Population genomic studies are increasing in the last decade, showing great potential to understand the evolutionary patterns in a great variety of organisms, mostly relying on RAD sequencing techniques to obtain reduced representations of the genomes. Among them, 2b-RAD can provide further secondary reduction to adjust study costs by using base-selective adaptors, although its impact on genotyping is unknown. Here we provide empirical comparisons on genotyping and genetic differentiation when using fully degenerate and base-selective adaptors and assess the impact of missing data. We built libraries with the two types of adaptors for the same individuals and generated independent and combined datasets with different missingness filters according to their presence (100%, 75% and 50%). Exploring locus-by-locus, we found 92% of identical genotypes between both libraries of the same individual when using loci present in 100% of the samples, which decreased to 35% when working with loci present in at least 50% of them. We show that missing data is a major source of individual genetic differentiation. The loci discordant by genotyping were in low frequency (7.67%) in all filtered files. Only 0.96% were directly attributable to base-selective adaptors, and 6.44% underestimated heterozygosity in NN libraries, of which ca. 70% had <10 reads per locus indicating that sufficient read depth should be ensured for a correct genotyping. Our work confirms that 2b-RAD libraries using base-selective adaptors are a robust tool to use in population genomics of species with large genome sizes.

DNA methylation is one of the most relevant epigenetic modifications. It is present in eukaryotes and prokaryotes and is related to several biological phenomena, including gene flow and adaptation to environmental conditions. The widespread use of third-generation sequencing technologies allows direct and easy detection of genome-wide methylation profiles, offering increasing opportunities to understand and exploit the epigenomics landscape of individuals and populations. Here, we present MeStudio, a pipeline which allows to analyse and combine genome-wide methylation profiles with genomic features. Outputs report the presence of DNA methylation in coding sequences (CDS) and noncoding sequences, including both intergenic sequences, and sequences upstream to CDS. We show the usage and performances of MeStudio on a set of single-molecule real time sequencing outputs from strains of the bacterial species Sinorhizobium meliloti. MeStudio is freely available under an open source GPLv3 license at https://github.com/combogenomics/MeStudio

Potential subdivision events in populations can have a wide range of causes: from natural disasters like bushfires that isolate communities, to anthropogenic disturbances like infrastructure projects cutting through a population’s habitat. Due to the unpredictability inherent in events like bushfires, or even for predictable events such as property development, populations affected by these potential subdivisions are often not studied until after the event, making it extremely hard to assess negative conservation impacts without the benefit of prior data. This paper aims to apply population genetics methods to assess whether it is possible to accurately assess the impact a potential subdivision event can have on the genetic makeup of a population, especially when one has no data prior to such an event. Differentiation measures, such as Fst, might be used for detecting whether a population has been subdivided. However, these measures often take dozens of generations to show a significant change from zero (i.e., no differentiation), especially in larger populations. In this paper we present a more sensitive method, which is suitable for detecting subdivision effects within a few generations of the event and which can be applied without prior data. We test this method using both simulated data, and genetic data from a population of koalas impacted by a railroad infrastructure development.

Knowledge of the persistent state of environmental DNA (eDNA) particles in water and modeling its particle size distribution (PSD) is crucial to refine the eDNA sampling strategy. Previous studies measured eDNA concentrations at different size fractions to infer eDNA PSDs, whereas such discrete PSDs greatly depend on filter pore sizes used and may potentially bias our understanding of eDNA states. Here, I evaluated the Weibull distribution model as a tool for parameterizing eDNA PSDs. First, of the selected models developed for PSDs, the Weibull model was fitted most accurately to the eDNA PSD. Second, the Weibull parameters significantly varied depending on target genetic regions (mitochondrial/nuclear), time passages, and species, which also substantially changed the eDNA capture efficiency at a given filter pore size. According to the results, the higher detectability of multi-copy nuclear eDNA than mitochondrial one could partly be accounted for differences in their PSDs, a larger pore size filter could be suitable to detect the eDNA releases associating incidental events, and the optimum filter pore size for sufficient eDNA detection could be different among the taxa. Although further consideration is needed, this study provided the groundwork for optimizing the strategy of aqueous eDNA sampling, which contributes to efficient biodiversity conservation and ecosystem management.

Mitochondrial DNA sequences (mtDNA) are often found as byproduct in hybrid enrichment data sets originally created to capture anchored hybrid enrichment (AHE) or ultra-conserved element (UCE) nuclear loci. The mtDNA sequences in these data sets are currently rarely used, even though mitochondrial genes such as COI, ND5, CytB, and 16S are of general interest and often not yet known and deposited in public databases. We developed MitoGeneExtractor to extract mitochondrial genes of interest from genomic libraries. Gene sequences are reconstructed through multiple sequence alignments of sequencing reads to an amino acid reference. We applied MitoGeneExtractor to recently published data created for UCE enrichment and were able to extract complete or nearly complete COI and ND5 sequences for a large proportion of the sequencing libraries. MitoGeneExtractor can be used to extract mitochondrial protein coding genes from a wide range of next generation sequencing data sets.

Environmental DNA (eDNA) is emerging as a potentially powerful tool to assess aquatic ecosystems, and to monitor fish assemblages. We conducted a pilot eDNA water sampling program at 5 Reserves within the National Estuarine Research Reserve System from temperate and tropical estuaries to better understand how eDNA data can support detection of fish species within an standardized monitoring network. Samples were collected in coordination with an existing long term monitoring program, and the sampling plan was designed to minimize additional work for field teams. We found that results varied; in some estuaries the number and type of fish species was consistent with expected occurrence, and eDNA analysis detected fish that often eluded traditional sampling methods. In warm turbid waters, however, we detected fewer species than expected, suggesting that additional processing is required at these sites. Managers interested in applying standardized eDNA monitoring across a range of sites may want to consider a decision tree protocol, where a baseline methodology is developed for all sites, enhanced by additional laboratory or analysis steps when specific conditions are present.

Reliable abundance estimation is a primary challenge in environmental DNA (eDNA) analysis, which has been addressed by considering the effects of eDNA transport and degradation. However, these eDNA spatial dynamics depend on the cellular and molecular structure of eDNA, with its persistence state (particle size and DNA fragment length) being essential for improved abundance estimation. This existing knowledge gap is bridged by utilizing datasets obtained from two types of aquarium experiments (targeting zebrafish [Danio rerio] and Japanese jack mackerel [Trachurus japonicus]) and comparing the relationships between eDNA concentration and species abundance among different eDNA size fractions and target marker lengths. We reared the fish in experimental tanks with different individual numbers or biomass densities, filtered rearing water using different pore size filters, and quantified eDNA concentrations targeting different fragment lengths or genetic regions. Consequently, both experiments showed that the accuracy and sensitivity in abundance estimation were improved (i.e., R2 values and slopes of linear regressions increased) when targeting eDNA at the 3–10-µm size fraction. On the other hand, targeting eDNA at the >10 µm size fraction yielded a lower R2 value. This result indicates that an “appropriately” larger eDNA particle is vital for improving abundance estimation accuracy and sensitivity. Conversely, the target marker length negatively affected the R2 value. This study proposes that the relationship between eDNA concentration and species abundance relies on the complex interactions between the particle size, persistence, and spatial heterogeneity of eDNA in water.

As human activities on our planet persist, causing widespread and irreversible environmental degradation, the need to biomonitor ecosystems has never been more pressing. These circumstances have required a renewal in monitoring techniques, encouraged by necessity to develop more rapid and accurate tools which will support timely observations of ecosystem structure and function. The World Exposition (from now ‘EXPO2015’) hosted in Milan from May to October 2015 was a global event that could be categorized as a mega-event, which can be defined as an acute environmental stressor, possibly generating biodiversity alteration and disturbance. During the six months of EXPO2015, exhibitors from more than 135 countries and 22 million visitors insisted on a 1.1 million square meters area. Faced with such a massive event, we explore the potential of DNA metabarcoding using three molecular markers to improve the understanding of anthropogenic impacts in the area, both considering air and water monitoring. Furthermore, we explore the effectiveness of the taxonomy assignment phase considering different taxonomic levels of analysis and the use of data mining approaches to predict sample origin. Unless the degree of taxa identification still remains open, our results showed that DNA metabarcoding is a powerful genomic-based tool to monitor biodiversity at the microscale, allowing us to capture exact fingerprints of specific event sites and to explore in a comprehensive manner the eukaryotic community alteration. With this work, we aim to disentangle and overcome the crucial issues related to the generalization of DNA metabarcoding in order to support future applications.

Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communites of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing and evaluated the performance of three different methods for generation of operational taxonomic units (OTUs). The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus’s time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic-dry part that has a species rich grass-dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigare how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30-40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.

Environmental DNA (eDNA) analysis is a promising tool for non-disruptive and cost-efficient estimation of species abundance. However, its practical applicability in natural environments is limited because it is unclear whether eDNA concentrations actually represent species abundance in the field. Although the importance of accounting for eDNA dynamics, such as transport and degradation, has been discussed, the influences of eDNA characteristics, including production source and state, and methodology, including collection and quantification strategy and abundance metrics, on the accuracy of eDNA-based abundance estimation were entirely overlooked. We conducted a meta-analysis using 56 previous eDNA literature and investigated the relationships between the accuracy (R2) of eDNA-based abundance estimation and eDNA characteristics and methodology. Our meta-regression analysis found that R2 values were significantly lower for crustaceans than fish, suggesting that less frequent eDNA production owing to their external morphology and physiology may impede accurate estimation of their abundance via eDNA. Moreover, R2 values were positively associated with filter pore size, indicating that selective collection of larger-sized eDNA, which is typically fresher, could improve the estimation accuracy of species abundance. Furthermore, R2 values were significantly lower for natural than laboratory conditions, while there was no difference in the estimation accuracy among natural environments. Our findings shed a new light on the importance of what characteristics of eDNA should be targeted for more accurate estimation of species abundance. Further empirical studies are required to validate our findings and fully elucidate the relationship between eDNA characteristics and eDNA-based abundance estimation.

The horned gall aphid Schlechtendalia chinensis, is an economically important insect that induces galls valuable for medicinal and chemical industries. S. chinensis manipulates its host plant to form well-organized horned galls during feeding. So far, more than twenty aphid genomes have been reported; however, all of those are derived from free-living aphids. Here we generated a high-quality genome assembly of S. chinensis, representing the first genome sequence of a galling aphid. The final genome assembly was 280.43 Mb, with 97% of the assembled sequences anchored into thirteen chromosomes. S. chinensis presents the smallest aphid genome size among available aphid genomes to date. The contig and scaffold N50 values were 3.39 Mb and 20.58 Mb, respectively. The assembly included 96.4% of conserved arthropod and 97.8% of conserved Hemiptera single-copy orthologous genes based on BUSCO analysis. A total of 13,437 protein-coding genes were predicted. Phylogenomic analysis showed that S. chinensis formed a single clade between the Eriosoma lanigerum clade and the Aphidini+Macrosiphini aphid clades. In addition, salivary proteins were found to be differentially expressed when S. chinensis underwent host alternation, indicating their potential roles in gall formation and plant defense suppression. A total of 36 cytochrome P450 genes were identified in S. chinensis, considerably fewer compared to other aphids, probably due to its small host plant range. The high-quality S. chinensis genome assembly and annotation provide an essential genetic background for future studies to reveal the mechanism of gall formation and to explore the interaction between aphids and their host plants.

During the history of range expansion, the populations encounter with variety of environments. They respond to the local environments by modifying the mutually interacting traits. Therefore, to understand the whole life history of the populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the coadaptation of the multiple traits. Toward this end, we provide an exploratory analysis based on the features of populations: site frequency spectra of populations, population-specific FST, association between genes and environments, positive selections on traits mapped on the admixture graph, and GWAS results. Correspondence analysis of genes, environments, and traits provides a bird’s-eye view of the history of population differentiation and range expansion and various types of environmental selections at the times. Principal component analysis of the estimated trait-specific polygenic adaptations mapped on the admixture graph enables to understand the coadaptation of multiple traits. The potential usefulness was confirmed by analyzing a public dataset of wild poplar in northwestern America. In response to the northern cold temperature and longer daylength, the populations increased the photosynthetic activity and nutrient use efficiency at the expense of the risk of pathogen invasion, and in response to warm temperature, they increased the growth. At higher altitude, they shifted the maximum activity to earlier period in spring to reduce the activity in dry summer. The R codes for our representation method and simulations of population colonization used in this study are available as supplementary script.

Knowledge of the sex determination system and sex-associated genes has important implications in physiology, ecology and evolution, but the mechanisms underlying sexual determination systems in turtles has not been fully elucidated, due to their remarkable variability and a lack of reference genomes in some species. In this report, we describe the first genome assembled at the chromosome scale for Mauremys mutica using Illumina and high-throughput chromatin conformation capture (Hi-C) technology. A total of 280.42 Gb of clean data were generated using the Pacific Biosciences platforms, which represented approximately 119× coverage of the Mauremys mutica genome. The assembly comprised 2.23 Gb with a contig N50 of 8.53 Mb and scaffold N50 of 141.98 Mb. Genome Hi-C scaffolding resulted in 26 pseudochromosomes containing 99.98% of the total assembly. Genome annotation predicted 24,751 protein-coding genes, and 97.23% of them were annotated. Comparative genomics analysis indicated that the lizard-snake-tuatara clade diverged from the bird-crocodilian-turtle clade at approximately 283.1-320.5 Mya. Additionally, positive selection of genes and gene families related to calcium signaling, neuroactive ligand-receptor interaction, and expansion of the hormone signaling pathway were identified, implicating their roles of sex regulation inMauremys mutica. High-quality genome assembly may provide a valuable genomic resource for further research investigating gene-environment interactions in Mauremys mutica.

A combination of next-generation sequencing technologies and mate-pair libraries of large insert sizes is used as a standard method to generate genome assemblies with high contiguity. The third-generation sequencing techniques also are used to improve the quality of assembled genomes. However, both mate-pair libraries and the third-generation libraries require high-molecular-weight DNA, making the use of these libraries inappropriate for samples with only degraded DNA. An in silico method that generates mate-pair libraries using a reference genome was devised for the task of assembling target genomes. Although the contiguity and completeness of assembled genomes were significantly improved by this method, a high level of errors manifested in the assembly, further to which the methods for using reference genomes were not optimized. Here, we tested different strategies for using reference genomes to generate in silico mate-pairs. The results showed that using a closely related reference genome from the same genus was more effective than using divergent references. Conservation of in silico mate-pairs by comparing two references and using those to guide genome assembly reduced the number of misassemblies (18.6% – 46.1%) and increased the contiguity of assembled genomes (9.7% – 70.7%), while maintaining gene completeness at a level that was either similar or marginally lower than that obtained via the current method. Finally, we compared the optimized method with another reference-guided assembler, RaGOO. We found that RaGOO produced longer scaffolds (17.8 Mbp vs 3.0 Mbp), but resulted in a much higher misassembly rate (85.68%) than our optimized in silico mate-pair method.

A proliferation in environmental DNA (eDNA) research has increased the reliance on reference sequence databases to assign unknown DNA sequences to known taxa. Without comprehensive reference databases, DNA extracted from environmental samples cannot be correctly assigned to taxa, limiting the use of this genetic information to identify organisms in unknown sample mixtures. For animals, standard metabarcoding practices involve amplification of the mitochondrial Cytochrome-c oxidase subunit 1 (CO1) region, which is a universally amplifyable region across majority of animal taxa. This region, however, does not work well as a DNA barcode for plants and fungi, and there is no similar universal single barcode locus that has the same species resolution. Therefore, generating reference sequences has been more difficult and several loci have been suggested to be used in parallel to get to species identification. For this reason, we developed a multi-gene targeted capture approach to generate reference DNA sequences for plant taxa across 20 target chloroplast gene regions in a single assay. We successfully compiled a reference database for 93 temperate coastal plants including seagrasses, mangroves, and saltmarshes/samphire’s. We demonstrate the importance of a comprehensive reference database to prevent species going undetected in eDNA studies. We also investigate how using multiple chloroplast gene regions impacts the ability to discriminate between taxa.

Purple butter clam (Saxidomus purpuratus) is an economically important bivalve shellfish. This species belongs to the subclass Heterodonta that diverged in calcite seas with low magnesium concentrations. We sequenced and assembled its genome and performed an evolutionary comparative analysis. A total of 911 Mb assembly of S. purpuratus was anchored into 19 chromosomes and a total of 48,090 protein-coding genes were predicted. We identified its repeat-based expanded genes that are associated with the sodium/potassium-exchange ATPase complex. In addition, different types of ion transporters were enriched in the common ancestor of Heterodonta (calcium, sulfate, and lipid transporters) and the specific evolution of S. purpuratus (calcium and sodium transporters). These differences seem to be related to the divergence times of Heterodonta (calcitic sea) and Veneraidea (aragonitic sea). Furthermore, we analyzed the evolution of scavenger receptor (SR) proteins in S. purpuratus, which are involved in a wide range of immune responses, and compared them to the closely related Cyclina sinensis. We showed that a small number of SR proteins, exhibited collinearity between the two genomes, which is indicative of independent gene evolution. Our genomic study provides an evolutionary perspective on the genetic diversity of bivalves and their adaptation to historical changes in the marine environment.

Whole genomes are commonly assembled into a collection of scaffolds and often lack annotations of autosomes, sex chromosomes and, and organelle genomes (i.e., mitochondrial and chloroplast). As these chromosome types can have highly disparate evolutionary histories, it is imperative to take this information into account when analyzing genomic variation. Here we assessed the accuracy of four methods for identifying the homogametic sex chromosome using two whole genome sequenced (WGS) and 133 RAD sequenced white-tailed eagles (Haliaeetus albicilla): i) difference in read depth per scaffold, ii) heterozygosity per scaffold in a male and female bird, iii) mapping to a reference genome of a related species (chicken) with identified sex chromosomes, and iv) an analysis of SNP-loadings from a principal components analysis (PCA), based on low-depth RADseq data from 133 individuals. In i and ii, the WGS were mapped to a reference genome consisting of 1142 assembled scaffolds from the golden eagle (Aquila chrysaetos) with no identified chromosomes. The read depth per scaffold identified 86.41% of the homogametic sex chromosome (Z) with few false positives. The SNP-loading scores found 78.6% of the Z-chromosome but had a false positive discovery rate of more than 10%. The heterozygosity per scaffold did not provide clear results due to a lack of diversity in both the Z and autosomal chromosomes, and potential interference from the heterogametic sex chromosome (W).

Cylas formicarius is one of the most important pests of sweet potato worldwide, causing considerable ecological and economic damage. To improve the effect of comprehensive management and understanding of genetic mechanisms, the genetic functions of C. formicarius have been the subject of intensive study. Using Illumina and PacBio sequencing, we obtained a chromosome-level genome assembly of adult weevils from lines inbred for 15 generations. The high-quality assembly obtained had a size of 338.84 Mb, with contig and scaffold N50 values of 14.97 Mb and 34.23 Mb, respectively. In total, 157.51 Mb of repeat sequences and 11,907 protein-coding genes were predicted. A total of 337.06 Mb of genomic sequences was located on the 11 chromosomes, and the sequence length that could be used to determine the sequence and direction accounted for 99.03% of the total length of the associated chromosome. Comparative genomic analysis showed that C. formicarius was sister to Dendroctonus ponderosae, and C. formicarius diverged from D. ponderosae approximately 138.89 million years ago (Mya). Many important gene families that were expanded in the C. formicarius genome were involved in the chemosensory system. In an in-depth study, the binding assay results indicated that CforOBP4-6 had strong binding affinities for sex pheromones and other ligands. Overall, the high-quality C. formicarius genome provides a valuable resource to reveal the molecular ecological basis, genetic mechanism and evolutionary process of major agricultural pests, deepen the understanding of environmental adaptability and apparent plasticity, and provide new ideas and new technologies for ecologically sustainable pest control.