Development and testing of an optimized method for DNA-based identification of jaguar (Panthera onca) and puma (Puma concolor) faecal samples for use in ecological and genetic studies

Article (PDF Available)inGenetica 136(3):505-512 · July 2009with290 Reads
DOI: 10.1007/s10709-008-9347-6
Abstract
The elusive nature and endangered status of most carnivore species imply that efficient approaches for their non-invasive sampling are required to allow for genetic and ecological studies. Faecal samples are a major potential source of information, and reliable approaches are needed to foster their application in this field, particularly in areas where few studies have been conducted. A major obstacle to the reliable use of faecal samples is their uncertain species-level identification in the field, an issue that can be addressed with DNA-based assays. In this study we describe a sequence-based approach that efficiently distinguishes jaguar versus puma scats, and that presents several desirable properties: (1) considerably high amplification and sequencing rates; (2) multiple diagnostic sites reliably differentiating the two focal species; (3) high information content that allows for future application in other carnivores; (4) no evidence of amplification of prey DNA; and (5) no evidence of amplification of a nuclear mitochondrial DNA insertion known to occur in the jaguar. We demonstrate the reliability and usefulness of this approach by evaluating 55 field-collected samples from four locations in the highly fragmented Atlantic Forest biome of Brazil and Argentina, and document the presence of one or both of these endangered felids in each of these areas.

Figures

Development and testing of an optimized method for DNA-based
identification of jaguar (Panthera onca) and puma
(Puma concolor) faecal samples for use in ecological
and genetic studies
Taiana Haag Æ Anelisie S. Santos Æ Carlos De Angelo Æ
Ana Carolina Srbek-Araujo Æ De
ˆ
nis A. Sana Æ
Ronaldo G. Morato Æ Francisco M. Salzano Æ Eduardo Eizirik
Received: 27 June 2008 / Accepted: 11 December 2008 / Published online: 10 January 2009
Ó Springer Science+Business Media B.V. 2009
Abstract The elusive nature and endangered status of
most carnivore species imply that efficient approaches for
their non-invasive sampling are required to allow for
genetic and ecological studies. Faecal samples are a major
potential source of information, and reliable approaches are
needed to foster their application in this field, particularly
in areas where few studies have been conducted. A major
obstacle to the reliable use of faecal samples is their
uncertain species-level identification in the field, an issue
that can be addressed with DNA-based assays. In this study
we describe a sequence-based approach that efficiently
distinguishes jaguar versus puma scats, and that presents
several desirable properties: (1) considerably high ampli-
fication and sequencing rates; (2) multiple diagnostic sites
reliably differentiating the two focal species; (3) high
information content that allows for future application in
other carnivores; (4) no evidence of amplification of prey
DNA; and (5) no evidence of amplification of a nuclear
mitochondrial DNA insertion known to occur in the jaguar.
We demonstrate the reliability and usefulness of this
approach by evaluating 55 field-collected samples from
four locations in the highly fragmented Atlantic Forest
biome of Brazil and Argentina, and document the presence
of one or both of these endangered felids in each of these
areas.
Keywords Faecal DNA Mitochondrial DNA
Panthera onca Puma concolor Species identification
Introduction
The jaguar (Panthera onca) and puma (Puma concolor) are
the only large felids currently present in the Neotropics.
Both species are now threatened by habitat loss and frag-
mentation, along with direct persecution by ranchers due to
conflict over livestock depredation (Nowell and Jackson
1996). Resilience to human disturbance varies between
them, with the jaguar being considerably more sensitive to
anthropogenic threats (Polisar 2002; Novack 2003; Silveira
2004). To maintain viable populations of these felids,
urgent conservation efforts are needed in several areas, and
T. Haag
Programa de Po
´
s-Graduac¸a
˜
o em Gene
´
tica e Biologia Molecular,
Universidade Federal do Rio Grande do Sul,
Porto Alegre, Rio Grande do Sul, Brazil
T. Haag A. S. Santos E. Eizirik (&)
Laborato
´
rio de Biologia Geno
ˆ
mica e Molecular, Faculdade de
Biocie
ˆ
ncias, Pontifı
´
cia Universidade Cato
´
lica do Rio Grande do
Sul, Av. Ipiranga 6681, CEP 90619-900 Porto Alegre,
Rio Grande do Sul, Brazil
e-mail: eduardo.eizirik@pucrs.br
C. De Angelo
National Research Council of Argentina (CONICET)
and Asociacio
´
n Civil Centro de Investigaciones del Bosque
Atla
´
ntico (CeIBA), Buenos Aires, Argentina
A. C. Srbek-Araujo
Instituto Ambiental Vale, Linhares, Vitoria, Espı
´
rito Santo,
Brazil
D. A. Sana R. G. Morato E. Eizirik
Instituto Pro
´
-Carnı
´
voros, Atibaia, Sa
˜
o Paulo, Brazil
R. G. Morato
CENAP/ICMBio, Atibaia, Sa
˜
o Paulo, Brazil
F. M. Salzano
Departamento de Gene
´
tica, Universidade Federal do Rio Grande
do Sul, Porto Alegre, Rio Grande do Sul, Brazil
E. Eizirik
Laboratory of Genomic Diversity, NCI, Frederick, MD, USA
123
Genetica (2009) 136:505–512
DOI 10.1007/s10709-008-9347-6
a first step towards this goal is the establishment of reliable
methods to assess the presence and abundance of each of
these species in different areas, so as to allow the adequate
design of ecological, behavioral and genetic studies.
A major impediment to the development of such efforts
is the difficulty and cost of obtaining direct information on
these animals throughout their geographic range, given
their low density and elusive behavior (Johnson et al.
2001). As a consequence, intensive studies such as those
based on capture and radio-telemetry data are expensive
and restricted to some focal areas, which in most cases still
lack long-term monitoring of populations. In recent years,
technical and analytical advances in approaches such as
camera trapping have allowed a substantial increase in the
number of studies investigating the presence of these spe-
cies. In spite of the relevance of this approach, it still lacks
the ability to provide biological samples of the identified
individuals, or to provide information on important eco-
logical and behavioral aspects such as diet, hormonal levels
or interactions with pathogens. Biological samples are also
critical for the development of genetic studies, which have
the potential to illuminate issues such as loss of allelic
diversity in fragmented populations, evolutionary history
of demographic units, social interactions among individu-
als and patterns of territoriality and dispersal. In this
context, recent advances in molecular biology have per-
mitted the use of noninvasive samples (e.g. scats, hairs) as
a reliable source of DNA, allowing genetic studies of free-
ranging animals to be performed without having to capture
or even observe them (Taberlet et al. 1999).
Analyses based on faecal DNA are now widespread, and
have been applied to a broad array of taxa to address a
variety of questions (Reed et al. 1997; Wasser et al. 1997;
Kohn et al. 1999; Sloane et al. 2000; Parsons 2001; Palo-
mares et al. 2002; Adams et al. 2003; Ernest et al. 2003;
Wan et al. 2003; Pilgrim et al. 2005; Bergl and Vigilant
2007). Once scats are collected in the field, a first step for
their use in genetic or ecological studies is their identifi-
cation at species level. Morphology-based criteria have
been shown to often be unreliable (Farrell et al. 2000;
Davison et al. 2002; Reed et al. 2004), leading to a growing
concern regarding the development of rigorous approaches
for species-level assignment of field-collected scats. This is
particularly relevant when the focal species for a field study
occurs in sympatry with related taxa, whose scat size,
morphology and scent may be quite similar. In such cases,
molecular methods based on DNA sequences, nuclear
VNTRs, PCR-RFLP or haplotype-specific Polymerase
chain reaction (PCR) have been shown to successfully
identify carnivore species, leading to a reliable alternative
to traditional means of identification (Farrell et al. 2000;
Davison et al. 2002; Palomares et al. 2002; Bhagavatula
and Singh 2006; Lucentini et al. 2007; Pilot et al. 2007).
In the specific case of jaguar and puma, these felids are
sympatric over almost all the range of the former species. It
is thus unlikely that field studies focusing on jaguars will
be carried out in areas devoid of pumas, so that both spe-
cies will probably be sampled in most scat collection
efforts. This issue is compounded by the established
knowledge that it is difficult to distinguish jaguar and puma
scats on the basis of their morphological features (Emmons
1987; Farrell et al. 2000; Chame 2003), rendering the
problem of species-level identification a critical impedi-
ment for reliable field studies on these felids. It is thus very
important to devote attention to the development of DNA-
based assays that reliably discriminate these species, and
that also present other desirable features such as high PCR
success rate and no co-amplification of prey DNA.
Although several assays based on mitochondrial DNA
(mtDNA) data have so far been applied to carnivore scat
identification (Farrell et al. 2000; Novack et al. 2005;
Bhagavatula and Singh 2006; Weckel et al. 2006; Lucen-
tini et al. 2007; Miotto et al. 2007), very few studies have
included Neotropical species, and puma and jaguar in
particular. Farrell et al. (2000) developed primers targeting
the mtDNA cytochrome b gene (cyt b) to identify carnivore
scats in the context of an ecological study in Venezuela
investigating four species (P. concolor, P. onca, Leopardus
pardalis and Cerdocyon thous). In that study, 20 of 34 scats
(59%) were successfully amplified and sequenced, allow-
ing species-level identification. More recently, Miotto et al.
(2007) used those same primers in a study to determine the
presence of pumas and their estimated minimum popula-
tion in two protected areas in Brazil, and also achieved
60% success rate in amplification and sequencing. Higher
amplification success rates (83% and 85%, respectively)
were observed with cyt b markers in the studies of Adams
et al. (2003) and Onorato et al. (2006) in different areas of
the United States. However, both of these papers reported a
certain amount of prey DNA amplification using these
primers (13 and 8%, respectively), which suggests that
additional marker development is desirable to maximize
the efficiency of assays capable of identifying carnivore
species.
In the case of the jaguar, a complicating factor for the
development of mtDNA-based assays is the presence in all
five Panthera species of a large nuclear insertion (numt)
containing most of the mitochondrial genome. A detailed
study investigating this Panthera numt suggested that it
encompasses a long segment spanning eight protein coding
genes (including cyt b), two rRNA genes, 17 tRNA genes,
and the control region (Kim et al. 2006). Since the
amplification (or coamplification) of a numt is a known
complication that can hamper or confound genetic analyses
(Zhang and Hewitt 1996; Kim et al. 2006), including DNA-
based identification, it is important to develop markers that
506 Genetica (2009) 136:505–512
123
target mtDNA regions that are not contained in this
translocation.
In this paper we describe a DNA-based assay for the
identification of jaguar and puma faecal samples that bears
the following assets: (1) high specificity, as sequence-based
identification leads to multiple diagnostic characters
between the two species; (2) high sensitivity, as the
amplified fragment is short and leads to high amplification
rates; (3) avoidance of numt amplification by targeting a
mtDNA region not included in this translocation; and (4)
no detected amplification of prey DNA. In addition to
accomplishing successful discrimination between jaguar
and puma scats, the method proposed here has a potential
for much broader application in carnivores, as its sequence-
based diagnosis allows for multiple sympatric species to be
reliably identified.
Materials and methods
In order to develop an effective molecular approach for
reliable identification of P. onca scats, especially with
respect to distinguishing it from and P. concolor, we used
52 jaguar reference samples that spanned the geographic
distribution of this species (Table 1), including blood
(n = 15), tissue (n = 1), hair (n = 2) and faeces (n = 34)
collected from captive animals. For comparison, we ana-
lyzed nine reference samples of pumas (eight blood
samples and one tissue), representing multiple geographic
regions where it is sympatric with the jaguar. Additional
analyses included a marker test using a faecal sample of
domestic cat (Felis catus) collected in Argentina, paired
samples of blood and scat collected from four captive
jaguars (see Table 1), and comparisons with multiple
sequences available in GenBank or generated in our labo-
ratory for other purposes.
In addition to samples originated from known specimens,
we analyzed 55 scats collected by field researchers in dif-
ferent areas, and identified as ‘large felid’ based on
morphological features such as diameter, size and shape, as
well as associated tracks. ‘Large felid’ scats are usually
interpreted as originating from either jaguar or puma, and
discriminating between the two is a known and recurrent
challenge in most cases. All 55 samples were collected from
areas in the Atlantic Forest biome where both species are
thought to occur, and where field projects addressing eco-
logical aspects of one or both of them are currently being
carried out. The field sites were: Reserva Natural Vale,
Espı
´
rito Santo State, Brazil (RV; n = 9); Parque Estadual do
Rio Doce, Minas Gerais State, Brazil (RD; n = 1); Parque
Estadual das Va
´
rzeas do Rio Ivinhema, Mato Grosso do Sul
State, Brazil (PI; n = 3), and several forested locations in
the Misiones Province, Argentina (MP; n = 42).
Blood samples were preserved in a salt-saturated solution
(100 mM Tris, 100 mM EDTA, 2% SDS) and tissues and
hairs were kept in ethanol 96%. Approximately 6 g of faeces
were collected and stored in a 15 ml vial containing silica gel
at a 4 g silica/g faeces ratio (Wasser et al. 1997). All samples
were stored at -20°C prior to DNA extraction. Genomic
DNA was extracted from blood and tissue samples using a
standard Proteinase-K digestion and phenol-chloroform-iso-
amyl alcohol protocol (Sambrook et al. 1989). Extractions
from the hair samples were performed with the Puregene
DNA Purification Kit (GENTRA), and those of scat samples
used the QIAamp DNA Stool Mini Kit (QIAGEN), following
the manufacturers’ instructions. Scat DNA extractions were
carried out in a separate laboratory area, in a UV-sterilized
laminar flow hood dedicated to the analysis of DNA from
noninvasive samples. Each batch of extractions (n = 10)
included one negative extraction control to monitor the
occurrence of contamination with extrinsic DNA.
In order to avoid amplification of the Panthera numt and
to aim for high amplification success and sequence vari-
ability, we targeted a short segment of the mtDNA ATP
synthase subunit 6 (ATP6) gene, including its overlapping
portion with the ATP8 gene. Previous studies indicated that
this segment was quite variable in carnivores (Trigo et al.
2008; E. Eizirik et al., unpublished), and that it lay outside
of the numt (Kim et al. 2006). We employed the reverse
primer ATP6-DR1 (5
0
-CCAGTATTTGTTTTGATGTTAG
TTG-3
0
), originally reported by Trigo et al. (2008), and
designed two new forward primers: ATP6-DF2 (5
0
-ATGA
ACGAAAATCTATTCGC-3
0
) and ATP6-DF3 (5
0
-AACG
AAAATCTATTCGCCTCT-3
0
). These forward primers
anneal at very similar positions (PCR product size in
combination with ATP6-DR1 is 175 bp for DF2 and
172 bp for DF3), and were designed to maximize the
probability of successful amplification in carnivores, given
a preliminary alignment including sequences drawn from
GenBank that represented the major lineages of this
mammalian order (i.e. Feliformia, Arctoidea, and Cynoi-
dea). On the basis of initial tests, both forward primers
appeared to perform well in carnivores (not shown), and
primer ATP6-DF2 was employed throughout this study.
Polymerase chain reactions were performed in a final
volume of 20 ll, containing 19 PCR buffer (Invitrogen),
2.0–2.5 mM MgCl
2
, 200 lM dNTPs, 0.2 lM of each pri-
mer, 0.5 unit of regular Taq DNA polymerase (Invitrogen) or
Platinum Taq DNA polymerase (Invitrogen) and 1–6 llof
empirically diluted template DNA. The reaction profile was
as follows: 10 cycles (Touchdown) of 94°C for 45 s, 60–
51°C for 45 s, 72°C for 1.5 min, followed by 30 cycles of
94°C for 45 s, 50°C for 45 s, 72°C for 1.5 min, and a final
extension at 72°C for 3 min. Products were visualized on a
1% agarose gel stained with GelRed (Biotium), purified with
>PEG8000, sequenced using the DYEnamic ET Dye
Genetica (2009) 136:505–512 507
123
Table 1 Samples utilized as reference in the present study
ID Sample Geographic origin Institution/Contact
Panthera onca
bPon-01 Blood Parana
´
state, Brazil Proj. Carnı
´
voros-Ibama—P. G. Crawshaw Jr.
bPon-03 Blood Mato Grosso do Sul state,
Brazil
Proj. Porto Primavera—P. G. Crawshaw Jr.
bPon-13 Blood Amazonas state, Brazil CIGS, Manaus
bPon-15
a
, bPon-27
a
, bPon-32
a
Blood and
faeces
Mato Grosso do Sul state,
Brazil
CENAP/ICMBio; I. Pro
´
-Carnı
´
voros;
Ilha Solteira Zoo; V. Queiro
´
s
bPon-16, bPon-35 Blood Mato Grosso do Sul state,
Brazil
CENAP/ICMBio; Pro
´
-Carnı
´
voros
bPon-18
a
Blood and
faeces
Sa
˜
o Paulo state, Brazil CENAP/ICMBio; I. Pro
´
-Carnı
´
voros; Ilha Solteira Zoo; V.
Queiro
´
s
bPon-24, bPon-51 Blood Sa
˜
o Paulo state, Brazil Inst. Pesquisas Ecolo
´
gicas (IPE)/L. Cullen and A. Nava
bPon-34 Muscle French Guiana Benoit de Thoisy
bPon-55
a
, bPon-56
a
, bPon-102
a
,
bPon-103
a
, bPon-104
a
Faeces Captivity, Brazil Sapucaia do Sul Zoo/R. von Hohendorff
bPon-59
a
Faeces Parana
´
state, Brazil CASIB/W. de Moraes
bPon-66 Blood Mato Grosso do Sul state,
Brazil
Embrapa-Pantanal/G Moura
˜
o
bPon-81
a
, bPon-82
a
Faeces Captivity, Brazil Americana Zoo/M. Falcade;
Limeira Zoo/A. C. A. Sorg
bPon-83
a
Faeces Amazonas state, Brazil Limeira Zoo/A. C. A. Sorg
bPon-90
a
, bPon-91
a
, bPon-93
a
,
bPon-94
a
Faeces Captivity, Brazil Parque Municipal ‘Danilo Galafassi’’/L. E. S. Delgado
bPon-95
a
, bPon-96
a
, bPon-97
a
Faeces Captivity, Brazil CEBUS/C. D. P. Coelho and L. C. Silva
bPon-98
a
, bPon-99
a
Faeces Captivity, Brazil Goiania Zoo/R. F. de Carvalho and D. Nogueira
bPon-100
a
Faeces Captivity, Brazil Campinas Zoo/E. F. Santos
bPon-101
a
Faeces Acre state, Brazil Campinas Zoo/E. F. Santos
bPon-105
a
, bPon-112
a
Faeces Captivity, Brazil Guarulhos Zoo/C. E. Bolochio
bPon-107
a
Faeces Acre state, Brazil Parque Ambiental Chico Mendes/J. O. Guimara
˜
es
bPon-108
a
Faeces Rondonia state, Brazil Parque Ambiental Chico Mendes/J. O. Guimara
˜
es
bPon-114
a
, bPon-115
a
Faeces Captivity, Brazil Parque Cyro Gevaerd/M. R. Achutti
bPon-116
a
Faeces Amazonas state, Brazil Curitiba Zoo/M. L. Javorouski
bPon-117
a
Faeces Santa Catarina state, Brazil Curitiba Zoo/M. L. Javorouski
bPon-120
a
Faeces Amazonas state, Brazil Pomerode Zoo (Fund. Hermann Weege)/C. H. Maas
bPon-123
a
Faeces Mato Grosso do Sul state,
Brazil
Pomerode Zoo (Fund. Hermann Weege)/C. H. Maas
bPon-126 (Pon-31) Blood San Luis Potosı
´
, Mexico Leon Zoo
bPon-127 (Pon-50) Blood Chaco, Paraguay Itaipu, Paraguay
bPon-128 (Pon-54) Blood Amazonas, Venezuela Las Delicias
P31-1 Hair Misiones Province,
Argentina
Proyecto Yaguarete
´
, CeIBA/Carlos De Angelo
P3-2 Hair Misiones Province,
Argentina
Proyecto Yaguarete
´
, CeIBA./Carlos De Angelo
Puma concolor
bPco-72 (Pco-356) Blood Texas, USA Laboratory of Genomic Diversity (LGD), USA
bPco-73 (Pco-541) Blood Panama Laboratory of Genomic Diversity (LGD), USA
bPco-74 (Pco-556) Blood Guatemala Laboratory of Genomic Diversity (LGD), USA
bPco-75 (Pco-560) Blood Argentina Laboratory of Genomic Diversity (LGD), USA
bPco-76 (Pco-700) Blood Paraiba state, Brazil Laboratory of Genomic Diversity (LGD), USA
bPco-77 (Pco-704) Blood Venezuela Laboratory of Genomic Diversity (LGD), USA
bPco-78 (Pco-707) Blood Bolivia Laboratory of Genomic Diversity (LGD), USA
508 Genetica (2009) 136:505–512
123
Terminator Sequencing Kit (GE Healthcare), and analyzed
in a MegaBACE 1000 automated sequencer (GE Health-
care). To assess the performance of a fast, straightforward
and lower-cost identification strategy, PCR products were
routinely sequenced for only one strand (using the forward
primer ATP6-DF2), and only bases that could be reliably
scored were kept in the data set. To verify sequence accuracy
and to ascertain the validity of only using a single strand for
identification purposes, we sequenced the reverse strand for
one jaguar and one puma sample (bPon-127 and bPco-014,
respectively), in both cases confirming the original result.
Sequences were visually checked and manually corrected
using CHROMAS 2.0 (http://www.technelysium.com.au/
chromas.html). All haplotypes identified here have been
deposited in GenBank (Accession numbers FJ596283-
FJ596287).
DNA sequences were aligned with the CLUSTALW
algorithm implemented in MEGA 3.1 (Kumar et al. 2004).
MEGA was also used to identify identical haplotypes, to
assess the presence and consistency of diagnostic sites
between the two species, and to perform phylogenetic
analyses. In most cases the identification of samples could
be performed with a direct, character-based approach, as
field-collected scats often contained identical haplotypes to
those observed in reference samples. To test whether dif-
ferent haplotypes observed in each species could be reliably
grouped, so as to provide an easy and consistent identifica-
tion tool even in cases where haplotypes were not identical,
we performed several phylogenetic analyses. These included
maximum parsimony and distance-based approaches using
the Neighbor-Joining algorithm (Saitou and Nei 1987) with
various types of genetic distances. The reliability of inferred
nodes was assessed using 1,000 bootstrap replications. In
addition to the data generated here, phylogenetic analyses
also included sequences from other felid species available in
GenBank, especially domestic cat (U20753) and cheetah
(Acinonyx jubatus; AY463959).
Results and discussion
The use of PCR primers ATP6-DF2 and ATP6-DR1 led to
excellent amplification success (100%) using blood, tissue
or hair as DNA sources. High quality DNA sequences
could thus be obtained from all reference samples. PCR
products were 175 bp long in both species, yielding a
130 bp long analyzable segment after removal of primer
sequences. When employing only forward sequences for
streamlined and lower-cost sample identification, 96 sites
could be reliably used after additional removal of bases that
did not present high quality scores with this DNA strand
alone. However, if the reverse strand was also used, all 130
sites could be reliably scored. Even considering only the 96
sites scored with the forward strand, species-level identi-
fication was found to be highly accurate, with a minimum
of 15 diagnostic sites identified between any jaguar and
puma sequence (Table 2).
Two different haplotypes, differing by a single nucleo-
tide, were observed in the P. onca samples. Among pumas,
two variable sites were identified, leading to three different
haplotypes (Table 2). In addition to the character-based
analysis that clearly diagnosed pumas versus jaguars,
phylogenetic analyses also demonstrated that the two spe-
cies could be easily differentiated using this mtDNA
segment (Fig. 1).
PCR amplification of this segment from faecal samples
also led to promising results. Thirty-four out of 39 fresh
scat samples collected from captive jaguars were success-
fully amplified and sequenced, corresponding to an 87%
success rate. Field-collected scats yielded variable success
rates, likely due to heterogeneity in environmental condi-
tions as well as sample age (when collected) and storage
time. A success rate of 78% (seven out of nine samples)
was obtained with scats from the RV location (see Mate-
rials and methods), in contrast to only 50% (21 out of 42
scats) for the samples from Misiones (MP). We examined
the underlying causes of this lower success rate with the
MP samples by partitioning these scats into different cat-
egories of field-assigned quality (‘‘freshness’’) and storage
time. If only samples categorized as ‘fresh’ were consid-
ered, the success rate became 89% (8/9 scats) for scats
from MP. Within the group categorized in the field as
‘intermediate’ in freshness, a 59% success rate (10/17)
was obtained for samples that had been stored for \1 year
prior to extraction, and only 22% (2/9) for those stored for
C1 year. Finally, the group of samples categorized as ‘low
Table 1 continued
ID Sample Geographic origin Institution/Contact
bPco-79 (Pco-7) Blood Oregon, USA Laboratory of Genomic Diversity (LGD), USA
bPco-014 Tissue Sa
˜
o Paulo state, Brazil F. Olmos
Felis catus
F3-120
a
Faeces Buenos Aires, Argentina Proyecto Yaguarete
´
, CeIBA/Carlos De Angelo
a
Faeces collected in captivity
Genetica (2009) 136:505–512 509
123
quality’’ in the field did exhibit the lowest success rate (1/7
scats, i.e. 14%), indicating that this initial assessment at the
collection stage did predict to some extent the success rate
of PCR and sequencing. Scats from other locations pre-
sented satisfactory success rates (1/1 for RD; 2/3 for PI),
though their smaller sample size precludes a more detailed
assessment of local variables.
Throughout all analyses of this mtDNA segment from
scat samples, no evidence of prey DNA amplification was
observed. If affirmed by further sampling and analyses of
other carnivore communities, this result would indicate that
the markers proposed here might have advantages in terms
of identification performance relative to others that have
been published previously (e.g. see Adams et al. 2003 and
Onorato et al. 2006). In addition, we also saw no evidence
of numt amplification or co-amplification (e.g. double
peaks, high sequence background) in the jaguar samples,
supporting the prediction that this segment excludes this
nuclear insertion. This would represent an additional
advantage of employing this marker relative to the cyt b
gene, targeted by other studies (e.g. Farrell et al. 2000).
Since very little variation was observed within each
species (see Fig. 1), most field-collected scats bore iden-
tical haplotypes to those observed in reference samples
(Table 2), leading to straightforward identification. In the
few cases where a unique haplotype was identified in scat
samples (e.g. PcoH2 and PcoH3, see Table 2), the presence
of multiple diagnostic sites made species-level identifica-
tion conclusive with either a character-based or a
phylogenetic approach (see Fig. 1). Employing this assay,
we where thus able to confidently detect the presence of
one or both species in all surveyed field locations (see
Table 2 Mitochondrial DNA ATP6 haplotypes identified from jaguar (PonH1, PonH2) and puma (PcoH1, PcoH2, PcoH3) samples
Haplotype ID Variable sites Known samples Field samples
b
112344556667889
369067025013692171
PonH1 TCACACTCGGTCTTACGT bPon01, 03, 13, 15
a
, 16, 18
a
, 24, 27
a
,
32
a
, 34, 35, 51, 55, 56, 59, 66, 82,
83, 90, 91, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105,
107, 108, 112, 114, 115, 116, 117,
120, 123, 126, 127, 128; P3-2, 31-1
RV04, 06, 07, 08, 09, 19, 31;
PI32, 40; MP5-9, 5-10, 5-12,
4-120, 23-18, 43-200
PonH2 ...........T...... bPon81
PcoH1 CTCTGACAAACTCCGGAC bPco14, 72, 73, 74, 75, 76, 77, 78, 79 RD01; MP18-31, 38-55, 56-26,
57-1, 2-635, 2-636, 2-711,
2-659
PcoH2 C.NTGACAAACTCCGGAC MP48-2, 61-3, 12-101
PcoH3 C.CT.ACAAACTCCGGAC MP4-13
Only variable sites are shown. Site numbers (vertical notation) refer to the aligned position in our 96 bp data set. Known samples (see Table1)
and field-collected scats bearing each haplotype are also indicated
a
Individuals for which paired blood and faecal samples were analyzed, in every case leading to identical results
b
Field samples consist of scats collected in the following locations: Reserva Natural Vale, Espı
´
rito Santo state, Brazil (RV); Parque Estadual do
Rio Doce, Minas Gerais state, Brazil (RD); Parque Estadual das Va
´
rzeas do Rio Ivinhema, Mato Grosso do Sul state, Brazil (PI); and Misiones
Province, Argentina (MP)
Fig. 1 Phylogenetic tree depicting the evolutionary relationships
among mtDNA haplotypes sampled in jaguars (PonH), pumas (PcoH)
and other carnivores (see text and Table 2). The tree is based on
96 bp of the mtDNA ATP6 gene, and was constructed using the
Neighbor-joining algorithm on the basis of a p-distance matrix.
Numbers above branches represent bootstrap support values generated
with 1,000 replicates
510 Genetica (2009) 136:505–512
123
Table 2). For MP, which presented the largest available
sample size, 12 scats were identified as originating from
pumas and six from jaguars, allowing the use of these
samples in downstream analyses addressing fragment
occupation, diet and population genetics. Interestingly, all
seven scats that could be sequenced from the RV site were
identified as jaguars, providing the first genetic samples of
P. onca collected from a Coastal Atlantic Forest location.
Further sampling from this area will be extremely impor-
tant to allow for an initial survey of the genetic diversity
and evolutionary distinctiveness of this isolated and criti-
cally endangered remnant population.
Finally, in addition to its diagnostic power for pumas
and jaguars, the informative content of this mtDNA
segment holds promise to provide reliable identification
for other carnivore species. For example, as a control in
this study, we amplified and sequenced DNA from a
domestic cat faecal sample, which was easily grouped with
the reference sequence for this species available in
GenBank (see Fig. 1). In the context of the inherent
uncertainty of field-based scat identification, especially in
ecosystems harboring multiple sympatric species, the use-
fulness of a sequence-based assay is noteworthy. One
graphic example was observed among the samples inves-
tigated in this study. Of the 21 sequenced samples from the
MP location, all of which had been identified in the field as
originating from a ‘large felid’’, 18 were identified as
jaguar or puma (see above and Table 2). Three others were
found to bear considerably different ATP6 haplotypes, and
were subsequently identified as originating from ocelots
(L. pardalis) by comparison with a multi-species data base
(P. B. Chaves et al. unpublished data). The finding that
ocelot samples were identified as belonging to a ‘large
felid’ by experienced field researchers highlights the
urgent issue of procuring accurate and standardized faecal
identification methods, so as to provide a reliable basis for
the development of non-invasive ecological and genetic
investigations on jaguars and pumas, and on carnivore
species in general.
Acknowledgments We would like to thank all the institutions and
people who helped with collection of biological samples used in this
study, including those mentioned in Table 1, as well as Warren E.
Johnson, Stephen J. O’Brien, Peter G. Crawshaw Jr., Laury Cullen Jr.,
Alessandra Nava, Leonardo R. Viana, Adriano G. Chiarello, Cristian
Corio, Esteban Hasson, Agustı
´
n Paviolo and Mario Di Bitteti. We
especially thank Paulo B. Chaves for technical assistance and sug-
gestions, as well as access to unpublished data on ocelot ATP6
sequences. We are also grateful to the Centro Nacional de Pesquisas
para a Conservac¸a
˜
o de Predadores Naturais (CENAP/ICMBio), In-
stituto Pro
´
-Carnı
´
voros, Companhia Energe
´
tica de Sa
˜
o Paulo (CESP)
and Instituto Ambiental Vale for having supported this project, and
the Laboratorio de Evolucio
´
n, Departamento de Ecologı
´
a, Gene
´
tica y
Evolucio
´
n, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, for help in the analysis of scat samples from Argentina.
T. Haag is supported by a fellowship from Coordenac¸a
˜
o de Aper-
feic¸oamento de Pessoal de
´
vel Superior (CAPES), Brazil.
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    • "As part of effective wildlife conservation and management programs, genetic studies are often initiated to investigate many aspects of a species' ecology (Frankham 2003). In pumas, genetic studies have concentrated either on the examination of gene flow on continental (Culver et al. 2000) and local scales (e.g., Loxterman [2011]; Andreasen et al. [2012]; Balkenhol et al. [2014]), or on the identification of individuals (Haag et al. 2009; Miotto et al. 2011; Naidu et al. 2011). Microsatellites have been the genetic marker of choice, and numerous loci derived from either domestic cats Felis catus (Menotti- Raymond and O'Brien 1995; Menotti-Raymond et al. 1997, 1999) or directly from pumas (Kurushima et al. 2006; Rodzen et al. 2007) have been developed and used in many studies (Figure 1). "
    [Show abstract] [Hide abstract] ABSTRACT: Pumas Puma concolor are one of the most studied terrestrial carnivores because of their widespread distribution, substantial ecological impacts, and conflicts with humans. Over the past decade, managing pumas has involved extensive efforts including the use of genetic methods. Microsatellites have been the most commonly used genetic markers; however, technical artifacts and little overlap of frequently used loci render large-scale comparison of puma genetic data across studies challenging. Therefore, a panel of genetic markers that can produce consistent genotypes across studies without the need for extensive calibrations is essential for range-wide genetic management of puma populations. Here, we describe the development of PumaPlex, a high-throughput assay to genotype 25 single nucleotide polymorphisms in pumas. We validated PumaPlex in 748 North American pumas Puma concolor couguar, and demonstrated its ability to generate reproducible genotypes and accurately identify individuals. Furthermore, in a test using fecal deoxyribonucleic acid (DNA) samples, we found that PumaPlex produced significantly more genotypes with fewer errors than 12 microsatellite loci, 8 of which are commonly used. Our results demonstrate that PumaPlex is a valuable tool for the genetic monitoring and management of North American puma populations. Given the analytical simplicity, reproducibility, and high-throughput capability of single nucleotide polymorphisms, PumaPlex provides a standard panel of markers that promotes the comparison of genotypes across studies and independent of the genotyping technology used.
    Full-text · Article · Jun 2016
    • "Comparative fecal DNA preservation studies for carnivores were conducted primarily for canids and ursids (e.g., Wasser et al. 1997, Murphy et al. 2000, 2002, Panasci et al. 2011). For felids, a wide variety of fecal DNA methods have been applied, including freezing (e.g., Ernest et al. 2002, Sugimoto et al. 2006), air drying (e.g., Farrell et al. 2000, Weckel et al. 2006), silica desiccation (e.g., Haag et al. 2009, Jane cka et al. 2011), or liquid storage using buffer solutions (e.g., 20% dimethyl sulfoxide buffer, Vynne et al. 2012) or ethanol (EtOH; e.g., Mondol et al. 2009, Michalski et al. 2011, see also online Supporting Information Table S1). Yet, only a handful of comparative fecal DNA preservation studies examined the effectiveness of different methods on amplification of fecal DNA for felids (e.g., for mtDNA for wild tigers [Panthera tigris]; Bhagavatula and Singh 2006) and nuclear DNA (nDNA) markers for captive tigers (Reddy et al. 2012). "
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    Full-text · Article · Apr 2015
    • "Photographic surveys are also logistically challenging in such landscape studies as they require equipment, skilled personals, intensive effort in low animal density areas, particularly outside protected zones. The molecular tools developed in this study thus provide certain advantages over earlier approaches used in carnivore studies (Cossíos and Angers 2006; Sugimoto et al. 2006; Bidlack et al. 2007; Livia et al. 2007; Haag et al. 2009; Roques et al. 2010). The approaches proposed here facilitate rapid screening of large number of samples using fewer steps in sample processing (DNA extraction, single multiplex PCR, electrophoresis), reduced species/sex misidentifications , and are cheaper than PCR–RFLP (Nagata Bidlack et al. 2007) or sequencing (Bhagavatula and Singh 2006; Perez et al. 2006; Pandey et al. 2007; Busby et al. 2009) approaches. "
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