Factors affecting multiple paternity: insights from the eastern quoll (Dasyurus viverrinus)

Factors affecting multiple paternity: insights from the eastern quoll (Dasyurus viverrinus) | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 August 2025 V1 Latest version Share on Factors affecting multiple paternity: insights from the eastern quoll (Dasyurus viverrinus) Authors : Brittany Brockett 0000-0003-2310-5393 [email protected] , Linda Neaves , Maldwyn Evans 0000-0001-7025-2287 , Iain Gordon , Jennifer Pierson , Belinda Wilson 0000-0002-7314-6401 , Claire Wimpenny , and Adrian Manning 0000-0003-0989-9203 Authors Info & Affiliations https://doi.org/10.22541/au.175552785.55623007/v1 Published Ecology and Evolution Version of record Peer review timeline 280 views 159 downloads Contents Abstract Highlights Abstract Introduction Materials and Methods Results and Discussion Conclusions Tables and Figures Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract not-yet-known not-yet-known not-yet-known unknown Breeding systems can impact population dynamics and genetic diversity, especially when a population is small or isolated. Here, we used a long-term dataset of demographic data combined with 1,745 single nucleotide polymorphisms to investigate mating patterns within a population of the eastern quoll (Dasyurus viverrinus) reintroduced to a conservation-fenced reserve. Pedigree reconstruction revealed that 47.62% of eastern quoll litters exhibited multiple paternity, containing up to three sires. Trait-based generalised linear mixed-modelling showed reproductive success was significantly correlated with bodyweight, age, and colour-morph. A cross-species comparison within the Dasyuridae (which includes the eastern quoll) showed that shorter lifespan increased the rate of multiple paternity, but the degree of sexual size-dimorphism or intersex aggression does not. Our study indicated that individual traits can predict reproductive success, but the likelihood of multiple paternity can depend on the evolutionary/life history of the species (e.g., lifespan). Further research should focus whether the interaction between breeding system and life history may predict patterns of multiple paternity within a species. Highlights • Eastern quolls ( Dasyurus viverrinus ) exhibit multiple paternity in approximately half of all litters • Heavier weight, youth, and being black rather than fawn colour-morph were correlated with higher reproductive success for the eastern quoll • High rates of multiple paternity within the Dasyuridae were correlated with shorter lifespan and increased rates of polyandry, but not sexual size-dimorphism Abstract Breeding systems can impact population dynamics and genetic diversity, especially when a population is small or isolated. Here, we used a long-term dataset of demographic data combined with 1,745 single nucleotide polymorphisms to investigate mating patterns within a population of the eastern quoll ( Dasyurus viverrinus ) reintroduced to a conservation-fenced reserve. Pedigree reconstruction revealed that 47.62% of eastern quoll litters exhibited multiple paternity, containing up to three sires. Trait-based generalised linear mixed-modelling showed reproductive success was significantly correlated with bodyweight, age, and colour-morph. A cross-species comparison within the Dasyuridae (which includes the eastern quoll) showed that shorter lifespan increased the rate of multiple paternity, but the degree of sexual size-dimorphism or intersex aggression does not. Our study indicated that individual traits can predict reproductive success, but the likelihood of multiple paternity can depend on the evolutionary/life history of the species (e.g., lifespan). Further research should focus whether the interaction between breeding system and life history may predict patterns of multiple paternity within a species. Introduction Breeding systems range from monogamy to promiscuity, from generating one offspring at a time to mass production, and there are many sub-classifications and combinations of these broad groupings. Studies into breeding systems often aim to understand their effects on population demographics and genetics, and the complex interactions between breeding systems, life history, and natural selection processes. Multiple paternity is one of these relatively well studied systems - a promiscuous, polyandrous mating system in which a female will mate with multiple males, and wherein a single litter is sired by multiple fathers (Sugg & Chesser, 1994; Taylor et al. , 2014). Polyandry has been shown to lower average relatedness of offspring (Glen et al. , 2009), and increase offspring survival rates, increase a female’s lifetime reproductive fitness, and reduce the likelihood that a small number of males dominate the breeding pool (Fisher et al. , 2006a). Polyandry is often attributed to the avoidance of intersex aggression and infanticide (Wolff & Macdonald, 2004), or increased offspring quality, elevated genetic diversity, and females bet-hedging around the most beneficial mate (Jennions & Petrie, 2000). It is likely that the effect of polyandry on a species will be greatest in small populations (Pearse and Anderson, 2009), but if population density and encounter rates are extremely low then this may result in reduced levels of multiple-male mating (Sale et al. , 2013). Multiple paternity adds an extra layer of complexity to polyandry, with diverse life history and population interactions. A review by Correia et al. (2021) summarised evidence for multiple paternity increasing the strength of sexual selection, reducing sexual selection, increasing post-copulatory sperm competition, and affecting social cooperation. While polyandry may be predictable through the occurrence of sexual size dimorphism (Weckerly, 1998), multiple paternity has been predicted to occur at the lowest frequency in populations with large sexual dimorphism (Dobson et al. , 2024). These complexities mean that it is important to view multiple paternity within the context of species-specific traits that may affect breeding systems to fully understand the effects on population dynamics and evolution. In their meta-analysis spanning 59 species across multiple mammalian taxa, Dobson et al. (2018) found that increasing litter size was weakly correlated with an increasing probability of multiple paternity and mean number of fathers per litter. Correia et al. (2021) proposed that behaviours that restrict female polyandry (e.g. male mate guarding and territoriality) are likely key factors in limiting the frequency at which multiple paternity occurs in a population, and that there is generally a positive association between litter size and the probability of multiple paternity. The results of Correia et al. (2021) also indicate that with increasingly uneven siring success (i.e. where sexual selection is stronger) populations are less likely to have observed rates of multiple paternity based on litter size alone. In Australia, polyandry is widespread in the marsupial Dasyuridae family, members of which often exhibit the unique life history of semelparity (total die-off of the adult male population after the breeding season). The occurrence of semelparity is thought to be a secondary result of maximising success in an intensely competitive breeding season through increased effort, and associated stress on the individual (Naylor et al. , 2008; Fisher et al. , 2013). The result of this intense competition is decreased male health and survival because they spend less time resting and more time active than females (Gaschk et al. , 2023), and in extreme cases such as seen in the Antechinus genus, the stress and increased testosterone levels of the breeding system affects multiple internal organ systems and results in the death of the majority of males (Naylor et al. , 2008). Where male reproductive success is highly competitive, their breeding lifespans are often shortened compared to females and males are rendered more susceptible to environmental stressors (Clutton-Brock, 2021). Based on predictions by Correia et al. (2021), this highly competitive breeding system should result in lower-than-expected rates of multiple paternity in the family compared to other mammalian taxa. Here, we describe the breeding system of the eastern quoll ( Dasyurus viverrinus ) and present a detailed analysis of the key drivers of reproductive success. We compare our findings to life history, behavioural, and physiological data for other species within the Dasyuridae family and reveal new potential predictors of multiple paternity. Materials and Methods Dataset generation Reintroduction monitoring procedures were adapted from the protocols developed for eastern bettong (see Manning et al. , 2019). Morphometric data collected included body weight, foot length (PES), head length, a pouch description (including presence/number of pouch young), an age estimate, and a general condition score. In addition to this, radiotracking was carried out for select individuals during translocations from 2016 to 2018 (see Wilson et al. , 2020. This allowed for the targeted trapping of den sites for newly translocated females and meant that some offspring could be assigned a known mother. In all monitoring sessions, individuals were marked by a microchip at first capture, and an ear biopsy was taken. This biopsy was sent to Diversity Arrays Technology (DArT) as per the company’s specifications (www.diversityarrays.com/orderinstructions). Extraction and sequencing at DArT used Dasyurid DArTseq 1.0, an optimised complexity reduction technique modified from Kilian et al. (2012). The resulting single nucleotide polymorphism (SNP) data was processed using DArT’s proprietary analytical pipelines, and candidate SNPs were returned for project-specific filtering. Few individuals from 2020 are represented in the dataset because individuals from earlier cohorts were prioritised to capture post-founding processes in detail. SNP-data filtration Dasyurid DArTseq 1.0 returned 7142 candidate SNPs, which were filtered for quality and informativeness in R v 4.1.0 (R-Core-Team, 2021) using the dartR package (Grueber et al. , 2018; Mijangos et al. , 2022). Firstly, locus call-rate and minimum acceptable repeatability thresholds of 0.95 were applied, ensuring that loci were represented in 95% of samples and that SNPs were not the result of sequencing errors. A minor allele frequency threshold of 0.05 was set, with various thresholds tested for their downstream population structure effects (Brockett et al. , 2022). The minimum allowable sequence depth was set to 10, and an allele depth ratio maximum of two was implemented to remove SNPs where one allele was sequenced at much greater depths than the other. The inbuilt dartR function gl.filter.secondaries was used with the method set to “best”, to retain only the SNPs with the highest polymorphic information content if multiple were present in a single tag. Following this locus-based filtering, 1746 candidate SNPs remained. Next, individuals were filtered using a call-rate of 0.90, removing seven samples. Finally, any monomorphic loci created by other filtering processes were removed. Hardy-Weinberg Equilibrium tests were not performed due to samples being taken from multiple, non-admixed populations with large amounts of immigration – breaking many of the equation’s assumptions. The resultant dataset contained 1745 SNPs across 164 individuals. Pedigree construction Both demographic and genetic data were used to construct a pedigree of 164 eastern quolls in Colony v 2.0.6.6, a program which uses maximum likelihood methods to simultaneously infer parental and sibling relationships (Jones & Wang, 2010). The mating system was set to allow polygamy for both males and females and, because the population was not thought to have high levels of inbreeding, the non-inbreeding model was used. A long run using full-likelihood methods was used to construct the pedigree. Allele frequencies were inferred by the program and were not updated throughout the pedigree’s construction based on inferred relationships, both to save computational time and because families were not thought to be extremely large. A weak sibship-prior was set, with maternal and paternal priors set to 1 as the number of offspring likely to be produced by each sex was unknown. Since full sibships were thought to consist of a small number of individuals (<20), sibship scaling was set to ‘no’. A single run was used, and the random seed was set to 1234. The 1745 SNPs remaining after dataset filtration were provided as co-dominant genetic markers, with an error rate of 0.02. Demographic data were aligned with the SNP dataset to assign sex to each individual, and to exclude unlikely assignments of paternity and maternity. Where maternal sibships were known from field studies, the identity of the mother and offspring were included. No known paternal sibs existed. Parentage exclusions began by eliminating all other individuals in the dataset as potential parents for the Tasmanian founders. Next, birth years were assigned based on estimated age-at-capture, and parents were excluded where they would not yet have reached sexual maturity (one year old) in the breeding season preceding an individual’s birth, or were likely to be dead (over four years old for males, and five years old for females). Finally, known maternity was assigned for litters in which the mother and offspring had been caught together during post-translocation monitoring, and age-at capture data was used to exclude parentage in years preceding an individuals’ birth. Investigation of reproductive parameters Reproductive parameters are a collection of traits that describe how, when, and how often a species reproduces. Most obviously, this study aimed to investigate whether the eastern quoll exhibited multiple paternity, and if so, to what degree. To facilitate this, the birth year and sex of each eastern quoll born in MFWS were considered alongside the pedigree-assigned mother and father. Female eastern quolls produce only one litter per year (Godsell, 1983). This allowed us to manually group offspring born in MFWS into generational cohorts by birth year, and within each of these cohorts, assign individuals from the same mother as siblings from a single litter. Then, by investigating the number of fathers assigned to each litter, the occurrence of multiple paternity was assessed. To understand the occurrence and extent of multiple paternity in the eastern quoll, the percentage of litters exhibiting polyandry, the average number of sires per litter, and, where multiple sires were present, the proportion of the litter sired by each male were all calculated. Other reproductive parameters of interest in this study included at which age, over how many years, and with how many partners offspring were produced. For the purposes of this analysis ”successful” reproduction was recruiting offspring into the population as independent individuals, rather than measuring mating success, conception/birth rates, or pouch occupancy. This was because parents must have successfully raised young through to independence and capture during monitoring (and hence genetic sampling) for young to be represented in our dataset. It is well established that eastern quolls give birth to around 20 partially developed young despite having only six teats available (Hill and O’Donoghue, 1913). Given that survival rates for young from birth to adolescent dispersal is between 68-90% (Godsell, 1983), it is likely that many pouch young were not successfully recruited. Nevertheless, by assessing drivers of recruitment, we discover the factors that lead to an individuals’ successful contribution to the population. Breeding Success Investigation into drivers of reproductive success within the population was carried out through generalised linear mixed modelling in the R package glmmTMB (Brooks et al. , 2017). A single individual (88C7E) was removed from the dataset due to potentially spurious breeding data (being a single male that was recorded as being 200 grams in weight when first calculated to have reproduced, which is highly unlikely and attributed to a scribing error). A binomial model was fitted with the fixed-effects sex, morph, individual heterozygosity, interactive fixed-effects weight and age, and the random effects of individual identity. A quadratic fixed-effect for weight was also included as an interactive variable with age. Continuous variables (weight, age, individual heterozygosity) were scaled to allow direct comparison. The model was tested for homogeneity, overdispersion, collinearity, and overall model fit (assessed using the performance package; Lüdecke et al. , 2021). Using the dredge function of the MuMIn package (Barton, 2023), model selection tested all possible combination of fixed-effect variables, facilitating selection of the ‘best’ model. Note that because the model was constructed to fail if NAs were present, each model considered by the dredge function used the same dataset and was therefore comparable. The model with the lowest AIC and minimal complexity (fewest parameters) was selected for further analysis, with models within delta AIC < 5 being considered for comparison. To visualise the effects of significant predictors on breeding success, predictions were generated using manual matrix multiplication of model coefficients. Confidence intervals (95%) were calculated on the link scale using standard errors before transformation to the response scale. The results of the matrix multiplication were used to visualise the breeding success of eastern quolls via the ggplot2 package (Wickham, 2011). Number of Offspring Individuals with more offspring recruited to the population are presumably the ‘fitter’ individuals within a population. To determine whether there were traits predicting differences in offspring recruitment among successfully breeding individuals, a separate GLMM was used to investigate factors impacting reproductive output within the eastern quoll population at MFWS. Initially, a zero-inflated Poisson model was tested incorporating the same fixed and random effects as the binomial model. However, due to the limited sample size when weight data were required, a simplified Poisson model was fitted using a larger dataset that excluded weight as a predictor. This model included the fixed-effects age, morph, sex, and cluster, with random effects of year and individual identity. The model selection method described for the binomial success/failure model was implemented, and the effects of significant variables on offspring number were visualised using ggplot2. This approach allowed us to utilise a larger sample size while still examining key biological predictors of reproductive output. Comparison to other species In order to investigate factors that might affect the occurrence and pattern of multiple paternity within a species, data was from the available literature on 11 species within the Dasyuridae family ( Antechinus agilis, A. minimum, A. stewartii, Dasykaluta rosamondae, Dasyurus geoffroyii, D. hallucatus, D. maculatus, D. viverrinus, Phascogale calura, P. tapoatafa, Sarcophilis harrisii ) and two outgroups of non-dasyurid Australian marsupials for which multiple paternity information was available ( Acrobates pygmaeus, Tarsipes rostratus ). Google Scholar was manually searched using combinations of the terms polyandry, semelparity, life history, breeding systems, multiple paternity, Dasyuridae, and dasyurids. A partial snowball-search method was used, wherein references within selected papers were in-turn selected for investigation. The online tool Litmaps was used to check the citation network generated by this search method for gaps, and relevant suggestions were incorporated (Litmaps, 2024). For our analysis we selected traits that are generally expected to predict polyandrous breeding systems, such as antagonistic breeding behaviours and infanticide, and/or significant sexual dimorphism (Wolff & Macdonald, 2004). These we represented through subjective categorisation of intersex aggression (low, moderate, or high) and a ratio of male:female bodyweight difference. We also incorporated various attributes of breeding system (litter size, percentage of litters sired by multiple fathers, maximum number of sires, relative paternity percentage of fathers) and lifespan (in years). A spearman’s rank correlation was conducted to test the degree to which the average number of sires, female-to-male weight ratio, male lifespan, female lifespan, and aggression was impacted the number of litters sired by multiple fathers. To facilitate this, the qualitative value of aggression was scored so that low = 1, moderate = 2, high = 3. Where values were midway, these were averages (e.g. low-moderate = 1.5). Then, a two-sample t-test was carried out for each comparison to test whether the relationship was significant. Results and Discussion The breeding system of the eastern quoll We investigated the breeding system of a reintroduced population of eastern quolls at Mulligans Flat Woodland Sanctuary (MWFS; Canberra, Australian Capital Territory, Australia) to determine whether morphological and genetic factors affect individual reproductive success. We identified 42 eastern quoll litters that were successfully recruited into the reintroduced population between 2016 and 2020. The eastern quoll pedigree revealed that 47.62% of these successful eastern quoll litters (20/42) in MFWS were sired by multiple males (Table 1). Of the 20 polyandrous litters detected, we found that 75% had two sires (15/20), 20% had three sires (4/20), and 5% had four sires (1/20) (Table 1). Larger litters tended to have more sires. Nine known-age mothers successfully reared a litter after their first year (eight at two years, one at three years), and only two females had more than one litter. The average age of a reproductively successful female eastern quoll was 1.22 years (median = 1 year). Males also successfully recruited most offspring within the first adult year (average of age fathers was 1.14 years, median = 1 year). Nine known-age sires recruited litters at two years old, and a single male recruited a litter at three years old. Eastern quolls in Tasmania also exhibit the greatest reproductive success in their first two years (Godsell, 1983), and recent study by Morrison et al. (2025) found that three of seven sampled litters in Tasmania had either two sires (two litters) or four sires (one litter). These results are encouraging because they provide evidence that the eastern quoll breeding system within the 485-hectare conservation-fenced MFWS is similar to that of remnant, wild populations in Tasmania. Our results showed that eastern quolls possess partial semelparity, as observed in other dasyurid species. From 2018 onwards approximately 33.2% of the population was aged two, and in 2019 and 2020 approximately 14.15% was three years old. We observed two females having more than one litter, eight males that bred in two years, and two males that recruited young across three years. The eastern quoll therefore does not possess true obligate semelparity as seen in many antechinus species (wherein all adults die following a single breeding season), but rather exhibit partial semelparity as seen in the kaluta ( Dasykaluta rosamondae ; Hayes et al. , 2019) and northern quoll ( Dasyurus hallucatus ; Oakwood, 1997). The survival rate for eastern quolls was high in comparison to other dasyurids. Where approximately 5% of kaluta and 10% of northern quolls survive for more than one year, with unknown reproductive success, our findings indicate that approximately 47% of the adult population may be over one year old each year. There is some evidence that semelparity does not always occur at a fixed-rate – in a fragmented system with few breeding opportunities and abundant resources, up to 50% of male northern quolls were found to survive to a second year (Cowan et al. , 2024). The MFWS eastern quoll population was established in 2016 in good conditions (high resource availability, no predators; Wilson et al , 2021), but the high survival rate was observed to be consistent through time and the 2019/2020 drought conditions (Wilson et al. , 2022). We therefore do not expect that the rates of semelparity would increase significantly in the MFWS population under changed environmental conditions. However, we propose to include within the definition of semelparity not only death after one year , but also failure to reproduce beyond one year . This is because if an individual cannot successfully reproduce and recruit young into the population they are effectively ‘dead’ to the gene pool, and we have shown here that beyond age one the likelihood of reproductive success was extremely low. In this way, though eastern quoll individuals had high survival rates, their effective rate of partial semelparity was similar to that of kaluta and northern quolls because few individuals bred successfully beyond one year of age. We note that this reproduction-only lens is appropriate only in species which non-reproductive individuals do not otherwise contribute to the population (e.g. socially through cooperative rearing or teaching of young). Within the MFWS population there was a tendency for uneven siring success; a single male often sired approximately 60% of young in a litter (Table 1). Siring unevenness has not been extensively studied in dasyurids, but is well known in at least the agile antechinus ( Antechinus agilis ), where a single male will sire up to 60% of the young in a litter (Fisher et al. , 2006b). While detailed observations of male–female interactions were not possible in our study, Fisher et al. (2006b) determined that for the agile antechinus it was always the last male mated with that would sire most of a females’ litter. This ‘last-male dominance’ is evidence of behavioural mechanisms males exhibit to maximise breeding success within a system driven by sperm competition. Other behavioural mechanisms to overcome the effects of sperm competition include long copulatory periods (agile antechinus; Shimmin et al. , 2000) and repeated matings (swamp antechinus A. minimus ; Sale et al. , 2013). Given that female eastern quolls are known to store sperm for up to 14 days (Taggart et al. , 2003), equal to storage times in the agile antechinus (Shimmin et al. , 2000), it seems highly likely that at least some of these behavioural mechanisms would be present. Pedigree reconstruction demonstrated that females tended to only produce one litter and have an average of 2.63 young in their lifetime (median 2, range 1–7), while males recruited an average of 3.33 offspring (median 2, range 1–16). The male average was heavily influenced by three individuals that fathered more than six young each. One of these males, who successfully recruited eight young, was a founding individual in the first trial reintroduction in 2016 (Wilson et al., 2021). The other two high-output males, who had 12 and 16 young each, were born in the first generation. Early cohorts likely faced lower rates of competition, because founder numbers were low (see Wilson et al. , 2020) and the percentage of both males and females successfully breeding decreased markedly following the second breeding year along with the number of offspring per individual (Supplementary Figure 3). We observed that litter size decreased over time from an average of 2.75 young per litter in 2016 to 1.8 in 2020 (spearman rank-correlation 0.005), but that there was little change in the average number of sires per litter (1.5 per litter in both 2016 and 2019, spearman rank-correlation -0.052). That is, the eastern quoll continued to display similar levels of multiple paternity despite smaller litter sizes. This may indicate there was a preference for polyandry in the eastern quoll similar to what has been observed in the banded mongoose Mungos mungo (Cant, 2000) and agile antechinus (Parrott et al. , 2015). It is likely that the highly competitive breeding system of the eastern quoll favours promiscuity. Not only due to bet-hedging by females to produce maximally fit offspring (Jennions & Petrie, 2000) or avoid harassment and infanticide (Wolff & Macdonald, 2004), but by males to maximise the likelihood of producing offspring at all. Kraaijeveld et al. (2003) demonstrated that for a male agile antechinus, when there was approximately 20% offspring survival and 70% female survival, a male would need to mate with at least six females to have a greater than 80% chance of recruiting offspring to the next generation. Thus, either the likelihood of offspring attrition or low female survival would result in increasing rates of promiscuity, and hence multiple paternity. There was no apparent pattern in which females raised multiple litters and, considered in conjunction with modelling results (as described below), it is likely that success in raising more offspring than average is due largely to unaccounted for factors. The significant downwards trends observed from 2017 to 2019 in both number of offspring and percentage of adult population successfully breeding (Supplemental Figure 3) indicated an increasingly competitive environment within the MFWS population. Increasing competition between individuals for den or food resources may be more important to how successful a mother is in raising her young than her individual physiological traits. It is also possible, even likely, that random chance plays a large part in whether a mother successfully raised her offspring to independence. However, the period from 2017-2019 is also notable for a severe drought that affected the eastern coast of Australia BOM, 2021. This drought drastically reduced resource availability within MFWS and would have naturally resulted in lower breeding success. Continued monitoring of reproductive patterns during times of abundance would be needed to fully understand the selective pressures driving reproductive success. Another complicating factor may be that the individual, and litter, sample size that were available for our study may not have had high enough resolution to detect patterns in the population. Predictors of reproductive success in the eastern quoll To determine predictors of eastern quoll reproductive success, we assessed (1) the drivers of reproductive success or failure, and (2) the factors affecting the number of offspring successfully recruited into the population by an individual using generalised linear mixed models (GLMMs). We found that individual traits significantly affected the likelihood of breeding success, with weight, age, and colour-morph (i.e. whether an individuals had black or fawn fur colouration) all significant predictors (Figure 1). Traits found to not significantly affect reproductive success or fecundity included individual heterozygosity, individual identity, or year of sampling. There was no sex-specific link between age and reproductive success, but population-wide data showed that youth was a highly significant predictor for both reproductive success (OR 0.3, p=0.003) and number of offspring (OR 0.26, p < 0.001). The strongly negative effect of age showed that for each year, an individual was 70% less likely to reproduce successfully (OR = 0.30, p = 0.003), and 74% less likely to produce more than one offspring (Table 1 , Figure 1 , Figure 3 ). Individuals over two years have less than a 50% likelihood of reproducing at all (Figure 1 ), and those over 3.4 years old were predicted to have no offspring (Figure 3 ). Weight had a strongly positive effect, with heavier individuals being approximately 12 times more likely to successfully recruit offspring into the population as compared with lighter individuals (OR = 12.24, p < 0.001). However, extremely heavy individuals were found to be less likely to reproduce successfully (OR = 0.50, p = 0.017). Noting that individuals get heavier with age, we attribute the presence of an optimal weight range to the strong effect of age on reproductive success. For both sexes, the predicted likelihood of breeding success exceeded 50% when an individual weighed approximately 900 grams or more (Figure 2 ), consistent with the overall trend of seen in Figure 1 . The effect of weight was near significant for males (OR 7.15, p=0.052) and significant for females (OR 1.42, p=0.006), though the effect size was much greater for males (Table 1 ). We found that sanctuary-born females were heavier on average (0.9 kg, maximum 1.6 kg) than those from Tasmania (averaging 0.7–0.8 kg, maximum 1.1 kg; Jones et al. , 2001). The weight difference may be a result of increased resource availability within MFWS, because the mammalian mesopredator niche was unfilled prior to the species’ reintroduction. We did not observe this weight difference in males, and despite relatively low competition in early years we believe that this was likely attributable to the high stress of the breeding season that eastern quolls go through, which often affects male dasyurids more than females. Fawn individuals within the MFWS population were predicted to have lower reproductive success than their dark-morph counterparts (OR = 0.09, p = 0.003), which aligns with demographic monitoring data showing a population-wide reduction in fawn-morph frequency from 83% to 7% between 2016 and 2019. In the brush-tailed possum ( Trichosaurus vulpecula ), coat colour variation was found to be regulated by mutations within the Agouti Signalling Protein (ASIP) coded for on chromosome 3 – missense substitutions within the ASIP gene prevented ASIP production and resulted in blacker colouration of the fur (Bond et al. , 2024). It has been posited by Bond et al. (2024) that dasyurids would be more prone to exhibiting these mutations because of the family’s rapid evolution, which may be supported by Sauermann et al. (2025) assertion that loss-of-function mutations have occurred repeatedly within the marsupials and represent an important mechanism underpinning coat colour variation. Hartley et al. (2024) provided strong evidence that the ASPI gene is important in regulating colour-morph in the eastern quoll, finding a deletion within the ASIP gene that they proposed underpins colour differences in both the eastern quoll and Tasmanian devil. Sauermann et al . (2025) propose that the selection for darker morph individuals may be a result of predator avoidance, because the eastern quoll is a nocturnal species. However, there is little evidence of whether colour morph, or the ASIP gene, is linked to other physiological or behavioural traits that may affect individual fitness, and drawing conclusions as to the cause black-morph dominance is not yet possible with much certainty. While it may be is unclear what is driving the difference in reproductive success between the colour morphs, our results show that there is a clear selective pressure negatively affecting fawn-morph individuals within the MFWS population. There were 30 incidences of black-morph male x black-morph female crosses, 20 black x fawn, 13 fawn x black, and 5 fawn x fawn (Supplementary Table 1). The reciprocal cross data in Supplementary Table 1 indicates that the inheritance of morph is likely poly-genic, and potentially sex-linked (paternal morph may have more effect that maternal. However, more research is needed to understand the drivers of fitness, including morph inheritance, within eastern quoll populations. Polyandry in the Dasyuridae A meta-analysis by Dobson et al. (2018) found that polyandrous species often exhibit multiple paternity in 25-50% of all litters, and posited that mating-associated behaviours and ecological factors such as population density could prevent a positive, linear correlation between the number of sires and litter sizes. However, members of the Dasyuridae have been recorded as having up to 97.8% of litters with multiple sires (average 67.95%, Supplemental Figure 2). Further, dasyurids tend to share similar life histories (semelparity, short maturation periods and lifespans, aggressive mating behaviour, and size-dimorphism) but these vary in the extremes observed between taxa. This variation means Dasyuridae can provide a useful case study for examining the drivers of multiple paternity frequency because the family displays highly variable rates of multiple paternity, is relatively well studied, and contains comparable life-history strategies. In our cross-species comparison, the availability of information for the 11 dasyurid ( Antechinus agilis, A. minimum, A. stewartii, Dasykaluta rosamondae, Dasyurus geoffroyii, D. hallucatus, D. maculatus, D. viverrinus, Phascogale calura, P. tapoatafa, Sarcophilis harrisii ) and two non-dasyurid species ( Acrobates pygmaeus, Tarsipes rostratus ) was variable, despite being relatively well-studied taxa. We encountered difficulties obtaining comparable values for many ecological and behavioural characteristics that could feasibly be linked to the occurrence of multiple paternity, notably home range size, population density, and sperm competition or retention. Male–female aggression was also not readily available in a quantitative format, but enough information was available to qualitatively categorise (i.e., low, medium, or high) aggression for subsequent analyses. Our comparison revealed that sexual size dimorphism had no impact on how many litters in a population would express multiple paternity (Spearman’s rank-correlation p = 0.523), while male-to-female aggression was positively correlated with multiple paternity (p = <0.001). This is consistent with findings from Wolff and Macdonald (2004), who found that paternity confusion and aggression avoidance can select for multiple paternity. A quantitative evaluation of aggression in species with multiple paternity would be needed to confirm this. Our results also indicated that the ‘live fast–die young’ strategy of the assessed species may extend to maximising offspring fitness through increased multiple paternity. Shorter lifespans correlated significantly with multiple paternity within a species for both males and females, as did the maximum number of sires per litter (p = 0.004, p = <0.001, p = <0.001, respectively). Finally, we found that the eastern quoll had the second lowest rate of polyandry within the assessed species, which ranged from 20% in the chuditch ( Dasyurus geoffroii ; Manning et al. , 2022) to an exceptionally high 97.8% in the agile antechinus ( Antechinus agilis ; Kraaijeveld-Smit et al. , 2002) (Supplemental Figure 2). It has been shown that there is little sperm competition in the eastern quoll (Taggart et al. , 2003), and high sperm competition in the agile antechinus ( Antechinus agilis ; Shimmin et al. , 2000). As raised by Dickman (1994) and Taggart et al. (2003), sperm competition is a likely key feature of dasyurid reproductive strategies, and we theorise that differing levels of sperm competition may be linked to the prevalence of multiple paternity. Further investigation is required as to whether sperm competition, or any other biological or behavioural characteristic, are part of shorter lives leading to more multiple paternity. Conclusions Here, we revealed that the eastern quoll possesses a polyandrous breeding system with a high rate of multiple paternity. We furthered understanding of the species’ reproductive patterns and discovered key physiological traits correlated with reproductive fitness. Our cross-species analysis exploring the biological factors influencing multiple paternity revealed that both behavioural and physiological traits (i.e., aggression, lifespan, and maximum possible number of sires in a litter) significantly affect the prevalence of multiple paternity litters in the Dasyuridae. We recommend that future studies quantify behavioural traits such as copulatory frequency and duration, and inter-sex aggression, and conduct additional genetic sampling to allow more detailed investigation into the factors underpinning the eastern quoll’s life history. Tables and Figures Table 1 Model effects table for the generalised linear mixed model investigating the effects of individual traits of the likelihood of reproductive success. Note that here, reproductive success equates to recruitment of offspring into the population. Odds ratios correspond to the relative likelihood Figure 1. Predicted effects of individual traits on the likelihood of reproductive success* in the eastern quoll (Dasyurus viverrinus) population at Mulligans Flat Woodland Sanctuary, ACT, Australia. A) The predicted likelihood of reproduction in each age class. Age 1 represents the first year following dispersal from the natal den, within which sexual maturity is reached (i.e. individuals within age class 1 are approximately between 0 and 1 years old). Shaded areas represent a 68% confidence interval. B) The predicted likelihood of reproduction relative to an individual’s body weight in kilograms. Shaded areas represent a 68% confidence interval. C) The predicted likelihood of reproduction for individuals displaying either of the two possible colour morphs for this species, black and fawn. Error bars represent standard error. *Note that reproductive success equates to recruitment of offspring into the population, as detected by capture and genetic sampling during regular population monitoring. 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Trends Ecol Evol , 19 (3), 127-134. https://doi.org/10.1016/j.tree.2003.12.009 Information & Authors Information Version history V1 Version 1 18 August 2025 Peer review timeline Published Ecology and Evolution Version of Record 30 Apr 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords breeding system eastern quoll multiple paternity polyandry Authors Affiliations Brittany Brockett 0000-0003-2310-5393 [email protected] Australian National University Fenner School of Environment and Society View all articles by this author Linda Neaves Murdoch University View all articles by this author Maldwyn Evans 0000-0001-7025-2287 Australian National University View all articles by this author Iain Gordon Australian National University Fenner School of Environment and Society View all articles by this author Jennifer Pierson Australian Wildlife Conservancy View all articles by this author Belinda Wilson 0000-0002-7314-6401 Australian National University Fenner School of Environment and Society View all articles by this author Claire Wimpenny ACT Government View all articles by this author Adrian Manning 0000-0003-0989-9203 Australian National University View all articles by this author Metrics & Citations Metrics Article Usage 280 views 159 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Brittany Brockett, Linda Neaves, Maldwyn Evans, et al. Factors affecting multiple paternity: insights from the eastern quoll (Dasyurus viverrinus). Authorea . 18 August 2025. DOI: https://doi.org/10.22541/au.175552785.55623007/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 . 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