Molecular phylogeny of Megasternini terrestrial water scavenger beetles (Hydrophilidae) reveals repeated continental interchange during Paleocene-Eocene thermal maximum EMMANUEL ARRIAGA-VARELA, VÍT SÝKORA and MARTIN FIKÁČEK Supplementary file S3 Time tree analysis of the family Hydrophilidae Material and methods Taxonomic and molecular sampling We assembled a dataset for Hydrophilidae in order to enable secondary calibrations for the Sphaeridiinae part of the tree for which no fossils are known and hence no fossil calibration points are available. Since the subfamily Sphaeridiinae was underrepresented in previous studies (Short & Fikaček, 2013; Bloom et al., 2014; Toussaint & Short, 2018), our principal aim was to improve the sampling in this clade: we selected 62 species of the Megasternini originally sequenced for the Megasternini analysis, and newly sequenced 7 species of Coelostomatini and 3 species of Omicrini (Table 1 below). We used the same laboratory protocols as specified for the Megasternini-only dataset. These newly acquired data were combined with sequenced provided by Short & Fikaček (2013), Minoshima et al. (2018), Deler-Hernández et al. (2018), Toussaint & Short (2018), Fikáček et al. (2018) and Seidel et al. (2020). The final dataset includes 236 taxa sampled for the following gene fragments: nuclear protein-coding genes histone 3 (H3), arginine kinase (ArgK) and topoisomerase I (TopI), nuclear 18S and 28S rDNA, mitochondrial protein-coding cytochrome oxidase I (COI 3' region) and cytochrome oxidase II (COII), and mitochondrial 16S rDNA. We did not attempt to amplify arginine kinase (ArgK) used by Short & Fikaček (2013) and Toussaint & Short (2018) for the newly sequenced samples. Instead, we included the topoisomerase I (TopI) fragment to improve the topology resolution for the Megasternini clade. Since the inclusion of the non-hydrophilid outgroup taxa resulted in problems with the alignment of the ribosomal genes, we only included the representatives of the Hydrophilidae in the final dataset, and used the earliest branching clade (Hydrophilinae) to root the tree, following the results of Short & Fikáček (2013) and Toussaint & Short (2018). Table 1 summarizes the composition of the dataset. All newly generated sequences are deposited in GenBank. The sequences used for the analysis and their GenBank accession numbers are specified in Table 2 below. Table 1. Composition of the Hydrophilidae dataset used for the time tree analysis. Gene Length Spec. Spec. w/o Data fragment (bp) with data data coverage (%) 18S 1406 184 52 78.0 28S 1101 142 94 60.2 cox1 754 201 35 85.2 cox2 659 162 74 68.6 16S 825 67 169 28.4 H3 290 84 152 35.6 ArgK 705 64 172 27.1 topoI 627 48 188 20.3 Table 2. List of taxa included in the Hydrophilidae analysis with data on the available sequences and their GenBank accession numbers. ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI AES0004 Helocombus bifidus KC935272 KC992475 - KC935048 KC992576 KC935151 - - AES0007 Hybogralius hartmeyeri KC935277 - - KC935054 KC992582 KC935157 - - AES0013 Cymbiodyta semistriata KC935252 - - KC935029 KC992557 KC935136 - - AES0014 Ametor scabrosus KC935213 KC992499 - KC934988 KC992518 - - - AES0021 Limnoxenus semicylindricus KC935290 KC992496 KC992691 KC935070 KC992595 KC935172 - - AES0024 Limnohydrobius melaenus KC935280 KC992495 - KC935057 KC992585 - - - AES0025 Limnoxenus zealandicus KC935291 KC992494 - KC935071 KC992596 KC935173 - - AES0027 Limnoxenus niger KC935289 KC992493 - KC935069 KC992594 KC935171 - - AES0029 Hydrochara obtusata KC935281 - - KC935058 KC992586 KC935160 - - AES0030 Sperchopsis tessellata KC935322 KC992421 - KC935098 KC992631 KC935198 - - AES0032 Tropisternus affinis KC935329 - - KC935105 KC992636 KC935205 - - AES010 Limnocyclus punticeps KY554444 - - KY554460 KY554469 - - - COL119 Coelostomopsis picea KC935245 KC992507 - KC935022 KC992551 - - - COL1761 Adolopus helmsi MW649832 MW655902 - - MW648938 - - - COL1767 Cyloma pictus MW649833 MW655903 - - - - - - COL1772 Adolopus badius MW649834 MW655904 - - MW648939 - - - COL1775 Adolopus versicolor MW649835 MW655905 - - MW648940 - - - COL1777 Hydrostygnus frontalis - MW655906 - MW649987 MW648941 - MW655960 MW655960 COL1778 Cyloma stewarti MW649836 MW655907 - - MW648942 - - - COL1786 Saphydrus suffusus MW649837 MW655908 - - MW648943 - MW655961 MW655961 COL1789 Cyloma stewarti MW649838 MW655909 - - MW648944 - - - COL1791 Exydrus gibbosus MW649839 MW655910 - - MW648945 - - - COL1794 Rygmodus cyaneus MG920289 MW655911 - - - - MG920309 - COL1803 Adolopus nonstriatus MW649840 MW655912 - - MW648946 - - - COL1806 Tormissus linsi MW649841 MW655913 - - MW648947 - MW655962 MW655962 COL1841 Rygmodus modestus MG920292 MW655914 - MW649988 MW648948 - MG920315 - COL1845 Rygmodus tibialis MG920294 MW655915 - MW649989 MW648949 - MG920300 - COL2028 Eurygmus helocharoides - MW655916 - - MW648950 - MW655963 MW655963 COL481 Coelostoma fabrici MW649828 - - MW649985 MW648934 - - - COL666 Borborophorus tuberculus KC935232 KC992409 - KC935009 - - - - COL800 Rygmostralia sp. MW649829 MW655899 - - MW648935 - MW655957 MW655957 COL803 Pseudohydrobius flavus MW649830 MW655900 - - MW648936 - MW655958 MW655958 COL992 Dactylosternum marginale KF802003 - - KF801837 KF802166 - - - COL993 Microgioton huonus MW649831 MW655901 - MW649986 MW648937 - MW655959 MW655959 ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI CUB572 Sacosternum sp. MW593941 - - MW598562 MW598752 - MW625386 - D30 Chimaerocyon shimadai MW593957 MW655917 - MW598613 MW648951 - MW655964 MW655964 GenBank Anacaena globulus AM287086 AM287108 AM287064 AM287125 AM287133 - - - GenBank Anacaena limbata DQ155734 - - AY745586 JX173143 - - - GenBank Berosus luridus AM287087 AM287109 AM287065 AJ810721 AJ810756 - - - GenBank Cercyon ustulatus DQ155947 AM287115 AM287071 AM287129 AM287137 - - - GenBank Coelostoma orbiculare AM287094 AM287116 - EF213785 KC992549 KC935131 - - GenBank Enochrus quadripunctatus AM287090 AM287112 AM287068 AM287127 AM287135 - - - GenBank Enochrus testaceus AM287089 AM287111 AM287067 AJ810719 AJ810754 - - - GenBank Hydrocassis sp. KF128921 KF131553 - KF131613 KF131633 - - - GenBank Hydrophilus acuminatus KF128914 KF131546 - KF131606 KF131626 - - - GenBank Limnoxenus namalokama KY554445 - - KY554462 KY554471 - - - GenBank Limnoxenus waialeale KY554448 - - KY554465 KY554473 KY554455 - - MF1013 Phaenonotum laterale - - - - MG208614 - - - MF1025 Pelosoma sp. (ECU) MW649864 MW655944 - MW650009 MW648971 - MW655977 MW655986 MF1061 Phaenonotum sp. MG208567 MG208592 - MG208656 MG208615 - - - MF1062 Phaenonotum exstriatum - - - - MG208616 - - - MF1065 Phaenonotum sp. MG208570 MG208595 - - - - - - MF1066 Phaenostoma posticatus MG208571 MG208596 - MG208661 MG208619 - - - MF1094 Cercillum sp. indesct. MW593995 MW655945 - MW598663 MW598849 - MW625459 MW625749 MF1098 Cercyon sp. (RSA) MW593996 MW655946 - MW598664 MW598850 - MW625460 MW625750 MF1115 Phaenonotum sp. (CUB) MG208597 MG208572 - MG208662 MG208620 - - - MF1158 Peltocercyon sp. 2 MW649865 - - MW650010 MW648972 - MW655978 MW655987 MF1178 Cercyon ca. alinae (CHN) MW649866 - - MW650011 MW648973 - MW655979 MW655988 MF1189_2 Cercyon ca. nigriceps MW649867 - - MW650012 MW648974 - - MW655989 MF1195 Cercyon ca. udus (INDO) MW593999 MW655947 - MW598667 MW598853 - - MW625753 MF1226 Pseudoosternum maculatum MW593952 MW655948 MW648984 MW598599 MW598789 - - MW625698 MF1269 Chledocyon semiopacus MW593953 MW655949 - MW598601 MW598791 - MW625411 MW625699 MF1283 Cercyon sp. (AUS) MW594005 - - MW598675 MW598860 - MW625469 MW625757 MF1291 Cercyodes kingensis - - - MW598606 MW598796 - MW625416 MW625702 MF1308 Cercyon undulipennis MW594010 - MW648985 MW598683 MW598868 - MW625475 MW625762 MF1322 Cercyon sp. 2 (RSA) MW594013 MW655950 - MW598692 MW598877 - MW625483 MW625769 (Arcocercyon) MF1324 Cercyon sp. 3 (RSA) MW594014 - - MW598693 MW598878 - MW625484 MW625770 MF1331 Cercyon sp. 4 (RSA) MW594017 - - MW598696 MW598882 - MW625488 MW625774 MF1339 Pelosoma sp. MW594020 - - MW598700 MW598886 - MW625492 MW625777 ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI MF135 Psalitrus sp. MW649842 - - MW649991 MW648952 - - - MF136 Coelostoma sp. MW649843 MW655918 - MW649992 MW648953 - - - (Lachnocoelostoma) MF1388 Psalitrus sp. MW649868 - - MW650013 MW648975 - MW655980 MW655980 MF139 Peltocercyon sp. MW593958 - - MW598618 MW598805 - MW625423 MW625708 MF1484 Cyrtonion sp. MW649869 - - MW650014 MW648976 - MW655981 MW655990 MF1497 Tectosternum - MW594021 MW655951 - MW598701 MW598887 - MW625493 MW625778 MF158 Coelostoma lemuriense MW649844 MW655919 - MW649993 MW648954 - - - (Lachnocoelostoma) MF1620 Ceronocyton sp. - - - MW598598 MW598788 - MW625410 MW625697 MF1627 Bolbonotum sp. - - - MW598708 MW598894 - MW625497 MW625782 MF1632 Nitidulodes ca. jansoni Sharp - MW655952 - MW598711 MW598896 - MW625499 MW625784 MF1644 Pelosoma sp. - MW655953 - MW598718 MW598902 - MW625505 MW625787 MF1727_2 Relictorygmus trevornoahi MW649870 MW655954 - MW653257, - - - - MW653259 MF183 Cercyon sp. (femoral lines) MW649845 - - MW649994 MW648955 - - - MF188 Cercyon sp. lutosus group MW593961 - - MW598622 MW598808 - MW625426 MW625709 (Arcocercyon) MF198 Grodum sp. MW649846 MW655920 - MW649995 - - - - MF200 Cercyon sp. (CHN) MW593962 MW655921 - MW598623 MW598809 - - MW625710 MF204 Noteropagus sp. MH910640 - - - - - - - MF205 Pacrillum sp. MW593963 KC992489 - MW598625 MW598811 - MW625428 MW625712 MF206 Australocyon ca. puncticollis MW593955 - - MW598608 MW598799 - MW625419 MW625703 MF2073 Megasternum sp. MW649871 - - - - - MW655982 MW655982 MF2075 Agna zaragozai