Chromosomes of Some Op isthobranchiate Mollusks from Eniwetok Atoll,
W estern Pacific'
] . B. BURCH2 and R. NATARA] AN3
ABSTR ACT: Chromosome numbers are reported for nine species of opisthobranchiate mollusks from Eniwetok Atoll, Marshall Islands, western Pacific. In
the N udibranchia, both D endrod oris nigra (Den drodori didae) and H erviella
mietta (Favorinida e) were found to have 13 bivalents during male meiosis. In
the Anaspidea, D olabrif era d olabrifera and Stylocb eilus longicallda (Aplysiidae)
both had 17 bivalents during male meiosis. In the Ceph alaspidea, H amin oea linda
and H. musetta (Atyi dae) each had 17 pairs of chromosomes during male meiosis
and Latb opb tbalm us smaragd imts and Smaragdinella calyculata (Smaragdinellidae)
had 18 pairs. In the Soleolifera, Onchidella eoelinae had 18 bivalents during male
meiosis.
Th e extreme conservativeness of chromosome numbers in opisthobranchiate mollusks is demonstrated by that fact that all 18 nudibr anchs from 10 families studied
so far have the single haploid chromosome number 13, and that 18 of the 21 species of the orders Entomotaeniata, Anaspidea, Cephalaspidea, and Sacoglossa have
17 pairs of chromosomes. Th e haploid number 18 is here reported for the first
time for nonsoleoliferan opisthobranchiate mollusks. Th e more advanced, mostly
fresh-water, order Basommatophora, in which the haploid number 18 is the basic
number, may well have been derived from a taxon with in or related to this cephalaspid superfam ily (Philin acea) .
IN RECENT YEARS, detailed investigations have
been made on the chromosomes of many basommatophoran and stylommatophoran snails
(Burch, 1965) , but relatively few species of
the opisthobranchiate! orders have been studied,
1 The field work for th is investigation was sup ported by the D ivision of Biology and Medicin e,
U . S. Atomic Energy Commission . The research was
supp orted ( in par t) by grants from the N ational
Science Found ation , W ashingto n, D . C. ( GB 787 )
and the N ational In stitute of All ergy and Infectious
D iseases, U. S. Pu blic H ealt h Service ( 5 T1 AI
41-08 ) . Manuscript received March 14, 1966.
2 Mus eum and D epartment of Zoology, University
of Mi chigan, Ann Arbor . Supp orted by a Public
H ealth Service research career program award ( N o.
5-K 3-AI-19, 451-03 ) .
3 Museum of Zoology, Uni versity of Michigan,
Ann Ar bor.
4 Boett ger (1955 ) considers the orders Opis thobranchiata and Pulmona ta ( togeth er equivalent to the
subc lr-ss Euthyneura ) to be unnatu ral ones, and instead divides the Euthyne ura into seven orders . While
we do not disagree with th is, it is still convenien:
to speak of his five lower (m ainl y marine) euthyneur an ord ers (Nudibranchia, Soleolifera, Cephalaspidea, Sacoglossa, and Anaspidea) as " opistho -
mainly because of the difficulties they pr esent
in collection and identification. Relying on the
studies of various authors during the early part
of the pr esent century, Makino (195 1) listed
the chromosome numbers of 16 opisthobranchiate species, but recent investigations by Inaba
and our present studies indicate that the earlier
records are not dependable and so are obsolete.
Previous reliable reports on the chromosomes of
opisthobranchiate gastropods are those of Inaba
and H irota (1954, 1958), Inaba (1959a, 1959b,
1961) , N atarajan (1959, 1960), Mancino and
Sordi ( 1964a and b) , and Burch ( 1965) . These
authors give the chromosome numbers of 36
species belonging to 21 families and 7 orders
(Tables 1 and 2) . This is a very small number
when compared with the great multitude of
species currently recognized in the opisthobranchiate orders. Th e present pap er presents the
chromosome numbers of 9 opisthobranchiate
branchs" as distinguished from th e more advanced
Basommatop hora (m ainly freshwater) and Stylommatophora (l and inhabita nts).
252
Chromosomes of Eniwetok Opisthobranchiates-s-Bun cn AND NATARAJAN
253
TABLE 1
CHRO MOSO ME NUMB ERS PREVIO USLY R EPORT ED IN T HE O PI ST H OBRAN CHIAT E O RDERS
NOTASPID EA AN D N UDIBRAN CH IA
CHROMOSOME
N UM BER
SPECIES
20
n
LOCA LIT Y
24
12
J apan
Inaba, 1959a
26
26
13
13
13
13
13
J apan
J apan
J apan
J apan
It aly
In aba and H irota, 19 58
Inaba and H irot a, 1954 ; 1958
Inaba, 1959a
Inaba , 1959a
Mancino and Sordi, 1964a
26
26
13
13
J apan
Japan
Inaba and H irota, 1958
Inaba and H irot a, 195'8 ;
In aba, 1959b
26
13
13
J apan
Japan
In aba and H irota, 1958
In aba and H irota, 1958
13
J apan
Inaba and Hi rota, 1958
13
Ja pan
Inaba , 19 59a
13
J apan
Inaba, 1961
13
J apan
Inaba and H irota, 1958
13
J apan
In aba, 1961
13
13
J apan
Ja pan
Inaba and Hi rota, 1958
Inaba and H irota, 19 58
REFEREN CE
N OT ASPIDEA
Pleurobran chid ae
Pieu robrancbaea
nauaezealandiae
N UDIBRANC H IA
Dori didae
Glossodoris [estiua
Glossodoris pallescens
Rostanga arbutus
Discodoris pardalis
D oris uerrucosa
D endrodorididae
D endrodoris miniata
D endrodoris nigra
Trioph idae
Kaloplocamus ramosus
Plocamopberus tilesii
Goniodorididae
Okenia barnardi
Fimb riidae
Melibe papillosa
D otonidae
Doto bella
Arminidae
D ermatobrancbus striat us
Cuth on idae
Catriona pup illae
Facelinidae
Facelina ceylonica
Facelina [aponica
26
26
species belonging to 6 families and 4 orders
(Table 3) , which were collected in shallow
water s around thr ee island s of En iwetok Atoll
in the western Pacific by th e senior author and
Dr. W illiam H . H eard du ring early 1960.
MATERIAL AND METHODS
The species studied in this investigation and
the localitie s wh ere th ey were collected are
listed below. Duplicate specimens have been
deposited in the collections of the Mu seum of
Zoology at the Univers ity of M ichigan, the
University of Sao Paulo , and the University of
H awaii.
NUDlBRANCHIA
L D endrodoris nigra (St impson ) (Fig. 1).
N orth end of Japt an Island , und er loose p ieces
of dead coral. Ap ril 1, 1960 .
2 . H eroielln mietta M arcus and Burch ( Fig.
2 ) . N orth end of En iwetok Island on the lagoon side , in about 10 em of water at low tide,
under submerged pieces of dead coral. Ap ril 2,
1960.
ANASPIDEA
3. D olabrif era dolabrifera (Rang ) ( Fig. 3) .
Und er loose p ieces of coral on seaward tid e flat
at the north end of Parr y Island . M arch 25,
1960.
PACIFIC SCIENCE, Vol. XX I, April 1967
254
TA BLE 2
CHROMOSOME NUMBERS PREVIOUSLY REPORTED IN THE O PISTHOBRANCHIATE ORDERS
ENTOMOTAENIATA, CEPHALASPIDEA, ANASPIDEA, SACOGLOSSA, AND SOLEOLIFERA
CHRO MOSO ME
NUMBER
SPECIES
2n
ENTOMOTAENIATA
PyramideJlidae
T iberia fasciata
ANASPIDEA
Apl ysiidae
Petalifera punctulata
N otarcbus leacbii [reeri
CEPHALASPIDEA
Acteonidae
Cylicbn atys angusta
Philin idae
Pbiline japonica
Agl ajidae
A glaja gigliolii
SACOGLOSSA
Elysiidae
Elysia amaku sana
Elysia oiridis
Stil igeridae
Alderia nigra
H ermaeopsis oariopicta
Placida dendr itic«
Placida viridis
Stiliger uesiculosus
Juliid ae
Bertbelinia limax
Polybranchiid ae
Bosellia mim etica
SOLEOLIFERA
Ve roniceJlid ae
V eronicella fioridana
Laeuicaulis alte
On chidiidae
Oncidie ll« kur odai
Onch idium uerraculatu m
34
34
34
34
34
14
36
n
LOCALITY
17
J apa n
In aba, pers . comm .
17
17
J apan
J apan
Inaba, 19 59a
Inaba, 19 59a
17
J apan
Inaba, pers . comm .
17
J apan
Inaba, 19 59a
17
J apan
Inaba, 19 59a
17
17
J apan
It aly
Inaba, 19 59a
M ancino and Sordi, 1964b
17
17
17
17
17
J apan
It aly
It aly
It aly
It aly
In aba, 1961
M ancino and
Mancino and
Mancino and
M ancino and
17
J apan
In aba, 1961
7
It aly
Mancino and Sordi , 1964b
16
17
U .S.A.
India
Burch , 1965
N ataraj an, 1960
17
18
Japan
I ndia
In aba, 1961
N atar ajan, 19 59
4. Stylocheilus longicauda (Quoy and Gaimard ) (Fi g. 4) . In tide flats of Eniwetok Island. March 4, 1960.
CEPHA LASPIDEA
5. Haminoea linda Marcus and Burch (F ig.
5) . Parry Island, in sand, in about 2 m of water,
in lagoon, about 17 m from shore. March 31,
1960.
REFERE NCE
Sord i,
Sordi,
Sordi,
Sord i,
1964b
196 4b
1964b
1964 b
6. Haminoea musette Marcus and Burch
(Fig. 6) . Middle part of Parry Island on seaward tide flats. April 2, 1960.
7. Lathophthalmus smaragdinus ( Ruppel
and Leuckart) (Fig. 7) . Collected at the south
end of Parry Island, under loose pieces of coral
on seaward tide flat. March 15, 1960.
8. Smaragdinella calyett/ata (Broderip and
Chrom osomes of Eniwetok Opi stbob ranchiates-i-Buncn
255
AND N ATARAJAN
T A BLE 3
CHROMOSOME NUMB ERS OF O PIST H OBRAN CHS O BSERVED IN THIS STUDY
SPECIES
N UM BER OF
SP ECI MENS GIVING
RESU LTS
CHROMOSO ME
N U M BER ( n)
N UDIBRAN CH IA
D endrod ori didae
D endrodoris nigra
Favorin idae
H eruiella miette
13
13 ( 2n
5
= 26 )
AN ASPIDEA
Ap lysiidae
D olabrifera dolabrijera
Stylocbeilus longicauda
17
17
9
2
CE PH ALASPID EA
Atyidae
H aminoea linda
H aminoea musetta
Smaragdinellidae
Lathophthalmus smaregdinus
Srnaragdinella ealyeulata
17
1
17
2
18
18
2
2
18
2
SOLEOLI FERA
Onchidiidae
Oncbidella euelinae
Sowerby) (Fig. 8) . In lagoon at north end of
Eniwetok Island .
camera lucida and reproduced at a table-top
magni fication of 4260 X .
SO LEO LIFERA
9. Oncbidella euelinae Marcus and Burch
(Fi g. 9). In cracks in coral slabs above water
lin e ( at low tide) 0;1 the lagoon side at the
north end of Eniwetok Island . Apr il 5, 1960.
The materials examined consisted of ovotestes fixed in either N ewcomer's (1953 ) or
Carney's (1 887) fluids, or the fixative of Sanfelice (191 8) . The material fixed in Newcomer's or Carney's fluids was stained by the
acetic-orcein squash technique (L a Cour, 1941) ,
and reproductive tissues fixed in Sanf elice's
fluid were sectioned at either 8 or 10 micra and
stained with Newton's (1926) crystal violet.
All observations were made on meiotic cells of
spermatogenesis (ex cept in H eruiella mietta,
where spermatogonial cells were also stud ied )
with a Nikon (Nippon Koga ku) microscope
equipped with a 100 X (n .a. 1.25) oil immersion objective and lOX , 20 X , and 30X oculars. D rawings were made with the aid of a
OBSERVAT IONS
1. D endr odoris nigra (Fig. 10 ) . The five
indi viduals of this species on which we were
able to obtain satisfactory observations all had
13 bivalents dur ing prophase of the first meiotic
division.
2. H eruiella miette (Fi g. 11) . Only one
specimen of this species gave satisfactory results.
It had 13 bivalents durin g diakinesis and 26
chromosomes durin g metaphase in spermatogoni al cells.
3. D olabrifera dolabrifera (Fi gs. 12 and
13) . N ine specimens had meiotic cells that
were satisfactory for chromosome numb er determin ations. All had 17 bivalents dur ing Pro ph ase I and Metaphase 1. Th e chromosomes of
a cell durin g diakinesis from an acetic-orcein
squash preparation are shown in Figure 12.
Metaph ase I bivalents from a cell of paraffin
sectioned material are shown in Figure 13.
PACIFIC SCIENCE, Vol. XXI, April 1967
256
(\
I' \'e.\,
\
} '.:
;'
\\
...~
.
....
~~J~'it-
...0
('II
Chromosomes of Eniwetok Opisthobranchiates-i-Buaca
-.
...,.c.~
"~c;'c;.a
,
.~
-' ..t
257
AN D N ATARAJAN
~
°f'~
It
• • II .,.
••
""0"
........,
"
12
~
.
-..
•.
•••
..
.
..
.. ,
.•.-.
'.f
.....
V,.
4t't~""
.....
...-r,) ,.....
..,,"," , -.9•
,....", .-'-- . -..
10
,.
... ....
.•
•
11
. ,pit
$C
.~ AI~"
'V., 1) ~
15
14
f
13
al·"
•••••
••
19
ft. I ...
W t;
t'
...
'\~
16
.,
•
~
~
•'••••
II
18
20"
17
FIGS. 10- 20. Chromosomes of Eniwetok opisthobranchs. 10, Dendrodoris nigra. 11, Herviella mietta. 12
and 13, Dolabrif era dolabrifera. 14, Stylocbeilus longicauda, 15, Haminoea musette. 16, H . linda. 17, Lathophthalmus smaragdinus, 18, Smaragdinella calyculata. 19 and 20, Onehidella eoelinae.
Figs. 10-1 2, 14, 15, 17, and 20 are of male diakin esis bivalents; 13,1 6, 18, and 19 are of male Metaphase
I bivalents. Figs. 10-12, 14, 15, 17 , 18, and 20 are from acetic-orcein squash preparations; 13, 16, and 19
are from sectioned material stained with crystal violet. Measurement line divided into micra.
FIGS. 1- 9.
Eniwetok opisthobranchs used in this study. 1, Dendrodoris nigra. 2, H eruiella miette. 3,
Dolabrijera dolabrijera. 4, Stylocbeilus longieauda. 5, Hamlnoea linda. 6, H . musette. 7, Lathophthalmus
smaragdinus. 8, Smaragdinella calyculata. 9, Oncbidella euelinae. Measurement lines are divided into millim eters.
258
PACIFIC SCIENCE, Vol. XXI, April 1967
4. Stylocbeilus longicaude (Fig. 14). Satis- ferent family. Each of these three species had
factory results were obtained from two speci- a haploid number of 17. In the pr esent inmens. Both had 17 bivalents during diakinesis. vestigation two species from each of two addi5. H amin oea linda (Fig. 16). We were able tional families were studied . H amin oea linda
to obtain cells that were satisfactory for study and H. musett a (Bullacea, Atyidae) had a hapfrom only one specimen. These cells had 17 loid chromosome number of 17, but Smaragdin ella calyculata and Latb opbtbalmus smaragbivalents during Metaphase 1.
6. Haminoea musette (Fig. 15). The two dinus (Philinacea, Smaragdinellidae) each had
specimens studied both had 17 bivalents in cells the haploid numb er 18. It will be interesting to
at the diakinesis stage. The diakinesis bivalents see if species of the other families of the Ph ilinacea ( the Philinidae, Scaphandrid ae, Aglaof one such cell are shown in Figure 15.
7. Laibophtb almus smaragdin us (Fig. 17). jidae, Gastropteridae, and Runcinidae) also
Two specimens had meiotic cells that were satis- have 18 pairs of chromosomes. If so, this would
factory for chromosome number determinations. separate this superfamily from all other cephaAll dividing cells from which accurate counts laspideids and, addition ally, from all other cytocould be made had 18 bivalents.
logically known entomotaenids and anaspideids
8. Sm aragdin ella calyculata (Fig. 18) . Satis- and from most of the sacoglossans. The haplo id
factory results were obtained from two speci- number 18 in this group may have another
mens. Both had 18 bivalents during diakinesis significance in that it seems to strengthen Pelseneer's ( 1893) and Boettger's (1955) views
and Metaphase 1.
9. Oncbidella eoelinae ( Figs. 19 and 20). regarding the origin of the Basommatophora
The two individuals on which we were able to from the Cephalaspidea, since the haploid numobtain satisfactory observations both had 18 ber 18 is basic for the Basommatophora.
D olabrijera dolabrife ra and Stylocbeilus
bivalents during diakinesis and Metaphase 1.
longicaud« (Anaspidea, Aplysiidae) both had
the haploid number 17, as did the two species
DISCU SSION
studied from this family by Inaba (19 59a) .
The chromosome numbers of the eight genera
Onch idella eoelinae (Soleolifera, Onch idiiof opisthobranchi ate mollusks presented here dae) had a haploid number of 18, which is
add to the information previously obtained by one bivalent more than that reported by Inaba
reliable authors. Of the species studied five (1961 a) for O. kttrodai of the same genus from
belong to three families not studied in past re- Japan, but n=18 is the same number reported
ports, i.e., the Favorinid ae (Nudibranchia) , and by Natarajan (1959) for Oncbidium uerracuthe Atyidae and Smaragdinellidae (Cephala- laturn from India . Much more cytological
spidea).
information is desirable for the various species
D endrodoris nigra (Nudibranchia, Dendro- belonging to this aberrant order, which is somedorididae) was studied previously by Inaba and times placed with the "pulrn onates" ( e.g.,
Hi rota (1958) . We found the same number Baker, 1955) .
Th e conservativeness of chromosome number
of chromosomes (n=13) for this species from
Eniwetok as they reported it to have from in the opisthobranchs indicates that these molJapan. H eruiella mietta (Nudibranchia, Favor- lusks are extremely resistant to changes in
inidae) from Eniwetok also had a haploid chromosome number s, regardless of major
number of 13, which adds to the growing evolved morph ological diversities within the
information regarding the extreme conservative- various groups, and that certain major divisions
ness of chromosome numbers of most opistho- (i .e., the Nudibranchia and the orders with
branchs. All 16 species of nudibranchs (belong- n=17 and higher chromosome numb ers) have
ing to nine different families) stud ied so far probably been separated for an extremely long
have this same haploid number, n=13.
geological time. In this regard, B osellia mimetAmong the Cephalaspidea three species have ica is either an extremely aberrant species, or
been studied previously (In aba, 1959a and per- its cytological evolution has been much more
sonal communication) , each belonging to a dif - rapid than has been the evolution of its gross
Chromos omes of Eniwetok Opi sthob ranchiates-B u RCH AND NATARA]AN
morphology in respect to all other opist hobranchs so far studied.
ACKNOWLEDGM ENTS
Gr ateful acknowledgm ent is made to the
United States Atomic Energy Commission for
supporting the study by the senior author at
Eniwetok by pro vidin g tr avel funds, logistical
supp ort, and use of the facilities of the Eniwetok Marine Biological Laboratory. The cooperation of the U.S.A.E.C. Eniwetok Field Office
and of H olmes and N arver, Inc. greatly facilitated the field collecting . A note of gra titude
is due D r. I. Eugene W allen, formerly with th e
U.S.A.E.C., Dr. Robert W. Hiatt, Uni versity of
H awaii, and Pro f. Henry van der Schalie, Un iversity of M ichigan, for promoting th ese
studies ; to Dr. William H . H eard , Florida
State Un iversity, for assistance whil e at Eniwetok ; and to John 1. Tottenham, Staff Artist,
Museum of Zoology, Uni versity of M ichigan,
fo r pr eparing illustratio ns of the animals used
in this study.
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BOETTGER, C. R. 1955 . Di e Systematik der
euthyneuren Schnecken. Verh . dtsch. zool.
Ges., 1954, pp . 253-280.
BURCH, J. B. 1965 . Chr omosome numb ers and
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C AR NO Y , H . B. 1887. Confe rence donnee a la
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La Cellul e, Rec. Cyto!. H istol. Gen. 3(2 /3) :
227-273.
INABA, A. 1959a. Cytological studies in molluscs. III. A chromosome survey in the opisthobranchiate Gastropoda. Annot. Zool.
Japan 32 ( 2) :81- 88.
- -- 1959b. On the chro mosomes of a nudi-
259
branch gastropod, D endrodoris (D endrodoris) nigra (Stimpson) . J. Sci. Hiroshima
Uni v. 18 (9 ) :95-98.
- - - 1961. Chromosomes of some opistho branchs . [In Jap anese.] Dobutsugaku Z asshi
70:24.
- - - and R. HIROTA. 1954. On the chro mosomes of Gl ossodoris pallescens. [In Japanese.] Do butsugaku Zasshi 63 :437.
- -- - - 1958. A chromosome survey
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169-1 74.
MAKINO, S. 1956. An Atl as of the Chromosome
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MANCINO, G. and M. SORDI. 1964a. Ricerche
cariologiche in D oris uetrncosa Cuvier, 1804
(Gasterop odi, Op isthobranchi) del litorale
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- - - - -- 1964b . II corredo cromosom ico
di alcuni Op isthobranchi Sacoglossi del mar
Tirreno . Ibid. 71. 12 pp .
NATARA]AN, R. 1959. Chromosomes of the
slug Oncbidium uerraculatnrn Cuv. ( Mollusca: Gastropod a : Pul monata) . J. Zool. Soc.
India 11 (1 ) :30-33.
- - - 1960. Further cytological studies in
Pulmonata (Mollusca: Gastrop oda) . Ibid.
12(1) :69-79.
N EWCOMER, E. H . 1953. A new cytological and
histological fixing fluid. Science 118 ( 3058 ):
161.
N EWTON, W . C. F. 1926. Chromosome stud ies
in T ulip« and some related genera. J. Linn .
Soc. 47 ( 316) :339-354.
PELSENEER, P. 1893. Recherches sur divers
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SANFELICE, F. 1918. Recherches sur le G enese
des corpu scules du MollllSCll1IZ cont agiosnm .
Ann . l'Inst. Pasteur 32( 8 ) :36 3-371.