Subterranean Biology 7: 25-33, 2009 (2010)

of the congener T. cavicola Kollar, 1833; the two species are often syntopic but no evidence of hybridization has been reported as yet, suggesting that they might not be phylogenetically closely related (Ketmaier et al 2004). The same authors used 18 allozymic loci to describe pat-terns of gene fl ow in 12 populations of T. cavicola, six populations of T. neglectus and three populations of T. andreinii, a species limited to a few locations in Apu-lia (Southern Italy). Pattern of genetic structuring was mainly related to the limestone structure of the sampled areas rather than to the availability of epigean routes for dispersal indicating a substantial lack of gene fl ow even among relatively nearby populations.

We were therefore quite surprised by a few reports detailing the occurrence of T. neglectus in two localities, one in Lower Saxony (Germany) and one in Northern Bohemian (Czech Republic), not far away from each other but well outside the known geographical range of the species (Fig.1) (Chladek et al 2000; Brunk et al 2003; Chladek and Tryzna 2004). Not only the geographic po-sition of these localities was puzzling, but also the fact that there is no continuity of limestone areas between them and the northernmost edges of the species range (Southern Austria and Southern Bavaria).

In this study we used sequence polymorphisms of one mitochondrial (Cytochrome Oxidase subunit I, COI) and one nuclear (Internal Transcribed Spacer 1, ITS1) gene to ask whether the occurrence of T. neglectus in Central Europe is the resultant of a natural process or not. To this

INTRODUCTION

The family Rhaphidophoridae is worldwide distrib-uted and includes species mostly confi ned to damp en-vironments. In the peri-Mediterranean area it is repre-sented by two genera (Dolichopoda and Troglophilus), both confi ned to natural and artifi cial caves (but limited nocturnal epigean activity has been also documented; Novak and Kustor 1983; Pehani et al 1997). These gen-era share a similar Eastern Mediterranean distribution, which extends to Asia Minor. Dolichopoda is by far more species-rich than Troglophilus. In the last three decades these wingless cave crickets have been the objects of a good deal of genetic work aimed at understanding vari-ous evolutionary phenomena ranging from phylogenetic relationships among species to processes at the shallow-est population level (Allegrucci et al 2005; 2009; Cobol-li et al 1999; Ketmaier et al 2000; 2004; Sbordoni et al 1981; 1985; 1991; 2000 and references therein). These studies have shed light on the systematics of the group and on the processes of adaptation and speciation related to colonization of caves. As generally expected for cave organisms, species of these two genera are genetically structured with a high level of fragmentation.

Here we focus on Troglophilus neglectus Krauss, 1879, a relatively widespread species ranging from the southern portion of the Balkan Peninsula to North Italy and Southern Bavaria (Steiner and Schlick-Steiner 2000; Fig.1). Its distribution partially overlaps with that

Genetic divergence in the cave cricket Troglophilus neglectus(Orthoptera, Rhaphidophoridae): mitochondrial and nuclear DNA data

Valerio KETMAIER (1), Claudio DI RUSSO (2), Mauro RAMPINI (2) and Marina COBOLLI (2)

(1) Unit of Evolutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Lieb-knecht-Strasse 24-25, Haus 25, D-14476, Potsdam, Germany; email: ketmaier@uni-potsdam.de

(2) Dipartimento di Biologia Animale e dell’Uomo, Universita’ di Roma “La Sapienza”, V.le dell’Universita’ 32, I-00185, Rome, Italy

ABSTRACT

In this study we used sequence polymorphisms at one mitochondrial and one nuclear gene (Cytochrome Oxidase subunit I and Internal Transcribed Spacer 1, respectively) to assess the degree of genetic divergence among 21 populations of the cave cricket Troglophilus neglectus (Orthoptera, Rhaphidophoridae), a species whose currently known range extends from the Balkan Peninsula to Southern Bavaria. Nineteen populations were sampled in Northern Italy, Slovenia, Croatia and Bosnia and Herzegovina, while two populations came from Germany (Lower Saxony) and Czech Republic, thus well outside the species range. Molecular data revealed a high level of fragmentation, with most of the study populations bearing exclusive haplotypes, the sole exception being the German and Czech specimens, which carried haplotypes also occurring at Slovenian locations. Spatial distribution of genetic heterogeneity and pattern of genetic divergence argue in favor of a recent origin of the two Central European populations, possibly through man-mediated dispersal event(s). These populations being not considered, our data are in remarkable agreement with a previous study based on allozymes conducted on a subset of populations of the same species and, more generally, with what is known on the population genetics of peri-Mediterranean Rhaphidophorids.

Key words: Troglophilus neglectus, cave cricket, mitochondrial DNA, nuclear DNA, genetic divergence, dispersal.

26 V. Ketmaier, C. Di Russo, M. Rampini, M. Cobolli

aim, we compared samples from the German and Czech localities to those collected in 19 additional locations from Northern Italy, Slovenia, Croatia and Bosnia and Herzegovina. Under a natural dispersal hypothesis, we have to envision a northward expansion of the species, which followed the post-ice age amelioration of climate. This is a well-known phenomenon documented in many taxa but never demonstrated in terrestrial cave organisms (Taberlet et al 1998; Hewitt 1999). We expect to fi nd the Central European populations to be at least partially genetically divergent from the others as a consequence of a process of dispersal that could have not been rapid, given the low vagility of these crickets. Alternatively, we have to invoke a man-mediated dispersal to account for the occurrence of the species in Central Europe. If the latter hypothesis were true, we should be able to identify very little (if any) geographical component in the pattern of genetic variation between Central and Southern Euro-pean populations. Anthropocore dispersal, though not a very common phenomenon in cave organisms, has been described at least twice in Dolichopoda (Allegrucci et al 1982; Bernardini et al 1997) but never over a geographi-cal distance comparable to that considered in the present study. Finally, the sequence data collected here are also discussed in light of the allozyme results obtained by Ketmaier et al (2004) on a smaller subset of T. neglectus populations.

MATERIALS AND METHODS

SamplingTwenty-one populations (75 individuals) of T. ne-

glectus were sampled for the study; nineteen of these are from the known geographic range of the species (fi ve from Northern Italy, ten from Slovenia, three from Croa-tia and one from Bosnia and Herzegovina) while two have been sampled well outside it (Germany and Czech Republic). The species distribution and the geographical areas sampled for the study are shown in Fig. 1; Table 1 lists details of each sampling site. Animals were collect-ed by hand and stored in 80% ethanol; part of the caudal femoral muscle was used for molecular analyses.

Molecular markersTotal genomic DNA was extracted following Ket-

maier et al (2008). We amplifi ed by Polymerase Chain Reaction (PCR) a fragment of the mitochondrial region encoding for the Cytochrome Oxidase subunit I gene (COI) and a fragment of the nuclear Internal Transcribed Spacer 1 (ITS1). For COI we used the same set of prim-ers as in Ketmaier et al (2008) while for ITS1 we ad-opted the primer pair Cas5p8sB1d/ Cas18sF1 developed by Ji et al (2003). Double-stranded PCR amplifi cations were performed in a 50 μL reaction volume containing 10mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, each dNTP at 2.5 mM, each primer at 1 mM, genomic DNA (10-100 ng) and 5 units of Amplitaq (Applied Bio-

system). Each PCR cycle (for a total of 30) consisted of a denaturation step at 94°C for 1 min, annealing at 50°C (both genes) for 1 min and extension at 72 °C for 2 min; cycles were followed by a fi nal extension at 72 °C for 7 min. PCR products were purifi ed using the GenEluteTM PCR DNA Purifi cation kit from Sigma. Sequences were determined with an automated sequencer (Applied Bio-systems 373A) following the manufacturer’s protocols. To promote accuracy strands were sequenced in both directions for each individual using the same primers employed for PCRs.

Data analysisSequences were edited using Sequencher 4.6 (Gene

Code Corporation, Ann Arbor, MI). COI sequences were aligned by eye following the guide provided by the read-ing frame; for ITS1 we used ClustalX (Thompson et al 1997) with default parameters. The suitability of pool-ing sequence data from the two sequenced genes was assessed by the Incongruence Length Difference (ILD) test (Farris et al 1994) as implemented in PAUP* 4.0b10 (Swofford, 2002). The ILD test showed that the two re-gions are not phylogenetically incongruent (P = 0.760). We therefore present only analyses based on the two genes combined. We used PAUP* 4.0b10 to calculate base frequencies and to test for base frequency homoge-neity across taxa (c2 test).

Aligned sequences were analyzed phylogenetically by maximum parsimony (MP; heuristic searches, ACC-TRAN character-state optimization, 100 random step-wise additions, TBR branch-swapping algorithm) (Far-ris 1970), maximum likelihood (ML; heuristic searches,

Fig. 1- Gray shaded area indicates the presently known range of T. neglectus while the diagonally stripped areas are those sampled for the study. Black dots indicate the German and Czech localities (upper and lower, respectively). Details on each sampling locality are given in Table 1.

Genetic divergence in Troglophilus neglectus 27

100 random stepwise additions, TBR branch swapping algorithm) (Felsenstein 1981), Neighbor-Joining (NJ) (Saitou and Nei 1987) and Bayesian methods (Rannala and Yang 1996; Mau and Newton 1997; Larget and Si-mon 1999; Mau et al 1999; Huelsenbeck 2000). MP, ML and NJ analyses were performed using PAUP* 4.0b10 (Swofford 2002); Bayesian analysis was carried out us-ing MRBAYES 3.1 (Ronquist and Huelsenbeck 2003). MP searches were run giving equal weight to all sub-stitutions. We ran the ML analyses on PAUP* 4.0B10 after having determined the best model of DNA substitu-tions that fi t our data using MODELTEST (Posada and Crandall 1998). According to the results of this program,

we ran all our ML analyses using the GTR + I model (proportion of invariant sites = 0.782). NJ analyses were carried out on ML distances calculated with the same settings used for the ML analyses. For the Bayesian ap-proach, we employed the same model of sequence evo-lution as in the ML searches allowing site-specifi c rate variation partitioned by gene and, for COI, by codon positions. MRBAYES was run for 2 million genera-tions with a sampling frequency of 100 generations. We ran one cold and three heated Markov chains. From the 20000 trees found, we discarded the fi rst 10% (“burn-in”) in order to include only trees for which convergence of the Markov chain had been reached. The remaining

Table 1 - Sampling localities, sample sizes and population codes. Asterisks indicate populations screened for allozy-mic variation in Ketmaier et al (2004).

Locality Sampling size Code

Germany

Festung Königstein 5 Tn1

Czech Republic

Ceske’ Svycarsko, Hrensko 1 Tn2

Italy

Rovere’ 1000, Verona 5 Tn3*

Covoli di Velo, Verona 5 Tn4

Pasubio, Vicenza 5 Tn5

Priabona, Vicenza 5 Tn6

Sagrado, Gorizia 5 Tn7*

Slovenia

Vracka Zjalka, Dobravlje 5 Tn8*

Cerknika Jama, Ljubljana 5 Tn9

Jama Pod Cesto, Ljubljana 5 Tn10*

Jama Nad Kobilo, Idrija 2 Tn11

Rakov Skocjan, Rakek 5 Tn12

Podpeska Jama, Dobrepolje 1 Tn13

Kapiniska Jama, Novo Mesto 2 Tn14

Kozja Luknja, Sembije 5 Tn15

Jelsane 5 Tn16

Jama Zjot-Djud, Crnomelj 1 Tn17

Croatia

Skulja Sitnice, Vizinada 1 Tn18

Matesica pecina, Slunj 1 Tn19

Spilja u Bastu, Biokovo, Makarska 5 Tn20*

Bosnia and Herzegovina

Popovo polje, Zavala 1 Tn21

28 V. Ketmaier, C. Di Russo, M. Rampini, M. Cobolli

trees were used to construct a 50% majority rule con-sensus tree using PAUP* 4.0b10. The robustness of the phylogenetic hypotheses was tested by bootstrap repli-cates (1000 replicates for MP and NJ and 100 replicates for ML) (Felsenstein 1985). For the Bayesian analysis, the posterior probabilities were estimated only for those generations sampled after the burn-in. All phylogenetic searches were run with Troglophilus gajaci Us, 1974 (Cennet cave; Turkey) as the outgroup. Competing phy-logenetic hypotheses were evaluated statistically with the unbiased tree selection test (AU) as implemented in CONSEL (Shimodaira and Hasegawa 2001); tree topol-ogies were compared simultaneously (Shimodaira and Hasegawa 1999).

We used the statistical parsimony procedure imple-mented in the package TCS 1.13 (Clement et al 2000) to identify haplotypes and to derive the haplotype network. Finally, we used PAUP* 4.0B10 to calculate the absolute number of substitutions among haplotypes for each gene separately.

RESULTS

Sequence variationWe sequenced 865 base pair (bp) of DNA (457 bp for

COI and 408 bp for ITS1) for each individual included in the study. Sequences have been deposited to GenBank (Accession N. HM013857-HM013870). We observed a few gaps and only in the ITS1 alignment; these were mostly confi ned to outgroup vs. ingroup comparisons. Levels of sequence variability were higher in COI than in ITS1; there were 64 (14.01%) polymorphic sites in COI and 25 (6.13%) in ITS1; parsimony informative sites were 27 (5.9%) and 9 (2.20%) for the two genes. As expected, most of the variation resided in COI 3rd codon positions; 45 (29.6%) sites were variable and of these 22 (14.47%) were also parsimony informative. The com-bined data set had 89 (10.29%) and 36 (4.16%) variable and parsimony informative sites. COI sequences were generally A+T rich, a typical pattern for mitochondrial genomes; A+T percentages ranged from 48.4% in COI 1st codon positions to 86.5% in COI 3rd codon positions.

Table 2 reports details of sequence variation by gene, codon position (COI only) and on the combined data set. A c2 test of homogeneity of base frequencies among taxa did not reveal any signifi cant differences, irrespective of the data partition tested (both genes combined, each gene separately and COI 1st, 2nd, and 3rd codon positions separately).

Levels and patterns of genetic divergenceThe 150 sequences obtained for this study defi ned a

total of 15 unique haplotypes whose distribution and fre-quency in the 21 populations of T. neglectus are shown in Table 3. Thirteen haplotypes were unique to single populations while haplotypes H1 and H2 were shared among six and two populations, respectively. These two haplotypes co-occurred in the two populations sampled outside of species range (Tn1, Germany; Tn2, Czech Republic) and in additional fi ve Slovenian populations (Tn8, Tn14, Tn15, and Tn17 for H1 and Tn12 for H2). Divergence between haplotype pairs in terms of absolute number of substitutions ranged between 1 (multiple in-stances) and 59 (H3 vs. H10) (Table 4). Haplotypes H9 and H10 found in the Bosnian (Tn21) and in the south-ernmost Croatian (Tn20) population were by far the most divergent, being 34 ± 0.64 / 39.15 ± 0.59 (COI) and 13.35 ± 0.16 / 15.21 ± 0.23 (ITS1) substitutions away from the others; they were separated from each other by 28 and 13 mutational changes (COI and ITS1, re-spectively). The same pattern of differentiation emerges from the analysis of the pairwise ML distances based on the GTR + I model of sequence evolution selected by MODELTEST (Table 4).

Fig. 2 shows the haplotype Bayesian tree obtained for the combined data set using the GTR + I model (propor-tion of invariant sites = 0.782) and summarizes the results of the MP, ML, NJ and Bayesian searches in terms of sta-tistical support. Trees yielded by the different phyloge-netic methods were statistically indistinguishable with the AU test (P ≥ 0.781). Trees were poorly resolved; the two most divergent haplotypes (H9 and H10) are placed on a clearly separate branch as each other’s closest relative with maximum support. Two additional nodes received moderate support but never for the four phylogenetic

Table 2 - Percentage of variable and parsimony informative sites by gene and codon positions across all the popula-tions included in the study; A+T percentages are also shown.

Gene and codon positions % variable % informative % A+T N° of sites

COI-First 6.53 1.96 48.40 153

COI-Second 5.92 1.31 56.70 152

COI-Third 29.6 14.47 86.50 152

COI-All 14.01 5.90 63.80 457

ITS1 6.13 2.20 43.80 408

COI + ITS1 10.29 4.16 54.60 865

Genetic divergence in Troglophilus neglectus 29

methods simultaneously. The remaining haplotypes, in-cluding those two found in the German and Czech popu-lations (H1 and H2), are dispersed in a large unresolved group; no particular geographical pattern is evident here. Relationships among haplotypes are also depicted in the network of Fig. 3. Haplotypes H9 and H10 could not be connected to the network according to the 95% statisti-cal parsimony criterion. Haplotype H1 lies at the centre of the network surrounded by less frequent haplotypes, mostly of Slovenian and Croatian origin. Haplotypes oc-curring in the Italian populations are spread across the network; from two to fi ve missing haplotypes are neces-sary to connect most of the Italian haplotypes (H3, H4, H8, and H14) to the rest of the network.

DISCUSSION

Populations of cave species are usually genetically disconnected from one another, owing to their tight de-pendence on the habitat of election (Sbordoni et al 2000).

As a rule of thumb, the stricter the association of a given species to the subterranean environment the higher the chances for its populations to evolve as separate units. Detailed genetic studies have integrated this general sce-nario showing how continuity of the limestone system is a very important factor (Allegrucci et al 1997; Sbordoni et al 2000; Ketmaier et al 2004 and references therein). For those species capable to attempt surface dispersal, bioclimatic conditions outside caves also matter. This would be of particular relevance for rhaphidophorids, since cave crickets belonging to the family are potential-ly able to leave caves at night to forage. If outside condi-tions are favorable (in terms of presence of cover of me-sophilous woods), crickets could theoretically disperse from one cave to another and eventually determine fl ow of genes. However, only few of the very many genetic studies centered on these organisms could unequivocally detect ongoing gene fl ow, while data generally indicate that gene fl ow broke off sometime in the past (i.e. his-torical gene fl ow; Allegrucci et al 1997; Ketmaier et al 2004 and literature reviewed therein).

Table 3 - Haplotype frequencies (COI + ITS1) of all individuals of T. neglectus sequenced for the study. For popula-tion codes see Table 1.

Haplotypes

Pops. H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15

Tn1 4 1

Tn2 1

Tn3 5

Tn4 5

Tn5 5

Tn6 5

Tn7 5

Tn8 5

Tn9 5

Tn10 5

Tn11 2

Tn12 5

Tn13 1

Tn14 2

Tn15 5

Tn16 5

Tn17 1

Tn18 1

Tn19 1

Tn20 5

Tn21 1

30 V. Ketmaier, C. Di Russo, M. Rampini, M. Cobolli

If we follow the above generalizations, it is diffi cult to hypothesize the occurrence of T. neglectus in Germa-ny and Czech Republic be due to a natural phenomenon, since this would imply a northward expansion of the species. As a matter of fact, most of the current Central and Northern European fl ora and fauna re-colonized the area from Southern European refuges after the ameliora-tion of climate at the end of the Quaternary. This process has been extensively documented in a variety of species on molecular grounds; Taberlet et al (1998) and Hewitt (1999) reviewed these data and categorized patterns of northward expansion. The speed of expansion is large-ly dependant on the potential for dispersal intrinsic to each species and it can vary from slow to rapid (Hewitt 1999), with divergence between southern and northern populations/clades being in inverse proportion to it. To our knowledge, no additional populations of T. neglec-tus from areas comprised between the species range (as intended in Fig.1) and the German and Czech localities have been found so far. Thus, no potential stepping-stone populations between these geographically remote areas exist. Next, most of the land north of the Alps is fl at, crossed by large rivers and with karstic areas occurring in rare and localized patches. All this implies that we need to invoke long-distance dispersal over ecologically unsuitable areas and crossing of powerful geographical barriers if we want to explain the disjunct occurrence of T. neglectus in Germany and Czech Republic on natu-ral bases. The pattern of genetic variation found at the two loci sequenced for this study is not very much in line with a northward expansion hypothesis either. If it were true and assuming that dispersal was not very rapid given the ecological characteristics of the species, we

Table 4 - Below the diagonal: pairwise absolute number of substitutions (COI fi rst value, ITS1 second value) among the 15 haplotypes found in the 21 populations of T. neglectus included in the study. Above the diagonal the pairwise ML distances among haplotypes (based on the GTR +I model as selected by MODELTEST) are reported.

HaplotypesH1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15

H1 - 0.001 0.008 0.003 0.002 0.001 0.001 0.008 0.075 0.086 0.004 0.013 0.004 0.006 0.002

H2 1/0 - 0.007 0.004 0.003 0.002 0.002 0.007 0.073 0.084 0.006 0.012 0.003 0.007 0.001

H3 6/1 5/1 - 0.009 0.011 0.009 0.009 0.015 0.084 0.098 0.013 0.020 0.011 0.014 0.008

H4 2/1 3/1 8/0 - 0.006 0.004 0.004 0.012 0.081 0.091 0.008 0.017 0.008 0.009 0.006

H5 2/0 3/0 8/1 4/1 - 0.003 0.003 0.011 0.074 0.091 0.004 0.013 0.004 0.008 0.004

H6 1/0 2/0 7/1 3/1 3/0 - 0.002 0.009 0.077 0.088 0.006 0.015 0.006 0.007 0.003

H7 1/0 2/0 7/1 3/1 3/0 2/0 - 0.007 0.072 0.084 0.006 0.015 0.006 0.007 0.003

H8 7/0 6/0 11/1 9/1 9/0 8/0 6/0 - 0.068 0.075 0.011 0.015 0.008 0.015 0.008

H9 34/13 33/13 37/14 36/14 34/13 35/13 33/13 31/13 - 0.058 0.080 0.076 0.076 0.085 0.075

H10 39/15 38/15 43/16 40/16 41/15 40/15 38/15 34/15 28/13 - 0.091 0.083 0.086 0.094 0.086

H11 4/0 5/0 10/1 6/1 4/0 5/0 5/0 9/0 3/136 41/15 - 0.013 0.002 0.011 0.007

H12 9/2 8/2 13/3 11/3 9/2 10/2 10/2 10/2 35/13 38/15/ 9/2 - 0.011 0.020 0.013

H13 4/0 3/0 8/1 6/1 4/0 5/0 5/0 7/0 34/13 39/15 2/0 7/2 - 0.011 0.004

H14 3/2 4/2 9/3 5/3 5/2 4/2 4/2 10/2 37/15 40/17 7/2 12/4 7/2 - 0.006

H15 1/1 0/1 5/2 3/2 3/1 2/1 2/1 6/1 33/14 38/16 5/1 8/3 3/1 4/1 -

Germany

Czech Republic

Italy

Slovenia

Croatia

Bosnia and Herzegovina

Fig. 2- Bayesian haplotype phylogram based on the combined data set and the GTR + I model (proportion of invariant sites = 0.782) as selected by MODELTEST. Numbers at nodes are the statistical supports for MP, ML, NJ and Bayesian searches; only nodes with a statistical support ≥ 75% are labeled. Hap-lotype codes match those in Table 3; the geographic origin of haplotypes is also shown.

Genetic divergence in Troglophilus neglectus 31

Fig. 3- Haplotype networks derived from 865 bp of DNA (COI and ITS1). The relative size of the circles is proportional to the number of individuals carrying that particular haplotype; shadings identify the geographical origin of haplotypes as indicated in the bottom right corner. Haplotype codes are as in Table 3; numbers indicate how many individuals carried that particular haplotype. Black dots are missing haplotypes.

should have found a certain degree of genetic divergence between the German/ Czech populations and the others. This divergence should have been at least as large as that separating geographically nearby populations. However, German and Czech populations are more similar- and in some instances identical- to distant populations (i.e. Slovenian) than some of the populations sampled over restricted geographical areas are to one another (Table 4). The latter observation is more evident when we con-sider the Italian populations, which, although all located a relatively short distance away, are genetically quite di-vergent (see Fig. 3). With the exclusion of German and Czech populations, which are also the only two sharing haplotypes between them as well as with others, the pic-ture that emerges is one of a profound genetic subdi-vision, in line with what already known for peri-Medi-terranean raphidophorids (Ketmaier et al 2000; 2004; Sbordoni et al 1981; 1985; 1991; 2000). On the other hand, as already reported by Maran (1958), geographi-cally distant populations of T. neglectus show strong variability in taxonomically important characters such as the male X tergite.

All these considerations being done, we tentatively conclude that the occurrence of T. neglectus at the Ger-man and Czech locations is the consequence of a very recent dispersal, most probably related to human activi-ties. Our study case thus resembles that reported by Ber-nardini et al (1997) for one population of Dolichopoda laetitiae. This species belongs to the same family as Tro-

glophilus and occurs naturally in Central Italian caves south of the Po River. In 1991 a single isolated popula-tion was found in Northern Italy, in a cave system situ-ated well outside the geographic range of the species. By using a multidisciplinary approach, Bernardini et al (1997) concluded that most probably a recent anthropo-core dispersal originated gave origin to this population. We are well aware that neither the number of popula-tions included in our study nor the sample sizes are large enough to derive definitive conclusions on how and from where our German and Czech T. neglectus popula-tions originated. Nonetheless, we believe that a scenario similar to that hypothesized by Bernardini et al (1997) is still the most parsimonious. It is also worth remem-bering that the German locality (Festung Königstein) was used as a Nazi camp for Allied offi cers and as a deposit of war material during World War II. At that time goods important for the conduct of the war (i.e. iron but also fruits, vegetables and textiles) were moved in large quantity from North East Italy (one of the richest and most productive area of the country) to Germany. We speculate that unintentional transplantation of individu-als might have occurred at that time; a similar mech-anism has been invoked by Allegrucci et al (1982) to explain the spread of another Dolichopoda species (D. schiavazzii) in Tuscany.

As shown in Table 1, fi ve populations included in this study had been also screened for allozyme polymor-phisms (Ketmaier et al 2004). These nuclear markers

Germany

Czech Republic

Italy

Slovenia

Croatia

Bosnia and Herzegovina

32 V. Ketmaier, C. Di Russo, M. Rampini, M. Cobolli

identifi ed two groups of populations one from Croa-tia and one from North Italy/Slovenia, which diverged from each other by a genetic distance of DNei’78 = 0.121 ± 0.038, a value almost twice as large as the average intra-specifi c divergence (0.086 ± 0.053). Even thought the two data sets are not completely comparable be-cause of the different number of populations included in each of them (six vs. 21), it is worth noting that in either case the southernmost populations (Croatian and Bosnian-Herzegovinian) are also the most divergent. In-terestingly, a striking similar pattern of differentiation is also evident in the congener T. cavicola (Ketmaier et al 2004). We tentatively conclude that geographic dis-tance among caves is the major force promoting diver-gence. At the same time, further studies based on a much denser sampling (i.e. in areas comprised between those considered for the present study; see Fig.1) are needed to test whether the deep divergence between Northern Italian/Slovenian and Croatian/Bosnian-Herzegovinian populations might rather be part of a more clinal pattern of variation across the species range.

ACKNOWLEDGMENTS

This study would not have been possible without the help of people who generously donated us samples; we are therefore extremely grateful to I. Brunk, M. Tryzna, B. Sket and his working group. We also wish to thank M. Beckmann for technical assistance in the lab. This study was fi nancially supported by a PRIN grant (the Italian Program for Relevant National Researches) to MC.

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