Short-read Sequencing Technology Research Articles

Identification of grapevine clones via high-throughput amplicon sequencing: a proof-of-concept study.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of four important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot, and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Beyond assembly: the increasing flexibility of single-molecule sequencing technology.

The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein-DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.

Cryoxcellia borchgrevinki gen. nov., sp. nov., a new parasitic X-cell species in an Antarctic nototheniid fish, the bald notothen Trematomus borchgrevinki

X-cells were first described as an unknown cell type in northern hemisphere flatfish in 1969. Almost a decade later they were described in an Antarctic fish, the bald notothen Trematomus borchgrevinki, thus demonstrating their global distribution. Since this time, X-cells from various northern hemisphere fish species and from three other Antarctic fishes, the emerald notothen Trematomus bernacchii, the crowned notothen Trematomus scotti, and the painted notothen Nototheniops larseni have been identified as perkinsozoan parasites of the Family Xcellidae. Currently there are seven X-cell species described within this family. Here we report the morphology of X-cells isolated from the gill filaments of the bald notothen and include details of some of its division forms. Using short-read high-throughput DNA sequencing technology we have sequenced, assembled, and verified a 5347-bp region of the X-cell rRNA repeat unit that includes the complete 18S gene. In all cases, phylogenetic analyses identified this sequence as a distinct taxon and placed it among the perkinsozoan alveolates alongside other previously identified species in the X-cell family. Using a combination of morphological and genetic evidence we now describe a new X-cell genus and species, Cryoxcellia borchgrevinki gen. nov., sp. nov., from McMurdo Sound, Antarctica.

Molecular Surveillance of Malaria Using the PF AmpliSeq Custom Assay for Plasmodium falciparum Parasites from Dried Blood Spot DNA Isolates from Peru.

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2.

High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short-read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the rolling circle to concatemeric consensus (R2C2) method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, and regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli.

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short-read next-generation sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with ultraviolet (UV) irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Detection of insertions and deletions (InDels) by short-read next-generation sequencing (NGS) technology can be challenging because of frequent misaligned reads. A systematic analysis of short InDels (1 to 30 bases) and fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplications (ITDs; 6 to 183 bases) from 46 clinical cases of solid or hematologic malignancy processed with a clinical NGS assay identified misaligned reads in every case, ranging from 3% to 100% of reads with the InDel showing mismapped bases. Mismaps also increased with InDel size. As a consequence, the clinical NGS bioinformatics pipeline undercalled the variant allele frequency by 1% to 84%, incorrectly called simultaneous single-base substitutions along with InDels, or did not report an FLT3 ITD that had been detected by capillary electrophoresis. To improve the ability of the pipeline to better detect and quantify InDels, we utilized a software program called Assembly-Based ReAligner (ABRA2) to more accurately remap reads. ABRA2 was able to correct 41% to 100% of the reads with mismapped bases and led to absolute increases in the variant allele frequency from 1% to 61% along with correction of all of the single-base substitutions except for two cases. ABRA2 could also detect multiple FLT3 ITD clones except for one 183-base ITD. Our analysis has found that ABRA2 performs well on short InDels as well as FLT3 ITDs that are <100 bases.

Sequencing Bait: Nuclear and Mitogenome Assembly of an Abundant Coastal Tropical and Subtropical Fish, Atherinomorus stipes.

Genetic data from nonmodel species can inform ecology and physiology, giving insight into a species’ distribution and abundance as well as their responses to changing environments, all of which are important for species conservation and management. Moreover, reduced sequencing costs and improved long-read sequencing technology allows researchers to readily generate genomic resources for nonmodel species. Here, we apply Oxford Nanopore long-read sequencing and low-coverage (∼1x) whole genome short-read sequencing technology (Illumina) to assemble a genome and examine population genetics of an abundant tropical and subtropical fish, the hardhead silverside (Atherinomorus stipes). These fish are found in shallow coastal waters and are frequently included in ecological models because they serve as abundant prey for commercially and ecologically important species. Despite their importance in sub-tropical and tropical ecosystems, little is known about their population connectivity and genetic diversity. Our A. stipes genome assembly is about 1.2 Gb with comparable repetitive element content (∼47%), number of protein duplication events, and DNA methylation patterns to other teleost fish species. Among five sampled populations spanning 43 km of South Florida and the Florida Keys, we find little population structure suggesting high population connectivity.

The evolutionary history of Cardamine bulbifera shows a successful rapid postglacial Eurasian range expansion in the absence of sexual reproduction.

Sexual reproduction is known to drive plant diversification and adaptation. Here we investigate the evolutionary history and spatiotemporal origin of a dodecaploid (2n = 12x = 96) Eurasian deciduous woodland species, Cardamine bulbifera, which reproduces and spreads via vegetative bulb-like structures only. The species has been among the most successful range-expanding understorey woodland plants in Europe, which raises the question of the genetic architecture of its gene pool, since its hexaploid (2n = 6x = 48) but putatively outcrossing closest relative, C. quinquefolia, displays a smaller distribution range in Eastern Europe towards the Caucasus region. Cardamine bulbifera belongs to a small monophyletic clade of four species comprising also C. abchasica (2n = 2x = 16) and C. bipinnata (unknown ploidy) from the Caucasus region. We sequenced the genomes of the two polyploids and their two putative ancestors using Illumina short-read sequencing technology (×7-8 coverage). Covering the entire distribution range, genomic data were generated for 67 samples of the two polyploids (51 samples of C. bulbifera, 16 samples of C. quinquefolia) and 6 samples of the putative diploid taxa (4 samples of C. abchasica, 2 samples of C. bipinnata) to unravel the evolutionary origin of the polyploid taxa using phylogenetic reconstructions of biparentally and maternally inherited genetic sequence data. Ploidy levels of C. bulbifera and C. quinquefolia were analysed by comparative chromosome painting. We used genetic assignment analysis (STRUCTURE) and approximate Bayesian computation (ABC) modelling to test whether C. bulbifera represents genetically differentiated lineages and addressed the hypothesis of its hybrid origin. Comparative ecological modelling was applied to unravel possible niche differentiation among the two polyploid species. Cardamine bulbifera was shown to be a non-hybridogenous, auto-dodecaploid taxon of early Pleistocene origin, but with a history of past gene flow with its hexaploid sister species C. quinquefolia, likely during the last glacial maximum in shared refuge areas in Eastern Europe towards Western Turkey and the Crimean Peninsula region. The diploid Caucasian endemic C. abchasica is considered an ancestral species, which also provides evidence for the origin of the species complex in the Caucasus region. Cardamine bulbifera successfully expanded its distribution range postglacially towards Central and Western Europe accompanied by a transition to exclusively vegetative propagation. A transition to vegetative propagation in C. bulbifera is hypothesized as the major innovation to rapidly expand its distribution range following postglacially progressing woodland vegetation throughout Europe. Preceding and introgressive gene flow from its sister species C. quinquefolia in the joint refuge area is documented. This transition and ecological differentiation may have been triggered by preceding introgressive gene flow from its sister species in the joint East European refuge areas.

A Unique Mitochondrial Gene Block Inversion in Antarctic Trematomin Fishes: A Cautionary Tale.

Many Antarctic notothenioid fishes have major rearrangements in their mitochondrial (mt) genomes. Here, we report the complete mt genomes of 3 trematomin notothenioids: the bald notothen (Trematomus (Pagothenia) borchgrevinki), the spotted notothen (T. nicolai), and the emerald notothen (T. bernacchii). The 3 mt genomes were sequenced using next-generation Illumina technology, and the assemblies verified by Sanger sequencing. When compared with the canonical mt gene order of the Antarctic silverfish (Pleuragramma antarctica), we found a large gene inversion in the 3 trematomin mt genomes that included tRNAIle, ND1, tRNALeu2, 16S, tRNAVal, 12S, tRNAPhe, and the control region. The trematomin mt genomes contained 3 intergenic spacers, which are thought to be the remnants of previous gene and control region duplications. All control regions included the characteristic conserved regulatory sequence motifs. Although short-read next-generation DNA sequencing technology has allowed the rapid and cost-effective sequencing of a large number of complete mt genomes, it is essential in all cases to verify the assembly in order to prevent the publication and use of erroneous data.

Differentially expressed genes in Mythimna separata under chlorantraniliprole exposure and functional identification

The oriental armyworm, Mythimna separata has led to great economic losses of corn in China. The main method to control this pest relies on chemical insecticides. Plenty of genes in armyworm can be activated in response to insecticides. In this study, the critical genes involved in detoxification of chlorantraniliprole in armyworm were investigated using RNA-seq (transcriptome sequencing), digital gene expression profiles, and functional identification by RNA interference (RNAi) technology. The de novo assembly of the transcriptomes was achieved based on Illumina short-read sequencing technology in M. separata. There were 70,845 unigenes produced and 35,327 (49.86%) annotated. 748 unigenes were significantly up-expressed and 741 were significantly down-expressed in chlorantraniliprole treatment group compared to the control group. Silencing of cytochrome B6-F (CYB6), and long chain fatty acid CoA ligase 4 (CoA) of the top 18 highly expressed genes selected in chlorantraniliprole treatment group, resulted in more significant sensibility of M. separata to chlorantraniliprole, growth retardation and antifeedant phenomenon, which indicated that both CYB6 and CoA were most possibly responsible for chlorantraniliprole metabolism/detoxication.

Improved Whole-Genome Sequence of Fusarium meridionale, the Fungal Pathogen Causing Fusarium Head Blight in Rice.

Members of the Fusarium graminearum species complex (FGSC) cause extensive yield losses in cereal production worldwide, and food safety concerns due to the accumulation of Fusarium toxins in infected grains. Among these pathogens, F. meridionale is responsible for Fusarium head blight of wheat and rice, ear and stalk rot of maize, and pod blight of soybean. Here, we present an improved genome assembly of F. meridionale strain SR5 isolated from rice in China based on PacBio long-read sequencing and Illumina short-read sequencing technology. The assembled genome of SR5 has a total size of 36.82 Mb, an N50 scaffold length of 7.82 Mb, nine scaffolds, and encodes 12,409 predicted genes. These high-quality data expand FGSC genomic resources and provide a valuable resource for better understanding their genetic diversity and the molecular basis of pathogenesis, which will facilitate the development of an effective control strategy.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Access to ultra-long IgG CDRH3 bovine antibody sequences using short read sequencing technology

The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties – a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline VH, DH and JH allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

Bioacoustics Reveal Hidden Diversity in Frogs: Two New Species of the Genus Limnonectes from Myanmar (Amphibia, Anura, Dicroglossidae)

Striking geographic variation in male advertisement calls was observed in frogs formerly referred to as Limnonectes doriae and L. limborgi, respectively. Subsequent analyses of mtDNA and external morphological data brought supporting evidence for the recognition of these populations as distinct species. We describe two new frog species of the genus Limnonectes (i.e., L. bagoensis sp. nov. and L. bagoyoma sp. nov.) from Myanmar. Limnonectes bagoensis sp. nov. is closely related to L. doriae whereas L. bagoyoma sp. nov. is closely related to L. limborgi. Results of this integrative study provide evidence for the presence of additional undescribed species in these species complexes but due to the lack of bioacoustical data, we consider these additional diverging populations as candidate species that need further study to resolve their respective taxonomic status. Both new species are distributed in Lower Myanmar. Limnonectes doriae is restricted to southern Myanmar along the Malayan Peninsula whereas L. limborgi is known to occur in eastern Myanmar and northwestern Thailand. The remaining populations formerly referred to as either L. doriae or L. limborgi are considered representatives of various candidate species that await further study. We further provide a de novo draft genome of the respective holotypes of L. bagoensis sp. nov. and L. bagoyoma sp. nov. based on short-read sequencing technology to 25-fold coverage.

Analysis of Hi-C Data for Discovery of Structural Variations in Cancer.

Structural variations (SVs) are large genomic rearrangements that can be challenging to identify with current short read sequencing technology due to various confounding factors such as existence of genomic repeats and complex SV structures. Hi-C breakfinder is the first computational tool that utilizes the technology of high-throughput chromatin conformation capture assay (Hi-C) to systematically identify SVs, without being interfered by regular confounding factors. SVs change the spatial distance of genomic regions and cause discontinuous signals in Hi-C, which are difficult to analyze by routine informatics practice. Here we provide step-by-step guidance for how to identify SVs using Hi-C data and how to reconstruct Hi-C maps in the presence of SVs.

Feasibility of applying shotgun metagenomic analyses to grapevine leaf, rhizosphere and soil microbiome characterisation

Background and Aims The characterisation of plant microbiomes using metabarcoding strategies is expected to be progressively replaced by shotgun metagenomic (SMg) approaches. In the present report we explore the potential of applying SMg to grapevine leaf, rhizosphere and soil samples. Methods and Results Our strategy involved combining a single method for column-based DNA extraction of multiple tissues, with sequencing using the BGISEQ short-read next-generation sequencing platform. This study details aspects of DNA isolation, library construction, sequencing and early bioinformatics treatment of read data, including the use of subsampling as a proxy for detection of sample microbial proportions. The combination of an innovative sequencing platform with recent mapping tools allowed for the characterisation of the microbiome associated with the grapevine leaf, rhizosphere and adjacent soil. Conclusions We coupled a robust single extraction/library preparation protocol suitable for contrasting samples, with cost-effective BGISEQ short-read next-generation sequencing technology, to generate a landscape of grapevine-associated microbial diversity. Significance of the Study Shotgun metagenomics is emerging as a state-of-the-art approach to grapevine microbiome characterisation. The present report provides a detailed and technical roadmap targeting multiple aspects of this important approach.

Identification of differentially expressed microRNAs under imidacloprid exposure in Sitobion miscanthi

Imidacloprid is a neonicotinoid that targets sucking pests, such as aphids and the green leaf bug and has been widely applied in wheat fields to control wheat aphids in China. To investigate the involvement of miRNAs in imidacloprid resistance, we sequenced small RNA libraries of Sitobion miscanthi Fabricius, across two different treatments using Illumina short-read sequencing technology. As a result, 265 microRNAs (miRNAs), of which 242 were known and 23 were novel, were identified. Quantitative analysis of miRNA levels showed that 23 miRNAs were significantly up-regulated, and 54 miRNAs were significantly down-regulated in the nymphs of S. miscanthi treated with imidacloprid in comparison with those of the control. Modulation of the abundances of differentially expressed miRNAs, smi-miR-316, smi-miR-1000, and smi-miR-iab-4 by the addition of the corresponding antagomir/inhibitor to the artificial diet significantly changed the susceptibility of S. miscanthi to imidacloprid. Subsequently, the post-transcriptional regulatory mechanism was conducted, smi-miR-278 and smi-miR-316 were confirmed to be participated in the post-transcriptional regulation of nAChRα1A and CYP4CJ6, respectively. The results suggested that miRNAs differentially expressed in response to imidacloprid could play a critical regulatory role in the metabolism of S. miscanthi to imidacloprid.

Functional identification of the terpene synthase family involved in diterpenoid alkaloids biosynthesis in Aconitum carmichaelii

Aconitum carmichaelii is a high-value medicinal herb widely used across China, Japan, and other Asian countries. Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in Aconitum. Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the Aconitum species, the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A. carmichaelii transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified. In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs. Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate. Five AcKSLs separately reacted with ent-CPP to produce ent-kaurene, ent-atiserene, and ent-13-epi-sandaracopimaradie: a new diterpene found in Aconitum. AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis, with AcKSL1, AcKSL2s and AcKSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A. carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.

A taxonomic revision of the genus Phrynoglossus in Indochina with the description of a new species and comments on the classification within Occidozyginae (Amphibia, Anura, Dicroglossidae)

We revise the frogs of the genusPhrynoglossusfrom Indochina based on data of external morphology, bioacoustics and molecular genetics. The results of this integrative study provide evidence for the recognition of three distinct species, one of which we describe as new.Phrynoglossus martensiihas a vast geographic distribution from central and southern Thailand across southern China to Vietnam, Laos, and Cambodia.Phrynoglossus myanhesseisp. nov.is distributed in central Myanmar whereasPhrynoglossus magnapustulosusis restricted to the Khorat Plateau, Thailand. These three species occur in allopatry and differ in their mating calls, external morphology, and in genetic distances of the 16S gene of 3.8–5.9%. Finally, we discuss and provide evolutionary evidence for the recognition ofPhrynoglossusas a genus distinct fromOccidozyga. Members of both genera form reciprocal monophyletic groups in our analyses of mtDNA data (16S) and are well differentiated from each other in morphology and ecology. Furthermore, they differ in the amplexus mode withPhrynoglossushaving an inguinal amplexus whereas it is axillary inOccidozyga. We further provide ade novodraft genome of the holotype based on short-read sequencing technology to a coverage of 25-fold. This resource will permanently link the genetic characterization of the species to the name-bearing type specimen.

A taxonomic revision of the genus Phrynoglossus in Indochina with the description of a new species and comments on the classification within Occidozyginae (Amphibia, Anura, Dicroglossidae)

We revise the frogs of the genus Phrynoglossus from Indochina based on data of external morphology, bioacoustics and molecular genetics. The results of this integrative study provide evidence for the recognition of three distinct species, one of which we describe as new. Phrynoglossus martensii has a vast geographic distribution from central and southern Thailand across southern China to Vietnam, Laos, and Cambodia. Phrynoglossus myanhesseisp. nov. is distributed in central Myanmar whereas Phrynoglossus magnapustulosus is restricted to the Khorat Plateau, Thailand. These three species occur in allopatry and differ in their mating calls, external morphology, and in genetic distances of the 16S gene of 3.8–5.9%. Finally, we discuss and provide evolutionary evidence for the recognition of Phrynoglossus as a genus distinct from Occidozyga. Members of both genera form reciprocal monophyletic groups in our analyses of mtDNA data (16S) and are well differentiated from each other in morphology and ecology. Furthermore, they differ in the amplexus mode with Phrynoglossus having an inguinal amplexus whereas it is axillary in Occidozyga. We further provide a de novo draft genome of the holotype based on short-read sequencing technology to a coverage of 25-fold. This resource will permanently link the genetic characterization of the species to the name-bearing type specimen.