Chromosomes of Some Op isthobranchiate Mollusks from Eniwetok Atoll, W estern Pacific' ]. B. BURCH2 and R. NATARA] AN3

ABSTRACT: Chromosome numbers are reported for nine species of opistho­ branchiate mollusks from Eniwetok Atoll, Marshall Islands, western Pacific. In the N udibranchia, both D endrodoris nigra (Dendrodori didae) and H erviella mietta (Favorinidae) were found to have 13 bivalents during male meiosis.In the , D olabrifera dolabrifera and Stylocbeilus longicallda () both had 17 bivalents during male meiosis. In the , Haminoea linda and H. musetta (Atyidae) each had 17 pairs of chromosomes during male meiosis and Latb opb tbalmus smaragdimts 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 mol­ lusks is demonstrated by that fact that all 18 nudibranchs from 10 families studied so far have the single haploid chromosome number 13, and that 18 of the 21 spe­ cies of the orders Entomotaeniata, Anaspidea, Cephalaspidea, and have 17 pairs of chromosomes. The 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 within or related to this cepha­ laspid superfam ily (Philin acea) .

INRECENT YEARS, detailed investigations have mainly because of the difficulties they present been made on the chromosomes of many ba­ in collection and identification. Relying on the sommatophoran and stylommatophoran snails studies of various authors during the early part (Burch, 1965) , but relatively few species of of the present century, Makino (195 1) listed the opisthobranchiate! orders have been studied, the chromosome numbers of 16 opisthobran­ chiate species, but recent investigations by Inaba 1 The field work for this investigation was sup­ and our present studies indicate that the earlier ported by the D ivision of Biology and Medicine, U. S. Atomic Energy Commission. The research was records are not dependable and so are obsolete. supp orted ( in part) by grants from the N ational Previous reliable reports on the chromosomes of Science Found ation , W ashington, D . C. ( GB 787 ) opisthobranchiate gastropods are those of Inaba and the N ational Institute of Allergy and Infectious and Hirota (1954, 1958), Inaba (1959a, 1959b, Diseases, U. S. Pu blic H ealth Service ( 5 T1 AI 41-08 ) . Manuscript received March 14, 1966. 1961) , Natarajan (1959, 1960), Mancino and 2 Museum and D epartment of Zoology, University Sordi ( 1964a and b) , and Burch ( 1965) . These of Michigan, Ann Arbor. Supported by a Public authors give the chromosome numbers of 36 Health Service research career program award (No. species belonging to 21 families and 7 orders 5-K3-AI-19, 451-03 ) . (Tables 1 and 2) . This is a very small number 3 Museum of Zoology, Uni versity of Michigan, Ann Arbor. when compared with the great multitude of 4 Boettger (1955 ) considers the orders Opis tho­ species currently recognized in the opisthobran­ branchiata and Pulmona ta (together equivalent to the chiate orders. Th e present paper presents the subc lr-ss ) to be unnatural ones, and in­ chromosome numbers of 9 opisthobranchiate stead divides the Euthyne ura into seven orders. While we do not disagree with this, it is still convenien: to speak of his five lower (m ainly marine) euthy­ branchs" as distinguished from the more advanced neuran orders (Nudibranchia, Soleolifera, Cephala­ Basommatophora (m ainly freshwater) and Stylomma­ spidea, Sacoglossa, and Anaspidea) as "opistho- tophora (l and inhabitants). 252 Chromosomes of Eniwetok Opisthobranchiates-s-Buncn AND NATARAJAN 253

TABLE 1

CHROMOSOME NUMBERS PREVIO USLY R EPORT ED IN THE O PISTHOBRAN CHIAT E O RDERS NOTASPIDEA AN DN UDIBRAN CH IA

CHROMOSOME N UM BER

SPECIES 20 n LOCA LITY REFEREN CE

N OTASPIDEA Pleurobranchidae Pieurobrancbaea nauaezealandiae 24 12 Japan Inaba, 1959a N UDIBRANC H IA Dorididae Glossodoris [estiua 13 Japan Inaba and H irota, 1958 Glossodoris pallescens 13 Japan Inaba and H irota, 1954 ; 1958 Rostanga arbutus 26 13 Japan Inaba, 1959a Discodoris pardalis 13 Japan Inaba , 1959a uerrucosa 26 13 Italy Mancino and Sordi, 1964a miniata 26 13 Japan Inaba and Hirota, 1958 D endrodoris nigra 26 13 Japan Inaba and Hirota, 195'8; Inaba, 1959b Trioph idae Kaloplocamus ramosus 13 Japan Inaba and H irota, 1958 Plocamopberus tilesii 26 13 Japan Inaba and H irota, 1958 Goniodorididae Okenia barnardi 13 Japan Inaba and Hi rota, 1958 Fimbriidae Melibe papillosa 26 13 Japan Inaba , 1959a Dotonidae Doto bella 13 Japan Inaba, 1961 Arminidae D ermatobrancbus striat us 26 13 Japan Inaba and H irota, 1958 Cuthonidae Catriona pupillae 13 Japan Inaba, 1961 Facelina ceylonica 13 J apan Inaba and Hi rota, 1958 Facelina [aponica 13 Ja pan Inaba and H irota, 1958 species belonging to 6 families and 4 orders NUDlBRANCHIA (Table 3) , which were collected in shallow L Dendrodoris nigra (St impson ) (Fig. 1). waters around thr ee island s of Eniwetok Atoll N orth end of Japtan Island , und er loose pieces in the western Pacific by the senior author and of dead coral. Ap ril 1, 1960 . Dr. W illiam H . H eard during early 1960. 2. Heroielln mietta Marcus and Burch ( Fig. 2) .N orth end of Eniwetok Island on the la­ MATERIAL AND METH ODS goon side, in about 10 em of water at low tide, under submerged pieces of dead coral. Ap ril 2, The species studied in this investigation and 1960. the localities wh ere they were collected are listed below. Duplicate specimens have been ANASPIDEA deposited in the collections of the Mu seum of 3. Dolabrifera dolabrifera (Rang ) (Fig. 3) . Zoology at the University of Michigan, the Und er loose pieces of coral on seaward tid e flat University of Sao Paulo, and the University of at the north end of Parry Island . March 25, Hawaii. 1960. 254 PACIFIC SCIENCE, Vol. XX I, April 1967

TA BLE 2 CHROMOSOME NUMBERS PREVIOUSLY REPORTED IN THE O PISTHOBRANCHIATE ORDERS ENTOMOTAENIATA, CEPHALASPIDEA, ANASPIDEA, SACOGLOSSA, AND SOLEOLIFERA

CHRO MOSO ME NUMBER SPECIES 2n n LOCALITY REFE RE NCE

ENTOMOTAENIATA PyramideJlidae T iberia fasciata 17 Japan In aba, pers . comm . ANASPIDEA Apl ysiidae Petalifera punctulata 34 17 Japan Inaba, 19 59a N otarcbus leacbii [reeri 17 Japan Inaba, 1959a CEPHALASPIDEA Acteonidae Cylicbn atys angusta 17 Japan Inaba, pers . comm . Philinidae Pbiline japonica 17 Japan Inaba, 1959a Aglajidae A glaja gigliolii 34 17 Japan Inaba, 1959a SACOGLOSSA Elysiidae amakusana 17 Japan Inaba, 1959a Elysia oiridis 17 Italy Mancino and Sordi, 1964b Stiligeridae Alderia nigra 17 Japan In aba, 1961 H ermaeopsis oariopicta 17 Italy Mancino and Sord i, 1964b Placida dendr itic« 34 17 Italy Mancino and Sordi, 1964b Placida viridis 34 17 It aly Mancino and Sordi, 1964b Stiliger uesiculosus 34 17 Italy Mancino and Sord i, 1964b Juliidae Bertbelinia limax 17 Japan Inaba, 1961 Polybranchiidae mimetica 14 7 It aly Mancino and Sordi , 1964b SOLEOLIFERA VeroniceJlidae V eronicella fioridana 16 U .S.A. Burch, 1965 Laeuicaulis alte 17 India N ataraj an, 1960 On chidiidae Oncidiell« kurodai 17 Japan In aba, 1961 Onchidium uerraculatum 36 18 India N atar ajan, 1959

4. Stylocheilus longicauda (Quoy and Gai­ 6. Haminoea musette Marcus and Burch mard) (Fig. 4) .In tide flats of Eniwetok Is­ (Fig. 6). Middle part of Parry Island on sea­ land. March 4, 1960. ward tide flats. April 2, 1960. 7.Lathophthalmus smaragdinus (Ruppel CEPHA LASPIDEA and Leuckart) (Fig. 7) . Collected at the south 5. Haminoea linda Marcus and Burch (Fig. end of Parry Island, under loose pieces of coral 5) . Parry Island, in sand, in about 2 m of water, on seaward tide flat. March 15, 1960. in lagoon, about 17 m from shore. March 31, 1960. 8. Smaragdinella calyett/ata (Broderip and Chromosomes of Eniwetok Opistbobranchiates-i-Buncn AND N ATARAJAN 255

T ABLE 3

CHROMOSOME NUMBERS OF O PISTH OBRAN CHS O BSERVED IN THIS STUDY

N UM BER OF CHROMOSO ME SP ECIMENS GIVING SPECIES N UMBER (n) RESU LTS

N UDIBRANCHIA Dendrod orididae Dendrodoris nigra 13 5 Favorinidae Heruiella miette 13 ( 2n = 26) ANASPIDEA Aplysiidae Dolabrifera dolabrijera 17 9 Stylocbeilus longicauda 17 2 CE PH ALASPIDEA Atyidae Haminoea linda 17 1 Haminoea musetta 17 2 Smaragdinellidae Lathophthalmus smaregdinus 18 2 Srnaragdinella ealyeulata 18 2 SOLEOLIFERA Onchidiidae Oncbidella euelinae 18 2

Sowerby) (Fig. 8) . In lagoon at north end of camera lucida and reproduced at a table-top Eniwetok Island. magnification of 4260 X .

SOLEOLIFERA 9. Oncbidella euelinae Marcus and Burch OBSERVAT IONS (Fi g. 9). In cracks in coral slabs above water 1. D endrodoris nigra (Fig. 10). The five line (at low tide) 0;1 the lagoon side at the indi viduals of this species on which we were north end of Eniwetok Island. April 5, 1960. able to obtain satisfactory observations all had The materials examined consisted of ovo­ 13 bivalents during prophase of the first meiotic testes fixed in either N ewcomer's (1953) or division. Carney's (1887) fluids, or the fixative of San­ 2. Heruiella miette (Fi g. 11) . Only one felice (191 8) . The material fixed in New­ specimen of this species gave satisfactory results. comer's or Carney's fluids was stained by the It had 13 bivalents durin g diakinesis and 26 acetic-orcein squash technique (La Cour, 1941) , chromosomes durin g metaphase in spermato­ and reproductive tissues fixed in Sanfelice's gonial cells. fluid were sectioned at either 8 or 10 micra and 3. D olabrifera dolabrifera (Fi gs. 12 and stained with Newton's (1926) crystal violet. 13) . N ine specimens had meiotic cells that All observations were made on meiotic cells of were satisfactory for chromosome numb er de­ spermatogenesis (except in Heruiella mietta, termin ations. All had 17 bivalents during Pro­ where spermatogonial cells were also studied) phase I and Metaphase 1. Th e chromosomes of with a Nikon (Nippon Koga ku) microscope a cell durin g diakinesis from an acetic-orcein equipped with a 100 X (n .a. 1.25) oil immer­ squash preparation are shown in Figure 12. sion objective and lOX , 20X , and 30X ocu­ Metaphase I bivalents from a cell of paraffin lars. Drawings were made with the aid of a sectioned material are shown in Figure 13. 256 PACIFIC SCIENCE, Vol. XXI, April 1967

(\

I\'\'e.\, } '.: \\ . ;' ....~ .... ~~J~'it-

...0 ('II Chromosomes of Eniwetok Opisthobranchiates-i-Buaca ANDN ATARAJAN 257 -. ...,.c.~ -'..t ~ •• II .,. " , . ~ ""0"...••....., ~c;'c;.a ~ °f'~ " . 12 10 It 11 -.~ ,. •..••••• '\••• .... ' ~...... • .. ,pit. .. . . 16 ..- • $C • ., , • 4t't~"" '.f. ~ AI~" ..... 13 V,. 'V., 1) ~ al·"••••• 15 •• 14 19 ft...... I ... .. r,) t' .. W t; .- ,,,","..... , -.9• " .-'--~ .... , .II -.. ,- ... f 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, Lath­ ophthalmus smaragdinus, 18, Smaragdinella calyculata. 19 and 20, Onehidella eoelinae. Figs. 10-12, 14, 15, 17, and 20 are of male diakin esis bivalents; 13,16, 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, Heruiella 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 millimeters. 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 present in­ mens. Both had 17 bivalents during diakinesis. vestigation two species from each of two addi­ 5. Haminoea linda (Fig. 16). We were able tional families were studied. Hamin oea linda to obtain cells that were satisfactory for study and H. musetta (Bullacea, Atyidae) had a hap­ from only one specimen. These cells had 17 loid chromosome number of 17, but Smarag­ bivalents during Metaphase 1. din ella calyculata and Latb opbtbalmus smarag­ 6. Haminoea musette (Fig. 15). The two dinus (Philinacea, Smaragdinellidae) each had specimens studied both had 17 bivalents in cells the haploid number 18. It will be interesting to at the diakinesis stage. The diakinesis bivalents see if species of the other families of the Phili­ of one such cell are shown in Figure 15. nacea (the Philinidae, Scaphandridae, Agla­ 7. Laibophtbalmus 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 cepha­ All dividing cells from which accurate counts laspideids and, additionally, from all other cyto­ could be made had 18 bivalents. logically known entomotaenids and anaspideids 8. Smaragdinella calyculata (Fig. 18) . Satis­ and from most of the sacoglossans. The haploid 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 Pel­ and Metaphase 1. seneer's (1893) and Boettger's (1955) views 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 num­ obtain satisfactory observations both had 18 ber 18 is basic for the Basommatophora. bivalents during diakinesis and Metaphase 1. D olabrijera dolabrife ra and Stylocbeilus longicaud« (Anaspidea, Aplysiidae) both had the haploid number 17, as did the two species DISCUSSION studied from this family by Inaba (1959a) . The chromosome numbers of the eight genera Onch idella eoelinae (Soleolifera, Onchidii­ of 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 (1961a) for O. kttrodai of the same 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 uerracu­ the Atyidae and Smaragdinellidae (Cephala­ laturn from India . Much more cytological spidea). information is desirable for the various species Dendrodoris nigra (Nudibranchia, Dendro­ belonging to this aberrant order, which is some­ dorididae) was studied previously by Inaba and times placed with the "pulrnonates" (e.g., Hirota (1958) . We found the same number Baker, 1955) . of chromosomes (n=13) for this species from The conservativeness of chromosome number Eniwetok as they reported it to have from in the opisthobranchs indicates that these mol­ Japan. H eruiella mietta (Nudibranchia, Favor­ lusks are extremely resistant to changes in inidae) from Eniwetok also had a haploid chromosome numbers, 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 numbers) have ing to nine different families) studied so far probably been separated for an extremely long have this same haploid number, n=13. geological time. In this regard, Bosellia mimet­ Among 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 Chromosomes of Eniwetok Opisthob ranchiates-BuRCH AND NATARA]AN 259 morphology in respect to all other opistho­ branch gastropod, D endrodoris (Dendro­ branchs so far studied. doris) nigra (Stimpson) . J. Sci. Hiroshima Uni v. 18 (9) :95-98. --- 1961. Chromosomes of some opistho­ ACKNOWL EDGM ENTS branchs . [In Jap anese.] Dobutsugaku Zasshi Grateful acknowledgment is made to the 70:24. United States Atomic Energy Commission for - - - and R. HIROTA. 1954. On the chromo­ supporting the study by the senior author at somes of Glossodoris pallescens. [In Japa­ Eniwetok by pro viding travel funds, logistical nese.] Dobutsugaku Zasshi 63:437. support, and use of the facilities of the Eniwe­ ------1958. A chromosome survey tok Marine Biological Laboratory. The coopera­ in ten species of nudibranchs ( , tion of the U.S.A.E.C. Eniwetok Field Office ) . Japan. J. Zool. 12( 2) :157-162. and of Holmes and N arver, Inc. greatly facil­ LA COUR, 1. 1941. Acetic-orcein : A new stain­ itated the field collecting. A note of gratitude fixative for chromosomes. Stain Techno!. 16 : is due D r. I. Eugene W allen, formerly with the 169-174. U.S.A.E.C., Dr. Robert W. Hiatt, Uni versity of MAKINO, S. 1956. An Atl as of the Chromosome Hawaii, and Prof. Henry van der Schalie, Un i­ Numbers in . 2nd ed. Iowa State Col­ versity of Michigan, for promoting these lege Press, pp. 22-23. studies ; to Dr. William H . H eard, Florida MANCINO, G. and M. SORDI. 1964a. Ricerche State Un iversity, for assistance while at Eniwe­ cariologiche in D oris uetrncosa Cuvier, 1804 tok ; and to John 1. Tottenham, Staff Artist, (Gasteropodi, Opisthobranchi) del litorale Museum of Zoology, Uni versity of Michigan, livornese. Atti Soc. Toscana Sci. N at., Ser. B, for preparing illustrations of the animals used 71. 14 pp. in this study. ------1964b . II corredo cromosom ico di alcuni Op isthobranchi Sacoglossi del mar Tirreno. Ibid. 71. 12 pp . REFERENCES NATARA]AN, R. 1959. Chromosomes of the BAK ER, H . B. 1955. H eterurethrous and aula­ slug Oncbidium uerraculatnrn Cuv. (Mol­ copod. N autilus 68 :109-112. lusca: Gastropod a: Pulmonata) . J. Zool. Soc. BOETTGER, C. R. 1955. Die Systematik der India 11 (1 ) :30-33. euthyneuren Schnecken. Verh. dtsch. zool. - - - 1960. Further cytological studies in Ges., 1954, pp . 253-280. Pulmonata (Mollusca: Gastropoda) . Ibid. BURCH, J. B. 1965. Chromosome numb ers and 12(1) :69-79. systematics in euthyneuran snails. Proc. First N EWCOMER, E. H . 1953. A new cytological and European Malacol. Congr., pp. 215-241. histological fixing fluid. Science 118 ( 3058): C ARNOY, H . B. 1887. Confe rence donnee a la 161. Societe beIge de Microscopie. Appendice: I. N EWTON, W . C. F. 1926. Chromosome stud ies Les Globules polaires de l' A scaris clauata. in Tulip« and some related genera. J. Linn . La Cellule, Rec. Cyto!. H istol. Gen. 3(2 /3) : Soc. 47 ( 316) :339-354. 227-273. PELSENEER, P. 1893. Recherches sur divers INABA, A. 1959a. Cytological studies in mol­ Opisthobranches. Academie royale de Bel­ luscs. III. A chromosome survey in the opis­ gique. Pp. 1-157, pls. 1-25. thobranchiate Gastropoda. Annot. Zool. SANF ELICE, F. 1918. Recherches sur le Genese Japan 32( 2) :81- 88. des corpu scules du MollllSCll1IZ contagiosnm. --- 1959b. On the chromosomes of a nudi- Ann . l'Inst. Pasteur 32(8) :363-371.