Evolution, 57(7), 2003, pp. 1584±1598 RAPID SPECIATION AND ECOLOGICAL DIVERGENCE IN THE AMERICAN SEVEN-SPINED GOBIES (GOBIIDAE, GOBIOSOMATINI) INFERRED FROM A MOLECULAR PHYLOGENY LUKAS RUÈ BER,1,2 JAMES L. VAN TASSELL,3,4 AND RAFAEL ZARDOYA1,5 1Departamento de Biodiversidad y BiologõÂa Evolutiva, Museo Nacional de Ciencias Naturales, Jose GutieÂrrez Abascal 2, 28006 Madrid, Spain 2E-mail: [email protected] 3Department of Biology, Hofstra University, Hempstead, New York 11549 4E-mail: [email protected] 5E-mail: [email protected] Abstract. The American seven-spined gobies (Gobiidae, Gobiosomatini) are highly diverse both in morphology and ecology with many endemics in the Caribbean region. We have reconstructed a molecular phylogeny of 54 Gobio- somatini taxa (65 individuals) based on a 1646-bp region that includes the mitochondrial 12S rRNA, tRNA-Val, and 16S rRNA genes. Our results support the monophyly of the seven-spined gobies and are in agreement with the existence of two major groups within the tribe, the Gobiosoma group and the Microgobius group. However, they reject the monophyly of some of the Gobiosomatini genera. We use the molecular phylogeny to study the dynamics of speciation in the Gobiosomatini by testing for departures from the constant speciation rate model. We observe a burst of speciation in the early evolutionary history of the group and a subsequent slowdown. Our results show a split among clades into coastal-estuarian, deep ocean, and tropical reef habitats. Major habitat shifts account for the early signi®cant accel- eration in lineage splitting and speciation rate and the initial divergence of the main Gobiosomatini clades. We found that subsequent diversi®cation is triggered by behavior and niche specializations at least in the reef-associated clades. Overall, our results con®rm that the diversity of Gobiosomatini has arisen during episodes of adaptive radiation, and emphasize the importance of ecology in marine speciation. Key words. Adaptive radiation, Caribbean, diversi®cation rates, marine ®sh, mitochondrial DNA. Received November 8, 2002. Accepted January 24, 2003. Adaptive radiation, the evolution of ecological and phe- umenting ®sh adaptive radiations in lacustrine environments notypic diversity within a rapidly multiplying lineage has (e.g. Fryer and Iles 1972; Echelle and Korn®eld 1984; Schlu- engaged biologists for over a century (Osborne 1902; Simp- ter 1996), only a few have focused on marine ®shes (Johns son 1953; Schluter 2000). Adaptive radiations are excellent and Avise 1998; Eastman and McCune 2000). systems in which to study the factors that in¯uence speciation The American seven-spined gobies (Gobiidae: Gobioso- processes. The astonishing biological diversity of these sys- matini; proper spelling of the tribe from Gobiosomini to Go- tems may follow the evolution of key innovations in mor- biosomatini corrected by Smith and Baldwin 1999) represent phological, physiological, or behavioral traits (Stanley 1979; a possible case of a marine adaptive radiation (Hoese and Sanderson and Donoghue 1994). Alternatively, the coloni- Larson 1985). The Gobiosomatini display a great diversity zation of novel habitats is thought to provide new ecological in color, ecology, and behavior; include over 130 species in opportunity or release of competition, and hence enable lin- 24 genera; and represent some 40% of the total New World eages to colonize new adaptive zones (Simpson 1953; Schlu- gobioid genera and perhaps as many as 50% of the species ter 2000). Molecular phylogenies provide a robust framework (Birdsong and Robins 1995). The Gobiosomatini are endemic to study the temporal pattern of speciation and diversi®cation to the New World, and occur in the western Atlantic from in lineages that have undergone adaptive radiation (Johns and Massachusetts to Argentina, and in the eastern Paci®c from Avise 1998; Lovette and Bermingham 1999). The branching the northern Gulf of California to Chile, although most of pattern of a phylogenetic tree can be used to detect changes the species are found in the Caribbean region. Some genera in speciation rate through time, and to detect asymmetries are either restricted to the Atlantic or to the Paci®c, but most between contemporaneous clades in their number of descen- have representatives in both oceans. Members of the Gobio- dant species (Nee et al. 1994a; Nee et al. 1996; Sanderson somatini display a remarkable diversity in morphology and and Donoghue 1996; Pybus and Harvey 2000). Such infor- are highly selective with regard to habitat (BoÈhlke and Robins mation is needed to distinguish speciation bursts from sto- 1968). Some are found in shallow fresh waters, brackish wa- chastic background rates and to identify historical factors ters, and estuaries with mud, shell, gravel, or algae-covered underlying the emergence of ecological and phenotypic di- substrate. Others are found in coral or rocky reefs and shelf vergence within a lineage. slopes at depths exceeding 500 meters (Appendix). With the accumulation of phylogenetic data it has become Differences in dentitions indicate specialized diets, al- evident that a considerable amount of biological diversity has though little information for most species (especially the ben- arisen during episodes of adaptive radiation (Schluter 2000). thic genera) is available (BoÈhlke and Robins 1968). Some Approximately 13,000 marine teleosts are known, which ac- members of the subgenus Elacatinus are known to remove count for about 61% of the total teleost diversity (Nelson ectoparasites from other ®shes (cleaning behavior) whereas 1994). Nevertheless, in contrast to the wealth of studies doc- others are associated with sponges. One species (Nes longus) 1584 q 2003 The Society for the Study of Evolution. All rights reserved. EVOLUTION OF AMERICAN SEVEN-SPINED GOBIES 1585 FIG. 1. Map of the Caribbean region with the sampling localities for the Gobiosomatini taxa. Proximate sampling locations have been gathered into a single number (see Appendix for more details). lives commensally with snapping shrimp whereas a close were deposited in the American Museum of Natural History, association with sea urchins is found in Ginsburgellus nov- New York (Appendix; for identi®cation numbers, GenBank emlineatus and Tigrigobius multifasciatum (BoÈhlke and Rob- accession numbers, and museum voucher numbers, see the ins 1968). Electronic Appendix, available from the Evolution Editorial Phylogenetic hypotheses of the tribe based on morpholog- Of®ce at [email protected]). Here, we follow BoÈhlke and ical characters are rather inconclusive, perhaps because dif- Robins's (1968) classi®cation of the genus Gobiosoma into ferent authors have used distinct sets of characters and taxa, the subgenera Austrogobius, Elacatinus, Gobiosoma, Gar- and due to the potential of high levels of homoplasy resulting mannia, and Tigrigobius and use their criteria to assign sub- from body size reduction and/or adaptations to extreme hab- generic status to eastern Paci®c species not included in their itats (e.g. soft mud bottom, sponge-dwelling; BoÈhlke and study. Robins 1969; Hoese 1971; Van Tassell 1998). The aim of Total genomic DNA was isolated from white muscle tissue this study was to establish a mtDNA-based molecular phy- or ®n clips by proteinase K/SDS digestion, phenol-chloro- logeny of the American seven-spined gobies to study the form extraction, and ethanol precipitation (Kocher et al. dynamics of speciation in this ecomorphologically and be- 1989). Partial mitochondrial 12S and 16S rRNA genes were haviorally highly diverse group of marine ®shes. A robust polymerase chain reaction (PCR) ampli®ed using the uni- phylogenetic framework will permit us (1) to test for depar- versal primers L1091 and H1478 (Kocher et al. 1989), and tures from the constant speciation rate model and to identify 16Sar-L and 16Sbr-H (Palumbi et al. 1991), respectively. A peak periods of cladogenesis, (2) to detect asymmetries be- PCR product of approximately 1300 bp that connects the tween contemporaneous clades in their number of extant spe- above mentioned gene fragments (including the 39 end of the cies, and (3) to map habitat shifts onto the phylogeny and 12S rRNA, the complete tRNA-Val, and the 59 end of the test their association with divergence or diversi®cation events 16rRNA genes) was ampli®ed with the primers ®sh-12F1 (59- during the evolutionary history of the Gobiosomatini. TGA AGG AGG ATT TAG CAG TAA G-39) and ®sh-16SR1 (59-AAG TGA TTG CGC TAC CTT CGC AC-39). These MATERIAL AND METHODS primers also work in a variety of other acanthomorph ®sh (L. RuÈber, pers. obs.). The primer ®sh-12SF2 (59-TCT CTG Samples and Sequences TGG CAA AAG AGT-39) was used as an internal sequencing To assess the molecular phylogeny of the American seven- primer. Polymerase chain reaction ampli®cations were con- spined gobies, 65 individuals (plus four outgroups) were col- ducted in 25 ml reactions containing 75 mM tris-HCl (pH lected from 17 localities (Fig. 1 and Appendix). Whole ®sh 9.0), 2 mM MgCl2, 0.4 mM of each dNTP, 0.4 mM of each were preserved in 70±100% ethanol, and voucher specimens primer, template DNA (10-100 ng), and Taq DNA polymer- 1586 LUKAS RUÈ BER ET AL. ase (one unit, Biotools, Madrid, Spain), using the following (Sanderson 1997) as implemented in TreeEdit version 1.0 program: one cycle of 2 min at 948C, 35 cycles of 60 sec at (Rambaut and Charleston 2001). To roughly estimate diver- 948C, 60 sec at 48±548C, 90 sec at 728C, and ®nally, one gence times between major clades, we applied a molecular cycle of 5 min at 728C. clock using the Tv rate of the 12S and 16S rRNA genes of After PCR puri®cation using an ethanol/sodium acetate 0.14% per million years (Ritchie et al. 1996). precipitation, samples were cycle-sequenced with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Tests of Diversi®cation Rate Kit (V3.0) in 10 ml reactions, and following manufacturer's instructions (Applied Biosystems, Foster City, CA), with We investigated whether rates of cladogenesis have 3.25 pmol of primer, 3 ml of Terminator Ready Reaction Mix changed through time in the seven-spined gobies using the and 5% dimethyl sulfoxide.