1 Preprint of a manuscript accepted for publication in Zoologica Scripta
2
3 Tectonic vicariance versus Messinian dispersal in western
4 Mediterranean ground beetles (Carabidae Trechini and
5 Pterostichini Molopina)
6
7 1,2 3 4 5 1 8 Arnaud Faille , Achille Casale , Carles Hernando , Salah Aït Mouloud and Ignacio Ribera
9 1 10 Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Maritim de la
11 Barceloneta 37, 08003 Barcelona, Spain 2 12 MECADEV - UMR 7179 MNHN/CNRS, Paris, France 3 13 C/o Università di Sassari, Dipartimento di Scienze della Natura e del Territorio (Zoologia).
14 Private: Corso Raffaello 12, 10126 Torino, Italy. e-mail: [email protected] 4 15 P.O. box 118, 08911 Badalona, Catalonia, Spain
5 16 Université Mouloud-Mammeri, Tizi Ouzou, Algeria
17
18
19 Correspondence: A. Faille, Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra),
20 Barcelona, Spain. E-mail: [email protected] 21
1 22 ABSTRACT
23
24 The complex geological history of the western Mediterranean region complicates the
25 interpretation of the evolutionary history of its current fauna, as similar distribution patterns
26 may have very different temporal and geographical origins. Particularly intriguing are some
27 subterranean species in islands, which origin is usually difficult to interpret as their strongly
28 modified morphologies obscure their relationships. We studied subterranean taxa and their
29 likely relatives of two groups of ground beetles in the western Mediterranean: the Duvalius
30 lineage ("isotopic" Trechini) and Molopina (Pterostichini). We included specimens from the
31 islands of Mallorca, Sardinia and Sicily, plus mainland Europe and north Africa. Phylogenetic
32 relationships were reconstructed with a combination of mitochondrial and nuclear data, and
33 divergence dates were estimated with Bayesian methods using the same a priori molecular
34 evolutionary rates for the same gene fragments in the two groups. The subgenus Trechopsis,
35 which includes all the highly modified cave or nivicolous species, was found to be
36 polyphyletic: the species from Mallorca was found to be of Pleistocene origin and sister to the
37 less modified species of subgenus Duvalius from the same island, whereas the Algerian
38 species of Trechopsis were, on the contrary, related to the Sicilian Duvalius, indicating a
39 northern colonization route during the late Pliocene. Molopina was divided in three main
40 lineages: the genera Abax, Percus, and the Molops groups of genera. The basal diversification
41 of the latter was dated within a temporal window (35-25 Ma) fully congruent with the tectonic
42 opening of the western Mediterranean basin, and included six main lineages with uncertain
43 relationships among them: the epigean genera (1) Molops and (2) Tanythrix; and the
44 subterranean (3) Typhlochoromus (Eastern Alps), (4) Speomolops (Sardinia), (5) Henrotius
45 (Mallorca) and (6) a strongly supported clade including the Pyrenean genera Zariquieya,
46 Oscadytes and Molopidius. Despite sharing a similar distribution of some of the subterranean
47 taxa, the two studied groups show thus a strongly contrasting origin and mode of
48 diversification. While the Duvalius lineage had a recent origin, with complex colonization
49 patterns and widespread morphological convergence among the subterranean species, the
50 subterranean Molopina had an ancient vicariant origin resulting from the tectonic opening of
51 the western Mediterranean basin.
52
53 Keywords: Western Mediterranean region, dispersal, tectonic vicariance, Messinian,
54 subterranean Coleoptera, Carabidae, Duvalius, Trechini, Pterostichini, Molopina
55
2 56 1. Introduction 57 58 The western Mediterranean fauna has a long tradition of phylogenetic and
59 biogeographic studies trying to understand the complex relationships between the Balearic
60 and Tyrrhenian islands with the neighbouring mainland. Corsica and Sardinia are a classical
61 model (e.g. Ketmaier and Caccone, 2013 and references therein), sharing some endemics with
62 a Tyrrhenian distribution (Médail and Quézel, 1999; Brullo et al., 2001; Ketmaier et al., 2006;
63 Cicconardi et al., 2009), but still with very different faunas in many groups, something that is
64 often overlooked (see e.g. Casale and Vigna Taglianti, 1996 for groundbeetles, or Caccone &
65 Sbordoni 2001, Martinsen et al 2009 among others for other subterranean fauna). The fauna
66 and flora of Balearic Islands are in turn composed by numerous vicariant species from the
67 Iberian Peninsula (Moreno Sáiz et al., 1998), some of them with close relationships with
68 southern France endemics (e.g. Gielly et al., 2001) or Corso-Sardinian species
69 (Contandriopoulos and Cardona, 1984). There are also some well documented relationships of
70 the fauna of Sicily and Calabria with that of Algeria and Tunisia (Chapman and Abbott, 2005;
71 Cosson et al., 2005; Stöck et al., 2008b; Habel et al., 2009, 2010, 2011; Pfenninger et al.,
72 2010; Pabijan et al., 2012; Marrone et al., 2013), as well as relationships of western North
73 Africa with southeast Iberia (the Baetic-Rifean area, e.g. Dobson and Wright, 2000; Stöck et
74 al., 2008a; Molina-Venegas et al., 2013; Faille et al., 2014). However, despite the
75 biogeographic relationships of both Sicily and Sardinia with north Africa, they do not share
76 many endemic species, and their faunas are overall very different (see e.g. Vigna Taglianti et
77 al., 2002 for groundbeetles). The origin of the Kabylian biota has been much less studied, and
78 is still poorly understood.
79 Many of the relationships within the western Mediterranean basin have been
80 interpreted as the results of vicariant splits due to its tectonic opening during the Oligocene
81 and early Miocene (Rosenbaum, 2002; Meulenkamp and Sissing, 2003; Schettino and Turco,
82 2006). However, due to lack of adequate fossil record this was often done without strong
83 evidence neither of the relationships between the species nor their age (see e.g. the surprising
84 resolution of the paradigmatic case of Euproctus salamanders in Carranza and Amat, 2005).
85 The interpretation of the origin of the species is complicated by the possibility of dispersal
86 during the salinity crisis of the Messinian ending 5.3 Ma (Hsü et al., 1973; Roveri et al.,
87 2014), through land bridges that in many cases paralleled the tectonic connections of the
88 Oligocene plates. The wide temporal interval between the two potential connections of
89 emerged landmasses in the western Mediterranean (original tectonic vicariance, between 35-
3 90 25 Ma, and Messinian dispersal, between 7-5 Ma) allows the use of molecular data to
91 discriminate between the two scenarios, despite all the uncertainties associated with molecular
92 clock approaches (Kumar, 2005). This has allowed to reliably reconstruct the origin of some
93 groups as either having an old, vicariant origin (e.g. Ribera et al., 2010; Bidegaray-Batista and
94 Arnedo, 2011) or through dispersal during the salinity crisis (e.g. Carranza et al., 2008; Mora
95 et al., 2017). There are, however, still many groups for which their evolutionary origin is
96 largely unknown.
97 A particularly interesting case are groups with a general poor dispersal ability but with
98 species in some of the islands, as they have often been assumed to have an ancient, tectonic
99 origin. These notoriously include subterranean species, though to being unable to disperse
100 even through the land connections during the Messinian due to their general reduced mobility
101 and the lack of suitable habitat (e.g. Ribera et al., 2010). Different groups of insects contain
102 species with subterranean habits, but only a few of them have a marked tendency for repeated
103 colonisations of the underground. In the western Mediterranean this is the case of two groups
104 of terrestrial Coleoptera within the Carabidae ground beetles, the Trechini and Pterostichini
105 Molopina.
106
107 1.1. Trechini
108 Within the western Mediterranean, the Pyrenean Aphaenops lineage, shown to be
109 monophyletic by Faille et al. (2010), is the most species rich among the strictly subterranean
110 Trechini. Other genera of western Mediterranean Trechini with aphaenopsoid shape, e.g.
111 Sardaphaenops Cerruti & Henrot, 1956 (two species endemic to Sardinia) and Paraphaenops
112 Jeannel, 1916 (two species endemic to southern Spain), were excluded from the Aphaenops
113 lineage by Faille et al. (2011a). In addition to these radiations, belonging to the “anisotopic”
114 Trechini (i.e. with the male genitalia with an asymmetric copulatory piece laying in lateral
115 position, Jeannel, 1928), there is another group of genera with mostly subterranean species:
116 the Duvalius lineage, or "isotopic" Trechini (i.e. with the male genitalia with a symmetric
117 copulatory piece not twisted with respect to the body axis, Jeannel, 1926). In Faille et al.
118 (2011a, 2013) this "isotopic" lineage was shown to be monophyletic and not directly related
119 to the “anisotopic” lineages.
120 Within the "isotopic" Trechini, the genus Duvalius Delarouzée is the most species
121 rich, with more than 300 species described so far (Moravec et al., 2003). The highest diversity
122 is in the French and Italian Alps, Italian and Balkan peninsulas and the Carpathian area, but
123 the genus reaches its western limit in the western Mediterranean, the westernmost species
4 124 being an endemic from Catalonia (Jeannel, 1926, 1928). Other species are found in north
125 Algeria (Kabylia, four species), the Balearic Islands (Mallorca, two species), Sardinia (one
126 species), and Sicily (nine species). All of them are endogean or cave-dwelling (Jeannel, 1928;
127 Henrot, 1964; Lagar, 1976; Bellès 1987; Vigna Taglianti et al., 2002; Magrini et al., 2006,
128 2016) (Table S1). A previous molecular study on Alpine Trechini showed that some locally
129 restricted and highly modified troglobitic genera were nested within the wider genus Duvalius
130 (Faille et al., 2013).
131 In the current literature four species also with marked troglobiomorphic features are
132 included in Duvalius subgenus Trechopsis Peyerimhoff (Jeannel, 1928; Moravec et al., 2003),
133 leaving in Duvalius s.str. the less troglobiomorphic species. The relationships of the four
134 species of Trechopsis are so far unknown. It was originally described as a genus for a species
135 endemic to the Djurdjura, in Algeria (D. (T.) lapiei (Peyerimhoff)). This species occurs in
136 high altitude caves or pits with snow (“tesserefts”, Peyerimhoff, 1908). The second described
137 species, D. (T.) iblis (Peyerimhoff), was found in a cave, and although originally included in a
138 different genus (Aphaenops) its affinities with D. (T.) lapiei were already noted by its
139 descriptor (Peyerimhoff, 1910) and later confirmed by Jeannel (1928), who also relegated
140 Trechopsis to a subgenus of Duvalius. A third endogean species was later described from the
141 Babor range (D. (T.) baborensis Bruneau de Miré, 1955). Subsequently, D. (T.) ferreresi
142 Lagar was described from caves of Mallorca and considered to be very close to the Algerian
143 D. (T.) iblis (Lagar, 1976). Jeannel (1928) hypothesised that the north African Trechopsis are
144 old relicts from a period when the Kabylian massif was attached to the Tyrrhenian plate (the
145 "Tyrrhenis"). He also suggested that they might be related to some Duvalius from the eastern
146 Mediterranean (the "Égéide"), and that the ancestors of Trechopsis might have colonized
147 Africa from Sicily during the “Nummulitique” (Paleogene) (Jeannel, 1928).
148 The relationships of the species of Duvalius s.str. from the western Mediterranean
149 islands are also unknown. The only Duvalius s.str.from Mallorca, D. balearicus Henrot, was
150 considered to be close to the only Iberian Duvalius s.str. (D. berthae Jeannel) (Henrot, 1964).
151 Duvalius berthae has been shown to be related to other Duvalius from southeast France
152 (Faille et al., 2013), but its potential relationships with the Mallorcan D. balearicus have
153 never been testes. Jeannel (1928) noted that the only species of Duvalius s.str. from North
154 Africa (D. jurjurae Peyerimhoff) had strong morphological similarities with the Sicilian
155 species of Duvalius. Following Jeannel (1928), Vigna Taglianti (1982) suggested that the
156 Sicilian Duvalius species have a close relationships with some species from the central
157 Apennine. Finally, the only species from Sardinia, Duvalius sardous (Dodero), is endemic to
5 158 a small massif in the central-eastern part of the island. Jeannel (1928) hypothesised a close
159 relationship of this peculiar, isolated species with the D. brujasi Sainte-Claire Deville species
160 group from the Alpes Maritimes, whereas Vigna Taglianti (1982), hypothesized some
161 possible affinities with the Iberian D. berthae.
162
163 1.2. Molopina
164 Species of Molopina (Carabidae Pterostichini) have an Euro-Mediterranean
165 distribution, and include a number of epigean genera, but also some species with subterranean
166 habits, with different degree of troglomorphism. The taxonomic and geographic limits of
167 Molopina have been contentious. Jeannel (1948) proposed a close relationship between the
168 Euro-Mediterranean taxa attributed to “Pterostichidae Molopini” and some genera of South
169 Africa, Madagascar, Australia and New Zealand. However, Casale and Ribera (2010) showed
170 that the clade “Molopina” with the exclusion of the austral taxa (represented in this study by
171 several Madagascan genera) was strongly supported. Molopina thus included the genera
172 Molops Bonelli, Tanythrix Schaum (treated here as a distinct genus: see Vigna Taglianti,
173 2005), Percus Bonelli and Abax Bonelli, but excluding Styracoderus Chaudoir (a genus
174 currently attributed to this subtribe, e.g. Mateu, 1955).
175 Molopina are treated as a valid and distinct subtribe of Pterostichini (or tribe when
176 Pterostichinae is treated as subfamily) in some recent contributions and catalogues
177 (Brandmayr and Zetto Brandmayr, 1979, 1994; Vigna Taglianti, 2005; Serrano, 2013), but
178 was ignored by both Bousquet (2003) and Lorenz (2005). Molopina are mainly characterised
179 by the lack of discal setae on elytra, by the presence of a keel at the base of the seventh stria
180 (reduced in some species) and by a membranous band at the base of the first antennal joint of
181 the larvae. In some genera all species show developed parental care and pre-social behaviour.
182 Casale and Ribera (2010) did not study any subterranean species of Molopina, and neither did
183 any of the other published phylogenies of the group (Brückner, 2002, 2004a,b; Brückner and
184 Mossakowski, 2006; Sasakawa & Kubota, 2007). These subterranean taxa include in the
185 Western Mediterranean the genera Speomolops Patrizi (Sardinia), Henrotius Jeannel
186 (Mallorca), and Oscadytes Lagar, Molopidius Jeannel and Zariquieya Jeannel (central and
187 eastern Pyrenees), all of them monospecific except for Zariquieya, with two species (Faille et
188 al., 2011b) (Table S1). Three further taxa of Molopina from the eastern Alps and the Balkans
189 are treated either as subgenera of Molops (e.g. Lorenz, 2005) or as distinct genera (e.g.
190 Bousquet, 2003), including the subterranean Typhlochoromus Moczarski with two species in
191 the eastern Alps.
6 192
193 In this contribution we present the first estimation of the phylogenetic relationships of the
194 subterranean species of the Duvalius lineage and of Molopina in the western Mediterranean.
195 For the Duvalius lineage we include species from Mallorca, Sicily and Sardinia, plus species
196 from the European and North African mainland. For Molopina we include a sample of all
197 epigean genera plus all subterranean taxa from the western Mediterranean and one from the
198 eastern Alps. In addition to establish the phylogenetic relationships of the subterranean taxa,
199 we provide an approximate time framework for their diversification, and hypothesize a
200 scenario for their biogeographical origin.
201
202 2. Materials and methods
203 2.1. Taxon sampling and DNA sequencing
204 We sampled representatives of species of the genus Duvalius from the westernmost
205 part of its distribution area, including Spain, France, Italy, Algeria and Western
206 Mediterranean Islands: Mallorca, Sardinia and Sicily (Fig. 1; Table S2). As outgroups we
207 included 41 species of Western European Duvalius of different species groups (Faille et al.,
208 2013) as well as representatives of other genera of Western European Trechini, both isotopic
209 (Agostinia, Anophthalmus Sturm, Luraphaenops, Trichaphaenops) and anisotopic
210 (Aphaenops Bonvouloir, Geotrechus Jeannel, Trechus Clairville). Trees were rooted on the
211 split between the anisotopic and isotopic genera, following previous works on the group (e.g.
212 Faille et al., 2013).
213 For Molopina, we included examples of all the western Mediterranean genera of the
214 subtribe (Jeannel, 1948; Mateu, 1955; Casale and Ribera, 2010), as well as one species of
215 Typhlochoromus, a subgenus of Molops (Bousquet, 2003) considered to be a valid genus by
216 some authors (e.g. Vigna Taglianti, 2005; Lorenz, 2005; Casale and Vigna Taglianti, 2005)
217 (Fig. 2; Table S2). As outgroups we included a number of genera of Pterostichini, including
218 some Madagascan genera and Styracoderus, formerly included in Molopina. Trees were
219 rooted in these Madagascan genera, following Casale and Ribera (2010).
220 Specimens were collected by hand and immediately killed in 96% ethanol, or by
221 means of pitfall traps containing propylene glycol as preserving agent. DNA was extracted
222 non-destructively from whole specimens using a standard phenol chloroform extraction or
223 commercial extraction kits (mostly Qiagen, Hilden, Germany). Voucher specimens have been
224 deposited in IBE (Barcelona), ZSM (Munich) and MNHN (Paris); DNA aliquots are kept in
225 the DNA collections of ZSM (Munich) and IBE (Barcelona).
7 226 We sequenced two mitochondrial fragments, including four genes (3’end of partial
227 cytochrome c oxidase subunit I, COI; and a continuous fragment, rrnL+trnL+nad1, including
228 the 3’ end of the gene for the large ribosomal RNA subunit, the leucine transfer RNA gene,
229 and the 5’ end of the NADH dehydrogenase subunit 1 gene) and two nuclear genes (5’ end of
230 the small ribosomal RNA subunit gene, SSU; and an internal fragment of the large ribosomal
231 unit, LSU). For Molopina we also sequenced the mitochondrial protein coding genes NADH5
232 and an additional fragment of NADH1, and the ribosomal 12S (see Table S3 for the primers
233 used). Sequences were assembled and edited with Bioedit 7 (Hall, 1999), Sequencher 4.6
234 (Gene Codes Corporation, Ann Arbor, MI, USA) or Geneious v.6 (Biomatters Ltd, Auckland,
235 New Zealand). Some of the sequences were obtained from Faille et al. (2010, 2011a, 2012,
236 2013) for Trechini and from Düring and Brückner (2000), Brückner (2002, 2004a,b),
237 Brückner and Mossakowski (2006) and Casale and Ribera (2010) for Molopina. New
238 sequences (242) have been deposited in EMBL databases (see Table S1 for Accession
239 Numbers).
240
241 Phylogenetic analyses
242 Protein-coding and ribosomal genes were aligned with the online version of MAFFT v.6
243 (Katoh and Toh, 2008) using the EINS-i and QINS-i algorithms, respectively, with other
244 parameters set to their defaults.
245 Phylogenetic analyses were conducted with maximum-likelihood (ML) and Bayesian
246 inference (BI) using the CIPRES Science Gateway (Miller et al., 2010). ML trees were
247 obtained using RAxML v.7.2 (Stamatakis, 2006). Data sets were partitioned by gene, with the
248 16S divided in two fragments in Molopina as for some taxa the sequence data were
249 incomplete. We applied an independent GTR+G evolutionary model to each partition, and
250 obtained node support values with 1000 bootstrap replicates.
251 For the Bayesian analyses we used BEAST 1.8 (Drummond et al., 2012), initially with
252 the same partitions and evolutionary models as in the ML analyses. In the Duvalius
253 phylogeny we deleted duplicated terminals, combining the two specimens of D. berthae in a
254 single chimera. In all analyses we used a Yule speciation model as tree prior and established
255 an arbitrary age for the root. We set a lognormal relaxed clock with flat priors for all
256 partitions, and run 100 MY generations sampling every 5,000, assessing convergence with
257 TRACER v1.6 (Rambaut et al., 2014).
258 For the calibration analyses in BEAST we excluded outgroups and rooted the trees
259 according to the topologies obtained in the ML and BEAST analyses with outgroups. There
8 260 are no known fossils of Molopina, Duvalius or other isotopic genera, and the available fossils
261 of Trechus sensu lato are too distant to be used reliably here (see e.g. Schmidt and Faille,
262 2015; Schmidt et al., 2016a,b for some recent fossils described from Baltic amber). To
263 calibrate the trees we thus used the rates estimated for some of the same genes for the related
264 ground beetle genus Carabus Linnaeus by Andújar et al. (2012, 2014) using a combination of
265 fossils and biogeographic events. We used a normal prior distribution with values 0.0145
266 subs/branch/MY for the 3' end of COI (Molopina) or 0.0130 for a fragment combining the 5'
267 and 3' ends (Duvalius), 0.0016 for 16S, 0.0159 for NADH5 (only in Molopina) and 0.0013 for
268 28S, all with a standard deviation of 0.0001. Preliminary results (not shown) demonstrated
269 that the exclusion of NADH5 in the calibration did not had any substantial effect in the
270 estimated age of the root of the Molopina tree. We use flat priors for the rates of other genes.
271 For the mitochondrial genes we compared a strict or a lognormal relaxed clock using AICM
272 in TRACER, and used a relaxed clock for all nuclear genes. The use of the same evolutionary
273 rates and models for the same genes in the two groups allows for a direct comparison between
274 the estimated ages.
275
276 3. Results
277 3.1. Duvalius phyletic lineage
278 a) General topology (analyses with outgroups)
279 The BEAST analysis using a partition by gene failed to converge, so we used two
280 partitions, one for the mitochondrial genes with a GTR+G+I evolutionary model and one for
281 the nuclear, with a HKY+G+I evolutionary model (a GTR model also failed to converge
282 adequately).
283 The topologies of the ML (RAxML) and Bayesian analyses (BEAST) were very
284 similar, with the same well-supported nodes (Figs 3, S1). The monophyly of the group of
285 western Duvalius species plus the genera Anophthalmus, Trichaphaenops, Luraphaenops and
286 Agostinia (the "isotopics") was well supported, although neither Duvalius nor any of its two
287 subgenera (Duvalius and Trechopsis) was recovered as monophyletic. The species D.
288 delphinensis was sister to the species of Anophthalmus, and all the other subterranean genera
289 were nested within Duvalius (forming what we call Duvalius sensu lato from now on), in
290 agreement with Faille et al. (2013). Within Duvalius there were some well defined species
291 groups. First, a clade of Duvalius species from the Maritime and Ligurian Alps including
292 Agostinia, confirmed to be closer to the species of the gentilei species group than to the
9 293 carantii group. Duvalius sicardi Fagniez, D. cailloli (Sainte-Claire Deville) and D. perrinae
294 Giordan form a well-supported clade.
295
296 b) Calibrated Bayesian analyses
297 The run with a strict clock for the mitochondrial partitions had a better AICM than that
298 using a relaxed clock (Table 1), so we used a partitioned model by genes, all with a GTR+G+I
299 evolutionary model and a strict clock. For the nuclear genes we used a HKY+G+I model, as a
300 GTR model failed to converge adequately, with a relaxed lognormal clock and flat priors.
301 The root of the tree (crown diversification of the sampled Duvalius sensu lato +
302 Anophthalmus) was estimated to have occurred at the end of the Miocene, ca. 6 Ma (95%
303 confidence interval 7.5-4.8 Ma), with the origin of the two species from Mallorca (D.
304 (Trechopsis) ferreresi and Duvalius (s.str.) balearicus) at ca. 4.2 Ma (c.i. 5.2-3.2).
305 Surprisingly, and despite their strongly different degree of troglomorphism, these two species
306 were sisters, with a very recent separation dating from the Pleistocene (0.7-0.01 Ma) (Fig. 4).
307 All sampled Algerian, Sicilian and Sardinian species formed a well supported clade, with an
308 uncertain sister group (Fig. 4). The Algerian species (D. (Trechopsis) lapiei and D. (T.) iblis)
309 were sister, with a separation dating from ca. 2.5 Ma, and in turn sister to the two studied
310 Sicilian species (D. aliciae Magrini, Baviera and Petrioli and D. ribaudoi Magrini, Petrioli
311 and Degiovanni). The separation between Algerian and Sicilian species was estimated to have
312 occurred during the Pliocene, ca. 3.2 Ma (c.i. 4.2-2.5). In turn, the separation of the
313 Sicilian+Algerian species from the Sardinian D. sardous was estimated to have occurred ca.
314 4.2 Ma (c.i. 5.3-3.2), with a stem age for the whole clade of ca. 5 Ma (Fig. 4).
315
316 3.2. Molopina
317 a) General topology (analyses with outgroups)
318 In the ML and Bayesian analyses with outgroups Molopina was strongly supported
319 and divided in two equally well supported clades, the genus Percus and the remaining genera.
320 Within the latter Abax was sister to a group of genera including Molops, Tanythrix and all the
321 subterranean taxa (the Molops group) (Figs 5, S2). Within the Molops group only two
322 intergeneric nodes were well supported in the ML analysis: the sister relationship between the
323 Mallorcan Henrotius and the genus Molops, and the monophyly of all the central and eastern
324 Pyrenean subterranean genera: Molopidius, Oscadytes and Zariquieya (Fig. 5). In the
325 Bayesian analysis, however, most relationships were well supported, with the two epigean
326 genera (Molops and Tanythrix) sisters and sister to all the subterranean genera. Within the
10 327 later, the relationships of the two island endemics (Speomolops and Henrotius) with a clade
328 including all the mainland genera were unresolved (Fig. S2).
329 Within the genus Percus, with only epigean species, relationships were in general very
330 well supported. Percus villae Kraatz (French and Italian Maritime Alps) was sister to the rest
331 of the genus, which was divided in an Iberobalear lineage and a lineage including Corsica,
332 Sardinia, mainland Italy and north Africa. The Balearic endemic P. plicatus Dejean was sister
333 to all the Iberian species (traditionally included in a separate subgenus, Pseudopercus
334 Motschulsky). The sampled species present in Corsica and Sardinia (P. strictus Dejean and P.
335 grandicollis Audinet-Serville) were sister to P. lineatus Solier, from Sicily, Algeria and
336 Tunisia (Figs 5, S2).
337
338 b) Calibrated Bayesian analyses
339 In the BEAST runs we constrained the separation between Percus and the rest of
340 Molopina, according to the topology with outgroups. The best AICM corresponded to the
341 combination of a GTR+G+I evolutionary model for all partitions and a relaxed molecular
342 clock (Table 1). The topology of the ingroup was very similar to that obtained for the analyses
343 with outgroups, with only some internal nodes of the Molops group showing a different
344 topology (Fig. 6). Topological changes affected specially the placement of the two island
345 endemics, Speomolops and Henrotius.
346 The basal diversification of Molopina was estimated to have occurred in the upper
347 Eocene (ca. 41 Ma, Fig. 6). Within the Molops group of genera, the diversification (with the
348 exception of the Pyrenean lineage) was estimated to have occurred in a remarkably narrow
349 temporal window, starting at 35 Ma (c.i. 41-28) and ending in the origin of the six main
350 lineages between 30-28 Ma (Fig. 6). The diversification within the Pyrenean lineage of
351 subterranean Molopina was posterior, during the Miocene, between 17 Ma for the origin of
352 Molopidius and ca. 9 Ma for the separation between the two species of Zariquieya (Fig. 6).
353 Within the genus Percus, the basal separation between P. villae and the rest of species
354 was also dated at ca. 28.6 Ma (c.i. 35-23), simultaneous with the basal diversification of the
355 Molops group. The estimated age for the separation of the Balearic endemic (P. plicatus) from
356 the Iberian species was ca. 19 Ma (c.i. 24-15), and that of the group of species from the
357 Tyrrhenian islands, mainland Italy and north Africa 14.5 Ma (c.i. 19-11) (Fig. 6).
358
359 4. Discussion
11 360 Our results clearly establish two contrasting temporal and geologic scenarios for the origin of
361 the subterranean taxa of the Duvalius lineage and Molopina in the western Mediterranean
362 islands. Despite the lack of fossil record or other unambiguous calibration points, the use of
363 the same evolutionary rates in the same genes in both lineages allows for a clear
364 discrimination between an Oligocene origin associated to the tectonic opening of the western
365 Mediterranean basin in Molopina, and a Messinian or post-Messinian diversification in the
366 Duvalius lineage.
367
368 4.1. Duvalius phyletic lineage
369 The reconstructed topology of the western Duvalius group plus the genus Anophthalmus is
370 consistent with previous results (Faille et al., 2013), including the position of D. delphinensis
371 as sister species of Anophthalmus, supported also by some morphological characters (shape of
372 male genitalia and pubescence of the head).
373 The westernmost Mediterranean species of the Duvalius lineage, D. berthae from the
374 Catalan coastal range, was previously considered to be close to D. lespesi (Fairmaire) from
375 southern France (Jeannel, 1928), the Sardinian D. sardous (Vigna Taglianti, 1982), or the
376 Mallorcan D. balearicus. It was, however, confirmed to be related to D. raymondi
377 (Delarouzée) from Provence (Southern France) with strong support, with an estimated
378 separation in the Late Pliocene, a relationship already suggested in a recent work (Faille et al.,
379 2013). The two species of Duvalius from Mallorca were recovered as sister with strong
380 support despite their remarkable morphological differences. The relationships of these two
381 species are however uncertain, as the sister relationship with the Ibero-Provençal D. berthae-
382 D. raymondi clade in the ML and uncalibrated BEAST analyses was poorly supported, and
383 not recovered in the calibrated BEAST analysis. In any case, the estimated age of the
384 Mallorcan lineage is clearly posterior to the tectonic separation of the Balearic Islands from
385 continental Spain during the Oligocene, and more compatible with a Messinian origin as
386 suggested for other terrestrial and low dispersers groups (Chueca et al., 2017). The separation
387 between the two Mallorcan species was estimated to be of Pleistocene origin, which would
388 imply that D. (Trechopsis) ferreresi developed its troglomorphic features in a very short time
389 - and in parallel to that of the other species of Trechopsis from North Africa (see below).
390 Among the other island species, Duvalius sardous, traditionally considered to be close
391 to D. brujasi (south France), was found sister to a clade including the Algerian Trechopsis
392 and Sicilian Duvalius. The paraphyly of the island species with respect to the Algerian
393 implies that the separation between Sardinian and Sicilian species occurred prior to the
12 394 colonization of Africa by Duvalius. The northern origin of the Algerian Duvalius is in
395 agreement with the complete lack of species belonging to this group in other African regions,
396 which is rich in Trechini of other species groups (genus Trechus in the widest sense),
397 especially in the Moroccan, Ethiopian and East African mountains (Jeannel, 1927). A
398 colonization of north Africa from Sicily is also known in other organisms (e.g. firs of the
399 genus Abies, Sánchez-Robles et al., 2014), although the contrary pattern - i.e. a southern
400 origin of Sicilian fauna - is also found in e.g. some reptiles (Carranza et al., 2008) and
401 amphibians (e.g. Stöck et al., 2008a,b). The connection between Sicily and Tunisia might
402 have been facilitated by the lower sea level during the glacial phases, reducing the distance
403 between the two continents and causing the emergence of potential stepping-stone islands
404 (Husemann et al., 2014).
405 The separation between D. sardous and the Sicilian-Algerian species was estimated to
406 date from the early Pliocene, approximately at the same age than the two Mallorcan species.
407 Sicily does not belong to the Corso-Sardinian tectonic plate, but during the Tortonian and
408 Messinian it was linked to north Africa, Calabria and the southern Apennines, which in turn
409 were closely linked to Corsica, Sardinia and the Tuscan area (Rosenbaum et al., 2002). The
410 connection between Sicily and north Africa remained until the Pliocene (Rosenbaum et al.,
411 2002; Lo Presti and Oberprieler, 2011), in agreement with our estimations, but the Sicily strait
412 is considered to have been a strong barrier during the Pleistocene, separating the European
413 and African biota much more efficiently than the Gibraltar strait (Lo Presti and Oberprieler,
414 2011; Husemann et al., 2014).
415 The subgenus Trechopsis, which originally included some species from Southern
416 Greece and Western Anatolia (Jeannel, 1934a,b; Coiffait, 1973), is currently restricted to four
417 Western Mediterranean species highly specialised to the subterranean environment, with
418 “aphaenopsian” habitus (Casale, 1979; Casale and Laneyrie, 1982; Moravec et al., 2003). Our
419 results clearly demonstrated its polyphyly, with the Mallorcan D. (T.) ferreresi not directly
420 related to the Algerian species, implying that their similarities in body shape are the result of
421 evolutionary convergence. The putative relationships between the oriental and African species
422 formerly included in Trechopsis (now treated as Duvalius huetheri group sensu Casale and
423 Laneyrie, 1982) remains unexplored, although the polyphyly of Trechopsis suggests that their
424 similarity may also be the result of convergence. Duvalius djurjurae, the only Duvalius sensu
425 stricto in Africa, is known only from a couple of exemplars from the Djurdjura. Although we
426 failed to find an exemplar for the molecular analysis, the species will most likely be included
13 427 in the clade of the Algerian Trechopsis+Sicilian Duvalius, as its morphological proximity
428 with Sicilian species was already evidenced by Jeannel (1928).
429
430 4.2. Molopina
431 The distribution of subterranean Pterostichini Molopina presents a classical
432 "Tyrrhenian" pattern, with morphologically specialised species in the Iberian peninsula,
433 Pyrenees, Sardinia and Mallorca. No subterranean species is known so far from Sicily, the
434 Maghreb or the eastern Mediterranean, although the remains of a specimen sampled in a cave
435 in Southern Anatolia were attributed to Molopina (Vigna Taglianti, 1980; Casale and Vigna
436 Taglianti, 1999). The ancient origin of Molopina and an early differentiation of the genera
437 were already hypothesized by Brandmayr and Zetto Brandmayr (1979), based in part in the
438 study of the peculiar system of parental care in these pre-social groundbeetles. They
439 hypothesised that the pre-social behaviour is ancestral in Molops, as all species show strongly
440 reduced ovipositors and the same life specialisation (what they call "aestivation"), considered
441 to be of recent origin. However, what they consider to be the ancient Molopina, the stenotopic
442 endogeous or cave-dwelling genera with only one or few species, were thought to be the relics
443 of Oligocene or at least pre-Miocene forms, which were once living on the land masses
444 around the Tethys (Brandmayr and Zetto Brandmayr, 1979). Although the breeding behaviour
445 of the subterranean species is unknown, neither Speomolops nor Typhlochoromus or
446 Stenochoromus show reduced terminalia in the female genitalia, suggesting the lack of pre-
447 social habits. Amongst the troglobitic species, only the larval stages of Speomolops sardous
448 are described so far (Casale et al., 2010), from five third instar larvae collected 20–30 cm
449 below the surface of a sandy bank near one of the freshwater subterranean lakes where adults
450 were very abundant. The larvae were never observed walking on the surface. This
451 demonstrates that the larva of this species has obligate underground, fossorial behaviour, in
452 sandy soil periodically flooded by the subterranean river of the cave.
453 Our results unambiguously show that the subterranean genera (Molopidius,
454 Speomolops, Henrotius, Zariquieya, Oscadytes and Typhlochoromus) are members of this
455 subtribe. They were all also included in the Molops group, one of the three lineages within
456 Molopina (the other two, the genera Percus and Abax do not have any known subterranean
457 species). The intergeneric relationships within the Molops groups were, however, poorly
458 supported with the exception of the monophyly of the subterranean Pyrenean genera, which
459 were estimated to have diversified during the Miocene, after the opening of the western
460 Mediterranean basin. It is remarkable that all basal lineages of the Molops group, with
14 461 uncertain relationships between them, were estimated to have originated in a relatively narrow
462 temporal window which fully overlaps with the tectonic opening of the western
463 Mediterranean basin (Rosenbaum et al., 2002; Schettino and Turco, 2006). In the only
464 analysis with some supported basal nodes, the uncalibrated Bayesian (Fig. S2), all
465 subterranean taxa were recovered monophyletic and the cladogenetic order reflected the
466 temporal succession of the separation of the plates: first the Sardinian species (Speomolops),
467 then the Mallorcan (Henrotius) and, within the continental taxa, first the eastern species
468 (Typhlochoromus) and finally the diversification within the Pyrenean group. This scenario
469 would imply an early division between an eastern lineage formed by epigean forest species
470 (Molops and Tanythrix), with a distribution centred in the central and eastern Alps and the
471 Balkan peninsula, and a mostly western lineage with an ancestor with at least some
472 subterranean habits and some troglomorphic characters, likely similar to the current Pyrenean
473 Molopidius or the eastern Typhlochoromus. The monophyly of subterranean lineages
474 previously though to have independently colonised the subterranean environment is a
475 recurrent result in multiple groups (e.g. Ribera et al., 2010; Faille et al., 2010, 2011a, 2013),
476 but in the case of Molopina the evidence is still insufficient. Other than the low support of the
477 internal relationships in most analyses, the morphological and geographic evidence supports a
478 closer relationship of Typhlochoromus with the eastern lineages of the Molops group
479 (Busulini, 1957). There is also no molecular data available of the Balkan-Dinaric subterranean
480 genera Stenochoromus and Henrotiochoromus, which may provide valuable information on
481 the evolution of the eastern Molopina.
482
483 Acknowledgements
484 We are particularly grateful for their support in obtaining material and data to Giuseppe
485 Grafitti, Paolo Marcia, Alessandro Molinu and Carlo Onnis in Sardinia, Paolo Magrini in
486 Sicily, and Charles Bourdeau, Alexandra Cieslak, Javier Fresneda, Enrico Lana, Miquel
487 Palmer, Germana Rondolini, Mateu Vadell and all the collectors listed in Table S1 in
488 Mallorca and other areas. We thank Ana Izquierdo (MNCN, Madrid), Rocio Alonso and
489 Anabela Cardoso (IBE, Barcelona) for laboratory work. We also thank F. Ciampor
490 (Bratislava) and R. Panin (Lviv) for the habitus photographs of some Molopina. Zoological
491 researches of AC were supported by the Italian Ministero dell’Istruzione, dell’Università e
492 della Ricerca Scientifica e Tecnologica (MIUR-PRIN 2004057217 “Zoogeography of
493 Mediterranean - Southern African disjunct distributions by a multimethod approach”), and the
494 UE program Interreg Sardinia-Corsica-Tuscany on Biodiversity. Molecular work was
15 495 supported by projects DFG FA 1042/1-1, CGL2010-15755 and CGL2016-76705-P
496 (AEI/FEDER, UE).
497
498 Appendix A. Supplementary material
499 Supplementary data associated with this article can be found, in the online version, at
500 ...
501
502 Table S1: List of species of Isotopic Trechini and Molopina occuring on Western
503 Mediterranean Islands, Iberian Peninsula and North Africa.
504 Table S2: Material used in the study, with locality data, voucher number and accession
505 numbers of the sequences.
506 Table S3: Primers used in the amplification and sequencing reactions.
507
508 Fig. S1: uncalibrated Bayes tree Duvalius
509 Fig. S2: uncalibrated Bayes tree Molopina
510
511
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776
24 777 Table 1
778 Evolutionary model and mitochondrial clock comparisons for the Duvalius lineage and
779 Molopina, including AICM values. Best AICM values shown in bold, models that failed to
780 converge adequately by a dash.
781
782 evolutionary mitochondrial AICM model nuclear clock Duvalius GTR strict - HKY strict 18283.3 GTR relaxed - HKY relaxed 18378.8 Molopina GTR strict 45275.5 HKY strict 45033.2 GTR relaxed 45006.4 HKY relaxed 45108.8
783
784
25 785 Figure legends
786
787 Fig. 1. Distribution of the subterranean species of the Duvalius lineage in the western
788 Mediterranean.
789 Fig. 2. Distribution of the subterranean species of Molopina in the western Mediterranean.
790 (except the epigean genera Percus and Abax). 1, Oscadytes; 2, Zariquieya; 3, Molopidius; 4,
791 Henrotius; 5, Speomolops; 6, Typhlochoromus; 7, Henrotiochoromus; 8, Molops (including
792 Tanythrix and Stenochoromus). In red, hypogean genera.
793 Fig. 3. Phylogeny of the western Mediterranean species of the Duvalius lineage. Phylogram
794 obtained with ML in RAxML. Numbers in nodes, bootstrap support. In red, highly modified
795 troglomorphic species; in red bold species of the subgenus Trechopsis. See Table S1 for
796 details on the specimens.
797 Fig. 4. Bayesian calibrated phylogeny of the Duvalius lineage. Ultrametric tree obtained with
798 BEAST. Number inside nodes, estimated age; numbers above branches, posterior probability
799 of the node. Dotted line marks the end of the Messinian salinity crisis 5.3 Ma. In red, highly
800 modified troglomorphic species; in red bold species of the subgenus Trechopsis. See Table S1
801 for details on the specimens. Habitus photographs, A. Faille.
802 Fig. 5. Phylogeny of the western Mediterranean species of Molopina. Phylogram obtained
803 with ML in RAxML. Numbers in nodes, bootstrap support. In red, subterranean species. See
804 Table S1 for details on the specimens. Habitus photographs, A-C & H, F. Ciampor; D, R.
805 Panin, E-G & I, A. Faille.
806 Fig. 6. Bayesian calibrated phylogeny of Molopina. Ultrametric tree obtained with BEAST.
807 Number inside nodes, estimated age; numbers above branches, posterior probability of the
808 node. Dotted line marks the end of the Messinian salinity crisis 5.3 Ma; purple band marks the
809 tectonic opening of the western Mediterranean basin. In red, subterranean species. See Table
810 S1 for details on the specimens.
811
26
Duvalius carantii L542 100 Duvalius carantii L283 50 Duvalius lanai L476 80 Duvalius pecoudi L275 63 Duvalius roberti AF129 74 Duvalius cailloli L192 82 Duvalius perrinae L426 92 Duvalius sicardi L241
85 Duvalius joffrei L23 57 Duvalius ochsi L172 95 55 Duvalius waillyi L434 Agostinia launi L435 63 Duvalius gentilei L294
Duvalius opermanni L754 Duvalius boldorii L352
Duvalius klimai L462 A B Duvalius erichsonii L480
Duvalius goemeriensis L451 100 Duvalius bokori L450
Duvalius szaboi L453 100 Duvalius microphthalmus L456
Duvalius cadurcus L312 100 Duvalius lespesi L243 99 Duvalius exaratus L394
Duvalius raymondi L433 100 99 Duvalius raymondi L427 Duvalius berthae AF115 (E)
Mallorca Duvalius (T.) ferreresi L807 (C) 100 Duvalius balearicus L809 (D) Duvalius (T.) lapiei L619 (A) Algeria 67 C D Duvalius (T.) iblis L618 (B) 87 Duvalius ribaudoi L813 Sicily 55 68 Duvalius aliciae L812 (F) Sardinia Duvalius sardous L743
Trichaphaenops gounellei L17 52 Luraphaenops gaudini AF116
Duvalius convexicollis L171 63 Duvalius pastorellii L662
Duvalius diniensis L428 Duvalius lineage (“isotopics”) 100 100 Duvalius brujasi L429
Anophthalmus schmidti L695 85 Anophthalmus tolminensis L686
Duvalius delphinensis L422 E F
Trechus quadristriatus AF96 95 Trechus obtusus L28 100
100 Trechus fulvus AF98 Trechus aff. schaufussi AF101
100 Aphaenops cerberus AF30 “anisotopic” 96 Aphaenops ehlersi AF64 lineage 69 Geotrechus discontignyi AF92 100 Aphaenops leschenaulti AF1
0.02 1 Duvalius carantii L283 0.1 Ma 0.84 0.5 Ma Duvalius carantii L542 0.98 Duvalius lanai L476 0.8 Ma 0.90 Duvalius pecoudi L275 1.0 Ma 1 Duvalius roberti AF129 1.3 Ma 1 Duvalius cailloli L192 0.34 Ma 1 Duvalius perrinae L426 0.7 Ma Duvalius sicardi L241 1 2.3 Ma Duvalius ochsi L172 0.5 Ma 1 Duvalius waillyi L434 0.7 Ma 0.67 1.5 Ma Duvalius joffrei L23 3.3 Ma 0.99 Agostinia launi L435 1.8 Ma Duvalius gentilei L294
0.5 Duvalius opermanni L754 3.0 Ma Duvalius boldorii L352
1 Duvalius microphthalmus L456 0.34 0.76 4.0 Ma Duvalius szaboi L453 3.3 Ma 1 Duvalius goemeriensis L451 0.46 Duvalius bokori L450 3.6 Ma 1 Duvalius cadurcus L312 0.1 1 4.2 Ma 2.2 Ma Duvalius lespesi L243 3.7 Ma 0.53 Duvalius exaratus L394 Duvalius erichsonii L480 3.7 Ma Duvalius klimai L462 0.56 4.6 Ma Mallorca 1 Duvalius (T.) ferreresi L807 0.3 Ma Duvalius balearicus L809
1 Duvalius raymondi L433 0.4 Ma 1 3.2 Ma Duvalius raymondi L427 4.8 Ma Duvalius berthae AF115
0.87 Luraphaenops gaudini AF116 3.4 Ma Trichaphaenops gounellei L17 5.1 Ma Algeria 0.96 Duvalius (T.) lapiei L619 2.6 Ma 1 Duvalius (T.) iblis L618 3.2 Ma 0.82 Duvalius aliciae L812 1 2.8 Ma 4.2 Ma 1 Sicily Duvalius ribaudoi L813 Sardinia 4.9 Ma Duvalius sardous L743 5.4 Ma Duvalius convexicollis L171
1 Duvalius brujasi L429 1.2 Ma 6.0 Ma 0.53 4.4 Ma Duvalius diniensis L428 Duvalius pastorellii L662
1 Anophthalmus tolminensis L686 3.7 Ma 1 4.7 Ma Anophthalmus schmidti L695 Duvalius delphinensis L422 6 Ma 5 4 3 2 1 0
MIOCENE PLIOCENE PLEISTOCENE Zariquieya boumortensis GBK 83 93 Zariquieya troglodytes AI271 (A) Oscadytes rovirai AI270 (B) 100 Molopidius spinicollis AI269 (C) 100 B Molopidius spinicollis AI268 A Typhlochoromus marcelloi AI1291 (D) Molops AN288 100 84 Molops piceus AI362 (E) Molops group 87 Henrotius jordai AI1242 (F) Speomolops sardous AI1292 (G) 100 Speomolops sardous AI272 Tanythrix edurus GBK 100 Tanythrix senilis AI473 93 Abax oblongus GBK D 81 C Abax fiorii GBK 100 71 Abax exaratus AI1295 Abax parallelepipedus GBK 100 Abax Abax ovalis AN297 100 Abax carinatus GBK 88 Molopina Abax pyrenaeus AI267 (H) 100 Percus patruelis GBK 75 Percus politus AI266 100 Percus stultus GBK 57 100 Percus guiraoi GBK E F Percus plicatus RA1085 (I) 76 Percus strictus AI265 100 Percus 100 100 Percus grandicollis RA812 100 Percus lineatus GBK Percus villae GBK Sterocorax globosus AI474 100 93 Styracoderus atramentarius AI1181
100 Corax ghilianii AI1068 Pterostichus AN150 G Astigis salzmanni AI1293 89 Laemostenus atlanticus AN122 100 H 56 Laemostenus carinatus AN253 Platyderus presahariensis AI472 Eucamptognathus androyanus AI477 100 100 Eucamptognathus oopterus AI476 Eudromus striaticollis AI478
0.03
I 1 Zariquieya troglodytes AI271 10.5 Ma 1 16.3 Ma Zariquieya boumortensis GBK Pyrennes 1 Oscadytes rovirai AI270 19.1 Ma Molopidius spinicollis AI268 0.87 1 28.2 Ma 0.38 Ma Molopidius spinicollis AI269 eastern Alps Typhlochoromus marcelloi AI1291 32.6 Ma 1 Tanythrix edurus GBK 12.0 Ma 0.65 Tanythrix senilis AI473 29.0 Ma 0.61 Sardinia 1 Speomolops sardous AI1292 34.6 Ma 0.34 Ma Speomolops sardous AI272
1 Molops AN288 15.4 Ma 29.9 Ma Molops piceus AI362 Mallorca Henrotius jordai AI1242 1 38.3 Ma 0.77 Abax fiorii GBK 0.5 Ma 1 Abax oblongus GBK 1.0 Ma 10.9 Ma Abax exaratus AI1295 0.98 Abax parallelepipedus GBK 12.4 Ma 1 Abax ovalis AN297 16.6 Ma
1 Abax pyrenaeus AI267 12.4 Abax carinatus GBK 40.9 Ma 0.98 Percus politus AI266 9.3 Ma 1 Percus patruelis GBK 11.5 Ma 1 Percus stultus GBK 1 7.8 Ma 19.3 Ma Percus guiraoi GBK
1 Percus plicatus RA1085 22.7 Ma 1 Percus grandicollis RA812 2.0 Ma 1 1 28.6 Ma 14.5 Ma Percus strictus AI265 Percus lineatus GBK
Percus villae GBK
4 0 3 0 2 0 1 0 0 EOCENE OLIGOCENE MIOCENE PLIO. PLE.