Molecular phylogeny of the Mediterranean species of Philaenus (Hemiptera: Auchenorrhyncha: Aphrophoridae) using mitochondrial and nuclear DNA sequences

Systematic Entomology (2010), 35, 318–328 DOI: 10.1111/j.1365-3113.2009.00510.x

Molecular phylogeny of the Mediterranean speciesof Philaenus (Hemiptera: Auchenorrhyncha:Aphrophoridae) using mitochondrial and nuclearDNA sequences

A N N A M A R Y A N S K A - N A D A C H O W S K A1, S A K I S D R O S O P O U L O S2,D O R O T A L A C H O W S K A3, Ł U K A S Z K A J T O C H1 and V A L E N T I N AG . K U Z N E T S O V A4

1Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Krakow, Poland, 2Department of AgriculturalBiotechnology, Agricultural University, Athens, Greece, 3Deparment of Entomology, Institute of Zoology, JagiellonianUniversity, Krakow, Poland and 4Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia

Abstract. The phylogenies of all eight European species of Philaenus were estimatedfrom cytochrome oxidase subunit I, cytochrome B and internal transcribed spacer 2(ITS2) fragments of DNA using phylogenetic reconstruction methods: maximum par-simony (MP), maximum likelihood (ML) and Bayesian inference (BI) analyses. Basedon the topologies of all obtained phylogenetic trees, the monophyly of Philaenus iswell supported, being congruent with morphological, ecological and chromosomaldata. Three phylogenetic lineages were distinguished in the mitochondrial and com-bined (mtDNA with ITS2) trees. The first lineage is represented by only one species,Philaenus maghresignus, which inhabits Maghreb and southern Spain. Clade Aincludes three species: P. tarifa (Southern Iberia), P. italosignus (Sicily and SouthernItaly) and P. signatus (the Balkans and Middle East). In clade B two subclades wererecognized: B1 represented by P. loukasi (Southern Balkans) and P. arslani (MiddleEast), and B2 comprising P. spumarus (the most widespread Palaearctic species) andP. tesselatus (from Southern Iberia and Maghreb). These clades were also retrieved intrees reconstructed from nuclear sequences. However, four species (P. maghresignus,P. tarifa, P. italosignus and P. signatus) showed unresolved polytomy at the base ofthe nuclear tree. Clade A together with P. maghresignus clustered with the ‘signatus’group defined from morphology, and clade B with the ‘spumarius’ group; these mightbe considered separate subgenera. Genetic distances in mitochondrial DNA betweeningroup species ranged from 14.0% between P. signatus and P. spumarius to 2.4%between P. tesselatus and P. spumarius. By contrast, genetic divergence of ITS2between ingroup species was very low, at most 2.1%. The divergence of Philaenusspecies is estimated to have occcurred between 7.9 and 0.6 Ma. Possibly three mainspeciation events occurred: the first at the Miocene/Pliocene boundary (c. 5.5 Ma)for deeper splits; the second between 4.2 and 2.5 Ma in the Pliocene, when pairsof more closely related species diverged; and the most recent during the Pleistoceneglaciations, when the separation of P. tesselatus and P. spumarius took place.The species status of all Philaenus species is confirmed except for P. tesselatus.

Correspondence: Anna Maryanska-Nadachowska, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska17, 31-016 Krakow, Poland. E-mail: [email protected]

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Introduction

Systematic research in recent decades has shown that mul-tiple sources of molecular, morphological and ecologicaldata when analysed simultaneously produce better supportedresults, which is essential for understanding biodiversity.Among the Hemiptera, the Auchenorrhyncha (cicadas, spittle-bugs, leafhoppers, treehoppers and planthoppers) is a diversegroup rich in species. Species of Philaenus Stal, 1864 arespittlebugs (Cercopoidea, Aphrophoridae). The Aphrophoridaecomprise 38 genera in the Palaearctic (Nast, 1972). For manyyears, taxonomists and geneticists worked on the widespreadand often abundant spittlebug, Philaenus spumarius, becauseof its very well-developed colour polymorphism (e.g. refer-ences in Halkka & Halkka, 1990; Stewart & Lees, 1996;Drosopoulos, 2003). Until the late 1980s only three specieswere recognized: P. spumarius (Linnaeus, 1758), a widelydistributed Holarctic species, and two Mediterranean species,P. signatus Melichar, 1896 and P. tesselatus Melichar, 1889.The latter was often treated as a subspecies (Wagner, 1959)or synonymized with P. spumarius (Nast, 1972), but recentlyDrosopoulos & Quartau (2002) confirmed that it is a validspecies. Since the 1990s, intensive morphological studieson Philaenus undertaken in the Mediterranean region haveresulted in the description of five new species: P. loukasifrom Greece (Drosopoulos & Asche, 1991), P. arslani fromLebanon (Abdul-Nour & Lahoud, 1996), P. maghresignusfrom Morocco and southern Spain (Drosopoulos & Remane,2000), P. italosignus from southern Italy and Sicily (Drosopou-los & Remane, 2000), and P. tarifa from the southern IberianPeninsula (Remane & Drosopoulos, 2001). The Mediterraneanspecies of Philaenus are sympatric with the most widely occur-ring species P. spumarius, although they are partly allopatricwith each other. The species form three groups according tolarval food plant preferences, with larvae developing: (1) onlyon the lily, Asphodelus aestivus Brotero, 1804 (= A. micro-carpus) (P. signatus, P. italosignus, P. maghresignus, andP. tarifa); (2) on ‘arid’ plants (P. loukasi and P. arslani );and (3) on various kinds of dicotyledons (P. spumariusand P. tesselatus) (Drosopoulos, 2003). The morphology ofmale genitalia suggests two subgroups within the genus: the‘spumarius’ group consisting of P. spumarius, P. tesselatus,P. loukasi and P. arslani ; and the ‘signatus’ group of P. signa-tus, P. italosignus, P. maghresignus and P. tarifa (Drosopoulos& Remane, 2000).

Mitochondrial and nuclear genes are valuable sources ofinformation for taxonomic and evolutionary studies. Theyare frequently used to investigate phylogenetic relationshipsamong groups of related species. Molecular evolution is mostoften studied using sequence divergences of the mitochondrialgenes cytochrome oxidase subunit I and/or II (COI and/orCOII) and cytochrome B (CytB) (Luchetti et al., 2004;Allegrucci et al., 2005). Regarding nuclear markers, some ofthe most studied are the internal transcribed spacers 1 and 2(ITS1, ITS2) and elongation factor 1-alpha (EF-1α) (Depaquitet al., 2000), occasionally in simultaneous analyses (Esseghiret al., 2000; Sueur et al., 2007; Coeur d’acier et al., 2008;

Kim & Lee, 2008). These markers may also make possiblethe identification of new species lacking morphological andgenitalic differences (Fanciulli et al., 1997).

Combined molecular and chromosomal data or molecularand morphological data are very useful in resolving relation-ships of closely related insect species. Such a strategy has beenused for many Mediterranean insects to reconstruct their phy-logenies, and for the identification of species without goodmorphological characters, for example the phasmid, Leptyniaattenuata (Passamonti et al., 2004), Scaritina beetles (Galianet al., 1999) and Phlebotomus sandflies (Esseghir et al., 2000).In Philaenus species, morphological differences are now wellknown (Drosopoulos & Asche, 1991; Abdul-Nour & Lahoud,1996; Drosopoulos & Remane, 2000; Drosopoulos & Quar-tau, 2002), and the chromosomes of all species have beendescribed (Kuznetsova et al., 2003; Maryanska-Nadachowskaet al., 2008 a,b; Maryanska-Nadachowska et al., in prepartion),but molecular data are currently missing.

Here we attempt to clarify the taxonomy of Philaenus withthe first extensive molecular phylogenetic analysis within thegenus, and to compare the resulting molecular phylogenieswith previous morphological and chromosomal data.

Materials and methods

Taxon sampling

Representatives of each of eight European species ofPhilaenus were sampled. Specimens were collected between2003 and 2008 in various parts of the Mediterranean region(Table 1 and Fig. 1). They were preserved in 99.8% ethanol andstored at −20◦C. For outgroup comparisons, sequences fromthe more distantly related Cercopidae Lepyronia coleoptrata(COI: FJ516396 /CytB: FJ664105) and Aphrophora alni (COI:FJ516397/CytB: FJ664106/ITS2: FJ560716) were obtained.All voucher specimens are preserved in the Institute ofSystematics and Evolution of Animals, Polish Academy ofSciences.

DNA extraction, polymerase chain reaction amplification andsequencing

Total genomic DNA was extracted with the DNeasy TissueMini Kit (Qiagen), using one to three individuals from eachspecies. Phylogenies were estimated using data from threeDNA fragments: partial COI, partial CytB and partial ITS2of ribosomal DNA. A stretch of 400 bp of COI was poly-merase chain reaction (PCR)-amplified with primers C1-J-1718[5′-GGAGGATTTGGAAATTGATTAGTTCC-3′] and C1-N-2191 [5′-CCCGGTAAAATTAAAATATAAACTTC-3′] (Lox-dale & Lushai, 1998). A CytB fragment of 650 bp was ampli-fied using primers CB-J10747 [5′-TGTCGAGATGTAAATTATGGNTG-3′] and CB-N11526 [5′-TTCAACTGGTCGRGCTCCAATYCA-3′] (Stewart & Beckenbach, 2005). The ITS2ribosomal DNA region of 514 bp was amplified with primers

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320 A. Maryanska-Nadachowska et al.

Table 1. Species of Philaenus collected in the Mediterranean region.

No. Species Collection locality Host plants GenBank Acc. No. COI/CytB/ITS2

1 P. arslani Lebanon Echinops, Carduus, Cirsium, Cistus FJ516391/ J664100/FJ5607112 P. italosignus Sicily (Italy) Asphodelus aestivus FJ516388/ J664097/FJ5607083 P. loukasi Greece Eryngium spp. FJ516392/FJ664101/FJ5607124 P. maghresignus Southern Spain A. aestivus FJ516394/J664103/FJ5607145 P. signatus Greece A. aestivus FJ516390/ J664099/FJ5607106 P. spumarius Southern France Various dicotyledonous plant species FJ516395/FJ664104/FJ5607157 P. tarifa Southern Spain A. aestivus FJ516389/ J664098/FJ5607098 P. tesselatus Portugal Various dicotyledonous plant species FJ516393/ J664102/FJ560713

COI, cytochrome oxidase subunit I; CytB, cytochrome B; ITS2, internal transcribed spacer 2.

Fig. 1. Ranges of Philaenus species in the Mediterranean region and locations of sample collection places (arrows). Abbreviations in circles: SPU,P. spumarius ; TES, P. tesselatus ; TAR, P . tarifa; ITA, P. italosignus ; SIG, P. signatus; MAG, P. maghresignus ; ARS, P. arslani ; LOU, P. loukasi.Philaenus spumarius occurs wherever the other species are found.

CAS5p8sFc [5′-GCGAACATCGACAAGTCGAACGCACAT-3′] and CAS28sB1d [5′-TTGTTTTCCTCCGCTTATTAATATGCTTAA-3′] (Kim & Lee, 2008).

PCR was performed in 30-μL reaction volumes with 3.0 μLof 10 × PCR buffer, 3.0 μL of 25 mm MgCl2, 0.6 μL of dNTPmixture, each in a 10 mm concentration, 0.6 μL of each 15 mmforward and reverse primers, 1.0–3.0 μL of 100 ng of genomicDNA, 0.2 μL of Taq DNA polymerase (Qiagen) and sterile anddeionized water (up to 30.0 μL).

PCR conditions were as follows: 4 min at 95◦C followed by35 cycles of 30s at 95◦C, 1 min at 50◦C and 2 min at 72◦C,with 10 min at 72◦C after the last cycle. PCR products weresubjected to electrophoresis on a 1.0% agarose gel and stainedwith ethidium bromide. Molecular weight standards wererun along with the samples for reference. The amplificationproducts were purified using the QIAmp PCR purificationkit (Qiagen). Purified DNA was used for sequencing in twodirections using the same primers and the BigDye Terminator3.1 Cycle Sequencing Kit (Applied Biosystems) following themanufacturer’s instructions.

Sequence analyses and phylogenetic reconstructions

The sequences were edited using the BioEdit SequenceAlignment Editor 5.0.9. (Hall, 1999) and aligned usingClustalX 1.8. (Thompson et al., 1997). All alignmentswere verified and corrected by eye. For mitochondrial genes,sequences were also verified for protein-coding frame-shiftsto avoid pseudogenes using Mega 4.0. (Tamura et al., 2007)and compared with sequences from GenBank through a Blastsearch.

For the analyses, the appropriate nucleotide substitutionmodel was first determined using Modeltest 3.06 (Posada& Crandall, 1998) in conjunction with paup*. The partitionhomogeneity test (Farris et al., 1994, 1995), as implementedin paup*, was then used to determine the validity of combiningmitochondrial (COI, CytB) genes into a single analysis. Thetest was performed with 500 replications of MP search onthe randomly partitioned dataset, with five random taxonadditions and a limit of 5000 trees per replicate, and thisallowed further analyses on joined sequences. The partitionhomogeneity test was also used to check for incongruencebetween mitochondrial and nuclear (ITS2) sequences. We used

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three methods for phylogenetic reconstruction: the maximumparsimony (MP) criterion, the maximum likelihood (ML)approach, and Bayesian inference (BI) phylogenetic analysis.MP and ML analyses were computed using paup* 4.0b10(Swofford, 2002). For all MP analyses, heuristic searcheswith tree bisection–reconnection (TBR), branch swappingand 500 random addition sequences were conducted. Gapswere treated as a fifth character state. Heuristic ML searcheswere performed with 1000 replicates of random sequenceaddition and TBR branch swapping. For both MP and MLanalyses, node support was assessed using bootstrap analyseswith 5000 pseudoreplicates and TBR branch swapping.Reconstructions of phylogenies were analysed using MrBayes3.1 (Huelsenbeck et al., 2001; Huelsenbeck & Ronquist, 2001)with one cold and three heated Markov chains for three milliongenerations and trees were sampled every 100th generation.Each simulation was run twice. The first 580 sampled treeswere discarded as ‘burn-in’, and the remainder used toreconstruct a 50% majority rule consensus tree. Analyseswere first performed on each molecular marker separately, andafterwards on combined sequences. Pairwise distances werealso calculated using paup*, with an appropriate model ofevolution. All trees were reconstructed with TreeView 1.6.6(Page, 1996).

Mitochondrial clock

There are no fossil data that could be used for the calibrationof a molecular clock for Philaenus. Although particular speciesinhabit different areas around the Mediterranean, their presentdistributions do not allow an estimation of divergence timesfor any pair of species because no geological event can becorrelated with speciation. Instead, we estimated species agesusing an oft-employed arthropod mtDNA substitution rateof 1.5–2.3% (per lineage, per million years) as calculatedfor mtDNA in other insects (Brower, 1994; Caccone &Sbordoni, 2001; Farrell, 2001) and a Philaenus ML mtDNAtopology. This topology was derived using a substitution modelselected using Modeltest 3.6. Two methods for estimatingspecies ages were used. First, to test whether significant ratedifferences occur among the studied species of Philaenus, theTajima relative rate test (Tajima, 1993) was computed, usingthe 1D (all substitution combined) method, as implemented inmega 4.0 (Tamura et al., 2007). The Aphrophora alni sequencewas used as outgroup for 28 comparisons between Philaenuspairs of species. Substitution rates of insect mtDNA wereapplied to nucleotide divergences among Philaenus haplotypes(species), separately to COI, CytB and combined COI–CytBsequences, using Tamura & Nei (1993) distances to follow thesame model of molecular evolution as in Caccone & Sbordoni(2001). In addition, a linearized mtDNA (COI with CytB) treefor Philaenus was obtained using the semiparametric methodof Sanderson (2002), which allows evolutionary rates to varybetween branches within certain limits using a penalizedlikelihood function (PL) as implemented in r8s 1.7. Thisfunction includes a roughness penalty, which increases when

rate differences in adjacent branches across a tree are large, anda smoothing parameter, which controls the trade-off betweenthe smoothing of rate changes across adjacent branches andthe goodness of fit in the model. This was accomplishedby cross-validation using the ML tree reconstruction andestimated branch lengths as the starting topology, and thetruncated Newton algorithm, which evaluated 100 smoothingvalues at 0.1 increments starting from 0.0. The most basalnode (between the Philaenus clade and Aphrophora alni ) wasarbitrarily fixed at age 100.0 (r8s manual; Sanderson, 2003).Rooting the topology for this analysis required an outgroupmore distant than those used for phylogenetic analyses inorder to scale the most basal divergences in our tree, whichotherwise would be decomposed arbitrarily. For this purpose,Yemmalysus parallelus (Heteroptera; Berytidae), which clearlyrepresents a divergent lineage, sequences were obtained fromGenBank (accession number EU427346.1). Divergence timesof the nodes in the Philaenus tree were calibrated using aniterative approach by adjusting the age of the root until anaverage rate of substitution was found (calculated using theprogram r8s) comparable to the preliminary mtDNA clock rateof 1.5–2.3%.

Results

Sequence analyses

No polymorphism in mtDNA and ITS2 sequences wasdetected between specimens belonging to particular species,and only single sequences (haplotypes) of mtDNA and ITS2were used in further analyses for each species. The three DNAfragments are listed in Table 2. The polymorphism of the twomitochondrial genes was similar (about 20% variable sites and10–13% parsimony-informative sites). The sequences of theITS2 fragments were highly conserved (2.5% variable sitesand 1.8% parsimony-informative sites).

Phylogenetic analyses

Sequencing of COI and CytB fragments resulted in align-ments of 399 and 650 bp, respectively. The partition homo-geneity test did not detect significant incongruence betweenmitochondrial data, so analyses were conducted on thecombined dataset. MP heuristic searches resulted in one

Table 2. Characteristics of analysed sequences for Philaenus species.

Aligned Parsimony- Nucleotidesequence Variable sites informative composition (%)

Sequence (bp) (%) sites (%) A : C : G : T

COI 399 77 (19.3) 39 (9.8) 29:14:14:43CytB 650 141 (21.7) 85 (13.1) 36:14:11:39ITS2 514 13 (2.5) 9 (1.8) 36:14:10:40

COI, cytochrome oxidase subunit I; CytB, cytochrome B; ITS2,internal transcribed spacer 2.

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322 A. Maryanska-Nadachowska et al.

cladogram (length = 652, consistency index (CI) = 0.7531,retention index (RI) = 0.5649) based on 214 parsimony-informative characters. Modeltest identified the GTR + I +G model (proportion of invariable sites I = 0.5644, gamma dis-tribution shape parameter G = 2.0673) as the best nucleotidesubstitution model for ML and MrBayes analyses of the com-bined mitochondrial data. Heuristic searches resulted in oneML tree (−ln = 4155.62743). The ML, MP and Bayesiananalyses resulted in a congruent topology (Fig. 2). Thetopologies of trees obtained for COI and CytB separatelywere also similar (not shown). Based on the GTR + I +G nucleotide substitution model, genetic distances betweeningroup species ranged from 14% between P. signatus andP. spumarius to 2.4% between P. tesselatus and P. spumarius(Table 3).

ITS2 data were first analysed separately. As a result ofproblems with the alignment of ITS2 sequences with thosefrom GenBank (only three species of Hemiptera were depositedthere) and difficulties with the amplification of ITS2 fragmentsin Lepyronia coleoptrata, the analyses were performed usingonly one outgroup taxon. The length of the aligned sequenceswas 514 bp. The topologies obtained with ITS2 were generallyless resolved than analyses of mitochondrial DNA. MPheuristic searches resulted in three trees (length = 162, CI =0.9938, RI = 0.9500) based on nine parsimony-informativecharacters. The HKY model (the proportion of invariable sitesI = 0, equal rates for all sites) was the most appropriate onefor analysing the data. Heuristic search resulted in one MLtree (−ln = 1429.07166). Again, the Bayesian topology wasvery similar to the ML and the MP trees (Fig. 3). The geneticdivergence between ingroup species was very low, the highest,about 2.1%, being between P. tarifa and P. loukasi and alsobetween P. tarifa and P. arslani.

We detected congruence between mitochondrial and ITS2sequences in separate runs of the partition homogene-ity test, which allowed us to produce combined phylo-genetic trees. MP heuristic searches resulted in one tree(length = 694, CI = 0.8069, RI = 0.4981) based on 148parsimony-informative characters. The TIM + I + G model(the proportion of invariable sites I = 0.5053, gamma distri-bution shape parameter G = 0.2597) was the most appropriateone for analysing the data. ML and Bayesian methods resultedin similar topologies. Based on topologies of all obtained phy-logenetic trees (Figs 2–4), the monophyly of Philaenus iswell supported. Three groups were recovered in the mito-chondrial and combined (mtDNA with ITS2) trees. Philaenusmaghresignus, which inhabits the western limits of the genusin southern Iberia and Maghreb (Drosopoulos & Remane,2000), is sister to all remaining Philaenus species. The rest ofspecies are clustered into two clades. Clade A comprises threeallopatric species: P. tarifa in Iberia (Remane & Drosopou-los, 2001), P. italosignus in Italy and Sicily (Drosopoulos &Remane, 2000), and P. signatus in the Balkans (Drosopoulos& Asche, 1991). In clade B two well-supported subclades wereretrieved. Species belonging to B1 are limited to high-altitudehabitats: P. arslani occurs in a few localities in the MiddleEast in Lebanon (Abdul-Nour & Lahoud, 1996), Turkey andIraq (Drosopoulos, 2003) and P. loukasi is distributed in thesouthern Balkans (Drosopoulos, 2003). The most widespreadPalearctic species, P. spumarius and P. tesselatus from south-ern Iberia and Maghreb (Drosopoulos & Quartau, 2002), areascribed to clade B2. Relationships between P. maghresignusand the others species inferred from mitochondrial fragmentswere not resolved on the nuclear tree, probably as a resultof the low polymorphism of ITS2 sequences. However, themonophyly of clade B was confirmed in the ITS2 tree (Fig. 3).

Bootstrap support and posterior probabilities in all treesare high (mostly over 80% for both), with two exceptions in

Fig. 2. Phylogenetic reconstruction of Philaenus obtained from analyses of combined cytochrome oxidase subunit I and cytochrome B sequences.Bayesian topology and branch lengths are shown. Values are respectively (from top): maximum likelihood (ML) and maximum parsimony (MP)bootstraps and Bayesian inference posterior probabilities (all in %). (-) indicate nodes that collapsed in the ML or MP bootstrap consensuses owingto lack of support (bootstrap values <50%). Clades are labelled A, B1 and B2.

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Table 3. Mitochondrial DNA pairwise distances for all pairs of studied species.

Species 1 2 3 4 5 6 7 8 9 10

1 P. tesselatus –2 P. spumarius 2.4 –3 P. arslani 10.5 10.5 –4 P. loukasi 11.1 11.2 10.0 –5 P. italosignus 10.0 10.8 11.4 11.0 –6 P. tarifa 9.3 10.0 11.2 10.3 4.7 –7 P. signatus 13.8 14.0 13.4 12.6 10.1 9.8 –8 P. maghresignus 8.6 9.3 11.1 8.9 8.6 8.0 11.8 –9 L. coleoptrata 26.6 26.7 27.8 27.2 24.5 25.8 29.4 24.4 –

10 A. alni 25.4 25.4 26.7 25.8 25.5 24.5 28.0 23.5 21.9 –

Fig. 3. Phylogenetic reconstruction of Philaenus obtained from the analyses of internal transcribed spacer 2 sequences. Bayesian topology andbranch lengths are shown. Values are respectively (from top): maximum likelihood (ML) and maximum parsimony (MP) bootstraps and Bayesianinference posterior probabilities (all in %). (-) indicate nodes that collapsed in the ML or MP bootstrap consensuses owing to lack of support(bootstrap values <50%). Clades are labelled A, B1 and B2.

the combined mitochondrial genes tree: the branch connectingP. signatus + P. italosignus (57%) and the branch unitingclades A and B (54%) (Fig. 2). In the combined analyses tree(Fig. 4), these branches have collapsed

Dating the divergence times

Results from Tajima’s (1993) relative rate tests indicatedthat the molecular clock hypothesis could not be rejectedin the whole group of considered species, but only forCOI sequences. None of the 28 COI haplotype comparisonsperformed returned a statistically significant value (P rangingfrom 0.2 to 1.0). Only COI sequences were used in furtheranalyses of the first method. To date the splitting of Philaenusspecies we used substitution rates previously reported forother insect species for mtDNA (Brower, 1994; Caccone& Sbordoni, 2001; Farrell, 2001). As such a calculationis subject to large errors, the resulting divergence timescan be regarded only as very broad estimates (Grauer &Martin, 2004). The temporal windows for three pairs ofspecies that were identified in mitochondrial trees, namely

P. tesselatus –P. spumarius , P. tarifa –P. italosignus andP. loukasi –P. arslani , were 1.6–1.0 Ma, 3.1–2.0 Ma and6.6–4.0 Ma, respectively. Divergences times between theremaining species pairs were between 7.9 and 3.7 Ma.

The inferred root age in the second analyses of 12.7 Maindicates a mean substitution rate of about 0.01 substitu-tions per Myr. Using this calibration, the origin of Philaenuswas estimated to be 0.9–0.6 Ma for P. tesselatus –P. spumar-ius , 2.5–1.6 Ma for P. tarifa –P. italosignus , 3.7–2.4 Mafor P. loukasi –P. arslani , 4.2–2.8 Ma for P. tarifa +P. italosignus –P. signatus , 4.6–3.1 Ma for P. tesselatus +P. spumarius –P. loukasi + P. arslani and 3.7–5.6 Ma forP. maghresignus with Clade A and Clade B.

Discussion

Phylogeny of Philaenus

The relationships of Philaenus species reconstructed fromour analyses of molecular markers match quite well withprevious estimates based on morphology (Drosopoulos &Remane, 2000), host-plant preferences (Drosopoulos, 2003)

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324 A. Maryanska-Nadachowska et al.

Fig. 4. Comparison of combined mtDNA and internal transcribed spacer 2 Philaenus genealogy with morphological, ecological and chromosomaldata. Bayesian topology and branch lengths are shown. Values are respectively (from top): maximum likelihood (ML) and maximum parsimony(MP) bootstraps and Bayesian inference posterior probabilities (all in %). (-) indicate nodes that collapsed in the ML or MP bootstrap consensusesowing to lack of support (bootstrap values <50%). Circles with a number inside (1–4) represent subsequent transformations of sex-determinationcharacters and host-plant associations. Clades are labelled A, B1 and B2.

and chromosomes (Kuznetsova et al., 2003; Maryanska-Nadachowska et al., 2008a,b; Maryanska-Nadachowska et al.,in preparation). On the basis of mitochondrial DNA, threespecies, P. tarifa, P. signatus and P. italosignus, are placedin one group previously defined from morphology as the‘signatus’ group. According to Drosopoulos & Remane (2000),P. maghresignus should also be placed in this group. However,the mitochondrial DNA of this species suggests that it isthe sister to all other Philaenus species. Moreover, nuclear(ITS2) DNA data do not resolve the ‘signatus’ group either.A similar polytomy was detected in ITS2 trees of the mostderived species complex of Phlebotomus sandflies from theMediterranean region (Esseghir et al., 2000). Such apparentsimultaneous radiation is probably the result of the rapidspeciation of certain species groups. All species belonging tothe ‘signatus’ group are monophagous – their nymphs feedonly on the lily Asphodelus aestivus (Drosopoulos, 2003).Furthermore, they have the same number of autosomes (22)and XY-type of male sex determination, with the exceptionof P. italosignus, which is characterized by 20 autosomes andX1X2Y sex determination (Maryanska-Nadachowska et al., inpreparation).

Species from clade B (P. loukasi, P. arslani, P. tesselatusand P. spumarius) belong to the ‘spumarius’ group definedfrom male genitalia morphology. Philaenus spumarius and P.

tesselatus are polyphagous, whereas P. loukasi and P. arslanifeed exclusively on mountain arid plants, namely on vari-ous species of Eryngium (Drosopoulos, 2003), or on Eryn-gium, Echinops, Carduus, Cirsium and Cistus (Abdul-Nour &Lahoud, 1996), respectively. Philaenus loukasi and P. arslanialso share a distribution pattern, being restricted to high eleva-tions (mostly above 1200 m a.s.l.), and they have a low numberof colour morphs (three and one, respectively) (Drosopoulos,2003). Chromosome numbers support the division of clade Binto two subclades, B1 and B2. B1 consists of two species,P. loukasi and P. arslani, which are characterized by 18autosomes and the XY type of male chromosome sex deter-mination (Maryanska-Nadachowska et al., 2008; Maryanska-Nadachowska et al., in prepartion), whereas the chromosomalsets of P. tesselatus and P. spumarius consist of 22 auto-somes and the XO type of male sex determination (Kuznetsovaet al., 2003; Maryanska-Nadachowska et al., in prepartion).However, it cannot be determined from the molecular mark-ers whether P. tesselatus is conspecific with P. spumarius orbelongs to a separate species. In all reconstructed trees bothtaxa form a monophylum.

The divergence of mitochondrial genes between insectspecies is usually greater than 3% (Ferguson, 2002). Thesevalues seem applicable for a wide range of invertebrates(Hebert et al., 2003). By contrast, genetic divergence between

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groups within the same species is generally under 2%. Forexample, the two more distantly related groups of asexual andsexual clones of the aphid species Rhopalosiphum padi show1.3% distance divergence (Delmotte et al., 2003). The lowgenetic divergence between P. tesselatus and P. spumarius(2.4%) is not necessarily in conflict with the opinion thatP. tesselatus is a good biological species as assumed on thebasis of morphological criteria. Because rates of molecularevolution vary among different segments of the genome thereis no standard level of divergence for a particular gene that canbe used to establish species boundaries (Lipscomb et al., 2003;Will & Rubinoff, 2004). Among many taxa of hemipterans (i.e.belonging to the aphid genus Megoura; Kim & Lee, 2008)even lower genetic divergences have been observed betweenspecies. Further studies on the genetic distance between distantpopulations of P. spumarius from its entire range (especiallyisolated ones) and P. tesselatus from Iberia and Maghrebare needed to solve the systematic position of the lattertaxon. In addition, precise studies on the genetic variabilityof individuals of P. tesselatus and P. spumarius, as wellas of P. maghresignus and P. tarifa from southern Iberia,where all these species live in sympatry, may be helpfulnot only in solving the taxonomic position of P. tesselatusbut also in checking possible past and recent gene-flowevents occurring among all these species. The genetic distancebetween P. tarifa and P. italosignus is intermediate (about4.7%), which indicates that their separation is more recent thanthat of the other species, except for the pair P. tesselatus –P. spumarius . Genetic distances between other pairs ofPhilaenus species are between 8 and 13% in mitochondrialgenes, supporting their species status. The mean sequencedivergence between clades A, B and P. maghresignus rangesfrom 2.9 to 6.4%. Similar values of mean sequence divergence(from 3.6 to 5.9%) were obtained for species belongingto closely related subgenera of the aphid genus Uroleucon.The divergence of more distantly related subgenera reachedvalues of 5.5–6.8% (Moran et al., 1999). Similar distances(5.0–5.2%) have been observed in the case of another aphidgenus, Brachycaudus (Coeur d’acier et al., 2008). Consideringthe taxonomic status of Philaenus, ‘signatus’ and ‘spumarius’groups might be identified as separate subgenera. Such adivision is also supported by a number of morphological,ecological and chromosomal characters that likewise classifythe species into these two clusters.

Dating divergence times

Patterns of DNA variation at the species level havefrequently been interpreted in the context of range contractionsand expansions associated with palaeoclimatic and geologicalevents of the Miocene and Pliocene and glacial cycles ofthe Quaternary. Molecular clock estimates based on mtDNAsequences confirm that the origins of extant species of manygroups of insects and other animals broadly coincide with theseperiods (Hewitt, 1999, 2000; Avise, 2000). The influence ofclimate changes during the Pliocene and Pleistocene and of

geographic barriers (seas and mountains chains) on the presentdistribution of Mediterranean insects and their speciationhas frequently been demonstrated (e.g. Ribera et al., 2003;Ribera & Vogler, 2004; Allegrucci et al., 2005; Horn et al.,2006). However, it remains less clear how exactly thesefactors affected the speciation process. Glaciations woulddisrupt species geographical ranges, perhaps enhancing thespeciation rate, whereas higher levels of extinction, greaterrange changes and the resulting gene flow may have suppressedthe net speciation rate. The historical reconstruction of rangechanges has frequently been attempted, in particular for taxa inwestern Eurasia where the glacial refugia in the Mediterraneanpeninsulas of Iberia, Italy, the Balkans and Asia Minor arerelatively well known (Hewitt, 1999, 2000). These studies havedemonstrated a complicated distribution of haplotype lineages,which originated as a result of range movements from majorsouthern refugial regions (where endemic groups speciated) tothe north.

The divergence of Philaenus species is estimated to haveoccurred between 7.9 and 0.6 Ma. There were possibly threemain speciation events. Dates of divergence for basal splittingevents are about 5.5 Ma (in the range 7.9–3.7 Ma) orslightly later – 4.5 Ma (2.5–5.6 Ma) – at the Miocene andPliocene boundary or in the first part of the Pliocene. Thiscoincides with the Messinian episode (Mediterranean salinitycrisis, 5.4 Ma), when landmasses around the Mediterraneanbasin were reconnected as a result of the desiccation of theMediterranean (Boccaletti et al., 1990). Land bridges, whichappeared during this episode, may have facilitated the rangeexpansion of Philaenus ancestors. After the Mediterraneanwas flooded again, populations on peninsulas, islands and themainland were isolated and allopatric speciation may havebegun. Particular clades (B) or pairs of species (P. loukasiand P. arslani ) separated, not necessarily simultaneously,between 4.2 and 2.5 Ma. The third speciation event probablyhappened around the Pliocene/Pleistocene boundary (2.6 Ma)and during the Pleistocene glaciations. In these times thepairs of most closely related species diverged. Philaenus tarifawas separated from P. italosignus (3.1–2.0 or 2.5–1.6 Ma).A genetic connection between the two probably existed before3.0 Ma (their ancestral ranges were larger – from Italy toIberia – and gene flow was possible across the MediterraneanSea). Alternatively, the ancestor of one of these speciescolonized distant land and was then isolated there (but onthe basis of present knowledge it is impossible to showthe direction of that colonization). The youngest pair ofspecies, P. tesselatus –P. spumarius , separated during thePleistocene glaciation (1.6–1.0 or 0.9–0.6 Ma), probably indistinct refugia in Iberia.

The suggested pattern of Philaenus divergence is very sim-ilar to that of many other Mediterranean genera. Molecularclock analyses have shown that the divergence of Mediter-ranean species took place mostly in the Pliocene and Pleis-tocene, for example 0.8–1.4 Ma (between species from onesubgenus of Dolichopoda crickets) and 1.2–2.4 Ma (betweenspecies from different Dolichopoda genera) (Allegrucci et al.,2005). In addition, among Phlebothomus sandflies divergence

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326 A. Maryanska-Nadachowska et al.

occurred mostly at the Miocene/Pliocene boundary, and Pleis-tocene oscillations did not play an important role other than inthe isolation of geographical races (Esseghir et al., 2000).

Evolution of Mediterranean Philaenus

Closely related species often have neighbouring geographicranges, as in Dolichopoda crickets from the Mediterranean andCaucasus regions, where sister species are mostly distributed inadjacent areas (Allegrucci et al., 2005). However, sometimes,as in diving beetles (Dytiscidae) in Iberia (Ribera & Vogler,2004) and Philaenus in the Mediterranean region, sisterspecies are mostly allopatric and endemic. The evolutionof Mediterranean species of Philaenus is complicated. Theancestral species of Philaenus probably appeared in theMediterranean area before the Messinian Crisis in the lateMiocene, and underwent subsequent speciation events asa result of palaeogeographical and palaeoclimatic changesduring the Pliocene and Pleistocene. The earliest isolationand fragmentation of Philaenus ranges probably led to theseparation of three lineages, leading to the ancestors of: (1)P. maghresignus; (2) P. tarifa–P. signatus –P. italosignus(clade A); and (3) P. loukasi–P. arslani–P. tesselatus –P. spumarius (clade B). For P. maghresignus is it possible tosay that its ancestor entered northern Africa, and was separatedthere from other species by the Mediterranean Sea.

The separation between species from clade A was proba-bly a result of ancestor expansion into the three major Euro-pean Mediterranean peninsulas. Owing to their geographicalstructure and geological history, Iberia, Italy and the Balkansplayed an important role in the evolution of three distinctlineages from a common ancestor. At present, species ofclade A (P. tarifa–P. signatus –P. italosignus) are allopatric,probably as a result of their habitat preferences and climaticor orographic limitations. The Mediterranean peninsulas areseparated from the main continental landmass of Europe byhigh mountains, which, together with severe climates, mighthave been barriers for the expansion of strictly Mediterraneanspecies, such as P. tarifa, P. italosignus and P. signatus. It ispossible that the expansion of ancestors of these species tookplace when the Mediterranean Sea had contracted, resulting inwide land bridges linking the southern parts of the present-daypeninsulas.

The reconstruction of the speciation history of the clade Bis much more difficult. The ancestors of two subclades (B1and B2) probably expanded into the Mediterranean area, likethose of clade A, and then split into eastern (B1) and western(B2) groups. Philaenus loukasi and P. arslani (subclade B1)are probably relict species that were more widespread duringglaciations but recently became limited to higher elevationsas a result of their adaptation to colder and drier conditionsin mountains. These two species populate limited areas in theBalkans (P. loukasi ) and the Middle East (P. arslani ). Forthe latter species, which was first described from Lebanon,additional populations have recently been detected in distantareas in Turkey and Iraq (Drosopoulos, 2003), and it is

probable that P. arslani will also be found in other high-mountain chains of the Middle East. The ranges of both speciesbelonging to subclade B1 were probably wider in the past, buthave now contracted as a result of climatic and environmentalchanges. The separation of P. tesselatus from P. spumarius(subclade B2) probably took place quite recently. Based ontheir present distributions and small molecular differentiationit is probable that P. tesselatus separated from P. spumariusduring one of the major Pleistocene glaciations, in southernIberia. Philaenus spumarius is the only Philaenus speciesadapted to temperate and even arctic habitats. These wideecological tolerances allowed for its wide expansion in Eurasia.However, to investigate these hypotheses further, detailedphylogeographic and demographic studies on populations fromthe whole range of P. spumarius are needed.

It would be interesting to speculate on the characteristics ofthe ancestral Philaenus, such as their host-plant relationshipsand place of origin. Host-plant species distribution may bean important factor in speciation (Horn et al., 2006). Half ofthe present species of spittlebugs are monophagous and feedon only one species of the Mediterranean lily, Asphodelus,so it is probable that ancestral species were also dependenton that plant. Thus, this condition might be the ancestralstate for species from clade A and P. maghresignus. Thetwo species from high altitudes (P. loukasi and P. arslani )feed mostly on plants characteristic of arid mountain areas,for example Eryngium, Echinops, Carduus, Cirsium, Cistus,and this preference can be treated as a synapomorphic statefor them. By contrast, P. spumarius and its closest relativeP. tesselatus are adapted to various plant food sources,which can also be regarded as synaphomorphic. This changemade possible the postglacial expansion of P. spumarius intotemperate regions of Eurasia, and facilitated its introductioninto North America and oceanic islands.

Conclusions

The most striking result of this study is the confirmation of themonophyly of the genus Philaenus and the major congruenceof phylogenetic relationships with morphological, ecologicaland cytological data. The species status of all but one of thepresently known species is confirmed on the basis of significantgenetic differences. Further studies should focus on theimportance of host-plant association and the role of geographicand ecological barriers in the process of Philaenus speciation.Phylogenetic, phylogeographic and population studies arenecessary to resolve relations between P. tesselatus andP. spumarius and for verification of the systematic status ofdistant Palaearctic populations of P. spumarius.

Acknowledgements

Thanks are due to H. Abdul-Nour, V. D’Urso and A. Nada-chowski for their help in collecting material. Thanks are alsodue to M. F. Claridge, who kindly edited the text.

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Phylogeny of the Mediterranean species of Philaenus 327

This research was supported by the Ministry of Scienceand Higher Education, Poland, grant no. 30301731/0639. V.G.Kuznetsova was partly supported by the Russian Foundationfor Basic Research, grant 08-04-00-787.

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Accepted 16 October 2009First published online 2 February 2010

© 2010 The AuthorsJournal compilation © 2010 The Royal Entomological Society, Systematic Entomology, 35, 318–328