Blackwell Publishing LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066© 2006 The Linnean Society of London? 2006
89?
589604
Original Article
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
S. GEENEN
ET AL
.
Biological Journal of the Linnean Society, 2006, 89, 589–604. With 3 figures
Molecular systematics of the Carinarion complex
(Mollusca: Gastropoda: Pulmonata): a taxonomic riddle
caused by a mixed breeding system
SOFIE GEENEN1*, KURT JORDAENS1 and THIERRY BACKELJAU1,2
1
Evolutionary Biology Group, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium
2
Received 22 November 2004; accepted for publication 15 January 2006
The original description of the slugs Arion (Carinarion) fasciatus, Arion (Carinarion) silvaticus and Arion (Carinarion) circumscriptus was based on subtle differences in body pigmentation and genital anatomy. However, body
pigmentation in these slugs may be influenced by their diet, whereas the genital differences between the species
could not be confirmed by subsequent multivariate morphometric analyses. Hence, the status of the three nominal
morphospecies remains controversial, with electrophoretic studies based on albumen gland proteins and allozymes
also providing conflicting results. These studies suggested that Carinarion species are difficult to reconcile with the
biological species concept because there is evidence of interspecific hybridization in places where these predominantly self-fertilizing slugs apparently outcross. Therefore, in the present study, the three Carinarion species are
evaluated under a phylogenetic species concept, using nucleotide sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS-1) and the mitochondrial 16S rDNA. ITS-1 showed no species specific variation. However, 16S
rDNA yielded five haplotype groups. Three of these grouped haplotypes by species, whereas the two others joined
haplotypes 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. The
present data also confirmed that North American Carinarion populations are genetically impoverished and may be
not sufficiently representative with respect to the taxonomy of Carinarion. In conclusion, we currently regard Carinarion as a single species-level taxon, whose taxonomically deceiving, correlated phenotypic and genetic intraspecific variation is caused by sustained self-fertilization. © 2006 The Linnean Society of London, Biological Journal
of 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 species
concepts as tools to describe biodiversity (de Meeûs,
Durand & Renaud, 2003). In this context, more than
20 species concepts have been defined, each of which
reflects different philosophical and biological foundations (Mayden, 1997). However, no single species
concept seems generally applicable to all organisms
(Hey, 2001) and different species concepts may even
be mutually inconsistent (Mayden, 1997). Nevertheless, taxonomists often describe species operationally
*Corresponding author. E-mail: [email protected]
under one species concept but interpret them within
the framework of another. For example, many animal
species were originally described as some sort of ‘morphospecies’, whereas subsequent practice implicitely
treated these morphospecies as biological species
(sensu Mayr, 1940). In fact, most species descriptions
do not even mention which species concept(s) they
implement. This situation has at least three negative
consequences: (1) the interpretation of what species
actually represent may be confused; (2) the value of
species as descriptors or units of biodiversity may be
ill-defined (Hey, 2001); and (3) describing new species
may be easier than falsifying species (Backeljau et al.,
1994).
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
589
–
–
0.94
0.06
0.13 (0.06)
–
–
0.87 (0.05)
0.14 (0.06)
0.18 (0.08)
–
–
0.93 (0.03)
0.08 (0.04)
0.15 (0.07)
–
0.74
0.67 (0.06)
0.40 (0.09)
0.31 (0.10)
–
–
0.73 (0.04)
0.31 (0.06)
0.28 (0.08)
–
–
0.79 (0.06)
0.24 (0.08)
0.21 (0.10)
Standard deviations are given in parentheses.
0.65
–
0.81 (0.10)
0.22 (0.12)
–
North America
Ireland
Europe
Europe
Europe
McCracken & Selander (1980)
Foltz et al. (1982)
Backeljau et al. (1997)
Backeljau et al. (1997)
Jordaens et al. (2000)
I
I
I
D
D*
wS
wF
SvC
FvC
FvS
FvCvS
Region
Study
Species comparison
A particularly challenging example of these problems is provided by the terrestrial slugs of the arionid
subgenus Carinarion Hesse, 1926, which comprises
three predominantly selfing pulmonate slug species,
namely Arion (Carinarion) fasciatus (Nilsson, 1823),
Arion (Carinarion) circumscriptus Johnston, 1828 and
Arion (Carinarion) silvaticus Lohmander, 1937. The
three species are widely distributed over Europe (Kerney, Cameron & Jungbluth, 1983) and North America,
where they have been introduced by man (Chichester
& Getz, 1969, 1973). They were initially distinguished
by their body pigmentation and subtle genital differences (Lohmander, 1937; Waldén, 1955), and later
confirmed on the basis of fixed electrophoretic differences in albumen gland proteins (AGP) among North
American specimens (Chichester, 1967). Subsequently, McCracken & Selander (1980) reported that
each of the three species in North America consisted of
a single homozygous multilocus genotype (MLG)
(based on 18 allozyme loci). This result was interpreted as a strong indication of sustained selfing
(autogamy) (Selander & Ochman, 1983), whereas the
low genetic identities between the three MLGs
(Table 1) were seen as strong support for their specific
differentiation. A similar electrophoretic survey by
Foltz et al. (1982) (Table 1) of 13 allozyme loci in
A. circumscriptus and A. silvaticus from Ireland confirmed that A. circumscriptus consists of one MLG,
whereas A. silvaticus yielded two MLGs. To extend
these previous electrophoretic studies, Backeljau et al.
(1987, 1997) conducted electrophoretic surveys of the
three Carinarion species in a part of their native area
in western Europe. Backeljau et al. (1987) showed
that, in this area, the three species differed consistently in their overall electrophoretic profiles for
esterases (EST) and AGP, confirming the earlier work
of Chichester (1967) on North American material. By
contrast, the allozyme survey of Backeljau et al.
(1997), who screened other enzymatic proteins,
showed that each of the three species in western
Europe consisted of a series of homozygous MLGs.
This latter result, combined with the allozyme surveys
of Jordaens et al. (1998, 2000), showed that, in
Europe, the three Carinarion species consist of at
least 13 (A. fasciatus), nine (A. circumscriptus), and 24
(A. silvaticus) MLGs. Overall genetic distances and
identities among the three species as reported by
Backeljau et al. (1997) and Jordaens et al. (2000) are
compared in Table 1. Importantly, these studies also
demonstrated that, in some areas, Carinarion spp.
produce heterozygous individuals, suggesting the
occurrence of facultative outcrossing and possibly
even of interspecific hybridization (Jordaens et al.,
1996, 2000). Further analyses by Geenen et al. (2003)
showed that the different conclusions of the allozyme
studies of European and (introduced) North American
wC
S. GEENEN ET AL.
Table 1. Nei’s (1972) mean genetic identities (I) and mean genetic distances (D) or Nei’s (1978) mean genetic distance (D*) between (denoted by ‘v’) and within
(denoted by ‘w’) Arion fasciatus (F), Arion silvaticus (S), and Arion circumscriptus (C)
590
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
populations were not due to technical discrepancies,
but most probably to the lower amount of allozyme
variation in the North American populations. This
implies that the electrophoretic data of Chichester
(1967) and McCracken & Selander (1980) may be not
sufficiently representative with respect to the population structure and the specific status of Carinarion
spp. in its native area (i.e. Europe). Moreover, the
predominantly autogamous (i.e. self-fertilization)
breeding system of Carinarion spp. precludes the
application 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 doubt
on Carinarion spp. as different biological species.
This argument is further supported by the fact
that self-fertilization may promote allozymic differentiation by fixing alternative alleles (Backeljau
et al., 1997; Jordaens et al., 2000), a point that not
only underpins the production of well-differentiated
homozygous MLGs, but also that possibly can
explain the ‘species specific’ electrophoretic patterns
of AGP and EST or even the subtle ‘species specific’
morphological differences. Insofar as these features
are genetically determined, it appears plausible that
fixation of alternative alleles at the loci involved
could produce misleadingly consistent morphological
and protein electrophoretic differences, leaving the
source of remaining phenotypic variation for a large
part to the environment (Jordaens et al., 2001).
Finally, a multivariate morphometric comparison of
the genital features in Carinarion spp. did not support the morphological differentiation between the
three species as described by Lohmander (1937) or
Waldén (1955), except for a consistent overall size
difference between A. fasciatus and the two other
species (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 the
three taxa as ‘good species’. Because this practice is
neither consistent with the BSC, nor with a morphospecies concept (MSC), we here explore whether
phylogenetic data provide a more consistent basis for
the taxonomic interpretation of this complex. To this
end, we present a DNA sequence analysis of a fragment of the mitochondrial 16S rDNA gene (16S) and
the nuclear ribosomal internal transcribed spacer I
(ITS-1). More precisely, we aimed to assess whether:
(1) the three nominal Carinarion species are monophyletic taxa consistent with the phylogenetic species
concept (PSC); (2) North American Carinarion is
genetically impoverished and differentiated compared
to European populations; (3) putative geographical
patterns in breeding biology (Jordaens et al., 2000) are
reflected by patterns of mtDNA variation; and (4)
591
mtDNA variation allows to recognize phylogeographical patterns that may help to disentangle the current
taxonomic complexity of the Carinarion complex.
MATERIAL AND METHODS
SAMPLING
We surveyed 1990 animals from 91 European
(N = 1746) and nine North American (N = 244) populations. Specimens were identified on the basis of morphological criteria (i.e. body pigmentation and genital
features), following Lohmander (1937) and Waldén
(1955). Individuals with an intermediate morphotype
(‘0’; unidentified in Table 2) were not used in the analysis. DNA was extracted from individual foot muscle
tissue according to Pinceel et al. (2004).
SINGLE STRAND CONFORMATION POLYMORPHISM
(SSCP) ANALYSIS AND DNA SEQUENCING
All individuals were subjected to SSCP analysis of two
fragments of the mitochondrial 16S rDNA gene (16S1,
101 bp; 16S2, 156 bp). Specific primers were developed to amplify each fragment [16S1: L16SCA1 (5′AAGGTAGCAAAATAAATAGGC-3′) and H16SCA1
(5′-TCTTAGGGTCTTCTCGTCTT-3′); 16S2: L16SCA2
(5′-ATAAGACGAGAAGACCCTAA-3′) and H16SAR2
(5′-GTCCAACATCGAGGTCAC)]. Polymerase chain
reaction (PCR) and SSCP protocols were in accordance
with those of Pinceel et al. (2004), except for the recipe
of the polyacrylamide gels for the first fragment (gels
16S1: T = 14%, C = 2%, with 5% glycerol). In each population, all haplotype combinations of the two fragments detected with SSCP were sequenced on an ABI
373A automatic DNA sequencer (Applied Biosystems
Inc.) following Pinceel et al. (2004) or were submitted
for sequencing to the Flanders Interuniversity Institute for Biotechnology (VIB, Department of Molecular
Genetics, University of Antwerp). Primers to amplify
the 16S rDNA stretch comprising both SSCP fragments were 16SAR (5′-CGCCTGTTTAACAAAAACAT3′) and 16SBR (5′-CCGGTTTGAACTCAGATCAGAT
CACGT-3′) (Palumbi & Benzie, 1991). This stretch
comprised 395 bp and was used for phylogenetic
analysis. All 16S rDNA sequences were deposited in
GenBank under accession numbers AJ715324–75.
Variation outside the SSCP fragments was very low
and yielded only one additional haplotype (X5) that
differed from haplotype X4 by a single substitution.
Hence, all X4 and X5 individuals were sequenced for
the entire 395 bp 16S rDNA stretch.
A fragment of the nuclear ITS-1 rDNA gene of 12
specimens was also sequenced. These 12 specimens
were selected based on which mitochondrial group
(see Results) they belong to (Table 2). The fragment
was amplified under the following PCR conditions:
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
592
S. GEENEN ET AL.
Table 2. Country, sampling localities and locality abbreviation (Abb), number of individuals sampled (No), haplotype
frequencies, and morphospecies (MS: F, Arion fasciatus; S, Arion silvaticus; C, Arion circumscriptus; 0, undefined)
Phylogenetic groups
C
S
Haplotypes
Country
Locality
Abb.
No C1
Belgium
Houx
Yvoir
Eigenbrakel
Gent
Pittem
Sint-Antonius
Zoersel
Genk
Diepenbeek
Halle
Halle
Oudenaarde
Sint-Antonius
Zoersel
Tielt
Nukerke
Maffe
GomzéAndoumont
Soy-Fisenne
BE1
BE2
BE3
BE4
BE5
BE6
22
20
12
16
15
18
BE7
BE8
BE9
BE10
BE11
BE12
18
15
18
24
11
24
BE13
BE14
BE15
BE16
6
4
9
4
Ae Village
Humshaugh
Innerleithen
Worksop
Burnopfield
Cranham
Epping Forest
Temple Ewell
GB1
GB2
GB3
GB4
GB5
GB6
GB7
GB8
13
25
30
30
26
25
25
12
Germany
Görlitz
Görlitz
Görlitz
Innerkoy
Warmbronn
GE1
GE2
GE3
GE4
GE5
16
13
11
22
25
Switzerland
Langnau
SW1
Therwil
SW2
Münchenstein SW3
16
5
20
Etroubles
Caino
Garessio
Gignese
Macugnaga
Sardagna
Cortina
d’Ampezzo
IT1
IT2
IT3
IT4
IT5
IT6
IT7
30
23
25
29
17
30
34
Rencurel
Agnieres
FR1
FR2
8
31
Sweden
Genarp
Göteborg
Lund
SE1
SE2
SE3
23
17
37
0.04
Poland
Bialowice
Muszkowice
Zgorzelec
Zgorzelec
PO1
PO2
PO3
PO4
14
15
18
25
0.07
Námest
CR1
41
Great Britain
Italy
France
Czech
Republic
1
1
0.17
0.44
1
0.06
C2
C3
C4
C5
C6 C7 S1
S2
S3
S4
S5
S6
S7 S8
S9
S10 S11
S12 S13 S14
0.58 0.25
0.06
0.50*
0.44 0.50
1
1
1
0.96
0.04
1
0.54 0.46
1
0.75
0.25*
0.56 0.44
0.75
0.25
BE17 11
1
0.15
0.03
0.35
0.04
0.75
0.04
0.24
0.80
0.42
0.88
1
0.25
0.08
0.36
0.30
0.08
0.60
0.03 0.20
0.20
0.15
0.08
1
1
0.08 0.92
1
0.40 0.60
1*
1
1
1
1
0.29
1
0.10
0.22
0.68
0.04
0.67
0.33
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
593
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
F
F1
N
F2
F3
F4
F5
F6
F7
N1
X
N2
N3
N4
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
X12
X13
X14
X15
X16
X17
X18
MS
C
C
S-C
S-C
C
S-C
S
S
S
S
S
S
C
C
C
S-C
S
0.69
F-S-C
S-C
F-S-C
S-C
F-S-C
S-C
C
C
0.20
0.08
0.45
C
C
F
S
S
0.55
1
S
S
S
1
0.14
0.86
0.71
C
F-S-C-0
C
S-C-0
S
C
F-S
C
S-C
0.92
1
1
F-S-C
F
F
0.93
F-C
F-C
F
F
0.11
0.32
0.89
0.68
1
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
F
594
S. GEENEN ET AL.
Table 2. Continued
Phylogenetic groups
C
S
Haplotypes
Country
Locality
Abb.
No
Austria
Vienna
Bischofshofen
Altenburg
Graz
Graz
Graz
Graz
Graz
Horn
Kamp
Altenburg
Innerlaterns
Elsbethen
AU1
AU2
AU3
AU4
AU5
AU6
AU7
AU8
AU9
AU10
AU11
AU12
AU13
22
27
11
122
17
33
47
43
25
11
45
17
9
Sinaia
Poiana
Timisu de Sus
Miercurea
Azuga
Cozanesti
Holda
Bicaz
Lunca Visagului
Prundu
Racova
Podu Dimbovitei
Baile Felix
Covasna
Dealu Negru
RO1
RO2
RO3
RO4
RO5
RO6
RO7
RO8
RO9
RO10
RO11
RO12
RO13
RO14
RO15
39
8
26
5
33
8
19
7
2
2
6
4
3
9
1
Bulgaria
Pastra
Batak
Cujpetlovo
Beli Osam
Vratce
Razlog
Batak
Batak
Karnare
Gabrovo
Ribarica
BU1
BU2
BU3
BU4
BU5
BU6
BU7
BU8
BU9
BU10
BU11
33
32
22
12
11
5
3
1
3
1
2
Slovenia
Begunjena
Gorenjskem
SL1
30
Romania
Liechtenstein
Schaanwald
LI1
12
USA
North Carvon
Bellingham
West Barnstable
West Barnstable
West Barnstable
Sandwich
Woods Hole
Mansfield
Pomfret
NA1
NA2
NA3
NA4
NA5
NA6
NA7
NA8
NA9
50
22
35
14
8
60
9
32
14
C1
C2
C3
C4
C5
C6
C7
S1
S2
0.82
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
0.18
0.23*
0.03
0.08
0.09
0.52
0.43
0.92
0.08*
0.96
0.14
0.64
0.25
1
0.11
0.56
0.33
Populations and haplotype groups are indicated by an asterisk (*) if they comprised specimens screened for nuclear ribosomal internal transcribed spacer I.
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 with
5 min at 72 °C. The Thermo Sequenase II Dye Terminator Cycle Sequencing Premix Kit (Pharmacia
Biotech, Inc.) and an ABI 373A automatic DNA
sequencer (Applied Biosystems Inc.) were used to
sequence the fragment between the primers ITS1L
(TCCGTAGGTGAACCTGCGGAAGGAT) and 58C
(TGCGTTCAAGATATCGATGTTCAA)
(Hillis
&
Dixon, 1991), following Pinceel et al. (2004). All ITS-
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
F
F1
N
F2 F3
F4
F5
F6
F7
X
N1 N2
N3 N4 X1 X2 X3 X4
X5
X6
X7
X8
X9
X10 X11 X12 X13
X14 X15 X16 X17 X18 MS
1
F
F
F
F
F
F
F
F
F
S
S
S
S
1
0.91
0.04
0.71*
0.09
0.96
0.29
1
0.96 0.04
0.91
0.09
0.20
0.80*
1*
1
1
0.28
0.31
0.15
F-S-C-0
C-0
F-S-C-0
F
F-S-0
F
F-S-0
S-0
S-0
F-0
S-C-0
S
C-0
S-0
C
0.88 0.12
0.92
0.80
0.15
1
0.95
595
0.20
0.06
0.18
0.05
1*
1
0.50
0.50
1
1
1
0.44
0.56*
1
1*
1
1
1
1
1
1
1
1
1
1
0.20
F-S-0
F
F-S-0
S-0
S-0
S
S
F
S
S
S
0.37 F-S-C-0
S
0.04
1
0.86
0.36
0.75
F-C
F
F-S
F-C
F-S
S
C
F
C
1
1
1 rDNA sequences were deposited in GenBank
under accession numbers AJ15486–97. ITS-1 rDNA
sequences available in GenBank under accession
numbers AJ509068–74 were included in the
analyses.
DATA
ANALYSIS
Sequences were aligned using CLUSTALX, version
1.8 (Thompson et al., 1997) with default settings. This
alignment was checked for ‘unstable hence unreliable’
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
596
S. GEENEN ET AL.
alignment blocks with SOAP, version 1.2 alpha 4 (Löytynoja & Milinkovitch, 2001). Numbers of polymorphic
and parsimony informative sites were calculated in
DnaSP, version 4.00 (Rozas et al., 2003), whereas haplotype 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 substitution model for phylogenetic analysis. An index of
substitution saturation (Iss; Xia et al., 2003) was calculated and a plot of transitions and transversions
against genetic distance was made to check for saturation in the data in DAMBE, version 4.0.97 (Xia &
Xie, 2001). The program TREE.EXE in the ‘Random
cladistics’ package (Siddall, 1995) was used to test for
the presence of phylogenetic signal using the g1 statistic (Hillis & Huelsenbeck, 1992). Likelihood mapping in TREE-PUZZLE (Strimmer & von Haeseler,
1996; Strimmer, Goldman & von Haeseler, 1997) was
applied to assess to what extent internal branches are
supported and to estimate the phylogenetic content of
the sequence alignment, using quartet trees.
Phylogenetic trees were inferred using the substitution model suggested by MODELTEST and were constructed with several methods. A Neighbour-joining
(NJ) tree (Saitou & Nei, 1987) was calculated with
MEGA, version 2.1 (Kumar et al., 2001). Maximum
parsimony (MP) and maximum likelihood (ML) trees
were made in PAUP*, version 4.0b10 (Swofford, 1998)
using a heuristic search with the tree-bisectionreconnection branch-swapping algorithm and random
addition of taxa. ML trees were also constructed
with TREE-PUZZLE, version 5.0 (Strimmer & von
Haeseler, 1996; Strimmer et al., 1997). Branch support was assessed via nonparametric bootstrapping of
1000 bootstrap replicates (Felsenstein, 1985). Only
bootstrap values higher than 70% were considered as
meaningful (Hillis & Huelsenbeck, 1992). Branch support was also evaluated using Bayesian posterior
probabilities (Larget & Simon, 1999) applied to phylogenetic trees inferred by the program MRBAYES,
version 2.01 (Huelsenbeck & Ronquist, 2001). Bayesian analyses were launched with random starting
trees and run for 107 generations, sampling Markov
chains at intervals of 100 generations using a generaltime-reversible model of evolution as imposed by
MRBAYES (nst = 6) with gamma distributed rate variation (Γ = 0.23) and base frequencies estimated from
the data. To explore tree and parameter space more
thoroughly, four incrementally heated Markov chains
(using default heating values) were used. Stationarity
of the Markov chain was determined as the point
when sampled log-likelihood values plotted against
generation time reached a stable mean equilibrium
value. Sample points generated before this point were
discarded as ‘burn-in’ samples. The log-likelihood val-
ues for sampled trees stabilized after approximately
40 000 generations. Therefore, a burn-in value of 400
was used, implying that trees sampled from generation ’40 100’ onwards, were used to estimate Bayesian
posterior probabilities. To ensure that analyses were
not trapped in local optima, three independent replicates were run and inspected (Huelsenbeck & Bollback, 2001). If ≥95% of the sampled trees contained a
given clade, it was considered to be significantly supported by the data (Ranker et al., 2003; and references
therein). All tree reconstructions were made with a
combined outgroup comprising Arion franciscoloi
Boato, Bodon & Giusti, 1983, Arion hortensis Férussac, 1819 and Arion distinctus Mabille, 1868. For the
Bayesian trees, all the outgroup species were included
in the trees; however, each of these taxa had to be used
separately as an outgroup.
The assumption of a molecular clock was tested with
a likelihood ratio test (LRT) in TREE-PUZZLE, version 5.0 (Strimmer & von Haeseler, 1996; Strimmer
et al., 1997), but was rejected (LRT = 353.73, d.f. = 48
and P < 0.0001). Therefore no estimations of divergence times were made. Sequence divergences (Nei,
1987) within and between the three nominal morphospecies were calculated employing the p-distance
in MEGA, version 2.1 (Kumar et al., 2001).
To further evaluate the allozyme based genetic
impoverishment of the North American Carinarion
spp. (Geenen et al., 2003), nucleotide diversities (π)
(Nei, 1987) within the three nominal morphospecies in
Europe and North America were calculated based on
the p-distance in MEGA, version 2.1 (Kumar et al.,
2001) and compared for the two geographical regions.
A haplotype network was constructed using statistical parsimony (Templeton, Crandall & Sing, 1992) in
the program TCS 1.13 (Clement, Posada & Crandall,
2000). Indels were treated as a fifth character state
and ambiguous loops were resolved by applying the
topology criterion (Pfenninger & Posada, 2002) before
implementing a nested clade analysis (NCA). The
nesting of clades was performed using the rules set out
in Templeton, Boerwinkle & Sing (1987), Templeton &
Sing (1993), and Crandall (1996). Nonrandom geographical associations among haplotypes and clades
were tested statistically with a nested contingency
analysis (Templeton & Sing, 1993; Templeton, Routman & Phillips, 1995). The program GEODIS, version
2.0 (Posada, Crandall & Templeton, 2000) was used to
calculate the various NCA distance measures and
their statistical significance (based on 1000 random
permutations of the contingency data table for each
clade). 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 processes
responsible for all clades that showed significant geo-
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
graphical relationships as indicated from the contingency analysis.
RESULTS
SEQUENCE DATA OF THE MITOCHONDRIAL 16S RDNA
FRAGMENT
The 16S rDNA fragment yielded 50 haplotypes, whose
sequence lengths ranged from 391–395 bp. Sequences
were AT biased (AT/GC proportion: 0.64/0.36) and contained 13 indels, 94 polymorphic sites, and 89 parsimony informative sites. These figures raised to 142
polymorphic and 96 parsimony informative sites when
the combined outgroup was included. There were no
unstable alignment blocks, and all sites were used in
further analyses.
PHYLOGENETIC
INFERENCES BASED ON THE
16S
RDNA FRAGMENT
The most suitable substitution model, according to
MODELTEST, 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 invariable sites = 0; and gamma distribution shape
parameter = 0.2317.
Plotting numbers of transitions and transversions
against genetic distance showed that transversions
outnumber transitions for genetic distances greater
than 0.1654. This may be indicative of saturation but
it appears that the 16S rDNA data set contains significant phylogenetic signal because (1) neither the
numbers of transitions, nor the numbers of transversions reached a plateau; (2) the index of substitution
saturation (Iss = 0.184) was significantly lower
(P < 0.0001) than the critical index of substitution saturation Iss.c for symmetrical trees (IssSym = 0.790) or
for asymmetrical trees (IssAsym = 0.756); (3) the g1
value (−1.72) is far below the critical value (−0.10)
given by Hillis & Huelsenbeck (1992); and (4) likelihood mapping showed that most of the quartet trees
(75%) were concentrated in the three corners of the
triangle.
Although the overall topologies of the different trees
were similar (Fig. 1), branch support values sometimes differed. All trees showed four monophyletic
haplotype 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 unresolved
branches were joined at the same level and referred
to as the X group (18 haplotypes). This arbitrary
grouping was suggested by the clustering of the X
597
haplotypes in the haplotype network (Fig. 2D). The F,
S and C groups only comprised haplotypes of morphologically typical A. fasciatus, A. silvaticus, and
A. circumscriptus, respectively. The X group included
specimens of the three nominal morphospecies and
specimens whose morphological identification was
ambiguous. At least four X haplotypes were shared by
the three nominal species, whereas another seven X
haplotypes were shared by two nominal species.
Hence, in total, 22% of the haplotypes were shared
between species. Individuals of the N group were
either A. silvaticus or A. fasciatus (Fig. 1). The monophyly of Carinarion was supported in all trees, except
in the Bayesian trees (Fig. 1B). The F group was consistently supported in all trees, whereas the S, C, and
N groups were supported by the NJ, MP and the
TREE-PUZZLE ML trees. The PAUP* ML tree only
supported the groups F, C, and N, whereas the Bayesian trees only supported the F group. None of the
trees consistently resolved and/or supported the relations among the four groups or the relationships
within the groups.
Sequence divergences within and between the three
species, and within and between the five haplotype
groups are given in Table 3. The 16S rDNA suggests a
large (approximately 20%) divergence between F and
all other groups, whereas divergence estimates
amongst the latter were much lower (< 5.5%).
Sequence divergence within a group never exceeded
2.5%, such that differences among haplotypes within
groups are small compared to the sequences differences between groups. By contrast, divergence estimates between A. fasciatus and both A. silvaticus and
A. circumscriptus were much larger than the divergence between A. silvaticus and A. circumscriptus, but
divergences within morphospecies were as large as the
divergence between nominal morphospecies. This is no
surprise since the distances among haplotypes within
species are much larger than within haplotype groups
because individuals with X haplotypes occurred in all
three nominal morphospecies (Fig. 1A, B).
HAPLOTYPE
DISTRIBUTION AND DIVERSITIES
OF THE
16S RDNA
FRAGMENT
Haplotype frequencies per population are given in
Table 2. Haplotypes were considered to be ‘common’
when they occurred in more than 10% of the European populations. Haplotypes C1, C2, S1, and F1
met this threshold and occurred in, respectively,
18%, 13%, 21%, and 24% of the European populations. Forty-seven percent of the European populations contained more than one haplotype with a
maximum of six haplotypes in population U3. In
22% of the European populations, more than one
haplotype group co-occurred. The distribution of the
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
598
S. GEENEN ET AL.
Figure 1. Phylogenetic trees. A, 70% Neighbour-joining bootstrap consensus tree made in MEGA, version 2.1 (Kumar
et 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 different
morphospecies appearing in each haplotype (yellow, A. fasciatus; red, A. silvaticus; blue, A. circumscriptus).
haplotype groups is shown in Figure 3. Group F was
found in the British Islands and from Scandinavia,
central and eastern Europe to the Balkan, but was
absent from Belgium and France. Group S occurred
everywhere except Bulgaria. Group C was also
widely distributed but was not found in Bulgaria,
Romania, Switzerland, or Austria, whereas group N
was confined to southern Germany and northern
Austria. Finally, group X appeared to be restricted to
northern Italy and the Balkan.
In North America, we found six haplotypes, four of
which were common in Europe (i.e. C1, C2, S1, and
F1). The two other haplotypes (C3 and S2) occurred in
7% and 8% of the European populations, respectively.
Interestingly, all six haplotypes were frequent in
Great Britain. Out of eight British populations, C1
occurred in five populations, C2 in seven, C3 in four,
and S1, S2, and F1 in three populations. X and N
haplotypes were not found in North American
populations.
Nucleotide diversities for European individuals
within the three nominal morphospecies and within
the five haplotype groups are given in Table 3 (diagonal). Because the X and N group were absent in North
American populations, nucleotide diversities within
North American A. silvaticus (0.010 ± 0.005) and
A. circumscriptus (0.010 ± 0.004) correspond to the
diversities of the S and C group. This suggests that the
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
D
C
Figure 2. Haplotype networks made with TCS 1.13 computer software (Clement et al., 2000). The topology criterion (Pfenninger & Posada, 2002) was applied to
obtain the most likely connection between two haplotypes before implementing the nested clade design. The nesting of clades was performed using the rules set
out by Templeton et al. (1987), Templeton & Sing (1993), and Crandall (1996). Open circles indicate missing haplotypes. A, network of the S group; B, network of
the C group; C, network of the F group; D, network of the X group. Colours refer to the inferred population history for the network diagrams: green, Contiguous
Range Expansion; pink, Inadequate Geographical Sampling; purple, Inconclusive Outcome; orange, Restricted Gene Flow with Isolation by Distance; blue,
Insufficient Genetic Resolution to Discriminate between Range Expansion/Colonization and Restricted Dispersal/Gene Flow, Sampling Design inadequate to
Discriminate between Isolation by Distance (Short Distance Movements) vs. Long Distance Movements; red, Sampling Design inadequate to Discriminate between
Isolation by Distance (Short Distance Movements) vs. Long Distance Movements.
B
A
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
599
600
S. GEENEN ET AL.
Table 3. Within sequence divergence (diagonal) [equal to nucleotide diversities (π) for European individuals] and between
sequence divergence (below the diagonal) for the three nominal morphospecies and for the five haplotype groups (standard
errors based on 10000 bootstrap replicates in parentheses)
Morphospecies
Arion fasciatus
Arion silvaticus
Arion
circumscriptus
Arion fasciatus (15)
Arion silvaticus (33)
Arion circumscriptus (17)
0.102 (0.010)
0.100 (0.010)
0.097 (0.010)
0.033 (0.005)
0.036 (0.005)
0.028 (0.005)
Haplotype group
F
S
X
C
N
F (7)
S (14)
X (18)
C (7)
N (4)
0.010
0.182
0.170
0.178
0.182
0.018 (0.004)
0.042 (0.008)
0.054 (0.009)
0.010 (0.003)
0.054 (0.010)
0.023 (0.006)
(0.003)
(0.020)
(0.018)
(0.020)
(0.019)
0.011
0.045
0.043
0.053
(0.003)
(0.009)
(0.009)
(0.010)
Numbers of haplotypes are given in parentheses in the first column.
0
GB1
GB2
0
GB3
250 mi
250 km
SE2
SE3
GB5
SE1
PO1
GB4
BE4
BE13
GB6
BE6
GB7
BE5
BE12
GB8
BE14
FR2
BE11
BE9
BE10
BE1
BE2
BE7
BE8
BE16
BE15
BE17
GE1
GE2
GE3
PO3
PO4
PO2
CR1
AU11
AU10
AU3
GE5
AU9
BE3
AU1
SW2
GE4 AU13
SW3
AU12
AU2
AU4
SW1
LI1
AU5
IT7
AU6
IT5
AU7
IT6 SL1
IT4
IT1
AU8
IT2
FR1
IT3
RO6 RO7
RO8
RO10
RO11
RO9
RO2
RO15
RO14
RO4
RO12
RO5
RO3
RO1
BU5 BU4
BU11
BU3 BU9
BU10
BU1
BU6 BU2
BU7
BU8
RO13
Figure 3. Distribution of the five haplotype groups over all populations: , F; , S, , C; , N; , X. Population
abbreviations are given in Table 2.
nominal morphospecies in North America are genetically less diverse than in Europe.
NESTED CLADE ANALYSIS OF THE 16S RDNA FRAGMENT
The haplotypes could not be treated together in a single network. Separate networks for the F, S, C, and X
groups are shown in Figure 2. Most terminal haplotypes were restricted to single populations (29 of the 50
haplotypes only occurred in one population), whereas
the interior nodes involved haplotypes that were more
common and widespread (except for the X group,
where most interior nodes involved missing haplotypes). NCA showed a highly significant association
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
between genetic and geographical distances for the the
four haplotype groups F, C, S, and X. The inferred population history included ‘Restricted Gene Flow with
Isolation by Distance’ for groups C and S, and ‘Contiguous Range Expansion’ for groups F and X (Fig. 2).
THE
NUCLEAR
ITS-1
FRAGMENT
The nuclear ITS-1 fragment was 539–542 bp long. ITS1 sequences available in GenBank were included in the
analyses. The AT/GC proportion was 0.46/0.54. The
fragment comprised seven polymorphic sites when
gaps were included, (three indels) of which only one
was parsimony informative, showing a C for
sequences of group S (N = 5) in position 41 (gaps
excluded), whereas all other sequences had a G,
including A. silvaticus, A. fasciatus, and A. circumscriptus from the X group, as well as representatives of
groups F, C, and N. Again, the three morphospecies did
not appear as separate monophyletic units (Table 4).
DISCUSSION
Previous studies questioned the applicability of the
BSC to the three nominal Carinarion species (Backeljau et al., 1997; Jordaens et al., 2000). However, these
earlier allozyme data were unable to refute the possibility that the three nominal species might comply
with the PSC. The present study now questions the
applicability of this latter species concept to Carinarion as well because the 16S rDNA sequences suggest
that neither of the three nominal species represents a
monophyletic unit. Actually, Carinarion spp. comprises at least 32 different haplotypes grouped in at
least four well-supported clades, plus at least another
18 haplotypes whose relationships remain unresolved
(X group). This latter group consists of specimens of
the three nominal species and includes all shared haplotypes. It is unclear, however, whether these shared
601
haplotypes represent: (1) retained ancestral polymorphisms in the three nominal species; (2) signatures of
recent interspecific hybridization between the three
nominal species; or (3) simple intraspecific variation if
Carinarion is considered as a single species. For the
time being, we presume that either of the latter two
possibilities is more likely than the first because sharing 22% of 16S rDNA haplotypes between two or three
Carinarion species would seem to be an exceptionally
high degree of overlap for these supposedly welldifferentiated species.
The very high sequence divergence between F haplotypes and the other groups (up to approximately
20%) or between A. fasciatus (i.e. F group + several X
haplotypes) and the two other nominal species (up to
approximately 10%) may be indicative of a species
level difference. However, these estimates still fall
within the upper limits of intraspecific stylommatophoran mtDNA sequence divergences (Davison, 2002).
Moreover, in the absence of other data, mere levels of
sequence divergence are an inadequate basis for defining species (Sangster, 2000; Ferguson, 2002). Hence,
the high 16S rDNA sequence divergence between the
F group or A. fasciatus and the other groups or nominal species does not necessarily support an interspecific difference. Actually, all interspecific A. fasciatus
allozyme heterozygotes reported by Jordaens et al.
(2000), precisely belong to the F group (S. Geenen, K.
Jordaens & T. Backeljau, unpubl. data), suggesting
that, despite these high sequence divergence, in field
conditions, F animals may hybridize with animals
from the other groups or nominal species.
By contrast to the 16S rDNA data, the ITS-1
sequences revealed little variation and showed neither
evidence of any consistent grouping, nor of any differentiation between the three nominal species. Several
factors may be responsible for the discrepancy
between both gene trees (Ballard, Chernoff & James,
Table 4. Polymorphic sites for the nuclear ITS-1 rDNA fragment
Position (base pair)
ITS- allele
8–10
41
197
206
234
284
467
Haplotype
group
1
2
3
4
–
–
–
–
G
.
.
.
G
.
.
.
A
.
.
.
–
T
–
–
G
.
.
.
–
–
T
–
F
F
C
F, C, X, N
5
6
7
8
ACG
–
–
–
.
.
.
C
.
.
T
.
.
G
.
.
–
–
–
–
.
C
.
.
–
–
–
–
X
X
F
S
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
Morphospecies
Arion fasciatus
Arion fasciatus
Arion circumscriptus
Arion fasciatus Arion
circumscriptus
Arion silvaticus
Arion silvaticus
Arion silvaticus
Arion fasciatus
Arion silvaticus
602
S. GEENEN ET AL.
2002) and it has been argued that, because the effective population size of mtDNA is one-quarter that of
nDNA, the mtDNA genealogy has a higher probability
of tracking the species tree in fewer generations
(Moore, 1995). Nevertheless, the ITS-1 data may be
taxonomically more suggestive than the 16S rDNA
data because ITS sequences are generally considered
to be highly homogenous within species, but well-differentiated between species (Hillis & Dixon, 1991;
Korte & Armbruster, 2003; Vidigal et al., 2004). Therefore, the extremely low divergence between the ITS-1
sequences in Carinarion and the lack of consistent
ITS-1 differentiation among the three nominal species
both suggest that this complex involves only one common nuclear gene pool. These findings need to be verified by sreening additional genetic markers and by
increasing sample sizes because only 16S rDNA was
studied intensively, whereas ITS-1 was surveyed in
only a few specimens.
The present mtDNA data confirm that North American Carinarion populations are genetically impoverished compared to European populations (six vs. 50
haplotypes, respectively) (Geenen et al., 2003). This
implies that earlier protein electrophoretic data of
North American Carinarion (Chichester, 1967;
McCracken & Selander, 1980) may be not sufficiently
representative to decide about taxonomic issues.
Combining all previous arguments leads to the
tentative conclusion that the three morphologically
defined Carinarion species cannot be maintained as
well-defined biological or phylogenetic species.
Instead, we reinforce our earlier suggestion that the
alleged interspecific differences in their morphology
(colour, size), genital anatomy, and electrophoretic
patterns of AGP and EST, may be misleading as taxonomic markers (Backeljau et al., 1997; Jordaens
et al., 2000, 2001, 2002). Indeed, inasmuch as these
differences are truly consistent (but see Jordaens
et al., 2002), genetically determined and independent
from environmental influences (but see Jordaens
et al., 1999, 2001), they may reflect different allelic fixations due to sustained self-fertilization.
Unfortunately, the current data are not particularly
informative with respect to the phylogeographical history of the breeding systems and different haplotype
groups in Carinarion. NCA identified a pattern of contiguous range expansion in the F and X groups vs.
mostly a pattern of restricted gene flow with isolation
by distance in the S and C groups. Incidentally, these
two patterns coincide with the occurrence of outcrossing and shared mtDNA haplotypes in the F and X
groups vs. the (near complete) lack thereof in the S
and C groups. However, evaluating whether this
observation is meaningful will require a more extensive population and DNA sampling in areas that were
not sufficiently covered in the present analysis.
In conclusion, irrespective of which species concept
is implemented, the present DNA sequence data do
not support the subdivision of Carinarion into the
three currently recognized nominal species. Instead,
Carinarion either represents a single, polymorphic,
gene pool or involves a set (> 3) of phylogenetic species. Currently, we favour the single species interpretation because (1) the 16S rDNA and ITS-1 yielded
different pictures (2); a considerable part of the 16S
rDNA tree remained unresolved; and (3) applying the
PSC using the supposedly fast evolving stylommatophoran mtDNA (Davison, 2002) may uncover a large
number of phylogenetic species, whose heuristic
value may be questionable in the absence of other
diagnostic features. Hence, we regard Carinarion as
a single species-level taxon whose taxonomically
deceiving, correlated phenotypic and genetic
intraspecific variation among MLGs or morphospecies is due to sustained self-fertilization. However,
considering the remaining ambiguity in the interpretation of Carinarion, we think that any nomenclatural change is still premature. Nevertheless,
relieving Carinarion spp. from their interpretation as
three species appears to be the best way to refuel the
interest in this kind of provocative taxonomic complexes and the challenging evolutionary questions
that emanate from them.
ACKNOWLEDGEMENTS
We thank Gary Bernon, Sven Lardon, and Jan Pinceel
for providing part of the material. S.G. held an IWT
scholarship. This research was supported by FWOgrant G.0003.02 and OSTC project MO/36/003 to T.B.
and by RAFO project JORDKKP02 to K.J. The comments of two anonymous referees improved the manuscript considerably.
REFERENCES
Backeljau T, Ahmadyar SZ, Selens M, Van Rompaey J,
Verheyen W. 1987. Comparative electrophoretic analyses of
three European Carinarion species (Mollusca, Pulmonata,
Arionidae). Zoologica Scripta 16: 209–222.
Backeljau T, De Bruyn L, de Wolf H, Jordaens K, van
Dongen S, Winnepenninckx B. 1997. Allozyme diversity
in slugs of the Carinarion complex (Mollusca, Pulmonata).
Heredity 78: 445–451.
Backeljau T, De Winter AJ, Martin R, Rodriguez T, De
Bruyn L. 1994. Genital and allozyme similarity between
Arion urbiae and A. anguloi (Mollusca: Pulmonata). Zoological Journal of the Linnean Society 110: 1–18.
Ballard JWO, Chernoff B, James AC. 2002. Divergence of
mitochondrial DNA is not corroborated by nuclear DNA,
morphology, or behavior in Drosophila simulans. Evolution
56: 527–545.
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
MOLECULAR SYSTEMATICS OF A SLUG SPECIES COMPLEX
Chichester LF. 1967. The zoogeography, ecology and taxonomy of arionid and limacid slugs introduced into northeastern North America. Unpublished PhD Thesis, University of
Connecticut.
Chichester LF, Getz LL. 1969. The zoogeography and ecology of arionid and limacid slugs introduced into northeastern
North America. Malacologia 7: 313–346.
Chichester LF, Getz LL. 1973. The terrestrial slugs of northeastern North America. Sterkiana 51: 11–42.
Clement M, Posada D, Crandall KA. 2000. TCS: a computer
program to estimate gene genealogies. Molecular Ecology 9:
1657–1659.
Crandall KA. 1996. Multiple interspecies transmissions of
human and simian T-cell leukemia/lymphoma virus type I
sequences. Molecular Biology and Evolution 13: 115–131.
Davison A. 2002. Land snails as a model to understand the
role of history and selection in the origins of biodiversity.
Population Ecology 44: 129–136.
Felsenstein J. 1985. Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39: 783–791.
Ferguson JWH. 2002. On the use of genetic divergence for
identifying species. Biological Journal of the Linnean Society
75: 509–516.
Foltz DW, Ochman H, Jones JS, Evangelisti SM,
Selander RK. 1982. Genetic population structure and
breeding 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 electrophoresis in detecting allozyme variation in pulmonate
land slugs. Electrophoresis 24: 622–627.
Hey J. 2001. The mind of the species problem. Trends in Ecology and Evolution 16: 326–329.
Hillis DM, Dixon MT. 1991. Ribosomal DNA − molecular
evolution and phylogenetic inference. Quarterly Review of
Biology 66: 410–453.
Hillis DM, Huelsenbeck JP. 1992. Signal, noise, and
reliability in molecular phylogenetic analyses. Journal of
Heredity 83: 189–195.
Huelsenbeck JP, Bollback JP. 2001. Empirical and hierarchical Bayesian estimation of ancestral states. Systematic
Biology 50: 351–366.
Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian
inference 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 Council
Symposium Proceedings 66: 13–20.
Jordaens K, Geenen S, Reise H, Van Riel P, Verhagen R,
Backeljau T. 2000. Is there a geographical pattern in the
breeding system of a complex of hermaphroditic slugs (Mollusca: Gastropoda: Carinarion)? Heredity 85: 571–579.
Jordaens K, Scheirs J, Verhagen R, Backeljau T. 1998.
Ecogenetic comparison of the selfing terrestrial slugs Arion
silvaticus and A. circumscriptus. Belgium (Mollusca: Pulmonata: Arionidae). Hereditas 129: 27–36.
Jordaens K, van Dongen S, Van Riel P, Geenen S, Verhagen R, Backeljau T. 2002. Multivariate morphometrics
603
of soft body parts in terrestrial slugs: comparison between
two datasets, error assessment and taxonomic implications.
Biological Journal of the Linnean Society 75: 533–542.
Jordaens K, Van Riel P, Geenen S, Verhagen R, Backeljau T. 2001. Food-induced body pigmentation questions the
taxonomic value of colour in the self-fertilizing slug Carinarion 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. and
their effects on taxonomic data analysis (Gastropoda,
Pulmonata, Arionidae). Electrophoresis 20: 473–479.
Kerney M, Cameron R, Jungbluth J. 1983. Die Landschnecken Nord- und Mitteleuropas. Hamburg: Paul Parey.
Korte A, Armbruster GFJ. 2003. Apomorphic and plesiomorphic ITS-I rDNA patterns in morphologically similar snails
(Stylommatophora: Vallonia), with estimates of divergence
time. Journal of Zoology 260: 275–283.
Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA2:
molecular evolutionary genetics analysis software. Bioinformatics 17: 1244–1245.
Larget B, Simon DL. 1999. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees.
Molecular Biology and Evolution 16: 750–759.
Lohmander H. 1937. Über die nordischen Formen von Arion
circumscriptus Johnston. Acta Societatis pro Fauna et Flora
Fennica 60: 90–112.
Löytynoja A, Milinkovitch MC. 2001. SOAP, cleaning
multiple alignments from unstable blocks. Bioinformatics
17: 573–574.
Mayden RL. 1997. A hierarchy of species concepts: the
denouement in the saga of the species problem. In: Claridge
MF, Dawah HA, Wilson MR, eds. Species: the units of biodiversity. London: Chapman & Hall, 381–424.
Mayr E. 1940. Speciation phenomena in birds. American Naturalist 74: 249–278.
McCracken GF, Selander RK. 1980. Self-fertilization and
monogenic strains in natural populations of terrestrial slugs.
Proceedings of the National Academy of Sciences of the
United 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 variation: mitochondrial gene trees versus nuclear-gene trees.
Evolution 49: 718–726.
Nei M. 1972. Genetic distance between populations. The American Naturalist 106: 283–292.
Nei M. 1978. Estimation of average heterozygosity and genetic
distance 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 differences between morphologically similar Penaeid shrimps.
Molecular Marine Biology and Biotechnology 1: 27–34.
Pfenninger M, Posada D. 2002. Phylogeographic history of
the land snail Candidula unifasciata (Helicellinae, Stylommatophora): Fragmentation, corridor migration, and secondary contact. Evolution 56: 1776–1788.
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604
604
S. GEENEN ET AL.
Pinceel J, Jordaens K, Van Houtte N, De Winter AJ,
Backeljau T. 2004. Molecular and morphological data
reveal cryptic taxonomic diversity in the terrestrial slug
complex Arion subfuscus/fuscus in continental Northwest
Europe (Mollusca, Pulmonata, Arionidae). Biological Journal of the Linnean Society 83: 23–38.
Posada D, Crandall KA. 1998. MODELTEST: testing the
model of DNA substitution. Bioinformatics 14: 817–818.
Posada D, Crandall KA, Templeton AR. 2000. GeoDis: a
program for the cladistic nested analysis of the geographical
distribution of genetic haplotypes. Molecular Ecology 9: 487–
488.
Ranker TA, Geiger JMO, Kennedy SC, Smith AR, Haufler
CH, Parris BS. 2003. Molecular phylogenetics and evolution of the endemic Hawaiian genus Adenophorus
(Grammitidaceae). Molecular Phylogenetics and Evolution
26: 337–347.
Rozas J, Sanchez-DeI, Barrio JC, Messeguer X, Rozas R.
2003. DnaSP, DNA polymorphism analyses by the coalescent
and other methods. Bioinformatics 19: 2496–2497.
Saitou N, Nei M. 1987. The Neighbor-joining method − a new
method for reconstructing phylogenetic trees. Molecular
Biology and Evolution 4: 406–425.
Sangster G. 2000. Genetic distance as a test of species boundaries in the citril finch Serinus citrinella: a critique and taxonomic reinterpretation. Ibis 142: 487–490.
Schneider S, Roessli D, Excoffier L. 2000. Arlequin, a
software for population genetic data analysis, Version 2.000.
Geneva: Genetics and Biometry Laboratory, Department of
Anthropology, University of Geneva.
Selander RK, Ochman H. 1983. The genetic structure of populations as illustrated by molluscs. Isozymes: Current Topics
in Biology and Medical Research 10: 93–123.
Siddall M. 1995. Random cladistics, the Ohio edition.
Program and Documentation, Version 4.0. University of
Toronto; program available from http://research.amnh.org/
~siddall/rc.html.
Strimmer K, Goldman N, von Haeseler A. 1997. Bayesian
probabilities and quartet puzzling. Molecular Biology and
Evolution 14: 210–211.
Strimmer K, von Haeseler A. 1996. Quartet puzzling: a
quartet maximum-likelihood method for reconstructing tree
topologies. Molecular Biology and Evolution 13: 964–969.
Swofford DL. 1998. PAUP*: phylogenetic analysis using parsimony (* and other methods), Version 4.0. Sunderland, MA:
Sinauer.
Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial
DNA in humans and chimpanzees. Molecular Biology and
Evolution 10: 512–526.
Templeton AR. 1998. Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history. Molecular Ecology 7: 381–397.
Templeton AR. 2004. Statistical phylogeography: methods of
evaluating and minimizing inference errors. Molecular Ecology 13: 789–809.
Templeton AR, Boerwinkle E, Sing CF. 1987. A cladistic
analysis of phenotypic associations with haplotypes inferred
from restriction endonuclease mapping. I. Basic theory and
an analysis of alcohol dehydrogenase activity in Drosophila.
Genetics 117: 343–351.
Templeton AR, Crandall KA, Sing CF. 1992. A cladistic
analysis of phenotypic associations with haplotypes inferred
from restriction endonuclease mapping and DNA sequence
data. III. Cladogram estimation. Genetics 132: 619–633.
Templeton AR, Routman E, Phillips CA. 1995. Separating
population structure from population history: a cladistic
analysis of the geographical distribution of mitochondrial
DNA haplotypes in the Tiger salamander, Ambystoma tigrinum. Genetics 140: 767–782.
Templeton AR, Sing CF. 1993. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction
endonuclease mapping. IV. Nested analyses with cladogram
uncertainty and recombination. Genetics 134: 659–669.
Thompson JD, Gibson TJ, Plewniak F, Higgins DG. 1997.
The ClustalX windows interface: flexible strategies for multiple 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. Analysis
of the first and second internal transcribed spacer sequences
of the ribosomal DNA in Biomphalaria tenagophila complex
(Mollusca: Planorbidae). Memorias do Instituto Oswaldo
Cruz 99: 153–158.
Waldén HW. 1955. The land Gastropoda of the vicinity of
Stockholm. Arkiv för Zoologi 7: 391–448.
Xia X, Xie Z. 2001. DAMBE: Software package for data analysis in molecular biology and evolution. Journal of Heredity
92: 371–373.
Xia XH, Xie Z, Salemi M, Chen L, Wang Y. 2003. An index
of substitution saturation and its application. Molecular
Phylogenetics and Evolution 26: 1–7.
© 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 589–604