Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

Phylogeographic inference of Sumatran ranids bearing gastromyzophorous tadpoles with regard to the Pleistocene drainage systems of Sundaland.

Rivers are known to act as biogeographic barriers in several strictly terrestrial taxa, while possibly serving as conduits of dispersal for freshwater-tolerant or -dependent species. However, the influence of river systems on genetic diversity depends on taxa-specific life history traits as well as other geographic factors. In amphibians, several studies have demonstrated that river systems have only minor influence on their divergence. Here, we assess the role of the paleodrainage systems of the Sunda region (with a focus on the island of Sumatra) in shaping the evolutionary history of two genera of frogs (Sumaterana and Wijayarana) whose tadpoles are highly dependent on cascading stream habitats. Our phylogenetic results show no clear association between the genetic diversification patterns of both anurans genera and the existence of paleodrainage systems. Time-calibrated phylogenies and biogeographical models suggest that these frogs colonized Sumatra and diversified on the island before the occurrence of the Pleistocene drainage systems. Both genera demonstrate phylogenetic structuring along a north-south geographic axis, the temporal dynamics of which coincide with the geological chronology of proto Sumatran and -Javan volcanic islands. Our results also highlight the chronic underestimation of Sumatran biodiversity and call for more intense sampling efforts on the island.

MMDIT: A tool for the deconvolution and interpretation of mitochondrial DNA mixtures.

Short tandem repeats of the nuclear genome have been the preferred markers for analyzing forensic DNA mixtures. However, when nuclear DNA in a sample is degraded or limited, mitochondrial DNA (mtDNA) markers provide a powerful alternative. Though historically considered challenging, the interpretation and analysis of mtDNA mixtures have recently seen renewed interest with the advent of massively parallel sequencing. However, there are only a few software tools available for mtDNA mixture interpretation. To address this gap, the Mitochondrial Mixture Deconvolution and Interpretation Tool (MMDIT) was developed. MMDIT is an interactive application complete with a graphical user interface that allows users to deconvolve mtDNA (whole or partial genomes) mixtures into constituent donor haplotypes and estimate random match probabilities on these resultant haplotypes. In cases where deconvolution might not be feasible, the software allows mixture analysis directly within a binary framework (i.e. qualitatively, only using data on allele presence/absence). This paper explains the functionality of MMDIT, using an example of an in vitro two-person mtDNA mixture with a ratio of 1:4. The uniqueness of MMDIT lies in its ability to resolve mixtures into complete donor haplotypes using a statistical phasing framework before mixture analysis and evaluating statistical weights employing a novel graph algorithm approach. MMDIT is the first available open-source software that can automate mtDNA mixture deconvolution and analysis. The MMDIT web application can be accessed online at https://www.unthsc.edu/mmdit/. The source code is available at https://github.com/SammedMandape/MMDIT_UI and archived on zenodo (https://doi.org/10.5281/zenodo.4770184).

International Wildlife Trafficking: A perspective on the challenges and potential forensic genetics solutions.

International wildlife trafficking (IWT) is a thriving and pervasive illegal enterprise that adversely affects modern societies. Yet, despite being globally recognized as a threat to biodiversity, national security, economy, and biosecurity, IWT remains largely unabated and is proliferating at an alarming rate. The increase in IWT is generally attributed to a lack of prioritization to curb wildlife crime through legal and scientific infrastructure. This review: (1) lays out the damaging scope and influence of IWT; (2) discusses the potential of DNA marker systems, barcodes, and emerging molecular technologies, such as long-read portable sequencing, to facilitate rapid, in situ identification of species and individuals; and (3) encourages initiatives that promote quality and innovation. Interdisciplinary collaboration promises to be one of the most effective ways forward to surmounting the complex scientific and legal challenges posed by IWT.

A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures.

Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture's genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic.

Numt identification and removal with RtN!

MOTIVATION: Assays in mitochondrial genomics rely on accurate read mapping and variant calling. However, there are known and unknown nuclear paralogs that have fundamentally different genetic properties than that of the mitochondrial genome. Such paralogs complicate the interpretation of mitochondrial genome data and confound variant calling. RESULTS: Remove the Numts! (RtN!) was developed to categorize reads from massively parallel sequencing data not based on the expected properties and sequence identities of paralogous nuclear encoded mitochondrial sequences, but instead using sequence similarity to a large database of publicly available mitochondrial genomes. RtN! removes low-level sequencing noise and mitochondrial paralogs while not impacting variant calling, while competing methods were shown to remove true variants from mitochondrial mixtures. AVAILABILITY AND IMPLEMENTATION: https://github.com/Ahhgust/RtN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phylogeography of montane dragons could shed light on the history of forests and diversification processes on Sumatra.

Biogeographical evidence, both, for and against the "regional endemism paradigm" hypothesis has been uncovered across the Greater Sunda Region (Sundaland) of Southeast Asia. Additionally, there are competing hypotheses regarding how Pleistocene forests may have impacted biological patterns and processes in Sumatra. Using montane agamid lizards from Sumatra, we derived and analyzed a phylogenetic dataset, genetic divergence estimates, and contemporary distributional patterns among species. We tested whether (1) Sumatra's highland Draconinae diversification fits the regional endemism paradigm hypothesis and (2) Draconinae phylogeography provides biological evidence for Pleistocene forest extent at various points in history. Our results suggest in situ diversification was the main driver behind montane Draconinae lizard diversification in Sumatra, rejecting the "regional endemism paradigm". Contemporary distribution of endemic species and their genetic relationships may potentially provide biologicalevidence for determining more precise elevational lower limits of montane forests during the Pleistocene epoch. Our data suggests montane forests did not retreat more than 700-750 m during glacial maxima because lower retreating forests would have become interconnected, allowing for widespread dispersal, exchange of gene flow and sympatric distributions contemporarily. To the contrary, our divergence estimates show that cloud forest dragons have been isolated for millions of years, suggesting there may have been a continuous disconnect between some areas, predating the Pleistocene. There may also be other ecological and evolutionary factors that impacted Draconinae distributions, such as competition, making this an excellent system for testing questions regarding montane biogeography. Additionally, we provide the first phylogeny for a wide range of Sundaland agamid species and identify some biogeographic pressures that may have triggered montane Draconinae diversification in Sumatra.

Molecular phylogenetics of Black Cobra (Naja naja) in Pakistan

Background: Snakes are found on every continent in the world except Antarctica, and on smaller land masses. Being ecologically important, they also cause a large number of bites, leading to millions of deaths. Mitochondrial and nuclear gene sequences are being used to identify, characterize, and infer genetic biodiversity among different snake species. Furthermore, phylogenetics helps in inferring the relationships and evolutionary histories among these species. Black cobra is one of the four most venomous snakes in Pakistan. Four mitochondrial (ND4, Cytochrome b, 12S rRNA, and 16S rRNA) and four nuclear (C-mos, RAG-1, BDNF, and NT3) genes were used to trace diversity and infer the phylogenetic relationship of black cobra in Pakistan. Results: Almost similar phylogenies were obtained through maximum likelihood and Bayesian inference, showing two species of cobra in Pakistan, namely, black cobra (Naja naja) and brown cobra (Naja oxiana). All Naja species were divided into three clades: black cobra (N. naja) and brown cobra (N. oxiana) cladding with different species of Naja; N. naja (Pakistan) cladding with N. naja from Nepal; and N. oxiana showed close relationship with Naja kaouthia from Thailand and Naja siamensis from Thailand. Conclusion: It was confirmed genetically that there are two cobra species in Pakistan, i.e., black and brown cobras. This study will help in not only genetic conservation but also developing anti-venom against snake species.

A novel phylogenetic approach for de novo discovery of putative nuclear mitochondrial (pNumt) haplotypes.

Current approaches for parsing true variation (i.e. signal) from noise, broadly involve estimating a baseline value of the latter, below which all sequence data are ignored. In an effort to deliver a more objective criterion for setting such thresholds, a novel approach based on phylogenetic principles is presented here., Our method deconstructs a special category of noise from true mitochondrial genome data, namely nuclear insertions of mitochondrial DNA (Numts). This bioinformatic approach leverages the relationship of massively parallel sequence reads and is capable of discovering putative Numts (pNumts) in absence of a reference genome. The new method was tested on a whole mitochondrial genome dataset (n = 41 individuals from an admixed population sample from Rio de Janeiro) and led to the discovery of 451 pNumt variants. Comparison of these pNumts haplotypes against an existing Numt database revealed 147 exact matches to previously discovered Numts, while 122 haplotypes differed only by a single base pair and none matched exclusively to the mitochondrial genome. In general, these sequences were considerably more divergent from the mitochondrial genome than from those of the Numt database, supporting that the novel pNumts were probably hitherto uncatalogued variants. Unlike previous techniques, our method appears to be able to detect both polymorphic and fixed Numt sequences. It was also found that the region containing the D-Loop and associated Promoters (DLP) in the human mitochondrial genome, which harbors markers of forensic genetics importance, is the origin of several Numts. Though currently designed for the mitochondrial genome, our novel approach has the potential to be expanded to other scenarios that might require construing signal from noise, including the deconvolution of mixtures, thus significantly improving how analytical thresholds may be established.

Diversification of bent-toed geckos (Cyrtodactylus) on Sumatra and west Java.

Complex geological processes often drive biotic diversification on islands. The islands of Sumatra and Java have experienced dramatic historical changes, including isolation by marine incursions followed by periodic connectivity with the rest of Sundaland across highland connections. To determine how this geological history influenced island invasions, we investigated the colonization history and diversification of bent-toed geckos (genus Cyrtodactylus) on Sumatra and west Java. We used mitochondrial and nuclear sequence data to explore species boundaries, estimate phylogenetic relationships and divergence times, and to reconstruct ancestral range evolution. We found that Sumatran and Javan Cyrtodactylus were closely related to species from the Thai-Malay Peninsula, rather than from Borneo, and that Cyrtodactylus most likely dispersed to Sumatra three times during the late Oligocene and early Miocene. Similarly, Cyrtodactylus invaded west Java from Sumatra once in the early Miocene. Our results suggest that despite isolation by marine incursions during much of the Miocene, Cyrtodactylus dispersed to and from Sumatra and west Java likely via land bridges, and that in situ diversification occurred several times on Sumatra.

Genetic surfing, not allopatric divergence, explains spatial sorting of mitochondrial haplotypes in venomous coralsnakes.

Strong spatial sorting of genetic variation in contiguous populations is often explained by local adaptation or secondary contact following allopatric divergence. A third explanation, spatial sorting by stochastic effects of range expansion, has been considered less often though theoretical models suggest it should be widespread, if ephemeral. In a study designed to delimit species within a clade of venomous coralsnakes, we identified an unusual pattern within the Texas coral snake (Micrurus tener): strong spatial sorting of divergent mitochondrial (mtDNA) lineages over a portion of its range, but weak sorting of these lineages elsewhere. We tested three alternative hypotheses to explain this pattern-local adaptation, secondary contact following allopatric divergence, and range expansion. Collectively, near panmixia of nuclear DNA, the signal of range expansion associated sampling drift, expansion origins in the Gulf Coast of Mexico, and species distribution modeling suggest that the spatial sorting of divergent mtDNA lineages within M. tener has resulted from genetic surfing of standing mtDNA variation-not local adaptation or allopatric divergence. Our findings highlight the potential for the stochastic effects of recent range expansion to mislead estimations of population divergence made from mtDNA, which may be exacerbated in systems with low vagility, ancestral mtDNA polymorphism, and male-biased dispersal.