1 Postprint, accepted in 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 hydraenid subfamily, with terrestrial species as well as species in almost all 25 types of 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 186 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 six genera: 36 Meropathus Enderlein, Ochthebius Leach, Protochthebius Perkins, Prototympanogaster 37 Perkins, Tympallopatrum Perkins and Tympanogaster Janssens. The genus Ochthebius 38 is here divided into nine subgenera in addition to Ochthebius s.str.: (1) O. 39 (Angiochthebius) Jäch & Ribera; (2) O. (Asiobates) Thomson; (3) O. (Aulacochthebius) 40 Kuwert; (4) O. (Cobalius) Rey; (5) O. (Enicocerus) Stephens; (6) O. (Gymnanthelius) 41 Perkins comb.n.; (7) O. (Gymnochthebius) Orchymont comb.n.; (8) O. (Hughleechia) 42 Perkins comb.n.; and (9) O. (Micragasma) Sahlberg. Within Ochthebius s.str., 17 43 species groups are proposed, five of them newly established (3, 9, 11, 13 and 16): (1) 44 andraei; (2) atriceps; (3) corrugatus; (4) foveolatus; (5) kosiensis; (6) lobicollis; (7) 45 marinus; (8) metallescens; (9) nitidipennis; (10) notabilis; (11) peisonis; (12) punctatus; 46 (13) quadricollis; (14) rivalis; (15) strigosus; (16) sumatrensis; and (17) vandykei. We 47 elevated to species rank two subspecies of Ochthebius: O. fallaciosus Ganglbauer stat.n. 48 (former subspecies of O. viridis Peyron) and O. deletus Rey stat.rest. (former subspecies 49 of O. subpictus Wollaston). 50 51 52 53 54

2 55 Introduction 56 57 Ochthebiinae, with ca. 650 species and 11 subspecies described, are the second 58 most diverse subfamily of the water beetle family Hydraenidae (Hansen, 1998; Jäch & 59 Balke, 2008; Tables 1, S1). They occur in all biogeographic regions, including the 60 Antarctic islands of Kerguelen and Heard, where they are the only Hydraenidae present 61 (Hansen, 1998). Ochthebiinae are the ecologically most diverse hydraenid subfamily, 62 with terrestrial species, species living in the interface between land and water, as well as 63 in most types of aquatic environments (Jäch et al., 2016). A large number of species are 64 tolerant to hypersaline waters, living either in coastal rockpools (e.g. Cobalius Rey or 65 Calobius Wollaston, Antonini et al., 2010; Sabatelli et al., 2016), coastal or inland 66 saltpans or in inland hypersaline streams (especially the O. notabilis group, but also 67 many other species in different groups, Abellán et al., 2009; Millán et al., 2011). 68 The external morphology of the species of Ochthebiinae is more heterogeneous 69 than in other Holarctic lineages of Hydraenidae (e.g. Hydraena Kugelann, Trizzino et 70 al., 2013, or Limnebius Leach, Rudoy et al., 2016). This has resulted in a more complex 71 taxonomy, with mostly all of the described genera or subgenera with uncertain 72 relationships. Thus, the genus Ochthebius Leach was divided in 16 subgenera by 73 Kuwert (1887), but all of them with the exception of Aulacochthebius Kuwert were later 74 synonymised (see Tables 1, S2 for a synopsis of the classification and Table S1 for a 75 complete checklist of Ochthebiini). 76 Ochthebiinae were divided by Perkins (1980) in two tribes, Ochthebiini and 77 Ochtheosini, the latter for the single terrestrial genus Ochtheosus Perkins, with two 78 species. The monotypic genera Edaphobates Jäch & Díaz and Ginkgoscia Jäch & Díaz, 79 for which we could not obtain fresh material for DNA extraction, were tentatively 80 hypothesized to be related to Ochtheosus by Jäch & Díaz (2003, 2004) and thus we 81 consider them within Ochtheosini. Perkins (1997) divided Ochthebiini in five newly 82 defined subtribes: Enicocerina (for the single genus Enicocerus Stephens), Meropathina 83 (Meropathus Enderlein, Tympallopatrum Perkins, Tympanogaster Perkins and the 84 recently described Prototympanogaster Perkins, Perkins, 2018), Neochthebiina 85 (Neochthebius Orchymont), Ochthebiina (Ochthebius, Gymnochthebius Orchymont, 86 Hughleechia Perkins, Gymnanthelius Perkins, Aulacochthebius and Micragasma 87 Sahlberg) and Protochthebiina (Protochthebius Perkins). Enicocerus was treated as a 88 subgenus by several subsequent authors (e.g. Jäch, 1998; Ribera et al., 2010; Jäch &

3 89 Skale, 2015), and Neochthebius was treated as a synonym of Ochthebius s.str. by Jäch 90 & Delgado (2014b), leaving ten genera in Ochthebiini, most of them described in the 91 20th century (Table 1). Ochthebius is the oldest available generic name (Leach, 1815; 92 Hansen, 1998; Table 1), grouped into four recognised subgenera with mostly Palaearctic 93 distribution: Asiobates Thomson, Calobius, Enicocerus and Ochthebius (Jäch & Skale, 94 2015; Tables 1, S1; see the detailed taxonomic history of subgenera and species groups 95 in the Discussion below). Within Ochthebius s.str., the most diverse subgenus, several 96 informal species groups have been defined, which have undergone important 97 modifications through their taxonomic history (Tables S1, S2). 98 The classification and proposed relationships within Ochthebiini have also 99 experienced many modifications during the last four decades. Perkins (1980) revised the 100 by then known American species, and proposed a phylogeny derived from the 101 examination of some morphological characters. Gymnochthebius was placed as sister to 102 the remaining taxa, which were divided in two lineages: (1) Meropathus plus 103 Neochthebius (currently a synonym of Ochthebius, Table S2) and (2) Ochthebius plus 104 Asiobates. Subsequently, Perkins (1997) synonymized four subgenera with Ochthebius 105 (Calobius, Cobalius, Liochthebius Sahlberg and Notochthebius Orchymont), and 106 described three additional genera (Tables 1, S2). Based mostly on the exocrine secretion 107 delivery system (ESDS), he divided the subfamily in two tribes, Ochtheosini for the 108 newly described Ochtheosus and Ochthebiini, divided in turn in subtribes, with 109 unresolved relationship among them. Ochtheosus was considered to have some 110 plesiomorphic characters similar to some southern African genera (e.g. antennae with 111 11 antennomeres, as in many Prosthetopinae, Perkins, 1997; see also Beutel et al., 112 2003), and did not share several of the most characteristic synapomorphies with the 113 remaining Ochthebiinae, in particular the structure of the tentorial arms, galea and 114 lacinia. 115 The first formal cladistic analysis of the family Hydraenidae was published by 116 Beutel et al. (2003), but sampling was too incomplete to resolve internal relationships 117 within Ochthebiinae other than the sister relationship of Meropathus with Ochthebius + 118 Gymnochthebius. There is no published global molecular phylogeny of the entire family 119 Hydraenidae or subfamily Ochthebiinae, but in recent years some detailed molecular 120 phylogenies for some lineages have been published, such as the Ochthebius notabilis 121 group (Abellán et al., 2009) and Enicocerus (Ribera et al., 2010). In Abellán et al. 122 (2009) an extensive phylogeny of Ochthebius and some related genera using only

4 123 mitochondrial markers was used to estimate the phylogenetic diversity of the Iberian 124 fauna. The sampling of some geographical areas was, however, very incomplete, as the 125 intention was not to produce a phylogenetic study. Still, most Palaearctic lineages were 126 represented, which allowed to establish the monophyly of most of the included 127 genera/subgenera and of the recognised species groups, although internal groups had 128 poor relationships between them. Sabatelli et al. (2016) used these data to study the 129 origin of species typical of rockpools, recovering basically the same relationships and 130 establishing a new species group for the South African O. capicola Péringuey. In the 131 same paper the subgenus Cobalius was found to be outside Ochthebius s.str., but 132 Calobius nested within it, referring to it as the “Calobius” lineage. 133 In this study we provide a comprehensive phylogeny of Ochthebiini, based on 134 mitochondrial and nuclear sequence data including representatives of most lineages. We 135 introduce several changes in the taxonomic classification to accommodate our 136 phylogenetic results, and provide a complete checklist based on our new classification 137 (Table S1). 138 139 Material and Methods 140 141 Taxon sampling 142 143 We studied 186 species and four subspecies of the 641 described species and 11 144 described subspecies of Ochthebiini, plus 29 specimens corresponding to undetermined 145 or still undescribed species (Tables S1, S3). For two species with an isolated or 146 unsupported placement (O. plesiotypus Perkins and O. peisonis Ganglbauer, see below) 147 we sequenced two specimens to test for possible sequencing mistakes. We included 148 examples of eight of the eleven genera currently recognised in the tribe, all subgenera 149 but two (within genus Tympanogaster), and all recognised species groups within the 150 genus Ochthebius but one (O. kosiensis group, Tables 1, S1, S3). The three missing 151 genera, Tympallopatrum (Australia), Protochthebius (Asia) and Prototympanogaster 152 (Lord Howe Island), have four, seven and a single species respectively (Table S1). 153 We used as outgroups 31 species of other Hydraenidae genera (Hydraena, 154 Laeliaena Sahlberg and Limnebius) and of Ptiliidae. Trees were rooted in the split 155 between Hydraenidae and Ptiliidae, considered to be sister groups both based on

5 156 molecular (e.g. Hunt et al., 2007; McKenna et al., 2015; Zhang et al., 2018) and 157 morphological evidence (Hansen, 1997; Lawrence et al., 2011). 158 159 DNA extraction and sequencing 160 161 Specimens were killed and preserved in absolute ethanol. DNA was extracted 162 with a standard phenol-chloroform extraction or by commercial extraction kits (mostly 163 Quiagen DNeasy Tissue Kit, Hildesheim, Germany) following the instructions of the 164 manufacturers. DNA samples and voucher specimens are kept in the collections of the 165 Institute of Evolutionary Biology (IBE, Barcelona), Museo Nacional de Ciencias 166 Naturales (MNCN, Madrid) and Naturhistorisches Museum Wien (NMW, Vienna). We 167 sequenced fragments of six genes in five sequencing reactions, three mitochondrial: (1) 168 5' end of the cytochrome c oxidase subunit 1 (the standard barcode, Hebert et al., 2003) 169 (COI-5’), (2) 3' end of cytochrome c oxidase subunit 1 (COI-3’), (3) 5’ end of 16S RNA 170 (16S) plus the Leucine tRNA transfer (tRNA-Leu) plus 5’ end of NADH dehydrogenase 171 subunit I (NAD1); and two nuclear: (4) an internal fragment of the large ribosomal unit, 172 28S RNA (28S) and (5) an internal fragment of the small ribosomal unit, 18S RNA 173 (18S) (see Table S4 for details on primers used and typical polymerase chain reaction 174 (PCR) conditions). Sequences were assembled and edited with GENEIOUS v10.1 175 (Kearse et al., 2012); new sequences (a total of 897) were deposited in the ENA 176 database with accession numbers LT990690-LT991586. 177 178 Phylogenetic analyses 179 180 Edited sequences were aligned using the online version of MAFFT v.7 with the 181 G-INS-I algorithm (Katoh et al., 2009). We used PartitionFinder v1.1.1 (Lanfear et al., 182 2012) to estimate the evolutionary model that best fitted the data, using one partition for 183 each gene fragment (six partitions in total), and using Akaike Information Criterion 184 (AIC) scores as selection criteria. Phylogenetic analyses were made using Bayesian 185 probabilities in BEAST 1.8 (Drummond & Rambaut, 2007), using the partition and 186 evolutionary models selected by PartitionFinder, with a Yule speciation process as tree 187 prior. There are few fossils usable for calibrating the phylogeny of Hydraenidae. The 188 oldest recognised members of the family are Ochthebiites Ponomarenko, from the 189 Jurassic (Arnol’di et al., 1991; Ponomarenko & Prokin, 2015; Yamamoto et al., 2017),

6 190 but they cannot confidently be placed in any extant lineage. One of the best preserved 191 hydraenid fossils is Archaeodraena cretacea Jäch & Yamamoto from Upper Cretaceous 192 Burmese amber (ca. 99 Ma, Yamamoto et al., 2017), which likely belongs to the crown 193 Hydraenidae. Both fossils are compatible with an estimate of ca. 170 Ma for the split 194 between Hydraenidae and Ptiliidae obtained in recent molecular phylogenies calibrated 195 with a range of fossils (Hunt et al., 2007; McKenna et al., 2015). We thus used this 196 estimation to calibrate our tree, with a normal distribution with a standard deviation of 1 197 Ma and an uncorrelated lognormal relaxed clock. A Middle Jurassic separation between 198 Hydraenidae and Ptiliidae is considerably younger than the estimation of Toussaint et 199 al. (2016) (Middle Triassic, 243 Ma), but older than the more recent estimation of 200 Zhang et al. (2018) (Upper Jurassic, ca. 150 Ma), both of which we consider to be less 201 plausible. In any case, it must be noted that the main objectives of our study do not 202 require an absolute calibration of the phylogeny of Ochthebiini, which is done only as a 203 preliminary exploration. 204 We ran the analyses for 100 million generations login results each 5,000, and 205 checked convergence to estimate the burn-in fraction with Tracer v1.6 (Drummond & 206 Rambaut, 2007). We ran an additional ML phylogenetic reconstruction with RAxML- 207 HPC2 (Stamatakis, 2006) in the CIPRES portal (Miller et al., 2010), using the same 208 partition scheme as in BEAST with a GTR+G model estimated independently for each 209 partition. Node supports values were estimated with 100 pseudoreplicas using a rapid 210 bootstrapping algorithm (Stamatakis et al., 2008). The same ML analysis was repeated 211 only with the nuclear sequence (18S and 28S). 212 213 Results 214 215 The final matrix included 252 terminals with 3,656 aligned characters. Protein 216 coding regions had no indels except for the 3' end of COI-3, where some species had an 217 additional codon. The best partitioning scheme obtained with PartitionFinder had six 218 partitions corresponding to (1) COI-5, (2) COI-3, (3) 16S+tRNA-Leu, (4) NAD1, (5) 219 18S and (6) 28S. The optimal evolutionary model was GTR+I+G for all partitions 220 except for NAD1 (best model TMV) and 28S (best model SYM). The BEAST run 221 implementing the best models did, however, not converge properly, mostly due to the 222 parameters related to the estimation of the branch lengths, especially for the genes 223 NAD1, 18S and 28S. We thus did a second run with simpler models for these genes

7 224 (HKY+G+I), which converged adequately. The topologies of the two Bayesian analyses 225 were, however, almost identical (Figs 1, S1), and unless specified we report only the 226 results of the analyses with the better parameter convergence (i.e. with the simpler 227 evolutionary models). 228 229 Molecular phylogeny 230 231 The topologies obtained in the ML and the two Bayesian analyses were very 232 similar, differing only in some poorly supported nodes (Figs 1, S1, S2), most notably in 233 the position of Hughleechia (see below). The ML tree with the nuclear sequence only 234 had a topology very similar to that obtained with the combined data, although with a 235 generally lower resolution and support. The main difference was the recovery of 236 Ochthebiini as paraphyletic, with the genus Hydraena as sister to Tympanogaster plus 237 Meropathus, although with very low support (bootstrap support, BS= 53%; Fig. S3). 238 Genera, subgenera and most species groups were, however, recovered as monophyletic 239 with strong support, with internal topologies very similar to that of the combined ML 240 tree (Figs S2, S3). 241 In the ML and Bayesian trees with the combined nuclear and mitochondrial data 242 the monophyly of Ochthebiini was strongly supported, as well as their separation in two 243 clades, (1) Meropathus plus Tympanogaster and (2) Ochthebius sensu lato (s.l. herein). 244 Meropathus was nested within a paraphyletic Tympanogaster in the ML tree (combined 245 and nuclear only) and in the Bayesian tree with the best models, and sister to 246 Tympanogaster with low support in the Bayesian tree with simpler models (posterior 247 probability, pp= 0.63; Figs 1a, S1, S2). 248 Within Ochthebius s.l. Asiobates and Aulacochthebius were sister groups in the 249 Bayesian tree with low support (pp= 0.85), and both sisters to the rest of Ochthebiini. In 250 the ML analysis Asiobates and Aulacochthebius were paraphyletic with respect to the 251 rest of Ochthebiini, also with low support (BS< 50%) (Figs 1a, S2). In both analyses 252 Ochthebiini minus Asiobates and Aulacochthebius were monophyletic with strong 253 support (BS= 80%; pp= 1; Fig. 1a). 254 The remaining Ochthebiini were divided in a series of well supported clades 255 corresponding to traditionally recognised genera or subgenera, but with poorly resolved 256 relationships among them. (1) Enicocerus, strongly supported and with well resolved 257 internal relationships, sister to the Australian Hughleechia in ML and the Bayesian

8 258 analysis with the simpler models (BS = 81%, pp= 0.76; Figs 1b, S2). In the Bayesian 259 analysis with the best models Hughleechia was sister to the clade formed by 260 Micragasma and Cobalius, with low support (pp= 0.88; Fig. S1). (2) A clade including 261 Gymnochthebius and Gymnanthelius, the latter as sister to Angiochthebius Jäch & 262 Ribera (Gymnochthebius plesiotypus group of Perkins, 1980; see Jäch & Ribera, 2018) 263 (BS= 94%, pp= 0.96). Within Gymnochthebius, the Australian and American species 264 were respectively monophyletic and sisters, with very strong support both in the ML 265 and Bayesian trees (Figs 1b, S2). (3) Cobalius, with a strongly supported monophyly 266 (BS= 98%, pp= 1) and sister to the only sequenced species of Micragasma, also with 267 strong support (BS= 100%, pp= 1) (Figs 1b, S2). (4) Ochthebius sensu stricto (s.str. 268 herein), including Calobius, strongly supported both in the ML (BS= 94%) and 269 Bayesian (pp= 1) trees (Figs 1b, S2). 270 Within Ochthebius s.str. most established Palaearctic species groups were 271 recovered as monophyletic (see Discussion, Figs 1b, 1c, S2). Their monophyly was 272 strongly supported in the ML and Bayesian trees, with the only exception of the group 273 of species related to the O. atriceps and O. notabilis groups in the ML analyses (see 274 below). The main difference with established groups was the expansion of the O. 275 marinus group to include the South African O. capicola and the American O. biincisus, 276 bisinuatus and interruptus groups of Perkins (1980). The O. foveolatus group of Jäch 277 (1991) was split in three clades: (1) O. foveolatus group, sister to the O. metallescens 278 group with strong support in both ML and Bayesian trees (BS= 71%, pp= 0.99); (2) O. 279 atriceps group and (3) O. corrugatus group. The latter two formed a clade with the 280 species of the O. notabilis and O. andraei groups, strongly supported in the Bayesian 281 tree (pp= 1) but not in the ML tree (BS< 50%), in which the group also included one of 282 the two sampled species of the O. rivalis group (Figs 1c, S2). Two coastal lineages, 283 Calobius and the O. vandykei group (formerly genus Neochthebius), were nested within 284 Ochthebius s.str., the former as sister to the O. lobicollis + O. strigosus groups (BS< 285 50%, pp= 0.92) and the latter as sister to the O. marinus group (BS< 50%, pp= 0.97) 286 (Figs 1b, 1c, S2). 287 According to our calibration scheme, with a separation between Hydraenidae 288 and Ptiliidae at 170 Ma, the estimated age of crown Hydraenidae was 106 Ma (HPD 289 122.8–90.2 Ma), and that of the crown Ochthebiini 93 Ma (HPD 109.7–80.8 Ma). The 290 basal diversification of Ochthebiini was reconstructed as having occurred in a relatively 291 short temporal window, with genera, subgenera and most species groups with an origin

9 292 between ca. 87–60 Ma (Fig. 1; see Table 2 for the estimated evolutionary rates of all 293 partitions). 294 295 Discussion 296 297 Our results strongly support the monophyly of Ochthebiini, but our sampling did 298 not allow to test for the monophyly of Ochthebiinae, or its position within Hydraenidae. 299 Within Ochthebiini our results recover two well supported clades: Meropathus plus 300 Tympanogaster, and Ochthebius s.l. (Fig. 2; see below for a detailed discussion of the 301 taxonomic classification of Ochthebiini). We did not find evidence to support the five 302 subtribes proposed by Perkins (1997), which are therefore not considered here. 303 We did not find evidence for a clear separation between the studied species of 304 Tympanogaster and Meropathus, in agreement with previous studies (Hansen, 1991). 305 However, we could not obtain material of the genera Prototympanogaster and 306 Tympallopatrum, considered to be closely related to Tympanogaster by Perkins (1997, 307 2018), and two of the subgenera of Tympanogaster (Plesiotympanogaster Perkins and 308 Topotympanogaster Perkins), so until more data become available we refrain from any 309 taxonomic change and consider Prototympanogaster, Tympallopatrum and 310 Tympanogaster as valid genera (Tables 1, S1; Fig. 2) (see Perkins, 2006 for a discussion 311 on the subgeneric classification of Tympanogaster). The species of Meropathus, 312 Prototympanogaster, Tympallopatrum and Tympanogaster are found in the Australian 313 Region, on two Antarctic islands (Kerguelen and Heard) and on several Subantarctic 314 islands, such as Campbell Island, Crozet Islands, Prince Edward Island and Falkland 315 Islands. 316 The second lineage, genus Ochthebius s.l., included all the non-Australian 317 Ochthebiini, as well as several Australian species. Our results agree remarkably well 318 with the currently recognised subgenera and many of the established species groups, 319 which were recovered as monophyletic with general strong support (Fig. 2). The 320 relationships between these lineages, however, do not confirm some previous 321 hypotheses on their relationships. Thus, Aulacochthebius was not found to be closely 322 related to Gymnochthebius, as proposed in Hansen (1991), but to Asiobates; 323 Micragasma and Hughleechia were not among the basal lineages and Cobalius and 324 Calobius were not closely related, as hypothesised in Perkins (1997). Novel 325 relationships found here are the possible sister relationship between Hughleechia and

10 326 Enicocerus, and the close relationship between Gymnochthebius and Gymnanthelius. 327 Interestingly, within the clade Gymnochthebius + Gymnanthelius + Angiochthebius 328 there are two cladogenetic events separating American from Australian species: one 329 within Gymnochthebius, dated at 73 Ma (95% HPD 87–60 Ma), and another separating 330 the Australian Gymnanthelius and the Chilean Angiochthebius, dated at 60 Ma (95% 331 HPD 78–51 Ma). Although a detailed biogeographic analysis is outside the scope of this 332 paper, it is interesting to note that these estimations are too recent for a tectonic split 333 between Australia and South America (i.e. west and east Gondwana), dated at ca. 130 334 Ma (McIntyre et al., 2017). Our calibration would thus require a different scenario, 335 probably through the colonization of some southern islands or the Antarctica. An older 336 age for these nodes is unlikely, given that our rate estimations are already slower than 337 most recent estimations for the same genes in other groups of Coleoptera (Table 2; see 338 e.g. Papadopoulou et al., 2010; Andújar et al., 2012; Cieslak et al., 2014). 339 340 Taxonomic classification of Ochthebiini Thomson, 1859 341 342 1. Genus Meropathus Enderlein, 1910 343 Type species: Meropathus chuni Enderlein, 1910, by monotypy. 344 Meropathus was described as genus, considered as subgenus of Ochthebius by 345 Orchymont (1938) and reinstated again as genus by Jeannel (1940). Bameul (1989) 346 redescribed the genus and recognised 12 species (in two species groups), transferring O. 347 schizolabrus Deane to Meropathus. Hansen (1991) noted the difficulty in establishing 348 clear distinctions within the Meropathus-Tympanogaster complex. Meropathus includes 349 seven New Zealand, Antarctic and Subantarctic species plus M. labratus Deane from 350 Queensland (Table S1). They are all found in coastal habitats, usually among debris and 351 algae (Bameul, 1989). 352 353 2. Genus Prototympanogaster Perkins, 2018 354 Type species: Prototympanogaster lordhowensis Perkins, 2018, by original designation. 355 Prototympanogaster was described by Perkins (2018) as a monotypic genus based on 356 two males collected in 2003 in Lord Howe Island. This genus seems to be closely 357 related to Tympanogaster, but without its characteristic glabrous tabella in the 358 metaventrite (Perkins, 2018) 359

11 360 3. Genus Tympallopatrum Perkins, 1997 361 Type species: Tympallopatrum longitudum Perkins, 1997, by original designation. 362 Tympallopatrum was described by Perkins (1997) as a monotypic genus within 363 Meropathina. Subsequently, Perkins (2004a) revised the genus and described three 364 additional species, all of them from western Australia (Table S1). We could not obtain 365 any representative of this genus for our study, and thus its phylogenetic placement 366 remains untested. 367 368 4. Genus Tympanogaster Janssens, 1967 369 Type species: Tympanogaster deanei Perkins, 1979 (replacement name for Ochthebius 370 longipes Deane, 1931), by monotypy. 371 Described by Janssens (1967) as a monotypic genus for O. longipes (= T. deanei 372 Perkins), Perkins (1997) redescribed Tympanogaster and transferred some species from 373 Meropathus. Perkins (2006) revised the genus and described three subgenera and 76 374 new species, raising the total number of the species in the genus to 84 (Tables 1, S1), all 375 distributed in Australia and Tasmania. 376 377 4.1. Subgenus Hygrotympanogaster Perkins, 2006 378 Type species: Tympanogaster maureenae Perkins, 2006, by original designation. 379 Hygrotympanogaster Perkins was described by Perkins (2006) as a subgenus of 380 Tympanogaster, to include mostly hygropetric species in south-western Australia. 381 Currently it includes 36 species (Perkins, 2006) (Table S1). 382 383 4.2. Subgenus Plesiotympanogaster Perkins, 2006 384 Type species: Tympanogaster thayerae Perkins, 2006, by original designation. 385 Plesiotympanogaster was described by Perkins (2006) as a subgenus of Tympanogaster 386 to include the type species plus Ochthebius costatus Deane (Table S1). Both species 387 were considered to have plesiomorphic characters within the genus. 388 389 4.3. Subgenus Topotympanogaster Perkins, 2006 390 Type species: Tympanogaster crista Perkins, 2006, by original designation. 391 Topotympanogaster was described by Perkins (2006) as a subgenus of Tympanogaster 392 to include eight Australian species, all described in Perkins (2006) (Table S1). We could

12 393 not obtain any representative of this and the previous subgenus for our study, and thus 394 their phylogenetic placement remain untested. 395 396 4.4. Subgenus Tympanogaster Janssens, 1967 397 Type species: Tympanogaster deanei Perkins, 1979 (replacement name for Ochthebius 398 longipes Deane, 1931), by monotypy. 399 Tympanogaster s.str. was revised by Perkins (2006), raising the total number of the 400 species to 38 (Tables 1, S1), all distributed in Australia and Tasmania. 401 402 5. Genus Ochthebius Leach, 1815 403 Type species: Helophorus marinus Paykull, 1798, fixed by Orchymont (1942). 404 The second well supported lineage within Ochthebiini includes the remaining 405 genera/subgenera with in some cases uncertain relationships among them. We consider 406 Ochthebius a single genus with 540 species and nine subspecies in ten well supported 407 subgenera, corresponding in most cases to currently recognised taxa (Fig. 2). One 408 additional species, O. eremita Knisch from Fiji, cannot be confidently placed in any of 409 the described subgenera, and it is left as incertae sedis within the genus Ochthebius 410 (Hansen, 1998; Table S1). 411 412 5.1. Subgenus Angiochthebius Jäch & Ribera, 2018 413 Type species: Gymnochthebius plesiotypus Perkins, 1980, by original designation. 414 The subgenus Angiochthebius was created for the Gymnochthebius plesiotypus species 415 group (sensu Perkins, 1980), which now includes three South American species (Jäch & 416 Ribera, 2018; Table S1). The species of the G. plesiotypus group were included within 417 Gymnochthebius by Perkins (1980) as they share a bifid apex of the aedeagus, but 418 external characters (e.g. the pubescent 5th abdominal ventrite) and some aedeagal 419 characters (Jäch & Ribera, 2018) as well as molecular data (Figs 1b, S2) warrant their 420 removal from Gymnochthebius and their status as a distinct subgenus of Ochthebius. 421 422 5.2. Subgenus Asiobates Thomson, 1859 423 Type species: Ochthebius rufimarginatus Stephens, 1829 (= O. bicolon Germar, 1824), 424 by monotypy. 425 Originally described as a genus, but downgraded to subgenus by Seidlitz (1875), and 426 treated as such by most authors (e.g. Jäch, 1990a; Hansen, 1991; Perkins, 1997). Jäch

13 427 (1990a) divided the Palaearctic species in the bicolon and minimus groups, which were 428 recovered as respectively monophyletic with strong support. The sampled American 429 species were divided in the puncticollis group of Perkins (1980), with only one sampled 430 species being sister to the rest of the subgenus with strong support (BS= 88%, pp= 1; 431 Figs 1a, S2), plus the discretus group of Perkins (1980). The placement of the studied 432 Nearctic species of the A. discretus group and two of the Afrotropical species (O. 433 andreiini Régimbart and O. andronius Orchymont) was uncertain both in the ML and 434 the Bayesian trees (Figs 1a, S2). We provisionally consider them within the A. minimus 435 group due to the similarities in their aedeagi and the morphology of the pronotum 436 (Orchymont, 1948; Perkins, 1980; Jäch, 1990a). The subgenus Asiobates currently 437 includes 105 described species and three subspecies occurring in all biogeographical 438 regions, except the Oriental and Australian Realms. While the puncticollis group is 439 restricted to the Nearctic Region, the bicolon and minimus groups are more widespread. 440 The former occurs in the Palaearctic and (with several undescribed species) Afrotropical 441 Regions, and the latter occurs in the Nearctic, Neotropical, Palaearctic, and Afrotropical 442 Regions. Many additional species of Asiobates await description, several of them 443 included in our phylogeny (Table S3). 444 445 5.3. Subgenus Aulacochthebius Kuwert, 1887. 446 Type species: Ochthebius exaratus Mulsant, 1844, by monotypy. 447 Considered as a subgenus until Perkins (1997) raised it to genus level. There are no 448 species groups defined within this subgenus, and our sampling is too incomplete to 449 draw firm conclusions. Currently the subgenus includes 13 Palaearctic, Oriental and 450 Afrotropical species (Table S1), although the taxonomy of the subgenus is in clear need 451 of revision and it is expected that the number of species will increase considerably 452 (Table S3). 453 454 5.4. Subgenus Cobalius Rey, 1886. 455 Type species: Ochthebius lejolisii Mulsant & Rey, 1861, fixed by Jäch (1989b). 456 Described as a subgenus of Ochthebius by Rey (1886), synonymised by Perkins (1997) 457 with Ochthebius s.str. and considered again as subgenus by Sabatelli et al. (2016). We 458 recovered it here as a strongly supported monophyletic lineage clearly outside 459 Ochthebius s.str., confirming its status as subgenus. Its nine recognised species and two 460 subspecies occur along the coasts of the Mediterranean Sea, the Black Sea and the

14 461 eastern Atlantic Ocean from Cape Verde to Scotland (Jäch, 1989b; Jäch & Skale, 2015; 462 Jäch & Delgado, 2017a). Its taxonomy is in need of revision (Sabatelli et al., 2016; Jäch 463 & Delgado, 2017a; unpublished results). 464 465 5.5. Subgenus Enicocerus Stephens, 1829. 466 Type species: Enicocerus viridiaeneus Stephens, 1829 (= Ochthebius exsculptus 467 Germar, 1824), by monotypy. 468 Enicocerus was originally described as a genus, downgraded to subgenus of Ochthebius 469 by Chenu (1851), reinstated again as genus by Perkins (1997) (within its own subtribe, 470 Enicocerina), but treated as a subgenus by subsequent authors (e.g. Jäch, 1998; Jäch & 471 Skale, 2015; Ribera et al., 2010). Our results support the exclusion of the East 472 Palaearctic and Oriental species, confirming Jäch (1998) and Skale & Jäch (2009), and 473 are in agreement with the phylogeny of Ribera et al. (2010). Enicocerus in its current 474 sense includes 16 species with a mostly Mediterranean distribution, with some species 475 reaching the British Isles, Central Europe, the Middle East and the Caucasus. One 476 species from eastern North America, O. benefossus LeConte, not included in our 477 phylogeny, is here tentatively placed in Enicocerus (following Perkins, 1980); it might, 478 however, instead belong to the O. (s.str.) nitidipennis group, or to a species group of its 479 own. 480 481 5.6. Subgenus Gymnanthelius Perkins, 1997 comb.n. 482 Type species: Ochthebius hieroglyphicus Deane, 1933, by original designation. 483 The genus Gymnanthelius was introduced by Perkins (1997) for O. hieroglyphicus. 484 Subsequently, Perkins (2004b) revised the genus and transferred to Gymnanthelius two 485 other Australian species described by Deane (1931, 1937) within Ochthebius (Table 486 S1). The eight described species are distributed mostly in south-eastern Australia, with 487 some reaching as far north as Queensland (Perkins, 2004b). 488 489 5.7. Subgenus Gymnochthebius Orchymont, 1943 490 Type species: Ochthebius nitidus LeConte, 1850 by original designation. 491 Gymnochthebius was originally described as a subgenus of Ochthebius (Orchymont, 492 1943) to place several American species described under Ochthebius that could not be 493 placed in any of the described subgenera, which had been established mostly for 494 Palaearctic species. Orchymont (1943) also included three Australian species for which

15 495 he could examine the aedeagus and confirmed that they had the same general structure 496 as the American species. Perkins (1980) revised the American species and Perkins 497 (2005) the Australian and Papuan species, recognising another four species in addition 498 to the three previously noted by Orchymont (1943) and raising the total number of 499 species in the subgenus to 58 (Table S1). The Australian and the American species of 500 the subgenus form two well supported clades, the O. australis and O. fossatus groups, 501 with 36 and 22 species respectively (Table S1). 502 503 5.8. Subgenus Hughleechia Perkins, 1981 comb.n. 504 Type species: Hughleechia giulianii Perkins, 1981, by original designation. 505 Originally described as a monotypic genus (Perkins, 1981), a second species was 506 described by Perkins (2007a). Both species inhabit coastal rockpools in southern 507 Australia and Tasmania, in the intertidal zone and (most frequently) above the tide 508 (Perkins, 2007a). 509 510 5.9. Subgenus Micragasma Sahlberg, 1900 comb.n. 511 Type species: Micragasma paradoxum Sahlberg, 1900, by monotypy. 512 Described as a monotypic genus for M. paradoxum (Sahlberg, 1900). Jäch (1997a) 513 redescribed the genus and transferred O. substrigosus Reitter to Micragasma. A third 514 species was recently described from Crete (Hernando et al., 2017), and there are two 515 additional undescribed species from Central Asia (unpublished data). Our results clearly 516 show that Micragasma is nested within Ochthebius s.l., and thus we consider it a 517 subgenus of Ochthebius. 518 519 5.10. Subgenus Ochthebius Leach, 1815 520 Type species: Helophorus marinus Paykull, 1798, fixed by Orchymont (1942). 521 Within Ochthebius s.str. we recovered, with strong support, most of the currently 522 recognised species groups as monophyletic. Most species groups are entirely 523 Palaearctic, or with mostly Palaearctic species, and thus the basis for the taxonomy of 524 the subgenus is the revisionary work of Jäch (e.g. 1989a, 1990a, 1991, 1992a), who 525 distinguished 13 groups and subgroups. With only one exception (O. jengi group), they 526 were all, with some modifications, recovered as monophyletic. According to our results, 527 the 322 described species and four subspecies of Ochthebius s.str. are separated in 17 528 species groups, five of them newly established herein (Fig. 2). A few species still have

16 529 an uncertain phylogenetic placement. This is particularly the case for O. belucistanicus 530 Ferro, O caudatus Frivaldszky, O. fissicollis Janssens and O. pierottii Ferro, which 531 presently cannot be confidently included in any of the recognised species groups, 532 mainly because their original descriptions lack information about relevant characters 533 (Table S1). 534 535 (1) O. andraei group: Defined and revised in Jäch (1992a), with additional species 536 described in Jäch (2002) and Jäch & Delgado (2010). Currently this group includes six 537 species and one subspecies of Palaearctic distribution (Table S1), typical of saline or 538 hypersaline habitats. We could study a single species (O. patergazellae Jäch & Delgado, 539 Table S3), included in a clade together with the species of the O. notabilis, corrugatus 540 and atriceps groups (Fig. 1). The close relationship between the species of the O. 541 andraei, corrugatus, notabilis and atriceps groups were already suggested in Jäch 542 (1991, 1992a). 543 544 (2) O. atriceps group: In Jäch (1991) the species of the O. foveolatus group were 545 divided in two subgroups, (A) foveolatus subgroup, sharing some characters with the 546 species of the O. metallescens group, and (B) atriceps subgroup, sharing some 547 characters with the species of the O. notabilis group. We recovered both subgroups as 548 respectively monophyletic, and confirm the suspected relationships proposed by Jäch 549 (1991) (see below). Ochthebius burjkhalifa Jäch & Delgado and O. despoliatus Jäch & 550 Delgado, both from the UAE and of uncertain affinities although hypothesised to be 551 related to the O. atriceps group (Jäch & Delgado, 2014a), were found to be sister to the 552 rest of the species of the group, with strong support in the Bayesian analysis (pp= 0.95) 553 but weaker in the ML (BS= 55%) (Figs 1, S2). With the inclusion of these two species 554 the O. atriceps group includes 20 species and one subspecies (Table S1). They have a 555 mostly Palaearctic distribution but extending to East Africa (Djibouti) (Jäch & Delgado, 556 2017b). 557 558 (3) O. corrugatus group: Jäch (1992a) suggested that O. corrugatus Rosenhauer, despite 559 being related to the species of the O. andraei and notabilis groups, could not be 560 included in either of them. Our results confirm this hypothesis, but extend the O. 561 corrugatus group to include two additional Mediterranean species previously included 562 in the O. atriceps subgroup (Jäch, 1991; Table S1).

17 563 564 (4) O. foveolatus group: The O. foveolatus group as here defined corresponds to the O. 565 foveolatus subgroup of Jäch (1991), recovered as sister of the O. metallescens group 566 with strong support (Fig. 1). After the additions and corrections of Delgado & Jäch 567 (2009) and Jäch & Delgado (2010, 2014b) it currently includes 27 species, all 568 Palaearctic (Fig. 1; Table S1). 569 570 (5) O. kosiensis group: Jäch (1997b) established this group for O. kosiensis Champion, 571 described within Ochthebius but originally not placed in any subgenus (Champion, 572 1920). Knisch (1924) placed it in Asiobates due to the resemblance of the general 573 habitus, although the male genitalia do not correspond to those of the species of 574 Asiobates (Jäch, 1997b). Jäch (2003) recognised the similarity between O. kosiensis, O. 575 strigosus and related species, and included both species in the strigosus subgroup of the 576 metallescens group. The study of some undescribed species deposited in the NMW 577 (M.A. Jäch, manuscript in prep.) suggests that the strigosus subgroup as defined in Jäch 578 (2003) should be divided in the kosiensis and strigosus groups, with two and 16 579 described species respectively (see below; Table S1). We could not obtain any species 580 of the kosiensis group suitable for DNA extraction, and thus their phylogenetic 581 relationships (and composition) remain untested. Based on described and undescribed 582 species the group is so far known from the Himalaya and Myanmar. 583 584 (6) O. lobicollis group: Jäch (1990b) revised the lobicollis group, with subsequent 585 additions by Jäch (1994) and Jäch et al. (1998). It currently includes 11 species with a 586 West Palaearctic distribution (Table S1; Fig. 1b). 587 588 (7) O. marinus group: The Palaearctic species of the O. marinus group, the most 589 speciose within Ochthebius s.str., were revised by Jäch (1992b). According to our 590 results it includes the species of the O. jengi group sensu Jäch (1998) and also species 591 from the Nearctic and Neotropical Regions (O. biincisus, bisinuatus and interruptus 592 groups of Perkins, 1980); the Afrotropical Region (O. extremus and salinarius groups of 593 Perkins & Balfour-Browne, 1994; Perkins, 2011; O. capicola group of Sabatelli et al., 594 2016), including Madagascar (O. alluaudi Régimbart; Perkins, 2017); the Oriental 595 Region (O. masatakasatoi Jäch; Jäch, 1992b; Jäch & Delgado, 2017a); and the 596 Australian Region (O. queenslandicus Hansen; Jäch, 2001a; Perkins, 2007b).

18 597 Two of the studied subspecies were not recovered as sisters to the nominal 598 subspecies in any of the analyses: O. viridis fallaciosus Ganglbauer (sister to O. 599 arefniae Jäch & Delgado and another specimen likely representing an undescribed 600 species), and O. subpictus deletus Rey (sister to O. marinus plus O. auropallens 601 Fairmaire), in both cases with strong support (Figs 1, S1, S2; Table S1). We thus 602 upgrade the two subspecies to species, O. fallaciosus Ganglbauer, 1901 stat.n. and O. 603 deletus Rey, 1885 stat.rest. (see Jäch, 1992b and Jäch & Delgado, 2008 for a detailed 604 description of the species). The O. marinus group as here defined includes 78 species, 605 plus two species of uncertain adscription (Table S1). Most of these species seem to be 606 associated to lentic habitats, frequently saline, especially those outside the Palaearctic, 607 coastal. 608 609 (8) O. metallescens group: The O. metallescens group was revised by Jäch (1989a). It is 610 well defined morphologically, but many species have variable isolated populations, 611 difficulting species recognition and diagnosis. This difficulty is reflected in the complex 612 taxonomic history of the group, with multiple changes in the status of some species (e.g. 613 Jäch, 1989a, 1999, 2001b). A number of species are typical of hygropetric habitats 614 covered by a thin film of water, such as seepages or the marginal areas of stony surfaces 615 in streams. The species group has currently 56 Palaearctic species and one subspecies 616 (Table S1). Due to the somewhat cryptic habits and restricted geographic ranges of 617 many species it is expected that this number will increase considerably. 618 619 (9) O. nitidipennis group: We include in the O. nitidipennis group the Asian species 620 formerly included in the subgenus Enicocerus (see above). As suggested by previous 621 authors (Jäch, 1989b; Skale & Jäch, 2009; Yoshitomi & Satô, 2011), morphological 622 similarities between these species and those of Enicocerus are the result of evolutionary 623 convergence, likely due to occupying similar microhabitats on the surface of rocks and 624 stones partially submerged in streams. The group currently includes 12 species in the 625 Himalaya Region and East Asia (Table S1). 626 627 (10) O. notabilis group: Jäch (1992a) recognised the O. notabilis group for species 628 formerly included in the subgenus Doryochthebius Kuwert, establishing its synonymy 629 with Ochthebius s.str. and differentiating the members of this group from the species of

19 630 Calobius (see below). The group includes 13 Palaearctic species, all typical of saline or 631 hypersaline habitats. 632 633 (11) O. peisonis group: Ochthebius peisonis was included in the O. marinus group by 634 Jäch (1992b). Our results, however, place the species in a very isolated and uncertain 635 position within Ochthebius s.str. We provisionally consider it in its own group, until 636 additional evidence clarifies its phylogenetic relationships. 637 638 (12) O. punctatus group: The punctatus group was defined by Jäch (1992c) to include 639 the species formerly considered under subgenus Bothochius Rey, with irregular elytral 640 punctation (Jäch, 1989c), and a series of species with similar morphological characters 641 but with regular elytral striae. The Ochthebius punctatus group includes 53 species and 642 one subspecies, mostly Palaearctic (reaching the Oriental Region) but with some 643 Afrotropical species, among them the namibiensis group of Perkins & Balfour-Browne 644 (1994) (Jäch, 1992c; Hansen, 1998; Perkins, 2011; Jäch & Delgado, 2017b; Table S1). 645 646 (13) O. quadricollis group: The O. quadricollis group corresponds to the genus 647 Calobius, described for C. heeri Wollaston from Madeira. The concept of Calobius was 648 expanded by subsequent authors to include species now in different species groups (e.g. 649 Reitter, 1886 included among them O. notabilis Rosenhauer and O. quadrifoveolatus 650 Wollaston), and was usually treated as a subgenus. It was revised by Jäch (1993), who 651 still considered it a subgenus, but was synonymised with Ochthebius s.str. by Perkins 652 (1997), who considered it to be closely related to Cobalius. Its status remained, 653 however, uncertain, with some authors treating it as a genus (e.g. Audisio et al., 2010) 654 or subgenus (e.g. Jäch & Skale, 2015). Finally, Sabatelli et al. (2016) provided evidence 655 of the phylogenetic position of Calobius, demonstrating its derived status within 656 Ochthebius s.str. and considering it as the “Calobius” lineage, named here the O. 657 quadricollis group for consistency with other species groups within Ochthebius s.str. 658 Sabatelli et al. (2016) also found that the group includes more than the five species 659 currently recognised (Table S1), in agreement with previous results from the Italian 660 species (e.g. Urbanelli et al., 2008; Audisio et al., 2010). Our results support this 661 impression, as happens with the subgenus Cobalius, which is also in need of taxonomic 662 revision. All species of the O. quadricollis group are found in coastal rockpools in the 663 Mediterranean basin and the islands of Madeira and the Canaries.

20 664 665 (14) O. rivalis group: Ochthebius rivalis Champion and two similar species were 666 originally considered to be a subgroup of the O. metallescens group (Jäch, 2003). Our 667 results, however, do not support a close relationship with the species of the O. 668 metallescens group, but with O. peisonis and the O. notabilis, corrugatus and andraei 669 groups (Figs 1, S2), with low support. In the Bayesian analysis the two sampled species 670 of the group were sister with strong support (pp= 1), but in the ML analysis they were 671 not placed together, although with low support (BS< 50%) (Fig. S2). Currently the 672 group includes four Asian species (including O. himalayae Jäch, originally described 673 within the O. metallescens group, Jäch, 1989a), distributed from the Himalaya to 674 Hainan Island (Table S1). 675 676 (15) Ochthebius strigosus group: Ochthebius strigosus Champion, described as 677 Ochthebius s.str., was included in the subgenus Asiobates by Jäch (1989b) based on the 678 study of female specimens only. After the discovery of males of a related species (O. 679 strigoides Jäch) they were placed in their own subgroup within the O. metallescens 680 group (Jäch, 1998). We found the only sequenced species of the group to be sister of the 681 O. lobicollis group with low support (BS= 56%, pp= 0.87), and we consider it here as a 682 distinct species group within Ochthebius s.str. The O. strigosus group currently includes 683 16 described plus some undescribed species, one of them included here (voucher IBE- 684 RA617). The group is distributed in the eastern Palaearctic, including Taiwan (Jäch, 685 2003; Table S1). 686 687 (16) Ochthebius sumatrensis group: In the original description, O. sumatrensis Jäch 688 could not be placed in any of the by then described groups, although some similarities 689 with O. jengi Jäch (currently in the marinus group) were noted (Jäch, 2001a). Several 690 undescribed species similar to O. sumatrensis have been collected in recent years (Jäch 691 et al. manuscript in prep.), among them the one from Hong Kong included here 692 (specimen voucher MNCN-AC16; Table S1), recovered as sister to the rest of the 693 species of Ochthebius s.str. with low support in the ML analysis (BS< 50%; Fig. S2) 694 and as sister to the punctatus group in the Bayesian analysis, also with low support (pp< 695 0.5; Fig. 1). They live in hygropetric surfaces, which makes them prone to evolutionary 696 convergence with non-related species sharing the same habitat, thus obscuring their

21 697 relationships. The group is distributed from the Himalaya to eastern China and 698 southward to Sumatra, where it is the only known species of Ochthebius s.str. 699 700 (17) O. vandykei group: The species of the O. vandykei group correspond to the former 701 Neochthebius, originally described as subgenus but raised to genus (within its own 702 subtribe, Neochthebiina) by Perkins (1997) based on peculiarities of their antennae and 703 the lack of ESDS. Jäch & Delgado (2014b), based on unpublished molecular data and 704 on aedeagal characters, synonymised Neochthebius and considered it as a species group 705 within Ochthebius s.str. The group currently includes eight species from the northern 706 Pacific coast, seven in Asia and one in North America (Jäch & Delgado, 2014b; Table 707 S1). They are all typical of rocky seashores or other coastal microhabitats. 708 709 5. Genus Protochthebius Perkins, 1997 710 Type species: Protochthebius satoi Perkins, 1997, by original designation. 711 The genus Protochthebius was described by Perkins (1997) for P. satoi and O. 712 jagthanae Champion, who erected also the subtribe Protochthebiina based on 713 peculiarities of the antennae and the ESDS. Subsequently, Jäch (1997b) and Perkins 714 (1998) described another two and three species respectively. All known seven species of 715 Protochthebius are found in the Himalaya Region, Meghalaya and Laos (Table S1). 716 Some of them have been found by sifting forest litter or moss (Jäch, 1997b; Perkins, 717 1998). 718 We could not obtain molecular data of any of the species of Protochthebius, and 719 thus the phylogenetic placement of the genus remains uncertain. Perkins (1997) noted 720 some presumably plesiomorphic characters of the pronotum and post-ocular area of the 721 head. The species of the genus have also a reduced ESDS system (Perkins, 1997), but 722 this might be a secondary loss due to their microhabitat preferences. Their male 723 genitalia are, however, typical of Ochthebius s.str., and when molecular data become 724 available the taxonomic status of Protochthebius may have to be changed to a subgenus 725 of Ochthebius or a species group within Ochthebius s.str., in which case P. satoi would 726 become a junior homonym. 727 728 Acknowledgements 729

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31 1034 Figure legends 1035 1036 Fig. 1: Majority rule consensus tree obtained with BEAST for the phylogeny of 1037 Ochthebiini, with the simple evolutionary models (see text). Numbers in nodes, 1038 posterior probabilities / bootstrap support values in RAxML. Names in nodes refer to 1039 the new classification. Habitus photographs correspond to species used in the analyses, 1040 with the addition of Limnebius papposus Mulsant, Hydraena riparia Kugelann (Fig. 1041 1a), Ochthebius (s.str.) bernhardi Jäch & Delgado and O. (Micrasgasma) minoicus 1042 Hernando, Villastrigo & Ribera (Fig. 1b). 1043 1044 Fig. 2: Summary tree of the phylogenetic relationships of the Ochthebiini main lineages. 1045 The width of the triangles reflects the number of species of the respective clade in the 1046 tree. Symbols in nodes: Circles, congruent topology in the maximum likelihood and 1047 Bayesian analyses; triangles: incongruent topologies; in black: nodes with good support 1048 (Bayesian posterior probability > 0.95 and maximum likelihood bootstrap support > 1049 70%) in both analyses; in grey: in one analyses only; in white: not supported nodes. Pie 1050 charts reflect the geographical distribution of the species of the respective clades. 1051 1052

32 1053 Supporting Information 1054 1055 Table S1: Checklist of the species of Ochthebiini, with the current (following Jäch & 1056 Skale, 2015 and Jäch et al., 2016) and new classifications. In bold, type species. phyl, 1057 species included in the phylogeny (in brackets, species for which the sequenced 1058 specimen was a female). Distribution: PAL, Palaearctic; AFR, Afrotropical; AUR, 1059 Australian; NAR, Nearctic; NTR, Neotropical; ORR, Oriental; ANR, Antarctic. In 1060 brackets, specimens considered to have a Palaearctic distribution in Jäch & Skale 1061 (2015), but including the Oriental or Afrotropical Regions in Hansen (1998). 1062 1063 Table S2: Current classification of Ochthebiini, with synonymies and type species 1064 (following Jäch & Skale, 2015 and Jäch et al., 2016). In bold, taxa included in the 1065 phylogeny. 1066 1067 Table S3: List of material used in the molecular phylogeny, including voucher numbers, 1068 accession numbers of the sequences and locality data. In bold, newly obtained 1069 sequences. 1070 1071 Table S4: (A) primers used for DNA amplification and sequencing reactions; (B) 1072 Typical conditions for the polymerase chain reaction. 1073 1074 Fig. S1: Majority rule consensus tree obtained with BEAST for the phylogeny of 1075 Ochthebiini with the best partition models. Numbers in nodes, posterior probabilities. 1076 1077 Fig. S2: Phylogeny obtained with RAxML, including current Ochthebiini classification. 1078 Numbers in nodes, bootstrap support values. 1079 1080 Fig. S3: Phylogeny obtained with RAxML with the nuclear genes only. Numbers in 1081 nodes, bootstrap support values. 1082

33 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