Evaluating genotyping-in-thousands by sequencing as a genetic monitoring tool for a climate sentinel mammal using non-invasive and archival samples.

Genetic tools for wildlife monitoring can provide valuable information on spatiotemporal population trends and connectivity, particularly in systems experiencing rapid environmental change. Multiplexed targeted amplicon sequencing techniques, such as genotyping-in-thousands by sequencing (GT-seq), can provide cost-effective approaches for collecting genetic data from low-quality and quantity DNA samples, making them potentially useful for long-term wildlife monitoring using non-invasive and archival samples. Here, we developed a GT-seq panel as a potential monitoring tool for the American pika (Ochotona princeps) and evaluated its performance when applied to traditional, non-invasive, and archival samples, respectively. Specifically, we optimized a GT-seq panel (307 single nucleotide polymorphisms (SNPs)) that included neutral, sex-associated, and putatively adaptive SNPs using contemporary tissue samples (n = 77) from the Northern Rocky Mountains lineage of American pikas. The panel demonstrated high genotyping success (94.7%), low genotyping error (0.001%), and excellent performance identifying individuals, sex, relatedness, and population structure. We subsequently applied the GT-seq panel to archival tissue (n = 17) and contemporary fecal pellet samples (n = 129) collected within the Canadian Rocky Mountains to evaluate its effectiveness. Although the panel demonstrated high efficacy with archival tissue samples (90.5% genotyping success, 0.0% genotyping error), this was not the case for the fecal pellet samples (79.7% genotyping success, 28.4% genotyping error) likely due to the exceptionally low quality/quantity of recovered DNA using the approaches implemented. Overall, our study reinforced GT-seq as an effective tool using contemporary and archival tissue samples, providing future opportunities for temporal applications using historical specimens. Our results further highlight the need for additional optimization of sample and genetic data collection techniques prior to broader-scale implementation of a non-invasive genetic monitoring tool for American pikas.

Reproductive behavior of Atlantic halibut (Hippoglossus hippoglossus) interpreted from electronic tags.

Reproductive timing, location, and behavior are important characteristics that determine marine population dynamics, structure, and resilience to threats, including fishing and climate change. It is challenging to evaluate factors driving variability in these reproductive traits in wild fishes because of the difficulty observing individuals in their natural environments. In the present study, we used high-resolution depth, temperature, and acceleration time series recorded by pop-up satellite archival tags to (1) identify and characterize patterns in depth and acceleration that may be indicative of spawning events in large Atlantic halibut (Hippoglossus hippoglossus), and (2) estimate the effects of individual traits (body size and sex) and environmental factors (location and temperature) on spawning time and frequency. Unique rapid rises observed in the winter depth profiles were interpreted as spawning events. The initiation of the first presumed spawning rise was negatively correlated to water temperature experienced during the prespawning season, suggesting that currently increasing water temperature in the Gulf of St. Lawrence may induce phenological change in halibut spawning time. The number of rises of batch-spawning females was unrelated to female body size. The present study demonstrates how electronic tagging can be used for in-depth characterization of timing, location, and behaviors associated with spawning in a large flatfish species. Such information can inform spatiotemporal management and conservation measures aiming to protect species from directed fishing and by-catch during spawning.

SNP Panel and Genomic Sex Identification in Atlantic Halibut (Hippoglossus hippoglossus).

The ability to identify sex is necessary in population biology for a proper understanding of the dynamics of a population. In Atlantic halibut, phenotypic sex identification is not possible due to the lack of significant external morphological differences. We developed an Illumina SNP panel for Atlantic halibut with 4000 SNPs spread evenly throughout the genome with a minor allele frequency MAF ≥ 0.4, except for N = 249 SNPs located in a sex-determining region on chromosome 12, N = 176 of these SNPs were selected to genetically identify male and female individuals using a DAPC analysis. The genomic identification of sex allows for non-lethal sex determination and validation of sex identification in the field. The SNP panel is a new genomic resource for Atlantic halibut that will make it possible to generate the genotypic data for the large number of individuals needed to estimate population abundance using genomics and the Close Kin Mark Recapture (CKMR) approach, an emerging component of fisheries management and stock monitoring.

Genomics reveal population structure, evolutionary history, and signatures of selection in the northern bottlenose whale, Hyperoodon ampullatus.

Information on wildlife population structure, demographic history, and adaptations are fundamental to understanding species evolution and informing conservation strategies. To study this ecological context for a cetacean of conservation concern, we conducted the first genomic assessment of the northern bottlenose whale, Hyperoodon ampullatus, using whole-genome resequencing data (n = 37) from five regions across the North Atlantic Ocean. We found a range-wide pattern of isolation-by-distance with a genetic subdivision distinguishing three subgroups: the Scotian Shelf, western North Atlantic, and Jan Mayen regions. Signals of elevated levels of inbreeding in the Endangered Scotian Shelf population indicate this population may be more vulnerable than the other two subgroups. In addition to signatures of inbreeding, evidence of local adaptation in the Scotian Shelf was detected across the genome. We found a long-term decline in effective population size for the species, which poses risks to their genetic diversity and may be exacerbated by the isolating effects of population subdivision. Protecting important habitat and migratory corridors should be prioritized to rebuild population sizes that were diminished by commercial whaling, strengthen gene flow, and ensure animals can move across regions in response to environmental changes.

Reference genome of lumpfish Cyclopterus lumpus Linnaeus provides evidence of male heterogametic sex determination through the AMH pathway.

Teleosts exhibit extensive diversity of sex determination (SD) systems and mechanisms, providing the opportunity to study the evolution of SD and sex chromosomes. Here we sequenced the genome of the common lumpfish (Cyclopterus lumpus Linnaeus), a species of increasing importance to aquaculture, and identified the SD region and master SD locus using a 70 K single nucleotide polymorphism array and tissue-specific expression data. The chromosome-level assembly identified 25 diploid chromosomes with a total size of 572.89 Mb, a scaffold N50 of 23.86 Mb and genome annotation-predicted 21,480 protein-coding genes. Genome-wide association analysis located a highly sex-associated region on chromosome 13, suggesting that anti-Müllerian hormone (AMH) is the putative SD factor. Linkage disequilibrium and heterozygosity across chromosome 13 support a proto-XX/XY system, with an absence of widespread chromosome divergence between sexes. We identified three copies of AMH in the lumpfish primary and alternate haplotype assemblies localized in the SD region. Comparison to sequences from other teleosts suggested a monophyletic relationship and conservation within the Cottioidei. One AMH copy showed similarity to AMH/AMHY in a related species and was also the only copy with expression in testis tissue, suggesting this copy may be the functional copy of AMH in lumpfish. The two other copies arranged in tandem inverted duplication were highly similar, suggesting a recent duplication event. This study provides a resource for the study of early sex chromosome evolution and novel genomic resources that benefits lumpfish conservation management and aquaculture.

RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis).

BACKGROUND: The lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging "imperforate leaves" do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called "PCD cells" that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called "NPCD cells" retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection. RESULTS: Transcriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes. CONCLUSIONS: RNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

Chromosome level reference of Atlantic halibut Hippoglossus hippoglossus provides insight into the evolution of sexual determination systems.

Changes in the genetic mechanisms that control sexual determination have occurred independently across the tree of life, and with exceptional frequency in teleost fishes. To investigate the genomic changes underlying the evolution of sexual determination, we sequenced a chromosome-level genome, multitissue transcriptomes, and reduced representation population data for the Atlantic halibut (Hippoglossus hippoglossus), which has an XY/XX sex determination mechanism and has recently diverged (0.9-3.8 Ma) from the Pacific halibut (Hippoglossus stenolepis), which has a ZZ/ZW system. We used frequency and coverage-based population approaches to identify a putative sex-determining factor, GSDF. We characterized regions with elevated heterozygosity and linkage disequilibrium indicating suppression of recombination across a nascent sex chromosome. We detected testis-specific expression of GSDF, the sequence of which is highly conserved across flatfishes. Based on evidence from genome-wide association, coverage, linkage disequilibrium, testis and brain transcriptomes, and sequence conservation with other flatfishes, we propose a mechanism for the recent evolution of an XY sex-determination mechanism in Atlantic halibut. Changes to the ancestral sex-determining gene DMRT1 in regulating the downstream gene GSDF probably coincided with GSDF, or a proximal regulatory element of it, becoming the primary sex-determining factor. Our results suggest changes to a small number of elements can have drastic repercussions for the genomic substrate available to sex-specific evolutionary forces, providing insight into how certain elements repeatedly evolve to control sex across taxa. Our chromosome-level assembly, multitissue transcriptomes, and population genomic data provide a valuable resource and understanding of the evolution of sexual systems in fishes.

Historical human activities reshape evolutionary trajectories across both native and introduced ranges.

The same vectors that introduce species to new ranges could move them among native populations, but how human-mediated dispersal impacts native ranges has been difficult to address because human-mediated dispersal and natural dispersal can simultaneously shape patterns of gene flow. Here, we disentangle human-mediated dispersal from natural dispersal by exploiting a system where the primary vector was once extensive but has since ceased. From 10th to 19th Centuries, ships in the North Atlantic exchanged sediments dredged from the intertidal for ballast, which ended when seawater ballast tanks were adopted. We investigate genetic patterns from RADseq-derived SNPs in the amphipod Corophium volutator (n = 121; 4,870 SNPs) and the annelid Hediste diversicolor (n = 78; 3,820 SNPs), which were introduced from Europe to North America, have limited natural dispersal capabilities, are abundant in intertidal sediments, but not commonly found in modern water ballast tanks. We detect similar levels of genetic subdivision among introduced North American populations and among native European populations. Phylogenetic networks and clustering analyses reveal population structure between sites, a high degree of phylogenetic reticulation within ranges, and phylogenetic splits between European and North American populations. These patterns are inconsistent with phylogeographic structure expected to arise from natural dispersal alone, suggesting human activity eroded ancestral phylogeographic structure between native populations, but was insufficient to overcome divergent processes between naturalized populations and their sources. Our results suggest human activity may alter species' evolutionary trajectories on a broad geographic scale via regional homogenization and global diversification, in some cases precluding historical inference from genetic data.