Lost reproductive value reveals a high burden of juvenile road mortality in a long-lived species.

Adult mortality is often the most sensitive vital rate affecting at-risk wildlife populations. Therefore, road ecology studies often focus on adult mortality despite the possibility for roads to be hazardous to juvenile individuals during natal dispersal. Failure to quantify concurrent variation in mortality risk and population sensitivity across demographic states can mislead the efforts to understand and mitigate the effects of population threats. To compare relative population impacts from road mortality among demographic classes, we weighted mortality observations by applying reproductive value analysis to quantify expected stage-specific contributions to population growth. We demonstrate this approach for snapping turtles (Chelydra serpentina) observed on roads at two focal sites in Ontario, Canada, where we collected data for both live and dead individuals observed on roads. We estimated reproductive values using stage-classified matrix models to compare relative population-level impacts of adult and juvenile mortality. Reproductive value analysis is a tractable approach to assessing demographically variable effects for applications covering large spatial scales, nondiscrete populations, or where abundance data are lacking. For one site with long-term life-history data, we compared demographic frequency on roads to expected general population frequencies predicted by the matrix model. Our application of reproductive value is sex specific but, as juvenile snapping turtles lack external secondary sex characters, we estimated the sex ratio of road-crossing juveniles after dissecting and sexing carcasses collected on roads at five sites across central Ontario, Canada. Juveniles were more abundant on roads than expected, suggesting a substantial dispersal contribution, and the road-killed juvenile sex ratio approached 1:1. A higher proportion of juveniles were also found dead compared with adults, and cumulative juvenile mortality had similar population-level importance as adult mortality. This suggests that the impact of roads needs to be considered across all life stages, even in wildlife species with slow life histories, such as snapping turtles, that are particularly sensitive to adult mortality.

Long-Term Resilience of Primary Sex Ratios in a Species with Temperature-Dependent Sex Determination after Decades of Climate Warming.

AbstractSpecies with environmental sex determination (ESD) have persisted through deep time, despite massive environmental perturbation in the geological record. Understanding how species with temperature-dependent sex determination (TSD), a type of ESD, persist through climate change is particularly timely given the current climate crisis, as highly biased sex ratios and extinction are predicted. Since 1982, we have studied primary sex ratios of a reptile with TSD (Chelydra serpentina). Primary sex ratios remained unchanged over time, despite warming in the environment. Resilience of the primary sex ratio occurred via a portfolio effect, realized through remarkable intra-annual variation in nest-level sex ratios, leading to a relatively consistent mean annual sex ratio. Intra-annual variation in nest-level sex ratios was related to variation in egg burial depth coupled with large clutch sizes, creating thermal gradients in the nest and promoting mixed-sex clutches. Furthermore, both locally and globally, sustained increases in nighttime air temperature contribute more to warming than increases in daily maximum temperature, but development rate was affected more strongly by maximum daily air temperature, conferring additional resilience to overall warming. Our study suggests that some TSD species may be resilient to warming and provides an example of how ESD may persist under environmental change.

Variation in space and time: a long-term examination of density-dependent dispersal in a woodland rodent.

Dispersal is a fundamental ecological process that can be affected by population density, yet studies report contrasting effects of density on propensity to disperse. In addition, the relationship between dispersal and density is seldom examined using densities measured at different spatial scales or over extensive time series. We used 51 years of trapping data to examine how dispersal by wild deer mice (Peromyscus maniculatus) was affected by changes in both local and regional population densities. We examined these patterns over both the entire time series and also in 10-year shifting windows to determine whether the nature and strength of the relationship changed through time. Probability of dispersal decreased with increased local and regional population density, and the negative effect of local density on dispersal was more pronounced in years with low regional densities. In addition, the strength of negative density-dependent dispersal changed through time, ranging from very strong in some decades to absent in other periods of the study. Finally, while females were less likely to disperse, female dispersal was more density-dependent than male dispersal. Our study shows that the relationship between density and dispersal is not temporally static and that investigations of density-dependent dispersal should consider both local and regional population densities.

Measurement and modelling of primary sex ratios for species with temperature-dependent sex determination.

For many oviparous animals, incubation temperature influences sex through temperature-dependent sex determination (TSD). Although climate change may skew sex ratios in species with TSD, few available methods predict sex under natural conditions, fewer still are based on mechanistic hypotheses of development, and field tests of existing methods are rare. We propose a new approach that calculates the probability of masculinization (PM) in natural nests. This approach subsumes the mechanistic hypotheses describing the outcome of TSD, by integrating embryonic development with the temperature-dependent reaction norm for sex determination. Further, we modify a commonly used method of sex ratio estimation, the constant temperature equivalent (CTE), to provide quantitative estimates of sex ratios. We test our new approaches using snapping turtles (Chelydra serpentina). We experimentally manipulated nests in the field, and found that the PM method is better supported than the modified CTE, explaining 69% of the variation in sex ratios across 27 semi-natural nests. Next, we used the PM method to predict variation in sex ratios across 14 natural nests over 2 years, explaining 67% of the variation. We suggest that the PM approach is effective and broadly applicable to species with TSD, particularly for forecasting how sex ratios may respond to climate change. Interestingly, we also found that the modified CTE explained up to 64% of variation in sex ratios in a Type II TSD species, suggesting that our modifications will be useful for future research. Finally, our data suggest that the Algonquin Park population of snapping turtles possesses resilience to biased sex ratios under climate change.

Altered spring phenology of North American freshwater turtles and the importance of representative populations.

Globally, populations of diverse taxa have altered phenology in response to climate change. However, most research has focused on a single population of a given taxon, which may be unrepresentative for comparative analyses, and few long-term studies of phenology in ectothermic amniotes have been published. We test for climate-altered phenology using long-term studies (10-36 years) of nesting behavior in 14 populations representing six genera of freshwater turtles (Chelydra, Chrysemys, Kinosternon, Malaclemys, Sternotherus, and Trachemys). Nesting season initiation occurs earlier in more recent years, with 11 of the populations advancing phenology. The onset of nesting for nearly all populations correlated well with temperatures during the month preceding nesting. Still, certain populations of some species have not advanced phenology as might be expected from global patterns of climate change. This collection of findings suggests a proximate link between local climate and reproduction that is potentially caused by variation in spring emergence from hibernation, ability to process food, and thermoregulatory opportunities prior to nesting. However, even though all species had populations with at least some evidence of phenological advancement, geographic variation in phenology within and among turtle species underscores the critical importance of representative data for accurate comprehensive assessments of the biotic impacts of climate change.

A new method of estimating thermal performance of embryonic development rate yields accurate prediction of embryonic age in wild reptile nests.

Temperature has a strong effect on ectotherm development rate. It is therefore possible to construct predictive models of development that rely solely on temperature, which have applications in a range of biological fields. Here, we leverage a reference series of development stages for embryos of the turtle Chelydra serpentina, which was described at a constant temperature of 20 °C. The reference series acts to map each distinct developmental stage onto embryonic age (in days) at 20 °C. By extension, an embryo taken from any given incubation environment, once staged, can be assigned an equivalent age at 20 °C. We call this concept "Equivalent Development", as it maps the development stage of an embryo incubated at a given temperature to its equivalent age at a reference temperature. In the laboratory, we used the concept of Equivalent Development to estimate development rate of embryos of C. serpentina across a series of constant temperatures. Using these estimates of development rate, we created a thermal performance curve measured in units of Equivalent Development (TPCED). We then used the TPCED to predict developmental stage of embryos in several natural turtle nests across six years. We found that 85% of the variation of development stage in natural nests could be explained. Further, we compared the predictive accuracy of the model based on the TPCED to the predictive accuracy of a degree-day model, where development is assumed to be linearly related to temperature and the amount of accumulated heat is summed over time. Information theory suggested that the model based on the TPCED better describes variation in developmental stage in wild nests than the degree-day model. We suggest the concept of Equivalent Development has several strengths and can be broadly applied. In particular, studies on temperature-dependent sex determination may be facilitated by the concept of Equivalent Development, as development age maps directly onto the developmental series of the organism, allowing critical periods of sex determination to be delineated without invasive sampling, even under fluctuating temperature.

Phenotypic plasticity of nest timing in a post-glacial landscape: how do reptiles adapt to seasonal time constraints?

Life histories evolve in response to constraints on the time available for growth and development. Nesting date and its plasticity in response to spring temperature may therefore be important components of fitness in oviparous ectotherms near their northern range limit, as reproducing early provides more time for embryos to complete development before winter. We used data collected over several decades to compare air temperature and nest date plasticity in populations of painted turtles and snapping turtles from a relatively warm environment (southeastern Michigan) near the southern extent of the last glacial maximum to a relatively cool environment (central Ontario) near the northern extent of post-glacial recolonization. For painted turtles, population-level differences in reaction norm elevation for two phenological traits were consistent with adaptation to time constraints, but no differences in reaction norm slopes were observed. For snapping turtle populations, the difference in reaction norm elevation for a single phenological trait was in the opposite direction of what was expected under adaptation to time constraints, and no difference in reaction norm slope was observed. Finally, among-individual variation in individual plasticity for nesting date was detected only in the northern population of snapping turtles, suggesting that reaction norms are less canalized in this northern population. Overall, we observed evidence of phenological adaptation, and possibly maladaptation, to time constraints in long-lived reptiles. Where present, (mal)adaptation occurred by virtue of differences in reaction norm elevation, not reaction norm slope. Glacial history, generation time, and genetic constraint may all play an important role in the evolution of phenological timing and its plasticity in long-lived reptiles.

Recurrent violations of invariant rules for offspring size: evidence from turtles and the implications for small clutch size models.

Smith and Fretwell's classic model predicts that parents can maximize fitness by dividing the energy available for reproduction into offspring of an optimal size. However, this model breaks down when clutch size is small (~1-10 offspring). Invariant rules are an extension of the Smith-Fretwell model, and these rules predict how offspring size will vary among and within individuals that produce small clutch sizes. Here, we provide a narrow test of invariant rules using three turtle species, then we synthesize and re-analyze existing data from 18 different species (comprising five Orders) to evaluate whether invariant rules are followed across broad taxa. We do not find support for most invariant rules in turtles, and our re-analysis demonstrates a general mismatch between observed and expected values across all taxa evaluated, suggesting that invariant rules fail to predict reproductive patterns in nature. Morphological constraints on offspring size and reproductive effort may be important reasons for disparities between theory and observation both in turtles and other taxa. Paradoxically, morphological constraints are most common in small-bodied species and individuals, but these same candidates are also those which produce the small clutch sizes that are necessary to test invariant rules, such that a fair test of invariant rules will often be challenging. Mismatches between theory and observation might also occur because theory assumes that mothers exert control over resource allocation to offspring. In fact, there is evidence of widespread genetic correlations among investment per offspring and reproductive effort, such that these traits are not independent.

Widespread reproductive variation in North American turtles: temperature, egg size and optimality.

Theory predicts the existence of an optimal offspring size that balances the trade-off between offspring fitness and offspring number. However, in wild populations of many species, egg size can still vary from year to year for unknown reasons. Here, we hypothesize that among-year variation in population mean egg size of freshwater turtles is partly a consequence of their gonadal sensitivity to seasonal temperatures, a physiological mechanism which principally functions to synchronize reproduction with a favorable time of year. As part of this process, among-year variation in seasonal temperatures modifies the extent of egg follicle development, and this may translate into variation in mean egg size among years (both at the individual and population level). To test this hypothesis, we applied an information-theoretic approach to model relationships between mean egg mass and the temperatures experienced during discrete periods of follicular development in wild populations of three turtle species (Chrysemys picta, Chelydra serpentina, Glyptemys insculpta) over 12 consecutive years. Because follicular development occurs in the fall for C. serpentina and G. insculpta, whereas it occurs both in the fall and spring for C. picta, we expected only fall temperatures would explain egg size variation in C. serpentina and G. insculpta, whereas both fall and spring temperatures would correlate with egg size variation in C. picta. These predictions were upheld. We then compared among-year variation in within-female egg and clutch sizes of each species in order to evaluate whether such variation might still be consistent with some tenets of optimal egg size theory. In all three species, we found that clutch sizes vary more than egg sizes in spite of temperature-induced egg size variation, and this pattern of relatively high clutch-size variation matches theoretical predictions. Future work should explore the roles of direct and indirect (i.e., nutritional) influences of temperature on egg size in natural settings.

A phylogeographic analysis of populations of the wood turtle (Glyptemys insculpta) throughout its range.

We investigated the phylogeography of wood turtles, Glyptemys insculpta, in North America using 750 bp of the mitochondrial control region from 117 individuals sampled at 29 localities across the species' range. A total of 21 haplotypes were identified and little genetic variation was found. The highest pairwise difference was 2%. From nested clade analysis (NCA), one main postglacial dispersal route was inferred along the east coast, with subsequent westward dispersal. NCA further revealed some patterns of restricted gene flow/dispersal. We propose that wood turtles experienced a combined effect of bottleneck during the Pleistocene as well as of selective sweep, which produced the low level of variation observed. Following the Pleistocene, wood turtles would have undergone a rapid northward expansion from a common southern refugium as glaciers retreated. These findings shed light on where to direct conservation priorities, on conservation strategies needed, and on the potential effects of interpopulation transfers for this vulnerable species.

Sexually dimorphic morphology of hatchling snapping turtles (Chelydra serpentina) from contaminated and reference sites in the Great Lakes and St. Lawrence River basin, North America.

Some organochlorine pesticides and industrial chemicals may alter sexually dimorphic traits through endocrine disruption. Therefore, we examined a sexually dimorphic trait, precloacal length, of hatchling snapping turtles (Chelydra serpentina) incubated from 31 clutches from a heavily contaminated site (Hamilton Harbour, ON, Canada; n = 14), a moderately contaminated site (Akwesasne Mohawk Territory; n = 3), and from a reference site (Algonquin Provincial Park, ON, Canada; n = 14). The mean sum polychlorinated biphenyls from Algonquin were low (20.33 ng/g wet wt), but were high from both Hamilton and Akwesasne (2,956.28 and 3,377.0 ng/g wet wt, respectively). Organochlorine pesticides, such as dichlorodiphenyldichloroethylene, were low from Algonquin (1.67 ng/g wet wt), moderate from Akwesasne (10.00 ng/g wet wt), and relatively high from Hamilton (135.14 ng/g wet wt). At all sites, the precloacal length of male hatchlings was larger than that of females by an equal amount at any given body size. However, the precloacal length of both males and females from Hamilton increased with body size at a slower rate than of males and females from the other two sites. Our results support an earlier study that found differences in sexually dimorphic morphology of adult snapping turtles among contaminated and uncontaminated sites. Furthermore, these alterations in secondary sexual characteristics previously observed in adults likely are initiated early in development, and may result in permanent organizational changes in morphology.

Octylphenol (OP) alters the expression of members of the amyloid protein family in the hypothalamus of the snapping turtle, Chelydra serpentina serpentina.

The gonadal estrogen estradiol-17beta (E(2)) is important for developing and regulating hypothalamic function and many aspects of reproduction in vertebrates. Pollutants such as octylphenol (OP) that mimic the actions of estrogens are therefore candidate endocrine-disrupting chemicals. We used a differential display strategy (RNA-arbitrarily primed polymerase chain reaction) to isolate partial cDNA sequences of neurotransmitter, developmental, and disease-related genes that may be regulated by OP or E(2) in the snapping turtle Chelydra serpentina serpentina hypothalamus. Hatchling and year-old male snapping turtles were exposed to a 10 ng/mL nominal concentration of waterborne OP or E(2) for 17 days. One transcript [421 base pairs (bp)] regulated by OP and E(2) was 93% identical to human APLP-2. APLP-2 and the amyloid precursor protein (APP) regulate neuronal differentiation and are also implicated in the genesis of Alzheimer disease in humans. Northern blot analysis determined that the turtle hypothalamus contains a single APLP-2 transcript of 3.75 kb in length. Exposure to OP upregulated hypothalamic APLP-2 mRNA levels 2-fold (p < 0.05) in month-old and yearling turtles. E(2) did not affect APLP-2 mRNA levels in hatchlings but stimulated a 2-fold increase (p < 0.05) in APLP-2 mRNA levels in yearling males. The protein beta-amyloid, a selectively processed peptide derived from APP, is also involved in neuronal differentiation, and accumulation of this neurotoxic peptide causes neuronal degeneration in the brains of patients with Alzheimer disease. Therefore, we also sought to determine the effects of estrogens on the expression of beta-amyloid. Using homology cloning based on known sequences, we isolated a cDNA fragment (474 bp) from turtle brain with 88% identity to human APP. Northern blot analysis determined that a single 3.5-kb transcript was expressed in the turtle hypothalamus. Waterborne OP also increased the expression of hypothalamic APP after 35 days of exposure. Our results indicate that low levels of OP are bioactive and can alter the expression of APLP-2 and APP. Because members of the APP gene family are involved in neuronal development, we hypothesize that OP exposure may disrupt hypothalamic development in young turtles.

Allocation of reproductive effort in female Arrenurus spp. water mites (Acari: Hydrachnidia; Arrenuridae).

Twenty-seven species of water mites of the genus Arrenurus were compared with respect to allocation of reproductive effort. Clutch volume was positively correlated to female volume, female volume was positively correlated with clutch size and with larval volume, while clutch size and larval volume were negatively correlated. In threespace, corresponding to female volume, clutch size and larval volume, species were arranged along two trajectories representing separate reproductive strategies. The strategy characterized by small larvae, large clutches and large females corresponded to species of the subgenus Arrenurus, known to be larval parasites of adult Odonata. Members of three subgenera, known to be larval parasites of adult Diptera, followed the second strategy of small clutch size and alrge larvae. Of nine species of mites tested for intraspecific relationships, only one significant relationship was found, between female volume and clutch size for A. (Megaluracarus) bartonensis.