TY - JOUR
AU - Herbig,, Hans-Georg
AB - Abstract It was Darwin that noted the large intraspecific diversity of the goose barnacle Lepas Linnaeus, 1758 and thought about distinct regional varieties. Today, biogeographic compartmentation is known from marine species, but data from globally occurring species remain scarce. We analysed inter- and intraspecific divergence within the epipelagic rafter Lepas from tropical and temperate oceans by means of two mitochondrial and one nuclear DNA marker. Besides phylogenetic relations, we resolved biogeography and controlling factors. Inhabiting the Southern Hemisphere, Lepas australis Darwin, 1851 shows separate populations from coastal Chile and from circum-Antarctic waters, most probably related to temperature differences in the current systems. The cosmopolitan Lepas anatifera Linnaeus, 1758 displays four regional subgroups (coastal Chile, Northeast Pacific/Oregon, the Southern Hemisphere Indopacific, and the Atlantic), and a global group, which might be an ancestral stem group. The differentiation reflects vicariance effects rooted in geological history: the closure of the Neogene Tethys in the Middle East and at the Panama Isthmus, the installation of the cool Benguela Current, differing Pleistocene currents and temperatures, and modern current systems. The extreme ecological generalists Lepas anserifera Linnaeus, 1767 and Lepas pectinata Spengler, 1793 are not differentiated, and might represent true global species. In conclusion, compartmentation of the oceans acts at the species level according to ecospace limits. For Lepas, the multitude of barriers favours allopatric speciation. Darwin, global species, molecular phylogeny, oceanic compartmentation, rafting, speciation, vicariance effects Introduction Before starting his work on the Origin of Species Charles Darwin spent several years studying barnacles (Cirripedia, Thoracica). He published four monographs on these cosmopolitan, highly derived, sessile crustaceans (Darwin, 1851a, b, 1854a, b). In the volume dedicated to lepadomorph (or goose) barnacles, Darwin (1851a: 68, 76) noted that ‘all the valves, even in the same species, are subject to considerable variation in shape’ and, concerning Lepas anatiferaLinnaeus, 1758, ‘from the foregoing description it will be seen how extremely variable almost every part of this species is’. This later led others to suspect either cryptic speciation or great morphological plasticity in at least the type species L. anatifera (Newman & Ross, 1971; Newman, 1972). In fact, the cosmopolitan epipelagic rafter Lepas might constitute a model genus to test speciation patterns in the open ocean and to verify the existence of truly cosmopolitan species. Since the beginning of the 21st century complexes of cryptic species have been discovered in a growing number of taxa by the application of the phylogenetic species concept. Although the most popular showcase might have been the African Elephant (Roca et al., 2001; Roca, Georgiadis & O'Brien, 2005), the marine environment harbours a large and diverse number of taxa for which cryptic species have been identified. In a now classic paper, Knowlton (2000) reviewed cryptic speciation in the marine environment and discussed the consequences for our understanding of the nature and age of species boundaries. She, and later (Westheide & Schmidt 2003) and Peijnenburg & Goetze (2013), for example, addressed the fact that the neglect of abundant cryptic speciation leads to the serious underestimation of global diversity, and thus has impact on present taxonomic usage and on the question of the existence of cosmopolitan species (Norris, 2000). On the other hand, (Coyne & Orr 2004) stated that ‘… it is not clear how barriers to gene flow operate in the open ocean’, and therefore the oceans of the world might constitute an interconnected environment where gene flow between populations might persist. Consequently, the question arises of whether the sea might harbour global/cosmopolitan species with little or no genetic differentiation (Hewitt, 2000), or whether divergence into geographically distinct cryptic species is common in most marine taxa, despite apparent morphological uniformity in some. A subsequent question tackles the problem of speciation mechanisms, i.e. speciation by sympatry in an apparently barrierless environment vs. speciation by allopatry in an environment with hitherto unrecognized barriers (see discussion in Norris & Hull, 2011). Taxa spending their whole life or very long times in the open ocean–such as holoplanktonic organisms, pelagic swimmers, drifters and rafters, and organisms with extended pelagic larval stages–might maintain gene flow over enormous distances in the oceanic environment. These taxa might have the potential to be truly cosmopolitan, but if no effective gene flow is maintained, diversification and evolution of regional variants should be expected. In that context, the matter of time is controversially discussed. Peijnenburg & Goetze (2013) postulated that rapid evolution into regional subtypes should be common in the zooplankton, but Norris & Hull (2011) stressed the importance of geological time for speciation. Surprisingly, to date, the majority of studies deal with geographically restricted benthic species, most with planktonic larvae. As these organisms must return to a suitable benthic environment within the finite larval lifetime, and must comply with the requirements of their often very complex lifecycles, they are no model candidates to resolve the question of cosmopolitanism. Among the many examples available, we mention the ascidian Ciona intestinalis (Linnaeus, 1767) (Caputi et al., 2007), the shallow-water temperate to boreal bryozoan Electra pilosa (Linnaeus, 1767) (Nikulina, Hanel & Schäfer, 2007), the pantropical seaweed Halimeda (Kooistra, Coppejans & Payri, 2002; Verbruggen et al., 2005), and the pantropical sea urchins Eucidaris and Tripneustes (Lessios et al., 1999; Lessios, Kane & Robertson, 2003), which all show separation by vicariance induced by land barriers and/or ocean currents. Among the pelagic organisms analysed, the hydrozoan Obelia geniculata (Linnaeus, 1758) showed previously unrecognized speciation between oceanic regions. It is, however, absent from some major oceanic regions (Govindarajan, Halanych & Cunningham, 2005), and therefore is not a suitable taxon for global assessment. A global phylogeographic study of the bryozoan Membranipora membranacea (Linnaeus, 1767), which is commonly attached to rafting substrata, indicated a high degree of regionalism induced either by temporal vicariance, i.e. allopatric speciation, as a result of climatic events or by founder populations dispersing to new suitable habitats (Schwaninger, 2008). Copepods are intensively studied and yielded differing results. Goetze (2003) found high levels of cryptic diversity at the species level in the Eucalanidae, and, concerning Rhincalanus nasutus Giesbrecht, 1888, geographically distinct populations within the Pacific, and between the Pacific and the Atlantic. Using the cytochrome c oxidase subunit I (COI) gene as a single marker, Goetze (2005) found that in two antitropical Eucalanus species, differentiation might result from habitat discontinuities induced by large oceanic gyre systems. Eberl et al. (2007) showed genetic diversification in the ‘pseudobenthic’ rafting copepod Macrosetella gracilis (Dana, 1847), but could not resolve the question of cryptic speciation, as the study is based on a single mitochondrial locus. Population differentiations within and between oceans were later also described in other copepod species (Goetze, 2011; Halbert, Goetze & Carlon, 2013; Norton & Goetze, 2013; Andrews et al., 2014; Cornils & Held, 2014). Yet, concerning the copepod Calanus finmarchicus (Gunnerus, 1770), entrapment in ocean current systems was questioned by Provan et al. (2009), based on genetic data and the modelling of the palaeodistribution during the Last Glacial Maximum. Also, data on the planktonic nudibranch Glaucus yielded different results on the species level: the Indo-Pacific Glaucus marginatus (Bergh, 1860) shows four regional populations, but the cosmopolitan Glaucus atlanticus Forster, 1777 appeared to be panmictic (Churchill et al., 2013). Refined analysis of G. atlanticus, however, showed the separation of two Indo-Pacific and two Atlantic haplotypes, related to the gyres of the northern and southern hemispheres, respectively (Churchill, Valdés & Ó Foighil, 2014). Concerning protists, extensive work on planktonic foraminifer genera show continuing cryptic speciation and regionalism from genetic and fossil data, from a local to global scale (Darling et al., 1999; De Vargas et al., 1999, 2002; Darling & Wade, 2008; Aurahs et al., 2009; Weiner et al., 2014). Results for the planktonic diatom Pseudo-nitzschia pungens (Grunow ex Cleve) G.R. Hasle, 1993 show limited gene flow and strong isolation by distance (Casteleyn et al., 2010). Protists, however, might be more strongly affected by temperature, salinity, chemistry, and other ecological factors, and/or higher speciation rates, and thus results might not be comparable with metazoans. All presently available data stress the fact that the planktonic environment is much more complex than previously anticipated (see also Darling & Wade, 2008). Most studies find consistent differences between the major ocean basins (e.g. Norton & Goetze, 2013), but high levels of connectivity have been reported for the Antarctic Ocean (Bortolotto et al., 2011). Reports about putative panmictic species in the epipelagic environment are scarce, and more research is necessary to understand the mechanisms that either permit cosmopolitan distribution or lead to cryptic speciation, as has already been emphasized (e.g. Saez & Lozano, 2005). The goose barnacles of the genus Lepas (Cirripedia, Thoracica) are ideal candidates to investigate this question because they are epipelagic surface rafters with abundant populations inhabiting all oceans (see Fig. 1 for the distribution of three important species). As stated above, reasons to suspect cryptic speciation in the genus go back to Darwin, and later to Newman & Ross (1971), who suggested the existence of subspecies in different oceanic regions. We tested the possible divergence within species of the genus on a global scale based on 26 sampling sites from temperate and tropical regions of the Pacific, Indian, and Atlantic oceans, relying on the putative cosmopolitans Lepas anatiferaLinnaeus, 1758; Lepas anseriferaLinnaeus, 1758; and Lepas pectinata Spengler, 1792, as well as Lepas australis Darwin, 1851, inhabiting the circum-Antarctic seas, and the rare Lepas testudinata Aurivillius, 1894. Figure 1. Open in new tabDownload slide Major oceanic current systems and the distribution of Lepas anatifera (blue), Lepas australis (green), and Lepas pectinata (orange, vertically ruled), after Hinojosa et al. (2006). Lepas anatifera is restricted to waters warmer than 15 °C, whereas L. australis is found in cooler water masses. Figure 1. Open in new tabDownload slide Major oceanic current systems and the distribution of Lepas anatifera (blue), Lepas australis (green), and Lepas pectinata (orange, vertically ruled), after Hinojosa et al. (2006). Lepas anatifera is restricted to waters warmer than 15 °C, whereas L. australis is found in cooler water masses. We show clear phylogenetic diversification, i.e. cryptic speciation, in two species, but also gene flow, respectively. panmixis between oceans in others. The extended geographic sampling allows for a discussion of pathways and barriers to gene flow in the open ocean. We find that allopatric speciation is the most likely explanation for divergence in certain species of Lepas, but do not rule out sympatric speciation as a possibility in the pelagic environment. Material and Methods Sampling and species identification Samples were collected from around the globe (Tables 1, 2) between summer 2007 and summer 2012. Specimens were preserved in ethanol (EtOH) immediately after collection. EtOH was exchanged upon sample reception, and the material was then stored at −20 °C. Individual specimens were again placed into EtOH and stored at −80 °C after DNA extraction. All original species identifications from local collectors were (re-)confirmed by the first author in accordance with descriptions from the literature (Darwin, 1851a; Gruvel, 1905; Nilsson-Cantell, 1930; Newman, 1972; Anderson, 1994; Hinojosa et al., 2006). Diagnostic features like the form and number of filamentary appendages, as well as shell structures (e.g. number, presence, and location of umbonal teeth; form of shell plates; ridges and impressions on the plates) were analysed with stereomicroscopes and photographed for sample subsets. Table 1. First sampling campaign Region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Coastal Chile Coquimbo 16 4 8 Gulf of Mexico Florida Coast 10 5 – – Spain Ria de Vigo 16 1 – 1 New Zealand N/A – – 6 6 Region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Coastal Chile Coquimbo 16 4 8 Gulf of Mexico Florida Coast 10 5 – – Spain Ria de Vigo 16 1 – 1 New Zealand N/A – – 6 6 Geographic location and number of specimens for which the sequences of three loci (18S, COI, and 16S) could be obtained. Open in new tab Table 1. First sampling campaign Region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Coastal Chile Coquimbo 16 4 8 Gulf of Mexico Florida Coast 10 5 – – Spain Ria de Vigo 16 1 – 1 New Zealand N/A – – 6 6 Region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Coastal Chile Coquimbo 16 4 8 Gulf of Mexico Florida Coast 10 5 – – Spain Ria de Vigo 16 1 – 1 New Zealand N/A – – 6 6 Geographic location and number of specimens for which the sequences of three loci (18S, COI, and 16S) could be obtained. Open in new tab Table 2. Geographic location of species in the second sampling campaign Major oceanic region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Lepas testudinata . East Pacific Coquimbo, Chile + – – + – Temperate/cool South Pacific Off the coast of southern Chile + – + – – New Zealand – + + + – North-east Pacific Oregon + – – – – North-west Pacific Japan – + – – – Equatorial South Pacific Tonga + – – – – New Caledonia + – – – – East Indic Western Australia + + + – + South-west Indic South Africa + + + + + Gulf of Mexico West coast of Florida + + – + – Atlantic Argentinia – – + – – Uruguay – + – – – Brazil + + – – – Azores – – – + – Senegal – + – – – Cape Verde + + – – – Madeira + – – – Atlantic Spain + + – – – Mediterranean Balearic Islands (Ibiza) – – – + – Major oceanic region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Lepas testudinata . East Pacific Coquimbo, Chile + – – + – Temperate/cool South Pacific Off the coast of southern Chile + – + – – New Zealand – + + + – North-east Pacific Oregon + – – – – North-west Pacific Japan – + – – – Equatorial South Pacific Tonga + – – – – New Caledonia + – – – – East Indic Western Australia + + + – + South-west Indic South Africa + + + + + Gulf of Mexico West coast of Florida + + – + – Atlantic Argentinia – – + – – Uruguay – + – – – Brazil + + – – – Azores – – – + – Senegal – + – – – Cape Verde + + – – – Madeira + – – – Atlantic Spain + + – – – Mediterranean Balearic Islands (Ibiza) – – – + – Open in new tab Table 2. Geographic location of species in the second sampling campaign Major oceanic region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Lepas testudinata . East Pacific Coquimbo, Chile + – – + – Temperate/cool South Pacific Off the coast of southern Chile + – + – – New Zealand – + + + – North-east Pacific Oregon + – – – – North-west Pacific Japan – + – – – Equatorial South Pacific Tonga + – – – – New Caledonia + – – – – East Indic Western Australia + + + – + South-west Indic South Africa + + + + + Gulf of Mexico West coast of Florida + + – + – Atlantic Argentinia – – + – – Uruguay – + – – – Brazil + + – – – Azores – – – + – Senegal – + – – – Cape Verde + + – – – Madeira + – – – Atlantic Spain + + – – – Mediterranean Balearic Islands (Ibiza) – – – + – Major oceanic region . Location . Lepas anatifera . Lepas anserifera . Lepas australis . Lepas pectinata . Lepas testudinata . East Pacific Coquimbo, Chile + – – + – Temperate/cool South Pacific Off the coast of southern Chile + – + – – New Zealand – + + + – North-east Pacific Oregon + – – – – North-west Pacific Japan – + – – – Equatorial South Pacific Tonga + – – – – New Caledonia + – – – – East Indic Western Australia + + + – + South-west Indic South Africa + + + + + Gulf of Mexico West coast of Florida + + – + – Atlantic Argentinia – – + – – Uruguay – + – – – Brazil + + – – – Azores – – – + – Senegal – + – – – Cape Verde + + – – – Madeira + – – – Atlantic Spain + + – – – Mediterranean Balearic Islands (Ibiza) – – – + – Open in new tab DNA extraction and chemistry Muscle tissue for DNA extraction was taken from the peduncle or the base of the cirri. DNA was extracted using commercially available kits. DNA was stored at −20 °C during the experimental phase, and later transferred to −80 °C. Polymerase chain reactions (PCRs) were conducted under the inclusion of high-fidelity polymerases. When necessary, 10% Trehalose or Betaine was added to enhance PCR yield and specificity. 16S and 18S primers, as described in Simon-Blecher, Huchon & Achituv (2007), were used. A new, slightly degenerated, forward primer (FwcoInt_Lep: 5′–ATAYTAATTCGTGCWGAACTMGG–3′) had to be designed for the COI locus while the ‘universal’ reverse primer (Hebert, Ratnasingham & deWaard, 2003) could be employed. PCR set-ups and cycle settings were optimized for each marker and depending on the quality of DNA extractions. PCR products were visualized on Agarose gels and products showing neither multiple bands nor excessive smearing on the gel were cleaned employing the Exo/Sap protocol (Werle et al., 1994), or commercial kits from various manufacturers. In few cases were multiple bands persisted after adaptation of the PCR protocols bands were gel-excised and purified with commercial kits. Sequences were either obtained through in-house Sanger sequencing and read out on ABI capillary DNA Analyzers at the Cologne Center for Genomics (CCG, University of Cologne), or by sending dried PCR products to Macrogen Inc. (Seoul, South Korea and Amsterdam, the Netherlands). Sequencing was in general conducted bi-directional and a subset of samples was repeatedly amplified and sequenced to control for possible sequencing errors. For 16S and COI the same forward and reverse primers as used in the PCR were employed. For the longer stretch of the 18S gene internal primers as described in Simon-Blecher et al., (2007) were used. An additional primer (18S_Fsq4_Lep: 5′-ATCGACTGGAGGGCAAGC), yielding a larger region of internal overlap, was designed. Genetic markers and out-group We constructed a three-loci phylogeny, sequencing fragments of one nuclear and two mitochondrial genes. We used the canonical barcoding gene cytochrome c oxidase subunit I (COI), which has been applied successfully in analyses of cryptic species complexes (Hebert et al., 2004). The 16S ribosomal DNA (16S) gene had been employed in recent studies on barnacle phylogeny (Simon-Blecher et al., 2007). To avoid depicting only the maternal site of evolution, we added a fragment of the conserved 18S rDNA [18S, small ribosomal subunit (SSU)] nuclear gene that previously had been used to establish a barnacle phylogeny (Simon-Blecher et al., 2007). From our initial analysis of the full sequence of the 18S gene (~1800 bp) we were able to define a smaller region that contained enough informative sites to robustly delimit species, and subsequently amplified this shorter fragment. We obtained specimens of Dosima fascicularis (Ellis & Solander, 1786) from Argentina and Heteralepassp. from Madeira as out-group species for the phylogenetic analyses. Phylogenetic analysis Sequences were compared with barnacle sequences stored at the US National Center for Biotechnology Information (NCBI) nucleotide database with BLAST to detect possible contamination with foreign DNA. All sequences obtained were submitted to GenBank and may be retrieved under the accession numbers: FJ906772–FJ906777, GU993588–GU993704, and KT947133–KT947465. Acquired sequence traces were visually inspected with the programs CODON CODE ALIGNER 2.0.6 and GENEIOUS 5 and 6. The PHRED (Ewing et al., 1998) quality-assessment algorithm was applied in CODON CODE ALIGNER to control the chromatograms. Assemblies were then built using a PHRAP subprogram and visually re-inspected for ambiguous base calls, which were then corrected or the sequence was discarded. For GENEIOUS we used stand-alone PHRED if necessary, and then assembled the reads with the implemented algorithm. We initially re-constructed a phylogeny for the limited set of samples available to us at that time using maximum-likelihood (ML) and Bayesian approaches, as described in Appendix S1. After acquiring our final set of global samples we combined these with the initial alignments of each single locus in GENEIOUS and re-aligned with CLUSTALΩ 1.0 (Sievers et al., 2011). We visually inspected and edited the alignments, where necessary, in GENEIOUS and SEAVIEW 4 (Galtier, Gouy & Gautier, 1996). GENEIOUS was also used to create a combined all-loci alignment. We used SPLITSTREE 4 (Kloepper & Huson, 2008) on the combined alignment to explore phylogenetic signal in our data and assess potential conflicts between loci and sample groups. We then chose to analyse all loci individually. We ran PhyML (release 20131016; Guindon & Gascuel, 2003) on our data, choosing the general time-reversible (GTR) + gamma model, and used RAxML 7.7.2 (Stamatakis, 2006), running for 20 iterations, as a second validation on some alignments. Bootstrapping was conducted for 1000 generations in PhyML. The program was also used to re-confirm the inference of our initial geographically restricted data set, originally performed in PAUP*. We tested our alignments with MODELGENERATOR 0.85 (Keane et al., 2006) and implemented the evolutionary models in MRBAYES 3.2.2 (Huelsenbeck & Ronquist, 2005) to infer Bayesian phylogenies. We ran this program for 5 million generations in four parallel runs on the local computer cluster CHEOPS. For the L. anatifera population we constructed haplotype genealogies using FITCHI (Matschiner, 2015), which also provided us with fixation index (FST) values for haplotypes in different oceans. For the FST calculations we compared results when assigning specimens to their sampling regions and to biogeographic regions. Results All L. pectinata and L. australis specimens shared morphological features as described in the literature, and could therefore be assigned to respective species. Lepas australis specimens from Coquimbo in Chile were much smaller than other individuals of this species, and were therefore considered as juveniles. Lepas testudinata specimens were identified by A. Biccard (Cape Town). Specimens of L. anatifera from the Southeast Pacific were also juveniles, differing in size from specimens collected from other regions. Interestingly, all L. anatifera from the Gulf of Mexico, as well as some specimens from other regions, show at least one line of brownish squares across the scuta (and sometimes across the terga as well), and slightly more elevated radial lines (or ribs) on the scuta (for shell nomenclature, see Fig. 1). Both features were not present to the same extent in the remaining samples. Nevertheless, all morphs were clearly assigned to L. anatifera, as the varying features had previously been described (see especially Newman, 1972). Initial phylogenetic analysis: indications from the Atlantic and Southern Pacific In a first assay we analysed specimens from three oceanic regions (Schiffer & Herbig, 2008): the North Atlantic (including the Gulf of Mexico); the south-east Pacific (Coquimbo, Chile); and the south-west Pacific (New Zealand). From these sampling sites genomic DNA from 76 specimens was suitable to sequence all three loci (Tables 1 and S1). Across all species, out of the 76 specimens 74 differing mitochondrial haplotypes for both marker loci (16S and COI) were obtained, whereas only six 18S haplotypes were retrieved for the whole sample set. The length of the alignment of all loci was 2792 bp. Both ML and Bayesian inference for the all-loci alignment yielded the same tree topology on genus, species, and intraspecies levels. Our later recalculation of the ML tree with PhyML showed no deviation in topology from the first inferences. Only minor differences are present in the placing of single individuals within biogeographical subgroups between ML and Bayesian inference. The monophyly of L. pectinata, L. anatifera, and L. anserifera received 100% bootstrap and posterior probability support, whereas L. australis is supported with 99.5 and 100%, respectively (Fig. 2). Figure 2. Open in new tabDownload slide Initial phylogeny based on three genes (18S, 16S, and COI) from a subset of samples from the major oceans (PAUP*, re-confirmed with PhyML and MrBayes). A split in subtypes is found in Lepas anatifera and Lepas australis. The red arrows indicate ‘outliers’ in L. anatifera and Lepas pectinata. In L. anatifera, these were later found to belong to a global group, whereas in L. pectinata no obvious biogeographic subgroup was found. Figure 2. Open in new tabDownload slide Initial phylogeny based on three genes (18S, 16S, and COI) from a subset of samples from the major oceans (PAUP*, re-confirmed with PhyML and MrBayes). A split in subtypes is found in Lepas anatifera and Lepas australis. The red arrows indicate ‘outliers’ in L. anatifera and Lepas pectinata. In L. anatifera, these were later found to belong to a global group, whereas in L. pectinata no obvious biogeographic subgroup was found. On an intraspecies level a split was found between the L. anatifera populations from the Southeast Pacific (monophyletic, with 100% bootstrap support and 96% posterior probability) and populations from the north-east Atlantic/Gulf of Mexico (71.7 and 98%; Fig. 2). A group of specimens from the Gulf of Mexico (both 100%) was set apart from the remaining individuals of the north-east Atlantic/Gulf of Mexico clade (99.8 and 100%). One L. pectinata specimen from the north-east Atlantic and south-west Pacific was set apart from all other individuals of this taxon (Fig. 2), albeit only weakly supported (76 and 50%). Two distinct biogeographical monophyletic clusters were found in L. australis, where specimens from the south-east Pacific form one group (99.9 and 99%), and specimens from the south-west Pacific (99.9 and 100%) form a second group (Fig. 2). Refined phylogenetic analysis: towards a global biogeography Nuclear phylogeny To map the diversity indicated by our first analysis more finely on a global scale, and to resolve population structures, we tried to acquire samples from all major oceans (Fig. 3; Table 2). As in the first assay, most specimens could be collected for L. anatifera and at least for L. pectinata (Table S1). Figure 3. Open in new tabDownload slide Phylogenetic (maximum-likelihood and Bayesian) tree based on the analysis of a fragment of the 18S ribosomal gene, including two out-group species. Posterior probabilities and bootstrap values are indicated; where support is maximal, only posterior probabilities are given. A neighbour net constructed in SplitsTrees indicates a clear tree-like signal in the sequence data, in conflict with the data for Lepas anserifera, which led us to analyse mitochondrial loci independently. Figure 3. Open in new tabDownload slide Phylogenetic (maximum-likelihood and Bayesian) tree based on the analysis of a fragment of the 18S ribosomal gene, including two out-group species. Posterior probabilities and bootstrap values are indicated; where support is maximal, only posterior probabilities are given. A neighbour net constructed in SplitsTrees indicates a clear tree-like signal in the sequence data, in conflict with the data for Lepas anserifera, which led us to analyse mitochondrial loci independently. From the initial phylogenetic analyses we were able to define a shorter region of the 18S gene that carried enough signal for species and possibly subspecies discrimination. We acquired 18S sequences from 140 additional specimens that could be incorporated into our biogeographic analysis. In this set we found two 18S variants in L. anatifera, two in L. australis, and one each in L. pectinata, L. anserifera, and in the out-groups. Specimens morphologically identified as L. testudinata were very similar in the 18S gene sequence to the L. anatifera specimens. The phylogenetic analysis of these sequences yielded a species tree (Fig. 3) that confirmed our initial findings from fewer sampling sites (see above), but includes the out-groups D. fascicularis and Heteralepassp. The tree shows a split into geographical subgroups in L. anatifera and L. australis, but not in the other species. Posterior probabilities strongly support the placement and monophyly of all species, but indicate that the groupings of L. australis and L. anatifera, as well as L. pectinata and D. fascicularis, as sister species cannot be resolved with this locus alone. As expected when using a shorter and highly conserved gene sequence, bootstrap values drop in comparison with what was described above for our initial phylogenetic inference; however, L. australis and L. anatifera still receive very strong support, and the other species are far from being weakly supported (Fig. 3). In L. australis we found two groups, one encompassing specimens exclusively from the Chilean coast and a second group containing samples from New Zealand (sampled at different times), Argentina, and from the western coast of Australia; additionally, a single sample from South Africa is included. The coherent group inferred for L. pectinata includes specimens from New Zealand and Chile (both from different sampling campaigns), from the Western Mediterranean (Balearic Islands/Ibiza), and the Azores. The coherent group found for L. anserifera holds samples from the North Atlantic (Gulf of Mexico, north-east Spain, Senegal, and the Cape Verde Islands), the South Atlantic (Brazil, Uruguay, and South Africa), and from New Zealand, western Australia, and Japan. Using the 18S tree as an indication of possible intraspecies divergence and a phylogenetic backbone, we tested our mitochondrial data for potential conflict in the phylogenetic signal. A neighbour net constructed from the all-loci alignment in SPLITSTREE (Fig. 3) shows a tree-like structure in most of its branches; however, some potential conflicts were indicated (e.g. for L. anserifera in a Japanese sample and in the L. testudinata subgroup). Furthermore, it appeared likely that the COI and 16S loci evolve at different speeds. Therefore, we decided to infer individual phylogenies for each of the two mitochondrial loci. Mitochondrial phylogenies The COI and 16S trees support all Lepas and out-group species with high posterior probabilities and bootstrap support, but differ in the positioning of single species (Fig. 4). In particular in the Bayesian analyses the out-group D. fascicularis (from the coast of Uruguay) is positioned next to L. australis and L. anserifera in the 16S tree, whereas it is found as a sister group to L. testudinata and thus close to the L. anatifera group in the COI tree. Lepas anserifera is also found closer to L. anatifera in the COI tree, where it is nested inside of a split between L. pectinata and L. australis. By contrast, in the 16S tree L. pectinata is closest to L. testudinata, which is an out-group to L. anatifera. These general groupings also slightly deviate in the ML inference. Figure 4. Open in new tabDownload slide Comparsion of mitochondrial single locus phylogenies based on PhyMl and MrBayes. All major groupings are retrieved from both loci. The faster evolving COI gene provides more intra-group resolution. Bootstrapping values are given above branches; posterior probabilities are given below branches. Figure 4. Open in new tabDownload slide Comparsion of mitochondrial single locus phylogenies based on PhyMl and MrBayes. All major groupings are retrieved from both loci. The faster evolving COI gene provides more intra-group resolution. Bootstrapping values are given above branches; posterior probabilities are given below branches. Both mitochondrial genes support a split in L. australis, as already described for the 18S gene, with one group from coastal Chile and a second group encompassing specimens from the other sampling sites of the Southern Ocean. The split is far more pronounced in the COI gene, indicating the higher substitution rate of this locus. The mitochondrial genes support the evolution of L. anatifera into several biogeographical subgroups, but disagree in the placement of groups in relation to each other. Consequently, branches leading towards single groups have strong support, whereas branches separating groups have weaker posterior probabilities and bootstrap values. The coastal Chilean subgroup, already inferred with the 18S gene, is retrieved in both mitochondrial gene trees. It is clearly separated from the other groups, but is supplemented by a single sample from eastern South Africa. Conversely, one single individual from the Chilean coast is plotted in a group with samples from the Indo-Pacific region (Fernandez Islands off Chile, Easter Islands, Tonga, South Africa). Two further groups were identified: one contains specimens from the Oregon Coast (the Northeast Pacific); and the second includes specimens from the Atlantic Ocean. The Atlantic group had already been inferred in our initial survey (Schiffer & Herbig, 2008), and is now supplemented by specimens from Brazil, Madeira, and further samples from Spain (Cádiz). In our initial survey we also recognized two specimens from the Gulf of Mexico, which were set apart from all other Atlantic L. anatifera (Fig. 1). They are now placed in a globally distributed subgroup known from Tonga, Cape Verde Islands, western Australia, and with one sample from South Africa. To further elucidate the complex biogeographic pattern found in the mitochondrial L. anatifera phylogenies, we calculated FST values and haplotype networks for the COI gene. We did not rely on reticulation networks to resolve haplotype connectivity, but used Fitchi (Matchiner, 2015), a method based on Salzburger, Ewing & von Haeseler (2011) to construct haplotype genealogies (Fig. 5). It incorporates the phylogenetic results, and should generate a biologically more meaningful picture allowing to compare monophyletic subgroups. We analysed 576 residues in the COI gene (Atlantic, 25 samples; Gulf of Mexico, ten samples; South Africa, seven samples; coastal Chile, 19 samples; Equatorial Pacific, six samples; East Indic, five samples; South Pacific, three samples; Oregon coast, three samples; out-group L. testudinata, six samples; for all pairwise results, see Table S2). Panmixis between the eastern Atlantic and the Gulf of Mexico is confirmed with very low FST values. The separation of the Oregon and coastal Chilean subgroups from other regions is firmly established. Results also support persistent gene flow between South Africa, the Southern Pacific (Easter Islands), and Equatorial Pacific (Tonga), confirming the Indo-Pacific subgroup of the phylogenetic analysis (Fig. 4). As the regionally dispersed individuals of the ‘global subgroup’ might influence the FST calculations if assigned to their respective sampling regions, these specimens were put into a single unit. The recalculated pairwise FST values between individuals of the ‘global subgroup’ and those of all other subgroups are high (0.80–0.94; Table S2), and stress the existence of a de facto global subgroup. Figure 5. Open in new tabDownload slide Haplotype genealogy of the COI genes from Lepas anatifera and Lepas testudinata populations, computed with Fitchi. A separation of oceanic regions described in the main text and depicted in Figure 6 can be seen, as well as one global group in L. anatifera. The L. testudinata haplotype recovered from Australia is set apart from the South African haplotypes. Figure 5. Open in new tabDownload slide Haplotype genealogy of the COI genes from Lepas anatifera and Lepas testudinata populations, computed with Fitchi. A separation of oceanic regions described in the main text and depicted in Figure 6 can be seen, as well as one global group in L. anatifera. The L. testudinata haplotype recovered from Australia is set apart from the South African haplotypes. As indicated above, the nuclear 18S gene and both mitochondrial genes place L. testudinata as an out-group to L. anatifera. The mitochondrial genes show a split between individuals from South Africa and Australia. In L. pectinata we retrieved a rather unstructured tree topology for both mitochondrial genes, but in the COI a tentative Atlantic–Mediterranean subgroup might be indicated, including our samples from South Africa, the Azores, north-east Spain, and the Balearic Islands (Ibiza). Discussion Barnacles within the genus Lepas attach to flotsam (wood, algae, styrofoam, buoys, etc.), vessels, and swimming animals after spending up to 2 months drifting as larvae (Darwin, 1851aa; Anderson, 1994; Hinojosa et al., 2006). Lepas anatifera is the goose barnacle with the highest abundance worldwide (Young, 1990), as reflected in our sampling. It thrives where the sea surface temperature (SST) exceeds 18 °C (Hinojosa et al., 2006), and is thought to survive in waters warmer than 15 °C (Patel, 1959). Temporary settlements, as transient species at physiological limits outside the contiguous distribution, are known (Skinner & Barboza, 2014). Lepas pectinata, on the other hand, tolerates somewhat cooler temperatures (Zevina & Memmi, 1981). The geographic ranges of both species range from approximately 50–55°N to 40–45°S (Fig. 1), thus spanning both temperate and tropical regions of the oceans of the world. Lepas australis is a cold-water species inhabiting all of the oceans surrounding the Antarctic continent (Newman & Ross, 1971; Hinojosa et al., 2006). These are first-order ecological constraints that limit the global distribution of the species. Further pathways and barriers for gene flow, leading either to panmixis or the establishment of subgroups, will be discussed below. First of all, our results demonstrate differing evolutionary trajectories in different Lepas species. Lepas pectinata and L. anserifera show one global genotype in the nuclear 18S gene. Also the mitochondrial loci do not indicate clear geographic subdivision; however, in L. pectinata a distinct COI genotype of a potential Atlantic subgroup cannot be ruled out. In L. australis and L. anatifera local subgroups are developed based on nuclear and mitochondrial data. In L. australis all markers support two subgroups. These are a coastal Chilean subgroup and a second subgroup encompassing the remainder of samples from the Southern Ocean. The results concerning L. anatifera are more complex. The nuclear marker supports three genotypes: one including L. testudinata as a subtype; one from the Chilean coast; and one global subgroup, with specimens from all major oceans. Mitochondrial data indicate even stronger differentiation in geographically distinct groups (Fig. 4, 5), but also support the existence of the global subgroup. Lepas australis It is most surprising to find a geographic pattern in L. australis that is even supported by the slowly evolving nuclear locus. The species in general is restricted to cold-water masses linked with Antarctica, an area where longitudinal landmasses that could impede gene flow, and thus lead to the evolution of geographically confined populations, are missing. In contrast, the westerly wind-driven Antarctic Circumpolar Current (ACC) effectively mediates the transport of organisms around Antarctica. Within the ACC the Antarctic Polar Front (APF, or Antarctic Convergence; U.S. Geological Survey 2010/2012), which coincides approximately with the edge of the winter sea ice, and a sudden change in seawater surface temperature, acts as an effective latitudinal barrier between water masses, and also between marine organisms (Thornhill et al., 2008). Further north, the Subantarctic Front (or Subtropical Convergence; U.S. Geological Survey, 2010/2012), i.e. the northern boundary of the ACC, is a second major latitudinal water-mass barrier. The effectiveness of these fronts as barriers to gene flow has already been demonstrated, e.g. in the chaetognath Eukrohnia hamata (Möbius, 1875) (Kulagin et al., 2014). All our sampling sites are situated north of the ACC. There, we recognize a common population of L. australis in the cool to temperate waters north of the Subantarctic Front, herein termed the ‘Southern Ocean subgroup’. This population of L. australis is obviously caught in the current system adjoining north of the Subtropical Convergence, albeit also circling eastwards around Antarctica, and in the north-east deviating current systems, which sweep up the coasts of Western Australia and New Zealand (South Indian Current s.l.), respectively, Argentina (Falkland Current), and South Africa (Benguela Current) (Fig. 6). Figure 6. Open in new tabDownload slide Global biogeography of common species of Lepas showing major genetic subgroups, sampling locations (dots), and the inferred generalized distribution of the subgroups according to species occurrences from the GBIF database and our results. Major current systems responsible for dispersal (red, warm water currents; blue, cold water currents) and biogeographic barriers (red double arrows: 1, closure of the Tethys in the Middle East during the later Miocene; 2, closure of the Panama gateway during the Pliocene; 3, separation of the Indopacific and the Atlantic by water mass barrier; 4, separation of Chilean coast ʽwaterpocket' by water mass barrier) are shown. A, Lepas australis: dark green, coastal Chilean subgroup; light green, Southern Ocean subgroup; asterisk, questionable separate Subantarctic subgroup. B, Lepas anatifera: black dot, disputable ancient global stem group (= Lepas anatifera indica subgroup); yellow, Indopacific subgroup; blue, Atlantic subgroup; dark green, Oregon subgroup (= Lepas anatifera pacifica subgroup); green, coastal Chilean subgroup. No data are available for the north-western Pacific (question mark). C, Lepas anserifera and Lepas pectinata with undifferentiated panmictic populations; Lepas testudinata with differences between western Australian and South African subgroups. Figure 6. Open in new tabDownload slide Global biogeography of common species of Lepas showing major genetic subgroups, sampling locations (dots), and the inferred generalized distribution of the subgroups according to species occurrences from the GBIF database and our results. Major current systems responsible for dispersal (red, warm water currents; blue, cold water currents) and biogeographic barriers (red double arrows: 1, closure of the Tethys in the Middle East during the later Miocene; 2, closure of the Panama gateway during the Pliocene; 3, separation of the Indopacific and the Atlantic by water mass barrier; 4, separation of Chilean coast ʽwaterpocket' by water mass barrier) are shown. A, Lepas australis: dark green, coastal Chilean subgroup; light green, Southern Ocean subgroup; asterisk, questionable separate Subantarctic subgroup. B, Lepas anatifera: black dot, disputable ancient global stem group (= Lepas anatifera indica subgroup); yellow, Indopacific subgroup; blue, Atlantic subgroup; dark green, Oregon subgroup (= Lepas anatifera pacifica subgroup); green, coastal Chilean subgroup. No data are available for the north-western Pacific (question mark). C, Lepas anserifera and Lepas pectinata with undifferentiated panmictic populations; Lepas testudinata with differences between western Australian and South African subgroups. The ‘coastal Chilean subgroup’ differs from all other sampling sites. It matches biogeographic results of a separate eastern Pacific/Chilean province from other pelagic organisms. Examples are the copepod Rhincalanus nasutus (Goetze, 2003), which has a sister group along the North American coast, the rafting bryozoan Membranipora (Schwaninger, 2008), the giant kelp Macrocystis (Coyer, Smith & Andersen, 2001), or, most evident, the bull kelp Durvillaea antarctica (Chamisso) Hariot 1892 (Fraser et al., 2009). The latter shows distinct haplotypes along the central and northern Chilean coast. In analogy to results from New Zealand, they are most probably related to the continuous warming of seawater (Fraser et al., 2009; Fraser, Nikula & Waters, 2011). The observations on D. antarctica are consistent with further zoogeographic studies of littoral benthic and pelagic organisms, which provide evidence for three distinct, most probably SST-related, zoogeographical regions along the Chilean coast (Escribano, Fernández & Aranís, 2003; Hinojosa et al., 2006). As goose barnacles from the southern hemisphere are abundantly rafting on detached Macrocystis and D. antarctica (Thiel & Gutow, 2004; Hinojosa et al., 2006), the coastal Chilean subgroup appears to be very plausible. In addition to extreme genetic divergence between populations of the bull kelp D. antarctica from Chile and New Zealand, and further genetic differentiation within both regions, a genetically homogeneous population exists further south within the ACC (Fraser et al., 2009, 2011). Unified Antarctic genotypes were also described for the chaetognath Eukrohnia hamata and the Antarctic krill Euphausia superba Dana, 1850 (Bortolotto et al., 2011; Kulagin et al., 2014). Therefore, it might be hypothesized that a third, truly subantarctic L. australis subtype might exist south of the Subtropical Convergence. One tiny L. australis specimen collected from floating Durvillaea kelp at around 50°S off the Chilean coast does not belong to the postulated Subantarctic subgroup, nor to the coastal Chilean subgroup, but to the Southern Ocean subgroup. Lepas anatifera Based on mitochondrial DNA we found five distinct subgroups, which can be clustered into three major divisions. A subgroup collected off the Chilean coast during several sampling campaigns is set apart from all other globally sampled specimens by divergence in mitochondrial and nuclear markers, except for a single specimen. It shares the global nuclear genotype, but is retrieved inside the Chilean group based on its mitochondrial genes. This specimen might have been introduced by human activity. Distinct Atlantic, Indo-Pacific, and Northeast Pacific subgroups based on differing mitochondrial loci within one nuclear genotype. A global subgroup containing specimens from dispersed sampling sites. Division 1: the coastal Chilean subgroup The coastal Chilean subgroup is equivalent to our findings in L. australis (Fig. 6). Obviously, the cold-water system of the Humboldt Current and the associated upwelling system along the Chilean margin prevent exchange between confined populations of certain organisms, and induce a Chilean phylogeographic province of strongly restricted permeability. Internally it is subdivided by increasing SSTs (see above). The influence of the Humboldt Current is obvious, as the warmer water species L. anatifera is not recorded south of 33°S (Hinojosa et al., 2006). The divergence of L. australis and L. anatifera in both mitochondrial and nuclear markers indicates an ancient origin of that ‘Chilean waterpocket’, which might be also seen in the present-day tendency of floating debris to accumulate in the South Pacific, west of Chile (Lebreton, Greer & Borrero, 2012). Division 2: regional populations with mitochondrial differences The well-individualized Atlantic subgroup of L. anatifera (Fig. 6) includes specimens from the Gulf of Mexico and from the Spanish Atlantic coast (Galicia, Gulf of Cádiz), with the Gulf Stream apparently mediating genetic exchange across the Atlantic. Specimens further south from Madeira and from the Cape Verde Islands demonstrate continued rafting within the Canary Current, and thus show well-expressed circling within the North Atlantic gyre. Finally, specimens from the Brazilian coast (Recife) demonstrate trans-equatorial exchange with the Southern Hemisphere, and thus provide evidence of a single large trans-equatorial Atlantic population. In the relatively narrow, north–south-orientated Atlantic Ocean, the North Equatorial Countercurrent is seasonally less pronounced, and water masses of both hemispheres are exchanged by the western, north-flowing Guiana Current in boreal winter and spring, although some through-flow also occurs in summer (Csanady, 1990; Condie, 1991). That current system is also in line with data on the distribution of L. pectinata in the Atlantic (see below). A similar pan-Atlantic population has also been observed in the ocean skater Halobates micans Eschscholtz, 1822 (Andersen et al., 2000), and in the scyphozoan Pelagia noctiluca (Forsskal, 1775) (Miller, von der Heyden & Gibbons, 2012). The most favourable tropical gateway between Pacific and Atlantic marine populations was blocked by the formation of the Panama land bridge about 3.5–3.1 Mya (Duque-Caro, 1990). The resulting genetic divergence in marine taxa has previously been described by Knowlton & Weigt (1998), and more recently by Bacon et al. (2015). As a result of SSTs as low as 8 °C (Boyer et al., 2005), drifting around Cape Horn is most unlikely for the warmer water species L. anatifera. In fact, along the Chilean coast L. anatifera dominates at 23°S, at a mean SST of 21.9 °C, but fades towards 33°S, at an SST of 18.2 °C (Hinojosa et al., 2006). This southern limit at the Chilean coast is in accord with the GBIF database (GBIF, 2015), which lists only a single specimen, without collecting data further south, at 43.88°S. Although somewhat variable, SSTs around the southern tip of South America have not been substantially higher through geological time (Mashiotta, Lea & Spero, 1999; King & Howard, 2000; Feldberg & Mix, 2002), and therefore this passage has apparently been blocked continuously. The only feasible remaining genetic exchange between populations from the Indo-Pacific and Atlantic would be around the Cape of Good Hope. The warm Agulhas Current transports organisms from the Indian Ocean to the southern tip of Africa, but is then reflected back eastwards, thus impeding the transport of rafting organisms into the Atlantic. The retroflection of the Agulhas Current is caused by West Wind drift and the Benguela Current, which transports cold water masses across the southern Atlantic towards the south-west African coast, feeding the upwelling system along the south-west African (Namibian) coast (Lange et al., 1999). The SST of the Benguela Current close to the coast is around 15 °C (Clement & Gordon, 1995). Towards the open ocean, this rises up to 19–20 °C during summer, but does not exceed 17–18 °C in winter (e.g. GES DISC, 2012). These relatively cold waters might be outside of the preferential ecospace of L. anatifera, or warm-water seasons might be too short for successful rafting across the area. Besides the general current reflection these seem to be additional factors impeding gene flow. In fact, L. anatifera is reported in a diversified assemblage of goose barnacles from the Cape Town peninsula (Whitehead, Biccard & Griffiths, 2011), at the westernmost waning of the Agulhas Current, but no record from the Namibian coast is known to us from literature or databases; only three specimens from offshore Luanda (Angola) are listed in the GBIF database (GBIF, 2015), and are the only ones found from along the African coast south of Dakar (Senegal), and south of our samples from approximately the same latitude from the Cape Verde Islands, which are plotted in the global subgroup (see below). The formation of Indo-Pacific and Atlantic echinoid species within the genus Tripneustes was also related to the Benguela Current (Lessios et al., 2003). Although direct comparison with Lepas might be somewhat problematic, as the pantropical shallow-water echinoids and their planktonic larvae are more sensitive to cold water, the current systems appears to be an ancient and impermeable barrier to other organisms, like the ocean skater H. micans (Andersen et al., 2000), the nudibranch G. atlanticus (Churchill et al., 2014), and warm–temperate fish (Henriques et al., 2014). The general bottleneck for marine plankton, caused by the complicated current systems at the southern tip of Africa, was recently elucidated by Villar et al. (2015). The Benguela Current System is ancient. It was established about 10–12 Mya (Diester-Haass, Meyers & Vidal, 2002; Heinrich et al., 2011). During the Last Glacial Maximum the southern 15 °C SST isotherm, herein considered as the minimum threshold temperature for L. anatifera, was considerably farther north than it is today, effectively touching the African and West Australian Coasts (Williams & Benzie, 1998; for present-day SST isotherms, see Fig. 1). Similar shifts have to be assumed for the preceding Pleistocene glacials. These extended intervals of globally decreased SSTs provided first-order barriers to gene flow in L. anatifera: the ‘Algulhas Leakage’ during Pleistocene interglacials that allowed certain species to enter the Atlantic from the Indic (Vermeij, 2012) obviously did not work for L. anatifera, and nor for G. atlanticus (Churchill et al., 2014). In summary, the present-day Atlantic subpopulation of the species (Brazil, Gulf of Mexico, Atlantic Spain, and Madeira) is related to different ancient vicariance events, namely: (1) plate tectonics (closure of the Panama land bridge); (2) installation and modern persistence of current systems (Benguela Upwelling System); (3) climate variability in time, resulting in shifting ecospace limits (i.e. the extension of low SSTs towards the north during glacial periods) as well as permanent climate barriers (Cape Horn passage). The Oregon coast subgroup of L. anatifera appears to be related to the North Pacific gyre (Fig. 6). It seems to be effectively separated from the Southern Pacific and Indic regions by the Equatorial Countercurrent (FST 0.93 and 0.94). Notably, the endemic Northeast Pacific species Lepas pacifica Henry, 1940 appears to be very similar to L. anatifera (Newman & Abbott, 1980, and references therein). As molecular data firmly establish a separate Northeast Pacific subgroup inside L. anatifera, we suggest that L. pacifica might be regarded as a subspecies, L. anatifera pacifica; however, as our specimens (conserved in in a solution containing dimethyl sulphoxide, disodium EDTA, and saturated NaCl: ‘DESS’, Yoder et al., 2006) were not suitable for in-depth anatomical comparisons, further studies are needed to resolve the taxonomy. Unfortunately, samples of L. anatifera from Japan are missing, and we cannot comment on relationships between north-western and north-eastern Pacific populations. The separate Oregon coast subgroup supports the importance of the Pacific gyre systems for L. anatifera, however, just opposite to L. anserifera (see below). Species of the ocean skater Halobates, as well as genetically discriminated populations of H. micans and Halobates sericeus Eschscholtz, 1822 (Andersen et al., 2000; Leo, Cheng & Sperling, 2012), also prove the existence of disjunct northern and southern populations in the Pacific. The rafting bryozoan Membranipora shows a much higher degree of regionalization, but the northern and the southern Pacific clades are clearly separated (Schwaninger, 2008). Like Membranipora, eucalanid copepods display strong regional diversification (Goetze, 2003, 2005). Rhincalanus nasutus has five genetically distinct populations in the Pacific (Goetze, 2003). As for L. anatifera, a north-eastern Pacific population is discerned, but additionally a north-western Pacific population is present. In the southern Pacific, R. nasutus shows separate south-eastern and south-western Pacific populations supplemented by a Sulu Sea population. In contrast to the different R. nasutus populations, L. anatifera forms a spatially extended, huge Indo-South Pacific subgroup, with specimens found from the Juan Fernandez Islands, off Chile, to South Africa, similar to the nudibranch G. atlanticus (Churchill et al., 2014). Oceanic surface flow in the ‘Southern Hemisphere supergyre’ (Speich, Blanke & Cai, 2007) was also evidenced by surface drifting tracer buoys (Van Sebille, England & Froyland, 2012), and this supports our observations. Division 3: the global group–a dispersed L. anatifera stemgroup? Most puzzling is a globally distributed subgroup with specimens from the Gulf of Mexico, the Cape Verde Islands, South Africa (Cape Town region), Western Australia, and Tonga (Fig. 4, 5). One could speculate that this is the most ancestral group, originating from the circumequatorial Tethys before its disruption by Miocene plate tectonic collisions in the Middle East and later closure of the Isthmus of Panama, in the early Pliocene, about 3.5–3.1 Mya (Duque-Caro, 1990). Vicariance through both events is also discussed for other taxa, such as the ocean skater Halobates micans (Andersen et al., 2000) and the green alga Halimeda (Hillis, 2001). The same appears to be supported by our FST values from the rapidly evolving COI gene, indicating a lack of gene flow between the subgroup and other L. anatifera subgroups. Interestingly, the specimens acquired from the Western Australian Museum were putatively determined as Lepas indica Annandale, 1909 (A. Hosie, pers. comm.). As they share the global nuclear L. anatifera genotype, they do not belong to a distinct species. The original determination of Annandale (1909) as L. anatifera indica might thus be taxonomically correct, but further in-depth analysis of type material is needed for clarification, as is the case for ‘L. anatifera pacifica’ discussed above. Lepas testudinata In general, L. testudinata seems to be a rare species. From our nuclear data (18S gene) we retrieved it as an in–group to L. anatifera. By contrast, Rees et al. (2014) found the species as a sister group to L. australis in their phylogram of barnacle phylogeny, based on 28S sequences. According to the morphological similarity of both species, we assume that single-gene genealogies are not suitable to resolve their relationships, and propose a future phylogenomic study. Mitochondrial data suggest the existence of two subgroups, in South African and western Australian waters. This is in contrast to the cool-water species L. australis, and to the warmer water species L. anatifera also. The different population structure remains unexplained. Lepas anserifera and Lepas pectinata Our 18S data place L. anserifera as an out-group to L. anatifera and L. australis, and L. pectinata as an out-group to all three (Fig. 3). Our data show that northern and southern hemisphere populations of L. pectinata are not genetically distinct. Previous observations from the Atlantic report finding the species in temperate and tropical waters from the north of Ireland to Cape Horn (Gruvel, 1905; Young, 1990; see also data from the GBIF database). This means that a split into distinct antitropical populations (Hinojosa et al., 2006: fig. 6; see our Fig. 1) is not realized, and that panmixis between oceans is likely. Tolerance of cooler waters is known for L. pectinata (Zevina & Memmi, 1981; see also Hinojosa et al., 2006; our Fig. 1). This means that transit from the Indopacific into the Atlantic around South Africa should be possible, in spite of the Benguela upwelling system, whereas the plate tectonic-induced barrier at the Isthmus of Panama and the climate-induced barrier at Cape Horn will persist. In L. anserifera a panmictic population in both hemispheres is documented by our samples from the north-equatorial Atlantic (Cabo Verde, Senegal), from South Africa, and from Japan. It appears that similar ecological constrains explain the undifferentiated biogeographic distribution of L. pectinata and L. anserifera. Concerning the Indopacific realm, the difference between L. anserifera and L. anatifera is striking. The latter, like other pelagic invertebrates, is strongly related to the hemispherical gyre systems, which are effectively separated by the eastward-directed equatorial countercurrent; however, a study using tracer buoys and subsequently modelling the development of the huge garbage patches that accumulate in the gyre systems of the oceans on centennial timescales (Van Sebille et al., 2012) demonstrated the leakiness of these systems and interchange between all of the oceans. Most important in this respect appear to be non-linear mesoscale eddies (Chelton, Schlax & Samelson, 2011). Trans-equatorial relationships are also well known for other Pacific marine organisms. In a study on the calcareous sponge Leucetta ‘chagosensis’ Dendy, 1913, Wörheide, Hooper & Degnan (2002) proved a trans-equatorial clade, including individuals from the northern and central Great Barrier Reef as well as from Guam and Taiwan. Benzie & Williams (1997) also found strongly related haplotypes of the giant clam Tridacna maxima Röding, 1798 from the Philippines and the Great Barrier Reef. Finally, Williams & Benzie (1998) noted remarkable analogies in the starfish Linckia laevigata (Linnaeus, 1758) between several south-western Pacific, Philippine, and Japanese occurrences. Trans-equatorial dispersal was also mentioned by Schwaninger (2008) concerning the Neogene dispersal of Membranipora, rafting on kelp southwards along the western coasts of the Americas, and later northwards in the Atlantic–a hypothesis in contrast to the current system stressed by Van Sebille et al. (2012). Thus, additional pathways seem to have existed in the Pacific seasonally, episodically, or during times of changed climate or sea level in the geological past. It might be speculated that during Pleistocene glacial periods and strongly restricted Indonesian throughflow towards the Indian Ocean, a western trans-hemispherical current was developed, comparable with the present-day Guiana Current along the western margin of the Atlantic. In fact, Benzie & Williams (1997) and Williams & Benzie (1998) showed that the routes of gene flow in the western Pacific are not consistent with present-day ocean currents. They inferred dispersal during lowered Pleistocene sea levels and colder climate, which affected wind and current systems in multiple ways, and the general extension of species originating in the western Pacific in the north-western direction, towards Southeast Asia. Conclusion Our results show that a multitude of inherited and persistent barriers exist in the present-day open ocean. They act on the species level, according to the ecospace realized by a taxon in the geological past, and today, with the following anticipated consequences. Speciation in the pelagic realm has its roots in geological time (for example, see Andersen et al., 2000; Fraser et al., 2009; Norris & Hull, 2011; Leo et al., 2012). The hypothesis of rapid diversification and regionalization of zooplanktic species (Peijnenburg & Goetze, 2013) cannot be regarded as a general valid rule. Allopatric speciation is of prime importance, even in apparently barrierless oceanic regions like the southern Pacific. Sympatric speciation remains an option in the vast pelagic realms, but has yet to be proven. There is no general pattern for cryptic speciation, with respect to the formation of populations in the pelagic realm. Studies on the species level are necessary for different taxa, in order to counterbalance ecological limits and the corresponding barriers operating in time and space. The existence of truly cosmopolitan taxa in the pelagic environment is not yet settled. Only very few taxa, such as L. pectinata and L. anserifera, which are extreme ecological generalists and are capable of long-distance dispersal, might be able to maintain gene flow throughout the oceans of the world. Both species might be model taxa to clarify the problem, by extending regional data and using second-generation sequencing approaches. Anthropogenic transport and swimming mammals further complicate the distributional patterns, as seen in genetically isolated specimens. Such dispersal modes have been known for more than half a century (e.g. Bishop, 1951; Zevina & Memmi, 1981); however, they are apparently still overridden by natural provincialism, as evidenced by the distinct populations. In fact, Darwin (1851a) started the discipline of phylogeography. He was the first to note the morphological plasticity within species–notably within Lepas–and the importance of (land) barriers for species separation. Some 120 years later, Newman (1972) recognized regional diversification, with the formation of subspecies within L. anatifera based on morphological differences. His results are partly supported by our genetic data. Still, our results from Lepas and the discussed comparisons with other taxa illustrate that biotic dynamics in the world oceans are far from being understood. Being the largest biotope of the world, and probably the most valuable resource for mankind, such data are indispensable, however, not least concerning questions of conservation in times of global change (Wright et al., 2015). Acknowledgements The initial results on the phylogeography of Lepas were obtained during work on the unpublished diploma thesis of P.S. (2009), but the results presented herein were achieved in the course of other projects by low-cost research, unfunded by research foundations. The authors acknowledge continued financial and laboratory support, however, from the Volkswagen Stiftung (P.S.) and Universität zu Köln (P.S., H.G.H.). We are especially thankful to Martin Thiel (Coquimbo, Chile) for providing specimens and valuable comments on earlier versions of the article. We particularly want to thank Wim Damen and Einhard Schierenberg, who provided lab space and consumables, and Michael Kroiher for technical advice. We thank A. Kraemer-Eis for help with PCRs and sequencing, and also for help with drawings. We are grateful to all colleagues who provided samples: P. Wirtz, Madeira; A. Biccard and C. Griffith, Cape Town; A. Hosie, Wellington and Perth (at the Western Australian Museum and acquired with help of M. Türkay, Frankfurt); E. Schwindt, Puerto Madryn; J.M. Guerra Garcia, Sevilla; and others. Last but not least, we would like to thank the two reviewers for their valuable comments, which helped to improve our article. 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TI - Endorsing Darwin: global biogeography of the epipelagic goose barnacles Lepas spp. (Cirripedia, Lepadomorpha) proves cryptic speciation
JO - Zoological Journal of the Linnean Society
DO - 10.1111/zoj.12373
DA - 2016-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/endorsing-darwin-global-biogeography-of-the-epipelagic-goose-barnacles-0VTs7sDhbA
SP - 507
VL - 177
IS - 3
DP - DeepDyve
ER -