Cladistics

Cladistics 24 (2008) 563–590 10.1111/j.1096-0031.2007.00192.x

Phylogeny and diversification of diving (Coleoptera: )

Ignacio Riberaa,*, Alfried P. Voglerb,c and Michael Balked

aDepartamentode Biodiversidad y Biologı´a Evolutiva, Museo Nacional de Ciencias Naturales, Jose´ Gutie´rrez Abascal 2, Madrid 28006, Spain; bDepartment of Entomology, Natural History Museum, Cromwell Road, London SW7 5BD, UK; cDivision of Biology, Department of Life Sciences, Imperial College London, Silwood Park Campus, Ascot SL5 7PY, UK; dZoologische Staatssammlung, Muenchhausenstrasse 21, D-81247 Mu¨nchen, Germany Accepted 30 July 2007

Abstract

Dytiscidae is the most diverse family of beetles in which both adults and larvae are aquatic, with examples of extreme morphological and ecological adaptations. Despite continuous attention from systematists and ecologists, existing phylogenetic hypotheses remain unsatisfactory because of limited taxon sampling or low node support. Here we provide a phylogenetic tree inferred from four gene fragments (cox1, rrnL, H3 and SSU, 4000 aligned base pairs), including 222 species in 116 of 174 known genera and 25 of 26 tribes. We aligned ribosomal genes prior to tree building with parsimony and Bayesian methods using three approaches: progressive pair-wise alignment with refinement, progressive alignment modeling the evolution of indels, and deletion of hypervariable sites. Results were generally congruent across alignment and tree inference methods. Basal relationships were not well defined, although we identified 28 well supported lineages corresponding to recognized tribes or groups of genera, among which the most prominent novel results were the polyphyly of ; the grouping of Pachydrini with Bidessini, Peschetius with Methlini and Coptotomus within ; the monophyly of all Australian Hydroporini (Necterosoma group), and their relationship with the Graptodytes and Deronectes groups plus Hygrotini. We found support for a clade formed by plus Laccophilini, and their sister relationship with Cybistrini and Copelatinae. The tree provided a framework for the analysis of species diversification in Dytiscidae. We found a positive correlation between the number of species in a lineage and the age of the crown group as estimated through a molecular clock approach, but the correlation with the stem age was non-significant. Imbalances between sister clades were significant for several nodes, but the residuals of the regression of species numbers with the crown age of the group identified only Bidessini and the Coptotomus + Agaporomorphus clade as lineages with, respectively, above and below expected levels of species diversity. The Willi Hennig Society 2008.

Approximately 25 families in three of four suborders They also occur in specialized habitats such as ground- of Coleoptera are typically aquatic in some of their life water aquifers, high altitude lakes and streams, or stages (Beutel and Leschen, 2005). Among these, the bromeliad water tanks in the forest canopy (Franciscolo, Dytiscidae (predaceous diving beetles), with some 4000 1979; Balke, 2005). Although most species are tightly described species (Nilsson, 2001, 2003a, 2004; Nilsson associated with their aquatic habitat throughout their and Fery, 2006) represent the perhaps most conspicuous life cycle except in the pupal stage, many species are group. Dytiscids range in size from about 1–50 mm, and strong flyers and able to disperse readily over land. are predators both as larvae and adults. They are found Diving beetles have been used as model organisms in in virtually any aquatic freshwater ecosystems, from a variety of ecological and evolutionary subjects, such as lakes, streams, springs, wet rock surfaces and puddles. coexistence and competition of closely related taxa (e.g., Juliano and Lawton, 1990a,b; Scheffer and Nes, 2006), *Corresponding author: the evolution of the stygobiontic fauna (Leys et al., E-mail address: [email protected] 2003, 2005), the role of habitat constraints in large-scale

The Willi Hennig Society 2008 564 I. Ribera et al./Cladistics 24 (2008) 563–590 macroecological patterns (Ribera et al., 2001, 2003b), or Hydroporinae (Wolfe, 1985, 1988). The position of the mechanics and functional morphology of swimming Laccornellus and Canthyporus within Hydroporini was behavior (Nachtigall, 1961; Ribera and Nilsson, 1995). also questioned (e.g., Roughley and Wolfe, 1987), as They are also among the best known groups of beetles, well as the composition of Bidessini and Hyphydrini being studied by an active research community, and (Bistro¨m, 1988; Bistro¨m et al., 1997). recent comprehensive revisions of many genera and an The most recent comprehensive taxonomic ordination up-to-date world catalog are available (Nilsson, 2001). of the family is that of Miller (2001a) (Table 1), based Despite this wide research interest, very few studies have on a phylogenetic analysis of morphological characters addressed phylogenetic relationships at the level of the and followed in the world catalog of Nilsson (2001). entire family using morphological (Burmeister, 1976; Main novelties were the recognition of Matinae and its Miller, 2001a) or molecular (Ribera et al., 2002b) placement as sister to the remaining Dytiscidae, the characters. These studies were limited in terms of taxon separation of from Colymbetinae, and the sampling or the set of characters used (mostly female establishment of Hydrodytinae for some inconspicuous genitalia; small subunit rRNA, SSU), and support levels taxa previously considered within Copelatinae. The were generally low. taxon sampling and phylogenetic resolution within The most recent taxonomic revision of the whole Hydroporinae were generally limited, and no major family was that of Sharp (1882), separating two major changes were proposed. In a subsequent analysis groups according to the articulation between the meta- centered on Hydroporinae (Miller et al., 2006), and thoracic anepisternum and the mesocoxa: the ‘‘Dytisci- despite the better sampling, the phylogenetic relation- dae Fragmentati’’ (no contiguous articulation), ships were equally poorly supported. In the first family including what are now families Hygrobiidae and wide molecular phylogenetic study of Dytiscidae Noteridae plus and Vatellini, and ‘‘Dyti- (Ribera et al., 2002b), based on 70 full SSU scidae Complicati’’ (contiguous articulation), including sequences, support was also generally low, although Amphizoidae and the rest of current Dytiscidae. Within some groups of genera within Hydroporinae were the latter, the major divisions separated the Hydropo- strongly supported (e.g., and Graptodytes rinae (excluding Vatellini) from the ‘‘Colymbetides’’ and groups, Hygrotini, Bidessini). what is the Dytiscinae in recent classifications. Here, we expand this molecular analysis of Dytiscidae In his monograph of the Palaearctic Dytiscidae, to include all major supra-generic groups and nearly Zimmermann (1930, 1933) already considered Hygro- 70% of the genera, based on four genes (4000–5000 biidae and Amphizoidae as separate families, and aligned nucleotides). Specific emphasis of taxonomic divided Dytiscidae in five subfamilies: Noterinae (now sampling was on the subfamily Hydroporinae, and in Noteridae), Laccophilinae, Hydroporinae, Colymbeti- particular Hydroporini, where the constitution and nae and Dytiscinae. The composition of these groups relationships of lineages remain particularly poorly has remained largely unchanged except for the large known. We aim to provide a phylogenetic framework Colymbetinae, which has experienced successive refine- for future evolutionary and systematic studies of Dytisc- ments by removing taxa with deviating morphologies idae, by identifying the major monophyletic lineages and no apparent close relatives. Thus, the Nearctic within the family and some of their phylogenetic Coptotomus was considered by Bo¨ving and Craighead relationships. The resulting tree was also used to study (1931) to form the subfamily Coptotominae, mostly relative diversification rates in different groups of because of the unusual larvae (with lateral gills on the Dytiscidae, as a first step of establishing the causes of abdomen). Burmeister (1976) recognized the non-mono- their high species richness relative to other aquatic phyly of the remaining ‘‘Colymbetines’’, associating lineages of beetles. Agabetes to Laccophilinae and placing the bulk of Copelatinae in an unresolved position within Dytisci- dae. Subsequent authors removed Lancetes (Ruhnau Materials and methods and Brancucci, 1984) and Hydrotrupes (Beutel, 1994) from the ‘‘Colymbetine’’ pool. Taxon sampling The phylogeny of the most diverse group of Dytisci- dae, the Hydroporinae, remains poorly understood The here follows the most recent world (Miller et al., 2006; Michat and Alarie, in press). Several catalog of Dytiscidae (Nilsson, 2001), with the following major lineages have been recognized, with, for example, modifications: (1) Tribe Carabdytini (comprising Carab- Zimmermann (1930, 1933) considering six tribes: Vatel- dytes) was considered part of Colymbetini, as shown by lini, Methlini, Hyphydrini, Hydrovatini, Bidessini and Balke (2001), Balke et al. (2007), and confirmed here. (2) Hydroporini. The first cladistic analyses showed that Tribe Hydroporini was divided in several groups of Hydroporini as defined at the time was polyphyletic, as genera (named after the oldest valid genus name), based Laccornis was hypothesized to be sister to the remaining on previous works and according to our results here: I. Ribera et al./Cladistics 24 (2008) 563–590 565

Table 1 Current classification of Dytiscidae (following Nilsson, 2001 and updates, see Text), with the modifications used in this study, and the number of genera, species described (up to February 2007), and sampled taxa. The number of species used in the analyses (Figs 5 and 6; Table 7) differ due to the exclusion of genera for which there were no molecular data or of genera of uncertain placement, and to the inclusion of known undescribed species of Copelatinae (M. Balke, unpublished data, 2007). Taxa marked with asterisks are groups of genera currently included in tribes Hydroporini and Carabhydrini (see Methods). Genus Lioporeus (two species, not sampled), currently in Hydroporini, is placed among ‘‘Incertae sedis’’

No. Sampled No. Sampled Subfamily Tribe genera genera species species Agabinae Agabini 10 8 391 21 Colymbetinae Anisomeriini 2 0 2 0 Colymbetini 8 7 131 10 Matinae Matini 3 1 8 2 Copelatinae Copelatini 6 6 585 11 Coptotominae Coptotomini 1 1 5 2 Dytiscinae Aciliini 7 5 69 5 Cybistrini 7 5 134 10 Dytiscini 1 1 27 3 Hyderodini 1 1 2 1 Eretini 1 1 4 2 Hydaticini 2 2 139 3 Aubehydrini 1 1 2 1 Hydrodytinae Hydrodytini 2 1 4 1 Hydroporinae Canthyporus*11 354 Deronectes gr* 6 6 183 27 Graptodytes gr* 7 6 46 10 Hydroporus gr* 6 6 260 11 Laccornellus*11 22 Necterosoma gr* 11 10 125 9 Peschetius*1191 Bidessini 38 17 609 27 Hygrotini 4 2 133 10 Hydrovatini 2 1 207 3 Hyphydrini 14 10 316 16 Laccornini 1 1 10 2 Methlini 2 2 41 4 Pachydrini 2 1 14 2 Vatellini 2 2 57 4 Incertae sedis 10 0 31 0 Laccophilinae Laccophilini 12 7 402 15 Agabetini 1 1 2 1 Lancetinae Lancetini 1 1 22 2 Totals 33 174 116 4007 222

Canthyporus group (Roughley and Wolfe, 1987), Dero- genera of Dytiscidae (as of February 2007; Table 1), we nectes group (Nilsson and Angus, 1992), Graptodytes included 116 or 67%. Twelve of the missing genera are group (Seidlitz, 1887; Ribera et al., 2002b), Hydroporus stygobiontic, with a total of 16 species, and two genera group (Ribera et al., 2002b), and Necterosoma group are secondarily terrestrial, with four species. Some of (see Table 1 and Appendix 1). (3) Tribe Carabhydrini these are likely to be highly modified species derived (comprising Carabhydrus) was included within the from within larger genera, as already established in the Necterosoma group of genera, based on the current case of stygobiontic species of Papuadytes (Balke et al. study and in previous molecular results (Balke and 2004b), Limbodessus (Balke and Ribera, 2004) and Ribera, 2004). (4) Tribe Pachydrini was considered Microdytes (Wewalka et al., 2007). A missing genus of valid, as originally proposed by Bistro¨m et al. (1997) uncertain relationships is Lioporeus, with two Nearctic and confirmed in Ribera and Balke (2007). species, which could be a plesiomorphic lineage within All recognized subfamilies and tribes of Dytiscidae Hydroporini without close living relatives (Wolfe, 1985). were included in the study, with the only exception of The missing genera have a combined diversity of 220 Anisomeriini (two genera with one species each from species, less than 6% of the total species diversity of the Juan Ferna´ndez and Tristan da Cunha; Nilsson, 2001). Dytiscidae. These two taxa are likely to be highly modified species of Outgroups included the three most closely related Rhantus in the R. signatus group (M. Balke, unpublished families of Dytiscidae (Ribera et al., 2002a,b; observations, 2006). Out of 174 currently recognized Balke et al., 2005; Beutel et al., 2006): Hygrobiidae, 566 I. Ribera et al./Cladistics 24 (2008) 563–590

Amphizoidae and Aspidytidae. Noteridae was consid- perform any sensitivity analyses in this sense, using the ered to be more distantly related and sister to all other default settings (which are usually optimized) except for families of Dytiscoidea, both according to morphology GB (permitted gap positions 50%). We performed and molecules (Ribera et al., 2002a; Balke et al., 2005). additional rounds of secondary refinement in MS until It was not included here due to their greater sequence no apparent change was detected. divergence, which would likely introduce an additional Bayesian analyses were conducted on a combined difficulty in sequence alignment and tree searches. data matrix with MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001), using four partitions (corresponding DNA extraction, amplification and sequencing to the four genes) and evolutionary models as estimated prior to the analysis with ModelTest 3.7 (Posada and Specimens were typically preserved in 96% ethanol in Crandall, 1998). MrBayes ran for 10 · 106 (MS and PR) the field, and kept refrigerated until extraction. Most or 15 · 106 (GB) generations using default values, extractions were non-destructive, using a standard saving trees each 100 or 1000 generations. ‘‘Burn-in’’ phenol–chloroform method or the DNeasy Tissue Kit values were established after visual examination of a (Qiagen GmbH, Hilden, Germany). Vouchers and DNA plot of the standard deviation of the split frequencies samples are kept in the collections of the Natural between two simultaneous runs. History Museum, London (NHM), Museo Nacional de For comparative purposes we also conducted parsi- Ciencias Naturales, Madrid (MNCN) and the Zoolog- mony searches in PAUP 4.0b10 (Swofford, 2002), with ische Staatssammlung, Munich (ZSM) (Appendix 1). 1000 replicates of random addition of taxa and saving We amplified four genes, two mitochondrial and two multiple trees. Support was measured with 1000 boot- nuclear, with one each ribosomal and protein coding: strap pseudoreplicates (Felsenstein, 1985) of 25 tree- the 3¢ of cytochrome oxidase subunit I (cox1, primers bisection-reconnection (TBR) searches each, not saving Jerry–Pat; Simon et al., 1994), the 5¢ end of the multiple trees. mitochondrial 16S rRNA (rrnL, primers 16SaR)16Sb; Simon et al., 1994), a fragment of histone 3 (H3, primers Estimation of diversification rates H3aF–H3aR; Colgan et al., 1998), and the small ribo- somal subunit (SSU). Of the latter, 600 bp of the 5¢ The comparison of the diversification rates among end were sequenced for all specimens (primers 5¢–b5.0; sister lineages requires fully resolved topologies, and Shull et al., 2001), and the full gene (or most of it) for a hence we used the single tree with the highest posterior representative sample (Appendix 1) (see Shull et al., probability (PP) for the MS and PR alignments as 2001 and Ribera et al., 2002a,b for primers and poly- obtained with MrBayes. To estimate branch lengths we merase chain reaction conditions). Sequences were used only the markers with nearly complete representa- assembled and edited with Sequencher TM 4.1.4 (Gene tion in the data set (cox1, rrnL, H3, 5¢-SSU; Appendix Codes, Inc., Ann Arbor, MI, USA). New sequences 1), retaining between 2200 and 2400 bp depending on have been deposited in GenBank with accession num- the alignment. We estimated the ML branch lengths for bers AJ850306–AJ850675 (273 sequences) and each of the four fragments in PAUP using the model EF670007–EF670317 (311 sequences) (Appendix 1). parameter values obtained in the initial Bayesian anal- Sequences of two species of Bidessini were obtained ysis (the parameters for the SSU were obtained using the from GenBank (Nirripirti and Gibbidessus, Appendix 1). whole sequence). The final branch lengths were the sum of branch lengths obtained from the four individual Phylogenetic analyses partitions. For comparison, we also estimated parame- ter values directly with PAUP using the same evolu- The two protein-coding genes (cox1 and H3) had no tionary model for each of the genes separately, and for indels, and aligning of sequences was trivial. For the combined sequence in a single partition with a aligning of the ribosomal genes we used three different GTR+I+G model. Branch lengths for missing se- approaches: multiple progressive pair-wise alignment quences were estimated through multiple regression: with secondary refinement using MUSCLE version 3.52 using only the taxa with full data, the branch lengths of (Edgar, 2004; ‘‘MS’’ in the following); multiple progres- each gene was regressed against the three others, and the sive alignment modeling the evolution of indels with missing values estimated with the regression model. PRANK (Loytynoja and Goldman, 2005; ‘‘PR’’); and Three taxa had two missing markers (Nirripirti, deleting the hypervariable regions using Gblocks version Gibbidessus and Papuadessus; Appendix 1); for them, 0.91b (Castresana, 2000; ‘‘GB’’). The latter was based we first estimated the value of the branch length of cox1, on the starting alignment produced by MS. This and then that of SSU using the estimated cox1 plus the multiple approach was preferred over ‘‘sensitivity ana- two empirical values. Four branch length estimates were lyses’’ sensu Wheeler (1995), i.e., exploring the param- thus obtained for each of the two topologies (MS and eter space within one particular method. We did not PR): (1) branch lengths estimated in PAUP with the I. Ribera et al./Cladistics 24 (2008) 563–590 567 combined data (single partition, GTR+I+G model); (2) Table 2 branch lengths with parameters estimated in PAUP for Number of nucleotids per gene and alignment, and total number of the individual genes; (3) branch lengths estimated with parsimony informative sites the parameters obtained in MrBayes for the individual Gene PRANK MUSCLE Gblocks genes, with missing data; (4) the same as (3), but with cox1 752 752 737 missing data estimated through multiple regression. H3 310 310 310 Path lengths, i.e., the total length of the branches from rrnL 677 552 456 the root to the tip for each terminal taxon, were 5¢-SSU 691 632 563 calculated for all eight trees in TreeStat v1.0 (http:// full SSU 2278 1533 – total 4708 3779 2066 evolve.zoo.ox.ac.uk). informative 1274 1231 782 To estimate relative rates of diversification we con- structed linearized trees using the penalized likelihood method of Sanderson (2002), as implemented in the of invariable sites (I) and unequal rates (G) was preferred software r8s 1.71. We used the Truncated Newton by ModelTest. Bayesian inference chains on the full data algorithm and did a cross-validation procedure with sets were slow to converge for each of the three smoothing factors between 1 and 105 (after elimination alignments. In the case of GB, the standard deviation of the outgroups), with the two topologies (MS and PR) of the split frequencies between the two simultaneous and the branch lengths obtained according to the runs remained above 0.08, despite running for more protocol (4) above. generations (Table 3). The number of trees in the 95% We tested the relative diversification of the different probability set was large, and individual trees had very lineages of the tree using the Slowinski and Guyer (1989) low posterior probabilities (Table 3). test for non-nested nodes. Species numbers were The protein-coding genes had no indels, and there- obtained from Nilsson (2001), updated with Nilsson fore the data matrices were identical for the MS and (2003a, 2004) and Nilsson and Fery (2006). To visualize PR analyses. The estimates of substitution rates and the diversification trends of the overall tree, and to among-site rate variation in the runs of the different compare the estimations obtained from the PR and MS alignments could be taken as a control for conver- alignments, we obtained lineage-through-time (LTT) gence among MCMC chains: they were highly con- plots from linearized trees using Genie v3.0 (Pybus and sistent across alignments, and mainly within the 95% Rambaut, 2002). confidence interval of each other (Appendix 2). The only difference between these analyses and the corre- sponding GB-based analysis was the proportion of Results invariable sites (Appendix 2), presumably because the procedure in GB removed terminal ends of the Phylogenetic analyses alignment affected by missing data for some taxa. For the ribosomal rRNA genes, the estimate of a for The final data matrix included 229 taxa (Table 1, the SSU partition had non-overlapping 95% confi- Appendix 1). The cox1 and H3 fragments could not be dence intervals for the three alignments (Appendix 2, amplified for six taxa each, and the 5¢-SSU for five taxa Fig. 1). The estimation of the proportion of invariant (Appendix 1). The full, or nearly full, length SSU was sites, p(I), for SSU in PR was also non-overlapping available for 79 species, and 26 had additional incom- with those of MS and GB. The estimations of plete fragments, ranging from 396 (Batrachomatus wingii substitution probabilities between nucleotides for the Clark) to 996 bp (Celina sp.2). As expected (Loytynoja rrnL had values fully within the 95% interval of each and Goldman, 2005), the longest alignment was that other, with the only exception of the GMT substitu- produced by PR (Table 2). For all gene fragments, the tion (which had very reduced 95% intervals). The GTR model (Rodrı´guez et al., 1990) with a proportion substitutions for the SSU typically had non-over-

Table 3 Details of the MrBayes and parsimony runs

Bayes Parsimony

Alignment Set of trees PP best No. method No. generations· 106 Burnin· 106 SD P ¼ 0.95 tree trees Length CI PRANK 10 9.8 0.06 1053 0.007 185 23751 0.12 MUSCLE 10 9.5 0.05 2872 0.003 210 24885 0.12 Gblocks 15 12 0.09 3928 0.002 172 20594 0.08 568 I. Ribera et al./Cladistics 24 (2008) 563–590

Fig. 1. Estimation of the parameters a and proportion of invariable sites in the model GTR + I + G of the different alignments of the genes rrnL and SSU in the MrBayes runs. Alignments: PR, PRANK; MS, MUSCLE; GB, Gblocks. lapping 95% confidence intervals between GB and terminal taxon, 20 were supported at PP > 0.95 under either PR or MS (Appendix 2). all three alignments (Fig. 2). These lineages mostly Differences in topology resulting from various tree correspond to recognized supra-generic taxa (tribes or construction methods (Bayesian and parsimony) and subfamilies, Table 1), in some cases corresponding to alignment procedures (MS, PR and GB) were also single species poor genera showing deviating morpho- comparatively minor, and mostly affected the degree of logies and uncertain relationships (Fig. 2, Table 1; see, resolution and support, but did not lead to strong e.g., Miller, 2001a). Among these, Agabetes forms the conflict. Parsimony trees had low support (bootstrap monotypic tribe Agabetini, currently placed in Lacco- < 50%) for most deep nodes, and the resolution and philinae but never associated with this subfamily here. support increased generally from the GB alignment to The only genus of subfamily Coptotominae, the Nearc- MS and PR (see Figs 2 and 3 for a summary of the tic Coptotomus, was found to be sister to the Neotrop- results). Very few nodes were contradicted under various ical Agaporomorphus (Copelatinae) in all analyses, with alignments and tree building procedures, and most of strong support (Fig. 2). Finally, the genus Hydrodytes, these had low support (PP < 0.8; parsimony bootstrap forming the recently described Hydrodytinae together < 50%). There were only two relatively well supported with Microhydrodytes, stands in an isolated position nodes in Bayesian analyses (PP > 0.90) contradicted near the base of Dytiscidae. between alignments, including (1) the placement of the The relationships among these 28 lineages were in Australian Hydroporini (the Necterosoma group), and general poorly supported, although we consistently (2) that of the species Notaticus fasciatus Zimmermann recovered some clades throughout under various (tribe Aubehydrini) (see below). alignment and tree construction methods (Fig. 2). The largest of these lineages grouped Hydroporinae, Phylogeny of Dytiscidae Laccophilini, Copelatinae (including Coptotomus)and Cybistrini, and was found in two of the alignments There were 28 well supported non-nested lineages in with Bayesian methods (PR and MS) (Fig. 2). Lacco- the tree, defined by the most inclusive nodes present in philini was nested within Hydroporinae (with high the Bayesian analyses of all three alignments, with support only in the GB alignment), rendering the latter PP ¼ 0.95 for at least two of them and PP ¼ 0.90 for paraphyletic. Copelatinae was monophyletic with the the third (Fig. 2). Of the 23 lineages with more than one inclusion of Coptotomus, and sister to Hydropori-

Fig. 2. Phylogeny of the major lineages of Dytiscidae, as a consensus from the results of the analyses using the three alignments (PR; MS and GB) (see Table 1 and Appendix 1 for the taxa included in the terminal groups). Support for all terminal groups is PP ¼ 1.0 for the tree alignments, except for those marked with dashed lines (Methlini, PP ¼ 0.98 ⁄ 0.97 for PR ⁄ MS, respectively, 0.81 for GB with the inclusion of Peschetius; gr. PP ¼ 0.96 ⁄ 1.0 ⁄ 0.90 for PR ⁄ MS ⁄ GB, respectively). Thin lines, groups with a single sampled specimen (see Table 1). Numbers above branches, PP · 100; below branches, presence of the node in the parsimony analyses (PR ⁄ MS ⁄ GB): ‘‘+’’, bootstrap > 50% in the parsimony analyses; ‘‘–’’, node unresolved or with very low support (PP < 0.7 in the Bayesian analyses; bootstrap < 50% in the parsimony analyses); ‘‘x’’ contradictory node (only when PP ¼ 0.7 in the Bayesian analyses, or bootstrap > 50% in the parsimony analyses). Numbers 1–28, most inclusive lineages considered to be well supported (see Results). I. Ribera et al./Cladistics 24 (2008) 563–590 569 570 I. Ribera et al./Cladistics 24 (2008) 563–590

Fig. 3. Phylogram of the tree obtained with the Bayesian analyses of the PRANK alignment. See Appendix 1 for the abbreviation of the species. Numbers in nodes, 28 well-supported lineages (see Fig. 2). Black circles: nodes with PP > 0.95 in the Bayesian analyses of the three alignments (PR, MS and GB); gray circles: nodes with PP > 0.95 in the Bayesian analyses of two of the alignments (PR plus either MS or GB); white circles: nodes with PP > 0.95 in the Bayesian analyses of one alignment (PR). See Fig. 2 for the support of the deeper nodes. I. Ribera et al./Cladistics 24 (2008) 563–590 571

Fig. 3. Continued. nae + Laccophilini, a clade also found in the parsi- Other notable relationships not reflected in Fig. 2 are mony search of the PR alignment. Dytiscinae was the sister relationship of Matinae and Hydrodytinae in never monophyletic, and separated into three distinct the Bayesian analysis of the PR alignment (PP ¼ 0.77), lineages: Dytiscini, Cybistrini and ‘‘Hydaticini sensu and also in the parsimony searches with the PR and MS lato’’ (i.e., Aubehydrini, Hyderodini, Hydaticini, Acil- alignments (also with low support). In the parsimony iini and Eretini), the latter with high support in two of search of both the PR and MS alignments Methlini + the alignments (PR and MS). Other deep nodes with Peschetius + Laccornellus + Laccornis form a clade, lower support (only one alignment with PP ¼ 0.95, a which is unresolved in GB (with two nodes, Celina + second alignment with PP < 0.90) group Agabini with Peschetius and Laccornellus + Laccornis + Methles). Colymbetinae, Agabetini with Lancetinae, and a monophyletic Platynectes group sensu Nilsson (2000) Internal phylogeny of selected lineages (including Hydrotrupes). Within the Hydroporinae plus Laccophilini clade In the following we provide detailed comments on the there were two main lineages: (1) Laccophilini, Bides- phylogeny of the most diverse (and well sampled) sini, Pachydrini, Hydrovatini and Vatellini; and (2) lineages (Figs 2 and 3). Hydroporini, Hygrotini, Hyphydrini, Methlini and Colymbetinae (lineage 6). The internal relationships Laccornini. The latter includes taxa formerly linked to of Colymbetinae were not well resolved. The most Hydroporini, but subsequently removed by different prominent result was the polyphyly of the large genus authors (e.g., Sharp, 1882; Guignot, 1959). Rhantus (with more than 100 species), with the inclusion 572 I. Ribera et al./Cladistics 24 (2008) 563–590

Fig. 3. Continued. of the South African R. cicurius (Fabricius) in a clade Hydrotrupes, Andonectes and Leuronectes, suggesting with the morphologically deviating monospecific genera the need for a deep taxonomic revision (as noted by Carabdytes (New Guinea) and Melanodytes (Mediterra- Nilsson, 2000). nean). It is interesting to note the inclusion of the genus ‘‘Hydaticini s.l.’’ (lineages 11–14). This clade in- Agabinus (currently considered a synonymy of Platam- cluded the current Aubehydrini, Hyderodini, Hydati- bus, Nilsson, 2000, 2001) within Colymbetinae in all cini, Aciliini and Eretini, all recovered as reciprocally parsimony searches, but as sister to Agabini in all monophyletic except for the inclusion of Eretes (the only Bayesian searches (with PP ¼ 1.0, Fig. 3). genus of Eretini, Table 1) within Aciliini. The same Agabinae (lineages 7–10). Agabinae was informally clade was recovered by Miller (2001a), and with a divided in two groups of genera by Nilsson (2000), the similar topology to that found here. The inclusion of Agabus and the Platynectes groups. We recovered Notaticus (formerly considered to constitute a separate these two lineages, although not as sisters: the Agabus subfamily, Aubehydrinae) was also supported with the group was placed as sister to Colymbetinae, and analyses of full SSU alone (Ribera et al., 2002b) and the Platynectes group unresolved at the base of morphology (Miller, 2000, 2001a; Miller et al., 2007b). Dytiscidae. Within the Agabus group the generic However, under GB Notaticus was sister to the Platy- division of Nilsson (2000) was confirmed, in agree- nectes gr.1 with PP ¼ 0.98, while under both MS and ment with the more detailed results of Ribera et al. PR it was included among the ‘‘Hydaticini s.l.’’ with (2004). Within the Platynectes group (including PP ¼ 1.0 (Fig. 2). In the parsimony searches, only MS Hydrotrupes, as suggested in Nilsson, 2000) the placed Notaticus together with the ‘‘Hydaticini s.l.’’ genus Platynectes was paraphyletic with respect to (with PR and GB its position was unresolved). The I. Ribera et al./Cladistics 24 (2008) 563–590 573 genus Prodaticus was nested within Hydaticus, suggest- Hyphydrus; and (4) a clade formed by all endemic South ing the need of a taxonomic rearrangement. African genera plus the Malagassy Hovahydrus. Copelatinae including Coptotomus (lineages 15– ‘‘Hygrotini s.l.’’ (lineage 28). This includes the tribe 17). Our trees were congruent with the more detailed Hygrotini, plus the Necterosoma, Graptodytes and results of Balke et al. (2004a), confirming the status of Deronectes groups of genera. These four lineages had Liopterus and Papuadytes as separate genera, and the strong support and were recovered consistently across need for a revision of the status of Aglymbus (nested alignments and tree construction methods. With the PR within ; Fig. 3b). alignment, the Necterosoma group was placed as sister Cybistrini (lineage 18). This was one of the best to the rest of ‘‘Hygrotini s.l.’’ with PP ¼ 0.95, but with supported lineages of Dytiscidae. Within Cybistrini, the MS, the Graptodytes group was sister to the rest with the Australian genera (Spencerhydrus, Austrodytes and same PP ¼ 0.95 (with GB the four lineages were Onychohydrus) were monophyletic and sister to unresolved) (Fig. 2). In the parsimony searches the four plus (Palaearctic, Ethiopian and in the groups were each recovered as monophyletic in all Americas), in agreement with Miller et al. (2007a). alignments, but they formed a clade only in PR (in MS However, and contrary to the latter work, we found and GB they were part of a large polytomy that also Megadytes nested within Cybister, mostly due to an included other lineages). undescribed species from Peru with intermediate mor- Within Hygrotini, the genera Herophydrus and Hy- phological characters (NHM-IR57, see Appendix 1, and grotus were nested with respect to each other, suggesting I. Ribera, unpublished results, 1999). the need for a taxonomic reordination (as noted by Laccophilini (lineage 19). Generic relationships with- Bistro¨m and Nilsson, 2002). The subgenus Coelambus, in Laccophilini were fully congruent with the results of as currently understood (Nilsson, 2001), was also not Alarie et al. (2000). There were three main clades within monophyletic. The Necterosoma group included all the tribe, keeping in mind that a number of genera were Australian Hydroporini, with Carabhydrus as highly not included in the analyses (Table 1): (1) a South derived within the clade and sister to Sternopriscus. The American and Ethiopian clade (genera Australphilus, stygobiontic genus Nirripirti was closely related to Laccodytes and an undescribed new genus from South Paroster, as noted by Leys et al. (2003). The generic America, MB1189, Appendix 1); (2) the cosmopolitan classification of the Deronectes group is also in need of genus Laccophilus; and (3) a mostly Australian and revision (Ribera, 2003), due to the para- or polyphyly of Oriental clade (Neptosternus and Philaccolilus), includ- the genus Stictotarsus and possibly Oreodytes (the latter ing also the genus Philaccolus from Madagascar. with lower support). Within the Graptodytes group, the Bidessini plus Pachydrini (lineage 22). This sister close relationship between the stygobiontic Iberoporus relationship (found in all searches except for the and the rheobiontic Rhithrodytes suggests the need for parsimony analysis of MS, in which they were unre- taxonomic revision in this group, too, although the lack solved), and the monophyly of Bidessini, were among of some key taxa (e.g., Siettitia) does not allow to draw the most robust results of our analyses. Within Bidessini firm conclusions from the trees presented here. many of the nodes were highly supported, and very consistent across alignments and analyses. Diversification Peschetius plus Methlini (lineage 24). This sister relationship was found in all the Bayesian searches Net diversification rates among the lineages of Dytisc- and in the parsimony search with PR, although only idae were estimated on clock-constrained trees based on with PP ¼ 0.81 in GB (Figs 2 and 3). the MS and PR alignments. The first step in this Hydroporus group (lineage 26). The Hydroporus procedure was to obtain an estimate of branch lengths, group of genera was split in two sister lineages, the which could be affected by different parameter estima- Nearctic Sanfilippodytes, Heterosternuta and , tions, tree topologies, and rates among the four mar- and the Holarctic Hydrocolus and Hydroporus. The kers. Branch lengths of the individual genes computed Palaearctic genus Suphrodytes was nested within under the GTR+I+G model using the parameters Hydroporus, in accordance with the traditional placement estimated in PAUP and those using the parameters prior to Angus (1985), but the only studied species of obtained by MrBayes were highly correlated, for the Hydrocolus was found as sister to Hydroporus (including four genes and the two alignments (r > 0.99 for all Suphrodytes), in agreement with Larson et al. (2000). cases except for cox1, with r ¼ 0.95 for MS and r ¼ 0.96 Hyphydrini (lineage 27). The internal topology of for PR, n ¼ 456). Hyphydrini was identical to that found in Ribera and Therefore, for simplicity only the parameter values Balke (2007) in a more comprehensive analysis of the estimated with MrBayes were used in subsequent tribe, with a split in four main lineages: (1) the genus analyses. Correlations of branch lengths between the Desmopachria (American); (2) an Oriental clade with the individual genes were also highly significant in all genera Allopachria and Microdytes; (3) the genus cases (P<0.001), in particular between the two 574 I. Ribera et al./Cladistics 24 (2008) 563–590

Table 4 estimations for the missing data in some partitions Correlations among the branch lengths estimated in PAUP for the obtained through multiple regression (using the param- individual genes, using the model GTR+I+G and the parameter eters of Table 5). values estimated in MrBayes (n ¼ 456; P < 0.001 for all) (PR ⁄ MS) Branch lengths obtained in this way for the trees cox1 H3 rrnL generated with each of the two alignments were H3 0.38 ⁄ 0.40 – subjected to rate smoothing using penalized likelihood rrnL 0.52 ⁄ 0.57 0.36 ⁄ 0.39 – to construct a clock-constrained tree, which can subse- SSU 0.20 ⁄ 0.23 0.23 ⁄ 0.26 0.43 ⁄ 0.42 quently be used to analyze the evolutionary dynamics of species diversification. The cross-validation procedure mitochondrial partitions cox1 and rrnL, but lower implemented in r8s to identify optimum smoothing between cox1 and SSU (Table 4). All multiple regres- parameters selected a value of 1000 for the MS sions of the branch lengths of each individual gene with alignment, while multiple failures prevented the compu- the other three were highly significant, as were all the tation for the PR alignment, in particular for smoothing pair-wise regressions (with the only exception of the parameters of 104 and above. As branch lengths in both SSU for the estimation of the cox1 branch length; alignments were highly correlated for smoothing param- Table 5). Finally, total path lengths in each of the eight eters 103 and 104 (r ¼ 0.999), but not when the trees obtained using MS and PR (four per alignment, see smoothing parameter was set to 105 (r < 0.4), we used Methods) were highly correlated (Table 6). When com- a value of 103 for both MS and PR, and an arbitrary age paring the two alignment procedures, the highest pair- for the root set to 100. wise correlations between the path lengths were those The dynamics of species build-up in Dytiscidae was computed using the parameters estimated in MrBayes initially investigated using lineage-through-time (LTT) for the four separate genes (Table 6). These were used in plots (Nee et al., 1992). The inferred trajectories of net all subsequent analyses, with the inclusion of the diversification through time were very similar for both

Table 5 Parameters and F-values of the regression of the branch lengths of each individual gene with the other two

cox1 H3 SSU F multiple < 0.0001 < 0.0001 < 0.0001 regression F cox1 NA < 0.0001 < 0.0001 F H3 < 0.0001 NA < 0.0001 F rrnL < 0.0001 < 0.0001 < 0.0001 F SSU NS < 0.05 ⁄ < 0.01 NA intercept 0.0412 ⁄ 0.0407 0.0081 ⁄ 0.0085 0.0017 ⁄ 0.0017 coef. cox1 NA 0.1207 ⁄ 0.1275 )0.0206 ⁄ )0.0049 coef. H3 0.4850 ⁄ 0.4628 NA 0.0684 ⁄ 0.0559 coef. rrnL 0.4432 ⁄ 0.3887 0.0793 ⁄ 0.0675 0.1250 ⁄ 0.0692 coef. SSU )0. 1818 ⁄ )0.0888 0.1502 ⁄ 0.2817 NA

PR ⁄ MS (whenever different). NS, not significant; NA, not applicable (same gene).

Table 6 Correlation between the path lengths of the trees for the PR and MS alignments

MS PR

sumByE sumBy allPA sumPA sumByE sumBy allPA MS sumByE 1 sumBy 0.996 1 allPA 0.92 0.92 1 sumPA 0.96 0.97 0.90 1 PR sumByE 0.61 0.61 0.59 0.57 1 sumBy 0.60 0.61 0.59 0.57 0.996 1 allPA 0.56 0.56 0.56 0.52 0.92 0.92 1 sumPA 0.56 0.57 0.56 0.55 0.96 0.97 0.89

sumByE, branch lengths estimated with the parameters obtained in MrBayes for the individual genes, with missing data estimated through regression; sumBy, branch lengths estimated with the parameters obtained in MrBayes for the individual genes, with missing data; allPA, branch lengths estimated in PAUP with the combined data; sumPA, branch lengths with parameters estimated in PAUP for the individual genes (see Methods). In bold, highest and lowest correlations between alignments. I. Ribera et al./Cladistics 24 (2008) 563–590 575

Fig. 4. Lineage through time plots of the linearized trees with the highest posterior probability in the Bayesian analyses of the PRANK and MUSCLE alignments (see Figs 5 and 6). Grey, PR alignment; black, MS alignment. Arrow, approximate location of the inflection point. Insert, plot in semilogarithmic scale.

alignments, with an apparent uniform decrease in two lineages would be significantly different in diversi- diversification rate towards the recent on a semiloga- fication rates (Table 7), but this node was not present in rithmic scale, and an inflection point at 60–70 units of the majority rule consensus tree (PP < 0.5; Fig. 2), and time when plotted on a linear scale (Fig. 4). This point therefore was not further considered. To correct for corresponds roughly to the early diversification within multiple comparisons, we pooled the contrasts for the 11 the main lineages, i.e., the origin of most extant genera non-nested lineages present in the tree approximately at (Figs 5 and 6). This analysis would not provide infor- the same depth as the three significant comparisons mation on the dynamics of lineage diversification using the combined probability of independent tests beyond this portion of the plot because our sampling ()2Slnpi, i ¼ [1k], d.f. ¼ 2k, Sokal and Rohlf, 1995, p. regime focused on comprehensive coverage of genera 794). The 11 simultaneous contrasts of sister species but minimal within-genus sampling (Table 1). numbers were also globally significant (P<0.01) We performed direct comparisons of the known (Table 7). extant species diversity of the various lineages by testing The Slowinski and Guyer (1989) test does not for shifts in rates at particular nodes using the test of determine whether the differences are due to an increase Slowinski and Guyer (1989) for unequal rates in sister in the rate of diversification, a decrease, or a combina- taxa. There were no significantly unbalanced nodes tion of both (Vogler and Ribera, 2003). To characterize common to all alignments with the exception of these shifts in diversification rates more precisely, we Agaporomorphus + Coptotomus versus Copelatinae regressed the logarithm of the extant species number (P<0.02), and Pachydrini versus Bidessini (P< against the relative age of both the stem and the crown 0.03) (Table 7). In some cases, unbalanced nodes were groups in each of the above 11 lineages. While the limited to one of the two alignments and had low nodal regression with the stem age was not significant (tree support. Thus, when Laccornini was resolved as sister to from PR alignment: P ¼ 0.4; MS: P ¼ 0.9; n ¼ 11), the the rest of an ‘‘Hydroporini s.l.’’ (i.e., including Hygro- age of the crown group was significantly correlated with tini, Hyphydrini and Methlini), as in the PR alignment species numbers (PR: P < 0.01; MS: P < 0.05; n ¼ 11) (PP ¼ 0.72), the contrast between these two clades was (Fig. 7). The residuals of this regression suggested a also significant (P<0.01) (Table 7) but this node was disproportionately low diversity of the Agaporomorphus not present in the trees obtained from the MS and GB plus Coptotomus clade, but an unexceptional diversity of alignments (Fig. 2). Similarly, the basal node in the MS- Copelatinae. The highest positive residuals were those of derived tree with the highest PP (Fig. 6) placed Dytiscini Bidessini and ‘‘Hydroporini s.l.’’ for the MS and PR as sister to the remaining Dytiscidae, in which case the alignments, respectively (Fig. 8). For this assessment it 576 I. Ribera et al./Cladistics 24 (2008) 563–590

Fig. 5. Linearized tree with the highest posterior probability in the Bayesian analyses of the PR alignment, obtained in r8s with a cross-validation value of 1000. In brackets, number of species of the terminal taxa (Table 1), excluding the species of the missing genera with dubious placement. For the Copelatus gr. the number includes an estimation of known undescribed species; and ‘‘Agabini’’ does not include the species of the Platynectes group (see text). Terminal taxa were pruned to correspond to those used in Fig. 2. Thick lines (with numbers) refer to the 28 well-supported lineages of Fig. 2. Numbers in circles correspond to the 11 lineages used for the contrast of diversification rates (Table 7). Filled gray circles mark nodes with significant differences in the diversification rate present in all alignments, according to the Slowinski and Guyer (1989) test (Table 7). Empty gray circles mark nodes with significant differences in the diversification rate present only in the PR and GB alignments. The scale bar gives a relative distance from the root, as determined in r8s. The vertical dashed line corresponds to the inflection point in the LTT (see Fig. 4). I. Ribera et al./Cladistics 24 (2008) 563–590 577

Fig. 6. Linearized tree with the highest posterior probability in the Bayesian analyses of the MS alignment, obtained in r8s with a cross-validation value of 1000. In brackets, number of species of the terminal taxa (Table 1), excluding the species of the missing genera with dubious placement. Species numbers as in Fig. 5. Thick lines, terminal taxa in Fig. 2. Numbers, 28 well-supported lineages (Fig. 2). In brackets, lineage not recovered as monophyletic in this tree, but with a PP ¼ 0.59 in the majority consensus rule (Fig. 2). All other analyses and symbols as in Fig. 5. 578 I. Ribera et al./Cladistics 24 (2008) 563–590

Table 7 Slowinski and Guyer (1989) test for comparison of diversification rates between sister lineages. (a) PRANK alignment; (b) MUSCLE alignment. See Figs 5 and 6 for the numbers of the lineages and species. With asterisks, comparisons present in the tree used, but with a posterior probability of the node lower than 0.5 (see text)

No. Lineage N sister N P (a) 1 ‘‘Hydaticini s.l.’’ 216 rest Dytiscidae 3775 NS 2 ‘‘Colymbetinae s.l.’’ 572 sister lineage 3203 NS 3 Coptotomus + Agaporomorphus 12 4-‘‘Copelatinae s.str.’’ 620 0.04 5 Cybistrini 134 Hydroporinae + Laccophilini 2437 NS 6 Laccophilini 402 sister lineage 875 NS 7 Hydrovatini + Vatellini 264 Pachydrini + Bidessini 611 NS 8 Pachydrini 14 9-Bidessini 597 0.05 10 Laccornini 10 11-‘‘Hydroporini s.l.’’ 1150 0.02 (b) 1 Dytiscini 27 rest Dytiscidae 3972 0.01* 2 Matinae + Hydrodytinae + Lancetinae 34 sister lineage 3938 0.02* 3 ‘‘Colymbetinae s.l.’’ + ’’Hydatycini s.l.’’ 735 sister lineage 3203 NS 4 Coptotomus + Agaporomorphus 12 5-‘‘Copelatinae s.str.’’ 620 0.04 6 Cybistrini 134 Hydroporinae + Laccophilini 2437 NS 7 Laccophilini 402 sister lineage 668 NS 8 Vatellini 57 Pachydrini + Bidessini 611 NS 9 Pachydrini 14 10-Bidessini 597 0.05 11 ‘‘Hydroporini s.l.’’ + Laccornini +Hydrovatini 1367 sister lineage 1070 NS is important to note that our taxon sampling was well space of a single method (i.e., sensitivity analysis sensu suited to these analyses because for most lineages Wheeler, 1995). The problem of homology assignments unsampled genera are likely to be placed within the remains a major unresolved question in molecular sampled crown group, not affecting the estimation of the phylogenetics, and the behavior and performance of crown age, with the possible exception of Pachydrini due some recent methods is still not fully understood (see, to the lack of examples of Heterhydrus (five species) e.g., Kjer et al., 2007). For multiple alignment of diver- (Table 1). In the tribe Laccornini the absence of North gent ribosomal sequences, iterative algorithms such as American species of Laccornis is not likely to affect the MUSCLE have been shown to consistently outperform estimation of the crown age, as the two European previous methods (Wilm et al., 2006). The suppression of species included here encompass the whole variation hypervariable regions by Gblocks resulted in topologies within the genus (Wolfe and Roughley, 1990). with fewer resolved nodes and overall lower support, but where nodes were recovered, they were largely congruent with those obtained from the full sequences. Similarly, Discussion preliminary results using only the 5¢ end of the SSU gene (not shown), complete in the data matrix for nearly all Phylogeny of Dytiscidae species, were largely compatible with the results reported here, but with lower overall support and resolution, in We found a generally high degree of congruence agreement with similar observations on other data among phylogenetic methods and different alignments, sets (for a review see Wiens, 2006). with most incongruent nodes showing low support. Differences between the PR and MS alignments were Although trees exhibited some incongruence and gener- apparently large, affecting the homology assignments in ally low backbone support, 54% of the 228 possible 1000 characters, although the number of informative nodes had a Bayesian PP equal to, or higher than 0.95 characters was very similar (a difference of 50), and for all three alignments, and this value rose to more than the topologies were largely compatible. The largest effect 60% when considering nodes supported by a minimum of the alignment method was in the estimation of the of two alignments. Node support was lower for the parameters in MrBayes, particularly for the SSU. The parsimony searches, but if node stability across methods elimination of the hypervariable regions with Gblocks is taken as a measure of confidence (Giribet, 2003), the had the effect of increasing the proportion of invariable number and composition of the main lineages and their sites and the value for a, the shape parameter of the rate internal topology can be considered as very robust. heterogeneity distribution. With smaller values of a the We opted for the use of different methodological distribution of rates becomes more uneven, with most approaches to address the alignment of the ribosomal sites exhibiting rates close to zero but a few sites with genes, rather than exploring a fraction of the parameter high rates (Felsenstein, 2004). The PR alignment greatly I. Ribera et al./Cladistics 24 (2008) 563–590 579

a

b

Fig. 7. Plot of the natural logarithm of the number of species versus the depth of the stem origin (white circles, dashed line) and crown diversification (black circles, solid line) of the 11 lineages used to test the relative diversification rates (Table 7, Figs 5 and 6). (a) MUSCLE alignment; (b) PRANK alignment. reduced the estimated proportion of invariable sites in variation that coincide with divergent groups identified the SSU, although this did not result in a substantial by the DNA-based tree. increase in informative characters (see above). Some of the traditionally recognized supra-generic We found 28 consistently well supported lineages groups have not been recovered in previous cladistic within the Dytiscidae recovered under all alignment and analyses using morphological characters. This was the phylogenetic inference methods, corresponding to tra- case for the tribe Hygrotini, and the Necterosoma, ditionally well characterized groups including many Hydroporus, Graptodytes and Deronectes groups of tribes or small subfamilies. The relationships at deeper genera (e.g., Alarie et al., 1999; Miller, 2001a; Alarie levels, defining relationships among clades mostly rec- and Challet, 2006; Miller et al., 2006; Michat and ognized as subfamilies in the current classification Alarie, in press; and references therein). The lack of (Nilsson, 2001), were in general poorly supported. Yet, easily detectable morphological synapomorphies did not it is interesting to note that most of the clades which are prevent knowledgeable authors to recognize these clades well-supported by molecular data had also been recog- based on overall similarity (e.g., Sharp, 1882; Seidlitz, nized in the taxonomic literature. These taxa would not 1887; Falkenstro¨m, 1939), but precluded their recovery correspond to a particular age (although they seem to be in formal phylogenetic analyses with incomplete taxon clustered around certain genetic divergences), but sampling or based on restricted data sets. Paradoxically, instead reflect significant gaps in the morphological the intuitively recognized groups of early dytiscid 580 I. Ribera et al./Cladistics 24 (2008) 563–590

a

b

Fig. 8. Plot of the residuals of the regression of the natural logarithm of the number of species of the 11 lineages used to test the relative diversification rates and the estimation of the crown diversification age (Table 7, Figs 5 and 6). (a) MUSCLE alignment; (b) PRANK alignment. workers show greater agreement with formal analyses of and thus compatible with Dytiscinae monophyly, but molecular, rather than morphological data. Cybistrini was usually included in a clade with Hydro- Despite the low resolution among the deeper nodes of porinae, Copelatinae (including Coptotomus) and Lac- the trees, we found compelling evidence for the rela- cophilini, although with high support only in the tionships among certain major clades of Dytiscidae. Bayesian analyses of the MS alignment. Most striking was the non-monophyly of the subfamily The well supported Cybistrini (Sharp, 1882; Ferreira, Dytiscinae, which we found separated into three distinct 2000; Miller, 2001a; Miller et al., 2007a) have previously clades including Cybistrini, Dytiscini and a clade formed been placed as sister to the remaining Dytiscinae (Miller by the tribes Aciliini + Eretini + Hydaticini + Hy- et al., 2007a). Interestingly, Sharp (1882) also divided derodini + Aubehydrini (our ‘‘Hydaticini s.l.’’). Sister the current Dytiscinae in the same three lineages relationships of the latter two clades were unresolved (Cybistrini, Dytiscini and ‘‘Hydaticides’’, corresponding I. Ribera et al./Cladistics 24 (2008) 563–590 581 to our ‘‘Hydaticini s.l.’’), with the only difference of The genus Hydrotrupes includes two madicolous Hyderodes being included in Dytiscini, as was the species, and its placement remained contentious for general opinion until Miller (2001a). Wolfe (1985) noted more than a century (Nilsson, 2000, 2003b; Miller, the absence of galea in both the larvae of Cybistrini and 2001a; Balke, 2005). It has been considered a separate most of the Hydroporinae, but interpreted this similarity subfamily, mostly based on larval characters (Beutel, as the product of an independent loss, a view reinforced 1994; Larson et al., 2000), or within Agabini, mostly by the occurrence of a reduced galea in Celina, Laccor- based on adult characters (Nilsson, 2000, 2001; Miller, nis, Hydrovatus and Canthyporus (Michat and Alarie, 2001a), but also larval chaetotaxy (Alarie, 1998). We in press). found evidence linking Hydrotrupes with the Platynectes The current concept of Dytiscinae (Nilsson, 2001) had group, a position suggested by Nilsson (2000) and already been adopted by Erichson (1832, 1837), defined compatible with Miller (2001a), who placed Hydrotrupes on the basis of the five-segmented tarsi of all legs and the and Platynectes at the base of Agabini (although not as male protarsal adhesive discs. Dytiscinae is one of the sisters). The Platynectes group has been considered part nodes with highest support in Miller (2001a), but with a of Agabini, as redefined by Nilsson (2000). It includes single unambiguous synapomorphy, the ‘‘Dytiscinae- the widespread genus Platynectes (absent from the type’’ female genitalia (Burmeister, 1976; Miller, 2000, Nearctic and Afrotropics), and the Neotropical endemic 2001a), as the traditional characters to define the group genera Agametrus, Andonectes and Leuronectes (e.g., Erichson, 1837) are either plesiomorphic (five- (Gue´orguiev, 1971, 1972). Based on morphology, the segmented tarsi) or highly homoplastic (male protarsal latter three genera are separated from Platynectes only adhesive discs). A non-monophyletic Dytiscinae would by different combinations of character reductions, i.e., imply the independent origin of several of the most loss of lateral pronotal bead and ⁄or loss of metacoxal spectacular modifications of diving beetles, usually lines (Gue´orguiev, 1971, 1972; Pederzani, 1995). Based interpreted as adaptations to high-speed swimming on our DNA sequence data, both Agametrus and (Nachtigall, 1961; Ribera and Nilsson, 1995). Leuronectes would be subordinated within Platynectes, The family Dytiscidae has a number of morpholog- confirming the need of a revision of the taxonomic ically divergent genera that have traditionally been status of the genera of the group. The deviating recognized as separate at the level of subfamilies or morphological characteristics of Hydrotrupes, such as tribes, such as Coptotomus (five species in North the lack of a ventral fringe of swimming hairs on the America), Agabetes (two species, in North America adult hind tibia and tarsus, or the lack of larval and Iran), Hydrotrupes (two species, in western North mandibular channel, seem to represent reversals due to America and eastern China) and Notaticus (two Neo- the shift to hygropetric habitat rather than an isolated tropical species) (Nilsson, 2001, 2003a, 2004). Coptoto- phylogenetic position (Ribera et al., 2003a). In all our mus, the only genus in the subfamily Coptotominae, was analyses, the relationships of the Platynectes group placed by Miller (2001a) in a clade together with (including Hydrotrupes) within Dytiscidae was unre- Laccophilinae, Copelatinae, Hydrodytinae and Hydro- solved or very weakly supported. porinae, which, except for the inclusion of Hydrodyti- The genus Agabinus, synonymized with Platambus by nae, is in agreement with our results. The strongly Nilsson (2000), was recovered as a separate lineage at supported sister relationship between Coptotomus and the base of Agabini in the Bayesian analysis, confirming Agaporomorphus was highly unexpected. The Neotrop- evidence from larval morphology (Alarie and Larson, ical Agaporomorphus is currently included in Copelati- 1998). However, Agabinus was placed inside Colymbe- nae (Nilsson, 2001), and considered to be sister to the tini when using parsimony optimization. Its position remaining species based on morphological evidence remains ambiguous, but we suggest reinstating Agabinus (Miller, 2001a,b), having a deviating female genital as a valid genus, in agreement with the results of Miller apparatus (without bursa copulatrix). (2001a) and Ribera et al. (2004). Agabetes was originally considered a separate sub- Within the most diverse group of Dytiscidae, the family (Agabetinae), but linked to Laccophilinae based Hydroporinae (with more than 50% of the genera and on similarities of the female genitalia and larval species of the whole family; Table 1), we find strong chaetotaxy (Burmeister, 1976; Nilsson, 1989; Alarie support for several higher lineages. In agreement et al., 2002). We found some evidence linking it to with Wolfe (1985), there was some evidence for a Lancetinae (mostly Neotropical), but never to Lacco- major split in a clade with a predominantly southern philini. The north–south vicariance defined by this hemisphere distribution (Bidessini, Pachydrini, Hydro- sister relationship would be similar to that of Agapor- vatini and Vatellini), and a clade including all omorphus plus Coptotomus, although this parallelism lineages with a mostly northern distribution, but also is compromised by a species from Iran included in some southern ones (Hydroporini, Hygrotini, Meth- Agabetes, and a representative of Lancetes from Aus- lini, Laccornini and Hyphydrini in some Bayesian tralia (Nilsson, 2001). analyses). 582 I. Ribera et al./Cladistics 24 (2008) 563–590

The genera Pachydrus and Heterhydrus were previously (Nilsson and Angus, 1992; Alarie and Watts, traditionally considered linked to Hyphydrini, until 2004), but usually the incomplete sampling of these Bistro¨m et al. (1997) recognized their morphological studies did not allow general conclusions. The Austra- distinctiveness and placed them in a separate tribe, lian genera of Hydroporini form a monophyletic clade Pachydrini. Both tribes were later merged again based (the Necterosoma group), including the morphologically on a wider taxon sampling of Dytiscidae (Miller, 2001a; enigmatic Carabhydrus, which to date was assigned to its Miller et al., 2006; see also Alarie and Challet, 2006; own tribe, Carabhydrini (Nilsson, 2001). Recent molec- Michat and Alarie, in press). However, we never found ular work has already shown the inclusion of Carabhy- any relationship between the two groups, in agreement drus among the Australian Hydroporini (Balke and with Ribera et al. (2002b) and Ribera and Balke (2007). Ribera, 2004). The only Australian Hydroporini genus In all our analyses, Pachydrus (the only genus of not included in our analyses is Sekaliporus, considered Pachydrini included) was sister to Bidessini, with strong to be closely related to both Tiporus and Antiporus support. (Watts, 1997). Preliminary analyses with recently col- Hydroporini as currently defined has been shown to lected specimens show that it does belong within the be highly polyphyletic by different authors (e.g., Miller Necterosoma group of genera (L. Hendrich and M. et al., 2006; Michat and Alarie, in press), mostly because Balke, unpublished results, 2007). Our newly found several genera with southern distribution were incor- scenario of a monophyletic Australian Hydroporini rectly placed in this group, including Canthyporus suggests a single, ancient colonization from the north. (Ethiopian), Peschetius (Ethiopian and Oriental) and Remarkably, Hydroporini do not occur in the Oriental Laccornellus (Neotropical). All of them have been region: the geographically nearest extant taxa only occur placed recently outside of Hydroporini, as already in the Eastern Palaearctic (Nilsson, 2001). Australian noted. Similarly, the genera currently in Hygrotini were Hydroporini now contain 10 or 11 morphologically and traditionally considered part of Hydroporini, but are ecologically rather diverse genera (Nirripirti might be now considered a separate tribe (Nilsson and Holmen, synonymized with Paroster, C.H.S. Watts, personal 1995). communication, 2006) with more than 120 species, The genus Peschetius is currently included in Hydro- constituting a good example of an adaptive radiation. porini (Nilsson, 2001; Bistro¨m and Nilsson, 2003). In a The Australian genera are represented by very few recent study it was hypothesized to be related to species in New Guinea (Balke, 1995), New Caledonia Bidessini, based mostly on the common presence of a and Fiji (Nilsson, 2001). spine in the spermatheca (Miller et al., 2006), but instead we found it always linked to Methlini. Bidessini, Evolution of species richness as redefined by Bistro¨m (1988) includes all species of Hydroporinae with bi-segmented male lateral lobes, and LTT plots and shifts in clade size between sister taxa is one of the best defined and supported lineages of provided insight into the evolutionary build-up of Dytiscidae, both with morphological and molecular species diversity in Dytiscidae, as the basis for future characters (Miller, 2001a; Ribera et al., 2002b; Miller studies that may link species diversification to ecomor- et al., 2006; and this study). We would not favor the loss phological traits (Ribera and Nilsson, 1995) or spatial of this coherence to expand its concept to include species arrangement of habitat patches and biogeographical with non-segmented parameres of more uncertain phy- regions (Ribera et al., 2003b; Vogler and Ribera, 2003; logenetic position, as proposed in Miller et al. (2006). Balke et al., 2007). These estimations were possible The two species of Laccornellus were formerly based on (1) a largely complete sampling of basal included in the genus Laccornis, but in Roughley and lineages, permitting assessments of net diversification Wolfe (1987) they were suggested to be more closely rates during the early phase of dytiscid evolution from related to the Ethiopian Canthyporus. All Bayesian LTT plots, and (2) a set of exemplars representing basal analyses placed Laccornellus as sister to Canthyporus,in branches within these major groups for age estimates of agreement with the latter authors, although with low early splits within subfamilies and groups of genera support in the MS alignment. But in all parsimony (crown age); their known total species numbers was then analyses (and in the tree with the highest PP of the MS used for an estimate of average diversification rates. alignment; Fig. 6), Laccornis and Laccornellus were A prerequisite for these analyses was an estimate of placed together with Methlini and Peschetius in a clock-constrained branch lengths, here scaled with the monophyletic clade, more according to the traditional penalized likelihood method. Various estimation meth- taxonomic hypothesis. ods of branch lengths gave very consistent results, with We find strong support for a clade including the high correlations between branches and similar LTT Deronectes, Graptodytes and Necterosoma groups plus plots, suggesting that the general structure of the trees Hygrotini (our ‘‘Hygrotini s.l.’’). A relationship between was very similar, despite topological differences. This the Deronectes and Necterosoma groups was suggested was also supported by the similar number of informative I. Ribera et al./Cladistics 24 (2008) 563–590 583 characters and estimates of rate variation across align- question would be not ‘‘Why so many species?’’ but ments, which seemed little affected by the large differ- ‘‘Why so little morphological diversity?’’. On the con- ences in the total number of aligned characters (Fig. 1 trary, the large positive residual in the significant and Appendix 2). contrast between Bidessini and Pachydrini suggests that Our sampling was virtually complete for the basal Bidessini have experienced a true speed-up in species splits of the tree, up to the origin of groups of genera, diversification. corresponding to 60 time units in the LTT plots. This limit also corresponds to the basal diversification within the well supported terminal lineages. In any case, in Acknowledgments quantitative terms the sampling starts to be substantially incomplete only within some of the terminal lineages We particularly thank all people mentioned in Appen- (mainly Bidessini and Laccophilini, Table 1), and in the dix 1 for providing material for study, and the South diversification within genera. This corresponds to an African National Parks (Albertus Lewis) for collecting inflection point in the linear LTT plot for both the MS permits to I.R. We also thank D.R. Madisson for and PR alignments. Given the complete sampling of the providing DNA of Hydrotrupes palpalis; James Hogan, basal part of the tree, a surprising result is the lack of Marta Albarra´n, Elisa Ribera (NHM London), and Ana correlation between the number of species and the stem Izquierdo (MNCN Madrid) for help with laboratory age of the lineages. The expected positive correlation of work; A.N. Nilsson for updated compiled data on clade age and species numbers (McPeek and Brown, species numbers; Y. Alarie for providing manuscripts in 2007) was only obtained when ages were calculated for press; and A.N. Nilsson and J. Go´mez-Zurita for the crown groups (i.e., since the first nodal split within a comments to earlier versions of the manuscript. This clade). This might suggest that major extinction events diving project has been funded since 1998 by grants pruned multiple basal lineages in a non-uniform way. to IR (Marie Curie fellowship; Leverhulme Trust; However, this interpretation is hampered by the uncer- Spanish MEC CGL2004-00028), APV (Leverhulme tainties in the topology of the basal nodes, which will Trust; NERC) and MB (Marie Curie fellowship; largely affect the estimation of the stem but not the SYNTHESYS ES-TAF 193, 2197; DFG BA2152 ⁄3-1, crown age of well-supported lineages, as noted above. 3-2; Fazit Foundation) held at the NHM and Imperial There were only two consistent significant contrasts of College (London), MNCN (Madrid) and ZSM (Munich). diversification rates as measured with the Slowinski and Guyer (1989) test: Copelatinae versus Coptoto- mus + Agaporomorphus, and Bidessini versus Pachyd- References rini. This is a very conservative test, but other methods evaluating full trees could not be implemented due to the Alarie, Y., 1998. Phylogenetic relationships of Nearctic Colymbetinae incomplete sampling in the tips of the tree (Mooers and (Coleoptera: : Dytiscidae) based on chaetotaxic and porotaxic analysis of head capsule and appendages of larvae. Can. Heard, 2002). When only the (almost) complete part of Entomol. 130, 803–824. the tree was used, among the few remaining lineages Alarie, Y., Challet, G.L., 2006. Larval description and phylogenetic ( 40 maximum), no significant whole tree asymmetries placement of the South Africa endemic genus Andex Sharp were found (results not shown). (Coleoptera: Adephaga: Dytiscidae). Ann. Entomol. Soc. Am. 99, The residuals of the expected relationship between 743–754. Alarie, Y., Larson, D.J., 1998. Larvae of Agabinus Crotch: generic number of extant species and crown age of the group characteristics, description of A. glabrellus (Motschulsky), and may give an indication of the absolute trend of the comparison with other genera of the subfamily Colymbetinae diversification rate of each of the individual lineages (Coleoptera: Adephaga: Dytiscidae). Col. Bull. 52, 339–350. (McPeek and Brown, 2007). In the case of the Copelat- Alarie, Y., Watts, C.H.S., 2004. Larvae of the genus Antiporus inae, the significance of the Slowinski and Guyer (1989) (Coleoptera: Dytiscidae) and phylogenetic implications. Invertebr. Syst. 18, 523–546. test seems to be due to the low diversity of the Alarie, Y., Nilsson, A.N., Hendrich, L., 1999. Larval morphology of Coptotomus + Agaporomorphus clade (with a negative the Palaearctic genera Deronectes Sharp and Scarodytes Gozis residual), not to the high diversity of the remaining (Coleoptera: Dytiscidae: Hydroporinae), with implications for the Copelatinae, which seems to have an ‘‘average’’ diver- phylogeny of the Deronectes-group of genera. Entomol. Scand. 30, sity. The known number of species of both groups may, 173–195. Alarie, Y., Nilsson, A., Hendrich, L., Watts, C.H.S., Balke, M., 2000. however, be vastly underestimated, because many spe- Larval morphology of four genera of Laccophilinae (Coleoptera: cies from tropical regions remain undescribed. The Adephaga: Dytiscidae) with an analysis of their phylogenetic species of Copelatinae are morphologically very uni- relationships. Syst. Evol. 31, 121–164. form, and they have been traditionally placed in very Alarie, Y., Spangler, P.J., Steiner,W.E. Jr, 2002. Larval morphology of Agabetes Crotch (Coleoptera: Adephaga: Dytiscidae): the hypoth- few genera, suggesting the possibility of an ‘‘explosive’’ esis of sister-group relationship with the subfamily Laccophilinae radiation of 600 described species (Balke et al., revisited. Col. Bull. 56, 547–567. 2004a). However, according to our results, the pertinent 584 I. Ribera et al./Cladistics 24 (2008) 563–590

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