Summary1. The recognition of management units (MUs) that respect interpopulation distinctions in management needs is central to many biological applications addressing species conservation, biological invasions and ecosystem processes. 2. We present a methodological approach for the evaluation of population-level differences in the host compatibility of natural populations of affiliate (dependent) species. Two experiments were performed to diagnose the sources of variability in the relationships between an endangered freshwater mussel Unio crassus and its host fish species in a fragmented river system in Central Europe. 3. Despite the common approach applied to the conservation of U. crassus throughout the study area, we identified differences in the ability of U. crassus to infest particular host fish species between nearby and recently isolated mussel populations. These differences could strongly influence their reproductive success under a constant conservation regime. 4. The observed differences were accompanied by genetic and morphometric distinctions, as revealed by microsatellite, mtDNA and elliptic Fourier descriptor analyses. The genetic data indicated substantial reproductive isolation, which could have resulted in adaptive or random changes responsible for the differences in host compatibility. 5. We showed that experimental testing of physiological host compatibility can be effectively used for the detection of different MUs of U. crassus, with direct implications for the management of host populations. In combination with traditional genetic and morphometric techniques, this approach may provide a general framework for the recognition and care of management units of affiliate species. 6. Synthesis and applications. Population-level evaluations of host compatibility should be utilized in management unit recognition and the formulation of management targets for affiliate species. We demonstrated that small-scale cross-compatibility testing can be effectively used to diagnose the sources of variability in host relationships with direct management implications. This approach can enhance the targeting of management actions in many biological applications addressing species conservation, biological invasions and ecosystem processes.

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We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e. a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in the previous studies on the microbial community of tardigrades as well as Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.

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.