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PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America's federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dobbins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

With limited sampling, geographic variation within a single species can be difficult to distinguish from interspecific variation, confounding our ability to draw accurate species boundaries. We argue that thorough sampling and analysis of contact zones between putative taxa can determine if assortative mating or selection against hybrids exists (supporting the presence of two distinct species), or alternatively if mating is random among genotypes and admixture among adjacent populations is gradual and continuous (supporting geographic variation within a single species). Here, we test two alternative hypotheses for two pairs of named taxa at contact zones within the American milksnake (Lampropeltis triangulum) complex. A prior morphological analysis found areas of gradual intergradation among named taxa, and concluded that the taxa represented geographical races of a single polytypic species. In contrast, a subsequent analysis of gene sequence data, but with limited sampling near the contact zones, hypothesized distinct boundaries between species at the contact zones. At the contact zone between proposed species L. triangulum and Lampropeltis gentilis, we examined a $\sim$700 km-wide transect across the states of Kansas and Missouri, with thorough sampling and reduced-representation genomic-level sequencing, to test the two opposing taxonomic hypotheses. Our transect analyses included examinations of population structure, fixed differences, cline-fitting, and an admixture index analysis. These analyses all supported a gradual and continuous geographic cline across a broad intergrade zone between two geographic forms of L. triangulum, thus providing strong support for a single species in this region (and no support for the recognition of L. gentilis as a distinct species). At a second contact zone between proposed species L. triangulum and Lampropeltis elapsoides (but variously treated as species or subspecies by different researchers) in Kentucky and Tennessee, we re-evaluated morphological data. In this case, the contact zone analysis indicated sympatry and reproductive isolation of the two taxa, and thus strongly supported L. triangulum and L. elapsoides as distinct species. We conclude that detailed studies of contact zones, based on either genetic or morphological data, are essential for distinguishing intraspecific from interspecific variation in the case of widely and continuously distributed taxa. [Contact zones; speciation; species concepts; species delimitation; taxonomy.]., (© The Author(s) 2022. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In most animals, mitochondrial DNA is strictly maternally inherited and non-recombining. One exception to this pattern is called doubly uniparental inheritance (DUI), a phenomenon involving the independent transmission of female and male mitochondrial genomes. DUI is known only from the molluskan class Bivalvia. The phylogenetic distribution of male-transmitted mitochondrial DNA (M mtDNA) in bivalves is consistent with several evolutionary scenarios, including multiple independent gains, losses, and varying degrees of recombination with female-transmitted mitochondrial DNA (F mtDNA). In this study, we use phylogenetic methods to test M mtDNA origination hypotheses and infer the prevalence of mitochondrial recombination in bivalves with DUI. Phylogenetic modeling using site concordance factors supported a single origin of M mtDNA in bivalves coupled with recombination acting over long evolutionary timescales. Ongoing mitochondrial recombination is present in Mytilida and Venerida, which results in a pattern of concerted evolution of F mtDNA and M mtDNA. Mitochondrial recombination could be favored to offset the deleterious effects of asexual inheritance and maintain mitonuclear compatibility across tissues. Cardiida and Unionida have gone without recent recombination, possibly due to an extension of the COX2 gene in male mitochondrial DNA. The loss of recombination could be connected to the role of M mtDNA in sex determination or sexual development. Our results support that recombination events may occur throughout the mitochondrial genomes of DUI species. Future investigations may reveal more complex patterns of inheritance of recombinants, which could explain the retention of signal for a single origination of M mtDNA in protein-coding genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Restriction-site-associated DNA sequencing (RADseq) has become an accessible way to obtain genome-wide data in the form of single-nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non-model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well-supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony-informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments., Competing Interests: The authors declare that they have no conflicts of interest. This publication is based, in part, on work by DCC while serving at the National Science Foundation. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the U.S. government., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri . We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri . Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri . We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri . Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

SignificanceTo adapt to arboreal lifestyles, treefrogs have evolved a suite of complex traits that support vertical movement and gliding, thus presenting a unique case for studying the genetic basis for traits causally linked to vertical niche expansion. Here, based on two de novo-assembled Asian treefrog genomes, we determined that genes involved in limb development and keratin cytoskeleton likely played a role in the evolution of their climbing systems. Behavioral and morphological evaluation and time-ordered gene coexpression network analysis revealed the developmental patterns and regulatory pathways of the webbed feet used for gliding in Rhacophorus kio .