Journal of
The Malacological Society of London
Molluscan Studies
Journal of Molluscan Studies (2016) 82: 305– 313. doi:10.1093/mollus/eyv062
Advance Access publication date: 15 December 2015
Radiation of Grossuana Radoman, 1973 (Caenogastropoda: Truncatelloidea)
in the Balkans
Andrzej Falniowski1, Dilian Georgiev2, Artur Osikowski3,4 and Sebastian Hofman3
1
Department of Malacology, Institute of Zoology, Jagiellonian University, Gronostajowa 9, Krako´w 30-387, Poland;
Department of Ecology and Environmental Conservation, University of Plovdiv, Tzar Assen Str. 24, Plovdiv BG-4000, Bulgaria;
3
Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, Gronostajowa 9, Krako´w 30-387, Poland; and
4
Department of Animal Anatomy, Institute of Veterinary Science, University of Agriculture in Krakow, Al. Mickiewicza 24/28, Krako´w 30-059, Poland
2
Correspondence: A. Falniowski; e-mail: [email protected]
(Received 10 July 2015; accepted 6 November 2015)
ABSTRACT
The aims of the study were to infer phylogeographic relationships between the populations of the
minute, dioecious, spring-inhabiting snail Grossuana from the Balkans, and to interpret the resulting
pattern in the context of geological history of the region. The cytochrome c oxidase subunit I gene was
sequenced from 23 previously unstudied populations of Grossuana from Bulgaria and analysed together
with published sequences from the other populations of Grossuana from the Balkans. In Bulgaria, six
clades or putative clades (lacking statistical support) were identified. Within the clades the p-distances
were in the range 0.2–0.9% and between the clades 1.6 –3.4%. Among all 33 studied populations, 42
haplotypes were found (haplotype diversity ¼ 0.955; nucleotide diversity p ¼ 0.059). All of the haplotypes from Bulgaria and Romania formed a clade that was distinct from all of the Serbian and Greek
haplotypes. At the estimated divergence time of 3.60 + 0.58 ma a sea connection between the
Pannonian Sea and Aegean Sea (at the site of the present Velika Morava Valley) formed a dispersal
barrier for these freshwater snails. The nucleotide diversity within the Bulgarian/Romanian lineage was
lower ( p ¼ 0.019, 41 polymorphic sites) that within the Serbian/Greek group (p ¼ 0.049, 70 polymorphic sites), perhaps as a result of bottlenecks during the Pleistocene glaciations. Within the
Bulgarian populations, all of the diversity originated in the Pleistocene, during the Calabrian (estimated time 1.26 –1.42 ma). During the Pleistocene, the unstable system of rivers and lakes in southwestern Bulgaria, with glaciers in the Pirin and Rila Mountains, probably resulted in the extinction of
Grossuana in SW Bulgaria. Subsequently, this territory was likely recolonized from eastern Bulgarian
populations.
INTRODUCTION
The rich fauna of the Balkans, including hydrobioid gastropods,
has been considered as a product of the complicated geological
history of the region, which has served as a refugium during glaciations and been subjected to numerous sea-level fluctuations
(Creutzburg, 1963; Kougioumoutzis, Simaiakis & Tiniakou, 2014).
Against this background we have studied phylogeographic diversification of the minute, hydrobioid spring snail Grossuana and
here attempt to interpret the observed pattern in the context of
the complex geological history of the region.
The freshwater truncatelloideans may have appeared in the
Early Carboniferous (Kabat & Hershler, 1993) and they are therefore suitable models for the evaluation of old (pre-Pleistocene)
biogeographic relationships. The distributions of freshwater
Truncatelloidea have been interpreted in terms of Neogene
drainage patterns and subsequent fragmentation by changes in
climate and landscape. In particular, spring snails have been
discussed in this way (Falniowski & Szarowska, 2011a), as assuming that the springs inhabited by those obligatorily aquatic
animals are stable habitats and that gene flow among the springs
is very low. There have been many studies on the phylogeny,
population genetic structure and gene flow of the spring fauna, including gastropods (e.g. Colgan & Ponder, 1994; Ponder, Eggler
& Colgan, 1995; Bilton, Freeland & Okamura, 2001; Finston &
Johnson, 2004; Hershler & Liu, 2004a, b; Falniowski, Szarowska
& Sirbu, 2009; Osikowski et al., 2015). Most of the studies point to
low levels of gene flow and high levels of endemism in spring
snails (e.g. Colgan & Ponder, 1994; Ponder et al., 1995; Finston &
Johnson, 2004), although some of the species are rather widespread, with much gene flow among their populations (Falniowski
et al., 1998; Falniowski, Mazan & Szarowska, 1999; Hershler,
Mulvey & Liu, 2005).
The truncatelloidean fauna of the Balkan Peninsula includes
many taxa with a simplified shell lacking characteristic traits, a
penis with a more or less prominent double or single lobe on the
# The Author 2015. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
A. FALNIOWSKI ET AL.
left edge and female reproductive organs with a loop of the
oviduct, bursa copulatrix and two seminal receptacles. Such
morphological characters have long been used for the descriptions of new species belonging to this group. However, molecular
studies have revealed that anatomical traits do not provide sufficient evidence to establish phylogenetic relationships between
higher taxa or to distinguish closely related species (Falniowski
et al., 2012).
Grossu (1946) described a new species of minute hydrobioid
gastropod, Paladilhiopsis codreanui, from a spring at Techirghiol
Lake in Romania, although first collected near Balčic in Bulgaria
(Grossu, 1986). Radoman (1966) described a new species of
Pseudamnicola vurliana from Kamena Vurla in Greece. Subsequently,
Radoman (1973) described a new genus, Grossuana, for which
the type species was the newly described G. serbica. He assigned
both P. codreanui and P. vurliana to the genus Grossuana and
described three additional new species of the genus, which were
later (Radoman, 1983) considered to be subspecies of G. serbica.
Grossuana ranges from Serbia through Macedonia to northern
Greece, Bulgaria and southeastern Romania (Radoman, 1985)
(Fig. 1). Szarowska et al. (2007) added two Greek species to this
genus, G. haesitans (Westerlund, 1881) from the spring of the
Louros River and G. delphica (Radoman, 1973) from the spring
at Delphi. In a broader study, Falniowski et al. (2012) found
that the distribution of Grossuana is disjunct, with part of the distribution covering areas of Serbia, Bulgaria and Romania, while
another covers northeastern Greece. The latter includes the
spring of Achilles northwest of Lamia, two springs on the Volos
peninsula (Pilion Mt.) and one on Evvoia Island [these three
identified as G. marginata (Westerlund, 1881)] and the spring of
Athena in the Thembi Valley [G. hohenackeri (Küster, 1853)]
(Fig. 1). Falniowski et al. (2012) demonstrated that using morphological data alone (the shell as well as the reproductive organs)
it was not possible either to determine or distinguish species or to
separate representatives of Grossuana and Radomaniola Szarowska,
2006, despite the fact that the genera are not sister taxa
(Szarowska, 2006).
Additional species of Grossuana have been described more recently. Considering shell characters as well as the penis, Glöer &
Georgiev (2009) described G. angeltsekovi Glöer & Georgiev, 2009
(from springs in the West Rhodopes Mts and the lower slopes
of the Pirin Mts in the Mesta River Valley) and G. thracica Glöer
& Georgiev, 2009 (from Chirpan Bunar spring, in the Upper
Thracian Lowland, southern Bulgaria). Georgiev (2012) described
G. aytosensis Georgiev, 2012 (from a water source near Aytos,
eastern Stara Planina Mts) and G. radostinae Georgiev, 2012 (from
a stream near Madara, northeastern Bulgaria). Georgiev & Glöer
(2013) described two additional species, G. slavyanica Georgiev
& Glöer, 2013 (from Slavyanka Mts., southwest Bulgaria) and
G. derventica Georgiev & Glöer, 2013 (from Dervent Heights, southeastern Bulgaria) and Georgiev et al. (2015) described G. falniowskii
Georgiev et al., 2015 (from spring of the Bedechka River,
Krayrechen Park, Stara Zagora, central Bulgaria).
The aim of this study was to infer, using the mitochondrial
cytochrome c oxidase gene (COI) as a molecular marker, phylogeographic relationships between all the populations of Grossuana
studied so far and to interpret the resulting pattern in the context
of the geological history of the region. It was not our intention to
evaluate the validity of species-level taxa in this genus.
described by D.G. (G. angeltsekovi, G. aytosensis, G. codreanui, G. falniowskii, G. radostinae and G. slavyanica, mostly paratypes) and one
Radomaniola species (R. bulgarica). The snails were collected by
hand or with a sieve. Individuals to be used for molecular analyses were washed in 80% ethanol, in which they were left to
stand for c. 12 h. The ethanol was subsequently changed twice
over 48 h and finally transferred to 96% ethanol after a few days.
Samples were stored at 220 8C prior to DNA extraction. Shells
were photographed under a Nikon SMZ18 stereomicroscope with
dark field illumination using a Canon EOS 50D digital camera.
DNA extraction and sequencing
DNA was extracted from foot tissue using a Sherlock extraction
kit (A&A Biotechnology) and dissolved in 20 ml of tris-EDTA
buffer. Polymerase chain reaction (PCR) was performed in
a reaction mixture with a total volume of 50 ml using the
primers LCOI490 (50 -GGTCAACAAATCATAAAGATATT
GG-30 ) (Folmer et al., 1994) and COR722b (50 -TAAACTTCA
GGGTGACCAAAAAATYA-30 ) (Wilke & Davis, 2000) for
COI. The PCR conditions were as follows: an initial denaturation step of 4 min at 94 8C, followed by 35 cycles at 94 8C for
1 min, 55 8C for 1 min and 72 8C for 2 min, with a final extension of 4 min at 72 8C. A 10 ml sample of the PCR product was
run on a 1% agarose gel to check the quality of the PCR product.
The PCR product was purified using Clean-Up columns (A&A
Biotechnology). The purified PCR product was then sequenced
in both directions using BigDye Terminator v. 3.1 (Applied
Biosystems), following the manufacturer’s protocol and using the
primers indicated above. The products of the sequencing reaction
were purified using ExTerminator Columns (A&A Biotechnology),
and the sequences were read using an ABI Prism sequencer.
Data analysis
Sequences were aligned and edited in Bioedit v. 7.1.3.0 (Hall,
1999). Basic sequence statistics, including haplotype polymorphism and nucleotide divergence, were calculated in DnaSP v. 5.10
(Librado & Rozas, 2009). The saturation test was performed
using DAMBE (Xia, 2013).
In a phylogenetic analysis, 15 other sequences from GenBank
were used as a reference (Table 1) and Daphniola exigua (GenBank
JF916470; Falniowski & Szarowska, 2011b), the type species of
the genus phylogenetically closest to Grossuana, was used as the
outgroup. The data were analysed using approaches based on
Bayesian inference and maximum likelihood (ML). We applied
the GTR þ I þ G model, which is the only nucleotide substitution model implemented in RaxML (Stamatakis, 2014).
The Bayesian analyses were run using MrBayes v. 3.2.3
(Ronquist et al., 2012) with the default priors. Two simultaneous
analyses were performed, each of which lasted 10,000,000 generations, with one cold chain and three heated chains, starting from
random trees and sampling the trees every 1,000 generations. The
first 25% of trees were discarded as burn-in. The analyses were
summarized as a 50% majority-rule tree.
A ML approach was applied in RAxML v. 8.0.24. One thousand searches were initiated with starting trees obtained through
the randomized stepwise addition maximum parsimony method.
The tree with the highest likelihood score was considered as the
best representation of the phylogeny. Bootstrap support was calculated with 1,000 replicates and summarized on the best ML
tree. RAxML analyses were performed using the free computational resource CIPRES Science Gateway (Miller, Pfeiffer &
Schwartz, 2010).
Median-joining calculations, as implemented in NETWORK
v. 4.6.1.1 (Bandelt, Forster & Röhl, 1999), was used to infer the
COI haplotype network. To test the validity of the molecular
clock assumption, the likelihoods for trees with and without the
MATERIAL AND METHODS
Sample collection and fixation
One of us (D.G.) collected Grossuana snails from 23 localities from
throughout Bulgaria (Fig. 1, Table 1) and determined them by
means of morphological characters and their localities. The
samples included representatives of six Grossuana species recently
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Figure 1. Map of all localities sampled in phylogenetic analyses. Localities B1 –B23 are represented by new COI sequences; localities A– J represented
by published sequences (Szarowska et al., 2007; Falniowski et al., 2012). Hatched areas: Grossuana range (after Radoman, 1985). Geographical features
discussed in the text are indicated. See Table 1 for locality details.
molecular clock were calculated with PAUP in a likelihood ratio
test (LRT) (Nei & Kumar, 2000). The relative rate test (RRT)
(Tajima, 1993) was performed in MEGA6 (Tamura et al.,
2013). As Tajima’s RRTs and the LRT test rejected an equal
evolutionary rate throughout the tree for Grossuana, time estimates were calculated using a penalized-likelihood method
(Sanderson, 2002) in r8s v. 1.7 for Linux (Sanderson, 2003). To
calibrate the molecular clock, two hydrobiids, Peringia ulvae
(AF478401) and Salenthydrobia ferreri (AF478410) were used as
outgroups. The divergence time between those two species (proposed by Wilke, 2003, with correction by Falniowski et al.,
2008), callibrated the nodes within Grossuana.
307
A. FALNIOWSKI ET AL.
Table 1. Sampling localities of Grossuana with their geographical coordinates and the haplotypes of the COI gene detected at each locality.
Locality
‘taxon’
B1
Site
Coordinates
Kavarna town, Bulgaria
43824′ 47′′ N
28821′ 04′′ E
HB1A × 2, HB1B
′
Kovach spring, Krepcha village, Bulgaria
43826 58 N
26806′ 51′′ E
HB2A, HB2B × 2
Balchik town, Bulgaria
43813′ 37′′ N
28800′ 08′′ E
HB1A × 4
Aladzha monastery, Bulgaria
43816′ 39′′ N
28801′ 04′′ E
HB1A × 3
Madara village, Bulgaria
43809′ 36′′ N
27803′ 36′′ E
HB1A
B6
Tserovo village, Bulgaria
43800′ 25′′ N
23820′ 33′′ E
HB6 × 2
B7
Iskrec village, Bulgaria
42859′ 46′′ N
23814′ 00′′ E
HB6 × 9
No. of Aytos town, Bulgaria
42842′ 52′′ N
27816′ 09′′ E
HB8A, HB8B, HB8C × 3
B9
Pekeyuka, karst spring, Bulgaria
42847′ 16′′ N
22859′ 26′′ E
HB6 × 2
B10
Bosnek village, water source, Bulgaria
42830′ 11′′ N
23810′ 57′′ E
HB10A × 2, HB10B
B11
Bosnek village, Popov izvor spring, Bulgaria
42830′ 22′′ N
23810′ 36′′ E
HB10A, HB11 × 2
B2
B3
Grossuana codreanui
B4
B5
B8
Grossuana radostinae
Grossuana aytosensis
′′
COI haplotypes
B12
Radomaniola bulgarica
Ostra Mogila village, Bulgaria
42827′ 11′′ N
25828′ 27′′ E
HB12 × 2
B13
Grossuana falniowskii
Stara Zagora city, spring, Bulgaria
42826′ 52′′ N
25838′ 03′′ E
HB13 × 6
B14
Smolichane village, Bulgaria
42807′ 58′′ N
22848′ 25′′ E
HB11 × 2
B15
Vaksovo village, spring, Bulgaria
42809′ 23′′ N
22851′ 28′′ E
HB11 × 2
Belashtitza village, spring, Bulgaria
42803′ 13′′ N
24844′ 10′′ E
HB16A × 2, HB16B, HB16C × 4
B17
Krichim town, Bulgaria
42801′ 22′′ N
24828′ 08′′ E
HB17 × 2
B18
Bachkovo town, water source, Bulgaria
41856′ 08′′ N
24851′ 46′′ E
HB16C × 2
B19
Vodnata dranchi dupka, Bulgaria
42802′ 55′′ N
26832′ 15′′ E
HB19A × 3, HB19B, HB19C
B20
Vitanovo spring, Bulgaria
42800′ 39′′ N
27825′ 03′′ E
HB20 × 5
B21
Gotse Deltchev town, karst spring, Bulgaria
41835′ 00′′ N
23841′ 37′′ E
HB21A, HB21B, HB21C, HB21D, HB21E
B22
Petrovo village, karst spring, Bulgaria
41825′ 42′′ N
23831′ 34′′ E
HB22A, HB22B
41825′ 53′′ N
23835′ 19′′ E
HB22B
B16
Grossuana angeltsekovi
B23
Grossuana slavyanica
Goleshovo village, Bulgaria
A
Grossuana codreanui
Jasenovo, Bulgaria (EF061920)
B
Grossuana codreanui
Techirghiol Lake, Romania (EF061919)
43859′ 37′′ N
28832′ 46′′ E
HB1A
C
Grossuana serbica
Serbia - Raška river Spring—(EF061921)
43806′ 57′′ N
20822′ 15′′ E
HS
D
Grossuana marginata
Spring between Loutsa and Steni, Evvoia
38835′ 16′′ N
23848′ 57′′ E
HG1
HB
island, Greece (KC011765)
E
Grossuana delphica
Kastalia spring, Delphi, Greece—(EF061922)
38828′ 59′′ N
22830′ 19′′ E
HG2
F
Grossuana hohenackeri
Spring of Athena, Tembi Valley, Greece
39858′ 26′′ N
22838′ 17′′ E
HG3, HG4, HG5
(KC011748 – KC011750)
G
Grossuana vurliana
Spring of Louros River, Greece (EF061923)
39825′ 56′′ N
20850′ 30′′ E
HG6
H
Grossuana sp.
Spring of Achilles, ESE of Kalamakion, Greece
38859′ 13′′ N
22822′ 43′′ E
HG7
39824′ 49′′ N
23809′ 23′′ E
HG8, HG9, HG10
39823′ 36′′ N
23802′ 33′′ E
HG11, HG12
(KC011746)
I
Grossuana sp.
Spring E of Anilion, Oros Pilion, Greece
(KC011767 – KC011769)
J
Grossuana sp.
Spring NW of Dhrakia, Oros Pilion, Greece
(KC011770 – KC011771)
Sequences from GenBank are also included (labelled A – J, with GenBank numbers; Szarowska et al., 2007; Falniowski et al., 2012).
This corresponds to an estimated divergence time between the six
Bulgarian clades of 1.26–1.42 Mya. The p-distances within the
clades were small, ranging from 0.2 to 0.9%. Clade I from southwestern Bulgaria was characterized by14 haplotypes, but most of
them differed by only one substitution from the closest haplotype
(Figs 2B, 3). Within Clade I, the most divergent haplotypes correspond to G. slavyanica (Fig. 2A). Putative Clades II and III,
from southeastern Bulgaria (Fig. 3), were unsupported and their
phylogenetic relationships were not resolved (Figs 2, 3); they may
represent one clade, although the haplotype network (Fig. 2B)
suggests their distinctness. Haplotypes belonging to both of these
putative clades were found at one of the localities (B8, including a
paratype of G. aytosensis). Putative Clade III also included haplotypes from localities B19 and B20.
Clades IV and V (the latter unsupported; Fig. 2A) were each
represented at a single locality: Clade IV from northern Bulgaria
(locality B2) and putative Clade V from central Bulgaria (locality
B13) (Fig. 3). In addition to population B13 (described as
RESULTS
In total we obtained 79 COI sequences of length 552 bp
(GenBank Accession numbers KU201035–KU201113). No saturation was revealed by the test of Xia et al. (2003). Among all
of the Grossuana COI sequences analysed, 42 haplotypes (haplotype diversity h ¼ 0.955; nucleotide diversity p ¼ 0.059) were
identified. The Bulgarian haplotypes (including one sequence
from Romania) formed a lineage that was clearly separated
from the Serbian/Greek Grossuana (bootstrap probability ¼ 70%;
Bayesian posterior probability ¼ 0.89), with an estimated separation time of 3.60 + 0.58 ma (Clades I–VI; Fig. 2). The nucleotide diversity within the Bulgarian Grossuana lineage was much
lower (p ¼ 0.019, 41 polymorphic sites) in comparison with that
within the Serbian/Greek clade (p ¼ 0.049, 70 polymorphic sites).
The Bulgarian Grossuana haplotypes formed six main clades
(Clades I–VI; Figs 2A, B, 3), some lacking statistical support,
between which p-distances ranged from 1.3 to 3.4% (Table 2).
308
GROSSUANA IN THE BALKANS
Figure 2. A. Maximum-likelihood phylogram of Grossuana COI haplotypes. Haplotypes sequenced in present work are indicated in bold; previously
published sequences are shown in plain text. See Table 1 for localities of haplotypes. Bootstrap support and Bayesian posterior probabilities are shown
(when greater than 50% or 0.5, respectively). B. Median joining tree for Bulgarian COI haplotypes.
309
A. FALNIOWSKI ET AL.
Figure 3. Geographical distribution of Bulgarian COI clades. Colour scheme of clades as in Figure 2. See Table 1 for locality details.
(Supplementary Material Fig. S1I–L). Wide variability of the
shell proportions, especially spire height, was observed in population B11, while in population B9 all of the shells were low-spired
with a broad aperture. However, these two populations of Clade I
encompass all of the shell variability observed in the Bulgarian
Grossuana (cf. Supplementary Material Figs S1, S2). The shells
of the other clades (Supplementary Material Fig. S2), found in
one to four populations each, showed less variation within each
clade. In Clade II (population B8: Supplementary Material
Fig. S2A–D) the shells were broad, with moderately high spires,
and the shells of Clade III (population B19: Supplementary
Material Fig. S2E, F) were similar. In Clade IV (population B2:
Supplementary Material Fig. S2G–I) the shells were relatively
high-spired, whereas in Clade V (population B13: Supplementary
Material Fig. S2J–N) the spire was low and the mouth broad. In
Clade VI (population B3: Supplementary Material Fig. S2O–Q),
the mouth was less broad, but the spire was high or low.
Table 2. COI p-distances between the main Grossuana clades and
putative clades (I– VI).
I
II
III
IV
V
I
0.009
II
0.019
0.002
III
0.025
0.013
0.006
IV
0.026
0.023
0.018
0.004
V
0.029
0.018
0.016
0.017
0.007
VI
0.034
0.016
0.028
0.024
0.018
VI
0.006
Italics indicate within-group genetic differentiation.
G. falniowskii), putative Clade V also included a published
Bulgarian sequence (HB haplotype, assigned to G. codreanui by
Szarowska et al., 2007); the p-distance between these two haplotypes was 0.7%.
Haplotypes from locality B12 (described as Radomaniola bulgarica),
situated close to B13, were assigned to Clade VI (Figs 2A, 3). The
remaining haplotypes forming this clade came from northeastern
Bulgaria. The p-distance between haplotype B12 and haplotypes
from northeastern populations was 0.008. Published sequences
from Romania were also assigned to Clade VI. Our samples identified as G. codreanui and G. radostinae carried the same haplotype in
Clade VI.
The shells of Grossuana belonging to the molecularly inferred
clades and putative clades are shown in Supplementary Material
Figures S1 and S2. The shells of Clade I (Supplementary Material
Fig. S1), represented by 10 populations, are illustrated for 2 populations: B11 (Supplementary Material Fig. S1A–H) and B9
DISCUSSION
Like other members of the rich European fauna of minute snails
belonging to the superfamily Truncatelloidea, the genus Grossuana
was long described only with reference to its morphological characters (e.g. Grossu, 1946, 1986; Radoman, 1973, 1983, 1985; Glöer &
Georgiev, 2009; Georgiev, 2012, 2013; Georgiev et al., 2015).
However, the application of molecular data in taxonomic and
phylogeographic studies of Grossuana (Szarowska et al., 2007;
Falniowski et al., 2012) has shown that when the morphology of
the shell and the reproductive organs is used alone, it is impossible
to achieve even species determination within this group. Thus,
310
GROSSUANA IN THE BALKANS
molecular markers must be applied to distinguish the true biological entities.
Our molecular results distinguished a major clade of Grossuana
in Bulgaria and Romania, which was clearly separated from the
other taxa of the genus known from Serbia and Greece. Similar
distinctness of the Bulgarian vs Dinaric haplotypes have been
found in the western capercaillie (Bajc et al., 2011), a turtle
(Fritz et al., 2006) the nose-horned viper (Ursenbacher et al.,
2008) and in newts (Wielstra et al., 2013).
The modern post-Alpine topography of the Mediterranean
emerged in the late Tortonian (8 ma) (Kostopoulos, 2009).
According to Popescu et al. (2009) and Suc et al. (2011), the
Balkan region suffered drastic environmental changes during the
Messinian salinity crisis. During the Miocene and Pliocene, a
shallow sea, the Pannonian Basin, filled the part of Central
Europe currently known as the Pannonian Plain. The Pannonian
Basin was connected through the Iron Gates with the Dacic Basin,
a vast water body that filled the area between the Carpathians
and the Balkan mountains (Popov et al., 2004, 2006; Clauzon et al.,
2005; Popescu et al., 2009). As a part of the Paratethys system, the
Pannonian and Dacic Basins were connected with the Euxinian
Basin to the east and directly with the present Aegean Sea to the
south. This saltwater connection, known as the Balkan Gateway,
existed through the graben valley of the present Velika (Big) and
Južna (South) Morava rivers, which was formed during the
Neogene as a result of tectonic movements (Stoyanov & Gachev,
2012). In the Miocene, Pliocene and lower Pleistocene, this region
was submerged by the Pannonian Sea. Finally, the present Velika
Morava Valley was cut into the floor of a former bay of the
Pannonian Sea. The presence of a saltwater connection between
the Pannonian Basin and the Aegean Sea (5.60–1.8 ma) and,
more recently, a wide river valley, may explain the distinctness of
the Serbian/Greek populations of Grossuana from the Bulgarian/
Romanian populations. Considering the present relatively
restricted geographical range of Grossuana in comparison with
Bythinella or Pseudamnicola, for example, the dispersal potential of
Grossuana appears to be low. Thus, the sea that existed in the past
and even the broad river valley with no calcium-rich springs
formed an effective barrier. The isolating role of graben valleys
was strengthened by the fact that glaciers were moving through
these landforms during certain intervals (Stoyanov & Gachev,
2012), which presumably made them uninhabitable for freshwater
snails.
According to a relaxed molecular clock, the separation of the
Bulgarian/Romanian populations from the Serbian/Greek
populations took place 3.6 + 0.58 ma. This estimated time of divergence coincides with the existence of the connection between
the Paratethys and the Aegean Sea described above, which likely
formed a dispersal barrier for Grossuana. Our estimate was computed from the 5.9% p-distance between the two groups and using
the rate 1.83 + 0.21% per myr for COI provided by Wilke (2003).
A similar estimate, of 1.62% per myr, was obtained by Hershler &
Liu (2008), which also places the divergence time during the existence of the Paratethys/Aegean connection. Following this isolation,
the Bulgarian/Romanian and Greek/Serbian groups evolved independently. The evidently lower diversity within the Bulgarian/
Romanian group may reflect bottlenecks during the Pleistocene
glaciations, as the glacial conditions in this northern region were
presumably more severe than in Greece, which is situated further
to the south.
Based on the topology of the COI tree and the geographical
distribution of the Bulgarian/Romanian clades, we suggest that
three main Grossuana groups can be distinguished: the Rhodopean
(Clade I), Strandzhan (Clades II and III) and Balkan (Clades
IV–VI) groups. Only the first of these is monophyletic in the
COI analyses (Fig. 2) and the phylogenetic relationships between
them are not resolved in our analyses. We speculate that the explanation for the separation of the Rhodopean and Strandzhan
groups could be the ecological barrier formed by the Maritsa
River Valley, a smaller-scale analogue of the Velika and Južna
Morava Valleys discussed above.
The estimated time of divergence between the six Bulgarian/
Romanian clades is 1.26–1.42 ma, corresponding to the Calabrian
during the Pleistocene. In the Pleistocene, the unstable fluviolacustrine system in southwestern Bulgaria, with glaciers present in the
Pirin and Rila Mountains (Zagorchev, 2007), probably formed
effective, temporary barriers for Grossuana and caused its extinction
in a large area of southwestern Bulgaria. Based on the available
data, the small genetic differences among the Bulgarian populations of Clade I reflect the short history of Grossuana in the area,
which was subsequently recolonized from elsewhere, perhaps from
regions presently inhabited by Clades II–VI (this hypothesis
could be tested by a more well-resolved phylogeny). In Clade I,
the presence of relatively high haplotype diversity, coupled with
low nucleotide diversity, agrees with the model of rapid population
growth from an ancestral population with a small evolutionarily
effective size (Avise, 2000). There must have been sufficient time
for the recovery of haplotype variation via mutations, yet not
enough time for the accumulation of larger sequence differences.
This general interpretation appears to be appropriate for these
spring-dwelling gastropods, whose populations may be established
by a few immigrants, passively transported by birds or as passengers on windblown leaves.
It must be emphasized that species delimitation was not an objective of this study, for which larger samples, independent genetic
markers and morphological data would be desirable. The study
did not yield any strong evidence for the occurrence of more than
one species at any one spring. While the presence of more than
one truncatelloid species in one spring is not common, it has been
noted for Bythinella (Radoman, 1976; Falniowski et al., 2009;
Falniowski & Szarowska, 2011a), including Bulgarian species
(Osikowski et al., 2015). We note that at locality B8 two haplotypes
were recorded with a divergence of 1.3%, which is close to the
interspecific threshold for COI of 1.5% suggested for Bythinella
(Bichain et al., 2007). Georgiev (2012) described specimens from
this locality as G. aytosensis, based on morphological characters,
and the presence of such divergent haplotypes requires further
study.
ACKNOWLEDGEMENTS
The study was supported by a grant from the National Science
Centre (2012/05/B/NZ8/00407) to Magdalena Szarowska. We
would like also to thank Associate Editor Robert Hershler,
Editor David G. Reid and two anonymous reviewers for their
valuable comments and suggestions.
SUPPLEMENTARY MATERIAL
Supplementary material is available at Journal of Molluscan
Studies online.
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