Phylogeny of Sphaerium Solidum (Bivalvia) Based on Karyotype and Sequences of 16S and ITS1 Rdna
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Cent. Eur. J. Biol. • 6(1) • 2011 • 105–117 DOI:DOI 10.2478/s11535-010-0101-6 Central European Journal of Biology Phylogeny of Sphaerium solidum (Bivalvia) 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 Sphaerium solidum, 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: Sphaeriidae • Sphaerium solidum • Sphaerium corneum • Cyrenastrum • Karyotype • B chromosomes • Haplotype • 16S mitochondrial ribosomal DNA • ITS1 nuclear rDNA sequence • Phylogeny ©VersitaSp.zo.o. 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 animals 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 eutrophication 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 taxonomy [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;