Molecular Phylogenetics and Evolution 155 (2021) 106982

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Repeated hybridization increased diversity in the door snail complex

Charpentieria itala in the Southern Alps
Jie Xu, Bernhard Hausdorf *
Center of Natural History, Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The door snail species complex Charpentieria itala is widely distributed in the Southern Alps and subdivided into
ddRAD several morphologically differentiated subspecies. Thus, it can be used as a model group for understanding
Charpentieria migration and differentiation processes in the Southern Alps. We generated genome-wide double digest Re­
Southern Alps
striction Site Associated DNA (ddRAD) sequencing data for 166 specimens from 36 populations of the door snail
Phylogeography
Hybridization
Charpentieria itala and for 8 specimens of the other three Charpentieria species to reconstruct their evolutionary
Land snails history and phylogeography. Phylogenetic and STRUCTURE analyses based on the ddRAD data indicated that the
repeated separation of the populations in western and eastern groups by the Garda glacier during the glacials was
the process that most strongly shaped the population structure of C. itala. This process may also explain a similar
phylogeographic boundary in many other southern Alpine animal and plant species. Our study revealed that
some populations that resemble Charpentieria stenzii morphologically and ecologically, the ‘stenzioid’ subspecies,
originated by a hybridization event with Charpentieria stenzii. A further hybridization event between stenzioid
populations that survived the glacials in mountain refuges and non-stenzioid populations that probably came into
contact with stenzioid populations as a result of climate warming during an interglacial resulted in the origin of a
hybrid subspecies that is adapted to intermediate altitudes. Our study demonstrated that the origin of new
differentiated taxa by hybridization, is more frequent than previously assumed.

1. Introduction
Climate changes often cause shifts or expansions of distribution areas
of many species that may result in contact between closely related
species that were previously geographically isolated (Hewitt, 2000;
Schmitt, 2007; Taberlet et al., 1998). This is true for the repeated cycles
of climate changes during the Pleistocene, but also for the current
human-induced climate changes. The encounter of closely related spe­
cies may result in hybridization (Chunco, 2014; Hewitt, 2000; Schmitt,
2007; Taberlet et al., 1998). In mountain areas geographical range
changes may further be complicated by altitudinal range shifts. During
the Pleistocene climactic cycles, population groups became frequently
isolated, begun to diverge and came into contact again in a later climatic
cycle. Hybridization of incompletely isolated groups can result in com­
plex population structures and morphological patterns. The evolu­
tionary outcomes of hybridization range from the introgression of genes
for adaptive traits (Abbott et al., 2013; Arnold and Kunte, 2017; Becker
et al., 2013; Hedrick, 2013; Minder and Widmer, 2008) to hybrid
speciation (Abbott et al., 2013; Koch et al., 2016; Mallet, 2007; Mavárez
and Linares, 2008; Nieto Feliner et al., 2017; Schumer et al., 2014, 2018)
or the extinction of one or both parental species (Gómez et al., 2015;
Muhlfeld et al., 2014; Rhymer and Simberloff, 1996; Seehausen et al.,
2008). The frequency of the different outcomes (Buerkle et al., 2003) has
still to be assessed across different taxa, different regions and different
environmental histories to be able to better predict the probable con­
sequences of the current human-induced climate changes.
The few markers that could be screened in non-model organisms
until recently did hardly allow the reconstruction of complex hybridi­
zation processes. The accessibility of genomic data for non-model or­
ganisms by next generation sequencing (Andrews et al., 2016; Franchini
et al., 2017; Peterson et al., 2012) and a plethora of new bioinformatics
tools for analysing admixture (Patterson et al., 2012; Pickrell and
Pritchard, 2012) have opened new possibilities to detect and reconstruct
hybridization and introgression.
The door snail genus Charpentieria includes four species which are
mainly distributed in the Southern Alps and adjacent mountain regions
(Nordsieck, 1963; Welter-Schultes, 2012) and can be used as a model
group for understanding migration and differentiation processes in the

* Corresponding author.
E-mail address: hausdorf@zoologie.uni-hamburg.de (B. Hausdorf).

https://doi.org/10.1016/j.ympev.2020.106982
Received 18 June 2020; Received in revised form 6 August 2020; Accepted 2 October 2020
Available online 13 October 2020
1055-7903/© 2020 Elsevier Inc. All rights reserved.
J. Xu and B. Hausdorf
Southern Alps. Charpentieria itala is the most widespread species that
extends from the Lago Maggiore to the Dolomites (Supplementary
Fig. S1) and which is represented by one subspecies in the Alpes-
Maritimes, the Ligurian Alps and the Northern and Central Apennines.
Charpentieria dyodon is restricted to the western Southern Alps from
Hautes-Alpes in France to Ticino. Charpentieria stenzii is spread from the
Dolomites to the Karawanks and Julian Alps in Slovenia. Charpentieria
ornata occurs from the Southeastern Alps in Austria southwards to
Croatia and in an isolate in the Sudetes.
Beside the four undisputed Charpentieria species there is a complex of
taxa in the Bergamasque, Brescia and Garda Prealps that combine fea­
tures of C. itala and of C. stenzii. Whereas typical C. itala live on humid
rocks covered with vegetation, usually in forests or shaded by bushes,
and can also be found directly on trees, the stenzii-like or “stenzioid”
subspecies, clavata, variscoi, balsamoi, triumplinae, trepida, tiesenhauseni,
and lorinae (Supplementary Fig. S1), usually dwell on vertical surfaces of
more exposed calcareous rocks as C. stenzii does (Nordsieck, 1963).
Charpentieria stenzii and the stenzioid subspecies are characterized by a
more or less protruded aperture with a continuous peristome. This is
presumably an adaptation to life on vertical surfaces which enables
these snails to attach the aperture more tightly to the rock surface while
resting or to carry the shell more easily in a vertical position (Scheel and
Hausdorf, 2012). Because of the similarities with C. stenzii, Käufel
(1928) classified the stenzioid taxa as subspecies of C. stenzii. However,
Nordsieck (1963) found shared characteristics in the genitalia between
the stenzioid subspecies and C. itala and intermediate populations be­
tween them. Thus, he suggested that the stenzioid taxa are not related to
C. stenzii, but classified them as subspecies of C. itala. Furthermore,
Nordsieck (1963) supposed that the stenzioid taxa originated poly­
phyletically from typical C. itala ancestors in adaptation to the open
limestone rock faces.
The stenzioid subspecies, and the non-stenzioid C. itala latestriata and
C. itala albopustulata are distributed in a mosaic pattern in the Berga­
masque, Brescia and Garda Prealps (Nordsieck, 1963; Supplementary
Fig. S1). The stenzioid subspecies occur at higher altitudes almost
exclusively in mountain ranges that were not glaciated during the gla­
cials (Scheel and Hausdorf, 2012). In the western Bergamasque Prealps
they are replaced by the non-stenzioid subspecies C. itala latestriata at
intermediate altitudes and C. itala albopustulata at the lowest altitudinal
levels. Populations attributed to the latter subspecies also populate the
valleys of the Brescia and Garda Prealps, where they are connected with
stenzioid subspecies by transitional populations. Nevertheless, Nord­
sieck (2002, 2007) separated the stenzioid subspecies from C. itala as a
distinct species, Charpentieria clavata, because of sympatric occurrences
of stenzioid and non-stenzioid populations without intermediates in the
western Bergamasque Prealps (Nordsieck, 1963).
The distribution and the differences in morphology and ecology
between stenzioid and non-stenzioid subspecies of Charpentieria itala can
be explained by different hypotheses. The stenzioid subspecies may have
a common origin and may represent relicts of an early colonization wave
that survived the ice ages in isolated mountain refuges within the Alps,
whereas the non-stenzioid subspecies may have been displaced from the
Alps during the glacials and re-colonized the south-alpine valleys only
postglacially (Käufel, 1928). In contrast, Nordsieck (1963) suggested
that the geographically isolated stenzioid subspecies evolved through
parallel adaptation of C. itala populations to life on vertical surfaces of
exposed calcareous rocks. Later, Nordsieck (1984) himself rejected the
hypothesis that the stenzioid subspecies originated by convergent evo­
lution and suggested that their similarity originated from an introgres­
sion with an extinct species.
A previous study of the relationships of southern Alpine C. itala
populations based on mitochondrial DNA sequences and nuclear AFLP
markers confirmed the classification of the stenzioid subspecies in the
C. itala complex and revealed monophyletic groups of western and
eastern stenzioid subspecies, but could not resolve their relationships to
each other and to other C. itala population groups (Scheel and Hausdorf,
2
Molecular Phylogenetics and Evolution 155 (2021) 106982
2012).
In the current study, we used double-digest restriction site associated
DNA (ddRAD) sequencing (Franchini et al., 2017; Peterson et al., 2012)
to generate a genomic dataset for all Charpentieria species and pop­
ulations from the complete range of the C. itala complex in the Southern
Alps to clarify the evolutionary history and phylogeography of the
C. itala complex in the Southern Alps and its relations to other Char­
pentieria species. We used new approaches to detect introgression events
to better understand the roles of parallel adaptation versus adaptive
introgression in the evolution of this species complex.
2. Material and methods
2.1. Sampling
Charpentieria populations in the Southern Alps between Lake Lugano
and the Dolomites were sampled during expeditions in 2009, 2011 and
2017. All samples were stored in 100% isopropanol at − 20 ◦ C. The
populations were classified according to Nordsieck (1963, 2007) and
Nardi (2011). For a comparison of the classifications of Nordsieck
(2007) and the classification proposed in this study see Supplementary
Table S1. We chose 163 specimens from 35 populations of the C. itala
complex from the Southern Alps (Fig. 1). Additionally, three specimens
from an introduced population in Weinheim in Germany, the type lo­
cality of C. itala braunii, were investigated. As outgroups, specimens of
the three other Charpentieria species, C. ornata, C. stenzii and C. dyodon,
and of representatives of three further genera of the Delimini, Delima
laevissima, Papillifera papillaris and Siciliaria grohmanniana, were
included. The morphological classification and collection data of the
specimens used in this study are compiled in Supplementary Table S1.
2.2. DNA extraction, ddRAD library preparation and sequencing
Total genomic DNA was extracted from tissue samples of the foot
following the protocol proposed by Sokolov (2000) with slight modifi­
cations as detailed in Scheel and Hausdorf (2012). DNA concentrations
were measured using the Qubit v2.0 fluorometer (Life Technologies,
Carlsbad, CA, USA) with the dsDNA HS assay kit (Thermo Fisher Sci­
entific, Waltham, MA, USA).
For library preparation, we used the modified double-digest restric­
tion-site-associated DNA (quaddRAD) sequencing protocol proposed by
Franchini et al. (2017) with slight modifications. In brief, 200 ng
genomic DNA were digested with the restriction enzymes FastDigest PstI
and MspI (Thermo Fisher Scientific) and ligated to adaptors with inner
barcodes and 4 random bases for the recognition of PCR duplicates in the
same reaction. The products were purified and double size-selected with
0.45x and 0.35x AMPure XP (Beckman Coulter, Brea, USA) beads. Then
equal amounts by weight of twelve samples each with different inner
barcode combinations were pooled. In the following step, each pool of
the digested/ligated DNA is amplified by PCR with Phusion High-
Fidelity Polymerase (Thermo Fisher Scientific, Waltham, MA, USA)
and primers with Illumina indices. After a purification step with 0.8x
AMPure XP, the DNA concentration and fragment length distribution of
each pool was determined on a TapeStation (Agilent Technologies,
Santa Clara, USA) and the amount of DNA of each pool in the range
400–500 bp was calculated. Aliquots of 16 pools with equimolar
amounts of fragments in this size range were pooled into one library.
Finally, fragments in the range 400–500 bp were selected using a Blue
Pippin electrophoresis system (Sage Science, Beverly, USA). Paired-end
next-generation sequencing (2 × 150 bp) was performed at Macrogen
Inc. (Seoul, Korea) in one Illumina HiSeq X lane. Raw RAD-seq data of
this study have been deposited in the European Nucleotide Archive
(ENA) at EMBL-EBI under accession number PRJEB41328, using the
data brokerage service of the German Federation for Biological Data
(GFBio, Diepenbroek et al., 2014).
J. Xu and B. Hausdorf Molecular Phylogenetics and Evolution 155 (2021) 106982

Fig. 1. Sampled Charpentieria itala populations in the Southern Alps and number of private SNPs (restricted to one sampled population)/ mean number of individuals
genotyped per locus (pSNPs/mN). Red ellipse: approximate ancestral area based on populations with high numbers of private SNPs. Encircled numbers refer to
localities listed in Supplementary Table S1, where also the classification of the populations is specified. Background map (Geologische Bundesanstalt, 2013) shows
maximum extension of the glaciers at the last glacial maximum. pSNPs/mN was not calculated for population 18, because only one specimen was sampled. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.3. quaddRAD locus assembly
The raw fastq files obtained from the Illumina run were demulti­
plexed by the sequencing provider using the outer dual Illumina indices.
We further processed the data using the STACKS software pipeline, version
2.4 (Catchen et al., 2013). First, we identified and removed PCR dupli­
cates based on the random four-base stretch incorporated into the
adaptors using the clone_filter module (option –inline_inline). If these
four bases at the beginning of each read and the rest of the sequence (six-
base inner barcode and DNA template) of these reads were identical, a
single copy, stripped off the four random bases at the 5′ end of each
paired reads, was retained. The retained reads were then demultiplexed
by the dual inner barcodes using the process_radtags module (option
–inline_inline). Two mismatches were allowed in the barcodes (option
–barcode_dist_1 –barcode_dist_2) and mutated barcodes were rescued
(option -r). Low-quality reads (options -c -q), reads shorter than 120 bp
(options -len_limit) and reads with an average quality score below 15
(option -s) within a sliding window were discarded.
The demultiplexed reads were assembled using the de novo assembly
pipeline of STACKS. We followed the recommendations of Paris et al.
(2017) to determine the most suitable assembly parameters for our
applied to call genotypes with the gstacks program. Loci were selected
that were present in more than 80% of all individuals using the pop­
ulations module (option -R). SNPs with a minimum minor allele fre­
quency of 0.05 (option –min-maf) and a maximum observed
heterozygosity of 0.5 (option –max-obs-het) were called. For the popu­
lation genetic analyses and the Neighbor-net, we used only one random
SNP per locus (option –write-random-snp) to minimize linkage. This
SNP data set can be found in Supplementary Table S2. Summary genetic
diversity statistics for populations of C. itala were calculated with STACKS
based on a dataset including one random biallelic SNP per locus with a
minimum minor allele frequency of 0.01 to record also private alleles, i.
e. alleles restricted to a population.
2.4. Population genetic structure
Individual-based clustering and admixture analyses of the SNP data
were performed with STRUCTURE 2.3.4 (Falush et al., 2007; Pritchard et al.,
2000). To minimize the effects of linkage, we only used one random SNP
per locus. The input files for the STRUCTURE analyses were generated by
STACKS populations module (option –structure). A total of 20 runs with

dataset. As recommended, we required that there were at least 3 iden­
tical reads in an individual sample to create a stack (option -m in
ustacks). Following the r80 rule of Paris et al. (2017), we varied the
number of mismatches allowed between stacks within individuals (op­
tion -M in ustacks) and selected the value that maximized the number of
polymorphic loci found in 80% of the samples. As further recommended
by Paris et al. (2017), we varied the number of mismatches allowed
between stacks between individuals (option -n in cstacks) as n = M ± 1.
Again, we selected the value that maximized the number of polymorphic
loci found in 80% of the samples. The default Marukilow model was
180,000 iterations after a burn-in period of 20,000 iterations for K = 1 to
K = 10 were carried out with the admixture model allowing for corre­
lated allele frequencies between populations. We used the mean prob­
abilities L(K) of the data for each K and the ad hoc quantity ΔK proposed
by Evanno et al. (2005) computed with STRUCTURE HARVESTER (Earl and
vonHoldt, 2012) to estimate the number of clusters. In addition, we
carried out a longer run for the optimal value of K with 800,000 gen­
erations after a burn-in period of 200,000 generations. To assess the
substructure within the primary groups, subsequent analyses were
conducted within each of the primary clusters, using the same param­
eters as described above. The STRUCTURE results were visualized using

3
J. Xu and B. Hausdorf
CLUMPAK (Kopelman et al., 2015).
2.5. Phylogenetic and network analyses
For RAXML, BEAST and TREEMIX analyses, we first generated all sequences
for each sample by the populations module of STACKS (option –fasta-
samples). Then, we computed consensus sequences of the two alleles of
each individual of each locus using a custom script and concatenated the
alignments of the consensus sequences with the program FASCONCAT (Kück
and Meusemann, 2010). The alignment files and phylogenetic trees are
available from TreeBASE (study accession number 26911).
Maximum likelihood (ML) analyses based on the concatenated se­
quences of the ddRAD loci were performed with RAXML version 8.2.12
(Stamatakis, 2014) using the GTR-GAMMA model. Branch support
values were computed by bootstrapping with a stopping criterion that
automatically determines if enough bootstrap replicate searches were
conducted for obtaining stable support values (Pattengale et al., 2010).
We used ASTRAL-III, version 5.6.3 (Zhang et al., 2018) to estimate the
species tree from unrooted gene trees under the multi-species coalescent
model. Gene trees were generated by RAXML with the GTR-GAMMA
model.
We dated the divergences of the major lineages within Charpentieria
using the Bayesian algorithm implemented in BEAST version 2.4.1
(Bouckaert et al., 2014) with the GTR + G model. A linked uncorrelated
relaxed lognormal molecular clock was used for the BEAST analysis (a
strict clock was rejected at a 1% significance level using the test
implemented in MEGA X; Kumar et al., 2018). As tree prior the birth-death
model was chosen and the analysis was run for 10,000,000 generations
with a sampling frequency set to 1000. Tracer v1.6 was used to assess
convergence of runs and 10% of generations were discarded as burn-in.
A maximum clade credibility tree with median node heights was
calculated with TreeAnnotator 2.1.3. According to the dated phylogeny
of the Clausiliidae shown by Uit de Weerd and Gittenberger (2013),
Charpentieria separated from the other Delimini (in our dataset repre­
sented by Delima laevissima, Papillifera papillaris and Siciliaria groh­
manniana) approximately 15 Ma ago. Thus, we calibrated the tree using
a lognormal-distributed prior assuming an age of 15 Ma for the sepa­
ration of Charpentieria from the other Delimini to get a rough time frame
for the evolution of Charpentieria.
A Neighbor-net (Bryant and Moulton, 2004) was constructed based
on shared allele distances (Bowcock et al., 1994) between Charpentieria
itala individuals using SPLITSTREE4 version 4.14.3 (Huson and Bryant,
2006). We calculated the shared allele distances based on the SNP data
using the function ’alleledist’ of PRABCLUS (Hennig and Hausdorf, 2020),
an add-on package for the statistical software R (R Core Team, 2020).
We used TREEMIX version 1.1 (Pickrell and Pritchard, 2012) to infer
the history of population splits and gene flow within Charpentieria,
allowing up to five migration events. The input file was generated by
STACKS populations program (–treemix). This method first constructs a
bifurcating maximum-likelihood tree of populations. It then identifies
populations that are poor fits to the tree model, and models migration
events involving these populations.
3. Results
3.1. Sequencing and SNP calling
We obtained a total of 886 million paired-end sequences with read
length of 151 bp. The raw data consisted of 16 pools (including 12 in­
dividuals each) with unique barcode combination. 5.4–10.2% of the
reads of each pool were identified as PCR duplicates and removed. The
output number of paired-end reads for each individual varied from
792,660 to 11,829,670. Samples with adapter barcode i501 had fewer
output reads then others indicating a quality problem with that adaptor.
After a test run with the de novo assembly pipeline of STACKS, we removed
8 samples due to low read coverage per locus (mean coverage, weighted
4
Molecular Phylogenetics and Evolution 155 (2021) 106982
by the number of samples present at that locus < 10). In addition, one
C. ornata individual was removed because of a high number of missing
loci (twice as much as in the other two C. ornata individuals).
We determined the most suitable parameters for the de novo assem­
bly of loci with STACKS for two datasets. For phylogenetic analyses with
RAXML, ASTRAL-III and BEAST, we generated a dataset including 3 individuals
of Delimini other than Charpentieria, 3 individuals of C. stenzii, 2 of
C. ornata and 3 of C. dyodon as well as 14 individuals of the C. itala
complex representing different subspecies. We optimized the STACKS pa­
rameters for this dataset (m = 3, M = 19, n = 19; see Supplementary
Fig. S2) and compiled 843 loci (in total 214,730 bp) present in 80% of
the individuals. For all other analyses except TREEMIX, we optimized the
STACKS parameters with a dataset including all C. itala samples (m = 3, M
= 7, n = 8; see Supplementary Fig. S2). With these parameters and minor
allele frequency of 0.05 3379 loci (in total 884,090 bp) present in 80% of
the individuals were found. An overview about the amount of sequence
data for the different datasets can be found in Supplementary
Table S3–4. For the calculation of summary genetic diversity statistics
for the populations of the C. itala complex, we set the minimum minor
allele frequency to 0.01 to record also private SNPs (Fig. 1, Supple­
mentary Table S5). The resulting dataset included one random biallelic
SNP each from 3457 loci. For the TREEMIX analyses, a dataset including
one random biallelic SNP each from 2457 loci present in 80% of the
individuals of all Charpentieria individuals was generated with the same
STACKS parameters.
3.2. Population genetic structure
The number of SNPs restricted to a population divided by the number
of scored individuals is highest in some populations from the Brescia,
Garda and western Venetian Prealps (Fig. 1; Supplementary Table S5). It
decreases westwards in the Bergamasque Prealps and eastwards in the
eastern Venetian Prealps. With a few exceptions, e.g. in the Berga­
masque Prealps and south of Meran, it is low in areas that were glaciated
during the last glacial maximum (Fig. 1).
The mean probability L(K) of the data increased from K = 1 to K = 6
(Supplementary Fig. S3A) and the ad hoc quantity ΔK based on the
output of the STRUCTURE analyses of the SNP data had its highest value for
K = 2 (Supplementary Fig. S3B). A longer run with 800,000 generations
after a burn-in period of 200,000 generations with K = 2 essentially
agreed with the shorter runs and indicated a division of the C. itala
complex into western and eastern populations with a border at Lake
Garda (Fig. 2A). Some populations close to Lake Garda showed admix­
ture up to 50%.
To assess the substructure within the two primary groups, subse­
quent analyses were conducted within both primary clusters. The mean
probabilities L(K) of the data increased from K = 1 to K = 4 (Supple­
mentary Fig. S3C, E) and the ΔK had its highest value for K = 2 for both
subgroups (Supplementary Fig. S3D, F). The western populations were
subdivided into stenzioid and non-stenzioid populations (Fig. 2B). The
populations of the western stenzioid subspecies (C. clavata clavata,
C. clavata variscoi and C. clavata balsamoi) showed no admixture with
non-stenzioid populations, whereas the populations of eastern stenzioid
subspecies (C. itala triumplinae, C. itala trepida, C. itala tiessenhauseni, and
C. itala lorinae) are composed of genomic shares of stenzioid as well as
non-stenzioid origin. Some stenzioid shares were also found in the
C. itala latestriata populations from the Bergamasque Prealps and in the
C. itala allatollae population from the Garda Prealps. The eastern pop­
ulation group was subdivided into the easternmost populations classi­
fied as C. itala serravalensis versus the other eastern populations with
some admixture (Fig. 2B).
3.3. Evolutionary history
The maximum-likelihood tree calculated with RAXML based on the
concatenated alignments of 843 loci including all outgroup samples and
J. Xu and B. Hausdorf Molecular Phylogenetics and Evolution 155 (2021) 106982

Austria

22 Switzerland
Weinheim
Germany

24 25 28 30 36

1 4 6 9 10 13 14 16 17 19 23 26 31 34 35

2 3 5 7 8 11 12 15
18 20 21 27 29 32 33

A 50 km

Austria

22 Switzerland
Weinheim
Germany

24 25 28 30 36

1 4l 6s 9 10 13s 14 16s 17 19 23 26 31 34 35

2 3s 5s 7l 8s 11 12s 15s
18 20 21 27 29 32 33

B 50 km

Fig. 2. A. STRUCTURE plots based on 3379 random SNPs (one per locus) of 166 individuals from 36 populations of the Charpentieria itala complex showing their
estimated ancestries in two assumed genetic clusters. B. STRUCTURE plot showing the estimated ancestries in two subclusters within the western group and two
subclusters within the eastern group. s = stenzioid subspecies; l = Charpentieria itala latestriata. Numbers refer to localities listed in Supplementary Table S1, where
also the classification of the populations is specified.

a subset of the C. itala individuals showed that the C. itala complex and
C. dyodon as well as C. stenzii and C. ornata are sister species (Fig. 3).
Within the C. itala complex, the deepest split separated the populations
from west of Lake Garda from those from east of Lake Garda. The
stenzioid subspecies form a clade that is nested in populations classified
as C. itala albopustulata from west of Lake Garda. Also the populations
classified as C. itala latestriata form a clade that is nested in this group.
Among the stenzioid subspecies, the western subspecies, C. clavata
clavata, C. clavata variscoi and C. clavata balsamoi form a clade. Char-
pentieria c. clavata is sister to C. clavata variscoi plus C. clavata balsamoi.
The population of C. clavata variscoi from Val Taléggio (population 5; see
Figs. 1 and 2) are more closely related to C. clavata balsamoi from Ambria
(population 8) than to C. clavata variscoi from Valtorta (population 6). In
the eastern clade, C. itala baldensis from higher altitudes of Mount Baldo
(population 20) is sister to all other populations. Charpentieria i. itala,
C. itala braunii and C. itala rubiginea are not separated in the tree. The

5
J. Xu and B. Hausdorf
Fig. 3. Trees based on sequences of 843 loci of 14 specimens of the Charpentieria itala complex and C. ornata, C. stenzii, C. dyodon, Delima laevissima, Papillifera
papillaris and Siciliaria grohmanniana. Left side: maximum likelihood tree based on concatenated sequences calculated with RAXML with bootstrap support values. Right
side: species tree based on gene trees calculated with ASTRAL-III with posterior probabilities. Encircled numbers refer to populations and colours refer to groups within
the Charpentieria itala complex (see Fig. 1 and Supplementary Table S1).
easternmost populations classified as C. itala serravalensis (populations
31–36) form a clade that is nested in the other eastern populations. The
relationships within the C. itala complex were largely confirmed in a
second maximum-likelihood tree based on the concatenated alignments
of 3379 loci of all individuals of the C. itala complex, rooted according to
the maximum-likelihood tree with the outgroups (Supplementary
Fig. S4).
The coalescent-based species tree estimated with ASTRAL-III based on
842 gene trees including all outgroup samples and a subset of the C. itala
individuals (Fig. 3) agreed largely with maximum likelihood tree.
However, C. itala latestriata was sister to the stenzioid subspecies in the
maximum likelihood tree based on the concatenated sequences, whereas
it was sister to non-stenzioid C. itala albopustulata populations in the
coalescent-based species tree.
The Neighbor-net based on shared allele distances calculated with
3379 SNPs of the individuals of the C. itala complex confirmed the
subdivision of the complex into a western and an eastern group of
populations separated by Lake Garda and the distinctness of the clusters
including the stenzioid populations and the C. itala latestriata pop­
ulations, respectively, within the western group (Supplementary
Fig. S5).
The BEAST analysis based on the concatenated alignment (Fig. 4)
indicated that the divergence between the C. itala complex and
C. dyodon on the one hand and C. stenzii and C. ornata on the other
started at the end of the Middle Miocene (11.3 Ma before present, 95%
highest posterior density interval (HPD): 8.4–14.2 Ma) and that the
divergence of each species pair occurred only little later at the beginning
6
Molecular Phylogenetics and Evolution 155 (2021) 106982
of the Late Miocene (C. itala – C. dyodon 10.4 Ma ago, 95% HPD:
7.8–13.4 Ma; C. stenzii – C. ornata 10.3 Ma ago, 95% HPD: 7.6–13.1 Ma).
The western and the eastern lineage of the C. itala complex diverged
probably at the beginning of the Pliocene (5.3 Ma ago; 95% HPD:
3.8–6.9 Ma). The origin of the stenzioid subspecies (c. 3.9 Ma ago; 95%
HPD: 2.8–5.0 Ma) also predated the Pleistocene.
Also the population tree calculated with TREEMIX (Fig. 5) based on one
random biallelic SNP each from 2457 loci with C. stenzii and C. ornata as
outgroups confirmed the relationships of the major groups within the
C. itala complex inferred with the other approaches. Charpentieria itala
latestriata is nested in the non-stenzioid C. itala albopustulata pop­
ulations. However, the TREEMIX analysis showed in addition gene flow
between C. stenzii and the clade including the stenzioid subspecies. It
indicated also several mixture events within the C. itala complex. The
migration edge with the highest weight goes from the stenzioid
C. clavata variscoi plus C. clavata balsamoi clade to the C. itala late-striata
group indicating that C. itala latestriata originated by hybridization be­
tween this stenzioid lineage and C. itala albopustulata. Furthermore, the
Lugano population (population 1) received admixture from an older
lineage, and there was gene flow from a population from the northern
Val di Non (population 24) to a population in the Eisack valley (popu­
lation 30) and from an older lineage to a C. itala serravalensis population
in the Valsugana (population 33).
J. Xu and B. Hausdorf Molecular Phylogenetics and Evolution 155 (2021) 106982

Fig. 4. Dated maximum clade credibility tree of Charpentieria based on concatenated sequences of 843 ddRAD loci. Values at the nodes represent median ages of
nodes in million years (Ma). The arrow indicates the node used for calibration. Stars at the branches indicate posterior probabilities * > 0.95 and ** >0.99. Numbers
refer to individuals, encircled numbers refer to populations and colours refer to groups within the Charpentieria itala complex (see Fig. 1 and Supplemen­
tary Table S1).

4. Discussion
4.1. Origin and differentiation of Charpentieria itala along the Garda line
A high number of private SNPs per individual, an indicator for the
longstanding of populations, in many of the populations from the
Brescia, Garda and western Venetian Prealps (Fig. 1, red ellipse; Sup­
plementary Table S5) indicated that these regions were part of the
ancestral area of the C. itala complex. This is also corroborated by the
position of populations from these regions close to the root within
C. itala in all phylogenetic analyses (Figs. 3–5; Supplementary Fig. S4).
All phylogenetic analyses (Figs. 3–5; Supplementary Fig. S4) as well
as the STRUCTURE analyses (Fig. 2A) indicated a primary division of the
Alpine populations of the C. itala complex into a western and an eastern
group at Lake Garda. According to the BEAST analysis (Fig. 4), the
divergence between these groups predated the Pleistocene (the highest
posterior density interval of the estimated divergence time does not
overlap with the beginning of the Pleistocene). However, the admixture
between the eastern and the western populations (Fig. 2A) and the
intermingling of eastern and western lineages in the mitochondrial gene
tree (Scheel and Hausdorf, 2012: Fig. 3) demonstrated that gene flow
across the contact zone occurred. Thus, the repeated geographical sep­
aration of these populations by the Garda glacier, which was the glacier
that extended furthest southwards in the central part of the Alps during
the glacials (Fig. 1; Geologische Bundesanstalt, 2013; Penck and
Brückner, 1909; Seguinot et al., 2018; Voges, 1995), was essential for
their continued divergence. During the long glacials western and eastern
population groups could differentiate by genetic drift and gene flow
between adjacent populations resulted in a homogenization within the
population groups separated by the Garda glacier. Only in the shorter
interglacial periods, the retreat of the Garda glacier enabled gene flow
between western and eastern metapopulations.
Similar phylogeographic breaks into western and eastern pop­
ulations in the Southern Alps along the Garda-Etsch-Brenner line have
been found in several other animals and plants (Cornetti et al., 2015;
Haubrich and Schmitt, 2007; Marchesini et al., 2017; Schönswetter
et al., 2002; Thiel-Egenter et al., 2009, 2011; Vernesi et al., 2016). This
line has already been recognized as a border between west and east
Alpine plant species by Kerner (1871) and has sometimes been called the
“Brenner zone” or “Brenner line” (Cornetti et al., 2015; Marchesini et al.,
2017; Thiel-Egenter et al., 2011; Vernesi et al., 2016). However, because
of the essential role of the Garda glacier for separating western and
eastern populations, it is more appropriate to call the boundary the
“Garda line” rather than the “Brenner line”. Some of the species that
show the pattern, like C. itala, do not occur up to the Brenner pass, which
itself is not a barrier.

7
J. Xu and B. Hausdorf
Fig. 5. Tree of 35 Charpentieria populations with five mixture events inferred
with TREEMIX. Migration arrows are coloured according to their weight and
indicate the gene flow direction. Horizontal branch lengths are proportional to
the amount of genetic drift that has occurred on the branch. The scale bar shows
ten times the average standard error of the entries in the sample covariance
matrix. Encircled numbers refer to populations and colours refer to groups
within the Charpentieria itala complex (see Fig. 1 and Supplementary Table S1).
Thiel-Egenter et al. (2011) suggested that this break zone repre­
sented a dispersal barrier for eastern and western genetic lineages of
alpine plants during post-glacial recolonization. They assumed that the
low and open valleys and high elevations above the permanent snowline
in this break zone can be considered as barriers because these areas lack
or only marginally harbour suitable habitats for alpine plants. For
C. itala, however, the valleys do not represent barriers. Charpentieria
itala can be found on the valley bottom as well as on adjacent mountain
slopes. Actually, the species spread through the Etsch and Eisack valley
upwards to Franzensfeste (Nordsieck, 1963) after the retreat of the
glaciers in the Holocene (and probably also during the interglacials). We
suggest that also in most other animal and plant groups the repeated
geographical separation of western and eastern populations during the
longer glacials by the Garda glacier was more important for their dif­
ferentiation than the lack of suitable habitats in the contact zone during
the shorter interglacials.
4.2. Increase of diversity by repeated hybridizations
Although hybridization between species was known for a long time,
it has often been supposed that it usually does not result in introgression
(Mayr, 1942, 1963, 1992). However, there is increasing evidence for
introgression and its evolutionary importance (Abbott et al., 2013;
Arnold and Kunte, 2017; Hedrick, 2013; Mallet, 2007; Mavárez and
Linares, 2008). Also in Charpentieria the discordant distribution of
characters of the genitalia and the shell in C. itala, C. stenzii and the
8
Molecular Phylogenetics and Evolution 155 (2021) 106982
stenzioid subspecies were first explained by convergent adaptation to
the life on exposed rocks by Nordsieck (1963). However, later Nordsieck
(1984) supposed that the character discordances were caused by hy­
bridization with an extinct species. We could resolve the cause of the
observed discordances with our genomic data and new tools for the
detection of introgression like TREEMIX (Pickrell and Pritchard, 2012).
The maximum-likelihood tree as well as the coalescent-based species
tree (Fig. 3) showed that C. dyodon is the sister species of the C. itala
complex as has been supposed by Nordsieck (2002) based on the simi­
larity of their genitalia, whereas C. stenzii is sister to C. ornata.
The stenzioid subspecies formed a monophyletic group within the
C. itala complex in all phylogenetic analyses (Figs. 3–5; Supplementary
Fig. S4). Thus, we can reject the hypothesis that the stenzioid subspecies
evolved through parallel adaptation of C. itala populations to life on
vertical surfaces of exposed calcareous rocks as supposed by Nordsieck
(1963). Instead, the TREEMIX analysis of the ddRAD data indicated
introgression from C. stenzii to the stenzioid subspecies (Fig. 5). This
introgression of C. stenzii genes is most likely the cause for the
morphological and ecological similarities between the stenzioid sub­
species and C. stenzii. Currently, there are no populations of C. stenzii in
the region occupied by the stenzioid populations. The geographically
next population of C. stenzii can be found approximately 50 km towards
the northeast of the easternmost stenzioid populations. The origin of the
stenzioid subspecies probably predated the Pleistocene (Fig. 4). We
suggest that propagules of C. stenzii that spread further west before the
Pleistocene were lost by the hybridization with C. itala populations that
resulted in the origin of the stenzioid populations. Nordsieck (1984) was
the first to suggest that introgression was involved in the origin of the
stenzioid subspecies. However, our TREEMIX analysis (Fig. 5) showed that
his assumption that an unknown, extinct species was involved is not
necessary but that introgression from C. stenzii can explain the similar­
ities of the stenzioid populations with C. stenzii best.
The ability of the stenzioid subspecies to live at exposed rocks with a
sparse cover of lichens, similar as C. stenzii, enabled the stenzioid sub­
species to colonize higher altitudes than the non-stenzioid populations
and to survive the glacials in unglaciated mountain areas, “massifs de
refuge” (Holdhaus, 1954; Stehlik, 2000; Tribsch, 2004; Tribsch and
Schönswetter, 2003). They are still geographically restricted to regions
that remained unglaciated during the glacials (Scheel and Hausdorf,
2012). Their occurrences indicate the locations of mountain refuges. In
contrast, most non-stenzioid populations probably survived the glacials
at lower altitudes at the margin of the Alps as suggested by Käufel
(1928).
The hybridization between C. itala and C. stenzii that resulted in the
origin of the stenzioid subspecies was not the only hybridization event
that increased the genetic and morphological diversity within the C. itala
complex. The stenzioid subspecies had a common origin, but their
evolutionary trajectories were different. The STRUCTURE analyses for the
western group of C. itala populations (Fig. 2B) showed that the western
subspecies with stenzioid morphology, C. clavata clavata, C. clavata
variscoi and C. clavata balsamoi, are genetically homogeneous, whereas
the eastern subspecies with stenzioid morphology have a high propor­
tion of ancestry in non-stenzioid populations. This is in accordance with
the fact that there are few morphologically intermediate specimens in
the west, whereas the eastern stenzioid subspecies are connected with
non-stenzioid populations by a range of morphologically transitional
populations (e.g. the populations named C. itala allatollae; see Nord­
sieck, 1963). In the west, stenzioid and non-stenzioid populations even
co-occur at a few sites on the same rocks without mixing (Nordsieck,
1963). One hypothesis to explain this is that the western stenzioid
subspecies had more time to differentiate from C. itala than the eastern
ones. The eastern stenzioid subspecies are distributed in the west of the
approximate ancestral area of C. itala in the Brescia, Garda and western
Venetian Prealps (see above; Fig. 1). Thus, they probably came earlier
and more often in contact with non-stenzioid populations. These con­
tacts resulted in admixture and counteracted the build-up of isolation
J. Xu and B. Hausdorf
mechanisms. In contrast, the western stenzioid subspecies are far from
the ancestral area of C. itala and could differentiate for a longer time
without contact to non-stenzioid populations. The continuous admixture
between neighbouring populations of stenzioid and non-stenzioid pop­
ulations in the east was not shown in the TREEMIX analysis (Fig. 5) as an
admixture event, but may be the reason for the placement of the eastern
stenzioid populations at the base of the stenzioid clade, close to the
neighbouring non-stenzioid populations.
TREEMIX revealed an additional hybridization event between the
stenzioid C. clavata variscoi/balsamoi lineage and neighbouring non-
stenzioid populations (Fig. 5). Probably during an early interglacial
the C. clavata variscoi/balsamoi populations came into contact with non-
stenzioid populations that colonized the valleys after the retreat of the
glaciers and hybridized with them. This was the origin of C. itala lates­
triata that was classified as a non-stenzioid subspecies so far. The
contribution of the stenzioid populations to the genome of C. itala late-
striata is also evident from the STRUCTURE analyses (Fig. 2B). C. itala
latestriata occurs at intermediate altitudes. It became probably isolated
from the stenzioid subspecies by the advancing glaciers in the next
glacial. How it was isolated from other non-stenzioid populations is less
clear. One possibility is that no other non-stenzioid populations
remained in this region and that C. itala albopustulata, which is found at
lower altitudes in that region now, re-colonized this region only later.
Our analyses revealed two instances in the C. itala complex in which
hybridization and introgression contributed to the evolution of new
differentiated taxa. The finding of an introgression from C. stenzii into
C. itala and the change of morphological and ecological characteristics
of exactly the clade that received the introgression towards a phenotype
similar to C. stenzii, makes it very likely that these changes are attrib­
utable to introgressive alleles of C. stenzii. Hybridization does not only
act as an additional source of adaptive genetic variation within pop­
ulations that may contribute more to adaptation than mutation (Abbott
et al., 2013; Arnold and Kunte, 2017; Grant and Grant, 1994; Hedrick,
2013) but the increase in variation contributes essentially to differen­
tiation of population groups and the evolution of new taxa. However, the
hybridization events themselves probably did not led to the emergence
of reproductive isolation, a criterion required in some definitions of
hybrid speciation (Schumer et al., 2014, 2018; but see Nieto Feliner
et al., 2017). The above described contrast in the isolation of western
and eastern stenzioid populations from adjacent non-stenzioid pop­
ulations either indicates that reproductive isolation between stenzioid
and non-stenzioid populations evolved only in the western stenzioid
populations after the hybridization or that it was (partly) associated
with the hybridization, but broke down in the eastern stenzioid pop­
ulations. The extent of gene flow between the hybrid subspecies C. itala
latestriata and the neighbouring C. itala albopustulata is unclear. Despite
there is no evidence that the hybridization directly led to the emergence
of reproductive isolation, hybridization contributed to an increase of
biodiversity in both cases. Depending on the perspective, this may be
considered more relevant for an evaluation of the importance of hy­
bridization than the question whether a hybridization event directly
resulted in reproductive isolation, i.e. hybrid speciation in the strict
sense.
4.3. Delimitation of species and subspecies
Not surprisingly, the delimitation of species and subspecies in a
group that has undergone repeated cycles of isolation, secondary contact
and hybridization poses problems. Nordsieck (1963) suggested classi­
fying the stenzioid populations as subspecies despite he was aware that
some of them co-occur locally with non-stenzioid populations without
mixing. Later, Nordsieck (2002, 2007) changed his mind and separated
the stenzioid subspecies from C. itala as a distinct species, C. clavata.
However, this necessitates an arbitrary subdivision of the continuous
series of intermediate populations in the Brescia and Garda Prealps.
Thus, Scheel and Hausdorf (2012) preferred to maintain the
9
Molecular Phylogenetics and Evolution 155 (2021) 106982
classification of the stenzioid taxa as subspecies of C. itala. None of the
two classifications reflects both, the sympatric co-occurrences of sten­
zioid and non-stenzioid populations without fusing in the Bergamasque
Prealps and the continuous intergradation of stenzioid and non-
stenzioid populations in the Brescia and Garda Prealps.
Our study demonstrated that the stenzioid taxa had a common origin
(Figs. 3–5; Supplementary Fig. S4), but the STRUCTURE analyses (Fig. 2B)
also showed that the trajectories of the stenzioid populations in the
Bergamasque Prealps and the Brescia and Garda Prealps were different.
The stenzioid taxa from the Bergamasque Prealps themselves show no
admixture with neighbouring non-stenzioid populations (Fig. 2B),
although they locally co-occur with them. Accordingly, we suggest
classifying them as a separate species C. clavata including the subspecies
C. c. clavata, C. clavata variscoi and C. clavata balsamoi (for a comparison
of the classification proposed by Nordsieck (2007) and the classification
proposed here see Supplementary Table S1). C. clavata variscoi proved to
be paraphyletic with regard to C. clavata balsamoi. Nevertheless, the
separation of these subspecies may be maintained because they are
morphologically well differentiated and are distributed in disjunct
mountain regions separated by the Val Brembana so that they will
probably continue to diverge.
In accordance with the presence of morphologically intermediate
populations between stenzioid and non-stenzioid populations in the
Brescia and Garda Prealps, the STRUCTURE analyses (Fig. 2B) have shown
that the eastern stenzioid populations share a large part of the genepool
with non-stenzioid C. itala. In contrast, there is no evidence for recent
gene flow between the stenzioid populations in the Bergamasque Prealps
and in the Brescia and Garda Prealps in accordance with the large
geographical gap between their ranges. Consequently, we suggest clas­
sifying the eastern stenzioid populations as a subspecies of C. itala (see
Supplementary Table S1). The stenzioid populations from the Brescia
Prealps are genetically not separated from those of the Garda Prealps,
although they were separated by the Chiese glacier during the glacials.
This is in agreement with the fact that the populations from the Brescia
Prealps do not share morphological characteristics that distinguish them
from those of the Garda Prealps (Nordsieck, 1963). On both sides of the
Chiese valley ribbed populations (C. i. tiesenhauseni in the Garda Prealps
and C. i. trepida in the Brescia Prealps) as well as populations with more
or less obsolete ribbing (C. i. lorinae in the Garda Prealps and C. i. tri­
umplinae in the Brescia Prealps) occur. Thus, we follow Nordsieck (1963)
in including all stenzioid populations from the Brescia and Garda Pre­
alps in C. i. lorinae. Later, Nordsieck (2007) and Nardi (2011) recognized
the mentioned named local forms and even some populations that are
intermediate between stenzioid and non-stenzioid populations (C. i.
allatollae) as distinct subspecies. This splitting obscures the close re­
lationships and minor genetic differentiation of the stenzioid pop­
ulations from the Brescia and Garda Prealps.
The morphologically distinct non-stenzioid populations from the
Bergamasque Prealps that originated by a hybridization event with
C. clavata (see above) were classified as C. itala latestriata by Nordsieck
(1963, 2007). Given the unique origin and morphologically distinctness
of these populations, classifying them as a distinct subspecies might be
acceptable. However, their relations to neighbouring non-stenzioid
populations should be investigated in more detail.
The other non-stenzioid populations belonging to the western group
of C. itala largely corresponds to C. itala albopustulata as delimited by
Nordsieck (1963). However, Nordsieck (1963) included also some
populations from east of Lake Garda as well as populations considered
transitional forms between C. itala albopustulata and C. itala rubiginea by
him, but later classified as separate subspecies C. itala baldensis and
C. itala braunii by Nordsieck (2007), in C. itala albopustulata. These
populations were placed in the eastern group in the phylogenetic and
admixture analyses (Figs. 2–5, Supplementary Fig. S4). Later, Nordsieck
(2007) classified these taxa as separate subspecies. However, the ranges
of C. itala braunii and C. itala rubiginea as given by Nordsieck (1963)
overlap (Supplementary Fig. S1) and the alleged characteristics like
J. Xu and B. Hausdorf
shell size and shape, ribbing of the upper whorls and continuous peri­
stome vary continuously and show no clear geographical pattern. The
molecular phylogenies also showed that the populations that were
classified as C. itala braunii and C. itala rubiginea do not form coherent
units (Figs. 3–5; Supplementary Fig. S4). Rather, these populations form
a morphologically diverse paraphyletic group from which the popula­
tion groups classified as C. itala itala and C. itala serravalensis by Nord­
sieck (1963) are derived. Monte Baldo was isolated by the Garda und the
Etsch glacier during the glacials, but remained free of ice. The high
number of private alleles found in the population on Monte Baldo
(population no. 20; Fig. 1) indicated that the C. itala population survived
the glacials in this mountain refuge. The Monte Baldo population rep­
resents the sister group to the remaining populations of the eastern
group of C. itala. Nordsieck (1963) considered it as a transitional form
between C. itala albopustulata and C. itala rubiginea. Actually, it shows
admixture with C. itala albopustulata (Fig. 2A). Although similar small
morphotypes can also be found in other regions, Nordsieck (2007)
classified C. itala baldensis as a subspecies. The populations from the
foothills of the Alps (Monte Berici and Colli Euganei) represent the
nominotypical C. itala itala. They are somewhat variable. Generally,
they do not differ from C. itala albopustulata except in the on average
larger shell, but some of the populations resemble C. itala serravalensis.
The populations from the Valsugana eastwards were classified as
C. itala serravalensis by Nordsieck (1963), but differ morphologically
hardly from some of the neighbouring populations classified as C. itala
itala and C. itala rubiginea. These populations form a monophyletic group
in the phylogenetic trees (Fig. 5; Supplementary Fig. S4) and are
recognized as a distinct cluster in the STRUCTURE analyses (Fig. 2B) of the
eastern C. itala subgroup with some admixture with the neighbouring
cluster in the lower Valsugana and elsewhere.
We propose to subsume all C. itala populations east of the Garda zone
into C. itala itala (see Supplementary Table S1). The alternative would be
to divide these populations into a morphologically and genetically
highly variable western group (C. itala itala) and a morphologically and
genetically uniform eastern group (C. itala serravalensis). The low
numbers of private alleles in the eastern group (populations no. 31–36;
Fig. 1; Supplementary Table S5) showed that these populations do not
represent a group that evolved separately for a long time, but that their
homogeneity is the result of a recent colonization of the eastern region.
Moreover, they are connected with the western group by morphologi­
cally intermediate populations. Thus, we prefer not to separate these
populations as a separate subspecies from the other C. i. itala
populations.
Author contribution
B.H. conceptualized the study; B.H. and J.X. collected the samples; J.
X. performed the laboratory analyses; J.X. and B.H. analysed the data; B.
H. and J.X. wrote the manuscript.
Acknowledgements
We are grateful to K.-L. Nägele, H. Nordsieck and F. Walther for
samples, P. Franchini for information about the quaddRAD protocol, D.
Granse for help with flow cytometry and S. Bamberger for comments on
the manuscript. This work was funded by the Land­
esforschungsförderung Hamburg and benefitted from the sharing of
expertise within the DFG priority program SPP 1991 Taxon-Omics and
support from DFG HA 2763/6-1.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ympev.2020.106982.
10
Molecular Phylogenetics and Evolution 155 (2021) 106982
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