Journal of
The Malacological Society of London
Molluscan Studies
Journal of Molluscan Studies (2013) 79: 241– 248. doi:10.1093/mollus/eyt017
Advance Access publication date: 14 May 2013
LOCAL ADAPTATION, REFUGIAL ISOLATION AND SECONDARY
CONTACT OF ALPINE POPULATIONS OF THE LAND SNAIL
ARIANTA ARBUSTORUM
MARTIN HAASE 1, SUSANNE ESCH 2 AND BERNHARD MISOF 2
1
Vogelwarte, Zoologisches Institut und Museum, Universität Greifswald, Soldmannstraße 23, D-17489 Greifswald, Germany; and
2
Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany
Correspondence: Martin Haase; e-mail: [email protected]
(Received 16 August 2012; accepted 3 April 2013)
ABSTRACT
Depressed-shelled populations of the land snail Arianta arbustorum occur in rocky Alpine habitats,
whereas globular shell forms are ubiquitous in lowland habitats of central Europe. It has been proposed
that Alpine, steep, rocky habitats are the ancestral habitat and depressed-shelled snails the ancestral
form of A. arbustorum. According to this hypothesis, the globular shell form evolved when A. arbustorum
adapted to damp lowland habitats during the Pliocene and depressed-shelled forms survived the
Pleistocene glaciations in isolated Alpine habitats like nunataks throughout the Alps. Alternatively, the
depressed shells could be a more recent adaptation to steep rocky habitats, derived from the widespread
globular-shelled lowland A. arbustorum. To test these alternatives, we compared globular and depressedshelled morphotypes and their molecular differentiation between two geologically similar massifs in the
Austrian Alps, the Totes Gebirge and the Gesäuse. These massifs are the centre of distribution of the
nominal depressed-shelled subspecies A. a. styriaca and lie about 50 km apart. While the Gesäuse was
at the edge of the Pleistocene ice-cover with numerous ice-free habitats, the Totes Gebirge lay under
glaciers offering only nunataks as possible refugia. Using geometric morphometrics and phylogenetic
analyses based on DNA sequences of COI, we asked (1) if the forms from the Gesäuse and Totes
Gebirge were morphometrically sufficiently similar to support common ancestry; (2) if there was
molecular evidence for common ancestry of shell forms; (3) if there was evidence for in situ survival on
nunataks during the Pleistocene and (4) if the ancestral shell shape of A. arbustorum could be inferred
based on the answers to questions 1–3. The depressed-shelled morphotypes of both massifs were significantly different in shape and size rejecting common ancestry. Additionally, snails with either depressed
or globular shells were not monophyletic based on COI sequences. Lastly, we found entire clades
unique to the Totes Gebirge indicating nunatak survival of snail populations. Our data also suggest
that populations of A. arbustorum experienced recent gene flow between morphotypes and both massifs.
Since the globular shells from the vicinity of Totes Gebirge and Gesäuse are morphologically indistinguishable in contrast to the locally strongly differentiated depressed-shelled morphotypes, we favour –
at least on a local scale – the more parsimonious hypothesis of a globular-shelled ancestor locally
evolving depressed-shelled forms.
INTRODUCTION
Many species of land snails including Arianta arbustorum
(Linnaeus, 1758) inhabit a large range of habitat types and
exhibit a pronounced polymorphism of shell shape, often on a
small geographical scale (e.g. Gould & Woodruff, 1978;
Teshima et al., 2003; Haase et al., 2003; Gittenberger, Piel &
Groenenberg, 2004; Elejalde et al., 2008; Fiorentino,
Manganelli, & Giusti, 2008a; Fiorentino et al., 2008b; Haase &
Misof, 2009; Stankowski, 2011). Such polymorphisms are most
likely adaptive, e.g. for balance on different substrates or for the
ability to withdraw into crevices (Goodfriend, 1986; Heller,
1987; Okajima & Chiba, 2011). It is known that they sometimes
change very rapidly over geological time scales (Stankowski,
2011). Although phenotypic plasticity is known to influence
shell shape in A. arbustorum (Baur, 1984; Burla, 1984), marked
shell shape differences are generally considered to be genetically
controlled (Gittenberger, Piel, & Groenenberg, 2004; the evidence for A. arbustorum is summarized by Haase & Misof 2009).
# The Author 2013. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
M. HAASE ET AL.
Numerous infraspecific taxa of A. arbustorum have been based
on more or less depressed-shelled and globular-shelled populations. Depressed-shelled populations occur in rocky habitats of
isolated massifs of the Pyrenees and in particular within the
Alps, while the globular-shelled nominate form has a wide
range throughout central Europe and southern Scandinavia
(Gittenberger, Piel, & Groenenberg, 2004). Gittenberger
(1991) and Gittenberger, Piel, & Groenenberg (2004) argued
that the ancestor of A. arbustorum was of a depressed-shelled
type and preferred high altitudes, like all other members of the
subfamily Ariantinae except the cylindrical Cylindrus obtusus
that is endemic to the East-Austrian Alps and the depresssedshelled, keeled Helicigona lapicida that is widespread in lowland
habitats. Gittenberger, Piel, & Groenenberg (2004) further
assumed that the globular-shelled morphotype evolved when
ancestral populations adapted to damp lowland habitats
during the Pliocene. They consider the few extant depressedshelled populations scattered throughout the Alps and
Pyrenees as relics that survived the unfavourable conditions in
ice-free, rocky habitats such as nunataks. According to their
hypothesis, newly available grassland habitats up to 3000 m asl
(Ehrmann, 1933) were recolonized by the globular-shelled
form after the retreat of the glaciers. Alternatively, Baminger
(1997) proposed that the depressed-shelled form of the nominal
subspecies A. a. styriaca (Kobelt, 1876) from the eastern Alps
evolved locally when the Pleistocene glaciations drove snail
populations into ice-free habitats including steep, rocky
terrain, a view compatible with the model of rapid shell evolution (Stankowski, 2011).
Previous phylogenetic analyses based on mtDNA markers did
not unambiguously support either hypothesis (Haase et al., 2003;
contra Gittenberger, Piel, & Groenenberg, 2004). Haase & Misof
(2009) focused on the Gesäuse massif where three morphotypes
occur in parapatry (one globular and two subcategories of
depressed, viz. intermediate and flat; see Material and Methods).
They were unable to resolve the direction of shell evolution
despite integrating phylogenetics, population genetics and morphometrics. It could only be shown that shell polymorphism
is most likely maintained by natural selection, despite gene
flow among snails of different shell morphotypes (Haase & Misof,
2009). However, this study did provide evidence for Pleistocene
refugia for the flat-shelled form at the periphery of the Alps.
In the present study, we compared shell morphometrics and
genetic differentiation of populations of the Gesäuse (Haase &
Misof, 2009) with populations of the Totes Gebirge, another
massif approximately 50 km WNW. Both mountain massifs
provide similar habitats, i.e. rough, rocky conditions with steep
faces, the same bedrock consisting of dolomite and limestone,
and they cover the centre of the distribution of A. a. styriaca
(see Klemm, 1974). In contrast to the Gesäuse, the Totes
Gebirge was entirely covered with ice during the last glaciations
except for some nunataks (van Husen, 1987). Both massifs
are among the areas predicted as potential Alpine refugia based
on theoretical considerations (Schönswetter et al., 2005).
Specifically, we asked (1) if the depressed-shelled morphotypes
from the Gesäuse and Totes Gebirge were sufficiently similar to
support common ancestry; (2) if there is molecular evidence for
common ancestry of snails from the Totes Gebirge and the previously identified clades from the Gesäus; (3) if there is evidence
for in situ survival during the Pleistocene, e.g. a high degree
of COI sequence variability in local populations or the presence
of local allele endemism; and (4) if we can infer the direction of
shell shape evolution of A. arbustorum – at least in the area under
consideration – based on the answers to questions 1–3.
MATERIAL AND METHODS
Sampling
Living snails, both adult and juvenile, as well as empty, fully
grown shells recognizable by a reflected apertural lip were collected near and in the Totes Gebirge in August 2004 and 2005
(Fig. 1, Table 1). We collected all snails and shells encountered
in an area estimated to be occupied by a single population
(Baur, 1993). Animals were preserved in 96% ethanol after a
hole had been made in the shell to allow rapid penetration of the
Figure 1. Localities of Arianta arbustorum in and near the Totes Gebirge. Squares and circles indicate globular- and depressed-shelled populations,
respectively. For acronyms, see Table 1. The inset shows Austria and the position of both massifs, the Totes Gebirge and the Gesäuse.
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ALPINE POPULATIONS OF ARIANTA ARBUSTORUM
tpsDig v. 2.0 (Rohlf, 2004b). Statistical analyses were performed
in the IMP suite of programs (http://www3.canisius.edu/
~sheets/morphsoft.html) and with PAST v. 2.0 (Hammer,
Harper & Ryan, 2001). Morphotypes were compared in
principal component and canonical variates analyses (PCA,
CVA). For pairwise comparisons, we used Goodall’s F-test.
Transformations of shapes were studied by thin-plate splines.
Allocation of samples to morphotypes was based on the mean
spire index (height– width ratio). Following Haase & Misof
(2009), we subdivided the category ‘depressed shells’ into intermediate and flat. Flat-shelled samples had a spire index ,0.62;
intermediate-shelled samples .0.62 and ,0.69, and globular
samples had an index .0.70. As pointed out previously (Haase
& Misof, 2009), assigning individual shells to a certain morphotype may seem ambiguous considering the apparent continuous
variation, but populations can fairly well be characterized by
simple descriptive statistics (Kothbauer et al., 1991; Bisenberger,
1993; Baminger, 1997).
Table 1. Localities of Arianta arbustorum in and near Totes Gebirge.
Acronym
ATE
Locality
Ascent towards Elm
Latitude,
Elevation
longitude
(m)
Ng
Ns
47.677178N,
1785
2
0
660
2
4
1900
16
0
2000
3
0
1517
13
9
1720
12
4
1588
1
9
1638
43
18
2000
21
1
2000
21
10
1750
9
7
2070
12
7
2125
1
2
720
1
24
1850
19
3
1950
1
0
1135
0
2
13.962808E
BDA
Bad Aussee
47.610568N,
13.783068E
ELM
Descent from Elm
47.676858N,
13.964278E
HBR
Hochbrett
47.695158N,
13.953258E
LAH
Lahngangsee
47.668338N,
13.924448E
NRL
Near Rollsattel
47.692788N,
13.976118E
OGA
Obere Gößler Alm
47.660568N,
13.912228E
PHT
Pühringer Hütte
47.687798N,
RGS
Rotgschirr
47.692738N,
RKS
Rotkogelsattel
47.690008N,
Sequencing and phylogenetic analyses
13.967598E
DNA isolation, PCR and sequencing of a 663-bp fragment of
COI on a Beckman Coulter CEQ 8000 sequencer followed
Haase & Misof (2009). The final alignment comprised 630 bp
and contained 77 unique haplotypes from the Totes Gebirge
(GenBank accession numbers JX025653-JX025729), 48 haplotypes from the Gesäuse and two outgroup sequences (Helicigona
lapicida EF398130, Chilostoma achates EF398132). Phylogenetic
trees were reconstructed by maximum likelihood (ML) and
Bayesian inference (BI). The best-fitting substitution model was
estimated using jModeltest v. 0.1 (Posada, 2008) allowing all 11
substitution schemes, variable base frequencies and substitution
rates as well as invariant sites. According to the Akaike information criterion corrected for small sample sizes, HKY þ G was
selected. ML was conducted using Phyml v. 3.0 (Guindon &
Gascuel, 2003), and BI using MrBayes v. 3.1.2 (Ronquist &
Huelsenbeck, 2003). The ML analysis was based on a BioNJ
starting tree and rearrangements were made by nearestneighbour interchange (NNI) or subtree-pruning and regrafting
(‘best of’). Node support was calculated as bootstrap (BS)
values from 500 replicates and by an approximate
likelihood-ratio test (aLRT) (Anisimova & Gascuel, 2006). The
BI was conducted for 20 million generations with a conservative
burnin of 25% of all sampled trees. Every 100th tree was
sampled and the posterior distribution summarized by a 50%
majority-rule consensus tree. Tree space was searched in two
parallel runs with eight chains each, one cold and seven heated.
Convergence of parameter estimates was monitored using the
statistics provided by MrBayes as well as the program Tracer
v. 1.5 (Rambaut & Drummond, 2009). The average standard
deviation of split frequencies of the parallel runs remained below
0.01 after roughly 17.5 million generations and all effective
sample sizes were .200. Measures of node support were compared using Spearman’s rank correlations.
14.00028E
13.991948E
RLS
Rollsattel
47.694728N,
13.980568E
SOF
Salzofen
47.680498N,
13,938228E
SUE
Summit of Elm
47.673678N,
13.962638E
TOP
Toplitzsee
47.642788N,
13.915568E
TRK
Ascent towards
Rotkogelsattel
TTR
Track to Rotgschirr
47.687998N,
13.918558E
47.695008N,
13.990838E
UGA
Untere Gößler Alm
47.651398N,
13.906948E
Ng, number of genetically analysed specimens (includes juveniles); Ns,
number of measured adult shells (includes empty shells). BDA, LAH, OGA,
TOP and UGA are globular-shelled populations. All other populations are
depressed (intermediate) shelled.
alcohol. A total of 177 snails were available for genetic analyses
and 100 fully grown shells for morphometrics. For the morphological comparisons with individuals from the Gesäuse, we used
all the adult shells used by Haase & Misof (2009) except those of
A. a. picea (a total of 359). For the phylogenetic analyses, we
selected 48 haplotypes representing all clades found in the foregoing study restricted to the Gesäuse.
RESULTS
Morphometrics
Morphometrics
Adult shells were photographed by MH in apertural view using
a Nikon D-70s camera equipped with an AF Nikkor 28 –
105 mm lens (Nikon Corp., Tokyo, Japan), all at the same scale.
We did not use the pictures of the Gesäuse shells taken for our
previous investigation, because they were taken with a different
camera. Analyses were conducted in the framework of geometric
morphometrics (Zelditch et al., 2004) in a similar way to Haase
& Misof (2009) except that we now placed 11 landmarks
(Fig. 2), again using the programs tpsUtil (Rohlf, 2004a) and
Among the populations from the Totes Gebirge, mean spire
index was never below 0.62, the threshold for inclusion in the category ‘flat’. Most populations exhibited an intermediate index,
while only the samples collected at a lower elevation than PHT
(1638 m), namely BDA (660 m), LAH (1517 m), OGA
(1588 m), TOP (720 m) and UGA (1135 m) had globular shells
(Fig. 1, Table 1). As in the foregoing account (Haase & Misof,
2009), morphotypes from the Gesäuse were continuously
arranged with individuals from flat-shelled populations lying
243
M. HAASE ET AL.
Figure 2. Plot of canonical variates analysis based on 11 landmarks on shells of Arianta arbustorum (shown on the globular shell from the Gesäuse).
Phylogenetic analysis
almost entirely to the left of the second CVA axis and those of
globular-shelled populations almost entirely to the right (a plot
of a PCA not requiring a priori designation of individuals to
groups as in a CVA looked practically identical). However, while
the globular-shelled morphotype from the Totes Gebirge
could be conceived as a subset of that from the Gesäuse, shells of
populations with intermediate spire index from the Totes
Gebirge filled the space between the flat- and globular-shelled
populations from the Gesäuse rather than overlapping with their
intermediate-shelled counterparts from the Gesäuse (Fig. 2).
Statistically, individuals of the intermediate-shelled morphotype
from the Totes Gebirge differed significantly from both the
intermediate- and flat-shelled morphotypes from the Gesäuse
(Goodall’s F-tests, P , 0.001 in both cases) and also differed from
the globular-shelled morphotype collected in and near the Totes
Gebirge (P , 0.001) (Fig. 3). Flat shells from the Gesäuse had
both a lower spire and particularly lower last whorl than intermediate shells from the Totes Gebirge, both contributing to the
lower spire index of the former (Fig. 3A). Transformations of
globular to intermediate shells from the Totes Gebirge (Fig. 3B)
and flat shells from the Gesäuse (Fig. 3C), respectively, are different. In the Totes Gebirge, the spire has become flatter and narrower while the last whorl has largely experienced an extension
in width (or the other way round). In the Gesäuse the spire has
only become flatter and the last whorl both flatter and wider (or
the other way round). Centroid sizes used as proxies for overall
size differed significantly across the five groups (Kruskal–Wallis
test, H ¼ 78.53, P , 0.001). Shells from the Totes Gebirge had
the larger median values (Fig. 4). In ten pairwise comparisons
(Mann–Whitney U-tests) only globular and intermediate morphotypes from both massifs and globular shells from the Gesäuse
and intermediate ones from the Totes Gebirge could not be
distinguished in terms of size.
Alphanumeric labels of terminal clades previously identified in
the Gesäuse were adopted from Haase & Misof (2009) and new
labels added to new clades. Terminal and more inclusive clades
also received numeric labels. The topologies of BI and ML analyses were identical, except for the arrangement of the subclades
of clade 6 (Fig. 5). In the ML tree, clade G was sister group to
(H, I). Haplotypes from the Gesäuse and the Totes Gebirge
were not reciprocally monophyletic. The large clades (A, B)/4
and C/10 from the Gesäuse had clades 5 and 11, respectively,
from the Totes Gebirge as sister groups. Most terminal clades
consisted of haplotypes of one geographic origin (Fig. 5). Only
clades D/12, I/6d and B were of mixed origin, the latter containing a single individual from the Totes Gebirge. Similar to the
Gesäuse (Haase & Misof, 2009), morphotypes were not monophyletic in the Totes Gebirge (Fig. 5). However, clades 5 and 11
as well as all individuals from the Totes Gebirge of clade I/6d
were from the Pühringer Hütte (1638 m asl) and higher elevations. Only clade D/12 contained samples from the lowlands,
the ascent and from the mountains as well. The only populations
not represented in clade D/12 were SOF and TTR. Of the
latter, however, we sequenced only a single individual.
Support was generally high for all clades descending from
node 2. The deep nodes 2 and 7 received rather low support, like
most clades descending from the latter. In general, BS values
(scaled to %) were, as expected, considerably lower than posterior probabilities (PP) or paLRT. The difference between pp and
paLRT was .0.05 at six nodes (37.5%). The highest discrepancies were found at nodes 10 (PP ¼ 0.54, paLRT ¼ 0.90) and
5 (PP ¼ 0.93, paLRT ¼ 0.64). All three measures were significantly correlated (Spearman’s rank correlation, P , 0.001 in all
pairwise comparisons); i.e. their relative performances were
similar. The highest accordance with rs of 0.808 was found
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ALPINE POPULATIONS OF ARIANTA ARBUSTORUM
Figure 4. Box plots of centroid size used as proxy for overall shell size of
Arianta arbustorum. Ges, Gesäuse; glob, globular; inter, intermediate; Tot,
Totes Gebirge.
spire index. The multivariate comparisons based on landmarks
clearly distinguished the depressed-shelled morphotype from the
Totes Gebirge from the depressed-shelled morphotypes of the
Gesäuse. These observations indicate that the spire index is not a
good proxy for shell shape, which does not come as a surprise.
There is obviously more than one possibility for the coiling of a
shell within the rather vague framework of a fixed proportion of
height to width.
We show that in both massifs shell shape changed along different trajectories from globular to flat or intermediate or the other
way round. The fact that the globular-shelled morphotype is
widely distributed and not significantly different between
studied areas suggests a common ancestry, so it is parsimonious
to assume local, independent transformations of a globularshelled and widespread morphotype to locally differentiated
depressed-shelled morphotypes (Fig. 2). Haase & Misof (2009)
have already suggested that the intermediate-shelled morphotype
in the Gesäuse is not a transitory or hybrid stage between
globular- and depressed-shelled forms, but does have an independent evolutionary origin as well.
Our interpretation is compatible with the fossil record,
because the earliest fossils of Arianta arbustorum had globular
shells and date back to the late Pliocene (Wenz, 1923; Ložek,
1964). Transitions from one morphotype to the other may therefore have occurred already long before the last ice age.
Our interpretation is in obvious contradiction to the conclusions of Gittenberger, Piel, & Groenenberg (2004). Of course,
our conclusions can in the first place only hold for the area
under consideration. Isolated, depressed-shelled populations
occur across wide parts of the Alps and Pyrenees (Gittenberger,
Piel, & Groenenberg, 2004). Considering that the sampling
design of Gittenberger, Piel, & Groenenberg (2004) covered the
entire distribution range of A. arbustorum, it is well possible that
changes in shell shape occurred repeatedly in either direction.
The geographically fine-scaled approach of Haase & Misof
(2009) and the present account indicate that the evolutionary
and demographic history of A. arbustorum is more complex.
Our molecular analysis indicates that all globular- and some
depressed-shelled individuals from the area of the Totes Gebirge
belong to haplotype clade D, in which globular-shelled
Figure 3. Thin-plate splines illustrating transformations between means
of two morphotypes of Arianta arbustorum. A. Intermediate shells from
Totes Gebirge to flat shells from Gesäuse. B. Globular shells from Totes
Gebirge to intermediate shells from Totes Gebirge. C. Globular shells
from Gesäuse to flat shells from Gesäuse. Exaggeration ¼ 2 in all three
splines.
between PP and BS, followed by PP and paLRT (rs ¼ 0.752)
and BS and paLRT (rs ¼ 0.742).
DISCUSSION
Multiple origin of depressed-shelled morphotypes
All populations from the Totes Gebirge inhabiting rocky conditions (PHT and above, see Table 1, Fig. 1) had an intermediate
245
M. HAASE ET AL.
Figure 5. Phylogenetic tree for sampled Arianta arbustorum. Fifty per cent majority-rule consensus tree summarizing posterior distribution of BI with
node support expressed as posterior probabilities (BI PP) as well as probabilities from approximate likelihood ratio test (ML aLRT) and bootstrap
values (ML BS) resulting from ML analysis. Alphanumeric labels of clades were taken from Haase & Misof (2009). Lower case acronyms denote
localities in the Gesäuse (Haase & Misof 2009), upper case acronyms represent samples from the Totes Gebirge (see Table 1). The colour code is
identical to that in Figure 2 with orange and pink identifying intermediate- and globular-shelled samples from the Totes Gebirge, and red, blue and
green standing for flat-, intermediate- and globular-shelled populations in the Gesäuse, respectively. In addition, black labels identify individuals not
used in morphometrics due to small sample sizes. Note that the individuals from the Gesäuse were not selected in proportion to morphotypes
represented in the original clades of Haase & Misof (2009).
246
ALPINE POPULATIONS OF ARIANTA ARBUSTORUM
individuals predominantly of lowland habitats of the Gesäuse
are also present (see Haase & Misof, 2009). Other haplotype
clades containing individuals from the Totes Gebirge consist of
exclusively depressed-shelled individuals from populations
above 1630 m asl. The Gesäuse is an area east of the Totes
Gebirge, which was at the periphery of the former Pleistocene
Alpine glaciers and was not entirely covered with ice during the
last glaciation (van Husen, 1987; Haase & Misof, 2009). In contrast, the lowlands at the Totes Gebirge were entirely glaciated
during the last glaciation period (van Husen, 1987) and it is thus
very likely that they were recolonized by globular-shelled
A. arbustorum populations from the east. If the lowlands of the
Totes Gebirge were recolonized by autochthonous, originally
depressed-shelled, Alpine populations we would expect additional haplotypes of these populations within the lowland
population of the Totes Gebirge. The mixed composition of
globular- and depressed-shelled morphotypes within haplotype
clade D is most plausibly explained by uphill migration. A
similar interpretation can be put forward for the haplotype
clade 11, only represented by individuals of depressed-shelled
morphotype of the Totes Gebirge. This clade is sistergroup to a
haplotype clade dominated by the globular-shelled morphotype
of the Gesäuse (Fig. 5; Haase & Misof, 2009). Our tree reconstruction implies that the globular-shelled morphotype was the
ancestral condition also in this case.
Together the distribution of morphotypes and haplotypes in
the Totes Gebirge and Gesäuse is puzzling, but best compatible
with the hypothesis of a multiple local origin of several
depressed-shelled morphotypes derived from globular-shelled
populations, coupled with the presence of flat- to intermediateshelled relic forms in refugia of the Totes Gebirge and Gesäuse.
Finally, if the shell shapes in Gesäuse and Totes Gebirge are not
identical and are probably of separate origin, there is no longer
a justification for them to be classified together in the infraspecific taxon A. a. styriaca (see also Kothbauer et al., 1991).
Connectivity
The mixed composition of clades B, D and I suggests several
events of migration between the Gesäuse and the Totes Gebirge
in the more recent past. The presence of a single haplotype from
the Totes Gebirge in specimens of clade B, which is dominated
by depressed-shelled individuals, even suggests that at least one
snail has been directly transported from a trench in the Gesäuse
to the Totes Gebirge without a ‘stop over’ in between. Potential
vectors might be birds such as the common raven Corvus corax
(Haffer, 1993; Baumgartner, personal communication).
CONCLUSIONS
Our analyses of shell shapes of Arianta arbustorum have demonstrated that a proxy of shape such as the spire index may be an
oversimplification, wrongly suggesting similarity or even identity
of shape. Using geometric morphometrics based on 11 landmarks we were able to show that different shapes may be realized
within a certain range of the spire index. Different populations
of A. arbustorum separated by only 50 km have apparently
adapted to similar mountainous, rocky conditions in different
ways. This results in a much more complex picture of shell
evolution than that suggested by Gittenberger (1991) and
Gittenberger, Piel, & Groenenberg (2004), who assumed a flatshelled ancestor occurring throughout the Alps and Pyrenees.
Since the globular shells from the vicinity of Totes Gebirge
and Gesäuse are practically inseparable, our analyses favour
Baminger’s (1997) hypothesis of a globular-shelled ancestor
locally evolving flat forms. Considering our spatially restricted
analysis, a scenario of repeated, independent changes in shape
in either direction cannot be excluded.
The evidence for northern and even Alpine refugia is still
growing and their importance for the preservation of morphological and genetic diversity through adverse climatic conditions
is evident. Yet the locations of actual refugia and their extent are
far from being identified in their entirety. The Totes Gebirge is
among the areas predicted as potential refuges based on theoretical considerations (Schönswetter et al., 2005). However, evidence has been lacking. Studying the genetic and morphological
differentiation of A. arbustorum, we here provide evidence for
Alpine refugia within the Totes Gebirge.
The presence of refugia
An identifiable refuge is characterized by the presence of rare or
unique haplotypes in comparison with recolonized areas. In the
Totes Gebirge haplotypes of the entire clades 5 and 11 and all
haplotypes belonging to clade 6d occurred only at high elevations and only clade D consisted of haplotypes that were also
found in the lowlands. This suggests that haplotypes of clades
5, 11 and 6d have endured the Pleistocene glaciations in Alpine
refugia of the Totes Gebirge. Indeed, practically all peaks and
ridges of the Totes Gebirge were probably ice free, i.e. nunataks,
during the Würm glaciation (van Husen, 1987). One of our localities, SOF, carries a very thick layer of Dryas octopetala suggesting continuous existence and growth for many thousands of
years (de Witte et al., 2012). This locality is thus a plausible
refuge for A. arbustorum. This suggestion is supported by our
phylogenetic analysis, with individuals from SOF appearing at
the base of clade 5, paraphyletic to only a few snails from other
sites. Haase & Misof (2009) have identified peripheral Alpine
refugia in the Gesäuse. Here we provide the first evidence for
inner Alpine ice-age refugia for a land snail. This evidence is not
absolutely compelling, because there were no samples available
from potential source populations for post-Pleistocene recolonization from north of the Totes Gebirge. Pleistocene survival on
Alpine nunataks has been known for a few species of plants
(Stehlik et al., 2002; Bettin et al., 2007; Parisod & Besnard, 2007;
Garcı́a et al., 2012); however, among animals we are only aware
of the carabid genus Trechus and the bristletail Machilis pallida
having partly persisted during glacial periods on ice-free sites
within the glacial cover of the southern Alps (Lohse, Nicholls, &
Stoner, 2011; Wachter et al., 2012).
ACKNOWLEDGEMENTS
The material was collected during two excursions into the Alps
in August 2004 and 2005. We thank all participants for their
collecting efforts, in particular Joe Dambach for his enormous
patience in supervising students. The presentation of complex
issues has been improved based on the reviewers’ comments.
Financial support was received from the German Science
Foundation (MI 649/5-1). The Alexander Koenig-Gesellschaft
provided additional support to SE for finishing her Diploma
thesis.
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