Unravelling abrupt alterations in the gut microbiota of wild species associated with nutritional stress is imperative but challenging for wildlife conservation. This study assessed the nutritional status of wild red deer during winter on the basis of changes in faecal nitrogen (FN) and urea nitrogen/creatinine (UN: C) levels and identified gut microbes associated with nutritional status via nutritional control experiments and metagenomic sequencing. The FN of wild red deer in winter 2022 was significantly lower than that in winter 2021 (p < 0.05, winter 2021: 1.37 ± 0.16% and winter 2022: 1.26 ± 0.22%), and the UN: C ratio increased (winter 2021: 2.19 ± 1.65 and winter 2022: 3.05 ± 3.50). Similar trends were found in late winter, which indicated greater nutritional pressure in winter (2022) and late winter. Compared with winter 2021, abundances of Ructibacterium and Butyrivibrio significantly increased, and Acetatifactor and Cuneatibacter significantly decreased during winter 2022 (p < 0.05). Compared with early winter, the cell growth and death pathways increased and lipid metabolism and its subpathway of secondary bile acid synthesis (ko00121) significantly decreased during late winter (p < 0.05), which was similar to the changes in malnourished experimental red deer. Abrupt alterations in the gut microbiota should receive increased attention when monitoring the nutritional health of wild ungulates. This study provides new insights and critical implications for the conservation of wild ungulate populations.

This study focuses on the Siberian roe deer population in the Greater Khingan Mountains, Northeast China. The cold temperate forest ecosystem in this area is distinctive. The Siberian roe deer is a crucial ecological indicator species, and its living conditions hold significant importance for ecological balance. From the winter of 2019 to 2022, 269 fecal samples of Siberian roe deer were collected from four protected areas in the northern part of the Greater Khingan Mountains, Heilongjiang Province. These samples were comprehensively analyzed using mitochondrial DNA and microsatellite markers, combined with conservation genetics evaluation methods. The results revealed that 244 individuals were identified in the fecal samples. The results of a Cyt b genetic analysis of the samples indicated that the haplotype and nucleotide diversity were 88.1% and 20.3%, respectively. The evaluation of 14 pairs of microsatellite loci showed that the average number of alleles was 11.2, and the average expected and observed heterozygosity were 0.672 and 0.506, respectively. Therefore, the overall genetic diversity level is high, but some populations of Siberian roe deer are at risk. AMOVA analysis and STRUCTURE Bayesian clustering confirmed the existence of obvious genetic differentiation among the populations. Historical studies have shown that the HZ and SH populations underwent the earliest diffusion events, and the BJC and SL populations also exhibited related signs (HZ: Huzhong Nature Reserve in the Greater Khingan Mountains; SH: Shuanghe National Nature Reserve in Heilongjiang Province; BJC: Heilongjiang Beijicun National Nature Reserve; SL: Songling District in Heilongjiang Province). Mismatch distribution and neutral tests indicated no expansion events or bottleneck effects in the population, and the inbreeding coefficient was positive, suggesting the possibility of inbreeding. The development potential of the population in the future varies among the various local populations. This study supports the biodiversity of Siberian roe deer at the genetic level to save the population and provides important scientific basis and reference for the protection and management of Siberian roe deer.