IUCN distribution of platypuses (grey shading in insert and main figure) and sampling locations for this study (BR Border Rivers, SR Snowy River, UM Upper Murray River) and other published or unpublished studies (see Table 1 for numbers related to publications and other available sources).

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... chemistry data from Tasmania, Victoria, and New South Wales. In addition, we sourced unpublished data from the ZIMS species360 database (www. speci es360. org), a global online database used by zoos for captive animals. In total, we used 12 different platypus studies and locations, including the three catchments that we sampled in this study ( Fig. 1 and Table 1). To investigate whether hematological or blood chemistry analytes varied non-linearly across the species' latitudinal range, we used the mean for each analyte and a Generalized Linear Model weighted by sample size 101 , fitted with a second order polynomial relationship, to allow a non-linear association, with latitude as ...

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... non-linear association, with latitude as a predictor variable, implemented in R 93 . Associations were considered significant if P ≤ 0.05. The 'ggplot2′ 94 package was used to represent the data. To further future research into population health of platypus, our data is made open source via Dryad (https:// doi. org/ 10. 5061/ dryad. brv15 dv9d). Fig. 1). Values are listed as mean ± SD (min to max), with sample size for particular analyte listed. N/A -unavailable data. ...

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... volume index (TVI) was not significantly associated to PCV or any of the tested serum chemistry analytes (Appendix S1), although there was some suggestive negative association with levels of triglycerides (P = 0.074) and urea (P = 0.064). Across the three catchments, TVI was not associated with month (Jan-May), sex, age, or elevation (Fig. 2, Appendix S1). On the Snowy River, TVI did not differ between the sexes, but did significantly www.nature.com/scientificreports/ fluctuate in male platypuses (P = 0.001) and marginally in females (P = 0.086), increasing towards the onset of the breeding season (October) and reaching a minimum by April (Fig. 3, Appendix S2). All hematology and serum ...

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... S2). All hematology and serum chemistry analytes, apart from urea, were significantly related to catchment (Fig. 2, Appendix S1). Platypuses from the Border Rivers catchment had lower PCV (P = 0.001), and lower concentrations of albumin (P = 0.006), and triglycerides (P = 0.001), compared to those from the Snowy and Upper Murray Rivers catchments (Fig. 2, Appendix S1). Also, TP (P = 0.02), globulin (P = 0.001), and creatinine (P = 0.005) concentrations were significantly higher in platypuses from the Border Rivers catchment, compared to the Snowy and Upper Murray Rivers catchments (Fig. 2, Appendix S1). Urea levels did not vary significantly among rivers, although there was some evidence for lower ...

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... = 0.006), and triglycerides (P = 0.001), compared to those from the Snowy and Upper Murray Rivers catchments (Fig. 2, Appendix S1). Also, TP (P = 0.02), globulin (P = 0.001), and creatinine (P = 0.005) concentrations were significantly higher in platypuses from the Border Rivers catchment, compared to the Snowy and Upper Murray Rivers catchments (Fig. 2, Appendix S1). Urea levels did not vary significantly among rivers, although there was some evidence for lower concentrations in platypuses from the Snowy Rivers catchment, compared to individuals from the other two catchments (P = 0.087), (Appendix S1). Platypuses in the geographically proximal Snowy and Upper Murray River catchments (Fig. 1) had ...

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... (Fig. 2, Appendix S1). Urea levels did not vary significantly among rivers, although there was some evidence for lower concentrations in platypuses from the Snowy Rivers catchment, compared to individuals from the other two catchments (P = 0.087), (Appendix S1). Platypuses in the geographically proximal Snowy and Upper Murray River catchments (Fig. 1) had similar levels for all hematology and serum chemistry analytes (Table S1). Further, no hematology nor serum chemistry analytes were related to elevation (Appendix ...

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... and concentrations of TP, albumin, and urea were positively (max P = 0.02) associated with month (Jan to May), while triglycerides concentrations were negatively associated (P = 0.001), and creatinine concentrations were not related to month (P = 0.226) (Fig. 2, Appendix S1). Globulin had a slight negative association with month (P = 0.07). Over a period of a year in the Snowy River catchment, there were no significant fluctuations in concentrations of albumin, urea or triglycerides (Appendix S2). In contrast, PCV (P = 0.03), TP (P = 0.01), and globulin (P = 0.01) concentrations of females in the Snowy ...

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... all available data from northern New South Wales (Lat -29.24) to Tasmania (Lat -43.05) were analyzed (Figs. 1 & 4, Table 1, Appendix S3), ranges of hematology and serum chemistry analytes in platypus from this study were slightly higher than those of platypus from Tasmania (Table 2). Albumin and PCV decreased with latitude (P = 0.03 and P = 0.001), TP and Triglycerides also showed a slight association (P = 0.06 and P = 0.08), triglycerides however increased with latitude. ...

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... southern extent, (Fig. 2, Table 1), may indicate poorer health 44,47,50 , possibly a result of lower food availability, but may also reflect other environmental effects. The significant differences in evaluated hematology and serum chemistry analytes in platypuses in the Border Rivers catchment, the most northerly river catchment surveyed (Fig. 1) may reflect naturally lower levels associated with the climate, indicative of possible habitat-related stressors or some combination of both [105][106][107] . The sensitivity of platypuses to high temperatures, with limited cooling options 108-111 is likely a key factor limiting their distribution into the lowland parts of the rivers ...

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... of possible habitat-related stressors or some combination of both [105][106][107] . The sensitivity of platypuses to high temperatures, with limited cooling options 108-111 is likely a key factor limiting their distribution into the lowland parts of the rivers of the Murray-Darling Basin, despite being widely distributed in connected upper parts (Fig. 1) 78,[112][113][114] . Thermal stress can directly impact health, and lead to changes in hematology and serum chemistry analytes 105,106,[115][116][117] . In addition, indirect effects on health may be caused by changes in temperature shifting food sources and availability 18 . Platypuses rely on aquatic macroinvertebrates for food and ...

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... 2. The seven hematology and serum chemistry variables (sample size), Reference Intervals (RI) (where N ≥ 120) using non-parametric methods 98,99 , mean, SD, and lower and upper 90% confidence intervals, partitioned by age and sex, for platypus captured from New South Wales and Victorian catchments (Snowy, Upper Murray, and Border Rivers) (see Fig. 1). *Robust method with Cox-Box transformation used due to sample size < ...