Multiple DNA Markers Research Articles

Phylogenetic Analysis of Rare and Endangered Tulipa Species (Liliaceae) of Kazakhstan Based on Universal Barcoding Markers.

In Kazakhstan, the genus Tulipa is represented by 35 species, 18 of which are listed in the Red Data Book of Kazakhstan and protected by the state. Recent studies of tulip specimens from regions bordering Kazakhstan emphasize the significance of species inventory and report the discovery of several hybrids. In this study, eight tulip species were identified based on morphological characteristics and using DNA barcoding methods. Molecular genetic markers, including nrDNA (ITS) and cpDNA markers (rbcL, matK), of the studied species were sequenced and analyzed using the Bayesian inference and maximum likelihood phylogenetic analysis methods. Our work demonstrates that DNA barcodes based on the ITS, rbcL, and matK marker regions have successful practical applicability, with ITS being the most informative at the intragenic level. However, for distinguishing closely related taxa, the most effective approach would be to use a combined dataset of sequences from multiple DNA markers. The results showed discrepancies in the placement of several taxa (T. kaufmanniana, T. patens), likely due to introgression and natural spontaneous hybridization. The molecular phylogenetic analysis suggests the existence of a previously undescribed hybrid between T. patens and T. alberti. Further detailed population studies are needed to validate this hypothesis.

Gene-flow investigation between garden and wild roses planted in close distance.

Rose is a major ornamental plant, and a lot of cultivars with attractive morphology, color and scent have been generated by classical breeding. Recent progress of genetic modification produces a novel cultivar with attractive features. In both cases, a major problem is the gene-flow from cultivated or genetically modified (GM) plants to wild species, causing reduction of natural population. To investigate whether gene-flow occurs in wild species, molecular analysis with DNA markers with higher efficient technique is useful. Here we investigated the gene-flow from cultivated roses (Rosa×hybrida) to wild rose species planted in close distance in the field. The overlapping flowering periods and visiting insects suggest that pollens were transported by insects between wild and cultivated roses. We examined the germination ratio of seeds from wild species, and extracted DNA and checked with KSN and APETALA2 (AP2) DNA markers to detect transposon insertions. Using two markers, we successfully detected the outcross between wild and cultivated roses. For higher efficiency, we established a bulking method, where DNA, leaves or embryos were pooled, enabling us to that check the outcross of many plants. Our results suggest that wild species and garden cultivars can cross in close distance, so that they should be planted in distance, and checked the outcross with multiple DNA markers.

First report of emerging fungal pathogens of Cordyceps militaris in Vietnam

Cultivation of Cordyceps militaris, a valuable medicinal and edible fungus, has dramatically increased in Vietnam since 2010. During industrial production, parasitic white molds were found to infect the mycelia and fruiting bodies of C. militaris causing significant quality and yield losses. Two different fungal strains were obtained from the mycelia and fruiting bodies of C. militaris in Danang mushroom farms and were characterized by morphological and multiple DNA markers analysis. The sequence alignment of ITS, LSU and rpb2 markers revealed that the pathogens are related to the type species Lecanicillium coprophilum and Calcarisporium cordycipiticola with more than 99% sequence identities. The growth characteristics and pathogenic activities of the two isolated species on their host C. militaris were also investigated. The phylogenetic analysis based on the ITS sequences showed that L. coprophilum WF2611 is closer to its host C. militaris than C. cordycipiticola NT1504. To our knowledge, this is the first worldwide report of C. militaris infected by L. coprophilum which would be an useful information on prevention and control of the disease and be helpful for the industrial cultivation of C. militaris.

DNA Barcoding and Species Classification of Morchella.

True morels (Morchella) are a well-known edible fungi, with economically and medicinally important values. However, molecular identification and species taxonomy of the genus Morchella have long been controversial, due to numerous intermediate morphologies among species. In this study, we determined the identification efficiency of DNA barcoding and species classification of 260 individuals from 45 Morchella species, on the basis of multiple nuclear DNA markers. DNA barcoding analysis showed that the individual DNA fragment has a lower resolution of species identification than that of combined multiple DNA markers. ITS showed the highest level of species discrimination among the individual genetic markers. Interestingly, the combined DNA markers significantly increased the resolution of species identification. A combination of four DNA genes (EF1-α, RPB1, RPB2 and ITS) showed a higher species delimitation than that any combination of two or three markers. Phylogenetic analysis suggested that the species in genus Morchella could have been divided into two large genetic clades, the Elata Clade and Esculenta Clade lineages. The two lineages divided approximately 133.11 Mya [95% HPD interval: 82.77–197.95] in the early Cretaceous period. However, some phylogenetic species of Morchella showed inconsistent evolutionary relationships with the traditional morphological classifications, which may have resulted from incomplete lineage sorting and/or introgressive hybridization among species. These findings demonstrate that the interspecific gene introgression may have affected the species identification of true morels, and that the combined DNA markers significantly improve the resolution of species discrimination.

Two New Species of Euscorpius (Scorpiones, Euscorpiidae) from Skyros and Andros Islands, Greece

Two new scorpion species are described from Skyros and Andros Islands (Greece), Euscorpius triantisi sp. n. and E. simaiakisi sp. n. respectively, based on morphological and molecular evidence. Identity and level of divergence of these taxa are confirmed by a phylogeny based on multiple DNA markers (Parmakelis et al., 2013 b). Euscorpius triantisi sp. n. forms a sister clade to E. mylonasi Fet et al., 2014 from Euboea; the new species is characterized primarily by higher trichobothrial numbers (Pv = 8 and Pe-et = 6). E. simaiakisi sp. n. forms a sister clade to E. kritscheri Fet et al., 2013 from Tinos; the new species is primarily characterized by lower trichobothrial numbers (Pv = 7 and Pe-et = 5).

Molecular Phylogeny of Cryptonanus (Didelphidae: Thylamyini): Evidence for a recent and complex diversification in South American open biomes

Systematic revisions of South American marsupials have contributed to our knowledge about genus and species diversity in the last decades, including studies of the most recently described genus Cryptonanus (Didelphidae), currently comprising four recognized species. Herein we provide the first phylogeny for these mouse opossums based on comprehensive sampling, including representatives from all nominal taxa, encompassing most of the geographic distribution of the genus while also extending its known range. The taxonomic status of Cryptonanus species was explored by analyses of multiple mitochondrial and nuclear DNA markers to assess phylogenetic relationships and to provide divergence time estimates, species delimitations and biogeographical hypotheses. Cryptonanus monophyly remained highly supported despite the inclusion of abundant new data from more than a hundred specimens, comprising 10 independent evolutionary lineages. Species-complexes within valid nominal taxa reveal higher species richness in the genus. Based on divergence estimates from a dated phylogeny, we suggest that Cryptonanus diversified along the Quaternary, with speciation events occurring well into the Pleistocene. The best supported biogeographical hypothesis endorses speciation by vicariance and subset speciation across open formations in shaping the evolutionary history of this didelphid genus, strongly associated with dry tropical landscapes of South America.

Metabarcoding From Microbes to Mammals: Comprehensive Bioassessment on a Global Scale

Global biodiversity loss is unprecedented, and threats to existing biodiversity are growing. Given pervasive global change, a major challenge facing resource managers is a lack of scalable tools to rapidly and consistently measure Earth's biodiversity. Environmental genomic tools provide some hope in the face of this crisis, and DNA metabarcoding, in particular, is a powerful approach for biodiversity assessment at large spatial scales. However, metabarcoding studies are variable in their taxonomic, temporal, or spatial scope, investigating individual species, specific taxonomic groups, or targeted communities at local or regional scales. With the advent of modern, ultra-high throughput sequencing platforms, conducting deep sequencing metabarcoding surveys with multiple DNA markers will enhance the breadth of biodiversity coverage, enabling comprehensive, rapid bioassessment of all the organisms in a sample. Here, we report on a systematic literature review of 1,563 articles published about DNA metabarcoding and summarize how this approach is rapidly revolutionizing global bioassessment efforts. Specifically, we quantify the stakeholders using DNA metabarcoding, the dominant applications of this technology, and the taxonomic groups assessed in these studies. We show that while DNA metabarcoding has reached global coverage, few studies deliver on its promise of near-comprehensive biodiversity assessment. We then outline how DNA metabarcoding can help us move toward real-time, global bioassessment, illustrating how different stakeholders could benefit from DNA metabarcoding. Next, we address barriers to widespread adoption of DNA metabarcoding, highlighting the need for standardized sampling protocols, experts and computational resources to handle the deluge of genomic data, and standardized, open-source bioinformatic pipelines. Finally, we explore how technological and scientific advances will realize the promise of total biodiversity assessment in a sample—from microbes to mammals—and unlock the rich information genomics exposes, opening new possibilities for merging whole-system DNA metabarcoding with (1) abundance and biomass quantification, (2) advanced modeling, such as species occupancy models, to improve species detection, (3) population genetics, (4) phylogenetics, and (5) food web and functional gene analysis. While many challenges need to be addressed to facilitate widespread adoption of environmental genomic approaches, concurrent scientific and technological advances will usher in methods to supplement existing bioassessment tools reliant on morphological and abiotic data. This expanded toolbox will help ensure that the best tool is used for the job and enable exciting integrative techniques that capitalize on multiple tools. Collectively, these new approaches will aid in addressing the global biodiversity crisis we now face.

Phylogeography and taxonomic reassessment of Arabidopsis halleri – a montane species from Central Europe

Evolutionary histories of plants from the mid-elevation (montane) zone of European mountain ranges have only rarely been documented, standing in contrast to those of well-researched inhabitants of (sub-)alpine and foothill zones. To fill this gap, we have reconstructed the phylogeography of Arabidopsis halleri, a species preferring coniferous woodlands and corresponding secondary habitats in the montane zone of the Alps, Carpathians, Hercynian massif and Dinaric Alps. Based on range-wide sampling and finer-scale analyses of multiple multilocus DNA markers, we have addressed phylogeographic patterns among the Carpathian populations and inferred their relationships to A. halleri from neighbouring mountain ranges. We also present a taxonomic re-evaluation of the species in Europe, based on the revealed genetic structure complemented by morphological data. Besides two distinct Alpine groups, we identified a major phylogeographic split between the Western and South-Eastern Carpathians. Interestingly, Western and South-Eastern Carpathian populations were genetically closer to populations from neighbouring mountain ranges (the Hercynian massif and the Dinaric Alps for the Western and South-Eastern Carpathians, respectively) than they were to each other, likely reflecting long-term isolation in different parts of the Carpathians or different (re)colonization pathways during the Holocene. In spite of the considerable environmentally correlated variation, the five major European genetic groups exhibited distinctive morphological characters, and we therefore propose treating them as separate subspecies: A. halleri subsp. halleri (Western Europe, Hercynian massif), A. halleri subsp. tatrica (Western Carpathians), A. halleri subsp. ovirensis (Eastern Alps), A. halleri subsp. occidentalis (Western Alps) and A. halleri subsp. dacica (Eastern and Southern Carpathians and Dinaric Alps).

Effects of Geological and Environmental Events on the Diversity and Genetic Divergence of Four Closely Related Pines: Pinus koraiensis, P. armandii, P. griffithii, and P. pumila.

The effects of mountain uplift and environmental oscillations on nucleotide variability and species divergence remain largely unknown in East Asia. In this study, based on multiple nuclear DNA markers, we investigated the levels and patterns of nucleotide diversity and interspecific divergence in four closely related pines in China, i.e., Pinus koraiensis, P. armandii, P. griffithii, and P. pumila. The four pine taxa shared low levels of nucleotide polymorphisms at the species level. P. pumila had the highest silent nucleotide diversity (πsil = 0.00661) whereas P. griffithii had the lowest (πsil = 0.00175), while the levels of genetic polymorphism in P. armandii (πsil = 0.00508) and P. koraiensis (πsil = 0.00652) were intermediate between the other two species. Population genetic structure analysis showed that variations primarily existed within populations of the four pine species, presumably due to habitat fragmentation or the island-like distributions of Pinus species. Population divergence (FST) analysis showed that the genetic divergence between P. griffithii and P. koraiensis was much greater than that between P. koraiensis and the other two pines species. Isolation-with-migration analysis suggested that asymmetric gene flow had occurred between any two pairs of pine species. Phylogenetic analyses indicated that the four allied species split into two groups about 1.37 million years ago, where P. armandii and P. pumila were closer and clustered as sister species, whereas P. koraiensis and P. griffithii were clustered on another branch. Our results and those obtained in previous studies suggest that mountain uplift and geological climate oscillations may have led to the patterns of genetic divergence and nucleotide variations in these four pine species.

Multiple DNA markers for evaluation of resistance against Potato virus Y, Potato virus S and Potato leafroll virus

Molecular markers within or close to genes of interest play essential roles in marker-assisted selection. PCR-based markers have been developed for numerous traits in different plant species including several genes conferring resistance to viruses in potato. In the present work, rapid and reliable approaches were developed for the simultaneous detection of Ryadg and Ry-fsto, Ns, and PLRV.1 genes conferring resistance to Potato virus Y, Potato virus S and Potato leafroll virus, respectively, on the basis of previously published and newly modified markers. The sequence characterized amplified region (SCAR) markers for Ryadg, Ns and PLRV1 and the newly modified cleaved amplified polymorphic sequences (CAPS) marker for Ry-fsto were amplified in one PCR reaction which could simply characterize Ryadg and PLRV.1 resistance. Additional digestion of amplicons with EcoRV and MfeI for genotyping the Ry-fsto and Ns resistance genes, respectively, was needed. The effectiveness of genotyping in triplex and tetraplex PCRs was tested on 35 potato varieties used for potato seed production and breeding programs.

Assessment of the Precision ID Ancestry panel.

AbstractThe ability to provide accurate DNA-based forensic intelligence requires analysis of multiple DNA markers to predict the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of biological evidence. Massively parallel sequencing (MPS) enables the analysis of hundreds of DNA markers in multiple samples simultaneously, increasing the value of the intelligence provided to forensic investigators while reducing the depletion of evidential material resulting from multiple analyses. The Precision ID Ancestry Panel (formerly the HID Ion AmpliSeq™ Ancestry Panel) (Thermo Fisher Scientific) (TFS)) consists of 165 autosomal SNPs selected to infer BGA. Forensic validation criteria were applied to 95 samples using this panel to assess sensitivity (1 ng-15 pg), reproducibility (inter- and intra-run variability) and effects of compromised and forensic casework type samples (artificially degraded and inhibited, mixed source and aged blood and bone samples). BGA prediction accuracy was assessed using samples from individuals who self-declared their ancestry as being from single populations of origin (n = 36) or from multiple populations of origin (n = 14). Sequencing was conducted on Ion 318™ chips (TFS) on the Ion PGM™ System (TFS). HID SNP Genotyper v4.3.1 software (TFS) was used to perform BGA predictions based on admixture proportions (continental level) and likelihood estimates (sub-population level). BGA prediction was accurate at DNA template amounts of 125pg and 30pg using 21 and 25 PCR cycles respectively. HID SNP Genotyper continental level BGA assignments were concordant with BGAs for self-declared East Asian, African, European and South Asian individuals. Compromised, mixed source and admixed samples, in addition to sub-population level prediction, requires more extensive analysis.

Large-scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae)

Southeast Asia and southern China (SEA-SC) harbor a highly diverse and endemic flora and fauna that is under increasing threat. An understanding of the biogeographical history and drivers of this diversity is lacking, especially in some of the most diverse and threatened groups. The Asian leaf-litter frog genus Leptolalax Dubois 1980 is a forest-dependent genus distributed throughout SEA-SC, making it an ideal study group to examine specific biogeographic hypotheses. In addition, the diversity of this genus remains poorly understood, and the phylogenetic relationships among species of Leptolalax and closely related Leptobrachella Smith 1928 remain unclear. Herein, we evaluate species-level diversity based on 48 of the 53 described species from throughout the distribution of Leptolalax. Molecular analyses reveal many undescribed species, mostly in southern China and Indochina. Our well-resolved phylogeny based on multiple nuclear DNA markers shows that Leptolalax is not monophyletic with respect to Leptobrachella and, thus, we assign the former to being a junior synonym of the latter. Similarly, analyses reject monophyly of the two subgenera of Leptolalax. The diversification pattern of the group is complex, involving a high degree of sympatry and prevalence of microendemic species. Northern Sundaland (Borneo) and eastern Indochina (Vietnam) appear to have played pivotal roles as geographical centers of diversification, and paleoclimatic changes and tectonic movements seem to have driven the major divergence of clades. Analyses fail to reject an “upstream” colonization hypothesis, and, thus, the genus appears to have originated in Sundaland and then colonized mainland Asia. Our results reveal that both vicariance and dispersal are responsible for current distribution patterns in the genus.

Exploring species boundaries with multiple genetic loci using empirical data from non‐biting midges

Over the past decade, molecular approaches to species delimitation have seen rapid development. However, species delimitation based on a single locus, for example, DNA barcodes, can lead to inaccurate results in cases of recent speciation and incomplete lineage sorting. Here, we compare the performance of Automatic Barcode Gap Discovery (ABGD), Bayesian Poisson tree processes (PTP), networks, generalized mixed Yule coalescent (GMYC) and Bayesian phylogenetics and phylogeography (BPP) models to delineate cryptic species previously detected by DNA barcodes within Tanytarsus (Diptera: Chironomidae) non‐biting midges. We compare the results from analyses of one mitochondrial (cytochrome c oxidase subunit I [COI]) and three nuclear (alanyl‐tRNA synthetase 1 [AATS1], carbamoyl phosphate synthetase 1 [CAD1] and 6‐phosphogluconate dehydrogenase [PGD]) protein‐coding genes. Our results show that species delimitation based on multiple nuclear DNA markers is largely concordant with morphological variation and delimitations using a single locus, for example, the COI barcode. However, ABGD, GMYC, PTP and network models led to conflicting results based on a single locus and delineate species differently than morphology. Results from BPP analyses on multiple loci correspond best with current morphological species concept. In total, 10 lineages of the Tanytarsus curticornis species complex were uncovered. Excluding a Norwegian population of Tanytarsus brundini which might have undergone recent hybridization, this suggests six hitherto unrecognized species new to science. Five distinct species are well supported in the Tanytarsus heusdensis species complex, including two species new to science.

Advances of Community-Level Plant DNA Barcoding in China.

DNA barcoding is a commonly used bio-technology in multiple disciplines including biology, environmental science, forensics and inspection, etc. Forest dynamic plots provide a unique opportunity to carry out large-scale, comparative, and multidisciplinary research for plant DNA barcoding. The paper concisely reviewed four previous progresses in China; specifically, species discrimination, community phylogenetic reconstruction, phylogenetic community structure exploration, and biodiversity index evaluation. Further, we demonstrated three major challenges; specifically, building the impetus to generate DNA barcodes using multiple plant DNA markers for all woody species at forest community levels, analyzing massive DNA barcoding sequence data, and promoting theoretical innovation. Lastly, we raised five possible directions; specifically, proposing a “purpose-driven barcode” fit for multi-level applications, developing new integrative sequencing strategies, pushing DNA barcoding beyond terrestrial ecosystem, constructing national-level DNA barcode sequence libraries for special plant groups, and establishing intelligent identification systems or online server platforms. These efforts will be potentially valuable to explore large-scale biodiversity patterns, the origin and evolution of life, and will also facilitate preservation and utilization of biodiversity resources.

REDESCRIPTION OF EUSCORPIUS TAURICUS (C.L. KOCH, 1837), WITH THE DESCRIPTION OF TWO NEW RELATED SPECIES FROM GREECE (SCORPIONES: EUSCORPIIDAE)

Euscorpius tauricus (C. L. Koch, 1837) was previously known only from the Crimea Peninsula, Ukraine. We report an unexpected presence of this species in the Cyclades Islands (Greece) and northwestern Anatolia (Turkey). In addition we designate a neotype for this species. We synonymize Euscorpius carpathicus aegaeus Di Caporiacco, 1950 syn. n., from Antiparos Island and Euscorpius rahsenae Yağmur et Tropea, 2013 syn. n., from Anatolia, with E. tauricus. In addition, we describe two new species related to E. tauricus, from the Cyclades Islands: E. curcici sp. n., from Ios and Sikinos Islands, and E. amorgensis sp. n., from Amorgos Island. Identity and level of divergence of these taxa is confirmed by multiple DNA markers.

Eutrema tianshanense (Brassicaceae), a new species from Tian Shan Mountains of central Asia

Eutrema tianshanense, a new species of Brassicaceae growing near to the permanent glaciation area in the Tian Shan Mountains of central Asia, is described. It is quite similar morphologically to E. heterophyllum, and E. racemosum from the Qinghai-Tibet Plateau and adjacent regions, and to E. edwardsii of Qinghai-Tibet Plateau, Central and North Asia, and northern North America, but with rounder and smaller leaves. In addition to its long-distance disjunction from the ranges of the above three species, phylogenetic analyses based on sequence variations of multiple chloroplast DNA markers and nuclear ITS suggested that E. tianshanense and these species represent two independently evolved lineages in Tian Shan Mountains and the Qinghai-Tibet Plateau. Their morphological similarities may have resulted from the same selection pressures in the alpine habitats where they grow.

Abstract 4338: Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin

Abstract Purpose of the study: Molecular tests have the potential to improve current non-invasive faecal immunochemical test (FIT) screening for colorectal cancer (CRC) and advanced precancerous lesions. We examined the performance of a panel of faecal DNA (sDNA) markers and FIT in archival samples from an invitational CRC screening population. Methods: Whole stool samples were prospectively collected from individuals participating an invitational primary colonoscopy-screening program (COCOS trial). Only participants that provided stool, performed FIT (OC-Sensor) and underwent colonoscopy were selected. The sDNA panel included quantitative molecular assays for KRAS mutations and for aberrant NDRG4 and BMP3 methylation. The performance of the sDNA plus FIT panel was compared to the FIT results alone, by Receiver Operator Characteristic (ROC) analyses. Results: A total of 1047 individuals (51% male) with a median age of 60 years (range 50-75) were included, of which 7 (0.7%) had colorectal cancer and 104 (9.9%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps ≥ 1 cm). The combination of sDNA and FIT was more sensitive than FIT alone for detecting advanced precancerous lesions (49% (50/102) and 25% (26/102), respectively). Specificities among individuals with non-advanced or negative findings (controls) were 89% and 96% for sDNA and FIT testing, respectively. ROC analysis of CRC and advanced precancerous lesions compared to controls revealed an Area Under the Curve (AUC) of 0.75 for the sDNA plus FIT test, compared to 0.68 for FIT alone. At an equal specificity of 95%, advanced precancerous lesions were detected with a higher sensitivity by the sDNA plus FIT test compared to FIT alone (36% vs 28%, p = 0.08). Conclusions: In an invitational colorectal cancer screening cohort, combining stool DNA markers with FIT detected more advanced neoplasia than FIT alone, primarily due to detecting more advanced adenomas. Citation Format: Linda J.W. Bosch, Veerle Melotte, Sandra Mongera, Kathleen L.J. Daenen, Veerle H.M. Coupe, Sietze T. van Turenhout, Esther M. Stoop, Thomas R. de Wijkerslooth, Chris J.J. Mulder, Ernst J. Kuipers, Evelien Dekker, Michael Domanico, Graham P. Lidgard, Barry M. Berger, Beatriz Carvalho, Manon van Engeland, Gerrit A. Meijer. Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4338.

Phylogeographic Structure of the White-Footed Mouse and the Deer Mouse, Two Lyme Disease Reservoir Hosts in Québec.

Modification of a species range is one of many consequences of climate change and is driving the emergence of Lyme disease in eastern Canada. The primary reservoir host of the bacteria responsible for Lyme disease, Borrelia burgdorferi, is the white-footed mouse (Peromyscus leucopus), whose range is rapidly shifting north into southern Québec. The deer mouse, P. maniculatus, is occurring over most Québec province and is a less competent host for B. burgdorferi. Here, we compared the phylogeographic structure of both Peromyscus species in Québec. Using a combination of multiple mitochondrial DNA markers and phylogeographic methods, we detected an ongoing and rapid expansion of P. leucopus, while P. maniculatus appears more stable. Haplotype and populations networks indicated that populations of P. maniculatus exhibit more genetic structure than P. leucopus across the study area. Furthermore, significant and consistent genetic divergences between populations of the two species on both sides of the St. Lawrence River suggest that distinct lineages of P. leucopus and P. maniculatus with different ancestral origins colonized Southern Québec following the Last Glacial Maximum. The phylogeographic structure of pathogens is expected to mirror the structure observed in their reservoir hosts. As different strains of Borrelia burgdorferi may be associated with different levels of pathogenicity and immune responses of their hosts, our results are helpful at better understanding the pattern of spread of Lyme disease in a zone of emergence, and associated risk for human populations.

A review of Galaxiella pusilla (Mack) (Teleostei: Galaxiidae) in south-eastern Australia with a description of a new species.

The dwarf galaxias, Galaxiella pusilla (Mack), is a small, threatened freshwater fish from coastal south-eastern Australia. Recent genetic studies, using multiple nuclear and mitochondrial DNA markers, found substantial differences between populations in western Victoria and south Australia ('west region') compared to eastern Victoria, Flinders Island, and Tasmania ('east region') that suggest the presence of a cryptic species. Morphological measurements and meristic counts from multiple populations within each region were undertaken to investigate potential differences between regions. Several characters, found to discriminate between individuals in the regions and to be diagnostic for two taxa, were used to describe a new species, Galaxiella toourtkoourt, for the west region. This is only the second species in the Galaxiidae to exhibit sexual dimorphism. The original description of Galaxiella pusilla, based on five specimens, is revised following examination of a large number of individuals. Both species are considered nationally threatened and are categorised as 'endangered'; the revised distribution of G. pusilla s.s. is reduced by approximately 60%. A number of inconsistencies in the most recent revision of the genus Galaxiella are also corrected.

Forensic soil DNA analysis using high-throughput sequencing: a comparison of four molecular markers.

Soil analysis, such as mineralogy, geophysics, texture and colour, are commonly used in forensic casework to link a suspect to a crime scene. However, DNA analysis can also be applied to characterise the vast diversity of organisms present in soils. DNA metabarcoding and high-throughput sequencing (HTS) now offer a means to improve discrimination between forensic soil samples by identifying individual taxa and exploring non-culturable microbial species. Here, we compare the small-scale reproducibility and resolution of four molecular markers targeting different taxa (bacterial 16S rRNA, eukaryotic18S rRNA, plant trnL intron and fungal internal transcribed spacer I (ITS1) rDNA) to distinguish two sample sites. We also assess the background DNA level associated with each marker and examine the effects of filtering Operational Taxonomic Units (OTUs) detected in extraction blank controls. From this study, we show that non-bacterial taxa in soil, particularly fungi, can provide the greatest resolution between the sites, whereas plant markers may be problematic for forensic discrimination. ITS and 18S markers exhibit reliable amplification, and both show high discriminatory power with low background DNA levels. The 16S rRNA marker showed comparable discriminatory power post filtering; however, presented the highest level of background DNA. The discriminatory power of all markers was increased by applying OTU filtering steps, with the greatest improvement observed by the removal of any sequences detected in extraction blanks. This study demonstrates the potential use of multiple DNA markers for forensic soil analysis using HTS, and identifies some of the standardisation and evaluation steps necessary before this technique can be applied in casework.