Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066© 2006 The Linnean Society of London? 200689?589604Original Article

MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEXS. GEENEN

ET AL

.

*Corresponding author. E-mail: sofie.geenen@ua.ac.be

Molecular systematics of the

Carinarion

complex (Mollusca: Gastropoda: Pulmonata): a taxonomic riddle caused by a mixed breeding system

SOFIE GEENEN

1

*, KURT JORDAENS

1

and THIERRY BACKELJAU

1,2

1

Evolutionary Biology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium

2

Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium

Received 22 November 2004; accepted for publication 15 January 2006

The original description of the slugs

Arion

(

Carinarion

)

fasciatus

,

Arion

(

Carinarion

)

silvaticus

and

Arion

(

Cari-narion

)

circumscriptus

was based on subtle differences in body pigmentation and genital anatomy. However, bodypigmentation in these slugs may be influenced by their diet, whereas the genital differences between the speciescould not be confirmed by subsequent multivariate morphometric analyses. Hence, the status of the three nominalmorphospecies remains controversial, with electrophoretic studies based on albumen gland proteins and allozymesalso providing conflicting results. These studies suggested that

Carinarion

species are difficult to reconcile with thebiological species concept because there is evidence of interspecific hybridization in places where these predomi-nantly self-fertilizing slugs apparently outcross. Therefore, in the present study, the three

Carinarion

species areevaluated under a phylogenetic species concept, using nucleotide sequences of the nuclear ribosomal internal tran-scribed spacer 1 (ITS-1) and the mitochondrial 16S rDNA. ITS-1 showed no species specific variation. However, 16SrDNA yielded five haplotype groups. Three of these grouped haplotypes by species, whereas the two others joinedhaplotypes of different species and included all haplotypes that were shared by species (22% of all haplotypes).Hence, the three nominal

Carinarion

species appear to be inconsistent with a phylogenetic species concept. Thepresent data also confirmed that North American

Carinarion

populations are genetically impoverished and may benot sufficiently representative with respect to the taxonomy of

Carinarion

. In conclusion, we currently regard

Car-inarion

as a single species-level taxon, whose taxonomically deceiving, correlated phenotypic and genetic intraspe-cific variation is caused by sustained self-fertilization. © 2006 The Linnean Society of London,

Biological Journalof the Linnean Society

, 2006,

89

, 589–604.

ADDITIONAL KEYWORDS:

16S rDNA – biological species concept – ITS-1 – morphospecies concept –

phylogenetic species concept – phylogenetics – self-fertilization – slugs.

INTRODUCTION

Recently, there has been a renewed interest in speciesconcepts as tools to describe biodiversity (de Meeûs,Durand & Renaud, 2003). In this context, more than20 species concepts have been defined, each of whichreflects different philosophical and biological founda-tions (Mayden, 1997). However, no single speciesconcept seems generally applicable to all organisms(Hey, 2001) and different species concepts may evenbe mutually inconsistent (Mayden, 1997). Neverthe-less, taxonomists often describe species operationally

under one species concept but interpret them withinthe framework of another. For example, many animalspecies were originally described as some sort of ‘mor-phospecies’, whereas subsequent practice implicitelytreated these morphospecies as biological species(

sensu

Mayr, 1940). In fact, most species descriptionsdo not even mention which species concept(s) theyimplement. This situation has at least three negativeconsequences: (1) the interpretation of what speciesactually represent may be confused; (2) the value ofspecies as descriptors or units of biodiversity may beill-defined (Hey, 2001); and (3) describing new speciesmay be easier than falsifying species (Backeljau

et al

.,1994).

.

A particularly challenging example of these prob-lems is provided by the terrestrial slugs of the arionidsubgenus

Carinarion

Hesse, 1926, which comprisesthree predominantly selfing pulmonate slug species,namely

Arion

(

Carinarion

)

fasciatus

(Nilsson, 1823),

Arion

(

Carinarion

)

circumscriptus

Johnston, 1828 and

Arion

(

Carinarion

)

silvaticus

Lohmander, 1937. Thethree species are widely distributed over Europe (Ker-ney, Cameron & Jungbluth, 1983) and North America,where they have been introduced by man (Chichester& Getz, 1969, 1973). They were initially distinguishedby their body pigmentation and subtle genital differ-ences (Lohmander, 1937; Waldén, 1955), and laterconfirmed on the basis of fixed electrophoretic differ-ences in albumen gland proteins (AGP) among NorthAmerican specimens (Chichester, 1967). Subse-quently, McCracken & Selander (1980) reported thateach of the three species in North America consisted ofa single homozygous multilocus genotype (MLG)(based on 18 allozyme loci). This result was inter-preted as a strong indication of sustained selfing(autogamy) (Selander & Ochman, 1983), whereas thelow genetic identities between the three MLGs(Table 1) were seen as strong support for their specificdifferentiation. A similar electrophoretic survey byFoltz

et al

. (1982) (Table 1) of 13 allozyme loci in

A. circumscriptus

and

A. silvaticus

from Ireland con-firmed that

A. circumscriptus

consists of one MLG,whereas

A. silvaticus

yielded two MLGs. To extendthese previous electrophoretic studies, Backeljau

et al

.(1987, 1997) conducted electrophoretic surveys of thethree

Carinarion

species in a part of their native areain western Europe. Backeljau

et al

. (1987) showedthat, in this area, the three species differed consis-tently in their overall electrophoretic profiles foresterases (EST) and AGP, confirming the earlier workof Chichester (1967) on North American material. Bycontrast, the allozyme survey of Backeljau

et al

.(1997), who screened other enzymatic proteins,showed that each of the three species in westernEurope consisted of a series of homozygous MLGs.This latter result, combined with the allozyme surveysof Jordaens

et al

. (1998, 2000), showed that, inEurope, the three

Carinarion

species consist of atleast 13 (

A. fasciatus

), nine (

A. circumscriptus

), and 24(

A. silvaticus

) MLGs. Overall genetic distances andidentities among the three species as reported byBackeljau

et al

. (1997) and Jordaens

et al

. (2000) arecompared in Table 1. Importantly, these studies alsodemonstrated that, in some areas,

Carinarion

spp.produce heterozygous individuals, suggesting theoccurrence of facultative outcrossing and possiblyeven of interspecific hybridization (Jordaens

et al

.,1996, 2000). Further analyses by Geenen

et al

. (2003)showed that the different conclusions of the allozymestudies of European and (introduced) North American

Tab

le 1

.

Nei

’s (

1972

) m

ean

gen

etic

ide

nti

ties

(

I

) an

d m

ean

gen

etic

dis

tan

ces

(

D

) or

Nei

’s (

1978

) m

ean

gen

etic

dis

tan

ce (

D

*) b

etw

een

(de

not

ed b

y ‘

v

’) an

d w

ith

in(d

enot

ed b

y ‘

w

’)

Ari

on f

asci

atu

s

(F

),

Ari

on s

ilva

ticu

s

(S

), an

d

Ari

on c

ircu

msc

ript

us

(C

)

Spe

cies

com

pari

son

wF

wS

wC

Stu

dyR

egio

nF

vCvS

FvS

FvC

SvC

I

McC

rack

en &

Sel

ande

r (1

980)

Nor

th A

mer

ica

0.65

––

––

––

I

Fol

tz

et a

l

. (19

82)

Irel

and

––

–0.

74–

––

I

Bac

kelj

au

et a

l

. (19

97)

Eu

rope

0.81

(0.

10)

0.79

(0.

06)

0.73

(0.

04)

0.67

(0.

06)

0.93

(0.

03)

0.87

(0.

05)

0.94

D

Bac

kelj

au

et a

l

. (19

97)

Eu

rope

0.22

(0.

12)

0.24

(0.

08)

0.31

(0.

06)

0.40

(0.

09)

0.08

(0.

04)

0.14

(0.

06)

0.06

D

*Jo

rdae

ns

et a

l

. (20

00)

Eu

rope

–0.

21 (

0.10

)0.

28 (

0.08

)0.

31 (

0.10

)0.

15 (

0.07

)0.

18 (

0.08

)0.

13 (

0.06

)

Sta

nda

rd d

evia

tion

s ar

e gi

ven

in

par

enth

eses

.

populations were not due to technical discrepancies,but most probably to the lower amount of allozymevariation in the North American populations. Thisimplies that the electrophoretic data of Chichester(1967) and McCracken & Selander (1980) may be notsufficiently representative with respect to the popula-tion structure and the specific status of

Carinarion

spp. in its native area (i.e. Europe). Moreover, thepredominantly autogamous (i.e. self-fertilization)breeding system of

Carinarion

spp. precludes theapplication of the biological species concept (BSC) and,even if applied, the natural occurrence of putative‘interspecific’ hybrids between

A. fasciatus

and

A. silvaticus

(Jordaens

et al

., 1996) would cast doubton

Carinarion

spp. as different biological species.This argument is further supported by the fact

that self-fertilization may promote allozymic differ-entiation by fixing alternative alleles (Backeljau

et al

., 1997; Jordaens

et al

., 2000), a point that notonly underpins the production of well-differentiatedhomozygous MLGs, but also that possibly canexplain the ‘species specific’ electrophoretic patternsof AGP and EST or even the subtle ‘species specific’morphological differences. Insofar as these featuresare genetically determined, it appears plausible thatfixation of alternative alleles at the loci involvedcould produce misleadingly consistent morphologicaland protein electrophoretic differences, leaving thesource of remaining phenotypic variation for a largepart to the environment (Jordaens

et al

., 2001).Finally, a multivariate morphometric comparison ofthe genital features in

Carinarion

spp. did not sup-port the morphological differentiation between thethree species as described by Lohmander (1937) orWaldén (1955), except for a consistent overall sizedifference between

A. fasciatus

and the two otherspecies (Jordaens

et al

., 2002; S. Geenen, K.Jordaens & T. Backeljau, unpubl. data).

Even if the taxonomic interpretation of

Carinarion

is highly confused, many authors continue to treat thethree taxa as ‘good species’. Because this practice isneither consistent with the BSC, nor with a mor-phospecies concept (MSC), we here explore whetherphylogenetic data provide a more consistent basis forthe taxonomic interpretation of this complex. To thisend, we present a DNA sequence analysis of a frag-ment of the mitochondrial 16S rDNA gene (16S) andthe nuclear ribosomal internal transcribed spacer I(ITS-1). More precisely, we aimed to assess whether:(1) the three nominal

Carinarion

species are mono-phyletic taxa consistent with the phylogenetic speciesconcept (PSC); (2) North American

Carinarion

isgenetically impoverished and differentiated comparedto European populations; (3) putative geographicalpatterns in breeding biology (Jordaens

et al

., 2000) arereflected by patterns of mtDNA variation; and (4)

mtDNA variation allows to recognize phylogeograph-ical patterns that may help to disentangle the currenttaxonomic complexity of the

Carinarion

complex.

MATERIAL AND METHODS

S

AMPLING

We surveyed 1990 animals from 91 European(

N

=

1746) and nine North American (

N

=

244) popu-lations. Specimens were identified on the basis of mor-phological criteria (i.e. body pigmentation and genitalfeatures), following Lohmander (1937) and Waldén(1955). Individuals with an intermediate morphotype(‘0’; unidentified in Table 2) were not used in the anal-ysis. DNA was extracted from individual foot muscletissue according to Pinceel

et al

. (2004).

S

INGLE

STRAND

CONFORMATION

POLYMORPHISM

(SSCP) ANALYSIS AND DNA SEQUENCING

All individuals were subjected to SSCP analysis of twofragments of the mitochondrial 16S rDNA gene (16S1,101 bp; 16S2, 156 bp). Specific primers were devel-oped to amplify each fragment [16S1: L16SCA1 (5′-AAGGTAGCAAAATAAATAGGC-3′) and H16SCA1(5′-TCTTAGGGTCTTCTCGTCTT-3′); 16S2: L16SCA2(5′-ATAAGACGAGAAGACCCTAA-3′) and H16SAR2(5′-GTCCAACATCGAGGTCAC)]. Polymerase chainreaction (PCR) and SSCP protocols were in accordancewith those of Pinceel et al. (2004), except for the recipeof the polyacrylamide gels for the first fragment (gels16S1: T = 14%, C = 2%, with 5% glycerol). In each pop-ulation, all haplotype combinations of the two frag-ments detected with SSCP were sequenced on an ABI373A automatic DNA sequencer (Applied BiosystemsInc.) following Pinceel et al. (2004) or were submittedfor sequencing to the Flanders Interuniversity Insti-tute for Biotechnology (VIB, Department of MolecularGenetics, University of Antwerp). Primers to amplifythe 16S rDNA stretch comprising both SSCP frag-ments were 16SAR (5′-CGCCTGTTTAACAAAAACAT-3′) and 16SBR (5′-CCGGTTTGAACTCAGATCAGATCACGT-3′) (Palumbi & Benzie, 1991). This stretchcomprised 395 bp and was used for phylogeneticanalysis. All 16S rDNA sequences were deposited inGenBank under accession numbers AJ715324–75.Variation outside the SSCP fragments was very lowand yielded only one additional haplotype (X5) thatdiffered from haplotype X4 by a single substitution.Hence, all X4 and X5 individuals were sequenced forthe entire 395 bp 16S rDNA stretch.

A fragment of the nuclear ITS-1 rDNA gene of 12specimens was also sequenced. These 12 specimenswere selected based on which mitochondrial group(see Results) they belong to (Table 2). The fragmentwas amplified under the following PCR conditions:

Table 2. Country, sampling localities and locality abbreviation (Abb), number of individuals sampled (No), haplotypefrequencies, and morphospecies (MS: F, Arion fasciatus; S, Arion silvaticus; C, Arion circumscriptus; 0, undefined)

Country Locality Abb. No

Phylogenetic groups

C S

Haplotypes

C1 C2 C3 C4 C5 C6 C7 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14

Belgium Houx BE1 22 1Yvoir BE2 20 1Eigenbrakel BE3 12 0.17 0.58 0.25Gent BE4 16 0.44 0.50* 0.06Pittem BE5 15 1Sint-Antonius

ZoerselBE6 18 0.06 0.44 0.50

Genk BE7 18 1Diepenbeek BE8 15 1Halle BE9 18 1Halle BE10 24 0.96 0.04Oudenaarde BE11 11 1Sint-Antonius

ZoerselBE12 24 0.54 0.46

Tielt BE13 6 1Nukerke BE14 4 0.75 0.25*Maffe BE15 9 0.56 0.44Gomzé-

AndoumontBE16 4 0.75 0.25

Soy-Fisenne BE17 11 1

Great Britain Ae Village GB1 13 0.15 0.08 0.08Humshaugh GB2 25 0.04 0.36 0.60Innerleithen GB3 30 0.03 0.24 0.30 0.03 0.20Worksop GB4 30 0.80 0.20Burnopfield GB5 26 0.35 0.42 0.15Cranham GB6 25 0.04 0.88 0.08Epping Forest GB7 25 1Temple Ewell GB8 12 0.75 0.25

Germany Görlitz GE1 16 1Görlitz GE2 13 1Görlitz GE3 11Innerkoy GE4 22Warmbronn GE5 25 0.08 0.92

Switzerland Langnau SW1 16 1Therwil SW2 5 0.40 0.60Münchenstein SW3 20 1*

Italy Etroubles IT1 30 1Caino IT2 23Garessio IT3 25 1Gignese IT4 29Macugnaga IT5 17 1Sardagna IT6 30 1Cortina

d’AmpezzoIT7 34 0.29

France Rencurel FR1 8 1Agnieres FR2 31 0.10 0.22 0.68

Sweden Genarp SE1 23 0.04 0.04Göteborg SE2 17Lund SE3 37

Poland Bialowice PO1 14 0.07Muszkowice PO2 15 0.67 0.33Zgorzelec PO3 18Zgorzelec PO4 25

Czech Republic

Námest CR1 41

MS

F N X

F1 F2 F3 F4 F5 F6 F7 N1 N2 N3 N4 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18

CCS-CS-CCS-C

SSSSSS

CCCS-C

S

0.69 F-S-CS-C

0.20 F-S-CS-C

0.08 F-S-CS-CCC

CC

0.45 0.55 F1 S

S

SSS

C1 F-S-C-0

C0.14 0.86 S-C-0

SC

0.71 F-S

CS-C

0.92 F-S-C1 F1 F

0.93 F-CF-C

0.11 0.89 F0.32 0.68 F

1 F

Country Locality Abb. No

Phylogenetic groups

C S

Haplotypes

C1 C2 C3 C4 C5 C6 C7 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14

Austria Vienna AU1 22Bischofshofen AU2 27Altenburg AU3 11Graz AU4 122Graz AU5 17Graz AU6 33Graz AU7 47Graz AU8 43Horn AU9 25Kamp AU10 11Altenburg AU11 45Innerlaterns AU12 17 0.82 0.18Elsbethen AU13 9

Romania Sinaia RO1 39 0.23* 0.03Poiana RO2 8Timisu de Sus RO3 26 0.08Miercurea RO4 5Azuga RO5 33 0.09 0.52Cozanesti RO6 8Holda RO7 19Bicaz RO8 7Lunca Visagului RO9 2Prundu RO10 2Racova RO11 6Podu Dimbovitei RO12 4Baile Felix RO13 3Covasna RO14 9Dealu Negru RO15 1

Bulgaria Pastra BU1 33Batak BU2 32Cujpetlovo BU3 22Beli Osam BU4 12Vratce BU5 11Razlog BU6 5Batak BU7 3Batak BU8 1Karnare BU9 3Gabrovo BU10 1Ribarica BU11 2

Slovenia BegunjenaGorenjskem

SL1 30 0.43

Liechtenstein Schaanwald LI1 12 0.92 0.08*

USA North Carvon NA1 50 0.96Bellingham NA2 22West Barnstable NA3 35 0.14West Barnstable NA4 14 0.64West Barnstable NA5 8 0.25Sandwich NA6 60 1Woods Hole NA7 9 0.11 0.56 0.33Mansfield NA8 32Pomfret NA9 14

Populations and haplotype groups are indicated by an asterisk (*) if they comprised specimens screened for nuclear ribosomal internal transcribed spacer I.

Table 2. Continued

5 min at 95 °C, then 30 cycles of 1 min at 95 °C,1 min at 55 °C and 2 min at 72 °C and ending with5 min at 72 °C. The Thermo Sequenase II Dye Ter-minator Cycle Sequencing Premix Kit (PharmaciaBiotech, Inc.) and an ABI 373A automatic DNA

sequencer (Applied Biosystems Inc.) were used tosequence the fragment between the primers ITS1L(TCCGTAGGTGAACCTGCGGAAGGAT) and 58C(TGCGTTCAAGATATCGATGTTCAA) (Hillis &Dixon, 1991), following Pinceel et al. (2004). All ITS-

MS

F N X

F1 F2 F3 F4 F5 F6 F7 N1 N2 N3 N4 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18

1 F1 F

0.91 0.09 F0.04 0.96 F0.71* 0.29 F

1 F0.96 0.04 F

0.09 0.91 F0.20 0.80* F

1* S1 S

S1 S

0.28 0.31 0.15 F-S-C-00.88 0.12 C-0

0.92 F-S-C-00.80 0.20 F0.15 0.06 0.18 F-S-01 F0.95 0.05 F-S-0

1* S-01 S-0

0.50 0.50 F-01 S-C-0

1 S1 C-0

0.44 0.56* S-01 C

1* F-S-01 F

1 F-S-01 S-0

1 S-01 S1 S

1 F1 S

1 S1 S

0.20 0.37 F-S-C-0

S

0.04 F-C1 F0.86 F-S0.36 F-C0.75 F-S

SC

1 F1 C

1 rDNA sequences were deposited in GenBankunder accession numbers AJ15486–97. ITS-1 rDNAsequences available in GenBank under accessionnumbers AJ509068–74 were included in theanalyses.

DATA ANALYSIS

Sequences were aligned using CLUSTALX, version1.8 (Thompson et al., 1997) with default settings. Thisalignment was checked for ‘unstable hence unreliable’

alignment blocks with SOAP, version 1.2 alpha 4 (Löy-tynoja & Milinkovitch, 2001). Numbers of polymorphicand parsimony informative sites were calculated inDnaSP, version 4.00 (Rozas et al., 2003), whereas hap-lotype frequencies were calculated in ARLEQUIN,version 2.00 (Schneider, Roessli & Excoffier, 2000).

MODELTEST, version 3.06 (Posada & Crandall,1998) was used to select the most appropriate substi-tution model for phylogenetic analysis. An index ofsubstitution saturation (Iss; Xia et al., 2003) was cal-culated and a plot of transitions and transversionsagainst genetic distance was made to check for satu-ration in the data in DAMBE, version 4.0.97 (Xia &Xie, 2001). The program TREE.EXE in the ‘Randomcladistics’ package (Siddall, 1995) was used to test forthe presence of phylogenetic signal using the g1 sta-tistic (Hillis & Huelsenbeck, 1992). Likelihood map-ping in TREE-PUZZLE (Strimmer & von Haeseler,1996; Strimmer, Goldman & von Haeseler, 1997) wasapplied to assess to what extent internal branches aresupported and to estimate the phylogenetic content ofthe sequence alignment, using quartet trees.

Phylogenetic trees were inferred using the substitu-tion model suggested by MODELTEST and were con-structed with several methods. A Neighbour-joining(NJ) tree (Saitou & Nei, 1987) was calculated withMEGA, version 2.1 (Kumar et al., 2001). Maximumparsimony (MP) and maximum likelihood (ML) treeswere made in PAUP*, version 4.0b10 (Swofford, 1998)using a heuristic search with the tree-bisection-reconnection branch-swapping algorithm and randomaddition of taxa. ML trees were also constructedwith TREE-PUZZLE, version 5.0 (Strimmer & vonHaeseler, 1996; Strimmer et al., 1997). Branch sup-port was assessed via nonparametric bootstrapping of1000 bootstrap replicates (Felsenstein, 1985). Onlybootstrap values higher than 70% were considered asmeaningful (Hillis & Huelsenbeck, 1992). Branch sup-port was also evaluated using Bayesian posteriorprobabilities (Larget & Simon, 1999) applied to phy-logenetic trees inferred by the program MRBAYES,version 2.01 (Huelsenbeck & Ronquist, 2001). Baye-sian analyses were launched with random startingtrees and run for 107 generations, sampling Markovchains at intervals of 100 generations using a general-time-reversible model of evolution as imposed byMRBAYES (nst = 6) with gamma distributed rate vari-ation (Γ = 0.23) and base frequencies estimated fromthe data. To explore tree and parameter space morethoroughly, four incrementally heated Markov chains(using default heating values) were used. Stationarityof the Markov chain was determined as the pointwhen sampled log-likelihood values plotted againstgeneration time reached a stable mean equilibriumvalue. Sample points generated before this point werediscarded as ‘burn-in’ samples. The log-likelihood val-

ues for sampled trees stabilized after approximately40 000 generations. Therefore, a burn-in value of 400was used, implying that trees sampled from genera-tion ’40 100’ onwards, were used to estimate Bayesianposterior probabilities. To ensure that analyses werenot trapped in local optima, three independent repli-cates were run and inspected (Huelsenbeck & Boll-back, 2001). If ≥95% of the sampled trees contained agiven clade, it was considered to be significantly sup-ported by the data (Ranker et al., 2003; and referencestherein). All tree reconstructions were made with acombined outgroup comprising Arion franciscoloiBoato, Bodon & Giusti, 1983, Arion hortensis Férus-sac, 1819 and Arion distinctus Mabille, 1868. For theBayesian trees, all the outgroup species were includedin the trees; however, each of these taxa had to be usedseparately as an outgroup.

The assumption of a molecular clock was tested witha likelihood ratio test (LRT) in TREE-PUZZLE, ver-sion 5.0 (Strimmer & von Haeseler, 1996; Strimmeret al., 1997), but was rejected (LRT = 353.73, d.f. = 48and P < 0.0001). Therefore no estimations of diver-gence times were made. Sequence divergences (Nei,1987) within and between the three nominal mor-phospecies were calculated employing the p-distancein MEGA, version 2.1 (Kumar et al., 2001).

To further evaluate the allozyme based geneticimpoverishment of the North American Carinarionspp. (Geenen et al., 2003), nucleotide diversities (π)(Nei, 1987) within the three nominal morphospecies inEurope and North America were calculated based onthe p-distance in MEGA, version 2.1 (Kumar et al.,2001) and compared for the two geographical regions.

A haplotype network was constructed using statis-tical parsimony (Templeton, Crandall & Sing, 1992) inthe program TCS 1.13 (Clement, Posada & Crandall,2000). Indels were treated as a fifth character stateand ambiguous loops were resolved by applying thetopology criterion (Pfenninger & Posada, 2002) beforeimplementing a nested clade analysis (NCA). Thenesting of clades was performed using the rules set outin Templeton, Boerwinkle & Sing (1987), Templeton &Sing (1993), and Crandall (1996). Nonrandom geo-graphical associations among haplotypes and cladeswere tested statistically with a nested contingencyanalysis (Templeton & Sing, 1993; Templeton, Rout-man & Phillips, 1995). The program GEODIS, version2.0 (Posada, Crandall & Templeton, 2000) was used tocalculate the various NCA distance measures andtheir statistical significance (based on 1000 randompermutations of the contingency data table for eachclade). The updated inferences key (available at http://inbio.byu.edu/Faculty/kac/crandall_lab/geodis.htm),based on Templeton et al. (1995) and Templeton (1998,2004), was used to infer the biological processesresponsible for all clades that showed significant geo-

graphical relationships as indicated from the contin-gency analysis.

RESULTS

SEQUENCE DATA OF THE MITOCHONDRIAL 16S RDNA FRAGMENT

The 16S rDNA fragment yielded 50 haplotypes, whosesequence lengths ranged from 391–395 bp. Sequenceswere AT biased (AT/GC proportion: 0.64/0.36) and con-tained 13 indels, 94 polymorphic sites, and 89 parsi-mony informative sites. These figures raised to 142polymorphic and 96 parsimony informative sites whenthe combined outgroup was included. There were nounstable alignment blocks, and all sites were used infurther analyses.

PHYLOGENETIC INFERENCES BASED ON THE 16S RDNA FRAGMENT

The most suitable substitution model, according toMODELTEST, was the Tamura & Nei (1993) model(–lnL = 2199.4604), with the following parameters:base frequencies A = 0.3558, C = 0.1329, G = 0.1554,T = 0.3460; substitution model rate matrix R(a)[A–C] = 1.0000, R(b)[A–G] = 4.0816, R(c)[A–T] = 1.0000,R(d)[C–G] = 1.0000, R(e)[C–T] = 7.6174, R(f)[G–T] =1.0000; among-site rate variation proportion of invari-able sites = 0; and gamma distribution shapeparameter = 0.2317.

Plotting numbers of transitions and transversionsagainst genetic distance showed that transversionsoutnumber transitions for genetic distances greaterthan 0.1654. This may be indicative of saturation butit appears that the 16S rDNA data set contains sig-nificant phylogenetic signal because (1) neither thenumbers of transitions, nor the numbers of transver-sions reached a plateau; (2) the index of substitutionsaturation (Iss = 0.184) was significantly lower(P < 0.0001) than the critical index of substitution sat-uration Iss.c for symmetrical trees (IssSym = 0.790) orfor asymmetrical trees (IssAsym = 0.756); (3) the g1value (−1.72) is far below the critical value (−0.10)given by Hillis & Huelsenbeck (1992); and (4) likeli-hood mapping showed that most of the quartet trees(75%) were concentrated in the three corners of thetriangle.

Although the overall topologies of the different treeswere similar (Fig. 1), branch support values some-times differed. All trees showed four monophyletichaplotype groups, referred to as F, S, C, and N (Fig. 1,Table 2), with seven, 14, seven, and four haplotypes,respectively. In addition, a series of unresolvedbranches were joined at the same level and referredto as the X group (18 haplotypes). This arbitrarygrouping was suggested by the clustering of the X

haplotypes in the haplotype network (Fig. 2D). The F,S and C groups only comprised haplotypes of morpho-logically typical A. fasciatus, A. silvaticus, andA. circumscriptus, respectively. The X group includedspecimens of the three nominal morphospecies andspecimens whose morphological identification wasambiguous. At least four X haplotypes were shared bythe three nominal species, whereas another seven Xhaplotypes were shared by two nominal species.Hence, in total, 22% of the haplotypes were sharedbetween species. Individuals of the N group wereeither A. silvaticus or A. fasciatus (Fig. 1). The mono-phyly of Carinarion was supported in all trees, exceptin the Bayesian trees (Fig. 1B). The F group was con-sistently supported in all trees, whereas the S, C, andN groups were supported by the NJ, MP and theTREE-PUZZLE ML trees. The PAUP* ML tree onlysupported the groups F, C, and N, whereas the Baye-sian trees only supported the F group. None of thetrees consistently resolved and/or supported the rela-tions among the four groups or the relationshipswithin the groups.

Sequence divergences within and between the threespecies, and within and between the five haplotypegroups are given in Table 3. The 16S rDNA suggests alarge (approximately 20%) divergence between F andall other groups, whereas divergence estimatesamongst the latter were much lower (< 5.5%).Sequence divergence within a group never exceeded2.5%, such that differences among haplotypes withingroups are small compared to the sequences differ-ences between groups. By contrast, divergence esti-mates between A. fasciatus and both A. silvaticus andA. circumscriptus were much larger than the diver-gence between A. silvaticus and A. circumscriptus, butdivergences within morphospecies were as large as thedivergence between nominal morphospecies. This is nosurprise since the distances among haplotypes withinspecies are much larger than within haplotype groupsbecause individuals with X haplotypes occurred in allthree nominal morphospecies (Fig. 1A, B).

HAPLOTYPE DISTRIBUTION AND DIVERSITIES OF THE 16S RDNA FRAGMENT

Haplotype frequencies per population are given inTable 2. Haplotypes were considered to be ‘common’when they occurred in more than 10% of the Euro-pean populations. Haplotypes C1, C2, S1, and F1met this threshold and occurred in, respectively,18%, 13%, 21%, and 24% of the European popula-tions. Forty-seven percent of the European popula-tions contained more than one haplotype with amaximum of six haplotypes in population U3. In22% of the European populations, more than onehaplotype group co-occurred. The distribution of the

haplotype groups is shown in Figure 3. Group F wasfound in the British Islands and from Scandinavia,central and eastern Europe to the Balkan, but wasabsent from Belgium and France. Group S occurredeverywhere except Bulgaria. Group C was alsowidely distributed but was not found in Bulgaria,Romania, Switzerland, or Austria, whereas group Nwas confined to southern Germany and northernAustria. Finally, group X appeared to be restricted tonorthern Italy and the Balkan.

In North America, we found six haplotypes, four ofwhich were common in Europe (i.e. C1, C2, S1, andF1). The two other haplotypes (C3 and S2) occurred in7% and 8% of the European populations, respectively.

Interestingly, all six haplotypes were frequent inGreat Britain. Out of eight British populations, C1occurred in five populations, C2 in seven, C3 in four,and S1, S2, and F1 in three populations. X and Nhaplotypes were not found in North Americanpopulations.

Nucleotide diversities for European individualswithin the three nominal morphospecies and withinthe five haplotype groups are given in Table 3 (diago-nal). Because the X and N group were absent in NorthAmerican populations, nucleotide diversities withinNorth American A. silvaticus (0.010 ± 0.005) andA. circumscriptus (0.010 ± 0.004) correspond to thediversities of the S and C group. This suggests that the

Figure 1. Phylogenetic trees. A, 70% Neighbour-joining bootstrap consensus tree made in MEGA, version 2.1 (Kumaret al., 2001). B, Bayesian inference of phylogeny assessed with MRBAYES, version 2.01 (Huelsenbeck & Ronquist, 2001).Only support values above 95% are displayed. Branches of both trees were coloured corresponding to the differentmorphospecies appearing in each haplotype (yellow, A. fasciatus; red, A. silvaticus; blue, A. circumscriptus).

Fig

ure

2.

Hap

loty

pe n

etw

orks

mad

e w

ith

TC

S 1

.13

com

pute

r so

ftw

are

(Cle

men

t et

al.

, 200

0). T

he

topo

logy

cri

teri

on (

Pfe

nn

inge

r &

Pos

ada,

200

2) w

as a

ppli

ed t

oob

tain

th

e m

ost

like

ly c

onn

ecti

on b

etw

een

tw

o h

aplo

type

s be

fore

im

plem

enti

ng

the

nes

ted

clad

e de

sign

. Th

e n

esti

ng

of c

lade

s w

as p

erfo

rmed

usi

ng

the

rule

s se

tou

t by

Tem

plet

on e

t al

. (19

87),

Tem

plet

on &

Sin

g (1

993)

, an

d C

ran

dall

(19

96).

Ope

n c

ircl

es i

ndi

cate

mis

sin

g h

aplo

type

s. A

, net

wor

k of

th

e S

gro

up;

B, n

etw

ork

ofth

e C

gro

up;

C, n

etw

ork

of t

he

F g

rou

p; D

, net

wor

k of

th

e X

gro

up.

Col

ours

ref

er t

o th

e in

ferr

ed p

opu

lati

on h

isto

ry f

or t

he

net

wor

k di

agra

ms:

gre

en, C

onti

guou

sR

ange

Exp

ansi

on;

pin

k, I

nad

equ

ate

Geo

grap

hic

al S

ampl

ing;

pu

rple

, In

con

clu

sive

Ou

tcom

e; o

ran

ge,

Res

tric

ted

Gen

e F

low

wit

h I

sola

tion

by

Dis

tan

ce;

blu

e,In

suffi

cien

t G

enet

ic R

esol

uti

on t

o D

iscr

imin

ate

betw

een

Ran

ge E

xpan

sion

/Col

oniz

atio

n a

nd

Res

tric

ted

Dis

pers

al/G

ene

Flo

w,

Sam

plin

g D

esig

n i

nad

equ

ate

toD

iscr

imin

ate

betw

een

Iso

lati

on b

y D

ista

nce

(Sh

ort

Dis

tan

ce M

ovem

ents

) vs.

Lon

g D

ista

nce

Mov

emen

ts; r

ed, S

ampl

ing

Des

ign

inad

equ

ate

to D

iscr

imin

ate

betw

een

Isol

atio

n b

y D

ista

nce

(S

hor

t D

ista

nce

Mov

emen

ts)

vs. L

ong

Dis

tan

ce M

ovem

ents

.

AB

CD

nominal morphospecies in North America are geneti-cally less diverse than in Europe.

NESTED CLADE ANALYSIS OF THE 16S RDNA FRAGMENT

The haplotypes could not be treated together in a sin-gle network. Separate networks for the F, S, C, and X

groups are shown in Figure 2. Most terminal haplo-types were restricted to single populations (29 of the 50haplotypes only occurred in one population), whereasthe interior nodes involved haplotypes that were morecommon and widespread (except for the X group,where most interior nodes involved missing haplo-types). NCA showed a highly significant association

Figure 3. Distribution of the five haplotype groups over all populations: �, F; �, S, �, C; �, N; �, X. Populationabbreviations are given in Table 2.

0 250 mi

250 km0GB3

GB6

AU12

GB4

GB7

GB2GB1

GB5

GB8

SE1

SE2

SE3

PO1

PO4PO3

GE2GE1

GE3

GE4GE5

FR1

FR2

SW3SW2

SW1LI1

CR1AU11AU3AU9

AU10

AU1

AU8AU7AU6AU5AU4AU2

AU13

SL1

RO15RO14

RO13

RO12

RO11RO10

RO9RO8

RO7RO6

RO5

RO4

RO3

RO2

RO1

BU1BU2BU7

BU6

BU5 BU4

BU3 BU9BU10

BU11

IT1IT2

IT3

IT4IT5

IT7

BE1

BE3

BE4

BE5 BE6BE7BE8

BE9BE10

BE11

BE12

BE13

BE14BE15BE16

BE17

BU8

PO2

IT6

BE2

Table 3. Within sequence divergence (diagonal) [equal to nucleotide diversities (π) for European individuals] and betweensequence divergence (below the diagonal) for the three nominal morphospecies and for the five haplotype groups (standarderrors based on 10000 bootstrap replicates in parentheses)

Morphospecies Arion fasciatus Arion silvaticusArioncircumscriptus

Arion fasciatus (15) 0.102 (0.010)Arion silvaticus (33) 0.100 (0.010) 0.033 (0.005)Arion circumscriptus (17) 0.097 (0.010) 0.036 (0.005) 0.028 (0.005)

Haplotype group F S X C N

F (7) 0.010 (0.003)S (14) 0.182 (0.020) 0.011 (0.003)X (18) 0.170 (0.018) 0.045 (0.009) 0.018 (0.004)C (7) 0.178 (0.020) 0.043 (0.009) 0.042 (0.008) 0.010 (0.003)N (4) 0.182 (0.019) 0.053 (0.010) 0.054 (0.009) 0.054 (0.010) 0.023 (0.006)

Numbers of haplotypes are given in parentheses in the first column.

between genetic and geographical distances for the thefour haplotype groups F, C, S, and X. The inferred pop-ulation history included ‘Restricted Gene Flow withIsolation by Distance’ for groups C and S, and ‘Contig-uous Range Expansion’ for groups F and X (Fig. 2).

THE NUCLEAR ITS-1 FRAGMENT

The nuclear ITS-1 fragment was 539–542 bp long. ITS-1 sequences available in GenBank were included in theanalyses. The AT/GC proportion was 0.46/0.54. Thefragment comprised seven polymorphic sites whengaps were included, (three indels) of which only onewas parsimony informative, showing a C forsequences of group S (N = 5) in position 41 (gapsexcluded), whereas all other sequences had a G,including A. silvaticus, A. fasciatus, and A. circum-scriptus from the X group, as well as representatives ofgroups F, C, and N. Again, the three morphospecies didnot appear as separate monophyletic units (Table 4).

DISCUSSION

Previous studies questioned the applicability of theBSC to the three nominal Carinarion species (Backel-jau et al., 1997; Jordaens et al., 2000). However, theseearlier allozyme data were unable to refute the possi-bility that the three nominal species might complywith the PSC. The present study now questions theapplicability of this latter species concept to Carinar-ion as well because the 16S rDNA sequences suggestthat neither of the three nominal species represents amonophyletic unit. Actually, Carinarion spp. com-prises at least 32 different haplotypes grouped in atleast four well-supported clades, plus at least another18 haplotypes whose relationships remain unresolved(X group). This latter group consists of specimens ofthe three nominal species and includes all shared hap-lotypes. It is unclear, however, whether these shared

haplotypes represent: (1) retained ancestral polymor-phisms in the three nominal species; (2) signatures ofrecent interspecific hybridization between the threenominal species; or (3) simple intraspecific variation ifCarinarion is considered as a single species. For thetime being, we presume that either of the latter twopossibilities is more likely than the first because shar-ing 22% of 16S rDNA haplotypes between two or threeCarinarion species would seem to be an exceptionallyhigh degree of overlap for these supposedly well-differentiated species.

The very high sequence divergence between F hap-lotypes and the other groups (up to approximately20%) or between A. fasciatus (i.e. F group + several Xhaplotypes) and the two other nominal species (up toapproximately 10%) may be indicative of a specieslevel difference. However, these estimates still fallwithin the upper limits of intraspecific stylommato-phoran mtDNA sequence divergences (Davison, 2002).Moreover, in the absence of other data, mere levels ofsequence divergence are an inadequate basis for defin-ing species (Sangster, 2000; Ferguson, 2002). Hence,the high 16S rDNA sequence divergence between theF group or A. fasciatus and the other groups or nom-inal species does not necessarily support an interspe-cific difference. Actually, all interspecific A. fasciatusallozyme heterozygotes reported by Jordaens et al.(2000), precisely belong to the F group (S. Geenen, K.Jordaens & T. Backeljau, unpubl. data), suggestingthat, despite these high sequence divergence, in fieldconditions, F animals may hybridize with animalsfrom the other groups or nominal species.

By contrast to the 16S rDNA data, the ITS-1sequences revealed little variation and showed neitherevidence of any consistent grouping, nor of any differ-entiation between the three nominal species. Severalfactors may be responsible for the discrepancybetween both gene trees (Ballard, Chernoff & James,

Table 4. Polymorphic sites for the nuclear ITS-1 rDNA fragment

ITS- allele

Position (base pair)Haplotypegroup Morphospecies8–10 41 197 206 234 284 467

1 – G G A – G – F Arion fasciatus2 – . . . T . – F Arion fasciatus3 – . . . – . T C Arion circumscriptus4 – . . . – . – F, C, X, N Arion fasciatus Arion

circumscriptusArion silvaticus

5 ACG . . . – . – X Arion silvaticus6 – . . G – C – X Arion silvaticus7 – . T . – . – F Arion fasciatus8 – C . . – . – S Arion silvaticus

2002) and it has been argued that, because the effec-tive population size of mtDNA is one-quarter that ofnDNA, the mtDNA genealogy has a higher probabilityof tracking the species tree in fewer generations(Moore, 1995). Nevertheless, the ITS-1 data may betaxonomically more suggestive than the 16S rDNAdata because ITS sequences are generally consideredto be highly homogenous within species, but well-dif-ferentiated between species (Hillis & Dixon, 1991;Korte & Armbruster, 2003; Vidigal et al., 2004). There-fore, the extremely low divergence between the ITS-1sequences in Carinarion and the lack of consistentITS-1 differentiation among the three nominal speciesboth suggest that this complex involves only one com-mon nuclear gene pool. These findings need to be ver-ified by sreening additional genetic markers and byincreasing sample sizes because only 16S rDNA wasstudied intensively, whereas ITS-1 was surveyed inonly a few specimens.

The present mtDNA data confirm that North Amer-ican Carinarion populations are genetically impover-ished compared to European populations (six vs. 50haplotypes, respectively) (Geenen et al., 2003). Thisimplies that earlier protein electrophoretic data ofNorth American Carinarion (Chichester, 1967;McCracken & Selander, 1980) may be not sufficientlyrepresentative to decide about taxonomic issues.

Combining all previous arguments leads to thetentative conclusion that the three morphologicallydefined Carinarion species cannot be maintained aswell-defined biological or phylogenetic species.Instead, we reinforce our earlier suggestion that thealleged interspecific differences in their morphology(colour, size), genital anatomy, and electrophoreticpatterns of AGP and EST, may be misleading as tax-onomic markers (Backeljau et al., 1997; Jordaenset al., 2000, 2001, 2002). Indeed, inasmuch as thesedifferences are truly consistent (but see Jordaenset al., 2002), genetically determined and independentfrom environmental influences (but see Jordaenset al., 1999, 2001), they may reflect different allelic fix-ations due to sustained self-fertilization.

Unfortunately, the current data are not particularlyinformative with respect to the phylogeographical his-tory of the breeding systems and different haplotypegroups in Carinarion. NCA identified a pattern of con-tiguous range expansion in the F and X groups vs.mostly a pattern of restricted gene flow with isolationby distance in the S and C groups. Incidentally, thesetwo patterns coincide with the occurrence of outcross-ing and shared mtDNA haplotypes in the F and Xgroups vs. the (near complete) lack thereof in the Sand C groups. However, evaluating whether thisobservation is meaningful will require a more exten-sive population and DNA sampling in areas that werenot sufficiently covered in the present analysis.

In conclusion, irrespective of which species conceptis implemented, the present DNA sequence data donot support the subdivision of Carinarion into thethree currently recognized nominal species. Instead,Carinarion either represents a single, polymorphic,gene pool or involves a set (> 3) of phylogenetic spe-cies. Currently, we favour the single species interpre-tation because (1) the 16S rDNA and ITS-1 yieldeddifferent pictures (2); a considerable part of the 16SrDNA tree remained unresolved; and (3) applying thePSC using the supposedly fast evolving stylommato-phoran mtDNA (Davison, 2002) may uncover a largenumber of phylogenetic species, whose heuristicvalue may be questionable in the absence of otherdiagnostic features. Hence, we regard Carinarion asa single species-level taxon whose taxonomicallydeceiving, correlated phenotypic and geneticintraspecific variation among MLGs or morphospe-cies is due to sustained self-fertilization. However,considering the remaining ambiguity in the interpre-tation of Carinarion, we think that any nomencla-tural change is still premature. Nevertheless,relieving Carinarion spp. from their interpretation asthree species appears to be the best way to refuel theinterest in this kind of provocative taxonomic com-plexes and the challenging evolutionary questionsthat emanate from them.

ACKNOWLEDGEMENTS

We thank Gary Bernon, Sven Lardon, and Jan Pinceelfor providing part of the material. S.G. held an IWTscholarship. This research was supported by FWO-grant G.0003.02 and OSTC project MO/36/003 to T.B.and by RAFO project JORDKKP02 to K.J. The com-ments of two anonymous referees improved the manu-script considerably.

REFERENCES

Backeljau T, Ahmadyar SZ, Selens M, Van Rompaey J,Verheyen W. 1987. Comparative electrophoretic analyses ofthree European Carinarion species (Mollusca, Pulmonata,Arionidae). Zoologica Scripta 16: 209–222.

Backeljau T, De Bruyn L, de Wolf H, Jordaens K, vanDongen S, Winnepenninckx B. 1997. Allozyme diversityin slugs of the Carinarion complex (Mollusca, Pulmonata).Heredity 78: 445–451.

Backeljau T, De Winter AJ, Martin R, Rodriguez T, DeBruyn L. 1994. Genital and allozyme similarity betweenArion urbiae and A. anguloi (Mollusca: Pulmonata). Zoolog-ical Journal of the Linnean Society 110: 1–18.

Ballard JWO, Chernoff B, James AC. 2002. Divergence ofmitochondrial DNA is not corroborated by nuclear DNA,morphology, or behavior in Drosophila simulans. Evolution56: 527–545.

Chichester LF. 1967. The zoogeography, ecology and taxon-omy of arionid and limacid slugs introduced into northeast-ern North America. Unpublished PhD Thesis, University ofConnecticut.

Chichester LF, Getz LL. 1969. The zoogeography and ecol-ogy of arionid and limacid slugs introduced into northeasternNorth America. Malacologia 7: 313–346.

Chichester LF, Getz LL. 1973. The terrestrial slugs of north-eastern North America. Sterkiana 51: 11–42.

Clement M, Posada D, Crandall KA. 2000. TCS: a computerprogram to estimate gene genealogies. Molecular Ecology 9:1657–1659.

Crandall KA. 1996. Multiple interspecies transmissions ofhuman and simian T-cell leukemia/lymphoma virus type Isequences. Molecular Biology and Evolution 13: 115–131.

Davison A. 2002. Land snails as a model to understand therole of history and selection in the origins of biodiversity.Population Ecology 44: 129–136.

Felsenstein J. 1985. Confidence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783–791.

Ferguson JWH. 2002. On the use of genetic divergence foridentifying species. Biological Journal of the Linnean Society75: 509–516.

Foltz DW, Ochman H, Jones JS, Evangelisti SM,Selander RK. 1982. Genetic population structure andbreeding systems in arionid slugs (Mollusca: Pulmonata).Biological Journal of the Linnean Society 17: 225–241.

Geenen S, Jordaens K, Castilho R, Backeljau T. 2003.Congruence between starch gel and polyacrylamide gel elec-trophoresis in detecting allozyme variation in pulmonateland slugs. Electrophoresis 24: 622–627.

Hey J. 2001. The mind of the species problem. Trends in Ecol-ogy and Evolution 16: 326–329.

Hillis DM, Dixon MT. 1991. Ribosomal DNA − molecularevolution and phylogenetic inference. Quarterly Review ofBiology 66: 410–453.

Hillis DM, Huelsenbeck JP. 1992. Signal, noise, andreliability in molecular phylogenetic analyses. Journal ofHeredity 83: 189–195.

Huelsenbeck JP, Bollback JP. 2001. Empirical and hierar-chical Bayesian estimation of ancestral states. SystematicBiology 50: 351–366.

Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesianinference of phylogenetic trees. Bioinformatics 17: 754–755.

Jordaens K, de Wolf H, Verhagen R, Backeljau T. 1996.Possible outcrossing in natural Carinarion populations(Mollusca, Pulmonata). British Crop Production CouncilSymposium Proceedings 66: 13–20.

Jordaens K, Geenen S, Reise H, Van Riel P, Verhagen R,Backeljau T. 2000. Is there a geographical pattern in thebreeding system of a complex of hermaphroditic slugs (Mol-lusca: Gastropoda: Carinarion)? Heredity 85: 571–579.

Jordaens K, Scheirs J, Verhagen R, Backeljau T. 1998.Ecogenetic comparison of the selfing terrestrial slugs Arionsilvaticus and A. circumscriptus. Belgium (Mollusca: Pulmo-nata: Arionidae). Hereditas 129: 27–36.

Jordaens K, van Dongen S, Van Riel P, Geenen S, Ver-hagen R, Backeljau T. 2002. Multivariate morphometrics

of soft body parts in terrestrial slugs: comparison betweentwo datasets, error assessment and taxonomic implications.Biological Journal of the Linnean Society 75: 533–542.

Jordaens K, Van Riel P, Geenen S, Verhagen R, Backel-jau T. 2001. Food-induced body pigmentation questions thetaxonomic value of colour in the self-fertilizing slug Carinar-ion spp. Journal of Molluscan Studies 67: 161–167.

Jordaens K, Van Riel P, Verhagen R, Backeljau T. 1999.Food-induced esterase electromorphs in Carinarion spp. andtheir effects on taxonomic data analysis (Gastropoda,Pulmonata, Arionidae). Electrophoresis 20: 473–479.

Kerney M, Cameron R, Jungbluth J. 1983. Die Land-schnecken Nord- und Mitteleuropas. Hamburg: Paul Parey.

Korte A, Armbruster GFJ. 2003. Apomorphic and plesiomor-phic ITS-I rDNA patterns in morphologically similar snails(Stylommatophora: Vallonia), with estimates of divergencetime. Journal of Zoology 260: 275–283.

Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA2:molecular evolutionary genetics analysis software. Bioinfor-matics 17: 1244–1245.

Larget B, Simon DL. 1999. Markov chain Monte Carlo algo-rithms for the Bayesian analysis of phylogenetic trees.Molecular Biology and Evolution 16: 750–759.

Lohmander H. 1937. Über die nordischen Formen von Arioncircumscriptus Johnston. Acta Societatis pro Fauna et FloraFennica 60: 90–112.

Löytynoja A, Milinkovitch MC. 2001. SOAP, cleaningmultiple alignments from unstable blocks. Bioinformatics17: 573–574.

Mayden RL. 1997. A hierarchy of species concepts: thedenouement in the saga of the species problem. In: ClaridgeMF, Dawah HA, Wilson MR, eds. Species: the units of biodi-versity. London: Chapman & Hall, 381–424.

Mayr E. 1940. Speciation phenomena in birds. American Nat-uralist 74: 249–278.

McCracken GF, Selander RK. 1980. Self-fertilization andmonogenic strains in natural populations of terrestrial slugs.Proceedings of the National Academy of Sciences of theUnited States of America 77: 684–688.

de Meeûs T, Durand P, Renaud F. 2003. Species concepts:what for? Trends in Parasitology 19: 425–427.

Moore WS. 1995. Inferring phylogenies from mtDNA varia-tion: mitochondrial gene trees versus nuclear-gene trees.Evolution 49: 718–726.

Nei M. 1972. Genetic distance between populations. The Amer-ican Naturalist 106: 283–292.

Nei M. 1978. Estimation of average heterozygosity and geneticdistance from a small number of individuals. Genetics 89:583–590.

Nei M. 1987. Molecular evolutionary genetics. New York, NY:Columbia University Press.

Palumbi SR, Benzie J. 1991. Large mitochondrial DNA dif-ferences between morphologically similar Penaeid shrimps.Molecular Marine Biology and Biotechnology 1: 27–34.

Pfenninger M, Posada D. 2002. Phylogeographic history ofthe land snail Candidula unifasciata (Helicellinae, Stylom-matophora): Fragmentation, corridor migration, and second-ary contact. Evolution 56: 1776–1788.

Pinceel J, Jordaens K, Van Houtte N, De Winter AJ,Backeljau T. 2004. Molecular and morphological datareveal cryptic taxonomic diversity in the terrestrial slugcomplex Arion subfuscus/fuscus in continental NorthwestEurope (Mollusca, Pulmonata, Arionidae). Biological Jour-nal of the Linnean Society 83: 23–38.

Posada D, Crandall KA. 1998. MODELTEST: testing themodel of DNA substitution. Bioinformatics 14: 817–818.

Posada D, Crandall KA, Templeton AR. 2000. GeoDis: aprogram for the cladistic nested analysis of the geographicaldistribution of genetic haplotypes. Molecular Ecology 9: 487–488.

Ranker TA, Geiger JMO, Kennedy SC, Smith AR, HauflerCH, Parris BS. 2003. Molecular phylogenetics and evolu-tion of the endemic Hawaiian genus Adenophorus(Grammitidaceae). Molecular Phylogenetics and Evolution26: 337–347.

Rozas J, Sanchez-DeI, Barrio JC, Messeguer X, Rozas R.2003. DnaSP, DNA polymorphism analyses by the coalescentand other methods. Bioinformatics 19: 2496–2497.

Saitou N, Nei M. 1987. The Neighbor-joining method − a newmethod for reconstructing phylogenetic trees. MolecularBiology and Evolution 4: 406–425.

Sangster G. 2000. Genetic distance as a test of species bound-aries in the citril finch Serinus citrinella: a critique and tax-onomic reinterpretation. Ibis 142: 487–490.

Schneider S, Roessli D, Excoffier L. 2000. Arlequin, asoftware for population genetic data analysis, Version 2.000.Geneva: Genetics and Biometry Laboratory, Department ofAnthropology, University of Geneva.

Selander RK, Ochman H. 1983. The genetic structure of pop-ulations as illustrated by molluscs. Isozymes: Current Topicsin Biology and Medical Research 10: 93–123.

Siddall M. 1995. Random cladistics, the Ohio edition.Program and Documentation, Version 4.0. University ofToronto; program available from http://research.amnh.org/~siddall/rc.html.

Strimmer K, Goldman N, von Haeseler A. 1997. Bayesianprobabilities and quartet puzzling. Molecular Biology andEvolution 14: 210–211.

Strimmer K, von Haeseler A. 1996. Quartet puzzling: aquartet maximum-likelihood method for reconstructing treetopologies. Molecular Biology and Evolution 13: 964–969.

Swofford DL. 1998. PAUP*: phylogenetic analysis using par-simony (* and other methods), Version 4.0. Sunderland, MA:Sinauer.

Tamura K, Nei M. 1993. Estimation of the number of nucle-otide substitutions in the control region of mitochondrialDNA in humans and chimpanzees. Molecular Biology andEvolution 10: 512–526.

Templeton AR. 1998. Nested clade analyses of phylogeo-graphic data: testing hypotheses about gene flow and popu-lation history. Molecular Ecology 7: 381–397.

Templeton AR. 2004. Statistical phylogeography: methods ofevaluating and minimizing inference errors. Molecular Ecol-ogy 13: 789–809.

Templeton AR, Boerwinkle E, Sing CF. 1987. A cladisticanalysis of phenotypic associations with haplotypes inferredfrom restriction endonuclease mapping. I. Basic theory andan analysis of alcohol dehydrogenase activity in Drosophila.Genetics 117: 343–351.

Templeton AR, Crandall KA, Sing CF. 1992. A cladisticanalysis of phenotypic associations with haplotypes inferredfrom restriction endonuclease mapping and DNA sequencedata. III. Cladogram estimation. Genetics 132: 619–633.

Templeton AR, Routman E, Phillips CA. 1995. Separatingpopulation structure from population history: a cladisticanalysis of the geographical distribution of mitochondrialDNA haplotypes in the Tiger salamander, Ambystoma tigri-num. Genetics 140: 767–782.

Templeton AR, Sing CF. 1993. A cladistic analysis of pheno-typic associations with haplotypes inferred from restrictionendonuclease mapping. IV. Nested analyses with cladogramuncertainty and recombination. Genetics 134: 659–669.

Thompson JD, Gibson TJ, Plewniak F, Higgins DG. 1997.The ClustalX windows interface: flexible strategies for mul-tiple sequence alignment aided by quality analysis tools.Nucleic Acids Research 25: 4876–4882.

Vidigal THDA, Spatz L, Kissinger JC, Redondo RAF,Pires ECR, Simpson AJG, Carvalho OS. 2004. Analysisof the first and second internal transcribed spacer sequencesof the ribosomal DNA in Biomphalaria tenagophila complex(Mollusca: Planorbidae). Memorias do Instituto OswaldoCruz 99: 153–158.

Waldén HW. 1955. The land Gastropoda of the vicinity ofStockholm. Arkiv för Zoologi 7: 391–448.

Xia X, Xie Z. 2001. DAMBE: Software package for data anal-ysis in molecular biology and evolution. Journal of Heredity92: 371–373.

Xia XH, Xie Z, Salemi M, Chen L, Wang Y. 2003. An indexof substitution saturation and its application. MolecularPhylogenetics and Evolution 26: 1–7.