Supplementary Material s23

Supplementary material

Table S1. Sampling locations of A. minutum isolates in the Mediterranean Sea and eastern Atlantic.

Sample* Sample Acronym Geographical coordinates Regional Sea Collection year number

Trieste TS 45°39'16''N, 13°42'52''E N Adriatic 1996; 1997 9

Marotta MAR 43°46'19''N, 13°09'24''E Central Adriatic 2005-2008 23

Siracusa SIR 37°03'04''N, 15°17'02''E Ionian 2001; 2007; 2008 19

Olbia OLB 40°57'11''N, 09°39'04''E Tyrrhenian 2002; 2007 24

Arenys de Mar AM 41°34'32''N, 02°33'30''E Catalan 2002 21

Palma PAL 39°33'25''N, 02°38'52''E Balearic 1995; 2008 11

Vigo VI 42°12'43''N, 08°52'29''W Atlantic 1987-2003 9 *The sample number contained also isolates collected from different localities, but included in the same regional Sea: Ionian Sea included 4 isolates from Taranto; Tyrrhenian Sea included two isolates from Porto Torres and one isolate from Oliveri; Catalan Sea included two isolates from Estartit. Table S2. List of Alexandrium minutum isolates and sequences obtained from this and other studies. Species Strain Geographical origin and collecting period Isolator Accession No. Reference

A. minutum LBMAL1T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. AM292216 [1] A. minutum LBMAL3T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. Not sequenced A. minutum LBMAL4T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. Not sequenced A. minutum LBMAL5T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. AM292215 [1] A. minutum LBMAL7T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. Not sequenced A. minutum LBMAL8T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. AM292214 [1] A. minutum LBMAL9T Trieste, N Adriatic Sea, Mediterranean, Italy, May 1997 Beran A. Not sequenced A. minutum LAC27 Trieste, N Adriatic Sea, Mediterranean, Italy, May 1996 Honsell G. AJ005050 [2] A. minutum AMITA Trieste, N Adriatic Sea, Mediterranean, Italy, May 1996 Honsell G. FR668142 This study A. minutum CBA2 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2005 Ingarao C. Not sequenced A. minutum CBA3 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2006 Ingarao C. Not sequenced A. minutum CBA4 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2007 Casabianca S. FN995984 This study A. minutum CBA5 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2007 Casabianca S. FR668025 This study A. minutum CBA6 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2007 Casabianca S. FR668024 This study A. minutum CBA37 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668026 This study A. minutum CBA38 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668027 This study A. minutum CBA39 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668028 This study A. minutum CBA40 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668029 This study A. minutum CBA41 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668030 This study A. minutum CBA52 Marotta, C Adriatic Sea, Mediterranean, Italy, May 2008 Casabianca S. FR668089 This study A. minutum CBA42 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668090 This study A. minutum CBA43 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668091 This study A. minutum CBA44 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668092 This study A. minutum CBA47 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668093 This study A. minutum CBA48 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668094 This study A. minutum CBA49 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668095 This study A. minutum CBA53 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668096 This study A. minutum CBA54 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668097 This study A. minutum CBA55 Marotta, C Adriatic Sea, Mediterranean, Italy, April 2008 Casabianca S. FR668098 This study Table S3. Genetic variability based on 7 microsatellite loci from 6 regional seas of the Mediterranean and Atlantic.

Regional sea Aminu11 Aminu22 Aminu29 Aminu41 Aminu43 Aminu44 Aminu48 Mean all loci (SD)

Adriatic N 32 32 29 27 26 24 31 28.71 (3.15) Na 9 9 9 17 11 11 10 10.86 (2.64) H 0.84 0.88 0.84 0.96 0.91 0.91 0.89 0.89 (0.04) A 6.67 7.04 6.88 10.77 8.12 8.18 7.51 7.88 (1.40) Ionian N 17 19 18 15 15 19 18 17.29 (1.70) Na 3 4 2 7 4 4 5 4.14 (1.46) H 0.32 0.66 0.50 0.87 0.68 0.57 0.71 0.62 (0.17) A 2.66 3.64 2.00 6.30 3.78 3.57 4.37 3.76 (1.36) Tyrrhenian N 23 24 24 20 24 21 24 22.86 (1.68) Na 6 3 5 10 5 5 5 5.57 (1.99) H 0.81 0.59 0.73 0.92 0.77 0.63 0.49 0.71 (0.14) A 5.32 2.82 4.41 8.16 4.67 4.11 3.86 4.76 (1.68) Catalan N 21 21 21 19 21 21 21 20.71 (0.76) Na 7 3 4 3 7 6 1 4.43 (2.30) H 0.78 0.64 0.61 0.68 0.81 0.81 0.00 0.62 (0.28) A 5.68 2.96 3.25 2.99 5.69 5.11 1.00 3.81 (1.75) Balearic N 11 11 11 8 11 11 11 10.57 (1.13) Na 5 2 2 5 6 4 4 4.00 (1.41) H 0.78 0.55 0.55 0.89 0.80 0.67 0.76 0.71 (0.13) A 4.81 2.00 2.00 5.00 5.74 3.87 3.99 3.92 (1.45) Atlantic N 8 9 9 8 9 9 8 8.57 (0.53) Na 5 4 5 4 5 4 5 4.57 (0.53) H 0.79 0.58 0.81 0.75 0.81 0.58 0.89 0.74 (0.12) A 5.00 3.98 4.98 4.00 4.98 3.98 5.00 4.56 (0.54) N, number of individual; Na, number of alleles observed per locus; H, gene diversity [4]; A, allelic richness. Table S4. Analysis of Molecular Variance (AMOVA) based on allele frequency for 7 microsatellite loci among A. minutum groups.

Degree of Fixation Analysis Percentage of variation freedom index Significance: ***, p < 0.001. Among groups 5 18.93 F = 0.189*** ST Significance was tested with 10 000 Within groups 226 81.07 permutations. Total 231 Table S5. Pairwise FST values for 15 pairwise comparisons among the 6 groups of

A. minutum based on 7 microsatellite loci.

Adriatic Ionian Tyrrhenian Catalan Balearic Atlantic

Adriatic - Ionian 0.170*** - Tyrrhenian 0.135*** 0.251*** - Catalan 0.176*** 0.267*** 0.215*** - Balearic 0.124*** 0.208*** 0.130*** 0.149*** - Atlantic 0.113*** 0.302*** 0.216*** 0.297*** 0.237*** -

Significance: ***, p < 0.001. Significance was tested with 10 000 permutations. Table S6. Average pairwise similarity values (H-values) for replicate of Structure v. 2.3 runs generated by using CLUMPP v. 1.1.2. Replicate runs shown below were performed for K = 2, 3 and 4 populations for 7 loci microsatellites.

Runs H’- values

K = 2 10 0.8659 K = 3 10 0.7333 K = 4 10 0.9248

Table S7. Percentage assigned genotype to regional seas.

Regional Endemic Allochthonous Mixed Tyrrhenian Catalan Ionian Adriatic sea

Adriatic 62 19 19 16 3 - -

Ionian 84 5 11 - 5 - -

Tyrrhenian 67 8 25 - 4 4 -

Catalan 90 5 5 - - - 5

References

[1] Penna, A., Fraga, S., Masò, M., Giacobbe, M.G., Bravo, I., Vila, M., Garcés, E., Bertozzini, E., Andreoni, F., Luglié, A., Vernesi, C. 2008 Phylogenetic relationships among the Mediterranean Alexandrium (Dinophyceae) species based on sequences of 5.8S gene and internal transcribed spacers of the rRNA operon. Eur. J. Phycol. 43, 163-178. (doi: 10.1080/09670260701783730)

[2] Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garcés, E. & Magnani, M. 2004. Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Appl. Environ. Microbiol. 70, 1199-1206.

[3] Vila, M., Giacobbe, M.G, Masò, M., Gangemi, E., Penna, A., Sampedro, N., Azzaro, F., Camp, J. & Galluzzi, L. 2005 A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas. Harmful Algae 4, 673-695. (doi:10.1016/j.hal.2008.08.006)

[4] Nei, M. 1987 Molecular Evolutionary Genetics. New York: Columbia University Press. DETAILED ANALYSES OF STRUCTURE

Using 20 iterations with a burnin of 500,000 and chain length of 2,000,000, we first set K = 2. The following Table S8 describes the results obtained in terms of proportion of membership of the original populations to the two inferred groups.

Table S8. Inferred groups from original populations by setting K = 2.

Population IG1 IG2

Ionian 0.059 0.941

Adriatic 0.918 0.082

Majorca 0.170 0.830

Catalan 0.079 0.921

Tyrrhenian 0.399 0.601

E Atlantic 0.891 0.109

It emerged that the inferred Group 1 is formed by just two populations: Adriatic and E Atlantic

(Vigo, Spain). The remaining 4 populations were clearly assigned to IG2, with only Tyrrhenian showing a value of membership below 0.8. Then, IG1 and IG2 were analysed separately with

STRUCTURE. Ten iterations for each value of K between 1 and 4 were run with burnin of 500,000 and chain length of 2,000,000 for IG1. The analysis of the second group (i.e. Ionian, Catalan,

Majorca and Tyrrhenian) were conducted with the same settings, but we investigated K from 1 to 6. Fig. S1. Mean log likelihood values of ten runs of STRUCTURE for each K value from 1 to 4.

Fig. S2. Second order statistics Delta K of the ten runs of STRUCTURE for each K value from 1 to 4.

The graph of the mean likelihood (figure S1) didn’t allow to take a final decision about the most likely number of inferred groups. From the second order statistics, DeltaK, we concluded that two was the most likely number of inferred groups (figure S2). The associated table of proportion of membership is presented below (table S9): Table S9. Inferred groups from two populations by setting K from 1 to 4.

Population IG1 IG2

Adriatic 0.682 0.318

E Atlantic 0.529 0.471

In this case, it is clear that while Adriatic could be assigned to IG1, E Atlantic showed a situation of mixed membership. In other terms, while it seemed possible to identify Adriatic as a single homogeneous genetic group with some evident signs of mixing, E Atlantic appeared as an admixed group. The analyses of the second group (i.e. Ionian, Catalan, Majorca and Tyrrhenian) were conducted with the same settings, but K from 1 to 6 was investigated.

Fig. S3. Mean log likelihood values of ten runs of STRUCTURE for each K value from 1 to 6. Fig. S4. Second order statistics Delta K of the ten runs of STRUCTURE for each K value from 1 to 6.

Also in this case the mean likelihood values were uninformative, while DeltaK revealed a clear peak at K = 3 (figures S3, S4).

These were the results in terms of proportion of membership with K= 3 (table S10).

Table S10. Inferred groups from 4 populations by setting K = 3. Population IG1 IG2 IG3

Ionian 0.123 0.091 0.786

Majorca 0.543 0.231 0.227

Catalan 0.963 0.011 0.026

Tyrrhenian 0.133 0.801 0.067

Again, considering a threshold of about 0.8, Ionian, Catalan and Tyrrhenian were assigned to three distinct homogeneous groups, while Majorca can be better considered a mixed group with significant proportion of each inferred group.

To conclude, the hierarchical STRUCTURE analyses gave important results, highlighting the presence of at least four different groups that are: Ionian, Catalan, Tyrrhenian and Adriatic. This was exactly the result obtained with our previous no hierarchical analyses (both STRUCTURE and

CLUMPP), further corroborated by the Principal Coordinates Analyses.

To summarize, the adoption of the suggested hierarchical approach confirmed previous results, thus giving more confidence to the K = 4 scenario.