Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans

This is an electronic reprint of the original article. This reprint may differ from the original in pagination and typographic detail. Author(s): Title: Year: Version: Please cite the original version: All material supplied via JYX is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans Kesäniemi, Jenni; Rawson, Paul D.; Lindsay, Sara M.; Knott, Emily Kesäniemi, J., Rawson, P., Lindsay, S., & Knott, E. (2012). Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans. Ecology and Evolution, 2 (5), 994-1007. doi:10.1002/ece3.226 Retrieved from http://onlinelibrary.wiley.com/doi/10.1002/ece3.226/full 2012 Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans J. E. Kes¨ aniemi1,P.D.Rawson 2,S.M.Lindsay 2& K. E. Knott1 1Department of Biological and Environmental Science, University of Jyv¨ askyl¨ a, P.O. Box 35, FI-40014, Finland 2School of Marine Sciences, University of Maine, Orono, Maine 04469-5751 Keywords COI, developmental mode, larvae, population structure. Correspondence Jenni E. Kes¨ aniemi, Department of Biological and Environmental Science, University of Jyv¨ askyl¨ a, P.O. Box 35, FI-40014, Finland. Tel: +358 40 805 3872; E-mail: [email protected] Funded by a grant from the Jenny and Antti Wihuri Foundation and the Centre of Excellence in Evolutionary Research (University of Jyv¨ askyl¨ a). K. E. K. acknowledges fruitful discussions with members of the Marie Curie Initial Training Network Speciation funded by the European Union. Received: 26 October 2011; Revised: 16 January 2012; Accepted: 19 January 2012 Ecology and Evolution 2012; 2(5): 994–1007 doi: 10.1002/ece3.226 Abstract Development in marine invertebrate species can take place through a variety of modes and larval forms, but within a species, developmental mode is typically uniform.Poecilogonyreferstothepresenceofmorethanonemodeofdevelopment within a single species. True poecilogony is rare, however, and in some cases, apparent poecilogony is actually the result of variation in development mode among recently diverged cryptic species. We used a phylogenetic approach to examine whether poecilogony in the marine polychaete worm, Pygospio elegans,istheresult of cryptic speciation. Populations of worms identified as P. elegans express a variety of developmental modes including planktonic, brooded, and intermediate larvae; these modes are found both within and among populations. We examined sequence variation among partial mitochondrial cytochrome c oxidase subunit I sequences obtained for 279 individual worms sampled across broad geographic and environmental scales. Despite a large number of unique haplotypes (121 haplotypes from 279 individuals), sequence divergence among European samples was low (1.7%) with most of the sequence variation observed within populations, relative to the variation among regions. More importantly, we observed common haplotypes that were widespread among the populations we sampled, and the two most common haplotypes were shared between populations differing in developmental mode. Thus, our results support an earlier conclusion of poecilogony in Pelegans. In addition, predominantly planktonic populations had a larger number of population-specific low-frequency haplotypes. This finding is largely consistent with interspecies comparisons showing high diversity for species with planktonic developmental modes in contrast to low diversity in species with brooded developmental modes. Introduction Most marine invertebrates have complex life cycles and show a diverse range of larval developmental modes. Developmental mode is often defined as discrete categories describing characteristics of larvae, or larval types (Levin and Bridges 1995). For example, larvae can be planktonic (pelagic) or benthic, feeding or nonfeeding, brooded or free-living, and a combination of multiple descriptors is often necessary for a complete definition of developmental mode (e.g., McEdward and Janies 1993; Collin 2003; Raff and Byrne 2006). Developmental mode is an important aspect of invertebrate life histories, with wide-ranging consequences affecting, for example, development time, mortality, and dispersal potential (Levin and Bridges 1995). Understanding the consequences and evolution of different developmental modes is, on one hand, aided by our tendency to categorize it as discrete types. On the other hand, such definitions may also lead us to overlook intermediate or facultatively varying forms that do not fit definitions of discrete developmental modes (Allen and Pernet 2007). Many different developmental modes may be observed within genera or larger taxonomic groups, but typically only one developmental mode exists within a single species. In rare cases, species may express two or more development modes. The term poecilogony (Giard 1905 cited in Krug 994 c 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution Non Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. J. E. Kes¨ aniemi et al. No Cryptic Speciation in Pygospio elegans 2009) has been used to describe such developmental mode polymorphism. In poecilogonous species, multiple developmental modes are observed, either within or among different populations of a single species. True poecilogony has been documented within spionid worms (e.g., Streblospio benedicti, Levin 1984, and Boccardia proboscidia, Gibson 1997; Oyarzun et al. 2011) and in sacoglossan sea slugs (reviewed in Krug 2007, 2009). However, in a number of cases, what were originally described as poecilogonous species have turned out to be morphologically cryptic species with species-specific developmental modes (see Hoagland and Robertson 1988). The rarity of true poecilogony has led some authors to suggest that there are costs associated with polymorphic development and that poecilogony is a transient stage of speciation co-occurring with developmental mode transitions (Gibson and Gibson 2004; Ellingson and Krug 2006). Alternatively, poecilogony might be an advantageous plastic response, and a potential bet-hedging strategy, to enhance offspring success in the face of changing environmental conditions (Krug 2007). One possible poecilogonous species is Pygospio elegans Clapar` ede, a small, sedentary, tube-building spionid polychaete worm, widely distributed in the northern hemisphere (Muus 1967; Anger 1984). After internal fertilization (Hannerz 1956), females deposit embryos and yolky nurse eggs in capsules inside the maternal tube. Different larvae emerge from the capsules depending on the relative number of embryos and nurse eggs laid by the mother; there are no initial differences in embryo size (S¨ oderstr¨ om 1920; Hannerz 1956; Rasmussen 1973; Anger et al. 1986; Blake and Arnofsky 1999, pers. obs.). Here, we define planktonic larvae as those that emerge when they are 3-setigers long (typically >20 embryos laid per capsule with few or no nurse eggs). The larvae develop long swimming setae and actively swim and feed in the water column (Hannerz 1956). Brooded larvae, on the other hand, do not have swimming setae and remain inside the capsules for a longer period subsisting only on nurse eggs (typically one to two embryos laid per capsule, Fig. 1). These larvae lack a pelagic phase during development and metamorphose into juveniles soon after their emergence from the capsules at 14–20 setigers. An intermediate type of larva also occurs (4–10 embryos laid per capsule; Hannerz 1956, pers. obs.). After emergence at approximately 10 setigers, these larvae have a short pelagic phase. Despite their differences, all larval types metamorphose into morphologically and ecologically identical adults. Monitoring reproduction in P. elegans is laborious and has been done exhaustively in only a few populations. There have been some observations of different larval forms simultaneously within a single population (Rasmussen 1973; Gudmundsson 1985, pers. obs.), providing some evidence that P. elegans is a true poecilogonous species. However, whether or not a single individual can produce multiple larFigure 1. Brooded Pygospio elegans larvae in capsules (from ¨ Angs ¨ o, Finland). The capsules (approx. 0.5 mm long, each containing one to two larvae) are visible after breaking down the sand tube. Photo credit: Jenni Kes¨ aniemi. val types is not clear (but, see Fig. 30 in Rasmussen 1973). Hannerz (1956) and Rasmussen (1973) hypothesized that developmental mode polymorphism in P. elegans is in fact variation within a single developmental mode, reflecting plastic responses to environmental variation. This hypothesis was based on observations that in some populations different larvae are produced seasonally. However, neither simultaneous nor seasonal production of different larvae in a single population is universal. More commonly, among population differences in developmental mode are noted, and some populationshaveevenbeenconsidered“fixed”foraparticular developmental mode since no other modes have been observed during repeated sampling from these populations (Anger 1984; Morgan et al. 1999; Bolam 2004, pers. obs.). The presence of “fixed” populations differing in developmental mode raises suspicion that cryptic species may be present. This suspicion was strengthened when Anger (1984) found that experimental exposure of worms from several “fixed” populations to different salinities and temperatures did not induce a change in developmental mode. No correlations between other environmental variables and developmental mode have been noted in the literature, but few experimental tests have been performed. Changes in density and food supply did not induce changes in developmental mode in P. elegans collected from Somme Bay, France (Morgan 1997), but in North America, low density has apparently increased the frequency of asexual reproduction in P. elegans (Wilson 1983). To clarify the species status of P. elegans populations, Morgan and colleagues (1999) examined population c 2012 The Authors. Published by Blackwell Publishing Ltd. 995 No Cryptic Speciation in Pygospio elegans J. E. Kes¨ aniemi et al. structure among four potentially “fixed” populations in the English Channel differing in developmental mode. They found high genetic similarity and potentially high gene flow among the P. elegans populations, and concluded that the species is poecilogonous. Nevertheless, due to the limited scope of their study and the rarity of poecilogony, the question of poecilogony versus cryptic speciation still remains. We addressed this question by surveying variation in a portion of the mitochondrial gene cytochrome c oxidase subunit I using haplotype network and phylogenetic methods, and using a DNA sequence-based criterion advocated in DNA barcoding studies to assess the presence of cryptic species. Our samples covered both a broad geographical area and a range of environmental conditions. For some populations, there were also data available regarding the predominant developmental mode among individuals. The large dataset also allowed us to investigate within-population diversity in our study populations. We hypothesized that P. elegans is indeed a poecilogonous species, despite apparent divergence of populations in developmental mode. Materials and Methods Sample collection and molecular methods Adult P. elegans were collected between 2007 and 2010 from 14 locations in Europe (Fig. 2) and three locations in the United States (east coast: Maine and west coast: Washington). In Europe, populations from the Baltic Sea (Finland, Germany, Denmark, Sweden), Wadden Sea (the Netherlands, Schiermonnikoog Island), North Sea (Edinburgh, UK), the English Channel (Plymouth, UK, and Somme Bay, France), White Sea (Russia), and the North Atlantic Ocean (Iceland) were sampled (Fig. 2, Table 1). Several colleagues enabled the collecting effort (see Acknowledgements). At most locations, the samples were collected from the shallow intertidal zone (0.1–1 m). The two samples from the Finnish archipelago (¨ Angs¨ oandF ˚ ar¨ o) were collected by scuba from 2–5 m deep water. Samples from Germany were collected from 18-m depth. At the time of collecting, the adult worms and sand tubes were examined for signs of larvae or egg capsules and then preserved in ethanol (94–99%). Using these observations, and information from previous studies of P. elegans’reproduction and development (i.e., Rasmussen 1973; Morgan et al. 1999; Bolam 2004), we characterized the sampling locations by the different larval developmental modes observed (Table 1). This characterization is tentative, since we were unable to survey all populations exhaustively, but represents our best knowledge of the predominant developmental mode in the populations. Additional sampling at the same sites has confirmed our characterization of developmental mode (pers. obs.) but at some sites we have not observed any signs of sexual reproduction and so a predominant developmental mode is not known. From the European samples, genomic DNA was extracted using the DNeasy Blood and Tissue extraction kit (Qiagen, Germany) and a KingFisher magnetic processor (ThermoScientific, MA, USA). A 600-bp fragment of the cytochrome c oxidase subunit I (COI) gene was amplified using speciesspecific primers (PeCox1 F 5– TAT AGG CCT TTG ATC AGG AAC – 3,PeCox1R5 – AGG GTC TCC GCC TCC TGT – 3). Polymerase chain reactions (PCRs) were performed in 20 μL reactions containing 1 μLoftheDNAextract, 3 mM MgCl2(Biotools, Spain), 200 μM of each dNTP (Fermentas, Germany), 0.5 μMofeachprimer(TAGCopenhagen, Denmark), 0.1 U of Taq polymerase, and 1 X of PCR Buffer (Biotools). Reaction conditions included an initial denaturation step at 94◦Cfor2min,then35cyclesofdenaturation at 94◦C for 15 s, annealing at 55◦Cfor15s,and extension at 72◦C for 45 s, followed by a final extension at 72◦C for 2 min. For sequencing, the PCR products were treated with Exonuclease I and Shrimp alkaline phosphatase (Fermentas), cycle sequenced in both directions using the BigDye v.3.1 kit, and visualized with an ABI 3130xl Genetic Analyzer and Sequencing Analysis v.5.2. software (all Applied Biosystems, CA, USA). DNA extraction, amplification, and sequencing of the North American samples followed similar protocols, but sequencing artifacts at the 5endoftheresultingsequences reduced the length of high-quality sequence reads for these samples. To be conservative, we analyzed a shorter fragment of the COI gene (567 bp) when North American samples were included. In analyses involving only the European samples, the 600-bp fragment was used. Haplotype network and phylogenetic analyses Sequences were aligned using the ClustalW option of MEGA 4 (Tamura et al. 2007). For these analyses, the 567-bp fragment of the COI gene was used and all individuals were included. To examine the relationship between the haplotypes, a minimum spanning network was constructed with Arlequin v.3.5.1.2. (Excoffier and Lischer 2010) and visualized with HapStar (Teacher and Griffiths 2010). For phylogenetic analyses, a single representative of each haplotype was used. JModeltest (Posada 2008) was used to find the optimal model of sequence evolution for the COI data (selected using the Akaike information criterion, AIC). The general time reversible model with invariant positions and gamma-distributed rates (GTR + I + G) was selected and used in tree reconstruction. Sequence divergence was estimated with MEGA 4 using a gamma shape parameter of 0.637 (according to JModeltest) and the Tamura Nei 996 c 2012 The Authors. Published by Blackwell Publishing Ltd. J. E. Kes¨ aniemi et al. No Cryptic Speciation in Pygospio elegans Figure 2. European sampling sites labeled according to their abbreviations in Table 1. Sites FIA ( ¨ Angs ¨ o) and FIF (F˚ ar ¨ o) are located in the Finnish archipelago, approximately 20 km apart. Regional grouping of populations for the hierarchical AMOVA analysis are indicated with numbered superscripts: 1. Northern Baltic Sea: Finland, 2. Southern Baltic Sea: Denmark, Germany, Sweden, 3. North Sea + Wadden Sea + English Channel: UK, France, the Netherlands, and 4. North Atlantic Ocean: Iceland. substitution model since the GTR model is not available in MEGA 4. For tree reconstruction, we explored both maximum likelihood and Bayesian analyses. Bayesian analysis was conducted with MrBayes v.3.1.2. (Ronquist and Huelsenbeck 2003). MCMC (Markov Chain Monte Carlo) chains (one cold and three heated chains) were run for 4 million generations, trees were sampled every 100 generations, and 25% of the trees were discarded in the burnin. All parameters were estimated in the analysis. Posterior probabilities were used to assess clade support, with 80% used as the minimum cutoff. Maximum likelihood analysis was conducted with PhyML 3.0. (Guindon and Gascuel 2003). All parameters were estimated in the analysis except the gamma shape parameter, which was set to 0.637 according to the results from JModeltest. Bootstrap analysis with 1000 replicates provided an estimate of clade support, with 70% used as the minimum cutoff. After analysis, trees were rooted along the lineage leading to most of the North American haplotypes (also the longest branch). Trees were visualized using FigTree v.1.2.2. (http://tree.bio.ed.ac.uk/software/figtree/). Analysis of genetic diversity Our genetic diversity analyses focused on populations with sufficient sample sizes for making robust estimates, so the c 2012 The Authors. Published by Blackwell Publishing Ltd. 997 No Cryptic Speciation in Pygospio elegans J. E. Kes¨ aniemi et al. Table 1. Sampling location information, population codes, diversity measurements, and observed larval modes of the populations. N=number of individuals in the genetic analysis, H=haplotype diversity, and π=nucleotide diversity. Box indicates the European populations with sufficient sample size used in diversity and demographic analyses as well as hierarchical analyses of population structure (AMOVA). Groups (regions) defined for AMOVA analysis are shaded. Region Location Code N*No. of haplotypes HπObserved larval mode Europe Northern Baltic Sea ¨ Angs ¨ o, Finland FIA 22 4 0.260 0.0017 Brooded F˚ ar ¨ o, Finland FIF 21 10 0.890 0.0129 Not known Hanko, Finland FIH 19 6 0.778 0.0128 Not known Southern Baltic Sea Germany GER 22 16 0.909 0.0118 Not known Vellerup, Denmark DKV 20 12 0.916 0.0141 Brooded, intermediate Herslev, Denmark DKH 20 11 0.916 0.0154 Brooded, intermediate Rorvig, Denmark DKR 21 6 0.710 0.0116 Intermediate, planktonic Gullmarfjord, Sweden SWE 21 5 0.633 0.0080 Not known Wadden sea the Netherlands NET 23 14 0.822 0.0107 Intermediate, planktonic North sea Drum sands, UK UKD 20 19 0.995 0.0130 Planktonic English Channel Somme Bay, France FRA 23 22 0.996 0.0141 Planktonic Plym Bay, UK UKP 24 20 0.975 0.0153 Planktonic Atlantic Ocean Iceland ICE 20 3 0.511 0.0009 Brooded, intermediate White Sea Russia RUS 3 1 0.000 0.0000 Not known North America Atlantic (east) Lubec, ME NAE1 7 3 0.733 0.0360 Not known Lowe’s cove, ME NAE2 6 3 0.810 0.0210 Not known Pacific (west) False Bay, WA NAW 7 1 0.000 0.0000 Not known *Based on 600 bp COI fragment in European populations, but based on 567-bp fragment in North American populations. Russian sample (n=3) and the North American samples (n=6–7) were excluded. In these analyses, the 600-bp fragment of the COI gene was used. Haplotype diversity and nucleotide diversity for each population were calculated with Arlequin v.3.5.1.2. (Excoffier and Lischer 2010), which was also used to estimate population structure (ST)viaahierarchical analysis of molecular variance (AMOVA). In the AMOVA analysis, sequences were grouped according to geographical regions (four groups: Northern Baltic Sea; Southern Baltic Sea; North Sea + Wadden Sea + English Channel; and North Atlantic Ocean; 10,000 permutations). Population structure was also investigatedusing BAPS 5.3 (Corander and Tang 2007), a Bayesian model-based clustering method that can use sequence data. In these analyses, the maximum number of clusters (K) was set from two to 13, and for each the analysis was run 10 times. In the end, the Kwith the highest likelihood was chosen to describe the samples. Exploratory analyses tested whether differences in haplotype and nucleotide diversity measures were evident among the European populations with different developmental mode. Here, planktonic populations (UKP, UKD, FRA, see Table1)werecomparedtopopulationsthatproducebrooded or intermediate type larvae (FIA, DKV, DKH, DKR, NET, ICE). This comparison is contingent on our definition of predominant developmental mode (see Table 1), so populations where developmental mode is not known (FIF, FIH, GER, SWE, and RUS) were excluded. For these comparisons, Mann–Whitney Utests were performed using PASW Statistics 18 (SPSS, Inc., 2009, Chicago, IL, www.spss.com). To assess if European populations (excluding RUS) have gone through a recent population expansion, Fu’s Fsneutrality test was calculated. Fu’s test (which is based on the haplotype distribution; Fu 1997) was used because it is thought to be better at revealing signs of population expansion than Tajima’s Dtest (Fu 1997; Schneider and Excoffier 1999). Tajima’s D(Tajima 1989) and Fu and Li’s F(Fu and Li 1993) were also calculated to test for neutrality of the sequences. Mismatch distributions, the frequencies of observed pairwise differences between haplotypes, were calculated for each European population. Also R2(Ramos-Onsins and Rozas 2002) and raggedness statistics (rg, Harpending 1994) with confidence intervals based on coalescent simulations were calculated to detect expansion (10,000 permutations and theta estimated from the data were used in the coalescent simulations). Lower R2and rg values are expected for a population growth scenario (Harpending 1994; Ramos-Onsins and Rozas 2002). These analyses were performed in DnaSP 4.0 (Rozas et al. 2003). Results Polymorphism and haplotype diversity A total of 279 P. elegans individuals from 14 European locations were sequenced. From this sample, 121 unique 998 c 2012 The Authors. Published by Blackwell Publishing Ltd. J. E. Kes¨ aniemi et al. No Cryptic Speciation in Pygospio elegans haplotypes of the COI gene fragment (600 bp) were identified. An expanded dataset included 20 additional individuals from three North American locations for a total of 299 sequences, 567 bp in length, with 123 unique haplotypes (GenBank accession numbers JN033571–JN033693). The most common haplotype, EUNA10, was shared by 36 European individuals and was found in all three Danish populations, Iceland, Sweden, and Plym Bay in the UK (English Channel). This haplotype was also observed in worms sampled from both populations on the East Coast of the United States (NAE). The second most common haplotype, EU11, was found in Denmark, Finland, France, the Netherlands, and the White Sea, Russia (35 individuals). Note that both EUNA10 and EU11 were found in populations differing in developmental mode (see Table 1). These two most common haplotypes also were found within the whole sample range in Europe and comprise 25% of all individuals sequenced. We observed a large number of low frequency haplotypes within locations in Europe. Out of 121 haplotypes, 98 were detected only once in the European dataset (from only one individual of the 279 sequenced). Ninety percent of the haplotypes (109 out of 121) were found in only one population (11ofthesewerefoundfrommorethanoneindividual).Populations from the North Sea, English Channel, and Wadden Sea had the highest percentage of population-specific lowfrequency haplotypes. The Baltic Sea populations (Finland, Denmark, Germany, Sweden)sharedmanyhaplotypes(seven out of 12 shared haplotypes are found only in the Baltic Sea), and only one haplotype (EU8) was shared exclusively among the three populations in the UK and France. In most populations, haplotype diversity was high (Table 1). However, two European populations had low diversity with most individuals sharing the same haplotype. In ¨ Angs¨ o, Finland, 19 of 22 individuals sampled (86%) shared an identical haplotype (EU6) and in the sample of 20 individuals from Iceland, 13 shared haplotype EU1 and six shared haplotype EUNA10. Overall, populations with predominantly planktonic larvae had higher haplotype diversity than populations that also produced other larval types (N=9, U=0.000, z=–2.334, P=0.020). However, nucleotide diversity was not significantly different (N=9, U=4.5, z=–1.167, P=0.243). Haplotype diversity in the North American samples was somewhat lower than in most of the European samples (Table 1), but North American sample sizes were also relatively small and so estimates of diversity from these populations may not be reliable. Mean sequence divergence (Tamura Nei model) within the total European dataset was 1.7%. Divergence between the European and North American haplotypes was noticeably higher: 5.3% (or 6.1% when excluding EUNA10, the haplotype that is shared with the European samples). Mean sequence divergence within the total North American dataset was 3.1%, higher than what we observed from the European sample. Haplotype network and phylogenetic analyses Figure 3 shows the minimum spanning haplotype network as calculated in Arlequin. The low sequence divergence among haplotypes is reflected in the network and haplotypes from different populations are intermingled. Arlequin detected many alternative connections among the European haplotypes due to the low level of divergence between them, but graphing all possible alternative connections would have made the network unreadable. The most common haplotype, EUNA10, was found from almost all populations and other linked haplotypes came from Iceland, the North Sea, the English Channel, and the Southern Baltic Sea, but not from Finland. The other common haplotype, EU11, is multiple mutational steps away from EUNA10. Moreover, the other North American haplotypes were not connected closely to EUNA10 and were clearly different from the European sequences. The European haplotype closest to the cluster of North American haplotypes is from the Netherlands (Fig. 3), and alternative connections (also 26 mutational steps to NAW NAE) are from the UK (two haplotypes from UKP, one from UKD; not shown). Overall, the minimum spanning network included a large number of small nodes depicting the high frequency of singleton haplotypes noted earlier, and some medium frequency haplotypes (observed in two to eight individuals) that were detected in one population only. These singleton and low-frequency haplotypes are widespread throughout the network. Phylogenetic analyses resulted in similar tree topologies regardless of which tree reconstruction method was used, therefore, only the results from the maximum likelihood analysis are discussed and shown (Fig. 4). As in the haplotype network, there was a clear separation between the European haplotypes and most North American haplotypes (other than EUNA10) and the European clade was well supported by bootstrap analysis (Fig. 4). Within the European clade, there was very little divergence and only a few groups were clearly resolved with high bootstrap support (Fig. 4, dots at supported nodes). The lack of bootstrap support at most nodes indicates limitations of the data for resolving relationships of the P. elegans haplotypes. However, this analysis also reveals clusters detected in the haplotype network. For example, one well-supported group contains almost all the Finnish ¨ Angs¨ o haplotypes (3 out of 4; EU6, FIA43, FIA44). Another well-supported group contains individuals from Germany and all of the three Danish populations, even though other haplotypes from these populations were also distributed elsewhere in the phylogenetic tree. In addition, most Swedish (except one) and all Icelandic haplotypes were included in a c 2012 The Authors. Published by Blackwell Publishing Ltd. 999 No Cryptic Speciation in Pygospio elegans J. E. Kes¨ aniemi et al. Figure 3. Haplotype network of the 123 COI haplotypes detected in P. elegans. Circle size is proportional to haplotype frequency and haplotypes with more than one individual are also named. Small black circles represent undetected intermediate haplotypes and lines connecting circles represent one mutational step unless otherwise specified. Circles are colored to represent sampling sites. Haplotypes found from more than one location are colored as pie charts with proportionally sized wedges representing the haplotype frequency in each population. Ovals with dashed outlines encircle clusters in the haplotype network which were also detected in phylogenetic analyses with strong (70% or greater) bootstrap support. well-supported group, which also included EUNA10, one of the most common haplotypes also sampled from North America. The two most commonly encountered haplotypes (EUNA10 and EU11) did not group together (Fig. 4, asterisks). Population structure and demographic analyses For the test of regional subdivision of sequence diversity in Europe (AMOVA), the populations were arranged into four groups according to geographical region (1. Northern Baltic Sea: Finland, 2. Southern Baltic Sea: Denmark, Germany, Sweden, 3. North Sea +Wadden Sea +English Channel: UK, France, the Netherlands, and 4. North Atlantic Ocean: Iceland, see Fig. 2). These results (Table 2) showed that most of the variation was found within populations (69.8%, P> 0.001) and that differentiation among the regions was significant although small (accounting for 8% of the molecular variance, P=0.001). Additional significant variation among populations within each region (22.2%, P>0.001) indicated that structure may also be present on smaller spatial scales. Analysis using the program BAPS detected seven genetic clusters in our data (with the probability of 0.99). Clusters were not based on sampling location, each cluster containing individuals from four to ten sampling locations. Two clusters were strictly Baltic, one containing most of the individuals sampled from the Finnish ¨ Angs¨ o site and the other containing most of the German samples, although German individuals were also placed into four other clusters (Table 3). Demographic analyses suggested there has been recent population expansion in the populations from the UK and France. In these populations, Fu’s Fs values were negative and significant. Unimodal mismatch distribution curves for these populations also indicate that a recent population expansion 1000 c 2012 The Authors. Published by Blackwell Publishing Ltd. J. E. Kes¨ aniemi et al. No Cryptic Speciation in Pygospio elegans Figure 4. Maximum likelihood tree of COI haplotypes detected in P. elegans. Nodes marked with black dots indicate clades resolved with bootstrap values of 70% or higher. Dashed lines connect haplotype names to the branches and should not be interpreted as branch length. Numbers in the brackets following the haplotype name indicate the number of individuals observed with that haplotype. When no number is indicated, the haplotype was sampled only once. Asterisks indicate the two most common haplotypes: EUNA10 and EU11. Central color wheel indicates region of sampling: light gray =Baltic Sea (Northern + Southern); dark gray =Iceland; black =North Sea, English Channel and Wadden Sea; and white =shared European haplotypes. Note, the North American haplotypes in the circular phylogeny are in the center of the color wheel. c 2012 The Authors. Published by Blackwell Publishing Ltd. 1001