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Microsatellite null alleles are commonly encountered in population genetics studies, yet little is known about their impact on the estimation of population differentiation. Computer simulations based on the coalescent were used to investigate the evolutionary dynamics of null alleles, their impact on FST and genetic distances, and the efficiency of estimators of null allele frequency. Further, we explored how the existing method for correcting genotype data for null alleles performed in estimating FST and genetic distances, and we compared this method with a new method proposed here (for FST only). Null alleles were likely to be encountered in populations with a large effective size, with an unusually high mutation rate in the flanking regions, and that have diverged from the population from which the cloned allele state was drawn and the primers designed. When populations were significantly differentiated, FST and genetic distances were overestimated in the presence of null alleles. Frequency of null alleles was estimated precisely with the algorithm presented in Dempster et al. (1977). The conventional method for correcting genotype data for null alleles did not provide an accurate estimate of FST and genetic distances. However, the use of the genetic distance of Cavalli-Sforza and Edwards (1967) corrected by the conventional method gave better estimates than those obtained without correction. FST estimation from corrected genotype frequencies performed well when restricted to visible allele sizes. Both the proposed method and the traditional correction method have been implemented in a program that is available free of charge at http://www.montpellier.inra.fr/URLB/. We used 2 published microsatellite data sets based on original and redesigned pairs of primers to empirically confirm our simulation results. [PUBLICATION ABSTRACT]

The Common House Crow ( Corvus splendens ) exhibits remarkable ecological adaptability, enabling its rapid expansion across continents. However, despite its wide distribution, there is a need for genetic studies to clarify its evolutionary history and population structure. This research employs DNA barcoding, focusing on the mitochondrial gene cytochrome oxidase subunit I ( Cox1 ), which is effective for species identification and phylogenetic analysis. Blood samples were collected from 70 C. splendens specimens across seven cities in Punjab, Pakistan: Lahore, Kasur, Sialkot, Narowal, Pakpattan, Gujranwala, and Bahawalpur. Genomic DNA extraction was performed, and a partial sequence of the COX1 gene was amplified using PCR techniques. Sequencing of the Cox1 marker from 10 randomly selected specimens revealed nine distinct genetic variants. Interspecific analysis positioned our C. splendens sequences alongside various Corvus species available in GenBank, while intraspecific analysis identified a total of 15 genetic variants. These variants showed nucleotide identity rates ranging from 98.7 to 99.8%, with genetic distances between 0.002 and 0.013. The analysis indicated that the C. splendens group consists of a single heterogeneous clade with variants from multiple countries, including Pakistan, Tanzania, Nepal, South Africa, Malaysia, Sri Lanka, Bangladesh, Kenya, Australia, and Singapore. This study significantly enhances our understanding of genetic diversity and evolutionary relationships within C. splendens populations, highlighting the necessity of genetic research to inform conservation strategies. Further research employing advanced molecular techniques and broader geographic sampling is essential to assess the genetic diversity and population dynamics of this adaptable species.

Background Bats are known reservoirs for various pathogens, many of which can infect other animals through blood-feeding arthropods. Over 100 bat species have been identified as hosts for kinetoplastid protozoans, including ≥ 30 distinct Trypanosoma spp. However, bat trypanosomes remain relatively understudied owing to the nocturnal behavior of their hosts and legal restrictions on their capture for research. In Southeast Asia, particularly Thailand, only one study has investigated bat trypanosomes, leaving their distribution and transmission pathways largely unexplored. Methods Between April 2021 and November 2023, bats were captured at ten locations across four provinces in Thailand. Blood samples were collected, examined microscopically, and screened for Trypanosoma DNA targeting the SSU rRNA and gGAPDH genes. Phlebotomine sand flies from bat sampling sites were collected and analyzed for Trypanosoma DNA and blood meal sources. Sequences were identified using BLASTn searches, while genetic relationships were assessed through pairwise genetic distance, phylogenetic reconstruction, and TCS haplotype network analyses. In addition, species delimitation was conducted to validate unidentified sequences at the species level. Results Out of 368 bats, 40 (10.9%) tested positive for four Trypanosoma species (including two previously named: T. dionisii and T. noyesi ). Out of 189 sand flies, a single one tested positive for an unnamed anuran trypanosome from a gravid female ( Phlebotomus stantoni ), and the study was unable to detect the blood source of this sand fly. In total, 37 pools (189 specimens) of female sand flies—comprising 159 unfed, 29 gravid, and 1 engorged specimen—were analyzed for vertebrate blood meals, but none tested positive. Multiple analyses (BLASTn, phylogenetics, haplotype networks, pairwise genetic distances, and species delimitation) also confirmed a Trypanosoma sp. in a gravid sand fly, along with T. dionisii and T. noyesi , plus two uncharacterized bat-associated species. Conclusions The Trypanosoma spp. detected in the present study aligns with prior reports of diverse trypanosomes in bat populations, reinforcing their role as key reservoirs. Notably, a single sand fly ( Phlebotomus stantoni ) tested positive for an unnamed anuran trypanosome, but its blood meal source could not be determined, leaving unresolved questions about potential transmission pathways. Graphical abstract

Currently, the lowest formal taxon in virus classification is species; however, unofficial lower-level units are commonly used in everyday work. Tick-borne encephalitis virus (TBEV) is a species of mammalian tick-borne flaviviruses that may cause encephalitis. Many known representatives of TBEV are grouped into subtypes, mostly according to their phylogenetic relationship. However, the emergence of novel sequences could dissolve this phylogenetic grouping; in the absence of strict quantitative criterion, it may be hard to define the borders of the first TBEV taxonomic unit below the species level. In this study, the nucleotide/amino-acid space of all known TBEV sequences was analyzed. Amino-acid sequence p-distances could not reliably distinguish TBEV subtypes. Viruses that differed by less than 10% of nucleotides in the polyprotein-coding gene belonged to the same subtype. At the same time, more divergent viruses were representatives of different subtypes. According to this distance criterion, TBEV species may be divided into seven subtypes: TBEV-Eur, TBEV-Sib, TBEV-FE, TBEV-2871 (TBEV-Ob), TBEV-Him, TBEV-178-79 (TBEV-Bkl-1), and TBEV-886-84 (TBEV-Bkl-2).

The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21–39.68%, 60.12–65.19% for the 16S marker gene, and 72.44–80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

Background Bayesian MCMC has become a common approach for phylogenetic inference. But the growing size of molecular sequence data sets has created a pressing need to improve the computational efficiency of Bayesian phylogenetic inference algorithms. Results This paper develops a new algorithm to improve the efficiency of Bayesian phylogenetic inference for models that include a per-branch rate parameter. In a Markov chain Monte Carlo algorithm, the presented proposal kernel changes evolutionary rates and divergence times at the same time, under the constraint that the implied genetic distances remain constant. Specifically, the proposal operates on the divergence time of an internal node and the three adjacent branch rates. For the root of a phylogenetic tree, there are three strategies discussed, named Simple Distance, Small Pulley and Big Pulley. Note that Big Pulley is able to change the tree topology, which enables the operator to sample all the possible rooted trees consistent with the implied unrooted tree. To validate its effectiveness, a series of experiments have been performed by implementing the proposed operator in the BEAST2 software. Conclusions The results demonstrate that the proposed operator is able to improve the performance by giving better estimates for a given chain length and by using less running time for a given level of accuracy. Measured by effective samples per hour, use of the proposed operator results in overall mixing more efficient than the current operators in BEAST2. Especially for large data sets, the improvement is up to half an order of magnitude.

Combining ability along with heterosis was elucidated in the 21 F1 hybrid population of rice derived from a 7 × 7 diallel mating design. Furthermore, to formulate a strategy for marker-based prediction of heterosis, the molecular genetic diversity (GD) was then surveyed among the seven parental lines with RAPD markers. Analysis of combining ability revealed significant GCA and SCA variances and the ratio between the aforesaid genetic parameter was more than unity for most of the traits signifying the preponderance of additive gene action. Hybrid combination Double Rice × Pokkali was the best specific combiner for the traits pollen fertility percentage (%), panicle length (cm), filled grains/panicle (no.) and grain yield/hill (g) while Muktagacha × BRRI dhan29 and Basmati × Double Rice were the best specific combiners for grain length (mm) and 1000-grain weight (g), respectively. Regarding the magnitude of heterosis, Double Rice × Pokkali exhibited maximum heterobeltiosis for panicle length (33.33%) and the number of filled grains/panicle (144.01%). Furthermore, Pokkali × Chinigura was verified for producing the highest heterobeltiosis for tillers/hill (173.17%), as well as grain yield/hill (71.05%). The heatmap relying on molecular genetic diversity exposed the highest genetic distance between Chinigura and Pokkali followed by Muktagacha and Pokkali. Unweighted neighbor-joining trees clustered the parental lines into three groups indicating the presence of considerable genetic diversity among those seven parental genotypes. A positive and significant correlation was explored between molecular GD with specific combining ability as well as heterosis for all of the traits revealing the huge potentiality of hybrid performance prediction using RAPD markers with the present set of individuals. Thus, exploration of definite genomic regions underlying the candidate genes for respective traits relying on such RAPD-generated data following an advanced molecular marker approach would pave the path of mining out yielder heterotic combinations.

ContextLandscape genetics provides powerful tools to quantify the effects of landscape features on population connectivity, but robust results are imperative to inform conservation planning.ObjectivesThe robustness of landscape genetic inferences was assessed using the case of the northern crested newt (Triturus cristatus) in Luxembourg. Specifically, the effect of different study designs and genetic distance metrics was tested in terms of model convergence and misspecification rates (Type I error).MethodsThe optimisation of resistance surfaces was performed in ResistanceGA, using individual- and population-based sampling designs and 16 genetic distance metrics inferred from 897 multilocus genotypes from 85 locations. Empirical results were complemented with simulations to assess Type I error rates and correlation between ‘true’ and optimised resistance surfaces.ResultsIndividual-based optimisations seemed prone to overfitting, with little convergence among empirical resistance surfaces from different sets of individuals. Simulations showed significant differences in performance among population genetic distance metrics. Linear topographical features exhibited higher Type I error rates (83.3%) than continuous features (44.9%), suggesting potential underestimation of road-induced fragmentation effects. Jost’s D, FST, and PCA axes 1–45 were the top three genetic distance metrics for recovering true resistance features. Topographic roughness consistently drove spatial genetic clustering of T. cristatus, but variability existed among conductivity maps derived from optimised resistance surfaces.ConclusionsThese findings underscore the importance of carefully selecting genetic distance metrics and addressing potential sources of uncertainty in resistance surface optimisation. By doing so, we can enhance the effectiveness of conservation planning efforts for T. cristatus and species with similar ecological considerations.

This study establishes the first core collection (CC) for Taiwanese Phalaenopsis orchids to preserve genetic diversity and key traits essential for breeding and research, thereby enhancing breeding efficiency without the need for a large maintained parent population. We examined 207 commercial orchid cultivars from ten nurseries, characterized by two phenotypes and genotypic data from eight simple sequence repeat markers. Multiple imputation was applied to estimate missing phenotypes, minimizing potential uncertainties and ensuring the reliability of population structure analysis. Weighted k -means clustering identified seven distinct clusters, highlighting substantial genetic diversity. We proposed a two-step phenotype and genotype strategy and modified genetic distance algorithms to effectively preserve both phenotypic and genetic diversity while retaining key features. Consequently, 22 core accessions were selected, distributed across seven clusters, and representing the orchid germplasm collection. Our evaluation revealed significant diversity preservation, particularly in distinct characteristics and rare features, outperforming other methodologies. Pedigree background analysis further confirmed the representativeness of the CC in maintaining diverse genetic materials. We emphasized the importance of evaluating the CC by detailing the criteria and statistical analyses used to ensure the quality, representativeness, and effectiveness of the selected accessions. This study contributes to orchid breeding, conservation efforts, and sustainable agricultural practices by providing a valuable and comprehensive resource. In conclusion, our research establishes a groundbreaking CC, offering insights into the genetic landscape of Taiwanese Phalaenopsis orchids and highlighting potential advancements in breeding commercially desirable varieties.

Distant hybridization is key to trait innovation and speciation, with Cyprinidae hybrid phylogeny helping to clarify diversification mechanisms. Yet, a major gap persists in Cyprinidae studies: the stabilization mechanisms of interspecific distant hybrid lineages. To address this, we systematically analyzed the molecular phylogeny of seven Cyprinidae distant hybrid lineages and their parental species, using an integrative genetic framework encompassing four mitochondrial genes (Cytb, COI, 16S rRNA, D-loop) and five nuclear genes (EGR2b, IRBP2, RAG1, RAG2, RH2). Homologous sequences of 41 representative Cyprinidae species (85 samples) were retrieved from GenBank to supplement the dataset. Phylogenies were reconstructed from concatenated sequences, complemented by haplotype networks. Intra-/interspecific divergence was quantified using two mitochondrial genes (COI, Cytb) and two nuclear (RAG1, RH2). The results showed that these hybrid lineages exhibited variation patterns analogous to other Cyprinidae species. Both ML and BI trees reconstructed exhibited congruent topologies with high support (bootstrap/BPP > 80%), resolving genus/species-level relationships. While most hybrids clustered intermediately between their parental species, they typically displayed maternal affinity. A notable exception was the 2nNCRC (a homodiploid hybrid from Cyprinus carpio ♀ × Megalobrama amblycephala ♂), which displayed convergent evolution toward Carassius auratus. COI-based K2P genetic distance analysis revealed 2nNCRC had a much closer relationship with C. auratus (0.0119) than with its parents (0.1249 to C. carpio, 0.1552 to M. amblycephala). These nine genes elucidate the genetic relationships between Cyprinid hybrid lineages and progenitors, serving as pivotal molecular markers for parentage tracing and genetic dissection of distant hybridization mechanisms. The integrated mitochondrial–nuclear marker system in this study advances understanding of cytonuclear coadaptation and the stabilization of interspecific distant hybrid lineages in Cyprinidae. Specifically, it provides a precise tool for parentage tracing, Cyprinid germplasm conservation, and targeted regulation of hybrid breeding—laying a foundation for exploring hybrid speciation and developing elite aquaculture germplasms.