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    Temporal monitoring of the Floreana Island Galapagos giant tortoise captive breeding program
    (2022) Gray, Rachel; Fusco, Nicole; Miller, Joshua M.; Tapia, Washington; Mariani, Carol; Caccone, Adalgisa; Jensen, Evelyn L.
    Captive breeding programs benefit from genetic analyses that identify relatedness between individuals, assign parentage to offspring, and track levels of genetic diversity. Monitoring these parameters across breeding cycles is critical to the success of a captive breeding program as it allows conservation managers to iteratively evaluate and adjust program structure. However, in practice, genetic tracking of breeding outcomes is rarely conducted. Here, we examined the first three offspring cohorts (2017–2020) of the genetically informed captive breeding program for the Floreana Island Galapagos giant tortoise, Chelonoidis niger. This captive breeding program is unique as the Floreana tortoise has been extinct since the 1800s, but its genome persists, in part, in the form of living hybrids with the extant Volcano Wolf tortoise, C. becki. Breeding over the study period took place at the Galapagos National Park Directorate breeding facility in four corrals, each containing three females and two males. Using 17 microsatellite markers, we were able to assign parentage to 94 of the 98 offspring produced over the study period. We observe that despite the addition of more founders since the pilot breeding program, the effective population size remains low, and changes to the arrangements of breeding corrals may be necessary to encourage more equal reproductive output from the males. This study demonstrates the value of hybrids for species restoration and the importance of continually reassessing the outcomes of captive breeding.
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    A roadmap to robust discriminant analysis of principal components
    (2023) Cullingham, Catherine; Peery, Rhiannon M.; Miller, Joshua M.
    Identification of population structure is a common goal for a variety of applications, including conservation, wildlife management, and medical genetics. The outcome of these analyses can have far reaching implications; therefore, it is important to ensure an understanding of the strengths and weaknesses of the methodologies used. Increasing in popularity, the discriminant analysis of principal components (DAPC) method incorporates combinations of genetic variables (alleles) into a model that differentiates individuals into genetic clusters. However, users may not have a full understanding of how to best parameterize the model. In this issue of Thia (Molecular Ecology Resources, 2022) looks under the hood of the DAPC. Using simulated data, he demonstrates the importance of careful parameter selection in developing a DAPC model, what the implications are for over-fitting the model, and finally, how best to evaluate the results of DAPC models. This work highlights the issues that can arise when over-parameterizing the DAPC model when gene flow is high among clusters and provides important guidelines to ensure researchers are making conclusions that are biologically relevant.
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    Effects of larval host and natural microsporidian infection on adult life history traits of the forest tent caterpillar (Lepidoptera: Lasiocampidae)
    (2023) Preti, Flavio; Flaherty, Leah; Evenden, Maya L.
    Host affiliation and entomopathogenic infections play a major role in shaping population dynamics of the forest tent caterpillar (FTC), Malacosoma disstria Hübner (Lepidoptera: Lasiocampidae). The effect of these individual factors has been studied, but it is unknown whether interactions between these factors significantly impact FTC life history traits. In the laboratory, we investigated a tritrophic interaction among larval diet, larval microsporidian infection, and FTC life history traits. Larvae were reared on foliage of trembling aspen, Populus tremuloides Michx (Malpighiales: Salicaceae) or sugar maple, Acer saccharum Marshall (Sapindales: Sapindaceae), or an artificial diet. Natural levels of microsporidian infection were assessed through microscopy and categorized as none (0 spores), low (1–100 spores), or high (>100 spores). Microsporidian infection and larval diet individually, but not interactively, impacted FTC life history traits. Moths with high infection had smaller wings, but infection did not increase the probability of wing malformations. Wings of FTC reared on fresh maple foliage were significantly smaller, had a higher probability of wing malformation, and a lower likelihood of cocoon production than FTC reared on other diets, but displayed higher overall survival. While microsporidian infection did not influence FTC-diet interactions, we provide further evidence on how these main effects may individually contribute to shaping FTC adult life history traits, and, ultimately, cyclical population dynamics. Future research should consider how larval mortality, distinct infection levels, and geographical source of FTC populations affect this tritrophic interaction.
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    Independent and interactive effects of diet and entomopathogenic microsporidia on an outbreaking forest insect defoliator
    (2024) Flaherty, Leah; Preti, lavio; Ishangulyyeva, Guncha; Erbilgin, Nadir; Whidden, Taylar; Evenden, Maya
    1. Insect herbivore diet mediates interactions with entomopathogens, yet this is relatively unexplored for microsporidia. Here, we examine a diet-mediated tri-trophic interaction between an outbreaking forest defoliator, forest tent caterpillar (FTC) Malacosoma disstria Hübner and Nosema sp. microsporidia. 2. We conducted two experiments where diet quality was manipulated by incorporating lyophilized aspen (Populus tremuloides Michaux) foliage into an artificial diet, which was compared to a standard artificial diet. Diet quantity varied between fully fed and partially starved conditions, simulating an outbreak scenario. Microsporidia infection occurred naturally or was induced via experimental inoculation. We assessed FTC survival, microsporidia infection and load, and sublethal effects of treatments on FTC traits. 3. Plant secondary metabolite concentrations in aspen-augmented diets varied between experiments. In Experiment 1, the aspen-augmented diet contained lyophilized aspen foliage with low concentrations of secondary metabolites, which increased FTC survival and reduced microsporidia infection. Diet quality and infection load also interactively influenced adult wing traits in Experiment 1. In Experiment 2, the aspen-augmented diet contained lyophilized aspen foliage with higher concentrations of secondary metabolites, which negatively affected FTC. No diet mediated interactions with microsporidia were observed in Experiment 2. 4. Diet quality (Experiments 1 and 2), diet quantity (Experiment 2) and microsporidia infection (Experiments 1 and 2) directly influenced FTC survival and/or had sublethal effects on FTC that may have cascading effects on population dynamics and dispersal. 5. We demonstrated that diet quality can mediate interactions between FTC and microsporidia, but these interactions depend on the defensive chemistry of the FTC diet. To our knowledge, this is the first study to report diet-mediated interactions between an outbreaking forest pest and microsporidia and one of only a few studies to examine this tri-trophic interaction among Lepidoptera.
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    Sex-specific effects of capital resources on reproductive timing and success in red squirrels
    (2022) Haines, Jessica A.; Delaney, David M.; Wishart, Andrea E.; McAdam, Andrew G.; Coltman, David W.; Lane, Jeffrey E.; Dantzer, Ben; Boutin, Stan
    Reproduction is an energetically expensive activity for both sexes. However, if males and females differ in their annual timing of reproduction, such that peak investment for one sex occurs during a more resource-limited period, there is an opportunity for sex-specific selection to act on the acquisition of energetic resources. Both male and female North American red squirrels (Tamiasciurus hudsonicus) cache conifer cones, although males typically have larger caches than females. Peak energetic investment in reproduction occurs for males during the mating season in winter and early spring (when squirrels rely almost exclusively on cached resources) and for females during lactation (which can align with fresh food availability). We provide evidence that suggests sex differences in cache size are likely driven by a stronger positive connection between cached resources and components of fitness for males than for females. Specifically, males with larger caches have greater siring success than males with smaller caches, whereas for females, only early breeding females experience a positive effect of cache size on the number of recruits produced. We also show that males sire pups and females give birth earlier in the year if they have larger caches compared with squirrels of the same sex with smaller caches. Sexual selection can thus extend beyond traits directly connected to mating behavior, and can act on traits related to acquiring resources needed to fuel reproduction that are expressed months or years in advance of breeding efforts.
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    Sexual dimorphism in the badlands cricket (Orthoptera, Gryllinae, Gryllus personatus)
    (2023) Madera, Esperance M.; Judge, Kevin
    Sexual dimorphism (SD) is a common phenomenon in sexual species and can manifest in a variety of ways. Sexual size dimorphism (SSD) is commonly investigated, but it can be confounded with sexual shape dimorphism (SShD) if multivariate measures of size are not used. Univariate studies may also overestimate the prevalence or direction of SSD when the sexes are strikingly different in shape, which may be an issue in taxa such as Orthoptera and other terrestrial arthropods where maximum body size is strongly constrained. Here we tested for the occurrence of both SSD and SShD in the badlands cricket Gryllus personatus (Orthoptera, Gryllinae). We measured four body size dimensions—maxillae span, head width, pronotum length, and mean hind femur length—and used multivariate methods to test whether male and female adult badlands crickets were sexually dimorphic in size and/or shape. All the univariate dimensions were sexually dimorphic, with males having wider heads and maxillae than females and females having longer pronota and hind femora than males, which indicates SShD. However, multivariate methods failed to detect SSD, instead confirming that the sexes primarily differ in body shape. We show how a simple ratio of head width to pronotum length captures SShD in badlands crickets and apply it to iNaturalist, a citizen science platform, to broaden our findings. We propose that orthopterists studying SD minimally measure head width, pronotum length, and hind femur length as a standard that will allow a more repeatable and generalizable assessment of the prevalence and direction of both SSD and SShD.
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    Five things I learned from squirrels
    (2023) Haines, Jessica A.
    I’m a wildlife biologist with a passion for squirrels. I’m currently working on Franklin’s ground squirrels in collaboration with Nature Alberta (check out the Winter 2023 edition of Nature Alberta Magazine for more info on that project). But before that, I spent several years working on red squirrels with the Kluane Red Squirrel Project based in the Yukon. Living and working in such a beautiful, remote place was thrilling, but what surprised me was how much I fell in love with red squirrels. They taught me a lot, and I would like to share with you some of the things I’ve learned.
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    Nature kids: my big Alberta backyard - tracking animals in winter
    (2023) Haines, Jessica A.
    Alberta is a great place to live. It’s a big, beautiful province full of all kinds of natural wonders. In My Big Alberta Backyard, we introduce you to the unique and interesting wild spaces that you can find in your province, and the diverse wildlife that live there. This time, let’s talk about tracking animals in the winter.
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    Squirrels of Alberta
    (2023) Haines, Jessica A.; Kaplan, Cora
    Squirrel identification guide covering the 14 species of squirrels in Alberta.
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    The value of team-based learning in a pandemic and five simple tips to get started
    (2023) Hills, Melissa
    Team-Based Learning (TBL) can be easily applied to different learning outcomes in various courses. This approach builds community and provides peer support for students in both in-person and online learning environments. When used for formative assessment, it can promote student learning while reducing the quantity of grading for instructors. Five simple tips to provide structure and flexibility for the successful implementation of Team-Based Learning are described using an example of a recent second-year principles of genetics course.
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    BMP3 is a novel locus involved in the causality of ocular coloboma
    (2022) Fox, Sabrina C.; Widen, Sonya A.; Asai-Coakwell, Mika; Havrylov, Serhiy; Benson, Matthew; Prichard, Lisa; Baddam, Pranidhi; Graf, Daniel; Lehmann, Ordan J.; Waskiewicz, Andrew J.
    Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, these data suggest that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.
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    Global urban environmental change drives adaptation in white clover
    (2022) Santangelo, James S.; Ness, Rob W.; Cohan, Beata; Flaherty, Leah
    Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.
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    User testing for serious game design: improving the player experience
    (2022) Shaw, Ross; Sperano, Isabelle; Andruchow, Robert; Cobzas, Dana
    This case study reflects on our use of user testing during a research project in which we designed a serious video game, “Life on the Edge.” The target audience of the game is first-year post-secondary biology students. As we designed the game, user testing was a critical component that allowed us to identify issues. Any issues that interfere with the flow or enjoyment of a video game can be distracting to players. In what follows, we will describe the research design and discuss the processes for testing a serious video game that will allow you to identify game issues successfully. How you recruit participants, test players, and prioritize player feedback is a component of effective user testing and improving your game. With user testing, we were able to identify problems in the game, prioritize them, and address them. By using variable user testing methods, you can adapt to the changing needs of your game project and develop a successful serious video game.
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    Ancient hybridization patterns between bighorn and thinhorn sheep
    (2021) Santos, Sarah H. D.; Peery, Rhiannon M.; Miller, Joshua M.; Dao, Anh; Lyu, Feng-Hua; Li, Xin; Li, Meng-Hua; Coltman, David W.
    Whole-genome sequencing has advanced the study of species evolution, including the detection of genealogical discordant events such as ancient hybridization and incomplete lineage sorting (ILS). The evolutionary history of bighorn (Ovis canadensis) and thinhorn (Ovis dalli) sheep present an ideal system to investigate evolutionary discordance due to their recent and rapid radiation and putative secondary contact between bighorn and thinhorn sheep subspecies, specifically the dark pelage Stone sheep (O. dalli stonei) and predominately white Dall sheep (O. dalli dalli), during the last ice age. Here, we used multiple genomes of bighorn and thinhorn sheep, together with snow (O. nivicola) and the domestic sheep (O. aries) as outgroups, to assess their phylogenomic history, potential introgression patterns and their adaptive consequences. Among the Pachyceriforms (snow, bighorn and thinhorn sheep) a consistent monophyletic species tree was retrieved; however, many genealogical discordance patterns were observed. Alternative phylogenies frequently placed Stone and bighorn as sister clades. This relationship occurred more often and was less divergent than that between Dall and bighorn. We also observed many blocks containing introgression signal between Stone and bighorn genomes in which coat colour genes were present. Introgression signals observed between Dall and bighorn were more random and less frequent, and therefore probably due to ILS or intermediary secondary contact. These results strongly suggest that Stone sheep originated from a complex series of events, characterized by multiple, ancient periods of secondary contact with bighorn sheep.
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    A phylogeographic contact zone for Arctic grayling (Thymallus arcticus) in Alberta, Canada
    (2021) Reilly, Jessica R.; Miller, Joshua M.
    Arctic Grayling Thymallus arcticus are a salmonid with a Holarctic distribution, extending from north-eastern Eurasia through north-western North America. Throughout their range, Arctic Grayling face a number of threats including angling mortality, habitat fragmentation and loss and climate change. Thus, there is a need to protect the species through targeted management actions. Genetic information can assist in determining the appropriate scale for these actions through description of Designatable Units (DUs). Here we use newly collected mitochondrial DNA sequence data to assess the phylogeographic structure of Arctic Grayling in Alberta, Canada and link these with previously collected mitochondrial and microsatellite data to determine how many DUs may exist across Canada. Our assessment of 831 base pairs of sequence data in 96 individuals found two deeply divergent lineages in Alberta. When combined with 22 previously collected sequences our results highlight that Alberta is a contact zone for the observed lineages of Arctic Grayling in North America. Reassessment of nine microsatellites genotyped in 1,116 individuals further highlighted inter-basin divergence, likely the result of historical processes. Given the divergence and geographic distribution of the genetic diversity, Arctic Grayling in Canada merit consideration for separate DUs in future species status assessments and management plans. Continuing research should aim to expand sampling geographically (e.g. regions east of Great Slave Lake and along the Arctic coastline) to clarify possible colonization routes, and add to or synthesize work on Arctic Grayling behaviour, morphology, and life-history to address the limited understanding of local adaptions within this species.
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    The crucial role of genome-wide genetic variation in conservation
    (2021) Kardos, Marty; Armstrong, Ellie E.; Fitzpatrick, Sarah; Hauser, Samantha; Hedrick, Philip W.; Miller, Joshua M.; Tallmon, David A.; Funk, W. Chris
    The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on conserving genome-wide genetic variation, and that the field should instead focus on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations towards extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide genetic variation on long-term population viability will only worsen the biodiversity crisis.
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    Linking genetic, morphological, and behavioural divergence between inland island and mainland deer mice
    (2022) Miller, Joshua M.; Garant, Dany; Perrier, Charles; Juette, Tristan; Jameson, Joël W.; Normandeau, Eric; Bernatchez, Louis; Réale, Denis
    The island syndrome hypothesis (ISH) stipulates that, as a result of local selection pressures and restricted gene flow, individuals from island populations should differ from individuals within mainland populations. Specifically, island populations are predicted to contain individuals that are larger, less aggressive, more sociable, and that invest more in their offspring. To date, tests of the ISH have mainly compared oceanic islands to continental sites, and rarely smaller spatial scales such as inland watersheds. Here, using a novel set of genome-wide SNP markers in wild deer mice (Peromyscus maniculatus) we conducted a genomic assessment of predictions underlying the ISH in an inland riverine island system: analysing island-mainland population structure, and quantifying heritability of phenotypes thought to underlie the ISH. We found clear genomic differentiation between island and mainland populations and moderate to high marker based heritability estimates for overall variation in traits previously found to differ in line with the ISH between mainland and island locations. FST outlier analyses highlighted 12 loci associated with differentiation between mainland and island populations. Together these results suggest that the island populations examined are on independent evolutionary trajectories, the traits considered have a genetic basis (rather than phenotypic variation being solely due to phenotypic plasticity). Coupled with the previous results showing significant phenotypic differentiation between island and mainland groups in this system, this study suggests that the ISH can hold even on a small spatial scale.
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    Ewe are what ewe wear: bigger horns, better ewes and the potential consequence of trophy hunting on female fitness in bighorn sheep
    (2022) Deakin, Samuel; Festa-Bianchet, Marco; Miller, Joshua M.; Pelletier, Fanie; Coltman, David W.
    In polygynous species, secondary sexual traits such as weapons or elaborate ornaments have evolved through intrasexual competition for mates. In some species, these traits are present in both sexes but are underdeveloped in the sex facing lower intrasexual competition for mates. It is often assumed that these underdeveloped sexually selected traits are a vestige of strong sexual selection on the other sex. Here, we challenge this assumption and investigate whether the expression of secondary sexual traits is associated with fitness in female bighorn sheep. Analyses of 45 years of data revealed that female horn length at 2 years, while accounting for mass and environmental variables, is associated with younger age at primiparity, younger age of first offspring weaned, greater reproductive lifespan and higher lifetime reproductive success. There was no association between horn length and fecundity. These findings highlight a potential conservation issue. In this population, trophy hunting selects against males with fast-growing horns. Intersexual genetic correlations imply that intense selective hunting of large-horned males before they can reproduce can decrease female horn size. Therefore, intense trophy hunting of males based on horn size could reduce female reproductive performance through the associations identified here, and ultimately reduce population growth and viability.
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    Global genetic diversity status and trends:towards a suite of Essential BiodiversityVariables (EBVs) for genetic composition
    (2022) Hoban, Sean; Archer, Frederick I.; Bertola, Laura D.; Bragg, Jason G.; Breed, Martin F.; Bruford, Michael W.; Coleman, Melinda A.; Ekblom, Robert; Funk, W. Chris; Grueber, Catherine E.; Hand, Brian K.; Jaffé, Rodolfo; Jensen, Evelyn L.; Johnson, Jeremy S.; Kershaw, Francine; Liggins, Libby; MacDonald, Anna J.; Mergeay, Joachim; Miller, Joshua M.; Muller-Karger, Frank; O'Brien, David; Paz-Vinas, Ivan; Potter, Kevin M.; Razgour, Orly; Vernesi, Cristiano; Hunter, Margaret E.
    Biodiversity underlies ecosystem resilience, ecosystem function, sustainable economies, and human well-being. Understanding how biodiversity sustains ecosystems under anthropogenic stressors and global environmental change will require new ways of deriving and applying biodiversity data. A major challenge is that biodiversity data and knowledge are scattered, biased, collected with numerous methods, and stored in inconsistent ways. The Group on Earth Observations Biodiversity Observation Network (GEO BON) has developed the Essential Biodiversity Variables (EBVs) as fundamental metrics to help aggregate, harmonize, and interpret biodiversity observation data from diverse sources. Mapping and analyzing EBVs can help to evaluate how aspects of biodiversity are distributed geographically and how they change over time. EBVs are also intended to serve as inputs and validation to forecast the status and trends of biodiversity, and to support policy and decision making. Here, we assess the feasibility of implementing Genetic Composition EBVs (Genetic EBVs), which are metrics of within-species genetic variation. We review and bring together numerous areas of the field of genetics and evaluate how each contributes to global and regional genetic biodiversity monitoring with respect to theory, sampling logistics, metadata, archiving, data aggregation, modeling, and technological advances. We propose four Genetic EBVs: (i) Genetic Diversity; (ii) Genetic Differentiation; (iii) Inbreeding; and (iv)Effective Population Size (Ne). We rank Genetic EBVs according to their relevance, sensitivity to change, generalizability, scalability, feasibility and data availability. We outline the workflow for generating genetic data underlying the GeneticEBVs and review advances and needs in archiving genetic composition data and metadata. We discuss how GeneticEBVs can be operationalized by visualizing EBVs in space and time across species and by forecasting Genetic EBVsbeyond current observations using various modeling approaches. Our review then explores challenges of aggregation, standardization, and costs of operationalizing the Genetic EBVs, as well as future directions and opportunities to maximize their uptake globally in research and policy. The collection, annotation, and availability of genetic data has made major advances in the past decade, each of which contributes to the practical and standardized framework for large-scale genetic observation reporting. Rapid advances in DNA sequencing technology present new opportunities, but also challenges for operationalizing Genetic EBVs for biodiversity monitoring regionally and globally. With these advances, genetic composition monitoring is starting to be integrated into global conservation policy, which can help support the foundation of all biodiversity and species’ long-term persistence in the face of environmental change. We conclude with a summary of concrete steps for researchers and policymakers for advancing operationalization of Genetic EBVs. The technical and analytical foundations of Genetic EBVs are well developed, and conservation practitioners should anticipate their increasing application as efforts emerge to scale up genetic biodiversity monitoring regionally and globally.
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    A new lineage of Galapagos giant tortoises identified from museum samples
    (2022) Jensen, Evelyn L.; Quinzin, Maud C.; Miller, Joshua M.; Russello, Michael A.; Garrick, Ryan C.; Edwards, Danielle L.; Glaberman, Scott; Chiari, Ylenia; Poulakakis, Nikos; Tapia, Washington; Gibbs, James P.; Caccone, Adalgisa
    The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.