Substantial Sequence Divergence Research Articles

Understanding epistatic networks in the B1 β-lactamases through coevolutionary statistical modeling and deep mutational scanning.

Throughout evolution, protein families undergo substantial sequence divergence while preserving structure and function. Although most mutations are deleterious, evolution can explore sequence space via epistatic networks of intramolecular interactions that alleviate the harmful mutations. However, comprehensive analysis of such epistatic networks across protein families remains limited. Thus, we conduct a family wide analysis of the B1 metallo-β-lactamases, combining experiments (deep mutational scanning, DMS) on two distant homologs (NDM-1 and VIM-2) and computational analyses (in silico DMS based on Direct Coupling Analysis, DCA) of 100 homologs. The methods jointly reveal and quantify prevalent epistasis, as ~1/3rd of equivalent mutations are epistatic in DMS. From DCA, half of the positions have a >6.5 fold difference in effective number of tolerated mutations across the entire family. Notably, both methods locate residues with the strongest epistasis in regions of intermediate residue burial, suggesting a balance of residue packing and mutational freedom in forming epistatic networks. We identify entrenched WT residues between NDM-1 and VIM-2 in DMS, which display statistically distinct behaviors in DCA from non-entrenched residues. Entrenched residues are not easily compensated by changes in single nearby interactions, reinforcing existing findings where a complex epistatic network compounds smaller effects from many interacting residues.

Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis.

The digestive organs of terrestrial isopods harbour bacteria of the recently proposed mollicute family Hepatoplasmataceae. The only complete genome available so far for Hepatoplasmataceae is that of 'Candidatus Hepatoplasma crinochetorum'. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepatoplamsataceae members: 'Candidatus Tyloplasma litorale' identified from the semiterrestrial isopod Tylos granuliferus, 'Candidatus Hepatoplasma vulgare' identified from the common pill bug Armadillidium vulgare, and 'Candidatus Hepatoplasma scabrum' identified from the common rough woodlouse Porcellio scaber. Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. The 16S ribosomal RNA gene sequences and phylogenomic analysis showed that 'Candidatus Tyloplasma litorale' and other semiterrestrial isopod-associated mollicutes represent the placeholder genus 'g_Bg2' in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harbouring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.

High-quality assembly and methylome of a Tibetan wild tree peony genome (Paeonia ludlowii) reveal the evolution of giant genome architecture.

Tree peony belongs to one of the Saxifragales families, Paeoniaceae. It is one of the most famous ornamental plants, and is also a promising woody oil plant. Although two Paeoniaceae genomes have been released, their assembly qualities are still to be improved. Additionally, more genomes from wild peonies are needed to accelerate genomic-assisted breeding. Here we assemble a high-quality and chromosome-scale 10.3-Gb genome of a wild Tibetan tree peony, Paeonia ludlowii, which features substantial sequence divergence, including around 75% specific sequences and gene-level differentials compared with other peony genomes. Our phylogenetic analyses suggest that Saxifragales and Vitales are sister taxa and, together with rosids, they are the sister taxon to asterids. The P. ludlowii genome is characterized by frequent chromosome reductions, centromere rearrangements, broadly distributed heterochromatin, and recent continuous bursts of transposable element (TE) movement in peony, although it lacks recent whole-genome duplication. These recent TE bursts appeared during the uplift and glacial period of the Qinghai-Tibet Plateau, perhaps contributing to adaptation to rapid climate changes. Further integrated analyses with methylome data revealed that genome expansion in peony might be dynamically affected by complex interactions among TE proliferation, TE removal, and DNA methylation silencing. Such interactions also impact numerous recently duplicated genes, particularly those related to oil biosynthesis and flower traits. This genome resource will not only provide the genomic basis for tree peony breeding but also shed light on the study of the evolution of huge genome structures as well as their protein-coding genes.

Divergent amplifications of CYP9A cytochrome P450 genes provide two noctuid pests with differential protection against xenobiotics

Here, we provide mechanistic support for the involvement of the CYP9A subfamily of cytochrome P450 monooxygenases in the detoxification of host plant defense compounds and chemical insecticides in Spodoptera exigua and Spodoptera frugiperda. Our comparative genomics shows that a large cluster of CYP9A genes occurs in the two species but with significant differences in its contents, including several species-specific duplicates and substantial sequence divergence, both between orthologs and between duplicates. Bioassays of CRISPR-Cas9 knockouts of the clusters show that, collectively, the CYP9As can detoxify two furanocoumarin plant defense compounds (imperatorin and xanthotoxin) and insecticides representing three different chemotypes (pyrethroids, avermectins, and oxadiazines). However, in vitro metabolic assays of heterologously expressed products of individual genes show several differences between the species in the particular CYP9As with activities against these compounds. We also find that the clusters show tight genetic linkage with high levels of pyrethroid resistance in field strains of the two species. We propose that their divergent amplifications of the CYP9A subfamily have not only contributed to the development of the broad host ranges of these species over long evolutionary timeframes but also supplied them with diverse genetic options for evolving resistance to chemical insecticides in the very recent past.

The genomics of linkage drag in inbred lines of sunflower

Crop wild relatives represent valuable sources of alleles for crop improvement, including adaptation to climate change and emerging diseases. However, introgressions from wild relatives might have deleterious effects on desirable traits, including yield, due to linkage drag. Here, we analyzed the genomic and phenotypic impacts of wild introgressions in inbred lines of cultivated sunflower to estimate the impacts of linkage drag. First, we generated reference sequences for seven cultivated and one wild sunflower genotype, as well as improved assemblies for two additional cultivars. Next, relying on previously generated sequences from wild donor species, we identified introgressions in the cultivated reference sequences, as well as the sequence and structural variants they contain. We then used a ridge-regression best linear unbiased prediction (BLUP) model to test the effects of the introgressions on phenotypic traits in the cultivated sunflower association mapping population. We found that introgression has introduced substantial sequence and structural variation into the cultivated sunflower gene pool, including >3,000 new genes. While introgressions reduced genetic load at protein-coding sequences, they mostly had negative impacts on yield and quality traits. Introgressions found at high frequency in the cultivated gene pool had larger effects than low-frequency introgressions, suggesting that the former likely were targeted by artificial selection. Also, introgressions from more distantly related species were more likely to be maladaptive than those from the wild progenitor of cultivated sunflower. Thus, breeding efforts should focus, as far as possible, on closely related and fully compatible wild relatives.

Horizontal Gene Transfer, Fitness Costs and Mobility Shape the Spread of Antibiotic Resistance Genes into Experimental Populations of Acinetobacter Baylyi.

Horizontal gene transfer (HGT) is important for microbial evolution, but how evolutionary forces shape the frequencies of horizontally transferred genetic variants in the absence of strong selection remains an open question. In this study, we evolve laboratory populations of Acinetobacter baylyi (ADP1) with HGT from two clinically relevant strains of multidrug-resistant Acinetobacter baumannii (AB5075 and A9844). We find that DNA can cross the species barrier, even without strong selection, and despite substantial DNA sequence divergence between the two species. Our results confirm previous findings that HGT can drive the spread of antibiotic resistance genes (ARGs) without selection for that antibiotic, but not for all of the resistance genes present in the donor genome. We quantify the costs and benefits of horizontally transferred variants and use whole population sequencing to track the spread of ARGs from HGT donors into antibiotic-sensitive recipients. We find that even though most ARGs are taken up by populations of A. baylyi, the long-term fate of an individual gene depends both on its fitness cost and on the type of genetic element that carries the gene. Interestingly, we also found that an integron, but not its host plasmid, is able to spread in A. baylyi populations despite its strong deleterious effect. Altogether, our results show how HGT provides an evolutionary advantage to evolving populations by facilitating the spread of non-selected genetic variation including costly ARGs.

Unravelling the Phylogeny of a Common Intestinal Protist: Intrageneric Diversity of Endolimax

Endolimax nana is a common endobiont of the human intestine, but members of the genus have also been reported in non-human hosts and in non-intestinal organs. Limited information is available regarding the genetic diversity of Endolimax, which is necessary to delineate species, host specificity and potential differences in clinical impact on the host. Here, we used cloning of PCR products followed by Sanger sequencing and next-generation PacBio Sequencing to obtain Endolimax-related nuclear ribosomal gene sequences and undertook a phylogenetic analysis to gain additional insight into the taxonomy of Endolimax and related organisms. The new sequences confirmed that E. nana forms a discrete clade within the Archamoebae and is related to Endolimax piscium and Iodamoeba. However, we identified substantial sequence divergence within E. nana and evidence for two distinct clades, which we propose to name E. nana ribosomal lineage 1 and E. nana ribosomal lineage 2. Both of the sequencing approaches applied in the study helped us to improve our understanding of genetic diversity across Endolimax, and it is likely that wider application of next-generation sequencing technologies will facilitate the generation of Endolimax-related DNA sequence data and help complete our understanding of its phylogenetic position and intrageneric diversity.

Morphometric and Phylogenetic analysis of morphotypes in Artemia franciscana Kellogg, 1906 (Crustacea: Anostraca)

We document morphology and genetic relatedness of morphotypes within the Artemia franciscana that has colonized Covelong saltern in Kelambakkam (South India). They exhibits high phenotypic plasticity and intra-population variation in a supposedly panmictic population. Morphometric analysis was carried out on 18 traits in males and 15 in females and subjected to multivariate analysis. Three types each in males (M1, M3 and M4) and females (F1, F2 and F3) were singled out. The axes in the plot explain 77.9% in males and 73.9% in females of the variation through discriminant function analysis. The sequence divergence of the population was evaluated through mtDNA (16S rRNA) and nDNA (p26 gene) markers. The substantial sequence divergence was observed between the morphotypes through nDNA and appearing sequence divergence was recognized by mtDNA. Likewise, phylogenetic analyses of maximum likelihood and Bayesian analysis through nDNA revealed a maximum similarities between M1, M4 with F1 & F2 and M3 with F3. Further, haplotype distribution of nDNA revealed unique haplotype between M1 and M4 with F1 and F2 morphotypes. This study thus confirms the occurrence of morphotypes with specific characteristics inside a seemingly homogeneous population.

Extensive sequence divergence between the reference genomes of two zebrafish strains, Tuebingen and AB.

The zebrafish (Danio rerio) is one of the most widely used model organisms for studying vertebrate gene function and human disease, given the 70% conserved protein-coding genes between zebrafish and human. Two of the most common laboratory zebrafish strains are Tuebingen and AB. Despite the fact that the zebrafish reference genome is derived from the Tuebingen strain, the AB strain is still widely used although a high-quality genome comparable to Tuebingen is lacking. Here, we report a 1.40-Gb representative de novo genome assembly of the AB strain (DrAB1), with contig N50length of 21Mb, by integrating Illumina short-read sequencing, Nanopore long-read sequencing and HiC-based chromatin mapping. Compared with the published zebrafish Zv11 reference genome (GRCz11), this genome assembly shows considerable improvements in both contiguity and completeness. In addition, substantial structural differences and extensive sequence divergence of unprecedented resolution have been uncovered, especially with respect to 9,029,929single nucleotide polymorphisms, 2,376,812 InDels, 32,623 insertions, 22,089 deletions and 220 inversions, which constitute ~2.6% of the DrAB1genome. Many of these variants may have potential functional effects on phenotype, which should be considered in further experimental designs. Consequently, our study provides additional genomic resources and a high-resolution structural variation map based on whole-genome alignment for the zebrafish community, which could also be an indispensable reference genome from a model species in future research on fish phylogenetic genomics, comparative genomics and adaptive evolution.

Plasticity in ligand recognition at somatostatin receptors.

Somatostatin is a signaling peptide that plays a pivotal role in physiologic processes relating to metabolism and growth through its actions at somatostatin receptors (SSTRs). Members of the SSTR subfamily, particularly SSTR2, are key drug targets for neuroendocrine neoplasms, with synthetic peptide agonists currently in clinical use. Here, we show the cryogenic-electron microscopy structures of active-state SSTR2 in complex with heterotrimeric Gi3 and either the endogenous ligand SST14 or the FDA-approved drug octreotide. Complemented by biochemical assays and molecular dynamics simulations, these structures reveal key details of ligand recognition and receptor activation at SSTRs. We find that SSTR ligand recognition is highly diverse, as demonstrated by ligand-induced conformational changes in ECL2 and substantial sequence divergence across subtypes in extracellular regions. Despite this complexity, we rationalize several known sources of SSTR subtype selectivity and identify an additional interaction for specific binding. These results provide valuable insights for structure-based drug discovery at SSTRs.

Complete mitochondrial genomes of Conogethes punctiferalis and C. pinicolalis (Lepidoptera: Crambidae): Genomic comparison and phylogenetic inference in Pyraloidea

• We sequenced complete mitogenomes of Conogethes pinicolalis and C. punctiferalis . • We compared to each other and to 54 available mitogenomes of Pyraloidea. • The two congeneric species showed substantial sequence divergence (mean 5.92%). • Phylogenetic tree provided strong supports for lower groups within Pyraloidea. Conogethes punctiferalis Guenée, 1854 (Lepidoptera: Crambidae) is a serious polyphagous pest that attacks more than one hundred species of plants. Previously, C. punctiferalis was determined to be composed of two ecotypes; later, Conogethes pinicolalis , was described as a separate species. Due to the prolonged negligence of C. pinicolalis as an independent species, the genetic perspective of the two species is limited. Thus, in this study, 15,332 and 15,336 bp-long complete mitochondrial genome (mitogenome) of the two species were sequenced and compared to each other and to 54 available mitogenomes of Pyraloidea. The comparison of each protein-coding gene (PCG) and rRNA gene of the two congeneric species showed substantial sequence divergence, ranging from 3.13% ( ATP8 ) to 8.3% ( COIII ), with an average of 5.92%. Phylogenetic analyses using concatenated sequences of 13 PCGs and 2 rRNAs (12,458 bp including gaps), both by maximum likelihood (ML) and Bayesian inference (BI) methods, consistently supported the monophyly of each family (Crambidae and Pyralidae) and subfamily, generally with the highest nodal supports. The subfamilial relationships of ((((Acentropinae + Schoenobiinae) + (Scopariinae + Crambinae)) + Evergestinae) + (Spilomelinae + Pyraustinae)) in Crambidae and the subfamilial relationships of ((((Pyralinae + Epipaschiinae) + Phycitinae) + Galleriilinae)) in Pyralidae were obtained in both analyses. However, nodal supports were substantially low in this study, mainly due to limited taxa.

Evolutionary Characterization of the Short Protein SPAAR.

Microproteins (<100 amino acids) are receiving increasing recognition as important participants in numerous biological processes, but their evolutionary dynamics are poorly understood. SPAAR is a recently discovered microprotein that regulates muscle regeneration and angiogenesis through interactions with conserved signaling pathways. Interestingly, SPAAR does not belong to any known protein family and has known homologs exclusively among placental mammals. This lack of distant homology could be caused by challenges in homology detection of short sequences, or it could indicate a recent de novo emergence from a noncoding sequence. By integrating syntenic alignments and homology searches, we identify SPAAR orthologs in marsupials and monotremes, establishing that SPAAR has existed at least since the emergence of mammals. SPAAR shows substantial primary sequence divergence but retains a conserved protein structure. In primates, we infer two independent evolutionary events leading to the de novo origination of 5′ elongated isoforms of SPAAR from a noncoding sequence and find evidence of adaptive evolution in this extended region. Thus, SPAAR may be of ancient origin, but it appears to be experiencing continual evolutionary innovation in mammals.

A taxonomic revision ofPennellaOken, 1815 based on morphology and genetics (Copepoda: Siphonostomatoida: Pennellidae)

AbstractThe taxonomy of the genus PennellaOken, 1815, a large parasitic copepod, is in a deplorable state. We performed morphological and genetic analyses on 52 Pennella individuals collected from 12 species of definitive hosts. Based on the morphological characteristics and their hosts, 29 individuals were determined or suggested to be large-size species (P. balaenoptera, P. filosa, P. instructa, and P. benzi), whereas 20 were considered intermediate- or small-size species. The remaining three small individuals, presumed to be newly attached, exhibited insufficient morphological characteristics for species identification or grouping. Mitochondrial DNA cytochrome c oxidase subunit 1 (COI) sequence analysis detected 154 haplotypes from 52 individuals classified into Groups I–IV. Sequences of Group IV were determined to be pseudogenes. Levels of divergence between all groups were significant (FST = 0.488–0.830, P &amp;lt; 0.001). All haplotypes of Group I were detected in intermediate- and small-size groups possessing large and well-branched antennary processes on the cephalothorax. Although Groups I and II exhibited the smallest difference in nucleotide sequence divergence (FST = 0.488 and K2P = 1.485 ± 0.355%), the morphological characteristics of Group I appeared to be distinct from those of the other groups. The substantial sequence divergence (FST = 0.830 and K2P = 4.123 ± 0.719%) between Groups II and III also supported their species status, but no characteristic morphological difference was observed between individuals in these groups. Results suggest that Pennella may contain only two or three species. and that some morphological characteristics and definitive host species previously used for species identification are not valid.

Fine mapping of Ne1, the hybrid necrosis gene complementary to Ne2 in common wheat (Triticum aestivum L.).

Apart from confinement of Ne1 to a 4.45 Mb genomic segment, markers closely linked to Ne2 were identified and incomplete dominance of both genes in conditioning necrosis severity was shown. Hybrid necrosis in plants is characterized by premature death of leaves or plants in F1 hybrids. Interaction of two complementary dominant genes Ne1 and Ne2 in wheat (Triticum aestivum L.) is known to cause hybrid necrosis. However, the mechanism underlying this necrosis is still elusive. To obtain markers closely-linked to these two genes, Ne1-carrying cultivar Zheng891 was crossed with Ne2-carrying cultivar Pan555. Using BC1F1 plants derived from crosses of the F1 plants with the two parental lines, Ne1 and Ne2 were mapped to a 2.2cM interval and a 2.3cM interval with newly developed markers, respectively. Ne1 was further delimited to a 0.19cM interval using 2015 Ne2-carrying F2 plants. Xwgrc3146, Xwgrc3147 and Xwgrc3150, three of the four markers co-segregating with Ne1, were all Zheng891-dominant, suggesting that, compared with Pan555, Ne1 is located in a region with substantial sequence diversity. The Ne1 interval is syntenic to chromosomes 5H, 4, 9 and 2 of barley, Brachypodium distachyon, rice and sorghum, respectively, and corresponds to a 4.45Mb Chinese Spring sequence. Variations in necrosis severity of the F2 plants differing in Ne1 and Ne2 genotypes implied that these two genes are incompletely dominant in determining the timing and severity of necrosis.

A new bat species from southwestern Western Australia, previously assigned to Gould’s Long-eared Bat Nyctophilus gouldi Tomes, 1858

A distributional isolate in southwestern Western Australia previously assigned to Gould’s Long-eared Bat Nyctophilus gouldi Tomes, 1858 is demonstrated to be a distinct and previously unnamed cryptic species, based on a lack of monophyly with eastern populations and substantial DNA sequence divergence (5.0 %) at the mitochondrial gene COI. Morphologically both species are alike and overlap in all measured characters but differ in braincase shape. The new species has one of the most restricted geographic ranges of any Australian Vespertilionidae and aspects of its ecology make it vulnerable to human impacts.

Hospitalization and mortality associated with SARS-CoV-2 viral clades in COVID-19

The COVID-19 epidemic of 2019–20 is due to the novel coronavirus SARS-CoV-2. Following first case description in December, 2019 this virus has infected over 10 million individuals and resulted in at least 500,000 deaths world-wide. The virus is undergoing rapid mutation, with two major clades of sequence variants emerging. This study sought to determine whether SARS-CoV-2 sequence variants are associated with differing outcomes among COVID-19 patients in a single medical system. Whole genome SARS-CoV-2 RNA sequence was obtained from isolates collected from patients registered in the University of Washington Medicine health system between March 1 and April 15, 2020. Demographic and baseline clinical characteristics of patients and their outcome data including their hospitalization and death were collected. Statistical and machine learning models were applied to determine if viral genetic variants were associated with specific outcomes of hospitalization or death. Full length SARS-CoV-2 sequence was obtained 190 subjects with clinical outcome data. 35 (18.4%) were hospitalized and 14 (7.4%) died from complications of infection. A total of 289 single nucleotide variants were identified. Clustering methods demonstrated two major viral clades, which could be readily distinguished by 12 polymorphisms in 5 genes. A trend toward higher rates of hospitalization of patients with Clade 2 infections was observed (p = 0.06, Fisher’s exact). Machine learning models utilizing patient demographics and co-morbidities achieved area-under-the-curve (AUC) values of 0.93 for predicting hospitalization. Addition of viral clade or sequence information did not significantly improve models for outcome prediction. In summary, SARS-CoV-2 shows substantial sequence diversity in a community-based sample. Two dominant clades of virus are in circulation. Among patients sufficiently ill to warrant testing for virus, no significant difference in outcomes of hospitalization or death could be discerned between clades in this sample. Major risk factors for hospitalization and death for either major clade of virus include patient age and comorbid conditions.

The D2 and D3 Sublineages of Human Papilloma Virus 16-Positive Cervical Cancer in Guatemala Differ in Integration Rate and Age of Diagnosis.

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. SIGNIFICANCE: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

An evolution-based model for designing chorismate mutase enzymes.

The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.

Exploring neighborhoods in large metagenome assembly graphs using spacegraphcats reveals hidden sequence diversity

Genomes computationally inferred from large metagenomic data sets are often incomplete and may be missing functionally important content and strain variation. We introduce an information retrieval system for large metagenomic data sets that exploits the sparsity of DNA assembly graphs to efficiently extract subgraphs surrounding an inferred genome. We apply this system to recover missing content from genome bins and show that substantial genomic sequence variation is present in a real metagenome. Our software implementation is available at https://github.com/spacegraphcats/spacegraphcatsunder the 3-Clause BSD License.

Re-evaluation of deep-sea dogfishes (genus Squalus) in Japan using phylogenetic inference

Japan is known for its marine biodiversity and diverse ecosystem due to its unique geological composition and water currents creating suitable habitats for a wider range of marine species. A rich deep-sea shark community with high endemism occurs on the continental shelf. Yet, the deep-sea shark taxonomy is still under development and poorly documented.Here, a DNA barcoding technique was adopted using the mitochondrial cytochrome oxidase I (COI) and NADH2 genes of samples collected from Japan with additional samples from genetic databases to draft out a phylogenetic tree of the deep-sea shark genus Squalus (dogfishes). The morphological divergence of congeners was further examined. Bayesian and maximum-likelihood-based species-level phylogenetic analyses of mitochondrial COI, ND2 and combined sequences provided strong support for distinct clades corresponding to the parental species with substantial sequence divergence between parental species. The combined dataset of the COI and ND2 gene was most suitable for barcoding of Squalus dogfishes. A multivariate analysis of morphological traits was largely consistent with the genetic data showing small but discrete differences in phenotypic characteristics allowing to separate Squalus species.Genetic samples of the taxonomically problematic Shortspine spurdog ‘Squalus mitsukurii’ from the Atlantic Ocean and Taiwan did not match with the type material of S. mitsukurii Jordan & Snyder, 1903 from mainland Japan. Squalus mitsukurii once considered a globally distributed species is a single, unique spurdog restricted to temperate Japan and Korea and may present an endemic in the Northwestern Pacific. This study further resolved two genetically and phenotypically distinct species of Squalus dogfishes in Japan, one in sympatry with S. mitsukurii in mainland Japan and one in sympatry with subtropical waters associated congeners in the Ryukyu Archipelago. The occurrence of populations of the ‘high-fin’ Taiwan spurdog, Squalus formosus, in subtropical Japan is herein confirmed.