Using non – invasive genetic techniques to assist in maned wolf conservation in a remnant fragment of the Brazilian Cerrado

Using non–invasive genetic techniques to assist in maned wolf conservation in a remnant fragment of the Brazilian Cerrado. The maned wolf is a South American canid considered a keystone species of the Cerrado. We performed a genetic assessment of maned wolves that inhabit a small remnant fragment of the Cerrado in Brazil. We collected 84 fecal samples over a year and also included two tissue samples from road–killed animals. We successfully identified the species, sex, and individuals using molecular markers. Using microsatellite loci analysis we identified 13 different individuals, eight females and five males. The genetic variability level found and the high number of individuals detected indicates the presence of an open population.


Introduction
The maned wolf is the keystone canid species of the Brazilian Cerrado, the largest and richest tropical savanna in the world, and one of the most threatened biomes in South America (Klink and Machado, 2005).The IUCN listed it as Near Threatened because of the drastic reduction of suitable habitats for their survival.In Brazil it is categorized as Endangered (De Paula et al., 2008).The Cerrado is considered 'the central range of this species', and has lost approximately 50 % of its native vegetation (Klink and Machado, 2005).
In this scenario, fragmented and isolated protected areas are too small for their long-term viability (Do Passo Ramalho et al., 2014;Lion et al., 2011).It is therefore critical to understand the amount of movement in and out of these areas.However, only two studies of maned wolves have been carried out to monitor population size and genetic diversity at a local scale in these protected areas (Do Passo Ramalho et al., 2014;Lion et al., 2011).Do Passo Ramalho et al. ( 2014) analyzed a maned wolf population in Jataí Ecological Station (São Paulo).The geographic distance between Jataí and our study area, the Estação Ecológica de Itirapina (EEI), is about 180 km.Habitat loss and fragmentation could lead to a rapid and dramatic decline if maned wolves are unable to disperse from one Cerrado patch to another.
Our aim was to assess the population genetic diversity of maned wolves in the EEI, an area that contains a remnant fragment of the Cerrado biome in São Paulo (Brazil) near other protected areas where maned wolf population status have previously been assessed.Our results provide valuable information to evaluate the population dynamics of maned wolves at a fine, local scale.
We conducted monthly, six-day sampling surveys using the linear transect method.Each transect was 3 m wide and 200 m long.From March 2007 to February 2008, we collected 84 feces samples.Samples were stored in 50 ml sterile plastic tubes with 100 % ethanol and deposited in a freezer at -20 ºC until DNA extraction.Two tissue samples were collected from road-killed animals of opposite sexes.
DNA extractions and PCR amplifications were conducted under sterile conditions with negative controls in Núcleo de Pesquisa e Conservação de Cervídeos (UNESP, Brazil).We used DNA Stool Mini Kit (Qiagen Inc.Valencia-California), following the manufacturer's instructions.DNA extractions from tissues were conducted following González et al. (2015a) procedures.
For species identification, we amplified a speciesspecific D-loop fragment of mitochondrial DNA (mtD-NA) using the protocols of González et al. (2015b).The PCR products were purified using Zymo Research DNA Clean and Concentrator TM and sequenced in Ma-crogen and Institute Pasteur-Montevideo.Sequences were compared with the GenBank database using BLAST.We used MEGA 5 software to construct the alignment by Clustal X (Tamura et al., 2011).
Sex was determined through Real Time-PCR (RT-PCR) amplification of a fragment of the ZFX-ZFY genes, and High Resolution Melting Analysis (HRMA) (González et al., 2015a).Experiments were conducted in duplicate in a Rotor Gene 6000® (Corbett Research) (software version 1.7, Qiagen, UK).
We used twelve microsatellite loci that were previously established to reliably amplify in maned wolves and had sufficient levels of polymorphism and adequate non-exclusion probabilities of identity to assign parent pairs (Mannise et al., 2017).Multiplex PCR reactions and thermal profile were conducted as suggested by Mannise et al. (2017).PCR products were run using the LIZ 500 size standard on an ABI PRISM 3100 sequencer.We analyzed fragment size and genotypes in GENMARKER V1.75 (SoftGenetics).PCRs were repeated three and two times for homozygotes and heterozygotes, respectively.
We identified capture histories and the number of maned wolves resampled from multiple scats using GENECAP software (Wilberg and Dreher, 2004).We detected matching genotypes with a probability of 0.01, assuming that individuals could be siblings (Wilberg and Dreher, 2004).The probability of identity (P(id)) was calculated for Hardy-Weinberg equilibrium (HWE) and for sibling presence.
Genotyping errors were estimated with MICRO-CHECKER (Van Oosterhout et al., 2004); genotypes were corrected following Brooksfield´s (1996) estimation if genotyping errors were detected.We used GENEPOP (Raymond and Rousset 1995) to compute HWE, linkage disequilibrium (LD) and inbreeding coefficients (Fis).In case of disequilibrium, Bonferroni corrections were applied.We calculated number of alleles, expected and observed heterozygosity and allele frequencies using CERVUS (Marshall, 1998(Marshall, -2007)).We applied the ML-RELATE software to analyze the relatedness coefficients and pedigree relationships among individuals (Kalinowski et al., 2006).

Results
We identified 58 fecal samples as being from maned wolves and we determined the presence of three Dloop haplotypes previously described in other maned wolf populations from Brazil (table 1) (González et al., 2015b).Of the twelve microsatellite loci tested, one (AHTK253) showed low amplification success.Samples with at least six reliable genotypes after replicates were included in further analyses.
We assigned eight females and five males based on the melting curve pattern (table 1).The mean melting curve was 82.95 ºC (SD = 0.22) for ZFX, and 82.08 ºC (SD = 0.17) for ZFY.The observed sex ratio was not significantly different from the expected 1:1 (x 2 = 0.69, p = 0.41).
The presence of null alleles in FH2561 has been previously detected in other maned wolf populations, for this reason we recommend that this locus should not be used in future studies.
We identified a minimum of three family groups (table 1).One comprised a mother with her son, two aunts and an uncle (table 1).Another included two full-siblings (brother and sister), and the third had two half-sisters (table 1).

Discussion
Our non-invasive genetic protocols allowed us to reliably identify species, gender, and individuals.We successfully amplified fecal DNA using microsatellite multiplex protocols to effectively genotype 67 % of the samples.Maned wolves in the EEI had high levels of polymorphism and similar levels of observed and expected heterozygosity (table 2).
We found that maned wolves in the EEI have moderate levels of mitochondrial genetic diversity and high levels of nuclear genetic diversity.Mean levels of heterozygosity (0.646) were slightly lower than other population heterozygosity estimates of maned wolf populations in Brazil (0.71 and 0.72) (Do Passo Ramalho et al., 2014;Lion et al., 2011).
The presence of three family groups in the EEI area during our surveys suggests that if dispersal in and out of this area were restricted, it would result in inbreeding and loss of genetic variability.In strong support of a recent genetic analysis conducted in maned wolves throughout their range (Mannise et al., 2017), our results here suggest that wolves maintain high levels of genetic diversity and may avoid inbreeding by dispersing through cultivated lands.Furthermore, these high levels of genetic variability coupled with the large number of individuals detected indicate the presence of an open population in the EEI.Effective conservation management strategies can benefit from approximate estimates of population size and non-invasive genetic studies such as this one (Kohn et al., 1999).
Our study provides valuable information regarding the genetic diversity of an imperiled canid species in a human-dominated landscape.It suggests that this small fragmented remnant of Cerrado can maintain several maned wolf family groups with adequate levels of genetic variability, and it highlights the essential need to protect the Cerrado habitat for their conservation.Future studies that include non-invasive   1 mi surveys outside these protected areas will be crucial to evaluate movement and corridor connectivity within this fragmented landscape.

Fig. 1 .
Fig. 1.Samples collected in the EEI.Each symbol represents a different individual; same shape represents re-sampled individuals.Road-killed animals and its re-sampled feces are shown by balloons.