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Journal: Molecular ecology resources


Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single-nucleotide polymorphism (SNP) markers in M. truncatula through large-scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina’s GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.

Concepts: Bioinformatics, Allele frequency, Genetic genealogy, Evolution, Population genetics, DNA, Gene, Genetics


Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non-invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH-psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH-psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.

Concepts: Aquatic plant, Organism, Plant, DNA, Water, Gene, Biology, Species


With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non-model species. Here, we describe a successful approach to a genome-wide medium density Single Nucleotide Polymorphism (SNP) panel in a non-model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP-chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP-chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP-chip to demonstrate the ability of such genome-wide marker data to detect population sub-division, and compared these results to similar analyses using microsatellites. The SNP-chip will be used to map Quantitative Trait Loci (QTL) for fitness-related phenotypic traits in natural populations.

Concepts: Gene, Spanish Sparrow, Passer, Eurasian Tree Sparrow, Sparrow, House Sparrow, Genetics, DNA


Species-level identification and delimitation of bryophytes using the proposed general barcode markers for land plants has been challenging. Bryophyta (mosses) is the second most species-rich group of land plants after angiosperms, and it is thus of great importance to find useful barcoding regions also for this group of plants. We investigated how the plastid regions atpF-atpH, rbcL and trnH-psbA and the nuclear ITS2 region performed as barcode markers on closely related bryophyte taxa of selected moss (Bartramia, Distichium, Fissidens, Meesia and Syntrichia) and liverwort (Blepharostoma) genera from boreal and arctic regions. We also evaluated how sequencing success of herbarium specimens is related to length of the sequenced fragment, specimen age and taxonomic group. Sequencing success was higher for shorter fragments and younger herbarium specimens, but was lower than expected in the genera Distichium and Fissidens, indicating imperfect universality of the primers used. None of the studied DNA barcode regions showed a consistent barcode gap across the studied genera. As a single locus, the region atpF-atpH performed slightly better than rbcL and ITS2 and much better than trnH-psbA in terms of grouping conspecific sequences in monophyletic groups. This marker also gave a higher percentage of correct hits when conducting blast searches on a local database of identified sequences. Concatenated data sets of two and three markers grouped more conspecific sequences in monophyletic groups, but the improvement was not great compared with atpF-atpH alone. A discussion of recent studies testing barcode regions for bryophytes is given. We conclude that atpF-atpH, rbcL and ITS2 are to be the most promising barcode markers for mosses.

Concepts: Sequence, Species, Hornwort, Embryophyte, Moss, Bryophyte, Plant, Marchantiophyta


As for many other regions, environmental and biodiversity monitoring of the brackish Baltic Sea suffers from low species resolution for several taxa. One such case is the benthic larvae of midges Chironomidae (Diptera), which are estimated to constitute about 30% of the macrozoobenthos species of the Baltic Sea and are important indicators of environmental quality. We assessed the usefulness of COI (cytochrome oxidase I) gene barcoding to improve species resolution and its potential for implementation in monitoring programmes. Neighbour-Joining, Maximum parsimony and Bayesian-inference analyses all provided high congruency with morphological analyses of adult males for almost all 42 species studied. Barcoding was helpful to elucidate some cases of taxonomical difficulties, such as synonyms. In contrast to the high identification accuracy when using our local database, there were a number of cases where matching with GenBank and BOLD provided puzzling results. For reliable species identification at least 15-30 specimens from 5-10 well-distributed sites within the geographical range of the species might be needed in a database to adequately cover the intraspecific variability of chironomids. Implementation of DNA barcoding, as applied here, in monitoring would result in an increase from at present less than 10% to more than 90% successful chironomid species identification of Baltic Sea benthic samples, as it also would for many nearby lakes. Routine monitoring of benthic environmental samples based on Next-Generation sequencing techniques would provide a cost effective way to obtain a taxonomically much more complete assessment of environmental quality and biodiversity, as required by EU directives and national legislation.

Concepts: Poland, Latvia, Estonia, Species, Identification, DNA barcoding, Taxonomy, Baltic Sea


We present a DNA barcoding study on the insect order Orthoptera that was generated in collaboration between four barcoding projects in three countries, viz. Barcoding Fauna Bavarica (Germany), German Barcode of Life, Austrian Barcode of Life, and Swiss Barcode of Life. Our dataset includes 748 COI sequences from 127 of the 162 taxa (78.4%) recorded in the three countries involved. 93 of these 122 species (76.2%, including all Ensifera), can be reliably identified using DNA barcodes. The remaining 26 caeliferan species (families Acrididae and Tetrigidae) form ten clusters that share barcodes among up to five species, in three cases even across different genera, and in six cases even sharing individual barcodes. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the species concerned are phylogenetically young and hybridization has been previously observed. We also highlight the problem of nuclear mitochondrial pseudogenes (numts), a known problem in the barcoding of orthopteran species, and the possibility of Wolbachia infections. Finally, we discuss the possible taxonomic implications of our barcoding results and point out future research directions. This article is protected by copyright. All rights reserved.

Concepts: Evolution, Biology, Austria, Grasshopper, DNA, Species, DNA barcoding, Orthoptera


Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine if reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next we designed P. bivittatus specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species and we found that P. bivittatus DNA was consistently detectable up to 96 hours. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus this method is a new and promising technique for the management of invasive reptiles. This article is protected by copyright. All rights reserved.

Concepts: Gene, Molecular biology, Polymerase chain reaction, Copyright, Python sebae, Water, Cytochrome b, DNA


Climate change is a major threat to global biodiversity that will produce a range of new selection pressures. Understanding species responses to climate change requires an interdisciplinary perspective, combining ecological, molecular and environmental approaches. We propose an applied integrated framework to identify populations under threat from climate change based on their extent of exposure, inherent sensitivity due to adaptive and neutral genetic variation and range shift potential. We consider intraspecific vulnerability and population-level responses, an important but often neglected conservation research priority. We demonstrate how this framework can be applied to vertebrates with limited dispersal abilities using empirical data for the bat Plecotus austriacus. We use ecological niche modelling and environmental dissimilarity analysis to locate areas at high risk of exposure to future changes. Combining outlier tests with genotype-environment association analysis we identify potential climate-adaptive SNPs in our genomic dataset and differences in the frequency of adaptive and neutral variation between populations. We assess landscape connectivity and show that changing environmental suitability may limit the future movement of individuals, thus affecting both the ability of populations to shift their distribution to climatically suitable areas and the probability of evolutionary rescue through the spread of adaptive genetic variation among populations. Therefore a better understanding of movement ecology and landscape connectivity is needed for predicting population persistence under climate change. Our study highlights the importance of incorporating genomic data to determine sensitivity, adaptive potential and range shift potential, instead of relying solely on exposure to guide species vulnerability assessments and conservation planning. This article is protected by copyright. All rights reserved.

Concepts: Copyright, Natural selection, Scientific method, Environmental niche modelling, Evolution, Ecological niche, Biodiversity, Ecology


Museum specimens provide a wealth of information to biologists, but obtaining genetic data from formalin-fixed and fluid-preserved specimens remains challenging. While DNA sequences have been recovered from such specimens, most approaches are time-consuming and produce low data quality and quantity. Here we use a modified DNA extraction protocol combined with high-throughput sequencing to recover DNA from formalin-fixed and fluid-preserved snakes that were collected over a century ago and for which little or no modern genetic materials exist in public collections. We successfully extracted DNA and sequenced ultraconserved elements (x̄ = 2,318 loci) from 10 fluid-preserved snakes and included them in a phylogeny with modern samples. This phylogeny demonstrates the general use of such specimens in phylogenomic studies and provides evidence for the placement of enigmatic snakes, such as the rare and never-before sequenced Indian Xylophis stenorhynchus. Our study emphasizes the relevance of museum collections in modern research and simultaneously provides a protocol that may prove useful for specimens that have been previously intractable for DNA sequencing. This article is protected by copyright. All rights reserved.

Concepts: Human Genome Project, Copyright, Human genome, Sequence, DNA sequencing, Molecular biology, Biology, DNA


New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR-free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (3 different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome-skimming) respectively. We found that even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read-biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for 8 out of 13 (amplicons B1FR-450bp, FF130R-130bp) or 4 out of 13 (amplicon FFFR, 658bp) species. Combining the results of all three COI amplicons (multi-amplicon approach) improved the read-biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment. This article is protected by copyright. All rights reserved.

Concepts: Species, Copyright, Mitochondrion, Mitochondrial DNA, Invertebrate, DNA sequencing, Molecular biology, DNA