2005 Blackwell Verlag, Berlin Accepted on 18 January 2005 JZS doi: 10.1111/j.1439-0469.2005.00307.139–148

1Department of Biochemistry and Molecular Biology and 2Department of Biology, Faculty of Biology, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain

Phylogeny of of the genus : evidence of monophyly of Iberian endemics and of the existence of relict species in Pyrenean refuges

J. Quinteiro1,J.RodrI´guez-Castro1,J.Castillejo2,J.Iglesias-Pin˜eiro2 and M. Rey-Me´ndez1

Abstract The Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. The present study investigated Arion on the basis of analyses of the mitochondrial ND1 gene and nuclear internal transcribed spacer 1 (ITS1) sequences. The Iberian endemic species were monophyletically clustered in two divergent sister clades. The topotype specimens of Arion lusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an ÔAtlanticÕ clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a ÔContinental– MediterraneanÕ clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around the Pliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic species with distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Pliocene and survived through the Pleistocene in geographically confined small populations. Arion lusitanicus showed up to be polyphyletic: specimens, sampled outside the geographic range of the topotype in the north-western Iberian Peninsula, were included in a non-monophyletic clade together with the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arion hortensis, Arion fagophilus and Arion intermedius) were located in basal positions in all topologies. The evolutionary history of these slug species (highly sensitive to climatic factors, with capacity for both outcrossing and selfing, and with low dispersal ability) appears to have been moulded by Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolation during periods of glaciation alternating with interglacial expansions limited by geographic barriers.

Key words: Arion – Iberian phylogeography – endemic species – relict species – ND1 gene – internal transcribed spacer

Introduction A. fagophilus, and the three closely related selfing species of the The genus Arion includes land slug species with a Paleartic subgenus Carinarion, A. (Carinarion) fasciatus, A. (C.) circum- distribution throughout the European continent, the Iberian scriptus and A. (C.) silvaticus]; (iii) a group of species present Peninsula being the geographic area with the highest species in the Pyrenees and adjacent areas (A. anthracius, A. iratii, diversity, as expected in view of its refuge role in the major A. lizarrustii, A. molinae); (iv) an Iberian endemic group Pliocene–Pleistocene and recent ice ages, and its diverse (A. baeticus, A. fuligineus, A. hispanicus, A. nobrei, A. paula- topography and climate. rensis, A. urbiae and A. wiktori); and finally (v) peripheral taxa The taxonomy of Arion is mainly based on morphological such as the Azorean species, A. pascalianus or the Siberian characters of the genital system, which nevertheless show species A. sibiricus (Kerney et al. 1983; Wiktor 1983; Backeljau intraspecific variability among close morphs, reflecting both and De Bruyn 1990; Backeljau et al. 1995; Garrido et al. 1995; internal and environmental effects. Accurate identification of Backeljau et al. 1997; Castillejo 1997, 1998). arionid species is typically difficult: it is essential to take into As noted, species and morph identification of arionids is a account variability in reproductive organ shape in juvenile, difficult task, and this is reflected in diverse problematic issues adult and senile specimens of the same species, in the same area in Arion taxonomy. For example, A. lusitanicus have been and at the same time of year. Moreover, integument colour widely cited throughout Europe on the basis of different polymorphism, another species-diagnostic character, displays morphological characters to those described for the topotype habitat- and season-correlated variation (Backeljau et al. specimens. In the case of two taxa, A. ater and A. rufus, there is 2001). In addition, within this genus, various breeding systems no consensus about their species status. On the other hand, have been observed and demonstrated with genetic markers, Iberian species with morphological singularities have been include outcrossing, self-fertilization, and both outcrossing recently described. Moreover, distribution areas of many and self-fertilization (McCracken and Selander 1980; Foltz species are not definitively known: for example, it is unclear et al. 1982). As a result, Arion systematics is complex and whether A. flagellus occurs outside the Iberian Peninsula controversial, without a clearly defined number of sensu stricto (Castillejo 1992). species, species complexes or subgenera. Molecular phylogenies of gastropods, based on analysis of Nevertheless, four broad biogeographic species groups can the ribosomal RNA gene cluster have suggested that the be usefully recognized within this genus: (i) a Lusitanian or divergent family is monophyletic with the Limaco- Atlantic group distributed along the European Atlantic idea taxon as sister clade (Wade et al. 2001). The few border, including the Iberian Peninsula, France, Great Britain molecular studies performed to date at lower systematic levels, and Ireland (A. flagellus, A. owenii, A. hortensis); (ii) a have mainly focused on population genetics (Backeljau et al. European sensu lato group of species occurring in a wide 2001). Within the genus Arion, genetic variation in enzymes variety of regions throughout the continent [A. ater, A. rufus, indicates that the nine species occurring in Great Britain and A. distinctus, A. intermedius, A. lusitanicus, A. subfuscus, Ireland form heterozygous populations, or monogenetic

JZS (2005) 43(2), 139–148 140 Quinteiro, RodrI´guez-Castro, Castillejo, Iglesias-Pin˜eiro and Rey-Me´ndez

Table 1. Specimens included in the present study

Accession number Species Specimen Location Date (ND1, ITS1)

Arion (Arion) ater AATE 39A Caldas de Gereˆ s, Portugal 31/10/84 AY316228, AY316268 (Linnaeus, 1758) AATE 39E Valporquero Cave, Leon, Spain 1/10/91 AY316229, AY316269 Arion (Arion) rufus ARUF 40A Sova Valley, Spain 17/09/91 AY316230 (Linnaeus, 1758) ARUF 40G Mont. Noire, Central Massif, France 6/09/92 AY316231, AY316270 Arion (Mesarion) nobrei ANOB 41A Luso, Portugal 28/01/86 AY316232, AY316271 Pollonera, 1889 ANOB 41B Luso, Portugal 28/01/86 AY316233, AY316272 Arion (Mesarion) lusitanicus ALUS 42A Serra da Arra´bida, Portugal 4/12/84 AY316234, AY316273 Mabille, 1868 ALUS 42B Serra da Arra´bida, Portugal 4/12/84 AY316235, AY316274 ALUS 42C Serra da Arra´bida, Portugal 4/12/84 AY316336, AY316275 ALUS 42G Alpi Carniche, Rivolato, Italy 10/08/86 AY316237, AY316276 ALUS 42H Surrey, UK 25/07/86 AY316238 ALUS 62E Mont. Noire, Central Massif, France 6/09/92 AY316239, AY316289 ALUS 70A Girona, Spain 17/11/90 AY316240 ALUS 70B Girona, Spain 17/11/90 AY316241 ALUS 70C Girona, Spain 17/11/90 AY316242, AY316290 Arion (Mesarion) fuligineus AFUL 43A Sao Silvestre, Portugal 15/12/85 AY316243, AY316277 Morelet, 1845 AFUL 43B Ponte da Lima, Portugal 15/12/85 AY316244 Arion (Mesarion) flagellus AFLA 44A Branley Bark, Croydon, UK 20/09/86 AY316245, AY316278 Collinge, 1893 AFLA 44B Branley Bark, Croydon, UK 20/09/86 AY316245 AFLA 65E Santiago de Compostela, Spain 25/11/89 AY316246 AFLA 66B Lugo, Spain AY316247 Arion (Mesarion) subfuscus ASUB 45G Coulsdon Wood, Surrey, UK 29/08/86 AY316248 (Draparnaud, 1805) ASUB 45A Montagne Noire, France 6/09/92 AY316279 Arion (Mesarion) iratii AIRA 46B Irati Forest, Navarra, Spain 24/09/91 AY316249 Garrido, Castillejo et Iglesias, 1995 Arion (Mesarion) lizarrustii ALIZ 47C Lizarrusti, Spain 19/9/94 AY316250, AY316280 Garrido, Castillejo et Iglesias, 1995 Arion (Mesarion) molinae AMOL 48B Serra del Cadı´, Barcelona, Spain 16/09/91 AY316251 Garrido, Castillejo et Iglesias, 1995 AMOL 48A Serra del Cadı´, Barcelona, Spain 16/09/91 AY316281 Arion (Mesarion) gilvus Torres AGIL 49A Serra del Pandols, Tarragona, Spain AY316252, AY316282 Mı´nguez, 1925 AGIL 49D Denia, Alicante, Spain 17/10/91 AY316253 Arion (Mesarion) urbiae AURB 50A AY316283 De Winter, 1986 AURB 50C Urbasa Sierra, Navarra, Spain 12/12/86 AY316254 Syn:A.(Mesarion) anguloi AANG 73A Burgos, Spain 16/03/86 AY316267, AY316291 Martı´netGo´mez, 1988 Arion (Mesarion) paularensis APAU 51A Guadarrama Sierra, Segovia, Spain 18/10/92 AY316255 Wiktor et Parejo, 1989 APAU 51D Moncayo Sierra, Zaragoza, Spain 1/11/91 AY316256, AY316284 Arion (Mesarion) hispanicus AHIS 52A Gouveia, Portugal 29/11/84 AY316257 Simroth, 1886 AHIS 52B Ca´ceres, Spain 3/05/91 AY316258, AY316285 Arion (Mesarion) baeticus ABAE 53A Huelva, Spain 10/03/92 AY316259 Garrido, Castillejo et Iglesias, 1994 ABAE 53C Cuenca Sierra, Cuenca, Spain 27/10/91 AY316260 Arion (Kobeltia) hortensis AHOR 54A S. Salvador de Bianya, Girona, Spain 13/11/89 AY316261 Fe´russac, 1819 Arion (Kobeltia) fagophilus AFAG 55C Lizarrusti, Navarra, Spain 19/09/94 AY316262 De Winter, 1986 Arion (Kobeltia) intermedius AINT 56D Aran Valley, Lleida, Spain AY316263, AY316286 Normand, 1852 AINT 56G Coulsdon Wood, Surrey, UK 7/10/86 AY316264 Arion (Kobeltia) anthracius AANT 57A Vale` ncia d’Aneu, Lleida, Spain 10/09/91 AY316265 Bourguignat, 1866 Arion (Kobeltia) wiktori AWIK 58A Demanda Sierra, Burgos, Spain 3/11/91 AY316287 Parejo et Martı´n, 1990 AWIK 58C Urbio´n Mountains, Soria, Spain 2/11/91 AY316266, AY316288 Dedoceras sp. DBEN 60A Bossot, Viella, Le´rida, Spain 11/11/89 AY316292 strains, or both types of populations with probable hybridiza- Isabellaria genus (Clausilidae) – has allowed the identification tion between them (Foltz et al. 1982). Allozyme analyse have of polyphyletic groups and demonstrated that the morpholo- also been used to investigate the status of the Azorean species gical characters traditionally used, the clausilial structures, are (Backeljau et al. 1992) and ecogenetic aspects of the Central of limited taxonomic value (Schilthuizen et al. 1995). European Arion fasciatus complex species, within the subgenus The mitochondrial NADH dehydrogenase 1 gene codes for Carinarion (Backeljau et al. 1997; Jordaens et al. 1998). To an enzyme subunit involved in the electron transport chain; its date there have been no DNA-based studies of Arion phylo- sequence is currently only available for three pulmonate geny or population genetics, although some populations of gastropod species (Yamazaki et al. 1997). The use of this large arionids have been differentiated using RAPD analysis mitochondrial NADH dehydrogenase 1 (ND1) DNA sequence (Noble and Jones 1996). In other gastropods, DNA sequence for phylogenetic reconstruction is infrequent, mainly due to analysis – specifically, analysis of internal transcribed spacer the absence of ÔuniversalÕ primers. The mitochondrial ND1 (ITS) sequence variation in Albinaria and the closely related does not contain iron–sulphur centres of NADH coenzyme Q 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 Phylogeny of Arion species 141 reductase (complex I), so that we can expect less strict An individual from the related genus Dedoceras sp. was analysed for functional constraints on nucleotide and aminoacid variability. use as outgroup (Wade et al. 2001). Specimens had originally been Reasonable high variability is a pre-requisite for phylogenetic collected 7–17 years ago, and had been preserved in formalin or ethanol and stored at room temperature. Total DNA extraction, from reconstruction at the genus level. However, mtDNA intro- heart or gonad, was performed using the DNeasy Tissue Kit (Qiagen, gression could have occurred in closely related species of Hilden, Germany). A pair of primers with degenerated positions was arionids, resulting in incongruent phylogenetic inferences from designed to amplify the first half of the mitochondrial ND1 gene: nuclear and mitochondrial data sets (Ferris et al. 1983). As MOL-NAD1F (5¢-CGRAARGGMCCTAACAARGTTGG-3¢) and noted, the internal transcribed spacer 1 (ITS1) nuclear MOL-NAD1R (5¢-GGRGCACGATTWGTCTCNGCTA-3¢). Am- sequences, has proved useful for inferring phylogenetic rela- plifications were performed using AmpliTaq polymerase in PCR tionships in other gastropod genera (Schilthuizen et al. 1995), Buffer II, in a GenAmp 9700 PCR System (Applied Biosystems, Foster City, CA, USA). The PCR thermal profile comprised an initial and as a source of complementary nuclear data to search for denaturation step at 94C, followed by 35 cycles of denaturation at putative introgressive hybridization events. 94C for 30 s, annealing at 50C for 30 s, and extension at 72C for Within the genus Arion, a number of phylogeographic and 60 s. MgCl2 concentration was 2.5 mM. In the few cases in which systematic issues remain unresolved or uncertain in the light of effective amplification was not obtained, annealing temperature was morphological data. These issues include (a) the species and reduced to 45C and MgCl2 concentration was tested over the range sub-genus status of recently redescribed species, initially 1.5–3.5 mM until amplification was achieved. In addition, nuclear ITS sequences were amplified with the primers BAB18S1F (5¢-GCTGGC- described on the questionable systematics studies in the 19th CGAGAAGAAGCTC-3¢) and BAB28SR (5¢-GACACTGAGGGAT- century (Castillejo 1998); (b) the evolutionary origins of and TCGGTGC-3¢), with the same PCR thermal profile except that relationships among species with distributions limited to the extension was for 2 min. Purified PCR products were directly Iberian Peninsula, or to small refuges in the Pyrenees; and (c) sequenced, in both directions, with the BigDye sequencing kit version the relationships between these endemic species and more 3.0 (Applied Biosystems). Fluorescent extension products were separ- widely distributed species. These issues are here addressed with ated and detected with an ABI PRISM 377 automated sequencer (Applied Biosystems). Revision of electropherograms, manual se- DNA sequence data. We discuss the utility of mitochondrial quence alignment, translation, and homology search by local BLAST ND1 gene for the study of close intra-genus relationships in in a mollusc DNA database, were performed with BioEdit v. 5.0.2 gastropods, and the role played by the Iberian Peninsula – (Hall 1999). All sequences were deposited in GenBank/EMBL/DDBJ characterized by its pronounced geographical barriers and under accession no. AY316228–AY316292. historical climatic conditions – in the speciation of these Hypotheses about phylogenetic relationships were established by organisms with low dispersal ability and high sensitivity to distance, maximum parsimony, and maximum likelihood methods biotic parameters, such as vegetation, humidity and tempera- using PAUP* version 4.0b10 (Swofford 1998) for nucleotide sequences and PHYLIP version 3.6 (Felsenstein 1993) for data sets ture. including translated amino acid sequences. To test for saturation by multiple substitutions, the observed number of transitions and transversions in pairwise comparisons were plotted against distance Materials and methods values. Because transition saturation was detected in ND1 sequences, DNA samples were isolated from 46 specimens, belonging to 20 species we additionally tried, two alternative approaches to avoid the of the genus Arion (Fig. 1, Table 1). negative effects of homoplasy in phylogenetic reconstruction:

Fig. 1. Distribution areas of the Arion species considered and sample location 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 142 Quinteiro, RodrI´guez-Castro, Castillejo, Iglesias-Pin˜eiro and Rey-Me´ndez

(a) consideration of transversional changes only; and (b) analysis of This sequence is included in the first half of the mitochond- translated amino-acid sequences. The nucleotide substitution model rial NADH dehydrogenase subunit 1 gene, and its translation that best fit the data was evaluated by the likelihood-ratio test resulted in a 145-aminoacid (aa) fragment. In the mitochond- (Huelsenbeck and Crandall 1997) with the aid of Modeltest version rial genome of Cepaea nemoralis, the sequence is located 3.0 (Posada and Crandall 1998), selecting a GTR + E´+ C model with proportion of invariable sites (I) ¼ 0.1857 and gamma distri- between position 5233 and 5660 (accession no.: U23045) in the bution (C) of variable sites with shape parameter a ¼ 0.6296. Based ND1 gene, which contains a total of 291 aa residues. There is a on this model we constructed a distance matrix and a neighbour- nucleotide composition bias towards T at all codon sites, and joining (NJ) tree. In addition, an NJ tree was constructed using only nucleotide frequencies mean (A ¼ 0.28, C ¼ 0.15, G ¼ 0.14, transversion-based distances. Using the above model parameters, a T ¼ 0.43) showed homogeneity among the taxa considered maximum likelihood (ML) tree was obtained by heuristic search with (v2 ¼ 119.94, df ¼ 123, P ¼ 0.56). In the alignment of the the tree-bisection-reconnection (TBR) branch-swapping algorithm. A consensus tree was also obtained under parsimony criterion from the Arion partial ND1 sequences we identified 304 variable sequence data set using a heuristic parsimony search with closest positions (69.6%), suggesting that this sequence is highly addition sequence. Starting trees were obtained by stepwise addition. variable with a constancy in the substitution rate among Confidence in nodes was evaluated by bootstrapping (Felsenstein lineages (LRT: P > 0.01). As expected for a coding sequence, 1985) with 1000 replicates. The transition/transversion ratio was the third codon position is the most variable, followed by the estimated by maximum likelihood. The hypothesis of monophyly of first and second positions. The transition/transversion ratio endemic Iberian species was tested using both the topology-depend- ent permutation tail probability test (T-PTP) (Faith 1991) and the estimated using ML topology was 0.99. At relatively low likelihood ratio test of monophyly (LRT) (Huelsenbeck et al. 1996). distances values such as 0.3, transversions outnumbered The Shimodaira–Hasegawa test was used to test whether the transitions, both overall and at each codon position (Fig. 2). difference between the estimated alternative topologies was significant The t-test indicated incomplete saturation [t ¼ 5.11, p ¼ 0.0], (Shimodaira and Hasegawa 1999). The constancy in the substitution but the evidence for relative saturation of transitions observed rate among lineages was evaluated with the likelihood ratio test using in the plot (Fig. 2), indicates that phylogenetic reconstruction DNAML and DNAMLK programs (PHYLIP package) (Felsenstein is best based on transversion changes only, thus avoiding 1993) and Modeltest version 3.0 (Posada and Crandall 1998). The analysis of protein sequences on the basis of distance, maximum homoplasy and its negative effect on both distance estimation likelihood and maximum parsimony was performed with the PROT- and tree reliability. DIS, PROTML and PROTPARS programs, respectively, included in The observed high variability in ND1 sequences was appar- the PHYLIP package. Distances between translated sequences were ent in the overall mean uncorrected pairwise distance value of based on the Jones et al. (1992) model of amino acid substitution. The 0.284 (SE ¼ 0.012). Inter-specific sequence divergence between Dedoceras sp. individual was used as outgroup. the Iberian species ranged from 4 to 35%, versus up to 40% considering non-Iberian species. In the Atlantic group (inclu- Results ding the divergent A. hispanicus together with A. lusitanicus, A. nobrei, A. fuligineus and A. flagellus), divergence ranged A 437-bp sequence was obtained for 46 individuals belonging from 4 to 31%, whereas in the Continental–Mediterranean to 20 Arion species and the outgroup, Dedoceras sp.

0.30 010. 00.1 v (first) v (second) s(a)ll s (first) s (second) v (a)ll 05.2

005. 05.0

0.20

05.1 000. 0.00 0.0. 0 04000 .8 00.0.0040.08 0.15 Substitutions v (third)

00.1 s (third) 010.

05.0 0.05

00.0 00.0.002.004.006.0 80 000. Distance 000. 040 . 0 .80

Fig. 2. Relationships between the number of substitutions (transversions and transitions) and genetic distance (Tamura–Nei), considering all nucleotide positions and each of the codon positions of the partial ND1 sequences, for pairwise comparisons of Arion species 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 Phylogeny of Arion species 143 group (A. wiktori, A. paularensis, A. urbiae, A. baeticus, whereas that Mesarion is paraphyletic (Fig. 3). A terminal A. gilvus), divergence ranged from 7 to 20%. In both groups, clade (Fig. 3) comprised a group of closely related species, the mean value was 15–20%, with the mean divergence between including A. flagellus, A. hispanicus, A. nobrei, A. fuligineus and groups being 26% (net distance ¼ 8.7%). The two species of the specimens of A. lusitanicus collected from the Portuguese the A. ater complex (A. ater and A. rufus) showed divergence of type localities in the Iberian Peninsula. This clade contained 20%, i.e. about the mean value. Divergences between geo- species with an Atlantic distribution, mainly in the Iberian graphically close Pyrenean species (A. iratii, A. lizarrusti, Peninsula, with the exception of specimens of A. flagellus A. molinae and A. anthracius) were unexpectedly high, ranging collected in the UK. Surprisingly, within the set of specimens from 32% to 37%. In particular, two sister species (A. iratii, identified as A. lusitanicus, those collected outside the area A. lizarrusti), with small (<50 km2) and geographically close described for the topotype (west and northwest Iberian (<100 km) distributed areas in wooded habitats, showed Peninsula) are included in a divergent clade together with divergence of 32%. This divergence is comparable to that A. rufus and A. ater. The topotype specimens of A. lusitanicus obtained (31–40%) for the species with a wide distribution in showed the lowest observed inter-specific divergences, with Europe (A. ater/A. rufus, A. subfuscus, A. hortensis and respect to A. nobrei and A. fuligineus (4 and 8%, respectively). A. intermedius). Intra-specific divergence values ranged from The specimens of A. flagellus, from Spain (65E and 66B) and 0% in various species to 9% in A. baeticus (n ¼ 2), 21% in the UK (44A and 44B), showed a mean intra-specific diver- A. rufus (n ¼ 2), and 26% in A. flagellus (n ¼ 4). gence of 23%, considerably higher than the mean inter-specific A number of clades, with high boostrapping values, were distance within this clade. With the exception of the A. his- observed in all reconstructed topologies; however, relation- panicus species, which shows alternative branch positions, this ships between them were not fully resolved. In addition, the clade comprised the A. lusitanicus complex of Castillejo (1997) subdivision of the Iberian species of the genus Arion into three (Fig. 3). subgenera (Castillejo 1997) is not completely supported by our A well-supported sister clade contained another set of molecular data. Excluding A. wiktori and the non-topotypical endemic Iberian species. In this clade, A. paularensis was A. lusitanicus, Arion and Kobeltia subgenera are monophyletic, paraphyletic with respect to the closely related species

Fig. 3. The 50% majority-rule con- sensus tree from 437 bp of the NADH1 gene, based on a distances matrix estimated under the GTR model with variation in rates between sites (a ¼ 0.6296) and the proportion on invariable sites (pinvar ¼ 0.1857), obtained by the neighbour-joining (NJ) method considering only transversions, or both transitions and transversions (dotted lines). Plotted branch lengths were estimated considering only transversions. Alternative branching patterns, obtained using MP and ML methodologies, are indicated with dashed lines and dot-dash lines respectively. Num- bers above nodes indicate boot- strap values (1000 replicates) in NJ(TV)/NJ(TS + TV)/MP, only significant values and values >50; bootstrap values for the terminal nodes are indicated by , >75%; , <50%; , 50–75%. The length of the parsimony consensus tree was 1840 (CI ¼ 0.357, RI ¼ 0.618). The log L for the maximum like- lihood tree was )7560.4. A Dedoceras sp. individual was used as outgroup 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 144 Quinteiro, RodrI´guez-Castro, Castillejo, Iglesias-Pin˜eiro and Rey-Me´ndez

A. wiktori. The two divergent specimens of A. baeticus were grouped monophyletically only in the NJ analysis considering transversions only, not in the MP and ML topologies. In addition, a well-supported clade contained the sister species A. gilvus and A. urbiae (Fig. 3). These two clades thus contained only endemic Iberian species, with the above-mentioned exception of the A. flagellus specimens collected in the UK. In topologies obtained by the different methods (NJ, MP and ML), they are defined as monophyletic sister clades. Despite this consensus, there is no strong bootstrap support for the hypothesis that the Iberian endemics are monophyletic. However, bootstrap does not reflect accuracy but, rather, repeatability or internal consis- tency of data sets. Therefore, to evaluate the confidence of this basic and problematic node, two independent specific tests for monophyly were used. The node (i.e. a monophyletic clade including endemic Iberian species) was supported by both tests (T-PTP test, P < 0.01; likelihood-ratio test, P < 0.01). Another clade, with boostrapping values of 100%, included all specimens of A. ater, A. rufus and A. lusitanicus collected outside NW Iberia. However, relationships within this clade were unclear, with alternative topologies for each reconstruc- tion methodology and data set used. The relationships between the other species, located in a basal position in the topologies, were not unambiguously resolved. This basal and divergent group comprised both taxa with a wide European distribution (A. hortensis, A. interme- dius, A. subfuscus and A. fagophilus) and Pyrenean taxa (A. lizarrusti, A. iratii, A. anthracius and A. molinae), initially Fig. 4. Parsimony consensus tree based on ITS1 sequences obtained included in an A. subfuscus complex. These species showed a from a limited subset of species of the genus Arion. The arrows indicate similar pattern, including high divergence values, a basal those well-supported clades containing the topotype and non-topotype samples of the polyphyletic A. lusitanicus. Tree length ¼ 194, Consis- position in reconstructed topologies, and rare insertion in well- tency index (CI) ¼ 0.8557, Homoplasy index (HI) ¼ 0.1443, Retent- supported clades. The mean uncorrected divergence among all ion index (RI) ¼ 0.8382. Rescaled consistency index (RC) ¼ 0.7172. basal species was 35%, similar to the values obtained for Pyrenean (35%) and European (37%) taxa. Table 2. Estimates of whether the difference between each observed Amplification of the ITS1 rDNA gave a sequence of about tree and the best tree is statistically significant, by means of the 440 bp. The mean divergence within the Arion genus was 2%, Shimodaira–Hasegawa test with sequence variability mostly due to short insertion-deletion events. This gap variation and the low divergence values limit Difference Significantly ) ) the phylogenetic use of these sequences, but the parsimony Tree ln L ln L p-value worse? topology derived from ITS1 was largely congruent with the Topologies based on nucleotide sequences ND1 topologies. The non-topotype specimens of A. lusitanicus NJ 7585.7 25.3 0.051 No were grouped with A. ater/A. rufus, whereas the Portuguese MP 7578.2 17.8 0.118 No individuals were grouped in the clade with Atlantic distribu- ML 7560.4 Best tion, with the exception of A. hispanicus. The main variation Topologies based on aminoacid sequences with respect to the ND1 topologies was the placement of the NJ 3963.1 16.5 0.667 No Pyrenean taxa in a monophyletic clade, with the lowest inter- MP 3962.2 15.6 0.480 No ML 3946.6 Best specific divergence with respect to A. subfuscus (Fig. 4). When comparing protein sequences across the Arion species, 96 of the 145 (66.2%) aa residues were variable, and 81 were Discussion parsimony-informative. Topologies obtained from analysis of the amino acid data set with different methodologies were Molecular phylogeny and taxonomic congruence highly congruent with those obtained from nucleotide analysis. The present molecular data contributes to elucidating diverse The hypothesis of monophyly of Iberian species, was suppor- unresolved issues in Arion taxonomy. Three different groups of ted by all topologies, including maximum likelihood, although Iberian endemic species, besides those species widely distri- with low bootstrap values in the analysis based on NJ and MP buted in Europe, can be defined on the basis of both genetic methods. divergence and geographic vicinity or sympatry. A group of The use of these alternative methods of phylogenetic sympatric species, exhibiting clear overlap in distribution reconstruction resulted in minor topological variations, with (Fig. 1), showed the lowest divergences, another group of no tree significantly worse than the best one, either using the species belonging to different geographic areas (Atlantic and nucleotide or the amino acid data set (Table 2). These Continental/Mediterranean) showed intermediate distance variations mainly involved the problematic nodes noted above values; finally, a geographically closer group of Pyrenean for the nucleotide-based topologies. species with the highest genetic divergence values. 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 Phylogeny of Arion species 145

The specimens of A. lusitanicus were sampled from diverse sequence data indicating a terminal position, not a basal sites throughout its wide distribution range in Europe, position such as is observed for species with a wide distribu- including Portugal, NW Spain, France, Italy and UK; all tion. Alternatively, the UK population might be Iberian in specimens were identified as A. lusitanicus only after exhaustive origin, its presence in the UK being associated with human morphological examination. The congruence between the activities. There are various examples of human-mediated nuclear and mitochondrial data on A. lusitanicus branching dispersion of : Arion ater is widespread in southern British pattern rule out hybridisation event involving A. lusitanicus Columbia (Rollo and Wellington 1975), and other European and A. ater/A. rufus species, and the transfer of mtDNA species (e.g. Arion intermedius, Limax maximus) are present in haplotypes of one species into the nuclear gene background of North America, New Zealand and/or South Africa (Roth another species, resulting in erroneous phylogenetic inference 1986). The human-mediated introduction of A. flagellus to the (see Ferris et al. 1983; Moore 1995). The absence of concor- UK, likely with agricultural products, is thus certainly a dance between the species classification of some specimens possibility. Similar considerations may explain the identity of analysed and the species identification based on nuclear and the sequences of the specimens from the UK and France mitochondrial molecular data is partially attributable to errors misidentified as A. lusitanicus (42H and 62E, respectively), of method and interpretation in the work of the historical belonging to the Arion ater/rufus complex. pioneers of Arion taxonomy in the 19th century. Arion The Arion ater complex comprises two morphological lusitanicus has been widely cited in Europe; however, both forms. The assignment of the nominal species Arion ater and external and genital morphology of non-Iberian individuals A. rufus to these forms is a source of taxonomic controversy. classified as A. lusitanicus is very different from the morphol- This complex showed both high genetic variation and high ogy of the Portuguese topotype specimens (Castillejo 1998). As morphological variability: indeed, some of the specimens a result, the validity of the A. lusitanicus denomination for genetically identified as A. ater/rufus had been assigned to non-Iberian taxa is debated (van Regteren Altena 1955; Davies other species, such as A. lusitanicus. In view of the molecular 1987). In an attempt to clarify this issue, the Portuguese data, this species complex must be reconsidered, with consid- topotype of the Arion lusitanicus species has been redescribed, eration of the non-topotype A. lusitanicus species and the while the names A. fuligineus and A. nobrei have been polyphyly of A. ater and A. rufus. As a result of these rehabilitated (Castillejo and Rodrı´guez 1993a,b). The present considerations, we can expect increased morphological vari- data contribute with molecular characters to the A. lusitanicus ation within this taxon, which can be correctly structured by redescription, and indicate that its distribution is restricted to means of population analysis of a representative number of the north-west Iberian Peninsula, from where the topotype individuals sampled from throughout the distribution range of specimens were originally collected. this complex. Likely, the taxonomic uncertainty about this The so-called A. lusitanicus complex includes medium to clade reflects an absence of distinct biological species and the large slugs, mostly with similar morphology. In addition to the existence of various ecotypes. Similar findings have been Portuguese A. lusitanicus, this complex comprises the species obtained for the species of the echinoderm genus Ophiothrix, A. nobrei, A. fuligineus and A. flagellus (Castillejo 1998). Its which shows marked ecomorphological plasticity (Baric and validity as full species is supported by its mitochondrial Sturmbauer 1999). monophyly in the majority of the topologies obtained in the The significant polymorphism observed in the reproductive present study, although a larger number of specimens and system of specimens assigned to the A. subfuscus complex has localities need to be sampled to verify its species status, led to the description of the new species A. iratii, A. lizarrustii particularly with respect to A. nobrei and A. fuligineus. The and A. molinae (Garrido et al. 1995). Our mitochondrial DNA interspecific distances observed in this complex are lower than data confirm the validity of these taxa, which are highly the intra-specific distances observed in species such as A. flag- divergent from the other species and, in particular, from the ellus. Likewise, the sister sets of endemic Iberian species, European specimen of A. subfuscus. However, analysis on the A. paularensis/A. wiktori and A. gilvus/A. urbiae also showed basis of our nuclear data resulted in lower interspecific very low interspecific distances, without clear monophyly. The distances, grouping the Pyrenean species with the closely divergence between the sympatric species without known related A. subfuscus species in a common clade. There are ecological differentiation (Castillejo 1997) may arise from diverse possible explanations for discrepancies in divergence changes between the different reproductive strategies observed levels and branching pattern between nuclear and mitochond- in this genus (McCracken and Selander 1980; Foltz et al. 1982) rial sequences, including the small effective population size of and the emergence of prezygotic reproductive isolating barriers the mitochondrial gene and the expected rapid rate of mtDNA (Avise 1994). lineage sorting in the reproductively isolated Pyrenean species Our analysis suggests that this group of species, with the (Neigel and Avise 1986), the mating system (Graustein et al. inclusion of A. hispanicus, constitutes a well-supported mon- 2002), population bottlenecks, the selection at the nuclear ophyletic clade with Atlantic distribution. With the exception locus and the retention of an ancestral mitochondrial poly- of A. flagellus, redescribed from UK specimens (Davies 1987), morphism by lineage sorting (Moore 1995). the rest of species are Iberian endemics. The UK and south- west European populations of A. flagellus may represent two currently isolated areas of a much wider European Atlantic Evolutionary history ancestral distribution, similar to those of A. intermedius or Breeding systems, including outcrossing and self-fertilization, A. hortensis. However, the intra-specific genetic divergence might be considered as an important factor in the evolutionary observed among the A. flagellus specimens, sampled from history of the Arion species. However, information about Iberian locations separated by about 100 km and from the specific mating systems and the relative contributions of the UK, is not correlated with geographic distance, and this various possible reproductive modes is limited. Allozymic species is closely related to the endemic species with our studies have revealed that the majority of arionid species are 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 146 Quinteiro, RodrI´guez-Castro, Castillejo, Iglesias-Pin˜eiro and Rey-Me´ndez outcrossers, with the exception of the selfing A. intermedius Mediterranean clade can similarly be dated at about 4.8– and the species of the A. fasciatus complex. A mixed breeding 2.4 My BP, at the Pliocene. Speciation events occurring within system was identified in A. ater and in a morphological form of these endemic Iberian clades were dated to the Pleistocene. The A. subfuscus (McCracken and Selander 1980; Foltz et al. 1982, absence of Arion species in north Africa, close related to 1984). Consequences of self-fertilization are the recombination Iberian endemics, is in congruence with these estimates, since constraint, a higher probability of maintaining coadapted gene the separation of the European and African continents by the complexes (Avise 1994), and an overall loss of genetic Straits of Gibraltar is dated to about 5.5 My BP. (Hsu¨et al. polymorphism (Jarne 1995). In part because of our small 1977), prior to the Arion speciation events. sample sizes, few of our results can be related to breeding Thus the current speciation and distribution of European system. For example, the widely distributed A. intermedius arionids probably reflects the climatic history and physical showed a separate basal position, and the specimens from geography of the Pliocene–Pleistocene. Terrestrial slugs can be distant locations (Spain and UK) showed little divergence. If expected to have been strongly affected by the dramatic this is indeed a selfing species (McCracken and Selander 1980; changes in climatic conditions occurring from the late Tertiary Foltz et al. 1982), the selfing system may be responsible for the onwards. Both palaeoclimate and biogeographic data suggest low observed genetic variability across a wide geographic that the Iberian Peninsula acted as a climate refuge during the range. In contrast, individuals of A. flagellus from similar Pliocene–Pleistocene in southern Europe. The climatic oscilla- locations showed higher divergence values. Various models tions led to cyclic population events, including dispersion, have been proposed to explain the greater reduction in nuclear extinction, survival in refuge and expansion, with evident than in mitochondrial sequence diversity in selfing species. consequences for species distribution and speciation (Hewitt Graustein et al. (2002) has suggested that mitochondrial 1999, 2000, 2001). During major glaciations, the Iberian sequence variation is typically about 10-fold higher than Peninsula probably constituted a refuge for Arion species, and nuclear sequence variation in selfing. In line with this, we a source of species for northern latitudes in warm periods, as found that mean interspecific distance in the A. subfuscus reflected by the currently high species diversity in the Iberian complex was about 28% on the basis of mitochondrial Peninsula, and low genetic divergence between Iberian and sequence, versus only 2% on the basis of nuclear sequence. more northerly individuals of widely distributed species. Selfing systems positively affect the colonizing ability of Several European case studies have reported similar genetic terrestrial slugs, since colonization typically involves small consequences of these variable climatic scenarios (Garcı´a-Parı´s populations of closely related individuals (Foltz et al. 1984). and Jockusch 1999; Hewitt 1999; Paulo et al. 2001). Such a scenario is expected for the Pyrenean species belonging The divergence of basal species, including the European to the A. subfuscus complex. A mixed mating system, detected sensu lato species and the Pyrenean species, can be traced back in this complex, may have played a decisive role in the survival to the late Miocene and Pliocene, and probably involved both of relict species in Pyrenean refuges, through historical geological events, such as the uplift of the Pyrenees (late population bottlenecks in these small and isolated mountain Miocene) (Plaziat 1981), and climatic events, such as the populations. On other hand, basal positions in the topologies Messinian salinity crisis (5.3 My BP) and related glaciations. A suggest that both the Pyrenean species and the A. intermedius similar pre-Pleistocene divergence dating has been proposed lineage may have ancestral origin, which may have been for other European terrestrial invertebrates such as of Iberian favoured by the selfing system due to its advantages as regards lizard, Lacerta schreiberi (Paulo et al. 2001), and the aspersa maintaining populations and lineages at low densities across and maxima lineages of the land snail Helix aspersa in the time, including the maintenance of local adapted and general- Western Mediterranean (Guillier et al. 2001). The split of the purpose genotypes and the assurance of reproduction even two lineages of Iberian endemic species, dated in the Pliocene, when isolated (Jarne and Sta¨dler 1995). appears to have been a response not only to climatic events but The fossil record of the Arionidae family indicates a possible also geological events, including the emergence of the principal first appearance the Lower Miocene (23.3–16.3 Mya) and Iberian hydrogeographic basins (1.8–2.5 My BP) (Calvo et al. definitive presence in the Upper Miocene (10.4–5.2 Mya). 1993), and to differences in soil, vegetation and aridity Extant fossil species include Craterarion pachyostracon (Cali- conditions between the eastern and western Iberian Peninsula. fornia, USA) and Geomalacus indifferens (Germany) (Tracey A comparable pattern has been described for two endemic et al. 1993). The highest distance value within the Arion genus species of Iberian frogs, Discoglossus galganoi and D. jeanneae, (0.41), calculated only from transversions, was slightly lower whose distributions roughly match those of our Atlantic and than the highest value estimated between the sister genera Continental–Mediterranean clades respectively (see Garcı´a- Arion and Dedoceras (0.45). These values allow a rough Parı´s and Jockusch 1999). The glaciation cycles in the Iberian calibration of the transversion mutation rate in the ND1 Peninsula during the Pleistocene period resulted in population partial sequence, assuming first appearance in the Upper isolations and interglacial expansion processes that may be Miocene, as 0.02–0.04 Tamura–Nei distance units/My. In responsible for the patterns of species diversity observed within addition, we calibrated the ND1 mutation rate using the well- each clade. characterized vicariant event of the rise of the Isthmus of In the European region, once the last glaciation (i.e. the Panama (Coates et al. 1992) for marine vetigastropod species Wu¨rm) ended approximately 11 000–10 000 years BP, a series with distribution on both sides of the isthmus. The estimated of warming stages occurred (Polunin and Walters 1985), rate for Atlantic Fissurella nimbosa and Pacific Fissurella causing aridity throughout much of the Iberian Peninsula. species is 0.03 transversional Tamura–Nei distance units/My. Consequently, the Iberian fauna retreated northward; but met Applying this calibration, based on independent and roughly the physical barrier of the Pyrenees. However, this barrier also congruent estimates, the speciation of the Pyrenean taxa can constituted a cooler refuge, resembling the original favourable be dated at about to 14–7 My BP, at the end of Miocene. The habitat for the ancestral and currently Pyrenean-endemic divergence between the Atlantic clade and the Continental– species: A. molinae, A. lizarrustii, A. iratii and A. anthracius. 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 Phylogeny of Arion species 147

Similar scenarios have been suggested for various invertebrate schra¨nkten Populationen. Die im Nordwesten der Iberischen Halbinsel groups throughout the Mediterranean region (Larsen 1987; gesammelten Exemplare von Arion lusitanicus clustern zusammen mit Pittaway 1995). These relict species persist today as small and den weit verbreiteten Arten A. ater und A. rufus in einer nicht- monophyletischen Gruppierung. Arten mit einer europaweiten Verbrei- isolated populations associated with habitats located mainly tung (A. subfuscus, A. hortensis, A. fagophilus und A. intermedius) stehen along the mountain river valleys of the Pyrenees. This an der Basis aller Topologien. Die Evolutionsgeschichte dieser Nackt- mountain area maintains a high level of genetic diversity, schnecken-Arten (stark abha¨ngig von klimatischen Faktoren, mit der reflecting not only probable slow expansion by vertical Fa¨higkeit zur Auskreuzung und Selbstbefruchtung und geringer Ver- displacement (Hewitt 1999), but also the confluence in this breitungskapazita¨t) scheint durch die klimatischen Ereignisse am refuge of diverse lineages previously wide spread in diverse U¨bergang vom Plioza¨n zum Pleistoza¨n und durch die scharfen Gegen- habitats in Southern Iberia. In addition, the low dispersal sa¨tze in der Topographie Su¨deuropas geformt worden zu sein. Sie verursachten wiederholte Zyklen der Isolierung von Populationen capacity in a mountainous scenario favours the subdivision of wa¨hrend alternierender Perioden der Vergletscherung und zwis- the population into isolated demes, increasing the probability cheneiszeitlicher Ausdehnungen, die zur Ausbildung geographischer of persistence of founding lineages (Guillier et al. 2001). Barrieren fu¨hrten. Studies of reproductive systems in these relict species might be useful in the elucidation of the role played by self- fertilization in the evolutionary viability of very small popu- References lations in microhabitats. Avise, J. C., 1994: Molecular Markers, Natural History and Evolution. Species richness in the Iberian Peninsula, may be associated New York: Chapman & Hall. with climatic stability in the peninsula from the late Tertiary to Backeljau, T.; De Bruyn, L., 1990: On the infrageneric systematics of the genus Arion,Fe´russac, 1819 (, ). Bull. 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2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148 148 Quinteiro, RodrI´guez-Castro, Castillejo, Iglesias-Pin˜eiro and Rey-Me´ndez

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