High‐resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile

Abstract Easy, economic, precise species authentication is currently necessary in many areas of research and diagnosis in molecular biology applied to conservation studies of endangered species. Here, we present a new method for the identification of three fox species of the Lycalopex genus in Chile. We developed an assay based on high‐resolution melt analysis of the mitochondrial cytochrome B gene, allowing a simple, low cost, fast, and accurate species determination. To validate the assay applicability for noninvasive samples, we collected fecal samples in the Atacama Desert, finding unexpectedly one species outside of its known distribution range. We conclude that the assay has a potential to become a valuable tool for a standardized genetic monitoring of the Lycalopex species in Chile.

The methods most commonly used for studying carnivores are direct methods like sightings, traps, carcasses, and review of collections; indirect methods traditionally used are odoriferous patterns, camera traps, response to vocalizations, trace transects and excreta or fecal collection (Gese, 2004). In studies of the behavioral ecology and feeding habits of carnivores, species are generally identified by the size, form, smell, and/or diet composition of the feces (Johnson, Aldred, Clinite, & Kutilek, 1981;Major, Johnson, Davis, & Kellog, 1980); however, this method of identification may be of limited use and lead to identification errors in areas where carnivores co-occur (Bulinski & McArthur, 2000;Davison, Birks, Brookes, Braithwait, & Messenger, 2002;Farrell, Roman, & Sunquist 2000;Fernandes, Ginja, Pereira, Bruford, & Santos, 2008;Hansen & Jacobsen, 2006;Onorato, White, Zager, & Waits, 2006;Wittwer, 2009). Identification of hairs in the feces, ingested during self-cleaning, has also been used; however, patterns may change in different carnivores, preventing the standardized use of this technique (Harrison, 2002;Onorato et al., 2006). The use of molecular methods, such as the bar code of mitochondrial DNA (mtDNA) and the genotyping of nuclear DNA, has made it possible to reduce errors in the identification of species, as well as to resolve phylogenetic relationships by revealing distribution patterns of wild canids. (Ochoa, 2011;Tchaicka et al., 2016;Torés, 2007) to the more recent use of barcode regions of cytochrome B (Behrens-Chapuis et al., 2018;Fernandes, Costa, Oliveira, & Mafra, 2017;Jeon, Anderson, Won, Lim, & Suk, 2017;Wang et al., 2016;Yacoub, Fathi, & Mahmoud, 2013).
A noninvasive way of obtaining mtDNA is through fecal collection, since this does not imply capturing animals to gain, blood or other tissues as DNA sources (Torés, 2007).
High-resolution melting (HRM) is a sensitive genotyping method, based on the thermal denaturation characteristics of the amplicons.
This method has higher performance information, never achieved by classical DNA melting curve analysis. HRM is performed using a fluorescent, double-stranded DNA dye that can be applied in fully saturating conditions (Wittwer, 2009). The amplicon is analyzed by gradual denaturation through increasing temperature and decreased fluorescence caused by the release of intercalating dye from the DNA. The melting temperature (T m ) and specific shape of the melting curve result from the DNA sequence, GC content, and amplicon length, (Vossen, Aten, Roos, & Dunnen, 2009), so it is a useful technique for obtaining species-specific genotypes. Due to the increased demand for rapid, economic, easy, high-throughput genotyping analyses, there has been a considerable focus on HRM, which can detect sequence variants without the use of sequencing or hybridization procedures (Reed & Wittwer, 2004;Tindall, Petersen, Woodbridge, Schipany, & Hayes, 2009 to the geomorphological characteristics of the stream and then from the prospective terrain to the study area to identify potential habitats such as trails, caves, and bushes used by foxes. Twentyseven sampling stations were selected in scrub, rocky, and sandy environments, which were georeferenced and where camera traps were installed to constantly monitor places and target species.
With this information, a map where each sampling point was plotted. For the collection of samples in the field, the feces were distinguished from those of other carnivores by their characteristic shape, excluding the white or dry appearance, to ensure to collect feces from foxes.

| References samples
Fresh frozen skin tissues from each of the taxonomically determined Chile-inhabiting species L. culpaeus, L. fulvipes, and L. griseus were used to obtain reference DNA for the PCR end-time and Real-Time reactions with HRM.

| Unknown samples from feces
DNA was extracted from 200 fecal samples, with the Isolate Fecal DNA kit (Bioline), following the manufacturer's instructions. As input material we used an amount of 150 mg taken from initial sample of the external contour of the feces. The DNA concentration was estimated by standard fluorometric methods, using the Qubit 2.0 (Thermo Fisher Scientific) following the manufacturer's instructions. The integrity of the extracted DNA was checked by agarose gel electrophoresis in 0.8% agarose gel. The concentration of the DNA samples was normalized to 25 ng/µl to dilute potentially present PCR-inhibitors and stored at −20ºC for further use.

| End point PCR standardization of the cytochrome B gene
DNA amplification using end point PCR was performed using the Applied Biosystems™ SimpliAmp™, 0.2 µM forward primer, 0.2 µM reverse primer, and 1 µl of 25 ng/µl DNA. The primer pair used for the cytochrome B gene was: LC CYTB-F: 5′TTCCAGCACCATCCAATATTTCCGC 3′ and LC CYTB-R: 5′GGCGCCGTTTGCATGTATGTAACG 3′. (Quinga, 2012), using an initial denaturing step at 96°C for 4 min followed by 35 cycles of 96°C for 30 s, 66°C for 30 s, and 72°C for 30 s. The amplicons were electrophoresed in agarose gel 2% TAE 1X and stained with GelRed (Biotium). These primers amplify a region of the mitochondrial cytochrome B gene. For general specificity validation, we performed a primer BLAST search (Ye et al., 2012) confirming that the primers are specific for Lycalopex as indicated in Quinga (2012). Amplification tests were performed by end point PCR to standardize the reaction conditions. It is optimized according to the size of the amplicon.

| Sequence analysis
PCR products were directly sequenced in two directions for each product using a BigDye Terminator V3.1 Cycle Kit in an automated Genetic Analyzer 3500 xl (Applied Biosystems, HITACHI), with software Data Collection v3. Sequences were aligned and proofread using the software MEGA 5 and submitted to GenBank.

| End point PCR standardization of the cytochrome B gene
Of the 200 DNA samples extracted, 150 were successfully amplified. These 150 samples were used for the HRM analysis. The samples analyzed were amplified approximately to a 200-bp product, as previously reported for the cytochrome B gene (Yacoub et al., 2013) (Quinga, 2012). (data not shown).

| Species determination of reference and unknown samples using HRM analysis of cytochrome B gene
Melting curves (Figure 1a

| D ISCUSS I ON
Significant improvements have been reported based on fecal DNA analysis for both extraction and quantification of nucleic acids as part of genotyping and species identification (Kanthaswamy, Premasuthan, Ng, Satkoski, & Goyal, 2012). Molecular analysis of feces is a noninvasive method with a high degree of integrity and quality when using specific commercial kits for these complex matrices (Deshpande, Villarreal, & Mills, 2016;Ramón-Laca, Soriano, Gleeson, & Godoy, 2015;Tende, Hansson, Ottosson, & Bensch, 2014). The main challenge of this study was to obtain DNA of good quality from fecal samples collected in the Atacama Desert. The DNA samples obtained from these matrices were used to obtain information about the presence and distribution of Lycalopex species in the Atacama Desert, which is of great importance for promoting and orienting conservation measures.
Feces contain epithelial cells from the intestine, mucus, and hairs.
DNA can be extracted from this material and amplified by PCR.
Although there are previous studies that have used mtDNA from feces (Chaves, Graeff, Lion, Oliveira, & Eizirik, 2012;Quinga, 2012;Ray & Sunquist, 2001;Taberlet, Waits, & Luikart, 1999), there are no reports of the use of mtDNA to analyze the presence and distribution of Lycalopex species in Chile and South America. We developed an assay based on HRM analysis of the mitochondrial cytochrome B gene, used for identifying genetic variations in nucleic acid sequences.
We also describe the HRM method for genotyping Lycalopex species, which shows the potential of the mitochondrial cytochrome B gene DNA barcode coupled with the HRM method to provide a fast and very accurate method of taxonomic identification and species identification.
We were able to identify all the three species of Lycalopex foxes inhabiting Chile using the developed assay; this is the first report of the presence of L. fulvipes foxes in northern Chile. The Chilean species of this genus are in different conservation categories. There are no reports on hybridization between species of the Lycalopex genus, only observations and stories of local people in the field, which refer to the possible hybridization without viability of the offspring.
The melting curves obtained from amplified fecal DNA samples are grouped or positioned with respect to the curves obtained from the control reference samples of the taxonomically determined species (three species of the Lycalopex genus that live in Chile). We can also add that our evidence indicates that an approximate proportion of 60% was obtained for L. griseus and approximately 40% for L. culpeus. (Medel & Jaksic, 1988).
Surprisingly, we could detect one L. fulvipes sample, which was not expected to be present in our study area. This is the first report of the presence of foxes of L. fulvipes in northern Chile. Therefore, it is important to determine their presence and distribution in different ecosystems throughout the country. Although the presence of L. fulvipes may be less frequent in these latitudes, the deepening of monitoring of Lycalopex species in northern Chile is very important because due to possible unknown distribution ranges of the Lycalopex species. A possible cause for migrations into new habitats could be a response to climate change or events such as forest fires, increasingly common in southern Chile and specifically in the ecosystems where L. fulvipes lives.
There are no previous reports of the use of mtDNA coupled to HRM analysis to establish the presence and distribution of Lycalopex species in Chile and South America. This work detects the bases for further studies of the distribution of these species in Chile, to understand their movement flows in different geographical areas and their adaptation to diverse environments and ecosystems.

ACK N OWLED G M ENTS
We thank the consultant Tierra del Sol for the supply of the samples and the Vice-Rector for Research and postgraduate studies at the Catholic University of Temuco for funding this work.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.