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Species with wide-range distributions usually display high genetic variation. This variation can be partly explained by historical lineages that were temporally isolated from each other and are back into secondary reproductive contact, and partly by local adaptations. The smooth newt (Lissotriton vulgaris) is one of the most widely distributed amphibians species across Eurasia and forms a species complex with a partially overlapping distribution and morphology. In the present study, we explored the population genomic structure of smooth newt lineages in the Carpathian Basin (CB) relying on single-nucleotide polymorphisms. Our dataset included new and previously published data to study the secondary contact zone between lineages in the CB and also tested for the barrier effect of rivers to gene flow between these lineages. We confirmed the presence of the South L. v. vulgaris Lineage distributed in Transdanubia and we provided new distribution records of L. v. ampelensis inhabiting the eastern territories of the CB. High genetic diversity of smooth newts was observed, especially in the North Hungarian Mountains and at the interfluves of the main rivers in the South with four distinct lineages of L. v. vulgaris and one lineage of L. v. ampelensis showing a low level of admixture with the spatially closest L. v. vulgaris lineage. Moreover, admixture detected at the interfluve of the main rivers (i.e. Danube and Tisza) suggested a secondary contact zone in the area. Finally, we found that the river Danube has a very weak effect on population divergence, while the river Tisza is a geographical barrier limiting gene flow between smooth newt lineages. As the range boundaries of L. v. vulgaris and L. v. ampelensis in the CB coincide with the river Tisza, our study underpins the influence of rivers in lineage diversification.

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This is the first detailed study assessing the morphological features of the testis and testicular sperm of members of the freshwater blood flukes Sanguinicola sp. from Leuciscus idus (Cyprinidae) and for S. volgensis from Pelecus cultratus (Cyprinidae), and the marine blood fluke Aporocotyle simplex from Hippoglossoides platessoides (Pleuronectidae). The present study reports a unique feature in the distribution of germinal cellular components in freshwater Sanguinicola sp., showing the presence of the individual spermatocytes or their clusters in the testicular lobes, and the gathering of spermatid rosettes and spermatozoa within middle testicular lumen, which extends along the entire length of the testis. In contrast, each testis of marine A. simplex contains the usual mixed distribution of germ cells at various stages of development. The first TEM data on spermatozoon character of studied species has shown, unusual for digenean sperm structure, the absence of cortical microtubules in sperm principal region. Moreover, a variation in axoneme patterns is revealed in the studied aporocotylids, belonging to the different aporocotylid lineages a 9 + 0 axonemal type observed for freshwater teleost-infective species, Sanguinicola sp. and S. volgensis, and a 9 + '1' axonemal type revealed in spermatozoa of marine teleost-infective species A. simplex. The results discussed with the published data on the digenean sperm structure and the testicular patterns in the Aporocotylidae likely represent additional characteristics supporting the divergent evolutionary lineages of freshwater and marine aporocotylids. We anticipate future morphological studies of the sperm structure in aporocotylids of three lineages for an understanding of their phylogenetic relationships.

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The genetic diversity of the USA honey bee (Apis mellifera L.) populations was examined through a molecular approach using two mitochondrial DNA (mtDNA) markers. A total of 1,063 samples were analyzed for the mtDNA intergenic region located between the cytochrome c oxidase I and II (COI-COII) and 401 samples were investigated for the NADH dehydrogenase 2 (ND2) coding gene. The samples represented 45 states, the District of Colombia and two territories of the USA. Nationwide, three maternal evolutionary lineages were identified: the North Mediterranean lineage C (93.79%), the West Mediterranean lineage M (3.2%) and the African lineage A (3.01%). A total of 27 haplotypes were identified, 13 of them (95.11%) were already reported and 14 others (4.87%) were found to be novel haplotypes exclusive to the USA. The number of haplotypes per state/territory ranged between two and eight and the haplotype diversity H ranged between 0.236-0.763, with a nationwide haplotype diversity of 0.597. Furthermore, the honey bee populations of the USA were shown to rely heavily (76.64%) on two single haplotypes (C1 = 38.76%, C2j = 37.62%) of the same lineage characterizing A. m. ligustica and A. m. carnica subspecies, respectively. Molecular-variance parsimony in COI-COII and ND2 confirmed this finding and underlined the central and ancestral position of C2d within the C lineage. Moreover, major haplotypes of A. m. mellifera (M3a, M7b, M7c) were recorded in six states (AL, AR, HI, MO, NM and WA). Four classic African haplotypes (A1e, A1v, A4, A4p) were also identified in nine states and Puerto Rico, with higher frequencies in southern states like LA, FL and TX. This data suggests the need to evaluate if a restricted mtDNA haplotype diversity in the US honey bee populations could have negative impacts on the beekeeping sustainability of this country.

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The integration of genetic, morphological, behavioral, and ecological information in the analysis of species boundaries has increased, allowing integrative systematics that better reflect the evolutionary history of biological groups. In this context, the goal of this study was to recognize independent evolutionary lineages within Euphonia affinis at the genetic, morphological, and ecological levels. Three subspecies have been described: E. affinis godmani, distributed in the Pacific slope from southern Sonora to Guerrero; E. affinis affinis, from Oaxaca, Chiapas and the Yucatan Peninsula to Costa Rica; and E. affinis olmecorum from Tamaulipas and San Luis Potosi east to northern Chiapas (not recognized by some authors). A multilocus analysis was performed using mitochondrial and nuclear genes. These analyses suggest two genetic lineages: E. godmani and E. affinis, which diverged between 1.34 and 4.3 My, a period in which the ice ages and global cooling fragmented the tropical forests throughout the Neotropics. To analyze morphometric variations, six morphometric measurements were taken, and the Wilcoxon Test was applied to look for sexual dimorphism and differences between the lineages. Behavioral information was included, by performing vocalization analysis which showed significant differences in the temporal characteristics of calls. Finally, Ecological Niche Models were estimated with MaxEnt, and then compared using the method of Broennimann. These analyses showed that the lineage distributed in western Mexico (E. godmani) has a more restricted niche than the eastern lineage (E. affinis) and thus we rejected the hypotheses of niche equivalence and similarity. Based on the combined evidence from genetic, morphological, behavioral, and ecological data, it is concluded that E. affinis (with E. olmecorum as its synonym) and E. godmani represent two independent evolutionary lineages.

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Pseudomonas aeruginosa filamentous (Pf) bacteriophages are important factors contributing to the pathogenicity of this opportunistic bacterium, including biofilm formation and suppression of bacterial phagocytosis by macrophages. In addition, the capacity of Pf phages to form liquid crystal structures and their high negative charge density makes them potent sequesters of cationic antibacterial agents, such as aminoglycoside antibiotics or host antimicrobial peptides. Therefore, Pf phages have been proposed as a potential biomarker for risk of antibiotic resistance development. The majority of studies describing biological functions of Pf viruses have been performed with only three of them: Pf1, Pf4, and Pf5. However, our analysis revealed that Pf phages exist as two evolutionary lineages (I and II), characterized by substantially different structural/morphogenesis properties, despite sharing the same integration sites in the host chromosomes. All aforementioned model Pf phages are members of the lineage I. Hence, it is reasonable to speculate that their interactions with P. aeruginosa and impact on its pathogenicity may be not completely extrapolated to the lineage II members. Furthermore, in order to organize the present numerical nomenclature of Pf phages, we propose a more informative approach based on the insertion sites, that is, Pf-tRNA-Gly, -Met, -Sec, -tmRNA, and -DR (direct repeats), which are fully compatible with one of five types of tyrosine integrases/recombinases XerC/D carried by these viruses. Finally, we discuss possible evolutionary mechanisms behind this division and consequences from the perspective of virus-virus, virus-bacterium, and virus-human interactions.

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The influence of genetic diversity and exposure to xenobiotics on the prevalence of pathogens was studied within the context of a voluntary epidemiological study in Spanish apiaries of Apis mellifera iberiensis, carried out during the spring season of years 2014 and 2015. As such, the evolutionary lineages of the honey bee colonies were identified, a multiresidue analysis of xenobiotics was carried out in beebread and worker bee samples, and the Toxic Unit (TUm) was estimated for each sampled apiary. The relationship between lineages and the most prevalent pathogens (Nosema ceranae, Varroa destructor, trypanosomatids, Black Queen Cell Virus; and Deformed Wing Virus) was analysed with contingency tables, and the possible relationships between TUm and the prevalence of these pathogens were studied by using a factor analysis. The statistical analysis supported the associations between V. destructor and Deformed Wing Virus (DWV), and between N. ceranae and Black Queen Cell Virus (BQCV), but the association between these pathogens and trypanosomatids was not observed. TUm values varied between 5.5 × 10-6 and 3.65 × 10-1. When TUm < 3.35 × 10-4, it was mainly determined by coumaphos, tau-fluvalinate and/or chlorfenvinphos. At higher values, other insecticides also contributed to TUm, although a clear predominance was not seen up to TUm ≥ 1.83 × 10-2, when it was mainly defined by acrinathrin, spinosad and/or imidacloprid. The possible cumulative effect from the joint action of xenobiotics was >10% in the 63% of the cases. The prevalence of pathogens did not appear to be influenced by the distribution of evolutionary lineages and, while the prevalence of V. destructor was not found to be determined by TUm, there was a trend towards an increasing prevalence of N. ceranae when TUm ≥ 23 10-4. This study is an example of using TUm approach beyond the field of the ecotoxicology.

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Chemical constituents of cryptic species detected within the liverwort Aneura pinguis were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fibre coating with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. A total of 48 samples of A. pinguis were analysed. The studied plants were identified genetically based on barcode DNA sequences and represented three cryptic species (A, B and F) of A. pinguis. Cryptic species A and B are genetically diverse; both represent three evolutionary lineages: A1, A2, A3 and B1, B2, B3, respectively. The cryptic species F that was recently detected is not diverse. The most characteristic compounds in analysed samples were sesquiterpene hydrocarbons (up to 17.7% for A1; 15.7% for A2; 20.6% for A3; 7.7% for B1; 2.0% for B2; 3.7% for B3; 10.2% for F), oxygenated sesquiterpenoids (up to 68.0% for A1; 54.7% for A2; 52.6% for A3; 63.5% for B1; 88.7% for B2; 82.7% for B3; 78.8% for F), and linear aliphatic hydrocarbons (up to 14.8% for A1; 1.1% for A2; 12.1% for A3; 6.9 for B1; 5.2% for B2; 1.1% for B3; 7.0% for F). The dominant compound in the studied samples was pinguisone. The second dominant compound present in the tested plant material was deoxopinguisone, except for lineage B2, where only a small relative concentration of this compound was found. A high content of deoxopinguisone in cryptic species A (lineages A1, A2 and A3) was accompanied by the presence of isopinguisone and methyl norpinguisonate, whereas these two compounds were not detected in cryptic species B (lineages B1 and B3) and F. The chemical compounds detected in the studied samples of A. pinguis were subjected to multivariate statistical analysis. The results showed that the chemical composition depends mainly on the genotype of the plant and slightly on the habitat. However, there was no clear correlation between the volatile compounds and the date of collection of the studied plants.

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Identifying the evolutionary and ecological mechanisms that drive lineage diversification in the species-rich tropics is of broad interest to evolutionary biologists. Here, we use phylogeographical and demographic analyses of genome-scale RADseq data to assess the impact of a large geographical feature, the Amazon River, on lineage formation in a venomous pitviper, Bothrops atrox. We compared genetic differentiation in samples from four sites near Santarem, Brazil, that spanned the Amazon and represented major habitat types. A species delimitation analysis identified each population as a distinct evolutionary lineage while a species tree analysis with populations as taxa revealed a phylogenetic tree consistent with dispersal across the Amazon from north to south. Phylogenetic analyses of mitochondrial DNA variation confirmed this pattern and suggest that all lineages originated during the mid- to late Pleistocene. Historical demographic analyses support a population model of lineage formation through isolation between lineages with low ongoing migration between large populations and reject a model of differentiation through isolation by distance alone. The results provide a rare example of a phylogeographical pattern demonstrating dispersal over evolutionary timescales across a large tropical river and suggest a role for the Amazon River as a driver of in situ divergence both by impeding (but not preventing) gene flow and through parapatric differentiation along an ecological gradient.

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The answer to the proffered question, "What is a species?" is considered one of the fundamental issues of biological science, as well as one of the most polarizing and sometimes acrimonious problems. Dozens of species concepts have been defined, but none are universal for implementation across all taxa. Within the past thirty years, the ability to analyze DNA data has progressed to the point that multiple methodologies can be simultaneously applied to the same evolutionary questions. The use of restriction fragment length polymorphisms, microsatellites, and mitochondrial (mtDNA) and nuclear DNA (nucDNA) sequence data has unarguably changed how we look at diversity and intensified the concept debate through the proliferation of species descriptions. Over the past two decades, Madagascar's biodiversity has gone through a tremendous taxonomic expansion by the elevation of subspecies to species and through novel descriptions, especially within the nocturnal lemurs. With the tremendous continuous loss of habitat, exponential human population growth, and stochastic changes predicted over coming decades, elucidating the earth's biodiversity will never be more important than now. Here, we examine species concepts and their attendant criteria. We predict how technological advances will alter, improve and, we hope, fully consolidate the unity of thoughts related to this central topic of evolutionary biology and its numerous interconnected disciplines.

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