Taxonomic diversity of the Hipposideros larvatus species complex (Chiroptera: Hipposideridae) in mainland Asia.

Hipposideros larvatus sensu lato constitutes a widespread species complex consisting of morphologically similar forms with a taxonomy that is in need of a revision. Here we present the results of a phylogenetic analysis of members of this species complex based on two mitochondrial (CytB and COI) and seven nuclear (ABHD11, ACOX2, COPS, RAG2, ROGDI2, SORBS2 and THY) genes, and a morphometric analysis. Morphological data, although showing certain diversity, does not allow an accurate delimitation of the taxonomic boundaries and occasionally contradict to genetic grouping. The molecular analyses based on mitochondrial data support the paraphyly of the H. larvatus species complex, with H. armiger recovered nested within. The nuclear data recovered all the lineages of H. larvatus sensu lato forming a monophyletic clade. Within it, apparently four different species-level clades could be revealed in Southeast Asia. One is distributed in northern Myanmar, southern China (including Hainan) and northern Vietnam, and also reaches into central Vietnam along the coast (Cham Islands). The valid name for this taxon must be H. poutensis Allen. The second clade is comprised by populations inhabiting the Malay Peninsula and the Sunda Islands, we identify this taxon as H. larvatus sensu stricto. The third taxon was identified only from southern Myanmar. The fourth taxon is distributed in southern and central Indochina. Its representatives were not related to any of the forms identified in Myanmar, and therefore, its previous identification with H. grandis sounds questionable. Since both the third and fourth taxa cannot now be unambiguously associated with a particular named form, we conventionally designate them as H. cf. grandis 1 and 2.

Utilizing the VirIdAl Pipeline to Search for Viruses in the Metagenomic Data of Bat Samples.

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.

Gene Loss, Pseudogenization in Plastomes of Genus Allium (Amaryllidaceae), and Putative Selection for Adaptation to Environmental Conditions.

Amaryllidaceae is a large family with more than 1,600 species, belonging to 75 genera. The largest genus-Allium-is vast, comprising about a thousand species. Allium species (as well as other members of the Amaryllidaceae) are widespread and diversified, they are adapted to a wide range of habitats from shady forests to open habitats like meadows, steppes, and deserts. The genes present in chloroplast genomes (plastomes) play fundamental roles for the photosynthetic plants. Plastome traits could thus be associated with geophysical abiotic characteristics of habitats. Most chloroplast genes are highly conserved and are used as phylogenetic markers for many families of vascular plants. Nevertheless, some studies revealed signatures of positive selection in chloroplast genes of many plant families including Amaryllidaceae. We have sequenced plastomes of the following nine Allium (tribe Allieae of Allioideae) species: A. zebdanense, A. moly, A. victorialis, A. macleanii, A. nutans, A. obliquum, A. schoenoprasum, A. pskemense, A. platyspathum, A. fistulosum, A. semenovii, and Nothoscordum bivalve (tribe Leucocoryneae of Allioideae). We compared our data with previously published plastomes and provided our interpretation of Allium plastome genes' annotations because we found some noteworthy inconsistencies with annotations previously reported. For Allium species we estimated the integral evolutionary rate, counted SNPs and indels per nucleotide position as well as compared pseudogenization events in species of three main phylogenetic lines of genus Allium to estimate whether they are potentially important for plant physiology or just follow the phylogenetic pattern. During examination of the 38 species of Allium and the 11 of other Amaryllidaceae species we found that rps16, rps2, infA, ccsA genes have lost their functionality multiple times in different species (regularly evolutionary events), while the pseudogenization of other genes was stochastic events. We found that the "normal" or "pseudo" state of rps16, rps2, infA, ccsA genes correlates well with the evolutionary line of genus the species belongs to. The positive selection in various NADH dehydrogenase (ndh) genes as well as in matK, accD, and some others were found. Taking into account known mechanisms of coping with excessive light by cyclic electron transport, we can hypothesize that adaptive evolution in genes, coding subunits of NADH-plastoquinone oxidoreductase could be driven by abiotic factors of alpine habitats, especially by intensive light and UV radiation.

Back to life and to taxonomy: new record and reassessment of Myotis bucharensis (Chiroptera: Vespertilionidae).

Myotis bucharensis is one of the least studied Palaearctic bat species, known from only three localities in Tajikistan and Uzbekistan and not reported since 1965. In autumn 2019, a male Myotis captured in Zerafshan river basin in Tajikistan was later identified as M. bucharensis based on tail and tibia proportions and strongly displacement of posterior small premolars. The identification was then confirmed by morphometric analyses supporting that M. bucharensis is specifically different but represents a part of the Myotis frater complex. Analyses of one mitochondrial (cyt b) and one nuclear gene (RAG2) were performed for the first time for M. bucharensis. According to these genetic results, this form is indeed a member of the «daubentonii¼ clade, which includes all known frater-like Myotis, and most likely represents a sister species to M. longicaudatus. Record of the alive specimen M. bucharensis has valuable implication for bat conservation in Tajikistan.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.