1 Preprint submitted to Systematic Entomology

2

3 A molecular phylogeny of the tribe Ochthebiini (Coleoptera,

4 Hydraenidae, Ochthebiinae)

5 6 ADRIÁN VILLASTRIGO1, MANFRED A. JÄCH2, ANABELA CARDOSO1, LUIS F. 7 VALLADARES3, and IGNACIO RIBERA1 8 9 1Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain, 10 2Naturhistorisches Museum Wien, Vienna, Austria, 3Departamento de Biodiversidad y 11 Gestión Ambiental (Zoología), Facultad de Ciencias Biológicas y Ambientales, 12 Universidad de León, León, Spain 13 14 15 Correspondence: Ignacio Ribera, Institute of Evolutionary Biology (CSIC-Universitat 16 Pompeu Fabra), Passeig Maritim de la Barceloneta 37, 08003 Barcelona, Spain. E-mail: 17 [email protected] 18 19 20

1 21 Abstract 22 Ochthebiinae, with ca. 650 species distributed worldwide, are the second most speciose 23 subfamily of the aquatic beetle family Hydraenidae. They are the ecologically most 24 diverse subfamily, with terrestrial species as well as species in almost all types of 25 aquatic habitats, including hypersaline waters. Ochthebiinae include the tribes 26 Ochtheosini (four species in three genera) and Ochthebiini. We provide here the first 27 comprehensive phylogeny of the tribe Ochthebiini, based on 188 species and four 28 subspecies from most genera, subgenera and species groups. We obtained sequence data 29 for a combination of mitochondrial and nuclear gene fragments including the 5’ and 3’ 30 ends of the cytochrome c oxidase subunit 1, the 5’ end of 16S RNA plus the Leucine 31 tRNA transfer plus 5’ end of NADH dehydrogenase subunit I, and internal fragments of 32 the large and small ribosomal units. The analyses with maximum likelihood and 33 Bayesian probabilities consistently recovered a generally well supported phylogeny, 34 with most currently accepted taxa and species groups as monophyletic. We provide a 35 new classification of the tribe based on our phylogenetic results, with three genera: 36 Meropathus Enderlein, Ochthebius Leach and Protochthebius Perkins. Meropathus in 37 its new sense includes five subgenera in addition to Meropathus s.str.: (1) M. 38 (Hygrotympanogaster) Perkins comb.n.; (2) M. (Plesiotympanogaster) Perkins comb.n.; 39 (3) M. (Topotympanogaster) Perkins comb.n. (the last three transferred from 40 Tympanogaster); (4) M. (Tympallopatrum) Perkins comb.n.; and (5) M. 41 (Tympanogaster) Janssens comb.n. (both currently considered as genera). The genus 42 Ochthebius is here divided into nine subgenera in addition to Ochthebius s.str.: (1) O. 43 (Angiochthebius) Jäch & Ribera; (2) O. (Asiobates) Thomson; (3) O. (Aulacochthebius) 44 Kuwert; (4) O. (Cobalius) Rey; (5) O. (Enicocerus) Stephens; (6) O. (Gymnanthelius) 45 Perkins comb.n.; (7) O. (Gymnochthebius) Orchymont comb.n.; (8) O. (Hughleechia) 46 Perkins comb.n.; and (9) O. (Micragasma) Sahlberg. Within Ochthebius s.str., 17 47 species groups are proposed, five of them newly established (3, 9, 11, 13 and 16): (1) 48 andraei; (2) atriceps; (3) corrugatus; (4) foveolatus; (5) kosiensis; (6) lobicollis; (7) 49 marinus; (8) metallescens; (9) nitidipennis; (10) notabilis; (11) peisonis; (12) punctatus; 50 (13) quadricollis; (14) rivalis; (15) strigosus; (16) sumatrensis; and (17) vandykei. We 51 could not obtain sequences of any species of Protochthebius, which is therefore 52 maintained as a distinct genus of Ochthebiini. We elevated to species rank two 53 subspecies of Ochthebius: O. fallaciosus Ganglbauer stat.n. (former subspecies of O.

2 54 viridis Peyron) and O. deletus Rey stat.rest. (former subspecies of O. subpictus 55 Wollaston). 56 57 Introduction 58 59 Ochthebiinae, with ca. 650 species and 11 subspecies described, are the second 60 most diverse subfamily of the water beetle family Hydraenidae (Hansen, 1998; Jäch & 61 Balke, 2008; Tables 1, S1). They occur on all biogeographic regions, including the 62 Antarctic islands of Kerguelen and Heard, where they are the only Hydraenidae present 63 (Hansen, 1998). Ochthebiinae are the ecologically most diverse hydraenid subfamily, 64 with terrestrial species, species living in the interface between land and water, as well as 65 in most types of aquatic environments (Jäch et al., 2016). A large number of species are 66 tolerant to hypersaline waters, living either in coastal rockpools (e.g. Cobalius Rey or 67 Calobius Wollaston, Antonini et al., 2010; Sabatelli et al., 2016), coastal or inland 68 saltpans or in inland hypersaline streams (especially the O. notabilis group, but also 69 many other species in different groups, Abellán et al., 2009; Millán et al., 2011). 70 The external morphology of the species of Ochthebiinae is more heterogeneous 71 than in other Holarctic lineages of Hydraenidae (e.g. Hydraena Kugelann, Trizzino et 72 al., 2013, or Limnebius Leach, Rudoy et al., 2016). This has resulted in a more complex 73 taxonomy, with mostly all of the described genera or subgenera with uncertain 74 relationships. Thus, the genus Ochthebius Leach was divided in 16 subgenera by 75 Kuwert (1887), but all of them with the exception of Aulacochthebius Kuwert were later 76 synonymised (see Tables 1, S2 for a synopsis of the classification and Table S1 for a 77 complete checklist of Ochthebiini). 78 Ochthebiinae were divided by Perkins (1980) in two tribes, Ochthebiini and 79 Ochtheosini, the latter for the single terrestrial genus Ochtheosus Perkins, with two 80 species. The monotypic genera Edaphobates Jäch & Díaz and Ginkgoscia Jäch & Díaz, 81 for which we could not obtain fresh material for DNA extraction, were tentatively 82 hypothesized to be related to Ochtheosus by Jäch & Díaz (2003, 2004) and thus we 83 tentatively consider them within Ochtheosini. Perkins (1997) divided Ochthebiini in 84 five newly defined subtribes: Enicocerina (for the single genus Enicocerus Stephens), 85 Meropathina (Meropathus Enderlein, Tympallopatrum Perkins and Tympanogaster 86 Perkins), Neochthebiina (Neochthebius Orchymont), Ochthebiina (Ochthebius, 87 Gymnochthebius Orchymont, Hughleechia Perkins, Gymnanthelius Perkins,

3 88 Aulacochthebius and Micragasma Sahlberg) and Protochthebiina (Protochthebius 89 Perkins). Enicocerus was treated as a subgenus by several subsequent authors (e.g. Jäch, 90 1998; Ribera et al., 2010; Jäch & Skale, 2015), and Neochthebius was treated as a 91 synonym of Ochthebius s.str. by Jäch & Delgado (2014b), leaving ten genera in 92 Ochthebiini, most of them described in the 20th century (Table 1). Ochthebius is the 93 oldest available generic name (Leach, 1815), with 540 species (Hansen, 1998; Table 1) 94 grouped into four recognised subgenera with mostly Palaearctic distribution: Asiobates 95 Thomson, Calobius, Enicocerus and Ochthebius (Jäch & Skale, 2015; Tables 1, S1; see 96 the detailed taxonomic history of subgenera and species groups in the Discussion 97 below). Within Ochthebius s.str., the most diverse subgenus, several informal species 98 groups have been defined, which have undergone important modifications through their 99 taxonomic history (Tables S1, S2). 100 The classification and proposed relationships within Ochthebiini have also 101 experienced many modifications during the last four decades. Perkins (1980) revised the 102 by then known American species, and proposed a phylogeny derived from the 103 examination of some morphological characters. Gymnochthebius was placed as sister to 104 the remaining taxa, which were divided in two lineages: (1) Meropathus plus 105 Neochthebius (currently a synonym of Ochthebius, Table S2) and (2) Ochthebius plus 106 Asiobates. Subsequently, Perkins (1997) synonymized four subgenera with Ochthebius 107 (Calobius, Cobalius, Liochthebius Sahlberg and Notochthebius Orchymont), and 108 described three additional genera (Tables 1, S2). Based mostly on the exocrine secretion 109 delivery system (ESDS), he divided the subfamily in two tribes, Ochtheosini for the 110 newly described Ochtheosus and Ochthebiini, divided in turn in subtribes, with 111 unresolved relationship among them. Ochtheosus was considered to have some 112 plesiomorphic characters similar to some southern African genera (e.g. antennae with 113 11 antennomeres, as in many Prosthetopinae, Perkins, 1997; see also Beutel et al., 114 2003), and did not share several of the most characteristic synapomorphies with the 115 remaining Ochthebiinae, in particular the structure of the tentorial arms, galea and 116 lacinia. 117 The first formal cladistic analysis of the family Hydraenidae was published by 118 Beutel et al. (2003), but sampling was too incomplete to resolve internal relationships 119 within Ochthebiinae other than the sister relationship of Meropathus with Ochthebius + 120 Gymnochthebius. There is no published global molecular phylogeny of the entire family 121 Hydraenidae or subfamily Ochthebiinae, but in recent years some detailed molecular

4 122 phylogenies for some lineages have been published, such as the Ochthebius notabilis 123 group (Abellán et al., 2009) and Enicocerus (Ribera et al., 2010). In Abellán et al. 124 (2009) an extensive phylogeny of Ochthebius and some related genera using only 125 mitochondrial markers was used to estimate the phylogenetic diversity of the Iberian 126 fauna. The sampling of some geographical areas was, however, very incomplete, as the 127 intention was not to produce a phylogenetic study. Still, most Palaearctic lineages were 128 represented, which allowed to establish the monophyly of most of the included 129 genera/subgenera and of the recognised species groups, although internal groups had 130 poor relationships between them. Sabatelli et al. (2016) used these data to study the 131 origin of species typical of rockpools, recovering basically the same relationships and 132 establishing a new species group for the South African O. capicola Péringuey. In the 133 same paper the subgenus Cobalius was found to be outside Ochthebius s.str., but 134 Calobius nested within it, referring to it as the “Calobius” lineage. 135 In this study we provide a comprehensive phylogeny of Ochthebiini, based on 136 mitochondrial and nuclear sequence data including representatives of most lineages. We 137 introduce several changes in the taxonomic classification to accommodate our 138 phylogenetic results, and provide a complete checklist based on our new classification 139 (Table S1). 140 141 Material and Methods 142 143 Taxon sampling 144 145 We studied 188 species and four subspecies of the 641 described species and 11 146 described subspecies of Ochthebiini, plus 27 specimens corresponding to undetermined 147 or still undescribed species (Table S3). For two species with an isolated or unsupported 148 placement (O. guerreroi Jäch & Ribera and O. peisonis Ganglbauer, see below) we 149 sequenced two specimens to test for possible sequencing mistakes. We included 150 examples of eight of the ten genera currently recognised in the tribe, all subgenera but 151 two (within genus Tympanogaster), and all recognised species groups within the genus 152 Ochthebius but one (O. kosiensis group, Tables 1, S1, S3). The two missing genera, 153 Tympallopatrum (Australia) and Protochthebius (Asia), have four and seven species 154 respectively (Table S1).

5 155 We used as outgroups 31 species of other Hydraenidae genera (Hydraena, 156 Laeliaena Sahlberg and Limnebius) and of Ptiliidae. Trees were rooted in the split 157 between Hydraenidae and Ptiliidae, considered to be sister groups both based on 158 molecular (e.g. Hunt et al., 2007; McKenna et al., 2015) and morphological evidence 159 (Hansen, 1997; Lawrence et al., 2011). 160 161 DNA extraction and sequencing 162 163 Specimens were killed and preserved in absolute ethanol. DNA was extracted 164 with a standard phenol-chloroform extraction or by commercial extraction kits (mostly 165 Quiagen DNeasy Tissue Kit, Hildesheim, Germany) following the instructions of the 166 manufacturers. DNA samples and voucher specimens are kept in the collections of the 167 Institute of Evolutionary Biology (IBE, Barcelona), Museo Nacional de Ciencias 168 Naturales (MNCN, Madrid) and Naturhistorisches Museum Wien (NMW, Vienna). We 169 sequenced fragments of six genes in five sequencing reactions, three mitochondrial: (1) 170 5' end of the cytochrome c oxidase subunit 1 (the standard barcode, Hebert et al., 2003) 171 (COI-5’), (2) 3' end of cytochrome c oxidase subunit 1 (COI-3’), (3) 5’ end of 16S RNA 172 (16S) plus the Leucine tRNA transfer (tRNA-Leu) plus 5’ end of NADH dehydrogenase 173 subunit I (NAD1); and two nuclear: (4) an internal fragment of the large ribosomal unit, 174 28S RNA (28S) and (5) an internal fragment of the small ribosomal unit, 18S RNA 175 (18S) (see Table S4 for details on primers used and typical polymerase chain reaction 176 (PCR) conditions). Sequences were assembled and edited with GENEIOUS v10.1 177 (Kearse et al., 2012); new sequences (a total of 897) were deposited in the ENA 178 database with accession numbers XXX–XXX. 179 180 Phylogenetic analyses 181 182 Edited sequences were aligned using the online version of MAFFT v.7 with the 183 G-INS-I algorithm (Katoh et al., 2009). We used PartitionFinder v1.1.1 (Lanfear et al., 184 2012) to estimate the evolutionary model that best fitted the data, using one partition for 185 each gene fragment (six partitions in total), and using Akaike Information Criterion 186 (AIC) scores as selection criteria. Phylogenetic analyses were made using Bayesian 187 probabilities in BEAST 1.8 (Drummond & Rambaut, 2007), using the partition and 188 evolutionary models selected by PartitionFinder, with a Yule speciation process as tree

6 189 prior. To obtain a general calibration of the tree we dated the root (i.e. the split between 190 Hydraenidae and Ptiliidae) with a normal distribution with an average of 170 Ma and a 191 standard deviation of 1 Ma following recent estimates based on a range of fossils (Hunt 192 et al., 2007; McKenna et al., 2015), and used an uncorrelated lognormal relaxed clock. 193 We ran 100 million generations login results each 5,000, and checked convergence to 194 estimate the burn-in fraction with Tracer v1.6 (Drummond & Rambaut, 2007). We ran 195 an additional ML phylogenetic reconstruction with RAxML-HPC2 (Stamatakis, 2006) 196 in the CIPRES portal (Miller et al., 2010), using the same partition scheme as in 197 BEAST with a GTR+G model estimated independently for each partition. Node 198 supports values were estimated with 100 pseudoreplicas using a rapid bootstrapping 199 algorithm (Stamatakis et al., 2008). The same ML analysis was repeated only with the 200 nuclear sequence (18S and 28S). 201 202 Results 203 204 The final matrix included 252 terminals with 3,656 aligned characters. Protein 205 coding regions had no indels except for the 3' end of COI-3, where some species had an 206 additional codon. The best partitioning scheme obtained with PartitionFinder had six 207 partitions corresponding to (1) COI-5, (2) COI-3, (3) 16S+tRNA-Leu, (4) NAD1, (5) 208 18S and (6) 28S. The optimal evolutionary model was GTR+I+G for all partitions 209 except for NAD1 (best model TMV) and 28S (best model SYM). The BEAST run 210 implementing the best models did, however, not converge properly, mostly due to the 211 parameters related to the estimation of the branch lengths, especially for the genes 212 NAD1, 18S and 28S. We thus did a second run with simpler models for these genes 213 (HKY+G+I), which converged adequately. The topologies of the two Bayesian analyses 214 were, however, almost identical (Figs 1, S1), and unless specified we report only the 215 results of the analyses with the better parameter convergence (i.e. with the simpler 216 evolutionary models). 217 218 Molecular phylogeny 219 220 The topologies obtained in the ML and the two Bayesian analyses were very 221 similar, differing only in some poorly supported nodes (Figs 1, S1, S2), most notably in 222 the position of Hughleechia (see below). The ML tree with the nuclear sequence only

7 223 had a topology very similar to that obtained with the combined data, although with a 224 generally lower resolution and support. Main difference was the recovery of 225 Ochthebiini as paraphyletic, with the genus Hydraena as sister to Tympanogaster plus 226 Meropathus, although with very low support (bootstrap support, BS= 53%; Fig. S3). 227 Genera, subgenera and most species groups were, however, recovered as monophyletic 228 with strong support, with internal topologies very similar to that of the combined ML 229 tree (Figs S2, S3). 230 In the ML and Bayesian trees with the combined nuclear and mitochondrial data 231 the monophyly of Ochthebiini was strongly supported, as well as their separation in two 232 clades, (1) Meropathus plus Tympanogaster and (2) Ochthebius sensu lato (s.l. herein). 233 Meropathus was nested within a paraphyletic Tympanogaster in the ML tree (combined 234 and nuclear only) and in the Bayesian tree with the best models, and sister to 235 Tympanogaster with low support in the Bayesian tree with simpler models (posterior 236 probability, pp= 0.63; Figs 1a, S1, S2). 237 Within Ochthebius s.l. Asiobates and Aulacochthebius were sister groups in the 238 Bayesian tree with low support (pp= 0.85), and both sisters to the rest of Ochthebiini. In 239 the ML analysis Asiobates and Aulacochthebius were paraphyletic with respect to the 240 rest of Ochthebiini, also with low support (BS< 50%) (Figs 1a, S2). In both analyses 241 Ochthebiini minus Asiobates and Aulacochthebius were monophyletic with strong 242 support (BS= 80%; pp= 1; Fig. 1a). 243 The remaining Ochthebiini were divided in a series of well supported clades 244 corresponding to traditionally recognised genera or subgenera, but with poorly resolved 245 relationships among them. (1) Enicocerus, strongly supported and with well resolved 246 internal relationships, sister to the Australian Hughleechia in ML and the Bayesian 247 analysis with the simpler models (BS = 81%, pp= 0.76; Figs 1b, S2). In the Bayesian 248 analysis with the best models Hughleechia was sister to the clade formed by 249 Micragasma and Cobalius, with low support (pp= 0.88; Fig. S1). (2) A clade including 250 Gymnochthebius and Gymnanthelius, the latter as sister to Angiochthebius Jäch & 251 Ribera (Gymnochthebius plesiotypus group of Perkins, 1980; see Jäch & Ribera, 2018) 252 (BS= 94%, pp= 0.96). Within Gymnochthebius, the Australian and American species 253 were respectively monophyletic and sisters, with very strong support both in the ML 254 and Bayesian trees (Figs 1b, S2). (3) Cobalius, with a strongly supported monophyly 255 (BS= 98%, pp= 1) and sister to the only sequenced species of Micragasma, also with 256 strong support (BS= 100%, pp= 1) (Figs 1b, S2). (4) Ochthebius sensu stricto (s.str.

8 257 herein), including Calobius, strongly supported both in the ML (BS= 94%) and 258 Bayesian (pp= 1) trees (Figs 1b, S2). 259 Within Ochthebius s.str. most established Palaearctic species groups were 260 recovered as monophyletic (see Discussion, Figs 1b, 1c, S2). Their monophyly was 261 strongly supported in the ML and Bayesian trees, with the only exception of the group 262 of species related to the O. atriceps and O. notabilis groups in the ML analyses (see 263 below). The main difference with established groups was the expansion of the O. 264 marinus group to include the south African O. capicola and the American O. biincisus, 265 bisinuatus and interruptus groups of Perkins (1980). The O. foveolatus group of Jäch 266 (1991) was split in three clades: (1) O. foveolatus group, sister to the O. metallescens 267 group with strong support in both ML and Bayesian trees (BS= 71%, pp= 0.99); (2) O. 268 atriceps group and (3) O. corrugatus group. The latter two formed a clade with the 269 species of the O. notabilis and O. andraei groups, strongly supported in the Bayesian 270 tree (pp= 1) but not in the ML tree (BS< 50%), in which the group also included one of 271 the two sampled species of the O. rivalis group (Figs 1c, S2). Two coastal lineages, 272 Calobius and the O. vandykei group (formerly genus Neochthebius), were nested within 273 Ochthebius s.str., the former as sister to the O. lobicollis + O. strigosus groups (BS< 274 50%, pp= 0.92) and the latter as sister to the O. marinus group (BS< 50%, pp= 0.97) 275 (Figs 1b, 1c, S2). 276 According to our calibration scheme, with a separation between Hydraenidae 277 and Ptiliidae at 170 Ma, the estimated age of crown Hydraenidae was 106 Ma (HPD 278 122.8–90.2 Ma), and that of the crown Ochthebiini 93 Ma (HPD 109.7–80.8 Ma). The 279 basal diversification of Ochthebiini was reconstructed as having occurred in a relatively 280 short temporal window, with genera, subgenera and most species groups with an origin 281 between ca. 87–60 Ma (Fig. 1; see Table 2 for the estimated evolutionary rates of all 282 partitions). 283 284 Discussion 285 286 Our results strongly support the monophyly of Ochthebiini, but our sampling did 287 not allow to test for the monophyly of Ochthebiinae, or its position within Hydraenidae. 288 Within Ochthebiini our results recover two well supported clades, which we consider 289 here as genera: Meropathus and Ochthebius (Fig. 2; see below for a detailed discussion

9 290 of the taxonomic classification of Ochthebiini). We did not find evidence to support the 291 five subtribes proposed by Perkins (1997), which are therefore not considered here. 292 We did not find evidence for a clear separation between the studied species of 293 Tympanogaster and Meropathus, in agreement with previous studies (Hansen, 1991). 294 We could not obtain material of the genus Tympallopatrum, considered to be closely 295 related to Tympanogaster by Perkins (1997), and two of the subgenera of 296 Tympanogaster (Plesiotympanogaster Perkins and Topotympanogaster Perkins), so 297 until more data become available we consider Tympanogaster s.str., the other subgenera 298 of Tympanogaster, and Tympallopatrum as subgenera of Meropathus (Tables 1, S1; Fig. 299 2) (see Perkins, 2006 for a discussion on the subgeneric classification of 300 Tympanogaster). The species of Meropathus are found in the Australian Region, on two 301 Antarctic islands (Kerguelen and Heard) and on several Subantarctic islands, such as 302 Campbell Island, Crozet Islands, Prince Edward Island and Falkland Islands. 303 The second lineage, genus Ochthebius s.l., included all the non-Australian 304 Ochthebiini, as well as several Australian species. Our results agree remarkably well 305 with the currently recognised subgenera and many of the established species groups, 306 which were recovered as monophyletic with general strong support (Fig. 2). The 307 relationships between these lineages, however, do not confirm some previous 308 hypotheses on their relationships. Thus, Aulacochthebius was not found to be closely 309 related to Gymnochthebius, as proposed in Hansen (1991), but to Asiobates; 310 Micragasma and Hughleechia were not among the basal lineages and Cobalius and 311 Calobius were not closely related, as hypothesised in Perkins (1997). Novel 312 relationships found here are the possible sister relationship between Hughleechia and 313 Enicocerus, and the close relationship between Gymnochthebius and Gymnanthelius. 314 Interestingly, within the clade Gymnochthebius + Gymnanthelius + Angiochthebius 315 there are two cladogenetic events separating American from Australian species: one 316 within Gymnochthebius, dated at 73 Ma (95% HPD 87–60 Ma), and another separating 317 the Australian Gymnanthelius and the Chilean Angiochthebius, dated at 60 Ma (95% 318 HPD 78–51 Ma). Although a detailed biogeographic analysis is outside the scope of this 319 paper, it is interesting to note that these estimations are too recent for a tectonic split 320 between Australia and South America (i.e. west and east Gondwana), dated at ca. 130 321 Ma (McIntyre et al., 2017). Our calibration would thus require a different scenario, 322 probably through the colonization of some southern islands or the Antarctica. An older 323 age for these nodes is unlikely, given that our rate estimations are already slower than

10 324 most recent estimations for the same genes in other groups of Coleoptera (Table 2; see 325 e.g. Papadopoulou et al., 2010; Andújar et al., 2012; Cieslak et al., 2014). 326 327 Taxonomic classification of Ochthebiini Thomson, 1859 328 329 1. Genus Meropathus Enderlein, 1910 330 Type species: Meropathus chuni Enderlein, 1910, by monotypy. 331 Meropathus was described as genus, considered as subgenus of Ochthebius by 332 Orchymont (1938) and reinstated again as genus by Jeannel (1940). Bameul (1989) 333 redescribed the genus and recognised 12 species (in two species groups), transferring O. 334 schizolabrus Deane to Meropathus. Hansen (1991) noted the difficulty in establishing 335 clear distinctions within the Meropathus-Tympanogaster complex. Perkins (1997) 336 erected the subtribe Meropathina for the genera Meropathus, Tympanogaster and the 337 newly described Tympallopatrum, the latter two including the Australian and 338 Tasmanian species. Subsequently, Perkins (2004a, 2006) revised Tympallopatrum and 339 Tympanogaster respectively, and described three new subgenera and 76 new species in 340 the latter. As defined here, Meropathus includes 96 species in six subgenera (Fig. 2; 341 Table S1). 342 343 1.1. Subgenus Hygrotympanogaster Perkins, 2006 comb.n. 344 Type species: Tympanogaster maureenae Perkins, 2006, by original designation. 345 Hygrotympanogaster Perkins was described by Perkins (2006) as a subgenus of 346 Tympanogaster, to include mostly hygropetric species in south-western Australia. 347 Currently it includes 36 species (Perkins, 2006) (Table S1). 348 349 1.2. Subgenus Meropathus Enderlein, 1910 350 Type species: Meropathus chuni Enderlein, 1910, by monotypy. 351 Meropathus s.str. includes the species of the former genus Meropathus that were not 352 transferred to Tympanogaster by Perkins (1997, 2006), i.e. all seven New Zealand, 353 Antarctic and Subantarctic species plus M. labratus Deane from Queensland (Table S1). 354 355 1.3. Subgenus Plesiotympanogaster Perkins, 2006 comb.n. 356 Type species: Tympanogaster thayerae Perkins, 2006, by original designation.

11 357 Plesiotympanogaster was described by Perkins (2006) as a subgenus of Tympanogaster 358 to include the type species plus Ochthebius costatus Deane (Table S1). Both species 359 were considered to have plesiomorphic characters within the genus. 360 361 1.4. Subgenus Topotympanogaster Perkins, 2006 comb.n. 362 Type species: Tympanogaster crista Perkins, 2006, by original designation. 363 Topotympanogaster was described by Perkins (2006) as a subgenus of Tympanogaster 364 to include eight Australian species, all described in Perkins (2006) (Table S1). 365 366 1.5. Subgenus Tympallopatrum Perkins, 1997 comb.n. 367 Type species: Tympallopatrum longitudum Perkins, 1997, by original designation. 368 Tympallopatrum was described by Perkins (1997) as a monotypic genus within 369 Meropathina. Subsequently, Perkins (2004a) revised the genus and described three 370 additional species, all of them from western Australia (Table S1). We could not obtain 371 any representative of this and the two previous subgenera for our study, and thus their 372 phylogenetic placement within Meropathus remains untested. 373 374 1.6. Subgenus Tympanogaster Janssens, 1967 comb.n. 375 Type species: Tympanogaster deanei Perkins, 1979 (replacement name for Ochthebius 376 longipes Deane, 1931), by monotypy. 377 Described by Janssens (1967) as a monotypic genus for O. longipes Deane (= T. deanei 378 Perkins), Perkins (1997) redescribed Tympanogaster and transferred some species from 379 Meropathus. Perkins (2006) revised the genus and described four subgenera (see 380 Meropathus above) and raised the total number of the species in the here defined 381 subgenus Tympanogaster to 38 (Table S1), all distributed in Australia and Tasmania. 382 383 2. Genus Ochthebius Leach, 1815 384 Type species: Helophorus marinus Paykull, 1798, fixed by Orchymont (1942). 385 The second well supported lineage within Ochthebiini includes the remaining 386 genera/subgenera with in some cases uncertain relationships among them. We consider 387 Ochthebius a single genus with 540 species and nine subspecies in ten well supported 388 subgenera, corresponding in most cases to currently recognised taxa (Fig. 2). One 389 additional species, O. eremita Knisch from Fiji, cannot be confidently placed in any of

12 390 the described subgenera, and it is left as incertae sedis within the genus Ochthebius 391 (Hansen, 1998; Table S1). 392 393 2.1. Subgenus Angiochthebius Jäch & Ribera, 2018 394 Type species: Ochthebius guerreroi Jäch & Ribera, 2018, by original designation. 395 The subgenus Angiochthebius was created for the Gymnochthebius plesiotypus species 396 group (sensu Perkins, 1980), which now includes four South American species (Jäch & 397 Ribera, 2018; Table S1). The species of the G. plesiotypus group were included within 398 Gymnochthebius by Perkins (1980) as they share a bifid apex of the aedeagus, but 399 external characters (e.g. the pubescent 5th abdominal ventrite) and some aedeagal 400 characters (Jäch & Ribera, 2018) as well as molecular data (Figs 1b, S2) warrant their 401 removal from Gymnochthebius and their status as a distinct subgenus of Ochthebius. 402 403 2.2. Subgenus Asiobates Thomson, 1859 404 Type species: Ochthebius rufimarginatus Stephens, 1829 (= O. bicolon Germar, 1824), 405 by monotypy. 406 Originally described as a genus, but downgraded to subgenus by Seidlitz (1875), and 407 treated as such by most authors (e.g. Jäch, 1990a; Hansen, 1991; Perkins, 1997). Jäch 408 (1990a) divided the Palaearctic species in the bicolon and minimus groups, which were 409 recovered as respectively monophyletic with strong support. The sampled American 410 species were divided in the puncticollis group of Perkins (1980), with only one sampled 411 species being sister to the rest of the subgenus with strong support (BS= 88%, pp= 1; 412 Figs 1a, S2), plus the discretus group of Perkins (1980). The placement of the studied 413 Nearctic species of the A. discretus group and two of the Afrotropical species (O. 414 andreiini Régimbart and O. andronius Orchymont) was uncertain both in the ML and 415 the Bayesian trees (Figs 1a, S2). We provisionally consider them within the A. minimus 416 group due to the similarities in their aedeagi and the morphology of the pronotum 417 (Orchymont, 1948; Perkins, 1980; Jäch, 1990a). The subgenus Asiobates currently 418 includes 105 described species and three subspecies occurring in all biogeographical 419 regions, except the Oriental and Australian Realms. While the puncticollis group is 420 restricted to the Nearctic Region, the bicolon and minimus groups are more widespread. 421 The former occurs in the Palaearctic and (with several undescribed species) Afrotropical 422 Regions, and the latter occurs in the Nearctic, Neotropical, Palaearctic, and Afrotropical

13 423 Regions. Many additional species of Asiobates await description, several of them 424 included in our phylogeny (Table S3). 425 426 2.3. Subgenus Aulacochthebius Kuwert, 1887. 427 Type species: Ochthebius exaratus Mulsant, 1844, by monotypy. 428 Considered as a subgenus until Perkins (1997) raised it to genus level. There are no 429 species groups defined within this subgenus, and our sampling is too incomplete to 430 draw firm conclusions. Currently the subgenus includes 13 Palaearctic, Oriental and 431 Afrotropical species (Table S1), although the taxonomy of the subgenus is in clear need 432 of revision and it is expected that the number of species will increase considerably 433 (Table S3). 434 435 2.4. Subgenus Cobalius Rey, 1886. 436 Type species: Ochthebius lejolisii Mulsant & Rey, 1861, fixed by Jäch (1989b). 437 Described as a subgenus of Ochthebius by Rey (1886), synonymised by Perkins (1997) 438 with Ochthebius s.str. and considered again as subgenus by Sabatelli et al. (2016). We 439 recovered it here as a strongly supported monophyletic lineage clearly outside 440 Ochthebius s.str., confirming its status as subgenus. Its nine recognised species and two 441 subspecies occur along the coasts of the Mediterranean Sea, the Black Sea and the 442 eastern Atlantic Ocean from Cape Verde to Scotland (Jäch, 1989b; Jäch & Skale, 2015; 443 Jäch & Delgado, 2017a). Its taxonomy is in need of revision (Sabatelli et al., 2016; Jäch 444 & Delgado, 2017a; unpublished results). 445 446 2.5. Subgenus Enicocerus Stephens, 1829. 447 Type species: Enicocerus viridiaeneus Stephens, 1829 (= Ochthebius exsculptus 448 Germar, 1824), by monotypy. 449 Enicocerus was originally described as a genus, downgraded to subgenus of Ochthebius 450 by Chenu (1851), reinstated again as genus by Perkins (1997) (within its own subtribe, 451 Enicocerina), but treated as a subgenus by subsequent authors (e.g. Jäch, 1998; Jäch & 452 Skale, 2015; Ribera et al., 2010). Our results support the exclusion of the East 453 Palaearctic and Oriental species, confirming Jäch (1998) and Skale & Jäch (2009), and 454 are in agreement with the phylogeny of Ribera et al. (2010). Enicocerus in its current 455 sense includes 16 species with a mostly Mediterranean distribution, with some species 456 reaching the British Isles, Central Europe, the Middle East and the Caucasus. One

14 457 species from eastern North America (O. benefossus LeConte, Perkins, 1980), not 458 included in our phylogeny, possibly belongs to Enicocerus as well. 459 460 2.6. Subgenus Gymnanthelius Perkins, 1997 comb.n. 461 Type species: Ochthebius hieroglyphicus Deane, 1933, by original designation. 462 The genus Gymnanthelius was introduced by Perkins (1997) for O. hieroglyphicus. 463 Subsequently, Perkins (2004b) revised the genus and transferred to Gymnanthelius two 464 other Australian species described by Deane (1931, 1937) within Ochthebius (Table 465 S1). The eight described species are distributed mostly in south-eastern Australia, with 466 some reaching as far north as Queensland (Perkins, 2004b). 467 468 2.7. Subgenus Gymnochthebius Orchymont, 1943 469 Type species: Ochthebius nitidus LeConte, 1850 by original designation. 470 Gymnochthebius was originally described as a subgenus of Ochthebius (Orchymont, 471 1943) to place several American species described under Ochthebius that could not be 472 placed in any of the described subgenera, which had been established mostly for 473 Palaearctic species. Orchymont (1943) also included three Australian species for which 474 he could examine the aedeagus and confirmed that they had the same general structure 475 as the American species. Perkins (1980) revised the American species and Perkins 476 (2005) the Australian and Papuan species, recognising another four species in addition 477 to the three previously noted by Orchymont (1943) and raising the total number of 478 species in the subgenus to 57 (Table S1). The Australian and the American species of 479 the subgenus form two well supported clades, the O. australis and O. fossatus groups, 480 with 36 and 21 species respectively (Table S1). 481 482 2.8. Subgenus Hughleechia Perkins, 1981 comb.n. 483 Type species: Hughleechia giulianii Perkins, 1981, by original designation. 484 Originally described as a monotypic genus (Perkins, 1981), a second species was 485 described by Perkins (2007a). Both species inhabit coastal rockpools in southern 486 Australia and Tasmania, in the intertidal zone and (most frequently) above the tide 487 (Perkins, 2007a). 488 489 2.9. Subgenus Micragasma Sahlberg, 1900 comb.n. 490 Type species: Micragasma paradoxum Sahlberg, 1900, by monotypy.

15 491 Described as a monotypic genus for M. paradoxum (Sahlberg, 1900). Jäch (1997a) 492 redescribed the genus and transferred O. substrigosus Reitter to Micragasma. A third 493 species was recently described from Crete (Hernando et al., 2018), and there are two 494 additional undescribed species from Central Asia (unpublished data). Our results clearly 495 show that Micragasma is nested within Ochthebius s.l., and thus we consider it a 496 subgenus of Ochthebius. 497 498 2.10. Subgenus Ochthebius Leach, 1815 499 Type species: Helophorus marinus Paykull, 1798, fixed by Orchymont (1942). 500 Within Ochthebius s.str. we recovered, with strong support, most of the currently 501 recognised species groups as monophyletic. Most species groups are entirely 502 Palaearctic, or with mostly Palaearctic species, and thus the basis for the taxonomy of 503 the subgenus is the revisionary work of Jäch (e.g. 1989a, 1990a, 1991, 1992a), who 504 distinguished 13 groups and subgroups. With only one exception (O. jengi group), they 505 were all, with some modifications, recovered as monophyletic. According to our results, 506 the 322 described species and four subspecies of Ochthebius s.str. are separated in 17 507 species groups, five of them newly established herein (Fig. 2). A few species still have 508 an uncertain phylogenetic placement. This is particularly the case for O. belucistanicus 509 Ferro, O. fissicollis Janssens and O. pierottii Ferro, which presently cannot be 510 confidently included in any of the recognised species groups, mainly because their 511 original descriptions lack information about relevant characters (Table S1). 512 513 (1) O. andraei group: Defined and revised in Jäch (1992a), with additional species 514 described in Jäch (2002) and Jäch & Delgado (2010). Currently this group includes six 515 species and one subspecies of Palaearctic distribution (Table S1), typical of saline or 516 hypersaline habitats. We could study a single species (O. patergazellae Jäch & Delgado, 517 Table S3), included in a clade together with the species of the O. notabilis, corrugatus 518 and atriceps groups (Fig. 1). The close relationship between the species of the O. 519 andraei, corrugatus, notabilis and atriceps groups were already suggested in Jäch 520 (1991, 1992a). 521 522 (2) O. atriceps group: In Jäch (1991) the species of the O. foveolatus group were 523 divided in two subgroups, (A) foveolatus subgroup, sharing some characters with the 524 species of the O. metallescens group, and (B) atriceps subgroup, sharing some

16 525 characters with the species of the O. notabilis group. We recovered both subgroups as 526 respectively monophyletic, and confirm the suspected relationships proposed by Jäch 527 (1991) (see below). Ochthebius burjkhalifa Jäch & Delgado and O. despoliatus Jäch & 528 Delgado, both from the UAE and of uncertain affinities although hypothesised to be 529 related to the O. atriceps group (Jäch & Delgado, 2014a), were found to be sister to the 530 rest of the species of the group, with strong support in the Bayesian analysis (pp= 0.95) 531 but weaker in the ML (BS= 55%) (Figs 1, S2). With the inclusion of these two species 532 the O. atriceps group includes 21 species and one subspecies (Table S1). They have a 533 mostly Palaearctic distribution but extending to East Africa (Djibouti) (Jäch & Delgado, 534 2017b). 535 536 (3) O. corrugatus group: Jäch (1992a) suggested that O. corrugatus Rosenhauer, despite 537 being related to the species of the O. andraei and notabilis groups, could not be 538 included in either of them. Our results confirm this hypothesis, but extend the O. 539 corrugatus group to include two additional Mediterranean species previously included 540 in the O. atriceps subgroup (Jäch, 1991; Table S1). 541 542 (4) O. foveolatus group: The O. foveolatus group as here defined corresponds to the O. 543 foveolatus subgroup of Jäch (1991), recovered as sister of the O. metallescens group 544 with strong support (Fig. 1). After the additions and corrections of Delgado & Jäch 545 (2009) and Jäch & Delgado (2010, 2014b) it currently includes 27 species, all 546 Palaearctic (Fig. 1; Table S1). 547 548 (5) O. kosiensis group: Jäch (1997b) established this group for O. kosiensis Champion, 549 described within Ochthebius but originally not placed in any subgenus (Champion, 550 1920). Knisch (1924) placed it in Asiobates due to the resemblance of the general 551 habitus, although the male genitalia do not correspond to those of the species of 552 Asiobates (Jäch, 1997b). Jäch (2003) recognised the similarity between O. kosiensis, O. 553 strigosus and related species, and included both species in the strigosus subgroup of the 554 metallescens group. The study of some undescribed species deposited in the NMW 555 (M.A. Jäch, manuscript in prep.) suggests that the strigosus subgroup as defined in Jäch 556 (2003) should be divided in the kosiensis and strigosus groups, with two and 16 557 described species respectively (see below; Table S1). We could not obtain any species 558 of the kosiensis group suitable for DNA extraction, and thus their phylogenetic

17 559 relationships (and composition) remain untested. Based on described and undescribed 560 species the group is so far known from the Himalaya and Myanmar. 561 562 (6) O. lobicollis group: Jäch (1990b) revised the lobicollis group, with subsequent 563 additions by Jäch (1994) and Jäch et al. (1998). It currently includes 11 species with a 564 West Palaearctic distribution (Table S1; Fig. 1b). 565 566 (7) O. marinus group: The Palaearctic species of the O. marinus group, the most 567 speciose within Ochthebius s.str., were revised by Jäch (1992b). According to our 568 results it includes the species of the O. jengi group sensu Jäch (1998) and also species 569 from the Nearctic and Neotropical Regions (O. biincisus, bisinuatus and interruptus 570 groups of Perkins, 1980); the Afrotropical Region (O. extremus and salinarius groups of 571 Perkins & Balfour-Browne, 1994; Perkins, 2011; O. capicola group of Sabatelli et al., 572 2016), including Madagascar (O. alluaudi Régimbart; Perkins, 2017); the Oriental 573 Region (O. masatakasatoi Jäch; Jäch, 1992b; Jäch & Delgado, 2017a); and the 574 Australian Region (O. queenslandicus Hansen; Jäch, 2001a; Perkins, 2007b). 575 Two of the studied subspecies were not recovered as sisters to the nominal 576 subspecies in any of the analyses: O. viridis fallaciosus Ganglbauer (sister to O. 577 arefniae Jäch & Delgado and another specimen likely representing an undescribed 578 species), and O. subpictus deletus Rey (sister to O. marinus plus O. auropallens 579 Fairmaire), in both cases with strong support (Figs 1, S1, S2; Table S1). We thus 580 upgrade the two subspecies to species, O. fallaciosus Ganglbauer, 1901 stat.n. and O. 581 deletus Rey, 1885 stat.rest. (see Jäch, 1992b and Jäch & Delgado, 2008 for a detailed 582 description of the species). The O. marinus group as here defined includes 78 species, 583 plus two species of uncertain adscription (Table S1). Most of these species seem to be 584 associated to lentic habitats, frequently saline and, especially those outside the 585 Palaearctic, coastal. 586 587 (8) O. metallescens group: The O. metallescens group was revised by Jäch (1989a). It is 588 well defined morphologically, but many species have variable isolated populations, 589 difficulting species recognition and diagnosis. This difficulty is reflected in the complex 590 taxonomic history of the group, with multiple changes in the status of some species (e.g. 591 Jäch, 1989a, 1999, 2001b). A number of species are typical of hygropetric habitats 592 covered by a thin film of water, such as seepages or the marginal areas of stony surfaces

18 593 in streams. The species group has currently 56 Palaearctic species and one subspecies 594 (Table S1). Due to the somewhat cryptic habits and restricted geographic ranges of 595 many species it is expected that this number will increase considerably. 596 597 (9) O. nitidipennis group: We include in the O. nitidipennis group the Asian species 598 formerly included in the subgenus Enicocerus (see above). As suggested by previous 599 authors (Jäch, 1989b; Skale & Jäch, 2009; Yoshitomi & Satô, 2011), morphological 600 similarities between these species and those of Enicocerus are the result of evolutionary 601 convergence, likely due to occupying similar microhabitats on the surface of rocks and 602 stones partially submerged in streams. The group currently includes 12 species in the 603 Himalaya Region and East Asia (Table S1). 604 605 (10) O. notabilis group: Jäch (1992a) recognised the O. notabilis group for species 606 formerly included in the subgenus Doryochthebius Kuwert, establishing its synonymy 607 with Ochthebius s.str. and differentiating the members of this group from the species of 608 Calobius (see below). The group includes 13 Palaearctic species, all typical of saline or 609 hypersaline habitats. 610 611 (11) O. peisonis group: Ochthebius peisonis was included in the O. marinus group by 612 Jäch (1992b). Our results, however, place the species in a very isolated and uncertain 613 position within Ochthebius s.str. We provisionally consider it in its own group, until 614 additional evidence clarifies its phylogenetic relationships. 615 616 (12) O. punctatus group: The punctatus group was defined by Jäch (1992c) to include 617 the species formerly considered under subgenus Bothochius Rey, with irregular elytral 618 punctation (Jäch, 1989c), and a series of species with similar morphological characters 619 but with regular elytral striae. The Ochthebius punctatus group includes 53 species and 620 one subspecies, mostly Palaearctic (reaching the Oriental region) but with some 621 Afrotropical species, among them the namibiensis group of Perkins & Balfour-Browne 622 (1994) (Jäch, 1992c; Hansen, 1998; Perkins, 2011; Jäch & Delgado, 2017b; Table S1). 623 624 (13) O. quadricollis group: The O. quadricollis group corresponds to the genus 625 Calobius, described for C. heeri Wollaston from Madeira. The concept of Calobius was 626 expanded by subsequent authors to include species now in different species groups (e.g.

19 627 Reitter (1886) included among them O. notabilis Rosenhauer and O. quadrifoveolatus 628 Wollaston), and was usually treated as a subgenus. It was revised by Jäch (1993), who 629 still considered it a subgenus, but was synonymised with Ochthebius s.str. by Perkins 630 (1997), who considered it to be closely related to Cobalius. Its status remained, 631 however, uncertain, with some authors treating it as a genus (e.g. Audisio et al., 2010) 632 or subgenus (e.g. Jäch & Skale, 2015). Finally, Sabatelli et al. (2016) provided evidence 633 of the phylogenetic position of Calobius, demonstrating its derived status within 634 Ochthebius s.str. and considering it as the “Calobius” lineage, named here the O. 635 quadricollis group for consistency with other species groups within Ochthebius s.str. 636 Sabatelli et al. (2016) also found that the group includes more than the five species 637 currently recognised (Table S1), in agreement with previous results from the Italian 638 species (e.g. Urbanelli et al., 2008; Audisio et al., 2010). Our results support this 639 impression, as happens with the subgenus Cobalius, which is also in need of taxonomic 640 revision. All species of the O. quadricollis group are found in coastal rockpools in the 641 Mediterranean basin and the islands of Madeira and the Canaries. 642 643 (14) O. rivalis group: Ochthebius rivalis Champion and two similar species were 644 originally considered to be a subgroup of the O. metallescens group (Jäch, 2003). Our 645 results, however, do not support a close relationship with the species of the O. 646 metallescens group, but with O. peisonis and the O. notabilis, corrugatus and andraei 647 groups (Figs 1, S2), with low support. In the Bayesian analysis the two sampled species 648 of the group were sister with strong support (pp= 1), but in the ML analysis they were 649 not placed together, although with low support (BS< 50%) (Fig. S2). Currently the 650 group includes four Asian species (including O. himalayae Jäch, originally described 651 within the O. metallescens group, Jäch, 1989a), distributed from the Himalaya to 652 Hainan Island (Table S1). 653 654 (15) Ochthebius strigosus group: Ochthebius strigosus Champion, described as 655 Ochthebius s.str., was included in the subgenus Asiobates by Jäch (1989b) based on the 656 study of female specimens only. After the discovery of males of a related species (O. 657 strigoides Jäch) they were placed in their own subgroup within the O. metallescens 658 group (Jäch, 1998). We found the only sequenced species of the group to be sister of the 659 O. lobicollis group with low support (BS= 56%, pp= 0.87), and we consider it here as a 660 distinct species group within Ochthebius s.str. The O. strigosus group currently includes

20 661 16 described plus some undescribed species, one of them included here (voucher IBE- 662 RA617). The group is distributed in the eastern Palaearctic, including Taiwan (Jäch, 663 2003; Table S1). 664 665 (16) Ochthebius sumatrensis group: In the original description, O. sumatrensis Jäch 666 could not be placed in any of the by then described groups, although some similarities 667 with O. jengi Jäch (currently in the marinus group) were noted (Jäch, 2001a). Several 668 undescribed species similar to O. sumatrensis have been collected in recent years (Jäch 669 et al. manuscript in prep.), among them the one from Hong Kong included here 670 (specimen voucher MNCN-AC16; Table S1), recovered as sister to the rest of the 671 species of Ochthebius s.str. with low support in the ML analysis (BS< 50%; Fig. S2) 672 and as sister to the punctatus group in the Bayesian analysis, also with low support (pp< 673 0.5; Fig. 1). They live in hygropetric surfaces, which makes them prone to evolutionary 674 convergence with non-related species sharing the same habitat, thus obscuring their 675 relationships. The group is distributed from the Himalaya to eastern China and 676 southward to Sumatra, where it is the only known species of Ochthebius s.str. 677 678 (17) O. vandykei group: The species of the O. vandykei group correspond to the former 679 Neochthebius, originally described as subgenus but raised to genus (within its own 680 subtribe, Neochthebiina) by Perkins (1997) based on peculiarities of their antennae and 681 the lack of ESDS. Jäch & Delgado (2014b), based on unpublished molecular data and 682 on aedeagal characters of several newly described species, synonymised Neochthebius 683 and considered it as a species group within Ochthebius s.str. The group currently 684 includes eight species from the northern Pacific coast, seven in Asia and one in North 685 America (Jäch & Delgado, 2014b; Table S1). They are all typical of rocky seashores or 686 other coastal microhabitats. 687 688 3. Genus Protochthebius Perkins, 1997 689 Type species: Protochthebius satoi Perkins, 1997, by original designation. 690 The genus Protochthebius was described by Perkins (1997) for P. satoi and O. 691 jagthanae Champion, who erected also the subtribe Protochthebiina based on 692 peculiarities of the antennae and the ESDS. Subsequently, Jäch (1997b) and Perkins 693 (1998) described another two and three species respectively. All known seven species of 694 Protochthebius are found in the Himalaya Region, Meghalaya and Laos (Table S1).

21 695 Some of them have been found by sifting forest litter or moss (Jäch, 1997b; Perkins, 696 1998). 697 We could not obtain molecular data of any of the species of Protochthebius, and 698 thus the phylogenetic placement of the genus remains uncertain. Perkins (1997) noted 699 some presumably plesiomorphic characters of the pronotum and post-ocular area of the 700 head. The species of the genus have also a reduced ESDS system (Perkins, 1997), but 701 this might be as a secondary loss due to their microhabitat preferences. Their male 702 genitalia are, however, typical of Ochthebius s.str., and when molecular data become 703 available the taxonomic status of Protochthebius may have to be changed to a subgenus 704 of Ochthebius or a species group within Ochthebius s.str., in which case P. satoi would 705 become a junior homonym. 706 707 Acknowledgements 708 709 We thank all collectors mentioned in Table S3 for allowing us to study their material, 710 and Ana Izquierdo (MNCN) and Rocío Alonso (IBE) for laboratory work. A.V. has a 711 FPI-MINECO PhD grant from the Spanish Government. This work has been partly 712 funded by project CGL2013-48950-C2 (AEI/FEDER, UE) to I.R. 713 714 References 715 716 Abellán, P., Millán, A., Ribera, I. (2009) Parallel habitat-driven differences in the 717 phylogeographical structure of two independent lineages of Mediterranean saline 718 water beetles. Molecular Ecology, 18, 3885–3902. doi:10.1111/j.1365- 719 294X.2009.04319.x 720 Andújar, C., Serrano, J., & Gómez-Zurita, J. (2012) Winding up the molecular clock in 721 the genus Carabus (Coleoptera: Carabidae): assessment of methodological 722 decisions on rate and node age estimation. BMC Evolutionary Biology, 12(1), 723 40. https://doi.org/10.1186/1471-2148-12-40 724 Antonini, G., Audisio, P., Mancini, E., de Biase, A., Tronci, C., Rossetti, G., Trizzino, 725 M. (2010) Molecular phylogeography of two Italian sibling species of Calobius 726 (Coleoptera, Hydraenidae, Ochthebiinae) inhabiting Mediterranean marine rock- 727 pools. Marine Biology, 157, 371–381. doi:10.1007/s00227-009-1324-9 728 Audisio, P., Trizzino, M., de Biase, A., Rossetti, G., Mancini, E., & Antonini, G. (2010)

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30 996 Figure legends 997 998 Fig. 1: Majority rule consensus tree obtained with BEAST for the phylogeny of 999 Ochthebiini, with the simple evolutionary models (see text). Numbers in nodes, 1000 posterior probabilities / bootstrap support values in RAxML. Names in nodes refer to 1001 the new classification. Habitus photographs correspond to species used in the analyses, 1002 with the addition of Limnebius papposus Mulsant, Hydraena riparia Kugelann (Fig. 1003 1a), Ochthebius (s.str.) bernhardi Jäch & Delgado and O. (Micrasgasma) minoicus 1004 Hernando, Villastrigo & Ribera (Fig. 1b). 1005 1006 Fig. 2: Summary tree of the phylogenetic relationships of the Ochthebiini main lineages. 1007 The width of the triangles reflects the number of species of the respective clade in the 1008 tree. Symbols in nodes: Circles, congruent topology in the maximum likelihood and 1009 Bayesian analyses; triangles: incongruent topologies; in black: nodes with good support 1010 (Bayesian posterior probability > 0.95 and maximum likelihood bootstrap support > 1011 70%) in both analyses; in grey: in one analyses only; in white: not supported nodes. Pie 1012 charts reflect the geographical distribution of the species of the respective clades. 1013 1014

31 1015 Supporting Information 1016 1017 Table S1: Table S1: Checklist of the species of Ochthebiini, with the current (following 1018 Jäch & Skale, 2015 and Jäch et al., 2016) and new classifications. In bold, type species. 1019 phyl, species included in the phylogeny (in brackets, species for which the sequenced 1020 specimen was a female). Distribution: PAL, Palaearctic; AFR, Afrotropical; AUR, 1021 Australian; NAR, Nearctic; NTR, Neotropical; ORR, Oriental; ANR, Antarctic. In 1022 brackets, specimens considered to have a Palaearctic distribution in Jäch & Skale 1023 (2015), but including the Oriental or Afrotropical Regions in Hansen (1998). 1024 1025 Table S2: Current classification of Ochthebiini, with synonymies and type species 1026 (following Jäch & Skale, 2015 and Jäch et al., 2016). In bold, taxa included in the 1027 phylogeny. 1028 1029 Table S3: List of material used in the molecular phylogeny, including voucher numbers, 1030 accession numbers of the sequences and locality data. 1031 1032 Table S4: (A) primers used for DNA amplification and sequencing reactions; (B) 1033 Typical conditions for the polymerase chain reaction. 1034 1035 Fig. S1: Majority rule consensus tree obtained with BEAST for the phylogeny of 1036 Ochthebiini with the best partition models. Numbers in nodes, posterior probabilities. 1037 1038 Fig. S2: Phylogeny obtained with RAxML, including current Ochthebiini classification. 1039 Numbers in nodes, bootstrap support values. 1040 1041 Fig. S3: Phylogeny obtained with RAxML with the nuclear genes only. Numbers in 1042 nodes, bootstrap support values. 1043

32 Asiobates sp. AN74 0.95/66 Asiobates lederi AN63 Fig. 1b 1/94 0.89/- Asiobates peregrinus AN206 Asiobates bicolon RA1171 0.53/- 1/100 Asiobates arator AN185 1/79 Asiobates striatus AI787 Asiobates crenulatus AH159 1/65 0.99/59 Asiobates cantabricus cf. AI1027 1/66 Asiobates ferroi PB28 1/100 Asiobates lenkoranus AN75 Ochthebius s.l. Asiobates stygialis AN160 1/94 1/97 0.62/- Asiobates auriculatus AH154 0.43/- Asiobates jaimei RA1081 1/100 Asiobates immaculatus AN434 1/100 Asiobates irenae AI986 1/100 Asiobates figueroi AN23 bicolon group 0.99/79 Asiobates bellieri AN290 1/100 Asiobates dilatatus AI792 1/94 Asiobates gagliardii RA121 1/100 Asiobates corsicus AN159 0.45/83 Asiobates opacus AI389 Asiobates heydeni AI390 1/89 0.99/92 Asiobates bonnairei AI1272 Asiobates montanus AN207 Asiobates sp. AN97 1/100 1/100 1/94 Asiobates sp. AN93 Asiobates remotus AI1030 1/62 Asiobates rugulosus AI637 1/100 Asiobates minimus AI447 1/73 Asiobates sanabrensis AH75 1/96 Asiobates flavipes RA437 1/100 Ochthebiini Asiobates aeneus AI914 1/79 1/76 1/88 Asiobates hokkaidensis AF213 Asiobates 1/96 Asiobates alpinus RA1114 minimus group 1/100 Asiobates andreinii cf. AN104 1/100 Asiobates andreinii AN20 0.94/34 Asiobates andronius AI498 Asiobates sp. AN292 puncticollis group Asiobates discretus AI503 0.85/- Asiobates puncticollis AI1274 Aulacochthebius sp. RA1155 1/63 1/90 Aulacochthebius sp. AI501 Aulacochthebius exaratus AI453 Aulacochthebius narentinus AF161 1/99 Aulacochthebius sp. AI499 1/99 Aulacochthebius sp. AN390 0.88/45 Aulacochthebius libertarius AI421 Aulacochthebius 1/99 0.91/71 Aulacochthebius perlaevis AI548 0.97/75 Aulacochthebius sp. AI519 1/100 Aulacochthebius sp. AI520 Tympanogaster modulatrix AN183 Tympanogaster 1/100 0.63/- Tympanogaster deanei AI372 1/100 Tympanogaster schizolabra AF167 Meropathus zelandicus AI715 Meropathus Limnebius millani AI920 0.97/65 Limnebius hieronymi AR22 0.97/89 Limnebius zaerensis AC14 1/96 0.76/36 Limnebius levantinus RA731 Limnebius cordobanus PA275 0.66/65 Limnebius crinifer AR55 1/93 1/100 Limnebius doderoi AI1174 Limnebius arenicolus AI466 Limnebius wewalkai RA108 1/100 1/49 1/100 Limnebius dioscoridus RA728 1/100 1/96 Limnebius acupunctus AI582 1/64 1/100 Limnebius extraneus RA89 Limnebius pollex RA1019 Laeliaena sahlbergi HI19 Hydraena hayashii AI691 0.49/76 0.98/74 Hydraena dochula AI518 1/99 Hydraena pygmaea AI346 0.89/75 Hydraena riberai AI568 1/94 Hydraena altamirensis AI425 1/- 0.98/- Hydraena croatica RA52 Hydraena putearius RA95 1/100 0.98/- Hydraena atrata AI314 Hydraena rugosa AI392 1/- 1/- Hydraena arenicola AI504 1/99 Hydraena impressicollis AI541 Hydraena palawanensis AH133 Cylindroselloides dybasi AI564 0.99/75 0.77/49 Rioneta uluguruensis AI415 Acrotrichis sp. AI413 1/100 Ptiliolum sp. AI649 1/71 Ptenidium pusillum AI515

-175 -150 -125 -100 -75 -50 -25 0 Fig. 1c Ochthebius corrugatus AI56 1/- 1/100 Ochthebius corrugatus cf. RA118 corrugatus group 1/100 1/50 Ochthebius perpusillus AN323 0.69/- Ochthebius gauthieri AI55 Ochthebius patergazellae RA735 1/- andraei group Ochthebius gereckei PA141 0.98/36 1/79 Ochthebius halophilus AN22 Ochthebius salinator AI53 notabilis group Ochthebius glaber PA30 1/99 Ochthebius normandi PA253 1/95 Ochthebius notabilis AI38 0.73/- Ochthebius lanarotis PA32 peisonis group 1/100 Ochthebius peisonis AN64 Ochthebius peisonis AF168 rivalis group Ochthebius himalayae AI1270 1/- Ochthebius rivalis AF81 Ochthebius rectus cplx AN291 0.98/60 Ochthebius interruptus AN219 Ochthebius s.str. 0.91/75 1/94 1/98 Ochthebius rectus cplx AN152 0.99/70 Ochthebius rectus AI464 Ochthebius uniformis AN437 0.98/50 1/100 Ochthebius gruwelli RA304 1/84 Ochthebius arizonicus AN177 Ochthebius sculptoides AN221 0.95/43 1/85 1/100 Ochthebius aztecus AN222 Ochthebius bisinuatus AN153 Ochthebius batesoni AI690 0.85/33 1/100 Ochthebius arefniae AN72 1/96 Ochthebius arefniae cf. AN446 Ochthebius viridis fallaciosus AI918 1/85 Ochthebius viridis viridis AN5 1/100 1/100 Ochthebius viridescens RA40 Ochthebius pusillus AI1028 Ochthebius evanescens AN66 0.98/58 Ochthebius lineatus AN223 1/86 0.92/- Ochthebius lineatus cplx RA1129 1/96 Ochthebius lividipennis AN71 0.92/59 Ochthebius costatellus cf. RA1119 0.75/36 Ochthebius nipponicus HI27 1/45 Ochthebius queenslandicus AN18 0.44/- 0.46/- Ochthebius chappuisi cf. AN475 0.71/42 Ochthebius mesoamericanus RA58 0.87/44 Ochthebius salinarius AN79 marinus group Ochthebius meridionalis RA373 1/76 1/44 1/100 Ochthebius marinus AI615 1/100 Ochthebius auropallens AN217 1/100 Ochthebius subpictus deletus AN433 Ochthebius subpictus subpictus AI452 0.97/29 Ochthebius pedalis AH98 1/98 1/45 Ochthebius spinasus AH100 Ochthebius capicola RA854 vandykei group Ochthebius vandykei AF159 1/100 Ochthebius yoshitomii AF121 Cobalius celatus AN441 0.58/72 1/100 1/100 Cobalius adriaticus adriaticus AN787 Cobalius subinteger AI432 0.97/59 0.97/74 Cobalius Cobalius lejolisii AI513 1/98 Cobalius freyi RA1197 1/100 Cobalius serratus AI1194 Micragasma paradoxum AF116 Micragasma Enicocerus exsculptus cf. AI925 1/98 1/94 Enicocerus exsculptus AI374 0.89/51 Enicocerus colveranus AI791 1/72 Enicocerus halbherri AH190 1/88 Enicocerus legionensis AI507 1/98 Enicocerus aguilerai AI387 1/80 1/97 Enicocerus gibbosus AI365 Enicocerus 1/100 Enicocerus melanescens AI344 Enicocerus granulatus AI427 0.76/81 0.84/94 Hughleechia Enicocerus saboorii RA739 Hughleechia giulianii AI716 Gymnochthebius setosus AF165 1/100 Gymnochthebius australis AI583 australis group 1/97 1/100 Gymnochthebius probus AI584 Gymnochthebius lividus AF163 Gymnochthebius 1/100 Gymnochthebius germaini AI454 1/100 0.83/98 Gymnochthebius sp. AI569 1/100 Gymnochthebius fossatus AN498 0.96/94 fossatus group Gymnochthebius peruvianus AI689 Angiochthebius 1/100 Angiochthebius plesiotypus AI562 Angiochthebius plesiotypus AI502 1/94 Gymnanthelius Gymnanthelius opacicollis AF162 1/100 Fig. 1a Gymnanthelius porchi AF164

-175 -150 -125 -100 -75 -50 -25 0 0.99/98 Ochthebius metallescens metallescens AI376 1/100 Ochthebius metallescens plato RA1057 0.56/- Ochthebius morettii RA1173 Ochthebius serpentinus AI819 1/100 Ochthebius maxfischeri AN164 Ochthebius albacetinus RA1181 1/95 1/99 Ochthebius judemaesi RA1179 Ochthebius diazi RA595 0.8/- Ochthebius sp. AN77 0.99/47 Ochthebius poweri AC26 1/100 Ochthebius pedroi RA1082 Ochthebius hivae RA744 metallescens group 1/100 Ochthebius gayosoi AN311 1/100 1/88 Ochthebius semotus RA1180 Ochthebius griotes AI943 1/100 Ochthebius semisericeus AI1064 0.82/56 0.98/15 Ochthebius preissi AN448 Ochthebius scopuli AN382 0.81/54 Ochthebius wurayah RA733 1/100 Ochthebius magnannulatus AN328 0.99/71 Ochthebius mediterraneus AI422 1/61 Ochthebius satoi AF210 0.75/54 Ochthebius elisae RA746 0.98/50 Ochthebius sidanus AF132 1/89 Ochthebius pedicularius AN809 1/82 0.99/49 Ochthebius harteni RA705 foveolatus group Ochthebius hajeki RA1231 1/100 1/93 Ochthebius foveolatus AI801 Ochthebius marginalis AN522 1/100 0.97/94 Ochthebius virgula AF134 1/44 Ochthebius merinidicus RA1023 1/100 Ochthebius eyrei AN600 0.92/67 Ochthebius lobicollis RA242 Ochthebius caesaraugustae AI1195 0.99/62 1/100 Ochthebius quadrifossulatus AI226 0.63/57 Ochthebius delgadoi AN364 lobicollis group 1/100 Ochthebius lapidicola AN126 Ochthebius basilicatus AN801 0.87/56 1/96 Ochthebius tivelunus AI420 strigosus group Ochthebius strigosus group RA617 Ochthebius brevicollis AN440 0.92/25 1/99 0.9/42 Ochthebius steinbuehleri AI517 Ochthebius quadricollis AI431 1/94 quadricollis group Ochthebius quadricollis cplx2 AI514 1/99 1/100 Ochthebius quadricollis cplx1 AN11 Ochthebius heeri AN200 Ochthebius sp. RA104 1/100 0.82/- Ochthebius klapperichi AI1269 Ochthebius ragusae AI1029 1/45 Ochthebius quadrifoveolatus AI636 0.98/82 Ochthebius nobilis AF133 1/49 Ochthebius sp. AF191 1/100 Ochthebius punctatus RA286 1/100 Ochthebius tudmirensis AI467 1/94 Ochthebius lanuginosus AN204 0.98/98 1/100 0.97/86 Ochthebius grandipennis AI616 1/100 Ochthebius pilosus AN363 Ochthebius joosti AF169 Ochthebius inermis RA795 Ochthebius monseti RA124 1/94 Ochthebius montesi AI491 punctatus group 1/81 1/94 Ochthebius mahmoodi RA126 1/94 Ochthebius micans AN474 1/99 Ochthebius difficilis cplx AN76 1/100 0.93/- Ochthebius difficilis AI944 Ochthebius nilssoni AH76 Ochthebius pagotrichus AI497 0.81/- Ochthebius silfverbergi RA1021 0.76/67 1/100 Ochthebius bifoveolatus AN381 1/94 Ochthebius cuprescens PA276 Ochthebius nanus PA277 sumatrensis group Ochthebius sumatrensis group AC16 nitidipennis group Ochthebius japonicus HI26 1/100 0.79/- Ochthebius hasegawai AI1289 1/99 Ochthebius thermalis AN451 1/100 Ochthebius tacapasensis baeticus AN365 Ochthebius dentifer PA290 1/94 Ochthebius loulae AN476 Ochthebius andalusicus PA296 1/95 Ochthebius anxifer AI945 1/100 0.84/81 Ochthebius atriceps AN210 atriceps group 0.72/95 Ochthebius depressionis AF171 Ochthebius burjkhalifa RA737 0.95/55 Fig. 1b Ochthebius despoliatus RA736

-175 -150 -125 -100 -75 -50 -25 0 metallescens gr.

Palaearctic foveolatus gr.

Nearctic lobicollis gr. strigosus gr. Neotropical quadricollis gr. Afrotropical

punctatus gr. Oriental

Australian sumatrensis gr. subgenus Ochthebius Antarctic nitidipennis gr.

atriceps gr.

corrugatus gr. andraei gr. notabilis gr. peisonis gr. rivalis gr.

marinus gr.

vandykei gr. subg. Cobalius subg. Micragasma

subg. Enicocerus subg. Hughleechia

australis gr. subgenus Gymnochthebius fossatus gr.

subg. Angiochthebius

genus Ochthebius subg. Gymnanthelius

bicolon gr. subgenus Asiobates

Ochthebiini minimus gr.

puncticollis gr.

subg. Aulacochthebius

genus Tympanogaster subg. Tympanogaster

subg. Hygrotympanogaster

Hydraenidae genus Meropathus

Limnebius Laeliaena Hydraena Ptiliidae