The illegal pet trade facilitates the global dispersal of invasive alien species (IAS), providing opportunities for new pests to establish in novel recipient environments. Despite the increasing threat of IAS to the environment and economy, biosecurity efforts often lack suitable, scientifically-based methods to make effective management decisions, such as identifying an established IAS population from a single incursion event. We present a proof-of-concept for a new application of a stable isotope technique to identify wild and captive histories of an invasive pet species. Twelve red-eared slider turtles (Trachemys scripta elegans) from historic Australian incursions with putative wild, captive and unknown origins were analysed to: (1) present best-practice methods for stable isotope sampling of T. s. elegans incursions; (2) effectively discriminate between wild and captive groups using stable isotope ratios; and (3) present a framework to expand the methodology for use on other IAS species. A sampling method was developed to obtain carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios from the keratin layer of the carapace (shells), which are predominantly influenced by dietary material and trophic level respectively. Both δ¹³C and δ¹⁵N exhibited the potential to distinguish between the wild and captive origins of the samples. Power simulations demonstrated that isotope ratios were consistent across the carapace and a minimum of eight individuals were required to effectively discriminate wild and captive groups, reducing overall sampling costs. Statistical classification effectively separated captive and wild groups by δ¹⁵N (captive: δ¹⁵N‰ ≥ 9.7‰, minimum of 96% accuracy). This study outlines a practical and accessible method for detecting IAS incursions, to potentially provide biosecurity staff and decision-makers with the tools to quickly identify and manage future IAS incursions.

biosecurity, invasive species, pet trade, provenancing, stable isotopes, Trachemys scripta elegans, wildlife trade

Wildlife trade, in particular the legal and illegal pet trade, facilitates the worldwide movements of invasive alien species (IAS), providing novel introduction pathways into new environments (Russello et al. 2008, Lockwood et al. 2019). Accidental escapes or intentional release of alien pets provide numerous opportunities for these species to establish, particularly where the propagule pressure is high from repeated or mass releases (Vall-Ilosera and Cassey 2017). If new populations are not detected rapidly, complete eradication is unlikely and often extremely costly and resource-intensive (Mack et al. 2000). IAS are a key threatening process to global biodiversity loss, thus the prevention of further establishment is critical (Lodge et al. 2006, Baillie et al. 2010). Current methods for discriminating between a recently escaped or released captive individual and an individual from an established population are ineffective and rarely identify the threat early enough for effective eradication (Schmidt et al. 2017). Here, we explore the potential for a novel application of carbon and nitrogen biogeochemistry for determining the provenance of a vertebrate IAS incursion.

The relative abundance of stable isotopes within a material is a function of its synthesis and environmental history, which, in the case of vertebrate animals, predominantly relates to their diet (Camin et al. 2016). In vertebrate animal tissue, stable carbon isotope ratios (a measure of the relative abundance of ¹³C/¹²C, reported as δ¹³C) are linked to the δ¹³C of the animal’s diet, which, in turn, is strongly influenced by the relative proportion of the C3 and C4 plants an animal directly or indirectly consumes. Nitrogen stable isotope ratios (a measure of the relative abundance of ¹⁵N/¹⁴N, reported as δ¹⁵N) are also influenced by the animal’s diet; specifically, they indicate the trophic position of the animal. Given that a difference in the diet between wild and captive animals is extremely likely, stable isotope ratios can potentially utilise these differences to provide information on the origin of the animal (Ziegler et al. 2018).

Stable isotopes are a well-established forensic technique and are a strong candidate for identifying the origin of IAS incursions (Cerling et al. 2016). Environmental research using stable isotopes include tracing vertebrate movements, including tracking migratory animals where satellite trackers cannot be used (e.g. MacKenzie et al. 2011, Madigan et al. 2017) and identifying diets and niche positions (e.g. Haubrock et al. 2020, Pearson et al. 2013). Previous studies have explored stable isotope tools to distinguish between wild and captive animals with success, including, but not limited to: short-beaked echidnas (Tachyglossus aculeatus) (Brandis et al. 2018); wolves (Canis lupis) (Kays and Feranec 2011); African grey parrots (Psittacus erithacus) (Alexander et al. 2019, Symes et al. 2017); reticulated pythons (Python reticulatus) (Natusch et al. 2017); and crocodile lizards (Shinisaurus crocodilurus) (van Schingen et al. 2016, Ziegler et al. 2018). In all of these cases, stable isotopes have proved efficient at identifying environmental histories and diets. Yet, with the exception of insects (Holder et al. 2014, Hood-Nowotny et al. 2016), the use of stable isotopes for determining the provenance of IAS, early in the incursion process, is relatively unexplored.

Trachemys scripta elegans (red-eared slider turtles) were selected as a case study to test the efficacy of δ¹³C and δ¹⁵N for biosecurity applications. As one of the world’s top 100 most invasive species, T. s. elegans have the potential to establish and spread in urban and semi-rural areas worldwide (IUCN 2000, Rodder et al. 2009, Banha et al. 2017). T. s. elegans are the most-traded turtle species in the world, where more than 50 million individuals were exported from USA between 1989 to 1997 to supply the global pet trade (Telecky 2001) and the trade continues illegally despite being restricted in most regions (Kitowski and Pachol 2009; García-Díaz et al. 2015). These animals grow large quickly, resulting in being intentionally released into waterways when they become undesirable as pets. As a consequence, T. s. elegans have established nearly 200 identified breeding populations worldwide (Kikillus et al. 2010). They are a significant threat to biodiversity, as they compete with native turtles for food and shelter (Pearson et al. 2013, Balzani et al. 2016) and carry exotic diseases including Ranavirus and Chlamydia spp. (Johnson et al. 2007, Mitura et al. 2017). As T. s. elegans are omnivorous, the proportions of meat and plant material that may vary between wild and captive diets are likely to drive differences in δ¹³C and δ¹⁵N, making them good candidates for our case study.

New methodologies are urgently needed to provide early identification of incursions as distinct from established populations, to allow for quick and effective eradication (Lodge et al. 2006). Here, we present a new application of δ¹³C and δ¹⁵N using historical Australian incursion samples of T. s. elegans of putative wild and captive origins to: (i) determine best-practice methods for sampling T. s. elegans incursions; (ii) evaluate the use of δ¹³C and δ¹⁵N to discriminate between wild and captive individuals; and (iii) provide a framework to expand the methodology for use on other IAS species.

Captive and wild T. s. elegans are effectively differentiated by their δ¹⁵N. Sampling scute proved to be a simple method; no specialist equipment was required for collection and samples could be taken anywhere on the scute and across multiple scutes with minimal variation within the individual. Although individuals were dissected for this study, the use of scute shavings is potentially a non-invasive method. This makes the technology accessible for non-specialist practitioners, such as biosecurity or veterinary staff and for samples to be collected and sent to a laboratory for analysis and determination of their origins. Furthermore, the power simulations demonstrated that minimal sampling per individual is required, reducing the overall sampling costs in time, effort and welfare, as well as monetary cost.

As material of high biosecurity risk is inherently difficult to obtain, the availability of T. s. elegans and other reptile IAS is limited, while information surrounding an animal’s history is not always accessible. Wild T. s. elegans specimens are rare in Australia, as at-large populations have only been confirmed in Sydney (Burgin 2006, Robey et al. 2011; Mo 2019) and Queensland (O’Keeffe 2005). Furthermore, T. s. elegans are illegal to import or keep in Australia without a licence, limiting available samples to those confiscated from illegal keeping (Department of Agriculture and Water Resources 2017). Here, we used a selection of samples for which we were initially confident in the assignment of status to an individual. Furthermore, the use of a power analysis demonstrated that a minimum of eight (n ≥ 8) individuals of known origin was sufficient to obtain a detectable effect size to effectively separate wild and captive groups.

Separation of wild and captive groups used a simple classification tree model, which effectively differentiated wild and captive individuals with minimal misclassification error. As samples with relatively high status confidence were used, this classification tree can be adopted as a set of best-practice methods and model to determine the origins of T. s. elegans individuals found in wild-states. However, further refinement of the model is required, such as including a wider range of locations of samples to improve the discrimination power.

Differences in the δ¹³C and δ¹⁵N composition of scutes from different status groups are likely primarily influenced by different proportions and sources of plant and animal material within a turtle’s diet, as well as varied sources of these food groups (Balzani et al. 2016). The trophic level of captive turtles, as inferred from δ¹⁵N, is consistently higher than for wild populations, despite potential δ¹⁵N enrichment from agricultural fertilisers in wild environments (Hofmeister et al. 2013). This is likely influenced by a higher consumption of meat-based products by captive turtles, including commercial turtle food (Mazumder et al. 2018). Commercial turtle food often contains marine origin proteins, which may increase δ¹³C and δ¹⁵N (Schoeninger and DeNiro 1984). A similar result has been identified in studies on other reptiles, such as crocodile lizards (Shinisaurus crocodilurus) and monitor lizards (Varanus spp.), where captive animals possessed enriched δ¹⁵N (van Schingen et al. 2016, Natusch et al. 2017, Ziegler et al. 2018). Therefore, δ¹⁵N is a strong candidate for the expansion of this forensic technique into other reptile groups, but likely requires species-specific validation.

The δ¹³C exhibited little power for separating wild and captive groups. As with δ¹⁵N, δ¹³C is influenced by a variety of environmental factors. However, δ¹³C was identified as the most significant separator for wild and captive juvenile T. s. scripta by Aresco and James (2005). Juvenile turtle W1 showed enrichment in δ¹³C , which may be due to an ontogenetic shift in diet by adult T. s. elegans (Reed and Krysko 2014). While δ¹³C had some separating power and may be applied for a more detailed analysis of T. s. elegans dietary behaviour in wild and captive states, we did not find it informative as a biosecurity tool for separating adult incursions.

For each turtle, where shed scute was available, the δ¹⁵N and δ¹³C exhibited significant differences between successive active seasons. However, as there was no consistent direction of change in the isotope data, it is unlikely the changes are due to tissue degradation and instead likely reflected temporal variability in the turtle’s diet. The variance in δ¹⁵N was sufficiently small to ensure that the specimen remained within the same status group, based on the δ¹⁵N‰ ≥ 9.7‰ discrimination value.

It is important to note that the status assignment refers to the confidence that the turtle was wild or captive for the entirety of the scute growth period. The natal origin (birthplace) of the turtle cannot be determined using scute growth alone as scutes are shed yearly (Alibardi 2005). Natal origin determination requires sampling of a tissue which has remained inert since hatching, such as bone, as explored by Holder et al. (2014). Determining a tissue which has remained inert since hatching in T. s. elegans and a comparison to scute material will be extremely useful for future biosecurity efforts.

The exploration of additional biogeochemical tracers may be useful to create a more diverse set of methods and potentially obtain greater evidence of environmental origin. Stable isotopes relating to the animal’s water source such as hydrogen (²H/¹H) and oxygen (¹⁷O/¹⁶O or ¹⁸O/¹⁶O) may provide useful information on the animal’s geographical origin and have been used in other animal tracking applications (Bowen et al. 2005, Hobson and Wassenaar 2018). Furthermore, controlled experiments to determine the scute-diet fractionation factor in T. s. elegans may provide greater information on the animals’ diets.

The values of δ¹³C and δ¹⁵N in scute keratin are effective at filling the requirement for the urgent need for effective forensic techniques to quickly identify the origin of T. s. elegans (red-eared slider turtle) incursions and has promising potential for applications on other high-risk IAS species (Lodge et al. 2006, McFadden et al. 2017). Stable isotope ratios in the scutes of T. s. elegans provide long-term information on individual environmental histories and, thus, provide an effective forensic method for identifying the origins of individuals found in wild-states. This study provides a set of best-practice, relatively accessible methods for sampling IAS incursions and subsequent analysis, including a classification tree, to determine the risk of future incursions. These approaches, using an emerging and effective forensic technique, contribute to the continuing development of various forensic techniques that are crucial for effective biosecurity efforts.

Turtle carcasses were kindly provided through scientific loans by the Queensland Museum (Patrick Couper), Department of Primary Industries and Regions, South Australia (Lindell Andrews) and Australian Museum’s Herpetology Collection (Jodi Rowley and Stephen Mahoney). For sample collection and seizure information, we thank the Primary Industries and Regions SA (Lindell Andrews), the Department of Primary Industry, NSW (Alyssa Trotter, Nathan Cutter), the Elizabeth Macarthur Agricultural Institute (Brendon O’Rourke, Naomi Porter) and Department of Jobs, Precincts and Regions/Agriculture Victoria (Jesse Miller). We are extremely grateful to Mark Rollog for assistance with CF-IRMS data collection, Jennifer Pistevos for her work on pilot analyses and Talia Wittmann for research assistance. This research was supported by the University of Adelaide Environment Institute, by ARC FT110100793 to F.A. McInerney and by Invasive Animals CRC (Project 1L4) and Centre for Invasive Species Solutions (Project PO1-I-002) funding to P. Cassey. An ARC LIEF grant (LE120100054) funded the IRMS used for analyses.