Molecular phylogeny of terrestrial water scavenger () 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 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 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 sp. MW649871 - - - - - MW655982 MW655982 MF2075 Agna zaragozai MW594030 MW655955 - MW598734 MW598918 - MW625521 MW625798 MF223 Peratogonus reversus KC935314 KC992408 - - KC992621 - - - MF224 gr. soricoides KC935306 KC992513 - MW598570 MW598761 - MW625392 - MF226 Cercyon sp. 2 (CHN) MW649847 - - MW649996 MW648956 - MW655966 MW655983 MF247 Chaetarthria sp. KC935237 KC992478 - -- KC992543 - - - MF249 Psalitrus sp. KC935316 KC992410 - KC935092 KC992625 - MW655967 MW655967 MF25 Dactylosternum sp.3 - - - MW649990 - - - - MF257 Anacaena lanzhujii KC935218 KC992455 - KC935002 KC992523 - - - MF265 Dactylosternum hydrophiloides MW649848 MW655922 ------MF283 Pachysternum apicatum MW593965 MW655923 - MW598627 MW598813 - - MW625714 MF284 Omicrogiton insularis KC935295 - - - KC992602 - MW655968 MW655968 MF285 Cercyon sp. 3 (CHN) MW649849 MW655924 - MW653258, MW648957 - MW655969 MW655984 MW653260 ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI MF288 Cercyon gr. nigriceps MW593967 - - MW598629 MW598815 - - MW625716 MF294 Cercyon sp. 4 (CHN) MW593968 MW655925 - MW598630 MW598816 - MW625431 MW625717 MF326 Coelostoma phallicum MW593938 KC992398 - MW598561 MW598749 - MW625383 - MF36 Phaenonotum caribense KC935315 KC992399 - - KC992623 - - - MF37 Phaenostoma sp. MG208550 - - MG208639 MG208598 - - - MF436 pygmaeum - MW655926 - MW598634 MW598820 - MW625434 MW625720 MF437 Peltocercyon sp. 2 MW593970 - - MW598635 MW598821 - MW625435 MW625721 MF455 Phaenonotum delgadoi MG208552 MG208583 - MG208641 MG208600 - - - MF457 Dactylosternum indicum MW649850 MW655927 - MW649997 MW648958 - - - MF461 Gillisius sp. MW593971 - - MW598637 MW598823 - MW625436 MW625723 MF472 Cercyon sp. (LAO) - - - MW649998 MW648959 - MW655970 MW655985 MF473 Cercyon sp. 1 (LAO) MW593976 - - MW598642 - - - MW625728 MF488 Coelostoma stultum MW649851 - - MW649999 MW648960 - - - MF49 Chaetarthria seminulum KC935238 KC992503 - KC935015 KC992542 - - - MF515 Cylomissus sp. - MW655928 - - MW648961 - MW655971 MW655971 MF530 Dactylosternum abdominale MW649852 MW655929 - MW650000 - - - - MF540 Austrotypus nothofagi MW649853 MW655930 - - MW648962 - MW655972 MW655972 MF576 Cercyon sp. 5 (CHN) MW593980 MW655931 - MW598646 MW598830 - MW625443 MW625732 MF599 Aculomicrus sp. MW649854 MW655932 - MW650001 - - - - MF63 Dactylosternum auripes KC935253 - - - KC992558 - - - MF656 Sphaerocetum arboreum KM874806 - - KM874807 KM874809 - - - MF665 Cylominae sp. MW649855 ------MF680 Cercyon haemorrhoidalis MW593982 MW655933 - MW598649 MW598833 - MW625446 MW625735 MF74 Aculomicrus reticulatus - KC992396 - - KC992514 - MW655965 MW655965 MF771 Cercyon laminatus MW593983 MW655934 - MW598650 MW598834 - MW625447 MW625736 MF772 Lachnodacnum luederwaldti MG208554 - - MG208643 MG208602 - - - MF790 Cylorygmus lineatopunctatus MW649856 MW655935 - - MW648963 - MW655973 MW655973 MF791 Pseudorygmodus versicolor KM262052 KM262053 - KM262050 KM262051 - - - MF792 Andotypus ashworthi MW649857 - - - MW648964 - MW655974 MW655974 MF793 Anticura flinti KM262054 - - KT447582 MW648965 - MW655975 MW655975 MF811 Dactylosternum dytiscoides MW649858 MW655936 - MW650002 MW648966 - - - MF817 Merosoma sp. MW593945 MW655937 - MW598585 MW598775 - MW625399 MW625689 MF818 Paromicrus sp. MW649859 - - MW650003 MW648967 - MW655976 MW655976 MF821 Cercyon sp. (SOL IS) MW593984 - - MW598651 MW598835 - MW625448 MW625737 MF829 Cercyon sp. (PER) MW593985 MW655938 MW648981 MW598653 MW598837 - MW625449 - MF830 Motonerus inca MW593943 MW655939 - MW598565 MW598755 - - MW625679 ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI MF831 Cercyon sp. 2 (PER) MW593986 - MW648982 MW598654 MW598838 - MW625450 MW625738 MF833 Pelosoma sp. (PER) MW649860 - - MW650004 MW648968 - - - MF834 Oosternum sp. (PER) MW593944 - - MW598571 MW598762 - MW625393 MW625683 MF837 Cycrillum sp. - - - - MW598841 - MW625452 MW625741 MF842 Cyclotypus sp. MG208557 MG208585 - MG208646 MG208605 - - - MF846 Phaenonotum sp. MG208560 MG208587 - MG208649 MG208608 - - - MF852 Pelosoma sp. (PER) MW593988 MW655940 MW648983 MW598657 MW598842 - MW625453 MW625742 MF855 Phaenostoma cf. kontax MW649861 - - MW650005 MW648969 - - - MF856 Phaenonotum sp. MW649862 MW655941 - MW650006 - - - - MF861 Phaenonotum sp. MW649863 MW655942 - MW650007 - - - - MF896 Cycreon floricola MW593989 MW655943 - MW650008 MW598843 - MW625454 MW625743 MF964 Cercyon convexiusculus MW593990 - - MW598658 MW598844 - MW625455 MW625744 MF980 Phaenonotum ondreji - - - - MW648970 - - - MSC1815 Crenitulus solstitialis KC935223 - KC992647 KC934998 KC992528 KC935119 - - MSC1816 Crenitulus suturalis KC935226 - KC992649 KC935001 KC992531 KC935120 - - MSC1818 Anacaena parvula KC935222 - - KC934997 KC992527 KC935118 - - MSC1820 Notionotus liparus KC935293 -- - KC935073 KC992598 KC935174 -- - MSC1821 Horelophopsis hanseni KC935274 -- - KC935051 KC992579 KC935154 -- - SLE0004 Oocyclus madidus KC935298 KC992435 - KC935077 KC992604 KC935177 - - SLE0005 Oocyclus petra KC935300 KC992442 - KC935078 KC992606 KC935179 - - SLE0008 Tropisternus collaris KC935330 KC992419 KC992714 KC935106 KC992637 KC935206 - - SLE0014 Berosus garciai KC935231 KC992441 KC992652 KC935007 KC992536 - - - SLE0015 Laccobius reflexipennis KC935287 KC992439 KC992689 KC935067 KC992592 KC935169 - - SLE0016 Regimbartia attenuata KC935317 KC992423 KC992704 KC935093 KC992626 KC935192 - - SLE0017 Sternolophus marginicollis KC935325 KC992416 KC992711 KC935101 KC992632 KC935202 - - SLE0020 Cercyon variegatus KC935234 KC992484 - KC935011 KC992539 - - - SLE0024 Guyanobius lacuniventris KC935267 KC992406 KC992676 KC935043 KC992571 KC935148 - - SLE0025 Enochrus mastersii KC935259 KC992472 - KC935035 - - - - SLE0027 Globulosis sp. KC935266 KC992433 - KC935042 KC992570 KC935147 - - SLE0030 Oocyclus sapphirus KC935302 KC992444 - - KC992608 KC935181 - - SLE0031 Helobata sp. KC935269 KC992453 KC992678 KC935045 KC992573 KC935149 - - SLE0032 Pelthydrus minutus gr. KC935313 KC992422 - KC935087 KC992620 - - - SLE0033 Oocyclus pico KC935301 KC992447 - - KC992607 KC935180 - - SLE0034 Helochares sp. KC935270 KC992471 - KC935046 KC992574 KC935150 - - SLE0035 Tobochares sulcatus KC935327 KC992465 KC992713 KC935103 KC992634 KC935204 - - SLE0037 Oocyclus trujillo KC935305 KC992448 KC992699 - KC992611 KC935184 - - ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI SLE0040 Paracymus limbatus KC935310 KC992425 KC992701 KC935083 KC992616 KC935186 - - SLE0041 Derallus sp. KC935256 KC992411 KC992666 KC93503 KC992561 KC935139 - - SLE0044 Paracymus pygmaeus KC935311 KC992473 KC992702 KC935084 KC992617 KC935187 - - SLE0046 Amphiops mater KC935214 KC992462 - KC934989 KC992519 KC935113 - - SLE0048 Enochrus talamanca KC935263 KC992486 KC992672 KC935039 KC992567 KC935144 - - SLE0049 Oocyclus funestus KC935297 KC992445 KC992693 KC935076 KC992603 KC935176 - - SLE0050 Oocyclus ornatus KC935299 KC992446 KC992694 - KC992605 KC935178 - - SLE0051 Hemiosus selva - KC992412 KC992680 KC935049 KC992577 KC935152 - - SLE0057 Laccobius serratus KC935288 KC992450 KC992690 KC935068 KC992593 KC935170 - - SLE0058 Cryptopleurum minutum KC935248 KC992491 - KC935024 KC992553 KC935132 - - SLE0063 Paroosternum saundersi - - - KC935086 KC992619 KC935189 - - SLE0066 Pachysternum nigrovittatum KC935308 KC992492 - KC935081 KC992614 - - - SLE0067 Hydrophilomima sp. - KC992438 KC992686 KC935063 KC992589 KC935164 - - SLE0070 Chaetarthria incisa - KC992479 KC992655 KC935014 KC992541 KC935126 - - SLE0071 Hydrobiomorpha spinicollis gr. KC992583 KC992418 - KC935055 KC992583 - - - SLE0073 Arabhydrus galagheri KC935227 KC992449 KC992650 KC935003 KC992532 - - - SLE0074 Hydrophilus albipes - - - KC935064 KC992590 - - - SLE0076 Tylomicrus costatus KC935332 KC992426 - - KC992639 KC935208 - - SLE0077 Chasmogenus sp. KC935241 KC992468 KC992658 - KC992546 KC935128 - - SLE0078 Chasmogenus sp. KC935242 KC992470 KC992659 KC935018 KC992547 KC935129 - - SLE0079 Chasmogenus ruidus KC935240 KC992469 KC992657 KC935017 KC992545 KC935127 - - SLE0080 Chasmogenus sp. KC935243 KC992466 - KC935019 KC992548 KC935130 - - SLE0082 Allocotocerus yalumbaboothbyi - KC992402 KC992641 KC934986 KC992517 KC935111 - - SLE0083 Enochrus cinctus KC935257 KC992464 KC992667 - KC992562 KC935140 - - SLE0084 Enochrus fimbriatus KC935258 KC992456 KC992668 KC935034 KC992563 KC935141 - - SLE0085 Oocyclus sp. KC935303 KC992436 KC992698 - KC992609 KC935182 - - SLE0087 Berosus ornaticollis - KC992424 KC992653 KC935008 KC992537 KC935123 - - SLE0091 Scoliopsis sp. KC935321 KC992477 KC992708 KC935097 KC992630 KC935196 - - SLE0092 Notionotus sp. KC935294 KC992508 KC992692 KC935074 KC992599 KC935175 - - SLE0093 Opthalmocyclus sp. KC935307 - KC992700 KC935080 KC992613 KC992613 MH317831 - SLE0094 Cercyodes laevigatus KC935233 KC992504 - KC935010 KC992538 KC935124 - - SLE0095 Cyloma sp. KC935249 - KC992661 KC935026 KC992554 KC935133 - - SLE0096 Ametor latus KC935212 KC992498 - KC934987 - KC935112 - - SLE0101 Tobochares sp.15a - KC992474 KC992684 KC935061 KC992600 - - - SLE0102 Tobochares sp. 10 KC935283 KC992487 KC992685 KC935060 KC992601 KC935162 - - SLE0107 Cyloma sp. KC935250 KC992506 KC992662 KC935027 KC992555 KC935134 - - ID Genus Species COI COII 16S 18S 28S ArgK H3 TopoI SLE0126 Destostethus scitus KC935255 KC992480 KC992665 KC935032 KC992560 KC935138 - - SLE0127 Thysanarthria sp. KC935326 KC992432 KC992712 KC935102 KC992633 KC935203 - - SLE0128 Moraphilus sp. KC935292 KC992457 - KC935072 KC992597 - - - SLE0129 Rygmodus sp. KC935318 KC992505 KC992705 KC935094 KC992627 KC935193 - - SLE0130 Nanosaphes tricolor KC935282 KC992488 - KC935062 KC992588 KC935163 - - SLE0132 Horelophus walkeri KC935275 KC992430 - KC935052 KC992580 KC935155 - - SLE0240 Helochares sp. KC935271 KC992460 - KC935047 - - - - SLE0241 Enochrus sp. KC935262 KC992485 KC992671 KC935038 KC992566 KC93514 - - SLE0242 Paranacaena lindi KC935220 KC992502 - KC934995 KC992525 - - - SLE0244 Tropisternus lateralis KC935331 KC992417 KC992715 KC935107 KC992638 KC935207 - - SLE0246 Omniops sp. - KC992500 - KC934996 KC992526 KC935114 - - SLE0247 Paranacaena lucida KC935221 - KC992646 KC934992 - KC935117 - - SLE0248 Enochrus ochraceus KC935260 KC992481 KC992669 KC935036 KC992564 - - - SLE0251 Cetiocyon incantatus MW593942 KC992454 - MW598564 MW598754 KC935125 - - SLE0252 Agraphydrus sp. KC935211 - KC992640 KC934985 - KC935110 - - SLE0295 Tormus sp. KC935328 - - KC935104 KC992635 - - - SLE0297 Protosternum sp. - KC992403 KC992703 KC935091 KC992624 KC935191 - - SLE0298 Sphaeridium bipustulatum KC935323 KC992458 - KC935099 - KC935200 - - SLE0301 Sphaeridium lunatum KC935324 - - KC935100 - KC935201 - - SLE0346 Crenitulus sp. - - KC992648 - - - - - SLE0369 Crenitulus hirsutus KC935217 KC992482 KC992644 KC934993 KC992522 - - - SLE0407 Amphiops sp. KC935215 KC992461 KC992642 KC934990 KC992520 - - - SLE0408 Amphiops sp. KC935216 KC992463 KC992643 KC934991 KC992521 - - - SLE0409 Enochrus piceus KC935261 KC992428 KC992670 KC935037 KC992565 - - - SLE0411 Guyanobius sp. KC992428 KC992670 KC935037 KC992565 KC992428 - - - SLE0872 Tritonus crenulatus KU711849 - - - KU711853 - - - Phylogenetic analyses

Sequences were edited and assembled and aligned in Geneious software (Kearse et al., 2012) using ClustalW algorithm with default settings. The final dataset of the total length of 6,366 bp is composed of 8 genes: 18S (1,406 bp), 28S (1,101 bp), COI (753 bp), COII (659 bp), 16S (825 bp), H3 (290 bp), ArgK (705 bp) and TopI (627 bp). An independent partition was set for each ribosomal gene and for each codon position of the protein coding genes. Best-fitting models of substitution were selected using PartitionFinder 2 (Lanfear et al., 2016). The concatenated dataset was analysed using MrBayes 3.2.6 (Ronquist et al., 2012) implemented on the CIPRES Science Gateway 3.3 (Miller et al., 2010) using independent parameters for each partition. Four runs with six chains each were run for 25 million generations, with trees being sampled every 12,500 generation. The convergence of the runs and their parameters were assessed in Tracer 1.7 (Rambaut et al., 2018). A 25% burn-in fraction was applied to the data.

Divergence dating

The divergence dating analysis was carried out in BEAST 2.4.7. We linked the substitution models in five partitions corresponding to mitochondrial ribosomal (16S), mitochondrial protein coding (COI+COII), nuclear protein coding (H3+ArgK+TopI), and nuclear ribosomal genes (18S+28S). A lognormal relaxed clock model was applied to all five partitions, with clock rates left with uniform priors. The birth-death model was used for the tree prior. A set of 8 fossils was used for calibrations (Table 2). Calibration points largely agree with those used by Toussaint & Short (2018), but are modified to include the findings by Fikáček et al. (2017) and Arriaga-Varela et al. (2019): Cercyon sp. is removed, and Crenitis profechuyi and Cretocrenis burmanicus are included to date the minimum age of the MRCA of Crenitulus + Horelophus and the stem age of Anacaenini, respectively. We used exponential priors with minimum age of each fossil calibration based on the age of given fossil and maximum age based on the maximum age estimate of the Hydrophiloidea (273 Ma) by Toussaint & Short (2018) (Table 2). We ran pre-burnin for 100 million generations followed by the MCMC chain set to 250 million generations, logging parameters every 12,500 generations. The convergence of all parameters was checked in Tracer 1.6., and the consensus tree was obtained by Tree Annotator 2.4.7 and visualized in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Table 2. Fossil taxa used for time-calibration of nodes and the settings of their truncated exponential priors.

Results

Phylogeny

The resulting topology inferred under BI agrees with those obtained by Short & Fikacek (2013) and Toussaint & Short (2018) in the support for the monophyly of all subfamilies and tribes (see Fig. 1 for the complete tree). Posterior probability values (pp) for the basal bifurcations are 1.0, except for the position of Acidocerinae as sister of the terrestrial lineage (Cylominae+Sphaeridiinae) (pp = 0.75). The internal topology in Megasternini tribe supports the division of the clade in two groups corresponding to the “Oosternum” and “Cercyon” groups of genera of Fikaček (2010) and Fikaček & Short (2010). The genus Cercyon Leach is found to be polyphyletic, recovered in the cladogram in more than 10 different positions. Parallely, Oosternum, the second most diverse genus in the tribe Megasternini was also found to be polyphyletic.

Divergence dating

The divergence dating resulted in a crown age for the family Hydrophilidae of 181.1 mya (162.2– 204.9) (Fig. 2). Our results stand between the crown-ages of the family given by Bloom et al. (2014) (205 mya) and Toussaint & Short (2018) (170 mya). We obtained similarly intermediate results for the crown-age of the terrestrial lineage (Cylominae + Sphaeridiinae), 162 mya (143.8– 183.4). Conversely, the age of crown Sphaeridiinae, 159.1 mya (140.0–179.0), is in closer agreement with that of Bloom et al. (2014) (161 mya) than to that of Toussaint & Short (2018) (137.4 mya). The age of the MRCA of Sphaeridiini+Megasternini was estimated 134.1 mya (105.7– 189.2), while the age of the MRCA of Megasternini is recovered to be 120.3 mya (93.2–177.4). The age of the MRCA of Megasternini was estimated to be 100.4 mya by Bloom et al (2014) and 80.0 by Toussaint & Short (2018). The older age estimates for Megasternini divergence events in our tree compared to those of Bloom et al. (2014) and Toussaint & Short (2018) is likely the result of the updated set of fossils used as calibration points, specifically the removal of the Baltic amber fossil identified previously as Cercyon sp. which age (44 mya) is significantly younger than all available age estimates for the Megasternini (Arriaga-Varela et al., 2020). Similarly, the MRCA of Crenitulus and Horelophus, calibrated using †Crenitulus palaeodominicus from Dominican amber (Fikáček & Engel, 2011) by Toussaint & Short (2018) and Bloom et al. (2014) was now dated using an older fossil, †Crenitis profechuyi, from Baltic amber (44 mya). Our results correspond with the finding of Toussaint et al. (2016) that the age estimates are strongly influenced by the selection of fossils used for the calibration. Another reason for older age estimates of the Megasternini clade may be the significantly improved taxon sampling: the analyses of Bloom et al. (2014) and Toussaint & Short (2018) only contained 16 taxa of the Megasternini, our analysis contains 62 taxa coverning all principal clades recognized in the main Megasternini analysis.

References

Arriaga-Varela, E., Brunke, A., Girón, J.C., Szawaryn, K., Bruthansová, J. & Fikáček, M. (2019) Micro-CT reveals hidden morphology and clarifies the phylogenetic position of Baltic amber water scavenger beetles (Coleoptera: Hydrophilidae). Historical Biology, DOI:10.1080/08912963.2019.1699921 Bloom, D.D., Fikáček, M. & Short, A.E.Z. (2014) Clade age and diversification rate variation explain disparity in species richness among water scavenger (Hydrophilidae) lineages. PLoS ONE, 9, 6, e98430. https://doi.org/10.1371/journal.pone.0098430 Deler-Hernández, A., Sýkora, V., Seidel, M., Cala-Riquelme, F. & Fikáček, M. (2017) Multiple origins of the Phaenonotum beetles in the Greater Antilles (Coleoptera: Hydrophilidae): phylogeny, biogeography and systematics. Zoological Journal of the Linnean Society, 183, 1, 97–120. https://doi.org/10.1093/zoolinnean/zlx071 Fikáček, M. (2010) Hydrophilidae: The genus Kanala Balfour-Browne (Coleoptera). In: Jäch, M.A. & Balke, M. (eds) Water Beetles of New Caledonia, Vol. 1. Monographs of Coleoptera, 3, 365– 394. Fikáček, M. & Engel, M.S. (2011) An aquatic water scavenger beetle in Early Miocene amber from the Dominican Republic (Coleoptera: Hydrophilidae). Annales Zoologici, 61, 4, 621–628. https://doi.org/10.3161/000345411X622462 Fikáček, M., Liang, W.-R., Hsiao Y., Jia F.-L. and Vondráček, D. 2018. Biology and morphology of immature stages of banana-associated Protosternum beetles, with comments on the status of Taiwanese endemic P. abnormale (Coleoptera: Hydrophilidae). Zoologischer Anzeiger, 277, 85– 100. https://doi.org/10.1016/j.jcz.2018.10.001 Fikáček, M. & Short, A.E.Z. (2010) A revision of the Neotropical genus Sacosternum Hansen (Coleoptera: Hydrophilidae: Sphaeridiinae). Zootaxa, 2538, 1–37. https://doi.org/10.11646/zootaxa.2538.1.1 Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C. & Thierer, T. (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 12, 1647–1649. https://doi.org/10.1093/bioinformatics/bts199 Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T. & Calcott, B. (2016) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution, 34, 3, 772–773. https://doi.org/10.1093/molbev/msw260 Miller, M.A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE), New Orleans, LA, 2010, pp. 1–8. http://dx.doi.org/10.1109/GCE.2010.5676129 Minoshima, Y.N., Seidel, M., Wood, J.R., Leschen, R.A., Gunter, N.L. & Fikáček, M. (2018) Morphology and biology of the flower‐visiting water scavenger beetle genus Rygmodus(Coleoptera: Hydrophilidae). Entomological Science, 21, 4, 363–384. https://doi.org/10.1111/ens.12316 Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67, 5, 901–904. https://doi.org/10.1093/sysbio/syy032 Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 3, 539–542. https://doi.org/10.1093/sysbio/sys029 Short, A.E.Z. & Fikáček, M. (2013) Molecular phylogeny, evolution, and classification of the Hydrophilidae (Coleoptera). Systematic Entomology, 38, 723–752. https://doi.org/10.1111/syen.12024 Toussaint, E.F., Hendrich, L., Escalona, H.E., Porch, N. & Balke, M. (2016) Evolutionary history of a secondary terrestrial Australian diving beetle (Coleoptera, Dytiscidae) reveals a lineage of high morphological and ecological plasticity. Systematic Entomology, 41, 3, 650–657. https://doi.org/10.1111/syen.12182 Toussaint, E.F. & Short, A.E.Z. (2018) Transoceanic stepping–stones between Cretaceous waterfalls? The enigmatic biogeography of pantropical Oocyclus cascade beetles. Molecular Phylogenetics and Evolution, 127, 416–428. https://doi.org/10.1016/j.ympev.2018.04.023

Figure 1. Maximum credibility tree of the MrBayes topology analysis of the Hydrophilidae dataset. Node labels = posterior probability. Megasternini are marked in red.

Amphiops mater (SLE0046) 1 1 Amphiops sp. (SLE0407) Amphiops sp. (SLE0408) Hydrobiomorpha spinicollis gr. (SLE0071) Hydrochara obtusata (AES0029) 1 0.73 Hydrophilus acuminatus (GB) 1 0.7 Hydrophilus albipes (SLE0074) Sternolophus marginicollis (SLE0017) 0.99 Tropisternus lateralis (SLE0244) 1 Tropisternus affinis (AES0032) 1 0.56 Tropisternus collaris (SLE0008) 1 Sperchopsis tessellata (AES0030) 1 Hydrocassis sp. (GB) 0.99 Ametor scabrosus (AES0014) 0.87 Ametor latus (SLE0096) 1 Hybogralius hartmeyeri (AES0007) 1 Limnohydrobius melaenus (AES0024) 1 Limnoxenus niger (AES0027) Limnocyclus punticeps (AES010) 1 0.82 Limnoxenus zealandicus (AES0025) 0.55 0.73 Limnoxenus semicylindricus (AES0021) 1 Limnoxenus namalokama (GB) 1 Limnoxenus waialeale (GB) Allocotocerus yalumbaboothbyi (SLE0082) 1 Regimbartia attenuata (SLE0016) 1 1 Derallus sp. (SLE0041) Hemiosus selva (SLE0051) 0.99 Berosus ornaticollis (SLE0087) 1 Berosus luridus (GB) 0.59 Berosus garciai (SLE0014) 0.2 Paracymus limbatus (SLE0040) 1 Paracymus pygmaeus (SLE0044) 0.99 Scoliopsis sp. (SLE0091) 1 Tritonus crenulatus (SLE0872) 0.29 Tormus sp. (SLE0295) Oocyclus madidus (SLE0004) 1 Oocyclus sp. (SLE0085) 0.96 1 Arabhydrus galagheri (SLE0073) 0.99 Pelthydrus minutus gr. (SLE0032) 0.7 1 Hydrophilomima sp. (SLE0067) Laccobius reflexipennis (SLE0015) 1 Laccobius serratus (SLE0057) 0.83 Opthalmocyclus sp. (SLE0093) 1 Oocyclus pico (SLE0033) 1 Oocyclus petra (SLE0005) 0.58 Oocyclus trujillo (SLE0037) 0.89 Oocyclus sapphirus (SLE0030) 1 Oocyclus funestus (SLE0049) 1 Oocyclus ornatus (SLE0050) Anacaena limbata (GB) 1 0.53 Anacaena globulus (GB) 0.88 Anacaena lanzhujii (MF257) 0.31 Grodum sp. (MF198) Anacaena parvula (MSC1818) 1 Paranacaena sp. (SLE0247) 1 1 Paranacaena lindi (SLE0242) 0.34 Omniops sp. (SLE0246) Pseudorygmodus versicolor (MF791) 0.53 Crenitulus hirsutus (SLE0369) 1 Crenitulus sp. (SLE0346) 1 Crenitulus suturalis (MSC1816) 0.87 0.93 Crenitulus solstitialis (MSC1815) 1 Horelophus walkeri (SLE0132) Chaetarthria incisa (SLE0070) 0.99 Chaetarthria sp. (MF247) 0.99 Thysanarthria sp. (SLE0127) 1 0.55 Chaetarthria seminulum (MF49) Guyanobius sp. (SLE0411) 1 Guyanobius lacuniventris (SLE0024) Enochrus piceus (SLE0409) 0.99 Enochrus ochraceus (SLE0248) 0.95 Enochrus testaceus (GB) 0.99 0.76 Enochrus quadripunctatus (GB) Enochrus fimbriatus (SLE0084) 0.73 0.97 Enochrus cinctus (SLE0083) Notionotus sp. (SLE0092) Enochrus sp. (SLE0241) 0.75 1 0.37 Enochrus talamanca (SLE0048) 1 0.11 Cymbiodyta semistriata (AES0013)

0.91 Enochrus mastersii (SLE0025) Notionotus liparus (MSC1820) 0.28 Helocombus bifidus (AES0004) Nanosaphes tricolor (SLE0130) 0.64 0.56 Tobochares sp.15a (SLE0101) 0.36 Tobochares sulcatus (SLE0035) 0.63 Tobochares sp.10 (SLE0102)

0.58 Globulosis sp. (SLE0027) Agraphydrus sp. (SLE0252) 1 Horelophopsis hanseni (MSC1821) 0.75 Chasmogenus ruidus (SLE0079) 1 1 Chasmogenus sp. (SLE0078) 1 Chasmogenus sp. (SLE0077) 0.75 Chasmogenus sp. (SLE0080) Helochares sp. (SLE0240) 0.66 1 0.99 Helobata sp. (SLE0031) Helochares sp. (SLE0034) Cyloma sp. (SLE0107) 0.53 0.98Cyloma sp. (SLE0095) 0.56 Cyloma stewarti (COL1789) 1 Cyloma pictus (COL1767) Cyloma stewarti (COL1778) 1 Adolopus badius (COL1772) 1 0.93 Adolopus helmsi (COL1761) 1 Adolopus versicolor (COL1775) Adolopus nonstriatus (COL1803) Rygmodus modestus (COL1841) 1 0.7 0.95 Rygmodus sp. (COL1794) 1 Rygmodus sp.1 (COL1845)

0.61 Rygmodus sp. (SLE0129) Pseudohydrobius flavus (COL803) 0.57 0.81 0.41 Rygmostralia sp. (COL800) 0.5 Coelostomopsis picea (COL119)

0.75 Borborophorus tuberculus (COL666) 0.46 Strange larva (Strange larva) 0.99 0.96 Saphydrus suffusus (COL1786) Eurygmus helocharoides (COL2028) Relictorygmus trevornoahi (MF1727_2) Tormissus linsi (COL1806) 1 1 1 Hydrostygnus frontalis (COL1777) 1 Exydrus gibbosus (COL1791) 0.99 Austrotypus nothofagi (MF540)

1 Andotypus ashworthi (MF792) Anticura flinti (MF793) 0.62 1 Cylomissus sp. (MF515) Cylorygmus lineatopunctatus (MF790) Psalitrus sp. (MF249) 0.99 Psalitrus sp. (MF135) 0.98 Psalitrus sp. (MF1388) 0.59 Psalitrus sp. (COL668) 0.98 Omicrogiton insularis (MF284) 1 1 Mircogioton huonus (COL993) Tylomicrus costatus (SLE0076) 0.77 Peratogonus reversus (MF223) 1 1 Noteropagus sp. (MF204) 0.54 Paromicrus sp. (MF818) 1 1 Aculomicrus reticulatus (MF74) Aculomicrus sp. (MF599) Phaenonotum sp. (CUB) (MF1115) 1 0.83 Phaenonotum sp. (MF1061) 0.48 Phaenonotum exstriatum (MF1062) Phaenonotum sp. (MF1065) 1 Phaenonotum laterale (MF1013) 0.93 1 0.92 Phaenonotum ondreji (MF980) Phaenonotum delgadoi (MF455) 1 Phaenonotum sp. (MF856) 1 Phaenonotum sp. (MF861) 0.64 Phaenonotum sp. (MF846)

0.38 Lachnodacnum luederwaldti (MF772) 1 Phaenostoma posticatus (MF1066) 1 0.93 Phaenostoma sp. (MF37) Phaenostoma cf. kontax (MF855) 0.52 0.98 Phaenonotum caribense (MF36) Cyclotypus sp. (MF842) 0.93 Dactylosternum sp.3 (MF25) 1 Dactylosternum abdominale (MF530) 0.61 0.58 Dactylosternum marginale (COL992) Dactylosternum auripes (MF63) 0.67 Dactylosternum dytiscoides (MF811) 0.99 1 Dactylosternum hydrophiloides (MF265) Dactylosternum indicum (MF457) 1 1 Coelostoma (Lachnocoelostoma) lemuriense (MF158) 1 Coelostoma (Lachnocoelostoma) sp. (MF136) Coelostoma phallicum (MF326) 1 Coelostoma (Holocoelostoma) stultum (MF488) 0.5 1 Coelostoma orbiculare (GB) 0.98 Coelostoma fabricii (COL481) Protosternum sp. (SLE0297) 1 Sphaerocetum arboreum (MF656) Sphaeridium lunatum (SLE0301) 1 Sphaeridium bipustulatum (SLE0298) Ceronocyton sp. (MF1620) 1 0.99 0.99 Pseudoosternum maculatum (MF1226) 1 Merosoma sp. (MF817)

1 Chledocyon semiopacus (MF1269) Cercyodes laevigatus (SLE0094) 1 Cercyodes kingensis (MF1291) 1 0.71 Oosternum sp. (PER) (MF834) 0.48 0.64 Motonerus inca (MF830) 0.27 CUB572 - Sacoosternum sp. (CRIC) 0.75 0.75 Oosternum gr. soricoides (MF224) Agna zaragozai (MF2075) Cetiocyon incantatus (SLE0251) Paroosternum saundersi (SLE0063) 0.64 1 Australocyon ca. puncticollis (MF206) Peltocercyon sp.2 (MF1158) 1 1 Peltocercyon sp. (MF139) Peltocercyon sp.2 (MF437) 0.75 Gillisius sp. (MF461) Cercyon ca. alinae Ryndevich (CHN) (MF1178) 0.36 Cercyon sp.4 (RSA) (MF1331) 0.52 1 Cercyon (Arcocercyon) sp. lutosus group (MF188) 1 Cercyon sp. (RSA) (MF1098) 0.75 1 0.11 Cercyon sp. (AUS) (MF1283) Cercyon ustulatus (GB) 0.55 Chimaerocyon shimadai (D30) 0.36 Cercyon sp. 3 (CHN) (MF285) 0.4 Cercyon sp. (SOL IS) (MF821) 0.94 Bolbonotum sp. (MF1627) 0.95 0.54 Destostethus scitus (SLE0126) 0.58 Cercyon sp. 1 (LAO) (MF473) 0.95 Cercyon sp. 2 (CHN) (MF226) 1 Cercyon sp. (LAO) (MF472) Cercyon undulipennis Ryndevich et al. (CHN) (MF1308) 0.98 Megasternum sp. (MF2073) Cercyon sp. (femoral lines) (MF183) 1 Cercyon haemorrhoidalis (MF680) 1 Cercyon laminatus (MF771) 0.56 1 Cercyon gr. nigriceps (MF288) 0.98 Cercyon ca. nigriceps (MF1189_2) Pacrillum sp. (MF205) 0.91 0.69 Tectosternum sp. (MF1497) 0.99 Cycrillum sp. (MF837) Pachysternum apicatum (MF283) 0.88 1 Pachysternum nigrovittatum (SLE0066) 1 Cryptopleurum pygmaeum (MF436) 1 Cryptopleurum minutum (SLE0058) 0.46 0.9 Cercillum sp. indesct. (MF1094) 1 Cyrtonion sp. (MF1484) Cercyon sp. 3 (RSA) (MF1324) Cycreon floricola (MF896) 1 Cercyon convexiusculus (MF964) 1 1 Cercyon (Arcocercyon) sp. 2 (RSA) (MF1322) Cercyon sp. 4 (CHN) (MF294) 0.84 1 Cercyon sp. 5 (CHN) (MF576) 0.66 Cercyon sp. (CHN) (MF200) 1 Cercyon ca. udus D'Orchymont (INDO) (MF1195) 0.94 Cercyon variegatus (SLE0020) 1 Moraphilus sp. (SLE0128) Pelosoma sp. (MF1644) 1 0.72 Pelosoma sp. (PER) (MF833) 0.6 1 Pelosoma sp. (PER) (MF852) Cercyon sp. 2 (PER) (MF831) 0.8 Nitidulodes ca. jansoni Sharp (MF1632) 0.73 Pelosoma sp. (MF1339) 1 Cercyon sp. (PER) (MF829) 0.96 Pelosoma sp. (ECU) (MF1025)

0.2 Figure 2. Fossil dated tree resuklting from the BEAST analysis of the Hydrophilidae dataset. Node labels = node ages. Megasternini are marked in red.

Amphiops sp. (SLE0408) 95.06 Amphiops mater (SLE0046) 70.8 Amphiops sp. (SLE0407) Hydrobiomorpha spinicollis gr. (SLE0071) Hydrochara obtusata (AES0029) 109.17 89.28 Hydrophilus acuminatus (GB) 62.01 103.57 Hydrophilus albipes (SLE0074) Sternolophus marginicollis (SLE0017) 81.54 Tropisternus lateralis (SLE0244) 32.1 Tropisternus affinis (AES0032) 139.72 29 162.33 Tropisternus collaris (SLE0008) Sperchopsis tessellata (AES0030) 64.53 Hydrocassis sp. (GB) 50.09 Ametor scabrosus (AES0014) 35.1 106.6 Ametor latus (SLE0096) Hybogralius hartmeyeri (AES0007) 48.36 Limnohydrobius melaenus (AES0024) 69.46 Limnoxenus niger (AES0027) Limnoxenus zealandicus (AES0025) 48.66 35.74 Limnocyclus punticeps (AES010) 152.12 41.88 Limnoxenus semicylindricus (AES0021) 25.8 Limnoxenus namalokama (GB) 14.9 Limnoxenus waialeale (GB) Allocotocerus yalumbaboothbyi (SLE0082) 100.48 Regimbartia attenuata (SLE0016) 80.4 121.4 Derallus sp. (SLE0041) Hemiosus selva (SLE0051) 98.8 Berosus ornaticollis (SLE0087) 74.73 Berosus luridus (GB) 62.1 Berosus garciai (SLE0014) 150.52 Paracymus limbatus (SLE0040) 106.34 Paracymus pygmaeus (SLE0044) 133.22 Scoliopsis sp. (SLE0091) 79.04 Tritonus crenulatus (SLE0872) 148.05 Tormus sp. (SLE0295) Oocyclus madidus (SLE0004) 57.87 Oocyclus sp. (SLE0085) 134.52 101.05 Arabhydrus galagheri (SLE0073) 91.18 Pelthydrus minutus gr. (SLE0032) 65.82 116.41 Hydrophilomima sp. (SLE0067) Laccobius reflexipennis (SLE0015) 66.78 181.84 Laccobius serratus (SLE0057) 106.58 Opthalmocyclus sp. (SLE0093) 85.81 Oocyclus pico (SLE0033) 66.21 Oocyclus petra (SLE0005) 61.29 Oocyclus trujillo (SLE0037) 51.29 Oocyclus sapphirus (SLE0030) 41.93 Oocyclus funestus (SLE0049) 18.19 Oocyclus ornatus (SLE0050) Guyanobius lacuniventris (SLE0024) 51.28 Guyanobius sp. (SLE0411) 149.83 Chaetarthria sp. (MF247) 85.05 Chaetarthria incisa (SLE0070) 129.24 Chaetarthria seminulum (MF49) 81.72 Thysanarthria sp. (SLE0127) 159.04 Horelophus walkeri (SLE0132) Crenitulus solstitialis (MSC1815) 126.94 45.18 Crenitulus suturalis (MSC1816) 53.28 Crenitulus sp. (SLE0346) 36.16 Crenitulus (SLE0369) 147.74 Pseudorygmodus versicolor (MF791) 108.83 Omniops sp. (SLE0246) 66.13 Paranacaena lindi (SLE0242) 47.64 117.96 Paranacaena sp. (SLE0247) Anacaena parvula (MSC1818) 109.71 Grodum sp. (MF198) 84.13 Anacaena lanzhujii (MF257) 72.49 Anacaena globulus (GB) 16.81 Anacaena limbata (GB) Helocombus bifidus (AES0004) 93.39 Notionotus liparus (MSC1820) 106.01 Enochrus mastersii (SLE0025) 180.3 93.21 Cymbiodyta semistriata (AES0013) 83.66 Enochrus talamanca (SLE0048) 133.06 21.64 Enochrus sp. (SLE0241) Notionotus sp. (SLE0092) Enochrus cinctus (SLE0083) 122.82 61.09 Enochrus fimbriatus (SLE0084) 94.39 Enochrus quadripunctatus (GB) 30.29 Enochrus testaceus (GB) 62.91 Enochrus ochraceus (SLE0248) 53.53 Enochrus piceus (SLE0409) Helochares sp. (SLE0034) 123.21 Helobata sp. (SLE0031) 113.14 Helochares sp. (SLE0240) Chasmogenus sp. (SLE0080) 138.94 92.72 Chasmogenus sp. (SLE0077) 66.77 Chasmogenus sp. (SLE0078) 36.37 121.1 Chasmogenus ruidus (SLE0079) 172.74 Horelophopsis hanseni (MSC1821) 70 Agraphydrus sp. (SLE0252) 107.41 Globulosis sp. (SLE0027) 94.29 Tobochares sp. (SLE0102) 79.87 Tobochares sulcatus (SLE0035) 64.11 Tobochares sp.15a (SLE0101) 37.75 Nanosaphes tricolor (SLE0130) Cylorygmus lineatopunctatus (MF790) Cylomissus sp. (MF515) 110.28 71.76 Anticura flinti (MF793) 93.4 Andotypus ashworthi (MF792) 80.16 Austrotypus nothofagi (MF540) 71.12 Exydrus gibbosus (COL1791) 58.11 119.23 Hydrostygnus frontalis (COL1777) 45.55 Tormissus linsi (COL1806) 169.83 Relictorygmus trevornoahi (MF1727) Eurygmus helocharoides (COL2028) 81.53 Saphydrus suffusus (COL1786) 64.11 109.93 strange larva (MF665) Borborophorus tuberculus (COL666) 83.19 Coelostomopsis picea (COL119) 101.59 Rygmostralia sp. (COL800) 73.6 50.62 Pseudohydrobius flavus (COL803) 65.28 Rygmodus sp. (SLE0129) 24.92 Rygmodus sp.1 (COL1845) 86.18 17.77 Rygmodus sp. (COL1794) 7.62 Rygmodus modestus (COL1841) Adolopus nonstriatus (COL1803) 64.51 Adolopus versicolor (COL1775) 56.03 Adolopus helmsi (COL1761) 40.51 73.22 Adolopus badius (COL1772) Cyloma stewarti (COL1778) 42.51 Cyloma pictus (COL1767) 36.56 Cyloma stewarti (COL1789) 162.91 24.39 Cyloma sp. (SLE0095) 11.48 Cyloma sp. (SLE0107) Aculomicrus sp. (MF599) Noteropagus sp. (MF204) 135.2 74.1 Peratogonus reversus (MF223) 118.98 Aculomicrus reticulatus (MF74) 62.52 Paromicrus sp. (MF818) 149.69 Tylomicrus costatus (SLE0076) 108.11 Mircogioton huonus (COL993) 79.92 Omicrogiton insularis (MF284) 133.28 Psalitrus sp. (COL668) 62.06 Psalitrus sp. (MF1388) 73.77 Psalitrus sp. (MF135) 31.08 Psalitrus sp. (MF249) Coelostoma fabricii (COL481) 65.36 Coelostoma orbiculare (GB) 60.1 85.01 Coelostoma (Holocoelostoma) stultum (MF488) Coelostoma phallicum (MF326) 74.15 Coelostoma (Lachnocoelostoma) sp. (MF136) 64.1 Coelostoma (Lachnocoelostoma) lemuriense (MF158) 54.45 159.16 Dactylosternum indicum (MF457) 137.78 Dactylosternum hydrophiloides (MF265) 100.56 Dactylosternum dytiscoides (MF811) 117.67 Dactylosternum auripes (MF63) 98.11 Dactylosternum marginale (COL992) 76.23 Dactylosternum abdominale (MF530) 130.71 Dactylosternum sp.3 (MF25) 69.53 Cyclotypus sp. (MF842) Phaenonotum caribense (MF36) 126.64 101.75 Phaenostoma cf. kontax (MF855) 116.64 Phaenostoma posticatus (MF1066) 18.62 Phaenostoma sp. (MF37) 108.23 Lachnodacnum luederwaldti (MF772) 102.05 Phaenonotum sp. (MF846) 155.48 91.31 Phaenonotum sp. (MF861) 75.86 Phaenonotum sp. (MF856) Phaenonotum delgadoi (MF455) 66.69 44.23 Phaenonotum laterale (MF1013) 31.92 51.08 Phaenonotum ondreji (MF980) Phaenonotum sp. (MF1065) 46.52 Phaenonotum exstriatum (MF1062) 39.61 Phaenonotum sp. (MF1061) 22.73 Phaenonotum sp. (CUB) (MF1115) Sphaerocetum arboreum (MF656) 62.06 Protosternum sp. (SLE0297) Sphaeridium bipustulatum (SLE0298) 63 Sphaeridium lunatum (SLE0301) 149.29 Cetiocyon incantatus (SLE0251) Agna zaragozai (MF2075) 87.81 Oosternum gr. soricoides (MF224) 107.58 82.64 Sacoosternum sp. (CUB572) 72.14 134.15 Motonerus inca (MF830) 65.03 101.87 Oosternum sp. (PER) (MF834) Cercyodes kingensis (MF1291) 31.58 Cercyodes laevigatus (SLE0094) 88.19 Chledocyon semiopacus (MF1269) 77.08 Merosoma sp. (MF817) 120.26 69.38 Pseudoosternum maculatum (MF1226) 43.38 Ceronocyton sp. (MF1620) Australocyon ca. puncticollis (MF206) 96.27 Paroosternum saundersi (SLE0063) Peltocercyon sp.2 (MF437) 65.71 Peltocercyon sp.2 (MF1158) 9.58 Peltocercyon sp. (MF139) 114.81 Gillisius sp. (MF461) Cercyon ca. alinae Ryndevich (CHN) (MF1178) 82.41 Cercyon sp. 4 (RSA) (MF1331) 97.58 46.11 Cercyon (Arcocercyon) sp. lutosus group (MF188) 13.9 Cercyon sp. (RSA) (MF1098) 107.96 2.87 93.02 Cercyon sp. (AUS) (MF1283) Cercyon ustulatus (GB) 71.81 Chimaerocyon shimadai (D30) 64.11 Cercyon sp. 3 (CHN) (MF285) 85.61 Bolbonotum sp. (MF1627) 58.37 Cercyon sp. (SOL IS) (MF821) 77.94 104.07 Deltostethus scitus (SLE0126) 70.13 Cercyon sp. 1 (LAO) (MF473) 45.23 Cercyon sp. 2 (CHN) (MF226) 2.59 Cercyon sp. (LAO) (MF472) Cercyon undulipennis Ryndevich et al. (CHN) (MF1308) 66.77 Megasternum sp. (MF2073) Cercyon sp. (femoral lines) (MF183) 80.22 Cercyon haemorrhoidalis (MF680) 70.93 Cercyon laminatus (MF771) 101.15 59.76 Cercyon ca. nigriceps (MF1189) 50.58 Cercyon gr. nigriceps (MF288) Tectosternum sp. (MF1497) 66.22 97.98 Pacrillum sp. (MF205) 85.52 Cycrillum sp. (MF837) Pachysternum apicatum (MF283) 77.11 36.53 Pachysternum nigrovittatum (SLE0066) 67.66 Cercillum sp. indesct. (MF1094) 47.86 Cyrtonion sp. (MF1484) 65.63 95.21 Cryptopleurum pygmaeum (MF436) 45.49 Cryptopleurum minutum (SLE0058) Cercyon sp. 3 (RSA) (MF1324) Cycreon floricola (MF896) 72.16 Cercyon (Arcocercyon) sp. 2 (RSA) (MF1322) 90.62 52.03 Cercyon convexiusculus (MF964) Cercyon sp. 4 (CHN) (MF294) 87.67 57.87 Cercyon sp. 5 (CHN) (MF576) 54.08 Cercyon ca. udus D'Orchymont (INDO) (MF1195) 20.11 Cercyon sp. (CHN) (MF200) 83.9 Cercyon variegatus (SLE0020) 46.37 Moraphilus sp. (SLE0128) Pelosoma sp. (MF1644) 78.54 57.78 Pelosoma sp. (PER) (MF833) 48.73 69.22 Pelosoma sp. (PER) (MF852) Cercyon sp. 2 (PER) (MF831) 62.63 Nitidulodes ca. jansoni Sharp (MF1632) 59.04 Pelosoma sp. (MF1339) 51.1 Pelosoma sp. (ECU) (MF1025) 40.85 Cercyon sp. (PER) (MF829)

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