1966 109

Chromosomes of Some Archaeopulmonata(Mollusca: Basommatophora)1

R. Natarajan and J. B. Burch

Museum and Department of Zoology, University of Michigan, Ann Arbor, Michigan, U. S. A.

Received March 5, 1965

Cytological studies of aquatic snails belonging to the higher limnic Basommatophora (Euthyneura: Branchiopulmonata) are rather numerous (for reviews see Burch 1960b, 1965), but reports on the lower basommatophoran snails (Archaeopulmonata) are relatively few. Publications to date are limited to the papers by Inaba (1950, 1953), Meyer (1955), Natarajan (1958), Burch (1960a, b, 1962) and Burch and Patterson (1963). These authors studied 11 species (Table 1). The present paper presents information on 7 additional species from the United States, Bermuda, Uruguay and the western Pacific.

Materials and techniques The specimens used in this study are listed below:

Family Siphonariidae Siphonaria (Siphonaria) alternata (Say). Bermuda. Collected by James N. Cather, August, 1964. Siphonaria (Siphonaria) guamensis Quoy and Gaimard. On rocks in the intertidal zone on the lagoon side, southwest end of Parry Island, Eniwetok Atoll, Marshall Islands, western Pacific. Collected by J. B. Burch and W. H. Heard, March 7, 1960. Siphonaria (Siphonaria) laciniosa (Linnaeus). Intertidal rocks at the south end of the beach at Anse Vata, Noumea, New Caledonia. Collected by J. P. E. Morrison, November 22, 1960. Siphonaria (Mouretus) pectinata (Linnaeus). Miami, Florida. Collected by J. A. Weber, February 23, 1959.

Family Ellobiidae Cassidula vespertilionis (Lesson). Mangrove swamp along the road on the east side of Boulari Bay, New Caledonia. Collected by J. P. E. Morrison, January 9, 1961. Melampus coffeus (Linnaeus). West of Tybee Island (Savannah Beach), Georgia, U. S. A. Collected by Harold W. Harry, August 29, 1960. Melampus ? coffeus Five miles west of Jekyll Island, Georgia, U. S. A., on highway U. S. 17. Collected by Harold W. Harry, August 28, 1960. Melampus bidentatus lineatus Say. Under logs and other debris on the shore of Greenvale Greek, Rocky Neck Farm, Mollusk, Lancaster County, Virginia, U. S. A. Collected by J. P. E. Morrison, October 26, 1958.

1 This investigation was supported (in part) by research grants GB 787 from the National Science Foundation, Washington, D. C. and 5 Tl AI 41-07 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service, and (in part) by a Public Health Service research career program award (number 5-K3-Al-19, 451-02) to the second author.

Cytologia 31, 1966 8 110 R. Natarajan and J. B. Burch Cytologia 31

Family Chilinidae Chilina fluviatilis (Maton). Bay of Colonia, La Plata River, Colonia, Uruguay. Collected by Miss Mirta Calcaterra, November 12, 1962, via Miguel A. Klappenbach.

Table 1. Chromosome numbers in Archaeopulmonata

The material examined consisted of ovotestes killed , fixed and preserved in Newcomer's fluid for acetic-orcein squash preparations (La Cour 1941) and in the fluid of Sanfelice

(1918) for sectioning. Sectioned material was stained with 1% aqueous crystal violet

(Newton 1926, Huskins 1927, Conn 1936). All observations were on cells of spermatogenesis

and were made with Nikon microscopes using 100•~ (n . a. 1.25) oil immersion objectives and 10, 20 and 30•~ oculars. All drawings of chromosomes were made with the aid of a camera

lucida and reproduced at a table top magnification of 5700•~ . 1966 Chromosomes of Some Archaeopulmonata 111

Observations Family Siphonariidae The only previous chromosome report on a siphonariid limpet is that of Inaba (1953) in which the numbers n=16, 2n=32 were reported for Siphon aria japonica from Japan. We have studied 4 additional species listed below. Our material came from Florida (U. S. A.), Bermuda, Eniwetok Atoll in the western Pacific and New Caledonia. Siphonaria (Siphonaria) alternata (Say). Five animals fixed in New comer's fluid were examined, but only 2 gave satisfactory results. Chromo somes were most easily studied during late prophase of the first meiotic division (diakinesis) of spermatogenesis. Such cells clearly had 16 bivalents (Fig. 1). No satisfactory mitotic figures were found.

Figs. 1-13. Chromosomes from spermatogenesis cells of some Archaeopulmonata. 1, Si

phonaria alternata (•‰diakinesis). 2, S. guamensis (•‰diakinesis). 3, S. laciniosa (•‰dia kinesis). 4, S. laciniosa (•‰Telophase I dyads). 5, S. pectinata (•‰diakinesis). 6, Chilina

fluviatilis (•‰diakinesis). 7, Melampus coffees (•‰Metaphase I). 8, M. bidentatus lineatus (•‰ Metaphase I). 9, M. ? coffeus (•‰diakinesis-tetraploid). 10, C. fluviatilis (spermato

gonial metaphase). 11, Cassidula vespertilionis (spermatogonial metaphase). 12, M. coffeus

(spermatogonial metaphase). 13, M. bidentatus lineatus (spermatogonial metaphase). Scale; one micron.

Siphonaria (Siphonaria) guamensis Quoy and Gaimard. A total of 22 specimens were examined; 15 animals were sectioned and the ovotestes of 7 specimens were used to make squash preparations. Eleven animals, 8 fixed

8* 112 R. Natarajan and J. B. Burch Cytologia 31 in the fluid of Sanfelice and 3 fixed in Newcomer's fluid, had cells satis factory for cytological studies. Only cells with chromosomes at the diakinesis and Metaphase I stages were suitable for critical examination, and all of these had 16 bivalents present (Fig. 2). Siphonaria (Siphonaria) laciniosa (Linnaeus). Five specimens were examined by the acetic-orcein squash technique, of which 3 gave satisfactory results. Cells in diakinesis had 16 bivalents (Fig. 3) and those cells at Telophase I each had 2 sets of 16 dyads (Fig. 4). Siphonaria (Mouretus) pectinata (Linnaeus). Six specimens fixed in New comer's fluid were studied, but only 2 had meiotic cells from which accurate counts could be made. Cells at diakinesis clearly had 16 bivalents present (Fig. 5). Family Ellobiidae Previous studies on ellobiid snails are those of Meyer (1955) on Phytia myosotis (n=18), Natarajan (1958) on Pythia plicata, Cassiduta mustelina (both with n=17, 2n=34) and Melampus ceilonicus (n=18, 2n=36), and Burch (1960a, b) on Phytia myosotis marylandica, Melampus bidentatus lineatus and Detracia floridana (all 3 with n=18, 2n=36). We have studied 2 additional species and accurately determined the caryotype of one of the species previously studied. These observations, all from squash preparations, are given below. Cassidula vespertilionis (Lesson). Ten animals were examined, of which only 2 had cells satisfactory for study. Spermatogonial cells had 34 chromo somes (Fig. 11). No meiotic stages suitable for chromosome counts were found. Melampus coffeus (Linnaeus). Seven specimens were examined, but only 2 were satisfactory for cytological studies. Thirty-eight chromosomes were found in spermatogonial cells (Fig. 12) and 19 bivalents in meiotic cells (Fig. 7) of both animals. This is the first time this number has been re ported in the Ellobiidae. Melampus ? coffeus. The shell morphology of the specimens in this popu lation deviated enough from more typical ill. coffeus to cause the collector (Dr. Harold W. Harry) to doubt whether or not they were the same species as Al. coffeus. We examined 9 specimens, all with active stages of gameto genesis. Eight specimens clearly had 19 elements present in meiotic cells and 19 pairs of chromosomes in spermatogonial cells . The other specimen, obviously a tetraploid, had 38 elements present in cells at diakinesis (Fig . 9). Melampus bidentatus lineatus Say . The chromosome number for this species, n=18, 2n=36, has already been established by Burch (1960a, b). Our present study confirms the earlier report and adds information on the caryotype of the species. We examined 20 specimens of a 1st year population , of which 14 were 1966 Chromosomes of Some Archaeopulmonata 113 satisfactory for study. All specimens had 18 bivalents present in dividing cells of the 1st meiotic division (Fig. 8) and 36 chromosomes in spermato gonial cells (Fig. 13). According to position of the centromere, the mitotic metaphase chromosomes fall roughly into 2 groups (Fig. 14): 7 pairs have medianly placed centromeres and in 11 pairs the centromeres are submedianly placed.2 In 8 of the submetacentric pairs the centromeres are rather close to the middle of the chromosomes, but in the other 3 pairs , the centromeres are almost close enough to the ends of the chromosomes to be considered subterminal. One pair of chromosomes, a slightly submedianly constricted pair, is noticeably larger than all the rest (a similar pair of much larger chromosomes is found in a great variety of euthyneuran snails, and accounts for the meiotic "heterochromosome" of Perrot (1930)). In the 2 cells in which we took measurements, these 2 largest chromosomes measured 2.7 - 2.8 micra. The other chromosomes grade gradually in size from 2.1 for the largest to 1.0 micra for the smallest. Chromosomes of the smallest pair are submedianly constricted; the next 3 pairs are nearly equal in size and are metacentrics, measuring 1.2 micra in length.

Fig. 14. Spermatogonial mitotic chromosomes of two cells paired and arranged according to decreasing lengths. M marks the metacentric chromosomes. Scale; one micron.

Family Chilinidae There are no other chromosome studies on snails of the primitive South American freshwater family Chilinidae. Burch and Patterson (1963) studied Latta neritoides of the closely related New Zealand family Latiidae3 and found it to have 18 pairs of chromosomes. Chilina fluviatilis (Maton). Nine specimens were examined, all with active stages of gametogenesis. Thirty-six chromosomes were found in spermatagonial cells (Fig. 10) and 18 bivalents in meiotic cells (Fig. 6).

Discussion

Chromosome numbers have now been determined for 17 species and 1 subspecies belonging to 6 families of the Archaeopulmonata. Five different 2 Some of the chromosomes appear to be almost subterminally constricted, but we are calling all chromosomes submedianly constricted that have short arms less than 1/2 but 1/3 or more their total length. 3 Boettger (1955) includes Latia in the family Chilinidae but as a separate subfamily, the Latiinae. He regards Chilina to be the most primitive of the freshwater Basom matophora because in its nervous system it has a crossed chain of visceral ganglia. As a result of flattened limpet-shape, Latia has apparently lost this visceral chain. 114 R. Natarajan and J. B. Burch Cytologia 31

chromosome numbers have been found, n=16, n=17, n=18, n=19 and n=38. The latter number is undoubtedly a tetraploid derivative from the haploid number 19. The 5 species of the Siphonariidae all have 16 pairs of chromosomes. The fact that they represent 2 different subgenera and are from widely separated localities (Japan, Marshall Islands, New Caledonia, Bermuda and Florida, U. S. A.) points to a cytological conservation in these gastropods that has been demonstrated in numerous other snail groups (see Burch 1965). The lower chromosome numbers of the Siphonariidae may indicate that they are the most primitive family of the suborder, if the suggestion (Burch 1961) that changes in chromosome numbers in the Euthyneura usually involve an increase rather than a decrease in numbers, and that higher numbers are nearly always found in the phylogenetically more advanced taxa. Four chromosome numbers occur in the 8 species of ellobiids so far studied. The haploid number 18 is the most common, occurring in 5 of the species and subspecies. Pythia plicata and the 2 species of Cassidula have the haploid number 17, while Melampus coffeus has the haploid number 19. Morton (1955) places Pythia, Ovatella (=Phytia), Ophicardelus and Cassidula in the "primitive section" (Subfamily Pythiinae) of the Ellobiidae. He considers Melampus (and also presumably Detracia) to belong to one of the most advanced groups of the family. It is perhaps significant that the 3 species with the lower number (n=17) are found in the more primitive taxon. One of the 9 specimens of "Melampus ? coffeus" inspected from 5 miles west of Jekyll Island, Georgia (U. S. A.), had 38 bivalents present at prophase and metaphase of the first meiotic division, whereas the other 8 specimens had 19 bivalents. Two facts lead us to believe that this was perhaps a hybrid population: 1) the shell morphology of these specimens was different enough from more typical M. coffeus to cause Dr. Harry some doubt as to whether they were the same species; and 2) the appearance of the tetraploid individual in such a population would perhaps suggest amphipolyploidy . It would be desirable to study many more individuals from this population , both cyto logically and morphologically, in order to establish its biological relationship to other populations of M. coffeus in the Georgia region . A single species has been studied in each of the families Amphibolidae , Chilinidae, Latiidae and Acroloxidae. Each species has the haploid number 18, and in this way they do not differ from the higher limnic Basommatophora (Branchiopulmonata).

Summary This paper presents the chromosome numbers of 7 species of primitive basommatophoran snails and discusses these numbers in relation to systematics and to those numbers reliably reported for the Archaeopulmonata by other authors. 1966 Chromosomes of Some Archaeopulmonata 115

1. Sixteen bivalents were observed in cells of the first division of meiosis in the 4 species of Siphonariidae studied, Siphonaria alternata, S. guamensis, S. laciniosa and S. pectinata. 2. Several different chromosome numbers were observed in the two species of Ellobiidae studied. Seventeen bivalents were observed in Cassidula vespertilionis and 19 pairs of chromosomes were found in Melampus coffees and in a population (of hybrids ?) resembling this species but differing slightly on shell morphology. One specimen in this latter population was a tetra ploid individual with 38 bivalents. 3. The one species of Chilinidae examined, Chilina fluviatilis, had 18 pairs of chromosomes. 4. The caryotype of Melampus bidentatus lineatus consists of 7 pairs of metacentric chromosomes and 11 pairs of submetacentric chromosomes. This is the first accurate report of the caryotype of an ellobiid snail.

Acknowledgments Grateful acknowledgment is made to the following colleagues for sending us cytologi cal material: Mirta Calcaterra, James N. Cather, Harold W. Harry, Miguel A. Klappenbach, J. P. E. Morrison and J. A. Weber. We are also grateful to C. M. Patterson for technical assistance and to Ernst Marcus for identifying Siphonaria alternata.

Literature cited Boettger, C. R. 1955. Die Systematik der euthyneuren Schnecken. Verhandl. Deutsch. Zool. Ges. Tubingen 1954: 253-280. Burch, J. B. 1960a. Chromosome morphology of aquatic pulmonate snails (Mollusca: Pulmonata). Trans. Amer. Micrcs. Soc. 79 (4): 451-461. - 1960b. Chromosome studies of aquatic pulmonate snails. Nucleus 3(2): 177-208. - 1961. The chromosomes of Planorbarius corneus Linnaeus, with a discussion on the value of chromosome numbers in snail systematics. Basteria 25(4/5): 45-52. - 1962. Cytotaxonomic studies of freshwater limpets (Gastropoda: Basommatophora). I. The European Lake Limpet, Acroloxus lacustris. Malacologia 1(1): 55-72. - 1965. Chromosome numbers and systematics in euthyneuran snails. Proc. First Europ. Malacol. Cong. pp. 215-241.- and Patterson, C. M. 1963. Cytotaxonomic studies of freshwater limpets (Gastropoda: Basommatophora). II. The New Zealand river limpet Latia neritoides. Malacologia 1(2): 313-319. Conn, H. J. 1936. Biological stains: A handbook on the nature and uses of the dyes employed in the biological laboratory. Commission on Standardization of Biological Stains, pp. 276 (p. 237). Huskins, C. L. 1927. On the genetics and cytology of fatuoid or false wild oats. J. Genet. 18(3): 315-364 (p. 318). Inaba, A. 1950. Chromosome numbers in some pulmonate mollusks (in Japanese). Jap. J. Genetics 25(1/2): 58-59. - 1953. Cytological studies in molluscs. I. Chromosomes in basommatophoric Pulmonata. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 14(16): 221-228. La Cour, L. 1941. Acetic-orcein: a new stain fixative for chromosomes. Stain Techn. 16: 169-174. Meyer, K. O. 1955. Naturgeschichte der Strandschnecke Ovatella myosotis (Draparnaud). Arch. Molluskenk. 84(1/3): 1-43. 116 R. Natarajan and J. B. Burch Cytologia 31

Morton, J. E. 1955. The evolution of the Ellobiidae with a discussion on the origin of the Pulmonata. Proc. Zool. Soc. London 125(1): 127-168. Natarajan, R. 1958. Chromosomes of some estuarine basommatophorous snails (Mollusca: Pulmonata) from Portonovo. J. Zool. Soc. India 10 (2): 103-107. Newcomer, E. H. 1953. A new cytological and histological fixing fluid. Science 118 (3058): 161. Newton, W. C. F. 1926. Chromosome studies in Tulipa and some related genera. J. Linn. Soc. 47 (316): 339-354. Perrot, J. L. 1930. Chromosomes et heterochromosomes chez les Gasteropodes pulmones. Rev. suisse Zool. 37 (20): 397-434. Sanfelice, F. 1918. Recherches sur le genese des corpuscules du Molluscum contagiosum. Ann. 1'Instit. Pasteur 32 (8): 363-371.