Characterization of the complete mitochondrial genome of the brazilian cownose ray Rhinoptera brasiliensis (Myliobatiformes, Rhinopteridae) in the western Atlantic and its phylogenetic implications.

BACKGROUND: The Brazilian cownose ray, Rhinoptera brasiliensis has undergone a global population reduction and is currently classified by IUCN as Vulnerable. This species is sometimes confused with Rhinoptera bonasus, the only external diagnostic characteristic to distinguish between both species is the number of rows of tooth plates. Both cownose rays overlap geographically from Rio de Janeiro to the western North Atlantic. This calls for a more comprehensive phylogenetic assessment using mitochondria DNA genomes to better understand the relationships and delimitation of these two species. METHODS AND RESULTS: The mitochondrial genome sequences of R. brasiliensis was obtained by next-generation sequencing. The length of the mitochondrial genome was 17,759 bp containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a non-coding control region (D-loop). Each PCG was initiated by an authoritative ATG codon, except for COX1 initiated by a GTG codon. Most of the PCGs were terminated by a complete codon (TAA/TAG), while an incomplete termination codon (TA/T) was found in five out of the 13 PCGs. The phylogenetic analysis showed that R. brasiliensis was closely related to R. steindachneri whereas the reported mitogenome as R. steindachneri (GenBank accession number KM364982), differs from multiple mitocondrial DNA sequences of R. steindachneri and is nearly identical to that of R. javanica. CONCLUSION: The new mitogenome determined in this study provides new insight into the phylogenetic relationships in Rhinoptera, while providing new molecular data that can be applied to population genetic studies.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Genetic Insights into the Giant Keyhole Limpet (Megathura crenulata), an Eastern Pacific Coastal Endemic: Complete Mitogenome, Phylogenetics, Phylogeography, and Historical Demography.

BACKGROUND: The giant keyhole limpet Megathura crenulata is a gastropod mollusk (Fissurella superfamily) that is endemic to the eastern Pacific coast from southern California, USA, to Baja California Sur, Mexico. M. crenulata is socioeconomically important as it produces a potent immune-stimulating protein, called Keyhole Limpet Hemocyanin, which is extracted in vivo and utilized for vaccine development. However, ecological studies are scarce and genetic knowledge of the species needs to be improved. Our objectives were to assemble and annotate the mitogenome of M. crenulata, and to assess its phylogenetic relationships with other marine gastropods and to evaluate its population genetic diversity and structure. METHODS: Samples were collected for mitogenome assembly (n = 3) spanning its geographic range, Puerto Canoas (PCA) and Punta Eugenia (PEU), Mexico, and California (CAL), USA. Total DNA was extracted from gills sequenced using Illumina paired-end 150-bp-read sequencing. Reads were cleaned, trimmed, assembled de novo, and annotated. In addition, 125 samples from eight locations were analyzed for genetic diversity and structure analysis at the 16s rRNA and COX1 genes. RESULTS: The M. crenulata mitogenomes had lengths of 16,788 bp (PCA) and 16,787 bp (PEU) and were composed of 13 protein-coding regions, 22 tRNAs, two rRNAs, and the D-Loop region. In terms of phylogeographic diversity and structure, we found a panmictic population that has experienced recent demographic expansion with low nucleotide diversity (0.002), high haplotypic diversity (0.915), and low φST (0.047). CONCLUSIONS: Genetic insights into the giant keyhole limpet provides tools for its management and conservation by delimiting fishing regions with low genetic diversity and/or genetically discrete units.

A draft of the genome of the Gulf Coast tick, Amblyomma maculatum.

The Gulf Coast tick, Amblyomma maculatum, inhabits the Southeastern states of the USA bordering the Gulf of Mexico, Mexico, and other Central and South American countries. More recently, its U.S. range has extended West to Arizona and Northeast to New York state and Connecticut. It is a vector of Rickettsia parkeri and Hepatozoon americanum. This tick species has become a model to study tick/Rickettsia interactions. To increase our knowledge of the basic biology of A. maculatum we report here a draft genome of this tick and an extensive functional classification of its proteome. The DNA from a single male tick was used as a genomic source, and a 10X genomics protocol determined 28,460 scaffolds having equal or more than 10 Kb, totaling 1.98 Gb. The N50 scaffold size was 19,849 Kb. The BRAKER pipeline was used to find the protein-coding gene boundaries on the assembled A. maculatum genome, discovering 237,921 CDS. After trimming and classifying the transposable elements, bacterial contaminants, and truncated genes, a set of 25,702 were annotated and classified as the core gene products. A BUSCO analysis revealed 83.4% complete BUSCOs. A hyperlinked spreadsheet is provided, allowing browsing of the individual gene products and their matches to several databases.

Comparison of genetic variation between rare and common congeners of Dipodomys with estimates of contemporary and historical effective population size.

Species with low effective population sizes are at greater risk of extinction because of reduced genetic diversity. Such species are more vulnerable to chance events that decrease population sizes (e.g. demographic stochasticity). Dipodomys elator, (Texas kangaroo rat) is a kangaroo rat that is classified as threatened in Texas and field surveys from the past 50 years indicate that the distribution of this species has decreased. This suggests geographic range reductions that could have caused population fluctuations, potentially impacting effective population size. Conversely, the more common and widespread D. ordii (Ord's kangaroo rat) is thought to exhibit relative geographic and demographic stability. We assessed the genetic variation of D. elator and D. ordii samples using 3RAD, a modified restriction site associated sequencing approach. We hypothesized that D. elator would show lower levels of nucleotide diversity, observed heterozygosity, and effective population size when compared to D. ordii. We were also interested in identifying population structure within contemporary samples of D. elator and detecting genetic variation between temporal samples to understand demographic dynamics. We analyzed up to 61,000 single nucleotide polymorphisms. We found that genetic variability and effective population size in contemporary D. elator populations is lower than that of D. ordii. There is slight, if any, population structure within contemporary D. elator samples, and we found low genetic differentiation between spatial or temporal historical samples. This indicates little change in nuclear genetic diversity over 30 years. Results suggest that genetic diversity of D. elator has remained stable despite reduced population size and/or abundance, which may indicate a metapopulation-like system, whose fluctuations might counteract species extinction.

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment.

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

Unveiling the Gut Microbiota and Resistome of Wild Cotton Mice, Peromyscus gossypinus, from Heavy Metal- and Radionuclide-Contaminated Sites in the Southeastern United States.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

A High-Quality Reference Genome Assembly of the Saltwater Crocodile, Crocodylus porosus, Reveals Patterns of Selection in Crocodylidae.

Crocodilians are an economically, culturally, and biologically important group. To improve researchers' ability to study genome structure, evolution, and gene regulation in the clade, we generated a high-quality de novo genome assembly of the saltwater crocodile, Crocodylus porosus, from Illumina short read data from genomic libraries and in vitro proximity-ligation libraries. The assembled genome is 2,123.5 Mb, with N50 scaffold size of 17.7 Mb and N90 scaffold size of 3.8 Mb. We then annotated this new assembly, increasing the number of annotated genes by 74%. In total, 96% of 23,242 annotated genes were associated with a functional protein domain. Furthermore, multiple noncoding functional regions and mappable genetic markers were identified. Upon analysis and overlapping the results of branch length estimation and site selection tests for detecting potential selection, we found 16 putative genes under positive selection in crocodilians, 10 in C. porosus and 6 in Alligator mississippiensis. The annotated C. porosus genome will serve as an important platform for osmoregulatory, physiological, and sex determination studies, as well as an important reference in investigating the phylogenetic relationships of crocodilians, birds, and other tetrapods.

Population genetics of two chromatic morphs of the Chagas disease vector Rhodnius pallescens Barber, 1932 in Panamá.

Rhodnius pallescens is the principal vector of Chagas disease in Panama. Recently a dark chromatic morph has been discovered in the highlands of Veraguas Province. Limited genetic studies have been conducted with regards to the population structure and dispersal potential of Triatominae vectors, particularly in R. pallescens. Next generation sequencing methods such as RADseq and complete mitochondrial DNA (mtDNA) genome sequencing have great potential for examining vector biology across space and time. Here we utilize a RADseq method (3RAD), along with complete mtDNA sequencing, to examine the population structure of the two chromatic morpho types of R. pallescens in Panama. We sequenced 105 R. pallescens samples from five localities in Panama. We generated a 2216 SNP dataset and 6 complete mtDNA genomes. RADseq showed significant differentiation among the five localities (FCT = 0.695; P = .004), but most of this was between localities with the dark vs. light chromatic morphs (Veraguas vs. Panama Oeste). The mtDNA genomes showed a 97-98% similarity between dark and light chromatic morphs across all genes and a 502 bp insert in light morphs. Thus, both the RADseq and mtDNA data showed highly differentiated clades with essentially no gene flow between the dark and light chromatic morphs from Veraguas and central Panama respectively. We discuss the growing evidence showing clear distinctions between these two morpho types with the possibility that these are separate species, an area of research that requires further investigation. Finally, we discuss the cost-effectiveness of 3RAD which is a third of the cost compared to other RADseq methods used recently in Chagas disease vector research.

Co-occurrence of antibiotic, biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site.

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim's Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment.

Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD).

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

Extensive mitochondrial heteroplasmy in the neotropical ants of the Ectatomma ruidum complex (Formicidae: Ectatomminae).

We assembled mitogenomes from 21 ant workers assigned to four morphospecies (E. ruidum spp. 1-4) and putative hybrids of the Ectatomma ruidum complex (E. ruidum spp. 2x3), and to E. tuberculatum using NGS data. Mitogenomes from specimens of E. ruidum spp. 3, 4 and 2 × 3 had a high proportion of polymorphic sites. We investigated whether polymorphisms in mitogenomes are due to nuclear mt paralogues (numts) or due to the presence of more than one mitogenome within an individual (heteroplasmy). We did not find loss of function signals in polymorphic protein-coding genes, and observed strong evidence for purifying selection in two haplotype-phased genes, which indicate the presence of two functional mitochondrial genomes coexisting within individuals instead of numts. Heteroplasmy due to hybrid paternal leakage is not supported by phylogenetic analyses. Our results reveal the presence of a fast-evolving secondary mitochondrial lineage of uncertain origin in the E. ruidum complex.

Complete mitochondrial genome of the porbeagle shark, Lamna nasus (Chondrichthyes, Lamnidae).

We report for the first time, the complete mitochondrial genome sequence of the porbeagle shark, Lamna nasus, from a specimen collected from offshore waters of New England, USA in the western North Atlantic Ocean. The genome structure of this species is similar to the other reported shark mitogenomes. The genome sequence has a total length of 16,697 bases; similar in size to the mtDNA genomes reported for other lamnid species. A Bayesian phylogenetic tree was reconstructed for the Lamnidae family using mitogenome sequences available in the Genbank.

Complete mitochondrial DNA genome of bonnethead shark, Sphyrna tiburo, and phylogenetic relationships among main superorders of modern elasmobranchs.

Elasmobranchs are one of the most diverse groups in the marine realm represented by 18 orders, 55 families and about 1200 species reported, but also one of the most vulnerable to exploitation and to climate change. Phylogenetic relationships among main orders have been controversial since the emergence of the Hypnosqualean hypothesis by Shirai (1992) that considered batoids as a sister group of sharks. The use of the complete mitochondrial DNA (mtDNA) may shed light to further validate this hypothesis by increasing the number of informative characters. We report the mtDNA genome of the bonnethead shark Sphyrna tiburo, and compare it with mitogenomes of other 48 species to assess phylogenetic relationships. The mtDNA genome of S. tiburo, is quite similar in size to that of congeneric species but also similar to the reported mtDNA genome of other Carcharhinidae species. Like most vertebrate mitochondrial genomes, it contained 13 protein coding genes, two rRNA genes and 22 tRNA genes and the control region of 1086 bp (D-loop). The Bayesian analysis of the 49 mitogenomes supported the view that sharks and batoids are separate groups.