phylogeny and diversification of diving beetles (coleoptera

Phylogeny and diversification of diving beetles (Coleoptera:Dytiscidae)

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

aDepartamentode Biodiversidad y Biologıa Evolutiva, Museo Nacional de Ciencias Naturales, Jose Gutierrez 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 Munchen,

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 extrememorphological and ecological adaptations. Despite continuous attention from systematists and ecologists, existing phylogenetichypotheses remain unsatisfactory because of limited taxon sampling or low node support. Here we provide a phylogenetic treeinferred from four gene fragments (cox1, rrnL, H3 and SSU, � 4000 aligned base pairs), including 222 species in 116 of 174 knowngenera and 25 of 26 tribes. We aligned ribosomal genes prior to tree building with parsimony and Bayesian methods using threeapproaches: progressive pair-wise alignment with refinement, progressive alignment modeling the evolution of indels, and deletionof hypervariable sites. Results were generally congruent across alignment and tree inference methods. Basal relationships were notwell defined, although we identified 28 well supported lineages corresponding to recognized tribes or groups of genera, among whichthe most prominent novel results were the polyphyly of Dytiscinae; the grouping of Pachydrini with Bidessini, Peschetius withMethlini and Coptotomus within Copelatinae; the monophyly of all Australian Hydroporini (Necterosoma group), and theirrelationship with the Graptodytes and Deronectes groups plus Hygrotini. We found support for a clade formed by Hydroporinaeplus Laccophilini, and their sister relationship with Cybistrini and Copelatinae. The tree provided a framework for the analysis ofspecies diversification in Dytiscidae. We found a positive correlation between the number of species in a lineage and the age of thecrown 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 thecrown age of the group identified only Bidessini and the Coptotomus + Agaporomorphus clade as lineages with, respectively, aboveand below expected levels of species diversity.

� The Willi Hennig Society 2008.

Approximately 25 families in three of four subordersof Coleoptera are typically aquatic in some of their lifestages (Beutel and Leschen, 2005). Among these, theDytiscidae (predaceous diving beetles), with some 4000described species (Nilsson, 2001, 2003a, 2004; Nilssonand Fery, 2006) represent the perhaps most conspicuousgroup. Dytiscids range in size from about 1–50 mm, andare predators both as larvae and adults. They are foundin virtually any aquatic freshwater ecosystems, fromlakes, streams, springs, wet rock surfaces and puddles.

They also occur in specialized habitats such as ground-water aquifers, high altitude lakes and streams, orbromeliad water tanks in the forest canopy (Franciscolo,1979; Balke, 2005). Although most species are tightlyassociated with their aquatic habitat throughout theirlife cycle except in the pupal stage, many species arestrong flyers and able to disperse readily over land.

Diving beetles have been used as model organisms ina variety of ecological and evolutionary subjects, such ascoexistence and competition of closely related taxa (e.g.,Juliano and Lawton, 1990a,b; Scheffer and Nes, 2006),the evolution of the stygobiontic fauna (Leys et al.,2003, 2005), the role of habitat constraints in large-scale

*Corresponding author:E-mail address: [email protected]

� The Willi Hennig Society 2008

Cladistics

10.1111/j.1096-0031.2007.00192.x

Cladistics 24 (2008) 563–590

macroecological patterns (Ribera et al., 2001, 2003b), orthe mechanics and functional morphology of swimmingbehavior (Nachtigall, 1961; Ribera and Nilsson, 1995).They are also among the best known groups of beetles,being studied by an active research community, andrecent comprehensive revisions of many genera and anup-to-date world catalog are available (Nilsson, 2001).Despite this wide research interest, very few studies haveaddressed phylogenetic relationships at the level of theentire family using morphological (Burmeister, 1976;Miller, 2001a) or molecular (Ribera et al., 2002b)characters. These studies were limited in terms of taxonsampling or the set of characters used (mostly femalegenitalia; small subunit rRNA, SSU), and support levelswere generally low.

The most recent taxonomic revision of the wholefamily was that of Sharp (1882), separating two majorgroups according to the articulation between the meta-thoracic anepisternum and the mesocoxa: the ‘‘Dytisci-dae Fragmentati’’ (no contiguous articulation),including what are now families Hygrobiidae andNoteridae plus Laccophilinae and Vatellini, and ‘‘Dyti-scidae Complicati’’ (contiguous articulation), includingAmphizoidae and the rest of current Dytiscidae. Withinthe latter, the major divisions separated the Hydropo-rinae (excluding Vatellini) from the ‘‘Colymbetides’’ andwhat is the Dytiscinae in recent classifications.

In his monograph of the Palaearctic Dytiscidae,Zimmermann (1930, 1933) already considered Hygro-biidae and Amphizoidae as separate families, anddivided Dytiscidae in five subfamilies: Noterinae (nowNoteridae), Laccophilinae, Hydroporinae, Colymbeti-nae and Dytiscinae. The composition of these groupshas remained largely unchanged except for the largeColymbetinae, which has experienced successive refine-ments by removing taxa with deviating morphologiesand no apparent close relatives. Thus, the NearcticCoptotomus was considered by Boving and Craighead(1931) to form the subfamily Coptotominae, mostlybecause of the unusual larvae (with lateral gills on theabdomen). Burmeister (1976) recognized the non-mono-phyly of the remaining ‘‘Colymbetines’’, associatingAgabetes to Laccophilinae and placing the bulk ofCopelatinae in an unresolved position within Dytisci-dae. Subsequent authors removed Lancetes (Ruhnauand Brancucci, 1984) and Hydrotrupes (Beutel, 1994)from the ‘‘Colymbetine’’ pool.

The phylogeny of the most diverse group of Dytisci-dae, the Hydroporinae, remains poorly understood(Miller et al., 2006; Michat and Alarie, in press). Severalmajor lineages have been recognized, with, for example,Zimmermann (1930, 1933) considering six tribes: Vatel-lini, Methlini, Hyphydrini, Hydrovatini, Bidessini andHydroporini. The first cladistic analyses showed thatHydroporini as defined at the time was polyphyletic, asLaccornis was hypothesized to be sister to the remaining

Hydroporinae (Wolfe, 1985, 1988). The position ofLaccornellus and Canthyporus within Hydroporini wasalso questioned (e.g., Roughley and Wolfe, 1987), aswell as the composition of Bidessini and Hyphydrini(Bistrom, 1988; Bistrom et al., 1997).

The most recent comprehensive taxonomic ordinationof the family is that of Miller (2001a) (Table 1), basedon a phylogenetic analysis of morphological charactersand followed in the world catalog of Nilsson (2001).Main novelties were the recognition of Matinae and itsplacement as sister to the remaining Dytiscidae, theseparation of Agabinae from Colymbetinae, and theestablishment of Hydrodytinae for some inconspicuoustaxa previously considered within Copelatinae. Thetaxon sampling and phylogenetic resolution withinHydroporinae were generally limited, and no majorchanges were proposed. In a subsequent analysiscentered on Hydroporinae (Miller et al., 2006), anddespite the better sampling, the phylogenetic relation-ships were equally poorly supported. In the first familywide molecular phylogenetic study of Dytiscidae(Ribera et al., 2002b), based on � 70 full SSUsequences, support was also generally low, althoughsome groups of genera within Hydroporinae werestrongly supported (e.g., Hydroporus and Graptodytesgroups, Hygrotini, Bidessini).

Here, we expand this molecular analysis of Dytiscidaeto include all major supra-generic groups and nearly70% of the genera, based on four genes (4000–5000aligned nucleotides). Specific emphasis of taxonomicsampling was on the subfamily Hydroporinae, and inparticular Hydroporini, where the constitution andrelationships of lineages remain particularly poorlyknown. We aim to provide a phylogenetic frameworkfor future evolutionary and systematic studies of Dytisc-idae, by identifying the major monophyletic lineageswithin the family and some of their phylogeneticrelationships. The resulting tree was also used to studyrelative diversification rates in different groups ofDytiscidae, as a first step of establishing the causes oftheir high species richness relative to other aquaticlineages of beetles.

Materials and methods

Taxon sampling

The taxonomy here follows the most recent worldcatalog of Dytiscidae (Nilsson, 2001), with the followingmodifications: (1) Tribe Carabdytini (comprising Carab-dytes) was considered part of Colymbetini, as shown byBalke (2001), Balke et al. (2007), and confirmed here. (2)Tribe Hydroporini was divided in several groups ofgenera (named after the oldest valid genus name), basedon previous works and according to our results here:

564 I. Ribera et al. / Cladistics 24 (2008) 563–590

Canthyporus group (Roughley and Wolfe, 1987), Dero-nectes group (Nilsson and Angus, 1992), Graptodytesgroup (Seidlitz, 1887; Ribera et al., 2002b), Hydroporusgroup (Ribera et al., 2002b), and Necterosoma group(see Table 1 and Appendix 1). (3) Tribe Carabhydrini(comprising Carabhydrus) was included within theNecterosoma group of genera, based on the currentstudy and in previous molecular results (Balke andRibera, 2004). (4) Tribe Pachydrini was consideredvalid, as originally proposed by Bistrom et al. (1997)and confirmed in Ribera and Balke (2007).

All recognized subfamilies and tribes of Dytiscidaewere included in the study, with the only exception ofAnisomeriini (two genera with one species each fromJuan Fernandez and Tristan da Cunha; Nilsson, 2001).These two taxa are likely to be highly modified species ofRhantus in the R. signatus group (M. Balke, unpublishedobservations, 2006). Out of 174 currently recognized

genera of Dytiscidae (as of February 2007; Table 1), weincluded 116 or 67%. Twelve of the missing genera arestygobiontic, with a total of 16 species, and two generaare secondarily terrestrial, with four species. Some ofthese are likely to be highly modified species derivedfrom within larger genera, as already established in thecase of stygobiontic species of Papuadytes (Balke et al.2004b), Limbodessus (Balke and Ribera, 2004) andMicrodytes (Wewalka et al., 2007). A missing genus ofuncertain relationships is Lioporeus, with two Nearcticspecies, which could be a plesiomorphic lineage withinHydroporini without close living relatives (Wolfe, 1985).The missing genera have a combined diversity of 220species, less than 6% of the total species diversity of theDytiscidae.

Outgroups included the three most closely relatedfamilies of Dytiscidae (Ribera et al., 2002a,b;Balke et al., 2005; Beutel et al., 2006): Hygrobiidae,

Table 1Current classification of Dytiscidae (following Nilsson, 2001 and updates, see Text), with the modifications used in this study, and the number ofgenera, 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 tothe exclusion of genera for which there were no molecular data or of genera of uncertain placement, and to the inclusion of known undescribedspecies of Copelatinae (M. Balke, unpublished data, 2007). Taxa marked with asterisks are groups of genera currently included in tribes Hydroporiniand Carabhydrini (see Methods). Genus Lioporeus (two species, not sampled), currently in Hydroporini, is placed among ‘‘Incertae sedis’’

Subfamily TribeNo.genera

Sampledgenera

No.species

Sampledspecies

Agabinae Agabini 10 8 391 21Colymbetinae Anisomeriini 2 0 2 0

Colymbetini 8 7 131 10Matinae Matini 3 1 8 2Copelatinae Copelatini 6 6 585 11Coptotominae Coptotomini 1 1 5 2Dytiscinae Aciliini 7 5 69 5

Cybistrini 7 5 134 10Dytiscini 1 1 27 3Hyderodini 1 1 2 1Eretini 1 1 4 2Hydaticini 2 2 139 3Aubehydrini 1 1 2 1

Hydrodytinae Hydrodytini 2 1 4 1Hydroporinae Canthyporus* 1 1 35 4

Deronectes gr* 6 6 183 27Graptodytes gr* 7 6 46 10Hydroporus gr* 6 6 260 11Laccornellus* 1 1 2 2Necterosoma gr* 11 10 125 9Peschetius* 1 1 9 1Bidessini 38 17 609 27Hygrotini 4 2 133 10Hydrovatini 2 1 207 3Hyphydrini 14 10 316 16Laccornini 1 1 10 2Methlini 2 2 41 4Pachydrini 2 1 14 2Vatellini 2 2 57 4Incertae sedis 10 0 31 0

Laccophilinae Laccophilini 12 7 402 15Agabetini 1 1 2 1

Lancetinae Lancetini 1 1 22 2Totals 33 174 116 4007 222

565I. Ribera et al. / Cladistics 24 (2008) 563–590

Amphizoidae and Aspidytidae. Noteridae was consid-ered to be more distantly related and sister to all otherfamilies of Dytiscoidea, both according to morphologyand molecules (Ribera et al., 2002a; Balke et al., 2005).It was not included here due to their greater sequencedivergence, which would likely introduce an additionaldifficulty in sequence alignment and tree searches.

DNA extraction, amplification and sequencing

Specimens were typically preserved in 96% ethanol inthe field, and kept refrigerated until extraction. Mostextractions were non-destructive, using a standardphenol–chloroform method or the DNeasy Tissue Kit(Qiagen GmbH, Hilden, Germany). Vouchers and DNAsamples are kept in the collections of the NaturalHistory Museum, London (NHM), Museo Nacional deCiencias Naturales, Madrid (MNCN) and the Zoolog-ische Staatssammlung, Munich (ZSM) (Appendix 1).

We amplified four genes, two mitochondrial and twonuclear, with one each ribosomal and protein coding:the 3¢ of cytochrome oxidase subunit I (cox1, primersJerry–Pat; Simon et al., 1994), the 5¢ end of themitochondrial 16S rRNA (rrnL, primers 16SaR)16Sb;Simon et al., 1994), a fragment of histone 3 (H3, primersH3aF–H3aR; Colgan et al., 1998), and the small ribo-somal subunit (SSU). Of the latter, � 600 bp of the 5¢end were sequenced for all specimens (primers 5¢–b5.0;Shull et al., 2001), and the full gene (or most of it) for arepresentative sample (Appendix 1) (see Shull et al.,2001 and Ribera et al., 2002a,b for primers and poly-merase chain reaction conditions). Sequences wereassembled and edited with Sequencher TM 4.1.4 (GeneCodes, Inc., Ann Arbor, MI, USA). New sequenceshave been deposited in GenBank with accession num-bers AJ850306–AJ850675 (273 sequences) andEF670007–EF670317 (311 sequences) (Appendix 1).Sequences of two species of Bidessini were obtainedfrom GenBank (Nirripirti and Gibbidessus, Appendix 1).

Phylogenetic analyses

The two protein-coding genes (cox1 and H3) had noindels, and aligning of sequences was trivial. Foraligning of the ribosomal genes we used three differentapproaches: multiple progressive pair-wise alignmentwith secondary refinement using MUSCLE version 3.52(Edgar, 2004; ‘‘MS’’ in the following); multiple progres-sive alignment modeling the evolution of indels withPRANK (Loytynoja and Goldman, 2005; ‘‘PR’’); anddeleting the hypervariable regions using Gblocks version0.91b (Castresana, 2000; ‘‘GB’’). The latter was basedon the starting alignment produced by MS. Thismultiple approach was preferred over ‘‘sensitivity ana-lyses’’ sensu Wheeler (1995), i.e., exploring the param-eter space within one particular method. We did not

perform any sensitivity analyses in this sense, using thedefault settings (which are usually optimized) except forGB (permitted gap positions 50%). We performedadditional rounds of secondary refinement in MS untilno apparent change was detected.

Bayesian analyses were conducted on a combineddata matrix with MrBayes 3.1.2 (Huelsenbeck andRonquist, 2001), using four partitions (correspondingto the four genes) and evolutionary models as estimatedprior to the analysis with ModelTest 3.7 (Posada andCrandall, 1998). MrBayes ran for 10 · 106 (MS and PR)or 15 · 106 (GB) generations using default values,saving trees each 100 or 1000 generations. ‘‘Burn-in’’values were established after visual examination of aplot of the standard deviation of the split frequenciesbetween two simultaneous runs.

For comparative purposes we also conducted parsi-mony searches in PAUP 4.0b10 (Swofford, 2002), with1000 replicates of random addition of taxa and savingmultiple trees. Support was measured with 1000 boot-strap pseudoreplicates (Felsenstein, 1985) of 25 tree-bisection-reconnection (TBR) searches each, not savingmultiple trees.

Estimation of diversification rates

The comparison of the diversification rates amongsister lineages requires fully resolved topologies, andhence we used the single tree with the highest posteriorprobability (PP) for the MS and PR alignments asobtained with MrBayes. To estimate branch lengths weused only the markers with nearly complete representa-tion in the data set (cox1, rrnL, H3, 5¢-SSU; Appendix1), retaining between 2200 and 2400 bp depending onthe alignment. We estimated the ML branch lengths foreach of the four fragments in PAUP using the modelparameter values obtained in the initial Bayesian anal-ysis (the parameters for the SSU were obtained using thewhole sequence). The final branch lengths were the sumof branch lengths obtained from the four individualpartitions. For comparison, we also estimated parame-ter values directly with PAUP using the same evolu-tionary model for each of the genes separately, and forthe combined sequence in a single partition with aGTR+I+G model. Branch lengths for missing se-quences were estimated through multiple regression:using only the taxa with full data, the branch lengths ofeach gene was regressed against the three others, and themissing values estimated with the regression model.Three taxa had two missing markers (Nirripirti,Gibbidessus and Papuadessus; Appendix 1); for them,we first estimated the value of the branch length of cox1,and then that of SSU using the estimated cox1 plus thetwo empirical values. Four branch length estimates werethus obtained for each of the two topologies (MS andPR): (1) branch lengths estimated in PAUP with the


combined data (single partition, GTR+I+G model); (2)branch lengths with parameters estimated in PAUP forthe individual genes; (3) branch lengths estimated withthe parameters obtained in MrBayes for the individualgenes, with missing data; (4) the same as (3), but withmissing data estimated through multiple regression.Path lengths, i.e., the total length of the branches fromthe root to the tip for each terminal taxon, werecalculated for all eight trees in TreeStat v1.0 (http://evolve.zoo.ox.ac.uk).

To estimate relative rates of diversification we con-structed linearized trees using the penalized likelihoodmethod of Sanderson (2002), as implemented in thesoftware r8s 1.71. We used the Truncated Newtonalgorithm and did a cross-validation procedure withsmoothing factors between 1 and 105 (after eliminationof the outgroups), with the two topologies (MS and PR)and the branch lengths obtained according to theprotocol (4) above.

We tested the relative diversification of the differentlineages of the tree using the Slowinski and Guyer (1989)test for non-nested nodes. Species numbers wereobtained from Nilsson (2001), updated with Nilsson(2003a, 2004) and Nilsson and Fery (2006). To visualizethe diversification trends of the overall tree, and tocompare the estimations obtained from the PR and MSalignments, we obtained lineage-through-time (LTT)plots from linearized trees using Genie v3.0 (Pybus andRambaut, 2002).

Results

Phylogenetic analyses

The final data matrix included 229 taxa (Table 1,Appendix 1). The cox1 and H3 fragments could not beamplified for six taxa each, and the 5¢-SSU for five taxa(Appendix 1). The full, or nearly full, length SSU wasavailable for 79 species, and 26 had additional incom-plete fragments, ranging from 396 (Batrachomatus wingiiClark) to 996 bp (Celina sp.2). As expected (Loytynojaand Goldman, 2005), the longest alignment was thatproduced by PR (Table 2). For all gene fragments, theGTR model (Rodrıguez et al., 1990) with a proportion

of invariable sites (I) and unequal rates (G) was preferredby ModelTest. Bayesian inference chains on the full datasets were slow to converge for each of the threealignments. In the case of GB, the standard deviationof the split frequencies between the two simultaneousruns remained above 0.08, despite running for moregenerations (Table 3). The number of trees in the 95%probability set was large, and individual trees had verylow posterior probabilities (Table 3).

The protein-coding genes had no indels, and there-fore the data matrices were identical for the MS andPR analyses. The estimates of substitution rates andamong-site rate variation in the runs of the differentalignments could be taken as a control for conver-gence among MCMC chains: they were highly con-sistent across alignments, and mainly within the 95%confidence interval of each other (Appendix 2). Theonly difference between these analyses and the corre-sponding GB-based analysis was the proportion ofinvariable sites (Appendix 2), presumably because theprocedure in GB removed terminal ends of thealignment affected by missing data for some taxa.For the ribosomal rRNA genes, the estimate of a forthe SSU partition had non-overlapping 95% confi-dence intervals for the three alignments (Appendix 2,Fig. 1). The estimation of the proportion of invariantsites, p(I), for SSU in PR was also non-overlappingwith those of MS and GB. The estimations ofsubstitution probabilities between nucleotides for therrnL had values fully within the 95% interval of eachother, with the only exception of the GMT substitu-tion (which had very reduced 95% intervals). Thesubstitutions for the SSU typically had non-over-

Table 2Number of nucleotids per gene and alignment, and total number ofparsimony informative sites

Gene PRANK MUSCLE Gblocks

cox1 752 752 737H3 310 310 310rrnL 677 552 4565¢-SSU 691 632 563full SSU 2278 1533 –total 4708 3779 2066informative 1274 1231 782

Table 3Details of the MrBayes and parsimony runs

Alignmentmethod

Bayes Parsimony

No. generations· 106 Burnin· 106 SDSet of treesP ¼ 0.95

PP besttree

No.trees Length CI

PRANK 10 9.8 0.06 1053 0.007 185 23751 0.12MUSCLE 10 9.5 0.05 2872 0.003 210 24885 0.12Gblocks 15 12 0.09 3928 0.002 172 20594 0.08


lapping 95% confidence intervals between GB andeither PR or MS (Appendix 2).

Differences in topology resulting from various treeconstruction methods (Bayesian and parsimony) andalignment procedures (MS, PR and GB) were alsocomparatively minor, and mostly affected the degree ofresolution and support, but did not lead to strongconflict. Parsimony trees had low support (bootstrap<50%) for most deep nodes, and the resolution andsupport increased generally from the GB alignment toMS and PR (see Figs 2 and 3 for a summary of theresults). Very few nodes were contradicted under variousalignments and tree building procedures, and most ofthese had low support (PP < 0.8; parsimony bootstrap<50%). There were only two relatively well supportednodes in Bayesian analyses (PP > 0.90) contradictedbetween alignments, including (1) the placement of theAustralian Hydroporini (the Necterosoma group), and(2) that of the species Notaticus fasciatus Zimmermann(tribe Aubehydrini) (see below).

Phylogeny of Dytiscidae

There were 28 well supported non-nested lineages inthe tree, defined by the most inclusive nodes present inthe Bayesian analyses of all three alignments, withPP ¼ 0.95 for at least two of them and PP ¼ 0.90 forthe third (Fig. 2). Of the 23 lineages with more than one

terminal taxon, 20 were supported at PP > 0.95 underall three alignments (Fig. 2). These lineages mostlycorrespond to recognized supra-generic taxa (tribes orsubfamilies, Table 1), in some cases corresponding tosingle species poor genera showing deviating morpho-logies and uncertain relationships (Fig. 2, Table 1; see,e.g., Miller, 2001a). Among these, Agabetes forms themonotypic tribe Agabetini, currently placed in Lacco-philinae but never associated with this subfamily here.The only genus of subfamily Coptotominae, the Nearc-tic Coptotomus, was found to be sister to the Neotrop-ical Agaporomorphus (Copelatinae) in all analyses, withstrong support (Fig. 2). Finally, the genus Hydrodytes,forming the recently described Hydrodytinae togetherwith Microhydrodytes, stands in an isolated positionnear the base of Dytiscidae.

The relationships among these 28 lineages were ingeneral poorly supported, although we consistentlyrecovered some clades throughout under variousalignment and tree construction methods (Fig. 2).The largest of these lineages grouped Hydroporinae,Laccophilini, Copelatinae (including Coptotomus) andCybistrini, and was found in two of the alignmentswith Bayesian methods (PR and MS) (Fig. 2). Lacco-philini was nested within Hydroporinae (with highsupport only in the GB alignment), rendering the latterparaphyletic. Copelatinae was monophyletic with theinclusion of Coptotomus, and sister to Hydropori-

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 rrnLand SSU in the MrBayes runs. Alignments: PR, PRANK; MS, MUSCLE; GB, Gblocks.

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, exceptfor 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; Liopterus 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 tobe well supported (see Results).


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: nodeswith PP > 0.95 in the Bayesian analyses of one alignment (PR). See Fig. 2 for the support of the deeper nodes.


nae + Laccophilini, a clade also found in the parsi-mony search of the PR alignment. Dytiscinae wasnever monophyletic, and separated into three distinctlineages: Dytiscini, Cybistrini and ‘‘Hydaticini sensulato’’ (i.e., Aubehydrini, Hyderodini, Hydaticini, Acil-iini and Eretini), the latter with high support in two ofthe alignments (PR and MS). Other deep nodes withlower support (only one alignment with PP ¼ 0.95, asecond alignment with PP < 0.90) group Agabini withColymbetinae, Agabetini with Lancetinae, and amonophyletic Platynectes group sensu Nilsson (2000)(including Hydrotrupes).

Within the Hydroporinae plus Laccophilini cladethere were two main lineages: (1) Laccophilini, Bides-sini, Pachydrini, Hydrovatini and Vatellini; and (2)Hydroporini, Hygrotini, Hyphydrini, Methlini andLaccornini. The latter includes taxa formerly linked toHydroporini, but subsequently removed by differentauthors (e.g., Sharp, 1882; Guignot, 1959).

Other notable relationships not reflected in Fig. 2 arethe sister relationship of Matinae and Hydrodytinae inthe Bayesian analysis of the PR alignment (PP ¼ 0.77),and also in the parsimony searches with the PR and MSalignments (also with low support). In the parsimonysearch of both the PR and MS alignments Methlini +Peschetius + Laccornellus + Laccornis form a clade,which is unresolved in GB (with two nodes, Celina +Peschetius and Laccornellus + Laccornis + Methles).

Internal phylogeny of selected lineages

In the following we provide detailed comments on thephylogeny of the most diverse (and well sampled)lineages (Figs 2 and 3).

Colymbetinae (lineage 6). The internal relationshipsof Colymbetinae were not well resolved. The mostprominent result was the polyphyly of the large genusRhantus (with more than 100 species), with the inclusion

Fig. 3. Continued.


of the South African R. cicurius (Fabricius) in a cladewith the morphologically deviating monospecific generaCarabdytes (New Guinea) and Melanodytes (Mediterra-nean). It is interesting to note the inclusion of the genusAgabinus (currently considered a synonymy of Platam-bus, Nilsson, 2000, 2001) within Colymbetinae in allparsimony searches, but as sister to Agabini in allBayesian searches (with PP ¼ 1.0, Fig. 3).

Agabinae (lineages 7–10). Agabinae was informallydivided in two groups of genera by Nilsson (2000), theAgabus and the Platynectes groups. We recoveredthese two lineages, although not as sisters: the Agabusgroup was placed as sister to Colymbetinae, andthe Platynectes group unresolved at the base ofDytiscidae. Within the Agabus group the genericdivision of Nilsson (2000) was confirmed, in agree-ment with the more detailed results of Ribera et al.(2004). Within the Platynectes group (includingHydrotrupes, as suggested in Nilsson, 2000) thegenus Platynectes was paraphyletic with respect to

Hydrotrupes, Andonectes and Leuronectes, suggestingthe need for a deep taxonomic revision (as noted byNilsson, 2000).

‘‘Hydaticini s.l.’’ (lineages 11–14). This clade in-cluded the current Aubehydrini, Hyderodini, Hydati-cini, Aciliini and Eretini, all recovered as reciprocallymonophyletic except for the inclusion of Eretes (the onlygenus of Eretini, Table 1) within Aciliini. The sameclade was recovered by Miller (2001a), and with asimilar topology to that found here. The inclusion ofNotaticus (formerly considered to constitute a separatesubfamily, Aubehydrinae) was also supported with theanalyses of full SSU alone (Ribera et al., 2002b) andmorphology (Miller, 2000, 2001a; Miller et al., 2007b).However, under GB Notaticus was sister to the Platy-nectes gr.1 with PP ¼ 0.98, while under both MS andPR it was included among the ‘‘Hydaticini s.l.’’ withPP ¼ 1.0 (Fig. 2). In the parsimony searches, only MSplaced Notaticus together with the ‘‘Hydaticini s.l.’’(with PR and GB its position was unresolved). The

Fig. 3. Continued.


genus Prodaticus was nested within Hydaticus, suggest-ing the need of a taxonomic rearrangement.

Copelatinae including Coptotomus (lineages 15–17). Our trees were congruent with the more detailedresults of Balke et al. (2004a), confirming the status ofLiopterus and Papuadytes as separate genera, and theneed for a revision of the status of Aglymbus (nestedwithin Copelatus; Fig. 3b).

Cybistrini (lineage 18). This was one of the bestsupported lineages of Dytiscidae. Within Cybistrini, theAustralian genera (Spencerhydrus, Austrodytes andOnychohydrus) were monophyletic and sister to Cybisterplus Megadytes (Palaearctic, Ethiopian and in theAmericas), in agreement with Miller et al. (2007a).However, and contrary to the latter work, we foundMegadytes nested within Cybister, mostly due to anundescribed species from Peru with intermediate mor-phological characters (NHM-IR57, see Appendix 1, andI. Ribera, unpublished results, 1999).

Laccophilini (lineage 19). Generic relationships with-in Laccophilini were fully congruent with the results ofAlarie et al. (2000). There were three main clades withinthe tribe, keeping in mind that a number of genera werenot included in the analyses (Table 1): (1) a SouthAmerican and Ethiopian clade (genera Australphilus,Laccodytes and an undescribed new genus from SouthAmerica, MB1189, Appendix 1); (2) the cosmopolitangenus Laccophilus; and (3) a mostly Australian andOriental clade (Neptosternus and Philaccolilus), includ-ing also the genus Philaccolus from Madagascar.

Bidessini plus Pachydrini (lineage 22). This sisterrelationship (found in all searches except for theparsimony analysis of MS, in which they were unre-solved), and the monophyly of Bidessini, were amongthe most robust results of our analyses. Within Bidessinimany of the nodes were highly supported, and veryconsistent across alignments and analyses.

Peschetius plus Methlini (lineage 24). This sisterrelationship was found in all the Bayesian searchesand in the parsimony search with PR, although onlywith PP ¼ 0.81 in GB (Figs 2 and 3).

Hydroporus group (lineage 26). The Hydroporusgroup of genera was split in two sister lineages, theNearctic Sanfilippodytes, Heterosternuta and Neoporus,and the Holarctic Hydrocolus and Hydroporus. ThePalaearctic genus Suphrodytes was nested withinHydroporus, in accordance with the traditional placementprior to Angus (1985), but the only studied species ofHydrocolus was found as sister to Hydroporus (includingSuphrodytes), in agreement with Larson et al. (2000).

Hyphydrini (lineage 27). The internal topology ofHyphydrini was identical to that found in Ribera andBalke (2007) in a more comprehensive analysis of thetribe, with a split in four main lineages: (1) the genusDesmopachria (American); (2) an Oriental clade with thegenera Allopachria and Microdytes; (3) the genus

Hyphydrus; and (4) a clade formed by all endemic SouthAfrican genera plus the Malagassy Hovahydrus.

‘‘Hygrotini s.l.’’ (lineage 28). This includes the tribeHygrotini, plus the Necterosoma, Graptodytes andDeronectes groups of genera. These four lineages hadstrong support and were recovered consistently acrossalignments and tree construction methods. With the PRalignment, the Necterosoma group was placed as sisterto the rest of ‘‘Hygrotini s.l.’’ with PP ¼ 0.95, but withMS, the Graptodytes group was sister to the rest with thesame PP ¼ 0.95 (with GB the four lineages wereunresolved) (Fig. 2). In the parsimony searches the fourgroups were each recovered as monophyletic in allalignments, but they formed a clade only in PR (in MSand GB they were part of a large polytomy that alsoincluded other lineages).

Within Hygrotini, the genera Herophydrus and Hy-grotus were nested with respect to each other, suggestingthe need for a taxonomic reordination (as noted byBistrom and Nilsson, 2002). The subgenus Coelambus,as currently understood (Nilsson, 2001), was also notmonophyletic. The Necterosoma group included allAustralian Hydroporini, with Carabhydrus as highlyderived within the clade and sister to Sternopriscus. Thestygobiontic genus Nirripirti was closely related toParoster, as noted by Leys et al. (2003). The genericclassification of the Deronectes group is also in need ofrevision (Ribera, 2003), due to the para- or polyphyly ofthe genus Stictotarsus and possibly Oreodytes (the latterwith lower support). Within the Graptodytes group, theclose relationship between the stygobiontic Iberoporusand the rheobiontic Rhithrodytes suggests the need fortaxonomic revision in this group, too, although the lackof some key taxa (e.g., Siettitia) does not allow to drawfirm conclusions from the trees presented here.

Diversification

Net diversification rates among the lineages of Dytisc-idae were estimated on clock-constrained trees based onthe MS and PR alignments. The first step in thisprocedure was to obtain an estimate of branch lengths,which could be affected by different parameter estima-tions, tree topologies, and rates among the four mar-kers. Branch lengths of the individual genes computedunder the GTR+I+G model using the parametersestimated in PAUP and those using the parametersobtained by MrBayes were highly correlated, for thefour genes and the two alignments (r > 0.99 for allcases except for cox1, with r ¼ 0.95 for MS and r ¼ 0.96for PR, n ¼ 456).

Therefore, for simplicity only the parameter valuesestimated with MrBayes were used in subsequentanalyses. Correlations of branch lengths between theindividual genes were also highly significant in allcases (P < 0.001), in particular between the two


mitochondrial partitions cox1 and rrnL, but lowerbetween cox1 and SSU (Table 4). All multiple regres-sions of the branch lengths of each individual gene withthe other three were highly significant, as were all thepair-wise regressions (with the only exception of theSSU for the estimation of the cox1 branch length;Table 5). Finally, total path lengths in each of the eighttrees obtained using MS and PR (four per alignment, seeMethods) were highly correlated (Table 6). When com-paring the two alignment procedures, the highest pair-wise correlations between the path lengths were thosecomputed using the parameters estimated in MrBayesfor the four separate genes (Table 6). These were used inall subsequent analyses, with the inclusion of the

estimations for the missing data in some partitionsobtained through multiple regression (using the param-eters of Table 5).

Branch lengths obtained in this way for the treesgenerated with each of the two alignments weresubjected to rate smoothing using penalized likelihoodto construct a clock-constrained tree, which can subse-quently be used to analyze the evolutionary dynamics ofspecies diversification. The cross-validation procedureimplemented in r8s to identify optimum smoothingparameters selected a value of 1000 for the MSalignment, while multiple failures prevented the compu-tation for the PR alignment, in particular for smoothingparameters of 104 and above. As branch lengths in bothalignments were highly correlated for smoothing param-eters 103 and 104 (r ¼ 0.999), but not when thesmoothing parameter was set to 105 (r < 0.4), we useda value of 103 for both MS and PR, and an arbitrary agefor the root set to 100.

The dynamics of species build-up in Dytiscidae wasinitially investigated using lineage-through-time (LTT)plots (Nee et al., 1992). The inferred trajectories of netdiversification through time were very similar for both

Table 4Correlations among the branch lengths estimated in PAUP for theindividual genes, using the model GTR+I+G and the parametervalues estimated in MrBayes (n ¼ 456; P < 0.001 for all) (PR ⁄MS)

cox1 H3 rrnL

H3 0.38 ⁄0.40 –rrnL 0.52 ⁄0.57 0.36 ⁄0.39 –SSU 0.20 ⁄0.23 0.23 ⁄0.26 0.43 ⁄0.42

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

cox1 H3 SSU

F multipleregression

< 0.0001 < 0.0001 < 0.0001

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

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

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

MS PR

sumByE sumBy allPA sumPA sumByE sumBy allPA

MS sumByE 1sumBy 0.996 1allPA 0.92 0.92 1sumPA 0.96 0.97 0.90 1

PR sumByE 0.61 0.61 0.59 0.57 1sumBy 0.60 0.61 0.59 0.57 0.996 1allPA 0.56 0.56 0.56 0.52 0.92 0.92 1sumPA 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 throughregression; sumBy, branch lengths estimated with the parameters obtained in MrBayes for the individual genes, with missing data; allPA, branchlengths estimated in PAUP with the combined data; sumPA, branch lengths with parameters estimated in PAUP for the individual genes (seeMethods). In bold, highest and lowest correlations between alignments.


alignments, with an apparent uniform decrease indiversification rate towards the recent on a semiloga-rithmic scale, and an inflection point at � 60–70 units oftime when plotted on a linear scale (Fig. 4). This pointcorresponds roughly to the early diversification withinthe main lineages, i.e., the origin of most extant genera(Figs 5 and 6). This analysis would not provide infor-mation on the dynamics of lineage diversificationbeyond this portion of the plot because our samplingregime focused on comprehensive coverage of generabut minimal within-genus sampling (Table 1).

We performed direct comparisons of the knownextant species diversity of the various lineages by testingfor shifts in rates at particular nodes using the test ofSlowinski and Guyer (1989) for unequal rates in sistertaxa. There were no significantly unbalanced nodescommon to all alignments with the exception ofAgaporomorphus + Coptotomus versus Copelatinae(P < 0.02), and Pachydrini versus Bidessini (P <0.03) (Table 7). In some cases, unbalanced nodes werelimited to one of the two alignments and had low nodalsupport. Thus, when Laccornini was resolved as sister tothe rest of an ‘‘Hydroporini s.l.’’ (i.e., including Hygro-tini, Hyphydrini and Methlini), as in the PR alignment(PP ¼ 0.72), the contrast between these two clades wasalso significant (P < 0.01) (Table 7) but this node wasnot present in the trees obtained from the MS and GBalignments (Fig. 2). Similarly, the basal node in the MS-derived tree with the highest PP (Fig. 6) placed Dytiscinias sister to the remaining Dytiscidae, in which case the

two lineages would be significantly different in diversi-fication rates (Table 7), but this node was not present inthe majority rule consensus tree (PP < 0.5; Fig. 2), andtherefore was not further considered. To correct formultiple comparisons, we pooled the contrasts for the 11non-nested lineages present in the tree approximately atthe same depth as the three significant comparisonsusing the combined probability of independent tests()2Slnpi, i ¼ [1k], d.f. ¼ 2k, Sokal and Rohlf, 1995, p.794). The 11 simultaneous contrasts of sister speciesnumbers were also globally significant (P < 0.01)(Table 7).

The Slowinski and Guyer (1989) test does notdetermine whether the differences are due to an increasein the rate of diversification, a decrease, or a combina-tion of both (Vogler and Ribera, 2003). To characterizethese shifts in diversification rates more precisely, weregressed the logarithm of the extant species numberagainst the relative age of both the stem and the crowngroups in each of the above 11 lineages. While theregression with the stem age was not significant (treefrom PR alignment: P ¼ 0.4; MS: P ¼ 0.9; n ¼ 11), theage of the crown group was significantly correlated withspecies numbers (PR: P < 0.01; MS: P < 0.05; n ¼ 11)(Fig. 7). The residuals of this regression suggested adisproportionately low diversity of the Agaporomorphusplus Coptotomus clade, but an unexceptional diversity ofCopelatinae. The highest positive residuals were those ofBidessini and ‘‘Hydroporini s.l.’’ for the MS and PRalignments, respectively (Fig. 8). For this assessment it

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


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


Fig. 6. Linearized tree with the highest posterior probability in the Bayesian analyses of the MS alignment, obtained in r8s with a cross-validationvalue 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 asmonophyletic 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.


is important to note that our taxon sampling was wellsuited to these analyses because for most lineagesunsampled genera are likely to be placed within thesampled crown group, not affecting the estimation of thecrown age, with the possible exception of Pachydrini dueto the lack of examples of Heterhydrus (five species)(Table 1). In the tribe Laccornini the absence of NorthAmerican species of Laccornis is not likely to affect theestimation of the crown age, as the two Europeanspecies included here encompass the whole variationwithin the genus (Wolfe and Roughley, 1990).

Discussion

Phylogeny of Dytiscidae

We found a generally high degree of congruenceamong phylogenetic methods and different alignments,with most incongruent nodes showing low support.Although trees exhibited some incongruence and gener-ally low backbone support, 54% of the 228 possiblenodes had a Bayesian PP equal to, or higher than 0.95for all three alignments, and this value rose to more than60% when considering nodes supported by a minimumof two alignments. Node support was lower for theparsimony searches, but if node stability across methodsis taken as a measure of confidence (Giribet, 2003), thenumber and composition of the main lineages and theirinternal topology can be considered as very robust.

We opted for the use of different methodologicalapproaches to address the alignment of the ribosomalgenes, rather than exploring a fraction of the parameter

space of a single method (i.e., sensitivity analysis sensuWheeler, 1995). The problem of homology assignmentsremains a major unresolved question in molecularphylogenetics, and the behavior and performance ofsome recent methods is still not fully understood (see,e.g., Kjer et al., 2007). For multiple alignment of diver-gent ribosomal sequences, iterative algorithms such asMUSCLE have been shown to consistently outperformprevious methods (Wilm et al., 2006). The suppression ofhypervariable regions by Gblocks resulted in topologieswith fewer resolved nodes and overall lower support, butwhere nodes were recovered, they were largely congruentwith those obtained from the full sequences. Similarly,preliminary results using only the 5¢ end of the SSU gene(not shown), complete in the data matrix for nearly allspecies, were largely compatible with the results reportedhere, but with lower overall support and resolution, inagreement with similar observations on other datasets (for a review see Wiens, 2006).

Differences between the PR and MS alignments wereapparently large, affecting the homology assignments in� 1000 characters, although the number of informativecharacters was very similar (a difference of � 50), andthe topologies were largely compatible. The largest effectof the alignment method was in the estimation of theparameters in MrBayes, particularly for the SSU. Theelimination of the hypervariable regions with Gblockshad the effect of increasing the proportion of invariablesites and the value for a, the shape parameter of the rateheterogeneity distribution. With smaller values of a thedistribution of rates becomes more uneven, with mostsites exhibiting rates close to zero but a few sites withhigh rates (Felsenstein, 2004). The PR alignment greatly

Table 7Slowinski and Guyer (1989) test for comparison of diversification rates between sister lineages. (a) PRANK alignment; (b) MUSCLE alignment. SeeFigs 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 thenode lower than 0.5 (see text)

No. Lineage N sister N P

(a)1 ‘‘Hydaticini s.l.’’ 216 rest Dytiscidae 3775 NS2 ‘‘Colymbetinae s.l.’’ 572 sister lineage 3203 NS3 Coptotomus + Agaporomorphus 12 4-‘‘Copelatinae s.str.’’ 620 0.045 Cybistrini 134 Hydroporinae + Laccophilini 2437 NS6 Laccophilini 402 sister lineage 875 NS7 Hydrovatini + Vatellini 264 Pachydrini + Bidessini 611 NS8 Pachydrini 14 9-Bidessini 597 0.0510 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 NS4 Coptotomus + Agaporomorphus 12 5-‘‘Copelatinae s.str.’’ 620 0.046 Cybistrini 134 Hydroporinae + Laccophilini 2437 NS7 Laccophilini 402 sister lineage 668 NS8 Vatellini 57 Pachydrini + Bidessini 611 NS9 Pachydrini 14 10-Bidessini 597 0.0511 ‘‘Hydroporini s.l.’’ + Laccornini +Hydrovatini 1367 sister lineage 1070 NS


reduced the estimated proportion of invariable sites inthe SSU, although this did not result in a substantialincrease in informative characters (see above).

We found 28 consistently well supported lineageswithin the Dytiscidae recovered under all alignment andphylogenetic inference methods, corresponding to tra-ditionally well characterized groups including manytribes or small subfamilies. The relationships at deeperlevels, defining relationships among clades mostly rec-ognized as subfamilies in the current classification(Nilsson, 2001), were in general poorly supported. Yet,it is interesting to note that most of the clades which arewell-supported by molecular data had also been recog-nized in the taxonomic literature. These taxa would notcorrespond to a particular age (although they seem to beclustered around certain genetic divergences), butinstead reflect significant gaps in the morphological

variation that coincide with divergent groups identifiedby the DNA-based tree.

Some of the traditionally recognized supra-genericgroups have not been recovered in previous cladisticanalyses using morphological characters. This was thecase for the tribe Hygrotini, and the Necterosoma,Hydroporus, Graptodytes and Deronectes groups ofgenera (e.g., Alarie et al., 1999; Miller, 2001a; Alarieand Challet, 2006; Miller et al., 2006; Michat andAlarie, in press; and references therein). The lack ofeasily detectable morphological synapomorphies did notprevent knowledgeable authors to recognize these cladesbased on overall similarity (e.g., Sharp, 1882; Seidlitz,1887; Falkenstrom, 1939), but precluded their recoveryin formal phylogenetic analyses with incomplete taxonsampling or based on restricted data sets. Paradoxically,the intuitively recognized groups of early dytiscid

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) PRANKalignment.


workers show greater agreement with formal analyses ofmolecular, rather than morphological data.

Despite the low resolution among the deeper nodes ofthe trees, we found compelling evidence for the rela-tionships among certain major clades of Dytiscidae.Most striking was the non-monophyly of the subfamilyDytiscinae, which we found separated into three distinctclades including Cybistrini, Dytiscini and a clade formedby the tribes Aciliini + Eretini + Hydaticini + Hy-derodini + Aubehydrini (our ‘‘Hydaticini s.l.’’). Sisterrelationships of the latter two clades were unresolved

and thus compatible with Dytiscinae monophyly, butCybistrini was usually included in a clade with Hydro-porinae, Copelatinae (including Coptotomus) and Lac-cophilini, although with high support only in theBayesian analyses of the MS alignment.

The well supported Cybistrini (Sharp, 1882; Ferreira,2000; Miller, 2001a; Miller et al., 2007a) have previouslybeen placed as sister to the remaining Dytiscinae (Milleret al., 2007a). Interestingly, Sharp (1882) also dividedthe current Dytiscinae in the same three lineages(Cybistrini, Dytiscini and ‘‘Hydaticides’’, corresponding

b

a

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 relativediversification rates and the estimation of the crown diversification age (Table 7, Figs 5 and 6). (a) MUSCLE alignment; (b) PRANK alignment.


to our ‘‘Hydaticini s.l.’’), with the only difference ofHyderodes being included in Dytiscini, as was thegeneral opinion until Miller (2001a). Wolfe (1985) notedthe absence of galea in both the larvae of Cybistrini andmost of the Hydroporinae, but interpreted this similarityas the product of an independent loss, a view reinforcedby the occurrence of a reduced galea in Celina, Laccor-nis, Hydrovatus and Canthyporus (Michat and Alarie,in press).

The current concept of Dytiscinae (Nilsson, 2001) hadalready been adopted by Erichson (1832, 1837), definedon the basis of the five-segmented tarsi of all legs and themale protarsal adhesive discs. Dytiscinae is one of thenodes with highest support in Miller (2001a), but with asingle unambiguous synapomorphy, the ‘‘Dytiscinae-type’’ female genitalia (Burmeister, 1976; Miller, 2000,2001a), as the traditional characters to define the group(e.g., Erichson, 1837) are either plesiomorphic (five-segmented tarsi) or highly homoplastic (male protarsaladhesive discs). A non-monophyletic Dytiscinae wouldimply the independent origin of several of the mostspectacular modifications of diving beetles, usuallyinterpreted as adaptations to high-speed swimming(Nachtigall, 1961; Ribera and Nilsson, 1995).

The family Dytiscidae has a number of morpholog-ically divergent genera that have traditionally beenrecognized as separate at the level of subfamilies ortribes, such as Coptotomus (five species in NorthAmerica), Agabetes (two species, in North Americaand Iran), Hydrotrupes (two species, in western NorthAmerica and eastern China) and Notaticus (two Neo-tropical species) (Nilsson, 2001, 2003a, 2004). Coptoto-mus, the only genus in the subfamily Coptotominae, wasplaced by Miller (2001a) in a clade together withLaccophilinae, Copelatinae, Hydrodytinae and Hydro-porinae, which, except for the inclusion of Hydrodyti-nae, is in agreement with our results. The stronglysupported sister relationship between Coptotomus andAgaporomorphus was highly unexpected. The Neotrop-ical Agaporomorphus is currently included in Copelati-nae (Nilsson, 2001), and considered to be sister to theremaining species based on morphological evidence(Miller, 2001a,b), having a deviating female genitalapparatus (without bursa copulatrix).

Agabetes was originally considered a separate sub-family (Agabetinae), but linked to Laccophilinae basedon similarities of the female genitalia and larvalchaetotaxy (Burmeister, 1976; Nilsson, 1989; Alarieet al., 2002). We found some evidence linking it toLancetinae (mostly Neotropical), but never to Lacco-philini. The north–south vicariance defined by thissister relationship would be similar to that of Agapor-omorphus plus Coptotomus, although this parallelismis compromised by a species from Iran included inAgabetes, and a representative of Lancetes from Aus-tralia (Nilsson, 2001).

The genus Hydrotrupes includes two madicolousspecies, and its placement remained contentious formore than a century (Nilsson, 2000, 2003b; Miller,2001a; Balke, 2005). It has been considered a separatesubfamily, mostly based on larval characters (Beutel,1994; Larson et al., 2000), or within Agabini, mostlybased on adult characters (Nilsson, 2000, 2001; Miller,2001a), but also larval chaetotaxy (Alarie, 1998). Wefound evidence linking Hydrotrupes with the Platynectesgroup, a position suggested by Nilsson (2000) andcompatible with Miller (2001a), who placed Hydrotrupesand Platynectes at the base of Agabini (although not assisters). The Platynectes group has been considered partof Agabini, as redefined by Nilsson (2000). It includesthe widespread genus Platynectes (absent from theNearctic and Afrotropics), and the Neotropical endemicgenera Agametrus, Andonectes and Leuronectes(Gueorguiev, 1971, 1972). Based on morphology, thelatter three genera are separated from Platynectes onlyby different combinations of character reductions, i.e.,loss of lateral pronotal bead and ⁄or loss of metacoxallines (Gueorguiev, 1971, 1972; Pederzani, 1995). Basedon our DNA sequence data, both Agametrus andLeuronectes would be subordinated within Platynectes,confirming the need of a revision of the taxonomicstatus of the genera of the group. The deviatingmorphological characteristics of Hydrotrupes, such asthe lack of a ventral fringe of swimming hairs on theadult hind tibia and tarsus, or the lack of larvalmandibular channel, seem to represent reversals due tothe shift to hygropetric habitat rather than an isolatedphylogenetic position (Ribera et al., 2003a). In all ouranalyses, the relationships of the Platynectes group(including Hydrotrupes) within Dytiscidae was unre-solved or very weakly supported.

The genus Agabinus, synonymized with Platambus byNilsson (2000), was recovered as a separate lineage atthe base of Agabini in the Bayesian analysis, confirmingevidence from larval morphology (Alarie and Larson,1998). However, Agabinus was placed inside Colymbe-tini when using parsimony optimization. Its positionremains ambiguous, but we suggest reinstating Agabinusas a valid genus, in agreement with the results of Miller(2001a) and Ribera et al. (2004).

Within the most diverse group of Dytiscidae, theHydroporinae (with more than 50% of the genera andspecies of the whole family; Table 1), we find strongsupport for several higher lineages. In agreementwith Wolfe (1985), there was some evidence for amajor split in a clade with a predominantly southernhemisphere distribution (Bidessini, Pachydrini, Hydro-vatini and Vatellini), and a clade including alllineages with a mostly northern distribution, but alsosome southern ones (Hydroporini, Hygrotini, Meth-lini, Laccornini and Hyphydrini in some Bayesiananalyses).


The genera Pachydrus and Heterhydrus weretraditionally considered linked to Hyphydrini, untilBistrom et al. (1997) recognized their morphologicaldistinctiveness and placed them in a separate tribe,Pachydrini. Both tribes were later merged again basedon a wider taxon sampling of Dytiscidae (Miller, 2001a;Miller et al., 2006; see also Alarie and Challet, 2006;Michat and Alarie, in press). However, we never foundany relationship between the two groups, in agreementwith Ribera et al. (2002b) and Ribera and Balke (2007).In all our analyses, Pachydrus (the only genus ofPachydrini included) was sister to Bidessini, with strongsupport.

Hydroporini as currently defined has been shown tobe highly polyphyletic by different authors (e.g., Milleret al., 2006; Michat and Alarie, in press), mostly becauseseveral genera with southern distribution were incor-rectly placed in this group, including Canthyporus(Ethiopian), Peschetius (Ethiopian and Oriental) andLaccornellus (Neotropical). All of them have beenplaced recently outside of Hydroporini, as alreadynoted. Similarly, the genera currently in Hygrotini weretraditionally considered part of Hydroporini, but arenow considered a separate tribe (Nilsson and Holmen,1995).

The genus Peschetius is currently included in Hydro-porini (Nilsson, 2001; Bistrom and Nilsson, 2003). In arecent study it was hypothesized to be related toBidessini, based mostly on the common presence of aspine in the spermatheca (Miller et al., 2006), butinstead we found it always linked to Methlini. Bidessini,as redefined by Bistrom (1988) includes all species ofHydroporinae with bi-segmented male lateral lobes, andis one of the best defined and supported lineages ofDytiscidae, both with morphological and molecularcharacters (Miller, 2001a; Ribera et al., 2002b; Milleret al., 2006; and this study). We would not favor the lossof this coherence to expand its concept to include specieswith non-segmented parameres of more uncertain phy-logenetic position, as proposed in Miller et al. (2006).

The two species of Laccornellus were formerlyincluded in the genus Laccornis, but in Roughley andWolfe (1987) they were suggested to be more closelyrelated to the Ethiopian Canthyporus. All Bayesiananalyses placed Laccornellus as sister to Canthyporus, inagreement with the latter authors, although with lowsupport in the MS alignment. But in all parsimonyanalyses (and in the tree with the highest PP of the MSalignment; Fig. 6), Laccornis and Laccornellus wereplaced together with Methlini and Peschetius in amonophyletic clade, more according to the traditionaltaxonomic hypothesis.

We find strong support for a clade including theDeronectes, Graptodytes and Necterosoma groups plusHygrotini (our ‘‘Hygrotini s.l.’’). A relationship betweenthe Deronectes and Necterosoma groups was suggested

previously (Nilsson and Angus, 1992; Alarie and Watts,2004), but usually the incomplete sampling of thesestudies did not allow general conclusions. The Austra-lian genera of Hydroporini form a monophyletic clade(the Necterosoma group), including the morphologicallyenigmatic Carabhydrus, which to date was assigned to itsown tribe, Carabhydrini (Nilsson, 2001). Recent molec-ular work has already shown the inclusion of Carabhy-drus among the Australian Hydroporini (Balke andRibera, 2004). The only Australian Hydroporini genusnot included in our analyses is Sekaliporus, consideredto be closely related to both Tiporus and Antiporus(Watts, 1997). Preliminary analyses with recently col-lected specimens show that it does belong within theNecterosoma group of genera (L. Hendrich and M.Balke, unpublished results, 2007). Our newly foundscenario of a monophyletic Australian Hydroporinisuggests a single, ancient colonization from the north.Remarkably, Hydroporini do not occur in the Orientalregion: the geographically nearest extant taxa only occurin the Eastern Palaearctic (Nilsson, 2001). AustralianHydroporini now contain 10 or 11 morphologically andecologically rather diverse genera (Nirripirti might besynonymized with Paroster, C.H.S. Watts, personalcommunication, 2006) with more than 120 species,constituting a good example of an adaptive radiation.The Australian genera are represented by very fewspecies in New Guinea (Balke, 1995), New Caledoniaand Fiji (Nilsson, 2001).

Evolution of species richness

LTT plots and shifts in clade size between sister taxaprovided insight into the evolutionary build-up ofspecies diversity in Dytiscidae, as the basis for futurestudies that may link species diversification to ecomor-phological traits (Ribera and Nilsson, 1995) or spatialarrangement of habitat patches and biogeographicalregions (Ribera et al., 2003b; Vogler and Ribera, 2003;Balke et al., 2007). These estimations were possiblebased on (1) a largely complete sampling of basallineages, permitting assessments of net diversificationrates during the early phase of dytiscid evolution fromLTT plots, and (2) a set of exemplars representing basalbranches within these major groups for age estimates ofearly splits within subfamilies and groups of genera(crown age); their known total species numbers was thenused for an estimate of average diversification rates.

A prerequisite for these analyses was an estimate ofclock-constrained branch lengths, here scaled with thepenalized likelihood method. Various estimation meth-ods of branch lengths gave very consistent results, withhigh correlations between branches and similar LTTplots, suggesting that the general structure of the treeswas very similar, despite topological differences. Thiswas also supported by the similar number of informative


characters and estimates of rate variation across align-ments, which seemed little affected by the large differ-ences in the total number of aligned characters (Fig. 1and Appendix 2).

Our sampling was virtually complete for the basalsplits of the tree, up to the origin of groups of genera,corresponding to � 60 time units in the LTT plots. Thislimit also corresponds to the basal diversification withinthe well supported terminal lineages. In any case, inquantitative terms the sampling starts to be substantiallyincomplete only within some of the terminal lineages(mainly Bidessini and Laccophilini, Table 1), and in thediversification within genera. This corresponds to aninflection point in the linear LTT plot for both the MSand PR alignments. Given the complete sampling of thebasal part of the tree, a surprising result is the lack ofcorrelation between the number of species and the stemage of the lineages. The expected positive correlation ofclade age and species numbers (McPeek and Brown,2007) was only obtained when ages were calculated forthe crown groups (i.e., since the first nodal split within aclade). This might suggest that major extinction eventspruned multiple basal lineages in a non-uniform way.However, this interpretation is hampered by the uncer-tainties in the topology of the basal nodes, which willlargely affect the estimation of the stem but not thecrown age of well-supported lineages, as noted above.

There were only two consistent significant contrasts ofdiversification rates as measured with the Slowinski andGuyer (1989) test: Copelatinae versus Coptoto-mus + Agaporomorphus, and Bidessini versus Pachyd-rini. This is a very conservative test, but other methodsevaluating full trees could not be implemented due to theincomplete sampling in the tips of the tree (Mooers andHeard, 2002). When only the (almost) complete part ofthe tree was used, among the few remaining lineages(� 40 maximum), no significant whole tree asymmetrieswere found (results not shown).

The residuals of the expected relationship betweennumber of extant species and crown age of the groupmay give an indication of the absolute trend of thediversification rate of each of the individual lineages(McPeek and Brown, 2007). In the case of the Copelat-inae, the significance of the Slowinski and Guyer (1989)test seems to be due to the low diversity of theCoptotomus + Agaporomorphus clade (with a negativeresidual), not to the high diversity of the remainingCopelatinae, which seems to have an ‘‘average’’ diver-sity. The known number of species of both groups may,however, be vastly underestimated, because many spe-cies from tropical regions remain undescribed. Thespecies of Copelatinae are morphologically very uni-form, and they have been traditionally placed in veryfew genera, suggesting the possibility of an ‘‘explosive’’radiation of � 600 described species (Balke et al.,2004a). However, according to our results, the pertinent

question would be not ‘‘Why so many species?’’ but‘‘Why so little morphological diversity?’’. On the con-trary, the large positive residual in the significantcontrast between Bidessini and Pachydrini suggests thatBidessini have experienced a true speed-up in speciesdiversification.

Acknowledgments

We particularly thank all people mentioned in Appen-dix 1 for providing material for study, and the SouthAfrican National Parks (Albertus Lewis) for collectingpermits to I.R. We also thank D.R. Madisson forproviding DNA of Hydrotrupes palpalis; James Hogan,Marta Albarran, Elisa Ribera (NHM London), and AnaIzquierdo (MNCN Madrid) for help with laboratorywork; A.N. Nilsson for updated compiled data onspecies numbers; Y. Alarie for providing manuscripts inpress; and A.N. Nilsson and J. Gomez-Zurita forcomments to earlier versions of the manuscript. Thisdiving beetle project has been funded since 1998 by grantsto IR (Marie Curie fellowship; Leverhulme Trust;Spanish MEC CGL2004-00028), APV (LeverhulmeTrust; NERC) and MB (Marie Curie fellowship;SYNTHESYS ES-TAF 193, 2197; DFG BA2152 ⁄3-1,3-2; Fazit Foundation) held at the NHM and ImperialCollege (London),MNCN (Madrid) andZSM (Munich).

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Appendix 1

List of specimens and sequences used in the analyses.For two species (I. meridionalis and M. coriacea) thecombined sequence was a composite of two differentspecimens. For other two it was a composite of twoclosely related species (N. dispar–N. penicillatum; C.feryi–C. meridionalis). For vouchers or DNA depositedin the Natural History Museum (London) a BMNHaccession number is included. 3¢-SSU bp, length of theSSU sequence in addition to the 5¢-�600 bp fragmentcommon to all species. ‘‘x-[No]’’, 5¢ incomplete; ‘‘[No]-x’’, 3¢ incomplete.


phylogeny and diversification of diving beetles (coleoptera

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