Non-invasive genetic sampling reveals a habitat use extension of Chrysocyon brachyurus and Leopardus guttulus inside a protected area of Southeastern Brazil / Amostragem genética não-invasiva revela uma extensão do uso de habitat por Chrysocyon brachyurus e Leopardus guttulus em uma área protegida do Sudeste do Brasil

Abstract Anthropogenic activities have modified landscapes leading to environmental damages and to a threatened biodiversity. As a result, protected areas have become the last refuge for many species. Protected areas surrounded by a highly modified landscape may accumulate species, especially large mammals, which may alter their habitual habitat use. Here we used non-invasive DNA analysis, molecular species identification, and landscape analyses to assess the habitat use of carnivore species in an isolated protected area, Itatiaia National Park (PNI). Two species were by far the most sampled within PNI, Chrysocyon brachyurus and Leopardus guttulus. The spatial distribution of each species was assessed to the following landscape variables: altitude; land use/land cover; slope and Euclidean distances from water and from urban buildings. The habitat use of C. brachyurus and L. guttulus was related to altitude and land use/land cover. We tested whether there were differences in the environmental indicators considering both species, which showed that higher altitudes, forest and/or grassland formation were indeed associated with them. We highlighted the unprecedented presence of both species at altitudes up to 2,631 meters. Our results suggest a habitat use extension for both species inside the PNI that could be a consequence of the highly modified landscape where PNI is inserted. Therefore, the results can be helpful for better understanding the species dynamics and their conservation in the face of landscape changes. Further, this study may be of help for management and conservation policies of this emblematic protected area.

Resumo As atividades antropogênicas modificaram as paisagens levando a danos ambientais e a uma biodiversidade ameaçada. Como resultado, as áreas protegidas se tornaram o último refúgio para muitas espécies. Áreas protegidas cercadas por uma paisagem altamente modificada podem acumular espécies, especialmente grandes mamíferos, o que pode alterar o uso de seu habitat habitual. Neste trabalho, usamos análise de DNA não-invasivo, identificação molecular de espécies e análises de paisagem para avaliar o uso de habitat de carnívoros em uma área protegida isolada, o Parque Nacional de Itatiaia (PNI). Duas espécies foram de longe as mais amostradas dentro do PNI, Chrysocyon brachyurus e Leopardus guttulus. A distribuição espacial de cada espécie foi avaliada com as seguintes variáveis paisagísticas: altitude; uso e cobertura do solo; declividade e distâncias euclidianas da água e de construções urbanas. O uso de C. brachyurus e L. guttulus no habitat estava relacionado à altitude e ao uso e cobertura do solo. Testamos se existiam diferenças nos indicadores ambientais considerando ambas as espécies, o que mostrou que altitudes mais elevadas, formação de florestas e/ou campestres estavam de fato associadas a elas. Destacamos a presença incomum de ambas as espécies em altitudes de até 2.631 metros. Nossos resultados sugerem uma extensão do uso do habitat conhecido para ambas as espécies como possível consequência da paisagem altamente modificada onde o PNI está inserido. Portanto, os resultados podem ser úteis para entender melhor a dinâmica das espécies e sua conservação diante das mudanças na paisagem. Além disso, este estudo pode ser útil para as políticas de manejo e conservação desta emblemática área protegida.

Bonobos' saliva remaining on the pith of terrestrial herbaceous vegetation can serve as non-invasive wild genetic resources.

Evaluating the genetic diversity of natural populations of endangered species is important for conservation. Although the genetic analysis of wildlife usually requires collecting DNA non-invasively, the variety of non-invasive DNA sampling methods is limited for each species. We present a method to obtain DNA of an endangered species, the bonobo (Pan paniscus), in which the pith of the terrestrial herbaceous vegetation (THV) that they consumed was newly utilized. We investigated the (1) frequency of encountering remnant saliva on three types of THV pith; (2) concentrations of DNA in the saliva samples by the real-time quantitative PCR; and (3) rates of positive PCR, accurate genotyping, and allelic drop out by analyzing two autosomal microsatellite loci (D7s817 and D9s910). The number of remnant saliva samples was recorded by following the bonobo groups on a daily basis. The frequency of encountering DNA samples was higher in saliva samples than in fecal samples. More than half of the saliva samples remaining on two types of THV pith provided sufficient concentrations of bonobo DNA (> 200 pg/µl). Rates of positive PCR and accurate genotyping were high, and allelic drop out rate was low when the amount of template DNA was above 200 pg per reaction. Our results suggest that the remnants of bonobo saliva on the pith of THV are a potential resource for obtaining DNA, and better than other kinds of samples from the perspective of the abundant sampling opportunities.

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species.

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non-invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore-using species, such as myriapods, arachnids and insects.

Mitochondrial DNA analyses revealed low genetic diversity in the endangered Indian wild ass Equus hemionus khur.

The Indian wild ass Equus hemionus khur, belonging to ass-like equid branch, inhabits the dry and arid desert of the Little Rann of Kutch, Gujarat. The E. h. khur is the sole survivor of Asiatic wild ass species/subspecies in South Asia. To provide first ever insights into the genetic diversity, phylogeny, and demography of the endangered Indian wild ass, we sampled 52 free-ranging individuals from the Little Rann of Kutch by using a non-invasive methodology. The sequencing of 230 bp in cytochrome b (Cyt b) and displacement loop (D-loop) region revealed that current ∼4000 extant population of Indian wild ass harbours low genetic diversity. Phylogenetic analyses confirmed that E. h. khur, E. h. onager, and E. h. kulan belong to a single strict monophyletic clade. Therefore, we suggest the delimitation of the five E. hemionus subspecies in vogue to a single species E. hemionus. The application of molecular clock confirmed that the Asiatic wild ass had undergone diversification 0.65 Million years ago. Demographic measurements assessed using a Bayesian skyline plot demonstrated decline in the maternal effective population size of the Indian wild ass during different periods; these periods coincided with the origin and rise of the Indus civilization in the northwest of the Indian subcontinent during the Neolithic. In conclusion, maintaining high genetic diversity in the existing isolated population of 4000 Indian wild asses inhabiting the wild ass sanctuary is important compared with subspecies preservation alone.

Second-generation non-invasive high-throughput DNA sequencing technology in the screening of Down's syndrome in advanced maternal age women.

The aim of the present study was to evaluate the efficacy of using non-invasive DNA testing technology in screening Down's syndrome among women of advanced maternal age (AMA) and to provide evidence for prenatal screening of Down's syndrome. With a double-blind design, 8 ml of peripheral venous blood samples were collected from 87 women aged ≥35 years after 12 weeks of pregnancy. All cases were recorded with unique identification cards with clinical details and followed up until delivery. All the non-invasive prenatal testing results were confirmed by amniotic fluid fetal karyotyping (the gold standard of aneuploidy test), follow-up examination by neonatologists or neonatal blood karyotyping. The sensitivity, specificity and other indicators of non-invasive DNA testing technology were calculated based on the data of 87 women of AMA. Among the 87 women of AMA, 5 were cases with abnormal numbers of chromosomes (3 cases of trisomy 21, 1 case of trisomy 18 and 1 case of 47, XXX). The sensitivity and specificity reached 100% for trisomy 21, trisomy 18 and 47, XXX. The present study supports that non-invasive DNA testing is a useful method of AMA screening of Down's syndrome with 100% accuracy. Therefore, it can be used as an important alternative screening method for Down's syndrome in women of AMA.