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RESEARCH ARTICLE
Comments on species divergence in the
genus Sphaerium (Bivalvia) and phylogenetic
affinities of Sphaerium nucleus and S.corneum
var. mamillanum based on karyotypes and
sequences of 16S and ITS1 rDNA
Romualda Petkevičiūtė*
☯
, Virmantas Stunz
ˇėnas
☯
, Graz
ˇina Stanevičiūtė
☯
P. B. S
ˇivickis Laboratory of Parasitology, Nature Research Centre, Vilnius, Lithuania
☯These authors contributed equally to this work.
*romualda@ekoi.lt
Abstract
Chromosome, 16S and ITS1 rDNA sequence analyses were used to obtain reliable diag-
nostic characters and to clarify phylogenetic relationships of sphaeriid bivalves of the genus
Sphaerium. The species studied were found to be diploid, with modal number 2n = 28 in S.
nucleus and 2n = 30 in S.corneum var. mamillanum. Small, biarmed, C- negative B chromo-
somes were found in all studied populations of both species. Karyological and molecular
markers revealed no differences between S.corneum s. str. and S.corneum var. mamilla-
num. No intraspecific differences were found in the basic karyotype of S.nucleus. Molecular
analyses, however, uncovered three genetically distinct ITS1 lineages: one comprised of
samples from Lithuania, Slovakia, and Russia, another from Czech, and a third from
Ukraine. Additionally to known 16S haplotype from Ukraine, three new 16S haplotypes of S.
nucleus were detected: one in the samples from Lithuania and Russia, one in Slovakian and
one in Czech population. In the ITS1 phylogenetic tree, all branches of S.nucleus clustered
in one clade. In the 16S phylogenetic tree, however, the haplotype of Czech S.nucleus
formed a separate branch, distant from three other haplotypes of S.nucleus. Molecular
results indicate that in the context of the Evolutionary Species Concept the S.nucleus mor-
phospecies may represent a complex of separate taxa, however referring on the Biological
Species Concept the genetic lineages could represent the intraspecific variability.
Introduction
The cosmopolitan bivalve family Sphaeriidae represents one of the most widespread molluscan
groups, inhabiting different freshwater habitats [1–3]. Estimation of sphaeriid species diversity
has been greatly hampered by the highly variable shell morphologies exhibited by many taxa
and the lack of reliable morphological traits for species differentiation [4–5]. Different taxo-
nomic significance has been attributed to all levels of morphological variation by different
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 1 / 17
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OPEN ACCESS
Citation: PetkevičiūtėR, Stunz
ˇėnas V, Stanevičiūtė
G (2018) Comments on species divergence in the
genus Sphaerium (Bivalvia) and phylogenetic
affinities of Sphaerium nucleus and S.corneum
var. mamillanum based on karyotypes and
sequences of 16S and ITS1 rDNA. PLoS ONE 13
(1): e0191427. https://doi.org/10.1371/journal.
pone.0191427
Editor: Daniel Rittschof, Duke University Marine
Laboratory, UNITED STATES
Received: May 9, 2017
Accepted: January 4, 2018
Published: January 23, 2018
Copyright: ©2018 Petkevičiūtėet al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by the
Research Council of Lithuania grant No MIP-43/
2015 (http://lmt.lt/download/6113/mip%20gtm_vi
%20kv%20%20finansuojamu%20projektu%
20sarasas_galutinis.pdf) awarded to V.S. and by
SYNTHESYS (http://www.synthesys.info/network-
activities/synthesys2-na2/), European Commission
taxonomic schools (see comments in [3,6–8]. Therefore, the taxonomic status of species within
the traditional genus Sphaerium Scopoli 1777 and intrageneric grouping have generated con-
siderable discussion for over a century. Based on the last taxonomic revision [3], the genus is
represented in Europe by seven valid species. The most variable member of the genus is the
type species S.corneum (L., 1758). Because of wide shell variability, several forms or varieties
of the species have been distinguished. Sphaerium nucleus (Studer, 1820) is usually considered
an intraspecific variety of S.corneum by Western specialists [2,9]. However, some concho-
logical and anatomical characters to support the distinctness of S.nucleus were provided by
Korniushin [3,10], with the shape of nephridium considered the most reliable of them. Never-
theless, anatomical characters are not widely used in sphaeriid taxonomy, and S.nucleus still
is poorly known due to confusion with S.corneum. It is believed that the geographic range
of the species comprises the major part of Europe [3], but only quite recently S.nucleus was
reliably recorded in some Central European countries [11–14] and in Britain [15–16]. The
exact geographic range of S.nucleus needs to be evaluated on the basis of new diagnostic char-
acters. Sphaerium corneum var. mamillanum is considered an intraspecific variation by West-
European malacologists [2,17] and a distinct species in Russian publications [10,18]. After a
comprehensive morphological analysis, Korniushin [3] concluded that S.corneum var. mamil-
lanum could not be definitely separated from typical S.corneum. Nevertheless, the problem of
the taxonomic status of these two forms is still not conclusively resolved.
In cases where traditional taxonomy gives problematic results, species distinctness and the
phylogenetic relationship of certain forms may be supported using karyological and/or molec-
ular data. Unfortunately, the number of karyologically studied sphaeriid taxa is still very lim-
ited and the data for many of them are incomplete. Among the species that have been
examined, highly polychromosomal nuclei are the rule, with chromosome numbers ranging to
above 200 (see review in [19–20]). Prior to this study, only three sphaeriid species were known
to be diploid: Palaearctic S.corneum and S.solidum and Nearctic S.rhomboideum [19,21–22].
Previous attempts to karyotype S.nucleus in order to find species-specific karyological charac-
ters and to compare it to S.corneum were unsuccessful [23].
This study is the first to characterize the mitotic chromosomes of S.nucleus and S.corneum
var. mamillanum. We describe the karyotypes of S.nucleus obtained from three different popu-
lations in Central Europe and of S.corneum var. mamillanum from one population in Estonia
using conventional karyometric analysis and C-banding. We also use molecular markers
based on the nuclear ITS1 and mitochondrial 16S ribosomal gene fragment sequences that
have been recently developed for numerous Holarctic sphaeriid species, for phylogenetic
reconstructions [22,24–25]. These two regions of rDNA of S.nucleus and S.corneum var.
mamillanum were sequenced from different populations, and the resulting alignments were
used for comparative phylogenetic analyses to obtain species-specific markers.
Materials and methods
Samples of S.nucleus were collected from three locations in Central Europe: in South Slovakia
(48˚25´32´´ N; 20˚01´34´´ E, the sampling place indicated by Kos
ˇel [12]), in Czech, South
Moravia (48˚44´58´´ N; 17˚00´14´´ E, the sampling place indicated by Korinkova [11]), and in
Lithuania from a marshy coast of Lake Terpez
ˇys (55˚ 15’ 29.69" N; 25˚ 53’ 51.17" E) in the
Labanoras Regional Park. This species should be considered comparatively rare in Lithuania,
as a number of favourable habitats were checked for its presence during 2006–2009, but the
species was found only in the above-mentioned location. One specimen of S.nucleus was
received from Russia (Moscow region) and used for comparative DNA analysis. Samples of S.
corneum var. mamillanum were collected from the stream between Lake Liinjarv and Lake
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 2 / 17
grant No DE-TAF-3965 awarded to R.P. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Suurjarv (57˚43’35.64" N; 26˚55’41.00" E) in Estonia. Also, further samples of S.corneum s. str.
were collected from two water bodies in Estonia, Lake Mustjarv (57˚56’6.41" N; 27˚20’23.76"
E) and River Vaike-Emajogi (57˚59’8.64" N; 26˚ 2’55.28" E), and used for molecular analysis.
The specimens were identified on conchological characters suitable for species identification
according to Korniushin [3;10]. It was found that shell pore density is one of the most reliable
diagnostic characters for preliminary differentiation of S.nucleus and S.corneum. According
to the International Union for Conservation of Nature (IUCN) information there are no
known conservation actions known for S.nucleus and S.corneum, and none are considered
necessary. The populations are thought to be stable [26–27]. No permissions are required for
their collection and further use for research. The field-collected species were sampled in free
access water bodies, where no permission is needed. Voucher specimen shells from each of
these samples have been deposited in the collection of the P.B. S
ˇivickis Laboratory of Parasitol-
ogy, Institute of Ecology of Nature Research Centre.
Brooding animals were found at the time of collection. For karyological analysis, whole
intact living animals were incubated in 0.01% colchicine in well-water during 3 to 5 h. The
bodies were removed from the shells under a dissecting microscope and treated for 50–60 min
in distilled water for hypotony. The fixation was made in three changes (20 min each) of a
freshly prepared fixative of ethanol-acetic acid (3: 1). Chromosome preparations were made
with a cell suspension air-drying technique [21]. Each slide was made from the tissues of a sin-
gle individual. Slides were stained in 4% Giemsa-Romanowski dye in phosphate buffer (pH
6.8) for 30–40 min. Chromosomes in suitable metaphases were counted and the best spreads
were photographed using an Olympus BX51 light microscope supplied with a digital camera.
The lengths of the short and long arms of chromosomes were measured in ten karyotypes
from different individuals obtained from each population. Data analysis was performed with
an Excel macro-program. Means and standard deviations of the absolute and relative lengths
(100 x absolute chromosome pair length divided by the total length of the haploid comple-
ment) and the centromeric index (100 x length of the short arm divided by the total chromo-
some length) were calculated for each pair of chromosomes. Terminology relating to the
centromere position follows that of Levan et al. [28], but a binary terminology was adapted
when the 95% confidence limits of the centromeric index mean covered two chromosome cat-
egories. Data were analysed using the independent two-sample Student’s ttest, and the results
were considered significant when P<0.05. C-banding was carried out according to the Sumner
[29] modified method, i.e., slides were treated with saturated Ba(OH)2 for 15 min, briefly
washed in distilled water, 0.2 N HCl, distilled water again, incubated in 2 x SSC (0.3 M NaCl,
0.03 M Na3C6H5O7) for 90 min at 60˚ C, and stained for 1 h in a 5% Giemsa solution buffered
to pH 6.8.
Total DNA for molecular analysis was isolated from the tissues of the same specimens used
for cytogenetic studies according to the protocol of Stunz
ˇėnas et al. [22]. A nucleotide frag-
ment ~480 bp of the mitochondrial large ribosomal subunit (16S) DNA was amplified using
primers 16Sar (5’-CGC CTG TTT ATC AAA AAC AT-3’) and 16Sbr (5’-CCG GTC
TGA ACT CAG ATC ACG T-3’) according to Palumbi [30]. An entire nuclear internal
transcribed spacer 1 (ITS1) sequence (~560 bp) was amplified following the protocol of
Stunz
ˇėnas et al. [22] and using primers from White [31] annealing to flanking regions of 18S
and 5.8S genes; these primers were, respectively, 18SWF (5’-TAA CAA GGT TTC CGT
AGG TG-3’) and 5_8_SWR (5’-AGC TRG CTG CGT TCT TCA TCG A-3’). The PCR
product was purified and sequenced in both directions at Macrogen Inc. (Seoul, Korea).
Sequence confirmation was accomplished by comparing complimentary DNA strands. Editing
of the DNA sequences, contig assembly, and the alignment of the consensus sequences were
carried out using the software program Sequencher 4.7 (Gene Codes Corporation).
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 3 / 17
Additional sequences were downloaded from GenBank and included in the phylogenetic
analysis: Sphaerium nucleus from Ukraine (AY093537, AY093573), S.corneum (AY792316,
AY792317, AY792319, AY792320, AY792321, AY093535, AF152037), S.solidum (FJ874903,
FJ874904, GU123690, FJ874907, FJ874908, FJ874909), S.rhomboideum (AF152038, AY093538),
S.occidentale (AF152046, AY093542), S.baicalense (AY093534). Sequences of Pisidium dubium
(AF152027, AY093533) and P.variabile (AF152030, AY093530) were included as the outgroup
taxa.
For phylogenetic analyses, the sequences of ITS1 dataset were aligned using ClustalW [32]
with an open gap penalty of 15 and gap extension penalty of 6.66. Multiple Sequence Align-
ment Software MAFFT version 7 [33] with iterative refinement method of G-INS-i were used
to align sequences of 16S dataset, because MAFF produced better parsimony-informative
alignment comparing with ClustalW (34 vs 32 parsimony informative sites). The best-fit
model of sequence evolution for phylogenetic analysis was estimated using jModeltest v. 0.1.1
software [34]. Ambiguously aligned positions were excluded from phylogenetic analysis.
Nucleotide by nucleotide distance between sequences was estimated in MEGA6 [35] using
model No. of differences with pairwise deletion of gaps/missing data and inclusion of all sub-
stitutions (transitions and transversions). Maximum likelihood phylogenetic trees were
obtained and analysed using MEGA6. Branch support was estimated by bootstrap analyses
with 1000 replicates. The phylogenetic trees were obtained using general time reversible model
with a gamma distribution of rates and a proportion of invariant sites (GTR + G + I) for both
the ITS1 and the 16S gene datasets. Gamma shape and number of invariant sites were esti-
mated from the data.
Results
Karyotype of Sphaerium nucleus
A total of 304 mitotic metaphase spreads from 33 individuals of S.nucleus (15 from Lithuania,
11 from Slovakia, and 7 from Czech) were analysed, and the modal diploid chromosome num-
ber of 2n = 28 was revealed (Table 1). A representative karyotype is shown in Fig 1. The chro-
mosomes in the karyotype show a regular decrease in size, except for the last pair, which is
strikingly smaller than the others. Table 2 indicates the absolute length, relative length, centro-
meric index (CI), and classification of the chromosome pairs in each of the three populations.
The chromosomes ranged in size from 1.16 μm to 9.47 μm. The mean total length of the hap-
loid complement (TCL) ranged from 61.83 μm in the Slovak population to 79.34 μm in the
Lithuanian population. Differences in the absolute length of chromosomes may be partially
accounted for by different chromosome condensation on the slides studied. The karyotype
consisted of all biarmed, metacentric, meta-submetacentric, and submetacentric chromo-
somes. The lowest CI value was estimated in chromosomes pair 12, and they were classified as
submetacentric. Within the karyotype of S.nucleus, pairs of homologous chromosomes could
be distinguished by their morphology, except for pairs 6 and 7, and pairs 9 and 10, both sets of
which had similar relative lengths and centromeric indices (see Table 2). Comparative study
revealed no significant (P<0.05) interpopulation differences in relative lengths and CI values
of the corresponding chromosomes of the basic complement.
Significant numbers of metaphase spreads with more than 28 chromosomes (from 14% to
35%) were observed in all three populations. The analysis of the corresponding karyotypes sug-
gested the presence of a variable number of comparatively small (the mean length was 2.5 μm),
biarmed, supernumerary (B) chromosomes, typically with 4 or 8 per cell (Fig 2A and 2B). In
the Lithuanian population cells with 8 supernumerary chromosomes were found most often,
but in the Slovakian population hyperdiploid cells contained either 4 or 8 B’s, and in the Czech
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 4 / 17
population only cells with 4 supernumerary chromosomes were observed (see Table 1). Poly-
ploid (4n) sets were rare; they were found in four cells (3 from Lithuanian and 1 from Czech
population).
Results of the C-banding procedure were studied in five animals from different populations.
Small but conspicuous pericentromeric constitutive heterochromatin blocks were always pres-
ent on 11 chromosome pairs 1–11 (Fig 2B). Heterochromatin blocks were not observed (C-
negative) on chromosome pairs 12, 13 and 14. B chromosomes also were C-negative.
Karyotype of Sphaerium corneum var. mamillanum
A total of 86 mitotic metaphase spreads from ten individuals were analysed. The modal diploid
chromosome number was 2n = 30 (Table 1). A representative karyotype is shown in Fig 3A.
The chromosomes ranged in size from 2.7 μm to 9 μm (Table 3). The TCL reached 75.56 μm.
The karyotype consisted of all biarmed elements and, according to the centromere position, 13
Table 1. Chromosome numbers in mitotic metaphases of Sphaerium nucleus and S.corneum var. mamillanum from different populations in Europe (S, Slovak, L,
Lithuanian, C, Czech, E, Estonian population).
Species
/population origin/ number of specimens
Chromosome number (main complement and supernumerary Bs) in mitotic metaphase
27 28 30 32 34–35 36 4n
S.nucleus /L/ 15 8
(4.32%)
150
(81.1%)
- - 4
(2.16%)
20
(10.8%)
3
(1.62%)
S.nucleus /S/ 11 2
(2.6%)
48
(62.3%)
- 15
(19.5%)
1
(1.3%)
11
(14.3%)
-
S.nucleus /C/ 7 4
(9.52%)
31
(73.8%)
- 6
(14.3%)
- - 1
(2.38%)
S.corneum var mamillanum /E/ 10 - - 60
(69.77%)
2
(2.33%)
20
(23.25%)
4
(4.65%)
-
https://doi.org/10.1371/journal.pone.0191427.t001
Fig 1. A mitotic metaphase chromosome spread and the karyotype of Sphaerium nucleus, 2n = 28. Scale
bar = 10 μm.
https://doi.org/10.1371/journal.pone.0191427.g001
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 5 / 17
chromosome pairs (pair number 1–3, 5, 6, 8–15) were classified as metacentric, and two pairs,
4 and 7, represented intermediates between the meta- and submetacentric structure. A
Table 2. Measurements (mean±SD) and classification of modal diploid (A) chromosomes of Sphaerium nucleus (S, Slovak, L, Lithuanian, C, Czech population).
Chromosome number Absolute lenght (μm) Relative length (%) Centromeric index Classification
1 S 7.86±1.34 12.70±0.97 47.15±2.48 m
L 9.47±2.03 11.86±0.65 44.90±2.88
C 8.80±1.76 12.14±0.77 45.27±2.78
2 S 6.95±1.18 11.19±0.56 45.23±2.74 m
L 8.43±1.61 10.61±0.60 42.35±3.61
C 8.00±1.04 11.15±0.50 44.97±3.24
3 S 5.36±0.81 8.67±0.58 37.59±1.39 m-sm
L 7.19±1.41 9.05±0.51 38.06±1.53
C 6.60±1.16 9.15±050 37.33±3.07
4 S 4.93±0.70 7.96±0.43 44.70±2.70 m
L 6.44±1.22 8.11±0.37 43.32±3.85
C 5.91±0.94 8.20±0.29 40.86±3.18
5 S 4.76±0.62 7.71±0.47 42.45±3.99 m
L 6.32±1.16 7.97±0.37 41.63±4.14
C 5.68±0.76 7.91±0.31 40.15±3.57
6 S 4.55±0.67 7.36±0.50 44.68±3.62 m
L 5.97±1.13 7.51±0.20 44.58±2.25
C 5.40±0.76 7.51±0.26 44.38±2.97
7 S 4.38±0.59 7.08±0.45 38.98±4.99 m-sm
L 5.79±0.97 7.32±0.29 37.51±3.27
C 5.23±0.75 7.27±0.31 39.24±3.19
8 S 4.20±0.62 6.79±0.40 44.24±2.69 m
L 5.66±1.05 7.13±0.34 44.21±4.34
C 5.14±0.79 7.13±0.32 43.05±3.30
9 S 4.06±0.48 6.58±0.31 46.15+2.06 m
L 5.51±1.16 6.92±0.35 42.62±3.69
C 4.96±0.69 6.89±0.21 44.73±3.32
10 S 3.88±0.42 6.31±0.58 46.98±1.49 m
L 4.98±0.96 6.28±0.52 42.28±4.50
C 4.69±0.65 6.53±0.25 43.60±2.47
11 S 3.59±0.40 5.80±0.26 37.60±3.12 sm-m
L 4.81±0.86 6.08±0.35 36.60±4.00
C 4.31±0.62 5.98±0.31 37.01±4.36
12 S 3.04±0.48 4.91±0.28 32.19±3.50 sm
L 3.89±0.71 4.91±0.34 28.78±3.03
C 3.30±0.49 4.60±0.45 28.80±2.34
13 S 2.84±0.42 4.60±0.26 45.95±4.38 m
L 3.07±0.42 3.91±0.26 42.78±2.47
C 2.81±0.40 3.92±0.37 42.77±3.08
14 S 1.43±0.15 2.33±0.32 50.00±3.46 m
L 1.81±0.18 2.35±0.44 42.91±4.55
C 1.16±0.10 1.62±0.24 43.07±1.63
m, metacentric; sm, submetacentric chromosome
https://doi.org/10.1371/journal.pone.0191427.t002
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 6 / 17
comparative study of centromeric indexes and relative lengths revealed no significant
(P<0.05) differences in the basic karyotype structure of this S.corneum var. mamillanum pop-
ulation from the population of S.corneum s. str., described in an earlier study [21].
Twenty-six of the 86 studied cells (30.23%) contained more than the modal number of
chromosomes, with a maximum of 36. The supernumerary (B) chromosomes observed in
these hyperdiploid cells were small biarmed elements and showed intra-individual variation
from 0 to 6. The modal number of B’s, 4 per cell, was found in 18 (20.93%) of the studied meta-
phases (Fig 3A).
C-banding revealed that all of the chromosomes of the basic complement (A) showed a
bright heterochromatic band in the centromeric region. No heterochromatin blocks were
observed on any of the B chromosomes in the analysed metaphases (Fig 3B).
Molecular and phylogenetic analyses
The 17 complete nucleotide sequences obtained during this study have been deposited in Gen-
Bank (Table 4, in bold). Partial sequences of mitochondrial 16S rDNA and nuclear ITS1 rDNA
were determined for the specimens from Lithuania, Slovakia, Czech (S. Moravia), and Russia.
Particular differences between sequences of related groups of Sphaerium spp. with pairwise
deletion of gaps/missing data and inclusion of all substitutions (transitions and transversions)
are indicated in the Tables 5and 6. All individuals of S.nucleus from Lithuania, Slovakia, and
Russia characterized for nuclear ITS1 rDNA sequences had identical genotypes. The individu-
als collected in Czech, however, were different from the S.nucleus collected in Lithuania, Slo-
vakia, and Russia, as well as from the Ukrainian specimen sequenced by Lee & O
´Foighil [24]:
the sequences differed by 4 bp and by 2 bp, respectively, in the ITS1 alignment of 556 sites
Fig 2. Mitotic metaphases and respective karyotypes of Sphaerium nucleus with different numbers of B
chromosomes: a, 2n = 28 + 4B, and b, C-banded chromosomes, 2n = 28 + 8B. Scale bars = 10 μm.
https://doi.org/10.1371/journal.pone.0191427.g002
Fig 3. Chromosomes of Sphaerium corneum var. mamillanum: a, conventionally stained mitotic metaphase and
karyotype, 2n = 30 + 4B, and b, C-banded mitotic metaphase and karyotype, 2n = 30 + 2B. Scale bars = 10 μm.
https://doi.org/10.1371/journal.pone.0191427.g003
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 7 / 17
(Table 5). Also, the ITS1 sequences of the S.nucleus with different genotypes have different
lengths. The identical lengths of the ITS1 sequences of S.corneum and S.solidum were shorter
than the ITS1 sequences of S.nucleus. All 16S sequences of S.nucleus from different popula-
tions have almost identical length but nucleotide differences varied from 6 bp to 13 bp
Table 3. Measurements (mean±SD) and classification of modal diploid (A) chromosomes of Sphaerium corneum var. mamillanum.
Chromosome number Absolute length (μm) Relative length (%) Centromeric index Classification
1 9.02±0.83 11.97±0.40 48.06±1.20 m
2 8.07±0.95 10.68±0.09 43.45±4.94 m
3 7.23±1.32 9.54±0.55 41.61±1.89 m
4 6.45±0.31 8.58±0.67 38.84±4.72 m-sm
5 6.25±1.67 8.19±1.18 44.06±2.76 m
6 5.47±0.56 7.25±0.17 43.12±3.52 m
7 4.56±0.55 6.04±0.03 40.25±5.26 m-sm
8 4.28±0.68 5.65±0.19 40.77±1.35 m
9 4.29±0.62 5.67±0.11 40.88±4.06 m
10 3.80±0.84 5.00±0.48 44.40±4.42 m
11 3.56±0.28 4.73±0.22 44.04±1.77 m
12 3.46±0.37 4.59±0.09 42.20±1.29 m
13 3.29±0.48 4.35±0.09 41.55±4.16 m
14 3.08±0.04 4.10±0.46 40.23±2.25 m
15 2.74±0.02 3.66±0.49 40.65±2.55 m
m, metacentric; sm, submetacentric chromosomes.
https://doi.org/10.1371/journal.pone.0191427.t003
Table 4. Sphaerium spp. subjected to molecular phylogenetic analysis with information of their host, locality and GenBank accession numbers.
Species Locality GenBank No
and a source if it is not from this study
16S 18S-ITS1-5.8S
Sphaerium nucleus Ukraine AY093573 [24] AY093537 [24]
Sphaerium nucleus Slovakia: a fen marsh near Vysna Pokoradz village HM208267, HM208268, HM208269 HM208261
Sphaerium nucleus Czechia: a marsh near Tvrdonice, South Moravia HM208271, HM208272, HM208273 HM208262
Sphaerium nucleus Russia: Moscow region HM208270 HM208263
Sphaerium nucleus Lithuania: Lake Terpez
ˇys HM208264, HM208265, HM208266 HM208260
Sphaerium corneum Germany AF152037 [24] AY093535 [24]
Sphaerium corneum France: Rennes AY093547 [24]
Sphaerium corneum Lithuania: a pond in the North part of Vilnius AY792316, AY792317 [21] AY792319 [21]
Sphaerium corneum Lithuania: River Vilnelėin Vilnius AY792320 [21] AY792321 [21]
Sphaerium corneum Estonia: Lake Mustjarv GU128620, GU128621 KU863151
Sphaerium corneum Estonia: River Vaike-Emajogi GU128617 KU863152
Sphaerium corneum, var. mamillanum Estonia: stream between Lake Liinjarv and Lake Suurjarv GU128618, GU128619 KU863153
Sphaerium baicalense Russia: Lake Baykal AY093534 [24]
Sphaerium solidum Lithuania: Curonian Lagoon FJ874903, FJ874904 [22] GU123690 [22]
Sphaerium solidum Hungary: Danube River FJ874907, FJ874908, FJ874909 [22] GU123689 [22]
Sphaerium rhomboideum USA: Michigan AF152038 [24] AY093538 [24]
Sphaerium occidentale USA: Michigan AF152046 [24] AY093542 [24]
Sequences obtained in this study are marked in bold
https://doi.org/10.1371/journal.pone.0191427.t004
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 8 / 17
(Table 6). In contrast, both of the sequences of S.corneum from Estonia were identical to S.
corneum var. mamillanum.
These two different sets of DNA sequences produced different tree topologies in the phylo-
genetic analyses (Figs 4and 5). The genetically different groups of S.nucleus formed separated
branches in both trees. In the ITS1 tree (Fig 4), all branches of S.nucleus clustered into one
clade and the specimens from Czech, Lithuania, Slovakia, Russia, and Ukraine formed a sub-
clade in a wellsupported clade with S.corneum,S.baicalensis, and S.solidum. In the 16S tree
(Fig 5), the Czech S.nucleus formed a distinct branch separated from all other S.nucleus and
from a clade of S.corneum and S.solidum specimens.
Discussion
While S.nucleus and S.corneum seem to possess only a few discriminative morphological char-
acters, a comparative karyological analysis separates these species because they differ both in
diploid numbers (2n = 28 and 2n = 30, respectively) and in the morphology and C-banding
patterns of some chromosome pairs. On the other hand, although S.corneum var. mamillanum
is a morphologically distinct form characterized by the presence of distinct embryonic shells
on the umbones, our analysis revealed no significant differences (P<0.05) between the karyo-
type structure of this form and S.corneum s. str., previously studied by Petkevičiūtėet al. [21].
Conservatism in chromosome numbers is noticeable in the bivalve taxa listed by Nakamura
[36] and Thiriot-Quievreux [37]. The evolution of unionid mussels has generally proceeded
without change in chromosome number [38–39]. The first data on chromosome numbers for
sphaeriid species were reported by Keyl [40], who found n = 18 and 2n = 36 in male meiosis of
S.corneum. The subsequent rate of karyological descriptions in Sphaeriidae has been low. It is
now known that the genus Sphaerium is characterized by an extreme karyotypic diversifica-
tion, with mitotic chromosome numbers varying from 28 to 247 [19–22,41–43, this study].
Sphaerium could be considered a typical example of explosive speciation related to a high
number of chromosomal reorganizations.
Most of the existing cytogenetic studies of sphaeriids have been focused on the number of
chromosomes, and only 4 species have been studied for chromosome morphology [19,21–22].
The scarcity of comprehensive cytogenetic studies on sphaeriid species may be associated with
the exceptionally high mitotic chromosome numbers found in most species analysed (see
review in [19–20]). In addition, the presence of a variable number of supernumerary chromo-
somes was revealed in some species [21–22].
It is worth noting that all diploid sphaeriid species studied so far are representatives of the
genus Sphaerium. Even in the diploid Sphaerium clams, karyotype composition varies from
species to species, but different groups of species follow different patterns. Two species, S.soli-
dum and S.corneum, have a stable karyotype morphology, with the same modal diploid chro-
mosome number (2n = 30), a complement of biarmed metacentric and submeta-metacentric
Table 5. Average number of nucleotide differences between ITS1 dataset sequences of closest related groups of
Sphaerium spp. with pairwise deletion of gaps/missing data and inclusion of all substitutions (transitions and
transversions).
Groups, sequence length 1. 2. 3. 4.
1. S.nucleus (Ukraine), 550 bp
2. S.nucleus (Lithuania, Slovakia, Russia), 554 bp 4
3. S.nucleus (Czech), 556 bp 4 2
4. S.corneum, 542 bp 3 3 3
5. S.solidum, 542 bp 4 4 4 1
https://doi.org/10.1371/journal.pone.0191427.t005
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 9 / 17
chromosomes of gradually decreasing size, and no significant interspecific karyotypic differ-
ences [22]. Nearctic species were regarded as highly polychromosomic, but Petkevičiūtėet al.
[19] showed that S.rhomboideum is diploid, with 2n = 44.
As described in this study, S.nucleus has the lowest chromosome number (2n = 28) of the
sphaeriids studied to date. Karyotypes with low chromosome numbers, 2n = 28 and 2n = 30,
are exclusively composed of biarmed meta- and submetacentric chromosomes, while uni-
armed telo- and subtelocentric chromosomes are present in the karyotype of S.rhomboideum
Table 6. Average number of nucleotide differences between 16S dataset sequences of closest related groups of Sphaerium spp. with pairwise deletion of gaps/missing
data and inclusion of all substitutions (transitions and transversions).
Groups,sequence length 1. 2. 3. 4. 5. 6. Within groups
1. S.nucleus (Ukraine), 475 bp -
2. S.nucleus (Lithuania, Russia), 475 bp 6 0
3. S.nucleus (Slovakia), 475 bp 10 4 0
4. S.nucleus (Czech), 474 bp 13 8 12 0
5. S.corneum (2n = 36), 474 bp 9 9 13 14 0
6. S.corneum, 474 bp 14.14 10.14 14.14 8.14 9.43 0.86
7. S.solidum, 474 bp 13.29 9.29 13.29 7.29 12.29 5.14 1.43
https://doi.org/10.1371/journal.pone.0191427.t006
Fig 4. Phylogenetic tree obtained from ITS1 sequences of nuclear rDNA and basedon the analysis of 520 sites. Bootstrap support given for maximum
likelihood analysis (bootstrap replications = 1000, complete deletion of gaps/missing data). Bootstrap support values lower than 70% are not shown. Names
of the target species are in bold. Pisidium dubium and P.variable were included as outgroups.
https://doi.org/10.1371/journal.pone.0191427.g004
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 10 / 17
(2n = 44) and in the karyotypic form (2n = 36) from species group of S.corneum [19,21]. Dif-
ferences in the number and morphology of chromosomes lend support to the assumption that
Robertsonian translocations are involved in the cytogenetic divergence of species. Reduction
in chromosome numbers by Robertsonian rearrangements was previously suggested in the
marine bivalve families Mytilidae and Pectinidae [44–45].
The second karyological peculiarity of all studied European species of Sphaerium s. str. is
the presence of mitotically unstable B chromosomes. Sphaerium rhomboideum differs in this
regard because no B chromosomes have been found in its cells [19]. B chromosomes of Sphaer-
ium spp. are small metacentric elements; in the cells of S.corneum and S.solidum they are sig-
nificantly smaller than any of the basic (A) chromosomes, while in S.nucleus Bs are larger than
the smallest chromosomes of the basic set. Furthermore, different degrees of numerical stabili-
zation and interpopulation differences in frequency of B chromosomes were revealed. B chro-
mosomes of S.corneum and S.solidum showed a more dispersed distribution, varying from 0
to 10, and from 0 to 6, respectively, but the even number of Bs (mostly four or eight) was more
often observed than any odd number [21–22]. During this study, eight B chromosomes were
commonly observed in the Lithuanian population of S.nucleus, while four or eight Bs were
Fig 5. Phylogenetic tree for 16S haplotypes based on the analysis of 471 sites of mitochondrial 16S rDNA sequences. Bootstrap support given for maximum
likelihood analysis (bootstrap replications = 1000, complete deletion of gaps/missing data). Bootstrap support values lower than 70% are not shown. Names of the
target species are in bold. Pisidium dubium and P.variable were included as outgroups.
https://doi.org/10.1371/journal.pone.0191427.g005
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 11 / 17
present with approximate frequencies in cells of clams from Slovakia, and four Bs were
recorded in hyperdiploid cells in the Moravian population.
The occurrence and persistence of B chromosomes in a lineage probably has a genomic
explanation and is thus of evolutionary significance [46]. B chromosomes are an intriguing
class of chromosomes. They are additions to the standard (A) chromosome complement and
follow their own evolutionary pathway. The term B chromosomes include very heterogeneous
types of chromosomes; their only consistent feature is that they are not essential for survival of
an individual and are present in some individuals from some populations in some species [46–
48]. Data for B chromosomes of bivalve mollusc species are very scarce. Variable numbers of B
chromosomes were recorded in clonal lineages of marine clams of the genus Lasea (Vener-
oida) [49–50]. Presence of 1–3 small supernumerary chromosomes was observed in Cerasto-
derma edule (Veneroida) and they were presumed to be B chromosomes [51]. Later analysis
using restriction enzyme banding demonstrated that those B chromosomes were, in fact, the
result of chromosomal fission involving the largest submetacentric chromosome pair [52].
For S.nucleus, the C-banding technique showed heterochromatic (C-positive) regions near
the centromeres of chromosomes pairs 1–11, but no C-blocks were revealed in chromosomes
pairs 12–14 of the main complement. All B chromosomes were C-negative in the S.nucleus
samples. In the karyotype of S.corneum var. mamillanum, conspicuous C-positive regions
were revealed in all chromosomes of the main complement and, as with S.nucleus, the B chro-
mosomes were all C-negative. B chromosomes are heterochromatic in many organisms, but
they can be C-negative as well [48,53]. In most animals heterochromatin is detected at the peri-
centromeric region [54]. C-banding analyses in marine bivalves Ostrea denselamellosa,O.
angasi,O.conchaphila,Mytilus edulis,M.galloprovincialis,M.trossulus and Crassostrea angu-
lata indicated that pericentromeric heterochromatin is not common in these species, although
telomeric and interstitial heterochromatin is [55–60]. Supposedly, the karyotypes with higher
telomeric heterochromatin must have an older phylogenetic status [57].
The results of our study show that S.corneum s. str. and form mamillanum share identical
ITS1 and 16S sequences. Both the karyological and the molecular evidence fail to support the
independent taxonomic status of S.corneum var. mamillanum. While the karyological analysis
of S.nucleus in this study revealed the same basic karyotype structure for representatives of
three populations, the comparisons of the ITS1 and 16S sequences indicated the different line-
ages within this species. The phylogenetic analyses and differences in the 16S sequences clearly
separated Czech S.nucleus from the other populations studied. Also, there was a clear diver-
gence in 16S between samples from the Lithuanian, Russian populations and Slovakian popu-
lation, and S.nucleus from Ukraine. DNA sequence analyses of S.solidum showed that only
one site was different from ITS1 of S.corneum [22]. Moreover, ITS1 was found to be identical
in S.corneum and endemic of Lake Baikal S.baicalense [24]. The ITS1 differences among S.
nucleus samples are more significant: 2–4 bp and the different lengths of ITS1 in all three line-
ages of S.nucleus. So, in the context of the Evolutionary Species Concept, one could treat the
three lineages of S.nucleus as three good species.
Morphological and molecular studies of sphaeriid phylogeny are incongruent (see [61]). At
the species level, however, S.corneum and S.nucleus represent closely related sister taxa, both
morphologically and in molecularly based studies (see [8,24]). In the morphologically based
analysis of Korniushin & Glaubrecht [8], five Sphaerium species (S.corneum,S.solidum,S.niti-
dum,S.nucleus, and S.rhomboideum) form a monophyletic group recognized as Sphaerium s.
str. Recent karyological and molecular studies [22] confirmed the close relationships of S.cor-
neum and S.solidum. Although the molecular data did not support the placement of North
American S.rhomboideum as sister to European S.nucleus and strongly suggested that S.rhom-
boideum be reassigned to the subgenus Herringtonium [24], recent karyological analysis [19]
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 12 / 17
gave an unexpected result–the chromosome set of S.rhomboideum is diploid. So, the morpho-
logically based intergeneric division of Sphaerium species is correlated with karyotypic pat-
terns; in the Sphaerium s. str. group, all karyologically studied species have diploid
chromosome sets, including Palaearctic S.corneum,S.solidum,S.nucleus, and Nearctic S.
rhomboideum.
Freshwater habitats have relatively discrete boundaries, suggesting that populations of
freshwater invertebrates should also be discrete [62]. Furthermore, ecological peculiarities of
sphaeriid clams, together with their odd system of reproduction, could lead to a low rate of
genetic exchange and to manifestation of founder effect followed by formation of highly iso-
lated populations. Regarding their reproduction, sphaeriids appear as specialized freshwater
molluscs, being simultaneous hermaphrodites and viviparous–they broods embryos up
through the juvenile stage in the suprabranchial chamber [6,25,63–65]. Even a single individ-
ual can give origin to a distinct and often isolated population. Sphaerium nucleus lives in small,
often temporal water bodies, so, considerable changes in population size and rapid differentia-
tion of populations under dissimilar selective regimes is predictable. Ecological heterogeneity
may have been a key-factor responsible for genetic divergence [66], but the genetic identity of
the S.nucleus specimens from the Slovakian, Lithuanian, and Russian populations do not cor-
relate with ecological or ecotypic similarity. The genetic divergence in S.nucleus is unlinked to
any apparent pattern of karyological and morphological variation or ecological preference.
This highlights a disconnection between molecular, karyological and morphological evolution.
Our findings demonstrate that reliance on the current morphological taxonomy underesti-
mates the underlying genetic diversity. The increasing availability of DNA sequences and utili-
zation of molecular markers in taxonomic and phylogenetic studies reveal that a broad
spectrum of taxa contains sets of morphologically similar, but genetically distinct, lineages
[67]. The use of a genetic yardstick, however, might be problematic. The number of methods
available for delimiting species markedly increases in recent years and different approaches to
species delimitation exist [68–71] however each have a unique set of challenges [70], so they
must be used with caution. It is difficult to calibrate the minimum threshold of divergence to
establish interspecific separations between organisms with inadequate taxonomies, such as
sphaeriid bivalves, which are also characterised by odd systems for reproduction, extraordi-
nary dispersal abilities and populations commonly found in isolated unstable environments.
According to the estimation of experts, the inferences drawn from species delimitation studies
should be conservative [25,70–71] and it is better refer to monophyletic groups as lineages
than falsely delimit ‘species’. A plausible and acceptable statistical method to recognise cryptic
species in sphaeriid bivalves has not been applied and still doesn’t exist. Moreover, all haplo-
types of S.nucleus share the same basic karyotype structure and there are no karyotypic barri-
ers (meiotic constrains) for interbreeding of individuals with distant haplotypes, or as
indicated Rannala [71], genetic isolation alone does not prove that the lineages are incapable
of interbreeding, and referring on the Biological Species Concept (the requirement of repro-
ductive incompatibility between species) such lineages do not represent actual species. We
hope that data on Sphaerium species diversity could be useful in creating a statistical method
able to recognise cryptic species and, herewith, do not fail to separate genetically closely related
species, such as S.corneum and S.solidum.
In general, studies that incorporate molecular, morphological and/or karyological data will
provide much better descriptions and interpretations of biological diversity than those that
focus on just one approach. Considering the genetic diversity uncovered in the S.nucleus com-
plex within the limited range studied here, it is likely that more cryptic diversity is present.
Our data show that many questions about this complex of species remain to be answered.
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 13 / 17
Acknowledgments
The authors want to express their appreciation to Dr Tereza Korinkova for helping in collect-
ing Sphaerium spp. in the Czech, and to Dr Tarmo Timm and Dr Henn Timm, Estonia. We
also thank the Charles University, Praha, and Centre for Limnology of Estonian University of
Life Sciences, for providing laboratory facilities. Dr Daniel P. Molloy (Great Lakes Centre at
SUNY Buffalo State College) kindly checked the English of the manuscript.
Author Contributions
Conceptualization: Romualda Petkevičiūtė.
Data curation: Virmantas Stunz
ˇėnas, Graz
ˇina Stanevičiūtė.
Formal analysis: Virmantas Stunz
ˇėnas.
Funding acquisition: Romualda Petkevičiūtė, Virmantas Stunz
ˇėnas.
Investigation: Romualda Petkevičiūtė, Virmantas Stunz
ˇėnas, Graz
ˇina Stanevičiūtė.
Methodology: Romualda Petkevičiūtė, Virmantas Stunz
ˇėnas.
Project administration: Virmantas Stunz
ˇėnas.
Resources: Graz
ˇina Stanevičiūtė.
Writing – original draft: Romualda Petkevičiūtė, Virmantas Stunz
ˇėnas, Graz
ˇina Stanevičiūtė.
Writing – review & editing: Romualda Petkevičiūtė, Virmantas Stunz
ˇėnas, Graz
ˇina
Stanevičiūtė.
References
1. Burch JB. Freshwater sphaeriacean clams (Mollusca, Pelecypoda) of North America. Hamburg, Michi-
gan: Malacological publications; 1975.
2. Piechocki A. The Sphaeriidae of Poland (Bivalvia, Eulamellibranchia). Ann Zool. 1989; 42: 249–320.
3. Korniushin AV. Taxonomic revision of the genus Sphaerium sensu lato in the Palaearctic Region, with
some notes on the North American species (Bivalvia: Sphaeriidae). Arch Mollusk. 2001; 129: 77–122.
4. Bailey RC, Anthony EH, Mackie GL. Environmental and taxonomic variation in fingernail clam (Bivalvia:
Pisidiidae) shell morphology. Can J Zool. 1983; 61: 2781–2788.
5. Dyduch-Falniowska A. The conchological variability of Sphaerium corneum (Linnaeus, 1758) in the Pol-
ish waters. Folia Malacol (Krakow). 1988; 2: 83–96.
6. Cooley LR, O
´Foighil D. Phylogenetic analysis of the Sphaeriidae (Mollusca: Bivalvia) based on partial
mitochondrial 16S rDNA gene sequences. Invertebr Biol. 2000; 119: 299–308.
7. Korniushin AV. Review of the studies on freshwater bivalve mollusk systematics carried out by the Rus-
sian taxonomic school. Malacol Rev Suppl. 1998; 7: 65–82.
8. Korniushin A, Glaubrecht M. Phylogenetic analysis based on the morphology of viviparous freshwater
clams of the family Sphaeriidae (Mollusca, Bivalvia, Veneroida). Zool Scripta. 2002; 31: 415–459.
9. S
ˇivickis P. [Mollusks of Lithuania and their identification]. Vilnius: State Press of Political and Scientific
Literature; 1960 (in Lithuanian).
10. Korniushin AV. [Bivalve mollusks of the superfamily Pisidioidea in the Palaearctic region: Fauna, sys-
tematics, phylogeny]. Kiev: Schmalhausen Institute of Zoology; 1996 (in Russian).
11. Korinkova T. First reliable records of Sphaerium nucleus (Mollusca: Bivalvia: Sphaeriidae) in the Czech
Republic. Acta Soc Zool Bohem. 2006; 69: 293–297.
12. Kos
ˇel V. The first record of Sphaerium nucleus (Bivalvia) in Slovakia. Biologia (Bratisl). 2006; 61: 524.
13. Korinkova T, Beran L, Horsak M. Recent distribution of Sphaerium nucleus (Studer, 1802) (Bivalvia:
Sphaeriidae) in the Czech Republic. Malacol Bohemoslov. 2008; 7: 26–32.
14. Falkner G. Sphaerium (Nucleocyclas)nucleus in Bayern. Heldia. 2000; 3: 11–18.
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 14 / 17
15. Killeen I, Aldridge D, Oliver G. Freshwater bivalves of Britain and Ireland. Occasional Publication 82.
Shrewsbury: FSC Publications; 2004.
16. Williams C, Gormally M, Anderson R. Sphaerium nucleus (Studer) (Mollusca: Bivalvia) from turlough
systems in Cos Galway and Roscommon. Ir Nat J. 2010; 31: 64–65.
17. Glo
¨er P, Meier-Brook C. Su¨sswassermollusken. 12
th
ed. Hamburg: DJN; 1998.
18. Andreev NI, Andreeva SI, Krasnogorova AN. Findings of Sphaerium mamillanum (Westerlund, 1871)
(Mollusca, Bivalvia, Sphaeriidae) in waterbodies of the Urals and Western Siberian Plain. Inland Water
Biol. 2011; 4: 264–266.
19. PetkevičiūtėR, StanevičiūtėG, Stunzˇėnas V, Lee T, O
´Foighil D. Pronounced karyological divergence
of the North American congeners Sphaerium rhomboideum and S.occidentale (Bivalvia: Veneroida:
Sphaeriidae). J Mollusc Stud. 2007; 73: 315–321.
20. Korinkova T, Moravkova A. Does polyploidy occur in central European species of the family Sphaeriidae
(Mollusca: Bivalvia)? Cent Eur J Biol. 2010; 5: 777–784.
21. PetkevičiūtėR, Stunz
ˇėnas V, StanevičiūtėG. Polymorphism of the Sphaerium corneum (Bivalvia,
Veneroida, Sphaeriidae) revealed by cytogenetic and sequence comparison. Biol J Linnean Soc. 2006;
89: 53–64.
22. Stunz
ˇėnas V, PetkevičiūtėR, StanevičiūtėG. Phylogeny of Sphaerium solidum (Bivalvia) based on kar-
yotype and sequences of 16S and ITS1 rDNA. Cent Eur J Biol. 2011; 6: 105–117.
23. Korinkova T, Kral J. Structure and meiotic behaviour of B chromosomes in Sphaerium corneum/S.
nucleus complex (Bivalvia: Sphaeriidae). Genetica. 2010; 139: 155–165. https://doi.org/10.1007/
s10709-010-9533-1 PMID: 21120681
24. Lee T, O
´Foighil D. Phylogenetic structure of the Sphaeriinae, a global clade of freshwater bivalve mol-
luscs, inferred from nuclear (ITS-1) and mitochondrial (16S) ribosomal gene sequences. Zool J Linnean
Soc. 2003; 137: 245–260.
25. Guralnick RP. Life-history patterns in the brooding freshwater bivalve Pisidium (Sphaeriidae). J Mollusc
Stud. 2004; 70: 341–351.
26. Killeen I, Seddon MB. Sphaerium nucleus. The IUCN Red list of threatened species. 2011;e.
T155874A4859030. Available from: http://www.iucnredlist.org/details/155874/1.
27. Van Damme D, Killeen I. Sphaerium corneum. The IUCN Red list of threatened species 2012;e.
T155511A731550. Available from: http://www.iucnredlist.org/details/155511/0.
28. Levan A, Fredga K, Sandberg AA. Nomenclature for centromere position in chromosomes. Hereditas.
1964; 52: 101–220.
29. Sumner AT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res. 1972;
75: 304–306. PMID: 4117921
30. Palumbi SR. Nucleic acid II: the polymerase chain reaction. In: Hillis DM, Moritz C, Mable B, editors.
Molecular systematics. Sunderland, Massachusetts: Sinauer Associates; 1996. pp. 205–247.
31. White L, Mcpheron B, Stauffer JR. Molecular genetic identification tools for the unionids of French
creek, Pennsylvania. Malacologia. 1996; 38: 181–202.
32. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res. 1994; 22: 4673–4680. PMID: 7984417
33. Katoh K, Standley DM. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in per-
formance and usability. Mol Biol Evol. 2013; 30: 772–780. https://doi.org/10.1093/molbev/mst010
PMID: 23329690
34. Posada D. jModelTest: Phylogenetic modelling averaging. Mol Biol Evol. 2008; 25: 1253–1256. https://
doi.org/10.1093/molbev/msn083 PMID: 18397919
35. Tamura K, Stecher G, Peterson N, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Anal-
ysis Version 6.0. Mol Biol Evol. 2013; 30: 2725–2729. https://doi.org/10.1093/molbev/mst197 PMID:
24132122
36. Nakamura HK. A review of molluscan cytogenetic information based on the CISMOCH–computerised
index system for molluscan chromosomes. Bivalvia, Polyplacophora and Cephalopoda. Venus. 1985;
44: 193–225.
37. Thiriot-Quie
´vreux C. Review of the literature on bivalve cytogenetics in the last ten years. Cah Biol Mar.
2002; 43: 17–26.
38. Park GM, Burch JB. Karyotype analysis of six species of North American freshwater mussels (Bivalvia,
Unionidae). Malacol Rev. 1995; 28: 43–61.
39. Jenkinson JJ. Chromosomal characteristics of North American and other naiades (Bivalvia: Unionida).
Malacologia. 2014; 57: 377–397.
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 15 / 17
40. Keyl HG. Boobachtungen u¨ber die ♂-meiose der Muschel Sphaerium corneum. Chromosoma. 1956; 8:
12–17.
41. Burch JB, Park GM, Chung EY. Michigan’s polyploid clams. Mich Acad. 1998; 30: 351–352.
42. Lee T. Polyploidy and meiosis in the freshwater clam Sphaerium striatinum (Lamarck) and chromosome
numbers in the Sphaeriidae (Bivalvia, Veneroida). Cytologia. 1999; 64: 247–252.
43. Lee T, O
´Foighil D. 6-Phosphogluconate dehydrogenase (PGD) allele phylogeny is incongruent with a
recent origin of polyploidization in some North American Sphaeriidae (Mollusca, Bivalvia). Mol Phylo-
genet Evol. 2002; 25: 112–124. PMID: 12383755
44. Libertini A, Boato A, Panozzo M, Fogato V. Karyotype and genome size in some species of Mytilidae
(Bivalvia, Mollusca). La Kromosomo II. 1996; 82: 2819–2827.
45. Insua A, Lo
´pez-Piño
´n MJ, Me
´ndez J. Characterization of Aequipecten opercularis (Bivalvia: Pectinidae)
chromosomes by different staining techniques and fluorescent in situ hybridization. Genes Genet Syst.
1998; 73: 193–200. PMID: 9880917
46. Camacho JPM, Sharbel TF, Beukeboom LW. B-chromosome evolution. Philos Trans R Soc London B.
2000; 355: 163–178.
47. Jones RN, Rees H. B chromosomes. New York: Academic Press; 1982.
48. Vujos
ˇevićM, BlagojevićJ. B chromosomes in populations of mammals. Cytogenet Genome Res. 2004;
106: 247–256. https://doi.org/10.1159/000079295 PMID: 15292599
49. O
´Foighil D, Thiriot-Quievreux C. Ploidy and pronuclear interaction in north-eastern Pacific Lasaea
clones (Mollusca: Bivalvia). Biol Bull. 1991; 181: 222–231. https://doi.org/10.2307/1542093 PMID:
29304641
50. O
´Foighil D, Thiriot-Quie
´vreux C. Sympatric Australian Lasaea species (Mollusca: Bivalvia) differ in
their ploidy levels, reproductive modes and developmental modes. Zool J Linnean Soc. 1999; 127:
477–494.
51. Insua A, Thiriot-Quie
´vreux C. Karyotypes of Cerastoderma edule,Venerupis pullastra and Venerupis
rhomboides (Bivalvia, Veneroida). Aquat Living Resour. 1992; 5: 1–8.
52. Leitão A, Chaves R, Joaquim S, Matias D, Ruano F, Guedes-Pinto H. Supernumerary chromosomes
on Southern Europe populations of the cockle Cerastoderma edule: Consequence of environmental
pollution? Estuar Coast Shelf S. 2008; 79: 152–156.
53. Jones RN. B chromosomes in plants. New Phytol. 1995: 131, 411–434.
54. Sumner AT. Chromosome banding. London: Unwin Hyman Ltd; 1990.
55. Insua A, Thiriot-Quie
´vreux C. The characterization of Ostrea denselamellosa (Mollusca, Bivalvia) chro-
mosomes: karyotype, constitutive heterochromatin and nucleolus organizer regions. Aquaculture.
1991; 97: 317–325.
56. Martinez-Lage A, Gonzalez-Tizon A, Mendez J. Characterization of different chromatin types in Mytilus
galloprovincialis and restriction endonuclease treatments. Heredity. 1994; 72: 242–249.
57. Martinez-Lage A, Gonzalez-Tizon A, Mendez J. Chromosomal markers in three species of the genus
Mytilus (Mollusca: Bivalvia). Heredity. 1995; 74: 369–375.
58. Li XX, Havenhand JN. Karyotype, nucleolus organiser region and constitutive heterochromatin in
Ostrea angasi (Mollusca: Bivalvia): evidence of taxonomic relationships within the Ostreidae. Mar Biol.
1977; 127: 443–448.
59. Leitão A, Chaves R, Santos S, Boudry P, Guedes-Pinto H, Thiriot-Quievreux C. Cytogenetic study of
Ostrea conchaphila (Mollusca: Bivalvia) and comparative karyological analysis within Ostreinae. J
Shellfish Res. 2002; 21: 685–690.
60. Cross I, Diaz E, Sanchez I, Rebordinos L. Molecular and cytogenetic characterization of Crassostrea
angulata chromosomes. Aquaculture. 2005; 247: 135–144.
61. Graf DL. Patterns of freshwater bivalve global diversity and the state of phylogenetic studies on the
Unionida, Sphaeriidae and Cyrenidae. Am Malacol Bull. 2013; 31: 135–153.
62. Bohonak AJ, Jenkins DG. Ecological and evolutionary significance of dispersal by freshwater inverte-
brates. Ecology Lett. 2003; 6: 783–796.
63. Odhner NH. On the anatomical characteristics of some British Pisidia. Proc Malacol Soc London. 1922;
15: 155–161.
64. Heard WH. Reproduction of fingernail clams (Sphaeriidae: Sphaerium and Musculium). Malacologia.
1977; 16: 421–455.
65. Korniushin AV. Growth and development of the outer demibranch in freshwater clams (Mollusca, Bival-
via): a comparative study. Ann Zool (Warsaw) 1996; 46: 111–124.
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 16 / 17
66. Schliewen UK, Tautz D, Pa
¨a
¨bo S. Sympatric speciation suggested by monophyly of crater lake cichlids.
Nature. 1994; 368: 629–632. https://doi.org/10.1038/368629a0 PMID: 8145848
67. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K et al. Cryptic species asa window on
diversity and conservation. Trends Ecol Evolut. 2007; 22: 148–155.
68. Birky CW Jr, Adams J, Gemmel M, Perry J. Using population genetic theory and DNA sequences for
species detection and identification in asexual organisms. PLoS ONE. 2010; 5(5): e10609. https://doi.
org/10.1371/journal.pone.0010609 PMID: 20498705
69. Birky CW Jr. Species detection and identification in sexual organisms using population genetic theory
and DNA sequences. PLoS ONE. 2013; 8(1): e52544. https://doi.org/10.1371/journal.pone.0052544
PMID: 23308113
70. Carstens BC, Pelletier TA, Reid NM, Satler JD. How to fail at species delimitation. Mol Ecol. 2013; 22:
4369–4383. https://doi.org/10.1111/mec.12413 PMID: 23855767
71. Rannala B. The art and science of species delimitation. Curr Zool. 2015; 61(5): 846–853.
Species divergence in the genus Sphaerium
PLOS ONE | https://doi.org/10.1371/journal.pone.0191427 January 23, 2018 17 / 17
