Feces Of Mammals Research Articles

Metabarcoding analysis provides insight into the link between prey and plant intake in a large alpine cat carnivore, the snow leopard.

Species of the family Felidae are thought to be obligate carnivores. However, detection of plants in their faeces raises questions about the role of plants in their diet. This is particularly true for the snow leopard (Panthera uncia). Our study aimed to comprehensively identify the prey and plants consumed by snow leopards. We applied DNA metabarcoding methods on 90 faecal samples of snow leopards collected in Kyrgyzstan, employing one vertebrate and four plant markers. We found that argali (Ovis ammon) was detected only from male snow leopards. Myricaraia sp. was the most consumed among 77 plant operational taxonomic units found in snow leopard samples. It frequently appeared in samples lacking any prey animal DNA, indicating that snow leopards might have consumed this plant especially when their digestive tracts were empty. We also observed differences in the patterns of plant consumption between male and female snow leopards. Our comprehensive overview of prey and plants detected in the faeces of snow leopards and other sympatric mammals will help in formulating hypotheses and guiding future research to understand the adaptive significance of plant-eating behaviour in felids. This knowledge supports the enhancement of their captive environments and the conservation planning of their natural habitats.

Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY–NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Comparison of DNA quantity and quality from fecal samples of mammals transported in ethanol or lysis buffer

Using fecal microbial community profiles through sequencing approaches helps to unravel the intimate interplay between health, wellness, and diet in wild animals with their environment. Ensuring the proper preservation of fecal samples before processing is crucial to ensure reliable results. In this study, we evaluated the efficiency of two different preservation methods, considering the following criteria: DNA yield, quality and integrity, and microbial community structure based on Oxford Nanopore amplicon sequencing of the V3-V4 region of bacterial 16S rRNA and protozoa 18S rRNA genes. Eighteen matched pairs of mammalian fecal samples were collected and transported in 99.8% ethanol and lysis buffer; processing occurred between 55 and 461 days post-collection. Wilcoxon signed-rank tests were used to analyze quantitative measurements for paired samples. The A260/280 ratio, a measure of nucleic acid purity, was assessed descriptively for each media, and the Bartlett test evaluated dispersion of this ratio. A Fisher test was performed to compare the number of positive reactions for DNA extraction or PCR amplification of the 16S and 18S rRNA genes between both media. The concentration of total DNA and amplicons, as well as the number of reads obtained in sequencing, was significantly higher in the samples preserved with lysis buffer compared to ethanol, with magnitudes up to three times higher. Electrophoretic analysis of total DNA and amplicons further confirmed superior DNA integrity in lysis buffer preserved samples. The A260/280 values obtained using the lysis buffer were of optimal purity (mean: 1.92) and with little dispersion (SD: 0.27); on the other hand, the ethanol samples also presented an excellent average quality (mean: 1.94), but they were dispersed (SD: 1.10). For molecular studies using mammalian feces, the lysis buffer reagent proved to be a reliable solution for their collection, conservation, and storage.

A simple and effective method to enrich endogenous DNA from mammalian faeces.

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing.

Millipede consumption and interaction between food preferences and sex in dung beetle Chalconotus convexus (Coleoptera: Scarabaeinae)

AbstractDung beetles feed mainly on scarce and ephemeral mammalian faeces making them vulnerable to declines of mammal populations. Therefore, studying how diverse groups like the Scarabaeinae subfamily utilize alternative food sources, such as decomposing invertebrates, enhances our understanding of their distribution and resilience in defaunated areas. One poorly studied aspect of the feeding habits of dung beetles is the potential differences between the sexes and their predatory behaviour. We evaluated how the sex (male and female) of individuals of Chalconotus convexus affected individual resource attraction to different bait types. Since C. convexus is known to feed on faeces, carrion and dead millipedes, we also test whether it can predate live individuals of millipedes. We used a combination of two field samplings and one laboratory experiment, using pitfall traps baited with faeces, carrion, millipedes and millipede's defensive compounds. While the preference for millipede was not associated with sex, we found a significant relationship between faeces and carrion, where females of C. convexus are significantly less attracted to faeces when compared to males and also males are significantly less attracted to carrion when compared to females. Furthermore, we also show that while actively consuming dead millipedes, C. convexus is unable to predate on live individuals. Our study illustrates that the versatility of food sources may help to explain the high abundance and wide distribution of C. convexus in Africa.

First record of the largest copro-necrophagous beetle in South America (Coprophanaeus lancifer) feeding on fruits

ABSTRACT Coprophanaeus lancifer is a copro-necrophagous beetle endemic to the Amazon region. Although beetles belonging to the subfamily Scarabaeinae primarily feed on dung, some have a greater diet plasticity that includes carrion, fungi, rotten fruits and invertebrates. The diet of the scavenger beetle C. lancifer mainly consists of vertebrate carcasses, although it is also attracted to mammal faeces. Here, we describe the first record of this species feeding on fruits of the genus Nectandra.

Forest conversion into pasture selects dung beetle traits at different biological scales depending on species pool composition.

The conversion of forests into open areas has large effects on the diversity and structure of native communities. The intensity of these effects may vary between regions, depending on the existence of native species adapted to open habitats in the regional pool or the time since habitat change.We assess the differences in species richness and functional diversity of dung beetle communities (Coleoptera: Scarabaeinae) between native forests and novel pasturelands of the Atlantic Forest and the Cerrado, two biomes with contrasting histories of human occupation in Brazil. We conducted standardized surveys in seven forest fragments and adjacent pastures in each region and measured 14 traits in individuals collected in each type of habitat at each particular site. We calculated functional richness, functional evenness, functional divergence, and community-weighted mean of traits for each area, and analyzed individual variation through nested variance decomposition and Trait Statistics.Communities were richer and more numerous at the Cerrado. We did not find any consistent relationship between functional diversity and forest conversion beyond the changes in species diversity. Although landscape changes were more recent at the Cerrado, the colonization of the new habitat by native species already adapted to open habitats lessens the functional loss in this biome. This indicates that habitat change's effects on trait diversity depend on the regional species pool rather than on time since land conversion.Forest conversion effects were primarily due to internal filtering. The effects of external filtering only appear at the intraspecific variance level, with contrasting differences between the Cerrado, where traits related to relocation behavior and size are selected, and the Atlantic Forest, where selection operates for traits related to relocation behavior and flight. These results evidence the importance of considering individual variance to address the responses of dung beetle communities to forest conversion.

Potential Probiotic Properties of Blautia producta Against Lipopolysaccharide-Induced Acute Liver Injury.

Blautia is a genus of anaerobic microbe extensively present in the intestine and feces of mammals. This study aims to investigate the influence of Blautia producta to prevent lipopolysaccharide (LPS)-induced acute liver injury (ALI) and elaborate on its hepatoprotective mechanisms. B. producta D4 and DSM2950 pretreatment decreased the activities of serum aspartate transferase (AST), and alanine transaminase (ALT) in mice with LPS treatment significantly decreased the levels of inflammatory tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) and increased the activities of antioxidative superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Compared with the model group, B. producta D4 and B. producta DSM2950 pretreatment slightly increased the levels of cecal propionic acid, isobutyric acid, butyric acid, valeric acid, and isovaleric acid (p > 0.05). Metagenomic analysis showed that B. producta D4 and DSM2950 pretreatment remarkably increased the relative abundance of [Eubacterium] xylanophilum group, Lachnospira, Ruminiclostridium, Ruminiclostridium 9, Coprococcus 2, Odoribacter, Roseburia, Alistipes, and Desulfovibrio in ALI mice, and their abundance is negatively related to the levels of inflammatory TNF-α, IL-1β, and IL-6 as revealed by Spearman's correlation analysis. Moreover, transcription and immunohistochemistry analysis revealed that B. producta D4 and B. producta DSM2950 intervention remarkably suppressed the transcription and expression levels of hepatic Tlr4, MyD88, and caspase-3 (p < 0.05). These data indicated that B. producta may be a good candidate for probiotics in the prevention of ALI.

Attractiveness of dung beetles (Coleoptera: Scarabaeinae) to faeces from native mammals in different trophic guilds

AbstractDung beetles are predominantly coprophagous and use mammalian faeces as their main food resource and for offspring rearing. However, these resources are generally ephemeral and scarce. The objective of this study was to verify the attractiveness to dung beetles of the faeces of nine native mammal species of the Cerrado biome in different trophic guilds. This study was conducted during the rainy season (October 2016 to March 2017) in two sensu stricto areas of the Cerrado in the Brasília National Park (PNB), Federal District, Brazil. Pitfalls containing faeces of nine mammals native to the region were used as bait (from three herbivores, three carnivores and three omnivores) for the collection of dung beetles. We collected 203 individuals and 19 species, and the most abundant species were Uroxys aff. thoracalis, Oxysternon palemo, Coprophanaeus spitzi and Diabroctis mirabilis. The most attractive faecal bait was from the omnivorous Chrysocyon brachyurus (32.67% of the individuals and 13 species). This study reveals variation in the attraction of dung beetles to native mammal dung of the Cerrado, with more abundance in the faeces of omnivores and carnivores and less in the faeces of herbivores. The species composition in the carnivore baits did not differ from that of the omnivore baits. These data indicate that changes in Scarabaeinae assemblages result from changes in mammal species such as those that have occurred in the Cerrado biome with the conversion to cattle production, which in recent years has caused intense fragmentation of habitats, expansion of agricultural and livestock activities and the introduction of exotic species. The mammal C. brachyurus, vulnerable to extinction in Brazil, should be a focus for future research, since its faeces support a great diversity of dung beetles in the Cerrado.

Capture of mammal excreta by Nepenthes is an effective heterotrophic nutrition strategy.

While isotopic enrichment of nitrogen (15N) and carbon (13C) is often used to determine whether carnivorous plant species capture and assimilate nutrients from supplemental sources such as invertebrate prey or mammal excreta (heterotrophic nutrition), little is known about how successful the different strategies deployed by carnivorous plants are at obtaining supplemental nutrition. The collection of mammalian faeces by Nepenthes (tropical pitcher plants) is the result of a highly specialized biological mutualism that results in heterotrophic nitrogen gain; however, it remains unknown how effective this strategy is in comparison to Nepenthes species not known to collect mammalian faeces. We examined how isotopic enrichment varied in the diverse genus Nepenthes, among species producing pitchers for invertebrate capture and species exhibiting mutualisms for the collection of mammal excreta. Enrichment factors were calculated from δ15N and δ13C values from eight Nepenthes species and naturally occurring hybrids along with co-occurring reference (non-carnivorous) plants from three mountain massifs in Borneo: Mount Kinabalu, Mount Tambuyukon and Mount Trus Madi. All Nepenthes examined, except N. edwardsiana, were significantly enriched in 15N compared to co-occurring non-carnivorous plants, and 15N enrichment was more than two-fold higher in species with adaptations for the collection of mammal excreta compared with other Nepenthes. The collection of mammal faeces clearly represents a highly effective strategy for heterotrophic nitrogen gain in Nepenthes. Species with adaptations for capturing mammal excreta occur exclusively at high elevation (i.e. are typically summit-occurring) where previous studies suggest invertebrate prey are less abundant and less frequently captured. As such, we propose this strategy may maximize nutritional return by specializing towards ensuring the collection and retention of few but higher-value N sources in environments where invertebrate prey may be scarce.

Good news! Sampling intensity needed for accurate assessments of dung beetle diversity may be lower in the Neotropics

Ecological studies with Scarabaeinae dung beetles have increased exponentially over the past 30 years, using lethal pitfall traps baited with mammal feces or carrion as the preferred sampling method. Different studies have determined the distance between pitfall traps for effective sampling, but the number of traps is often subjective, leading to excessive or poor sampling. This study provides quantitative guidelines for establishing the sample size for optimal completeness of dung beetle diversity by systematically reviewing the relationship between sampling intensity and sampling coverage, habitat type, and the journal impact factor in peer-reviewed research. We gathered 94 studies covering a range from México to Argentina. Sampling was conducted mainly in forested habitats, followed by treeless agriculture and agroforestry systems, with a median value of 50 pitfall traps per sampled habitat. Sampling completeness was above 0.9 in 95% of the studies. Oversampling ranged from 1 to more than 96,000 individuals, and sampling deficit varied between 2 and 3,300 specimens. Sampling intensity and the journal impact factor were significantly and positively correlated with oversampling, but these variables did not explain the sampling deficit. The positive correlation between journal impact factor and oversampling may reflect a publication bias where high-impact journals and researchers seek more generalizable information obtained with a higher sampling intensity. Dung beetle oversampling was not homogeneous between habitats, being highest in old-growth forests and lowest in disturbed habitats such as pastures and forest edges. Our results show that the collection intensity used in dung beetle studies should be reconsidered carefully. By incorporating ethical principles used in animal science, we suggest sampling guidelines for a robust sampling scheme of dung beetle diversity, which would also prevent oversampling. Consciously reducing sampling intensity will make resource use more cost-effective. We suggest increasing the number of independent sampling units rather than intensifying subsampling, thereby increasing the predictive power of statistical models to obtain more robust evidence of the phenomena under study.

Negative effects of forest gaps on dung removal in a full-factorial experiment.

Ecosystem functioning may directly or indirectly-via change in biodiversity-respond to land use. Dung removal is an important ecosystem function central for the decomposition of mammal faeces, including secondary seed dispersal and improved soil quality. Removal usually increases with dung beetle diversity and biomass. In forests, dung removal can vary with structural variables that are, however, often interrelated, making experiments necessary to understand the role of single variables on ecosystem functions. How gaps and deadwood, two main outcomes of forest management influence dung removal, is unknown. We tested if dung removal responds to gap creation and deadwood provisioning or if treatment effects are mediated via responses of dung beetles. We expected lower removal rates in gaps due to lower dung beetle biomass and diversity. We sampled dung beetles and measured dung removal in a highly-replicated full-factorial forest experiment established at 29 sites in three regions of Germany (treatments: Gap, Gap + Deadwood, Deadwood, Control). All gaps were experimentally created and had a diameter of around 30 m. Dung beetle diversity, biomass and dung removal were each lower in gaps than in controls. Dung removal decreased from 61.9% in controls to 48.5% in gaps, irrespective of whether or not the gap had deadwood. This treatment effect was primarily driven by dung beetle biomass but not diversity. Furthermore, dung removal was reduced to 56.9% in the deadwood treatment. Our findings are not consistent with complementarity effects of different dung beetle species linked to biodiversity-ecosystem functioning relationships that have been shown in several ecosystems. In contrast, identity effects can be pronounced: gaps reduced the abundance of a large-bodied key forest species (Anoplotrupes stercorosus), without compensatory recruitment of open land species. While gaps and deadwood are important for many forest organisms, dung beetles and dung removal respond negatively. Our results exemplify how experiments can contribute to test hypotheses on the interrelation between land use, biodiversity and ecosystem functioning.

Ingestion of plastics by terrestrial small mammals

The exposure of wildlife to waste plastic is widely recognised as an issue for aquatic ecosystems but very little is known about terrestrial systems. Here, we addressed the hypothesis that UK small mammals are ingesting plastics by examining faecal samples for the presence of plastic using micro Fourier Transform infrared microscopy. Plastic polymers were detected in four out of the seven species examined (European hedgehog (Erinaceus europaeus), wood mouse (Apodemus sylvaticus); field vole (Microtus agrestis); brown rat (Rattus norvegicus)). Ingestion occurred across species of differing dietary habits (herbivorous, insectivorous and omnivorous) and locations (urban versus non-urban). Densities excreted were comparable with those reported in human studies.The prevalence of confirmed plastics in the 261 faecal samples was 16.5 % (95 % CI 13 %, 22 %). Most (70 %) of the 60 plastic fragments were <1 mm (microplastics). Polyester, likely to be derived from textiles, accounted for 27 % of the fragments and was found in all plastic-positive species except for the wood mouse. The high prevalence of polyester in terrestrial ecosystems was unexpected and suggests that evaluation is needed of practices likely to transfer this plastic into the environment (such as sewage sludge application to farmland). Polynorbornene, which is likely to be derived from tyre wear, and polyethylene were also commonly detected polymers. ‘Biodegradable’ plastics formed 27 % (n = 12) of the particles found in wild mammal faeces, warranting further research to assess their persistence in the environment.

The Eurasian badger-generated seed rain drives the natural (re)colonization of vacant human-altered areas by a keystone pioneer palm

Seed dispersal is an essential ecological process for plant (re)colonization, especially in intensively human-altered habitats such as old-fields (i.e. abandoned farmlands) where seed arrival is often limited. Assessment of spatial patterns of mammal-generated seed rain and their matching with the spatial distribution of adult plants provides essential information on the patterns and pace of the (re)colonization processes. For instance, increased seedling survival far from adult plants could indicate density-dependent mortality (Janzen-Connell) effects whereas increased survival close to adult plants could suggest facilitative effects. Here, we characterized during two dispersal seasons the spatial distribution of feces from four frugivorous mammals and quantified its spatial association with the distribution of adult plants of the pioneer Mediterranean dwarf palm (Chamaerops humilis) in two old-fields in southwestern Spain. We also estimated the dispersal kernels of both emerged and surviving seedlings and assessed potential evidence for Janzen-Connell and/or facilitative effects. Using a spatially explicit approach, we revealed strong differences between study sites in the strength and scale of spatial associations between mammal feces with C. humilis seeds and adult dwarf palms, being strongly positive at small scales in one site and slightly positive at larger scales in the other one. Further, we found some evidence of both Janzen-Connell and facilitative effects depending on the study site. Altogether, our results emphasize the central role as seed disperser of the Eurasian badger (Meles meles) in the natural (re)colonization of Mediterranean old-fields and the spatial variations of the underlying mechanisms and demographic consequences for plant populations.

Validation and effects of drying and prey hair on fecal hormone concentrations in spotted hyenas

Abstract As fecal steroid methods increasingly are used by researchers to monitor the physiology of captive and wild populations, we need to expand our validation protocols to test the effects of procedural variation and to identify contamination by exogenous sources of steroid hormones. Mammalian carnivore feces often contain large amounts of hair from the prey they consume, which itself may contain high concentrations of hormones. In this study, we report first a validation of two steroid hormone antibodies, corticosterone and progesterone, to determine fecal concentrations of these hormones in wild spotted hyenas (Crocuta crocuta). Next, we expand on these standard validation protocols to test two additional metrics: (i) whether hair from consumed prey or (ii) the specific drying method (oven incubation vs. lyophilization) affect steroid hormone concentrations in feces. In the first biological validation for the progesterone antibody in this species, progesterone concentrations met our expectations: (i) concentrations of plasma and fecal progesterone were lowest in immature females, higher in lactating females, and highest in pregnant females; (ii) across pregnant females, fecal progesterone concentrations were highest during late pregnancy; and (iii) among lactating females, fecal progesterone concentrations were highest after parturition. Our additional validation experiments indicated that contamination with prey hair and drying method are hormone-specific. Although prey hair did not release hormones into samples during storage or extraction for either hormone, its presence appeared to “dilute” progesterone (but not corticosterone) measures indirectly by increasing the dry weight of samples. In addition, fecal progesterone, but not corticosterone, values were lower for lyophilized than for incubated samples. Therefore, in addition to the standard analytical and biological validation steps, additional methodological variables need to be tested whenever we measure fecal hormone concentrations, particularly from predatory mammals.

On the origin of feces: Fungal diversity, distribution, and conservation implications from feces of small mammals

AbstractFungi are highly diverse, but only a small fraction of the total estimated species have been characterized. Often, the extent of diversity and distribution of fungal communities is difficult or near impossible to assess due to the fact that many fungi are cryptic and persist predominantly hidden within substrates such as soil or plant material. This is particularly true for hypogeous sporocarps, such as truffles and false truffles, which are extremely difficult to survey in a systematic manner. However, hypogeous fungi have evolved traits that make them highly attractive to animals, such as small mammals, which ingest and disperse fungal spores through defecation. Here, samples of feces from 138 small mammal museum vouchers collected in the western United States were assessed for total fungal diversity using a dual‐index metabarcoding approach. Our findings exhibit many identifications within the mushroom‐forming fungi (Agaricomycetidae), with 65 of the 138 samples containing sequences belonging to several species of the false truffle‐forming genus Rhizopogon. Metadata for each collection, such as geospatial coordinates, can be used as a proxy for the presence or absence of Rhizopogon species identified in their feces. Utilizing these proxy data from only a few years of sampling, these records quadrupled the rate of observations of Rhizopogon made over the past 100 years, including some species that have only been recorded once previously. This substantial increase in datapoints has implications for how fungal distributions are interpreted, with direct impact on standard assessment tools for fungal conservation.

Faeces of marine birds and mammals as substrates for microbial plankton communities

The chemical composition of the seawater soluble fraction (WSF) of yellow-legged gulls and harbour seal faeces and their impact on microbial plankton communities from an eutrophic coastal area have been tested. After characterisation of the C:N:P stoichiometry, trace metals content and organic molecular composition of the faeces, significant differences between species have been observed in all parameters. Seagull faeces present about three times larger N content than seal faeces and are also richer in trace elements except for Cu and Zn. Organic nitrogen in seagull faeces is dominated by uric acid, while the proteins are the main N source in seal faeces. It is remarkable that seagull faeces are five times more soluble in seawater than seal faeces and present a much higher N content (48.0 versus 3.5 mg N in the WSF per gram of dry faeces), >85% of which as dissolved organic nitrogen, with C:N molar ratios of 2.4 and 13 for seagull and seal faeces, respectively. Based on this contrasting N content, large differences were expected in their impact on microbial populations. To test this hypothesis, a 3-day microcosm incubation experiment was performed, in which coastal seawater was amended with realistic concentrations of the WSF of seagull or seal faeces. A significant and similar increase in bacterial biomass occurred in response to both treatments. In the case of phytoplankton, the impact of the treatment with seagull faeces was significantly larger that the effect of the treatment with seal faeces. Our data suggest that the distinct competitive abilities of phytoplankton and bacteria largely influence the potential impact of distinct animal faeces on primary productivity in coastal ecosystems. Impacts on the microbial plankton communities do not affect only this trophic level, but the whole trophic chain, contributing to nutrient recycling in coastal areas where large populations of these species are settled.

Seed size and pubescence facilitate secondary dispersal by dung beetles

AbstractIn tropical forests, primary dispersal by animals is the most important form of seed dispersal. Dung beetles are secondary seed dispersers attracted to mammal feces. When they bury dung of frugivorous mammals, they move seeds to new sites, possibly protecting them from seed predation or pathogens, or moving to better micro‐climates and away from conspecifics. As a result, secondary dispersal by dung beetles potentially increases rates of seed survival and germination. Previous studies examined how dung beetles filter seeds by size. However, other seed traits have not been examined. We discovered that pubescent seeds covered with hairs on their surface hold a thin layer of dung and possibly “trick” the dung beetles into burying them like a dung ball. In a lowland tropical forest (Chocó Ecuador), we collected dung balls from dung beetles (Canthon angustatus, Oxysternon conspicillatum, Sulcophanaeus noctis, and Scybalocanthon trimaculatus), and fecal samples from brown‐headed spider monkeys (Ateles fusciceps fusciceps) and mantled howler monkey (Alouatta palliata). We characterized the traits of seed morphospecies found within samples and counted them. Our data show that larger size is coupled with a higher proportion of pubescence in seeds. The association between seed size and pubescence may extend beyond our study area supported by an analysis of the literature data for neotropical seeds at the genus level. Large pubescent seeds were more likely to be included in dung balls than smooth large seeds. Our results are consistent with the hypothesis that secondary dispersal by dung beetles exerts some selection pressure on the phenotype of endozoochorous seeds.

Multiproxy analysis of permafrost preserved faeces provides an unprecedented insight into the diets and habitats of extinct and extant megafauna

The study of faecal samples to reconstruct the diets and habitats of extinct megafauna has traditionally relied on pollen and macrofossil analysis. DNA metabarcoding has emerged as a valuable tool to complement and refine these proxies. While published studies have compared the results of these three proxies for sediments, this comparison is currently lacking for permafrost preserved mammal faeces. Moreover, most metabarcoding studies have focused on a single plant-specific DNA marker region. In this study, we target both the commonly used chloroplast trnL P6 loop as well as nuclear ribosomal ITS (nrITS). The latter can increase taxonomic resolution of plant identifications but requires DNA to be relatively well preserved because of the target length (∼300–500 bp). We compare DNA results to pollen and macrofossil analyses from permafrost and ice-preserved faeces of Pleistocene and Holocene megafauna. Samples include woolly mammoth, horse, steppe bison as well as Holocene and extant caribou. Most plant identifications were found using DNA, likely because the studied faeces contained many vegetative remains that could not be identified using macrofossils or pollen. Several taxa were, however, identified to lower taxonomic levels uniquely with macrofossil and pollen analysis. The nrITS marker provides species level taxonomic resolution for commonly encountered plant families that are hard to distinguish using the other proxies (e.g. Asteraceae, Cyperaceae and Poaceae). Integrating the results from all proxies, we are able to accurately reconstruct known diets and habitats of the extant caribou. Applying this approach to the extinct mammals, we find that the Holocene horse and steppe bison were not strict grazers but mixed feeders living in a marshy wetland environment. The mammoths showed highly varying diets from different non-analogous habitats. This confirms the presence of a mosaic of habitats in the Pleistocene ‘mammoth steppe’ that mammoths could fully exploit due to their flexibility in food choice.

Faecal proteomics as a novel method to study mammalian behaviour and physiology.

Mammalian faeces can be collected noninvasively during field research and provide valuable information on the ecology and evolution of the source individuals. Undigested food remains, genome/metagenome, steroid hormones, and stable isotopes obtained from faecal samples provide evidence on diet, host/symbiont genetics, and physiological status of the individuals. However, proteins in mammalian faeces have hardly been studied, which hinders the molecular investigations into the behaviour and physiology of the source individuals. Here, we apply mass spectrometry‐based proteomics to faecal samples (n = 10), collected from infant, juvenile, and adult captive Japanese macaques (Macaca fuscata), to describe the proteomes of the source individual, of the food it consumed, and its intestinal microbes. The results show that faecal proteomics is a useful method to: (i) investigate dietary changes along with breastfeeding and weaning, (ii) reveal the taxonomic and histological origin of the food items consumed, and (iii) estimate physiological status inside intestinal tracts. These types of insights are difficult or impossible to obtain through other molecular approaches. Most mammalian species are facing extinction risk and there is an urgent need to obtain knowledge on their ecology and evolution for better conservation strategy. The faecal proteomics framework we present here is easily applicable to wild settings and other mammalian species, and provides direct evidence of their behaviour and physiology.