afro-eurasia and the americas present barriers to gene flow for the cosmopolitan neustonic...

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Mar Biol (2014) 161:899–910DOI 10.1007/s00227-014-2389-7

OrIgInal PaPer

Afro‑Eurasia and the Americas present barriers to gene flow for the cosmopolitan neustonic nudibranch Glaucus atlanticus

Celia K. C. Churchill · Ángel Valdés · Diarmaid Ó Foighil

received: 16 September 2013 / accepted: 21 December 2013 / Published online: 21 January 2014 © Springer-Verlag Berlin Heidelberg 2014

results indicate that G. atlanticus is not globally panmictic, but that populations appear to be panmictic within ocean basins. We detected several topologically ectopic haplo-types in the atlantic Ocean, but the molecular clock analy-sis indicates that these have diverged from closely related Indo-Pacific haplotypes over 1.2 MYa, coinciding with cooling in waters around in the southern tip of africa and resulting oceanographic changes. These data and the fact that G. atlanticus is not known from polar latitudes suggest that gene flow between ocean basins is hindered by physi-cal barriers (supercontinents) and water temperatures in the arctic and Southern Oceans.

Introduction

after decades of treating the pelagic ocean as a vast, homo-geneous environment and its constituent taxa as genetically interconnected (Day 1963; Mcgowan 1971; Finlay 2002), our understanding of open-ocean population structuring has recently become much more nuanced. Proposed speciation models for passive drifters contradict each other, ranging from the ubiquity hypothesis, in which huge population sizes eliminate dispersal as a limit to gene flow—thereby also eliminating endemism (Finlay 2002), to allopatric hypotheses of potential barriers to gene flow, such as per-sistent currents and continental land masses (Palumbi 1994; Dawson and Hamner 2008). Population genetic data for some pelagic microorganisms are broadly consistent with the former (de Vargas et al. 1999; Darling et al. 2000; Bucklin et al. 2003; Taniguchi et al. 2004; ely et al. 2005; goetze 2005). Yet, others show either subtle (Cowen et al. 2007) or pronounced (de Vargas et al. 1999; Darling et al. 2000; goetze 2003, 2005; Selje et al. 2004; Dawson et al. 2011) genetic structuring within- and among-ocean basins

Abstract Pelagic species have been traditionally thought to occupy vast, genetically interconnected, geographic ranges in an essentially homogeneous environment. although this view has been challenged recently for some mesopelagic planktonic taxa, the population structure of hyponeustonic (surface-drifting) species remains unknown. Here, we test the hypothesis of panmixis in Glaucus atlan-ticus, a cosmopolitan neustonic nudibranch, by assessing the genetic differentiation of multiple representatives from a global neustonic sampling effort. Specimens were col-lected from all subtropical oceanic gyre systems (north atlantic, South atlantic, north Pacific, South Pacific, and Indian Ocean). We sequenced a fragment of the mitochon-drial cytochrome oxidase I gene for 98 individuals and performed population structure, differentiation (analysis of molecular variance, spatial analysis of molecular vari-ance, FST, Jost’s D), and molecular clock analyses. Our

Communicated by T. reusch.

Electronic supplementary material The online version of this article (doi:10.1007/s00227-014-2389-7) contains supplementary material, which is available to authorized users.

C. K. C. Churchill · D. Ó Foighil Museum of Zoology and Department of ecology and evolutionary Biology, University of Michigan, 1109 geddes avenue, ann arbor, MI 48109-1079, USa

Present Address: C. K. C. Churchill Marine Science Institute, University of California, Santa Barbara, Santa Barbara, Ca 93106, USa

Á. Valdés (*) Department of Biological Sciences, California State Polytechnic University, 3801 West Temple avenue, Pomona, Ca 91768, USae-mail: [email protected]

http://dx.doi.org/10.1007/s00227-014-2389-7

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and subtropical ocean gyres. Pelagic diversification pro-cesses are taxon-specific. Moreover, we now have empiri-cal evidence that within- and among-basin water mass boundaries may be differentially permeable even to sister taxa (goetze 2005), due to variation in details of ecology and life history, as predicted by early dispersal models (gaylord and gaines 2000).

a related obstacle to understanding the genesis of plank-tonic diversity is that not all drifters are the same. Most planktonic research focuses on species in the water column, but a subset of marine plankton, the neuston, is only associ-ated with the air–water interface at the ocean surface (Mar-shall and Burchardt 2005). neustonic taxa drift on wind-driven surface currents and have no vertical dispersal (e.g., diel migration, temperature-related mixing). In nonpolar latitudes, the neuston comprises a discrete community of species residing there temporarily (e.g., larval fish) and widespread, holoneustonic taxa (Marshall and Burchardt 2005). How are holoneustonic populations structured? available data are almost completely restricted to the five epineustonic (above the water surface) insect species of Halobates, the oceanic sea skater, which show restricted gene flow among gyres within species, including prolonged reciprocal monophyly between the north atlantic, north Pacific, and Indian subtropical gyres for the widespread species H. micans dated to 1–3 MY (andersen et al. 2000). Because no adjacent subtropical gyres were sampled, it is impossible to determine whether equatorial surface cur-rents versus land masses act as barriers to gene flow. The five oceanic Halobates species stem from two distinct colo-nizations by different coastal ancestral lineages (Damgaard et al. 2000), and there is little range overlap among recent sister taxa (andersen et al. 2000). Inferred speciation pat-terns from Halobates gene tree topologies, and present day distributions, are most consistent with allopatric speciation. given that the differential permeability of ocean basin and gyre boundaries to pelagic microorganisms is related to life

history, however, Halobates spp. populations may be very differently structured than their hyponeustonic (below the water surface) counterparts. Halobates spp. lay eggs on driftwood, whereas hyponeustonic taxa have pelagic larval stages. Do hyponeuston also show trenchant genetic struc-turing by ocean basin or subtropical gyre?

We address this question by focusing on a member of the marine hyponeuston, Glaucus atlanticus, a nudibranch sea slug that floats upside down at the surface of subtropi-cal gyre systems by storing gulped air inside its muscular stomach (lalli and gilmer 1989). a recent phylogenetic study of Glaucus revealed that the Indo-Pacific congener G. marginatus constitutes a species complex containing two cryptic pairs of sister species with overlapping dis-tributions (Churchill et al. 2013). a parallel change in the reproductive morphology has occurred once in each cryp-tic species pair, suggesting that a nongeographic isolation mechanism—reproductive character differentiation—may be a primary driver of cladogenesis in this species complex. On the other hand, molecular phylogenetic analyses of the cosmopolitan G. atlanticus did not recover trenchant clado-genetic structuring nor evidence of cryptic species within- and/or among-ocean basins (Churchill et al. 2013).

Here, we revisit Churchill et al. (2013) G. atlanticus mitochondrial dataset using more sensitive population genetics methodologies and a number of new sequences representing all five subtropical gyres (including the South atlantic for the first time) to test three general biogeo-graphic hypotheses for this species: (1) global panmixis (null hypothesis)—rates of global historical gene flow have been sufficient to result in the absence of genetic struc-turing among the five subtropical gyre systems (Fig. 1a). allopatric speciation processes are therefore impeded. (2) Ocean basin panmixis—neustonic exchange occurs readily among adjacent subtropical gyre systems, but not among-ocean basins, producing three ocean basin-specific clades, each potentially capable of proceeding toward allopatric

A B C

Fig. 1 Hypothetical networks of global subtropical gyre genetic structuring. a global panmixis. b Panmixis only within-ocean basins. c Pan-mixis only within-ocean gyres. NA north atlantic; SA South atlantic; NP north Pacific; SP South Pacific; IN Indian

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speciation (Fig. 1b). (3) Within-gyre panmixis—each sub-tropical gyre population is a discrete and isolated gene pool. Prolonged isolation is predicted to produce reciprocal monophyly, and eventually speciation, of gyre populations (Fig. 1c).

Materials and methods

Sample collection, Dna extraction, amplification, and sequencing

Glaucus atlanticus specimens were collected as a part of a global sampling of neustonic invertebrates from 2006 to 2012 (Fig. 2). Specimens were either collected by hand as they washed up on beaches, or in the open ocean via neus-ton net tows. all specimens were preserved in 95 % ethanol and identified by external morphology. at least 15–20 indi-viduals, when possible, were chosen arbitrarily from each ocean gyre for genetic analysis, but only five were available from the South atlantic (Table 1).

approximately 30 mg of tissue was sampled from the left ceratal cluster or foot. genomic Dna was extracted using the e.Z.n.a. Mollusc Dna Kit (Omega Bio-Tek) according to manufacturer’s protocols. Mitochondrial cytochrome oxidase I (COI) was amplified using the uni-versal primers lCO1490 and HCO2198 (Folmer et al. 1994) using the general PCr protocol [2 min at 95 °C; 35 cycles of 30 s at 94 °C, 30 s at X °C, 1 min at 72 °C; 5 min at 72 °C], where X = 45 °C. PCr products were sequenced directly with PCr primers using an aBI 3730xl automated sequencer (applied Biosystems, Inc.) by the University of Michigan Dna Sequencing Core. Forward and reverse primer chromatograms were aligned using the MUSCle algorithm (edgar 2004) implemented in Codon-Code aligner (CodonCode Corporation) and checked by

eye. COI sequences were aligned according to amino acid translations using the mitochondrial code for Mytilus edulis (nCBI-genBank).

Phylogenetic analyses

The sequence matrix (658 nucleotides of mitochondrial COI) was analyzed in jModelTest (Posada 2008) using likelihood calculations performed in PhyMl (guin-don and gascuel 2003) to determine the best-fit models of nucleotide substitution by akaike information crite-rion (HKY + Γ). Bayesian Markov Chain Monte Carlo (MCMC) analyses were performed in BeaST 1.7.2 (Drummond et al. 2012) using the HKY model with empir-ical base frequencies, gamma site heterogeneity with four rate categories, and nucleotides were partitioned using the SrD06 model (Shapiro et al. 2006). all parameters were linked, and the clock model was strict with substitu-tion rates estimated for nudibranch COI (1 %/MY; Shields 2009). Starting trees were generated randomly, and the tree priorly assumed the coalescent process of constant size. MCMC analyses were 10 million generations, logging parameters every 1,000 generations, and the first 25 % of trees were discarded as burn-in. Convergence was con-firmed by eye using the “Trace” function in Tracer v. 1.5 (rambaut and Drummond 2009) and by repeating all anal-yses three times. Three analyses were combined to gener-ate summary statistics and maximum clade credibility trees (median node heights and >0.5 posterior probabilities).

Phylogeographic analyses

To visualize the genetic structure of G. atlanticus, a hap-lotype network was constructed using TCS 1.21 (Clem-ent et al. 2000) with a 95 % connection limit. To identify genetic subdivisions between ocean basins and between

Fig. 2 Glaucus atlanticus and collecting localities. a G. atlanticus individual. Scale bar = 1.0 cm. b Map of collecting localities show-ing subtropical gyre boundaries. Collecting sites are indicated with

red squares. Subtropical gyres are color-coded: blue north atlantic; green South atlantic; red north Pacific; black South Pacific; yellow Indian

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Table 1 Material examined in this study, including isolate code, museum voucher number, coordinates (decimal degree), gyre system location, and genBank accession numbers

Isolate Voucher latitude longitude gyre system genBank #

01gaIWa WaM S59354 −32.0333333 115.7500000 Indian JQ699594





06gaIWa WaM S59356 −32.0000000 115.7500000 Indian KF961612












01gnagu UMMZ 302975 27.4965000 −084.9965000 north atlantic KF961623







08gnagu UMMZ 302975 27.4965000 −084.9965000 north atlantic JQ699574


11gnagu no voucher 27.4965000 −084.9965000 north atlantic KF961631










22gnaDO UMMZ 302975 27.4988000 −085.9983000 north atlantic KF961641













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Table 1 continued Isolate Voucher latitude longitude gyre system genBank #
















03ganPKo UMMZ 302983 19.3550000 −156.7216667 north Pacific JQ699588

05ganPKo UMMZ 302981 19.3550000 −156.7216667 north Pacific JQ699585

07ganPKo UMMZ 304388 19.3550000 −156.7216667 north Pacific KF961667







16ganPWH UMMZ 304391 19.1250000 −153.3616667 north Pacific KF961674

01gaSa UMMZ 303481 −34.1372000 018.4336000 South atlantic KF961675



04gasa UMMZ 303481 −34.1372000 018.4336000 South atlantic KF961678


01gaSPWH UMMZ 302980 −04.2650000 −141.7150000 South Pacific JQ699584

02gaSPPC aMS C.462956 −33.7380000 151.3100000 South Pacific JQ699587




06gaSPPC aMS C.462956 −33.7380000 151.3100000 South Pacific KF961680













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subtropical gyres, haplotypes were assigned to five popula-tions (north atlantic, South atlantic, north Pacific, South Pacific, and Indian Ocean) and grouped by ocean basin (atlantic, Indian, and Pacific). These subdivisions were tested for genetic structure using the analysis of molecu-lar variance (aMOVa) implemented in arlequin 3.5 (excoffier and lischer 2010). Molecular diversity indices were calculated in arlequin 3.5. arlequin 3.5 was also used to calculate FST values as a measure of pairwise differences between all population groups. The significance of the pairwise FST values was estimated by performing 16,000 permutations, and the nominal p values were subsequently corrected for multiple test biases by the Bonferroni stand-ard correction. Because F statistics and their relatives, such as GST, greatly underestimate differentiation when applied to highly polymorphic markers (Hedrick 2005; Jost 2008; Meirmans and Hedrick 2011), we also calculated the D statistic (Jost 2008), which is independent of within popu-lation diversity. genalex 6.5 (Peakall and Smouse 2006, 2012) was used to calculate Jost’s D by treating the mtDna haplotype data as genotype data (assuming homozygosity in all loci). The significance of the pairwise Jost’s D values was estimated by performing 9,999 permutations, and the nominal p values were subsequently corrected for multiple test biases by the Bonferroni standard correction. addition-ally, a spatial analysis of molecular variance (SaMOVa) was performed using SaMOVa version 1.0 (Dupanloup et al. 2002), which aims to define groups of samples that are maximally differentiated from each other yet geograph-ically homogenous. Because many sampling locations con-tained a single individual, locations that were geographi-cally close (within 3° of longitude or latitude) and in the same gyre system were merged together and their coordi-nates averaged, resulting in five populations: north atlan-tic, north Pacific, South atlantic, South Pacific 1 (aus-tralia), South Pacific 2 (Marquesas Islands), and Indian Ocean. a SaMOVa was performed for all five populations with 100 simulated annealing processes. The aMOVa and SaMOVa analysis results were compared for the a priori hypotheses of two groups (atlantic, Indo-Pacific) and three groups (atlantic, Indian, Pacific) to determine whether they produced similar clustering of genetic variation. Demo-graphic histories were characterized using mismatch analy-ses in arlequin 3.0 and by comparing Bayes factors for two demographic models in BeaST: coalescent with constant size, and coalescent exponential.

Molecular clock analyses

Date estimates for selected uncalibrated nodes in the BeaST Bayesian tree were derived by using r8s 1.7 (Sanderson 2003) using the finalization of the closure of the Isthmus of Panama as the calibration point, estimated

in 3.5 MYa (Coates et al. 1992; Coates and Obando 1996). The nodes tested represent the divergence between several haplotypes of Indo-Pacific affinity found in the atlantic from their Indo-Pacific closest relatives. analyses using the langley–Fitch and penalized likelihood methods were run. For the langley–Fitch method, analyses were run using the Powell and Truncated newton as well as the local molecu-lar clock procedure. The penalized likelihood analysis was run with the Powell algorithm.

Results

Phylogenetic reconstruction

Phylogenetic analysis of within-species G. atlanticus mito-chondrial haplotype diversity produced a poorly resolved tree (appendix S1) lacking significant support for most branches.

Phylogeographic analyses

Figure 3 shows the haplotype network of relationships in G. atlanticus, which is largely consistent with the results of the phylogenetic analyses, but incorporates non-bifurcating genealogical information derived from population-level divergences. Most atlantic haplotypes, north and south, clustered together; in fact, all five South atlantic specimens shared a haplotype with a north atlantic conspecific. at least 10 individuals were haplotyped from each ocean gyre except for the South atlantic (N = 5). Haplotype diversity (h) was maximal (1.00 within each ocean gyre population), and so an additional 30 individuals were genotyped from one ocean gyre (north atlantic) to investigate whether the addition of more individuals might reach saturation, but north atlantic haplotype diversity (h) remained at 1.00 (Table 2). This is almost entirely due to synonymous sub-stitutions in the third codon position, as evidenced by the much higher rate of evolution (2.54 vs. 0.23) of the third codon position generated in the Bayesian phylogenetic reconstruction. average pairwise diversity (π) was 6.37–7.96 for all gyre populations except the north Pacific, which was higher (π = 10.42). Tajima’s D values are nega-tive for all gyre populations, significantly so for all except South atlantic (Table 2); this further indicates an excess of rare haplotypes.

Hierarchical aMOVa analyses comparing variance between ocean gyres and ocean basins show that ϕST (between gyre) and ϕCT (between ocean basin) values are similar and somewhat low: 0.26 and 0.25, respectively (Table 3; ϕST is significant). In one additional nonhierarchi-cal aMOVa (Table 3), samples were grouped by atlantic basin versus Pacific + Indian basins, (ϕST = 0.29, p = 0.00),

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and in this case, ϕST is higher than in the original aMOVa. In both cases, the great majority of the variation (70–75 %) is explained by variation within populations (oceanic gyres) and almost 0 % is explained by variation among gyres within groups (ocean basins, i.e., north and South atlan-tic, north and South Pacific). These results probably reflect the presence of six haplotypes of Indo-Pacific affinity in the atlantic Ocean and one haplotype (13ganPKo) of atlantic affinity in the Pacific Ocean (Fig. 3).

The SaMOVa analysis of two groups confirmed the a priori hypothesis of an atlantic Indo-Pacific differentia-tion (Table 4). The results of the analysis are similar to the aMOVa results (Table 3), with approximately 70 % of the haplotype diversity found within populations, but an appre-ciable amount of haplotype diversity (28.09 %) separates groups. The differences among populations within regions are very small (0.52 %). as in the aMOVa analysis, ϕST (within populations) and ϕCT (among groups) values are

Fig. 3 Haplotype network of G. atlanticus mtCOI sequences. Subtropical gyres are color-coded: blue north atlantic; green South atlantic; red north Pacific; black South Pacific; yellow Indian

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similar and somewhat low: 0.28 and 0.28, respectively (ϕST is significant). The SaMOVa analysis of three groups failed to separate the remaining populations by ocean basins (atlantic, Pacific, Indian) as originally hypothesized, but instead, it separated them into the following three groups: atlantic, (Indian, north Pacific, South Pacific 1), and South Pacific 2. In this case, the haplotype diversity among groups is slightly smaller than in the two-group analysis (27.52 %).

The analysis of mismatch distributions generated in arlequin resulted in θ1 upper limit values of 99,999 for all populations (ocean gyres). This indicates that the population sizes are so large that there are no recent coa-lescent events and that demographic history cannot be estimated precisely by this method with these data (Sch-neider and excoffier 1999); thus, those results are not shown.

Table 2 Molecular diversity indices for ocean gyre populations of G. atlanticus

number of specimens haplotyped (N), nucleotide diversity—mean number of pairwise differences (π), haplotype diversity (h), and Tajima’s D with associated p values for all populations examined

Population Sample size (N) Haplotype diversity (h) nucleotide diversity (π) Tajima’s D p value

north atlantic 47 1.0000 ± 0.0044 6.799260 ± 3.260492 −2.23902 0.00094

South atlantic 5 1.0000 ± 0.1265 7.000000 ± 3.963246 −0.20309 0.49844

north Pacific 10 1.0000 ± 0.0447 10.42222 ± 5.197645 −1.68011 0.02831

South Pacific 18 1.0000 ± 0.0185 6.366013 ± 3.164781 −2.00876 0.00881

Indian 17 1.0000 ± 0.0202 7.955882 ± 3.891067 −1.92848 0.01394

Table 3 aMOVa tests of regional (ocean basin) and population (ocean gyre) genetic structure in G. atlanticus with two different data partitions

group 1, atlantic Ocean (2 populations); group 2, Pacific Ocean (2 populations); group 3, Indian Ocean (1 population)

Partitioning into three groups (ocean basins): atlantic, Pacific, Indian

df Sum of squares Variance components % Of variation F statistics

among groups (ocean basins) 2 79.145 1.21792 Va 24.93 ϕCT = 0.24930, p = 0.00000

among populations (gyres) within groups (ocean basins)

2 7.845 0.02564 Vb 0.52 ϕSC = 0.00699, p = 0.30594

Within populations (gyres) 92 335.041 3.64175 Vc 74.54 ϕST = 0.25455, p = 0.00000

Partitioning into two groups (ocean basins): atlantic, Pacific + Indian

df Sum of squares Variance components % Of variation F statistics

among groups (ocean basins) 1 74.147 1.43171 Va 27.94 ϕCT = 0.27943, p = 0.10238

among populations (gyres) within groups (ocean basins)

3 12.844 0.05023 Vb 0.98 ϕSC = 0.01361, p = 0.15446

Within populations (gyres) 92 335.041 3.6415 Vc 71.08 ϕST = 0.28923, p = 0.00000

Table 4 SaMOVa tests of genetic structure in G. atlanticus with group partitions into two and three groups

IN Indian Ocean; NA north atlantic; NP north Pacific; SA South atlantic; SP1 South Pacific 1 (australia); SP2 South Pacific 2 (north of Mar-quesas)

Partitioning into three groups: na + Sa, SP2, nP + SP1 + In

df Sum of squares Variance components % of variation F statistics

among groups 2 77.548 1.40182 Va 27.52 ϕCT = 0.27518, p = 0.02248

among populations within groups 2 7.845 0.02564 Vb 0.52 ϕSC = 0.01246, p = 0.00000

Within populations 92 335.041 3.64175 Vc 74.54 ϕST = 0.28422, p = 0.00000

Partitioning into two groups: na + Sa, SP1 + SP2 + nP + In

df Sum of squares Variance components % of variation F statistics

among groups 1 74.146 1.43903 Va 28.09 ϕCT = 0.28087, p = 0.06647

among populations within groups 4 16.073 0.03819 Vb 0.75 ϕSC = 0.01037, p = 0.00000

Within populations 91 331.031 3.64629 Vc 71.17 ϕST = 0.28087, p = 0.00000

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The genetic structure of G. atlanticus recovered in the haplotype network was tested using FST and Jost’s D pair-wise analyses (Tables 5, 6). The results indicate that north atlantic and north Pacific populations are not signifi-cantly different from each other. also, north Pacific, South Pacific, and Indian Ocean populations are not significantly different. However, all atlantic populations are signifi-cantly different from Indo-Pacific populations, suggesting limited gene flow between ocean basins. Our G. atlanticus data therefore reject hypotheses of exclusive within-gyre panmixis, and of global panmixis, but provide a mixed outcome for among-ocean basin panmixis: rejection for

atlantic/Indo-Pacific, but not for Indian/Pacific Ocean populations.

Molecular clock analyses

The molecular clock analyses revealed divergence times varying between 4.25–3.74 MYa and 1.47–1.2 MYa among atlantic haplotypes of Indo-Pacific affinity and their closest Indian Ocean haplotypes (Table 7). assum-ing that the temporal calibration point is correct (see “Dis-cussion”), this means that one north atlantic haplotype (37gnagu) diverged from its co-clustering Indo-Pacific haplotypes (Fig. 3) before the closure of the Isthmus of Panama, about 3.5 million years ago (Coates and Obando 1996), whereas the rest of the topologically ectopic north atlantic haplotypes (25gnagu, 49gnagu, 12gnagu, 43gnagu) diverged more recently, between 1.51 and 1.2 MYa. Our dataset lacks evidence of atlantic/Indo-Pacific gene flow within the last 1.2 million years.

Discussion

Glaucus atlanticus is not globally panmictic

Our data were inconsistent with all three hypotheses out-lined in Fig. 1, but best matched with Fig. 1b: panmixis only within-ocean basins. This is only partially consistent

Table 5 Matrix of the population group comparisons results, with FST values (lower triangular) and associated p values (upper triangular)

after Bonferroni correction (10 comparisons), significant values are p < 0.005. Significantly distinct FST values in bold, significant p values marked with an asterisk

NA north atlantic; SA South atlantic; NP north Pacific; SP South Pacific; IN Indian Ocean

na Sa nP SP In

na – 0.47312 ± 0.0045 0.00000 ± 0.0000* 0.00000 ± 0.0000* 0.00000 ± 0.0000*

Sa −0.00635 – 0.00020 ± 0.0001* 0.00000 ± 0.0000* 0.00000 ± 0.0000*

nP 0.24921 0.23789 – 0.14048 ± 0.0035 0.15840 ± 0.0034

SP 0.32328 0.40346 0.01473 – 0.06871 ± 0.0025

In 0.28405 0.31773 0.01302 0.01780 –

Table 6 Matrix of the population group comparisons results, with Jost’s D values (lower triangular) and associated p values (upper tri-angular)

after Bonferroni correction (10 comparisons), significant values are p < 0.005. Significantly distinct Jost’s D values in bold, significant p values marked with an asterisk

NA north atlantic; SA South atlantic; NP north Pacific; SP South Pacific; IN Indian Ocean

na Sa nP SP In

na – 0.492 0.001* 0.001* 0.001*

Sa 0.000 – 0.001* 0.001* 0.001*

nP 0.018 0.022 – 0.214 0.191

SP 0.023 0.032 0.000 – 0.060

In 0.021 0.027 0.001 0.001 –

Table 7 Divergence time ranges (in millions of years) between atlantic haplotypes of Indo-Pacific affinity (37gnagu, 25gnaDO, 49gnagu, 12gnagu, and 43gnagu) and their closest Indo-Pacific

haplotypes, estimated with r8s using different methods, algorithms, and calibration dates

TN truncated newton algorithm; PL penalized likelihood method

37gnagu 25gnaDO 49gnagu 12gnagu43gnagu

Mean rate variation Standard deviation

langley–Fitch/Tn 3.74 1.28 1.20 1.55 0.002602 1.93e−09

langley–Fitch/Powell 3.73 1.28 1.20 1.52 0.00261 2.212e−09

langley–Fitch/local 3.73 1.28 1.20 1.52 0.00261 2.212e−09

Pl/Powell 4.25 1.51 1.47 1.87 0.00204 0.0005069

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with Halobates micans (andersen et al. 2000), but pro-vides additional resolution that surface currents at gyre boundaries alone do not act as barriers to hyponeustonic gene flow. although the lack of resolution in the phyloge-netic tree and the mosaic topology of the haplotype net-work are consistent with global panmixis, the haplotype network recovered a group of mainly atlantic haplotypes and a group of mainly Indo-Pacific haplotypes with some limited topological intermixing, suggesting a certain level of ancestral gene flow between ocean basins. limited gene flow also probably accounts for the low ϕCT values in the aMOVa analysis. However, the FST and Jost’s D analyses clearly show that populations in one ocean basin (atlan-tic) are genetically distinct from those in the other two (Indian and Pacific) and do not support the hypothesis of global panmixis. Furthermore, the FST and Jost’s D analy-ses indicate that individual gyre populations in the atlantic and Indo-Pacific are not genetically distinct, rejecting the hypothesis of exclusive within-ocean gyre panmixis and supporting the hypothesis of within-ocean basin panmixis. On the contrary, the within-ocean basin panmixis, hypoth-esis is not supported by the SaMOVa analysis that main-tained Indian, north Pacific, and South Pacific 1 popula-tions in the same group, while separating South Pacific 2 into a group on its own. This, however, could be an artifact, because the South Pacific 2 population was represented by a single sequence. Both SaMOVa and aMOVa analyses indicate that most of the haplotype diversity is found within each population with an appreciable amount of diversity separating ocean basis (among groups). These results com-bined seem to indicate that G. atlanticus is not a panmictic species, although total or partial within-ocean basin pan-mixis cannot be rejected.

The nonpolar distribution of Glaucus likely reflects its inability to survive the cold waters of the arctic and Southern Oceans, thereby limiting its capacity to maintain gene flow between the atlantic and Indo-Pacific basins. However, available mtDna evidence failed to reveal structure within-ocean basins or gyres in G. atlanticus, suggesting that this species is a capable disperser through-out physically connected tropical and subtropical oceans. Intriguingly, its sister group, the Glaucus marginatus spe-cies complex, is composed of species with much more limited geographic ranges (Churchill et al. 2013) even though both groups share similar life histories and behav-iors. although sexual selection seems to be an important force resulting in speciation in the G. marginatus species complex, speciation events are most consistent with sym-patry and do not explain their smaller ranges or absence of this species complex from the atlantic Ocean. Further research on the feeding behavior or larval development of Glaucus may explain the different dispersal abilities of these species.

Cape of good Hope disjunction

One unexpected pattern found in our G. atlanticus data is the clustering of South atlantic specimens (sampled near Cape Town, South africa), exclusively in the atlantic group instead of the Indo-Pacific group, which are found only about 1,850 km eastward, in Durban. above the subtropi-cal convergence zone, the agulhas current flows southwest down the eastern side of South africa and enters a retro-flection region off the Cape of good Hope. On the western side, the Benguela current flows northeast from the retro-flection region (Walker 1989). either the South atlantic sample size was too low (N = 5) to recover expected global clade lineages, our sequence data are not sufficiently varia-ble to provide needed resolution to separate the South afri-can population from the rest of the atlantic, or this further supports that physical models, in this case of ocean surface currents, are inadequate in accurately predicting neustonic gene flow.

Historic gene flow between ocean basins

The molecular clock analysis indicates that all atlantic haplotypes of Indo-Pacific affinity are long diverged from their closest Indo-Pacific haplotypes: There is no evidence of gene flow between ocean basins for the last 1.2 MY. The main mechanism by which Indian Ocean marine organ-isms have migrated into the atlantic during the Pleistocene is the so-called agulhas leakage (Vermeij 2012), which is the exchange of fauna between the Indo-Pacific and the southwest atlantic. This faunal exchange is promoted by an enhanced agulhas current around the southern tip of africa, which oscillates in intensity with global tempera-tures (Peeters et al. 2004; Biastoch et al. 2009) and allows some dispersal of Indian Ocean species into the atlantic during interglacial periods (Vermeij 2012). Fish stocks are thought to have colonized the north atlantic and Mediter-ranean during these episodic oceanic interchanges (alva-rado Bremer et al. 2005), including top predators such as the great white shark (gubili et al. 2011) and the (marine mammal) orca (Foote et al. 2011). However, the sedimen-tary record in the southern tip of africa suggests that the pattern of glacial-interglacial contrasts was established 1.2 MYa (Diekmann and Kuhn 2002), making the agulhas leakage more episodic after a global cooling event dur-ing the mid-Pleistocene. In the case of Glaucus, it appears that gene flow between the Indo-Pacific and the atlantic may have been ceased during this mid-Pleistocene cooling period suggesting that the late Pleistocene agulhas leak-age may be less permissive for passive drifters than for active swimmers. This may also be a factor in the absence of the Indo-Pacific G. marginatus species complex from the atlantic Basin.

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However, the results of the molecular analysis must be interpreted cautiously. Without external calibration points for neustonic taxa (e.g., given global surface current con-nectivity, it is impossible to say with certainty that the closure of the Panamanian Isthmus completely separated north atlantic and north Pacific neuston populations), the divergence times are only as accurate as the molecu-lar clock estimates. Shields (2009) reviews the support for using 1 %/MY in nudibranchs from the ross Sea—the rate is based upon a fossil-calibrated bivalve phylogeny of trans-isthmanian species pairs (Marko 2002), and it has also been recommended for circumtropical gastropods in a fossil-calibrated phylogeny (Frey and Vermeij 2008) and for invertebrate mitochrondrial COI in general (Dawson et al. 2011).

The biogeographic relationship of Glaucus spp. to other hyponeustonic taxa depends on critical genetic data from their prey species, the neustonic hydrozoans Physalia, Vele-lla, and Porpita, and a co-occurring neustonic mollusk lin-eage, the bubble-rafting snails Janthina janthina. Unfortu-nately, there are almost no data available on the population genetics of these organisms, but a recent study suggests that there is substantial cryptic diversity among Physalia present in new Zealand coastal waters (Pontin and Cruick-shank 2012). Considering the morphological variation in Physalia between larger regions, there is the possibility that this group contains a substantial amount of cryptic variation.

global panmixis as a model of planktonic population structure

Few studies address planktonic population structure at the global scale, but the overwhelming majority that do have found no support for true panmixis. Macrogeographic IBD signals have been recovered in populations of a nearshore rotifer (Mills et al. 2007), two sister species of oceanic copepods (goetze 2005), a superspecies of high-dispersal diatoms (Casteleyn et al. 2010), and eight mophospecies of planktonic foraminiferans (reviewed in Darling and Wade 2008), for example. In general, hypotheses of planktonic gene flow are conceived as large-scale versions of studies of smaller areas (e.g., seas, offshore regions) where pan-mixis is common. Our study adds to the small, but growing body of evidence that global planktonic panmixis occurs chiefly in theoretical frameworks.

Acknowledgments We thank the following for their assistance: USa: J. lyczkowski-Shultz (Southeast area Monitoring and assess-ment Program) and r. Humphreys (Pacific Islands Fisheries Science Center) of the national Oceanographic and atmospheric admin-istration, the students and crew of Sea Semester (www.sea.edu), T. lee (University of Michigan Museum of Zoology); australia: P. Colman (australian Museum), l. Beckley (Murdoch University), S.

Slack-Smith and C. Whisson (Western australian Museum); South africa: r. van der elst (Oceanographic research Institute), D. Herbert (natal Museum), Mark gibbons (University of the Western Cape), W. Florence and e. Hoenson (Iziko Museums of Cape Town). D. riek photographed live glaucinins. Funding for this research comes from nSF award OCe 0850625 and national geographic Society award 8601-09 to D.ÓF.

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