Molecular Phylogenetics and Evolution 54 (2010) 97–106 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev A molecular phylogeny shows the single origin of the Pyrenean subterranean Trechini ground beetles (Coleoptera: Carabidae) A. Faille a,b,*, I. Ribera b,c, L. Deharveng a, C. Bourdeau d, L. Garnery e, E. Quéinnec f, T. Deuve a a Département Systématique et Evolution, ‘‘Origine, Structure et Evolution de la Biodiversité” (C.P.50, UMR 7202 du CNRS/USM 601), Muséum National d’Histoire Naturelle, Bât. Entomologie, 45 rue Buffon, F-75005 Paris, France b Institut de Biologia Evolutiva (CSIC-UPF), Passeig Maritim de la Barceloneta 37-49, 08003 Barcelona, Spain c Museo Nacional de Ciencias Naturales (CSIC), José Gutiérrez Abascal 2, 08006 Madrid, Spain d 5 chemin Fournier-Haut, F-31320 Rebigue, France e Laboratoire Evolution, Génomes, Spéciation, CNRS UPR9034, Gif-sur-Yvette, France f Unité ‘‘Evolution & Développement”, UMR 7138 ‘‘Systématique, Adaptation, Evolution”, Université P. & M. Curie, 9 quai St–Bernard, F-75005 Paris, France article info abstract Article history: Trechini ground beetles include some of the most spectacular radiations of cave and endogean Coleoptera, Received 16 March 2009 but the origin of the subterranean taxa and their typical morphological adaptations (loss of eyes and Revised 1 October 2009 wings, depigmentation, elongation of body and appendages) have never been studied in a formal phylo- Accepted 5 October 2009 genetic framework. We provide here a molecular phylogeny of the Pyrenean subterranean Trechini based Available online 21 October 2009 on a combination of mitochondrial (cox1, cyb, rrnL, tRNA-Leu, nad1) and nuclear (SSU, LSU) markers of 102 specimens of 90 species. We found all Pyrenean highly modified subterranean taxa to be monophyletic, to Keywords: the exclusion of all epigean and all subterranean species from other geographical areas (Cantabrian and Subterranean environment Iberian mountains, Alps). Within the Pyrenean subterranean clade the three genera (Geotrechus, Convergence Endogean Aphaenops and Hydraphaenops) were polyphyletic, indicating multiple origins of their special adaptations Troglobitic to different ways of life (endogean, troglobitic or living in deep fissures). Diversification followed a Trechinae geographical pattern, with two main clades in the western and central-eastern Pyrenees respectively, Aphaenops and several smaller lineages of more restricted range. Based on a Bayesian relaxed-clock approach, and using as an approximation a standard mitochondrial mutation rate of 2.3% MY, we estimate the origin of the subterranean clade at ca. 10 MY. Cladogenetic events in the Pliocene and Pleistocene were almost exclusively within the same geographical area and involving species of the same morphological type. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction (Caccone, 1985). Amongst insects, many groups of Coleoptera have repeatedly colonised subterranean habitats, but two of them are The origin and evolution of cave organisms has fascinated evo- particularly diverse: Leiodidae (especially subfamily Cholevinae) lutionists and biologists for more than two hundred years, since in the suborder Polyphaga, and Carabidae of the subfamily Trechi- the discovery of the first troglobitic species (Proteus anguinus, de- nae in the suborder Adephaga (Casale et al., 1998). Subterranean scribed by Laurenti, 1768). Organisms living in a subterranean species of both groups share morphological modifications consid- environment tend to show a highly modified morphology and biol- ered to be adaptations to a subterranean lifestyle: loss of metatho- ogy, and a mixture of losses (eye degeneration, depigmentation) racic wings, eyes and pigment, similar changes in body shape and and adaptations (development of sensory organs, changes in the size (Jeannel, 1926a,b; Vandel, 1964; Barr and Holsinger, 1985), life cycle and metabolism, body shape modifications) (Racovitza, and modifications in their way of life (Deleurance, 1958). The 1907; Vandel, 1964; Culver et al., 1990). Troglobitic invertebrates extensive convergence in morphological characters obscures the isolated in karstic areas are also very good models to study speci- phylogenetic relationships among species (Marquès and Gnaspini, ation and diversification, because of the isolation of populations in 2001; Desutter-Grandcolas et al., 2003), which has resulted in a well-defined karstic units with highly restricted gene flow high number of taxonomic arrangements with non-monophyletic taxa (see e.g. Fresneda et al., 2007 for an example with a lineage of Leiodidae cave beetles). * Corresponding author. The Pyrenean Chain is known to be one of the main world hot- E-mail addresses: [email protected], [email protected] (A. Faille), igna spots for subterranean invertebrate fauna (Culver et al., 2006). The [email protected] (I. Ribera), [email protected] (L. Deharveng), Lionel.Gar [email protected] (L. Garnery), [email protected] (E. Quéinnec), deuve phylogenetic relationships among the subterranean species of @mnhn.fr (T. Deuve). Pyrenean Trechini, one of the groups which have experienced 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.10.008 98 A. Faille et al. / Molecular Phylogenetics and Evolution 54 (2010) 97–106 extensive diversification in the area (Jeannel, 1941), are poorly species (A. schmidtii Sturm). Putzeys (1870) transferred these known, and studies have so far been based on morphological char- species to the genus Trechus Clairville, which also includes epigean acters only (Jeannel, 1941; Casale et al., 1998; see below). species. Bonvouloir (1862) erected the genus Aphoenops for the The subterranean Trechini of the Pyrenees include ca. 80 species subterranean species A. leschenaulti Bonvouloir on the basis of the in three genera, Geotrechus, Aphaenops and Hydraphaenops (Mora- non-dilated protarsi of the male, a character currently considered vec et al., 2003; see Appendix for nomenclatorial remarks). All of reduced phylogenetic relevance (Bedel and Simon, 1875). See the species are Pyrenean endemics (most of them with very nar- the appendix for the use of Aphaenops in place of Aphoenops. row distributions), with different adaptations for life in subterra- The current concept of the genus Aphaenops includes 41 species nean habitats. They are all completely blind and apterous, with a on both sides of the Pyrenees, all of them highly modified and slender body form, and (in some species) an extreme elongation exclusive to karst areas, living either in deep cavities or, in some of the head, pronotum and appendages (Jeannel, 1941; Casale cases, in the Superficial Hypogean Compartment (‘‘Milieu souter- et al., 1998), resulting in a very characteristic appearance, the rain superficiel”, MSS, Juberthie and Bouillon, 1983). Diagnostic ‘‘aphaenopsian” morphological type (Jeannel, 1941; Vandel, characters are the presence of incomplete frontal furrows (vs. com- 1964)(Fig. 1). Many subterranean insects around the world have plete in Geotrechus), very elongated legs and antennae, body pale, independently developed similar characteristics, and ‘‘aphaenop- completely depigmented, and a pronounced narrowing (a ‘‘neck”) sian”, ‘‘aphaenopsoid” or ‘‘Aphaenops-like” is commonly used to re- at the base of the head (Coiffait, 1962)(Fig. 1). It is subdivided in fer to this syndrome in other groups of Carabidae (Barr, 1979; six subgenera (for the taxonomic ordination of the group we follow Deuve, 2001; Ortuño et al., 2004; Uéno and Clarke, 2007), and even the recent catalogue of Moravec et al., 2003, although we do not other insects (e.g. Hymenoptera, Roncin and Deharveng, 2003). consider subspecies unless otherwise stated): In this study we provide for the first time a phylogenetic frame- work obtained with numerical algorithms to study the origin and (1) Aphaenops Bonvouloir, 1862: 10 species, mainly found in the diversification of the subterranean species of Pyrenean Trechini, western Pyrenees. based on a combination of nuclear and mitochondrial genes. We (2) Geaphaenops Cabidoche, 1965: 7 species, also in the western include a broad sample of the three subterranean genera (51 spe- Pyrenees. All the species of this group seem to be endogean, cies, some with repeated examples), plus a representation of other and their external morphology is very homogeneous. troglobitic species and potential relatives living on the surface in (3) Cerbaphaenops Coiffait, 1962: 16 species, mainly found in the the Pyrenees and other west Mediterranean areas. Our specific central and eastern Pyrenees, between Bagnères-de-Bigorre aims were to (1) determine the origin of the subterranean genera and the Ariège River. This is also a group with a very homo- and their relationships with epigean species, (2) investigate the geneous morphology, although no clear diagnostic charac- monophyly of traditional taxa (genera and subgenera), established ters were given by Coiffait (1962) (pubescent head, short on external morphological characters, and (3) investigate the rela- mandibles). tionship between endogean and cave species. (4) Pubaphaenops Genest, 1983: a single species from a cave in Ariège, A. laurenti Genest, fully pubescent. 2. Materials and methods (5) Arachnaphaenops Jeanne, 1967: three species, one in the western Pyrenees, two in Ariège and Haute-Garonne respec- 2.1. Historical and taxonomic background of Pyrenean subterranean