Phylogenetic Analyses and Outgroup Choice

Appendix 1: Detailed molecular methods

Phylogenetic analyses and outgroup choice

The optimal model of evolution (COI: TrN + I + G; 18S: TrN) was selected in MODELTEST v.3.7 (Posada & Crandall 1998), under the Akaike Information Criterion (Posada & Buckley 2004). The NJ analysis was performed in MEGA v.4 (Tamura et al. 2007), with clade support estimated using 1000 bootstrap replicates. The BI analysis was performed in MRBAYES v.3.1 (Ronquist & Huelsenbeck 2003), with priors left at default values. We performed four independent runs of four chains for 10 x 106 generations, with trees sampled every 100th generation, and the first 25% of trees discarded as burn-in. Posterior probabilities were calculated as the percentage of trees recovering a particular node. Tamura-Nei corrected (Tamura & Nei 1993) divergences were estimated in MEGA.

For the COI phylogeny, we used Botrylloides leachii (formerly classified as Botryllus tyreus; Ascidiacea World Database http://www. marinespecies.org/ascidiacea/) as an outgroup (GenBank Accession DQ365851). This represents the member in the Styelidae family most closely related to B. schlosseri, for which COI data is available in GenBank. Additional exploratory runs using COI sequences from the styellid species Polycarpa pomarina (GenBank Accession AY600984), and Styela clava (GenBank Accession HQ730809) revealed a stable tree topology, regardless of which outgroup or outgroup combinations were used. For the 18S phylogeny, the congeneric Botryllus planus (GenBank Accession DQ346653) was used as an outgroup.

STRUCTURE analysis

We used the admixture model with independent allele frequencies, and allowed admixture proportions to vary for each population (i.e. we inferred a separate α for each population). Each of five independent STRUCTURE runs consisted of 106 generations, with a burn-in of 105 generations, with the number of clusters (K) ranging from 1 to 30 (total sites). Additionally, we inferred a suitable value for λ, an approach that is recommended when allele frequencies are skewed towards low/high frequencies (Pritchard et al. 2000). The most likely value of K was determined using the methods of Evanno et al. (2005) and Pritchard et al. (2000). Finally, STRUCTURE results were visualized in DISTRUCT v.1.1 (Rosenberg 2004).

Appendix 2: Additional Botryllus schlosseri COI sequences used for the phylogenetic reconstructions, with haplotype code, sampling location, and GenBank Accession numbers; (a) Lopez-Legentil et al. (2006); (b) A. Lacoursière-Roussel 2011, unpublished data; (c) Stach & Turbeville (2002).

Haplotype code / Location(s) / GenBank
Accession nos.
Clade A
HG / Canet Harbour, France / DQ340208(a)
HI / Graña Harbour, Spain / DQ340210(a)
HJ / Graña Harbour, Blanes Harbour, Spain / DQ340211(a)
HK / Cubelles, Spain / DQ340212(a)
HP / Estaque Harbour, France / DQ340217(a)
HQ / Woods Hole, MA, USA / DQ340222(a)
HR / Woods Hole, MA, USA / DQ340223(a)
HS / Woods Hole, MA, USA / DQ340224(a)
HV / Ste. Marie La Mer, France / DQ340221(a)
Bs13 / Point Tupper, NS, Canada / N/A(b)
Bs14 / Mahone Bay, NS, Canada / N/A(b)
Bs15 / Point Tupper, NS, Canada / N/A(b)
Bs16 / Sydney, NS, Canada / N/A(b)
Bs17 / Dartmouth, NS, Canada / N/A(b)
Clade B
HU / Vilanova, Spain / DQ340219(a)
Clade C
HM / Fornelos, Spain / DQ340214(a)
HT / Vilanova, Spain / DQ340218(a)
Clade D
HL / Fornelos, Spain / DQ340213(a)
ST / Roscoff, France / AY116601(c)
Clade E
HC / Cadaqués, Spain / DQ223768(a)
HE / Roses Harbour, Spain / DQ340206(a)
HF / Roses Harbour, Canet Harbour, Spain / DQ340207(a)
HN / Fornelos, Spain / DQ340215(a)
HW / Palamós, Spain / DQ340220(a)

Appendix 3: Statistical parsimony network of Botryllus schlosseri cytochrome c oxidase subunit I (COI) haplotypes. Circle size is proportional to haplotype frequency, and small black dots indicate unsampled haplotypes inferred from the data.

Appendix 4: Pairwise ФST comparisons for Botryllus schlosseri clade A populations using the mitochondrial COI marker. Asterisk indicates values that are significant after sequential Bonferroni correction (Rice 1989). Populations with N < 10 individuals were removed from this analysis. Site IDs are defined in table 1.

VEN / ETQ / PAL / CER / BLA / ADM / ALI / FAL / HEL / PLT / SAL / WDH / FRC / LSM / BRN
16
VEN
ETQ / 0.52*
PAL / 0.49* / -0.06
CER / 0.54* / -0.04 / -0.01
BLA / 0.49* / 0.03 / 0.00 / 0.08
ADM / 0.72* / 0.22 / 0.27* / 0.16 / 0.45*
ALI / 0.65* / 0.07 / 0.14 / 0.07 / 0.31* / -0.01
FAL / 0.52* / -0.06 / -0.03 / -0.01 / 0.04 / 0.23* / 0.11
HEL / 0.73* / 0.85* / 0.75* / 0.73* / 0.73* / 0.93* / 0.88* / 0.77*
PLT / 0.24* / 0.37 / 0.35* / 0.44* / 0.34* / 0.70* / 0.59* / 0.38* / 0.40*
SAL / 0.01 / 0.64* / 0.60* / 0.64* / 0.60* / 0.85* / 0.77* / 0.62* / 0.84* / 0.17
WDH / 0.63* / 0.71* / 0.64* / 0.66* / 0.62* / 0.88* / 0.81* / 0.66* / 0.03 / 0.20 / 0.68*
FRC / 0.61* / 0.75* / 0.59* / 0.60* / 0.48* / 0.91* / 0.82* / 0.63* / 1.00* / 0.50* / 0.81* / 0.87*
LSM / 0.55* / 0.64* / 0.49* / 0.54* / 0.40* / 0.87* / 0.77* / 0.54* / 0.94* / 0.36* / 0.71* / 0.76* / 0.03
BRN / 0.49* / 0.53* / 0.43* / 0.50* / 0.35* / 0.83* / 0.72* / 0.47* / 0.75* / 0.18 / 0.57* / 0.49 / 0.32 / 0.09
BBY / 0.44* / 0.03 / 0.03 / 0.08 / 0.06 / 0.28* / 0.19 / 0.05 / 0.55* / 0.27* / 0.50* / 0.46* / 0.40* / 0.34* / 0.28

Appendix 5: Pairwise ФST comparisons for Botryllus schlosseri clade E populations using the mitochondrial COI marker. Asterisk indicates values that are significant after sequential Bonferroni correction (Rice 1989). Populations with N < 10 individuals were removed from this analysis. Site IDs are defined in table 1.

BRE / PEN / FAL / PLY
BRE
PEN / 0.28*
FAL / 0.29* / -0.05
PLY / 0.34* / 0.24* / 0.26*
BRI / 0.47* / 0.37* / 0.40* / -0.01

Appendix 6: Genetic diversity at 10 microsatellite loci for 16 sites of Botryllus schlosseri clade A. N, sample size; NA, number of alleles; A, allelic richness (with rarefaction to 10 individuals); HO, observed heterozygosity; HE, expected heterozygosity; PHW, P-value for the Hardy-Weinberg equilibrium test, with significant departures from equilibrium after sequential Bonferroni correction (Rice 1989) in bold. Populations with N < 10 individuals were removed from this analysis. Site IDs are defined in table 1.

Sites / BS321 / PB29 / PB49 / PB41 / PBC1 / Bsm1 / Bsm2 / Bsm4 / Bsm6 / Bsm9
VEN
(N = 28) / NA /A / 8/5.5 / 4/3.8 / 8/7.2 / 5/4.1 / 18/11.6 / 3/2.6 / 6/5.5 / 7/5.4 / 6/4.2 / 5/3.4
HO / 0.583 / 0.423 / 0.214 / 0.286 / 0.391 / 0.240 / 0.679 / 0.440 / 0.423 / 0.200
HE / 0.612 / 0.637 / 0.839 / 0.569 / 0.929 / 0.536 / 0.766 / 0.737 / 0.600 / 0.292
PHW / 0.226 / 0.042 / 0.000 / 0.000 / 0.000 / 0.004 / 0.136 / 0.005 / 0.001 / 0.014
ETQ
(N = 15) / NA /A / 4/4.0 / 5/4.8 / 10/9.4 / 5/4.7 / 7/6.8 / 4/3.6 / 5/4.7 / 7/6.4 / 2/1.7 / 3/2.8
HO / 0.100 / 0.600 / 0.583 / 0.667 / 0.667 / 0.182 / 0.500 / 0.643 / 0.067 / 0.083
HE / 0.553 / 0.646 / 0.913 / 0.777 / 0.862 / 0.398 / 0.714 / 0.810 / 0.067 / 0.301
PHW / 0.000 / 0.026 / 0.000 / 0.409 / 0.049 / 0.019 / 0.024 / 0.281 / 1.000 / 0.007
PAL
(N = 19) / NA /A / 4/3.9 / 6/4.7 / 14/11.4 / 6/5.2 / 10/8.7 / 4/3.8 / 6/4.9 / 5/4.8 / 2/2.0 / 2/2.0
HO / 0.154 / 0.529 / 0.733 / 0.579 / 0.385 / 0.235 / 0.444 / 0.611 / 0.000 / 0.000
HE / 0.406 / 0.677 / 0.920 / 0.765 / 0.843 / 0.569 / 0.695 / 0.754 / 0.189 / 0.173
PHW / 0.004 / 0.071 / 0.000 / 0.012 / 0.000 / 0.000 / 0.005 / 0.158 / 0.050 / 0.047
CER
(N = 34) / NA /A / 9/5.5 / 10/5.3 / 16/10.7 / 5/4.8 / 11/8.7 / 5/3.8 / 9/5.8 / 6/5.2 / 7/4.9 / 3/2.7
HO / 0.423 / 0.567 / 0.690 / 0.576 / 0.310 / 0.375 / 0.567 / 0.677 / 0.120 / 0.179
HE / 0.579 / 0.656 / 0.915 / 0.761 / 0.874 / 0.532 / 0.733 / 0.764 / 0.542 / 0.382
PHW / 0.062 / 0.002 / 0.000 / 0.185 / 0.000 / 0.003 / 0.063 / 0.017 / 0.000 / 0.003
BLA
(N = 24) / NA /A / 9/6.2 / 5/4.4 / 13/10.0 / 5/4.2 / 6/5.8 / 4/3.7 / 9/7.5 / 6/5.2 / 4/3.6 / 3/2.5
HO / 0.438 / 0.652 / 0.636 / 0.625 / 0.143 / 0.348 / 0.565 / 0.708 / 0.235 / 0.190
HE / 0.488 / 0.693 / 0.904 / 0.704 / 0.772 / 0.693 / 0.855 / 0.686 / 0.651 / 0.292
PHW / 0.082 / 0.218 / 0.000 / 0.509 / 0.000 / 0.001 / 0.001 / 0.935 / 0.000 / 0.061
ADM
(N = 38) / NA /A / 5/4.1 / 6/4.2 / 15/10.1 / 6/4.8 / 11/8.0 / 3/2.9 / 7/5.6 / 6/4.9 / 3/2.4 / 5/2.7
HO / 0.273 / 0.686 / 0.576 / 0.553 / 0.367 / 0.314 / 0.500 / 0.737 / 0.094 / 0.063
HE / 0.579 / 0.665 / 0.904 / 0.762 / 0.837 / 0.469 / 0.775 / 0.692 / 0.228 / 0.179
PHW / 0.000 / 0.272 / 0.000 / 0.012 / 0.000 / 0.002 / 0.001 / 0.674 / 0.001 / 0.002
ALI
(N = 33) / NA /A / 6/5.3 / 4/3.1 / 11/8.1 / 5/4.3 / 8/7.0 / 4/3.7 / 6/5.4 / 5/4.7 / 3/2.5 / 5/3.5
HO / 0.375 / 0.515 / 0.571 / 0.594 / 0.273 / 0.194 / 0.774 / 0.813 / 0.080 / 0.107
HE / 0.740 / 0.536 / 0.862 / 0.692 / 0.844 / 0.650 / 0.787 / 0.757 / 0.222 / 0.321
PHW / 0.001 / 0.935 / 0.009 / 0.130 / 0.000 / 0.000 / 0.142 / 0.086 / 0.001 / 0.000
FAL
(N= 18) / NA /A / 2/1.9 / 4/4.0 / 6/5.3 / 4/3.6 / 6/5.6 / 2/2.0 / 5/4.6 / 6/5.7 / 2/1.7 / 2/1.8
HO / 0.133 / 0.353 / 0.333 / 0.235 / 0.143 / 0.059 / 0.533 / 0.471 / 0.071 / 0.000
HE / 0.129 / 0.704 / 0.743 / 0.690 / 0.780 / 0.401 / 0.692 / 0.738 / 0.071 / 0.114
PHW / 1.000 / 0.002 / 0.000 / 0.000 / 0.000 / 0.001 / 0.119 / 0.043 / 1.000 / 0.031
HEL
(N = 10) / NA /A / 1/1.0 / 1/1.0 / 3/3.0 / 1/1.0 / 4/4.0 / 3/3.0 / 4/4.0 / 3/3.0 / 2/2.0 / 1/1.0
HO / NA / NA / 0.500 / NA / 0.500 / 0.100 / 0.200 / 0.700 / 0.300 / NA
HE / 0.000 / 0.000 / 0.416 / 0.000 / 0.616 / 0.647 / 0.432 / 0.568 / 0.521 / 0.000
PHW / NA / NA / 1.000 / NA / 0.625 / 0.000 / 0.028 / 0.547 / 0.244 / NA
PLT
(N = 12) / NA /A / 3/2.8 / 5/5.0 / 5/4.5 / 1/1.0 / 5/5.0 / 3/2.8 / 6/5.8 / 3/3.0 / 2/1.8 / 1/1.0
HO / 0.250 / 0.200 / 0.500 / NA / 0.200 / 0.750 / 0.667 / 0.500 / 0.083 / NA
HE / 0.236 / 0.800 / 0.435 / 0.000 / 0.663 / 0.562 / 0.812 / 0.620 / 0.083 / 0.000
PHW / 1.000 / 0.000 / 1.000 / NA / 0.000 / 0.239 / 0.500 / 0.250 / 1.000 / NA
SAL
(N = 11) / NA /A / 2/2.0 / 3/3.0 / 3/3.0 / 1/1.0 / 4/4.0 / 3/3.0 / 4/3.8 / 3/3.0 / 2/1.9 / 1/1.0
HO / 0.200 / 0.200 / 0.100 / NA / 0.100 / 0.273 / 0.545 / 0.273 / 0.091 / NA
HE / 0.442 / 0.195 / 0.595 / 0.000 / 0.658 / 0.498 / 0.502 / 0.455 / 0.091 / 0.000
PHW / 0.129 / 1.000 / 0.002 / NA / 0.000 / 0.039 / 0.508 / 0.064 / 1.000 / NA
WDH
(N = 14) / NA /A / 4/3.9 / 4/3.4 / 3/3.0 / 3/2.7 / 7/6.9 / 3/3.0 / 6/5.8 / 4/3.9 / 2/2.0 / 2/2.0
HO / 0.231 / 0.214 / 0.231 / 0.143 / 0.091 / 0.615 / 0.929 / 0.571 / 0.143 / 0.071
HE / 0.588 / 0.458 / 0.668 / 0.553 / 0.775 / 0.594 / 0.810 / 0.653 / 0.254 / 0.198
PHW / 0.004 / 0.004 / 0.002 / 0.001 / 0.000 / 0.218 / 0.583 / 0.577 / 0.216 / 0.108
FRC
(N = 26) / NA /A / 4/2.9 / 5/3.9 / 4/3.3 / 4/3.8 / 5/4.8 / 3/2.4 / 3/2.6 / 4/3.8 / 1/1.0 / 1/1.0
HO / O.227 / 0.217 / 0.100 / 0.154 / 0.727 / 0.769 / 0.320 / 0.423 / NA / NA
HE / 0.555 / 0.647 / 0.353 / 0.502 / 0.756 / 0.497 / 0.340 / 0.673 / 0.000 / 0.000
PHW / 0.003 / 0.000 / 0.000 / 0.000 / 0.000 / 0.002 / 0.034 / 0.000 / NA / NA
LSM
(N = 14) / NA /A / 2/2.0 / 3/3.0 / 3/2.7 / 4/4.0 / 4/4.0 / 3/2.7 / 3/2.7 / 5/4.5 / 1/1.0 / 1/1.0
HO / 0.333 / 0.000 / 0.357 / 0.700 / 0.917 / 0.143 / 0.714 / 0.769 / NA / NA
HE / 0.391 / 0.695 / 0.315 / 0.763 / 0.772 / 0.442 / 0.500 / 0.738 / 0.000 / 0.000
PHW / 1.000 / 0.000 / 1.000 / 0.000 / 0.009 / 0.006 / 0.228 / 0.000 / NA / NA
BRN
(N = 11) / NA /A / 3/3.0 / 5/5.0 / 6/6.0 / 3/3.0 / 6/6.0 / 3/3.0 / 3/2.9 / 4/3.9 / 1/1.0 / 1/1.0
HO / 0.100 / 0.200 / 0.300 / 0.000 / 0.900 / 0.400 / 0.545 / 0.636 / NA / NA
HE / 0.279 / 0.574 / 0.447 / 0.653 / 0.763 / 0.584 / 0.515 / 0.619 / 0.000 / 0.000
PHW / 0.053 / 0.002 / 0.045 / 0.000 / 0.863 / 0.243 / 1.000 / 0.761 / NA / NA
BBY
(N = 36) / NA /A / 5/3.5 / 6/4.8 / 8/6.7 / 3/2.5 / 7/4.6 / 4/3.8 / 4/3.7 / 4/3.5 / 2/1.7 / 5/3.3
HO / 0.600 / 0.361 / 0.824 / 0.353 / 0.500 / 0.500 / 0.588 / 0.444 / 0.030 / 0.333
HE / 0.595 / 0.775 / 0.830 / 0.514 / 0.648 / 0.655 / 0.608 / 0.556 / 0.088 / 0.428
PHW / 0.004 / 0.000 / 0.130 / 0.006 / 0.421 / 0.016 / 0.008 / 0.019 / 0.045 / 0.126

Appendix 7: Pairwise FST comparisons for Botryllus schlosseri clade A populations using 10 microsatellite markers. Asterisk indicates values that are significant after sequential Bonferroni correction (Rice 1989). Populations with N < 10 individuals were removed from this analysis. Site IDs are defined in table 1.

VEN / ETQ / PAL / CER / BLA / ADM / ALI / FAL / HEL / PLT / SAL / WDH / FRC / LSM / BRN
16
VEN
ETQ / 0.06
PAL / 0.05 / -0.01
CER / 0.05 / -0.01 / 0.01
BLA / 0.05 / 0.02 / 0.03 / 0.02
ADM / 0.04 / 0.00 / 0.01 / 0.00 / 0.00
ALI / 0.05* / 0.00 / 0.01 / 0.01 / 0.02 / 0.01
FAL / 0.05 / 0.04 / 0.06 / 0.05 / 0.00 / 0.02 / 0.02
HEL / 0.22* / 0.09 / 0.17 / 0.16* / 0.11* / 0.11* / 0.14* / 0.14
PLT / 0.18* / 0.09 / 0.14* / 0.14* / 0.13* / 0.14* / 0.08* / 0.11 / 0.18
SAL / 0.29* / 0.18* / 0.27* / 0.23* / 0.23* / 0.24* / 0.20* / 0.21* / 0.23 / 0.07
WDH / 0.14* / 0.11* / 0.17* / 0.13* / 0.12* / 0.15* / 0.11* / 0.08 / 0.20* / 0.03 / 0.06
FRC / 0.24* / 0.12 / 0.19 / 0.20* / 0.18* / 0.16* / 0.17* / 0.20* / 0.04 / 0.19* / 0.27* / 0.24*
LSM / 0.19* / 0.10 / 0.19* / 0.16* / 0.15* / 0.15* / 0.15* / 0.15* / 0.13* / 0.10* / 0.13* / 0.09 / 0.11
BRN / 0.18* / 0.06 / 0.13 / 0.13* / 0.10* / 0.09* / 0.11* / 0.14 / 0.03 / 0.16* / 0.28* / 0.22* / 0.02 / 0.08
BBY / 0.15* / 0.04 / 0.11* / 0.09* / 0.07* / 0.06* / 0.10* / 0.09* / 0.04 / 0.16* / 0.22* / 0.18* / 0.10* / 0.08 / 0.02

Appendix 8: Selection of STRUCTURE model

The ΔK statistic predicted 2 clusters