Table Of Content©EntomologicaFennica.11October2006
Molecular data supports the inclusion of Ildobates neboti
Español in Zuphiini (Coleoptera: Carabidae: Harpalinae)
IgnacioRibera,SergioMontagud,SantiagoTeruel&XavierBellés
Ribera,I.,Montagud,S.,Teruel,S.&Bellés,X.2006:Moleculardatasupports
the inclusion of Ildobates neboti Español in Zuphiini (Coleoptera: Carabidae:
Harpalinae).—Entomol.Fennica17:207–213.
The phylogenetic relationships of Ildobates neboti Español (Coleoptera:
Carabidae:Harpalinae)wereinvestigatedbasedonthreenucleargenes(full18S
rRNA,andafragmentofeach28SrRNAandwingless).Wecompiledadataset
usingpublishedsequencesof32membersofHarpalinaeincludingoneexample
eachofDryptini(genusDesera),Galeritini(Galerita)andZuphiini(Thalpius),
plus three Brachininae as outgroups. These three tribes form the “Dryptitae”,
withinwhichvariousrelationshipsofIldobateshadbeenproposed.Theanalyses
ofthedatamatrixusingparsimony(withequallyweightedandreweightedchar-
acters) and Bayesian posterior probabilities all support the monophyly of the
three tribes in “Dryptitae”, as well as a closest relationship of Ildobates with
ThalpiustotheexclusionofDeseraplusGalerita.Thisconfirmsthepreviousin-
clusion of Ildobates among the Zuphiini, and corroborates current taxonomic
classificationsbasedonmorphologicalcriteria.
I.Ribera,DepartamentodeBiodiversidadyBiologíaEvolutiva,MuseoNacional
deCienciasNaturales,JoséGutiérrezAbascal2,28006Madrid,Spain;E-mail:
[email protected]
S. Montagud and S. Teruel, Museu Valencià d’Història Natural (Fundación
EntomológicaTorresSala).PaseodelaPechina15,46008Valencia,Spain;E-
mail:[email protected]
X. Bellés, Department of Physiology and Molecular Biodiversity, Institut de
BiologiaMoleculardeBarcelona(CID,CSIC),JordiGirona18,08034Barce-
lona,Spain;E-mail:[email protected]
Received12December2005,accepted6April2006
1.Introduction MediterraneancoastofSpain,intheprovinceof
Castellón (Ortuño et al. 2004). Although in the
Among the diverse fauna of Coleoptera of the originaldescriptionthespecieswasidentifiedasa
Iberian peninsula, Ildobates neboti Español “Dryptidae”(Español1966),itspreciserelation-
stands out as one of the most emblematic taxa, shipswerecontentious,withcharactersrelatingit
both because of its morphology [with extreme to several of the tribes within the group [see
modificationsforcavelife(Español1966,Bellés Ortuñoetal.(2004)forasummaryofthetaxo-
1987)] and its rarity, being known for only a nomic history of the species]. The current con-
handful of specimens from three caves in the sensus view seems to include Ildobates among
208 Riberaetal. (cid:127) ENTOMOL.FENNICAVol.17
Table1.Sequencesusedinthestudy.TaxonomicordinationfollowsLöbl&Smetana(2003).
No Subfamily Tribe Species 28SrRNA wingless 18SrRNA
1 BRACHININAE BrachinushirsutusBates AF398693 AF398572 AF012478
2 PheropsophusaequinoctialisLinné AF398678 AF398619 AF012477
3 AptinusdisplosorDufour AF398638 AF398569 AF012480
4 HARPALINAE Anthiini Cypholobasp. AF398695 AF398584
5 Calophaenini Calophaenasp.n. AF398666 AF398575
6 Catapieseini CatapiesisbrasiliensisGray AF398645 AF398577 AF012476
7 Chlaeniini ChlaeniusruficaudaChaudoir AF398680 AF398578 AF002777
8 Cnemalobini CnemalobussulciferPhilippi AF398706 AF398580 AF012474
9 Ctenodactylini LeptotrachelusdorsalisFabricius AF398646 AF398599
10 Cyclosomini TetragonoderuslatipennisLeConte AF398653 AF398631 AF012471
11 Graphipterini GraphipteruscordigerDejean AF398711 AF398593
12 Harpalini DiscoderuscordicollisHorn AF398652 AF398588 AF002776
13 Pelmatellussp. AF398690 AF398615 AF398720
14 Helluonini Omphrasp. AF398657 AF398610
15 Lachnophorini CalybelaetulaLeConte AF398705 AF398576 AF002772
16 Lebiini CymindispuntigeraLeConte AF398651 AF398583 AF002773
17 Licinini DicaelusambiguusLaFerté-Sénectère AF398655 AF398586
18 Loxandrini Loxandrusnr.amplithoraxStraneo AF398661 AF398600 AF002778
19 Metriini Metriussp. AF398654 AF398604 AF012475
20 Morionini MorionaridusAllen AF398698 AF398606 AF002783
21 MoriosomusseticollisMacLeay AF398701 AF398607 AF398721
22 Odocanthini ColliurispennsylvanicaLinné AF398712 AF398581
23 Oodini StenocrepiselegansLeConte AF398668 AF398627
24 Orthogoniini Orthogoniussp. AF398709 AF398611 AF398719
25 Panagaeini PanagaeussalleiChaudoir AF398691 AF398612
26 Peleciini Peleciumnr.sulcipenneChaudoir AF398672 AF398614 AF398715
27 Perigonini PerigonanigricepsDejean AF398665 AF398617
28 Platynini AgonumextensicolleSay AF398643 AF398564 AF002775
29 Pseudomorphini Pseudomorphanr.angustataHorn AF398714 AF398622 AF002782
30 Sphallomorphasp. AF398679 AF398636 AF398717
31 Pterostichini PterostichusmelanariusIlliger AF398707 AF398623 AF002779
32 Zabrini AmaraapricariaPaykull AF398694 AF398565 AF002774
33 “Dryptitae” Galeritini GaleritaleconteileconteiDejean AF398686 AF398590 AF002780
34 Dryptini DeseraaustralisPéringuey AF398659 AF398585 per.com.
35 Zuphiini Thalpiusnr.rufulusLeConte AF398697 AF398632 AF002781
36 IldobatesnebotiEspañol AM051084 AM051083 DQ130051
theZuphiini,asreflectedinthemostrecenttaxo- aimistoprovideforthefirsttimeasoundphylo-
nomic catalogues [both Iberian (Serrano 2003) genetic ground for the taxonomic placement of
and Palaearctic (Löbl & Smetana 2003)] and in thisemblematicspecies,aswellastomakefeasi-
morphological studies (Ortuño et al. 2004). At ble future studies on its evolutionary and geo-
present,however,therearenophylogeneticstud- graphicoriginbytheidentificationofitspotential
ies(eithermorphologicalormolecular)including closestlivingrelatives.
Ildobates,thereforealldiscussionsonitsrelation-
shipshavebeenpurelydescriptive.
Inthiscontributionwepresentthefirstformal 2.Materialandmethods
phylogenetic study including Ildobates neboti,
based on sequences of three nuclear gene frag- 2.1.DNAextractionandsequencing
mentsandincludingagoodtaxonomiccoverage
ofHarpalinae(withoneexampleeachofthethree GenomicDNAwasobtainedthroughastandard
tribestowhichIldobateshasbeenrelated).Our phenol-chloroform extraction using abdominal
ENTOMOL.FENNICAVol.17 (cid:127) PhylogeneticpositionofIldobatesneboti 209
tissue(voucherspecimenkeptinX.Belléscoll., constructed with only the species for which all
DNA aliquots kept in the MNCN ref. 6409). genes were available (26 including Ildobates),
Three nuclear fragments were amplified, those andallsubsequentanalyseswereconductedwith
for which there were enough published se- thisdataset.
quences of related species: the full 18S rRNA,
andafragmentofeachwinglessand28SrRNA.
The18SrRNAsequencewasobtainedbydi- 2.3.Phylogeneticanalyses
rect sequencing of four overlapping fragments
amplified with internal primers (see Shull et al. Three hyper-variable regions of the 18S rRNA
2001 for details of the primers and PCR condi- gene and six of the 28S rRNA gene were ex-
tionsused).Bothforwardandreversesequences cludedfromtheanalyses,andtheremainingse-
were obtained for each fragment, which were quencewasalignedbyhand.Toalignthewing-
contigged and edited using Sequencher 4.2 less gene we translated the nucleotide sequence
(GeneCodesCorporation).Thewinglessand28S with McClade 4.0 (Maddison & Maddison
rRNAsequenceswereobtainedusingtheprimers 2000),andalignedtheaminoacidsequenceman-
andthePCRprotocoldescribedinOber(2002). ually.Thenucleotidesequencewasthusaligned
Accession numbers for the sequences are usingtheaminoacidsequenceasatemplate.An
DQ130051(18SrRNA),AM051083(wingless) additionaldatasetwasconstructedwiththeonly
andAM051084(28SrRNA)(Table1). fourspeciesof“Dryptitae”includedintheanaly-
ses(Table1),withthefullsequences(including
hypervariable regions), which were aligned by
2.2.Additionalmoleculardata hand.Thiswasaimedtoincreasethesupportfor
theinternalnodeswithinthegroup.
WerestrictedouranalysestoHarpalinae, asthe ParsimonyanalysiswasconductedinPAUP*
inclusionofIldobateswithinthiscladeisnotcon- version4.0b10(Swofford2002)usingTBRheu-
tentious.WeconsideredBrachininaeastheout- risticsearcheswith1,000randomsequenceaddi-
group, following Ober (2002) and Ribera et al. tionreplicates.Inallsearchesgapswerecodedas
(2005).Sequenceswerecompiledforallspecies amissingcharacterstate.Toincreaseresolution,
of Harpalinae and Brachininae for which the characters were re-weighted according to the
three studied fragments were available (25 spe- rescaled consistency index (Farris 1969), and
cies),plusoneexampleofeachoftheHarpalinae new heuristic searches conducted starting with
tribesforwhichno18SrRNAwasavailable(ten thetreesobtainedwithequallyweightedcharac-
additionalspecies;Table1).Sequenceswereob- ters. Node support was measured with non-
tainedfromMaddisonetal.(1998)(GenBankac- parametricbootstrappingwith1,000pseudo-rep-
cession numbers AF002772–AF002783), Mad- licatesof100randomsequenceadditionseach.
dison et al. (1999) (7 sequences among Previous analyses including the sequences
AF012471–AF012480) and Ober (2002) (5 se- used in this study (Maddison et al. 1998, 1999,
quences among AF398715–AF398721) (Table Ober2002,Riberaetal.2005)showedthelikely
1).Winglessand28SrRNAsequenceswereob- presenceofartefactsduetothedistortingeffects
tained fromOber (2002) (35 sequences of each of long branches (Felsenstein 1978). Therefore,
among AF398569–AF398636 and AF398638– we conducted additional analyses using model-
AF398564, respectively) (Table 1). The 18S based phylogenetic methods, which should in
rRNA sequence of Desera australis Peringuey principlebelesssensitivethanparsimonytothe
(Harpalinae, Dryptini) was obtained from D. biases introduced by highly saturated or homo-
Maddison(personalcommunication;June2005). plasiousdata(e.g.Swoffordetal.1996,Felsen-
Preliminarysearcheswereconductedwiththe stein2004).
28SrRNAandwinglessgenesalonetotestforthe Theoptimalmodelofnucleotidesubstitution
possibilitythatIldobatescouldberelatedtoany was determined with Modeltest 3.7 (Posada &
ofthetribeswithmissing18SrRNAsequences. Crandall1998),forthethreegenesseparately.In
As this was not the case, a reduced dataset was all cases a generalised time reversible (GTR)
210 Riberaetal. (cid:127) ENTOMOL.FENNICAVol.17
Fig.1.Phylogramofthe
singlemostparsimoni-
oustreeresultingfrom
theheuristicsearchon
there-weightedcom-
bineddataset.Num-
bersabovebranches,
bootstrapsupportval-
uesontheequally
weighteddataset;be-
lowbranches,bootstrap
supportvaluesonthe
re-weighteddataset;in-
sidenodes,posterior
Bayesianprobabilities
ofthesearchin
MrBayes(×100)(see
Methodsfordetails).
model (Tavaré 1986) with gamma distributed searcheswereconductedusingthedefaultpriors
amongsiteratevariationandestimatingthepro- (uniform probabilities) starting with random
portion of invariable sites (Yang 1993) was se- trees, with three heated and one cold Markov
lected as the best fit to the data. To analyse the chainsfor2,000,000generations,sampledatin-
dataweusedBayesianprobabilities(Rannala& tervalsof100generations.Todeterminethepoint
Yang1996)asimplementedinthecomputerpro- atwhichtheMarkovchainsreachedstationarity,
gram MrBayes 3.1 (Huelsenbeck & Ronquist the log-likelihood scores were plotted against
2001, Ronquist & Huelsenbeck 2003), as it al- generation time, and visually determined when
lows the estimation of different evolutionary thelog-likelihoodvaluesreachedastableequilib-
models for the user-defined data partitions. The rium. Theparameter estimations (including tree
parameters of the three partitions (i.e., genes) topologies) obtained before reaching the
wereestimatedindependently.Twoindependent stationarityarediscardedasa“burnin”,andonly
ENTOMOL.FENNICAVol.17 (cid:127) PhylogeneticpositionofIldobatesneboti 211
thetreessampledafterthatpointareconsidered
(Huelsenbeck&Ronquist2001).
Posterior probabilities were used to assess
node stability. Although generally higher than
bootstrap support values, posterior probabilities
abovethestandard95%thresholdcanbetakenas
indicativeofstrongnodestability(Suzukietal.
2002, Alfaro et al. 2003, Douady et al. 2003,
Simmons et al. 2004, Huelsenbeck & Rannala
2004).
3.Results
The matrix with the 36 species with 28S rRNA
andwinglesssequences(Table1)had1,187char-
acters,ofwhich359wereparsimonyinformative.
A heuristic search in PAUP resulted in two
equally shortest trees of 2,669 steps, in which
Ildobates was included in a clade with Desera,
ThalpiusandCatapiesiswith69%bootstrapsup-
port,noneofthemwith18SrRNAmissing(Table
1). The more inclusive clades containing Fig.2.Un-rootedtreeoftheexhaustivesearchonthe
Ildobates had bootstrap support values lower combineddataset(wingless,18SrRNA,28SrRNA)of
thefourspeciesof“Dryptitae”,includinghyper-vari-
than50%.
ableregionsoftheribosomalgenes.
Astherewasnoevidenceofacloserelation-
shipofIldobateswithany ofthespecieswitha
missing18SrRNAsequence(Table1),anewma- ofIldobatesandThalpius(Fig.1).
trixwasbuildwiththe26specieswithfulldata, The data matrix of the full sequences of the
with 2,959 characters of which 422 were parsi- four species of “Dryptitae” (Galerita, Desera,
mony informative. The heuristic parsimony ThalpiusandIldobates)had3,846characters,of
searchresultedin11shortesttreesof2,385steps which 114 were parsimony informative. In the
(CI: 0.382, RI: 0.312), with “Dryptitae” mono- shortestofthethreepossibleunrootedtrees,with
phyletic (with only 56% bootstrap support) and 772 steps, the closest relative of Ildobates was
Galerita sister to the rest, which formed a Thalpius, with exclusion of Galerita + Desera
polytomy (not shown). After re-weighting the (i.e., a monophyletic Zuphiini). The same rela-
characters,theheuristicsearchonthe11original tionship was found with a Bayesian probability
treesresultedinasingletree,inwhichThalpius search,withaposteriorprobabilityof1.0(Fig.2).
was sister to Ildobates (i.e., a monophyletic
Zuphiini),alsowithverylowsupport(bootstrap
54%, Fig. 1). The bootstrap support for “Dryp- 4.Discussion
titae”usingre-weightedcharacterswasveryhigh
(100%,Fig.1).ThetreeobtainedwithBayesian Despite the limited taxon sampling (with only
posteriorprobabilitieshadalargepolytomyatthe one example of each of the potentially relevant
baseofHarpalinae,althoughthenodesthatwere groups), all our analyses recovered the mono-
resolved were identical to those of the re- phylyoftribesZuphiini,GaleritiniandDryptini
weightedparsimonytree(Fig.1).Thenodesde- inwhatcouldbecalledthesupertribe“Dryptitae”
finingtherelationshipofIldobateshadingeneral [i.e., the subfamily Dryptinae of e.g. Serrano
highersupport,withaposteriorprobabilityof1.0 (2003)].Thiswasoneofthefewnodeswithhigh
for“Dryptitae”and0.94forthesisterrelationship support among the Harpalinae, both with parsi-
212 Riberaetal. (cid:127) ENTOMOL.FENNICAVol.17
mony and Bayesian posterior probabilities. The Casale, A., Vigna-Taglianti, A. & Juberthie, CH. 1998:
resolutionwithin“Dryptitae”ishoweverlow,al- ColeopteraCarabidae.–In:Juberthie,C.&Decu,V.
(eds.), Encyclopaedia Biospéologica 2: 1047–1081.
thoughallanalysespointtoasisterrelationshipof
SociétedeBiospéologie,Moulis-Bucarest.
the only included Zuphiini (i.e. Thalpius) and
Douady,C.J.,Delsuc,F.,Boucher,Y.,Doolittle,W.F.&
Ildobates, confirmingtheinclusionofIldobates Douzery, J. P. 2003: Comparison of Bayesian and
among the Zuphiini, as considered in Ortuño et maximumlikelihoodbootstrapmeasuresofphylogen-
al.(2004)basedonamorphologicalanalysis,and eticreliability.—MolecularBiologyandEvolution
20:248–254.
inagreementwiththetraditionaltaxonomicclas-
Español, F. 1966: Interesantes descubrimientos bio-
sification (e.g., Löbl & Smetana 2003, Serrano
espeleológicosenlaprovinciadeCastellón.—Publ.
2003). Inst.Biol.Aplic.Barcelona40:67–79.[InSpanish.]
Zuphiiniisatribewithanalmostcosmopoli- Farris,J.S.1969:Asuccessiveapproximationsapproach
tandistribution,withnumerousspeciesoccupy- to character weighting. — Systematic Zoology 18:
374–385.
ingthesubterraneanmedium(bothendogeanand
Felsenstein,J.1978:Casesinwhichparsimonyorcompat-
troglobitic)(Moore1995,Casaleetal.1998).Itis
ibilitymethodswillbepositivelymisleading.—Sys-
remarkablethatsomeofthesetroglobiticspecies tematicZoology27:401–410.
in very distant areas have a strikingly similar Felsenstein, J. 2004: Inferring phylogenies. — Sinauer,
overallappearance,suchase.g.Speozuphiumand Sunderland.664pp.
Huelsenbeck,J.P.&Rannala,B.2004:Frequentistproper-
SpeothalpiusinAustralia(Moore1995,Casaleet
tiesofBayesianposteriorprobabilitiesofphylogen-
al.1998).Ourresultsdemonstratetheinclusion
etictreesundersimpleandcomplexsubstitutionmod-
ofIldobatesamongZuphiini,butaresilentabout els.—SystematicBiology53:904–913.
any further detail on relationships within the Huelsenbeck,J.P.&Ronquist,F.2001:MrBAYES:Bay-
tribe. It would be most interesting to know if esian inference of phylogenetic trees. — Bio-
informatics17:754–755.
Ildobates is most closely related to similar
Löbl,I.&Smetana,A.(eds.)2003:CatalogueofPalaearc-
troglobiticformsindistantgeographicalareas–
tic Coleoptera. 1: Archostemata–Myxophaga–Ade-
in which case it could be appropriately called a phaga.—ApolloBooks,Stenstrup,Denmark.819pp.
“relict” species, as it is usually considered Maddison,D.R.,Baker,M.D.&Ober,K.A.1998:Apre-
(Casaleetal.1998,Ortuñoetal.2004).Alterna- liminaryphylogeneticanalysisof18SribosomalDNA
ofcarabidbeetles(Insecta:Coleoptera).—In:Ball,G.
tively,itcouldbemostcloselyrelatedwithsome
E.,Casale,A.&Vigna-Taglianti,A.(eds.),Phylogeny
oftheEuropeanspecies,inwhichcasethesimi-
and classification of Caraboidea (Coleoptera: Ade-
laritiesinexternalmorphologywithdistantcave phaga):229–250.MuseoRegionalediScienzeNatu-
specieswouldbeaninterestingcaseofparallelor rali,Torino.543pp.
convergentevolution. Maddison,D.R.,Baker,M.D.&Ober,K.A.1999:Phy-
logenyofcarabidbeetlesasinferredfrom18Sribo-
somalDNA(Coleoptera:Carabidae).—Systematic
Acknowledgements. We greatly thank David Maddison Entomology24:103–138.
(Arizona)fortheunpublishedsequenceofDeseraaustra- Maddison,W.P.&Maddison,D.R.2000:MacClade,ver-
lis,andA.Izquierdo(MNCN,Madrid)andM.D.Piulach sion4.0.—Sinauer,Sunderland,Massachussets.
(CID,Barcelona)forhelpinthesequencingwork.Wealso Moore,P.1995:Tworemarkablenewgeneraandspecies
thankAlbertoSendra(Valencia)andVicenteOrtuño(Ma- oftroglobiticCarabidae(Coleoptera)fromNullarbor
drid)fortheirhelpinobtainingthespecimensforstudy. Caves.—JournalofAustralianEntomologicalSoci-
ety34:159–161.
Ober, K. A. 2002: Phylogenetic relationships of the
References carabidsubfamilyHarpalinae(Coleoptera)basedon
molecularsequencedata.—MolecularPhylogenetics
andEvolution24:228–248.
Alfaro, M. E., Zoller, S. & Lutzoni, F. 2003: Bayes or Ortuño, V. M., Sendra, A., Montagud, S. & Teruel, S.
Bootstrap?Asimulationstudycomparingtheperfor- 2004:Systématique etbiologie d’une espèce paléo-
manceofbayesianMarkovchainMonteCarlosam- endémique hypogée de la péninsule Ibérique: Ildo-
pling and bootstrapping in assessing phylogenetic bates neboti Español 1966 (Coleoptera: Carabidae:
confidence.—MolecularBiologyandEvolution20: Dryptinae).—Ann.Soc.Entomol.Fr.(n.s.)40:459–
255–266. 475.[InFrench.]
Bellés, X. 1987: Fauna cavernícola i intersticial de la Posada,D.&Crandall,K.A.1998:Modeltest:testingthe
Península Ibèrica i les Illes Balears. — Ed. Moll, model of DNA substitution. — Bioinformatics 14:
Mallorca.207pp.[InCatalan.] 817–818.
ENTOMOL.FENNICAVol.17 (cid:127) PhylogeneticpositionofIldobatesneboti 213
Rannala,B.&Yang,Z.1996:Probabilitydistributionof meaningfulareBayesiansupportvalues?—Molecu-
molecularevolutionarytrees:Anewmethodofphylo- larBiologyandEvolution21:188–199.
geneticinference.—JournalofMolecularEvolution Suzuki,Y.,Glazko,G.V.&Nei,M.2002:Overcredibility
43,304–311. of molecular phylogenies obtained by Bayesian
Ribera,I.,Mateu,J.&Bellés,X.2005:Phylogeneticrela- phylogenetics.—ProceedingsoftheNaturalAcad-
tionshipsofDalyatmirabilisMateu,2002,withare- emyofScience,U.S.A.99:16138–16143.
visedmolecularphylogenyofgroundbeetles(Coleop- Swofford,D.L.,Olsen,G.J.,Waddell,P.J.&Hillis,D.M.
tera,Carabidae).—JournalofZoologicalSystematics 1996: Phylogenetic inference. — In: Hillis, D. M.,
andEvolutionaryResearch43:284–296. Moritz,C.&Mable,B.K.(eds.),MolecularSystemat-
Ronquist, F., & Huelsenbeck, J. P. 2003: MrBayes 3: ics. 2nd edition: 407–514. Sinauer & Associates,
Bayesianphylogeneticinferenceundermixedmodels. Sunderland,Massachusetts.
—Bioinformatics19:1572–1574. Swofford,D.L.2002:PAUP*.PhylogeneticAnalysisus-
Serrano,J.2003:CatálogodelosCarabidae(Coleoptera) ingParsimony(*andothermethods),Version4.0b10.
delaPenínsulaIbérica.MonografíasS.E.A.,Vol.9.— —Sinauer&Associates,Sunderland,Massachusetts.
Sociedad Entomológica Aragonesa, Zaragoza. 130 Tavaré,S.1986:Someprobabilisticandstatisticalprob-
pp.[InSpanish.] lemsontheanalisisofDNAsequences.—In:Miura,
Shull,V.L.,Vogler,A.P.,Baker,M.D.,Maddison,D.R.& R.M.(ed.),Somemathematicalquestionsinbiology–
Hammond,P.M.2001:Sequencealignmentof18Sri- DNAsequenceanalysis:57–86.AmericanMathSoci-
bosomal RNA and the basal relationships of Ade- ety,Providence,RodeIsland.
phagan beetles:Evidence formonophyly ofaquatic Yang,Z.1993:Maximumlikelihoodestimationofphylog-
familiesandtheplacementofTrachypachidae.—Sy- enyfromDNAsequenceswhensubstitutionratesdif-
st.Biol.50:945–969. fer over sites: Approximate methods. — Molecular
Simmons,M.P.,Pickett,K.M.&Miya,M.2004:How BiologyandEvolution10:1396–1401.
