The use of non-invasive molecular techniques to confirm the presence of mountain bongo <Emphasis Type="Italic">Tragelaphus eurycerus isaaci</Emphasis> populations in Kenya and preliminary inference of their mitochondrial genetic variation

The mountain bongo antelope Tragelaphus eurycerus isaaci has rapidly declined in recent decades, due to a combination of hunting, habitat degradation and disease. Endemic to Kenya, mountain bongo populations have shrunk to approximately 100 individuals now mainly confined to the Aberdares mountain ranges. Indirect observation of bongo signs (e.g. tracks, dung) can be misleading, thus methods to ensure reliable species identification, such as DNA-based techniques, are necessary to effectively study and monitor this species. We assessed bongo presence in four mountain habitats in Kenya (Mount Kenya National Park, Aberdare National Park, Eburu and Mau forests) and carried out a preliminary analysis of genetic variation by examining 466 bp of the first domain of the mtDNA control region using DNA extracted from faecal samples. Of the 201 dung samples collected in the field, 102 samples were molecularly identified as bongo, 97 as waterbuck, one as African buffalo and one as Aders’ duiker. Overall species-identification accuracy by experienced trackers was 64%, with very high error of commission when identifying bongo sign (37%), and high error of omission for waterbuck sign (82%), suggesting that the two species’ signs are easily confused. Despite high variation in the mtDNA control region in most antelope species, our results suggest low genetic variation in mountain bongo as only two haplotypes were detected in 102 samples analyzed. In contrast, the analysis of 63 waterbuck samples from the same sites revealed 21 haplotypes. Nevertheless, further examination using nuclear DNA markers (e.g. microsatellites) in a multi-locus approach is still required, especially because the use of mitochondrial DNA can result in population overestimation as distinct dung samples can potentially be originated from the same individual.     

Estimating population sizes for elusive animals: the forest elephants of Kakum National Park,Ghana

African forest elephants are difficult to observe in the dense vegetation, and previous studies have relied upon indirect methods to estimate population sizes. Using multilocus genotyping of noninvasively collected samples, we performed a genetic survey of the forest elephant population at Kakum National Park, Ghana. We estimated population size, sex ratio and genetic variability from our data, then combined this information with field observations to divide the population into age groups. Our population size estimate was very close to that obtained using dung counts, the most commonly used indirect method of estimating the population sizes of forest elephant populations. As their habitat is fragmented by expanding human populations, management will be increasingly important to the persistence of forest elephant populations. The data that can be obtained from noninvasively collected samples will help managers plan for the conservation of this keystone species.     

Some coprophilous fungi from Kenya

The occurrence of coprophilous fungi on samples of antelope, buffalo, zebra and hippopotamus dung collected from the Marula Estates in Kenya was recorded. A hundred and seventy three fungal isolates representative of 40 genera and 59 species were identified. Among these species, 23 were Ascomycetes (39%), 30 Deuteromycetes (50.8%), 5 Zygomycetes (8.5%) and 1 Basidiomycete (1.7%). Ascobolus immersus, Coprotus niveus, Iodophanus carneus, Lasiobolus lasioboloides, Podospora anserina, P. australis and Sporormiella minima were the dominant species occurring in the dung of these animals. Notes on infrequent or interesting Ascomycetes include Kernia nitida, Saccobolus versicolor, Sordaria fimicola and others. The interdependence of the vegetation of this area with the fungi occurring on the dung of these animals is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Species identification of Tanzanian antelopes using DNA barcoding

Efficient tools for consistent species identification are important in wildlife conservation as it can provide information on the levels of species exploitation and assist in solving forensic-related problems. In this study, we evaluated the effectiveness of the mitochondrial cytochrome c oxidase subunit I (COI) barcode in species identification of Tanzanian antelope species. A 470 base-pair region of the COI gene was examined in 95 specimens representing 20 species of antelopes, buffalo and domestic Bovidae. All the Tanzanian species showed unique clades, and sequence divergence within species was <1%, whereas divergence between species ranged from 6.3% to 22%. Lowest interspecific divergence was noted within the Tragelaphus genus. Neighbour-joining phylogenetic analyses demonstrated that the examined COI region provided correct and highly supported species clustering using short fragments down to 100 base-pair lengths. This study demonstrates that even short COI fragments can efficiently identify antelope species, thus demonstrating its high potential for use in wildlife conservation activities.     

Distinguishing dung from blue, red and yellow-backed duikers through noninvasive genetic techniques

Dung counts have been widely used to estimate duiker abundance and densities in tropical African forests. However, one of the major limitations of this method is that species' identification of dung based on morphological characteristics is extremely difficult in most cases. Some authors made the assumption that dung pellets could at least be distinguishable between the blue duiker, the red duikers and the yellow-backed duiker. However, this study is the first attempt to test the reliability of field identification. In this study, we suggest a method based on a noninvasive genetic protocol to identify duiker species. The comparison of field and genetic identifications shows that only dung from Cephalophus silvicultor was identified in the field without error. The rate of error for red duikers and the blue duiker is high and any attempt to distinguish among those species will result in wrong estimations for each species. We recommend the use of DNA tests to ensure reliable species' identification when duiker censuses based on dung counts are used. This methodological input will probably strengthen the dung count technique and increase its reliability for duiker species abundance estimations and spatial distribution studies.     

Molecular Characterization of Coprophilous Fungal Communities Reveals Sequences Related to Root-Associated Fungal Endophytes

This paper reports the use of molecular methods to characterize the coprophilous fungal communities (CFC) that inhabit the dung of four species of mammalian herbivores at two sites, Sevilleta National Wildlife Refuge (SNWR) in New Mexico and Wind Cave National Park (WCNP) in South Dakota. Results reveal that CFC from domesticated cattle (Bos taurus) at SNWR, and bison (Bison bison) and black-tailed prairie dogs (Cynomys ludovicianus) at WCNP were diverse but dominated primarily by members within eight taxonomic orders, including the rarely cultured and anaerobic order Neocallimastigales. In addition, 7.7% (138 of 1,788) of the sequences obtained from all dung samples were at least 97% similar to root-associated fungal (RAF) sequences previously described from blue grama (Bouteloua gracilis), a common forage grass found throughout North America and growing at both study sites. In contrast, 95.8% (295 of 308) of the sequences and four of the total seven operational taxonomic units obtained from pronghorn antelope (Antilocapra americana) dung belonged to the Pleosporalean order. We hypothesize that some herbivore vectors disperse non-systemic (non-clavicipitaceous) fungal endophytes. These dispersal events, it is argued, are most likely to occur via herbivores that occasionally forage and masticate root tissue, especially in arid regions where aboveground vegetation is sparse. The results of this study suggest that some (possibly many) members of the RAF community can expand their ecological role to include colonizing dung.     

Potential of endozoochorous seed dispersal by sheep in calcareous grasslands: correlations with seed traits

Questions: What is the potential of sheep to serve as seed dispersers via ingestion and defecation in calcareous grasslands? Is the presence of viable seeds from dung correlated with specific seed traits? Location: Calcareous grasslands, South Limburg, the Netherlands/Belgium. Methods: Dung samples (n=24) from sheep were collected between September 2006 and November 2007 from five sites with Mesobromion plant communities, and communities of Nardo‐Galion saxatilis. Germinability and identity of seeds in the dung samples were ascertained from germination of seedlings under glasshouse conditions. Seed traits of species with viable seeds in dung were compared with those present in the local species pool. Results: Seventy‐two plant species from 23 plant families had viable seeds in sheep dung. The plant families encountered most frequently were Gramineae and Compositae. The most abundant and frequently recorded plant species in dung samples was Urtica dioica, accounting for >80% of the total number of seeds. Mean seed density in sheep dung was 0.8 seeds g^?1 dry matter. Seeds with low seed mass and a high seed longevity index were over‐represented in dung. Viable seeds >2.5 mg were infrequent in the dung samples. Conclusions: We conclude that sheep are potentially important dispersers of plant species in Dutch calcareous grasslands. Although smaller seeds were relatively abundant in sheep dung, it cannot be excluded that this was mainly caused by differences in seed abundance.     

Dung beetles as secondary seed dispersers in a temperate grassland

The two-phase dispersal event in which dung beetles move seeds after endozoochory is often assumed to be advantageous for plant regeneration. Because seeds are expected to end up in favourable and safe germination sites, it is considered as an example of directed dispersal. However, literature so far is restricted to tropical rain forest ecosystems, while data for temperate regions are lacking. In this study, the effect of dung beetles on seedling establishment of endozoochorically dispersed seeds is evaluated for a temperate grassland ecosystem. We performed a field experiment in which cages excluded dung beetles from horse and cattle dung samples with mixed-in grass seeds. Seed germination from these samples was significantly higher than that from samples which were accessible to dung beetles. This indicates that the effect of dung beetles on short-term seedling establishment was negative, which contrasts with the patterns found for large-seeded species used in tropical studies. This is most likely attributed to the lack of roller species and the larger depth at which tunneling Geotrupes species bury seeds.     

The problem of precision and trend detection posed by small elephant populations in West Africa

The precision of elephant estimates from aerial sample surveys and dung counts is inversely proportional to abundance. West African elephant populations are already small, and the power of a monitoring programme to detect changes in abundance diminishes as the population shrinks in size. Thus it will be difficult to evaluate the effects on elephant numbers of new management policies in West Africa. The same will be true of monitoring schemes for antelope and primate populations that are hunted for bushmeat. Elephant estimates from dung counts are more precise than those from aerial sample surveys, and changes in elephant numbers are more likely to be detected in the subregion by dung counts than by aerial sample surveys.     

Abrupt species loss of the Amazonian dung beetle in pastures adjacent to species-rich forests

Anthropogenic disturbances, such as conversion of forests to pastures, are considered one of the major causes of Amazonian biodiversity loss. Pastures are hostile environments for forest species that are not adapted to the adverse conditions of open areas. The aim of this study was to analyze the effects of forest replacement by cattle pastures in the north-eastern part of the Amazonian rainforest using dung beetles (Scarabaeidae: Scarabaeinae) as indicators. Therefore, we determined edge effects and differences in dung beetle species richness, composition, and abundance between forest and neighbouring cattle pastures. A total of 6723 individuals of 63 species of dung beetle were collected. In comparison to forests, adjacent pastures were sharply less rich and abundant in dung beetles. The forests had 6604 individuals and 59 species, while the pastures had 119 individuals and 12 species. The replacement of forests by pastures results in an increase of dung beetle species turnover. Only 12% of dung beetle species were shared by both forest and pasture environments. We highlight that most Amazonian dung beetles are forest species sensitive to abrupt human-driven modification of habitats and are only moderately affected by edge effects.     

Medicinal Plants Recommended by the World Health Organization: DNA Barcode Identification Associated with Chemical Analyses Guarantees Their Quality

Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines.     

Morphometric analyses of non-invasive fecal samples of the Korean long-tailed goral (Naemorhedus caudatus) for species and age identification

The Korean long-tailed goral (Naemorhedus caudatus) is at risk of population decline due to habitat loss and fragmentation. Therefore, it is essential to ascertain its presence and/or the identity of individuals of the goral using non-invasive fecal samples for its conservation and management. In this study, we examined the morphology of fecal samples to provide the baseline data that can be used to distinguish species and age of goral individuals. We detected a significant difference in the length-to-width ratios of feces among the five ungulate species found in Korea. Also, we detected a significant difference in the length-to-width ratios of feces of gorals depending on the age groups. To assess the accuracy of species and age identification based on the fecal morphology, we conducted a series of blind comparison between the mean length-to-width ratios of the fecal pellets and the reference mean ratio values of the fecal pellets. Using 20 fecal pellets, our results showed 73%–86% probability of correct identification of the three species (gorals, goats, and roe deer), and 83%–90% probability of correct identification of >5 year-old goral individuals. The use of fecal morphometric analyses will be useful for the studies of Korean ungulate species, particularly the endangered gorals.     

Estimating brocket deer (<Emphasis Type="Italic">Mazama gouazoubira</Emphasis> and <Emphasis Type="Italic">M. americana</Emphasis>) abundance by dung pellet counts and other indices in seasonal Chiquitano forest habitats of Santa Cruz,Bolivia

We assessed dung and track counts for indexing brocket deer abundance in seasonal habitats on a ranch where hunting is not practiced in the Bolivian lowlands. Surveys were replicated along four 10-km trails (totaling 180 km in the wet season and 90 km in the dry season) through four semideciduous forest habitats. Dung pellets and tracks were identified as belonging to Mazama gouazoubira or M. americana by size and shape. Pellet groups lasted more than 1 month during the dry season, but decayed within 1–2 weeks in the wet season. Mean density estimates based on dung counts varied widely between seasons for gray brockets (from 6.77±4.0 to 30.57±23.5 ind/km²; mean±SD) but not for reds (from 3.52±4.6 to 6.98±7.2 ind/km²). These values were probably too high due to underestimation of daily deposition rate, and were reduced during the wet season because of dung decay. We found consistently more dung in the dry season and more tracks in the wet season. Sightings of red brockets were too few for line-transect analysis (n=6), but those of gray brockets (n=42) produced an overall estimate of 5.6 ind/km² (95% CI=3.5–9.0 ind/km²). Different estimates indicated that gray brockets were more abundant than reds in all situations, except perhaps in the riverine forest. Environmental factors affected these indices of abundance differently, and while we recommend the use of dung counts in dry-season scenarios, we think that index reliability should be assessed locally before conducting population comparisons.     

Do riparian reserves support dung beetle biodiversity and ecosystem services in oil palm-dominated tropical landscapes?

Agricultural expansion and intensification are major threats to global biodiversity, ecological functions, and ecosystem services. The rapid expansion of oil palm in forested tropical landscapes is of particular concern given their high biodiversity. Identifying management approaches that maintain native species and associated ecological processes within oil palm plantations is therefore a priority. Riparian reserves are strips of forest retained alongside rivers in cultivated areas, primarily for their positive hydrological impact. However, they can also support a range of forest‐dependent species or ecosystem services. We surveyed communities of dung beetles and measured dung removal activity in an oil palm‐dominated landscape in Sabah, Malaysian Borneo. The species richness, diversity, and functional group richness of dung beetles in riparian reserves were significantly higher than in oil palm, but lower than in adjacent logged forests. The community composition of the riparian reserves was more similar to logged forest than oil palm. Despite the pronounced differences in biodiversity, we did not find significant differences in dung removal rates among land uses. We also found no evidence that riparian reserves enhance dung removal rates within surrounding oil palm. These results contrast previous studies showing positive relationships between dung beetle species richness and dung removal in tropical forests. We found weak but significant positive relationships between riparian reserve width and dung beetle diversity, and between reserve vegetation complexity and dung beetle abundance, suggesting that these features may increase the conservation value of riparian reserves. Synthesis and applications: The similarity between riparian reserves and logged forest demonstrates that retaining riparian reserves increases biodiversity within oil palm landscapes. However, the lack of correlation between dung beetle community characteristics and dung removal highlights the need for further research into spatial variation in biodiversity–ecosystem function relationships and how the results of such studies are affected by methodological choices.     

The Use of DNA Barcoding in Identification and Conservation of Rosewood (Dalbergia spp.)

The genus Dalbergia contains many valuable timber species threatened by illegal logging and deforestation, but knowledge on distributions and threats is often limited and accurate species identification difficult. The aim of this study was to apply DNA barcoding methods to support conservation efforts of Dalbergia species in Indochina. We used the recommended rbcL, matK and ITS barcoding markers on 95 samples covering 31 species of Dalbergia, and tested their discrimination ability with both traditional distance-based as well as different model-based machine learning methods. We specifically tested whether the markers could be used to solve taxonomic confusion concerning the timber species Dalbergia oliveri, and to identify the CITES-listed Dalbergia cochinchinensis. We also applied the barcoding markers to 14 samples of unknown identity. In general, we found that the barcoding markers discriminated among Dalbergia species with high accuracy. We found that ITS yielded the single highest discrimination rate (100%), but due to difficulties in obtaining high-quality sequences from degraded material, the better overall choice for Dalbergia seems to be the standard rbcL+matK barcode, as this yielded discrimination rates close to 90% and amplified well. The distance-based method TaxonDNA showed the highest identification rates overall, although a more complete specimen sampling is needed to conclude on the best analytic method. We found strong support for a monophyletic Dalbergia oliveri and encourage that this name is used consistently in Indochina. The CITES-listed Dalbergia cochinchinensis was successfully identified, and a species-specific assay can be developed from the data generated in this study for the identification of illegally traded timber. We suggest that the use of DNA barcoding is integrated into the work flow during floristic studies and at national herbaria in the region, as this could significantly increase the number of identified specimens and improve knowledge about species distributions.     

Effects of Degradation and Replacement of Southern Brazilian Coastal Sandy Vegetation on the Dung Beetles (Coleoptera: Scarabaeidae)

In this paper we address the effects of anthropogenic disturbance and replacement of Brazilian Coastal sandy vegetation (restingas) on dung beetles communities. We sampled dung beetles in the four main vegetative physiognomies of Guriri Island, Espírito Santo State: forest restinga, restinga Clusia, disturbed restinga (from burning events), and pastures. We placed four sets of two pitfall traps (baited with horse and human dung) in four independent areas of each vegetation type, and collected 14,534 individuals of 13 dung beetle species. Neither log₁₀ of individuals nor log₁₀ of species richness were good predictors of restinga disturbance. However, a significant amount of variation in dung beetle abundance and richness could be explained by bait type. Ordination of these sites using hybrid multidimensional scaling revealed a gradient of habitat disturbance from undisturbed restinga samples to pasture. Dung beetle communities along this gradient demonstrated a complete turnover in species composition, from restinga‐specialists to invasive and generalists species respectively. This complete turnover signals the local extirpation of forest‐adapted species in disturbed and converted areas. Only a single dung beetle species in preserved restingas is protected by Brazilian law (Dichotomius schiffleri). Given the extent of the clearing of restinga habitat, the conservation status of dung beetles associated with restinga forest gives cause for concern.     

El Niño impacts on human-modified tropical forests: Consequences for dung beetle diversity and associated ecological processes

Our knowledge of how tropical forest biodiversity and functioning respond to anthropogenic and climate-associated stressors is limited. Research exploring El Niño impacts are scarce or based on single post-disturbance assessments, and few studies assess forests previously affected by anthropogenic disturbance. Focusing on dung beetles and associated ecological functions, we assessed (a) the ecological effects of a strong El Niño, (b) if post-El Niño beetle responses were influenced by previous forest disturbance, and (c) how these responses compare between forests impacted only by drought and those affected by both drought and fires. We sampled 30 Amazonian forest plots distributed across a gradient of human disturbance in 2010, 2016, and 2017—approximately 5 years before, and 3–6 and 15–18 months after the 2015–16 El Niño. We found 14,451 beetles from 98 species and quantified the beetle-mediated dispersal of >8,600 seed mimics and the removal of c. 30 kg of dung. All dung beetle responses (species richness, abundance, biomass, compositional similarity to pre-El Niño condition, and rates of dung removal and seed dispersal) declined after the 2015–16 El Niño, but the greatest immediate losses (i.e., in 2016) were observed within fire-affected forests. Previous forest disturbance also influenced post-El Niño dung beetle species richness, abundance, and species composition. We demonstrate that dung beetles and their ecological functions are negatively affected by climate-associated disturbances in human-modified Amazonian forests and suggest that the interaction between local anthropogenic and climate-related stressors merits further investigation.     

The effect of seeds of exotic species transported via horse dung on vegetation along trail corridors

It has been suggested that exotic species will colonize within forests more frequently by the continual introduction of seeds through horse dung deposited along trails. Whether or not these exotic species have the ability to spread into and establish in the forest interior has been disputed. To address this, horse dung and soil samples were collected from trails during Autumn 1994 and Summer 1995 from three areas in southern Illinois, USA open to recreational horse travel. In addition, deer dung samples were collected from each of the study areas. Vegetation data were collected from each of the trail systems as well as from a trail along which horse travel was prohibited. The density of vascular plants in 0.25 m² quadrats placed at varying distances from the trail center to 5 m into the forest interior were recorded. Finally, dung samples were placed in situ along horse trails at one site to examine seedling germination in natural conditions. While 23 exotic species germinated from samples of horse dung placed out in a greenhouse, only one of these exotic species was also found in trail plots (Kummerowia striata). Similarly, while there were empirically more exotic species found along the trails allowing horse travel than there were on the trail lacking horse travel, the relative importance of those species was negligible along both trails. These results suggest that the emigration of exotic species via horse dung does not pose an immediate threat to the plant communities adjacent to trails in these forest systems. Nevertheless, the large number of exotic species in horse dung reflects the constant threat to any system from these species. Care must be taken, when allowing horseback use in areas, to anticipate invasion by exotic species from horse dung