The Patterns and Puzzles of Genetic Diversity of Endangered Freshwater Mussel Unio crassus Philipsson, 1788 Populations from Vistula and Neman Drainages (Eastern Central Europe).

Mussels of the family Unionidae are important components of freshwater ecosystems. Alarmingly, the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species identifies almost 200 unionid species as extinct, endangered, or threatened. Their decline is the result of human impact on freshwater habitats, and the decrease of host fish populations. The Thick Shelled River Mussel Unio crassus Philipsson, 1788 is one of the examples that has been reported to show a dramatic decline of populations. Hierarchical organization of riverine systems is supposed to reflect the genetic structure of populations inhabiting them. The main goal of this study was an assessment of the U. crassus genetic diversity in river ecosystems using hierarchical analysis. Different molecular markers, the nuclear ribosomal internal transcribed spacer ITS region, and mitochondrial DNA genes (cox1 and ndh1), were used to examine the distribution of U. crassus among-population genetic variation at multiple spatial scales (within rivers, among rivers within drainages, and between drainages of the Neman and Vistula rivers). We found high genetic structure between both drainages suggesting that in the case of the analyzed U. crassus populations we were dealing with at least two different genetic units. Only about 4% of the mtDNA variation was due to differences among populations within drainages. However, comparison of population differentiation within drainages for mtDNA also showed some genetic structure among populations within the Vistula drainage. Only one haplotype was shared among all Polish populations whereas the remainder were unique for each population despite the hydrological connection. Interestingly, some haplotypes were present in both drainages. In the case of U. crassus populations under study, the Mantel test revealed a relatively strong relationship between genetic and geographical distances. However, in detail, the pattern of genetic diversity seems to be much more complicated. Therefore, we suggest that the observed pattern of U. crassus genetic diversity distribution is shaped by both historical and current factors i.e. different routes of post glacial colonization and history of drainage systems, historical gene flow, and more recent habitat fragmentation due to anthropogenic factors.

Detection of bacterial endosymbionts in freshwater crustaceans: the applicability of non-degenerate primers to amplify the bacterial 16S rRNA gene.

Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.

First insight into microbiome profile of fungivorous thrips Hoplothrips carpathicus (Insecta: Thysanoptera) at different developmental stages: molecular evidence of Wolbachia endosymbiosis.

Insects' exoskeleton, gut, hemocoel, and cells are colonized by various microorganisms that often play important roles in their host life. Moreover, insects are frequently infected by vertically transmitted symbionts that can manipulate their reproduction. The aims of this study were the characterization of bacterial communities of four developmental stages of the fungivorous species Hoplothrips carpathicus (Thysanoptera: Phlaeothripidae), verification of the presence of Wolbachia, in silico prediction of metabolic potentials of the microorganisms, and sequencing its mitochondrial COI barcode. Taxonomy-based analysis indicated that the bacterial community of H. carpathicus contained 21 bacterial phyla. The most abundant phyla were Proteobacteria, Actinobacteria, Bacterioidetes and Firmicutes, and the most abundant classes were Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Betaproteobacteria, with different proportions in the total share. For pupa and imago (adult) the most abundant genus was Wolbachia, which comprised 69.95% and 56.11% of total bacterial population respectively. Moreover, similarity analysis of bacterial communities showed that changes in microbiome composition are congruent with the successive stages of H. carpathicus development. PICRUSt analysis predicted that each bacterial community should be rich in genes involved in membrane transport, amino acid metabolism, carbohydrate metabolism, replication and repair processes.

Instances of erroneous DNA barcoding of metazoan invertebrates: Are universal cox1 gene primers too "universal"?

The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer's "universal" primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer's primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.

The complete maternal and paternal mitochondrial genomes of Unio crassus: Mitochondrial molecular clock and the overconfidence of molecular dating.

The availability of a rapidly growing number of complete mitochondrial genome sequences provokes high confidence dating approaches. However, even if the congruence between mitochondrial and nuclear markers is reasonable, the resulting topologies are frequently questionable. The unique opportunity to study the evolutionary history of two independent mitochondrial genomes in one phylogenetic context exists in the freshwater mussels family Unionidae. The two lineages function under doubly uniparental inheritance since well before the emergence of the family. Despite the relatively high number of available complete sequences of maternally inherited genomes, comparative analyses are limited by the small number of sequences of counterpart paternally inherited genomes. We have sequenced for the first time the representative set of five sequences (two maternal and three paternal) from the species Unio crassus. Comparative analysis of the phylogenies reconstructed using relevant mitogenomic data available in GenBank (13 species in total) reveal that single - genome inferences are congruent only if the relaxed clock is assumed.

Gender-Associated Mitochondrial DNA Heteroplasmy in Somatic Tissues of the Endangered Freshwater Mussel Unio crassus (Bivalvia: Unionidae): Implications for Sex Identification and Phylogeographical Studies.

Some bivalve species possess two independent mitochondrial DNA lineages: maternally (F-type) and paternally (M-type) inherited. This phenomenon is called doubly uniparental inheritance. It is generally agreed that F-type mtDNA is typically present in female somatic and gonadal tissues as well as in male somatic tissues, whereas the M-type mtDNA occurs only in male germ line and gonadal tissue. In the present study, the mtDNA heteroplasmy (for both F and M genomes) in male somatic tissues of Unio crassus (Philipsson, 1788), species threatened with extinction, has been confirmed. Taking advantage from the presence of Mcox1 marker only in male somatic tissues, we developed a new method of sex identification in this endangered species, using nondestructive tissue sampling. Probability of correct sex identification was estimated at 97.5%. The present study is the first report on gender-associated mitochondrial DNA heteroplasmy in male somatic tissues of thick-shelled river mussel and first approach to U. crassus sex identification at molecular level. Our study also confirmed the utility of paternally inherited Mcox1 gene fragment as a complementary molecular tool for resolving phylogeographical relationships among populations of thick-shelled river mussel.

Identification and characterization of the first microsatellite loci for the thick-shelled river mussel Unio crassus (Bivalvia: Unionidae).

The thick-shelled river mussel Unio crassus is critically endangered throughout its range as a result of increasing human activity and habitat loss. Next generation DNA sequencing was used to develop a set of microsatellite markers that can be used for future ecological and population genetics studies of U. crassus. A total of 11 polymorphic loci were identified and characterized using 57 individuals from two Polish populations. Numbers of alleles ranged from 2 to 15 and expected heterozygosity levels ranged from 0.069 to 0.899. Deficiency of heterozygous genotypes was observed in four loci. Marker independence was confirmed with tests for linkage disequilibrium, however, analyses indicated evidence of null alleles in four loci. The microsatellite markers developed specifically for U. crassus provide a valuable tool for future ecological, population genetic assessments, and conservation management of this species.

A new view on dam lines in Polish Arabian horses based on mtDNA analysis.

Polish Arabian horses are one of the oldest and the most important Arab populations in the world. The Polish Arabian Stud Book and the Genealogical Charts by Skorkowski are the main sources of information on the ancestors of Polish Arabs. Both publications were viewed as credible sources of information until the 1990s when the data regarding one of the dam lines was questioned. The aim of the current study was to check the accuracy of the pedigree data of Polish dam lines using mtDNA analysis. The analyses of a 458 bp mtDNA D-loop fragment from representatives of 15 Polish Arabian dam lines revealed 14 distinct haplotypes. The results were inconsistent with pedigree data in the case of two lines. A detailed analysis of the historical sources was performed to explain these discrepancies. Our study revealed that representatives of different lines shared the same haplotypes. We also noted a genetic identity between some lines founded by Polish mares of unknown origin and lines established by desert-bred mares.

Genetic differences among several species of Tricladida from the relict Lake Ohrid as revealed by enzyme electrophoresis.

Six endemic and two widely distributed species living in Lake Ohrid were studied. In general, these hermaphroditic animals displayed no signs of departure from Hardy-Weinberg equilibrium. Genetic variation in all but one of the endemic species was of the same extent as that in geographically wide ranging invertebrates. On the other hand, the Lake Ohrid population of the common European species Dendrocoelum lacteum was monomorphic at all loci examined. D. sanctinaumi, one of the endemic species, exhibited a clear genetic subdivision into spring and littoral subpopulations. The genetic differentiation of Crenobia alpina alpina and C. a. montenigrina proved commensurable to that of well separated species from other genera. The data suggest that the separation of particular lineages in the set of Lake Ohrid endemics was widely dispersed over time.