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Presumptive rapid speciation after a founder event in a laboratory population of Nereis: allozyme electrophoretic evidence does not support the hypothesis.

Some theories of speciation propose that population bottlenecks, or "founder events" are particularly conducive to the origin of a new species (Mayr 1963; Carson 1975; Templeton 1980). One possible example concerns the marine polychaete Nereis acuminata. A laboratory population ("Lab") started with five or six individuals collected near Los Angeles has been maintained in the laboratory since 1964. Tests conducted after some 75 generations (25 y) demonstrate that the Lab population is reproductively isolated from two populations ("P1" and "P2") collected near the site where the original worms were obtained. On the basis of reproductive isolation tests, it has been suggested that speciation occurred in the Lab population following the founder event (Weinberg et al. 1992). If speciation on the time scale involved had actually occurred, this would be a very important result, because there are few examples of rapid speciation.

A critical assumption in Weinberg's experiment is that the P1 and P2 populations are, in fact, representatives of the natural population from which the Lab population hypothetically had diverged and speciated in the laboratory. We have tested this hypothesis by assaying 18 electrophoretic gene loci in the Lab, P1 and P2 populations and in an Atlantic population of a different species, used as a reference control. If the Lab population had speciated from P1 or P2, we would expect that randomly selected electrophoretic markers should be largely similar between the Lab and P1 or P2 populations. However, no common alleles between Lab and P1 or P2 are found in 13 (72%) loci, and at two more loci the alleles fixed in Lab are at low frequencies in P1 and P2. The genetic distances between Lab and P1 or P2, are 1.75 [+ or -] 0.51 and 1.76 [+ or -] 0.52, larger than between most pairs of congeneric species in many sorts of organisms; and roughly similar to the distance between P1 or P2 and the reference population from the Atlantic (D = 1.36 [+ or -] 0.40). The Lab population is genetically depauperate, most likely as a consequence of the founder event, but this reduced variability contributes only trivially (about 1%) to the genetic differentiation between the populations. We conclude that the Lab population was already a species different from P1 and P2 at the time when it was originally sampled in 1964.

MATERIALS AND METHODS

Collecting methods, sites, dates, and laboratory culture conditions are given in Weinberg et al. (1990, 1992). The populations studied were verified to be Nereis acuminata [or its synonyms (Day 1973) Nereis (Neanthes) arenaceodentata, and Nereis (Neanthes) caudata] based on morphology of denticles, setae, ligules, cirri, and head shape (Ehlers 1868; Moore 1903; Pettibone 1963; Day 1973). In July 1964, Dr. D. Reish, of California State University at Long Beach, started a culture from five or six individuals (sex ratio unknown) collected from Los Angeles Harbor. In December 1986, one of us (J.R.W.) transported four pairs of these worms to the Woods Hole Oceanographic Institution (WHOI), where the Lab culture was started from them. In 1987-1988, more than 100 individuals collected from subtidal sands at the mouth of the San Gabriel River, Long Beach, California and from the upper bay at Newport Beach, California, were used to start the P1 and P2 populations at WHOI. Additional individuals were collected and added to the cultures in 1988, 1989, and 1990 to reduce inbreeding. In July 1992, the Lab, P1, P2 populations were moved from WHOI to the Marine Biological Laboratory (MBL). In June and August 1993, individuals were collected from a field site in W. Falmouth Harbor, Massachusetts, for comparison with the three Pacific populations, which had been in culture five to 28 years. This Falmouth population differs from the three California populations in having a very different karyotype and number of chromosomes (Weinberg et al. 1990, 1992), complete pre-mating isolation (Weinberg et al. 1990), and smaller maximum size and lower fecundity (Weinberg, unpubl. data). Individual specimens of laboratory cultured (Lab, P1, P2) and field-collected (Falmouth) worms were frozen at -80[degrees]C, and shipped on dry ice to the University of California at Irvine for isozyme analysis.

The same continuous buffer was used to separate all enzyme systems. Single polychaetes were homogenized at 4[degrees]C, the homogenates centrifuged for 5 min at 8000 rpm, and the supernatant absorbed with two 1 x 0.4 cm pieces of filter paper (Whatman #1), which were each inserted into a different 12% starch-gel block (19.8 x 17.5 x 1 cm). After electrophoresis, the gels were sliced horizontally into seven 1.5 mm-thick slices, and the bottom six slices were used for six different enzyme assays. The staining procedures and other conditions were essentially the same as described by Brewer (1970) and Ayala et al. (1972).

Genetic variability within populations was assessed by computing the unbiased estimates of expected and observed single-locus heterozygosity ([h.sub.j]), and its average (H) over all loci (Nei 1972, 1978, 1987). Genetic divergence between populations was measured according to Nei (1978).

RESULTS

We have analyzed 12 enzyme systems encompassing 18 different loci in four populations of the marine polychaete Nereis (Table 1). In 13 out of 18 loci studied (Aco-1, Aco-2, Ak, [A.sub.o], Got-1, Got-2, Hk-2, Idh, Ldh-1, Ldh-2, Mdh-2, Me-1, and Pgm), the Lab population does not share any alleles with either the P 1 or P2 populations, which are those considered by Weinberg et al. (1992) to be representatives of the natural population from which Lab hypothetically speciated in the laboratory. Moreover, in two additional loci (Acph and Hk-1), the Lab population is fixed for alleles that have low frequency in P1 and P2. The two populations Pl and P2 exhibit the same alleles at similar frequencies in all loci (except for Ldh-[2.sup.120] and Pgm[degrees]86, which are present at low frequencies in P1 and P2, respectively). A chi-square contingency test of allele frequencies indicates that P1 and P2 can be considered the same population. Falmouth, the Atlantic population, does not share alleles with the Lab, P1 or P2 in eight loci (Got-1, Got-2, Hk-1, Hk-2, Idh-2, Mdh-1, Mdh-2, and Me-1); in addition, it has no alleles in common with Lab at five loci (Aco-1, Aco-2, Ak, Ldh-2, and Pgi), and with P1 or P2 at three other loci (Acph, Ao, and Ldh-1).

The Lab population does not show variability at any locus (H = 0). This result is not surprising because Lab has recently gone through two successive bottlenecks, and the initial level of variability was likely low, as is the case in the other three populations. The average expected heterozygosity is 0.027 [+ or -] 0.017 in P1, 0.021 [+ or -] 0.009 in P2, and 0.051 [+ or -] 0.034 in Falmouth. The lower heterozygosities of P1 and P2 might be a consequence of founder effects and population bottlenecks while these populations were kept in the laboratory. The difference between average expected heterozygosities is significant for the pair Lab-P2 (P [less than] 0.05).

Table 2 gives the pairwise genetic distance, D, between populations. The D values are large between Lab and P1, P2, or Falmouth, as well as between Falmouth and P1 or P2, of a magnitude within the range of interspecific differentiation (Ayala 1975; Nei 1987; Avise 1994). Although Lab seems to be more similar to Falmouth than to P1 or P2, a t-test based on mean identities (Nei 1987) is not statistically significant. Between P1 and P2, D = 0.00 [+ or -] 0.24, consistent with the interpretation that both are local populations of the same species.

In the presence of bottlenecks, genetic-distance increases at a rate inversely proportional to the magnitude of the bottleneck (Chakraborty and Nei 1977). To obtain a better picture of the evolutionary relationship between Lab and the other' populations before the bottlenecks, we have made a correction for bottleneck effect (Chakraborty and Nei 1977). The corrected D values (given at the bottom of Table 2 in parentheses) are smaller than those without the correction, but the difference is trivially small.

DISCUSSION

The most significant result of this study is that 13 (72%) out of the 18 loci surveyed do not exhibit any common alleles between the Lab population and either the P1 or P2 populations, which were considered by Weinberg et al. (1992) to be representative of the natural population from which Lab hypothetically had speciated in the laboratory. Moreover, in two of the remaining five loci, the allele fixed in Lab is the one with lowest frequency in P1 and P2. The high number of nonshared alleles between Lab and P1 or P2 might conceivably be a consequence of (1) the low level of polymorphism exhibited by this kind of organism (H ranges between 2.1 and 5%) plus (2) the relatively small size of our samples. But doubling the sample size by pooling P1 and P2 does not increase the small proportion of alleles they share with Lab population. It seems likely either that Lab does not share any alleles with P1 or P2 at a majority of loci or that any shared alleles are at frequencies around or less than 1%.

Estimates of intertaxa divergence for allozyme loci (Ayala 1975; Nei 1987) indicate that the genetic differentiation observed between P1 or P2 and the Lab population (D = 1.75 [+ or -] 0.51 and 1.74 [+ or -] 0.52 for the pairs Lab-P1 and Lab-P2. respectively) is typical of that observed between species and much larger than between populations of the same species. Indeed, D between Lab and either P1 or P2 is of the same magnitude as between any of the three populations and Falmouth, a distinct species. The standard error of D is large. because it depends on the relatively small number of loci tested. Yet, if we assume an average rate of codon substitutions detectable by electrophoresis of [Alpha] = [10.sup.-7] (Nei 1987), D between Lab and either P1 or P2 corresponds to 8.7 [+ or -] 2.5 million years of independent evolution. The genetic distances reported here are among the highest found among sibling species of marine invertebrates (see Knowlton 1993). Sibling species of sympatric capitellid polychaetes have been found to be fixed for alternate alleles at several loci (Grassle and Grassle 1976), much like the results found here. Fong and Garthwaite (1994) computed D among three allopatric species of nereid polychaetes of the genus Hediste from different parts of the world; the greatest distance (D = 1.28) was between a German population of Hediste diversicolor and a US Pacific Coast population of Hediste limnicola.

The genetic differentiation between our populations is consistent with the observation that Lab exhibits postmating reproductive isolation from P1 and P2; but it is unexpected as a consequence of two bottlenecks and chance differentiation [TABULAR DATA FOR TABLE 1 OMITTED] [TABULAR DATA FOR TABLE 2 OMITTED] during the brief 28-y history of the Lab population. The large D between P1 or P2 and the Lab population supports rather the inference that the population sampled in 1964 by D. J. Reish belonged to a species different from P1 and P2.

Extensive field searches (Weinberg et al. 1992) of suitable marine habitats in the Los Angeles area over a 4-y period have failed to locate this species. These searches were limited to intertidal and shallow subtidal zones as well as the fouling communities on the undersides of floating boat docks, the same habitat where Reish collected the founders of his laboratory culture in 1964. Thus, either the Lab species has become extremely rare over the past three decades, or it occurs only in deeper waters of bays and harbors in the Los Angeles area.

One consequence of population bottlenecks is that genetic distance increases rapidly between taxa in inverse proportion to the size of the bottleneck population (Chakraborty and Nei 1977). The bottlenecks experienced by Lab cannot well account for the observed differentiation, because reductions in effective population number would eliminate infrequent alleles, not the common ones. In any case, we can assess the probability that the observed divergence between P1 or P2 and Lab is due to the founder events. If we focus only on the process of gene sampling in the founding population and ignore the subsequent history of drift and selection, the question is reduced to a simple one: what is the probability that the alleles found in the Lab population were present in the sample collected by Reish in 1964? We shall assume conservative conditions that increase the probability of sampling infrequent alleles: random mating; the frequency of the Lab alleles in the source natural population was 0.03; and the maximum possible effective size at the bottleneck, [N.sub.e] = 6. The probability of sampling at least one of the observed alleles is then given by the binomial [Sigma](12!/[(12-n)!n!][(0.97).sup.12-n][(0.03).sup.n] = 0.306, with n = 1 . . . 12, for each locus. Because we have 15 out of 18 differentiated loci, and supposing that they are independent, the final probability becomes 5.3 x [10.sup.-6]. This estimate is conservative because the probability of sampling rare alleles under any common system of nonrandom mating is even smaller. Moreover, we have assumed a threshold of 3% as the frequency of rare alleles although, in 13 out of 15 diagnostic loci, none of the Lab alleles were detected in either P1 or P2; and the mean frequency of rare alleles is 0.011 [+ or -] 0.005 in P1 and P2. It must be noted that the estimated probability is more sensitive to lower assumed frequencies of the rare alleles than to larger ones because these frequencies are multiplied in calculating the probability. Finally, we have assumed a favorable effective size at the bottleneck of [N.sub.e] = 6, with equal number of males and females, although the effective number could be smaller because Reish did not record the sex ratio in his sample (nor do we know that all produced progeny).

Furthermore, it is possible to gain some information about how much the bottleneck has contributed to the dissimilarity observed between Lab and P1 or P2. To do this, we have computed a new measure of distance that corrects for the founder effect (Chakraborty and Nei 1977). If the founder event caused the large differentiation observed between Lab and P1 or P2, this should be reflected in the corrected measure of genetic divergence. Although the new distances are smaller (see Table 2), the genetic distance is reduced only by 1%.

In light of results from previous studies (Weinberg et al. 1990, 1992), it is surprising that D between Lab and P1 (or P2) is as great as that between the California and Falmouth populations. Worms from all three California populations pair bond and mate with one another in experimental trials (although offspring from crosses between Lab and either P1 or P2 do not survive); in contrast, worms from California and Falmouth are so aggressive to each other that pair bonding never takes place between them. All three California populations have nine pairs of relatively large chromosomes, most of which are metacentric; the Falmouth karyotype is very different, consisting of 11 pairs of small acrocentric chromosomes. The California karyotype is also distinct from that of the mid-Pacific island of Hawaii where worms have 14 pairs of chromosomes (Weinberg et al. 1992). Finally, adults from California grow to larger size, and females have higher fecundity, than worms from Falmouth, when all are raised in a standard environment (Weinberg, unpubl. data).

The entire process of speciation rarely has been observed and it is indeed notoriously difficult to assess (Provine 1989). Thus, it is perhaps not surprising that there is a growing body of untested theory and controversy about modes of speciation, their prevalence, and genetic consequences (Templeton 1980; Futuyma 1983; Barton and Charlesworth 1984; Coyne and Barton 1988; Barton 1989; Coyne 1990). Numerous models have postulated genetic drift as the critical factor in speciation (Mayr 1963; Carson 1970, 1975; Lande 1980; Templeton 1980, 1989; Hedrick 1981; Lyttle 1989). Carson's model of founder-flush speciation has received support from some laboratory tests performed with Drosophila pseudoobscura (Powell 1978; Dodd and Powell 1985), but not from experiments with Drosophila simulans (Ringo et al. 1985; Ringo 1987) and the housefly (Meffert and Bryant 1991). Moreover, a very extensive recent experiment with D. pseudoobscura has also failed to support the founder-flush-crash model (Galiana et al. 1993, 1994). This latter experiment was largely conducted in the laboratory of one of us (F. J. Ayala) as part of a broad research program focused on the causes and patterns of speciation. Thus, our interest was engaged by early evidence suggesting that speciation had occurred in Nereis as a consequence of founder events (Weinberg et al. 1992). The results herein reported do not support this hypothesis.

ACKNOWLEDGMENTS

We are grateful to A. Kuzirian, J. Hanley, and the Marine Biological Laboratory, Woods Hole, for support in the culture of polychaetes since July 1992. We appreciate critical comments made by H. Lessios, V. Starczak, and A. Kuzirian during the study.

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Author:Rodriguez-Trelles, Francisco; Weinberg, James R.; Ayala, Francisco J.
Publication:Evolution
Date:Feb 1, 1996
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