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, Haifa 31080, Israel 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 Gibraltar (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 Resale or republication not permitted without written consent of the publisher 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. schlos seri 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