Many turtle species have temperature-dependent sex determination (TSD), raising the prospect that climate change could impact population dynamics by altering sex ratios. Understanding how climate change will affect populations of animals with TSD requires a reliable and minimally invasive method of identifying the sexes of young individuals. This determination is challenging in many turtles, which often lack conspicuous external sexual dimorphism until years after hatching. Here, we explore four alternatives for sexing three age classes of captive-reared young gopher tortoises (Gopherus polyphemus), a terrestrial turtle of conservation concern native to the southeastern United States: (1) naive testosterone levels, (2) testosterone levels following a follicle stimulating hormone (FSH) challenge, (3) linear morphological measurements, and (4) geometric morphometrics. Unlike some other turtle species, male and female neonatal gopher tortoises have overlapping naive testosterone concentration distributions, justifying more complicated methods. We found that sex of neonates (<7 days old) is best predicted by a “random forest” machine learning model with naive testosterone levels and morphological measurements (8% out-of-bag error). Sex of hatchlings (4–8 months old) was predicted with 11% error using a simple threshold on naive testosterone levels, or with 4% error using a simple threshold on post-FSH testosterone levels. Sex of juveniles (approximately 3.5 years old) was perfectly predicted using a simple threshold on naive testosterone levels. Sexing hatchlings at >4 months of age is the easiest and most reliable non-surgical method for sex identification. Given access to a rearing facility and equipment to perform hormone assays, these methods have the potential to supplant laparoscopic surgery as the method of choice for sexing young gopher tortoises.
Biotic and abiotic factors drive assortative mixing (preference for or sorting with individuals with similar characteristics) in animal populations on a landscape, with implications for dispersal, population structuring, and other ecological and evolutionary processes. However, patterns and generative mechanisms of assortative mixing are overlooked in amphibians outside of specific life history events such as reproduction. The aims of this project were to determine whether there is assortative mixing by size and life history category in eastern spadefoots (Scaphiopus holbrookii), whether these patterns are preserved across time and spatial scale, and quantify the nature and relative role of various habitat and soil features in explaining observed patterns in spatial organization of individuals. We conducted field surveys in southeastern Virginia, USA, in 2016 and 2017 during nonbreeding periods to create spatial networks of S. holbrookii. We quantified spatial assortativity by size and life history stage and evaluated the roles of multiple landscape features in explaining spatial organization of S. holbrookii. We found that S. holbrookii sorted spatially by size and sex outside of breeding periods, with juveniles and adults less likely to sort with each other. Within each life history stage, S. holbrookii sorted by size. These patterns were similar across time and spatial scale. Soil and habitat types had no effect on assortativity. Instead, the distance to nearest breeding pool, wetland, and meadow were related to life history stage assortativity, as well as size assortativity in males and subadults. Adult males and females displayed affinity for breeding pools and meadows and avoidance of other types of wetlands, while subadults and nonbreeding adults showed opposite patterns compared with breeding adults. Our results indicate that (1) previously established guidelines for the minimum size of buffer zones to protect wetland-breeding amphibians may be inadequate, (2) nonbreeding wetlands may be important core habitat for subadults, and (3) the upland spatial organization of amphibians may be used to predict locations of undetected breeding pools.
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