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It is widely accepted in prokaryotic systematics that a 95–96% ANI (average nucleotide identity) value is equivalent to 70% dDDH (digital DNA–DNA hybridization) value in prokaryotic systematics. However, we recently found that a 70% dDDH value was equivalent to an approximately 96.7% ANIm value in the genus Micromonospora based on a correlation analysis between dDDH and ANIm from a total of 1770 pairs of type Micromonospora strains (60 type strains). Therefore, we proposed that 96.7% ANIm (ANI based on the MUMmer algorithm) value could act as the threshold value in delineating Micromonospora species. Meanwhile, the taxonomic status of an actinobacterial strain HUAS LYJ1^T, isolated from the rhizosphere soil of Camellia oleifera, was determined by using a polyphasic method. A 16S rRNA gene sequence analysis indicated that strain HUAS LYJ1^T shared the highest similarity to Micromonospora wenchangensis CCTCC AA 2012002^T (99.3%). Phylogenetic trees based on 16S rRNA gene and whole-genome sequences demonstrated that strain HUAS LYJ1^T was most closely related to M. wenchangensis CCTCC AA 2012002^T. However, the ANIm value between them was 95.77%, below the 96.7% cut-off point recommended above; the dDDH value between them was 63.2%, also far below the 70% threshold value in delineating bacterial species. Based on these molecular data, as well as phenotypic and chemotaxonomical features, it is concluded that strain HUAS LYJ1^T represents a novel Micromonospora species, for which the name Micromonospora cynarisoli sp. nov. is proposed. In addition, it was found that the ANIm and dDDH values of Micromonospora haikouensis DSM 45626^T, Micromonospora harpali NEAU-JC6^T and Micromonospora oryzae DSM 102119^T were 97.21–97.86% and dDDH 73.9–79.6%, respectively, above the 96.6% ANIm and 70% dDDH threshold value in delineating Micromonospora species. Consequently, according to rule 42 of the International Committee on Systematics of Prokaryote Code, we propose that M. harpali Fang et al. 2015 and M. oryzae Kittiwongwattana et al. 2015 are later heterotypic synonyms of M. haikouensis Xie et al. 2012. Full article

This work introduces an approach to evolutionary analysis in which information encoded in amino-acid sequences is converted into a specific type of image, termed a genetic image. Genetic images derived from the amino-acid sequences of cytochrome b and cytochrome c oxidase subunit I are shown to be suitable for identifying evolutionary similarities between organisms. Furthermore, artificial neural networks are demonstrated to recognize these genetic images, enabling identification of species evolution. The results indicate the similarity of the genetic images of organisms belonging to species that emerged earlier during Earth’s evolutionary history to the genetic images of organisms belonging to species that emerged later. This finding indicates that genetic images are inherited and undergo gradual modification during evolutionary processes. The phenomenon of inheritance and modification of genetic images suggests that evolution tends to change the already existing functionalities of organisms, which allows for the ordering of organisms belonging to different species from ancient forms, through species that appeared successively during evolution, to those belonging to species that have developed more recently, up to Homo sapiens. Moreover, unlike analyses based on phylogenetic trees, the method presented in this article does not require computing hypothetical taxonomic units to study evolution. Combined with analyses of the inheritance of genetic images, it can support the interpretations of phylogenetic trees and evolutionary research. Full article

The ‘Tenuifoliae irises’ are a distinctive group of beardless, rhizomatous perennial irises, which are characterised by their somewhat vertical rhizomes, typically clothed at the apex with long maroon-brown, sharp fibrous remains of leaf sheaths; perianth tube long, filiform to scapiform; stigma bilobed; capsules often trigonous to six-ribbed, apically beaked; and seeds angulose to subcubic or pyriform, lacking fleshy appendages, and with testa hard, irregularly wrinkled. The representatives of the aggregate are mostly native to the dry steppes and grasslands from lowland to high mountain habitats of Central and Eastern Asia, extending westwards to the Black Sea and Caspian regions. Morphological classification of the ‘Tenuifoliae irises’ recognises about ten to eleven species, which are arranged into two genera, Sclerosiphon and Cryptobasis. Diverse molecular research recovered members of the ‘Tenuifoliae irises’ in contrasting placements within the ‘Iris-flower clade’. Sometimes, Sclerosiphon was sister to Eremiris, but Cryptobasis aligned with the ‘Spuria irises’ (Chamaeiris) and the ‘Spanish irises’ (Xiphion and related genera); in other cases, both Sclerosiphon and Cryptobasis formed a clade sister to Chamaeiris, or Cryptobasis alone was identified as the basal member of the Iris s.l. clade, positioned immediately after Siphonostylis. To examine these taxonomic discrepancies within a rigorous molecular systematic framework and using 12 reliably authenticated specimens, we generated 24 sequences of the matK gene (12) and the trnL (UAA)–trnF (GAA) loci (12) from members of the ‘Tenuifoliae irises’. These sequences were subsequently incorporated into a comprehensive dataset of the ‘Iris-flower clade’, enabling a broader analytical assessment. The obtained three-taxon statement hierarchy of patterns and maximum likelihood phylogenetic trees both recover the ‘Tenuifoliae irises’ as monophyletic and sister to Chamaeiris, and in turn to the ‘Xiphion s.l. clade’. We also found Sclerosiphon and Cryptobasis as sister genera. The morphological and karyological data supporting those relationships are discussed, which allow getting back to Rodionenko’s sources and recovering Sclerosiphon in his original sense, alongside Cryptobasis. Furthermore, the molecular results allow us to expand Sclerosiphon to include the Eastern Chinese members of the aggregate. In consequence, five new combinations (one series and four species) are established in the genus, one lectotype is designated, and data on nomenclature, distribution and ecology of the accepted species are reported. Full article

Background: Pangolins are critically endangered mammals, and a comprehensive understanding of their genetic diversity is crucial for effective conservation. The mitochondrial genome serves as a vital molecular marker for phylogenetic and population genetic studies. Obtaining genetic material from these elusive animals non-invasively remains a challenge. This study aimed to sequence and characterize the complete mitochondrial genome of Manis javanica and explore the phylogenetic relationships among pangolin species. Methods: The complete mitochondrial genome was sequenced from a saliva-derived sample. Standard procedures for DNA extraction, amplification, and sequencing were employed. The genome was assembled and annotated using bioinformatic tools. Phylogenetic analysis was conducted based on the cytochrome c oxidase subunit I (COXI) gene sequences from nine pangolin species, with the resulting tree constructed using the maximum-likelihood method. Results: The complete mitochondrial genome of M. javanica (GenBank accession: PP110760) is a circular molecule of 16,573 bp, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The overall base composition showed a lower GC content (43.83%) than AT content (56.17%). Phylogenetic analysis based on COXI sequences delineated the nine species into three distinct genera: Manis, Phataginus, and Smutsia. Within the genus Manis, Manis pentadactyla was identified as the closest relative to M. javanica. The newly described species Manis mysteria was found to be closer to Manis culionensis and Manis crassicaudata than to other congeners. Furthermore, the analysis indicated that African pangolins diverged earlier than Asian pangolins. Conclusions: This study successfully demonstrates the feasibility of extracting and sequencing the complete mitochondrial genome from saliva samples, providing a valuable non-invasive method for future genetic studies on pangolins. The genomic data and phylogenetic results offer significant molecular insights that will benefit the genetic management and conservation of critically endangered pangolin resources. Full article

Avian reovirus (ARV) causes viral arthritis and leads to considerable economic losses in the poultry industry. In this study, six ARV strains of distinct genotypes (FJNP01–FJNP06) were isolated from commercial broiler farms. Through gene sequencing and pathogenicity assessment, we analyzed the genetic evolution and pathogenic characteristics of the σC, P10, σB, μB, and λC genes. Pathogenicity tests revealed that inoculation with FJNP01–FJNP06 by footpad or oral gavage induced symptoms in specific-pathogen-free (SPF) chickens, including mortality and growth retardation. Among the isolates, FJNP04 (genotype IV) showed the highest pathogenicity, causing increased mortality, weight loss, and severe lesions in the footpads and bursa of Fabricius, followed by FJNP05 and FJNP02. The pathogenicity of FJNP06 varied by inoculation route, with enhanced pathogenicity observed following oral gavage. In contrast, FJNP01 and FJNP03 demonstrated relatively low pathogenicity. Identity analysis indicated that σC and P10 were highly variable, σB was relatively conserved, while μB and λC displayed considerable divergence. Phylogenetic analysis placed FJNP01–FJNP06 into genotypes I to VI, respectively, forming six distinct branches on the σC and P10 phylogenetic trees, yet clustering more closely on the σB, μB, and λC trees. The pathogenicity of different genotypes of ARV varies, among which FJNP04 (genotype IV) exhibits the strongest pathogenicity. Genetic sequence analysis revealed that σC and P10 are highly variable, σB is relatively conserved, while μB and λC display a wide range of variation. This study provides insights into the genetic variation and pathogenic characteristics of ARV and serves as a reference for future research. Full article

The genus Acanthosaura is characterized by a high level of cryptic species diversity and is subdivided into several species complexes. The phylogenetic relationships within the A. coronata complex remain unresolved due to the presence of cryptic lineages and limited molecular data for several species. In this study, these relationships are clarified using a molecular genetic analysis that integrates newly collected field samples and historical museum specimens with previously uncertain identification. Three mitochondrial genes (cyt b, COI, and ND2) from samples, including fresh collections of A. murphyi from Phu Yen Province (Vietnam) and museum specimens from Vietnam and Myanmar, were analyzed. In addition, morphological characters of the examined specimens with diagnostic traits of known species were compared. Phylogenetic analyses confirmed the distinct species status of A. murphyi and enabled the taxonomic reassignment of previously undetermined museum specimens to this species. Specimens from Vietnam and Myanmar formed a single, well-supported clade, suggesting a broader distribution for A. murphyi than previously recognized. It is demonstrated for the first time that A. murphyi belongs to the A. coronata complex, together with A. coronata and A. cuongi, a result consistently supported by both genetic distances and phylogenetic tree topology. Full article

Objective: In this study, the heat shock transcription factor (HSF) gene family in Ganoderma lucidum was systematically characterized. Using genomic and transcriptomic data, we identified HSF family members and investigated their expression patterns under temperature stress and their potential regulatory roles in triterpenoid biosynthesis. Methods: A genome-wide identification of HSF genes in G. lucidum was performed using bioinformatic approaches. A phylogenetic tree was constructed, and conserved motifs, gene structures, and protein tertiary structures were predicted. The relative expression levels of HSF genes and key mevalonate (MVA) pathway enzyme genes were examined by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in mycelia subjected to temperature stress. Total triterpenoid content in fermented mycelia under temperature stress was determined using the vanillin–glacial acetic acid method. Results: Eight HSF family members (GlHSF1–GlHSF8) were identified in G. lucidum. Phylogenetic analysis revealed that GlHSF proteins were closely related to PoHSF from Pleurotus ostreatus. Transcriptomic analysis showed that HSF genes exhibited relatively high expression levels during the mature stage while being barely expressed during the mycelial stage. Under heat stress (42 °C), most GlHSF genes peaked at 18 h, with GlHSF2 showing the most pronounced response (approximately 13-fold upregulation). Downstream MVA pathway genes, including IDI, PMK, and MVD, were significantly upregulated at 24 h, whereas the upstream rate-limiting enzyme gene HMGR was continuously suppressed. Despite HMGR suppression, total triterpenoid content did not decrease significantly, likely due to the activation of downstream genes. Under cold stress (14 °C), the expression of most GlHSF and MVA pathway genes decreased, accompanied by a significant reduction in total triterpenoid content. Conclusions: The HSF gene family was identified in the G. lucidum genome. Based on expression analysis, GlHSF2 showed the strongest response under heat stress, and its expression peak was correlated with the sequential activation of downstream genes in the MVA pathway. This suggests that GlHSF2 acts as a potential key regulatory node, differentially regulating upstream and downstream MVA pathway genes to influence triterpenoid biosynthesis under heat stress. These findings provide a theoretical basis for future research on the biological functions of GlHSF homeostasis. Full article

Ipê trees (Bignoniaceae), mainly belonging to the genus Handroanthus, are widely used in urban landscaping and reforestation programs in Brazil. Anthracnose, typically associated with species of Colletotrichum, represents one of the major diseases affecting ipê seedlings and ornamental trees. However, the etiological agents involved have not yet been fully clarified using modern phylogenetic tools. In this study, we identified Colletotrichum species associated with anthracnose in ipê trees from Pernambuco, Brazil. A total of 22 isolates were obtained from symptomatic leaves of Handroanthus impetiginosus and H. chrysotrichus. Species identification was based on multilocus phylogenetic analyses using CAL, GAPDH, GS, and TUB2 loci. The isolates were assigned to three species: Colletotrichum siamense, C. tropicale, and C. karsti. Colletotrichum siamense was the most prevalent species (50%), followed by C. tropicale (36.3%), while C. karsti represented 13.7% of the isolates. Pathogenicity tests confirmed that all isolates were pathogenic to both ipê species, producing typical anthracnose symptoms. Aggressiveness differed between hosts, with H. impetiginosus showing higher susceptibility, as indicated by larger lesion development, whereas H. chrysotrichus exhibited lower disease aggressiveness. Thus, our findings represent the first multilocus-based identification of Colletotrichum species causing anthracnose in ipê trees, providing new insights into the diversity and epidemiology of this disease in urban environments. Full article

The plant mitochondrial genome has become a current research hotspot as an independent genetic model. Nevertheless, mitochondrial genome information for most Dendrobium species remains unknown. In this study, the assembly of mitochondrial genome of Dendrobium nobile Lindl.,1830 and Dendrobium denneanum Kerr., 1933 was conducted through the application of second- and third-generation sequencing technologies, with the mitochondrial genome of D. denneanum Kerr. being reported first. The results revealed that the mitochondrial genomes of the two species possessed a multi-chromosome circular structure. Their total lengths were 641,414 bp and 558,760 bp, consisting of 21 and 19 contigs, respectively. A total of 67 and 72 genes, 993 and 1491 repeat sequences, and 549 and 553 RNA editing sites were identified. Gene loss was observed. A total of 26 and 36 homologous fragments were detected between the mitochondrial and the chloroplast genome, accounting for 5.09% and 4.93% of the total lengths, respectively, indicating intracellular gene transfer. Synteny and phylogenetic analyses revealed that the two species shared extensive collinear regions and clustered together in a distinct clade of the phylogenetic tree, indicating a close sister relationship. These findings enrich the mitochondrial genome database and provide valuable insights to guide future research on species identification and molecular evolution of the genus Dendrobium. Full article

Avian orthoavulavirus 1 (AOaV-1), commonly known as Newcastle disease virus (NDV), despite widespread vaccination, remains a significant threat to domestic chickens (Gallus gallus domesticus). Currently available live-attenuated NDV vaccines are derived from genotypes I and II lentogenic strains, whereas genetically divergent velogenic strains predominantly caused recent NDV outbreaks. This study examined the extent of genotypic divergence between NDV vaccine strains and field strains using phylogenetic and multivariate analyses of two major antigenic and virulence-associated genes: fusion (F) and haemagglutinin-neuraminidase (HN). A total of 121 full-length NDV-F and 81 NDV-HN gene sequences, representing reported NDV genotypes, were downloaded from GenBank and analysed using maximum-likelihood (ML) phylogenetic trees and principal coordinates analysis (PCoA). The phylogeny revealed genotype-specific clustering for both genes, consistent with current NDV classification. NDV vaccine strains belonging to genotypes I and II formed distinct clades, segregated from the majority of NDV field strains, including velogenic or virulent NDV genotypes. The principal coordinates analysis of both genes further confirmed the phylogenetic clustering of NDV genotypes, indicating increased genomic heterogeneity. These findings suggest genetic segregation of divergent velogenic or virulent genotypes from lentogenic NDV vaccines, requiring biological experiments for determining their efficacy against field strains. This study highlights the importance of molecular surveillance of NDV to monitor its genomic diversity, which is crucial for developing strategies to combat NDV outbreaks in domestic chickens. This study provides an updated, NDV-glycoprotein-gene-based comparative analysis across reported NDV genotypes using phylogenetic and multivariate approaches. Full article

Background: Elaeagnus oxycarpa Schltdl. (Elaeagnaceae) is a vital sand-fixing tree species in the arid, semi-arid, and desert regions of China, holding significant ecological and economic value. However, its chloroplast (cp) genome has not been previously characterized. Results: In this study, we sequenced the complete cp genome of Elaeagnus oxycarpa using Illumina high-throughput sequencing technology. We performed de novo assembly, annotation, and comparative genomic and phylogenetic analyses with six other Elaeagnaceae species. The results revealed a typical quadripartite structure for the E. oxycarpa cp genome, with a total length of 150,567 bp and a GC content of 36.90%. Annotation identified 132 genes, comprising 86 protein-coding genes (PCGs), 38 tRNA genes, and 8 rRNA genes. Codon usage bias analysis indicated a preference for A/U endings, with leucine codons being the most frequent (9.5%). Additionally, 77 simple sequence repeat (SSR) loci were detected, predominantly mononucleotide repeats (71.4%). Comparative genomic analysis demonstrated high sequence conservation among the seven Elaeagnus species, with nucleotide variations primarily concentrated in non-coding regions and intergenic spacers of genes such as rps16, ycf1, and trnC-GCA. These variable regions and SSR loci represent valuable molecular markers for future population genetics and species identification studies on Elaeagnus. Phylogenetic analysis strongly supported the notion that E. oxycarpa and Elaeagnus angustifolia form a sister clade, indicating their close genetic relationship. Conclusions: Our findings provide crucial genomic resources and a theoretical foundation for the species identification and elucidation of the evolutionary history of Elaeagnaceae. Full article

Background/Objectives: Castanopsis tibetana Hance (C. tibetana) is a valuable timber species in southern China. Its chloroplast and nuclear genomes have been characterized, but its mitochondrial genome (mitogenome) remains unknown. This study assembles and characterizes the first complete mitogenome of C. tibetana, elucidating its structural and evolutionary features. Methods: A hybrid approach combining Oxford Nanopore long reads and Illumina short reads was used. The mitogenome was assembled via iterative seed-based mapping and annotated via GeSeq and tRNAscan-SE. Repeats were identified via MISA, TRF, and REPuter. The RNA editing sites were predicted with the PREP suite. Phylogenetic analysis was performed on 14 conserved protein-coding genes from 13 species via maximum likelihood and Bayesian inference. Results: The mitogenome is a 554,078 bp circular molecule (GC 45.27%) encoding 51 genes (32 PCGs, 16 tRNAs, 3 rRNAs). It contains 202 simple sequence repeats (37.1% tetrameric). We predicted 53 C-to-U RNA editing sites, most frequently in nad7 and nad5. Codon usage showed bias, with 28 codons having RSCU > 1. Twenty fragments (6001 bp, 1.08% of the mitogenome) were transferred from the chloroplast. Phylogenomic analysis placed C. tibetana within Fagaceae, close to other Castanopsis species. Conclusions: This study provides the first comprehensive characterization of the C. tibetana mitogenome, revealing its structural architecture, repetitive landscape, RNA editing profile, and phylogenetic placement. These findings offer valuable genomic resources for understanding mitogenome evolution in Fagaceae and support future research on the conservation genetics and molecular breeding of this important tree species. Full article

Codon usage bias (CUB) is a universal feature in both mitochondrial and ribosomal genes, shaped by the combined forces of mutation and selection, and serves as a valuable indicator of evolutionary processes and phylogenetic signals. However, comprehensive analysis of CUB is lacking in the Leuciscinae family. We sequenced and annotated the complete mitochondrial genome of Abramis brama orientalis and examined codon usage patterns in all Leuciscinae species, subsequently finding the dominant evolutionary forces and phylogenetic relationships. We performed a PR2 bias plot, neutrality plot, effective number of codons (ENC) vs. GC3, relative synonymous codon use (RSCU) clustering and Ka/Ks calculations for 22 Leuciscinae species. We constructed the phylogenetic trees of Leuciscinae using Bayesian inference and maximum likelihood on concatenated mitochondrial sequences. The complete mitochondrial genome of Abramis brama orientalis was 16,607 bp, with typical vertebrate structure and high A + T bias. The codons of protein-coding genes in Leuciscinae have a preference for ending in A/C. All protein-coding genes were under strong purifying selection (Ka/Ks < 1). RSCU patterns and phylogenetic analyses showed two lines of Leuciscinae in the RSCU, with A. brama orientalis being a monophyletic group with A. brama. The results demonstrate the strong role for selection in shaping mitochondrial codon usage in Leuciscinae, despite mutational biases. The study clarified the taxonomic status of A. brama orientalis and provided a framework for understanding molecular evolution in this ecologically important freshwater fish family. Full article

In ruminant nutrition, the lignocellulosic complex is a primary constraint limiting the utilization of dietary fiber. The objective of this study was to evaluate the effects of inoculating lignin-degrading bacteria (LDB) isolated from the ruminant rectum on in vitro rumen fermentation characteristics. Rectal fecal samples were collected from healthy beef cattle, dairy cattle, buffaloes, and goats (n = 4 per species) using the grab sampling technique. Twenty-eight bacterial colonies were isolated through enrichment and screening on media containing sodium lignosulfonate. Lignin degradation efficiency was assessed spectrophotometrically, while laccase activity was determined using a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation assay. Seven isolates exhibiting ligninolytic activity (1.4–5.6% degradation efficiency) were selected to evaluate their effects on in vitro rumen fermentation using a completely randomized design with four replicates. LDB treatments were standardized to a concentration of 2.4 × 10⁵ colony-forming units/mL of rumen fluid medium, while the control received an equal volume of a 0.85% sterile NaCl solution. A rice straw-based total mixed ration served as the substrate, with rumen fluid collected from beef cattle. All treatments were incubated for 48 h. Notably, isolate BC3 consistently enhanced in vitro dry matter digestibility (23.1%), total gas production (18.6%), and total volatile fatty acid concentrations (13.2%) relative to the control and other LDB isolates (p < 0.01). All seven LDB isolates were identified as Gram-negative, rod-shaped, facultative anaerobic bacteria that exhibit catalase activity and tolerate moderately acidic conditions. Phylogenetic tree analysis based on 16S rRNA gene sequencing identified isolate BC3 as being closely related to Escherichia coli strains. These findings demonstrate that the ruminant hindgut is a promising source of LDB with the functional potential to enhance feed digestibility and fermentation end-products in the rumen. Future research should prioritize in vivo trials to evaluate the safety and efficacy of LDB as a direct-fed microbial, specifically focusing on its impact on animal performance and health. Full article

Avocado (Persea americana Mill.) is one of the most widely consumed fruit tree crops worldwide, with cultivation expanding rapidly beyond its Mesoamerican and northwest South America center of origin. In emerging secondary diversity centers such as East Africa, farmers have long propagated seedling naturalized populations that may hold valuable reservoirs of genetic diversity, yet these resources remain underexplored. To help fill this gap, this study developed the first genomic resources for avocados in Tanzania, where avocado has a long history of introduction and diversification dating to the first Arab incursions and Catholic missionary missions. Low-coverage whole-genome resequencing (lcWGS) data were obtained from 95 trees sampled in Tanzania across the low- to mid-altitude Morogoro region (n = 25) and the Southern Highlands—i.e., the Iringa (n = 20), Mbeya (n = 30) and Ruvuma (n = 20) regions. In order to guide racial assignation, sequences were merged with NCBI-available lcWGS data from 205 avocado trees, including 42 commercial varieties, with reported ancestry. Population stratification as inferred via maximum likelihood phylogenetic inference, genetic principal component analysis, and ADMIXTURE unsupervised clustering suggested that the sampled Tanzanian avocado trees were genetically closer to the West Indian race and more distant from the northwest South American Caribbean and Andean groups. Additionally, while the trees from the low- to mid-altitude region of Morogoro were almost exclusively West Indian type, some trees from the Southern Highlands aligned more closely with West Indian × Guatemalan and West Indian × Mexican hybrids. These trends were equally supported by a subset of 10,460 high-coverage (10×) SNP markers. Together these findings clarify the dynamics of avocado diversification in a secondary center in East Africa, spanning recent introductions from a single Mesoamerican race, adaptation to a wide range of locally geographic conditions, and farmer-driven selection matching local tribal preferences. Characterizing these locally adapted resources is key for identifying underrepresented yet promising provenances, developing resilient and sustainable horticultural production systems, and safeguarding the species’ global genetic heritage. Full article