JAMES W. VALENTINE Department of Geology, University of California, Davis, Davis, California 95616 DENNIS HEDGECOCK Department of Genetics, University of California, Davis, Davis, California 95616 GARY S. ZUMWALT Department of Geology, University of California, Davis, Davis, California 95616 FRANCISCO J. AY ALA Department of Genetics, University of California, Davis, Davis, California 95616

Mass Extinctions and Genetic Polymorphism in the "Killer Clam," Tridacna

ABSTRACT the optimal genotypes are eventually elimi- nated by selection. Depauperate gene pools, Mass extinctions of marine invertebrates then, would be the rule among vast numbers have been attributed to genetic depauperation of lineages inhabiting long-stable environ- in specialized lineages. Tridacna maxima is a ments. If the environmental regime then be- plausible modern analog of the lineages that came less stable, however, these lineages would were commonly associated with mass extinc- lack genetic materials for rapid evolutionary tions; it is restricted to a relatively stable development of the required functional versa- biogeographic province, lives in shallow water, tility and would tend to become extinct. Thus, is highly specialized, and is associated with if most of the world's shallow seas were envi- reef communities. Our studies show, however, ronmentally stable for a long period of time that it is highly polymorphic and heterozygotic, and then became significantly less stable, mass and thus fails to support the depauperate gene- extinction would result. Bretsky (1968) noted pool hypothesis of mass extinction. that elements of shallow-water (relatively un- stable) Paleozoic shelf communities were less INTRODUCTION: THE PROBLEM beset by extinction than elements of deeper The reality of the great mass extinctions water (relatively stable) shelf communities, that are registered in the fossil record seems and this suggested that the shallow-water forms well established, although no single explanation were more flexibly adapted; this supports the has achieved general acceptance. Extinction, hypothesis. whether associated with the waves of mass Whether a living population has high or low extinctions or occurring as part of a sort of genetic variability cannot be determined by background level, is a most enigmatic event. measuring its morphological variability, but This is partly because it is so difficult to test can be determined by studying its enzyme most extinction hypotheses. polymorphism. Thus, various kinds of tests of One relatively recent hypothesis of mass the Bretsky-Lorenz hypothesis may be con- extinction (Bretsky and Lorenz, 1969, 1970) ducted. Schopf and Gooch (1972), for example, suggests that these extinctions result from studied samples of several species of deep-sea genetic strategies that have the effect of pre invertebrates. Their results suggest that those venting organisms from adapting successfully populations are substantially variable geneti- to changing environmental conditions. In envi- cally, well within the range of variation of the ronments that are relatively stable for a long majority of other animal species that have been time, the argument goes, the pooled genetic studied. According to the Bretsky-Lorenz variability of the average population declines. hypothesis, they should have very low genetic Only a small range of genotypes would be re- variability, and thus the Schopf and Gooch quired for adaptation to the narrow range of observations are at variance with the expecta- conditions, and therefore all genes but those in tions of the Bretsky-Lorenz hypothesis.

Geological Society of America Bulletin, v. 84, p. 3411—3414, October 1973 3411

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However, there are reasons for further work cally; the other, A.fatua, is more polymorphic on this topic. The deep-sea communities are genetically but substantially less variable mor- highly diverse (Hessler and Sanders, 1967), yet phologically than A. barbata (fain and Mar- the very resources that are partitioned in shall, 1967; see also Jain, 1969). In general, equally diverse shallow-water communities the lineages that became extinct near the seem to be widely shared in the deep sea. close of the Paleozoic were not particularly Dayton and Hessler (1972) suggest that deep- invariant morphologically. Many of the sea population sizes are limited by unselective productoid brachiopods, for example, were predation, thus freeing resources for additional partially buried in or pressed close to the populations; but such restrictions are diversity- substrate (like T. maxima) and were rather independent (Valentine, 1972) and can be over- variable in form, while the richthofeniaceans come by natural selection. Thus, the evolu- and many other reef-dwelling lineages were tionary ecology of the deep sea is not well morphologically quite plastic. understood, and until we have convincing We collected a sample cf 107 individuals explanations of the adaptive strategies of from shallow water (0 to -3 ft tidal datum) deep-sea organisms, which may well include at Eniwetok Atoll, Marshall Islands, during special reproductive and genetic mechanisms, July 1972, and studied allelic variation at 30 we cannot evaluate the evidence of genetic loci by starch-gel electrophoresis (for a de- polymorphism. scription of the techniques employed, see Furthermore, the lineages that are known to Ayala and others, 1972). From each specimen, participate in mass extinctions are nearly all samples of five tissues were assayed (adductor from shallow water, and many are intertidal or muscle, gill, kidney, stomach, and mantle shallow subtidal. A great number are associated ventral to the zooxanthellae). The results are with fossil reef complexes, are highly specialized summarized in Table 1. At 25 of the 30 loci,

members of their clades, and are frequently TABLE 1. GENETIC VARIATION OF 30 GENE widespread in what appears to have been the LOCI OF TRIDACNA MXIMA

tropics of their times. The Permian-Triassic Gene* Sample Number of Frequency of Observed frequency extinction, which on balance is the most im- sizet alleles most comnon of heterozygous observed allele individuals portant of the mass extinctions, is character- Adk-1 150 1 1.00 0.000 ized by the disappearance of just these sorts of AS:-a 184 4 0.90 0.174 lineages. In addition to deep-sea forms, modern Adk-4 204 4 0.92 0.137 Adk-S 198 3 0.99 0.020 analogs of the sorts of shallow-water lineages Adk-S 116 4 0.81 0.31C that actually became extinct should also be Esi-B 174 5 0.69 0.414 Est—4 116 4 0.88 0.241 studied. Est-5 214 4 0.99 0.026 Est- e 134 5 0.86 0.254 Gdh-S 176 3 0.92 0.13E GZodtl 214 4 0.70 0.477 PRESENT WORK Ida 144 4 0.82 0.333 L&i 210 3 0.96 0.086 Lao-l 210 4 0.78 0.257 We selected Tridacna maxima, the "killer Lap-3 202 4 0.61 0.327 clam," as such an analog. It is a highly special- Kdh-1 214 1 1.00 0.000 Mdh-S 206 6 0.71 0.456 ized reef-dwelling cardioid pelecypod (Stasek, Mdh-1 176 4 0.87 0.239 Kdh-l 62 1 1.00 0.000 1962) that is restricted to the Indo-Pacific m 204 2 0.97 0.059 province (Rosewater, 1965). It contains abun- Odh 110 3 0.84 0.327 Pgrn-1 214 4 0.62 0.439 dant zooxanthellae in mantle tissues, which Pgv-i 212 4 0.83 0.283 provide an energy supplement, creating an Pt-1 208 1 1.00 0.000 it- 2 212 4 0.67 0.415 unusually stable trophic resource base. Tridacna Pt-3 150 2 0.97 0.053 Pt-4 184 1 1.00 0.000 is a protandric functional hermaphrodite but To 210 4 0.98 0.038 does not self (Wada, 1954). T. maxima is some- Tpi-1 214 3 0.92 0.168 Tpi-Z 64 4 0.72 0.375 what variable phenotypically, but this is not Average 176 + 8 3.3 ± 0..2 4 0.202 +0.029 necessarily an indication of genetic variability. * Symbols used for genes are: Ad}:, adenylate kinase; Indeed, populations with low genetic varia- Est, esterase; Gdh, glutamic dehydrogenase: G3pdh, glyceraldehyde-3-phosphate dehydrogenasei Idh, lsocitrate bility may have considerable morphological dehydrogenase; Ldh, lactate dehydroijenase; Lap, leucine plasticity. A particularly interesting example airizzopeptidase; i 'dh, malate dehydrogenase; Me, malic enzyme; Odh, octinol dehydrogenase; Pgm, phosphoglucoiw- is furnished by two congeneric species of oats. tase; pt, non-enzymatic protein; To. tetrazolium oxidase; One, Avena barbata, is largely monomorphic Tpi, triose-phosphate isomerase. t Sample size is the number of genes sampled, or twice genetically but is quite variable morphologi- the number of individuals.

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or 83.3 percent, we found at least two alleles. stead, we have found only between one and If we use a rather restrictive criterion of poly- six alleles at each locus, many of them in morphism, namely that the most common moderate frequencies. It should be pointed allele must have a frequency no greater than out that these alleles with intermediate fre- 0.95, the number of polymorphic loci was 19, quencies contribute most of the variation or 63.3 percent. The average number of alleles found in our sample. per locus found in our sample was 3.33 ± 0.24. A second possible explanation of the high The most precise measure of genetic variation level of genetic variation observed in Tridacna for an outbreeding sexual organism is the is that the variation is adaptively neutral proportion of loci at which an individual is (Kimura and Ohta, 1971). This explanation heterozygous; this statistic, in our sample, was disposes of the whole problem because a 20.2 ± 2.9 percent. This is very high; indeed, polymorphism of adaptively equivalent alleles T. maxima has a proportion of polymorphic is irrelevant to adaptation. If the variation were loci and a proportion of heterozygous loci per neutral to natural selection, however, we individual as large as have ever been detected would expect at each locus either many alleles in organisms for which an adequate number of all at low frequencies or one allele at a very loci have been studied. This result is at vari- high frequency and all others at very low fre- ance with the predictions of the depauperate quencies (Ewens, 1972). Instead, we find at gene-pool hypothesis of extinction. most loci one allele at high frequency and There are three obvious possibilities to several others at intermediate frequencies. explain why this population of T. maxima The third possible explanation is that the should contain so much genetic variation. variation is adaptively significant and is being First, Eniwetok Atoll was the site of a number maintained by balancing natural selection. of nuclear tests between 1946 and 1958. A This is, of course, the classical neo-Darwinian species of Tridacna (T. gigas) is known to explanation. It may be accepted as a working concentrate radioisotopes, both in its soft hypothesis because of the improbability of parts (Co60; Weiss and Shipman, 1957) and in either of the other two explanations, and its shell (Sr90; Bonham, 1965). The shell of because work with other organisms seems to our largest specimen was examined by a high- support the hypothesis that most enzyme volume, high-resolution Ge(Li) gamma de- variation is adaptively significant (Ayala, tector in an approximate 2ir geometry, but 1972). proved to contain no unusual concentration of radioisotopes. However, the ancestral clams CONCLUSIONS of the specimens in our sample must have Our results do not support the hypothesis been exposed to massive doses of high-fre- that some massive extinctions registered in the quency radiations. Such radiations and (or) fossil record may have been due to the scarcity the mutagens accumulated in the specimens' of genetic variation in populations which were tissues may have created an enormous amount adapted to stable environments and which of genetic variation in the reproductive tissues. were unable to cope genetically with rising The variation that we have observed would, environmental instability. The adaptive strate- then, consist of recently induced mutations gies of ancient organisms appear to have been that have not yet been eliminated by natural affected by increasing instabilities of ancient selection. Such an explanation is unlikely for environments and to have been associated two reasons. First, as Table 1 shows, the amount with extinctions (Valentine and Moores, 1972), of heterozygosity per locus is rather evenly but how genetic strategies were involved in distributed between 0 and 48 percent, except these events is uncertain. Much more relevant for an excess of loci (eight) with little or no information must be accumulated before this genetic variation (less than 5 percent hetero- question can be resolved. zygous individuals). If the detected variation had been induced by the random process of ACKNOWLEDGMENTS mutation, it should be normally distributed Sample collecting was made possible by a around a mean value of heterozygosity per grant from the Atomic Energy Commission locus. Second, if the variation were induced by administered through the Eniwetok Marine radiation, we would expect at each locus many Biological Laboratory. We thank Lori Barr mutants, each with very low frequency. In- for technical help with the enzyme assays, Ted

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DeLaca for his extensive aid in sample collec- diversity in the deep-sea: Deep-Sea Research, tion and preparation, and Thomas Cahill and v. 14, p. 65-78. the trace-element group, Crocker Nuclear Jain, S. K., 1969, Comparative ecogenetics of two Laboratory, University of California, Davis, Avena species occurring in central California: Evolutionary Biol., v. 3, p. 73-118. for radioisotope analyses. Jain, S. K., and Marshall, D. R„ 1967, Population studies in predominantly self-pollinating spe- REFERENCES CITED cies. X. Variation in natural populations of Avena fatua and A. barl'ata: Am. Naturalist, Ayala, F. J., 1972, Darwinian versus non-Dar- v. 101, p. 19-33. winian evolution in natural populations of Drosophila, in Le Cam, L. M., and others, Kimura, M., and Ohta, T., 1971, Protein poly- eds., Darwinian, neo-Darwinian and non- morphism as a phase of molecular evolution: Darwinian evolution: Berkeley, Univ. Cali- Nature, v. 229, p. 467-4(59. fornia Press. Rosewater, J., 1965, The family Tridacnidae in Ayala, F. J., Powell, J. R., Tracey, M. L., Mourao, the Indo-Pacific: Indo-Pacific Mollusca, v. 1, C. A., and Perez-Salas, S., 1972, Enzyme p. 347-396. variability in the Drosophila willistoni group. Schopf, T.J.M., and Gooch, L L„ 1972, A natural IV. Genie variation in natural populations of experiment to test the hypothesis that loss Drosophila mllistoni: Genetics, v. 70, p. of genetic variability was responsible for mass 113-139. extinctions of the fossil record: Jour. Geology, v. 80, p. 481-483. Bonham, K., 1965, Growth rate of giant clam Tridacna gigas at Bikini Atoll as revealed by Stasek, C. R., 1962, The form, growth and evolu- autoradiography: Science, v. 149, p. 300-302. tion of the Tridacnidae (;*iant clams): Archives Bretsky, P. W., 1968, Evolution of Paleozoic ma- de Zoology Expérimentale et Générale, v. rine invertebrate communities: Science, v. 101, p. 1-40. 159, p. 1231-1233. Valentine, J. W., 1972, Conceptual models of Bretsky, P. W„ and Lorenz, D. M., 1969, Adaptive ecosystem evolution, in Schopf, T.J.M., ed., response to environmental stability: A unify- Models in paleobiology: San Francisco, Free- ing concept in paleoecology: N. Am. Paleon- man, Cooper & Co., p. 192-215. tology Convention, Chicago, Proc., pt. E, Valentine, J. W„ and Moore;;, E. M., 1972, Global p. 522-550. tectonics and the fossil lecord: Jour. Geology, v. 80, p. 167-184. 1970, An essay on genetic-adaptive strategies and mass extinctions: Geol. Soc. America Wada, S. K., 1954, Spa wring in the tridacnid Bull., v. 81, p. 2449-2456. clams: Japanese Jour. Zool., v. 11, p. 273-285. Dayton, P. K., and Hessler, R. R., 1972, Role of Weiss, H. V., and Shipman, W. H., 1957, Biological biological disturbance in maintaining diversity concentration by killer clams of cobalt 60 in the deep sea: Deep-Sea Research, v. 19, from radioactive fallout: Science, v. 125, p. 199-208. p. 695. Ewens, W. J., 1972, The sampling theory of selec- tively neutral alleles: Jour. Theoretical Popu- MANUSCRIPT RECEIVED BY THE SOCIETY FEBRUARY lation Biol., v. 3, p. 87-112. 5, 1973 Hessler, R. R„ and Sanders, H. C., 1967, Faunal REVISED MANUSCRIPT RECEIVED APRIL 9, 1973

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