Plant DNA sequences from feces: potential means for assessing diets of wild primates

Analyses of plant DNA in feces provides a promising, yet largely unexplored, means of documenting the diets of elusive primates. Here we demonstrate the promise and pitfalls of this approach using DNA extracted from fecal samples of wild western gorillas (Gorilla gorilla) and black and white colobus monkeys (Colobus guereza). From these DNA extracts we amplified, cloned, and sequenced small segments of chloroplast DNA (part of the rbcL gene) and plant nuclear DNA (ITS-2). The obtained sequences were compared to sequences generated from known plant samples and to those in GenBank to identify plant taxa in the feces. With further optimization, this method could provide a basic evaluation of minimum primate dietary diversity even when knowledge of local flora is limited. This approach may find application in studies characterizing the diets of poorly-known, unhabituated primate species or assaying consumer-resource relationships in an ecosystem.     

From feces to data: A metabarcoding method for analyzing consumed and available prey in a bird‐insect food web

Diets play a key role in understanding trophic interactions. Knowing the actual structure of food webs contributes greatly to our understanding of biodiversity and ecosystem functioning. The research of prey preferences of different predators requires knowledge not only of the prey consumed, but also of what is available. In this study, we applied DNA metabarcoding to analyze the diet of 4 bird species (willow tits Poecile montanus, Siberian tits Poecile cinctus, great tits Parus major and blue tits Cyanistes caeruleus) by using the feces of nestlings. The availability of their assumed prey (Lepidoptera) was determined from feces of larvae (frass) collected from the main foraging habitat, birch (Betula spp.) canopy. We identified 53 prey species from the nestling feces, of which 11 (21%) were also detected from the frass samples (eight lepidopterans). Approximately 80% of identified prey species in the nestling feces represented lepidopterans, which is in line with the earlier studies on the parids' diet. A subsequent laboratory experiment showed a threshold for fecal sample size and the barcoding success, suggesting that the smallest frass samples do not contain enough larval DNA to be detected by high‐throughput sequencing. To summarize, we apply metabarcoding for the first time in a combined approach to identify available prey (through frass) and consumed prey (via nestling feces), expanding the scope and precision for future dietary studies on insectivorous birds.     

Sex identification assay useful in great apes is not diagnostic in a range of other primate species.

The ability to identify the sex of individuals from noninvasive samples can be a powerful tool for field studies. Amelogenin, a nuclear gene proximate to the pseudoautosomal region of mammalian sex chromosomes, has a 6 base-pair (bp) size difference between human X and Y chromosomes that can be PCR-amplified and sized to distinguish male from female DNA. We examined whether this test can be used to identify sex from different DNA sources across a number of nonhuman primate taxa. Using human amelogenin primers, we were able to amplify diagnostic products from the four great ape species tested, but products from five other primate species were not sexually dimorphic.     

Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples

Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal DNA quality and COII amplification varied according to storage solution (formalin, ethanol, LB and GTB), extraction method (LB-based and GTB-based) and primate species (chimpanzee, baboon, human). It is recommended that fecal samples be collected in LB for DNA analysis. However, GTB-based protocols are suitable when total RNA is needed for epidemiological studies of viral diseases or gene expression analysis.     

Noninvasive Saliva Collection for DNA Analyses From Free‐Ranging Tibetan Macaques (Macaca thibetana)

Cryptic and endangered fauna, including many primate taxa, pose challenges for noninvasive collection of biomaterials. As a result, application of noninvasive genotyping to primates has been limited to the use of samples such as feces and hair for the extraction of PCR‐amplifiable DNA. We present a method for noninvasive collection of saliva from habituated, free‐ranging monkeys. The method utilizes a low‐cost apparatus that controls for contamination and is usable with individual, free‐ranging primates. Saliva samples were collected from 18 individuals in a population of Tibetan macaques (Macaca thibetana) in the Valley of Wild Monkeys in Huangshan, People's Republic of China. DNA was extracted from these samples and PCR‐amplified for both mitochondrial and nuclear genes, Cytochrome B and MHC‐DR Beta 1, respectively. These results indicate this is an effective technique for the noninvasive collection of saliva across age and sex class, and dominance rank in a free‐ranging, terrestrial primate species. This device could have wide application for obtaining high‐quality saliva samples from free‐ranging primate populations for use in epidemiological studies, hormonal analyses of HPA axis function, pathogen screening, noninvasive genotyping, and behavioral genetics. Am. J. Primatol. 74:1064‐1070, 2012. © 2012 Wiley Periodicals, Inc.     

Methods of estimating marine mammal diets: A review of validation experiments and sources of bias and uncertainty

Diet estimation in marine mammals relies on indirect methods including recovery of prey hard parts from stomachs and feces, quantitative fatty acid signature analysis (QFASA), stable isotope mixing models, and identification of prey DNA in stomach contents and feces. Experimental evidence (9 species/13 studies) shows that digestion strongly influences the proportion and size of otoliths that can be recovered in feces. Number correction factors (NCF) and digestion coefficients have been experimentally determined to reduce the biases in fecal analysis. Correction factors and coefficients have not been determined for diet estimated from stomach contents. QFASA estimates which prey species and amounts must have been eaten to account for the fatty acid composition of the predator. Experimental studies on mammals and seabirds (9 species/10 studies) indicate that accurate estimates of diet can be determined using QFASA. Stable isotope mixing models provide rather coarse taxonomic resolution of diet composition. Prey DNA analysis shows promise as a method to estimate the species composition of diet, but further development and testing is needed to validate its use. Obtaining a representative sample from marine mammal populations is a significant challenge. Therefore, the use of complementary methods is recommended to obtain the most informative results.     

Noninvasive collection of fresh hairs from free‐ranging howler monkeys for DNA extraction

The use of noninvasive collected samples as source of DNA in studies of wild primate populations has increased in recent years. Fresh‐plucked hairs represent an important source of DNA, with relatively high quality and concentration. In this study, we describe a low‐cost noninvasive technique for collecting fresh‐plucked hairs used to obtain DNA samples from free‐ranging black howler monkey populations (Alouatta pigra). We designed and manufactured darts made of wooden dowels, with the anterior part smeared with glue, which were projected with blowpipes to trap howler monkey hairs. All of the materials to make the darts are inexpensive and are available locally. We collected 89 samples from 76 individuals residing in 15 troops, and the total number of hairs obtained was 754. We found no differences in the number of hairs collected among sex–age classes or among localities but the percentage of darts recovered with sample varied among localities. Preliminary results indicate that over 96% of samples yielded DNA suitable for polymerase chain reaction‐based microsatellite marker analysis. The technique proved successful for collecting fresh‐plucked hairs of free‐ranging black howler monkeys without any trauma to the animals and can be easily adapted to obtain samples from other wild primate and mammal species. Am. J. Primatol. 71:359–363, 2009. © 2009 Wiley‐Liss, Inc.     

Quantification and genetic profiling of DNA isolated from free-floating feces of the North Atlantic right whale (Eubalaena glacialis)

Fecal analysis from the highly endangered North Atlantic right whale provides valuable information about health and reproductive parameters of individual animals. Genetically profiling the feces facilitates this connection when the sample originator is unknown. Although genetic analysis of feces collected in terrestrial systems has become well established, genetic studies of cetacean DNA are rare. Here, the use of free‐floating feces as a source of right whale DNA and the reliability of the genotypes produced are examined. On average, fecal extracts yielded 25 ng of DNA/mg dry weight, but less than 1% was right whale DNA. Although all samples were amplified using genus‐specific mitochondrial control region primers, the quantity of right whale DNA present was over estimated when compared to amplifications using nuclear primers. No correlation was found between the quantity of right whale DNA recovered and the duration the sample sat in the water. Composite microsatellite profiles from multiple amplifications of 28 fecal samples of known origin were consistent with profiles of the same individuals obtained from skin biopsies, however, the rate of allelic dropout varied depending on the amount of right whale DNA added. A screening and genotyping protocol for reliable genetic profiling based on fecal DNA quantification is presented.     

Japanese macaque microsatellite PCR primers for paternity testing

Capture and blood sampling in wild primate populations are difficult. For this reason, we need to use DNA extracted from the hair or feces of target animals. The polymerase chain reaction (PCR) method, which amplifies small volumes of DNA, provides an ideal means for studying DNA variations in wild populations. Three sets of PCR primers which amplify highly polymorphic (GT/AC)_n dinucleotide repetitive regions were synthesized from DNA sequences of Japanese macaques (Macaca fuscata). One of the primer pairs detected at least seven alleles in one captive Japanese macaque group. Also, the fathers of four offspring whose mothers had died in a captive group of Japanese macaques were identified. In such cases, the father cannot be determined by the previous DNA fingerprinting method based on the polymorphism of minisatellite DNA. These primers were further tested with some species of the Cercopithecidae, e.g. grivet monkeys (Cercopithecus aethiops tantalus) and hamadryas baboons (Papio hamadryas). The results obtained suggest that these primers can detect stably inherited polymorphic regions in each species.     

Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape

The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.     

Molecular scatology: the use of molecular genetic analysis to assign species, sex and individual identity to seal faeces

Seals and commercial fisheries are potential competitors for fish and cephalopods. Research into the diet of British seal species has been based on conventional dietary analyses, but these methods often do not allow assignment of species identity to scat samples. We present a protocol for obtaining DNA from seal scat (faecal) samples which can be used in polymerase chain reactions to amplify both nuclear and mitochondrial DNA. This can provide a method of identifying the species, sex and individual identity of the seal, from a particular scat sample. Combined with conventional dietary analyses these techniques will allow us to assess sources of variation in seal diet composition.
Scat samples have been collected from intertidal haul-out sites around the inner Moray Firth, north-east Scotland. We have assessed methods to extract and purify faecal DNA, a combination of DNA from the individual seal, prey items, and gut bacteria, for use in PCR. Controls using faecal and blood samples from the same individual have enabled microsatellite primer sets from four pinniped species to be tested. Approximately 200 scat samples have been examined for species identity and individual matches. This study will provide essential information for the assessment of interactions between seals and commercial or recreational fisheries.     

Identifying Species in Chimpanzee (Pan troglodytes) Feces: A Methodological Lost Cause?

Ascertaining the full range of dietary constituents of a primate population allows the identification of habitats with important food resources and can assist efforts to conserve primates. For unhabituated populations, we can acquire otherwise unobtainable dietary information from macroscopic inspection of fecal samples. This method has made a significant contribution to understanding food intake in various primate species. Increasing knowledge of the omnivorous diet of our closest living relatives, the common chimpanzee (Pan troglodytes) and the bonobo (P. paniscus), which range and forage in various habitats to meet daily nutrient requirements, provides more scope to assess human omnivory and its evolution from our last common ancestor. However, macroscopic inspection may lead to bias toward undigested and therefore identifiable food items, e.g., fruit seeds, vs. pulverized components, e.g., leaves, that are unidentifiable at this level. This study seeks to validate findings from macroscopic inspection by comparing species identified in fecal samples from select individuals vs. data from direct observations of their feeding. We collected data from 10 adult chimpanzees of the Kanyawara community in Kibale National Park. We identified 86% of species from which fruit had been eaten vs. only 21% from which leaves had been eaten in fecal samples analyzed. This study provides empirical support for previous assumptions and confirms the limitations of macroscopic inspection of feces for identifying the nonfrugivorous dietary elements to species level. However, valuable insights into seasonality of diet can be gleaned from macroscopic inspection. Also, if we combine data on species identified in feces with direct observation of food intake, we can establish when food items were eaten, which enables estimations of gut passage rates for wild populations. Finally, analyzing fecal samples collected from various group members can provide insight into the dietary repertoire at the individual level.     

地黄属植物的DNA条形码研究

地黄属(Rehmannia)为玄参科(Scrophulariaceae)药用植物,广泛分布于中国中东部及北部地区。由于地黄属植物经历了快速成种,导致其属内物种间形态性状差异较小,运用传统的形态学分类方法已难以准确地鉴定物种,近年来迅速发展起来的DNA条形码技术为快速、准确地鉴别物种提供了新思路。本研究选用3个叶绿体DNA非编码区片段(trn L-trn F、trn M-trn V和trn S-trn G)及核基因ITS片段,运用PWG-distance和TreeBuilding两种方法对地黄属5个物种75个个体进行了DNA条形码分析。结果表明:单个叶绿体DNA片段或核基因ITS片段对地黄属物种的鉴别率较低(0%~20%),组合的叶绿体DNA片段分辨能力虽然高于单个DNA片段,但并不能将地黄属5个物种完全区分开;trn S-trn G+ITS片段组合的分辨率可达100%,能够将地黄属5个物种准确区分,与所有叶绿体DNA片段和核基因ITS片段组合(trn L-trn F+trn M-trn V+trn S-trn G+ITS)的辨别率相同,因此推荐trn S-trn G+ITS作为地黄属植物的标准条形码。此外,利用DNA条形码鉴别物种时,可采用叶绿体DNA片段和核DNA片段组合的方法来提高物种鉴定的成功率。     

Genome Size and Pollen Viability as Taxonomic Criteria: Application to the Genus Hosta

Abstract: Genome size (C values) and pollen viability staining were applied as new criteria to investigate the taxonomy of the genus Hosta Tratt. (Hostaceae). Nearly all species of the genus Hosta have the same basic chromosome number (2n = 2x = 60). However, the nuclear DNA contents, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 17.2 to 26.6 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. Therefore, nuclear DNA content is a very relevant taxonomic trait that can be measured simply by flow cytometry. In addition, differences in overall DNA composition were demonstrated by comparing to DAPI fluorescence. In general, genome size data confirmed the division into three subgenera. The geographical distribution of genome sizes indicates the migration pattern of Hosta throughout East Asia. The species belonging to the mainly Korean subgenus Bryocles, with a low nuclear DNA content (17.2 - 19.3 pg), can now largely be distinguished from the mainly Japanese species of the subgenus Giboshi (21.3 - 26.5 pg). The exception is H. longissima, that with only 19.6 pg provides a nice example of a decrease in DNA content. On the mainland, as well as on Honshu, species with increased and decreased DNA content have evolved independently. The usefulness of pollen viability to detect hybrids in Hosta was demonstrated in a large series of artificial crosses between bona fide species. Consequently, pollen viability was measured in all available Hosta described as species. Several had low pollen viability and were concluded to be hybrids. Morphology and DNA content confirmed this in most cases. The resulting 23 species approximate the number of Hosta species that follows from the combined studies by Fujita (1976¹⁸) on the Japanese species and Chung (1991 a¹¹) on the Korean species.     

Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1 beta and an increase of interleukin-10.     

When the excrement hits the fan: Fecal surveys reveal species-specific bat activity at wind turbines

The reasons why bats are coming into contact with wind turbines are not yet well understood. One hypothesis is that bats are attracted to wind turbines and this attraction may be because bats perceive or misperceive the turbines to provide a resource, such as a foraging or roosting site. During post-construction fatality searches at a wind energy facility in the southern Great Plains, U.S., we discovered bat feces near the base of a wind turbine tower, which led us to hypothesize that bats were actively roosting and/or foraging at turbines. Thus over 2 consecutive years, we conducted systematic searches for bat feces on turbines at this site. We collected 72 bat fecal samples from turbines and successfully extracted DNA from 56 samples. All 6 bat species known to be in the area were confirmed and the majority (59%) were identified as Lasiurus borealis; a species that also comprised the majority of the fatalities (60%) recorded at the site. The presence of bat feces provides further evidence that bats were conducting activities in close proximity to wind turbines. Moreover, feces found in areas such as turbine door slats indicated that bats were using turbines as night or foraging roosts, and further provided evidence that bats were active near the turbines. Future research should therefore aim to identify those features of wind turbines that bats perceive or misperceive as a resource, which in turn may lead to new minimization strategies that effectively reduce bat fatalities at wind farms.     

Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis

Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.     

Empirical evaluation of preservation methods for faecal DNA

We evaluate the relative effectiveness of four methods for preserving faecal samples for DNA analysis. PCR assays of fresh faecal samples collected from free-ranging baboons showed that amplification success was dependent on preservation method, PCR-product size, and whether nuclear or mitochondrial DNA was assayed. Storage in a DMSO/EDTA/Tris/salt solution (DETs) was most effective for preserving nuclear DNA, but storage in 70% ethanol, freezing at –20°C and drying performed approximately equally well for mitochondrial DNA and short (<200 bp) nuclear DNA fragments. Because faecal DNA is diluted and degraded, repeated extractions from faeces may be necessary and short nuclear markers should be employed for genotyping. A review of molecular scatology studies further suggests that three to six faeces per individual should be collected.     

Testing and using complete plastomes and ribosomal DNA sequences as the next generation DNA barcodes in Panax (Araliaceae)

Complete plastid genome (plastome) sequences and nuclear ribosomal DNA (nrDNA) regions have been proposed as candidates for the next generation of DNA barcodes for plant species discrimination. However, the efficacy of this approach still lacks comprehensive evaluation. We carried out a case study in the economically important but phylogenetically and taxonomically difficult genus Panax (Araliaceae). We generated a large data set of plastomes and nrDNA sequences from multiple accessions per species. Our data improved the phylogenetic resolution and levels of species discrimination in Panax, compared to any previous studies using standard DNA barcodes. This provides new insights into the speciation, lineage diversification and biogeography of the genus. However, both plastome and nrDNA failed to completely resolve the phylogenetic relationships in the Panax bipinnatifidus species complex, and only half of the species within it were recovered as monophyletic units. The results suggest that complete plastome and ribosomal DNA sequences can substantially increase species discriminatory power in plants, but they are not powerful enough to fully resolve phylogenetic relationships and discriminate all species, particularly in evolutionarily young and complex plant groups. To gain further resolving power for closely related species, the addition of substantial numbers of nuclear markers is likely to be required.