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Bandicoot rats (genus Bandicota), widely known as rodent pests, are abundant and widespread throughout the continental part of the Indo-Malayan realm. However, their evolutionary history is not yet well understood. The molecular phylogenetic relationships of the three bandicoot rat species, Bandicota bengalensis, Bandicota indica, and Bandicota savilei, were assessed based on the gene sequences of the specimens collected from Myanmar, where all three species occur along with database sequences. Early divergence of B. savilei (1.5–1.7 million years ago) was inferred from the mitochondrial cytochrome b (Cytb) gene and the nuclear interphotoreceptor retinoid-binding protein (Irbp), and melanocortin 1 receptor (Mc1r) gene sequences. The Cytb lineage of B. bengalensis from Sri Lanka was distinct from the monophyletic lineage of the continental lineages of B. bengalensis and B. indica. This can be explained by the preservation of ancient mitochondrial DNA (mtDNA) in the insular population owing to female philopatry and male dispersal, given that no substantial intraspecies geographic subdivision was observed in the nuclear markers. The paraphyletic relationship of B. bengalensis with B. indica may be explained by introgression of the mtDNA from B. bengalensis to B. indica, but further investigation is required to confirm this. B. bengalensis Cytb sequences from a wide area of Myanmar had limited nucleotide diversity (π = 0.00079), implying that the genetic diversity of B. bengalensis in Myanmar was acquired through Holocene human activities.

Made available in DSpace on 2020-12-10T19:51:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Araucaria Foundation (Fundacao Araucaria de Apoio e Desenvolvimento Cientifico e Tecnologico do Estado do Parana) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Astyanax is a species-rich, non-monophyletic genus composed of several supraspecific taxa that are poorly delimited. The Astyanax fasciatus complex is one of these taxa and shows high taxonomic complexity. To elucidate the evolutionary history of the A. fasciatus complex from southern South America, we conducted cytogenetic, molecular and morphological analyses in specimens from the Uruguay River basin. Cytogenetic characters demonstrated two closely related operational taxonomic units: Astyanax sp. 1 (8m+22sm+10st+6a), Astyanax sp. 2 (8m+24sm+10st+4a) and natural hybrids (8m+23sm+8st+5a). 5S ribosomal DNA sites were found in two pairs of m chromosomes and one pair of a chromosomes in Astyanax sp. 1, two pairs of a chromosomes and one pair of m chromosomes in Astyanax sp. 2, and three m chromosomes and three a chromosomes in hybrids. As51 sites were found in three chromosomes in Astyanax sp. 1 and in five chromosomes in Astyanax sp. 2 and hybrids. Mitochondrial sequence analyses did not separate the two units and hybrids. Morphological analyses revealed differences between Astyanax sp. 2 and hybrids. This secondary contact with gene flow between lineages that diverged long ago might slow or reverse the differentiation/speciation process. These results help us to understand the evolutionary history of this highly complex clade of Astyanax in southern South America. Univ Estadual Oeste Parana, Ctr Ciencias Biol & Saude, Cascavel, Parana, Brazil Univ Estadual Maringa, Posgrad Biol Comparada, Maringa, Parana, Brazil Univ Estadual Maringa, Nucleo Pesquisas Limnol Ictiol & Aquicultura NUPE, Maringa, Parana, Brazil Univ Estadual Maringa, Dept Biol, Maringa, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Morfol, Lab Biol & Genet Peixes, Botucatu, SP, Brazil Univ Estadual Oeste Parana, Grp Pesquisas Recursos Pesqueiros & Limnol GERPEL, Pos Grad Recursos Pesqueiros & Engn Pesca, Pos Grad Conservacao & Manejo Recursos Nat, Toledo, Parana, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Morfol, Lab Biol & Genet Peixes, Botucatu, SP, Brazil

Background African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. Methodology We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. Results We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ2 = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly’s age based on wing fray category (χ2 = 22.374, P T. vivax (6.62%), T. congolense (2.57%), T. brucei and T. simiae each at 0.73%. Blood meal analyses revealed five principal vertebrate hosts, namely, cattle (Bos taurus), humans (Homo sapiens), Nile monitor lizard (Varanus niloticus), African mud turtle (Pelusios chapini) and the African Savanna elephant (Loxodonta africana). Conclusion We found an infection rate of 10.8% in the tsetse sampled, with all infections attributed to trypanosome species that are causative agents for AAT. However, more verification of this finding using large-scale passive and active screening of human and tsetse samples should be done. Cattle and humans appear to be the most important tsetse hosts in the region and should be considered in the design of control interventions.

Ramularia leaf spot caused by the fungus Ramularia collo-cygni, has recently become widespread in Estonian barley fields. Currently, disease control in barley fields relies on SDHI and DMI fungicides, which might be threatened by R. collo-cygni isolates that are well-adapted to fungicide pressure. In a two-year study, 353 R. collo-cygni isolates were collected from spring barley fields in Estonia. A total of 153 R. collo-cygni isolates were examined for sensitivity to azoles (DMIs, prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs, boscalid, fluxapyroxad). Epoxiconazole was the least effective and a new fungicide mefentrifluconazole was the most effective DMI. Among SDHIs, fluxapyroxad was more effective than boscalid. Also, single R. collo-cygni isolates with high resistance to tested fungicides occurred, which could affect fungicide control of the pathogen. The entire collection of R. collo-cygni was analysed for mutations in fungicide target proteins. Six mutations were identified in CYP51 gene, the most dominant being I381T, I384T, and S459C. Also, numerous point mutations in the SdhC gene were present. The mutation G143A in strobilurin target protein CytB dominates in over 80% of the R. collo-cygni population, confirming the low efficacy of strobilurin fungicides in barley disease control.

Background. The exponential growth of research concerning a role of morphological modularity and integration in evolution has taking place from the beginning of the century. It was especially noted that inter-level integration should manifest itself, first of all, in the congruent variability of the modules of different structural levels. We analyzed congruence between the interspecific variability of the first lower molar (m1) masticatory surface and the mtDNA Cytb gene in ten species of the gray voles Microtus s.l. from the point of view of the modular organization. Materials and methods. In total, 5306 pairs of chewing surface contours of vole molar m1 were investigated. Thirty one different morphotypes and 187 their different combinations are identified: 30 – symmetric and 157 – asymmetric. 576 sequences of the Cytb mtDNA gene from the GenBank database are used. Climatic data are taken from the website Climate:Date.org. Data are processed using a DJ-method. The morphogenetic matrix of Euclidean distances between species is obtained from the frequencies of m1 morphotypes co-occurrence from the right and left sides of the lower jaw, and the molecular-genetic one from the frequencies of synonymous codon substitutions. The algorithm is realized in the Jacobi 4 package. Results. A high correlation ( r = 0.847) between the first principal component of the molecular-genetic distance matrix and second principal component of the morphogenetic one is found. From the standpoint of the modular organization of the phenotype, the principal components of these matrices are treated as variability modules. The molecular-genetic module is caused by change of frequencies of the codons ACC and GCA along geo-climatic gradient, and morphogenetic one – various aspects of the m1 asymmetry. Conclusions. The proposed approach allowed to identify two congruently varying modules from different trait systems of the studied species along the geo-climatic gradient.

Avian haemosporidian infections are widespread and can result in the decline of wild bird populations or in some cases contribute to extinction of species. We determined the prevalence and genetic diversity of avian haemosporidia in 93 samples from 22 landbird species from South Africa (N = 76) and West Africa (N = 17), of which six are intra-African migrants and one is a Palearctic migrant. The samples were analysed for the presence of avian haemosporidian DNA using real-time quantitative PCR (qPCR) and nested PCR assays targeting specific mitochondrial genes of these parasites. The cytochrome b (cytb) gene was sequenced for all samples that tested positive and phylogenetic analysis was conducted in order to determine the relationship of the new sequences with previously published sequences from the MalAvi database. The overall prevalence of avian haemosporidiosis was 68.82% (95% CI: 56.4%–78.87%) and 82.80% (95% CI: 65.68%–86.11%) as determined by qPCR and nested PCR respectively. Eighteen (19.36%; 95% CI; 10.78%–29.97%) samples had mixed infections. Infection prevalence of all haemosporidian spp. were significantly higher (p, Graphical abstract Image 1, Highlights • Avian haemosporidian infections in free ranging birds are common and diverse. • The prevalence was 68.82% and 82.80% by qPCR and nested PCR assays respectively. • Eighteen (19.36%) samples had mixed infections. • Seventeen cytochrome b lineages were identified from 14 avian species.

Although there are several nuisance species of serpulids reported worldwide, the present study shows that some of them have been overlooked even in geographic areas that are considered well studied. We report for the first time in Majorca the invasive species Ficopomatus enigmaticus and the first records for the Balearic Islands of the also non-native species Hydroides dianthus, H. dirampha, H. elegans and H. nigra. The most abundant species were F. enigmaticus, found at higher densities in Portixol (with up to 280 ind. m–2), and H. elegans, found at highest densities in the port of Palma (with up to 270 ind. m–2). Species have been identified after morphological examination and corroborated by mitochondrial DNA sequence data: cytochrome c oxidase subunit 1 (COI) and cytochrome b (Cytb). Molecular data show that the same haplotypes have been found in distant and disjoint biogeographic areas worldwide, which is only explicable by unintentional translocation of specimens through vectors (i.e. ship hulls or ballast water). Species delimitation analyses support previous findings that species complexes are common in both Ficopomatus and Hydroides. In fact, only among the Majorcan samples were we able to detect three species of the F. enigmaticus species complex, two of the H. elegans species complex and two of the H. dianthus species complex. The genetic distances between members of the F. enigmaticus species complex are 8.7% to 16.7% in COI sequences. The species of Hydroides hold 5.4% to 47.6 % genetic divergence between species of the same complex., Hay constancia de especies alóctonas de serpúlidos reportadas en el mundo. Pero el presente estudio muestra que algunas de ellas han sido pasadas por alto incluso en áreas geográficas consideradas bien estudiadas. En este trabajo se cita por primera vez en Mallorca la especie invasora Ficopomatus enigmaticus y se registra también por primera vez para las Islas Baleares las también especies exóticas Hydroides dianthus, H. dirampha, H. elegans e H. nigra. Las especies más abundantes fueron F. enigmaticus, encontrada en mayores densidades en Portixol (con hasta 280 ind. m–2) y H. elegans, encontrada en mayores densidades en el puerto de Palma (con hasta 270 ind. m–2). Las especies han sido identificadas después su estudio morfológico y corroboradas por las secuencias de ADN mitocondrial: subunidad 1 de la citocromo c oxidasa (COI) y citocromo b (Cytb). Los datos moleculares muestran que se han encontrado los mismos haplotipos en áreas biogeográficas distantes e inconexas de a nivel mundial, lo que solo se explica por la translocación involuntaria de ejemplares a través de vectores (es decir, cascos de barcos o agua de lastre). Los análisis de delimitación de especies respaldan resultados previos acerca de la existencia de complejos de especies en ambos géneros Ficopomatus e Hydroides. De hecho, sólo entre las muestras mallorquinas pudimos detectar tres especies del complejo de especies F. enigmaticus, dos del complejo de especies de H. elegans y dos del complejo de especies de H. dianthus. Las distancias genéticas entre los miembros del complejo de especies de F. enigmaticus son del 8.7 al 16.7% en las secuencias COI. Las especies de Hydroides tienen un 5.4-47.6% de divergencia genética entre especies del mismo complejo.

Babesia gibsoni is considered as an emerging protozoan parasite of dogs in North America and Europe. However, no data have been published on its prevalence, molecular-phylogenetic characteristics and associated co-infections in dogs used for illegal fighting (i.e. predisposed to acquiring this piroplasm via biting) in Europe. In this study, blood samples from 79 American Staffordshire Terrier dogs, confiscated for illegal dog fights, were molecularly analyzed for tick-borne pathogens. Babesia gibsoni was detected in 32 dogs, i.e. with a prevalence of 40.5%. In addition, Babesia vulpes was found in 8 samples (prevalence of 10.1%), for the first time in dogs in Hungary. Canine hemoplasmas were also identified in 49 samples (62%): only Mycoplasma haemocanis in 32 (40.5%) dogs, only “Candidatus Mycoplasma haematoparvum” in 9 (11.4%) dogs, and both hemoplasmas in 8 (10.1%) dogs. Thus, hemoplasma infections also showed a particularly high prevalence in this dog population. Based on a partial fragment of the 18S rRNA gene, B. gibsoni from Hungary exhibited complete sequence identity with conspecific strains reported from Europe and Asia. The cytochrome c oxidase subunit 1 (cox1) gene sequence of this isolate showed the closest identity with B. gibsoni reported from Japan but had a nonsynonymous mutation (M33I). Furthermore, the 11 B. gibsoni-positive samples analyzed for sequence variants of the cytochrome b (cytb) gene showed the presence of a common mutation (P310S). Most importantly, B. gibsoni had two further nonsynonymous mutations, M121I and F258L, in a dog with severe and relapsing anemia following atovaquone treatment. Phylogenetically, both cytb sequence variants clustered together, with a clear geographical pattern showing the closest relationship of both haplotypes identified in Hungary with those from China and Japan. To the best of our knowledge, this is the first cox1 and cytb characterization of B. gibsoni in Europe, as well as the first report on the emergence of this piroplasm and hemoplasmas with high prevalence among “fighting dogs” north of the Mediterranean Basin.

Cubilose, a valuable traditional Chinese medicine, is mainly composed of the saliva by several species of Aerodramus or Collocalia in the Apodidae. Due to rarity, high economic value and huge market demand, its fake or adulteration is frequently found in the market. Therefore, it is urgent to establish a simple and accurate method for authenticating cubilose. DNA barcoding, which is an easy, quick and reliable method, is widely used to trace the origin of traditional Chinese medicine. For identifying the original species of cubilose, cytb gene of 18 cubilose samples including 15 officer cubilose and 3 feather cubilose were amplified and entered into the GenBank database using the BLAST search tool. The genetic distances among 18 cubilose samples were calculated based on the Kimura two parameter (K2P) model. To construct the reference database, 18 cytb sequences of Aerodramus or Collocalia were downloaded from GenBank. The neighbor-joining (NJ) and unweighted pair group method with arithmetic average (UPGMA) trees were constructed based on sequences from GenBank and our dataset. Blast analysis showed that all cubilose samples had the highest similarity with A. fuciphagus, and the sequence similarity reached over 99%. Genetic distance of 18 cubilose samples ranged from 0.000–0.010. Trees constructed by NJ and UPGMA gave similar topology: all cubilose samples clustered together with A. fuciphagus. These result demonstrated that the original species of all 18 cubilose samples were A. fuciphagus, and cytb gene is a good candidate for identifying cubilose.

Calanoid copepod Peudodiaptomus marinus Sato, 1913 was first recorded in Sevastopol Bay in the northern Black Sea in September 2016. We performed regular observations of this new invasive species between October 2016 and December 2018. We conducted bi-weekly plankton sampling at three stations located within or adjacent to Sevastopol Bay. This is the first paper to combine (i) a detailed morphological study, (ii) molecular genetic analysis, and (iii) an investigation of P. marinus seasonal dynamics and interannual abundance variability in the coastal Black Sea. Our morphological research showed similarities between Pseudodiaptomus specimens and existing P. marinus illustrations and descriptions. Our morphological analysis results were confirmed using molecular genetic studies. Based on the genetic variability of ITS2 and cytb, we found that all investigated specimens from Sevastopol Bay belonged to P. marinus. Investigations of P. marinus seasonal and interannual abundance variability showed the same seasonal patterns throughout the studied period, with a higher seasonal abundance from October to November and one pronounced density peak in autumn. The highest abundances (2,000 ind m–3 at the mouth of the bay and more than 5,000 ind m–3at its centre) were recorded in November 2018. In the samples, we found adults of both sexes, including ovigerous females, copepodites I–V, and nauplii, suggesting that the species reproduce in Sevastopol Bay. Our research indicated that P. marinus is a new non-indigenous species (NIS) in the Black Sea, and we will discuss a possible vector of its introduction into this basin.

Background The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Methods Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. Conclusions The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Konosirus punctatus inhabits areas of significant commercial importance in Chinese coastal waters. Twelve populations distributed all along the Chinese coast were analyzed using two mtDNA genes. The cytochrome oxidase subunit I (COI) and cytochrome b (Cytb) genes were amplified from 242 and 259 individuals, respectively, and fragments of 999 and 415 bp sequence in length were used for analysis. COI and Cytb denoted a high haplotype diversity and low nucleotide diversity. The COI mtDNA gene revealed a high molecular variance within populations and low variance among populations, whereas Cytb showed a high variance among populations and low within populations. FST values obtained for COI and Cytb showed low genetic differentiation among the populations, except for the Zhongshan population where the Cytb gene showed high FST values. The constructed phylogenetic tree and haplotype network tree showed a single lineage, however, Cytb gene data showed that the Zhongshan population has a greater difference level within a population. The present investigation contributed to existing knowledge of the genetic structure of K. punctatus and can be useful for its future conservation.

Seafood is an important component of the human diet. With depleting fish stocks and increasing prices, seafood is prone to fraudulent substitution. DNA barcoding has illustrated fraudulent substitution of fishes in retail and restaurants. Whether substitution also occurs in other steps of the supply chain remains largely unknown. DNA barcoding relies on public reference databases for species identification, but these can contain incorrect identifications. The creation of a high quality genetic reference database for 42 European commercially important fishes was initiated containing 145 Cytochrome c oxidase subunit I (COI) and 152 Cytochrome b (cytB) sequences. This database was used to identify substitution rates of Atlantic cod (Gadus morhua) and common sole (Solea solea) along the fish supply chain in Belgium using DNA barcoding. Three out of 132 cod samples were substituted, in catering (6%), import (5%) and fishmongers (3%). Seven out of the 41 processed sole samples were substituted, in wholesale (100%), food services (50%), retailers (20%) and catering (8%). Results show that substitution of G. morhua and S. solea is not restricted to restaurants, but occurs in other parts of the supply chain, warranting for more stringent controls along the supply chain to increase transparency and trust among consumers. ispartof: FOOD AND CHEMICAL TOXICOLOGY vol:141 ispartof: location:England status: published

Background Several members of genus Babesia are important pathogens causing babesiosis in dogs. In China, at least five Babesia species have been described in dogs or ticks. This study sought to determine the prevalence and molecular characteristics of various Babesia spp. in dogs in cities in Shaanxi Province in China, including Xi’an and Hanzhong. Methods A total of 371 blood samples were collected from pet dogs presenting to veterinary clinics in the cities of Xi’an and Hanzhong in Shaanxi, China. Babesia spp. DNA was detected via amplification of partial 18S rRNA genes by semi-nested PCR. Almost full-length 18S rRNA, ITS, partial TRAP and complete cytb genes were recovered for analysis of the genetic characteristics and relationships with known isolates. Results A single species, Babesia gibsoni, was identified in dogs in Xi’an and Hanzhong. Consistently, B. gibsoni was also detected in 14 ticks collected from positive dogs. Sequence similarities and phylogenetic analysis suggested that the isolates identified herein showed a closer genetic relationship with isolates from East Asian countries rather than India, Bangladesh, or the USA. Sequence analysis based on tandem repeat analysis of the TRAP gene further revealed that specific haplotypes were circulating in both Xi’an and Hanzhong, with no specific regionality. In addition, 10.9% of all isolates with atovaquone (ATV)-resistance were identified because of M121I mutation in the deduced cytb protein. Conclusions This study revealed a high prevalence rate of Babesia infection. Babesia gibsoni was the only Babesia species identified in cases of canine babesiosis in the cities of Xi’an and Hanzhong cities in Shaanxi, China. In addition, the TRAP gene presented high genetic diversity across isolates. Such information is useful for elucidating the epidemiological characteristics of canine babesiosis, as well as the overall genetic diversity of Babesia spp. circulating in dog populations in Shaanxi Province.

Monoxenous parasites may easily infect animals in captivity, and nematodes belonging to the genus Trichuris are commonly reported in zoological gardens worldwide. Infections in captive animals should be accurately monitored and the characterization of pathogens is highly advisable, as a tool to infer possible routes of intra- and interspecific transmission pathways and to assess the related zoonotic potential. Whipworms are usually identified on the basis of few morphological features of adults males and eggs and by an host-affiliation criterion. Given the strong morphological convergence of adaptive traits and the possible occurrence of hybridization and/or cross-infections events, the use of molecular methods is of great utility. Here, we analysed two partial mitochondrial loci, the cytochrome c oxidase I and the cytochrome b regions, in Trichuris spp. infecting four animal species hosted in the Bioparco Zoological Garden of Rome. Results from molecular systematics, compared to previous data, suggested that the five Trichuris taxa recovered were well separated, showing a significant degree of host affiliation (herbivorous, primates/swine and rodents/canids). The screw horn antelopes and the camels were infected with two variants of Trichuris ovis; Trichuris sp. similar to those observed in rodents from South America was infecting the Patagonian maras. Moreover, Trichuris from the ring-tailed lemur showed a great similarity to Trichuris infecting the Japanese macaque previously analysed from the same zoological garden, and clustering together with Trichuris trichiura, posing a potential zoonotic threat for visitors and workers.

[Background]: The Pantosteus plebeius-nebuliferus species-group is a group of freshwater fishes distributed in endo- and exorheic drainage basins in the Mexican Sierra Madre Occidental mountain range system and central North Mexico. The geological history of this region is considered an important factor in explaining the evolutionary history of low vagility animals like freshwaters fishes. The aim of this study was to examine the phylogenetic relationships and describe the evolutionary history of the species-group. We hypothesized that the genetic structure and distribution of the main clades of Pantosteus plebeius-nebuliferus are associated with the geological history of Northern Mexico. To this end, we obtained DNA sequences of mitochondrial and nuclear genes and performed phylogenetic and phylogeographic analyses. Divergence time estimation and ancestral area reconstruction were also carried out to propose a biogeographical hypothesis, and species boundaries within the species-group were also tested., [Results]: We identified four clades within the Pantosteus plebeius-nebuliferus species-group in both markers. Divergence ranged from 5.9% to 9.2% for cytb and 0.1% to 0.9% for GHI. We observed significant genetic structure and no shared haplotypes between clades. We estimated that the clades diverged during the last 5.1 Myr, with a biogeographic scenario suggesting eight vicariant and four dispersal events through the historic range of the species-group. We found that the best species-delimitation model is when four species are assumed, which correspond to the main clades. We identified nine evolutionary significance units (ESUs), pertinent to the conservation of the group, each representing populations present in distinct drainage basins., [Conclusions]: The evolutionary history of the Pantosteus plebeius-nebuliferus species-group is characterized by vicariant post-dispersal processes, linked to geological changes in the Sierra Madre Occidental and central Northern Mexico since the Pliocene. This is congruent with biogeographic patterns described for other co-distributed fish species. We propose a new phylogenetic hypothesis for the species-group, clarifying the taxonomy of this evolutionarily complex group. Our results suggest that the species-group consists of at least four clades with independent evolutionary histories, two of which may represent new undescribed species. Our identification of ESUs provides a basis upon which conservation measures can be developed for the species-group., This work was partial supported by the Coordinación de la Investigación Científica (CIC-UMSNH 2014), the Ministerio de Economía y Competitividad of Spain (grant reference CGL2013-41375-P). Both resources were used to finance fieldwork, laboratory (DNA isolation and amplification of molecular markers) and sequencing service, as well as for the publication of this work. The Consejo Nacional de Ciencia y Tecnología CONACYT with sabbatical grant and the scholarship to ODD (grant number: 260596) and DKCS (number grant: 254270/411621) respectively.

Over millions of years of evolution, the genomes of modern insects have accumulated a significant number of mutations, which often can lead up a blind alley when carrying out phylogenetic research. Genomic differences between some representatives belonging to the same family or group are often so great that they demand using nonconventional methods of the phylogenetic analysis. It is known that molecular evolution goes by the way of not only single nucleotide substitutions, but also by larger genomic reorganizations, such as insertion or deletion of large genome fragments, and even changing the order of genes. Mitochondrial DNA genes (mtDNA) are quite often used as markers for phylogenetic research into many organisms including arthropods, because mtDNA is multicopied, is inherited maternally, does not undergo recombination and accumulates mutations quickly enough (relative to the nuclear genome). To date, a large number of full nucleotide sequences of mitogenomes (thousands of organisms) has been deposited in public databases; however, their phylogenetic analysis has obstacles, especially for representatives of the insects (Insecta), whose evolution takes a considerable part of geological time. In this work we describe the application and a comparison of two ways of the phylogenetic analysis for different groups of insects. The first method uses the variability of the nucleotide sequence of mtDNA, and the second one analyses the order of genes in full mitochondrial genomes of insects that can be used as an additional marker in phylogenetic research into representatives of the order Hymenoptera.

18 pages, 4 figures., In this study, 111 ready-to-eat anchovy products were collected on the Italian market. The products were molecularly identified through a BLAST analysis of a highly informative cytb fragment amplified by a newly designed primer pair for the genus Engraulis spp. and the mislabelling rate was assessed. In addition, the labels were analysed in the light of the current EU law. Despite only one mislabelling case was observed (mislabelling rate 0.9%), which involved the substitution of the European anchovy (Engraulis encrasicolus) with the low-valuable Peruvian/Chilean anchoveta (Engraulis ringens), the molecular technique developed in this study was proved as suitable tool for detecting species in processed anchovy products. It could be therefore applied to carry out more extensive EU survey aimed at evaluating the mislabelling rate of such products, still poorly covered by a targeted and clear legislation system.

We report a new locality for Monodelphis handleyi, a rare short-tailed opossum species, previously known only from its type locality, in Loreto, northeastern Peru. One adult male was collected using pitfall trap disposed in Humaita Amazonian savanna of southern Amazonas state, Brazil. Voucher specimen had their identification confirmed by molecular data (mitochondrial gene Cytochrome b) and morphological comparison. We provide external and cranial measurements of this specimen and comment on its morphology. The specimen reported here represents the first record of M. handleyi in Brazil and is the second known locality for the species, and there is high level of divergence found between the two distant localities (5.1%). This record extends the species range at least 1200 km eastern beyond the type locality and denotes the importance of use of complementary methods for sampling small nonvolant mammals.

SummaryGerm plasm-related structures (GPRS) are known to accompany meiotic cell differentiation but their dynamics are still poorly understood. In this study, we analyzed the ultrastructural mechanisms of GPRS transformation during oogenesis and spermatogenesis of the bivalve molluscRuditapes philippinarum(Manila clam), exploring patterns of GPRS activity occurring at meiosis onset, sex-specific difference/similarity of such patterns, and the involvement of mitochondria during GPRS-assigned events. In the two sexes, the zygotene–pachytene stage of meiosis is anticipated by three shared steps. First, the dispersion of germ plasm granules containing the germ line determinant VASA occurs. Second, the VASA protein deriving from germ plasm granules enters neighbouring mitochondria and appears to induce mitochondrial matter release, as supported by cytochrome B localization outside the mitochondria. Third, intranuclear VASA entrance occurs and the protein appears involved in chromatin reorganization, as supported by VASA localization in synaptonemal complexes. In spermatogenesis, these three steps are sufficient for the normal course of meiosis. In oogenesis, these are followed by the action of ‘germ plasm granule formation complex’, a novel type of structure that appears alternative to the Balbiani body. The possibility of germ plasm involvement in reproductive technologies is also suggested.

Comparar la distribución geográfica actual y pasada de una especie puede revelar importantes conocimientos sobre los patrones de la biodiversidad, incluyendo su respuesta a los cambios ambientales. Mediante análisis morfológicos y genéticos de especímenes de museo y muestras recientemente recolectadas en el campo, documentamos la ocurrencia histórica de una población de tuco-tuco colonial (Ctenomys sociabilis) en Laguna Nahuelquir, provincia del Chubut, Argentina. La confirmación de la población de C. sociabilis en la provincia del Chubut representa una expansión significativa del rango geográfico de esta especie, que se creía endémica de la Sierra de Cuyín Manzano en la provincia del Neuquén, ~130 km al norte de la Laguna Nahuelquir. Los estudios de campo en la localidad de Laguna Nahuelquir no revelaron la presencia de poblaciones existentes de C. sociabilis; aunque el hábitat en este lugar es generalmente consistente con las áreas ocupadas por C. sociabilis en el Cuyín Manzano, el sitio Laguna Nahuelquir está ahora ocupado por el tuco-tuco patagónico, C. haigi. Este cambio en las especies de Ctenomys es consistente con los patrones de cambio de fauna revelados por el análisis de depósitos de finales del Pleistoceno y Holoceno temprano en la Sierra de Cuyín Manzano y apoya la hipótesis de que los cambios en las condiciones ambientales locales han contribuido a la pérdida de C. sociabilis en estas localidades. Por lo tanto, la comparación de las muestras actuales e históricas de Ctenomys proporciona evidencias de retracción del rango geográfico del tuco-tuco colonial a través de diferentes escalas temporales y se suman a un creciente cuerpo de evidencia sobre los efectos aparentes de los cambios ambientales en la fauna pequeños mamíferos del norte de la Patagonia. Comparisons of a species’ modern and historic geographic distributions can yield important insights into patterns of biodiversity, including faunal responses to environmental change. Using morphological and genetic analyses of museum specimens and samples recently collected in the field, we document the historical occurrence of a population of colonial tuco-tucos (Ctenomys sociabilis) from Laguna Nahuelquir in Chubut Province, Argentina. This locality represents a significant increase in the geographic distribution of this species, which had been thought to be endemic to the Sierra de Cuyín Manzano located ~130 km to the north in Neuquén Province. Field surveys of the Laguna Nahuelquir locality failed to reveal extant populations of C. sociabilis; although the habitat at this location is generally consistent with areas occupied by C. sociabilis in the Cuyín Manzano, the Laguna Nahuelquir site is now occupied by the Patagonian tuco-tuco, C. haigi. This apparent replacement of ctenomyid species is consistent with patterns of faunal turnover revealed by analyses of late Pleistocene–early Holocene cave deposits near the Sierra de Cuyín Manzano and supports the hypothesis that changes in local environmental conditions have contributed to the loss of C. sociabilis from these localities. Thus, comparisons of historical and modern specimens of ctenomyids provide evidence of range retractions by colonial tuco-tucos over multiple time scales and add to a growing body of evidence regarding the apparent effects of environmental change on the small mammal fauna of northern Patagonia. Fil: Tammone, Mauro Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Diversidad y Evolución Austral; Argentina Fil: Lavin, Brian R.. Sonoma State University; Estados Unidos Fil: Pardiñas, Ulises Francisco J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Diversidad y Evolución Austral; Argentina Fil: Lacey, Eileen Anne. University of California; Estados Unidos

Phylogenetic analyses provide information that can be useful in the conservation of genetic variation by identifying intraspecific genetic structure. Reconstruction of phylogenetic relationships requires the use of markers with the appropriate amount of variation relative to the timeframe and purpose of the study. Here, genetic structure and clustering are inferred from comparative analyses of three widely used mitochondrial markers, the CR, cytb and the COI region, merged and separately, using Eurasian reindeer as a model. A Bayesian phylogeny and a MJ network, both based on the merged dataset, indicate several distinct maternal haplotype clusters within Eurasian reindeer. In addition to confirm previously described clusters, two new subclusters were found. When comparing the results from the merged dataset with the results from analyses of the three markers separately, similar clustering was found in the CR and COI phylogenies, whereas the cytb region showed poor resolution. Phylogenetic analyses of the merged dataset and the CR revealed congruent results, implying that single sequencing analysis of the CR is an applicable method for studying the haplotype structure in Eurasian reindeer.

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification. This work was supported in part by Instituto de Salud Carlos III (MPY-1248/12) grant and (PI14CIII/00016) funded by AESI. Ivonne Pamela Llanes-Acevedo was granted a student fellowship by COLCIENCIAS/COLFUTURO. Sí

International audience; Mitochondrial genetic markers have been extensively used to study the phylogenetics and phylogeography of many birds, including seabirds of the order Procellariiformes. Evidence suggests that part of the mitochondrial genome of Procellariiformes, especially albatrosses, is duplicated, but no DNA fragment covering the entire duplication has been sequenced. We sequenced the complete mitochondrial genome of a non-albatross species of Procellariiformes, Puffinus lherminieri (Audubon’s shearwater) using the long-read MinION (ONT) technology. Two mitogenomes were assembled from the same individual, differing by 52 SNPs and in length. The shorter was 19 kb long while the longer was 21 kb, due to the presence of two identical copies of nad6, three tRNA, and two dissimilar copies of the control region (CR). Contrary to albatrosses, cob was not duplicated. We further detected a complex repeated region of undetermined length between the CR and 12S. Long-read sequencing suggests heteroplasmy and a novel arrangement within the duplicated region, indicating a complex evolution of the mitogenome in Procellariiformes.

Fishes are, by far, the most diverse group of vertebrates. Their classification relies heavily on morphology. In practice, the correct morphological identification of species often depends on personal experience because many species vary in their body shape, color and other external characters. Thus, the identification of a species may be prone to errors. Due to the rapid development of molecular biology, the number of sequences of fishes deposited in GenBank has grown explosively. These published data likely contain errors owing to invalid or incorrectly identified species. The erroneous data can lead to downstream problems. Thus, it is critical that such errors get identified and corrected. A strategy based on DNA barcoding can detect potentially erroneous data, especially when intraspecific K2P variation exceeds interspecific K2P divergence. Analyses of the most used DNA marker for fishes (mitochondrial Cytb) discovers that intraspecific differences of fishes are generally less than 1%, while interspecific differences are generally higher than 10%. Based on this ruler, our analyses identify 1,303 potential problematic Cytb sequences of fishes in GenBank and point to taxonomic problems, errors in identification, genetic introgression and other concerns. Care must be taken to avoid the perpetuation of errors when using these available data.

Background Anopheles stephensi, the major vector of urban malaria in India, was recently detected for the first time in Sri Lanka in Mannar Island on the northwestern coast. Since there are different biotypes of An. stephensi with different vector capacities in India, a study was undertaken to further characterise the genotype and biotype of An. stephensi in Mannar Island. Methods Mosquito larvae were collected in Pesalai village in Mannar and maintained in the insectary until adulthood. Adult An. stephensi were identified morphologically using published keys. Identified adult An. stephensi were molecularly characterized using two mitochondrial (cox1 and cytb) and one nuclear (ITS2) markers. Their PCR-amplified target fragments were sequenced and checked against available sequences in GenBank for phylogenetic analysis. The average spiracular and thoracic lengths and the spiracular index were determined to identify biotypes based on corresponding indices for Indian An. stephensi. Results All DNA sequences for the Mannar samples matched reported sequences for An. stephensi from the Middle East and India. However, a single nucleotide variation in the cox1 sequence suggested an amino acid change from valine to methionine in the cox1 protein in Sri Lankan An. stephensi. Morphological data was consistent with the presence of the Indian urban vector An. stephensi type-form in Sri Lanka. Conclusions The present study provides a more detailed molecular characterization of An. stephensi and suggests the presence of the type-form of the vector for the first time in Sri Lanka. The single mutation in the cox1 gene may be indicative of a founder effect causing the initial diversification of An. stephensi in Sri Lanka from the Indian form. The distribution of the potent urban vector An. stephensi type-form needs to be established by studies throughout the island as its spread adds to the challenge of maintaining the country’s malaria-free status. Electronic supplementary material The online version of this article (10.1186/s13071-017-2601-y) contains supplementary material, which is available to authorized users.

Tuna is the second largest fishery commodity in Indonesia after the shrimp. Since thehigh demand and the limited stock of tuna resulted in fraudulent chance. Authenticationis required to meassure consumers regarding the accuracy of its labeling and foodsafety. In this study, the authentication was based on protein and DNA barcoding usingcytochrome-b gene (cyt-b) of the mitochondrial DNA as the target of gene. Primer ofcyt b gene was designed based on the tuna species. This study aimed to identify theauthenticity of tuna fresh and its processed products through protein using SDS-PAGE and DNA barcoding techniques. The phases of this research were protein electrophoresisby SDS-PAGE, DNA extraction, PCR amplification, electrophoresis and sequencing.Samples of fresh fish (Tu1, Tu2, Tu3, Tu4, and Tu5) and processed tuna (canned andsteak) were successfully extracted. Result showed that SDS-PAGE proved the damage ofproteins in the processed tuna, so this method was not appropriate if it is used to identifythe authenticity of tuna. PCR electrophoresis results showed that the samples of tuna,tuna steak, sushi, meat ball, abon, and caned tuna were successfully amplified in the rangeof 500-750 bp except Ka3, which was in line with the target of DNA (620 bp). Resultedsequences of Tu2, Tu3, Tu4 and Tu5 were identified according the results of morphometricnamely T. albacares, while Tu1 was identified as T. obesus with homology level of 99%.Processed tunas (steak and canned tuna) were identified as T. albacares, as stated on thelabels.Keywords: Authentication, cytb, DNA barcoding, design primer, SDS-PAGE

Accurate species identification based on visual cues can be challenging due to morphological similarities and the cryptic nature of certain species. Thus a more conclusive method of identification is required, namely DNA barcoding. This is the case regarding two South African otter species, Cape Clawless otter ( Aonyx capensis) and the spotted necked otter ( Lutra maculicollis ). Due to the cryptic nature of these animals faecal samples, known as spraints, are the easiest way of confirming the presence of the animal in an area. In this study, we compared results obtained for universal and partial CytB primer sets on collected spraint and tissue control samples. Universal CytB primers revealed a low percentage of amplified otter species from faecal samples (species specific amplification success of 10.9%) whereas, the partial CytB primer set resulted in successful amplification of 45 out of 55 (82%) samples. We were thus able to positively differentiate between the two otter species using the partial CytB primer set developed in this study. The ability to accurately identify species using partial DNA will be beneficial in understanding numerous aspects of the behaviour and ecological importance of animals in their environment.

The complete mitochondrial genome sequence was determined for a specimen of Calotomus japonicus, a temperate parrotfish endemic to coastal East Asia. It was compared phylogenetically with previously published partial sequences from this species and other parrotfishes. The obtained tree indicated that the three cytb sequences of C. japonicus from a recent molecular study (LC068806-8) probably resulted from introgression through intergeneric hybridization, or possibly from sample confusion. Taking the presently obtained mitogenome as representative of C. japonicus, the species most closely related to this one among congeners is C. zonarchus, which is endemic to the Hawaiian islands.

Background Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. Results The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. Conclusions This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be useful in detecting infection and for follow-up during therapy. Electronic supplementary material The online version of this article (10.1186/s13071-017-2374-3) contains supplementary material, which is available to authorized users.

The taxonomy of American deer has been established almost entirely on the basis of morphological data and without the use of explicit phylogenetic methods; hence, phylogenetic analyses including data for all of the currently recognized species, even if based on a single gene, might improve current understanding of their taxonomy. We tested the monophyly of the morphology-defined genera and species of New World deer (Odocoileini) with phylogenetic analyses of mitochondrial DNA sequences. This is the first such test conducted using extensive geographic and taxonomic sampling. Our results do not support the monophyly of Mazama, Odocoileus, Pudu, M. americana, M. nemorivaga, Od. hemionus, and Od. virginianus. Mazama contains species that belong to other genera. We found a novel sister-taxon relationship between “Mazama” pandora and a clade formed by Od. hemionus columbianus and Od. h. sitkensis, and transfer pandora to Odocoileus. The clade formed by Od. h. columbianus and Od. h. sitkensis may represent a valid species, whereas the remaining subspecies of Od. hemionus appear closer to Od. virginianus. Pudu (Pudu) puda was not found sister to Pudu (Pudella) mephistophiles. If confirmed, this result will prompt the recognition of the monotypic Pudella as a distinct genus. We provide evidence for the existence of an undescribed species now confused with Mazama americana, and identify other instances of cryptic, taxonomically unrecognized species-level diversity among populations here regarded as Mazama temama, “Mazama” nemorivaga, and Hippocamelus antisensis. Noteworthy records that substantially extend the known distributions of M. temama and “M.” gouazoubira are provided, and we unveil a surprising ambiguity regarding the distribution of “M.” nemorivaga, as it is described in the literature. The study of deer of the tribe Odocoileini has been hampered by the paucity of information regarding voucher specimens and the provenance of sequences deposited in GenBank. We pinpoint priorities for future systematic research on the tribe Odocoileini.

SUMMARYLeishmaniasis is a complex of zoonotic diseases caused by parasites of the genus Leishmania, which can develop in domestic as well as wild animals and humans throughout the world. Currently, this disease is spreading in rural and urban areas of non-endemic regions in Brazil. Recently, bats have gained epidemiological significance in leishmaniasis due to its close relationship with human settlements. In this study, we investigated the presence of Leishmania spp. DNA in blood samples from 448 bats belonging to four families representing 20 species that were captured in the Triangulo Mineiro and Alto Paranaiba areas of Minas Gerais State (non-endemic areas for leishmaniasis), Brazil. Leishmania spp. DNA was detected in 8·0% of the blood samples, 41·6% of which were Leishmania infantum, 38·9% Leishmania amazonensis and 19·4% Leishmania braziliensis. No positive correlation was found between Leishmania spp. and bat food source. The species with more infection rates were the insectivorous bats Eumops perotis; 22·2% (4/18) of which tested positive for Leishmania DNA. The presence of Leishmania in the bat blood samples, as observed in this study, represents epidemiological importance due to the absence of Leishmaniasis cases in the region.

We examined sequence variation in mitochondrial and nuclear genes of seven species of the genus Martes (Mustelidae, Carnivora): Martes americana (American marten), Martes flavigula (yellow-throated marten), Martes foina (beech marten), Martes martes (pine marten), Martes melampus (Japanese marten), Martes pennanti (fisher) and Martes zibellina (sable), focusing on the phylogenetic history of the Hokkaido subspecies of the sable, Martes zibellina brachyura. Nucleotide sequence analysis of the mitochondrial cytochrome b gene confirmed the view that the Hokkaido sable population has lower genetic diversity. In contrast, network analysis of a nuclear gene related to coat colour, melanocortin-1 receptor (Mc1r), revealed two different haplogroups for this population: one shared with that of Russian sables and the other specific to this population but with a close relationship with the American and Japanese martens, implying that these endemic haplotypes are composed of uncharacterised ancestral lineages of a past population. We also examined the sequence variation in a neighbouring nuclear gene, transcription factor 25 (Tcf25), located about 5 kb upstream from the Mc1r gene, and found similar trends. The sable genome leaves the impression that Hokkaido hosted ancient marten lineages, with subsequent recent migrations from the continent. The validity of a candidate Mc1r mutation for the entirely yellow coat observed on Hokkaido sables was also discussed.

The number of goatfish species has increased recently, thanks in part to the application of molecular approaches to the taxonomy of a family with conservative morphology and widespread intraspecific color variation. A new subspecies Mulloidichthys flavolineatus flavicaudus Fernandez-Silva & Randall is described from the Red Sea and Arabian Sea, including Socotra and Gulf of Oman. It is characterized by a yellow caudal fin, 25–28 gill rakers, and 37–38 lateral-line scales and it is differentiated from nominal subspecies Mulloidichthys flavolineatus flavolineatus by 1.7% sequence divergence at the mitochondrial cytochrome b gene. The morphometric examination of specimens of Mulloidichthys flavolineatus flavolineatus revealed variation in head length, eye diameter, and barbel length, in western direction from the Hawaiian Islands, South Pacific, Micronesia, and the East Indies to the Indian Ocean. The population of Mulloidichthys flavolineatus flavicaudus subsp. n. in the Gulf of Aqaba differs from that of the remaining Red Sea by shorter barbels, smaller eyes, shorter head, and shorter pelvic fins. We present a list of 26 endemic fishes from the Gulf of Aqaba and discuss the probable basis for the endemism in the light of the geological history of this region.

We investigated mitochondrial and nuclear DNA genotypes in nominal Littorina sitkana samples from 2 localities in Eastern Hokkaido, northern Japan. Our results indicated the existence of cryptic species. In the analysis of partial mitochondrial Cytchrome b gene sequences, haplotypes of L. sitkana samples were monophyletic in a phylogenetic tree with orthologous sequences from other Littorina species, but were apparently separated in 2 clades. One included typical L. sitkana (CBa clade) samples, which formed a clade with an allopatric species, L. horikawai. The other, CBb, was independent from CBa and L. horikawai. Haplotypes of the mitochondrial 16S rRNA gene also separated into 2 clades. We additionally examined intron sequence of the heat shock cognate 70 (HSC70) nuclear gene and identified 17 haplotypes. These were also separated into 2 clades, HSCa and HSCb. Among the examined Hokkaido samples, 60% of individuals were heterozygotes. However, each heterozygote consisted of haplotypes from the same clade, HSCa or HSCb, and no admixture of HSCa and HSCb haplotypes was observed. These results indicate reproductive isolation between the 2 clades. Among the genotyped Hokkaido samples, 93% of individuals had CBa + HSCa or CBb + HSCb genotypes, and 7% had CBb + HSCa genotypes. The discrepancy between the mtDNA and nuclear DNA haplotypes in a few individuals may have been caused by genetic introgression due to past hybridization.

The Japanese white-toothed shrew (Crocidura dsinezumi) is a species endemic to Japan. For this species, only minimal phylogeographic investigations have been conducted. We obtained DNA sequences of mitochondrial cytochrome b and control region and nuclear ApoB genes for 191 individuals of C. dsinezumi from 107 locations collected throughout its known range. In the phylogenetic trees based on mitochondrial DNA sequences, two haplogroups (Eastern and Western Clades) were recognized, and the demarcation line between them was located in central Honshu without an overlapping area. The estimated divergence time between the two major clades indicated that they could have diverged prior to the final geologic division of Hondo and the Asian Continent (100–150 KYA). For the ApoB gene, Types A, G, and R (heterozygote) were recognized although there was a single site mutation. Type A mainly occurs in eastern and central Japan and Types G and R in central and western Japan. It was suggested in the present study that some shrews in Hokkaido were introduced recently from eastern Honshu (possibly the Tohoku Region) whereas others might have been distributed there naturally, and that population in Jeju (South Korea) was introduced recently from Kyushu.

International audience; Here, we describe the development of an oomycete-specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter- and intra-specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete-specific mitochondrial markers currently available.

Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer’s disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan’s spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin–proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin–protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders.

International audience; To date, studies on host preferences and blood meal identification have been conducted for Culicoides species using molecular-based methods such as PCR techniques to amplify only a fragment from universal vertebrate mitochondrial genes such as cytochrome c oxidase subunit I or cytochrome b (Cyt b). The vertebrate prepronociceptin gene (PNOC) was also tested in this field. However, the choice of molecular marker to identify blood meal is critical. The objective of our study is to compare the ability of Cyt b and PNOC as molecular markers for blood meal identification depending on the stage of blood meal digestion. In order to determine whether these Cyt b and PNOC could provide a positive result, 565 blood-fed females of Culicoides spp were collected and morphologically identified. The samples were collected between 2012 and 2014, in two localities in France. The collection localities were near either livestock or a forest. To catch the specimens, we used UV CDC miniature light traps. PNOC sequence of donkeys (Equus asinus) was sequenced and submitted because it was missing in GenBank. Our findings emphasize that the PNOC marker is not suitable to separate closely related Equid species such as horses and donkeys. The Cyt b marker was able to identify 204 more samples when compared to PNOC (99.55% of specimens). Cyt b appears to be better able to detect the origin of blood meals from females with digested blood in their abdomens. We conclude that Cyt b is a good marker as it increases the accuracy of blood meal identification of engorged females containing digested blood in their abdomens. The host opportunist behavior of Culicoides, especially that of C. obsoletus and C. scoticus, the main vectors of BTV in Europe was also highlighted.

Several phylogeographic studies of seabirds have documented low genetic diversity that has been attributed to bottleneck events or individual capacity for dispersal. Few studies have been done in seabirds on the Brazilian coast and all have shown low genetic differentiation on a wide geographic scale. The Kelp Gull is a common species with a wide distribution in the Southern Hemisphere. In this study, we used mitochondrial and nuclear markers to examine the genetic variability of Kelp Gull populations on the Brazilian coast and compared this variability with that of sub-Antarctic island populations of this species. Kelp Gulls showed extremely low genetic variability for mitochondrial markers (cytb and ATPase) and high diversity for a nuclear locus (intron 7 of the β-fibrinogen). The intraspecific evolutionary history of Kelp Gulls showed that the variability found in intron 7 of the β-fibrinogen gene was compatible with the variability expected under neutral evolution but suggested an increase in population size during the last 10,000 years. However, none of the markers revealed evidence of a bottleneck population. These findings indicate that the recent origin of Kelp Gulls is the main explanation for their nuclear diversity, although selective pressure on the mtDNA of this species cannot be discarded.

Contact: tixier@supagro.inra.fr; The public database GenBank is an increasingly important source of sequence data for diagnostic and phylogenetic research; however, not all deposited sequences are necessarily correctly ascribed to a source species. We considered the example of the mite family Phytoseiidae to determine how the corresponding sequences could be accurately exploited. Phytoseiidae mites are well known worldwide for their ability to control certain mite and insect pests. The number of molecular approaches, especially for diagnostic purposes, has increased over the past decade, leading to an increase in the number of sequences registered in the GenBank database. The aim of the present paper was to evaluate the validity of the DNA sequences presently assigned to Phytoseiidae species in this database. Three hundred and fifty-one sequences, corresponding to the four most frequently registered DNA fragments (ITS, COI, Cytb and 12S rRNA), were considered. DNA extraction, amplification and sequencing were performed for the fragments 12S rRNA and ITS for Amblyseius andersoni, A. swirskii, Iphiseius degenerans, Euseius ovalis, E. stipulatus, Neoseiulus cucumeris and Typhlodromus pyri, as some identifications were questionable. Numerous problems were evident based on genetic distance analyses of these sequences. First, nomenclatural problems were encountered, preventing the correct identification of the taxa sequenced in one case. Suspected misidentifications were frequent, stressing the importance of voucher specimen availability. For the 12S rRNA fragment, sequences assigned to three Phytoseiidae species were those of their prey (Astigmata), underlining the care that must be taken when manipulating the DNA of such predators (sterile conditions and specific PCR primers). Finally, sequences of two regions of the COI mtDNA were encountered, leading to alignment problems between sequences of a same gene and same species. These results are discussed in relation to responsibilities of authors in terms of taxon identification and the great utility of open access DNA sequence databases, such as GenBank, for improving taxonomic identifications and advancing scientific research.

Background Wild animals’ meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species. Results The combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as ‘near threatened’; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species. Conclusions The reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices. Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.