from Domoni and Mutsamudu
(Menaché 1954) identified thermoclines between
150-200 m, with temperatures between 23—26°C

20 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE 4.

in the upper 100 m and 15°C below 200 m. Un-
published bathythermograph data collected by
the Vancouver Aquarium at Iconi, Grande Co-
more, convey a similar deep and stable ther-
mocline at 115-125 m, and temperatures of 15—
17°C below 200 m.

It is important to note that the temperature at
depth, measured by a suspended bathythermo-
graph, is not necessarily the temperature regime
inhabited by Latimeria. The freshwater aquifers
are generally cooler than the surrounding salt-
water and might, for that reason, be selected for
by Latimeria to permit them to extend their
range of thermal tolerance into the relatively
richer shallow reef area for feeding.

COELACANTH PREY ITEMS

The dentition, jaw structure, and reduced gut
length of Latimeria are adaptations for a pred-
atory feeding behavior. Published records of gut
contents are limited to entire cuttlefish (Mc-

Allister 1971) and the lanternfish Diaphus me-
topoclampus (Millot and Anthony 1958). The
examination of recent specimens has resulted in

Coelacanth fishermen fishing along the dropoff for oilfish and coelacanths near Itsandra, Grande Comore.

additional observations of prey species, allowing
further insight into coelacanth behavior.

The large female Latimeria at the United
States National Museum of Natural History
(C49, 165 cm, USNM 205871) contained the gill
arches, hyoid, dentary, cleithrum and several
vertebrae of a Stout Beardfish, Polymixia noblis
(family Polymixiidae). A comparison of the re-
mains with the jaws of intact P. noblis (USNM
202120) indicated that the prey specimen was ca.
17-18 cm in standard length. Wheeler (1975)
states that P. noblis is a near-bottom dweller,
widely distributed in all tropical oceans at
depths of 183-640 m.

The male Latimeria at the California Acad-
emy of Sciencesa(@79; 108 ‘em, 1CASP 33111)
contained two intact, but partially digested,
specimens (7-8 cm) of deepwater snappers,
Symphysanodon sp. (family Lutjanidae). I am
advised by G. D. Johnson that they are the sec-
ond known Indian Ocean specimens and prob-
ably represent a new species. Elsewhere,
species of Symphysanodon are from moderate
depths (119-476 m) around islands (Anderson
1970) and live on or just above the bottom.

McCOSKER: COELACANTH NATURAL HISTORY

heenad

Latimeria
caught

S ON OD J F
month

FIGURE 5.

MAM JS J A

Correlation of mean monthly rainfall at Maoueni, Grande Comore, during 1961-1970 with the monthly capture

of coelacanths (82 specimens) in the archipelago. Figure by K. O'Farrell.

No identifiable remains were discovered with-
in the stomach of the small, male Latimeria at
the Scripps Institution of Oceanography (C78,
103 cm, SIO 75-347). Small, apparent fish frag-
ments, including otoliths and scales, were found
in the spiral valve by C. L. Hubbs.

The adult, male Latimeria at the California
Academy of Sciences (C59, 122 cm, CAS 24862)
contained two specimens of deepwater cardi-
nalfishes (family Apogonidae), Coranthus poly-
acanthus. The specimens, 12—14 cm in standard
length, were partially digested, but conclusively
identified by G. D. Johnson on the basis of their
peculiar osteology. Coranthus is monotypic and
was previously known only from two deepwater
(150 m) specimens from Réunion Island (Fraser
1972).

The above-listed prey species are typical in-
habitants of insular deep reefs, associated with
but living slightly above the substrate. All prey
species appeared to have been swallowed entire,
presumably the result of a grouper-like rapid-in-

halation feeding method. It is important to note
that open water (non-reef associated) bathype-
lagic or mesopelagic prey items have not been
observed among coelacanth gut contents.

CONCLUSIONS

An hypothetical life history of Latimeria cha-
lumnae can be constructed on the basis of its
anatomy, diet, catch records, and Comoran
oceanographic and meteorological data. On that
basis, it appears that Latimeria behaves like a
large, reef-associated piscivorous grouper.

The ovoviviparous reproductive system is a
specialization related to the retention of urea,
not unlike that of the ovovivaparous elasmo-
branchs. The large 163 cm female of 5 January
1972 contained 20 eggs, 8.5—9 cm in diameter
and 300-344 g in weight (Millot and Anthony
1974). Although it is possible that some may
have aborted during capture, it appears that they
represent the normal complement of a pregnant
female. The only known embryos were near

22 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

term and indicate that length at birth is more
than 32 cm (Smith et al. 1975). The estimated
gestation period is approximately 13 months (not
unlike many elasmobranchs) with births pre-
sumably occurring in February (Smith et al.
1975; Hureau and Ozouf 1977). The juvenile
Latimeria are probably predatory as indicated
by their dentition and jaw structure. The allo-
metric growth of the upper jaw and head length
(McAllister and Smith 1978) would assist the
predatory behavior of young fish. The smallest
Latimeria captured on hook and line is 42.5 cm
long and weighs 800 g. Its age, first estimated to
be 3% years by Anthony and Robineau (1976),
has been reinterpreted to be one year or less
(Hureau and Ozouf 1977). Age and growth es-
timates made from scale analysis suggest that
the largest females (180 cm) are nearly 11 years
old. It appears that females attain a larger length
and weight than males (McAllister and Smith
1976; and my data), a condition shared by cer-
tain sharks and probably related to the repro-
ductive commitment to ovoviviparity and preg-
nancy.

The limited geographic distribution of Lati-
meria must in some way be related to the re-
duced vagility which accompanies its live-bear-
ing reproductive mode. The lower depth limit of
Latimeria distribution has not been delimited,
but it is quite likely that the seamount chain be-
tween the Comores and the African coast is
traversable by juveniles and/or adults. The
chance discovery of a Latimeria off South Af-
rica in 1938 has not been repeated, in spite of an
actively continuing and reasonably informed
fishery. It is likely that coelacanths exist uncom-
monly along the offshore seamounts and banks
of the western Indian Ocean, but this has not
been confirmed due to the capture difficulties
and the lack of a prolonged exploratory fishing
effort. Their presence might most effectively be
explored using shallow depth submersibles.

The seasonality and lunar periodicity of coel-
acanth capture indicates that their behavior and/
or presence in shallow water fluctuates. Presum-
ing that Latimeria presence in shallow water is
affected by rainfall-fed submarine aquifers, it
seems likely that the fat-investment of the swim-

bladder is an adaptation which assists vertical
migration. Dead, intact specimens are slightly
denser than seawater. The high, extracellular

lipid and wax ester content of the muscles com-

pensates somewhat for the lack of swimbladder
function (Nevenzel et al. 1966). The fish is there-
fore slightly negatively ‘“‘buoyant’’ (not unlike
large groupers, cirrhitids, and blennioids), al-
lowing it to perch on a reef platform and lunge
short distances to capture prey items. Its body
shape and fin size and location are adapted for
such a feeding method. The intercranial mobil-
ity, subcephalic musculature, and jaw angle
(Thomson 1966, 1970, 1973; Alexander 1973)
also contribute to rapid prey capture and en-
gulfment.

The coelacanth eye is adapted to moderate
depths in clear, tropical waters. The retina pos-
sesses numerous, densely-packed rods; cones
(single type) are very rare and possess a single
oil droplet (Ali and Anctil 1976). The visual pig-
ment maximum absorbance is at 473 nm (Dart-
nall 1972). The large, nearly color-blind eye is
therefore adapted to low light levels (indicative
of a primarily nocturnal activity pattern?) and
similar in habit and structure to elasmobranchs
which occupy a similar habitat (Millot and Ca-
rasso 1955). The relatively large eye of the em-
bryos and juvenile specimen evidence an allo-
metric growth (McAllister and Smith 1978)
which presumably would allow young fish to
occupy the same photic horizon as adults.

If the natural history of the living coelacanth
is as I have inferred, then it is quite likely that
Latimeria is amenable to aquarium captivity. Its
lack of a functional swimbladder would allow its
existence at ambient pressure and a minimal re-
duction in temperature and light level might be
the only modifications necessary. I look forward
to the solution of the political problems within
the Comores so that an expedition may be
mounted and a coelacanth might finally be cap-
tured in a living, healthy condition. At that time,
Latimeria will be given an opportunity to agree
or disagree with the life history portrait that its
students have hypothesized.

ACKNOWLEDGMENTS

Many individuals have assisted in this study.
I am particularly grateful to: Jean Anthony and
Daniel Robineau, Paris Museum of Natural His-
tory, for allowing me to examine coelacanth
specimens; Francis Debuissy, then of the Com-
oran Department of Production, for assisting me
during the 1975 Expedition and providing infor-
mation about coelacanth fishermen; the Vancou-

McCOSKER: COELACANTH NATURAL HISTORY

ver Aquarium, for permission to cite their un-
published temperature data; G. David Johnson,
then of the Scripps Institution of Oceanography,
for identifying coelacanth prey items; William
N. Eschmeyer, California Academy of Sciences,
and the staff of the U.S. National Museum of
Natural History, for assistance with specimens
in their fish collections; and many citizens of
Grande Comore for their hospitality, informa-
tion, and assistance during and since the 1975
Expedition. Particular thanks are due President
Ahmed Abdallah, Prince Nacr-Ed-Dine, Chief
Mohammed Ali Chabane, Abdallah Said Omar,
and Said Bacar Moussa.

LITERATURE CITED

ALEXANDER, R. M. 1973. Jaw mechanisms of the coelacanth
Latimeria. Copeia 1973(1):156—-158.

Ati, M. A., AND M. ANcTIL. 1976. Retinas of fishes. Spring-
er-Verlag, Berlin. 284 pp.

ANDERSON, W. D. 1970. Revision of the genus Symphysan-
odon (Pisces: Lutjanidae) with descriptions of four new
species. Fish. Bull. 68:325—346.

ANONYMOUS. 1978. A man and his dog. Time Magazine, 21
August, p. 37.

ANTHONY, J., AND D. ROBINEAU.
acteres juveniles de Latimeria chalumnae. C. R. Acad. Sci.
283: 1739-1742.

DaRTNALL, H. J. A. 1972. Visual pigment of the coelacanth.
Nature 239:341-342.

Forster, G. R. 1974. The ecology of Latimeria chalumnae
Smith: results of field studies from Grande Comore. Proc.
R. Soc. London, Ser. B 186:291—296.

Fraser, T. H. 1972. Comparative osteology of the shallow
water cardinal fishes (Perciformes: Apogonidae) with ref-
erence to the systematics and evolution of the family. Ich-
thyol. Bull., J. L. B. Smith Inst. Ichthyol., No. 34. 105 pp.

HurReEAU, J.C., AND C. OzouF. 1977. Determination de l’age

1976. Sur quelques car-

et croissance du coelacanthe Latimeria chalumnae. Cy-
bium, 3° sér. 2:129-137.

Locket, N. A., AND R. W. GRIFFITH.
of a living coelacanth. Nature 237:175.

MCALLIsTeER, D. E. 1971. Old Fourlegs, a ‘‘living fossil.’’
National Museums of Canada Odyssey Series. 25 pp.

, AND C. L. SmitH. 1978. Mensurations morpholo-
giques, dénobrements meristiques et taxonomie du coel-
acanthe, Latimeria chalumnae. Nat. Can. 105:63—76.

McCosker, S., AND J. E. McCosker. 1976. To the islands
of the moon. Pac. Discovery 29:19-32.

MENACHE, M. 1955. Etude hydrologique sommaire de la ré-
gion d’Anjouan en rapport avec la peéche de trois coel-
acanthes. Mem. Inst. Sci. Madagascar, Sér. A 9:151-185.

MILLor, J. 1955. First observations on a living coelacanth.
Nature 175:362-363.

, AND J. ANTHONY. 1958. Latimeria chalumnae, der-

nier des crossoptérygiens. Pages 2553-2597 in P. P. Grassé,

ed., Traite de Zoologie, Vol. 13. Masson, Paris.

, AND 1974. Les oeufs du coelacanthe. Sci.

Nat. Paris, No. 121, pp. 3-4.

5 , AND D. RoBINEAU. 1972. Etat commenté des
captures de Latimeria chalumnae ... effectuées jusqu’au
mois d’octobre 1971. Bull. Mus. Nat. Hist. Nat. 39:533-
548.

MILLoT, J., AND N. Carasso. 1955. Note preliminaire sur
Voeil de Latimeria (Coelacanthidae). C. R. Acad. Sci.
241:576-577.

NEVENZEL, J. C., W. RoDEGKER, J. F. MEAD, AND M. S.
GORDON. 1966. Lipids of the living coelacanth, Latimeria
chalumnae. Science 152:1753—1755.

SmitH, C. L., C. S. RAND, B. SCHAEFFER, AND J. W. Atz.
1975. Latimeria, the living coelacanth, is Ovoviviparous.
Science 190:1105—1106.

THOMSON, K. S. 1966. Intercranial mobility in the coel-
acanth. Science 153:999-1000.

1970. Intercranial movement in the coelacanth Lat-

imeria chalumnae Smith (Osteichthyes, Crossopterygii).

Postilla 149:1-12.

. 1973. New observations on the coelacanth fish, Lar-
imeria chalumnae. Copeia 1973(4):813-814.

WHEELER, A. 1975. Fishes of the world. MacMillan Pub.
Co., New York. 366 pp.

1972. Observations

APPENDIX 1: COELACANTH CAPTURE LIST

This list is a continuation of the list compiled by Millot et
al. (1972). These data were provided by the Grande Comore
Direction de la Production and several informants from Iconi,
Moroni, and Mitsamiouli.. As mentioned in the text, the pur-
ported depth of capture and distance from shore are fisher-
mans’ estimates; they should not be considered to be accu-

OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

rate. There is a discrepancy of three specimens between this
list and that in Millot et al., such that C67 here would be C70
by their system. This is due to two specimens which they note
in a footnote (p. 547) and a taxidermied adult (ca. 100 cm
length) captured near Mitsamiouli and attached to the wall of
the bar in the Maloudja Hotel. Abbreviations are: A, Anjouan;
GC, Grande Comore.

Distance

Speci- from shore Depth Weight Length

men Date Time Location (m) (m) (kg) (cm) Sex
C70 5172 0100 A: Domoni 2,000 400 78 163 2
C71 22 Ill 72 0200 GC: Iconi 1,000 300 10 90 fe)
C72 12 V 72 2300 GC: Iconi 800 90 40 140 9?
C73 12 VIII 72 0300 CG: Iconi 400 100 — 90 ?
C74 16 X 72 — A: Dzindni 1,000 350 30 120 3?
C75 6 VII 73 — GC: Mitzoudje 500 — 35 132 3
C76 27 VII 73 — GC: Iconi 100 100 10 86 fo)
C77 6 XI 73 0200 A: Vouani — 175 32 120 3?
C78 22 XI 73 2100 GC: Iconi 800 180 24 103 3
C79 27 XI 73 0330 GC: M’Bachili 400 225 30 110 3
C80 14 II 74 0100 A: Miroutsy — 220 40 139 3
C81 17 V 74 2100 GC: Vanamboini 300 150 40 139 ?
C82 17 VII 74 1645 GC: Iconi — 180 0.8 42.5 &
C83 18 X 74 —_ GC: Iconi — 240 45 180 g
C84 9 XI 74 = GC: Iconi 2,000 250 37 145 2?
C85 22175 — A: Mromouhouli 3,000 300 —_ 165 Q
C86 27176 —_— GC: Iconi — — = = =
C87 IX 76 — A: Domoni — — 11 90 —
C88 III 77 — GC: Iconi — = = os =

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 20 pages

December 22, 1979

THE COELACANTH AND THE CHONDRICHTHYES AS SISTER
GROUPS: A REVIEW OF SHARED APOMORPH CHARACTERS AND
A CLADISTIC ANALYSIS AND REINTERPRETATION

By

Michael D. Lagios

Children’s Hospital,
San Francisco, California 94118, and
Steinhart Aquarium, California Academy of Sciences,
San Francisco, California 94118

CHAPTER I

The principal expectation generated by Ms.
Latimer’s finding of that singular blue fish in a
discarded trawler haul was that the soft parts,
biochemistry and physiology of the living coel-
acanth might mirror that of its long extinct but
morphologically similar brethren. The signifi-
cance of this lay not in studying these features
in a living fossil alone but rather in what these
studies might imply for its extinct sister group,
the Rhipidistia, and the immediate preterrestrial
condition.

Coelacanths and Rhipidistia are convention-
ally classified as sister taxa and share numerous
seemingly homologous features including lobed
fins, scales, lungs and most importantly a pe-
culiar intracranial joint (Romer 1966, 1968;
Thomson 1969). This conventional classifica-
tion of the coelacanth as a Crossopterygian re-
flects a selection bias of calcified tissues and ex-
ternal form, since prior to 1938 that was all the
evidence available for review, and because sys-
tematists have been more accustomed to count-
ing vertebrae than determining vascularization,

biochemistry, and protein structure. It was with
this bias that the coelacanth has been studied by
numerous independent investigators hoping to
look backward into the Devonian through a
beast little changed since they and our preter-
restrial ancestors presumably diverged. Yet
these expectations reflect the romance of the
coelacanth more than the reality of the dead oily
fish putrifying in the hot summer sun of East
London where Marjorie Courtney Latimer dis-
covered it in 1938.

This paper will present a reappraisal of the
systematic position of the coelacanth which sug-
gests that this fish is in no way related to the
Rhipidistia or their tetrapod descendents and in
fact probably represents an archaic sister group
to the Chondrichthyes—or briefly, a palaeozoic
shark-like fish.

This reappraisal is predominantly based on
three complex characters: 1) The pituitary
gland: pars distalis structure and organization;
2) Cation excreting tissue: the presence of a ho-
mologous rectal gland; and 3) Osmotic adaption:
specifically urea and TMAO retention in the
marine environment.

26 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

In a cladistic analysis traits may appear to be
similar because: 1) they are common primitive
or plesiomorphic features of the group, 2) be-
cause of independent convergent evolution, and
3) finally, because they are homologous special-
ized characters or synapomorphic. Only the lat-
ter are useful in evaluating systematic relations.
My thesis is that the coelacanth and Chondrich-
thyes share a number of independent synapo-
morphic characters which make an association
with the Rhipidistia and their tetrapod descen-
dents untenable. In order to support this con-
clusion, this presentation will establish that the
similarities in the several characters I present
are: 1) truly specialized, that is apomorphic and
not primitive or baseline features of an ancestral
group; 2) exclusive to the proposed sister groups,
that is the Chondrichthyes and the Coelacanthi-
ni; and 3) most likely homologous rather than
examples of convergent evolution.

Pituitary Complex

The pituitary complex, derived from an evag-
ination of the roof of the stomodeum and a cor-
ollary growth of the overlying hypothalamic
floor, is a constant and diagnostic feature of the
Vertebrata. Both its morphology and the peptide
sequences of its various hormones indicate a
very conservative rate of evolution. Amino acid
sequence analysis of the neurohypophysial hor-
mones, arginine vasotocin and subsequent sister
derivatives (Acher et al. 1968; Sawyer 1969,
1977) and more recent analysis of the pars dis-
talis glycoproteins, TSH, LH (Fontaine and
Burzawa-Gerard 1977) and to some extent
growth hormone (Hayashida et al. 1975; Hay-
asnhida 1977; Wallis 1975), have been utilized by
comparative biochemists and systematists to es-
tablish protein phylogenies and to aid systematic
group analysis. Analysis of the morphology of
the pituitary, its innervation, vascularization
and distribution of cell types, has generated an
enormous literature in comparative endocrinol-
ogy over the last fifty years and has provided
many signal discoveries in biology.

Despite this intense interest in pituitary struc-
ture, however, only a few basic patterns of or-
ganization, i.e., the way in which the neurohy-
pophysis and adenohypophysis relate to each
other in terms of innervation, vascularization
and distribution of specific cell types, have been
documented. These patterns are summarized in
the accompanying table and schematic diagram

(Fig. 1). Some of the variations are rather minor
in character, and all save the tetrapod pattern,
are limited to fishes. Each pattern is so distinc-
tive that it is diagnostic for the group which pos-
sesses it.

The extant petromyzonts and myxinoids dem-
onstrate pituitary organization in rather simple
form. The adenohypophysis is a relatively flat,
compact discoid mass of cells which shows close
approximation to the neurohypophysis poste-
riorly to form a neurointermediate lobe, a char-
acteristic feature of all aquatic vertebrate
groups. There is no direct innervation of the an-
terior adenohypophysis or equivalent pars dis-
talis. In the petromyzonts numbers of vessels
occur in the thin fibrous tissue separating the
pars distalis from the overlying hypothalamic
floor and although no hypothalamo-hypophysial
portal system occurs, perivascular connective
tissue may serve as a diffusion gradient for neu-
rotransmittor substances (Gorbman 1965; Tsu-
neki and Gorbman 1975; Goossens et al. 1977).
In the myxinoids the vascular arrangements are
less well developed but similar (Henderson
1972; Kobayashi and Uemura 1972).

All gnathostomes, fishes and tetrapods have
a well developed hypothalamo-hypophysial por-
tal vascular system through which specific neu-
rotransmittors, identified only recently in mam-
mals, control the secretory function of specific
adenohypophysial cell types. The majority, in-
cluding relic actinopterygians, dipnoans and tet-
rapods, retain a compact pars distalis with only
modest evidence of regional differences in dis-
tribution of specific cell types within the gland,
most clearly evident in birds (Wingstrand 1966;
Hayashida and Lagios 1969; Lagios 1968a,
1968b, 1970; Hansen 1971; Polenov 1968).

In contrast to both the agnathans and the fore-
going group of gnathostomes, which have re-
tained a number of plesiomorph features in pi-
tuitary organization, are the teleosts and
Chondrichthyes.

Teleosts are unique among vertebrates for the
number of radical innovations in pituitary or-
ganization which they manifest. Among these
are direct synaptic innervation of specific pars
distalis cell types with a corollary suppression
of the hypothalamo-hypophysial portal system,
an extreme segregation of cell types such that
the gland becomes compartmentalized, and an
interdigitation of the neurohypophysis with the
pars distalis (Vollrath 1967; Lagios 1970; Zam-

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 27

ACTINOPTERYGII

TELEOST

TETRAPOD

FIGURE 1.

PETROMYZON

+ PRO

Patterns of vertebrate pituitary organization. Schematic diagrams of representative vertebrate types in midsagittal

section. Anterior, left. Neurointermediate lobe (pars intermedia and associated posterior peptidergic neurohypophysis) shaded.
Vascular systems and separate blood supplies of neurointermediate lobe (Dipnoi) and pars nervosa (Tetrapod) indicated as
schematic vessels. Specific hormone-producing cell types of pars distalis indicated by key. Chondrichthyes: Note the separate
ventral lobe with gonadotrope and thyrotrope sequestration and its separate vascular supply.

brano 1971). These features occur among phy-
letically more primitive teleost groups, e.g., the
clupeomorphs, elopomorphs and ostariophy-
sines, but are best developed in the more ad-
vanced recent groups, e.g., the perciforms. Te-
leost pituitary organization is decidedly
apomorphic, probably more so than any other
vertebrate group. This would appear to be of
very recent origin.

Although more remote in origin, the Chon-
drichthyes similarly exhibit unique features in
pituitary organization as compared to a more
conservative baseline pituitary structure. These
unique features are exemplified by the elasmo-
branchs in which the pars distalis is peculiarly
divided into a number of separate cell masses or
lobes, each of which sequesters one or more
functional cell types and shows specific special-
izations in vascularization. Although both the
rostral and proximal partes distalis, the more
conventional portions of the elasmobranch pi-

tuitary, show vascular specializations—with
each area receiving portal venous drainage from
a specific region of the hypothalamic floor (Mel-
linger 1960; Meurling 1967a)—it is in the devel-
opment of a ventral lobe that this tendency oc-
curs in its most extreme form.

The ventral lobe is a physically separated
ventral portion of the pars distalis which may be
connected by a persistent tubular remnant of the
hypophysial cavity (= Rathke’s pouch equiva-
lent) with the more dorsally located median lobe
of the pars distalis (Norris 1941; Alluchon-Ger-
ard 1971; Knowles and Vollrath 1975). The lobe
itself is closely associated with and often adher-
ent to the carotid anastomosis, a characteristic
feature of elasmobranchs (Norris 1941), and fre-
quently lies in arecess within the chondrocranial
floor (Figs. 2 and 3). The ventral lobe has a char-
acteristic branching tubular structure which
contrasts with the more compact arrangement
of follicles and cords of the remainder of the

28 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Ficures 2 and 3. Fig. 2. Notorynchus maculatus (Seven-gill shark), midsagittal diagram. Anterior, right. Pituitary: dark

shading. Note the ventral lobe separated from the remainder of the adenohypophysis (A) in a recess within the floor of the
chondrocranium (light shading). Portion of hypophysial duct connecting ventral lobe with remainder of pars distalis is visible.
Vessels in the recess represent carotid anastomosis. OX, optic chiasm. Scale 5mm. Fig. 3. Heterodontus francisci (Hornshark),

midsagittal diagram. Anterior, right. Ventral lobe lies in a shallow recess within chondrocranium. Note associated carotid
anastomosis and hypophysial veins. Scale 5 mm.

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 29

pituitary gland. Uniquely among vertebrates,
the ventral lobe receives a direct arterial supply
from the internal carotids and is independent of
the venous effluent of the hypothalamo-hypo-
physial portal system (Mellinger 1960; Meurling
1967a). This physically separated, independent-
ly vascularized portion of the pars distalis se-
questers in large part, if not exclusively, the
gonadotropic activities of the pituitary in elas-
mobranchs. Evidence for this has been estab-
lished from extirpation experiments (Dodd 1975;
Dobson and Dodd 1977) bioassays using chick
testis (Scanes et al. 1972) and Xenopus oocyte
systems (Firth and Vollrath 1973), and activa-
tion of specific ventral lobe adenyl cyclase using
mammalian LH-FSH-RF (Deery and Jones
1975). These studies corroborate immunohisto-
chemical localization (Mellinger and Dubois
1973) based on a specific reaction with anti-
ovine LH limited to the elasmobranch ventral
lobe. Firth and Vollrath (1973) did identify some
LH-like activity in the median lobe of Scylio-
rhinus, but the mean value was only one tenth
that present in ventral lobe extracts.

Such hormonal sequestration is characteristic
of the other lobes of the elasmobranch pars dis-
talis. Evidence indicates that TSH is restricted
to the ventral lobe along with LH-like activity,
while ACTH activity is localized in the rostral
lobe (Jackson and Sage 1973; Mellinger and Du-
bois 1973).

For a long while considerable speculation was
generated concerning the functional significance
of a vascular arrangement which would appear
to exclude gonadotropic function from central
nervous control in the elasmobranchs. Seasonal
studies however, have clearly shown that go-
nadotropic activity must be correlated with CNS
control in this group. More recently, elegant ex-
periments have shown specific activation of ven-
tral lobe adenyl cyclase with elasmobranch hy-
pothalamic homogenates (Deery and Jones
1975). This evidence suggests that elasmo-
branchs regulate gonadotropic function by a
specific neurotransmittor as in other verte-
brates, and that the peculiar morphology of the
system is a ‘nonsense’ feature in terms of en-
docrine control and function, as witness the re-
productive success of elasmobranchs over the
last 300 million years. Since the survival value
of a “‘nonsense’’ feature is null, and by defini-
tion it is outside the influence of selection pres-

sure, its potential value in a cladistic analysis is
increased.

Among the Holocephali, the sister group of
the extant elasmobranchs, pituitary organization
is similarly unique although more bizarre (Fig.
4). The equivalent ventral portion of the pars
distalis remains in close association with the
homologue of the internal carotids, but both the
lobe and the homologous vessels remain outside
the chondrocranium in the roof of the oral cavity
(Fahrenholz 1828; Sathyanesan 1965). The in-
ternal carotid homologues form an anastomosis
in some forms (Allis 1912; Meurling 1967b;
Jasinski and Gorbman 1966) and in others show
ipsilateral development, but do supply the ex-
tracranial portion of the pars distalis, or Rach-
endach-hypophysis. Embryologically the Rach-
endach-hypophysis shows a tenuous tubular
connection through a defect in the chondrocran-
ium with the persistent hypophysial cavity of the
more conventional intracranial pars distalis
(Honma 1967), a condition entirely comparable
to that of the sharks Notorynchus and Hetero-
dontus. Like the elasmobranch ventral lobe the
holocephalan Rachendach-hypophysis appears
to sequester gonadotropic function, based on
seasonal studies of gonad maturation, and shows
a histochemical reaction consistent with a di-
sulfide containing glycoprotein secretion com-
parable to that of the known gonadotropes of
the elasmobranch ventral lobe (Sathyanesan
1965). Recently Dodd and Dodd (1979) have
confirmed high levels of gonadotropic activity in
the Rachendach-hypophysis of Hydrolagus ina
radio-immunoassay using an antibody to a par-
tially purified elasmobranch gonadotropin.

Apart from prior brief general descriptions
(Millot and Anthony 1955, 1958, 1965) studies of
the coelacanth pituitary are limited to a few in-
dividual specimens, only two of which received
proper histologic fixation (Lagios 1975; van Ka-
menade and Kremers 1975). These latter two
studies represent two specimens, a small im-
mature female, 85 mm total length, and a large
ovigerous mature female, 167 cm. Despite the
fact that van Kamenade and Kremers (1975)
were limited by their material to the more prox-
imal portion of the pituitary, their independent
observations and those of the present author
(Lagios 1972, 1975) are remarkably similar.

The most striking feature of the coelacanth
pituitary complex is the great anterior elongation

30 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Ficures 4 and 5. Fig. 4. Hydrolagus colliei (Ratfish), midsagittal diagram. Anterior to right. Separated portion of the pars
distalis, the Rachendach-hypophyse (R) lies outside the chondrocranium surrounded by lymphoid tissue, L, beneath the oral
mucosa, M. More conventional intracranial hypophysis, A, is depicted in relation to the optic chiasm, OX, and saccus vas-
culosus, SV. Scale 10 mm. Fig. 5. Latimeria chalumnae (Coelacanth). Sagittal diagram. Anterior, left. Rostral extension (pars
buccalis) of the pars distalis, PD, greatly attenuated and 83 mm long in this 140 cm adult male. Internal carotids, IC, converge
to anastomose in a deep recess in the basisphenoid, BS. Lateral vein, V. Notochord, N. PI, pars intermedia (neurointermediate
lobe); PA, preafferent arteriole of portal system; OX, optic chiasm. Scale 10 mm.

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 31

of the pars distalis (Fig. 5) (Millot and Anthony
1955). This elongate extension, variously termed
pars buccalis, or rostral division, consists of an
attenuated hypophysial cavity from which
branch numerous tubular structures whose ar-
chitecture is reminiscent of the holocephalan
Rachendach-hypophyse. The intermittent islets
of endocrine tissue noted previously (Millot and
Anthony 1955, 1958; Lagios 1972, 1975; van
Kamenade and Kremers 1975) represent vari-
able discontinuous clusters of branching tubules
arising from a continuous duct-like hypophysial
cavity (Fig. 6). Aside from the tubular hypophy-
sial cavity, this lobe, like the ventral lobe of
elasmobranchs, is physically separated from the
remainder of the pars distalis. At its rostral ex-
tremity it is intimately associated with an anas-
tomosis or convergence of the internal carotids
(Lagios 1972). Pars distalis follicles lie within the
adventitia of the carotids prior to their anasto-
mosis in the chondrocranial floor. Clearly evi-
dent in serial reconstruction of this region are
direct arterial vessels which arise obliquely from
the carotids and run retrograde before bifurcat-
ing within the lobe. Histochemical studies (La-
gios 1975; van Kamenade and Kremers 1975)
have shown the presence of disulfide-containing
glycoprotein secretion granules within the cells
of this lobe. Only thyrotropes and gonadotropes
among definitively identified functional pituitary
cell types show this histochemical reaction. Cor-
roboration from endocrine ablation experi-
ments, bioassay, radio-immunoassay and im-
munofluorescent reactions to specific hormones
have not yet been performed in the coelacanth.

The cytology of the small disulfide containing
cells in the immature female (Lagios 1975) con-
trasts markedly with large vacuolated cells of
this same lobe in the sexually mature ovigerous
female reported by van Kamenade and Kremers
(1975) and noted by M. Olivereau (pers. comm.)
who studied the bulk of the pars buccalis of the
same 5 January 1972 specimen. Both investi-
gators note the similarity of the cytology of these
cells to known gonadotropes. Their change with
sexual maturation and their histochemistry are
both consistent with this interpretation.

CHAPTER II
Rectal Gland

Cells specialized for the active transport of
excess cation are a common feature of marine

FiGure 6. Latimeria chalumnae (Coelacanth). Schema-

tized frontal section through conventional proximal pituitary
complex in Latimeria. Level is ventral to median eminence.
Residual hypophysial cavity (C) extends as a tubular process
through pars distalis (PD) and continues rostrad. Note inter-
mittent islet of rostral extension (= ventral lobe homologue)
arising from tubular cavity. V, ventricle of neurohypophysis,
NH; SV, saccus vasculosus; PI, pars intermedia. Dark shad-
ing: connective tissue. Scale, 1 mm.

vertebrates. The most widely distributed and
best known are the so-called *‘chloride cells’ in
the gills of fishes. Although originally described
in teleosts, “‘chloride cells’*> occur among lam-
preys, elasmobranchs and holocephalans as
well as bony fishes. Such a wide distribution
would suggest that this is a very plesiomorphic
feature of vertebrates.

Materials

The present account is based on rectal gland
gross anatomy, histology and ultrastructure of
the following sharks: Squalus acanthias, Par-
maturus xaniurus, Mustelus henlei, Charchar-
odon charcharias, Carcharhinus melanopterus,
C. amblyrhynchus, the holocephalan Hydrola-

32 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES. No. 134

FIGURE 7.

Hydrolagus colliei (Ratfish). Midsagittal diagram of distal (postvalvular) hindgut. SV, spiral valve, RV, rectal

valve (= distalmost plica of spiral valve), RG, intramural cation-excreting glands, homologue of those of elasmobranchs.

Scale 0.5 mm.

gus colliei, and the coelacanth Latimeria cha-
lumnae. The sharks were made available to me
courtesy of John E. McCosker, Steinhart
Aquarium, California Academy of Sciences and
John Stephens, Occidental College, Los Ange-
les; Hydrolagus courtesy of Professor Stephens
and George and Susan Brown, Department of
Fisheries, The University of Washington, Seat-
tle; and coelacanth material, courtesy of the late
Earl S. Herald and the living John E. McCosker,
Steinhart Aquarium; Robert Griffith, then of the
Peabody Museum of Natural History, Yale Col-
lege; James Atz and C. Lavett Smith, American
Museum of Natural History; Charles Rand,
Long Island University, New York; and Robert
Lavenberg, Los Angeles County Museum of
Natural History.

The Chondrichthyes have an additional spe-
cialized cation-excreting epithelium exclusive to
the group, arranged as a gland derived from the
terminal (post-valvular) hindgut. Among sharks
this structure was recognized as the rectal, dig-
itiform or cecal gland long before its function
was realized. The gland exists in its simplest
form among the Holocephali where it occurs as

a circumferentially disposed group of separate
racemose glands, each with its own duct, within
the submucosa of the terminal hindgut. This por-
tion of the gut is supplied by a discrete rectal
artery which enters the dorsal aspect of the gut.
In Hydrolagus colliei the majority of the sepa-
rate submucosal glands lie in the dorsal portion
of the gut wall, while in Chimaera monstrosa
they are restricted to this site (Crofts 1925;
Fange and Fugelli 1963; Lagios and Stasko-Con-
cannon 1979).

In Hydrolagus colliei a bluish discoloration of
the serosa of the terminal hindgut, which ap-
pears related to vascularization, belies the pres-
ence of the intramural rectal glands in a nar-
rowed post-valvular gut segment. Their location
can be determined with certainty in the living
specimen by noting the point of entry of the dor-
sal rectal artery. Sections of the hindgut reveals
discrete 1-2 mm yellow-beige submucosal
glands in the bowel wall between the rectal valve
proximally (the terminal plica of the spiral
valve) and the thin, muscular segment of hindgut
distally. The latter exits the body directly (Fig.
7); there is no cloaca.

es)
ies)

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS

FiGures 8 and 9. Fig. 8. Parmaturus xaniurus (Filetail Cat Shark). Schematic drawing of rectal gland relationships. Left
lateral exposure, body wall excised, rectum (R) and liver lobe (L) retracted ventral. Rectal gland (RG) consists of a single
fusiform gland supported by a dorsal mesorectum (M). Dorsal rectal artery enters gland at cephalic pole, then courses with
duct of rectal gland to reach dorsal surface of rectum. Kidney (K). Scale 10 mm. Fig. 9. Latimeria chalumnae (Coelacanth).
Sagittal diagram. 140 cm adult male. Rectum (R) is partially cut away to reveal position of rectal gland, RG, in relation to more
distal cloaca, C. Bladder (B) is bilobed. K, Kidney; L, Liver; T, Testis and meso-orchium: LG, fat-filled vestigial lung.

34

OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ces i‘

IP « hamagtens yatta in pa
easels Pipaenmhel MAS G aie! yer oes, aon ==
ar ait aes iphiad ore se an 9S (10)
~. aii

Ficures 10 and 11. Fig. 10. Latimeria chalumnae. Right lateral diagrammatic views of rectal gland in an adult male (LACM
6824-1). Distal rectum (R), rectal gland (RG), bladder (B) and ventral floor of abdomen have been hemisected to expose duct
of rectal glance urse of right urethra into posterior rectal wall, and entrance of right ureter (arrow) in bladder. Right testis
(T) and ductus deferens course ventral far posterior to rectum but exit was not traced in this specimen. Fig. 11. Latimeria

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 35

Among elasmobranchs the rectal cation-ex-
creting tissue becomes a single, compact, extra-
mural compound gland, lying in the dorsal me-
sorectum and emptying by a single duct into the
dorsal wall of the post-valvular gut (Fig. 8). The
relationship of the gland and dorsal rectal artery,
evident in the Holocephali, becomes clearer,
since it directly supplies the gland. Despite the
common appellation ‘‘digitiform™’ the majority
of elasmobranch rectal glands are fusiform and
appendix-like in shape.

The extant coelacanth, Latimeria, has a sim-
ilar gland, termed “‘post-anal’’ by Miullot and
Anthony (1972, 1973a). The present description
which differs from that of the latter authors, is
based on the examination of three adult speci-
mens: two large mature males (C59, 122 cm total
length, California Academy of Sciences CAS
24862, and 121 cm total length, Los Angeles
Museum of Natural History LACM 6824-1), and
a mature ovoviviparous female (C26, 160 cm to-
tal length, American Museum of Natural History
AMNH 32949) and one of her contained em-
bryos. Histologic material was available from
both adult males and a second coelacanth em-
bryo loaned by Charles S. Rand, M.D. Ultra-
structural studies were limited to C68, the same
specimen which provided the material for pub-
lished ATPase studies (Griffith and Burdick
1976).

The coelacanth rectal (“‘post-anal’’) gland lies
within the dorsal mesorectum, close to the dor-
sal wall of the posterior-most portion of the rec-
tum, just as it angles steeply through the ventral
body wall to exit. In the mature male, the an-
terior face of the gland is adherent to the surface
of the rectum itself (Fig. 9). A single duct leads
from the ventral portion of the gland, paralleling
the rectal contour, before entering the dorso-
posterior wall of same, 3 cm proximal to the
rectal orifice in C26 (Fig. 10).

The anatomical relationships of the coel-
acanth rectal (‘‘post-anal’’) gland are virtually
indistinguishable from those of the rectal glands
of sharks, although Millot and Anthony (1972)
claim significant differences between the topog-

raphy, anatomy and histology of the gland of
Latimeria and those of elasmobranchs. They
have interpreted the rectal (‘‘post-anal”’) gland
of the coelacanth as emptying into a cloaca
which receives a common urogenital papilla
more distally on its posterior wall. The coel-
acanth rectal gland is therefore not comparable
to elasmobranchs in this view since it is not
strictly rectal, that is proximal to the anal ori-
fice. However, dissection of the LACM speci-
men clearly shows paired urethrae entering the
posterior wall of the rectum but separate from
the ductus deferens (archinephric ducts) which
course far caudad of the rectum. Dingerkus et
al. (1978) have clearly shown a separate exit of
the conjoined ductus deferens posterior to the
rectum. Thus the male “cloaca” in Latimeria
could not be confirmed. In the ovoviviparous
AMNH female, the rectum, urethrae and ovi-
duct exit separately into a shallow scale-bearing
depression (Fig. 11) and the rectal gland clearly
empties by a single median duct into the pos-
terior wall of the rectum.

Crofts (1925) has shown a similar close prox-
imity of the rectal gland duct, ureters and ovi-
ducts in Chlamydoselache and in Heptanchus
embryos. In these sharks the rectal gland duct
enters the rectum so posteriorly that its actual
exit can be described as anal, i.e. the point of
demarcation between the cloaca and terminal
hindgut. Elasmobranchs show considerable
variation in the relationships of the duct to the
rectal wall, and this apparently relates to the
length of the postvalvular segment (Crofts 1925)
rather than differences in embryologic origin.
Given this variation in elasmobranchs, and the
greater differences in the homologous intramural
glands of the Holocephali, the small differences
between the gland in Latimeria and the predom-
inant situation in sharks falls within the degree
of variation evident in extant elasmobranchs,
and well within the variation in Chondrichthyes.

Millot and Anthony also cite the absence of
a well developed central lumen in the coelacanth
gland as evidence indicating non-homology with
those of elasmobranchs, in which the lumen is

—

chalumnae. Left lateral diagrammatic view of rectal gland, R in ovoviviporous (AMNH) female specimen. M, mesorectum: B,
left bladder; K, kidney; OV, dominant right oviduct viewed through rent in mesorectum. Inset, ventral view of vent, rostrad
(superior in figure) is the large separate rectal opening, caudad, a funnel-shaped common oviduct and to either side the small

paired urethral openings.

OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Ficure 12. Coelacanth rectal gland EM. A. A group of tubules are seen in cross section. Note the numerous mitochondria
(arrow). Nucleated red blood cells, R, lie in capillary between adjacent tubule profiles. N, nucleus of tubular cell, L, lumen.
B. Basal cisternae of a rectal gland cell. Note deep infoldings of the basal cell membrane (arrow), producing the basal cisternae
between which lic mitochondria, M. The ultrastructure is identical to other epithelia engaged in active ion transport and ts
indistinguishable from rectal glands of the Chondrichthyes.

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 37

FiGure 13. Comparable magnifications of Ratfish (Hydrolagus colliei). Labels as in Fig. 12.

38 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGurReE 14.

present. The latter would appear to be an em-
bryologic remnant of the enteric diverticulum
which produces the gland Anlage. However,
among the Holocephali, the separate submuco-
sal homologous rectal glands lack such a central
gut lumen.

The rectal glands of the Chondrichthyes and
that of the extant coelacanth are virtually iden-
tical in their histology and ultrastructure. The
terminal, functional portions of the gland are
composed of a single layered columnar epithe-
lium which displays the characteristic arrange-
ment of cell surface, organelles, and histoen-
zymology of active cation transport. A complex
system of basal and lateral cisternae, represent-
ing deep plate-like parallel invaginations of the
basal and lateral cell membranes, enormously
increase the surface area in contact with the ex-
tracellular compartment. These cisternae are as-
sociated with numerous mitochondria, which
generate the energy required to drive the active
transport of cations. Junctional complexes,
comprising multiple maculae adhaerentes and
zonulae adhaerentes, bind the luminal aspects
of the cell surfaces (Figs. 12A, B, 13A, B and

Seven-gill Shark (Notorynchus maculatus) right, rectal gland. Labels as in Fig. 12.

14). In sharks the role of these glands in cation
excretion has been confirmed by physiologic ex-
periments. Similar experiments have not been
performed on the Holocephali or the coelacanth,
but both demonstrate similar sodium-potassium
activated ATPase within the gland, comparable
to that of the better known elasmobranch struc-
ture (Griffith and Burdick 1976; Lagios and Stas-
ko-Concannon 1979).

CHAPTER III

One of the first biochemical surprises to
emerge from studies of the coelacanth was a
high level of serum urea and hepatic-urea cycle
enzymes comparable to those of elasmobranchs.
At the time of this discovery (Brown and Brown
1967; Pickford and Grant 1967) however, it was
interpreted as further evidence for an associa-
tion of the coelacanth with the lungfish, Protop-
terus, in which retention of urea is an important
adaptation to estivation. Due to the prevailing
bias which saw the coelacanth as “Old Four
Legs,’’ this biochemical evidence tended to con-
firm the systematic position of the coelacanth as

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 39

a member of the Sarcopterygil, a taxon created
by the late Alfred Sherwood Romer.

There are three basic methods of water and
electrolyte regulation observed in living marine
organisms. Simplest and most pervasive of these
is the ionic regulation seen in all invertebrates
and in the hagfishes—but not in lampreys. Plas-
ma is iso-osmolar to seawater and contains very
similar ionic concentrations. Divalent cations,
such as calcium, magnesium, etc. are regulated
as is sodium and potassium in the hag. Since this
particular mechanism is so widely distributed
among invertebrates, including the primitive
chordates, and also present in the hags, it prob-
ably represents an unspecialized, primitive or
plesiomorphic condition.

The second and next most pervasive mecha-
nism of marine adaptation is characteristic of
most gnathostomes (including modern bony
fishes) and present in the more ancient lamprey.
Tetrapods retain a similar osmolality and elec-
trolyte composition. In this group, exemplified
by modern marine teleosts, serum is maintained
hypo-osmolar to the environmental salinity.
Body water is obtained by drinking seawater and
excreting large amounts of cations through a
branchial excretory mechanism, the chloride
cells of the gills. In euryhaline forms, both the
total osmolality and the ionic composition are
rigorously maintained independent of environ-
mental salinity.

The third, least common and the most restrict-
ed in its distribution of the adaptive mechanisms
is urea retention. This adaptation is seen only
among chondrichthyian fishes—sharks, rays and
chimaeras—and in the extant coelacanth among
fishes with a long palaeontologic history of ma-
rine residence. Large quantities of urea are re-
tained in the blood to achieve an osmolality vir-
tually identical to seawater. Excess salt is
excreted through a rectal gland, although a bran-
chial salt excreting apparatus and chloride cells
are present in Chondrichthyes. The presence of
similar cells in the coelacanth gill is highly prob-
able but has not been confirmed.

On the basis of its very limited distribution in
marine vertebrates, virtually confined to a single
group, the Chondrichthyes, and its absence in
marine invertebrates, hagfish and lampreys, it
would be parsimonious to accept urea retention
as a specialized or apomorphic condition. Some,
however, have argued that since other forms
have similarly developed urea retention, specif-

ically the marine and estuarian amphibians Bufo
marinus and Rana cancrivora, that this feature
is without systematic value. I disagree. Urea re-
tention in two species of amphibians which can
adapt to limited marine habitats is obviously a
specialized secondary feature in that group and
can best be interpreted as isolated convergence.
The ability to synthesize urea is a primitive fea-
ture in vertebrates; it is the retention of urea in
marine environment which is apomorphic.

Similarly, urea retention in the burrowing toad
Scaphiopus and the modern estivating lungfish-
es are specializations. The contrary argument
that urea retention as a marine adaptation is
primitive for sharks and coelacanths ignores the
limited distribution of this biochemical mecha-
nism and its absence in relic agnathans. Fur-
thermore, there is evidence that protein function
(specifically hemoglobin in sharks) is dependent
on the maintenance of high levels of urea (Bon-
aventura et al. 1974) which may account for the
retention of serum urea of 170 mM/I in the
freshwater Lake Nicaragua Bull Shark Carchar-
hinus leucas (Thorson et al. 1973).

In addition to retention of large amounts of
urea, the chondrichthyian fishes and the coel-
acanth share one other biochemical peculiarity
not seen in either marine or estivating amphibia.
This is a high level of trimethylamine oxide
(TMAO) in both groups. Among sharks and the
coelacanth the levels of TMAO contribute very
significantly to the total serum osmolality. Con-
version by liver explants of C'* labelled choline
and TMA to TMAO in both shark and coel-
acanth occur at rates sufficient to account for
the blood levels. Although TMAO levels may
also be substantial in marine teleosts, evidence
from hatchery raised salmonids and cyprinids
suggests that TMAO levels in these fishes may
be entirely of dietary origin. Biochemical evi-
dence of endogenous synthesis of TMAO in te-
leosts 1s contradictory.

Griffith and Burdick (1976) and Pang et al.
(1977) have argued that despite the superficial
similarities of urea retention among elasmo-
branchs and the coelacanth, two significant dif-
ferences exist: the coelacanth is slightly hypo-
tonic (ca. 100 mOsm) to seawater sampled at the
capture site; and the urea concentration in blad-
der urine and serum were virtually identical.
Pang et al. (1977) interpreted the relative serum
hypotonicity of the specimen relative to sea-
water as an indication of a fundamentally differ-

40 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ent method of adaptation. However their results
are perhaps an artifact in that the specimen, a
small, 85 cm immature female, remained in the
surf at a 1 M depth in a chicken wire cage for
6-8 hours prior to serum sampling. The speci-
men was caught during the monsoon season
when large freshwater aquifers drain into the lit-
toral, reducing the salinity considerably. Al-
though these were not sampled by Griffith, I
have encountered these same freshwater aqui-
fers in the littoral and in deeper water of the reef
face at 30-40 M depth (see McCosker, this vol-
ume). Native Comorans employ this tidepool
freshwater runoff to wash themselves and their
clothing. The 100 mOsm difference between the
serum of the damaged and dying specimen and
that of the offshore water at its capture site may
simply represent transient cation loss in hypo-
tonic media. Similarly, much has been made of
the high bladder urea level. This has been inter-
preted to mean that coelacanths lack an active
urea resorption. More probably, lack of urea re-
sorption reflects the moribund condition of the
specimen rather than fundamental differences in
the physiology or urea excretion in sharks and
coelacanths.

CHAPTER IV

Epple and Brinn (1975) in an extensive study
of the relationships of islets of Langerhans
among vertebrates point out another peculiar
character shared by the Chondrichthyes and the
coelacanth. In both groups the endocrine islet
tissue occurs as a system of tubular structures
within a compact extra-intestinal pancreas, an
arrangement not identified in any other agnathan
or gnathostome. Although the authors designate
this arrangement as a ‘‘primitive gnathostome
type,” it differs significantly from those seen in
the extant Agnatha which shows islet tissue sep-
arate from the exocrine pancreas, occurring
within the intestinal mucosa in Myxinoidea and
intramurally in the Petromyzontoidea. An un-
expected result of their study was the parallel-
ism between the groupings based on pituitary
and islet organization. The same vertebrate
groups which exhibit a specific organization of
islet tissue exhibit a distinctive pattern of pitu-
itary Organization.

CONCLUSION

shares with the Chondrich-
thyes several unrelated, apomorphic and prob-
haracters which are difficult

1© COCiaCanin

to reconcile with the conventional systematic
interpretation of the Actinistia as a sister group
to any of the extant Osteichthyes, let alone the
Rhipidistia. These characters are reflected in
pituitary and endocrine islet organization, the
development of a rectal cation excreting gland
and urea retention in the marine environment.

The pituitary complex is specialized in a man-
ner entirely comparable to the Chondrichthyes,
particularly the more plesiomorphic hexanchid
sharks. Both groups share an independently vas-
cularized, separate portion of the pars distalis
which sequesters gonadotrope function and is
closely associated with a carotid anastomosis.
Further, both show a similar compartmentali-
zation of the remainder of the distalis in refer-
ence to acidophil cell types. The pituitary com-
plex alone thus encompasses several independent
apomorphic features.

In a peculiar, parallel manner (Epple and
Brinn 1975), both the extant coelacanth and the
Chondrichthyes show a comparable and distinc-
tive organization of the endocrine islet tissue,
with duct-like islets distributed within a compact
extra-intestinal pancreas. Evidence accumulat-
ed from both living agnathan subgroups indicate
that the coelacanth/chondrichthyian pattern of
islet tissue is derived and distinct from that of
other gnathostome groups.

Both the coelacanth and the Chondrichthyes
share the development of an accessory cation-
excreting tissue derived from the rectum which
augments salt-excretion by the more plesio-
morphic branchial *‘chloride cells.’’ Both groups
show a specialized urea retention in the marine
environment, an adaptation not utilized by any
other piscine vertebrate group albeit indepen-
dently developed by two singular euryhaline,
tropical amphibian species. Although multiple
independent convergences may account for
these shared derived characters, it is far easier
to suggest that they represent synapomorphies.

On the other hand, the two groups, coelacanth
and Chondrichthyes, obviously differ in many
respects and in numerous superficial features the
coelacanth resembles the primitive Osteichthy-
es. Striking among the latter are the presence of
bone, scales, lobed fins, an opercular apparatus
and a vestigial lung in the coelacanth and their
absence in the Chondrichthyes. A number of
these similar characters are decidedly plesio-
morphic, however, and cannot contribute to a
cladistic analysis.

LAGIOS: COELACANTHS AND CHONDRICHTHYES AS SISTER GROUPS 4]

Bone

Bone is the oldest documented vertebrate tis-
sue, and forms the only Ordovician evidence of
the group. The absence of bone in the extant
Chondrichthyes, notwithstanding Moss (1970)
who argues for the presence of cellular mem-
branous bone about teeth in Squalus acanthias,
is a secondary or derived feature of the group.
Jarvik (1977) has recently reappraised the Acan-
thodii and has concluded that they and the ex-
tant sharks are related and should be included
in Chondrichthyes (Elasmobranchii). From the
viewpoint of this analysis, early Chondrich-
thyes, i.e., Acanthodii, had both dermal and en-
dochondral bone documenting the presence of
this calcified tissue in the ancestors of the extant
Chondrichthyes.

Scales

Scales, once the hallmark of the Osteichthyes,
would thus become a plesiomorphic character
since a well developed shagreen of non-imbri-
cating plates clothed the Acanthodii (Moy-
Thomas and Miles 1971). Zangerl (1968) has not-
ed in Pennsylvanian sharks composite ‘‘scales”’
of dermal membranous bone which supported
elaborate clustered denticles. The latter pattern
is not fundamentally dissimilar from the ‘‘scales”’
of Latimeria which represent thin lamellae of
dermal bone with loosely attached odontodes
(Smith et al. 1972; Miller, this volume).

Lung

Lungs, or more generally, air sacs, are widely
distributed among Osteichthyes but also appear
to have been present in placoderms, a group
whose chondrichthian features have been de-
bated (Miles and Young 1977). Thomson (1971)
has theorized that the absence of lungs in extant
Chondrichthyes may relate to their marine and
free-swimming habitus. This same argument can
account for the great reduction in calcified tissues
and secondary loss of the airbladder as adapta-
tions to a highly mobile and pelagic existence.

Opercular Apparatus

The absence of an opercular apparatus in the
elasmobranchs may represent a specialization
rather than retention of a more plesiomorphic
condition. Holocephalans have an analagous
opercular arrangement although it is clearly in-
dependently derived and not homologous with

those of the Osteichthyes (see W. Miller, this
volume). It is not clear, however, whether the
opercular apparatus in the coelacanth is homol-
ogous with the osteichthyean structure.

Intracranial Kinesis

Apart from the general problem of the affini-
ties of the coelacanth with the Osteichthyes,
there is a more particular problem of their re-
lationship to the Sarcopterygii (Romer 1966), a
group including both the Dipnoi and the Rhipi-
distia. The coelacanth and Rhipidistia were pre-
viously considered to represent sister groups
predominantly on the basis of a single character,
the intracranial kinesis. The latter was consid-
ered a synapomorphic feature which united the
two otherwise superficially similar groups. Two
recent developments contradict this interpreta-
tion. Bjerring (1973) has shown that the intra-
cranial kinesis in coelacanths and Rhipidistians
is not homologous; it has different relationships
to the segmentation of cranial somites in the two
groups and thus is clearly not a synapomorphy.
Additionally, Nelson (1969) has shown an anal-
ogous system of subcephalic muscles which
work the ventral portion of the kinesis in the
extant polypterids, and Gardiner and Bartram
(1977) have reviewed the subcephalic fissure in
fossil palaeonisciforms, documenting the fact
that such intracranial kineses were probably
more widely distributed among palaeozoic fishes
than previously suspected (Thomson 1969).

During the course of reading Compagno’s re-
buttal to this presentation, I became aware of
Lgvtrup’s independent analysis of coelacanth
relationships (Lg@vtrup 1977). This investigator
came to the same conclusions regarding sister
group status of the coelacanth and the Chon-
drichthyes as the present author. Interestingly,
he did so without reference to the structure of
the pituitary gland or of the endocrine pancreas,
which evidence would have strengthened his ar-
gument.

In sum then, recent palaentologic reappraisal
has demonstrated that features once considered
synapomorphic, thereby uniting the coelacanth
with the extinct Rhipidistia, are in fact plesio-
morphic characters. These more recent reap-
praisals corroborate the conclusion obtained
from the present investigations that the coel-
acanth cannot be a sister group of the Rhipidis-
tia, at least as judged by their more plesiomor-
phic tetrapod descendents. The shared charac-

42 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ters between the coelacanth and the Chondrich-
thyes, which I interpret as derived (apomor-
phic), were completely unexpected, but repre-
sent specializations in rather conservative organ
systems—pituitary gland, the endocrine pan-
creas, and in cation excretion and urea retention
in the marine environment. Such signal special-
izations shared only by the extant coelacanth and
the Chondrichthyes strongly suggest that these
are synapomorphic characters of great antiquity
despite the traditional folklore of *“‘Old Four
Legs,’ the pretetrapod cousin.

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OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 8 pages

December 22, 1979

COELACANTHS: SHARK RELATIVES OR BONY FISHES?

By

Leonard J. V. Compagno

Department of Biological Sciences,
Stanford University, Stanford, California 94305

Because of their extensive fossil record,
stretching in time from the middle Devonian to
the upper Cretaceous, coelacanths (Actinistia)
were relatively well-known morphologically be-
fore the capture of the sole living representative
of the group, Latimeria chalumnae, in 1938.
Before and since that time coelacanths were
generally classified as lobe-finned fishes or Cros-
sopterygia, along with the extinct rhipidistian
fishes, and placed in the class (or subclass) Os-
teichthyes or bony fishes. However, recent in-
vestigations on the physiology and soft-part
morphology of Latimeria have suggested a rad-
ically divergent interpretation of their relation-
ships to some observers, as a sister-group to the
class Chondrichthyes (Fig. 1) or cartilaginous
fishes (sharks, rays and chimaeras). L@vtrup
(1977) listed a number of characters common to
Chondrichthyes and Actinistia, which he im-
plied were shared derived characters of the two
groups. These include common presence of a
fatty liver; a rectal gland; similarities in eye
structure; similar patterns of gray matter distri-
bution and absence of Mauthner’s fibers in the
spinal cord; stalked olfactory bulbs in the brain;
similar structure of the thymus and thyroid
glands; very large eggs; and osmoregulation
through retention of high concentrations of urea
and trimethylamine oxide in the blood plasma

45

and tissue fluids. Lagios (this volume) points to
detailed similarities in pituitary gland anatomy,
histology and histochemistry between Latimeria
and cartilaginous fishes, which he regards as
shared derived characters of Chondrichthyes
and Actinistia.

Lgvtrup (1977) was sufficiently impressed by
these similarities in soft-part morphology to sug-
gest that no serious alternative could be found
to ranking the Actinistia and Chondrichthyes as
sister groups. However, this is true only if evi-
dence from skeletal morphology is ignored or
slighted, evidence which is contrary to the hy-
pothesis of a chondrichthyian relationship for
coelacanths but strongly supports their conven-
tional placement (Romer 1966; Moy-Thomas
and Miles 1971; Andrews 1973; Miles 1977) in
the class Osteichthyes as ‘fleshy-finned’ fishes
or Sarcopterygii (a group including the lungfish-
es or Dipnoi, the coelacanths, and the rhipidis-
tians).

One practical difficulty in using soft-part mor-
phology in interpreting the phylogeny of gnatho-
stome fishes is that it restricts the number of
groups that can be compared. No information
on the soft-part characters cited above exists for
the classes Placodermi or Acanthodii. In the
class Osteichthyes there is no information on
these characters for early lungfishes, primitive

46 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ACANTHODII

ACTINISTIA

ee ee THYES

ACTINOPTERYG!I
DIPNO!
POROLEPIFORMES
OSTEOLEPIFORMES

Figure 1. Cladogram illustrating the hypothesis of im-
mediate sister-group relationship of coelacanths (Actinistia)
and chondrichthyians, after Lgvtrup (1977, figs. 5.7-5.8).
Placodermi were not placed in this cladogram, but other major
groups of gnathostome fishes are included.

ray-finned fishes (palaeoniscoid actinopterygi-
ans) or rhipidistians, although their condition in
the first two groups can be inferred from living
lungfishes, sturgeons (acipenserids), paddlefish-
es (polyodontids) and bichirs (polypterids). In
the case of rhipidistians, or at least Osteolepi-
formes (which most writers agree gave rise to
tetrapods or land vertebrates), the states of
these soft-part characters can be inferred from
living tetrapods (especially amphibians), but
with the danger that many of them were changed
in the transition from water to land and by sub-
sequent evolution on land. In contrast, the skel-
etal morphology of gnathostome fishes, includ-
ing extinct groups, has been intensively
investigated in the past 100 years and is rela-
tively well known.

Most of this account is a summary of some of
the evidence from skeletal morphology and oth-
er characters that indicates an osteichthyian and
sarcopterygian placement of coelacanths. Some
coelacanth characters are listed that occur in
other osteichthyians and sarcopterygians, are
lacking in chondrichthyians and often acantho-
dians and placoderms, and which are probably
shared derived characters that indicate a com-
mon ancestry for coelacanths and other sarcop-
terygians and osteichthyians.

DERIVED OSTEICHTHYIAN CHARACTERS OF
COELACANTHS

Osteichthyian characters of coelacanths that
are apparently shared derived characters of the
class Osteichthyes were compiled from Schaef-
fer (1968), Moy-Thomas and Miles (1971), Miles

(1973), and Gardiner (1973). Some of these were
used by Miles (1968, 1973) and Moy-Thomas and
Miles (1971) to support the placement of the spi-

ny-finned Acanthodii as the sister group of Os-
teichthyes in a common group of Teleostomi.
However, Jarvik (1977) has recently challenged
the teleostome hypothesis and presented evi-
dence that he interprets as supporting an elas-
mobranch (shark and ray) or shark relationship
for acanthodians. It is beyond the scope of this
account to consider the relative merits of an
elasmobranch or osteichthyian relationship for
acanthodians, except to note that some of the
characters cited by Jarvik as uniting sharks and
acanthodians may be primitive gnathostome or
chondrichthyian + teleostome characters, while
others (especially similarities between the orbit-
al palatoquadrate articulations in squalomorph
sharks and acanthodians) may be convergent
between these groups. To avoid taking up the
question of acanthodian relationships here, tel-
eostome derived characters are included with
the following list of derived characters shared
by coelacanths and other osteichthyians:

1. The neurocranium or braincase has a pair
of lateral occipital fissures separating the occip-
ital region from the otic region, which are pres-
ent in acanthodians but apparently absent in
chondrichthyians and placoderms.

2. The neurocranium has a ventral fissure that
basally and transversely divides it into anterior
prechordal and posterior parachordal parts,
which is also present in acanthodians but absent
in chondrichthyians and placoderms.

3. The neurocranium has a pair of spiracular
grooves in the basisphenoid region, which are
lacking in chondrichthyians and placoderms and
possibly present in acanthodians.

4. The neurocranium is more or less tropi-
basic, with a narrow basal plate, no suborbital
shelves, and relatively compressed orbital walls
in the orbital region. Acanthodians also have
tropybasic neurocrania, but placoderms and
primitive chondrichthyians have broad-based or
platybasic crania, with a broad basal plate, sub-
orbital shelves, and wide-spaced orbital walls.

5. There is a skull roof of large and small der-
mal bones superficially covering the neurocra-
nium, which has a similar pattern of homologous
elements in different osteichthyian groups.
Apart from the dermal bones of the secondary
upper jaw, opercle, and palate, these include
small rostral bones on the dorsal surface of the
snout; a paired longitudinal series on the dorsal
surface of the head, including nasals, frontals,
parietals, and postparietals; circumorbital bones,

COMPAGNO: COELACANTHS: SHARKS OR BONY FISHES? 47

including lachrimals, infraorbitals and postorbit-
als: small elements bordering the posterior mar-
gin of the skull roof, the extrascapulars; ele-
ments lateral to the longitudinal, dorsal series,
the supratemporals, intertemporals, and tabu-
lars; and elements of the cheek region anterior
to the opercle, the squamosals and quadratoju-
gals. There are problems in homologizing some
bones of the coelacanth skull roof with their
equivalents in actinopterygians and rhipidistians
(Schaeffer 1952; Moy-Thomas and Miles 1971),
but those definitely present include rostrals,
postrostrals, a frontonasal series, postparietals
(intertemporals), tabulars, extrascapulars, lac-
rimojugals, postorbitals, and squamosals. Acan-
thodians primitively have the head covered with
numerous small dermal bones resembling the
small, non-imbricated scales of the body, chon-
drichthyians primitively have the head covered
with dermal denticles similar to those elsewhere
on the body, while placoderms have a skull roof
of large bony plates that are not homologous
with those of osteichthyians.

6. Dermal bony plates are present on the pal-
ate, including paired vomers and a medial para-
sphenoid bone (absent in non-osteichthyians).

7. The palatoquadrates, or primary upper
jaws, have basal or basitrabecular articulations
with the neurocranium, posterior to their pala-
tine (or orbital) articulations with the cranium.
Early coelacanths have the basitrabecular artic-
ulation but later ones (including Latimeria) have
lost it. Sharks primitively have orbital articula-
tions with the cranium, but these occupy a ba-
sitrabecular position in many squalomorph
sharks, and are lost in rays. Chimaeras (Holo-
cephali) and lungfishes have the palatoquadrates
fused to the cranium, and hence have any joints
with the cranium obliterated. The nature of the
acanthodian palatoquadrate articulation is dis-
puted (see Jarvik 1977).

8. The palatoquadrates are expanded medi-
ally to form most of the palatal roof. These are
more laterally restricted in Chondrichthyes, but
their condition is uncertain in Acanthodii. The
palatoquadrates of placoderms are often highly
modified (see Stensid 1963), but apparently are
not expanded medially also.

9. The palatoquadrates are functionally re-
placed by dermal bones lateral to them that form
part of the superficial dorsal armor of the skull,
the maxillae and premaxillae. These form sec-
ondary upper jaws, with an arcade of teeth lat-

eral to those of the palatoquadrates. However,
maxillae were apparently lost in coelacanths
(which have premaxillae), premaxillae were also
lost in Dipnoi, and chondrichthyians, placo-
derms and acanthodians lack these bones. Plac-
oderms have upper tooth plates (anterior and
posterior superognathals) that are not homolo-
gous but are functionally similar to maxillae and
premaxillae.

10. The primary lower jaws, or Meckel’s car-
tilages, are functionally replaced by a number of
dermal and cartilage-replacement bones forming
a similar pattern in osteichthyian groups. These
include the tooth-bearing dentaries, splenials,
prearticulars, coronoids, articulars, surangulars,
and angulars (the last two fused in coelacanths).
All of these are absent in acanthodians, chon-
drichthyians and placoderms, although many of
the latter have lower tooth plates (inferogna-
thals) that partially replace the Meckel’s carti-
lages.

11. Dorsal elements of the hyoid visceral arch
are modified to form a hyomandibula, with a
proximal or dorsal articulation on the lateral
commissures of the neurocranium, lateral to and
partly or entirely above the lateral head vein. A
hyomandibula is absent in holocephalians but
present in elasmobranchs, at least some placo-
derms, and acanthodians. The neurocranial ar-
ticulation of the hyomandibula is below the lat-
eral head vein in placoderms and elasmobranchs,
but its position is disputed in acanthodians. The
different articulations of the hyomandibula in
elasmobranchs and osteichthyians and its ab-
sence in holocephalians (in which the dorsal ele-
ments of the hyoid arch are unmodified and sim-
ilar to dorsal branchial arch elements) suggest
that the hyomandibula was separately evolved
at least in chondrichthyians and osteichthyians.

12. The hyoid arch has an intermediate ele-
ment (interhyal or stylohyal) between the hy-
omandibula and ceratohyal on each side, a sym-
plectic below the hyomandibula and lateral to
the interhyal, a hypohyal between basihyal and
ceratohyal, and a glossohyal behind the basi-
hyal. An interhyal may be present in acantho-
dians, but interhyals, symplectics, hypohyals
and glossohyals are otherwise absent in non-os-
teichthyians.

13. The gill cover or hyoid septum of the
hyoid arch is greatly expanded posteriorly as an
operculum that covers all the gills. It has at least
two large dermal bones, the opercular and sub-

48 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

opercular, and branchiostegal plates ventrally.
It functionally replaces the interbranchial septa
of the gills (which are greatly reduced) as a
pumping element of the respiratory apparatus.
Elasmobranchs lack an expanded hyoid oper-
culum but have separate external gill openings
and unreduced interbranchial septa (probably a
primitive condition among gnathostome fishes).
Acanthodians have a partial hyoid operculum
with small bony plates similar to branchioste-
gals, holocephalians have a soft hyoid opercu-
lum covering the partially reduced interbranchi-
al septa, and placoderms have an internal gill
chamber with a single external aperture on each
side, but none of these fishes have an operculum
of osteichthyian type.

14. The gill arches have suprapharyngobran-
chials dorsally in addition to the infrapharyn-
gobranchials present in other fishes.

15. The shoulder girdle has a series of dermal
bones, attached to the scapulocoracoid or pri-
mary shoulder girdle and functionally replacing
it in part, with a dorsal attachment on each side
to the dermal cranium. The dermal shoulder gir-
dle of osteichthyians includes posttemporals,
supracleithra, cleithra, clavicles, and in some
groups anocleithra and postcleithra. The scapu-
locoracoid only is present in acanthodians and
chondrichthyians, but in placoderms an analo-
gous but very different dermal shoulder girdle
is present, which probably evolved separately
from the osteichthyian one.

16. The incurrent and excurrent apertures of
the nostrils are separate openings. In chondrich-
thyians and probably placoderms these are parts
of a single opening, but their condition is uncer-
tain in acanthodians.

17. A swim bladder or lungs are present, but
lacking in chondrichthyians and uncertain in
placoderms or acanthodians.

18. The vertebrae have dorsal ribs, which are
absent in non-osteichthyians as far as is known.

19. The fins are primarily supported by bony
fin rays or lepidotrichia, which are absent in
non-osteichthyians.

20. The scales are relatively large, bony
plates that are arranged in diagonal rows on the

body, with the anterior ends of the scales of each
row deeply inserted in the skin beneath the pos-
terior ends of the scales of the next most anterior
row (imbric. ted). Acanthodians have small bony
scales and chondrichthyians dermal denticles, in
both groups sot imbricated or in diagonal rows.

Placoderms are naked or have small to large
scales that are not imbricated and either not in
rows or in transverse rows.

21. At least some cranial elements are endo-
chondrally ossified, in addition to the perichon-
dral ossification found in non-osteichthyians
with bone in the endoskeleton. Chondrichthyi-
ans lack perichondral bone but have it replaced
by perichondral calcified nodules.

22. The inner ear has large, regular otoliths,
possibly also present in acanthodians.

DERIVED SARCOPTERYGIAN CHARACTERS OF
COELACANTHS

- Within the Osteichthyes coelacanths are mor-

phologically closest to the rhipidistian fishes, in-
cluding the Osteolepiformes, Rhizodontiformes,
Porolepidormes, and Onychodontiformes, and
with them are generally placed in a major group
(often ranked as a subclass or superorder of Os-
teichthyes), the Crossopterygii. The lungfishes
or Dipnoi, although specialized and aberrant in
many characters, are morphologically closer to
rhipidistians and coelacanths than actinopteryg-
ians and are sometimes included with coel-
acanths and rhipidistians in a common group,
the Sarcopterygii (Romer 1966), which is ranked
as a subclass of Osteichthyes coordinate with
Actinopterygii. Derived sarcopterygian charac-
ters linking these fishes are compiled from Ro-
mer (1966) and Miles (1977):

1. The neurocranium has a lateral crest on
each side that supports the edges of the dermal
skull roof, roofs the gill chambers, and laterally
defines the dorsal or supraotic muscle fossae.

2. The basal plate of the neurocranium lacks
aortic canals, which are present in early acti-
nopterygians as well as acanthodians, placo-
derms, and early chondrichthyians.

3. The neurocranium has an adotic process
on the lower edge of the jugular groove near the
first gill arch.

4. The head of the hyomandibula has a broad
articular condyle or pair of condyles, with a lat-
eral articulation on the lateral commissure that
straddles the lateral head vein.

5. Epaxial lepidotrichia are present in the
caudal fin, forming a variably developed epi-
chordal lobe (absent in Actinopterygii).

6. The pectoral fins, and usually the pelvic
fins also, have fleshy, lobate bases. The pectoral
basals and radials are very narrow and project
into the fin. Paired fins are primitively broad-

COMPAGNO: COELACANTHS: SHARKS OR BONY FISHES? 49

based and not lobate in actinopterygians, acan-
thodians, early chondrichthyians, and apparent-
ly placoderms, but lobate fins have indepen-
dently and repeatedly evolved in several
actinopterygian groups, in extinct xenacanth
sharks and some neoselachian groups, in the ex-
tinct holocephalian Chondrenchelys and living
chimaeras, and in antiarch placoderms.

There are a number of derived characters in
coelacanths that suggest a close relationship to
rhipidistians and which are crossopterygian
characters of these groups. These are compiled
from Millot and Anthony (1958), Moy-Thomas
and Miles (1971), Gardiner (1973), and Andrews
(1973):

1. The lateral occipital fissures of the neuro-
cranium are partially filled.

2. The neurocranial ventral fissure is expand-
ed and modified as an intracranial joint, with the
notochord enlarged anteriorly and expanded for-
ward to the basisphenoid ossification, the basi-
occipital bone laterally excavated, and a ventral
basicranial muscle present and connecting the
cranial halves.

3. The neurocranium lacks an ossified prootic
bridge.

4. The hyomandibula has two articular con-
dyles, a dorsal one and a ventral one, the latter
opposite the lateral head vein.

Also, Andrews (1977) notes that the vertebral
structure of coelacanths, as exemplified by Lar-
imeria, is basically similar to that of rhipidis-
tians, and differs from that of other fishes, but
she was uncertain whether this common verte-
bral type is a shared derived character of cros-
sopterygians.

The exact relationship of coelacanths to other
sarcopterygians remains uncertain. Three hy-
potheses of coelacanth relationships seem most
likely and are expressed here as cladograms
(Figs. 2-4). The first, suggested by the classifi-
cation of Romer (1966), is that rhipidistians and
actinistians are sister groups (Fig. 2), both
groups are sister to the Dipnoi, and that all three
groups (the Sarcopterygii) are sister to the Ac-
tinopterygii. The grouping of rhipidistians and
coelacanths in a common crossopterygian group
primarily depends on the interpretation of the
intracranial joint as a unique derived character
of the Crossopterygii, with the condition of the
ventral fissure of early Dipnoi considered prim-
itive. The ranking of rhipidistians and coela-
canths as sister groups rests on the presence of

ACTINOPTERYGII
DIPNOI

SARCOPTERYGI!
CROSSOPTERYGI

ACTINISTIA
RHIPIDISTIA

FiGuRE 2. Cladogram of osteichthyian relationships, illus-
trating the hypothesis of sister-group relationship of coel-
acanths (Actinistia) and rhipidistians; from the classification of
Romer (1966).

many divergent characters in coelacanths, in-
cluding their lack of choanae, reduced dermal
cheek bones, lack of maxillae, quadratojugals,
and mandibular bones, reduction in number and
increase in size of sensory canal pores, devel-
opment of dorsal antotic articulations of the pal-
atoquadrates to the neurocranium, presence of
extracleithra in the dermal shoulder girdle, and
presence of relatively few, unbranched lepido-
trichia in the fins (listed from Andrews 1973).
However, many of these characters, except per-
haps absence of choanae, may be unique derived
characters of coelacanths and may not preclude
the descent of coelacanths from some rhipidis-
tian group.

Andrews (1973) accepted the grouping of coel-
acanths and rhipidistians as crossopterygians,
but did not discuss the relationships of crossop-
terygians to other osteichthyians. She suggested
a new hypothesis (Fig. 3), that coelacanths are
the sister group of porolepiform rhipidistians
and that both form the sister group of osteo-
lepiform, rhizodontiform, and onychodontiform
rhipidistians. This was based on the common
presence of a specific ‘binostian’ type of ‘‘skull
table’’ (the postparietal, tabular and extratem-
poral bones of the dermal skull roof) in Porolep-
iformes and Actinista, as contrasted by the
‘quadrostian’ type in Rhizodontiformes, Osteo-
lepiformes and Onychodontiformes. Andrews
also mentioned primitive, undescribed Devonian
coelacanths with rhipidistian or even porolepi-
form characters, but did not consider coel-
acanths as straight porolepiform derivatives be-
cause known coelacanths showed no sign of loss
of dentine folding in their teeth (folding present
in porolepiform teeth), symphysial tooth whorls
of Porolepiformes, and the elongated, dipnoan-
like pectoral fin axes of Porolepiformes (all of
which characters she considered as unique de-
rived features of Porolepiformes).

Miles (1977), in a recent study of primitive
Devonian lungfishes, suggested a third alterna-

50 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

TINISTIA
BINOSTIA porns
POROLE PIFORMES
ONYCHODONTIFORMES
QUADROSTIA RHIZODONTIFORMES

OSTEOLEPIFORMES

FiGure 3. Cladogram of crossopterygian relationships, il-

lustrating the hypothesis of sister-group relationship of coel-
acanths (Actinistia) and Porolepiformes, after Andrews (1973,
fig. 5).

tive, that Dipnoi and rhipidistians (or “‘choa-
nates’’) are sister groups, both form a sister
group to coelacanths, and that all three groups
(the Sarcopterygii) are the sister group of Actin-
opterygii (Fig. 4). He bases his hypothesis of
interrelationships within the Sarcopterygii on
the shared possession in early lungfishes and
rhipidistians of a supraotic cavity in the neuro-
cranium, cosmine on the scales and dermal
bones, and submandibular bones, which he re-
gards as shared derived characters of these
groups that are lacking in coelacanths. This hy-
pothesis requires the intracranial joint of coela-
canths and rhipidistians (as well as other *‘cros-
sopterygian’’ characters of these groups listed
above) to be primitive for sarcopterygians (and
a unique derived character of the group), and
that lungfishes have secondarily lost the joint.
However, the condition of the ventral fissure in
the primitive lungfishes studied by Miles does
not suggest secondary loss of an intracranial
joint, but rather the primitive osteichthyian or
even teleostome condition. This would support
the hypothesis that the intracranial joint of coel-
acanths and rhipidistians is a shared derived
character of these fishes, but requires that coel-
acanths would have secondarily lost the su-
praotic cavity, submandibular bones, and cos-
mine of lungfishes and rhipidistians, and that
these characters be considered as primitive ones
for Sarcopterygii. Miles (1977) initially support-
ed the hypothesis (Fig. 2) ranking coelacanths
and rhipidistians as sister groups, but eventually
supported the hypothesis of lungfishes and rhip-
idistians as sister groups (Fig. 4). He rejected
Andrews’ (1973) arrangement of Porolepiformes
and Actinista as sister groups because of the ab-
sence of the characters uniting lungfishes, Po-
rolepiformes, and other rhipidistians in coel-
acanths. Miles accepted Andrews’ phylogeny of
rhipidistians (with coelacanths deleted) and sug-
gested that the ‘binostian’ condition in Porole-

ACTINOPTERYGII

| ACTINISTIA
a

RHIPIDISTIA

FIGURE 4.

trating the hypothesis of sister-group relationship of coel-

acanths (Actinistia) and rhipidistians + Dipnoi, after Miles
(1977, fig. 158).

Cladogram of osteichthyian relationships, illus-

piformes and coelacanths may be a primitive
condition of sarcopterygians and hence of no
weight in supporting the placement of these fish-
es In a Common group.

A radically different interpretation of coel-
acanth relationships was proposed by Bjerring
(1973), who thought that coelacanths are not re-
lated to rhipidistians because their intracranial
joints are not homologous. His arguments are
complex and detailed but primarily hinge on his
interpretations of the embryonic components of
the posterior part of the neurocranium in these
fishes, based in turn on extrapolations from cra-
nial development of other fishes and other ver-
tebrates and hypotheses on the vertebral com-
ponents of the gnathostome neurocranium (but
not on direct morphogenetic evidence from the
cranium of coelacanths or rhipidistians). It also
depends on his view that an intracranial! joint of
crossopterygian type (or types), the associated
basicranial muscle, and the greatly expanded
anterior end of the notochord in coelacanths and
rhipidistians are primitive gnathostome charac-
ters, and that their absence in other gnathostome
fishes is derived. However, the presence of a
ventral fissure but absence of an intracranial
joint in early actinopterygians (Gardiner 1973;
Gardiner and Bartram 1977) and Dipnoi (Miles
1977, but see above), the absence of the joint in
acanthodians (Miles 1968, 1973; Jarvik 1977),
placoderms (Stensid 1963; Moy-Thomas and
Miles 1971) and all known chondrichthyians,
and the suite of derived osteichthyian characters
uniting the Dipnoi, Actinista, Rhipidistia, and
Actinopterygii all strongly suggest that an intra-
cranial joint of crossopterygian (or sarcopteryg-
ian) type is derived (Gardiner 1973; Miles 1977).
The observed morphological differences be-
tween rhipidistian and coelacanth intracranial
joints may support the hypothesis that coel-
acanths cannot be derived from any known rhip-
idistian type, without negating their placement

COMPAGNO: COELACANTHS: SHARKS OR BONY FISHES? 5]

as sister groups of either the Rhipidistia or Rhip-
idistia + Dipnoi.

OTHER CHARACTERS SEPARATING
COELACANTHS AND
CHONDRICHTHYIANS

In addition to shared derived characters plac-
ing them with Sarcopterygii and Osteichthyes,
coelacanths have many other differences from
Chondrichthyes. These include unique derived
characters of Chondrichthyes as well as char-
acters of uncertain polarity. The following list
is hardly exhaustive as it includes mostly skel-
etal characters and can be expanded by com-
parisons of other systems in coelacanths and
chondnrichthyians.

1. Chondrichthyian neurocrania have lateral-
ly expanded ethmoid regions with globular nasal
capsules but no medial rostral cavity and organ,
the cranial cavity communicating with the ex-
terior through an anterior fontanelle, and an en-
dolymphatic or parietal fossa on the cranial roof,
with paired perilymphatic and endolymphatic
foramina. Coelacanth crania have a medial ros-
tral cavity and organ in the ethmoid region,
which is not expanded laterally with globular
nasal capsules, no anterior fontanelle, and no
parietal fossa on the cranial roof.

2. In chondrichthyians the inner ears com-
municate with the exterior through paired en-
dolymphatic pores on the dorsal surface of the
head, but not in coelacanths.

3. The palatoquadrates of chondrichthyians
usually have anteriomedial palatine processes
that meet in an upper symphysis at the midline
of the mouth, but those of coelacanths (and oth-
er osteichthyians) lack these processes and do
not meet in an upper symphysis. The palato-
quadrates of coelacanths (and rhipidistians)
have several parts separated by sutures, those
of chondrichthyians form a single unit. The man-
dibular joint (posterior joints of palatoquadrates
and Meckel’s cartilages) is double in chondrich-
thyians (and acanthodians) but single in coela-
canths (and other osteichthyians).

4. The gill arches of coelacanths (and oste-
ichthyians generally) have small bony plates
with small teeth, while those of chondrichthyi-
ans have dermal denticles, denticle gill rakers,
papillar gill rakers, or chondropapillar gill rak-
ers. The basibranchial skeleton of coelacanths
has a single basibranchial element present, but

multiple basibranchials are primitively present
in chondrichthyians.

5. The nostrils are ventral on the snout of
chondrichthyians (as some placoderms and pos-
sibly acanthodians), dorsolateral in coelacanths
(as in other osteichthyians generally).

6. Chondrichthyians generally have subter-
minal mouths, coelacanths terminal mouths (as
in other osteichthyians generally).

7. Chondrichthyians have highly differentiat-
ed teeth with distinct roots and crowns, bound
to the primary jaws by the dental membrane
only and arranged in transverse rows or tooth
families. Coelacanths have simple conical teeth
that are fused to the jaw bones and are not ar-
ranged in rows.

8. Chondrichthyians have paired intromittant
organs or claspers developed as posterior exten-
sions of the pelvic fins, which are lacking in
coelacanths (and other osteichthyians).

9. Chondrichthyians have bone, when pres-
ent, confined to the roots of denticles and teeth
but otherwise lack it; it is replaced by calcified
nodules in the endoskeleton. Coelacanths, as
with other osteichthyians, placoderms, and
acanthodians, have bone in the endoskeleton
and the external dermal covering.

FALSIFICATION OF THE HYPOTHESIS OF
COELACANTH-CHONDRICHTHYIAN
RELATIONSHIPS

The numerous differences between coel-
acanths and chondrichthyians and similarities
between coelacanths, rhipidistians, dipnoans,
and actinopteryians make it unlikely that simi-
larities in soft-part morphology between Actin-
istia and Chondrichthyes reflect an immediate
common ancestor for them shared by no other
group of gnathostome fishes. Acceptance of the
cladogram implied by these soft-part similarities
(Fig. 1) requires the acceptance of either of two
grossly unparsimonious hypotheses: 1. The
osteichthyian, sarcopterygian and crossoptery-
gian characters of coelacanths, if regarded as
derived, were separately evolved in these fishes
from a primitive, chondrichthyian-like ancestor
and are closely convergent in these characters
with dipnoans, rhipidistians, and actinopterygi-
ans. Coelacanths, at their onset in the Devonian,
are no more chondnichthyian-like in these char-
acters than their Mesozoic and living descen-
dents, and if anything are less divergent from

other sarcopterygians. 2. The osteichthyian, sar-
copterygian and crossopterygian characters of
coelacanths are a result of common ancestry
with rhipidistians, dipnoans and actinopterygi-
ans, but chondrichthyians are still the immediate
sister group of coelacanths. The absence, in this
case, of osteichthyian, sarcopterygian and cros-
sopterygian characters in chondrichthyians is
the result of secondary loss without a trace.
However, there is no evidence from living or
fossil chondrichthyians that they ever had a ven-
tral fissure, intracranial joint, spiracular groove,
primitively tropibasic neurocranium, dermal
head, palatine, jaw, and shoulder bones of os-
teichthyian type, an osteichthyian type of pala-
toquadrate or hyomandibular, symplectics, in-
terhyals, hypohyals, urohyals, an osteichthyian
hyoid operculum, suprapharyngobranchials,
dorsal ribs, a swim bladder or lungs, separate
narial incurrent and excurrent apertures, lepi-
dotrichia, or scales or otoliths of osteichthyian
type. Chondrichthyes, from Devonian to Re-
cent, show no sign of a shift from an osteich-
thyian morphotype, and at most are related to
the Osteichthyes at a basal level, as a sister
group of acanthodians and osteichthyians
(Schaeffer 1975; Schaeffer and Williams 1977).
In the light of the great divergence between coel-
acanths and chondrichthyians, it seems likely
that some of their similarities in soft-part mor-
phology may reflect primitive gnathostome (or
chondrichthyian + teleostome) traits, and other
convergences between these fishes.

ACKNOWLEDGMENTS

I would especially like to thank Bobb Schaef-
fer (Department of Paleontology, American Mu-
seum of Natural History) and Warren Freihofer
(then of the Department of Ichthyology, Cali-
fornia Academy of Sciences) for reviewing an
early draft of this paper, and for offering helpful
suggestions. I would also like to thank Michael
Lagios (Children’s Hospital of San Francisco)
for allowing me to read a manuscript copy of his
paper on coelacanth relationships, and John

52 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

McCosker (Steinhart Aquarium, California
Academy of Sciences) for making it possible for
me to contribute this account.

LITERATURE CITED

ANDREWS, S. M. 1973. Interrelationships of crossopterygi-
ans. Zool. J. Linn. Soc., London 53(supp. 1):137—177.

. 1977. The axial skeleton of the coelacanth, Latime-
ria. Linn. Soc. London Symp. (4):271-288.

BJERRING, H. C. 1973. Relationships of coelacanths. Zool.
J. Linn. Soc., London 53(supp. 1):179-204.

GARDINER, B. G. 1973. Interrelationships of teleostomes.
Zool. J. Linn. Soc., London 53(supp. 1):105—135.

, AND A. W. H. BARTRAM. 1977. The homologies of
ventral cranial fissures in osteichthyians. Linn. Soc. Lon-
don Symp. (4):227-245.

JARVIK, E. 1977. The systematic position of acanthodian fish-
es. Linn. Soc. London Symp. (4):199-225.

Lgvrrup, S. 1977. The phylogeny of vertebrata. John Wiley
& Sons, London, New York. xii + 330 pp.

MILEs, R. S. 1968. Jaw articulation and suspension in Acan-
thodes and their significance. Pages 109-127 in T. Orvig,
ed., Current problems of lower vertebrate phylogeny. Pro-
ceedings of 4th Nobel Symposium. Interscience, New
York.

1973. Relationships of acanthodians. Zool. J. Linn.

Soc., London 53(supp. 1):63-103.

1977. Dipnoan (lungfish) skulls and the relationship
of the group: a study based on new specimens from the
Devonian of Australia. Zool. J. Linn. Soc., London 61(1—
3):1-328.

MILLoT, J.,. AND J. ANTHONY. 1958. Anatomie de Latimeria
chalumnae. Tome 1. Squelette, muscles et formations de
soutien. C. N. R. S., Paris. 122 pp.

Moy-TuHomas, J. A., AND R. S. Mires. 1971. Palaeozoic
fishes, 2nd ed. W. B. Saunders Co., Philadelphia, Toronto.
x1 + 259 pp.

Romer, A. S. 1966. Vertebrate paleontology, 3rd ed. Uni-
versity of Chicago Press, Chicago, London. viii + 468 pp.

SCHAEFFER, B. 1952. The Triassic coelacanth fish Diplurus,
with observations on the evolution of the Coelacanthini.
Bull. Am. Mus. Nat. Hist. 99(2):25-78.

. 1968. The origin and basic radiation of the Osteich-

thyes. Pages 207-222 in T. Orvig, ed., Current problems of

lower vertebrate phylogeny. Proceedings of 4th Nobel Sym-
posium. Interscience, New York.

. 1975. Comments on the origin and basic radiation of

the gnathostome fishes with particular reference to the feed-

ing mechanism. Collog. Int. C. N. R. S. (218):101-109.

, AND M. WILLIAMS. 1977. Relationships of fossil and
living elasmobranchs. Am. Zool. 17:293-302.

STENSIO, E. A. 1963. Anatomical studies on the arthrodiran
head. Part I. K. Sven. Vetensk. Akad. Handl., Ser. 4,
9(2): 1-419.

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 3 pages

December 22, 1979

REPLY TO THE REBUTTAL OF LEONARD COMPAGNO.
*“COELACANTHS: SHARK RELATIVES OR BONY FISHES’”’

A significant advantage to a comparative and
in particular a systematic analysis based on cal-
cified tissues, to wit the skeletal and calcified
dermal structures of fishes, is the ability to uti-
lize such material from the fossil record. It must
be emphasized, however, that this is perhaps the
only advantage to such an approach. The other
various organ systems, peptide sequences of
hormones and other proteins, and biochemical
specializations which characterize a living or-
ganism, are necessarily excluded from consid-
eration by this traditional method of systematic
analysis. Moreover, many extant taxa poorly
represented in the fossil record can hardly be
evaluated by this method. This is not to say that
such a traditional method has not been success-
fully used, but only that it presents a very lim-
ited viewpoint. The systematics developed on
the basis of calcified tissues are more useful,
moreover, when the members of the respective
taxa show substantial divergence so that a pat-
tern of baseline or plesiomorphic character traits
can be distinguished from subsequent special-
izations. When, as in the case of the coelacanth,
one is limited to a single surviving species and
a rather homogeneous fossil record, it becomes
necessary to evaluate evidence other than pa-
leontologic remains.

By
Michael D. Lagios

Children’s Hospital,
San Francisco, California 94118

53

Compagno notes that the extinct classes Plac-
odermi and Acanthodii cannot be evaluated by
a comparative approach using organ systems
other than calcified tissues. This is true, but it
must be recalled that traditional systematics
has done little to clarify the relationships of
these two groups. Indeed, numerous papers
have attempted to place the Acanthodii in the
Osteichthyes or Chondrichthyes, depending on
the interpretation of the data. Ten of the 17 de-
rived skeletal characters which Compagno cites
for the coelacanth and the Osteichthyes were
present in the Acanthodul, which Jarvik (1977)
regards as a Chondrichthian group.

Many of the characters which have been uti-
lized to distinguish bony fishes from the Chon-
drichthyes reflect their divergent specializations
over the last 300 million years rather than base-
line differences of the groups. The Chondrich-
thyes are generally characterized as benthic
‘nose’ ’-fishes with subterminal mouths, a sha-
green of denticles, claspers, unrestricted noto-
chord with neither centra nor ribs, and serially
replaced teeth which are loosely held to the
jaws. Yet the archetypal palaeozoic shark Clad-
oselache is an obvious non-benthic ‘eye’ ’-pred-
ator with reduced rostrum, terminal mouth,
composite multicuspid scales, an absence of

54 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

claspers, and, as speculated by Keith Thomson
(1972), a probable hydrostatic organ. In con-
trast, the *‘Teleostome’’ (= Osteichthyes +
Acanthodii) Acanthodes shared with Cladosel-
ache an unrestrictive notochord with calcified
neural arches but no ribs, a similar jaw suspen-
sion, and in some forms, composite scales or
denticles on a base of acellular bone, and serial
whorls of multicusped teeth attached to the jaw
cartilages by connective tissue.

Among the derived sarcopterygian characters
of coelacanths which Compagno cites are the
intracranial kinesis which has traditionally been
used as the most important synapomorph char-
acter linking the coelacanth with the rhipidis-
tian fishes. As discussed at length in my paper
(this volume), the significance of this feature has
been interpreted very differently and recent evi-
dence suggests that such kineses including the
ventral fissure are widely distributed in archaic
gnathostome groups.

The superficial similarities of the coelacanth
lobate fin with those of the Rhipidistia, and with
some imagination early amphibian groups, has
also traditionally been used as evidence for an
association. As Compagno himself notes, such
limb structure independently evolved in several
other groups including certain mesozoic sharks.

The question of bone in the Chondrichthyes.
As noted in my discussion, bone is an archaic
plesiomorphic character of vertebrates and its
virtual absence in modern sharks no doubt re-
lates to secondary adaptation. Similar great re-
ductions in mineralized tissues have occurred in
true bony fishes, particularly those that are me-
sopelagic in habitat, and has frequently been
noted in the evolution of several different fish
taxa. The skeletal bone as well as the dermal
ornamentation of the living coelacanth is itself
greatly reduced and much of the bone including
that of the dentary (see Miller, this volume) is
largely acellular and membranous in type.
Should Jarvik’s interpretation of the Acanthodii
(1977) be sustained, it would make the entire
question of bone in modern sharks moot, as ear-
ly acanthodians have both well developed min-
eralized skeletal tissue, including endochondral
bone and dermal ornamentation.

Compagno chooses to consider the ‘‘scales”’
or the branchial arches of bony fishes as distinct
from the similarly located ‘‘denticles’’ of the
chondrichthian gill arches, although both are
epidermal derivatives composed of dentine. The

‘“‘scales’’ of the coelacanth gill arch are entirely
comparable to the odontodes which ornament
the body scales. Their gross appearance and
structure are most ‘“‘chondrichthian-like’’ and
are reminiscent of the elaborate composite
scales described by Zanger! (1968) in mesozoic
sharks.

Characteristically, in a cladistic analysis no
weight is given to particular traits in analyzing
differences between taxa. Although this makes
a great deal of sense in terms of the internal logic
of cladism, it ignores the empirically-observed
degrees of conservation and polymorphism for
particular character traits. Forexample, somatic
color pattern can be subjected to cladistic anal-
ysis but the diversity of this trait is so enormous
that no reasonable weight can be given it except
on the interspecific level. Pigmentation obvious-
ly reflects specific adaptation and as such is a
very labile character trait. Although I don’t wish
to suggest that calcified tissues represent an or-
gan system or similar lability, they are nonethe-
less decidedly less stabile as compared to the
limited patterns of endocrine organization. Nu-
merous examples of convergence in cranial mor-
phology and locomotor skeletal structures are
known, e.g., the caudal peduncle of thunnid
fishes and isurid sharks, the limb structure of
elephants and brontosauri, etc. Such conver-
gence obviously reflects adaptation to specific
environmental needs. In contrast endocrine or-
ganization represents a ‘“‘nonsense’’ feature in
terms of function. Insulins produced by the dis-
crete and separate islet tissue, the Brockmann’s
bodies, of teleost fishes and those of the diffuse
small intrapancreatic islets of mammals are not
only functionally equivalent in their respective
taxa, but, due to marked similarities in amino
acid sequence, show considerable biologic ac-
tivity in heterologous taxa as well. The gonad-
otropins of sharks, synthesized by cells in a dis-
crete, isolated ventral lobe are entirely equivalent
functionally to those of other vertebrate groups.
Although comparative work on the gonadotro-
pins of the coelacanth and other vertebrates has
not been performed it is clear that the growth
hormone of the coelacanth shows numerous an-
tigenic similarities with those of tetrapods and
preserves significant biologic activity in the lat-
ter (Hayashida, this volume).

In summary, the coelacanth has historically
been interpreted as a crossopterygian predom-
inantly on the basis of a number of superficial

LAGIOS: REPLY TO REBUTTAL OF COMPAGNO

skeletal similarities on which Compagno has
based his rebuttal. These similarities include
many, such as lobed fins, scales, and cranial ki-
nesis, which have been independently evolved
by various taxa and could represent examples
of convergence in the coelacanth. Analysis of
organ systems other than skeletal tissues, such
as endocrine and visceral anatomy, which em-
pirically show a greater degree of morphologic
conservatism among extant taxa, suggest that
the sister group of the coelacanth is to be found
among the Chondrichthyes. Modern Chondrich-
thyes, with their great reduction in calcified tis-
sues, simplified scales and serially replaced
teeth, hardly seem a likely sister taxon to “‘Old
Four Legs,’ yet most of the *“‘diagnostic”’ char-

ss

acter traits of the Chondnchthyes represent spe-
cializations for an active, predaceous habitus.

The conclusions which have been reached on
the basis of endocrine organization, soft part
morphology and biochemistry in the coelacanth
are admittedly at odds with the prevailing con-
ception of its systematic position. Though soft
part morphology cannot be compared among
extinct taxa, it nonetheless carries significant
weight in any systematic analysis. In the opinion
of this observer more so than the more labile
calcified tissues which have conventionally been
used to classify the coelacanth as a sister group
to the Rhipidistia and as a member of the Os-
teichthyes.

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 12 pages

December 22, 1979

VENTRAL GILL ARCH MUSCLES AND THE PHYLOGENETIC
RELATIONSHIPS OF LATIMERIA

By
E. O. Wiley

Museum of Natural History,
The University of Kansas,
Lawrence, Kansas, 66045

ABSTRACT

The phylogenetic relationships of Latimeria chalumnae as evidence by ventral gill
arch musculature is reviewed. I conclude that Latimeria and, by inference, other ac-
tinistians (coelacanths) are the sister group of all other osteichthyans. Osteological
evidence which supports this hypothesis is reviewed. Two alternate views are examined:
(1) that actinistians are sarcopterygians and (2) that actinistians are relatives of Chon-
drichthyans. Alternate (1) is reasonable if the intracranial joint is considered synapo-
morphic (derived) for actinistians and rhipidistians. Alternate (2) is supported only by
characters which can be shown to be plesiomorphic (primitive) or of uncertain quality.
I conclude that (a) Latimeria is an osteichthyan and (b) that Latimeria and other
actinistians are the sister group of all other osteichthyans (the Euosteichthyes).

INTRODUCTION

The phylogenetic relationship of Latimeria
chalumnae has been debated as much by past
investigators as it is debated in this book. Wes-
toll (1949) placed coelacanths with the sarcop-
terygian groups Dipnoi (lungfishes) and choa-
nata (Rhipidistia and Tetrapoda). He considered
the intracranial joint a primitive character of
Sarcopterygii. Romer (1966) and Thomson

56

(1969) considered Latimeria the closest living
relative to the tetrapods and closely related to
the extinct rhipidistians. The alignment of coel-
acanths with rhipidistians and tetrapods has
most recently been advocated by Andrews
(1973) and Miles (1975). Other investigators
have expressed doubts. Although Nelson (1969)
grouped Latimeria in Sarcopterygil, he suggest-
ed that Latimeria might be derived from a non-

WILEY: GILL ARCH MUSCLES

sarcopterygian teleostome.! Von Wahlert (1968)
suggested that coelacanths were the sister-group
of choanates + actinopterygians and that Dip-
noi was the sister-group of the three (Fig. 1a).
Bjerring (1973) questioned the homology of the
intracranial joints of rhipidistians and coel-
acanths and suggested that the relationships of
coelacanths were unknown. Andrews (1977)
came to a similar conclusion, pointing out that
the vertebrae of Latimeria invite comparison
with all the major osteichthyan groups. Miles
(1977) concluded that coelacanths were sarcop-
terygians, but presented evidence that Dipnoi
was more closely related to choanates than are
coelacanths (Fig. 1b). Lg@vtrup (1977) came to
an entirely different conclusion—that coel-
acanths are the sister-group of chondrichthyans
(Fig. lc). A similar viewpoint is expressed in
this volume by Lagios.

My own work with the ventral gill arch mus-
cles of gnathostomes (Wiley 1979; specimens
examined are listed in that paper) led me to con-
clusions that differ in one respect or another
from all the above authors. I concluded that
coelacanths are the sister-group of all other Re-
cent osteichthyans (Fig. 1d). Below I will review
the evidence supporting this hypothesis and at-
tempt to resolve the apparent character conflicts
between this hypothesis and competing hypoth-
eses.

Phylogenetic Analysis

Phylogenetic (or ‘‘cladistic’’) analysis is a
method to assess the relative genealogical rela-
tionships between organisms. It is designed to
ask if two groups of organisms shared a common
ancestral species not shared by another organ-
ism. If these two groups show similarities in
morphology or other attributes not shown by
other groups, then the method assumes it 1s
more parsimonious to consider these similarities

' A word about nomenclature is in order. Teleostomi in-
cludes, in this paper, acanthodians, coelacanths, dipnoans,
rhipidistians, tetrapods and actinopterygians: in short, all
non-chondrichthyan and non-placoderm gnathostomes.
Osteichthyans are all teleostome groups except acanthodians.
Sarcopterygii, in the common usage, includes choanates (rhip-
idistians and tetrapods), Dipnoi and coelacanths. But, coela-
canths are excluded from the Sarcopterygii in this paper for
reasons explained below. Thus, ‘‘Sarcopterygii’’ used without
qualification refers only to Choanata and Dipnoi. The term
‘‘Euosteichthyes” refers to Sarcopterygii (as defined herein)
plus Actinopterygii. These terms follow Wiley (1979).

LY iS SS 9 ~ => S is ~ ~
OS OS SS OS an ee
Sy Q) 1S) 1S) NE (Se {S) 9 1S) Na

fe) b.

SS S i~ x We Ss S < ~
OQ) (@) Oo To oS oT VD LY & >
GG oO oS = GS soos ND

C. de
FiGure 1. Four hypotheses of the interrelationships of

chondrichthyans (Chon), dipnoans (Dipn), actinistians (coel-
acanths, Coel), choanates (Choa) and actinopterygians (Acti):
(a) from von Wahlert (1968); (b) from Miles (1978); (c) from
Levtrup (1977); (d) from Wiley (1979).

as having developed in the common ancestor of
the two groups than it is to assume that these
similarities are the result of convergence or par-
allelism. Characters hypothesized to demon-
strate a unique common ancestry relationship
are synapomorphous or derived characters.
These may be defined as homologous characters
developed in the ancestral species of the taxa
showing them and in no other ancestral species.
A number of alternate hypotheses based on dif-
ferent characters may result from such an anal-
ysis. In that case the phylogeneticist attempts to
resolve the conflict by demonstrating (1) that
supposed synapomorphies are actually primitive
or plesiomorphous characters or (2) that sup-
posed synapomoprhies are actually non-homol-
ogies. Failing this, the hypothesis with the great-
est number of hypothesized synapomorphies is
considered the best estimate of phylogenetic de-
scent. For those interested in the finer aspects
of this method and justification for using the
method rather than alternates I can recommend
Hennig (1966) and various articles in the journal
Systematic Zoology. My thoughts on the meth-

58 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE 2.

Diagrammatic ventral views of the gill arch hypobranchial muscles of (a) a generalized shark; (b) a generalized

dipnoan; (c) a generalized actinopterygian. Bone and cartilage stippled, basibranchial copulae not shown. Abbreviations: Cl,
ceratobranchial 1; CA, coracoarcualis; CB1, 2, coracobranchiales | and 2; CM, coracomanbibularis; HA, hyoid arch; LJ, lower

jaw; PG, pectoral girdle; STH, sternohyoideus.

od are outlined in Wiley (1975, 1976) and En-
gelmann and Wiley (1977).

Ventral Gill Arch Muscles

Branchial and hypobranchial ventral gill arch
muscles serve to abduct and adduct the visceral
arches, to open and aid in opening the lower jaw
and to aid in moving the pectoral girdle. Hypo-
branchial muscles are derived from the ventral
portions of the more anterior post-otic body
myotomes and are innervated by one or more
spino-occipital (post-vagus) nerves. These mus-
cles are shown in Table 1. Branchial muscles are
derived from lateral plate mesoderm. Those of
the first arch are innervated by a branch of the
glossopharyngeal (IX) and/or a branch of the
vagus (a branch of X,). Branchial muscles of
more posterior arches are innervated by the

TABLE |.
Wiley (1979).

post-trematic branch of the vagus for their re-
spective arch (i.e., the X,—X, for those species
with five arches). These muscles are shown in
Table 2. Note in the discussion that some of the
muscles shown in these tables are homologues
of others and that none are found in all taxa.

Hypobranchial Muscle Patterns

Sternohyoideus Muscles.—Latimeria cha-
lumnae has a pair of sternohyoidei. The sterno-
hyoideus of each side originates on the clavicle
medial and dorsal to the coracomandibularis. It
inserts on the ‘“‘urohyal’’ (not homologous with
the teleost urohyal) just anterior to the insertion
of the transversus ventralis 2.

All gnathostomes have sternohyoidei. These
muscles in Latimeria are unique in their inser-
tion. The usual insertion for primitive members

HyPOBRANCHIAL MUSCLES ASSOCIATED WITH THE VENTRAL GILL ARCHES OF GNATHOSTOMES. Names follow

Muscle Origin

Insertion

Coracoarcualis Pectoral girdle

Coracomanbibularis Pectoral, girdle, and/or

Sternohyoides

Lower jaw

sternohyoides, or 3rd hypobranchial

Sternohyoideus Pectoral girdle

Coracobranchiales Pectoral girdle (last CB only)

Hyoid arch or urohyal

Ceratobranchial of respective arch

and medial hypobranchial muscles

WILEY: GILL ARCH MUSCLES

59

TABLE 2. BRANCHIAL MUSCLES ASSOCIATED WITH THE VENTRAL GILL ARCHES OF GNATHOSTOMES. Names follow Wiley

(1979).

Muscle

Origin

Interarcualis |
Interarcualis 2
Interarcualis 3
Interarcualis 4
Interarcualis 5
Pharyngoclavicularis

Ph. externus

Ph. internus
Obliquus ventralis |
Obliquus ventralis 2
Obliquus ventralis 3
Obliquus ventralis 4
Rectus communis
Rectus ventralis
Subarcualis rectus

Hyoid arch
Ceratobranchial 1
Ceratobranchial 2
Ceratobranchial 3
Ceratobranchial 4
Pectoral girdle

Hyoid arch
Hypobranchial 2
Hypobranchial 3
Hypobranchial 4
Variable
Ceratobranchial 5
Ceratobranchial 5

Transversus ventralis 2 Midline
Transversus ventralis 3 Midline
Transversus ventralis 4 Midline
Transversus ventralis Midline

posterior
Ventral superficial
constrictors

fascia of arch

Insertion Innervation
Ceratobranchial 1 DXGEIENG
Ceratobranchial 2 x
Ceratobranchial 3 X,
Ceratobranchial 4 >
Ceratobranchial 5 xX

4

Ceratobranchial 5 X, and/or recurrens
Ceratobranchial 5 xe
Ceratobranchial 1* IX + X,
Ceratobranchial 2
Ceratobranchial 3
Ceratobranchial 4
Variable
Ceratobranchial 3
Ceratobranchial 2,3
Ceratobranchial 2
Ceratobranchial 3
Ceratobranchial 4
Ceratobranchial 5

4

XX MK KKM RH

=

respective arch DXGtopxge=

and/or midline

* Hypobranchial | in actinopterygians.
** For species with five arches.

of other gnathostome groups is on the lower part
of the hyoid arch (Figs. 2a, b and c). The ster-
nohyoidei provide no insight into the phyloge-
netic relationships of Latimeria because, where
they differ from other gnathostomes (in inser-
tion), they are unique.

Coracomandibularis Muscles.—The cora-
comandibulares of Latimeria originate on the
lower lateral side of the clavicles and insert on
the lower jaws dorsal to the intermandibularis
musculature and ventral to the sternohyoidei.

Primitive members of all gnathostome groups
have corcomandibulares. The condition in Lati-
meria is similar to that of many chondrichthyans
(Fig. 2a) and all dipnoans (Fig. 2b). Actinop-
terygians (including polypterids) differ in having
the origins of these muscles on the third hypo-
branchial (Fig. 2c, thus the term branchioman-
dibularis). In teleosts and gars the muscle is
modified or absent. This is also true for am-
niotes. Since both the primitive sarcopterygian
and chondrichthyan conditions are similar to
each other and to Latimeria, this muscle also
provides no insight into the phylogenetic rela-
tionship of Latimeria.

Coracobranchialis Muscles.—Coracobran-

chiales are found only in chondrichthyans (Fig.
2a). Their absence in osteichthyans and in ag-
nathans supports the idea that the presence of
these muscles in chondrichthyans is a synapo-
morphy for that group. The lack of coracobran-
chiales in Latimeria, therefore, neither supports
nor refutes any hypothesis concerning its rela-
tionships.

Coracoarcualis Muscles.—Coracoarcuales are
well developed in chondrichthyans (Fig. 2a) and
are probably found in osteichthyans as part of
the sternohyoidei or coracomandibulares (Wiley
1979). There are indications of coracoarcuales
in Neoceratodus but none in Latimeria (at least
according to Millot and Anthony 1958). At best,
the presence or absence of coracoarcuales are
ambiguous characters because the “‘loss’’ could
support a monophyletic Osteichthyes or the
‘gain’? could support a monophyletic Chon-
drichthyes.

Summary of Hypobranchial Muscle Pat-
terns.—Hypobranchial muscle patterns are use-
ful in supporting hypotheses of the monophyly
of actinopterygians (coracomandibularis origin
on the third hypobranchial) and chondrichthy-
ans (presence of coracobranchiales). The ab-

60 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FIGURE 3.

Diagrammatic ventral views of the gill arch branchial muscles of (a) a generalized shark; (b) a hypothetical

primitive dipnoan; (c) Latimeria chalumnae; and (d) a primitive actinopterygian. Bone and cartilage stippled, basibranchial

copulae not shown. Abbreviations: HA, hyoid arch; IV1, 2,

interarcuales ventrales | and 2; OVI, 4: obliqui ventrales

1 and 4; PC, pharyngoclavicularis; SC1, 3, 5, superficial constrictors 1, 3, and 5; SR, subarcualis rectus; TV2, 3, 4, transversi

ventrales 2, 3, and 4; TVP, transversus ventralis posterior.

sence of coracoarcuales in Latimeria may sup-
port a monophyletic Osteichthyes, but since
agnathans do not have comparable hypobran-
chial muscles, it is not possible to evaluate
whether the absence or reduction of coracoar-
cuales in osteichthyans is primitive or derived.
I conclude that hypobranchial muscles are not
useful in evaluating the phylogenetic relation-
ships of coelacanths.

Branchial Muscle Patterns

General Observations.—There are two basic
sources for branchial muscles. Both are onto-
genetic derivatives of lateral plate mesoderm.
The first of these sources is the muscle sheet
associated with each gill arch. The second is the

oesophageal musculature. The muscle fibers as-
sociated with each gill arch give rise to various
muscles in various gnathostome groups. They
include the superficial constrictors, transversi
ventrales and their ontogenetic derivatives, and
the interarcuales ventrales. The oesophageal
musculature gives rise to the posterior transver-
sus and the pharyngoclavicularus and its deriv-
atives. The following discussion is based on
Wiley (1979) and references therein.

Superficial Constrictor Muscles.—Latimeria
chalumnae has four pairs of superficial constric-
tors, one for each of the first four gill arches
(Fig. 3b). The presence of superficial constric-
tors on the first four gill arches is characteristic
of dipnoans (Fig. 3c), amphibians (larvae), and

WILEY: GILL ARCH MUSCLES

actinopterygians (where the superficial constric-
tors are present as the interbranchial muscula-
ture). Chondrichthyans differ in having superfi-
cial constrictors on all arches (i.e., on the fifth,
sixth, or seventh depending on the number of
arches, Fig. 3a). On ontogenetic and phyloge-
netic grounds it is reasonable to postulate that
the lack of superficial constrictors on the last gill
arch is a secondary loss character. Therefore,
this character is derived for Osteichthyes and
evidence that Latimeria is more closely related
to other osteichthyans than to chondrichthyans.
Transversus Ventralis and Obliquus Ventra-
lis Muscles.—Latimeria chalumnae has two
anterior transversus muscles, one associated
with the second gill arch (transversus ventralis
2) and one associated with the third gill arch
(transversus ventralis 3). Latimeria also has a
pair of posterior transversus muscles associated
with the fifth gill arch (transversus ventralis pos-
terior). These muscles are illustrated in Fig. 3b.
Dipnoans, tetrapods (amphibian larvae) and
actinopterygians have the same two anterior
transversus muscles as Latimeria (Figs. 3c, d).
(Actinopterygians have obliqui ventrales, onto-
genetic derivatives of the transversi ventrales.)
In contrast, chondrichthyans lack anterior trans-
versus muscles. In chondrichthyans the super-
ficial constrictors either meet in an undifferen-
tiated condition at the midline (Fig. 3a) or (in
some sharks and holocephalians) end with the
holobranchs. This represents the more primitive
phylogenetic condition. Thus, I interpreted the
presence of transversi ventrales 2 and 3 as
shared derived characters linking Latimeria
with Osteichthyes (Wiley 1979). I note that dip-
noans, amphibian larvae and actinopterygians
have an obliquus ventralis 1 and a transversus
ventralis 4 (Figs. 3c, d). Neither muscle is found
in chondrichthyans or Latimeria (Figs. 3a, b).
I conclude that this evidence supports a mono-
phyletic Euosteichthyes and excludes Latimeria
from being a sarcopterygian (Wiley 1979). Fi-
nally, a transversus ventralis posterior is char-
acteristic of all gnathostomes and therefore use-
less in assessing phylogenetic relationships.
Pharyngoclavicularis Muscles.—Latimeria
chalumnae lacks paired pharnygoclaviculares.
This similarity is shared with agnathans and
chondrichthyans and is primitive (Wiley 1979;
Figs. 3a, b). In contrast, euosteichthyans have
pharyngoclaviculares. In sarcopterygians, there
are multiple pharyngoclaviculares on each side

61

(Fig. 3c) while in actinopterygians there is a sin-
gle pharyngoclavicularis on each side (Fig. 3d).
The presence of a pharyngoclavicularis muscle
system can be interpreted as a derived character
and evidence of a monophyletic Euosteichthyes
(Wiley 1979).

Interarcualis Ventralis Muscles.—Latimeria
chalumnae has interarcuales ventrales connect-
ing the hyoid arch with the first gill arch and
each succeeding gill arch (Fig. 3b). This similar-
ity is shared with dipnoans (Fig. 3c) and, in a
reduced condition (i.e., some are missing), with
tetrapods (amphibian larvae). These muscles are
missing in chondrichthyans and in actinopteryg-
lans (Figs. 3a, d). Without considering other evi-
dence, the presence of interarcuales ventrales is
a unique similarity shared among dipnoans, tet-
rapods and Latimeria. Thus, it could be inter-
preted as a synapomorphy linking Latimeria
with Sarcopterygii. However, when other mus-
cles are considered it is more parsimonious to
consider the presence of interarcuales ventrales
a derived character of Osteichthyes and thus
primitive within Osteichthyes. This calls for the
ad hoc hypothesis that the lack of interarcuales
ventrales in actinopterygians is a loss character
derived for that group rather than homologous
with the lack of interarcuales ventrales in chon-
drichthyans. The lack of these muscles in chon-
drichthyans is interpreted as a primitive gna-
thostome character.

A SUMMARY OF THE EVIDENCE

The phylogenetic hypothesis presented in Fig.
4 summarizes the ventral gill arch muscle evi-
dence for the phylogenetic relationships of ma-
jor gnathostome groups. It also summarizes evi-
dence presented below and by Wiley (1979)
concerning the interrelationships of certain
groups within Actinopterygii and Sarcopterygil.
Gnathostomes differ from agnathans in having
ventral branchial gill arch musculature. Chon-
drichthyans share the presence of coracobran-
chiales. Osteichthyans share three derived char-
acters: 1) the presence of discrete transversi or
obliqui ventrales on the second and third gill
arches, 2) the presence of interarcuales ven-
trales (secondarily lost in Actinopterygii), and
3) the loss of superficial constrictors on the fifth
gill arch. Additionally, the loss or reduction of
the coracoarcuales might be derived condition.
Euosteichthyans share three derived characters

62 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Ficure 4. An hypothesis of vertebrate interrelationships.
Synapomorphies (black bars) uniting taxa are: (1), general
synapomorphies of gnathostomes; (2), ventral branchial mus-
cles present in the gill arches; (3), dermal bones associated
with the endochondral palate; (4), parasphenoid present; (5),
premaxilla present; (6), angular, prearticular and coronoid
bones present in the lower jaw: (7), cleithrum and clavicle
present in the pectoral girdle; (8), dermal operculum and car-
tilaginous suboperculum present; (9), accessory hyoid element
present; (10), interarcuales ventrales 1—5 present; (11), trans-
versi ventrales 2 and 3 present; (12), fifth superficial constric-
tor absent; (13), the lateral commissure in anterior position:
(14), ventral cranial fissure present; (15), dermal subopercu-
lum present; (16), maxilla present; (17), obliquus ventralis 1
present; (18), pharyngoclavicularis present; (19), transversus
ventralis 4 present; (20), origin of the coracomandibularis on
the third hypobranchial; (21), obliqui ventrales (rather than
transversi ventrali) present; (22), interarcuales ventrales ab-
sent; (23), interbranchiales (rather than superficial constric-
tors) present; (24), cosmine present; (25), presence of a su-
praotic cavity associated with the endolymphatic system; (26),
presence of submandibular bones. Synapomorphies are drawn
from several sources, see text.

not found in Latimeria: 1) the presence of trans-
versi or obliqui ventrales on the fourth gill arch,
2) the presence of obliqui ventrali connecting the
hyoid arch and the first gill arch, and 3) the pres-
ence of pharyngoclaviculari. Therefore, Lati-
meria and other actinistians are osteichthyans
and the sister-group of all other osteichthyans.
Of these characters, the acceptance of the pres-
ence of interarcuales ventrali as a synapomor-
phy of osteichthyans is ad hoc, a by-product of
considering the phylogenetic relationships of
Latimeria as evidenced by other characters.

Other Corroborating Evidence

Additional evidence relating to the problem of
actinistian relationships falls, naturally, into
those lines which tend to corroborate and those
lines which tend to refute the scheme of rela-
tionships presented above. Below, I will briefly
summarize some of the morphological evidence
which corroborates actinistians as osteichthyans
and as the sister-group of Euosteichthyans.

1. The Neurocranium.—Latimeria chalum-
nae and other actinistians are similar in general
ossification pattern to other teleostomes but dif-
fer from chondrichthyans and placoderms in a
number of neurocranial characters. These in-
clude:

a. The presence of a ventral cranial fissure
(the ventral part of the intracranial joint; Gar-
diner and Bartram 1977).

b. The lateral commissure is located in the
anterior part of the otic region, carries the hyo-
mandibular articulation and forms part of the tri-
gemino-facial chamber (Jollie 1971; Miles 1973).
This is distinctly similar to other osteichthyans
and distinctly different than chondrichthyans
(Jollie 1971).

2. Dermal Bones in the Braincase.—Os-
teichthyans among all vertebrates have a para-
sphenoid (Miles 1973) and dermal bones asso-
ciated with the endochondral palate (Schaeffer
1968; Gardiner 1973).

3. Hyoid and Visceral Arches.—Schaeffer
(1968) and Jollie (1971) have called attention to
the fact that only teleostomes among all verte-
brates have accessory elements between the
hyomandibular and ceratohyal (i.e., the inter-
hyal and/or sympletic, also see Miles 1973:72 for
discussion). Actinistians differ from acanthodi-
ans but resemble euosteichthyans in having both
supra- and infrapharyngobranchials (cf. Gardi-

WILEY: GILL ARCH MUSCLES

ner 1973) and in having anteriorly directed in-
frapharyngobranchials. All three conditions are
derived relative to non-osteichthyan gnathos-
tomes.

4. Lower Jaw.—The lower jaw of Latimeria
is composed of several elements. Ossifications
of Meckel’s cartilage include the mentomecke-
lian and the articular. Both are primitive based
on their presence in placoderms (Moy-Thomas
and Miles 1971). Dermal elements include a den-
tary, infradentary (or splenial), angular, prear-
ticular and a coronoid series (Nelson 1973). Of
these the dentary and angular may be primitive
based on the presence of similar bones in plac-
oderms (i.e., the intergnathal may represent
both, see illustration and discussion of Moy-
Thomas and Miles 1971). The remaining bones
are, to my knowledge, found only in Osteich-
thyes and represent unique similarities. The or-
ganization of the tooth-bearing bones of actinis-
tians is comparable only to primitive dipnoans
(Schultze 1969) and Nelson (1973) has suggested
that the organization of the actinistian lower jaw
is uniquely primitive. This is compatible with
the interpretation that actinistians are the sister-
group of all other osteichthyans.

5. Shoulder Girdle.—Latimeria, like other
osteichthyans, has a well-developed dermal
shoulder girdle which includes a clavicle, clei-
thrum and one dorsal element. Neither acantho-
dians nor placoderms have comparable ossifi-
cations. (Placoderms do have trunk shield
armour and acanthodians have dermal plates but
apparently neither is homologous to the dermal
elements of osteichthyans: Moy-Thomas and
Miles 1971; Miles and Young 1977.)

6. Upper Jaw.—Latimeria has a premaxilla
(Millot and Anthony 1965; von Wahlert 1968;
Miles 1977) but lacks any element which can be
called a maxilla. Attempts to identify a vestigial
maxilla (i.e. Nielsen 1936; as discussed by Miles
1977:188) are simply not convincing and I con-
clude that there is no good evidence that acti-
nistians ever had a maxilla. In contrast, dipnoans
have a series of tooth-plates which might be
homologized with the maxilla of other euos-
teichthyans (cf. discussion by Miles 1977:186—
188). I interpret the presence of a premaxilla as
a shared derived character of Osteichthyes
based on its absence in acanthodians, placo-
derms and chondrichthyans. I interpret the pres-
ence of a maxilla as a shared character of Euos-
teichthyes based on its absence in actinistians

63

and all other non-euosteichthyan vertebrate
groups.

7. The Opercular Series.—Patterson (1977)
has discussed the homology and development of
various skull bones which forms the basis for
this section. There are, among gnathostomes,
two series of opercular elements—one series is
cartilaginous and one is dermal. The dermal os-
sifications are neither the ontogenetic nor phy-
logenetic derivatives of the cartilaginous ele-
ments. Holocephalians have a large cartilaginous
operculum, sharks have numerous small hyoid
ray cartilages. Latimeria chalumnae has both a
cartilaginous and a dermal operculum, and it has
(at least according to Millot and Anthony 1965)
a separate cartilaginous suboperculum. The
presence of a dermal operculum and a cartilagi-
nous suboperculum are unique osteichthyan fea-
tures while the presence of dermal bone in the
opercular flap seems a unique teleostome feature
(acanthodians have many smaller dermal ele-
ments, Schaeffer 1968; Miles 1973: similar struc-
tures are missing in placoderms). Further, the
presence of a dermal operculum and cartilag-
inous suboperculum can be interpreted as
shared derived characters of osteichthyans.
Within Osteichthyes, Latimeria and other acti-
nistians are primitive in lacking a dermal sub-
operculum. Thus, a dermal suboperculum can
be interpreted as a shared derived character of
Euosteichthyes.

8. Dermal Skull Roofing Bones .—Graham-
Smith (1978) recently analyzed various skull
roofing bone patterns in placoderms and os-
teichthyans, excluding actinistians. His analyses
indicate that many patterns of fusion and frag-
mentation exist. While it is apparent that acti-
nistians differ in several respects from other os-
teichthyans, it is not clear to me what the
significance of this variation means in terms of
phylogenetic relationships.

Refuting Evidence

Refutation of the hypothesis that actinistians
are osteichthyans and the sister group of euos-
teichthyans falls into two basic classes. First, it
has been argued that actinistians are either the
sister group of choanates (Romer 1966; Andrews
1973; Miles 1975) or at least the sister group of
dipnoans + choanates (Miles 1977). Second, it
has been argued that actinistians are (among
Recent groups) the sister group of chondrich-
thyans (Lgvtrup 1977; Lagios, this volume).

64 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Actinistians as the Sister Group of Choanates

This may be termed the ‘‘traditional’’ hypoth-
esis and is based on such features as the orga-
nization of the archiopterygial fin, the presence
of an intracranial joint in actinistians, rhipidis-
tians and ichthyostegid amphibians, and the gen-
eral similarity of actinistians and rhipidistians.
However, Miles (1977) has presented evidence
that dipnoans are more closely related to choa-
nates than are actinistians. His hypothesis is
based on three characters shared by dipnoans
and choanates that are not shared by actinis-
tians: (1) the presence of cosmine, (2) the pres-
ence of a supraotic cavity associated with the
endolymphatic system in the cranium, and (3)
the presence of a series of submandibular bones.
Of these three characters, (1) and (2) are unique
to dipnoans and choanates while (3) is shared
with actinopterygians (Jessen 1968; Miles 1977
is of the opinion that the series in actinoptery-
gians is not homologous with that of choanates
and dipnoans).” Miles’ (1977) evidence supports
the conclusion that dipnoans are the closest Re-
cent relatives of tetrapods and the closest rela-
tives of choanates among all vertebrates.

Latimeria as a Sarcopterygian

The major line of evidence supporting this hy-
pothesis is the presence of an intracranial joint
in actinistians and rhipidistians (and perhaps in
some primitive tetrapods such as the ichthyo-
stegids). This joint separates the brain case into
two regions and is impressively similar in the
two groups. However, there are differences. In
the cranium, these differences involve the to-
pographic relationship of the joint and the tri-
germinal nerve (Schaeffer 1968; Bjerring 1973).
In rhipidistians, the maxillary and mandibular
branches of the trigeminal exit the posterior di-
vision of the braincase via a foramen which is
located posterior to the intracranial joint. In ac-
tinistians, these branches of the trigeminal exit
through the intracranial joint itself. There are
also differences between the topographic posi-
tion of the joint and the dermal skull roof (Bjer-
ring 1973). In rhipidistians, the joint’s external
position is between the parietals and frontals. In

* If, however, Actinistia is the sister group ofall other os-
teichthyans, there is no phylogenetic reason to consider the
submandibular series as anything but a synapomorphy of
Euosteichthyes and its absence in Latimeria as a primitive

character

actinistians, the external position of the joint is
between two bones of uncertain homology (var-
iously termed frontals and parietals or an ante-
rior portion of the frontal and a posterior portion
of the frontal fused with the parietal; cf. An-
drews 1973, and Bjerring 1973, respectively). In
actinistians the joint is anterior to the junction
of the infraorbital and supraorbital sensory ca-
nals while in rhipidistians the joint is posterior
to this junction.

Bjerring (1973) argued, on the basis of an ar-
cual theory of cranial ossification, that the intra-
cranial joint of actinistians is non-homologous
with that of rhipidistians. Arcual theories of cra-
nial ossification stem from Goethe (see DeBeer
1937) and state that various cranial ossifications
correspond to metameres in the cranium that are
serial equivalents of the vertebral metameres.
Bjerring (1973) identified the topographic posi-
tion of the actinistian joint as lying between the
presumptive mandibular (2nd) and hyoid (3rd)
metameres and the topographic position of the
rhipidistian joint as lying within the mandibular
metamere between the antotic pilae and the
acorochordals. However, there is a problem
with this hypothesis. Gardiner and Bartram
(1977) demonstrated that the ventral part of the
intracranial joint is the homologue of the ventral
otic fissure. The ventral fissure is a plesiomor-
phic osteichthyan feature present in acanthodians
actinopterygians and dipnoans as well as being
represented as part of the intracranial joint. This
means that the arcual hypothesis must be refut-
ed since it is tied to the hypothesis that the entire
joint (dorsal and ventral portions) is non-ho-
mologous in actinistians and rhipidistians. But,
this does not mean that the entire joint must be
thought of as homologous in the two groups.
Wiley (1979) suggested that the differences ex-
hibited in the dorsal part of the joints of actinis-
tians and rhipidistians might mean that the dor-
sal part of the joints were non-homologous.

Comments.—We are faced with three mu-
tually exclusive hypotheses. (1) Actinistians are
the sister group of Euosteichthyes. (2) Actinis-
tians are the sister group of choanates and dip-
noans are the sister group of the two. (3) Actinis-
tians are the sister group of dipnoans and
choanates. If we opt for hypothesis (1), then we
must reject the intracranial joint of actinistians
as being homologous with that of rhipidistians
and accept that it has been gained twice. If we
opt for hypothesis (2), then the lack of the var-

WILEY: GILL ARCH MUSCLES

ious Ossifications and muscles in Latimeria out-
lined above must be thought of as being lost
characters which are synapomorphous for the
Actinistia. Such an hypothesis might be reason-
able if it could be shown that the variation in
trigeminal nerve exits in rhipidistians (Thom-
son, 1967) made the differences between actinis-
tian and rhipidistian joints trivial and if enough
weight was placed on the character to preclude
the use of the characters embodied in (1) as syn-
apomorphies. Hypothesis (3) seems the most
uneconomical of the three. It assumes that the
intracranial joint of sarcopterygians was lost
twice and it assumes that all of the characters
uniquely exhibited by euosteichthyans have
been independently derived in actinopterygians
on the one hand and dipnoans + choanates on
the other hand.

All of this discussion, of course, rests on the
assumption that the rhipidistian fishes are the
sister group of tetrapods. If it could be shown
that rhipidistians are more primitive than dip-
noans, then other interpretations are possible,
not all of which are inconsistent with the inter-
pretation that Latimeria is a sarcopterygian.

Latimeria as the Sister Group of Chondrich-
thyes

The Pituitary Complex.—Lagios (1975, and
this volume) has compared the organization of
the pituitary gland of Latimeria with other
gnathostomes. Like chondrichthyans, the pars
distalis of Latimeria is organized into two dis-
tinct components, a ventral component (the ros-
tral lobe) and a dorsal component. The dorsal
component incorporates a dorsal lobe composed
primarily of acidiophilic cells and a proximal
lobe composed of mixed cell types. Lagios
(1975) suggests a homology between the rostral
lobe of Latimeria and what is usually termed
the ventral lobe of chondrichthyans. He rejects
a homology between the ventral lobe of Polyp-
terus and certain teleosts and the ventral lobe of
chondrichthyans and Latimeria because the
actinopterygian lobe contains cells which se-
crete ‘“‘prolactin’’ whereas the ventral lobes of
Latimeria and chondrichthyans lack these cells.

Comments.—I see no reason to reject the hy-
pothesis that the ventral lobe of the pars distalis
of chondrichthyans is homologous with the sim-
ilar structure found in Latimeria. However, I
think there is good reason to reject the notion
that this homology is a synapomorphy indicating

65

a sister group relationship between these
groups. Olsson (1968) has pointed out that the
ventral lobe of the pars distalis is an embryonic
remnant of Rathke’s pouch. Those gnatho-
stomes with ‘“‘compact”’ or ‘‘follicular’’ pituitar-
ies (cf. Olsson 1968) go through an embryonic
stage in which the adenohypophysis is formed by
an invagination of the stomodeum (the ectoder-
mal lining of the anterior pharyngeal cavity).
The adenohypophysis primordium pushes dorsal-
ly until it contacts the neurohypophysis. It then
loses its connection with the stomodeum and
may become ‘‘duct type,” ‘‘follicle type’ or
‘““compact type’? in its organization depending
on subsequent ontogenetic change. Thus, Lati-
meria, chondrichthyans, and polypterids (all of
which have the ‘‘duct type’’) can be interpreted
as having and ‘“‘incomplete’’ ontogeny of the
pars distalis that is the primitive expression of a
phylogenetically derived condition found in all
euosteichthyans except (to my knowledge) po-
lypterids. Following Olsson (1968), I conclude
that the bipartite organization of the pars dis-
talis of Latimeria, chondrichthyes and polyp-
terids is a primitive gnathostome feature and
thus not evidence for a sister group relationship
between Latimeria and chondrichthyans. In-
deed, if we follow Wingstrand (1966), this bi-
partite organization is a primitive vertebrate
feature and the compact pituitaries of lampreys
and hagfishes cannot be considered homologous
with the compact pituitaries of various euos-
teichthyan groups. Finally, the presence of
‘prolactin’? cells in the ventral lobes of acti-
nopterygians does not denote the non-homolo-
gous nature of this lobe to that of actinistians so
much as it suggests that these cells are derived
relative to the lack of ‘“‘prolactin’’ cells.

The Rectal Gland.—The presence of a rectal
gland in Latimeria and elasmobranchs has been
cited as evidence for a sister group relationship
between chondrichthyans and actinistians by
Lgvtrup (1977) and by Lagios (this volume). The
similarities are striking, but there are problems
with interpreting these similarities as synapo-
morphies. Holocephalians do not have rectal
glands. As pointed out by Lagios (this volume),
holocephalians do have cation excreting tissue
in the hindgut which may be reasonably consid-
ered as a phylogenetically more primitive
expression of the rectal gland of elasmobranchs.
The problem with interpreting the rectal glands
of Latimeria and elasmobranchs as synapomor-

66 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

phies is that we must accept the hypothesis that
Latimeria is more closely related to elasmo-
branchs than are holocephalians. In other
words, we must assume that Latimeria fits be-
tween holocephalians and sharks and not below
holocephalians and sharks in the phylogeny.
Since there is reasonable evidence at hand to
reject this hypothesis in favor of an alternate
hypothesis that among these three taxa, holo-
cephalians are more closely related to elasmo-
branchs than is Latimeria we are left with three
hypotheses. (1) The rectal gland is a primitive
gnathostome feature and thus no evidence for
relationships within the Gnathostomata. (2) The
rectal glands are non-homologous, and thus no
evidence of relationship at any level. (3) The
rectal glands are synapomorphic for Actinistia
+ Chondrichthyes and have been independently
reduced in holocephalians. The third hypothesis
is uneconomical for the same logical reasons
that I have given for Miles’ (1977) hypothesis
concerning the intracranial joint.

Other Evidence.—Without going into great
detail or out-group comparisons, L@vtrup (1977)
listed the following characters as similarities
shared by Latimeria and chondrichthyans: a fat-
ty liver, eye structure, shape of the gray matter
in the spinal chord, the absence of Manthner’s
fibers, the position of the first pair of cranial
nerves in a hollow stalk which is a continuation
of the telecephalic cavity, the structure of both
the thymus and thyroid, large eggs, and osmo-
regulation through urea retention.

Comments.—I would not pretend to be com-
petent to evaluate all of these characters. I
would point out that Lgvtrup’s (1977) list of
characters does not have the benefit of out-
group comparison. That is, critical comparisons
have not been made with agnathans. I note, for
example, that a cursory glance at lampreys (Jol-
lie 1973, Figs. 13-15) indicates they have a sim-
ilar pattern for the first two cranial nerves as
chondrichthyans and Latimeria. Thus a case
could be made for this character being primitive.
The distribution of the gray matter in the spinal
cord is probably a derived character for gna-
thostomes. I base this conclusion on the fact that
the condition in teleosts and amphibians is not
that much different from Latimeria and sharks
and that gnathostomes (in having distinct ‘‘X-
shaped” distribution of gray matter) differ from
cyclostomes and amphioxus. Indeed, the distri-
bution of gray matter in sharks seems to me to

be as similar to teleosts as to Latimeria and the
specific similarities between Latimeria and
chondrichthyans could be explained as the re-
tention of a primitive character. The simple
structure of the thymus and thyroid glands might
likewise be primitive, but comparisons will have
to be made with agnathans and euosteichthyans
before the status of these characters can be as-
sessed. Jollie (1973) mentions Mauthner’s cells in
teleosts and salamanders. What its distribution
is within Euosteichthyes and Agnatha is un-
known to me. Again, more comparisons will
have to be made. Other characters, such as a
fatty liver and large eggs can not be evaluated
without additional information (for example—is
the oil retained in the liver if similar biochem-
istry connoting similar and homologous bio-
chemical pathways’).

Finally, there is the question of osmoregula-
tion by urea. I have previously dismissed this
evidence because lungfishes also retain urea
(Wiley 1979). Thus, urea retention, if considered
homologous at al, can be interpreted as a prim-
itive character of gnathostomes (see Griffith and
Pang, this volume).

SUMMARY

In view of the evidence summarized in this
paper, Latimeria chalumnae may be considered
(1) an osteichthyan and (2) the sister group of all
other Recent osteichthyans. Twelve shared de-
rived characters (synapomorphies) corroborate
the first hypothesis and five shared derived char-
acters corroborate the second hypothesis (Fig.
4). The phylogenetic relationships of Latimeria
chalumnae advocated here are summarized in
the form of a classification, shown below.

INFRAPHYLUM Gnathostomata
SUPERCLASS Chondrichthyes
SUPERCLASS Teleostomi

CLASS Actinistia
ORDER Coelacanthiformes
Latimeria chalumnae
CLASS Euosteichthyes
SUBCLASS Sarcopterygii
SUBCLASS Actinopterygii

ACKNOWLEDGMENTS

I thank Bobb Schaeffer (American Museum
of Natural History) and Hans-Peter Schultze
(University of Kansas) for reading and providing
valuable comments on earlier drafts. I have ben-

WILEY: GILL ARCH MUSCLES

efited from numerous discussions with Donn
Rosen (AMNH) concerning sarcopterygian re-
lationships and with James Atz (AMNH) con-
cerning pituitary glands.-Review and discussion
does not imply acceptance of my hypothesis.

LITERATURE CITED

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1977. The axial skeleton of the coelacanth, Latime-
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and A. D. Walker, eds., Problems in vertebrate evolution.
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DeBeer, G. R. 1937. The development of the vertebrate
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BJERRING, H. C. 1973. Relationships of coelacanthiforms.
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ENGELMANN, G. F., AND E. O. WILEY. 1977. The place of
ancestor-descendant relationships in phylogeny reconstruc-
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GARDINER, B. G. 1973. Interrelationships of teleostomes.
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Patterson, eds., Interrelationships of fishes. Academic
Press, London.

, AND A. W. H. BARTRAM. 1977. The homologies of
ventral cranial fissures in osteichthyans. Pages 227-245 in
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lems in vertebrate evolution. Academic Press, London.

GRAHAM-SMITH, W. 1978. On the lateral lines and dermal
bones in the parietal region of some crossopterygian and
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HENNIG, W. 1966. Phylogenetic systematics. University of
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JESSEN, H. 1968. The gular plates and branchiostegal rays in
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phylogeny. Interscience Publications, New York.

Jotitie, M. 1971. Some developmental aspects of the head
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phol. 133:17—40.

. 1973. Chordate morphology. Robert E. Krieger Pub-
lishing Company, Huntington, New York.

Lacios, M. D. 1975. The pituitary gland of the coelacanth
Latimeria chalumnae Smith. Gen. Comp. Endrocrinol.
25:126-146.

1979. The coelacanth and the chondrichthyes as sis-
ter groups: a review of shared apomorph characters and a
cladistic analysis and re-interpretation. This volume.

Lgvrrup, S. 1977. The phylogeny of Vertebrata. John Wiley
and Sons, New York.

Mices, R. S. 1973. Relationships of acanthodians. Pages 63-
103 in P. H. Greenwood, R. S. Miles, and C. Patterson,
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. 1975. The relationships of the Dipnoi. Colloq. Int. C.

N.R. S. 218:133-148.

. 1977. Dipnoan (lungfish) skulls and the relationships

67

of the group: study based on new species from the Devonian

of Australia. Zool. J. Linn. Soc., London 61: 1-328.

. AND G. C. YOUNG. 1977. Placoderm interrelation-
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R. S. Miles, and A. D. Walker, eds., Problems in vertebrate
evolution. Academic Press, London.

MILLor, J., AND J. ANTHONY. 1958. Anatomie de Latimeria
chalumnae 1. Squelette, muscles et formations de soutien.
CANE ReesS-vRanis-

Moy-THomas, J. A., AND R. S. MILEs. 1971.
fishes. W. B. Saunders Company, Philadelphia.

NELSON, G. J. 1969. Gill arches and the phylogeny of fishes
with notes on the classification of vertebrates. Bull. Am.
Mus. Nat. Hist. 141:475-552.

1973. Relationships of clupeomorphs, with remarks
on the structure of the lower jaw in fishes. Pages 333-349
in P. H. Greenwood, R. S. Miles, and C. Patterson, eds.,
Interrelationships of fishes. Academic Press, London.

Oxtsson, R. 1968. Evolutionary significance of the *‘prolac-
tin’’ cells in teleostomean fishes. Pages 455-472 in T. Orvig,
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tebrate phylogeny. Interscience Publishers, New York.

PATTERSON, C. 1977. Cartilage bones, dermal bones and
membrane bones, or the exoskeleton versus the endoskel-
eton. Pages 77-121 in S. M. Andrews, R. S. Miles, and A.
D. Walker, eds., Problems in vertebrate paleontology. Ac-
ademic Press, London.

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of Chicago Press, Chicago.

SCHAEFFER, B. 1968. The origin and basic radiation of the
Osteichthyes. Pages 207-222 in T. Orvig, ed., Current prob-
lems of lower vertebrate phylogeny. Proceeding of 4th No-
bel Symposium. Interscience Publishers, New York.

SCHULTZE, H.-P. 1969. Griphognathus Gross, ein lang-
schnauziger Dipnoer aus dem Oberdevon von Bergish-Glad-
bach (Rheinisches schiefergebirge) und von Lettland. Geol.
Palaeontol. 3:21-61.

THOMSON, K. S. 1967. Mechanisms of intracranial kinetics
in fossil rhipidistian fishes (Crossopterygia) and their rela-
tives. J. Linn. Soc. (Zool.) 46:223-253.

. 1969. The biology of the lobe-finned fishes. Biol. Rev.
Cambridge Philos. Soc. 44:91-154.

VON WAHLERT, G. 1968. Latimeria und die Geschichte der
Wirbeltiere. Eine evolutions biologische Untersuchung.
Gustav Fischer Verlag, Stuttgart.

WesToLlL, T. S. 1949. On the evolution of the Dipnoi. Pages
121-184 in G. L. Jepsen, G. G. Simpson, and E. Mayr,
eds., Genetics, paleontology and evolution. Princeton Uni-
versity Press.

WiLey, E. O. 1975. Karl R. Popper, Systematics and clas-
sification: a reply to Walter Bock and other evolutionary
taxonomists. Syst. Zool. 24:233-243.

. 1976. The systematics and biogeography of fossil and

Recent gars (Actinopterygii: Lepisosteidae). Misc. Publ.

Mus. Nat. Hist., Univ. Kans., No. 64:1-111.

. 1979. Ventral gill arch muscles and gnathostome phy-
logeny, with a new classification of vertebrates. J. Linn.
Soc. London, in press.

WINGSTRAND, K. G. 1966. Comparative anatomy and evo-
lution of the hypophysis. Pages 58-126 in G. W. Harris and
B. T. Donovan, eds., The pituitary gland. Butterworths,
London.

Palaeozoic

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 11 pages December 22, 1979

OBSERVATIONS ON THE STRUCTURE OF MINERALIZED
TISSUES OF THE COELACANTH, INCLUDING THE
SCALES AND THEIR ASSOCIATED ODONTODES

By
William A. Miller
Department of Oral Biology,

State University of New York at Buffalo,
Buffalo, New York 14226

ABSTRACT

Pieces of dentary, premaxilla and pterygoid from five specimens of Latimeria cha-
lumnae have been examined radiographically and histologically. Scales from seven, in
addition to an embryo specimen (No. 1 from American Museum of Natural History),
were examined radiographically and grossly; histological examination was made of
scales from three of them.

Our original findings on bone structure are confirmed. Bone remodeling in some
bones results in primary and secondary haversian systems. Jaws are mainly composed
of outer dense plates or laminae supported by interlaminar struts, while the teeth sit
in shallow sockets in the bone and are attached by radiating smaller struts.

Examination of scale rings (annuli) indicates that their number is variable on the
same specimen, and even on the same scale depending along which axis the count is
made. This is true of both minor and major scale rings.

Embryo scales show that the initial ornamentation is by melanocytes, not odontodes,
and that their orientation is related to the fine radially oriented ridges on the scale
surface. Increase in thickness of scale is brought about by the addition of nonminer-
alized isopedine layers.

Adult scale growth may be divided into true increase in size of the scale at the
periphery and modifications to the ornamentation both at the periphery (posteriorly)
and on the anterior margin of the ornamental part where adjacent scales overlap. In
either case melanocytes appear to be established prior to the development of odontodes.

68

MILLER: COELACANTH MINERALIZED TISSUES

INTRODUCTION

Brief descriptions of the histological structure
of the mineralized tissues of Latimeria chalum-
nae Smith have appeared since the first speci-
men was caught nearly forty years ago (Bern-
hauser 1961; Miller and Hobdell 1968; Peyer
1968; Miller 1969; Hobdell and Miller 1969; Iso-
kawa, Toda and Kubota 1968; Grady 1970) al-
though the topographical anatomy of these
structures had been described in detail by Smith
(1939, 1940) and Millot and Anthony (1958).
Schaeffer (1977) has recently reviewed these
structures in relationship to the skeletal tissues
of other groups of fishes and Andrews (1977) has
discussed the axial skeleton in great detail.

The scales and their odontodes were de-
scribed initially by Smith (1940) and by Roux
(1942) and in greater detail by Smith, Hobdell
and Miller (1972). Orvig (1977) has described the
formation of successive generations of odon-
todes in Latimeria scales and Schaeffer (1977)
has discussed the tissues which make up the
odontodes.

MATERIALS AND METHODS

Jaw material was obtained for histological ex-
amination from the following specimens:

Birmingham (U.K.) University, dentary.

British Museum Natural History II, premaxilla
and dentary.

Peabody Museum, premaxilla and dentary and
pterygoid.

Field Museum II, premaxilla, pterygoid.

Royal Society of London, premaxilla, pterygoid.

Adult scales have been examined from:

British Museum Natural History II.

Field Museum II.

Peabody Museum.

Smithsonian Museum.

Steinhart Aquarium, California Academy of Sci-
ences.

Miscellaneous scales from specimens: Nos. 84
and 85 USNM 205871.

Photographs from Smith (1940).

Embryo scales from embryo number 1
(A.M.N.H.) were also studied (Smith et al.
1975)

Bone and tooth specimens and the whole head
of BMNH II were examined by X-ray micros-
copy (Hilger and Watts Intercol X-ray micro-

69

scope) at 50 or 60 KV 4 mA (Saunders and Ely
1965). Preliminary results were reported previ-
ously (Hobdell and Miller 1969). Some material
was recorded as stereo-pair radiographs for
three dimensional representation of the bony
structures. Other radiographs were taken of dis-
sected material and the scales using fine-grain
photographic film and a modified dermatological
X-ray source (target film distance 75 cm 70 KVP
9 mA) (Miller and Radnor 1970).

Selected specimens were subsequently de-
mineralized, with radiographic control, in 10%
formic acid and processed either through alco-
hols and paraffin or through Cellosolve and ester
wax. Serial or semiserial 8 um sections were
stained with a wide variety of histological stains.

Scales

For the major investigations two sets of scales
were taken from each specimen: A circumfer-
ential set around the left side of the body just
anterior to the root of the anterior dorsal fin; a
longitudinal (horizontal) set, one scale below the
lateral line (Smith, Hobdell and Miller 1972).

Similar sets were obtained from embryo no.
I and also other scales from along the lateral line
system including nos. 1, 3 and 5.

Adult scales were examined by X-ray micros-
copy, microradiography, histological techniques
and direct observation of cleared specimens by
transmitted and reflected light (Smith, Hobdell
and Miller 1972). Embryo scales were examined
by contact radiography, clearing and mounting
and histological techniques.

RESULTS AND DISCUSSION
Bone and Cartilage

The radiographic appearance of the jaws has
been briefly described previously (Hobdell and
Miller 1969). There is a single bony element in
each lower jaw although the dentaries are not
directly connected in the midline (Fig. 1). These
bones are composed of dense plates of mainly
acellular bone on their lateral and medial as-
pects, each made up of parallel lamellae. Large
struts pass between these bony plates. The larg-
er teeth are ankylosed by the bone of attachment
to the tooth bearing bone beneath them (Hobdell
and Miller 1969) which in turn is attached to the
jaw bone proper by a firm fibrous ligament in-
serted by Sharpey fibres into each bony com-
ponent (Miller and Hobdell 1968). At the labial

70 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

X-ray micrograph (Hilger and Watts) of den-

FIGURE 1.
taries of Latimeria chalumnae (BMNH II) prior to dissection.
The overall trabecular pattern is evident as are the paired-

tooth positions. The gular plates are visible below.

aspect a separate set of small bony elements
may be observed with one or two small teeth in
each (Fig. 2). These also are attached by fibrous
bands to the main bone of the jaw.

The teeth of the premaxilla are held in a way
similar to that of the other bony elements. Fine
radiating struts may be observed between tooth
and tooth bearing bone (Figs. 3 and 4) which is
connected to jaw bone proper by fibres. In turn
this bone is attached to large but apparently un-
mineralized cartilaginous elements by fibrous
bands (Fig. 5). The bone of the pterygoid is
much lighter in structure with minor condensa-
tions at the surface and around the larger tooth
pairs. Teeth are ankylosed in a way similar to
the other bones. Fine radiating struts were seen
where the bone articulated with adjacent bones.

Much of the bone studied histologically was
relatively acellular in the larger bony elements.

FiGu! X-ray micrograph of left dentary prior to dis-
section to show the tooth-bearing bone blocks at the periphery
of the major osseous component.

Pe

X-ray micrograph of premaxilla prior to dissec-

#
8

FIGURE 3.
tion. The strutted construction/arrangement is clearly visible.
Tooth positions with different stages of tooth development
may be observed (arrows).

However the development of primary haversian
systems to varying degrees, was often observed
within the spaces of the trabeculae (Fig. 6). An
irregular arrangement of osteocytes could be
seen along with many, presumably unmineral-
ized Sharpey fibres with which they might be
confused at first sight. Bone tissue was lined
with a tinctorially different osteoid which was
particularly wide in the trabecular bone (Fig. 5).
In some of the jaw bones secondary haversian
systems were observed in which the outer bor-
der of the bone unit was bounded by a scalloped
reversal line (Fig. 7), which indicated prior bone
resorption.

Most bone resorption was associated with the
attachment of new, developing teeth where it
was frequently extensive even though adjacent
to functioning teeth. Large craters were eroded

FIGURE 4.
from premaxilla. There is ankylosis of the tooth with a fine
surrounding trabecular pattern. The coarser trabecular pattern
of the rest of the bone is clearly visible. Flattened odontodes
can be seen on the external/facial side of the bone (arrows).

X-ray micrograph of slab cut parasagittally

MILLER: COELACANTH MINERALIZED TISSUES

FIGURE 5.

Photomicrograph of parasagittal section of pre-
maxilla. Major and minor trabecuale are present. Wide osteoid
seams can be seen (arrows). The fibrous attachments between
the bones and to the cartilage (C) are clearly visible. H & E
x 40.

prior to the attachment of the developing tooth
which was observed some distance above the
bone, solely within soft tissue (see below).
Gross examination of jaw pieces showed that on
occasion a large crater or socket could be seen
on the oral surface of the bone which implied
that much of the bone of attachment and pos-
sibly several teeth in a group along with their
tooth bearing bone had been shed. Considerable
epithelial proliferation may accompany such
tooth and bone loss (Miller and Hobdell 1968).
Resorption was accompanied by the presence of
multinculeate and mononucleate osteoclasts,
smaller than, though similar to, those seen in
mammalian bone remodeling.

FIGURE 6.
illary bone. Careful examination of the circular bodies around
the haversian system reveals most of them as Sharpey fibres
related to fibrous attachment. A few osteocytes are visible
(arrows). van Gieson x 100.

Primary haversian system (osteon) in premax-

71

FIGURE 7.
A peripheral scalloped outline is visible in each (arrows). La-
mellae and occasional osteocytes can be seen within each sys-
tem. H & E x100.

Secondary haversian system in pterygoid bone.

On the whole, bone remodeling was minimal
so that frequently there was incremental en-
largement of bone processes in one direction in
particular, with only slight accompanying re-
sorption on the opposite side of the bone. Dis-
tinct and asymmetrical resting lines were ob-
served not only histologically (Fig. 8) but were
clearly visible in microradiographs (Hobdell and
Miller 1968). Some of the illustrations of Smith
(1940) show similar but perhaps grosser rings on
the surface of bones. The subopercular bone is
such an example which in the first described
specimen of Latimeria chalumnae had 16 dis-
tinct major annuli and 40-44 minor ones. The
gular plate that I have examined showed similar
series of annular lines.

Spicule of premaxilla (BMNH-II) with nine

FIGURE 8.
major growth rings and several minor ones. Some resorption
has occurred at the lower edge. Lison x 100.

72 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGurRE 9. Tooth of dentary in situ. Dentine, cementum
and bone of attachment are clearly visible. The tinctorial
change associated with mesodermal enamel is visible at the
outer, coronal part of the dentine. It will be noticed that the
epithelium around the tooth stands away from the dentine.
This space in life would be partly occupied by highly miner-
alized true enamel. H & E x40.

The cartilage of the jaws of Latimeria cha-
lumnae shows an irregular arrangement of chon-
drocytes with irregular shape and staining of the
pericellular zones. There is an outer zone of pro-
liferation but this is far less regular than that
seen in mammalian cartilage. No areas of min-
eralization were observed as is frequently seen
in elasmobranch cartilage.

Tooth Structure

The teeth (Fig. 9) are made up of pseudo-
dentine (sensu Bertin 1958) rather than ortho-
dentine (Bernhauser 1961) with adjacent tubules
connecting at all levels. Incremental lines are
evident (Fig. 10); see also Miller and Hobdell
(1968). The outer surface is covered with enamel
which is distinct from the underlying dentine
(Miller and Hobdell 1968; Miller 1969). This was
confirmed by Grady (1970). Scanning electron
microscopy also shows this tissue as a distinct

FiGure 10. Photomicrograph of unerupted tooth. Bone of
attachment and cementum may be seen. Incremental lines are
visible with every fifth one more defined. These may be daily
or possibly lunar increments. The space above the dentine is
partly due to shrinkage and partly due to loss of highly min-
eralized true enamel during histological processing. Gomori
reticulin x40.

structure (Smith 1978). The outer layer of den-
tine has many characteristics of mesodermal
enamel (sensu Kvam 1946) (Miller 1969). Iso-
kawa et al. (1968) did not find enamel on gill-
arch teeth but it should be remembered that gill-
arch epithelium is endodermal in origin while
dentary epithelium is ectodermal. Gillarch teeth
may not have ectodermal enamel or perhaps it
was lost in preparation. Radicular dentine blend-
ed into a sheath of cementum-like tissue which
was less well calcified than the surrounding tis-
sues (Hobdell and Miller 1969). This was sepa-
rated from the true bone of attachment by a
PAS-positive and Gomori-negative line (Miller
and Hobdell 1968).

The tooth bearing bone is frequently observed
to have several incremental layers on its lower
surface (Fig. 11). These are asymmetrically ar-
ranged to one end of each tooth/bone unit im-
plying that there is a gradual tilting of this bone
unit as initial teeth become worn and others are
added at the end where incremental bone is
thickest.

The tooth bearing bones are held to the bone
of the jaws by dense fibrous ligaments. Fine
fibres are seen entering each bony component,
the fibres joining to become a coarse central
complex between the bones (Fig. 12).

MILLER: COELACANTH MINERALIZED TISSUES

‘

Base of tooth bearing bone of dentary; several

FiGure 11.
incremental lines are visible with resulting areal increase. This
seems to be associated with continued tooth development and
subsequent tilting of the bone. H & E x40.

Growth of Teeth

We may interpret the various stages of tooth
development in the following schema:

1. Initially all teeth develop on the underside
of the mucous membranes at first as a thickening
of the basement membrane when viewed after
PAS staining. Presumably this is related to
neural crest cells in the underlying mesenchyme
although the first obvious mesenchymal change
was early differentiation of odontoblasts. Mes-
enchymal condensation, so marked an early
change in mammals, did not occur until the ini-
tial mineralized tissue had formed. The cap stage
is formed by proliferation of the epithelium, but
a well marked dental lamina is not seen. At this
stage, bone well below the developing tooth
starts to resorb to form a cup.

2. There is rapid growth in length of the tooth
with little increase in thickness. This phenome-
non has been observed in various nonmammal-
ian teeth: Pristis cuspidatus (rostral teeth) (Mil-
ler 1974), Amia calva (Miller and Radnor 1970)
and Necturus maculosus (Miller and Rowe 1972).

3. Eruption of the tooth occurs and it is sub-
sequently held in place intraorally by soft tis-
sues. As a consequence, teeth may be observed
in the mouth which are approaching full height
but which are loose to the touch.

Details of fibrous attachment between tooth
Fine
fibres attach to the bones per se which in turn form a dense
coarser network between the bones. van Gieson x40.

FiGuReE 12.
bearing bone (above) and basal bone of jaw (below).

4. There is a subsequent further thickening of
the dentine and development of cementum and
bone of attachment as the tooth finally becomes
ankylosed and fully functional.

5. The tooth bearing bone may show several
incremental lines at its lower border where it is
attached to the subdontal ligament (Fig. 12).
This probably indicates further eruptive move-
ment of the tooth and bone of attachment after
ankylosis.

6. Other teeth may be added, usually on the
inner or lingual side to the same tooth bearing
bone. The older labial teeth may show signs of
wear or fracture. Some new teeth may attach to
the bone of attachment of previous teeth.

7. The presence of relatively large sockets in
various parts of the mouth leads one to suggest
that eventually a whole group of small teeth or
odontodes may be lost including their bone of
attachment and tooth bearing bone. Epithelial
proliferation reminiscent of mammalian socket
healing may then be seen (Miller and Hobdell
1968) which fills in the surface defect.

74 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Central portion of adult scale, stained in aliz-
arin Red S and cleared. Note two odontodes only have stained
and that the bone attachment of other odontodes has taken up
the stain to varying degrees (arrows). The overlap that might
be expected from Orvig’s theories (1977) is not observed. x6.

FiGuRE 13.

Scales

The basic structure of the adult Latimeria
scale has been described previously (Smith,
Hobdell and Miller 1972). Each consists of a
posterior third which is exposed and ornament-
ed with odontodes (Smith 1940; Roux 1942; Or-
vig 1977) and an anterior two-thirds overlapped
by neighboring scales. Many annular rings are
apparent at irregular intervals. These are pro-
duced by slight variations in regular smaller
ridges which radiate from the apex and are ap-
proximately perpendicular to the annuli.

It has been suggested that one may age indi-
vidual Latimeria by scale ring counts as is pos-
sible in various teleosts. However the number
of annuli is extremely variable. The counting of
minor annuli is very difficult and can be found
to vary depending on whether one starts to
count from the centre or from the periphery of
the scale. There is also variation between the
long and short axes of the scale which is accen-
tuated when the scale is elongate as compared
to oval. Even adjacent scales may vary, the
centres of the scales in particular having very
different numbers of annuli and yet the scales
being of similar size and form. It has been sug-
gested that this variation may be due to in vivo

FiGure 14. Central portion of embryo scale. Ridges can
be seen radiating out, in relation to a V-shaped line, in three
groups. Melanocytes of varying size are arranged in rows
which seem to be related to the scale ridges. An additional
small colony of small melanocytes is visible on the leading
edge of the ornamented part of the scale (arrow). Cleared scale
x25.

scale loss (possibly due to trauma) and subse-
quent replacement, but adjacent scales in em-
bryo No. | showed similar variation and these
could not have been subjected to trauma. The
scales of adult specimens are large and relatively
difficult to remove and I have never seen a short
scale which might be interpreted as a more rap-
idly growing, replacing scale in any of the spec-
imens so far examined, or in any illustrations of
Latimeria scales in the literature. One is struck
by the regular scale pattern although pigmenta-
tion does vary and many scales are missing post-
mortem.

The major annuli show similar but less pro-
nounced variation to the minor annuli. For in-
stance those scales illustrated in Smith (1940)
vary between I1 (scale 65) and 16 (scales 64 +
71). Specimen No. 84 varies between 7-10; No.
85, 10-17. The Peabody specimen to which
Thomson (1966) gives a scale age of about eight
years varied between 8 and 14.

Odontodes

At first sight odontodes appear very regular
in their arrangement. However, their orientation

MILLER: COELACANTH MINERALIZED TISSUES

varies from generally radial to sometimes cir-
cumferential. Adjacent groups may be at sharply
varying angles. When viewed radiographically
the bone of attachment can be seen to radiate
diffusely around each odontode and the radio-
graphic density of each is quite constant. Each
odontode is discrete and there is not the overlap
of image that one might expect from the inter-
pretation of Orvig (1977) of odontode regenera-
tion. However near to the apex of the scale and
occasionally elsewhere a residual image of the
bone of attachment was seen. Grossly a small
raised ring around a space was observed. Stain-
ing of several whole scales with alizarin red S
prior to clearing resulted in little staining of
odontodes—mature enamel and dentine do not
stain readily with this technique (Miller 1959).
However a few discrete odontodes had stained
(Fig. 13) indicating immature calcified tissues
and presumably a recently developed odontode
which may either have been replacement or as
is equally likely, to have been an adjustment for
growth of the animal and its scales (see below).
Bone of attachment also showed variations in its
staining density.

Vertical canals, which may be readily ob-
served, open as pores on the underside of the
ornamented part of the scale (Smith, Hobdell
and Miller 1972). These seem to be extremely
variable in their distribution being dense in some
parts and missing in others. This variation may
be either near the scale apex or occur periph-
erally but does not appear to be related to the
position or density of the odontodes on the ex-
ternal surface of the scale. Neither were the ca-
nals related to the major or minor annuli. It
might be assumed that there are considerable
but short-lived metabolic demands by the cells
forming the odontodes and that consequently
there would be a regular relationship between
canals and odontodes. This appears not to be
SO.

As pointed out previously, the mineralization
of this highly modified cosmoid scale is only
found in the odontodes and most superficial part
of the isopedine layer (Smith, Hobdell and Mil-
ler 1972). However this layer of mineralization
is thickened around the lateral canal to form a
lightly mineralized and presumably rigid tube.
In sections of embryo scales the layers of iso-
pedine were observed to divide around the canal
although they did not appear to be mineralized
as yet when studied radiographically. The meth-

75

od of enlargement with growth would be an in-
teresting study.

Initially the isopedine beneath the ornamen-
tation is not mineralized although its layers are
laid down with the same dimensions of thickness
as in the adult scale. Increase in thickness of the
scale takes place by the increase in the number
of isopedine layers and not by their interstitial
growth.

The adult scale shows variation in pigmenta-
tion, indeed melanocytes may be observed in
association with the external and internal sur-
face of odontodes in the oral cavity as well as
those on the scales. While in the adult the me-
lanocytes seem to be secondarily arranged to the
mineralized tissues, examination of embryo
scales shows that the initial “‘ornamentation”’ is
by melanocytes not by odontodes. Clearly dis-
tinguishable melanocytes may be seen radiating
in rows from the apex of the scale, usually as-
sociated with the ridges which themselves ra-
diate in three relatively distinct groups—two in
the covered, unornamented, part of the scale
and the third in the exposed ornamented portion
(Fig. 14). That the melanocytes are the initial
ornamentation is also apparent in the growth se-
quences of the adult scale (see below). The me-
lanocytes show great variation of size and ex-
pansion. Whether this is functional, related to
the fixation process or due to poor fixation per
se is difficult to determine from this specimen.

There is clearly an initial relationship between
the melanocytes and ridges. There is also a later
relationship between odontodes and melano-
cytes although a relationship does not apparent-
ly exist between odontodes and ridges (Smith,
Hobdell and Miller 1972). To an experimental
embryologist this is an extremely intriguing
question; what inductive processes are involved
in the formation and orientation of melano-
cytes—of neural crest origin—and also cells
which form the dentine of the odontodes—which
may be presumed to also be of neural crest or-
igin? Odontoblasts are certainly of neural crest
origin in the oral teeth of some of the higher
nonmammalian vertebrates (Horstadius 1950).

Scale Growth

Once the scale has been established it would
appear that most of the areal growth takes place
at the periphery of the scale and that odontodes
are added at the edge so that the oldest ones are
at the centre (apex) of the scale and the youngest

76 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Leading edge of ornamented portion of adult

FiGure 1S.
scale. A discrete colony of melanocytes is visible with no
associated odontodes. Cleared scale x6.

at the periphery. This is also probably true of
the melanocytes. Orvig (1977) has drawn atten-
tion to the replacement activity that may be ob-
served at the apex. Odontodes are shed centrally
leaving the bone of attachment as a flat shell.
New odontodes sometimes develop over these
remnants. Radiographs indicate that the dentine
crowns are always discrete and that the coronal
dentine of the first odontode does not become
involved in the attachment of its successor and
form an ‘‘odonto-complex’’ (sensu Orvig 1977).
However usually the new odontodes are distinct
from their predecessors (Fig. 13).

There are clearly two phases in scale growth:
first its enlargement and second, modifications
of the ornamentation related to that growth and
adjustments that occur between adjacent scales
as the animal grows overall.

Along the anterior edge of the ornamented
portion one may observe occasional young and
partially developed odontodes with no signs of
previous bone of attachment related to other
odontodes (Fig. 13). Many small melanocytes
may be seen in this region. It might be tempting
to suggest that as they were not exposed to the
external environment they were not expanded
and sc smaller than their neighbors. However in

Ornamented portion of adult scale denuded of

FiGure 16.
odontodes presumably due to trauma. A central group of firm-
ly attached odontodes can be seen. The small odontodes
around the traumatized area may be new structures “‘recolon-
izing’ the damaged scale. Cleared specimen x6.

fish living at any depth this light related phenom-
enon is unlikely. Similar observations on em-
bryo scales (Fig. 14) strengthen this conclusion.
In one specimen a relatively large “‘colony’’ of
melanocytes was observed expanding onto the
normally unornamented part of an adult scale.
Their orientation was possibly related to the
ridges of the unornamented portion (Fig. 15). It
would seem that new odontodes are preceded
by melanocyte proliferation in both initial de-
velopment (see above and Fig. 14) and in later
normal increase in area of the ornamented parts
of the scale.

Repair of the ornamented portion may also
occur. Figure 16 illustrates a new set of odon-
todes developing in the centre of a bare area.
The scale came from the more bulbous part of
the fish and may have been scraped against the
substrate with consequent loss of odontodes.
The colony was firmly attached and consisted of
mostly small odontodes, particularly visible in
the comparable radiograph. Around the trau-
matized area small odontodes were observed.
These may also have been recolonizing the area
centripetally.

We now pass onto the vexing question of how

MILLER: COELACANTH MINERALIZED TISSUES

does one age Latimeria. From the previous dis-
cussion it is evident that the counting of scale
annular rings is extremely uncertain and that
although one may be able to count rings they
are variable in number even between adjacent
scales. There is also variation between the long
axis and the short axis of the scale and this is
accentuated when one compares a round central
body scale with a more oval or elongated tail or
posterior scale or a belly scale. Even though one
can count with some degree of certainty major
scale rings, it is apparent that even this param-
eter is variable.

Dentine is laid down in increments but the
teeth are constantly being replaced. Are these
increments daily, or are they perhaps a reflec-
tion of the lunar migration that has been hinted
at by McCosker (this volume) in his description
of the possible lunar related movements of this
fish? Teeth per se are not useful for aging. For
accurate aging one needs an organ that shows
incremental changes but with minimal variabil-
ity.

If one examines the various easily observed
bones such as the subopercula (Smith 1940) or
the gular plates (Fig. 17) one is struck by the
regular incremental lines which are visible on
the surface. Radiographs should be even more
clear and the gulars are particularly appropriate.
One would suggest that such material would be
more appropriate than scale ring counts. Ed-
ward Brothers (1977, personal communication)
has suggested that the annuli of ear ossicles
might be most appropriate for assessing the age
of Latimeria specimens. However the techni-
calities may be too complicated for routine use
and cannot be used in the field. A gular or sub-
opercula bone may be readily examined unaid-
ed:

It is suggested that scale ring counts are not
appropriate for age determination but that the
major resting lines in certain easily investigated
bones such as the subopercula or gular plate
may be more suitable.

Lagios (1977) has suggested that the coel-
acanth and Chondrichthyes are sister groups. As
far as the skin and scales are concerned the great
morphological similarity between odontodes on
the scales of Latimeria and the placoid scales
of selachians is very striking. Smith, Hobdell
and Miller (1972) suggested however that there
were close similarities between Latimeria scales
and those of modern lungfish (Kerr 1955). While

Wd,

FiGuRE 17.
annular rings. Field Museum II.

Internal surface of gular plate showing 9-10

his data (Lagios 1972, 1974, 1975) are very per-
suasive, the data on scales and skin are not very
helpful. Smith (1978) has pointed out that true
dental enamel is found in mammals, reptiles,
amphibians, as well as actinistians (coelacanth)
while elasmobranchs and actinopterygians have
a distinctive enameloid or mesodermal enamel.
Thus the outward similarity between elasmo-
branch dermal denticles and coelacanth scale
odontodes is not found in their structure and
possibly their development. The oral teeth of
both groups are likewise dissimilar.

ACKNOWLEDGMENTS

I wish to thank my previous coauthors, Martin
H. Hobdell and Moya Meredith Smith, for past
cooperation in preparing some of the material
presented here; also the latter for making more
recent information on Latimeria enamel avail-
able to me while in manuscript form. I also wish
to thank K. Thomson (Peabody Museum, Yale
University), K. Liem (Field Museum, Chicago).
S. H. Weitzman (Smithsonian Institution,
Washington, D.C.) and most of all P. H. Green-
wood, who greatly encouraged these studies at
their beginning, for making material available for
study.

The more recent help and cooperation of
George Brown, John McCosker and Mike La-
gios has been a great pleasure. Dr. Lagios is to
be thanked for making the scales of embryo no.
1 (A.M.N.H.) available for study.

The technical assistance of Mona Everett,
Christopher Miller and Glenn Denis was invalu-
able as has been the photographic expertise of
Malcolm McQuaig and Hector Velasco. The
manuscript was typed by Mary Parkhill and
Rose Parkhill.

LITERATURE CITED

ANDREWS, S. M. 1977. The axial skeleton of the coelacanth,
Latimeria. Pages 271-288 in S. M. Andrews, R. S. Miles,

78 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

and A. D. Walker, eds., Problems in vertebrate evolution.
Academic Press, London.

BERNHAUSER, A. 1961. Zur Knocken- und Zahnhistologie
von Latimeria chalumnae Smith und einiger Fossil formen.
S. B. Ost Akad. Wiss., mat.-naturw. 170:119-137.

BERTIN, L. 1958. Tissues squelettiques. Pages 532-55 in P.
P. Grassé ed., Traité de zoologie, Vol. 13. Masson & Cie.,
Paris.

Hospe.l, M. H., AND W. A. MILLER. 1969. Radiographic
anatomy of the teeth and tooth supporting tissues of Lati-
meria chalumnae. Arch. Oral Biol. 14:855-858.

Horstapius, S. 1950. The neural crest, its properties and
derivatives in the light of experimental research. Oxford
Univ. Press, London and New York.

IsoKAWA, S., Y. TODA, AND K. KuBoTa. 1968. A histolog-
ical observation of a coelacanth (Latimeria chalumnae). J.
Nihon Univ. Sch. Dent. 14:102-114.

Kerr, T. 1955. The scales of modern lungfish. Proc. Zool.
Soc. Lond. 125:335-345.

Kvam, T. 1946. Comparative study of the ontogenetic and
phylogenetic development of dental enamel. Norski Tan-
dloege Foren. Tid. 56 (Suppl.): 1-129.

Lacios, M. D. 1972. Evidence for a hypothalamo-hypophys-
ial portal vascular system in the Coelacanth Latimeria cha-
lumnae Smith. Gen. Comp. Endocrinol. 18 (1):73-82.

. 1974. Granular epithelioid (Juxtaglomerular) cell and

renovascular morphology of the Coelacanth Latimeria cha-

lumnae Smith (Crossopterygii) compared with that of other

fishes. Gen. Comp. Endocrinol. 2296-307.

1975. The pituitary gland of the Coelacanth Lati-
meria chalumnae Smith. Gen. Comp. Endocrinol. 25:126—
146.

Locket, A.
70:456—458.

McCosker, J. E. 1979. Inferred natural history of the living
Coelacanth. This volume.

MILLER, W. A. 1959. Layering in dentin caries as demon-
strated by localization of dyes. M.S. Thesis, Univ. of IIli-
nois.

1976. A future for the coelacanth? New Sci.

. 1968. Preliminary report on the histology of the den-
tal and paradental tissues of Latimeria chalumnae (Smith)
with a note on tooth replacement. Arch. Oral Biol. 13:1289-
1291.

1974. Observations on the developing rostrum and
rostral teeth of sawfish: Pristis perottetei and P. cuspida-
tus. Copeia 1974 (2):311-318.

, AND C. J. P. RADNorR. 1970. Tooth replacement pat-

terns in young Caiman sclerops. J. Morphol. 130:501—510.

, AND D. J. Rowe. 1973. Variations in tooth replace-
ment patterns in adult Necturus maculosus. J. Morphol.
140:63-76.

MILLoT, J., AND J. ANTHONY. 1958. Latimeria chalumnae,
dernier des Crossopterygiens. Pages 2553-3597 in P. P.
Grasse, ed., Traite de zoologie, Vol. 13 (3). Masson et Cie.,
Paris.

OrviGc, T. 1967. Phylogeny of tooth tissues: evolution of
some calcified tissues in early vertebrates. Pages 45-110 in
A. E. W. Miles, ed., Structural and chemical Organization
of teeth. Academic Press, New York and London.

1977. A survey of odontodes (*“‘dermal teeth’) from
developmental, structural, functional and phyletic points of
view. Pages 53-75 in S. M. Andrews, R. S. Miles, and A.
D. Walker, eds., Problems in vertebrate evolution. Aca-
demic Press, London.

Peyer, B. 1968. Comparative odontology. Univ. of Chicago
Press, Chicago and London. 347 pp. and pls.

Romer, A. S. 1946. The early evolution of fishes. Q. Rev.
Biol. 21:33-69.

Roux, G. H. 1942. The microscopic anatomy of the Lati-
meria scale. S. Afr. J. Med. Sci., Suppl. 7, pp. 1-18.

SAUNDERS, R. L. DE C. H., AND R. V. Ety. 1965. Optique
des rayons X et microanalyse. R. Castaing, P. Deschamps,
and J. Philibert, eds., Herman, Paris.

SCHAEFFER, B. 1953. Latimeria and the history of coelacanth
fishes. Trans. N.Y. Acad. Sci. 15:170-178.

. 1977. The dermal skeleton in fishes. Pages 25—S2 in
S. M. Andrews, R. S. Miles, and A. D. Walker, eds., Prob-
lems in vertebrate evolution. Academic Press, London.

Situ, C. L., C. S. RAND, B. SCHAEFFER, AND J. W. ATZz.
1975. Latimeria, the living coelacanth, is ovoviviparous.
Science 90:1105—1106.

SmitH, J. L. B. 1939. A surviving fish of the order Actinistia.
Trans. R. Soc. S. Afr. 27:47-S0.

1940. A living coelacanthid fish from South Africa.
Trans. R. Soc. S. Afr. 28:1-106.

SmitH, M. M. 1978. Enamel in the oral teeth of Latimeria
chalumnae: a scanning electronmicroscope study. J. Zool
Lond. 185:355-369.

, M. H. HoBDELL, AND W. A. MILLER. 1972. The
structure of the scales of Latimeria chalumnae. J. Zool.
Lond. 167:501—509.

THomson, K. S. 1966. Intracranial mobility in the coel-
acanth. Science 153:999-1000.

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134 15 pages December 22, 1979

MECHANISMS OF OSMOREGULATION IN THE COELACANTH:
EVOLUTIONARY IMPLICATIONS

By
Robert W. Griffith

Department of Biology,
Southeastern Massachusetts University,
North Dartmouth, Massachusetts 02747

and
Peter K. T. Pang

Department of Pharmacology and Therapeutics,
Texas Tech University School of Medicine,
Lubbock, Texas 79409

ABSTRACT

Latimeria maintains a blood osmolarity near that of its sea water environment by
retaining urea and trimethylamine oxide in concentrations similar to those of chon-
drichthians. Ion levels are fairly low, as in teleosts, and evidence is presented suggesting
that the rectal gland is involved as a sodium secreting organ. Bladder urine of Latimeria
is isouremic with the serum, contrasting with the elasmobranchs which reabsorb most
of the filtered urea. The evolutionary implications of the similarity between the methods
of osmoregulation in Latimeria and in the chondrichthians are discussed. Because of
(1) the lack of the characteristic chondrichthian mechanism of renal urea reabsorption,
(2) the use of different mechanisms for internal fertilization, a necessary concomitant
of the urea retention habitus in fishes and (3) the independent occurrence of urea
retention in the euryhaline amphibian, Rana cancrivora it is suggested that urea reten-
tion in Latimeria evolved independently of its occurrence in the cartilaginous fishes.
The factors which might favor the appearance of urea retention as an adaptation to
the marine environment rather than the hyposmotic mechanism used by teleost fishes
are discussed.

79

80 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

INTRODUCTION

According to traditional views on the phylog-
eny of vertebrates, the living coelacanth, Lati-
meria chalumnae, occupies a crucial position.
Although controversy surrounds the exact po-
sition of the coelacanths relative to other cros-
sopterygians and the Dipnoi (Miles 1975; Gar-
diner 1973), few students of the subject doubt
that the coelacanths branched off close to the
origin of the tetrapods. This fact, plus the ro-
mance surrounding the discovery of the living
coelacanth by J. L. B. Smith in 1938 have made
biologists in general and physiologists in partic-
ular keenly interested in the coelacanths as a
potential index of the biology of the prototetra-
pod.

Hence, it was considerably surprising when
Pickford and Grant (1967) and Brown and
Brown (1967) reported that the coelacanth pos-
sessed an osmoregularoty mechanism apparent-
ly identical with that of sharks and their kin and
unlike that of other fishes and tetrapods [see also
Lutz and Robertson (1971); Griffith et al.
(1974)]. That mechanism is the possession of a
blood osmolarity similar to that of seawater by
virtue of the presence of very high urea levels.
This feature was long thought to be a unique
(and ubiquitous) adaptation of the elasmobranch
fishes and represented such a characteristic ele-
ment of their biology that the eminent renal
physiologist Homer Smith suggested (perhaps
not facetiously) that the sharks and their kin
would more aptly be known by a taxonomic ap-
pellation which reflected the urea retention hab-
itus rather than by the less inclusive and exclu-
sive term Elasmobranchii (Smith 1953).

From this arises the question: is the posses-
sion of a common feature such as urea retention
an indication of some special evolutionary link
between the sharks and the coelacanth; could it
be an ancestral feature possessed by all early
fishes and subsequently lost in groups other than
the sharks and coelacanth; or is the linkage a
spurious One due to the independent acquisition
of the feature in the two groups? More recently,
a variety of soft tissue features have been de-
scribed unique to elasmobranchs and the coel-
acanth (Lagios 1975; Epple and Brinn 1975;
Chavin 1976; see also Lagios, this volume)
which lends some credence to the notion that
the elasmobranchs and Latimeria share a com-
mon and unique ancestry.

In this report we intend to analyze the os-
moregulatory mechanisms in Latimeria and
sharks in search of clues to determine if the fea-
ture was independently derived in the two
groups or is truly homologous. We also intend
to see whether any of the other features that
have been proposed as unique specializations of
the two groups might not be consequences of
urea retention. Surely such a major biochemical
adaptation must affect other features of the bi-
ology and physiology of Latimeria and the
sharks. Finally, we would like to suggest an ex-
planation for the adoption of the urea retention
method of osmoregulating in some fishes and the
adoption of hyposmotic mechanisms in other
groups of fishes. Most of the actual data pre-
sented here have been previously published else-
where (Griffith et al. 1974, 1975, 1976; Griffith
and Burdick 1976), but the format of this sym-
posium permits a synthesis and a much deeper
consideration of the evolutionary implications of
this work.

PRESENTATION OF DATA
Blood Chemistry

A comparison of the principal osmotically-ac-
tive solute concentrations in the blood serum of
Latimeria with those of representatives of other
marine fish groups is given in Table 1. An in
depth and specific comparison of different
groups of fishes for each blood component
would be beyond the scope of this discussion
since a variety of factors including stress, sex,
environmental salinity, diet and species vari-
ability can influence levels considerably. The
reader is referred to Griffith et al. (1974) for a
discussion of these problems as they relate to
the blood composition of Latimeria. Some very
general conclusions can be safely drawn, how-
ever.

For inorganic ions (Nat, Kt, Catt, Mgtt,
Ch. HCO; :SO,-; 20.3) Latimeria most
closely resembles Fundulus and other teleost
fishes (see review by Homes and Donaldson,
1969). This is especially true of the monovalent
ions Na‘ and Cl which are near sea water levels
in Myxine (ca. 500 mM) around 300 mM in hol-
ocephalans and elasmobranchs and under 200
mM in teleosts and Latimeria. In contrast, the
total blood osmolarity of Latimeria places it
with Myxine, holocephalans and elasmobranchs
with levels around the osmolarity of sea water

GRIFFITH & PANG: COELACANTH OSMOREGULATION

TABLE 1. BLOoD CHEMISTRY OF SOME SELECTED Ma-

RINE FISHES.

Rat- Coel-
Hag- fish! Shark? acanth* Teleost*
fish! Chi- Squa- Lati- Fundu-
Myxine —maera lus meria lus
glu- mon- acan- cha- hetero-
tinosa strosa thias lumnae clitus
Nat 487 338 296 197 183
Kt 8.4 11.7 Ted 5.8 4.8
(Caceh 4.8 4.3 310 4.8 2.3
Mg** 9.3 6.1 3.5 553 75)
cl” 500 353 276 187 146
HCO, ee 2.6 — 9.6 13.3
SO, S7) S22 3.1 4.8 —
PO, 0.4 — 2.4@ 551 523)
Urea 2ASe 332 308 377 4.0**
TMAO 38 0.0 72 122 ae
AA’s “ — 11.6 16.0 Sens
Osmol. 969 1,046 998 932 363

' After Robertson (1976 and 1966).

* After Robertson (1975).

3 After Griffith et al. (1974).

4 After Pickford et al. (1969).

* Total non-protein nitrogen = 38 mg N/kg (TMAO prob-

ably not included).

** Total non-protein nitrogen = 46.3 mg N/l TMAO prob-
ably not included).

(@ Total phosphate.

(ca. 1,000 mOsm/l) and distinguishes it from te-
leosts, euryhaline lampreys in sea water and
[with one exception, the crab-eating frog (Gor-
don et al. 1961)], from marine tetrapods. Note,
however, that osmolarity in Latimeria is ac-
tually slightly less than that of environmental
water (932 vs. 1,035 mOsm/l). As is now well
known, high blood osmolarity is obtained by
virtue of high blood levels of nitrogenous com-
pounds, principally urea and trimethylamine ox-
ide (TMAO), in the coelacanth, elasmobranchs
and holocephalans but by having high ion levels
in Myxine.

Hence, the blood chemistry of Latimeria
shows some points of similarity to that of acti-
nopterygian fishes (blood ion levels), and others
to chondrichthian fishes (high osmolarity, high
urea and TMAO). With respect to the nitroge-
nous compounds, we suspect that high urea is
the more meaningful feature: trimethylamine ox-
ide levels are apparently strongly influenced by
the diet (Groninger 1959), the feature is not
ubiquitous in the chondrichthians since holo-
cephalans lack it or have low levels (Read 1971;

81

TABLE 2. SODIUM-POTASSIUM ACTIVATED ATPASE IN
Latimeria AND ELASMOBRANCH TISSUES. (Values in «M P;,/
60!/mg protein.)

Coel-
Dogfish Skate acanth
Mustelus Raja Latimeria
canis erinacea chalumnae
Rectal gland Sel! 4.5 5.1
Kidney 2.3 ite Ded
Rostral organ — — 3.0
Muscle 1S) 1.0 0.6
Gills 0.7 0.9 =
Liver — — 0.6

After Griffith and Burdick (1976).

Robertson 1976); it is an important intracellular
osmotic component in many teleosts (Norris and
Benoit 1945) and it is found in substantial quan-
tities in the blood serum of some deep sea and
inshore marine teleosts (Griffith, unpublished).

Rectal Gland Function

In elasmobranchs, a substantial portion (but
not all) of the excess sodium and chloride that
enters the fish via passive routes is secreted by
a specialized organ, the rectal gland (Burger and
Hess 1960). Present evidence suggests that al-
ternative routes of salt secretion can compen-
sate for the rectal gland in its absence (Haywood
1975a; Chan et al. 1967). The coelacanth pos-
sesses a rectal gland in the same anatomical po-

TABLE 3. URINE COMPOSITION IN SOME SELECTED Ma-
RINE FISHES. (Values are in mM/I: numbers in parentheses are
urine/plasma ratios.)

Hagfish Coel- Teleost
Epta- Shark acanth Lophius
tretus Squalus Latimeria pisca-
stouti acanthias — chalumnae torius
Nat 553 (1.0) 240 (1.0) 184 (0.9) 11 (0.1)
Kt 11 (1.6) 2 (0.5) 9 (1.5) 2 (0.4)
Cais 4 (0.8) 3 (0.9) 2 (0.4) UX CXS)
Mg** 15 (1.2) 40 (33.3) 30 (5.7) 137 (54.5)
Cl 548 (1.0) 240 (1.0) 15 (0.1) 132 (0.9)
PO, 9 (3.9) 33 (34.0) 38 (5.5) 2 (0.3)
SO, 7 (8.6) 70 (140) 104 (21.6) 42 (35.0)
Urea 9 (1.8) 100 (0.3) 384 (1.0) 0.6 (1.9)
TMAO — 10 (0.1) 94 (0.8) 13 (1.5)
Osmol. — 800 (0.8) 962 (1.0) 406 (0.9)

Modified after Griffith et al. (1976). Original data from
Munz and McFarland (1964); Burger (1967); Brull and Nizet
(1953); Brull and Cuypers (1954) and Griffith et al. (1976).

82 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

sition (Millot and Anthony 1972) and with iden-
tical ultrastructural features (Lemire 1976) as
that of elasmobranchs. Investigations of the bio-
chemistry of the rectal gland of Latimeria (Grif-
fith and Burdick 1976) have demonstrated levels
of (Na+, K*)-ATPase which show a very close
parallel to the high levels found in the rectal
glands of representative elasmobranchs (Table
2). (Na*, K*)-ATPase is, of course, an enzyme
closely implicated in active salt transport in the
rectal gland of elasmobranchs and in the salt se-
creting organs of other animals (Bonting 1966).
It must be pointed out that a homology between
the rectal glands of elasmobranchs and Lati-
meria has yet to be demonstrated; the rectal
gland of the holocephalans differs considerably
in structure from that of the elasmobranchs
proper (Fange and Fugelli 1963; Read 1971;
Stahl 1967).

Kidney Function in Latmeria

One of the most crucial physiological mech-
anisms permitting the elasmobranch fishes to
maintain high internal urea concentrations is the
renal reabsorption of most of the urea that is
filtered through the glomerulus (see reviews by
Smith 1936; Hickman and Trump 1969; Schmidt-
Nielsen 1972). This is also true of the holoceph-
alans (Read 1971), but apparently not of the
crab-eating frog which also manages to maintain
high blood levels of urea (Schmidt-Nielsen and
Lee 1962). In elasmobranchs, urea reabsorption
appears to involve a countercurrent exchange
mechanism in the kidney (Boylan 1972; Deetjen
et al. 1972; Stolte et al. 1977), and also to be
linked with sodium reabsorption (Schmidt-Niel-
sen et al. 1972).

Data on the solute composition of coelacanth
bladder urine are compared with that of a selec-
tion of other marine fishes in Table 3. We have
also included values for the ratio of levels in
urine to serum which may be used as an index
of which substances are conserved and which
eliminated. Although the coelacanth urine was
admittedly from a severely stressed fish (Locket
and Griffith 1972), many features are as antici-
pated: high polyvalent ion (Mgt+, SO,=, PO,=>)
U/S ratios are found as is characteristic of the
urine of most marine animals, and monovalent
ion levels (Na* and Cl) are below serum levels
as is true of most fishes. Osmolarity is identical
to that of serum, a condition approached in
many healthy marine fishes of various groups

(Hickman and Trump 1969). Rather unexpect-
edly, the coelacanth appears to be unable to
reabsorb urea and the urine is characterized by
levels of TMAO only slightly lower than blood
serum.

That the urine urea data in Latimeria are not
a pathological consequence of stress is suggest-
ed by the calculation of Phosphate/Nitrogen ra-
tios (Smith 193la) which come out close to those
of normal carnivorous vertebrates (Griffith et al.
1976) and contrast with those of elasmobranchs
which have high urinary P/N ratios since the
bulk of the urea is lost through extrarenal routes
(Smith 193la, 1931b; Chan and Wong 1977).
Also, even severe stress never elicits isouremia
in elasmobranchs (Forster et al. 1972). The U/P
ratio of 0.8 for TMAO in Latimeria may (1) be
indicative of some reabsorption of this com-
pound, (2) may be a consequence of limited glo-
merular permeability to TMAO or (3) more like-
ly may be attributable to elevated blood levels
during stress not being fully reflected in the blad-
der urine which presumably included some urine
formed prior to severe stress. Muscle levels of
TMAO are much higher in Latimeria than blood
levels as is also true of elasmobranchs, holoce-
phalans and teleosts (Lutz and Robertson 1971;
review by Lombardini et al., this volume), and
could act as a source of blood TMAO during
severe muscular exercise.

DISCUSSION
How the Coelacanth Osmoregulates

Although based upon a single moribund spec-
imen (Locket and Griffith 1972) the data we have
presented here give us considerable insight into
the mechanisms the coelacanth uses to regulate
solutes and water. If the blood osmolarity is in-
deed lower than environmental values as our
data suggest, the coelacanth must be losing
water to its environment and presumably drinks
sea water to replace the lost fluid as do teleosts
(Smith 1930a; Conte 1969). Preliminary data on
the composition of gut water (Griffith, unpub-
lished) suggests that this is so; sodium and chlo-
ride levels drop from the stomach to the large
intestine and rectum whereas osmolarity rises,
possibly reflecting an ion-linked water transport
in the gut comparable to that of teleosts. How-
ever, it has been recently suggested that the true
habitat of Latimeria may be in subterranean
caves with a dilution of the media by aquifers

GRIFFITH & PANG: COELACANTH OSMOREGULATION

(McCosker, this volume). The hyposmotic blood
of Latimeria might be regarded as circumstan-
tial evidence favoring this hypothesis.

Latimeria regulates its blood osmolarity at
levels near that of its environment while main-
taining total electrolytes at levels only 40% those
of sea water. We have seen that this is accom-
plished by building up high blood concentrations
of organic compounds, principally urea and
TMAO. Although the mechanisms involved in
the maintenance of these high concentrations of
nitrogenous products remain a topic for specu-
lation, it appears that renal reabsorption is not
a factor. Rather, we suspect that the mainte-
nance of high blood urea and TMAO is a con-
sequence of one or more of the following fac-
tors: (1) low glomerular filtration rate, (2)
extremely low branchial and epithelial perme-
ability to urea and (3) high production rates of
urea from hepatic ornithine-urea cycle enzy-
matic pathways. Although Latimeria kidneys
appear to be well supplied with moderately large
glomeruli (Millot and Anthony 1973: Lagios
1974), their somewhat hyposmotic condition
places them in negative water balance which
should lead to a low GFR. Although conclusive
demonstrations of an important role of the renin-
angiotensin system in osmorregulation of other
fishes is lacking, the presence of a renin-angio-
tensin system in Latimeria (Nishimura et al.
1973) might provide a mechanism for control of
renal function (Lagios 1974). The gill area of
Latimeria relative to body weight is the lowest
reported for any fish (Hughes 1972, 1976;
Hughes and Morgan 1973) and Latimeria is a
large animal offering, of course, a low surface
area for urea loss relative to the volume of urea
synthesizing tissue. The activity levels of en-
zymes of the ornithine-urea cycle determined in
slowly frozen Latimeria liver are comparable to
those of elasmobranchs (Brown and Brown
1967; Goldstein et al. 1973), but it is feasible that
even higher levels might characterize coelacanth
tissue if it had been frozen more promptly or
assayed when fresh.

The regulation of electrolytes in the coel-
acanth would seem to involve the kidneys, the
rectal gland and, possibly, the gills. The kidneys
function primarily in the excretion of polyvalent
electrolytes such as Mg**, SO, and PO, and
in the selective elimination of certain organic
substances such as glucuronides and creatine
and the conservation of others (glucose, lactate,

83

protein etc.). Although there is no direct evi-
dence that the rectal gland functions in the se-
cretion of NaCl, this would seem to be a likely
conclusion in view of the high (Na*, K*)-ATP-
ase levels (Griffith anc» Burdick 1976), the ultra-
structural similarity to the elasmobranch rectal
gland which functions in this way (Lemire 1976)
and the apparent inability of the kidney to con-
centrate NaCl. There is no evidence for or
against a role of the gills in ion secretion in Lati-
meria, although the gills do seem to play a sig-
nicant role in ion transport in agnathans, elas-
mobranchs and teleosts (review by Conte 1969)
and such a role would not be unexpected.
‘Chloride’ cells are sparse in the gills of Lati-
meria as they are in elasmobranchs (Hughes
1976).

Interpreting Shared Features

In comparing the osmoregulatory mechanisms
of Latimeria with those of other marine fishes,
perhaps the most notable feature is the unique
occurrence of high blood osmolarity by virtue
of high blood urea levels in the coelacanth and
in the elasmobranch fishes and related holoceph-
alans. For a comparative biologist this obser-
vation immediately invites the question of the
evolutionary significance of urea retention. Like
any other physiological or morphological fea-
ture, there are three possible explanations for
urea retention being a uniquely shared charac-
ter: (1) that the feature is a primitive one that
has been lost in other more advanced lines, (2)
that it is a specialized feature developed inde-
pendently in the two groups and tells us nothing
of their phylogeny and (3) that it is a specialized
feature which evolved only once and is evidence
of a uniquely shared phylogenetic relationship
between the two groups (see also Atz 1973; Grif-
fith et al. 1974; Lagios, this volume).

Vertebrate Phylogenies

Traditional phylogenetic schemes (e.g. Romer
1966; Moy-Thomas and Miles 1971; Schaeffer
1968; Gardiner 1973; Miles 1973) portray the
coelacanth as being close to the origin of the
tetrapods, sharing a common ancestry with oth-
er bony fishes including the dipnoans and acti-
nopterygians and having only a remote relation-
ship to the elasmobranchs and holocephalans.
The restriction of urea retention to the coel-
acanth and chondrichthians may be concordant
with traditional views on vertebrate phylogeny

84 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ELASMOBRANCH TELEOST COELACANTH TETRAPOD

LL

ELASMOBRANCH TELEOST COELACANTH TETRAPOD

| ia

FIGURE lI.

ELASMOBRANCH COELACANTH TELEOST TETRAPOD

ae

TELEOST TETRAPOD COELACANTH ELASMOBRANCH

uae

Urea retention (indicated by shading) and some alternate vertebrate phylogenies: Possibility 1. A traditional

scheme with urea retention as an ancestral trait which Is lost in teleosts and tetrapods. Possibility 2. A traditional scheme with
the independent acquisition of urea retention in elasmobranchs and coelacanths. Possibility 3a. A heretical scheme with urea
retention being a unique specialization evolved in the common ancestor of coelacanths and elasmobranchs. Possibility 3b.
Another heretical scheme with urea retention being a unique specialization which evolved in the common ancestor of coelacanths

and elasmobranchs.

if the aforementioned possibilities #1 (loss of it
as a primitive feature in other groups) or #2 (in-
dependent acquisition) were to be the case, but
if #3 were true (urea retention is a unique spe-
cialization evolved only once) then the tradi-
tional scheme must be discarded. This may be
visualized in Figure 1, where possibilities #1
and #2 are shown in terms of traditional phy-
logenies and possibility #3 is shown with two
alternative phyletic schemes. We might point
out that although possibility #3 (unique special-
ization) directly invalidates the traditional phy-
logeny, possibilities #1 (loss in other groups)
and #2 (independent acquisition) do not unique-
ly support the traditional phylogeny since the
“‘heretical’’ phylogenies will fit equally as well.
It is unfortunately the case that the fossil record
is ambiguous both in regards to the precise re-
lationships of the coelacanths relative to the
chondrichthians and other major groups of fish-
es (cf. Bjerring 1973; Jarvik 1968a, 1968b) and
in terms of their early environmental history rel-
ative to the marine environment (Thomson 1969,
1971; Moy-Thomas and Miles 1971).

How can we decide which of the three alter-
native explanations for the appearance of urea
retention in chondrichthians and Latimeria is

correct? Although neither will provide incon-
trovertible proof, two approaches may be taken.
First, we can see whether the bulk of the other
characters supports the “‘heretical’’ phylogeny
based upon distribution of urea retention. This
would provide circumstantial evidence for or
against urea retention as a unique specialization.
Secondly, we might analyze the mechanisms in-
volved in urea retention and see whether they
are the same in the two groups and also see
whether logical concomitants of urea retention
are accomplished by the same mechanism in the
two groups. This approach might provide a more
direct means of judging whether urea retention
is truly homologous and, hence will provide evi-
dence for or against urea retention as an inde-
pendently derived character state.

A Consideration of Features Other than Urea
Retention

Recent studies on the soft tissue biology of
Latimeria have come up with an impressive
number of features which Latimeria and the
chondrichthians have uniquely in common (see
also Lagios, this volume). Among them are (1)
a duct-associated pancreatic islet tissue (Epple
and Brinn 1975), (2) adenohypophysial anatomy

GRIFFITH & PANG: COELACANTH OSMOREGULATION

(Lagios 1975), (3) extra sulfonation in chondro-
itin sulfate (Mathews 1966, 1967) and (4) a rectal
gland of apparent ion secretory function (Millot
and Anthony 1972; Griffith and Burdick 1976;
Lemire 1976). A number of other features are
shared by the coelacanth and elasmobranchs but
not necessarily uniquely including high TMAO
levels (Griffith et al. 1974), high levels of en-
zymes of the ornithine-urea cycle and trimethy-
lamine oxidase (Brown and Brown 1967; Gold-
stein et al. 1973), an ovoviviparous breeding
mode with extremely large eggs (Griffith and
Thomson 1973; Smith et al. 1975), a single me-
dian compact thyroid gland (Chavin 1976) and,
not trivially, both the elasmobranchs and Lati-
meria are large fish. On the other hand, there
are a variety of features which characterize La-
timeria and other bony fishes that do not occur
in chondrichthians including (1) true scales (2)
internal endochondral bone (3) an operculum (4)
a gas-filled diverticulum off the gut, the lung or
gas bladder and (5) blood levels of monovalent
ions (Griffith et al. 1974), and there are a number
of characters which link the coelacanth and fos-
sil rhipidistians with the earliest tetrapods in-
cluding the structure of the limbs and girdles,
the arrangement of the bones of the skull, and
the presence of a kinetic skull with a joint sys-
tem between the orbitotemporal and otic regions
of the endocranium.

In fact, arguments may be made for essen-
tially all of the above characters as being (1)
independently acquired (2) primitive features
lost in other lines or (3) unique specializations.
A compilation of the number of features held in
common between the two groups actually pro-
vides us with little insight into true phylogenetic
relationships at the level of major groups as we
are dealing with here. Most structural and func-
tional relationships will change much more rap-
idly in evolutionarily labile groups such as te-
leosts and tetrapods while remaining in a near-
primitive state in more static groups such as the
lungfishes, coelacanths and chondrosteans. On
the other hand, one should avoid making the
assumption that, since in other features two evo-
lutionary lines remain static, the occurrence of
what can only be regarded as a highly special-
ized feature that is uniquely shared in the two
groups is a priori evidence of a close relation-
ship. Different character complexes may evolve
at different rates depending upon environmental
pressures (see DeBeer 1954).

85

Evidence for the Non-independence of Charac-
ters

Returning again to urea retention, is it possi-
ble that any of the other features linking Lati-
meria and the chondrichthians might be inti-
mately related to urea retention rather than
being truly independent characters? Unques-
tionably, some are. The retention of high TMAO
levels in the blood is probably related to the re-
tention of urea, since a reduced loss of one com-
pound across surface areas (and/or through the
kidney) is probably due to a rather general re-
duction in permeability towards solutes. Elas-
mobranchs are characterized by a rather low
permeability to ions (Haywood 1975; Payan and
Maetz 1973; Carrier and Evans 1972; Pang, Grif-
fith and Maetz, unpublished) as well as to urea.
High activities of enzymes of the ornithine-urea
cycle would seem to be a prerequisite for main-
taining high internal urea concentrations in the
face of some unavoidable loss through the kid-
neys and across the body surfaces, and high
titres of TMAOase (Goldstein et al. 1973) prob-
ably have a similar cause. Provision of a pro-
tected environment for the embryo during its
early development by ovoviviparity or viviparity
and large adult size are also closely associated
with the urea retention habitus for reasons elab-
orated below (see also Griffith and Thomson
1973). The presence of sulfonated uronate resi-
dues in the cartilage appears to be related to
uremia since the feature is absent from fresh-
water elasmobranchs with low blood urea (Ma-
thews 1962; Thorson et al. 1967; Griffith et al.
1973) and elasmobranch high sulfate cartilage
chondroitin sulfate protein does not dissociate
in the presence of very high urea concentrations
as do the cartilage proteins of other vertebrates
(Mathews 1967). It is possible that the appear-
ance of an ion-secreting organ that empties into
the posterior part of the gut is also closely tied
in with urea retention although, admittedly, this
is speculation with little hard data behind it. One
might argue that the necessary reduction in the
permeability of the gills to solutes which is a
consequence of urea retention eliminates them
as a major site for the active secretion of ions
and, in the absence of such *‘preadaptations’’ as
tear, salivary or sweat glands, which are avail-
able to amniotes as ion secreting organs, a urea-
retaining fish must secrete excess salt into the
terminal part of the gut or urogenital system.

86 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

The appearance of such ion secreting organs in
plotosid catfish (Lennep 1968; Kowarsky 1973)
and in the lungfish Protopterus (Lagios and
McCosker 1977) are suggestive if not particular-
ly enlightening.

Although we have suggested some bases for
the interdependence of many of the characters
shared by the chondrichthians and Latimeria,
it is obvious that other features such as pituitary
morphology and pancreatic islet associations
cannot readily be regarded as consequences of
urea retention. It is apparent that the analysis of
other characters provides little insight into
whether urea retention is independently ac-
quired or homologous in Latimeria and the elas-
mobranchs, and we should instead analyze the
mechanisms involved in urea retention and see
whether they are homologous in the two groups.

Analysis of Mechanisms Involved in Urea Re-
tention and of its Concomitants

We believe that there are three lines of evi-
dence which suggest that urea retention in chon-
drichthians and Latimeria was independently
derived. One is the absence of renal reab-
sorption of urea in Latimeria, the second is the
utilization of different modes of internal fertil-
ization in the two groups, and the third is the
independent appearance of urea retention as an
osmoregulatory device in vertebrate groups oth-
er than Latimeria and the chondrichthians.

We have already considered the difference
between Latimeria and the elasmobranchs in
kidney function. In elasmobranchs up to 95% of
the filtered urea is reabsorbed and U/P ratios
under 0.30 are usual (reviews by Schmidt-Niel-
sen 1972; Pang et al. 1977). This renal conser-
vation of urea is of such obvious selective ad-
vantage to a urea retaining fish that we were
greatly surprised when we found that Latimeria
lacked the ability to reabsorb urea. Urea reab-
sorption is such a ubiquitous feature in chon-
drichthians (including the holocephalans with a
long separate evolutionary history, Read 1971)
and involves such complex morphological (Boy-
lan 1972; Stolte 1977) and physiological (Schmidt-
Nielsen 1972; Hays et al. 1977) adaptations that
it is difficult for us to conceive of it developing
under conditions other than a severe challenge
such as the initial movement of the ancestor of
the elasmobranchs and holocephalans from
fresh water to the marine environment with the
concomitant development of urea retention. We

regard the absence of renal urea reabsorption in
Latimeria as evidence that it was never present
in coelacanths but rather that they adopted dif-
ferent mechanisms for conserving urea and
evolved the urea retention habitus independent-
ly from the chondnrichthians.

The second point of evidence, the mechanism
for internal fertilization, involves somewhat
complex reasoning but is an equally strong ar-
gument for the independent acquisition of urea
retention. We have already suggested that inter-
nal fertilization and an ovoviviparous or vivip-
arous reproductive mode are obligatorily linked
with urea retention which in fishes is a specific
adaptation to osmoregulation in marine habitats,
but some discussion may be necessary before
this is obvious. The converse argument that urea
retention arose secondarily to the evolution of
a protected fetal environment as a means of
dealing with the toxic buildup of ammonium in
an analogous manner to the amniotes would
seem to be discredited by the elimination of urea
retention in freshwater stingrays without a
change in their reproductive mode.

The loss of urea in elasmobranchs occurs
principally across the branchial and other sur-
face epithelia (Smith 1931b; Payan et al. 1971,
1973; Boylan 1967; Chan and Wong 1977);
whereas its synthesis takes place within the liver
which, of course, represents a portion of the
body volume. In very small animals such as ear-
ly embryos the surface area will be very large
relative to the volume of the liver and urea syn-
thesis will not be able to keep up with urea loss.
For this reason, urea retention is not economical
in very small embryos and we observe that all
chondrichthians (Wourms 1977) either maintain
their young internally to an advanced stage of
development or secrete a case around the egg
which acts as a barrier to urea loss [albeit not
a complete one, see Read (1968), Price and Dai-
ber (1967)]. Such developmental patterns re-
quire internal fertilization; the intromittant or-
gans being the claspers, modified pelvic fins, in
all chondrichthians (Wourms 1977) and presum-
ably being erectile caruncules surrounding the
‘cloaca’ in the coelacanth (Griffith and Thom-
son 1973; Millot and Anthony 1960). If a pro-
tected embryonic environment with internal fer-
tilization is a necessary prerequisite for urea
retention as we suspect that it is, then the dif-
ference between coelacanths and chondrichthi-
ans in the method of internal fertilization is evi-

GRIFFITH & PANG: COELACANTH OSMOREGULATION

dence that both the reproductive mode and urea
retention were independently acquired in the
two groups. A common ancestor of coelacanths
and chondrichthians possessing urea retention
but not internal fertilization would be a contra-
diction to our argument and an intermediate
state between claspers and erectile cloacal ca-
runcules is difficult to imagine.

The final argument for independent acquisi-
tion both provides direct evidence that urea re-
tention can arise more than once and also pro-
vides some support for the validity of the two
preceding arguments. Like Latimeria and the
chondrichthians, the crab-eating frog, Rana
cancrivora, builds up high levels of urea in its
blood to maintain blood osmolarity near that of
its marine environment (Gordon et al. 1961). All
evidence is that the ranid frogs are of relatively
recent origin (Estes and Reig 1973; Lynch 1973)
and, like other amphibia, are originally of fresh-
water habitat and lack the urea retention habitus
per se, although some amphibians do possess
the capacity to build up urea (Funkhouser and
Goldstein 1973; Schoffeniels and Tercefs 1966;
Schlisio et al. 1973; Jungreis 1974). Unquestion-
ably, Rana cancrivora did acheive the urea re-
tention mechanism independently of that of Lat-
imeria and the chondrichthians. Like Latimeria,
and unlike the chondrichthians, Rana cancriv-
ora lacks the ability to reabsorb urea in the renal
tubules (Schmidt-Neilsen and Lee 1962) but, in
apparent contrast to Latimeria, it possesses the
capacity to reabsorb urea across the urinary
bladder (Schmidt-Nielsen and Lee 1962; Gordon
and Tucker 1968; Balinsky et al. 1972; Hew et
al. 1972; Dicker and Elliot 1973). Hence, al-
though it has reached the same solution as the
elasmobranchs and Latimeria in terms of blood
urea levels, Rana cancrivora uses an entirely
different mechanism from either of them to do
so. The problem of the loss of urea in very small
embryos also has a unique solution in Rana can-
crivora (Gordon and Tucker 1965). Like most
amphibians, Rana cancrivora lays a large num-
ber of small, unprotected eggs but does breed in
saline waters. Up to the point of metamorphosis
the tadpoles osmoregulate like marine teleost
fishes by being hyposmotic to the environment
and do not build up urea. Although this solution
to the problem is probably dictated by the nor-
mal switchover from an ammonotelic to a ureo-
telic excretion during metamorphosis (reviews
by Campbell 1973; Goldstein 1972), we think it

87

also supports our view that urea retention, in-
ternal fertilization and a protected embryonic
milieu are obligatorily coupled in fishes where
metamorphosis does not occur.

Choice of Strategy: The Energetics of Urea Re-
tention and Hyposmotic Regulation

Having concluded that urea retention proba-
bly arose at least three separate times in verte-
brates when freshwater animals entered a ma-
rine environment, the question naturally arises
as to why these animals chose urea retention
whereas all actinopterygian fishes and the petro-
myzontid agnathans opted for an hyposmotic
mechanism for osmoregulating when they en-
tered the marine environment.

First of all, we should consider the energy ex-
penditure that might be involved in the different
mechanisms for osmoregulating. The teleost
method of osmoregulation, maintaining the
blood hyposmotic to the environment, involves
active, energy-requiring sodium transport at two
sites; an inward transport of sodium in the gut
which is coupled with the uptake of water to
replace that lost across the epithelia, and an out-
ward transport across the gills to get rid of the
sodium that enters through the gut and also that
which enters passively across the epithelial sur-
faces (Smith 1929; reviews by Potts 1968; Conte
1969). Elasmobranchs are hyperosmotic or isos-
motic and do not drink sea water or actively
move sodium across the gut, but they must ex-
pend energy synthesizing urea to replace that
lost across epithelial surfaces and also must ex-
pend energy in eliminating excess sodium that
enters passively by secreting it through the rec-
tal gland or across the gills (review by Pang et
al. 1977). Very crude estimates of the osmo-
regulatory energy expenditure in terms of ATP
which is hydrolyzed both in active sodium trans-
port (ca. 0.4 mM ATP/mM Na’*) and in urea syn-
thesis through the ornithine urea cycle (the
equivalent of 4 mM ATP/mM urea) may be made
for the elasmobranch Poroderma africanum
based on the data of Haywood (1974, 1975b) and
on representative teleosts based on the summary
of Maetz and Bornancin (1975). In Poroderma,
urea turnover is 0.12 mM/kg-hr and sodium ex-
change 1s 0.185 mM/kg-hr of which about 0.050
mM/kg-hr may be via rectal gland secretion.
Somewhat higher urea and sodium exchanges
have been reported for other elasmobranchs
(Burger and Tosteson 1966; Chan et al. 1967;

88 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Carrier and Evans 1972; Goldstein and Forster
1971; Payan and Maetz 1971). In teleosts bran-
chial sodium excretion ranges from 3.8 mEq/Kg-
hr in Salmo gairdneri to 78 in Mugil captio with
26 mEq/Kg-hr being a more-or-less typical value
for marine species. Thus, the total energy ex-
pended on osmoregulation may be estimated for
our ‘“‘typical’’ teleost at 10.4 mM ATP/Kg-hr
based solely on sodium excretion and for Po-
roderma at 0.55 mM ATP/Kg-hr by adding the
0.48 mM used for urea synthesis and the 0.07
mM used for overall sodium excretion. From
these very rough calculations it would seem that
the elasmobranch method of osmoregulating is
more economical although, as we will point out,
many other factors will determine whether urea
retention is both feasible and economical.

Factors Predisposing Fishes Towards the De-
velopment of Urea Retention

Among the factors influencing the develop-
ment of urea retention, we can suggest a number
of predisposing features (prerequisites or pre-
adaptations), four features which must be de-
veloped simultaneously with urea retention but
are not necessarily prerequisites, and one con-
cept that may act to prevent the ready switch-
over from a hyposmotic mechanism to urea re-
tention and vice versa.

The first prerequisite for urea retention is the
presence of a complete ornithine-urea cycle.
Most authors now agree that a complete O-U
cycle was found in the ancestral gnathostome
(see reviews by Campbell 1973; Goldstein 1972).
However, it is absent in living agnathans (Read
1968, 1970, 1975) and, probably secondarily, is
low or incomplete in many teleosts (Huggins et
al. 1969; Wilson 1973; Gregory 1977). Groups
lacking a complete O-U cycle may be prevented
a priori from developing urea retention unless
alternative pathways for urea synthesis, as
through the metabolism of purines, can be used.
A second predisposing feature is large size.
Since very small fish have large surface/volume
ratios, the loss of urea across the epithelia will
exceed the capacity of the liver to synthesize it
or, at any rate, will make the mechanism of urea
retention uneconomical. Hyposmotic regulation
will be no more expensive for small fish than for
large fish since both sodium intake and sodium
extrusion will take place across the surface ep-
ithelia. A related predisposing feature, at least
in fishes which adopt urea retention as a per-

manent habitus, is internal fertilization since the
early embryonic stages would be unable to syn-
thesize enough urea to replace that lost across
their relatively large surface areas. Another fac-
tor which might influence the development of
urea retention is the life history of the animal
and two aspects of this are worth considering;
activity patterns and the exposure to changing
salinities. A very active fish requires a large gill
area and a close contact between the branchial
blood supply and the environmental water for
the rapid exchange of gases (review by Hughes
and Morgan 1973). It follows that these adap-
tations would interfere with the development of
a urea impermeable epithelium. Hence, it would
be more likely for a sluggish than for an active
fish to develop urea retention, particularly with-
in smaller body sizes. We think it is something
more than a coincidence that the more active
elasmobranchs such as the lamnid sharks are in-
variably large both at birth and as adults where-
as sluggish elasmobranchs such as the batoids
may be small in size. Air breathing, as in adult
Rana cancrivora, might be a helpful preadap-
tation to urea retention. We have earlier sug-
gested (Griffith et al. 1973) that the teleostean
method of osmoregulating might be more effi-
cient for survival in conditions of rapidly chang-
ing salinity since the high osmolarity inherent in
urea retention becomes increasingly disadvan-
tageous as salinities drop below sea water levels
and in elasmobranchs physiological modifica-
tions of blood solute levels are both slow and
incomplete (Smith 193la; Price and Creaser
1967; Urist 1962; Thorson 1967; Thorson et al.
1973). Although there are some euryhaline elas-
mobranchs (Smith 1936; Boeseman 1964; Thor-
son and Watson 1975; Thorson 1976), these are
exceptions and most euryhaline fishes are te-
leosts.

The Evolution of the Process of Urea Retention

Other than the exceptional occurrence of urea
retention in estivating lungfishes (Smith 1930b),
we cannot conceive of any evolutionary path-
way for the development of urea retention in
fishes other than the movement from fresh water
into the marine environment. The very fact that
urea retaining fishes have blood ion levels lower
than the environment is incontestible evidence
of their freshwater ancestry. Virtually all marine
invertebrates and certainly all that are primary
marine groups, have extracellular fluid electro-

GRIFFITH & PANG: COELACANTH OSMOREGULATION

lyte levels that differ only in minor points from
sea water and this holds true even for such high-
ly organized groups as the crustaceans and
cephalopod molluscs.

In going from a freshwater to a marine en-
vironment, a fish which becomes a urea retainer
must undergo several modifications of his basic
physiology as well as possess the proper com-
bination of prerequisites for urea retention as
described in the preceding section. Specifically,
if not already present, there must be (1) devel-
opment of surface epithelia impermeable to
urea, (2) modification of renal function so that
urea is conserved in the urine, (3) elevation of
activities of enzymes of the O-U cycle to pro-
duce sufficient amounts of urea, and (4) devel-
opment of a tissue tolerance to elevated urea
levels. We should point out that tissue tolerance
to high urea is probably not a serious impedi-
ment to the development of urea retention: urea
levels comparable to those in the tissues of elas-
mobranchs are not invariably toxic to teleost
fishes (Hugoneng and Florence 1921; Rasmus-
sen and Rasmussen 1977; Griffith et al., in press)
although biochemical and osmoregulatory prob-
lems often ensue.

The Non-interconvertibility of Hyposmotic Reg-
ulation and Urea Retention

Although there is no a priori reason to believe
that if is impossible for a marine teleost to de-
velop urea retention or for a marine elasmo-
branch to relinquish urea retention and become
a hyposmotic regulator, we know empirically
that this has not occurred. With the incredible
diversity of bony fishes, one might think that
unless there were some strong opposing force,
that certain large, sluggish teleosts with internal
fertilization (e.g. the zoarcids or brotulids) de-
velop urea retention and one could also imagine
that there must be some ecological niche for a
small active shark for which urea retention
might be an energetic disadvantage. We are con-
vinced that the reason these switchovers have
not occurred is a phenomenon we might desig-
nate as evolutionary channelization. Simply ex-
pressed, this means that once an evolutionary
line has adopted a complex solution to an en-
vironmental problem and has successfully
adapted to it, that line must retain that solution
and all concomitant features that go with it until
a new and different environmental challenge
presents itself that would make the original so-

89

lution disadvantageous. In addition to urea re-
tention, we might suggest that other physiolog-
ical adaptations such as low blood electrolytes
in vertebrates and homeothermy in birds and
mammals are good examples of the operation of
the phenomenon.

The only environmental change that has re-
sulted in dechannelization of urea retention is
the return to fresh water of such groups as the
Amazon River rays of the family Potamotrygon-
idae. Not only are blood urea levels low (Thor-
son et al. 1967; Junqueira et al. 1968; Griffith et
al. 1973) but enzymes of the ornithine urea cycle
have declined (Goldstein et al. 1971; Gerst and
Thorson 1977), the rectal gland has become non-
functional (Griffith et al. 1973; Gerst and Thor-
son 1977), renal reabsorption of urea is absent
(Gerst and Thorson 1977), the cartilage has lost
the extra sulfonation characteristic of elasmo-
branchs (Mathews 1962) and the ability to tol-
erate sea water is no longer present (Thorson
1970; Griffith et al. 1973; Gerst and Thorson
1977). It is interesting to speculate about the
mechanisms that might be adopted were the po-
tamotrygonids ever to become euryhaline again
and reinvade the marine environment.

SUMMARY

The coelacanth, Latimeria chalumnae, dis-
plays an osmoregulatory strategy similar in
many respects to that of the elasmobranch and
chimaerid fishes. Total serum osmolarity ap-
proaches that of the marine environment, while
concentrations of most major ions are consid-
erably lower, the bulk of the remaining solutes
consisting of urea and trimethylamine oxide. As
do elasmobranchs, the coelacanth possesses a
rectal gland of presumptive ion secreting func-
tion. However, in contrast to the elasmobranchs
and holocephalans, Latimeria lacks the ability
to reabsorb urea across the renal tubules and
also appears to have a serum osmolarity some-
what below that of the environment, putting it
in negative water balance.

Based upon three lines of evidence, we sug-
gest that the urea retention habitus in Latimeria
and the cartilaginous fishes was acquired inde-
pendently. (1) The absence of the characteristic
chondrichthian renal reabsorption of urea in the
coelacanth indicates that the urea retention hab-
itus is only superficially similar in the two lin-
eages and is not homologous. (2) Fishes that use
urea retention as an osmoregulatory strategy

90 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

must make some provision to prevent excessive
loss of urea in small embryos whose large sur-
face/volume ratios preclude a free existence.
Although coelacanths, like many sharks, main-
tain the young internally to an advanced state of
development, the mechanisms for internal fer-
tilization are radically different indicating that
the reproductive mode, and by inference urea
retention, were independently derived. (3) The
euryhaline amphibian, Rana cancrivora, builds
up urea like Latimeria and the chondrichthians,
but both the mechanisms of urea retention in the
urinary system (bladder but not tubular reab-
sorption) and the reproductive strategy (larvae
osmoregulate like marine teleosts) differ from
both lineages, demonstrating both that urea re-
tention is not a unique strategy of the coelacanth
and cartilaginous fishes and that the other cri-
teria we used as evidence for independent de-
velopment of the mechanism in the two groups
of fishes are valid.

A number of features predispose fishes to-
wards the urea retention habitus rather than hy-
posmotic regulation when they move from fresh
water to marine environments. Among them are:
(1) the presence of a complete ornithine-urea
cycle, (2) internal fertilization, (3) large size, (4)
sluggish life style, and (5) invasion of stable ma-
rine environments rather than the variable salin-
ity of estuaries. Concomitant with the develop-
ment of high blood urea must be: (1) urea
impermeable epithelia, (2) some mechanism for
reduction of urea loss through the urine, and (3)
tolerance of elevated blood and tissue urea
levels which would seem to include a number of
biochemical adaptations. We suggest that once
either urea retention of hyposmotic regulation
has been adopted as the strategy for osmoregu-
lating in a marine environment, the alternative
strategy is no longer feasible unless a major en-
vironmental reversal occurs.

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No. 134, 17 pages

OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

SOME BIOCHEMICAL PARAMETERS IN THE COELACANTH:

VENTRICULAR AND NOTOCHORDAL FLUIDS

By
Lois E. Rasmussen

Oregon Graduate Center,
19600 NW Walker Road,
Beaverton, Oregon 97005

ABSTRACT

The coelacanth, Latimeria chalumnae, has a distinct evolutionary appearance.
Knowledge of its biochemical constituents may clarify this position and at the same
time aid in the understanding of its physiological functioning. Therefore, utilizing a
variety of microtechniques, a spectrum of biochemical parameters was measured in
the notochordal fluid, and, for the first time, in the ventricular fluid of the coelacanth.
These included inorganice and selected organics—urea, lipids, proteins, and enzymes,
and involved both total assays and separations—electrophoretic and thin layer chro-
matography. The results were repetitively reproducible despite conditions prior to
assay. Although only one specimen was available for study, the results were precise,
without appreciable replicate variation, allowing valid comparisons between previous
coelacanth notochordal fluid and serum data and other marine fish brain fluids. These
comparisons allow some speculation on the phylogenetic place of the coelacanth and
its unique and/or unusual biochemical physiology.

December 22, 1979

INTRODUCTION

Certain, but not all, of the more primitive ma-
rine, or partially marine, fishes are among the
small coterie of animals which possess high (by
mammalian standards) urea tissue and fluid
levels. The investigation in these hyperuremic
animals of two distinct urea functions, the well-
documented one of osmoregulation, and a pos-

94

sible additional function in the maintenance of
protein and/or enzyme stability seemed of rele-
vance.

Pursuing this goal, a spectrum of biochemical
constituents was measured in the ventricular
brain fluid (for the first time) and in notochordal
fluid of the coelacanth, Latimeria chalumnae;
the assay of urea was given special attention.

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 95

Previous investigations established certain nor-
mal urea ranges in a chimaera—Hydrolagus col-
liei, several elasmobranchs and selected Isopon-
dyli (Rasmussen and Rasmussen 1977), and
included tracer experiments involving experi-
mentally induced transitory hyperuremia (Ras-
mussen 1971, 1974b; Rasmussen and Rasmussen
1977).

The second purpose was a comparison of our
data with data for another coelacanth notochor-
dal fluid (Griffith et al. 1975) to give an indication
of the range of variation between individual
coelacanths, recognizing that the data compari-
son was numerically limited as our values were
obtained from only a single fish. Fishes are
known to have recognizable individual varia-
tions, including species variations, differences
among individuals within a given species (Mus-
telus canis, Rasmussen and Rasmussen 1967b),
and even individual variations dependent on en-
vironmental factors such as salinity and temper-
ature (M. canis, Rasmussen and Rasmussen
1967a and 1967b), and/or physiological factors
such as estivation states (Protopterus, Lock-
wood 1963), reproductive states (salmon, Urist
and Van de Putte 1967), and/or stress (salmon
and sharks, Idler et al. 1959; Rasmussen and
Rasmussen 1967b). Nevertheless the data are
valuable in comparison to the previously re-
ported notochordal fluid values (Griffith et al.
1975), and provide a comparison of these no-
tochordal fluid values with the first time mea-
surements in the ventricular brain fluid.

METHODS

Coelacanth #79 can be regarded as a “‘sec-
ond-best’’ specimen (biochemically). Within
hours after capture, the fish was frozen whole,
and stored at —10 to (0)°C for 18 months. After
transport to the United States the still frozen
fish was thawed and then dissected (May 1975)
at San Francisco (see Acknowledgments). The
question of the degree of stress experienced dur-
ing capture or the holding period was discussed
by Dr. Lagios during the June 1977 AAAS meet-
ing and is briefly discussed at the end of this
paper. Subsequent tissue conditions were as fol-
lows. After removal, the fluids were rapidly re-
frozen. Unlike the procedures followed for other
fish fluids no NADH was added (although sub-
sequently a few additional unfrozen, refrozen
aliquots did have this stabilizer). The material
received was subdivided into aliquots at the time

of the assay of the most labile substances. By
the time of assay or actual separative analyses,
the fluids were thawed 2-3 times. All of these
separative analyses were on different aliquots
from a single sample of both notochordal and
ventricular fluids from the same coelacanth.
Multiple assays were done on replicate samples,
a factor to keep in mind during the data analysis.
However, despite its age (3+ years) and these
conditions, the excellence of the physiological
condition of at least the ventricular fluid is evi-
denced by the lack of visible hemolysis, the lack
of very high Ca, Mg, or K levels, and reason-
ableness of the assayed enzyme levels. Ex-
tremely high levels might have been indicative
of possible cell damage.

Inorganics measured included the following:
Total osmolarity by Precision Systems os-
mometer, vapor pressure OSsmometer, and melt-
ing point osomometer, Cl using Hg* plus the
indicator diphenyl carbazone (Hawk et al. 1954),
Na by Stone and Goldzieher (1949), and Ca by
a modification of Yanagisawa (1955) and Schales
and Schales (1941).

Organics measured for their total values in-
cluded the following lipids: Total /ipid after lipid
extraction (Folch et al. 1957) determined by the
dry weight of the lipid chloroform-methanol ex-
tract (Zollner et al. 1966) and/or the colorimetric
determination using sulfo-phospho-vanillin re-
agent (Fring and Dunn 1970); total lipid phos-
phorus by the method of Lowry and Tinsley
1974: and triglycerides assayed by the dienzyme
method of Wakayama and Swanson 1977 with
Calbiochem reagents (glycerolkinase, glycerol-
3-phosphate dehydrogenase). Cholesterol was
assayed both by a slight modification of Peder-
sen, 1973 CSF method and a precise tri-enzy-
matic method utilizing cholesterol ester hydro-
lase, cholesterol oxidase, and horse radish
peroxidase (Witte et al. 1974). Urea was assayed
by Archibald, 1945 method using phenyl 1,2 pro-
panedione 2 oxime. Total glycolipids were as-
sayed by method of Jatzkewitz and Mehl 1960.
Proteins were measured by method of Lowry et
al. 1951). Data involving more than two repli-
cates were expressed as the mean + SE (num-
ber). Differences where P was less than 0.05
were considered significant.

Separation Methods included thin layer chro-
matography (TLC) for lipids and several vari-
eties of electrophoretic separation techniques
for the proteins. Three types of proteins were

96 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

examined—soluble proteins, lipoproteins, and
glycoproteins, by 3—4 types of electrophoresis.

Cellulose acetate electrophoresis (CAE), agar
gel electrophoresis (AGE), starch gel electro-
phoresis (SGE), and polyacrylamide gel electro-
phoresis (PAGE) of at least 3 and up to 10 rep-
licates of coelacanth notochordal and spinal
fluids soluble proteins both ‘‘au naturale’’ and
concentrated between 2-10 fold by pressure di-
alysis in special collodion tubes were run. Both
the visibly clear supernatant and the gently cen-
trifuged more precipitate, fiber containing frac-
tions were separated. The CAE technique uti-
lized was the Microzone System (Beckman
Bulletin #7033), SGE (Smithies 1959), AGE
(Uriel 1964), 7% w/v PAGE (Pratt and Danger-
field 1963). Stains for soluble proteins were Fast
Green and Amido Black (Davis 1964).

Lipoproteins were stained after AGE or
PAGE with lipid crimson, with Sudan Black in
ethanoldiol (McDonald and Ribeiro 1959), or by
Sudan Black pre-incubated for |—2 hours before
electrophoresis (Fring et al. 1971), as appropri-
ate.

Glycoproteins were separated by AGE (Uriel
1964) and stained by azure A producing a purple-
red color due to an asymmetric dimethyl thioin
(Uriel 1964), or the periodic acid (specifically
oxidizing 1,2 glycol groups)—Schiff (less specif-
ic, reacting with slow lipoproteins, ketones, and
unsaturated groups) reaction (PAS) until a pink
color appeared (Zacharius et al. 1969). This PAS
technique demonstrated both glyco- and lipo-
proteins.

Groups of lipids were separated using thin lay-
er chromatography (TLC). Lipids were removed
from the lipoproteins by ethanol diethyl ether
and acetone was used to separate glycolipids
from phospholipids. The primary solvent system
used was CCl, 85, MeOH 15, HAc 10, and H,O
4 v/v with a somewhat different solvent system
used for specific cholesterol separations. Cho-
lesterol was visualized on silicic acid coated
plates, after separation by 50% H.SO, spray-
stain, followed by heating (Jatzkewitz and Mehl
1960), or H,SO, with dichromate, or 2% FeCl.,.
The highly specific spray-stain, diphenylamine,
was used to resolve the glycolipids (Christie
1973; Freeman and West 1966).

Several coelacanth tissues, especially the liv-
er, have been analyzed for enzyme levels. The
results of Solomon and Brown (1975) with fro-
zen coelacanth liver, demonstrating high levels

of the following enzymes: peptidase, diaphorase,
acid phosphatase, esterase, LDH, and peroxi-
dase (the latter four having residual activity after
8 years), were used as a guide toward choosing
the enzymes assayed in this investigation. Ac-
cordingly, two of these lactic dehydrogenase
(LDH), and acid phosphatase (AcP), and a third
enzyme—alpha-amylase were assayed. For each
enzyme assay, 5 replicates per assay were used,
and the assays carried out at least three times.
The methods used were: amylase—(Cseska et
al. 1959); AcP—(Modder 1973); LDH—(Amador
et al. 1965). The rate reaction was employed be-
cause isozymes of LDH from different species
and tissues may have different affinities for py-
ruvate (Hochachka 1965), and because of its su-
perior precision. A unit of LDH activity equals
an increase in absorbance at 340 my of 0.001 per
minute per milliliter of serum or fluid at 25°C.
Additional LDH assays were carried out at
varying urea concentrations.

RESULTS
!. Total Osmolarity and Inorganics

A background spectrum of serum data in rel-
evant vertebrate species for total osmolarity and
inorganic ions, especially chloride is listed in
Table 1. For comparative purposes, values were
averaged somewhat in Table 1. The total serum
osmolarity mOsm/I of marine hagfishes (1,152),
chimaeras (942), coelacanth (1,181) (Griffith et
al. 1974), and elasmobranchs (973) was 2-3 fold
that of lampreys (317), marine teleosts (500)
(Schmidt-Nielsen 1976), and human serum (300).
The sodium levels in the elasmobranchs and chi-
maeras were noticeably higher than coelacanth
and marine teleost levels (Table 1); specifically
the chimaera value was 268, the elasmobranchs
255, but the coelacanth was 197 and the teleosts
180. Chlorides showed a similar species pattern.
Mg coelacanth levels were of the elasmobranch
magnitude, and Ca levels were similar to chi-
maera and elasmobranch levels, but 2 fold
higher than teleosts.

In the nervous system associated fluids (Grif-
fith et al. 1975 and coelacanth #79 Rasmussen)
the total osmolarity was highest in the coel-
acanth, slightly lower in elasmobranchs, notice-
ably lower in chimaeras, and significantly lower
in the teleosts. The total osmolarity was greater
in the CSF than the serum, and the total os-
molarity was greater in the fluids than seawater

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS Oy

TaBLe 1. Electrolytes (meq/I).

Serum Fluids
OsMo Na Cl Mg Ca OsMo Na Cl Mg Ca
Ventricular brain fluid
1,181 197 187 523 4.8 Coelacanth 1,048 185 125
Notochordal fluid
250 ilo?
1,000 190? 2047 ES eZ
Ventricular brain fluid
300! 306! 7.8 4.8 Chimaera 1,670! 307! 3141 3.88! 3.03!
942 268 272 801 285 283 3.9
Ventricular brain fluid
973 255 239 3.0 5.0 Elasmobranch 9904 270 264 1 3
1,0008
Extra brain fluid
270 244 1 3
Ventricular brain fluid
500 i80 160 1.4 2.6 Teleost low 180 120 3
Amphibian
830° R. cancrivora?
220 109 71 Dal R. exculenta®
Seawater 930°
' Read, L. 1971.

* Griffith et al. 1975.

’ Schmidt-Nielsen 1976.

4 Total osmolarity slightly higher in ventricular fluid than serum.

> Total osmolarity greater in fluids than seawater and slightly higher than serum.
® Potts and Parry 1964.

(Table 1). The coelacanth notochordal chlorides Table 2 presents a comparison of previously
were higher than teleost or coelacanth CSF reported coelacanth notochordal fluid data (Grif-
chlorides, but lower than elasmobranchs and fith et al. 1975) and the current Coelacanth #79

chimaera (Table 1). data. Critical values for P (less than 0.05) were
TABLE 2.
mOsm/] mM/]
Total osmolarity Na Cl K Mg

Coelacanth

notochordal fluid 1,058 + 2 (3)! 188.7 + .2 (3)! 204.2 + 3.8 (3)! Mleteh se 3 iD: = 2022

1,062 + 4 (5) 190.1 + .3 (10) ele S610) aI) Se of! 1.48 + .04

ventricular fluid 1,048 + 2 (4) 185.2 + THS (10) 125.9 + 4.3 (10) Wie} Se"=5) 1235-02
Chimaera

ventricular fluid 801 + 3 (8) 240 + 3.2 (20)

Elasmobranch

ventricular fluid 990

I+

5 (7) 264

I+

5.1 (20)

' Griffith et al. 1975.
* Mean + S.E. (number of replicate samples).
Significant variation at p < 0.05 level.

98 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

TABLE 3. UREA mM/I.
Fluids

Extra O-U
Serum Ventricular brain Notochordal Spinal cord Cycle

S/T) se 7 420 + 13 (3)! Coelacanth CSF > Serum +

540 + 8 (6) 410 + 7 (10) (Higher totals than
elasmobranchs)
3034 325 + 5 (20) 425 + 6 (20) 400 + 7 (20) Chimaera CSF > Serum +

300-50 380 —
380 420

wal
ie)

(equivalent or
lower totals than
elasmobranchs)

Elasmobranch CSF = Serum +
— S. acanthias +
— R. eglanteria CSF > Serum +
— Teleost? Serum > CSF low

' Griffith et al. 1975.

* Bentley 1971.

3 Forster and Bergland 1956.

4 Urist and Van de Putte 1967.

noted and demonstrated both the lack of varia-
tion and the precision of the methods. Especially
to be noticed were the lower chloride ventricular
fluid values contrasted to notochordal fluid
levels. Ten replicate samples of both notochor-
dal and ventricular fluids demonstrated this
close correlation and precision.

2. Organics
Urea

Historically hemolyzed serum was one of the
first coelacanth tissues available for biochemical
study; analysis yielded urea values of 2,264 mg%
(377 mM/l) (Pickford and Grant 1967). In sub-
sequent measurements of unhemolyzed serum,
urea values were, not surprisingly, close (Grif-
fith et al. 1974). Many other vertebrate serum
values are about 10 mM/I, including teleosts at
about 5 mM/I (Bentley 1971). However other
marine fishes have similar values, ratfishes, 303—
350 mM/I, and marine elasmobranchs (250-420,
ave. 350 mM/l). Relevantly noted on Table 3 was
the general presence or absence of the ornithine-
urea cycle and its enzymes (Prosser 1972).

The CSF/serum urea ratios are also listed on
Table 3. In elasmobranchs the ratios were equiv-
alent, whereas in the ratfish CSF the values were
greater than the serum with the absolute values
equivalent to or lower than elasmobranch levels:
whereas in the coelacanth, the ventricular levels

were greater than the serum and also higher than
elasmobranchs numerical values (Table 3).

Urea numerical values for coelacanth fluids
included: intraocular—303 mM/I (Cole 1973);
notochordal—410 mM/I (Griffith et al. 1975) and
#79 420 mM/I ; ventricular fluid—S40 mM/I (Ta-
ble 3).

In the teleosts and elasmobranchs brain fluid
and serum, urea values were equivalent to each
other (although different between species) but
in both the coelacanth and chimaera the levels
were higher in the fluids than in the serum. Total
brain fluid levels were higher in the coelacanth
than in the chimaeras and elasmobranchs (Table
SE

Table 3 delineates differences and similarities
in various types of brain fluids. In elasmo-
branchs the urea levels were identical in the CSF
and EDF. In both the brain and notochordal
fluids of the coelacanth the actual values were
higher than elasmobranch fluids. Chimaera CSF
values (325 mM/l) were lower (by 33%) than
EDF (425 mM/l). Spinal cord fluid (SpC.F.) chi-
maera values (400 mM/Il) were similar to EDF
levels. In the coelacanth, notochordal fluid val-
ues were 24% lower than ventricular fluid val-
ues. Coelacanth notochordal fluid urea values
were higher than chimaera CSF, lower than
EDF, and about the same as chimaera spinal
cord fluid values (Table 3).

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 99

TABLE 4. Lipips (mg%).
Coelacanth #79 Chimaera Elasmobranchs Human
Noto- Ven- Ven- Ven- Teleost Ven-
chordal tricular tricular tricular tricular
Serum fluid fluid Serum fluid Serum fluid Serum Serum fluid
Total 500+7 5.0 755) 7008 700
Triglycerides 322 32 42-200 10-100
% Total 64%
140-215
Cholesterol 151 0.06 0.01 320 0.6 150 0.35 210! 140-215 0.25-0.6
4743 86-214! 350°
975 7004
662
% Total 1.2% 0.2%

*Spleen
*Spleen
13%

! Pacific Cod, Gadus macrocephalus.

* Ling Cod, Ophiodon elongatus.

8 Carp, Cyprinus carpio, Field et al. 1943.

4 Larsson and Fange 1977.

° Urist and Van de Putte 1967.

® Nevenzel et al. 1966.

7 Estimated from reported values, Griffith et al. 1974.

3. Lipids

Total lipid content was 2.5 mg% in the ven-
tricular fluid and about double—S mg% in the
notochordal fluid (Table 4). This compared with
the following other measured biological fluids:
human serum 700 mg%, elephant serum 260
mg%, marine teleosts serum 700 mg%, elephant
temporal gland fluid 75 mg% (Buss et al. 1976),
coelacanth serum 151 mg% (Pickford and Grant
1967), and human CSF (Griffith et al. 1975) 0.1
mg% (Table 4).

Triglycerides in the notochordal fluid were 3.2
mg%; low levels compared to human serum (10-
100 mg%), elephant serum (30 mg%), ratfish se-
rum (32 mg%), elasmobranch serum (42-200
mg%), and even elephant temporal gland fluid
(5-10 mg%) (Table 4). In the coelacanth the per-
cent of triglycerides/total lipids was 63%, com-
pared with 6% values for spleen and 78% for
liver (Table 4). Human serum triglycerides were
only 30%.

Another lipid component, cholesterol was de-

tectable at low levels—O.01 mg% in the ventric-
ular fluid, and 0.06 mg% in the notochordal fluid.
The possible loss of some of the cholesterol and/
or its ester due to the initial handling proce-
dures, subsequent freezings and thawings, and
its storage in plastic containers are unknown
factors. The accuracy and sensitivity were noted
by the closeness of the two assay methods. Ta-
ble 5 depicts the detailed values of notochordal
fluid cholesterol levels, which were low (0.06

TABLE 5. CHOLESTEROL (mg@%).
Ventricular brain fluid

Average Range
Coelacanth 0.006 0.1—0.02
Chimaera 0.6 0.8—0.4
Elasmobranch 0.36 0.42—0.30
Teleost
Human! 0.44 0.29-0.59

' Cevallos et al. 1595.

100 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

Cholesterol Glycolipids

--------- x

15 Ee) sCHOL- STD)
©
(PTC-sTD) a
: Se
GUE are ORIGIN====—=+—>-=—
FeClo Diphenylamine
spray spray

THIN LAYER CHROMATOGRAPHY

Ficure 1. Diagram depicting TLC of two types of lipids:
cholesterol spots on left and glycolipids on the right. Migration
distances of standards are depicted in the center. Spray-stains
are indicated.

mg%) even compared to human CSF (average
0.44 mg%; range 0.29-0.59 mg%, Cevallos et al.
1959), elasmobranchs CSF (average 0.36, range
0.2-0.5 mg%), and chimaera CSF (0.5 mg%;
range 0.8—0.4 mg%) (Table 5). The cholesterol/
total lipid content was compared in various
species and tissues; contrasting to the 13% in
the coelacanth spleen (Nevenzel et al. 1966). To
be noted with interest were the high serum levels
in some teleosts, Gadus macrocephalus and
Ophiodon elongatus. The high chimaera values
(Table 4) are contrasted to the relatively low
elasmobranch levels (Table 4).

In addition to the total cholesterol levels mea-
sured, by TLC two spots, specifically stained
for cholesterol, migrated the same distance as
purified, commercial cholesterol (Fig. 1). Other
several general lipid components migrated less
far. The two spray stains relatively specific for
cholesterol, 2% FeCl, and 50% H.SO, (at times
with dichromate) and charring, demonstrated
two cholesterol spots, the slower migrating,
quantitatively greater one probably cholesterol,
and the one migrating at the solvent front, its
ester (Fig. 1).

Several other lipids were analyzed by TLC.
17- ketosteriods, demonstrable in the ratfish se-
rum and fluids (Rasmussen, unpublished) were
not detected in the coelacanth fluids. Glycolipids
were readily detectable by TLC at low fluid con-
centrations. Staining with diphenylamine dem-

TABLE 6. PROTEINS OF NERVOUS SYSTEM ASSOCIATED
FLUIDS.
Mg% Ratios
Ven- Noto-
tricular chordal
Ven- Noto- fluid fluid
tricular chordal
fluid fluid serum serum
Chimaera 20-50
Elasmobranch 50! 1:30
Coelacanth 30) s2 2(G)) ite) se OS) 1g 1:16
210?
Teleost Sculpin 300
Human 15—40 1:300

' Certain species up to 200 mg%.
> Griffith et al. 1975.

onstrated 2 glycolipid components in the clear
notochordal fluid, with an 18 cm advancing sol-
vent front, two glycolipid components migrated
3 cm and 6 cm from the origin. The faster mi-
grating component migrated the same distance
as the phosphatidyl choline standard. TLC of
the fiber containing notochordal fluid fraction
demonstrated two glycolipids at 5 and 2 cm.

4. Proteins

Protein values, the mean of five replicates,
are listed in Table 6. For coelacanth #79 ven-
tricular brain fluid values were 130 + 2 mg% (5),
and notochordal fluid levels were 180 + 9 (5).
Contrasted to human CSF, the coelacanth fluid
protein values were high and compared with
other fish brain and nervous system associated
fluids these values were also high. The ratio of
protein in these fluids/serum is listed in the last
column in Table 6. The ratios were: human CSF,
1:300; elasmobranch ventricular fluid, 1:30;
coelacanth ventricular fluid, 1:21; and notochor-
dal fluid, 1:16.

a. Soluble Proteins.—Electrophoretic sepa-
ration of the soluble proteins, lipoproteins, and
glycoproteins revealed a variety of proteins.
First, the soluble proteins in the notochordal
fluid, by CAE separated into 3 main peak areas,
20% of the protein in peak 1, 75% in peak 2, and
5% in peak 3. The middle peak 2 corresponded
approximately to the general extended region of
the beta-globulins. This contrasted to human se-
rum and CSF where 73% (peak 1) was albumin-
like proteins plus a-globulins. Coelacanth dis-
tribution was more similar to elasmobranchs.

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 101

Figure 2. Soluble proteins (SP), separated by AGE,
stained with Amido Black, of the notochordal fluid.

Table 7 also lists the number of bands re-
solved by AGE, SGE, and PAGE in the noto-
chordal fluid. First, by AGE, stained with
Amido Black, 4 bands were discernible in the
notochordal fluid (Fig. 2). Compared to the spi-
nal fluid, Fig. 3, the patterns were different. By
SGE in the ventricular fluid Fig. 4 at least 6
clearly recognizable bands were separated; in
the notochordal fluid, utilizing two stains a

TABLE 7.

FIGURE 3.

Soluble proteins, separated by AGE, stained
with Amido Black, of the notochordal fluid (right) and ventric-
ular fluid (left). Origin is faint line in the middle.

greater number (8—10) of faster migrating bands
were resolved, stained with Amido Black (Fig.
5) and Fast Green (Fig. 6). Quantitatively, the
most amount of protein and qualitatively the
greatest number of bands was seen in the beta-
globulin region.

Multi-concentrated soluble proteins of coel-
acanth notochordal fluid (middle), cat serum

FLUID PROTEINS.

% type of protein (CAE)

Peak | Peak 2 Peak 3
Human (CSF) 13 17 10
Elasmobranchs (CSF)
S. acanthias 25 67 8
C. taurus 34 aif 9
Coelacanth (Noto.) 20 75 5

# of bands resolved by

AGE SGE PAGE

4-5 6 8 Soluble proteins
— 2 5 Lipoproteins
3-4 2 4 Glycoproteins

102 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGure 4. Soluble proteins, separated by SGE, stained
with Amido Black of the ventricular fluid. Origin is at bottom.

(left), and marine elasmobranch ventricular fluid
(right) were separated by SGE runs of short du-
ration (Fig. 7). Both the coelacanth notochordal
fluid and the elasmobranch ventricular fluid
exhibited dominant bands in the beta-globulin
region with some minor (1-2) bands similar in
mobility to albumin (Fig. 7).

PAGE of the soluble proteins of the spinal
fluid demonstrated 7-8 bands stained with Fast
Green or Amido Black Table 7, Fig. 8 (left), rep-
resented diagrammatically in Fig. 9.

PAGE of the notochordal fluid resolved into
6 bands in the 1x concentrated material stained
with Amido Black (Fig. 9), into 8 bands in the 3x

COELACANTH NOTOCHORD FLUID

PROTEINS
Amido Black

Ficure 5. Soluble proteins, separated by SGE, stained
with Amido Black of the notochordal fluid. Origin is at right.

concentrate (Fig. 8—middle strip) and in the 6x
concentrated material, stained with Fast Green
(Fig. 8—right strip), and also in the 8x concen-
trate stained with Amido Black (Fig. 9). In the
ventricular fluid simultaneous gels of 8x con-
centrated material stained with amido black also
demonstrated 8 bands; the predominance of
protein was in the beta-globulin region. Noto-
chordal fluid also demonstrated predominantly
beta-globulin migrating proteins with several ad-
ditional definitive bands in the a and y-globulin
regions (Fig. 9). Compared to published protein
patterns in several marine fish sera (Drilhon
1959), the patterns were distinct, bearing a re-
semblance to chimaera serum and spinal fluid
patterns (Rasmussen, unpublished), and elas-
mobranch fluids (Rasmussen and Rasmussen
1967a).

b. Lipoproteins.—In human serum 3% of the
total proteins are lipoproteins which migrate
electrophoretically with the alpha-globulins, and
5% with the beta-globulins (Harper 1963). Ven-
tricular fluid lipoproteins, somewhat concen-
trated, were separated by PAGE after prestain-
ing with Sudan Black in ethylene glycol; as
diagrammed in Fig. 9, the ventricular fluid re-
solved into more bands than the notochordal
fluid, but the percent of the faster migrating ones
was greater in the notochordal fluid. Fig. 10 (left
strip) is the actual gel of the 4x concentrated

+i &= —-.

COELACANTH NOTOCHORD FLUID J
FiGure 6. Soluble proteins, separated by SGE, stained

with Fast Green, of the notochordal fluid. Origin is at right.

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 103

FIGURE 7.
time run), of cat serum (left), coelacanth ventricular fluid
(middle), and elasmobranch CSF (right), stained with Amido
Black.

Soluble proteins, separated by SGE (shorter

notochordal lipoproteins ‘stained with Sudan
Black; Fig. 10 (right strip) is the same material
stained with lipid crimson.

c. Glycoproteins.—Glycoproteins are hexos-
amines (less than 5%) containing proteins, mi-
grating electrophoretically predominantly with
the albumin and alpha,-globulin fractions in
mammalian serum. In human CSF the glycopro-
tein was 2.5—7 mg%, being somewhat less than
15% of the total protein (Harper 1963). In the
coelacanth notochordal fluid the total glycopro-

FIGURE 8.
ventricular fluid (left), notochordal fluid (3 concentrated)
(middle), and 8x concentrated (right), stained with amido
black and fast green respectively. Origin is at bottom.

Soluble proteins, separated by PAGE of the

tein was 9 mg% (5% of the total protein), and in
the ventricular fluid 1-2 mg% (1.5% of total pro-
tein) (Table 8).

Table 8 also compares published coelacanth
notochordal fluid values (Griffith et al. 1975),
and the current coelacanth #79, both the num-
ber of bands resolved on PAGE, and the molec-
ular weights measured by Griffith et al. 1975. By
the various electrophoretic methods, glycopro-
tein bands were visualized. By AGE and azure
A staining 4 glycoprotein bands were resolved
in the notochordal fluid, whereas AGE, followed
by PAS staining of the ventricular fluid at the
same concentration resolved 2—3 bands, proba-

104 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

POLYACRYLAMIDE GEL ELECTROPHORESIS
COELACANTH FLUIDS

Ventricular brain fluid Notochordal fluid
Soluble proteins Lipoproteins Soluble proteins Lipoproteins Glycoproteins
(Ix) (8x)
8
6
4
2
CM
Amido Sudan Amido black Sudan Azure A
black black black

FiGure 9. Composite diagram of the PAGE separation of the soluble proteins, lipoproteins, and glycoproteins of the
notochordal and ventricular fluids.

TABLE 8. GLYCOPROTEINS.

Bands resolved

AGE SGE PAGE
Total % GP Se
mg% total Azure A PAS Az.A FeCl, Az.A
Human (CSF) =I 15
Coelacanth
Ventricular fluid =? 1S fixe} 3 1 3
Notochordal fluid! 9 5 4 3-4 2 1 4

' Molecular weights: 50,000—100,000 (Griffith et al. 1975).

TABLE 9. COELACANTH.

Enzymes Ventricular fluid Notochordal fluid
Amylase
Somogyi Units
SSS 50 + 4(5 250 + 6(5
( ara ) (5) (5)
Acid phosphatase
(wm/hr) 0.38 + 0.03 (5)! 0.42 + 0.02 (5)
LDH Detectable?” Detectable

(std. units)
Alteration of LDH levels

M Urea
0.5 0.6 0.4 0.3 0.1

Decreasing % of maximum values

100 61 59 45 15 %

' Elasmobranch, S$. acanthias 0.13 + 0.02 (5).
* Slightly higher than notochordal fluid.
3 §. acanthias, 185 units; H. colliei, detectable.

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 105

FiGure 10. Lipoproteins of the notochordal fluid separat-
ed by PAGE stained with Sudan Black (left) and lipid crimson
(right). Origin is at bottom.

bly mixture of glyco- and lipoproteins (Table 8).
AGE, by the latter method, of the notochordal
proteins also demonstrated 3—4 lipo- and/or gly-
coproteins (Fig. 11). SGE, followed by azure A
stain of more concentrated samples of ventric-
ular fluid also resolved several bands and in the
notochordal fluid 2 bands (Table 8). FeCl, stain-
ing after SGE demonstrated a predominant band
near origin both in the ratfish and coelacanth
fluids. PAGE resolved 3 bands in the ventricular
fluid, and 4 in the notochordal (Table 8, Fig. 9,

FiGure 11.
rated by AGE PAS staining. Origin is faint line about mid-
point.

Glycoproteins of the notochordal fluid sepa-

Fig. 12), with the notochordal glycoproteins
generally being less negative in their migration
pattern than those of the ventricular fluid.

Enzymes

The results from the three enzyme assays
were as follows: acid phosphatase levels were
0.42 wM/hr/mg protein (1 uM = 0.139 mg nitro-
phenol) in the notochordal fluid and 0.38 .M/hr/
mg protein in the ventricular fluid, levels not of
significant difference; contrastingly elasmo-
branch brain fluid levels were 0.13 and ratfish
4.0, whereas human values ranged from 0.8—2.3
(Table 9). Amylase levels in the notochordal
fluid were considerably higher—250 Somgyi
units—than the ventricular fluid levels—50 Som-
gyi units, which were within the limits of the
human serum values (Table 9).

The third enzyme LDH, as measured by the
rate of NADH, depletion per minute was de-
tectable in both fluids at values less than 30
units. The notochordal fluid was somewhat low-
er than the ventricular fluid, and low compared
to other brain fluids such as S$. acanthias (185),
and human (40) (Richterich 1969), but was of
comparable values to chimaera brain fluids (Ta-
ble 9). Comparative serum values were human
serum (225 units), and the high salmon serum
(4,000 units). The detectability of LDH in the
spinal fluid samples frozen, without the stabiliz-

106 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

FiGure 12. Glycoproteins, separated by PAGE of the
notochordal fluid (right) and ventricular fluid (left) stained
with azure A. Origin is at bottom.

ing NADH, additive, and thawed three times
prior to assay (Table 9) was remarkable.
Observing the effects of varying urea concen-
trations on the levels of LDH and taking 100%
equal to the highest total assay value obtained
at 0.5 M urea, progressive loss of LDH level
was observed on either side of this urea opti-
mum. Specifically at 0.4 M, the LDH level was
only 59% of the total, and at 0.6 M only 61%;
at 0.3 M only 45%, and at 0.1 M only 15%. Tak-
ing into account the 3% precision of the rate

reaction as utilized, the differences are signifi-
cant (Table 9).

DISCUSSION

Several results from this biochemical investi-
gation of coelacanth #79 notochordal and ven-
tricular brain fluids are worthy of further em-
phasis. First, among the inorganic components,
chloride values in the coelacanth ventricular
fluid were equivalent to the chloride levels in
chimaera and elasmobranch ventricular fluids.
As seen in Table 2, notochordal chloride values
were relatively close, but significantly different
from previously reported values (Griffith et al.
1975), and notochordal chloride values were def-
initely significantly different from ventricular
levels.

Among the simple organic compounds, urea
levels were of interest. The relative importance
in the coelacanth of the ornithine-urea cycle and
its enzymes (Brown and Brown 1967) and the
probable functioning of the whole coelacanth
organism as a urea space (Brown and DiJulio
1976) are relevant to a discussion of the high
coelacanth urea levels. Coelacanth liver values
were high—284 mM/I—(DiJulio and Brown
1976): high urea values were also found in the
ventricular and notochordal associated fluids.
The values for the coelacanth ventricular fluid
were among the highest values reported for ver-
tebrates, 540 mM/I, considerably higher than the
notochordal fluid values or the 350 mM/Ifor the
ventricular fluid of the hyperuremic marine elas-
mobranchs; like the chimaeras, the values were
higher in the ventricular fluid than the serum.
Numerically, the values were the same order of
magnitude as the chimaera extradural brain and
spinal cord fluids. These high urea values are
especially intriguing in view of some initial en-
zyme results.

The detection of the glycolipids in the CSF
was of interest in view of the low amount of
albumin in both this fluid and serum, and cor-
related with the resolution of glycoproteins in
the fluids by various electrophoretic methods.

Of interest is the higher total soluble protein
content in the notochordal fluid than in the ven-
tricular fluid; not unexpected as the notochordal
canal lining is composed of secretory type cells.
Compared to fluids in other species, protein
levels were numerically high.

That the majority of the soluble proteins mi-
grated with the mobility of the beta-globulins,

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 107

and that the albumins detected were in very low
concentration and had a different mobility from
mammalian albumins was confirmed by all the
electrophoretic methods. Obviously the proteins
of the coelacanth brain fluids were different in
type-proportions and within a given type, dif-
fered in size and mobility from mammalian and
other vertebrate forms. Compared to other fish
species, the most similarity was noticed to the
chimaera fluids and then next to the elasmo-
branchs fluids.

The demonstration of the variety of lipopro-
teins and glycoproteins was of interest. The no-
tochordal lipoproteins were distinctive in pat-
tern when compared to the ventricular fluid
proteins. Glycoproteins were also distinctive in
the notochordal fluid, resolving into, at the same
concentration, a greater number of bands than
the ventricular fluid. These coelacanth glycopro-
teins, were of different mobility and of higher
molecular weight than the serum glycoproteins
of certain Antarctic teleosts (DeVries et al.
1970). Data (Griffith et al. 1975) indicated some
similarities in carbohydrate content to human
CSF, namely, glucosamine, mannose, galactose,
and sialic acid (Gottschalk 1966). Interestingly,
the observations (Qureschi et al. 1977) on the
more even distribution of various electropho-
retic zones of Oncorhynchus nerka, sockeye
salmon, than the cow was somewhat apparent
by less quantitative methodology in the coel-
acanth fluids, especially the notochordal.

Other complex organics such as the total lip-
ids were low in comparison to serum but higher
than human CSF, notochordal fluid being twice
that of ventricular fluid; triglycerides were rel-
atively high, more than 50% of the total lipids,
and cholesterol levels were between 0.2—1.2%.
Both the assay of low levels of total cholesterol
(0.06 mg%) and the detection by TLC of two
cholesterol spots was possible because of our
use of an ultrasensitive measurement technique;
these levels may be influenced by the handling
procedures or unknown degrees of stress to the
animal. Our percent cholesterol results correlat-
ed with the moderately high reported levels for
coelacanth serum (Pickford and Grant 1967),
and compared to high serum cholesterol in var-
ious marine fishes (Larsson and Fange 1977),
including the ling and Pacific cod (Table 4).

The cholesterol content was comparable to
that of marine elasmobranchs (Griffith et al.
1975). Serum cholesterol levels may have some

correlation to FFA levels in turn related to both
the triglyceride content (Nevenzel et al. 1967)
and the rate of lipase activity. A link between
these lipid levels and observations on LDH may
have a corollary in the pulmonate land snails,
which can survive long periods of anoxia by de-
pleting glycogen and utilizing anaerobic glycol-
ysis and have the resultant end products of FFA
(Storey 1977). As a relationship exists between
high FFA and triglycerides and like other marine
fishes utilizing muscle and liver for triglyceride
storage (Larrson and Fange 1977), 78% of the
lipids in the coelacanth liver are in the form of
triglycerides (Nevenzel et al. 1967), the inves-
tigation of whether the coelacanth funnels its
LDH reaction toward FFA end products rather
than lactate, and the measurement of FFA is
projected.

The presence of a pyruvate inhibited H-type
LDH partially accounts for this channelling in
the snail. The habitat of the coelacanth is in
question; it may live in oxygen deficient regions
or in a freshwater cave habitat. Yet two fishes—
the halibut and lamprey dwelling in rather an-
aerobic environments possessed only M-type
LDH (Wilson et al. 1964). It would be of rele-
vance to determine the predominant LDH type
(M or H), and the relative percent of isozymes.

The measurement of total LDH levels, the
determination of the optimal pyruvate concen-
tration for maximum activity, and the establish-
ment of the relative percent of the isozymic
forms may provide information on this habitat
and relevant biochemical information on the
functioning of carbohydrate metabolism in the
coelacanth. Total LDH was present at detect-
able levels in the frozen-thawed coelacanth
fluids (Rasmussen 1977), and in the liver (DiJulio
and Brown 1976). The latter two aspects are cur-
rently being investigated.

The exciting detectability of measurable levels
of enzymes in quite old frozen fish tissue pre-
sents some interesting speculation regarding en-
zyme stability. The enzymes were assayable in
both the concentrated and unconcentrated fluids
of notochordal and ventricular fluids; AcP levels
were similar or slightly higher than levels re-
ported for elasmobranchs; amylase levels were
higher in the notochordal fluid than the spinal
fluid; but LDH interestingly was higher in the
spinal fluid than the notochordal. Because op-
timal LDH levels appeared to be at high critical
urea levels as seen in the supplemental urea ex-

108 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

periments, it is possible that in these primitive
fishes the urea may have a functional stabil-
ization role in the preservation of certain en-
zyme activity levels. The interplay that the high
levels of trimethylamine oxide (TMAO) reported
(Griffith et al. 1975) may have in correlation with
the urea or with another aspect of cellular me-
tabolism is under consideration.

SUMMARY

By a variety of techniques, including total as-
says and electrophoretic and TLC separations,
inorganic and organic constituents of the coel-
acanth notochordal and ventricular fluids result-
ed in some interesting observations both about
the coelacanth itself and in comparison to other
marine fishes. Because of numerically repro-
ducible results with less than expected varia-
tions from replicate samples and, despite a dif-
ferent coelacanth specimen obtained, stored,
dissected, and assayed under different condi-
tions, valid comparisons could be made with the
notochordal fluid from the other previous coel-
acanth specimen, for with several exceptions the
data from these two coelacanths, in parameters
dually measured, were close.

Measurements of special interest and rele-
vance included:

1. Chloride measurements in the notochordal
fluid were considerably higher than the ventric-
ular fluid levels (and also were significantly
higher than Griffith et al. 1975 values), and
slightly lower than levels in marine elasmo-
branch ventricular fluid.

2. The urea level value (540 mM/l) in the ven-
tricular fluid was significantly higher than the
410 mM/I in the notochordal fluid, and the 350
mM/I in elasmobranch ventricular fluid. The
nearest values to the high ventricular fluid val-
ues were found in ratfish EDF (425 mM_/l).

3. Lipid fluid analysis showed respectively in
the ventricular and notochordal fluids, total lip-
ids 2.5 mg% and 5 mg%:; triglycerides less than
3.2 mg%, and cholesterol 0.01 mg% and 0.06
mg%. The total triglycerides were low, but the
% per total lipids was high. Separation by sev-
eral TLC techniques demonstrated two choles-
terol components and two glycolipid compo-
nents.

4. Total proteins were respectively in the no-
tochordal and ventricular fluids, 180 and 130
mg%. The protein content was comparatively

high, three fold than of elasmobranch brain
fluid, which was higher than human CSF. This
is demonstrated by the ventricular fiuid/serum
ratio values respectively, coelacanth 1:16 and
1:21, shark 1:30, and human 1:300.

5. Soluble proteins were predominantly in the
beta-globulin region as seen by CAE; again a
similarity to elasmobranchs and a dissimilarity
to humans. By four electrophoretic techniques
up to 8 bands were resolved and differences not-
ed between the notochordal and ventricular
fluids, with a similarity noted to the proteins of
the ratfish (unpublished).

6. The lipoproteins demonstrated more bands
in the ventricular fluid (6) than the notochordal.
The hexosamine containing glycoproteins were
greater in the notochordal fluid (9 mg) compared
to the ventricular fluid (2.5—7 mg). The data were
in accord with Griffith et al. (1975). At least four
bands were resolved by several techniques, in
the notochordal fluid.

7. Enzyme level measurements demonstrated
higher levels of amylase in the notochordal fluid
compared to the ventricular fluid, AcP similar
levels, and LDH slightly higher levels in the
ventricular fluid. Data were presented showing
the effect of urea as an additive.

8. Possibilities regarding the role of urea and
its relation to coelacanth physiological function-
ing in its natural environment are discussed.

ACKNOWLEDGMENTS

Numerous acknowledgements are necessary
to give proper credit to this multi-supported ef-
fort. Foremost, George Brown, College of Fish-
eries, University of Washington is recognized as
the prime mover in the obtaining of good phys-
iological quality coelacanth tissue for biochem-
ical analysis through the initial organization and
laborious building up of SPOOF (Society for the
Protection of Old Fishes). Prime financial sup-
port for the particular coelacanth #79 used in
this study was generously awarded by the Char-
line H. Breeden Foundation through CAS 33111.
Special logistics and the actual obtaining of the
specimen was due to the efforts of M. Lagios
and J. McCosker. I. O. Buss, L. Kirschner, and
H. Went, Department of Zoology, Washington
State University are thanked for their generous
use of laboratory facilities. The expert, deft re-
moval of the brain and notochordal fluids was
performed by Susan Brown.

RASMUSSEN: COELACANTH VENTRICULAR AND NOTOCHORDAL FLUIDS 109

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OCCASIONAL PAPERS

OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 17 pages

December 22, 1979

CHORDATE CYTOGENETIC STUDIES: AN ANALYSIS OF THEIR
PHYLOGENETIC IMPLICATIONS WITH PARTICULAR REFERENCE
TO FISHES AND THE LIVING COELACANTH

By

Guido Dingerkus

Department of Ichthyology,
The American Museum of Natural History,
New York, New York 10024

INTRODUCTION

Since the early 1960's, cytogenetic studies
have become a favored method of gaining insight
into the evolutionary relationships of verte-
brates. Although it is sometimes assumed that
they provide more basic data than do osteolog-
ical and other anatomic approaches, I doubt that
cytogenetic analysis is intrinsically any more in-
formative than the more traditional anatomic
studies. Such studies, however, do permit in-
vestigation of another independent set of char-
acters which are unavailable to traditional ex-
amination, and these may complement the
contribution of the morphologic methods toward
analysis of phylogenetic relationships.

The first examination of fish chromosomes (by
Retzius 1890, on Myxine glutinosa) and subse-
quent studies until 1964 were limited to histo-
logically sectioned and prepared material. Al-
though microscopic sections provide a
reasonably accurate chromosome number, ac-
tual three-dimensional chromosome morphology
or karyotype cannot be determined. It was not
until the introduction of ‘squash’ preparations
(Roberts 1964) and flame-dry methods (Denton

111

and Howell 1969) that both fish chromosome
number and morphology could be documented.
Flame-dry and similar air-dry techniques are to-
day the most widely-used techniques to prepare
fish chromosomes and permit the preparation of
reliable chromosome karyotypes and idiograms.
Although karyotypes have been described for
perhaps only 400 of the estimated 20,000 species
of living fishes, representative species of most
of the major phyletic groups have been studied.
These morphologic methods have been supple-
mented significantly by the introduction of tech-
niques such as fluorometric assay (Hinegardner
1971), which permit DNA-per-cell values to be
determined and compared for most of the major
phyletic groups of fishes.

Latimeria has never been karyotyped, but its
nucleoli, in interphase nuclei, have been exam-
ined (Dingerkus 1977; Dingerkus et al., unpub-
lished data) and its DNA/cell content has been
measured (Vialli 1957; Pedersen 1971; Cimino
and Bahr 1974).

On the basis of these data from Latimeria,
and the same information, as well as karyotypic
data from other chordate groups, the cytogenet-

112 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ic relationships of Latimeria can be evaluated
and a hypothetical Latimeria karyotype pro-
posed.

CHROMOSOME NUMBERS AND MORPHOLOGY

The following terms will be used to describe
chromosome morphology. Microchromosomes
are small, usually less than 0.5 uw in the greatest
dimension, and without discernible centromere
or chromosome arms. Macrochromosomes are
larger than 0.5 w with a discernible centromere
and chromosome arms. On the basis of centro-
mere position, macrochromosomes may be sep-
arated into three groups: acrocentric, metacen-
tric, and submetacentric, in which the centromere
is located respectively at or extremely close to
the terminal end of the chromosome, at or near
the median of the chromosome, or in an inter-
mediate position.

Figure 1 shows comparative idiograms of
chromosomes of all the principal chordate
groups. An attempt has been made to draw these
as accurately as possible to the scale indicated
on the first idiogram so that size and morphology
can be compared easily. However, because of
their extremely large size the idiogram of Nec-
turus maculosus has been drawn at half scale.
Illustrated idiograms of representative species
appear in parentheses in the text to facilitate re-
ferral to the figures.

Tunicates (represented by Ciona intestinalis)
have a karyotype of 8 to 32 small microchro-
mosomes and no macrochromosomes. Although
reports of 4 to 40 chromosomes exist in the lit-
erature, it is believed that these are due to the
inaccuracies of older techniques. The true basic
chromosome number of tunicates seems to be
close to 24 (Taylor 1967; Colombera 1973). Am-
phioxi (represented by Branchiostoma floridae)
have a karyotype of 38 microchromosomes, but
these are somewhat larger than those of tuni-
cates (Howell and Boschung 1971). This would
suggest that the primitive chordate karyotype
may consist of microchromosomes.

The hagfishes (Eptatretus stoutii) have a chro-
mosomal makeup of 46 to 52 acrocentric chro-
mosomes (Taylor 1967; Potter and Robinson
1973). Nygren and Jahnke (1972a) found an ex-
tremely variable number of chromosomes in
Myxine glutinosa. 1 believe that much of the
latter variation arose from a loss of chromo-
somes or from overlapping spreads as a result
of the technique that was used.

Lampreys of the northern hemisphere (/ch-
thyomyzon gagei) have a much higher number
of chromosomes, ranging from 142 to 174. In
some of these, it is extremely difficult to detect
the centromere, and they may be true micro-
chromosomes. The rest are all acrocentric
(Howell and Duckett 1971; Potter and Robinson
1973). The southern hemisphere lampreys (Mor-
dacia mordax) have a chromosome number of
76 and all chromosomes are metacentrics (Pot-
ter, Robinson and Walton 1968; Potter and Rob-
inson 1973).

In the holocephalians or chimaeras, chromo-
some numbers of 58 and 86 have been found for
Hydrolagus colliei and Chimaera monstrosa,
respectively. Both have a mixture of acrocentric
chromosomes and microchromosomes (Ohno et
al. 1969b; Nygren and Jahnke 1972b).

Among the skates and rays, composite chro-
mosome numbers also exist. In the family Raj-
idae (Raja clavata) a range of 98 to 104 chro-
mosomes was found, consisting of a mixture of
a few metacentric chromosomes, many acrocen-
tric chromosomes, and many microchromo-
somes ( Nygren and Jahnke 1972b). Stingrays,
family Dasyatidae (Dasyatis sabina), have 68 to
84 chromosomes. Most of these are metacen-
trics, the rest being acrocentric, or just barely
submetacentric (Donahue 1974). Electric rays,
family Torpedinidae (Narcine brasiliensis), have
a very low number of 28, all chromosomes being
metacentric or submetacentric and most of them
large in size (Donahue 1974).

Only two species of sharks have been reliably
karyotyped, Squalus acanthias and Etmopterus
spinax (Nygren and Jahnke 1972b). The latter
has a karyotype of 86 chromosomes, made up
of acrocentrics and microchromosomes. S.
acanthias has a karyotype of 78 chromosomes,
comprising a mixture of metacentrics and acro-
centrics but no microchromosomes.

In the Chondrostei, consisting of paddlefish-
es, family Polyodontidae (Polyodon spathula),
and the sturgeons, family Acipenseridae, a high
number of chromosomes has been found, rang-
ing from 112 to 240. These are a mixture of meta-
centrics, submetacentrics, acrocentrics, and mi-
crochromosomes (Ohno et al. 1969a; Fontana
and Colombo 1974; Dingerkus and Howell
1976).

Among the gars, family Lepisosteidae, only
Lepisosteus oculatus (= productus) has been
karyotyped. It has 68 chromosomes with a mix-

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

ture of metacentrics, acrocentrics, and micro-
chromosomes (Ohno et al. 1969). The bow fin,
Amia calva, has 46 chromosomes, a mixture of
submetacentrics to metacentrics and acrocen-
trics but with no microchromosomes (Ohno et
al. 1969).

In the Teleostei, the most speciose of all the
fish groups, a diverse array of chromosome
numbers has been found (see the tabulation of
chromosome numbers in Chiarelli and Capanna
1973; and Denton 1973). Among the teleosts, no
species exhibits microchromosomes. In the os-
teoglossids (Osteoglossum bicirrhosum), there
is a variety of chromosomal compositions, rang-
ing from 34 to 56, including various mixtures of
metacentric and acrocentric chromosomes
(Uyeno 1973). Clupeids (Alosa pseudoharengus )
have 48 acrocentric chromosomes (Mayers and
Roberts 1969). The Euteleostei, or higher te-
leosts, have chromosomes ranging from 14 to
140, with various assortments of metacentrics,
submetacentrics and acrocentrics. The basic
chromosome number has been suggested to be
48 by Ohno (1970a).

The Brachiopterygii, comprised solely of the
Polypteridae (Polypterus palmas), have 36 rath-
er large, submetacentric to metacentric chro-
mosomes (Denton and Howell 1973).

The Dipnoi or lungfishes (Lepidosiren para-
doxa) are similar to polypterids in having 38 sub-
metacentric to metacentric chromosomes, but
they are huge in size (Ohno and Atkin 1966).
Older studies (Wickbom 1945) indicate this also
holds true for Neoceratodus and Protopterus.

The amphibians exhibit very divergent karyo-
types among the three groups: Urodela (sala-
manders), Anura (frogs and toads), and Apoda
(caecilians). The Urodela (Necturus maculosus)
have a chromosome number between 22 and 64,
the majority of species having mostly metacen-
tric to submetacentric chromosomes with some
acrocentrics, although some species have no ac-
rocentrics (Morescalchi 1973). Their chromo-
somes are extremely large, and with the dip-
noans share the distinction of the largest known
animal chromosomes. Anurans (represented by
Ascaphus truei) generally have a chromosome
number that ranges from 14 to 48, but a few
species have 104 chromosomes (Morescalchi
1973). The more primitive species (such as as-
caphids and discoglossids) have a mixture of
metacentrics, submetacentrics, acrocentrics or
microchromosomes. In contrast, the advanced

113

species (such as bufonids, hylids, ranids and
rhacophorids) have a mixture of metacentrics
to submetacentrics. The Apoda (Geotrypetes

seraphinii) have a range of 20 to 42 chromo-

somes, with a mixture of metacentrics to sub-
metacentrics and some very small acrocentrics
or microchromosomes (Stingo 1974; Morescal-
chi 1973).

Reptiles can be divided into three karyotype
groups. The tuatara, Sphenodon punctatus, has
36 chromosomes. These are mostly large meta-
centrics to submetacentrics, with a few very
small metacentrics and one pair of very small
acrocentrics or microchromosomes (Wylie et al.
1968). The crocodilians (Alligator mississipien-

sis), of which all species have been karyotyped

(Cohen and Gans 1970), have from 30 to 42 chro-
mosomes. In some species, presumably the
more primitive ones, the karyotype is made up
of a number of large metacentrics, fairly large
acrocentrics, and very small metacentrics. In
other species, the acrocentrics appear to have
fused, resulting in a karyotype consisting entire-
ly, or almost entirely, of metacentrics which
range in size from large to very small. The liz-
ards and snakes (the Squamata) and the turtles
have very similar chromosomal makeups. The
lizards (Agama pallida), have from 22 to 63
chromosomes (see review by Gorman 1973).
The species with higher numbers have a mixture
of mainly acrocentrics and microchromosomes;
those with lower numbers have more metacen-
trics, probably resulting from fusions of acro-
centrics. This same general pattern is shown by
the snakes (Drymarchon corais), with 24 to 50
chromosomes (see review by Gorman 1973), and
again in the turtles (Chelydra serpentina) in
which chromosome numbers from 26 to 68 are
found (Killebrew 1977; Gorman 1973). In turtle
karyotypes, however, there are more metacen-
trics.

The birds have a pattern very similar to the
latter reptile groups, but with higher numbers of
chromosomes, that is, from 56 to 90 (see review
by Ray-Chauduri 1973). The loon, Gavia stel-
lata, as can be seen from the idiogram, has a
combination of mostly acrocentrics, one pair of
submetacentrics, and many microchromosomes.
Other birds with lower numbers have more
metacentrics, again probably arising from fusion
of acrocentrics.

The monotremes (Tachyglossus aculeatus)
have a chromosome number from 54 to 64, con-

114 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

sisting of submetacentric and metacentric chro-
mosomes. Marsupials (Didelphis marsupialis)
have 14 to 22 chromosomes, the species with
higher numbers having all acrocentrics and
those with lower numbers proportionally more
metacentrics (Sharman 1973).

Among the eutherian mammals, between 6
and 84 chromosomes have been found (see re-
view by Matthey 1973). Once again, the species
with higher numbers have a karyotype consist-
ing mostly of acrocentrics, and in the lower-
numbered species there are more and larger
metacentrics. Especially in the lemurs (Lemur
catta), a series of very small chromosomes has
been described. These have been considered to
be very small acrocentrics, but their centro-
meres are very difficult if not impossible to vis-
ualize, and I refer to them as 8 microchromo-
somes. In the fox, a few ‘“‘microchromosomes”’
have been observed (Low and Benirschke 1972).
These differ in appearance and number among
individuals of this species, however, and prob-
ably are 8 chromosomes rather than true micro-
chromosomes. Evidence indicates these 8 chro-
mosomes represent deleted segments from a
chromosomal rearrangement. They occur in
varying low numbers and are not present in all
individuals. Therefore, these 8 chromosomes
are not to be confused with true microchromo-
somes which are found in constant, and usually
high, numbers in all individuals of a species.

DNA PER CELL CONTENT

It is well established that cells from the same
individual at the same state of the cell cycle, and
also cells from other individuals of the same
species, contain the same amount of DNA. Be-
cause this uniformity exists, DNA/cell values
have become another basis for comparison be-
tween different species and higher systematic
categories. When used in conjunction with
karyotypes, DNA/cell values can yield infor-
mation as to whether karyotypic differences are
due to minor changes, such as chromosomal
rearrangement, or major changes such as tetra-
ploidy. Moreover, since DNA is the basic ge-
netic material, it is believed that these DNA/cell
comparisons can give an estimate of the funda-
mental similarity or difference between groups.

In the following discussion and in Figure 2, the
DNA values given are those per haploid cell. In
the graph, each bar indicates the range of DNA
values found within a systematic group but

every value within a particular bar is not nec-
essarily represented by a species possessing that
value.

Tunicates have the lowest DNA/cell of any
chordate. These values range from 0.15 to 0.21
picograms (pcg) for various species. Amphiox-
us, Branchiostoma lanceolatus, has 0.6 pcg of
DNA per cell. Eptatretus stoutii, a hagfish, has
a rather high value of 2.8 pcg. The northern
hemisphere lampreys exhibit between 1.4 and
2.5 peg of DNA per cell.

The only holocephalian that has been studied,
Hydrolagus colliei, has 1.5 to 1.6 pcg. Sharks
and rays range between 2.75 and 9.8 pcg.

For sturgeons, values of 1.75 to 5.1 peg have
been obtained. No evaluation has yet been made
for either species of paddlefish. The gar, Lepi-
sosteus oculatus (= productus) has a value of
1.35 pcg, the alligator gar, Atractosteus spathu-
la (= Lepisosteus ferox), has 1.2 peg, and the
bowfin, Amia calva, has 1.23 pcg.

Teleosteans exhibit a wide range of DNA val-
ues, viz. 0.4 to 4.4 pcg.

In Latimeria, Vialli (1957) obtained a value of
2.8 pcg, Pedersen (1971) one of 3.5, and Cimino
and Bahr (1974) one of 3.61 pcg. Cimino and
Bahr (1974) have also reinterpreted Vialli and
Pedersen’s data to values of 3.465 and 3.8 pcg,
respectively. It seems valid to conclude that the
DNA/cell of Latimeria lies somewhere between
these values.

In the brachiopterygians the DNA/cell value
has been estimated at 4.7 to 5.8 pcg (Bachmann,
Goin, and Goin 1972; Hinegardner 1976).

The species of living dipnoans range between
80.2 and 140 pcg of DNA/cell. The lungfishes
probably have the largest amount of DNA/cell
of any living animal.

Urodelans also have extremely high values
ranging from 10 to 95 pcg per cell. Anurans have
lower values, from 0.8 to 10.5 peg, and caecili-
ans are similar with 3.7 to 13.9 pcg.

No DNA/cell measurement has been made on
Sphenodon, the tuatara. Snakes and lizards both
range from 2.1 to 2.4 peg. Turtles and crocodil-
ians share ranges from 2.8 to 3.0.

Birds have surprisingly low amounts of DNA,
ranging from 1.54 to 2.0 pcg—especially in view
of their high number of chromosomes. Mammals
have a fairly uniform range, with monotremes
exhibiting from 3.2 to 3.4 pcg, marsupials from
3.4 to 3.5, and eutherian mammals from 3.1 to
3:5:

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

NUCLEOLI AND NUCLEOLAR ORGANIZER
REGIONS (NORs)

Nucleolar organizer regions (NORs) are re-
gions of certain chromosomes that are com-
posed of highly repetitive ribosomal DNA
(rDNA), which code for ribosomal RNA (rRNA)
and in the interphase cell forms the nucleolus or
nucleoli (Howell 1977). The NORs and nucleoli
can be selectively stained using an ammoniacal
silver technique (Denton et al. 1976; Howell and
Denton 1974; Howell et al. 1975). Recent studies
of nucleoli and NORs in fishes (Dingerkus 1977;
Dingerkus et al., unpublished data) show that di-
ploid species have 2 NORs, usually with two
small nucleoli in the interphase nucleus; tetra-
ploid species have four NORs, usually with four
nucleoli in the interphase nucleus; and species
with high amounts of tandemly duplicated genes
have two NORs, usually with two very large
nucleoli in a large interphase nucleus.

Representative photographs of these three
states are shown in Figure 3. The barb, Barbus
tetrazona, paddlefish, Polyodon spathula, and
lungfish, Lepidosiren paradoxa, represent, re-
spectively, the diploid, tetraploid, and tandem
gene-duplicated states. The interphase nucleus
of Latimeria has been shown to contain two
small nucleoli, like that of the barb, indicating
on this basis that Latimeria is a typical diploid
species.

Recent studies on NORs in mammals have
shown that different species have from one to
ten NORs per cell (Goodpasture and Bloom
1975; Yates et al. 1976). Since there is no evi-
dence of ploidy among mammals, this variation
in NORs cannot be correlated with states of
ploidy, as it can in fishes. The variability in NOR
numbers among different species of mammals
must be due either to a holdover from ploidies
undergone in chordate evolution long ago, or to
rearrangements in the mammalian genome. The
fact that the number of NORs can vary from cell
to cell in the same individual tends to indicate
that the variable numbers of NORs in mammals
results from genomic rearrangement. As more
species are studied in this way, we may be able
to further understand the significance and evo-
lution of NORs.

DISCUSSION

The following discussion includes a brief re-
view of the apparent trends in chromosomal

115

evolution. These have been described and dis-
cussed by Ohno, Wolf, and Atkin (1968), Ohno
(1970a, 1974), and Koulischer (1973).

The primitive chromosome type appears to
be the microchromosome. Some microchromo-
somes later fuse into acrocentrics. These acro-
centrics can then undergo robertsonian fusion
to form metacentrics or submetacentrics.
Which of these two types of chromosome is
formed depends, respectively, on whether the
fusing elements are of equal or unequal size.
These changes do not necessarily occur in all
the chromosomes of a given complement. Hence
the karyotype of a species may be a composite
of varying degrees of these changes, and may
have a mixture of microchromosomes, acro-
centrics, submetacentrics, and metacentrics.

Since tunicates are the most primitive of
living chordates, it is not surprising that they
have a primitive karyotype consisting entirely
of microchromosomes, and a low level of DNA/
cell. Some of the differences in chromosome
number among tunicates can be explained by
fusions to account for lower numbers. On the
other hand, incidents of tetraploidy may ac-
count for the higher ones. I believe that the
basic tunicate chromosome number is 20 to 24.
Amphioxus has almost twice as many chromo-
somes, and one might conclude that a tetra-
ploidy has occurred. If this is true, amphioxus
should have twice as much DNA, but it actually
has more than double the DNA/cell value of
tunicates.

Hagfish, with their chromosome range of 46
to 52, may have arisen from the basic amphioxus
karyotype by the way of another tetraploidy.
Their number is somewhat lower than expected,
but some of their chromosomes are much larger
than the others, and this would indicate the
fusion of some of the original chromosomes. If
this hypothesis is true, the DNA/cell of hagfish
should be approximately twice that of amphiox-
us. It is, however, much higher than that. The
lampreys of the northern hemisphere have a
very high number of chromosomes, and this
could be due to a tetraploidy of the basic hagfish
karyotype. Southern hemisphere lampreys,
however, have approximately half this number,
that is, in the range of the hagfish. Chromosome
morphology reveals that hagfish have small ac-
rocentrics to microchromosomes, while south-
ern hemisphere lampreys have metacentrics that
are roughly twice as large as the acrocentrics of

116 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

their northern hemisphere relatives. The south-
ern hemisphere lampreys probably arose from
a karyotype like that of the northern hemisphere
lampreys by fusion of all the acrocentrics into
metacentrics. The DNA/cell of southern hemi-
sphere lampreys has not been measured, but I
predict that they will be found to have approx-
imately the same number of DNA/cell as do the
northern ones. The latter have a DNA/cell con-
tent that is roughly four times that of amphioxus.
One might speculate that the hagfish karyotype
originally arose by tetraploidy from a basic am-
phioxus karyotype, that this basic hagfish karyo-
type then gave rise to a basic lamprey karyotype
through another tetraploidy and that after this
event, the hagfish lineage underwent consider-
able tandem gene duplication. The common lam-
prey karyotype eventually gave rise to the
southern hemisphere lampreys by robertsonian
fusion of the acrocentrics. A test of this hypoth-
esis would be to determine whether hagfish ex-
hibit a high level of ribosomal gene multiplicity,
since this would indicate a high level of tandem
gene duplication. Some indication that this is in-
deed the case has been provided by the fact that
Ohno and Morrison (1966) found that one
species of hagfish has four independent gene loci
for monomeric haemoglobin polypeptides, while
Adinolfi et al. (1959) found only two in the lam-
prey Lampetra planeri. Another possibility is
that after the splitting of the lamprey lineage,
hagfish underwent a secondary tetraploidy in-
stead of tandem gene duplication. The low chro-
mosome number of hagfish would tend to argue
against this, but such a low number could be due
to chromosomal fusions in the hagfish.

The holocephalians, or chimaeras, exhibit a
mixture of acrocentrics and microchromosomes,
with approximately the same amount of DNA/
cell as in the lampreys. Such a karyotype could
have been formed by a fusion of some of the
small acrocentrics and microchromosomes of
the basic lamprey karyotype. This hypothesis
finds support in their high number of chromo-
somes (which is, however, less than the number
found in the lampreys), and in the size of their
acrocentrics (which are proportionately larger
than those of the lampreys). The holocephalian
karyotype, however, could also have arisen
from a basic hagfish karyotype by way of a tet-
raploidy, followed by secondary chromosomal
fusions. Further biochemical studies and chro-

mosome banding patterns may shed light on this
problem.

The Elasmobranchia (skates, rays, and sharks)
have approximately twice as much DNA/cell as
do the chimaeras. This may indicate that a tet-
raploidy occurred between the basic holoce-
phalian and elasmobranch karyotypes. This is
supported by the rajid karyotype which contains
approximately twice as many chromosomes as
in the chimaeras and consists mostly of acro-
centrics and microchromosomes. The dasyatid
karyotype, which has a lower number of chro-
mosomes than rajids, but has a greater propor-
tion of metacentric chromosomes seems to have
arisen from a basic chondrichthyan karyotype
through robertsonian fusion of some of the ac-
rocentrics. The torpedinid lineage appears to
have undergone a greater amount of chromo-
somal fusion since they have a low number of
very large, mostly metacentric-submetacentric
chromosomes. Unfortunately there are karyo-
type data on only two species of sharks. Etmop-
terus has a karyotype very similar to that of a
rajid but with fewer chromosomes. The karyo-
type of Squalus has a low chromosome number
with metacentrics, no true microchromosomes,
and some very small acrocentrics. This karyo-
type could have developed by the fusion of some
of the elements of an Etmopterus karyotype,
and Etmopterus probably more closely repre-
sents the basic shark karyotype. Preliminary
studies on the chromosomes of Ginglymostoma
cirratum, Sphyrna tiburo, and Negaprion bre-
virostris Support this (Dingerkus, unpublished
data). I hypothesize that rajids and some sharks
possess a karyotype most like the basic elas-
mobranch karyotype. Chromosome banding
patterns again may show which of these is most
closely related to the holocephalian lineage.
Some elasmobranchs have fairly high amounts
of DNA/cell (up to 9.8 pcg). This would suggest
that during their evolution these forms under-
went secondary tandem gene duplication follow-
ing their tetraploid ongin.

The Actinopterygii (ray-finned fishes) may be
divided into three main groups: Chondrostei,
Holostei, and Teleostei. The chondrosteans
have a very high chromosome number, including
metacentrics, submetacentrics, acrocentrics,
and a high number of microchromosomes. It has
been argued that they have a tetraploid origin
(Dingerkus and Howell 1976). This is upheld by

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

the presence of four nucleoli per interphase cell
in Polyodon (Dingerkus 1977). Since chondros-
teans and elasmobranchs have approximately
the same amount of DNA/cell, however, it can
be argued that the basic chondrostean karyotype
did not arise by tetraploidy from a chondrich-
thyan karyotype. Instead, the lineage probably
arose by tetraploidy from some sort of holoce-
phalian-like karyotype. Since the chondrosteans
already have a number of metacentrics among
their large number of chromosomes, and these
metacentrics occur in sets of four, it may be
concluded that the karyotype from which the
chondrostean karyotype arose already pos-
sessed metacentrics and was already more ad-
vanced than the basic holocephalian karyotype.
In some species of sturgeons, a chromosome
number is found that is twice that of other stur-
geons. These are almost certainly due to a tet-
raploidy of the already tetraploid sturgeon
karyotype, and therefore they could be consid-
ered as octoploids. The holostean gars, with a
lower number of chromosomes than chondros-
teans, originated from a karyotype in which
chromosomes must have been lost and/or fused.
The lowered DNA/cell level found in holosteans
agrees with this hypothesis. Gars exhibit only
two nucleoli per nucleus (Dingerkus 1977), and
this together with their lower chromosome num-
ber and DNA/cell level, indicates that a diploid-
ization of the basic tetraploid chondrostean
karyotype gave rise to the holostean karyotype.
Amia, with a still lower number of chromo-
somes, no true microchromosomes (the last pair
of very small acrocentrics might be argued to be
microchromosomes), and less DNA/cell, seems
to have arisen from such a basic holostean
karyotype and to have undergone even more
chromosome losses and fusions than have the
gars.

At first glance, the teleosts appear to be ina
chaotic state with regard to chromosome num-
bers and DNA/cell values. With further obser-
vation, however, distinct trends can be ob-
served. Ohno (1970a) has attributed this variation
as “‘a reflection of nature’s great experiment
with gene duplication.”’ I tend to agree with
Ohno’s statement, but would add gene deletion
to duplication. Evidence from both karyotypes
and DNA/cell values has shown that numerous
species and lineages have either undergone or
arisen through tetraploidy. Uyeno and Smith

117

(1972) showed that the catostomids arose, as a
lineage, through tetraploidy from a cyprinid
ancestor. Ohno et al. (1967) showed that nu-
merous cyprinids have evolved by tetraploidy,
most notably Carassius auratus. This is also the
case in clupeid and salmonid fish (Ohno et al.
1969b). These cases are confirmed by observa-
uions of nucleoli (Dingerkus 1977). Hinegardner
and Rosen (1972), based on DNA/cell values,
have suggested that ploidy has played an im-
portant role in the evolution of various catfish,
gobies, and parrotfish. They also have suggested
a correlation between more specialized species
and lowered levels of DNA/cell. I believe this
lower level of DNA/cell is probably correlated
with a reduction in number of chromosomes.
Since the teleosts are such a diverse and spe-
close group, it is not surprising to find that both
of these mechanisms appear to have been used
widely in evolving the present species of te-
leosts, since we can account for species of te-
leosts with high chromosome numbers and
DNA/cell levels as well as species with low
numbers of chromosomes and DNA/cell levels.
Somewhere between the extremes must lie the
basic teleost karyotype. No species of teleost
possesses microchromosomes, and microchro-
mosomes must have been lost in the transition
between holosteans and teleosteans. Ohno
(1970) argues that the basic teleost karyotype is
48 acrocentric chromosomes, mainly because
this configuration is widely found among the te-
leosts. If we look at the karyotypes of the os-
teoglossids, which are currently viewed as the
most primitive living teleost group (Patterson
and Rosen 1977), we see that they have a higher
chromosome number, with metacentrics present
in some species (Uyeno 1973). Neither DNA/cell
nor nucleoli studies indicate any tetraploidy
(Hinegardner and Rosen 1972; Dingerkus 1977).
I think that Osteoglossum with 56 chromo-
somes, mostly acrocentrics, probably exhibits
a karyotype that is most like the basic teleost
karyotype. From such a karyotype, the other
lower-numbered karyotypes, which in many
cases include numerous metacentrics, probably
arose by chromosomal rearrangements, fusions,
and deletions. The anguilloid eels and clupeids,
which have an apparent basic number of 48,
probably underwent chromosomal fusion and
deletion. This is indicated by similarities in
DNA/cell values among osteoglossids, anguil-

118

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—

loids, and clupeids. The most primitive living
forms of the Euteleostei, most notably the cyp-
rinids (excluding the tetraploids), have a chro-
mosome number of 48. However, many of these
species possess metacentrics. For example, in
the idiogram of Carassius auratus (Fig. 1) there
are numerous metacentrics, and although Car-
assius is tetraploid, it apparently arose from a
karyotype that already included several meta-
centrics. From this it would appear that the ba-
sic teleost karyotype already included metacen-
trics, or had a higher number of acrocentrics that
fused into metacentrics. The basic DNA/cell
value of teleosts appears to be about 1.0-1.2
pcg. This value is fairly constantly retained
among osteoglossids, clupeids, and primitive eu-
teleosts. I believe that the basic teleost chro-
mosome number was roughly 60 and that at least
a few of these were metacentrics. This karyo-
type most likely arose by fusions and deletions
from a basic, gar-like, holostean karyotype and
from this point underwent further deletions, fu-
sions, and ploidies to produce the myriad of
karyotypes found in teleosts today, with an ap-
parent convergence to 48 chromosomes in nu-
merous groups.

As discussed earlier, there is considerable evi-
dence that dipnoans underwent massive tandem
gene duplication in their evolution. The brachi-
opterygians also appear to have undergone tan-
dem gene duplication, but not to an extent com-
parable with the dipnoans. At one time the

119

brachiopterygians were considered chondros-
teans (Romer 1959). On the basis of karyotypes,
Denton and Howell (1973) argued that they are
more closely related to dipnoans. Their relative-
ly high amount of DNA/cell would support the
view that they have undergone tandem gene du-
plication, and this is reinforced by nucleoli stud-
ies (Dingerkus 1977). Among lungfishes a graded
series has been found with Neoceratodus having
the lowest amount of DNA/cell and apparently
the smallest chromosomes, Protopterus a higher
DNA level and larger chromosomes and Lepi-
dosiren the highest DNA level and largest chro-
mosomes (Wickbom 1945: Pedersen 1971; Ohno
and Atkin 1966). This indicates that evolution
among the lungfishes has proceeded in conjunc-
tion with additional tandem gene duplication.
Since brachiopterygians have the same chro-
mosome types and similar chromosome num-
bers as dipnoans, and have also undergone some
tandem gene duplication, one might speculate
that the dipnoan karyotype arose from a basic
brachiopterygian karyotype through further tan-
dem gene duplication. At what point could this
joint brachiopterygian-dipnoan karyotype have
arisen? It does not seem possible that it arose
from a teleostean or holostean karyotype on the
basis of what is known about piscine phylogeny
as well as the number of metacentrics exhibited
by these more recently evolved groups. Could
it have arisen from a basic actinopterygian (os-
teichthyan) karyotype? Assuming that this an-
cient karyotype was similar to that of the pad-
dlefish or sturgeon, then the basic chromosomal
configuration consisted of approximately 40
metacentrics to submetacentrics, 8 acrocentrics,
and 72 microchromosomes. If, in this configu-
ration, the microchromosomes were deleted or
fused into macrochromosomes and the acrocen-
trics fused into metacentrics, the result would
have been approximately 44 metacentrics. Al-
lowing for the loss of some chromosomes by
specialization from one lineage to another and
for differing amounts of tandem gene duplica-
tion, the final result could be the 36 to 38 large
metacentric chromosomes we find in brachiop-
terygians and dipnoans. A test for this hypoth-
esis 1s not presently available. When banding of
fish chromosomes becomes better established,
however, homologous bands might be shown to
exist among chondrosteans, brachiopterygians,
and dipnoans, and this would support the above
hypothesis.

120 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

DNA per 150
haploid 140
cell
content
in
picograms: 110

fo) (2) () ©) Io

FIGURE 2.

Among amphibians, the Urodela have ex-
tremely large chromosomes resembling those of
the dipnoans. They also have extremely high
amounts of DNA/cell like the dipnoans. This in-
dicates that the urodelans underwent tandem
gene duplication in their evolution, but does it
show a close phylogenetic relationship between
urodelans and dipnoans? The other amphibian
groups of Anura and Apoda have different
karyotypes and DNA/cell values. In some re-
spects lungfishes were considered to be close to
the ancestors of the tetrapod lineage, but this
theory now has been rejected. However, it
should be reconsidered. Although the basic
chromosome number of salamanders appears to
be 38, as in the lungfish, salamanders have a
number of acrocentrics in their karyotypes. This
may result from their having arisen from a
brachiopterygian-dipnoan karyotype in which all
acrocentrics were not fused or that they may
have undergone a centromeric fission of some
metacentrics.

An alternative hypothesis is that the salaman-
der karyotype originated from an anuran or apo-
dan karyotype. Both of these groups have basic
karyotypes that consist of a mixture of meta-
centrics, submetacentrics, acrocentrics, and

Graphs of DNA per cell ranges found in the different chordate groups. Based on data identified in the text.

very small acrocentrics which could be argued
to be microchromosomes. Among the anurans,
there is a trend to fuse the remaining acrocen-
trics into metacentrics, and this leads to anurans
in which the karyotypes have only metacentrics.
There are also species with lower-numbered
karyotypes that have undergone chromosome
losses. Still other species and groups have
undergone ploidy to produce species with high
numbers of chromosomes (Becak et al. 1966;
Wasserman 1970; Bogart and Wasserman 1972).
Caecilians do not seem to have undergone as
much secondary chromosomal change. The cae-
cilian karyotype could have originated from a
basic anuran karyotype by fusions of acrocen-
trics into metacentrics. The DNA levels of cae-
cilians indicate that they have undergone tan-
dem gene duplication, but not as much as in the
dipnoans or urodelans.

It is possible that the urodelans also under-
went fusion of the acrocentrics in the basic an-
uran karyotype with secondary, extensive tan-
dem gene duplication. The similarity between
dipnoan and urodelan chromosomes would then
be the results of convergence between the two
groups. This is the view held by Ohno (1974).
Among dipnoans there seems to be an increase

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

of DNA from the presumably more primitive
Neoceratodus to the more derived Protopterus
and Lepidosiren. In urodelans, however, the
trend seems to be the opposite, with more prim-
itive groups, such as the Proteidae and Am-
phiumidae, exhibiting higher DNA levels, and
more derived groups such as the Ambystomidae
and Plethodontidae, lower ones. This indicates
that the group originated through massive tan-
dem gene duplication and later became more
specialized through the loss of DNA and chro-
mosomal rearrangements. One might speculate
that since the lungfishes and primitive salaman-
ders occupy a similar semi-aquatic and semi-ter-
restrial ecological niche, it was necessary for
both of them to produce large quantities of DNA
to evolve and adapt to this new type of habitat.
Once they had adapted to a new terrestrial type
of life, they could lose much of the redundant
DNA that had allowed them to make the tran-
sition. This appears to have happened in the
more advanced, more completely terrestrial sal-
amanders.

At present it is difficult to decide from which
group the urodelans arose. With the advent of
banding studies of dipnoan, urodelan, anuran,
and apodan chromosomes, homologous bands
may show which groups are more closely relat-
ed. I propose that the term megachromosomes
be applied to the huge chromosomes found in
these two groups. Not only are they most prob-
ably the largest chromosomes found in any liv-
ing animal, but they also have a common origin
in having been produced by massive amounts of
tandem gene duplication. Thus, megachromo-
somes are defined as very large chromosomes
(more than 15 w long), that have originated by
means of tandem gene duplication. Megachro-
mosomes are not to be confused with the ‘‘giant
chromosomes,” found in Drosophila and other
dipterans. These ‘“‘giant chromosomes’”’ are not
somatic chromosomes but are unique chromo-
somes, found only in the salivary glands of these
flies, and they originate through polytene-puffs.
Megachromosomes are somatic chromosomes
and do not have such an origin.

Returning to the Anura and Apoda, how could
their karyotype have originated? I pointed out
previously that the basic apodan karyotype
could have originated from the basic anuran one
by fusion of acrocentrics into metacentrics and
secondary tandem gene duplication. Anurans
also seem to have undergone fusions of chro-

121

mosomes. Since the anurans have a number of
very small acrocentrics, which may be micro-
chromosomes, it seems logical to suggest that
they arose from a basic karyotype that had mi-
crochromosomes. Allowing for tetraploid species
and species that have lost DNA by chromosomal
deletion, it appears that the basic DNA/cell val-
ue for anurans is probably between 1.0 and 1.5
pcg. The basic anuran karyotype appears to
have had about 60 chromosomes, a mixture
mostly of acrocentrics with some metacentrics-
submetacentrics and microchromosomes. The
karyotype that could have given rise to such a
makeup is a basic actinopterygian one that has
undergone diploidization.

Three of the five subdivisions of the reptiles
have microchromosomes: the chelonians, snakes,
and lizards. However, the crocodilians and the
tuatara also have a few very small chromosomes
that could be considered as microchromosomes,
or at least as originating from a fusion of micro-
chromosomes. Chelonians have the largest num-
ber of chromosomes and chromosome arms of
any reptile. They therefore may be considered
to have the most primitive living reptile karyo-
type and one which is probably representative
of the basic reptilian karyotype. The reptiles ex-
hibit a higher amount of DNA/cell than do the
anurans but with only a limited increase in num-
ber of chromosomes. If the basic reptilian
karyotype is envisioned as arising from the an-
uran one, it may be concluded that between the
two an increase of DNA by tandem gene dupli-
cation occurred. Another plausible hypothesis
(supported by evidence presented later) is that
the basic reptilian karyotype arose from the ba-
sic anuran karyotype by tetraploidy. This is in
accord with the increased DNA level in reptiles.
Secondary fusion and/or deletions of chromo-
somes could account for the lowered chromo-
some numbers found in living reptiles. Biochem-
ical and chromosome banding studies eventually
may show which of these hypotheses is more
likely to be correct. From a basic reptilian
karyotype, the crocodilian karyotype of almost
all metacentrics could have arisen by fusion of
acrocentrics and microchromosomes. The very
similar amounts of DNA/cell found in chelonians
and crocodilians would be expected according
to this theory. The karyotype of Sphenodon can
be envisioned as being brought about by tandem
gene duplication which would increase the size
of the chromosomes. It would be useful, of

122 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

course, to have a DNA/cell value for Spheno-
don. If this turns out to be a higher value than
in other reptiles, tandem gene duplication would
be strongly indicated. Some of the chromosomes
in Sphenodon are large enough to be considered
megachromosomes.

In contrast, the lizards and snakes do not have
a karyotype that can be accounted for complete-
ly by fusions occurring in a basic reptilian karyo-
type. Because of their low chromosome num-
bers, they are better explained by rearrangements
and deletions. This view finds support in their
level of DNA/cell which is lower than in any
other reptile group. The basic lizard karyotype
consists of all, or almost all, acrocentrics and
microchromosomes. This may be the result of
centromeric fissions of the metacentrics in the
basic reptilian karyotype. The basic snake
karyotype could have originated from a basic
lizard karyotype by the robertsonian fusion of
acrocentrics. Evidence from both of these lin-
eages (and to a lesser extent among turtles and
crocodilians) indicates that the more advanced
species have evolved from the primitive ones by
fusion of chromosomes. In certain species this
included the loss of all microchromosomes.
Only among the lizards does it appear that some
species have undergone ploidy (Lowe et al.
1970).

The similarity between avian and reptilian
karyotypes has been noted several times (Becak
et al. 1964; Takagi and Sasaki 1974; Takagi et
al. 1972). However, the more primitive birds
have karyotypes that are higher in number than
those of reptiles. The ratites (ostrich, rhea, emu,
and cassowary) have chromosome numbers in
the range of 80 to 82. Gavia, the loon, has 90
chromosomes with one pair of submetacentrics
and the rest acrocentrics, and microchromo-
somes. The ratite karyotype, with its metacen-
trics, may have arisen by fusion from a karyo-
type similar to that of Gavia, which I believe
resembles the basic bird karyotype. Such a basic
bird karyotype with its acrocentrics and high
number of microchromosomes can be envi-
sioned giving rise to other living bird karyotypes
by fusions and deletions. Because of their high
number of chromosomes, birds might be consid-
ered to have arisen from reptiles in part by tet-
raploidy. If this hypothesis is true, birds should
have about twice as much DNA as reptiles, but
they actually have less DNA/cell than any rep-

tile. There are two plausible explanations for
this situation: |. Birds originated from a reptile
with a karyotype that had a higher number of
chromosomes than does any living reptile (ex-
cept for tetraploids) and thus all living reptiles
exhibit deleted karyotypes. This hypothesis
would tend to uphold a tetraploid origin of the
basic reptile karyotype. The lowered DNA level
in birds could then be accounted for by DNA
losses—not unexpected since they must have
undergone considerable specialization to attain
their present morphotype. 2. The bird karyotype
originated from a basic reptilian karyotype by
tetraploidy and secondarily lost considerable
amounts of DNA by specialization. This is also
not unreasonable since birds probably required
considerable duplicate DNA (as would be pro-
duced by such a tetraploidy) to evolve into their
present morphotype. This evolution could also
have resulted in considerable DNA loss. Takagi
and Sasaki (1974) have shown that the G-band-
ing patterns of the first six chromosomes of nu-
merous birds and one turtle are nearly the same.
This argues against a tetraploid origin of the ba-
sic bird karyotype, for had the bird karyotype
arisen through tetraploidy, it should show twice
as many sets of similarly-banded chromosomes
as does that of reptiles. We can conclude that
the basic bird karyotype originated from a basic
reptile karyotype with a high number of chro-
mosomes. Since the crocodilians and birds are
considered living archosaurs and turtles living
examples of the more primitive cotylosaurs,
crocodilians should exhibit the same chromo-
some banding pattern as do birds and the turtle.
If they do not, then the chromosome bands of
birds and turtles must be considered a conver-
gence rather than a homology. However, if the
same banding pattern is found in crocodilians,
it must be considered a primitive reptilian con-
dition. Karyotypic studies of kiwis and tinamous
may be useful in further determining the basic
bird karyotype and additional banding studies of
reptiles and birds should help explain karyotypic
relations among reptiles and birds.

Because monotremes have a high number of
all metacentric to submetacentric chromosomes,
it appears that mammals originated from a lin-
eage that had a high number of chromosomes.
It is very difficult, however, to imagine how the
basic marsupial karyotype, which appears to
consist of 22 acrocentric chromosomes, could

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

arise from a monotreme karyotype. Among the
eutherian mammals, 48 is the predominant chro-
mosome number, most of the chromosomes
being metacentric to submetacentric in form.
How can we explain this seemingly contradic-
tory evidence? Since lemurs have a considerable
number of microchromosomes, the basic mam-
mal karyotype must have included microchro-
mosomes. This can be concluded because all
evidence indicates that once microchromosomes
have been lost in a group of animals, they cannot
be re-evolved in relatively high, constant num-
bers. Since lemurs also have a relatively high
number of acrocentrics, it can also be concluded
that the basic mammal karyotype had a high
number of acrocentrics. The monotreme karyo-
type can be envisioned as having arisen from a
basic karyotype having microchromosomes.
The small metacentrics and the small arms of
the submetacentrics could have originated from
fusions of, and with, microchromosomes. The
typical metacentrics could have arisen from fu-
sions of acrocentrics. The basic karyotype, con-
sisting mostly of acrocentrics and microchro-
mosomes, which gave rise to the monotreme
karyotype must have had a high number of chro-
mosomes, in the neighborhood of at least 100.
Such a basic mammal karyotype could have
arisen from the basic reptile one. This, however,
again argues for a tetraploid origin of the basic
reptile karyotype. DNA/cell values for mammals
are in agreement with this hypothesis, as it is
probable that the living reptiles have lost DNA
from the basic reptile configuration described
above. Moreover, mammals do not seem to lose
DNA through chromosomal rearrangements and
fusions, as indicated by their diverse chromo-
some numbers and configurations yet rather
constant DNA/cell values. Could a high-num-
bered basic karyotype of mostly acrocentrics
with some microchromosomes give rise to living
mammal karyotypes? I have already discussed
how it could be changed into a monotreme
karyotype. The basic marsupial karyotype could
have been obtained by fusions that produced
only large acrocentrics. A lemur karyotype
could have been obtained by retaining some
microchromosomes and by a fusion of acrocen-
trics to produce the large pair of metacentrics
and the other metacentrics and submetacentrics.
Other eutherian mammal karyotypes, such as
that of Homo sapiens, could have been pro-

123

duced by inclusion of microchromosomes into
macrochromosomes and then further rearrange-
ments and fusions to yield the other chromo-
somes.

What is the place of Latimeria in the above-
outlined pattern? The coelacanth has a DNA/cell
value between 2.8 and 3.8 pcg and its nucleoli
indicate that it is diploid. Clearly its affinities do
not seem to lie with living brachiopterygians,
dipnoans, or urodelans. Latimeria is believed
by some to be a crossopterygian and a descen-
dant of a lineage closely related to the lineage
that supposedly gave rise to the tetrapods. Be-
cause of the numbers of microchromosomes
found in all tetrapod groups, except the urode-
lans, the lineage from which the tetrapods arose
must have had a relatively high number of mi-
crochromosomes. On the whole it seems that
most tetrapods have at least some macrochro-
mosomes that are larger than those found in oth-
er chordates, except brachiopterygians and dip-
noans. Since some view the Sarcopterygii to
include brachiopterygians, dipnoans, and tetra-
pods, the generality can be advanced that most
of the sarcopterygians have some macrochro-
mosomes that are larger than the macrochro-
mosomes of other chordates. The only excep-
tions are that Narcine exhibits some unusually
large macrochromosomes and that some birds
have macrochromosomes all of rather small
size. If Latimeria is indeed a sarcopterygian, it
should have at least some fairly large macro-
chromosomes. Its fairly high DNA value, with
no indication of ploidy, tends to indicate this. If
we can assume that the basic anuran karyotype
was also the basic tetrapod karyotype, then it
must also approximate the karyotype of the fish
lineage from which the tetrapods arose. If Lati-
meria is a living relative of that lineage, it should
exhibit a karyotype of about 60 chromosomes,
most acrocentric, with some microchromo-
somes, and at least a few large macrochromo-
somes. This model presents some problems,
however. Latimeria appears to have too much
DNA/cell in proportion to the basic anuran con-
figuration. This might be explained by DNA loss
in the living anurans. Their ancestral stock
would have had more DNA, that is, an amount
more like that of Latimeria. Or the lineage of
Latimeria may have undergone secondary tan-
dem gene duplication. The first hypothesis might
be more favored, since Latimeria has a DNA

124 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

—

FiGure 3. Silver-stained nucleoli in interphase nuclei of
the barb Barbus tetrazona (A): paddlefish Polyodon spathula
(B); coelacanth Latimeria chalumnae (C); and lungfish Lepi-
dosiren paradoxa (D). A and C are diploid species, B is a
tetraploid, and D is a species that underwent considerable
tandem gene duplication. Scale bar equals 10 pw.

value very similar to most reptiles and mam-
mals. If Latimeria does NoT show the predicted
karyotype, however, it cannot be concluded that
it is not a sarcopterygian. The Latimeria lineage
secondarily may have undergone further karyo-
typic evolution and have acquired a substantial-
ly different karyotype. Even after Latimeria has
been karyotyped, I believe it will be necessary
to use chromosomal banding patterns to show
which group is most closely related to the only
living coelacanth.

CONCLUSIONS

A provisional chordate cladogram (Fig. 4) can
be produced on the basis of cytogenetic data,
primarily chromosome size. Sharing the largest
chromosomes and DNA/cell values of living
chordate groups, the dipnoans and urodelans
are classified as synapomorphic sister groups.
Polypterids, with the next largest chromosomes

among chordates, form a sister group to the
dipnoan-urodelan taxon. Tetrapods exclusive
of the Urodela jointly have the next largest
chromosomes and form a polychotomous sister
group to the common polypterid-dipnoan-uro-
dele assemblage. The latter assemblage and tet-
rapods exclusive of the Urodela can be termed
the Sarcopterygil.

Actinopterygians have chromosomes next
largest in size to the Sarcopterygii as defined
above, and form their sister group. The actinop-
terygians can be divided into chondrosteans,
holosteans, osteoglossids, and non-osteoglossid
teleosts on the basis of chromosome number.
The chondrichthyans, which as a group have
the next largest chromosomes, become the sis-
ter group of the Osteichthyes, i.e., the actinop-
terygians and sarcopterygians as defined above.
Employing comparative data from karyotype
analysis, chimaeras (=holocephalians) and elas-
mobranchs form sister groups within the Chon-
drichthyes.

Lampreys, on the basis of their smaller chro-
mosome size, can be classified as the sister
group of the gnathostomes. On the basis of
karyotype (all acrocentrics versus all metacen-
trics), the Northern Hemisphere and Southern
Hemisphere lampreys are sister groups to one
another. Hagfish, having the smallest chromo-
somes of any vertebrate group but with some
macrochromosomes, become the sister group
of all other vertebrates. The Amphioxi, having
the largest microchromosomes but a karyotype
devoid of macrochromosomes, become the sis-
ter group of all vertebrates. Tunicates, with
their entire karyotype composed of very small
microchromosomes, are classified as the sister
group of the Cephalochordata and Vertebrata.

Although the position of Latimeria in this
cladogram is speculative (without actual data
on karyotype and chromosome size), on the
basis of cell size, DNA/cell values and the pre-
dicted hypothetical karyotype, it would be clas-
sified as the sister group of all other sarcop-
terygians. If Latimeria is a living representative
of the sister group of rhipidistians which gave
rise to the tetrapods, it should exhibit a karyo-
type with approximately 60 chromosomes con-
sisting of a mixture of mostly acrocentrics with
microchromosomes, and with some relatively
large macrochromosomes. On the other hand,
on the basis of the available cytogenetic data
(primarily DNA/cell values), Latimeria is indis-

DINGERKUS: CHORDATE CYTOGENETIC STUDIES

CHONDRICHTHYES

NORTHERN HEMISPHERE LAMPREYS
SOUTHERN HEMISPHERE LAMPREYS

wn
=
Oo
z
[%) n <<
w _ xt a
= x< == a {ee}
x ro) 7) uw e)
Oo = Ss <x =
7 5 UL >= wn
2 a oO = <
2 = xt = —
- < - oO lu
00
= iS)
oy
60
Q
i) ~
a.
va) oN
y “So
a.
Coal cq !
~N w
. ' ™
' a) N
im _
=
FIGURE 4.

discussed in the text.

tinguishable from the extant elasmobranchs,
chondrosteans or polypterids.

It is apparent that reliable descriptions of the
banding patterns of fish chromosomes will be
essential to enable us to reconstruct the evolu-
tion of fish karyotypes. It is hoped that the nec-
essary techniques will soon be developed.

ACKNOWLEDGMENTS

I would like to thank Drs. W. Mike Howell,
Donn E. Rosen, James W. Atz, Gareth J. Nel-
son, Lowell D. Uhler, Charles H. Uhl, Michael
D. Lagios, William L. Brown, Jr., John E.
McCosker, C. Lavett Smith, Howard E. Evans,
and Perry W. Gilbert for assistance, advice, and
enlightening discussions in the preparation of
this paper. I would also like to thank Celeste
Roman, Peter Spencer, Thomas Lindsay, Larry
Herbst, Frank Bartalone, Bernice Churnetski,
Tina Segalla, U. Erich Friese, Weysan Dun and
my mother, Lieselotte Dingerkus for assistance
in the preparation of this manuscript.

125
SARCOPTERYGI I
oD
3) 0 * —
Soe Gar mon 2
i=) iss —¢
wn x WwW =
ol OV uu
ACTINOPTERYGII S
Va) a
. io) i=) =
D ae eee us
=< D —
= = 2 2
op) [) > a wo ve a
io) = = uw Zz < On
oe 22) O — = ay e>
Q io) = o fo) w os
z n <x WwW i] > z fa) “Zo
=) ss = w S| a s) — x
aie <x >= WwW Oo fo) = ao we
oO oO <x = ) a A =) rw
i
u )
| oe
! un
5D ~O
S)
iss 1
co
xn

ee Sr ere

Chordate cladogram based on cytogenetic data. Numbers relate to DNA/cell values for respective taxa as

This work was funded through a grant from
the Alabama Academy of Science, while at Sam-
ford University; the Cornell University Office of
Academic Funding and a Sigma Xi Grant-In-Aid
of research, while at Cornell University; and the
Department of Ichthyology, the American Mu-
seum of Natural History.

The coelacanth tissues used for nucleoli stud-
ies came from a preserved female specimen in
the American Museum of Natural History
(AMNH 32949), and from a fresh-frozen male
specimen in the California Academy of Sciences
(CAS 33111), provided through the courtesy of
J. E. McCosker from the CAS 1975 Coelacanth
Expedition, supported by a grant from the Char-
line H. Breeden Foundation.

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Chromosome
Chromosoma

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 14 pages

December 22, 1979

IMMUNOCHEMICAL AND BIOLOGICAL STUDIES WITH GROWTH
HORMONE IN EXTRACTS OF PITUITARIES FROM
EXISTING PRIMITIVE FISHES

By

Tetsuo Hayashida

Department of Anatomy and the Hormone Research Laboratory,
University of California,
San Francisco, California 94143

ABSTRACT

In confirmation of earlier reports growth hormone in pituitary extracts of modern
bony fishes were found to be incapable of or were extremely poor in eliciting significant
biological activity in the mammal on the basis of the rat tibia assay. In contrast, those
of existing primitive fishes including chondrosteans, holosteans, elasmobranchs, a dip-
noan and the coelacanth, showed positive stimulation. These results slowed a general
correlation with immunochemical relatedness of growth hormone or a growth hormone-
like substance in the pituitary extracts from these major groups of fishes, when com-
pared to purified growth hormones of tetrapods.

INTRODUCTION

In the present investigations we have em-
ployed two general methods for studying the re-
latedness of growth hormone (GH) or a GH-like
substance in pituitary extracts (PEs) of various
fishes with respect to pituitary GH of tetrapods.
The well known tibia assay in hypophysecto-
mized rats (Greenspan et al. 1949) was used for
the bioassay, and the immunochemical studies
were conducted utilizing monkey antisera to
purified tetrapod GHs employing the techniques

of immunodiffusion (Ouchterlony 1953) and ra-
dioimmunoassay, the details of which have been
previously presented (Hayashida 1969, 1970).

I. BIOASSAYS

For many years it was thought that fish pitu-
itary GH was not capable of stimulating growth
in the mammal on the basis of the rat tibia assay
(see Knobil and Hotchkiss 1964; and Geschwind
1967), in spite of the fact that PEs of amphibian,
avian and reptilian species tested were all ca-

HAYASHIDA: GROWTH HORMONE STUDIES

129

TABLE 1. TiBIAL PLATE STIMULATING EFFECTS OF PITUITARY EXTRACTS FROM VARIOUS FISHES.
Daily Animal Tibial plate width
Group dosage (no.) (um + S.E.) Significance (*)
1. Controls — 6 150.8 + 3.3 --
2. Bovine GH 2.5 pg 6 173.0 + 4.8 levse2ps0l
3. Bovine GH 5.0 ug 6 19S O33 26 2vs.3 p< .001
4. Bovine GH 10.0 ug 6 PBN dae Syop) 3 vs.4 p< .001
5. Mackerel 0.6 mg 4 SDE 92 V5 5 WsSt
6. Mackerel 2.4 mg 4 153: 9 r4e2 WSO Wess
7. Salmon 0.6 mg 4 163.3 + 4.5 IW ES
8. Salmon 3.6 mg 4 162.6 + 10.2 1RVSSOuneS:
9. Paddlefish 1.2 mg 4 tot S2 Gye! LVS Oper Oil
10. Paddlefish 2.4 mg 4 186.3 + 9.1 1 vs. 10 p < .01
11. Paddlefish 3.6 mg 4 202.9 + 6.8 Sivse litpy <= -05
12. Sturgeon 0.6 mg 4 NWe3) 32 3G 1 vs. 12 p < .001
13. Sturgeon 1.2 mg a L9GESy=5 7/54 1 vs. 13 p < .001
14. Sturgeon 2.4 mg 4 P| PIAS) = TES) 12 vs. 14 p < .001

Female rats of the Long-Evans strain were hypophysectomized at 28 days of age, injected subcutaneously daily for 4 days
and sacrificed on the Sth day. Pituitary extracts of the various fishes represent milligrams equivalent of whole pituitary tissue.
(*) Significance of the difference between mean values in terms of p values of Fisher. n.s. indicates that difference is not
significant. From Hayashida and Lagios (1969), Gen. Comp. Endocrinol. 13:403, by permission of the editors.

pable of showing positive stimulation in this as-
say. However, an examination of the literature
revealed that practically all of the fish PEs or
purified GH preparations utilized previously had

TABLE 2. TIBIAL PLATE STIMULATING EFFECTS OF Ex-
TRACTS OF PITUITARIES FROM THE LUNGFISH, STRIPED BASS
AND SALMON.*

Daily
dosage No. of Tibial plate width
Group (mg) rats (um + S.E.)
1. Lungfish (a) 0.32 4 167.0 + 6.8
0.63 4 185.3 + 6.9
1.26 4 ZOQES ==) 1113
2252 3 22821 = 1164
(b) 0.63 4 lo 2295)
1.26 3 i} S= P57)
DeS2 3 23273) = 1354
2. Salmon (a) 0.63 4 1I55-2;-== 16:3
1.26 4 155.9 + 4.0
DeS2 4 146.7 + 4.4
3.78 4 162.6 + 9.1
(b) 0.63 4 16335-3457,
3. Striped bass (a) 0.63 4 14322, 5:9
1.26 4 S/O) 3533}
2E52 4 161.2 + 10.6
5.04 4 161.6 + 9.7
(b) 2.52 4 162.3 + 10.3

* Mean tibial plate width for hypophysectomized control
rats was approximately 150 wm.

Lungfish—African lungfish, Protopterus aethiopicus; salm-
on—silver salmon, Oncorhynchus kisutch,; striped bass, Mo-
rone saxatilis. From Hayashida (1970), Gen. Comp. Endocri-
nol. 15:432, with permission of the editors.

originated from pituitaries of modern bony fish-
es, perhaps simply because of their ready avail-
ability.

I would like to present the results of some our
own studies conducted over the past several
years. The first table (Hayashida and Lagios
1969) shows the results obtained in the rat tibia
assay with PEs of two modern bony fishes
(salmon, Oncorhynchus kisutch and mackerel,
Scomber scombrus) compared to results ob-
tained with PEs of primitive bony fishes (the
sturgeon, Acipenser transmontanus and paddle-
fish, Polyodon spathula). Injections were given
subcutaneously daily for 4 days and the rats
were sacrificed on the Sth day to obtain the tibias
to determine the extent of stimulation of the
widening of the tibial epiphyseal cartilage plate,
determined with the aid of a microscope
equipped with an ocular micrometer. A purified
bovine GH was used as a reference standard and
it may be seen that it gave a good dose-response.
Neither teleost PEs gave any significant stimu-
lation, in confirmation of the earlier reports. On
the other hand, comparable doses of PEs from
the primitive bony fishes induced significant
stimulation of the rat tibial plate with that of the
sturgeon showing a slightly greater potency on
a wet tissue weight basis.

Table 2 shows the results obtained in another
rat tibia assay (Hayashida 1970) in which a PE
of a dipnoan, the African lungfish (Protopterus
aethiopicus) was tested in addition to PEs of the

130 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

\“ y
I G/RAFFE
HORSE @

ff

f

& MECTURUS y
/
RGH

FIGURE 1. Ouchterlony plate showing precipitin reactions between monkey antiserum to rat GH (RGH) and GH in extracts
of pituitaries (PE) from mammals, birds, reptiles, amphibians, and teleost fishes. Each plate contains 0.15 ml undiluted antiserum
in the central well. All wells containing RGH have 8 yg of the hormone in a volume of 50 yl, unless otherwise indicated. Each
PE represents a volume of 50 yl. (A) The HGH well contains 10 wg of the hormone in 50 wl. Each PE represents 0.25—0.5 mg
of pituitary tissue in 50 zl of phosphate-saline buffer. Photographed at 24 hours. (B) RGH wells contain 10 wg hormone/S50 wl.
Each PE representing 0.5 mg of pituitary tissue except that of the penguin, which represents 1.0 mg. Photographed at 42 hours.
(C) Each PE represents 1.0 mg of tissue except duck PE which represents 0.5 mg. Note the reactions of identity between PEs
of the duck, crocodile, and turtle, and the reactions of partial identity between Necturus and turtle PEs and between Necturus
and duck PEs indicated by spur formations. Photographed at 24 hours. (D) Necturus and bullfrog PEs represent 1.0 mg of
pituitary tissue each, and each of the PEs in other wells represent 2.0 mg of tissue. Photographed at 24 hours. (From Hayashida,
1970, Gen. Comp. Endocrinol. 15:432, by permission of the editors.)

salmon and striped bass (Morone saxatilis). The
mean tibial plate width for the hypophysecto-
mized control group was again approximately
150 uw. Each PE was tested twice using separate
pools of pituitaries on two different occasions.

It may be seen from the data that the lungfish
PE gave a very definite stimulation showing a
good dose-response on each occasion, very sim-
ilar to that obtained with the purified bovine GH
standard. Again, the PEs of the teleost species

HAYASHIDA: GROWTH HORMONE STUDIES

ie) Pituitary Tissue

063 25 1.0 40 158 64.0
1 i

= 2 Mackerel
Salmon A. Fishes
\S is
So AS SS Lungfish
~S ~ g
Wy a
\N ~
YS ‘ SSS Pas Bull Frog
\ SSS Ss
L SS SSS <u = Mud puppy> B. Amphibians
SS
To sol SS SESS. PRN
O SESS X
[a4 SSS NS Ss
le St eae ~ Lizard
a SESS
w ie Ne ~S S
= SOS
rr if NSSISES chick ;
faa) SS ASAA rekon C. Reptiles
SO \ AS Turtle and
ro) b \ sS Crocodile Birds
= \ puck
ray
7 30;-—
=a) L
(=
Z L
Ww
S li Opossum
tu -20;- RGH
|_ Standard Sea Ox
Gui
Goat
L b Horse
10;—
ESS i | Se It l al a
2 8 32 125 500 2000

myg equivalent of RGH

FIGURE 2.
relatedness of GH in pituitary extracts from representatives

Composite diagram showing immunochemical

of various vertebrate classes based on radioimmunoassay with
antiserum to RGH. (From Hayashida 1970, Gen. Comp. En-
docrinol. 15:432, by permission of the editors.)

(salmon and striped bass) gave no significant
stimulation at comparable and higher doses.

I]. COMPARATIVE IMMUNOCHEMICAL STUDIES

Before going on to the discussion of results
obtained in bioassays with PEs of other primi-
tive fishes I would like to review the results of
immunochemical studies which we were con-
ducting at the same time using the same PEs
employed in the above bioassays.

In contrast to results obtained in our earlier
investigations with the use of rabbit antisera to
purified mammalian GHs in which the findings
suggested the existence of a high degree of im-
munochemical species specificity for GH even
among mammals themselves (Hayashida and Li
1958a, 1958b and 1959), the subsequent use of
antisera prepared in rhesus monkeys gave us a
very useful tool for crossing species barriers
(Hayashida and Contopoulos 1967; Hayashida
1970).

A. Immunodiffusion Studies

The results of the immunodiffusion studies
employing monkey antiserum to purified rat GH

131

DaDDLetisH
6 ween

SALMUN.

FIGURE 3. Ouchterlony plate showing immunochemical
relatedness of GH in PEs of primitive bony fishes to rat GH
and the lack of detectable relatedness of GH in PEs of modern
bony fishes to RGH. RPE indicates 0.3 mg equivalent of an
adult male rat pituitary. The sturgeon and paddlefish PEs rep-
resent 1.0 mg and 1.5 mg of tissue, respectively, while the
salmon and mackerel PEs represent 2.0 mg of tissue each.
Photographed at 24 hours. (From Hayashida 1970, Gen.
Comp. Endocr. 15:432, by permission of the editors.)

are shown in Figure |. These studies were car-
ried out with crude extracts of pituitaries rep-
resenting each of the major vertebrate classes.
In the first agar plate we can see that PEs of
species representing several different orders of
mammals show reactions of identity with each
other and with the purified rat GH reference. In
the second plate the PEs of avian species gave
reactions of identity with each other, but reac-
tions of partial identity with respect to the rat
GH indicated by the formation of spurs, sug-
gesting that avian GHs do not share all of the
immunoreactive determinants possessed by rat
GH. In the next plate we see that an avian PE
shows a reaction of identity with PEs of two
different reptilian species (snapping turtle, Chel-
ydra serpentina and a crocodilian, Caiman
sclerops), and that an amphibian PE (mud pup-
py, Necturus) shows a reaction of partial iden-
tity compared to the avian and reptilian species
as well as with respect to rat GH. In the fourth
plate of this series we again see that the Nec-

132 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

turus PE gives a reaction of partial identity with
rat GH and that another amphibian PE (bullfrog,
Rana catesbeiana) almost gives a reaction of
identity with the Necturus PE. On the other
hand, the lungfish PE (Protopterus aethiopicus )
as well as the teleost PEs (striped bass, Morone
saxatilis, and carp, Cyprinus carpio) gave no
detectable reaction with this technique although
higher levels of the PEs were employed.

B. Radioimmunoassay Studies

In the radioimmunoassay (RIA) studies, high-
ly purified rat GH was labeled with I'*° (Green-
wood et al. 1963) and used as the labeled hor-
mone as well as for standards. Preliminary tests
had shown that unlabeled, purified rat GH would
competitively inhibit the binding of the labeled
hormone with the antibody to rat GH. The avail-
able purified GHs (mammalian species only) as
well as the GHs in the PEs from the various
vertebrate classes were then employed to test
their relative abilities to compete in this system
(Hayashida 1969, 1970). The results showed
(Fig. 2) that all mammalian GHs or PEs were
the most efficient as indicated by the steepness
of their inhibition slopes, and that PEs of reptiles
and birds were the next most efficient while
those of amphibians were still less efficient and
those of fishes including the African lungfish and
two teleost species showed the least ability to
compete in this system. The PEs of two other
teleost species including the mackerel (Scember
scombrus) and the carp (Cyprinus carpio) were
also found to be virtually inactive in this system.
The GHs or PEs thus fell into major immuno-
chemical groupings which corresponded to the
results obtained by immunodiffusion and cor-
related well with their major phylogenetic clas-
sifications.

C. Studies with Chondrostean PEs (Sturgeon
and Paddlefish)

Immunodiffusion studies employing the mon-
key antiserum to rat GH (Fig. 3) showed that
purified rat GH and a rat PE gave reactions of
identity, while the PE of the sturgeon (Acipenser
transmontanus) gave a Clear line of partial iden-
tity with rat GH or PE. Paddlefish (Polyodon
spathula) PE showed a slightly weaker reaction
than that of the sturgeon, but a reaction of iden-
tity with the latter. The two teleost PEs again
were negative. The results of RIA were in ac-
cord with those obtained by immunodiffusion.

yg Pituitary Tissue

(0632S 1.0 40 15.8 64.0
60 rae Mackerel
Salmon

50 Paddlefish

ro)
Sturgeon
ae
O
SAO
uw
O
O
2
fa)
FA Si0)r
Para)
—
Zz
Ww
U
a
mm 20);
——-<ARot PE
10-- RGH
L a a a eee ee eee Es
) 2B 4h 9G 32 128 500 2000
myg UNLABELLED RGH
FiGure 4. Cross-reactions of GH in PEs of primitive and

modern bony fishes based on radioimmunoassay with monkey
antiserum to RGH. The relative ability of GH in the various
fish PEs to competitively inhibit the binding of labelled RGH
is represented. (From Hayashida and Lagios 1969, Gen.
Comp. Endocrinol. 13:403, by permission of the editors.)

Both chondrostean PEs gave a low, but signifi-
cant degree of cross-reaction while those of the
teleosts were essentially nil (Fig. 4).

The rat tibia assay was utilized to determine
whether or not the monkey antiserum to rat GH
could neutralize the biological activity of stur-
geon PE. Table 3 shows that this antiserum was
indeed capable of neutralizing the rat tibial plate
stimulating activity of sturgeon PE suggesting
that the substance in this PE responsible for this
activity is immunochemically related to rat GH.
As a test for antiserum toxicity or specificity a
monkey antiserum to rat pituitary prolactin was
utilized in another group of rats injected with
sturgeon PE. This control antiserum, used at the
highest dose level, did not show any detectable
neutralization providing additional evidence for
the specificity of the antiserum for GH.

HAYASHIDA: GROWTH HORMONE STUDIES

TABLE 3.
THE RAT TIBIA ASSAY.

133

CROSS-NEUTRALIZATION OF GH IN STURGEON PITUITARY EXTRACT WITH MONKEY ANTISERUM TO RAT GH IN

No. of Serum Tibial plate width

Group rats (ml) (um + S.E.) Significance (*)
1. Hypohysectomized controls 8 — 154.4 + 6.1
2. Normal monkey serum (NMS) 3 0.3 160.1 + 6.4
3. Sturgeon PE + NMS 5 0.2 PAM ICSY ae C12 2 vs. 3 p < 0.001
4. Sturgeon PE + NMS 3) 0.3 206.2 + 10.2
5. Sturgeon PE + AS 3 0.1 204.2 + 6.7
6. Sturgeon PE + AS 3) 0.2 184.8 + 7.8 3 vs. 6n.s.
7. Sturgeon PE + AS 4 0.3 160.5 + 10.8 3 vs. 7p < 0.01
8. Sturgeon PE + AS (RP) 4 0.3 YDS) 2= S57)

PE, pituitary extract; AS, monkey antiserum; AS (RP), monkey serum to rat prolactin. (*) Significance of difference between
mean values in terms of p values of Fisher; n.s., not significant. From Hayashida (1970), Gen. Comp. Endocrinol. 15:432, with

permission of editors.

Ill. BloLOGICAL AND IMMUNOCHEMICAL
STUDIES WITH PES OF ELASMOBRANCHS
AND HOLOSTEANS

A. Elasmobranchs

Extracts were prepared from the freshly fro-
zen pituitaries of two species of sharks (smooth
dogfish, Mustelus canis and spiny dogfish,
Squalus acanthias) and one species of skate
(clear-nosed skate, Raja eglanteria) and were
subjected to testing in the rat tibia assay and in

immunochemical studies (Hayashida 1973).
Each PE was tested at 2 or 3 dose levels. An
ovine GH standard was employed as a refer-
ence. The results showed that the shark PEs
were Clearly active in stimulating growth in the
mammal on the basis of the rat tibia assay (Table
4). The potencies of the PEs from the two
species of shark appeared to be about the same.
The skate PE was somewhat less active than the
shark PEs but still showed significant stimula-
tion. In a separate experiment the simultaneous

TABLE 4. Rat TiBIAL PLATE STIMULATING EFFECTS OF PITUITARY EXTRACTS FROM CARTILAGINOUS FISHES (ELASMO-

BRANCHS).°
Animal Tibial plate width
Group? Daily dosage no. (jem) ==)S2E2) Significance®

1. Hypophysectomized controls — 9 IS923-= 3-8 _—

2. Sheep GH 5.0 ug Uf 205-2355 I vs. 2 p < 0.001
3. Sheep GH 10.0 ug 7 22.655) = Sail 2 vs. 3 p < 0.01
4. Sheep GH 20.0 ug 8 245.6 + 5.2 3 vs. 4p < 0.02
5. Squalus acanthias 1.3 mg U RS7e3) == 256 l vs. Sp < 0.001
6. Squalus acanthias 2.5 mg 7 204.3 + 3.0 5 vs. 6 p < 0.01
7. Squalus acanthias 5.0 mg 3 210.2 + 7.6

8. Mustelus canis 1.3 mg 7 186.7 + 2.6 I vs. 8 p < 0.001
9. Mustelus canis 2.5 mg 7 206.0 + 4.8 8 vs. 9p < 0.01
10. Mustelus canis 5.0 mg 6 204.9 + 3.8
11. Raja eglanteria 2.5 mg i 178.0 + 3.7 I vs. 11 p < 0.01
12. Raja eglanteria 5.0 mg 8 200.3 + 4.4 I vs. 12 p < 0.001

Il vs. 12 p < 0.01

* Female rats of the Long-Evans strain were hypophysectomized at 28 days of age and injected sc daily for 4 days, beginning
10-12 days postoperatively. The animals were killed on day 5. Pituitary extracts of the various fish represent milligram equiv-

alents of whole pituitary tissue.

» The assay was performed in two parts, utilizing all animal groups each time. Since the results for the corresponding groups

were very similar to each other in the two assays, the data were combined in the above table. Squalus acanthias (spiny
dogfish), Mustelus canis (smooth dogfish), Raja eglanteria (clear-nose skate).

© Significance of the difference between mean values in terms of p values of Fisher.

From Hayashida (1973), Gen. Comp. Endocrinol. 20:377, with permission of editors.

134 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

SKATE ID @ SKATE LT

&

> /

SQUALUS & PIVSTELUS /

FIGURE 5.

skate
SKaTe IL SQ Musrexus

SQUALUS SQUALUS

ste

Ouchterlony plates demonstrating the reaction of GH in pituitary extracts (PEs) of elasmobranchs with monkey

antiserum to rat GH. Antiserum (0.15 ml) is located in the center well. Each of the Squalus and Mustelus extracts represents
the extract from 1.5 mg of tissue, and the skate extract represents 2.0 mg of tissue in each case. All extracts in 50 ul of
phosphate-saline buffer. Photographed after 48 hours of immunodiffusion at room temperature. (A) RGH indicates highly
purified rat GH (2.7 USP units/mg, 7.5 wg in SO wl volume). Note clear reactions of partial identity with RGH shown by the
Squalus and Mustelus PEs (GHs). (B) Note reactions of qualitative identity between the PEs (GHs) of Mustelus and Squalus.
(From Hayashida 1973, Gen. Comp. Endocrinol. 20:377, by permission of the editors.)

daily injection of antiserum to rat GH, complete-
ly blocked the tibial plate stimulating activity of
both shark and skate PEs, indicating that this
biological activity of the elasmobranch PEs was
due to GH or a GH-like substance present in the
extracts, which was immunochemically related
to mammalian GH (rat).

Immunodiffusion studies (Fig. 5A, B) re-
vealed the presence of a substance in PEs of the
sharks which was partially related to rat GH,
although the skate PE did not show a detectable
precipitin line. The results of RIA (Fig. 6) with
these extracts indicated that the shark PEs con-
tain a GH or GH-like substance which shows a
low, but significant degree of relatedness to rat
GH, while the skate PE showed the weakest
cross-reaction as revealed by its even lesser
slope. Thus the results of RIA again correlated
well with those obtained by immunodiffusion.

The results of all of the studies reported thus
far have shown that PEs of all existing primitive
fishes which we have studied to date including
the African lungfish, elasmobranchs, and chon-
drosteans, are capable of significantly stimulat-
ing growth in the mammal on the basis of the rat
tibia assay, whereas the PEs of several different
species of teleosts were found to be incapable
of stimulation. Moreover, immunochemical

tests have indicated that the PEs (GHs) from the
above primitive fishes show a significant degree
of immunochemical relatedness to tetrapod GHs
while the PEs (GHs) of the several species of
teleosts studied, show little or no such related-
ness at least on the basis of the methods em-
ployed.

B. Holosteans

Holosteans would be of particular interest in
these studies since they are generally considered
to be intermediate in their level of development
toward the teleosts. Extracts were prepared
from the pituitaries of the bowfin (Amia calva)
and the gar (Lepisosteus spp.) which included
both long and short-nosed gars. Immunodiffu-
sion tests showed that PEs (GHs) of both of
these holosteans gave reactions of partial iden-
tity with rat GH, with that of the gar being slight-
ly weaker than the precipitin line due to the
bowfin. Both PEs, however, gave reactions of
identity with each other (Hayashida 1971). The
results of the rat tibia assay indicated that the
bowfin PE (GH) was capable of significant stim-
ulation in this assay although, perhaps, slightly
less active than that due to equivalent amounts
of PEs of the shark or sturgeon. Again, RIA
revealed that both holostean PEs (GHs) showed

HAYASHIDA: GROWTH HORMONE STUDIES

sg Pituitary Tissue

0 16 64

250 1000
lial

50t—
x e@
O
(B%
4 4—4 Skate
= e e
im 40 —~e Mustelus
[=a}
<
=
uw e Squalus
O b SS Mustelus
Squalus
O 30}—
Zz L
{a)
Za te
ray
—
7h. 20}—
Ww
U i= e
ao
jee)
é I Xe
10 ee
° RGH
@—»— 6 Standard
JRE
Ue Jay | 16 64 250 1000 4000 16006
mg RGH standard
FiGuRE 6. Radioimmunoassay with monkey antiserum to

rat GH (RGH) showing the relatedness of GH in pituitary
extracts of Squalus, Mustelus, and Raja (skate) to each other
and to RGH. Two different pituitary extracts are represented
for each species of shark. Note that the skate extract (GH)
shows the least cross-reaction indicated by its lower slope.
(From Hayashida 1973, Gen. Comp. Endocrinol. 20:377, by
permission of the editors.)

low but significant relatedness to rat GH with
their slopes approximating that of sturgeon PE
observed in previous studies.

IV. RECENT USE OF MONKEY ANTISERUM
TO TURTLE GH

In our more recent studies carried out in col-
laboration with Drs. Susan Farmer and Harold
Papkoff of the Hormone Research Laboratory
(Hayashida et al. 1975; Farmer et al. 1976b) it
was found that an antiserum prepared in the
monkey against GH from the snapping turtle
(Chelydra serpentina) representing a species
phylogenetically lower than the mammal, con-
siderably broadened the extent of cross-reactiv-
ities observed with the GH antiserum. To illus-
trate, in immunodiffusion studies GHs of
reptiles and birds now gave reactions of identity

135

or near identity with those of mammals (Fig. 7A,
B, C). However, GHs or PEs of species below
the reptilian level (turtle), continued to give re-
actions of partial identity in a “‘step-laddering”’
fashion as we had previously observed with the
use of antiserum to rat GH. For example, we
can see in Fig. 7D that sturgeon PE (GH) (Aci-
penser transmontanus) gave a reaction of partial
identity with turtle PE, and shark PE (Mustelus
canis) or purified shark GH (Prionace glauca)
(U. J. Lewis) in turn gave a reaction of partial
identity with GH in sturgeon PE. Bowfin (Amia
calva) PE also gave a reaction of partial identity
with turtle PE, while the PE of the teleost rep-
resented by the striped bass (Morone saxatilis)
was again negative.

Radioimmunoassay was performed to test
these and other PEs employing the turtle GH
serum and using rat GH as the labeled hormone
and for standards. The results were in complete
accord with those obtained by immunodiffusion.
All mammalian, reptilian and avian GHs and
PEs tested (Fig. 8A, B) gave slopes of inhibition
which were parallel to the rat GH standard,
while an amphibian GH preparation (bullfrog,
Rana catesbeiana) (Fig. 8B) gave a curve with
a lesser slope and PEs of the sturgeon and bow-
fin (Fig. 8A) gave curves of even lesser slope,
while the shark PE showed a still lesser slope.
A highly purified shark GH preparation (U. J.
Lewis) gave a slope that was essentially identi-
cal to that shown by the shark PE.

Both the monkey antiserum to snapping turtle
GH as well as the antiserum to shark GH were
found to be capable of completely neutralizing
the rat tibial plate stimulating activity of shark
PE (Hayashida and Lewis, unpublished obser-
vations).

V. STUDIES WITH COELACANTH AND
RATFISH PEs

In a recent investigation we had the oppor-
tunity to test the PE of a coelacanth (Latimeria
chalumnae Smith) in the rat tibia assay and de-
termine whether or not it contains a substance
immunochemically related to tetrapod GHs uti-
lizing monkey antiserum to turtle GH (Hayashi-
da 1977). The fish was a gift from the Comoran
government to the California Academy of Sci-
ence Coelacanth Expedition of 1975 and is iden-
tified as Latimeria No. 79, CAS 33111. It was
a relatively large male specimen which mea-
sured 110 cm in length and weighed 30 kilos.

136 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ALLIGHIOR2 *

@*~

FIGURE 7.

} PGT

!/ BGA e

°o.
o

\ SOHO. PE POSS: PE |

Immunochemical relatedness of purified somatotropins or somatotropins in pituitary extracts from various ver-

tebrate species based on immunodiffusion tests. Monkey antiturtle somatotropin serum (0.15 ml) is in the center well of each
agar plate. The amounts of pituitary somatotropin (GH) in (A) are 10 ug for snapping turtle (T); 10 wg each for bovine (B),
porcine (P), and whale (W); 7.5 wg for rat (R). The amounts in (B) are 10 wg each for T, B, P. The amounts in (C) are 40 pg
for monkey (M), 15 ug each for chicken and duck. The amount in (D) is 10 wg for shark (Prionace glauca) somatotropin. All
pituitary extracts (PE) are the equivalent of 2.0 mg wet tissue weight per well in all agar plates. All peripheral wells contain
antigen in a volume of 0.05 ml, except those in Fig. 7C, in which the volumes are 0.2 ml each. The plates were photographed
at 43-46 hour incubation, except Fig. 7A, at 24 hr. (From Hayashida et al. 1975, Proc. Nat. Acad. Sci. 72:4322, by permission

of the editors.)

The whole gland weighed 60 mg not including
the long, slender rostral extension described by
Lagios (1975).

The results of the rat tibia assay (Table 5) in-
dicated that the coelacanth PE was indeed ca-
pable of showing significant stimulation in this
assay. Only one dose level of the extract was
employed utilizing only 2 rats due to the short-
age of the precious material. Because of this,
the relative potency of the extract cannot be ex-

pressed in terms of the ovine GH used for stan-
dards. However, if we assume that the dose-re-
sponse for this PE were parallel to that of the
standard, had enough material been available,
the degree of response shown by the 5.0 mg dai-
ly dose of the PE would be equivalent to that
induced by about 5 to 6 ywg/day of ovine GH.
Immunodiffusion studies employing the mon-
key antiserum to turtle GH (Fig. 9) showed that
sturgeon PE gave a reaction of partial identity

HAYASHIDA: GROWTH HORMONE STUDIES 137

Microgram pitutary tissue

Os O66 25 O AO Ib @ 20
i T T T | yl |

60

=
B
A.
530) |=
ac SS
& 40h 2 S
e . = S
= 5
o 2 N Shark GH (Lewis)
Oo (Prionace glauca)
o) aN Zz
oye ® Shark PE a
[a) ® (Mustelus) Z
Zz fos
a be
g \ :
& O
U +— Monotreme PE p a
fs 2AdiF \ + (Echidna) Bowfin PE ne a 4 Amphibian GH
= (Amia) 4 (Bullfrog)
A Reptilian PE Ovine GH
(Alligator) O Sturgeon PE
+ (Acipenser) Bovine GH
Marsupial PE—> ‘\ ae
(Opossum) ~~ Mammalian GH — <+— Mammalian GH
(RGH Standard) (RGH Standard)
ee Nl Mt ya | al | | J Jie Se ieee = | J
OD5105alieeza 4 16 64 250 1000 4000 OWPHOS Ww 2! 16 64 250 1000 4000
Nanogram equivalent of RGH Nanograms of purified GH

FiGuRE 8. Radioimmunoassay with monkey antiserum to turtle somatotropin (GH) showing immunochemical relatedness
between purified somatotropins in pituitary extracts (PE) from various vertebrate species. Rat somatotropin (RGH), used as
the reference standard, competitively inhibited the binding of '*'I-labeled RGH as shown by the standard curve. (A) Results
obtained with pituitary extracts compared to the RGH standard. (B) results obtained with purified somatotropins compared to
the RGH standard. Note that all mammalian, reptilian, and avian somatotropins or pituitary extracts gave the steepest inhibition
slopes, which paralleled or nearly paralleled the RGH standard curve, and that somatotropins or pituitary extracts of an
amphibian and relatively primitive fishes gave curves with lesser slopes, which showed significant relatedness to those of the
phylogenetically higher species. Alligator pituitary extracts and shark somatotropin curves are represented by broken lines.
These preparations were run in other assays in which the total binding of labeled RGH and the slope of the RGH standard
curves were essentially identical to those shown here, and were therefore entered in this figure to illustrate their relative
positions and slopes with respect to the standard curves. (From Hayashida et al. 1975, Proc. Nat. Acad. Sci. 72:4322, by
permission of the editors.)

with turtle GH and the shark PE in turn gave a__ by the more sensitive method of RIA (Fig. 10),
similar type of reaction with sturgeon PE al- the coelacanth PE did show a low but significant
though weaker, while both coelacanth and lung- degree of relatedness to tetrapod GHs and a
fish PEs gave no detectable reaction. However, closer relatedness to sturgeon PE and a still

TABLE 5. TiBiAL PLATE STIMULATING EFFECT OF COELACANTH PITUITARY EXTRACT.#

Daily Animal Tibial plate width
Group dosage (no.) (um + SE) Significance”
1. Hypophysectomized controls = 8 193235-= S259
2. Ovine GH 7.0 wg 6 244.8 + 6.20 I vs. 2 p < 0.001
3. Ovine GH 14.0 pg 4 268.1 + 1.74
4. Ovine GH 28.0 wg 4 302.6 + 5.11
5. Coelacanth PE 5.0 mg 2 23329 = 1B ALO I vs. 5p < 0.01

4 The assay was performed in female Sprague-Dawley rats, hypophysectomized at 28 days and used beginning 9 days
postoperatively.

» Significance of difference between mean values in terms of p values of Fisher. From Hayashida (1977), Gen. Comp.
Endocrinol. 32:221, with permission of editors.

138 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

closer degree of relatedness to shark and lung-
fish PEs. Striped bass and ratfish (Hydrolagus
colliei) PEs showed only a very smali degree of
cross-reaction based on the relative flatness of
their curves with respect to those of tetrapods.

Ratfish PE has shown certain significant dif-
ferences from shark PE by both bioassay and by
immunochemical studies. When tested in the rat
tibia assay ratfish PE showed only a slight, al-
though statistically significant degree of stimu-
lation (Table 6), whereas in the case of shark
PEs (Squalus acanthias and Mustelus canis) it
was previously demonstrated that they are ca-
pable of showing greater stimulation at compa-
rable dosage levels of PE (refer to Table 4). In
immunodiffusion studies the ratfish PE did not
show a precipitin line with monkey antiserum to
rat GH or turtle GH, whereas the shark PEs
readily did so (Fig. 5). This correlates well with
the above findings based on RIA, in which rat-
fish PE showed only a small degree of immu-
nochemical relatedness to tetrapod GHs, much
less than that exhibited by shark PEs or purified
shark GH (Fig. 10).

GENERAL SUMMARY

1. In confirmation of earlier reports we have
found that PEs of several teleost species are
not capable of eliciting significant biological
activity in the mammal on the basis of the rat
tibia assay which is the most widely em-
ployed bioassay for detection and measure-
ment of pituitary growth hormone (GH).

FIGURE 9.
munodiffusion with the antiserum to turtle GH. The amounts
in each well (50 ul volume) are as follows: porcine and turtle
GH, 10 wg each; sturgeon PE, 1.0 mg; shark PE, 1.6 mg;
coelacanth and lungfish PEs, 2.0 mg each. Photographed after
48 hr of immunodiffusion. (From Hayashida 1977, Gen.
Comp. Endocrinol. 32:221, by permission of the editors.)

Ouchterlony plate showing the results of im-

2. In contrast to these findings, PEs of several
species of existing primitive fishes were ca-
pable of showing significant stimulation in the
rat tibia assay. These include PEs of the

TABLE 6. TIBIAL PLATE STIMULATING EFFECT OF PITUITARY EXTRACT FROM THE RATFISH.**”
Daily Animal Tibial plate width
Group dosage (no.) (um + SE) Significance‘
1. Hypophysectomized
controls — 11 Sie =e _

2. Bovine GH 5.0 ug 8 197.8 + 4.9 I vs. 2 p < 0.001
3. Bovine GH 10.0 ng 4 220.0 + 5.1 2 vs.3 p < 0.01
4. Bovine GH 20.0 ug 8 249.0 + 3.8 3 vs.4p < 0.01
5. Ratfish 0.6 mg 4 Wop) IL se 720)

6. Ratfish 1.2 mg 8 17O6)-=31

7. Ratfish 2.5 mg 8 Li Nee 220 I vs. 7 p < 0.001
8. Ratfish 3.7 mg 8 177.4 + 4.1 Il vs. 8p < 0.01

4 Ratfish (Hydrolagus colliei).

® This assay was performed in female Long-Evans rats, hypophysectomized at 27 days of age, and utilized beginning 8 days
postoperatively. The assay was conducted on two separate occasions. Since the results obtained in each of the corresponding
groups were quite similar, the data were combined in the above table.

© Significance of the difference between mean values in terms of p values of Fisher.

From Hayashida (1977), Gen. Comp. Endocrinol. 32:221, with permission of the editors.

HAYASHIDA: GROWTH HORMONE STUDIES

MICROGRAMS PITUITARY TISSUE
06395-25) O40 116) 64) 250
a T T T T =|

o
= g
oO 8 “N&
~ 40
<
oa B COELACANTH PE
a
w
=
a s
= 30+ a
=
= ; Ne
N
oO
Ze BOVINE GH
OQ 20- a B LUNGFISH PE
eZ
eS a
= SHARK PE
Zz STURGEON PE
si SN
& ob Is © BULLFROG GH
w ©
RAT GH STANDARD
(ae 4 = — sill

: am)
OSI 24 16 64 250 1000 4000

NANOGRAMS OF PURIFIED
GROWTH HORMONE (somatotropin)

139

MICROGRAMS PITUITARY TISSUE
063.25 10 40 16 64 250 1000
f T T Ts T Th T 1

509
© “ol 8 Pe RATFISH PES
e \ ..
< SS
[oa x
a 8 COELACANTH PE
=
a 30 i
<
=
N
© 20+
Zé
a
Ze
faa)
—
rq Oe
cS) NG
ira] ALLIGATOR PE
a ©.

Ne RATGH STANDARD
Jt} i's 1 4E L — a= =|
225) Selina 4 16 64 250 1000 4000 16000

NANOGRAMS OF PURIFIED
RAT GH (somatotropin)

FiGure 10. The immunochemical relatedness of a substance in pituitary extracts of the coelacanth and other existing primitive
fishes with respect to GH of tetrapods based on radioimmunoassay. The antiserum consisted of a monkey anti-turtle GH serum,
employed at a final dilution of 1:90,000. (From Hayashida 1977, Gen. Comp. Endocrinol. 32:221, by permission of the editors.)

chondrosteans, elasmobranchs, a dipnoan,
holosteans and the only surviving member of
the Crossopterygii, the coelacanth.

All of these PEs gave dose-response slopes
that were less steep than that due to mam-
malian GH standards, with the exception of
lungfish PE which gave a slope similar to
mammalian GH. No slope was established
for the coelacanth due to insufficient mate-
rial.

3. Immunochemical studies utilizing immuno-
diffusion in agar gel and RIA employing mon-
key antisera to rat GH or turtle GH have
demonstrated significant immunochemical
relatedness of GH or a GH-like substance in
the PEs of the primitive fishes, with respect
to GH of tetrapods.

Such a substance was demonstrated in the
PE of a single coelacanth by RIA employing
the antiserum to turtle GH. The results sug-
gested that it is more closely related to GH
or a GH-like substance in the pituitaries of
the African lungfish and the shark, than to
that of the sturgeon.

4. By both bioassay and immunochemical test-
ing ratfish PE (GH) appeared to show signif-
icant differences from shark PE (GH).

DISCUSSION

In the series of studies reported here both im-
munochemical and biological techniques have
been employed to examine the relatedness of
growth hormones (GHs) and pituitary extracts
(PEs) of existing primitive fishes and modern
bony fishes, with respect to highly purified pi-
tuitary GHs of tetrapods. The ability of GH or
a GH-like substance in the PEs of primitive fish-
es to stimulate growth in the mammal was clear-
ly demonstrated on the basis of the tibia assay
in hypophysectomized rats. These included PEs
of elasmobranchs, a dipnoan (African lungfish),
chondrosteans, as well as holosteans. A purified
preparation of shark GH has been recently
shown to be capable of significant stimulation in
this assay (Hayashida and Lewis, manuscript in
preparation). On the other hand PEs of several
modern bony fishes were all found to be incap-

140 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

able of showing such stimulation. However, a
highly purified GH from this group of fishes
(Tilapia) did show a small but significant degree
of stimulation in this assay, but only when a very
large dose was employed (Farmer et al. 1976a).
The demonstration that antisera to tetrapod GHs
(rat or turtle) are capable of completely neutral-
izing the rat tibial plate stimulating activity of
elasmobranch and sturgeon PEs strongly sug-
gests that such activity of the PEs from these
primitive fishes is due to a GH or GH-like sub-
stance that is immunochemically related to tet-
rapod GHs. The immunochemical studies in-
cluding the use of immunodiffusion and
radioimmunoassay, have indeed shown that
there is a substance in the PEs from primitive
fishes which is immunochemically related to
purified tetrapod GHs. This has proven to be
true for an elasmobranch in that PEs of the
shark, Mustelus canis, reacted in a manner
identical to that of a purified shark GH (Prionace
glauca) by both immunodiffusion and radioim-
munoassay (Hayashida et al. 1975).

The findings obtained thus far with PEs of
primitive fishes and modern bony fishes, have
suggested that there must be significant differ-
ences in the structure of GH from these two
groups of fishes. Although recent studies have
emphasized the notable similarities in molecular
weights, amino acid composition, etc., among
the GHs of several species of tetrapods (Farmer
et al. 1976b) and the purified GHs of a modern
bony fish (Tilapia) (Farmer et al. 1976a) as well
as that of the shark (Lewis et al. 1972), closer
examination of the amino acid composition data
reveal a number of significant differences as
well. For example, Tilapia GH contains signif-
icantly higher amounts of serine and significant-
ly lower amounts of methionine and phenylala-
nine than tetrapod GHs; and shark GH shows
that the percent composition of at least five out
of eighteen amino acid residues is more like that
of tetrapods than shown by Tilapia GH, while
in another five of the eighteen residues the ami-
no acid compositions are similar for shark, Ti-
lapia and tetrapod GHs. These findings are very
suggestive that structural sequence studies will
most likely reveal the existence of significant
differences between GHs of modern bony fishes
and those of existing primitive fishes and that
the GHs from the latter group of fishes will re-
semble more closely the GH of tetrapods. The
confirmation of these speculations must await

the results of amino acid sequence studies which
are now only in the preliminary stages. Only
when more information becomes available in
this area of research can we be in a position to
learn more about the determinants responsible
for immunochemical and biological relatedness.

There are some interesting observations con-
cerning relatedness among the PEs of the prim-
itive fishes. Although shark and ratfish (Hydro-
lagus colliei) are classified as members of the
Chondrichthyes, the results of the present stud-
ies suggest that these are two quite separate
groups. For example, GH in shark PEs could be
readily demonstrated by the immunodiffusion
technique, employing either antiserum to rat GH
or turtle GH, while precipitin lines were never
detected with ratfish PEs even at higher concen-
trations. Ratfish PEs did show a slight cross-re-
action by the more sensitive radioimmunoassay
procedure, while shark PEs as well as purifed
shark GH, showed a partial but substantial de-
gree of cross-reaction (Fig. 8A, B). Moreover,
the results of the rat tibia bioassay revealed that
elasmobranch PEs including two species of
shark and the clear-nosed skate (Raja eglanter-
ia) are capable of substantial stimulation al-
though the dose-response slopes were very low,
while ratfish PE at similar dose levels showed
only a slight degree of stimulation and no dose-
response slope.

As far as the coelacanth PE is concerned the
immunochemical findings suggest that the GH
or GH-like substance in this PE is more closely
related to GH in lungfish and shark PEs than it
is to sturgeon PE (GH), and that it is more
closely related to sturgeon PE (GH) than it is to
the GHs of tetrapods. The results of the bioas-
say indicate that coelacanth PE (GH) was defi-
nitely capable of stimulating the growth of the
rat tibial plate, similar to the results obtained
with PEs of the lungfish, shark and sturgeon.
Thus immunochemically and biologically one
can state that the GH in PEs of existing primitive
fishes, including the coelacanth, lungfish, shark
and sturgeon, and extending to the holosteans
as well, are more closely related to GH of tet-
rapods than are the GHs of modern bony fishes.
These findings provide additional evidence that
the former group of fishes are less divergent
from the main line of vertebrate evolution lead-
ing to the tetrapods, than are the modern bony
fishes which represent by far the most numerous
of living vertebrates today.

HAYASHIDA: GROWTH HORMONE STUDIES

LITERATURE CITED

FARMER, S. W., H. PAPKOFF, AND T. HAYASHIDA. 1976a.
Purification and properties of teleost growth hormone. Gen.
Comp. Endocrinol. 30(1):91—190.

F , AND 1976b. Purification and prop-
erties of reptilian and amphibian growth hormones. Endo-
crinology 99(3):692—700.

GESCHWIND, I. I. 1967. Molecular variation and possible
lines of vertebrate evolution of peptide and protein hor-
mones. Am. Zool. 7:89—108.

GREENSPAN, F. S., C. H. Li, M. E. SIMpson, AND H. M.
Evans. 1949. Bioassay of hypophyseal growth hormones:
the tibia test. Endocrinology 45(5):455—463.

GREENWooD, F. C., W. M. HUNTER, AND J. S. GLOVER.
1963. The preparation of '*!-labeled human growth hormone
of high specific radioactivity. Biochem. J. 89(1):114—123.

HAYASHIDA, T. 1969. Relatedness of pituitary growth hor-
mone from various vertebrate classes. Nature (London)
222(5190):294-295.

1970. Immunological studies with rat pituitary

growth hormone (RGH). II. Comparative immunochemical

investigation of GH from representatives of various verte-
brate classes with monkey antiserum to RGH. Gen. Comp.

Endocrinol. 15(3):432-452.

1971. Biological and immunochemical studies with

growth hormone in pituitary extracts of holostean and chon-

drostean fishes. Gen. Comp. Endocrinol. 17(2):275—280.

1973. Biological and immunochemical studies with

growth hormone in pituitary extracts of elasmobranchs.

Gen. Comp. Endocrinol. 20(2):377-385.

1977. Immunochemical and biological studies with

growth hormone in a pituitary extract of the coelacanth,

141

Latimeria chalumnae Smith. Gen. Comp. Endocrinol.
32(2):22 1-229.

, AND A. N. CoNntopouLos. 1967. Immunological
studies with rat pituitary growth hormone. Gen. Comp. En-
docrinol. 9(2):217—226.

, S. W. FARMER, AND H. Papkorr. 1975. Pituitary
growth hormone: Further evidence for evolutionary con-
servatism based on immunochemical studies. Proc. Nat.
Acad. Sci. 72(11):4322-4326.

, AND M. LaGios. 1969. Fish growth hormone: A bi-
ological, immunochemical, and ultrastructural study of stur-
geon and paddlefish pituitaries. Gen. Comp. Endocrinol.
13(3):403-411.

, AND C. H. Li. 1958a. Immunological investiga-
tions on bovine pituitary growth hormone. Endocrinology
63(4):487-497.

, AND

1958b. An immunological investigation
of human pituitary growth hormone. Science 128:1276—
1277.

, AND

1959. A comparative immunological
study of pituitary growth hormone from various species.
Endocrinology 65(6):944—956.

KNoBIL, E., AND J. HOTCHKISS.
Annu. Rey. Physiol. 26:47-74.

Lacios, M. D. 1975. The pituitary gland of the coelacanth
Latimeria chalumnae Smith. Gen. Comp. Endocrinol.
25(2): 126-146.

Lewis, U. J., R. N. P. SINGH, B. K. SEAvey, R. LASKER,
AND G. PICKFORD. 1972. Growth hormone- and prolactin-
like proteins of the blue shark (Prionace glauca). Fish. Bull.
70(3):933-939.

OUCHTERLONY, O. 1953. Antigen-antibody reactions in gels:
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1964. Growth hormone.

OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 18 pages

December 22, 1979

EVOLUTION OF THE CREATINE KINASE ISOZYME SYSTEM
IN THE PRIMITIVE VERTEBRATES

By

Suzanne E. Fisher and Gregory S. Whitt!

Department of Genetics and Development,

University of Illinois,
Urbana, Illinois 61801

ASTRACT

The number of creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential

tissue expressions were determined for species in 72 families in the groups Urochordata,
Cephalochordata, Petromyzontiformes, Elasmobranchiomorphi, Crossopterygii, Dip-
noi, Chondrostei, Holostei, Teleostei, Amphibia, Reptilia, and Mammalia. Urochor-
dates have a single creatine kinase locus. The appearance of the second CK locus in
the cephalochordates correlated with a reported threefold higher DNA content in these
species compared to certain urochordate species (Atkins and Ohno 1967). All non-
teleostean fishes examined have two creatine kinase loci (A and C). The differential
patterns of expression of these loci among specific tissues vary greatly among these
fishes. The Amphibia and Reptilia also have two homologous CK loci which are also
expressed in a variety of tissues. In the successful and diverse teleost fishes a very
different strategy of CK gene evolution and isozyme expression has been followed. As
many as four functional CK loci are present and the differential expression of these

loci has been increasingly restricted to specific cell types.

INTRODUCTION

There are many approaches to the study of
the relationships of the primitive vertebrates.
Investigations based on morphological elements
can encompass both fossil and extant forms. In-
vestigations of embryology may reveal relation-

' Please address all correspondence concerning this manu-
script and reprint requests to G.S.W.

142

ships that are not obvious from a study of adult
morphology; one example is the many similari-
ties of cephalochordates and the ammocoete lar-
va of cyclostomes (Romer 1966). Although a
comparative biochemical approach is limited to
studying extant species, it can be valuable in
resolving questions about the systematics of
higher taxa (Zuckerkandl and Pauling 1965).
Within the primitive vertebrates there are many
examples of once species-rich groups being rep-

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 143

resented today by one or a few species. In many
cases, and certainly the coelacanth is a prime
example, the extant species are morphologically
very similar to their ancestors. Much of the
work reported in this paper is based on the
premise that where there is considerable struc-
tural similarity between ancient and modern
groups, it is reasonable to expect some conser-
vation of the ancient genome structure and reg-
ulation as well. Specifically, we are assuming
that the number of structural genes, their ho-
mologies, and the patterns of regulation of these
genes reflect the ancestral condition in much the
same way extant species reflect the ancestral
morphologies and life histories. Of course mo-
lecular divergence has occurred through time
because of the inevitable incremental changes in
nucleotide sequences resulting in amino acid
substitutions in the corresponding proteins
(Langely and Fitch 1974; Wilson et al. 1977).
While it is not possible to directly determine the
biochemical attributes of such extinct ancestral
forms as the placoderm or acanthodian fishes,
the existence of relic species such as the bowfin,
lungfish, and coelacanth provide a unique op-
portunity to make phylogenetic inferences about
the origins of once numerous taxa.

Although the functional macromolecules in
the earliest period of molecular evolution were
presumably formed by fortuitous and random
combinations of smaller units, subsequent mo-
lecular and cellular evolution is thought to have
been more constrained and to have proceeded
by modifications of pre-existing, functionally
specific genes. The evolution of a given gene to
a new function might be constrained if the older
but also essential functions must be retained.
This dilemma can be avoided by the formation
of duplicate copies of the gene. The concept of
evolution by gene duplication has been devel-
oped by Susumu Ohno (1967, 1970, 1974) and
others (Watts and Watts 1968a, 1968b; Watts,
R. L. 1971). Most simply stated, Ohno has pro-
posed that the major advance to the vertebrate
grade must have required a large increase in ge-
nome size. Additional copies of structural genes
and regulatory elements were then free to accept
mutations and to evolve to new functions. Ohno
(1974) has argued that tandem duplications fre-
quently produce additional copies of structural
genes that are under the control of the same reg-
ulatory element, which makes divergence in
these duplicate loci more difficult. [However,

there is evidence that two closely linked paral-
ogous loci such as the Ldh B and C genes in
birds (Zinkham et al. 1969) can be under quite
separate control.| For this reason as well as evi-
dence derived from measurements of DNA con-
tent and examination of karyotypes, Ohno has
proposed that the amplification of the genome
that preceded the radiation of the vertebrates
was a polyploidization event.

The fact that cephalochordates have haploid
DNA contents (Amphioxus lanceolatus 0.60 pg)
three times higher than certain tunicate species
(Ciona intestinalis 0.21 pg) provides consider-
able evidence for at least one major genome am-
plification event early in the evolution of the
chordate line (Atkin and Ohno 1967). In addi-
tion, in several cases among the vertebrates the
loci encoding homologous polypeptides are lo-
cated on separate chromosomes (Kucherlaptai
et al. 1974; Wheat et al. 1972, 1973; Whitt et al.
1976). These observations are consistent with
the postulate that a polyploidization event oc-
curred early in chordate evolution. Obviously
most extant species subsequently have under-
gone extensive diploidization. The alternative
interpretation, that these homologous loci were
originally formed by tandem duplications and
have since been placed on separate chromo-
somes through chromosomal rearrangements, 1s
unlikely in view of the karyotypic conservation
observed in many lower vertebrates (Ohno 1974;
Morizot et al. 1977; Bush et al. 1977).

Isozymes [multiple molecular forms of the
same enzyme (Markert and Moller, 1959)], have
proved to be powerful tools for studying genome
evolution. Those isozyme systems which are the
products of homologous gene loci are well suited
for studying the evolution of gene structure,
function, and regulation (Markert 1975; Markert
et al. 1975; Whitt et al. 1975; Fisher et al. 1976).
A phylogenetic survey of a multilocus isozyme
system allows a partial reconstruction of the
probable times of the gene duplication events
and the inferred course of their regulatory di-
vergence. The significance of evolutionary
changes in gene regulation is only starting to be
realized. Most vertebrates presumably possess
similar arrays of structural genes; the differ-
ences between species of vertebrates therefore
appears to be primarily due to changes in the
timing and the level of expression of these genes
during development. Subtle changes in gene reg-
ulation can lead to dramatic changes in mor-

144 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

phology (Wilson et al. 1974a, 1974b, 1977; King
and Wilson 1975). Monitoring the tissue speci-
ficity of expression of homologous isozyme loci
through phylogeny is a simple and direct method
of ascertaining the probable sequential changes
in patterns of gene regulation during the course
of evolution.

The most extensively studied multilocus iso-
zyme system is lactate dehydrogenase (LDH,
EC 1.1.1.27). A survey of LDH gene expression
in the fishes revealed an increase from one to
three loci and considerable increases in the spa-
tial and temporal specificity of gene expression
(Horowitz and Whitt 1972; Shaklee et al. 1973;
Whitt et al. 1973, 1975; Markert et al. 1975). A
few families of teleosts (Salmonidae, Catostom-
idae) have undergone a more recent genome am-
plification (tetraploidization) which has resulted
in the reduplication of the three LDH loci as
well as all other loci (Allendorf et al. 1975; Engle
et al. 1975; Ferris and Whitt 1977). The pattern
of gene evolution that is inferred from the stud-
ies of LDH is that the structure and expression
of a duplicate locus is initially identical to the
expression of the locus from which it arose.
Through time, mutations are accepted at both
the structural genes and the DNA regulatory se-
quences controlling them, resulting in a differ-
ential regulation of the duplicated loci.

A more limited survey of glucosephosphate
isomerase (GPI, EC 5.3.1.9) gene expression in
the bony fishes showed that a gene duplication
event probably occurred early in that line and
that the higher teleosts have evolved a tissue
specific pattern of expression of the two GPI
genes (Avise and Kitto 1973).

The creatine kinase (CK, EC 2.7.3.2) multi-
locus isozyme system provides an excellent op-
portunity to study the evolution of homologous
structural genes and the evolution of gene reg-
ulation. Creatine kinase is a dimeric enzyme
with a molecular weight of 80,000 daltons. It cat-
alyzes the readily reversible conversion of ATP
and creatine to ADP and phosphocreatine (Daw-
son et al. 1967; Masters and Holmes 1975). Con-
sequently it is very important in maintaining en-
ergy balance in a variety of cells. There is recent
evidence that certain CK isozymes may fulfill
roles in cell structure as well as catalytic roles
(Turner et al. 1973; Eppenberger et al. 1975).
Three CK loci are expressed in mammals; two
loci (which in the past have been called M and
B) encode isozymes localized in the cytosol and

an additional locus which encodes an isozyme
localized in the mitochondria (Masters and
Holmes 1975). As many as four CK loci have
been detected in the advanced teleost fishes
(Eppenberger et al. 1971; Perriard et al. 1972;
Scholl and Eppenberger 1972, Champion et al.
1975; Champion and Whitt 1976; Shaklee et al.
1975; Fisher and Whitt 1975, 1977).

The CK system is valuable not only because
of the proliferation of isozyme loci during the
evolution of the fishes and the evolution of high-
ly specific patterns of gene expression, but also
because the origins of this enzyme can be traced
back to a different but related enzyme in the
invertebrates. There are clear homologies be-
tween the creatine kinase isozymes of chordates
and the phosphagen kinases of invertebrates.
Watts and others (Robin 1974; Watts, D. C.
1968, 1971, 1975) have reported that inverte-
brates utilize a variety of phosphagen kinases,
the most common enzyme being arginine kinase
(ARG K, EC 2.7.3.3). Coelenterata, Platyhel-
minthes, and Arthropoda have monomeric ar-
ginine kinases. Monomeric and dimeric arginine
kinases are found in different species of mol-
luscs. Dimeric creatine and arginine kinase iso-
zymes are found in the echinoderms and uro-
chordates (Virden and Watts 1966; Moreland et
al. 1967). In the echinoderms there is an evolu-
tionary progression from some species having
only arginine kinase, to those having both argi-
nine kinase and creatine kinase, to those having
only creatine kinase. The active site peptides of
creatine and arginine kinase are very similar
(Watts, D. C. 1971). Immunological similarities
have been reported between denatured forms of
creatine kinase and a monomeric arginine kinase
(Robin et al. 1976). Furthermore, it is possible
through reversible in vitro denaturation to form
a hybrid protein containing a functioning echi-
noderm arginine kinase subunit and a function-
ing mammalian creatine kinase subunit (Watts
et.al. 1972):

The present paper is a partial report on a sur-
vey of creatine kinase gene expression in 72
families from the echinoderms through mam-
mals concentrating, however, on the phylogeny
of expression of CK genes in relatively **primi-
tive’’ groups of fish—Petromyzontiformes,
Elasmobranchiomorphi, Crossopterygii, Dipnoi,
Chondrostei, and Holostei. Within these groups,
many of which are represented today by a single
or a few species, there is evidence of consider-

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 145

able diversity in the patterns of tissue expression
of the two creatine kinase loci among the adult
tissues. In the two main evolutionary lines de-
rived from these primitive fishes—the teleost
fishes and the land vertebrates—two very dif-
ferent strategies of creatine kinase gene prolif-
eration and expression have been observed.

MATERIALS AND METHODS
Nomenclature

ithe icreatme kinase (CK, EC 2-7-3.2) iso-
zymes and the corresponding CK loci have been
named according to the rules of the IUPAC-IUB
Commission on Biological Nomenclature (1973)
and previous citations in the literature (Cham-
pion and Whitt 1976). The alphabetical desig-
nations A, B, C, and D reflect the order in which
the loci have been described and not the evo-
lutionary order of appearance of the four iso-
zyme loci.

Preparation of Tissue Extracts

All fish used in this study were obtained as
fresh or frozen specimens and stored at —20°C
until use. Whenever possible ten tissues were
examined: white skeletal muscle, heart, eye,
brain, stomach muscle, gill, liver, spleen, gonad
and kidney. The dissected tissue samples were
homogenized in an equal volume of 0.1 M so-
dium phosphate buffer, pH 7.0, using a motor-
driven Potter Elvehjem homogenizer. The ho-
mogenates were centrifuged at 12,000 ¢ for 30
minutes at 4°C and the resulting supernatants
recentrifuged at 27,000 g for 30 minutes at 4°C.
The final supernatants were used for electropho-
resis.

Electrophoresis

Vertical starch gel electrophoresis (Buchler
Instruments Inc., Fort Lee, New Jersey) was
performed at 6°C with 15% starch gels (Elec-
trostarch lots 371, 302, and 307, Electrostarch
Co., Madison, Wisconsin).

The buffer system used was the pH 8.6
EDTA-boric acid-Tris (EBT) buffer system de-
scribed by Shaklee et al. (1973). 2,000 units of
sodium heparin were added to each gel. Elec-
trophoresis was performed at 250 volts for 16—
24 hours.

Histochemical Staining

After electrophoresis the gels were sliced
lengthwise and stained according to Shaw and

Prasad (1970). One slice was stained using all
the reagents for creatine kinase and a second
slice from the same gel was stained using all the
staining reagents except the substrate phospho-
creatine. The omission of phosphocreatine was
employed as a control to detect the presence of
adenylate kinase (AK, EC 2.7.4.3) activity
which appears as an artifact of the creatine ki-
nase staining procedure. All staining reagents
were obtained from Sigma Chemical Company,
St. Louis, Missouri.

Purification

The details of the purification of the individual
CK isozyme will be published elsewhere (Fisher
and Whitt, in preparation). Three homodimeric
isozymes (A,, B, and C,) of the green sunfish
(Lepomis cyanellus) and the A, isozyme of the
spotted gar (Lepisosteus oculatus) were purified
utilizing gel filtration on Sephacryl S-200, affin-
ity chromatography on Blue Sepharose 6B
(Easterday and Easterday 1973), and prepara-
tive starch gel electrophoresis.

Preparation and Use of Antibodies

Antibodies were formed against the four pur-
ified creatine kinase isozymes in adult male New
Zealand white rabbits according to the following
schedule: on day 0 purified CK in 0.5 ml of 0.1
M sodium phosphate pH 7.0 buffer was emul-
sified in 1.0 ml Freund’s complete adjuvant and
then injected into the footpads. Depending on
the isozyme used as antigen individual rabbits
were injected with as little as 20 wg up to as
much as 1,000 wg. On day 10 the same amount
of antigen as initially employed was emulsified
in 1.0 ml Freund’s incomplete adjuvant and in-
jected into the footpads. On day 17 the same
amount of antigen dissolved in buffer was in-
jected without adjuvant into the thigh and scap-
ular muscles. Blood was collected from the mar-
ginal ear vein twice a week on a continuous basis
starting on day 20 and continuing for about 8
weeks. The antisera were titred according to the
enzymatic equivalence point technique de-
scribed by Markert and Holmes (1969). The as-
say system for creatine kinase was that of Oliver
(1955). All enzyme activity assays were per-
formed in duplicate on a Beckman Kintrac VII
spectrophotometer. Controls for adenylate ki-
nase were carried out by omitting phosphocrea-
tine. The antisera were concentrated by precip-
itation with 33% ammonium sulfate.

146 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

PURPLE SEA URCHIN
(Arbacia punctulata )

ARGININE KINASE
®

Arg K C Aad -e

FiGureE |. Cross reaction of the single arginine kinase of
the purple sea urchin, Arbacia punctulata with antiserum
formed against the A, creatine kinase isozyme of green sun-
fish, Lepomis cyanellus. This result is consistent with the pos-
tulated origin of creatine kinase from a duplicated arginine
kinase locus.

The antibodies were used in the immunopre-
cipitation plus electrophoresis technique devel-
oped by Markert and Holmes (1969). Increasing
amounts of antisera were added to aliquots of a
given enzyme extract. The enzyme-antibody
mixtures were incubated for 2 hr at 4°C, then
centrifuged to remove precipitable antibody-en-
zyme complexes. The supernatants were elec-
trophoresed to remove any remaining enzyme-
antibody complexes through the molecular siev-

CREATINE KINASE
LEATHERY SEA SQUIRT
(Styela plicata)

Origin
Be ae Os oe owe C. x = 2%
7/0 359900 25 85%055%0
Of0K AK Se «KT Ko

FiGurReE 2. Reaction of anti CK A,, and anti CK B,, and
anti CK C, sera (of similar titres) with the single creatine ki-
nase of Styela plicata. Only the anti A, serum cross-reacts,
which suggests that the first creatine kinase of the chordates
is homologous to the A, isozyme of teleosts.

ing action of the starch gel. Electrophoresis and
histochemical staining were performed in the
usual manner. The reduction of staining inten-
sity on the gel as a result of incubation with
increasing antibody concentrations is an indi-
cation of cross-reactivity. Controls for dilution
of enzyme activity by antiserum addition were
carried out with normal (pre-immunization) se-
rum. The high level of sensitivity of this immu-
noprecipitation and electrophoresis technique
has been documented and discussed elsewhere
(Holmes and Scopes 1974).

RESULTS

Patterns of differential creatine kinase gene
expression were analyzed for over 100 species
in 72 families of chordates. Evidence for the ho-
mology of the arginine kinase of echinoderms
and the creatine kinase of vertebrates is provid-
ed in Figure |. Antiserum formed against the
purified CK A, isozyme of green sunfish clearly
cross-reacted with the single arginine kinase of

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 147

KINASE ISOZYMES OF THE SPOTTED GAR
(Lepistosteus oculatus )

CREATINE KINASE

.

®

ee

Origin —» ee ee CO

Y Qo, oO “yy dg el og
S.9,e8 Y S

Wile >» ONCA6O.F
47 7? % an ek

FIGURE 3.

ADENYLATE KINASE

e
Sai
ve

eo a

Creatine kinase isozymes of the tissues of the spotted gar (Lepisosteus oculatus). Any bands or smears appearing

on both gels are adenylate kinase isozymes. Only the two bands unique to the CK zymogram are creatine kinase isozymes.

the purple sea urchin (Arbacia punctulata).
Equal volumes of sea urchin enzyme extract
were mixed with two levels of antibodies. A di-
lution control with normal serum was also per-
formed for the highest amount of antiserum
added. A comparison of the results in the third
(SO ul anti CK A,) and fourth (50 yl normal se-
rum) slots reveals that the anti CK A, serum
precipitates the arginine kinase. A single argi-
nine kinase was also detected in the striped sea
cucumber (Thyonella gemnata) and the red-
footed sea cucumber (Pentacta pygmaea). The
arginine kinase of the sea cucumbers did not
clearly cross-react with the antisera formed
against any of the creatine kinase isozymes of
the sunfish.

The urochordates are considered to be the
most primitive members of the chordate line.
Hemichordates are currently considered a sep-
arate phylum that has affinities with both the

chordates and echinoderms (Kluge 1977). The
urochordate Styela plicata (leathery sea squirt)
expresses a single CK locus (Fig. 2). When anti-
sera (of similar titres) formed against each of the
A,. B,, and C, isozymes of the green sunfish
were separately mixed with the tunicate creatine
kinase, only the anti CK A, serum cross-reacted
with this single creatine kinase. These results
suggest that the A locus of higher fishes still re-
tains considerable structural homology with the
original creatine kinase locus of chordates. The
tunicates and perciform fishes last shared a com-
mon ancestor more than 500 million years ago
and considerable caution is necessary when in-
terpreting the specificity of immunochemical re-
actions over such large evolutionary distances.

The cephalochordates are the most primitive
taxon in which the expression of two CK loci
has been detected. After histochemical staining
of the starch gels three bands of CK activity,

148 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

KINASE /SOZYMES OF THE COEWACANTH
(Latimeria chalumnae/

CREATINE KINASE

FIGURE 4.

ADENYLATE KINASE

CREATINE KINASE
Cs)

AK- — Hy *

Creatine kinase isozymes of the tissues of the coelacanth (Latimeria chalumnae). The two zymograms on the

left show the creatine kinase isozymes (and the adenylate kinase control) in the tissues examined. The bands slightly anodal
to the main CK band in skeletal muscle are sub-bands or conformational isozymes. The zymogram on the right shows that
antiserum formed against the CK-A, isozyme of green sunfish clearly cross-reacts with the CK of coelacanth skeletal muscle.

indicating the presence of two homodimers and
a heterodimer of intermediate electrophoretic
mobility, were detected for each individual of
amphioxus (Branchiostoma floridae) analyzed.
Immunological reactions indicate that one locus
is more closely related to the A locus of higher
fishes and the second locus is more closely re-
lated to the C locus of higher fishes. Thus, it
appears that the first two creatine kinase loci
were those which have evolved to the A and C
loci of perciform fishes. The alphabetical des-
ignations of the CK isozymes and the corre-
sponding loci were made before the evolutionary
history of this isozyme system was known.
Since the specimens of amphioxus we analyzed
were quite small (less than 2 cm in length) they
were homogenized whole and we cannot make
a definitive statement on the tissue patterns of
gene expression in this group. However, there

was considerably more CK A, activity than CK
CF

Analyses of CK isozyme activity among the
tissues of fish in the groups Petromyzonti-
formes, Elasmobranchiomorphi, Crossoptery-
gil, Dipnoi, Chondrostei, and Holostei indicate
that two creatine kinase loci are exhibited in all
species examined. However, there is consider-
able variation among species in the patterns of
expression of the A and C loci among the var-
ious tissues. The creatine kinase isozymes of the
spotted gar (Lepisosteus oculatus) are shown in
Figure 3. The staining activity seen in both gels
stained for creatine kinase and adenylate kinase
represents the adenylate kinase isozymes. Ad-
enylate kinase is not a phosphagen kinase and
is not directly related to creatine kinase or ar-
ginine kinase. Adenylate kinase catalyzes the
conversion of 2 ADP molecules to ATP and

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 149

AMP and its presence results in extra stained
bands on the gel stained for CK purely as an
artifact of the staining procedure used. Only the
bands unique to the creatine kinase zymogram
are in fact creatine kinase isozymes. Of these
two creatine kinases in the gar, the A, isozyme
exhibits the greatest activity in skeletal muscle
and to a lesser extent in kidney, liver, heart,
eye, and gills. The C, isozyme activity was de-
tected predominantly in stomach muscle and go-
nad and to a lesser extent in eye, brain and kid-
ney.

A zymogram of the creatine kinase isozymes
of the coelacanth tissues (Latimeria chalumnae
#79) is shown in Figure 4. Although the stan-
dard set of ten tissues we examined for other
species was not available, it is clear that the
coelacanth has at least two creatine kinase loci.
The bands slightly anodal to the main band in
skeletal muscle are sub-bands or conformational
variants; they have previously been reported for
the coelacanth (Hamoir et al. 1973) and are ob-
served in many other species. It is difficult to
interpret the lack of creatine kinase activity in
gill, liver, testis, and kidney. It may be that the
creatine kinase isozymes of the coelacanth are
labile to heat and/or cold storage and that the
CK activity was detected only in those tissues
which initially had very high levels of the en-
zyme. Alternatively, certain tissues of this fish
may possess low or no levels of CK in vivo.
However, in most other species we have found
that most tissues have detectable levels of at
least one of the CK isozymes. The zymogram
to the right in Figure 4 shows that the antiserum
formed against the CK A, isozyme of green sun-
fish cross-reacted with the CK isozyme of coel-
acanth skeletal muscle. Antisera formed against
the CK C, and B, isozymes of green sunfish do
not cross-react with the CK isozyme of coel-
acanth muscle, which by inference is the A, CK.

A summary of our data on the tissue specific
patterns of expression of the CK loci in different
taxa of fishes which express two creatine kinase
loci is in Table 1. This table documents the di-
versity in the patterns of gene expression which
exists among species in the primitive fishes.
There is not a consistent relationship of relative
electrophoretic mobilities of the C, and A, iso-
zymes. In the sea lamprey (Petromyzon mari-
nus), pallid sturgeon (Scaphirhynchus albus)
and paddlefish (Polyodon spathula) the C, iso-
zyme is highly anodal. In the gar (Lepisosteus)

and bowfin (Amia calva) the C, isozyme is
slightly more anodal than the A, isozyme. In
some sharks and rays, as well as coelacanth,
lungfish (Lepidosiren paradoxa) and reedfish
(Calamoichthys calabaricus) the A, isozyme is
more anodal than the C, under our electropho-
retic conditions. The tissue specificity of expres-
sion of the two loci varies considerably among
species. The only constant feature of CK gene
expression which emerges for all species is that
the only isozyme detected in skeletal muscle is
the A,. In some chondrosteans (sturgeon and
paddlefish) the A, isozyme is detected only in
skeletal muscle and not in other tissues. In the
reedfish, which is classified in a separate order
in the same division, the A, isozyme is present
in highest levels in the skeletal muscle but can
also be detected in all other tissues examined.
Similarly, the C, isozyme in some species can
also be quite restricted in its expression among
the adult tissues. For example, the C, isozyme
is detected only in heart and brain in the lung-
fish. In other species such as the sturgeon and
gar, the C, is detected in many tissues. The tis-
sues in which the C, isozyme activity is most
commonly found are eye, brain, stomach and
kidney. The conclusion we draw from the data
presented in Table 1 is that there is no single
characteristic pattern of expression of the two
creatine kinase loci in the primitive fishes.

Although there is some debate as to the exact
ancestral taxa involved it is generally accepted
that two separate lines—the amphibians and the
teleost fishes—had their origins in ancestors of
fish which we have shown currently to possess
two creatine kinase loci. However, the number
of CK loci and patterns of CK gene expression
among adult tissues are very different between
these two lines.

We have examined the creatine kinase iso-
zyme patterns of eight amphibian species; Am-
bystoma tigrinum, Amphiuma means, Bufo

americanus, Bufo marinus, Necturus maculo-
sus, Rana pipiens, Triturus viridescens, and
Xenopus laevis. A typical tissue specific cre-
atine kinase pattern for amphibian tissues is
shown in Figure 5. The CK isozyme pattern of
the marine toad (Bufo marinus) indicates that
two loci are present. It is probable that the single
locus expressed in skeletal muscle is homolo-
gous to the A locus of fishes. For the sake of
simplicity we will refer to the second locus as
the C locus, even though its orthology with the

OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

150

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+ 7 - — +++
++ +4 +4 + +44
++ ++ ++ + F+4+4
+ + 7 + +44
++ = ~ - +

+ + + + +++
+ ++ + — +++
= - — ++
++ + + + +
++ + + +
++ = + + +++
. - Se ee
YI dg Wah tS) WS

sndo] D Jo AWANOYy

++

++

++

++

+++

Aq

Cis

*POUIWIeXd JON
quasqy —
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“AWANIB YBIH + + +

AWIGOW [epouy aaneeay WV

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SOULIOJIWY
Brees ate = ae J + ae site ak at dete +++ YW<<9O snuojsojvjd snajsosida]
eer te = Je it au de + Sets = dels Jit ae V<)O SNASSO SNIISOSIdAT
aL = 43 aL ee ae ae a Ete A ++ +++ YW<9<O SHIDINIO SNasosidaT
SOWIOJNOUOIWIAS
NALSOTOH
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souojiiaid A]og
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+4 ie +++ W<9Q snq]p snyoudayalydvoy
SOWOJLIgsuadIoy
IH LSOYANOHO
ES eet sents ab - = ++ +++ 9O<YV pxoppvavd uasisopidaT
SOWOFJIUDIISOpIdaT
IONdIG
= = = = = +++ 9J<V ADUUN]DYO DIUIUNDT
SOWIOJIYJURIL[IOD
HWOAAALdAOSSOUO
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SOWIOJNRQOIAW
= 4 ft ae aL aT +++ Q<V UDUADS DIDY
SoUOsIey
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SOWAIOJIUIPId4o [,
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SOWIOJIUIYIBY IIe
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snso] Y Jo AVANOY

“HSI, NVALSOA TALNON AO SANSSIL NI lOO] ASVNIM ANILVAYND OM] AO NOISSHYdXA *] AIEAV

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 151

KINASE ISOZYMES OF THE MARINE TOAD
(Bufo marinus)

CREATINE KINASE

o**

®

ADENYLATE KINASE

sO
ADC 0, % "Sesser One ‘on 2
Or% 1 2 oP ee ae D In? 4-8, FS
Ye” ae “7 Cf ra
FiGuReE 5. Creatine kinase isozymes of the tissues of the marine toad (Bufo marinus). Only in heart are both loci expressed

and consequently a heterodimer is found.

C locus of fishes has yet to be definitively de-
cided. The antibodies formed against the puri-
fied CK isozymes of the green sunfish cross-re-
acted in a somewhat ambiguous fashion with the
amphibian CK isozymes. Both the anti A, and
anti C, sera precipitate both amphibian iso-
zymes. Anti B, shows a slight cross-reaction
with the amphibian C, isozyme but does not
react with the A, isozyme. When we employed
antiserum formed against the A, isozyme of a
more primitive fish, the gar, the amphibian A,
isozyme cross-reacted at lower concentrations
of antiserum than the amphibian C, isozyme.
Only the CK A, isozyme was detected in skel-
etal muscle in all eight species of amphibians we
examined. The C, isozyme was detected in a
variety of tissues, but not including skeletal
muscle. There was no discernible evolutionary
pattern in the different relative electrophoretic
mobilities of the A, and C, isozymes in the am-

phibians. In three of the eight species the A,
isozyme was more anodal than the C,.

The other evolutionary line which arose from
the primitive fishes is the highly successful and
diverse teleost fishes. In the most primitive te-
leosts we have examined (the true eels, order
Anguilliformes) a third creatine kinase locus is
found. This third locus, designated the B locus,
consistently encodes a protein with a high net
negative charge at pH 8.6. In the relatively prim-
itive groups of teleosts (Clupeiformes, Salmon-
iformes, and Siluriformes) the A, isozyme is
found mainly in skeletal muscle, the C, in stom-
ach muscle, and the B, isozyme in several tis-
sues. In all other orders of teleosts the B locus
is restricted in its expression and B subunits are
detected only in eye and brain. A typical cre-
atine kinase isozyme pattern for the majority of
teleosts that we have examined is shown in Fig-
ure 6. The tissue specificity of differential CK

152 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

KINASE ISOZYMES OF THE YELLOW BASS
(Morone mississippiensis )

CREATINE KINASE

FIGURE 6.

gene expression is shown for the yellow bass
(Family Percichthyidae, Morone mississippien-
sis). The A, isozyme has greatest activity in
skeletal muscle. The B, isozyme is found only
in eye and brain. The C, isozyme predominates
in stomach muscle. Although the B, isozyme
characteristically possesses a highly anodal mo-
bility in teleost species, the relative electropho-
retic mobilities of the A, and C, isozymes are
often reversed among different species. A fourth
CK locus (D) encoding a testis specific CK iso-
zyme has been found in several orders (Semio-
notiformes, Clupeiformes, Cypriniformes, Ath-
eriniformes, and Perciformes).

The A,, B,, C, and D, creatine kinase iso-
zymes are each encoded by a different locus.
Allelic variants have been observed for all four
loci and allow us to reject the hypothesis that
one or more of these isozymes have been formed
by some epigenetic modification of a single gene
product. In most cases where heterozygotes for

ADENYLATE KINASE

Creatine kinase isozymes of the tissues of the yellow bass (Morone mississippiensis).

the different loci were detected the expected
three isozyme pattern was seen. In some species
of fish a heterodimer is not seen in individuals
heterozygous at the CK A locus. In all cases the
allelic variation only altered the electrophoretic
mobilities of the CK isozymes containing the
polypeptide with the genetic alteration of its net
charge.

Our postulated phylogeny of the creatine ki-
nase loci is shown in Figure 7. This diagram in-
dicates the gene duplication events which are
assumed to have occurred during the evolution
of the creatine kinase genes. A duplication of an
arginine kinase locus prior to the echinoderms
presumably gave rise to two arginine kinase loci,
one of which subsequently evolved to a creatine
kinase locus. These two different but homolo-
gous enzyme loci still coexist in certain echi-
noderm species (Watts, D. C. 1971). Two lines
of evidence suggest that the single creatine ki-
nase locus seen in urochordates is most closely

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 153

PHYLOGENY OF CREATINE KINASE LOCI

A C B

TETRAODONTIFORMES
PLEURONECTIFORMES

e PERCIFORMES
SCORPAENIFORMES
GASTEROSTEIFORMES
BERYCIFORMES

° ATHERINIFORMES
GADIFORMES
LOPHIIFORMES
BATRACHOIDIFORMES
SILURIFORMES

e CYPRINIFORMES
SALMONIFORMES
OSTEOGLOSSIFORMES

e CLUPEIFORMES
ANGUILLIFORMES

AMIIFORMES

° SEMIONOTIFORMES
POLYPTERIFORMES
ACIPENSERIFORMES

LEPIDOSIRENIFORMES
COELOCANTHIFORMES

MYLIOBATIFORMES
RAJIFORMES

PETROMY ZONTIFORMES

CEPHALOCHORDATA

UROCHORDATA ae eae

= = =
ARGININE CREATINE

ECHINODERMATA KINASE KINASE

e Testis specific creatine kinase isozyme (D locus ?)

FiGURE 7. Phylogeny of creatine kinase genes in the fish-
es. e Indicates orders in which testis specific creatine kinase
isozymes have been observed.

related to the A locus of higher fishes. First,
when we employed antisera of similar titres to
the A,, B, and C, isozymes of green sunfish,
only anti CK A, precipitated this enzyme. Sec-
ond, phylogenetically the A locus is the first lo-
cus to achieve the tissue specific pattern of reg-
ulation common to all fishes. Even in the
primitive fishes it is the only isozyme found in
skeletal muscle.

The appearance of the C locus in the cephalo-
chordates correlates well with the threefold in-
crease in cellular DNA content Atkin and Ohno
(1967) have reported for this group relative to
the urochordates. The isozyme found in skeletal
muscle of the primitive fishes is clearly immu-
nologically identified as an A, isozyme. All three
antisera to green sunfish isozymes (anti A., B,,
and C,) precipitate the second CK isozyme of
primitive fish, although the anti A, and anti C,
are effective at lower levels than the anti B,.
Antiserum to the gar A, precipitates both the A,
and C, isozymes of primitive fish.

The third locus, the B locus, is first detected
in the teleosts. Its appearance is not accom-
panied by any large increases in cellular DNA
content or a substantial increase in chromo-
some number. It is more likely the result of a
regional gene duplication. In several groups of
primitive teleosts the B locus is expressed in a
variety of tissues. In the most advanced teleosts
the B, isozyme is present only in eye and brain.
Several lines of evidence indicate that the B lo-
cus arose as a duplication of the C locus. Anti-
sera made against the A,, B, and C, isozymes
of the green sunfish cross-react with the A,, B,
and C, isozymes of the green sunfish as follows:
1) The anti A, antiserum precipitates A, iso-
zymes most effectively. The anti A, serum pre-
cipitates C, isozymes at lower levels than those
required to precipitate B, isozymes. 2) The anti
C, serum precipitates C, isozymes most effec-
tively, and will also cross-react with A, iso-
zymes at lower levels than those required for B,
isozymes. 3) The anti B, serum primarily pre-
cipitates the B, isozyme but also cross-reacts
with the A, and C, isozymes. The sequence in
which the loci achieve a tissue restricted pattern
of expression during phylogeny is A (prete-
leosts), C (primitive teleosts), and B (advanced
teleosts). In the primitive teleosts which have a
generalized expression of the CK B locus, B,
isozymes are generally found in the same tissues
as C, isozymes. In the teleosts the A, isozyme
is quite heat labile, while the B, and C, isozymes
are both considerably more resistant to heat de-
naturation. Therefore, on the bases of the anti-
genic and physical properties of the isozymes as
well as their tissue distribution, we postulate
that the B locus arose from a duplication of the
C locus. Figure 7 also indicates the orders of
fish in which we have seen a testis specific cre-
atine kinase isozyme (D,). Further study is
needed to pinpoint the exact time(s) of origin of
this locus and the extent of its expression in the
fishes. Obviously the D locus exhibits the most
tissue restricted pattern of gene expression seen
for the homologous CK loci. Immunochemical
studies indicate that the D locus arose as a du-
plication of the C locus.

DISCUSSION

The multiple locus creatine kinase isozyme
system provides many opportunities to study the
mode of evolution of homologous genes. The
immunological data presented here provide fur-

154 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

ther evidence that creatine kinase arose from a
duplicated arginine kinase locus (Robin et al.
1976: Watts, D. C. 1971; Watts et al. 1972). In
many echinoderms arginine kinase is the phos-
phagen kinase found in somatic tissues, while
creatine kinase is located only in sperm. Only
the most advanced echinoderms (Ophiuroidea)
possess creatine kinase exclusively (Watts
1968). Watts (1968, 1971, 1975) has suggested
that there was considerable selective advantage
to completely separating amino acid metabolism
and energy storage in the sperm. Using phos-
phocreatine instead of phosphoarginine removes
competition for arginine for both histone or oth-
er protein synthesis and energy storage.

In the evolutionary progression from the echi-
noderms to the tunicates it is obvious that there
have been considerable changes in the regula-
tion of the creatine locus. Creatine kinase is con-
sistently found in somatic tissues of tunicates.
Our studies and those of Moreland et al. (1967)
indicate that tunicates have a single CK locus.

A wide variety of organisms (Cephalochor-
data, Petromyzontiformes, Elasmobranchiom-
orphi, Crossopterygii, Dipnoi, Chondrostei, Ho-
lostei and Amphibia) express two creatine
kinase loci. These taxa have divergence times
spread over at least 150 million years (Dayhoff
1972). These groups encompass major evolu-
tionary changes—jawless to jawed vertebrates,
cartilaginous to bony fishes, lobe fin to ray fin
fishes, aquatic to air breathing vertebrates.
Therefore it should not be surprising to find con-
siderable variation in the patterns of expression
of the two paralogous creatine kinase loci in this
assemblage. The problem, then, is to construct
a framework within which this variation can be
meaningfully interpreted. To varying extents,
these primitive taxa constitute a limited repre-
sentation of once far more numerous groups.
However, the persistence of one or a few mem-
bers of a major group allows significant bio-
chemical inferences to be made. In many cases
the relict species are morphologically very sim-
ilar to their ancestors. In these cases it seems
reasonable to assume that these species still re-
tain biochemical and physiological similarities to
their ancestors as well. We are also assuming
that duplicate genes for creatine kinase are not
readily lost once their products occupy different
essential metabolic niches and their genes come
under differential regulatory control. With these
assumptions it is possible to investigate a mul-

tilocus isozyme system in various remnant taxa
and to make inferences about patterns of gene
regulation at very different evolutionary levels.
The diversity in expression of two creatine ki-
nase loci presented in Table | may represent the
diversity of metabolic strategies that were tried
early in vertebrate evolution following a major
genome amplification event. It is not necessary
to argue that this variation in patterns of gene
expression in the primitive fish is due to the dif-
ferent environments in which these fish now ex-
ist. In the advanced teleost fishes, which exhibit
tremendous morphological and habitat diversity,
the pattern of regulation of the CK loci is re-
markably constant. Once two (or more) homol-
ogous structural loci are formed, they can di-
verge to a variety of patterns of differential gene
regulation. The success of these patterns would
be expected to vary considerably. The patterns
of regulation of the two CK loci in the primitive
fishes are diverse, but two overall evolutionary
trends emerge. First, the expression of the A
locus is greatest in skeletal muscle. Second, the
C locus is most frequently expressed in eye,
brain, stomach and kidney.

For many years it was accepted that the Cros-
sopterygil, of which the coelacanth is the only
living representative, is a sister group to the
Rhipidistia which gave rise to the amphibians
(Romer 1966; Pang et al. 1977). However, more
recently as seen in several of the papers in this
volume and elsewhere (Jarvik 1968a, 1968b; Ep-
ple and Brinn 1975; Lagios 1975) this view has
been challenged. It has been proposed that the
Crossopterygians may not be a sister group to
the Rhipidistia but have closer affinities to the
sharks. Our data are not able to exclude either
of these alternative views because the elasmo-
branchs, coelacanth, and amphibia all have two
creatine kinase loci. Future studies employing
a variety of fresh coelacanth tissues are required
for a complete picture of the number of CK loci
and the pattern of differential gene expression
in the tissues of this species. Detailed reciprocal
immunochemical studies using antisera to the
creatine kinase isozymes and other proteins of
coelacanth, sharks, and amphibia would also
prove valuable.

Lungfish (Dipnoi) are noted for their unusu-
ally high DNA content (Mirsky and Ris 1951).
This amplification of DNA has not been paral-
leled by an increase in the number of creatine
kinase loci. A survey of fourteen other enzyme

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 155

systems did not reveal any additional structural
genes in the South American lungfish (Lepido-
siren paradoxa) (Fisher et al. 1976). Ohno (1970)
has suggested that the lungfish and Amphiuma
[an amphibian with 28 times the DNA of humans
(Mirsky and Ris 1951; Comings and Berger
1969)] are an example of how extensive regional
gene duplication may not generate new struc-
tural gene functions. Duplicate genes formed by
this mechanism may have been silenced early in
evolutionary history or they may not have been
free to diverge to new functions. By contrast,
the family Catostomidae (Cypriniformes) was
formed by a polyploidization event about 50
million years ago (Uyeno and Smith 1972). After
this relatively short span of time an average of
40% of the duplicate locus sets are still retained.
Some of these duplicate loci already show di-
vergence in function and regulation (Ferris and
Whitt 1977). The contrasting fates of duplicate
gene expressions in true polyploid groups and
those of the lungfish point out the importance of
considering coevolution of the structural genes
and their control elements.

Although the actual ancestral taxa have not
been satisfactorily identified, there is no doubt
that both the amphibians and the teleost fishes
had separate origins from those ancestral fish
which possessed two creatine kinase loci. The
land vertebrates and teleost fishes have subse-
quently followed two very different paths of cre-
atine kinase gene evolution.

The amphibians also possess two creatine ki-
nase loci. Neither locus shows a highly restrict-
ed pattern of tissue expression in any of the eight
species that we have examined. In skeletal mus-
cle only the A locus is expressed. In other tis-
sues both loci are active to varying degrees. The
CK isozymes of the reptiles have not been ex-
tensively investigated. We have looked at only
one species (Anolis carolinensis) and it has two
CK loci. One is expressed almost exclusively in
skeletal muscle. The second, which codes for a
quite anodal isozyme, is activated in many tis-
sues. Mammals have two loci coding for cyto-
plasmic forms of creatine kinase (Masters and
Holmes 1975). The isozymes are often referred
to as MM (found predominantly in skeletal mus-
cle), BB (the brain form) and MB (a heteropoly-
mer found in heart and other tissues). This ter-
minology is quite misleading. It incorrectly
implies that these isozymes are restricted in
their expression to those tissues. The highly an-

odal isozyme seen in eye and brain is also found
in significant amounts in heart, stomach, lung,
liver, spleen, ovary, testis and kidney. The two
creatine kinase loci in mammals are presumably
homologous to the A and C loci of the primitive
fishes. The time of origin of a third locus which
encodes the mitochondrial CK isozyme and the
gene from which it is derived are uncertain. The
results of our present study, based on a limited
sample size, suggest that the land vertebrates
have maintained both the ancestral state of at
least two creatine kinase loci and a generalized
pattern of expression of these loci.

In the teleost fishes we have observed the
most extensive proliferation of creatine kinase
loci of all the vertebrates, as well as the greatest
tissue specificity of CK gene expression. One
conclusion that might be drawn from this study
is that as additional homologous isozyme loci
are formed there are greater possibilities for the
isozymes to evolve more specific metabolic
roles and more specific cellular and subcellular
localizations. At the same time there would also
be an increased possibility for a more highly spe-
cialized control of these loci. In primitive fishes
the C locus is most frequently expressed in eye,
brain, stomach, and kidney. In the highly suc-
cessful and diverse teleost fishes two separate
loci encode the isozymes found in stomach (C,)
and eye and brain (B,). While the C, isozyme in
stomach may vary in its electrophoretic mobil-
ity, the more recently derived B locus always
codes for a highly anodal isozyme. The D locus
shows the most dramatic restriction of gene
expression. Our study confirms the earlier ob-
servations of Eppenberger’s group (Scholl and
Eppenberger 1971) of testis specific CK iso-
zymes in Barbus nigrofasciatus (Cypriniformes,
Cyprinidae), Platypoecilus variatus, and Xi-
phophorus helleri helleri (both Atheriniformes,
Poeciliidae). They postulated that these iso-
zymes were either epigenetically derived or un-
der separate genetic control. We have found al-
lelic variation which supports the postulate of a
separate genetic basis. Testis and sperm specific
isozymes have been observed for a number of
enzyme systems (Goldberg 1977). It has ob-
viously been advantageous for the teleost fishes
to maintain several CK loci each encoding an
isozyme uniquely suited to a few cell types.

It is interesting to note that in both the fishes
and the mammals one of the creatine kinase loci
codes for a highly anodal isozyme which its pre-

156 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

dominately synthesized in neural tissues—the B
locus in fishes and the C locus in mammals. This
correspondence in net charge of the isozymes
with their tissue specificity of synthesis in quite
different taxa at two different loci is probably
due to convergent evolution for surface charge.
A large, net negative charge may be important
for a specific subcellular localization in the cells
of neural tissues.

In many respects the evolution of the creatine
kinase isozyme system in the vertebrates is sim-
ilar to the evolution of other multilocus isozyme
systems, particularly the extensively studied
LDH system (Markert et al. 1975; Whitt et al.
1975). In both CK and LDH isozyme systems
two loci appear to have been formed by an early
polyploidization event (as postulated by Ohno
1970) and additional loci by subsequent regional
gene duplications. [In a few orders of fishes
(Salmoniformes, Cypriniformes) more recent
tetraploidization events have resulted in another
round of duplication of isozyme loci.] In general,
when a locus first appears through duplication
by either mechanism it is initially regulated in
the same manner as its duplicate gene. At an
intermediate stage of gene evolution a duplicate
gene has a generalized and often variable pattern
of expression among tissues and can be quite
variable in its tissue expression among species.
In the later stages of gene evolution the speci-
ficity of expression is narrowed. It is possible to
infer a fundamental metabolic significance of the
isozymes from our observation of the highly
temporally and spatially restricted expression of
homologous isozyme loci. These patterns of
gene expression are virtually identical for many
species which are occupying quite different hab-
itats and which possess quite divergent mor-
phologies. Although an important adaptive value
for this specialization of gene expression can be
assumed, the specific metabolic role for any giv-
en isozyme within a multilocus isozyme system
has yet to be definitively identified. The specific
kinetic differences and the physiological conse-
quences among fish possessing two, three, or
four creatine kinase loci are not known at this
time. It will be valuable if future investigations
elucidate the reason for the teleosts possessing
more isozyme loci for several enzyme systems
than either the primitive fishes or the higher ver-
tebrates. Certainly at the level of molecular evo-
lution and the evolution of gene regulation there
are many questions to be asked about the rela-

tionships of the primitive vertebrates which can
be efficiently studied employing multilocus iso-
zyme systems.

SUMMARY

The process of evolution by gene duplication
can be effectively studied through a phyloge-
netic analysis of multilocus isozyme systems.
We have examined the expression of isozyme
loci in a variety of tissues in a comprehensive
survey of primitive vertebrates and have docu-
mented increases in the number of isozyme loci
as Well as an increasing restriction of gene reg-
ulation. A basic assumption for this approach 1s
that extant species that are morphologically sim-
ilar to their ancestors retain genetic and molec-
ular homologies with their ancestors as well.
Specifically, one would expect the number of
homologous loci and patterns of regulation of
these genes to reflect the ancestral numbers and
patterns. Thus, while many major and once
highly successful groups of early vertebrates are
represented today by only a few species, these
extant fish provide a unique opportunity to gain
insights into the evolution of multilocus systems
as well as the phylogenetic relationships of ver-
tebrates.

We have investigated the number and tissue
expression of creatine kinase isozymes in over
100 species from 72 different families in echi-
noderms through mammals. There is persuasive
evidence that creatine kinase arose as a dupli-
cation of an arginine kinase locus (Watts, D. C.
1971; Watts et al. 1972). Our immunological
studies which revealed that the anti-CK of te-
leosts cross-reacts with the arginine kinase of
the echinoderm Arbacia punctulata provide fur-
ther support for this hypothesis.

The urochordates have a single creatine ki-
nase locus. The appearance of a second creatine
kinase locus in the cephalochordates is corre-
lated with a corresponding threefold increase in
DNA content in this group when compared to
the urochordates as reported by Atkin and Ohno
(1967). In the nonteleostean fishes there is con-
siderable variation in the expression of the A
and C creatine kinase loci. This diversity may
reflect the diversity of patterns of gene regula-
tion that were present early in vertebrate evo-
lution following a major genome amplification
event. Within this diversity of gene expression
two general trends are seen in the wide range of

FISHER & WHITT: CREATINE KINASE ISOZYME EVOLUTION 157

fish possessing two creatine kinase loci: the A,
isozyme is the only creatine kinase found in
skeletal muscle and the C, isozyme is most fre-
quently detected in eye, brain, stomach, and
kidney.

The amphibians and reptiles also have two
creatine kinase loci with a relatively generalized
pattern of expression. In mammals a mitochon-
drial CK isozyme is found in addition to the two
creatine kinase isozymes found in the cytoplasm
(Masters and Holmes 1975). Our analysis indi-
cates that the two cytoplasmic CK loci of mam-
mals also have a generalized pattern of expres-
sion.

The advanced teleost fishes have up to four
creatine kinase loci which are expressed in a
highly tissue specific manner. The A, isozyme
is found mainly in skeletal muscle, the B, iso-
zyme is detected only in eye and brain, the C,
isozyme predominates in stomach muscle, and
the D, isozyme is found exclusively in mature
testis. The large number of isozymes and the
distinctive temporal and spatial specificity of
their synthesis suggests that a very refined
mechanism has evolved to differentially regulate
these closely related loci.

ACKNOWLEDGMENTS

Specimens used in this survey were obtained
from a variety of commercial and private
sources. We would like to thank William Child-
ers of the Illinois Natural History Survey, David
Philipp of the University of Illinois, James Shak-
lee of the Department of Zoology of the Uni-
versity of Hawaii, and Donald Buth and Steve
Ferris of the University of Illinois for providing
many of the species of fish used. The Coelacanth
tissues were from fish #79 obtained in the 1975
California Academy of Sciences expedition. We
are grateful to John McCosker for providing
these samples and the specimen of Mustelus
henleii.

The Sepharose Blue 6B was a gift of Phar-
macia Fine Chemicals.

This research was supported by National Sci-
ence Foundation Grants GB 43995 and PCM 76-
08383 to G.S.W.; a NSF predoctoral fellowship
and a NIH traineeship in Cell Biology to S.E.F.

We would like to thank Steve Ferris and Da-
vid Philipp for their constructive comments dur-
ing the preparation of this manuscript.

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OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 10 pages December 22, 1979

AMINO ACIDS AND TAURINE IN INTRACELLULAR
OSMOREGULATION IN MARINE ANIMALS

By
J. B. Lombardini and Peter K. T. Pang
Department of Pharmacology and Therapeutics,

Texas Tech University School of Medicine,
Lubbock, Texas 79409

and
Robert W. Griffith

Department of Biology,
Southeastern Massachusetts University,
North Dartmouth, Massachusetts 02747

ABSTRACT

Organic molecules, especially taurine and other amino acids and trimethylamine
oxide, are important in both invertebrates and vertebrates in maintaining intracellular
osmolarity similar to that of the extracellular fluids. In this study we have analyzed
muscle tissues for their amino acid and taurine content in five marine invertebrates
(the shrimp Penaesus setiferus, the blue crab Callinectes sapidus; the sea hare Aplysia
californica; the sea squirt Thyonella gemnata and the sea cucumber Styela plicata), four
marine vertebrates (the stingray Dasyatis sabina, the smooth dogfish Mustelus canis,
the coelacanth Latimeria chalumnae and the killifish Fundulus grandis) and two fresh-
water vertebrates (the tiger salamander Ambystoma tigrinum and the South American
lungfish Lepidosiren paradoxa). The results indicate that while taurine is the predom-
inant free amino acid in the muscles of certain species such as shrimp (25%), sea squirt
(67%), stingray (70%), killifish (68%) and dogfish (42%), it is only a minor constituent
in the blue crab (12%), sea hare (10%), sea cucumber (0.4%) and coelacanth (15%).
Taurine levels were also measured in the internal organs of the listed species. It is
reported in this study that certain species also contain large quantities of free specific
protein amino acids in their muscle tissues, although no clear evolutionary or ecological
trends are evident. The importance and role of taurine and amino acids in marine
animals are reviewed.

160

LOMBARDINI, PANG & GRIFFITH: INTRACELLULAR OSMOREGULATION 161

INTRODUCTION

The osmoregulatory apparatus of advanced
aquatic animals may be regarded as a concen-
tric, three compartment system: the outer ring
comprising the environment, the middle the ex-
tracellular fluid, and the inner sphere the tissues.
The organism must first of all be capable of reg-
ulating the composition of the extracellular fluid
different from that of the external environment
by using specialized osmoregulatory organs
such as the gills and kidneys. Secondly, the
membranes of tissue cells must maintain an in-
tracellular fluid composition that is radically dif-
ferent from that of the extracellular fluid.

For marine animals, the environment is high
in solutes (specifically, in ions such as Na* and
Cl-). The extracellular fluids may be lower (as
in teleost fishes) or nearly identical in total sol-
utes with the environment (as in most inverte-
brates, hagfish and elasmobranchs). The isos-
motic condition may be achieved either by
having high ion content (invertebrates and hag-
fish) or by building up organic solutes such as
urea (elasmobranchs and Latimeria). The total
amount of solutes is almost always identical be-
tween extra- and intracellular fluids in animals,
but invariably there is a higher concentration of
electrolytes in the extra- than in the intracellular
fluid. This difference in ion concentration is
compensated for by building up organic mole-
cules such as TMAO, amino acids, taurine or
others in the intracellular fluid (cf. review by
Pang et al. 1977).

During fluctuations in environmental salini-
ties, both marine invertebrate and vertebrate
species must be capable of preventing large
changes in the volume of cell water which, un-
less carefully controlled, will lead to cell de-
struction. Thus osmoregulatory mechanisms are
necessary, especially for those organisms which
inhabit estuaries and other areas which undergo
periodic changes in salinity. These mechanisms
may involve homeostatic adaptations that per-
mit the extracellular fluids to remain more-or-
less constant in composition and they may also
include adaptations to maintain the cell volume
constant during changes in extracellular fluid
composition that involve modifying the intra-
cellular solute composition. Generally the sol-
utes that are adaptively changed in response to
salinity are organic, since electrolytes play such
crucial roles in membrane transport and bio-
electrical phenomena.

The use of free pools of non-protein nitroge-
nous substances as intracellular osmoregulatory
agents in the tissues of marine invertebrates has
been amply described (Shaw 1958; Bricteux-
Gregoire et al. 1964; Clark 1964). Furthermore,
more discriminating techniques have indicated
that specific amino acids along with the sulfonic
amino acid, taurine, are intimately involved with
maintaining the equilibrium between the intra-
cellular content of marine invertebrate tissues
and that of the external medium. One of the
more interesting studies to test whether free
amino acids are involved in osmoregulation was
performed by Kaneshiro et al. (1969). These in-
vestigators adapted the marine ciliate, Miamien-
sis avidus, to different salinities and noted that
the total amino acid levels increased 5 fold when
the salinity was increased from 25 to 200% sea-
water for a period of 20 to 90 minutes. Individual
amino acids were also measured and alanine,
glycine and proline were found to be the pre-
dominant amino acids accounting for 73% of the
total. However, taurine was not detected. The
rapid change of the amino acid content of the
marine ciliate cells reported in this study is also
noteworthy. In this limited time period (20 to 90
minutes) de novo synthesis of amino acids is
probably not possible and thus the authors spec-
ulate that there is a mobilization of amino acids
from protein or from other bound states within
the cell.

There are numerous studies involving differ-
ent species of molluscs which adapt to changes
in the salinity of their environment by adjusting
both the levels of free amino acids and taurine
(Lange 1963; Lynch and Wood 1966; Bedford
1971; Pierce 1971; Gilles 1972; Kasschau 1975;
Turgeon 1976). In the cited study by Kasschau
(1975) utilizing the mudflat snail, Nassarius ob-
soletus, it was dramatically demonstrated that
a change in the salinity of seawater from 12 to
30%c at 25 C markedly increased the taurine con-
tent of the digestive gland from 162 u~moles of
taurine/g protein to 355 wmoles. In either envi-
ronmental condition (12 or 30%c) taurine was ap-
proximately 50% of the total free amino acid
pool. Similar results were obtained when the
animals were maintained at 10 C. A second ex-
ample of the ability of molluscs to osmotically
regulate their tissue cells against the external
environment is that of the snail, Melanopsis tri-

fasciata. Bedford (1971) demonstrated that the

foot tissue of Melanopsis increased its content

162 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

of alanine by 10 fold, glutamic acid by 3.6 fold,
serine by 2 fold and taurine by 45% when placed
in 75% sea water as compared to fresh water.
Previous investigators had also noted that the
total free amino acid content of marine animals
had decreased when placed in reduced salinities
(Shaw 1958: Potts 1958; Duchateau et al. 1959;
Schoffeniels 1960; Duchateau-Bosson et al.
1961; Lynch and Wood 1966), however, there
are few literature reports of an increase in free
amino acid content of vertebrate tissues in re-
sponse to increased salinity (Gordon and Tucker
1968).

A notable exception to the observed trend that
taurine content increases with salinity is the
study of Boone and Claybrook (1977) who ob-
served that the mud crab, Panopeus herbstii,
did not alter its taurine content in muscle, he-
patopancreas, or gill when exposed to 10%c or
30%c. However, the levels of certain of the other
free amino acids did markedly change. Thus, it
appears that while the protein amino acids are
often utilized as intracellular osmoregulatory
agents in invertebrates, taurine, a non-protein
sulfonic amino acid, is not always utilized. In
the hagfish, Myxine glutinosa, amino acids again
appear to be osmoregulatory agents when the
salinity of the environment is modified (Cholette
and Gagnon 1973). In the hagfish the amino acids
which are quantitatively most important are pro-
line, alanine, valine, leucine, and threonine. Un-
fortunately, taurine content was not measured.

Taurine levels in the parietal muscle of eels
adapted from fresh water to sea water do not
change, however, the total ninhydrin positive
material does increase significantly (Huggins
and Colley 1971). The increase was accounted
for by changes in alanine, glutamine, glutamic
acid, glycine, proline, and cystathionine.

Marine elasmobranchs have also been sub-
jected to environmental dilution experiments
and their tissue amino acids analyzed. Studies
by Forster and Goldstein (1976) demonstrated
that the intracellular amino acid levels in muscle
tissue of the skate, Raja erinacea, decreased
from 214 to 144 mmole/liter during adaption to
50% sea water, moreover, urea levels fell from
398 to 264 and trimethylamine oxide from 64 to
36 mmole/liter. These investigators also report-
ed that total amino acid, urea, and TMAO levels
of muscle from the stingray, Dasyatis ameri-
cana, also decrease significantly upon adaption

to 50% sea water. In a subsequent study by
Boyd et al. (1977) who analyzed various tissues
of the skate, Raja erinacea, for specific amino
acids it was reported that taurine content de-
creased in erythrocytes and brain, remained the
same in heart, and slightly increased in the wing
muscle when the animals were placed in 50%
sea water. These investigators also measured
the levels of sarcosine (44.1 «moles/g wet tis-
sue), B-alanine (40.7 uwmoles/g), and creatinine
(19.8 «moles/g) which were the major amino
acids of the wing muscle. Two of these non-pro-
tein amino acids, sarcosine and #-alanine de-
creased significantly when the skates were
adapted to half-strength sea water.

Thus the osmoregulatory effects of free amino
acids in marine invertebrates and some marine
vertebrates are well established. On the other
hand there is only limited documentation con-
cerning the role of free amino acids or taurine
in controlling the osmolality of tissue cells in
teleosts. One of the first literature reports dem-
onstrating the involvement of TMAO and total
free ninhydrin positive substances in teleosts
was that of Lange and Fugelli (1965) on the
flounder, Pleuronectes flesus, and the stickle-
back, Gasterosteus aculeatus. A study by Cow-
ey et al. (1962) involving free amino acids in both
muscle and plasma of the tissues of the Atlantic
salmon, Salmo salar, during spawning migration
suggests that, at least in this species, amino
acids do not change. On the contrary, two eu-
ryhaline fishes, the thick-lipped mullet, Creni-
mugil labrosus, and the southern flounder, Par-
alichthys lethostigma, when adapted to
freshwater or 200% sea water show dramatic
changes in parietal muscle levels of the so-called
non-essential amino acids (glycine, alanine, as-
partic acid, glutamic acid, serine, and proline)
and taurine (Lasserre and Gilles 1971). In these
two fish the taurine content of the muscle ac-
counts for approximately 44-70% of the total
free amino acids.

More recently the studies of Colley et al.
(1974) demonstrated that the agonid Agonus
cataphractus also alters its muscle free amino
acid and taurine pools in response to changes in
the salinity of its surroundings. Once again, as
in the previous studies of Lasserre and Gilles
(1971) involving the mullet and flounder, taurine
is the predominant free amino acid. The percent
level of taurine in the muscle of this agonid

LOMBARDINI, PANG & GRIFFITH: INTRACELLULAR OSMOREGULATION 163

species, accounting for approximately 90% of
the non-protein nitrogenous constitutents, is the
highest yet reported in the animal kingdom.

A non-protein amino acid that has been gen-
erally neglected heretofore in studies involving
osmoregulation is y-aminobutyric acid. How-
ever, in the erythrocytes of the flounder (Plat-
ichthys flesus) y-aminobutyric acid is a principal
component of the free amino acid pool second
only to taurine (Fugelli and Zachariassen 1976).
Flounders adapted to seawater had 31.9 mmole/
kg intracellular water of taurine and 15.3 mmole
of y-aminobutyric acid in their erythrocytes.
When adapted to freshwater the taurine and
y-aminobutyric acid content of the erythrocytes
was 19.0 and 6.8 mmole/kg intracellular water,
respectively. Interestingly the other amino
acids, while very low when compared to taurine
and a-aminobutyric acid, all increased during
freshwater adaptation. The only exception was
methionine which remained constant.

Perhaps the most elegant study to date is that
of Ahokas and Sorg (1977) who studied both the
effects of salinity and temperature on the intra-
cellular osmoregulation and muscle free amino
acid levels in the killifish (Fundulus diaphanus).
In all experimental conditions, i.e., changes in
salinity and temperature, taurine levels were not
altered. When the animals were maintained at
0.5 C the total amino acid content of muscle in
fresh water was 67.5 mmoles/kg tissue water
whereas in 30%c salinity the levels increased to
79.1 mmoles. However, when the animals were
maintained at 15 C the muscle total free amino
acid levels were 74.6 mmoles in fresh water and
91.3 mmoles in 30%c salinity. Since taurine levels
did not change upon adaptation to different sa-
linities it was reasoned that this compound does
not function as an intracellular osmoregulatory
agent in this species. The amino acids that did
change making up the differences in the total
amino acid levels were alanine, glycine, serine,
proline and aspartic acid.

METHODS

The following species were obtained from
Gulf Specimen Company, Inc., Panacea, Flori-
da: shrimp (Penaeus setiferus), a blue crab (Cal-
linectes sapidus), sea cucumber (Thyonella
gemnata), sea squirt (Styela plicata) and sting-
ray (Dasyatis sabina). Sea hares (Aplysia cali-
fornica were purchased from Pacific Bio-Marine

Supply Co., Venice, California. The two species
of killifish used in this study, Fundulus grandis
and Fundulus heteroclitus, were purchased
from Gulf Specimen Co. and a supplier in
Queens, N.Y. respectively. Male rats (140-160
grams) were purchased from Sprague-Dawley,
Madison, Wisconsin.

The marine animals were used upon receipt.
The tiger salamanders and South American
lungfish were kept in fresh water at 23 C. The
rats were housed in plastic cages with wood
shavings and fed Purina laboratory chow ab li-
bitum.

All coelacanth tissues were shipped to the au-
thors frozen in dry ice.

The free tissue amino acids found normally in
protein (aspartic acid, glutamic acid, threonine,
serine, proline, glycine, alanine, valine, methi-
onine, isoleucine, leucine, tyrosine, phenylala-
nine, lysine, histidine, arginine) were analyzed
on a Beckman 121 amino acid analyzer utilizing
AA-15 resin. The column temperature was
maintained at 5S C. The buffers used in eluting
the amino acids were 0.2 M sodium citrate, pH
3.25 (applied for 110 min); 0.2 M sodium citrate,
pH 4.25 (for 42 min); and 0.2 M sodium citrate,
pH 6.40 (for 120 min). Taurine was also mea-
sured by the amino acid analyzer technique uti-
lizing 0.2 M sodium citrate buffer, pH 2.4. Tau-
rine eluted at 27 minutes under these conditions.

The total amino acid content of muscle tissue
was estimated by summing the levels of the in-
dividual amino acids.

RESULTS
Taurine and Amino Acid Levels in Muscle of
Invertebrate and Vertebrate Species
The analyses of taurine and total free amino

acid levels in muscle of various invertebrate and
vertebrate species reveals that taurine is of ma-

jor quantitative importance in only certain

species (Table 1). Three of the invertebrates
tested, the shrimp (Penaeus setiferus), blue crab
(Callinectes sapidus) and sea squirt (Styela pli-
cata) contain extremely high levels of taurine in
the range of 30 to 49 wmole/gm wet tissue while
the sea hare (Aplysia californica) and sea cu-
cumber (Thyonella gemnata) have low values
(1.7 and 0.4). When the muscle tissue of these
animals was analyzed for total amino acid con-
tent a wide variation in the levels of the total

164 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

TABLE l.

CONCENTRATION OF TAURINE (4MOLE/GM WET TISSUE) AND TOTAL FREE AMINO ACIDS (4MOLE/GM WET TISSUE)

IN MUSCLE OF VARIOUS INVERTEBRATE AND VERTEBRATE SPECIES.

Total amino

Species Taurine acids % Taurine

Shrimp (Penaeus setiferus) 48.8 + 3.1 (8) 193.2 + 7.8 (4) Dee
Blue Crab (Callinectes sapidus ) SO 2ZE= 32016) 242.9 + 11.2 (4) 12.4
Sea Hare (Aplysia californica) Le= 1025) GES) = 23 (©) 10.3
Sea Cucumber (Thyonella gemnata) O42 051 (6) [0618 == 25396) 0.4
Sea Squirt (Styela plicata) 38.8 + 3.1 (8) She) sx Tai (4!) 67.1
Stingray (Dasyatis sabina) 39D 12-256) 56.3 + 65.8 (2) 69.7
Killifish (Fundulus grandis) 209) 9) a= 3)(0) (5) 66.8 + 2.4 (5) 63.3
Killifish (Fundulus heteroclitus) S/o 22 ily (3)
Dogfish (Mustelus canis) 11.60; 7.86 (2) 26:6; LON (2) 43.4; 41.2
Coelacanth (Latimeria chalumnae)* ESS ies (2) 14299 7231(2) 10.5; 18.8
Tiger Salamander (Ambystoma tigrinum) 0.84; 1.76 (2)
South American Lungfish (Lepidosiren

paradoxa) 0.05; (1)
Rat (Rattus norvegicus) 10.9 + 0.7 (10)

Numbers in parentheses represent number of animals utilized in the analyses.

All data are reported as means + SEM.

* First observation is from Coelacanth specimen #79; second observation is from the coelacanth captured by the joint British-

French-American expedition.

pool was also noted. The blue crab contained
the highest level (242.9) and the sea hare the
lowest total free amino acid level (16.5). Of the
five invertebrate muscle tissues analyzed for
taurine and free amino acids the sea cucumber
had the lowest percentage of taurine (0.4%).
However, the percentage of total free amino
acids in the other invertebrate species that con-
sisted of taurine is quantitatively more signifi-
cant. For example, the taurine content is 10.3%
of the total amino acids in the sea hare, 12.4%
in the blue crab, 25.2% in the shrimp, and 67.1%
in the sea squirt. Taurine is clearly the dominant
amino acid in the latter species.

Concentrations of taurine and total free amino
acids were also determined in the stingray
(Dasyatis sabina) and in the killifish (Fundulus
grandis). Both of these animals had high levels
of muscle taurine (39.2 and 42.2 wmole/gm wet
tissue) which constituted 69.7 and 63.3% of the
total free amino acids. The taurine content of
muscle tissue obtained from F. heteroclitus was
quite similar to that of F. grandis. However,
when muscle tissue from two smooth dogfish
(Mustelus canis) were analyzed the taurine
levels were found to be in an intermediate range
(11.6 and 7.9 wmole/gm wet tissue). Neverthe-
less, the percent of the total free amino acids
that are represented by taurine in this species is
quite high, above 40%. On the contrary, the

coelacanth (Latimeria chalumnae) muscle had
very low taurine content (1.6 and 1.4 umole)
which accounted for only 10 to 20% of the total
free amino acid pool. The taurine content of
muscle from two freshwater vertebrates, the
tiger salamander (Ambystoma tigrinum) and the
South American lungfish (Lepidosiren para-
doxa), was also measured. In both of these
species the levels of taurine were low, 0.8 and
1.8 «mole/gm wet tissue in the muscle of the
salamander and 0.1 in the lungfish. For compar-
ison purposes the taurine content of rat muscle
(10.9) is presented.

Concentration of Amino Acids in Muscle of
Some Invertebrate and Vertebrate Species

A profile of the constituents of the free, pro-
tein amino acid pool of muscle tissues from var-
ious invertebrate and vertebrate species is pre-
sented in Tables 2 and 3. Data for twelve amino
acids are reported. Table 2 contains the level of
two acidic amino acids, glutamic and aspartic
acids, and four neutral amino acids, glycine, al-
anine, proline and threonine. Table 3 contains
data for three basic amino acids, arginine, lysine
and histidine, and three neutral amino acids, va-
line, isoleucine and tyrosine.

The predominant amino acids in the muscle
of shrimp are glycine (96.1 wmole/gm wet tissue)
and arginine (29.4). The blue crab has quite high

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INTRACELLULAR OSMOREGULATION

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LOMBARDINI, PANG & GRIFFITH

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166 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

TABLE 4. DISTRIBUTION OF TAURINE (4MOLE/GM WET TISSUE) IN TISSUES OF SOME MARINE INVERTEBRATES.
Sea hare Sea cucumber Sea squirt Blue crab
Tissue (Aplysia californica) (Thyonella gemnata) (Styela plicata) (Callinectes sapidus)

Digestive gland Da eile (6) 3.4 + 0.8 (6) 44.4 + 6.7 (5)
Gill 1728) =10:9) (6) 27.5 + 2.4 (8) 20.3 + 4.0 (6)
Capsule oil SSO sil (7A)
Heart 40.3 + 13.2 (6) 43.3 + 4.3 (6)
Albumin gland 26.9 + 1.6 (6)
Kidney 4°3' += 0! (6)
Ovotestes 40.2 +5.7 (6)

Numbers in parentheses represent numbers of animals utilized in the analyses.

All data are reported as means + SEM.

levels of glycine (90.0) and arginine (51.4) and
moderately high levels of proline (22.2), alanine
(15.3) and threonine (10.4). The blue crab has
the highest total free amino acid pool (242.9) of
all the muscle tissues from the species that were
analyzed (Table 1).

Sea hare muscle is quite low in its total free
amino acid content (16.5 wmole/gm wet tissue,
Table 1) and consequently does not have high
levels of any specific amino acid. Glutamic acid
(2.4) and aspartic acid (3.56) are found in the
highest quantity in sea hare muscle of any of the
17 amino acids analyzed (all data not reported).

The free amino acid pools of sea cucumber
muscle is composed primarily of glutamic acid
(85.0 xmole/gm wet tissue) which accounts for
approximately 78% of the total pool. Arginine
(8.6), while quantitatively less important than
glutamic acid, comprises almost 8% of the free
amino acid pool in this species.

Analyses of the muscles from sea squirts,
stingrays, and dogfish indicate that there is no
single protein amino acid that contributes to a

major portion of the free amino acid pool. How-
ever, glycine (2.9 wmole/gm wet tissue) and al-
anine (3.7) levels are slightly higher than the oth-
er amino acids in the sea squirt; glycine (2.4)
and lysine (1.7) levels are higher in the stingray;
and glycine (3.4), alanine (3.0), and perhaps ly-
sine (1.8) are higher in the dogfish.

Three amino acids, lysine (6.4 wmole/gm wet
tissue), glycine (5.6), and threonine (4.2) are rel-
atively high in muscle tissue of the killifish (F.
grandis). Of all the muscle tissues of the various
species that were analyzed the total free amino
acid pool of the coelacanth is the lowest (14.9,
7.3, Table 1). No single protein amino acid (Ta-
bles 2 and 3) appears to be quantitatively more
important in the coelacanth as was the case in
some of the invertebrate species such as the
shrimp or blue crab.

Distribution of Taurine in Tissues of Some In-
vertebrates and Vertebrates

Tissues other than muscle were also analyzed
for their taurine content. The distribution of

TABLE 5. DISTRIBUTION OF TAURINE (4MOLE/GM WET TISSUE) IN TISSUES OF SOME VERTEBRATES.
Killifish Lungfish Tiger salamander Rat
Sting ray Killifish (Fundulus (Lepidosiren (Ambystoma (Rattus

Tissue (Dasyatis sabina) (Fundulus grandis) heteroclitus) paradoxa) tigrinum) norvegicus )
Liver 39.4 + 4.3 (3) 45,2 = 3:2) (5) 32.0 + 2.2 (8) 0.11 (1) 0.41 + 0.18 (3) 14+ 0.1 (9)
Heart 53}, 22 10L7/ (B) 52.7 + 8.8 (5) 39.4 + 1.8 (8) 0.07 (1) 0.04 + 0.02 (3) 20.1 + 1.3 (10)
Kidney AW syese EI ((B)) 49.3 + 5.6 (8) ND* 0.06 (1) 11.6 + 0.7 (10)
Testes (ley se CRS (P)) 36.3 = 2.9)(3) 30.4 + 0.9 (8) ND* 3.2 + 0.2 (10)
Gill ANS) AS} 25 33.3) (3) 34.0 + 1.8 (5)

Spleen WS3es) == Tt. (GB) 64.5 + 7.4 (5) SAIS 25188) 0.09 + 0.05 (3) 11.8 + 1.2 (10)
Brain G2FIG== 22803) 32745-10315) 24.7 + 1.4 (8) 5-22 10'2(110)
Lung 0.10 (1) 0.09 + 0.06 (3) 11.5 + 0.8 (10)

ND* = Not detectable.

Numbers in parentheses represent numbers of animal tissues utilized in the analyses.

All data are reported as means + SEM.

LOMBARDINI, PANG & GRIFFITH: INTRACELLULAR OSMOREGULATION 167

taurine in tissues of marine invertebrate species
is shown in Table 4. The highest levels of taurine
in sea hare tissues were found in the heart (40.3
wmole/gm wet tissue) and ovotestes (40.2) while
the kidney had the lowest (4.3). The albumin
gland, digestive gland, and gill had intermediate
values. Taurine content varied greatly in the
digestive glands from the sea cucumber (3.4),
blue crab (44.4), and sea hare (22.5) whereas the
taurine levels of gill tissue from the sea squirt
(27.5), blue crab (20.3), and sea hare (17.8) were
somewhat similar as was heart tissue from the
blue crab (43.3) and sea hare (40.3). Capsule tis-
sue analyzed from the sea squirt was very low
in taurine (1.1).

Accordingly, the distribution of taurine in tis-
sues of some vertebrate species was measured
(Table 5). Every tissue that was examined in the
stingray (Dasyatis sabina) and the two species
of killifish (F. grandis, F. heteroclitus) had high
levels of taurine ranging from 24.7 to 71.7
wmole/gm wet tissue. However, when two fresh-
water vertebrates, the South American lungfish
(Lepidosiren paradoxa) and the tiger salamander
(Ambystoma tigrinum), were examined taurine
levels were extremely low or not detectable. Rat
tissues had intermediate taurine levels ranging
from 1.4 in the liver to 20.1 in the heart.

Taurine Levels in Some Coelacanth Tissues

Data showing the distribution of taurine in tis-
sues of the coelacanth (Latimeria chalumnae)
are presented in Table 6. The spleen and liver
contain the highest taurine levels (38.8 and 22.9
mole/gm wet tissue) whereas the other tissues,
stomach, pancreas, lung and muscle are all quite
low in taurine content (3.4 to 1.4).

DISCUSSION

When one considers the possible osmoregu-
latory role of taurine and other amino acids in
marine animals their most likely function would
be to maintain intracellular osmolarity equal to
that of the extracellular fluid. Invariably, the ex-
tracellular composition has a higher concentra-
tion of ions than the intracellular compartment
and the difference must be compensated with
organic molecules.

The properties of a compound used in intra-
cellular osmoregulation are: 1) limited diffusi-
bility through cell membranes, 2) relative inert-
ness, that is, they are not rapidly metabolized in
the tissues concerned, and, 3) availability from

TABLE 6. DISTRIBUTION OF TAURINE IN VARIOUS TISSUES
OF THE COELACANTH (Latimeria chalumnae).*

umole/gm

Tissue wet tissue
Spleen 38.8
Liver 22.9
Stomach 3.4
Pancreas 2)
Lung* 23)

Muscle (lateral body) eG alie4!

* Data were obtained from coelacanth specimen #79 with
exception of second observation for muscle.
* Fat filled body.

the environment or from metabolic pathways.
The principal solutes demonstrated to play such
a role are the amino acids, taurine, and trime-
thylamine oxide. Although urea may constitute
a very large proportion of the intracellular or-
ganic solutes in some animals, it 1s almost al-
ways in equilibrium between the extracellular
and intracellular fluids and does not play a role
in intracellular osmoregulation. Parenthetically,
we might mention that varying the pool of intra-
cellular organic solutes is a common mechanism
of adapting to changing salinities in many inver-
tebrate species.

In terms of the phylogenetic distribution of
the mechanism of intracellular osmoregulation,
no firm conclusions can be drawn from the lit-
erature. What is important to know is whether
the principal solute used is TMAO, taurine, or
other amino acids such as glycine, alanine, as-
partic acid, betaine and so forth.

We have looked at a cross section of verte-
brate and invertebrate species (including, of
course, the coelacanth) and have drawn some
conclusions about the relationship between the
importance of amino acids and other organic
molecules in intracellular osmoregulation and
the environment and evolutionary history of the
animals. Firstly, does taurine play a role in in-
tracellular osmoregulation in animals, and sec-
ondly, what influences its importance in osmo-
regulation?

Unquestionably taurine does play a significant
role in osmoregulation in some animals. High
levels can be demonstrated in the muscle of a
variety of vertebrates and invertebrates includ-
ing blue crab, sea squirt, killifish, and some elas-
mobranchs. Negligible values of taurine are

168 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

characteristic of extracellular fluids in animals
generally, so the substance, taurine is obviously
being used to maintain intracellular osmolarity
equal to that of the extracellular compartment.

What appears to be the most likely determi-
nants of tissue levels of taurine are 1) differences
between total ion levels in the plasma and tissue
fluids which will dictate the total amount of or-
ganic solutes present in the tissue fluids and 2)
the levels of other non-diffusible organic solutes,
principally other amino acids, TMAO, and be-
taine. A variety of physiological factors may in-
fluence these parameters including salinity, diet,
the kind of tissue with which we are dealing, and
perhaps phylogeny.

The role of salinity is clearly evident from the
virtual absence of taurine in the tissues of strict-
ly freshwater animals such as lungfish and tiger
salamander, and the high levels of taurine that
characterize many (but not all) marine animals.
The influence of salinity on taurine levels could
either be through effects on blood and tissue ion
concentrations: on the dietary availability of the
solute or its precursor; or by effects of salinity
on metabolic pathways.

Marked differences in taurine levels are found
between tissues in the same individual. Could
this be related to cell diffusibility, and/or metab-
olism of one or another of the organic molecules
involved?

Finally, phylogenetic differences may well ex-
ist in the importance of taurine as an intracel-
lular osmoregulatory device, although the data
fail to show a clear-cut picture. Both high and
low levels of taurine are found in marine rep-
resentatives of the deuterostomes [high in tuni-
cate and many fishes, low in echinoderm (sea
cucumber) and coelacanth] and the protostomes
(high in shrimp and crab, low in sea hare). And
even within one closely knit class, the elasmo-
branchs, both moderately low taurine levels in
Mustelus and quite high levels in the stingray
may be found.

It should not be overlooked that the coel-
acanth, at least from the limited amount of data
available, does not display the same kind of in-
tracellular osmoregulation as do elasmobranchs.
Instead of having high concentrations of amino
acids (including taurine) and other compounds
such as betaine within the cells in addition to
the TMAO, the coelacanth appears to rely al-
most entirely on TMAO to balance the intracel-
lular deficit in ions.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Wilbur
G. Sawyer, Department of Pharmacology, Co-
lumbia University College of Physicians and
Surgeons, for the South American lungfish (Lep-
idosiren paradoxa) and Dr. Francis L. Rose,
Department of Biology, Texas Tech University,
for the tiger salamanders (Ambystoma tigrinum).
Tissues from one of the coelacanths, specimen
#79, was kindly provided by Dr. John E.
McCosker, Director, Steinhart Aquarium, San
Francisco, California. The California Academy
of Sciences 1975 Coelacanth Expedition was
supported by a grant from the Charline H. Bree-
den Foundation. Tissues from the second coel-
acanth were supplied by one of us (R.W.G.).
This specimen was caught March 22, 1972, off
Iconi, Grand Comore, by the joint British-
French-American expedition.

The valuable technical expertise of Ms.
Evangeline V. Medina is greatly appreciated.
The authors also thank Mr. Harvey O. Olney of
the Department of Biochemistry, Texas Tech
University School of Medicine, for the amino
acid analyses. This study was supported in part
by NIH grant 1-ROI-NS-11406 and NSF grant
TCM 76-82239.

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OCCASIONAL PAPERS
OF THE

CALIFORNIA ACADEMY OF SCIENCES

The Biology and Physiology of the Living Coelacanth

No. 134, 6 pages

December 22, 1979

RECAPITULATION OF THE DISCUSSION AT THE CLOSE OF THE
15 JUNE 1977 SYMPOSIUM ON THE RELATIONSHIPS OF
PRIMITIVE FISHES, WITH PARTICULAR
REFERENCE TO THE COELACANTH

Transcribed by

Susan and George Brown

Society for the Protection Of Old Fishes,
College of Fisheries,

University of Washington,
Seattle, Washington 98195

McCosker: .. . Well, I suggest we can discuss
this now if you would like, or we could con-
tinue over at the aquarium. Are there any oth-
er questions that anyone else has to bring up
before these two (Lagios and Griffith) face
off?

Male from audience: You indicated that Lati-
meria gills had a smaller volume?

Griffith: Apparently the surface area is much
smaller than any other fish that George
Hughes measured. I never measured it, but

Same male voice: You mean there is just a quan-
titative withholding of urea?

Griffith: Well, presumably . . . I mean, urea lost
will be across surface area, and respiratory
surface area is presumably the best way to
lose it. Now if you cut down the respiratory
surface area... of course, it means that you
are also cutting down the amount of oxygen
coming in. So it means you can’t be a very
active animal.

B. Jeanne Davis: How big are these gills?

170

Griffith: The filaments are really short.

McCosker: They're really quite like elasmo-
branch gills. I was very struck to see how sim-
ilar they were when we dissected the speci-
men.

Griffith: Well, of course, elasmobranchs face the
same problem. They want to cut down loss
across the gills, too.

McCosker: I would be hard pressed to relate
surface of gill area to body area.

Griffith: It may depend upon the size of the an-
imals involved. Relative to an active teleost,
I would think they’re quite small (reference
George Hughes).

McCosker: I was really struck by the statement
that the osmolality of the coelacanths was
slightly below seawater. Can you conceive of
a situation whereby what you’ve measured is
an artifact? That is, you've measured surface
seawater salinity, and we're talking about a
fish that has been captured at or near an aqul-
fer at depth.

Griffith: Quite possibly. This is an iso-osmolar

BROWN & BROWN:

urine, also an isO-uremic urine. What it seems
to be doing, if the urine is doing anything, is
regulating nothing but ions. Regulating diva-
lent ions, such as sulfate and magnesium, and
getting rid of a little bit of glucuronates. You
would probably find other things: creatine,
left-over metabolites, phosphate.

McCosker: A stressed shark seems to display
the same syndrome as a captured coelacanth.

Griffith: Most of the data that has been done on
elasmobranch osmoregulation has probably
been done under similar stress situations.

McCosker: It would be nice to put one in fresh
water for a short period of time and see if it
makes some compensation.

Griffith: Why do you put them in a dilute envi-
ronment? They don’t have to. Urea levels re-
main fairly high, but the total osmolarity will
drop, and ion levels will also drop... .

Rasmussen: There’s a time lag, too. After a
while, urea levels will start to drop.

G. Brown: What is the situation on water reab-
sorption? Do you know anything about that?

Griffith: Well, from the data that we have here,
it doesn’t look like anything’s happening. It
could be the thing is there’s no way to get any
clearance data.

G. Brown: What about the structure of the kid-
ney?

Griffith: It seems to be well developed.

Lagios: Its very much like an elasmobranch
kidney.

G. Brown: That's what I wanted to know.

Lagios: . . . Except for one small incidental fea-
ture. I was going to ask you about two inter-
esting points. One is, I remember you telling
me that elegant and remarkable story, when
I met you in April 1972 and went back with
all the tissues at New Haven, about the actual
capture of the coelacanth, and if I recall cor-
rectly, Chabane (chief of the village of Iconi)
came in about two o'clock in the morning and
woke you up, and the coelacanth had been
captured and brought in by fishermen and was
put in a chicken-wire cage.

Griffith: Right.

Lagios: In the surf zone at Iconi where you

Griffith: | don’t want that to get out ! (Laughter).
Lagios: The explanation for high urea. . . . The
Comoros are twelve degrees south, and it fol-
lows that, of course, in the tropics the sun
doesn't really rise until around 7:30 and there

1977 SYMPOSIUM RECAPITULATION 171

is not enough light to take pictures until eight
o'clock. Is that about right?

Griffith: Right.

Lagios: So that the animal was really about five
or six hours in the surf zone. I think the way
you put it together is very nice. I am glad that
we at least agree that urea retention is a spe-
cialized feature. I think we may have to go
back and catch another coelacanth to see
whether it really doesn’t resorb urea.

Griffith: It would be nice if we could just get one
alive in the lab and put a canula in it.

Lagios: What about urea reabsorption in the
elasmobranch kidney? It that an active trans-
port system?

Griffith: Apparently they are designed to do it
passively. I’m not quite sure how it works.

Lagios: All the counter-current systems really
require a very rigid organization of the kidney
in terms of nephrons and loops, and we only
see that in certain birds and mammals.

G. Brown: Don't you have the same concentra-
tion of urea in the plasma as in the bladder?
Presumably, why would you need to have any
active transport?

Lagios: Well, if sharks resorb urea from urine,
how do they do it?

G. Brown: | would say that is is an active pro-
cess, but you have a different urine to plasma
ratio for urea. But Griffith's data indicate that
it iS isoosmotic.

Lagios: That’s exactly my point. If it is indeed
an active transport system and if we assume
that the coelacanth has an active transport
system for urea and you bang it around in the
surf zone for five and a half hours, the active
transport system may have turned off. As a
matter of fact, that’s not uncommonly seen in
human patients who have active renal trans-
port systems but go into shock.

Rasmussen: Could it be a carrier-mediated func-
tion?

Griffith: Something like that.

Rasmussen: Yes, that gets around the active
transport problem.

Griffith: But there may be structural features in-
volved.

Lagios: The kidney I looked at looked very
much like other primitive fishes.

Griffith: Well, you would have to microdissect
an individual tubule. Could you do that?

Lagios: What it requires to have the ability to
do passive transport using a counter-current

172 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

system is a rigid organization such as mam-
malian kidney . . . loops of Henle, and have
a gradient system: This you don’t have in a
fish. Maybe the coelacanth doesn’t have an
active transport system, then banging it
around in the surf for five and a half hours
would be more than likely to turn it off.

Griffith: The thing is, some of the transport sys-
tems of that specimen were clearly function-
ing as indicated by chloride, sulfate, and phos-
phate concentrations. There is no way you
can get that without an active transport sys-
tem.

Lagios: But they may be differentially separat-
ed, that is damaged.

Griffith: Right.

G. Brown: I think the higher lactate concentra-
tions may suggest a certain amount of anaer-
obic metabolism going on, and that might
knock out active transport requiring ATP.

Lagios: That’s right. You mentioned the lactate
was high.

Griffith: There’s no question. But we also don't
know how long the urine was sitting in the
bladder.

Miller: Is is true that urea is one feature that you
lose in severe shock? I recall that something
in severe situations would void its urine.

Lagios: Well, the lactate concentration in the
urine was about what it was in the blood,
wasn't it?

Griffith: Lower.

Lagios: A lot lower?

Griffith: Oh, not a lot lower, maybe sixty per-
cent. It is mostly reabsorbed. So, this proba-
bly is an indication of that (urea) passed over
the threshhold (in the coelacanth specimen).

McCosker: In terms of a marine ancestry, I
haven't heard anything “‘physiological”’ that
anyone has said which would refute it... .
Some coelacanths, Undina for example, have
gone into fresh water, and so have the fresh-
water stingrays.

From audience: What about the ancestral coel-
acanths? Do we still consider them to be close
relatives sharing common ancestry with Lat-
imeria?

McCosker: There’s no reason not to. The mere
fact that they were in freshwater deposits is
a reflection of the conditions in which fossils
can be preserved. Most fish fossils occur in
freshwater deposits for that reason. I see no
reason to exclude the common ancestry, of

course, based on that saltwater situation. But
I think that should give us some insight into
the relevance as either a specialized or a con-
vergent feature of this urea retention business.
I’m not convinced that it is not a specialized
feature. I hate to side with Mike (Lagios) on
this, but I think he’s got something there.

Griffith: 'm not so sure.

McCosker: One stressed animal makes it diffi-
cult to evaluate the actual physiology.

Griffith: Urea retention may be a convergent
feature.

McCosker: But it would then require two evo-
lutionary losses for the frogs to possess that
ability. If in the evolution of a single line, you
brought a single lineage all the way up to tet-
rapods including Rana, we would require only
one offshoot for elasmobranchs. Lagios would
say that coelacanths are a sister group, and
then we're going to require a couple of lin-
eages as with dipnoans and teleosts who
would have to lose urea retention which is
entirely likely. The fact is that teleosts went
off and tried something entirely different very
early in their evolution, and the Dipnoi have
been in the freshwater ever since.

Male from audience: Then you are talking about
a plesiomorph characteristic?

McCosker: That is a plesiomorph characteristic,
not apomorphic.

Lagios: Except that you have to also explain the
teleost-like serum of the lamprey.

Griffith: You know, that’s only based ona single
observation.

Lagios: Actually, there are several. George
Brown talked about how tadpoles develop
(urea cycle enzymes)... . It has been shown
that when freshwater sharks or migrating
sharks go back and forth from fresh water to
the marine environment their urea levels go
up and down.

McCosker: Well, is this a plesiomorph feature,
a very primitive feature that has been retained
throughout lower vertebrate evolution? It
would be nice, in fact, if Rana did it quite
differently ....

Griffith: Actually, Rana does it entirely differ-
ently. It doesn’t have kidney reabsorption,
but it has bladder reabsorption (of urea). It’s
not either like the coelacanth or like the shark.

Male from aduience: (Urea) It’s probably just
a convenient organic compound that can be
used for osmotic purposes.

BROWN & BROWN:

Griffith: | think the same thing holds true for
trimethylamine oxide. What they’re doing is
retaining some bulk solute for osmotic (pur-
poses).

G. Brown: | think there is something more fun-
damental than just the fact that because it’s
there they can use it, because you can make
urea in several different ways. You can break
down purines. All organisms can make it from
hydrolysis of arginine, and some groups can
synthesize it de novo. And it would seem to
me in talking about origins and so forth, you
want to look at the mechanism for the syn-
thesis of that compound. And then, superim-
posed upon that, then the adaptive uses of the
(compound).

Griffith: | think that is very true. And I think
you would probably agree that at least in
everything, the ancestor of everything after
the Agnatha would probably have had suffi-
ciently high levels to be functional.

G. Brown: Yes, that’s my view. I'm still puz-
zled by the reports of levels of the really im-
portant enzyme of the cycle—they’re all im-
portant, of course—carbamolyphosphate
synthetase, has been found in a number of
teleostean fishes. We don’t know of any sit-
uation where teleostean fishes made use of
urea in an osmotic sense, do we?

Griffith: I was suspecting that maybe something
that’s air-breathing, or is out of water for a
while might.

G. Brown: These would be the species to pin-
point.

Rasmussen: There must be some—the mud-
skipper, isn’t that the one?

G. Brown: The mudskipper’s been looked at,
but it doesn’t have the cycle.

Lagios: When was that abstract by R. M. Matler
on Amia (dissertation abstracts, 1967) on the
bowfin. He suggested urea retention in that
species.

Griffith: Didnt someone recently assay it in
some of the holosteans?

G. Brown: Carbamolyphosphate synthetase of
a different type than found in the pprimitive
fishes was found by Paul Anderson at Min-
nesota—in the smallmouth bass—and that en-
zyme has some different properties, and he’s
in the process now of characterizing it and
making comparisons. There’s been reported
maybe five different types of enzymes for the
synthesis of carbamolyphosphate, if they re-

1977 SYMPOSIUM RECAPITULATION 173

quire different metals (cofactors) then there
has been some evolutionary change in that,
and I think somebody, somehow, is going to
have to surface out and push it all back, and
see what the ancestral molecule was. And
then maybe things would fit into place as far
as your conceptions here are concerned. But
I’m in agreement with that.

Griffith: Good! (Laughter).

McCosker: 1 don’t know if it’s convergent or
not, but as I recall, only Latimeria and certain
frogs have a bilobed bladder. Does that have
anything to do with it? I think the ovovivi-
parity business requires some further exami-
nation, and, that is, coelacanths obviously are
doing it. There’s still a lot of coelacanths
around, and it has been shown their internal
fertilization is by bony carbuncles that can be
extended ne

Griffith: (sub-rosa): Hypothesized.

McCosker: (continuing) . . . the mere fact that
they don’t have claspers I think might be a
secondary loss.

Griffith: Do you really think they could have
them and lose them?

McCosker: That ancestral line was very produc-
tive. (Laughter). The chimaera has developed
two more, as well as another structure, and
no one knows what they do with it. But mod-
ern ichthyologists consider the chimaeras as
a sister group to sharks and rays. And, I think
that this would be acceptable to most ichthy-
ologists (goes to blackboard) ... although
you did question that viewpoint . . . chimae-
ras... I think Patterson’s work has been re-
futed by recent evidence, that sharks and rays
and chimaeras were sister groups, and I would
be willing to support Lagios by placing Lati-
meria here, and all you have to do is have a
deletion right here of claspers and evolve
another means of fertilization.

Griffith: That’s a hard thing to believe, I think.

McCosker: 1 think another approach, of course,
is to consider Latimeria the next branching
point, not sharing this (elasmobranch) com-
mon ancestor, which however, would place
Lagios’ pituitary information as a primitive
condition. And that would be hard for him to
swallow.

Lagios: Vl try to summarize. Any feature that
you analyze can be either convergent, ances-
tral, or specialized. And the idea is to try to
develop evidence that’s going to clearly tell

174 OCCASIONAL PAPERS OF THE CALIFORNIA ACADEMY OF SCIENCES, No. 134

you one way or the other. Fortunately, we
have living agnathans, both hagfishes and lam-
preys, to look at in terms of pituitary struc-
ture. We didn’t delve into it at any great
length, but they had a compact pituitary struc-
ture. They have a poorly developed vascular
supply, but it is a single vascular supply. In
the sense of their endocrine structure, the pi-
tuitary, they can be accepted as primitive. It
certainly differs very little from what we see
in primitive bony fishes.

Male from audience: Have you looked at the
embryogenesis of the shark pituitary?

Lagios: | have not. Honma has indeed done the
embryology of the Hydrolagus pituitary. The
Rachendach-hypophysis (of Hydrolagus) and
the ventral lobe of sharks are homologous
structures, both derived off the pars distalis,
and both get hung up in the carotid anasto-
mosis. In the shark, it is hung up with the
carotid anastomosis. But the pituitary (as a
plesiomorph feature) is a little difficult to
swallow because we're really not talking
about one feature, but a whole complex of
features. I was going to ask Bob (Griffith) that
he discuss the rectal gland in terms of urea
retention.

Griffith: If you're cutting off loss across the gills
by really restricting surface area, maybe by
using an ion pump across the gills ... it’s a
way to lose urea, too.

Lagios: I am sure it is related, that there was a
need to have a cation-excreting gland some-
where. The interesting point is that other an-
imals have developed cation-excreting glands
in all sorts of different tissues: nasal glands,
lacrimal glands, anal papillomata.

Griffith: However, I think in all fishes it tends
to be either the gills or somewhere associated
with the gut . . . rather than eyes or in facial
glands. Head (cation-excreting) glands seem
to be a reptilian (characteristic).

Lagios: It is also associated with the bladder and

cloaca in some, as I can show you with Pro-
topterus. There are many groups of special-
ized characters. Each one, any specialized
character, can be either an indication of com-
mon ancestry or convergence. But in the coel-
acanth you have several independent features
which appear to be synapomorphic with those
of sharks. Let’s assume that urea metabolism
and the rectal gland evolved together—al-
though really they don’t. Similar pituitary and
the duct-associated islet (of Langerhans) tis-
sue structure would make it more difficult to
accept as convergences.

Griffith: Actually there’s another one. . . which
you brought up, Dr. Miller, and that is carti-
lage composition.

Miller: It has been done.

Griffith: Morton B. Mathews at Chicago did it
in 1962.

Miller: This accomplished something for what?

Griffith: For coelacanths and all sorts of sharks.
And God knows what else. Apparently, they
have a cartilage composition in common:
there’s an extra sulfonation in chondroitin sul-
fate, and it’s found in all elasmobranchs and
coelancanths and in no other animals. The ex-
ceptions to the elasmobranchs are the fresh-
water stingrays, the Potomotrygonidae, which
he also measured.

Miller: This is then yet another similarity.

Griffith: Yes. It seems to be closely related to
urea retention because if you put chondroitin
sulfate in high urea concentrations, unless it
has the extra sulfonation, it dissociates. So it
seems to be tied to it.

Miller: | wasn’t only interested in the ground
substance, but also in the collagen and in the
interrelationship between ground substances
and the cells that are producing collagen and
ground substance.

McCosker: I think it would be wisest to carry
this on later this evening, over wine and fillet
of coelacanth.

BROWN & BROWN:

1977 SYMPOSIUM RECAPITULATION

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POLIO AE NATH 89 9

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