Temporal dynamics of the gut microbiota of wild animals are generally poorly understood, due primarily to the difficulty of repeatedly collecting samples from known individuals over an extended period [1, 2]. Recently there has been a surge in time-series investigations in wild animals and humans [2–7]. Most longitudinal studies in wild animals have focused on primates, with diets encompassing a wide variety of plant species, and incorporating multiple plant parts (e.g., foliage, bark, and fruit) in their diet. These studies have included western lowland gorillas (Gorilla gorilla gorilla) and mountain gorillas (Gorilla beringei beringei) , black howler monkeys (Alouatta pigra) , and Udzungwa red colobus monkeys (Procolobus gordonorum) . Others have focused on primates with more omnivorous diets, including chimpanzees (Pan troglodytes schweinfurthii) , baboons (Papio cynocephalus) , ring-tailed lemurs (Lemur catta) . The results of these studies, particularly those focusing on wild herbivores, have shown that seasonal shifts in food availability (e.g., of ripe fruit) can drive gut microbiota composition. In contrast, studies in humans have found that diet and host lifestyle influence the gut microbiota at a finer (e.g., daily) scale [1, 4, 12]. Overall, temporal variability of the gut microbiota varies from animal to animal and can be heavily influenced by diet.
The koala (Phascolarctos cinereus), a specialist folivore, has recently been listed as endangered due to threatening processes including habitat fragmentation , climate change , and disease (e.g., chlamydiosis and koala retrovirus) [15–17]. The koala is an obligate dietary specialist  which relies almost exclusively on eucalypt foliage (from the genera Eucalyptus, Corymbia and Angophora, family Myrtaceae) . Eucalypt foliage is a chemically complex and nutritionally challenging food source  and its consumption and digestion is thought to be facilitated by the fermentative ability of gut microbiota and by the host’s physiological capacity to detoxify and eliminate plant toxins. Given recent calls that the gut microbiota could be linked to host health and conservation [21, 22], there has been a surge of interest in understanding the role of the gut microbiota in koala diet choice and health. Recently Brice et. al.,  found that within a single koala population, the gut microbiota of koalas differed according to which of two Eucalyptus species, manna gum (Eucalyptus viminalis) and messmate (E. obliqua), were eaten. They concluded that the koala gut microbiota adjusts to individual diet, presumably enabling optimal degradation and extraction of available nutrients. This population of koalas had experienced a period of exponential growth, resulting in overbrowsing and die-back of their preferred food species, manna gum . In 2013, hundreds or thousands of koalas died of starvation, due in part to their reluctance to feed on available messmate patches . In a captive experiment, Blyton et. al.,  altered the microbial community composition of manna gum-feeding koalas to resemble more closely that of messmate-feeding koalas, using faecal inoculations. Individuals with more substantially altered microbiota subsequently increased their consumption of messmate. This demonstrated that an individual koala's gut microbiota influences feeding behaviour and suggests that the microbiota might limit the ability of koalas to feed from messmate during mass starvation events. However, we currently lack a detailed understanding of the stability of the koala gut microbiota through time, particularly in wild koalas. Such knowledge could be useful for disentangling the effects of diet and other treatment effects from natural variations in koalas.
To fill this gap, we characterised the gut microbiota from faecal pellets collected over a one-month period from wild koalas rescued from extensive wildfires that occurred on Kangaroo Island, South Australia (SA), from December 2019 - January 2020, and rehomed at Cleland Wildlife Park, SA, as well as from mainland South Australian koalas housed at Cleland. We also undertook a seven-month time-series study to determine the stability of the faecal microbiota of wild koalas living in a mixed eucalypt forest at Mountain Lagoon, in and adjoining Wollemi National Park, NSW, Australia. We aimed to address the questions: 1) how much intra-individual variation in faecal microbiota diversity and composition is there for koalas over days and months, and 2) is there significant inter-individual variation in gut microbial community composition between koalas?