Lake Dali Nur, located in Inner Mongolia, North China, is alkaline, with Triplophysa dalaica one of the three fish species that not only survive, but thrive, in the lake. To investigate the presence of molecular mutations potentially responsible for this adaptation, the whole genome sequence of the species endemic to the lake was sequenced. A total of 126.5 Gb and 106 Gb data, covering nearly 200X of the estimated genome, were generated using long-read sequencing and Hi-C technology, respectively. De novo assembly generated a genome totalled 607.91 Mb, with a contig N50 of 9.27 Mb. Nearly all whole genome sequences were anchored and oriented onto 25 chromosomes, with telomeres for most chromosomes also being recovered. Repeats comprised approximately 35.01% of the whole genome. A total of 23,925 protein-coding genes were predicted, within which, 98.62% could be functionally annotated. Through comparisons of T. dalaica, T. tibetana, and T. siluroides gene models, a total of 898 genes were identified as likely being subjected to positive selection, with several of them potentially associated with alkaline adaptation, such as sodium bicarbonate cotransporter, SLC4A4. Demographic analyses suggested that the Dali population might have diverged from endemic freshwater Hai River populations, approximately 1 million years ago. The high-quality T. dalaica genome, sequenced in this study, not only aids in the analyses of alkaline adaptation, but may also assist in revealing the mysteries of the highly divergent genus Triplophysa.

The amphibian chytrid fungus (Bd) has caused extinction of amphibian populations worldwide. Early and accurate Bd detection is essential for management and treatment of susceptible anurans. We analyzed the effectiveness of an in-situ DNA extraction along with handheld mobile quantitative PCR (qPCR) thermocycler to detect Bd on skin frog swabs, and to detect Bd in water samples using environmental DNA (eDNA). We collected duplicate eDNA samples and skin swabs from three Bd positive Rana sierrae populations. We processed one set of samples using a field protocol (a handheld thermocycler), and the other half using a standard lab protocol. We detected Bd DNA in all R. sierrae swabbed across all three sites using both the field and lab protocols. We also detected Bd DNA in eDNA samples collected at two of the three sites using both the field and lab protocols although the field and lab protocol failed to detect Bd eDNA at separate sites. The probability of detecting Bd DNA in the technical replicates was lower for the field protocol compared to samples extracted using the lab protocol, suggesting the field protocol has reduced sensitivity and may not detect low quantities of DNA. Our results suggest the field extraction protocol using a handheld qPCR platform is a promising tool for rapid detection of Bd in susceptible amphibian populations. The field protocol yielded accurate results in less than 60 minutes. However, the applied field protocol may be prone to false negatives when analyzing low-quantity DNA samples (i.e. eDNA).

Environmental DNA (eDNA) metabarcoding is a common tool for measuring and cataloguing biodiversity, yet standard methodological approaches to generate metabarcoding data sets have yet to emerge, in part due to challenges understanding the biological and technical biases that affect eDNA profiles. Here, we explore how two experimental choices – depth of sequencing of PCR amplicon libraries and the number of PCR replicates – influence estimates of α and β diversity. We extracted DNA from six soil samples from three ecologically distinct locations, performed 24 PCR replicates from each using two common metabarcodes, and sequenced each to an average depth of 83,898 reads. We found PCR replicates are consistent in composition and relative abundance of abundant taxa, allowing differentiation of samples and sites. However, rare taxa were unique to one or a few replicates, suggesting that even large numbers of experimental replicates may be insufficient to catalogue biodiversity fully. We recommend that to differentiate sites, separately sequencing only a minimum of two PCR replicates to a depth that allows 1,000 reads identified to taxa, is sufficient to differentiate sites. We also conclude that metabarcoding is impractical for exhaustive taxonomic inventory and, because rare taxa are not amplified consistently, taxonomic tallies that rely on consensus among replicates artificially lower richness estimates. These findings provide new considerations for eDNA experimental design and data interpretation.

Stings from certain species of cubozoan jellyfish are dangerous to humans and their seasonal presence in tropical marine waters poses a significant risk to coastal communities. The detection of cubozoans is difficult due to high spatial and temporal variation in their occurrence and abundance. Environmental DNA (eDNA) has the potential to detect rare species and therefore offers potential to detect cubozoans, not only pelagic medusae, but presence of cryptic polyp life-stages. The objective of this study was to validate the use of eDNA as a viable detection method for four cubozoan species (Chironex fleckeri, Copula sivickisi, Carybdea xaymacana and Carukia barnesi). Species-specific primers were developed for each of these four cubozoans and an eDNA approach validated utilising both laboratory and field trials. Laboratory DNA degradation experiments demonstrated that C. sivickisi DNA degraded quickly but could still be detected in sea water for up to 9 days post-jellyfish removal. Positive detection was found for C. fleckeri, C. xaymacana and C. sivickisi medusae in the waters surrounding Magnetic Island, Queensland, in the Austral spring/summer (September-January). Based on visual surveys there was a poor relationship between concentration of eDNA and abundance of jellyfish. Positive eDNA amplification was also shown near the substratum when C. sivickisi medusae were absent. This can only be explained by the detection of polyps. Consequently, eDNA is an effective tool to detect both the medusae and polyps of cubozoans. This approach provides the means to reduce the risk of envenomation to swimmers and enhance our knowledge of cubozoan ecology.

The accurate determination of the spatial trends on the variability of the gene pool of a species is essential to elucidate the underlying demographic-evolutionary events and, ultimately, to unravel the microevolutionary history of the species or population under examination. In this work, we present a new software tool called GenoCline, which is essentially devised to assist in detecting genetic clines from allele and phenotypic frequency data, and even from genome-wide data. This program package allows identifying the geographic orientation of clinal genetic variation through a system of iterative rotation of a virtual coordinate axis. At the same time, GenoCline can also be used to carry out complementary statistical analyses aimed at clarifying the potential origin of the genetic clines found, among which stand out spatial autocorrelation, isolation by distance, centroid method, multidimensional scaling and Sammon projection. Among the main advantages of this software are those related to ease in data entry and potential interconnection with other programs. Data entry is user-friendly. Genetic frequencies and geographic coordinates can be easily entered in spreadsheet table formatting, while genome-wide data can be imported in Eigensoft format. Genetic frequencies can also be exported in a format compatible with other programs dealing with population genetics and evolutionary biology. All illustrations of results are saved in eps format so that there will be high quality and easily editable vectorial graphs available for the researcher. GenoCline is implemented in Java, so it can be used with different operating systems.

Plants host diverse microbial communities, but there is little consensus on how we sample and characterize these communities, and this has unknown consequences. Using root and leaf tissue from 20 showy milkweed (Asclepias speciosa) plants in the field, we compared two common sampling strategies by: 1) homogenizing after subsampling a small proportion of tissue (30 mg), and 2) homogenizing bulk tissue before subsampling 30 mg. Due to potential differences in richness and spatial distributions among microorganisms, we targeted bacteria, arbuscular mycorrhizal (AM) fungi and non-AM fungi in roots, as well as foliar fungal endophytes (FFE) in leaves. We also sampled FFE twice across the season using sampling strategy 1 to assess temporal dynamics, and we extracted DNA from all remaining homogenized bulk leaf tissues to determine the extent of potential undersampling. Bacterial richness was higher under sampling strategy 2, and all microbial groups except AM fungi differed in composition. Community overlap between the two sampling strategies increased when rare taxa were removed, but FFE and bacterial communities remained more different than alike and showed largely non-overlapping communities within individual plants. Increasing the extraction mass 10x also increased FFE richness ~10x, confirming the severe undersampling indicated in the sampling strategy comparisons. Even so, seasonal patterns in FFE communities were apparent, suggesting that strong drivers may be identified despite severe undersampling. Our findings highlight that current sampling practices poorly characterize many microbial groups and that increased sampling intensity is necessary to identify subtler patterns and to increase the reproducibility of studies.

Lactobacillus (L.) plantarum is widely distributed in the natural environments and previous studies failed to identify habitat-specific genomic signatures. Thus, comparative genomic analysis together with genome-wide association study of 551 L. plantarum genomes were performed to investigate how natural habitats shaped the bacterial genomes. Firstly, the study constructed a phylogenic tree using a strict core-genome of L. plantarum, followed by refining the clades with genome-wide single nucleotide polymorphisms (SNPs). Our data showed that plant-originated isolates dispersed evenly across the phylogenetic tree, supporting a previously reported model of nomadic lifestyle. Isolates from animal and dairy sources showed significant biases in clade distribution, suggesting close association between bacterial genomes and habitats. The animal-originated isolates distributed mainly to clade A and were characterized with more carbon utilization-associated genes. The dairy isolates distributed mainly to subclades B2 and B3, which harbor more nitrogen utilization-associated genes. Both animal and dairy isolation sources were directly linked with the development of human civilization in the aspects of food culture and food industry. Furthermore, based on five population genetic signals, subclade B2 had the highest genetic diversity and SNP distance. Subclade B1 had the highest mutation rate, possibly resulted from recombination events. The animal-originated isolates had lower nucleotide diversity and higher recombination/mutation ratio, which might be the genetic trail remained from the adaptation process. Our data have provided an explanation for the high versatility of L. platnarum on the genomic level and showed that the L. plantarum species is more genetically diverse than previously expected.

The analysis of environmental DNA (eDNA) allows efficient surveys of freshwater species and is being increasingly used. However, most studies generally have a limited sampling plan. Because different methods are used by different authors, all the data produced with eDNA cannot be easily pooled together for a global overview. Nevertheless, one of the promising perspectives is the standardization of the methods and protocols, for long-term monitoring, early detection of invasive species, and rare species detection. We here present the biggest dataset using eDNA metabarcoding with standardized methods for freshwater bivalves. Sampling was performed mainly in France, with over 350 localities spread over all major basins. Metabarcoding was performed using two new sets of primers, one for the Unionida and one for the Venerida. The species distributions inferred from eDNA sampling are then compared with those currently known. Results give an insight into the valuable data that eDNA analysis can provide if used at a large scale. Some species were found outside of their known range, especially invasive species but also species of conservation interest. Others were found in a surprisingly low number of localities, although currently considered widespread and of no conservation interest. This can be explained by the databases currently used to establish conservation status, which are often biased by including ancient data, shell-only data, and potentially misidentified species. The present study shows that our eDNA metabarcoding approach will be an essential tool to unveil the true species distributions, to better evaluate their status and improve conservation policymaking.

Conservation genomics is an important tool to manage threatened species under current biodiversity loss. Recent advances in sequencing technology mean that we can now use whole genomes to investigate demographic history, local adaptation, inbreeding, and more in unprecedented detail. However, for many rare and elusive species only non-invasive samples such as faeces can be obtained, making it difficult to take advantage of whole genome data. We present a method to extract DNA from the mucosal layer of faecal samples to reconstruct high coverage whole genomes using standard laboratory techniques, therefore in a cost-effective and efficient way. We use wild collected faecal pellets collected from wild caribou (Rangifer tarandus), a species undergoing declines in many parts of its range in Canada and subject to comprehensive conservation and population monitoring measures. We compare four faecal genomes to two tissue genomes sequenced in the same run. Quality metrics were similar between faecal and tissue samples with the main difference being the alignment success of raw reads to the reference genome likely due to differences in endogenous DNA content, affecting overall coverage. One of our faecal genomes was only reconstructed at low coverage (1.6X), however the other three obtained between 7 and 15X, compared to 19 and 25X for the tissue samples. We successfully reconstructed high-quality whole genomes from faecal DNA and, to our knowledge, are the first to obtain genome-wide data from wildlife faecal DNA in a non-primate species, representing an important advancement for non-invasive conservation genomics.

Analysis of biodiversity in natural environments based on environmental DNA (eDNA) has been applied to a wide range of ecosystems and species. The combination of high-throughput sequencing technologies and eDNA analysis is a powerful tool that enables comprehensive non-invasive monitoring of species present in the environment. Quantitative data of the eDNA from each species is essential for understanding species abundance but until recently required individual assays targeting each species. Recently developed quantitative sequencing (qSeq) allows simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. The eDNA extracted from aquaria with five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus) across 4 days was quantified by microfluidic digital PCR using a TaqMan probe and qSeq. The eDNA abundance quantified by qSeq was consistent with dPCR for each fish species at each sampling time. However, the relative abundances of sequences obtained from high throughput sequencing did not follow the same trend as the quantitative analyses, probably due to different PCR amplification efficiencies for each species. The correlation coefficients between qSeq and dPCR were 1.052, 1.074, and 1.114 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA. The application of qSeq to eDNA of other species will provide comprehensive quantitative data that could deepen our knowledge of natural ecosystems.

DNA barcoding identification needs a good characterization of intra-specific genetic divergence to establish the limits between species. Yet, the number of barcodes per species is many times low and geographically restricted. A poor coverage of the species distribution range may hamper identification, especially when undersampled areas host genetically distinct lineages. If so, the genetic distance between some query sequences and reference barcodes may exceed the maximum intra-specific threshold for unequivocal species assignation. Taking a group of Quercus herbivores (moths) in Europe as model system, we found that the number of DNA barcodes from southern Europe is proportionally very low in the Barcoding of Life Data Systems (BOLD). This geographical bias complicates the identification of southern query sequences, due to their high intra-specific genetic distance with respect to barcodes from higher latitudes. Pairwise intra-specific genetic divergence increased along with spatial distance, but was higher when at least one of the sampling sites was in southern Europe. Accordingly, GMYC (General Mixed Yule Coalescent) single threshold model retrieved clusters constituted exclusively by Iberian haplotypes, some of which could correspond to cryptic species. The number of putative species retrieved was more reliable than that of multiple threshold GMYC but very similar to results from ABGD and jMOTU. Our results support GMYC as a key resource for species delimitation within poorly inventoried biogeographic regions in Europe, where historical factors (e.g. glaciations) have promoted genetic diversity and singularity. Future European DNA barcoding initiatives should be preferentially performed along latitudinal gradients, with special focus on southern peninsulas.