Journal of Biogeography (J. Biogeogr.) (2013) 40, 1861–1873
ORIGINAL ARTICLE
Early Pliocene range expansion of a clade of subterranean Pyrenean beetles Valeria Rizzo1, Jordi Comas2, Floren Fadrique2, Javier Fresneda2,3 and Ignacio Ribera1*
1
Animal Biodiversity and Evolution, Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain, 2Museu de Ciencies Naturals (Zoologia), Barcelona, Spain, 3Ca de Massa, E-25526 Llesp-El Pont de Suert, Lleida, Spain
ABSTRACT
Aim To investigate the possibility of range expansion and diversification within the subterranean environment in a genus of troglobiont beetles of the family Leiodidae (Troglocharinus), which have a disjunct distribution between the Pyrenees and the Catalonian coast. Location North-eastern Iberian Peninsula. Methods We sequenced 4 kb of five mitochondrial and two nuclear genes of 50 specimens of 12 of the 18 species of Troglocharinus, plus several outgroups. We reconstructed a phylogeny using Bayesian inference and maximum likelihood, estimated divergence times using Bayesian probabilities and an a priori evolutionary rate, compared the diversification of the main clades within the genus, and reconstructed their ancestral distribution using maximum likelihood. Results We found strong support for the monophyly of Troglocharinus and the clades in each of the geographical areas, which diverged in the early Pliocene. The coastal clade was further divided into geographically well-defined lineages, separated by Quaternary deposits. The origin of the coastal clade was a single colonization in the early Pliocene from the central Pyrenees. The diversification of the Pyrenean clade followed a constant rate, while the diversification rate of the coastal clade significantly decreased through the Plio-Pleistocene transition. Main conclusions Troglocharinus expanded its range from its ancestral area in the central Pyrenees to the coast of Catalonia and subsequently diversified, probably within the subterranean environment. Our favoured scenario is a stepping-stone migration, with possibly short-distance dispersals through the surface, along the eastern margin of the north-eastern Ebro basin. The range expansion took place in a narrow temporal window with favourable conditions between the early Pliocene and the onset of the Mediterranean climate by the mid-Pliocene. Surface dispersal was probably severely limited afterwards, as shown by the fragmentation of the coastal lineage.
*Correspondence: Ignacio Ribera, Institute of Evolutionary Biology (IBE), Passeig Maritim de la Barceloneta, 37–49, 08003, Barcelona, Spain. E-mail:
[email protected]
Keywords Dispersal, diversification, Leptodirini, Pyrenees, subterranean environment, troglobites, Troglocharinus.
INTRODUCTION The fauna of caves has merited the continuous attention of biologists since the description of the first troglobites in the early 19th century (for example see Romero, 2009, and Culver & Pipan, 2009, for historical reviews). The subterranean ª 2013 John Wiley & Sons Ltd
environment seems well suited for evolutionary and biogeographical studies, as it has extreme but very homogeneous conditions, facilitating the identification of morphological and physiological adaptations of the organisms living in it. Species adapted to this environment usually have very poor dispersal abilities and are confined to well-defined geological http://wileyonlinelibrary.com/journal/jbi doi:10.1111/jbi.12139
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V. Rizzo et al. or geographical units and the general stability and homogeneity of the habitat has permitted the long-term persistence of some lineages (Culver & Pipan, 2009; Juan et al., 2010). Some groups are also taxonomically very diverse, allowing the study of multiple replicates of the same evolutionary processes. Despite all these advantages, the study of the subterranean fauna has lagged behind that of other systems that could be considered similar, such as islands, probably because of the difficulties in access and the taxonomy of many of the groups (Culver, 1970; Barr & Holsinger, 1985). Recently, the application of molecular methods has allowed for the first time tests of some of the traditional hypotheses on the origin and distribution of the subterranean fauna (Juan et al., 2010). There are, however, many questions that remain open, especially regarding the possibility of geographical expansion and diversification of species adapted to the subterranean environment (Culver et al., 2009; Juan et al., 2010; Ribera et al., 2010). The traditional view on subterranean terrestrial species is that they are evolutionary dead ends, usually of recent (mostly Pleistocene) origin, that rarely disperse or diversify once fully adapted to life in caves or soil (Vandel, 1964; Barr & Holsinger, 1985). The existence in caves of ancient relict species (‘living fossils’) has also been recognized for a long time (e.g. Assmann et al., 2010; Bauza-Ribot et al., 2012), but these species are viewed as isolated ancient survivors of otherwise extinct lineages, not as the origin of extended radiations. The geographical range of subterranean species is usually very restricted, especially for terrestrial organisms, and traditionally it was assumed that, even for closely related taxa, subterranean species originated multiple times independently from epigean ancestors not showing the morphological characters usually associated with life in the subterranean environment (Jeannel, 1943; Vandel, 1964; Belles, 1987). This multiple origin was postulated irrespective of their mode of speciation, i.e. whether they speciated in geographical isolation by extinction of their surface ancestors (‘climate relict’ hypothesis), or with partial gene flow with their surface ancestors during a limited time (‘habitat shift’ hypothesis), the two commonly accepted alternatives for the origin of subterranean species (Howarth, 1973; Desutter-Grandcolas & Grandcolas, 1996; Juan et al., 2010). The possibility that subterranean terrestrial species could expand their ranges and diversify once fully adapted to the subterranean environment is rarely contemplated, and usually only for closely related species at a reduced geographical scale (e.g. Barr, 1968; Crouau-Roy, 1989; Holsinger, 2000; Culver et al., 2009). Only recently have large-scale diversifications within the subterranean environment been advocated for ancient (i.e. pre-Pleistocene) groups, on the basis of the monophyly of lineages of Miocene origin including only subterranean species and occupying a well-defined geographical area (Faille et al., 2010; Ribera et al., 2010). Although well grounded in phylogenetic results, these hypotheses face the difficulty of explaining how species that are generally assumed to have reduced dispersal abilities (they are all blind 1862
and apterous), and a limited tolerance to high temperature and low humidity, could disperse to areas far from the karstic system in which they originated. Particularly puzzling are the cases in which apparently closely related taxa have disjunct distributions separated by extensive areas not suitable for subterranean species, such as compact, sedimentary soils older than their assumed separation. Prominent among these is the beetle genus Troglocharinus, part of the Pyrenean lineage of subterranean Leptodirini (the Speonomus group; Ribera et al., 2010). The genus Troglocharinus as currently understood is distributed in two disjunct areas (Fig. 1): some mountain systems south of the central area of the Pyrenees, and the coastal karstic formations between Tarragona and Barcelona (Salgado et al., 2008). The shortest distance between the two is c. 60–70 km, and they are separated by extensive sedimentary basins of marine or continental origin which were deposited during the Palaeogene (Fig. 1; Vera, 2004) and are unsuitable for the formation of a deep subterranean environment. Disjunct distributions like that of the genus Troglocharinus are very rare among the strictly subterranean genera, which tend to occupy a well-defined and restricted geographical region, usually within a continuous subterranean environment (Holsinger, 2005). Many genera of subterranean Pyrenean Coleoptera previously considered to have disjunct distributions have recently been shown to be polyphyletic, with their closest relatives among taxa in the same geographical area (Faille et al., 2010; Ribera et al., 2010). Troglocharinus currently includes 18 species and 19 subspecies (see Appendix S1a in Supporting Information). They are all considered to be highly adapted to the subterranean environment, living exclusively in the deepest part of caves in total darkness and very constant temperature and humidity (Salgado et al., 2008). The current concept of the genus was only recently established by Fresneda (1998) based on the structure of the male aedeagus, and includes species previously considered to belong to different genera (Troglocharinus, Speophilus, Antrocharidius and Speonomus; Appendix S1a). In Ribera et al. (2010), only one Pyrenean species of the genus was studied (Troglocharinus fonti), which was found to be deeply nested within a clade including other subterranean genera from the Pyrenees. According to the calibration in Ribera et al. (2010), based on the tectonic separation of the Corso-Sardinian plate, the origin of the Pyrenean group of Leptodirini (the Speonomus group) would be in the early Oligocene, c. 34 Ma. All known species in this group show the typical adaptations to a subterranean environment, i.e. they are anophthalmous, apterous and depigmented, although some can be found in relatively superficial environments (e.g. some species of Bathysciola in deep layers of forest litter or under deeply buried stones; Salgado et al., 2008). Within this group, the clade to which Troglocharinus belongs was estimated to date from the early Miocene (c. 21 Ma, node 8 in Ribera et al., 2010), and includes only strictly troglobiont species (sensu Sket, 2008) from the central and western Pyrenees, either in caves or in the mesovoid shallow Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
Pliocene range expansion of subterranean beetles
Figure 1 Distribution of the sampled localities of the genus Troglocharinus, overlaid on a tectonic map of north-eastern Spain (modified from Rodrıguez et al., 2004), with the geographical areas used in Lagrange (see Fig. 3 and Table 2). Sampled localities: triangles, Pyrenean area (P); squares, Garraf massif (F); diamonds, area north of the Anoia river (K); black circles, area between the rivers Anoia, Foix and Francolı (Penedes depression, E); asterisks, area south of the Francolı river (subgenus Antrocharidius, area A); diamond with white circle, Cova del Pas, in which specimens VR7 and VR8, belonging to the clade of T. elongatus, and VR18, belonging to the clade of T. kiesenwetteri coexist (see Figs 2 & 3 and Appendix S1b). Yellow areas, Tertiary and Quaternary sedimentary basins; green areas, folded Mesozoic cover; light grey areas, filling of Miocene and Quaternary fractures; pink and purple areas, Hercynian and Palaeozoic substratum. Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
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V. Rizzo et al. substratum (MSS; the superficial subterranean environment). There are no species of the Speonomus group outside the Pyrenees other than the coastal species of Troglocharinus (Salgado et al., 2008; Ribera et al., 2010). On the other hand, the coastal species of Troglocharinus do not overlap with the range of any species of Leptodirini other than Bathysciola zariquieyi Bolıvar, 1919, which is found in forest litter or under stones and belongs to a different lineage. In this study we aim to: (1) investigate the phylogenetic and geographical origin of the coastal species of Troglocharinus and the possibility of a range expansion; (2) estimate a time framework for the divergence of the main lineages within the genus; and (3) explore potential scenarios for the range expansion. MATERIALS AND METHODS
We obtained sequences of five mitochondrial and two nuclear genes: 3′ end of the cytochrome c oxidase subunit I (COI); 5′ end of the large ribosomal unit plus the leucine transfer RNA plus the 3′ end of NADH dehydrogenase subunit 1 (rrnL + trnL + nad1); an internal fragment of cytochrome b (cob); the 5′ end of the small ribosomal unit, 18S rRNA (SSU); and an internal fragment of the large ribosomal unit, 28S rRNA (LSU) (see Appendix S2 for PCR conditions and primers). Owing to the generally low levels of variability, only a representative sample of nine taxa of Troglocharinus was sequenced for the SSU and LSU genes. The protein-coding sequences had no internal stop codons, and preliminary searches showed no sign of possible contaminations or sequencing errors. New sequences (a total of 190) were deposited in the EMBL database under accession numbers HF912453–HF912642 (Appendix S1b).
Taxon sampling
Phylogenetic analyses
We studied 50 specimens of 12 species and 8 subspecies of Troglocharinus, including representatives of both geographical areas (Appendix S1b). For some taxa, we included examples of different caves to test the monophyly of the recognized species and the geographical variation. We used two data sets for the phylogenetic analyses. 1. An extended data set, to test whether coastal species of Troglocharinus are more related to other Mediterranean lineages of Leptodirini (or even to other European lineages within the subfamily Cholevinae) than to the Pyrenean lineage. We added to the data set of Fresneda et al. (2011) a representative sample of the genus (five species of the Pyrenean and coastal areas each), chosen among the specimens with the most complete sequence data (Appendix S1b). 2. A reduced data set, to study in detail the phylogenetic relationships, the temporal diversification and the geographical origin of the species of Troglocharinus. We selected this data set according to the results of the previous analysis, adding all genera shown to be closely related to Troglocharinus included in Ribera et al. (2010) plus three additional genera and three species newly sequenced for this work (Appendix S1b). All genera of Leptodirini known from the Pyrenees were thus represented, many of them by more than one species.
To align the gene fragments, we used multiple progressive pairwise alignment with secondary refinement using mafft online 6 (http://mafft.cbrc.jp/alignment/server/) with the default settings. The protein-coding genes had no insertions/ deletions and were aligned using a fast FFT-NS-1 algorithm. The ribosomal genes were aligned with the Q-INS-i algorithm, which considers the secondary structure (Katoh & Toh, 2008). Bayesian analyses of the reduced data set were conducted on a combined data matrix with MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001), using two partition schemes: (1) six partitions corresponding to the sequenced genes (the fragment rrnL + trnL was treated as a single partition), referred to as ‘partition by genes’; and (2) a partition by codon position for the protein-coding genes (COI, cob and nad1), a ribosomal mitochondrial partition (rrnL + trnL) and a ribosomal nuclear partition (LSU + SSU), referred to as ‘partition by codon’. The evolutionary model of each partition was estimated prior to the analysis with jModelTest 3.7 (Posada, 2008) using the Akaike information criterion (AIC). The burn-in fraction was established as the number of generations for which the standard deviation of the split frequencies between the two simultaneous runs reached values < 0.01. Trees were saved each 1000 generations, and MrBayes was left running after reaching convergence (the burn-in value) until there were enough trees to have reliable estimates of the parameters, as measured with the effective sample size in Tracer 1.5 (Drummond & Rambaut, 2007). For the two data sets, we conducted maximum-likelihood (ML) searches in the parallel version of RAxML 7.0.4 (Stamatakis et al., 2008), with an estimated GTR+I+G model and the same partition by genes as was used in MrBayes. We chose the best of 100 replicates as our preferred topology. Node support was measured with 1000 fast bootstrap replicates using the CAT approximation (Stamatakis et al., 2008). For the reduced data set, the same RAxML analyses were repeated for the mitochondrial and nuclear sequences sepa-
DNA extraction, amplification and sequencing Specimens were collected in caves with the use of baits (mostly old cheese that was spread within the cave), baited pitfall traps with propylene glycol as the preservative, or through manual searches. DNA extractions of single specimens were nondestructive, using either a phenol–chloroform method (Sambrook et al., 1989) or commercial kits (mostly DNeasy Tissue Kit, Qiagen, Hilden, Germany) following the manufacturer’s instructions. Vouchers and DNA samples are kept in the Museo Nacional de Ciencias Naturales, Madrid (MNCN) and the Institute of Evolutionary Biology, Barcelona (IBE). 1864
Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
Pliocene range expansion of subterranean beetles rately, partitioning by gene, to compare the resolution and topology obtained with each. For the analyses of the nuclear sequences, we excluded specimens with only the SSU gene, due to the low variation within the ingroup (Appendix S1b). For the extended data set, trees were rooted with the species of Staphylinidae. For the reduced data set, we rooted the trees according to the topology found in the analyses of the extended data set and in Ribera et al. (2010). Estimation of ages of divergence To obtain an ultrametric calibrated tree of the reduced data set, we used Bayesian inference as implemented in beast 1.7 (Drummond et al., 2012). We used an uncorrelated lognormal relaxed molecular clock, which allows variation in substitution rates among branches. We enforced a GTR+I+G evolutionary model and used only the combined mitochondrial sequences, as the rate we used was estimated for mitochondrial genes (see below). We constrained the respective monophyly of the outgroup and ingroup, and used a Yule speciation model sampling each 1000 generations. We ran two analyses for 60 9 106 generations, with 10 9 106 used as burn-in. We used Tracer 1.5 to determine convergence and measure the effective sample size of each parameter. Priors and other parameters were left with default values, with the exception of the prior of the evolutionary rate. Because of the absence of fossil record to calibrate the trees, we used an a priori rate of 0.01 substitutions site 1 Myr 1 (standard deviation 0.002), estimated in Ribera et al. (2010) for the same combination of mitochondrial genes and group of beetles and using the tectonic separation of the Corso-Sardinian plate as a calibration point. Consensus trees were compiled with TreeAnnotator 1.5.4 (Drummond & Rambaut, 2007) to estimate the median of the posterior distribution of node ages. Diversification analyses We estimated the rate of diversification using the log-lineage through time approach (LTT) (Harvey et al., 1994), using the ultrametric tree obtained in beast after pruning the outgroups and the duplicated specimens of the same taxa. We used the c-statistic (Pybus & Harvey, 2000) for measuring the relative timing of the diversification, i.e. whether there is a constant diversification through the tree, or if the interior nodes are closer to the tips or to the root than expected under a pure birth process (i.e. without extinction). The c-values of complete reconstructed phylogenies follow a standard normal distribution. If c < 0, the internal nodes can be said to be closer to the root than expected under a pure birth process, and vice versa (Pybus & Harvey, 2000). To test the significance of the c-statistic we generated a null distribution of 10,000 random simulations using a pure birth process including the known missing taxa in a random position in the tree, and tested the observed c against it (Pybus & Harvey, 2000). Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
We tested the adequacy of our data to different diversification models with likelihood methods. The models tested were a pure birth (Yule), a birth–death with constant diversification rate, two models with variable diversification rates (logarithmic and exponential), and a pure birth model with a shift in the diversification rate. We checked the significance of the results by comparing the observed AIC value for constancy of diversification rates against a null distribution of the statistic (see Rabosky, 2006). The test for the significance of the c-statistic described in Pybus & Harvey (2000) assumes that missing species are randomly distributed in the tree, which is generally not the case (H€ ohna et al., 2011). We therefore constructed a composite tree adding missing species in the most likely position according to morphological similarity of the male genitalia and their geographical distribution, known to be of phylogenetic relevance in this group of cave beetles (e.g. Fresneda et al., 2007; Salgado et al., 2012). As their position could not always be unambiguously established, we created polytomies and generated a set of 1000 randomly resolved trees in Mesquite 2.74 (Maddison & Maddison, 2010), with nodes placed in the middle of the connecting branch. The c-statistic was computed for the whole set of 1000 trees, as well as the significance of the maximum and minimum values. All diversification tests were performed using the R libraries ape (Paradis et al., 2004) and laser (Rabosky, 2006). Ancestral area reconstruction We estimated the ancestral areas of distribution with Lagrange, a model-based ML inference method that considers branch lengths and an a priori matrix of dispersal probabilities between the recognized areas (Ree & Smith, 2008). We established the areas of distribution according to the results of the phylogenetic analyses, and used the ultrametric tree obtained with beast. We performed a sensitivity analysis with alternative scenarios for the probability of dispersion between zones to test for the robustness of the results: (1) all pairwise dispersions between areas had equal probability, and equal to 1; (2) the probability of dispersal between areas that had another in between was 0; (3–5) the probability of dispersal between non-contiguous areas was set between 0 and 1, proportionally to the minimum distance between them, in three different combinations. RESULTS Phylogeny The analysis of the extended data set recovered all species of Troglocharinus as monophyletic and within the central Pyrenean clade of Leptodirini, with very strong support (Appendix S3). The internal topology of the genus was, however, not well resolved, with a polytomy formed by: (1) the Pyrenean species; (2) T. ferreri; and (3) a clade comprising all the coastal species except T. ferreri. 1865
V. Rizzo et al. For the reduced data set, we included all taxa in the least inclusive clade with strong support (bootstrap > 90%), using two genera outside this clade as outgroups (Appendix S3), and adding the newly sequenced taxa (see Materials and Methods). The molecular data matrix of the reduced data set included 68 taxa and 3608 aligned characters. The models implemented in MrBayes according to the results of jModelTest were GTR+G+I for the mitochondrial fragments, HKY+I for the SSU gene, and GTR+I for the LSU gene. In the partition by codon, we implemented GTR+G+I for each codon position, GTR+G for the rrnL + trnL fragment (ribosomal mitochondrial), and GTR+I for the SSU + LSU fragment (ribosomal nuclear). For the partition by gene, the runs of MrBayes reached a standard deviation of the split frequencies lower than 0.01 at 15 9 106 generations, which was considered the burn-in fraction of a total of 19 9 106 generations. For the partition by codon, the burnin to reach the same level of convergence was established at 17 9 106 out of 27 9 106 generations. The topologies of the trees obtained with the two partition schemes of the Bayesian probabilities and that obtained in RAxML were almost identical, only differing in the position within the same clade of two specimens of T. ferreri (IBEAF172 and IBE-VR34) and one of T. elongatus mateui (IBEVR5) between the RAxML and the MrBayes runs. The two partitions of MrBayes resulted in identical topologies, although the partition by codon had in general lower node support (Fig. 2). The analysis of the nuclear sequences with RAxML included 27 specimens (Appendix S1b) and showed a general lack of support for most nodes except for closely related species or species groups. The topology was largely congruent with that obtained with the mitochondrial sequences and the combined tree, except for the internal topology of the genus Troglocharinus. The species T. senenti, at the extreme south-west of the Pyrenean group, was recovered as sister to the rest of the Pyrenean plus the coastal species, with strong support (BT 96%, Appendix S3). This support was provided by the gene LSU only. The analysis of the mitochondrial sequences resulted in a tree with identical topology and similar node support values to those of the combined tree (Appendix S3). All the results strongly support the monophyly of the genus Troglocharinus, which was sister to a clade formed by part of the central Pyrenean genera (Fig. 2). In the analyses of the combined and mitochondrial data, the species in the two geographical areas of distribution of Troglocharinus were respectively monophyletic, with strong support for the Pyrenean clade, but only moderate support for the coastal clade (Fig. 2). Within each of the two lineages of Troglocharinus, there were well-supported clades, with deep divergences within some of the species and widespread paraphyly. In some cases, these clades could be matched with named subspecies (e.g. within the species T. kiesenwetteri), but in others there was no clear correspondence between the current taxonomy of the group and the phylogenetic lineages (e.g. T. elongatus, T. fonti). 1866
Within the coastal group, the main clades are distributed across different mountain massifs separated by rivers on Quaternary sediments, considered as distributional areas in the Lagrange analyses (Fig. 1). The first split separated off the species T. ferreri, distributed in the Garraf massif close to Barcelona and isolated by the Llobregat river in the northeast, the Anoia river in the north, and the Foix river in the south-west (area F, Fig. 1). The clade of T. kiesenwetteri and T. patracoi included all species living north of the Llobregat and Anoia rivers, with the inclusion of the massif of Montserrat (area K, Fig. 1). Two specimens (vouchers VR7 and VR8) collected in the same cave as some specimens of T. kiesenwetteri, and at the edge of their distributional area (Fig. 1), were placed within the T. elongatus clade, with which they were more similar in external morphology. The clade including the T. elongatus complex, T. orcinus and allies is distributed along a continuous karst of Permian–Triassic origin south-west of the Anoia and Foix rivers (area E, Fig. 1). The only species of the subgenus Antrocharidius, T. (A.) orcinus, was nested deep within this clade (Fig. 2), occupying the extreme south-west area, and separated from the rest by the Francolı river (area A, Fig. 1). The Pyrenean clade (area P, Fig. 1) was distributed across a geologically more continuous area, with Alpine deformation of a Cretaceous substratum without major discontinuities produced by Quaternary sedimentary basins (Fig. 1). This is reflected in the lack of a clear geographical distribution of the clades, although the higher number of missing species in this group could obscure some patterns. Diversification The estimated age of the stem lineage of Troglocharinus was 10 Ma, very similar to that estimated in Ribera et al. (2010), with a wide confidence interval (Fig. 3). There was an interval of c. 6 Myr between the origin of the genus and the last common ancestor of the two clades, which was estimated to have occurred c. 4 Ma, at the beginning of the Pliocene. The three main clades within the coastal lineage diversified at the end of the Pliocene (the T. ferreri complex in the Garraf massif) or beginning of the Pleistocene (the clades separated by the Anoia river), in agreement with the estimated dates for the formation of the sedimentary basins (Losantos et al., 2002). Most of the current taxa originated during the Pleistocene, both in the coastal area and the Pyrenean area. For the analyses of diversification, we used the whole genus and the two clades corresponding to the two geographical areas, to test for possible differences in diversification patterns between them. In the analyses of the whole genus with missing species randomly distributed through the tree, the c-statistic was negative but not significantly different from zero (Table 1). The data best fitted a pure birth diversification model, i.e. a constant rate of speciation (Table 1). The inclusion of the missing species in their most likely phylogenetic position according to morphology (Appendix Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
Pliocene range expansion of subterranean beetles T. elongatus elongatus VR1
98/1/1
T. elongatus mateui VR4 T. elongatus mateui VR5
50/0.58/0.75 100/1/1
T. elongatus VR24 T. elongatus ollai VR17 T. espanoli VR23 100/1/1 T. orcinus orcinus IRC42 95/1/1 T. orcinus orcinus VR9 61/0.75/ 0.65
5
T. orcinus VR10 T. orcinus acevedoi VR21
99/1/1
99/1/1 100/0.99/1
T. orcinus acevedoi VR20
Troglocharinus sp. VR7
Coastal clade
Troglocharinus sp. VR8 90/0.91/0.9
61/0.58/0.54
T. elongatus AF115 T. elongatus ollai VR15
91/0.91/0.93
T. elongatus pinyareti VR38
4
T. jacasi VR2
95/1/1
91/0.88/0.85 T. jacasi VR22
T. kiesenwetteri sanllorensi VR12
99/1/1 100/1/1
T. kiesenwetteri sanllorensi VR11 T. kiesenwetteri sanllorensi VR14
71/0.69/-
T. kiesenwetteri kiesenwetteri VR39
87/1/1
T. kiesenwetteri kiesenwetteri VR18
99/1/1
98/1/1
T. patracoi VR16
3
T. kiesenwetteri andresi VR13
73/0.84/0.81
68/0.76/0.6 T. ferreri VR28 100/0.99/0.98
T. ferreri VR26 T. ferreri VR29
0.7 (upper branch of node 5, Fig. 3). DISCUSSION Our results confirm the monophyly of the genus Troglocharinus – first hypothesized mostly on the basis of the structure of the male genitalia by Fresneda (1998), but never tested 1869
V. Rizzo et al. with independent data – and its inclusion within the Pyrenean clade of subterranean Leptodirini. This was irrespective of the data set and method used for phylogenetic reconstruction, and always with very good support. According to the reconstructed distribution of the clade, it can also be affirmed with confidence that the ancestral area of distribution of the genus was the central Pyrenees. The conclusion should thus be that the genus expanded its range from the central Pyrenees to some coastal karstic areas in which no species of the same lineage were previously found (Salgado et al., 2008; Ribera et al., 2010). Other species of Leptodirini in the Mediterranean coast of Spain (the Spelaeochlamys group) are confined south of the distribution area of the coastal Troglocharinus by a patch of Palaeozoic substratum without subterranean environment, most likely long before the arrival of Troglocharinus to the coast (Fig. 1; Ribera et al., 2010). According to our results, the extant species of the coastal area are monophyletic, so it must also be concluded that there was a single colonization with a subsequent diversification to form the currently recognized taxa. The incongruence between the topologies obtained with the nuclear and mitochondrial genes could indicate that the coastal species are nested within the Pyrenean clade, more closely related to the eastern than the western species – in agreement with our proposed scenario of range expansion (see below). At present, there are not enough data to discriminate among alternative possibilities, but the paraphyly of the Pyrenean clade would in any case only strengthen the argument for range expansion. Within the lineage to which Troglocharinus belongs, the most recent common ancestor to all species strictly living in MSS or caves dates to the late Oligocene–early Miocene, and that of all the species with all modifications typically associated with subterranean life (i.e. lack of eyes, lack of wings and depigmentation) to the early Oligocene (Ribera et al., 2010). The most parsimonious explanation is thus that the species expanded their range and diversified once fully adapted to the subterranean environment, both in morphology and habitat (i.e. they were truly troglobionts in the sense of Sket, 2008). Although a simultaneous extinction of hypothetical surface ancestors due to Pleistocene glaciations can never be fully discarded, there is no supporting evidence for that hypothesis. It must be noted, however, that this would not only require the extinction of all possible intermediate forms, but also that the extinct surface species either shared some of the morphological characteristics of their subterranean relatives that are usually considered an adaptation to the subterranean environment (such as their body shape), or that each of the lineages independently colonizing the subterranean environment within the same genus evolved the same characteristics in a concerted way. Irrespective of the morphology and habitat of the ancestral species of the coastal Troglocharinus, the temporal reconstruction shows that the divergence of the two main clades post-dates the formation of the sedimentary basin that 1870
separates their respective areas of distribution, which originated when the Ebro river opened to the Mediterranean during the Miocene (Friend & Dabrio, 1996). There are three possible scenarios for the range expansion from the central Pyrenees to the coastal area: (1) a direct dispersal through the same geological layers below the sedimentary basin; (2) a direct dispersal through the surface of the sedimentary basin; (3) a stepping-stone dispersal bordering the sedimentary basin. The sedimentary layers at the edge of the Ebro basin are typically c. 400–500 m deep (Friend & Dabrio, 1996). It is possible to find species of Leptodirini at even deeper levels in caves (Salgado et al., 2008), but the extensive presence of aquifers (Tourıs & Montua, 1992) and the probably unfavourable conditions in the non-submerged areas makes the first possibility highly unlikely. Similarly, although a hypothesis of direct surface dispersal through the shortest distance cannot be discarded, it seems implausible given the lack of potential refugial areas in an essentially flat landscape formed by compact sediment. The most likely scenario thus seems to be a route that initially heads east, crossing the head of the river Llobregat in the southern slopes of the Cadi mountain system, and then turns south, passing west of the river Ter and the plain of Vic (Fig. 1). Along the first part of the route, there are several calcareous massifs that could have acted as stepping stones, although the head of the Llobregat river would have had to be crossed. The area is rich in caves, with species of the genus Speonomites (used as outgroup, Fig. 2 and Ribera et al., 2010), but no specimen of Troglocharinus has ever been found despite intensive searches for more than 100 years. Through the second part of the hypothesized itinerary (east of the Llobregat river), there are some areas formed by conglomerates and gypsum. In these types of substratum, the formation of a strict subterranean environment is rare, and no species of Leptodirini has ever been found in the few known natural cavities (Salgado et al., 2008). In the coastal area, the estimated age of divergence of the different clades is compatible with fragmentation due to the formation of the Quaternary basins of the rivers that separate them. More detailed studies of the age of the sedimentary basins and the phylogeography of the different groups would be necessary to rigorously test this hypothesis (V. Rizzo et al., in preparation), but according to our results the only likely dispersal through one of the current basins could have been the east–west crossing of the Llobregat river within the area of the T. kiesenwetteri clade (area K, Fig. 1). In this area, the Llobregat river cuts deeply through a narrow band of the Mesozoic cover, with very shallow or non-existent sedimentary deposits (Fig. 1). This makes it a favourable area for dispersal across the river either during periods of low flow or by changes of the river course. It is interesting to note that the geological origin of the area of distribution of some taxa of the T. kiesenwetteri clade is different from that of other areas in which the genus is distributed, being the only ones with a Tertiary origin (Fig. 1). It is possible that at the time of colonization of the coastal region (early Pliocene), there Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
Pliocene range expansion of subterranean beetles was still no well-developed subterranean environment in the area currently occupied by the T. kiesenwetteri clade, which could only be colonized subsequently. This interpretation is supported by the results of the ancestral area reconstruction, which, although inconclusive, point towards the Garraf massif as the ancestral area of the whole coastal clade, with a later colonization of the areas currently north of the rivers Llobregat and Anoia, despite being geographically closer to the Pyrenees. There is also the possibility that some populations of the T. elongatus clade (area E) crossed the headwaters of the river Anoia, coexisting with populations of T. kiesenwetteri in the same cave at the western edge of area K (Fig. 1). However, owing to its small size, the river does not form a sedimentary basin in this area, and thus there are no discontinuities of the subterranean environment. The climatic conditions in the north-east of the Iberian peninsula during the early Pliocene, when the range expansion of Troglocharinus took place according to our estimations, were generally cooler and wetter than during the previous period (Messinian). Estimations from pollen reconstructions show an annual precipitation in the Garraf massif of 1100–1600 mm (versus 600 mm today) and temperatures of 15–20 °C, lower than at the end of the Miocene but more similar to the current temperatures (Suc & Cravatte, 1982; Jimenez-Moreno et al., 2010). These conditions could have facilitated surface displacements through deep valleys or the crossing of small rivers, especially below snow or ice plates or in the debris at the edge of cold streams flowing from the Pyrenees. There is anecdotal evidence to support the tolerance of some species of Troglocharinus to surface conditions under certain circumstances. Thus, T. senenti was observed in the cave Querant Gran de Pa us (Vilanova de Meia, Pyrenean area) at the base of the entrance pit, at c. 10 m but with direct sunlight, where it was commonly observed running over the moss covering the walls (J. Fresneda, pers. obs. in 2002). There are also multiple records of the occasional occurrence of other troglobiont organisms outside caves (Sket, 2008). With the onset of the Mediterranean climate c. 3.2 Ma (Suc, 1984), these conditions changed, with increasing aridity due to pronounced summer dry periods and the spread of xerophytic communities (Barr on et al., 2010; Carri on et al., 2010). This increase of aridity could have prevented any further displacement through the intermediate areas, resulting in the current isolation between the Pyrenean and coastal clades. In the Pyrenean area, the diversification was not geographically structured, although the higher number of missing species and the taxonomic uncertainties make the interpretation of the results difficult. In this area, there is also a higher continuity of the subterranean environment, without river basins filled with Quaternary sediments that could act as strong barriers (Fig. 1; Losantos et al., 2002). Within the Pyrenean area, there are also several cases in which two species of Troglocharinus coexist in the same cave (Salgado et al., 2008, pp. 676, 694), indicating a higher geographical mobility of Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
the species. These differences may be reflected in the observed contrast in the diversification patterns: while the coastal clade shows a decrease of the diversification rate with time (with a significantly negative c-statistic), suggesting a saturation of the available space due to the isolation of small areas, there is no sign of saturation in the Pyrenean clade, with a non-significant c-statistic and a constant speciation rate (pure birth) as the preferred model. CONCLUSIONS We have shown that the ancestor of the extant coastal Troglocharinus expanded its range from the central Pyrenees to the coastal area of Catalonia in the early Pliocene, where it subsequently diversified during the late Pliocene and the Pleistocene. This expansion could have been limited to a narrow temporal window of favourable conditions, in which species that may have been fully adapted to the subterranean environment could cross the headwaters of some rivers or non-karstified areas over the surface. Our results add to the growing evidence of the possibility of range expansions and diversification in lineages fully adapted to the subterranean environment, without the need to invoke multiple extinct surface ancestors to explain the extraordinary diversity and geographical distribution of the subterranean fauna. ACKNOWLEDGEMENTS We thank the Sie-Aliga speleogroup of Barcelona, Josep Pastor and Julia Gonzalez of Grup d’Espeleologia de Badalona (GEB), and Enric Lleopart for their help in the field, as well as other collectors mentioned in Appendix S1b. We also thank Alexandra Cieslak and Arnaud Faille for comments and support in multiple ways, Michele Spada for informatics support, Mauro Gobbi of Museo delle Scienze of Trento for comments on biogeographical issues, and three anonymous referees for comments. This work was funded by a postgraduate grant to V.R. (Universita La Sapienza, Roma) and projects CGL2007-61943 (to A. Cieslak) and CGL2010-15755 (to I.R.). REFERENCES Assmann, T., Casale, A., Drees, C., Habel, J.C., Matern, A. & Schuldt, A. (2010) Review – the dark side of relict species biology: cave animals as ancient lineages. Relict species: phylogeography and conservation biology (ed. by J.C. Habel and T. Assmann), pp. 91–103, Springer, Heidelberg. Barr, T.C. Jr (1968) Cave ecology and the evolution of troglobites. Evolutionary Biology, 2, 35–105. Barr, T.C. Jr & Holsinger, J.R. (1985) Speciation in cave faunas. Annual Review of Ecology and Systematics, 16, 313–317. Barr on, E., Rivas-Carballo, R., Postigo-Mijarra, J.M., AlcaldeOlivares, C., Vieira, M., Castro, L., Pais, J. & Valle-Hernan1871
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Pliocene range expansion of subterranean beetles Ree, R.H. & Smith, S.A. (2008) Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Systematic Biology, 57, 4–14. Ribera, I., Fresneda, J., Bucur, R., Izquierdo, A., Vogler, A.P., Salgado, J.M. & Cieslak, A. (2010) Ancient origin of a Western Mediterranean radiation of subterranean beetles. BMC Evolutionary Biology, 10, 29. Rodrıguez, L.R., Bellido, F., Dıez, A., Gonzalez, E., Heredia, N., L opez, F., Marın, C., Martın-Parra, L.M., Martın-Serrano, A., Matas, J., Montes, M., Nozal, F., Quintana, L., Roldan, F., Rubio, F. & Salazar, A. (2004) Mapa tectonico de Espa~ na, con la inclusion de Portugal continental y Pirineos franceses. Escala 1:2.000.000. SGE-IGME, Madrid, Spain. Romero, A. (2009) Cave biology: life in darkness. Cambridge University Press, Cambridge, UK. Salgado, J.M., Blas, M. & Fresneda, J. (2008) Fauna Iberica. Vol. 31: Coleoptera: Cholevidae. Consejo Superior de Investigaciones Cientıficas, Madrid, Spain. Salgado, J.M., Luque, C.G., Labrada, L., Fresneda, J. & Ribera, I. (2012) Revisi on del genero Cantabrogeus Salgado, 2000, con la descripci on de tres nuevas especies hipogeas endemicas de la Cordillera Cantabrica (Coleoptera, Leiodidae, Cholevinae, Leptodirini). Animal Biodiversity and Conservation, 35, 27–50. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sket, B. (2008) Can we agree on an ecological classification of subterranean animals? Journal of Natural History, 42, 1549–1563. Stamatakis, A., Hoover, P. & Rougemont, J. (2008) A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology, 57, 758–771. Suc, J.-P. (1984) Origin and evolution of the Mediterranean vegetation and climate in Europe. Nature, 307, 429–432. Suc, J.-P. & Cravatte, J. (1982) Etude palynologique du Pliocene de Catalogne (nord-est de l’Espagne). Paleobiologie Continentale, 13, 1–31.
Journal of Biogeography 40, 1861–1873 ª 2013 John Wiley & Sons Ltd
Tourıs, i. & Montua, R. (eds) (1992) Mapa d’arees hidrogeologiques de Catalunya 1:250 000. 2nd edn. Servei Geol ogic de Catalunya, Barcelona. Vandel, A. (1964) Biospeologie: la biologie des animaux cavernicoles. Gauthier-Villars, Paris. Vera, J.A. (ed.) (2004) Geologıa de Espa~ na. SGE-IGME, Madrid. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Appendix S1 Additional data on the studied specimens: (a) taxonomy; (b) locality data and sequence accession numbers. Appendix S2 Additional methods: (a) list of primers used for sequencing; and (b) general PCR conditions. Appendix S3 Additional phylogenetic results: (a) extended data set; (b) nuclear sequences of the reduced data set; (c) mitochondrial sequences of the reduced data set; (d) calibrated tree with estimated placement of missing species. BIOSKETCH Valeria Rizzo is interested in conservation and evolutionary biology, using cave beetles as model species and genetic and bioinformatics methods. Her PhD is focused on the phylogeny and historical biogeography of a genus of Leptodirini (Coleoptera, Leiodidae) and the conservation of its threatened subterranean habitat in the Garraf massif (Barcelona, Spain). Author contributions: V.R., J.F. and I.R. conceived the work; V.R., J.C., F.F. and J.F. led the specimen collection; V.R. obtained the molecular data; V.R. and I.R. analysed the data and led the writing; all authors contributed to the discussion of results and the writing.
Editor: Melodie McGeoch
1873
Late Pliocene range expansion in a clade of subterranean Pyrenean beetles Valeria Rizzo, Jordi Comas, Floren Fadrique, Javier Fresneda and Ignacio Ribera Appendix S1 Additional data on the studied specimens: (a) taxonomy, (b) locality data and accession numbers. (a) Checklist of the species and subspecies of Troglocharinus, with their geographical distributions (P: Pyrenean; C: coastal). Genus Troglocharinus Reitter, 1908 Subgenus Antrocharidius Jeannel, 1910 1. Troglocharinus (Antrocharidius) orcinus (Jeannel, 1910) ssp. acevedoi (Español, 1953) C: Serra de Prades, south of Brugent river, Tarragona ssp. lagari (Español, 1953) C: southeast of Serra de la Mussara, Tarragona ssp. orcinus C: Serra de la Mussara, Tarragona Subgenus Troglocharinus Reitter, 1908 2. Troglocharinus (Troglocharinus) espanoli (Jeannel, 1930) C: Pla de Cabra, Tarragona 3. Troglocharinus (Troglocharinus) ferreri (Reitter, 1908) ssp. abadi Lagar, 1981 C: Sant Pau d’Ordal y Subirats, Macizo de Garraf, Barcelona ssp. ferreri C: Macizo de Garraf-Ordal, Barcelona ssp. pallaresi Bellés, 1973 C: southwestern side of La Mola de Montmany de Cervelló, Barcelona 4. Troglocharinus (Troglocharinus) fonti (Jeannel, 1910) ssp. fonti P: Serra del Boumort (Lleida), between Segre and Noguera Pallaresa rivers ssp. infernus (Jeannel, 1911) P: western side of Serra de Boumort, Lleida ssp. schuettei (Español, 1955) P: eastern side of Serra de Boumort, Lleida ssp. zariquieyi (Jeannel, 1924) P: northwestern side of Serra de Boumort, Lleida 5. Troglocharinus (Troglocharinus) hustachei Jeannel, 1911 P: northern side of Serra del Montsec de Rúbies, Lleida 6. Troglocharinus (Troglocharinus) impellitierii Español, 1955 P: Serra del Boumort and western-hand of Noguera Pallaresa river, Lleida
7. Troglocharinus (Troglocharinus) jacasi (Lagar, 1966) C: La Llacuna, Barcelona 8. Troglocharinus (Troglocharinus) kiesenwetteri (Dieck, 1869) ssp. andresi (Escolà, 1966) C: Montserrat massif, Esparraguera, Barcelona ssp. kiesenwetteri C: Montserrat massif, western side of Llobregat river, Barcelona ssp. sanllorensi (Zariquiey, 1924) C: Sant Llorenç del Munt and Serra de l’Obac, Barcelona 9. Troglocharinus (Troglocharinus) ludovici Bellés y Deliot, 1983 P: western extreme of the Serra del Port del Comte, Lleida 10. Troglocharinus (Troglocharinus) olerdolai Lagar, 1952 C: northwestern extreme of Macizo de Garraf, Barcelona 11. Troglocharinus (Troglocharinus) patracoi (Zariquiey, 1922) C: Serra de Rubió, Esparreguera, Barcelona 12. Troglocharinus (Troglocharinus) quadricollis (Jeannel, 1911) P: Serra de Lleràs, Lleida 13. Troglocharinus (Troglocharinus) rovirai Lagar, 1975 P: Solana valley, Huesca 14. Troglocharinus (Troglocharinus) schibii (Español, 1972) C: Serra d’Ancosa, La Llacuna (Barcelona) 15. Troglocharinus (Troglocharinus) senenti Escolà, 1967 P: southeastern side of the Sierra del Montsec de Rúbies, Lleida 16. Troglocharinus (Troglocharinus) subilsi (Español, 1966) P: Sierra de Odén, Solsonès and Alt Urgell, Lleida 17. Troglocharinus (Troglocharinus) elongatus Zariquiey, 1950 ssp. abenzai (Lagar, 1972) C: Pontons, Alt Penedès, Barcelona ssp. mateui Zariquiey, 1950 C: Querol, Tarragona ssp. ollai Zariquiey, 1950 C: El Montmell, Aiguaviva, Tarragona ssp. pinyareti Zariquiey, 1950 C: El Montmell, El Vendrell, Tarragona ssp. portai Zariquiey, 1950 C: San Quintì de Mediona, Alt Penedes, Barcelona ssp. elongatus Zariquiey, 1950 C: La Llacuna, Plana d’Ancosa, Barcelona 18. Troglocharinus (Troglocharinus) vinyasi (Escolà, 1971) P: both side of Segre river, from Pons to Organyà, Lleida
Journal of Biogeography SUPPORTING INFORMATION Late Pliocene range expansion in a clade of subterranean Pyrenean beetles Valeria Rizzo, Jordi Comas, Floren Fadrique, Javier Fresneda and Ignacio Ribera Appendix S1 (b) List of studied material with locality, collector, voucher reference and accession numbers. in bold, new sequences data set: E, extended; R, reduced *: data from two individuals of T. Mestrei were combined to form a chimeric sequence new sequences for the fragment rrnL+trnL+nad1 were submitted separately for the ribosomal and protein-‐coding genes all
data set E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E, R E E E E E E E E E E, R E E, R E E, R R E, R R E
country China Ecuador Falkland Islands Spain Spain Spain Spain Morocco Spain Spain Spain France Spain France France Morocco Russia Spain Spain Spain Spain Spain USA USA Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain France Spain France Spain France Spain Spain Spain Spain Spain France Spain Spain France Spain Spain France France Spain
Locality Yunnan Orellana, Reserva Nacional de Yasuni, Estación Biológica
Salgado, 1980 (Bolívar, 1917) Salgado & Fresneda, 2005 (Coiffait, 1965) (Salgado, 1982) Jeannel, 1930 Zariquiey, 1940 Dieck, 1870 Salgado & Giachino, 1991 (Sharp, 1873) Abeille de Perrin, 1878 Español, 1972 (Abeille de Perrin, 1904) Salgado & Fresneda, 2001 Fresneda & Fery, 2009 (Sharp, 1873) (Auroux y Bellés, 1974) (Español, 1967) Dupré, 1991 (Bolívar, 1921) Giachino & Guéorguiev, 1989 (Bolívar, 1921) (Zariquiey, 1924) Jeannel, 1924 (Lagar, 1974) (Jeannel 1911) Coiffait, 1952 Foures 1954 (Fairmaire, 1860)
voucher IBE-‐RA187 IBE-‐AF219 BMNH-‐673231 MNCN-‐AI592 NHM-‐IRC38 IBE-‐AF56 MNCN-‐HI21 IBE-‐AF215 IBE-‐AF72 NHM-‐IRC43 IBE-‐AF30 MNCN-‐AI550 IBE-‐AF32 IBE-‐AF144 IBE-‐AF197 IBE-‐AF216 IBE-‐AF67 MNCN-‐AI574 MNCN-‐AI927 IBE-‐AF222 NHM-‐IRC13 MNCN-‐AI1255 IBE-‐AF217 IBE-‐AF218 MNCN-‐AI662 NHM-‐IRC4 MNCN-‐AI573 NHM-‐IRC3 MNCN-‐AI1075 NHM-‐IRC1 NHM-‐IRC37 NHM-‐IRC31 MNCN-‐AI664 MNCN-‐AI1067 MNCN-‐AI594 NHM-‐IRC17 MNCN-‐AI540 NHM-‐IRC14 MNCN-‐AI601 NHM-‐IRC12 NHM-‐IRC21 MNCN-‐AI597 NHM-‐IRC15 NHM-‐IRC16 MNCN-‐AI525 NHM-‐IRC19 NHM-‐IRC30 MNCN-‐AI533 NHM-‐IRC29 IBE-‐AF123 MNCN-‐AI539 IBE-‐AF184 NHM-‐IRC35
Córdoba, Luque, Abuchite, L-‐14 Cordoba, Priego de Cordoba, Cueva de los Mármoles Málaga, Antequera, Complejo del Romeral Jaén, Sierra de Cazorla, alrededores de Linarejos Taza, Ghar Chiker Córdoba, Cabra, La Nava Lleida, El Pont de Suert, Cova de la Carretera Cantabria, Bustablado, Cueva la Cañuela Ariège, Illartein, Ravin de la Tire, MSS Huesca, Seira, MSS Haute-‐Garonne, Izaut de l'Hotel, Grotte de la Maouro Haute-‐Garonne, Tibiran, Grotte de Tignahuste Aït Mohammed, Ifri N'Caid Primorsky Kr., Anisimovka village Málaga, Antequera, Sierra de las Cabras, N331 Km133 Albacete, N Sierra de Segura, Rio Tús Cantabria, Carcabal Merilla, Cueva de Covallarco Lleida, Es Bordes, Goells deth Joeu Barcelona, Sant Celoni, Serra de Montnegre CA: Calaveras Co., Stanislaus OR: Hood River Co., Mt. Hood, N.F., N of Barlow / Pass Asturias, Caravia Alta-‐Arriondas, Cueva de Entrecuevas Cantabria, Sámano, Cueva de la Lastrilla Cantabria, Santoña, Barrio del Dueso, Cueva del Polvorín Guipúzcoa, Olatz, Sima Kobeta Asturias, Viboli, Cueva de los Moros Castellón, Montan, Cova Cirat Tarragona, Hospitalet de l'Infant, Avenc de la Julia Alicante, Cocentaina, Cova de les Meravelles Asturias, San Tirso-‐San Román de Candamo, Cueva del Ferradal Navarra, Zegama, Cueva de Orobe Ariège, Alzen, Grotte de Ferrobach Guipúzcoa, Mendaro, Cueva del Viento Pyrénées-‐Atlantiques, Arbailles, Grotte d'Istaurdy Navarra, Arive, Cueva de Mendia Landa Ariège, Galey, Grotte d'Escarchein Navarra, Zegama, Dolina de Orobe Huesca, Sercué, Cueva de Aso Girona, Olopte, Cova d'en Manent Navarra, Donamaria, Cueva de Iguntsoro Navarra, Atalo, Cueva Malkorrandi Ariège, Seix, Bordes de Crues Guipúzcoa, Aia, Cueva de Pagoeta Lleida, Os de Balaguer, Cova Joan d'Os Ariège, Galey, Grotte d'Escarchein Huesca, Foradada del Toscar, Inflas de Naspún Lleida, Sort, Cueva Saverneda Ariège, Seix, Grotte Les Souleillos Ariège, Bordes-‐sur-‐Lez, Aven du Trapech d'en Haut Girona,Terrades, Bauma de Taleixà
2002 2002 2008 2006 2009 2007 2002 2008 2004 2008 2003 2009 2009 2008 2004 2003 2009 2001 2006 2006 2006 2006 1998 2003 1998 2006 1998 2001 2002 2005 2006 2004 2000 2004 2000 2004 1998 2001 2004 2000 2000 2004 2000 2001 2004 2001 2009 2004 2003 2002
Zariquiey, 1924 Coiffait, 1947 Laneyrie, 1988 (Zariquiey, 1922) (Jeannel, 1911) Español, 1970 Jeannel, 1919 (Escolà, 1972)
NHM-‐IRC40 IBE-‐AF27 MNCN-‐AI912 NHM-‐IRC41 MNCN-‐AI587 MNCN-‐AI580 NHM-‐IRC11 MNCN-‐AI1074
E E E E E, R E E E, R
Spain France France Spain Spain Spain Spain Spain
Girona, Queralbs, Coves Encantades Pyrénées-‐Atlantiques, Barlanès, Grotte de Barlanès Pyrénées-‐Atlantiques, Esterencuby, Grotte du Renard Girona, Roses, Cau del Lliri Huesca, Santa Olaria de Ara, Ginuabel, Forato de los Moros Guipuzcoa, Usurbil, Guardetxe Aurre ko Leizia Guipúzcoa, Albiztur, Cueva de Mendikute Lleida, Gerri de la Sal, Baen, Cova dels Porredons
2001 F. Fadrique E. Dupré 2006 C. Bourdeau 2001 F. Fadrique 2002 J. Fresneda 2002 I. Zabalegui 1998 J. Fresneda 2006 J. Fresneda
family Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae
subfamily Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae
tribe Anemadini Anemadini Anemadini Anemadini Anemadini Anemadini Anemadini Anemadini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Cholevini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini
series
species Anemadus cf. smetanai Dissochaetus sp. Falkonemadus cf. sphenisci Speonemadus angusticollis Speonemadus angusticollis Speonemadus bolivari Speonemadus clathratus Speonemadus maroccanus Catops andalusicus Catops fuliginosus Catops sp. Catops subfuscus Catops ventricosus Choleva angustata Choleva cisteloides Choleva kocheri Fusi cf. nyujwa Nargus algiricus Nargus velox Sciodrepoides watsoni Bathysciola Bathysciola catalana Bathysciola Bathysciola zariquieyi Platycholeina Platycholeus opacellus Platycholeina Platycholeus sp. Quaestus Breuilites eloyi Quaestus Espanoliella jeanneli Quaestus Espanoliella luquei Quaestus Quaestus (Quaesticulus) noltei Quaestus Quaestus (Quaestus) jeannei pongai Spelaeochlamys Anillochlamys subtruncata Spelaeochlamys Paranillochlamys velox Spelaeochlamys Spelaeochlamys ehlersi Speonomidius Notidocharis laurae Speonomidius Speonomidius crotchi crotchi Speonomus Antrocharis querilhaci dispar Speonomus Aranzadiella leizaolai Speonomus Bathysciella jeanneli Speonomus Bathysciola diegoi Speonomus Bathysciola mystica Speonomus Bathysciola rugosa Speonomus Bellesia espanyoli Speonomus Ceretophyes riberai Speonomus Euryspeonomus (Euryspeonomus) beruetei Speonomus Euryspeonomus (Urbasolus) ciaurrizi Speonomus Gesciella delioti Speonomus Josettekia mendizabali Speonomus Lagariella colominasi Speonomus Machaeroscelis infernus arbasanus Speonomus Naspunius eseranus Speonomus Pallaresiella pallaresana Speonomus Paraspeonomus vandeli Speonomus Paratroglophyes carrerei Speonomus Parvospeonomus delarouzeei
authority
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae
Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae
Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini
Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus
Perriniella bofilli Phacomorphus (Phacomorphoides) sioberi sioberi Phacomorphus (Phacomorphus) duprei Pseudospeonomus raholai Salgadoia brieti Speocharidius (Kobiella) galani Speocharidius (Speocharidius) breuili Speonomites antemi
(Kraatz, 1870) (Kraatz, 1870) (Jeannel, 1922) (Perris, 1864) (Jeannel, 1936) Heyden, 1870 Erichson, 1837 Kellner, 1846 (Weise, 1877) (Fabricius, 1781) (Frölich, 1799) Henrot, 1962 Portevin, 1903 (Spence, 1813) (Spence, 1813) Coiffait, 1959 Bolívar, 1919 Fall, 1909
year leg. 2010 V.V. Grebennikov 2009 J.M. Salgado A. Moreno & M. Baena J. Fresneda G.E. Villacarrillo M. Baena A. Faille M. Baena J. Fresneda J.M. Salgado, A. Faille, J. Fresneda, I. Ribera & A. Cieslak Ph. Déliot & A. Faille J. Fresneda via A. Faille C. Bourdeau & J. Fresneda A. Faille V. Grebennikov M. Baena V. Assing & P. Wunderle J.M. Salgado & C.G. Luque J. Fresneda C. Hernando A. Newton & M. Thayer A. Newton & M. Thayer J.M. Salgado J. Fresneda J.M. Salgado J. Fresneda J.M. Salgado J. Fresneda F. Fadrique J. Fresneda J.M. Salgado J. Fresneda A. Faille J. Fresneda A. Faille J. Fresneda Ph. Déliot & A. Faille J. Fresneda J. Fresneda Ph. Déliot & A. Faille J. Fresneda J. Fresneda J. Fresneda J. Fresneda J. Fresneda Ph. Déliot & A. Faille J. Fresneda J. Fresneda, C. Bourdeau & A. Faille C. Bourdeau & A. Faille via J. Fresneda F. Fadrique
cox1
rrnL+trnL[+nad1]
cob
SSU
LSU
HE572819 HF912461 EF214358 HE572809 HE572818 HE572787 HE572815 HE572796 HE572791 AJ890079/GU356860 HE572785 GU356861 HE572786 HE572794 HE572795 HE572797 HE572789 GU356871 GU356870 HE572802 GU356854 GU356857 HE572798 HE572799 GU356859 GU356863
HE576718 HF912611 EF214126
HF912508
HF912581
HF912593
HE576717
HE572888
HE576715 HE576699 HE576694 GU356756 HE576691 GU356757 HE576692 HE576697 HE576698 HE576700 HE576693 GU356767 GU356766 HE576705 GU356750 GU356753 HE576701 HE576702 GU356755 GU356759
HE572886
HE576716 GU356782 GU356745 GU356770 GU356784 GU356769 GU356788 HE576711 GU356747 GU356748 GU356749 HE576712 GU356751 GU356754 GU356758 GU356761 GU356762 GU356763 GU356764 GU356765 GU356791 GU356768 HF912607 GU356771
HE572887 HE572884 GU356802 GU356823 GU356833 GU356822 GU356837
HE572816 GU356884 GU356850 GU356874 GU356886 GU356873 GU356890 HE572810 GU356852 GU356853 HE572811 GU356855 GU356858 GU356862 GU356865 GU356866 GU356867 GU356868 GU356869 GU356893 GU356872 HF912455 GU356875 HF912460 HE572817 GU356877 HE572784 HE572814 GU356880 GU356885 GU356887 GU356888 GU356891
nad1
HE572871
HE572830 HE572826 GU356811 HE572882
HE572878
GU356820 HE572880 GU356806 HE572885
GU356810 GU356813
GU356804
GU356912 HE572822 HE572828 HE572829 HE572831 HE572824 GU356919 HE572835
HE572832 GU356911 GU356914 GU356928 GU356929 GU356904 GU356922 GU356931 GU356921 GU356934 GU356906 GU356907
GU356805 GU356807 GU356809 GU356812 GU356815 GU356816 GU356818 GU356819
HF912544
GU356821 HF912505 HE572881 HF912507
HE572852 HE572848 GU356950 HE572865 GU356951 HE572844 HE572850 HE572851 HE572853 HE572846 GU356962 HE572857 GU356945 GU356947 HE572866 HE572854 GU356949 GU356955 GU356956 HE572869 GU356976 GU356941 GU356965 GU356964 GU356981 GU356943 GU356944
GU356910 GU356913 GU356915 GU356916 GU356917 GU356918 GU356936 GU356920 HF912578
GU356948 GU356952 GU356957 GU356958 GU356959 GU356960 GU356961 GU356984 GU356963
HF912580
HE572867 HF912592 HE572870
GU356773 HE576690
GU356825 HE572874
GU356924 HE572821
GU356967 HE572843
GU356778 GU356783 GU356785 GU356786 GU356789
GU356828 GU356832 GU356834 GU356835 HE572883
GU356926 GU356930 GU356932
GU356972 GU356977 HE572868 GU356979 GU356982
GU356935
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 85 86 87 88 89 90 91 92 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139
Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Leiodidae Staphylinidae
Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Cholevinae Leiodinae Platypsyllinae Platypsyllinae Micropeplinae
Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Leptodirini Ptomaphagini Ptomaphagini Ptomaphagini Ptomaphagini Sciaphyini Sciaphyini Sciaphyini Sciaphyini
Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus Speonomus
Speonomites tincatincensis Speonomus (Speonomus) abeillei Speonomus (Speonomus) carrerei Speonomus (Speonomus) diecki Speonomus (Speonomus) fagniezi Speonomus (Speonomus) gaudini arivensis Speonomus (Speonomus) orgibetensis Speonomus (Speonomus) stygius stygius Speonomus (Speonomus) zophosinus Stygiophyes akarsticus Stygiophyes hansferyi Trapezodirus carrodillae Trapezodirus cerberus Trocharanis mestrei* Trocharanis mestrei* Troglocharinus (Antrocharidius) orcinus acevedoi Troglocharinus (Antrocharidius) orcinus acevedoi Troglocharinus (Antrocharidius) orcinus orcinus Troglocharinus (Antrocharidius) orcinus orcinus Troglocharinus (Antrocharidius) orcinus ssp. Troglocharinus (Troglocharinus) elongatus Troglocharinus (Troglocharinus) elongatus Troglocharinus (Troglocharinus) elongatus elongatus Troglocharinus (Troglocharinus) elongatus mateui Troglocharinus (Troglocharinus) elongatus mateui Troglocharinus (Troglocharinus) elongatus ollai Troglocharinus (Troglocharinus) elongatus ollai Troglocharinus (Troglocharinus) elongatus pinyareti Troglocharinus (Troglocharinus) espanoli Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri Troglocharinus (Troglocharinus) ferreri ferreri Troglocharinus (Troglocharinus) ferreri ferreri Troglocharinus (Troglocharinus) ferreri pallaresi Troglocharinus (Troglocharinus) fonti Troglocharinus (Troglocharinus) fonti Troglocharinus (Troglocharinus) fonti fonti Troglocharinus (Troglocharinus) fonti schuettei Troglocharinus (Troglocharinus) hustachei Troglocharinus (Troglocharinus) impellitierii Troglocharinus (Troglocharinus) impellitierii Troglocharinus (Troglocharinus) jacasi Troglocharinus (Troglocharinus) jacasi Troglocharinus (Troglocharinus) kiesenwetteri andresi Troglocharinus (Troglocharinus) kiesenwetteri kiesenwetteri Troglocharinus (Troglocharinus) kiesenwetteri kiesenwetteri Troglocharinus (Troglocharinus) kiesenwetteri sanllorensi Troglocharinus (Troglocharinus) kiesenwetteri sanllorensi Troglocharinus (Troglocharinus) kiesenwetteri sanllorensi Troglocharinus (Troglocharinus) patracoi Troglocharinus (Troglocharinus) quadricollis Troglocharinus (Troglocharinus) senenti Troglocharinus (Troglocharinus) senenti Troglocharinus (Troglocharinus) sp. Troglocharinus (Troglocharinus) sp. Adelopsis sp. Ptomaphagus pyrenaeus Ptomaphagus tenuicornis Ptomaphagus troglodytes Sciaphyes shestakovi Sciaphyes shestakovi Sciaphyes sibiricus Sciaphyes sibiricus Eucatops sp. Agathidium sp. Leptinus testaceus Silphopsyllus desmanae Micropeplus sp.
(Escolà, Bellés & Comas, 1985) (Saulcy, 1872) Fourès, 1954 (Saulcy, 1872) Jeannel, 1910 Jeannel, 1930 Gers, 1986 (Dieck, 1869) (Saulzy 1872) (Escolà, 1980) Fresneda & Escolà, 2001 (Jeannel, 1911) (Jeannel 1911) (Abeille 1878) (Abeille 1878) (Español, 1953) (Español, 1953) (Jeannel, 1910) (Jeannel, 1910) (Jeannel, 1910) Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 Zariquiey, 1950 (Jeannel, 1930) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) (Reitter, 1908) Bellés, 1973 (Jeannel, 1910) (Jeannel, 1910) (Jeannel, 1910) (Español, 1955) Jeannel, 1911 Español, 1955 Español, 1955 (Lagar, 1966) (Lagar, 1966) (Escolà, 1966) (Dieck, 1869) (Dieck, 1869) (Zariquiey, 1924) (Zariquiey, 1924) (Zariquiey, 1924) (Zariquiey, 1922) (Jeannel, 1911) Escolà, 1967 Escolà, 1967
Jeannel, 1934 (Rosenhauer, 1856) Blas & Vives, 1983 Fresneda et al., 2011 Fresneda et al., 2011 (Reitter, 1887) (Reitter, 1887)
Müller, 1817 Olsufiev, 1923
NHM-‐IRC6 NHM-‐RB2 MNCN-‐AI530 MNCN-‐AI536 IBE-‐AF125 NHM-‐IRC18 MNCN-‐AI526 MNCN-‐AI534 MNCN-‐AI667 NHM-‐IRC7 NHM-‐IRC39 NHM-‐IRC9 MNCN-‐AI600 IBE-‐AC112* IBE-‐AC169* IBE-‐VR20 IBE-‐VR21 IBE-‐VR9 NHM-‐IRC42 IBE-‐VR10 IBE-‐AF115 IBE-‐VR24 IBE-‐VR1 IBE-‐VR4 IBE-‐VR5 IBE-‐VR15 IBE-‐VR17 IBE-‐VR38 IBE-‐VR23 IBE-‐VR26 IBE-‐VR28 IBE-‐VR29 IBE-‐VR30 IBE-‐VR31 IBE-‐VR32 IBE-‐VR33 IBE-‐VR34 IBE-‐VR35 IBE-‐AF172 MNCN-‐AI1065 IBE-‐VR3 IBE-‐AF130 IBE-‐AF181 NHM-‐IRC26 IBE-‐VR37 IBE-‐VR36 IBE-‐AF182 NHM-‐IRC22 IBE-‐VR2 IBE-‐VR22 IBE-‐VR13 IBE-‐VR18 IBE-‐VR39 IBE-‐VR11 IBE-‐VR12 IBE-‐VR14 IBE-‐VR16 MNCN-‐AI578 IBE-‐VR6 MNCN-‐AI585 IBE-‐VR7 IBE-‐VR8 IBE-‐AF221 IBE-‐RA261 MNCN-‐AI760 NHM-‐IRC33 IBE-‐AF108 IBE-‐AF66 IBE-‐AF107 IBE-‐AF68 IBE-‐AF220 MNCN-‐AI1305 IBE-‐AF82 IBE-‐RA265 MNCN-‐AI521
E, R E, R E, R E, R R E E E, R R E, R E, R E, R R R R R R R E, R R R R R E, R R R R R R R R R R R R R R R R E, R R R R E, R R R R E, R R E, R R R R E, R R R R E, R E, R R R R E E E E E E E E E E E E E
Spain France France France France Spain France France France Spain Spain Spain Spain France France Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Ecuador France Spain Spain Russia Russia Russia Russia Ecuador Spain Spain Russia Bhutan
Lleida, Altron, Forat del Tincatinc Ariège, Le Mas d'Azil, Grotte de Peyrounard Ariège, Illartein, Ravin de la Tire, MSS Ariège, Moulis, Grotte d'Aubert Aude, Saint-‐Paul-‐de-‐Fenouillet, Grotte de la Madeleine Navarra, Arive, Cueva de Mendia Landa Ariège, Illartein, Ravin de la Tire, MSS Ariège, Rogalle, Grotte d'Ardet F-‐09, Massat, Grotte Ker Lleida, Altron, Forat del Tincatinc Lleida, El Pont de Suert, Cova de la Carretera Huesca, Estadilla, Grallera de Estadilla Huesca, Bonansa, Esplluga des Tosses F-‐09, Belesta, Trou du Vent de Pedrous Aude, Coudons, Grotte du Bac de Lacaune Tarragona, La Riba, Cova d'en Cartanyà Tarragona, La Riba, Cova d'en Cartanyà Tarragona, La Febrò, Cova Gran de la Febró, Tarragona, La Febró,Cova Gran de la Febrò Tarragona, Reus, Cova Can Masiet Barcelona, Alt Penedès, Cv. De la Sensada, Orpí Barcelona, Igualada, Mines de Pontons 1 Barcelona, La Llacuna, Av. De la Plana d'Ancosa Tarragona, Alt Camp, Querol, Cv. Del Garrofet, Tarragona, Alt Camp, Querol, Cv. Del Garrofet Barcelona, Albinyana, Cova de Vallmajor Barcelona, Aiguaviva (Montmell), Cova de la Olla Tarragona, El Montmell, Aiguaviva, Avenc dels Pinyarets Tarragona, Pla de Cabra, Cova del Traça Barcelona, Garraf, La Plana Novela, Avenc del Corral Nou Barcelona, Garraf, La Plana Novela, Avenc del Corral Nou Barcelona, Garraf, La Plana Novela, Avenc del Corral Nou Barcelona, Garraf, Castelldefells, Av. Dells Guerrillers Barcelona, Garraf, Castelldefells, Av. Dells Guerrillers Barcelona, Ordal, Llerdoner, Cova de Coll Verdaguer Barcelona, Ordal, Llerdoner, Cova de Coll Verdaguer Barcelona, Ordal, Llerdoner, Cova de Coll Verdaguer Barcelona, Ordal, Llerdoner, Cova de Coll Verdaguer Barcelona, Baix Llobregat, Av. De la Bardissa, Ordal Barcelona, Begues, Avenc del Sant Roc Barcelona, Corbera, Av. de Can Montmany Lleida, La Guardia d'Ares, Av. Pla Fornesa Lleida, La Guardia d'Ares, Av. Pla Fornesa Lleida, Montanisell, Cova d'Ormini Lleida, Hortoneda, Serra de Boumort, Cova Font Mentidora Lleida, Vilanova de Meià, Noguera, Forat del Gel Lleida, La Guardia d'Ares, Av. Pla Fornesa Lleida,Taús, Cova Palomera Barcelona, La Llacuna, Cova de les Rondes Barcelona, La Llacuna, Cova de les Rondes Barcelona, Collbatò, Avenc del Clast (?) Barcelona, Castellolì, (Anoia), Cova del Pas Barcelona, Collbatò, Baix Llobregat, Cova Freda Barcelona, Matadepera, Coves de Mura Barcelona, Matadepera, Coves de Mura Barcelona, Matadepera, Cova S. Agnes Barcelona, Esparraguera, Cova Patracoi Lleida, Tremp, Graller de Castellet Lleida, Vilanova de Meià, Querant Gran de Paús Lleida, Vilanova de Meià, Querant Gran de Paús Barcelona, Castellolí, (Anoia), Cova del Pas Barcelona, Castellolí, (Anoia), Cova del Pas Pichincha, Unión de Toachi Pyrénées-‐Atlantiques, Iriberri, MSS Lleida, El Pont de Suert, Llesp Granada, Gualchos, Cueva de las Campanas Primorsky Kray. Chuquevskiy Primorsky Kray. Chuquevskiy Primorsky Kray Gamova pen. Primorsky Kray Anisimovka village Pichincha, Unión de Toachi Córdoba, Cabra, Via Verde Cantabria, El Bosque, Entrambasaguas, Cueva de las Enjanas Russia, Lipetsk distr., Plavitsariv. BHUTAN loc. 32: Mo Chhu [river], Punakha Prov./Timphu Prov.
1999 2004 2004 2004 2009 2001 2004 2004 2006 1999 2002 1999 2004 2009 2009 2010 2010 2009 2002 2010 2009 2010 2009 2009 2009 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2009 2006 2009 2009 2009 2001 2010 2010 2009 2001 2009 2010 2010 2010 2010 2010 2010 2010 2010 2003 2002 2002 2009 2009 2009 2010 2006 2002 2008 2008 2008 2008 2009 2006 2007 2007 2005
J. Fresneda Ph. Déliot & A. Faille Ph. Déliot & A. Faille A. Faille C. Bourdeau J. Fresneda Ph. Déliot & A. Faille C. Bourdeau C. Bourdeau J. Fresneda J. Fresneda J. Fresneda J. Fresneda C. Vanderbergh C. Bourdeau F. Fadrique F. Fadrique V. Rizzo F. Fadrique V. Rizzo & F. Fadrique L. Auroux & J. Comas V. Rizzo & J. Comas J. Comas J. Comas J. Comas V. Rizzo & J. Comas J. Comas & V.Rizzo J.M. Victoria F. Fadrique V. Rizzo V. Rizzo V. Rizzo V. Rizzo V. Rizzo V. Rizzo V. Rizzo V. Rizzo V. Rizzo J. Comas J. Fresneda J. Comas J. Fresneda, A. Faille & C. Bourdeau C. Bourdeau & A. Faille J. Fresneda J. Fresneda, I. Ribera & V. Rizzo A. Meseguer C. Bourdeau & A. Faille J. Fresneda J.M. Victoria J. Comas & V.Rizzo V. Rizzo & J. Comas J. Comas & V.Rizzo J. Comas V. Rizzo V. Rizzo V. Rizzo V. Rizzo & J. Comas J. Fresneda J. Fresneda J. Fresneda V. Rizzo & J. Comas V. Rizzo & J. Comas J.M. Salgado E. Dupré J. Fresneda J. Fresneda V. Grebennikov V. Grebennikov V. Grebennikov V. Grebennikov J.M. Salgado A. Castro C.G. Luque A. Zemlanukhin M.A. Jäch
GU356892 GU356894 GU356895 GU356896 HF912456 GU356897 HE572805 HF912500 GU356899 GU356900 GU356901 HF912499
HF912472 HF912473 HF912495 HF912502 HF912463 HF912454 HF912476 HF912462 HF912491 HF912492 HF912468 HF912470 HF912489 HF912475 HF912477 HF912478 HF912479 HF912481 HF912482 HF912483 HF912484 HF912485 HF912486 HF912457 HF912496 HF912480 HF912458 GU356902 HF912488 HF912487 HF912459 HF912501 HF912474 HF912466 HF912471 HF912490 HF912464 HF912465 HF912467 HF912469 HF912497 HF912493 HF912498 HF912494 HE572801 HE572820 GU356881 GU356882 HE572793 HE572788 HF912453 HE572790 HE572800 GU356849 HE572792 HE572804
GU356790 GU356792 GU356793 GU356794 HF912608 GU356795 GU356796
GU356983
HF912545
HF912616 GU356798 GU356799 GU356800 HF912615
HF912551
HF912604
HF912541
HF912550
GU356839 GU356840 GU356841 HF912506 GU356842 GU356843 HF912513 GU356845 GU356846 GU356847 HF912512 HF912503
GU356937 GU356938 HF912579 GU356939 HF912584 GU356940
HF912583
GU356985 GU356986 HF912591 GU356987 GU356988 HF912598 GU356990 GU356991 GU356992 HF912597
HF912576 HF912527
HF912642 HF912618 HF912620 HF912606
HF912575 HF912552 HF912554 HF912543
HF912540 HF912515 HF912517 HF912504
HF912619 HF912638 HF912639 HF912625 HF912627 HF912636 HF912630 HF912631
HF912553 HF912571 HF912572 HF912559 HF912561 HF912569 HF912563 HF912564
HF912516 HF912537 HF912522 HF912524 HF912535 HF912529 HF912530
HF912588
HF912633
HF912566
HF912532
HF912589
HF912634
HF912567
HF912610 HF912612 HF912632 HF912609
HF912547 HF912565 HF912546
HF912509 HF912531
HF912582
HF912594 HF912601
GU356848 HF912534 HF912533
HF912585
GU356993
GU356801 HF912635
HF912568
HF912617 HF912629 HF912623 HF912628 HF912637 HF912621 HF912622 HF912624 HF912626 HF912613 HF912640 HF912614 HF912641
HF912557 HF912562 HF912570 HF912555 HF912556 HF912558 HF912560 HF912548 HF912573 HF912549 HF912574
HF912514 HF912526 HF912528 HF912520 HF912525 HF912536 HF912518 HF912519 HF912521 HF912523 HF912510 HF912538 HF912511
HF912590 HF912600 HF912586 HF912577
HF912602
HF912599
HF912587
HF912595 HF912603 HF912596
HF912539 HE576704
HE572834
GU356779 GU356780 HE576696 HF912605 HE576703 GU356744 HE576695 HE576719 HE576707
HE572856 HE572872 GU356973 GU356974
GU356829 GU356830
GU356927
HE572875
HE572823
HE572845
HE572876 HE572879
HE572825 HE572833 GU356903 HE572827
HE572847 HE572855
HF912542
HE572877
HE572837
HE572849 HE572873 HE572859
140 141 142 143 144 145
Staphylinidae Staphylinidae Staphylinidae Staphylinidae Staphylinidae Staphylinidae
Omaliinae Oxytelinae Paederinae Phloeocharinae Staphilininae Tachyporinae
Lesteva sp. Thinodromus sp. Paederus sp. Phloeocharis diecki Coprophilus striatulus Tachyporus hypnorum
(Saulcy, 1870) (Fabricius, 1793) (Fabricius, 1775)
MNCN-‐AI450 MNCN-‐AI554 MNCN-‐AI552 MNCN-‐AI880 MNCN-‐AI839 MNCN-‐AI555
E E E E E E
Spain Switzerland Italy Spain England Switzerland
Avila, Puerto Pico de Gredos Pfyn Liguria, Cosio di Arriscia Guipuzcoa, Pagoeta, Legarrola Norfolk, Ludham, How Hill Marsh Pfyn
2005 2005 2005 2006 2006 2005
I. Ribera I. Ribera & A. Cieslak I. Ribera & A. Cieslak C. Hernando I. Ribera I. Ribera & A. Cieslak
HE572803 HE572807 HE572806 HE572813 HE572812 HE572808
HE576706 HE576709 HE576708 HE576714 HE576713 HE576710
HE572836 HE572839 HE572838 HE572842 HE572841 HE572840
HE572858 HE572861 HE572860 HE572864 HE572863 HE572862
Late Pliocene range expansion in a clade of subterranean Pyrenean beetles Valeria Rizzo, Jordi Comas, Floren Fadrique, Javier Fresneda and Ignacio Ribera Appendix S2 Additional methods: (a) list of primers used for sequencing and (b) general PCR conditions.
(a) List of primers: Gene cox1
cob rrnL-nad1
SSU LSU
Name Jerry (M202) Pat (M70) Chy Tom Tom-2 CB3 CB4 16saR (M14) 16Sa 16Sb 16SAlf1 ND1A (M223) 5' b5.0 Ka Kb
Sense F R F R R F R F R R R R F R F R
Sequence CAACATTTATTTTGATTTTTTGG TCCA(A)TGCACTAATCTGCCATATTA T(A/T)GTAGCCCA(T/C)TTTCATTA(T/C)GT AC(A/G)TAATGAAA(A/G)TGGGCTAC(T/A)A A(A/G)GGGAATCATTGAATAAA(A/T)CC GAGGAGCAACTGTAATTACTAA AAAAGAAA(AG)TATCATTCAGGTTGAAT CGCCTGTTTA(A/T)CAAAAACAT ATGTTTTTGTTAAACAGGCG CCGGTCTGAACTCAGATCATGT GCATCACAAAAAGGCTGAGG GGTCCCTTACGAATTTGAATATATCCT GACAACCTGGTTGATCCTGCCAGT TAACCGCAACAACTTTAAT ACACGGACCAAGGAGTCTAGCATG CGTCCTGCTGTCTTAAGTTAC
Reference Simon et al. (1994) Simon et al. (1994) Ribera et al. (2010) Ribera et al. (2010) Ribera et al. (2010) Barraclough et al. (1999) Barraclough et al. (1999) Simon et al. (1994) Simon et al. (1994) Simon et al. (1994) Vogler et al. (1993) Simon et al. (1994) Shull et al. (2001) Shull et al. (2001) Ribera et al. (2010) Ribera et al. (2010)
(b) General PCR conditions: 1 2 3 4 5 6 7
96ºC…...……………..3´ 96ºC…..…………….30-45´´ 47-50ºC……..……….30´´-1´ 72ºC………………….1´ Go to step 2, repeat 34-44x 72ºC………………….7-10´ Hold………………..4ºC
References: Barraclough, T.G., Hogan, J.E. & Vogler, A.P. (1999) Testing whether ecological factors promote cladogenesis in a group of tiger beetles (Coleoptera: Cicindelidae). Proceedings of the Royal Society, Series B, Biological Sciences, 266, 1061–1067.
Ribera, I., Fresneda, J., Bucur, R., Izquierdo, A., Vogler, A.P., Salgado, J.M. & Cieslak, A. (2010) Ancient origin of a western Mediterranean radiation of subterranean beetles. BMC Evolutionary Biology, 10, 29. Shull, V.L., Vogler, A.P., Baker, M.D., Maddison, D.R. & Hammond, P.M. (2001) Sequence alignment of 18S ribosomal RNA and the basal relationships of adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae. Systematic Biology, 50, 945–969. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. & Flook, P. (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reaction primers. Annals of the Entomological Society of America, 87, 651–701. Vogler, A.P., DeSalle, R., Assmann, T., Knisley, C.B. & Schultz, T.D. (1993) Molecular population genetics of the endangered tiger beetle Cicindela dorsalis (Coleoptera, Cicindelidae). Annals of the Entomological Society of America, 86, 142–152.
Late Pliocene range expansion in a clade of subterranean Pyrenean beetles Valeria Rizzo, Jordi Comas, Floren Fadrique, Javier Fresneda and Ignacio Ribera Appendix S3 Additional results: (a) extended dataset; (b) nuclear sequence of the reduced dataset; (c) mitochondrial sequence of the reduced dataset.
(a) Phylogram of the best of 100 replicated topologies obtained with the analysis of the extended dataset with RAxML using a partition by gene, with the geographical distribution of the main clades of Iberian Leptodirini. Numbers in nodes, bootstrap support values. (b) Phylograms of the best of 100 replicated topologies obtained with the analyses of the nuclear genes of the reduced dataset with RAxML using a partition by gene, with the geographical distribution of the main clades of Troglocharinus. Numbers in nodes, bootstrap support values. (c) Phylograms of the best of 100 replicated topologies obtained with the analyses of the mitochondrial genes of the reduced dataset with RAxML using a partition by gene, with the geographical distribution of the main clades of Troglocharinus. Numbers in nodes, bootstrap support values. (d) Pruned, calibrated tree with the inclusion of the missing species in the most likely phylogenetic position according to the morphology of the male genitalia and their geographic distribution, used in the diversification analyses.
T. orcinus orcinus IRC42
59 92
T. elongatus mateui VR4
99
T. jacasi VR22
T. kiesenwetteri sanllorensi VR11
Troglocharinus
T. senenti VR6
92 91
100
T. impellitieri IRC22
T. fonti fonti IRC26
100
T. quadricollis AI578
T. ferreri AI1065
Lagariella colominasi IRC30
62
97
Stygiophyes hansferyi IRC39
Stygiophyes akarsticus IRC7
94 100
61
Naspunius eseranus IRC29
Salgadoia brieti AI587
Trapezodirus carrodillae IRC9
Paraspeonomus vandeli AI539
68
Speonomus carrerei AI530
Speonomus stygius AI534
100
100
Speonomus diecki AI536
Pyrenees
Speonomus abeillei RB2
Speonomites antemi AI1074
85
98
Speonomites tincatincensis IRC6
Antrocharis querilhaci AI594
100
Gesciella delioti AI525
70
Ceretophyes riberai AI597
99
96
Perriniella bofilli IRC40
Bathysciella jeanneli AI540
64
100
Phacomorphus duprei AI912
Phacomorphus sioberi AF27
100 100
Bellesia espanyoli IRC21
100
57
Machaeroscelis infernus AI533
Speonomus gaudini IRC18
100
Speonomus orgibetensis AI526
Parvospeonomus delarouzeei IRC35
100
Pseudospeonomus raholai IRC41
99
Speocharidius galani AI580
80
Speocharidius breuili IRC11
100
80
Leptodirina
Josettekia mendizabali IRC19
Euryspeonomus ciaurrizi IRC16 Bathysciola mystica AI601
62
Bathysciola rugosa IRC12
Bathysciola diegoi IRC14
100
Espanoliella luquei AI573
Espanoliella jeanneli IRC4
100
Quaestus jeannei p AI1075
100
Breuilites eloyi AI662
Anillochlamys subtruncata c IRC1
64
97
Paranillochlamys velox IRC37
Bathysciola zariquieyi AI1255
61
39
Notidocharis laurae AI664
Platycholeus sp. AF218
100
99
100
north Mediterranean coast & Pyrenees
Bathysciola catalana IRC13 100
100
central Mediterranean coast
Spelaeochlamys ehlersi IRC31
100 81
Cantabrian mountains
Quaestus noltei IRC3
99
Leptodirini
Euryspeonomus beruetei IRC15
82
92
Aranzadiella leizaolai IRC17
Speonomidius crotchi AI1067
Platycholeus opacellus AF217
Cantabrian mountains
Platycholeina
Ptomaphagus pyrenaeus RA261
Ptomaphagini
Ptomaphagus tenuicornis AI760
Ptomaphagus troglodytes IRC33
Adelopsis AF221
Speonemadus angusticollis IRC38 100
CHOLEVINAE
98
100
Speonemadus bolivari AF56
Speonemadus clathratus HI21
Anemadus cf. smetanai RA187
78 Sciaphyes shestakovi AF66
Sciaphyes shestakovi AF108
100
84
Sciaphyes sibiricus AF68
100
100
Catops fuliginosus IRC43
Catops ventricosus AF32
Catops AF30
97
Catops subfuscus AI550
Sciodrepoides watsoni AF222
100
Fusi cf. nyujwa AF67
56 100
100
Choleva kocheri AF216
Nargus velox AI927
Nargus algiricus AI574
Falkonemadus BMNH673231
59
Dissochaetus AF219
Eucatops AF220
98
Agathidium AI1305
79 100
Silphopsyllus desmanae RA265
Leptinus testaceus AF82
LEIODINAE
PLATYPSYLLINAE
Phloeocharinae Phloeocharis AI880
87
Tachyporinae Tachyporus AI555 98
83
Cholevini
Choleva angustata AF144
Choleva cisteloides AF197
100
100
Sciaphyini
Catops andalusicus AF72
52
LEIODIDAE
Anemadini
Speonemadus maroccanus AF215
100
96
Speonemadus angusticollis AI592
Oxytelinae Thinodromus AI554
Staphylininae Coprophilus AI839
Micropeplinae Micropeplus AI521
Omalinae Lesteva AI450
STAPHYLINIDAE
Paederinae Paederus AI552 0.2
84
T. ferreri AI1065 T. ferreri pallaresi VR3 T. quadricollis AI578
96
T. elongatus mateui VR4 91
Troglocharinus
coastal clade
T. impellitieri IRC22
50
85
T. fonti fonti IRC26
T. orcinus orcinus IRC42 100
Pyrenean clade
T. senenti VR6 T. senenti AI585 Trapezodirus cerberus AI600
98
Trapezodirus carrodillae IRC9 Salgadoia brieti AI587
50
Speonomus stygius AI529
78
Speonomus diecki AI536 Speonomus fagniezi AF125
68
70
Paraspeonomus vandeli AI539 Speonomus carrerei AI530
Speonomus zophosinus AI667 Stygiophyes akarsticus IRC7 Stygiophyes hansferyi IRC39 Lagariella colominasi IRC30
100
Naspunius eseranus IRC29 97
Speonomites antemi AI1074 Speonomites tincatincensis IRC6
87
Antrocharis querilhaci AI588 Paratroglophyes carrerei AF184
100
Gesciella delioti AI525 0.03
T. elongatus elongatus VR1 67 T. elongatus mateui VR4 T. elongatus mateui VR5 51 T. elongatus VR24 99 T. elongatus ollai VR17 T. espanoli VR23 100 T. orcinus orcinus IRC42 96 T. orcinus orcinus VR9 65 T. orcinus VR10 99 T. orcinus acevedoi VR21 T. orcinus acevedoi VR20 98 100 Troglocharinus sp. VR8 91 Troglocharinus sp. VR7 T. elongatus AF115 62 T. elongatus pinyareti VR38 92 T. elongatus ollai VR15 88 T. jacasi VR22 97 coastal clade T. jacasi VR2 99 T. kiesenwetteri sanllorensi VR12 100 T. kiesenwetteri sanllorensi VR11 T. kiesenwetteri sanllorensi VR14 66 T. kiesenwetteri kiesenwetteri VR18 88 99 T. kiesenwetteri kiesenwetteri VR39 97 T. patracoi VR16 T. kiesenwetteri andresi VR13 77 73 T. ferreri VR26 100 T. ferreri VR28 T. ferreri VR29 T. ferreri VR32 88 T. ferreri VR33 97 T. ferreri VR35 99 T. ferreri ferreri AF172 T. ferreri VR34 100 100 T. ferreri VR31 Troglocharinus 100 90 T. ferreri VR30 100 T. ferreri AI1065 T. ferreri pallaresi VR3 T. senenti VR6 98 T. hustachei VR36 T. senenti AI585 54 94 T. fonti AF181 79 T. impellitieri AF182 Pyrenean clade T. fonti AF130 69 T. impellitieri IRC22 90 38 T. fonti schuettei VR37 T. quadricollis AI578 99 T. fonti fonti IRC26 Speonomus diecki AI536 99 100 Speonomus fagniezi AF125 Speonomus stygius AI529 89 Speonomus abeillei RB2 96 Speonomus zophosinus AI667 Paraspeonomus vandeli AI539 71 57 Speonomus carrerei AI530 Stygiophyes hansferyi IRC39 99 Stygiophyes akarsticus IRC7 Lagariella colominasi IRC30 93 Naspunius eseranus IRC29 100 92 Salgadoia brieti AI587 Trapezodirus carrodillae IRC9 100 Trapezodirus cerberus AI600 Pallaresiella pallaresana AF123 100 97 Speonomites tincatincensis IRC6 Speonomites antemi AI1074 Trocharanis mestrei AC112-169 Paratroglophyes carrerei AF184 Gesciella delioti AI525 Antrocharis querilhaci AI588 98
100
83 100
0.08
T. elongatus elongatus VR1 T. elongatus mateui VR4 T. olerdolai T. schibii T. elongatus portai T. elongatus abenzai T. elongatus ollai VR17 T. espanoli VR23 T. orcinus acevedoi VR20 T. orcinus VR10 T. orcinus lagari T. elongatus ollai VR15 T. elongatus pinyareti VR38 T. elongatus AF115 T. sp VR8 T. jacasi VR22 T. kiesenwetteri kiesenwetteri VR18 T. patracoi VR16 T. kiesenwetteri sanllorensi VR14 T. kiesenwetteri andresi VR13 T. ferreri VR30 T. ferreri pallaresi VR3 T. ferreri VR26 T. ferrei abadi T. fonti schuettei VR37 T. impellitieri C22 T. fonti infernus T. fonti zariquieyanus T. fonti AF130 T. senenti VR6 T. hustachei VR36 T. rovirai T. quadricollis AI578 T. fonti C26 T. vinyasi T. subilsi T. ludovici 4.0
3.0
2.0
1.0
0.0