Search Results (31)

Background: The enhanced sensitivity of next-generation sequencing (NGS) for assessing hepatitis B virus (HBV) quasispecies heterogeneity over clone-based sequencing (CBS) is well documented. However, its comparative reliability for genotype determination remains an open question. Objective: This study aimed to directly compare the performance of NGS and CBS for genotyping HBV using the entire viral genome. Methods: We selected five challenging clinical samples that previously could not be subgenotyped or showed conflicting results when using direct sequencing of the S open reading frame (ORF). The full HBV genome from these subjects was amplified and then analyzed in parallel by both NGS and CBS. Phylogenetic analysis was subsequently used to assign genotypes. Results: Both methods identified a range of genotypes, including B, C, and I, as well as aberrant and recombinant forms. For three of the five subjects, genotyping results were identical between the two platforms. In the remaining two cases, however, CBS revealed greater complexity, identifying additional subgenotypes and recombinant/aberrant strains not detected by NGS. Notably, for three individuals, the genotypes determined by both modern methods contradicted earlier results from 2011 based on direct S ORF sequencing. Furthermore, the specific mutations detected were incongruent between the platforms, with CBS identifying a higher number of variants than NGS. Conclusions: Our findings indicate that genotyping results from NGS and CBS can be discordant. Contrary to expectations, CBS may uncover more genetic diversity, including a greater number of subgenotypes and mutations, than NGS in certain contexts. The study also confirms that genotyping based solely on direct sequencing of the S ORF can be unreliable and lead to misclassification. Full article

Background: Helicobacter pylori is associated with a wide range of gastroduodenal diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. Eradication efforts are challenged by increasing antimicrobial resistance rates, particularly in Southeast Asia. We sequenced the whole genomes of clinical H. pylori isolates from Southern Thailand to elucidate their resistance profiles, virulence determinants, and evolutionary relationships. Methods: Three clinical H. pylori isolates (004, 117, and 189) were subjected to whole-genome sequencing, phenotypic antimicrobial susceptibility testing, and comparative genomic analyses. Results: All strains exhibited high-level resistance to metronidazole. Additionally, H. pylori 117 was resistant to both amoxicillin and levofloxacin, classifying it as multidrug-resistant. Genomic analysis revealed mutations in rdxA, frxA, and rpoB, as well as in penicillin-binding protein genes (pbp2 and pbp3), supporting the phenotypic findings. While all isolates harboured clarithromycin resistance mutations (A2142G and A2143G in the 23S rRNA gene), they were phenotypically susceptible, highlighting a potential discordance that requires further investigation. Virulence gene profiling identified 115–118 conserved genes per strain, including cagA, vacA, oipA, babA, and flagellar, urease, and lipopolysaccharide biosynthesis genes. Phylogenetic analysis using core-genome single-nucleotide polymorphisms demonstrated that these strains formed a distinct Southern Thai monophyletic clade, suggesting localised clonal expansion driven by regional selective pressures. Conclusions: Region-specific surveillance strategies and treatment guidelines are urgently needed in Thailand. The combination of high-risk virulence genes and rising antimicrobial resistance in H. pylori strains necessitates tailored therapeutic approaches, the integration of genomic surveillance into clinical diagnostics, and expanded studies linking genotype to clinical outcomes in diverse populations. Full article

Porcine reproductive and respiratory syndrome virus remains one of the most economically significant pathogens in swine production, with PRRSV-2 being the dominant variant in the United States. While lineage classification has traditionally relied on ORF5 sequencing, recent studies suggest that this single-gene approach may overlook key evolutionary events such as recombination. In this study, we performed whole-genome sequencing and phylogenetic analysis of seven PRRSV-2 isolates collected in the U.S. between 2006 and 2024. Using reference-guided assembly, lineage assignment, and recombination detection with RDP5 and SIMplot, we identified discordant phylogenetic placements between ORF5 and whole genomes in four of the seven isolates. These discordances were explained by multiple recombination events affecting different genomic regions, particularly ORF2–ORF7. In contrast, three isolates showed phylogenetic concordance and no strong evidence of recombination. Our findings demonstrate that recombination plays a significant role in shaping PRRSV-2 evolution and highlight the limitations of ORF5-based lineage classification. Whole-genome surveillance is therefore essential to accurately track viral diversity, detect recombinant strains, and inform control strategies. This work underscores the need for a broader adoption of full-genome analysis in routine PRRSV surveillance and research. Full article

Species characterized by undetermined clade affiliations, limited research coverage, and deficient systematic investigation serve as enigmatic entities in plant and animal taxonomy, yet hold critical significance for exploring phylogenetic relationships and evolutionary trajectories. Subgenus Cyathophora (Allium, Amayllidaceae), a small taxon comprising approximately five species distributed in the Qinghai–Tibet Plateau (QTP) and adjacent regions might contain an enigmatic species that has long remained unexplored. In this study, we collected data on species from subgenus Cyathophora and its close relatives in subgenus Rhizirideum, as well as the enigmatic species Allium siphonanthum. Combining phylogenomic datasets and morphological evidence, we investigated species relationships and the underlying mechanism of phylogenetic discordance. A total of 1662 single-copy genes (SCGs) and 150 plastid loci were filtered and used for phylogenetic analyses based on concatenated and coalescent-based methods. Furthermore, to systematically evaluate phylogenetic discordance and decipher its underlying drivers, we implemented integrative analyses using multiple approaches, such as coalescent simulation, Quartet Sampling (QS), and MSCquartets. Our phylogenetic analyses robustly resolve A. siphonanthum as a member of subg. Cyathophora, forming a sister clade with A. spicatum. This relationship was further corroborated by their shared morphological characteristics. Despite the robust phylogenies inferred, extensive phylogenetic conflicts were detected not only among gene trees but also between SCGs and plastid-derived species trees. These significant phylogenetic incongruences in subg. Cyathophora predominantly stem from incomplete lineage sorting (ILS) and reticulate evolutionary processes, with historical hybridization events likely correlated with the past orogenic dynamics and paleoclimatic oscillations in the QTP and adjacent regions. Our findings not only provide new insights into the phylogeny of subg. Cyathophora but also significantly enhance our understanding of the evolution of species in this subgenus. Full article

Ancient introgression is an infrequent evolutionary process often associated with conflicts between nuclear and organellar phylogenies. Determining whether such conflicts arise from introgression, incomplete lineage sorting (ILS), or other processes is essential to understanding plant diversification. Previous studies have reported phylogenetic discordance in the placement of Xanthoceras, but its causes remain unclear. Here, we analyzed transcriptome data from 41 Sapindaceae samples to reconstruct phylogenies and investigate this discordance. While nuclear phylogenies consistently placed Xanthoceras as sister to subfam. Hippocastanoideae, plastid data positioned it as the earliest-diverging lineage within Sapindaceae. Our coalescent simulations suggest that this cyto-nuclear discordance is unlikely to be explained by ILS alone. HyDe and PhyloNet analyses provided strong evidence that Xanthoceras experienced ancient introgression, incorporating approximately 16% of its genetic material from ancestral subfam. Sapindoideae lineages. Morphological traits further support this evolutionary history, reflecting characteristics of both contributing subfamilies. Likely occurring during the Paleogene, this introgression represents a rare instance of cross-subfamily gene flow shaping the evolutionary trajectory of a major plant lineage. Our findings clarify the evolutionary history of Xanthoceras and underscore the role of ancient introgression in driving phylogenetic conflicts, offering a rare example of introgression-driven diversification in angiosperms. Full article

Camphora Fabr. is a genus in the family Lauraceae, comprising over 20 tropical and subtropical tree species. Since the genera Camphora and Cinnamomum Schaeff. were described, there has been a long-lasting controversy regarding the phylogenetic relationships among taxa in both genera. In particular, phylogenetic inferences derived from plastid data remain debated, with varying hypotheses proposed and occasional disputes concerning the monophyly of Camphora taxa. To further investigate the relationships, We analyzed plastomes and nuclear ribosomal cistron sequences (nrDNA) of 22 Camphora taxa, 15 Cinnamomum taxa, and 13 representative taxa of related genera. The Camphora plastomes range from 152,745 to 154,190 bp, with a GC content of 39.1% to 39.2%. A total of 128 genes were identified in the Camphora plastomes, including 84 protein-coding genes, 8 rRNA genes, and 36 tRNA genes. A total of 1130 SSR loci were detected from plastomes of Camphora, and A/T base repeats looked like the most common. Comparative analyses revealed that the plastomes of Camphora exhibit high similarity in overall structure. The loci ycf1, ycf2, trnK (UUU), psbJ-psbL, and ccsA-ndhD were identified as candidate DNA barcodes for these taxa. Plastome phylogenetic analysis revealed that Camphora is not monophyletic, whereas the nrDNA dataset supported the monophyly of Camphora. We propose that intergeneric hybridization may underlie the observed discordance between plastid and nuclear data in Camphora, and we recommend enhanced taxonomic sampling and precise species identification to improve phylogenetic resolution and accuracy. Full article

The genus Pourthiaea Decne., a deciduous woody group with high ornamental value, belongs to the family Rosaceae. Here, we reported newly sequenced plastid genome sequences of Pourthiaea beauverdiana (C. K. Schneid.) Hatus., Pourthiaea parvifolia E. Pritz., Pourthiaea villosa (Thunb.) Decne., and Photinia glomerata Rehder & E. H. Wilson. The plastomes of these three Pourthiaea species shared the typical quadripartite structures, ranging in size from 159,903 bp (P. parvifolia) to 160,090 bp (P. beauverdiana). The three Pourthiaea plastomes contained a pair of inverted repeat regions (26,394–26,399 bp), separated by a small single-copy region (19,304–19,322 bp) and a large single-copy region (87,811–87,973 bp). A total of 113 unique genes were predicted for the three Pourthiaea plastomes, including four ribosomal RNA genes, 30 transfer RNA genes, and 79 protein-coding genes. Analyses of inverted repeat/single-copy boundary, mVISTA, nucleotide diversity, and genetic distance showed that the plastomes of 13 Pourthiaea species (including 10 published plastomes) are highly conserved. The number of simple sequence repeats and long repeat sequences is similar among 13 Pourthiaea species. The three non-coding regions (trnT-GGU-psbD, trnR-UCU-atpA, and trnH-GUG-psbA) were the most divergent. Only one plastid protein-coding gene, rbcL, was under positive selection. Phylogenetic analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences strongly supported the monophyly of Pourthiaea. As for the relationship with other genera in our phylogenies, Pourthiaea was sister to Malus in plastome phylogenies, while it was sister to the remaining genera in nrDNA phylogenies. Furthermore, significant cytonuclear discordance likely stems from hybridization events within Pourthiaea, reflecting complex evolutionary dynamics within the genus. Our study provides valuable genetic insights for further phylogenetic, taxonomic, and species delimitation studies in Pourthiaea, as well as essential support for horticultural improvement and conservation of the germplasm resources. Full article

The genus Rorippa belongs to the family Brassicaceae, and its members usually have high medicinal value. The genus consists of approximately 75 species and mainly grows in the Northern Hemisphere, occurring in every continent except Antarctica. The taxonomy and phylogenetic relationships of Rorippa are still unsettled, largely due to complex morphological variations in Rorippa, which were caused by frequent hybridization events. Here, we sequenced four complete plastid genomes of Rorippa species by Illumina paired-end sequencing. The four new plastid genomes of Rorippa ranged in total size from 154,671 bp for R. palustris to 154,894 bp for R. sylvestris. There are 130 genes in the four plastomes, embodying 8 rRNA, 37 tRNA, and 85 protein-coding genes. Combining with six published plastid genomes, we carried on comparative and phylogenetic analyses. We found that the ten Rorippa plastid genomes were conservative in gene number and order, total size, genomic structure, codon usage, long repeat sequence, and SSR. Fourteen mutational hotspot regions could be selected as candidate DNA barcoding to distinguish Rorippa plants. The phylogenetic trees clearly identified that ten Rorippa species displayed monophyletic relationships within the tribe Cardamineae based on plastomes and nrDNA ITS sequences. However, there are significant cytonuclear discordances in the interspecific relationships within Rorippa, as well as the intergeneric relationships between Rorippa and its related genera. We inferred that the cytonuclear discordance is most likely a result of interspecific hybridization within Rorippa, as well as intergeneric hybridization with its related genera. These plastid genomes can offer precious information for studies of species authentication, evolutionary history, and the phylogeny of Rorippa. Full article

Evergreen sclerophyllous oak forests (ESOFs) in southwestern China are a special vegetation type developed in response to the expansion of arid habitats after the uplift of the Himalayas. Here, we used chloroplast (cp) DNA and nuclear ribosomal (nr) DNA to investigate the fine-scale genetic variation patterns of six sympatric oaks (Quercus, Fagaceae) in the hot-dry valley ESOFs of the Jinsha River, southwestern China. Three cp genomes were assembled for each species. Nine cp genome haplotypes and 16 nrDNA haplotypes were identified based on single-nucleotide variants and indels. Our results demonstrated that discordance existed between the cpDNA and nrDNA phylogenies of the sclerophyllous oaks in section Ilex. The nrDNA phylogeny was consistent with species boundaries, while the cpDNA phylogeny was decoupled from taxonomy. Interspecific sharing of closely related cp genome haplotypes was detected between Quercus cocciferoides and the other two sclerophyllous oaks, Q. longispica and Q. franchetii. Specifically, Q. cocciferoides and Q. longispica sampled in a mixed stand exhibited two haplotypes that differed by a 9 bp indel. The local distribution of the two highly similar haplotypes suggested that they may have arisen from ancient introgression. Given that the two species have diverged for a long time, it is possible that the ancestral cp genome of one species was captured by another species through asymmetric introgression in early times, and an indel event occurred subsequently. Phylogenetic analyses using more previously published cp genome sequences indicated that Q. cocciferoides and Q. franchetii shared multiple cpDNA lineages of Ilex oaks, which may be caused by shared ancestral polymorphism and/or ancient introgression. Our study showed that at least three highly variable regions (ψycf1, ndhF-rpl32, and trnK^UUU-rps16 or rpl32-trnL^UAG) can distinguish the nine haplotypes identified by whole-cp genome sequences. These markers are useful for the evolutionary studies of the maternal lineages of oaks in hot-dry valley ESOFs. Full article

A range-wide phylogenetic/phylogeographic study of the three-toed jerboas of the genus Stylodipus is conducted using the mitochondrial cytb gene and fragments of several nuclear genes. The genus has been believed to include three species: S. telum (W Central Asia, SE Europe), S. andrewsi (E Central Asia), and S. sungorus (Dzungar basin). Our data support the dichotomy between S. andrewsi and the other taxa forming S. telum species group. Within the latter, both mtDNA and nuclear loci indicate a species-level divergence between S. telum and the S. t. birulae lineage (Zaisan depression, NE Kazakhstan), previously considered a subspecies of S. telum and here elevated to full species. S. sungorus is recovered as a close sister group to S. birulae on the basis of nuclear data but clustered with S. telum in the mitochondrial tree. The latter taxon is the most variable and includes two closely related eastern and western sublineages, separated by the Volga-Ural sands and joined by a more divergent S. t. karelini lineage (E Kazakhstan). The observed mitonuclear discordance is hypothesized to occur due to mtDNA introgression resulting from hybridization between S. sungorus and S. t. karelini, which highlights the important role of reticulations in the evolution of Dipodidae. Full article

Marattiaceae is a phylogenetically isolated family of tropical eusporangiate ferns including six genera with more than one-hundred species. In Marattiaceae, monophyly of genera has been well-supported phylogenetically. However, the phylogenetic relationships among them were elusive and controversial. Here, a dataset of 26 transcriptomes (including 11 newly generated) were used to assess single-copy nuclear genes and to obtain the organelle gene sequences. Through phylotranscriptomic analysis, the phylogeny and hybridization events of Marattiaceae were explored and a robust phylogenomic framework for the evolution of Marattiaceae was provided. Using both concatenation- and coalescent-based phylogenies, the gene-tree discordance, incomplete lineage sorting (ILS) simulations, and network inference were examined. Except the low support with mitochondrial genes of Marattiaceae, nuclear genes and chloroplast genes strongly supported a sister relationship between Marattiaceae and leptosporangiate ferns. At the genus level, all phylogenetic analysis based on nuclear genes datasets recovered five genera in Marattiaceae as monophyletic with strong support. Danaea and Ptisana were the first two diverged clades in turn. Christensenia was a sister clade to the clade Marattia + Angiopteris s.l. In Angiopteris s.l., three clades (Angiopteris s.s., the Archangiopteris group, and An. sparsisora) were well identified with maximum support. The Archangiopteris group was derived from Angiopteris s.s. at ca. 18 Ma. The putative hybrid species An. sparsisora between Angiopteris s.s. and the Archangiopteris group was verified by the species network analyses and the maternal plastid genes. This study will improve our understanding for using the phylotranscriptomic method to explore phylogeny and investigate hybridization events for difficult taxa in ferns. Full article

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely. Full article

Plastids and mitochondria are organelles of plant cells with small genomes, which may exhibit discordant microevolution as we earlier revealed in pea crop wild relatives. We sequenced 22 plastid and mitochondrial genomes of Pisum sativum subsp. elatius and Pisum fulvum using Illumina platform, so that the updated sample comprised 64 accessions. Most wild peas from continental southern Europe and a single specimen from Morocco were found to share the same organellar genome constitution; four others, presumably hybrid constitutions, were revealed in Mediterranean islands and Athos Peninsula. A mitochondrial genome closely related to that of Pisum abyssinicum, from Yemen and Ethiopia, was unexpectedly found in an accession of P. sativum subsp. elatius from Israel, their plastid genomes being unrelated. Phylogenetic reconstructions based on plastid and mitochondrial genomes revealed different sets of wild peas to be most related to cultivated P. sativum subsp. sativum, making its wild progenitor and its origin area enigmatic. An accession of P. fulvum representing ‘fulvum-b’ branch, according to a nuclear marker, appeared in the same branch as other fulvum accessions in organellar trees. The results stress the complicated evolution and structure of genetic diversity of pea crop wild relatives. Full article

A recent field inventory focusing on catfishes of the trichomycterine genus Cambeva detected the occurrence of two morphotypes, C. barbosae and C. cubataonis, in the Guaratuba-Babitonga-Itapocu area (GBIA) of southern Brazil, reporting some discordance with results of coalescent-based approaches for species delimitation that indicated different estimates of species number. Contrastingly, based on examination of characters taken from the external morphology and osteology, we here recognised six species of Cambeva in GBIA: C. cf. botuvera, a polymorphic and geographically widespread species; C. cubataonis, endemic to the Rio Cubatão do Norte; and four new species, two endemic to the Rio Itapocu basin, one endemic to the Baía de Babitonga system and one endemic to the Baía de Guaratuba system. We performed a molecular phylogenetic analysis indicating that Cambeva comprises three major clades, the alpha-, beta- and gama-clades, with C. cf. botuvera and a clade comprising C. cubataonis and three new species belonging to the beta-clade and another new species belonging to the gama-clade. We concluded that species here recognised are not in fact incongruent with results of that recent study when taxa are correctly identified by a representative sample of morphological characters, highlighting the importance of osteological characters for delimiting trichomycterine species. Full article

Juglans mandshurica has strong freezing resistance, surviving temperatures as low as −40 °C, making it an important freeze tolerant germplasm resource of the genus Juglans. APETALA2/ethylene responsive factor (AP2/ERF) is a plant-specific superfamily of transcription factors that regulates plant development, growth, and the response to biotic and abiotic stress. In this study, phylogenetic analysis was used to identify 184 AP2/ERF genes in the J. mandshurica genome, which were classified into five subfamilies (JmAP2, JmRAV, JmSoloist, JmDREB, and JmERF). A significant amount of discordance was observed in the 184 AP2/ERF genes distribution of J. mandshurica throughout its 16 chromosomes. Duplication was found in 14 tandem and 122 segmental gene pairs, which indicated that duplications may be the main reason for JmAP2/ERF family expansion. Gene structural analysis revealed that 64 JmAP2/ERF genes contained introns. Gene evolution analysis among Juglandaceae revealed that J. mandshurica is separated by 14.23 and 15 Mya from Juglans regia and Carya cathayensis, respectively. Based on promoter analysis in J. mandshurica, many cis-acting elements were discovered that are related to light, hormones, tissues, and stress response processes. Proteins that may contribute to cold resistance were selected for further analysis and were used to construct a cold regulatory network based on GO annotation and JmAP2/ERF protein interaction network analysis. Expression profiling using qRT-PCR showed that 14 JmAP2/ERF genes were involved in cold resistance, and that seven and five genes were significantly upregulated under cold stress in female flower buds and phloem tissues, respectively. This study provides new light on the role of the JmAP2/ERF gene in cold stress response, paving the way for further functional validation of JmAP2/ERF TFs and their application in the genetic improvement of Juglans and other tree species. Full article