Molecular Phylogenetics and Evolution 37 (2005) 153–164 www.elsevier.com/locate/ympev 1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.04.022 Molecular phylogeography of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae): A scenario suggested by mitochondrial DNA Giuliana Allegrucci ¤ , Valentina Todisco, Valerio Sbordoni Department of Biology, University of Rome Tor Vergata, Via della Ricerca ScientiWca, 00133 Rome, Italy Received 8 November 2004; revised 29 April 2005 Available online 16 June 2005 Abstract This study focuses on the phylogenetic relationships among a number of West-Mediterranean cave crickets species belonging to Dolichopoda; primarily a Mediterranean genus, distributed from eastern Pyrenees to Caucasus. In this paper, 11 Dolichopoda species from the French Pyrenees (D. linderi), the island of Corsica (D. bormansi and D. cyrnensis), and northern, central, and southern Italy (D. ligustica, D. schiavazzii, D. aegilion, D. baccettii, D. laetitiae, D. geniculata, D. capreensis, and D. palpata) were studied. Two more species, one from the Caucasus, D. euxina, and one from Greece, D. remyi, were also included in the analyses, together with more dis- tant species within the same family to be used as outgroups. Fifteen hundred base pairs of mitochondrial DNA, corresponding to the small subunit of the ribosomal RNA (16S rRNA) and to the subunit I of the cytochrome oxidase I (COI), were sequenced in order to clarify the phylogenetic relationships and biogeography of this group of Mediterranean cave crickets. The molecular data are con- gruent with a phylogeographic pattern; with the geographically close species also the most related ones. Based on mtDNA diver- gence, the present-day distribution of genetic diversity seems to have been impacted by climatic events due to glacial and interglacial cycles that have characterized the Pleistocene era. 2005 Elsevier Inc. All rights reserved. Keywords: Phylogeography; Biogeography; Cytochrome oxidase I; 16S rRNA; Mitochondrial DNA; Divergence times; Cave crickets; Dolichopoda 1. Introduction The family Rhaphidophoridae (Orthoptera, Gryllac- ridoidea) includes a large number of cave-adapted gen- era and species with a worldwide distribution. Morphologically, the cave species are quite similar: all species have long legs and antennae, small eyes and poor pigmentation. Several species also occur in epigean habi- tats, being mostly forest dwellers in tropical areas. Cave- adapted species occur in temperate Holarctic (included in the subfamilies Dolichopodinae, Rhaphidophorinae, and Ceuthophilinae), Afrotropical, Neotropical, and Australian regions (Macropathinae) and they are thought to derive from sylvicolous ancestors (Di Russo and Sbordoni, 1998; Hubbel and Norton, 1978; Leroy, 1967). Fossil representatives of such fauna have been recorded in Europe from Baltic ambers dating back to the Oligocene. Species within the Mediterranean genera, Troglophilus and Dolichopoda, are thought to originate from such ancestors (Chopard, 1936). However, phyletic relations within Rhaphidophoridae are controversial (Gorochov, 2001). Object of this study are the West-Mediterranean species of genus Dolichopoda belonging to the subfam- ily Dolichopodinae. This subfamily includes also the Nearctic genera Hadenoecus and Euhadenoecus (Hub- bel and Norton, 1978). Dolichopoda is a circum-Medi- terranean genus, consisting of around 30 species distributed throughout the North Mediterranean regions from the Pyrenees to Turkish Armenia and the Caucasus. Some Dolichopoda species are strictly * Corresponding author. Fax: +39 06 72595965. E-mail address: [email protected](G. Allegrucci).
Transcript
Molecular Phylogenetics and Evolution 37 (2005) 153–164
www.elsevier.com/locate/ympev
Molecular phylogeography of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae): A scenario suggested by mitochondrial DNA
Department of Biology, University of Rome Tor Vergata, Via della Ricerca ScientiWca, 00133 Rome, Italy
Received 8 November 2004; revised 29 April 2005Available online 16 June 2005
Abstract
This study focuses on the phylogenetic relationships among a number of West-Mediterranean cave crickets species belonging toDolichopoda; primarily a Mediterranean genus, distributed from eastern Pyrenees to Caucasus. In this paper, 11 Dolichopoda speciesfrom the French Pyrenees (D. linderi), the island of Corsica (D. bormansi and D. cyrnensis), and northern, central, and southern Italy(D. ligustica, D. schiavazzii, D. aegilion, D. baccettii, D. laetitiae, D. geniculata, D. capreensis, and D. palpata) were studied. Two morespecies, one from the Caucasus, D. euxina, and one from Greece, D. remyi, were also included in the analyses, together with more dis-tant species within the same family to be used as outgroups. Fifteen hundred base pairs of mitochondrial DNA, corresponding to thesmall subunit of the ribosomal RNA (16S rRNA) and to the subunit I of the cytochrome oxidase I (COI), were sequenced in order toclarify the phylogenetic relationships and biogeography of this group of Mediterranean cave crickets. The molecular data are con-gruent with a phylogeographic pattern; with the geographically close species also the most related ones. Based on mtDNA diver-gence, the present-day distribution of genetic diversity seems to have been impacted by climatic events due to glacial and interglacialcycles that have characterized the Pleistocene era. 2005 Elsevier Inc. All rights reserved.
The family Rhaphidophoridae (Orthoptera, Gryllac-ridoidea) includes a large number of cave-adapted gen-era and species with a worldwide distribution.Morphologically, the cave species are quite similar: allspecies have long legs and antennae, small eyes and poorpigmentation. Several species also occur in epigean habi-tats, being mostly forest dwellers in tropical areas. Cave-adapted species occur in temperate Holarctic (includedin the subfamilies Dolichopodinae, Rhaphidophorinae,and Ceuthophilinae), Afrotropical, Neotropical, andAustralian regions (Macropathinae) and they arethought to derive from sylvicolous ancestors (Di Russo
1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.04.022
and Sbordoni, 1998; Hubbel and Norton, 1978; Leroy,1967). Fossil representatives of such fauna have beenrecorded in Europe from Baltic ambers dating back tothe Oligocene. Species within the Mediterranean genera,Troglophilus and Dolichopoda, are thought to originatefrom such ancestors (Chopard, 1936). However, phyleticrelations within Rhaphidophoridae are controversial(Gorochov, 2001).
Object of this study are the West-Mediterraneanspecies of genus Dolichopoda belonging to the subfam-ily Dolichopodinae. This subfamily includes also theNearctic genera Hadenoecus and Euhadenoecus (Hub-bel and Norton, 1978). Dolichopoda is a circum-Medi-terranean genus, consisting of around 30 speciesdistributed throughout the North Mediterraneanregions from the Pyrenees to Turkish Armenia andthe Caucasus. Some Dolichopoda species are strictly
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allopatric and are well diVerentiated both morphologi-cally and karyologically. On the other hand, low mor-phological diVerentiation is associated with identicalor variable chromosome numbers (Baccetti, 1982; Sal-tet, 1967) in species that show quasi-parapatric ranges.The highest species diversity is present in insular andpeninsular Greece and Italy. In particular, nine speciesof Dolichopoda occur in Italy, where they range fromthe Maritime Alps to the southern tip of the Italianpeninsula (Fig. 1). Most species of this genus arestrictly dependent upon caves. However, especially inthe northern part of the range, several populationsinhabit cave-like habitat such as rock-crevices andravines and individuals are often observed outside inmoist or mesic woods. In peninsular Italy, Dolichopodapopulations often live in cellars, catacombs, aqueducts,Etruscan tombs, and other similar man-made hypo-gean environments. Population sizes can be small andconstant over long periods, at least in natural caves(Carchini et al., 1983; Sbordoni et al., 1987).
Based on morphology three subgenera have beendescribed in insular and peninsular Italy: Dolichopoda(Dolichopoda), D. (Chopardina), and D. (Capraiacris)(Baccetti, 1975; Baccetti and Capra, 1959, 1970). One ofthe main morphological diVerences between these sub-genera is presence or absence of spinulation on theappendages. The subgenus Dolichopoda includes the
highest number of species distributed throughout therange of the genus, except for some coastal areas and it ischaracterized by the presence of spines on the anteriortibiae. Species belonging to Chopardina subgenus aremostly found in insular and peninsular Tuscany in Italyand in Corsica, with a single species found also in Greece(Macedonia). Chopardina species are characterized bythe presence of several spines also on the hind femurs.The subgenus Capraiacris includes only two species,restricted to the Giglio Island and Monte Argentario inthe Tuscan archipelago. Its two species (D. aegilion andD. baccettii) are distinct from the species in the other twosubgenera because of the lack of spines on the anteriortibiae and on the hind femur.
For the present study we sequenced 1500 bp of amitochondrial DNA, corresponding to two fragments ofthe small subunit of the ribosomal RNA (16S rRNA)and to the subunit I of cytochrome oxidase I (COI) inorder to clarify the phylogenetic relationships and thehistorical processes that shaped the current geographicdistributions of the 11 Dolichopoda species that occur inthe Western Mediterranean area from the FrenchPyrenees to the southernmost of Italian peninsula(Fig. 1). To attempt to encompass a larger geographicwindow we also included two species from the eastern-Mediterranean region, one from Greece and one fromCaucasus (D. remyi and D. euxina, respectively). These
Fig. 1. Sampling sites and ranges of the West-Mediterranean Dolichopoda species studied. The symbol shape denotes species; shading of a givensymbol denotes subspecies.
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two species are formally included in the Chopardina andDolichopoda subgenera, respectively. To understand therelation of the genus Dolichopoda within the familyRhaphidophoridae we included in the analyses fouradditional taxa, belonging to four diVerent genera withinthe family. These species are considered the closest livingrelative to the genus Dolichopoda (Hubbel and Norton,1978).
Based on genetic divergence, we attempted toreconstruct possible ancient scenarios explaining thepresent-day distribution of the West-MediterraneanDolichopoda species. The Mediterranean region is wellsuited for testing biogeographic hypotheses. Its faunaand Xora has evolved through a complex interplay ofgeological and paleoclimatic vicariance events (Blondeland Aronson, 1999). The geological and paleontologicalscenarios are here compared with genetic divergencedata in order to test alternative hypotheses such as dis-persal and/or vicariance. These two mechanisms havebeen identiWed as the principal ones responsible for theformation of biogeographic patterns. Cave crickets asDolichopoda are favorable organisms to test both dis-persal and vicariance hypotheses, since genetic diVerenti-ation of populations and speciation could have beeninXuenced by active dispersal during the moist and warminterglacials, and by vicariance during the glacial ones.During these periods refugial populations experiencedextremely low dispersal opportunities, because of thewidespread steppic environment, caused by the dry andcold climate.
2. Materials and methods
2.1. Taxon sampling
A total of 52 individuals representing 25populations of 13 species of the genus Dolichopodaand eight individuals of four outgroup species weresampled in this study (Table 1). The species withinthe subgenus Dolichopoda included in this study are:D. linderi from the Pyrenees, D. ligustica from theMaritime Alps, D. laetitiae from the northern-centralApennines, D. geniculata from the central-southernApennines, D. capreensis endemic from the island ofCapri, D. palpata (syn. D. calabra Galvagni) from thesouthernmost part of the Apennines, and D. euxinafrom the Caucasus (Russia, not included in Fig. 1). Weanalyzed all the species within the subgenus Chopar-dina: D. bormansi and D. cyrnensis from the island ofCorsica, D. schiavazzii from the coastal Tuscany (Fig.1), and D. remyi from Macedonia (Greece, notincluded in Fig. 1). Similarly, we studied the two spe-cies included in the subgenus Capraiacris: D. aegilionfrom the island of Giglio and D. baccettii from MonteArgentario, both in Tuscany (Fig. 1). As outgroups we
used Ceuthophilus gracilipes, Troglophilus cavicola,Hadenoecus cumberlandicus, and Euhadenoecus insol-itus. These taxa were chosen as representatives ofdiVerent subfamilies of Rhaphidophoridae. TheNearctic H. cumberlandicus and E. insolitus belong tothe same subfamily Dolichopodinae as the specieshere studied, while the Palaearctic T. cavicola and theNearctic C. gracilipes belong to the Troglophilinaeand Ceuthophilinae subfamilies, respectively.
Two individuals from each population were assayedto provide an approximate idea of intra-speciWcvariation, but mostly verify the accuracy of the sequenc-ing results.
2.2. Laboratory procedures
DNA was isolated from leg muscle using a C-TABprotocol (Doyle and Doyle, 1987). Liquid nitrogen wasused during the homogenization phase. Fresh, frozen or95% ethanol-preserved specimens usually gave the samequality and quantity of DNA. Two overlapping frag-ments of cytochrome oxidase I gene (COI, total of971 bp) and a 527-bp fragment of the 16S rRNA genewere ampliWed through the polymerase chain reaction(PCR) from each individual DNA sample. Primers usedwere J1751, N2191 (Simon et al., 1994), UEA1, UEA5,UEA8, and UEA10 (Lunt et al., 1996) for the COI gene,and 16Sa (Kocher et al., 1989) and 16Sb (Palumbi, 1996)for the 16S rRNA gene.
Double-stranded ampliWcations were performedwith a Perkin-Elmer-Cetus thermal cycler in 50 �l of asolution containing genomic DNA (10–100 ng), 1.5 mMMgCl2, 2.5 mM of each dNTP, 0.5 �M primer, 1 U ofAmplitaq (Perkin-Elmer-Cetus) and the buVer suppliedby the manufacturer. Optimal cycling parameters var-ied for each primer pair used. To obtain two overlap-ping fragments of COI (total of 971 bp) we performed anested-PCR. In the Wrst PCR we used UEA1, UEA10primers to obtain an invisible fragment of about1500 bp. This fragment was then used as template toobtain two partially overlapped DNA fragments,480 bp (using J1751 and N2198 primers) and 650 bp(using UEA5 and UEA8 primers) long. The Wrst PCRincluded a 10 min denaturation at 94 °C followed by 5cycles at 98 °C for 15 s, 48 °C for 45 s, and 72 °C for2 min 30 s and 30 cycles at 92 °C for 15 s, 52 °C for 45 s,and 62 °C for 2 min 30 s with a Wnal extension at 62 °Cfor 7 min. The nested-PCRs included a 10 min denatur-ation at 94 °C followed by 30 cycles at 95 °C for 30 s,50 °C for 30 s, and 72 °C for 1 min 30 s with Wnal exten-sion at 72 °C for 10 min. 16S PCR was performed asfollows: after a 5 min denaturation step at 94 °C, eachcycle of PCR consisted of denaturation for 30 s at95 °C, annealing for 30 s at 48 °C, and extension for 45 sat 72 °C. After 35 cycles, a 5 min extension step at 72 °Cfollowed. Double-stranded ampliWed products were
da Species and outgroup taxa included in this study
Collection locality
shilus gracilipes Hamden, CT, USAhilus cavicola Covoli di Veroli Cave, Veneto, Northern Italyecus cumberlandicus Bat Cave, Carter Cave State Park, Carter Co., KY, USAnoecus insolitus Indian Grave Point Cave, De Kalb Co., TN, USA
Central-West Italyvazzii caprai Fichino Cave (FIC), Tuscany, Central-West Italyansi Brando Cave (BRA), Corsica Island, France; Sisco Cave (SIS), Corsica Island, France
nsis Valletto Cave (VLT), Corsica Island, France; Sabara Cave (SAB), Corsica Island, France
G. Allegrucci et al. / Molecular Phylogenetics and Evolution 37 (2005) 153–164 157
checked for the expected size by electrophoresis of 1/10of the product through a 1% agarose gel. PCRfragments were puriWed by using the GFXTM DNAand Gel Band puriWcation kit (Amersham PharmaciaBiotech), directly sequenced (in both directions) usingthe BigDye terminator ready-reaction kit, and resolvedon either ABI 310 or 3100 Genetic Analyzer (PEApplied Biosystems), following the manufacturer’sprotocols. Sequence data were edited and compiledusing Sequencher version 4.1 (Gene Codes). Allsequences were submitted to GenBank (Accession Nos.AY793561–AY793640).
2.3. Phylogenetic analysis
For the 16S rRNA fragment, DNA sequences werealigned using CLUSTAL X 1.81 (Thompson et al., 1997)with opening gap D 10 and extending gap D 0.10. Thealignment, also checked by eye, did not require furtherimprovement by considering a secondary structuremodel. Cytochrome oxidase I nucleotide sequences wereassembled, aligned, and translated with Sequencher 4.1(Gene Codes). Phylogenetic structure in the data result-ing from both COI and 16S genes was assessed using thepermutation tail probability (PTP test; Faith, 1991) asimplemented in PAUP* version 4.0b10 (SwoVord, 2000),with 1000 random matrices and randomizing of ingrouptaxa only.
Phylogenetic analyses were performed usingmaximum-parsimony (MP; Farris, 1970), and maxi-mum-likelihood (ML; Felsenstein, 1981) methods, asimplemented in PAUP*. A Bayesian analysis was per-formed using MRBAYES version 3.0 (Ronquist and Huel-senbeck, 2003).
MP trees were inferred with a heuristic search usingstepwise addition of taxa with 10 random replicationsand ACCTRAN character-state optimization. Gaps weretreated as missing data. The consistency index (CI;Kluge and Farris, 1969), calculated after the exclusionof uninformative characters (Sanderson and Donog-hue, 1989), was used to examine overall homoplasy lev-els. MP searches were run using both all substitutionsun-weighted and using only transversions (Tv) in thirdposition for the COI data set. The latter was usedbecause saturation analyses revealed a small amount ofsaturation of third codon transitions (analysis avail-able from the authors). The appropriate model ofDNA substitution for ML analysis was determinedusing MODELTEST version 3.06 (Posada and Crandall,1998) for each of the two genes considered, separately.Bayesian analysis was performed using four searchchains for 1,000,000 generations, sampling trees every100 generation and considering the same model as inML analysis. The Wrst 1000 trees were discarded asburn-in. Data were partitioned by gene and, for COI,by codon position. Site-speciWc rate variation was also
calculated. Parameter stability was estimated by plot-ting log-likelihood values against generation time, anda consensus tree with posterior probabilities was thengenerated in PAUP*.
To check if the data from the two mtDNA fragmentscould be combined we used the partition-homogeneitytest (Farris et al., 1994, 1995) as implemented in PAUP*,with 10,000 iterations including and excluding uninfor-mative sites.
Bootstrap supports for the resulting topologies werecalculated using 1000 replicates for MP and 100 repli-cates for the ML tree, as implemented in PAUP*.
Twelve competing phylogenetic hypotheses weretested using the approximately unbiased (AU; Shimoda-ira, 2002) tree selection test in the software package CON-SEL (Shimodaira and Hasegawa, 2001). For comparisonwe also performed the Shimodaira–Hasegawa test (SH;Shimodaira and Hasegawa, 1999).
A Mantel test (1967), considering all ingroup taxa,was carried out to assess possible correlation betweengenetic and geographic distances.
To compare the mtDNA data with previously studiedallozymes data (Allegrucci et al., 1992, 1997; Di Russo,1993 and unpublished data) we carried out a neighborjoining (NJ; Saitou and Nei, 1987) analysis, using ML dis-tances based on the parameters inferred by MODELTEST.Allele frequencies were combined from the diVerent stud-ies and a NJ tree was constructed on the basis of Nei’s(1978) genetic distance index.
2.4. Dating of the cladogenetic events
To test whether signiWcant rate diVerence occursamong the whole group of considered species, wecarried out for each gene, 240 Tajima’s relative ratetests (1993), using both the 1D (all substitutionscombined) and 2D (Ti and Tv considered separately)methods, as implemented in MEGA version 2.1 (Kumaret al., 2001).
To approximate absolute ages of divergence amonghaplotypes, we applied substitution rates previouslyreported for insect mitochondrial COI and 16S genesand calibrated using geological evidence. Two COI sub-stitution rates (i.e., 1.2 and 2.3% per lineage, per millionyears) were used. The Wrst one was calculated for cavebeetles (Coleoptera, Bathyiscinae, Caccone and Sbor-doni, 2001) and the second one was obtained by combin-ing multiple rates from a variety of insects (Brower,1994). Only one substitution rate was used for 16S gene(i.e., 1.1% per lineage, per million years). For this rate,reported in Brower (1994), no molecular evolutionarymodel was available. These rates were applied to nucleo-tide divergences among haplotypes estimated usingTamura and Nei (1993) distances to follow the samemodel of molecular evolution as in Caccone andSbordoni (2001).
158 G. Allegrucci et al. / Molecular Phylogenetics and Evolution 37 (2005) 153–164
3. Results
3.1. Levels of sequence variation in the 16S and COI genes
A total of 971 bp from COI gene was sequenced foreach individual. Percent of sequence divergence rangedfrom 19.6 to 3% between species and from 0.1 to 1%within species, with one to three haplotypes found ineach species. The highest inter-speciWc values were usu-ally associated to comparisons including D. euxina and/or D. remyi, the two Eastern Mediterranean species.Among all considered species, 332 sites are variable and267 are parsimony-informative. Out of 267 parsimony-informative sites, 215 are found in the third positions.The transitions/transversions (Ti/Tv) ratio ranged from0.6 to 6. Ti’s accounted for about 52 or 67% of all substi-tutions, if outgroups are included or excluded,respectively. This suggests some Ti’s saturation due tomultiple substitutions, especially when the outgroupcomparisons were included. This is also revealed by theplot of the number of Ti’s and/or Tv’s vs. geneticdistances (data available from the Wrst author).However, signiWcant phylogenetic structure was detectedwith PTP test (P D 0.001).
Total alignment length for the 16S rRNA sequenceswas 552 bp (obtained using CLUSTAL X). Percent ofsequence divergence ranged from 0.2 to 5% between spe-
cies and from 0 to 0.6% within species, with onehaplotype found in each species. When all species wereconsidered 157 sites were variable and 97 parsimony-informative, the Ti/Tv ratio ranged from 0.39 to 7.
The partition-homogeneity test did not reject the nullhypothesis that COI and 16S data sets are any diVerentfrom random partitions of the pooled data (P D 0.998),this was true also when we tested codon positions withinCOI (P D 0.996). Thus, we combined both data for allsubsequent analyses. Results from separate data set werecongruent to the results of the combined data set andavailable from the authors upon request. The combineddata set included 1523 sites, 504 of which were variableand 360 were parsimony-informative. SigniWcant phylo-genetic structure was detected by the PTP test(P D 0.001).
MODELTEST indicated the general time reversiblemodel with among-site rate heterogeneity and propor-tion of invariant sites (GTR + I + �; Gu et al., 1995; Lan-ave et al., 1984; Yang, 1994) as the best Wt for both genesand the combined data set. Rate matrix parameters forthe combined data set were: A–C D 2.646, A–G D 15.640,A–T D 10.715, C–G D 2.103, C–T D 50.026, G–T D 1.000.Base frequencies were: A D 0.325, C D 0.141, G D 0.156and T D 0.379. Among-site rate variation was approxi-mated with the gamma distribution shape parameter�D 0.812. Proportion of invariant sites was 0.539. These
Fig. 2. Relationships among species of Dolichopoda, inferred by maximum likelihood (ML) from mtDNA sequences. Values above and belowbranches indicate bootstrap percentage for MP and ML methods and posterior probabilities derived from Bayesian analysis (Wrst, second, and third
value, respectively). Only bootstrap values higher than 50% are reported.
G. Allegrucci et al. / Molecular Phylogenetics and Evolution 37 (2005) 153–164 159
parameters were used for subsequent phylogeneticanalyses.
3.2. Phylogenetic analysis of the combined mtDNA data
The MP search using equal weights for all substitu-tion produced two most parsimonious trees 1226 stepslong (CI D 0.582). Fig. 2 shows the tree obtained underML analysis. On each node bootstrap values (BP), rela-tive to MP or ML, and posterior probability (PP) valuesare reported. Generally, this topology is stronglysupported in all performed analyses.
Within the Italian species, three main groups could bedistinguished (Fig. 2). The Wrst group included the conti-nental species from Liguria to Southern Italy (D. ligus-tica, D. laetitiae, and D. geniculata). The second groupwas represented by the southernmost species D. palpataand its sister taxon D. capreensis, endemic of the islandof Capri. All these species belong to subgenus Dolicho-poda. The third group consisted of the species belongingto the subgenera Capraiacris and Chopardina, from con-tinental and insular Tuscany and Corsica (D. aegilion,D. baccettii, D. schiavazzii, D. cyrnensis, and D. bor-mansi). Each of these three clusters is strongly supportedwith BP values ranging from 65 to 100% and PP valuesranging from 95 to 100%. D. euxina (subgenus Dolicho-poda) from Caucasus was the most diVerentiated species.The Pyrenean species, D. linderi, clustered as the sistertaxon of the rest of Italian-Corsican species, even if itbelongs to subgenus Dolichopoda. The Greek species,D. remyi (subgenus Chopardina), linked outside the Ital-ian-Corsican group of species. D. euxina (subgenus Doli-chopoda) from the Caucasus clustered as basal to all theother Dolichopoda species included in this study. Allthese nodes were highly supported both from BP and PPvalues and suggested that both Dolichopoda and Chopar-dina subgenera are polyphyletic. This is also conWrmedby results from AU and SH likelihood ratio tests carriedout for a set of alternative trees, suggested by traditionaltaxonomy, and compared to our overall optimal tree.The AU and SH tests are employed to determine theconWdence set of trees (Shimodaira, 2002; Shimodairaand Hasegawa, 1999). The AU test is a powerful test forgeneral tree selection problems involving a priori and aposteriori hypotheses. The SH test is biased againstrejecting trees when comparing many trees and it is pro-vided here for comparison. Eight of the 12 suboptimaltrees can be rejected in favor of optimal tree using AUtest, while Wve can be rejected using the more conserva-tive SH test (data available from the Wrst author). In par-ticular, topologies considering D. euxina as sister relativeto the other species belonging to subgenus Dolichopodaor D. remyi as sister of the other species belonging tosubgenus Chopardina are rejected by both AU and SHtests in favor of our optimal tree (P < 0.001, in all cases).Within subgenus Dolichopoda, three alternative topolo-
gies were considered. The Wrst one considers D. capreen-sis/D. palpata clade as sister relative to the D. geniculatapopulations and it is rejected by both AU and SH tests(P < 0.05), while in the second one this clade is sister toD. geniculata/D. laetitiae/D. ligustica cluster and it isaccepted by both AU and SH tests (P D 0.279 and 0.923,respectively). Placement of D. linderi as sister relative toD. ligustica or to the Tuscan/Corsican species is not sup-ported by AU test (P < 0.001). Finally, topology consid-ering D. ligustica as sister of Chopardina–Capraiacrisspecies is not accepted by both AU and SH tests(P < 0.05).
Results from Mantel (1967) test, carried out toexplore a possible correlation between geographic andgenetic distance in all studied taxa, suggested a strongphylogeographic pattern with correlation coeYcient,r D 0.785 (� < 0.05).
3.3. Comparison of diVerent genetic markers
In our laboratory, Dolichopoda cave crickets havebeen the object of extensive genetic research. Allozymevariation was thoroughly investigated in several popula-tions of D. laetitiae, D. geniculata, D. baccettii, D. aegi-lion (Sbordoni et al., 1985), D. linderi (Di Russo, 1993;Sbordoni et al., 2000), D. schiavazzii (Allegrucci et al.,1997), D. ligustica, D. capreensis, and D. palpata (unpub-lished data). Allozyme data were not available only forD. euxina and D. remyi. Combining allele frequencies for22 loci from diVerent studies we obtained the NJ treeillustrated in Fig. 3A. This tree, based on Nei’s (1978)genetic distance index, is compared to the NJ tree frompresent data set including exactly the same populationsand species (Fig. 3B). Generally, the two topologies arealmost identical but, as shown by branch lengths thereare striking diVerences at the inter- and intra-speciWclevel, with allozymes 15 times running faster thanmtDNA.
3.4. Dating of cladogenetic events
Results from the Tajima’s (1993) relative rate testsindicated that the molecular clock hypothesis could notbe rejected in the whole group of considered species.None of the 480 comparisons performed returned a sta-tistically signiWcant value (P ranging from 0.100 to 1.00,data available from Wrst author).
To date the main lineage-splitting events in Dolicho-poda group of species, we used substitution ratespreviously reported in other insect species for mitochon-drial COI and 16S genes (Brower, 1994; Caccone andSbordoni, 2001; see also Section 2). Given the large errorassociated with the use of this approach, resulting diver-gence times must be treated with caution as tentativeestimates (Grauer and Martin, 2004). In particular,according to our estimates based on a range of available
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rates (from 1.1 to 2.3%, per lineage, per million years)divergence times between the coastal group (subgeneraCapraiacris and Chopardina) and the inland group ofspecies (Dolichopoda spp.) range from 2.4 to 1.2 millionyears (Myr) ago. The same temporal window is outlinedwhen divergence time is estimated between D. Capraia-cris–D. Chopardina and D. linderi species. Narrowertemporal windows are delineated when comparison aremade within the coastal group or the inland group ofspecies, the divergence time estimates ranging from 2.1and 0.9 and from 1.6 to 0.8 Myr ago, respectively.
4. Discussion
4.1. Phylogenetic reconstruction
As outlined in Section 3, three main groups can bedistinguished within the West-Mediterranean species(Fig. 2). The Wrst one includes the inland species distrib-uted from Liguria to southern Italy, the second one com-prises the coastal species occurring in Corsica and incoastal Tuscany, and the third one consists of the south-ernmost species occurring in Calabria and on the Islandof Capri.
Within the Wrst group, the relationships among popu-lations of D. geniculata are not completely resolved. Atleast some populations of this species are closely relatedto D. laetitiae, and results from hybridization studies inlaboratory and in nature, indicated the lack of pre-mat-ing barriers (Allegrucci et al., 1982; Sbordoni et al.,1987). Allozyme studies indicated that peripheral popu-
lations of D. geniculata Xanking the Tyrrhenian and theAdriatic coasts were genetically more diVerentiated fromeach other, unlike the inland, montane group of popula-tions. This group, consisting of both D. laetitiae andD. geniculata populations, is genetically more homoge-neous, regardless of their taxonomic assignment and/ortheir geographic distance (Allegrucci et al., 1987; Cesa-roni et al., 1997; Sbordoni et al., 1985). Our choice tosample coastal populations of D. geniculata for thisstudy was based on these hints. However, results fromCOI and 16S data sets, while emphasizing the close linkbetween D. laetitiae and D. geniculata, do not clearlyresolve relationships among D. geniculata populations.Comparison of the results from the diVerent data sets(allozymes, morphology, and mitochondrial DNA) sug-gests that D. geniculata is a complex of sibling speciesgradually divergent from D. laetitiae.
Dolichopoda ligustica, a species distributed in north-west Italy, appears to be more closely related to the laeti-tiae–geniculata group than to the other species includedin this study. This is supported by high BP and PP valuesin the ML and Bayesian analyses (75 and 96%, respec-tively, Fig. 2). These results along with the AU test falsifythe hypothesis of D. ligustica as a sister taxon of thePyrenean species D. linderi. Actually, D. ligustica is thewesternmost species among the Italian ones and it couldbe considered as putative sister taxon to D. linderi, basedon geographical considerations.
A controversial point concerns the relationshipsbetween the Tuscany insular endemics D. aegilion andD. baccettii, for which the subgenus Capraiacris has beenerected, and the Tuscan-Corsican species included in the
Fig. 3. Genetic relationships in diVerent populations and species of Dolichopoda based (A) on allozymes and (B) on mtDNA data. Values at nodes arebootstrap percentage of 1000 replications for neighbor joining method. Only bootstrap values higher than 50% are reported.
G. Allegrucci et al. / Molecular Phylogenetics and Evolution 37 (2005) 153–164 161
subgenus Chopardina (D. schiavazzii, D. bormansi, andD. cyrnensis). These two subgenera were erected byBaccetti (1975) on the basis of the absence/presence ofseries of spines on the femurs and the dorsal margin ofthe protibiae. However, despite these diVerences, previ-ous studies based on allozymes and scn DNA–DNAhybridization suggested a close aYnity between thesetwo groups (Allegrucci et al., 1992), a result strongly sup-ported by the present study. Within the Tuscan andCorsican species group, two main clusters can be distin-guished; one constituted by the Tuscan species (D. aegi-lion, D. baccettii, and D. schiavazzii) and the other by theCorsican species (D. cyrnensis and D. bormansi).D. remyi, another putative Chopardina species occurringin Greece, branches outside the Italian-Corsican groupof species, suggesting that the subgenus Chopardina ispolyphyletic. This result is also conWrmed by AU andSH tests; suboptimal topologies considering D. remyi assister of the other species belonging to subgenus Chopar-dina are rejected in favor of our optimal tree. Since themain morphological character deWning the subgenusChopardina is the occurrence of a series of spines on theinferior margin of metafemura, it could be concludedthat the presence/absence of spines on femurs issubjected to homoplasy. We argue that lack of spines isan adaptive character state, tracing the evolution to caveenvironment, as we can also observe in other species ofGryllidae where spines on femur become lost in caveobligate species (Desutter-Grandcolas, 1993; Leroy,1967). It could be speculated that, in epigean crickets,occurrence of spines on legs represents a defence againstvertebrate predators, which are absent in caves.
The third clearly distinct clade in our phylogeneticreconstruction (Fig. 2) includes two other species of thesubgenus Dolichopoda, D. capreensis and D. palpata.Their relatedness as sister taxa was already highlightedat morphological level, from male genitalia morphology(Capra, 1968); however, relationships of this clade withthe other species are not resolved, as also indicated bythe AU and SH tests.
The Pyrenean species, D. linderi, is the sister taxon tothe three clades described above, suggesting an olderdiVerentiation of this species from the Italian ones(Fig. 2).
DiVerent mitochondrial haplotypes clustered follow-ing a geographic pattern, strongly supported by BP, PPanalyses and Mantel test. This phylogeographical pat-tern typically results from isolation due to environmen-tal or geographical barriers. The results shown in thisstudy are in broad agreement with previous studies onWve Italian Dolichopoda species based on diVerentgenetic markers (allozymes, DNA–DNA hybridization,and RFLP mtDNA; Allegrucci et al., 1992; Venanzettiet al., 1993), since clustering trees produced with all thesemarkers produced topologies reXecting the same topo-logical relationships amongst the Wve species as obtained
in the present work. For a larger population data set,which included multiple populations of all the ingrouptaxa in this study but for D. euxina and D. remyi, wecould directly compare divergence levels between allo-zyme and mtDNA data (Fig. 3). Also for this data set thepattern of divergence between the two genetic markers isquite concordant, the main diVerence between the twotrees being on the relative branch lengths (the mtDNAtree branches are 15 times shorter than the allozymic treeones) rather than on topology. This result is not unex-pected given our knowledge on the evolutionary rates inthe two compartments of the genome tracked by thesetwo markers.
4.2. Timing of the cladogenetic events
Estimation of molecular rates is, in principle, a pow-erful tool to trace the tempo and mode of cladogeneticevents; however, the reliability of such estimates stronglydepends on the accuracy by which genetic distances areestimated and on the appropriateness of the calibrationmethod (Arbogast et al., 2002; Bromham et al., 1999;Bromham and Penny, 2003; Grauer and Martin, 2004).The choice of calibration events is therefore crucial tothe accuracy of molecular dating. In the present case,diVerent scenarios would be suitable to calibrate ourmolecular clock, based on diVerent interpretations of thecomplex geological history of the Mediterranean basin.
The current distribution of Dolichopoda in Corsica andon the islands oV the coast of Tuscany could be the out-come of vicariance events dating back to the Messinian(5.5Myr ago), during the Mediterranean salinity crisiswhen landmasses around the Mediterranean basin werereconnected as a result of the desiccation of the Mediter-ranean Sea (Boccaletti et al., 1990). Alternatively, the pres-ence of Dolichopoda in Corsica and in Tuscany could bedue to active dispersal during the Plio-Pleistocene (2–0.5Myr ago), when recurrent marine regressions led to theformation of land bridges, or a series of stepping stoneislands, between Corsica and Tuscany (Burgassi et al.,1983; La Greca, 1990; Lipparini, 1976). More ancientpaleogeographic events, such as the separation of theCorsican-Sardinian plate from the Pyrenees, making theWrst move during the Oligocene–Miocene transition(between 29 and 24Myr ago; Alvarez, 1972; Bellon et al.,1977; Carmignani et al., 1995), could, at least specula-tively, explain the occurrence of Dolichopoda in Corsicaand the Pyrenees. The present distribution of the Italianinland Dolichopoda species could be the outcome of vicar-iance and/or dispersal events caused by the alternation ofglacials and interglacials during the whole Quaternary.
Even if in our taxon samples several insular Dolicho-poda are represented, the recurrent occasions for isola-tion and dispersal occurred from Miocene to Pleistocenemake diYcult tracing a particular calibration event.Therefore, rather than exploiting paleogeographic
162 G. Allegrucci et al. / Molecular Phylogenetics and Evolution 37 (2005) 153–164
information to calibrate the molecular clock, we decidedto use molecular rates previously calculated for insects inboth COI and 16S genes (Brower, 1994; Caccone andSbordoni, 2001). By this procedure, we attempt to con-sider geological and paleoclimatic scenarios for specia-tion as dependent variables, rather than predeterminedbases for calibration.
Based on these molecular rates, ranging from 1.1 to2.3% (per lineage, per million years), the approximatetiming of divergences among major haplotype clusterswould correspond to the paleoclimatic events that inXu-enced the West Mediterranean region during the Pleisto-cene. Whatever COI or 16S rate we consider, the samebroad temporal window is outlined. The most importantspeciation events, driving the subdivision of speciesbelonging to diVerent subgenera would have occurredbetween 2.4 and 1.2 Myr ago. These events date back tothe Plio-Pleistocene era, when repeated changes betweenmarine regressions and transgressions might havefavored dispersal and allopatric separation of the Chop-ardina–Capraiacris species, respectively. Interestingly, acomparable time of divergence was hypothesized for evo-lutionary splitting of the isopod Stenasellus racovitzaispecies group, which is distributed in Corsica and Sardi-nia islands and in coastal Tuscany. According to Ketma-ier et al. (2003) the presence of Stenasellus in coastalTuscany might be due to dispersal through a continuoushydrographic system on a land bridge that connectedthese regions during the Quaternary. Also the presence ofsister taxa of Dolichopoda in Corsica and in coastal Tus-cany might be due to active dispersal through habitatsuited corridors connecting the surfaced lands, during thePleistocene. The subsequent isolation and speciation ofinsular taxa could have occurred during one of the recur-ring marine transgressions. Most of these speciation epi-sodes should have started between 1.2 and 0.9 Myr ago.At this time, around the Calabrian–Ionian transition animportant marine transgression took place, lasting atleast 500,000 years. This transgression represents a likelyevent driving genetic diVerentiation in isolation of thecoastal and the insular Dolichopoda in the Capraiacris–Chopardina group. Moreover, allozyme data indicated adivergence time between the two Capraiacris species,D. aegilion and D. baccettii, corresponding to 800,000years ago (Allegrucci et al., 1992) and between D. baccettiiand D. schiavazzii dating to 940,000 years ago (Sbordoniet al., 1985), in excellent agreement with present mito-chondrial DNA dating, if 2.3%, per lineage, per millionyears, (for COI) and 1.1%, per lineage, per million years,(for 16S) molecular rates are considered. Divergence timebetween D. linderi and the Chopardina–Capraiacris specieswould date back to 1.4–1.1Myr, in disagreement with thehypothesis that the current distribution of Dolichopoda inCorsica and in Pyrenees derived from the much older con-nection of the Corsican-Sardinian plate to Pyrenees, andtheir subsequent separation in early Miocene.
Within the subgenus Dolichopoda, assuming 2.3% (perlineage, per million years) for COI and 1.1% (per lineage,per million years) for 16S molecular rates, the splitbetween D. ligustica and D. laetitiae–D. geniculata cladewould have occurred between 1.3 and 0.9 Myr ago, whiledivergence time estimates between D. laetitiae and D. gen-iculata are 0.8 Myr in all cases. These estimates date againback to the Pleistocene, when continental populationsexperienced repeated instances of active dispersal, duringinterglacial periods in mesic woods, alternating to episodesof population fragmentation and reduction of gene Xowduring the dry cold climatic phases. During these periodsancestral epigean forest populations of Dolichopoda wereforced to use forest remains and caves as refugia.
In conclusion, we can observe that the temporalsequence of cladogenetic events is in good agreementwith the geographic distribution of the studied taxa, sup-porting the issue that speciation events have been strictlyallopatric and mostly determined by isolation of diVer-ent populations in isolated cave systems, following therefugium model (Barr and Holsinger, 1985; Sbordoni,1982; Sbordoni et al., 2000). This model assumes frag-mentation of a widespread epigean species and isolationof small populations in cave refugia, where divergence ismainly driven by genetic drift. Actually, the vast major-ity of Dolichopoda species is allopatric and comprisesgenetically well-diVerentiated populations, as indicatedby low levels of current and historical gene Xow amonggeographically close conspeciWc populations (Cesaroniet al., 1997, and unpublished data).
Acknowledgments
The authors express their gratitude to Claudio DiRusso, Mauro Rampini, and Nikolai Mugue for collect-ing samples of D. linderi, D. remyi, and D. euxina, respec-tively. Gisella Caccone kindly provided the DNA fromthe North American cave crickets. Stefano De Feliciassisted us in the use of UNIX system. We thank SaverioVicario for valuable comments on the manuscript.
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