Lack of Mitochondrial Genetic Structure in Hamlets

Molecular Ecology (2003) 12, 2975–2980 doi: 10.1046/j.1365-294X.2003.01966.x

BlackwellLack Publishing Ltd. of mitochondrial genetic structure in hamlets (Hypoplectrus spp.): recent speciation or ongoing hybridization?

MARINA L. RAMON,*† PHILLIP S. LOBEL* and MICHAEL D. SORENSON† *Boston University Marine Program, Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543, †Department of Biology, Boston University, Cummington Street, Boston, MA 02215, USA

Abstract Species in the genus Hypoplectrus (hamlet fish) have been recognized primarily on the basis of colour morphology, which varies substantially. Limited differentiation in other morphological characters, however, has led to ongoing debate about their taxonomy. Our analysis of mtDNA sequences demonstrates neither reciprocal monophyly nor significant genetic differentiation among hamlet colour morphs. These data are potentially consistent with a model of recent speciation due to sexual selection on colour morphology. The presence within hamlets of two divergent mtDNA clades, however, suggests a longer history during which hybridization and gene flow have prevented the differentiation of hamlet colour morphs, at least in neutral genetic markers. Keywords: Hypoplectrus, lineage sorting, mtDNA, phylogeography, Serranidae, speciation Received 2 January 2003; revision received 17 April 2003; accepted 17 July 2003

identified, including ‘Tan’ (Thresher 1978; Domeier 1994) Introduction and a variant of H. gemma found in Belize that we refer to Molecular analyses have contributed to a renewed interest as ‘Belize Blue’ (Domeier 1994; Heemstra et al. 2002). among evolutionary biologists in the roles of diversifying Hamlets are distributed widely in the Caribbean and ecological and/or sexual selection in speciation (e.g. Schluter West Indies and many species (or colour morphs) are 2000; Via 2001). Particularly in freshwater cichlids, there broadly sympatric (Randall 1968; Barlow 1975; Domeier has been considerable interest in how such processes might 1994). Up to seven colour morphs may occur in close result in sympatric speciation (e.g. Wilson et al. 2000; Lande proximity on the same reef (Randall 1968; Thresher 1978; et al. 2001; Schliewen et al. 2001). We examined the sys- Serviss 1982). Given their morphological similarity, ichthy- tematics and phylogeography of hamlet fish, a marine ologists have been divided over the issue of whether group in which similar processes may account for a rapid hamlet colour morphs are distinct species (see Domeier 1994 diversification of distinct forms. for a review). Field observations suggest strongly assort- Hamlets (genus Hypoplectrus, family Serranidae) include ative mating with respect to colour (Barlow 1975; Fischer 13 or more species of predatory reef fish in the New World 1980a; Lobel & Neudecker 1985), and in captive experi- tropics (Acero & Garzón-Ferreira 1994; Domeier 1994; ments hamlet colour morphs mated with like individuals Heemstra et al. 2002). They exhibit little differentiation in in 95–100% of cases (Domeier 1994). These observations, in morphometric and meristic measures and historically have combination with somewhat different geographical distri- been distinguished solely on the basis of colour patterns butions for different colour morphs, led Domeier (1994) to (Randall 1968; Thresher 1978; Acero & Garzón-Ferreira suggest that hamlets be recognized as ‘good species’. Hamlets, 1994; Domeier 1994). To date, 10–12 hamlet colour morphs however, are broadcast spawners in which all early life have been described as species while others have been stages are planktonic (Fischer 1980b), a life history that may be conducive to extensive dispersal and gene flow. Correspondence: Marina L. Ramon. Present address: Department of Ecology and Evolutionary Biology, Long Marine Laboratory, Do hamlets provide an example of rapid sympatric University of California Santa Cruz, 100 Shaffer Road, COH speciation, as has been suggested for haplochromine cich- 247, Santa Cruz, CA 95064, USA. Fax: 831 4593383; E-mail: lids (Wilson et al. 2000; Lande et al. 2001), or do hamlets [email protected] represent a single, highly polymorphic species in which

2976 M. L. RAMON ET AL. interbreeding between colour morphs, while perhaps and the 5′ half of tRNA-glu was amplified and sequenced with rare, is frequent enough to prevent genetic divergence and primers, HND5.F1 (5′-CAAAATGCTTGACCAAACCTG- speciation? While potentially difficult to distinguish, a first 3′) and HtGlu.R1 (5′-GTTGAATAACAACGGTGGTTYTTC- test of these alternatives is to evaluate whether hamlet 3′). Polymerase chain reaction (PCR) and sequencing colour morphs are differentiated significantly for neutral protocols were as described in previous studies (Sorenson genetic markers, a result that would provide evidence of et al. 1999). We used ND6 because initial attempts to amplify historical reproductive isolation (Avise 2000) and potentially the control region failed. Subsequent sequencing revealed independent evolutionary trajectories (de Queiroz 1998). a major gene rearrangement involving the control region in Using allozyme markers, Graves & Rosenblatt (1980) found Hypoplectrus and Serranus (M.L. Ramon & M.D. Sorenson, no genetic differentiation among hamlet colour morphs unpubl. data). All sequences have been deposited in and concluded that hamlets comprise a single species with GenBank (Accession nos AY262168–AY262254). several colour morphs adapted for aggressive-mimicry Given the generally small number of differences observed of prey species. Although indicating clearly that hamlet among haplotypes, we constructed an unrooted tree using colour morphs are derived recently, the general lack of information from both parsimony analyses with equal allozyme variation provided little opportunity to test for weights for all changes in paup* (version 4.0b10, Swofford genetic structure (only five of 32 loci were variable, with 2002) and the statistical parsimony program tcs (Clement overall heterozygosity = 0.0005). et al. 2000). We used analysis of molecular variance We tested for genetic differentiation among hamlet (amova) (Excoffier et al. 1992) as implemented in arlequin species (or colour morphs) from Belize, Jamaica and Florida (version 2.000, Schneider et al. 2000) to test for significant and investigated the phylogeography of this group using genetic structure between geographical regions and between mtDNA sequences. Results are discussed in relation to hamlet species. For the geographical analysis, all hamlets hamlet life history and behaviour and alternative models from a given region (Belize, Florida and Jamaica, respec- for the evolution and maintenance of colour morphs. tively) were treated as a single population. Significance tests were based on 10 000 random permutations of the data set. Materials and methods Hamlets collected in Belize, Florida and Jamaica were Results identified to species based on colour morphology using Randall’s (1968) and Domeier’s (1994) descriptions (Table 1). Among 87 individuals sampled, we found 39 unique Collection sites included Pelican Cayes and Glovers Atoll haplotypes, 65 variable sites of which 44 were parsimony in Belize, Summerland Key in Florida and Discovery informative, and no length variation in the 703 bp mtDNA Bay in Jamaica, with a smaller number of fish from fragment. A large number of equally parsimonious trees other localities (see Ramon 2000 for more information on (n = 1143; length = 778; CI = 0.885) were found, but these specimens). trees comprise a relatively small number of alternative DNA was extracted from muscle or fin tissue with a connections in the context of an unrooted network (Fig. 1). DNeasy Tissue Kit (Qiagen). A 703 base pair (bp) mtDNA The network of hamlet mtDNA haplotypes has three main fragment comprising the 3′ end of the ND5 gene, all of ND6, features: (1) most of the fish sampled carry one of a set of closely related haplotypes that form a star-like phylogeny in which the length of almost all branches is one step. Table 1 Numbers and locations of samples for genetic analysis. No samples were available for H. gummigatta and H. providencianas. Uncorrected genetic distances among these haplotypes One individual appeared to be a hybrid between H. unicolor and range from 0.1% to 1.1% (1.1% = 8/703). (2) Five fish from H. puella Florida have unique haplotypes that are similar to each other but are 3.7% to 4.8% divergent from the other fish Belize Jamaica Florida Total sampled. (3) There is no obvious structuring of mtDNA variation either among hamlet species or geographical H. puella 5104 19 H. unicolor 8* 2 14 24 regions (Fig. 1). H. nigricans 10 2 3 15 amova analyses also indicate little or no structuring of H. gemma 00 1313 genetic variation either between geographical regions or H. indigo 24 1 7species. A comparison of all samples from Belize, Jamaica H. aberrans 12 0 3 Φ and Florida yields a ST value of 0.048 (P = 0.025). This H. guttavarius 01 0 1significant result, however, is due entirely to the five indi- ‘Belize Blue’ 3 0 0 3 viduals from Florida with divergent haplotypes (Fig. 1). ‘Tan’ 2 0 0 2 Total 31 21 35 87 With these five excluded, haplotype frequencies do not dif- Φ ≈ fer among the three regions ( ST 0, P = 0.61). Considering

MITOCHONDRIAL PHYLOGEOGRAPHY OF HAMLETS 2977

than the MRCA of the less common haplotype group detected only in Florida.

Discussion Genetic data for hamlets are difficult to reconcile with their morphology, behaviour and geographical distributions and raise questions about the nature of hamlet species and how they evolved. Given strongly assortative mating and somewhat different geographical distributions (Barlow 1975; Fischer 1980a; Lobel & Neudecker 1985; Domeier 1994), we expected at least some degree of genetic structure among hamlet colour morphs. While we found substantial variation in hamlet mtDNA sequences, there was limited evidence for differentiation in haplotype frequencies either between colour morphs or geographical regions, a result that is consistent with previous work based on less variable allozyme data (Graves & Rosenblatt 1980). The only result suggesting some level of genetic differentiation among colour morphs was a difference in haplotype frequencies in H. indigo compared to four other species. Given the small sample of H. indigo in our analysis, however, this result Fig. 1 Unrooted parsimony network of Hypoplectrus mitochondrial should be tested in future studies. It should also be noted haplotypes (length = 78 steps, CI = 0.885). Each geometric shape that larger sample sizes might reveal slight but significant represents an individual fish coded according to species (or colour differences in haplotype frequencies among other colour morph) and geographical location. Individuals within a box have morphs as well, providing some evidence of limited inter- identical haplotypes. Each line segment indicates a single change breeding, but the general pattern of mtDNA haplotypes in DNA sequence, except for a branch of length 25 separating a group of five divergent haplotypes from all others. Dotted lines being broadly shared among species would remain. indicate alternative, equally parsimonious connections between The general lack of mtDNA differentiation in hamlets haplotypes. An apparent hybrid between H. puella and H. unicolor can be explained by two quite different historical models, carried the most common haplotype. both of which are potentially consistent with the recognition of hamlets as distinct species. We describe each model and the kinds of data that will be needed to test them. the five hamlet species for which seven or more samples From one viewpoint, hamlet species evolved so recently were sequenced (Table 1), there is also no significant genetic and rapidly that there has not been enough time for lineage differentiation among species (with the five divergent sorting to produce significant differentiation in neutral Φ Florida haplotypes included, ST = 0.008, P = 0.29; with genetic markers (Avise 2000). Speciation in hamlets might Φ these five excluded, ST = 0.025, P = 0.11). In pairwise com- trace back to the most recent glaciation cycle during which parisons, however, H. indigo is significantly different from lower sea levels may have isolated populations in different Φ H. puella, H. unicolor, H. nigricans and H. gemma ( ST rang- areas. Domeier (1994) found that several hamlet colour ing from = 0.15–0.20, P = 0.014–0.032). Although repres- morphs have distinct population centres and suggested ented by only seven samples, H. indigo appears to have a that these might represent the original regions in which limited subset of Hypoplectrus haplotypes — six of the seven each evolved. With or without geographical isolation, H. indigo we sampled, including fish from Florida, Belize sexual selection also may have played a role in the differ- and Jamaica, had haplotypes within one to four steps of entiation of hamlet colour morphs. Assortative mating by each other (Fig. 1). colour provides a potential mechanism for the isolation of A final result of interest is the somewhat different level sympatric populations and rapid speciation (Via 2001). of nucleotide diversity in the two mtDNA clades within This model, however, raises questions about the develop- Hypoplectrus. Treating the five divergent haplotypes from ment of mating preferences. Social imprinting mechanisms, Florida as one group and all other haplotypes as a second important in determining mating preferences in some group, nucleotide diversity is higher in the former group organisms (Bolhuis 1991), presumably do not operate in (0.0077) than in the latter (0.0037), suggesting that the most species with no parental care and pelagic larvae. None the recent common ancestor (MRCA) of the more common less, empirical data indicate strongly assortative mating and and broadly distributed haplotype group is more recent a potential role for sexual selection in the diversification of

2978 M. L. RAMON ET AL. hamlets, whatever the mechanisms underlying the develop- back 2 million years or more — Bermingham et al. (1997) ment of mate preferences. estimated the rate of mtDNA evolution in geminate marine A second alternative is that the genetic similarity of fishes for ND2 at just 1.3% divergence per million years. hamlets is due to ongoing hybridization and the transfer This split may reflect earlier isolation of hamlet popula- of neutral genetic markers across species or population tions in the late Pliocene or early Pleistocene, perhaps due boundaries. In this case, the mtDNA haplotype phylogeny to changes in sea level. More recently, dispersal by pelagic for hamlets does not reflect the history of speciation in larvae has resulted in the movement of common haplotypes the group, a possibility that has been long recognized but from the Caribbean to Florida, where haplotypes charac- rarely given much consideration. Theory suggests that a teristic of an ancestral Florida population have drifted relatively low level of ongoing hybridization between a towards extinction. The northward direction of prevailing pair of species is sufficient for introgressed mtDNA haplo- currents near Florida (see Shulman & Bermingham 1995) types eventually to drift to fixation such that both species suggests that such dispersal has been predominantly from share similar mtDNA (Takahata & Slatkin 1984). The south to north and helps to explain the lack of the diver- potential result is that the MRCA of the mtDNA lineages gent Florida haplotypes in the Caribbean. found in two species is more recent than the divergence Such dispersal may also be important in introducing of the species, a pattern exactly opposite to that expected colour morphs evolved in one area to another region. A in the standard vicariant model of speciation with no question of interest in this regard is the presence in Belize subsequent gene flow (e.g. Edwards & Beerli 2000). In this of hamlets similar in appearance to H. gemma, a Florida model, sexual and/or ecological selection might maintain endemic. These ‘Belize Blue’ hamlets are distinct in mor- hamlet colour morphs as distinct populations, while regu- phology from sympatric H. indigo which, like H. gemma, lar but infrequent hybridization allows introgression of may be associated with and prey on blue chromis Chromis neutral genetic markers and causes the lineage sorting cyanea (Randall & Randall 1960; Thresher 1978; Fischer process to occur across the species complex as a whole. 1980b). Do ‘Belize Blue’ hamlets reflect the dispersal of H. Empirical observations suggest hybridization in hamlets gemma larvae from north to south (opposite the prevailing may be sufficiently frequent for this kind of process to direction) or the evolution in Belize of a novel colour occur. Fischer (1980a) observed mating between colour morph through hybridization or local adaptation? Deci- morphs in seven of 189 cases, while Domeier (1994) sug- phering the genetics of colour determination in hamlets gested that 2% of matings involved hybrid pairs. Although may be necessary to answer questions such as these, but small from a behavioural perspective, hybridization in 2– further fieldwork is also needed to determine if ‘Belize 4% of matings may have significant population genetic Blue’ hamlets represent an established population with consequences (Avise 2000). Assuming hybrids are viable positive assortative mating, and to assess their ecological and can reproduce successfully, there may be substantial similarities and differences with H. gemma and H. indigo. gene flow among hamlet colour morphs. An alternative to the above models, which presume The unusual mitochondrial genome of hamlets may also at least some level of current reproductive isolation, is contribute to the homogenization of mtDNA haplotypes that hamlet colour morphs are the expression of a complex across hamlet species. A novel gene rearrangement is found genetic polymorphism within a single biological species. in Hypoplectrus and Serranus tortugarum, but not other ser- Thresher (1978) suggested that colour morphs in hamlets ranids, suggesting a recent change in gene order (Ramon & might be maintained by balancing selection associated Sorenson, unpubl. data). After such a significant mutation with mimicry of nonpredatory reef fish. This model sug- event, subsequent mutations (such as deletions of nucle- gests, however, that matings between colour morphs are otides from a remnant control region in the original loca- frequent and generally produce offspring with one of the tion) may be more likely to be selectively advantageous as parental phenotypes. A small proportion of individuals the genome ‘adjusts’ to its new gene order. Positive selec- with hybrid phenotypes, however, are observed in the tion for mtDNA haplotypes that are more efficient in field (Fischer 1980a; Domeier 1994), consistent with limited replication, for example, would reduce the frequency of breeding between colour morphs. hybridization required to homogenize mtDNA haplotypes As discussed above, ongoing hybridization and retained across species boundaries. ancestral polymorphism following recent speciation pro- Perhaps the most interesting aspect of our results argues vide two possible explanations for the genetic similarity of for the above model rather than one of very recent and hamlet colour morphs, but discriminating between these rapid speciation. Five individuals from Florida represent- alternatives will be difficult at best. The presence within ing three different species had haplotypes that are diver- hamlets of two divergent mtDNA clades presents a poten- gent from the other hamlets we sampled. The average tial problem for the recent speciation hypothesis, although genetic distance between the two mtDNA clades is greater recent diversification of colour morphs in the Caribbean than 4%, suggesting a split between these lineages dating followed by introgression of both colour genes and mtDNA

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