Noninvasive genetic sampling of endangered muriqui (Primates, Atelidae): efficiency of fecal DNA extraction

Abstract

The muriqui (Brachyteles) is one of the most endangered primates in the world, however little is known about the viability of the remaining populations. We evaluated the technique of extracting DNA from wild muriqui feces for PCR applications. In order to determine the effect of the DNA in subsequent amplifications, we analyzed five different extracts. The importance of the recommended BSA and the HotStarTaq DNA polymerase was tested. The minimal conditions to successfully amplify highly degraded fecal DNA were determined, showing that the recommended reagents are not required. We envision that this method may be useful in further conservation management studies.Keywords: Brachyteles, conservation genetics, endangered species, fecal DNA, noninvasive sampling. The genus Brachyteles (muriqui) represents the largest neotropical nonhuman primate and comprises two endemic species occurring in the Brazilian Atlantic Rainforest. The species B. hypoxanthus (Kuhl, 1820) or northern muriqui can be found in the States of Bahia, Minas Gerais and Espírito Santo, and B. arachnoides (É. Geoffroy, 1806) or southern muriqui is distributed along over the States of Rio de Janeiro, São Paulo and Paraná (Aguirre, 1971;Lemos de Sá et al., 1990, Lemos de Sá et al., 1993Martuscelli et al., 1994). Their small population size and the deforestation of the Atlantic Forest have led to the classification of muriqui as an "endangered" primate since 1982, and as "critically endangered" since 2000 (Rylands et al., 2003). B. hypoxanthus is also listed as one of the 25 most endangered primates of the world since the year 2000 (Mittermeier et al., 2005). Historically, the muriqui species roamed throughout the Atlantic Brazilian Rainforest, but now it is estimated that there are no more than 1200 individuals living in a few dozen remaining forest fragments Fonseca, 1996/1997). Thus, questions about the consequences of habitat fragmentation in the genetic structure of populations, gene flow and probability of extinction are frequently addressed with regard to muriqui (Leigh and Jungers, 1994;Strier, 1995;Strier, 2000). DNA assessment is critical for investigating these questions, and genotyping for molecular markers like microsatellites is essential to conduct more realistic population viability analyses (PVAs), improving the currently available data (Strier, 1995).Until recently, molecular genetic analyses of primates have been limited by the availability of blood or tissue samples for DNA extraction (Surridge et al., 2002). Despite the fact that noninvasive DNA sampling usually yields low quantities of DNA (Taberlet et al., 1996;Taberlet et al., 1997;Constable et al., 2001), the advent of the polymerase chain reaction (PCR) technique has been successfully used to assess the genetic composition of social groups and populations, and to evaluate both species and genealogical relationships based on such small samples (Höss et al., 1992;Morin et al., 1994;Constable et al., 1995;Gerloff et al., 1995;Taberlet et al., 1996;Reed et al., 1997;Cons...

Improving PCR detection of prey in molecular diet studies: importance of group‐specific primer set selection and extraction protocol performances

“…The QIAamp ® DNA stool mini kit (QIAGEN), hereafter EP1, was specifically designed to extract DNA from faecal samples with Proteinase K (PK) and currently remains the most widely used commercial kit in molecular studies conducted on faecal or stomach contents (Eggert et al 2005;Chaves et al 2006;Zhang et al 2006;Oehm et al 2011). The main advantage of this kit is the tablet called 'inhibitEX' used to absorb and pellet PCR inhibitors and DNA-damaging substances in the same step.…”

A PCR‐RFLP assay for gender assignment in the three‐toed sloths (Bradypus, Pilosa, Bradypodidae)

“…Current sex determination methods of sloths, such as direct inspection of the genitals (Lara-Ruiz & Chiarello 2005) and karyotyping (Pinder 1993) have proven unmanageable especially because they often rely on captures and extraction of blood samples, which are also stressful procedures for the animals. By contrast, molecular sexing holds a potential to overcome these drawbacks, particularly after the advent of noninvasive methods for obtaining DNA from sources such as hairs and faeces (Fernando & Melnick 2001;Shaw et al 2003;Morin et al 2005;Chaves et al 2006). In this study, we standardized a reliable and easy method for molecular sex determination of Bradypus sloths using Restriction Fragment Length Polymorphisms after Polymerase Chain Reaction (PCR-RFLP) of the X-(ZFX) and Y-linked (ZFY) genes encoding a zinc finger protein.…”

Genetic structure in two northern muriqui populations (Brachyteles hypoxanthus, Primates, Atelidae) as inferred from fecal DNA

“…Standardization of a non-invasive method for DNA extraction from muriqui feces is an important tool for assessing the conservation status and behavioral ecology of these monkeys (Chaves et al, 2006). Furthermore, data on the levels of genetic variability and differentiation in muriqui populations as well as pedigree reconstruction and information regarding the relatedness between individuals and the extent of inbreeding can contribute to the planning of effective conservation strategies for these species.…”

“…Fecal samples from 126 free-ranging northern muriquis (Brachyteles hypoxanthus Kuhl 1820) were collected immediately after defecation and stored at 4°C or room temperature (~24-28°C) in 50 mL polypropylene vials containing a layer, about 1 mm to 4 mm deep, of desiccated silica beads to dehydrate the feces (Chaves et al 2006). Each individual sampled was identified by its natural markings by the experienced field researchers who collected the fecal samples within the ambit of a collecting license (number 363/2001) issued by the Brazilian National Environmental Agency (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis -IBAMA).…”

“…Analysis of the human HVRI restriction sites excluded cross-contamination of muriqui PCRs with human DNA (Figure 2). The PCR was carried out in a final volume of 50 μL of 1X Taq buffer containing 3 mM MgCl 2 , 0.4 mM of each dNTP, 0.4 μM of each primer, 2.5 units of Platinum Taq DNA polymerase (Invitrogen) and 50 ng of DNA (Chaves et al, 2006). Amplifications were conducted at 92°C for 5 min, followed by 35-37 cycles of 92°C for 60 s, 47°C to 52°C for 30 s to 60 s and 72°C for 30 s to 60s, with a final extension at 72°C for 5 min.…”