divergent evolutionary histories of two sympatric spruce...

Divergent evolutionary histories of two sympatric sprucebark beetle species

CORALIE BERTHEAU,* HANNES SCHULER,* WOLFGANG ARTHOFER,† DIMITRIOS N. AVTZIS,‡FRANC!OIS MAYER,§ SUSANNE KRUMB€OCK,* YOSHAN MOODLEY¶1 and CHRISTIAN STAUFFER*1

*Department of Forest and Soil Sciences, Institute of Forest Entomology, Forest Pathology and Forest Protection, Boku,University of Natural Resources and Life Sciences, Vienna, Austria, †Molecular Ecology Group, University of Innsbruck,Innsbruck, Austria, ‡Forest Research Institute, NAGREF, Vasilika, Thessaloniki, Greece, §Lutte Biologique et Ecologie Spatiale,Universit"e Libre de Bruxelles, Brussels, Belgium, ¶Department of Integrative Biology and Evolution, Konrad Lorenz Instituteof Ethology, University of Veterinary Medicine, Vienna, Austria

Abstract

Ips typographus and Pityogenes chalcographus are two sympatric Palearctic bark beetlespecies with wide distribution ranges. As both species are comparable in biology, lifehistory, and habitat, including sharing the same host, Picea abies, they provideexcellent models for applying a comparative approach in which to identify commonhistorical patterns of population differentiation and the influence of species-specificecological characteristics. We analysed patterns of genetic diversity, genetic structureand demographic history of ten I. typographus and P. chalcographus populationsco-distributed across Europe using both COI and ITS2 markers. Rather than similari-ties, our results revealed striking differences. Ips typographus was characterised bylow genetic diversity, shallow population structure and strong evidence that all extanthaplogroups arose via a single Holocene population expansion event. In contrast,genetic variation and structuring were high in P. chalcographus indicating a longerand more complex evolutionary history. This was estimated to be five times older thanI. typographus, beginning during the last Pleistocene glacial maximum over100 000 years ago. Although the expansions of P. chalcographus haplogroups also dateto the Holocene or just prior to its onset, we show that these occurred from at leastthree geographically separated glacial refugia. Overall, these results suggest that themuch longer evolutionary history of P. chalcographus greatly influenced the levels ofphylogeographic subdivision among lineages and may have led to the evolution of dif-ferent life-history traits which in turn have affected genetic structure and resulted inan advantage over the more aggressive I. typographus.

Keywords: comparative phylogeography, COI, Ips typographus, ITS2, Pityogenes chalcographus,species-specific characters

Received 25 June 2012; revision received 7 February 2013; accepted 13 February 2013

Introduction

An important issue in evolutionary biology is whethergeneral and predictable relationships exist between thephylogeographic structure of species, their environmentalrequirements and species-specific ecology (Avise 2000).

Comparative phylogeography is a powerful approachto match historical patterns of gene flow, divergenceand speciation mechanisms among co-distributed taxathat overlap in space and time, but which are indepen-dently confronted with the same historical events,submitted to the same or different ecological processesand presenting similar or distinct intrinsic life-historytraits (Taberlet et al. 1998; Avise 2000; Hickerson et al.2010). This multispecies approach offers a deeperunderstanding of the evolutionary processes affecting

Correspondence: Coralie Bertheau, Fax: +43 1 3686352/97;E-mail: [email protected] contributing senior authors.

© 2013 John Wiley & Sons Ltd

Molecular Ecology (2013) 22, 3318–3332 doi: 10.1111/mec.12296

phylogeographic patterns among sympatric species(Bermingham & Moritz 1998; Zink 2002). Concordantgeographic distributions among species lineages haveenabled the detection of climatic refugia, postglacialre-colonization routes or zones of contact (Taberlet et al.1998; Hewitt 2000), while unrelated phylogeographicpatterns among species highlighted the influence ofenvironmental factors, life-history and/or ecologicaltraits which affect dispersal capacities, host specializa-tion or adaptability to new environments (Avise et al.1987; Bowen & Avise 1990; Peterson & Dennot 1998).Although the number of comparative genetic studies oninsects has increased in recent years, most deal withhost-parasite interactions (Whiteman et al. 2007; Renet al. 2008; Borer et al. 2012; among others) or closelyrelated species (Solomon et al. 2008; Papadopoulou et al.2009; Morgan et al. 2011; among others), and few havebeen carried out in Europe (Brouat et al. 2004; Hayward& Stone 2006; Kerdelhu"e et al. 2006; Esp"ındola &Alvarez 2011).Here, we present a comparative study of two sympatric

insect species with similar life histories, biology andhabitats to determine how these have been influencedby their evolutionary histories. Ips typographus (L.) andPityogenes chalcographus (L.) are two Palearctic scolytidbeetles belonging to the tribe Ipini. They have a widedistribution range concordant with the distribution oftheir main host, the Norway spruce, Picea abies (L. H.Karst.) (Pfeffer 1995). The two species specialize inexploiting weakened spruce trees especially afterextreme climatic events, such as storms, snow breakageand droughts. These events favour population growththat can lead to extensive ecological and economicaldamage, making the beetles the most serious pests tospruce forests in Europe (Gr"egoire & Evans 2004).Among the reasons for their breeding success are theirefficient pheromone-mediated infestation and aggrega-tion behaviour (Byers 2004), their strong associationwith blue stain fungus (Kirisits 2004; Six & Wingfield2011) and their flexible reproductive cycles which allowfor up to three generations in particularly warm years(Jurc et al. 2006; J€onsson et al. 2011). Both species arepolygamous with an endophytic life cycle as they boregalleries into the bark of their host where larval devel-opment and most often adult maturation take place.They are mostly found together on the same trees(Bertheau et al. 2009a). Ips typographus is confined to themid- and lower parts of the trunk (Hedgren 2004;Wermelinger 2004), whereas P. chalcographus segregatespreferentially into the thinner bark of the tree’s upperreaches and branches but it is not rare to find it in thethicker bark (Gr€unwald 1986). While both speciespreferentially infest P. abies, only P. chalcographus isconsidered oligophagous, with the ability to colonize

and develop in other Pinaceae species (F€uhrer &Muehlenbrock 1983; Bertheau et al. 2009a,b). Conse-quently, after strong storms across Europe in 1990,P. chalcographus was responsible for the destruction ofeight million m3 timber of spruce and other conifers,whereas the monophagous I. typographus destroyedfour times that amount exclusively in spruce (Gr"egoire& Evans 2004).The phylogeography of both species has been rela-

tively well investigated in Europe (see Avtzis et al. 2012for review). Ips typographus has been the subject of sev-eral studies using a variety of molecular markers. Anoriginal analysis of a fragment of the mitochondrialgene cytochrome c oxidase subunit I (COI) from 18spruce populations revealed only eight haplotypes butidentified two potential glacial refugia—one in thesouth Alps and the other in the Moscow region (Staufferet al. 1999). Contrary to knowledge that spruce recolon-ized Scandinavia from a refuge on the Russian plain(Tollefsrud et al. 2008), Stauffer et al. (1999) concludedthat I. typographus re-colonized Scandinavia only fromsouthern Europe because Russian and Lithuanian popu-lations did not share haplotypes with Scandinavia.Nuclear allozyme and microsatellite studies highlightedlow diversity and lack of structure in I. typographuspopulations due to high gene flow (Stauffer et al. 1999;Sall"e et al. 2007; Gugerli et al. 2008). However, therecent discovery of cryptic nuclear copies (numts) thatappear very similar to authentic mitochondrial DNA(mtDNA) in I. typographus (Bertheau et al. 2011) createddoubts in the interpretation of Stauffer et al. (1999). Avt-zis et al. (2008) studied P. chalcographus in 39 Europeanspruce populations, sequencing almost the completeCOI gene, and discovered 58 haplotypes that were par-titioned into three major clades. Two of these clades arethought to have diverged 70 000–100 000 years ago(kya) from central to northeastern European refugia andare now found in northern and central Europe withunidirectional reproductive incompatibility (F€uhrer1976; Avtzis et al. 2008). Southern Europe comprisedthe remaining genetic variation with glacial refugia sug-gested in the Apennines and the Dinaric Alps.While much work has been carried out, the numt

problem, the lack of a fully comparative assessmentand of an informative nuclear DNA (nuDNA) markercurrently preclude a common synthesis of bark beetlephylogeography in Europe.Ips typographus and P. chalcographus share many eco-

logical and life characteristics, but they show variablelevels of specialization to P. abies. As the geographicdistribution of phytophagous insects is necessarilyembedded within the range of their host plants(Simonato et al. 2007), one might expect the geneticstructure of the monophagous I. typographus to more


COMPARATIVE GENETIC STUDY OF BARK BEETLES 3319

closely resemble that of P. abies than the oligophagousP. chalcographus. Dr#es & Mallet (2002) showed that hostspecificity and host availability play a major role in thepartitioning of genetic variation and structure in phy-tophagous insects. Given a fully comparative approachand considering previous findings, one would expectthat populations of a monophagous species would bemore differentiated than an oligophagous speciesbecause the scarce and patchy distribution of its specifichost may lead to population isolation and a reductionof gene flow (Peterson & Dennot 1998; Kelley et al.2000). Alternatively, the rarity of appropriate hostplants may force specialized I. typographus individualsto disperse in search of new suitable hosts, leading tohigher gene flow with a reduction of species-widegenetic structure (Lieutier 2002). To test these hypothe-ses, we employed a multispecies-multi-marker strategyfor the combined analysis of phylogeographic patternsin I. typographus and P. chalcographus. We sampled bothspecies extensively at ten key locations and quantifiedgenetic diversity, structure and demographic historyusing the mitochondrial COI and nuclear internaltranscribed spacer two region (ITS2) fragments. Thisapproach provided the unique opportunity of unravel-ling two distinctive evolutionary trajectories. We usethis information in combination with that of host spe-cies, P. abies, to discuss the relative benefits conferredthrough differences in life-history strategies over evolu-tionary time and how these may have affected geneticstructure.

Materials and methods

Sampling

Adults of I. typographus and P. chalcographus were col-lected at ten different locations from ten countries in con-tinental Europe covering the natural distribution area oftheir main host P. abies (Table 1, Fig. 1). Between 1995and 2010, 394 I. typographus and 476 P. chalcographusspecimens were sampled from P. abies trees. Only oneindividual per mother gallery-the gallery system con-structed by a single female after mating-was taken toprevent the sampling of siblings. All beetles were storedin absolute ethanol at !20 °C.

DNA protocols

DNA was extracted from whole beetles for a subset of15-48 individuals from each population using the GenE-lute Mammalian Genomic DNA miniprep kit (Sigma)according to the manufacturer’s protocol. MitochondrialCOI gene was amplified via PCR using the sense primerdescribed by Juan et al. (1995) and the anti-sense primerUEA10 (Lunt et al. 1996) for I. typographus (629 bp) andthe sense primer PcCOIF 5′- ATTATTAACAGACCGAAACG-3′ and the anti-sense primer UEA10 (Lunt et al.1996) for P. chalcographus (950 bp). The full ITS2, includ-ing the end of the 5.8S and the beginning of the 28Sribosomal gene, was amplified using standard oligonu-cleotide primers ITS2F/ITS2R (Campbell et al. 1993) for

Table 1 Sampling sites, abbreviations of Ips typographus and Pityogenes chalcographus populations, year of capture and geographicalcoordinates

Species Country Location Code Date Latitude Longitude

Ips typographus Austria Rothwald It-AtRo 1996 47° 45′N 15° 04′ECroatia Vrhovine It-CrVr 2009 44° 51′N 15° 25′EFinland Joensuu It-FiJo 2009 62° 35′N 29° 45′EFrance Dole It-FrDo 2007 47° 05′N 05° 29′EGreece Drama It-GrDa 2010 41° 08′N 24° 09′EItaly Abetone It-ItAb 2009 44° 08′N 10° 39′EPoland Hajnowka It-PlHa 2010 52° 44′N 23° 34′ERomania Belis! It-RoBe 1996 46° 04′N 23° 01′ERussia Moscow It-RuMo 1995 55° 45′N 37° 36′ESweden H€ogberget It-SwHo 1996 60° 27′N 15° 05′E

Pityogenes chalcographus Austria Rothwald Pc-AtRo 2004 47° 45′N 15° 04′ECroatia Saborsko Pc-CrSa 2009 44° 59′N 15° 28′EFinland Jarvenp€a€a Pc-FiJa 2004 60° 28′N 25° 06′EFrance Dole Pc-FrDo 2007 47° 05′N 05° 29′EGreece Drama Pc-GrDa 2004 41° 08′N 24° 09′EItaly Abetone Pc-ItAb 2009 44° 08′N 10° 39′ELithuania Vilnius Pc-LiVi 2004 54° 04′N 25° 20′ERomania Bistra Pc-RoBi 2004 46° 22′N 23° 06′ERussia Sverdlovsk Pc-RuSv 2009 58° 53′N 61° 51′ESweden Overkalix Pc-SwOv 2004 66° 19′N 22° 50′E


3320 C. BERTHEAU ET AL.

both species. Reactions were carried out in 25 lL totalvolumes, containing 19 the reaction buffer providedwith the polymerase, 2 mM MgCl2, 100 lM dNTPs, 0.4

and 0.2 lM of the COI and ITS2 primers, respectively, 1unit of Taq polymerase (Fermantas) and ~50 ng templateDNA. PCR was performed with an initial denaturation

(A)

(C)

(B)

(D)

Fig. 1 Geographical distribution of mitochondrial haplogroups (COI) and nuclear alleles (ITS2) of two European spruce bark beetles.(A) Geographical mapping of the three COI haplogroups and the four ITS2 alleles detected in Ips typographus populations and (B) ofthe six haplogroups and ten ITS2 alleles detected in Pityogenes chalcographus populations. Population frequencies are approximatedby the area of the circle. Haplotype and allele distributions are accompanied by Bayesian skyline plots showing changes in effectivepopulation size over evolutionary time. MtDNA COI sequences of the I. typographus (C) and P. chalcographus (D) were subjected toBayesian skyline analysis using the software BEAST v. 1.6.1. The dashed lines correspond to the 95% confidence limits while the fulllines correspond to the median value. Populations are abbreviated according to Table 1.



step at 94 °C for 3 min followed by 30 cycles of94 °C-60 s, 48 °C (COI) or 51 °C (ITS2)-60 s, 72 °C-90 s,followed by a final extension at 72 °C for 10 min. PCRproducts were purified using the GenElute PCR Clean-Up kit (Sigma-Aldrich) and sequencing was performedat the Cancer Research Center DNA Sequencing Facility(University of Chicago, Chicago, IL, USA). COI and ITS2sequences were visualized using Bioedit v7.0.5. (Hall1999) and aligned using Clustal W (Thompson et al.1994) as implemented in Bioedit.Due to the recent detection of cryptic numts in

I. typographus, only haplotypes with clear, unambiguoussequence chromatograms and confirmed by sequencing-independent PCR amplicons were used for phylogeneticanalysis (see Bertheau et al. 2011). For P. chalcographus,all sequences were free from ambiguous sites and pre-mature stop codons, consistent with true mitochondrialorigin (see Zhang & Hewitt 1996 for review).The ITS2 chromatograms of most individuals of

I. typographus (89.6%) displayed numerous double peaksat four ambiguous positions and continuously from the417th nucleotide until the end of the fragment due toinsertion/deletion polymorphisms; 58% of the individu-als of P. chalcographus showed double peaks at tenambiguous positions. These double peaks suggested thesuperposition of two sequences reflecting heterozygos-ity. The different allele sequences were reconstructed bycomparing chromatograms for the forward and reverseprimers following Flot et al. (2006). A confirmation ofthe alleles was obtained by cloning PCR products offour heterozygote I. typographus individuals from threepopulations (Austria, France and Italy) and ten hetero-zygote P. chalcographus individuals from six populations(Croatia, Finland, Greece, Italy, Lithuania and Roma-nia). PCR products were ligated into the pTZ57R/T vec-tor (Fermentas) and transformed into competent JM109Escherichia coli cells according to the instructions of themanufacturer. Plasmid DNA of two to eight clones perindividual was sequenced using the universal M13 for-ward primer.

Data analyses

Gene diversity Hd, nucleotide diversity p and meannumber of pairwise differences were calculated usingArlequin 3.5 (Excoffier & Lischer 2010) for both COIand ITS2 sequences. Allelic richness r was computed forboth species populations and for both COI and ITS2markers using the rarefaction method proposed by Petitet al. (1998) using Contrib (http://www.pierroton.inra.fr/genetics/labo/Software/Contrib/).Bayesian phylogenies were reconstructed using the

software BEAST 1.6.1 (Drummond & Rambaut 2007) forboth mtDNA and nuDNA data. Trees were generated

using the model of nucleotide substitution that best fitsthe data under the hierarchical likelihood-ratio test(hLRT) criterion, determined with Modeltest v3.7(Posada & Crandall 1998). The most appropriate modelsof COI sequence evolution were HKY+G (Hasegawa et al.1985) for I. typographus and K81+G+I (Kimura 1981) forP. chalcographus. For the ITS2 locus, we used the JC69model (Jukes & Cantor 1969) for both species. We used aYule process tree prior, with base frequencies estimatedand the gamma distribution described by ten categories.We performed 350 million MCMC simulations, loggingto file every 350 000 iterations and discarded 10% asburn-in. A likelihood-ratio test (LRT, Felsenstein 1988)supported a molecular clock model for both I. typogra-phus (v2 = 8.77, d.f. = 28, P < 0.05) and P. chalcographus(v2 = 71.15, d.f. = 119, P < 0.05). COI and ITS2 sequencesof Ips cembrae (GenBank KC514452, KC514464), Ipsamitinus (KC514451, KC514463), Pityogenes bidentatus(KC514453, KC514465) and Pityogenes quadridens(KC514454, KC514466) were used as outgroups. We datedthe clock-like COI phylogenies by applying a globalmutation rate of 1.87%/Myr, which is an average of threerecently calibrated Coleopteran COI mutation rates rang-ing from 2.5 to 1.5%/Myr (Borer et al. 2010). Mutationrates for the ITS2 locus do not currently exist for the Cole-optera, precluding the dating of nuclear phylogenies.Statistical parsimony networks were computed, for

both mitochondrial and nuclear markers, using TCS

version 1.21 (Clement et al. 2000). To solve the fewcladogram ambiguities that occurred, we used topologi-cal, geographical and frequency criteria (Crandall &Templeton 1993).We investigated geographical structure in our data

sets by conducting spatial analyses of molecular vari-ance (SAMOVA 1.0, Dupanloup et al. 2002), which identifygroups of populations that are geographically homoge-neous and maximally differentiated. The methodrequires the a priori definition of the number of groups(K) of populations that exist and generates F-statistics(FSC, FST and FCT) using an AMOVA approach. Thegenetic differentiation among groups, expressed by theFCT value associated with the K groups, was computedusing pairwise differences between DNA sequences asmolecular distance (Dupanloup et al. 2002). Theprogram was run for 10 000 permutations from 100random initial conditions for two to nine differentiatedgroups (K = 2 to K = 9). Occurrence of significant phy-logeographic structure was also assessed by testingwhether Gst (coefficient of genetic variation over allpopulations) was significantly smaller than Nst (equiva-lent coefficient taking into account the similaritiesbetween haplotypes) fusing 1000 permutations (see Pons& Petit 1996) in the program Permut (http://www.pierroton.inra.fr/genetics/labo/Software/Permut/).



Population historical events were inferred using bothmitochondrial and nuclear data sets. The frequency-based indicators of a population expansion (or selectionin non-neutral markers) Tajima’s D (Tajima 1989) andFu’s Fs (Fu 1997) were calculated with Arlequin 3.5(Excoffier & Lischer 2010). Finally, we used coalescentsimulations in BEAST 1.6.1 to reconstruct extended Bayes-ian skyline plots, which uses the coalescent to modelchanges in the effective population size. As with thedating of phylogenetic lineage splits, we were only ableto perform these analyses for the COI locus, due a gen-eral lack of ITS mutation rates. We performed theseanalyses for each species using 350 million MCMCiterations, logging to file every 350 000 iterations anddiscarding 10% as burn-in.

Results

Genetic diversity

A homologous 564 bp fragment of the COI mtDNAgene was analysed in both species. Among the 394I. typographus individuals sequenced, 22 transitions andthree transversions resulted in 30 haplotypes (GenBankITU82589, AF036150, AF036156, JN133853-JN133879;Table S1, Supporting information). The two most com-mon were shared by 222 and 110 individuals, respec-tively (Table S1, Supporting information). Total genediversity was 0.60 " 0.02, while nucleotide diversity was0.0013 " 0.0010. Contrastingly, mitochondrial geneticdiversity among the 476 P. chalcographus individuals washigher revealing 61 transitions, 18 transversions and 94haplotypes (KC514357-KC514450, Table S2, Supportinginformation). While haplotype sharing was also high inthis species (two haplotypes were shared by 119 and132 individuals, Table S2, Supporting information),gene diversity (0.87 " 0.05) and nucleotide diversity(0.012 " 0.006) were significantly higher in P. chalcogra-phus than in I. typographus. Nuclear variation within theITS2 region showed similar intraspecific trends, but withdecreased diversity. Four alleles were identified among 96I. typographus individuals (KC514467-KC514470, Table S1,Supporting information) with a gene diversity of0.51 " 0.01 and a nucleotide diversity of 0.003 " 0.002,whereas 95 P. chalcographus individuals returned tenalleles (JQ066307, JQ066310, KC514455–KC514462; TableS2, Supporting information) with a gene diversity of0.57 " 0.02 and a nucleotide diversity of 0.001 " 0.001.

Relatedness and divergence

Bayesian phylogenetic trees (Fig. 2) and haplotype/allele networks (Fig. 3) were reconstructed for mito-chondrial and nuclear markers. Both species showed

very similar patterns of relatedness. MtDNA trees com-prised two major lineage splits at the point of speciescoalescence, yet slower evolving nuDNA was able todistinguish ancestral alleles B and I in I. typographusand P. chalcographus respectively, and a more derivedclade (Fig. 2). Haplotype networks resolved three majormtDNA haplogroups in both species, each of whichconsisted of one to three haplotypes at high frequency,with all other haplotypes radiating mainly from theseby one or two base pairs (Fig. 3). In P. chalcographusone of the three mtDNA haplogroups (Pc-III) was struc-tured into four subgroups, two of which occur at lowfrequency (Fig. 2B, 3B). Nuclear allele networks showedthat both species consisted of ancestral and derivedclades (Fig. 3C,D).We dated the time to the most recent common ances-

tor (tMRCA) of all mitochondrial haplogroups to~19 kyr (CI95%, 11-28 kyr) in I. typographus and to101 kyr (CI95%, 68-132 kyr) in P. chalcographus (Table 3).Divergence times were consistent whether both or onlya single outgroup sequence was used. The tMRCAs foreach haplogroup varied considerably within each spe-cies, however, only those among I. typographus couldconceivably (with 95% confidence) have evolved duringthe Holocene (in the last 12 kyr).

Species geographic structure

The distribution of genetic diversity was also strikinglysimilar in both species. Generally, western Europetended to be more diverse than either northern or south-eastern Europe in both species (Table 2). Northern andsoutheastern European populations consisted of a single,but different mtDNA haplotype for I. typographus orhaplogroup for P. chalcographus at high frequency.In I. typographus, mtDNA haplogroup It-A was more

widely represented in north and central Europe, It-Bwas distributed at highest frequency in Croatia andGreece, whereas It-C appeared at lower frequenciesacross Europe (Fig. 1A, Table S1, Supporting informa-tion). Accounting for geography, a SAMOVA returned amaximally significant FCT statistic (0.497, P < 0.05) forthe two-lineage phylogenetic hypothesis whichdecreased consistently for all K > 2 (Fig. S1A, Support-ing information). One of the two groups was made upof Croatian and Greek populations, and the secondcomprising all other populations. However, Gst (0.39)and Nst (0.35) did not differ significantly, possiblybecause of the close relationships among the threemajor haplogroups. Even lower variation at the ITS2was unable to resolve geographic structure, with theGst (!0.050) not differing significantly from Nst(!0.052). This is likely because alleles A and B wereboth widely distributed at high frequency across most



of Europe, and alleles C and D occurred at low fre-quency only in France and Sweden, respectively.The higher genetic diversity of P. chalcographus gave

rise to a more complex phylogeographic structure,although still very similar to that of I. typographus. Of

the three most common and star-shaped haplogroups,Pc-I and Pc-IIIa were distributed in opposing north-southgradients, being most frequent in northern/easternEurope, and in Greece/western Europe respectively.The third major haplogroup Pc-IIId was at highest

(A) (B)

(C)

(D)

Fig. 2 Mitochondrial and nuclear Bayesian phylogenetic trees of the two European spruce bark beetles. (A) Ips typographus COI, (B)Pityogenes chalcographus COI, (C) I. typographus ITS2, (D) P. chalcographus ITS2. All branches shown on the trees are present in 100%of the posterior sample. Clock-like mtDNA phylogenies (A and B) were linearized and dated by applying an averaged coleopteranCOI mutation rate of 1.87%/Myr (calculated from Borer et al. 2010).



frequency in the Dinaric Alps of Croatia, but alsooccurred at low frequency in western Europe (Fig. 1B).The remaining less frequent haplogroups (II, IIIb andIIIc) were all endemic to the Apennine population ofItaly (Figs 1B and 3B), despite each being of indepen-dent phylogenetic origin (Fig. 2B). The P. chalcographus

mitochondrial data set was therefore significantly geo-graphically structured [Gst (0.12) < Nst (0.17), P < 0.01]and a SAMOVA returned a maximum FCT value (0.339,P < 0.05) for K = 3 (Fig. S1A, Supporting information).This equated to one group composed of the Croatianpopulation, the second group with the diverse Italian

(A) (B)

(C)

(D)

Fig. 3 Mitochondrial and nuclear networks of the two European spruce bark beetles. (A) Ips typographus COI, (B) Pityogenes chalcogra-phus COI, (C) I. typographus ITS2, (D) P. chalcographus ITS2. Each circle corresponds to one haplotype; circle size gives the proportionof individuals belonging to the haplotype. Each link between circles indicates one mutational event. Populations are abbreviatedaccording to Table 1.



population and the third group comprising all otherpopulations. Similar to I. typographus, the less diversenuDNA sequences were more homogeneously distrib-uted across Europe with the Gst (0.020) and Nst(!0.001) not significantly different. A SAMOVA, however,returned a maximum and significant FCT value (0.089,P < 0.05) for K = 3 (Fig. S1B, Supporting information),partitioned similarly to the mitochondrial groups above,the exception being that one group consisted of Greecerather than Croatia. This result supports the observationthat the two most common ITS2 alleles occur at highestfrequency in Italy (allele I) and Romania and Greece(allele II).

Population history

Overall the significant values for both Tajima’s D andFu’s Fs statistics (Table 2) point to a hypothesis of his-torical population expansions in both species. However,while overall Tajima’s D was significant (P < 0.05) forP. chalcographus, none of the sampled populationsshowed highly negative or highly significant values(Table 2). This, in contrast to several I. typographus pop-ulations with highly significant population expansionsignatures (Table 2), suggests that demographic eventsshaping population structure in P. chalcographus haveoccurred earlier than in I. typographus. Negative valuesfor both D and Fs for the less diverse nuclear ITS2sequences also lend support to a population expansionhypothesis in both species, although only Fs was signifi-cant in I. typographus. We used extended Bayesian sky-line simulations to reconstruct the changes in effectivepopulation size occurring through evolutionary timeand found that while both species were characterised bya rapid increase in effective population size, the I. typog-raphus expansion occurred more recently, dating back tothe Holocene (~7-15 kya, Fig. 1C). In contrast, popula-tion expansions among P. chalcographus haplogroupsoccurred prior to the onset of the Holocene, between 18and 26 kya (Fig. 1D). Both population expansion eventspostdate haplogroup divergence times. While Bayesianskyline plots may be sensitive to heavily structured pop-ulations (Ho & Shapiro 2011), employment of thismethod is reasonable in the case presented here, giventhe relatively low level of structure observed in bothstudy species and recent species expansion events.

Discussion

Our comparative study of two sympatric bark beetle spe-cies across European forests revealed striking differencesin genetic variation, population structure, evolutionaryand demographic history. In general, I. typographusexhibited low genetic diversity, shallow structure, which

resulted from a recent divergence, and an early- to mid-Holocene population expansion, whereas P. chalcogra-phus was structured into six haplogroups, was highlydiverse and underwent a longer, more complex evolu-tionary history.

Diversity

Lower genetic diversity in I. typographus supports previ-ous findings (Stauffer et al. 1999; Avtzis et al. 2008),although the much larger sample sizes used in the pres-ent study considerably increase the number of detectedhaplotypes-I. typographus 30 vs. 8; P. chalcographus 94 vs.56-allowing for higher resolution evolutionary inference.Conversely, the greater diversity observed in the pres-ent study may be attributable to the presence of numts(Bensasson et al. 2001; Song et al. 2008). We argueagainst this possibility because unlike in I. typographuswhere numts could potentially be widespread,nuclear copies of mtDNA have never been identified inP. chalcographus (Arthofer et al. 2010), yet mtDNA ofthis species is about ten times more diverse than inI. typographus (Table 2). Furthermore, even if numtshave contaminated the present study, the three mainhaplotypes observed here (HTI, HTII and It1) were pre-viously validated as originating strictly from mitochon-dria (Bertheau et al. 2011).

Evolutionary history

The genetic structure of these bark beetle speciesreflects both their origins and their demographic histo-ries. Low variation at mtDNA and nuDNA in I. typogra-phus suggests high gene flow in this species and isconsistent with previous allozyme and microsatelliteresults (Stauffer et al. 1999; Sall"e et al. 2007). Despite thislow diversity, we found that I. typographus populationsare indeed structured across Europe. However, thethree star-shaped mtDNA haplogroups were linked toeach other by single mutations and one to four stepmutations were found to link all 30 I. typographus haplo-types. The two most common nuclear ITS2 alleles(A and B) were both distributed at high frequency inalmost all populations. The two main mtDNA haplo-groups (It-A and It-B) both evolved at or just before theend of the last glacial maximum, and expanded in theHolocene (Fig 1C). We suggest the most parsimoniousexplanation for these results is a very recent species ori-gin and expansion from a single late-Pleistocene glacialrefuge as it is unlikely that haplogroups would remainso closely related if they have been isolated in two ormore refugia. The three closely related haplogroupsmust therefore have evolved due to postexpansiondemographic processes. The differing frequencies of



Tab

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Indices

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speciesan

dmolecu

larmarker,Tajim

a’sD

andFu’s

Fsstatistics.Pop

ulationsareab

breviatedaccord

ingto

Tab

le1

Sp.

Pop.

COI

ITS2

N#H

TH

d"

SDr

MNPD

"SD

p"

SDTajim

a’D

Fu’s

Fs

N#A

lH

d"

SDr

MNPD

"SD

p"

SDTajim

a’D

Fu’s

Fs

Ips typographu

s

It-A

tRo

4211

0.57

"0.09

4.158[15]

0.84

"0.61

0.0015

"0.0012

!1.73*

!8.59***

102

0.52

"0.05

0.99

[6]

1.57

"0.98

0.003"

0.002

1.51

NS

4.21

NS

It-C

rVr

473

0.20

"0.07

1.118[15]

0.36

"0.36

0.0006

"0.0007

!0.98

NS

!0.31

NS

102

0.53

"0.04

0.99

[6]

1.58

"0.98

0.003"

0.002

1.57

NS

4.40

NS

It-FiJo

456

0.29

"0.09

2.047[15]

0.31

"0.32

0.0005

"0.0006

!1.82**

!5.25***

102

0.53

"0.04

0.99

[6]

1.58

"0.98

0.003"

0.002

1.56

NS

4.40

NS

It-FrD

o34

60.70

"0.05

3.302[15]

1.18

"0.78

0.0021

"0.0015

!0.55

NS

!1.00

NS

103

0.56

"0.08

1.33

[6]

1.57

"0.98

0.003"

0.002

0.17

NS

2.35

NS

It-G

rDa

365

0.30

"0.10

2.064[15]

0.37

"0.36

0.0007

"0.0007

!1.51*

!3.19**

102

0.53

"0.04

0.99

[6]

1.58

"0.98

0.003"

0.002

1.55

NS

4.32

NS

It-ItA

b35

30.48

"0.06

1.428[15]

0.50

"0.44

0.0009

"0.0009

0.06

NS

0.16

NS

102

0.51

"0.06

0.99

[6]

1.54

"0.97

0.003"

0.002

1.43

NS

4.07

NS

It-PlH

a44

90.53

"0.09

3.901[15]

0.73

"0.55

0.0013

"0.0011

!1.69*

!6.09***

102

0.53

"0.04

0.99

[6]

1.58

"0.98

0.003"

0.002

1.57

NS

4.40

NS

It-RoBe

485

0.40

"0.08

2.276[15]

0.43

"0.40

0.0008

"0.0008

!1.20

NS

!2.47*

102

0.53

"0.04

0.99

[6]

1.58

"0.98

0.003"

0.002

1.57

NS

4.40

NS

It-RuMo

153

0.26

"0.14

2.000[15]

0.27

"0.31

0.0005

"0.0006

!1.49

NS

!1.55*

62

0.55

"0.07

0.99

[6]

1.64

"1.04

0.003"

0.002

1.44

NS

3.56

NS

It-SwHo

484

0.12

"0.06

0.938[15]

0.13

"0.20

0.0002

"0.0004

!1.70*

!4.22***

103

0.58

"0.06

1.28

[6]

1.81

"1.09

0.003"

0.002

!0.38

NS

2.82

NS

Total

394

300.60

"0.02

0.76

"0.56

0.0013

"0.0010

!2.09***

!3.4E

+38***

964

0.51

"0.01

1.53

"0.92

0.003"

0.002

!0.07

NS

!1.01

NS

Pityogenes

chalcographu

s

Pc-AtRo

4720

0.90

"0.03

19.00[47]

5.08

"2.50

0.009"

0.005

!0.56

NS

!5.26*

102

0.53

"0.05

1.00

[9]

0.53

"0.46

0.001"

0.001

1.53

NS

1.39

NS

Pc-CrSa

4821

0.92

"0.02

19.71[47]

6.32

"3.05

0.011"

0.006

!0.32

NS

!4.30

NS

104

0.66

"0.08

2.20

[9]

0.80

"0.61

0.002"

0.001

!0.40

NS

!0.82

NS

Pc-FiJa

4710

0.54

"0.08

09.00[47]

2.36

"1.31

0.004"

0.003

!1.33

NS

!1.38

NS

94

0.64

"0.07

2.06

[9]

0.75

"0.58

0.001"

0.001

!0.44

NS

!0.83

NS

Pc-FrD

o48

170.84

"0.05

15.81[47]

5.29

"2.60

0.009"

0.005

!0.54

NS

!2.40

NS

93

0.59

"0.07

1.56

[9]

0.65

"0.53

0.001"

0.001

0.20

NS

0.11

NS

Pc-GrD

a48

90.55

"0.08

07.92[47]

3.50

"1.81

0.006"

0.004

!1.06

NS

0.88

NS

102

0.46

"0.11

1.00

[9]

0.46

"0.43

0.001"

0.001

0.95

NS

0.98

NS

Pc-ItAb

4817

0.92

"0.02

15.86[47]

6.93

"3.31

0.012"

0.007

!0.23

NS

!0.99

NS

103

0.41

"0.15

1.62

[9]

0.54

"0.48

0.001"

0.001

!0.46

NS

!0.41

NS

Pc-LiV

i47

70.64

"0.05

06.00[47]

2.23

"1.25

0.004"

0.002

!1.44

NS

0.61

NS

93

0.60

"0.09

1.69

[9]

0.69

"0.56

0.001"

0.001

0.21

NS

0.05

NS

Pc-RoBi

488

0.74

"0.03

06.90[47]

2.71

"1.46

0.005"

0.003

!0.77

NS

0.60

NS

104

0.57

"0.13

2.26

[9]

0.78

"0.60

0.001"

0.001

!0.53

NS

!0.96

NS

Pc-RuSv

4811

0.75

"0.04

09.90[47]

3.56

"1.83

0.006"

0.004

!1.02

NS

!0.37

NS

94

0.63

"0.08

2.06

[9]

0.72

"0.57

0.001"

0.001

!0.53

NS

!0.92

NS

Pc-Sw

Ov

4713

0.70

"0.07

12.00[47]

2.95

"1.57

0.005"

0.003

!1.12

NS

!2.67

NS

92

0.53

"0.05

1.00

[9]

0.53

"0.47

0.001"

0.001

1.50

NS

1.32

NS

Total

476

940.87

"0.05

6.93

"3.23

0.012"

0.006

!1.73*

!24.97***

9510

0.57

"0.02

0.65

"0.50

0.001"

0.001

!1.53

NS

!10.38***

N:Number

ofindividualsan

alysed;#H

T:Number

ofhap

lotypes

per

pop

ulation;#A

l:Number

ofallelesper

population;H

d:Gen

ediversity

anditsstan

darddev

iation;r:Allelic

rich

nessafterrarefaction;MNPD:Meannumber

pairw

isedifferencesan

ditsstan

dard;p:

Nucleo

tidediversity

anditsstan

darddev

iation;NS:

Nonsignificant.

*P<0.05,**

P<0.01,***P

<0.001.



each haplogroup among locations probably reflect vary-ing levels of gene flow, and/or genetic drift. However,the fact that several populations contain at least two ofthe three haplogroups at high frequency makes a deter-mination of its origin difficult. Furthermore, shallowgenetic structure of I. typographus is incongruent withthat of its host P. abies, which is highly structuredacross Europe (Tollefsrud et al. 2008). These findingscorroborate the view of Sall"e et al. (2007) who usedmicrosatellite markers to show that local insect–tree relationships and co-adaptations are unlikely to bethe main forces shaping the evolutionary history ofI. typographus.Conversely, the high mtDNA genetic variation found

within P. chalcographus is structured into six haplo-groups, consistent with Avtzis et al. (2008), but our datashow that the two most frequent haplogroups (Pc-I andPc-IIIa) are also distributed as far east as European Rus-sia. We also find a reduction in mtDNA diversity ofeastern relative to western European populations(Fig. 1, Table 2) which suggests several different poten-tial glacial refugia for this species. Comparatively, thecoalescence of P. chalcographus mitochondrial lineages isfive times older than I. typographus, dating back to~101 kya, which is just after the peak of the last Pleisto-cene inter-glacial maximum, where temperatures wereeven warmer than in the Holocene (Jouzel et al. 2007).The extant phylogeographic structure of P. chalcographusmust therefore be the result of an initial expansion fromthe species origin during this last Pleistocene intergla-cial period, followed by glacial contraction and, finally,recent expansion just prior to the Holocene (Fig. 1D).The most probable origin of the species is theApennines as this is the only population harbouringendemic mtDNA haplotypes and it contains the ances-tral ITS2 allele I at highest frequency. This originalpopulation, consisting of the precursors of mtDNAhaplogroups Pc-I/Pc-II and Pc-III as well as the ances-tral ITS2 allele I, then expanded at least as far east asEuropean Russia, diversifying into the mitochondriallineages leading to haplogroups Pc-I/Pc-II, Pc-IIIa, Pc-IIIb and Pc-IIIc/Pc-IIId and simultaneously into nuclearalleles II, IV, VIII. Of these one-step mutational variants,only allele II is not endemic to western Europe, and itshigh frequency across the entire species suggests that itmust have evolved very soon after the initial Pleisto-cene expansion. At ~80 kya (Table 3), a strengtheningglacial period would have contracted populations toglacial refugia. Considering haplogroup/allelic distribu-tions, frequencies and the glacial distribution of P. abies(Tollefsrud et al. 2008), upon which P. chalcographus isdependent, we propose the following glacial refugia:the Apennines for the precursors of mtDNA haplo-groups Pc-II, Pc-IIIb and PcIIIc/PcIIId, central Europe

(either the Carpathians or Bulgaria) for haplogroupPc-IIIa and the Russian plain for haplogroup Pc-I. Theisolation of P. chalcographus in different refugia is alsoreflected by nuclear alleles. All refugia are presumed tohave contained nuclear alleles I and II, the latter ofwhich mutated independently in different refugia togive rise to the derived nuclear clade. Thus, the geneticstructure of the oligophagous P. chalcographus appearsmore congruent with its host species than does that ofthe monophagous I. typographus, probably due to theformer species’ longer evolutionary history. Accordingto Tollefsrud et al. (2008), P. abies in southern refugiaexpanded rapidly at the advent of the Holocene, recon-necting western European refugia by 9 kya, and expan-sion over the entire northern range from a single refugeon the Russian plain was complete by 6 kya, with bothpopulations coming into present day secondary contactin Poland. This is compatible with the expansions ofPc-IIId from Apennines, Pc-IIIa from central Europeanrefugia, and with Pc-I expanding out of the Russianplain. It is interesting to note that the two newlyevolved haplogroups endemic to the Apennines (Pc-IIIband Pc-IIIc) do not seem to have participated in recol-onization. Alleles of the derived nuclear clade wouldalso be distributed across the species range throughrecolonization.

Species-specific life-history traits and genetic structure

Pityogenes chalcographus must have shared one of itsglacial refugia with the newly emerging I. typographus,where the two species may have come under directcompetition with each other. However, P. chalcographushad already been subjected to different environments asit established itself throughout Europe during its initialPleistocene expansion and then to severe selection pres-sure at the onset of the last Pleistocene glacial periodwhich funnelled its pan-European distribution intothree isolated refugia. We propose that several of thedifferences in life-history characteristics between P. chal-cographus and I. typographus may have arisen, eitherthrough gain or loss of traits, in response to the harshclimate of the late Pleistocene. Whether products ofinheritance, neutral evolution or adaptation, thesespecific life traits appear to have given P. chalcographusan advantage over the more aggressive I. typographus.This would explain the faster postglacial dispersal ofP. chalcographus (see Fig.1C,D), which would have influ-enced its genetic structure and led to the contrastingphylogeographic patterns we observe between the twobark beetle species. One example of species-specificlife-history traits that could potentially affect dispersalis the ability of P. chalcographus to over-winter atany ontological stage (Postner 1974), whereas only adult



I. typographus are able to survive a winter (Austar$a &Midtgaard 1986; Christiansen & Bakke 1988; Hrasovecet al. 2011). This may also explain the loss of large tractsof spruce forests due to I. typographus in recent years, asless severe European winters tend to promote popula-tion explosions (J€onsson et al. 2009). Additionally, theability of P. chalcographus to colonize other Pinaceaespecies and to thrive on different parts of the tree is alife-history trait that not only limits the interspecificcompetition between the two species, but may also haveinfluenced genetic structure. Host specialization hasbeen associated with extreme reduction in geneticdiversity in scolytid beetles (Kelley et al. 2000). A simi-lar association with the lower genetic diversity of themonophagous I. typographus, relative to the high diver-sity of the oligophagous P. chalcographus was observedin this study. However, this may be more attributableto the younger evolutionary history of I. typographusrather than a reduction of its diversity due to hostspecialization. While Kelley et al. (2000) argued that iso-lated host patches tend to hinder gene flow and pro-mote genetic differentiation among specialized species,we show that I. typographus conforms more to Lieutier’s(2002) alternative hypothesis that patchy host distribu-tion would force highly specialized species to undergorecurrent long-distance migration, thus increasing geneflow between populations and reducing species-wideneutral genetic structure. The converse that the abilityto colonize alternative host tree species promotes differ-entiation is unlikely to be the case for P. chalcographus,as its mitochondrial haplogroup structure was notfound to correlate with host tree species (Bertheau et al.2012). It is possible that beneficial life-history traitsevolved in one geographic area, whether through popu-

lation expansion or contraction, may have been transmit-ted via introgression and divergent selection to otherareas upon secondary contact, as was recently demon-strated in Heliconius butterflies (Dasmahapatra et al. 2012;Nadeau et al. 2012). This would confer unto a speciessuch as P. chalcographus, the capacity to shift on alterna-tive host species, while maintaining the ability to exploitits natural host (Bertheau et al. 2012).

Conclusions

The unique findings of this study highlight the potentialof a multispecies approach. By combining knowledgeon host and insect species with mitochondrial andnuclear DNA markers, we were able to unravel the evo-lutionary histories of two prominent and economicallyimportant bark beetle species. These results provide aplatform from which the relationship between geneticstructure and intrinsic life-history factors may bedefined, both physiologically and by genome-widescreening for signals of positive selection and speciationislands. A variation to this approach, using transcripto-mes, has already been applied to another closely relatedspecies, the mountain pine beetle, Dendroctonus ponderosae(Keeling et al. 2012) yielding positive results.

Acknowledgements

We thank the Austrian Science Fund (P21147-B17), EuropeanTerritorial Co-operation Austria-Czech Republic 2007–2012,European Union Seventh Framework Programme FP7 2007–2013 (KBBE 2009-3) under grant agreement 245268 ISEFOR andFederal Ministry of Agriculture, Forestry, Environment andWater Management, Austria for financial support; B. Heinze,

Table 3 Ips typographus and Pityogenes chalcographus lineage divergence times (in kyr)

Sp. Haplogroups Distance tMRCA95% confidenceinterval

Ips typographus Haplogroups It-A, B, C 0.00072 19 269 11° 108–28° 066Haplogroup It-A 0.00069 18 434 10° 722–27° 164Haplogroup It-B 0.00050 13 424 7247–21° 036Haplogroup It-C 0.00032 8 634 3322–15° 081

Pityogenes chalcographus Haplogroups Pc-I, II, III 0.00376 100 616 68° 098–132° 249Haplogroups Pc-I, II 0.00313 83 752 51° 559–124° 359Haplogroups Pc-III 0.00334 89 523 58° 695–123° 535

Haplogroups Pc-IIIb, c, d 0.00314 84 051 55° 774–122° 598Haplogroup Pc-I 0.00190 50 881 35° 166–71° 025Haplogroup Pc-II 0.00122 32 742 12° 819–55° 503

Haplogroup Pc-IIIa 0.00204 54 737 35° 273–73° 736Haplogroup Pc-IIIb 0.00164 44 025 22° 457–69° 100Haplogroup Pc-IIIc 0.00117 31 229 14° 486–52° 750Haplogroup Pc-IIId 0.00233 62 268 34° 831–94° 552

tMRCA: The most recent common ancestor.



H. Mihalciuc, M. Pernek, N. I. Ukhova, H. Viiri, R. Wegensteinerand P. Zolubas for providing populations; A. Drummond forhis advice on the Bayesian skyline plots; Godfrey Hewitt, SeanSchoville and three anonymous referees for their comments onearlier drafts of the manuscript.

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This study is the result of a joint European TerritorialCo-operation Austria-Czech Republic 2007–2012, EFREproject of C.S.’s and Y.M.’s institute. This work is a partof C.B.’s post-doctoral research focused on ecology andevolution of plant–insect relationships, especially conif-erous - forest insects. H.S. is post-doctoral researcher

focusing mainly on the biology and evolution of endos-ymbionts in Tephritids. W.A. is assistant professor at theMolecular Ecology Group at the University of Innsbruckand interested in all aspects of arthropod evolution andsymbiosis. D.N.A’s research focuses on studying hostassociation of forests pests and resolving phylogeo-graphic patterns within pest species, particularly barkbeetles, in an endeavour to identify evolutionary forcesthat shape divergence. F.M. is a PhD candidate in ForestEntomology working on bark beetles’ ecology andgenetic. S.K. is a technical assistant. Y.M. is a populationand evolutionary geneticist with a broad interest inhybrid speciation, species-wide phylogeography andconservation. C.S.’s research focuses on the phylogeogra-phy of scolytids and tephritids, and on host-parasiteco-evolution.

Data accessibility

COI: Ips typographus GenBank accession numbers:ITU82589, AF036150, AF036156, JN133853–JN133879;Pityogenes chalcographus: KC514357–KC514450 and out-group species KC514451–KC514454.ITS2: Ips typographus GenBank accession numbers:

KC514467–KC514470, Pityogenes chalcographus: JQ066307,JQ066310, KC514455–KC514462 and outgroup speciesKC514463–KC514466.GenBank accession numbers of each COI haplotypes

and ITS2 alleles for both species uploaded as onlinesupporting information Tables S1 and S2 (Supportinginformation).

Supporting information

Additional supporting information may be found in the onlineversion of this article.

Table S1 Mitochondrial haplotypes and nuclear alleles foundin Ips typographus populations. Codes for populations are givenin Table 1.

Table S2 Mitochondrial haplotypes and nuclear alleles foundin Pityogenes chalcographus populations. Codes for populationsare given in Table 1.

Fig. S1 Plots of the FCT for different values of K, the number ofpopulation groups, for both Ips typographus and Pityogenes chal-cographus generated using SAMOVA (Dupanloup et al. 2002).(A) COI marker, (B) ITS2 marker. FCT plot in solid line forI. typographus and in dotted line for P. chalcographus.



divergent evolutionary histories of two sympatric spruce...

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