1

Supporting information

A combined genetic-morphometric analysis unravels the complex biogeographic history

of Polyommatus icarus and P. celina Common Blue butterflies

Vlad Dincă, Leonardo Dapporto, Roger Vila

Fig. S1 Representation of fixed landmarks (yellow circles) and sliding semilandmarks (red circles) used for geometric morphometry analyses of (a) wings underside, (b) falces and (c) valvae (lateral view).

2

Fig. S2 Maximum likelihood tree based on COI sequences of Polyommatus icarus, P. celina and several other Polyommatus s. str. taxa. Bootstrap supports (>50%) are shown above recovered branches. Grey cases indicate the position of P. icarus and P. celina in the tree. Specimens in bold were differently identified based on COI and ITS2 respectively.

3

Fig. S3 Bayesian trees based on the analysis of COI haplotypes of P. icarus and P. celina. (a) Bayesian tree based on COI haplotypes of P. icarus. (b) Bayesian tree based on COI haplotypes of P. celina. Numbers at nodes indicate Bayesian posterior probabilities (shown if higher than 50%).

4

Fig. S4 Maximum likelihood tree based on ITS2 sequences of Polyommatus icarus, P. celina and several other Polyommatus s. str. taxa. Bootstrap supports (>50%) are shown above recovered branches. Grey cases indicate the position of P. icarus and P. celina in the tree. Specimens in bold were differently identified based on COI and ITS2 respectively.

5

Fig. S5 Maximum likelihood tree based on the combined dataset of COI and ITS2. Bootstrap supports and Bayesian posterior probabilities (>50%) are shown above recovered branches. Grey cases indicate the position of P. icarus and P. celina in the tree.

6

Fig. S6 Discriminant analyses displaying relative positions of specimens belonging to the 16 different areas for (a) all traits, (b) for genitalia PCs and aedeagus length, (c) for wing PCs. Numbered black squares represent group centroids. (d) Relationship between values for falces PCw and forewing centroid size (wing size) in P.

celina (white squares) and P. icarus (black squares); symbols represent mean values for each of the 16 areas.

7

Fig. S7 Median-joining haplotype network inferred for P. icarus using the program Network 4.6.0.0.

8

Fig. S8 Median-joining haplotype network inferred for P. celina using the program Network 4.6.0.0.

9

Table S1 Specimens of Polyommatus icarus, P. celina and other Polyommatus s. str. taxa that were analyzed in this study. Specimens of P. icarus and P. celina with GenBank sequences used in the analyses are also included in the list. Samples marked in bold represent specimens with discordant mitochondrial and nuclear DNA-based identification.

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

MZUFalg1 P. celina x Aflou Algeria MZUFalg2 P. celina x Aflou Algeria MZUFalg3 P. celina x Mecheria, Oran Algeria MZUFalg4 P. celina x Mascara Algeria MZUFsar11 P. celina x Tempio Pausania, Sardinia Italy RVcoll.09-T503 hc22 JN084663 x JN084773 P. celina x Gennargentu, env. Broncu Spina, Sardinia Italy MZUFsar13 P. celina x Tempio Pausania, Sardinia Italy RVcoll.09-T570 hc22 x JN084766 P. celina x Gonnosfanadiga, Sardinia Italy RVcoll.09-T502 hc26 JN084665 x JN084765 P. celina x Gennargentu, env. Broncu Spina, Sardinia Italy MZUFsic11 P. celina x Cuaccia, Sicily Italy MZUFsic10 P. celina x Cuaccia, Sicily Italy RVcoll.186 hc17 JN084676 x JN084790 P. celina x Santa Lucia del Mela, Sicily Italy RVcoll.09-T513 hc18 P. celina x Ficuzza, Bosco della Ficuzza, Sicily Italy RVcoll.09-T512 hc19 JN084681 P. celina x Ficuzza, Bosco della Ficuzza, Sicily Italy RVcoll.183 hc20 JN084678 x JN084789 P. celina x Santa Lucia del Mela, Sicily Italy RVcoll.Lip1 hc18 JN084677 x JN084769 P. celina x Lipari island Italy LDcoll.lip3 P. celina x Lipari island Italy RVcoll.Lip2 hc21 JN084679 x JN084770 P. celina x Lipari island Italy GU559745 x P. celina Tourchte, High Atlas, Marrakech Morocco GU559740 x P. celina Oukaimeden, High Atlas, Marrakech Morocco GU559746 x P. celina Oukaimeden, High Atlas, Marrakech Morocco RVcoll.06-A087 hc10 JN084674 x JN084755 P. celina x Oukaimeden, High Atlas, Marrakech Morocco AY556968 (MW02006) hc12 x P. celina Oukaimeden, High Atlas, Marrakech Morocco RVcoll.06-A065 hc14 JN084671 x JN084754 P. celina x Gorges de l'Oued-el-Abid, Azilal, Middle Atlas Morocco GU559739 hc5 P. celina Col du Zad, S. Timahdite, Middle Atlas Morocco RVcoll.06-A046 hc5 JN084666 x JN084775 P. celina x Ouled Ayad, Azilal, Middle Atlas Morocco FJ428805 (ILL044) x x P. celina Tafraout, Antiatlas Morocco GU559742 hc1 P. celina Tizi-n-Tarakatine, E Tafraoute, Antiatlas Morocco GU559737 hc13 P. celina Betancuria, Fuerteventura, Canary Islands Spain RVcoll.09-X299 x JN084768 P. celina Fuerteventura, Canary Islands Spain

10

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

RVcoll.08-H191 hc22 x JN084756 P. celina x Llimpa, Maó, Menorca Spain RVcoll.08-H195 hc22 x JN084782 P. celina Llimpa, Maó, Menorca Spain RVcoll.08-H177 hc23 P. celina x Llimpa, Maó, Menorca Spain RVcoll.08-H192 hc25 JN084662 x JN084781 P. celina x Llimpa, Maó, Menorca Spain RVcoll.08-H172 hc22 P. celina x Es Grau, Maó, Menorca Spain RVcoll.09-X105 hc22 x JN084753 P. celina x Cala de Tirant, Fornells, Menorca Spain RVcoll.09-X106 hc22 x JN084771 P. celina x Cala de Tirant, Fornells, Menorca Spain

RVcoll.09-X114 hc22 x JN084772

P. celina x Parque Albufera des Grau, Menorca Spain

RVcoll.08-P297 hc22 P. celina Genova, Mallorca Spain RVcoll.08-P310 hc22 P. celina Sant Elm, Andratx, Mallorca Spain

RVcoll.08-P244 hc22 x JN084778

P. celina x Puig d’en Galileu, Mallorca Spain

RVcoll.08-P246 hc24 JN084664 x JN084763 P. celina x Puig d’en Galileu, Mallorca Spain RVcoll.08-P282 hc22 x JN084786 P. celina x S’Albufera, Mallorca Spain

RVcoll.08-H637 hc15 x JN084779 P. celina x La Linea de la Concepcion, playa de Santa Margarita, Cádiz

Spain

RVcoll.08-L622 hc7 x JN084785

P. celina Algeciras, Arroyo del Prior, Cádiz Spain

RVcoll.06-K695 hc7 P. celina x Guillimona, La Sagra, Huéscar, Granada Spain RVcoll.09-V558 hc3 JN084675 x JN084752 P. celina x Collado de las Sabinas (Sierra Nevada), Granada Spain RVcoll.08-L002 hc9 JN084670 x JN084762 P. celina x Carretera al Veleta (Sierra Nevada), Granada Spain RVcoll.09-V503 hc4 JN084668 x JN084776 P. celina x Sierra de Alfacar, Granada Spain RVcoll.08-L063 hc4 P. celina x Venta del Molinillo, Huetor de Santillan, Granada Spain RVcoll.09-V916 hc11 JN084673 x JN084730 P. celina x Sierra de la Sagra, Granada Spain RVcoll.09-V483 hc7 JN084672 x JN084767 P. celina x Sierra de la Sagra, Granada Spain RVcoll.09-V900 hc7 x JN084777 P. celina x Sierra de la Sagra, Granada Spain RVcoll.08-H634 hc4 x JN084758 P. celina Benadalid, Málaga Spain RVcoll.08-H977 hc8 x JN084774 P. celina Hoyocasero, Venta Rasquilla, Ávila Spain RVcoll.08-J926 hc16 JN084680 x JN084751 P. celina x Acceso Plain de Jugurtha, Kalaat Es Senan Tunisia RVcoll.08-J920 hc6 JN084667 x JN084759 P. celina x Acceso Plain de Jugurtha, Kalaat Es Senan Tunisia RVcoll.08-J937 hc2 JN084669 x JN084783 P. celina x Tableau de Jugurtha, Kalaat Es Senan Tunisia RVcoll.08-J900 hc7 x JN084780 P. celina x Sebkhet de Sidi El Hani, Kerker Tunisia RVcoll.08-J940 hc7 x JN084760 P. celina x Arroyo en Montes de Kroumirie, Ain Draham Tunisia

11

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

RVcoll.08-J945 hc7 x JN084761 P. celina x Arroyo en Montes de Kroumirie, Ain Draham Tunisia GU377278 x P. celina Mahdia Tunisia MZUFaus1 P. icarus x Portschach Austria MZUFbel1 P. icarus x Hockay Belgium FJ428817 x P. icarus Iskr, Pasarel Bulgaria MZUFcroat1 P. icarus x Sabbioncello Croatia RVcoll.08-L303 hi14 JN084695 x JN084745 P. icarus Hammer Bakker Denmark RVcoll.07-C710 hi11 JN084694 x JN084733 P. icarus Uusimaa Loviisa Harmaakallio Finland MZUFfin1 P. icarus x Riekkalansari Finland RVcoll.09-V243 hi1 P. icarus x Domaine de la chasse de Puits de Rians, Provence France RVcoll.09-X260 hi20 JN084698 P. icarus x Bédoine, Provence France RVcoll.09-X210 hi7 P. icarus x Oraison, Provence France RVcoll.09-X222 x JN084688 x JN084788 P. icarus x Sault, Provence France RVcoll.08-P209 hi7 x JN084787 P. icarus x Courbassil, Languedoc-Roussillon France MZUFfra6 P. icarus x Puybelliard France MZUFfra7 P. icarus x Vence France LDcoll.cor7 P. icarus x Corte, Corsica France RVcoll.07-C552 hi13 JN084693 x JN084717 P. icarus x Hungen Steinheim, Hessen Germany MZUFger2 P. icarus x Schleiden Germany RVcoll.150308PP10 hi22 JN084691 x JN084747 P. icarus x Montes de Ornos, Crete Greece RVcoll.150308PP67 hi22 x JN084750 P. icarus x Montes de Ornos, Crete Greece RVcoll.150308PP62 hi23 JN084692 x JN084749 P. icarus x Montes de Ornos, Crete Greece RVcoll.150308PP22 x JN084709 x JN084748 P. icarus x Bahía de Almyros, Almyros, Crete Greece AY556866 (JC00063) hi18 x P. icarus Mt. Falakro Greece FJ428819 (ILL027) x x P. icarus Dodoni, near Igoumenista Greece MZUFgre7 P. icarus x Olympus Greece MZUFgre9 P. icarus x Olympus Greece MZUFgre10 P. icarus x Olympus Greece MZUFgre13 P. icarus x Olympus Greece MZUFgre14 P. icarus x Olympus Greece MZUFgre15 P. icarus x Olympus Greece MZUFgre5 P. icarus x Salonika Greece MZUFgre6 P. icarus x Salonika Greece MZUFgre8 P. icarus x Salonika Greece

12

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

MZUFgre11 P. icarus x Salonika Greece MZUFgre12 P. icarus x Salonika Greece MZUFgre16 P. icarus x Salonika Greece MZUFgre17 P. icarus x Salonika Greece AY556927 (MW00412) hi17 x P. icarus Hajiabad, Golestan Iran EU597142 x P. icarus N. Torbat-e-Heydariyeh, Khorasan Iran FJ428823 x P. icarus Nir Sannich Abad, Yazd Iran FJ428824 x P. icarus S. Lalehzar, Kerman Iran RVcoll.292 hi1 x JN084743 P. icarus x Aspromonte, Santuario Polsi, Calabria Italy RVcoll.07-D899 hi1 x JN084718 P. icarus x Serra del Prete, Mont Pollino, Calabria Italy RVcoll.07-E051 hi1 x JN084719 P. icarus x P.N. La Sila, Calabria Italy RVcoll.07-E207 hi2 JN084703 x JN084737 P. icarus x Ozein-Visyes, Cogne Valley, Valle d'Aosta Italy RVcoll.07-D812 hi3 JN084700 x JN084742 P. icarus x Ostia Antica, Roma Italy RVcoll.07-D884 hi9 JN084705 P. icarus x Carpineto Romano, Monti Lepini, Roma Italy RVcoll.07-E062 hi5 JN084702 P. icarus x Monte Sirino, Potenza, Basilicata Italy RVcoll.07-D864 hi1 x JN084738 P. icarus x Filettino, Monti Simbruini, Frosinone, Lazio Italy EU597139 (ILL018) x x P. icarus near Trento Italy MZUFelba2 P. icarus x Poggio, Elba island Italy MZUFelba3 P. icarus x Poggio, Elba island Italy MZUFelba4 P. icarus x Poggio, Elba island Italy MZUFelba5 P. icarus x Poggio, Elba island Italy MZUFelba6 P. icarus x Marciana Alta, Elba island Italy LDcoll.capri2 P. icarus x Salerno, Capri island Italy FJ663998 x P. icarus Oktyabrsky v. Kazakhstan FJ663999 x P. icarus Oktyabrsky v. Kazakhstan RVcoll.08-J150 hi1 P. icarus x Ponte Pedrinha, Beça, Boticas Portugal RVcoll.08-J127 hi1 P. icarus x Seculca, Beca, Boticas Portugal RVcoll.08-J199 hi1 P. icarus x Serra do Larouco, Padornelos, Montalegre Portugal RVcoll.07-D104 hi12 P. icarus x Ciolanu, Buzău Romania RVcoll.06-M943 x P. icarus x Istrita hill, Breaza, Buzău Romania RVcoll.07-C366 hi13 P. icarus Sadova, pas Pașcanu, Suceava Romania RVcoll.08-M374 hi13 P. icarus x Buciumeni forest (Rădăcinești), Bacău Romania RVcoll.07-D439 hi13 P. icarus x Suatu, Cluj Romania RVcoll.06-V653 hi19 P. icarus x Făget forest, Râtu Penii, Cluj Romania

13

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

RVcoll.08-M230 hi13 P. icarus x 2 Km E of Moreni, Dâmbovița Romania RVcoll.07-D053 hi15 P. icarus 4 Km SE of Enisala, Tulcea Romania RVcoll.08-M425 hi16 P. icarus x Gura Dobrogei, Constanța Romania RVcoll.07-C964 hi19 P. icarus Baile Herculane, Pecinisca, Caraș-Severin Romania RVcoll.08-M630 hi19 P. icarus Scorota (Retezat Mts.), Hunedoara Romania EU597140 x P. icarus Ukhta, Komi Republic Russia EU597141 x P. icarus Ukhta, Komi Republic Russia FJ428801 x P. icarus Azov, Rostov-on-Don Russia FJ428802 x P. icarus Azov, Rostov-on-Don Russia FJ428803 x P. icarus Dugino, Azov, Rostov-on-Don Russia FJ428820 x P. icarus Dugino, Azov, Rostov-on-Don Russia FJ428822 (ILL041) x x P. icarus Dugino, Azov, Rostov-on-Don Russia FJ428821 (ILL030) x x P. icarus Sochi, Krasnodar Russia FJ428825 (ILL049) x x P. icarus Zav’yalovsky, Udmurtia Russia GQ885173 (ILL054) x x P. icarus Belokalitvensky District Russia RVcoll.08-H410 hi1 x JN084757 P. icarus x Cantoblanco, Madrid Spain RVcoll.08-H434 hi1 P. icarus x Campo Real, Madrid Spain RVcoll.08-L092 hi1 P. icarus x Ruta de la Sima, El Vallecillo, Teruel Spain RVcoll.09-X521 hi1 x JN084732 P. icarus x 2 km NW El Vallecillo, Teruel Spain

RVcoll.08-L276 hi1 P. icarus Mas de Cap del Pla a Mas del Pont, Vistabella del Maestrazgo, Castellón

Spain

RVcoll.07-F062 hi1 x JN084734 P. icarus x 10 km S. of Morella, Castellón Spain RVcoll.08-R025 hi1 P. icarus Sant Vicent, Lliria, Valencia Spain

RVcoll.08-L281 hi24 JN084687 x JN084725 P. icarus x Los Collados - Benimaurell, Vall de Laguart, Alicante

Spain

RVcoll.06-G507 hi1 P. icarus x Sierra de Alcubierre, Aragón Spain RVcoll.08-H255 hi1 x JN084721 P. icarus x Mequinenza, Huesca Spain RVcoll.07-C463 hi1 P. icarus x UAB, Bellaterra, Barcelona Spain RVcoll.08-H232 hi1 P. icarus UAB, Bellaterra, Barcelona Spain RVcoll.06-G433 hi20 P. icarus Can Rovira, El Brull, Barcelona Spain RVcoll.09-V396 hi7 P. icarus x La Tancada, Delta de l'Ebre, Tarragona Spain RVcoll.08-M674 hi1 P. icarus Serra Major, Morera de Montsant, Tarragona Spain RVcoll.08-L473 hi7 P. icarus x Ivars de Noguera, Lleida Spain RVcoll.08-P076 hi7 P. icarus x Torrent de Ridolaina, Cerdanya, Girona Spain

14

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

RVcoll.07-C623 hi7 P. icarus x Meranges, Girona Spain RVcoll.08-H592 hi1 x JN084744 P. icarus x Gualda, La Alcarria, Guadalajara Spain RVcoll.08-J962 hi25 x JN084723 P. icarus x Fuente Albergue de San Francisco, Güejar Sierra Spain RVcoll.08-J960 hi25 JN084689 x JN084722 P. icarus x Fuente Albergue de San Francisco, Güejar Sierra Spain RVcoll.09-V446 hi25 x JN084726 P. icarus x San Juan (Sierra Nevada), Granada Spain RVcoll.08-J968 hi25 x JN084724 P. icarus near Veleta, Monachil, Sierra Nevada, Granada Spain RVcoll.06-K697 hi7 P. icarus Guillimona, La Sagra, Huéscar, Granada Spain RVcoll.06-K698 x P. icarus Guillimona, La Sagra, Huéscar, Granada Spain RVcoll.09-V902 hi21 JN084699 x JN084728 P. icarus x Sierra de la Sagra, Granada Spain RVcoll.09-V941 hi21 x JN084741 P. icarus Sierra de la Sagra, Granada Spain RVcoll.09-V486 hi7 JN084707 x JN084735 P. icarus x Sierra de la Sagra, Granada Spain RVcoll.09-V488 hi7 x JN084727 P. icarus x Sierra de la Sagra, Granada Spain RVcoll.09-V905 hi7 P. icarus x Sierra de la Sagra, Granada Spain RVcoll.09-V907 hi7 x JN084729 P. icarus x Sierra de la Sagra, Granada Spain RVcoll.08-J899 hi1 P. icarus Vega de Espinareda, León Spain RVcoll.08-P416 hi6 JN084704 x JN084764 P. icarus x Burón, Picos de Europa, León Spain RVcoll.08-P653 hi7 P. icarus x Valle (Valle-Zurea), Lena, Asturias Spain RVcoll.08-R458 hi7 P. icarus x Padrón, Picarana-Rio Tinto, Galicia Spain RVcoll.08-R488 hi8 P. icarus x Tui, Rio Louro-Magdalena, Galicia Spain AY556949 hi4 P. icarus Ubierna, Burgos Spain MZUFsvez1 P. icarus x Sweden Sweden RVcoll.07-C177 hi16 JN084696 x JN084746 P. icarus x 13 km N. of Saimbeily, Adana Turkey RVcoll.07-C179 hi16 x JN084715 P. icarus x 13 km N. of Saimbeily, Adana Turkey RVcoll.07-F274 hi16 x JN084720 P. icarus x 13 km N. of Saimbeily, Adana Turkey RVcoll.07-C260 hi16 x JN084716 P. icarus x Lake Tuzla, Kayseri Turkey RVcoll.07-F178 hi16 x JN084736 P. icarus x Kiskaçli, Kayseri Turkey RVcoll.07-F225 hi16 P. icarus Ski resort, Erciyes Mountain, Kayseri Turkey AY556994 x P. icarus Dedegol Gecidi, Isparta Turkey MZUFtur6 P. icarus x Malatya Turkey MZUFtur7 P. icarus x Malatya Turkey MZUFtur8 P. icarus x Malatya Turkey MZUFtur9 P. icarus x Malatya Turkey MZUFtur10 P. icarus x Ak. Chehir Turkey RVcoll.09-V960 hi19 JN084697 x JN084731 P. icarus N Pirin Mt., Banderitsa nut-vihren hut road Bulgaria

15

Sample ID/GenBank

Accession number

COI

haplotype

GenBank

COI ITS2

GenBank

ITS2 Taxon

Morpho-

metry Locality data Country

RVcoll.09-V959 x JN084708 x JN084784 P. icarus N Pirin Mt., Banderitsa nut-vihren hut road Bulgaria

RVcoll.09-T515 hi1 JN084701 x JN084791

P. icarus x Bonifacio, Corsica France

RVcoll.09-T560 hi1 P. icarus Salerno, Capri island Italy RVcoll.09-T559 hi10 JN084706 x JN084740 P. icarus x Salerno, Capri island Italy RVcoll.09-T572 hi1 P. icarus x Monte Perone Elba island Italy

FJ428818 (ILL043) x x P. icarus

fuchsi Nov. Chara Russia

GU244491 (DB08003) x x P. icarus juno Mt. Hermon Israel

FJ428826 x P. icarus

zelleri Mount Tavor Israel

AY556864 (JC00061) hi11 x P. icarus

[andronicus] Mt. Falakro Greece

RVcoll.07-W163 x JN084686 x JN084739 P. eros Vallibierna, Huesca Spain

RVcoll.07-D815 x JN084685 x JN084711 P. eros Filettino, Monti Simbruini, Sky Resort, Frosinone, Lazio

Italy

RVcoll.09-X293 x JN084684 x JN084714 P. eros Simplon Pass, Valais Switzerland RVcoll.09-V961 x JN084683 x JN084712 P. eroides N Pirin Mt., Bunderitse nut-vihren hut road Bulgaria RVcoll.07-D671 x JN084682 x JN084710 P. eroides Rila mountains, Rila/Samokov Bulgaria RVcoll.09-X075 x JN084690 x JN084713 P. menelaos Mt. Taiyetos, Peloponnesos Greece