Supporting the recovery of large carnivores is a popular yet challenging endeavour. Estuarine crocodiles in Australia are a large carnivore conservation success story, with the population having extensively recovered from past heavy exploitation. Here, we explored if dietary changes had accompanied this large population recovery by comparing the isotopes δ 13 C and δ 15 N in bones of crocodiles sampled 40 to 55 years ago (small population) with bones from contemporary individuals (large population). We found that δ 13 C and δ 15 N values were significantly lower in contemporary crocodiles than in the historical cohort, inferring a shift in prey preference away from marine and into terrestrial food webs. We propose that an increase in intraspecific competition within the recovering crocodile population, alongside an increased abundance of feral ungulates occupying the floodplains, may have resulted in the crocodile population shifting to feed predominantly upon terrestrial food sources. The number of feral pigs consumed to sustain and grow crocodile biomass may help suppress pig population growth and increase the flow of terrestrially derived nutrients into aquatic ecosystems. The study highlights the significance of prey availability in contributing to large carnivore population recovery.

1. Introduction

Across the globe, large-bodied carnivores have been extirpated from much of their original ranges [1]. Most have experienced substantial population declines and range contractions due to conflict with humans, loss of habitat, and reduction in prey availability [2–5]. Large carnivore loss is an issue because there is a growing body of evidence that healthy populations of these animals are necessary to maintain biodiversity and ecosystem function through ‘top-down’ forces, such as predation, intraspecific competition, and trophic cascades; as well as ‘bottom-up’ forces, such as primary production, nutrient dynamics, and energy cycles [1,6–9]. Moreover, large carnivores tend to be iconic species, and as such, there is a considerable global effort to conserve and recover their populations [10].

Nevertheless, numerous cases remain where large carnivores have not recovered despite concerted efforts. In some cases, the reasons for this are precipitated by humans, while for others, recovery has been unsuccessful due to increased competition for limited resources as the population grows [11].

The estuarine crocodile (Crocodylus porosus) is Australia's large carnivore restoration success story. Unregulated hunting in the mid-twentieth century drove the species to the brink of extinction, with only a few thousand individuals existing in the wild by the 1970s [12]. Regulation in the global trade of crocodile skins and national protection has allowed the Australian estuarine crocodile population to recover to pre-exploitation levels with little additional intervention [13]. The recovery rates in crocodile populations around Australia have been mixed. Some regions, like the Northern Territory (NT), have seen rapid recoveries [14], while populations in other states like Queensland (QLD) and Western Australia (WA) have shown limited recovery over the same period [15,16]. Crocodile population abundance is strongly linked with habitat type, catchment areas, and climate seasonality [17]. Nonetheless, the quality and quantity of the diet presumably play a significant role in the rate of recovery. Understanding the role prey has played in allowing the estuarine crocodile to achieve this population growth may assist us to better understand the relationship between prey competition and large carnivore population recovery.

Estuarine crocodiles are a large-bodied (greater than 500 kg adult body wt.) generalist carnivore, and although they lack the high metabolic requirements of endothermic carnivores, an estuarine crocodile must feed upon the equivalent of approximately 4% of its body weight every week to maintain body mass [18]. Therefore, a 1000 kg crocodile would need to eat approximately 40 kg of protein per week for maintenance, growth and reproduction [18]. Crocodiles' biomass in the NT rivers has increased over the past 50 years, from virtually zero in the early 1970s to almost 500 kg of crocodile per kilometre of river in 2020 [14]. This level of prey protein needs to have been acquired somewhere.

Here, we assessed if the dietary isotopic niche of the estuarine crocodile population has changed from a time when population densities were very low (less than a few thousand) to the current population size (greater than 100 000 adults) [14]. We measured the ratio of stable isotopes (δ13C and δ15N) in bone from museum specimens collected 40 to 55 years ago and compared this with contemporary individuals and potential prey items. Isotopic variations in bones have been used to study animal diets [19], including the balance between freshwater, marine, and terrestrial food sources [20,21]. We hypothesized that as populations grew, the increased competition between crocodiles may have resulted in a greater reliance upon readily available alternative prey sources.

2. Methods

(a) Sample collection

Estuarine crocodile bone samples were collected from north-western areas of the NT (Australia) between Darwin Harbour and the East Alligator River (approx. 300 km apart). Historical bone samples were collected from the Museum and Art Gallery of the NT (MAGNT, Darwin, NT, Australia). Those bones were from crocodiles caught and killed between 1968 and 1986 from various parts of the NT (n = 22). Some crocodiles were very large and were likely older than 50 years of age upon death [22]. Sampled crocodile bones were from animals with total body lengths ranging between 120 and 513 cm. The sampled bones were not treated or preserved and cleaned by macerating the bones in water. Samples were taken from the left front leg humerus. First, an approximately 0.2 mm outer bone layer was scraped away using a scalpel, and then approximately 0.2 g of bone was scraped out and placed in a sample vial for the analysis.

The contemporary cohort of bone samples was collected from crocodiles trapped and removed from around Darwin as part of the crocodile management programme in 2016 [23]. These crocodiles ranged from 115 to 330 cm in total body length (n = 24). A small section from the left humerus bone was removed and prepared as the museum specimens. The bones were not acidified before analysis as carbonate removal has been shown to have minimal effect upon reptile bone δ13C, but instead, a correction factor was used (×1.2 + 2.1; [24]). The mean C/N ratios for the bone samples was 3.4 ± 0.2 s.d. (range = 3.1 to 4.3), and only three out of 48 samples had C/N ratios above a proposed threshold of 3.6 for intact collagen [25].

Bone samples from both cohorts were freeze-dried at −40°C (Dynavac Freeze Dryer) for 48 h and then homogenized to a fine powder using an electric ball-mill grinder (RETSCH Mixer Mill MM400). A small sub-sample from each was weighed (0.8–1.0 mg) into tin capsules and analysed for stable isotopes (δ13C and δ15N) at the Stable Isotope Core Laboratory at Washington State University (WA, USA). The δ13C and δ15N values from those bone samples were corrected using diet–tissue discrimination factors (1.4‰ and 3.0‰, respectively) [24,26,27]. The carbon isotope values were adjusted for the Suess effect by applying a correction factor to the ‘historic’ cohort based on the atmospheric CO 2 [28] for each crocodile's year of death.

Body size is known to influence δ13C and δ15N values in crocodilians. Our ‘contemporary’ cohort had a reduced range of body sizes compared to the ‘historic’ cohort. To provide a more comparative range of body sizes, we sourced additional stable isotope values from Adame et al. [26]. These estuarine crocodiles (n = 41, size range = 83.5 to 420 cm, details in the electronic supplementary material, table S1) were captured from the same region between 2012 and 2014 (details on scute tissue isotopic discrimination in [26]). We first tested if it was a valid assumption to combine these two groups of crocodiles, whose collagen had been sampled from different tissues (bone and scute). The first method performed was an analysis of covariance (ANCOVA) in R [29]. The models assessed the dependent variables δ13C and δ15N against the interaction between the factors ‘group’ and ‘total body length’ (covariate). The second method was a Bayesian framework to assess the stable isotope niche region and pairwise niche overlap. Isotopic values were plotted using the package nicheROVER [30] in R (electronic supplementary material, figure S1). Given the lack of statistical difference (electronic supplementary material, table S2 and figure S1), the stable isotope data from all contemporary crocodiles were combined (n = 65) for comparison with the ‘historic’ cohort.

ANCOVA was used to test for differences in δ13C and δ15N between the historic and contemporary samples across the size ranges, with the stable isotope values as dependent variables and the interaction between cohort and total body length (covariate) as factors (table 1). Isotopic niche widths and overlap were calculated using the package SIBER (Stable Isotope Bayesian Ellipses in R) [31] in R (electronic supplementary material, figure S2).

Table 1. Summary of performed statistics tests (ANCOVA). Collapse model coefficients dependent variable: δ15N dependent variable: δ13C sum Sq d.f.d F-value pe sum Sq d.f.d F-value pe intercepta 81.1 1 85.496 <0.001 4728.2 1 1044.606 <0.001 cohortb 5.6 1 5.937 0.017 77.2 1 17.054 <0.001 body lengthc 16.7 1 17.567 <0.001 105.7 1 23.355 <0.001 cohort : body length 0.7 1 0.782 0.379 5.3 1 0.167 0.283

3. Results and discussion

Our key finding was that contemporary estuarine crocodiles have significantly lower δ13C and δ15N values than those sampled 40 to 55 years ago (figure 1). Collagen has a slow tissue turnover rate, and these isotopic values reflect the individual's broad diet over several years [24,32]. Estuarine crocodiles are highly mobile individuals and regularly travel over 400 km in a few weeks, connecting populations across broad regions [33,34]. In the study region, individuals regularly travel between coastal, estuarine and freshwater environments [35]. Therefore, our SIA results present a broad picture of the isotopic landscape of the estuarine crocodile population over two different periods. We found that individuals became more 13C- and 15N-enriched as they grew, and this relationship was similar between ‘historical’ and ‘contemporary’ cohorts (figure 1 and table 1). Ontogenetic changes in isotopic values are common in crocodylians [27,36–39] and other large carnivores that show indeterminate growth [40] and are suggested to reflect changes in diet and metabolism as the animal grows [38,41]. This evidence suggests that the δ15N of estuarine crocodiles scaled similarly with body size class under high (contemporary) and low (historic) population densities. However, the baseline shifts in both δ13C and δ15N suggest that the contemporary diets of crocodiles include a significantly greater contribution from terrestrial sources (figure 2). Figure 1. Relationship between (a) δ15N (‰) and (b) δ13C (‰) and total body length of estuarine crocodiles (Crocodylus porosus). Purple circles denote specimens collected between 1968 and 1986 (n = 22), and blue triangles represent samples collected between 2012 and 2016 (n = 65). Shaded area denotes 95% confidence intervals. Figure 2. Stable isotope biplot of estuarine crocodiles (Crocodylus porosus). Purple denotes samples collected between 1968 and 1986 (n = 22) and blue represents samples collected between 2012 and 2016 (n = 65). Marginal density plots represent the data distribution and ellipses show the standard Bayesian areas corrected for the sample size for each cohort. Standardized ellipses area and Layman metrics are shown in the electronic supplementary material (figure S2). The isotopic ratios for wallaby Macropus agilis (n = 19), buffalo B. bunalis (n = 8), pig S. scrofa (n = 57), mullet Liza ordensis (n = 82), barramundi Lates calcarifer (n = 170) and catfish Netuma thalassina (n = 5) were sourced from [26]. Data points are mean ± s.d.

The observed decreases in both δ13C and δ15N values of contemporary crocodiles (figure 1) are inconsistent with potential isotopic shifts in terrestrial and floodplain basal resources (e.g. invasion of C4 grasses [42]). Similarly, they are inconsistent with a shift in δ15N baseline values from rising point-source nitrogen loads in the region [43,44]. We argue that the observed differences in isotope values between historical and contemporary crocodiles reflect a major shift in prey from predominantly riverine–marine to predominantly terrestrial food resources (figure 2). As a population, historic crocodiles appear to have fed upon a broader diversity of prey than contemporary individuals. Crocodiles are generalized feeders and eat whatever prey is available to them. While this may reflect a decreased availability of marine-based and other prey, we propose that the dietary shift was more likely due to increased availability of a single terrestrial prey (e.g. feral pigs, Sus scrofa) [18,26,45].

Over the last 50 years, management interventions have significantly reduced the local population of Asian water buffalo (Bubalus bubalis; 5.6 to less than 0.1 buffalo per km2) [46]. There are sparse survey data for feral pig abundance over a comparative period. Still, anecdotal observations suggest feral pigs are at far higher densities than before buffalo eradication [47], and empirical studies demonstrate that buffalo removal results in growth in local feral pig abundance [48]. Buffalo are large herding animals and could be eaten only by very large crocodiles, whereas the reduced size of feral pigs and their wallowing behaviour near waterbodies make them ideal prey across a broader size of estuarine crocodiles. Pigs are prolific breeders compared to buffalo and native animals like kangaroos, providing a quickly replenished and highly nutritious food supply for crocodiles.

If feral pigs are indeed the primary prey source maintaining and growing this large estuarine crocodile population, then the increase in crocodile biomass would be accompanied by terrestrial sourced nutrients being deposited into the freshwater environment. This is supported by stable isotope values reflecting high terrestrial inputs (specifically feral pigs), even for crocodiles with a body size too small to capture live pigs [26]. Our results reveal that this terrestrial contribution was not as high 40 to 50 years ago, supporting the hypothesis that crocodylians create habitat linkages and nutrient fluxes between terrestrial and aquatic food chains [49–51]. Although feral pig abundance has been reported to have increased in the past 40 years [47], it is plausible that crocodiles may suppress feral pig populations. Therefore, estuarine crocodiles may mitigate feral pigs' adverse ecological and agricultural impacts [52].

In conclusion, the extensive recovery of the estuarine crocodile population in the study area appears to have been supported by access to an increased abundance of invasive terrestrial prey (i.e. feral pigs). The differences in population recovery rates in other parts of Australia [15,16] may be attributed to lower terrestrial prey densities, where riparian habitats do not support such large feral pig populations. As the crocodile population grew, interspecific competition caused individuals to move back into inland floodplains, where they were previously extirpated by hunters [12,14,53]. Before the arrival of large exotic terrestrial vertebrates, crocodiles likely fed upon native terrestrial herbivores. However, these do not occur in similar densities within floodplains nor inhabit swamp areas that would make them as accessible to crocodiles. Further research is required to better understand the link between prey availability, bioenergetics and crocodile population growth. Crocodile prey choice may be enriching oligotrophic freshwater systems with terrestrially derived nutrients, like what has been observed for large vertebrates in other ecosystems [54]. What this means for the ecology of Australian river-floodplain ecosystems remains unknown and requires future investigation.

Ethics

The historical cohort of estuarine crocodile bones used in the present study was sampled from museum specimens (Museum and Art Gallery of the Northern Territory; MAGNT). The contemporary cohort of bone samples was donated by the Department of Environment, Parks and Water Security (DEPWS) from crocodiles removed for human safety [23,55]. The sampling of crocodile scute was conducted under the Griffith University animal's ethics protocol approved by the Animal Ethics Committee (ENV/08/11/AEC) and Kakadu National Park permit guidelines (RK 786) [26].

Data accessibility

All relevant data are within the paper and its electronic supplementary material. Dataset files are available from the Dryad repository linked to https://doi.org/10.5061/dryad.j3tx95xgm [56].

The data are provided in the electronic supplementary material [57].

Authors' contributions

M.A.C.: conceptualization, data curation, formal analysis, investigation, methodology, project administration, validation, visualization, writing—original draft and writing—review and editing; V.U.: formal analysis, funding acquisition, methodology, validation, visualization, writing—original draft and writing—review and editing; Y.F.: funding acquisition, resources, writing—original draft and writing—review and editing; R.K.K.: funding acquisition, writing—original draft and writing—review and editing; T.D.J.: formal analysis, funding acquisition, investigation, methodology, validation, writing—original draft and writing—review and editing; S.E.B.: data curation, funding acquisition, investigation, validation, writing—original draft and writing—review and editing; H.A.C.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, validation, writing—original draft and writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This work was supported by the Australian Research Council (project ID: DP210103369).

Acknowledgements We thank Gavin Dally from the Museum and Art Gallery of the Northern Territory (MAGNT, Darwin, NT, Australia) and the Northern Territory Department of Environment, Parks and Water Security (DEPWS) for the crocodile tissues, and Maria Fernanda Adame for providing published crocodile data.

Footnotes

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5942391.