Unexpected geographic variability of the free running period in the linden bug, Pyrrhocoris apterus

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1 Supplementary Online Material: Unexpected geographic variability of the free running period in the linden bug, Pyrrhocoris apterus Lenka Pivarciova 1,2,, Hanka Vaneckova 1,, Jan Provaznik 1,2, Bulah Chia-hsiang Wu 1,2, Martin Pivarci 1,2, Olga Peckova 1, Olga Bazalova 1,2, Stepan Cada 1, Petr Kment 3, Joanna Kotwica-Rolinska 1,4 and David Dolezel 1,2* 1 Institute of Entomology, Biology Centre of Academy of Sciences of the Czech Republic, Branisovska 31, Ceske Budejovice, , Czech Republic 2 Faculty of Science, University of South Bohemia, Branisovska 31, Ceske Budejovice, , Czech Republic, Ceske Budejovice, Czech Republic 3 National Museum, Cirkusová 1740, Praha 9, Czech Republic 4 Department of Animal Physiology, Institute of Zoology, Faculty of Biology, University of Warsaw, 1 Miecznikowa Str., Warsaw, Poland These authors contributed equally to this work. *corresponding author Phone: (420) david.dolezel@entu.cas.cz

2 Table S1. Field-lines used in this study Field-line Collection [month, year] Original size for establishing the line Latitude Longitude Altitude [m.a.s.l.] Sex measured [M, F] FRP mean [h] SD [h] SEM [h] Rhythmic individuals [%] Toila animals M Stockholm #2 VI, animals M Stockholm #1 IV, animals M Valka V, animals M Saule V, animals M Riga V, animals M Vilnius V, animals M Brok animals M F Płock VI, animals M Berlin animals M Wegrow VI, animals M Otterlo VI, animals M Slawa VII, animals M Orenburg animals M Skarzysko-Kamienna VI, animals M Dresden animals M Koln VII, animals M Jena animals M Karpniki animals M Ksiaz animals M Horbach VII, animals M Zacler IV, animals M Dvur Kralove IV, animals M Keilberg VII, animals M Oldrichovec IV, animals M Kuncice pod Ondrejnikem animals M Chelcice animals M Chelcice-nd (non-diapause established from Chelcice in 40 th single pair -//- -//- -//- M mutant strain) generation Nova Sedlica animals M Rudolfov animals M Gif-sur-Yvette VI, animals M Cabov VIII, animals M Kleinwetzdorf VIII, animals M Linz IV, animals M Measured individuals [n]

3 Bratislava VIII, animals M Pervomaisk IX, animals M Mliecany animals M Konstanz IV, animals M Sevelen IV, animals M Szeged animals M Roana animals M F Zagreb IV, animals M Lyon-red (wild type color) 1992 unknown M Lyon-yellow (body color mutant) established from Lyon- red in 131 th generation (100 generatioins prior measurments single pair M F Padova X, animals M Novi Sad VI, animals M Deliblatska Pescara animals M Nikita IX, animals M Marseille VI, animals M Marseille-nd (non-diapause XI, 2004, established from Marseille single pair M mutant strain) in 5 th generation Villabona X, animals M Sofia animals M Cantalice, Rieti XII, animals M Lleida animals M Kozani VI, animals M Cordoba animals M Glyfada VIII, male, 2 females M Safed animals M F Jerusalem animals M Insects we considered as rhythmic, if the the PN was 15 (Lomb-Scargle periodogram analysis in ActogramJ). PN value is determined automatically in ActogramJ software by following formula (see also ActoJ manual:

4 Figure S1. Examples of representative actograms

5 Figure S2. The free running period (y-axis) is independent on the percentage of rhythmic bugs (x-axis): Pearson product-moment correlation coefficient (Pearson's r) = ; p = Figure S3. The number of rhythmic individuals in field-lines (x-axis) is independent on the latitude of their origin: Pearson product-moment correlation coefficient (Pearson's r) = ; p = Color codes for FRP.

6 Figure S4. High percentage of arrhythmic individuals (x-axis) correlates with larger SEM (yaxis): Pearson product-moment correlation coefficient (Pearson's r) = ; p < 10-6 Figure S5. Comparison of the FRP between males and females. Statistic analysis revelaed that the FRP of males and females did not differ in Brok, Roana and Safed field-lines. Only Lyon-yellow males and females possessed significantly different FRPs.

7 Figure S6. Phylogenetic analysis of selected field-lines. Circa 1kb intronic region of period gene was PCR amplified and directly sequenced using primers published previously (Dolezel et al., 2005). RAxML tree was constructed in GENEious 8.1 program (Biometters, Ltd). Numbers under branch nodes indicate bootstrap values in % (based on 1000 replicates). Figure S7. The free running period (y-axis) plotted over the lattitude (x-axis). Color of dots corresponds to the phylogeographic cluster of particular population. FRP correlates with the latitude: Pearson product-moment correlation coefficient (Pearson's r) = 0.508; p < 10-3 (for all field-lines), Pearson's r = 0.763; p < 10-8 (for only diapause-entering field-lines, when ndlines were omitted from the analysis). Genetic clustering is based on the phylogenetic analysis shown in the Figure S6.