73( MALACOLOGIA, 1(1): 55-72 CYTOTAXONOMIC STUDIES OF FRESHWATER LIMPETS (GASTROPODA: BASOMMATOPHORA) I. THE EUROPEAN LAKE LIMPET, ACROLOXUS LACUSTRIS 1 by J. B. Burch 2 ABSTRACT Acroloxus lacustris (Linnaeus) is a freshwater limpet common to Europe, northern Asia and Caucasia. It has nearly always been assigned to the basom- matophoran family Ancylidae, and hence is generally regarded as one of the most specialized and phylogenetically advanced basommatophorans. It is shown in this paper that in regard to certain details ot cytology, A. lacustris should not be considered closely related to other Ancylidae, but rather placed in a family by itself, the Acroloxidae, a conclusion corroborated by other authors on morphological grounds. Indeed, the various cytological differences would tend to further separate Acroloxus from other Basommatophora. The dif- ferences observed consist in the large size of the various cells of spermatogenesis, the greater volume ratio of chromatin to cytoplasm, the relatively large size of the chromosomes and the morphology of the mature sperm, whose heads are long and thread-like, not bullet- or turnip-shaped like those found in other basommatophor - an snails. In addition, the chromosome number (n=18), although characteristic of the Basommatophora in general, is different from that found in other freshwater limpets (x or basic haploid number-45 in the Ancylinae-Ferrissiinae; n=17 in the Laevapecinae). The mitotic chromosomes of A. lacustris are metacentric as characteristic of all Basommatophora; 6 pairs (including the 2 largest and the smallest) are medianly constricted; the other 12 pairs are submedianly or subterminally con- stricted. This is the first time the caryotype of any Euthyneuran snail has been accurately determined and figured. The phylogenetic position of the Acroloxidae may be close to the base of the Basommatophora as suggested by Bondesen and Hubendick, but at the present state of knowledge the evidence which would tend to support such a conclusion can not be found in details of cytology. But, contrary to earlier views reached on purely ana- tomical grounds, the position of the Ancylidae, as determined by their chromosome numbers, should also be near the base of the Basommatophora, but not close to the Acroloxidae because of the other cytological differences. INTRODUCTION Most molluscan systematists have placed Acroloxus in the basommatophoran Acroloxus lacustris (Linnaeus) is a family Ancylidae which originally contain- freshwater limpet common to Europe, ed all freshwater limpets. However, Bon- northern Asia and Caucasia. It is often deson (1950) and Burch (1961b) have con- called the "lake limpet" because of its tended that the differences found between preference for a lentic environment in Acroloxus and other ancylids in regard contrast to the other common freshwater to egg-capsule morphology and spermato- limpet of its region, Ancylus flu viatilis genesis are great enough to warrant its Muller3, which inhabits rivers. separation as a distinct family. These authors were not the first to appreciate 'This investigation was supported (in part) by a research grant, 2E-41, from the National Institute of Allergy and Infectious Diseases, 3Generic designations given here are in accord U. S. Public Health Service. with Opinion 363 of the International Commis- 2 Museum and Department of Zoology, Univer- sion on Zoological Nomenclature (Hubendick, sity of Michigan, Ann Arbor, Michigan, U.S.A. 1952; Hemming, 1955). (55) Reprinted J. B. BURCH 1 2 3 40' . • 5 10 8 9 FIGS. 1-5. Chromosomes of Acroloxus lacustris. Figs. 1-3. Spermatogonial metaphase chromo- somes. The chromosomes in Fig. 1 are excessively contracted. A camera lucida drawing of these chromosomes is shown in Fig. 16. The chromosomes in Figs. 2 and 3 have divided. Camera lucida drawings of Figs. 2 and 3 are shown in Figs. 18 and 24. Fig. 4. Late prophase I (diakinesis) chromosomes. Fig. 5. Metaphase I chromosomes. FIG. 6. Spermatogonial late prophase chromosomes of Rhodacmea cahawbensis. FIG. 7. Spermatogonial metaphase chromosomes of Laevapex fuscus. A camera lucida drawing of these chromosomes is shown in Fig. 17. CYTOTAXONOMY OF ACROLOXUS 57 the differences exhibited by Acroloxus, taken along the River Thames at Sonning as indicated by Walker's (1923) precedent (near Reading) on April 13, 1959, and of placing it in a subfamily by itself from 7 specimens collected near East among the Ancylidae. Most recently Bergholt, Suffolk, on June 26, 1959. The Hubendick (1962) has also argued for tissues were killed, fixed and preserved familial status for Acroloxus on morpho- in Newcomer's (1953) fluid and stainedby logical grounds. the acetic-orcein squash technique (La Chromosome studies of freshwater lim- Cour, 1941) for chromosome studies, or pets are rather sparse and to date are stained with haematoxylin and eosin for restricted to only six publications (Le a general histological study of gameto- Calvez and Certain, 1950; Burch 1959a, b, genesis . Tissues for the latter study 1960a, c; Burch, Basch and Bush, 1960). were washed in absolute alcohol, cleared The present report presents cytological in chloroform, embedded in paraffin and information obtained in our laboratory sectioned at 15 micra. Shells of dupli- on the common European limpet, Acro- cate specimens (from the Thames River loxus lacustris, and discusses the signi- at Sonning) have been deposited in the ficance of these findings in respect to collection of the Museum of Zoology, Uni- commonly held concepts of systematics versity of Michigan (UMMZ cat. no. and phylogeny in freshwater limpet-like 207600). mollusks. Observations were made with a Tiyoda- Grateful acknowledgement is made to microscope using a 90X (n.a. 1.25) oil Dr. Dorothea Franzen, Illinois Wesleyan immersion objective and 10-30X oculars. University, Bloomington, Illinois, U. S. A., The chromosomes in Figs. 12-26 were and to Mr. Jack Hayworth, University of drawn with the aid of a camera lucida Reading, Reading, England, for supplying and reproduced at a table top magnifica- me with the cytological material of Acro- tion of 4650X. Photographs (Figs. 1-11) loxus lacustris. I am also indebted to were taken using a 20X ocular, oil im- Dr. Henry van der Schalie, Museum of mersion objective, a Kodak Wratten 57A Zoology, University of Michigan for faci- (green) filter, and Kodak High Contrast lities and many kindnesses, to Mrs. Eliz- Copy and Ektachrome Type F films. abeth Poulson, also from our Museum, for technical assistance during part of OBSERVATIONS the work, and to Mrs. Anne Gismann for critically reading the manuscript. 1. Cytology of Acroloxus lacustris a. Spermatogonial Divisions MATERIALS AND METHODS Thirty-six chromosomes are easily Specimens used in this study were ob- counted during metaphase of spermato- tained by Dr. Dorothea Franzen and Mr. gonial divisions of Acroloxus lacustris Jack Hayworth from two localities in (Figs. 1-3, 16, 18, 24). Primary con- England. The material examined con- strictions can easily be observed in these sisted of ovotestes from 3 specimens metaphase chromosomes, and in addition FIG. 8-9. Chromosomes of Ancylus fluviatilis. Fig. 8. Late Prophase I (diakinesis) chromo- somes. A camera lucida drawing of the chromosomes of a cell similar to this one is shown in Fig. 23. Fig. 9. Metaphase I chromosomes. A camera lucida drawing of these chromosomes is shown in Fig. 26. FIG. 10. Metaphase I chromosomes of Rhodacmea cahawbensis. FIG. 11. Metaphase I chromosomes of Laevapex fuscus. A camera lucida drawing of these chromosomes is shown in Fig. 22. Figures 1-11 X1470. N N it !I ntl aft to n inleLueei%e# lest fig , UIi it IN it, U ii ill ,4 ,9e, oaec)3 0 e* a cbt3 41) to 0 4?EiPoife T on El t..3 Z 0 0 •EE 0 c4 cl 0 3 t•J c:3 n A:6 &tz3 0c° eAi. op a & H31111£1 - 1) c. )43 gibe L A1 .„) 2 t 60% 64412 00379+4 4.1PW CYTOTAXONOMY OF ACROLOXUS 59 to their constrictions they are often also 2.8, 2.7, 2.7, 2.7, 2.5, 2.5, 2.5, 2.5, 2.3, very noticeable because they are non- 2.2, 1.9. Similar highly contracted chro- staining or only lightly staining (Fig. 1; mosomes have been found in other spe- the non-staining character associated with cies of basommatophoran snails and have the primary constrictions is not shown been discussed by Burch (1960a). in Fig. 16, and only in the shaded chro- mosomes in Fig. 12). b. Meiotic Divisions During their period of maximum con- Eighteen bivalents can be readily ob- traction, spermatogonial metaphase chro- served during late prophase (diakinesis) mosomes appear to be divided into 6 and metaphase of the first meiotic di- pairs of medianly or nearly medianly visions of spermatogenesis. The pairing constricted homologues (shaded in Figs. behavior of the bivalents appeared to be 12-14) and 12 pairs of distinctly sub- normal, and during diakinesis the paired medianly or subterminally constricted chromosomes were held together by one homologues (solid in Figs. 12-14). The or more chiasmata. Details of the chro- two largest pairs and the smallest pair mosome cycle of Acroloxus lacustris of chromosomes are medianly constricted. during meiosis appear to be similar in One pair of chromosomes has secondary most respects to that of other basomma- constrictions (the 10th pair shown in Fig. tophoran snails as described by Burch 12). (1960c). There is considerable variation in the degree of contraction of spermatogonial c. Mature Sperm metaphase chromosomes. The extent of The mature sperm of Acroloxus locus- this variability can readily be observed tris were first described by Retzius in Figs. 1-3, 12-14, 16, 18, 24. Mea- (1906) and the present observations do surements in micra for the homologous not add additional information but merely pairs of excessively contracted chromo- confirm his report. The mature sperm somes shown in Fig.