Population Genetic Structure of North American Ophiactis Spp. Brittle Stars Possessing Hemoglobin

Mar Biol (2008) 154:755–763 DOI 10.1007/s00227-008-0968-1

ORIGINAL PAPER

Population genetic structure of North American Ophiactis spp. brittle stars possessing hemoglobin

Ana B. Christensen · Eric F. Christensen · David W. Weisrock

Received: 8 October 2007 / Accepted: 25 March 2008 / Published online: 8 April 2008 © Springer-Verlag 2008

Abstract With the discovery of previously unreported Introduction populations of hemoglobin-possessing Ophiactis from the Texas coast in the Gulf of Mexico, an investigation into its In an age of substantial human-mediated movement of population structure, including populations of O. simplex marine organisms, especially invertebrates from fouling from the PaciWc coast of California and O. rubropoda from communities (e.g., Roy and Sponer 2002; Martela et al. the Atlantic coast of Florida, was undertaken using DNA 2004; Mackie et al. 2006; May et al. 2006; Cardigos et al. sequence data from the mitochondrial COI gene. The 2007), understanding the genetic structure and evolutionary reconstructed haplotype network suggests that California history of newly discovered populations is of great impor- populations contain the ancestral source of mtDNA varia- tance. The invasive transfer of individuals can reestablish tion, and there is no evidence of recent introductions into gene Xow among previously diverging populations (e.g., Texas. Population genetic analyses reveal the California, incipient species on opposite sides of the Panamanian Isth- Florida, and Texas Ophiactis populations to each be mus), leading to a reduction in lineage diversity. Further- signiWcantly diVerentiated from one another. Sequence more, the establishment of exotic populations can have a divergence among the three areas is shallower than would signiWcantly negative eVect on the native community (Ross be predicted given biogeographic history. Texas and et al. 2003). In contrast, newly discovered marine popula- Florida populations are equally genetically diverged from tions may result from previously limited natural history California populations as they are to one another, despite studies, and instead represent an extension of a species’ the greater potential for gene Xow between these areas. range that has had a long and undetected evolutionary The genetic distinctiveness of the Texas populations and the history. Analysis of genetic data within a phylogenetic and concordance of this pattern with phylogeographic patterns population-genetic framework is becoming increasingly in other brittle star systems indicate an isolated and inde- important in addressing these alternative scenarios, espe- pendent evolutionary history and we hypothesize that cially in marine organisms displaying limited morphologi- the three geographic regions included in this study each cal variation (Sponer and Roy 2002). serve as hypotheses of population-level lineages that Of the 2,000 extant species of brittle stars (Echinoder- remain to be tested with independent sources of data. mata: Ophiuroidea), four species, all belonging to family Ophiactidae, are unique in possessing hemoglobin con- tained within coelomocytes in their water vascular system: Communicated by M.I. Taylor. Ophiactis virens (Foettinger 1880; Cuénot 1891), O. simplex (Christensen 1998), O. rubropoda (Ruppert and Fox A. B. Christensen (&) · E. F. Christensen Department of Biology, Lamar University, 1988), and Hemipholis elongata (Hajduk and Cosgrove PO Box 10037, Beaumont, TX 77710, USA 1975; Christensen et al. 2003). The presence of hemoglobin e-mail: [email protected] imparts a bright red color to the tube feet and provides a clear diagnostic character that can rapidly distinguish these D. W. Weisrock Department of Biology, University of Kentucky, species from other co-occurring brittle stars. However, Lexington, KY 40506, USA further morphological discrimination between three of the 123 756 Mar Biol (2008) 154:755–763 species (O. simplex, O. virens, and O. rubropoda) is their great geographic distance from other known coastal complicated by their ability to asexually reproduce via North American populations, may be genetically distinct Wssion. In this mode of clonal reproduction, individuals tear from other hemoglobin-possessing ophiactids. To investi- themselves into two pieces, with each “half” regenerating gate these possible alternatives, we studied the geographic the missing body parts (Mladenov and Burke 1994). genetic structuring of North American populations of Positive morphological identiWcation of Wssiparous species Wssiparous, hemoglobin-possessing ophiactids. We col- typically requires individuals that have completed regener- lected and analyzed mitochondrial DNA (mtDNA) sequence ation and have reached a size conferring sexual maturity data within a phylogenetic and population-genetic frame- (disc diameter > 3 mm). This may be diYcult as the majority work to assess the levels of genetic divergence among hapl- of collected individuals are often small (disc diameter < 2 mm) otypes sampled from North American populations and and in some stage of regeneration (Mladenov and Emson tested alternative hypotheses regarding gene Xow and 1984; Christensen 2004). A feature that is more obvious in genetic divergence among these populations. larger specimens is the lack of bursal slits in O. virens (Simroth 1876). In the absence of these states, positive species identiWcation of Wssiparous brittle stars can be Materials and methods extremely challenging. Despite the lack of obvious diagnostic characters, diVerentiating the hemoglobin possessing Collection of animals species has not been problematic as they are reported to have separate geographic ranges: O. simplex is distributed Animals were collected from a total of six localities (Fig. 1, in the eastern PaciWc from the Channel Islands to Panama Table 1); two from the newly discovered Texas Ophiactis, and the Galapagos Islands (Nielsen 1932; Lonhart and one from the Atlantic and Caribbean species, O. rubropoda, Tupen 2001), O. rubropoda is distributed along the coasts and three from the California species O. simplex. Texas and of Florida and the Caribbean (Singeltary 1973; Hendler Florida collections were made by scraping tunicate colonies et al. 1995), and O. virens is distributed throughout the and sponge from the hard substrate and manually removing Mediterranean Sea (Hansson 2001) and Canary Islands the brittle stars from the material. Diablo Cove specimens (Tuset et al. 1996). The fourth species possessing hemoglo- were collected by scuba and breaking oV bits of rock. Most bin, Hemipholis elongata, is much larger and morphologi- animals were kept alive in aquaria until DNA isolation. San cally distinct from the others, relies strictly on sexual Diego and Long Beach specimens were collected from reproduction, and inhabits a diVerent type of habitat (it bur- fouling communities and preserved in ethanol (70–90%) rows in soft mud and sand) (Christensen and Colacino prior to arrival in the laboratory. An average of 13 individu- 2000). als per locality was used for genetic analysis with a range of In 2001 a small Wssiparous brittle star was collected from 5–21. the fouling communities of the rock jetties of Port Aransas, Outgroup sequences for phylogenetic analysis were col- TX (Christensen 2004). A striking feature of this popula- lected from Hemipholis elongata (from South Carolina) tion was the red color of the tube feet. However, no hemo- and from O. savignyi (from Florida). Additional COI out- globin-containing, Wssiparous brittle star had previously group sequences were retrieved from GenBank: O. algicola been reported for the Gulf of Mexico. The majority of indi- (AF331527), O. lymanii (AF331528), O. quinqueradia viduals were very small (average disc diameter < 2 mm, (AF331529), and O. savignyii (AF331557 and AF331601). maximum = 3.8 mm). Specimens were initially identiWed as O. rubropoda given the proximity to Florida and the Caribbean; however, further comparisons of morphological features with museum specimens of the same size classes of O. simplex and O. rubropoda failed to yield a conclusive identiWcation (Hendler, personal communication). Given the great dispersal potential of many marine organisms with pelagic, free living larvae (Highsmith 1985; Silberman et al. 1994; Hendler et al. 1999; Uthicke and Benzie 2003), and the increasing human-mediated introduction of species into new ranges (Carlton and Geller 1993; Roy and Sponer 2002), the newly discovered Texas Gulf populations may represent a recent range expansion of O. simplex or O. rubropoda. Alternatively, these popula- Fig. 1 Map showing the general sampling localities used in this study. tions may have a long and undetected history, and given Localities are numbered according to Table 1 123 Mar Biol (2008) 154:755–763 757

Table 1 Sampling localities for Locality # Species na Locality description all samples used in this study 1 Ophiactis simplex 20 Diablo Canyon, San Luis Obispo County, CA 2 Ophiactis simplex 10 Near the mouth of Alamitos Bay, Long Beach, CA 3 Ophiactis simplex 9 Mission Bay Channel entrance, San Diego, CA 4 Ophiactis rubropoda 5 The pier at Rosenstiel School of Marine and Atmospheric Science, Miami, FL 5 Ophiactis sp. 21 South jetty, Port Aransas, TX 6 Ophiactis sp. 12 North jetty at Isla Blanca State Park, a Number of individuals used in South Padre Island, TX genetic analyses

DNA isolation, ampliWcation and sequencing models for use in Bayesian analysis were assessed using the Akaike Information Criterion in MODELTEST v3.6 (Posada Genomic DNA was isolated from animals using a modiWca- and Crandall 1998). Four Markov chains were used with the tion of the Chelex protocol outlined by Medeiros-Bergen temperature parameter set at 0.2 giving the three heated et al. (1998). Ethanol preserved specimens were Wrst chains temperatures of 0.83, 0.71, and 0.62. Default priors allowed to air dry to remove the ethanol and then soaked in were used in all analyses and random trees were used to start two changes of TE buVer for 30 min each. Whole animals each Markov chain. Chains were run for ten million genera- were ground in 500 l of TE buVer with mortar and pestle; tions with topology and model parameter estimates sampled whole animals were used as many of the specimens were every 1,000 generations. The Wrst Wve million generations small (disc diameter < 2 mm). About 300 l of a 5% Che- were discarded as burn-in yielding a posterior distribution of lex 100 (BioRad) was added and samples were incubated at 5,000 sampled trees. Mean log likelihood (lnL), branch 55°C for 35 min. The samples were then boiled for 8 min, lengths and topologies were compared across three replicate cooled to room temperature, vortexed for 20 s followed by analyses for each data set to insure that a stable posterior dis- 1 min centrifugation at 14,000£g. The supernatant was tribution was reached. Sampled trees were parsed with decanted and kept refrigerated until use in PCR reactions. MrBayes to construct a phylogram based upon mean branch A portion of the mitochondrial COI gene was PCR ampli- lengths and to calculate posterior probabilities (PPs) of all Wed and sequenced using newly designed ophiuroid speciWc branches using a majority-rule consensus. primers: forward 5Ј-ATTAATTCTCCCCGGGTTTG-3Ј and reverse 5Ј-GGGAARAATGTNAGGTTTACTCC-3Ј. Population-genetic analysis Thermal cycling consisted of a 45 s denaturation at 94°C, 45 s annealing at 51–53°C, and 1 min extension at 72°C; The number of segregating nucleotide sites, the average this was repeated 35 times. Successful PCR reactions were number of nucleotide diVerences per site between two identiWed on 2% agarose gels stained with ethidium bro- sequences (), and the proportion of segregating polymor- mide and compared to a molecular weight standard. PCR phic sites ( =4N) were calculated using DnaSP v4.0 products were separated from excess primers and dNTPs (Rozas et al. 2003). Departure from neutral evolution for using Sigma Spin columns (Sigma Aldrich). PuriWed individual loci was assessed using Tajima’s D statistic product was then used as a template for DNA sequencing (Tajima 1989). Haplotype networks for each locus were reaction using ampliWcation primers and the BigDye constructed using statistical parsimony in TCS v1.2.2 Terminator v3.1 cycle sequencing kit (Applied Biosys- (Clement et al. 2000). tems). Automated sequencing was performed on an ABI Population structure among sampling localities of the 3100 Genetic Analyzer (Applied Biosystems). Identity of California, Florida, and Texas localities of Ophiactis was the sequences was conWrmed by performing BLAST assessed using analysis of molecular variance (AMOVA) to searches on sequences stored in GenBank. Sequences were assess the geographic partitioning of nucleotide sequence checked and edited using SeqMan II (DNASTAR) and then variation (ExcoYer et al. 1992). Unweighted squared Euclid- manually aligned. Sequence alignment was straightforward ean distances were calculated for all pairwise combinations due to a lack of length heterogeneity among sequences. of haplotypes. The total variance of these distances was parti- tioned into three components with respect to regional group- Phylogenetic analysis ings: (1) variance among the regional groups, (2) variance among populations within these regional groups, and (3) var- A bifurcating gene tree was reconstructed using Bayesian iance among individuals within a population. These variance Y phylogenetic analysis using the parallel processor version of components were used to calculate ST (Exco er et al. W MrBayes v3.04 (Altekar et al. 2004). Best- t evolutionary 1992), an analog of Wright’s FST, which characterizes 123 758 Mar Biol (2008) 154:755–763 nucleotide variation in a single population relative to all from the Bayesian posterior distribution were G M T = 1.0, sampled populations. We considered genetic structuring C M T=34.36, CM G=0.18, AM T=11.72, AM G= V across three di erent regional scenarios: (1) a scenario in 22.33, A M C = 13.81, A = 0.274, C = 0.294, G = 0.177, W which populations were grouped into Paci c versus Gulf T = 0.253, = 0.128, and proportion of invariant regions, separated by the Panamanian Isthmus, (2) a scenario sites = 0.224. The Bayesian analysis resolved O. rubro- in which populations were grouped by species with Texas poda, O. simplex, and the Texas Ophiactis populations as a populations forming a third undescribed species, and (3) a strongly supported clade (Fig. 2; PP = 0.97). InsuYcient scenario in which Texas populations are grouped with California O. simplex versus the Florida O. rubropoda population. All analyses were performed using the program ARLEQUIN version 2.0 (Schneider et al. 2000). SigniW- cance of the variance components was determined using a random permutation test with 1,000 replicates. We estimated migration among populations using a Bayesian approach implemented in the program LAMARC (Kuhner et al. 2005). These analyses used Xat logarithmic priors for each parameter and used the default upper and lower boundaries for sampling of each parameter. Our sampling strategy used 10 initial chains each with trees sampled every 50 generations for 5,000 sampling events following a burnin of 2,000 trees. Parameter estimates from the Wnal short chain were used as starting estimates for 2 subsequent long chains that sampled trees every 50 generations for 100,000 sampling events following a burnin of 5,000 trees. The most-probable estimates of migration and population size were used to calculate the population-migration rate

(Nm) among populations.

Results

About 469 bp of COI mtDNA sequence data were collected from 77 Ophiactis ingroup individuals. All sequences Fig. 2 The mtDNA COI Bayesian consensus phylogram resulting are deposited in GenBank under accession numbers from a majority rule consensus of trees sampled from the Bayesian posterior distribution. Branch lengths are depicted as the average EU583136-EU583215. Nucleotide polymorphism among across the posterior distribution. Numbers above the branches repre- the Ophiactis ingroup samples was generally low, with 16 sent posterior probabilities. Haplotype labels in the Bayesian tree in- polymorphic sites found across the Atlantic, Gulf, and clude the locality number from which a haplotype was sampled (in PaciWc populations (Table 2). A positive Tajima’s D statis- parentheses). A statistical-parsimony haplotype network for COI haplotypes sampled from O. rubropoda, O. simplex, and Texas Ophiactis tic was estimated (Table 2); however, this was not signiW- populations is presented showing the resolution of mutational relation- cant indicating that the COI locus is evolving in an ships. Open circles represent recovered haplotypes, bold black dots eVectively neutral fashion. represent unsampled or extinct haplotypes, and single lines represent a Bayesian phylogenetic analysis yielded a posterior dis- single mutational events. Open circles are drawn proportionally to the number of individuals containing a particular haplotype. For reference, tribution with an average log likelihood of ¡2,098.86 and a haplotypes COI-4 and COI-8 are each represented by a single sampled variance of 24.44. The mean model parameters estimates individual

Table 2 Nucleotide polymorphism parameters measured across populations of North American Ophiactis Locus # of Haplotypes Polymorphic sites Nucleotide diversity ()a Nucleotide diversity ()b Tajima’s Dc

COI 9 16 0.01069 0.00694 1.56494 a Nucleotide diversity based on the average number of nucleotide diVerences per site between two sequences b Nucleotide diversity based on the number of segregating sites (Watterson estimator; =4N, where N =eVective population size and = neutral mutation rate) c Not signiWcant at P <0.05 123 Mar Biol (2008) 154:755–763 759 outgroup sampling prohibited an assessment of monophyly vs. Texas Ophiactis) was strongly signiWcant (P <0.001) in for the genus Ophiactis. However, branch lengths indicated partitioning genetic variation among these three higher that our ingroup clade is substantially genetically diverged level groups (Table 4). This model received an accordingly W from the outgroup Ophiactis samples. Statistical parsimony high and signi cant CT estimate of 0.639. Alternative analysis connected all ingroup haplotypes in a single models in which the Texas Ophiactis are grouped with network with a 95% connection limit of 9 mutational steps. either the California O. simplex populations or the Florida Haplotypic divergence within the Ophiactis ingroup clade O. rubropoda populations did not signiWcantly explain the was especially low, with haplotypes separated at most by geographic partitioning of genetic variation at the among- six mutational events in the statistical parsimony network group level. The AMOVA results also found a highly sig- (Fig. 2). Uncorrected and corrected sequence divergences niWcant partitioning of variation at the among group and were almost identical, revealing a maximum pairwise within-population levels across all three models (Table 4). estimate of sequence divergence of 2.1% between haplo- These results were paralleled by high SC and ST values types sampled from California and Texas (Table 3). Each (Tables 4, 5), indicating highly restricted gene Xow among of the three North American coastal regions contain an populations. exclusive set of haplotypes (Fig. 2). The California O. sim- Estimates of migration revealed low levels of gene Xow plex populations contain the highest haplotypic diversity, within the California and Texas geographic regions X with some of these haplotypes occupying internal positions (Nm ¿ 1), and nearly non-existent gene ow between geo- in the statistical parsimony network, suggesting that the graphic regions (Table 6). Texas populations of Ophiactis California populations may represent the ancestral source appeared to have little to no gene Xow with other geo- of mtDNA diversity. However, we note that the greater graphic regions, with Nm values no greater than 0.052 esti- genetic diversity within the California region may also be mated into the California Diablo Canyon population. All due to a biased sampling across more distantly sampled other non-Texas comparisons were substantially lower than localities. this. The sampled population of O. rubropoda appears to be

Analysis of molecular variance using a model of three highly genetically isolated from other populations with Nm separate population groupings (O. rubropoda vs. O. simplex estimates close to 0 in most comparisons.

Table 3 Pairwise uncorrected sequence divergences among haplotypes COI-1 (TX) COI-2 (TX) COI-3 (FL) COI-4 (CA) COI-5 (CA) COI-6 (CA) COI-7 (CA) COI-8 (CA) COI-9 (CA)

COI-1 (TX) – COI-2 (TX) 0.002 – COI-3 (FL) 0.021 0.019 – COI-4 (CA) 0.008 0.011 0.013 – COI-5 (CA) 0.011 0.013 0.015 0.002 – COI-6 (CA) 0.015 0.017 0.015 0.011 0.013 – COI-7 (CA) 0.013 0.011 0.017 0.008 0.011 0.015 – COI-8 (CA) 0.013 0.015 0.017 0.004 0.002 0.011 0.013 – COI-9 (CA) 0.017 0.019 0.017 0.013 0.015 0.002 0.017 0.013 –

Table 4 Analysis of molecular variance under three diVerent models of population partitioning Population groupings Partitioning of molecular variance (% total) Statistics

Among Among populations Within CT SC ST groups within groups populations

Atlantic + gulf populations (FL and TX) 38.20 36.40 25.39 0.382 0.589 0.746 versus paciWc populations (CA) O. rubropoda (FL) versus O. simplex (CA) 63.96 12.18 23.85 0.639 0.338 0.761 versus Texas Ophiactis PaciWc + gulf populations (CA and TX) 41.49 39.31 19.21 0.414 0.671 0.807 versus Atlantic populations (FL) Italic numbers represent signiWcant values with a P <0.001 123 760 Mar Biol (2008) 154:755–763

Table 5 Pairwise estimates of ST among sampling localities the California, Florida, and Texas populations are each genetically disjunct from one another. Alternative models 1 (CA) 2 (CA) 3 (CA) 4 (FL) 5 (TX) 6 (TX) in which two of these regions are lumped together do not 1 (CA) – signiWcantly account for the geographic partitioning at the 2 (CA) 0.334 – among region level, and no matter which geographic model W 3 (CA) 0.135 0.085 – is used, all yield signi cant SC values indicating substan- 4 (FL) 0.589 0.874 0.728 – tial population genetic structure across sampled populations 5 (TX) 0.636 0.909 0.819 0.972 – within regions. 6 (TX) 0.608 0.924 0.823 1.0 0.754 – Interestingly, this pattern is both concordant and discor- dant with phylogeographic patterns found across the range Population numbers refer to those listed in Table 1. Italic numbers are signiWcant with a P <0.05 of the widespread brittle star, O. savignyi (Roy and Sponer 2002). In this system western Atlantic/Caribbean popula- Table 6 Estimates of the population-migration parameter (Nm) based tions contain two major mtDNA haplotype lineages. One of on Bayesian estimates of migration and population size ( ) these (Lineage A in Roy and Sponer 2002) is exclusive to 1 (CA) 2 (CA) 3 (CA) 4 (FL) 5 (TX) 6 (TX) the western Atlantic, presumably evolving through isolation following the closing of the Panamanian Isthmus (see 1 (CA) – 0.242 0.328 0.013 0.051 0.052 below for further discussion). However, the other lineage 2 (CA) 0.404 – 0.412 0.034 0.018 0.013 (Lineage B in Roy and Sponer 2002) contains haplotypes 3 (CA) 0.1 0.069 – 0.007 0.011 0.001 shared with Indo-PaciWc populations, providing strong » » » » » 4 (FL) 0 0 0– 0 0 evidence for recent human-mediated dispersal across the 5 (TX) 0.023 0.004 0.01 0.004 – 0.068 Panamanian Isthmus. Our results Wnd concordance with 6 (TX) 0.002 0.001 0.001 0.001 0.009 – O. savignyi lineage A in detecting an evolutionary history Population numbers refer to those listed in Table 1. The direction of of isolation across the Panamanian Isthmus. gene Xow is from the column population into the row population It is important to note, however, that we did not sample across the entire range of O. simplex; it is reported to occur in the eastern PaciWc from southern California down through Panama and Ecuador (Nielsen 1932). The possibil- Discussion ity exists that the COI haplotypes uncovered in our Texas populations may come from outside our sampling range of The Wssiparous Ophiactis species possessing hemoglobin O. simplex in the PaciWc. Nonetheless, our data are indica- (O. simplex, O. rubropoda, and the Texas Ophiactis popu- tive of substantial genetic structure among our sampled lations) form a divergent clade when compared to other regions, even between regions not separated by apparent ophiactid species, including other hemoglobin-producing physical barriers (Texas and Florida). species (i.e., Hemipholis elongata) (Fig. 2). Within this A factor leading to the high genetic structure across sam- clade genetic divergences are very shallow, indicating a pled populations may lie with Ophiactis’ ability to repro- recent evolution of this group. However, each of the focal duce asexually. While it is believed to seasonally produce a geographic regions in this study (California, Florida, and pelagic larvae with an approximate of 3 week duration in Texas) appears to be genetically diVerentiated from one the water column (Hendler, personal communication), another, exhibiting very low levels of gene Xow and distinct locally it may rely more heavily on Wssion as a means of assemblages of haplotypes. reproduction. The proportion of regenerating individuals in While our mtDNA data resolve North American Ophiac- the Port Aransas population can be as high as 80% during tis as a shallowly diverged clade, the current evidence summer months (Christensen 2004). Environmental condi- argues strongly against the newly discovered Texas populations in the sampled populations may increase the rate of tions being established through recent human inXuence. Wssion. All populations, except for Diablo Canyon, are Contemporary human-established populations are expected intertidal and subject to periodic air exposure and high to contain a number of genetic signatures, including shared wave action. These types of habitat are highly unstable, genetic variation with source populations (e.g., Kolbe et al. more hostile, and correlated with high rates of asexual 2004) and reduced population-genetic structure across reproduction (Mladenov and Emson 1984, 1990). Conse- native and introduced populations (e.g., Roy and Sponer quently, long distance dispersal may occur at very reduced 2002; Mackie et al. 2006). Neither of these patterns are rates across Ophiactis populations and contribute to the supported by our data. No haplotypes are shared across regional genetic substructure we see in our data. the California, Florida, and Texas populations and Production of a pelagic larva, however, is not a guaran- the AMOVA results signiWcantly favor a model in which tee of long distance dispersal (for recent review Levine 123 Mar Biol (2008) 154:755–763 761

2006) and other factors may limit gene Xow. The Loop So do the California, Florida, and Texas populations of Current—a potential driver of gene Xow between the Texas Ophiactis each represent a separate species or a single and Florida populations—enters the Gulf of Mexico genetically diverse species? Population genetic analyses between Cuba and the Yucatan Peninsula and exits via the strongly suggest that the three regions are genetically Florida Straits (Hofmann and Worley 1986). However, the isolated from each other. However, the lack of reciprocal Loop Current produces eddies that are likely to constrain monophyly (California appears to contain ancestral pelagic larvae within the western Gulf. Larvae caught in the mtDNA variation) renders their diagnosis as separate phy- northeastern current running along the coastline would logenetic species diYcult. Our characterization of these encounter a barrier to migration due to large volumes of regions as separate species is also limited by the use of a freshwater entering the Gulf from the Mississippi River. single genetic marker. Morphological variation is extremely This coastal current also reverses direction and Xows south- limited among these populations and clear diagnostic ward during the summer months. These phenomena are morphological characters for the currently described thought to inXuence the dispersal and recruitment of O. rubropoda and O. simplex do not exist (Hendler, per- scleractinian coral larvae in the Flower Gardens Bank and sonal communication). If we are to focus solely on the use Florida Keys (Lugo-Fernandez et al. 2001; Shearer and of genetic data in the assessment of these geographic CoVorth 2006). The same factors that would restrict larval regions as phylogenetic species it would require genetic dispersal would also restrict juvenile/adult dispersal due to data from at least one additional independent marker to rafting. This mode of dispersal has been reported for juve- assess either genealogical concordance or concordance in nile brittle stars (Hendler et al. 1999) and O. simplex in patterns of genetic isolation (Sites and Marshall 2004). particular (Christensen 2004; Thiel and Gutow 2005). Nonetheless, concordance in the resolution of genetic Without evidence for recent gene Xow, the focal Ophi- diVerentiation between PaciWc and Gulf/Atlantic popula- actis populations of this study provide an interesting tions in this study with the resolution of distinct mtDNA scenario in which the California PaciWc, Texas Gulf, and haplotype lineages on either side of the Panamanian Isth- Florida Atlantic populations represent isolated and geneti- mus in O. savignyi (Roy and Sponer 2002) appears to be cally distinct groups, yet display low levels of genetic strong evidence for a shared historical vicariant event and divergence from one another. This is particularly note- an independent evolution of Gulf and Atlantic brittle star worthy in the comparison of the California populations populations from those in the PaciWc. As such, populations with those from Texas and Florida where the average of O. simplex in the PaciWc are hypothesized to serve as a sequence divergence is just 1.4% (§0.3%). This number distinct phylogenetic species. The roughly equivalent levels is just slightly lower than (but overlaps with) the 2.6% of sequence divergence detected between Texas Gulf, Flor- (§1.3%) estimate of COI divergence detected between ida Atlantic, and California PaciWc populations also seems the western Atlantic Lineage A and PaciWc haplotypes in to suggest that Texas Ophiactis and Florida O. rubropoda O. savignyi (Roy and Sponer 2002). The Isthmus of Pan- populations are also distinct phylogenetic species. These ama provides a very clear barrier to gene Xow, but its for- tentative hypotheses stand to be tested with both greater mation approximately three million years ago (Coates and population sampling, and with additional genetic markers. Obando 1996) suggests an expected level of mtDNA divergence much greater than seen in Ophiactis. Studies Acknowledgments We would like to thank Jay Carroll, Constance of mitochondrial COI divergence between 15 sister-spe- Gramlich, and Dr. Bruno Pernet for collection of California populations; David Powell and the crew of the RV Walton Smith for aid in cies pairs of snapping shrimp separated by the Isthmus O. rubropoda collections; Dr. Gordon Hendler for morphological reveal a minimum sister-species likelihood-corrected examination of specimens; Dr. Michael Roy for initial primer design; divergence of approximately 5% (Knowlton and Weigt and Dr. Bob Fjellstrom for technical advice. 1998). Similar levels of genetic divergence are seen in a variety of other sister-species pairs split across the Isthmus (e.g., Bermingham et al. 1997; Metz et al. 1998; References Lessios et al. 2001; Wares and Cunningham 2001; but see Marko 2002). 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