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University Micrdfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8400224
Jenkinson, John Joseph
MITOTIC AND CHROMOSOMAL CHARACTERISTICS IN THE NORTH AMERICAN NAIADES (BIVALVIA:UNIONACEA)
The Ohio State University Ph.D. 1983
University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106 MITOTIC AND CHROMOSOMAL CHARACTERISTICS
IN THE NORTH AMERICAN NAIADES
(BIVALVIArUNIONACEA)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
John Joseph Jenkinson, B.S., M.S.
* * * * *
The Ohio State University
1983
Reading Committee: Approved By
Dr. David H. Stansbery, Adviser
Dr. Ted M. Cavender
Dr. Barry D. Valentine Advi ser Department of Zoology ACKNOWLEDGEMENTS
In my mind this document would not be complete without the grateful
acknowledgement of contributions made by others. The taxonomic breadth of
this survey would have been considerably more narrow had it not been for
the assistance of many collectors. Their names appear in the Appendix;
however, those citations do not include sufficient indication of the
personal time and expense they gave to this project. The extended
collecting trip to Alabama and surrounding states was made possible by
receipt of a Mary H. Osburn Memorial Fund Summer Fellowship from the
Graduate School of The Ohio State University. This also is an appropriate
place to thank Dr. David H. Stansbery for the wealth of malacological and
ecological information he has helped me learn during the last 20 years.
In more recent years, Tennessee Valley Authority staff members
Ben Jaco, Gordon Hall and, lately, Billy Carroll have refused to let me forget that this task was incomplete and made time and resources available to support it. Agency professionals Dr. William C. (Clay) Barr and
Thomas A. McDonough patiently assisted with the statistical analyses and interpretations, at times serving largely as informed sounding boards to help me develop my ideas. Back at Ohio State, Dr. Ted M. Cavender asked thought-provoking questions and made several suggestions that substan tially improved the quality of this report.
Finally, there is my wife Carolyn. She collected specimens, helped prepare slides, contributed useful suggestions, provided financial support, and typed and proofread the manuscript. Her encouragement and technical help have been extremely important in bringing this long-standing project to completion.
iii VITA
October 16, 1942 ...... Born - Columbus, Ohio
1963 - 1965 ...... Museum Assistant, Department of Natural History, Ohio Historical Society, Columbus, Ohio
1964...... B. S., Zoology, The Ohio State University, Columbus, Ohio
1965...... Resident Summer Naturalist, Wahkeena Nature Preserve, Fairfield County, Ohio
1966 - 1969 ...... Active Duty, U. S. Navy: Officer Candidate School, Newport, Rhode Island USS Bayfield, Homeport Long Beach, California Naval Communications Station, Exmouth, Western Australia
1970 - 1971 ...... Assistant Curator for Science Education, Ohio Historical Society, Columbus, Ohio
1972 ...... Graduate Library Assistant, Auburn University, Auburn, Alabama
1973 ...... Graduate Teaching Assistant, Department of Zoology-Entomology, Auburn University, Auburn, Alabama
1973 ...... M. S ., Zoology, Auburn University, Auburn, Alabama
1975 - 1978 ...... Graduate Teaching Associate, Department of Zoology, The Ohio State University, Columbus, Ohio
1975 ...... Mary H. Osburn Memorial Fund Summer Fellow, Graduate School, The Ohio State University, Columbus, Ohio
iv 1977 ...... Research Associate, Department of Zoology, The Ohio State University, Columbus, Ohio
1978 - present ...... Biologist (Malacologist), Fisheries and Aquatic Ecology Branch, Tennessee Valley Authority, Knoxville, Tennessee
Pub!ications
Jenkinson, John J. 1975. Topic 19 Ecology. Pages 71-75 _IN Grubb, T. C., ed., A Manual for Zoology, 2nd., Burgess Publishing Company, Minneapolis, Minnesota.
______. 1975. The fall line as a barrier to the distribution of some unionids. Bulletin of the American Malacological Union for 1974:30-31.
. 1977. Chromosome numbers of some North American naiades (Bivalvia:Unionacea). Bulletin of the American Malacolog ical Union for 1975:16-17.
______and F. L. Kokai. 1978. Villosa lienosa in Ohio. Bulletin of the American Malacological Union for 1977:82-83.
______. 1979. The occurrence and spread of Corbicula manilensis (Philippi, 1841) in east-central Alabama. Nautilus, 93(4):149-153.
______.. 1981. The Tennessee Valley Authority Cumberlandian Mollusk Conservation Program. Bulletin of the American Malacolog ical Union for 1980:62-63.
______. 1982. Endangered or threatened aquatic mollusks for the Tennessee River system. Bulletin of the American Malacological Union for 1981:43-45.
______. 1982. Cumberlandian Mollusk Conservation Program. Hi Miller, A. C., compiler. Report of Freshwater Mollusks Workshop 19-20 May 1981. U.S. Army Engineer Waterway Experiment Station, Environmental Laboratory, Vicksburg, Mississippi. Pg. 95-103.
______. (1983). Status report on the Tennessee Valley Authority Cumberlandian Mollusk Conservation Program. IN Miller, A. C., compiler. Report of Freshwater Mollusks Workshop 26-27 October 1982. U.S. Army Engineer Waterway Experiment Station, Environmental Laboratory, Vicksburg, Mississippi. In Press.
v Fields of Study
Major field: Zoology
Studies in Systematic Malacology: Dr. David H. Stansbery
Studies in Freshwater Biology: Dr. David H. Stansbery TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... i1
VITA...... iv
LIST OF TABLES ...... ix
LIST OF FIGURES...... xvii
INTRODUCTION ...... 1
Naiades and their Classification ...... 3 Naiad Chromosomes ...... 8 Chromosome and Cytogenetics Background ...... 11
METHODS...... 14
Slide Preparation Technique ...... 15 Identification and Disposition of Specimens ...... 21 Unresolved Problems ...... 21 Location and Analysis of Chromosome Spreads ...... 23
RESULTS...... 25
Species Accounts ...... 32
DISCUSSION I: NAIAD MITOTIC ACTIVITY ...... 131
Temperature Effects ...... 135 Annual C y c l e ...... 138 Holding T i m e ...... 143 Synthesis ...... 157
DISCUSSION II: NAIAD CHROMOSOMES ...... 160
Chromosome Number ...... 160 Supernumerary Chromosomes ...... 162 Chromosome Morphology ...... 163 Matrix Comparisons ...... 166 Measurement Error ...... 169 Composite Analysis ...... 171 Alasmidonta-1ike Analysis ...... 183 Lampsilis-1ike Analysis ...... 188
vi i TABLE OF CONTENTS (Continued)
Other Suprageneric Groups ...... 194 Synthesis...... 194
SUMMARY AND CONCLUSIONS ...... 203
APPENDIX...... 207
LITERATURE CITED ...... 221
v i i i LIST OF TABLES
Table Page
1. Species by species summary of the categories of information recorded while locating usable chromosome spreads. The taxa are arranged alphabetically by genus, then by species. Distinctions between the column headings and more complete treatments of each species are presented in the species accounts ...... 26
2. Separation of the chromosomes of Actinonaias ligamentina form carinata using values of "%TCL" and "r" derived from measurements of three spreads all from specimen JJJ:52. The first and fourth entries in each section of the table are based upon different sets of measure ments of the same (unoutlined) photograph ...... 35
3. Separation of the chromosomes of Alasmidonta marginata using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:1. The firs t and second entries in each section of the table are based upon different photographs (and measurements) of the same spread. Abbreviations are explained in the footnote to table 2 ...... 38
4. Separation of the chromosomes of Anodonta grandis using values of "%TCL" and "r" derived from measurements of eight spreads from specimens JJJ:9 and 106. Abbrevia tions are explained in the footnote to table 2 ...... 42
5. Separation of the chromosomes of Anodontoides ferussacianus using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:14. Abbreviations are explained in the footnote to table 2 ...... 44
6. Separation of the chromosomes of El 1iptio crassidens using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:104. Abbrevi ations are explained in the footnote to table 2 ...... 47
ix LIST OF TABLES (Continued)
Table Page
7. Separation of the chromosomes of Elliptio dilatatus using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ:191. Abbreviations are explained in the footnote to table 2 ...... 49
8. Separation of the chromosomes of Epioblasma torulosa rangiana using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:214. Abbreviations are explained in the footnote to table 2 ...... 52
9. Separation of the chromosomes of Fusconaia barnesiana using values of "%TCL" and "r" derived from measure ments of three spreads all from specimen JJJ:114. Abbreivations are explained in the footnote to table 2 ...... 54
10. Separation of the chromosomes of Fusconaia flava using values of "%TCL" and "r" derived from measure ments of two spreads both from JJJ:79. Abbreviations are explainedin the footnote to table 2 ...... 56
11. Separation of the chromosomes of Gonidea angulata using values of "%TCL" and "r" derived from measure ments of four spreads all from specimen JJJ.-55. Abbreviations are explained in the footnote to table 2 ...... 59
12. Separation of the chromosomes of Lampsilis australis using values of '^TCL11 and "r" derived from measure ments of one spread from specimen JJJ.-156. Abbrevia tions are explained in the footnote to table 2 61
13. Separation of the chromosomes of Lampsilis fasciola using values of "%TCL" and "r" derived from measure ments of three spreads all from specimen JJJ:48. Abbreviations are explained in the footnote to table 2 ...... 61
14. Separation of the chromosomes of Lampsilis higginsi using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ:223. Abbreviations are explained in the footnote to table 2 ......
x LIST OF TABLES (Continued)
Table Page
15. Separation of the chromosomes of Lampsilis radiata luteola using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:99. Abbreviations are explained in the footnote to table 2 ...... 67
16. Separation of the chromosomes of Lampsilis subanqulata using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:137. Abbrevia tions are explained in the footnote to table 2 ...... 69
17. Separation of the chromosomes of Lampsilis ventricosa using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:35. Abbrevia tions are explained in the footnote to table 2 ...... 71
18. Separation of the chromosomes of Lasmiqona complanata using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:67. Abbrevia tions are explained in the footnote to table 2 ...... 73
19. Separation of the chromosomes of Lasmiqona costata using values of "%TCL" and "r" derived from measure ments of nine spreads from specimens JJJ:23 and 47. Abbreviations are explained in the footnote to table 2 ...... 76
20. Separation of the chromosomes of Lemiox rimosus using values of "%TCL" and "r" derived from measure ments of two spreads from specimens JJJ:94 and 102. Abbreviations are explained in the footnote to table 2 ...... 78
21. Separation of the chromosomes of Leptodea fraqilis using values of "%TCL" and "r" derived from measure ments of four spreads from specimens JJJ:236 and 237. Abbreviations are explained in the footnote to table 2 ...... 80
22. Separation of the chromosomes of Lexinqtonia dolabelloides using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ: 111. Abbreviations are explained in the footnote to table 2 ...... 82
23. Separation of the chromosomes of Liqumia nasuta using values of "TCL" and "r" derived from measurements of four spreads all from specimen JJJ:238. Abbreviations are explained in the footnote to table 2 ...... 85 xi LIST OF TABLES (Continued)
Table Page
24. Separation of the chromosomes of Ligumia recta using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ.-227. Abbreviations are explained in the footnote to table 2 ...... 87
25. Separation of the chromosomes of Margaritifera margaritifera form falcata using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:56. Abbreviations are explainedin the footnote to table ...2...... 87
26. Separation of the chromosomes of Medionidus conradicus using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ.-217. Abbreviations are explained in the footnote to table 2 ...... 90
27. Separation of the chromosomes of Obovaria olivaria using values of "%TCL" and "r" derived from measure ments of four spreads from specimens JJJ:226 and 229. Abbreviations are explained in the footnote to table 2 ...... 93
28. Separation of the chromosomes of Oboravia subrotunda using values of "%TCL" and "r" derived from measure ments of seven spreads all from specimen JJJ:31. Abbreviations are explained in the footnote to table 2 ...... 96
29. Separation of the chromosomes of Pleurobema coccineum using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:105. Abbrevia tions are explained in the footnote to table 2 98
30. Separation of the chromosomes of Pleurobema rubrum using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:115. Abbrevia tions are explained in the footnote to table 2 ...... 100
31. Separation of the chromosomes of Potamilus alatus using values of "%TCL" and "r" derived from measure ments of four spreads from specimens JJJ: 15, 17 and 81. Abbreviations are explained in the footnote to table 2 ...... 103
xi i LIST OF TABLES (Continued)
Page
Separation of the chromosomes of Pt.ychobranchus fasciolaris using values of "%TCL" and "r" derived from measurements of five spreads from specimens JJJ:21, 30 and 84. Abbreviations are explained in the footnote to table 2 ...... 105
Separation of the chromosomes of Quadrula pustulosa using values of "%TCL" and "r" derived from measure ments of one spread from specimen JJJ:225. Abbrevia tions are explained in the footnote to table 2 ...... 108
Separation of the chromosomes of Quincuncina infucata using values of "%TCL" and "r" derived from measure ments of one spread from specimen J JJ: 147. Abbrevia tions are explained in the footnote to table 2 ...... 110
Separation of the chromosomes of Toxolasma lividus glans using values of "%TCL" and "r" derived from measurements of one spread from specimen J«JJ:28. Abbreviations are explained in the footnote to table 2...... 112
Separation of the chromosomes of Toxolasma parva using values of "%TCL" and "r" derived from measure ments of three spreads from specimens JJJ:33 and 38. Abbreviations are explained in the footnote to table 2 ...... 114
Separation of the chromosomes of Tritongonia verrucosa using values of "^TCL" and "r" derived from measure ments of one spread from specimen J JJ: 11. Abbrevia tions are explained in the footnote to table 2 ...... 116
Separation of the chromosomes of Villosa iris iris using values of "%TCL" and "r" derived from measure ments of four spreads all from specimen JJJ:4. Abbrevia tions are explained in the footnote to table 2 ...... 119
Separation of the chromosomes of Villosa iris nebulosa. using values of "%TCL" and "r" derived from measure ments of one spread from specimen J J J :222. Abbrevia tions are explained in the footnote to table 2 ...... 121
Separation of the chromosomes of Villosa lienosa using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ:10. Abbrevia tions are explained in the footnote to table 2 ...... 123
x i i i LIST OF TABLES (Continued)
Table Page
41. Separation of the chromosomes of Villosa taeniata punctata using values of "%TCL" and "r" derived from measurements of nine spreads from specimens JJJ:27 and 39. Abbreviations are explained in the footnote to table 2 ...... 126
42. Separation of the chromosomes of Villosa trabalis using values of "%TCL" and "r" derived from measure ments of ten spreads from specimens JJJ-.29, 40, 41 and 49. Abbreviations are explained in the footnote to table 2 ...... 128
43. Assignment of the naiad genus-level taxa included in this project to higher taxonomic groups as proposed in six classification systems. Classification references are discussed in the Introduction and are listed in the Literature C ite d ...... 133
44. Mean number of mitotic figures observed on slides from naiades collected at water temperatures between 21 and 27°C. The number of slides contributing to each mean (n) is indicated below the calculated value followed by twice the standard error (in parentheses). Temperature or overall mean values different from each other (P < 0.05) are indicated by asterisks ( * ) ...... 136
45. Month-by-month and summary data on the number of mitotic figures observed on slides from animals processed within four days after they were collected. The mean values are followed by the number of observations (n) and twice the standard error (in parentheses). Mean values for June, August, and September that were found to be different from the others (P <0.1) are indicated by asterisks (* ) ...... 140
46. Counts, mean values and approximate 95 percent confi dence intervals for each of the days naiad specimens were processed after they were collected. The exponential values have been back calculated from base 10 logarithms . . 144
47. Comparison of the tests for linearity, equation para meters and correlation coefficients for the mitotic figure count by holding time data (days 0-17) when sorted by various taxonomic groupings. Equation parameters and correlation coefficients are given only for relationships significant at the 0.05 level .... 147
xiv LIST OF TABLES (Continued)
Table Page
48. Results of a covariance analysis on seven actual or combination genera in the Lampsilis-1ike group (typically the subfamily Lampsilinae) ...... 152
49. Results of several covariance analyses comparing equations based upon different sortings of naiad mitotic figure count by holding time data. The day zero means of equations with different slopes were compared using equal variance t tests. These values are enclosed in brackets in the ta b l e ...... 154
50. Hypothetical example of the comparison of two naiad taxa based upon the interaction of r and %TCl values. The (hypothetical) mean values for the taxa are presented in opposite corners of each table section and to ta l ...... 168
51. Comparison of means (>7), standard deviations (s) and coefficients of variation (lOOs/x) for two pairs of subtelocentric chromosomes from measurements of 10 Lampsilis spreads and '26 Villosa spreads. Units for X and s are percent complement length, for (100s/x) they are percentage...... 170
52. Species-by-species listing of the number of analyzed spreads, the number of analyzed spreads with 38 chromosomes and the suprageneric groups in which the species have been placed. The abbreviations are explained in the caption to table 43 (page 1 3 3 ) ...... 172
53. Mean numbers of chromosomes from 79 analyzed spreads present in each section of the r by %TCL matrix. Also included are the rounded even numbers of chromosomes which form the overall mean karyotype based upon these s p r e a d s ...... 174
54. Sectional mean values and rounded karyotype numbers for six suprageneric groups: Alasmidonta-like (A- 16 spreads), Gonidea angulata (G-3 spreads), Lampsilis- like (L-51 spreads), Margaritifera margaritifera form falcata (M-l spread), Pleurobema-!ike (P-6 spreads) and Quadrula-1 ike (Q-2 spreads) ...... 176
55. Number of apparent chromosomal modifications in each suprageneric group and the number of shared differences for each pair of groups from the overall mean karyotype . . . 178
xv LIST OF TABLES (Continued)
Table Page
56. Sectional mean values and rounded karyotype numbers for four Alasmidonta-1ike species: Alasmidonta marginata (Am-3 spreads; identical to the Alasmidonta- like mean), Anodonta qrandis (Ag-6 spreads), Anodon-~ toides ferussacianus (Af-2 spreads), and Lasmiqona costata (Lc-5 spreads) ...... 184
57. Number of apparent chromosomal modifications for each species and the number of shared differences for each pair of species from the Alasmidonta-1ike mean karyotype...... 185
58. Sectional mean values and rounded karyotype numbers for 11 Lampsilis-like species and a composite for the genus Lampsilis. The order of species, species abbreviations, and the number of analyzed spreads per species are as follows: Actinonaias ligamentina form carinata (Ac-3), Leptodea fraqilis (Lf-2), Ligumia nasuta (Ln-4; identical to the Lampsilis- like mean), Lfqumia recta (Lr-2), Obovaria subrotunda (Os-6), Potamilus alatus (Pa-2), Pt.ychobranchus fasciolaris (Pf-4), Toxolasma parva (Tp-2), Villosa iris ir is TVi-4), Villosa taeniata punctata (Vp-7), Villosa trabalis (Vt-5), Lampsilis composite (Lc-4) 189
59. Numbers of apparent chromosomal modifications for each species (or composite) and the number of shared differences for each pair of species from the Lampsilis-like mean karyotype ...... 191
xvi LIST OF FIGURES
Figure Page
1. Karyotype of Actinonaias ligamentina form carinata based upon an analysis of "%TCL" and "r" values from three chromosome spreads, all from specimen JJJ:52. This is the spread that was not outlined but was measured twice ...... 37
2. Karyotype of Alasmidonta marginata based upon an analysis of "%TCL" and "r" values from two chromo some spreads, both from J JJ: 1 ...... 39
3. Karyotype of Anodonta qrandis based upon an analysis of "%TCL" and "r" values from eight chromosome spreads from JJJ:9 and 106. This spread (from JJJ:106) includes the dot chromo some mentioned in the t e x t ...... 43
4. Karyotype of Anodontoides ferussacianus based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from JJJ:14. Darker stain areas at the position of the centromeres indicate that this spread came from a slide treated to show "C" banding patterns ...... 45
5. Karyotype of Elliptio crassidens based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:104 ...... 48
6. Karyotype of Elliptio dilatatus based upon an analysis of "%TCL" and "r" values from two chromo some spreads, both from JJJ:191 ......
7. Possible karyotype of Epioblasma torulosa rangiana based upon "%TCL" and "r" values from a chromosome spread of specimen JJJ:214. Several pairs of separated chromatids, a few difficult overlaps and two apparently missing chromosomes may have confused the analysis of this spread ......
8. Karyotype of Fusconaia barnesiana based upon an analysis of "%TCL" and "r" values from three chromosome spreads, all from specimen JJJ-.114 . .
xvi i LIST OF FIGURES (Continued)
Figure Page
9. Karyotype of Fusconaia flava based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:79 ...... 57
10. Karyotype of Gonidea angulata based upon an analysis of "%TCL" and "r" values from four chromosome spreads, all from specimen JJJ:5 5 ...... 60
11. Karyotype of Lampsilis australis based upon an analysis of "%TCL" and "r" values from a chromosome spread from specimen JJJ:156 62
12. Karyotype of Lampsilis fasciola based upon an analysis of "%TCL" and "r" values from three chromosome spreads all from JJJ:48. Two chromosomes somewhat removed from the rest of this spread were not photographed ...... 63
13. Karyotype of Lampsilis higginsi based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from JJJ:223 ...... 65
14. Karyotype of Lampsilis radiata luteola based an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:99 68
15. Karyotype of Lampsilis subangulata based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:137 ...... 70
16. Karyotype of Lampsilis ventricosa based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:35 72
17. Karyotype of Lasmiqona complanata based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:67 74
18. Karyotype of Lasmiqona costata based upon an analysis of "%TCL" and "r" values from nine chromosome spreads from specimens JJJ:23 and 47 ...... 77
19. Karyotype of Lemiox rimosus based upon an analysis of "%TCL" and "r" values from two chromosome spreads from specimens JJJ:94 and 1 0 2 ...... 79
xvi i i LIST OF FIGURES (Continued)
Figure Page
20. Karyotype of Leptodea fragilis based upon an analysis of "%TCL" and "r" values from four chromosome spreads from specimens JJJ:236 and 237 ...... 81
21. Karyotype of Lexingtonia dolabelloides based upon an analysis of '^TCL" and ''r'1 values from a chromosome spread of specimen J J J : 1 1 1 ...... 83
22. Karyotype of Ligumia nasuta based upon an analysis of "%TCL" and "r" values from four chromosome spreads, all from specimen JJJ:238 86
23. Karyotype of Ligumia recta based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:227 ...... 88
24. Karyotype of Margaritifera margaritifera form falcata based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:56 89
25. Karyotype of Medionidus conradicus based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:217 ...... 91
26. Karyotype of Obovaria olivaria based upon an analysis of "%TCL" and "r" values from four chromosome spreads from specimens JJJ:226 and 229 94
27. Karyotype of Obovaria subrotunda based upon an analysis of "%TCL" and "r" values from seven chromosome spreads, all from specimen JJJ:31 ...... 97
28. Karyotype of Pleurobema coccineum based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ:105 ...... 99
29. Karyotype of Pleurobema rubrum based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ:115 ...... 101
30. Karyotype of Potamilus alatus based upon an analysis of "%TCL" and "r" values from four chromosome spreads from specimens JJJ:15, 17 and 81 104
x ix LIST OF FIGURES (Continued)
Figure Page
31. Karyotype of Pt.ychobranchus fasciolaris based upon an analysis of "%TCL" and "r" values from five chromosome spreads from specimens JJJ:21, 30 and 84 106
32. Karyotype of Quadrula pustulosa based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ:225 ...... 109
33. Karyotype of Quincuncina infucata based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ: 147. This contracted spread includes only 34 chromosomes ...... Ill
34. Karyotype of Toxolasma lividus glans based upon an analysis of '‘%TCL" and "r" values from a chromosome spread of specimen JJJ:28 ...... 113
35. Karyotype of Toxolasma parva based upon an analysis of "%TCL" and "r" values from three chromosome spreads from specimens JJJ:33 and 3 8 ...... 115
36. Karyotype of Tritogonia verrucosa based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ: 1 1 ...... 117
37. Karyotype of Villosa iris iris based upon an analysis of "%TCL" and "r" values from four chromosome spreads, all from specimen JJJ:4 ...... I20
38. Karyotype of Villosa iris nebulosa based upon an analysis of "%TCL" and "r" values from a chromo some spread of specimen JJJ:222 ...... 122
39. Karyotype of Villosa lienosa based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:10 124
40. Karyotype of Villosa taeniata punctata based upon an analysis of "%TCL" and "r“ values from nine chromosome spreads from specimens JJJ:27 and 39 ...... 127
41. Karyotype of Villosa trabalis based upon an analysis of "%TCL" and "r" values from ten chromosome spreads from specimens JJJ:29, 40, 41 and 4 9 ...... l 2^
xx LIST OF FIGURES (Continued)
Figure page
42. Relationships between temperature and naiad mitotic figure counts from 67 chromosome slides sorted by subfamily (after Ortmann). The large confidence intervals may mask differences between the groups...... 137
43. Monthly mean counts of mitotic figures separated by subfamily. The monthly and total values for the Lampsilinae and Unioninae were not statistically different and have been combined. Mean values for the Anodontinae in June, August, September and the full year are different (P < 0.1) from the other subfam ilies ...... 141
44. Daily mean values and the best f it (exponential) equation for the relationship between mitotic figure count and holding t i m e ...... 146
45. Linear plots of the log transformed equations describing the relationships between mitotic figure count and holding time. The slopes and means of the Lampsilis- and the Pleurobema-1ike equations are not dissimilar (P < 0.8T! Neither are the slopes and means of the Alasmidonta- and Quadrula-like equations (P < 0.8) T T ...... 156
46. Tally of chromosome counts made during this project. The mode (38) includes 504 of the 774 counts (65.12%). . . . 161
47. Distribution of naiad arm ratio (r) values as indicated by four category means from 121 measured chromosome spreads ...... 165
48. Distribution of naiad percent total complement length (%TCL) values as indicated by four category means from 121 measured chromosome spreads ...... 165
49. Relationships of six suprageneric naiad groups based upon an analysis of chromosome morphology. Data used . to prepare this figure are contained in tables 54 and 55; the detailed interpretation is included in the text. Abbreviations are explained in the heading of table 54. Underlined abbreviations indicate groups repre sented by 10 or more analyzed spreads ...... 180
xxi LIST OF FIGURES (Continued)
Figure Page
50. Relationships of four Alasmidonta-1ike species based upon an analysis of chromosome morphology. Data used to prepare this figure are contained in tables 56 and 57; the interpretation is included in the text. Abbreviations are explained in the heading of table 5 6 ...... 185
51. Relationships of 11 Lampsilis-like species and a composite for the genus Lampsilis based upon an analysis of chromosome morphology. Data used to prepare this figure are contained in tables 58 and 59; the interpretation is included in the text. Abbreviations are explained in the heading of table 58. Underlined abbreviations indicate taxa represented by five or more analyzed s p r e a d s ...... 192
52. Relationships of the suprageneric groups of North American naiades based solely upon analysis of chromosomal morphologic data. Two alternatives are presented: a - assuming that the overall mean karyotype is the ancestral form, and b - assuming that the karyotype most similar to all of the suprageneric group means is ancestral. Letter codes for suprageneric groups are explained in the footnote to table 43 (page 1 3 3 )...... 197
53. Relationships of suprageneric groups of North American naiades (only) as proposed in five classifications: a - Ortmann, 1910; b - Model!, 1942; c - Morrison, 1955; d - Heard and Guckert, 1970; and e - Davis and Fuller, 1981. Letter and number codes for suprageneric groups are explained in the footnote to table 43 (page 133) ...... 199
xx i i INTRODUCTION
This project was undertaken to determine if chromosomal charac teristics of the naiades (Bivalvia:Unionacea) could be examined as a routine procedure and if the study of these characteristics would provide information useful in the systematics of this group. No previous study has compared chromosomal characteristics between groups of naiades or has reported chromosome numbers for any North Amer ican species. Comparisons of chromosome numbers have been made for some marine bivalves (Menzel, 1968) and chromosomal features in many gastropod groups have been studied in some detail (Patterson, 1969;
Murray, 1975). Incorporation of chromosomal characteristics into a classification system for naiades could help identify closely related groups of species and separate unrelated species that exhibit similar shell or anatomical features.
This project required the development of a new preparation tech nique because the sources of mitotic chromosomes used in most other molluscan studies -- developing embryos -- are rarely observed and, apparently, only occur in one- to two-week periods per year for many species. Once a usable preparation technique was developed, the remainder of the project consisted of gathering data and comparing mitotic activity patterns, chromosome numbers, and chromosome morphology from as many North American naiad species as could be sampled. This broad spectrum survey establishes the general characterises of naiad 1 mitotic and chromosomal patterns which will support and tie together future analyses of relationships among species, genera,,and supra- generic groups. Suggestions for improving the preparation technique and specimen handling procedures that can be derived from this project set the stage for research using chromosomal banding methods and other advanced procedures which may be required to address many of the lingering questions of naiad systematics.
The organization of this document differs only slightly from the typical scientific paper format. The remainder of the Introduction provides a brief synopsis of the naiades and the classification systems that have been proposed to organize them, a review of the logic which lead to the selection of this project and the meager literature on naiad chromosomes, and a quick review of the generalities and terms used in cytogenetic studies. An extended description of the slide making procedure developed during this project is included in the Methods section, along with details of the more routine procedures followed after the slides had been made.
The Results section presents total and species-by-species summaries of the data that were collected. This section also includes an alphabetically-arranged set of species accounts in which all of the information gathered on each species is collated for future reference.
Two separate discussion sections are included: one dealing with variations in mitotic activitiy, and the other dealing with chromosomal characteristics. The mitotic activity discussion addresses variations in the observed number of dividing cells as they relate to temperature, month of the year, and holding time. Major topics included in the second discussion are chromosome number and chromosomal morphology, A
brief Summary and Conclusions section completes the text followed by an
Appendix listing the locations of all of the collection sites.
Naiades and Their Classification
"The naiades, or pearly fresh-water mussels, have a universal distribution throughout the ponds, lakes, and streams of the world, not only on the continents, but on most of the larger and some of the smaller islands. Some of the genera have probably extended back with but little change to the beginning of Mesozoic or possibly well into Paleozoic time; hence their study is an extremely inter esting one, which may help us in obtaining a knowledge of the distribution of other life, and the mutations of land and sea in time past." (Simpson, 1896:265)
This paragraph, written almost 90 years ago, still provides a succinct introduction to the naiades and states or implies several reasons why taxonomists, zoogeographers, evolutionary biologists, paleontologists and others continue to study them. A variety of naiad species occur in every zoogeographic province but these animals are all restricted to fresh (or nearly fresh) water. Nearly all of the species that have been studied have life histories including a brief period as an obligate parasite on specific fish hosts. Many naiades only occur in parts of extensive river systems but at least one species has a circum- polar distribution, spanning many river systems.
The recent world-wide fauna of large, freshwater bivalves with nacreous shells (the naiades -- also called mussels, clams and unionids by some) has been estimated to include approximately 1000 species
(Simpson, 1900). Over 500 of the species in this lis t occur in North
America, most from east of the great plains. The extant species from around the world have typically been considered as part of the same family or superfamily (Unionidae or Unionoidea); however, some authors have suggested that this may be a polyphyletic group (Simpson, 1896;
McMichael and Hiscock, 1958).
Although naiades are relatively large animals, are (or were) generally accessible to local naturalists and received taxononmic attention since well before 1758 (bibliography in Lea, 1870), the classification of these animals remains a matter of continuing debate.
This appears to be due primarily to the fact that most proposed class ifications have used shell characteristics as their sole or major crite ria even though shell features have long been known to show ecological variations (Say, 1817) and, more recently, are suggested to be the products of strong parallel or convergent evolution (Hannibal, 1912;
Davis and Fuller, 1981). During much of the last century, information about the soft parts (the animal) or other characteristics of these species were generally not available. Say described the situation and its effect on taxomony quite plainly:
"While we study shells, without regarding the animal, we are aware they are but considered partially. The animals that inhabit them should guide us in our researches; they alone are the fabricators of the shell, and the shell is only their habitation, to which they give the form, the bulk, hardness, colours, and all the peculiarities of elegance we admire. If we were to examine these new and almost unknown beings, we should discover a number of parts as remarkable for their structures as their functions, and an infinite variety of curious and interesting particulars relative to their general habits and manners of life. It is a subject worthy of the serious contemplation and atten tion of the naturalist, and should never be neglected when an opportunity offers. But a system of conchology, founded entirely on the structure of the animals must, probably, ever remain one of the desiderata of natural science." (Say, 1817) Lea (1870) provides an extended comparison of most of the early classification systems proposed for the naiades. Most of these early classifications (Phillipsson, 1788; Sowerby, 1822; Swainson, 1840; Lea,
1836, 1838, 1852, 1870; etc.) are based solely on hinge tooth, shell form and sculpture characters. Later systems (D'Orbigny, 1839-47; Troschel,
1847; Stimpson, 1851; Agassiz, 1852; etc.) use some soft part features
(the siphons, the foot, attachment of the gills to the foot and the location of egg storage) as primary criteria for separating groups of species.
One elaborate early classification system was specifically excluded from Lea's review. "The entire uncertainty of the divisions of
Mr. Rafinesque, or their multiplied, useless, and incomprehensible groups, are not deemed important to insert here." (Lea, 1870:xxiv). In his 1820 monograph, Rafinesque divided the naiad family Pedifera into five subfamilies, eleven genera, 17 subgenera, 92 species and 42 vari eties. (One subfamily, one genus, four subgenera and eight species included in these totals are the subfamily Cycladia which appears to be synonymous with the families Sphaeriidae Dali, 1895 and Psidiidae Gray,
1857). Each of these taxa are described in an hierarchial sequence based upon shell shape, tooth structure and beak sculpture. Under standing this organization of the species requires precise definitions of several terms (not ever presented in text by Rafinesque) and the realization that neither this monograph nor the accompanying visual characterizations of the species were frivolously prepared. This publi cation should be viewed as the firs t serious attempt to organize an obvi ously large group of previously unknown naiad species; however, when 6
this monograph became known to American workers, they failed to recognize
the precise nature of its organization and panned the sketchy nature of
the species-level descriptions and figures. Inconsistencies between the
descriptions and specimens later identified by Rafinesque added to the
confusion. The necessary introduction and glossary for this monograph
have never been prepared, even though more and more authors are beginning
to recognize the seniority of the specific and higher category names that
were presented.
Between 1890 and 1920, naiad classification changed drastically.
Von Ihering (1893) used the presence of a glochidium larva in many well
known species and the presence of a lasidium larva in some South Ameri
can species to suggest a sound basis for separating the Family Mutelidae
from the Unionidae. Simpson (1896) corroborated the separation between
Unionidae and Mutelidae using shell characters and, in 1900, following
suggestions made by Sterki (1895, 1898), began to separate the Unionidae
using characteristics of the marsupium — the portion of the gills where
glochidia are stored during their development. Sterki (1903) suggested
that the presence of large ventral spines on some glochidia and varia
tions in the length of time the glochidia are held by the females also could be useful in classification.
In a series of straightforward and complete papers, Ortmann
(chiefly 1910, 1911a, 1911b, 1912, 1919 and 1921) took the innovations of
Sterki, Simpson and others, added his own wealth of material and atten tion to detail, and produced a classification of naiades based solely on soft parts, reproductive and glochidial characteristics. In its final form (Ortmann, 1921), Ortmann1s classification consisted of one superfamily (Naiades), three families (Margaritanidae, Unionidae and
Mutelidae) and five subfamilies (three in Unionidae: Unioninae, Anodon-
’tinae and Lampsilinae; and two in Mutelidae: Hyriinae and Mutelinae).
Nearly all classifications proposed since 1910 are based upon
Ortmann's work. Most recent classifications accept the super-, sub-, or family status of the Hyriidae, Mutelidae and the Margaritiferidae
(=Margaritanidae); however, several proposals separate family or sub family level groupings for the unionines which carry glochidia in all four gills as opposed to only in the outer gills (Hannibal, 1912;
Morrison, 1955, 1973; Haas, 1969a and b; Heard and Guckert, 1970; and
Davis and Fuller, 1981). Some systems also segregate the Alasmidonta- like genera from the remainder of the Anodontinae (Frierson, 1927;
Morrison, 1955, 1973; Haas, 1969a and b; Clarke, 1973), or the genera
Unio and Gonidea from the other unionines [Hannibal, 1912; Morrison,
1955 (Unio separation only ?); Heard and Guckert, 1970; Davis and Fuller,
1981 ( Gonidea separation only)]. Most of these modifications were mentioned by Ortmann as possibilities; "However, I refrain at present from working this out in detail, since there are yet many, chiefly exotic
(Asiatic) genera, which require further study." (Ortmann, 1916:53). Most of these Ortmann-based classification systems are variously weighted reassessments of the North American species without the addition of sub stantial new information. The two exceptions are Davis et al (1978) and
Davis and Fuller (1981) who include a statistical analysis of protein characteristics in their work and Parodiz and Bonetto (1963) who include anatomical and larval studies in their revision of the Southern Hemi sphere forms. The classification of Model! (1942, 1949 and 1964) offers a sharp
contrast to its Ortmann-based contemporaries. Modell believed that the
value of soft anatomy had been over extended and attempted an unbiased
new analysis of shell, soft-part, ecological, and palaeontological data.
"It is almost as if I had before me the material obtained on an expedition to an unexplored planet and I have used on it the experiences of a biological nature which I have obtained in better than 20 years of collecting." (Modell, 1942 - in translation)
The classification system which emerged is largely based on beak
sculpture and tooth structure but i t shows a strong interest in suspected
phylogenetic relationships. The fauna is divided into four families
(Mutelidae, Elliptionidae, Margaritiferidae and Unionidae) which include
39 subfamilies, with only the Lampsilinae reasonably comparable to a
similar grouping proposed by Ortmann.
To summarize, the relatively long sequence of naiad classification
systems can be thought of as a few major revisions, each occasioned by
the availability of new sets of potentially useful characteristics and a
larger number of less extensive refinements using various weightings of
characters. Shell features dominated the various systems until approx
imately 1900 when characteristics of the soft parts, reproductive physi
cal adaptations and larval differences had been studied sufficiently to
be incorporated successfully. Since that time, the only apparent addi
tion has been an examination of protein similarities.
Naiad Chromosomes
If naiad taxonomic revisions and refinements occur when new sets of characteristics become available, the logical approach for a 9 naiadologist interested in clarifying or testing existing classifica tions would be to explore character sets not yet (or not sufficiently) examined. As this project was being conceived, several fruitful areas of investigation appeared to exist. At this (later) date, these include:
- fish host relationships - The taxonomic relationships of the
various fish species used as parasitic hosts could be used
to clarify naiad systematic relationships if they were
known.
- larval anatomy - The two types of electron microscopes
seem to remove most of the impediments to studying the shells
and soft parts of these poorly known life stages.
- chromosome number and comparative morphology - The number
and similarity of naiad chromosomes could show degrees of
relationship and could suggest sequences of evolution.
- DNA sequencing - Recent quantum jumps in technique suggest
that it may be possible to identify and compare nucleoprotein
sequences along the lengths of chromosomes.
Of these possible avenues of discovery, chromosome studies were selected as the subject of this project for two major reasons. When this project was first being discussed, the only naiad chromosome number that could be found in the literature was a report of 16 chromosomes for Unio complanata (Lillie, 1901). Secondly, by the mid 1970's a number of biologists were beginning to realize that chromosomal banding techniques, which had led to the detailed recognition of each human chromosome (Paris
Conference, 1971), could be adapted for systematic use with other mammals
(i.e.: Zech et a l, 1972; Stock and Hsu, 1973; Yosida and Sagai, 1975), 10 other classes of vertebrates (Bogart, 1973; Takagi and Saski, 1974) and with other animals and plants (Babrakzai et a l , 1976; Merker, 1973).
Taken together, these observations suggested that chromosomal informa tion about naiades might provide an easily accessible, essentially independent set of taxonomically useful characteristics which could be used to test, correct or augment previous naiad classification systems.
As this study progressed, a few naiad chromosome references were found along with several pertinent comments about molluscan chromosomes, largely derived from research on gastropods. Patterson (1969) tabulated many of the previous reports of mollusk chromosome numbers, listing
1,100 for gastropod species, 2 for cephalopods and 22 for pelecypod species.
None of the recent bivalve chromosome numbers cited by Patterson were for naiades. The pattern indicated by much of the work on gastropods and the scanty work on other classes suggests that mollusks show a remarkable degree of conservatism in chromosome number (Murray, 1975).
Family groups rarely vary more than one or two chromosome pairs from other members of higher taxonomic groups.
Apparently Lillie (1901) was the firs t to give a number of naiad chromosomes. In his extensive study of naiad eggs and their development,
Lillie saw many sectioned chromosome spreads and he counted 16 haploid chromosomes for Unio complanata ( = El 1iptio complantus). McMichael and
Hiscock (1958) mentioned that three Australian naiades all were found to have 34 diploid chromosomes. More recently, van Griethuysen et al (1969) described 38 diploid chromosomes for two European species and Nadamitsu and Kanai (1975; 1978?) reported 38 diploid chromosomes for three
Japanese species. A preliminary report on this project (Jenkinson, 1977) n in which 38 was given as the diploid chromosome number for 15 North
American species, apparently is the only other chromosome study on naiades to appear in the literature. Other pertinent information con tained in these naiad references will be included at appropriate points
in Discussion II.
Chromosome and Cytogenetics Background
In the way of a brief review of chromosomes and cytogenetics, most general biology texts (i.e ., Hickman et a l , 1974) will describe how the inheritable factors which provide the genetic basis for the develop ment, form and functioning of an organism (the genes) are joined together in long nucleoprotein threads (the chromosomes). When seen during cell division, typical chromosomes resemble short rods with a constriction
(the centromere) dividing each one into two "arms". The number of chromosomes varies among species but is nearly always constant within a species. Most multi cellular organisms have two nearly identical (homol ogous) sets of chromosomes (they are diploid), one member of each pair of chromosomes (a haploid complement) coming from each parent. In normal cell division (mitosis), each chromosome is replicated and the identical pieces (chromatids) separate into the daughter cells. During the formation of gametes (meiosis), cell division occurs twice but the chromosomes replicate only once. Each gamete receives one chromosome representing each homologous pair. The joining of gametes at f e r tili zation restores the full complement.
Genetics or cytogenetics texts (i.e., Brown, 1972) expand on this base to discuss how the linear arrangement of genes on chromosomes produces linkage groups and how, during meiosis, genes or linkage groups
can be exchanged between homologous chromosomes (crossing over) when
they are tangent to each other during this process. Rarely during meiosis, linkage groups can be inverted, duplicated, deleted or exchanged with another chromosome pair (translocation). It is possible that one chromosome could break into two (Robertsonian fission) or that two chromosomes could fuse together (Robertsonian fusion). And it also is possible for diploid gametes to be produced or other situations to occur which might lead to triploid, tetraploid or other polyploid organisms.
A haploid chromosome complement with linkage group rearrangements, translocations or chromosome fusions or fissions will not match up well with an unmodified complement. An apparently healthy organism possessing substantially mismatched haploid complements will produce defective gametes because normal meiosis cannot occur. Substantial differences in the arrangement of the genetic material on the chromosomes could lead to effective sterility of the individual.
For comparative purposes, the chromosome complement of a species is usually presented as a karyotype, where photographs or drawings of the diploid chromosomes are arranged in pairs by the position of the centromeres, then by overall length. Sometimes an idiogram is used for this purpose; in this case bar-like representations of the haploid complement are arranged along a comparison line which marks the location of the centromere on each chromosome. When the chromosomes of several specimens or species are measured and/or arranged in either of these ways, similarities and differences between individual chromosomes can be compared. Similar numbers of chromosomes with few apparent 13 rearrangements of the genetic material can be assumed to imply recent common ancestry. Conversely, substantially different chromosome numbers and little similarity among the chromosomes may be assumed to indicate distant common ancestry. With some care, sequences of apparent rearrangements can be described to relate a number of karyotypes.
The relatively recent addition of chromosomal banding adds immeasurably to this interpretative process. Chromosomes, banded using various staining techniques (Zech, 1973; Yunis and Sanchez, 1973;
Arrighi and Hsu, 1974; etc.), are marked by sequences of bands all along their lengths. High quality, consistent preparations permit the recog nition of chromosome segments regardless of where they occur in the chromosome complement. Careful analysis of banded karyotypes can be used to show degrees of similarity between taxa and to identify the specific types of rearrangements which constitute their differences.
The primary purpose of this project was to determine if chromosomal characteristics will provide new information that can be useful in naiad systematics. If sufficient chromosomal information became available, a secondary goal of the project was to determine how the analysis of this data set compared with the more important naiad classification systems that have been proposed. METHODS
For chromosomal information to be used as a common and versatile tool in naiad identification and taxonomy, preparation methods must be designed to meet a number of procedural and use criteria. In the field, chromosome preparation techniques must provide useful information from specimens collected at any time throughout the year and must be simple enough to interfere very little with other activities. In the laboratory, chromosome preparations must provide readily retrievable, independent information about the specimens from which they were taken; they must be permanent, reusable records; they should be adaptable enough to be reanalyzed when new comparison techniques are discovered; and, ideally, they should be applicable to specimens already housed in a research collection. Obviously this set of requirements is not likely to be completely met by any technique. In fact, all techniques require some commitment of time and expertise—commodities always in short supply during field activities—and none proposed so far are applicable to preserved mater ial. Most extant chromosome preparation techniques do not attempt to produce permanent or widely usable slides. The aceto-orcein squash technique widely used in non-mammalian chromosome work (LaCour, 1941) produces pre-stained, temporary slides that are difficult to transport or to convert into more-permanent preparations.
14 15 Slide Preparation Technique
At the beginning of this project, it became apparent that the most detailed chromosome comparisons then being conducted were the largely- mammalian studies involving comparisons of various types of chromosome banding patterns. Examination of the procedures employed to produce banded chromosomes showed that many of them involved a relatively simple basic process, produced unstained permanent slides and appeared to be adaptable to molluscan subjects. Arrighi and Hsu (1974) describe the basic steps in this process, essentially as follows:
-if the number of mitoses is low, treat cell preparations
or tissues with a metaphase arresting agent.
-treat the cell preparation with a hypotonic solution.
-fix cells in 1 glacial acetic acid: 3 methanol.
-place cells on a microscope slide and either air-dry or flame-dry.
Evolution of a technique for naiad specimens started with these basic steps and resulted in the following procedure which, although not totally satisfactory, generally yields some chromosome spreads from most specimens,
-sever both adductor muscles at their insertion on one valve
of the shell, pull the mantle loose from the shell and open
the living naiad.
-remove all of the labial palp and/or part of a non-gravid gill
from one side.
-cut the tissue sample into 5-10 mm pieces and place them in
demineralized double distilled water for 30 minutes.
-remove the tissue samples from the water, accept any water that 16 will come off easily, then flood the samples with freshly-mixed
1 glacial acetic acid: 3 absolute methanol fixative.
- change the fixative twice during 30 - 45 minutes.
- gently swab a piece of tissue over most of one side of a labeled,
clean, dry microscope slide.
- dry the slide by waving it in the air, blowing on it (mouth,
hairdryer, etc.) or by heating it gently high over an alcohol
lamp.
(The slides are permanent at this point and can be stored for
later staining.)
- for unbanded stained chromosomes, heat the slides at 60°C for
one hour, stain them in one percent Giemsa blood stain for
5 - 7 minutes, rinse under the tap and allow to dry.
- mount stained slides with an oversize covers!ip using Permount
or some other permanent mounting medium.
The modifications from the mammalian process that are included
in this naiad chromosome procedure were derived from a series of trial- and-error and more specific experiments on a fairly large number of naiad specimens. The bases for these modifications are discussed in the fol
lowing paragraphs.
Tissue Choice - Considerable early effort was invested in determining which naiad tissue would provide the highest number of chromosome spreads and the lowest amount of cellular or environmental debris on the slides. Tissues or structural areas examined at one time or another included: unmodified mantle, anterior free edge of the mantle, siphonal area of the mantle, labial palps, non-gravid g ill, foot, gonad, kidney 17 and developing embryos. The edges of the mantle and the foot were apparently too firm to release many cells when swabbed onto slides. The gonad and kidney tissues were so soft that globs of material came off on the slides and individual nuclei or chromosome spreads were obscured by this debris. Developing embryos were observed and the preparation of chromosome slides were attempted only on two occasions, both of which yielded some chromosome spreads; however, this source of chromosomal material is so rarely available that it was abandoned as an option.
The relative usefulness of the three remaining tissues (unmod ified mantle, labial palps and non-gravid gill) were compared in the process of gathering chromosome spreads from a number of naiades.
Unmodified mantle tissue gave some spreads; however, it often deposited a good deal of debris on the slides. Both labial palp and non-gravid gill material routinely produced clean slides; however, labial palp preparations almost always contained more chromosome spreads (average of 13 pairs of comparisons: palp - 12.8 spreads per slide, gill - 5.0),
Pretreatment - Most chromosome preparation techniques include one or more pretreatment steps to accumulate mitotic figures (Arrighi and Hsu,
1971; Chambers, 1982). From the beginning, this project was complicated by the very low numbers of chromosome spreads on the slides. Several experiments with Velban (vinblastine sulphate), one of the mitotic arresting agents in common usage, failed to show any increase in spread number but did seem to cause chromosome contraction in some specimens.
A single experiment with phytohemaglutanin, a mitotic inducer, also failed to affect the number of spreads. Further experimentation with these and similar agents may identify dosage levels or treatment 18 procedures that would increase the chromosome spread yield; however, it now appears that such procedures would be more complex than could be accomodated by most field crews.
A further complication to using these pretreatments is that the effective dosage must be related to the animal body weight. Comparisons of animal to total weight for a number of naiades quickly indicated that species-specific animal/total weight ratios would have to be generated before uniform dosages of these chemicals could be applied. From there, experiments on the effective dosage levels and exposure times could begin. This subject remains for some future student to investigate.
Hypotonic Treatment - Typical hypotonic treatments for vertebrate animals are dilutions of one percent sodium citrate (Arrighi and Hsu,
1971). Although a number of dilutions of this material were tried, no recognizable chromosome spreads were observed until demineralized double distilled water was used alone as the hypotonic treatment. With regard to the treatment time, tissue samples of one animal were fixed after 0, 10, 20, 30, 40, 50, and 60 minutes in demineralized double distilled water. Although the number of chromosome spreads continued to increase with treatment time, the edges of individual chromosomes became increasingly fuzzy in treatments longer than 30 minutes.
Transition into Fixative - For some time after this technique was sup posed to have become standardized, variations continued to occur in the size of interphase nuclei and, apparently, in the number of chromosome figures. Careful monitoring of all aspects of the procedure indicated that water on the tissue samples when they were flooded with fixative reduced the size of nuclei on the resulting slides. This problem was 19 overcome by touching the tissue samples to dry paper towels as they came out of the hypotonic treatment and then placing them in a dry vial where they were flooded with the fixative. Some care also was taken to keep the absolute methanol tightly closed to prevent it from absorbing moisture from the air.
Fixation - No substantial change was made in the composition of the fix ative or the length of fixation time because experimental results indi cated that some fixation was required but that long fixation time did not change the number of chromosome spreads observed on slides. In one case chromosome spreads were observed on slides made 32 days after the tissue samples were fixed. Similar tests using tissue samples that had been in fixative for one and two years failed to produce spreads or recognizable cell nuclei.
Slide-making Technique - The use of tissue samples as the source of cellular material for chromosome slide preparations simplifies a number of procedural steps but it also places more of the burden of producing chromosome spreads on the actual preparation of the slides.
Experimentation with this aspect of the procedure was concerned with how to get cells on the slides and how best to dry the liquid preparations.
Several tests were run "dabbing" or "swabbing" tissue samples onto dry or wet slides. The best technique involved lightly rubbing the tissue sample over the surface of a dry slide, making sure to keep the sample wet with fixative. Lightly dabbing the tissue sample on the slide did not deposit enough cellular material. Rubbing a drying piece of tissue across a dry slide deposited so much material that nuclei and spreads often were obscured. Slides wet with water sometimes produced shriveled nuclei if not dried very quickly. 20 Three types of drying techniques were tried, without determining significant differences. Some slides were air-dried — dried by fanning them or waving them around in the air. This technique works well in relatively warm, dry environments; however, it is slow and tiring in cool, humid conditions not uncommon along a stream bank.
The other standard drying technique -- flame-drying -- involves passing the slide through or over an alcohol (or similar) flame. This technique was found to be quite rapid but could produce spots or streaks of nuclei and other material if the slide was dried so fast that the liquid beaded up. Holding the slide approximately 30 cm above an alcohol lamp flame was found to be sufficient to dry the slide in a short time, but was gentle enough to prevent streaking.
An intermediate drying technique was tried on a few occasions when air-drying was not working. These slides were dried by blowing on them by mouth or using a mechanical warming device such as a hair dryer or a desk lamp. In the laboratory, the use of a standardized warm- drying technique may help standardize some staining procedures.
Staining (unbanded) - All staining of these chromosome preparations so far has included the use of a stock solution of Giemsa blood stain
(150 g Giemsa stain, 10 ml glycerin and 10 ml methanol in 1 lite r of water). Experimental results suggest that three factors are involved in staining intensity: stain concentration, staining time and the initial dryness of the slides. Slides more than four days old that had been heated at 50° - 60° C for one hour, stain well during 5 - 7 minutes in a 1 - 2 percent stock solution. Slides less than four days old, with or without the heat treatment, generally take stain more slowly but may 21
have areas that will overstain. Very old slides (3 to 15 months) vary
considerably in their acceptance of stain regardless of the one hour
heat treatment; however, 5 - 7 minutes in 1 - 2 percent stain is likely
to give acceptable results for most of these slides.
Identification and Deposition of Specimens
After the various tissue samples had been removed and processed
for chromosomal use, the soft parts of each specimen were preserved in
70 percent ethanol. This usually was accomplished with one side of the
mantle still attached to the entire shell. Identification of each
specimen was made using shell and soft part characteristics and, for
nearly all specimens, was verified or corrected by Dr. David H. Stans-
bery. All specimens from which chromosome slides were made have been
deposited in the Bivalve Collection, Museum of Zoology, Ohio State
University (OSUM) and are in the process of being cataloged into that
collection. The cataloged specimens will retain the "JJJ" numbers used
during this project as well as a label notation that chromosome slides
had been prepared from them. Upon completion of this project, all of
the stained and unstained slides prepared from these specimens also will be deposited at OSUM.
Unresolved Problems
During the evolution of this chromosome preparation technique two
important problems were encountered that were not resolved. The more
important of these problems concerns the low number of chromosome
spreads that occur on most of these slides. After the slide preparation 22 techniques and tissue selection processes were completed, the entire surface of a typical slide could be expected to have between five and ten mitotic figures of which one or two would be spread well enough for the chromosomes to be counted. On a typical slide, none of the figures would be of sufficient quality for the details of chromosome structure to be analyzed.
Several factors probably affect this low recovery rate. Certainly the use of adult tissues as sources of mitotic figures could be expected to produce relatively few spreads. Further, this slide-making technique restricts the number of mitotic figures likely to be recovered to those that occur only near the surface of each tissue sample. Neither of these factors appeared to be correctable without sacrificing the simplicity or the utility of this preparation technique.
A second major unresolved problem discovered during the evolution of this technique involves the techniques required to produce banded chromosomes. A number of experiments using vertebrate banding tech niques did produce a few weakly-banded naiad spreads; however, these banded spreads were not sufficiently clear to be considered usable and the few banded spreads on any one slide differed from each other so much that the direction of appropriate modification to the staining technique was not apparent. Perfection of banding techniques will require exami nation of a large number of spreads exposed to a variety of subtle differences in pretreatments, stain pH and staining time. The pro duction and analysis of banded naiad karyotypes will have to wait until the number of spreads on a typical slide can be substantially increased. 23
Location and Analysis of Chromosome Spreads
Each chromosome slide prepared during this project was assigned
an identifying code number that included the consecutive number assigned
to the animal, a letter identifier for the tissue involved, and the
consecutive number of the slide made from that tissue. Chromosome
spreads were located by conducting a complete scan of the area under a
20 X 50 mm cover slip using a 200X lens combination on an Olympus phase contrast microscope. This time-consuming procedure was necessary because of the low number of spreads on the slides and their small size when compared to the large number of often-aggregated nuclei. When a spread was located, it was recorded on a form keyed to that slide. Mitotic figures that included uncountable spreads were simply noted. Better mitotic figures were located using the mechanical stage coordinates.
Each of these spreads was evaluated using a five step grading scale, counted at 900X and described briefly on the form.
Well separated chromosome spreads that included indications of centromeres or arms were photographed for further analysis using a
Pentax Spotmatic camera mounted on an Olympus phase contrast microscope with a built-in light source. All photographs were taken on Kodak High
Contrast Copy Film at 1500X at between 1 and 4 seconds for well-stained spreads or at 15 or 30 seconds for light spreads enhanced with a green filte r. All usable negatives were printed to the same scale to simplify comparisons between spreads. Individual chromosomes on the photographs were outlined and the centromeres located (when possible) while compar ing the photograph to the actual spread magnified to 1500X through the microscope. 24
Dial calipers were used to measure the outlined photographs of the chromosomes. Generally, the longer chromatid arm on each end of the chromosome was measured from the centromere to the tip. When the chromatid arm or the centromere could not be distinguished, the greatest length of the arm or the entire chromosome was used. All measurements for arms or entire chromosomes were recorded on a worksheet at the nearest 0.05 mm. Two statistics were calculated for each chromosome:
"%TCL" - the percentage of the total chromosome complement length, and
"r" - the length of the longer arm divided by the length of the shorter arm. The species by species accounts in the Results section include a tabular comparison of the "%TCL" and "r" statistics for any chromosome spreads that were measured. RESULTS
During this project, 250 naiades representing approximately 75 nominal species and 34 recognized genera were used as sources of mater ial for chromosome slides. These animals had been collected in ten of the east-central to southeastern United States, the western state of
Oregon,and Manitoba Province, Canada, between August, 1975 and May, 1978.
Three hundred eighty-one slides from 218 of these specimens (all 75 species and 34 genera) were examined under the microscope, resulting in a variety of counts of chromosome spreads and more detailed examinations.
Species-specific and total results for the categories of information collected are included in table 1.
Chromosome counts were obtained from one or more representatives of 66 species level taxa; however, fewer than five counts were obtained from 25 taxa. In all of the remaining 41 species (and many of the poorly studied ones), the clear modal diploid chromosome number was 38.
Two hundred five superior quality chromosome spreads were photographed,
130 of these were measured and 121 (representing 41 species) were analyzed. The information derived from the various levels of examination of these slides form the basis for the two discussion sections.
25 Table 1. Species by species summary of the categories of information recorded while locating usable chromosome spreads. The taxa are arranged alphabetically by genus, then by species. Distinctions between the column headings and more complete treatments of each species are presented in the species accounts.
Genera and Species Processed Specimens Examined Specimens SI ides SI Scanned Spreads Noted Noted or Observed Spreads Studied 38‘s Photographed Chromosome Counts Number of Number Spreads Spreads Measured Measurements Analyzed J
Actinonaias 1igamentina 8 5 13 109 46 21 15 5 3 4 pectorosa 2 2 4 19 5 0 0 0 0 0 Alasmidonta arcula 1 1 4 17 14 5 3 0 0 0 marginata 5 4 7 185 50 20 13 2 2 3 viridis 2 2 10 0 0 0 0 0 0 0 Amblema piicata 8 8 11 167 38 19 11 3 0 0 Anodonta grandis 5 4 5 120 46 40 22 12 10 8 imbecillis 1 1 2 8 8 6 4 2 0 0 oregonensis 2 2 5 6 5 3 3 1 0 0 Anodontoides ferussacianus 4 4 10 118 91 36 21 8 2 2 Arcidens confragosus 1 1 1 1 1 1 1 0 0 0 Cyclonaias tuberculata 4 4 5 37 7 2 1 0 0 0 El 1 i pti o buckleyi 2 2 2 0 0 0 0 0 0 0 crassidens 1 1 1 9 2 1 1 1 1 1 dariensis 3 3 6 166 31 13 8 7 0 0 dilatatus 11 6 10 51 15 5 5 3 3 2 ro al 1 (continued) 1 Table 1“ O “TJ m m cu o c ■o —i 3 3 to _ o . — j "O o o —j « in -5 o O 3 " —h cu to CU Q . to —h o c r o r+ c r to to to mmt. ■o CD rt* cu < -s _ o . cu c —I' 3 fD c= c cu 3 O —j "O - o "O o r+ fD CL cu 3 LQ co CO —J IO cu o cu 3 -s cu “S cu • •• rt* 3 cu rt* cu tO O rt* C o < fD 3 —I* cr to fD O fD 3 CU cu r+ -J. _ o . “S to __o _J. cu O fD cu — j 3 s» —s. “5 c+ CU 3 o cu cu to — J to o cu c_. C_. C_, - j . s cu 3 cu in _ o rt* to c —4. to c_, C_I C o 3 o fD CU wmJ. cu cu to CU cu c_. c_. C_. cu o cu CU to 3 3 3 c cu -$ 1 1 1 1 1 1 rt* Q. rt* Cu ( 7 \ CD fD 3 3 C/> O LO c ■a ~ o . fD fD CU Q. n 3 ■ J i CU fD CO
Specimens —• O V CO Processed
Specimens r o —< -P* — > c o — 1 -P» -P> — i — 1 — 1 w Examined
SI ides ' cy> ro —■ ro ro ro 1 0 r o cr> r o -£> '■o r o —■ — ' c o Scanned
Spreads
CO —1 CO — ' CO — 1 O l — 'C O -P» CO Noted or o > -0 -P» co —« cn cri (7> —1 o —1 co —■ —< co io Observed
co co ro —1> ro Spreads o o —1 ro cr> cn cn cn o -P* —» co o o o *-o Studied
Chromosome o u i o - 1 ro-P> cn o —1 —' -P* 'vi o o ro co Counts
Number of O CO O —* ' O CD O O ro 38’s
Spreads o co o o ro co o oi o o o Photographed
Spreads o ro o o ro co o r o o c o o o o o Measured
Measurements 1 o o ro co o r o o c o o o o o Analyzed LZ al 1 (continued) 1. Table 2 3 1— r~ r" r™ c~ f“ fD cu ml. (D fD fD cu CU O. -5 LQ X 3 3 to 3 u . LQ C _ l. c+ ml. 3 3 | 3 n O 3 cu -s 3 3 Q . 3 —h O -s o o O o ml. < r t to CO cr> fD o 3 CU "5 fD cu ml. O LQ -s Q- -J* X o O o LQ fD fD c —J. fD 3 3 S mi. O (/> cu r t cu fD =3 CO 3 3 O 3 - s c r —J 3 mi, -s Q - IQ r t r+ c CU O LQ cu o r t 3 3 3 r t fD cu — CD O CU C CU ml. cu r+ c r 3 ml. CO cu **5 — j CU - s CO 3 CO -S ml. Q. CO -s CU fD - j . —1 Cl r t fD CU ml. LQ cu m-1—»« —J. fD _J CU ml. CO CU CO 3 n C <»— —J O rt- -S —J (/) CO CU o mml n cu QJ c ml. cu o cu ct- CO CU o 3 rt to - h ml. cu cu c+ 3 Q. C fD Cl CU c+ cn fD __i, CO cu 00 3 *3 c n> fD o Q. fD (/>
r o -p* c n r o c n c o r o o —J ro Specimens Processed
r o -p* .p> r o c n t o r o c n — > —* o o —< r o Specimens Examined
ro cri o cn ro cn co ro —■ co —> Slides Scanned
Spreads r o —■ r o t n -P» t n -P> o o t n -P> t o r o —'O -P> tn—' —' -o -o to co -p to cn o Noted or Observed
—«rororo—*cn ^ —j -p* o •£» o < r o -p * — • cr> .p» c n c o r o Spreads Studied
ro -p. ro ro p . — 1 o a > t n 00 ro—■'o -sjrotn Chromosome Counts
—> ro 0 3 otn ro to to co o to to —> ro Number of 38' s
o ro ro top to >~j ro —1 o to w o n Spreads Photographed
o r o r o n p ro tn ro o o ro —1 o ro Spreads Measured
o ro ro rop - p r o t o o Measurements Analyzed 82 al 1 (continued) 1. Table -a “O -o *3 O o 3 rt O —J —j cr cr fD << ct fD CD o —■* LQ O CD c LQ < CD —j. cr CO 3* CD3 oo in -s tp •a o o -S —J in O CD -s . 0 CD c CU O -J.-U "3 c << < o O -J. O c —J -5 fD c O O fD cr (/) cr CD cr -5 fD o cr 3 —J cr —J. ~b CD*< 3 =3 rt o “5 rt C -5 3 —b o fD fD CD -5 < CD —j 7T CD fD fD CD C 10 ----./■—«c ~h c o 3 O O CD fD —it ~S 3 o 3 in C_. C-J 3 o 3 “5 3 CD _ j rt -5 X CD 3 0> CD rt O Cw c_. -s 3 fD CD c _j. CD ■J* 00 c CD 3* c_. c_. 3 fD c r+ 3 CD CD CD 3 -5 C fD 3 CD CL 3 3 in CD CD Q. 00 CTO CO —1 CO “OCO fD O —j* fD in
Oo c n —j — i r o •—1 —' co ro co ro ro cn Specimens Processed
i «vj -p» —i — i r o — 1 — ' go r o go r o r o c n Specimens Examined
ro —i cn —i —' ro — 1 — 1 w r o c o r o -P» c n Slides Scanned
Spreads < n -P» r o — i c o — > — < r o tn r o —' 00 CO —iQJiOOHOO oo to —1 Oo Noted or Observed
—< po ro ro ro ro ro o o — 1 co o co co -vi co cn ro ro Spreads Studied
4 ^ r o —j c o c o c n o o — ■ o •£» r o — 1 o cr o --j Chromosome Counts
ro—■ o o o —' ro o co —> —> coo o Number of 38's
'tn cn o O —* —* o o — 1 o coin O O Spreads Photographed
Spreads Measured
ocn p» o o —■ o o o — 1 o --J -p- o o Measurements Analyzed 62 al 1 (continued) 1. Table < c r •H —1 -H CO JZD jO —J. 3 3 3 O e+ cr cr —J —j. c —j* X 3 —j. cu ■—>J O 3 c+ o O 3 Q. —J. —J. “ +) o Q. 3 <-+ O < O ro t o ■—1 —-* c to ■a —J. OLQ to 3 o -5 3 QJ to fD fD fD t o CU cu cu 3 * 0 z r 3 cr C cr C fD •u. c r cu n 3 c: 3 o C 3 < to Q_• «->• - h 3 CU to —J =3 to to CU cr 3 —j 3 3 —4 < _J. 3 cr c+ C O Q. c+ cu a> —t _j. to o cu C cu cu CL cu —j —- C o _j. 3 c: 3 3 •J. —j . < cu o cu C cu C_, to cu 3 C —j CU fD 3 to c rt- o l/> rt- c_. rt- cu o c r to CU to C Cm cu cu to CU c l/> cu t o to • • cu 3 —J CL o cu t n to 3 r o GO cu to "O fD O _ i » fD to
o o r o • o o r o t o - j ^1 ro cn cn Specimens Processed
Specimens 0 0 • oo ro to ■ oo • Examined
Slides t n r o r o —* ro co oo cn on cn —1 r o t n Scanned
Spreads .£ » G j -£» — 1 ' ' i o r o o o c n o o cn —1 ro iv) —1 ~~j —iio o co co oo Noted or Observed
—' oo —' —1 cn oo —* oo—1 ro —1 o oo o ro oo oo to on co to oo-P» Spreads Studied
r o r o —* —j t n —» O o n o c n —• 4 ^ o n o o Oo -p* t o c n Chromosome Counts
c n o o cn o oo —‘-~joo —1 co —* r o 4=» Number of 38’ s
on O o iv) o o) iv) co —1 —> -c» ro Spreads Photographed
Spreads —' 4^> O O O —1 OOOIV) OO —> O Measured
Measurements —I -P» o o o Analyzed 0£ Table 1. (continued)
Genera and Species Specimens Processed Specimens Examined SI ides SI Scanned Noted or Noted Spreads Spreads Observed Spreads Studied Chromosome Counts 38's Number of Number Photographed Spreads Spreads Measured Measurements Analyzed
Villosa lienosa 3 3 6 27 19 14 8 3 2 2 sp. (JJJ:157) 1 1 2 4 2 2 1 0 0 0 sp. (JJJ:264 & 265) ' 2 1 1 8 3 1 1 0 0 0 taeniata punctata 4 3 5 40 40 27 21 11 9 9 trabalis 6 6 10 88 88 71 46 13 10 10 vibex 4 4 8 74 15 5 4 1 0 0 TOTALS Genera 34 Species 74 250 218 381 3,556 1,463* 774 504 205 130 121 ( + 7 sp.)
* The number of spreads studied amounted to 41.1 percent of the spreads noted or observed. ** The number of spreads found to include 38 chromosomes amounted to 65.1 percent of all the counts that were made. Species Accounts
Species-specific information concerning the number of animals
processed, the streams from which they were collected and the results of
the chromosomal examinations are presented in the following paragraphs.
The taxa are arranged alphabetically, first by genus, then by species.
Precise locale information is presented alphabetically by stream name in
the Appendix. In both of these sections, a number preceeded by "JJJ" is
the specimen-specific identifier assigned to associate the slides,
tissue samples, soft parts and shells of each animal studied during this
project.
Various terms are used in these accounts and in table 1 to indi cate different processing stages, intensity levels of microscopic exami nation or analysis. In a full sequence these levels would include:
- processed - Various tissues from an animal were used to
prepare chromosome slides.
- examined - One or more slides from the animal were stained and
examined under the microscope.
- scanned - Part or all of a slide was searched for chromosome
spreads under the microscope.
- noted - For all slides from specimen JJJ:1 through 72, only
mitotic figures with several separable chromosomes
were mentioned on the record form; all others were
ignored.
- observed - For each slide from specimen JJJ:73 through 265, the
entire area under a 22 X 50mm cover slip was systema
tically searched and all mitotic figures (regardless 33 of chromosome quality) were tallied on the record
form. These observations were typically made at
200X magnificaiton.
- studied - Mitotic figures which appeared to include identifi
able chromosomes were studied at 900X magnification
and were individually identified (using stage
coordinates) on the record form.
- counted - If the chromosomes were sufficiently well separated
to permit a complete and unequivocal count, this
number was entered on the record form. Counts for
spreads with groups of overlapping or unseparable
chromosomes were indicated on the form as "37?" or
"34+" and are not included in the "count" totals.
- photographed - Mitotic figures with well spread, seldom over
lapping chromosomes usually showing arms and centro
meres were indicated by a condition code on the
record form and were photographed for further
analysis.
- measured - Usable photographic prints were compared with the
actual chromosome spread. During this comparison,
centromeres and arms were located or outlined on the
print. If most centromeres could be located, the
spread was measured.
- analyzed - Nearly all sets of measurements were analyzed. The
ones that were not, included five or more chromo
somes on which the centromere had riot been located. 34 Actinonaias ligamentina (Lamarck, 1819)
Eight specimens representing the two forms of this species were
processed. Three of six specimens of the carinata (Barnes, 1823) form
were collected in the Green River, Kentucky, (JJJ:51, 52 and 53). The
other carinata form individuals came from French Creek, Pennsylvania
(JJJ:249); Meramec River, Missouri (JJJ:200); and the Black River,
Missouri (JJJ:206). The two specimens of the ligamentina form were
were collected in the Clinch River, Tennessee (JJJ:124 and 126).
One hundred nine chromosome spreads were noted or observed on
13 slides that were scanned (JJJ:51, 200 and 249 not included).
Forty-six spreads were studied in some detail, resulting in 21 counts of
the chromosomes. Chromosome counts ranged from 24 to 50 distributed as
follows: 24-1, 30-1, 36-1, 37-1, 38-15, 39-1, 50-1. Five of these
spreads were photographed and three photographs (all from specimen
JJJ-.52) were measured (one twice). The composite analysis of these
three measured spreads is presented in table 2 and the suggested karyo
type of one of them is presented in figure 1.
Actinonaias pectorosa (Conrad, 1834)
Two specimens were examined; one from Buck Creek in Kentucky (JJJ:
32) and the other from the Clinch River in Tennessee (JJJ:130). Scans of
four slides representing both animals yielded 19 chromosome spreads. Five
chromosome spreads were studied but none of them was sufficiently well
spread to provide a chromosome count. No spread was photographed.
Alasmidonta arcula (Lea, 1838)
One specimen (JJJ:75) from the Ohoopee River, Georgia was examined.
Of the seventeen spreads observed on four slides, chromosome counts were 35
Table 2. Separation of the chromosomes of Actinonaias ligamentina form carinata using values of "%TCL" and "r" derived from measurements of three spreads all from specimen JJJ:52. The first and fourth entries in each section of the table are based upon different sets of measurements of the same (unoutlined) photograph. *
* The abbreviations used in this and succeeding similar tables are: "%TCL" - percent total complement length (The sum of both arms of a chromosome divided by the sum of all chromosome arms measured in this spread.) Number ranges refer to the 0 to 2 percent complement length interval, the 2 to 3 percent interval, etc. "m" - metacentric chromosomes (r values between 1.0 and 1.7). "r" - ratio of the length of the long arm of a chromosome divided by the length of the short arm of that chromosome. "sm" - submetacentric chromosomes (r values between 1.7 and 3.0). "st" - subtelocentric chromosomes (r values between 3.0 and 7.0). "t" - telocentric chromosomes (r values greater than 7.0). "x" - arithmetic mean of the values present in this section of the table. Superscript numbers in the "r" totals row indicate chromosomes on which the centromere could not be located. These chromosomes have been included in the column means and in the grand mean (lower right) but are not included in any other sections.
t 36
Table 2 (continued)
% TCL
0 - 2 2 - 3 3 - 4 > 4 Totals
4 4 3 13 14 14 1 3 2 2 2 1 20 23 20 4 12 2 2 20 m x = 3.75 x = 13.25 x = 2.00 x = 1.75 x = 20.75
2 0 1 8 8 8 4 1 4 0 0 0 14 9 13 sm 3 8 4 0 15 x = 1.50 x = 8.00 x = 3.25 x = 0 x = 12.75 r 0 0 0 4 4 5 0 0 0 0 0 0 4 4 5 St 1 2 0 0 3 x = 0.25 x = 3.75 x = 0 x = 0 x = 4.00
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 m 6 4 4 25 26 27 5 4+1 6 2 2 1 38 36+1 38 ra 8 22 6 2 38 o x = 4.00 x = 25.00 x = 5.50 x = 1.75 x = 37.75 m I (: S HSU 8SS a Rll
> 4% (2) 3-4% (2) 2-3% (14) 0-2% (4) m I I u
s m 3-4% (4) Mil H U B is 2-3% ( 8 )
S t 10m H ft l 2-3% (4)
Figure 1. Karyotype of Actinonaias ligamentina form carinata based upon an analysis of "%TCL" and "r" values from three chromosome spreads, all from specimen JJJ:52. This is the spread that was not outlined but was measured twice.
CO 38 obtained from five, as follows: 25-1, 35-1, 38-3. None of these spreads was photographed or measured.
Alasmidonta marqinata Sa.y, 1818
Four specimens (JJJ:1, 98, 100, 172) from Big Darby Creek, Ohio and one (JJJ:246) from French Creek, Pennsylvania were processed and examined. Seven slides from all four animals were scanned. Of the 185 spreads observed, 50 were studied in detail. Chromosome counts were obtained from 20 as follows: 22-1, 27-1, 31-1, 32-1, 36-2, 38-13, 39-1.
Three of these spreads (all from JJJ:1) were photographed and two of the photographed spreads were measured (one twice from different photo graphs). The measured spreads are compared in table 3 and a suggested karyotype is presented in figure 2.
Table 3. Separation of the chromosomes of Alasmidonta marqinata using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:1. The firs t and second entries in each section of the table are based on different photographs (and measurements) of thfe same spread. Abbreviations are explained in the footnote to table 2.
% TCL 0 - 2 2 - 3 3 - 4 > 4 Totals
3 5 3 10 11 10 6 3 8 0 1 0 19 20 21 m x = 3.67 x = 10.33 x = 5.67 x = 0.33 x = 20.00 0 2 1 12 10 12 3 2 2 0 0 0 15 14 15 sm x = 1.00 x = 11.33 x = 2.33 x = 0 x = 14.67 0 0 0 3 4 2 1 0 0 0 0 0 4 4 2 st x = 0 x = 3.00 x = 0.33 x = 0 x = 3.33
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 in 3 7 4 25 25 24 10 5 10 0 1 0 38 38 38 +JIB O x = 24.67 x = 8.33 x = 0.33 x = 38.00 1— x = 4.67 m
3-4% (6) 2-3% (10) 0-2% (4) m
3-4% (2) s m 2-3% (12)
s t 2-3% (4) 10 |J
Figure 2. Karyotype of Alasmidonta marqinata based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ: 1.
c o v o 40 Alasmidonta viridis (Rafinesque, 1820)
Two specimens (JJJ:255 and 258) were processed and examined; both
were collected in Log Pond Run, Ohio. No chromosome spreads were
observed on 10 slides scanned from both specimens.
Amblema plicata (Say, 1817)
Eight specimens representing three subspecies or forms were
processed and examined. The form costata Rafinesque, 1820, was repre
sented by two specimens (JJJ:239 and 240), both collected from Lake Erie
in Ohio; the form or subspecies perplicata (Conrad, 1841) was represent
ed by two specimens (JJJ:163 and 174), both collected in the Cahaba
River, Alabama; and the form or subspecies plicata (Say, 1817) was
represented by four specimens, two of which (JJJ:77 and 120) were
collected in Big Darby Creek, Ohio; one (JJJ:186) from the Duck River,
Tennessee and one (JJJ:205) from the Black River, Missouri.
From these specimens, eleven slides were scanned and a total of
167 spreads were observed. Thirty-eight spreads were studied in detail,
of which 19 yielded counts as follows: 26-1, 29-1, 31-1, 33-1,
35-1, 38-11, 39-2, 40-1. Three of these spreads were photographed;
however, none of them was sufficiently distinct to be measured.
Anodonta grandis Say, 1829
Five specimens referable to this species were processed; two from
Big Darby Creek, Ohio (JJJ:106 and 183) and one from each of the follow
ing locations: (Big) Buffalo Creek, Ohio (JJJ:9); Lake Manitoba, Canada
(JJJ: 173); and Whitewater Creek, Georgia (JJJ:131).
One hundred twenty spreads were observed on five slides from all of these animals except JJJ:183. Forty-six spreads were studied in detail of which 40 yielded chromosome counts as follows: 20-1, 22-1,
24-1, 26-1, 27-1, 28-1, 30-1, 31-2, 32-1, 33-1, 36-1, 37-5, 38-22. Six
spreads from specimen JJJ:106 included what appeared to be a small "dot"
chromosome in addition to the normal-size complement. In two spreads,
38 chromosomes and the single dot could be counted.
Twelve spreads were photographed and ten of these (from specimens
JJJ:9 and 106) were measured. Separations based on eight of these sets
of measurements are compared in table 4. Figure 3 is the suggested
karyotype of one spread from JJJ:106 and includes the dot chromosome.
Anodonta imbecillis Say, 1829
The single specimen of this species that was processed and
examined (JJJ:24) was collected from Lake Erie in Ohio. On two slides
from this animal, eight spreads were noted. Six of these spreads were
counted, as follows: 24-1, 35-1, 38-4. Two of these spreads were
photographed; however, neither was measured.
Anodonta oregonensis Lea, 1838
Two specimens of this species were processed and examined. One
of these animals (JJJ:54) was collected in Devils Lake, Oregon while the
other (JJJ:58) came from the Willamette River, Oregon. Six spreads on
five slides from both of these animals were noted and chromosome counts
were made for three spreads (all 38). One of these spreads was
photographed but was not measured.
Anodontoides ferussacianus (Lea, 1834)
The four members of this species that were processed and examined were collected in two small Ohio streams. Three of these specimens 42
Table 4. Separation of the chromosomes of Anodonta qrandis using values of "%TCL" and "r" derived from measurements of eight spreads from specimens JJJ:9 and 106. Abbreviations are explained in the footnote to table 2.
% TCL 0 - 2 2 - 3 3 - 4 > 4 Totals
1 3 4 15 13 12 6 6 8 1 1 2 23 23 26 1 2 3/ 14 11 16 9 6 6 1 2 1 25 21 26 m 1 3 15 9 6 5 2 0 24 17 X = 2.25 X = 13.13 x = 6.50 X = 1. 25 X = 23.13 1 0 0 4 11 6 3 1 2 0 0 0 8 12 8 0 0 2 10 10 8 0 1 2 0 0 0 10 11 12 sm 1 1 8 12 1 3 1 0 11 16
X = 0.63 X = 8.63 x = 1.63 X = 0. 13 X = 11.00
0 0 1 7 3 2 0 0 0 0 0 0 7 3 3 0 1 0 1 5 0 0 0 0 0 0 0 1 6 0 r St 0 2 3 3 0 0 0 0 3 5 1 X O X = 0.50 X = 3.00 II X = 0 X = 3.50
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t 0 0 0 0 0 0 0 0 0 0 X = 0 X = 0 x = 0 X = 0 X = 0
2 3 5 26 27 20 9 7 10 1 1 2 38 38 37 >4% (2) 3-4% (6) 2-3% (14) 0 - 2% ( 2 ) m S t 2-3% (4) "dot" 1 0 |J Figure 3. Karyotype of Anodonta grandis based upon an analysis of "%TCL" and "r" values from eight chromosome spreads from specimens JJJ:9 and 106. This spread (from JJJ:106) includes the dot chromosome mentioned in the text. CO 44 (JJJ:14, 34 and 36) came from (Big) Buffalo Creek and the other specimen (JJJ:215) was found in Alum Creek. Ten slides from all four of these animals contained at least 118 chromosome spreads (some slide scans were incomplete). Ninty-one spreads were studied and the 36 counts of chromo somes ranged as follows: 19-1, 21-2, 23-2, 24-1, 26-1, 30-1, 33-2, 34-1, 36-1, 37-3, 38-21. Eight of these spreads were photographed and two were measured. The comparison of these measurements is presented in table 5 and a suggested karyotype is presented in figure 4. Arcidens confraqosus (Say, 1829) The single specimen of this species that was processed (JJJ:260) was collected in the Green River, Kentucky. One slide from this Table 5. Separation of the chromosomes of Anodontoides ferussaci- ar.us using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:14. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 2 8 8 3 6 1 0 15 16 m x = 2.50 >< = 8.00 x = 4.50 x = 0.50 x = 15.50 2 1 12 14 2 2 0 0 16 17 sm x = 1.50 x = 13.00 x = 2.00 x = 0 x = 16.50 0 0 4 5 2 0 0 0 6 5 St x = 0 x = 4.50 x = 1.00 x = 0 x = 5.50 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 w) 5 3 24+1 27 7 8 1 0 37+l 38 4->ro O I— x = 4.00 x = 26.00 x = 7.50 x = 0.50 x = 38.00 m 3-4% (6) 2-3% (8) 0 - 2% ( 2 ) m 3-4% (2) s m 2-3% (14) 0- 2% ( 2 ) Figure 4. Karyotype of Anodontoides ferussacianus based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:14. Darker stain areas at the position of the centromeres indicate that this spread came from a slide treated to show "C" banding patterns. cn 46 specimen was scanned and a single spread of 38 chromosomes was found. This spread was not photographed or measured. C.yclonaias tuberculata (Rafinesque, 1820) Three of the four specimens of C. tuberculata processed (JJJ:169, 170 and 187) were collected in the Duck River, Tennessee and the fourth specimen (JJJ:190) was found in Big Darby Creek, Ohio. Five slides from all four of these animals were scanned and 37 chromosome spreads were observed. Seven of these spreads were studied resulting in two chromosome counts: 27-1, 38-1. Neither spread was photographed or measured. El 1iptio buckleyi (Lea, 1843) The two specimens of this species that were processed (JJJ:259 and 261) had been purchased from Carolina Biological Supply Company and probably had been collected in Florida. Scans of one slide from each animal failed to show any chromosome spreads. El 1iptio crassidens (Lamarck, 1819) The specimen of E. crassidens that was processed (JJJ-.104) was collected in the Green River, Kentucky. One slide was scanned and nine chromosome spreads were observed. Two of these spreads were studied and one was counted (38 chromosomes). This spread was photographed and measured. Analysis of the data from this spread is presented in table 6 and the apparent karyotype is illustrated in figure 5. 47 El 1iptio dariensis (Lea, 1842) All three specimens of this species that were processed and examined (JJJ:68, 78 and 182) were collected in the Ohoopee River, Georgia. The forms on six slides from these three animals note 166 chromosome spreads, of which 31 were studied. Thirteen counts of the chromosomes were as follows: 18-1, 19-1, 26-1, 28-1, 38-8, 39-1. Seven of these spreads were photographed; however, none of them was measured. El 1iptio dilatatus (Rafinesque, 1820) Eleven specimens of E. dilatatus were processed. Specimens numbered JJJ:74, 107 and 184 were collected in Big Darby Creek, Ohio; Table 6. Separation of the chromosomes of Elliptio crassidens using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:104. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 11 4 2 20 m 0 16 1 0 17 sm 0 1 0 0 1 St 0 t 0 0 0 0 3 28 5 2 38 Totals m U*> M U 4% (2) 3-4% (4) 2 - 3% (10) 0- 2% (4) m MU issa s m 4% (2) 3% (14) 1 0 m i s t i i 3% (2) Figure 5. Karyotype of El 1iptio crassidens based upon an analysis of "%TCL" and "r" values from a chromosome spread o f specimen JJJ:104. 49 JJJ:191 and 192 came from Little Darby Creek, Ohio; JJJ:198 and 204 came from the Meramec River, Missouri; JJJ:232, 233 and 234 came from the Gasper River, Kentucky; and JJJ:248 came from French Creek, Pennsylvania. Ten slides from six of these animals were scanned (JJJ:198, and lower numbers) and 51 chromosome spreads were observed. Fifteen spreads were studied in detail and five were counted (all 38). Three of these spreads were photographed and all three were measured. A comparison of the data from two of these spreads is presented in table 7 and the suggested karyotype is shown in figure 6. Table 7. Separation of the chromosomes of Elliptio dilatatus using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:T91. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 4 1 15 9 4 6 2 2 25 18 x = 2.50 x = 12.00 x = 5.00 x = 2.00 x = 21.50 1 1 7 12 1 0 0 0 9 13 sm x = 1.00 x = 9.50 x = 0.50 x = 0 x = 11.50 0 0 2 1 0 2 0 0 2' 3 st x = 0 x = 1.50 x = 1,00 x = 0 x = 2.50 0 0 0 0 t 0 0 0 0 0 0 x = 0 x = 0 x = 0 x = 0 x = 0 cn 5 2 24 22 5+2 8 2 2 36+2 34 4-> — _ . O h- x = 3.50 x = 23.00 x = 7.50 x = 2.00 x = 36.00 >4% (2) 3-4% (6) 2-3% (14) 0 - 2% ( 2 ) m » i 3-4% (2) s m 2-3% (8) K7 UH 0 - 2% ( 2 ) S t J 1 0 jJ 2-3% (2) Figure 6. Karyotype of El 1iptio dilatatus based upon an analysis'%TCL" of and values from two chromosome spreads, both from specimen JJJ:'191. cn o Elliptio icterina (Conrad, 1834) Three specimens of this species were processed and examined. One of them (JJJ:135) was collected in Whitewater Creek, Georgia, and the other two (JJJ:139 and 148) came from Uchee Creek, Alabama. Three slides were scanned and a total of 49 spreads were observed. Seven spreads were studied and three were counted: 35-1, 38-2. One of these spreads was photographed but not measured. El 1iptio sp. Three processed animals collected from Alabama Gulf Coast drainage streams appeared to be members of the genus Elliptio; however, they were not identified to species. Specimen number JJJ: 151 was taken out of Cane Creek, and specimens JJJ:164 and 177 were found in the Cahaba River. One slide from the Cane Creek specimen yielded 34 chromo some spreads, seven of which were studied and two were counted (23-1, 38-1). From the Cahaba material, one slide of specimen JJJ:164 exhib ited three uncountable spreads and two slides of JJJ:177 yielded one uncountable spread. None of these spreads was photographed. Epioblasma torulosa ranqiana (Lea, 1839) Four specimens were processed and examined: two (JJJ:76 and 214) from Big Darby Creek, Ohio and two (JJJ:254 and 256) from the Allegheny River, Pennsylvania. Seven slides examined from these four animals included a total of 41 chromosome spreads, of which 20 were studied and seven were counted (21-2, 38-5). Three of these spreads were photo graphed and one was measured. The separation of this spread is presented in table 8 and the apparent karyotype is illustrated in figure 7. 52 Table 8. Separation of the chromosomes of Epioblasma torulosa rangiana using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:214. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 3 3 2 11 m 3 10 1 1 15 sm 0 3 2 0 5 st 0 0 1 0 1 t i 6+1 16+3 7 3 32+4 Totals Fusconaia barnesiana (Lea, 1838) Three of the five specimens of JF. barnesiana that were prepared (JJJ:103, 113 and 114) were collected in the Duck River, Tennessee; the other two specimens (JJJ:262 and 263) were collected in the Clinch River, Virginia. Four slides examined from these speciemns (one each, excluding JJJ:263) yielded 38 chromosome spreads. Eighteen spreads were studied, of which 14 could be counted. These counts were distributed as follows: 23-1, 26-1, 35-1, 37-1, 38-10. Five of these spreads were photographed and three of them were measured. The data from these spreads are compared in table 9 and a suggested karyotype is presented in figure 8. 4% (2) 3-4% (4) 2-3% (8) 0-2% (4) m %% 6H\f» s m 3-4% (2) aH iW U C U 1. 2-3% (12) s t 3-4% ( 2 ) 2-3% (4) 1 0 jJ Figure 7. Possible karyotype o f Epioblasma torulosa rangiana based upon "%TCL" and chromosome spread of specimen JJJ:214. Several pairs of separated chromatids, a few d iffic u lt overlaps and two apparently missing chromosomes may have confused the analysis of this spread. <_n co 54 Table 9. Separation of the chromosomes of Fusconaia barnesiana using values of "%TCL" and "r" derived from measurements of three spreads all from specimen JJJ:114. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 3 1 19 21 20 2 3 6 2 2 1 26 29 28 m x = 2.33 x = 20.00 x = 3.67 x = 1.67 x = 27.67 2 1 1 5 7 8 1 1 1 0 0 0 sm 8 9 10 x= 1.33 x = 6.67 x = 1.00 x = 0 x = 9.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 St x = 0 x = 0 x = 0 x = 0 x = 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x" = 0 x = 0 x = 0 . x = 0 x = 0 cn re 5+1 4 2 24+3 28 28 3 4 7 2 2 1 34+4 38 38 +j o h- x = 3.67 x = 27.67 x = 4.67 x = 1.67 x = 38.00 Fusconaia cuneolus (Lea, 1840) The specimen of £_. cuneolus that was processed and examined (JJJ:128) was collected in the Clinch River, Tennessee. Two slides contained a total of 11 chromosome spreads, of which only one was countable (38 chromosomes). It was photographed but not measured. Fusconaia flava (Rafinesque, 1820) Six specimens were processed; however, slides from only three of them have been examined. Specimen origin is as follows: JJJ:65, Little Barren River, Kentucky; JJJ:79 and 242, Lake Erie, Ohio; JJJ:93, Green River, Kentucky; JJJ:219, Black River, Missouri; and JJJ:251, Allegheny River, Pennsylvania. Six slides from specimens JJJ:65, 79 and 93 >4% (2) 3-4% (4) 2-3% (20) 0 - 2% ( 2 ) m s m 2-3% ( 8 ) 0 - 2 % ( 2 ) lOjU Figure 8. Karyotype of Fusconaia barnesiana based upon an analysis of "%TCL" and "r" values from three chromosome spreads, all from specimen JJJ:114. c n <_n 56 included 50 chromosome spreads, of which 24 were studied in detail. The chromosome count distribution for the 11 countable spreads is: 26-1 36-1, 38-9. Six of these spreads were photographed and two of them were measured. The data from these measured spreads are compared in table 10 and a suggested karyotype is given in figure 9. Fusconaia succissa (Lea, 1852) The specimen of this species that was processed and examined (JJJ:160) was collected in Burnt Corn Creek, Alabama. Two slides were scanned, resulting in 11 spreads, none of which could be counted. None were photographed or measured. Table 10. Separation of the chromosomes of Fusconaia flava using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:79. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 4 2 11 14 4 2 2 2 21 20 m >< = 3.00 x = 12.50 x = 3.00 x = 2.00 x = 21.50 2 3 6 9 5 4 o 13 16 sm I II X X x = 2.50 x = 7.50 x = 4.50 o o o o x = 14.50 1 X O 0 0 2 1 o o 0 0 2 1 St II O x = 0 x = 1.50 o x = 0 x = 1.50 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 i/i 2 2 36+1 37 (O 6 5 19+1 24 9 6 +J O 1— x = 5.50 x = 22.00 x = 7.50 x = 2.00 x = 37.00 m t o >4% (2) 3-4% (4) 2-3% (14) 0- 2% ( 2 ) m 3-4% (6) s m 2-3% (6) K liC s III He, v*0 - 2% ( 2 ) 2-3% (2) s t *«* IO jj Figure 9. Karyotype of Fusconaia Hava based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:79. 58 Gom'dea anqulata (Lea, 1838) All four of the specimens of G. anqulata processed (JJJ:55, 60, 61 and 62) were collected in the Williamette River, Oregon. Nine slides prepared from all four of these animals were scanned and 36 chromosome spreads were observed and studied. Twenty-four of these spreads were counted with the following results: 26-1, 32-2, 35-1, 36-1, 37-3, 38-16. Seven of these spreads were photographed and four of them were measured. The analysis of the data is presented in table 11 and the suggested karyotype is presented in figure 10. Lampsilis australis Simpson, 1900 One specimen was processed (0J0:156). It was collected in Burnt Corn Creek, Alabama. Two slides from this animal contained 16 chromo some spreads, five of which were studied and counted. The distribution of these counts is: 18-1, 19-1, 20-1, 38-2. One of these spreads was photographed and measured. The separation of this spread is presented in table 12 and the apparent karyotype in figure 11. Lampsilis fasciola Rafinesque, 1820 Three specimens of L_. fasciola were processed: one (JJJ:48) from Buck Creek, Kentucky; one (JJJ:185) from Big Darby Creek, Ohio; and one (JJJ:252) from the Allegheny River, Pennsylvania. Five spreads noted and studied on one slide from JJJ:48 yielded four chromosome counts (37-1, 38-3); however, no spreads were observed on one slide from JJJ:185. Slides from JJJ:252 were not scanned. Three of the counted spreads were photographed and all three of them were measured. A compar ison of the separation based upon these measurements is presented in table 13 and the suggested karyotype is illustrated in figure 12. 59 Table 11. Separation of the chromosome of Gonidea anqulata using values of "%TCL" and "r" derived from measurements of four spreads all from specimen JJJ:55. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 3 3 - 4 > 4 Totals 3 2 1 8 7 10 1 2 3 1 0 0 13 11 14 1 13 3 0 17 m x = 1.75 x = 9.50 x = 2.25 X = 0 25 X = 13.75 3 5 1 13 7 15 6 3 4 0 1 0 22 16 20 sm 1 12 7 0 20 x = 2.50 x = 11.75 x = 5.00 X = 0 25 X = 19.50 r 0 2 0 3 3 3 0 2 0 0 0 0 3 7 3 0 0 0 0 0 st x = 0.50 x = 2.25 x = 0.50 X = 0 X = 3.25 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 t x = 0 "x = 0.25 x = 0 X = 0 X = 0.25 7+2 7 i/> 6 9 2 24 17 +2 29 7 1 1 0 38 34+4 38 ro 2 25 10 0 37 +J 1— x = 4.75 x" = 24.25 x = 8.25 X = 0 . 50 X = 37.75 m 3-4% (2) 2-3% (10) 0 - 2% ( 2 ) m 9 * 3-4% (6) s m 2-3% (12) 0- 2% ( 2) 1 9 s t 2-3% (4) Alt 10 |J Figure 10. Karyotype of Gonjdea anqulata based upon an analysis of "%TCL" and "r" values from four chromosome spreads, a ll from specimen JJJ:55. CT> O 61 Table 12. Separation of the chromosomes of Lampsilis australis using values of "%TCL" and "r" derived from measurements of one spread from specimenJJJ:156. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals m 3 14 1 2 20 1 8 1 1 11 sm st 0 5 0 0 5 0 0 0 0 0 t 4 27+1 2+1 3 36+2 Totals Table 13. Separation of the chromosomes of Lampsilis fasciola using values of "%TCL" and "r" derived from measurements of three spreads all from specimen JJJ:48. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 0 4 5 14 10 13 5 3 1 1 1 2 20 18 21 m x = 3.00 x = 12.33 x = 3.00 x = 1.33 x = 19.67 0 2 0 6 10 9 4 2 5 0 0 0 10 14 14 sm x = 0.67 x = 8.33 x = 3.67 x = 0 x = 12.67 0 0 0 4 5 2 1 2 0 0 0 0 5 7 2 st x = 0 x = 3.67 x = 1.00 x = 0 x = 4.67 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 in 0 6 5 24 25 24+l 10 7 6 1 1 2 35 39 37+1 rO +J O x = 3.67 x = 24.33 x = 7.67 x = 1.33 x = 37.33 h - m J! || |I9)!I<< > 4% (2) 3-4% (2) 2-3% (14) 0-2% (4) m •till* if IV 3-4% (2) s m ||w m I1111)9)1 I A l l l l i 2-3% 10 M I 2-3% (6) s t if 8%lf Figure 11. Karyotype of Lampsilis australis based upon "%TCL" and "r" values from a chromosome spread o f specimen JJJ: 156. cr> f\3 m i p >4% (2) 3-4% (4) 2-3% (12) 0-2% (4) m H i m Kit 3-4% (4) s m KiSUtt 2-3% (8 ) s t 2-3% (4) lO jj Figure 12. Karyotype of Lampsilis fasciola based upon an analysis of "%TCL" and "r" values from three chromosome spreads, all from specimen JJJ:48. Two chromosomes somewhat removed from the rest of this spread were not photographed. CTi CO 64 Lampsilis higginsi (Lea, 1857) The single specimen of this species processed and examined (JJJ:223) was collected in the Mississippi River, Wisconsin. Two slides held a total of 31 chromosome spreads, of which six were studied and two counted (38-1, 39-1). Both of these spreads were photographed and measured. Table 14 presents a comparison of the analysis data and figure 13 presents the karyotype suggested. Lampsilis ornata (Conrad, 1835) A specimen (JJJ:143) collected in Uphapee Creek, Alabama was the only member of this species processed. The single slide scanned had 13 chromosome spreads, two of which were studied. One could be counted (38). This spread was not photographed or measured. Table 14. Separation of the chromosomes of Lampsilis higginsi using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:223. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 6 4 7 13 3 2 2 2 18 21 m x = 5.00 x = 10.00 x = 2.50 x = 2.00 x = 19.50 1 1 10 9 3 2 0 0 14 12 sm _ x = 1.00 x = 9.50 x = 2.50 x = 0 x = 13.00 0 0 0 2 1 0 0 0 1 2 |x |X |X o Oo o II II s i II x = 0.50 x = 1.50 I 0 0 0 0 0 0 0 0 0 0 I x = 0 x = 0 x = 0 x = 0 x = 0 to ra 7 5 17+3 24+4 7+l 4 2 2 33+4 35+4 o 1— x = 6.00 x = 24.00 x = 6.00 x = 2.00 x = 38.00 m » I U >4% (2) 3-4% (2) 2-3% (12) 0-2% (4) m f»it< llll 3-4% (4) s m 2-3% (10) Icit iiiiifuii tt 0 - 2% ( 2 ) 2-3% (2) s t 1 0 ju Figure 13. Karyotype of Lampsilis higginsi based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:223. CP> cn 66 Lampsilis ovata (Say, 1817) The Clinch River, Tennessee provided the single specimen of this species that was processed (JJJ:127). Two scanned slides yielded a total of four spreads, none of which could be counted. Lampsilis radiata luteola (Lamarck, 1819) Ten specimens of this common Interior Basin form were prepared for chromosome study. Seven of these animals (JJJ:2, 3, 5, 7, 21, 99 and 176) were collected in Big Darby Creek, one (JJJ:218) from Little Darby Creek, and one (JJJ:216) from Alum Creek (all Ohio). Specimen JJJ:64 was collected in the Little Barren River, Kentucky. The slides from specimens JJJ:2 and 3 were discarded early in the life of this project and the slides from JJO:216 and 218 have not been examined. For the remaining six animals, 16 slides included a total of 97 chromosome spreads. Thirty spreads were studied in some detail and 15 of these spreads could be counted, resulting in the following d istri bution pattern: 23-1, 33-1, 38-13. Three of the countable spreads were photographed and two of these were measured. The "r" by "%TCL" sorting of the data from one of these spreads is presented in table 15 and the suggested karyotype is presented in figure 14. Lampsi1 is straminea form claibornensis (Lea, 1838) Specimen JJJ:144 was the sole representative of this taxon pro cessed. This animal was collected in Uphapee Creek, Alabama. One slide was scanned and no chromosome spreads were observed. 67 Table 15. Separation of the chromosomes of Lampsilis radiata luteola using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:99. Abbreviations are explained in the foot note to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals m 5 14 0 2 21 0 12 3 0 15 sm 0 1 0 0 1 st 0 t 0 0 0 0 27+1 5+> 3 2 37 +2 Totals Lampsilis (Vi 11osa?) subanqulata (Lea, 1840) Two specimens of this species were processed (JJJ:137 and 146); both of them were collected in Lichee Creek in Alabama. Four slides from both of these animals included 20 chromosome spreads, of which 12 were studied in detail. Chromosome counts for five spreads were distributed as follows: 17-1, 22-1, 37-1, 38-2. Two of these spreads were photo graphed and both were measured. Table 16 presents the analysis of one of these spreads and figure 15 illustrates the suggested karyotype. m )hU!(U!l!8! >4% (2) 2-3% (14) 0-2% (4) m t i n s m 3-4% (4) tut 2-3% (12) s t fi 10 Jj 2-3% (2) Figure 14. Karyotype of Lampsilis radiata luteola based upon an analysis of "%TCL" " and values "i from a chromosome spread o f specimen JJJ:99. CT> 00 69 Table 16. Separation of the chromosomes of Lampsilis subanqulata using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:137. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 17 3 2 25 m sm 0 5 3 0 8 0 4 0 0 4 st 0 0 0 0 0 t 3+1 26 6 2 37 +1 Totals Lampsilis teres (Rafinesque, 1820) A specimen (JJJ:142) of L_. teres from Uphapee Creek, Alabama was the only member of this species processed. The single slide scanned included six chromosome spreads, of which three were studied and two were counted (25-1, 38-1). Neither of these spreads was photographed or measured. Lampsilis ventricosa (Barnes, 1823) Ten specimens of L_. ventricosa were processed; however, slides have been scanned for only eight of them. Three of these animals (JJJ:13, 20 and 42) were collected in Buck Creek, Kentucky; two (JJJ:70 and 108) were collected in Big Darby Creek, Ohio; two (JJJ:202 and 209) were collected in the Meramec River,Missouri; and one each came from 3-4% (2) 2-3% (18) 0-2% (4) m UWHCO Kit 3-4% (4) s m 2-3% (4) s t 2-3% (4) 1 0 | J Figure 15. Karyotype of Lampsilis subanqulata based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJO:137. O (Big) Buffalo Creek, Ohio (JJJ:35); the Gasconade River, Missouri (JJJ.-197); and French Creek, Pennsylvania (JJJ:247). Scans of 13 slides representing all of these animals except JJJ:197 and 247, yielded 139 chromosome spreads. Of these, 45 were studied in detail and 27 had the chromosomes sufficiently well separated so that they could be counted. The distribution of these counts is as follows: 23-1, 24-1, 25-1, 28-1, 30-1, 32-1, 33-4, 34-1, 35-1, 37-2, 38-13. Three of these counted spreads were photographed and one of them was measured. Analysis of the measured spread is presented in table 17. The apparent karyotype is presented in figure 16. Table 17. Separation of the chromosomes of Lampsilis ventricosa using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:35. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 16 2 2 23 m sm 0 11 2 0 13 0 2 0 0 2 st 0 0 0 0 0 t 3 29 4 2 38 Totals - *t/o \Cj 3-4% (2) 2-3% (16) 0-2% (4) m hit* 3-4% (2) s m 2-3% (10) S t 2-3% (2) 10 Jj Figure 16. Karyotype of Lampsilis ventricosa based upon an analysis of "%TCL" and "r" values from a chromosome spread of specimen JJJ:35. ro 73 Lasmigona complanata (Barnes, 1823) The single animal of this species that was processed (JJJ:67) was collected in Big Darby Creek, Ohio. Four slides were examined and a total of 44 chromosome spreads were noted. Seven of these spreads were counted giving the following distribution: 26-2, 27-1, 29-1, 38-3. Three of these spreads were photographed and two of them were measured. Table 18 presents the analysis of one set of these data and figure 17 presents the apparent karyotype. Lasmigona compressa (Lea, 1829) One specimen of I. compressa was processed (JJJ:245). It was collected in French Creek, Pennsylvania. The single slide from this animal that was scanned included 58 chromosome spreads, only one of Table 18. Separation of the chromosomes of Lasmigona complanata using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:67. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 6 8 0 17 m 2 4 4 0 10 sm 0 7 st 0 0 7 0 0 0 t 0 0 5 17+2 12+1 0 34+3 Totals m t u t n m * t t 3-4% (8) 2-3% (8) 0-2% (4) m i i «« 3-4% (4) s m 2-3% (4) HU n u i i 0 - 2% ( 2 ) s t 2-3% (8) >aiiciih 1 0 m Figure 17. Karyotype of Lasmigona complanata based upon an analysis of "%TCL" and "r" values from a chromosome spread o f specimen JJJ:67. 75 which could be counted (33). The chromosomes in this and many of the other spreads on this slide were quite short and none of them were photographed or measured. Lasmigona costata (Rafinesque, 1820) Chromosome slides were prepared from seven members of this species. Three of these animals (JJJ:23, 119 and 210) were collected in Big Darby Creek, Ohio; two of the animals (JJJ:46 and 47) were collected in Buck Creek, Kentucky; one animal (JJJ:129) came from the Clinch River, Tennessee and the seventh animal (JJJ:244) came from French Creek, Pennsylvania. Eleven slides (representing all of these animals except JJJ:244) were scanned and 89 chromosome spreads were noted or counted. Seventy-one spreads were studied and counts were obtained from 42 of these spreads, producing the following distribution: 23-1, 28-2, 31-1, 34-1, 36-4, 37-3, 38-29, 39-1. Eleven of these spreads were photographed and 10 were measured. Table 19 presents the analysis of nine measured spreads. Figure 18 presents a suggested karyotype. Lemiox rimosus (Rafinesque, 1831) Both specimens of this species that were processed (JJJ:94 and 102) were collected in the Duck River, Tennessee. One slide from each animal yielded a total of seven chromosome spreads. Four of these spreads were studied and counted with the following results: 37-1, 38-3. Two of these spreads were photographed and measured. The analysis of the measurement data on these spreads is given in table 20. The suggested karyotype of one spread is presented in figure 19. 76 Table 19. Separation of the chromosomes of Lasmigona costata usinq values of "%TCL" and "r" derived from measurements of nine spreads from specimens JJJ:23 and 47. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 2 4 7 6 4 4 5 8 2 1 1 16 14 17 3/ 4 3 4 8 5 3 6 5 1 0 0 11 18 13 m 6 6 4 7 5 9 5 4 7 0 0 0 18 15 20 x = 3.89 x = 6.11 x = 5.22 x = 0.56 x = 15.78 2 0 2 14 13 13 2 2 2 0 0 0 18 15 17 1 0 2 12 9 13 2 5 3 0 0 0 15 14 18 sm 0 1 1 8 13 8 3 4 2 0 0 0 11 18 11 x = 1.00 x = 11.44 x = 2.78 x = 0 x = 15.22 0 0 0 4 7 3 0 0 0 0 0 0 4 7 3 0 0 0 6 6 6 2 0 1 0 0 0 8 6 7 r st 0 0 0 6 5 5 1 0 0 0 0 0 7 5 5 x = 0 x = 5.33 x = 0.44 x = 0 x = 5.78 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t 0 0 0 1 0 0 1 0 0 0 0 0 2 0 0 x = 0 x = o.ll X = 0.11 x = 0 x = 0.22 5 2+1 6 25,,26+120 6„ 7 10 2 1 1 38+.36+^ 37 CO 4+1 4 !j>i 22 '23 24-j 7 U t 9 1 0 0 34 38 38 ro 0 0 0 38+138+l36+: +J 6 1 7 5 1 22 23 22 1 10 8 1 9 O 1— x = 5.22 x = 23.33 x = 8.89 x = 0.56 x = 38.00 m l i l l l 3-4% (6) 2-3% (6) 0-2% (4) m 881* 3-4% (2) s m 2-3% (12) HfiltiiSliS II0 - 2% ( 2 ) S t 114 i «i 10|J 2-3% (6) Figure 18. Karyotype of Lasmigona costata based upon an analysis of "%TCL" and "r" values from nine chromosome spreads from specimens JJJ:23 and 47. 78 Table 20. Separation of the chromosomes of Lemiox rimosus using values of "%TCL" and "r" derived from measurements of two spreads from specimens JJJ:94 and 102. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 0/ 5 8 10 6 2 0 1 14 18 m x = 2.50 x = 9.00 x = 4.00 x" = 0.50 x = 16.00 0 1 20 11 2 4 0 0 22 16 sm x = 0.50 x = 16.00 x = 2.50 x = 0 x = 19.00 0 0 2 2 0 0 0 0 2 2 St x = 0 x = 2.00 x = 0 x = 0 x = 2.00 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 in +2 0 6 30 23 8 6+1 0 1 38 36+3 4->fO O x = 3.00 x = 27.50 x = 7.00 x = 0.50 x = 38.50 1— Leptodea fragilis (Rafinesque, 1820) Three specimens (JJJ:236, 237 and 257) collected in Lake Erie, Ohio were processed. One slide from each of these animals was examined and a total of 147 chromosome spreads were observed. Fifty-two of these spreads were studied, resulting in 28 chromosome counts: 21-1, 23-1 (plus dot), 24-2, 28-2 (one plus dot), 29-1, 30-1, 32-1, 33-2, 36-1, 37-2, 38-13, 39-1. Seven counted spreads were photographed and five of these were measured. The analysis of four measured spreads is presented in table 21. A suggested karyotype is given in figure 20. Lexingtonia dolabelloides (Lea, 1840) All five specimens of j_. dolabelloides processed (JJJ.-95, 110, 111, 167 and 168) were collected in the Duck River, Tennessee. Five - m t m 3-4% (4) 2-3% (16) 14«akk 2-3% (2) 5 ‘ M 10 ju Figure 19. Karyotype of Lemiox rimosus based upon an analysis of "%TCL" and “r" values from two chromosome spreads from specimens JJJ:94 and 102. 80 Table 21. Separation of the chromosomes of Leptodea fragilis using values of "%TCL" and "r" derived from measurements of four spreads from specimens JJJ:236 and 237. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 2/ 1 0 11 8 10 3 4 2 1 1 2 17 14 14 2 11 3 2 18 m x = 1.25 x = 10.00 x = 3.00 x = 1.50 x = 15.75 2 5 3 12 10 11 5 2 4 0 0 0 19 17 18 sm 2 12 2 0 16 x = 3.00 x = 11.25 x = 3.25 x = 0 x = 17.50 r 0 0 0 1 3 4 1 1 0 0 0 0 2 4 4 St 0 2 2 0 4 x = 0 x = 2.50 x = 1.00 x = 0 x = 3.50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 X = 0 + 4 +1 in 4 6 3 24 21+^25+1 9 7 + Z 6 1 1 2 38 35 36 (O 4 25 7 2 38 4-> 1— x = 4.25 x = 24.50 x = 7.75 x = 1.50 x = 38.00 m it* >4% (2) 3-4% (2) 2-3% (10) il 0 - 2% ( 2 ) 3-4% (4) 2-3% (12) "Mill IMVIA 0 - 2% ( 2 ) *1 3-4% (2) 2-3% (2) - ill »A 1 0 JJ Figure 20. Karyotype o f Leptodea fr a g ilis based upon an analysis o f "%TCL" and " r" values from four chromosome spreads from specimens JJJ:236 and 237. CO 82 slides were examined (one from each animal) and 51 chromosome spreads were observed. Eleven of these spreads were studied, resulting in five chromosome counts: 19-1, 36-1, 37-1, 38-2. Three of these spreads were photographed and two of them were measured. Only one of these measured spreads could be analyzed in detail (table 22) because the other had many obscured centromeres. The apparent karyotype for this spread is presented in figure 21. Ligumia nasuta (Say, 1817) Both of the specimens processed (JJJ:87 and 238) were collected in Lake Erie, Ohio. Examination of two slides (one slide each) yielded 141 chromosome spreads of which 26 were studied. The chromosomes in 18 spreads were counted, as follows: 28-1, 30-1, 35-1, 38-15. Four of Table 22. Separation of the chromosomes of Lexingtonia dolabel loides using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ: 111. Abbreviations are explained in the foot note to table 2. % TCL 1 1 0 ro CO 2 - 3 4* > 4 Totals 3 11 6 2 22 m sm 0 10 2 0 12 1 0 0 0 1 st 0 0 0 0 0 t 4 21+1 8 2 35+1 Totals m >4% (2) 3-4% (6) 2-3% (14) 0-2% (4) m i m i r t u u i 3-4% (2) s m it ))) 2-3% (10) Figure 21. Karyotype of Lexingtonia dolabelloides based upon an analysis of "%TCL" and "r" values from a chromosome spread o f specimen JJJ: 111. 00 CO 84 these spreads were photographed and all four were measured. Table 23 presents the analysis of these measurements. Figure 22 presents a suggested karyotype. Ligumia recta (Lamarck, 1819) Five specimens were processed: two (J JJ:199 and 203) from the Meramec River, Missouri; one (JJJ:227) from the Mississippi River, Wis consin; one (JJJ:86) from Lake Erie, Ohio and one (JJJ:250) from French Creek, Pennsylvania. Six slides from four of these animals (all but JJJ:250) were scanned and 55 chromosome spreads were observed. Twenty of these spreads were studied resulting in counts from 10 of them (all 38). Three of these spreads were photographed but only two were suffic iently distinct to be measured. The analysis of these measurements is presented in table 24 and figure 23 gives the suggested karyotype. Marqaritifera marqaritifera form falcata (Gould, 1850) The four specimens processed (JJJ:56, 57, 59 and 63) were all collected in the Willamette River, Oregon. Ten slides made from all of these animals were examined and 24 chromosome spreads were noted. Eleven of these spreads yielded definite chromosome numbers: 24-1, 27-1, 33-1, 38-8. Two spreads were photographed and measured. The analysis of these measurements is presented in table 25. A suggested karyotype is given in figure 24. Medionidus conradicus (Lea, 1834) One of the two specimens of M. conradicus processed (J JJ:12) was collected in Buck Creek, Kentucky; the other (JJJ:217) came from Copper Creek, Virginia. Six slides from both of these animals were examined 85 Table 23. Separation of the chromosomes of Ligumia nasuta usinq values of "%TCL" and "r" derived from measurements of four spreads all from specimen JJJ:238. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 5 3 13 7 15 2 3 4 1 2 1 19 17 23 4 11 1 2 18 m x = 3.75 x = 11.50 x = 2.50 x = 1 50 x = 19.25 1 0 0 10 11 8 4 2 3 0 0 0 15 13 11 0 13 2 0 15 sm x = 0.25 x = 10.50 x = 2.75 x = 0 x = 13.50 r 0 0 0 3 6 3 1 1 1 0 0 0 4 7 4 0 5 0 0 5 St x = 0 x = 4.25 x = 0.75 x = 0 x = 5.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 CO 4 5+1 3 26 24 26 7 6 8 1 2 1 38 37+1 38 to 3 2 38 4-> 4 29 O 1- x = 4.25 x = 26.25 x = 6.00 x = 1. 50 x = 38.00 3-4% (2) 2-3% (12) 0-2% (4) m W4A 3-4% (4) s m 2-3% (10) S t 2-3% (4) 1 0 m Figure 22. Karyotype of Ligumia nasuta based upon an analysis of "%TCL" and "r" values from four chromosome spreads, all from specimen JJJ:238. CO CD 87 Table 24. Separation of the chromosomes of Ligumia recta using val ues of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:227. Abbreviations are explained in the footnote to table 2 . % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 2 2 14 18 3 3 2 0 21 23 m II x| o x = 2.00 x = 16.00 x = 3.00 o x = 22.00 0 0 10 6 4 3 0 0 14 9 sm _ _ x = 0 x = 8.00 x = 3.50 x = 0 x = 11.50 0 1 1 3 0 2 0 0 1 6 St II x| x = 0.50 x = 2.00 x = 1.00 o x = 3.50 2 0 0 0 0 0 0 0 2 0 Ix O o t II x = 0 x = 0 x = 0 x = 1.00 10 4 3 25 27 7 8 2 0 38 38 +j (—o )T = 3.50 x = 26.00 x = 7.50 x = 1.00 x = 38.00 Table 25. Separation of the chromosomes of Margaritifera margari- tifera form falcata using values of "%TCL" and "r" derived from measure ments of two spreads both from specimen JJJ:56. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 1 1 14 10 3 5 0 0 18 16 m x = 1.00 x = 12.00 x = 4.00 x = 0 x = 17.00 1 0 10 10 1 4 0 0 12 13 sm 7 = 0.50 x = 10.00 x = 2.50 x = 0 x = 12.50 0 0 4 2 4 3 0 0 8 5 St x = 0 x = 3.00 x = 3.50 x = 0 x = 6.50 0 0 0 0 0 0 0 1 0 1 t x = 0 x = 0 x = 0 x = 0.50 x = 0.50 tr> 2 1 28 22 8 12 0 1 38 36 ro O x = 1.50 x = 25.00 x = 10.00 x = 0.50 x = 37.00 1— m H ft K 3-4% (4) 2-3% (16) 0-2% (2) m $n (4) s m 8 a n n ( 8 ) 3-4% (2) S t 4\ft ft Si 10|J 2-3% (2) Figure 23. Karyotype of Ligumia recta based upon an analysis'%TCL" of and "r" values from two chromosome spreads, both from specimen JJJ:227. co co m m i tSliiscmtx 3-4% (4) 2-3% (12) 0-2% (2) m X t 3-4% (2) s m i it m u m i 2-3% (10) 3-4% (4) s t 2-3% (4) Illl m m 10 M Figure 24. Karyotype of Marqaritifera marqaritifera form falcata based upon an analysis of "%TCL" and 'r" values from two chromosome spreads, both from specimen JJJ-.56. kOoo 90 and 10 chromosome spreads were noted or observed. Four of these spreads were counted (23-1, 36-1, 38-2), two were photographed and both were measured. The resulting analysis of the data is presented in table 26 and led to the suggested karyotype presented in figure 25. Medionidus penicillatus (Lea, 1857) The single specimen processed (JJJ:132) was collected in White water Creek, Georgia. Two slides yielded 21 chromosome spreads, of which four were studied and two were counted (31-1, 38-1). No spread was photographed. Table 26. Separation of the chromosomes of Medionidus conradicus using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:217. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 3 14 10 2 7 1 1 20 21 m x = 3.00 x = 12.00 x = 4.50 x = 1.00 x = 20.50 3 1 6 1 4 4 1 0 14 6 sm x = 2.00 x = 3.50 x = 4.00 x = 0.50 x = 10.00 0 0 3 1 0 1 0 1 3 3 St x = 0 x = 2.00 x = 0.50 x = 0.50 x = 3.00 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 cn 23+1 12+ 2 6 12 2 2 + l 37+l 30+4 03 6 4+1 + j o x = 5.50 x = 19.00 x = 9.00 x = 2.50 x = 36.00 I— m >4% (2) 3-4% (4) 2-3% (12) 0-2% (4) m l®|f} J>Ot« 3-4% (4) s m ♦ 2-3% (6) > p I s 0 - 2% ( 2 ) S t 2-3% (4) P li 1 0 J J Figure 25. Karyotype of Medionidus conradicus based upon an analysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:217. 92 Megalonaias boykiniana (Lea, 1840) One specimen of this species (JJJ.-136) was processed from White water Creek, Georgia. One slide was examined and eight chromosome spreads were observed. Two spreads were studied, but only one spread could be counted (38). It was not photographed. Obliquaria reflexa Rafinesque, 1820 Four of the five specimens of this species that were processed (JJJ:88, 109, 122 and 123) were collected in the Ohio portion of Lake Erie. The other specimen (JJJ:118) came from the Green River, Kentucky. Examination of six slides from all of these animals yielded 21 chromo some spreads, only two of which could be studied in detail and none of which could be counted. No spreads were photographed. Obovaria olivaria (Rafinesque, 1820) Both of the specimens processed (JJJ-.226 and 229) were collected in the Mississippi River, Wisconsin. Two slides were examined from each animal and a total of 59 chromosome spreads were observed and studied. Counts were obtained from 16 spreads: 22-1, 30-2, 33-1, 34-1, 38-9 (one with an additional dot) and 40-2. Five of these spreads were photo graphed and four of them were measured. Table 27 presents the analysis of the measurements and figure 26 presents a suggested karyotype. Obovaria subrotunda (Rafinesque, 1820) One of the specimens of this species that was processed (JJJ:31) came from Buck Creek, Kentucky and the other specimen (JJJ:91) came from the Green River, Kentucky. Two slides were examined (one from each animal) and a total of 28 spreads v/ere noted or observed. Twenty of 93 Table 27^ Separation of the chromosomes of Obovaria olivaria usinq values of "%TCL" and "r" derived from measurements of four spreads from specimens JJJ:226 and 229. Abbreviations are explained in the footnote to table 2. of /O TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 1 2 1/ 13 12 9 1 3 ' 3 2 0 3 17 17 16 0 5 6 1 12 m 7 = 1.00 x = 9.75 x = 3.25 x = 1.50 x = 15.50 0 1 2 6 8 8 5 2 2 0 0 0 11 11 12 0 2 6 0 8 sm x = 0.75 x = 6.00 x = 3.75 x = 0 x = 10.50 r 1 2 0 6 5 1 0 0 2 0 0 0 7 7 3 0 3 3 1 7 St 7 = 0.75 x = 3.75 x = 1.75 x = 0.25 x = 6.00 0 1 0 1 1 1 0 0 0 0 0 0 1 2 1 0 0 0 0 0 t x = 0.25 x = 0.75 x = 0 x = 0 x = 1.00 in 2+1 6+2 3 26 26+2 19+1 6+1 5 7+1 2 0 3 36+237+432+2 ra 0 10+2 15+2 2 27+4 4-> O f— x = 3.50 x = 21.50 x = 9.25 x = 1.75 x = 36.00 m o lift >4% (2) 3-4% (4) 2-3% (12) 0 - 2% ( 2 ) m i* 3-4% (6) s m iii»i> illMi 2-3% (6) ' ililM Figure 26. Karyotype of Obovaria olivaria based upon an analysis of "%TCL" and "r" values from four chromosome spreads from specimens JJJ:226 and 229. 95 these spreads were counted, as follows: 26-1, 33-1, 34-1, 35-1, 36-2, 37-1, 38-13. Eight spreads were photographed and seven were measured. Table 28 presents the comparison of the measurements and figure 27 presents a suggested karyotype. Plagiola lineolata (Rafinesque, 1820) All three of the specimens processed (JJJ:90, 97 and 117) were collected in the Green River, Kentucky. Three slides from these animals (one from each) were found to include 19 chromosome spreads, of which seven were studied. The single spread which could be counted had 38 chromosomes. No spread was photographed. Pleurobema coccineum (Conrad, 1836) One processed specimen of this species (JJJ:105) was collected in the Green River, Kentucky and the other (JJJ:194) was collected in the Black River, Missouri. Ten chromosome spreads were observed on two slides (one from each animal), three of these spreads were studied and two were counted (31-1, 38-1). One spread was photographed and measured. The analysis is presented in table 29 and the apparent karyotype is presented in figure 28. Pleurobema oviforme (Conrad, 1834) The three specimens of ovi forme processed (JJJ:96, 112 and 166) were all collected in the Duck River, Tennessee. Three slides were examined (one from each animal) and 39 chromosome spreads were observed. Eight spreads were studied and counts were made for four of them (36-1, 38-3). None were of sufficient quality to be photographed. 96 Table 28. Separation of the chromosomes of Obovaria subrotunda using values of "%TCL" and "r" derived from measurements of seven spreads all from specimen JJJ:31. Abbreviations are explained in the footnote to table 2. % TCL C\1 o 1 1 2 - 3 CO > 4 Totals 4 7 2 11 10 14 4 3 1 1 0 2 20 20 19 2 2 3 14 14 10 6 4 6 0 1 1 22 .21 20 m 6 8 2 2 18 X = 3.71 x = 11.57 X = 3.71 X = 1. 00 x = 20.00 0 0 0 12 9 7 2 4 5 0 0 0 14 13 12 0 0 2 9 7 7 3 3 3 0 0 0 12 10 12 sm 0 10 3 0 13 X = 0.29 x = 8.71 X = 3.29 X = 0 x = 12.29 0 0 0 4 2 6 0 1 0 0 0 0 4 3 6 0 0 0 4 5 6 0 2 0 0 0 0 4 7 6 r St 0 6 0 0 6 3T= 0 x = 4.71 X = 0.43 X = 0 x = 4.86 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 00 000 0000 0 0 0 t 0 0 0 0 0 II X = 0 x = 0 X = 0 X = 0 X| o j 4 7 2 27 21+127 6 8+ 1 6 1 0 2 38 36+^ 37 2 2 5 27 26 23 9 9 9 0 1 1 38 ,38 38 6 24+1 5 2 37 4-Jra O _ — 1— X = 4.00 x = 25.29 X = 7.57 X = 1 .00 x = 37.86 m m i l u m i o 3-4% (4) 2-3% (12) 0-2% (4) m »K«« 3-4% (4) s m llltf ISiiSHStRl 2-3% (10) S t 2-3% (4) ft*. I 1 0 (J Figure 27. Karyotype of Obovaria subrotunda based upon an analysis of "%TCL" and "r" values from seven chromosome spreads, a ll from specimen JJJ:31. 98 Pleurobema plenum (Lea, 1840) One specimen of this species (JJJ: 116) from the Green River, Kentucky was processed and one slide was examined. Sixteen chromosome spreads were observed but none was studied or counted. Pleurobema pyriforme (Lea, 1857) One animal (JJJ: 140) collected in Uchee Creek, Alabama was processed and one slide was examined. Of the 29 spreads observed, eight were studied and four were counted (20-1, 32-1, 38-2). One of these spreads was photographed but so few details were present that it was not measured. Table 29. Separation of the chromosomes of Pleurobema coccineum using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:105. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 0 15 3 0 18 m sm 1 6 7 0 14 st 0 2 0 0 2 0 0 0 0 0 t 1 23+2 10 0 34+2 Totals s m 3-4% (6) 2-3% (8) 0 - 2% ( 2 ) S t 10 ju 2-3% (2) Figure 28. Karyotype o f Pleurobema coccineum based upon "%TCL" and "r" values from a chromosome spread o f specimen JJJ:105. 100 Pleurobema rubrum (Rafinesque, 1820) Both of the specimens of P_. rubrum processed (JJJ:92 and 115) were collected in the Green River, Kentucky. No chromosome spreads were observed on a slide from JJJ:92 but four chromosome spreads were observed on a slide from JJJ:115. One of these spreads was studied, counted (38), photographed and measured. The analysis is presented in table 30 and the apparent karyotype in figure 29. Pleurobema sp. Two supposed Pleurobema specimens from Gulf Coast drainage streams were processed; however, they have yet to be identified to species. One slide from the specimen collected in Whitewater Creek, Georgia (JJJ:133) held no chromosome spreads at all, while one slide Table 30. Separation of the chromosomes of Pleurobema rubrum using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ: 115. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 0 18 2 1 21 m sm 1 8 2 0 11 St 0 4 2 0 6 t 0 0 0 0 0 i 1 30 6 1 38 Totals m >4% (2) 3-4% (2) 2-3% (18) m i s m 3-4% (2) f l > 2-3% (8) S t 3-4% (2) 10 M 2-3% (4) Figure 29. Karyotype o f Pleurobema rubrum based upon "%TCL" and " r" values from a chromosome spread o f specimen JJJ:115. o 102 from the specimen collected in Burnt Corn Creek, Alabama (JJJ:161) held one uncountable spread. Potamilus alatus (Say, 1817) The five animals that were processed, were collected from three sites. Two animals (JJJ:15 and 17) came from Buck Creek, Kentucky; two (JJJ:207 and 211) came from the Black River, Missouri; and the fifth specimen (JJJ:81) came from the Ohio portion of Lake Erie. Six slides from four of the animals were scanned (all but JJJ:211) and 43 chromo some spreads were noted or counted. Twenty-two spreads were studied in some detail and 15 of them were counted (29-1, 30-1, 31-1, 37-2, 38-10). Five of these spreads were photographed and four were measured. Table 31 presents an analysis of the measurements and figure 30 presents a suggested karyotype. Ptychobranchus fasciolaris (Rafinesque, 1820) Eight specimens of this species were processed. Five of them (JJJ:21, 22, 72, 101 and 121) were collected in Big Darby Creek and two (JJJ:84 and 89) were collected in Little Darby Creek (both in Ohio). The other specimen (JJJ:30) was collected in Buck Creek, Kentucky. Eleven slides from seven of these animals (all except JJ0-.22) were scanned and 178 chromosome spreads were noted or observed. Seventy-two of these spreads were studied and 43 of them were counted with the following distribution: 22-1, 31-1, 33-1, 34-1, 36-4, 37-4, 38-31. Five of these spreads were photographed and all five were measured. The analysis of these measurements is presented in table 32. The suggested karyotype is presented in figure 31. 103 Table 31^ Separation of the chromosomes of Potamilus alatus usinq values of "%TCL" and "r" derived from measurements of four spreads from specimens JJJ:15, 17 and 81. Abbreviations are explained in the foot note to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 4/ 3 0/ 4 19 18 3 4 3 2 1 2 13 27 23 4 6 5 1 16 m x = 2.75 x = 11.75 x = 3.75 x = 1.50 x = 19.75 1 1 1 16 5 10 5 3 1 0 0 0 22 9 12 4 9 3 0 16 sm x = 1.75 x = 10.00 x = 3.00 x = 0 x = 14.75 r 0 0 0 3 1 1 0 1 1 0 0 0 3 2 2 St 2 2 2 0 6 x = 0.50 x = 1.75 x = 1.00 x = 0 x = 3.25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 O CO CO LO CO O — II «— 5+l ^ 1 23 25 29 + 2 1 2 38,938 37 fO 38 +-> 10 1 17 1 o 1. O O h- X x = 5.25 x = 23.50 x = 1.50 x = 38.25 1 • Hif > 4% (2) 3-4% (4) 2-3% (12) RAXX MX 0 - 2% ( 2 ) 3-4% (4) 2-3% (10) 0 - 2% ( 2 ) tin 2-3% (2) ' ft# 10 m Figure 30. Karyotype of Potamilus alatus based upon an analysis of "%TCL" and "r" values from four chromosome spreads from specimens JJJ:15, 17, and 81. 105 Table 32. Separation of the chromosomes of Ptychobranchus fascio- laris using values of "%TCL" and "r" derived from measurements of five spreads from specimens JJJ.-21, 30 and 84. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 3/ 3 8 8 9 3 4 2 3 1 1 17 16 15 3/ 1 5 9 4 6 0 0 12 16 m x = 2.60 x = 7.80 x = 3.80 x = 1 00 x = 15.20 3 3 1 10 13 15 3 2 5 0 0 0 16 18 21 20 19 sm 1 0 14 17 4 2 1 0 x = 1.60 x = 13.80 x = 3.20 x = 0 20 x = 18.80 r 0 0 0 3 3 0 0 1 1 0 0 0 3 4 1 St 0 0 3 0 1 0 0 0 4 0 x = 0 x = 1.80 x = 0.60 x = 0 x = 2.40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 lx O t II II o x = 0 x = 0 x = 0 X| V) 6 4 24 24+1 6+1 7 8 3 1 1 36+138 37+1 r — 21. o rc 4 1+1 22 26+2 9 8 1 0 36+235+3 O 1— x = 4.40 X = 24.40 x = 7.80 x = 1 20 x = 37.80 m >4% (2) 3-4% (4) 2-3% (8) 0 - 2% ( 2 ) m I S H K t 11 3-4% (4) s m 2-3% (14) «Sf(K10- 2% ( 2 ) II s t 2-3% (2) 1 0 m Figure 31. Karyotype of Ptychobranchus fasciolaris based upon an analysis of "%TCL" and "r" values from fiv e chromosome spreads from specimens JJJ:21, 30, and 84. O CT> 107 Ptychobranchus subtenum (Say, 1825) The single animal of this species processed (JJJ:125) was collected in the Clinch River, Tennessee. Two slides were scanned and 61 chromosome spreads were observed. Twelve of these spreads were studied with chromosome counts taken for three of them (23-1, 38-2). One spread was photographed but not measured. Quadrula pustulosa (Lea, 1831) Six specimens were processed; two each from Lake Erie, Ohio (JJJ:85 and 241); Meramec River, Missouri {JJJ:196 and 208); and Mississippi River, Wisconsin (JJJ:225 and 228). Six slides were scanned from four of these animals (not JJJ:196 or 228) and 83 chromo some spreads were observed. Fourteen of these spreads were studied resulting in six counts of the chromosomes: 25-1, 37-1, 38-4. Two of these spreads were photographed and one of them was measured (table 33). The apparent karyotype is illustrated in figure 32. Quadrula quadrula (Rafinesque, 1820) Four of the five specimens of Q. quadrula prepared (JJJ:6, 73, 171, and 180) were collected in Big Darby Creek, Ohio. The fifth specimen (JJJ:224) was collected in the Mississippi River, Wisconsin. Seven slides were scanned from four of these animals (all but JJJ;224) and 69 chromosome spreads were noted or observed. Thirty-eight of these spreads were studied and 19 chromosome counts were obtained. These counts ranged from 22 to 38 as follows: 22-1, 24-1, 32-1, 33-1, 34-1, 36-1, 37-1, 38-12. Four of these spreads were photographed but none was measured. 108 Table 33. Separation of the chromosomes of Quadrula pustulosa using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ.-225. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 1 16 3 2 22 m 1 7 2 0 10 sm 0 3 1 0 4 st 0 0 0 0 0 t 2+1 26+1 6 2 36+2 Totals Quincuncina infucata (Conrad, 1834) Both specimens (0JJ:138 and 147) were collected in Uchee Creek, Alabama. One slide was examined from each specimen and a total of 34 chromosome spreads were observed. Nine spreads were studied, resulting in four counts of chromosomes: 31-1, 36-2, 38-1. One of these spreads was photographed and measured (table 34, figure 33). Strophitus sp. A processed specimen (JJJ:152) that was collected in Cane Creek, Alabama appears to belong to the genus Strophitus but has not been assigned to a species. One slide from this animal was scanned and 23 chromosome spreads were observed. Thirteen of these spreads were studied with chromosome counts taken from eight of them: 17-1, 19-1, >4% (2) 3-4% (4) 2-3% (16) 0 - 2% ( 2 ) m ;;v. s m 3-4% ( 2 ) 2-3% ( 8 ) S t 1 0 JJ 2-3% (4) Figure 32. Karyotype of Quadrula pustulosa based '%TCL" upon and "r" values from a chromosome spread o f specimen JJJ:225. O <£> 110 Table 34. Separation of the chromosomes of Quincuncina infucata using values of "%TCL" and "r" derived from measurements of one spread from specimen 0JJ-.147. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 1 9 5 0 15 m 0 6 5 2 13 sm r 0 1 0 0 1 st 0 0 1 0 1 t i 1 16+4 11 2 30+4 Totals 22-1, 23-1, 25-1, 38-3. One of these spreads was photographed but it was not measured. Strophitus undulatus (Say, 1817) Three of the four specimens processed (JJJ:69, 80 and 179) were collected in Big Darby Creek, Ohio; while the fourth specimen (JJJ:253) was collected in the Allegheny River, Pennsylvania. Six slides were scanned from three of these animals (all but JJJ:253) and 100 chromosome spreads were noted or counted. Thirty-five of these spreads were studied, resulting in counts from 13 spreads (20-1, 30-1, 38-11). Three of these spreads were photographed but none was measured. 3-4% (6) 2-3% (12) 0-2% (2) m * s >4% (2) s m 3-4% (6) Hill 2-3% (6) S t * h 2-3% (2) i? 3-4% (2) 1 0 JJ Figure 33. Karyotype of Quincuncina infucata based upon "%TCL" and "r" values from a chromosome spread o f specimen JJJ:147. This contracted spread includes only 34 chromosomes. 112 Toxolasma lividus glans (Lea, 1831) The single specimen of this subspecies processed (JJJ:28) was collected in Buck Creek, Kentucky. Five slides from this animal were examined and nine chromosome spreads were noted and studied. Counts were possible for five of these spreads: 33-1, 36-1, 38-3. Two spreads were photographed and both were measured. The "r" by "%TCL" sorting of one of these spreads is presented in table 35 and the suggested karyo type is given in figure 34. Toxolasma parva (Barnes, 1823) Eight of the nine specimens of T. parva processed (JJJ:25, 33, 37, 38, 43, 44, 45 and 50) were collected in the Ohio portion of Lake Erie. The ninth specimen (JJJ:134) was collected in Whitewater Creek, Table 35. Separation of the chromosomes of Toxolasma lividus glans using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:28. Abbreviations are explained in the footnote to table 2. % TCL 1 ° r o 2 - 3 3 - 4 > 4 Totals 1 4 18 2 1 25 m 0 8 3 0 11 sm r 0 st 0 1 0 1 0 t 0 0 0 0 4 26+1 6 1 37+1 Totals m 88 KKSIU >4% (2) 3-4% (2) 2-3% (18) 0-2% (4) m ttssamsftc* s m 3-4% (4) 9 I ft 8 i ft 5 a 2-3% (8) 10ju Figure 34. Karyotype o f Toxolasma liv id u s glans based upon "%TCL" and "r" values from a chromosome spread o f specimen JJJ:28. 114 Georgia. Twenty-five slides from all of these animals were scanned and a total of 71 chromosome spreads were noted and studied. Chromosome counts for 24 spreads were distributed as follows: 33-1, 35-2, 36-2, 37-2, 38-17. Six of these spreads were photographed and three were measured. Table 36 presents an analysis comparison of the measurements of these spreads. Figure 35 presents a suggested karyotype. Toxolasma paula (Lea, 1840) The two specimens of this species that were processed (JJJ:150 and 154) were both collected in Cane Creek, Alabama. The three slides scanned from both specimens held seven chromosome spreads, three of which were studied. None of these spreads could be counted. Table 36. Separation of the chromosomes of Toxolasma parva using values of "%TCL" and "r" derived from measurements of three spreads from specimens JJJ:33 and 38. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 1 / 2 16 8 15 8 11 6 0 0 0 27 20 23 m x = 2.00 x = 13.00 x = 8.33 x = 0 x = 23.33 1 2 1 6 13 10 3 2 2 0 0 1 10 17 14 sin x = 1.33 x = 9.67 x = 2.33 x = 0.33 x = 13.67 r 0 0 0 0 0 1 1 0 0 0 0 0 1 0 1 St x = 0 x = 0.33 x = 0.33 x = 0 x = 0.66 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 r— 4 3 3 22 21 26' 12 13 8 0 0 1 38 37 38 4->1X3 o x = 11.00 x = 0.33 x » 37.67 1— x = 3.33 x = 23.00 fCJb? 3-4% (8) 2-3% (14) 0-2% (2) m UOJUc 3-4% (2) s m 2-3% (10) t 0 - 2% ( 2 ) 10 ju Figure 35. Karyotype o f Toxolasma parva based upon is of "%TCL" and "r" values from three chromosome spreads from specimens JJJ:33 and 38. 116 Tritogonia verrucosa (Rafinesque, 1820) Three animals were processed; two (JJJ:66 and 82) from the Little Barren River, Kentucky and one (JJJ:11) from Big Darby Creek, Ohio. Eight slides from all three specimens were scanned and 17 chromsome spreads were noted or counted. Tv/elve of these spreads were studied, resulting in six chromosome counts as follows: 30-1, 31-1, 32-1, 38-3. Two of these spreads were photographed and one of them was measured (table 37). The apparent karyotype is illustrated in figure 36. Truncilla truncata Rafinesque, 1820 The specimen of T. truncata processed (JJJ:26) was collected in the Ohio portion of Lake Erie. Two slides were scanned and a single uncountable chromosome spread was noted. Table 37. Separation of the chromosomes of Tritogonia verrucosa using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ: 11. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 0 19 3 1 23 m 1 10 0 0 11 sm r 1 0 0 0 1 st 0 0 0 0 0 t 2 29+3 3 1 35+3 Totals m >4% (2) 3-4% (2) 2-3% (22) m % s m 2-3% (10) 0 - 2% ( 2 ) 1 0 m Figure 36. Karyotype of Tritogonia verrucosa based upon "%TCL" and "r" values from a chromosome spread o f specimen JJJ: 11. 118 Uniomerus declivus (Say, 1831) The single specimen processed (JJJ:155) was collected in Cane Creek, Alabama. One slide was scanned. It yielded 42 chromosome spreads, of which 13 were studied. The five countable spreads all contained 38 chromosomes. One of these spreads was photographed but it was not measured. Villosa fabalis (Lea, 1831) The specimen processed (JJJ:243) was collected in French Creek, Pennsylvania. Two slides were scanned and two uncountable chromosome spreads were observed. Villosa iris iris (Lea, 1829) Two specimens of this subspecies or species were processed; one (JJJ:4) was collected in Big Darby Creek, the other (JJJ:193) was collected in Little Darby Creek (both in Ohio). Two slides were scanned from JJJ:4 (none from JJJ:193) and 31 chromosome spreads were noted. Twenty-one of these spreads could be counted as follows: 19-2, 26-1, 27-2, 28-1, 32-1, 33-1, 35-1, 37-1, 38-10, 39-1. Five of these spreads were photographed and four of them were measured. Table 38 presents a comparison of the analysis results and figure 37 presents a suggested karyotype. Villosa iris nebulosa (Conrad, 1834) Three specimens of this subspecies or species were processed; two of them (JJJ:213 and 222) were collected in Pitman Creek and the other (JJJ:18) was collected in Buck Creek (both in Kentucky). Five slides from all three specimens were scanned and 45 chromosome spreads were 119 ^ Table 38._ Separation of the chromosomes of Villosa iris iris usina values for "25TCL" and "r" derived from measurements of four spreads all from specimen JJJ:4. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 3 1 13 14 17 4 2 3 1 1 1 21 20 22 4 15 3 1 23 m x = 2.75 x = 14.75 x = 3.00 x = 1.00 x = 21.50 0 0 1 12 10 10 1 2 3 0 0 0 13 12 14 0 6 2 1 9 sm x = 0.25 x = 9.50 x = 2.00 x = 0.25 x = 12.00 r 0 1 0 4 3 2 0 1 0 0 0 0 4 5 2 St 0 4 0 0 4 x = 0.25 x = 3.25 x = 0.25 x = 0 x = 3.75 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t x = 0 x = 0 x = 0 x = 0 x = 0 to 3 4 2 29 27+1 29 5 o 5 6 1 1 1 38 _37+1 38 rO 5+1 2 36 +-> 4 25+1 O 1— x = 3.25 x = 28.00 x = 5.50 x = 1.25 x = 38.00 m A.WK8RK 3-4% (4) 2-3% (14) 0-2% (4) m A hkhkkkx HUHK s m 3-4% (2) A AJiAAflKKXJt* 2-3% (10) •• ft h P* ft 10|J 2-3% (4) Figure 37. Karyotype of Villosa iris iris based upon an analysis of "%TCL" and "r" values from four chromosome spreads, a ll from specimen JJJ:4. 121 noted or counted. Twelve of these spreads were studied, resulting in five chromosome counts -- all 38. Four of these spreads were photo graphed but only one was measured. The analysis results are presented in table 39 and the apparent karyotype in figure 38. Villosa 1ienosa (Conrad, 1834) Three specimens referrable to this species were processed: two of them (JJJ:8 and 10) from (Big) Buffalo Creek, Ohio and the other (JJJ:141) from Uchee Creek, Alabama. Six slides from all three speci mens were scanned and 27 chromosome spreads were noted or observed. Nineteen spreads were studied, resulting in 14 counts of the chromosomes: 30-1, 31-1, 33-1, 35-1, 36-1, 37-1, 38-8. Three of these spreads were photographed and two of them were measured. The analysis of the data Table 39. Separation of the chromosomes of Villosa iris nebulosa using values of "%TCL" and "r" derived from measurements of one spread from specimen JJJ:222. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3 13 4 2 22 m 0 11 1 0 12 sm r 0 3 1 0 4 St 0 0 0 0 0 t 3 27 6 2 38 Totals m >4% (2) 3-4% (4) 2-3% (12) 0-2% (4) m Km s m 3-4% (2) I 2-3% (10) 2-3% (4) ' | A & R 10 M Figure 38. Karyotype of Villosa iris nebulosa based upon "%TCL" and "r" values from a chromosome spread of specimen JJJ:222. ro PO 123 from these measured spreads are compared in table 40. The suggested karyotype of one of them is illustrated in figure 39. Villosa sp. Three specimens were processed that appear to belong to the genus Villosa; however, they have not been assigned to named species. Specimen JJJ:157was collected in Burnt Corn Creek, Alabama. Scans of two slides yielded 4 chromosome spreads, two of which were studied. One of these spreads included 38 chromosomes and the other had 43. Neither spread was photographed. Specimens JJJ:264 and 265 were collected in Clinch River, Virgin ia. One slide of JJJ:264 was scanned and eight spreads were observed. Three of these spreads were studied, resulting in one count of 33 Table 40. Separation of the chromosomes of Villosa lienosa using values of "%TCL" and "r" derived from measurements of two spreads both from specimen JJJ:10. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 2 6 12 9 5 5 1 1 20 21 m x = 4.00 x = 10.50 x = 5.00 7 = i.oo x = 20.50 0 1 10 7 3 3 0 2 13 13 sm x = 0.50 x = 8.50 x = 3.00 x = 1.00 x = 13.00 r 0 0 4 3 0 0 0 0 4 3 St x = 0 x = 3.50 x = 0 x = 0 x = 3.50 t 0 0 0 0 0 0 0 0 0 0 x = 0 x = 0 x = 0 x = 0 x = 0 2 7 26+119 8 8 1 3 37+l 37 +Jro o 7 = 4.50 x = 23.00 x = 8.00 x = 2.00 x = 37.50 1— m >4% (2) 3-4% (4) 2-3% (10) 0-2% (4) m t 'U r s m 3-4% (4) 2-3% (10) S t 2-3% (4) lOju Figure 39. Karyotype of Villosa lienosa based upon an anlaysis of "%TCL" and "r" values from two chromosome spreads, both from specimen JJJ:10. r o 125 chromosomes. It was not photographed. The slides from specimen JJJ:265 have not been scanned. Villosa taeniata punctata (Lea, 1865) Four specimens assigned to this subspecies were processed. Two of them (JJJ:27 and 39) were collected in Buck Creek and the other two (JJJ:212 and 221) were collected in Pitman Creek (both Kentucky). Five slides from three of these animals (not JJJ:221) were scanned and 40 chromosome spreads were noted or counted. All forty spreads were studied, resulting in 27 counts of the chromosomes as follows: 29-1, 30-1, 35-2, 37-2, 38-21. Eleven of these spreads were photographed and nine of them were measured. Table 41 presents the analysis comparison of the measurement data and figure 40 presents a suggested karyotype. Villosa trabalis (Conrad, 1834) All six specimens processed (JJJ:16, 19, 29, 40, 41 and 49) were collected in Buck Creek, Kentucky. Ten slides from all six animals were scanned and 88 chromosome spreads were noted. All of these spreads were studied, resulting in chromosome counts from 71 spreads as follows: 23-1, 25-1, 29-2, 33-1, 34-2, 35-6, 36-6, 37-6, 38-46. Thirteen of these spreads were photographed and 10 were measured. The results of the analysis of measurement data are compared in table 42 and a suggested karyotype is presented in figure 41. Villosa vibex (Conrad, 1834) Two of the four specimens of this species processed (JJJ:158 and 159) were collected in Burnt Corn Creek; another specimen (JJJ:149) was collected in Cane Creek and the fourth specimen (JJJ:145) was collected 126 Table 41. Separation of the chromosomes of Villosa taeniata punctata using values of "%TCL" and "r" derived from measurements of nine spreads from specimens JJJ:27 and 39. Abbreviations are explain ed in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 7 3 2 10 13 17 2 1 4 2 2 2 21 19 25 4 4 0 12 15 11 4 1 2 1 1 2 21 22 15 m 7 5/ 6 4 11 8 3 3 2 2 2 2 16 21 18 x = 4.22 X = 11 .22 X = 2.44 X = 1.89 x = 19.78 1 0 0 7 n 3 5 3 4 0 0 0 13 14 7 1 0 2 9 8 9 2 3 4 0 0 0 12 11 15 sm 1 1 0 10 7 13 3 4 3 0 0 0 14 12 16 X 0.67 X = 8. 56 X = 3.44 X = 0 x = 12.67 1 0 0 3 3 4 0 1 0 0 0 0 4 4 4 0 1 1 4 3 5 1 1 1 0 0 0 5 5 7 r st 1 0 1 6 4 3 0 0 0 0 0 0 7 4 4 X = 0.56 X = 3.89 X = 0.44 X = 0 x = 4.89 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 • 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IX O II j< = 0 X = 0 X = 0 X = 0 9 3 2 20 27 24 7 5 8 2 2 2 38 37 36 co 5 5 3 25 26 25+1 7 5 7 1 2 2 38 38 37+1 ro 9 6 7 20+122+1 24 6 7 5 2 2 2 37+137+1 38 o h- x = 5.44 X = 24 .00 X = 6.33 X = 1.89 x = 37.67 m >4% (2) 3-4% (2) 2-3% (12) 0-2% (4) m 3-4% (4) s m 2-3% (10) 2-3% (4) S t 10 p Figure 40. Karyotype of Villosa taeniata punctata based upon an analysis of "%TCL" values and "r1 from nine chromosome spreads from specimens JJJ:27 and 39. ro '•vi 128 Table 42. Separation of the chromosomes of Villosa trabalis using values of "%TCL" and "r" derived from measurements of ten spreads from specimens JJJ:29, 40, 41 and 49. Abbreviations are explained in the footnote to table 2. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 2/ 3 2/ 14 7 13 1 7 2 3 1 2 20 18 19 3 5 3 14 13 13 2 2 3 1 1 1 20 21 20 m 3 2 1/ 13 12 14 5 4 3 1 2 2 22 20 20 2 13 3 2 20 x = 2.60 x = 12.60 x = 3.20 x = 1.60 x = 20.00 1 2 1 9 11 9 0 3 3 0 0 0 10 16 13 0 0 0 10 5 10 4 4 3 0 0 0 14 9 13 sm 0 1 1 8 9 11 4 3 3 0 0 0 12 13 15 0 10 2 0 12 x = 0.60 x = 9.20 x = 2.90 x = 0 x = 12.70 1 0 0 1 3 5 2 1 0 0 0 0 4 4 5 0 0 0 3 5 4 1 2 1 0 0 0 4 7 5 0 0 0 4 4 3 0 0 0 0 0 0 4 4 3 r st 0 4 1 0 5 x = 0.10 x = 3.60 x = 0.80 x = 0 x = 4.50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 t 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 x = 0 x = 0 x = 0 x = 0 x = 0 4 5 3 24+121 27 3+1 11 5 3 1 2 34+^38 37 3 5 3 27 20 27 7 8 7 1 1 1 38 37 38 fO 3 3 2 25 25 28 9 7 6 1 2 2 38 37 38 +J o 2 27 6 2 37 h- x = 3.30 x = 25.20 x = 7.00 x = 1.60 x = 37.40 m >4% (2) 3-4% (4) 2-3% (12) 0-2% (4) m s m 3-4% (2) 2-3% (10) S t lOju 2-3% (4) Figure 41. Karyotype of Villosa trabalis based upon an analysis of "%TCL" andvalues from ten chromosome spreads from specimens JJJ:29, 40, 41 and 49. r o ID 130 in Uphapee Creek. All three streams are located in Alabama. Eight slides representing all four specimens were scanned and 74 chromosome spreads were observed. Fifteen of these spreads were studied resulting in chromosome counts from five of them as follows: 34-1, 38-4. One of these spreads was photographed but it was not measured. DISUCSSION I: NAIAD MITOTIC ACTIVITY The low number of mitotic figures generally present on these chromosome slides essentially required complete scans of the entire area under the 22 X 50 mm cover slips. Because these complete scans were made, and because all mitotic figures were counted on each slide (start ing with specimen JJJ:73), the data accumulated during this scanning exercise may be useful to compare variations in mitotic activity between the animals that were examined. Analysis, or, simply, recognition of these types of variations could indicate timing or handling improvements to this chromosomal preparation technique. Analysis of these variations also might suggest physiologic differences that could be useful in taxonomy. Not all of the variation in mitotic figure counts present on these slides should be expected to result from physiologic differences between taxa. Several sources of variation could be related to slight differ ences in preparation technique. While the number of mitotic figures on each slide was counted carefully, counts or estimates of the number of cell nuclei on the slides were not made. The size of the tissue sample being used, the amount of liquid on the slide and the physiologic condition of the animal when it was processed all could have affected the number of cells deposited and, as a subset of them, the number of mitotic figures. Another possible source of variation could involve differences in mitotic activity on the sides of the labial palp flaps. The probable 131 132 differences in function of the two surfaces of each palp flap (Allen, 1914) suggest that each side is likely to replace cells at different rates. In the process of making these slides, once the palp flaps were separated from each other and, in some cases, cut into smaller pieces, no attempt was made to distinguish between the inner and the outer surface or to consistently use one or the other side of a tissue piece as it was swabbed onto a slide. Variations in the number of mitotic figures caused by procedural inconsistencies or specimen handling trauma are likely to have occurred as these slides were prepared. However, each type of uncontrolled variation will have occurred at random throughout the full collection of slides. Therefore, estimates of variance in these data may be larger than for more controlled studies and differences between means may be harder to detect, but significant statistics can be taken to indicate real differences among the species. At times during both discussions, reference will be made to taxonomic groupings of the genera involved in this project as proposed by one or more authors. Five recent or substantially different classification systems were chosen to be compared. Table 43 was prepared to identify the group assignments for each of the 34 genera examined. The taxonomic groups used for each classification typically consisted of the three to eight family and/or subfamily level taxa established by the author. Group assignment was based upon either specific mention by the author that the genus fell within the group or, when all genera were not specifically mentioned, con formity of the genus with the apparent criteria of the group. In the case of Rafinesque (1820), species described at later dates were assigned to 133 Table 43. Assignment of the naiad genus-level taxa included in this project to higher taxonomic groups as proposed in six classification systems. Classification references are discussed in the Introduction and are listed in the Literature Cited. * * To facilitate comparison of these groupings, entries in the table use an alpha-numeric code based upon the placement of a few key genera. Taxonomic groupings which included identical lists of these 34 genera were given identical table entries. Groupings with the same key genus involved but including different cohorts, were given a table code made up of the same letter designation followed by number modifiers (assigned in no particular order). The codes used are: A - Alasmidonta-1i ke A1 - Anodontinae of Ortmann, Heard and Guckert, Davis and Fuller A2 - Alasmidontinae of Model 1 and Morrison A3 - Alasmidia of Rafinesque G - Gonidea-1ike - Gonideinae of Heard and Guckert, Gonideini of Davis and Fuller H - Mega!onaias-1ike - Megalonaiadinae of Heard and Guckert L - Lampsilis-like - Lampsilinae of Ortmann, Model!, Morrison Heard and Guckert; Lampsilini of Davis and Fuller M - Margaritifera-1ike - Margaritanidae of Ortmann and Morrison; Margaritiferidae of Model!; Margariti- ferinae of Heard and Guckert, Davis and Fuller N - Anodonta-1ike (when separated from Alasmidonta-1ike) N1 - Anodontidae of Rafinesque N2 - Anodontinae of Modell N3 - Anodontinae of Morrison P - Pleurobema-like (when separated from Amblema-like) PI - Pleurobeminae of Heard and Guckert P2 - Pleurobemini of Davis and Fuller P3 - Pleurobeminae of Modell P4 - Ambleminae of Modell (similar genera) Q - Quadrula-1ike (when separated from Amblema-like) Q1 - Ambleminae of Heard and Guckert Q2 - Amblemini of Davis and Fuller Q3 - Quadrulinae of Model! Q4 - Elliptioninae of Modell (similar genera) R - unique Rafinesquean groupings R1 - Uniodiae R2 - Amblemidia U - Amblema-like - Unioninae of Ortmann and Ambleminae of Morrison 134 Table 43 (continued) Genera 1820 1912 1942 1955 Fuller 1981 Rafinesque and Heard Guckert 1970 Davis and Since 1912 Ortmann Morrison Model! Agreement Actinonaias R1 LL LL L L Alasmidonta A3 A1 A2 A2 A1 A1 Amblema R2 U Q4 U Q1 Q2 Anodonta N1 A1 N2 N3 A1 A1 Anodontoides A3 A1 A2 N3 A1 A1 Arcidens R2 A1 A2 A2 A1 A1 Cyclonaias R1 U Q3 U PI Q2 Elliptio R1 U P4 U PI P2 Epioblasma R1 L L L L L L Fusconaia R1 P3 Q1 P2 Gonidea G G Lampsilis R1 L L L L L L Lasmigona A3 A1 A2 A2 A1 A1 Lemiox R1 L L L L L L Leptodea R1 L L LL L L Lexingtonia R2 P3 PI P2 Ligumia R1 L L LL L L Margaritifera R1 M MM Medionidus Rl L L L LLL Megalonaias R1 Q3 H Q2 Obiiquaria Rl L L L LLL Obovaria Rl L L L LLL Plagiola Rl L L LLL L Pleurobema R2 P3 PI P2 Potamilus Rl L LL LL L Ptychobranc.hus Rl L L LL L L Quadrula Rl Q3 Q1 Q2 Quincuncina R2 Q3 Q1 Q2 Strophitus N1 A1 A2 A2 A1 A1 Toxolasma Rl L L LLL L Tri togonia Rl Q3 Ql Q2 Truncilla Rl L L L L L L Uniomerus Rl P4 PI P2 Villosa Rl L L L LLL 2 Number of groups 4 4 8 5 7 6 represented (15 L, 1 M) 135 groups by using his subfamily descriptions. Following the interpretations of Stansbery (personal communication), shells were oriented along a line bisecting the umbonal swelling or each valve (the axis), then categorized as being transverse (=elongate) or longitudinal (=high) and with or with out cardinal and/or lamellar teeth (terminology from the translation by Poulson, 1832). Temperature Effects Water temperature was not taken on most visits to collection sites; however, water temperature readings are available for 51 animals that were processed on the day they were collected. Seventy-one slides were scanned from these 51 animals. The sampled temperature range includes one specimen (two slides) from 1° Celsius (C), two specimens (two slides) from 17°C and the remaining 48 animals (67 slides) from between 21° and 27°C. Most of these animals were collected and processed in late summer and fall 1976, many of them during an extended collecting trip to Georgia, Alabama and Tennessee. The specimen collected at 1°C was a Lasmigona costata (JJJ:210) found in six inches of flowing water under 14 inches of ice in Big Darby Creek, Ohio, February 1977. No indication of any mitotic activity occurred on the two slides that were scanned. At 17°C, two species were involved: a Quadrula quadrula (JJJ:171) and an Alasmidonta marginata (JJJ:172) both collected in Big Darby Creek, Ohio, October 3, 1976. The (£. quadrula slide contained eight mitotic figures and the A. marginata slide contained 46 mitotic figures. With out additional information, these two dissimilar observations are difficult to interpret. 136 Results from the 21° through 27°C range are presented in table 44. The highest mean number of mitotic figures (18.33) occurred at 23°C, with a sharp decrease at 1° and 2° lower temperatures but a more gradual decrease in the higher four degree range. Table 44 also includes the separation of these data into three subfamilies (following Ortmann, 1912). Sorting the data in this way appears to associate many of the slides with high numbers of mitotic fig ures (the Anodontinae), those with modest numbers (the Unioninae) and those with low numbers (the Lampsilinae). Figure 42 illustrates the relationships between these subfamily groupings and indicates the large confidence interval associated with each mean value. Comparisons of the Table 44. Mean number of mitotic figures observed on slides from naiades collected at water temperatures between 21 and 27°C. The number of slides contributing to each mean (n) is indicated below the calculated value followed by twice the standard error (in parentheses). Temperature or overall mean values different from each other (P < 0.05) are indicated by asterisks (*). Subfamily-level Overal1 Groups (after 21.0° 22.0° 23.0° 23.5° 25.0° 27.0° Group Ortmann, 1912) Means 36.25 11.00 27.83 Anodontinae 4 2 6 (26.68) (14.00) (20.27) 6.62 10.30 12.30 5.83* 5.60 8.77* Lampsilinae 8 10 10 6 5- 39 (4.45) (6.08) (10.18) (3.77) (4.22) (3.26) 6.50 4.00 19.20 5.50 19.40* 14.77* Unioninae 2 3 10 2 5 22 (7.00) (3.46) (9.25) (1.00) (7.94) (5.30) Overal1 6.50 5.91 18.33 11.14 12.00 5.60 12.45 Temperature 2 11 24 14 11 5 67 Means (7.00) (3.36) (7.03) (7.43) (5.80) (4.22) 137 Anodontinae 35 - Lamps i 1i nae Unioninae 30 - 25 - Li_ O 4-> O 4-> •r- s: 26 Temperature (°C) Figure 42. Relationships between temperature and naiad mitotic figure counts from 67 chromosome slides sorted by subfamily (after Ortmann). The large confidence intervals may mask differences between the groups. 138 subfamily degree-by-degree means and the overall taxonomic group means indicate that only the lampsiline and unionine values at 25°C and the lampsiline and unionine group means are different from each other (P<0.05). Other apparent differences between individual temperature means and the overall means are affected by the large variances. The heterogenous nature of the data and the fragmentary temper ature range coverage probably have concealed any actual correlations between temperature and mitotic activity. The five to seven degree temperature range with the best coverage (21-27°C), appears to indicate a mitotic peak at approximately 23°C with relatively higher mitotic activity continuing to approximately 25°C. The statistical separation of the Lampsilinae and the Unioninae at 25° and in the overall group means may indicate adaptations of groups of species to different temper ature regimes. The poorly-represented, non-significant tendency of high mitotic activity in the Anodontinae also may suggest basic physiologic differences between groups of species. Continued investigation into this subject could expand upon these apparent relationships. Annual Cycle Some naiades used for this project were collected during each month of the calendar year except January. While temperature and local climate are both likely to affect mitotic activity, it may be useful to sort these data by month to determine if mitotic activity varies through out the year and, if so, if there are different mitotic activity patterns among the species. 139 One hundred twenty-two labial palp slides representing the 90 animals processed in the firs t four days after being collected were used for this analysis. The month-by-month and summary means of these data are presented in table 45 along with separations of the observations into three subfamilies (after Ortmann, 1912). In a number of cases, when the data are segregated into these taxonomic groupings, the sample size becomes so small that single slide counts are the monthly means. Reliance on single counts may produce chaotic appearing month-to-month relationships and, effectively, could mask any pattern of mitotic activity. In a few cases, however, these groupings do associate data points that suggest mitotic activity cycles. The means for months with several observations (June, August and September) and the annual group means were compared statistically (standard and unequal variance t tests) to distinguish separable values. Anodontine means for June, August and the full year were dissimilar to each of the other values (P < 0.05 for all cases except P < 0.1 for Lampsilinae in August). All other means for the three months and the annual totals were not dissimilar P > 0.2). Figure 43 illustrates the two apparent patterns that emerge from this analysis. The Anodontine pattern begins with no mitotic activity in February, increases consistently to peak at 63 figures per slide in August, drops sharply in September and rebounds upward in October. Table 45. Month-by-month and summary data on the number of mitotic figures observed on slides from animals processed within four days after they were collected. The mean values are followed by the number of observations (n) and twice the standard error (in parentheses). Mean values for June, August, and September that were found to be different from the others (P < 0.1) are indicated by asterisks (*). Taxonomic Monthly Means Overall Groupings Group JF M AM JJ A S 0 N D Means Anodontinae - 0.00 - 18.00 - 33.00* - 63.33* 21.80* 46.00 -- 33.00* 1 1 2 3 5 1 13 (0.00) (13.38) (19.46) (13.98) Lampsilinae — “ 5.50 8.00 3.60 23.50 29.07 7.88 54.00 - - 13.46 2 1 5 4 14 41 1 68 (9.00) (4.80) (13.22) (15.72) (3.08) (4.55) Unioninae “ — 7.78 13.00 19.88 14.77 8.00 “ 14.02 9 1 8 22 1 41 (7.98) (14.49) (5.30) (4.47) Overall 0.00 5.50 18.00 8.00 9.62 21.40 30.24 11.13 36.00 “ 15.73 Monthly 1 2 1 1 16 5 25 68 3 122 Means (9.00) (6.54) (11.07) (11.14) (2.21) (28.38) (3.41) Lampsilinae 8.00 6.29 21.40 25.73 10.29 31.00 13.67 and 5.50 14 5 22 63 2 109 Unioninae 2 1 (9.00) (5.59) (11.07) (9.17) (2.97) (32.53) (3.26) Together Mitotic Figure Count otl ad oa vle fr h Lmslne n Uinne ee o saitcly differ statistically not Unioninae were and Lampsilinae the for values total monthly and n te ul er r dfeet P 01 fo te te subfamilies. other the from 0.1) < (P different September are August, year June, Anodontinae in full the the and for Mean values combined. been have and ent 40 0 - 30 - 50 - 60 0 - 70 Jan iue 3 Mnhy en ons f ioi fgrs eaae b sbaiy The subfamily. by separated figures mitotic Monthly of mean counts 43. Figure e Mr p My Jun May Apr Mar Feb Anodontinae apiine and nae Lampsi1i Unioninae aedr Month Calendar u u e Oct Sep Aug Jul Nov Dec 142 The joint pattern followed by the Lampsilinae and Unioninae differs both in timing and in magnitude. Mitotic activity is low and relatively stable from March through June, rises sharply in July and August (to 26 figures per slide), decreases in September and rebounds in October. At least two aspects of these apparent patterns raise intriguing questions. Why would the lampsiline and unionine species not start producing new cells until mid-summer, especially when the anodontine species start cell division at least by April? If the onset of massive cell division is related to other biological processes (such as reproduction), how do the physiologic cycles of these two major groups of species (especially the Unioninae and the Anodontinae) differ? Another question, probably more related to this data set than to mitotic activity, concerns the September decrease in both apparent patterns. Many of the animals contributing to the September data were collected in Georgia and Alabama - areas where the water temperature was still fairly warm (23-27°C). If this apparent substantial reduction in mitotic activity signals a metabolic shift to some other process (such as fat storage or gamete production), why did two out of the three animals collected in October have essentially a summer-like number of mitotic figures? Future research on the relationship between mitotic activity and time of year will be required to answer these physiologic questions. Such research also will begin to address the apparently unexplored topic of energy storage and utilization in the naiades. 143 Holding Time As indicated in the two preceeding subsections, some of the naiades used as sources of material for these chromosome slides were held for various lengths of time before they were processed. This had been done to provide a continuing source of animals for processing and to determine if chromosomal information could be acquired from specimens brought back alive from the field. When sorted by holding time, the mitotic count information can be used to address naiad mitotic response to captivity. If variations are found to occur, they may correlate with taxonomic groupings. Data concerning 147 specimens (190 full-scan labial palp slides) are available for this analysis. Fifty-nine specimens were processed on the day they were collected; the other 88 specimens were processed from 1 to 179 days post collection. In nearly all cases, the specimens that were processed more than a day or two after being collected were kept in 40 1 (10 gal.) well- aerated aquaria. These aquaria typically held guppies in addition to 5-20 naiades (no substrate) and stayed at approximately 20 - 22°C in a room with an approximate 12 hour light period per day. Maintenance consisted of adding commercial tropical fish food 3 to 5 times per week and removing dead fish or gaping naiades whenever they were observed. Table 46 presents the number of animals, slides, arithmetic and exponential mean values and approximate 95 percent confidence intervals for the various numbers of days the animals were held. When data from days 0 - 179 are considered, the number of mitotic figures on a slide 144 Table 46. Counts, mean values and approximate 95 percent confidence intervals for each of the days naiad specimens were processed after they were collected. The exponential values have been back calculated from base 10 logarithms. Days Number Number Arithmetic Exponential Until of of Processed Animals SI ides Mean (2SE) Mean - 2SE + 2SE 0 59 79 16.43 4.42 8.51 6.31 11.37 1 9 17 14.82 7.63 7.90 3.89 15.22 2 13 14 14.64 10.33 7.93 3.87 15.37 3 4 7 12.00 16.18 2.18 -0.16 11.10 4 5 5 16.00 12.54 11.24 4.11 28.31 5 1 1 7.00 - 7.00 -- 6 7 7 4.00 2.39 2.99 1.18 6.29 7 0 0 ---- 8 3 6 5.67 1.91 5.34 3.77 7.42 9 3 3 3.00 3.06 2.48 0.66 6.27 10 4 4 4.50 3.11 3.95 1.96 7.27 11 6 6 3.83 5.02 1.83 0.17 5.82 12 1 1 18.00 - 18.00 - - 13 5 5 7.20 5.71 4.49 0.96 14.37 14 0 0 -- - - - 15 3 4 0.75 1.50 0.41 -0.29 1.83 16 0 0 ----- 17 2 3 0.67 0.67 0.59 0.00 1.52 20 1 2 47.50 3.00 47.48 44.57 50.57 23 1 2 13.50 13.00 11.96 3.94 33.02 30 1 2 0.50 1.00 0.41 -0.29 1.83 31 1 1 7.00 - 7.00 - - 38 2 3 9.00 9.02 4.94 0.00 34.35 39 2 2 7.00 9.90 6.42 2.37 15.32 49 2 2 22.50 15.00 21.27 10.49 42.15 50 2 2 50.00 72.00 35.13 5.23 208.53 56 2 3 0.00 0.00 0.00 0.00 . 0.00 87 1 1 2.00 - 2.00 - - 90+ 1 1 22.00 - 22.00 -- 93 1 1 101.00 - 101.00 - - 106 1 1 26.00 - 26.00 -- 130 1 2 35.50 23.00 33.64 17.04 65.51 143 1 1 0.00 - 0.00 -- 179 2 2 21.00 20.00 18.60 6.35 51.26 Totals 147 190 145 and holding time show only weak linear or exponential relationships (P <0.1); however, if only the data from day 0 through day 17 are considered (162 slides - 85 percent of the total), mitotic figure count and holding time show strong linear and exponential relationships (P < 0.0001). A logarithmic transformation of the count values norma lized the variances and was f it by an exponential equation with a strong correlation coefficient ( r = 0.34, P < 0.01). This curve, the anti log means and confidence intervals are presented in figure 44. Day 17 was chosen as the cut-off point simply because the data were spaced at more than alternate-day intervals from there to day 179. The 28 data points for days 20 - 179 do not differ from a random distribution (P = 0.54). This analysis indicates that between eight and nine mitotic figures can be expected on an average slide processed on the day the animal was collected and that this number will decrease at a decreasing rate over the next 17 days. Beyond that point the number of mitotic figures on a slide appears to be a random function or, more likely, is substantially affected by specific conditions in the holding chamber. The 162 slides that were processed during days 0-17 include enough observations to permit statistical comparisons of most of the supra-generic groups presented in table 43. When the log transformed data are sorted by these groupings and are analyzed, a variety of relationships emerge (table 47). Eleven of the 18 groupings exhibit strong relationships (P <_0.05) between the counts of mitotic figures and holding time. The seven groupings that do not exhibit this relationship include all three of the Anodonta-1ike taxa (Nl, N2, N3), two of the four Quadrula-like taxa (Ql, Q3), a Pleurobema-1ike grouping Mitotic Figure Count 6 - 16 10 12 14 hp ewe mttc iue on ad odn time. holding and count figure between mitotic ship 2 0 - - 0 1 iue 4 Diy en aus n te et t epnnil euto fr h relation the for equation (exponential) it f best the and mean Daily values 44. Figure 5 9 7 5 3 I I I I I I I I I i odn Tm (days) Time Holding = 8.47(10) = y -0.04x 11 3 5 17 15 13 I I i I I I I cn 147 Table 47. Comparison of the tests for linearity, equation parameters and correlation coefficients for the mitotic figure count by holding time data (days 0-17) when sorted by various taxonomic groupings. Equation parameters and correlation coefficients are given only for relationships significant at the 0.05 level. Linearity of Significant Exponential Classification Log 10 Equations Group n Transformed y = a(10)bx (Table 43 Code) Data P = a . 3 r P < All data 162 20 .99 0.0001 8 47 -0 .04 0.34 0.01 Alasmidonta-1ike A1 23 11 .43 0.0028 20.26 -0 .08 0.59 0.01 A2 20 19.54 0.0003 21 75 -0 .10 0.72 0 .01 A3 15 11 .14 0.0053 19 55 -0 04 0.68 0 01 Anodonta-1ike N1 7 1 17 0.3295 N2 3 0 00 0.9585 N3 4 0 01 0.9174 Lampsilis-1ike L 79 5 45 0.0221 6 98 -0 03 0.26 0 05 Amblema-like U 60 9 66 0.0029 8 60 -.0 04 0.38 0 01 Pleurobema-like PI 34 5 35 0.0273 6 93 -0 03 0.38 0 05 P2 41 4 45 0.0413 6 73 -0 03 0.32 0 1 P3 25 0 22 0.6413 P4 16 6 19 0.0260 6 27 -0 05 0.55 0 05 Quadrula-like Q1 25 1 71 0.2035 Q2 19 4 20 0.0561 13 47 -0 08 0.44 0 1 Q3 13 0 19 0.6713 Q4 6 12 88 0.0230 22. 55 -0. 29 0.87 0 05 Rafinesquean Rl 115 12. 99 0.0005 7. 47 -0. 03 0.32 0 01 R2 25 1 97 0.1741 148 (P3) and a Rafinesquean grouping (R2). In some cases, the low number of slides available for analysis in these groupings may have affected the likelihood of relationship (n = 3, 4 and 7 for the Anodonta-like groupings); however, the other apparently non-interactive groupings include more slides than groupings which did show relationships between mitotic figure counts and holding time. The intercept and slope parameters of the significant relation ships (table 47) include a modest range of values among the proposed groupings of the genera but apparent similarity between similar groupings. In the following paragraphs, relationships within similar groupings are discussed first, followed by a discussion of the relationships among the groups. The three Alasmidonta-1ike groupings yield equation intercept values that range from 19.55 to 21.75 and slope component values that range from -0.04 to -0.10. Analysis of covariance indicates that both the intercepts and the slopes of these three curves are not dissimilar (P >0.5 for both tests). The different numbers of genera included in these three similar groupings come close to forming a stepwise addition sequence. The group A3 (Rafinesque subfamily Anodontidae) begins the sequence with 15 slides from two genera: Alasmidonta and Lasmiqona. Group A2 (Alasmidontinae of Modell and Morrison) adds five slides from Anodontoides, Arcidens, and Strophitus which increases the strength of the relationship (F value) and raises the intercept slightly, but does not affect the slope. The final addition to form group A1 (Anodontinae of Ortmann, Heard and Guckert, and Davis and Fuller) is three slides from the genus 149 Anodonta. This substantially decreases the F statistic, decreases the intercept by one and alters the slope. The magnitude of these Anodonta-related changes suggest that this genus might not exhibit the same mitotic response to holding time as the other genera; however, data from three extremely variable slides (day 0 - 6 spreads, day 2-74 spreads and day 12 - 18 spreads) neither form statistical or logical grounds for confirming such a distinction. The variability of these few Anodonta slides also may have prevented the recognition of any relationship in the Anodonta-1ike groupings. Anodonta alone comprises group N2 (Anodontinae of Model!) and is joined by one Anodontoides slide to form N3 (Anodontinae of Morrison) or four Strophitus slides to form N1 (Anodontidae of Rafinesque). In jigneral, these mitotic.figure count by holding time data support the similarity of five genera (Alasmidonta, Anodontoides, Arcidens, Lasmiqona, and Strophitus) that typically have been placed in the same naiad subfamily. The few data points available for the genus Anodonta suggest that it may not react to being held in aquaria like the others; however, additional data should be collected to determine the mitotic activity response Anodonta does exhibit. Various authors have proposed to separate the naiad subfamily Unioninae (Ortmann, 1912) into two groups: a Pleurobema-1ike group and a Quadrula-1ike group. Table 43 includes four similar Pleurobema-1ike groupings, one of which (P3 - Pleurobeminae of Modell) did not yield a significant relationship of the count by holding time data. Intercept and slope parameters for each of the other three groupings are s ta tis ti cally not dissimilar (P > 0.3 for both covariance tests). These 150 intercept values range from 6.27 to 6.93 and slope values range from -0.03 to -0.05. Group P4 (Elliptioninae of Modell) includes only the genera Elliptio and Uniomerus in this data set (16 slides). Group P2 (Pleurobemini of Davis and Fuller) adds Fusconaia, Lexinqtonia and Pleurobema (9, 5 and 11 slides respectively) which reduces the F statis tic, raises the intercept slightly (to 6.93) and reduces the slope parameter (to -0.03). Group PI (Pleurobeminae of Heard and Guckert) replaces Fusconaia with Cyclonaias (n = 2), which returns the F statistic essentially to its previous level of significance, drops the inter cept slightly (to 6.73), but does not alter the slope parameter. If there is an outlier in this group, it appears to be the genus Fusconaia. The substitution of two variable Cyclonaias slides (33 and 3, both day 0) for nine Fusconaia slides between groupings PI and P2 improves the f it of the data to the curve (r increases from 0.32 to 0.38). Of the four Quadrula-1ike groupings listed in table 43, only Q2 (Amblemini of Davis and Fuller) and Q4 (Ambleminae of Modell) showed strong indications of a relationship between these data. Group Q4 in this analysis is represented in six data points from the genus Amblema. These few observations certainly lie along a describable curve (intercept 22.55, slope parameter -0.29 ); however, whether this parti cular curve accurately portrays the typical Amblema mitotic response to being kept in aquaria probably should be evaluated when more data become available. As presently described, the slope of Q4 is different from Q2 (P < 0.04) but the day zero means are not dissimilar (P > 0.2). 151 Group Q2 is represented by five genera in this data set: Amblema (n = 6), Cyclonaias (n = 2), Megalonaias (n = 1), Quadrula (n = 8) and Quincuncina (n = 2). Tritoqonia also is included in Q2 but the only full scan count made was from a specimen processed 143 days after it was collected (a zero). The intercept of this curve is 13.47 and the slope parameter is -0.08. The 19 counts that make up the data base for Q2 are not numerous enough to indicate relationships between genera. When the six counts for Quadrula are considered separately they fail to show a relationship with holding time (P = 0.3858). No other genus in this group is represented by more than two counts. Group L in table 43 (Lampsilinae of Ortmann, Modell, Morrison, Heard and Guckert, and Lampsilini of Davis and Fuller) includes 15 genera which no author has proposed to subdivide at the supra-generic level. As a unit, these genera follow an exponential relationship quite similar to the Pleurobema-1ike group PI and P2. For group L, the inter cept is 6.98 and the slope parameter is -0.03. An analysis of covar iance of the genera within group L is reviewed in table 48. This analysis required the creation of four "combination genera", a process in which the data from two to four recognized genera were combined to give at least two observations from the full day 0 - day 179 data set. The genera that were combined are often placed near each other on phylo- genetically arranged lists (Ortmann, 1924; Stansbery, 1982). Covariance analysis indicates that the slopes of the equations for the genera are not statistically different from each other (P > 0.3) 152 Table 48. Results of a covariance analysis on seven actual or combination genera in the Lampsilis-like group (typically the subfamily Lampsilinae). Combined Analysis Degrees F Probabi1i ty Hypothesis of Freedom Statistic Level Slopes are not different 5, 66 1.23 0.3067 Means are not different 6, 71 2.79 0.0172 Comparison of Least Square Means "k 'k Epioblasma Villosa TOME Lampsilis PTOBAC PLOBLEPO* LILE* Mean Value 3.11 4.84 5.81 9.12 9.53 21.58 24.85 (Anti log) ** * Combination genus, decoded as follows: LILE = Ligumia and Lemiox PLOBLEPO = PI agio!a, Obovaria, Leptodea and Potamilus PTOBAC = Ptychobranchus, Obiiquaria and Actinonaias TOME = Toxolasma and Medionidus ** Mean values underlain by the same bar are similar at the 0.05 level. 153 but that not all of the generic equation means (and intercepts) are similar (P < 0.02). Comparison of the least square means for each equation (table 48) indicates that the combination genera PLOBLEPO (Plaqiola, Obovaria, Leptodea and Potamilus) and LILE (Ligumia and Lemiox) are different from the other five genera or combination genera involved (P < 0.05). The joint weighted (antilog) intercept value for PLOBLEPO and LILE is 25.97 and the similar value for the other genera is 8.96. This apparent separation of the Lampsilinae into two groups on the basis of mitotic activity appears to warrant further investigation of both mitotic activity patterns and other sets of characteristics. It is rather unlikely that the high counts recorded for the six genera combined into PLOBLEPO and LILE could all have been atypical; however, it is also rather unlikely that all six of the genera essentially blindly placed in these combination genera actually exhibit statistically similar high mitotic rates. Several of the preceeding paragraphs include discussions of covariance analyses that were run to compare the holding time data within previously proposed groupings of the genera included in this project. Slope and intercept covariance statistics for the subfamily- and tribe-level groupings already discussed are presented in table 49 along with similar statistics for some higher-level comparisons. For comparisons between the A, P, and Q subfamily level groupings, the best f it and, generally, most inclusive groupings (A1, PI and Q2) are used. 154 Table 49. Results of several covariance analyses comparing equations based upon different sortings of naiad mitotic figure count by holding time data. The day zero means of equations with different slopes were compared using equal variance t tests. These values are enclosed in brackets in the table. Hypotheses Groupings Compared Slopes are Equal Means are Equal F P < F F P < F Alasmidonta-like 0.23 0.7988 0.14 0.8675 Al, A2, A3 Pleurobema-like 0.39 0.6782 0.33 0.7197 PI, P2, P4 Quadrula-like 4.89 0.0382 * [t = -1.17 0.2796] Q2, Q4 Alasmidonta- and Lampsilis- like 3.03 0.0846* 2.13 0.1476 Al, L [t = 2.77 0.0081] * Alasmidonta- and Pleurobema- like 2.64 0.1101 3.77 0.0575 * Al, PI Alasmidonta- and Quadrula-like 0.00 0.9553 0.04 0.8520 Al, Q2 Lampsilis- and Pleurobema-like 0.00 0.9573 1.06 0.3044 L, PI Lampsilis- and Quadrula-like 1.26 0.2649 2.62 0.1088 L, Q2 Pleurobema- and Quadrula-like 1.10 0.2989 4.42 0.0407 t PI, Q2 Lampsilis-like, Pleurobema- like and Rafinesquean 0.00 0.9976 0.73 0.4816 L, PI, R1 * P < 0.1 * P < 0.05 155 Three covariance analyses compared the Alasmidonta-like group (Al - including Anodonta) individually with each of the others. The Alasmidonta- and Quadrula-1ike groups, Al and Q2, have quite similar slopes and mean values (P > 0.8). The slopes and means of the Alasmi donta- and Pleurobema-like groups are not dissimilar at the 95 percent confidence level; however, the means are dissimilar at the 90 percent level and the F value for the slope comparison is quite close to the 90 percent probability level (P = 0.1101). The Alasmidonta-like, Lampsilis-like (Al, L) slope comparison falls between the 0.1 and 0.05 probability levels (P = 0.0846). If the slopes are considered to be dissimilar, a t test indicates that the day zero means are dissimilar (P < 0.01). The pair of covariance analyses in which the Lampsilis-like group was compared with the Pleurobema-like and the Quadrula-like groups yield inconsistent results. Group L does not have a dissimilar slope or mean value from PI (P > 0.3 for both tests). The slopes for L and Q2 also are not dissimilar (P > 0.2) and the mean values are just not dissimilar at the 90 percent confidence level (P = 0.1088). The comparison of Pleurobema-like and Quadrula-like groups found no dissimilarity in slopes (P >0.2) but clear dissimilarity in mean values (P < 0.05). A final covariance run examined the relationship of group R1 (subfamily Uniodiae of Rafinesque) with its modern counterparts L and PI. The slopes and intercepts of these curves are statistically not dissimilar (P > 0.4). Figure 45 presents the linear plots (log transformations) of the exponential curves for Al, L, PI and Q2. It illustrates the similarity 156 -0.08x Alasmidonta-like y=20.26(10) 25 Lampsilis-like y = 6.98(10)“° ‘03x -0.03x 20 ------Pleurobema-1ike y = 6.93(10) ------Quadrula-like y = 13.47(10) •0.08x 15 10 5 - c 3 O o < u S- 3 o i 2 u •r- +J +° J 1 I 0 .5 - 4 6 8 10 12 14 16 Holding Time (days) Figure 45. Linear plots of the log transformed equations describ ing the relationships between mitotic figure count and holding time. The slopes and means of the Lampsilis- and the Pleurobema-1ike equations are not dissimilar (P < 0.8). Neither are the slopes and means of the Alasmidonta- and Quadrula-like equations (P < 0.8). 157 of the slopes and intercepts of the Alasmidonta-like and Quadrula-like equations and the different pattern shared by the Lampsilis-like and Pleurobema-like equations. Synthesis The three analyses of the naiad mitotic activity data just pre sented (temperature, annual cycle and holding time) form an increasingly complete image of naiad mitotic activity cycles and some of the factors that can affect them. The temperature analysis, which includes the smallest number of observations (71), provides a fragmentary notion of increasing mitotic activity to approximately 23°C followed by a slower decline. It also begins to identify differences between taxonomic groups. The annual cycle analysis includes more data (n = 121) and presents two apparent cycles of mitotic activity: an early onset of cell division accompanied by a high August peak in the Anodontinae, and a later initiation of mitotic activity and lower August peak in the Lampsilinae and Unioninae. The differences between these two groups are usually statistically significant when sufficient data are available. The holding time analysis includes the largest data set (n = 190) and describes the exponential decrease in mitotic activity during the first 17 days naiades are held in aquaria. More importantly, perhaps, this analysis demonstrates statistically significant differences between previously proposed taxonomic groupings of the genera and suggests that some relationships should be investigated further. 158 None of these three analyses was included in the initial scope of this project and no experimental design for any of them was developed until the project was essentially complete. Because these studies were not rigorously designed and because they lack statistical depth in many instances, .the mitotic activity and taxonomic relationships that they produced should be taken as suggestions to be confirmed rather than any sort of proofs. Suggestions about mitotic activity that have been touched upon in these analyses appear to fall into two categories: initiation/cessation, and relative rate. These three analyses indicate that cell division in naiades is affected by temperature, season (apparently), and other environmental factors, all of which fits the expected pattern for the vast majority of plants and animals. The interesting observation that emerges from these analyses is that the onset of cell division appar ently differs between groups of sympatric naiad species, perhaps suggesting different physiologic regimes and/or climatic adaptations. Such a train of thought could lead to the formulation and testing of a number of hypotheses about climatic or geographic points of origin of different taxa. Not unrelated to the initiation and cessation of mitotic activity is the observed difference in mitotic rate. Mitotic activity was found to be higher in the Anodontinae than in the Lampsilinae or the Pleurobema- like group within the Unioninae (which appear to be similar). Further investigation may support suggestions that the Quadrula-1ike genera and a few lampsiline genera have intermediate mitotic rates. It is possible that various lines of naiad research may correlate high mitotic rate and 159 rapid overall growth in some of these groups (especially the Anodontinae). Alternatively, high labial palp mitotic activity may be found to be a restricted, single tissue phenomenon that correlates with mechanical abrason in specific habitats. The taxonomic relationships suggested by these analyses are intriguing, regardless of how incomplete they may be. All three analyses suggest a distinction between the Alasmidonta-like genera (perhaps, including Anodonta) and some or all of the other species considered. (Gonidea is included in only the Unioninae of Ortmann and Morrison; Gonidea and Margaritifera are in separate subfamily- or tribe- level taxa proposed by the other authors and were not represented in this data set by enough samples to be analyzed. The holding time analysis indicates a statistical similarity between two pairs of groupings: the Alasmidonta- and Quadrula-like groups and the Lampsilis- and Pleurobema-like groups. These do not correspond exactly with any existing classification system; however, they do provide new support for the distinctiveness of the Alasmidonta-1ike and Quadrula-1ike groups. Appropriately designed experiments that gather sufficiently data on the same or additional taxa will clarify these general relationships and will begin to explore some suspected differences within these groups. Specifically, the associations of the genera Anodonta and Fusconaia should be investigated; both were represented in this data set by a few apparently unrelated observations. Also it would be interesting to see if additional data will confirm and expand upon the separation of groups with high and low mitotic activity levels within the Lampsilinae. DISCUSSION II: NAIAD CHROMOSOMES The original purposes of this project (stated in the Introduction) were to determine if naiad chromosomal characteristics include taxonomi- cally-useful data and, if possible, to compare the analysis of this kind of a data set with existing naiad classification systems. Both of these topics are covered in this discussion. Chromosome Number Table 1 and the information presented in the various species accounts indicate that the modal number of diploid chromosomes found on these slides is 38. Figure 46 reinforces this assertation by presenting the tally of 774 counts of chromosomes for all species studied during this project. The clear mode at 38 (65.12 percent of all counts), accompanied by the sharply skewed distribution, strongly suggests that 38 is the typical chromosome number present in complete spreads of North American naiades. On a species-by-species basis, each of the 41 species-level taxa with five or more chromosome counts had a modal number of 38. In addition, of the 25 species with between one and four counts,- the strong est suggestion of a mode other than 38 was for Quincuncina infucata (four counts: 31-1, 36-2, 38-1). These data lead to the conclusion that 38 is the diploid chromosome number for the 41 species with over five counts, and more than likely, 38 is the diploid number for the 25 160 nlds 0 o te 7 cut (65.12%). 774 counts the 504 of includes Number of Counts — 500 40 30 50 60 10 20 0 5 iue 6 Tly f hoooe ons ae uig hs rjc. The mode (38) project. chromosome of this made during counts Tally 46. Figure 1 2 2 2 2 2 3 3 3 3 3 40 38 36 34 32 30 28 26 24 22 20 18 1 6 hoooe Number Chromosome 43 50 162 species (including Quincuncina infucata) represented by between one and four counts each. Conservation in chromosome number among species within a family or superfamily appears to be typical of the Bivalvia (Menzel, 1968; Wada, 1978), if not the Mollusca (Patterson, 1969; Murray, 1975), This being the expectation, the reports of 38 diploid chromosomes for two European naiades (van Griethuysen, et a l, 1969) and for three Japanese naiades [Nadamitsu and Kamai, 1975; 1978(?)] appear to extend this North American study to characterize the fauna of the Northern Hemi sphere and, perhaps, the Ortmann-proposed families Margaritiferidae and Unionidae. If the 34 diploid number reported for three Australian species by McMichael and Hiscock (1958) is typical of endemic Southern Hemisphere groups, it could lend considerable support to speculation about the separate evolution of major naiad taxa (Simpson, 1896; McMichael and Hiscock, 1958). Supernumerary Chromosomes Three species accounts include mention of a chromosomal feature not included in table 1 or figure 46. One slide each of Anodonta grandis (JJJ:106:P:2), Leptodea fragilis (JJJ:236:P:3) and Obovaria olivaria (JJJ:226:P:2) contained a few spreads that included a small "dot" chromosome. Single dots were present in six of 14 spreads (43%) noted on the A. grandis slide, four of 21 spreads (19%) noted on the k- fragilis slide and one of 14 spreads (7%) noted on the 0. olivaria slide. The dots in two measured A. grandis spreads comprised 0.64 and 0.81 percent of the total complement length, less than half the length 163 of the next smallest chromosomes (figure 3). No centromere was observed on any of the dots. The dot chromosomes on these naiad slides appear to fit the general description of supernumerary chromosomes (Brown, 1972). As a group, supernumeraries are small chromosomes which occur infrequently in species and individuals and appear to act essentially as chromosomal parasites. In plants, where supernumerary chromosomes are more common, various lines of research have indicated that they cause little or no detectable phenotypic effect, may deviate from normal duplication and separation patterns during cell division (nondisjunction), and may occur in a variety of numbers within the cells of an organism. At times supernumerary chromosomes follow "preferential distribution" into specific tissues or cells, especially in reproductive areas. Patterson (1969) cites a few cases of supernumerary chromosomes in gastropods but none in other mollusks. Some of the cases presented appear to be similar to the rare occurence, non-uniform pattern found in plants and during this'project; however, other cases seem more likely to have been various types of disruptions of the normal chromosome complement. Chromosome Morphology Given a stable chromosome number among the naiad species involved in this project, chromosomal comparative features must be sought at a more detailed level. In many animal and plant species, variations in relative lengths of the chromosomes and in the location of the centro mere on each chromosome provide quantifiable features which can be 164 analyzed and compared. These morphologic studies are becoming more common than accounts of chromosome numbers and are supplying more useful data than was previously thought available from chromosomal work (Takagi and Sasaki, 1974; Bogart, 1973). Chromosome morphologic studies on mollusks were begun by Burch (1962) and Patterson (1965) and continue to increase in sophistication and application (Babrakzai and Miller, 1976; Chambers, 1982; Nakamura, 1982). As previously described (in Methods), arm ratio (r) and percent complement length (%TCL) values were calculated for 121 spreads repre senting 41 species. Tables 2 - 42 (in Results) present the species-by- species summaries of the relationships between these two parameters. Figures 47 and 48 illustrate the composite distributions of these parameters for all of the measured chromosome spreads. In addition to illustrating the distribution of r values in these naiad data, figure 47 also indicates that there is no analysis value in comparing "fundamental numbers" among these spreads. The concept of counting all of the chromosome arms was proposed by Matthey (1949) and has been used to advantage to help understand situations in which single-armed (telocentric) chromosomes exist in some species but fewer biarmed chromosomes exist in other species. Figure 47 indicates that virtually none of the measured naiad spreads included telocentric chromosomes. This observation, when combined with the uniform 38 diploid chromosome number, produces a uniform fundamental number of 76 for all of the species that were examined. 20 14 - 12 - 10 - :::: 1.00-1.70 1.71-3.00 3.01-7.00 > 7.00 Figure 47. Distribution of naiad arm ratio (r) values as indicated by four category means from 121 measured chromosome spreads. 20 - 0 . 00- 2.00 2.01-3.00 3.01-4.00 > 4.00 Figure 48. Distribution of naiad percent total complement length (%TCL) values as indicated by four category means from 121 measured chromosome spreads. 166 Matrix Comparisons In Results, the format of tables 2 through 42 was used as a convenient way to present both the arm ratio (r) and percent complement length (%TCL) data from analyzed chromosome spreads. Much of the following discussion makes use of another feature of this interaction of the r and %TCL relationship - its value as ananalytical tool. Arm ratio and percent complement length are virtually independent characteristics of any given chromosome. The r value describes the location of the centromere without regard to the length of the chromo some. The %TCL value compares the length of one chromosome to the aggregate length of all chromosomes in the spread without regard to the location of the centromere. These chrarcteristies are virtually independent because obscured folding of a chromosome or other measure ment errors will affect both the r and %TCL values. It is important to realize that both r and %TCL are continuous variables and that the division of the r by %TCL matrix (or table) into sections is an artificial phenomenon which must be accommodated by the type of analysis. The stabilization in arm ratio terminology proposed by Levan et al (1964) includes a clear description of the range of possible locations of the centromere along with the authors' logic for the mathematical break points between the metacentric (m; r =? 1.00-1.70), submetacentric (sm; r = 1.71-3.00), subtelocentric (st; r = 3.01-7.00) and acrocentric [=telocentric](t; r > 7.00) categories. No comparable separation of %TCL values is likely to be widely applicable because of the wide range in chromosome numbers (and, there fore, %TCL ranges) among animal and plant species. Early in this 167 project, exact interger values were arbitrarily set as the break points between %1CL sections; however, so few data points fell below 1 %TCL or above 5 %TCL that outlying sections were combined to produce the 0-2, 2-3, 3-4 and > 4 categories used in the tables. Table 50 presents a hypothetical example of this type of analysis and illustrates its usefulness. If the two taxa being compared are represented by large numbers of analyzed spreads, the mean numbers of chromosomes present in each section of the table approach the actual values and, where the means differ, the differences can be assumed to be significant at some confidence level. In this example, the only values in the table which differ occur in sections m 0-2 and sm 0-2. The total values which differ are m and sm. The result of this comparison indicates that one of the two taxa has two more chromosomes in matrix section m 0-2 and the other taxon has two more chromosomes in section sm 0-2. The simplest type of chromosomal modification that could have produced this observed difference is the relocation of a centromere on a chromosome (a peri centric inversion) followed by this difference being incorporated into the karyotype. Notice that the total for the single %TCL category in which this chromosomal difference occurs (0-2 %TCL) is not differenct but both the m and sm totals are different. In general, pericentric inversions and more complex relocations of centromeres produce changes in r values (vertical differences in these tables) while duplications, deletions and more complex modifications in chromosome length produce changes in %TCL values (horizontal differences in these tables). 168 Table 50. Hypothetical example of the comparison of two naiad taxa based upon the interaction of r and %TCL values. The (hypothe tical) mean values for the taxa are presented in opposite corners of each table section and total. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 4 10 0 2 16 m 2 10 0 2 14 0 10 2 0 12 sm 2 10 2 0 14 r 0 10 0 0 10 St 0 10 0 0 10 0 0 0 0 0 t 0 0 0 0 0 4 30 2 2 38 Totals 4 30 2 2 38 169 Measurement Error The following comparisons of naiad karyotypes are based solely upon measurement data. As indicated in Methods, these measurements weremade on photographs that had been outlined while being compared with the magnified spread. The r by %TCL tables were prepared under the assumption that whatever errors might be associated with the measure ments are not large enough to materially affect the results. The validation of this assumption requires an examination of measurement error. An ideal test of measurement error would consist of evaluating the sample mean and variance associated with measuring a previously- known object similar in size, character, and preparation to the primary subjects. In the absence of a known standard among naiad chromosomes, the closest approximation appeared to consist of comparing several measurements of a distinctive chromosome pair. Experience also indicated that relative measurements (%TCL) would have to be compared because of the considerable differences in chromosomal contraction observed among the spreads that had been studied. The species account tables indicate that several analyzed spreads include four distinctive subtelocentric chromosomes, typically in the 2-3 %TCL size range. These chromosomes generally are present in spreads of the closely related genera Lampsilis and Villosa (suggested relationship references: Ortmann, 1912; Stansbery, 1982) offering 36 replicates (spreads) for this analysis. The four chromosomes with the largest arm ratio (r) values in each spread were sorted into pairs based solely on their %TCL values. 170 Mean and standard deviation values were calculated for the long and short arms, and for the full length of each chromosome pair (table 51). If the means are taken to represent the actual proportional lengths of the chromosome segments and the standard deviations are taken to estimate the total effects of inconsistencies in outling, location of centromeres and measurements; then the Coefficient of Variation (lOOs/x) can be taken to estimate the incidence of error in the data (Steel and Torrie, 1960). The values presented in table 51 indicate that error was greatest (18 or 19 percent) for short chromo some segments (less than 0.5 %TCL) and approximately half that (9.3- 9.8 percent) for longer segments (2.0-2.7 %TCL). The average error value based upon these four arm estimates is 14.3 percent. Table 51 . Comparison of means (x), standard deviations (s) and coefficients of variation (lOCs/x) for two pairs of subtelocentric chromosomes from measurements of 10 Lampsilis spreads and 26 Villosa spreads. Units for x and s are percent complement length, for (lOOs/x) they are percentage. Segment n X s lOOs/x pair B - short arms 72 0.492 0.095 19.3 pair A - short arms 72 0.593 0.108 18.2 pair B - long arms 72 1.853 0.181 9.8 pair A - long arms 72 2.176 0.213 9.8 pair B 72 2.345 0.229 9.8 pair A 72 2.770 0.258 9.3 171 The short arms of these subtelocentric chromosomes were among the shortest typical segments encountered in each spread. Mechanical difficulties in measuring these very short segments (usually less than 5.0 mm on the photographs), accompanied by the persistent problem of identifying the ends of chromosomes, may have affected the precision of these measurements. A consistent error value would have affected measurements of shorter arms more than longer ones simply because the error component would make up a larger percentage of the length. With between 10 and 20 percent measurement error involved in these data, precise assocation of homologous chromosomes is not likely to be possible. The imprecise measurement data also will be likely to affect the assignment of all chromosomes to the proper sections of the r by %TCL matrix, producing some level of random noise in these analyses. In analyses which include few chromosome spreads, this random variation may hinder the recognition of differences between taxa and, generally, will weaken this analysis to suggesting relationships rather than confirming them. Composite Analysis Table 1 indicates that 121 chromosome spreads were analyzed and includes the names of the 41 species from which these spreads were taken. Unfortunately 42 of the analyzed spreads included fewer (33 spreads) or more than (9 spreads) 38 chromosomes. These incomplete, indistinct, inaccurately outlined, or otherwise imperfect spreads have been excluded from the following analyses, primarily to eliminate the abnormal %TCL data they would have contributed. Table 52 presents the 172 Table 52. Species-by-species listing of the number of analyzed spreads, the number of analyzed spreads with 38 chromosomes and the suprageneric groups in which the species have been placed. The abbreviations are explained in the caption to table 43(page 133). Genus and Species Spreads Exactly 38 Suprageneric Analyzed Chromosomes Group Actinonaias 1. form carinata 4* 3* L Alasmidonta marginata 3* 3* A Anodonta grandis 8 6 A Anodontoides ferussacianus 2 2 A Elliptio crassidens 1 1 P Elliptio dilatatus 2 1 P Epioblasma torulosa rangiana 1 0 - Fusconaia barnesiana 3 3 P Fusconaia flava 2 0 - Gonidea angulata 4 3 G Lampsilis australis 1 1 L Lampsilis fasciola 3 1 L Lampsilis higginsi 2 0 Lampsilis radiata luteola 1 0 Lampsilis subangulata 1 1 L Lampsilis ventricosa 1 1 L Lasmigona complanata 1 0 Lasmigona costata 9 5 Lemiox timosus 2 1 L Leptodea fragilis 4 2 L Lexingtonia dolabelloides 1 0 Ligumia nasuta 4 4 L Ligumia recta 2 2 L Margaritifera m. form falcata 2 1 Medionidus conradicus 2 1 L Obovaria olivaria 4 1 L Obovaria subrotunda 7 6 L Pleurobema coccineum 1 0 Pleurobema rubrum 1 1 P Potamilus alatus 4 2 L Ptychobranchus fasciolaris 5 4 L Quadrula pustulosa 1 1 Quincuncina infucata 1 0 Toxolasma lividus glans 1 1 L Toxolasma parva 3 2 Tritogonia verrucosa 1 1 Villosa iris iris 4 4 L Villosa iris nebulosa 1 1 L Villosa lienosa 2 1 L Villosa taeniata punctata 9 7 L Villosa trabalis 10 5 L A G L M P JL Total spreads 121 79 T6 3 51 1 6 2 Total species 41 33 4 1 21 1 4 2 Total genera 25 22 4 1 11 1 3 2 * includes a second set of measurements of one of the spreads. 173 species-by-species breakdown of the 79 "perfect" spreads which were analyzed. Data from all 79 spreads were used to calculate an overall mean karyotype. This karyotype was calculated by computing the mean number of chromosomes present in each section of the r by %TCL matrix and then rounding the section values to even numbers (for the diploid comple ment) which best f it the row and column totals and contributed to an overall total of 38 chromosomes. The section means based upon the 79 spreads and the suggested overall mean karyotype are presented in table 53. The suggested karyotype numbers presented in each section of table 53 must be considered with close attention to the information included in table 52. The 79 chromosome spreads used to calculate these adjusted average values were derived from 33 species in 22 genera out of an estimated North American total of 500 species (Simpson, 1900) in approximately 50 genera (Burch, 1973). This fragmentary sample of North American naiad diversity also is affected by the large number of Lampsilis-like spreads included (64.6%) and the large number of Mississippi River drainage species represented (29 of 33 species or 87.9%). Other assortments of species and genera including different taxonomic and geographic representation should be expected to yield relatively, or radically different mean karyotypes. Regardless of the relatively fluid nature of a mean karyotype, any reasonably robust set of r and %TCL values can be used as a basis for comparing the chromosome complements of the contributing groups of species. Table 54 presents the section-by-section calculated and rounded 174 Table 53. Mean numbers of chromosomes from 79 analyzed spreads present in each section of the r by %TCL matrix. Also included are the rounded even numbers of chromosomes which form the overall mean karyo type based upon these spreads. % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals 3.03 12.22 3.73 1.14 20.11 m 4 12 4 0 20 0.89 10.13 2.78 0.09 13.89 sm 0 10 4 0 14 r 0.19 3.28 0.48 0.00 3.95 St 0 4 0 0 4 0.03 0.03 0.00 0.00 0.05 t 0 0 0 0 0 4.13 25.65 7.00 1.23 38.00 Totals 4 26 8 0 38 175 mean chromosome numbers for the six suprageneric taxonomic groups represented by the 79 spreads. Within each suprageneric group, the numbers of chromosomes which fell in each section of the r by %TCL matrix were averaged together and rounded to even numbers that contributed to the total of 38 (the identical process used to calculate the overall mean karyotype). Examination of table 54 indicates that the suprageneric groups differ from each other in various matrix sections. These differences may represent the incorporation of detectable chromosomal inversions, translocations or more complex modifications in a majority of the species within each group. However, for groups represented by relatively few analyzed spreads, these differences also could have been affected by the inclusion of poor quality spreads or measurement errors. For the purpose of this analysis, the values in table 54 will be assumed to represent mean karyotypes of these taxa. Future studies will support, modify or refute these early approximations. The mean karyotypes presented in table 54 provide a way of relating the suprageneric groups to each other. The mean Lampsilis-like karyotype (based on 51 spreads) differs from the overall mean karyotype in two matrix sections [m 3-4 (-2) and m >4 (+2)]. The simplest type of chromosomal modification which could have produced this difference is the duplication of a chromosome segment followed by its incorporation into the karyotype. More-than-likely the close similarity of this group karyotype to the overall mean karyotype is due to the predominance of lampsiline spreads in the data set. 176 Table 54. Sectional mean values and rounded karyotype numbers for six suprageneric groups: Alasmidonta-like (A-16 spreads), Gonidea anqulata (G-3 spreads), Lampsilis-like (L-51 spreads), Margaritifera margaritifera form falcata (M-l spread), Pleurobema-like (P-6 spreads) and Quadrula-1ike (Q-2 spreads). % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals Groups A 2.94 4 10.06 10 5.50 6 0.75 0 19.19 20 G 2.00 2 8.33 9 2.00 2 0.33 0 12.67 12 L 3.29 4 12.29 12 3.31 2 1.25 2 20.14 20 m M 1 0 14 14 3 4 0 0 18 18 P 2.50 2 17.33 18 3.83 4 1.67 2 25.33 26 Q 1.00 0 17.50 18 3.00 4 1.50 2 23.00 24 A 1.00 0 10.81 12 2.25 2 0.06 0 14.13 14 G 3.00 4 12.33 12 5.00 6 0.33 0 20.67 22 L 0.71 0 9.90 10 3.12 4 0.10 0 13.82 14 sm M 1 2 10 10 1 0 0 0 12 12 P 1.00 2 9.00 8 1.17 2 0.00 0 11.17 12 Q 1.00 2 10.50 10 1.00 0 0.00 0 12.50 12 A 0.19 0 4.19 4 0.25 0 0.00 0 4.63 4 G 0.67 0 3.00 4 0.67 0 0.00 0 4.33 4 L 0.18 0 3.31 4 0.49 0 0.00 0 3.98 4 r st M 0 0 4 4 4 4 0 0 8 8 P 0.00 0 1.17 0 0.33 0 0.00 0 1.50 o Q 0.50 0 1.50 2 0.50 0 0.00 0 2.50 2 A 0.00 0 0.00 0 0.00 0 0.00 0 0.00 o G 0.00 0 0.33 0 0.00 0 0.00 0 0.33 o L 0.04 0 0.02 0 0.00 0 0.00 0 0.06 o t M 0 0 0 0 0 0 0 0 0.00 o P 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 Q 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 A 4.13 4 25.06 26 8.00 8 0.81 0 G 5.67 6 24.00 24 7.67 8 0.67 0 L CO 4.22 4 25.49 26 6.92 6 1.35 2 ro All +->o M 2 2 28 28 8 8 0 0 38.00 38 P 3.50 4 27.50 26 5.33 6 1.67 2 Q 2.50 2 29.50 30 4.50 4 1.50 2 177 The Alasmidonta-like mean karyotype (16 spreads) differs from the overall mean karyotype by four pairs of chromosomes in as many sections of the matrix [m 2-3 (-2), m 3-4 (+2), sm 2-3 (+2), and sm 3-4 (-2)]. The simplest chromosomal modification that could have produced these apparent differences is the translocation of a small arm segment from a chromosome in section m 2-3 to the small arm segment of a chromosome in section sm 3-4. After these modifications were incor porated into the karyotype, the altered chromosomes would plot in matrix sections sm 2-3 and m 3-4 (as observed). The Pleurobema-like group (six spreads) differs from the overall mean karyotype by ten pairs of chromosomes, indicating a minimum of five chromosome modifications. Similarly, the Quadrula-1ike group (two spreads) differs by ten chromosome pairs - five modifications; Gonidea angulata (three spreads) differs by eight pairs - four modifications; and Margaritifera margaritifera form falcata (one spread) differs by eight pairs - four modifications. These minimal numbers of chromosome modifications for the various suprageneric groups are listed in a left hand column of table 55. The remainder of table 55 is devoted to a more specific compari son of how the suprageneric groups differ from the overall mean karyo type. This comparison consisted of examining every section of the r by %TCL mean karyotype matrix (table 54) from the perspective of each possible pair of suprageneric groups. The entries in table 55 indicate the number of chromosome pairs by which both groups were simultaneously lower or higher than the overall mean values. 178 Table 55. Number of apparent chromosomal modifications in each suprageneric group and the number of shared differences for each pair of groups from the overall mean karyotype. Number of Shared Differences Suprageneric Group Apparent Apparent Number of Number Modifications Number of Spreads Number Gonidea angulata Gonidea Pleurobema-like Lampsilis-like Alasmidonta-like Margaritifera m. form falcata i Alasmidonta-like 16 2 Gonidea angulata 3 4 2 - Lampsilis-like 51 1 0 1 - Margaritifera m. form falcata 1 4 1 2 0 - Pleurobema-like 6 5 1 2 1 4 - Quadrula-1ike 2 5 1 2 1 4 8 179 For example, table 55 includes a "2" for the comparison of Pleurobema-like and Gonidea angulata. In table 54, although both groups vary considerably from the overall mean karyotype, the only matrix sections in which Pleurobema-like and CL angulata both have less than or more than the overall mean value are m 0-2 (-2 for each) and sm 0-2 (+2 for Pleurobema-like but +4 for (3. angulata). Had the Pleurobema- like value in sm 0-2 also been two chromosome pairs (four chromosomes) above the overall mean, the comparison value for these groups would have been "3" (one for section m 0-2 and two for section sm 0-2). The value of table 55 is that it associates groups which differ in apparently similar ways from the overall mean karyotype. Nothing in this exercise establishes that any groups share a specific chromosomal modification; it only indicates that chromosome numbers in specific sections of the matrix vary from the overall mean in the same positive or negative direction. Figure 49 is a graphic representation of the apparent relation ships of these suprageneric groups, based primarily upon the data in table 55. The concentric circles in this figure represent increasing numbers of chromosomal modifications from the overall mean karyotype in the center. The lines radiating out to the suprageneric groups represent apparent shared differences from the overall mean karyotype, then distinctions between the group means. The apparent relationships illustrated in this figure were deter mined by noting the specific matrix sections in which the suprageneric groups differed from the overall mean karyotype but were similar to each other. Groups which were similar only in one section of the matrix were 180 Figure 49. Relationships of six suprageneric naiad groups based upon an analysis of chromosome morphology. Data used to prepare this figure are contained in tables 54 and 55; the detailed interpretation is included in the text. Abbreviations are explained in the heading of table 54. Underlined abbreviations indicate groups represented by 10 or more analyzed spreads. 181 considered to be chance phenomena that were not the result of a shared chromosomal modification. The section-by-section values for groups with two or more shared differences were studied to identify the chromosomal modifications common to these and, perhaps, other groups. This procedure may be clarified by working through some examples. The Alasmidonta-like group shares one difference from the overall mean karyotype with M. m. form falcata, Pleurobema-like, and Quadrula-1ike (-2 in sm 3-4 -- indicated in table 55); however, there is no matrix section in which Alasmidonta-like shares the reciprocal difference (+2) with any of these groups. This lack of agreement in a pair of matrix sections suggests that the Alasmidonta-like group diverges from the overall mean karyotype in a different direction from the other groups. In a rather more complex example, G. angulata was found to share two differences from the overall mean karyotype with four of the other suprageneric groups (all except Lampsilis-1ike). The matrix sections in which Alasmidonta-like and (3. angulata shared differences from the overall mean [m 2-3 (-2) and sm 2-3 (+2)] were not the same sections in which G. angulata shared differences with the other suprageneric groups [m 0-2 (-2) and sm 0-2 (+2)]. While G. angulata might be more similar to Alasmidonta-like than the others, the most parsimonous arrangement is to associate angulata with the three suprageneric groups exhibiting what appears to be the same chromosomal modification ( a pericentric inversion in m 0-2 which now plots in sm 0- 2). Figure 49 illustrates the distinctions between the Alasmidonta- like, Lampsilis-like, and composite of other suprageneric groups by showing them radiating in different directions from the overall mean 182 (the center). The angles between groups or the right or left place ment of a group in this and succeeding similar figures is strictly arbitrary. The only controlled aspects of these figures are the numbers of chromosomal modifications by which each taxon differs from the mean and the shared differences of taxa joined together for some number of modifications away from the mean. The information in tables 54 and 55 (illustrated in figure 49) indicates that G. angulata shares only one chromosomal modification with M. m. form falcata, Pleurobema-1ike, and Quadrula-1ike (a pericentric inversion in m 0-2 which now plots in sm 0-2). The three subsequent modifications in G^. angulata appear to have consisted of a pericentric inversion in m 3-4 (which now plots in sm 3-4) and a small translocation between two chromosomes in m 2-3 which resulted in additions to both sm 0-2 and sm 2-3. Margaritifera m. form falcata also diverges from the others after sharing in the pericentric inversion in m 0-2. Subsequently, this species appears to have incorporated two pericentric inversions in sm 3-4 (both now plotting in st 3-4) and what may have been the dupli cation of a chromosome segment in m 0-2 (which plots in m 2-3). The Pleurobema-like and Quadrula-1ike groups appear to have shared two more modifications: a pericentric inversion in st 2-3 (now plots in m 2-3) and a duplication in sm 3-4 (plots in m >4). After that, the Pleurobema-1ike mean appears to have incorporated two additional pericentric inversions (one in sm 2-3 and the other in st 2-3, both now plotting in m 2-3). The Quadrula-1 ike mean apparently was modified in a 183 different way by incorporating a translocation in which a chromosome in sm 3-4 donated material to one in m 0-2 and both now plot in m 2-3. Alasmidonta-like Analysis The techniques used in the previous subsection to compare suprageneric groups also can be applied to genus or species level taxa. These analyses should be viewed with caution, however, because many of the taxa are represented by few analyzed spreads. Table 56 presents the section-by-section mean chromosome numbers and rounded karyotypes for the four species included in this study that are typically placed in Alasmidonta-1ike genera (refer to table 43). Anodonta grandis and Lasmigona costata are represented by six and five analyzed spreads, respectively, while Alasmidonta marqinata and Anodontoides ferussacianus are represented by three and two spreads, respectively. The rounded karyotype for A. marqinata is identical to the mean Alasmidonta-1ike karyotype but karyotypes for the other species vary from the mean. Table 57 presents the number of apparent chromosomal modifications from the Alasmidonta-like mean (one half the number of chromosome pairs by which each species differs from the mean) and the number of chromo some pairs by which each pair of species differs in the same way from the mean. This table indicates that the three species appear to differ from the mean (and A. marqinata) by two or three modifications and that A. ferussacianus shares one differnece with A. grandis (probably a random phenomenon) and two differences with L_. costa ta (perhaps, sugges ting a shared chromosomal modification). 184 Table 56. Sectional mean values and rounded karyotype numbers for four Alasmidonta-1ike species: Alasmidonta marqinata (Am-3 spreads; identical to the Alasmidonta-like mean), Anodonta grandis (Ag -6 spreads), Anodontoides ferussacianus (Af-2 spreads), and Lasmiqona costata (Lc- 5 spreads). % TCL 0 - 2 2 - 3 3 - 4 > 4 Totals Species Am 3.67 4 10.33 10 5.67 6 0.33 0 20.00 20 Ag 2.17 2 13.17 12 5.83 6 1.17 2 22.33 22 m Af 2.50 2 8.00 8 4.50 4 0.50 0 15.50 14 Lc 3.60 4 7.00 6 5.40 6 0.60 0 16.60 16 Am 1.00 0 11.33 12 2.33 2 0.00 0 14.67 14 Ag 0.83 0 8.83 10 1.83 2 0.17 0 11.67 12 sm Af 1.50 2 13.50 14 2.00 2 0.00 0 17.00 18 Lc 1.00 2 11.80 12 2.80 2 0.00 0 15.60 16 Am 0.00 0 3.00 4 0.33 0 0.00 0 3.33 4 Aq 0.50 0 3.50 4 0.00 0 0.00 0 4.00 4 st Af 0.00 0 4.50 4 1.00 2 0.00 0 5.50 6 Lc 0.00 0 5.60 6 0.20 0 0.00 0 5.80 6 Am 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 Ag 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 z Af 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 Lc 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 Am 4.67 4 24.67 26 8.33 8 0.33 0 25.50 26 7.67 8 1.33 2 Ag 3.50 2 All ro Af 4-> 26.00 26 7.50 8 0.50 0 O 4.00 4 38.00 38 1— Lc 4.60 6 24.40 24 8.40 8 0.60 0 185 Table 57. Number of apparent chromosomal modifications for each species and the number of shared differences for each pair of species from the Alasmidonta-like mean karyotype. Number of Shared Differences Species Number of Number Apparent Apparent Modifications ferussacianus Number of Spreads Number Anodonta grandis marginata Anodontoides Alasmidonta 1 Alasmidonta marginata 3 0 - Anodonta grandis 6 2 0 - Anodontoides ferussacianus 2 3 0 1 - Lasmigona costata 5 2 0 0 2 Af A m Figure 50. Relationships of four Alasmidonta-like species based upon an analysis of chromosome morphology. Data used to prepare this figure are contained in tables 56 and 57; the interpretation is included in the text. Abbreviations are explained in the heading of table 56. 186 A careful evaluation of table 56 indicates that it is unlikely for A. ferussacianus and IL. costata to share this suggested chromosomal modification. The transfer of a chromosome pair from the matrix section where these species are both less than the mean [m 2-3 (-2 or -4)] to the one in which they are both larger than the mean [st 0-2 (+2 each)] would involve a chromosomal deletion. This appears to be the case for L. costata because m 2-3 is the only matrix section in which this species is lower than the mean (-4) and the other modification indi cated is an apparent pericentric inversion (m 2-3 to plot in st 2-3). For A. ferussacianus, however, three matrix sections include fewer than the mean number of chromosomes (as well as three sections with more), offering a variety of possible modification scenarios. If the deletion in m 2-3 occurred in A. ferussacianus•(the resulting chromosome plotting in sm 0-2), the apparent karyotype would virtually require an off-setting duplication in m 0-2 (which would plot in sm 2-3). A much more parsimonous alternative for this species would be a pair of pericentric inversions in m 0-2 and m 2-3, plotting in sm 0-2 and sm 2-3 respectively. In addition, A. ferussacianus apparently has undergone a third pericentric inversion, this one in m 3-4 (plotting in st 3-4). To complete the apparent lis t of modifications from the Alasmidonta-1 ike mean, A_. grandis appears to have incorporated a peri centric inversion in sm 2-3 (now plotting in m 2-3) and a major dupli cation in m 0-2 (now plotting in m >4). The relationships of these species indicated by this analysis are illustrated in figure 50. 187 Statistical verification of the suggested differences in the section and row or column totals among these species was attempted using the Wilcoxon Rank Sum Test (Hollander and Wolfe, 1973). This nonparametric procedure was used to test for statistical differences between pairs of species by comparing the relative ranks of chromosome counts from each analyzed spread in each section of the r by %TCL matrix. At the 90 percent confidence level, this procedure recognized some differences between better-represented species (i.e.: between A. grandis and L_. costata in m 0-2, m 2-3, sm 2-3, and st 2-3), but did not substantiate the other differences between these species included in table 56. When species with few analyzed spreads were included, a few differences were recognized in Wilcoxon tests (i.e.: between A. marqinata and A. ferussacianus or I. costata in m 2-3) but fairly often wide ranging values in the poorly represented species effectively masked any substantiation of differences (i.e.: A. marginata totals for 2-3 %TCL - see table 3). Statistical verification of the differences suggested in table 56 (and elsewhere in this discussion) apparently will require larger numbers of analyzed spreads per species and/or a lower level of measurement error. Lampsilis-like Analysis Table 52 indicates that 51 analyzed spreads included in this data set are from species typically placed in Lampsilis-like genera (the subfamily Lampsilinae in many classifications). Ten of these spreads are single representatives of species, providing too little species- level information to be relied upon; however, four species often consid ered members of the genus Lainpsilis (L_. austral i s , J_. fasciola, 188 U subangulata and j.. ventricosa) are included in this group and these data have been combined to represent the genus in the analysis. Table 58 presents the mean chromosome numbers and the rounded karyotypes for 11 species and the genus Lampsilis. Four or more analyzed spreads contributed to the mean values for seven of these taxa while the other four species are each represented by two or three spreads. Data from four spreads of Ligumia nasuta produced a rounded karyotype identical to the rounded mean karyotype based upon all 51 Lampsilis-like spreads. The other taxa varied from this mean in two or more sections of the matrix. Table 59 includes the apparent numbers of chromosomal modifica tions for each taxon and the number of times each pair of taxa differed in the same direction from the Lampsilis-like mean. One of the possible interpretations of the relationships between these karyotypes is presented in figure 51. The seven taxa listed in table 59 as having one or two chromo somal' modifications are relatively easy to sort; however, the large number of chromosomal modifications that appear to have occurred in some species complicate the full interpretation of these data. Three species (Obovaria subrotunda, Villosa i_. iris and V. trabalis) apparently share a deletion in matrix section m >4 (to plot in m 3-4). -Obovaria subrotunda and \/. i_. iris then diverge from \/. trabal is and each other by different second chromosomal modifications: a pericentric inversion for 0. subrotunda in sm 2-3 (which now plots in st 2-3) and a deletion for V.- L- iris in sm 3-4 (which now plots in m 2-3). The four other one- or two-modification taxa each differ from the Lampsilis-like mean in 189 Table 58. Sectional mean values and rounded karyotype numbers for 11 Lampsilis-like species and a composite for the genus Lampsilis. The order of species, species abbreviations, and the number of analyzed spreads per species are as follows: Actinonaias 1igamentina form carinata (Ac-3) Leptodea fraqilis (Lf-2) Ligumia nasuta (Ln-4; identical to the Lampsilis-like mean) Ligumia recta (Lr-2) Obovaria subrotunda (Os-6) Potamilus alatus~(Pa-2) Ptychobranchus fasciolaris (Pf-4) Toxolasma parva (Tp-2) Villosa iris iris (Vi-4) Villosa taeniata punctata (Vp-7) Villosa trabalis (Vt-5) Lampsi1 is composite (Lc-4) al 5 (continued) 58Table :< Total s •e»U>C?ktOtO.£k-£k'C*CO*£k-£ka\ OOOOOOOO—'OOO oooooooooooo o o o o - J o o o ro ro u > ro 4s» ro ro ro c o4s»ro-C5»roco roroNNUiotntouiNoo oooooooooooo oo-virooooocnooco rOCJlCONOPOOWOMOO NOiSjsJtnsJuiOOOOCn cnotocnooocoocnoo oooooooooooo o o —*cnooooooou> tnooiuiomouooioo CD o —'UlOUlOOOOO'-J o o o o o o o o o|o o o o o o o o o o o o|o o o oooororoooojororo -e»-e»cn-tiropo.£»-e»-e»J-&r'o.£» rorororororoporororororo ■*»j ■fc»CDO>tnO03C0N|sjQC0tn oooooooooooo o o o o o o o o - nococjNrow-t»wcorow-£> ro-fc»rocj-vj-c*co-P*corooj- SIOMWOOOCOUIOOW oooooooooooo OQ-bwcnsjm uio^uiO sj ^£>0 ^ 1 roootn sjoio oo*NOOuiwocno(Ji cnotooooocoooo’-o oooooooooooo oococnocnooocnoo cnocooocnooocnoo OOC0CnOOOC0OO©'«J -c»cocr>cTiococococojcr»cocjv o o o o o o o o o|o o o o o o o o o o o rojo o o ro-&ro-c»co-Ci-fi-CiroJpo-c»ro ro— < o o —-o —■—<—<- oooooooooooo OOOOOOOOOOOO oooooooooooo ropooopocn-ocnCoocncncn oooooooooooo rooorocnrooooooo or\> 0ooocncncoocn.cn< aiooiuiomouooo^ oooooooooooo cnoocnocnoooooo OOOOOOOOJOOO- rooroooorooojrororo OOOOOOOO o|o o o o o o o o o o o o|o o o o o o o o o o o o[o o o NONOOONOO|rororo _ , _,_ , _ , _ , _ , _, r o ro ro ro ro ro ro _, ro OOOOOOOO—'OOO co -c» cn -e» -1 to ro cn co cn co CO rorococnro —* CO -o CO O CO —1cncno o roto o OOOOOOOOOOOO cn o —* O O O cn —1 cn o o O cno -fc» ro o cncnco cncncnO o o -fc. -«sj o cno cno cncno OOOOOOOOOOOO 0 0 - P » 0 0 0 0-n4 0 0 0 0 o o CO cno O o co O O O o o o CO cno O o o o O o O rororororo—»rororo|ro—*ro o o o o o o o o o|o o o o^^roroooiro roj-c. co ■ftOOroaiO'OO l\3|0 05 o Number of Shared Differences Species (O Apparent Apparent -> Number of Number S- Modifications ro Q. Number of Spreads Number L. L. fragilis L. recta L. L. nasuta 0. subrotunda P. alatus P. fasciolaris V. V. t. punctata A. A. 1. carinata V. i . s ri i V. trabal is Actinonaias liga- mentina carinata 3 1 - Leptodea fragilis 2 3 0 - Ligumia nasuta 4 0 0 0 - Ligumia recta 2 3 1 0 0 - Obovaria subrotunda 6 2 1 0 0 2 - Potamilus alatus 2 2 0 3 0 0 0 - Ptychobranchus fasciolaris 4 4 1 5 0 1 1 3 - Toxolasma parva 2 6 2 4 0 4 2 2 5 - Villosa iris iris 4 2 0 0 0 2 1 0 1 3 - Villosa taeniata punctata 7 1 0 1 0 0 0 1 1 0 0 - Villosa trabalis 5 1 0 1 0 1 2 0 1 1 2 0 - Lampsilis composite 4 2 1 0 0 3 1 0 0 4 2 0 0 192 Pa Os Vt p Figure 51. Relationships of 11 Lampsilis-1ike species and a composite for the genus Lampsilis based upon an analysis of chromosome morphology. Data used to prepare this figure are contained in tables 58 and 59; the interpretation is included in the text. Abbreviations are explained in the heading of table 58. Underlined abbreviations indicate taxa represented by five or more analyzed spreads. 193 in individual ways [Actinonaias ligamentina form carinata - a deletion in sm 2-3 (plots in sm 0-2), Villosa taeniata punctata - a deletion in m 2-3 (plots in m 0-2), others are discussed below]. The four remaining species differ from the Lampsilis-like mean in several matrix sections, allowing for a number of sets of possible relationships. The two species with the largest number of apparent modifications (Toxolasma parva - 6 and Ptychobranchus fasciolaris - 4) share five differences from the mean; however, Ligumia recta (three modifications) shares four differences with T. parva but only one difference with £. fasciolaris. Comparison of the section-by-section rounded karyotypes among all of the Lampsilis-like taxa suggests that P. fasciolaris, Leptodea fragilis and Potamilus alatus share a duplication in m 2-3 (now plots in m 3-4) and a pericentric inversion in st 2-3 (now plots in sm 2-3) while T. parva appears to share a trans location with the Lampsilis mean (from the long arm of a chromosome in sm 3-4 to a short arm in sm 2-3 making both plot in m 2-3). Once the association with_P. alatus is established, £. fasciolaris and I. fragilis also appear to share a second pericentric inversion in m 0-2 (which plots in sm 0-2). The final modification for £. fasciolaris apparently is a deletion in m >4 which makes that pair plot in sm 2-3. These proposed relationships associate three of the four outlying species with less modified taxa; however, this arrangement forces the separation of T. parva, £. fasciolaris, and L_. recta in apparent contra diction to the numbers of differences from the mean that they share. The inclusion of additional analyzed spreads from any of these Lampsilis- like taxa would improve the evaluation, but more data on T. parva and 194 L_. recta (both represented by only two spreads in this analysis) could substantially revise the apparent relationships. Other Suprageneric Groups No further analysis is possible for the four other suprageneric groups. Gonidea angulata and Margaritifera margaritifera form falcata are single species represented, respectively, by three and one analyzed spreads. The Pleurobema-1ike group includes four species in three genera; however, three of them are represented by single analyzed spreads. The Quadrula-1ike group is represented by single spreads from two species. Additional material from any of these taxa would improve the reliability of the overall analysis and could substantially modify the apparent relationships of the karyotypes. Synthesis The preceeding paragraphs have achieved one of the stated purposes of this discussion: to determine if taxonomically useful data are available from naiad chromosomes. The information presented on chromo some number appears sufficient to support a major taxonomic distinction between the unionid and hyriid naiades. Morphologic characteristics of the chromosomes were found to vary among the taxa studied, indicating that appropriate karyotypic analyses could assist in evaluating taxo nomic relationships. In addition, a technique was introduced which appears capable of performing this taxonomic evaluation. 195 The detailed interpretations that are presented, almost certainly, are over-extensions of the available data. No species in this data set is represented by more than seven analyzed chromosome spreads and only two of the six suprageneric groups are represented by more than ten analyzed spreads. More-than-1ikely, the inclusion of adequate numbers of analyzed spreads would obviate the necessity to propose so many inversions, deletions, and duplications to explain observed differences between apparent karyotypes. Adequate sample sizes also probably would reduce the number of lines of difference from each group mean. The comparison of apparent chromosomal relationships to existing naiad classifications was the second stated purpose of this discussion; however, the analysis results have yet to be considered from that perspective. The following paragraphs address this aspect of the project. Table 53 presents the mean numbers of chromosomes from all 79 analyzed spreads. An observation not made when that table was introduced, is that nine of the 16 matrix sections include essentially all (99 percent) of the chromosomes. Matrix sections virtually or actually without chromosomes are sm>4, st 0-2, st>4 and all four t sections. Obviously, all of the karyotypes are quite similar to each other, which adds support to the concept that this is a single evolutionary group. Tables 54, 55, and figure 49, indicate the apparent relationships between the six previously proposed suprageneric groups included in the data set. The available information indicates that the Alasmidonta- and Lampsilis-1ike mean karyotypes are only slightly different from the overall mean and each other. The Pleurobema- and Quadrula-1ike means also are similar but at some distance from Alasmidonta- and Lampsilis- 196 1 ike. Gonidea angulata and Margaritifera m. form falcata appear to join the others at a common midpoint. Figure 52 presents the relationships indicated in figure 49 as more conventional branching diagrams. The single difference between the content of these two figures is that figure 49 illustrates relationships without regard to evolutionary sequences while figure 52a and b each use a specific karyotype as the suggested common ancestor from which the groups may have evolved. The suggested common ancestor for the arrangement of suprageneric groups in figure 52a is the overall mean karyotype. This arrangement is most similar to the classification proposed by Ortmann in 1910 (figure 53a). The separation of Gonidea from the Pleurobema- and Quadrula-1ike groups along the right branch in figure 52a conforms with likely refine ments Ortmann discussed in 1916. Ortmann did not establish taxonomic distinctions between the Pleurobema- and Quadrula-1ike groups within his subfamily Unioninae; however, his key to the genera of this subfamily (1912) sorts the North American fauna into these two groups before characterizing any individual genus. The single difference between figure 52a and Ortmann's classifica tion concerns the placement of Margaritifera. Ortmann considered Margaritifera to be in a separate family, more primitive than the other North American naiades. In figure 52a, this group is included with Ortmann's Unioninae, diverging when Gonidea branches off. The point where Margaritifera and Gonidea are joined to the union- ine branch is interesting because it represents the karyotype most similar to all of the group means. If this point is considered to be the common 197 Figure 52. Relationships of the suprageneric groups of North American naiades based solely upon analysis of chromosomal morphologic data.* Two alternatives are presented: a - assuming that the overall mean karyotype is the ancestral form, and b - assuming that the karyotype most similar to all of the suprageneric group means is ancestral. Letter codes for suprageneric groups are explained in the footnote to table 43 (page 133). * Circled numbers identify apparent chromosomal modifications, as follows: 1 - duplication in m 3-4, plots in m > 4; 2 - translocation m 2-3 to sm 3-4, plot in sm 2-3 and m 3-4; 3a - pericentric inversion in m 0-2, plots in sm 0-2; 3b - reverse of 3a; 4 - two inversions in sm 3-4, both plot in st 3-4, and duplication in m 0-2, plots in m 2-3; 5 - translocation within m 2-3, plot in sm 0-2 and sm 2-3, and inversion in m 3-4, plots in sm 3-4; 6 - inversion in st 2-3, plots in m 2-3, and duplication in sm 3-4, plots in m > 4; 7 - inversion in sm 2-3, plots in m 2-3, and inversion in st 2-3, plots in m 2-3; 8 - translocation from sm 3-4 to m 0-2, both plot in m 2-3. Figure 52 (continued) 198 Overall Mean I Karyotype Most Similar I Karyotype 199 M L yA1 U M Q3 N2 A2 P3 Q4 ^4 L a M A 2 ^J3 u L MW Ai Pi L G Q1 H l^y Yc M Ai G Q 2 P2 L Figure 53. Relationships of suprageneric groups of North American naiades (only) as proposed in five classifications: a - Ortmann, 1910; b - Modell, 1942; c - Morrison, 1955; d - Heard and Guckert, 1970; and e - Davis and Fuller, 1981. Letter and number codes for suprageneric groups are explained in the footnote to table 43 (page 133). 200 ancestor (figure 52b), the Lampsilis- and Alasmidonta-like groups branch off together (briefly) while Margaritifera and Gonidea each form separate branches, no longer associated with the Pleurobema- and Quadrula-like branch. This arrangement would agree with Ortmann's level of separation for Margaritifera but would suggest more distance between Gonidea and the Pleurobema- and Quadrula-like groups than Ortmann proposed. The associa tion of the Lampsilis- and Alasmidonta-1ike groups would differ from the distince status of these groups in the Ortmann classification. The North American components of other recent classifications examined in Discussion I also are diagramed in figure 53. [No diagram is present for Rafinesque (1820) because he did not indicate relationships among the groups he recognized.] Both arrangements of suprageneric groups based on chromosomal data (figure 52) differ from each of these classifi cations in various ways. All of these classifications include the early separation of Margaritifera from the other groups,'which agrees with the karyotypic arrangement using the most similar karyotype as the common ancestor (figure 52b). Classifications proposed by Modell (figure 53b) and Heard and Guckert (figure 53d) include major separations of the Pleurobema- and Quadrula-like groups that are different from the close relationship of these groups indicated by the karyotypic data and the classifications of Morrison (figure 53c) and Davis and Fuller (figure 53e). The chromosomal data in no way support the three major lineages of North American naiades included in Model!'s classification (figure 53b). In general, the arrangements of suprageneric groups based upon chromosomal data are most similar to the classification proposed by Ortmann (1910) followed, in decreasing order, by the proposals of Davis 201 and Fuller (1981), Morrison (1955), Heard and Guckert (1970), and Modell (1942). Previous authors have not attempted to describe evolutionary relationships among smaller groups of naiad genera or species. The pattern set by Simpson (1900) and Ortmann (1912) has been to associate groups of similar genera and species in apparent order of (decreasing or increasing) specialization. The examination of chromosomal relationships among Alasmidonta-like species (tables 56, 57, and figure 50) includes only four species, three of which appear to share more karyotypic features with Alasmidonta marginata than with each other. No previous author has suggested relationships among these species or genera. Tables 58, 59, and figure 51 present the species-level evaluation of the Lampsilis-1ike group. With only three species represented by five or more analyzed spreads, the relationships indicated in figure 51 may not be extremely accurate. However, the association of two of the three Villosa species and, separately, Leptodea fragilis with Potamilus alatus conforms to associations based upon shell and anatomical features (Ortmann, 1912; Burch, 1973; Stansbery, 1982). Other associations indicated in figure 51, especially the rather solid placement of Ptycho- branchus fasciolaris with Leptodea and Potamilus, are considerably different from expectations. The preceeding comparison of naiad relationships based solely upon chromosomal data with classifications based upon other sets of characteristics has illustrated that karyotypic information can make a valuable contribution to naiad systematics. The relationships of suprageneric groups indicated by chromosomal data alone are not substantially different from some previously-proposed classifications based on many other sets of characteristics. The differences indicated by karyotypic analysis may include evolutionary refinements which cannot be recognized in previously explored sets of characteristics. On the generic and specific level, this type of karyotypic analysis appears to be able to associate taxa and suggest evolutionary relationships where other approaches (used to date) indicate only general trends. SUMMARY AND CONCLUSIONS This project was conducted to determine if chromosomal characteristics can provide new information useful in naiad systematics. In the process of acquiring material to use for this determinations a field-compatable slide preparation technique was developed and perfected to the point that a few chromosome spreads could be expected from most animals processed. Using this technique, chromosome slides were prepared from 250 naiades representing 34 genera and 74 species. While scanning slides from 178 of these animals, information was collected on naiad mitotic activity. Exploratory analysis of this impromptu data set indicated that mitotic activity varied with temper ature, month of the year, and the length of time an animal was held in captivity before being processed. Different patterns also were found for various suprageneric groups of naiades. This analysis supported the distinctions between some previously-proposed suprageneric groups (i.e. Alasmidonta-1ike and Lampsilis-1ike) but suggested associations (i.e. Alasmidonta-1ike and Quadrula-1ike) or separations (within Lampsilis-1ike) that should receive further study. The chromosomal analysis included two major components: an assessment of chromosome numbers and an evaluation of chromosomal morphologic features. Sixty-six species of North American naiades included in this project were found to maintain a constant 38 diploid chromosome number which is identical to reported diploid numbers of three 203 204 European and three Asian unionid naiades. Three Australian hyriid naiades are reported to have 34 diploid chromosomes, suggesting that they might have come from a different evolutionary line. The evaluation of chromosome morphology dealt with the relation ships between two measurement-based parameters: arm ratio (r) and percent total complement length (%TCL). This evaluation used a new analysis technique based upon a mean karyotype concept to perform comparisons of six suprageneric groups and species-level taxa within two of the groups. The overall mean karyotype produced fromthisdata set includes 20 meta- centric chromosomes (four in the 3-4 %TCL range, 12 in 2-3 %TCL, and four in 0-2 %TCL), 14 submetacentrics (four in 3-4 %TCL, and 10 in 2-3 %TCL), and four subtelocentrics (all in 2-3 %TCL). The suprageneric groups differ from the overall mean by one (Lampsilis-1ike), two (Alasmidonta-1ike), four (Gonidea angulata and Marqaritifera m. form falcata) , or five (Pleurobema-like and Quadrula-like) apparent chromosomal modifications. Relationships indicated by the locations of shared differences from the overall mean karyotype associate the Pleurobema- and Quadrula-1ike groups on one branch, joined near its base by Gonidea angulata and Marqaritifera m. form falcata. The Lampsilis- and Alasmidonta-1ike groups form independent branches. This set of relationships is similar to the classification proposed by Ortmann (1910; 1921) and only modestly differ ent from classifications proposed by Davis and Fuller (1981) and Morrison (1955). An alternative set of relationships based upon the karyotype most similar to each of the suprageneric groups also is most similar to the 205 Ortmann classification, then Davis and Fuller, then Morrison. Both sets of chromosomal relationships are considerably less similar to classifica tions proposed by Heard and Guckert (1970) and Model 1 (1942). The value of this entire project is that it has begun the exploration of mitotic activity and chromosomal characteristics as they exist in the naiades. Both areas appear to include virtually independent information that could be used to augment or orient other sets of naiad characteristics useful to systematics. The apparent conservative nature of naiad karyotypes suggests that chromosomal data may be especially well suited for addressing relationships among suprageneric groups. Additional data may demonstrate similar value of karyotypic evaluation in addressing species and generic level groups. Apart from any taxonomic use, mitotic activity data may lead to new insights regarding naiad growth patterns, metabolic cycles and energy budgets. Future work on naiad chromosomes may utilize some of these procedures; however, incorporation of some modifications would avoid many of the problems encountered during this project. The major problem to be overcome is the extremely low number of mitotic figures on these slides. The use of a cell culture system to provide material with a considerably higher mitotic index would prevent the field preparation of slides but would greatly facilitate all succeeding steps of the procedure. Once large numbers of mitotic figures are present on the slides, experimentation should begin on the perfection of naiad chromosomal banding techniques. The ability to identify individual chromosome segments that should occur when banding techniques are adapted for use with naiades, is likely to substantially modify and refine the comparison of various karyotypes. With or without the addition of banding techniques, continued study of naiad chromosomes appears likely to help resolve many otherwise unanswerable questions about the relationships, zoogeography, and evolution of this ancient group of bivalves. APPENDIX Collection and Catalog Data This appendix contains collection site data for the 250 naiad specimens included in this project. The collections are arranged alpha betically by stream or lake name then in chronologic order. An attempt has been made to preserve the content of the original collectors' locale data where it was known, and then to augment those data as necessary to meet acceptable standards. This has led to some apparent inconsistency in the use of metric and English units, track and range data, etc. The only exception to this attempt to preserve original collec tors' locales occurred when more than one collection had been made at the same site. In that case, each collection was listed using identical locale information, accompanied by the appropriate date and lis t of collectors. Latitude and longitude coordinates have been calculated for each collection site (or were part of the original locale data). When these coordinates were calculated from 7.5' topographic maps they are expressed in degrees, minutes and seconds; when the coordinates were calculated using less-detailed maps, they are expressed in degrees and minutes only. Whenever latitude and longitude coordinates were calculated by this author they are intended to locate the bridge, ford, or other landmark at the collection site. This landmark was also the feature to which distances were measured from nearby towns. 207 Following each set of collection data is a lis t of the naiad specimens that were processed from that v isit to the site. These specimens are listed by "JJJ number" and scientific name. If the specimen already has been cataloged into the Bivalve Collection, Ohio State University Museum of Zoology (OSUM), that number follows the scientific name. The specimens without catalog numbers given are in the process of being added to the collection. Allegheny River (Ohio River system) OSUM:1977:177 East side of Allegheny River for approx. 1 km below Pa. Rt. 127 bridge, opposite West Hickory, 30.5 km NE of Oil City, Lat 41°34'18"N, Long 79° 24'17"W, Forest Co., Pennsylvania 12 September 1977 Carolyn S. and John J. Jenkinson JJJ:251 Fusconaia flava (OSUM 40555) 252 Lampsilis fasciola (OSUM 40561) 253 Strophitus undulatus (OSUM 40551.1) 254 Epioblasma torulosa rangiana (OSUM 40562.6) 256 Epioblasma torulosa rangiana (OSUM 40562.5) Alum Creek (Ohio River system) Alum Creek at Co. Rt. 34 bridge, 4.2 km NNE of Kilbourne, 14.0 km NE of Delaware, [Lat 40°2r23"N, Long 82°55'18"W] Delaware Co., Ohio 12 April 1977 William H. LeGrande and Ichthyology Class JJJ:215 Anodontoides ferussacianus 216 Lampsilis radiata luteola Big Darby Creek (Ohio River system) Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39045'11"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 11 Augustl975 John J. Jenkinson JJJ:1 Alasmidonta marginata 2 Lampsil is radiata luteola 3 Lampsilis radiata luteola 4 Villosa iris 209 JJJ:5 Lampsilis radiata luteola 6 Quadrula quadrula 7 Lampsilis radiata luteola 11 Tri togonia verrucosa 21 Ptychobranchus fasciolaris 22 Ptychobranchus fasciolaris 23 Lasmigona costata Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 kn S of Orient, Lat 39°45 '11"N, Long 83o08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 15 June 1976 John J. Jenkinson JJJ:67 Lasmigona complanata 69 Strophitus undulatus 70 Lampsilis ventricosa 71 Lampsi1 is radiata luteola 72 Ptychobranchus fasciolaris 73 Quadrula quadrula 74 Elliptio dilatatus 76 Epioblasma torulosa rangiana 77 Amblema piicata Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39°45111"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 29 June 1976 John J. Jenkinson and Frank L. Kokai JJJ:80 Strophitus undulatus Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39°45111"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 22 August 1976 Carolyn S. and John J. Jenkinson JJJ:98 Alasmidonta marqinata 99 Lampsilis radiata luteola 100 Alasmidonta marqinata 101 Ptychobranchus fasciolaris 106 Anodonta grandis 107 Elliptio dilatatus 108 Lampsilis ventricosa 119 Lasmigona costata 120 Amblema piicata 121 Ptychobranchus fasciolaris Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39045'n"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 3 October 1976 John J. Jenkinson JJJ: 171 Quadrula quadrula 172 Alasmidonta marqinata 176 Lampsilis radiata luteola 179 Strophitus undulatus 180 Quadrula quadrula 210 JJJ:183 Anodonta grandis 184 Elliptio dilatatus 185 Lampsilis fasciola 190 Cyclonaias tuberculata Big Darby Creek below Commercial Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39°45'n"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio 10 February 1977 John J. Jenkinson JJJ:210 Lasmigona costata Big Darby Creek below Commerical Point Rd. bridge, 5.2 km ESE of Derby, 5.8 km S of Orient, Lat 39°45'11"N, Long 83°08'23"W, Scioto/Darby Twps., Pickaway Co., Ohio mid December 1976 John J. Jenkinson JJJ:214 Epioblasma torulosa rangiana Black River (Mississippi River system) Black River above Rt. 67 bridge, 1 km NW of Hendrickson, 18.1 km NNW of Poplar Bluff, Lat 36°53'N, Long 90°29'W, Butler Co., Missouri 13 November 1976 John J. Jenkinson JJJ:194 Pleurobema coccineum 205 Amblema piicata 206 Actinonaias 1igamentina carinata 207 Potamilus alatus 211 Potamilus alatus 219 Fusconaia flava Buck Creek (Cumberland River system) OSUM:1975:137 Buck Creek at Ky. Rt. 1677 bridge, 1.2 mi [1.9 km] W of Dahl, 10.5 mi. [16.9 km] NE of Somerset, [Lat 37°10'48"N, Long 84°27'21"W] Pulaski Co., Kentucky 31 October 1975 David H. Stansbery JJJ:12 Medionidus conradicus 13 Lampsilis ventricosa 15 Potamilus alatus 16 Villosa trabalis 17 Potamilus alatus 18 Villosa iris nebulosa 19 Villosa trabalis 20 Lampsilis ventricosa OSUM:1975:134 Buck Creek at Ky. Rt. 70 bridge, 1.5 mi [2.4 km] SW of Clarence, 13.1 mi [21.1 km] N of Somerset, [Lat 37°16'33"N, Long 84°3r57"W] Pulaski Co., Kentucky 30 October 1975 David H. Stansbery JJJ:27 Villosa taeniata punctata 211 JJJ:28 Toxolasma lividus plans 29 Vi 1losa trabalis 39 Villosa taeniata punctata 40 Vi 1losa trabalis 41 Villosa trabalis 42 Lampsilis ventricosa OSUM:1975:138 Buck Creek at Ky. Rt. 1003 bridge, 0.4 mi [0.6 km] NW of Ula, 9.5 mi [15.3 km] E of Somerset, [Lat 37°06'11"N, Long 84°26,08"W] Pulaski Co., Kentucky 31 October 1975 David H. Stansbery JJJ:30 Ptychobranchus fasciolaris 31 Obovaria subrotunda 32 Actinonaias pectorosa 46 Lasmigona costata 47 Lasmigona costata 48 Lampsilis fasciola 49 Villosa trabalis (Big) Buffalo Creek (Ohio River system) Buffalo Creek for approx. 1.5 km upstream from its mouth, 3.4 km NNW of Waterloo, Lat 38°43'46"N, Long 82°29'39"W, Symmes Twp,, Lawrence Co., Ohio 4 October 1975 John J. Jenkinson JJJ:8 Villosa lienosa 9 Anodonta grandis 10 Villosa 1ienosa 14 Anodontoides ferussacianus 34 Anodontoides ferussacianus 35 Lampsilis ventricosa 36 Anodontoides ferussacianus Burnt Corn Creek (Escambia River system) Burnt Corn Creek below d irt road bridge, 14.2 km WSW of Castleberry, 22.0 km NW of Brewton, Lat 31016'N, Long 87°10'W, Conecuh Co., Alabama 11 September 1976 John J. Jenkinson JJJ:156 "Lampsilis" austral is 157 Villosa sp. 158 Villosa vibex 159 Villosa vibex 160 Fusconaia succissa 161 Pleurobema sp. 212 Cahaba River (Mobile River system) Cahaba River above US Rt. 78 bridge, 6.3 km W of Leeds, 18.5 km ENE of Birmingham, Lat 32°30'16"N, Long 86°36'46"W, Jefferson Co., Alabama 13 September 1976 John J. Jenkinson JJJ:163 Amblema piicata perplicata 164 El 1iptio sp. 174 Amblema piicata perplicata 177 El 1iptio sp. Cane Creek (Escambia River system) Cane Creek above unnamed road bridge, NW of 1-65, 5.0 km NW of Evergeen, 68.2 km NE of Atmore, Lat 31°28'N, Long 86°59'W, Conecuh Co., Alabama 7 September 1976 Carolyn S. and John J. Jenkinson JJJ:149 Villosa vibex 150 Toxolasma paula 151 Elliptio sp. 152 Strophitus sp. 154 Toxolasma paula 155 Uniomerus declivus Clinch River (Tennessee River system) Clinch River above US Rt. 25E bridge, 5.6 km NW of Thorn Hill, 11.6 km SE of Tazewell, Lat 36°24'08"N, Long 83°27'04"W, Grainger/Claiborne Cos., Tennessee 1 September 1976 Carolyn S. and John J. Jenkinson JJJ: 124 Actinonaias ligamentina ligamentina 125 Ptychobranchus subtenum 126 Actinonaias ligamentina ligamentina 127 Lampsilis ovata 128 Fusconaia cuneolus 129 Lasmigona costata 130 Actinonaias pectorosa OSUM:1978:128 Clinch River at Va. Rt. 16A bridge, at River Jack, 2.0 mi [3.2 km] NW of Tazewell, [Lat 37°07,45"N, Long 81°33'01"W] Tazewell Co., Virginia 21 May 1978 David H. Stansbery JJJ:262 Fusconaia barnesiana 263 Fusconaia barnesiana 264 Villosa sp. 213 Copper Creek (Tennessee River system) Copper Creek for approx. 1 mi., 2.8 km E of Spivey Mi'll, 5.4 km NW of Gate City, [Lat 36°39'31"N, Long 82°37'30"W] Scott Co., Virginia 19 March 1977 William H. LeGrande, Larry T. McGeehan, Miles Coburn and Ted M. Cavender JJJ:217 Medionidus conradicus Devils Lake OSUM:1976:14 Devils Lake about 1 mi [1.6 km] E of Lincoln City (=Oceanlake), 1.5 mi [2.4 km] NNE of the mouth of Rock Creek, [Lat 44°59'N, Long 123°59'W] Lincoln Co., Oregon late May 1976 [prior to 25 May] Roger D. Meyerhoff JJJ:54 Anodonta oregonensis Duck River (Tennessee River system) OSUM:1976:126 Duck River at Li Hard Mill, at Mi 11 town, 14 mi [22.5 km] E of Columbia, 9 mi [14.5 km] N of Lewisburg, [Lat 35°35'24"N, Long 86°47'04"W] Marshall Co., Tennessee 14 August 1976 David H. Stansbery and Constance M. Boone JJJ:94 Lemiox rimosus (OSUM 38369.1) 95 Lexingtonia dolabelloides (OSUM 38361.63) 96 Pleurobema oviforme (OSUM 38376.1) 102 Lemiox rimosus (OSUM 38369.2) 103 Fusconaia barnesiana (OSUM 38359.8) 110 Lexingtonia dolabelloides (OSUM 38361.64) 111 Lexingtonia dolabelloides (OSUM 38361.65) 112 Pleurobema oviforme (OSUM 38376.3) 113 Fusconaia barnesiana (OSUM 38359.10) 114 Pleurobema rubruni (OSUM 38359.9) Duck River below Hardison Mill, below US Rt. 431 bridge, 18.2 km NNW of Lewisburg, 19.5 km E of Columbia, Lat 35°36'33"N, Long 86°49'26,,W, Maury/Marshall Cos., Tennessee 13 September 1976 Carolyn S. and John J. Jenkinson JJJ:166 Pleurobema oviforme 167 Lexingtonia dolabelloides 168 Lexingtonia dolabelloides 169 Cyclonaias tuberculata 170 C.yclonaias tuberculata 186 Amblema piicata 187 Cyclonaias tuberculata 214 Florida (St. Johns River system?) [peninsular Florida] purchased from Carolina Biological Supply Co., February 1977 JJJ:259 Elliptio buckleyi 261 Elliptio buckleyi French Creek (Ohio River system) OSUM:1977:176 French Creek just below and for approx. 1 km above US Rts. 6 and 19 bridge, 1.0 km NE of Venango, 15.6 km NNE of Meadville, Lat 41°46I21"N, Long 80°06'41"W, Crawford Co., Pennsylvania 9 September 1977 Carolyn S. and John J. Jenkinson JJJ:243 Villosa fabalis (OSUM 40543.1) 244 Lasmigona costata (OSUM 40534) 245 Lasmigona compressa (OSUM 40535.1) 246 Alasmidonta marginata (OSUM 40533) 247 Lampsilis ventricosa (OSUM 40545) 248 Elliptio dilatatus (OSUM 40539) 249 Actinonaias ligamentina carinata (OSUM 40541) 250 Ligumia recta (OSUM 40542] Gasconade River (Missouri River system) Gasconade River just below the mouth of Third Creek, 0.9 km NW of Cooper Hill, 28.9 km E of Westphalia, Lat 38°26'N, Long 91°40'W, Osage Co., Missouri 12 November 1976 Carolyn S. and John J. Jenkinson JJJ:197 Lampsilis ventricosa Gasper River (Ohio River system) OSUM:1977:101 Gasper River at Thompson Bridge (Ky. Rt. 626 bridge), 4.1 km S of Hadley, 15.0 km W of Bowling Green, [Lat 37°01'18"N, Long 86°36'20"W] Warren Co., Kentucky 17 August 1977 David H. Stansbery, John J. Jenkinson and Frank L. Kokai JJJ:232 Elliptio dilatatus (OSUM 40603) 233 Elliptio dilatatus (OSUM 40603) 234 Elliptio dilatatus (OSUM 40603) 215 Green River (Ohio River system) Green River above US Rt. 31W bridge, at Munfordville, 10.1 km N of Horse Cave, Lat 37°16'05"N, Long 85°52'41"W, Hart Co., Kentucky 20 March 1976 Carolyn S. and John J. Jenkinson JJJ:51 Actinonaias ligamentina carinata 52 Actinonaias 1iqamentina carinata 53 Actinonaias ligamentina carinata OSUM:1976:127 Green River at Rt. 185 bridge, 1 mi [1.6 km] SW of Glenmore, 11.0 mi [17.7 km] N of Bowling Green, [Lat 37°09'16"N, Long 86°24'30"W] Warren Co., Kentucky 16 August 1976 bought from commercial clammer by David H. Stansbery JJJ:90 Plagiola lineolata (OSUM 39185.9.) 91 Obovaria subrotunda (OSUM 39186.4) 92 Pleurobema rubrum (OSUM 39180.7) 93 Fusconaia flava (OSUM 39175.5) 97 Plagiola lineolata (OSUM 39185.7) 104 Elliptio crassidens (OSUM 39181.2) 105 Pleurobema coccineum (OSUM 39177.1) 115 Pleurobema rubrum (OSUM 39180.3) 116 Pleurobema plenum (OSUM 39178.5) 117 Plagiola lineolata (OSUM 39185.8) - 118 Obliquaria reflexa (OSUM 39183.7) OSUM:1977:182 Green River above Rt. 185 bridge, 1 mi [1.6 km] SW of Glenmore, 11.0 mi [17.7 km] N of Bowling Green, [Lat 37°09'16"N, Long 86°24'30"W] Warren Co., Kentukcy 2 November 1977 David H. Stansbery, Kathy G. Borror and William N. Kasson JJJ:260 Arcidens confragosus Lake Erie (St. Lawrence River system) OSUM:1975:328 Fishery Bay [of Lake Erie] near Terwillegar1s Pond, just S of Peach Point, [Lat 41°38'36"N, Long 82°49'35"W] Put-in-Bay Twp., Ottawa Co., Ohio 10 November 1975 Lynwood MacLean and Matthew Conklin JJJ:24 Anodonta imbecillis 25 Toxolasma parva 26 Truncilla truncata 33 Toxolasma parva 37 Toxolasma parva 38 Toxolasma parva 43 Toxolasma parva 44 Toxolasma parva 45 Toxolasma parva 50 Toxolasma parva OSUM:1976:71 (?) Fishery Bay [of Lake Erie, Lat 41°38'N, Long 82°49'W, Put-in-Bay Twp.,] Ottawa Co., Ohio 28 June 1976 John E. Zapotosky and Invertebrate Zoology Class JJJ:79 Fusconaia flava 81 Potamilus alatus OSUM:1976:72 Lake Erie beach at Locust Point, at Davis-Besse Nuclear Power Station, 9 mi [14.5 km] NW of Port Clinton, 22 mi [35.4 km] ESE of Toledo, Lat 41° 37'N, Long 83°04'W, Sec. 1, Carroll Twp., Ottawa Co., Ohio 12 August 1976 Charles A. Bowen JJJ:85 Quadrula pustulosa 86 Ligumia recta 87 Ligumia nasuta 88 Obiiquaria reflexa OSUM:1976:114 Lake Erie beach at Locust Point, at Davis-Besse Nuclear Power Station, 9 mi [14.5 km] NW of Port Clinton, 22 mi [35.4 km] ESE of Toledo, Lat 41° 37'N, Long 83°04'W, Sec. 1, Carroll Twp., Ottawa Co., Ohio 19 August 1976 Carol B. Stein and associates JJJ:109 Obiiquaria reflexa 122 Obiiquaria reflexa 123 Obiiquaria reflexa Lake Erie beach, 1.0 km NW of Port Clinton, Lat 41°31'05"N, Long 82°56' 48"W, Ottawa Co., Ohio 30 August 1977 John J. Jenkinson JJJ:236 Leptodea fragilis 237 Leptodea fragilis 238 Ligumia nasuta 239 Amblema piicata piicata 240 Amblema piicata piicata 241 Quadrula pustulosa 242 Fusconaia flava 257 Leptodea fragilis Lake Manitoba (St. Lawrence River sytem) Lake Manitoba beach at Delta Station, 22 km N of Portage la Prairie, 92 km NW of Winnipeg, Lat 50°1VN, Long 98°19'W, Manitoba Province, Canada ca. 24 September 1976 Charles E. Herdendorf JJJ:173 Anodonta grandis 217 L ittle Barren River (Ohio River system) Little Barren River at Ky. Rt. 88 bridge, 16.6 km WSW of Greensburg, 20.1 km ESE of Munfordville, Lat 37°13'34"N, Long 85°40'06"W, Green Co., Kentucky 20 March 1976 Carolyn S. and John J. Jenkinson JJJ:64 Lampsilis radiata luteola 65 Fusconaia flava 66 Tritogonia verrucosa 82 Tritogonia verrucosa Little Darby Creek (Ohio River system) Little Darby Creek at West Jefferson Island, just above US Rt. 40 bridge, in West Jefferson, 17.7 km ENE of London, Lat 39°56'46"N, Long 83°15' 42"W, Jefferson Twp., Madison Co., Ohio 15 May 1976 John J. Jenkinson JJJ:84 Ptychobranchus fasciolaris 89 Ptychobranchus fasciolaris 191 Elliptio dilatatus 192 Elliptio dilatatus 193 Villosa iris Little Darby Creek between Little Darby Rd. ford and the mouth of Spring Fork Creek, 9.6 km NW of West Jefferson, 4.0 km SE of Plumwood, Lat 39° 59'17"N, Long 83°22'34"W, Monroe Twp., Madison Co., Ohio 16' April 1977 John J. Jenkinson JJJ:218 Lampsilis radiata luteola Log Pond Run (Ohio River system) OSUM:1977:103 Log Pond Run at Twp. Rt. 123 (King Rd.) bridge (No. 888), 2.5 mi [4.0 km] SSW of Vanatta, .3.3 mi [5.3 km] NW of center of Newark, Lat ao °05'40"N, Long 82°26'45"W, T 2 N, R 12 W, Newton Twp., Licking Co., Ohio 3 September 1977 Kathy G. Borror and Augustus E. Spreitzer JJJ:255 Alasmidonta viridis 258 Alasmidonta viridis Meramec River (Mississippi River system) Meramec River at Times Beach, between 1-44 and US Rt. 50 bridges, 34.0 km SSW of St. Charles, Lat 38°30'N, Long 90°35'W, St. Louis Co., Missouri 12 November 1976 Carolyn S. and John J. Jenkinson JJJ:196 Quadrula pustulosa 198 Elliptio dilatatus" 199 Ligumia recta 200 Actinonaias ligamentina carinata 218 JJJ:202 Lampsilis ventricosa 203 Ligumia recta 204 El 1iptio dilatatus 208 Quadrula pustulosa 209 Lampsilis ventricosa Mississippi River (Mississippi River system) OSUM:1977:134 East channel of the Mississippi River at river mile 635.2-635.4, [above US Rt. 18 bridge, at Prairie-du-Chien, Lat 43°03'N, Long 91°08'W, Crawford Co.] Wisconsin 2 July 1977 Marian E. Havlik JJJ:223 Lampsilis hiqginsi 224 Quadrula quadrula 225 Quadrula pustulosa 226 Obovaria olivaria 227 Ligumia recta 228 Quadrula pustulosa 229 Obovaria olivaria Ohoopee River (Altamaha River system) OSUM:1976:327 Ohoopee River at Rt. 56 bridge, 3.6 mi [5.8 km] W of Reidsville, 8.2 mi [13.2 km] SW of Collins, [Lat 32°06'N, Long 82°10'W] Tattnall Co., Georgia 10 June 1976 Eugene P. Keferl JJJ:68 Elliptio dariensis OSUM:1976:313 Ohoopee River at Ga. Rt. 147 bridge, 5.7 mi [9.2 km] SSW of Reidsville, 14.3 mi [23.0 km] WNW of Glennville, [Lat32°00'N, Long 82°09'W] Tattnall Co., Georgia 10 June 1976 Eugene P. Keferl JJJ:75 Alasmidonta arcula (?)78 Elliptio dariensis 182 Elliptio dariensis Pitman Creek (Cumberland River system) OSUM:1977:154 Pitman Creek above US Rt. 80 bridges, 4.0 km NE of Somerset, 43.0 km W of London, Lat 37o06'41"N, Long 84°33'50"W, Pulaski Co., Kentucky 21 March 1977 John J. Jenkinson JJJ:212 Villosa taeniata punctata 213 Villosa "ir is " nebulosa 221 Villosa taeniata punctata 222 Villosa "iris" nebulosa 219 Uchee Creek (Apalachicola River system) Uchee Creek above Ala. Rt. 169 bridge, 9.3 km NNW of Seale, 15.8 km SW of Phenix City, Lat 32°21'N, Long 85°06'W, Russell Co., Alabama 4 September 1976 John J. Jenkinson and John C. Hurd JJJ:137 Lampsilis subangulata 138 Quincuncina infucata 139 Elliptio icterina 140 Pleurobema pyriforme 141 Villosa 1ienosa 146 Lampsilis subangulata 147 Quincuncina infucata 148 Elliptio icterina Uphapee Creek (Mobile River system) Uphapee Creek between Ala. Rt. 81 bridge and 1-85 bridge, 5.8 km NNE of Tuskegee, 9.4 km SSW of Notasulga, Lat 32°24'N, Long 85°41'W, Macon Co., Alabama 4 September 1976 John J. Jenkinson and John C. Hurd JJJ:142 Lampsilis teres 143 Lampsilis excavata 144 Lampsilis straminea claibornensis 145 Villosa nebulosa Whitewater Creek (Apalachicola River system) Whitewater Creek below Starr's Mill, 5.3 km NE of Senoia, 27.8 km ESE of Newnan, Lat 33°21'N, Long 84°30'W, Fayette Co., Georgia 3 September 1976 John J. Jenkinson JJJ:131 Anodonta sp. cf. grandis 132 Medionidus penicillatus 133 Pleurobema sp. 134 Toxolasma parva 135 El 1iptio icterina 136 Megalonaias boykiniana Willamette River (Columbia River system) OSUM:1976:104 Willamette River, W bank, about 2.3 mi [3.7 km] E of Greenberry, 8.5 mi [13.7 km] S of Corvallis, Lat 44°26'N, Long 123° 14'W, Sec. 13, T 13 S, R 5 W, Benton Co., Oregon 20 May 1976 David H. Stansbery and Richard Tubb JJJ:55 Gonidea angulata (OSUM 45213.1) 56 Margaritifera falcata (OSUM 45211.4) 57 Margaritifera falcata (OSUM 45211.1) :58 Anodonta oreqonensis (OSUM 45212) 59 Margaritifera falcata (OSUM 45211.3) 60 Gonidea angulata (OSUM 45213.2) 61 Gonidea angulata (OSUM 45213.3) 62 Gonidea angulata (OSUM 45213.4) 63 Margaritifera falcata (OSUM 45211.2) LITERATURE CITED *Agassiz, L. 1852. Uber die gattungen unter den nordamerikanischen najaden. Arch. Naturg. 18:41-52. Allen, W. R. 1914. The food and feeding habits of freshwater mussels. Biological Bulletin 27(3):127-147. 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