Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

"Type D" killer whale genomes reveal long-term small population size and low genetic diversity.

Genome sequences can reveal the extent of inbreeding in small populations. Here, we present the first genomic characterization of type D killer whales, a distinctive eco/morphotype with a circumpolar, subantarctic distribution. Effective population size is the lowest estimated from any killer whale genome and indicates a severe population bottleneck. Consequently, type D genomes show among the highest level of inbreeding reported for any mammalian species (FROH ≥ 0.65). Detected recombination cross-over events of different haplotypes are up to an order of magnitude rarer than in other killer whale genomes studied to date. Comparison of genomic data from a museum specimen of a type D killer whale that stranded in New Zealand in 1955, with 3 modern genomes from the Cape Horn area, reveals high covariance and identity-by-state of alleles, suggesting these genomic characteristics and demographic history are shared among geographically dispersed social groups within this morphotype. Limitations to the insights gained in this study stem from the nonindependence of the 3 closely related modern genomes, the recent coalescence time of most variation within the genomes, and the nonequilibrium population history which violates the assumptions of many model-based methods. Long-range linkage disequilibrium and extensive runs of homozygosity found in type D genomes provide the potential basis for both the distinctive morphology, and the coupling of genetic barriers to gene flow with other killer whale populations.

Limited movement of an avian hybrid zone in relation to regional variation in magnitude of climate change.

Studies of natural hybrid zones can provide documentation of range shifts in response to climate change and identify loci important to reproductive isolation. Using a temporal (36-38 years) comparison of the black-capped (Poecile atricapillus) and Carolina (P. carolinensis) chickadee hybrid zone, we investigated movement of the western portion of the zone (western Missouri) and assessed whether loci and pathways underpinning reproductive isolation were similar to those in the eastern portion of the hybrid zone. Using 92 birds sampled along the hybrid zone transect in 2016 and 68 birds sampled between 1978 and 1980, we generated 11,669 SNPs via ddRADseq. These SNPs were used to assess movement of the hybrid zone through time and to evaluate variation in introgression among loci. We demonstrate that the interface has moved ~5 km to the northwest over the last 36-38 years, that is, at only one-fifth the rate at which the eastern portion (e.g., Pennsylvania, Ohio) of the hybrid zone has moved. Temperature trends over the last 38 years reveal that eastern areas have warmed 50% more than western areas in terms of annual mean temperature, possibly providing an explanation for the slower movement of the hybrid zone in Missouri. Our results suggest hybrid zone movement in broadly distributed species, such as chickadees, will vary between areas in response to local differences in the impacts of climate change.

Population Genomics of New Zealand Pouched Lamprey (kanakana; piharau; Geotria australis).

Pouched lamprey (Geotria australis) or kanakana/piharau is a culturally and ecologically significant jawless fish that is distributed throughout Aotearoa New Zealand. Despite its importance, much remains unknown about historical relationships and gene flow between populations of this enigmatic species within New Zealand. To help inform management, we assembled a draft G. australis genome and completed the first comprehensive population genomics analysis of pouched lamprey within New Zealand using targeted gene sequencing (Cyt-b and COI) and restriction site-associated DNA sequencing (RADSeq) methods. Employing 16 000 genome-wide single nucleotide polymorphisms (SNPs) derived from RADSeq (n = 186) and sequence data from Cyt-b (766 bp, n = 94) and COI (589 bp, n = 20), we reveal low levels of structure across 10 sampling locations spanning the species range within New Zealand. F-statistics, outlier analyses, and STRUCTURE suggest a single panmictic population, and Mantel and EEMS tests reveal no significant isolation by distance. This implies either ongoing gene flow among populations or recent shared ancestry among New Zealand pouched lamprey. We can now use the information gained from these genetic tools to assist managers with monitoring effective population size, managing potential diseases, and conservation measures such as artificial propagation programs. We further demonstrate the general utility of these genetic tools for acquiring information about elusive species.

Receding ice drove parallel expansions in Southern Ocean penguins.

Climate shifts are key drivers of ecosystem change. Despite the critical importance of Antarctica and the Southern Ocean for global climate, the extent of climate-driven ecological change in this region remains controversial. In particular, the biological effects of changing sea ice conditions are poorly understood. We hypothesize that rapid postglacial reductions in sea ice drove biological shifts across multiple widespread Southern Ocean species. We test for demographic shifts driven by climate events over recent millennia by analyzing population genomic datasets spanning 3 penguin genera (Eudyptes, Pygoscelis, and Aptenodytes). Demographic analyses for multiple species (macaroni/royal, eastern rockhopper, Adélie, gentoo, king, and emperor) currently inhabiting southern coastlines affected by heavy sea ice conditions during the Last Glacial Maximum (LGM) yielded genetic signatures of near-simultaneous population expansions associated with postglacial warming. Populations of the ice-adapted emperor penguin are inferred to have expanded slightly earlier than those of species requiring ice-free terrain. These concerted high-latitude expansion events contrast with relatively stable or declining demographic histories inferred for 4 penguin species (northern rockhopper, western rockhopper, Fiordland crested, and Snares crested) that apparently persisted throughout the LGM in ice-free habitats. Limited genetic structure detected in all ice-affected species across the vast Southern Ocean may reflect both rapid postglacial colonization of subantarctic and Antarctic shores, in addition to recent genetic exchange among populations. Together, these analyses highlight dramatic, ecosystem-wide responses to past Southern Ocean climate change and suggest potential for further shifts as warming continues.

Runs of homozygosity in killer whale genomes provide a global record of demographic histories.

Runs of homozygosity (ROH) occur when offspring inherit haplotypes that are identical by descent from each parent. Length distributions of ROH are informative about population history; specifically, the probability of inbreeding mediated by mating system and/or population demography. Here, we investigated whether variation in killer whale (Orcinus orca) demographic history is reflected in genome-wide heterozygosity and ROH length distributions, using a global data set of 26 genomes representative of geographic and ecotypic variation in this species, and two F1 admixed individuals with Pacific-Atlantic parentage. We first reconstructed demographic history for each population as changes in effective population size through time using the pairwise sequential Markovian coalescent (PSMC) method. We found a subset of populations declined in effective population size during the Late Pleistocene, while others had more stable demography. Genomes inferred to have undergone ancestral declines in effective population size, were autozygous at hundreds of short ROH (<1 Mb), reflecting high background relatedness due to coalescence of haplotypes deep within the pedigree. In contrast, longer and therefore younger ROH (>1.5 Mb) were found in low latitude populations, and populations of known conservation concern. These include a Scottish killer whale, for which 37.8% of the autosomes were comprised of ROH >1.5 Mb in length. The fate of this population, in which only two adult males have been sighted in the past five years, and zero fecundity over the last two decades, may be inextricably linked to its demographic history and consequential inbreeding depression.

Roe deer on ice: Selection despite limited effective population size through the Pleistocene.

Roe deer (Capreolus spp.) are a little odd. They are one of only a few placental mammals-and the only genus among even-toed ungulates-capable of putting embryonic development "on ice", also known as embryonic diapause (Figure 1). It would seem such an unusual trait is probably the product of natural selection, but a big question is, how does selection for important traits, such as diapause, interact with the historical demography of a species? In a 'From the Cover' article in this issue of Molecular Ecology, de Jong et al. (2020) demonstrate that selection is acting on genes associated with reproductive biology in roe deer, despite heightened genetic drift due to reduced effective population size through the Pleistocene.

The association between mitochondrial genetic variation and reduced colony fitness in an invasive wasp.

Despite the mitochondrion's long-recognized role in energy production, mitochondrial DNA (mtDNA) variation commonly found in natural populations was assumed to be effectively neutral. However, variation in mtDNA has now been increasingly linked to phenotypic variation in life history traits and fitness. We examined whether the relative fitness in native and invasive common wasp (Vespula vulgaris) populations in Belgium and New Zealand (NZ), respectively, can be linked to mtDNA variation. Social wasp colonies in NZ were smaller with comparatively fewer queen cells, indicating a reduced relative fitness in the invaded range. Interestingly, queen cells in this population were significantly larger leading to larger queen offspring. By sequencing 1,872 bp of the mitochondrial genome, we determined mitochondrial haplotypes and detected reduced genetic diversity in NZ. Three common haplotypes in NZ frequently produced many queens, whereas the four rare haplotypes produced significantly fewer or no queens. The entire mitochondrial genome for each of these haplotypes was sequenced to identify polymorphisms associated with fitness reduction. We found 16 variable sites; however, no nonsynonymous mutation that was clearly causing impaired mitochondrial function was detected. We discuss how detected variants may alter secondary structures, gene expression or mito-nuclear interactions, or could be associated with nuclear-encoded variation. Whatever the ultimate mechanism, we show reduced fitness and mtDNA variation in an invasive wasp population as well as specific mtDNA variants associated with fitness variation within this population. Ours is one of only a few studies that confirm fitness impacts of mtDNA variation in wild nonmodel populations.

Demography or selection on linked cultural traits or genes? Investigating the driver of low mtDNA diversity in the sperm whale using complementary mitochondrial and nuclear genome analyses.

Mitochondrial DNA has been heavily utilized in phylogeography studies for several decades. However, underlying patterns of demography and phylogeography may be misrepresented due to coalescence stochasticity, selection, variation in mutation rates and cultural hitchhiking (linkage of genetic variation to culturally-transmitted traits affecting fitness). Cultural hitchhiking has been suggested as an explanation for low genetic diversity in species with strong social structures, counteracting even high mobility, abundance and limited barriers to dispersal. One such species is the sperm whale, which shows very limited phylogeographic structure and low mtDNA diversity despite a worldwide distribution and large population. Here, we use analyses of 175 globally distributed mitogenomes and three nuclear genomes to evaluate hypotheses of a population bottleneck/expansion vs. a selective sweep due to cultural hitchhiking or selection on mtDNA as the mechanism contributing to low worldwide mitochondrial diversity in sperm whales. In contrast to mtDNA control region (CR) data, mitogenome haplotypes are largely ocean-specific, with only one of 80 shared between the Atlantic and Pacific. Demographic analyses of nuclear genomes suggest low mtDNA diversity is consistent with a global reduction in population size that ended approximately 125,000 years ago, correlated with the Eemian interglacial. Phylogeographic analysis suggests that extant sperm whales descend from maternal lineages endemic to the Pacific during the period of reduced abundance and have subsequently colonized the Atlantic several times. Results highlight the apparent impact of past climate change, and suggest selection and hitchhiking are not the sole processes responsible for low mtDNA diversity in this highly social species.

The Influence of Selection on MHC DQA and DQB Haplotypes in the Endemic New Zealand Hector's and Maui Dolphins.

Strong balancing selection on the major histocompatibility complex (MHC) can lead to different patterns in gene frequencies and neutral genomic variation within species. We investigated diversity and geographic structure of MHC genes DQA and DQB, as well as their inferred functional haplotypes, from 2 regional populations (East and West Coast) of the endangered Hector's dolphin (Cephalorhynchus hectori hectori) and the critically endangered Maui dolphin (Cephalorhynchus hectori maui) (West Coast, North Island), and contrasted these results with patterns from neutral microsatellites. The Maui had the lowest number of alleles for DQA (2) and DQB (3), consistent with strong genetic drift acting on this remnant population. However, the 2 retained DQA alleles are among the most divergent combinations of all 4 alleles found across the Hector's metapopulation, potentially reflecting the retention of divergent alleles due to balancing selection. The high frequency of the divergent DQB*04 allele also gave this population the highest nucleotide diversity for DQB. Strong differentiation was evident for DQA, DQB, and DQA-DQB haplotypes between the regional populations of Hector's dolphins (FST > 0.213) and both subspecies (FST > 0.311). Differentiation was generally greater than observed at neutral microsatellite loci, suggesting the influence of selection between geographically proximate East and West Coast populations. This might be the result of spatial differences in directional selection on those opposite coastlines. In addition, measures of the ratio of nonsynonymous to synonymous substitutions (dN/dS) were consistent with balancing selection over evolutionary time. Together, these results suggest a complex interplay of balancing selection, directional selection, local fidelity, and genetic drift.

Species delimitation with gene flow: A methodological comparison and population genomics approach to elucidate cryptic species boundaries in Malaysian Torrent Frogs.

Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single-nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent-based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2, ABBA-BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean Fst  = 0.9) to differentiating populations involving recent gene flow (mean Fst  = 0.05; Nm  > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.

The Eurasian invasion: phylogenomic data reveal multiple Southeast Asian origins for Indian Dragon Lizards.

BACKGROUND: The Indian Tectonic Plate split from Gondwanaland approximately 120 MYA and set the Indian subcontinent on a ~ 100 million year collision course with Eurasia. Many phylogenetic studies have demonstrated the Indian subcontinent brought with it an array of endemic faunas that evolved in situ during its journey, suggesting this isolated subcontinent served as a source of biodiversity subsequent to its collision with Eurasia. However, recent molecular studies suggest that Eurasia may have served as the faunal source for some of India's biodiversity, colonizing the subcontinent through land bridges between India and Eurasia during the early to middle Eocene (~35-40 MYA). In this study we investigate whether the Draconinae subfamily of the lizard family Agamidae is of Eurasian or Indian origin, using a multi locus Sanger dataset and a novel dataset of 4536 ultraconserved nuclear element loci. RESULTS: Results from our phylogenetic and biogeographic analyses revealed support for two independent colonizations of India from Eurasian ancestors during the early to late Eocene prior to the subcontinent's hard collision with Eurasia. CONCLUSION: These results are consistent with other faunal groups and new geologic models that suggest ephemeral Eocene land bridges may have allowed for dispersal and exchange of floras and faunas between India and Eurasia during the Eocene.

What influences the worldwide genetic structure of sperm whales (Physeter macrocephalus)?

The interplay of natural selection and genetic drift, influenced by geographic isolation, mating systems and population size, determines patterns of genetic diversity within species. The sperm whale provides an interesting example of a long-lived species with few geographic barriers to dispersal. Worldwide mtDNA diversity is relatively low, but highly structured among geographic regions and social groups, attributed to female philopatry. However, it is unclear whether this female philopatry is due to geographic regions or social groups, or how this might vary on a worldwide scale. To answer these questions, we combined mtDNA information for 1091 previously published samples with 542 newly obtained DNA profiles (394-bp mtDNA, sex, 13 microsatellites) including the previously unsampled Indian Ocean, and social group information for 541 individuals. We found low mtDNA diversity (π = 0.430%) reflecting an expansion event <80 000 years bp, but strong differentiation by ocean, among regions within some oceans, and among social groups. In comparison, microsatellite differentiation was low at all levels, presumably due to male-mediated gene flow. A hierarchical amova showed that regions were important for explaining mtDNA variance in the Indian Ocean, but not Pacific, with social group sampling in the Atlantic too limited to include in analyses. Social groups were important in partitioning mtDNA and microsatellite variance within both oceans. Therefore, both geographic philopatry and social philopatry influence genetic structure in the sperm whale, but their relative importance differs by sex and ocean, reflecting breeding behaviour, geographic features and perhaps a more recent origin of sperm whales in the Pacific. By investigating the interplay of evolutionary forces operating at different temporal and geographic scales, we show that sperm whales are perhaps a unique example of a worldwide population expansion followed by rapid assortment due to female social organization.

A framework for identifying fertility gene targets for mammalian pest control.

Fertility-targeted gene drives have been proposed as an ethical genetic approach for managing wild populations of vertebrate pests for public health and conservation benefit. This manuscript introduces a framework to identify and evaluate target gene suitability based on biological gene function, gene expression and results from mouse knockout models. This framework identified 16 genes essential for male fertility and 12 genes important for female fertility that may be feasible targets for mammalian gene drives and other non-drive genetic pest control technology. Further, a comparative genomics analysis demonstrates the conservation of the identified genes across several globally significant invasive mammals. In addition to providing important considerations for identifying candidate genes, our framework and the genes identified in this study may have utility in developing additional pest control tools such as wildlife contraceptives.

Job prospects and career pathways for human anatomy graduates from the University of Otago: Implications for student support and professional development.

The Department of Anatomy (Anatomy) at the University of Otago delivers programs for students in diverse areas, including clinical anatomy, neuroscience, reproduction and biological anthropology. This study explored the experiences of alumni during their study and career pathways post-graduation through an online questionnaire distributed to department alumni. Most of the 190 participants studied anatomy as undergraduates (74.2%) and graduated in the past decade (56.8%). Reasons for taking anatomy included finding the topic interesting, a pathway into professional programs, or a degree requirement. Current employment differed between undergraduate (44.7% currently employed in clinical settings) and postgraduate alumni (26.4% currently employed in research, 19.5% in clinical settings). The main pathways for finding jobs were by direct search (38.6%), completing tertiary education (29.2%), and through social network connections (16.4%). Women alumni were less likely to feel that Anatomy prepared them for their careers than men. Themes related to positive and negative experiences included staff, course material/resources, social events, and peers. Suggestions to improve the departmental "sense of community" included increasing departmental events and resources. Alumni suggested that Anatomy should provide more potential career information, make available recent alumni profiles, and organize career fairs and networking opportunities. Postgraduate alumni were more likely to feel a "sense of belonging" in Anatomy than undergraduate alumni. Findings from this research provide an essential data point in the international evaluation of career prospects of anatomy graduates and provide a road map for other institutions to survey their alumni to obtain local insights.

A framework for identifying fertility gene targets for mammalian pest control.

Fertility-targeted gene drives have been proposed as an ethical genetic approach for managing wild populations of vertebrate pests for public health and conservation benefit.This manuscript introduces a framework to identify and evaluate target gene suitability based on biological gene function, gene expression, and results from mouse knockout models.This framework identified 16 genes essential for male fertility and 12 genes important for female fertility that may be feasible targets for mammalian gene drives and other non-drive genetic pest control technology. Further, a comparative genomics analysis demonstrates the conservation of the identified genes across several globally significant invasive mammals.In addition to providing important considerations for identifying candidate genes, our framework and the genes identified in this study may have utility in developing additional pest control tools such as wildlife contraceptives.

The admixed brushtail possum genome reveals invasion history in New Zealand and novel imprinted genes.

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits.

Paternity assignment and demographic closure in the New Zealand southern right whale.

The identification and characterization of reproductively isolated subpopulations or 'stocks' are essential for effective conservation and management decisions. This can be difficult in vagile marine species like marine mammals. We used paternity assignment and 'gametic recapture' to examine the reproductive autonomy of southern right whales (Eubalaena australis) on their New Zealand (NZ) calving grounds. We derived DNA profiles for 34 mother-calf pairs from skin biopsy samples, using sex-specific markers, 13 microsatellite loci and mtDNA haplotypes. We constructed DNA profiles for 314 adult males, representing 30% of the census male abundance of the NZ stock, previously estimated from genotypic mark-recapture modelling to be 1085 (95% CL 855, 1416). Under the hypothesis of demographic closure and the assumption of equal reproductive success among males, we predict: (i) the proportion of paternities assigned will reflect the proportion of the male population sampled and (ii) the gametic mark-recapture (GMR) estimate of male abundance will be equivalent to the census male estimate for the NZ stock. Consistent with these predictions, we found that the proportion of assigned paternities equalled the proportion of the census male population size sampled. Using the sample of males as the initial capture, and paternity assignment as the recapture, the GMR estimate of male abundance was 1001 (95% CL 542, 1469), similar to the male census estimate. These findings suggest that right whales returning to the NZ calving ground are reproductively autonomous on a generational timescale, as well as isolated by maternal fidelity on an evolutionary timescale, from others in the Indo-Pacific region.

Commonly used Hardy-Weinberg equilibrium filtering schemes impact population structure inferences using RADseq data.

Reduced representation sequencing (RRS) is a widely used method to assay the diversity of genetic loci across the genome of an organism. The dominant class of RRS approaches assay loci associated with restriction sites within the genome (restriction site associated DNA sequencing, or RADseq). RADseq is frequently applied to non-model organisms since it enables population genetic studies without relying on well-characterized reference genomes. However, RADseq requires the use of many bioinformatic filters to ensure the quality of genotyping calls. These filters can have direct impacts on population genetic inference, and therefore require careful consideration. One widely used filtering approach is the removal of loci that do not conform to expectations of Hardy-Weinberg equilibrium (HWE). Despite being widely used, we show that this filtering approach is rarely described in sufficient detail to enable replication. Furthermore, through analyses of in silico and empirical data sets we show that some of the most widely used HWE filtering approaches dramatically impact inference of population structure. In particular, the removal of loci exhibiting departures from HWE after pooling across samples significantly reduces the degree of inferred population structure within a data set (despite this approach being widely used). Based on these results, we provide recommendations for best practice regarding the implementation of HWE filtering for RADseq data sets.

Ultraconserved element (UCE) probe set design: Base genome and initial design parameters critical for optimization.

Targeted capture and enrichment approaches have proven effective for phylogenetic study. Ultraconserved elements (UCEs) in particular have exhibited great utility for phylogenomic analyses, with the software package phyluce being among the most utilized pipelines for UCE phylogenomics, including probe design. Despite the success of UCEs, it is becoming increasing apparent that diverse lineages require probe sets tailored to focal taxa in order to improve locus recovery. However, factors affecting probe design and methods for optimizing probe sets to focal taxa remain underexplored. Here, we use newly available beetle (Coleoptera) genomic resources to investigate factors affecting UCE probe set design using phyluce. In particular, we explore the effects of stringency during initial design steps, as well as base genome choice on resulting probe sets and locus recovery. We found that both base genome choice and initial bait design stringency parameters greatly alter the number of resultant probes included in final probe sets and strongly affect the number of loci detected and recovered during in silico testing of these probe sets. In addition, we identify attributes of base genomes that correlated with high performance in probe design. Ultimately, we provide a recommended workflow for using phyluce to design an optimized UCE probe set that will work across a targeted lineage, and use our findings to develop a new, open-source UCE probe set for beetles of the suborder Adephaga.