Target Enrichment Approach Research Articles

Evidence of Intraspecific Adaptive Variation in the American Pika (Ochotona princeps) on a Continental Scale Using a Target Enrichment and Mitochondrial Genome Skimming Approach.

Montane landscapes present an array of abiotic challenges that drive adaptive evolution amongst organisms. These adaptations can promote habitat specialisation, which may heighten the risk of extirpation from environmental change. For example, higher metabolic rates in an endothermic species may contribute to heightened cold tolerance, whilst simultaneously limiting heat tolerance. Here, using the climate-sensitive American pika (Ochotona princeps), we test for evidence of intraspecific adaptive variation amongst environmental gradients across the Intermountain West of North America. We leveraged results from previous studies on pika adaptation to generate a custom nuclear target enrichment design to sequence several hundred candidate genes related to cold, hypoxia and dietary detoxification. We also applied a 'genome skimming' approach to sequence mitochondrial DNA. Using genotype-environment association tests, we identified rare genomic variants associated with elevation and temperature variation amongst populations. Amongst mitochondrial genes, we identified intraspecific variation in selective signals and significant changes to the amino acid property equilibrium constant, which may relate to electron transport chain efficiency. These results illustrate a complex dynamic of adaptive variation amongst O. princeps where lineages and populations have adapted to unique regional conditions. Some of the clearest signals of selection were in a genetic lineage that includes pikas of the Great Basin region, which is also where recent localised extirpations have taken place and highlights the risk of losing adaptive alleles during environmental change.

Fine mapping of RNA isoform diversity using an innovative targeted long-read RNA sequencing protocol with novel dedicated bioinformatics pipeline.

Solving the structure of mRNA transcripts is a major challenge for both research and molecular diagnostic purposes. Current approaches based on short-read RNA sequencing and RT-PCR techniques cannot fully explore the complexity of transcript structure. The emergence of third-generation long-read sequencing addresses this problem by solving this sequence directly. However, genes with low expression levels are difficult to study with the whole transcriptome sequencing approach. To fix this technical limitation, we propose a novel method to capture transcripts of a gene panel using a targeted enrichment approach suitable for Pacific Biosciences and Oxford Nanopore Technologies platforms. We designed a set of probes to capture transcripts of a panel of genes involved in hereditary breast and ovarian cancer syndrome. We present SOSTAR (iSofOrmS annoTAtoR), a versatile pipeline to assemble, quantify and annotate isoforms from long read sequencing using a new tool specially designed for this application. The significant enrichment of transcripts by our capture protocol, together with the SOSTAR annotation, allowed the identification of 1,231 unique transcripts within the gene panel from the eight patients sequenced. The structure of these transcripts was annotated with a resolution of one base relative to a reference transcript. All major alternative splicing events of the BRCA1 and BRCA2 genes described in the literature were found. Complex splicing events such as pseudoexons were correctly annotated. SOSTAR enabled the identification of abnormal transcripts in the positive controls. In addition, a case of unexplained inheritance in a family with a history of breast and ovarian cancer was solved by identifying an SVA retrotransposon in intron 13 of the BRCA1 gene. We have validated a new protocol for the enrichment of transcripts of interest using probes adapted to the ONT and PacBio platforms. This protocol allows a complete description of the alternative structures of transcripts, the estimation of their expression and the identification of aberrant transcripts in a single experiment. This proof-of-concept opens new possibilities for RNA structure exploration in both research and molecular diagnostics.

All tangled up: Unraveling phylogenetics and reticulate evolution in the vining ferns, Lygodium (Schizaeales).

Reticulate evolution, often accompanied by polyploidy, is prevalent in plants, and particularly in the ferns. Resolving the resulting non-bifurcating histories remains a major challenge for plant phylogenetics. Here, we present a phylogenomic investigation into the complex evolutionary history of the vining ferns, Lygodium (Lygodiaceae, Schizaeales). Using a targeted enrichment approach with the GoFlag 408 flagellate land plant probe set, we generated large nuclear and plastid sequence datasets for nearly all taxa in the genus and constructed the most comprehensive phylogeny of the family to date using concatenated maximum likelihood and coalescence approaches. We integrated this phylogeny with cytological and spore data to explore karyotype evolution and generate hypotheses about the origins of putative polyploids and hybrids. Our data and analyses support the origins of several putative allopolyploids (e.g., L. cubense, L. heterodoxum) and hybrids (e.g., L. ×fayae) and also highlight the potential prevalence of autopolyploidy in this clade (e.g., L. articulatum, L. flexuosum, and L. longifolium). Our robust phylogenetic framework provides valuable insights into dynamic reticulate evolution in this clade and demonstrates the utility of target-capture data for resolving these complex relationships.

Sea anemone (Anthozoa, Actiniaria) diversity in Mo’orea (French Polynesia)

Sea anemones (Order Actiniaria) are a diverse group of marine invertebrates ubiquitous across marine ecosystems. Despite their wide distribution and success, a knowledge gap persists in our understanding of their diversity within tropical systems, owed to sampling bias of larger and more charismatic species overshadowing cryptic lineages. This study aims to delineate the sea anemone diversity in Mo’orea (French Polynesia) with the use of a dataset from the Mo’orea Biocode’s “BioBlitz” initiative, which prioritized the sampling of more cryptic and understudied taxa. Implementing a target enrichment approach, we integrate 71 newly sequenced samples into an expansive phylogenetic framework and contextualize Mo’orea’s diversity within global distribution patterns of sea anemones. Our analysis corroborates the presence of several previously documented sea anemones in French Polynesia and identifies for the first time the occurrence of members of genera Andvakia and Aiptasiomorpha. This research unveils the diverse sea anemone ecosystem in Mo’orea, spotlighting the area’s ecological significance and emphasizing the need for continued exploration. Our methodology, encompassing a broad BLAST search coupled with phylogenetic analysis, proved to be a practical and effective approach for overcoming the limitations posed by the lack of comprehensive sequence data for sea anemones. We discuss the merits and limitations of current molecular methodologies and stress the importance of further research into lesser-studied marine organisms like sea anemones. Our work sets a precedent for future phylogenetic studies stemming from BioBlitz endeavors.

A proof of concept for a targeted enrichment approach to the simultaneous detection and characterization of rickettsial pathogens from clinical specimens.

Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities.

A target enrichment approach for enhanced recovery of Synchytrium endobioticum nuclear genome sequences.

Potato wart disease is caused by the obligate fungal pathogen Synchytrium endobioticum. DNA extraction from compost, purified spores and crude wart tissue derived from tuber galls of infected potatoes often results in low S. endobioticum DNA concentration or highly contaminated with DNA coming from other microorganisms and the potato host. Therefore, Illumina sequencing of these samples generally results in suboptimal recovery of the nuclear genome sequences of S. endobioticum. A hybridization-based target enrichment protocol was developed to strongly enhance the recovery of S. endobioticum DNA while off-target organisms DNA remains uncaptured. The design strategy involved creating a set of 180,000 molecular baits targeting both gene and non-gene regions of S. endobioticum. The baits were applied to whole genome amplified DNA samples of various S. endobioticum pathotypes (races) in compost, from purified spores and crude wart tissue samples. This was followed by Illumina sequencing and bioinformatic analyses. Compared to non-enriched samples, target enriched samples: 1) showed a significant increase in the proportion of sequenced bases mapped to the S. endobioticum nuclear genome, especially for crude wart tissue samples; 2) yielded sequencing data with higher and better nuclear genome coverage; 3) biased genome assembly towards S. endobioticum sequences, yielding smaller assembly sizes but higher representation of putative S. endobioticum contigs; 4) showed an increase in the number of S. endobioticum genes detected in the genome assemblies. Our hybridization-based target enrichment protocol offers a valuable tool for enhancing genome sequencing and NGS-based molecular detection of S. endobioticum, especially in difficult samples.

A combined phylogenetic strategy illuminates the evolution of Goniodorididae nudibranchs (Mollusca, Gastropoda, Heterobranchia)

Goniodorididae is a family of small dorid nudibranchs distributed worldwide that feed on entoprocts, ascidians, and bryozoans. The evolutionary relationships between its taxa have been uncertain due to the limited taxa available for phylogenetic analyses; some genera being paraphyletic. The family includes a remarkable number of synonymized genera in which the species richness is unequally distributed, while some genera have dozens of species others are monospecific. Some clades are very uniform morphologically while others are considered highly variable. To increase backbone phylogenetic resolution a target enrichment approach of ultra-conserved elements was aimed at representative Goniodorididae species for the first time. Additionally, we increase species representation by including mitochondrial markers cytochrome c oxidase subunit I and ribosomal RNA 16S as well as nuclear Histone 3 and ribosomal RNA 18S from 109 Goniodorididae species, out of approximately 160 currently valid species. Maximum likelihood and Bayesian inference analyses were performed to infer the phylogeny of the family. As a result, two subfamilies and eleven genera were elucidated. The synonymized genera Bermudella, Cargoa, and Ceratodoris are here resurrected and a new genus, Naisdoris gen. nov., is described. The clades included taxa with shared prey preference, showing that trophic behavior could have driven species evolution and morphological uniqueness within the family Goniodorididae.

A novel target-enriched multilocus assay for sponges (Porifera): Red Sea Haplosclerida (Demospongiae) as a test case.

With declining biodiversity worldwide, a better understanding of species diversity and their relationships is imperative for conservation and management efforts. Marine sponges are species-rich ecological key players on coral reefs, but their species diversity is still poorly understood. This is particularly true for the demosponge order Haplosclerida, whose systematic relationships are contentious due to the incongruencies between morphological and molecular phylogenetic hypotheses. The single gene markers applied in previous studies did not resolve these discrepancies. Hence, there is a high need for a genome-wide approach to derive a phylogenetically robust classification and understand this group's evolutionary relationships. To this end, we developed a target enrichment-based multilocus probe assay for the order Haplosclerida using transcriptomic data. This probe assay consists of 20,000 enrichment probes targeting 2956 ultraconserved elements in coding (i.e. exon) regions across the genome and was tested on 26 haplosclerid specimens from the Red Sea. Our target-enrichment approach correctly placed our samples in a well-supported phylogeny, in agreement with previous haplosclerid molecular phylogenies. Our results demonstrate the applicability of high-resolution genomic methods in a systematically complex marine invertebrate group and provide a promising approach for robust phylogenies of Haplosclerida. Subsequently, this will lead to biologically unambiguous taxonomic revisions, better interpretations of biological and ecological observations and new avenues for applied research, conservation and managing declining marine diversity.

A phylogeny of the evening primrose family (Onagraceae) using a target enrichment approach with 303 nuclear loci

BackgroundThe evening primrose family (Onagraceae) includes 664 species (803 taxa) with a center of diversity in the Americas, especially western North America. Ongoing research in Onagraceae includes exploring striking variation in floral morphology, scent composition, and breeding system, as well as the role of these traits in driving diversity among plants and their interacting pollinators and herbivores. However, these efforts are limited by the lack of a comprehensive, well-resolved phylogeny. Previous phylogenetic studies based on a few loci strongly support the monophyly of the family and the sister relationship of the two largest tribes but fail to resolve several key relationships.ResultsWe used a target enrichment approach to reconstruct the phylogeny of Onagraceae using 303 highly conserved, low-copy nuclear loci. We present a phylogeny for Onagraceae with 169 individuals representing 152 taxa sampled across the family, including extensive sampling within the largest tribe, Onagreae. Deep splits within the family are strongly supported, whereas relationships among closely related genera and species are characterized by extensive conflict among individual gene trees.ConclusionsThis phylogenetic resource will augment current research projects focused throughout the family in genomics, ecology, coevolutionary dynamics, biogeography, and the evolution of characters driving diversification in the family.

Patterns of speciation in a parapatric pair of Saturnia moths as revealed by target capture.

The focus of this study has been to understand the evolutionary relationships and taxonomy of a widely distributed parapatric species pair of wild silk moths in Europe: Saturnia pavonia and Saturnia pavoniella (Lepidoptera: Saturniidae). To address species delimitation in these parapatric taxa, target enrichment and mtDNA sequencing was employed alongside phylogenetic, admixture, introgression, and species delimitation analyses. The dataset included individuals from both species close to and farther away from the contact zone as well as two hybrids generated in the lab. Nuclear markers strongly supported both S. pavonia and S. pavoniella as two distinct species, with hybrids forming a sister group to S. pavoniella. However, the Maximum Likelihood (ML) tree generated from mtDNA sequencing data presented a different picture, showing both taxa to be phylogenetically intermixed. This inconsistency is likely attributable to mitonuclear discordance, which can arise from biological factors (e.g., introgressive hybridization and/or incomplete lineage sorting). Our analyses indicate that past introgressions have taken place, but that there is no evidence to suggest an ongoing admixture between the two species, demonstrating that the taxa have reached full postzygotic reproductive isolation and hence represent two distinct biological species. Finally, we discuss our results from an evolutionary point of view taking into consideration the past climatic oscillations that have likely shaped the present dynamics between the two species. Overall, our study demonstrates the effectiveness of the target enrichment approach in resolving shallow phylogenetic relationships under complex evolutionary circumstances and that this approach is useful in establishing robust and well-informed taxonomic delimitations involving parapatric taxa.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

Recovery of complete genome sequences of Crimean-Congo haemorrhagic fever virus (CCHFV) directly from clinical samples: A comparative study between targeted enrichment and metagenomic approaches

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, endemic to the Balkans, Africa, Middle East and Asia. There are currently no licensed vaccines or effective antivirals against CCHF. CCHF virus (CCHFV) has a negative sense segmented tripartite RNA genome consisting of the small (S), medium (M) and large (L) segments. Depending on the segment utilised for genetic affiliation, there are up to 7 circulating lineages of CCHFV. The current lack of geographical representation of CCHFV sequences in various repositories highlights a requirement for increased CCHFV sequencing capabilities in endemic regions. We have optimised and established a multiplex PCR tiling methodology for the targeted enrichment of complete genomes of Europe 1 CCHFV lineage directly from clinical samples and compared its performance to a non-targeted enrichment approach on both short-read and long-read sequencing platforms. We have found a statistically significant increase in mapped viral sequencing reads produced with our targeted enrichment approach. This has allowed us to recover near complete S segment sequences and above 90% of the M and L segment sequences for samples with Ct values as high as 31.3. This study demonstrates the superiority of a targeted enrichment approach for recovery of CCHFV genomic sequences from samples with low virus titre. CCHFV is an important vector-borne human pathogen with wide geographical distribution. The validated methodology reported here adds value to front-line public health laboratories employing genomic sequencing for CCHFV Europe 1 lineage surveillance, particularly in the Balkan and Middle Eastern territories currently monitoring the spread of the pathogen. Tracking the genomic evolution of the virus across regions improves risk assessment and directly informs the development of diagnostics, therapeutics, and vaccines.

A hybridization target enrichment approach for pathogen genomics.

Emerging infectious diseases require continuous pathogen monitoring. Rapid clinical diagnosis by nucleic acid amplification is limited to a small number of targets and may miss target detection due to new mutations in clinical isolates. Whole-genome sequencing (WGS) identifies genome-wide variations that may be used to determine a pathogen's drug resistance patterns and phylogenetically characterize isolates to track disease origin and transmission. WGS is typically performed using DNA isolated from cultured clinical isolates. Culturing clinical specimens increases turn-around time and may not be possible for fastidious bacteria. To overcome some of these limitations, direct sequencing of clinical specimens has been attempted using expensive capture probes to enrich the entire genomes of target pathogens. We present a method to produce a cost-effective, time-efficient, and large-scale synthesis of probes for whole-genome enrichment. We envision that our method can be used for direct clinical sequencing of a wide range of microbial pathogens for genomic epidemiology.

Highly multiplexed targeted sequencing strategy for infectious disease surveillance

BackgroundGlobal efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).ResultsOur approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.ConclusionsOverall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.

New Records of the Cryptogenic Soft Coral Genus Stragulum (Tubiporidae) from the Eastern Caribbean and the Persian Gulf

The monotypic soft coral genus Stragulum van Ofwegen and Haddad, 2011 (Octocorallia: Malacalcyonacea: Tubiporidae) was originally described from Brazil, southwest Atlantic Ocean. Here, we report the first records of the genus from the eastern Caribbean and the Persian Gulf in the northwest Indian Ocean. We compare the morphological features of specimens, together with molecular data from three commonly used barcoding markers (COI, mtMutS, 28S rDNA) and 308 ultraconserved elements (UCE) and exon loci sequenced using a target-enrichment approach. The molecular and morphological data together suggest that specimens from all three localities are the same species, i.e., Stragulum bicolor van Ofwegen and Haddad, 2011. It is still not possible to establish the native range of the species or determine whether it may be an introduced species due to the limited number of specimens included in this study. However, the lack of historical records, its fouling abilities on artificial substrates, and a growing number of observations support the invasive nature of the species in Brazilian and Caribbean waters and therefore suggest that it may have been introduced into the Atlantic from elsewhere. Interestingly, the species has not shown any invasive behaviour in the Persian Gulf, where it has been found only on natural, rocky substrates. The aim of the present report is to create awareness of this taxon with the hope that this will lead to new records from other localities and help to establish its native range.

A first complete phylogenomic hypothesis for diploid blueberries (Vaccinium section Cyanococcus).

The true blueberries (Vaccinium sect. Cyanococcus; Ericaceae), endemic to North America, have been intensively studied for over a century. However, with species estimates ranging from nine to 24 and much confusion regarding species boundaries, this ecologically and economically valuable group remains inadequately understood at a basic evolutionary and taxonomic level. As a first step toward understanding the evolutionary history and taxonomy of this species complex, we present the first phylogenomic hypothesis of the known diploid blueberries. We used flow cytometry to verify the ploidy of putative diploid taxa and a target-enrichment approach to obtain a genomic data set for phylogenetic analyses. Despite evidence of gene flow, we found that a primary phylogenetic signal is present. Monophyly for all morphospecies was recovered, with two notable exceptions: one sample of V. boreale was consistently nested in the V. myrtilloides clade and V. caesariense was nested in the V. fuscatum clade. One diploid taxon, Vaccinium pallidum, is implicated as having a homoploid hybrid origin. This foundational study represents the first attempt to elucidate evolutionary relationships of the true blueberries of North America with a phylogenomic approach and sets the stage for multiple avenues of future study such as a taxonomic revision of the group, the verification of a homoploid hybrid taxon, and the study of polyploid lineages within the context of a diploid phylogeny.

Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells.

The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.

The genetic mechanisms underlying the convergent evolution of pollination syndromes in the Neotropical radiation of Costus L.

Selection together with variation in floral traits can act to mold floral form, often driven by a plant’s predominant or most effective pollinators. To investigate the evolution of traits associated with pollination, we developed a phylogenetic framework for evaluating tempo and mode of pollination shifts across the genus Costus L., known for its evolutionary toggle between traits related to bee and bird pollination. Using a target enrichment approach, we obtained 957 loci for 171 accessions to expand the phylogenetic sampling of Neotropical Costus. In addition, we performed whole genome resequencing for a subset of 20 closely related species with contrasting pollination syndromes. For each of these 20 genomes, a high-quality assembled transcriptome was used as reference for consensus calling of candidate loci hypothesized to be associated with pollination-related traits of interest. To test for the role these candidate genes may play in evolutionary shifts in pollinators, signatures of selection were estimated as dN/dS across the identified candidate loci. We obtained a well-resolved phylogeny for Neotropical Costus despite conflict among gene trees that provide evidence of incomplete lineage sorting and/or reticulation. The overall topology and the network of genome-wide single nucleotide polymorphisms (SNPs) indicate that multiple shifts in pollination strategy have occurred across Costus, while also suggesting the presence of previously undetected signatures of hybridization between distantly related taxa. Traits related to pollination syndromes are strongly correlated and have been gained and lost in concert several times throughout the evolution of the genus. The presence of bract appendages is correlated with two traits associated with defenses against herbivory. Although labellum shape is strongly correlated with overall pollination syndrome, we found no significant impact of labellum shape on diversification rates. Evidence suggests an interplay of pollination success with other selective pressures shaping the evolution of the Costus inflorescence. Although most of the loci used for phylogenetic inference appear to be under purifying selection, many candidate genes associated with functional traits show evidence of being under positive selection. Together these results indicate an interplay of phylogenetic history with adaptive evolution leading to the diversification of pollination-associated traits in Neotropical Costus.

An integrative taxonomic revision and redefinition of Gephyromantis (Laurentomantis) malagasius based on archival DNA analysis reveals four new mantellid frog species from Madagascar

The subgenus Laurentomantis in the genusGephyromantiscontains some of the least known amphibian species of Madagascar. The six currently valid nominal species are rainforest frogs known from few individuals, hampering a full understanding of the species diversity of the clade. We assembled data on specimens collected during field surveys over the past 30 years and integrated analysis of mitochondrial and nuclear-encoded genes of 88 individuals, a comprehensive bioacoustic analysis, and morphological comparisons to delimit a minimum of nine species-level lineages in the subgenus. To clarify the identity of the speciesGephyromantis malagasius, we applied a target-enrichment approach to a sample of the 110 year-old holotype ofMicrophryne malagasiaMethuen and Hewitt, 1913 to assign this specimen to a lineage based on a mitochondrial DNA barcode. The holotype clustered unambiguously with specimens previously namedG. ventrimaculatus. Consequently we propose to considerTrachymantis malagasia ventrimaculatusAngel, 1935 as a junior synonym ofGephyromantis malagasius. Due to this redefinition ofG. malagasius, no scientific name is available for any of the four deep lineages of frogs previously subsumed under this name, all characterized by red color ventrally on the hindlimbs. These are here formally named asGephyromantis fiharimpesp. nov.,G. matsilosp. nov.,G. oelkrugisp. nov., andG. portonaesp. nov.The new species are distinguishable from each other by genetic divergences of >4% uncorrected pairwise distance in a fragment of the 16S rRNA marker and a combination of morphological and bioacoustic characters.Gephyromantis fiharimpeandG. matsilooccur, respectively, at mid-elevations and lower elevations along a wide stretch of Madagascar’s eastern rainforest band, whileG. oelkrugiandG. portonaeappear to be more range-restricted in parts of Madagascar’s North East and Northern Central East regions. Open taxonomic questions surroundG. horridus, to which we here assign specimens from Montagne d’Ambre and the type locality Nosy Be; andG. ranjomavo, which contains genetically divergent populations from Marojejy, Tsaratanana, and Ampotsidy.

Unraveling Structural Rearrangements of the CFH Gene Cluster in Atypical Hemolytic Uremic Syndrome Patients Using Molecular Combing and Long-Fragment Targeted Sequencing

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.