Phylogenomic Analysis of Target Enrichment and Transcriptome Data Uncovers Rapid Radiation and Extensive Hybridization in Slipper Orchid Genus Cypripedium L.

BACKGROUND AND AIMS: Cypripedium is the most widespread and morphologically diverse genus of slipper orchids. Despite several published phylogenies, the topology and monophyly of its infrageneric taxa remained uncertain. Here, we aimed to reconstruct a robust section-level phylogeny of Cypripedium and explore its evolutionary history using target capture data for the first time. METHODS: We used the orchid-specific bait set Orchidaceae963 in combination with transcriptomic data to reconstruct the phylogeny of Cypripedium based on 913 nuclear loci, covering all 13 sections. Subsequently, we investigated discordance among nuclear and chloroplast trees, estimated divergence times and ancestral ranges, searched for anomaly zones, polytomies, and diversification rate shifts, and identified potential gene (genome) duplication and hybridization events. KEY RESULTS: All sections were recovered as monophyletic, contrary to the two subsections within sect. Cypripedium. The two subclades within this section did not correspond to its subsections but matched the geographic distribution of their species. Additionally, we discovered high levels of discordance in the short backbone branches of the genus and within sect. Cypripedium, which can be attributed to hybridization events detected based on phylogenetic network analyses, and incomplete lineage sorting caused by rapid radiation. Our biogeographic analysis suggested a Neotropical origin of the genus during the Oligocene (~30 Ma), with a lineage of potentially hybrid origin spreading to the Old World in the Early Miocene (~22 Ma). The rapid radiation at the backbone likely occurred in Southeast Asia around the Middle Miocene Climatic Transition (~15-13 Ma), followed by several independent dispersals back to the New World. Moreover, the Pliocene-Quaternary glacial cycles may have contributed to further speciation and reticulate evolution within Cypripedium. CONCLUSIONS: Our study provided novel insights into the evolutionary history of Cypripedium based on high-throughput molecular data, shedding light on the dynamics of its distribution and diversity patterns from its origin to the present.

Exploring waterborne viruses in groundwater: Quantification and Virome characterization via passive sampling and targeted enrichment sequencing.

Aquifers, which provide drinking water for nearly half the world's population, face significant challenges from microbial contamination, particularly from waterborne viruses such as human adenovirus (HAdV), norovirus (NoV) and enterovirus (EV). This study, conducted as part of the UPWATER project, investigates the sources of urban groundwater contamination using viral passive sampling (VPS) and target enrichment sequencing (TES). We assessed the abundance of eight viral pathogens (HAdV, EV, NoV genogroup I and II, rotavirus, influenza A virus, hepatitis E virus and SARS-CoV-2) and investigated the virome diversity of groundwater in the aquifer of the Besòs River Delta in Catalonia. Over a period of 7 months, we collected 114 samples from the aquifer using nylon and nitrocellulose membranes to adsorb viruses over a 10-day period. Human faecal contamination was detected in nearly 50 % of the groundwater samples, with mean HAdV total counts ranging from 1.23E+02 to 3.66E+03 GC, and occasional detections of EV and NoV GI and GII. In addition, deep sequencing revealed a diverse virome in the aquifer, with detection of human pathogens, including adenovirus, astrovirus, calicivirus, enterovirus, herpesvirus, papillomavirus and rotavirus. Time-integrated sampling using VPS increases the likelihood of virus detection and, when combined with TES, can provide a deeper understanding of virus prevalence in this important water compartment. This approach is expected to streamline long-term monitoring efforts and enable small communities or water managers with limited resources to effectively manage their groundwater reservoirs.

The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice.

Adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes integrate into the host genome, but the theoretical risk of tumorigenesis depends on vector regulatory features. A mouse model was used to investigate integration profiles of an AAV serotype 5 (AAV5) vector produced using Sf and HEK293 cells that mimic key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads were derived from episomes, and mean (± standard deviation) integration frequency was 2.70 ± 1.26 and 1.79 ± 0.86 integrations per 1,000 cells for Sf- and HEK293-produced vector. Longitudinal integration analysis suggested integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal and randomly distributed. No major differences in integration profiles were observed for either vector production platform, and no integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver (p = 1 × 10-4) and less enriched for genes of lower expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.

Efficient enrichment of free target sequences in an integrated microfluidic device for point-of-care detection systems.

Nucleic acid biomarker detection has great importance in the diagnosis of disease, the monitoring of disease progression and the classification of patients according to treatment decision making. Nucleic acid biomarkers found in the blood of patients have generated a lot of interest due to the possibility of being detected non-invasively which makes them ideal for monitoring and screening tests and particularly amenable to point-of-care (POC) or self-testing. A major challenge to POC molecular diagnostics is the need to enrich the target to optimise detection. In this work, we describe a microfabricated device for the enrichment of short dsDNA target sequences, which is especially valuable for potential detection methods, as it improves the probability of effectively detecting the target in downstream analyses. The device integrated a heating element and a temperature sensor with a microfluidic chamber to carry out the denaturation of the dsDNA combined with blocking-probes to enrich the target. This procedure was validated by fluorescence resonance energy transfer (FRET) technique, labelling DNA with a fluorophore and a quencher. As proof of concept, a 23-mer long dsDNA sequence corresponding to the L858R mutation of the EGFR gene was used. The qualitative results obtained determined that the most optimal blocking rate was obtained with the incorporation of 11/12-mer blocking-probes at a total concentration of 6 µM. This device is a powerful DNA preparation tool, which is an indispensable initial step for subsequent detection of sequences via nucleic acid hybridisation methods.

Complex but Clear Allopolyploid Pattern of Subtribe Tussilagininae (Asteraceae: Senecioneae) Revealed by Robust Phylogenomic Evidence, with Development of a Novel Homeolog-Sorting Pipeline.

Polyploidy is a significant mechanism in eukaryotic evolution and is particularly prevalent in the plant kingdom. However, our knowledge about this phenomenon and its effects on evolution remains limited. A major obstacle to the study of polyploidy is the great difficulty in untangling the origins of allopolyploids. Due to the drastic genome changes and the erosion of allopolyploidy signals caused by the combined effects of hybridization and complex post-polyploid diploidization processes, resolving the origins of allopolyploids has long been a challenging task. Here we revisit this issue with the interesting case of subtribe Tussilagininae (Asteraceae: Senecioneae) and by developing HomeoSorter, a new pipeline for network inferences by phasing homeologs to parental subgenomes. The pipeline is based on the basic idea of a previous study but with major changes to address the scaling problem and implement some new functions. With simulated data, we demonstrate that HomeoSorter works efficiently on genome-scale data and has high accuracy in identifying polyploid patterns and assigning homeologs. Using HomeoSorter, the maximum pseudo-likelihood model of Phylonet, and genome-scale data, we further address the complex origin of Tussilagininae, a speciose group (ca. 45 genera and 710 species) characterized by having high base chromosome numbers (mainly x = 30, 40). In particular, the inferred patterns are strongly supported by the chromosomal evidence. Tussilagininae is revealed to comprise two large groups with successive allopolyploid origins: Tussilagininae s.s. (mainly x = 30) and the Gynoxyoid group (x = 40). Two allopolyploidy events first give rise to Tussilagininae s.s., with the first event occurring between the ancestor of subtribe Senecioninae (x = 10) and a lineage (highly probably with x = 10) related to the Brachyglottis alliance, and the resulting hybrid lineage crossing with the ancestor of Chersodoma (x = 10) and leading to Tussilagininae s.s. Then, after early diversification, the Central American group (mainly x = 30) of Tussilagininae s.s., is involved in a third allopolyploidy event with, again, the Chersodoma lineage and produces the Gynoxyoid group. Our study highlights the value of HomeoSorter and the homeolog-sorting approach in polyploid phylogenetics. With rich species diversity and clear evolutionary patterns, Tussilagininae s.s. and the Gynoxyoid group are also excellent models for future investigations of polyploidy.

GeCKO: user-friendly workflows for genotyping complex genomes using target enrichment capture. A use case on the large tetraploid durum wheat genome.

BACKGROUND: Genotyping of individuals plays a pivotal role in various biological analyses, with technology choice influenced by multiple factors including genomic constraints, number of targeted loci and individuals, cost considerations, and the ease of sample preparation and data processing. Target enrichment capture of specific polymorphic regions has emerged as a flexible and cost-effective genomic reduction method for genotyping, especially adapted to the case of very large genomes. However, this approach necessitates complex bioinformatics treatment to extract genotyping data from raw reads. Existing workflows predominantly cater to phylogenetic inference, leaving a gap in user-friendly tools for genotyping analysis based on capture methods. In response to these challenges, we have developed GeCKO (Genotyping Complexity Knocked-Out). To assess the effectiveness of combining target enrichment capture with GeCKO, we conducted a case study on durum wheat domestication history, involving sequencing, processing, and analyzing variants in four relevant durum wheat groups. RESULTS: GeCKO encompasses four distinct workflows, each designed for specific steps of genomic data processing: (i) read demultiplexing and trimming for data cleaning, (ii) read mapping to align sequences to a reference genome, (iii) variant calling to identify genetic variants, and (iv) variant filtering. Each workflow in GeCKO can be easily configured and is executable across diverse computational environments. The workflows generate comprehensive HTML reports including key summary statistics and illustrative graphs, ensuring traceable, reproducible results and facilitating straightforward quality assessment. A specific innovation within GeCKO is its 'targeted remapping' feature, specifically designed for efficient treatment of targeted enrichment capture data. This process consists of extracting reads mapped to the targeted regions, constructing a smaller sub-reference genome, and remapping the reads to this sub-reference, thereby enhancing the efficiency of subsequent steps. CONCLUSIONS: The case study results showed the expected intra-group diversity and inter-group differentiation levels, confirming the method's effectiveness for genotyping and analyzing genetic diversity in species with complex genomes. GeCKO streamlined the data processing, significantly improving computational performance and efficiency. The targeted remapping enabled straightforward SNP calling in durum wheat, a task otherwise complicated by the species' large genome size. This illustrates its potential applications in various biological research contexts.

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.

Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach.

Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus-host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus's genetic landscape.

Benefits and Limits of Phasing Alleles for Network Inference of Allopolyploid Complexes.

Accurately reconstructing the reticulate histories of polyploids remains a central challenge for understanding plant evolution. Although phylogenetic networks can provide insights into relationships among polyploid lineages, inferring networks may be hindered by the complexities of homology determination in polyploid taxa. We use simulations to show that phasing alleles from allopolyploid individuals can improve phylogenetic network inference under the multispecies coalescent by obtaining the true network with fewer loci compared to haplotype consensus sequences or sequences with heterozygous bases represented as ambiguity codes. Phased allelic data can also improve divergence time estimates for networks, which is helpful for evaluating allopolyploid speciation hypotheses and proposing mechanisms of speciation. To achieve these outcomes in empirical data, we present a novel pipeline that leverages a recently developed phasing algorithm to reliably phase alleles from polyploids. This pipeline is especially appropriate for target enrichment data, where depth of coverage is typically high enough to phase entire loci. We provide an empirical example in the North American Dryopteris fern complex that demonstrates insights from phased data as well as the challenges of network inference. We establish that our pipeline (PATÉ: Phased Alleles from Target Enrichment data) is capable of recovering a high proportion of phased loci from both diploids and polyploids. These data may improve network estimates compared to using haplotype consensus assemblies by accurately inferring the direction of gene flow, but statistical non-identifiability of phylogenetic networks poses a barrier to inferring the evolutionary history of reticulate complexes.

A new nuclear phylogeny of the tea family (Theaceae) unravels rapid radiations in genus Camellia.

Molecular analyses of rapidly radiating groups often reveal incongruence between gene trees. This mainly results from incomplete lineage sorting, introgression, and gene tree estimation error, which complicate the estimation of phylogenetic relationships. In this study, we reconstruct the phylogeny of Theaceae using 348 nuclear loci from 68 individuals and two outgroup taxa. Sequence data were obtained by target enrichment using the recently released Angiosperm 353 universal probe set applied to herbarium specimens. The robustness of the topologies to variation in data quality was established under a range of different filtering schemes, using both coalescent and concatenation approaches. Our results confirmed most of the previously hypothesized relationships among tribes and genera, while clarifying additional interspecific relationships within the rapidly radiating genus Camellia. We recovered a remarkably high degree of gene tree heterogeneity indicative of rapid radiation in the group and observed cytonuclear conflicts, especially within Camellia. This was especially pronounced around short branches, which we primarily associate with gene tree estimation error. Our analysis also indicates that incomplete lineage sorting (ILS) contributed to gene-tree conflicts and accounted for approximately 14 % of the explained variation, whereas inferred introgression levels were low. Our study advances the understanding of the evolution of this important plant family and provides guidance on the application of target capture methods and the evaluation of key processes that influence phylogenetic discordances.

Divergence and reticulation in the Mexican white oaks: ecological and phylogenomic evidence on species limits and phylogenetic networks in the Quercus laeta complex (Fagaceae).

BACKGROUND AND AIMS: Introgressive hybridization poses a challenge to taxonomic and phylogenetic understanding of taxa, particularly when there are high numbers of co-occurring, intercrossable species. The genus Quercus exemplifies this situation. Oaks are highly diverse in sympatry and cross freely, creating syngameons of interfertile species. Although a well-resolved, dated phylogeny is available for the American oak clade, evolutionary relationships within many of the more recently derived clades remain to be defined, particularly for the young and exceptionally diverse Mexican white oak clade. Here, we adopted an approach bridging micro- and macroevolutionary scales to resolve evolutionary relationships in a rapidly diversifying clade endemic to Mexico. METHODS: Ecological data and sequences of 155 low-copy nuclear genes were used to identify distinct lineages within the Quercus laeta complex. Concatenated and coalescent approaches were used to assess the phylogenetic placement of these lineages relative to the Mexican white oak clade. Phylogenetic network methods were applied to evaluate the timing and genomic significance of recent or historical introgression among lineages. KEY RESULTS: The Q. laeta complex comprises six well-supported lineages, each restricted geographically and with mostly divergent climatic niches. Species trees corroborated that the different lineages are more closely related to other species of Mexican white oaks than to each other, suggesting that this complex is polyphyletic. Phylogenetic networks estimated events of ancient introgression that involved the ancestors of three present-day Q. laeta lineages. CONCLUSIONS: The Q. laeta complex is a morphologically and ecologically related group of species rather than a clade. Currently, oak phylogenetics is at a turning point, at which it is necessary to integrate phylogenetics and ecology in broad regional samples to figure out species boundaries. Our study illuminates one of the more complicated of the Mexican white oak groups and lays groundwork for further taxonomic study.

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches.

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing.

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa.

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.

Deqformer: high-definition and scalable deep learning probe design method.

Target enrichment sequencing techniques are gaining widespread use in the field of genomics, prized for their economic efficiency and swift processing times. However, their success depends on the performance of probes and the evenness of sequencing depth among each probe. To accurately predict probe coverage depth, a model called Deqformer is proposed in this study. Deqformer utilizes the oligonucleotides sequence of each probe, drawing inspiration from Watson-Crick base pairing and incorporating two BERT encoders to capture the underlying information from the forward and reverse probe strands, respectively. The encoded data are combined with a feed-forward network to make precise predictions of sequencing depth. The performance of Deqformer is evaluated on four different datasets: SNP panel with 38 200 probes, lncRNA panel with 2000 probes, synthetic panel with 5899 probes and HD-Marker panel for Yesso scallop with 11 000 probes. The SNP and synthetic panels achieve impressive factor 3 of accuracy (F3acc) of 96.24% and 99.66% in 5-fold cross-validation. F3acc rates of over 87.33% and 72.56% are obtained when training on the SNP panel and evaluating performance on the lncRNA and HD-Marker datasets, respectively. Our analysis reveals that Deqformer effectively captures hybridization patterns, making it robust for accurate predictions in various scenarios. Deqformer leads to a novel perspective for probe design pipeline, aiming to enhance efficiency and effectiveness in probe design tasks.

Compositae-ParaLoss-1272: A complementary sunflower-specific probe set reduces paralogs in phylogenomic analyses of complex systems.

Premise: A family-specific probe set for sunflowers, Compositae-1061, enables family-wide phylogenomic studies and investigations at lower taxonomic levels, but may lack resolution at genus to species levels, especially in groups complicated by polyploidy and hybridization. Methods: We developed a Hyb-Seq probe set, Compositae-ParaLoss-1272, that targets orthologous loci in Asteraceae. We tested its efficiency across the family by simulating target enrichment sequencing in silico. Additionally, we tested its effectiveness at lower taxonomic levels in the historically complex genus Packera. We performed Hyb-Seq with Compositae-ParaLoss-1272 for 19 Packera taxa that were previously studied using Compositae-1061. The resulting sequences from each probe set, plus a combination of both, were used to generate phylogenies, compare topologies, and assess node support. Results: We report that Compositae-ParaLoss-1272 captured loci across all tested Asteraceae members, had less gene tree discordance, and retained longer loci than Compositae-1061. Most notably, Compositae-ParaLoss-1272 recovered substantially fewer paralogous sequences than Compositae-1061, with only ~5% of the recovered loci reporting as paralogous, compared to ~59% with Compositae-1061. Discussion: Given the complexity of plant evolutionary histories, assigning orthology for phylogenomic analyses will continue to be challenging. However, we anticipate Compositae-ParaLoss-1272 will provide improved resolution and utility for studies of complex groups and lower taxonomic levels in the sunflower family.

Mitochondrial genome structure and composition in 70 fishes: a key resource for fisheries management in the South Atlantic.

BACKGROUND: Phylogenetic gaps of public databases of reference sequences are a major obstacle for comparative genomics and management of marine resources, particularly in the Global South, where economically important fisheries and conservation flagship species often lack closely-related references. We applied target-enrichment to obtain complete mitochondrial genomes of marine ichthyofauna from the Brazilian coast selected based on economic significance, conservation status and lack of phylogenetically-close references. These included sardines (Dorosomatidae, Alosidae), mackerels (Scombridae) croakers (Sciaenidae), groupers (Epinephelidae) and snappers (Lutjanidae). RESULTS: Custom baits were designed to enrich mitochondrial DNA across a broad phylogenetic range of fishes. Sequencing generated approximately 100k reads per sample, which were assembled in a total of 70 complete mitochondrial genomes and include fifty-two new additions to GenBank, including five species with no previous mitochondrial data. Departures from the typical gene content and order occurred in only three taxa and mostly involved tRNA gene duplications. Start-codons for all genes, except Cytochrome C Oxidase subunit I (COI), were consistently ATG, whilst a wide range of stop-codons deviated from the prevailing TAA. Phylogenetic analysis confirmed assembly accuracy and revealed signs of cryptic diversification within the Mullus genus. Lineage delimitation methods using Sardinella aurita and S. brasiliensis mitochondrial genomes support a single Operational Taxonomic Unit. CONCLUSIONS: Target enrichment was highly efficient, providing complete novel mitochondrial genomes with little sequencing effort. These sequences are deposited in public databases to enable subsequent studies in population genetics and adaptation of Latin American fish species and serve as a vital resource for conservation and management programs that rely on molecular data for species and genus-level identification.

A new genus of soft coral (Octocorallia, Malacalcyonacea, Cladiellidae) and three new species from Indo-Pacific coral reefs.

Molecular systematic studies of the anthozoan class Octocorallia have revealed widespread incongruence between phylogenetic relationships and taxonomic classification at all levels of the Linnean hierarchy. Among the soft coral taxa in order Malacalcyonacea, the family Alcyoniidae and its type genus Alcyonium have both been recognised to be highly polyphyletic. A recent family-level revision of Octocorallia established a number of new families for genera formerly considered to belong to Alcyoniidae, but revision of Alcyonium is not yet complete. Previous molecular studies have supported the placement of Alcyoniumverseveldti (Benayahu, 1982) in family Cladiellidae rather than Alcyoniidae, phylogenetically distinct from the other three genera in that family. Here we describe a new genus, Ofwegenumgen. nov. to accommodate O.verseveldticomb. nov. and three new species of that genus, O.coronalucissp. nov., O.kloogisp. nov., and O.collisp. nov., bringing the total number of species in this genus to four. Ofwegenumgen. nov. is a rarely encountered genus so far known from only a few locations spanning the Indian and western Pacific Oceans. We present the morphological characters of each species and use molecular data from both DNA barcoding and target-enrichment of conserved elements to explore species boundaries and phylogenetic relationships within the genus.

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.

Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya.

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as the double-stranded RNA Hubei chryso-like virus 1. Both viruses were found in the same Anopheles spp. homogenate extracted from a sample that showed CPE on both VeroB4 and C6/36 cells. The detection of both viruses in a single mosquito homogenate indicated coinfection. Phylogenetic analyses suggested that the Culex flavivirus sequence detected was closely related to a Culex flavivirus isolated from Uganda in 2008. All four Hubei chryso-like virus 1 segments clusters closely to Hubei chryso-like virus 1 strains isolated in Australia, China and USA. Two novel strains of insect-specific viruses in Anopheles mosquitoes were detected and characterized.