Peripatric speciation drives diversification and distributional pattern of reef hermit crabs (Decapoda: Diogenidae: Calcinus)

Maria Celia (Machel) Malay

The presence of boundaries to dispersal has been recently documented for many Indo-West Pacific (IWP) species with planktonic propagules and a widespread distribution. We studied the phylogeography of the mangrove crab Neosarmatium meinerti (Brachyura: Sesarmidae) and the phylogenetic relationship to its presumed sister species N. fourmanoiri in the IWP in order to compare intraspecific with interspecific diversity. Portions of the mitochondrial genes 16S and CoxI were sequenced for 23 specimens of N. meinerti and 5 N. fourmanoiri, while a fragment of the 28S was obtained for a subset of specimens. Genetic data are supplemented by morphometric and based on 37 adult males of N. meinerti and 9 males of N. fourmanoiri.The conserved nuclear 28S reveals the existence of a genetic break between the Indian and Pacific oceans. Otherwise, mitochondrial genes as well as morphometry clearly support the presence of a species complex within N. meinerti composed by four well structured and geographically defined lineages: East African coast; western Indian Ocean islands; South East Asia; and Australia.

The banded coral shrimp, Stenopus hispidus (Crustacea: Decapoda: Stenopodidea) is a popular marine ornamental species with a circumtropical distribution. The planktonic larval stage lasts ∼120–253 days, indicating considerable dispersal potential, but few studies have investigated genetic connectivity on a global scale in marine invertebrates. To resolve patterns of divergence and phylogeography of S. hispidus, we surveyed 525 bp of mitochondrial cytochrome c oxidase subunit I (COI) from 198 individuals sampled at 10 locations across ∼27,000 km of the species range. Phylogenetic analyses reveal that S. hispidus has a Western Atlantic lineage and a widely distributed Indo-Pacific lineage, separated by sequence divergence of 2.1%. Genetic diversity is much higher in the Western Atlantic (h = 0.929; π = 0.004) relative to the Indo-Pacific (h = 0.105; π < 0.001), and coalescent analyses indicate that the Indo-Pacific population expanded more recently (95% HPD (highest posterior density) = 60,000–400,000 yr) than the Western Atlantic population (95% HPD = 300,000–760,000 yr). Divergence of the Western Atlantic and Pacific lineages is estimated at 710,000–1.8 million years ago, which does not readily align with commonly implicated colonization events between the ocean basins. The estimated age of populations contradicts the prevailing dispersal route for tropical marine biodiversity (Indo-Pacific to Atlantic) with the oldest and most diverse population in the Atlantic, and a recent population expansion with a single common haplotype shared throughout the vast Indian and Pacific oceans. In contrast to the circumtropical fishes, this diminutive reef shrimp challenges our understanding of conventional dispersal capabilities of marine species.

ABSTRACTCoral reefs are the epitome of species diversity rivaled only by tropical rainforests on land. Yet, the number of described scleractinian coral species, the framework-builders of coral reefs, remains moderate by comparison. DNA sequencing studies are challenging this notion by uncovering a wealth of “cryptic diversity”, but the ecological relevance of this diversity remains largely unknown. Here, we present a comprehensive case study of one of the most geographically and environmentally ubiquitous coral species in the Indo-Pacific, Pachyseris speciosa. We provide an annotated draft genome and establish through extensive resequencing that its broad ecological distribution can be explained by the presence of at least three sympatric cryptic lineages. The lineages lack morphologically diagnostic characters yet exhibit genome-wide divergence with demographic modelling indicating an ancient divergence. Targeted genotyping surveys revealed differences in their ecological distribut...

O R I G I NA L A RT I C L E doi:10.1111/j.1558-5646.2009.00848.x PERIPATRIC SPECIATION DRIVES DIVERSIFICATION AND DISTRIBUTIONAL PATTERN OF REEF HERMIT CRABS (DECAPODA: DIOGENIDAE: CALCINUS) Maria Celia (Machel) D. Malay1,2 and Gustav Paulay1,3 1 Florida Museum of Natural History and Department of Biology, University of Florida, Gainesville, Florida 32611 2 E-mail: malay@flmnh.ufl.edu 3 E-mail: paulay@flmnh.ufl.edu Received March 23, 2009 Accepted August 6, 2009 The diversity on coral reefs has long captivated observers. We examine the mechanisms of speciation, role of ecology in speciation, and patterns of species distribution in a typical reef-associated clade—the diverse and colorful Calcinus hermit crabs—to address the origin of tropical marine diversity. We sequenced COI, 16S, and H3 gene regions for ∼90% of 56 putative species, including nine undescribed, “cryptic” taxa, and mapped their distributions. Speciation in Calcinus is largely peripatric at remote locations. Allopatric species pairs are younger than sympatric ones, and molecular clock analyses suggest that >2 million years are needed for secondary sympatry. Substantial niche conservatism is evident within clades, as well as a few major ecological shifts between sister species. Color patterns follow species boundaries and evolve rapidly, suggesting a role in species recognition. Most species prefer and several are restricted to oceanic areas, suggesting great dispersal abilities and giving rise to an ocean-centric diversity pattern. Calcinus diversity patterns are atypical in that the diversity peaks in the west-central oceanic Pacific rather than in the Indo-Malayan “diversity center.” Calcinus speciation patterns do not match well-worn models put forth to explain the origin of Indo-West Pacific diversity, but underscore the complexity of marine diversification. KEY WORDS: Allopatric speciation, biodiversity, biogeography, color pattern evolution, circumtropical speciation, coral reef, coral triangle, cryptic species, crustacea, ESU, Indo-Malayan hot spot, molecular phylogenetics, sympatric speciation. The marine tropics can be divided into four broad regions defined by largely endemic biotas: the East Atlantic (EA; West African tropical coastline and offshore islands, Mediterranean), West Atlantic (WA; East American tropical coastline, Caribbean, and offshore islands including Bermuda), East Pacific (EP; West American tropical coastline to offshore islands including Galapagos and Clipperton), and Indo-west Pacific (IWP; from East Africa to Easter Island) regions (Ekman 1953; Briggs 1974). Diversity is lowest in the EA and highest, by about an order of magnitude, in the IWP (Paulay 1997). Further patterns are evident within the vast IWP, where marine biodiversity peaks in the Indo-Malayan trian- C 2009 The Society for the Study of Evolution. 2009 The Author(s). Journal compilation Evolution 64-3: 634–662 C 634 gle bounded by the Philippines, Indonesia, and New Guinea and decreases in a striking manner toward the central Pacific (Stehli and Wells 1971). Hermit crabs show a similar diversity pattern, although this has not been systematically documented (see below). Much early work focused on this striking IWP diversity pattern and attempted to find single or at least dominant processes to explain diversification in the IWP based on this pattern. Although numerous hypotheses of diversification have been proposed (Rosen 1988), three have been most emphasized: the center of origin (Ekman 1953; Briggs 1974), center of overlap (Woodland 1983), and center of accumulation hypotheses (Ladd 1960; P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S Jokiel and Martinelli 1992). These three hypotheses have attributed the origin of IWP diversity to geographically localized diversity pumps: within the Indo-Malayan area, at the boundary between the Indian and Pacific basins, and on remote, peripheral islands, respectively; with subsequent range expansion from these areas. Increasing documentation of variation in spatial diversity patterns as well as modes of speciation have, however, led to the realization that multiple processes must be involved in generating the diversity (Palumbi 1997; Paulay 1997; Williams 2007). Molecular phylogenetics provides a powerful tool for understanding the origins of observed patterns of species richness. By analyzing diverse taxa, we can address questions of diversification from a quantitative, mechanistic perspective: what is the relative importance of different modes of speciation in generating species richness and spatial patterns of diversity? In the past, analyses of spatial patterns of diversity were largely inferential (i.e., top-down): by examining biota-level patterns, researchers inferred likely processes of diversification. In contrast a quantitative phylogenetic approach provides a mechanistic (i.e., bottom-up) perspective: by documenting numerous speciation events, we can investigate how regional-level diversity patterns arise. Such an approach necessitates thorough spatial and taxonomic sampling, so that most or all speciation events in a clade are identified and characterized. Thorough taxonomic coverage is also one of the most important factors in recovering an accurate tree topology, as shown by both empirical and simulation studies (e.g., Graybeal 1998; Zwickl and Hillis 2002; Soltis et al. 2004). The objectives of this study are to pursue a comprehensive phylogenetic and biogeographic analysis of the reef-associated hermit crab genus Calcinus, to: (1) determine spatial and temporal patterns of diversification, (2) evaluate the relative importance of different modes of speciation and how they gave rise to observed patterns of diversity and distribution, and (3) assess the roles of color and ecology in diversification. Calcinus is a diverse group of diurnal, colorful, and abundant diogenid hermit crabs. The Diogenidae is the second largest of the seven families that comprise the Paguroidea, the hermit crabs, with 19 genera currently recognized (McLaughlin 2003). Diogenids are most diverse in shallow tropical waters and most medium- to large-sized hermit crabs on coral reefs are diogenids. All known Calcinus species are tropical or subtropical, most live on coral reefs, and several are facultative coral associates, frequently encountered within branching corals. The genus is circumtropical, with 41 described species (Table 1): 33 in the IWP and two to four in each of the other tropical regions (EA, WA, EP). There is substantial variation in ranges among IWP species, with some extending from East Africa to Hawaii, whereas others are known from single islands or archipelagos (Table 1). Calcinus species are most readily identified from, and a few can only be reliably differentiated based on, their color pattern (e.g., Poupin and McLaughlin 1998; Poupin and Lemaitre 2003). Partly because colors fade in preserved specimens, coloration has been underutilized in crustacean taxonomy in the past. However more effective field methods, including SCUBA, field photography, increased sampling, and appreciation of color differences, have substantially improved our knowledge of Calcinus in recent decades. Alpha taxonomy and geographic distributions are now comparatively well documented (Poupin 2003), making Calcinus an excellent focus for evolutionary and biogeographic study. We constructed mitochondrial and nuclear gene phylogenies of Calcinus based on most described species in the genus, including samples from multiple locations spanning the known ranges of most widespread species. Sequence data provide evidence for substantial cryptic diversity in the genus. In some species, color pattern appears to have evolved so rapidly that sister species with strikingly different color patterns are only slightly or not genetically differentiated. Most young sister species pairs have allopatric distributions, indicating that allopatric speciation is the main or only mechanism for diversification. Isolation on remote island groups appears to be the most common cause of speciation. The diversity pattern of Calcinus is atypical in that it peaks in the central Pacific, a pattern driven by an apparent ecological preference of many species to oceanic habitats, great dispersal ability, and predominance of peripatric speciation. Materials and Methods SPECIMENS We sampled 37 of the 43 nominal species of Calcinus and nine additional, undescribed phylogenetic species recognizable on the basis of sequence data (Table 1). The species not sequenced are Calcinus urabaensis, known from a single specimen in Colombia, Calcinus kurozumii, known only from a single collection on Pagan Island (Marianas), C. tropidomanus, known from a single collection in Somalia, and C. sirius from Australia. We also did not sample “Calcinus” paradoxus, a species based on a single specimen collected in much deeper (500 m) water than any other Calcinus, whose generic assignment even its author questioned (Bouvier 1922); nor the dwarf species C. revi, suspected to be the juvenile of more common Calcinus species (J. Poupin, pers. comm.). Much of the material was collected by reef walking, snorkeling, or scuba-diving, fixed in 75–95% ethanol, and deposited in the Invertebrate Collections of the Florida Museum of Natural History, University of Florida (UF; Table 1). Additional specimens were borrowed from other institutions (Table 1). Whenever possible living animals were photographed to record color pattern. We identified specimens using Poupin’s (2003) interactive taxonomic key, the primary taxonomic literature, and in consultation with J. Poupin and P. McLaughlin. Data on geographic ranges and ecology of species were compiled from the EVOLUTION MARCH 2010 635 636 ESU count EVOLUTION MARCH 2010 1 2 3 4 Known species of Calcinus and new ESUs, including their geographic ranges and accession information for all the sequenced specimens (including outgroup specimens). Species Calcinus albengai Poupin and Lemaitre 2003—deep morph Calcinus albengai aff. Poupin and Lemaitre 2003—shallow morph Calcinus anani Poupin and McLaughlin 1998 Calcinus argus Wooster 1984 Reported geographic range Specimen info Museum Catalog no. COI seq 16S seq H3 seq H92 MNHN Pg.6378 + + + Australs H94 MNHN Pg.6385 + + + Bismarck Arch. (PNG) Marquesas Bismarck Arch. (PNG) Mascarenes Mascarenes N Marianas Hawaii Hawaii New Caledonia Mascarenes Mascarenes W Australia W Australia Marianas Mascarenes Tuamotus Lines Lines Hawaii Hawaii NW Hawaiian Ids. NW Hawaiian Ids. NW Hawaiian Ids. NW Hawaiian Ids. Hawaii Palau Philippines H77 UF 4808 + + + H95 H324 MNHN Pg.6357 UF 11740 + + + + H62 H114 H96b H146 H192 H203 H312 H313 H101 H133 H5 H67 IP31 H306 H317 H15 H113 H292 H293 H294 H304 H307 H42 H136 UF 5437 UF 5446 UF 5714 UF 7364 UF 8038 MNHN UF 12814 UF 13022 UF 6297 UF 6297 UF 325 UF 5504 UF 1351 UF 11487 UF 11204 UF 3216 UF 8350 UF 12060 UF 12064 UF 12064 UF 12068 UF 14838 UF 3924 UF 6744 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Region W E Specimen provenance Specimen no. IWP Australs Australs Australs IWP Australs Australs IWP Japan Tuamotus; Marquesas IWP Mascarenes Hawaii 5 Calcinus dapsiles Morgan 1989 IWP W Australia W Australia 6 Calcinus elegans (H. Milne Edwards 1836) IWP S Africa Tuamotus; Marquesas 7 Calcinus elegans aff.—Hawaii IWP Hawaii Hawaii 8 Calcinus gaimardii (H. Milne Edwards 1848) IWP Maldives Fiji Continued. M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Table 1. Table 1. Continued. ESU count Species 9 10 11 13 Calcinus guamensis Wooster 1984 Calcinus haigae Wooster 1984 Calcinus hakahau Poupin and Mclaughlin 1998 Calcinus hazletti Haig and McLaughlin 1984 Specimen info Region W E Specimen provenance Specimen no. IWP Lines Tuamotus Tuamotus Tuamotus Lines Samoa Hawaii Marquesas Societies Mascarenes Hawaii Samoa Marianas Tuamotus Tuamotus Tuamotus Lines Hawaii Hawaii Lines Tuamotus Marquesas Marquesas Hawaii NW Hawaiian Ids. NW Hawaiian Ids. N Marianas N Marianas Wake Atoll Easter Is. New Caledonia H25 IP5 H190 H23 H60b H49 IP44 H58 H142 H41 H83 H82 H120 H232 H175 H139 H230 H176 H231 H51 H117 H119 H295 H302 H79 H90 H191 H38 H107 IWP IWP Somalia Red Sea Hawaii; Marquesas Hawaii; Tuamotus IWP Marquesas Marquesas IWP Hawaii Hawaii 14 Calcinus hazletti aff.—Northern Marianas IWP Japan?/N Marianas N Marianas 15 16 Calcinus imperialis Whitelegge 1901 Calcinus inconspicuus Morgan 1991 IWP IWP E Australia E Australia Easter Is. New Caledonia Museum Catalog no. COI seq UF 1349 UF 1863 UF 8604 UF 3224 UF 3219 UF 5171 UF 1888 UF 5418 UF 3219 UF 3225 UF 5713 UF 1744 UF 1332 UF 9270 UF 8372 UF 8035 UF 8035 UF 8379 UF 9269 UF 5175 UF 5166 UF 8349 UF 12157 UF 12158 UF 5732 UF 5728 UF 8438 UF 3646 MNHN + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 16S seq H3 seq + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 637 Continued. P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S EVOLUTION MARCH 2010 12 Calcinus gouti Poupin 1997 Reported geographic range EVOLUTION MARCH 2010 Continued. ESU count Species 17 Calcinus isabellae Poupin 1997 18 19 20 Calcinus kurozumii Asakura and Tachikawa 2000 Calcinus laevimanus Randall 1840 Calcinus latens Randall 1840 Reported geographic range Specimen info Museum Catalog no. COI seq 16S seq H3 seq IP42 IP20 H174 H199 H228 UF 732 UF 1758 UF 8371 UF 8449 UF 10354 + + + + + + + + + H75 H66 IP28 H76 H13 H198 IP39 H316 H110 IP7 IP9 H322 H297 H320 H308 H298 H321 H16 H109 H299 H300 H81 H314 H39 H291 H303 UF 3221 UF 5426 UF 601 UF 1720 UF 3221 UF 8445 UF 460 UF 12564 UF 5450 UF 1712 UF 1712 UF 10805 UF 10339 UF 8440 UF 10686 UF 10339 UF 8440 UF 3217 UF 3217 UF 12066 UF 12066 UF 5428 UF 5416 UF 3625 UF 12059 UF 12278 + + + + + + + + + + + + + + + + + + + + + + + + + + + Region W E Specimen provenance Specimen no. IWP Marianas Hawaii; Pitcairn Marianas Tuamotus Lines Wake Atoll Cooks Hawaii Mascarenes Marianas Tuamotus Hawaii Wake Atoll Marianas Mascarenes Mascarenes Tuamotus Tuamotus Lines Cooks Wake Atoll Lines Cooks Wake Atoll Hawaii Hawaii NW Hawaiian Ids. NW Hawaiian Ids. Oman Oman Hawaii NW Hawaiian Ids. NW Hawaiian Ids. IWP N Marianas N Marianas IWP S Africa Hawaii; Tuamotus IWP Mozambique; Yemen Tuamotus 21 Calcinus latens aff.—Hawaii IWP Hawaii Hawaii 22 Calcinus latens aff.—Oman IWP Oman Oman 23 Calcinus laurentae Haig and McLaughlin 1984 IWP Hawaii Hawaii + + + + + + + + + + + + + + + + + + Continued. M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY 638 Table 1. Table 1. Continued. ESU count Species 24 Reported geographic range Specimen info H84 IP19 H137 H177 H86 IP32 H140 H149 H27 IP33 IP43 H130 H145 H147 H26 H121 H129 IP2 H50 H37 H40 H59 H135 H193 H194 H148 UF3255 UF 1322 UF 6990 UF 8600 UF3263 UF 1321 UF 6511 UF 6982 UF 3236 UF 1350 UF 652 UF 3992 UF 6995 UF 7237 UF 1334 UF 1334 UF 6886 UF 1347 UF 5177 UF 3648 UF 3890 UF 5430 UF 8357 UF 5396 UF 6531 UF 5553 + + + + + + + + + + + + + + + + + + + + + + + + + + H309 H144 UF 12741 UF 5430 + + H63 H118b H310 H305 UF 5427 UF 5435 UF 12781 UF 12635 + + + + E Specimen provenance Specimen no. Calcinus lineapropodus Morgan and Forest 1991 IWP Cocos Keeling Tuamotus 25 Calcinus minutus Buitendijk 1937 IWP Cocos Keeling Samoa 26 Calcinus morgani Rahayu and Forest 1999 IWP S Africa Tuamotus Samoa Marianas Ryukyus Lines Samoa Marianas Philippines Ryukyus Samoa Societies Marianas Palau Ryukyus Ryukyus Societies Societies Societies Tuamotus Marquesas Easter Is. Palau Mascarenes Philippines Micronesia Philippines Papua New Guinea (Milne Bay) Mascarenes Mascarenes Tuamotus Oman Oman Mascarenes Mascarenes 27 Calcinus nitidus Heller 1865 IWP Society Tuamotus 28 29 30 Calcinus orchidae Poupin 1997 Calcinus pascuensis Haig 1974 Calcinus pulcher Forest 1958 IWP IWP IWP Marquesas Easter Is. Seychelles Marquesas Easter Is. New Caledonia 31 Calcinus pulcher aff.—Mascarenes IWP Mascarenes Mascarenes 32 Calcinus revi Poupin and McLaughlin 1998 McLaughlin 1998) Heller 1861 Calcinus rosaceus IWP Japan Tuamotus IWP Red Sea Gulf of Oman; Mauritius 33 639 COI seq W 16S seq H3 seq + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Continued. P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S EVOLUTION MARCH 2010 Museum Catalog no. Region Table 1. Continued. Species 34 Reported geographic range Specimen info EVOLUTION MARCH 2010 Museum Catalog no. COI seq 16S seq H3 seq IP36 H14 UF 562 UF3223 + + + + + + Australs New Caledonia Cooks H93b H106 H229 MNHN Pg.6395 MNHN UF 10337 + + + + + + + + + N Marianas Ryukyus Philippines Cooks Cooks Mascarenes Mascarenes Mascarenes Mascarenes H88 H131 H132 H47 H301 H311 H318 H319 H80 UF 5742 UF 6992 UF 6748 UF 1377 UF 11702 UF 12634 UF 13011 UF 13011 UF 5425 + + + + + + + + + + + + + + + + + + + + + Mascarenes Baja CA Sur Baja CA Sur Clipperton Atoll Clipperton Atoll Panama Panama Clipperton Atoll H87 H98 H99 H179 H204 H105 H111 H178 UF 5412 UF 8367 UF 15221 MNHN Pg.7617 MNHN UF 8359 UF 8359 MNHN Pg.7622 + + + + + + + + + + + + + + + + + + + + + + Florida Florida Tobago H102 H103 H124 UF 8363 UF 8364 UF 8358 + + + + + + + Bermuda H138 UF 8365 + + + Cape Verde H206 MNHN + + Region W E Specimen provenance Specimen no. Calcinus seurati Forest 1951 IWP Somalia Hawaii; Tuamotus Marianas Hawaii 35 36 37 Calcinus sirius Morgan 1991 Calcinus sirius aff. Poupin 1997 Calcinus spicatus Forest 1951 IWP IWP IWP W Australia Australs E Australia E Australia Australs Pitcairn Is. 38 IWP Somalia Somalia 39 Calcinus tropidomanus Lewinsohn 1981 Calcinus vachoni Forest 1958 IWP Mascarenes Easter Is. 40 Calcinus vachoni aff.—Cook Islands IWP Cooks Cooks 41 Calcinus vachoni aff.—Réunion IWP Mascarenes Mascarenes 42 Calcinus vanninii Gherardi and McLaughlin 1994 IWP Mascarenes Mauritius 43 Calcinus californiensis Bouvier 1898 EP Baja California El Salvador 44 Calcinus explorator Boone 1930 EP Gulf of CA Galapagos 45 Calcinus obscurus Stimpson 1859 EP El Salvador Ecuador; Colombia 46 Calcinus mclaughlinae Poupin 2006 EP Clipperton Atoll 47 Calcinus tibicen Herbst 1791 WA Clipperton Atoll Belize 48 Calcinus urabaensis Campos and Lemaitre 1994 Calcinus verrilli Rathbun 1901 Calcinus paradoxus Bouvier 1922 Calcinus talismani A. Milne Edwards and Bouvier 1892 WA Colombia Colombia WA EA EA Bermuda Azores Cape Verde Bermuda Azores Guinea 49 50 51 Ubatuba Brazil Continued. M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY 640 ESU count + + + IP15 IP18 H45 H56 Marianas Tuamotus Hawaii Marquesas UF 3639 + + + + + + + + + + + + UF 1871 UF 5433 UF 1742 IP21 H68 H32 Marianas Mascarenes Tuamotus UF 326 UF 1760 UF 3507 + + + + + + + + + + + + UF 8361 UF 8361 H91 H97 Madeira Madeira taxonomic literature, Poupin’s (2003) website on the genus, the UF specimen database, and the authors’ field observations (see Supporting Information). The diogenid hermit crabs Ciliopagurus (e.g., C. strigatus, C. tricolor, and C. galzini) and Dardanus (e.g., D. lagopodes, D. sanguinocarpus, and D. longior) were chosen as the closest outgroup taxa based on a phylogenetic analysis (not shown) of a larger set of hermit crab taxa (including nine diogenid, four pagurid, and two coenobitid genera). Samples for sequencing were selected to span as much of the geographic range of each species as available material permitted (Table 1, Figs. 3–14). We collected DNA sequence data from 150 operational taxonomic units (OTUs). All but one (C. talismani) of the 150 OTUs were sequenced for the cytochrome oxidase I (COI) mitochondrial gene fragment. We generated phylogenetic trees for the COI-only dataset, and on the basis of these trees we selected a subset of 96 OTUs for further sequencing of 16S rDNA and Histone 3 (H3) genes. The 96-OTU subset was comprised of only the two genetically most divergent individuals in each species or genetically distinct putative new species. Thus, the full 150-OTU taxon set was used for constructing the COI-only tree whereas a “pruned” subset of 96 OTUs was used for constructing individual gene trees and a concatenated three-gene tree. The ILD test for data combinability (see below) was also performed on the 96OTU subset. Lastly, molecular clock analyses (see below) were performed on a further reduced 50-OTU taxon subset, to keep computations manageable. Dardanus sanguinocarpus Degener 1925 Dardanus longior Asakura 2006 Ciliopagurus strigatus Herbst 1804 Ciliopagurus tricolor Forest 1995 Ciliopagurus galzini Poupin and Malay 2009 Dardanus lagopodes Forskal 1775 MOLECULAR METHODS Outgroups Lebanon EA Calcinus tubularis Linnaeus 1767 52 Ascension Is.; Madeira Specimen provenance E W Region Species ESU count Table 1. Continued. Reported geographic range Specimen info Specimen no. Museum Catalog no. COI seq 16S seq H3 seq P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S DNA was extracted from muscle tissue using DNAzol and proteinase K following the protocol given in Meyer 2003. Sequence data were collected for two mitochondrial DNA markers (COI and 16S) and one nuclear marker (H3). Average length of the amplified fragments and PCR primers used are as follows: COI: ∼645 base pairs (bp), primers dgLCO (5′ -GGT CAA CAA ATC ATA AAG AYA TYG G-3′ ) and dgHCO (5′ -TAA ACT TCA GGG TGA CCA AAR AAY CA-3′ ; Meyer 2003). 16S: ∼550 bp, primers 16SAR (5′ -CGC CTG TTT ATC AAA AAC AT-3′ ) and 16SBR (5′ -GCC GGT CTG AAC TCA GAT CAC GT-3′ ; Palumbi 1996). H3: ∼350 bp, primers H3af (5′ -ATG GCT CGT ACC AAG CAG ACV GC-3′ ) and H3ar (5′ -ATA TCC TTR GGC ATR ATR GTG AC-3′ ; Colgan et al. 1998). PCR thermocycler profiles for COI and 16S were as in Meyer (2003), whereas the PCR profile for H3 followed Perez-Losada et al. (2004). PCR products were either (1) cleaned using Wizard PCR Preps (Promega, Madison, WI) and sequenced using the ABI Big Dye protocol and a Perkin-Elmer ABI Automated Sequencer (Perkin-Elmer Applied Biosystems Inc., Foster City, CA); or (2) cleaned using the exo-sap cleanup protocol and sequenced at the high-throughput sequencing facility of the University of Florida’s Interdisciplinary Center for Biotechnology research (ICBR) in a 96-well format using EVOLUTION MARCH 2010 641 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY BigDyeTerminator (Applied Biosystems, Foster City, CA) cycle sequencing reactions, employing an ABI-3730-XL for electrophoresis. Initially, mitochondrial DNA sequencing was done along both directions of a DNA fragment, and as our confidence in base calls increased in later stages, only one strand was sequenced (unless base ambiguities were noted, in which case the second direction was sequenced). Histone 3 sequencing was always done on both directions. SEQUENCE ANALYSIS Chromatograms of the sequences were manually checked and edited using the software Sequencher version 4.2 (Gene Codes Corporation, Ann Arbor, MI). Sequence alignment was done by eye using Se-Al version 2.0a11 (Rambaut, http://tree.bio.ed.ac.uk/software/seal/). Sequences are available in GenBank (accession nos. FJ620149-FJ620493, EF683559EF683561) and aligned sequences are available from the authors. We also included COI data from GenBank for Calcinus obscurus (AF436039). In all analyses, all sites were weighted equally, characters were unordered, and gaps were treated as missing data. We used two approaches to decide whether to pool the threegene fragments into a single analysis. First, we used the incongruence length difference (ILD) test (Farris et al. 1995), a parsimonybased statistical test of data combinability commonly employed in phylogenetic studies. We used PAUP∗ version 4.0b10 (Swofford 2002) to perform the ILD test simultaneously for the three data partitions. No significant incongruences were noted among the three gene trees. However, the usefulness of the ILD test for evaluating data combinability has been called into question (e.g., Yoder et al. 2001; Barker and Lutzoni 2002). To address these concerns and to explore our data further, we also visually compared Bayesian tree topologies resulting from independent searches for each of the three gene regions. The gene trees were not in conflict with each other or with the three-gene concatenated analysis (data not shown). Based on this evidence, we decided that a combined analysis was appropriate. We determined the simplest model of evolution that best fit our COI-only dataset as well as our three-gene dataset using the Akaike Information Criterion (AIC) as implemented by the program Modeltest 3.6 (Posada and Crandall 1998). Phylogenetic relationships were estimated using maximum likelihood (ML), maximum parsimony (MP), and Bayesian statistics (BS). Parsimony analyses were done using PAUP, ML analyses were implemented using both PAUP and GARLI version 0.951–1 (Zwickl 2006, http://www.bio.utexas.edu/faculty/ antisense/garli/Garli.html), whereas Bayesian analyses were implemented using MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). In the MP and ML analyses using PAUP, heuristic searches started with random addition of taxa replicated 10 times using the tree-bisection-reconnection (TBR) branch-swapping al- 642 EVOLUTION MARCH 2010 gorithm. Branch support in the MP analyses was estimated by bootstrap support values, calculated as above with 1000 (for the three-gene tree) or 200 (for the COI tree) replicates. ML branch support values were not calculated using PAUP due to computational constraints. In the ML analyses using GARLI, we used random starting trees and performed—five to seven independent runs to obtain the best tree. Branch support values were estimated in GARLI using 2200 and 1300 bootstrap replicates for the COI-only and three-gene datasets, respectively. In the Bayesian analyses, we ran two independent chains for 1 million generations each; each chain was sampled every 100 generations. The MCMC runs reached stationarity in 60k generations or less. We discarded the initial 25% of the trees as the burn-in phase. Bayesian posterior probabilities were calculated based on the remaining 75% of the trees. We calculated pairwise COI genetic distances for each sister species pair identified in our phylogenetic trees using PAUP. We used Kimura’s (1980) K2P distance metric to facilitate comparison with earlier studies. ANALYSIS OF SPECIATION AND BIOGEOGRAPHY Our analysis of speciation patterns focuses on described species as well as previously undescribed, but genetically distinct evolutionary significant units (ESUs; sensu Moritz 1994). ESUs are defined as reciprocally monophyletic populations for the locus investigated (here 16S and COI mtDNA; H3 was not considered due to low levels of interspecific divergence), that have at least one other independent, defining attribute such as distinct color pattern, structural morphology, distribution, or reciprocal monophyly in another, independent marker. ESUs satisfy the phylogenetic species concept, and are clades with an evolutionary history separate from other ESUs. Some ESUs are as morphologically and genetically distinctive as described species; conversely a few described species are not reciprocally monophyletic in mtDNA (see below). ESUs are thus species-level units which, unlike biological species, can be defined in allopatric as well as in sympatric settings without experimental tests of interbreeding. We call the divergence of ESUs from each other evolutionary significant events (ESEs). ESEs are to speciation what ESUs are to species: they are objectively defined diversification events that give rise to ESUs. To quantify the relative importance of different modes of diversification, we enumerated all identifiable ESEs that have given rise to at least one individual ESU (or described species). That is, we considered ESEs that have given rise to either two separate ESUs, or led to the separation of one ESU from a clade that subsequently further diversified. Species occurrence records were mapped in ArcGIS, and species ranges inferred by drawing a polygon around bordering record points. Species were considered allopatric when they had separate ranges; such ranges may end on adjacent islands, but are P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S List of ESUs used in biogeographic analyses, and their geographic distributions relative to each other. Table 2. Clade ESU pair Distribution I II II III III III III allopatric allopatric allopatric allopatric allopatric allopatric sympatric III IV IV V VI VI VI VI VII VII VII VII VIII IX IX IX IX X X X X C. verrilli–C. tubularis C. latens–C. aff. latens Hawaii C. latens–C. aff. latens Oman C. hazletti–C. aff. hazletti N Marianas C. minutus–C. rosaceus C. minutus–C. nitidus C. haigae–C. minutus/C. rosaceus/ C. nitidus C. inconspicuus–rest of clade III C. vachoni–C. aff. vachoni Cooks C. vachoni–C. aff. vachoni Mascarenes C. spicatus–C. pascuensis C. mclaughlinae–C. obscurus C. californiensis–C. mclaughlinae/ C. obscurus C. tibicen–C. talismani C. explorator–C. tibicen/C. talismani C. gaimardii–C. morgani C. elegans–C. aff. elegans Hawaii C. imperialis–C. vanninii C. isabellae–C. imperialis/C. vanninii C. laevimanus–C. seurati inferred to occur because of peripheral records, and (2) lack of sampling of marginal occurrence will lead to an underestimation of marginal range, but lack of sampling of central occurrence will not lead to an underestimation of central range. As a second method for estimating local diversity, we also assembled species lists for relatively well-studied areas and have indicated the number of species known from these on the contour maps. The latter method is prone to the biases of geographically varied sampling methods and efforts. MOLECULAR CLOCK ANALYSIS parapatric allopatric allopatric allopatric allopatric parapatric allopatric allopatric sympatric allopatric allopatric parapatric sympatric (depthseparated) C. pulcher–C. aff. pulcher Mascarenes allopatric C. hakahau–C. gouti allopatric C. laurentae–C. hakahau/C. gouti allopatric C. lineapropodus–rest of clade IX sympatric C. albengai–C. aff. albengai deep sympatric (depthseparated) C. dapsiles–C. albengai complex allopatric C. argus–C. aff. sirius allopatric C. anani–C. argus/C. sirius sympatric separated by an open ocean. Species ranges that truly abut, or overlap for <10% of the range of the sister taxon with the smaller distribution, were termed parapatric. Table 2 lists the ESUs used in the biogeographic analyses. Diversity contour maps were generated from this data by superimposing the inferred distributional range of each species. For each species inferred species ranges were represented by a polygon as described above. These ranges were superimposed on each other to give the total number of species inferred to occur at any locality. Iso-diversity contour lines were then drawn around areas within which a given number of species are expected to occur based on the stacked species ranges. Such diversity contour maps can be biased in that (1) diversity in interior areas can be overestimated when species are actually absent from there but We used BEAST 1.4.8 (Drummond and Rambaut 2007) to estimate divergence times of Calcinus sister taxa. We did a partitioned analysis for all three genes (3-nucleotide codon partitions for COI and H3 and 1 partition for 16S) using an uncorrelated, log-normal, relaxed clock. For each partition, we specified a GTR + I + G model of sequence evolution. We estimated the time to most recent common ancestor (TMRCA) of each pair of sister species using a Yule tree prior, a UPGMA starting tree, and two independent runs of 1 × 107 generations each. Posterior distributions were sampled every 1000 generations after removing the first 10% of the MCMC chain as the burn-in. Convergence of the results was checked by loading the posterior distributions into the program Tracer. The fossil record in this group is too poor for fossil-based calibration. Instead the analysis was calibrated by specifying a prior on the divergence date of the transisthmian species pair Calcinus tibicen and C. explorator. The timing of vicariance of transisthmian sister species varies substantially among taxa, with many falling around 3.1 my (Coates and Obando 1996), but others are older (cf. Knowlton and Weigt 1998; Lessios 2008). As a preliminary approximation, we set a prior with a lognormal distribution with a mean of 1.21352716 and standard deviation of 0.28012786. This approximates a normal distribution with a mean of 3.5 my and a standard deviation of 1.0 (a normal distribution was not used because a transisthmian divergence time of zero would have had a positive probability, which is unrealistic; A. J. Drummond, pers. comm.). Results SEQUENCE ATTRIBUTES The COI region sequenced was 609 base pairs (bp) long, with 368 invariable and 238 parsimony-informative sites. Mean base frequencies were: 0.25A, 0.17C, 0.23G, 0.35T, showing an A– T bias of 60%. The 16S gene fragment contained some regions that could not be confidently aligned across all taxa. We tested the importance of these hypervariable regions by running separate analyses with and without them. The inclusion or exclusion of hypervariable regions did not result in substantial topological differences, thus they were included in the final analyses. The EVOLUTION MARCH 2010 643 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY 16S gene fragment was 459 bp long, with 276 bp invariable and 125 bp parsimony-informative sites, and mean base frequencies of 0.32A, 0.18C, 0.13G, 0.36T (A–T bias 68%). The H3 gene fragment was 336 bp long, with 279 bp invariable and 53 bp parsimony informative sites, and mean base frequencies of 0.19A, 0.34C, 0.28G, 0.19T (A–T bias 38%). The best-fit models were GTR + I + G for COI, 16S, and the combined three-gene set, and GTR-I for H3. We observed 27 insertions and deletions (indels) in the 16S gene fragment whereas COI and H3 had no indels. PHYLOGENY RECONSTRUCTION AND SPECIES BOUNDARIES The three methods of phylogenetic analyses used (MP, ML, and BS) gave congruent results, and the topologies generated from the three-gene and COI-only datasets were likewise congruent (Fig. 1A and B). Bootstrap values were higher in the three-gene trees (particularly at the deeper nodes), as expected. We thus used the three-gene trees to identify supra-specific clades within Calcinus. Ten strongly supported clades were identifiable within the genus. We defined strong phylogenetic support as >70% bootstrap values in the MP and ML trees and >95% posterior probabilities in the BS trees (see clades I–X in Fig. 1A; the sole exceptions to our criteria for defining clades were clade VII, which had a 62% bootstrap value for the ML analysis; and clade IV, which was supported by both ML and BS analysis, but had no bootstrap support under MP, nonetheless this grouping was recovered in all methods of analysis used). Relationships of ESUs within these clades were generally well resolved, but the relationships of the clades to each other was generally poorly resolved. Thus, these clades served as the basic units for our analyses of speciation patterns. Because the COI analyses (Fig. 1B) covered more individuals from more geographic locations, these were used to delineate species and ESUs. Analyses revealed nine ESUs (22% of the sampled IWP fauna) that do not correspond to previously described species. Eight of these nine are allopatrically divergent populations of described species whereas one is codistributed with its sister-species but has a nonoverlapping depth range. Three described species were not reciprocally monophyletic: Calcinus minutus, C. nitidus, and C. rosaceus are interdigitated in a mostly unresolved species complex (Fig. 1A, B, Clade III). All other nominal species for which multiple individuals were sequenced were recovered as monophyletic units with high bootstrap/posterior probability support values. Thus most named species fulfilled the ESU criterion and phylogenetic species concept (Wheeler and Meier 2000). Note that the EA species C. talismani is not represented in the COI-only phylogeny because we were unable to amplify this gene region from the available specimen. Nonetheless in 16Sonly, two- and three-gene trees, C. talismani is recovered as sister to C. tibicen. 644 EVOLUTION MARCH 2010 Excluding the C. minutus complex (see Discussion), intraspecific K2P distances ranged from 0% to 6% (1.3 ± 1.0%), with only one outlier with K2P > 4%. Pairwise, interspecific K2P distances within clades ranged from 4% to 25% (K2P) (Fig. 2A). Thus, there was no barcoding gap (Hebert et al. 2003; Meyer and Paulay 2005), but also little overlap between intraspecific and interspecific distances. Including the C. minutus complex creates a much larger overlap between intra- and interspecific differences (Fig. 2B). Of 267 pairwise intraspecific K2P distance comparisons, 10% had values >2.7%. These were within C. argus, C. pulcher s.s., C. haigae, and C. anani. These species appear to exhibit substantial geographic structuring across their range : C. argus appears to have divergent populations in the Mascarenes and Hawaii (Fig. 1B, Clade X); C. pulcher has a distinct population in the Philippines (Fig. 1B, Clade IX); C. haigae shows divergence in the Tuamotus (except for 1 individual; Fig. 1B, Clade III); and C. anani from the Marquesas and Papua New Guinea appear genetically differentiated (Fig. 1B, Clade X). Interestingly, we observed distinct color morphs for a C. anani specimen from the Philippines (not sequenced) and for juvenile C. haigae from the Tuamotus (illustrated in Poupin 2003). However, the distinct groupings were not consistently supported across all methods of analysis and small sample sizes also limit our ability to further investigate differentiation within these species. DISTRIBUTION OF CALCINUS SPECIES Our surveys led to numerous new geographic records and substantial improvement in the documentation of the distribution of Calcinus species (see Supporting Information and http://www.flmnh.ufl.edu/scripts/dbs/malacol_pub.asp for source of records). Figures 3–14 show the presently known geographic range of each species. DIVERSITY PATTERNS IN CALCINUS The species richness of Calcinus is highest in the oceanic Pacific, and does not peak in the Indo-Malayan triangle (Fig. 15). Both projected and known diversity peak in the Mariana and Tuamotu Islands, in NW and SE Oceania. Sixteen species have been recorded from the Marianas and 15 from the Tuamotus. Diversity in the Indo-Malayan triangle is substantially lower, with eight species recorded from the Philippines, nine from Indonesia, and 10 from all of New Guinea. Only 12 species have been recorded from the entire Indo-Malayan archipelago, compared with 21 species from SE Polynesia. EVOLUTIONARILY SIGNIFICANT EVENTS Twenty-four ESEs were identified in the IWP and four in other regions. Six of the IWP ESEs separate sympatric sister taxa, others are geographically structured. Of the 22 geographically structured P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S ESEs, 20 separate allopatric sister taxa and two split parapatric sisters with narrow areas of distributional overlap. Sympatric sister taxa are generally separated by deeper genetic distances than allopatric or parapatric taxa (see the following paragraphs). All 21 pairs of allopatric or parapatric sister taxa appear to have adjacent ranges as far as current sampling can document. Geographically structured ESEs span the globe, but cluster in some areas (Fig. 16); most fall in areas previously recognized as potentially important in speciation, as evidenced by a high proportion of endemics. Within the IWP, four ESEs separate ESUs in Hawaii. Three ESEs each separate ESUs between the tropical and subtropical S Pacific, and across the Indian Ocean A Figure 1. Bayesian phylograms constructed using (A) three concatenated genes and (B) COI only. The values above the branches represent parsimony bootstraps, maximum likelihood bootstraps, Bayesian posterior probabilities, respectively. EVOLUTION MARCH 2010 645 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY B Figure 1. Continued. 646 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S A 120 intraspecific interspecific 100 Frequency 80 60 40 20 0 0.00 B 0.02 0.04 0.06 0.07 0.09 0.11 0.13 0.15 K2P distance 0.17 0.19 0.21 120 0.22 0.24 intraspecific interspecific 100 Frequency 80 60 40 20 0 0.00 Figure 2. 0.02 0.04 0.06 0.07 0.09 0.11 0.13 0.15 K2P distance 0.17 0.19 0.21 0.22 0.24 Frequency distribution of K2P distances for intraspecific variation and interspecific distances in Calcinus, without (A) and with (B) the C. minutus complex. (although the exact locations of the latter separations are poorly constrained because of sparse sampling in the Indian Ocean). Two ESEs each separate ESUs in the Marquesas, SE Polynesia, and at subtropical latitudes across Australia. Single ESEs separate ESUs in Arabia and Easter Island (Fig. 16). Outside the IWP, two ESEs separate ESUs between adjacent regions (EP–WA, and Bermuda–EA), and two separate sister taxa within the EP: along the central American coast, and between Clipperton Island and the central American coast. range = 2.2–10.2 my; Fig. 17; P > 0.05, t-test) and all young (<2.5 my) divergences were among allopatric sister taxa. There is considerable spread in TMRCAs, particularly for sympatric sister species pairs. There is no temporal gap dividing the ages of strictly allopatric species from sister species that have broadly overlapping geographic ranges (Fig. 17). Discussion SPECIES BOUNDARIES TIME TO ALLOPATRY The molecular clock analyses showed that allopatrically distributed sister species pairs were significantly younger than sympatric sister species (allopatric sister species: mean = 2.0 my, range = 0.4–6.3 my; sympatric sister species: mean = 5.8 my, Although there is a general correspondence between described species and genetically defined ESUs, 22% of the taxa examined were not concordant. Three described species were not reciprocally monophyletic whereas nine ESUs represent previously undescribed (and mostly unrecognized) forms. EVOLUTION MARCH 2010 647 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Calcinus minutus, C. nitidus, and C. rosaceus failed to sort into monophyletic units (Fig. 1A, B, Clade III). Most specimens in this complex form a tight cluster (K2P < 2%, with all species combinations represented at K2P ≤ 0.5%), except for one C. rosaceus from the Mascarene Islands (K2P = 10%). These three species have allopatric, abutting ranges and are very similar morphologically; with C. minutus and C. nitidus, in particular, nearly impossible to distinguish except by color (Fig. 5; Morgan 1991; Poupin and McLaughlin 1998; Poupin 2003). Several factors can cause species-level nonmonophyly (Funk and Omland 2003). First, there may be insufficient differences in the marker used to differentiate species. We consider this unlikely because mitochondrial gene regions used cleanly resolve other Calcinus species. Second, ancestral polymorphisms may have been retained because of a slow rate of evolution or recent speciation. Although there is no evidence for a slow-down in the rate of evolution in this lineage, species divergence may have been so recent that ancestral haplotypes have not had sufficient time to sort into monophyletic clades. The virtual lack of morphological differentiation (other than color) between C. nitidus and C. minutus is suggestive of recent divergence. Third, mitochondrial haplotypes could have introgressed across species boundaries. The occurrence of a divergent sequence in one C. rosaceus specimen, sister to all others in the complex, suggests that introgression is a plausible explanation. Morphologically as well as in color pattern C. rosaceus is closest to C. haigae, the sister taxon to this complex (Fig. 5; Poupin 2003; Asakura and Tachikawa 2003). This suggests that the divergent sequence may represent the original C. rosaceus genotype, which has largely been replaced by a sweep of C. minutus haplotypes. Independent markers could provide a test of this hypothesis. The H3 nuclear sequences are not variable across this complex, and appear to lack the power to resolve this problem. Color patterns are likely under genetic, nuclear control, thus they represent separate “genetic” markers; however color may be under selection and could thus have evolved more rapidly than potentially neutral mitochondrial markers (see below). Future work with other markers is needed to resolve the status of these species. In contrast, six previously described species show marked differentiation into two or three ESUs each. In two (C. albengai (Clade X, Fig. 14) and C. elegans (Clade VII, Fig. 9)), the differentiated ESUs are conspicuous and previously noted color Figures 3–14. Distributions, color patterns, and COI phylogeny of Clade I Calcinus species. Colored symbols represent specimens available in the FLMNH collection; unfilled/black symbols represent records derived from the literature. 648 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S Figure 4. Distributions, color patterns, and COI phylogeny of Clade II Calcinus species. Symbols follow Fig. 3. forms that have not been taxonomically recognized (Poupin and Lemaitre 2003; Haig and McLaughlin 1984). In the six others (C. hazletti (Clade III, Fig. 5), C. vachoni (Clade IV, Fig. 6), C. latens X2 (Clade II, Fig. 4), and C. pulcher (Clade IX, Fig. 13), no color differences were noted during collection, but are evident in four of the five for which live images were taken. Color differences could not be discerned only in photographs of C. hazletti ESUs in Micronesia and the Hawaiian Islands (although color polymorphism has been reported in this species in Japan; Asakura 2004). No images were available for the Cook Islands C. vachoni ESU. Much of the incongruence between morphology-based species and genetic ESUs results from changing taxonomic traditions, and reflects a lack of systematic revision. Historically, carcinologists hesitated to describe species distinguished solely by a color pattern; thus, the strikingly distinctive Hawaiian color form of C. elegans (Fig. 9) has not been named (Haig and McLaughlin 1984). More recently, workers have tended to recognize such structurally similar color forms, such as the Marquesan endemic C. hakahau (Fig. 12), as distinct species (Poupin and McLaughlin 1998). A well-executed revision should rectify alternate species concepts currently in use. Species boundaries can be defined based on a variety of criteria and characters (e.g., Wheeler and Meier 2000). When taxa are sympatric and co-occurring, species limits are usually straightforward, however species delimitations are more subjective for allopatric taxa not subjected to potential interbreeding. Genetics, color pattern, structural morphology, and/or geography can all inform taxonomic delineations. We defined ESUs as reciprocally monophyletic taxa in a genetic marker, which are also distinguishable by at least one additional independent character. Three described species (C. minutus, C. nitidus, and C. rosaceus) do not meet this definition, as they are not demonstrably reciprocally monophyletic with the genetic markers used. However these three forms do have other, independent characters that correlate: color pattern and geography, implying that they are on independent evolutionary trajectories. EVOLUTION OF COLOR PATTERNS The general correspondence between color forms and ESUs indicates that color patterns are almost always reliable and sufficient for differentiating Calcinus species. Color pattern-level differentiation between morphologically similar sister species is common among hermit crabs (e.g., Asakura and Paulay 2003; Lemaitre and Poupin 2003; Poupin and Malay 2009), other crustaceans (e.g., Knowlton 1993; Macpherson and Machordom 2001; Ravago and Juinio-Meñez 2003), as well as in other taxa, such as reef fish (e.g., EVOLUTION MARCH 2010 649 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Figure 5. Distributions, color patterns, and COI phylogeny of Clade III Calcinus species. Symbols follow Fig. 3. Figure 6. Distributions, color patterns, and COI phylogeny of Clade IV Calcinus species. Symbols follow Fig. 3. 650 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S Figure 7. Distributions, color patterns, and COI phylogeny of Clades V and VI Calcinus species. Symbols follow Fig. 3. McMillan et al. 1999; Bowen et al. 2006; reviewed in Knowlton 1993). Among reef fish, there have also been documented cases of closely related species that differ strikingly in color and yet show few (if any) structural differences and are not reciprocally monophyletic at the mitochondrial level (e.g., McMillan et al. 1999; Bowen et al. 2006). If the lack of monophyly is not due to introgression, these findings imply that the rate of color pattern evolution can equal or outpace mitochondrial sequence divergence, which suggests that differentiation in coloration may be driven by selection. In Calcinus, coloration is so conspicuous and varied that it can be reasonably assumed to serve a purpose and thus be acted upon by natural selection. For example, it has been demonstrated that the size of the white chelar patch in C. laevimanus (Fig. 11) influences success in interspecific agonistic encounters (Dunham 1978). It is likely that other Calcinus species use color patterns in adaptive ways. If coloration is involved in conspecific interactions, then strong selection on these visual cues could result in the rapid color evolution, and genetically isolated populations may diverge in these cues over relatively short periods of time. Moreover, if color patterns are used for species recognition, then divergence in color may lead to the development of reproductive isolation barriers and thus speciation. Color patterns have been shown to serve in species recognition and mate choice in other marine groups, including fiddler crabs (Detto et al. 2006) and fish (McMillan et al. 1999; Puebla et al. 2007; Seehausen et al. 2008). GEOGRAPHY OF SPECIATION Speciation appears to be largely or exclusively allopatric in Calcinus, as in most animals (Coyne and Orr 2004), and allopatric separation of sister taxa is retained for more than 2 my (Fig. 17). The narrowly allopatric to parapatric ranges of all young sister taxa imply either that the geography of the original speciation event has been maintained in these taxa and there has been little postspeciational changes in distribution, or that such changes were EVOLUTION MARCH 2010 651 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Figure 8. Distributions, color patterns, and COI phylogeny of Clade VIIa Calcinus species. Symbols follow Fig. 3. Figure 9. Distributions, color patterns, and COI phylogeny of Clade VIIb Calcinus species. Symbols follow Fig. 3. 652 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S Figure 10. Distributions, color patterns, and COI phylogeny of Clade VIIc Calcinus species. Symbols follow Fig. 3. reciprocal; i.e., expansion in the range of one ESU was associated with contraction in the range of the other. Although the latter hypothesis is difficult to falsify, the former is much more parsimonious and also more likely because boundaries between sister ESUs tend to fall at recognized zones of transition associated with major dispersal or ecological barriers. Narrowly allopatric ranges also imply that localized endemics are the result of speciation rather than reliction. Endemism Figure 11. can be high on peripheral island groups, but endemics can result from either local (typically peripatric) speciation (e.g., neoendemics) or reliction (paleoendemics; see Ladd 1960; Stehli and Wells 1971; Newman and Foster 1987). Reliction refers to the survival of formerly widespread taxa; often in remote, biologically less-intense, “safe” places (Vermeij 1987). As relicts are generally older taxa that have undergone substantial reduction in their range, they are not expected to be narrowly allopatric with their Distributions, color patterns, and COI phylogeny of Clade VIII Calcinus species. Symbols follow Fig. 3. EVOLUTION MARCH 2010 653 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Figure 12. Distributions, color patterns, and COI phylogeny of Clade IXa Calcinus species. Symbols follow Fig. 3. sister taxa, but to show disjunct or sympatric ranges. None (except C. albengai; Fig. 14) of the insular endemics are sympatric or have disjunct distribution with their sister taxa. The location of geographic speciation events span the globe, but are not randomly distributed. Peripatric speciation on remote Figure 13. islands is most prevalent, whereas speciation events between the Indian and Pacific ocean basins or within the Indo-Malayan triangle are rare/absent. Speciation across ecological gradients, such as latitude and depth (Fig. 16), and between the four tropical regions is also evident. Distributions, color patterns, and COI phylogeny of Clade IXb Calcinus species. Symbols follow Fig. 3. 654 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S Figure 14. Distributions, color patterns, and COI phylogeny of Clade X Calcinus species. Symbols follow Fig. 3. Isolation on remote islands and archipelagos appears to be the most prevalent cause of speciation in Calcinus: 60% of the ESEs have resulted in at least one of the sister taxa becoming restricted to a remote island group, lending some support to the center of accumulation hypothesis. This hypothesis posits that species predominantly originate in peripheral areas, and subsequently accumulate in Indo-Malaya by distributional expansion Figure 15. across the IWP, followed by reliction to Indo-Malaya (Ladd 1960; Jokiel and Martinelli 1992). Peripheral endemics may follow one of two trajectories: (1) range expansion after establishment of reproductive barriers with their sister species, leading to buildup of regional species diversity, or (2) maintenance of restriction until eventual extinction in their isolated ranges. It is difficult to test the importance of these two alternatives, although the predominance Spatial distribution of species richness. Contour lines represent number of species expected in area based on overlay of species ranges (Figs. 3–14, see methods). Contours are drawn for 4, 10, 13, and 17 species in increasingly darker shades. Numbers represent number of documented species within the following regions and archipelagoes: Australia, Austral-Rapa, Caroline, Cocos-Christmas, Cook, Fiji, Gilbert, Hawaii, Indonesia, Japan, Line, Madagascar, Mariana, Marquesas, Marshall, Mascarene, Nauru, New Caledonia, New Guinea, Ogasawara, Palau, Philippine, Pitcairn, Ryukyu, Samoa, Society, Solomon, Taiwan, Tonga, Tuamotu, Vanuatu, and Wake. Differences between the contour line and numbered estimates are presumably the result of local undersampling. EVOLUTION MARCH 2010 655 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY Figure 16. Approximate distribution of IWP ESEs. Boundaries separating the ranges of sister taxa—and thus the location of ESEs—are drawn as lines. Numbers indicate how many ESEs occur across each of these zones. of peripatric speciation in Calcinus, combined with high diversity of widespread taxa and substantially greater age of sympatric sister species, suggest that the first trajectory may occur reasonably frequently. In contrast, reliction to Indo-Malaya has not been an important process, as demonstrated by the paucity of IndoMalayan Calcinus endemics. Insular Calcinus endemics have evolved in Hawaii (4), Marquesas (2), SE Polynesia (2), Mascarenes (2), Easter Island (1), Clipperton (1), and Bermuda (1); an additional endemic putatively assigned to the genus (C. paradoxus; see above) in the Azores has not been sampled. These are some of the most remote islands in the world, renowned for high endemism (e.g., Briggs 1974; Randall 1998), thus it is not surprising that they also host endemic Calcinus. A similar pattern of predominantly peripatric speciation has been found in Thalassoma wrasses (Bernardi et al. 2004). Among fish, the highest levels of endemism in the IWP are encountered in peripheral areas: Easter, Hawaiian, Marquesas, Mascarene islands, and the Red Sea (4.4–23% endemics; Randall 1998; Allen 2007). Among these remote island groups, we have sampled the Hawaiian Islands most thoroughly, and have sequenced nine of 10 species known from there. Of the nine, four (44%) are endemic: C. laurentae, and endemic ESUs of the widespread C. hazletti, C. latens, and C. elegans. Calcinus isabellae (known from two Hawaiian records) remains untested. In the Marquesas, two of four recorded species are endemic whereas one of three from Easter Island are. However the status of widespread species in the Marquesas and Easter remain to be genetically evaluated. Four clades appear to have given rise to multiple peripheral endemics. In two, peripatric speciation from a widespread form appears to have been the source of these endemics whereas in two others, insular endemics appear to have undergone local diversification within a basin. Calcinus elegans (Fig. 1B Clade VII, Fig. 9) and C. latens (Fig. 1B Clade II, Fig. 4), both ranging from East Africa to Polynesia, gave rise to four peripatric endemics: three on remote islands and one on the Arabian peninsula. The wide-ranging ESU is a terminal branch in both clades, implying it was the source of successive peripheral endemics. In contrast the insular-endemic sister species C. laurentae-C. hakahau-C. gouti 7 allopatric Frequency of ESEs 6 sympatric 5 4 3 2 1 0 0 0.51 1.02 1.53 2.04 2.55 3.06 3.57 4.08 4.59 5.1 5.61 6.12 6.63 7.14 7.65 8.16 8.67 9.18 9.69 10.2 Time since separation (my) Figure 17. Age distribution (in million years, my) of Calcinus sister species pairs. 656 EVOLUTION MARCH 2010 P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S (Fig. 1B Clade IX, Fig. 12) and Hawaiian-Micronesian ESUs of C. hazletti (Fig. 1B Clade III, Fig. 5) represent lineages diversifying within the central Pacific, and are only more distantly related to widespread taxa (C. lineapropodus-pulcher and C. minutuscomplex, respectively). In contrast to the abundance of peripheral speciation, Calcinus show no diversification within the Indo-Malayan area: no ESEs are identified within the area, and only one species, C. gaimardii, is (largely) confined to it (Fig. 8). This contrasts with many marine taxa that have numerous endemics in Indo-Malaya, some with substantial in situ diversification (e.g., Paulay 1997; Meyer et al. 2005; Barber et al. 2006; Williams and Reid 2004), as predicted by the center of origin hypothesis. Overall, speciation along continental shorelines appears to be uncommon in Calcinus, with the divergence between the EP species C. californiensis (Gulf of California to El Salvador) and C. obscurus (El Salvador to Peru) the only known example (Fig. 1B Clade VI, Fig. 7). Calcinus species show little differentiation between the Indian and Pacific Ocean basins. In contrast the restricted seaway between the Indian and Pacific basins is one of the most important sites of speciation for other marine taxa, with numerous well-known as well as cryptic species-pairs differentiating across the boundary between these great basins (e.g., Randall 1998; Read et al. 2006; Barber et al. 2000), as predicted by the center of overlap hypothesis. In Calcinus only two ESEs are known that may fall in this area, i.e., in the C. pulcher (Clade IX, Fig. 13) and C. vachoni (Clade IV, Fig. 6) complexes. However the location of the boundary between western and eastern ESUs of both species is poorly constrained, as no samples have been genetically tested between the Philippines/Ryukyus and Mascarenes. In contrast none of the other five widespread species tested (C. laevimanus, C. argus, C. elegans, C. guamensis, C. latens) show much genetic differentiation between populations in the Indian and Pacific Ocean basins. The genetic homogeneity of such wide-ranging species, prevalence of peripatric speciation on remote archipelagos, and diverse Calcinus assemblages on the world’s most isolated islands imply that these crabs have great powers of dispersal, and that this has influenced their modes of speciation. INTERREGIONAL COMPARISONS Although Calcinus diversity in the IWP and EP are largely the result of in situ radiation, interregional speciation was the source of Atlantic diversity. All non-IWP species studied are in two clades (I and VI). Clade I (Fig. 3) is comprised of C. tubularis (EA) and C. verrilli (Bermuda). The eastward relationship of the Bermudan endemic is unusual, as the majority of marine organisms in Bermuda originated from the WA, a result of the Gulf Stream facilitating dispersal (Sterrer 1986; Smith-Vaniz et al. 1999; Floeter et al. 2008). Clade VI (Fig. 7) is comprised of four EP, one WA, and one EA species. Close connections between the EP, WA, and EA is a common pattern among marine organisms (Briggs 1974; Paulay 1997). Species in this clade are nearly morphologically identical, but are readily distinguished by color pattern. Calcinus tibicen (WA) is sister to C. talismani (EA), and this Atlanticspecies pair is sister to C. explorator (EP), a geminate species likely isolated by the emergence of the Isthmus of Panama. The other subclade is comprised of EP species only (C. californiensis, C. obscurus, and C. mclaughlinae). Calcinus mclaughlinae is endemic to Clipperton Island whereas C. californiensis and C. obscurus have parapatric ranges along the central American coast and are absent from EP oceanic islands. Offshore EP islands mostly harbor C. explorator, a species also present in and near the Gulf of California, but not along the continental coast further south (Fig. 7). ECOLOGY Species distributional boundaries can be set by ecological limitations as well as dispersal barriers (with dispersal barriers themselves a type of ecological limitation). A prevalent form of distributional restriction in the IWP is to “continental” or “oceanic” habitats (Abbott 1960; George 1974; Paulay 1994; Reid et al. 2006). Although both can be caused by dispersal as well as ecological limitations, ecological restriction is implied for species that range widely among remote islands, but are absent from nearby continents. Pacific-plate endemism (Springer 1982; Kay 1984) is a well-documented example of such ecological oceanic restriction (Paulay 1997). Continental and oceanic habitats differ in many ways, including levels of primary productivity, terrigenous influence, habitat diversity, and presence/absence of predators and competitors that are restricted to continental shores by dispersal limitations. Oceanic restriction is prevalent in Calcinus. Thus only seven of 17 species of Calcinus recorded from Australian territories are known from the continent, the remaining species are recorded only from offshore islands (Morgan 1991). Although 12 species are recorded from Cocos Keeling and Christmas Islands, small oceanic islands just SW of Indonesia, only nine species have been recorded from all of Indonesia, the most diverse marine archipelago in the world (Fig. 15). Similar continental avoidance (including greater diversity on nearby islands than in Australia or Indonesia) occurs in several groups of terrestrial crabs and hermit crabs (Paulay and Starmer, in press). Calcinus isabellae is a classic, widespread Pacific-plate endemic (Fig. 10), and five other species appear to be regionally widespread, yet largely confined to islands: C. sirius in the South Pacific (Fig. 14), C. argus (Fig. 14) and C. seurati (Fig. 11) across the IWP, C. explorator in the EP (Fig. 7), and C. talismani in the EA (Fig. 7). An additional 16 species are restricted to one or a few neighboring oceanic island EVOLUTION MARCH 2010 657 M . C . ( M AC H E L ) D. M A L AY A N D G . PAU L AY groups, but could be so restricted by dispersal limitation as well as ecology. Conversely, only three species show largely continental restriction: C. gaimardii in the IWP (Fig. 8) and C. californiensis and C. obscurus in the EP (Fig. 7). Calcinus also includes several species restricted to relatively cool, subtropical or moderately deep (100–300 m) waters. The following species are known only from subtropical latitudes in the southern IWP: C. sirius, C. aff. sirius, C. albengai, C. aff. albengai, C. dapsiles (clade X, Fig. 14), C. spicatus, C. pascuensis (clade V, Fig. 7), C. imperialis, and C. vanninii (clade VII; Fig. 10). The origin of these taxa is predominantly by in situ diversification within the subtropics. Similar latitude-based niche conservatism has been demonstrated in gastropods (Frey and Vermeij 2008; Williams et al. 2003; Williams 2007). Only the last clade has a relatively recent and thus readily identifiable origin in the tropics, sister to the parapatric C. isabellae. All deep water species investigated (C. anani, C. albengai, C. aff. sirius) are members of clade X (Fig. 14), suggesting that invasion of deep reef habitats may have occurred only once. Interestingly this clade also includes a large portion of subtropically restricted Calcinus, implying that temperature may be an important factor limiting their distribution. Our field observations show that even the two clade X members known from relatively shallow, tropical waters (C. argus and C. anani) are rare in those habitats, but also occur in the subtropics or deep water. Sequence and/or morphological data suggest incipient differentiation in four of five described species in this clade (C. anani, C. argus, C. sirius, C. albengai), the only exception being the geographically restricted W Australian endemic C. dapsiles. Moreover, subtropical and deep reef habitats remain substantially undersampled for Calcinus, and future explorations will likely result in discovery of numerous new forms and document additional radiation. The small number of samples on hand prevents detailed analysis of speciation in this clade. Another example of niche conservatism are the sister species C. tubularis and C. verrilli (Clade I, Fig. 3). These are the only Calcinus known with sexually dimorphic behavior; with females commonly adopting a sessile habit, living in tubes of sessile turritellid and vermetid gastropods (Markham 1977; Gherardi 2004). Although there is substantial niche conservatism in Calcinus, there is also evidence for interesting ecological shifts between sister species. Sister species C. seurati and C. laevimanus (Clade VIII, Fig. 11) are the only high intertidal/supratidal Calcinus, a habitat otherwise occupied by the related (but competitively inferior—Hazlett 1981) diogenid genus Clibanarius. However although C. laevimanus lives in the upper intertidal, C. seurati is restricted to supratidal splash pools. Two ESUs of C. albengai (clade X, Fig. 14) separate by depth: one ranging from shore to <50 m depths whereas the other is exclusively deep-water (50– 280 m; Poupin and Lemaitre 2003). The role of ecology versus 658 EVOLUTION MARCH 2010 geography in the divergence of these species deserves further attention; the latter, with both forms only known from one small island, is potentially a case of sympatric speciation through niche differentiation. Such depth-related sympatric differentiation has also been reported in cnidarians (Carlon and Budd 2002; Prada et al. 2008; Eytan et al. 2009). DIVERSITY PATTERNS Calcinus species diversity is about an order of magnitude higher in the IWP than other regions, a pattern typical for reef organisms (Paulay 1997). The IWP is home to 40 ESUs, the EP, WA, and EA to four, three, and two respectively. Local diversity shows similar interregional differences, with up to 16 species coexisting in one archipelago (Marianas) in the IWP, but at most two in other regions. Calcinus species richness does not peak within Indo-Malaya, but is highest in Oceania, peaking at two locations: the Mariana and Tuamotu Islands, with 16 and 15 species, respectively (Fig. 15). This contrasts with the majority of marine taxa that reach their diversity peak in Indo-Malaya, from where richness decreases in all directions, but most conspicuously across the Pacific basin (Stehli et al. 1967; Briggs 1974; Hoeksema 2007). Nevertheless, diversity patterns, especially the steepness of the diversity increase toward Indo-Malaya varies greatly among taxa, and in large groups it arises from the composite of varied, cladespecific patterns (see Fig. 4 in Paulay and Meyer 2006). Similarly, the unusual diversity pattern in Calcinus contrasts with hermit crabs as a whole, which show the typical diversity pattern, with diversity much higher in Indo-Malaya than in the oceanic Pacific (compare McLaughlin et al. 2007 [133 paguroid species in Taiwan] with Paulay et al. 2003 [64 spp. in Guam] and Poupin 1996 [45 spp. in French Polynesia]). We propose that the diversity pattern of Calcinus is a reflection of the genus’ affinity to oceanic conditions, combined with substantial dispersal ability that has allowed species to reach remote islands. The 10 species diversity-contour extends from the Mascarene to the Hawaiian Islands, and although 13 species are recorded from the total SE Asian area, only 10 are known from any one country in the Indo-Malayan archipelago. As noted above, several Calcinus species avoid continental habitats, such that diversity is higher on oceanic islands immediately outside the IWP diversity center than on the more terrigenous and continental settings of Indo-Malaya and Australia. Finally, the predominance of peripatric speciation has lead to local diversity hot spots in peripheral locations, like in SE Polynesia, where 21 species are known, with up to 15 species recorded from a single archipelago (Tuamotus). The distribution of diversity in Calcinus contrasts with hermit crabs as a whole, implying that the typical diversity pattern of hermit crabs is a composite of different clade-specific P E R I PAT R I C S P E C I AT I O N I N R E E F H E R M I T S patterns. Similar variance in the distribution of diversity and implied modes of speciation were demonstrated among groups of cowries by Paulay and Meyer (2006). Such variance in patterns of diversity and diversification among related clades implies that multiple processes are involved in generating diversity in larger taxa. Thus, although hypotheses such as the center of origin, overlap, or accumulation may be supported in small groups, they are not general or exclusive explanations for diversification in the IWP. Conclusions Our study has uncovered a wealth of unrecognized diversity in a relatively well-known reef dweller, Calcinus. The number of ESUs in the IWP was augmented by 22%. This large increase in ESUs was made possible by our approach of intensively sampling populations of every accessible species across their range. Through photo-documentation of live specimens, we conclude that differences in coloration correspond to boundaries between ESUs, thus color patterns are very important in species delineations. We show that differences in color pattern evolve rapidly, and hypothesize that coloration may serve an adaptive purpose, such as species recognition or mate selection. This hypothesis deserves further investigation, for instance by studying the evolution of genes responsible for differences in decapod coloration. The geographic distributions of Calcinus species are now well documented and illustrate several patterns atypical for reef fauna. Non-IWP species fall into two clades. One clade connects a Bermudan endemic with an EA species, a rarely observed pattern. The second non-IWP clade groups together species from EP, WA, and EA (including one geminate species pair). This clade contains the only known instances in Calcinus of speciation along a continental margin. Among IWP species, we show that the center of species diversity is not in the Indo-Malayan triangle, but further east in the Mariana Islands, with a second peak in SE Polynesia. This may be the result of a tendency in Calcinus to prefer oceanic habitats. We found no support for either center of origin or center of overlap theories. Instead, our results show generally high-dispersal abilities coupled with peripatric speciation in remote areas. The youngest sister species pairs all have narrowly allopatric distributions, and a substantial amount of time (>2 million years, usually much longer) is needed for sister species to develop sympatric distributions. Ecological factors have also played a role in Calcinus distribution and speciation. Distributions are shaped in part by restriction of species to oceanic environments (common) or continental environments (rare). Phylogenetic conservatism of ecological niches is common, however there are also a few cases of large ecological shifts between sister species. In one instance, a shift between a shallow-water and a deep-water morph may have occurred in sympatry. A diverse range of factors, including both historical and ecological mechanisms, influence species distributions in Calcinus. Given this complexity, biogeographers should expect different taxa to show nonidentical biogeographic patterns, reflecting the unique histories and ecological adaptations of different groups. The overall top-down picture of marine biodiversity is a summation of all these individual histories. ACKNOWLEDGMENTS We are deeply grateful to J. Poupin for his advice, encouragement, specimens, and for kind permission to use the images and information posted on his excellent website on Calcinus. D.L. Rahayu kindly lent us a paratype specimen, and R. Cléva facilitated access to the MNHN collections. We thank P. McLaughlin and J. Poupin for help with some of the identifications; C. Meyer, L. Kirkendale, and J. Light for advice on phylogenetic analyses; M. Bemis and J. Slapcinsky for curatorial support; and M. Gitzendanner and the University of Florida Phyloinformatics Cluster for High Performance Computing in the Life Sciences for computational support. We thank M. Hellberg and three anonymous reviewers for their helpful comments on the manuscript. We wish to thank the many people who contributed specimens to this study: D. Eernise, M. Frey, J. Hooper, L. Kirkendale, J. O’Donnell, B. Olaivar, C. Pittman, J. Poupin, L. Rocha, W. Sterrer, and P. Wirtz. Photo credits: J. Poupin (C. californiensis, C. explorator, imperialis, inconspicuus, mclaughlinae, orchidae, pascuensis, aff. sirius), L. Albenga (albengai, aff. albengai), J. Hoover (hakahau), W. Sterrer (verrilli), C. d’Udekem d’Acoz (tubularis), P. Wirtz (talismani), J. Okuno (anani), and D.L. Felder (obscurus). All other photos by G. Paulay and M. Malay. 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Hellberg Supporting Information The following supporting information is available for this article: Appendix S1. Calcinus_records_L_S_v2.xls Supporting Information may be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting informations supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. 662 EVOLUTION MARCH 2010

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