Eur. J. Entomol. 106: 325–334, 2009 http://www.eje.cz/scripts/viewabstract.php?abstract=1458 ISSN 1210-5759 (print), 1802-8829 (online)

Genetic diversity of the xerothermic inustus and Centricnemus leucogrammus (Coleoptera: ) in central Europe

1 2 3 àUKASZ KAJTOCH , DOROTA LACHOWSKA-CIERLIK and MIECZYSàAW MAZUR

1Institute of Systematics and Evolution of , Polish Academy of Science, Sáawkowska 17 St., Krakow, Poland, e-mail: [email protected] 2Institute of Zoology, Department of Entomology, Jagiellonian University, Ingardena 6 St., Krakow, Poland 3Institute of Biology, Pedagogical Academy, Podbrzezie 3, Krakow, Poland

Key words. Curculionidae, weevils, mtDNA, phylogeography, xerothermic habitats

Abstract. Phylogeography, genetic diversity, and demography of central European populations of two flightless xerothermic weevils, Polydrusus inustus and Centricnemus leucogrammus, were studied based on the polymorphism of three mtDNA genes (COII, CytB, and ND1). Results indicate that these xerothermic may have different origins. P. inustus is a recent migrant as the parthenogenetic form has a low level of genetic diversity and lacks a geographic population structure. This is probably a result of a recent (before the end of last glaciation) expansion and/or present dispersal mediated by humans. On the other hand, C. leuco- grammus appears to be a relic species as the populations of this species are much more genetically diverse (six distinct clades) and some of the populations are allopatric and others sympatric. They probably diverged and expanded during the last few glaciations. Genetic discontinuities were detected among localities that are now separated by gaps in the distribution. Boundaries (mountains or farmland) separate the populations into three groups: (1) Moravia and Slovakia, (2) the lower Vistula Valley in northern Poland and (3) south-eastern Poland together with western Ukraine. Evidence for recent gene flow was found only among populations from south-eastern Poland and western Ukraine, and between these two groups. One population from northern Poland was surprisingly related to populations in southern Poland, which may be due to extinction of intermediate populations.

INTRODUCTION sible that some forest or grassland species may have The present distribution of species in central Europe is adapted to these dry environments and colonized them. a result of their glacial and postglacial history and the An intermediate hypothesis claims that some xerothermic impact of anthropogenic transformations on environ- habitats are natural relicts whereas others are of anthropo- ments. The origin of many species is reconstructed on the genic origin. Consequently, some species should be basis of paleontological, chorological, and genetic data. native to these habitats, persisting for long periods of Most of the species studied occur in the temperate and time, while others should be new migrants. Unlike the boreal zones (Taberlet et al., 1998; Hewitt, 1999; Avise, changes in the distribution of temperate and boreal envi- 2000; Varga, 2003). In contrast, our knowledge of the ronments, steppe-like habitats were common during gla- history of species that inhabit dry environments on the ciations but contracted during warmer periods. Steppe continent (e.g. steppe), is based mainly on paleontological and xerothermic habitats regressed in the Holocene. It is and pollen data (Kowalski et al., 1989; Tarasov et al., possible that present xerothermic habitats are “warm- 2000). Steppe-tundra environments were common in stage refuges” for species that formerly inhabited steppe- Europe in areas between ice sheets in the north, and like environments (Bhagwat & Willis, 2008). mountains or forest zones in the south (Nehring, 1890; Among the most common xerothermic species are Willis & van Andel, 2004). After climate warming, these . Faunistic studies on xerothermic insects in central environments started to contract and move: tundra to the Europe are based mainly on the present distribution of north and higher altitudes, steppe to south-eastern areas grasshoppers (Orthoptera) and weevils (Curculionidae) of Europe. At present, areas of potential natural steppe (Liana, 1987; Mazur, 2001). The only examples of phylo- environment in Europe are limited to the Ukraine, Cas- geographic analyses of semi-steppe insects in Europe are pian area, and Pannonian Basin. Xerothermic habitats on butterflies (Lepidoptera), which are highly mobile so similar to steppe exist in many places in western and cen- might have survived glacial periods in more southern tral Europe and the Balkans. These habitats consist refugees and afterwards expanded their range northwards mostly of small patches such as isolated turfs and thickets (Bereczki et al., 2005; Gratton et al., 2008). On the other on steep slopes of hills and along river beds. The origin of hand, there is little knowledge on the history of flightless, these habitats is unclear. They have been considered as xerothermic insects. relicts of steppe-tundra environments. Alternatively, they This paper presents the results of a study of the genetic may have resulted from the deforestation that has diversity of central European populations of two species occurred over the last few thousand years. It is also pos- of weevils from the subfamily , Polydrusus inustus Germar, 1824 and Centricnemus leucogrammus

325 (Germar, 1824). P. inustus inhabits an area between the N-10920, N1-J-12533 and LR-N-12866 (Simon et al., 1994). Ural and Caucasus Mts in the east, and south-eastern The cycling profile for the PCR was: 95°C for 4 min, 35 cycles Poland and the eastern slopes of the Carpathians in the of 95°C for 30 s, 50°C for 1 min, 72°C for 2 min, and a final west (Korotyaev, 1996). This species does not live in the extension period of 72°C for 10 min. After purification PCR fragments (QIAquick PCR Purification Kit, Qiagen) were Pannonian Basin. Some isolated populations occur farther sequenced using the BigDye Terminator v.3.1. Cycle to the west and north in Poland (Mazur, 2001). Popula- Sequencing Kit (Applied Biosystems Inc., Foster City, USA) tions of this over most of its range consist of par- and an ABI 3100 Automated Capillary DNA Sequencer. All thenogenetic females and, at least in Poland, they are sequences were deposited in GenBank (accession numbers triploid (Lachowska et al., 2008). Bisexual populations FJ442860-FJ442930, strain numbers correspond to combined are known only from Georgia and Turkey (Korotyaev, haplotypes of COII-CytB-ND1 genes whose numbers are used 1996). These weevils live mostly on Rosacea in xero- in this paper). Sequences were checked and aligned using Bio- thermic habitats but sometimes are also found in dry Edit v.7.0.5.2 (Hall, 1999) and ClustalX (Thompson et. al., wastelands. The main range of C. leucogrammus stretches 1997). No indels (i.e. insertions or deletions) or stop codons from central Asia to southern Poland. A disjunctive part were observed. The three fragments of mtDNA genes obtained (COII, CytB, and ND1) were subjected to a partition- of its range is situated in the Pannonian Basin and along homogenity test (Farris, 1995) using PAUP* 4.10b (Swofford, the Danube valley. Isolated populations are also found 2002), which showed that further analyses could be performed farther to the north – in Pomerania in Poland (Mazur, on combined sequences. Haplotypes were identified and stan- 2001). This species is bisexual and diploid (Petryszak, dard genetic indices such as haplotype diversity (h), nucleotide 1977; Lachowska et al., 2006). It lives exclusively on diversity (S) and number of private haplotypes (Np) for popula- plants in dry and warm xerothermic turfs and steppe. tions were computed using the program DnaSP 4.20.2 (Rozas et These two species are flightless and their ability to dis- al., 2003). perse over great distances limited, especially currently, Phylogeny and phylogeography because of the fragmentation of xerothermic habitats in The Akaike information criterion in MODELTEST (Posada & central Europe. On the other hand, at least for P. inustus, Crandall, 1998) was used to determine the best-fitting nucleo- it is postulated that human mediated dispersal is possible tide substitution model. Parameters of 56 models of sequence as this species might survive in, e.g. strawberry fields evolution were estimated in PAUP* 4.10b (Swofford, 2002). (Mazur, 2001). Low mobility allows their expansion The HKY+I model was chosen for P. inustus and the HKY+I+G routes and range contractions to be assessed over a longer model for C. leucogrammus (Hasegawa et al., 1985). Unrooted perspective. They are also polyphagous, implying that haplotype trees were obtained for each species using Bayesian their present range was not limited by the availability of inference in MrBayes 3.1 (Huelsenbeck & Ronquist, 2001; Ron- quist & Huelsenbeck, 2003). A distribution of posterior prob- food in the past. abilities was produced by allowing four incrementally heated The aim of this study was to describe the phylogeogra- Markov chains (using default heating values) to proceed for 2 × phy, estimate genetic diversity and level of gene flow 107 generations, with sampling occurring every 1 × 103 genera- among central European populations of these two tions. The approximate number of generations needed to obtain weevils, and calculate divergence and expansion times for convergence of the sampled trees was estimated graphically, these populations. Comparison of the results for the two and was 500 for both species. These samples were discarded species allowed us to infer the history of flightless xero- (burn-in) from the analyses and the remaining samples were thermic beetles (both bisexual and parthenogenetic) in the used to produce a 50% majority rule consensus tree. For estima- north of central Europe closest to the area covered by the tion of divergence times between main clades, pairwise dis- tances among them were calculated according to t = (d – ice-sheet during the Pleistocene. xy 0.5*(dx+dy)*µ, where dx and dy denote the average sequence MATERIAL AND METHODS diversity within, and dxy the sequence diversity among clades, respectively (Nei, 1987) and µ is the mutation rate. There is no Both species were collected from most of their ranges in the data on divergence times or mutation rates for any xerothermic north of the Carpathian Mountains. For C. leucogrammus, mate- beetles because their chitin outer skeleton is not preserved in an rial was also collected from two localities in the south of the alkaline environment. Therefore, the mutation rate was assumed Carpathians (Table 1, Fig. 1a, b). Only parthenogenetic indi- to be 1.5–2.0% per million years as calculated for the mtDNA viduals of P. inustus were collected because bisexual popula- of other beetles (Juan et al., 1996; Farrell, 2001; Ribera et al., tions are unknown in Europe other than those in the Caucasus 2001; Caccone & Sbordoni, 2001; Barraclough & Vogler, Mountains. Adults of these beetles were collected on several 2002). In addition to Bayesian trees, haplotype networks were expeditions in 2005–2008. Samples were first preserved in 99% constructed using the statistical parsimony method (SP) (Tem- ethanol and then stored at –22°C. For DNA analyses, 5 indi- pleton et al., 1992) and TCS 1.21 program (Clement et al., viduals per population were taken, with the exception of the 2000). Based on these networks, nested clade phylogeographic HAL population of P. inustus for which only 2 specimens were analyses (NCPA) (Templeton, 1998, 2001; Emerson et al., available. DNA was extracted from whole bodies using 2001) were done using the program ANeCA (Panchal, 2007; the DNeasy Tissue Kit (Qiagen, Hilden, Germany). Amplifica- Panchal & Beaumont, 2007), which is composed of GEODIS tion of three fragments of mitochondrial DNA (mtDNA; total 2.4 (Posada et al., 2000) and Templeton’s inference key (Tem- length of more than 1600 bp), including cytochrome oxidase II pleton, 2004). The utilization of NCPA for reconstructing events (COII), cytochrome b (CytB), and nicotinamide adenine dinu- in the past history of populations was recently questioned (i.e. cleotide dehydrogenase 1 (ND1) along with the tRNALeu gene, Knowles & Maddison, 2002; Petit & Grivet, 2002; Beaumont & was performed using the following pairs of primers, Panchal, 2008). However, it was also indicated that this method respectively: TL2-J-3038 and TK-N-3782, CB-J-10933 and CB- may be useful providing any inferences are interpreted in the

326 TABLE 1. The localities and standard genetic indices (h – haplotype diversity; S – nucleotide diversity; Np – number of private haplotypes) of the populations of P. inustus and C. leucogrammus studied. Symbol of population a Geographic coordinates h ± SD S ± SD (%) Np Haplotypes (number of specimens) Polydrusus inustus KRA N 50°02´32,1˝, E 19°52´55,5˝ 0.4 ± 0.24 0.024 ± 0.01 1 P1 [4], P2 [1] DAL N 50°19´58,6˝, E 20°12´55,4˝ 0 ± 0 0 ± 0 0 P3 [5] CHO N 50°22´30,5˝, E 20°42´43,9˝ 0.7 ± 0.22 0.048 ± 0.02 1 P1 [1], P3 [3] BOC N 51°20´33,1˝, E 21°58´48,6˝ 0.7 ± 0.05 0.071 ± 0.03 1 P1 [3], P5 [1], P13 [1] GRO N 50°48´01,6˝, E 23°57´12,7˝ 0.6 ± 0.18 0.029 ± 0.01 1 P1 [3], P6 [2] KOR N 50°24´24,9˝, E 23°32´25,3˝ 0 ± 0 0 ± 0 0 P7 [5] MIE N 52°19´21,3˝, E 23°02´12,2˝ 0 ± 0 0 ± 0 0 P7 [5] ROZ N 49°43´03,4˝, E 22°46´49,7˝ 0.4 ± 0.24 0.048 ± 0.03 1 P1 [4], P8 [1] JAR N 49°58´34,3˝, E 22°38´14,2˝ 0 ± 0 0 ± 0 0 P1 [5] SZC N 49°56´28,6˝, E 20°53´06,9˝ 0.4 ± 0.24 0.024 ± 0.01 1 P1 [4], P9 [1] BIS N 49°58´06,5˝, E 20°06´55,5˝ 0 ± 0 0 ± 0 1 P10 [1] KAL N 51°59´14,4˝, E 21°13´59,3˝ 0.6 ± 0.18 0.036 ± 0.01 1 P1 [3], P11 [2] MOD N 52°25´59,9˝, E 20°41´25,7˝ 0 ± 0 0 ± 0 1 P12 [5] WLO N 52°39´44,4˝, E 19°04´11,6˝ 0 ± 0 0 ± 0 0 P7 [5] GOG N 50°29´30,8˝, E 18°02´59,5˝ 0 ± 0 0 ± 0 0 P7 [5] GLO N 51°40´50,6˝, E 16°04´27,4˝ 0 ± 0 0 ± 0 0 P7 [5] SAN N 50°41´26,0˝, E 21°48´40,5˝ 0.4 ± 0.24 0.048 ± 0.03 0 P1 [1], P13 [4] ZLO N 49°39´58,3˝, E 24°37´00,5˝ 0.4 ± 0.24 0.024 ± 0.01 1 P1 [4], P14 [1] HAL N 48°36´19,9˝, E 24°57´47,2˝ 0 ± 0 0 ± 0 1 P15 [2] Centricnemus leucogrammus DAL N 50°19´58,6˝, E 20°12´55,4˝ 0.7 ± 0.05 0.049 ± 0.02 2 C1 [3], C2 [1], C3 [1] CHO N 50°22´30,5˝, E 20°42´43,9˝ 0.8 ± 0.16 0.431 ± 0.12 1 C4 [2], C5 [2], C6 [1] DOB N 51°17´02,2˝, E 21°53´03,1˝ 0.8 ± 0.16 0.431 ± 0.12 1 C4 [2], C5 [2], C7 [1] GRO N 50°48´01,6˝, E 23°57´12,7˝ 0.4 ± 0.24 0.025 ± 0.02 2 C8 [4], C9 [1] GRE N 53°15´23,7˝, E 14°57´23,7˝ 0.4 ± 0.24 0.025 ± 0.02 2 C10 [4], C11 [1] BRA N 48°08´00,0˝, E 17°06´00,0˝ 0.9 ± 0.16 0.160 ± 0.05 4 C12 [2], C13 [1], C14 [1], C15 [1] NIE N 52°50´31,6˝, E 18°54´38,7˝ 0.6 ± 0.18 0.074 ± 0.02 2 C16 [3], C17 [2] PLU N 53°15´55,8˝, E 18°24´06,5˝ 0.7 ± 0.22 0.148 ± 0.07 3 C18 [3], C19 [1], C20 [1] SAN N 50°41´26,0˝, E 21°48´40,5˝ 1.0 ± 0.13 0.148 ± 0.04 5 C21 [1], C22 [1], C23 [1], C24 [1], C25 [1] ZLO N 49°39´58,3˝, E 24°37´00,5˝ 0.7 ± 0.13 0.148 ± 0.04 3 C26 [3], C27 [1], C28 [1] BRO N 49°55´27,7˝, E 25°02´01,6˝ 1.0 ± 0.13 0.320 ± 0.08 5 C29 [1], C30 [1], C31 [1], C32 [1], C33 [1] HAL N 48°36´19,9˝, E 24°57´47,2˝ 0.9 ± 0.16 0.357 ± 0.08 4 C34 [2], C35 [1], C36 [1], C37 [1] POU N 48°33´42,9˝, E 16°44´56,7˝ 0.9 ± 0.16 0.160 ± 0.04 4 C38 [1], C39 [2], C40 [1], C41 [1] aSymbols for the populations were derived from the names of cities or towns situated nearest to a particular population (these sym- bols are used in Fig. 1). Localities in Poland: KRA – Krakow; DAL – Dale; CHO – Chotel; BOC – Bochotnica; GRO – Gródek; KOR – Korhynie; MIE – Mielnik; ROZ – RoĪubowice; JAR – Jarosáaw; SZC – Szczepanowice; BIS – Biskupice; KAL – Góra Kal- waria; MOD – Modlin; WLO – Wáocáawek; GOG – Gogolin; GLO – Gáogów; SAN – Sandomierz; DOB – Dobre; GRE – GrĊdziec; NIE – Nieszawa; PLU – Páutowo. Localities in Ukraine: ZLO – Záoczów; BRO – Brody; HAL – Halicz. Locality in Czech Republic: POU – PouzdĜany. Locality in Slovakia: BRA – Bratislava. light of the ecology and past history of the species studied and C. leucogrammus were divided into 5 clusters: Maáopolska compared with results of other analyses (Garrick et al., 2008). In Uplands [DAL, CHO, DOB, SAN], Pomerania [GRE], Moravia- this study the NPCA results were verified in this way. Slovakia [BRA, POU], Vistula valley [PLU, NIE], and western Demography and population genetics Ukraine [ZLO, BRO, HAL] with eastern Poland [GRO]. A Mantel test (Mantel, 1967) was performed in ARLEQUIN 3.1 to Populations of each species were grouped according to their check if the genetic structure of the populations fits an isolation geographical locations (Table 1). FST indices (Wright, 1951) by distance model (IBD) (Slatkin, 1993), using pairwise FST were calculated using ARLEQUIN 3.1 (Schneider et al., 2000). values and straight-line geographic distances in kilometers. To Populations of P. inustus were divided into 6 clusters: test for the presence of contemporary or historical barriers Maáopolska Uplands [KRA, DAL, CHO, SAN], eastern Poland between populations, a spatial analysis of molecular variance [BOC, GRO, KOR, MIE], Carpathian Foothills [BIS, SZC, was conducted using the program SAMOVA (Dupanloup et al., JAR, ROZ], Vistula valley [KAL, MOD, WLO], Oder valley 2002). A mismatch distribution (MD) (Rogers & Harpending, [GOG, GLO], and western Ukraine [ZLO, HAL]. Populations of 1992) and Fu’s test (FS) (Fu, 1997) were calculated in ARLE-

327 Fig. 1. Maps of central Europe with distribution ranges of the weevils studied (dark grey area): P. inustus (a) and C. leuco- grammus (b). Symbols for populations (sampling sites), such as KRA, DAL, etc., were derived from the names of cities or towns situated nearest to that population (see Table 1 for full names). # – extinct; ? – probably extinct.

QUIN 3.1 in order to examine the demographic history, and spe- types varied from 1 in 2 populations to 5 in 2 populations cifically, test for historical (temporal) expansions of populations and except for one pair of populations (DAL and DOB of both species. The probable time of expansion (how long ago from Maáopolska Uplands), no haplotypes were shared the expansion occurred) was estimated by the parameter W = 2ut between populations (Table 1). (where u = mTµ; mT – length of analyzed sequence, µ – mutation rate; t – number of generations since expansion) obtained from Phylogeography the MD. Mutation rate was the same as that assumed for the For each species, the topologies of the gene trees (Figs divergence time (1.5–2.0% per million years) and t as 1 genera- 2a, 3a) and haplotype networks (Fig. 2b, 3b) were similar. tion / year. The level of migration between populations was esti- In P. inustus, haplotypes were grouped into 3 clades (Fig. mated using the frequency of private haplotypes (Np) (Slatkin, 2a, b). These clades and haplotypes were separated from 1985) and FST indices (Wright, 1951), for which values below 0.33 (for mtDNA) indicate high gene flow (Cockerham & Weir, each other only by single mutations. Clade 1-1 was lim- 1993). ited to 9 individuals from the Maáopolska Uplands. Clade 1-2 was broadly distributed – 31 individuals from this RESULTS clade were found in the eastern (Roztocze Uplands, cen- Genetic diversity of populations There were 15 haplotypes in the 92 individuals of P. inustus, 6 of which were found only in single specimens (Table 1). The 1643 bp long sequences showed 13 poly- morphic sites. Codon position polymorphism was as fol- lows: 5 (0.9%), 2 (0.3%), and 7 (1.3%) for 1st,2nd, and 3rd codon positions, respectively. Mean haplotype diversity (h) was 0.795 ± 0.028 and mean nucleotide diversity (S) was 0.071% ± 0.005 (Table 1). Ten populations were monomorphic (Table 1). The number of private haplo- types was 0 in 8 populations and 1 in 11 populations (Table 1). Two haplotypes were particularly widespread: P1 found in 32 individuals and P7 found in 25 individuals (Table 1). Fig. 2. Unrooted phylogenetic tree based on Bayesian Infer- Genetic diversity of C. leucogrammus populations was ence (a) and network of Statistical Parsimony (b) of 15 mtDNA much greater. There were 41 haplotypes in the 65 indi- haplotypes of P. inustus. Posterior probabilities are indicated on viduals of C. leucogrammus of which 29 were found only the tree and nested clades are shown in the network. Dashed once (Table 1). The 1626 bp long sequences showed 44 lines enclose clades (with probabilities <0.05) inferred by polymorphic sites. Codon position polymorphism was as NCPA: LDC/PF – Long distance colonization and/or past frag- follows: 9 (1.7%), 3 (0.6%), and 27 (5.0%) for 1st,2nd, and mentation, RD – Restricted long distance dispersal of non- sexual species, CRE – Contiguous range expansion. Dotted line 3rd codon positions, respectively. Mean haplotype diver- between P9 and P8 indicates a second possible connection sity (h) was 0.981 ± 0.006 and mean nucleotide diversity between these two haplotypes in the network. Each 1-step clade (S) was 0.564 % ± 0.017 % (Table 1). No population was is marked either by a square, circle or triangle, which corre- monomorphic (Table 1). The number of private haplo- spond to the symbols in Fig. 4a.

328 Fig. 3. Unrooted phylogenetic tree based on Bayesian Inference (a) and network Statistical Parsimony (b) of 41 mtDNA haplo- types of C. leucogrammus. Posterior probabilities are indicated on the tree and nested clades are shown in the network. Dashed lines enclose clades (with probabilities < 0.05) inferred by NCPA: LDC/PF – Long distance colonization and/or past fragmentation, CRE – Contiguous range expansion, RGFwIbD – Restricted gene flow due to isolation by distance, RGFwLDD – Restricted gene flow/dispersal but with some long-distance dispersal over areas not occupied by the species, PGF – Past gene flow followed by extinction of intermediate populations, AF – Allopatric fragmentation, IO – Inconclusive outcome. Each 3-step clade is marked either by a square, circle, triangle, pentagon, star or diamond, which correspond to symbols in Fig . 4b. tral Vistula Valley and Bug Valley) and the western parts found only in populations along the central part of the of Poland (lower Vistula Valley and Oder Valley). The basin of the Vistula river. most numerous clade, 1-3 (52 individuals), was repre- Haplotypes found in C. leucogrammus were grouped sented by samples from western Ukraine and eastern into 6 clades (3-step clades in NCPA analyses) (Fig. 3a, Poland, the Carpathian Foothills and the Vistula Valley b). Clade 3-1 did not have a compact range – 10 indi- (Fig. 4a). Individuals of P. inustus with mtDNA haplo- viduals with haplotypes from this clade were found in types from 2 clades (1-3 and 1-2 or 1-1 and 1-3) were southern Poland (CHO, DOB, DAL) and 3 individuals from one population from western Ukraine (HAL). Hap-

Fig. 4. Geographic distribution of mtDNA clades in central Europe, as indicated by haplotype trees (Fig. 2a, b) and networks (Fig. 3a, b), in populations of P. inustus (a) and C. leucogrammus (b). The symbols of clades (square, circle, triangle, pentagon, star or diamond) are the same as in Figs 2 and 3, respectively; dark grey area indicate species distribution range, broken lines – boundaries between genetically distinct groups and dotted arrows – possible migration routes of C. leucogrammus.

329 Fig. 5. Histograms of mismatch distributions (MD) for P. inustus (a) and C. leucogrammus (b). x axis – differences between pairs of haplotypes, y axis – frequency of differences, ov – observed values, ev – expected values.

lotypes from clade 3-2 were found only in Moravia was also confirmed by Fu’s test (FS = – 6.87629, P = (POU) and southern Slovakia (BRA) (in both 5 individu- 0.006). als). Ten individuals from the lower Vistula valley (NIE, The mean pairwise distance between clades of C. leuco- PLU) were grouped in clade 3-3. Clade 3-4 consisted of 8 grammus is between 0.3 and 0.6%, yielding a divergence individuals from western Ukraine (ZLO, BRO). Haplo- time of between 380 000 and 150 000 years ago. MD types from clade 3-5 were found in all populations from results showed that the expansion of central European western Ukraine (4 individuals) and eastern Poland populations of C. leucogrammus occurred between 280 (GRO) (5 individuals). Clade 3-6 grouped haplotypes 000 and 110 000 years ago (W = 9.823, 95% confidence from southern Poland (CHO, SAN, DOB) (10 intervals 7.129–13.631) (Fig. 5b). A significant result of individuals) and surprisingly from Pomerania (GRE) in Fu’s test (FS = –20.00875, P < 0.001) also confirms the northwestern Poland (5 individuals) (Fig. 4b). The only demographic expansion. differences between the tree and the network were the Isolation and migration positions of two intermediate haplotypes: C19 (situated in clade 3-3 in the tree and clade 3-6 in the network) and For most pairs of groups of P. inustus populations FST C32 (situated in clade 3-1 in the tree and clade 3-4 in the indices are lower than 0.33, which suggest moderate network). The most probable explanations of the present genetic differences between them (Table 2). The genetic geographic distribution of haplotypes and clades of both (FST) and geographic (km) distances between populations species obtained by NCPA analyses are presented in Figs of this weevil did not appear to be correlated (the result of 2b and 3b. Mantel test was not significant: r = 0.0355, P = 0.4) (Fig. 6a). According to the SAMOVA, the largest differences Divergence and expansion times between groups of populations (66.2%) occur when Because of the little divergence between clades of P. populations are divided into 5 clusters: Maáopolska inustus, their divergence times could not be calculated. Uplands (CHO, DAL) (1), vicinity of Krakow (BIS) (2), The MD results (W = 1.324; 95% confidence intervals vicinity of Warsaw (MOD) (3), eastern Poland (SAN, 0.787–2.725) for this species showed that the expansion KOR, MIE) (4) and all other populations (5). In this divi- of the populations studied occurred between 55 000 and sion of the populations, 11% of the genetic differences 12 000 years ago (Fig. 5a). The demographic expansion are ascribed among populations within groups and 22.8% within populations. This division does not delineate clear

TABLE 2. The FST indices (below diagonal) and their statistical significance (above diagonal) for pairs of populations of P. inustus and C. lecucogrammus. Grouping of populations into clusters is described in Material and methods (* – values of FST that are sig- nificant at P < 0.05).

Cluster of P. inustus Cluster of C. lecucogrammus populations 1 2 3 4 5 6 populations 1 2 3 4 5 1 0.00 * * * * * 1 0.00 * * * * 2 0.18 0.00 * * 2 0.40 0.00 * * * 3 0.24 0.33 0.00 * * * 3 0.46 0.68 0.00 * * 4 0.24 0.32 0.21 0.00 * * 4 0.60 0.86 0.68 0.00 * 5 0.19 0.31 0.11 0.17 0.00 * 5 0.29 0.49 0.44 0.51 0.00 6 0.18 0.28 0.17 0.17 0.18 0.00 1– Malopolska Uplands; 2 – eastern Poland; 3 – Carpathian Foothills; 1 – Malopolska Uplands; 2 – Pomerania; 3 – Moravia–Slo- 4 – Vistula Valley; 5 – Oder Valley; 6 – western Ukraine. vakia; 4 – Vistula Valley; 5 – western Ukraine.

330 Fig. 6. The relationship between genetic (FST) and geographic distance (km) for populations of P. inustus (a) and C. leucogrammus (b) (Mantel tests). borders between groups of populations because four of of the mainly parapatric clades of P. inustus suggests at them are located inside the range of cluster which include least two episodes of expansion of these weevils in cen- most of populations. tral Europe. The disjunctive range of clade 1-2 (which

FST indices for most pairs of regional groups of C. leu- occupied eastern Poland and isolated localities in western cogrammus populations are larger than 0.33 indicating Poland) probably resulted from individuals of clade 1-3 that these groups are isolated from each other (Table 2). spreading from the western Ukraine along the Carpathian

An FST value of 0.29 indicates possible migrations only foothills and through the Lubelska Uplands to the Vistula for populations from the Maáopolska Uplands and valley. Such a conclusion is also supported by NCPA western Ukraine-eastern Poland. Populations of C. leuco- results, which explain the present distribution of clades grammus fit the IBD model (significant result of Mantel 1-2 and 1-3 as dispersal restricted by distance and long test: r = 0.421, P = 0.003), i.e. generally populations more distance colonization and/or past fragmentation. Haplo- genetically distinct are more distant in space, however, types belonging to two clades were found in populations this correlation is rather weak (Fig. 6b). According to the along the central part of the basin of the Vistula River. SAMOVA, the largest differences between groups of Such population admixture may have originated as a populations (but only 34.5%) occur when populations are result of migration of specimens from different localities. divided into three clusters: western Ukraine with south- Haplotypes from clade 1-1 (found only at two localities in eastern Poland and one population from Pomerania (1), the Maáopolska Uplands) probably originated as a result Moravia with Slovakia (2) and lower Vistula valley (3). of a founder event and limited spread. Such a division is partially concordant with gaps in the The much greater genetic diversity of individuals and present range of the species. However, the high per- populations of C. leucogrammus and mixed distribution centage of genetic differences among populations within of its clades (allopatric and sympatric) is probably the groups (41.0%) and within populations (24.5%) showed result of the long-term existence of these weevils in cen- that almost all populations are significantly different from tral Europe. Estimated divergence times between main one another. clades (380 000 – 150 000 years ago) and expansion time (280 000 – 110 000 years ago) are nested in the second DISCUSSION part of the Pleistocene, which accords with the present Origin and phylogeography of xerothermic weevils in knowledge of the distribution of steppe-like habitats and central Europe species in central Europe. Even if these weevils (or other The low genetic differentiation of P. inustus popula- steppe species) existed in the area to the north of the tions in central Europe suggests they are of recent origin. Carpathian-Sudeten Mts before 430 000 BP, they could As bisexual populations of this weevil are known only not have survived the most extensive Sanian (Cromenian) from Black Sea-Caspian Sea area (Korotyaev, 1996) it is glaciation. During the second part of the Pleistocene most likely that its parthenogenetic form arose there and insects in temperate and boreal areas in Eurasia and North afterwards spread into eastern and central Europe. The America diverged (Stauffer et al., 1999; Ritzerow et al., origin of parthenogenesis in this species is not known but 2004; Laffin et al., 2005; Grapputo et al., 2005; Maroja et preliminary studies (Kajtoch et al., in press) suggest that al., 2007). Present spatial distribution of C. leuco- it is a result of hybridization. Expansion of the partheno- grammus clades is a result of several past events. Distri- genetic form of P. inustus took place during the Vistulian bution of haplotypes from clade 4-1 is probably a result (Weichselian) glaciation (55 000 – 12 000 years ago) of contiguous range expansion. This is not surprising when steppe-like habitats were more common. Dispersal because that clade was found in populations from western and colonization of new areas are ongoing because new Ukraine and south-eastern Poland – the western part of populations of this species were found recently in areas the main, continuous range of C. leucogrammus. On the where they had not been found earlier despite intensive other hand geographic distributions of clades 2-4, 2-5, investigations (Mazur, 1994). The geographic distribution 2-7, and 3-1 are explained by allopatric divergence or long distance colonization and past fragmentation. These

331 clades are represented by specimens from isolated groups mote the dispersal of P. inustus (Mazur, 1994). A similar of populations from the lower Vistula Valley or from lack of genetic differences between populations was separate populations from western Ukraine and eastern found in many highly mobile (often flying) species of Poland. Geographic localization of specimens belonging weevils and bark beetles, which inhabit the coniferous to the second main clade (4-2) is probably a result of forests of Eurasia (Stauffer et al., 1999; Faccoli et al., restricted gene flow with isolation by distance, long- 2005; Conord et al., 2006; Lakatos et al., 2007). These distance dispersal over areas not occupied by the species species are tree pests and spread only a few thousand or past gene flow followed by extinction of intermediate years ago after climate warming. The low genetic differ- populations. Haplotypes belonging to the 4-2 clade are ences among populations of these beetles may also be a found in three groups of populations, clearly isolated at result of human-aided dispersal. present: eastern (Polish-Ukrainian), southern (Moravian- Three groups of C. leucogrammus populations are Slovakian), and northern (from lower Vistula Valley). separated by wide breaks in the species range – areas Past gene flow followed by extinction of intermediate which are not suitable for this species at present (farm- populations is also the best explanation for the geographic land and forested mountains) but which might had been distribution of clade 3-6. That clade was found not only occupied in the past. On the other hand, the distribution in some populations from southern Poland but also in a of clades in some populations from southern and eastern population from Pomerania (GRE). It is isolated from Poland together with western Ukraine are the result of other localities by a wide break in the species range of long distance colonization and/or past fragmentation, about 300 km. The hypothesis of extinction of some restricted gene flow with isolation by distance and con- populations is also supported by the 33 haplotypes identi- tiguous range expansion. Evidence for gene flow between fied in the network but not present in the collected mate- populations in southern Poland, eastern Poland and rial. A similar allopatric and sympatric distribution of western Ukraine is also supported by FST values, which clades is observed in many insects from the temperate are lower than 0.33 only between these groups of popula- zone such as the bark , Dendroctonus rufipennis, tions, and by the presence of similar (closely related) hap- leaf beetle, Gonioctena pallida, and , lotypes in distant populations from the Maáopolska solieri (Mardulyn, 2001; Garnier et al., 2004; Uplands (DAL, CHO, DOB) and western Ukraine (HAL), Maroja et al., 2007), also the xerothermic snail Candidula eastern Poland (GRO) and western Ukraine (BRO, ZLO, unifasciata (Pfenninger & Posada, 2002; Pfenninger et HAL), and also between regional groups of populations al., 2003) and xerothermic butterfly Clouded Apollo Par- in the Maáopolska Uplands (DAL, CHO, DOB, SAN) and nassius mnemosyne (Gratton et al., 2008). These species western Ukraine (HAL, ZLO, BRO). These data and the each consist of few main clades, which survived the presence of a high frequency of private haplotypes (Np) Pleistocene in different refuges in which they diverged in most of the populations support migration in the past from one another. Afterwards these phylogenetic lineages rather than the present. Only in one case, in populations expanded and now occur sympatrically at some localities. DAL and DOB from the Maáopolska Uplands were the Isolation and migration same haplotypes detected, which is strong evidence for recent gene flow. The remaining populations, especially Our study revealed that five genetically distinct groups from distant localities, are now isolated. Such a scenario of P. inustus populations are not separated geo- is also congruent with the result of the Mantel test, which graphically. One group is particularly widespread and the indicate a weak but significant correlation between the rest are localized inside the range of the main cluster. geographic and genetic distances of populations. Such population division may be the result of a recent colonization of central Europe or migration between con- General conclusions temporary localities. In this case both explanations are According to Coope (1994), insects may react to probably correct because these beetles arrived in central changes in climate and environment in at least three ways. Europe at the end of the last ice-age and have been They may evolve (produce new forms better adapted to spreading ever since. Such a scenario is confirmed by the changing conditions), move (changes in distribution, NCPA, which inferred a continuous range expansion for populations in the northern part of the range may go all clades. Values of FST below 0.33 for most pairs of geo- extinct but survive in southern refuges) or become extinct graphic groups of populations, the low number of private (applies mostly to species which inhabit isolated and haplotypes (Np) in populations and lack of correlation small areas and are unable to migrate). The genetic diver- between geographic and genetic distances also confirm sity of the weevils studied showed that xerothermic spe- present migration and/or recent colonization. Despite cies exhibit at least two strategies for surviving climatic being flightless, individuals of P. inustus are able to move oscillations. Polydrusus inustus represents a group of and survive outside of xerothermic habitats (Mazur, asexual forms and/or highly mobile insects with a rela- 2001). Parthenogenesis may facilitate expansion because tively short history in central Europe. This insect survived only a single female or group of female clones is needed glaciations in southern refuges and spread to Eurasian for the foundation of a new population. Lack of genetic steppe-xerothermic habitats during warmer periods and is differences in mtDNA in several populations, in which a good example of Coope’s second strategy. On the other only one haplotype was found, is probably the result of a hand, Centricnemus leucogrammus is an example of a founder effect. Human-mediated dispersal may also pro- relic element and is probably representative of other

332 bisexual species with poor dispersal abilities, which have FACCOLI M., BLAĪENEC M. & SCHLYTER F. 2005: Feeding existed in central Europe since the end of the most exten- response to host and non-host compounds by males and sive Sanian glaciation (after 430 000 years ago). These females of the spruce bark beetle Ips typographus in a tunnel- species probably consist of many phylogenetic lineages, ling microassay. J. Chem. Ecol. 31: 745–759. FARRELL B.D. 2001: Evolutionary assembly of the milkweed which diverged during interglaciations in separate warm- fauna: cytochrome oxidase I and the age of tetraopes beetles. stage refuges (Bhagwat & Willis, 2008), such as steppe- Mol. Phylogen. Evol. 18: 467–478. like habitats in western Ukraine (Wolyn and Podole FARRIS J.S., KÄLLERSJÖ M., KLUGE A.G. & BULT C. 1995: Con- Uplands), southern Poland (Maáopolska Uplands), the structing a significance test for incongruence. Syst. Biol. 44: Pannonian Basin and maybe also further to the north. 570–572. They expanded during the following glaciations resulting FU X.-Y. 1997: Statistical tests of neutrality of mutations against in a partially overlapping distribution of clades. C. leuco- population growth, hitchhiking and background selection. grammus fits Coope’s first strategy but its clades have not Genetics 147: 915–925. had enough time to evolve into distinct taxonomic units. GARNIER S., ALIBERT P., AUDIOT P., PRIEUR B. & RASPLUS J.-Y. However, it is probable that such units exist somewhere 2004: Isolation by distance and sharp discontinuities in gene frequencies: implications for the phylogeography of an alpine within the wide range of this species. Further studies on insect species, Carabus solieri. Mol. Ecol. 13: 1883–1897. different species of xerothermic insects should reveal GARRICK R.C., DYER R.J., BEHEREGARAY L.B. & SUNNUCKS P. whether the results of this study are general for all steppe- 2008: Babies and bathwater: a comment on the premature like insect assemblages in Europe. obituary for nested clade phylogeographic analysis. Mol. Ecol. 17: 1401–1403. ACKNOWLEDGEMENTS. We would like to thank greatly GRAPPUTO A., BOMAN S., LINDSTRÖM L., LYYTINEN A. & MAPPES people who helped us during the successive stages of this work: J. 2005: The voyage of an invasive species across continents: M. Holecová, B. Petryszak, S. Knutelski, M. Mazur, W. Rizun, genetic diversity of North American and European Colorado D. Kubisz, and W. Babik. This work was supported by a grant potato beetle populations. Mol. Ecol. 14: 4207–4219. 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