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Phylogeography of the Pantropical Sea Urchin Eucidaris in Relation to Land Barriers and Ocean Currents

Phylogeography of the Pantropical Sea Urchin Eucidaris in Relation to Land Barriers and Ocean Currents

Evolution, 53(3), 1999, pp. 806-817

PHYLOGEOGRAPHY OF THE PANTROPICAL IN RELATION TO LAND BARRIERS AND OCEAN CURRENTS

H. A. LESSIOS,1,2 B. D. KESSING,1 D. R. ROBERTSON,1 AND G. PAULAY3 1SmithsonianTropical Research Institute,Box 2072, Balboa, Panama 2E-mail. lessiosh@ naos.si. edit 3MarineLaboratory, University of Guam, Mangilao, Guam 96923, USA E-mail: gpaulay@ uog9.uog. edu

Abstract.-Thepantropical sea urchingenus Eucidaris contains four currently recognized , all ofthem allopatric: E. metulariain theIndo-West Pacific, E. thouarsiin theeastern Pacific, E. tribuloidesin boththe westernand eastern Atlantic, and E. clavata at the central Atlantic islands of Ascension and St. Helena. We sequenced a 640-bp region of the cytochromeoxidase I (COI) gene of mitochondrialDNA to determinewhether this division of the genusinto species was confirmedby molecularmarkers, to ascertaintheir phylogenetic relations, and to reconstructthe history of possible dispersaland vicarianceevents that led to present-daypatterns of species distribution.We foundthat E. metulariasplit first from the rest of theextant species of thegenus. If COI divergenceis calibratedby theemergence of the Isthmusof Panama, the estimateddate of the separationof the Indo-WestPacific species is 4.7-6.4 million yearsago. This date suggeststhat the last available routeof geneticcontact between the Indo-Pacificand therest of the tropicswas fromwest to east throughthe EasternPacific Barrier, rather than through the Tethyan Sea or around the southerntip of Africa.The secondcladogenic event was the separationof easternPacific and Atlanticpopulations by the Isthmusof Panama.Eucidaris at the outereastern Pacific islands (Galapagos, Isla del Coco, ClippertonAtoll) belong to a separateclade, so distinctfrom mainland E. thouarsias to suggestthat this is a differentspecies, for whichthe name E. galapagensisis revivedfrom the older taxonomicliterature. Complete lack of sharedalleles in threeallozyme loci betweenisland and mainlandpopulations support their separate specific status. Eucidaris gala- pagensisand E. thouarsiare estimatedfrom their COI divergenceto have splitat aboutthe same timethat E. thouarsi and E. tribuloideswere being separatedby theIsthmus of Panama. Even thoughcurrents could easily conveylarvae betweenthe easternPacific islands and the Americanmainland, the two species do not appearto have invadedeach other'sranges. Conversely, the centralAtlantic E. clavata at St. Helena and Ascensionis geneticallysimilar to E. tribuloidesfrom the Americanand Africancoasts. Populationson these islands are eithergenetically connected to the coasts of the Atlanticor have been colonizedby extantmitochondrial DNA lineages of Eucidariswithin the last 200,000 years. Althoughit is hard to explain how larvae can cross the entirewidth of the Atlanticwithin their competentlifetimes, COI sequencesof Eucidaris from the west coast ofAfrica are very similar to thoseof E. tribuloides fromthe .FST statisticsindicate that gene flowbetween E. metulariafrom the Indian Ocean and fromthe westernand centralPacific is restricted.Low geneflow is also evidentbetween populations of E. clavatafrom Ascension and St. Helena. Rates of intraspecificexchange of genes in E. thouarsi,E. galapagenisis, and E. tribuloides,on the otherhand, are high.The phylogenyof Eucidaris confirms Ernst Mayr's conclusions that major barriers to thedispersal of tropicalechinoids have been thewide stretchof deep waterbetween central and easternPacific, the cold wateroff the southwestcoast of Africa,and the Isthmusof Panama. It also suggeststhat a colonizationevent in the eastern Pacifichas led to speciationbetween mainland and island populations. Key words.-Biogeography,cytochrome oxidase, gene flow,islands, mitochondrial DNA, ocean currents,sea urchins.

Received May 1, 1998. AcceptedJanuary 25, 1999.

In 1954 Ernst Mayr published an article on geographic raphy can provide informationdifficult to obtain fromother speciation of tropical echinoids (Mayr 1954). In this paper characters, such as the existence of sibling species and the he plotted the ranges of species belonging to 16 sea urchin, timing of splits between populations (Palumbi 1996, 1997). sand dollar, and heart urchin genera as given in Mortensen's The present paper is an attemptto apply molecular tools to (1928-1951) monograph, and reached the conclusion that the species of the Eucidaris to evaluate Mayr's con- theirgeographic distributionswere consistentwith allopatric clusions. speciation. Geographical barriers seen by Mayr as causing Eucidaris belongs to the subclass , sep- speciation were the wide stretchof deep water dividing the arated from all other extant Echinoidea at least since the eastern Pacific from the central Pacific (the Eastern Pacific (Durham 1966; Smith 1984). The fossil record of Barrier), the Isthmus of Panama, and the cold waters off Eucidaris dates back to the Upper Eocene (Fell 1966; Cutress southwest Africa. He wrote that the genus Eucidatris "illus- 1980), approximately50 million years ago (mya). The genus trates geographic speciation almost diagrammatically." is pantropical, with all of its species concentratedin shallow Mayr reached these conclusions while recognizing thathe water (< 570 m; Mortensen 1928-195 1). Its fourrecognized was dealing with morphospecies ratherthan biological spe- morphospecies have adjacent but nonoverlapping geograph- cies and that echinoid systematics were still at the stage of ical distributions. ranges from the east alpha . We now have the tools to investigate what coast of Africa to the central Pacific. Eucidaris thouarsi is Avise et al. (1987) have called "phylogeography," that is, found in the eastern Pacific. is distrib- intra- and interspecificphylogenies, which in combination uted fromthe Atlantic coast of tropical America to the west- with distributionalinformation can help deduce patternsand ern coast of Africa. Eucidaris clavata is endemic to the central causes of divergence and speciation. Molecular phylogeog- Atlantic islands of Ascension and St. Helena. The morpho- 806 ?) 1999 The Society forthe Study of Evolution. All rightsreserved.

This content downloaded on Thu, 17 Jan 2013 14:37:51 PM All use subject to JSTOR Terms and Conditions PHYLOGEOGRAPHY OF EUCIDARIS 807 logical differencesbetween the species are slight. There are DNA extractions, polymerase chain reaction (PCR) ampli- doubts in the taxonomic literatureas to whether the Gala- fication,PCR productpurification, and DNA sequencing were pagos populations of Eucidaris belong to E. thouarsi or carried out as described previously (Lessios et al. 1998). whether they constitute a separate species, E. galapagensis Primers for both PCR and sequencing were: either COI-f 5' (Mortensen 1928-195 1, vol. I, p. 399) and as to whetherE. (CCTGCAGGAGGAGGAGAYCC) or COI-p 5' (GGTCA- clavata is a separate species fromE. tribuloides(Mortensen CCCAGAAGTGTACAT) and COI-a 5' (AGTATAAGC- 1928-1951, vol. I, p. 411). Eucidaris tribuloides on the Af- GTCTGGGTAGTC). All individuals were sequenced in both rican coast has been recognized as a separate subspecies, E. directions. Phylogenies from mtDNA data were constructed tribuloides africana (Mortensen 1928-1951, vol. I, p. 406). withtest version 4.0d64 of PAUP*, writtenby David L. Swof- We used sequence data from a 640-bp fragmentof the ford and used with his permission, and with PUZZLE 4.0, cytochrome oxidase I (COI) region of mitochondrial DNA writtenby Strimmerand von Haeseler (1996). Other analyses (mtDNA) and isozymes to determinethe validity of species were performedwith SEQUENCER 4.0, an Apple Macintosh described on the basis of morphology and to ascertain their program writtenby B. D. Kressing and available fromhim. phylogenetic relations. Specifically, we were interested in Sequences have been deposited in GenBank under accession answering the following questions: (1) Is each geographic nos. AF063309-AF063399 and AF107700-AF107721. isolate a separate evolutionary lineage? (2) To what extent do the accepted morphospecies coincide withmtDNA clades? Isozymes (3) What is the order and timing of cladogenic events, and what can theytell us about the effectsof presumed geographic Because the COI sequence data indicated that Eucidaris barriers on the speciation of tropical marine organisms? from the outer eastern Pacific islands (Galapagos, Isla del Coco, and Clipperton) were a distinctlineage fromE. thouarsi

MATERIALS AND METHODS from the American coast (see results), isozymes were used to determine whether genetic discontinuities in the eastern Collections Pacific extended to the nuclear genome. Twelve loci were assayed in fromthe Galapagos and Isla del Coco in One hundredtwelve individuals were collected formtDNA the same buffersand with the same staining recipes as those sequencing at the following locations for each species (see of Berminghamand Lessios (1993). The data were compared Fig. 1): E. metularia: Reunion, Indian Ocean (n = 4); Ishi- with data from the same loci obtained previously by these gaki-Jima,Sakishima Islands, Japan (n = 2); Sesoko, Oki- authorsfrom coastal populations at Mexico and Panama. Pre- nawa-Jima,Ryukyu Islands, Japan (n = 4); Guam, west Pa- sumptive alleles were standardized by running individuals cific (n = 5); and Hawaii (n = 15, two specimens fromLanai, fromPanama on the same gels as those fromGalapagos and one fromMaui, 12 fromOahu); E. thouarsi: Galapagos (n = Isla del Coco. The assayed loci were: acid phosphatase 7, two specimens from each of the islands of Bartolome, (Acph), aspartate aminotransferase(Got-], Got-2), fructokin- Fernandina, and Genovesa, and one from Isabela); Isla del ase (Fk), hexokinase (Hk), malate dehydrogenase (Mdh-]), Coco (n = 6); Clipperton Atoll (n = 1); Guaymas, Sea of L-leucyl-L-tyrosine peptidase (Peplt-]), phosphoglucose Cortez, Mexico (n = 6); and Taboguilla Island, Bay of Pan- isomerase (Pgi), phosphoglucomutase (Pgm), octanol dehy- ama (n = 4); E. tribuloides: Carrie Bow Cay, Belize (n = drogenase (Odh), superoxide dismutatse (To), and xanthine 11); Cochino Pequefno,Bay Islands, Honduras (n = 2); San dehydrogenase (Xdh). Calculation and comparison of jack- Blas Islands, Panama (n = 10); Puerto Rico (n = 4); St. John, knifedaverage Nei's (1978) genetic distances were performed U.S. Virgin Islands (n = 3); Cayman Brac, Cayman Islands according to the method of Mueller and Ayala (1982), em- (n = 1); Tamandare, Brazil (n = 4); Sao Tome, Gulf of Guinea ploying program NEIC (Lessios 1990). (n = 10); Ada, Ghana, Gulf of Guinea (n = 1); E. clavata: St. Helena (n = 7); Ascension (n = 5). We rooted the phy- RESULTS logenetic tree of Eucidaris with one individual of Phylla- canthus imperialis from Rottnest Island, western Australia. Phylogenetic Groupings and Genetic Similarities Cladistic analysis of morphological characters suggests that Phyllacanthus is the sister genus of Eucidaris (Smith and Figure 2 presents a maximum-likelihoodreconstruction of Wright 1989). Samples were preserved in 95% ethanol, in the phylogeny of Eucidaris, as inferredfrom the determined high-salt DMSO buffer(Seutin et al. 1991), or in liquid N2. COI sequences. In addition to the presented tree produced Forty-twoindividuals were collected for isozyme analysis at by the quartet puzzling algorithmof Strimmerand von Hae- Isla del Coco (n = 20) and at Galapagos (n = 22, two in- seler (1996), we also reconstructed phylogeny using the dividuals fromIsabela, five fromBartolome, four fromGen- neighbor-joining algorithm of Saitou and Nei (1987), with ovesa, six from Marchena, two from Santiago, three from distances correctedby Hasegawa et al.'s (1985) model of base Fernandina); all were frozen in liquid N2. substitution. The neighbor-joining tree was bootstrapped 1000 times. When branches with less than 70% supportwere Mitochondrial DNA collapsed, the tree topologies produced by the two techniques were identical, except for some minor rearrangementsof the We amplifiedand sequenced 640 nucleotides fromthe COI terminalbranches. region, corresponding to positions 6448 (or, depending on There is strong support for the existence of four clades the primer,6499) to 7128 of the mitochondrial genome of (see Fig. 2). The Indo-Pacific E. metularia (clade A) is an Strongylocentrotuspurpuratus (Jacobs et al. 1988). Genomic outgroup to all otherEucidaris. We were able to obtain spec-

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Ascension-1,2 81 Ascension-3;St. Helena-i E. clavata St. Helena-2,3, 4, 5, 6, 7 St. John-I PuertoRico- I San Blas-I San Blas-2 Honduras-I Belize- 1, 2; Caymans-1; < Honduras-2;San Blas-3, 4, 5, 6; Belize-3 PuertoRico-2; St. John-2 55% Belize-4 Belize-5 Belize-6 Belize-7; PuertoRico-3 Belize-8 Brazil-1 J Brazil-2 PuertoRico-4; Sao Tom6-1; 74 Ghana Brazil-3,4; San Blas-7, 8 E. tribuloides Sao Tom6-2 Sao Tom6-3 Sao Tom6-4 Sao Tome-5 Sao Tome-6 Sao Torn6-7 Sao Tom6-8 Sao Tom6-9 Belize-9 San Blas-9 Belize-10 St. John-3 94 Belize-iI Sao Tom6-IO San Blas-IO Coco- 1; Galapagos-I Galapagos-2 Galapagos-3 Galapagos-4 Coco-3 C - iCoco-32 E. galapagensis Coco-4 Coco-5, 6; Galapagos-5, 6, 7 Clipperton-I 88 Mexico-i 94 Mexico-2 Mexico-3; Panama-I ______I B Mexico-4 E. thouarsi 100 Panama-2,3 Panama-4 Mexico-5 Mexico-6 80 Hawaii-l Hawaii-2 - Reunion-1 - Ishigaki-l Reunion-2 Reunion-3 Reunion-4 Hawaii-3 Hawaii-4

Hawaii-6 E. metularia Hawaii-7 Hawaii-8, 9 Hawaii-10 Hawaii-lI, 12, 13, 14, 15 Sesoko-1 Ishigaki-2 Guam-1, 2 Guam-3 Guam-4,5; Sesoko-2, 3, 4

Phyllacanthus imperialis

FIG. 2. Maximum-likelihoodtree of COI haplotypesof Eucidaris,generated by the quartetpuzzling technique of Strimmerand von Haeseler (1996), using Hasegawa et al.'s (1985) model of base substitution.Multiple labels nextto a branchindicate that the same haplotypewas observedin morethan one individual.Numbers next to nodes indicatepercent of quartetsthat support them. Only nodes withmore than 70% supportare shown.Letters next to branchesmark major clades.

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TABLE 1. Numberof sampledindividuals and gene frequenciesin TABLE 2. Averagejackknifed means of Nei's D distancesbetween 12 electrophoreticallyassayed loci of easternPacific populations easternPacific populations, assayed for 12 electrophoreticallyde- of Eucidaris.Locus abbreviationsare explainedin the text.Allele terminedloci. designationsindicate relative distance from the origin.Data forE. thouarsiare fromBermingham and Lessios (1993). Got-2,Odh, and Mexico Taboguilla Isla del Coco Xdh were monomorphicin all populations. Taboguilla 0.084 E. galapagensis Isla del Coco 0.639 0.580 E. thouarsi Galapagos 0.718 0.625 0.000 ______Isla del Locus Allele Mexico Taboguilla Coco Galapagos Acph n 30 28 18 22 sents E. thouarsi sensu stricto. Specimens from Galapagos, 90 0.133 0.196 0.472 0.546 Isla del Coco and the Clipperton Atoll cluster in a separate 100 0.833 0.786 0.528 0.455 110 0.033 0.018 0.000 0.000 clade (C), which is likely to be a differentspecies. Reviving Fk n 19 33 20 22 D6derlein's (1887) name, we have labeled it as E. galapa- 80 0.000 0.015 0.000 0.000 gensis. The 640 mtDNA bases we sampled contain 33 nu- 90 1.000 0.924 0.275 0.273 cleotide sites in which E. thouarsi and E. galapagensis are 98 0.000 0.000 0.650 0.568 consistently different.In keeping with the high degree of 100 0.000 0.061 0.000 0.000 120 0.000 0.000 0.075 0.159 conservatism of amino acid composition of COI, all of these Got-i n 32 76 16 22 fixed differences are silent. The single specimen we were 100 1.000 1.000 0.000 0.000 able to obtain from Clipperton Atoll is sufficientlydifferent 110 0.000 0.000 1.000 1.000 fromall others coming from eastern Pacific islands (Kimura Hk n 29 44 20 22 [1980] two-parametercorrected percent difference [K2] of 90 0.000 0.045 0.000 0.000 1.79%) to suggest that it may belong to an additional evo- 100 1.000 0.955 0.000 0.000 lutionarylineage. The differentiationof Eucidaris at this re- 110 0.000 0.000 1.000 1.000 mote Atoll fromother populations of the easternPacific needs Mdh-i n1 37 55 19 22 97 0.000 0.000 0.842 0.932 to be investigated further. 98 0.000 0.009 0.000 0.000 Isozyme results from eastern Pacific populations confirm 100 0.405 0.109 0.158 0.068 that Eucidaris populations from Isla del Coco and from the 103 0.595 0.855 0.000 0.000 Galapagos are very similar to each other and that no gene 105 0.000 0.027 0.000 0.000 flow has occurred between island and mainland populations Peplt-] n 33 56 20 22 80 0.879 0.000 0.000 0.000 during recent evolutionary time. The most common allele in 90 0.121 0.170 0.100 0.114 five of the 12 assayed loci is differentbetween the islands 100 0.000 0.750 0.900 0.886 and the American coast. In three of these loci there is com- 110 0.000 0.080 0.000 0.000 plete partitioningof alleles (Table 1). Nei's D between pop- Pgi n 30 26 19 ulations at Galapagos and Isla del Coco is less than 0.001 94 0.000 0.077 0.000 (Table 2), whereas the average jackknifed Nei's D between 95 0.000 0.077 0.105 97 0.050 0.000 0.000 E. galapagensis and E. thouarsi is 0.641, which is not sig- 100 0.900 0.692 0.711 nificantlydifferent (P > 0.05) from the value of Nei's D 103 0.050 0.154 0.184 (0.758) calculated from the same 12 loci (of 25 sampled by Pgm n 25 35 18 22 Bermingham and Lessios 1993) between E. thouarsi and the 70 0.020 0.057 0.000 0.000 Caribbean E. tribuloides. 90 0.100 0.157 0.028 0.023 100 0.640 0.671 0.944 0.977 Clade D in Figure 2 is composed of all individuals from 110 0.200 0.071 0.028 0.000 the Atlantic, irrespectiveof whetherthey belong to the west 120 0.040 0.043 0.000 0.000 Atlantic E. tribuloides,the African E. tribuloidesafricana, or To , 22 46 20 22 the central Atlantic E. clavata. Within this clade, there are 100 0.000 0.000 1.000 1.000 no resolved subclades, except for one joining all haplotypes 105 1.000 1.000 0.000 0.000 fromAscension and one from St. Helena (which is identical to one of the Ascension haplotypes). The low degree of phy- logenetic structureis the result of high haplotype similarity. imens of this species only from five localities, and with this There are no fixed differencesbetween Caribbean, Brazilian, limited sample it is difficultto draw conclusions regarding and AfricanE. tribuloides.A single transitionat a thirdcodon the number of distinct lineages that are trulyrepresented in position is diagnostic between E. clavata from Ascension this vast oceanic expanse. However, despite the long distance (and one individual fromSt. Helena) and E. tribuloidesfrom separating Reunion from Guam (a straight-linedistance of the American and African coasts. A thirdposition transition > 10,000 km) and from Hawaii (17,000 km), the mtDNA at a differentsite distinguishes the remaining six individuals lineages do not form separate clades, indicating that even from St. Helena fromcontinental E. tribuloides. Thus, some very distant populations of E. metularia have not evolved differentiationexists between E. clavata and E. tribuloides. independently. However, the mtDNA divergence between Eucidaris fromthe The data clearly indicate thatthere are two distinctlineages Caribbean and the central Atlantic islands (average K2 = of Eucidaris in the easternPacific. All coastal specimens from 0.49) is no larger than the differentiationseen between E. Mexico and Panama belong to one clade (B), which repre- clavata from St. Helena and from Ascension (K2 = 0.64).

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TABLE 3. FST-values(below the diagonal) and estimatednumber of femalepropagules (Nen; above thediagonal) betweenpopulations of Eucidaris in which at least fourindividuals were sampled.Calculations follow Hudson et al. (1992); ud, undefinedvalue of Nem because FST forthis comparison is negative.

Indo-WestPacific Species Region Reunion Ryukyu Guam Hawaii E. metularia Reunion 0.79 0.53 0.22 E. metularia Ryukyu 0.39 ud 0.88 E. metularia Guam 0.48 -0.09 1.23 E. metularia Hawaii 0.70 0.36 0.29 Eastern Pacific Species Region Panama Mexico Isla del Coco Galapagos E. thouarsi Panama ud 0.02 0.02 E. thouarsi Mexico -0.03 0.04 0.04 E. galapagensis Isla del Coco 0.96 0.93 ud E. galapagensis Galapagos 0.95 0.93 -0.06 Atlantic Species Region Ascension St. Helena Puerto Rico Belize San Blas Brazil Sao Torn6 E. clavata Ascension 0.19 0.51 1.44 0.66 0.55 1.11 E. clavata St. Helena 0.73 0.53 6.28 1.12 0.52 4.77 E. tribuloides Puerto Rico 0.50 0.49 ud ud ud ud E. tribuloides Belize 0.26 0.07 -0.36 ud ud 4.50 E. tribuloides San Blas 0.43 0.31 -0.10 -0.07 596.41 2.31 E. tribuloides Brazil 0.48 0.49 -0.08 -0.22 0.01 ud E. tribuloides Sao Tome 0.31 0.09 -0.16 0.10 0.18 -0.11

Similarly,the differencebetween E. clavata and E. tribuloides only negative, but also not significantlydifferent (P = 0.938) africana (K2 = 0.75) is only slightly larger than the differ- from that generated from 500 random reshufflingsof hap- ences within E. clavata. lotypes. There is also high apparent gene flow between the Estimates of gene flow withinthe Indo-West Pacific based American and the African coast. Each western Atlantic pop- on FST-values (Table 3) suggest that,despite the lack of dis- ulation of E. tribuloides exchanges genes with Sao Tome at tinct evolutionary lineages, some degree of genetic isolation rates higher than 2.3 females per generation (Table 3). How- exists between the Indian and the Pacific Oceans. Although ever, the genetic connections between E. tribuloides and E. populations fromGuam and the Ryukyu Islands are panmic- clavata seem more variable. High gene flow is estimated tic, and Hawaii and Guam are also connected by fairlyhigh between the African coast and the central Atlantic islands gene flow, Reunion, as might be expected, is more differ- and between the central Atlantic islands and Belize. Puerto entiated. Gene flow between this Indian Ocean island and the Rico, San Blas, and Brazil, despite being genetically con- western Pacific is less than the equivalent of one female nected with Belize and with the African coast, appear to be propagule per generation. Overall, the values of FST in E. contributingfewer genes toward connections withE. clavata. metularia are correlated with the straight-linedistance be- Interestingly,although Ascension and St. Helena are sup- tween localities. In the easternPacific, mtDNA differentiation posed to be both inhabited by E. clavata, and even though between localities within each presumed species is less than the distance between them (1300 km) is smaller than that diversitywithin each locality. However, estimated migration between the American and the African coast (a minimumof between mainland E. thouarsi and island E. galapagensis is about 2800 km), the estimated number of female propagules almost equal to zero, confirmingour conclusion that island exchanged between the two islands is the lowest for any and mainland populations must have remained isolated for a comparison within the Atlantic. long time. In the Atlantic,most populations are geneticallyconnected. Timing of Cladogenic Events The two populations in the Caribbean fromwhich we sampled 10 or more individuals, San Blas and Belize, contain hap- How long ago did these clades diverge from each other? lotypes that are more similar between than within localities, Mitochondrial DNA differentiation(see Table 4), under the leading to a negative FST-value. Eucidaris tribuloides from assumption of constant rates of evolution, can provide an Brazil, despite substantial distance (4000 km along the coast estimate. This estimate cannot be very precise, because in fromthe closest point in the Caribbean) and possible barriers Eucidaris evolution of the sequenced segment is not all that to echinoid dispersal due to the freshwaterdischarge of the constant. If there were no rate variation between lineages, E. Orinoco and the Amazon, is also connected to the Caribbean metularia would have been equidistant fromall otherspecies with high levels of gene flow. Indeed, the FST-values suggest and E. tribuloideswould be equidistant fromE. galapagensis that Eucidaris from the entire western Atlantic belong to a and E. thouarsi. However, the average K2 distance between single panmictic unit. The overall FST-value forcomparisons E. metularia and each of the otherspecies ranges from16.01% among all Caribbean localities plus Brazil (-0.143) is not (E. rnetularia-E. clavata) to 19.85% (E. rnetularia-E. gala-

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TABLE 4. Means of nucleotidepercent difference between species of Eucidaris.Values below thediagonal are percentdifference in all sites(K9), correctedwith Kimura's (1980) two-parametermodel. Values on thediagonal (in bold) areaverage K9-values between individuals withina species. Values above thediagonal are percentnucleotide differences in silentsites (Ks), estimatedfrom the equations of Pamilo and Bianchi (1993) and Li (1993).

Species Region E. clavata E. meti/aria E. galapagensis E. thouarsi E. tribidloides E. clavata CentralAtlantic 0.42 72.72 28.42 39.30 1.53 E. metularia Indo-WestPacific 16.01 1.12 131.74 90.52 74.55 E. galapagensis Centraleastern Pacific 8.29 19.85 0.58 31.12 29.21 E. thouarsi East easternPacific 10.83 18.78 9.11 0.72 40.28 E. tribuloides Caribbeanand Africancoast 0.54 16.34 8.49 11.05 0.52

pagensis), and the average differenceof E. tribuloidesfrom doubted that it deserved specific rank. More recent biogeo- E. thouarsi (11.05 %) is 1.3 times higher than the difference graphic (Maluf 1988, 1991) and ecological (Glynn et al. of E. tribuloides from E. galapagensis (8.49%). Thus, K2- 1979; Glynn 1994; Wellington 1997) works have all dealt values that would have been equal under a strictmolecular with Galapagos populations of Eucidaris as if they belonged clock hypothesiscan vary by up to 30%. Substitutionat silent to E. thouarsi. However, both our mtDNA and our isozyme sites is supposed to be more regular because of the absence data provide strongevidence thatE. galapagensis is a species of selective constraints,yet Ks-values show variation that is separate from E. thouarsi, from which it diverged at about even higher,probably because they are based on a fraction the same time that the eastern Pacific was being separated of all sites, and thus have larger sampling error (Table 4). fromthe Caribbean by the Central American isthmus. More- Thus, the cladogenic events in Eucidaris can only be dated over, this species is not a Galapagos endemic, but is also within broad time constraints. If we assume that the sepa- found at Isla del Coco and perhaps at Clipperton Atoll. ration of E. tribuloidesfrom E. thouarsi and E. galapagensis An interestingquestion is whetherE. galapagensis and E. occurred at the time of the final closure of the Isthmus of thouarsi are trulyallopatric. Given theirmorphological sim- Panama 3.1 million years ago (Coates and Obando 1996) and ilarity,it is possible that colonization of the mainland by E. equate this length of time with the average divergence be- galapagensis or of the islands by E. thouarsicould have gone tween all east Pacific and all Atlantic sequences (K2 = undetected in faunal lists of either region. Indeed, based on 9.52%), we obtain a calibration of 3.1 % sequence divergence morphology, we have erroneously reported E. thouarsi as per million years. By thiscalibration, and allowing fora range present at Isla del Coco and the Clipperton Atoll (see table of rate variation 15% on eitherside of the mean, E. metularia 1 in Lessios et al. 1996). Although the question cannot be diverged from all other Eucidaris in the late Miocene/early answered with certaintyuntil the western American coast is Pliocene between 4.7 and 6.4 million years ago (average K2 more extensively surveyed,our collections would be unlikely = 17.19%), and E. galapagensis separated fromE. thouarsi to have missed genotypes of the "wrong" species in localities in the Pliocene approximately 2.5-3.4 million years ago. If thatwere sampled. Our combined mtDNA and isozyme sam- we were to assume thatE. clavata no longer exchanges genes ples come to 45 individuals from the islands (some individ- with E. tribuloides,the estimated date of separation would uals were assayed forboth kinds of molecules) and 117 from be 148,000-200,000 years ago. the American mainland. Fifty-twoadditional E. thouarsifrom the Bay of Panama and 51 from the Gulf of Chiriqui were DISCUSSION sampled in previous isozyme studies (Lessios 1979, 1981) that included four of the loci diagnostic between E. thouarsi Correspondence between ntDNA Clades and and E. galapagensis. These studies would have noted the Biological Species presence of E. galapagensis genotypes in the mainland, yet What new insightsinto the phylogeny of Eucidaris do the none of them showed evidence of invasion of one lineage molecular data provide, and what general patternsof ocean into the geographical range of the other. That no island ge- biogeography can they address? The firstpoint genetic data notypes appear to be present on the American coast is not can establish, a point difficultto examine with previously easy to explain, given the relatively small distances involved available alpha taxonomic studies of the genus, is to dem- (Isla del Coco is 500 km west of Costa Rica) and the pre- onstratewhich populations exchange genes and which do not. vailing currentflow from west to east (Wyrtki 1965, 1966, In its extreme form of complete cessation of gene flow, this 1967; Abbott 1966; Tsuchiya 1974), but neitheris it all that is tantamountto the demonstrationof specific status. unusual. There are 17 species endemic to the As mentioned in the introduction,there has been disagree- Galapagos and nine endemic to Isla del Coco (Maluf 1991). ment as to whether the eastern Pacific harbors one or two The apparent absence of the reverse colonization, by E. species of Eucidaris. Although the name E. thonarsi is un- thouarsi from the coast to the islands, is more peculiar. All questionably applied by all authors to the populations on the otherspecies of sea urchinsfound commonly on the mainland westernAmerican coast, D6derlein (1887) described the form are also known from the outer islands. In at least one case, found in the Galapagos as a separate species, E. galapagensis. that of Diadenia nmexicanumn,sequencing of mtDNA has However, Clark (1925) stated that he would hesitate to rec- shown that this is not another case of an undetected sibling ognize it even as a "valid variety." Mortensen (1928-1951) species (Lessios et al. 1996). did accept E. galapagensis as a variety of E. thouarsi, but Despite the geographic analogies between the eastern Pa-

This content downloaded on Thu, 17 Jan 2013 14:37:51 PM All use subject to JSTOR Terms and Conditions PHYLOGEOGRAPHY OF EUCIDARIS 813 cific islands of Galapagos, Coco, and Clipperton and the cen- TABLE 5. Sample size and averagewithin-population percent nu- tral Atlantic islands of St. Helena and Ascension, the case cleotidedifference (K9) of COI in all populationsof Eucidaris in whichmore than two individualswere sampled. of E. clavata is opposite to that of E. galapagensis. As they have for E. galapagensis, echinoderm taxonomists have ex- Locality nI K2 pressed doubts as to whetherE. clavata should be treatedas Reunion 4 2.33 a species distinctfrom E. tribuloides.Mortensen (1928-1951, Ryukyu 4 0.25 vol. I, p. 411) considered whetherE. clavata should be just Guam 5 0.31 a variety of E. tribuloides, but decided to describe it as a Hawaii 15 0.63 separate species and continued to regard it as such in other Galapagos 7 0.44 Isla del Coco 6 0.33 publications (Mortensen 1932, 1933). However, he statedthat Bay of Panama 4 0.36 E. tribuloides is present at Ascension along with E. clavata Mexico 6 0.89 (Mortensen 1936). Pawson (1978) carried out measurements Belize 11 0.54 of specimens of Eucidaris fromAscension, St. Helena, West San Blas 10 0.31 Africa, and the West Indies, and, on the basis of morpho- St. John 3 0.85 PuertoRico 4 0.28 logical differences,he concluded thatE. clavata is a separate Brazil 4 0.34 species to which all Eucidaris from both Ascension and St. Sao Tome 10 0.72 Helena belong. In contrast to the case of E. galapagensis, St. Helena 7 0.14 however, the mtDNA similarities indicate that E. clavata is Ascension 5 0.25 more likely to be a peripheral deme of E. tribuloides. Although the spatial distances are formidable (Ascension to Brazil, 2300 km, Ascension to St. Helena, 1300 km, St. Helena is thought to be 13.3-15.3 million years old, but Helena to Africa, 1900 km), transportof larvae from the Ascension has only emerged in the last 1.5 million years continentsto the central Atlantic is not beyond the realm of (Baker 1970; Mitchell-Thome 1982). If we assume thatthere possibility. The larvae of E. thouarsi (Emlet 1988) and (pos- has not been repeated genetic contact, our calibration of the sibly) E. tribuloides(McPherson 1968) settlein the laboratory Eucidaris COI clock would estimate the colonization of these 25-30 days afterfertilization. The southernmostlimit of the islands as having occurred less than 200,000 years ago. Such range of E. tribuloidesextends to Rio de Janeiro(Bernasconi a recent date eliminates the possibility that colonization by 1955) and the easternmostlimit to the Gulf of Guinea (Chesh- extant mtDNA clades in the central Atlantic took place at a er 1966). The close genetic similaritybetween E. tribuloides time before sea-floor spreading had established the Atlantic from the Caribbean Sea and Brazil to the Gulf of Guinea in its present-daydimensions (Berggren and Hollister 1974; indicates that there has been recent gene flow between these Scheltema 1995). areas, despite the fact thatthe transittime across the tropical Haplotype diversity of Eucidaris in the central Atlantic Atlantic,estimated from the average velocity of the equatorial islands suggests that a single recent introductionis a pos- undercurrent(Metcalf et al. 1962), is 43-70 days (Chesher sibility.Palumbi et al. (1997) found thatEchinonietra mathaei 1966). The time estimated by Manning and Chace (1990) for in Hawaii shows 0% sequence heterogeneityin COI. They larval transitfrom the coast of Brazil to Ascension on the explained this lack of diversityas the result of a single recent equatorial counter-currentis 48 days. Travel in the opposite colonization event. Eucidaris at St. Helena has less haplotype direction,from the central Atlantic to the coast of Brazil on diversitythan any other population of this genus for which the westward-flowingsouth equatorial current (Longhurst we sequenced three or more specimens (see Table 5). Six 1962) is also a possibility. Edwards and Lubbock (1983) haplotypes are identical, while the seventh is similar to the report E. clavata from St. Paul's Rocks, which is 2000 km ones from Ascension (see Fig. 2). Diversity values at As- northwestof Ascension, and 960 km northeastof the Bra- cension are higher,but still among the lowest ones observed. zilian coast, so theremay be a stepping stone along the way. Thus, although Eucidaris in the central Atlantic islands is That FST-values suggest little genetic exchange between the not as genetically depauperate as E. mathaei at Hawaii and two central Atlantic islands is partlydue to the low mtDNA its haplotypes are closely related to those found at continental variability within each locality (see below), which tends to margins, it may be to a considerable extent cut off fromthe drive FST values upward (Charlesworth 1998). However, as mainland. pointed out, mtDNA sequence divergence between haplo- from the two islands is than differentiationbe- types larger Possible Causes of Isolation tween central and western Atlantic populations of Euicidaris. As the single shared haplotype indicates, migrationbetween Although Mayr (1954) was more inclined to thinkin terms St. Helena and Ascension does occasionally happen. How- of dispersal, the allopatric species of Eucidaris, with adjacent ever, each island represents a minuscule source and a mi- ranges spanning in their aggregate the entire tropics, have nuscule target for waterborne larvae, so it is not surprising always been consistentwith the hypothesisof a circumglobal that such events are infrequent. original common stock, which became fractionatedby the The alternativeexplanation to ongoing gene flow between closure of seaways and the formationof deep oceanic stretch- the central Atlantic islands and the continental margins is es. The phylogenetic informationadded by our study cannot thatsingle, improbable events of colonization established the distinguish between biogeographic models of dispersal and ancestors of the extantmtDNA haplotypes at each island and vicariance, but it can provide estimates of the approximate there has been little or no subsequent genetic exchange. St. age of separationbetween geographical isolates. This, in turn,

This content downloaded on Thu, 17 Jan 2013 14:37:51 PM All use subject to JSTOR Terms and Conditions 814 H. A. LESSIOS ET AL. allows informed guesses about the nature of the possible tact. Apparently the last time that east and west Pacific Eu- barriersthat resulted in the species as we see them today. exchanged genes was 4.7-6.4 million years ago. The firstvicariance event in the historyof extantEucidaris The cause of the next cladogenic event in the history of was the isolation of the Indo-Pacific species fromall others. extant species of Eucidaris is no mystery.The Isthmus of In this regard, the phylogeny of Eucidaris resembles that of Panama separated E. tribuloides from eastern Pacific stock. Echinometra,which also shows a deep split between species We cannot be certain whether this separation of Eucidaris from the western Pacific and those from the eastern Pacific was contemporaneous with the final closing of the isthmus and the Atlantic (Palumbi 1996). Cutress (1980, p. 165) sug- or whetherit occurred at some point duringits gradual shoal- gested that affinitiesbetween Atlantic and Indo-Pacific ci- ing (Coates and Obando 1996) because the transisthmianCOI daroids can be best explained as the result of west-to-east divergence reported here (9.5% sequence divergence when dispersal through the Tethyan Sea. Miocene fossils of Eu- distances between E. tribuloidesand the two east Pacific spe- cidaris from the Azores and the Canary Islands (areas in cies are averaged) is similar to that found between analo- which the genus is now extinct) support the view of unin- gously separated Atlantic and Pacific species of Echinometra terrupteddistribution from the Atlantic to the Indian Ocean. (9.8%), but twice as large as that of species of Diadema However, our estimated time of separation of E. nmetularia (4.7%; Lessios 1998). Although it is unclear whether the suggests that circumglobal genetic connections in Eucidaris lower values of transisthmiandivergence in Diaderna have continuedafter the closure of the TethyanSea. Althoughthere resulted from slower rate of COI evolution in this genus, is considerable disagreement regarding the time of the final breaching of the isthmus at times of high sea level stands closure of the Tethys (Adams 1967; McKenzie 1967; Rug- (Cronin and Dowsett 1996), continuing gene flow through gieri 1967; Luyendyk et al. 1972; Berggren and Hollister 1974; R6gl and Steininger 1984; Piccoli et al. 1987; Robba mangrove swamps at the time of isthmus completion (Keller 1987; Rosen and Smith 1988; Por 1989; Hallam 1994, p. et al. 1989), or circumglobal gene flow around the southern 178), we are aware of no estimate that is more recent than tip of Africa, the discrepancy points to the possibility that 12 million years ago; the most widely accepted date is the separation of eastern Pacific and western Atlantic Eucidaris early Miocene, approximately 20 million years ago (Rosen may be somewhat older than 3.1 million years ago. If so, the 1984; Winterbottomand McLennan 1993). Even with broad dates of othercladogenic events, dated by a calibration based allowances for rate variation, our oldest estimate from on this split, may also be older, but not by so much as to mtDNA forthe separation of E. metularia is 6.5 million years alter conclusions as to the possible causes of separation. One ago, in the late Miocene. Thus, Atlantic and eastern Pacific would need to postulate a calibration of COI evolution three Eucidaris were still exchanging genes with the Indo-Pacific times slower than our estimate to accept the closure of the afterthe shallow epicontinentalTethyan Sea ceased to exist. Tethys as a credible cause of speciation of E. nmetularia. In principle, connections were still possible in the late Mio- The final definitesplit in Eucidaris occurred in the eastern cene and early Pliocene around the southern tip of Africa, Pacific between mainland and island populations. There is because the Benguela cold water upwelling systemoff south- no obvious extrinsic barrier to gene flow that could have western Africa, which appeared in the Miocene (Diester- caused this split. Presumably, the shoaling of the Isthmus of Haass and Schrader 1979; Siesser 1980), was not established Panama affectedocean circulation (Maier-Reimer et al. 1990; as a continuous phenomenonuntil the late Pliocene (Shannon Haug and Tiedemann 1998), but it is not clear how it did so 1985). However, unlike other Indo-Pacific shallow-water in the eastern Pacific or how long before the final closure the echinoids, the ranges of which extend past Durban, E. nme- shoaling altered surface currents.It appears that, like many tularia only reaches Mozambique (Clark 1925; Clark and other marine species that are endemic to the Galapagos Courtman-Stock 1976). This suggests thatEucidaris may be (Walker 1966; Glynn and Wellington 1983; James 1984; a more "tropical" genus, the larvae of which are less likely Garth 1991; Kay 1991; Maluf 1991; Allen and Robertson to cross an area of even weak A more upwelling. probable 1994) and/orto other eastern Pacific islands (Hertlein 1963; last obstacle to migrationis the Eastern Pacific Barrier,the Garth 1991; Maluf 1991; Allen and Robertson 1994; Rob- 5000-km stretchof deep water separating the central from ertson and Allen 1996), E. galapagensis may have speciated the easternPacific (Ekman 1953; Briggs 1974; Vermeij 1978, because larvae reached the islands and then, for unknown p. 253, 1987; Grigg and Hey 1992). Although the Eastern reasons, were cut off fromthe mainland. That the age of the Pacific Barrier has possibly existed for the entire Cenozoic oldest present-day Galapagos islands (see Glynn and Wel- (Grigg and Hey 1992), in sea urchins, as in many other groups, it is functioningto the present day as a haphazard lington 1983, p. 162; Chavez and Brusca 1991 and references filter,which is subject to sporadic breaches by larvae (Lessios therein)roughly agrees with the threemillion years ago tim- et al. 1996), sometimes resultingin massive gene flow (Les- ing of the split between E. thouarsi and E. galapagensis is sios et al. 1998). The presence in the eastern Pacific of mi- probably a coincidence. The central eastern Pacific contains tochondrialhaplotypes that belong to central Pacific species several "drowned" islands, dating back to nine million years has been documented in other sea urchin genera, such as ago (Christie et al. 1992). Land vertebrateson the Galapagos Echinothrix(Lessios et al. 1998), Diaderna (Lessios et al. appear to have been separated fromtheir mainland ancestors 1996), and Echinonietra (Palumbi 1997). However, that cen- for much longer than three million years (Wyles and Sarich tral and eastern Pacific populations of some of these genera 1983; Rassmann 1997). Thus, the time available forperipatric have speciated in the firstplace indicates thatthere have also speciation of E. galapagensis is longer than the age of any been periods duringwhich theydid not maintain genetic con- extant island.

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Conclusions in the laboratory.The Administratorof Ascension, the Gov- ernmentof St. Helena, H. Pinto da Costa, and the Direcao Because of the lack of geographic overlap between the das Pescas of Sao Tome made collections in the central and species, the genus Eucidaris representsthe simplest possible eastern Atlantic possible. The captains and the crews of the case for reconstructingphylogeographic patternsin pantrop- R/V Benjamin, the M/S Marigold, the R/V Gyre, and the ical shallow water tropical genera withmoderately long-lived M/S Royal Star assisted withcollections in the easternPacific. larvae. Most of what we know about marine biogeography This study was supported by funds from the Smithsonian and barriersto dispersal formarine organisms has been based Molecular Evolution Program, by Smithsonian Scholarly on the traditionalapproach of studyingthe numberof species Studies Grant 1234S607, Baird Fund Grant 14404507, and that are found in common in differentgeographic regions a Japan Society for the Promotion of Science fellowship to (e.g., Ekman 1953; Briggs 1974; Vermeij 1978, 1987). Sev- DRR. eral featuresof the Eucidaris phylogeographypresented here have significance that may extend beyond this particularge- LITERATURE CITED nus. Despite the tremendousdistances involved, populations fromthe east and west Atlanticcoasts are connected by recent ABBOTT, D. P. 1966. Factorsinfluencing the zoogeographicaffin- ities of the Galapagos. Pp. 108-112 in R. I. Bowman,ed. The gene flow,as one would have surmised fromtheir assignment Galapagos. Univ. of CaliforniaPress, Berkeley. by traditionaltaxonomists into the same species. The central ADAMS, C. G. 1967. TertiaryForaminifera in the Tethyan,Amer- Atlantic islands of Ascension and St. Helena show only a ican, and Indo-Pacificprovinces. Syst. Assoc. Publ. no. 7:195- small degree of genetic isolation from the continental mar- 217. ALLEN, G. R., AND D. R. ROBERTSON. 1994. Fishes of thetropical the of these based on the gins. Estimates of isolation islands, easternPacific. Univ. of Hawaii Press,Honolulu. percentage of endemism (e.g., Mortensen 1933; Briggs 1974; AVISE, J. C., J. ARNOLD, R. M. BALL, E. BERMINGHAM, T. LAMB, Rosewater 1975; Pawson 1978; Lubbock 1980; Edwards J. E. NEIGEL, C. A. REEB, AND N. C. SAUNDERS. 1987. Intra- 1990; Manning and Chace 1990; Biernbaum 1996), may in specificphylogeography: the mitochondrialDNA bridge be- some cases be biased upward because of the erroneous ele- tween populationgenetics and systematics.Annu. Rev. Ecol. Syst. 18:489-522. vation of their populations to separate specific status. The BAKER, I. 1970. Geological historyof Saint Helena in relationto offshoreislands of the eastern Pacific have provided oppor- its floraland faunalcolonization. Mus. R. Afr.Cent. Tervuren tunities for speciation dating back to the Pliocene. Despite Belg. Ann. Ser. 8 Sci. Zool. 181:25-35. the existence of oceanographic conditions favoringdispersal BERGGREN, W. A., AND C. D. HOLLISTER. 1974. Paleogeography, of in between them and the the island and the paleobiogeographyand thehistory circulation theAtlantic American mainland, Ocean. Pp. 126-186 in W. W. Hay, ed. Studiesin Paleo-Ocean- mainland species have not invaded each other's ranges. This ography.Society of Economic Paleontologyand Mineralogy, isolation is not limited to the Galapagos, as suggested by Special Publication,no. 20. traditionalbiogeography (Ekman 1953, p. 45; Briggs 1974, BERMINGHAM, E. B., AND H. A. LESSIOS. 1993. 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