Development of an eDNA metabarcoding tool for surveying the world’s largest amphibian

Due to the overexploitation of farming, as well as habitat loss or degradation, the wild population of Chinese giant salamander Andrias davidianus (CGS), a species with seven genetically distinct lineages, has decreased by over 80% in the past 70 years. Traditional survey methods have proven to be unsuitable for finding this rare and elusive species. We evaluated the efficacy of environmental DNA (eDNA) sampling to detect CGS indirectly from its aquatic environment. We developed several species-specific primer sets; validated their specificity and sensitivity; and assessed their utility in silico, in the laboratory, and in two field sites having released farm-bred CGSs. We detected the presence of CGS DNA by using polymerase chain reaction (PCR) and Sanger sequencing. We also sequenced an amplicon mixture of seven haplotype-represented samples using high-throughput sequencing. Our eDNA methods could detect the presence of CGS at moderate densities reported across its range, proving them as a cost-effective way to establish broad-scale patterns of occupancy for CGS. In addition, our primers enabled the detection of a mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.

Oceanographic Drivers of Cuvier’s (Ziphius cavirostris) and Sowerby’s (Mesoplodon bidens) Beaked Whales Acoustic Occurrence along the Irish Shelf Edge

Cuvier’s and Sowerby’s beaked whales occur year-round in western Irish waters, yet remain some of the most poorly understood cetaceans in the area. Considering the importance of the area for anthropogenic activities and the sensitivity of beaked whales to noise, understanding their ecology is essential to minimise potential overlaps. To this end, fixed bottom-mounted autonomous acoustic recorders were deployed at 10 stations over four recording periods spanning from May 2015 to November 2016. Acoustic data were collected over 1934 cumulative days, for a total of 7942 h of recordings. To model the probability of presence of Cuvier’s and Sowerby’s beaked whales in the area as a function of oceanographic predictors, we used Generalised Additive Models, fitted with Generalised Estimating Equations to deal with temporal autocorrelation. To reflect prey availability, oceanographic variables acting as proxies of primary productivity and prey aggregation processes such as upwelling events and thermal fronts were selected. Our results demonstrated that oceanographic variables significantly contributed to the occurrence of Cuvier’s and Sowerby’s beaked whales (p-values between <0.001 and <0.05). The species showed similar preferences, with the exception of sdSST. The inclusion of a parameter accounting for the recorders location confirmed the existence of a latitudinal partitioning for those species in the area. This study provides a point of comparison for future research and represents an important step towards a better understanding of those elusive species.

Environmental DNA for conservation

Detection and monitoring of wildlife species of concern is a costly and time-consuming challenge that is critical to the management of such species. Tools such as lures and traps can cause unnecessary stress or other health impacts to sensitive species. Development and refinement of tools that provide means to detect rare and elusive species without requiring contact with them reduce such impacts. Further, the potential of detection after the target species has moved on from a sampling site could allow for higher potential for detection of rare species. The ability to amplify DNA from environmental samples (e.g. water, soil, air, and other substrates) has provided a non-invasive method for detection of rare or elusive species while reducing negative impacts to wildlife. Like other non-invasive methods, such as cameras, there are methodological pitfalls associated with environmental DNA (eDNA) sampling to consider. Each study system will provide unique challenges to adequate eDNA sampling. Thus, pilot studies are critical for successful implementation of a larger-scale detection and monitoring study. This chapter will describe the benefits and challenges of using eDNA, detail types of eDNA sampling, and provide guidance on designing appropriate study design and sampling schemes. Empirical studies using eDNA applied to wildlife conservation efforts will be highlighted and discussed.

New records on the distribution and habitat of the northern naked-tailed armadillo, Cabassous centralis (Mammalia, Cingulata, Chlamyphoridae) in Costa Rica

The northern naked-tailed armadillo, Cabassous centralis, is a rare and elusive species. It ranges from southern Mexico to northern South America. It has been detected in several types of habitats, but appears to prefer Tropical and Subtropical broadleaf forests. In Costa Rica, this species is difficult to observe and there are only eight records reported in the scientific literature. To search records of this armadillo, we used camera traps in north-western Costa Rica and visited several additional localities in the centre and the Caribbean lowlands of the country. We also examined and assessed records of this species from the Global Biodiversity Information Facility (GBIF) database. We added four new locality records for C. centralis in Costa Rica, based on photos from camera traps and field observations. We found only three localities (five records) in GBIF additional to the eight reported in literature. Habitat in these new Costa Rican localities reported here varied from mature dense forest (one site) to semi-urban areas (two sites). Additionally, two individuals were detected in secondary forest patches, one of them adjacent to mature riparian forest. Given the species’ scarcity, much additional information still is required to ground protection actions in a scientific framework.

First Locality Record of Melanistic Oncilla (Leopardus tigrinus) in Monteverde, Costa Rica

The persistence of the coat color polymorphism melanism has been reported for several tropical felids, but its evolutionary advantages remain an active area of research. Few publications have explored melanism in the elusive species, oncilla (Leopardus tigrinus) within the Neotropical part of their range in Costa Rica. Herein, I present the first record of a melanistic oncilla within the montane cloud forest of Monteverde, Costa Rica. Recent studies have found support for theories (e.g. Temporal Segregation Hypothesis and Gloger’s Rule) explaining the ecological advantages driving melanism in oncilla and tropical felid populations. However, it is unclear what is driving melanism in this Monteverde oncilla population due to the singular observation. Further research investigating whether melanism is occurring at a higher frequency in other individuals in the region is critical to better understanding the occurrence of melanism in local populations of this cryptic species. The montane cloud forest in Monteverde provides critical habitat to this vulnerable species within the small Neotropical part of their range. Melanistic individuals may be particularly threatened by land-use change and increasing human pressure if theories for the evolutionary advantages and ecological conditions motivating melanism are supported.

Elusive species criteria and paradigms of botany

The development of botanical science and the improvement of methods used to identify the boundaries of species lead to constant changes in the criteria and volume of species in various systematic groups of plants. Currently, to solve these problems, modern methods of geno- and chemosystematics (for example, use of PCR, ITSS, and other markers), methods of discriminant and factor analyses, assessment of boundaries of ecological and climatic niches of different species, approaches of chemosystematics and metabolomics are used. However, the emergence of a large number of different modern approaches to estimate the volume of species has not simplified the task of plant taxonomists. Instead, it gave them the task to select or reasonably combine reliable methods. The latter would allow to progress in solving this difficult tasks and set priorities for the development of an integrated methodological approach to the study criteria and volumes of species.

Limitations of using surrogates for behaviour classification of accelerometer data: refining methods using random forest models in Caprids

Background Animal-attached devices can be used on cryptic species to measure their movement and behaviour, enabling unprecedented insights into fundamental aspects of animal ecology and behaviour. However, direct observations of subjects are often still necessary to translate biologging data accurately into meaningful behaviours. As many elusive species cannot easily be observed in the wild, captive or domestic surrogates are typically used to calibrate data from devices. However, the utility of this approach remains equivocal. Methods Here, we assess the validity of using captive conspecifics, and phylogenetically-similar domesticated counterparts (surrogate species) for calibrating behaviour classification. Tri-axial accelerometers and tri-axial magnetometers were used with behavioural observations to build random forest models to predict the behaviours. We applied these methods using captive Alpine ibex (Capra ibex) and a domestic counterpart, pygmy goats (Capra aegagrus hircus), to predict the behaviour including terrain slope for locomotion behaviours of captive Alpine ibex. Results Behavioural classification of captive Alpine ibex and domestic pygmy goats was highly accurate (> 98%). Model performance was reduced when using data split per individual, i.e., classifying behaviour of individuals not used to train models (mean ± sd = 56.1 ± 11%). Behavioural classifications using domestic counterparts, i.e., pygmy goat observations to predict ibex behaviour, however, were not sufficient to predict all behaviours of a phylogenetically similar species accurately (> 55%). Conclusions We demonstrate methods to refine the use of random forest models to classify behaviours of both captive and free-living animal species. We suggest there are two main reasons for reduced accuracy when using a domestic counterpart to predict the behaviour of a wild species in captivity; domestication leading to morphological differences and the terrain of the environment in which the animals were observed. We also identify limitations when behaviour is predicted in individuals that are not used to train models. Our results demonstrate that biologging device calibration needs to be conducted using: (i) with similar conspecifics, and (ii) in an area where they can perform behaviours on terrain that reflects that of species in the wild.

Stable Isotope Analysis of Specimens of Opportunity Reveals Ocean-Scale Site Fidelity in an Elusive Whale Species

Elusive wildlife are challenging to study, manage, or conserve, as the difficulty of obtaining specimens or conducting direct observations leads to major data deficiencies. Specimens of opportunity, such as salvaged carcasses or museum specimens, are a valuable source of fundamental biological and ecological information on data-deficient, elusive species, increasing knowledge of biodiversity, habitat and range, and population structure. Stable isotope analysis is a powerful indirect tool that can be used to infer foraging behavior and habitat use retrospectively from archived specimens. Beaked whales are a speciose group of cetaceans that are challenging to study in situ, and although Sowerby's beaked whale (Mesoplodon bidens) was discovered &gt;200 years ago, little is known about its biology. We measured δ13C and δ15N stable isotope composition in bone, muscle, and skin tissue from 102 Sowerby's beaked whale specimens of opportunity collected throughout the North Atlantic Ocean to infer movement ecology and spatial population structure. Median δ13C and δ15N values in Sowerby's beaked whale bone, muscle, and skin tissues significantly differed between whales sampled from the east and west North Atlantic Ocean. Quadratic discriminant analysis that simultaneously considered δ13C and δ15N values correctly assigned &gt;85% of the specimens to their collection region for all tissue types. These findings demonstrate Sowerby's beaked whale exhibits both short- and long-term site fidelity to the region from which the specimens were collected, suggest that this species is composed of two or more populations or exhibits a metapopulation structure, and have implications for conservation and management policy. Stable isotope analysis of specimens of opportunity proved a highly successful means of generating new spatial ecology data for this elusive species and is a method that can be effectively applied to other elusive species.

Multi-sensor biologgers and innovative training allow data collection with high conservation and welfare value in zoos

Zoos are valuable resources for research, providing scientists with access to rare and elusive species in an easy to observe environment. Animal-attached loggers (aka biologgers) offer profound insight into animal behaviour. Their use in zoos has high yet largely untapped potential to collect data relevant for wild animal research and conservation but also welfare and enrichment monitoring of the zoo animals themselves. However, affixing biologgers to study animals can be problematic in captive settings, limiting the accessibility of this technology for use on zoo species which ordinarily need to be sedated for the fitting of such devices, including large carnivores. Here we show that biologging collars and crate-training allow collection of novel datasets on captive animals with high welfare and conservation value, using endangered African wild dogs (Lycaon pictus) tagged with tri-axial accelerometer and tri-axial magnetometer loggers, as a case study. Two yearling female wild dogs were fitted with biologging collars while sedated in preparation for translocation from London to Whipsnade Zoo, with data collected for 10–26 hours until collar detachment. Two adult male wild dogs at London Zoo were trained to accept collars in a modified crate in exchange for a food reward, which allowed fitting and detaching the collars without sedation, with data collected for 28 days. First, we show how accelerometer and magnetometer data allow detection of fine-scale individual differences in the recovery from sedation as well as within- and between-individual variation in activity patterns in relation to the type of food received (tong vs. rabbit and pony carcass). Using the vectorial dynamic body acceleration metric (VeDBA), a proxy for movement-related energy expenditure, further shows that daily energy expenditure was higher on days with partial pony carcass feeds compared to rabbit feeds but varied considerably between days where flesh pieces were fed with tongs. Using the dead-reckoning method allowed reconstruction of fine-scale (1 Hz locations) movement paths within enclosures, indoors and outdoors, allowing visualisation and quantification of fine-scale movement and space use differences between individuals and over time, for example in response to different enrichment methods. Using multi-sensor biologgers, combined with training captive animals to accept collars without the use of anaesthetic, can enable flexible, experimental approaches to data collection with minimal impact on study animals, providing novel understanding of relevance for both zoo and wild animals.