A molecular phylogeny shows the single origin of the Pyrenean

Transcription

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
a r t i c l e
i n f o
Article history:
Received 16 March 2009
Revised 1 October 2009
Accepted 5 October 2009
Available online 21 October 2009
Keywords:
Subterranean environment
Convergence
Endogean
Troglobitic
Trechinae
Aphaenops
a b s t r a c t
Trechini ground beetles include some of the most spectacular radiations of cave and endogean Coleoptera,
but the origin of the subterranean taxa and their typical morphological adaptations (loss of eyes and
wings, depigmentation, elongation of body and appendages) have never been studied in a formal phylogenetic framework. We provide here a molecular phylogeny of the Pyrenean subterranean Trechini based
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
the exclusion of all epigean and all subterranean species from other geographical areas (Cantabrian and
Iberian mountains, Alps). Within the Pyrenean subterranean clade the three genera (Geotrechus,
Aphaenops and Hydraphaenops) were polyphyletic, indicating multiple origins of their special adaptations
to different ways of life (endogean, troglobitic or living in deep fissures). Diversification followed a
geographical pattern, with two main clades in the western and central-eastern Pyrenees respectively,
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
The origin and evolution of cave organisms has fascinated evolutionists and biologists for more than two hundred years, since
the discovery of the first troglobitic species (Proteus anguinus, described by Laurenti, 1768). Organisms living in a subterranean
environment tend to show a highly modified morphology and biology, and a mixture of losses (eye degeneration, depigmentation)
and adaptations (development of sensory organs, changes in the
life cycle and metabolism, body shape modifications) (Racovitza,
1907; Vandel, 1964; Culver et al., 1990). Troglobitic invertebrates
isolated in karstic areas are also very good models to study speciation and diversification, because of the isolation of populations in
well-defined karstic units with highly restricted gene flow
* Corresponding author.
E-mail addresses: [email protected], [email protected] (A. Faille), igna
[email protected] (I. Ribera), [email protected] (L. Deharveng), Lionel.Gar
[email protected] (L. Garnery), [email protected] (E. Quéinnec), deuve
@mnhn.fr (T. Deuve).
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2009.10.008
(Caccone, 1985). Amongst insects, many groups of Coleoptera have
repeatedly colonised subterranean habitats, but two of them are
particularly diverse: Leiodidae (especially subfamily Cholevinae)
in the suborder Polyphaga, and Carabidae of the subfamily Trechinae in the suborder Adephaga (Casale et al., 1998). Subterranean
species of both groups share morphological modifications considered to be adaptations to a subterranean lifestyle: loss of metathoracic wings, eyes and pigment, similar changes in body shape and
size (Jeannel, 1926a,b; Vandel, 1964; Barr and Holsinger, 1985),
and modifications in their way of life (Deleurance, 1958). The
extensive convergence in morphological characters obscures the
phylogenetic relationships among species (Marquès and Gnaspini,
2001; Desutter-Grandcolas et al., 2003), which has resulted in a
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).
The Pyrenean Chain is known to be one of the main world hotspots for subterranean invertebrate fauna (Culver et al., 2006). The
phylogenetic relationships among the subterranean species of
Pyrenean Trechini, one of the groups which have experienced
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A. Faille et al. / Molecular Phylogenetics and Evolution 54 (2010) 97–106
extensive diversification in the area (Jeannel, 1941), are poorly
known, and studies have so far been based on morphological characters only (Jeannel, 1941; Casale et al., 1998; see below).
The subterranean Trechini of the Pyrenees include ca. 80 species
in three genera, Geotrechus, Aphaenops and Hydraphaenops (Moravec et al., 2003; see Appendix for nomenclatorial remarks). All
the species are Pyrenean endemics (most of them with very narrow distributions), with different adaptations for life in subterranean habitats. They are all completely blind and apterous, with a
slender body form, and (in some species) an extreme elongation
of the head, pronotum and appendages (Jeannel, 1941; Casale
et al., 1998), resulting in a very characteristic appearance, the
‘‘aphaenopsian” morphological type (Jeannel, 1941; Vandel,
1964) (Fig. 1). Many subterranean insects around the world have
independently developed similar characteristics, and ‘‘aphaenopsian”, ‘‘aphaenopsoid” or ‘‘Aphaenops-like” is commonly used to refer to this syndrome in other groups of Carabidae (Barr, 1979;
Deuve, 2001; Ortuño et al., 2004; Uéno and Clarke, 2007), and even
other insects (e.g. Hymenoptera, Roncin and Deharveng, 2003).
In this study we provide for the first time a phylogenetic framework obtained with numerical algorithms to study the origin and
diversification of the subterranean species of Pyrenean Trechini,
based on a combination of nuclear and mitochondrial genes. We
include a broad sample of the three subterranean genera (51 species, some with repeated examples), plus a representation of other
troglobitic species and potential relatives living on the surface in
the Pyrenees and other west Mediterranean areas. Our specific
aims were to (1) determine the origin of the subterranean genera
and their relationships with epigean species, (2) investigate the
monophyly of traditional taxa (genera and subgenera), established
on external morphological characters, and (3) investigate the relationship between endogean and cave species.
2. Materials and methods
2.1. Historical and taxonomic background of Pyrenean subterranean
Trechini
The first known Pyrenean cave ground-beetles were included in
the genus Anophthalmus, created for an eastern Alpine hypogean
species (A. schmidtii Sturm). Putzeys (1870) transferred these
species to the genus Trechus Clairville, which also includes epigean
species. Bonvouloir (1862) erected the genus Aphoenops for the
subterranean species A. leschenaulti Bonvouloir on the basis of the
non-dilated protarsi of the male, a character currently considered
of reduced phylogenetic relevance (Bedel and Simon, 1875). See
the appendix for the use of Aphaenops in place of Aphoenops.
The current concept of the genus Aphaenops includes 41 species
on both sides of the Pyrenees, all of them highly modified and
exclusive to karst areas, living either in deep cavities or, in some
cases, in the Superficial Hypogean Compartment (‘‘Milieu souterrain superficiel”, MSS, Juberthie and Bouillon, 1983). Diagnostic
characters are the presence of incomplete frontal furrows (vs. complete in Geotrechus), very elongated legs and antennae, body pale,
completely depigmented, and a pronounced narrowing (a ‘‘neck”)
at the base of the head (Coiffait, 1962) (Fig. 1). It is subdivided in
six subgenera (for the taxonomic ordination of the group we follow
the recent catalogue of Moravec et al., 2003, although we do not
consider subspecies unless otherwise stated):
(1) Aphaenops Bonvouloir, 1862: 10 species, mainly found in the
western Pyrenees.
(2) Geaphaenops Cabidoche, 1965: 7 species, also in the western
Pyrenees. All the species of this group seem to be endogean,
and their external morphology is very homogeneous.
(3) Cerbaphaenops Coiffait, 1962: 16 species, mainly found in the
central and eastern Pyrenees, between Bagnères-de-Bigorre
and the Ariège River. This is also a group with a very homogeneous morphology, although no clear diagnostic characters were given by Coiffait (1962) (pubescent head, short
mandibles).
(4) Pubaphaenops Genest, 1983: a single species from a cave in
Ariège, A. laurenti Genest, fully pubescent.
(5) Arachnaphaenops Jeanne, 1967: three species, one in the
western Pyrenees, two in Ariège and Haute-Garonne respectively, all with very long legs and antennae, which give them
the appearance of an arachnid.
(6) Cephalaphaenops Coiffait, 1962: two species, one in the western Pyrenees, the other in Ariège and Haute-Garonne, with
a large and pubescent head and long mandibles.
Fig. 1. Habitus of (1) Aphaenops alberti Jeannel (troglobitic), (2) Aphaenops pluto Dieck (troglobitic), (3) Hydraphaenops navaricus Coiffait & Gaudin (troglobitic), (4) Geotrechus
seijasi Español (endogean), and (5) Trechus quadristriatus (Schrank) (epigean). Scale bars, 1 mm. Photos 1–3 P. Déliot, 4 A. Faille, 5 U. Schmidt.
99
The genus Geotrechus was created by Jeannel (1919) for some
blind species with an ‘‘Anophthalmus-like” habitus, in opposition
to the species of Aphaenops. Diagnostic characters of Geotrechus
are the presence of frontal furrows and their more robust appearance, with short legs and antennae (Coiffait, 1962; Fig. 1). Jeannel
(1919) considered the genera Aphaenops and Geotrechus as two closely related but distinct lineages. Most of the 22 known species of
Geotrechus are endogean, and although some populations can be
locally abundant in caves, most of them seem to be more common
in the ground at the entrance of the cavities (Jeannel, 1926b, 1941).
Some species can also be found under large stones in forests, when
hydric conditions are favourable.
Hydraphaenops was first described as a subgenus of Aphaenops
by Jeannel (1926a), and subsequently upgraded by Coiffait
(1962). Currently it includes 18 species, characterised by an elongated and parallel-sided, almost cylindrical head, sharp, sickleshaped mandibles, short appendages and the body at least partially
covered with pubescence (Jeannel, 1941; Coiffait, 1962) (Fig. 1).
Most species are exceedingly rare, some of them being known from
only one or two specimens, and their biology is virtually unknown
(Cabidoche, 1966). They do seem to be highly hygrophilous, requiring a water-saturated atmosphere to colonise karstic areas (Deleurance-Glaçon, 1963). Some species are known at low altitude (e.g.
H. galani Español, found at sea level), while others have only been
found in high altitude shafts in direct contact with ice (e.g. H. penacollaradensis Dupré, H. mouriesi Genest) (Español, 1968; Dupré,
1991; Genest, 1983). As happens with Aphaenops, Hydraphaenops-like species are known in other lineages of subterranean Trechinae (Deuve, 2000; Casale, 2004).
2.2. Taxon sampling
Trechini species were collected in caves, shafts and MSS from
the Pyrenean chain, in France and Spain as listed in Suppl. Table
1. Single individuals were used for amplification and sequencing.
We included as outgroups several examples of Trechus from the
Pyrenees (mainly epigean, some of them hypogean), plus some
other genera from different geographical areas, including both epigean and subterranean species (Suppl. Table 1). To root the tree we
used one species of Anillini (Typhlocharis Dieck) and two of Bembidiini (Porotachys Netolitzky and Philochthus Stephens), which
are clearly outside Trechini (Grebennikov and Maddison, 2005;
Grebennikov, 2008). In total, we sampled 50 specimens of 32 species of Aphaenops, 11 specimens of 9 species of Hydraphaenops and
12 specimens of 10 species of Geotrechus (Table 1; Suppl. Table 1).
2.3. DNA extraction, PCR amplification and sequencing
Specimens were collected alive in the field and directly killed
and preserved in 96% ethanol. DNA was extracted from whole specimens by a standard phenol–chloroform extraction (Blin and Stafford, 1976). DNA extraction was usually non-destructive, to
preserve voucher specimens for subsequent morphometric and
morphological study (Pons, 2006; Gilbert et al., 2007; Rowley
et al., 2007). Specimens were incubated overnight in a mix of
500 ll of buffer (10 mM Tris, pH 8.0; 0.5% SDS; 0.1 M EDTA, pH
8.0) and 25 ll of proteinase K (20 mg/ml) at 55 °C, with the abdominal ventrites slightly opened to facilitate the action of the digestion
enzyme. The use of non-destructive methods allowed the molecular
study of very rare species, as even fragile structures of taxonomic
importance, like the chaetotaxy or the internal structures of the
aedeagus, were perfectly preserved after extraction (Pons, 2006;
Gilbert et al., 2007; Rowley et al., 2007). Voucher specimens are
kept in the MNHN (Paris), DNA aliquots are kept in the tissue collections of the MNHN and IBE (CSIC-UPF, Barcelona).
Table 1
Checklist of genera and subgenera of subterranean species of Pyrenean Trechini, with
total number of species and species included in the study. Taxonomy follows Moravec
et al. (2003) updated.
a
Genus
Subgenus
N. spp.
Sampled spp.
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Hydraphaenops
Geotrechus
Geotrechus
Trechus
Aphaenops
Geaphaenops
Cerbaphaenops
Cephalaphaenops
Arachnaphaenops
Pubaphaenops
Hydraphaenops
Geotrechus
Geotrechidius
Trechus
10
7
16
2
3
1
18
8
15
17a
8
2
14
1
3
1
8
4
6
7
Species occurring in the Pyrenees, 11 of them endemic.
We sequenced three mitochondrial (50 end of cytochrome c oxidase subunit 1, cox1; cytochrome b, cyb, 50 end of large ribosomal
unit plus the Leucine transfer plus the 30 end of NADH dehydrogenase subunit 1, rrnl+tRNA-Leu+nad1) and two nuclear (small ribosomal unit, SSU, large ribosomal unit, LSU) gene fragments (see
Table 2 for the primers used). Sequences were assembled and edited with Bioedit v. 7.00 (Hall, 1999) or Sequencher 4.6 (Gene
Codes, Inc., Ann Arbor, MI). New sequences have been deposited
in GenBank with Acc. Nos. GQ293502–GQ293896 (395 sequences)
(Suppl. Table 1). For some species, the final sequence is a chimera
of sequences obtained from different specimens (labelled with the
two voucher numbers in all Figures, see Suppl. Table 1). Protein
coding genes were not length variable, and the ribosomal genes
were aligned with the online version of MAFFT v.6 using the GINS-i algorithm and default parameters (Katoh et al., 2002; Katoh
and Toh, 2008).
2.4. Phylogenetic analyses
Bayesian analyses were conducted on a combined data matrix
with MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001), using five
partitions corresponding to the five sequenced fragments. Evolutionary models were estimated prior to the analysis with ModelTest 3.7 (Posada and Crandall, 1998). MrBayes ran for 6 106
generations using default values, saving trees each 500. ‘‘Burn-in”
values were established after visual examination of a plot of the
standard deviation of the split frequencies between two simultaneous runs.
We used two additional phylogenetic approaches for comparative purposes, maximum likelihood with a genetic algorithm
implemented in Garli v0.9 (Zwickl, 2006), using an estimated
GTR+I+G model for the combined sequence and the default settings, and parsimony in PAUP v4.b10 (Swofford, 2002), with
10,000 random replicates, swapping on best trees only and not saving multiple trees. Node support was measured with the posterior
probabilities in MrBayes, and 1000 bootstrap replicates (Felsenstein, 1985) in Garli and PAUP. To reduce computation time in Garli, the number of generations without improving the topology
necessary to complete each replica was reduced to 5000 instead
of the default 10,000. In PAUP we performed heuristic searches
with random addition of taxa with 10 repetitions for each of
1000 replications. Differences between alternative topologies were
evaluated using the tests of Templeton (1983) for parsimony and
Shimodaira and Hasegawa (1999) for maximum likelihood.
To check for possible topological incongruences we did maximum likelihood analyses in Garli with the nuclear sequence alone,
using the GTR+I+G evolutionary model and estimating node support with 1000 bootstrap replicas as above.
100
Table 2
Primers used in the study.
Marker
Primer
Sequence
Ref.
0
0
cox1
RON
HOBBES
TONYA
NANCY
JERRY
PAT
5 GGATCACCTGATATAGCATTCCC3
50 AAATGTTNGGRAAAAATGTTA30
50 GAAGTTTATATTTTAATTTTACCGG30
50 CCCGGTAAAATTAAAATATAAACTTC30
50 CAACATTTATTTTGATTTTTTGG30
50 TCCAATGCACTAATCTGCCATATTA30
Simon et al. (1994)
Monteiro and Pierce (2001)
Monteiro and Pierce (2001)
Simon et al. (1994)
Simon et al. (1994)
Simon et al. (1994)
cyb
CB1
CP1
TSERco
50 TATGTACTACCATGAGGACAAATATC30
50 GATGATGAAATTTTGGATC30
50 TATTTCTTTATTATGTTTTCAAAAC30
Simon et al. (1994)
Kergoat (pers. comm., 2004)
Simon et al. (1994)
rrnl+tRNA-Leu+nad1
NDIA
16SaR
50 GGTCCCTTACGAATTTGAATATATCCT30
50 CGCCTGTTTATCAAAAACAT30
Simon et al. (1994)
Simon et al. (1994)
LSU
D3
D1
50 GCATAGTTCACCATCTTTC30
50 GGGAGGAAAAGAAACTAAC30
Ober (2002)
Ober (2002)
SSU
18S-50
18S-b5.0
50 GACAACCTGGTTGATCCTGCCAGT30
50 TAACCGCAACAACTTTAAT30
Shull et al. (2001)
Shull et al. (2001)
2.5. Estimation of divergence times
To estimate the relative age of divergence of the lineages we
used the Bayesian relaxed phylogenetic approach implemented
in BEAST v1.4.7 (Drummond and Rambaut, 2007), which allows
variation in substitution rates among branches (Drummond et al.,
2006). We implemented a GTR+I+G model of DNA substitution
with four rate categories using the mitochondrial data set only
and pruning species with more than one missing gene fragment,
with an uncorrelated lognormal relaxed molecular clock model
to estimate substitution rates and the Yule process of speciation
as the tree prior. The main nodes of the topology were constrained
to match that of the tree obtained with the whole dataset (mitochondrial plus nuclear) in MrBayes. We ran two independent analyses for each group, sampling each 500 generations, and used
TRACER version 1.4 to determine convergence, measure the effective sample size of each parameter, and calculate the mean and
95% highest posterior density interval (HPD) for divergence times.
Results of the two runs were combined with LogCombiner v1.4.7
and the consensus tree compiled with TreeAnnotator v1.4.7
(Drummond and Rambaut, 2007).
The analyses were run for 25 106 generations, with the initial
10% discarded as burn-in. Because of the absence of fossil record
for both groups, to calibrate the trees we used as a prior a normal
distribution with average equal to the standard rate of 2.3% MY,
equivalent to a per-branch rate of 0.0115 substitutions/site/MY
(Brower, 1994), and a standard deviation of 0.0001. This rate is
lower to that obtained by Contreras-Díaz et al. (2007) for the genus
Trechus, using calibration points based on the colonisation of the
Canary islands (0.015 substitutions/site/MY), although the later
was based on cox1 and cox2 only, which have faster evolutionary
rates than the ribosomal rrnL (Ribera et al., 2001).
3. Results
3.1. Phylogenetic analysis
The aligned data matrix had 3653 characters, of which 932 were
parsimony informative. There was no length variation in the protein coding genes, and variation in the ribosomal genes was mostly
concentrated in the LSU, ranging from 862 (Typhlocharis) to 909 bp
(Perileptus), both among the outgroups. Length variation in the ingroup LSU was reduced to between 867 (G. saulcyi, G. seijasi) and
898 bp (Hydraphaenops galani, H. delicatulus). For the SSU fragment
there was only three bp maximum length difference, and for the
rrnL+tRNA-LEU fragment the maximum length difference was
14 bp between Geotrechus vandeli and some species of Aphaenops
(A. alberti, A. cabidochei, A. ochsi).
The optimal evolutionary model for the mitochondrial genes, as
measured with Modeltest under the Akaike information criterion,
was GTR+I+G. For the SSU the optimal model was TVMef+I, and
for the LSU TVM+I+G. The runs of MrBayes converged at ca.
2 106 generations, with a standard deviation of the split frequencies between the two runs of ca. 0.015. The two runs were interrupted at 5 106 generations (see the estimated parameters in
Suppl. Table 2). A heuristic search using PAUP and assuming an
equal weight for all characters resulted in 2151 trees of 4537 steps
(consistency index, CI = 0.41, retention index, RI = 0.60).
The topology of the tree, and the support for the main nodes,
were very similar for the three reconstruction methods (Bayesian
probabilities, maximum likelihood and parsimony) (Fig. 2, Suppl.
Fig. 1). In all cases the three subterranean genera of the Pyrenees
(Aphaenops, Hydraphaenops and Geotrechus) formed a clade with
exclusion of all epigean species, with strong support (Fig. 2, Suppl.
Fig. 1). The Pyrenean subterranean lineage was sister to a poorly
supported clade including all species of Trechus of different areas
(including the Pyrenees), plus some other subterranean taxa outside the Pyrenees (Apoduvalius, Cantabrian mountains; Duvalius,
Alps; Paraphaenops, Iberian System) (Suppl. Table 1; Fig. 2, Suppl.
Fig. 1). Basal relationships within this clade were not supported.
Within the Pyrenean subterranean clade, the three genera were
polyphyletic under all reconstruction methods, with at least one
well-supported node determining the polyphyly in each case
(Fig. 2). We constrained the monophyly of the three genera and
searched the best topology compatible with this constrain both
in PAUP using parsimony and in Garli with maximum likelihood.
The search in PAUP with the constraint of the monophyly of the
three subterranean genera resulted in 227 trees of 4691 steps
(CI = 0.39; RI = 0.57). The resulting topologies were significantly
worse, as tested both for parsimony (Templeton test, p < 0.0001)
and maximum likelihood (Shimodaira–Hasegawa test, p < 0.0005).
The basal nodes of the subterranean clade were not well-supported, but the best topologies in Bayesian analyses and maximum
likelihood placed a paraphyletic series of species of Geotrechus
from the Eastern Pyrenees at the base (Fig. 2), included in the subgenus Geotrechidius (the ‘‘vulcanus group” sensu Coiffait, 1962). The
rest of the species were included in two main well-supported
clades (pp = 1, bootstrap >70% in all analyses) plus some western
lineages of Hydraphaenops and Geotrechus (Figs. 2 and 3). The
two well-supported main lineages were (1) species of Aphaenops
distributed in the western Pyrenees (clade W), and (2) a clade of
species of Aphaenops and Hydraphaenops from the eastern Pyrenees
101
69/0.97/78
80/1/84
x/0.77/63
A. vandeli MNHN-AF43
59/0.96/67
A. bouiganensis MNHN-AF46
54/0.74/x
A. crypticola MNHN_AF52
84/0.99/79
A. parallelus MNHN_AF53
97/1/95
61/0.72/70
67/0.96/80
76/1/69
72/1/70
A. bouilloni MNHN_AF56
75/0.98/87
A. mariarosae MNHN_AF57
x/1/59
85/-/71
A. sp MNHN_AF133
68/0.62/67
A. sioberae MNHN_AF54
A. pluto MNHN_AF58
x/0.99/A. carrerei MNHN_AF34
x/0.57/x
A.
laurenti
MNHN_AF63
x/0.9/A. michaeli MNHN_AF35
x/0.94/A. bonneti MNHN_AF38
97/1/95
A. delbreili MNHN_AF37
100/1/99
A. cerberus MNHN_AF20_AF30
56/0.77/x
A. jauzioni MNHN_AF33
57/0.79/x
x/0.64/61
A.
hustachei
MNHN_AF39
52/1/53
A. aeacus MNHN_AF40
86/1/x
A.
crypticola
MNHN_AF134
95/1/97
A. sp MNHN_AF42
x/0.88/69
83/1/73
A. bucephalus MNHN_AF62
A. chappuisi MNHN_AF61
100/1/100
Eastern clade
A.
tiresias
MNHN_AF59_AF60
98/1/89
H. bourgoini MNHN_AF68
x/1/H. bourgoini MNHN_AF69
100/1/100
H. ehlersi MNHN_AF64
x/0.55/H. pecoudi MNHN_AF72
H. elegans MNHN_AF120
H. penacollaradensis MNHN_AF121
86/0.99/69
A. abodiensis MNHN_AF4
100/1/99
A. bessoni MNHN_AF122
100/1/100
A. loubensi MNHN_AF3
59/0.95/A. ludovici MNHN_AF15
100/1/100
A. rhadamanthus MNHN_AF13_AF14
98/1/96
A. jeanneli MNHN_AF11
66/0.99/63
A. orionis MNHN_AF9_AF10
97/1/88
A. alberti MNHN_AF12
A. cabidochei MNHN_AF5_AF6
58/0.95/Western clade
100/1/100
A. ochsi MNHN_AF7_AF8
74/0.99/76
A. catalonicus MNHN_AF2
x/0.63/A. leschenaulti MNHN_AF1
63/1/57
H. galani MNHN_AF67
77/1/78
H. vasconicus MNHN_AF65
99/1/93 G. jeanneli MNHN_AF77
71/0.92/x
G. gallicus MNHN_AF76
x/0.63/x
H. pandellei MNHN_AF70
100/1/99
100/1/100 H. pandellei MNHN_AF71
x/1/99/1/97
G. discontignyi MNHN_AF92
G. orcinus MNHN_AF85
59/0.81/x
G. trophonius MNHN_AF83
H. delicatulus MNHN_AF66
G. orpheus MNHN_AF79_AF81
99/0.78/100
Pyrenean hypogean
G. saulcyi MNHN_AF86
100/1/x
G.
saulcyi
MNHN_AF87
clade
G. vandeli MNHN_AF88
92/1/80
G. vulcanus MNHN_AF91
G. seijasi MNHN_AF89
64/0.94/x
T. escalerae MNHN_AF104
x/0.53/x
T. saxicola MNHN_AF100
68/1/58
A. alberichae MNHN_AF105
72/0.99/70
T. navaricus MNHN_AF103
x/0.53/T. uhagoni MNHN_AF102
53/1/92
x/0.84/x
Apoduvalius sp MNHN_AF106
T. fulvus MNHN_AF98
x/0.61/x
94/1/97
T. barnevillei MNHN_AF97
86/1/71
T. obtusus MNHN_AF126
87/1/71
T. ceballosi MNHN_AF128
100/1/100 T. distigma MNHN_AF94
T. quadristriatus MNHN_AF96
x/1/51
S. mayeti MNHN_AF107
73/0.98/97
100/1/99
T. comasi MNHN_AF127
T. schaufussi MNHN_AF101
x/0.91/x
P. breuilianus MNHN_AF108
x/0.68/x
Agostinia gaudini MNHN_AF116
90/1/x
x/0.6/x
D. berthae MNHN_AF114_AF115
100/1/100
D. roberti MNHN_AF129
A. robini MNHN_AF112
x/0.86/x
I. bolivari MNHN_AF111
L. deharvengi MNHN_AF117
.
.
.
P. areolatus MNHN_AF113
P. bisulcatus MNHN_AF131
x/0.7/x
x/1/x
P. lunulatus MNHN_AF118
Typhlocharis MNHN_AF119
0.09
Fig. 2. Phylogram of subterranean Trechini of the Pyrenees obtained with maximum likelihood in Garli, using the combined data matrix. Number in nodes, ML bootstrap/
Bayesian posterior probability, obtained in MrBayes/parsimony bootstrap (see Section 2 for details). ‘‘Western” and ‘‘Eastern” clades marked with ‘‘W” and ‘‘E” respectively
(see text). In red, species of Aphaenops; in green, species of Hydraphaenops; in blue, species of Geotrechus. Habitus, from top to bottom: Aphaenops pluto, A. bessoni, A. alberti,
Hydraphaenops galani, Geotrechus gallicus, G. seijasi, Trechus sp., Paraphaenops breuilianus, Duvalius berthae (see Suppl. Table 1). (For interpretation of colour mentioned in this
figure the reader is referred to the web version of the article.)
102
Fig. 3. Distribution of the main clades of subterranean Trechini of the Pyrenees, according to the phylogeny in Fig. 2. ‘‘Western” and ‘‘Eastern” clades marked with ‘‘W” and
‘‘E” respectively (see text).
(clade E). The Eastern group of Aphaenops species corresponds
mostly to the subgenus Cerbaphaenops sensu Coiffait (1962), plus
some morphologically characteristic species so far placed in the
subgenera Arachnaphaenops, Cephalaphaenops and Pubaphaenops
(A. bucephalus, A. laurenti, A. chappuisi, A. pluto and A. tiresias; Suppl.
Table 1).
The Western clade of Aphaenops included all species of the subgenus Geaphaenops (forming a monophyletic lineage) plus species
of Aphaenops s.str. and A. (Arachnaphaenops) alberti. The easternmost species of this clade is Aphaenops catalonicus, which is also
the southern-most species of Aphaenops, with records from the
Pre-Pyrenees in the Ribagorza valley (Fig. 3). It has morphological
affinities to the northern species and in particular to its sister A.
leschenaulti (specially the male genitalia, Faille et al., 2006).
Within the Eastern clade (Cerbaphaenops sensu lato), what is
currently known as A. crypticola is polyphyletic, with some lineages
associated to other Aphaenops species according to their geographic distribution. These affinities are also supported by morphological characters (see Section 4). Similarly, the only species
of Pubaphaenops (Genest, 1983), A. laurenti, with a peculiar morphology, is grouped in a clade (albeit with low support) with the
species in the same geographical area, between the Lez and the
Vicdessos valleys, at the eastern limit of the distribution of Aphaenops (Figs. 2 and 3).
In all trees the genera Hydraphaenops and Geotrechus (and the
subgenera Geotrechus and Geotrechidius of the later) were polyphyletic, with strong support (Fig. 2, Suppl. Fig. 1). The two Aphaenops
lineages were sister to some species of Hydraphaenops, while Geotrechus was split between a paraphyletic basal series and some
species in a lineage with Hydraphaenops (Fig. 2).
In the analyses of the nuclear sequence we excluded six specimens because of missing data (see Suppl. Table 1). The tree obtained with Garli with the combined SSU + LSU had the same
basic topology as the combined tree (Suppl. Fig. 2), with a wellsupported monophyletic lineage for all subterranean species from
the Pyrenees, and the polyphyly of all three genera. The main subterranean clades found in the combined tree (including the basal
paraphyly of species of Geotrechus) were also present with bootstrap values above 70%, although, due to the lower variability of
the nuclear genes, relationships within the two main clades (W
and E in Figs. 2 and 3) were not recovered.
3.2. Divergence time estimates
We combined the results of the two independent runs of Beast,
with a final estimation of the rate at 0.0115 ± 0.0002 substitutions/
site MY. The estimated age of the origin of the subterranean clade
was 9.7MY, with a 95% interval of confidence between 7.6 and
12.2MY (Fig. 4). The origin of the main clades (Eastern and Western), and that of the different lineages within each genus, was estimated to be in the Upper Miocene, before the end of the Messinian
(Fig. 4). Cladogenetic events within the Pliocene and Pleistocene
were almost exclusively within the same geographical area and
involving species of the same morphological type (i.e. within lineages of each of the traditional genera) (Fig. 4).
4. Discussion
4.1. Origin of the subterranean Pyrenean clade
The most remarkable result of our work was the finding that all
the highly modified species of subterranean Trechini from the
Pyrenees share a common origin, to the exclusion of all sampled
epigean species and all highly modified subterranean species considered by some authors to belong to the phyletic lineage of
Aphaenops from other geographical areas (Apoduvalius, Speotrechus,
Paraphaenops, Suppl. Table 1). Jeannel (1928) hypothesised a common origin for Aphaenops (plus Hydraphaenops) and Geotrechus,
well separated from the epigean Trechus, but included in this subterranean ‘‘phyletic series” other genera from outside the Pyrenees.
According to our results, these highly modified subterranean species from nearby areas, or less modified troglobitic species from
the Pyrenees, were nested within Trechus sensu lato, and not directly related with the subterranean clade. This was the case of
Speotrechus from the Cevennes (Jeannel, 1922), Apoduvalius from
the Cantabrian chain (Vives, 1980; although see Faille, 2006 for a
different view), Paraphaenops from the Iberian system, or the microphthalmous (but not blind) Trechus navaricus from the Pyrenees.
Other subterranean genera, such as Duvalius and Agostinia, have
traditionally being considered as part of a distinct lineage (the
‘‘Duvalius phyletic lineage”), not directly related to Aphaenops, in
agreement with our results (e.g. Jeannel, 1928; Casale et al., 1998).
There are several obvious possible caveats to this conclusion:
(1) there could be some un-sampled epigean species which could
belong to this clade, (2) there could be some un-sampled Pyrenean
subterranean species outside this clade (i.e. sharing a most recent
common ancestor with other epigean species, not with the subterranean clade), or (3) there could be some un-sampled non-Pyrenean subterranean species inside this clade. Based on previous
morphological analyses there are no obvious candidate species
for the first two cases, but for the third only the study of potential
candidates (e.g. Sardaphaenops, Italaphaenops, Allegrettia; Casale
103
0.2
[0.05,0.38]
[0.12,0.57]
0.33
[0.39,1.11]
0.73
0.27
[0.07,0.51]
[0.72,1.75]
1.2
[1.17,2.51]
[0.84,3.32]
3.35
A_laurenti_MNHN_AF63
A_cerberus_MNHN_AF20_AF30
A_jauzioni_MNHN_AF33
[0.72,2.51]
[2.3,4.38]
A_bucephalus_MNHN_AF62
[3.26,5.22]
0.67
5.19
2.6
[4.02,6.4]
[0.14,1.37]
[1.25,4.07]
1.05
[0.29,1.98]
4.76
[6.28,9.16]
7.69
H_penacollaradensis_MNHN_AF121
3.54
H_elegans_MNHN_AF120
[0.84,6.96]
0.86
[0.25,1.64]
3.71
W
[0.35,2.11]
6.29
1.45
[5.07,7.52]
7.93
[6.65,9.25]
A_loubensi_MNHN_AF3
A_jeanneli_MNHN_AF11
1.16
5.82
[0.52,2.53]
4.33
7.26
A_abodiensis_MNHN_AF4
A_alberti_MNHN_AF12
[2.27,5.23]
[5.97,8.6]
H_bourgoini_MNHN_AF69
H_pecoudi_MNHN_AF72
[0.81,3.39]
[4.59,7.07]
H_bourgoini_MNHN_AF68
H_ehlersi_MNHN_AF64
2.02
[2.99,6.47]
9.69
[7.62,12.25]
A_crypticola_MNHN_AF135
A_tiresias_MNHN_AF59_AF60
[5.47,8.13]
[7.11,9.83]
A_hustachei_MNHN_AF39
A_sp_MNHN_AF42
6.73
8.41
A_vandeli_MNHN_AF45
A_delbreili_MNHN_AF37
2.1
1.55
E
A_vandeli_MNHN_AF44
A_sp_MNHN_AF133
[2.4,4.16]
4.01
[3.06,4.95]
4.24
A_pluto_MNHN_AF58
[1.66,3.2]
3.26
A_mariarosae_MNHN_AF57
1.81
2.4
A_parallelus_MNHN_AF53
A_ludovici_MNHN_AF15
A_rhadamanthus_MNHN_AF13_AF14
A_cabidochei_MNHN_AF5_AF6
A_ochsi_MNHN_AF7_AF8
A_leschenaulti_MNHN_AF1
[2.27,6.12]
A_catalonicus_MNHN_AF2
6.28
H_vasconicus_MNHN_AF65
H_galani_MNHN_AF67
[4.3,8.12]
H_delicatulus_MNHN_AF66
6.01
G_gallicus_MNHN_AF76
G_trophonius_MNHN_AF83
[4.1,7.78]
H_pandellei_MNHN_AF70
3.05
7.7
[0,7.35]
[5.12,10.12]
G_vulcanus_MNHN_AF91
G_saulcyi_MNHN_AF86
G_orpheus_MNHN_AF79_AF81
1.0
Fig. 4. Ultrametric tree of the Phylogeny of subterranean Trechini of the Pyrenees obtained with Beast, using a standard mitochondrial rate (0.0115 substitutions/site/MY).
Number above nodes, estimated age (in MY); numbers below nodes, 95% confidence intervals.
and Laneyrie, 1982) can establish their phylogenetic relationships
with certain confidence.
The sister lineage of the subterranean clade was not well-defined in our analyses, as support for the basal nodes of the lineage
including Trechus and related (mostly subterranean) genera was
low. What seems clear from our analyses is that, under its current
concept, Trechus, with more than 440 species distributed in the
northern Hemisphere and the mountains of sub-Saharan Africa
(Casale and Laneyrie, 1982), includes epigean or weakly modified
subterranean species with a plesiomorphic morphology, forming
a largely paraphyletic series with numerous genera of highly modified species nested within. A thorough taxonomic revision of Trechus sensu lato (including the Pyrenean subterranean taxa) would
be highly desirable, but impossible until a more comprehensive
phylogeny is available.
The monophyly of all the highly modified subterranean species
of the Pyrenees strongly suggests a single origin of their shared
character states: loss of eyes, apterism, depigmented body, a subterranean life, and a requirement for high levels of humidity (e.g.
Jeannel, 1926a; Vannier and Thibaud, 1971). These are also traits
that have been linked with a reduced dispersal ability (Kane
et al., 1992; Barr and Holsinger, 1985; Caccone, 1985), and thus
run against the interpretation of a single origin of subterranean
adaptations with subsequent diversification over a relatively large
geographical area (ca. 360 km, from the Puigmal massif, Geotrechus
puigmalensis Lagar, 1981, to Guipuzcoa, Hydraphaenops galani
Español, 1968). The traditional solution to this dilemma was the
assumption that there have been multiple independent active colonisations of the subterranean environment restricted to a very
limited geographical area, each derived from different epigean
ancestors and with a secondary reduction of gene flow (the ‘‘adaptive shift hypothesis”, Howarth, 1982; Peck and Finston, 1993;
Chapman, 1993; Desutter-Grandcolas and Grandcolas, 1996; Rivera et al., 2002).
104
Under this scenario, one would expect to find multiple instances of species with ‘‘intermediate” morphologies (e.g. partly
depigmented bodies, reduced eyes) interspersed among the highly
modified or epigean ones. Although still based on a limited number
of species, this seems to be the case for the Cantabrian chain, with
species of Apoduvalius intermixed with epigean Trechus (Fig. 2),
and also of the Trechus radiation in the Canary islands, where the
subterranean species of the archipelago are closely related to the
epigean species from the same geographical area (Contreras-Díaz
et al., 2007; Borges et al., 2007). Recent molecular work on other
cave-dwelling species also suggests frequent multiple colonisations of the subterranean environment in the same geographic area
(Crustacean isopods, Rivera et al., 2002; Amphipods, Fišer et al.,
2008).
In our case, this interpretation would require the complete
extinction of all species with intermediate morphologies which
could be included in the Pyrenean subterranean clade. This has
been hypothesised for areas subjected to strong fluctuating climate, in particular in areas subjected to dry periods which could
result in the extinction of epigean hygrophilous species: the ‘‘climatic relict hypothesis” of Jeannel (1943) and Peck and Finston
(1993). According to our estimations based on a standard mitochondrial rate, the origin of the Pyrenean subterranean clade
would be mid-late Miocene, a time in when the general climate
in the area seems to have been warmer and wetter than today,
with extensive forested areas (Bruch et al., 2007; Jiménez-Moreno
and Suc, 2007). A possible dry period producing this generalised
extinction, and the separation between the main Eastern and Western clades, could have been the Messinian salinity crisis at the
Miocene–Pliocene boundary, although recent data suggest that
the vegetation of the north Mediterranean area may not have been
deeply affected (e.g. Bertini, 2006). In any case, it must be stressed
that these dates are based on a fixed rate estimated from a combination of genes in several arthropod groups (0.0115 substitutions/
site/MY, Brower, 1994), and thus have to be considered as merely
orientative. The only estimate of mitochondrial evolutionary rate
of a closely related group (0.015 substitutions/site/MY for the
genus Trechus) is based on the colonisation of the Canary islands,
(Contreras-Díaz et al., 2007). As already noted, this was based on
a combination of cox1 and cox2, known to have faster rates than
ribosomal genes, and thus not directly applicable to our dataset.
4.2. Diversification of the subterranean Pyrenean clade
Within the subterranean Pyrenean clade, the three currently
recognised genera (Aphaenops, Hydraphaenops and Geotrechus)
were found to be polyphyletic. These genera were originally defined according to their general body shape, especially the head
and elytra (Jeannel, 1926b; Coiffait, 1962; see Section 2 above).
These are likely to be characters reflecting different adaptations
to the subterranean environment: even if most species are only
known from caves, species of Geotrechus are mostly endogean, living in deep humid soil, while species of Aphaenops live in more
open subterranean spaces, such as caves or the interstices of the
MSS (Jeannel, 1926b; Juberthie and Bouillon, 1983). A particularly
interesting case is the apparently highly specialised habit of most
of the species of the genus Hydraphaenops, which seem to live in
the cracks of karstic massifs and are only occasionally found in
caves. They have a cylindrical head and long and sickled mandibles,
likely to be adapted to an unknown prey (Jeannel, 1926b; Deleurance-Glaçon, 1963). According to our results, it seems that the
general body shape is associated with the particular ecological
and physical conditions of the subterranean environment colonised by these species, with a high degree of homoplasy and convergence (Marquès and Gnaspini, 2001; Fišer et al., 2008).
The main lineages within the subterranean clade seem to be
geographically well differentiated, with successive splits between
the eastern and western Pyrenees resulting in several geographically well-defined clades (Fig. 3). Relationships among closely related species reflect geographical proximity more than general
morphological similarities, with morphologically highly divergent
species found in close proximity, as found for other subterranean
organisms (Fišer et al., 2008). This is for example the case of
Hydraphaenops pandellei and Geotrechus gallicus, of very different
morphology and ecology, or Aphaenops jeanneli and A. alberti. The
latter (Fig. 1) is a very distinct and scarce species endemic to the
Arbailles massif, in the western Pyrenees, previously assumed to
be related to some species from the Eastern clade (Cerbaphaenops
sensu lato), such as A. bucephalus (Jeannel, 1939; Coiffait, 1962)
or A. pluto (Jeanne, 1967). It occurs in the same caves with A. jeanneli, to which it is closely related according to our molecular data
despite its very different body shape, suggesting an ecological
differentiation.
On the other hand, species that were previously considered to
be closely related based on their general appearance, but occurring
in different geographical areas, were found to be included in their
local clades. Thus, the species of the subgenera Arachnaphaenops
(A. pluto, A. tiresias and A. alberti), with a very similar appearance,
were included in three different clades with other Aphaenops species according to their distributions. Similarly, according to our results what is currently considered as Aphaenops crypticola,
distributed from caves between Haute-Garonne and Hautes-Pyrénées, would be polyphyletic. The populations of the western part
of the range (Aure valley, Mont Né) are very close to A. crypticola
aeacus and A. hustachei from the same area, while populations from
the eastern part are subdivided in two groups delimited by the
Garonne valley: a western (A. crypticola MNHN-51 and 48) and
an eastern group (A. crypticola MNHN-47, 49, 52, 136). Due to
the lack of resolution of the nuclear data (Suppl. Fig. 2) it is not possible to discard the possibility of local introgression among some of
these closely related species, but there is no evidence of incongruence between the nuclear and mitochondrial genomes in any of the
lineages for which there is enough resolution, contrary to what
happens in other groups of Carabidae, in which introgression
through hybridisation is common (e.g. Sota and Vogler, 2001; Deuve, 2004; Streiff et al., 2005; Zhang and Sota, 2007). A re-examination of the morphology of the different populations of A. crypticola
in the light of our results revealed differences in the shape of the
aedeagus and some male secondary sexual characters consistent
with this geographical split (Faille, 2006).
We found clear differences in the pattern of diversification between the Western and Eastern clades. The Western clade, between Bagnères-de-Bigorre and the Arbailles massif (clade W,
Aphaenops s.str. plus A. (Arachnaphaenops) alberti and Geaphaenops), seems to be the oldest lineage of troglobitic species, with an
estimated Late Miocene basal diversification (Fig. 4). Some of the
species within this group have secondarily developed endogean
habits, with a reverse to a more stout (i.e. less ‘‘aphaenopsian”)
body shape (A. ludovici, A. rhadamanthus). They were included in
the subgenus Geaphaenops by Cabidoche (1965), together with
other endogean species of more uncertain relationships not included in our study (A. linderi Jeannel, 1938, A. rebereti Gaudin,
1947, and also A. cissauguensis Faille and Bourdeau, 2008).
The main clade of the Eastern Pyrenees, between Bagneres-deBigorre and the Ariege River (Cerbaphaenops plus the morphologically distinct species A. laurenti, A. bucephalus, A. chappuisi, A. pluto
and A. tiresias), seems to be of more recent origin, with a Pliocene–
Pleistocene diversification (Fig. 4) and species with a more homogeneous morphology (Coiffait, 1962). The sampling of this clade
was complete, with two exceptions: (1) A. bourdeaui Coiffait,
1976, considered as part of Cerbaphaenops despite being found in
the area of distribution of the W clade. It is only known from two
females collected the same day (Coiffait, 1976), but never found
again despite numerous visits to the cave. The lack of males and
its geographical distribution cast doubts about its affinities, which
could only be solved with molecular data. (2) A. hidalgoi Español
and Comas, 1985, also from the W Pyrenees. It was described as
Cerbaphaenops (Español and Comas, 1985), but it is a Hydraphaenops-like species, apparently close to H. penacolladarensis—which is
found in the same geographical area (Faille, unpublished observations). The Western and Eastern clades overlap in the Bigorre area,
where one species of each group occur sympatrically in a few
caves: Aphaenops leschenaulti (Eastern clade) and Aphaenops crypticola aeacus (Western clade) (Fresneda et al., 2009).
A potential explanation for the differences between the eastern
and western lineages of Pyrenean subterranean Trechini could be
the different pattern of the limestone areas in which they are
found. In the west, areas of suitable karstified habitat tend to be
larger and more homogeneous, frequently with continuous patches
of ca. 150 km2 (e.g. the Arbailles massif, Vanara, 2000). On the contrary, in the Eastern Pyrenees karstified limestone is highly fragmented, opening opportunities for the development of multiple
isolated local populations leading to allopatric speciation (Culver,
1970; Crouau-Roy, 1986; Faille and Déliot, 2007).
Acknowledgments
We wish to thank J.P. Besson, F. Brehier, E. Dupré, F. Fadrique, J.
Fresneda, G. Jauzion, J.M. Salgado, E. Ollivier, J. Raingeard,
C. Vanderbergh and all the collectors mentioned in the Suppl. Table
1 for their help during field work, the members of the Groupe
Spéléologique du Couserans and Groupe Spéléologique Minos,
without whom visiting some particularly difficult cavities would
have been impossible, G. Kergoat for the primer cp1, U. Schmidt
(http://www.kaefer-der-welt.de/) for the photo of T. quadristriatus,
A. Hassanin for help during the PhD of A.F., A. Cieslak, J. Fresneda,
A. Casale for multiple discussions on the evolution of the subterranean beetles, D.T. Bilton for reading the manuscript, and three
anonymous referees for useful comments on previous versions of
our work. A.F. and C.B. thank the Subterranean laboratory of Moulis
(currently Station d’écologie expérimentale du CNRS) for support
during field work. This study was funded in part by the project
Synthesys (ES-TAF-1540) to A.F. and I.R., the Société Entomologique de France (grants ‘‘Germaine Cousin”) to A.F., and the Spanish
MICINN project CGL2007-61665 to I.R. We dedicate this work to
Philippe Déliot, recently disappeared, without whom collecting
all the species necessary to this study could not have been
possible.
Appendix A
Bonvouloir (1862) described the genus Aphoenops for a species
collected in a cave of the central French Pyrenees, A. leschenaulti
Bonvouloir, 1862. Subsequently, the genus name was written as
Aphaenops by Grenier (1864), and this has been the spelling used
afterwards by all authors with the only exception of Abeille de Perrin (1872), Bedel and Simon (1875) and Peyerimhoff (1915). Recently Moravec et al. (2003) resurrected the original graphy
Aphoenops, which has been subsequently used by Lorenz (2005)
and some other authors. An opinion to the ICZN is in preparation
to conserve Aphaenops, based on the acceptance by the ICZN of
the equivalence of oe and ae for species-level names (not specified
for genus-level names), and the prevalence of use for near 150
years, with hundreds of examples of the use of Aphaenops and virtually no use of Aphoenops.
105
Appendix B. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2009.10.008.
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Supplementary Fig. 1. Phylogeny of subterranean Trechini of the Pyrenees obtained
with parsimony in PAUP, using the combined data matrix. Number in nodes,
bootstrap values (if above 50%). “Western” and “Eastern” clades marked with “W”
and “E”, respectively (see text).
Supplementary Fig. 2: Phylogeny of subterranean Trechini of the Pyrenees obtained
with maximum likelihood in Garli, using only the nuclear sequences (LSU+SSU).
Number in nodes, bootstrap values (if above 50%). “Western” clade marked with
“W”; “E+” Eastern clade plus some additional species (see text).
Suppl. Table 1. Sequenced specimens, with locality, collectors, sequence accession numbers and ecology (T: troglobitic, E: endogean, Ep: Epigean). Code of specimens used to build composite sequences marked with stars.
No
sp
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
62
63
64
65
Trechini
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Bonvouloir, 1862
Bonvouloir, 1862 (sensu stricto)
leschenaulti Bonvouloir, 1861
catalonicus Escolà & Canció, 1983
loubensi Jeannel, 1953
abodiensis Dupré, 1988
bessoni Cabidoche, 1961
cabidochei Coiffait, 1959
Aphaenops ochsi Gaudin, 1925
Aphaenops jeanneli (Abeille de Perrin, 1905)
Aphaenops orionis Fagniez, 1913
Geaphaenops Cabidoche, 1966
Aphaenops rhadamanthus (Linder, 1860)
Aphaenops ludovici Colas & Gaudin, 1935
Cerbaphaenops Coiffait, 1962
Aphaenops cerberus (Dieck, 1869)
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
Aphaenops
jauzioni Faille, Déliot & Quéinnec, 2007
carrerei Coiffait, 1953
michaeli Fourès, 1954
delbreili Genest, 1983
bonneti Fourès, 1948
hustachei Jeannel, 1916
sp.
sp.
crypticola aeacus (Saulcy, 1864)
sp.
vandeli Fourès, 1954
Aphaenops vandeli bouiganensis Fourès, 1954
Aphaenops crypticola (Linder, 1859)
Aphaenops parallelus Coiffait, 1954
Aphaenops sioberae Fourès, 1954
Aphaenops bouilloni Coiffait, 1955
Aphaenops sp.
Aphaenops mariaerosae Genest, 1983
Aphaenops chappuisi Coiffait, 1955
Arachnaphaenops Jeanne, 1967
Aphaenops pluto (Dieck, 1869)
Aphaenops tiresias (Piochard de La Brûlerie, 1872)
Aphaenops alberti Jeannel, 1939
Cephalaphaenops Coiffait, 1962
Aphaenops bucephalus (Dieck, 1869)
Pubaphaenops Genest, 1983
Aphaenops laurenti Genest, 1983
Hydraphaenops Jeannel, 1926
Hydraphaenops ehlersi (Abeille de Perrin, 1872)
Hydraphaenops vasconicus (Jeannel, 1913)
Hydraphaenops vasconicus delicatulus Coiffait, 1962
Hydraphaenops galani Español, 1968
Hydraphaenops bourgoini (Jeannel, 1945)
Hydraphaenops pandellei (Linder, 1859)
locality
collector
Grotte de Castelmouly - Bagnères-de-Bigorre (France-65)
Cova des Toscllosses - Bonansa (Spain-Huesca)
Salle de la Verna - Sainte-Engrâce (France-64)
Villanueva de Aezkoa - Sierra de Abodi - P70 (Spain-Navarra)
Gouffre du Col d’Aran 3 - Bielle (France-64)
Villanueva de Aezkoa - Sierra de Abodi - P70 (Spain-Navarra)
Grotte d’Ayssaguer - Larrau (France-64)
Sima de Garralda - P10 (Spain-Navarra)
Aven d’Istaurdy - Aussurucq (France-64)
Mine de Larrey - Montory (France-64)
Gouffre EL71 - Château-Pignon (France-64)
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
Bourdeau,
P. Déliot, A. Faille
P. Déliot, J. Fresneda
A. Faille, E. Quéinnec
E. Ollivier
A. Faille, E. Quéinnec
Doline de la Sablère - Castet (France-64)
Aven de Nabails - Arthez d'Asson (France-64)
Grotte d’Ambielle - Arette (France-64)
C. Bourdeau
C. Bourdeau, P. Déliot, A. Faille
Grotte du Sendé - Moulis (France-09)
Grotte de L'Estelas - Cazavet (France-09)
Grotte d’Artigouli - Estadens (France-31)
Gouffre du Trapech d’en Haut - Bordes-sur-Lez (France-09)
Grotte de Noël - Seix (France-09)
Gouffre du Petit Mirabat - Ercé (France-09)
Trou du Rantou - Suc-et-Sentenac (France-09)
Grotte de l'Eglise - Nistos (France-65)
Grotte de Frechet-Aure - Frechet-Aure (France-65)
Grotte de la Cascade - Sarrancolin (France-65)
Grotte de Castelmouly - Bagnères-de-Bigorre (France-65)
Tuto de la Cigalero - Ferrère (France-65)
Grotte de Payssa - Salsein (France-09)
MSS S100 - Illartein (France-09)
Grotte SL1 - Saint-Lary (France-09)
Grotte de L'Ournas - Saint-Lary (France-09)
Gouffre de Peyreigne - Tibiran-Jaunac (France-65)
Grotte d’Aron - Portet d’Aspet (France-31)
Grotte de Gouillou - Aspet (France-31)
Grotte de Terreblanque - Aspet (France-31)
Grotte de l’Haiouat de Pelou - Nistos (France-65)
Grotte de la Maouro - Izaut-de-l'Hôtel (France-31)
Grotte de la Buhadère - Coulédoux (France-31)
Grotte de Payssa - Salsein (France-09)
Grotte de Pétillac - Bordes-sur-Lez (France-09)
Grotte d'Aulignac - Bordes-sur-Lez (France-09)
Gouffre du Trapech d’en Haut - Bordes-sur-Lez (France-09)
C. Bourdeau, P. Déliot,
A. Faille
A. Faille
Grotte du Sendé - Moulis (France-09)
Gouffre de la Peyrère - Balaguères (France-09)
Grotte du Goueil-di-Her - Arbas (France-31)
Aven prox. Istaurdy - Aussurucq (France-64)
A. Faille
C. Bourdeau
Gouffre de la Peyrère - Balaguères (France-09)
A. Faille
A. Faille
A.
A.
A.
A.
Faille
Faille
Faille
Faille
A. Faille
biology
code
T
T
T
T
T
T
T
T
T
T
T
T
MNHN-AF1
MNHN-AF2
MNHN-AF3
MNHN-AF4
MNHN-AF122
MNHN-AF5*
MNHN-AF6*
MNHN-AF7*
MNHN-AF8*
MNHN-AF11
MNHN-AF10*
MNHN-AF9*
E
E
E
MNHN-AF14*
MNHN-AF13* GQ293506
MNHN-AF15*
T
T
T
T
T
T
T
T
T
T
T
T
T
MSS
T
T
T
T
T
T
T
T
MSS/T
T
T
T
T
T
MNHN-AF30*
MNHN-AF20*
MNHN-AF33
MNHN-AF34
MNHN-AF35
MNHN-AF37
MNHN-AF38
MNHN-AF39
MNHN-AF134
MNHN-AF135
MNHN-AF40
MNHN-AF42
MNHN-AF44
MNHN-AF43
MNHN-AF45
MNHN-AF46
MNHN-AF51
MNHN-AF52
MNHN-AF47
MNHN-AF50
MNHN-AF48
MNHN-AF49
MNHN-AF53
MNHN-AF54
MNHN-AF56
MNHN-AF133
MNHN-AF57
MNHN-AF61
SSU
LSU
GQ293593
GQ293508
GQ293555
cox1
rrnL
tRNA-Leu
nad1
GQ293629
GQ293739
GQ293757
GQ293822
GQ293674
GQ293699
GQ293756
GQ293821
cyb
GQ293886
GQ293660
GQ293863
GQ293627
GQ293862
GQ293554
GQ293556
GQ293667
GQ293520
GQ293890
GQ293741
GQ293778
GQ293831
GQ293740
GQ293777
GQ293830
GQ293666
GQ293521
GQ293601
GQ293594
GQ293507
GQ293892
GQ293661
GQ293891
GQ293664
GQ293885
GQ293592
GQ293677
GQ293717
GQ293776
GQ293827
GQ293895
GQ293550
GQ293676
GQ293716
GQ293775
GQ293828
GQ293896
GQ293589
GQ293646
GQ293718
GQ293779
GQ293835
GQ293871
GQ293581
GQ293640
GQ293512
GQ293572
GQ293641
GQ293515
GQ293585
GQ293722
GQ293768
GQ293815
GQ293513
GQ293570
GQ293720
GQ293773
GQ293804
GQ293721
GQ293774
GQ293805
GQ293711
GQ293751
GQ293844
GQ293710
GQ293749
GQ293842
GQ293526
GQ293638
GQ293571
GQ293514
GQ293573
GQ293636
GQ293877
GQ293643
GQ293576
GQ293635
GQ293574
GQ293750
GQ293845
GQ293637
GQ293696
GQ293752
GQ293843
GQ293884
GQ293584
GQ293657
GQ293706
GQ293762
GQ293808
GQ293869
GQ293705
GQ293761
GQ293807
GQ293709
GQ293765
GQ293812
GQ293653
GQ293708
GQ293764
GQ293811
GQ293868
GQ293582
GQ293652
GQ293707
GQ293763
GQ293810
GQ293865
GQ293587
GQ293639
GQ293575
GQ293644
GQ293648
GQ293656
GQ293510
GQ293516
GQ293590
GQ293654
GQ293580
GQ293642
GQ293591
GQ293651
GQ293577
GQ293671
GQ293579
GQ293650
GQ293578
GQ293655
GQ293856
GQ293867
GQ293855
GQ293866
GQ293858
GQ293859
GQ293870
GQ293586
GQ293645
GQ293694
GQ293758
GQ293806
GQ293888
GQ293568
GQ293649
GQ293704
GQ293760
GQ293809
GQ293860
GQ293525
GQ293563
GQ293684
T
T
T
T
MNHN-AF58
MNHN-AF59* GQ293527
MNHN-AF60*
MNHN-AF12
GQ293567
GQ293647
GQ293596
GQ293658
GQ293713
GQ293748
GQ293800
GQ293595
GQ293662
GQ293700
GQ293782
GQ293829
GQ293853
A. Faille
T
MNHN-AF62
GQ293588
GQ293675
GQ293693
GQ293747
GQ293814
GQ293876
Grotte de Bordes de Crues - Seix (France-09)
A. Faille
T
MNHN-AF63
GQ293569
GQ293634
GQ293719
GQ293767
GQ293813
GQ293873
Goueil-di-Her - Arbas (France-31)
Aven d’Istaurdy - Aussurucq (France-64)
Guardetxe Koleccia - Usurbil (Spain-Guipuzcoa)
Grotte de l'Eglise - Nistos (France-65)
Grotte d’Arréglade - Rébénacq (France-64)
C. Bourdeau
C. Bourdeau
T
T
T
T
T
T
T
MNHN-AF64
MNHN-AF65
MNHN-AF66
MNHN-AF67
MNHN-AF69
MNHN-AF68
MNHN-AF70
GQ293565
GQ293683
GQ293622
GQ293628
GQ293698
GQ293759
GQ293803
GQ293600
GQ293663
GQ293695
GQ293753
GQ293818
A. Faille
A. Faille
A. Faille
A. Faille
A. Faille
GQ293509
GQ293883
GQ293712
GQ293583
GQ293511
GQ293530
GQ293524
GQ293864
GQ293889
GQ293602
GQ293875
GQ293872
GQ293697
GQ293746
GQ293817
GQ293553
GQ293672
GQ293734
GQ293755
GQ293824
GQ293894
GQ293552
GQ293659
GQ293733
GQ293772
GQ293826
GQ293851
GQ293545
GQ293681
GQ293880
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
Hydraphaenops pecoudi (Gaudin, 1938)
Hydraphaenops elegans Gaudin, 1945
Hydraphaenops penacollaradensis Dupré, 1991
Geotrechus Jeannel, 1919
Geotrechus Jeannel, 1919 (sensu stricto)
Geotrechus discontignyi (Fairmaire, 1863)
Geotrechus orcinus (Linder, 1859)
Geotrechus orpheus (Dieck, 1869)
Geotrechus trophonius (Abeille de Perrin, 1872)
Geotrechidius Jeannel, 1947
Geotrechus gallicus (Delarouzee, 1857)
Geotrechus jeanneli Gaudin, 1938
Geotrechus saulcyi (Argod-Vallon, 1913)
Geotrechus seijasi Español, 1969
Geotrechus vandeli Coiffait, 1959
Geotrechus vulcanus (Abeille de Perrin, 1904)
Trechus Clairville, 1806
Trechus distigma Kiesenwetter, 1851
Trechus quadristriatus (Schrank, 1781)
Trechus barnevillei Pandellé, 1867
Trechus fulvus Dejean, 1831
Trechus saxicola Putzeys, 1870
Trechus schaufussi Putzeys, 1870
Trechus grenieri uhagoni Crotch, 1869
Trechus navaricus (Vuillefroy, 1869)
Trechus escalerai Abeille de Perrin, 1903
Trechus obtusus Erichson, 1837
Trechus comasi Hernando, 2001
Trechus ceballosi Mateu, 1953
Apoduvalius Jeannel, 1953
Apoduvalius alberichae Español, 1971
Apoduvalius sp.
Speotrechus Jeannel, 1922
Speotrechus mayeti (Abeille de Perrin, 1875)
Paraphaenops Jeannel, 1916
Paraphaenops breuilianus (Jeannel, 1916)
Iberotrechus Jeannel, 1920
Iberotrechus bolivari (Jeannel, 1913)
Duvalius Delarouzée, 1859
Duvalius berthae (Jeannel, 1910)
Duvalius roberti (Abeille de Perrin, 1903)
Agostinia Jeannel, 1928
Agostinia gaudini (Jeannel, 1952)
Laosaphaenops Deuve, 2000
Laosaphaenops deharvengi Deuve, 2000
Aepopsis Jeannel, 1922
Aepopsis robini (Laboulbène, 1849)
Perileptus Schaum, 1860
Perileptus areolatus (Creutzer, 1799)
Bembidiini
Philochthus Stephens, 1828
Philochthus lunulatus (Fourcroy, 1795)
Typhlocharis Dieck, 1869
Typhlocharis sp.
Porotachys Netolitzky, 1914
Porotachys bisulcatus (Nicolaï, 1822)
Grotte d' Ambielle - Arette (France-64)
C. Bourdeau, A. Faille
Gouffre du Barroti - Lacourt (France-09)
A. Faille
Subterranean river of Artigaléou-Arodets - Esparros (France-65) C. Bourdeau, E. Ollivier, E. Quéinnec
Aven El Sinistro, Villanúa (Spain-Huesca)
C. Bourdeau, E. Ollivier
T
T
T
T
MNHN-AF71
MNHN-AF72
MNHN-AF120
MNHN-AF121
GQ293546
GQ293566
GQ293673
GQ293754
GQ293816
GQ293702
GQ293771
GQ293823
GQ293744
GQ293789
GQ293802
A. Faille
E/T
E/T
E/T
E/T
E/T
MNHN-AF92
MNHN-AF85 GQ293519
MNHN-AF81* GQ293528
MNHN-AF79*
MNHN-AF83
GQ293560
Grotte de la Bouhadère - Saint-Pé-de-Bigorre (France-65)
Grotte du Ker - Rivérenert (France-09)
Gouffre du Barroti - Lacourt (France-09)
Cova d'en Manent - Isòvol (Spain-Girona)
Aven d'Anglade - Couflens (France-09)
Perte du Fustié - Saint-Martin-de-Caralp (France-09)
A. Faille
E
E
E/T
E/T
E/T
E
E/T
MNHN-AF76
MNHN-AF77
MNHN-AF87
MNHN-AF86
MNHN-AF89
MNHN-AF88
MNHN-AF91
Collau de la Plana del Turbón - Egea (Spain-Huesca)
Cueva del Pis - Penilla, Santiurde de Toranzo (Spain-Cantabria)
Braña Caballo - Piedrafita (Spain-León)
Ciudad Real-Navas de Estena-"El Boqueron" (Spain-Toledo)
Cueva de Orobe - Alsasúa (Spain-Navarra)
Grotte de Sare - Sare (France-64)
Cueva de Porro Covañona - Covadonga (Spain-Asturias)
Saint-Pé-de-Bigorre (France-65)
Cueva Basaura - Barindano (Spain-Navarra)
Aven de Licie Etsaut, Lanne-en Barétous (France-64)
P. Déliot, A. Faille, J. Fresneda
A. Faille
C. Bourdeau
C. Bourdeau
J.M. Salgado
J. Fresneda
Ep
Ep
Ep/T
Ep/T
Ep
E/MSS
T
T
T
Ep
T
Ep
MNHN-AF94
MNHN-AF96
MNHN-AF97
MNHN-AF98
MNHN-AF100
MNHN-AF101
MNHN-AF102
MNHN-AF103
MNHN-AF104
MNHN-AF126
MNHN-AF127
MNHN-AF128
Cova de Agudir - Cardano de abajo - Palencia (Spain-Asturias)
Cueva Requexada - Piloñeta (Spain-Asturias)
J.M. Salgado
J.M. Salgado
T
T
MNHN-AF105 GQ293536
MNHN-AF106 GQ293537
GQ293618
Perte du Rimouren - Saint-Montant (France-07)
J-Y. Bigot
T
MNHN-AF107 GQ293535
GQ293547
Cova Cambra - Tortosa (Spain-Tarragona)
T
MNHN-AF108 GQ293541
GQ293551
GQ293685
Ep/T
MNHN-AF111
GQ293615
GQ293679
Cova d’en Xoles - Pratdip (Spain-Tarragona)
Cova Massega - Llaberia (Spain-Tarragona)
Grotte de Peïra Cava - Peïra Cava (France-06)
C. Bourdeau, P. Déliot, F. Fadrique, A. Faille
C. Bourdeau, P. Déliot, F. Fadrique, A. Faille
A. Coache, J. Raingeard
T
T
T
MNHN-AF115*
MNHN-AF114* GQ293531
MNHN-AF129
GQ293606
GQ293626
Puits des Bauges - Dévoluy (France-05)
J-Y. Bigot
T
MNHN-AF116 GQ293543
GQ293604
Vang Vieng-Nam Xang Tai (Laos)
A. Bedos, L. Deharveng
T
MNHN-AF117 GQ293542
GQ293621
Plage du Toëno - Trébeurden (France-22)
A. Faille
Ep
MNHN-AF112 GQ293504
GQ293623
Immouzer des Ida Outanane (Maroc)
P. Aguilera, C. Hernando, I. Ribera
Ep
MNHN-AF113 GQ293503
GQ293625
A. Cieslak, I. Ribera
Ep
MNHN-AF118 GQ293505
E
MNHN-AF119 GQ293502
GQ293624
MNHN-AF131
GQ293544
Santa Almagrera (Spain-Almería)
C. Andujar
Grotte des Fées - Saint-Cricq-du-Gave (France-40)
Ep/T
GQ293878
GQ293703
GQ293564
Grotte du Tuco - Bagnères-de-Bigorre (France-65)
Gouffre de Peyreigne - Tibiran (France-65)
Grotte de la Quère - Mérigon (France-09)
Grotte de Montespan - Ganties (France-31)
Grotte de Tuto Heredo - Merigon (France-09)
Guadalajara - El Pobo de Dueñas (Spain-Guadalajara)
GQ293738
GQ293562
GQ293893
GQ293559
GQ293597
GQ293665
GQ293723
GQ293780
GQ293834
GQ293561
GQ293631
GQ293715
GQ293766
GQ293825
GQ293874
GQ293518
GQ293557
GQ293670
GQ293724
GQ293769
GQ293798
GQ293517
GQ293558
GQ293725
GQ293770
GQ293799
GQ293522
GQ293548
GQ293714
GQ293784
GQ293833
GQ293701
GQ293786
GQ293832
GQ293743
GQ293745
GQ293841
GQ293727
GQ293783
GQ293848
GQ293788
GQ293820
GQ293669
GQ293668
GQ293529
GQ293598
GQ293523
GQ293549
GQ293534
GQ293619
GQ293533
GQ293607
GQ293678
GQ293680
GQ293613
GQ293614
GQ293887
GQ293854
GQ293599
GQ293611
GQ293852
GQ293879
GQ293729
GQ293682
GQ293882
GQ293532
GQ293620
GQ293737
GQ293540
GQ293616
GQ293730
GQ293539
GQ293603
GQ293538
GQ293612
GQ293731
GQ293793
GQ293839
GQ293608
GQ293726
GQ293795
GQ293847
GQ293728
GQ293791
GQ293850
GQ293732
GQ293794
GQ293840
GQ293736
GQ293796
GQ293846
GQ293687
GQ293617
GQ293610
GQ293632
GQ293609
GQ293881
GQ293605
GQ293735
GQ293781
GQ293819
GQ293691
GQ293785
GQ293837
GQ293692
GQ293787
GQ293838
GQ293689
GQ293792
GQ293836
GQ293690
GQ293797
GQ293801
GQ293742
GQ293790
GQ293849
GQ293857
GQ293630
GQ293688
GQ293686
GQ293861
GQ293633
Suppl. Table 2. Estimated parameters in the MrBayes run.
Partition
Gene
1
LSU
2
cox1
3
cyb
4
SSU
5
rrnL-tRNA-Leu-nad1
Parameter
TL{all}
r(A<->C){1}
r(A<->G){1}
r(A<->T){1}
r(C<->G){1}
r(C<->T){1}
r(G<->T){1}
r(A<->C){2}
r(A<->G){2}
r(A<->T){2}
r(C<->G){2}
r(C<->T){2}
r(G<->T){2}
r(A<->C){3}
r(A<->G){3}
r(A<->T){3}
r(C<->G){3}
r(C<->T){3}
r(G<->T){3}
r(A<->C){4}
r(A<->G){4}
r(A<->T){4}
r(C<->G){4}
r(C<->T){4}
r(G<->T){4}
r(A<->C){5}
r(A<->G){5}
r(A<->T){5}
r(C<->G){5}
r(C<->T){5}
r(G<->T){5}
pi(A){1}
pi(C){1}
pi(G){1}
pi(T){1}
pi(A){2}
pi(C){2}
pi(G){2}
pi(T){2}
pi(A){3}
pi(C){3}
pi(G){3}
pi(T){3}
pi(A){5}
pi(C){5}
pi(G){5}
pi(T){5}
alpha{1}
alpha{2}
alpha{3}
alpha{5}
pinvar{1}
pinvar{2}
pinvar{3}
pinvar{4}
pinvar{5}
Mean
4.559693
0.049424
0.315247
0.230424
0.021475
0.31653
0.066901
0.040399
0.311296
0.048516
0.118613
0.46844
0.012735
0.047638
0.411278
0.037981
0.060626
0.4055
0.036978
0.01421
0.05063
0.861162
0.003883
0.064736
0.005379
0.021898
0.62562
0.115637
0.024756
0.144934
0.067155
0.318408
0.229642
0.1729
0.27905
0.358209
0.099804
0.086904
0.455084
0.393645
0.123165
0.05234
0.430851
0.388421
0.058252
0.08781
0.465517
0.384221
0.666526
0.887375
0.65994
0.221579
0.693532
0.546503
0.592707
0.496361
Variance
0.063014
0.000068
0.000824
0.00033
0.000042
0.000831
0.000136
0.00011
0.001581
0.000052
0.000945
0.002121
0.00003
0.000106
0.003426
0.000054
0.000519
0.00329
0.000132
0.00008
0.000178
0.000686
0.000004
0.000298
0.000018
0.000096
0.001727
0.000267
0.000488
0.000931
0.000222
0.000161
0.000138
0.000108
0.000141
0.000261
0.000049
0.000125
0.000241
0.000368
0.000088
0.000123
0.000311
0.000215
0.000063
0.000081
0.000239
0.00168
0.005281
0.027705
0.011901
0.001691
0.000355
0.001226
0.000356
0.0011
Lower
4.07
0.034689
0.261275
0.196747
0.010314
0.262069
0.045651
0.022005
0.234833
0.03531
0.065049
0.377717
0.004212
0.030334
0.297987
0.025427
0.023866
0.302696
0.017882
0.001436
0.030033
0.800711
0.000898
0.037439
0.000194
0.006474
0.538516
0.086505
0.000957
0.092562
0.040165
0.293197
0.207182
0.152807
0.256189
0.326175
0.086864
0.067076
0.424815
0.35688
0.105748
0.034735
0.396442
0.359728
0.044143
0.071917
0.435488
0.312122
0.531185
0.595748
0.47105
0.134414
0.654392
0.470943
0.554866
0.42531
Upper
5.043
0.066694
0.37451
0.267916
0.035718
0.374831
0.091642
0.063336
0.392138
0.063409
0.184674
0.555796
0.025192
0.070141
0.521927
0.053689
0.112859
0.520306
0.06153
0.035942
0.081783
0.903696
0.00901
0.103357
0.015923
0.044816
0.703213
0.150775
0.082658
0.212862
0.098204
0.343815
0.25343
0.193682
0.302581
0.390413
0.113943
0.110516
0.485691
0.43187
0.142669
0.076711
0.465501
0.417285
0.074907
0.107191
0.496042
0.473951
0.81887
1.240414
0.89661
0.297168
0.728343
0.606305
0.628952
0.555367
Median
4.559
0.049003
0.314323
0.230173
0.020852
0.315589
0.066263
0.039562
0.310238
0.048136
0.116375
0.468631
0.012044
0.046778
0.413283
0.037463
0.058092
0.402505
0.035806
0.012656
0.04883
0.864183
0.003525
0.062386
0.004471
0.020428
0.627552
0.114925
0.018881
0.142116
0.066398
0.318377
0.229434
0.172902
0.279116
0.358168
0.09955
0.086183
0.454603
0.393724
0.122806
0.050999
0.430869
0.388326
0.057884
0.087314
0.465426
0.381721
0.66541
0.876048
0.652117
0.223342
0.694088
0.549672
0.592955
0.498523
PSRF *
1.018
1
1
1
1
1
1
1.002
1.005
1.01
1.003
1.005
1
1.003
1.01
1.015
1
1.008
1.002
1.003
1.001
1.003
1
1
1
1
1
1
1
1
1
1.001
1
1
1
1.001
1
1.01
1.002
1.003
1.006
1.013
1.002
1
1.001
1
1
1.002
1
1.002
1
1
1
1.001
1.001
1.002

A molecular phylogeny shows the single origin of the Pyrenean

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