Phylogeny of slug species of the genus Arion: evidence of

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

Phylogeny of slug species of the genus Arion: evidence of monophyly of Iberianendemics and of the existence of relict species in Pyrenean refuges

J. Quinteiro1, J. RodrIguez-Castro

1, J. Castillejo2, J. Iglesias-Pineiro2 and M. Rey-Mendez1

AbstractThe Iberian Peninsula contains the majority of the Paleartic land slug species of the genus Arion, which exhibits diverse taxonomic problems. Thepresent study investigated Arion taxonomy 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 Arionlusitanicus and the closely related species Arion nobrei and Arion fuligineus, as well as Arion hispanicus and Arion flagellus, were grouped into an�Atlantic� clade, whereas Arion baeticus, Arion gilvus, Arion anguloi, Arion wiktori and Arion paularensis were included in a �Continental–Mediterranean� clade. Calibration of mutation rate in the ND1 gene suggested that the divergence of these two clades occurred around thePliocene–Pleistocene boundary, with subsequent speciation events during the Pleistocene. A group of ancestral and divergent endemic specieswith distribution centred in the Pyrenean mountain range (Arion molinae, Arion lizarrusti, Arion antrhacius and Arion iratii) arose in the Plioceneand 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 togetherwith the widely distributed species Arion ater and Arion rufus. The divergent species with a wide European distribution (Arion subfuscus, Arionhortensis, 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 mouldedby Pliocene–Pleistocene climate events and by the rugged topography of southern Europe, giving rise to repeated cycles of population isolationduring 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

The genus Arion includes land slug species with a Palearticdistribution throughout the European continent, the IberianPeninsula being the geographic area with the highest species

diversity, as expected in view of its refuge role in the majorPliocene–Pleistocene and recent ice ages, and its diversetopography and climate.

The taxonomy of Arion is mainly based on morphologicalcharacters of the genital system, which nevertheless showintraspecific variability among close morphs, reflecting both

internal and environmental effects. Accurate identification ofarionid species is typically difficult: it is essential to take intoaccount variability in reproductive organ shape in juvenile,adult and senile specimens of the same species, in the same area

and at the same time of year. Moreover, integument colourpolymorphism, another species-diagnostic character, displayshabitat- and season-correlated variation (Backeljau et al.

2001). In addition, within this genus, various breeding systemshave been observed and demonstrated with genetic markers,include outcrossing, self-fertilization, and both outcrossing

and self-fertilization (McCracken and Selander 1980; Foltzet al. 1982). As a result, Arion systematics is complex andcontroversial, without a clearly defined number of sensu stricto

species, species complexes or subgenera.Nevertheless, four broad biogeographic species groups can

be usefully recognized within this genus: (i) a Lusitanian orAtlantic group distributed along the European Atlantic

border, including the Iberian Peninsula, France, Great Britainand Ireland (A. flagellus, A. owenii, A. hortensis); (ii) aEuropean sensu lato group of species occurring in a wide

variety of regions throughout the continent [A. ater, A. rufus,A. distinctus, A. intermedius, A. lusitanicus, A. subfuscus,

A. fagophilus, and the three closely related selfing species of thesubgenus Carinarion, A. (Carinarion) fasciatus, A. (C.) circum-scriptus and A. (C.) silvaticus]; (iii) a group of species present

in the Pyrenees and adjacent areas (A. anthracius, A. iratii,A. lizarrustii, A. molinae); (iv) an Iberian endemic group(A. baeticus, A. fuligineus, A. hispanicus, A. nobrei, A. paula-

rensis, A. urbiae and A. wiktori); and finally (v) peripheral taxasuch as the Azorean species, A. pascalianus or the Siberianspecies A. sibiricus (Kerney et al. 1983; Wiktor 1983; Backeljau

and De Bruyn 1990; Backeljau et al. 1995; Garrido et al. 1995;Backeljau et al. 1997; Castillejo 1997, 1998).As noted, species and morph identification of arionids is a

difficult task, and this is reflected in diverse problematic issues

in Arion taxonomy. For example, A. lusitanicus have beenwidely cited throughout Europe on the basis of differentmorphological characters to those described for the topotype

specimens. In the case of two taxa, A. ater and A. rufus, there isno consensus about their species status. On the other hand,Iberian species with morphological singularities have been

recently described. Moreover, distribution areas of manyspecies are not definitively known: for example, it is unclearwhether A. flagellus occurs outside the Iberian Peninsula

(Castillejo 1992).Molecular phylogenies of gastropods, based on analysis of

the ribosomal RNA gene cluster have suggested that thedivergent Arionidae family is monophyletic with the Limaco-

idea taxon as sister clade (Wade et al. 2001). The fewmolecular studies performed to date at lower systematic levels,have mainly focused on population genetics (Backeljau et al.

2001). Within the genus Arion, genetic variation in enzymesindicates that the nine species occurring in Great Britain andIreland form heterozygous populations, or monogenetic

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

JZS (2005) 43(2), 139–148

strains, or both types of populations with probable hybridiza-tion between them (Foltz et al. 1982). Allozyme analyse havealso been used to investigate the status of the Azorean species(Backeljau et al. 1992) and ecogenetic aspects of the Central

European Arion fasciatus complex species, within the subgenusCarinarion (Backeljau et al. 1997; Jordaens et al. 1998). Todate there have been no DNA-based studies of Arion phylo-

geny or population genetics, although some populations oflarge arionids have been differentiated using RAPD analysis(Noble and Jones 1996). In other gastropods, DNA sequence

analysis – specifically, analysis of internal transcribed spacer(ITS) sequence variation in Albinaria and the closely related

Isabellaria genus (Clausilidae) – has allowed the identificationof polyphyletic groups and demonstrated that the morpholo-gical characters traditionally used, the clausilial structures, areof limited taxonomic value (Schilthuizen et al. 1995).

The mitochondrial NADH dehydrogenase 1 gene codes foran enzyme subunit involved in the electron transport chain; itssequence is currently only available for three pulmonate

gastropod species (Yamazaki et al. 1997). The use of thismitochondrial NADH dehydrogenase 1 (ND1) DNA sequencefor phylogenetic reconstruction is infrequent, mainly due to

the absence of �universal� primers. The mitochondrial ND1does not contain iron–sulphur centres of NADH coenzyme Q

Table 1. Specimens included in the present study

Species Specimen Location DateAccession number

(ND1, ITS1)

Arion (Arion) ater(Linnaeus, 1758)

AATE 39A Caldas de Geres, Portugal 31/10/84 AY316228, AY316268AATE 39E Valporquero Cave, Leon, Spain 1/10/91 AY316229, AY316269

Arion (Arion) rufus(Linnaeus, 1758)

ARUF 40A Sova Valley, Spain 17/09/91 AY316230ARUF 40G Mont. Noire, Central Massif, France 6/09/92 AY316231, AY316270

Arion (Mesarion) nobreiPollonera, 1889

ANOB 41A Luso, Portugal 28/01/86 AY316232, AY316271ANOB 41B Luso, Portugal 28/01/86 AY316233, AY316272

Arion (Mesarion) lusitanicusMabille, 1868

ALUS 42A Serra da Arrabida, Portugal 4/12/84 AY316234, AY316273ALUS 42B Serra da Arrabida, Portugal 4/12/84 AY316235, AY316274ALUS 42C Serra da Arrabida, Portugal 4/12/84 AY316336, AY316275ALUS 42G Alpi Carniche, Rivolato, Italy 10/08/86 AY316237, AY316276ALUS 42H Surrey, UK 25/07/86 AY316238ALUS 62E Mont. Noire, Central Massif, France 6/09/92 AY316239, AY316289ALUS 70A Girona, Spain 17/11/90 AY316240ALUS 70B Girona, Spain 17/11/90 AY316241ALUS 70C Girona, Spain 17/11/90 AY316242, AY316290

Arion (Mesarion) fuligineusMorelet, 1845

AFUL 43A Sao Silvestre, Portugal 15/12/85 AY316243, AY316277AFUL 43B Ponte da Lima, Portugal 15/12/85 AY316244

Arion (Mesarion) flagellusCollinge, 1893

AFLA 44A Branley Bark, Croydon, UK 20/09/86 AY316245, AY316278AFLA 44B Branley Bark, Croydon, UK 20/09/86 AY316245AFLA 65E Santiago de Compostela, Spain 25/11/89 AY316246AFLA 66B Lugo, Spain AY316247

Arion (Mesarion) subfuscus(Draparnaud, 1805)

ASUB 45G Coulsdon Wood, Surrey, UK 29/08/86 AY316248ASUB 45A Montagne Noire, France 6/09/92 AY316279

Arion (Mesarion) iratiiGarrido, Castillejo et Iglesias, 1995

AIRA 46B Irati Forest, Navarra, Spain 24/09/91 AY316249

Arion (Mesarion) lizarrustiiGarrido, Castillejo et Iglesias, 1995

ALIZ 47C Lizarrusti, Spain 19/9/94 AY316250, AY316280

Arion (Mesarion) molinaeGarrido, Castillejo et Iglesias, 1995

AMOL 48B Serra del Cadı, Barcelona, Spain 16/09/91 AY316251AMOL 48A Serra del Cadı, Barcelona, Spain 16/09/91 AY316281

Arion (Mesarion) gilvus TorresMınguez, 1925

AGIL 49A Serra del Pandols, Tarragona, Spain AY316252, AY316282AGIL 49D Denia, Alicante, Spain 17/10/91 AY316253

Arion (Mesarion) urbiaeDe Winter, 1986

AURB 50A AY316283AURB 50C Urbasa Sierra, Navarra, Spain 12/12/86 AY316254

Syn: A. (Mesarion) anguloiMartın et Gomez, 1988

AANG 73A Burgos, Spain 16/03/86 AY316267, AY316291

Arion (Mesarion) paularensisWiktor et Parejo, 1989

APAU 51A Guadarrama Sierra, Segovia, Spain 18/10/92 AY316255APAU 51D Moncayo Sierra, Zaragoza, Spain 1/11/91 AY316256, AY316284

Arion (Mesarion) hispanicusSimroth, 1886

AHIS 52A Gouveia, Portugal 29/11/84 AY316257AHIS 52B Caceres, Spain 3/05/91 AY316258, AY316285

Arion (Mesarion) baeticusGarrido, Castillejo et Iglesias, 1994

ABAE 53A Huelva, Spain 10/03/92 AY316259ABAE 53C Cuenca Sierra, Cuenca, Spain 27/10/91 AY316260

Arion (Kobeltia) hortensisFerussac, 1819

AHOR 54A S. Salvador de Bianya, Girona, Spain 13/11/89 AY316261

Arion (Kobeltia) fagophilusDe Winter, 1986

AFAG 55C Lizarrusti, Navarra, Spain 19/09/94 AY316262

Arion (Kobeltia) intermediusNormand, 1852

AINT 56D Aran Valley, Lleida, Spain AY316263, AY316286AINT 56G Coulsdon Wood, Surrey, UK 7/10/86 AY316264

Arion (Kobeltia) anthraciusBourguignat, 1866

AANT 57A Valencia d’Aneu, Lleida, Spain 10/09/91 AY316265

Arion (Kobeltia) wiktoriParejo et Martın, 1990

AWIK 58A Demanda Sierra, Burgos, Spain 3/11/91 AY316287AWIK 58C Urbion Mountains, Soria, Spain 2/11/91 AY316266, AY316288

Dedoceras sp. DBEN 60A Bossot, Viella, Lerida, Spain 11/11/89 AY316292

140 Quinteiro, RodrIguez-Castro, Castillejo, Iglesias-Pineiro and Rey-Mendez

� 2005 Blackwell Verlag, Berlin, JZS 43(2), 139–148

reductase (complex I), so that we can expect less strictfunctional constraints on nucleotide and aminoacid variability.Reasonable high variability is a pre-requisite for phylogenetic

reconstruction at the genus level. However, mtDNA intro-gression could have occurred in closely related species ofarionids, resulting in incongruent phylogenetic inferences from

nuclear and mitochondrial data sets (Ferris et al. 1983). Asnoted, the internal transcribed spacer 1 (ITS1) nuclearsequences, has proved useful for inferring phylogenetic rela-tionships in other gastropod genera (Schilthuizen et al. 1995),

and as a source of complementary nuclear data to search forputative introgressive hybridization events.

Within the genus Arion, a number of phylogeographic and

systematic issues remain unresolved or uncertain in the light ofmorphological data. These issues include (a) the species andsub-genus status of recently redescribed species, initially

described on the questionable systematics studies in the 19thcentury (Castillejo 1998); (b) the evolutionary origins of andrelationships among species with distributions limited to the

Iberian Peninsula, or to small refuges in the Pyrenees; and (c)the relationships between these endemic species and morewidely distributed species. These issues are here addressed withDNA sequence data. We discuss the utility of mitochondrial

ND1 gene for the study of close intra-genus relationships ingastropods, and the role played by the Iberian Peninsula –characterized by its pronounced geographical barriers and

historical climatic conditions – in the speciation of theseorganisms with low dispersal ability and high sensitivity tobiotic parameters, such as vegetation, humidity and tempera-

ture.

Materials and methods

DNA samples were isolated from 46 specimens, belonging to 20 speciesof the genus Arion (Fig. 1, Table 1).

An individual from the related genus Dedoceras sp. was analysed foruse as outgroup (Wade et al. 2001). Specimens had originally beencollected 7–17 years ago, and had been preserved in formalin orethanol and stored at room temperature. Total DNA extraction, fromheart or gonad, was performed using the DNeasy Tissue Kit (Qiagen,Hilden, Germany). A pair of primers with degenerated positions wasdesigned to amplify the first half of the mitochondrial ND1 gene:MOL-NAD1F (5¢-CGRAARGGMCCTAACAARGTTGG-3¢) andMOL-NAD1R (5¢-GGRGCACGATTWGTCTCNGCTA-3¢). Am-plifications were performed using AmpliTaq polymerase in PCRBuffer II, in a GenAmp 9700 PCR System (Applied Biosystems, FosterCity, CA, USA). The PCR thermal profile comprised an initialdenaturation step at 94�C, followed by 35 cycles of denaturation at94�C for 30 s, annealing at 50�C for 30 s, and extension at 72�C for60 s. MgCl2 concentration was 2.5 mM. In the few cases in whicheffective amplification was not obtained, annealing temperature wasreduced to 45�C and MgCl2 concentration was tested over the range1.5–3.5 mM until amplification was achieved. In addition, nuclear ITSsequences were amplified with the primers BAB18S1F (5¢-GCTGGC-CGAGAAGAAGCTC-3¢) and BAB28SR (5¢-GACACTGAGGGAT-TCGGTGC-3¢), with the same PCR thermal profile except thatextension was for 2 min. Purified PCR products were directlysequenced, in both directions, with the BigDye sequencing kit version3.0 (Applied Biosystems). Fluorescent extension products were separ-ated and detected with an ABI PRISM 377 automated sequencer(Applied Biosystems). Revision of electropherograms, manual se-quence alignment, translation, and homology search by local BLASTin a mollusc DNA database, were performed with BioEdit v. 5.0.2(Hall 1999). All sequences were deposited in GenBank/EMBL/DDBJunder accession no. AY316228–AY316292.Hypotheses about phylogenetic relationships were established by

distance, maximum parsimony, and maximum likelihood methodsusing PAUP* version 4.0b10 (Swofford 1998) for nucleotidesequences and PHYLIP version 3.6 (Felsenstein 1993) for data setsincluding translated amino acid sequences. To test for saturation bymultiple substitutions, the observed number of transitions andtransversions in pairwise comparisons were plotted against distancevalues. Because transition saturation was detected in ND1 sequences,we additionally tried, two alternative approaches to avoid thenegative effects of homoplasy in phylogenetic reconstruction:

Fig. 1. Distribution areas of the Arion species considered and sample location

Phylogeny of Arion species 141


(a) consideration of transversional changes only; and (b) analysis oftranslated amino-acid sequences. The nucleotide substitution modelthat best fit the data was evaluated by the likelihood-ratio test(Huelsenbeck and Crandall 1997) with the aid of Modeltest version3.0 (Posada and Crandall 1998), selecting a GTR + E + C modelwith proportion of invariable sites (I) ¼ 0.1857 and gamma distri-bution (C) of variable sites with shape parameter a ¼ 0.6296. Basedon this model we constructed a distance matrix and a neighbour-joining (NJ) tree. In addition, an NJ tree was constructed using onlytransversion-based distances. Using the above model parameters, amaximum likelihood (ML) tree was obtained by heuristic search withthe tree-bisection-reconnection (TBR) branch-swapping algorithm. Aconsensus tree was also obtained under parsimony criterion from thesequence data set using a heuristic parsimony search with closestaddition sequence. Starting trees were obtained by stepwise addition.Confidence in nodes was evaluated by bootstrapping (Felsenstein1985) with 1000 replicates. The transition/transversion ratio wasestimated by maximum likelihood. The hypothesis of monophyly ofendemic Iberian species was tested using both the topology-depend-ent permutation tail probability test (T-PTP) (Faith 1991) and thelikelihood ratio test of monophyly (LRT) (Huelsenbeck et al. 1996).The Shimodaira–Hasegawa test was used to test whether thedifference between the estimated alternative topologies was significant(Shimodaira and Hasegawa 1999). The constancy in the substitutionrate among lineages was evaluated with the likelihood ratio test usingDNAML and DNAMLK programs (PHYLIP package) (Felsenstein1993) and Modeltest version 3.0 (Posada and Crandall 1998).

The analysis of protein sequences on the basis of distance, maximumlikelihood and maximum parsimony was performed with the PROT-DIS, PROTML and PROTPARS programs, respectively, included inthe PHYLIP package. Distances between translated sequences werebased on the Jones et al. (1992) model of amino acid substitution. TheDedoceras sp. individual was used as outgroup.

Results

A 437-bp sequence was obtained for 46 individuals belongingto 20 Arion species and the outgroup, Dedoceras sp.

This sequence is included in the first half of the mitochond-rial NADH dehydrogenase subunit 1 gene, and its translationresulted in a 145-aminoacid (aa) fragment. In the mitochond-

rial genome of Cepaea nemoralis, the sequence is locatedbetween position 5233 and 5660 (accession no.: U23045) in theND1 gene, which contains a total of 291 aa residues. There is a

nucleotide composition bias towards T at all codon sites, andnucleotide frequencies mean (A ¼ 0.28, C ¼ 0.15, G ¼ 0.14,T ¼ 0.43) showed homogeneity among the taxa considered(v2 ¼ 119.94, df ¼ 123, P ¼ 0.56). In the alignment of the

Arion partial ND1 sequences we identified 304 variablepositions (69.6%), suggesting that this sequence is highlyvariable with a constancy in the substitution rate among

lineages (LRT: P > 0.01). As expected for a coding sequence,the third codon position is the most variable, followed by thefirst and second positions. The transition/transversion ratio

estimated using ML topology was 0.99. At relatively lowdistances values such as 0.3, transversions outnumberedtransitions, both overall and at each codon position (Fig. 2).

The t-test indicated incomplete saturation [t ¼ 5.11, p ¼ 0.0],but the evidence for relative saturation of transitions observedin the plot (Fig. 2), indicates that phylogenetic reconstructionis best based on transversion changes only, thus avoiding

homoplasy and its negative effect on both distance estimationand tree reliability.

The observed high variability in ND1 sequences was appar-

ent in the overall mean uncorrected pairwise distance value of0.284 (SE ¼ 0.012). Inter-specific sequence divergence betweenthe Iberian species ranged from 4 to 35%, versus up to 40%

considering non-Iberian species. In the Atlantic group (inclu-ding the divergent A. hispanicus together with A. lusitanicus,A. nobrei, A. fuligineus and A. flagellus), divergence ranged

from 4 to 31%, whereas in the Continental–Mediterranean

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Fig. 2. Relationships between the number of substitutions (transversions and transitions) and genetic distance (Tamura–Nei), considering allnucleotide positions and each of the codon positions of the partial ND1 sequences, for pairwise comparisons of Arion species



group (A. wiktori, A. paularensis, A. urbiae, A. baeticus,A. gilvus), divergence ranged from 7 to 20%. In both groups,the mean value was 15–20%, with the mean divergence between

groups being 26% (net distance ¼ 8.7%). The two species ofthe A. ater complex (A. ater and A. rufus) showed divergence of20%, i.e. about the mean value. Divergences between geo-

graphically close Pyrenean species (A. iratii, A. lizarrusti,A. molinae and A. anthracius) were unexpectedly high, rangingfrom 32% to 37%. In particular, two sister species (A. iratii,A. lizarrusti), with small (<50 km2) and geographically close

(<100 km) distributed areas in wooded habitats, showeddivergence of 32%. This divergence is comparable to thatobtained (31–40%) for the species with a wide distribution in

Europe (A. ater/A. rufus, A. subfuscus, A. hortensis andA. intermedius). Intra-specific divergence values ranged from0% in various species to 9% in A. baeticus (n ¼ 2), 21% in

A. rufus (n ¼ 2), and 26% in A. flagellus (n ¼ 4).A number of clades, with high boostrapping values, were

observed in all reconstructed topologies; however, relation-

ships between them were not fully resolved. In addition, thesubdivision of the Iberian species of the genus Arion into threesubgenera (Castillejo 1997) is not completely supported by ourmolecular data. Excluding A. wiktori and the non-topotypical

A. lusitanicus, Arion and Kobeltia subgenera are monophyletic,

whereas that Mesarion is paraphyletic (Fig. 3). A terminalclade (Fig. 3) comprised a group of closely related species,including A. flagellus, A. hispanicus, A. nobrei, A. fuligineus and

the specimens of A. lusitanicus collected from the Portuguesetype localities in the Iberian Peninsula. This clade containedspecies with an Atlantic distribution, mainly in the Iberian

Peninsula, with the exception of specimens of A. flagelluscollected in the UK. Surprisingly, within the set of specimensidentified as A. lusitanicus, those collected outside the areadescribed for the topotype (west and northwest Iberian

Peninsula) are included in a divergent clade together withA. rufus and A. ater. The topotype specimens of A. lusitanicusshowed the lowest observed inter-specific divergences, with

respect to A. nobrei and A. fuligineus (4 and 8%, respectively).The specimens of A. flagellus, from Spain (65E and 66B) andthe UK (44A and 44B), showed a mean intra-specific diver-

gence of 23%, considerably higher than the mean inter-specificdistance within this clade. With the exception of the A. his-panicus species, which shows alternative branch positions, this

clade comprised the A. lusitanicus complex of Castillejo (1997)(Fig. 3).A well-supported sister clade contained another set of

endemic Iberian species. In this clade, A. paularensis was

paraphyletic with respect to the closely related species

Fig. 3. The 50% majority-rule con-sensus tree from 437 bp of theNADH1 gene, based on a distancesmatrix estimated under the GTRmodel with variation in ratesbetween sites (a ¼ 0.6296) and theproportion on invariable sites(pinvar ¼ 0.1857), obtained by theneighbour-joining (NJ) methodconsidering only transversions, orboth transitions and transversions(dotted lines). Plotted branchlengths were estimated consideringonly transversions. Alternativebranching patterns, obtained usingMP and ML methodologies, areindicated with dashed lines anddot-dash lines respectively. Num-bers above nodes indicate boot-strap values (1000 replicates) inNJ(TV)/NJ(TS + TV)/MP, onlysignificant values and values >50;bootstrap values for the terminalnodes are indicated by , >75%;, <50%; �, 50–75%. The length

of the parsimony consensus treewas 1840 (CI ¼ 0.357, RI ¼ 0.618).The log L for the maximum like-lihood tree was )7560.4. ADedoceras sp. individual was usedas outgroup



A. wiktori. The two divergent specimens of A. baeticus weregrouped monophyletically only in the NJ analysis consideringtransversions only, not in the MP and ML topologies. In

addition, a well-supported clade contained the sister speciesA. gilvus and A. urbiae (Fig. 3).These two clades thus contained only endemic Iberian

species, with the above-mentioned exception of the A. flagellusspecimens collected in the UK. In topologies obtained by thedifferent methods (NJ, MP and ML), they are defined asmonophyletic sister clades. Despite this consensus, there is no

strong bootstrap support for the hypothesis that the Iberianendemics are monophyletic. However, bootstrap does notreflect accuracy but, rather, repeatability or internal consis-

tency of data sets. Therefore, to evaluate the confidence of thisbasic and problematic node, two independent specific tests formonophyly 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 collectedoutside NW Iberia. However, relationships within this cladewere unclear, with alternative topologies for each reconstruc-tion methodology and data set used.

The relationships between the other species, located in abasal position in the topologies, were not unambiguouslyresolved. 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

included in an A. subfuscus complex. These species showed asimilar pattern, including high divergence values, a basalposition in reconstructed topologies, and rare insertion in well-

supported clades. The mean uncorrected divergence among allbasal species was 35%, similar to the values obtained forPyrenean (35%) and European (37%) taxa.Amplification of the ITS1 rDNA gave a sequence of about

440 bp. The mean divergence within the Arion genus was 2%,with sequence variability mostly due to short insertion-deletionevents. This gap variation and the low divergence values limit

the phylogenetic use of these sequences, but the parsimonytopology derived from ITS1 was largely congruent with theND1 topologies. The non-topotype specimens of A. lusitanicus

were grouped with A. ater/A. rufus, whereas the Portugueseindividuals were grouped in the clade with Atlantic distribu-tion, with the exception of A. hispanicus. The main variationwith respect to the ND1 topologies was the placement of the

Pyrenean taxa in a monophyletic clade, with the lowest inter-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 wereparsimony-informative. Topologies obtained from analysis ofthe amino acid data set with different methodologies were

highly congruent with those obtained from nucleotide analysis.The hypothesis of monophyly of Iberian species, was suppor-ted by all topologies, including maximum likelihood, although

with low bootstrap values in the analysis based on NJ and MPmethods.The use of these alternative methods of phylogenetic

reconstruction resulted in minor topological variations, with

no tree significantly worse than the best one, either using thenucleotide or the amino acid data set (Table 2). Thesevariations mainly involved the problematic nodes noted above

for the nucleotide-based topologies.

Discussion

Molecular phylogeny and taxonomic congruence

The present molecular data contributes to elucidating diverse

unresolved issues in Arion taxonomy. Three different groups ofIberian endemic species, besides those species widely distri-buted in Europe, can be defined on the basis of both genetic

divergence and geographic vicinity or sympatry. A group ofsympatric species, exhibiting clear overlap in distribution(Fig. 1), showed the lowest divergences, another group ofspecies belonging to different geographic areas (Atlantic and

Continental/Mediterranean) showed intermediate distancevalues; finally, a geographically closer group of Pyreneanspecies with the highest genetic divergence values.

Fig. 4. Parsimony consensus tree based on ITS1 sequences obtainedfrom a limited subset of species of the genus Arion. The arrows indicatethose well-supported clades containing the topotype and non-topotypesamples of the polyphyletic A. lusitanicus. Tree length ¼ 194, Consis-tency index (CI) ¼ 0.8557, Homoplasy index (HI) ¼ 0.1443, Retent-ion index (RI) ¼ 0.8382. Rescaled consistency index (RC) ¼ 0.7172.

Table 2. Estimates of whether the difference between each observedtree and the best tree is statistically significant, by means of theShimodaira–Hasegawa test

Tree )ln LDifference)ln L p-value

Significantlyworse?

Topologies based on nucleotide sequencesNJ 7585.7 25.3 0.051 NoMP 7578.2 17.8 0.118 NoML 7560.4 Best

Topologies based on aminoacid sequencesNJ 3963.1 16.5 0.667 NoMP 3962.2 15.6 0.480 NoML 3946.6 Best



The specimens of A. lusitanicus were sampled from diversesites throughout its wide distribution range in Europe,including Portugal, NW Spain, France, Italy and UK; all

specimens were identified as A. lusitanicus only after exhaustivemorphological examination. The congruence between thenuclear and mitochondrial data on A. lusitanicus branching

pattern rule out hybridisation event involving A. lusitanicusand A. ater/A. rufus species, and the transfer of mtDNAhaplotypes of one species into the nuclear gene background ofanother species, resulting in erroneous phylogenetic inference

(see Ferris et al. 1983; Moore 1995). The absence of concor-dance between the species classification of some specimensanalysed and the species identification based on nuclear and

mitochondrial molecular data is partially attributable to errorsof method and interpretation in the work of the historicalpioneers of Arion taxonomy in the 19th century. Arion

lusitanicus has been widely cited in Europe; however, bothexternal and genital morphology of non-Iberian individualsclassified as A. lusitanicus is very different from the morphol-

ogy of the Portuguese topotype specimens (Castillejo 1998). Asa result, the validity of the A. lusitanicus denomination fornon-Iberian taxa is debated (van Regteren Altena 1955; Davies1987). In an attempt to clarify this issue, the Portuguese

topotype of the Arion lusitanicus species has been redescribed,while the names A. fuligineus and A. nobrei have beenrehabilitated (Castillejo and Rodrıguez 1993a,b). The present

data contribute with molecular characters to the A. lusitanicusredescription, and indicate that its distribution is restricted tothe north-west Iberian Peninsula, from where the topotype

specimens were originally collected.The so-called A. lusitanicus complex includes medium to

large slugs, mostly with similar morphology. In addition to the

Portuguese A. lusitanicus, this complex comprises the speciesA. nobrei, A. fuligineus and A. flagellus (Castillejo 1998). Itsvalidity as full species is supported by its mitochondrialmonophyly in the majority of the topologies obtained in the

present study, although a larger number of specimens andlocalities need to be sampled to verify its species status,particularly with respect to A. nobrei and A. fuligineus. The

interspecific distances observed in this complex are lower thanthe intra-specific distances observed in species such as A. flag-ellus. Likewise, the sister sets of endemic Iberian species,

A. paularensis/A. wiktori and A. gilvus/A. urbiae also showedvery low interspecific distances, without clear monophyly. Thedivergence between the sympatric species without knownecological differentiation (Castillejo 1997) may arise from

changes between the different reproductive strategies observedin this genus (McCracken and Selander 1980; Foltz et al. 1982)and the emergence of prezygotic reproductive isolating barriers

(Avise 1994).Our analysis suggests that this group of species, with the

inclusion of A. hispanicus, constitutes a well-supported mon-

ophyletic clade with Atlantic distribution. With the exceptionof A. flagellus, redescribed from UK specimens (Davies 1987),the rest of species are Iberian endemics. The UK and south-

west European populations of A. flagellus may represent twocurrently isolated areas of a much wider European Atlanticancestral distribution, similar to those of A. intermedius orA. hortensis. However, the intra-specific genetic divergence

observed among the A. flagellus specimens, sampled fromIberian locations separated by about 100 km and from theUK, is not correlated with geographic distance, and this

species is closely related to the endemic species with our

sequence data indicating a terminal position, not a basalposition such as is observed for species with a wide distribu-tion. Alternatively, the UK population might be Iberian in

origin, its presence in the UK being associated with humanactivities. There are various examples of human-mediateddispersion of slugs: Arion ater is widespread in southern British

Columbia (Rollo and Wellington 1975), and other Europeanspecies (e.g. Arion intermedius, Limax maximus) are present inNorth America, New Zealand and/or South Africa (Roth1986). The human-mediated introduction of A. flagellus to the

UK, likely with agricultural products, is thus certainly apossibility. Similar considerations may explain the identity ofthe sequences of the specimens from the UK and France

misidentified as A. lusitanicus (42H and 62E, respectively),belonging to the Arion ater/rufus complex.The Arion ater complex comprises two morphological

forms. The assignment of the nominal species Arion ater andA. rufus to these forms is a source of taxonomic controversy.This complex showed both high genetic variation and high

morphological variability: indeed, some of the specimensgenetically identified as A. ater/rufus had been assigned toother species, such as A. lusitanicus. In view of the moleculardata, this species complex must be reconsidered, with consid-

eration of the non-topotype A. lusitanicus species and thepolyphyly of A. ater and A. rufus. As a result of theseconsiderations, we can expect increased morphological vari-

ation within this taxon, which can be correctly structured bymeans of population analysis of a representative number ofindividuals sampled from throughout the distribution range of

this complex. Likely, the taxonomic uncertainty about thisclade reflects an absence of distinct biological species and theexistence of various ecotypes. Similar findings have been

obtained for the species of the echinoderm genus Ophiothrix,which shows marked ecomorphological plasticity (Baric andSturmbauer 1999).The significant polymorphism observed in the reproductive

system of specimens assigned to the A. subfuscus complex hasled to the description of the new species A. iratii, A. lizarrustiiand A. molinae (Garrido et al. 1995). Our mitochondrial DNA

data confirm the validity of these taxa, which are highlydivergent from the other species and, in particular, from theEuropean specimen of A. subfuscus. However, analysis on the

basis of our nuclear data resulted in lower interspecificdistances, grouping the Pyrenean species with the closelyrelated A. subfuscus species in a common clade. There arediverse possible explanations for discrepancies in divergence

levels and branching pattern between nuclear and mitochond-rial sequences, including the small effective population size ofthe mitochondrial gene and the expected rapid rate of mtDNA

lineage sorting in the reproductively isolated Pyrenean species(Neigel and Avise 1986), the mating system (Graustein et al.2002), population bottlenecks, the selection at the nuclear

locus and the retention of an ancestral mitochondrial poly-morphism by lineage sorting (Moore 1995).

Evolutionary history

Breeding systems, including outcrossing and self-fertilization,might be considered as an important factor in the evolutionary

history of the Arion species. However, information aboutspecific mating systems and the relative contributions of thevarious possible reproductive modes is limited. Allozymic

studies have revealed that the majority of arionid species are



outcrossers, with the exception of the selfing A. intermediusand the species of the A. fasciatus complex. A mixed breedingsystem was identified in A. ater and in a morphological form of

A. subfuscus (McCracken and Selander 1980; Foltz et al. 1982,1984). Consequences of self-fertilization are the recombinationconstraint, a higher probability of maintaining coadapted gene

complexes (Avise 1994), and an overall loss of geneticpolymorphism (Jarne 1995). In part because of our smallsample sizes, few of our results can be related to breedingsystem. For example, the widely distributed A. intermedius

showed a separate basal position, and the specimens fromdistant locations (Spain and UK) showed little divergence. Ifthis is indeed a selfing species (McCracken and Selander 1980;

Foltz et al. 1982), the selfing system may be responsible for thelow observed genetic variability across a wide geographicrange. In contrast, individuals of A. flagellus from similar

locations showed higher divergence values. Various modelshave been proposed to explain the greater reduction in nuclearthan in mitochondrial sequence diversity in selfing species.

Graustein et al. (2002) has suggested that mitochondrialsequence variation is typically about 10-fold higher thannuclear sequence variation in selfing. In line with this, wefound that mean interspecific distance in the A. subfuscus

complex was about 28% on the basis of mitochondrialsequence, versus only 2% on the basis of nuclear sequence.Selfing systems positively affect the colonizing ability of

terrestrial slugs, since colonization typically involves smallpopulations of closely related individuals (Foltz et al. 1984).Such a scenario is expected for the Pyrenean species belonging

to the A. subfuscus complex. A mixed mating system, detectedin this complex, may have played a decisive role in the survivalof relict species in Pyrenean refuges, through historical

population bottlenecks in these small and isolated mountainpopulations. On other hand, basal positions in the topologiessuggest that both the Pyrenean species and the A. intermediuslineage may have ancestral origin, which may have been

favoured by the selfing system due to its advantages as regardsmaintaining populations and lineages at low densities acrosstime, including the maintenance of local adapted and general-

purpose genotypes and the assurance of reproduction evenwhen isolated (Jarne and Stadler 1995).The fossil record of the Arionidae family indicates a possible

first appearance the Lower Miocene (23.3–16.3 Mya) anddefinitive presence in the Upper Miocene (10.4–5.2 Mya).Extant fossil species include Craterarion pachyostracon (Cali-fornia, USA) and Geomalacus indifferens (Germany) (Tracey

et al. 1993). The highest distance value within the Arion genus(0.41), calculated only from transversions, was slightly lowerthan the highest value estimated between the sister genera

Arion and Dedoceras (0.45). These values allow a roughcalibration of the transversion mutation rate in the ND1partial sequence, assuming first appearance in the Upper

Miocene, as 0.02–0.04 Tamura–Nei distance units/My. Inaddition, we calibrated the ND1 mutation rate using the well-characterized vicariant event of the rise of the Isthmus of

Panama (Coates et al. 1992) for marine vetigastropod specieswith distribution on both sides of the isthmus. The estimatedrate for Atlantic Fissurella nimbosa and Pacific Fissurellaspecies is 0.03 transversional Tamura–Nei distance units/My.

Applying this calibration, based on independent and roughlycongruent estimates, the speciation of the Pyrenean taxa canbe dated at about to 14–7 My BP, at the end of Miocene. The

divergence between the Atlantic clade and the Continental–

Mediterranean clade can similarly be dated at about 4.8–2.4 My BP, at the Pliocene. Speciation events occurring withinthese endemic Iberian clades were dated to the Pleistocene. The

absence of Arion species in north Africa, close related toIberian endemics, is in congruence with these estimates, sincethe separation of the European and African continents by the

Straits of Gibraltar is dated to about 5.5 My BP. (Hsu et al.1977), prior to the Arion speciation events.

Thus the current speciation and distribution of Europeanarionids probably reflects the climatic history and physical

geography of the Pliocene–Pleistocene. Terrestrial slugs can beexpected to have been strongly affected by the dramaticchanges in climatic conditions occurring from the late Tertiary

onwards. Both palaeoclimate and biogeographic data suggestthat the Iberian Peninsula acted as a climate refuge during thePliocene–Pleistocene in southern Europe. The climatic oscilla-

tions led to cyclic population events, including dispersion,extinction, survival in refuge and expansion, with evidentconsequences for species distribution and speciation (Hewitt

1999, 2000, 2001). During major glaciations, the IberianPeninsula probably constituted a refuge for Arion species, anda source of species for northern latitudes in warm periods, asreflected by the currently high species diversity in the Iberian

Peninsula, and low genetic divergence between Iberian andmore northerly individuals of widely distributed species.Several European case studies have reported similar genetic

consequences of these variable climatic scenarios (Garcıa-Parısand Jockusch 1999; Hewitt 1999; Paulo et al. 2001).

The divergence of basal species, including the European

sensu lato species and the Pyrenean species, can be traced backto the late Miocene and Pliocene, and probably involved bothgeological events, such as the uplift of the Pyrenees (late

Miocene) (Plaziat 1981), and climatic events, such as theMessinian salinity crisis (5.3 My BP) and related glaciations. Asimilar pre-Pleistocene divergence dating has been proposedfor other European terrestrial invertebrates such as of Iberian

lizard, Lacerta schreiberi (Paulo et al. 2001), and the aspersaand maxima lineages of the land snail Helix aspersa in theWestern Mediterranean (Guillier et al. 2001). The split of the

two lineages of Iberian endemic species, dated in the Pliocene,appears to have been a response not only to climatic events butalso geological events, including the emergence of the principal

Iberian hydrogeographic basins (1.8–2.5 My BP) (Calvo et al.1993), and to differences in soil, vegetation and aridityconditions between the eastern and western Iberian Peninsula.A comparable pattern has been described for two endemic

species of Iberian frogs, Discoglossus galganoi and D. jeanneae,whose distributions roughly match those of our Atlantic andContinental–Mediterranean clades respectively (see Garcıa-

Parıs and Jockusch 1999). The glaciation cycles in the IberianPeninsula during the Pleistocene period resulted in populationisolations and interglacial expansion processes that may be

responsible for the patterns of species diversity observed withineach clade.

In the European region, once the last glaciation (i.e. the

Wurm) ended approximately 11 000–10 000 years BP, a seriesof warming stages occurred (Polunin and Walters 1985),causing aridity throughout much of the Iberian Peninsula.Consequently, the Iberian fauna retreated northward; but met

the physical barrier of the Pyrenees. However, this barrier alsoconstituted a cooler refuge, resembling the original favourablehabitat for the ancestral and currently Pyrenean-endemic

species: A. molinae, A. lizarrustii, A. iratii and A. anthracius.



Similar scenarios have been suggested for various invertebrategroups throughout the Mediterranean region (Larsen 1987;Pittaway 1995). These relict species persist today as small and

isolated populations associated with habitats located mainlyalong the mountain river valleys of the Pyrenees. Thismountain area maintains a high level of genetic diversity,

reflecting not only probable slow expansion by verticaldisplacement (Hewitt 1999), but also the confluence in thisrefuge of diverse lineages previously wide spread in diversehabitats in Southern Iberia. In addition, the low dispersal

capacity in a mountainous scenario favours the subdivision ofthe population into isolated demes, increasing the probabilityof persistence of founding lineages (Guillier et al. 2001).

Studies of reproductive systems in these relict species mightbe useful in the elucidation of the role played by self-fertilization in the evolutionary viability of very small popu-

lations in microhabitats.Species richness in the Iberian Peninsula, may be associated

with climatic stability in the peninsula from the late Tertiary to

the present, through the generalized climatic changes of thePleistocene. However, there were and are marked climaticdifferences within Iberia, reflecting its geographic location andrugged topography, and resulting in the existence of diverse

climatic regions (Atlantic, Continental and Mediterranean)and thus favouring the appearance of isolated Pleistocenerefuges. The Iberian Arion species reflect the current habitat

diversity against a background of palaeoclimatic events. Theidentification of the Arion lusitanicus Atlantic endemism and ofthe relict species surviving only in small Pyrenean populations

has important implications for the conservation managementof Iberian slug species and their habitats, since a set ofevolutionary significant units, appropriate to biodiversity

preservation, has been determined.

Acknowledgements

We thank Alberto Olivares for the supply of DNA samples fromFissurella species, Burkhard Horstkotte and Hartmut Rehbein for thepreparation of the manuscript and two anonymous reviewers, forthoughtful comments on the manuscript. Sampling work was partiallysupported by projects PB87–0397 �Fauna Iberica I�, DGICYT (1988–1990) and PB89–0081 �Fauna Iberica II�, DGICYT (1990–1993).

Zusammenfassung

Phylogenie von Nacktschnecken der Gattung Arion: Nachweis derMonophylie iberischer Endemismen und des Vorkommens von Reliktar-ten in Ruckzugsregionen der Pyrenaen

Auf der Iberischen Halbinsel lebt die Mehrzahl der palaarktischenLandnacktschnecken-Arten der Gattung Arion, die vielfaltige taxo-nomische Probleme aufweist. Die vorliegende Studie untersuchte dieArion-Taxonomie auf der Basis einer Analyse des mitochondrialenND1-Gens und der nuklearen ITS1-Sequenz. Die endemischen iberi-schen Arten bilden zwei monophyletische Schwestergruppen. DerTopotyp von A. lusitanicus und die naher verwandten Arten A. nobreiund A. fuligineus sowie A. hispanicus und A. flagellus lassen sich einer�Atlantischen� Gruppe zuteilen, wohingegen A. baeticus, A. gilvus,A. anguloi,A.wiktoriundA. paularensis in eineKontinental-mediterraneGruppe gehoren. Die Kalibrierung der Mutationsrate im ND1-Gensuggeriert, dass die Abspaltung dieser beiden Gruppen beim Ubergangvom Pliozan zum Pleistozan geschah, mit nachfolgender Artbildungwahrend des Pleistozans. EineGruppe von ancestralen und unterschied-lich endemischen Spezies mit Verbreitung in den Pyrenaen (A. molinae,A. lizarrusti, A. antrhacius und A. iratii) tauchte wahrend des Pliozansauf und uberlebte wahrend des Pleistozans in geographisch einge-

schrankten Populationen. Die im Nordwesten der Iberischen Halbinselgesammelten Exemplare von Arion lusitanicus clustern zusammen mitden weit verbreiteten Arten A. ater und A. rufus in einer nicht-monophyletischen Gruppierung. Arten mit einer europaweiten Verbrei-tung (A. subfuscus,A. hortensis,A. fagophilus undA. intermedius) stehenan der Basis aller Topologien. Die Evolutionsgeschichte dieser Nackt-schnecken-Arten (stark abhangig von klimatischen Faktoren, mit derFahigkeit zur Auskreuzung und Selbstbefruchtung und geringer Ver-breitungskapazitat) scheint durch die klimatischen Ereignisse amUbergang vom Pliozan zum Pleistozan und durch die scharfen Gegen-satze in der Topographie Sudeuropas geformt worden zu sein. Sieverursachten wiederholte Zyklen der Isolierung von Populationenwahrend alternierender Perioden der Vergletscherung und zwis-cheneiszeitlicher Ausdehnungen, die zur Ausbildung geographischerBarrieren fuhrten.

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Authors’ addresses: Javier Quinteiro (for correspondence), JorgeRodrıguez-Castro and Manuel Rey-Mendez, Departamento de Bio-quımica e Bioloxıa Molecular Facultade de Bioloxıa, Universidade deSantiago de Compostela, 15782 Santiago de Compostela, Galicia,Spain, e-mail: [email protected]; Jose Castillejo and Javier Iglesias-Pineiro, Departamento de Bioloxıa Animal, Facultade de Bioloxıa,Universidade de Santiago de Compostela, 15782 Santiago de Com-postela, Galicia, Spain



Phylogeny of slug species of the genus Arion: evidence of

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