Cent. Eur. J. Biol. • 6(1) • 2011 • 105–117 DOI:DOI 10.2478/s11535-010-0101-6

Central European Journal of Biology

Phylogeny of solidum () based on karyotype and sequences of 16S and ITS1 rDNA

Research Article Virmantas Stunžėnas*, Romualda Petkevičiūtė, Gražina Stanevičiūtė

Institute of Ecology of Nature Research Centre, Vilnius LT-08412, Lithuania Received 07 May 2010; Accepted 20 September 2010

Abstract: Thepresent work represents the first karyological and molecular characterisation of , a rare European clam. SpecimensofS. solidumwerecollectedinLithuaniaandHungary.Themodaldiploidchromosomenumberfoundinbothpopulations was2n=30.Small,biarmedBchromosomeswerefoundin42.3%ofcellsstudiedinclamsfromLithuaniaandin11.8%ofcellsin clamsfromHungary.Comparativeanalysisrevealednosignificant(P<0.05)interspecificdifferencesinchromosomemorphology of S. solidum and that of previously studied S. corneum. DNA sequence analyses of S. solidum showed no interpopulation differences in ITS1; moreover,only one site was different from ITS1 of S. corneum. However,differences in mitochondrial 16S sequenceofS. solidumwererevealed:twohaplotypesinLithuaniaandthreeinHungarywereidentified.Thegeneticcharacteristics revealedinthisstudydonotsupportascriptionofS. solidumandS. corneumtodifferentsubgenus,CyrenastrumandSphaerium s. str.,respectively. Comparative cytogenetic analysis disclosed that the chromosome morphology could be conserved in some sphaeriidspeciesduringspeciationdespitethefactthatmostotherspeciesinthisfamilyundergoradicalkaryotypicdifferentiation.

Keywords: • Sphaerium solidum • • Cyrenastrum • Karyotype • B chromosomes • Haplotype • 16S mitochondrial ribosomal DNA • ITS1 nuclear rDNA sequence • Phylogeny

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1. Introduction malacological investigations of Schlesch and Krausp [10]. Recently, populations of S. solidum were recorded Clams of the family Sphaeriidae are cosmopolitan and in the Curonian Lagoon and Nemunas River Delta [11]. widespread in freshwater ecosystems, with maximum In Hungary, S. solidum was detected few years ago [12]. diversities in the Holarctic Region [1-3]. Sphaeriids exhibit Recently, numerous populations have been discovered interesting biological features – they are hermaphrodites in the Danube [13]. and brood their young within an inner demibranch of the Intrageneric relationships amongst Sphaeriidae ctenidia (gills) until their release as bentic juveniles [4,5]. are still poorly understood and controversial despite a Even single individuals of these can give rise to number of systematic hypotheses proposed based on distinct, and often isolated populations [6,7]. Sphaerium morphological or molecular data [3,14-16]. Sphaerium solidum (Normand, 1844) is known mostly from large solidum was included in the subgenus Cyrenastrum rivers, and less frequently from reservoirs and canals Bourguignat (1854) on the basis of several morphological in Central and Eastern Europe [3]. In some regions, features: solid shell with clear surface sculpture with S. solidum is on the verge of extinction. Its populations concentric ribs, broad hinge with hooked cardinal teeth, in Poland and Britain may be in decline, possibly as a separated siphonal retractor scars and open nephridium result of increased pollution and of rivers with elongated dorsal lobe [3]. Phylogenetic analyses [8,9]. Consequently, this species is protected in these based on a set of morphological characters have countries. In Lithuania, populations of S. solidum are revealed monophyly of a group including S. solidum and known in the Rivers Nemunas and Neris from early the species of the traditional Sphaerium s. str. subgenus

* E-mail: [email protected] 105 Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

[3,15]. Karyological features, as well as molecular data, P.B. Šivickis Laboratory of Parasitology, Institute of may indicate the evolutionary distance between different Ecology of Nature Research Centre, Vilnius, Lithuania taxa that may not be obvious at the morphological of the Institute of Ecology of Nature Research Centre, level. Unfortunately, the chromosomal composition of (collection numbers: SS3, SS4, SS10, SS12, SS13, S. solidum was unknown and no molecular markers SS14, SS15, SS21, SS440, SSE13, SSE14, SSE15, were available for this species thus far. SSE19, SSE42, SSE44, SSE46, SSE66). Karyological studies of sphaeriid species, although Initial phases of chromosome preparation were scarce, revealed the exceptionally high variability completed as soon as possible after the animals were of mitotic chromosome numbers, from 30 to 247 collected and identified. To obtain metaphases, intact [17]. Clearly, significant changes in chromosome living animals were maintained in 0.01% colchicine in number and structure occurred during the evolution of well water for 3-4 h. The bodies were then removed from Sphaeriidae. Only two diploid species are known. The the shells under a dissecting microscope and treated in first species studied cytogenetically was S. corneum distilled water for 40-50 min for hypotony. Tissues were (Linnaeus, 1758), for which Keyl [18] demonstrated the fixed by three incubations of 20 min each in a freshly mixed chromosome number n=18 and 2n=36. More recent modified Carnoy’s fixative (ethanol/acetic acid = 3:1). analyses of Lithuanian populations of S. corneum have Fixed material was kept refrigerated until it could be revealed intrapopulation and intraindividual variation in processed in the laboratory. Each slide preparation was chromosome numbers, associated with the existence made from one individual using an air-drying technique. of two different karyomorphs, 2n=30 and 2n=36, and This involved placing a small piece of fixed tissue on a with the variable number of B chromosomes in the cells clean microscope slide in a few drops of 50% acetic acid, of karyomorph 2n=30 [19]. During the recent studies smearing dissociated cells onto the slide and drying by of chromosome sets of North American sphaeriid heating to 40-50 ºC with an alcohol lamp. Preparations species, the diploid number of 2n=44 was reported for were then treated with 1N HCl for 10-15 min, rinsed three S. rhomboideum (Say, 1822) and this is the first record of times in distilled water, and stained for 30 min with 4% a diploid species in the highly polychromosomic Nearctic Giemsa solution made up in phosphate buffer, pH 6.8. sphaeriid fauna [17]. Close morphological affinities of The chromosome preparations were examined with a S. solidum and the diploid species of the Sphaerium s. BX51 (Olympus) microscope using a 100x oil immersion str. subgenus, S. corneum and S. rhomboideum, led to objective. Karyotypes were constructed by arranging the the presumption that S. solidum could be also diploid. chromosomes based on type (centromere position) and Previous studies have demonstrated that maximum in descending order by size. Chromosome arm lengths likelihood as well as maximum parsimony phylogenetic (short arm, p; long arm, q) were measured. For each analyses of 16S mitochondrial gene and ITS1 nuclear chromosome pair, mean and standard deviations of DNA sequence datasets yielded largely congruent and absolute length (p + q), relative length ((p + q)/total haploid well-resolved topologies, and robustly supported clades, length/100) and centromeric index (100 x p/(p + q)) which have been used to revise sphaeriid [16]. were calculated in a Microsoft Excel spreadsheet. In the present study we have karyotyped S. solidum and Terminology relating to the centromere position follows sequenced the 16S mitochondrial gene fragment, and the that of Levan et al. [20]. In situations where the standard nuclear ITS1 ribosomal sequence. These genetic markers deviation of the centromeric index was at the borderline have been utilized to characterise the species and to test between two chromosome types, the nomenclature for the phylogenetic relationships of S. solidum revealed by both was given. Data were analysed using the Student’s morphological traits in the previous studies [3,15]. t test. Results were considered significant when P<0.05. Total DNA was extracted from the tissues of the same specimens, which were prepared and used for 2. Experimental Procedures cytogenetic studies. In a previous study [21], we found that a tissue sample preparation for karyological analysis Specimens of S. solidum were collected from two using the colchicine treatment has no mutagenic or geographically distant regions (Figure 1): in Lithuania degradational effect on DNA sequences and this sample (in the Curonian Lagoon near Nemunas delta and in can be used for DNA sequencing via PCR. For the the water reservoir of the dammed up river Nemunas DNA extraction, a very rapid method without any lyses near Kaunas) and in Hungary (in the Danube River near buffer, only sterile Tris-borate-EDTA (TBE) buffer (this Budapest) in May-August 2008. Voucher specimens buffer is common for DNA agarose electrophoresis) from which DNA fragments and chromosomes were was used. In the previous study, this method allowed studied have been deposited in the collection of us to extract high quality DNA from tissue samples from

106 V. Stunžėnas et al.

Figure 1. Map of central Europe showing the collection sites in Lithuania and Hungary. Collection sites denoted by numbers 1 and 2 represent places in the Curonian Lagoon near the Nemunas delta and the dammed up river Nemunas near Kaunas, respectively; number 3 - in the Danube River, North from Budapest. The map was compiled from the few maps presented on d-maps.com website: http://d-maps.com/carte.php?lib=western_and_central_europe_map&num_car=2254&lang=en; http://d-maps.com/carte.php?lib=hungary_map&num_car=2289&lang=en; http://d-maps.com/carte.php?lib=lithuania_map&num_car=2365&lang=en insects [22,23], rodents [24] and trematodes [25] for ATC AAA AAC AT-3’) and 16Sbr (5’-CCG GTC TGA ACT the amplification and sequencing of mitochondrial and CAG ATC ACG T-3’) according to Palumbi [26]. An entire nuclear DNA fragments. Before the DNA extraction, nuclear internal transcribed spacer 1 (ITS1) sequence a small piece (1-3 mm3) of the mollusc foot tissue (~560 bp) was amplified using primers annealing to was placed in 500 µl of TBE buffer for 5 min to wash flanking regions of 18S and 5.8S genes: 18SWF (5’-TAA out the fixative. The mollusc tissue sample from the CAA GGT TTC CGT AGG TG-3’) and 5_8_SWR (5’-AGC washing TBE buffer was placed in 50 µl of TBE buffer TRG CTG CGT TCT TCA TCG A-3’) according to White, on a sterile microscope glass slides (76 x 26 mm). The Mcpheron and Stauffer [27]. All DNA fragments were tissue sample in TBE buffer was covered and crushed amplified via a standard Polymerase Chain Reaction to homogenate mass by other microscope slide. The (PCR) using Taq DNA Polymerase recombinant tissue homogenate was collected in a microtube and (Fermentas), under the following conditions: 36 cycles incubated at 95°C for 10 min in a thermocycler, then of 30s at 94ºC, 60 s at 55ºC and 120 s at 72ºC. The PCR placed on ice for 3 min, after, centrifuged at 12,000 g product was purified and sequenced in both directions for 5 min in a refrigerated centrifuge. The temperature at Macrogen (Seoul, Korea). Sequence confirmation of the refrigerated centrifuge was maintained between was accomplished by comparing complimentary DNA 4 and 7°C. The 25 µl of the supernatant with total strands. Editing of the DNA sequences, contig assembly, DNA was transferred to a fresh tube and stored at and alignment of consensus sequences were performed -20ºC. Fragments of mitochondrial and nuclear DNA using the software program Sequencher 4.7 (Gene sequences were amplified from the 2 µl of the total DNA Codes Corporation). The sequences were aligned solution in 50 µl of standard Polymerase Chain Reaction with ClustalW [28] with an open gap penalty of 15, a buffer (Fermentas). A nucleotide fragment ~480 bp of gap extension penalty of 6.66. An unrooted neighbour- the mitochondrial large ribosomal subunit (16S) DNA joining [29], minimum evolution (ME) and maximum was amplified using primers 16Sar (5’-CGC CTG TTT parsimony (MP) phylogenetic trees [30] were obtained

107 Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

and analysed using MEGA 4 [31]. The genetic distances S. (M.) lacustre (AF152035), S.(M.) securis (AF152033, were calculated by maximum composite likelihood [32] AY093509), S. rivicola (AY093539), Pisidium dubium and Kimura’s two-parameter [33] methods. To estimate (AF152027, AY093533) and P. variabile (AF152030, the relative branch support, bootstrap analysis with AY093530). 10,000 replicates was conducted. Unrooted maximum likelihood trees were obtained using PhyML 3.0 [34] with 4 rate categories. Gamma shape and number of 3. Results invariant sites were estimated from the data. Support to internal branches for maximum likelihood trees were An analysis of 78 mitotic metaphases from 14 estimated by bootstrap analyses with 1000 replicates. specimens collected in the Curonian Lagoon and 136 Bayesian inferences of phylogeny were performed with metaphases from 13 specimens collected in the Danube MrBayes 3.1.2 [35] by coupled Markov chain Monte River revealed the modal diploid chromosome number Carlo (MCMC) sampling, four simultaneous MC chains, 2n=30 (Table 1, Figure 2). A few spreads displaying a running 500,000 generations, and concluding with an chromosome number lower than 30 were encountered. average standard deviation of split frequencies below These might be attributed either to aneuploidies or, 0.01, sample frequency 100, burn-in 2500 generations. most likely, to technical shortcomings leading to loss of Modeltest 3.06 [36] was used to estimate the evolutionary chromosomes during the slide preparation. The number model that best fit the nucleotide data sets for maximum of polyploid cells was low; only two tetraploid cells likelihood and Bayesian inferences of phylogeny. Akaike were encountered in two specimens from the Danube information criteria (AIC) implemented in Modeltest River. In the remaining cells, variable numbers of small selected a General Time Reversible General Time metacentric supernumerary (B) chromosomes were Reversible model plus gamma (GTR+G) for ITS1 found. B chromosomes showed intraindividual numerical and 16S datasets of DNA sequences. The obtained variation from 0 to 6 and from 0 to 4 in specimens phylogenies were visualised using MEGA 4. from the Curonian Lagoon and the Danube River, Sequences of 16 species were downloaded from respectively (Table 1). Recognition of the supernumerary GenBank and included in the phylogenetic analysis: chromosomes was rather easy in chromosome sets of S. corneum (AF152037, AY792320, AY792317, S. solidum due to their small size. They do not exceed AY792316, AY093535, AY792321, AY792319, 1.9 μm in length and were apparently smaller than AY093547), S. baicalense (AY093534), S. nucleus the chromosomes of the last 15th pair of the basic set (AY093573, AY093537), S. rhomboideum (AF152038, (Figure 3). The numerical variation and morphological AY093538), S. occidentale (AF152046, AY093542), distinctness prove that these supernumerary S. novaezealandia (AF152047, AY093543), chromosomes were not aneuploidies. In the aneuploid S. tasmanicum (AY093544), S. striatinum (AF152041, cells the extra chromosomes are duplicates of one or AY093541, AY093545, AY093546), S. fabale more of the basic set. The modal number of B’s was 4 in (AY093536), S. simile (AF152040, AY093540), both populations (Table 1, Figure 3), but the frequency S. (Musculium) argentinum (AF152034, AY093503), of B’s showed significant interpopulation differences:

Chromosome number

2n 4n Population origin Total number of spreads 28 29 30 31 32 33 34 35 36

Curonian Lagoon

1 2 42 3 9 - 14 2 5 - 78 Spread number

Danube River

3 4 111 2 1 1 12 - - 2 136 Spread number

Table 1. Chromosome numbers in mitotic spreads of Sphaerium solidum from Lithuanian and Hungarian populations.

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Figure 2. Mitotic metaphase and karyotype of Sphaerium solidum from the Danube River, 2n=30. Scale bar = 10 μm. Note the secondary constrictions (arrowheads) in the karyotype. they were found in 33 (42.3%) cells studied in clams from the Curonian Lagoon and in 16 (11.8%) cells in clams from the Danube River (Table 1). Seven metaphases from 5 individuals obtained from the Curonian Lagoon population and 10 metaphases from 6 individuals from the Danube River have been karyotyped and measured. A summary of the results obtained after measuring is given in Table 2. The chromosomes were medium-sized and ranged from 2.5 μm to 9.3 μm. The mean total length of the haploid Figure 3. Two mitotic metaphases and two respective karyotypes of complement was 76.6 μm and 75.0 μm in Lithuanian and Sphaerium solidum from Curonian Lagoon with different Hungarian populations, respectively. The karyotype is numbers of B chromosomes: (a) 2n=30+4B and (b) 2n=30+5B. Scale bars = 10 μm. Note the secondary composed exclusively of bi-armed elements. Figures 2 constrictions (arrowheads) in the karyotype. and 3 show them arranged in order of decreasing size. As the size decreases gradually, it was impossible to the water reservoir of the dammed up River Nemunas and group the chromosomes in different classes by length. 5 specimens from the Danube River. These sequences are Chromosome pairs 1, 2, 3, 5, 14 and 15 are metacentric, identical for specimens of S. solidum from all investigated pairs 4 and 7 represent an intermediate between the populations and only one nucleotide base pair is different submeta- and metacentric type of structure, and the from ITS1 sequences of S. corneum studied by Lee and remaining pairs (6, 8, 9, 10, 11, 12 and 13) are of the Ó Foighil [16] and Petkevičiūtė et al. [19]. Phylogenetic meta-submetacentric type. Secondary constriction in analysis of the ITS1 sequences resulted in the neighbour- the short arms of the chromosomes constituting pair joining (Figure 4) and maximum likelihood (Figure 5) 14 could be observed in the most metaphase plates. trees. The phylogenetic trees constructed by the different A comparative study of centromeric indexes and relative methods were identical in the main topology. These lengths revealed no significant (P<0.05) interpopulation phylogenetic analyses revealed that S. solidum have differences in the basic karyotype structure. been clustered in one well-supported clade together with Complete nucleotide sequences are available in S. corneum, S. baicalense and S. nucleus (Figure 4 and GenBank under accession numbers FJ874903-09 and Figure 5). All other species have been included in more GU123689-91 (Table 3). Sequences of the nuclear distant clades. ribosomal ITS1 were determined for 6 specimens of Partial sequences of mitochondrial genes of the S. solidum from the Curonian Lagoon, one specimen from 16S large subunit were determined for 8 specimens

109 Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

Chromosome number Absolute lenght μm Relative length (%) Centromeric index Classification*

1 L 9.32 ± 1.52 12.17 ± 0.60 45.72 ± 1.65 m H 9.29 ± 1.35 12.36 ± 1.01 46.42 ± 3.05

2 L 8.36 ± 1.59 10.89 ± 0.81 45.00 ± 2.44 m H 8.42 ± 1.31 11.19 ± 0.77 43.57 ± 3.41

3 L 7.03 ± 1.22 9.16 ± 0.32 42.16 ± 4.39 m H 7.10 ± 0.62 9.50 ± 0.57 41.86 ± 3.58

4 L 6.41 ± 0.87 8.40 ± 0.48 36.75 ± 2.13 sm-m H 6.32 ± 0.94 8.40 ± 0.62 35.68 ± 3.48

5 L 5.90 ± 1.17 7.66 ± 0.61 39.68 ± 2.37 m H 5.75 ± 0.66 7.68 ± 0.48 39.08 ± 2.04

6 L 5.54 ± 0.82 7.25 ± 0.33 38.47 ± 3.77 m-sm H 5.48 ± 0.62 7.32 ± 0.45 39.99 ± 3.69

7 L 4.90 ± 0.70 6.42 ± 0.40 35.00 ± 3.78 sm-m H 4.76 ± 0.57 6.35 ± 0.46 36.61 ± 3.58

8 L 4.57 ± 0.95 5.93 ± 0.55 39.85 ± 4.83 m-sm H 4.38 ± 0.46 5.85 ± 0.38 39.38 ± 5.45

9 L 4.37 ± 0.48 5.74 ± 0.39 38.13 ± 4.67 m-sm H 4.17 ± 0.42 5.57 ± 0.20 39.65 ± 3.98

10 L 4.02 ± 0.47 5.27 ± 0.26 38.74 ± 3.22 m-sm H 3.89 ± 0.47 5.19 ± 0.26 38.43 ± 3.78

11 L 3.92 ± 0.47 5.12 ± 0.15 39.89 ± 3.24 m-sm H 3.52 ± 0.51 4.70 ± 0.36 37.27 ± 3.03

12 L 3.52 ± 0.47 4.62 ± 0.32 39.44 ± 4.45 m-sm H 3.50 ± 0.48 4.66 ± 0.30 38.45 ± 3.79

13 L 3.19 ± 0.47 4.18 ± 0.37 37.76 ± 3.63 m-sm H 3.14 ± 0.51 4.19 ± 0.51 37.79 ± 4.58

14 L 2.75 ± 0.48 3.58 ± 0.24 39.84 ± 3.22 m H 2.72 ± 0.20 3.64 ± 0.29 40.06 ± 3.88

15 L 2.76 ± 0.43 3.62 ± 0.35 39.99 ± 3.28 m H 2.55 ± 0.27 3.42 ± 0.32 42.31 ± 4.41

Table 2. Measurements (mean±SD) and classification of chromosomes of Sphaerium solidum (L, Lithuanian, H, Hungarian population).

* m, metacentric; sm, submetacentric chromosome

of S. solidum from Curonian Lagoon, including one to the first sampling place. Two haplotypes - Curo1 specimen from the water reservoir of the dammed and Curo2 – have been detected in the S. solidum up River Nemunas near Kaunas, and 8 specimens specimens from the Curonian Lagoon, and one of them from the Danube River. A total of 5 haplotypes have – Curo2 - is the same as a specimen from the water been detected among all analysed 16S sequences reservoir of the dammed up River Nemunas. Three (Table 3). The 16S haplotypes were named according different haplotypes have been detected in S. solidum

110 V. Stunžėnas et al.

GenBank numbers Collection numbers Sampling location Haplotype 16S ITS1

SS3, SS4 Curonian Lagoon Curo1 FJ874903 GU123690

SS12, SS13, SS14, SS15 Curonian Lagoon Curo2 FJ874904

SS10, SS21 Curonian Lagoon Curo2 FJ874905

SS440 Water reservoir on river Nemunas Curo2 FJ874906 GU123691

SSE13, SSE14, SSE15, SSE19 Danube River Duna1 FJ874907

SSE42, SSE44 Danube River Duna2 FJ874908

SSE46, SSE66 Danube River Duna3 FJ874909 GU123689

Table 3. Samples of Sphaerium solidum specimens subjected to DNA sequencing of the 16S and ITS1 loci. Collection numbers (collection of P.B. Šivickis Laboratory of Parasitology, Institute of Ecology of Nature Research Centre, Vilnius, Lithuania), the sampling location in Lithuania and Hungary, and the names of 16S haplotypes and GenBank accession numbers for 16S and ITS1 DNA sequences are provided.

Figure 4. Neighbour-joining phylogenetic tree obtained from ITS1 sequences of Sphaerium solidum and Sphaerium spp. based on the analysis of 496 sites (bootstrap replications = 10,000, complete deletion of gaps/missing data). The maximum composite likelihood model was used to calculate distances. Bootstrap percentages refer to NJ/ME/MP analysis. The phylogenetic trees constructed by these different methods were identical in the main topology. Kimura 2-parameter method was used for the determination distances between sequences in the ME analysis. Only bootstrap values above 70% are shown. Pisidium dubium and P. variabile were included as outgroups. Mitotic chromosome numbers are provided for cytogenetically studied taxa. Haplotype names are indicated for Sphaerium solidum specimens. Accession numbers are indicated for sequences obtained from GenBank. New sequence GenBank accession numbers are provided in Table 3.

111 Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

Figure 5. Maximum likelihood (ML) tree obtained from ITS1 sequences of Sphaerium solidum and Sphaerium spp. based on analysis of 496 sites. Pisidium dubium and P. variabile were included as outgroups. Bootstrap support (1000 replicates) for ML using GTR+G model and posterior probabilities estimated with MrBayes (500,000 generations, concluding with an average standard deviation of split frequencies below 0.01, sample frequency 100, burn-in 2500 generations) using the same model are given at the nodes (ML/Bayesian); only values above 70 are shown. The topology of the Bayesian tree was indentical to ML. Mitotic chromosome numbers are provided for cytogenetically studied taxa. Haplotype names are indicated for Sphaerium solidum specimens. Accession numbers are indicated for sequences obtained from GenBank. New sequence GenBank accession numbers are provided in Table 3.

from the Danube River: Duna1, Duna2 and Duna3. The 4. Discussion haplotypes Curo1, Curo2, Duna2 and Duna3 differ only in one base pair, while the haplotype Duna1 has more Chromosomal studies, together with molecular differences. The greatest difference was established analysis, may provide a unique perspective on between Duna1 and Duna2 – three base pairs, while the evolution and phylogenetic relationships of the comparison of Duna1 with Duna3 and Curo1 had sphaeriid clams. The present study revealed that showed differences in 2 base pairs. S. solidum is the diploid species in family Sphaeriidae The neighbour-joining and maximum likelihood trees including predominantly polychromosomic species. of 16S sequences is given in Figure 6 and Figure 7, The comparative karyological analysis provided respectively; the main topology of these trees was unexpected results - no significant differences (P<0.05) identical. Also, as with ITS1, the phylogenetic analysis in centromeric indexes or the relative length values of of 16S sequences revealed a well-supported S. solidum corresponding chromosomes of S. solidum and those subclade different from other sphaeriids in the both from the previously studied S. corneum, 2n=30 [19] was trees. This subclade was grouped in one well-supported found. The divergence of these two species proceeded clade comprising a subclade of S. corneum with 30 without changes in gross karyotype structure (number, chromosomes; while haplotypes of S. nucleus and size and shape of chromosomes). It is notable that the S. corneum with 36 chromosomes were clustered most molluscan groups are generally conservative together in another, well-supported sister clade. with regard to chromosome changes. For instance, the

112 V. Stunžėnas et al.

Figure 6. Neighbour-joining (NJ) phylogenetic tree for 16S haplotypes of Sphaerium solidum and Sphaerium spp. with known karyotypes. Pisidium dubium and P. variabile were included as outgroups. The tree was constructed by the maximum composite likelihood distance method, based on analysis of 472 sites of mitochondrial 16S rDNA sequences (bootstrap replications = 10,000, complete deletion of gaps/missing data). Bootstrap percentages refer to NJ/ME/MP analysis. The phylogenetic trees constructed by these different methods were identical in the main topology. The Kimura 2-parameter method was used for the determination of distances between sequences in the ME analysis. Only bootstrap values above 70% are shown. Mitotic chromosome numbers are provided for cytogenetically studied specimens. Haplotype names are indicated for new Sphaerium solidum sequences. Accession numbers are indicated for sequences obtained from GenBank. New sequence GenBank accession numbers are provided in Table 3. evolution of unionid mussels has proceeded without chromosomes were observed in chromosome sets of any change in chromosome number [37]. But this Cerastoderma edule (Veneroida); these were small, is not a case in sphaeriid clams, with chromosome much smaller than that of the smallest in the standard number ranging from 30 to 247 [17]. Moreover, the complement and occurred in number of 1 to 3 [46]. karyotypes of S. solidum and S. corneum are similar Two populations of S. solidum, studied herein, differ in the presence of small, metacentric and mitotically in frequency of B chromosomes. Significantly more unstable B chromosomes. The term, Bs (also named cells with B’s were found in the Lithuanian population. as supernumerary or accessory chromosomes), Ecological and historical factors could provide includes very heterogeneous types of chromosomes, complementary explanations for B chromosome differing in size, shape and molecular composition, and geographical distribution. Results of the survey of the only consistent feature of Bs is that they are not some plant and species led to the conclusion essential for survival of an individual and are present that the higher incidence of B chromosomes was in some individuals from some populations in some associated with more favourable environments species [38-41]. Intraindividual mosaicism caused by a [47-49]. The results of the survey of other species variable number of B chromosomes has been reported showed the opposite trend - higher frequencies of in various species [41-43]. Although B chromosomes animals with B’s in peripheral populations were found are found in numerous species of all major groups [50,51] and our data correspond to this trend, because of animals and plants, data on the occurrence of B the territory of Hungary was not covered with ice chromosomes in bivalve molluscs are so far very during the last glacial period [52,53] and Lithuanian scarce. Variable numbers of B chromosomes were populations of S. solidum could be regarded as more recorded in clonal lineages of marine clams of the peripheral ones, originating after last glaciation period. Lasea (Veneroida) [44,45]. Supernumerary Our data are too scarce for any generalizations, but

113 Phylogeny of Sphaerium solidum (Bivalvia) based on karyotype and sequences of 16S and ITS1 rDNA

Figure 7. Maximum likelihood (ML) tree for 16S haplotypes of Sphaerium solidum and Sphaerium spp. with known karyotypes. Pisidium dubium and P. variabile were included as outgroups. The tree is based on the analysis of 496 sites. Bootstrap support (1000 replicates) for ML using the GTR +G model and posterior probabilities estimated with MrBayes (500,000 generations, concluding with an average standard deviation of split frequencies below 0.01, sample frequency 100, burn-in 2500 generations) using the same model are given at the nodes (ML/ Bayesian); only values above 70 are shown. The main topology of the Bayesian tree was indentical to ML. Mitotic chromosome numbers are provided for cytogenetically studied taxa. Haplotype names are indicated for Sphaerium solidum specimens. Accession numbers are indicated for sequences obtained from GenBank. New sequence GenBank accession numbers are provided in Table 3.

the subsequent studies on additional populations of Petkevičiūtė et al. [19] in 16S based phylogenies of S. solidum could help to reveal the factors influencing Sphaerium spp. have revealed some intriguing points. the frequencies of B chromosomes. Although haplotypes of S. solidum and S. corneum The comparative analyses of sphaeriid gene represent the different phylogenetic branches in fragments supported previous findings that mitochondrial the phylogenetic trees (Figure 6 and Figure 7), the 16S DNA sequence is more variable and molecular differences between the S. solidum haplotype Duna1 substitutions accrue more rapidly in it than in nuclear and nearest haplotype of S. corneum (GenBank ITS1 DNA sequence. For some congeneric species, No. AF152037) exist as only in 3 base pairs and ITS1 was found to be identical, such as it is in European is equal to intraspecific differences between most S. corneum and endemic of Lake Baikal S. baicalense distinct haplotypes of S. solidum. All other genetically [16]. ITS1 of two North American taxa, Sphaerium studied sphaeriid species are phylogenetically more rhomboideum and S. occidentale, were found to be differentiated from S. solidum. Based on the genetic compellingly similar in contrast to essentially different analysis, the species distinctness of S. solidum may be karyotypes [16,17]. Identical ITS1 was also revealed for well concluded from 16S sequences. two karyomorphs of Lithuanian S. corneum, differing in Interestingly, according to Korniushin [3] and chromosome set structure and 16S sequences [19]. In Korniushin and Glaubrecht [15] morphologically our present analysis, ITS1 sequences of S. solidum differ S. corneum appeared more similar to S. nucleus from S. corneum only in one nucleotide pair site, but this and S. rhomboideum (the other member of the site was parsimonious informative, and the phylogenetic Sphaerium s. str. subgenus with diploid chromosome analysis well separated S. solidum from S. corneum. set), but S. solidum remained as a single species of Comparisons of the 16S sequences of S. solidum the subgenus Cyrenastrum Bourguignat, 1854. In the with those obtained by Lee and Ó Foighil [16] and 16S phylogenetic trees (Figure 6 and Figure 7), two

114 V. Stunžėnas et al.

karyomorphs of S. corneum were divided into two basal position of the diploid sphaeriid clade in the 16S different sister clades, there S. solidum and S. corneum trees (Figure 6 and Figure 7), but in the ITS1 trees, containing 30 chromosomes were clustered in S. rhomboideum occurs in one highly supported clade one of these sister clades. With the karyological with polychromosomic S. occidentale (Figure 4 and and molecular data now available it can be stated Figure 5). The phylogenetic position of S. rhomboideum that, despite undoubted morphological differences was well discussed in the previous publication [17]. and distinct ecological preferences (S. corneum is In summary, it is worthwhile to stress that while extremely eurytopic species, living in a wide range the family Sphaeriidae as whole is characterised by of lentic and lotic aquatic habitats), there is a very an extreme karyotypic diversification, the comparative close phylogenetic affinity between S. solidum and the analysis of chromosome set structure and DNA S. corneum with 2n=30. However, the karyotypic form sequences of S. solidum provides a particularly striking of S. corneum, 2n=36, clusters together with the other example of species divergence within Sphaeriidae. member of the subgenus Sphaerium s. str., S. nucleus, Although morphology, ecological preferences, ITS1 in 16S phylogenetic trees (Figure 6 and Figure 7). and 16S sequences demonstrate the distinctness of The discontinuity in the karyological as well as in the S. solidum and S. corneum These species retained molecular phylogenetic evidences obtained support stable gross chromosome set structure, 2n=30 +Bs. the assumption that S. corneum is a paraphyletic The new data, reported therein, constitutes a notable taxon. Moreover, with the data obtained it could be addition to the understanding of diversity and argued that karyotypic and mollecular distinctness of evolution of sphaeriid clams. Clustering of diploid karyotypic form 2n=36 is enough to regard it as a taxon and polychromosomic Sphaerium species into of its own. different clades could denote the relation between In the both 16S phylogenetic trees of (Figure 6 and chromosomal changes and the speciation process, Figure 7), Sphaerium spp. clustered in two different but more comprehensive studies on additional species clades with high bootstrap support: one with all known are required to reveal any consistent patterns of diploid Sphaerium spp and other with polychromosomic evolutionary changes in Sphaeriidae. Sphaerium spp. It is worth to note, that in the cluster of the species with polychromosomic sets the closely related Sphaerium spp. greatly differs in chromosome Acknowledgments numbers. The new molecular data did not changed the main topology of Sphaerium phylogeny that was This study was supported by the Lithuanian Science described in previous publications [16,17,19], but and Studies Foundation research grants C-07002 and data on phylogenetic position of S. solidum gave T-09075. The authors would like to thank to Erika Bódis strong support for the monophyly of Palaearctic diploid and Bence Tóth (Hungarian Danube Research Station Sphaerium spp. and leads to the presumption that of the Hungarian Academy of Sciences) for helping in S. nucleus and S. baicalense should be also diploid the collection of sphaeriid clams in the Danube River species. The Nearctic diploid, S. rhomboideum is the and Dr. Darius Daunys (Coastal Research and Planning only one exception from the diploid sphaeriid clustering. Institute, Klaipėda University) for information on exact This species was placed on the branch nearest to the S. solidum habitats in Curonian Lagoon.

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