Introduction

The genus Trechus (Coleoptera, Carabidae, Trechinae) includes more than 800 species, most of them in the Palearctic area (Moravec et al.2003; Lorenz 2005). This genus is known to contain many wingless short range endemic species (Jeannel 1927, Casale and Laneyrie 1982, Schmidt 2009) and is currently understood as polyphyletic (Faille et al. 2010a, 2011a).

Jeannel (1927) gathered seven species from the uhagoni group distributed from the French slope of the Pyrenees to the Cantabrian area: Trechus bonvouloiri Pandellé, 1867 (France: Hautes–Pyrénées), Trechus bordei Peyerimhoff, 1909 (France: Pyrénées–Atlantiques), Trechus brucki Fairmaire, 1862 (France: Hautes–Pyrénées, Pyrénées–Atlantiques), Trechus grenieri Pandellé, 1867 (France: Hautes–Pyrénées), Trechus navaricus Vuillefroy, 1867 (France: Pyrénées–Atlantiques), Trechus sharpi Jeannel, 1921 (Spain: Cantabria) and Trechus uhagoni Crotch, 1869 (Spain: Navarra). This group of species was also considered close to the group of Trechus angusticollis Kiesenwetter, 1850, a Pyreneo–Cantabrian group with nine species, all apterous, orophilous or troglobitic.

In this paper we describe a species collected by traps in a MSS (mesovoid shallow substratum, “Milieu Souterrain Superficiel” sensu Juberthie et al. 1980, Giachino and Vailati 2010) in the Sierras de Urbasa–Andía (Western pre–Pyrenees, Navarra, Spain) and a second orophilic species from the French Central Pyrenees. We study the phylogenetic relationships of the new species and provide a molecular phylogeny of the group, including all known species but four.

Historical background

Trechus uhagonii was described by Crotch in 1869 and dedicated to S. de Uhagon with whom he visited caves in the Alsasua area in June 1869. Trechus bruckii was first described under the name Trechus politus by Fairmaire (1862b). He renamed it one year later to bruckii because politus was already in use for an American species. The two names were corrected to uhagoni and brucki by subsequent authors, and recently renamed uhagonii and bruckii in catalogues (Lorenz 1998, 2005, Moravec et al. 2003, Queinnec and Ollivier 2011). As the names uhagonii and bruckii have not been used since their description, we choose to keep the prevailing usage of uhagoni and brucki in accordance with the article 33.3.1 of the International Code of Zoological Nomenclature on incorrect subsequent spellings (ICZN 1999).

In the Monographie des Trechinae, Jeannel (1927) erected the uhagoni group for the seven species of Trechus from the Pyreneo–Cantabrian area quoted above. Español (1970) described Trechus ortizi from a cave of Burgos province (Spain), and included it in the uhagoni group, close to Trechus bordei.

The uhagoni group sensu Jeannel (1927), although poorly defined morphologically, was enriched with 5 species by Casale and Laneyrie (1982) in their catalogue of species of world Trechinae: Trechus pecoudi Colas & Gaudin, 1935 (described first as a subspecies of Trechus brucki), Trechus ortizi, Trechus escalerae Abeille de Perrin, 1903 (considered by Jeannel (1927) to belong to the Trechus angusticollis group), Trechus enigmaticus Coiffait, 1971 and Trechus aubryi Coiffait, 1953. Trechus uhagoni was here considered as subspecies of Trechus grenieri. By describing Trechus baztanensis from a cave of Navarra, Dupré (1991) suggested that the peculiar genital morphology of Trechus bordei, Trechus navaricus, Trechus bonvouloiri and the new species should lead to their removal from the uhagoni group, and he created the bonvouloiri group for these species, opinion followed by Queinnec and Ollivier (2011). Toribio and Rodríguez (1997) added one species from Cantabria to the uhagoni group, Trechus carrilloi, a species collected in the MSS. Sciaky (1998) described Trechus jeannei from Cantabria, and included it in the uhagoni group, close to Trechus bordei and Trechus ortizi. Hernando (2002) described Trechus comasi from a cave of Navarra and suggested that it should be considered as sister species of Trechus brucki. Molecular and morphological evidence suggest that this species should be removed from the uhagoni group (Faille et al. 2010a, 2011a, Ortuño and Arribas 2010). Ortuño and Toribio (2005) described a new species of Trechus belonging to another group of species, indicating that 11 species belong to the uhagoni group in the Iberian Peninsula. Reboleira et al. (2010) considered that 10 species belong to this group in the Peninsula, without providing the list of taxa included.

Trechus brucki is an alpine species located at high altitude in the central and western Pyrenees, and it is until now not recorded from the Spanish slope of the chain (Serrano 2003). Colas and Gaudin described Trechus pecoudi in 1935 from the western Pyrenees (Pic d´Orhy) as a subspecies of Trechus brucki. Coiffait (1952) described 3 subspecies of Trechus brucki; Trechus brucki vandeli, Trechus brucki truilheti and Trechus brucki microthorax. The subspecies vandeli and truilheti were later related to Trechus pecoudi (Casale & Laneyrie, 1982) so that Trechus pecoudi counts three subspecies in the Catalogue of Palearctic Coleoptera (Moravec et al. 2003). Queinnec and Ollivier (2011) considered Trechus pecoudi as a subspecies of brucki restricted in the Anie and Orhy massifs, whereas the subspecies vandeli, described from Anie, was considered a synonym of Trechus brucki brucki together with the subspecies truilheti and microthorax.

Materials and methods Taxon sampling, Morphological study, DNA extraction and sequencing

Specimens were collected by hand or by means of pitfall traps containing water saturated in salt or propylene glycol, known to preserve DNA (Rubink et al. 2003, López and Oromí 2010) (Table 1). The protocol is detailed in Faille et al. (2010b):

Extractions of single specimens were non–destructive, using the DNeasy Tissue Kit (Qiagen GmbH, Hilden, Germany). After extraction, specimens were mounted on cards and genitalia stored in water–soluble dimethyl hydantoin formaldehyde resin (DMHF) on transparent cards, pinned beneath the specimen. Vouchers and DNA samples are kept in the collections of ZSM, IBE and MNHN.

We included examples of most species of the Trechus uhagoni group, with the exception of Trechus bruckoides sp. n., Trechus carrilloi and Trechus sharpi and some examples of Trechus of the angusticollis group sensu Jeannel (1927) and Casale and Laneyrie (1982) (Table 1). The tree was rooted with Aphaenops leschenaulti Bonvouloir, 1862, which is known to belong to a different group of Pyrenean Trechini (Jeannel 1927, Faille et al. 2010a).

We amplified fragments of four mitochondrial genes: 3’ end of cytochrome c oxidase subunit (cox1); a single fragment including the 3’ end of the large ribosomal unit (rrnL), the whole tRNA–Leu gene (trnL) and the 5’ end of the NADH dehydrogenase 1 (nad1);and one nuclear gene (internal fragment of the large ribosomal unit 28S rRNA, LSU) (see Table 2 for primers used). Sequences were assembled and edited using Sequencher TM 4.8 (Gene Codes, Inc., Ann Arbor, MI). Parts of the sequences for 14 of the species were taken from Faille et al. (2010a) and Faille et al. 2011a (Table 1).

New sequences have been deposited in the EMBL database with Accession Numbers HE817887–HE817940 (Table 1).

Phylogenetic analyses

We aligned the sequences using the MAFFT online v.6 and the Q–INS–i algorithm (Katoh and Toh 2008), a progressive pair–wise method with secondary refinement. We used Maximum Likelihood as implemented in the on–line version of RAxML (which includes an estimation of bootstrap node support, Stamatakis et al. 2008), using GTR+G as the evolutionary model and three partitions corresponding to the cox1, rrnL+trnL+nad1 and LSU fragments.

The aedeagus and genital duct were extracted and included in a drop of Canada balsam or dimethyl hydantoin formaldehyde resin (DMHF) on a transparent slide. Preparations were mounted below the specimen, on the same pin. Pictures were taken with microscopes Olympus ch and Olympus szx16, coupled with a camera Olympus c5060wz. Serial pictures were combined using the CombineZP software, and finally processed using Adobe Photoshop CS.

Institutional codes and abbreviations used in the taxonomic treatment and private collectors

IBE Institute of Evolutionary Biology (CSIC-UPF), Barcelona (Spain).

MNCN Museo Nacional de Ciencias Naturales (CSIC), Madrid (Spain)

MNHN Muséum National d´Histoire Naturelle, Paris (France).

MZB Museu de Ciències Naturals (Zoologia), Barcelona (Spain).

ZSM Zoologische Staatssammlung, München (Germany).

MFN Museum für Naturkunde, Berlin (Germany).

CAF coll. A. Faille (Paris, France).

CCB coll. C. Bourdeau (Rebigue, France).

CJF coll. J. Fresneda (Llesp, Spain).

CMT coll. M. Toribio (Madrid, Spain).

LE Length of elytra.

LP Length of pronotum.

WE Width of elytra.

WH Width of head.

WP Width of pronotum.

WPB Width of pronotal base.

Results Discussion

The molecular phylogeny (Fig. 34) suggests a well–supported clade gathering the following species:

Trechus beusti (Fig. 4), Trechus bouilloni sp. n.(Fig. 1), Trechus grenieri (Fig. 2), Trechus brucki (Fig. 7), Trechus pieltaini (Fig. 5) and Trechus uhagoni (Fig. 3). This result is in accordance with morphology: all the species of the clade share the aedeagal median lobe long and strongly curved just behind basal bulb, with terminal lamella well–developed. Moreover, the clade is supported by a strong synapomorphy: all the species share a strongly sclerotized part of the sperm duct, forming a second copulatory piece (Figs 15, 17, 19, 21, 23, 27: CP2). This synapomorphy is also present in Trechus bruckoides sp. n.(Fig. 25). Consequently, molecular and morphological results allow us to define the Trechus brucki group sensu novo: Trechus beusti, Trechus bouilloni sp. n., Trechus brucki, Trechus bruckoides sp. n., Trechus grenieri, Trechus uhagoni and Trechus pieltaini.

Two species, Trechus pieltaini and Trechus beusti, were included by Jeannel (1927) in the angusticollis group. Trechus carrilloi and Trechus sharpi are provisionally not included in the group because of the absence of sclerotization of the sperm duct (CP2). Moreover, concerning Trechus carrilloi, the apical hook is not a synapomorphy, as several other Trechus groups have this kind of hook (i.e. Trechus aubryi or the Tibetan species Trechus bastropi Schmidt, 2009 and Trechus damchungensis Deuve, 1997). As expected by Dupré (1991) for some of the species that he included in a “bonvouloiri group”, the following regional species previously considered part of the brucki group are clearly excluded here: Trechus escalerai, Trechus navaricus, Trechus bordei, Trechus jeannei, Trechus bonvouloiri. Although not included in our analyses, we also exclude of the group Trechus ortizi, Trechus baztanensis and Trechus enigmaticus, as those species were put into the uhagoni group because of close morphological affinities with Trechus bordei and Trechus navaricus (Español 1970, Coiffait 1971, Dupré 1991). Trechus jeannei, which was said to be close to Trechus bordei (Sciaky 1998), does not belong to the Trechus brucki clade and is not clearly related with the Trechus bordei clade. A study including more Iberian species should clarify its phylogenetic affinities. Trechus aubryi is excluded from the Trechus brucki clade and shares strong affinities with Trechus distinctus.

Our molecular results as well as genital morphology, in dorsal and lateral view and shape of copulatory piece (Figs 10, 19, 20), suggest that Trechus uhagoni could be considered a distinct species from Trechus grenieri. Morphological differences between the two species are the following:

– Trechus grenieri: aedeagus in dorsal view (Fig. 9) with subparallel sides, round apex with a short triangular tip; in lateral view (Fig. 17) basal third strongly rounded, median lobe slightly angular in the middle; apical hook with a thin tip. The copulatory piece is an asymmetrical gut slightly tapering and filled with a densely scaly area (Fig. 18).

– Trechus uhagoni: aedeagus in dorsal view (Fig. 10) with the left side narrowed or sinuate from the middle to apex, the left side of apical quarter deeply narrowed, forming a long triangular tip; in lateral view (Fig. 19) only the basal quarter rounded, median lobe without dorsal angle in the middle, short with apical hook with massive tip. The copulatory piece is similar to the one of grenieri but the gut is parallel and shortened in its apical part (Fig. 20).

With 7 subspecies recognized in the last catalogues (Moravec et al 2003, Queinnec and Ollivier 2011), Trechus grenieri currently lives in humid forests (1000 m) from Espinal to Iraty (ssp. ruteri), then from Gave de Pau river (north side of Pic de Montaigu) to the Neste d’Aure valley (ssp. grenieri). From Aure valley to the Salat, it is replaced by the subspecies despaxi, which crosses the Garonne river near Saint Béat (Haute Garonne). An isolated subspecies (prepyrenaeus) was described by Coiffait in the high Arize valley (Andronne and Bosc forests around 1000 m) (Coiffait 1974). Along the axial ridge, Trechus grenieri lives above 1500 m from high Garonne valley (ssp. bepmalei) to Mont Valier (ssp. aulaensis Aubry, 1981). Study of numerous specimens suggests that Trechus uhagoni and all the specimens of the subspecies ruteri are morphologically close, especially in the shape of the male genitalia, and should be considered as a distinct species. Moreover, we studied specimens from various localities of the Pyrenean range (see Distribution) and established that they share some morphological characters (color pale, brown, pronotum transverse with lateral margin regularly rounded, elytral striae superficial, weakly impressed) that justify keeping the status of ruteri as a subspecies of Trechus uhagoni. The subspecies ruteri should then be considered as belonging to uhagoni, so that grenieri is restricted to the area between Gave de Pau and Ariège valley, northern slope of Pyrenees. Trechus uhagoni ruteri n. comb. could be distinguished from Trechus uhagoni uhagoni by its color, usually paler brown, the pronotum transverse with lateral margin regularly rounded and the elytral striae superficial, weakly impressed. It is restricted to the western Pyrenees.

The study of specimens from the whole range of Trechus grenieri including all the subspecies, most of the types and material from intermediate localities (see distribution) leads us to conclude that the characters used to discriminate the subspecies (size, eyes size, shape of elytra and pronotum) are inconstant and overlapping between populations. The shape of the male genitalia is similar for all the populations between Gave de Pau and Ariège valley, including the one (ssp aulaensis) which was said to be different (Queinnec and Ollivier 2011). We consider then Trechus grenieri as a single species without any valid subspecies: Trechus grenieri grenieri Pandellé, 1867 = Trechus grenieri bepmalei Jeannel, 1921 = Trechus grenieri despaxi Jeannel, 1922 = Trechus grenieri aulaensis Aubry, 1981 = Trechus grenieri prepyrenaeus Coiffait, 1974, syn.n.

Trechus beusti was described by Schaufuss (1863). The type series is located in the Schaufuss collection in the Museum für Naturkunde, Berlin (M. Jaeger pers. com.). Trechus pieltaini was described by Jeannel (1920) from a cave of the Basque country, Cueva de Mairruelegorreta. Bolívar y Pieltain and Jeannel (1921) suggested that the peculiar morphology of the aedeagus of these two species indicates clear affinities with Trechus uhagoni and Trechus grenieri. Surprisingly, Jeannel (1927) in his Monographie des Trechinae considered that these two species belong to another group of species, the Trechus angusticollis group. This opinion was followed by subsequent authors (Español 1965, Casale and Laneyrie 1982, Ortuño and Marcos 2003). However, and in accordance with the morphology of the median lobe of the aedeagus, molecular results support Bolívar y Pieltain and Jeannel’s (1921) point of view and confirm the close affinities between Trechus beusti, Trechus pieltaini and thespecies of the Trechus brucki clade. Differences between the two species are weak: the apical part of the aedeagus is longer in Trechus pieltaini (Fig. 12, 23) than in Trechus beusti (Fig. 11, 21). The copulatory pieces are almost identical (Fig. 22, 24). Trechus beusti is larger, with elytra more oval and elytral striae less impressed (Fig. 4); Trechus pieltaini is smaller, narrower, and elongate with subparallel elytra and striae more impressed (Fig. 5).

Trechus brucki and Trechus bruckoides sp. n.do not have the peculiar hooked apex of the median lobe observed in the other species of the clade, but the apex is nevertheless strongly curved (Figs 25, 27).

The case of two further species remains doubtful: Trechus carrilloi was included by its descriptor in the uhagoni group especially because of the structure of the aedeagus, with an apex with an apical hook (Fig. 31). However, the secondary sclerotization of the ejaculatory duct is lacking in this species and it is characterized by a homogenous elytral pubescence which is present in other species of the area (Ortuño and Jimenez–Valverde 2011), but lacking in all the species of the Trechus brucki clade sensu novo. The presence of a hook at the apex of the aedeagus is also known in other Pyreneo–Cantabrian species like Trechus arribasi Jeanne, 1988, currently included in the Trechus fulvus group (Toribio 2001, Reboleira et al. 2010) or Trechus aubryi from Ariège. This character led its descriptor to include Trechus aubryi in the Trechus uhagoni group. Queinnec and Ollivier (2011) included the species in the Trechus angusticollis group. The species appears to be the sister species of Trechus distinctus (Fig. 34).

Finally, Trechus sharpi was included in the Trechus uhagoni group by Jeannel (1927), but the shape of the median lobe of the aedeagus and the copulatory piece, that shares some similarities with the Trechus bordei group, together with the lack of the sclerotization of spermiduct present in all the species of the Trechus brucki group sensu novo, cast doubts on its real phylogenetic affinities.

The species of the Trechus brucki clade are humicolous (grenieri, uhagoni), orophilous (grenieri, brucki, bruckoidessp. n.), or troglobitic/subterranean (bouilloni sp. n., pieltaini, beusti). We can notice a coincidence in the ecology of Trechini and Leptodirini with Basque–Pyrenean distribution: whereas the species are humicolous (or nivicolous for some Trechini) in the Pyrenees, the species occurring in the Basque country are mainly hypogean (Salgado et al. 2008, Ribera et al. 2010).

Biogeography of the Trechus brucki clade

If we use the standard mitochondrial mutation rate for insects of 2.3% divergence per Myr (0.0115 substitutions ⁄ site ⁄ Myr) (Brower 1994, Papadopoulou et al. 2010, Pons et al. 2010, Ribera et al. 2010), the isolation between the Trechus navaricus and Trechus brucki group seems to have occurred at the end of the Pliocene (Faille et al. 2011a). Pliocene climate was much warmer than the Present (Uriarte 2003): the interval between 3.3 Myr to 3 Myr was called Mid Pliocene warm Period, with an average temperature of about 3°C higher than at present and an annual average precipitation between 400 to 1000 mm higher than present. The transition to the Pleistocene (ca 2.7 Myr) is marked by the onset of marked climatic variability; the radiation of the Trechus brucki clade occured during the Pleistocene, following the rhythm of alternations of cold, warm/humid and dry periods that led to changes in biome composition (Salzmann et al. 2008).

Strong erosion leading to a deep excavation of Pyrenean valleys associated with climate variations led to the dispersal and diversification of the brucki clade. The main events are (Barrère 1963, Campos 1979, Serrat and Ventura 1993, Calvet 2004):

1. Persistence of the Ebro depression between the Basque–Pyrenean area and the Iberian central plateau. The persistence of the Ebro salty basin from the late Oligocene (25 Ma) until the late Miocene (6 Ma) isolated groups with an Iberian distribution from those with a Pyrenean or Basque–Pyrenean distribution. This flat and shallow lagoon area received the tributaries of the Ebro river, from Reinosa to the Mediterranean Sea.

2. Impact of Quaternary erosion on karst fragmentation. On the northern slope, the folds which have an east–west orientation are narrow and divided by north–south valleys. On the southern slope, orogenesis caused the formation of two folds with an east–west orientation (internal and external “sierras”) parallel to the axial chain. Similarly, Quaternary erosion separated these sierras by narrow north–south valleys. Near the Atlantic, these “sierras” meet with Basque folds which have a complex north–west/south–east orientation, divided by narrow north–south valleys, from Bilbao to Alsasua. Between Vitoria and Pamplona, these Basque “sierras” are separated by the Pre–pyrenean middle depression, a broad valley excavated by the Zadorra (westward) and Arakil (eastward) rivers (Fig 35). These rivers flow into the Ebro Basin, separating the northern massifs of Aralar, Urquilla and Gorbea from the southern Sierra of Urbasa–Andía. The hydrographic system was set mainly by significant erosion due to numerous glaciation cycles during the Pleistocene (2.5 Ma).

Our molecular study suggests that the brucki lineage could have originated in the area delimited by the northern sierras of Gorbea and Urquilla and the edge of the sierra de Andía. The sierras de Andía, Urbasa and Entzia form the exact border between the hypogean fauna of the Pyrenees and Iberia. North of this limit occur Trechus bouilloni sp. n., Troglorites breuili Jeannel, 1919 (Carabidae, Pterostichini) –Urbasa–Andía–Entzia, Aralar, Ernio and Pagoeta massifs, between the Deba and Urola rivers (Ortuño et al. 2010)–, Euryspeonomus eloseguii (Español, 1948), Bathysciola rugosa (Sharp, 1873), –Leiodidae, Cholevinae, Leptodirini which also belong to a clade of Basque–Pyrenean distribution (Ribera et al. 2010)– whereas south of this area (Sierras de la Demanda and Lóquiz, surrounding the Ebro basin) is characterized by a lack of Leptodirini and Troglorites. The only cave Coleoptera is Trechus schaufussi comasi (Basaula cave in Baríndano, south of Urbasa). Trechus schaufussi Putzeys, 1870 is a model of Iberian extensive distribution: it is widespread in the Iberian Peninsula, from Algarve in Portugal to Cantabria, Iberian Central System and the pre–pyrenean massif of Guara in Spain (Jeanne and Zaballos 1986, Zaballos and Jeanne 1994, Serrano 2003). This species is known to have separated early from Trechus sensu stricto (Faille et al. 2010a, 2011a).

Trechus bouilloni sp. n. has a subterranean lifestyle among the scree–covered northern slope (900 m) of Sierra de Andía, whereas the type locality of Trechus uhagoni is the Orobe doline (700 m), located at the eastern limit of Sierra de Urquilla. Early Pleistocene climate variations could have led to drastic changes in biome composition, limiting dispersal possibilities and leading to the isolation of the population of Trechus bouilloni sp. n. (potentially forestal), south of the Arakil River. One hypothesis could be that the hygrophilous species were colonizing high altitude or hypogean habitats during interglaciar warming as observed in other species of Coleoptera (Hernando et al. 1999, Faille et al. 2011b). These climate fluctuations might also have led to the western subterranean colonization of the two hypogean species, Trechus beusti (Sierra de Urquilla) and Trechus pieltaini (Sierra de Gorbea) while the group colonized the Pyrenean chain and diversified in numerous forms living in humid forests and alpine zones, from the Iraty Valley to the Ariège Basin. Migration toward East could have been possible along the small sierras of Tajonar and Labia, which link the Basque Mountains to the Pyrenees.

Trechus brucki lives in the alpine zone (above 1700 m) of the axial ridge from Pic d´Orhy to Col du Pourtalet, in the high Ossau Valley. On the north ridge, Trechus brucki can be encountered in the same biotopes, near snow tongues melting on scree-covered slopes, from Aspe to Gave de Pau Valleys. As for Trechus grenieri and Trechus uhagoni, both are mainly forestal and occur at lower altitude except for Trechus grenieri in the eastern part of the range (Mount Valier area). The Ariège Valley is the eastern limit of the group.

The distribution area of the Trechus brucki group coincides with the one of the Basque–Pyrenean Leptodirini clade (Fig 35). In the Pyrenees, both groups are made up of forestal, endogean, humicolous, lapidicolous or orophilous, but not hypogean, species. It is only in Basque relief, the western part of their distribution, that both groups include subterranean species. Regarding Leptodirini, the basal group of the Basque–Pyrenean clade is the Bathysciola schiodtei group (endogean/humicolous elements); its distribution area is extended from Ariège, Trechus mystica Fresneda & Fery, 2009 (France: Haute–Garonne and Ariège; Spain: Val d’Aran) to the Basque relief, Bathysciola breuili Bolívar, 1921 in Peña Gorbea or Bathysciola rugosa (Sharp, 1873) in Sierra de Urbasa and Urquilla. A high degree of troglobiomorphy is only found in some hypogean species of the Basque area: Aranzadiella Español, 1972 (basin of Deba River), Euryspeonomus Jeannel, 1919 (Aralar, Urbasa/Andía and Baztan Valley), Josettekia Bellés & Déliot, 1983 (Ernio and Aralar massifs), Nafarroa Fresneda & Dupré, 2010 (Kintoa Massif) and Speocharidius Jeannel, 1919 (between the Urola and Orio Rivers). In the Pyrenees, the species of the Trechus brucki clade are epigean, forestal (Trechus grenieri, Trechus uhagoni ruteri) or orophilous(Trechus brucki, Trechus bruckoides sp. n.). Pyrenean speciation events in the group are more recent and are probably closely related to late Pleistocene climatic changes, as already observed in alpine Trechus (Lohse et al. 2011). Troglobiomorphic features (depigmentation, microphthalmy) only occur in the hypogean Trechus beusti and Trechus pieltaini, both of them located in the Basque area. The two other species of this geographical area are located in wet and cold dolines (Trechus uhagoni) or subterranean environments (Trechus bouilloni sp. n.). Their general appearance (pigmented, well–developed eyes), similar to other epigean species, could be an indication of the recent colonization of this reduced habitat.

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