Nuclear mtDNA pseudogenes as a source of new variants of the mtDNA cytochrome <Emphasis Type="Italic">b</Emphasis> haplotypes: A case study of Siberian rubythroat <Emphasis Type="Italic">Luscinia calliope</Emphasis> (Muscicapidae,Aves)

Sequence polymorphism of the mitochondrial DNA cytochrome b gene fragment was analyzed in 21 specimens of subspecies Luscinia calliope calliope (Pallas, 1776) and two specimens of L. c. anadyrensis (Portenko, 1939). On sequence chromatograms, in 19 specimens of L. c. calliope, double peaks of heteroplasmy type in the taxon-specific positions were revealed. Moreover, two clone variants were identified. The first variant was the calliope mitochondrial cyt b gene and the second was the nuclear cyt b pseudogene, similar to the mitochondrial haplotype anadyrensis-camtschatkensis. In L. c. anadyrensis, four clone variants, represented by the mitochondrial calliope and anadyrensis-camtschatkensis cyt b genes and nuclear calliope and sachalinensis cyt b pseudogenes, were identified. Some nuclear cyt b pseudogenes were highly similar (98–99%) to the mitochondrial genes of the subspecies L. c. anadyrensis, L. c. camtschatkensis, and L. c. sachalinensis. In the same time, the majority of nuclear pseudogene sequences were characterized by a high level of polymorphism, caused by nonsynonymous substitutions (up to five substitutions per sequence), the presence of indels in some of the clones, and TAA and TGA stop codons. In our opinion, the mitochondrial haplotypes anadyrensis-camtschatkensis and sachalinensis occurred as a result of intergenomic homologous recombination. This finding provides a new insight into the colonization history of the northeastern part of the range by L. calliope, according to which populating the territory of Chukotka, Kamchatka, and Sakhalin took place at different times and along the independent pathways.     

Genetic structure of Schrenck newt <Emphasis Type="Italic">Salamandrella schrenckii</Emphasis> populations by mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> variation

The nucleotide sequence variation of the mitochondrial cytochrome b gene was studied in Schrenck newt Salamandrella schrenckii (Strauch, 1870) from populations of Primorye and the Khabarovsk region. Phylogenetic analysis revealed two haplotype clusters, southern cluster 1 and northern cluster 2, with a divergence of 3%. Analysis of the mtDNA and cytochrome b amino acid sequence variations made it possible to assume that the modern range of Schrenck newt was colonized from south Primorye northwards. In contrast to the southern cluster, the northern one demonstrated all the signs of demographic expansion (a unimodal distribution of pairwise nucleotide differences, specific results of tests for selective neutrality of mtDNA variation, and a good correspondence of genetic parameters to those expected from demographic expansion models).     

Species complex delimitation and patterns of population structure at different geographic scales in Neotropical silver catfish (<Emphasis Type="Italic">Rhamdia</Emphasis>: Heptapteridae)

The Neotropical catfish genus Rhamdia inhabits rivers and lakes from Mexico to Argentina. Previous studies have found that the taxonomy of this genus, as well as that of R. quelen, remains controversial. The present study aims to contribute to the understanding of Rhamdia systematics by delimiting putative species, and to elucidate the pattern of genetic differentiation of Rhamdia at different geographic levels within the cis-andean region. Species boundaries were defined by Generalized Mixed Yule Coalescent and Automatic Barcode Gap Discovery methods, and by phylogenetic analyses of cytochrome b (cyt b) sequences. Moreover, we performed phylogeographic analyses based on cyt b sequences and microsatellite markers. Patterns of differentiation were analyzed at three nested geographic levels: in the main cis-andean basins (macrogeographic scale); in the second major Neotropical basin system (mesogeographic scale), which encompasses La Plata basin, Patos-Merin basin, and the coastal lagoons draining to SW Atlantic Ocean; and finally, in the three most important coastal lagoons for artisanal fisheries in Uruguay (microgeographic scale). Sixteen species were found within Rhamdia, divided into two clades (cis- and trans-andean clades), each composed of eight putative species. Cis-andean Rhamdia species have probably diverged due to vicariance events occurring between and within basins since late Miocene-Pleistocene. Microgeographic scale analysis based on cyt b and microsatellite data revealed a high genetic structuring among the studied coastal lagoons. Mitochondrial and microsatellite markers enabled to identify three different populations, corresponding to the three coastal lagoons analyzed, which would have diverged recently and could be considered as different Management Units.     

<Emphasis Type="Italic">Basidiophora delawarensis</Emphasis>, a new downy mildew species infecting cultivated goldenrod (<Emphasis Type="Italic">Solidago sphacelata</Emphasis>) in the USA

Species in the genus Basidiophora (Oomycota, Peronosporales) are pathogens causing downy mildew disease on several closely related plant hosts in the family Asteraceae, including the genera Conyza, Erigeron, Solidago, and Symphyotrichum. Despite their widespread occurrence, little is known about the diversity, biology, and economic impact of downy mildew pathogens of ornamental or wild plants in the Asteraceae. In June 2017, Solidago sphacelata plants of the cultivar ‘Golden Fleece’ were found in the state of Delaware, USA, showing typical symptoms of downy mildew disease. Initial morphological observations indicated the specimen belonged to the genus Basidiophora. Phylogenetic analysis of a sequence matrix of the partial cox2 mitochondrial gene using Bayesian and maximum likelihood methods showed that the Basidiophora isolate from S. sphacelata represents an undescribed species that is here described as B. delawarensis. This new species constitutes the third accepted species in the genus Basidiophora and can be distinguished from other Basidiophora species by differences in size of sporangia and sporangiophores and nucleotide sequence divergence. This new species poses a potential threat to its host, warranting close monitoring and the implementation of disease management programs.     

Intraspecific Variation in Mitogenomes of Five <Emphasis Type="Italic">Crassostrea</Emphasis> Species Provides Insight into Oyster Diversification and Speciation

A large number of Crassostrea oysters are found in Asia-Pacific. While analyses of interspecific variation have helped to establish historical relationships among these species, studies on intraspecific variation are necessary to understand their recent evolutionary history and current forces driving population biology. We resequenced 18 and analyzed 31 mitogenomes of five Crassostrea species from China: Crassostrea gigas, Crassostrea angulata, Crassostrea sikamea, Crassostrea ariakensis, and Crassostrea hongkongensis. Our analysis finds abundant insertions, deletions, and single-nucleotide polymorphisms in all species. Intraspecific variation varies greatly among species with polymorphic sites ranging from 54 to 293 and nucleotide diversity ranging from 0.00106 to 0.00683. In all measurements, C. hongkongensis that has the narrowest geographic distribution exhibits the least sequence diversity; C. ariakensis that has the widest distribution shows the highest diversity, and species with intermediate distribution show intermediate levels of diversity. Low sequence diversity in C. hongkongensis may reflect recent bottlenecks that are probably exacerbated by human transplantation. High diversity in C. ariakensis is likely due to divergence of northern and southern China populations that have been separated without gene flow. The significant differences in mitogenome diversity suggest that the five sister species of Crassostrea have experienced different evolutionary forces since their divergence. The recent divergence of two C. ariakensis populations and the C. gigas/angulata species complex provides evidence for continued diversification and speciation of Crassostrea species along China’s coast, which are shaped by unknown mechanisms in a north–south divide.     

Genetic characterization of <Emphasis Type="Italic">Wolbachia</Emphasis> from Great Salt Lake brine flies

Wolbachia are intracellular prokaryotic endosymbionts associated with a wide distribution of arthropod and nematode hosts. Their association ranges from parasitism to mutualism, and there is growing evidence that Wolbachia can have dramatic effects on host reproduction, physiology, and immunity. Although all Wolbachia are currently considered as single species, W. pipientis, phylogenetic studies reveal about a dozen monophyletic groups, each designated as a supergroup. This study uses 16S rRNA gene sequences to examine the genetic diversity of Wolbachia present in three species of Great Salt Lake brine flies, Cirrula hians, Ephydra gracilis, and Mosillus bidentatus. The brine fly Wolbachia sequences are highly similar, with an average nucleotide sequence divergence among the three species of 0.00174. The brine fly Wolbachia form a monophyletic group that is affiliated with a subset of supergroup B, indicating that this supergroup may be more diverse than previously thought. These findings expand the phylogenetic diversity of Wolbachia and extend their host range to taxa adapted to a hypersaline environment.     

Phylogenetic relationships of Sakhalin taimen <Emphasis Type="Italic">Parahucho perryi</Emphasis> inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

Complete mitochondrial genome of threatened mahseer <Emphasis Type="Italic">Tor tor</Emphasis> (Hamilton 1822) and its phylogenetic relationship within Cyprinidae family

The mahseers (Tor, Neolissochilus and Naziritor) are an important group of fishes endemic to Asia with the conservation status of most species evaluated as threatened. Conservation plans to revive these declining wild populations are hindered by unstable taxonomy. Molecular phylogeny studies with mitochondrial genome have been successfully used to reconstruct the phylogenetic tree and to resolve taxonomic ambiguity. In the present study, complete mitochondrial genome of Tor tor has been sequenced using ion torrent next-generation sequencing platform with coverage of more than 1000 ×. Comparative mitogenome analysis shows higher divergence value at ND1 gene than COI gene. Further, occurrence of a distinct genetic lineage of T. tor is revealed. The phylogenetic relationship among mahseer group has been defined as Neolissochilus hexagonolepis ((T. sinensis (T. putitora, T. tor), (T. khudree, T. tambroides)).     

mtDNA-based identification of two widespread roach species (<Emphasis Type="Italic">Rutilus</Emphasis>, Cyprinidae) characterized by sympatric zone

Test systems using multiplex PCR and restriction analysis have been designed for the simple and rapid identification of two widespread species of roach, Rutilus rutilus and R. lacustris, characterized by the sympatric zone. This method is based on the variation of nucleotide sequences of the mitochondrial markers (cytochrome oxidase I subunit and cytochrome b); it allows identifying the species omitting the DNA sequencing.     

High Level of Interspecific Divergence in the <Emphasis Type="Italic">Salamandrella</Emphasis> Genus Based on Variability of the <Emphasis Type="Italic">RAG2</Emphasis> Gene

Previously, a very low level of divergence between the species of the genus Salamandrella—S. keyserlingii and S. schrenckii—was detected on the basis of variability of the nucleotide sequences of three genes of the nuclear genome (BDNF, POMC, and RAG1). Fixed interspecific differences were detected only in one nucleotide position of the RAG1 gene, and the level of interspecific divergence for this gene was only 0.07%. In this paper, we present the results of a study of the variability of the ENC1, MGAT4C, and RAG2 nuclear genes. The level of interspecific divergence for the MGAT4C gene was 0.14%, and for the RAG2 gene, it was 0.8%. The results of a phylogenetic analysis of the nucleotide sequences of the RAG2 gene in representatives of the family Hynobiidae indicate that the separation of the Salamandrella branch, which is basal for the genera Batrachuperus, Liua, Hynobius, and Pseudohynobius, occurred approximately 55 million years ago. The time of divergence between species of the Salamandrella genus was approximately 21 million years ago.     

Revision of the taxonomic position of the Olkhon mountain vole (Rodentia,Cricetidae)

The phylogenetic position of the Olkhon mountain vole (Alticola olchonensis Litvinov, 1960) was studied using the sequences of four nuclear (BRCA, GHR, LCAT, and IRBP) and one mitochondrial (cyt. b) gene. Until now multiple studies of the systematic position of this vole had been based exclusively on morphological data, while the major taxonomic references contain contradictory information regarding both the subgeneric and species status of this animal. It was established that the molecular data and morphology data allow us to concern the Lake Baikal vole unambiguously as a part of the nominative subgenus Alticola instead of Aschizomys.     

Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

Study on chloroplast DNA diversity of cultivated and wild pears (<Emphasis Type="Italic">Pyrus</Emphasis> L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (P_i), average number of nucleotide differences (k) and haplotype diversity (H_d) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.     

Microsatellite analysis supports the existence of three cryptic species within the bumble bee <Emphasis Type="Italic">Bombus lucorum sensu lato</Emphasis>

Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA ‘barcodes’ is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Genetic polymorphism of commander islands polar foxes <Emphasis Type="Italic">Alopex lagopus semenovi</Emphasis> Ognev, 1931 and <Emphasis Type="Italic">Alopex lagopus beringiensis</Emphasis> Merriam, 1902

The polymorphism of the mitochondrial DNA (mtDNA) control region sequence was examined in 30 polar foxes from Bering Island and 30 polar foxes from Mednyi Island. Seven haplotypes were revealed in polar foxes from Bering Island, and one, in polar foxes from Mednyi Island. The age of divergence of these populations (12 000 ± 6 000 years) was calculated based on a fragment of the D-loop. In Bering polar foxes, the sequence nucleotide diversity (π) was 0.003 (S.D. = 0.002), the haplotype diversity h in Bering polar foxes was 0.835 (S.D. = 0.037). The effective number of females n the Bering Island population was estimated as 105 animals.     

Tashkent riffle minnow <Emphasis Type="Italic">Alburnoides oblongus</Emphasis> belongs to the genus <Emphasis Type="Italic">Alburnus</Emphasis> (Osteichthyes: Cyprinidae) as inferred from mitochondrial and nuclear DNA markers

Sequences of mitochondrial (cytochrome b) and nuclear (recombination activating gene 1–RAG1) DNA markers were obtained for two species of the genus Alburnoides, the Taskent riffle minnow A. oblongus Bulgakov 1923 and the Terek spirlin A. gmelini Bogutskaya and Coad 2009. Phylogenetic analysis revealed that A. oblongus belongs to the genus Alburnus.     

Molecular evolution of glutathione peroxidase (<Emphasis Type="Italic">Gpx</Emphasis>) family genes in desert poplar (<Emphasis Type="Italic">Populus euphratica</Emphasis> Oliv.)

Populus euphratica Oliv. is a poplar species that is distributed mainly in deserts, making it an interesting model in which to investigate molecular mechanisms underlying different stress responses. Here, we used molecular population genetic methods to detect potential selection in candidate genes belonging to the P. euphratica glutathione (GSH) peroxidase (Gpx) family, which are associated with an enzymatic mechanism that combats oxidative damage caused by reactive oxygen species (ROS) in plant cells; earlier studies have shown that Gpx proteins play important roles in coping with increased ROS levels during biotic and abiotic stresses in plants. We analyzed the nucleotide diversity and divergence patterns of five loci encoding Gpx genes, and 16 reference loci used as controls, in order to detect departures from the neutral expectation. Gpx1 has an excess of mid-frequency alleles; high intraspecific nucleotide diversity, distributed in the upper tail of the simulated neutral model; and extensive LD, showing strong evidence of balancing selection/local adaptation. The Gpx3.2 gene exhibits very low nucleotide diversity and divergence, suggesting that it has evolved under strong purifying selection. We failed to detect any evidence for natural selection at the other loci (Gpx2, Gpx4, and Gpx5) compared with the reference loci. The results show that nucleotide diversity and/or divergence differ greatly between members of the Gpx gene family, resulting from differential selective pressure acting on genes, and that adaptive evolution influenced the distribution of P. euphratica in desert regions.