Unique Population or Unique Species? Genetic Insights into the Pygmy Freshwater Crocodiles of Northern Australia

Isolated populations of freshwater crocodiles (Crocodylus johnstoni) in elevated, rocky escarpment areas of the Northern Territory reach sexual maturity earlier and generally have an adult size half that of their downstream counterparts. There is debate over whether these ‘pygmy’ or ‘dwarf’ crocodile populations are the result of extreme phenotypic plasticity or the origins of a unique species through peripatric speciation. This debate is of conservation concern as these populations face immediate threats of invasive species and climate change, and species classification can affect conservation management strategies. We used mitochondrial DNA control region and cytochrome b sequences to investigate whether mtDNA haplotype variation supports any evidence of speciation between the pygmy and standard-size freshwater crocodiles. Additionally, we used genotyping by sequencing (dd-RADseq) to examine genetic clustering. Separated and concatenated control region and cytochrome b haplotypes were shared between pygmy and standard-size crocodile populations. Principle Component Analysis and STRUCTURE analyses on dd-RADseq data showed pygmy and standard-size crocodiles cluster by geographic location as much as by phenotype. We found no clear and objective genetic evidence to suggest that the pygmy freshwater crocodiles should be considered a separate species to any other population of the standard-size crocodiles. However, the isolation and environmental adaptability of these unique pygmy freshwater crocodile populations make them ecologically unique and valuable for conservation, and worth safeguarding against potential threats. Unique Population or Unique Species? Genetic Insights into the Pygmy Freshwater Crocodiles of Northern Australia Katherine Brittain 1, Torre Muhlbach 1, Rui Cao 1, Jose Luis Mijangos 1,2, Dean Yibarbuk 3, Ruchira Somaweera 4,5, Nancy N. FitzSimmons 6, Jaime Gongora 1 1 Sydney School of Veterinary Sciences, Faculty of Science, University of Sydney, NSW 2006, Australia 2 Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia 3 Warddeken Land Management Ltd, P.O. Box 785, Nightcliff NT0814 4 Stantec Australia, 226, Adelaide Tce, Perth WA 6000, Australia 5 Environmental and Conservation, Murdoch University, 90 South Street, Murdoch, WA 6150, Australia 6 Australian Rivers Institute, Griffith University, Nathan, QLD 4111, Australia Corresponding author: [email protected] Short title: Unique population or unique species?

Isolated populations of freshwater crocodiles ( Crocodylus johnstoni ) in elevated, rocky escarpment areas of the Northern Territory reach sexual maturity earlier and generally have an adult size half that of their downstream counterparts. There is debate over whether these ‘pygmy’ or ‘dwarf’ crocodile populations are the result of extreme phenotypic plasticity or the origins of a unique species through peripatric speciation. This debate is of conservation concern as these populations face immediate threats of invasive species and climate change, and species classification can affect conservation management strategies. We used mitochondrial DNA control region and cytochrome b sequences to investigate whether mtDNA haplotype variation supports any evidence of speciation between the pygmy and standard-size freshwater crocodiles. Additionally, we used genotyping by sequencing (dd-RADseq) to examine genetic clustering. Separated and concatenated control region and cytochrome b haplotypes were shared between pygmy and standard-size crocodile populations. Principle Component Analysis and STRUCTURE analyses on dd-RADseq data showed pygmy and standard-size crocodiles cluster by geographic location as much as by phenotype. We found no clear and objective genetic evidence to suggest that the pygmy freshwater crocodiles should be considered a separate species to any other population of the standard-size crocodiles. However, the isolation and environmental adaptability of these unique pygmy freshwater crocodile populations make them ecologically unique and valuable for conservation, and worth safeguarding against potential threats.

Wildlife conservation, Crocodylus johnstoni, dwarfism, dd-RADseq, phenotypic plasticity, speciation

The genus Crocodylus (i.e., true crocodiles) makes up approximately half of the extant diversity of the Order Crocodylia (crocodylians) (Uetz et al., 2025, Grigg, 2015, Oaks, 2011). True crocodiles span a diverse range of habitats, with three major evolutionary lineages distributed in Australasia, the Americas, and Africa (Meredith et al., 2011, Oaks, 2011). Two species of crocodylians occur in Australia, which are sympatric in parts of northern Australia. The larger of the two, the saltwater or estuarine crocodile ( Crocodylus porosus ), inhabits possibly all tidal waterways and many adjacent freshwater systems across tropical northern Australia, as well as small islands offshore (Webb et al., 1983). The smaller Australian freshwater crocodile ( Crocodylus johnstoni ) is endemic to tropical mainland northern Australia (in Western Australia, WA; Northern Territory, NT; and Queensland, QLD) and has not been reported from any offshore islands. Introduced populations exist from Townsville to Rockhampton in QLD and further south (Read et al., 2004). They typically inhabit freshwater habitats upstream of tidal influence within their range, while few populations extend into tidal, saline waters (Read et al., 2004, Webb et al., 1983). The males of this species can grow to over 3 m in length and the females over 2 m, and maximum recorded ages are 64 years for males and 54 years for females (Tucker et al., 2006, Webb and Manolis, 1989). In the 1980s, isolated populations of freshwater crocodiles were found in higher-elevation, escarpment habitats at Liverpool River in Arnhem Land (NT) that exhibit several morphological trait variations compared to the standard-size freshwater crocodile (Webb, 1985). These crocodiles generally grow to half the mature adult size of their downstream counterparts and reach sexual maturity at a smaller size (Webb, 1985). Consequently, these populations are referred to as ‘dwarf’, ‘stunted’, ‘stone country’, or ‘pygmy’ (hereafter pygmy) crocodiles by the popular media (Britton et al., 2013). A second population was reported in the upper reaches of the Bullo River (NT) in the 2000s that may have greater size variation, with the largest crocodile found at a total length of 1.7 m (Britton et al., 2013). There has been a public debate as to whether these pygmy freshwater crocodile populations are an example of phenotypic plasticity - the varying phenotypic expression of an organism in response to environmental stimuli or resource availability (Pfennig and West-Eberhard, 2021, West-Eberhard, 2005) - or whether speciation has occurred (McCue, 2014, Webb, 1985, Webber, 2015). While classifying what constitutes a species is an ongoing discussion (Baker and Bradley, 2006, Hillis, 2019) and beyond the scope of this paper, our study aims to produce genetic evidence to assist in making conservation and management decisions. A very limited amount of peer-reviewed research in ecology, biology or genetics exists in quantifying differences in pygmy freshwater crocodiles. Morphometric analyses have revealed overall reduced dimensions in pygmy populations compared to other freshwater crocodiles (Edwards et al., 2017). However, geographic variation in morphology is common among crocodylians (Labarre et al., 2017, Nestler, 2012, Webb and Messel, 1978), suggesting that exploring genetic differences could provide further insights. Classifying the pygmy freshwater crocodiles as a distinct species from the standard-size freshwater crocodile could have important implications for the conservation management of these populations. Currently, the freshwater crocodile is listed as ‘Least Concern’ on the IUCN red list, based on the total species population size, largely intact habitat and a wide distribution (Isberg et al., 2017). We investigated the genetic variation of the pygmy and standard-size freshwater crocodiles from representative and widely spread population across northern Australia using the hypervariable mitochondrial control region (CR) and cytochrome b (Cytb) sequences. The CR was chosen due to its high mutation rate, making it useful for determining intraspecific relationships and its common use in the literature to report population genetics for other crocodylian species. The Cytb gene was chosen to provide resolution at an inter-specific level, as it is more commonly used to report genetic relationships between species. We further investigated the genetic clustering and relatedness statistics of pygmy and standard-size freshwater crocodiles across a subset of the species distribution using the whole genome approach reduced representation sequencing (dd-RADseq), providing a higher resolution dataset.

Sample collection and DNA extraction Freshwater crocodile tissues were collected from 21 sampling locations across the species’ distribution in the northern river systems of Australia, including samples from pygmy freshwater crocodile populations (Appendix 1). All samples were collected for other studies and appropriated for this paper (Cao et al., 2020, FitzSimmons et al., 2000, Somaweera et al., 2019, Somaweera and Shine, 2011, Somaweera et al., 2013). In summary, individuals were captured and restrained using methods appropriate to their size (Cao et al., 2020), and dorsal tail scutes were cut using scissors or a knife dipped in 70% ethanol for marking the crocodiles, and the resulting tissues stored in Ethics Committee (AEC) (approvals 2017-18 and 2017-22) and the Department of Biodiversity Conservation and Attractions AEC (approval 2014/13), with relevant scientific research permits 08-000949-1, 08-000950-1, SC001387 and ECNR970004. DNA was extracted from the samples using the DNeasy Blood and Tissue extraction kit from Qiagen (Germany), the phenol-chloroform protocol (Green and Sambrook, 2012) or the Isolate II Genomic DNA kit from Bioline. The concentration and quality of DNA samples were determined using Qubit (Life Technologies, USA), Nanodrop and 1% agarose gels. Analysis of control region and cytochrome b To examine genetic distances and haplotype sharing between pygmy and standard-size freshwater crocodile populations, we used 85 of the collected samples to analyse two mtDNA markers. A 794 bp fragment of the mtDNA CR was amplified using primers L15463 and 5’ CAC TAA AAT TAC AGA AGA GCC GAC 3’ modified for freshwater crocodile specificity from universal crocodylian primer H16260 in FitzSimmons et al. (2002) and the following PCR thermal cycling conditions: initial denaturation for 2 minutes at 94˚C, 32 cycles of denaturation at 94˚C for 25 sec, annealing at 48˚C for 45 sec and extension at 72˚C for 45 sec, followed by one cycle of 72˚C for 5 minutes for the final extension. A 1347 bp fragment of the mtDNA Cytb gene including flanking regions was amplified using primers 5’ ACC AAG ACC TAG GGC ACG AAA AAC C 3’ and 5’ TCT GTC TTA CAA GGC CAG CGC TTT 3’ that were modified for freshwater crocodile specificity from universal crocodylian primers CP14126 and CP15546 in Meganathan et al. (2009). PCR conditions were: 94˚C for 5 min of initial denaturation followed by 30 cycles of denaturation at 95˚C for 1 min; annealing at 50˚C for 30 sec; extension at 72˚C for 30 sec. Amplification ended with a 5 min final extension step. Samples were sent to the Australian Genome Research Facility (AGRF) for PCR amplification and Sanger sequencing. Sequence reads were aligned using ClustalW (Thompson et al., 1994) and trimmed in BioEdit (Hall, 1999) to give a final sequence length of 685 bp for the CR and 1200 bp for the Cytb gene. Additionally 18 CR sequences from 18 Bullo River pygmy freshwater crocodiles were aligned with CR data generated here. As some sites only had one or two samples, sites were grouped into river basins for further analysis, except for the Bullo River samples (Victoria River Basin), which were analysed independently of the Victoria River Basin standard-size samples. We assessed whether the level of divergence among haplotypes found in standard-size versus pygmy freshwater crocodile phenotypes was greater than the level of divergence among haplotypes found in different standard-size crocodile river basin populations. This was done by calculating pairwise genetic distances of haplotypes between and within river basin populations for each of the CR and Cytb haplotype sets in MEGA-X (Kumar et al., 2018) using the Tamura-Nei substitution model. This model was chosen as the best fit for CR and Cytb analyses based on the Bayesian information criterion given in MEGA-X. To visualise the shared and unique haplotype distribution among river basins, median joining networks (MJN) and haplotype maps were created in PopART 1.7 (Leigh and Bryant, 2015). The CR and Cytb sequences (excluding Bullo River samples; n = 85) were then concatenated to give a single alignment, and an additional MJN was created for these data. Population clustering using genotyping by sequencing dd-RADseq To generate dd-RADseq data, seven of the pygmy freshwater crocodile samples from Liverpool River used in the mtDNA analysis and one additional pygmy freshwater crocodile sample from Liverpool River were used. Methods and analyses followed that of Cao et al. (2020), who analysed standard-size freshwater crocodiles in the east and west Kimberley region of Australia. Genomic DNA (200 ng) was double-digested using the enzyme pair ecoRI and NlaIII, with the ligation-compatible barcode adapters A and P2, and restriction site overhang. Fragmented DNA was enriched by PCR, size selected for 60 bp, and sequenced using the NextSeq platform (Illumina, USA) in four lanes. SNP discovery was done using a maximum likelihood model implemented in Stacks (Catchen et al., 2013) before being stored in a VCF file as described in Cao et al. (2020). To improve the quality of our analyses, the standard-size crocodile data (stored in the Sequence Read Archive under project number PRJNA551392) used in Cao et al. was refiltered along with the pygmy freshwater crocodile data as a single dataset. The dataset was filtered in the R package dartR (Gruber et al., 2018) to exclude individuals with more than 94% missing data and loci missing from more than 64% of individuals. These thresholds were chosen due to the overall poor SNP call rate of the dataset and to keep the small number of pygmy freshwater crocodile samples in the dataset. The dataset was also filtered in dartR to remove SNPs out of Hardy-Weinberg equilibrium in any sampling location using the ‘Out-Combo’ approach (Pearman et al., 2022) using p-values adjusted for multiple comparisons and other default parameters, or with a minor allele frequency below 0.05. To measure genetic variation within sample locations (Appendix 1), observed heterozygosity ( H o ), unbiased (i.e., corrected for sample size) expected heterozygosity ( uH e ) and inbreeding coefficient (F IS ) were calculated. To visualise spatial patterns of genetic variation in freshwater crocodiles, principal component analysis (PCA) was performed using the R package dartR (Gruber et al., 2018). Private alleles, which are exclusive to particular populations, and fixed differences, in which two populations share no alleles, can be used as indicators of gene flow (Georges et al., 2018, Szpiech and Rosenberg, 2011). To investigate gene flow between freshwater crocodile sample locations, dartR was used to calculate the number of private alleles and fixed differences between these locations. To use fixed differences to infer operational taxonomic units (OTU), iterative fixed difference analysis grouped populations with one or fewer fixed differences. Expected number of false positives for fixed differences adjusted for grouped population size was calculated in dartR and observed and expected false positives were tested for significance (P < 0.05). Inbreeding (mating of related individuals) is one factor that can increase genetic differentiation between populations, particularly if populations are small (Huang et al., 2009). To investigate the degree to which inbreeding may have influenced genetic structure in freshwater crocodile populations, the probability of identity of descent (IBD) was calculated across all loci that would result from all the possible crosses of the individuals that were sampled. IBD was calculated by an additive relationship matrix approach (Endelman and Jannink, 2012) as implemented in dartR and rrBLUP (Endelman, 2011). To further investigate levels of inbreeding, inbreeding coefficients were estimated for each individual using two different statistics as described in Keller et al. (2011). These were a) F alt, where a homozygous locus in an individual is weighted by the inverse of that allele’s frequency in the population using the software GCTA (Yang et al., 2011) and the command –ibc, and; b) F h, which is a deviation in homozygosity from its Hardy–Weinberg expectation using the software PLINK (Purcell et al., 2007) using the –het command (Earl and vonHoldt, 2012). Population clustering was further investigated using STRUCTURE (Pritchard et al., 2000). The ‘no admixture’ model with default settings was run with 50 000 burn-in steps and 50 000 replications for five iterations for each K (number of populations modelled) from 1 to 6. The most likely K was selected using the delta K method described by Evanno et al. (2005) and supported by the Ln Pr(X|K) method described by Pritchard et al. (2000). Plots for these methods were generated in Structure Harvester (Earl and vonHoldt, 2012). To determine substructure, the STRUCTURE model was repeated on the subset of data for each K found until no clear structure was identified. Final structure plots were generated using CLUMPP (Jakobsson and Rosenberg, 2007) and DISTRUCT (Rosenberg, 2004).

Mitochondrial cytochrome b and control region analyses Shared and unique Cytochrome b (Cytb) and Control Region (CR) haplotypes were found between the pygmy and standard-size freshwater crocodile populations (stored in BankIt with GenBank accession numbers PV102152 to PV102236 and PV102049 to PV102151 respectively). The partial Cytb gene contained 28 substitutions to give 16 distinct haplotypes, and the partial CR sequence contained 13 substitutions to give eight distinct haplotypes. The Cytb haplotypes were labelled Cytb_H1 through Cytb_H16 (Figure 1). Haplotype Cytb_H1 was not found in pygmy freshwater crocodiles but was found in standard-size crocodiles in eight of 13 river basins in Western Australia (WA), Queensland (QLD) and the Northern Territory (NT) (Figure 2). Cytb_H2 was differentiated from Cytb_H1 by eight mutations and was the predominant haplotype in the Liverpool River Basin pygmy freshwater crocodile population. This haplotype was also found in standard-size individuals in the Normanby River Basin (QLD) and Ord River Basin (WA). Haplotype Cytb_16 was identified only in the pygmy freshwater crocodile samples and differed from Cytb_H1 by a single mutation. Of the remaining 13 haplotypes, 12 were unique to their river basin, and only one was found across two West Australian river basins. The MJN analysis did not suggest any clear clustering patterns among haplotypes and geographical location, except a cluster of haplotypes (Cytb_H3-6) found only in the Ord River Basin that differed by at least seven mutations from any other haplotype. The CR haplotypes were labelled CR_H1 through CR_H8. The CR haplotypes, including those from the Bullo River samples, showed the same pattern as Cytb haplotypes of having one main haplotype. Haplotype CR_H1 was found in 12 of 14 river basins across the entire geographical range, including the Liverpool River Basin pygmy freshwater crocodile samples. CR_H2 was found in 4 of 12 river basins across the Northern Territory and Queensland, including the Bullo River and Liverpool River Basins pygmy freshwater crocodiles. The remaining six haplotypes were unique to a single river basin. Of the 18 Bullo River samples, 16 had the CR_H2 haplotype, and two had unique haplotypes (CR_H7, CR_H8). The Liverpool River Basin pygmy freshwater crocodiles had no unique haplotypes. The MJN did not show any clear clustering patterns among haplotypes and geographical location. Pairwise genetic distances between CR haplotypes in pygmy and standard-size freshwater crocodiles ranged from 0.15% to 1.21% (Table 1). The greatest pairwise distance occurred between haplotypes unique to standard-size freshwater populations in the Ord River Basin (WA) versus Normanby River Basin (QLD) or Roper River Basin (NT). Pairwise distances between the Cytb haplotypes ranged from 0.08% to 1.28%. The greatest pairwise distances (>1.19%) occurred between Cytb_H2, found in pygmy and standard-size freshwater crocodiles, and haplotypes Cytb_H3 -H6, which were all haplotypes unique to the Ord River Basin (WA). Population clustering using dd-RADseq Sequencing and variant calling generated 88 672 loci across 181 individuals. After filtering we retained 172 individuals and 1032 loci for downstream analyses. The dd-RADseq analyses indicated that populations clustered by river basin. Genetic diversity as measured by observed heterozygosity (H o ) was similar among sample locations while unbiased expected heterozygosity (uH e ) varied between locations (Figure 3). H e was lower in the groups sampled at Liverpool River (pygmy freshwater crocodiles) and at Lake Argyle, which also had a small sample size per locus. Inbreeding coefficient F IS was positive in all sample locations. Principal component analyses (PCA) showed that the dd-RADseq data segregated into four different clusters based on river basins (Figure 4). Samples from the Lakes Argyle and Kununurra within the Ord River Basin (east Kimberley area) formed an independent cluster. In contrast, the clusters formed by Fitzroy River Basin and Lennard River Basin (west Kimberley area) had some overlap. The pygmy freshwater crocodile samples from the Liverpool River Basin (Arnhem Land escarpment area) formed the fourth cluster, although individuals in this group were more spread than in the other clusters. The first principal component axis (PC1) explained 7.2% of the variance between samples; PC2 explained 2.9% of the variance and separated the pygmy individuals, sampled in the Liverpool River, from the rest of individuals in other groups, and PC3 explained 2% of the variance and separated individuals from Fitzroy River Basin from individuals sampled at Lennard River Basin. Estimates of private alleles (Table 2) show that the two groups with smaller sample size (pygmy freshwater crocodiles sampled at the Liverpool River, n = 8, and individuals sampled at Lake Kununurra, n = 6) had the largest number of private alleles when compared to the rest of the individuals. These results may be in part due to the small sample sizes of these groups, which would not have captured the extent of genetic diversity. The largest number of fixed differences, when each group was compared to the rest of the individuals, was observed in the pygmy freshwater crocodiles despite the small sample size. Pygmy freshwater crocodiles also had many private alleles compared to the rest of individuals, followed by the Kununurra sample, which also had a low sample size. Fixed difference analysis grouped populations into three operational taxonomic units (OTU), which aligned with defined geographic areas of west Kimberley, east Kimberley, and Arnhem Land escarpments in the Northern Territory (Table 3). The observed number of fixed differences between these OTUs significantly exceeded expected false positives in all pairwise comparisons (P = <0.001). Identity by descent (IBD) results (Figure 5) agreed with the results of the PCA analysis in identifying four clusters, each including related individuals. Pygmy freshwater crocodiles sampled in the Liverpool River had the highest IBD values. In agreement with IBD results, mean inbreeding coefficients by individual (Figure 6) were higher in the pygmy freshwater crocodile group sampled in the Liverpool River than in other groups, particularly in the statistic F alt . Clustering shown by sequential STRUCTURE analyses (Figure 7) using the DeltaK and Ln Pr(X|K) methods identified four genetic clusters (K = 4), which were consistent with the four river basins.

We analysed mitochondrial sequences and SNPs to inform the debate on whether the traits displayed by pygmy freshwater crocodiles in comparison to standard-size freshwater crocodiles are due to phenotypic plasticity or a result of an ongoing speciation process. We did not find strong evidence suggesting that speciation has occurred despite the phenotypic differences observed in the population of pygmy freshwater crocodiles in escarpment areas of the Liverpool River in Arnhem Land (Britton et al., 2013, Webb, 1985). We found that genetic difference between pygmy and standard-size crocodiles was smaller than the genetic difference among standard-size crocodiles populations sampled at different river basins. Population structure and genetic distance using mtDNA haplotypes Our results based on mtDNA do not support the hypothesis that pygmy freshwater crocodiles form a separate species from standard-size crocodiles. We observed that the maximum pairwise distance (1.28%) of Cytb haplotypes between pygmy and standard-size freshwater crocodiles was below the reported minimum uncorrected distance of 5.2% between the species of Central America/Colombian C. acutus and C. rhombifer, and closer to the intra-species maximum of 0.8% for C. acutus within Central America and Colombia (Bloor et al., 2015). For instance, previous work on species delimitation in mammals suggests that two groups of individuals can be considered the same species if the Cytb distance between the groups is below 2%; groups in which Cytb distance is between 2% and 11% require further study to determine species delimitation; and groups separated by a Cytb distance above 11% can be considered different species (Bradley and Baker, 2001). Although crocodylian nuclear genomes have a slower evolutionary rate compared to most other vertebrates (Green et al., 2014), crocodylian mitochondrial genomes have a faster evolutionary rate similar to mammals (Eo and DeWoody, 2010, Janke and Arnason, 1997). We could expect similar levels of genetic difference between species with similar divergence times. Few studies have used Cytb to estimate pairwise distance in crocodylian species, and while more data are available for CR, it is less reliable for identifying speciation. The maximum CR pairwise distance of 1.21% for all pygmy and standard-size freshwater crocodile haplotypes was within the intra-species ranges reported in other Crocodylus studies, ranging from 0.48% to 1.8% (Gratten, 2003, Luck et al., 2012, Ray et al., 2004). Studies that have reported on the inter-specific CR pairwise distance of Crocodylus species had minimum values of 5.53% (Gratten, 2003, Ray et al., 2004). Additionally, the predominant pygmy Cytb and CR haplotypes were both observed in standard-size populations. The most common haplotype in standard-size freshwater crocodiles, Cytb_H1, was not present in pygmy freshwater crocodiles. In contrast, the only haplotype unique to pygmy freshwater crocodiles, Cytb_16, differed from Cytb_H1 by a single mutation. Unique haplotypes were also found in other populations. The predominant pygmy freshwater crocodile haplotypes Cytb_H2 and CR_H2 differed from the most common haplotypes by eight and four mutations, respectively, indicating they may be different ancestral lineages. It is important to note that Cytb_H2 and CR_H2 are shared between pygmy and standard-size freshwater crocodiles. Additionally, a third lineage may be present in standard-size populations from the eastern Kimberley area, with a cluster of Cytb haplotypes unique to the area that differ from the common haplotype by eight mutations and a CR haplotype unique to the area that differs from the common haplotype by six mutations. The MJN created from concatenated Cytb, and CR sequences supported these conclusions but does not provide any further resolution (Appendix 2). Larger sample sizes from all populations could help resolve the dynamics among these populations. Population clustering using dd-RADseq The PCA and STRUCTURE graphs generated from dd-RADseq data supported genetic clustering by river basin, with clear independent clustering of the Ord River Basin (east Kimberley, WA) samples, the Lennard and Fitzroy River Basins (west Kimberley, WA) samples and the Liverpool River Basin (Arnhem Land escarpments, NT). There was no evidence of structure within river basins, given the limited sampling. Inbreeding coefficients, the relatedness matrix, private alleles, and fixed differences indicated restricted gene flow among river basins. This is expected given the limited overland movements and site fidelity behaviours of the freshwater crocodile (Lang, 1978, Webb et al., 1983), vast geographical distances (approximately 400-1100 km) and geographic barriers between the sampled basins. Further genomic studies could also incorporate data from Bullo River pygmy populations to see if these pygmy freshwater crocodiles share more genetic structure with the Liverpool River Basin pygmy freshwater crocodiles or standard-size freshwater crocodiles in their own Victoria River Basin. The sequential STRUCTURE analyses showed population structure occurring between the river basins but not within river basins, supporting the results of the PCA. However, our small sample size for the Liverpool River Basin and our lack of diverse sample sites within the river basins may affect the analyses’ ability to reflect true genetic structure. Private allele and fixed difference analyses can be used as an indicator of lack of gene flow between populations. While our results do show significant differences in allele fixation among our three geographic areas of west Kimberley area (Lennard and Fitzroy River Basins), west Kimberley area (Ord River Basin) and Arnhem Land escarpments (Liverpool River Basin), our analysis lacked the comprehensive coverage of populations (only one to three sample sites within each river basin) needed to accept this as an accurate representation of OTUs across the freshwater crocodile distribution (Georges et al., 2018). While the east and west Kimberley regions are adjacent to each other, neither is adjacent to the Arnhem Land escarpment area, and even within the Kimberley region, no sample sites were located along the east/west divide where we would most likely see cases of migration and gene flow if they were occurring. Evidence for speciation potential While there is little genetic evidence to support a separate taxonomic status between pygmy and standard-size freshwater crocodiles, the geographic isolation of the latter populations and the phenotypic differences indicate the potential for peripatric speciation. This is a type of allopatric speciation where the secondary population is smaller and, therefore, more likely to experience genetic drift and allele fixation than the first population. We can see possible evidence of genetic drift in the Cytb and CR haplotype maps, where the predominant haplotype differs among populations, and in the SNP data analyses where fixed difference analysis and STRUCTURE clusters occurred at geographic boundaries. Allopatric speciation is a process rather than being the result of a single event in time. It is not always a linear progression from ‘same species’ to ‘different species’ but could instead involve periods of allopatry mixed with periods of gene flow or introgression events (Georges et al., 2018). As a form of allopatric speciation, peripatric speciation encounters all the same challenges for genetic species delimitation and the ‘burden of proof’ required for diagnosing species from OTUs identified using molecular techniques such as fixed differences is greater in allopatric scenarios compared to sympatric or parapatric ones (Unmack et al., 2022). For cases where intermediate sample sites exist but have not been included in the analysis – as is the case with our pygmy and standard-size freshwater crocodiles – the burden of proof is higher still. As such, a greater distribution of sample sites, particularly along the contact zones between geological divisions and putative migration barriers, is needed to provide a more robust analysis of possible speciation. It is believed that the pygmy freshwater crocodile populations in the Liverpool River and Bullo River escarpment areas exhibit extreme, yet species-typical, phenotypic plasticity due to resource limitations. Crocodile fitness is partially influenced by the environments they inhabit, with limited food resources known to reduce growth rates and maximum size in reptiles, including crocodiles (Le Galliard et al., 2005, Stamps and Tanaka, 1981, Webb and Messel, 1978). Mark-and-recapture studies across the Kimberley region indicate that, on average, larger body sizes are observed in freshwater crocodiles living in permanent water bodies with a more abundant food supply (Somaweera, pers. obs.). For instance, the average body size of both male and female crocodiles from Lake Argyle and Lake Kununurra—where water remains deep throughout the year with abundant fish and invertebrate life—are larger than those from the Lennard and Fitzroy River systems which dries into smaller pools with limited food supply. Even within the Fitzroy River system, crocodiles from lower river sections, where both water and food are available year-round (e.g., Camballin Weir), tend to be larger than those from the upper reaches (e.g., Mornington). Although food abundance has not been empirically measured in the Liverpool and Bullo River habitats, field observations suggest a reduction in fish species (Webb, 1985), which is consistent with evidence of declining and unstable fish communities in higher-elevation river systems in the Northern Territory (Bishop et al., 1990). However, physical barriers (waterfalls, ravines) between the pygmy freshwater crocodiles’ escarpment habitats and nearby downstream freshwater crocodile populations also make a case as an isolating mechanism for allopatric speciation. The physical isolation of these pygmy populations, unique haplotypes, private alleles, and genetic structuring indicate that there has been limited gene flow between these populations and the standard-size freshwater populations, and further studies into movement of individual animals and more in-depth molecular analysis may help to uncover the extent of this. Implications for conservation Small population size and genetic drift leave populations at increased risk of threats, and this study shows that inbreeding and relatedness are high in a number of populations, particularly in pygmy freshwater crocodiles. The higher levels of inbreeding in the pygmy populations are of concern as they already face a number of heightened threats, that inbreeding and a loss of genetic diversity will likely make them more susceptible to. Invasive species such as cane toads have already been shown to affect the size and demographics of the pygmy freshwater crocodile population at Bullo River (Britton et al., 2013), and is it of pressing concern that the population at Liverpool River be similarly investigated as effects of cane toads can be highly site specific (Somaweera and Shine, 2011, Somaweera et al., 2013). The small body size of these crocodiles may also mean that they are more susceptible to seasonal changes in food availability (Britton et al., 2013). This could become more problematic as climate change in the Northern Territory is predicted to increase rainfall but also increase periods of drought, increase temperature, and increase carbon dioxide levels (Dunlop and Brown, 2008), all of which could affect resource availability. For these immediate and long-term conservation concerns, the pygmy freshwater crocodile could be considered an ‘ecological species’ (where barriers to gene flow between populations result from ecological mechanisms; Butlin et al., 2012) as these animals have adapted to the unique climate and resource conditions of their isolated river escarpment habitats. Such unique and often isolated populations of crocodylians are of considerable ecological and natural history interest, given their distinctive adaptations to utilize resources in different environments. These adaptations have been observed in several crocodylian species, including Australian freshwater crocodiles (Shirley et al., 2017, Somaweera et al., 2014). A targeted conservation program could be designed for these unique populations similar to what it has been done for other populations of freshwater crocodiles and lizards (de Novaes e Silva et al., 2014, Somaweera et al., 2019, Somaweera et al., 2013). Conserving these pygmy freshwater crocodile populations is crucial, as they represent the genetic variability that enables freshwater crocodiles to adapt to environmental changes. This adaptability will become increasingly important for the survival of the species in the face of growing threats of invasive species and climate change (Britton et al., 2013, Dunlop and Brown, 2008).

Maternal and bi-parental genetic analyses indicate that the pygmy freshwater crocodile population in the upstream Liverpool River escarpment is genetically distinct from standard-size freshwater crocodile populations. However, the level of differentiation is comparable to that observed among other standard-size crocodile populations, some of which may also be experiencing genetic isolation. As such, the pygmy freshwater crocodiles should be regarded as part of the same species as other standard-sized populations. Nonetheless, further research is needed to better understand the dynamics of this population, including studies on the pygmy crocodiles at Bullo River, a broader sampling distribution, more extensive genetic markers, and a closer examination of individual dispersal patterns. This would provide valuable insights into isolation, gene flow, and the processes of speciation. In the meantime, the pygmy freshwater crocodiles should be viewed as a unique ‘ecological species,’ whose isolation and environmental adaptability make them particularly important to conserve in the face of potential threats.

Queensland Museum allowed sampling of specimens lodged by NNF. Chris Banks and Margaret Webb of Melbourne Zoo provided samples from crocodiles collected from the pygmy Liverpool River crocodiles for captive rearing. Traditional owner Bardayal Nadjamerrek kindly gave his permission to collect samples from the Liverpool River crocodiles. Field collection was assisted by the Department of Biodiversity, Conservation and Attractions (DBCA), CSIRO, Bunuba and Miriwoong rangers, Adam Britton, Jack Djandjomerr, George Djandjomerr, Damien Fordham, Winston Kay, Rod Kennett, Jeff Miller, Matt Puaza, Mark Read, Dani Tikel, Tony Tucker, David Quammen and Graham Webb. Samples from the Bullo River were provided by Adam Britton. Authors’ contributions: Study was designed by JG and KB. RS provided samples from east and west Kimberley crocodile populations. NNF (through Queensland Museum) and DY provided samples for remaining crocodile populations. Data was generated by KB, TM, RC and NNF. Data analyses were done by KB and JLM. Data interpretation and manuscript preparation were done by KB, JLM, RS, NNF and JG. Data availability All data is available through the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra; project number PRJNA551392) or the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accession numbers PV102152 to PV102236 and PV102049 to PV102151) | King River | 2 | - | Ord | N/A | WA | | Lake Kununurra | 1 | 6 | Ord | East Kimberley | WA | | Lake Argyle | 6 | 34 | Ord | East Kimberley | WA | | Windjana Gorge | 8 | 97 | Lennard | West Kimberley | WA | | Geike Gorge | 3 | 34 | Fitzroy | West Kimberley | WA | | Drysdale River | 5 | - | Drysdale | N/A | WA | | Liverpool River-pygmy | 11 | 8 | Liverpool | Arnhem Land escarpments | NT | | Mann River | 2 | - | Liverpool | N/A | NT | | Mary River | 7 | - | Mary | N/A | NT | | Victoria River | 2 | - | Victoria | N/A | NT | | Bullo River-pygmy | 18 (CR only) | - | Victoria | N/A | NT | | Roper River | 9 | - | Roper | N/A | NT | | Hodgson | 5 | - | Roper | N/A | NT | | Daly River | 5 | - | Daly | N/A | NT | | Douglas River | 1 | - | Daly | N/A | NT | | McKinlay River | 10 | - | Mary | N/A | NT | | Louis Creek | 1 | - | Gilbert | N/A | QLD | | Townsville | 1 | - | Ross | N/A | QLD | | Lakefield National Park | 5 | - | Normanby | N/A | QLD | | Lawn Hill | 1 | - | Nicholson | N/A | QLD | Appendix 2 Figure A2 : Median joining network of concatenated cytochrome b and control region haplotypes from 85 Crocodylus johnstoni samples across 13 river basins. Pygmy sized samples from Liverpool River Basin are represented in purple. Mutations between haplotypes are represented by a dash. Supplementary Material File (figures_tables_final.docx) - Download - 865.63 KB

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Authors Metrics & Citations Metrics Article Usage 635views 261downloads Citations Download citation Katherine Brittain, Torre Muhlbach, Rui Cao, et al. Unique Population or Unique Species? Genetic Insights into the Pygmy Freshwater Crocodiles of Northern Australia. Authorea. 27 March 2025. DOI: https://doi.org/10.22541/au.174307689.98463113/v1 DOI: https://doi.org/10.22541/au.174307689.98463113/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.