Population genetics of Melampus bidentatus (Gastropoda: Pulmonata): the effect planktonic development on gene flow

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1 Population genetics of Melampus bidentatus (Gastropoda: Pulmonata): the effect planktonic development on gene flow of S. W. Schaeffer 1, E. C. Keller l, Jr. & N. E. Buroker 2 i Dept. of Biology, West Virginia University, Morgantown, WV26506, USA 2 Center for Environmental and Estuarine Studies, University of Maryland, Crisfield, Md, USA Abstract Melampus bidentatus has restricted gene flow of gametes and the potential for extensive gene flow of their planktonic larvae. We used genetic data to examine the influence of these opposing forces on the population structure of six Delmarva Peninsular populations. We analyzed the population structure of these six populations using twenty-three electrophoretic loci. Two ocean populations gave the appearance of large panmictic populations, while the other four populations appeared to be highly subdivided due to either restricted gene flow or age-structuring of the population. Two distinct groups of populations are apparent, which accounts for the extreme interpopulation heterogeneity. These data suggest that the direction of tides and currents limit the effectiveness of the planktonic dispersal stage of M. bidentatus in overcoming restricted gene flow of gametes. Introduction Successful gene flow requires dispersal of gametes or newly formed zygotes to a new population and subsequent establishment of these gametes or zygotes (Endler, 1977; Scheltema, 1975). McCracken & Bussard (1981) showed that restricted gene flow in terrestrial snails increased the genetic differences between populations. Other sedentary organisms show similar patterns (Levin & Kerster, 1974; Schaal, 1975; Workman & Niswander, 1970). On the other hand, extensive gene flow in marine invertebrates reduces genetic differences between populations (Buroker et al., 1979; Koehn et al ). These experimental results are consistent with Wright's (1943) isolation-by-distance model. Melampus bidentatus (Say) is a pulmonate snail which lives above the average high-tide mark of coastal and estuarine salt marshes along the Atlantic Ocean and Gulf of Mexico (Holle & Dineen, 1957). Hatching M. bidentatus larvae are washed out into the offshore plankton layer with the ebbing tide. The larvae spend two weeks developing into adults in the plankton layer. Following planktonic development, the adults settle into the salt marsh where they reside for their remaining two to three years of life (Russell-Hunter et al., 1972). The terrestrial existence of M. bidentatus should increase genetic differences between populations, but a planktonic larval stage has the potential to reduce these genetic differences. In this paper we examine the influence of these two opposing forces on the population structure of natural populations of M. bidentatus. Material and methods We sampled six natural populations ofm. bidentatus located on the peninsula formed by Delaware, Maryland and Virginia (Delmarva Peninsula). Three Atlantic Ocean localities (Fenwick Island, Delaware; Wallops Island, Virginia; and Cape Charles, Virginia) and three Chesapeake Bay sites Genetica 66, (1985). Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.

2 224 ~,. Surfacel~.--P7" Current I'-k,~... -~ y" "'Current I I k N I... Chesapeake - f B j, c v,,/. Y. '. \ ccv# / phoglucose isomerase (pgi), phosphoglucomutase (pgm), and superoxide dismutase (sod). The nomenclature of Ayala et al (1972) was used to describe our electrophoretic loci. We calculated allele frequencies for each polymorphic locus in each population. A locus was considered polymorphic if the frequency of the most common allele in any of the six populations sampled was less than or equal to An analysis of population structure compares the observed gene and genotypic frequencies with those expected under the assumption of random mating. Departures from random mating suggest that selection, inbreeding or other evolutionary forces are acting on the individuals of the population. We tested departures of genotypic frequencies from the expected proportions under random mating by means of chi-squared tests for goodness-of-fit. A chisquared test for homogeneity or gene frequency was used to detect departures from random mating between populations (Schaal, 1975; Workman & Niswander, 1970). Genetic similarities and genetic distances for all pairwise comparisons were calculated using Nei's (1972) formulas. We clustered Nei's genetic distances using the UPGMA algorithm of Sheath & Sokal (1973). Results Fig. 1. A map of the Delmarva Peninsula showing the six Melampus bidentatus sampling sites. The map also shows the currents along the Atlantic coast and in the Chesapeake Bay (after Scheletema, 1975). The six populations are: Fenwick Island (FI), Wallops Island (Wl), Cape Charles (CC), Eastville (EV), Cashville (CV), and Crisfield (CF). (Eastville, Virginia; Cashville, Virginia; and Crisfield, Maryland) were selected (Fig. 1). We collected 125 snails from each population and stored them at -20 o C for electrophoresis. Salinity measurements were taken at each site. Fourteen enzyme systems involving 23 loci were examined using horizontal starch gel electrophoresis (see Schaeffer, 1980). We stained for the following enzymes: acid phosphatase (acp), aldolase (aid), aminopeptidase (ap), aspartate aminotransferase (aat), esterase (est), isocitrate dehydrogenase (idh), leucine aminopeptidase (lap), malate dehydrogenase (mdh), malic enzyme (me), muscle protein (rap), 6-phosphogluconate dehydrogenase (6-pgd), phos- The six populations were monomorphic for the same electrophoretic allele at thirteen enzyme loci of the twenty-three loci scored for M. bidentatus. Ten loci examined were polymorphic and in some cases highly so with six electromorphs or more. The polymorphic loci were: acp-2, ald, ap, est-2, idh,!ap-1, lap-2, lap-3, 6-pgd, and sod. Our estimate of percent polymorphic loci, P = , was within the range observed for other marine and land snails (Nevo, 1978; Selander, 1976). The average individual heterozygosity, H = , Table 1. Average salinity ± standard error for six Delmarva Peninsular sampling sites. Salinity is given in parts per thousand. Population Salinity ± SE Fenwick Island Wallops Island Cape Charles Eastville 20.6 ± 1.3 Cashville Crisfield 18.3 ± 3.5

3 225 was greater than that reported in land or marine snails. Salinities for each site are given in Table!. We observed significant departures of our observed genotypic frequencies from those expected under random mating in the Fenwick Island, East- ville, Cashville, and Crisfield populations. These populations had a deficiency of heterozygotes at most polymorphic loci. The other two populations, Wallops Island and Cape Charles, exhibited few significant departures (Table 2). Table 2. Gene frequencies, number of alleles (N), relative mobilities (R M), observed heterozygosity (HO), and expected heterozygosity (HE) for six Delmarva Peninsular populations of Melampus bidentatus. Locus RM FI WI CC EV CV CF a~h-2 N 250*** *** HO HE am N *** HO HE ap N 250*** *** *** 108 0, I HO HE est-2 N 248*** *** 248*** HO HE idh N 248*** *** 244*

4 226 Table 2. Continued. Locus RM FI Wl CC EV CV CF 96 0, HO HE lap-i N "** 244*** , , HO , HE lap-2 N * 246*** 248*** I , , ,110 0, , HO 0, , HE lap-3 N 246*** *** 246*** , , , , , HO , HE pgd N " ,743 0, , , HO 0, HE sod N " 192"** *** 248*** , ,516 0, , , , , ,048 HO 0, , HE 0, Symbols: Fenwick Island (FI), Wallops Island (Wl), Cape Charles (CC), Eastville (EV), Cashville (CV), Crisfield (CF). Significant departures from genotypic proportions expected under rando m mating are denoted: * P < 0.05; * * - P < 0.01 ; *** - P < 0.00 I. N o data were available for the lap-i locus at Wallops Island which is denoted by ().

5 227 Table3. Chi-squared test for homogeneity of allele frequencies among six Delmarva Peninsular populations of Melampus bidentatus for ten polymorphic enzyme loci. Locus Chi-squared* DF aqj aid ap est idh lap lap lap pgd sod * All Chi-squared values have a P < Significant genic heterogeneity among populations was observed at all loci (Table 3). The UPGMA cluster of Nei's genetic distances shows two groups of populations, Wallops Island and Cape Charles, and Fenwick Island, Eastville, Cashville, and Crisfield (Fig. 2). Notice that the Wallops Island and Cape Charles populations have a genetic similarity expected for neighboring demes (Ayala, 1975), while the Wallops Island and Crisfield populations have a genetic similarity corresponding to subspecies. One curious feature is that the Fenwick Island population shows a greater genetic similarity to the Crisfield population than the nearby Wallops Island population (Table 4) (Fig. 2). Discussion We found departures of genotypic frequencies from expected proportions under random mating = t I L t I I / GENETIC DISTANCE (D)!- CAPE CHARLES WALLOPS ISLAND CRISFIELD CASHVILLE EASTVILLE FENWICK ISLAND Fig. 2. The dendrogram formed by the UPGMA cluster of Nei's genetic distances for six populations of Melampus bidentatus. Table 4. Nei's genetic similarity/genetic distance matrix for all pairwise comparisons of six Delmarva Peninsular populations of Melampus bidentatus. Genetic similarities are found above the diagonal; genetic distances are located below the diagonal. Site FI Wl CC EV CV CF FI WI CC EV CV CF Symbols: Fenwick Island (FI), Wallops Island (WI), Cape Charles (CC), Eastville (EV), Cashville (CV), Crisfield (CF). in the Crisfield, Cashville, Eastville, and Fenwick Island populations (Table 2). The deficiency of heterozygotes in these populations suggests that planktonic larval dispersal is insufficient to overcome the restricted gamete flow of adult snails between demes. When larvae are washed out of these populations the currents may be unidirectional so that few larvae settle into the marsh where they originated to increase gene flow between local demes. Thus, the Crisfield, Cashville, Eastville, and Fenwick Island populations give the appearance of highly subdivided populations (Wright, 1943). If, on the other hand, larvae do return to their origin, then what we may be observing is an age-structured population with several generations of snails each having different gene frequencies (Charlesworth, 1980). At this time we have no data to reject this hypothesis but it could be tested by aging snails according to shell size and electrophoresing individuals in the different size classes. Wallops Island and Cape Charles appear to be large panmictic populations since few departures from random mating were observed. In these populations, larval dispersal appears to be sufficient to overcome the effects of restricted gene flow of gametes. An alternative explanation for the severe heterozygote deficiency might be natural selection acting in a heterogeneous environment (Levene, 1953). Each salt marsh may be divided into small microgeographic environments. Sampling these microgeographic environments as one population will give the appearance of a subdivided population if selection is acting differentially on the same genotypes. At this time we have no evidence to support or reject this hypothesis.

6 228 The genic heterogeneity observed among the six populations suggests that planktonic larvae are not exchanged between ocean and bay populations. The flushing of the Chesapeake Bay may be so strong that planktonic larvae settle into marshes downstream from their origin site. Also, larvae from oceanic populations rarely penetrate the bay, although some oceanic genotypes are found in the Eastville population (Table 2) (Schaeffer, 1980). The net effect is that the bay populations are highly subdivided and distinct from the ocean populations. If currents and tides are responsible for the observed pattern in M. bidentatus, then the Fenwick Island population should be more closely related to the oceanic populations than the bay populations. This contradiction suggests that natural selection or some other evolutionary force is acting on these snails. The obvious environmental variable that could be responsible for the observed cline in gene frequency in M. bidentatus around the Delmarva Peninsula is salinity. The two genetic groups correlate well with the salinity of the environment in which they are found (Table 1) (Schaeffer, 1980) providing indirect evidence that selection is acting on allozyme genotypes. A direct proof would show that certain allozymic genotypes are favored in a more brackish environment (Koehn et al., 1980). The genetic similarity observed between the bay group and the ocean group suggests that these two groups could be subspecies. The geographic isolation of these snails could have occurred just by the nature of currents and tides near large estuaries. The currents around the peninsula may prevent mixing of the two groups of snails. Scheletema (1975) found that surface dwelling larvae rarely enter major estuaries along the Atlantic coast. Our data are consistent with a surface dwelling larval stage for M. bidentatus since little mixing occurs between the ocean populations and bay populations. Thus, bay snails became geographically isolated and differentiated from the ocean snails. Our study indicates that the extensive gene flow is dependent on the strength of good dispersal vectors, in this case the ocean currents. Gene flow appeared to be high when the ocean currents travelled between populations. On the other hand, gene flow was low when the currents were blocked. Acknowledgements We would like to thank the Center for Environmental and Estuarine Studies at Crisfield, Maryland and the Department of Biology at West Virginia University for supplying support for this research. Also, we would like to thank Wyatt W. Anderson for useful comments on this manuscript. R eferences Ayala, F. J., Genetic differentiation during the speciation process. In: Evolutionary biology 8: T. Dobzhansky, M. K. Hecht & W. C. Steere, eds, Plenum Press, New York. Ayala, F. L., Powell, J. R., Mourao, G. A. & Perez-Salas, S., Enzyme variability in the Drosophila willistoni group. IV. Genic variations in populations of Drosophila willistoni. Genetics 70: Buroker, N. E., Hershberger, W. K. & Chew, K. K., Population genetics of the family Ostreidae 1. lntraspecific studies of Crassostrea gigas and Saccostrea commercialis. Mar. Biol. 54: Charlesworth, B., Evolution in age-structured populations. Cambridge University Press, New York. Endler, J. A., Geographic variation, speciation and clines. Princeton University Press, Princeton, New Jersey. Holle, P. A. & Dineen, C. F., Life history of the salt marsh snail, Melampus bidentatus Say. Nautilus 70: Koehn, R. K., Bayne, B. L., Moore, M. N. & Siebenaller, J. F., Salinity related physiological and genetic differences between populations of Mytilis edulis. Biol. J. Linnean Society 14: Koehn, R. K., Milkman, R. & Mitton, J. B., Population genetics of marine pelecypods. IV. Selection, migration, and genetic differentiation in the blue mussel Mytilus edulis. Evolution 30: Levene, H., Genetic equilibrium when more than one ecological niche is available. Am. Nat. 87: Levin, D. A. & Kerster, H. W., Gene flow in seed plants. In: Evolution Biology 7: T. Dobzhansky & M. K. Hecht, eds, Plenum Press, New York. McCracken, G. F. & Brussard, P. F., The population biology of the white-lipped snail, Triodopsis albolabris: Genetic variability. Evolution 34: Nei, M., Genetic distance between populations. Am. Nat. 106: Nevo, E., Genetic variation in natural populations: Patterns and theory. Theor. Pop. Biol. 13: Russell-Hunter, W. D., Apley, M. L. & Hunter, R. D., Early life history of Melampus and the significance of semilunar synchrony. Biol. Bull. 143: Schaeffer, S. W., Genetic variation of the salt marsh snail (Melampus bidentatus) around the southern Delmarva peninsula. M.S. Thesis, West Virginia University, Morgantown, W.V. Schaal, B., Population structure and local differentiation in Liatris cylindracea. Am. Nat. 109:

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