Vol. 485: 143–154, 2013 MARINE ECOLOGY PROGRESS SERIES Published June 27 doi: 10.3354/meps10313 Mar Ecol Prog Ser

Population genetic structure and modes of dispersal for the colonial ascidian Botryllus schlosseri along the Scandinavian Atlantic coasts

Eitan Reem1,3,*, Ipsita Mohanty1, Gadi Katzir2,3, Baruch Rinkevich1

1Israel Oceanography and Limnological Research, National Institute of Oceanography, Tel Shikmona, PO Box 8030, 31080, 2Department of Marine Biology, Faculty of Science and Science Education, University of Haifa, Haifa 31905, Israel 3Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Haifa 31905, Israel

ABSTRACT: The colonial ascidian Botryllus schlosseri is a well-known cosmopolitan invader of sheltered temperate marine communities which has garnered major scientific attention. We ana- lyzed modes of dispersal and population genetic structures for 11 populations of B. schlosseri along the Scandinavian coasts, using 5 microsatellite loci. The analysis revealed high poly - morphism, resulting in 108 different alleles (of which 58 were private alleles), positive correlations between the number of sites shared by specific alleles and their mean frequencies, and lower genetic diversity values than in previously studied worldwide populations. A complex network of gene flow among sampled populations was revealed, with 2 clades, southeastern and northwest- ern, and higher genetic variation in the latter clade due to either restricted gene flow or more intensive genetic drift. A detailed analysis of allele frequencies revealed possible ancestral alleles. By using Bayesian analysis, 9 previously studied populations from Britain and European Atlantic coasts were compared, encompassing a single geographical entity along thousands of kilometers from (36° 8’ N) to Ålesund, Norway (62° 29’ N). Results showed a high connectivity among distant localities, most probably due to extensive human-mediated transport. This refutes isolation by distance, with a higher intensity of gene flow among Scandinavian sites compared to the other European sites. Bayesian clustering computation assembled the whole data set of 19 populations into 14 clusters and 2 major northern and southern clades.

KEY WORDS: Invasive species · Bayesian clustering · Ancestral alleles · Isolation by distance · Gene flow · Genetic diversity

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INTRODUCTION ting invasion routes, recording various levels of con- nectivity among populations of species with various Species and population expansion, as well as in- life history traits, and elucidating levels of genetic creasing biological invasions, have stimulated con- diversity within populations of introduced species siderable interest among biologists. Today, these (reviewed in Estoup & Guillemaud 2010). phenomena are being investigated using population The increase in ocean water temperatures, one of genetics tools (Kolbe et al. 2004, Stepien et al. 2005, the results of global climatic change (Anadón et al. Lavergne & Molofsky 2007, Roman & Darling 2007, 2007), has also affected the trajectories and mag - Puillandre et al. 2008). Such tools enable reconstruc - nitude of biological invasions, causing northward

*Email: [email protected] © Inter-Research 2013 · www.int-res.com 144 Mar Ecol Prog Ser 485: 143–154, 2013

movements of more southern species in the northern since the eighteenth century from Falmouth Bay in hemisphere, followed by the establishment of non- England (Pallas 1766), from the Faeroe Islands and indigenous species in both terrestrial and marine western and southern Norway to the Medi terranean, ecosystems (Franke & Gutow 2004, Parmesan 2006, Adriatic and Black seas (Van Name 1945, Berrill Anadón et al. 2007). In many coastal communities, 1950). Nevertheless, while there is wide knowledge invasion rates are enhanced by substantial global of the distribution of this species along the western shipping traffic, as many organisms of a wide spec- coasts of Europe, only a few studies have elucidated trum of taxa foul ship hulls, are conveyed in ballast its population genetic profiles in European waters water, or are transported on hauled edible inverte- (Rin kevich et al. 2001, Ben-Shlomo et al. 2006, Ló - brates, such as oysters (Ruiz et al. 2000, Lambert pez-Legentil et al. 2006, Bock et al. 2012). Despite the 2001, Dijkstra et al. 2007). current interest in the northward movement of ma- The colonial ascidian Botryllus schlosseri is a rine species, the distribution of B. schlosseri along the worldwide invader, commonly found in sheltered northern European coasts, such as along the western temperate marine communities, primarily marinas coasts of Scandinavia, has been little studied. Previ- and harbors, in the northern and southern hemi- ous population genetic studies of B. schlosseri using spheres (Berrill 1950, Ruiz et al. 2000, Lambert 2001, microsatellites (Stoner et al. 2002, Paz et al. 2003, Stoner et al. 2002, Paz et al. 2003, Ben-Shlomo et al. Ben-Shlomo et al. 2010) revealed high polymorphism 2006, 2010, Simkanin et al. 2012), and successfully and heterozygote deficiency as general attributes of reproducing at 11 to 28°C (Brunetti 1974, Grosberg all studied populations. Two of those studies (Stoner 1988, Chadwick-Furman &Weissman 1995, Rinke- et al. 2002, Ben-Shlomo et al. 2010) indicated pos - vich et al. 1998a,b). In the northern hemisphere, B. sible routes of invasion patterns and connectivity schlosseri populations are distributed from the south- between sites on the Pacific and Atlantic coasts of the ern coast of India (8° 22’ N latitude; Meenakshi & Americas, Europe, and New Zealand. Senthamarai 2006), where sea water temperature The presence of Botryllus schlosseri along the ranges from 24 to 29.5°C (Damotharan et al. 2010), to Scandinavian coasts has only been mentioned anec- the Norwegian sea ports (>62° N) with sea water dotally. Van Name (1945) and Berrill (1950) referred temperatures ranging between 3 and 17°C (Nair to it as appearing in ‘western and southern Nor- 1962). In natural environments, they can be found way’ (supported by the observations of Nair 1962 below and above stones, on exposed natural rocky and Dybern 1967). Nair (1962) mentioned it among habitats and down to 200 m depth (Ben-Shlomo et al. other fouling organisms from 4 sampling stations in 2006). In harbors and marinas, colonies are found the fjords of the Bergen area, demonstrating that submerged on hard substrata such as stones, pon- colonies are active only during the summer and fall toons, wharfs and ropes. The life history of B. periods when water temperatures are >9°C and dis- schlosseri includes a stage of short-lived (~1 h) pela - appear for the winter months, probably hibernating gic larvae that settle in close proximity to parental (Brunetti et al. 1980). Following this limited know- colonies (Grosberg 1987, Rinkevich & Weissman ledge, the aim of this study was to investigate popu- 1987), thus restricting their long-range dispersal. lation genetics of B. schlosseri along the Atlantic Adult colonies are dispersed mainly via attachment coasts of Scandinavia to elucidate patterns of connec- to ship hulls, floating objects and edible inverte- tivity and possible gene-flow trajectories among brates, such as crabs and oysters, which are shipped sampling sites from Öckerö on the southern west between distant sites (Paz et al. 2003, Bernier et al. coast of Sweden to Ålesund in Norway beyond 62° 2009, Lacoursière-Roussel et al. 2012). Recent studies latitude. In addition, based on information from Ben- (Cohen & Carlton 1995, Ruiz et al. 2006, Locke et al. Shlomo et al. (2006). We aimed to examine possible 2009) have revealed new B. schlosseri introductions connectivity routes between Scandinavian sites and in colder sites in the northern hemisphere. some European Atlantic and British sites. Despite ample information on the global distribu- tion of Botryllus schlosseri, there is no consensus for its site of origin. Citing Van Name (1945) and Berrill MATERIALS AND METHODS (1950), studies have claimed Mediterranean and Eu- ropean Atlantic waters as B. schlosseri sites of origin, Sampling whereas Carlton (2005) proposed a Pacific Ocean hub of origin. The distribution of B. schlosseri along Colonies of Botryllus schlosseri (N = 319) were the European Atlantic coasts has been documented sampled in the summer of 2005 from 5 sites along Reem et al.: Genetic structure and dispersal of Botryllus schlosseri in Scandinavia 145

Microsatellite typing

Five Botryllus schlosseri microsatellite loci, PBC-1, PB-29, PB-41, PB-49 (Stoner et al. 1997), and BS-811 (Pancer et al. 1994) were amplified by polymerase chain reaction (PCR) with specific fluorescent pri- mers (Agentech) using the following conditions: 94°C for 2 min, followed by 32 cycles at 94°C for 1 min, 52°C for 1 min and 72°C for 1 min, and a final exten- sion step at 72°C for 45 min followed by storage at 10°C. The 16 µl reaction mixture contained 8 µl PCR mix (R2523-100 RXNREDTaq ReadyMix, Sigma), 5 pmol fluorescent primer (Applied Biosystems), 6 µl DDW and 1 µl DNA solution (1:100). A loading cock- tail was prepared by adding 1 µl of PCR product to 12 µl formamide and 0.5 µl size standard (Genescan 400HD[ROX] 402985, Applied Biosystems). Vials with the cocktail were heated to 92°C for 2 min and cooled to 4°C. Microsatellite allele lengths of the samples were analyzed using 3130 Genetic Analyzer and Genotyper software (Applied Biosystems). To avoid scoring errors due to stutters appearing in the chro- Fig. 1. Sampled sites in Scandinavia matograms that might obscure the real reading of the microsatellite, we employed Micro-Checker software (Van Oosterhout et al. 2004) to score errors and null the Atlantic coast of Sweden, and in the summer of alleles. Suspected samples were re-genotyped and 2007 from 6 sites along the Atlantic coasts of Nor- double-checked by 2 researchers until a consensus way (Fig. 1, Table 1). Samples were taken from sub- was reached. merged parts of docks or ropes, 0.1 to 0.5 m below sea level and only from colonies that were >1 m apart, in order to avoid sampling kin colonies that Data analysis may be identical by descent (see Grosberg 1987), chimeras, or ramets of the same genotype. Sampling Data were analyzed by GenAlEx version 6.2 soft- efforts were aimed at collecting between 30 and 50 ware (Peakall & Smouse 2006) and by Tools for Pop- different colonies but in some of the sites colonies ulation Genetic Analyses (TFPGA) software version were rare, resulting in a reduced number of sam- 1.3 (Miller 1997). The significance level of population ples. Whole colonies or colony fragments were differentiation (pairwise analysis of all populations; removed from their substrates by single-edge razor exact test, Raymond & Rousset 1995) was determined blade. Each sample was placed separately in a after 1000 dememorization steps and 10 batches of 1.5 ml vial containing 200 µl lysis buffer (0.25 M Tris 2000 permutations per batch, using TFPGA; the Bon- borate, 0.1 M EDTA, 2% wt/vol SDS, 0.1 M NaCl, ferroni correction method for multiple comparisons adjusted to pH 8.2) and 40 µl NaClO4 (5 M). Sam- was applied. ples were homogenized and DNA was extracted The same software was employed to assess isola- with 200 µl phenol:chloroform:isoamyl alcohol solu- tion by distance Mantel test. Geographical distances tion (25:24:1 vol:vol:vol) (Graham 1978, Paz et al. were measured using Google Earth program with the 2003). The vials were shipped to the laboratory at application that enables drawing a line along the the National Institute of Oceanography in Israel for coastline contour between 2 points, resulting in di - further work. In the laboratory, DNA isolation was rect reading of the distance. In addition, the popula- completed as described by Paz et al. (2003). DNA tion differentiation Dest was calculated according to concentration was approximately ~5 µg µl−1. Sam- Jost (2008) and by SMOGD version 1.2.5 software ples were kept as stock at 4°C. For microsatellite (Crawford 2010). The genetic variation, Fst, and typing, we used DNA diluted 1:100 in sterile dou- overall inbreeding coefficient Fis were calculated ble-distilled water (DDW). via analysis of molecular variance (AMOVA) proce- 146 Mar Ecol Prog Ser 485: 143–154, 2013 is 4 F 0.439 8 ±0.037 erved heterozygosity; He: 1.249 ±1.108 ±0.691 ±0.157 standard error ±0.837 ±0.815 ±0.653 ±0.158 : inbreeding coefficient = 1 − (Ho/He); is F ; –1 ) 2 π 73 8 0.330 0.662 7.400 5.847 3.617 0.470 76 6 0.339 0.703 6.600 5.889 3.698 0.490 90 3 0.311 0.587 6.200 5.052 0497 0.420 82 9 0.312 0.630 0.760 6.589 4.294 0.514 7 84 7 0.341 5.562 6.400 5.052 3.051 0.396 22 88 6 0.290 0.668 5.000 4.869 3.326 0.548 28 78 1 0.342 0.623 7.200 5.524 3.385 0.377 29 76 7 0.300 0.597 7.600 5.738 3.767 0.471 27 79 6 0.353 0.658 6.800 4.816 3.288 0.494 22 88 4 0.456 0.636 5.000 4.712 2.860 0.232 22 92 1 0.282 0.490 4.800 4.125 2.519 0.421 Shared alleles No. of Mean diversity parameters and s with other sites private inbreeding coefficients ±SE ±0.079 ±0.113 ±1.020 ±0.769 ±0.724 ±0.147 ±0.046 ±0.091 ±2.379 ±1.703 ±0.769 ±0.072 ±0.105 ±0.125 ±1.691 ±1.251 ±0.880 ±0.115 ±0.090 ±0.128 ±1.720 ±1.319 ±1.190 ±0.121 ±0.089 ±0.098 ±1.319 ±1.042 ±0.497 ±0.133 ±0.089 ±0.139 ±1.200 ±0.997 ±0.565 ±0.101 ±0.081 ±0.057 ±0.837 ±0.771 ±0.418 ±0.170 No. % alleles Ho He Na AR Ne ±0.076 ±0.136 ±1.833 ±1.616 ±1.360 ±0.079 ±0.106 ±0.077 ±1.828 ±0.849 ±0.507 ±0.12 319 58 0.332 0.620 6.418 6.642 3.327 ±0.025 ±0.029 ±0.436 ±1.412 ±0.22 ); Na: number of different alleles; Ne: number of effective alleles = (Sum 2 π ±0.109 ±0.073 blades ±0.109 ±0.061 ± , Laminaria Laminaria ) × (1 − Sum Laminaria –1 . Sampling sites, allelic distribution and genetic diversity parameters for 11 Scandinavian populations. Abbreviations: Ho: obs AR: mean allelic richness over loci based on minimal sample size of 14 diploid individuals and the rarefaction approach; SE: Botryllus schlosseri Öckerö Island Risør also fresh water, Fevik also fresh water Hamburgsund from shallow water, Strömstad shallow and deep water, Fiskebäckskil from shallow water, Tananger from hard substrata Håkonshella samples collected from Florø many on Dolvik most on Ålesund Total Total Coordinates Description Date Size allele many colonies many colonies dispersed population many colonies bivalves), abundant (ropes, a pipe along the dock N E

No. Name Site Sample No. of 1 Sweden 57°42’ 11°39’ marina, on ropes 01/09/05 25 38 31 4 Sweden 58°56’ 11°10’ marina, from 09/03/05 48 32 2 5 Norway 58°43’ 9°14’ marina, 01/09/07 29 25 6 Norway 58°22’ 8°40’ marina, 31/08/07 30 24 3 Sweden 58°33’ 11°16’ marina, 06/09/05 18 36 2 Sweden 58°14’ 11°27’ marina, 30/08/05 22 38 7 Norway 58°56’ 5°34’ shallow water, harbor, 29/08/07 18 31 28 8 Norway 60°20’ 5°11’ Marina, many colonies, 24/08/07 40 37 27 9 Norway 60°19’ 5°15’ marina, many colonies, 24/08/07 22 33 25 10 Norway 61°31’ 5°02’ small dock, 26/08/07 35 25 11 Norway 62°28’ 6°09’ No data 25/07/07 32 34 unbiased expected heterozygosity = 2N·(2N − 1) Table 1. Table Reem et al.: Genetic structure and dispersal of Botryllus schlosseri in Scandinavia 147

dure among all sampling sites (each sampling site scored alleles (319 specimens, 5 microsatellite loci). was considered a group), following the methods of Locus BS-811 emerged as the most polymorphic Michalakis & Excoffier (1996) with 999 permutations with 49 alleles, while loci PB-49, PBC-1, PB-41 and using GenAlEx 6.2 software. The significance levels PB-29, comprised 20, 20, 12 and 7 alleles, respec- of Hardy Weinberg tests for heterozygote deficiency tively (Table S1 in the supplement at www.int- and for Fis were determined by Genepop ver. 4.0.10 res.com/articles/suppl/m485p143_supp.pdf). Each of (Raymond & Rousset 1995, Rousset 2008). Allelic the 11 sampling sites had 1 to 9 private alleles (8 to richness was estimated by FSTAT software (Goudet 27% of alleles per site) summing to 58 at all sites 2002) using the rarefaction approach (El Mousadik & (Table 1). Seventeen alleles were found to be shared Petit 1996) to avoid bias due to different sampling by 73% of the sites (8 out of 11 sites) and 15 by at sizes. Bayesian partitioning was calculated by using least 82% of the sites (8/11 and 9/11, respectively; BAPS v.5.3 software (Corander et al. 2009). Possible Table 2). The overall mean frequency of the alleles connectivity among sites was calculated and plotted that appeared in at least 8/11 and 9/11 of the popula- following Tang et al. (2009) and implemented in the tions was 0.26 and 0.30, respectively. Furthermore, in same software. Both calculations are based on group 49% of the cases presented in Table 2, the allele level mixture and admixture analyses. An additional frequency was >0.2. Thirty-two out of 1595 scored Bayesian analysis with Structure 2.3.1 software alleles, composing 2% of the total, failed to amplify (Pritchard et al. 2000) was employed to further assess and were defined as null alleles. the outcomes. Statistical normality of distribution test We found a positive correlation (Spearman rho = and t-test computations for differentiation signifi- 0.982, p < 0.01) between the number of sites sharing cance between genetic diversity parameters from dif- specific alleles and the mean frequency of these com- ferent geographical regions and for correlations were mon alleles. Private alleles showed a low mean fre- performed using SPSS 16 software. quency of 0.038, while common alleles in 10 and 11 Scandinavian sites showed mean frequencies >0.2, i.e. they were an order of magnitude more frequent. RESULTS Exact tests for population differentiation (Raymond & Rousset 1995) among all pair-populations showed All 5 microsatellite loci were highly polymorphic, highly significant results (p < 0.001), even after per- altogether showing 108 different alleles out of 1595 forming the Bonferroni correction for multiple test-

Table 2. Botryllus schlosseri. Alleles that appeared in 8 to 11 of the 11 sampling populations and their frequencies. Frequencies >0.25 are in bold, and frequencies between 0.20 to 0.25 are underlined. Abbreviations: Nor = Norway; Swd = Sweden; Hak = Håkonshella; Dol = Dolvik; Flo = Florø; Ale = Ålesund; Tan = Tananger; Fev = Fevik; Ris = Risør; Fis = Fiskebäckskil; Ock = Öckerö; Str = Strömstad; Ham = Hamburgsund. Based on all 5 microsatellite loci

Locus Allele Swd Swd Swd Swd Nor Nor Nor Nor Nor Nor Nor Overall Appea- size Ock Fis Ham Str Ris Fev Tan Hak Dol Flo Ale mean rance frequency ratio

PB41 169 0.660 0.386 0.156 0.313 0.875 0.950 0.611 0.885 0.909 0.943 0.683 0.670 11/11 PB29 162 0.720 0.318 0.583 0.229 0.446 0.883 0.333 0.750 0.705 0.571 0.875 0.583 11/11 PB29 155 0.260 0.591 0.361 0.771 0.536 0.117 0.667 0.238 0.136 0.386 0.094 0.378 11/11 BS811 174 0.063 0.357 0.308 0.489 0.115 0.466 0.306 0.150 0.071 0.561 0.387 0.297 11/11 PBC1 198 0.180 0.295 0.441 0.448 0.276 0.133 0.278 0.513 0.227 0.157 0.094 0.277 11/11 PB49 211 0.260 – 0.118 0.185 0.018 0.467 0.194 0.325 0.455 0.529 0.100 0.265 10/11 PBC1 210 0.260 – 0.118 0.125 0.345 0.217 0.556 0.113 0.159 0.286 0.328 0.251 10/11 BS811 178 0.396 0.214 0.077 0.152 0.519 0.172 – 0.200 0.286 – 0.129 0.238 9/11 PB49 209 0.280 0.452 0.176 0.239 0.036 0.017 0.389 0.163 0.182 0.162 0.150 0.204 11/11 PBC1 179 0.300 – 0.235 0.010 0.034 0.433 0.083 0.200 0.159 0.114 0.250 0.182 10/11 PB49 201 0.140 0.143 0.412 0.261 0.071 0.017 – 0.163 0.205 0.162 0.150 0.172 10/11 PB49 213 0.240 – – 0.033 0.018 0.317 0.278 0.150 0.136 0.029 0.317 0.169 9/11 PB49 205 0.020 0.214 0.235 0.228 0.411 0.167 0.028 0.075 – 0.059 0.217 0.165 10/11 BS811 186 0.021 0.071 0.154 0.033 0.038 0.121 0.361 0.088 0.095 0.182 0.226 0.126 11/11 PB41 171 – 0.045 0.125 0.271 0.036 – 0.222 – 0.045 0.057 0.017 0.102 8/11 BS811 176 0.042 – 0.154 0.033 0.077 0.069 – 0.125 – 0.076 0.097 0.084 8/11 PBC1 201 0.040 0.045 0.088 – 0.207 0.033 – 0.025 0.068 0.043 0.016 0.063 9/11 148 Mar Ecol Prog Ser 485: 143–154, 2013

ing. In addition, overall variation measure (Fst = 0.7 0.137; p < 0.001) and the overall differentiation mea- 0.6 sure (Dest = 0.265, CI = 0.229 to 0.331), point at first glance to a modest genetic structure in the entire 0.5 studied area. However, dividing the whole sampled 0.4 area into 2 regions, one including the 6 sites in south- eastern Scandinavia along the coasts of the Skager- 0.3 rak bight, and the other encompassing the north- 0.2 western sites along the Atlantic coastal fjords of Nei genetic distance 0.1 Norway, resulted in a higher Fst and Dest in the north- ern versus southern region: 0.129 (p < 0.001) versus 0 0 200 400 600 800 1000 0.087 (p < 0.001) for Fst, and 0.269 (CI = 0.183 to Geographic distance (km) 0.407) versus 0.153 (CI = 0.063 to 0.237) for Dest, respectively. Both population parameters, therefore, Fig. 2. Botryllus schlosseri. Mantel test for isolation by dis- show the same trend: higher genetic variation due to tance among populations. r = 0.158, p = 0.12, Z from original either restricted connectivity or higher genetic drift data: 8748.776, mean Z after 9999 permutations: 8312.651 among the northwest sites, which caused higher dif- ferentiation in the northwestern region as compared Mantel tests for association between geographic and to the southeastern region. Based on the same geo- genetic distances: one among the northwestern sam- graphical criterion, Nei genetic distance revealed pling sites and one among the southeastern sampling a similar outcome: mean genetic distance among sites. Surprisingly, the results were not significant southeastern sites was 0.192 versus 0.360 among in the northwest (p = 0.09) but significant (p < 0.05) northwestern sites (Tables S2 & S3 in the supple- in the southeast (Figs. S1 & S2 in the Supplement ment). Genetic diversity parameters, including mean a www. int-res. com/ articles/ suppl/m485 p143_supp. numbers of different (Na) and effective (Ne) alleles, pdf). observed and unbiased expected heterozygosity (Ho Bayesian group clustering analysis disclosed 10 and He, respectively), the mean inbreeding coeffi- clusters out of 11 sampling sites (Fig. 3a), whereas cients F is (also known as system of mating inbreed- the Norwegian and Swedish sites, Fevik and Ham- ing coefficient f [Templeton 2006]) values, and allelic burgsund, were grouped into one cluster, indicating richness (AR) based on the rarefaction approach (El a possible recent common ancestry or intensive inter- Mousadik & Petit 1996) were calculated (Table 1). change gene flow. Based on group level mixture Hardy Weinberg exact test resulted in heterozygote analysis, the log marginal likelihood of optimal parti- deficiency for all loci in all populations (p < 0.001 tion is −5115.62, with probabilities of 0.081 for 8 clus- Genepop ver. 4.0.10; Raymond & Rousset 1995, Rous- ters and 0.919 for 10 clusters (BAPS 5.3 software; set 2008), meaning that Botryllus schlosseri popu - Corander et al. 2009). Interestingly, but not surpri - lations in Scandinavia deviate from Hardy Wein- singly, this outcome is in accordance with the results berg equili brium, as already recorded in other B. of the pairwise differentiation test Dest (Jost 2008) schlosseri populations worldwide (Ben-Shlomo et al. and pairwise Nei genetic distance, which were found 2001, 2006, 2010, Stoner et al. 2002, Paz et al. 2003, to be highly and significantly correlated (Table S4, Johnson & Yund 2007), thus weakening the possi - Fig. S3 in the Supplement). Gene flow plots among bility for he terozygote deficiency due to null alleles. Scandinavian clusters, computed by Bayesian clus-

Overall Fis value calculated via AMOVA was 0.452 tering based on the parameters mentioned above (p < 0.001). Genetic diversity parameters He and AR (Fig. 3b), revealed a complex network of trajectories (Table 1) between southeastern and northwestern between clusters. However, 3 of the sites (Dolvik, Scandinavian sites revealed no statistical difference Tananger, Risør) were 100% self-seeded, while at (p = 0.06 for He; p = 0.99 for AR). Mantel test for cor- the other 8 sites, there was limited gene flow among relation between geographic and genetic distances the sites (90 to 97% self-seeding). (TFPGA; Miller 1997) also revealed (Fig. 2) that isola- Fig. 3b also reveals only a single gene-flow trajec- tion by distance is not characteristic for the sampled tory among 5 northwestern clusters, compared to 5 Scandinavian populations (p = 0.12 and r = 0.158), trajectories among the southeastern clusters and 10 further indicating that the effect of geographical trajectories between the northwest and southeast. distance does not explain the variance in genetic The intensity (mean ± SE) of gene flow among distance. We performed 2 additional independent southeas tern clusters was 0.023 ± 0.007 and 0.018 ± Reem et al.: Genetic structure and dispersal of Botryllus schlosseri in Scandinavia 149

a DISCUSSION

The results of this study, which con - centrated on 11 Scandinavian popula- tions, revealed (1) high polymorphism and genetic variability, (2) that specific alleles shared by most sites have high fre- quency, (3) no isolation by distance, (4) a complex, although with limited intensity, network of gene flow among most sam- pling sites, and (5) 2 suggested distinct northwestern and southeastern clades.

b Genetic diversity and possible ancestral alleles

Based on the same 5 microsatellite loci, genetic diversity values depicted for each of the Scandinavian populations (0.482 < He < 0.686; 4.13 < AR < 6.59) and their means (0.620 ± 0.029 and 5.29 ± 0.21, respectively; Table 1) were lower than values revealed in other Botryllus schlos- seri populations, such as the Santa Cruz harbor (California, USA) population that was sampled over a period of 13 yr (0.718 < He < 0.802; 5.642 < AR < 8.216; Reem et al. 2013), and 3 sites stu died along the Mediterranean coast of Israel (0.94 < He < 0.97; Paz et al. 2003). How- ever, the Scandinavian population values were higher than those recorded for north - ern European and English populations (0.17 < He < 0.61; Ben-Shlomo et al. 2006). Fig. 3. Botryllus schlosseri. Bayesian clustering computation results among Botryllus schlosseri populations proba- Scandinavian sampling sites. (a) Nei genetic distances on phylogenetic tree of the 10 Scandinavian clusters. (b) Gene-flow plot among Scandina- bly occurred in Scandinavia earlier than vian clusters. Arrows indicate direction of gene flow. Numbers on arrows the 1940s, when reported in southern represent the mean contributions of source clusters among the individuals Norway (Van Name 1945, Berrill 1950), assigned to a target cluster. Threshold value for gene flow was set to 1% and at the approximate time that they (0.01). Based on admixture analysis among clusters, using BAPS v.5.3 software (p < 0.05) were first recorded from California coasts (Van Name 1945, Cohen & Carlton 1995). A possible explanation for the lower 0.002 be tween northwest and southeast clusters, genetic polymorphism recorded in the Scandinavian pointing to stronger gene flow in the southeastern versus other populations is based on environmental clusters. The difference, however, was not statisti- conditions. While colonies of B. schlosseri in the cally significant (t-test; p = 0.34, df = 12, t = −0.998). Mediterranean Sea and in California are found year- Among the 10 northwestern and southeastern trajec- round (Chadwick-Furman & Weissman 1995, Stoner tories, 6 were from northwest to southeast with a et al. 2002, Paz et al. 2003), in Scandinavia the mean intensity of 0.016 ± 0.002 and 4 from southeast colonies are recorded only from July to December, to northwest with a mean intensity of 0.021 ± 0.003, when water temperatures are >9°C, and disappear albeit the difference was not statistically significant when the water temperature drops (Nair 1962), prob- (t-test; p = 0.19, df = 8, t = −1.419). ably going into hibernation (Bru netti et al. 1980). 150 Mar Ecol Prog Ser 485: 143–154, 2013

This shorter ecologically active period limits the Botryllus schlosseri is currently found in northern accumulation of substantial genetic diversity. An- sites from which it was probably absent in the past other viable explanation is that Scandinavian popu- (B. Rinkevich unpubl. data) and is likely moving lations were influenced by a small number of intro- north in Scandinavia as shown by the gene-flow duction events, partly supported by the constrained trajectories (Fig. 3b), reflecting the shifts in North gene flow between sites. Atlantic oscillation temperatures over the last 3 de - Analysis of allele distribution revealed 13 out of cades (Visbeck et al. 2001). An increase in sea water 108 different alleles that were common to most (10 to temperatures has been further documented in Scan- 11) Scandinavian sites, with overall mean frequen- dinavia in the last decade, associated with several cies between 0.13 and 0.67; in most of the cases the record heat waves (Mork et al. 2007). values were >0.2. This, coupled with the positive cor- Nevertheless, dispersal into new sites is most likely relation between the number of sites shared by the not the outcome of expanding vanguard populations, same alleles and mean frequency of those alleles but rather coincidental, depending on vessel traffic (Spearman rho Correlation coefficient = 0.982; p < trajectories in Scandinavia. Fishing activities are re- 0.01) suggest the possibility of these alleles being stricted by international agreements that prohibit ancestral, or founding alleles of the Scandinavian fisherman from crossing international marine borders. populations. Therefore, we assume that recreation and motor boats fouled with colonies of Botryllus schlos seri that cross the Skagerrak during the summer, mainly from Nor- Isolation by distance and gene flow among way and Denmark (H. Sköld, Gothenburg Univ., pers. Scandinavian sites comm.), and commercial ships from other parts of Eu- rope are the main dispersal vectors. The notion of an- Overall Mantel test (Fig. 2) indicated no isolation thropogenic dispersal of botryllid ascidians is further by distance among the Scandinavian localities and supported by other publications (Locke et al. 2009, connectivity among populations, a result supported Bock et al. 2011, Lacoursière-Roussel et al. 2012). by the Bayesian gene-flow outcomes among sampled Despite the extensive gene flow between Scandi- Scandinavian populations (Fig. 3b). This is clearly navian populations, the 11 examined populations illustrated when considering the closest Norwegian eventually formed 10 clusters and 2 clades (Fig. 3a). populations of Håkonshella and Dolvik (10 km The first included 5/6 southeastern populations and apart). The Håkonshella population is genetically one (Håkonshella; Norway) from the northwest, and closer to the clusters of the southern region (Öckerö, the second comprised the 4 remaining northwestern Fiskebäckskil, Strömstad and Hamburgsund-Fevik), populations and one (Risør; Norway) from the south- which are between 490 and 680 km away, than to the east. This division is consistent with the results of

Dolvik cluster (Fig. 3a). Similarly, Fevik and Risør the different Fst and Dest values in the northern (60 km apart) are more divergent than Risør and and southern regions of Scandinavia. An additional Tananger, which are 360 km apart (Fig. 3a). At first Bayesian clustering analysis with Structure 2.3.1 soft- glance, it seems that the Nei genetic distances in ware revealed similar, although not identical results Fig. 3a and the gene-flow plots in Fig. 3b are not con- (Fig. S4 in the Supplement). While less likely, it is gruent in 2 cases: in Fig. 3a, Alesund and Dolvik are possible that the 2 yr gap in sampling (Sweden — genetically similar, but no gene flow between these 2005, Norway— 2007) added some variation to the populations is shown in Fig 3b. The same applies to genetic structure among sites, due in part to changes Risør and Tananger. However, as genetic similarity that occurred over time, on top of the emerged in- can be achieved by either gene flow or common herent spatial differences at a single time point. ancestry of 2 groups that could have diverged rela- tively recently, the same outcome could reflect either cause. The gene-flow trajectory analysis (Fig. 3b) Comparisons between Scandinavian and western further supports restricted connectivity among the European sites northwestern compared to the southeastern clusters, and the absence of isolation by distance. The exten- The meta-population structure of the cosmopolitan sive gene flow among populations over geographical species Botryllus schlosseri is made up of many local scales of hundreds of kilometers is probably due to populations that have colonized distant sites on anthropogenically mediated dispersal, mainly con- different occasions, through dissimilar immigration tinuous traffic of fouled vessels. vectors, all subjected to anthropogenic activities (Paz Reem et al.: Genetic structure and dispersal of Botryllus schlosseri in Scandinavia 151

et al. 2003, Dijkstra et al. 2007, Bernier et al. 2009, La- sites in Scandinavia. Overall, Fst and Dest values for 8 coursière-Roussel et al. 2012). This should be taken western European sites combined were 0.419 (p < into consideration when evaluating population ge- 0.001) and 0.749 (CI = 0.729 to 0.790), respectively, netic properties and structures between remote exhibiting a re latively strong European population populations. Previous population genetic analyses of structure with higher differentiation among sites B. schlosseri from the southern Atlantic coasts of as compared to Scandinavia, thus emphasizing the Europe, the North Sea and the British Isles (Ben- higher connectivity among Scandinavian populations Shlomo et al. 2006; using 4/5 microsatellites and ex- in comparison to the connectivity among western Eu- cluding locus PBC-1) provide us with the opportunity ropean sites. Establishing a phylogenetic tree through to extend the comparisons over a geographical scale Bayesian clustering, 14 clusters emerged out of the 19 of thousands of kilometers. Data of 8 sites from Spain Scandinavian and western European sites studied, to Scotland and Germany were compared with the 11 with 2 distinct major clades (Fig. 4a). Of the 11 Scan-

Fig. 4. Botryllus schlosseri. Bayesian clustering computation results among western European and Scandinavian sites. (a) Nei genetic distances on phylogentic tree of all western European and Scandinavian clusters computed by Bayesian clustering method. Based on 4/5 microsatellites. English and European data are from Ben-Shlomo et al. (2006). (b) Gene-flow plot among Scandinavian and western European populations computed by Bayesian clustering. Arrows indicate direction of gene flow. Numbers on arrows represent the mean contributions of source clusters among the individuals assigned to a target cluster. Threshold value for gene flow was set to 1% (0.01). Based on admixture analysis among clusters, using BAPS v.5.3 software (p < 0.05) 152 Mar Ecol Prog Ser 485: 143–154, 2013

dinavian populations, 10 were clustered in a single Acknowledgements. We thank G. Paz for technical help and clade; the exception being Risør (Norway) which was figure preparation and J. Douek for helping in genotyping analyses. This study is part of E.R.’s PhD dissertation and clustered with the other western European clade, on was supported by grants from the Israel Science Foundation the same branch as Setubal (Portugal). Surprisingly, (1342/08 and 68/10). 3 populations from the south Ibe rian Peninsula (Faro, Portugal, Barbeta, Spain and Gibraltar) were LITERATURE CITED clustered together with the ‘Scandinavian cluster’ (Fig. 4a). These results suggest a recent gene flow or Anadón R, Danovaro R, Dippner JW, Drinkwater KF and common ancestry between Scandinavia and the others (2007) Impacts of climate change on the European south Iberian Peninsula, roughly 3000 km away when marine and coastal environment. 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Editorial responsibility: Karen Miller, Submitted: July 2, 2012; Accepted: February 20, 2013 Hobart, Tasmania, Australia Proofs received from author(s): June 11, 2013