A male-specific sex marker for the endangered western sawshelled turtle (Myuchelys bellii) using in silico whole-genome subtraction

Artificial incubation of eggs for the mass release of hatchlings is a common conservation intervention for imperilled turtle species. Programs designed to reinforce wild populations need to ensure that they are releasing appropriate male to female ratios into the wild. In many turtle species, the sex of juveniles cannot be identified using external morphology until they approach sexual maturity. For the endangered western sawshelled turtle, Myuchelys bellii, sexual dimorphism does not occur until at least 6 years of age. We aimed to develop a molecular test to identify the sex of M. bellii during the life stages where they cannot be sexed morphologically-embryos, hatchlings and small juveniles. We used in silico whole-genome subtraction of a female M. bellii (XX) from a male (XY) to identify a Y chromosome-specific sequence which we characterized and developed into a PCR sex test. Our research is the first to use a whole-genome subtraction method in-silico to successfully establish sex chromosome markers in a freshwater turtle species. Developing this technology provides an opportunity for conservation programs to ensure that populations are supplemented with a proportionate number of male and female hatchlings. Further, it allows large scale measurement of naturally occurring sex ratios in hatchlings and small juveniles, which in turn enables estimates of sex ratios within wild populations free from age-at-maturity bias. The application of sex-specific marker technology also provides an opportunity to quantify the influence of sex on behaviour, movement and survival in the segment of populations that cannot be morphologically sexed

Optimising the hatching success of artificially incubated eggs for use in a conservation program for the western saw-shelled turtle (Myuchelys bellii)

Artificial incubation of eggs and the release of hatchlings into the wild is a common conservation intervention designed to augment threatened turtle populations. We investigate a range of incubation temperatures to establish an optimal temperature for maximum hatching success of western saw-shelled turtle (Myuchelys bellii) eggs. We report on the influence of incubation temperature on incubation duration and hatching success and describe two experimental incubation methods which, for the same incubation temperature (27°C), resulted in 77% and 97% hatching success, respectively. Eggs were incubated at constant temperatures (27°C, 28°C and 29°C) to determine the influence of temperature on incubation period, hatchling morphology and external residual yolk. Incubation duration was negatively correlated with incubation temperature. We report on the morphology of eggs and hatchlings and show that their dimensions are positively correlated with maternal adult size and mass. A constant incubation temperature of 27°C produced the highest hatching success and smallest external residual yolk on hatching and is therefore recommended for incubation of eggs for population reinforcement programs. Our study is the first to optimise artificial incubation procedures for M. bellii and will be a valuable resource for M. bellii and other threatened freshwater turtle conservation initiatives

Towards a Diagnostic Tool for Turtle Ootaxonomy: Investigation of Microstructural Differences in the Eggshells of Australian Freshwater Turtles

Researchers frequently encounter turtle nests after the nest has been raided, finding only eggshell fragments that are difficult to identify. Valuable information on the breeding biology and needs of different species might still be collected from these nests if remains could be confidently assigned to a particular species. However, this can be difficult if there are multiple turtle species nesting within an area at a given time, and gross-scale characteristics such as egg size and shape are no longer available due to damage. This study examined eggshell microstructure, by scanning electron microscopy (SEM), among multiple species of freshwater turtle eggshell fragments, with the aim of potentially developing a diagnostic tool for species identification. Eggshell fragments were collected from known sources of four species of turtle native to eastern Australia: Eastern Long-necked Turtle (Chelodina longicollis), Murray River Turtle (Emydura macquarii), Bell's Turtle (Myuchelys bellii), and Bellinger River Turtle (M. georgesi). Fragments were scanned and analyzed for microstructural and ultrastructural features, and measured for differences in feature size across species. Central plaque diameter emerged as a potential diagnostic feature for separating species, with longneck turtle eggshells having no visible plaques, Murray River Turtle eggshells having relatively small mean plaque size (19.8 µm ± 10.28 SD), while Bell's Turtle (43.5 µm ± 14.16 SD) and Bellinger River Turtle eggshells (41.9 µm ± 15.41 SD) had relatively large plaques with little overlap between genera. Other measured features (shell unit size, basal knob size, and shell unit density) significantly differed in means among species but overlapped in range, or showed a lack of statistical difference, likely preventing the technique being definitive with a single measure. Some of this variance may be due to inherent confounding factors, such as the replicability of fragment preparation, condition of eggshells, and stage of embryonic development at the time of collection. These results suggest that use of measurements of microstructural features has promise as a means for distinguishing the provenance of eggshell fragments, and further investigation is warranted to develop site-specific protocols for use as a diagnostic tool for turtle ootaxonomy

Opportunities for research and conservation of freshwater turtles in Australia

Australia's freshwater turtles have high endemicity and many are threatened by extinction. Following a symposium held at the 2022 conference of the Australian Society of Herpetologists, we summarized the current status of research and conservation for Australian freshwater turtles and identified opportunities for future research. Eight species (32%) of Australia's 25 native freshwater turtles are listed as threatened by Australia's Federal Government. Symposium discussions on the primary gaps in research identified the lack of baseline data to inform population modelling as a key deficiency. Knowledge of the most effective conservation actions, the effectiveness of attempts to aid population recovery, and whether these actions are required at all, remains lacking for many species. A heavy bias exists between some well-studied species compared with others for which little or no information is published. Community science, engagement with First Nations people, advances in technology, and recognition of the importance of turtles are contributing to better knowledge

Nocturnal basking in freshwater turtles: A global assessment

Diurnal basking (“sunning”) is common in many ectotherms and is generally thought to be a behavioural mechanism for thermoregulation. Recent studies have reported the occurrence of nocturnal basking in a few distantly-related species of freshwater turtles, but the true extent of this behaviour is unknown, and it may be underreported due to sampling biases (e.g., not surveying for turtles at night). Therefore, we initiated a global, collaborative effort to systematically document and quantify basking activity (diurnal and nocturnal) across a wide range of freshwater turtle species and locations. We conducted camera trap or manual surveys in North America, the Caribbean, Europe, Asia, Africa, the Seychelles, and Australia. We collected 873,111 trail camera photographs (25,273 hrs of search effort) and obtained data on 29 freshwater turtle species from seven families. Nocturnal basking was documented in 13 species, representing six families (Chelidae, Emydidae, Geoemydidae, Kinosternidae, Pelomedusidae, and Trionychidae), including representatives in Central America, Trinidad and Tobago, Africa, the Seychelles, Asia, and Australia. Nocturnal basking was restricted to tropical and sub-tropical locations, suggesting that environmental temperature plays a role in this behaviour. However, the primary factors driving nocturnal basking are yet to be determined and may vary geographically and by species. The frequency and duration of nocturnal basking varied among species and seasons, but nocturnal basking events were often substantially longer than diurnal events. This is the first study to document a widespread occurrence of nocturnal basking, and our results suggest that nocturnal basking may be a common, although overlooked, aspect of many species’ ecology