Caleb J. Krueger

The spotted turtle (Clemmys guttata) is the sole extant member of the genus Clemmys and can be found in shallow wetlands across eastern North America. In recent decades, habitat alterations and poaching have reduced spotted turtle populations by more than 50% range-wide, resulting in federal protection in Canada and state-level protection within the United States. Here, we provide an annotated, chromosome-level assembly of C. guttata derived from long- and ultra-long read data as an addition to the conservation genomics resources for the species. The assembly comprises 2,286,936,868 base pairs, 98.5% of which are assembled in 25 contigs, consistent with published karyotypic data (n = 25) and including 16 gapless, telomere-to-telomere chromosome sequences. BUSCO scores indicate a highly complete assembly (99.4%). Annotation of the genome yielded 21,335 protein-coding genes with a BUSCO completeness score of 98.4%. Comparisons with a separate assembly derived from long-read and Hi-C data showed that substituting ultra-long for Hi-C sequencing can improve telomere assembly and reduce structural errors in this taxon at the cost of reduced haplotype phasing, though primary assemblies are highly syntenic overall. PSMC analyses of both assemblies independently revealed a long, essentially identical history of population declines in spotted turtles. Coupled with range-wide genomic sampling, these resources will provide critical data to understand genetic structure, patterns of local adaptation, and demographic history in this species and help stem additional declines.

Cellular hallmarks of aging have been discovered and characterized in a number of model species for studying aging biology - such as humans, mice, fruit flies, and nematodes. Whether these canonical age-related changes to cellular physiology are present across diverse species that have variable rates of demographic aging remains less studied. Here, we tested whether several ubiquitous cellular hallmarks of aging - mitochondrial function, reactive oxygen species generation, and inducible DNA damage - change with age and in a sex-dependent manner in a species with indeterminate growth and reproduction (painted turtles, Chrysemys picta). A further feature of their biology that recommends them for an ecological model of vertebrate aging is their female-biased longevity, despite an absence of genotypic sex determination. Thus lifespan and aging may be reliable features of sex-specific life-histories. We measured aspects of mitochondrial health (cellular basal, maximal, and spare oxygen consumption rates), cellular levels of reactive oxygen species, and aspects of DNA damage and repair from exposure to UVB. We used these measures across several physiological axes as proxies for age-related physiological dysfunction. We further assessed our measures across several populations of painted turtles. We found that sex explained the largest proportion of variation, with males differing from females in mitochondrial function, reactive oxygen species production, and inducible DNA damage. In several cases, age significantly interacted with sex, but the effect size was small relative to sex alone. Thus, we found that sex, rather than age or size, was a consistent predictor of cellular aging physiological in this species with where females live longer and age slower.

For some species, gonadal sex determination relies on environmental cues and thus ought to match local conditions, which can be accomplished through changes in maternal behavior and/or the sex-ratio reaction norm. Here, we investigate the evolution of the latter at broad spatial and phylogenetic scales for turtle species with temperature-dependent sex determination (TSD). We find evidence that pivotal temperatures (Tpiv, where a 1:1 offspring sex ratio is produced) and average temperatures during incubation increase together across the phylogeny, while on contemporary timescales, Tpiv increases with incubation temperature variability. Limited evidence also indicates that the breadth of the logistic-shaped reaction norm decreases with temperature variability, contradicting expectations and suggesting that this reaction norm feature may relate to intrinsic biological factors rather than to environmental conditions alone. In sum, these findings indicate that adaptive sex-ratio reaction norm variation within turtle species with TSD is driven by climate variability, while adjustments to the maternal behavior of nest-site choice may primarily compensate for differences in climate averages.

Extreme climate events, including rapid-onset flash droughts, are increasing with anthropogenic climate change. Flash droughts impact growth and survival of plants, microbes, and invertebrates, yet less is known about their ecological consequences in vertebrates. Although constant water deprivation during vertebrate embryonic development influences a range of offspring traits, the effects of acute, short-term hydric stress are less well-studied, particularly in the context of subsequent survival in the field. In this study, we combined experimental manipulation in the laboratory with a large-scale field experiment to examine the effects of the developmental timing of flash drought in an oviparous vertebrate model. We exposed common snapping turtle (Chelydra serpentina) eggs to simulated flash drought events at different stages of embryonic development and measured the effects on growth and offspring phenotypes. We then conducted an experimental release in the field to evaluate offspring survival during migration from the nest. Flash drought during mid-to-late development decreased egg mass, incubation time, and hatchling body size, while flash drought during late development substantially limited post-hatching survival in the field. This study is among the first to examine juvenile survival effects of flash drought during embryonic development in a vertebrate system. Our results suggest that early-life mortality is likely to increase as flash droughts intensify with climate change. This study contributes to a growing body of research on the ecological consequences of extreme climate events and highlights the importance of considering these events in a developmental context.

Accurate scientific information is critical for undertaking appropriate conservation and management practices for imperiled species. One source of concern is that research findings might vary for non-biological reasons, including experimental design and analytical methods. To illustrate, we provide detailed modern analysis of reproductive data for leatherback turtles (Dermochelys coriacea). This species exhibits significant fluctuations in nesting densities across different regions, possibly driven by local rather than global factors. Key factors influencing these changes include hatching success and sex determination, both sensitive to incubation temperatures (e.g., lower temperatures yield more males, higher temperatures yield females). This study updates the understanding of temperature-dependent sex determination (TSD) in this species using Bayesian statistics. Growth rate data from the West Pacific and Northwest Atlantic populations show a similar, monotone increase with temperature, affirming the reliability of the models used. The analysis of TSD patterns indicates that observed differences are more likely due to study methodologies and clutch-specific factors rather than regional differences. These findings challenge previous assumptions, showing that leatherback TSD does not conform to a simple on/off pattern but is influenced by multiple, interacting environmental factors. Population dynamics models must account for these complexities, recognizing that both sex ratios and hatching success are critical to understand the rapid changes observed in some leatherback populations.

Species with temperature-dependent sex determination (TSD) exhibit significant variation in the relationship between incubation temperatures and the sex ratios they produce, making this an ideal system for comparing processes producing variation above and below the species level. Furthermore, a deeper mechanistic understanding may help reveal the currently unknown adaptive significance of this variation or of TSD as a whole. Here we probe these topics by examining the macro- and microevolutionary dynamics of this sex-determining mechanism in turtles. Our ancestral state reconstructions of discrete patterns of TSD suggest that producing females at cool incubation temperatures is derived and potentially adaptive. However, the ecological irrelevance of these cool temperatures and a strong genetic correlation across the sex-ratio reaction norm in Chelydra serpentina both contradict this interpretation. We further find the phenotypic consequence of this genetic correlation in C. serpentina reflected across all turtle species, suggesting that a single genetic architecture underlies both intra- and interspecific variation in TSD in this clade. This correlated architecture can explain the macroevolutionary origin of discrete TSD patterns without assigning cool-temperature female production an adaptive value. However, this architecture may also constrain adaptive microevolutionary responses to ongoing climate change.

In contrast to genotypic sex determination (GSD), temperature-dependent sex determination (TSD) in amniotic vertebrates eludes intuitive connections to Fisherian sex-ratio theory. Attempts to draw such connections have driven over 50 years of research on the evolution of sex-determining mechanisms (SDM), perhaps most prominently among species in the order Testudines. Despite regular advancements in our understanding of this topic, no efforts have been published compiling the entirety of data on the relationships between incubation temperature and offspring sex in any taxonomic group. Here, we present the Reptilian Offspring Sex and Incubation Environment (ROSIE) database, a comprehensive set of over 7,000 individual measurements of offspring sex ratios in the order Testudines as well as SDM classifications for 149 species. As the name suggests, we plan to expand the taxonomic coverage of ROSIE to include all non-avian reptiles and will regularly release updates to maintain its comprehensive nature. This resource will enable crucial future research probing the ecology and evolution of SDM, including the presumed sensitivity of TSD to rapid environmental change.

Long-term demographic data are important for conserving populations of threatened and endangered species. Collecting data specific to the population of interest can improve conservation success by allowing managers to make informed decisions, evaluate the efficacy of habitat management, and assess extinction risk. Population viability analysis (PVA), a tool commonly used to estimate extinction risk in threatened populations, is most informative when precise, population-specific data are available. We used mark-recapture data spanning ten years (2008–2017) to estimate annual adult survival rates in a population of threatened Eastern Massasauga rattlesnakes (Sistrurus catenatus) in Barry County, Michigan. Using these survival estimates and ten additional parameter values obtained from our site and a nearby population, we modeled population viability over the next 100 years. We performed sensitivity analysis using Latin hypercube sampling, followed by logistic regression to assess the relative influence of m...