COMPARATIVE KARYOTYPE ANALYSES OF SELECTED MEMBERS OF THE GENUS ASHMUNELLA (MOLLUSCA: PULMONATA: POLYGYRIDAE)

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Authors Reeder, Richard LeRoy, 1945-

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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 76-1410 REEDER, Richard LeRoy, 1945- COMPARATIVE KARYOTYPE ANALYSES OF SELECTED MEMBERS OF THE GENUS ASHMUNELLA (MOLLUSCA: PULMONATA: POLYGYRIDAE). The University of Arizona, Ph.D., 1975 Zoology

Xerox University Microfilms, Ann Arbor, Michigan 48106

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. COMPARATIVE KARYOTYPE ANALYSES OF SELECTED MEMBERS OF THE GENUS ASHMUNELLA (MOLLUSCA: PULMONATA: POLYGYRIDAE)

by Richard LeRoy Reeder

A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOLOGICAL SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN ZOOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA

19 7 5 THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by Richard LeRoy Reeder

entitled COMPARATIVE KARYOTYPE ANALYSES OF SELECTED MEMBERS OF THE GENUS ASHMUNELLA (MOLLUSCA: PULMONATA: POLYGYRIDAE)

be accepted as fulfilling the dissertation requirement of the

degree of DOCTOR OF PHILOSOPHY

Dissertation Director*^ Date fj (

After inspection of the final copy of the dissertation, the

follox;ing members of the Final Examination Committee concur in

its approval and recommend its acceptance:"

19, '9/£-

(JSkt&kJi (iJt-4 /9 i°n<5 U rd '

TirU G-A—crv^

This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library, Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. ACKNOWLEDGMENTS

I would like to express my appreciation to Drs. Albert R. Mead and Walter B. Miller for their limitless aid in directing my program, guiding my research, producing this dissertation, and for their encouragement. I would also like to thank Drs. Peter E. Pickens, Charles T. Mason, and Willard Van Asdall for their suggestions and for critically reading this work, and Dr. Joseph C. Bequaert for comments, encouragement, and friendship. Thanks are extended to Dr. Walter B. Miller, Carl C. Christensen, Noorullah Babrakzai, David B. Richman, Peter N. D'Eliscu, and Roy S. Houston for their help and com­ panionship in the field. Special thanks go to Carl C. Christensen and Noorullah Babrakzai for numerous personal considerations, and to Margaret Vescovi for her diligent photography work over the past three years and for the illustrations in this dissertation. I would also like to extend my appreciation to my parents, Mr. and Mrs. E. L. Reeder, and to my wife's parents, Mr. and Mrs. A. G. Kimmell, for their support and encouragement. I owe much to all of those mentioned above, but my greatest thanks and appreciation go to my wife, Carolyn, for her support, encouragement, tenacity, love, and just plain

iii iv being there. Without her, this dissertation could not have been completed. TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS vi LIST OF TABLES vii ABSTRACT ix INTRODUCTION , . 1 MATERIALS AND METHODS 6 RESULTS 10 Ashmunella angulata Pilsbry ...... 10 Ashmunella bequaerti Clench and Miller ...... 10 Ashmunella chiricahuana (Dall) ..... 14 Ashmunella esuritor Pilsbry ..... 16 Ashmunella lenticula Gregg 24 Ashmunella lepiderma Pilsbry and Ferriss 24 Ashmunella levettei (Bland) 31 Ashmunella mogollonensis Pilsbry 31 Ashmunella proxima Pilsbry 38 DISCUSSION AND CONCLUSIONS 43 REFERENCES 55

V LIST OF ILLUSTRATIONS

Figure Page 1. Karyotype of Ashmunella angulata 11 2. Idiogram for Ashmunella angulata ...... 13 3. Karyotype of Ashmunella chiricahuana from below Winn Falls ...... 15 4. Diakinesis of Ashmunella chiricahuana from below Winn Falls 17 5. Idiogram for Ashmunella chiricahuana from below Winn Falls 19 6. Karyotype of Ashmunella chiricahuana from Reed's Mountain 20 7. Idiogram for Ashmunella chiricahuana from Reed's Mountain ...... 22 8. Karyotype of Ashmunella esuritor ...... 23 9. Idiogram for Ashmunella esuritor 26 10. Karyotype of Ashmunella lenticula 27 11. Idiogram for Ashmunella lenticula ...... 29 12. Karyotype of Ashmunella levettei ...... 32 13. Idiogram for Ashmunella levettei ... 34 14. Karyotype of Ashmunella mogollonensis 3 5 15. Idiogram for Ashmunella mogollonensis ..... 37 16. Karyotype of Ashmunella proxima ...... 39 17. Idiogram for Ashmunella proxima , . 41

vi LIST OF TABLES

Table Page 1. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella angulata 12 2. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella chiricahuana from below Winn Falls ' . 18 3. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella chiricahuana from Reed's Mountain 21 4, Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella esuritor 25 5. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella lenticula 28

6, Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of a second cell of Ashmunella lenticula , 30 7. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella levettei , . , . 33 8, Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella mogollonensis 36

vii viii

LIST OF TABLES—Continued Table Page 9. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella proxima 40 10. The relative number of chromosome types in seven species of Ashmunella 47 ABSTRACT

This study reports the chromosome morphology of seven species of the land snail genus Ashmunella from southeastern Arizona. Karyotypes and idiograms have been constructed for Ashmunella angulata Pilsbry, A. chiricahuana (Dall), A. esuritor Pilsbry, A. lenticula Gregg, A. levettei (Bland), A. mogollonensis Pilsbry, and A. proxima Pilsbry. For all of these species and two additional species, A. bequaerti Clench and Miller and A. lepiderma Pilsbry and Ferriss, the haploid and diploid numbers are 29 and 58, respectively. Heteromorphism in an homologous chromosome pair was found in one specimen of A. chiricahuana, and a primary trisomy was found in one specimen of A. lenticula. The value of karyotype studies as a taxonomic tool in Ashmunella is assessed in light of the strengths and weakness of the colchicine hypotonic squash technique. The evidence from such studies is found to be useful in some cases and conflicting in others. It is suggested that further refinements of the technique are needed before information from such studies is completely reliable when dealing with closely related species. A generic karyotype pattern has been established for Ashmunella for use in assessing the relationships it possesses with other polygyrid genera.

ix INTRODUCTION

The genus Ashmunella constitutes a distinct group of southwestern land snails belonging to the family Poly- gyridae. Its relationships to other genera in this family remain obscure. Pilsbry (1940) placed Ashmunella in the subfamily Triodopsinae, including also Allogona, Vespericola, and Triodopsis, owing to the presence of a penial sheath. However, the sheath is imperfect in Ashmunella and the spermatheca is long, narrow, and without a terminal sac. It shares an epiphallus with Allogona, Vespericola, and the subgenus Cryptomastix (of Triodopsis). All of these also possess a vestige of an epiphallic caecum and all (except Allogona) are confined to western North America. Webb (19 54) has proposed elevating Cryptomastix to generic status and creating a new subfamily, Ashmunellinae, to include it with Ashmunella and Allogona. This would leave the eastern Triodopsis and Xolotrema (which he raises to generic status) and the western Vespericola constituting the subfamily Triodopsinae. The wisdom of leaving Vespericola therein remains uncertain. The presence of the penial sheath would also seem to indicate that the subfamilies Triodopsinae and Ashmunellinae are more closely related to each other than to the Polygyrinae. The latter subfamily includes (according to Pilsbry, 1940) the genera Trilobopsis, Giffordius,

1 2

Praticolella, Polygyra, Stenotrema, and Mesodon. In Triolobopsis, the presence of a well-developed epiphallus, lack of a penial sheath and epiphallic caecum, as well as its isolated distribution (southern Oregon and northern California) make its subfamilial assignment questionable. Bequaert and Miller (1973) indicated that Ashmunella is most closely related to Polygyra. Indeed there are many parallels in shell structure and type, but the relation­ ship is difficult to defend on anatomical grounds. The multiplicity of species, subspecies, and local races in the isolated mountain ranges of southeastern Arizona and southern New Mexico make specific relationships within the genus no less confusing than the generic rela­ tionships discussed above. There has been remarkable parallel evolution of shell structure. For instance, toothless forms such as Ashmunella chiricahuana (Dall), A. mogollonensis Pilsbry, and A. ashmuni robusta Pilsbry have evolved in widely separated mountain ranges and were, at first, considered as one species. Anatomically, however, they are more closely related to the toothed forms in their respective ranges than to one another (Pilsbry, 1940). Anatomically the shape of the penis is probably the most useful taxonomic character. The relative lengths of the epiphallus, penis, and spermatheca may be of some value (Pilsbry, 1940). Pilsbry describes the genus as homogeneous with regard to anatomy. Some species, such as A. levettei 3

(Bland) have the upper and lower portions of the penis equal in size. Others, such as the species inhabiting the Chiricahua Mountains have the upper portion about half as wide as the lower. Still others are intermediate. A de­ tailed anatomical study of the genus is badly needed. In view of the difficulty in determining relation­ ships among members of Ashmunella and indeed between this genus and others, the present study was undertaken in an attempt to add a new dimension, i.e., a new tool, with which to approach the problem. This tool is the study of chromo­ somes. There have been numerous reports of chromosome studies in molluscs in recent years. Most of these dealt only with chromosome numbers. These works have been re­ viewed by Burch (1965) and Patterson (1969, 1973). Burch (1965 and elsewhere) has stated that there is a gradual increase in the number of chromosomes as one ascends the phylogenetic tree. This seems to be true when one con­ siders large taxa. Numbers, however, are of little value in groups such as families in which all of the genera and species have the same or closely similar haploid numbers. In the study of such taxa karyotype analysis must be employed. Karyotype analyses have been found quite useful in other groups of animals such as fish (Raicu, Taisecu, and Banarescu, 1973) and amphibians (Bogart, 1970). Karyo­ type studies in gastropods, however, are few. In 1973 4

Patterson reported that karyotypes of only about 25 species were known, and most of these are basoinmatophores or pro- sobranchs. Since that time, Babrakzai (1975a) has reported karyotypes of ten species in the land snail family Helmin- thoglyptidae and Babrakzai, Miller, and Ward (1975, pp, 4 -11) have reported on six species of Oreohelix. There have been a limited number of reports devoted to the Polygyridae. The most notable is that of Husted and Burch (1946) wherein they reported the chromosome numbers of 18 species and subspecies belonging to Stenotrema, Mesodon, and Triodopsis. Other studies include Pennypacker (1930) , Husted and Burch (1953), Ford (1962), Burch (1965), and Babrakzai and Miller (1974). The first reports dealing at length with Ashmunella appeared in 1974. Stern and Metcalf (1974) reported the chromosome numbers of 14 species and subspecies of Ashmunella. Reeder and Miller (1974) con­ firmed the numbers of some species discussed by Stern and Metcalf (1974) and reported on an additional species. Two karyotypes of ashmunellae have been reported by Reeder, Babrakzai, and Miller (1975) and chromosomal aberrations have been found in Ashmunella and other genera (Babrakzai, Reeder, and Miller, 1975). The above studies have established that, with the exception of Allogona, all of the polygyrid genera studied thus far have a haploid number of 29 and a normal diploid number of 58. Allogona has a haploid number of 26. The 5 report by Husted and Burch (194 6) that Triodopsis fraudulenta (Pilsbry) varied from n = 29 to n = 31 may be the result of supernumerary chromosomes. They found one individual in which some cells were n = 31 and others n = 32. Such variation within a single animal certainly lends support to the argument for supernumeraries in this case. Since there is little variation in the haploid chromosome number in polygyrids, and since relationships between genera can be argued in several ways, I undertook the present study with two goals in mind. First, to deter­ mine if karyotype techniques could be applied to Ashmunella and if karyotypic analysis could be useful as a taxonomic tool within the genus. Second, to establish, if possible, the basic karyotype pattern of Ashmunella for later com­ parison with that of other polygyrid genera. To this end, twelve species of Ashmunella were investigated. MATERIALS AND METHODS

The animals used in the present study were collected during numerous field trips over a period of three years. They were maintained in redwood terraria in the laboratory until treatment. Precise locality data are to be found under the discussion of each species. The squash technique used was that of Babrakzai and Miller (1974). The animal to be studied was weighed on a torsion balance and, with the aid of a dissecting needle, a small hole was placed in the second or third whorl of the shell. Through this orifice the animal was injected with 0.1 ml of 10 — 3M colchicine per gram of body weight (in­ cluding the shell) using a glass pack B-D 26G 3/8 1/2 cc syringe (Becton, Dickinson, & Co., New Jersey). Following injection, the animals were kept in finger bowls with moistened paper for 6 to 8 hours. They were then placed in a waxed petri dish and covered with a 0.65% sodium citrate solution with 10 -3M colchicine added. The spire of the shell was removed with forceps and the ovotestis dissected from the animal. This organ was then torn into small pieces and kept in sodium citrate (again with colchicine added) for 15 minutes in order to swell the cells. The tissue was then placed on a glass slide with a medicine dropper and the excess hypotonic medium drained off. Then, with the aid of

6 7 forceps, the tissue was placed in Newcomer's (1953) fluid for fixation. Fixation times varied from 72 hours to two weeks. Best results were obtained after 3 or 4 days. After fixation, the tissue was placed in 4 5% acetic acid for 4 to 15 minutes, depending on how long it had been in the fixative. The acetic acid removes the fixative from the tissue. The material was then placed on a glass slide and stained in bulk with lactic acetic orcein (Cooperrider and Morrison, 1967).for 15 minutes. Small pieces of tissue were then transferred to pre-cleaned (in 2% acid alcohol) glass slides and macerated in a small drop of stain with the aid of dissecting needles and fine forceps. The slides were then covered with number one, 22 x 22 mm coverslips (also pre-cleaned). For squashing, each slide was placed on a paper towel, coverslip down, and covered with another portion of the towel. Pressure was applied by rolling the thumb over the covered slide, carefully avoiding any lateral slippage. This pressure was sufficient to burst the cells and spread the chromosomes. Care was taken to avoid crushing the coverslip. Prepared slides were then stored in the refrigerator at temperatures below freezing. The slides were systematically examined using a Wild binocular microscope. Coordinates of suitable spreads were recorded. The selected chromosome spreads were then photographed through the microscope with a Pentax, single lens, reflex, 35 mm camera, using Kodak High Contrast Copy 8 film. Most photographs were taken using oil immersion. Photos were then enlarged on 8" x 10" Kodabromide paper to the maximum size possible while still maintaining good resolution. The images of the chromosomes were then cut from the photographs and placed in a sequence from largest to smallest. Each arm of each chromosome was measured in ram using a standard drafting divider. With the aid of these measurements and some visual features, homologous chromo­ somes were paired and comparative parameters computed. The following were used: q, the length of the long arm; p, the length of the short arm; p+q» the lotal length of the chromosome; q/p, the arm ratio; p/p+q, the centrometric index. The calculations of the arm ratio and centrometric index were based on the relative lengths of the chromosomes determined by dividing the length of each chromosome of the haploid set by the total length of the set, thus expressing each as a percentage. No attempt was made to measure actual lengths of chromosomes as these would vary with every cell studied. Chromosomes were categorized following the method of Levan, Fredga, and Sandberg (1964). Metacentric chromo­ somes with strictly median centromeres, i.e., with an arm ratio of 1.0 and centrometric index of 50.0, are termed M chromosomes. Those with arm ratios between 1.01 and 1.70 and a centrometric index between 49.9 and 37.0 are termed 9 m chromosomes. Chromosomes with arm ratios between 1.71 and 3.00 and a centrometric index between 36.9 and 2 5.0 are termed sm chromosomes. Chromosomes with an arm ratio greater than 3.01 and a centrometric index less than 24.9 are termed £t chromosomes. Using these data, idiograms were constructed illustrating the chromosomes graphically as a percentage of the total length of the haploid set. In so doing, the actual relative length percentages were multi­ plied by a factor of ten to enlarge the scale of the idio- gram. The chromosomes are arranged from largest to smallest within each category in both karyotypes and idiograms (after Bogart, 1970). RESULTS

Of the twelve species and subspecies of Ashmunella investigated, karyotypes were obtained from seven. The data from nine species are discussed below, the species being arranged alphabetically. Specimens from the other three, A. ferrissi, A. proxima harveyi, and A. levettei varicifera, did not yield usable chromosome spreads.

Ashmunella angulata Pilsbry

Locality: East facing rockslide, 8.3 miles up Horseshoe Canyon (from the mouth) at the "Red Box"; NW 1/4 Sect. 7, R. 31 E., T. 19 S.; ca. 6,300'; Chiricahua Mountains, Cochise County, Arizona. Two individuals representing this species were studied, with only one yielding suitable results. Two cells in diakinesis were observed, both with 29 bivalents. The meiotic figures were normal. Three mitotic metaphase spreads were obtained, all with 58 chromosomes. The karyo­ type (Fig. 1) consists of 5 M, 19 m, and 5 sm chromosome pairs. The data are summarized in Table 1 and illustrated graphically by idiogram in Fig. 2.

Ashmunella beguaerti Clench and Miller

Locality: The animals studied are terrarium descendants of the original collection of the species; rockslides

10 11

Fig, 1. Karyotype of Ashmunella angulatat 12

Table 1. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella angulata.

Chromosome Chromosome Number RL q/p CI Type

1 17.8 1.00 50.0 M 2 17.8 1.00 50.0 M 3 17.8 1.00 50,0 M 4 17.8 1.00 50,0 M 5 13. 4 1.00 50,0 M 6 111. 5 1.08 48.0 m 7 79.2 1.22 45,1 m 8 66.9 1.14 46.6 m 9 51.3 1.30 43, 5 m 10 46.8 1.34 42.7 m 11 46.8 1.10 47.6 m 12 39,0 1.69 37.2 m 13 36.8 1. 20 45.4 m 14 33.4 1. 51 39,8 m 15 27.9 1. 27 44.1 m 16 26.8 1.39 41.8 m 17 26.8 1.39 41,8 m 18 24,5 1. 21 45.3 m 19 24.5 1. 21 45.3 m 20 22.3 1. 51 39,9 m 21 22.3 1. 51 39,9 m 22 15.6 1.33 42,9 m 23 14,5 1,59 38,6 m 24 11.1 1.52 39,6 m 25 69.1 1.82 35,5 sm 26 40.1 2.02 33, 2 sm 27 31.2 1.81 35,6 sm 28 26.8 1,98 33,6 sm 29 20.1 2. 00 33,3 sm 120 -

Fig, 2. Idiogram for Ashmunella angulata.

CO 14

ca. 1/4 mile up Goat Cave Canyon, a tributary canyon to Little Aguja Canyon, near Buffalo Trail Boy Scout Camp at the base of the northeast slope of Black Mountain; 4,900'; Davis Mountains, Jeff Davis County,

Texas. Two individuals were investigated. Meiosis was observed in one. All of the diakinesis figures studied had 29 bivalents and appeared to be normal. No karyotype information was obtained.

Ashmunella chiricahuana (Dall)

Localities: (1) Along Cave Creek, 1/2 mile below Winn Falls; ca, 6,800'; Cave Creek Canyon, Chiricahua Mountains, Cochise County, Arizona, (2) Talus at the foot of the northeast slope of Reed's Mountain, near the base of Snowshed Trail; ca. 5,500'; Chiricahua Mountains, Cochise County, Arizona, (3) Under logs along trail on northwest slope of Fly Peak, 1/4 mile below summit; ca. 9,450'? Chiricahua Mountains, Cochise County, Arizona, A total of 11 individuals of this species was examined. Four cells in meiosis were suitable for counting. All have 29 bivalents. Three mitotic metaphase spreads were suitable for study, each possessing 58 chromosomes, The karyotype presented in Fig, 3 is from an indiyidual collected at the first locality above. It consists of 15

Fig, 3, Karyotype of Ashmunella chiricahuana from below Winn Falls. 16 6 M, 19 m, and 3 sm chromosome pairs and one st pair. Chromosome pair number 6 is heteromorphic. I chose the smaller of the pair to measure. This heteromorphic bivalent can also be observed in meios.is as shown in Fig, 4. This abnormality could not be found in either meiosis or mitosis of individuals from other localities. The data from this karyotype are summarized in Table 2 and idiogram is presented in Fig. 5. The karyotype of an individual collected from the Reed's Mountain locality is shown in Fig. 6, with the data given in Table 3 and the idiogram constructed in Fig. 7. This animal has 6 M, 16 m, and 7 sm chromosome pairs. Reconciliation of these two karyotypes will be dealt with in the discussion.

Ashmunella esuritor Pilsbry

Locality: In talus among aspen trees on the west slope of Buena Vista Peak, below Bar Foot Lookout; ca, 8,500'; Chiricahua Mountains, Cochise County, Arizona, Only one individual of this species was collected alive. From this single animal, one cell in diakinesis and one in mitosis were observed. The cell in diakinesis has 29 bivalents and appears to be normal. The mitotic metaphase figure has 58 chromosomes, The karyotype constructed from this latter spread is shown in Fig, 8, There are 5 M and 17

* &-

0

* n * a

Fig. 4. Diakinesis of Ashmunella chiricahuana from below Winn Falls. 18 Table 2. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella chiricahuana from below Winn Falls.

Chromosome Chromosome Number RL q/p CI Type

1 77,7 1.00 50,0 M 2 42.8 1.00 50,0 M 3 25,7 1,00 50,0 M 4 21,4 1.00 50.0 M 5 21,4 1.00 50,0 M 6 12,8 1. 00 50,0 M 7 113,4 1.12 47,2 m 8 68,4 1.29 43.7 m 9 55.6 1,16 46, 2 m 10 49.2 1. 55 39,2 m 11 42.8 1.22 45.1 m 12 38.5 1.25 44.4 m 13 36.4 1.43 41,2 m 14 36.4 1.43 41,2 m 15 32.1 1. 51 39,9 m 16 29,9 1.56 39,1 m 17 27. 8 1.17 46,0 m 18 27.8 1.17 46.0 m 19 27,8 1.17 46.0 m 20 25. 7 1.40 41,6 m 21 19.3 1. 24 44,6 m 22 15.0 1.34 42,7 m 23 15.0 1.34 42,7 m 24 15.0 1.34 42,7 m 25 15.0 1.34 42,7 m 26 25,7 1.99 33,5 sin 27 23. 5 1,77 36,2 sm 28 20,3 2,17 31,5 sm 29 38,5 3.48 22,3 St 120 -

100

80-

60-

JS 40

20- ] 0000 1 1100" 1 1 2 3 4 5 6 /1 7 G S ID 11 12 13 14 15 IS 17 13 ID 20 21 22 23 2i 25j\2S 27 2flj|29| v V SID St

Fig. 5, Idiogram for Ashmunella chiricahuana from below Winn Falls,

<£> Fig. 6. Karyotype of Ashmunella chiricahuana from Reed's Mountain, Table 3. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella chiricahuana from Reed's Mountain.

Chromosome Chromosome Number RL q/p CI Type

1 71.4 1.00 50.0 M 2 49.6 1.00 50.0 M 3 25.8 1.00 50.0 M 4 23.8 1.00 50.0 M 5 21.8 1.00 50.0 M 6 17.9 1.00 50.0 M 7 107.1 1.08 48.1 m 8 73.4 1.64 37.9 m 9 54.6 1.20 45.4 m 10 51,6 1. 26 44,2 m 11 40,7 1.27 44.0 m 12 32.7 1. 21 45,3 m 13 31.7 1.66 37. 5 m 14 27.8 1.34 42.8 m 15 27.8 1.34 42.8 m 16 26.8 1.33 42.9 m 17 22.8 1,30 43.4 m 18 21.8 1.20 45.4 m 19 19.8 1.51 39.9 m 20 15.9 1.65 37.7 m 21 15.9 1.27 44,0 m 22 14.9 1.48 40,3 m 23 43.7 1,74 36,4 sm 24 37,7 1.71 36,9 sm 25 33.8 2.38 29,6 sm 26 29,8 1.98 33,6 sm 27 28,8 1.88 34,7 sm 28 19,8 2,35 29,8 sm 29 10.9 1.73 36.7 sm 120-

100-

80-

60-

40-

20 *8X000/\ a /\ 0 i~) Q nrn 12 3 4 5 8 3 10 II 12 13 14 15 IS 17 18 19 2D 21 24 25 25 27 28 23j

sm

Fig. 7. Idiogram for Ashmunella chiricahuana from Reed's Mountain.

to to 23

IK IN *« 2 3 4 5

BP

XI w 12 15 tt

IS #C* 17 18 19 20 21 22

V 8 Xj # t *K 4S «» 23 25 26 27 28 29

, 8, Karyotype of Ashmunella esuritor, 24

24 m chromosome pairs. The data are summarized in Table 4 and the idiogram presented in Fig. 9.

Ashmunella lenticula Gregg

Locality; Rockslides on south side of Horseshoe Canyon, 2.2 miles from the mouth; NE 1/4 SE 1/4 Sect. 15, R. 31 E., T. 19 S„; ca. 5,100'; Chiricahua Mountains, Cochise County, Arizona. Six animals representing this species were examined. No meiosis was observed. Three cells in mitotic metaphase, all from the same individual, were studied. All three possess a primary trisomy and 59 chromosomes can be counted. The karyotype, presented in Fig. 10, consists of 5 M, 13 m, and 10 sm pairs and one sj: pair. The data are summarized in Table 5 and the idiogram is shown in Fig. 11, Data from a second cell are summarized in Table 6. The complement of this cell was calculated to be 5 M, 16 m, and 7 sm pairs and one st_ pair. No karyotype or idiogram is illustrated for this second cell,

Ashmunella lepiderma Pilsbry and Ferriss

Locality: Rockslides along White Tail Creek, 3.3 miles from the Jhus Canyon Road; ca. 5,400'; Chiricahua Mountains, Cochise County, Arizona, Five animals representing this species were studied. Only one suitable spread, a cell in diakinesis, was obseryed. It has 2 9 bivalents with no detectable abnormalities. 25

Table 4. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella esuritor.

Chromosome Chromosome Number RL q/p CI Type

1 27.4 1.00 50,0 M 2 27, 4 1.00 50,0 M 3 21,9 1.00 50,0 M 4 16,4 1.00 50,0 M 5 16.4 1,00 50,0 M 6 112,3 1.28 43.9 m 7 82. 2 1.14 46.7 m 8 73. 4 1.45 40,8 m 9 63.0 1.30 43.5 m 10 63.0 1.09 47.8 m 11 52.1 1,11 47.4 m 12 41.1 1. 51 39.9 m 13 41.1 1.51 39.9 m 14 32.9 1,40 41.6 m 15 32.9 1.40 41. 6 m 16 30,1 1,20 45.5 m 17 30.1 1.20 45,5 m 18 27.4 1.50 40,1 m 19 24.7 1.25 44.5 m 20 24,7 1.25 44.5 m 21 24.7 1. 25 44.5 m 22 24,7 1. 25 44.5 m 23 21.9 1.50 37.4 m 24 19.1 1,34 42.9 m 25 13.7 1.49 40,1 m 26 13.7 1,49 40.1 m 27 13,7 1.49 40.1 m 28 13.7 1. 49 40.1 m 29 13.7 1,49 40.1 m 120-

100-

-60-

1 2 3 4 51 6 7 0 S 13 11 12 13 M 15 IB 17 13 19 23 21 22 23 24 25 23 27 20 29 V

Fig. 9. Idiogram for Ashmunella esuritor.

to Fig. 10• Karyotype of Ashmunella lentjcula. 28

Table 5. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella lenticula.

Chromosome Chromosome Number RL q/p CI Type

1 24.0 1.00 50.0 M 2 14. 4 1.00 50.0 M 3 12.0 1.00 50.0 M 4 12.0 1.00 50.0 M 5 9.6 1.00 50.0 M 6 124.6 1.08 48.1 m 7 83.8 1.33 42,8 m 8 79.0 1,20 45.4 m 9 55.1 1.09 47.9 m 10 52.7 1.20 45, 5 m 11 46.7 1. 29 43.7 m 12 38.3 1.67 37. 6 m 13 35. 9 1.50 40.1 m 14 31.1 1.60 38.6 m 15 31.1 1.60 38.6 m 16 24. 0 1.50 40,0 m 17 24.0 1.50 40,0 m 18 16,8 1.33 42.9 m 19 57. 5 1,99 33,4 m 20 35.9 2.74 26.7 sm 21 28.7 2,00 33.4 sm 22 26,3 1.75 36,5 sm 23 26.3 1.75 36,5 sm 24 22, 8 1.71 36,8 sm 25 22,8 1,71 36.8 sm 26 14.4 2.00 33,3 sm 27 14.4 2,00 33.3 sm 28 14.4 2.00 33.3 sm 29 21, 6 3.50 22.2 St 120 -

100 -

Fig. 11. Idiogram for Ashmunella lenticula.

voto 30

Table 6. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of a second cell of Ashmunella lenticula.

Chromosome Chromosome Number RL q/p CI Type

1 60.3 1.00 50.0 M 2 32.4 1.00 50.0 M 3 32.4 1.00 50.0 M 4 18. 5 1.00 50.0 M 5 13.9 1.00 50.0 M 6 122.8 1.12 47.1 m 7 78.8 1.12 47.1 m 8 67.2 1.42 41.4 m 9 51.0 1,20 45.5 m 10 41.7 1.25 44. 4 m 11 27.8 1.40 41.7 m 12 26,7 1.30 43.4 m 13 25.5 1.20 45.5 m 14 24.3 1.64 37.9 m 15 20.9 1.25 44. 5 m 16 20,9 1.25 44.5 m 17 20.9 1.25 44.5 m 18 18.5 1. 68 37.3 m 19 16.2 1.35 42.6 m 20 16.2 1.35 42.6 m 21 11. 6 1, 52 39.7 m 22 37.1 2.99 25,1 sm 23 34,8 2.74 26.7 sm 24 34.8 2.00 33.3 sm 25 30.1 2.27 30.6 sm 26 27,8 1.99 33,4 sm 27 22.0 2.19 31.4 sm 28 13,9 2.02 33.1 sm 29 51,0 4.48 18,2 St 31

Ashmunella levettei (Bland)

Locality; Along Miller Canyon Road, 4.0 miles from Route 92; SE 1/4 Sect. 23, R. 20 E., T. 23 S,; ca. 7,700'; Huachuca Mountains, Cochise County, Arizona.

Eight individuals belonging to this species were studied. Meiosis was observed in two animals and mitosis in a third. The diakinesis figures each have 29 bivalents and are normal. The cell in mitosis has 58 chromosomes with the karyotype (Fig. 12) consisting of 5 M, 19 m, 3 sm, and 2 st chromosome pairs. The data for this cell are summarized in Table 7 and the idiogram presented in Fig, 13.

Ashmunella mogollonensis Pilsbry

Locality: Beneath rocks at the head of Salt House Trail near its junction with Route 666; NW 1/4 Sect. 33, R. 29 E,, T, 3 N,; ca, 9,200'; Apache National Forest, Greenlee County, Arizona, Three animals representing this species were studied, A total of three cells in meiosis and five in mitotic metaphase were examined. All of the meiotic cells have 29 bivalents and no abnormalities. Those observed undergoing mitosis each possess 58 chromosomes. The karyotype presented in Fig, 14 consists of 5 M, 16 m, 5 sm, and 3 sit chromosome pairs. The data are summarized in Table 8 and the idiogram is shown in Fig, 15, 32

HVS ** U

Fig. 12. Karyotype of Ashmunella levettei. 33

Table 7. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella levettei.

Chromosome Chromosome Number RL q/p CI Type

1 25,8 1.00 50.0 M 2 25.8 1.00 50.0 M 3 19.4 1.00 50,0 M 4 16,1 1.00 50.0 M 5 12.9 1.00 50.0 M 6 116.1 1.12 47.2 m 7 88.7 1.12 47,2 m 8 80. 6 1.08 48.0 m 9 69.4 1.26 44. 2 m 10 51.6 1. 28 43.8 m 11 38.7 1. 67 37.5 m 12 35.5 1,21 45. 4 m 13 32.3 1.50 39.9 m 14 24.2 1. 50 40.1 m 15 24.2 1.50 40,1 m 16 24.2 1.14 46.7 m 17 24,2 1.14 46,7 m 18 21.0 1.59 38.6 m 19 21,0 1.59 38.6 m 20 21.0 1.16 46.2 m 21 19.4 1. 40 41.8 m 22 16.1 1.52 39,8 m 23 16.1 1.52 39,8 m 24 11.3 1.35 42.5 m 25 37,1 1.88 34,8 sm 26 35,5 1,75 36,3 sm 27 27,4 1.82 35,4 sm 28 40.3 3.15 24.1 st 29 24,2 4,04 19,8 st 120 -

Fig, 13, Idiogram for Ashmunella levettei.

u> 35

mM wM V:j f J

m

y* if* »» »» 15 16 17

sm If XX us 24 25 26 ns % st

Fig, 14. Karyotype of Asfiatmnella ipogollonensis, 36

Table 8. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella mogollonens is.

Chromosome Chromosome Number RL q/p CI Type

1 83.0 1.00 50. 0 M 2 49,8 1.00 50,0 M 3 22,1 1.00 50.0 M 4 16.6 1. 00 50.0 M 5 16.6 1.00 50.0 M 6 118.9 1.26 44.2 m 7 69.2 1.08 48,0 m 8 60,9 1.20 45,5 m 9 49.8 1.57 39.0 m 10 36.0 1.59 38.6 m 11 36,0 1.17 46.1 m 12 36.0 1.17 46.1 m 13 30.4 1.20 45.4 m 14 30.4 1.20 45.4 m 15 22.1 1.66 37. 6 m 16 22.1 1. 66 37.6 m 17 20.7 1.49 40.1 m 18 19.4 1.34 42.8 m 19 15,2 1. 57 36.2 m 20 13.8 1.51 39.9 m 21 12.4 1.25 44, 4 m 22 47,0 2. 41 29.4 sm 23 24,9 2.00 33.3 sm 24 24. 9 2.00 33.3 sm 25 24.9 2.00 33,3 sm 26 19.4 1.77 36.1 sm 27 33.2 3,00 22,3 st 28 24,9 3.53 22.1 st 29 19.4 3.62 21.6 st Fig, 15. Idiogram for Ashmunella mogollonensis.

u> 38 Ashmunella proxima Pilsbry

Localities: (1) In talus among aspen trees on the west slope of Buena Vista Peak, below Bar Foot Lookout; ca, 8,500'; Chiricahua Mountains, Cochise County, Arizona,

(2) Under loose rocks on a steep slope, ca. 3/4 mile south-southeast of the head of Jhus Canyon and ca. 1.5 miles north of Onion Saddle; ca, 6,700'; Chiricahua Mountains, Cochise County, Arizona. Animals from the above localities are included here as A. proxima with some reservation. Subspecies in this complex are at present greatly confused. Animals from the first locality above were, until recently, thought to be h.* angulata, but they are now best referred to A. proxima, as will be explained in the discussion. The population at the second locality is probably A, proxima albicauda Pilsbry and Ferriss. These two populations are placed here together as a matter of convenience. Thirteen animals from the first locality were studied. Meiosis was observed in four individuals with a total of six spreads in all. Each has 29 bivalents and appears normal. Four cells in mitotic metaphase were examined from three different individuals. The karyotype shown in Fig, 16 consists of 5 M and 24 m chromosome pairs. The data are tabulated in Table 9 and the idiogram presented in Fig, 17, tt *ft 3 5

yr / ^ ...« ft'

0 -4H§>. 0 e4 # <1 12 13

ll S* If 17 18 • #* ML 19 20 21 22 23

m $% w A M U 24 25 28 29

Fig. 16, Karyotype of Ashrounella proxima. 40 Table 9. Relative length (RL), arm ratio (q/p), centromeric index (CI), and chromosome types of the haploid set of Ashmunella proxima.

Chromosome Chromosome Number RL q/p CI Type

1 31.8 1.00 50.0 M 2 31,8 1.00 50.0 M 3 25. 5 1.00 50.0 M 4 19.1 1.00 50.0 M 5 19.1 1,00 50.0 M 6 121.0 1.24 44.7 m 7 82.8 1.17 46.1 m 8 63,7 1.22 45,1 m 9 51.0 1.29 43.7 m 10 41.4 1.17 46.1 m 11 41.4 1.17 46.1 m 12 38.2 1.40 41.6 m 13 36. 6 1.30 43.4 m 14 33,4 1,63 38.0 m 15 33. 4 1,63 38.0 m 16 31.8 1,50 39.9 m 17 31,8 1.50 39.9 m 18 28.7 1.24 44.6 m 19 27.1 1.12 47.2 m 20 25, 5 1,65 37.6 m 21 22.3 1,32 43.0 m 22 22,3 1.32 . 43.0 m 23 22,3 1.32 43,0 m 24 22,3 1.32 43,0 m 25 22.3 1.32 43,0 m 26 20,7 1,58 38,6 m 27 20,7 1,58 38,6 m 28 19,1 1,39 41,9 m 29 12,7 1.70 37,0 m Pig. 17. Idiogram for Ashmunella proxima. Two individuals were investigated from the second locality, Meiosis was observed in one and mitosis in the other. Twenty-nine bivalents can be observed in diakinesis and each of the mitotic cells has 58 chromosomes. These cells were not suitable for karyotype analysis. DISCUSSION AND CONCLUSIONS

As one looks at the data presented in the previous section, it is striking that so few cells suitable for study were obtained. In the course of the project several hundred slides from numerous individuals were carefully examined. Several species not herein discussed,, such as Ashmunella ferrissi Pilsbry and A. proxima harveyi Pilsbry, were treated and examined without obtaining usable chromosome spreads. This indeed is one of the greatest problems encountered in the study. Cell division in preparation for reproduction is, of course, seasonal. Unfortunately, it varies among the species. Mitosis and subsequent meiosis begins while the animal is still in hibernation. Pre­ dicting the best time for injecting the animal is difficult, and the time varies even among individuals of the same species, For example, on several occasions three or four animals were activated and injected at the same time. Often, only one of the lot was undergoing any cell division. This problem is compounded by the fact that even in animals undergoing divisions the cells may not burst or the chromosomes may overlap or be clumped. In areas of intense cell division one may find up to 30 cells in various phases of mitosis in close proximity. It then becomes difficult to determine which chromosomes actually belong to which cell

43 44 It is a rare cell then that is suitable for study. For karyotyping purposes the cell must be in mitotic metaphase, relatively isolated, well-stained, and with no or little overlapping of chromosomes, Considering the number of man-hours required to obtain the karyotypes presented herein and the difficulty with which suitable spreads are found, extending the study to include 30 or 40 individuals of each species was considered infeasible. There were other problems encountered with regard to the technique. The chromosome categories, like any classification, are arbitrary. Borderline computations can present quite a dilemma. For instance, the data for A. lenticula differ slightly between two cells from the same animal (Tables 5 and 6), This is the result of two diffi­ culties. First, there are, in these animals, 10 to 12 small chromosomes of similar size and appearance. Without visual markers it is impossible to be sure that you are matching the same pair in each cell. Secondly, with these small chromosomes, a half millimeter difference in measurement Con the photographs) can change the category to which they belong. With regard to A, lenticula, the data in Table 5 were used to construct the karyotype. When one looks at the arm ratios Cq/p) in this table for chromosomes 12, 17, 22, 23, 24, and 25, and then looks at the arm ratio of chromosome 18 in Table 6, it can easily be seen that 45 adjusting the borderline groups would make the karyotypes more similar. A second case in point is that of A, chiricahuana. The karyotype of the animal from below Winn Falls (Fig. 3) consists of 6 M, 19 m, and 3 sm pairs and one st pair. A second individual, from a different population near Reed's Mountain, has 6 M, 16 m, and 7 sm chromosome pairs (Fig, 6), However, as I have noted, borderline arm ratios must be carefully considered. When one examines the arm ratios of chromosomes 23, 24, and 29 in Table 3 (Reed's Mountain animal) it can easily be seen that these three are quite close to falling into the m group. A minor difference in measurement can make the difference. The st chromosome is admittedly more difficult to explain, although an error in measurement or a fold of a chromosome would not have to be large to account for it. On the other hand, the two individuals may indeed be different with respect to the st chromosome, At any rate, when the borderline cases are analyzed, the karyotypes are quite similar. Several improvements in the technique are possible, Unfortunately I learned of them too late to adapt them to the present study. The number of mitotic cell divisions suitable for study can be increased significantly by squashing embryos (Babrakzai, 1975b), When removed from the egg at two or three millimeters, numerous cell divisions are available and the animal is easily squashed as no shell 46 has yet formed. This has the advantage that every slide you prepare is a different individual. If the use of embryos is coupled with some of the more recent banding techniques, particularly geimsa banding (Babrakzai, 197 5b), to mark the chromosomes, one can match them consistently in several cells from numerous individuals. A composite karyotype could then be constructed. One disadvantage to using embryos is that, of course, one cannot study meiosis. With the above limitations of method and suggested improvements in mind we can turn our attention to what the results of this study suggest. The data, as summarized in Table 10, point to a generic karyotype pattern in Ashmunella. A comparative look at the idiograms suggests further stimilarities, There are no strictly telocentric (/t) chromosomes, i.e., with terminal centromeres. There are no or few st chromosomes, and in all species investi­ gated, except A. chiricahuana, there are five M chromosomes. Further, a look at the number six (number seven in A, chiricahuana) chromosome in all of the species investigated shows it to be exceptionally long. Finally, most chromo­ somes fall into the m-sm grouping, An examination of the idiograms at first leads one to believe that the five M chromosomes are not necessarily the same chromosomes in every species. This is particularly evident in A, chiricahuana and A. mogollonensis, In the former species, however, if one eliminates the number one 47 Table 10, The relative number of chromosome types in seven species of Ashmunella.

Chromosome types Species M m sm st

Ashmunella angulata 5 19 5 Ashmunella ehiricahuana (Winn Falls) 6 19 3 Ashmunella chiricahuana (Reed's Mountain) 6 16 7 Ashmunella esuritor 5 24 0 Ashmunella lenticula (cell 1) 5 13 10 Ashmunella lenticula (cell 2) 5 16 7 Ashmunella levettei 5 19 3 Ashmunella mogollonensis 5 16 5 Ashmunella proxima '5 24 0 48 chromosome, the remaining five are more in line with those of other species, In the case of A, mogollonensis, it is widely separated geographically from the other species and, I think, could be expected to differ slightly,, The single long chromosome pair found in every species examined may be characteristic of the entire family Polygyridae, It can be observed in all photos of diakinesis presented for Ashmunella by Stern and Metcalf (1974), and Husted and Burch (1946:414-416) noted that in all of the species of Stenotrema, Mesodon, and Triodopsis which they investigated one bivalent was larger than all of the others. It would be interesting to see if this long bivalent appears in members of any other North or Central American family. This might provide a clue to the origin of the Polygyridae which, at present, is completely obscure. Having discussed the overall similarities we can now look at what the differences seem to indicate. In so doing we must keep in mind the points made previously with regard to A, lenticula and A, chiricahuana, Borderline differences must be analyzed when comparing two karyotypes. The species of the Chiricahua Mountains constitute a complex of isolated to semi-isolated populations, most of which are closely similar in appearance. To be suref there are differences, especially in size, position and size of apertural teeth, elevation of the spire, and in how angular the periphery of the body whorl happens to be, However, these are highly variable and grade into one another in different populations, Ashmunella chiricahuana and A, esuritor are easily separated from the other species in the range. The former is large (19 mm, in diameter) and without apertural teeth and the latter species is large (although smaller than A. chiricahuana) and without apertural teeth in most individuals. A few have a vestige of a lip tooth. The remaining species and subspecies of the range (see Pilsbry, 1940 for discussion) are small (8 to 12 mm. in diameter) with a large parietal tooth and three lip teeth. They present a confusing picture both in shell morphology and anatomy, all having basically the same reproductive anatomy. Large species merely have larger (not differing in detail) anatomies. All have the upper sac of the bipartite penis narrower than the lower sac. Thus far, examinations of the reproductive anatomy from numerous populations, including type localities, have failed to uncover any consistent, significant differences. In addi­ tion to the present karyotype studies, several inter­ breeding studies between these confusing populations are in progress. Obtaining data from this kind of project, however, will take several years. The present study of karyotypes, allowing realise tically for the limitations and borderline problems noted above, has given us a start in unraveling some of these relationships between the Chiricahuan species. A case in 50 point is A. angulata, This species was described (Pilsbry, 1905:244) from the South Fork of Cave Creek, In 1910, Pilsbry and Ferriss stated that it had been collected in the main Cave Creek Canyon, at the head of Turkey Creek, at the "Red Box" in Horseshoe Canyon, in Bar Foot Park (at their station la), and elsewhere. I have collected from the South Fork, "Red Box," and Bar Foot localities, and I have seen shells from below Winn Falls in the main Cave Creek Canyon, The shells from Bar Foot Park (Buena Vista Peak) differ from the others in being smaller, less angular, and in having the space between the basal lip teeth wider. Zoogeographically, the presence of A, angulata in Bar Foot Park is disturbing as the more northerly regions of the Chiricahua Range are dominated by the A, proxima complex, I have presented karyotypes of individuals from the "Red Box" and Buena Vista Peak (which, as far as we can determine, is station la in the 1910 paper noted above). In comparing the idiograms of these two (Figs. 2 and 17) we find that the snail from the "Red Box" has 5 sm chromosome pairs. The animal from Buena Vista Peak has no sm chromosomes, although number 29 is borderline. This is a strong indication, not conclusive proof, that these two populations belong to different species. The animals from Buena Vista Peak are then best referred to A, proxima as was done here in the chapter entitled Results. This argument gains support from 51

the fact that the type locality for A, proxima proxima is also given as Bar Foot Park (Pilsbry, 1940:954). Bequaert and Miller (1973) made both A, angulata and A, lenticula subspecies of A. proxima. However, when one looks at the karyotype of A. lenticula (Fig. 10), even considering the borderline m and sm chromosomes (Tables 5 and 6) , it is much different from either of the other two species (Figs. 1 and 16). Whether or not the entire popula­ tion of A, lenticula possesses a trisomy I do not know. The trisomy aside, however, A, lenticula still remains quite different, having more sm chromosomes than any of the other small, tridentate species studied. When this evidence is coupled with the small but consistent differences in shell morphology, I think it advisable to keep A, lenticula at the specific level, Bequaert and Miller (1973) also consider A. esuritor to be a subspecies of A. chiricahuana. A comparison of the idiograms (Figs, 5, 7, and 9) of these two species, however, indicates that they are probably distinct species„ A, esuritor has 5 M and 24 m chromosome pairs whereas A, chiricahuana has 6 M pairs, 3-7 sm pairs, and perhaps one st pair, There are instances, however, where one begins to have doubts, A comparison of the karyotypes of A, esuritor (Fig, 9) and A, proxima (Fig, 17), which species are sympatric on Buena Vista Peak, reveals that they are nearly 52 identical. Both have 5 M and 24 m chromosome pairs. These are the only two species found to be alike in this respect. The fact that they live together and are readily separated from one another morphologically casts doubt on the importance of differences and similarities between karyotypes. Thera are no intergrades between A. esuritor and A. proxima on Buena Vista Peak. They are clearly dis­ tinct species. This appears to be a case of convergent evolution of chromosomes in which arm ratios have become closely similar, This situation clearly illustrates that karyotypes alone can be misleading. Pilsbry (1940:953) felt that A, esuritor was not related to A, chiricahuana, but was the terminal member of the A. angulata group. Karyotype evidence seems to indicate that Pilsbry was correct with regard to esuritor's relationship with A. chiricahuana, but suggests that A, esuritor may be closer to the proxima group

than the angulata group. The question of the value of karyotype analysis is also raised when one examines the karyotype of A. levettei CFig. 12) from the Huachuca Mountains. This species is anatomically distinct from the Chiricahuan species, having the upper and lower sacs of the penis equal in size. However, the karyotype of A, levettei is closer to some species of the Chiricahuas than some of the latter are to one another. This seems to be evidence that karyotypes need 53 not be radically different between geographically separated, anatomically distinct species. There is, then, somewhat conflicting evidence. In some cases it appears that karyotypes are quite different between morphologically similar populations (e.g., A. proxima and A. lenticula). In other instances the karyotypes of quite distinct species are similar (e„g., A. proxima and A. esuritor). This study has established that karyotypes are variable intragenerically in Ashmunella and that, unfortunately, this variability is not consistent. The techniques need to be refined. One cannot always be sure, particularly in borderline cases, whether the variability is real or due to the technique. It is admitted then that my conclusions are tentative and I would, at present, be conservative with the use of karyotypes in comparing species within a genus, particularly closely related ones. In the one individual of A, lenticula and the two of A, chiricahuana from which karyotypes were obtained, those karyotypes are found to be quite similar when the borderline chromosomes are analyzed. The differences are probably largely due to technique. Tentative conclusions drawn in those cases where there are substantial chromosome differences are probably reliable when considered along with pertinent anatomical, morphological, and zoogeographical data, The reliability of karyotype data in refining generic relationships has yet to be established. The 54 present study has, however, produced a basic karyotype pattern for the genus Ashmunella and I think that the use of karyotypes in comparison of genera holds great promise, particularly if large series of cells can be obtained. Even here, however, one should not rely solely on karyotype information, but should consider all other taxonomic parameters available as well. REFERENCES

Babrakzai, Noorullah 1975a Chromosomes of Helminthoglyptidae (Gastropoda: Pulmonata), Ph, D, Dissertation, University of Arizona, 1975b Department of General Biology, University of Arizona, personal communication. Babrakzai, Noorullah and Walter B. Miller 197 4 A colchicine hypotonic squash technique for chromosome spreads of pulmonate land snails, Malacol, Rev,, 7_:3"7-38. Babrakzai, Noorullah, Walter B, Miller, and Oscar G, Ward 1975 Cytotaxonomy of some Arizona Oreohelicidae (Gastropoda: Pulmonata), Bull, Amer, Malacol. Union for 197 4. Babrakzai, Noorullah, Richard L, Reeder, and Walter B, Miller 1975 Chromosomal aberrations in southwestern pulmonate gastropods, J, Ariz, Acad, Sci,, 1(), Proc, Supple,, p, 30, Bequaert, Joseph C, and Walter B, Miller 1973 The mollusks of the arid Southwest, Univ. Ariz, Press, Tucson, Arizona, 271 pp.

Bogart, James P, 197 0 Systematic problems in the amphibian family Leptodactylidae (Anura) as indicated by karyotypic analysis. Cytogenetics, 2^:369-383.

Burch, John B. 1965 Chromosome numbers and systematics in euthyneuran snails. Proc. First Europ, Malacol, Congr,, 1962, pp, 215-241, Cooperrider, T, S, and J. H, Morrison 1967 Lactic acetic orcein as a chromosome stain, Michigan Botanist, 6:176-177,

55 56

Ford, James M. 1962 The chromosomes of Northwest pulmonate snails. Ph. D. Dissertation, Oregon State Univ., Corvalis, Oregon. Husted, Ladley and Paul R. Burch 194 6 The chromosomes of polygyrid snails, Amer. Nat., 80:410-429. 1953 The chromosomes of the polygyrid snail Allogona profunda. Va. J. Sci., 4_: 62-64 0 Levan, Albert, Karl Fredga, and Avery A. Sandberg 1964 Nomenclature for centromeric position on chromosomes0 Hereditas, 52;201-220. Newcomer, Earl H» 1953 A new cytological and histological fixing fluid. Science, 118(3058):161. Patterson, Charlotte M. 1969 Chromosomes of molluscs. Proc. Symp, Mollusca, Part 2, J. Mar. Biol. Assoc, India, pp, 635-686, 1973 Cytogenetics of gastropod mollusks. Malacol, Rev,, 6^:141-150, Pennypacker, Miriam I, 1930 The germ cells in the hermaphroditic gland of Polygyra appressa, J, Morph, Physiol,, 49(2): 415-453, Pilsbry, Henry A, 1905 Mollusca of the southwestern States, I, Urocoptidae; Helicidae of Arizona and New Mexico, Proc, Acad. Nat, Sci, Philadelphia, 57:211-290. 1940 Land mollusca of North America (north of Mexico), Acad, Nat, Sci, Philadelphia Monogr,, no, 3, vol, 1, pt, 2, pp, 575-994. Pilsbry, Henry A. and James H, Ferriss 1910 Mollusca of the southwestern states, XV, The Chiricahua Mountains. Proc, Acad. Nat, Sci, Philadelphia, 62:44-147,

Raicu, P,f Elena Taisecu, and P, Banarescu 1973 A comparative study of the karyotype in the genus Gobio (Pisces: Cyprinidae) , CytQlogia, 3^8:731- 736. 57

Reeder, Richard L. , Noorullah Babrakzai, and Walter B, Miller 1975 Cytotaxonomic studies in Polygyridae (Gastropoda: Pulmonata). J, Ariz. Acad, Sci., 1(), Proc. Supple,, p. 15,

Reeder, Richard L, and Walter B. Miller 1974 Cytotaxonomy of some Arizona Ashmunella (Gastropoda: Pulmonata). Amer, Zool., 14(4): 1264. Stern, Edward M. and Artie L. Metcalf 197 4 Chromosome numbers in Ashmunella (Gastropoda: Pulmonata: Polygyridae), Veliger, 17(1):19-22.

Webb, Glenn R, 19 54 The life history and sexual anatomy data on Ashmunella, with a revision of the triodopsin snails, Gastropodia, ]L(2):13-18,