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, 370 05, Czech Republic 2 Faculty of Science, University of South Bohemia, Branisovska 31, Ceske Budejovice, 370 05, Czech Republic, Ceske Budejovice, Czech Republic 3 National Museum, Cirkusová 1740, 193 00 Praha 9, Czech Republic 4 Department of Animal Physiology, Institute of Zoology, Faculty of Biology, University of Warsaw, 1 Miecznikowa Str., 02-096 Warsaw, Poland These authors contributed equally to this work. *corresponding author Phone: (420) 387775239 E-mail: david.dolezel@entu.cas.cz
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 2010 20-30 animals 59.4160 27.5173 44 M 25.33 3.29 0.48 24.1 191 Stockholm #2 VI, 2010 20-30 animals 59.2437 18.1159 29 M 26.72 1.83 0.27 46.4 97 Stockholm #1 IV, 2010 20-30 animals 59.20 18.1500 27 M 27.88 1.70 0.28 64.2 56 Valka V, 2010 20-30 animals 57.7733 26.0150 54 M 26.95 3.59 0.95 26.9 52 Saule V, 2010 10 animals 57.7 25.92 68 M 26.73 2.09 0.39 33.7 86 Riga V, 2010 20-30 animals 57.0048 24.2911 10 M 27.25 1.06 0.26 31.4 54 Vilnius V, 2010 10 animals 54.6862 25.2798 105 M 26.47 1.13 0.18 44.9 78 Brok 2004 20-30 animals 52.6965 21.8471 105 M 25.24 1.31 0.23 71.7 46 F 24.95 2.70 0.53 33.8 77 Płock VI, 2014 20-30 animals 52.5463 19.7065 102 M 25.58 1.51 0.35 27.9 68 Berlin 2014 20-30 animals 52.4483 13.2736 45 M 24.5 2.19 0.61 20 65 Wegrow VI,2014 20-30 animals 52.3995 22.0163 120 M 25.79 1.93 0.39 40.7 59 Otterlo VI, 2012 10 animals 52.0827 5.83338 37 M 26.2 1.86 0.23 52.1 144 Slawa VII, 2014 20-30 animals 51.8759 16.0718 60 M 25.54 1.96 0.48 24.6 69 Orenburg 2014 10 animals 51.7538 55.1140 93 M 24 2.08 0.43 35.4 65 Skarzysko-Kamienna VI,2014 20-30 animals 51.1121 20.8475 250 M 23.45 2.19 0.78 13.8 58 Dresden 2014 20-30 animals 51.0586 13.7850 118 M 25.66 2.09 0.51 30.3 56 Koln VII, 2014 20-30 animals 50.9363 6.95277 59 M 25.8 1.72 0.42 32.1 53 Jena 2010 20-30 animals 50.9201 11.5900 146 M 25.28 1.24 0.22 59.6 57 Karpniki 2013 20-30 animals 50.86 15.87 460 M 24.71 2.12 0.39 57.7 52 Ksiaz 2012 10 animals 50.8420 16.2918 391 M 24.79 1.87 0.27 58.1 86 Horbach VII, 2014 15 animals 50.8324 6.05045 160 M 24.5 3.35 0.87 26.7 56 Zacler IV, 2010 20-30 animals 50.6629 15.9106 620 M 25.52 1.74 0.30 35.1 94 Dvur Kralove IV, 2010 15 animals 50.4230 15.8083 288 M 24.35 2.39 0.44 30.2 96 Keilberg VII, 2014 20-30 animals 49.9799 9.25538 168 M 24.5 1.48 0.45 20 55 Oldrichovec IV, 2010 50 animals 49.6004 14.6628 662 M 24.93 1.43 0.18 69.1 94 Kuncice pod Ondrejnikem 2012 20-30 animals 49.5353 18.3028 481 M 24.34 1.16 0.19 64.4 59 Chelcice 2006 100 animals 49.1217 14.1692 456 M 25.13 1.69 0.23 53.6 97 Chelcice-nd (non-diapause established from Chelcice in 40 th single pair -//- -//- -//- M 25.84 0.41 0.07 72.3 47 mutant strain) generation Nova Sedlica 2012 20-30 animals 49.04 22.51 389 M 24.7 1.03 0.20 53.5 48 Rudolfov 2015 500 animals 48.9937 14.5417 480 M 24.6 1.35 0.14 37.2 239 Gif-sur-Yvette VI, 2013 20-30 animals 48.7058 2.13225 84 M 24.84 1.79 0.28 42 100 Cabov VIII, 2014 20-30 animals 48.8047 21.6403 225 M 23.83 1.28 0.28 37.5 56 Kleinwetzdorf VIII, 2014 20-30 animals 48.4964 15.9535 224 M 24.25 1.38 0.28 32.5 77 Linz IV, 2014 20-30 animals 48.3069 14.2858 261 M 24.75 2.08 0.43 24.5 98 Measured individuals [n]
Bratislava VIII, 2014 20-30 animals 48.1559 17.0704 165 M 25.8 1.40 0.26 50.8 59 Pervomaisk IX, 2013 10 animals 48.0414 30.8590 75 M 23.45 1.82 0.24 48.3 118 Mliecany 2011 10-30 animals 47.9674 17.6169 112 M 23.26 2.23 0.26 61.8 123 Konstanz IV, 2012 20-30 animals 47.6545 9.17724 397 M 23.8 0.97 0.18 77.5 40 Sevelen IV, 2010 20-30 animals 47.1222 9.48988 460 M 25.19 1.66 0.31 45.9 61 Szeged 2011 20-30 animals 46.2511 20.1514 84 M 22.55 2.83 0.56 45.6 57 Roana 2003 15 animals 45.8753 11.4626 967.3 M 24.21 0.67 0.07 82.6 101 F 24.51 2.11 0.41 41.9 62 Zagreb IV, 2014 20-30 animals 45.813 15.9779 134 M 23.5 1.78 0.41 18.8 101 Lyon-red (wild type color) 1992 unknown 45.7 4.8 150 M 22.86 0.71 0.09 86.8 68 Lyon-yellow (body color mutant) established from Lyon- red in 131 th generation (100 generatioins prior measurments single pair 45.7 4.8 150 M 21.09 0.96 0.1 82.9 114 45.7 4.8 150 F 21.55 0.85 0.15 58.9 56 Padova X, 2014 60 animals 45.4099 11.8877 17 M 23.58 1.39 0.15 63.5 140 Novi Sad VI, 2011 20-30 animals 45.2615 19.8554 80 M 23.48 1.67 0.19 46 176 Deliblatska Pescara 2014 20-30 animals 44.8740 21.0928 164 M 23.66 2.20 0.73 17.3 52 Nikita IX, 2013 20-30 animals 44.5068 34.2343 58 M 23.39 1.30 0.19 49.5 97 Marseille VI, 2004 10 animals 43.2933 5.35988 12 M 24.58 2.83 0.63 20.3 69 Marseille-nd (non-diapause XI, 2004, established from Marseille single pair 43.2933 5.35988 12 M 23.88 1.16 0.34 20.7 59 mutant strain) in 5 th generation Villabona X, 2012 10 animals 43.1779-2.0514 126 M 23.84 0.83 0.08 76.2 139 Sofia 2011 10 animals 42.70 23.32 550 M 24.32 1.83 0.31 41.1 85 Cantalice, Rieti XII, 2012 10 animals 42.4672 12.9037 621 M 23.82 1.74 0.19 67.8 121 Lleida 2012 20-30 animals 41.6278 0.5973 187 M 24.83 3.61 0.90 25 64 Kozani VI, 2011 20-30 animals 40.3001 21.6634 688 M 23.44 1.97 0.18 58.7 213 Cordoba 2012 20-30 animals 37.8893-4.7977 124 M 25.22 1.64 0.25 57.5 73 Glyfada VIII, 2009 1male, 2 females 37.87948333 23.7684 94 M 23.65 2.91 0.54 20.7 140 Safed 2010 10 animals 32.9587 35.4946 715 M 25.72 1.47 0.21 55.4 83 F 25.92 2.72 0.66 27.9 61 Jerusalem 2010 10 animals 31.7637 35.2133 742 M 25.5 1.89 0.21 61.1 126 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: http://132.187.25.13/actogramj/manual.html):
Figure S1. Examples of representative actograms
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) = -0.083; p = 0.136 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) = -0.150; p = 0.237. Color codes for FRP.
Figure S4. High percentage of arrhythmic individuals (x-axis) correlates with larger SEM (yaxis): Pearson product-moment correlation coefficient (Pearson's r) = 0.7680535; 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.
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.