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Abstract

Carnivores are important elements of Neotropical biomes that are in need of conservation efforts. However, successful conservation methods rely on the identification of accurate evolutionary taxa. Unfortunately, in the case of Procyonidae systematics, there has been little knowledge in some genera. Two of these genera are Nasuella and Nasua, also known as the coatis. Herein, we analyzed a dataset obtained in South America and Central America, containing sequences of three mitochondrial genes (ND5, Cyt-b, and D-loop) collected from 42 mountain coati (Nasuella olivacea) specimens, plus 50 white-nosed coati (Nasua narica) and 51 ring-tailed coati (Nasua nasua) (total sample of 143). Our results support four main findings. (1) We detected four significantly different groups of N. olivacea. There were two small groups, one distributed in the Central Colombian Andean Cordillera and Western Ecuadorian Andean Cordillera, and another in the Western Colombian and Ecuadorian Andean Cordilleras. The specimens of these small groups were phenotypically un-differentiable from N. olivacea, but their mtDNA were more related to that of N. nasua than to the mtDNA of the other N. olivacea. The other two groups of N. olivacea contained the major part of the specimens analyzed. One is in the Eastern Colombian Andean Cordillera and is molecularly un-differentiable from the proposed “new” endemic Venezuelan species, Nasuella meridiensis. The ancestor of this group gave origin to another expanded group in the Western and Central Colombian and Ecuadorian Andean Cordilleras. (2) Different analyses (network, temporal splits, genetic diversity analyses) showed that the mitochondrial haplotypes of N. nasua were the first to appear (temporal diversification during the Late Miocene, and Pliocene), followed by the haplotypes of the current groups of Nasuella (temporal diversification during the Pliocene and beginning of the Pleistocene), and then the haplotypes that of the Central American N. narica (temporal diversification during the Pleistocene). Within N. nasua, we detected, at least, four highly differentiated groups that contain cryptic species or highly differentiated subspecies. (3) All of the taxa we analyzed showed high levels of mitochondrial genetic diversity, but N. nasua showed the highest levels, whereas N. narica showed the lowest levels. (4) Some groups of N. olivacea, and N. narica showed Pleistocene population expansions, but all the taxa showed a very strong signal of population declination in the last 20,000 years ago (YA), which could be correlated with the drastic climatic changes in that epoch.
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Mammalian Biology
Zeitschrift für Säugetierkunde
ISSN 1616-5047
Mamm Biol
DOI 10.1007/s42991-020-00050-w
The phylogeographic structure of the
mountain coati (Nasuella olivacea;
Procyonidae, Carnivora), and its
phylogenetic relationships with other coati
species (Nasua nasua and Nasua narica) as
inferred by mitochondrial DNA
Manuel Ruiz-García, Maria Fernanda
Jaramillo, Carlos Herney Cáceres-
Martínez, et al.
1 23
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Vol.:(0123456789)
1 3
Mammalian Biology
https://doi.org/10.1007/s42991-020-00050-w
ORIGINAL ARTICLE
The phylogeographic structure ofthemountain coati (Nasuella
olivacea; Procyonidae, Carnivora), andits phylogenetic relationships
withother coati species (Nasua nasua andNasua narica) asinferred
bymitochondrial DNA
ManuelRuiz‑García1· MariaFernandaJaramillo1· CarlosHerneyCáceres‑Martínez2· JosephMarkShostell3
Received: 25 January 2020 / Accepted: 25 June 2020
© Deutsche Gesellschaft für Säugetierkunde 2020
Abstract
Carnivores are important elements of Neotropical biomes that are in need of conservation efforts. However, successful con-
servation methods rely on the identification of accurate evolutionary taxa. Unfortunately, in the case of Procyonidae systemat-
ics, there has been little knowledge in some genera. Two of these genera are Nasuella and Nasua, also known as the coatis.
Herein, we analyzed a dataset obtained in South America and Central America, containing sequences of three mitochondrial
genes (ND5, Cyt-b, and D-loop) collected from 42 mountain coati (Nasuella olivacea) specimens, plus 50 white-nosed coati
(Nasua narica) and 51 ring-tailed coati (Nasua nasua) (total sample of 143). Our results support four main findings. (1)
We detected four significantly different groups of N. olivacea. There were two small groups, one distributed in the Central
Colombian Andean Cordillera and Western Ecuadorian Andean Cordillera, and another in the Western Colombian and Ecua-
dorian Andean Cordilleras. The specimens of these small groups were phenotypically un-differentiable from N. olivacea,
but their mtDNA were more related to that of N. nasua than to the mtDNA of the other N. olivacea. The other two groups of
N. olivacea contained the major part of the specimens analyzed. One is in the Eastern Colombian Andean Cordillera and is
molecularly un-differentiable from the proposed “new” endemic Venezuelan species, Nasuella meridiensis. The ancestor of
this group gave origin to another expanded group in the Western and Central Colombian and Ecuadorian Andean Cordilleras.
(2) Different analyses (network, temporal splits, genetic diversity analyses) showed that the mitochondrial haplotypes of N.
nasua were the first to appear (temporal diversification during the Late Miocene, and Pliocene), followed by the haplotypes
of the current groups of Nasuella (temporal diversification during the Pliocene and beginning of the Pleistocene), and then
the haplotypes that of the Central American N. narica (temporal diversification during the Pleistocene). Within N. nasua,
we detected, at least, four highly differentiated groups that contain cryptic species or highly differentiated subspecies. (3)
All of the taxa we analyzed showed high levels of mitochondrial genetic diversity, but N. nasua showed the highest levels,
whereas N. narica showed the lowest levels. (4) Some groups of N. olivacea, and N. narica showed Pleistocene population
expansions, but all the taxa showed a very strong signal of population declination in the last 20,000years ago (YA), which
could be correlated with the drastic climatic changes in that epoch.
Keywords Central America· Coatis· Mitochondrial genes· Nasua narica· Nasua nasua· Nasuella meridiensis· Nasuella
olivacea· Phylogeography· Population expansions· Spatial genetic structure· South America
Handling editor: Laura Iacolina.
* Manuel Ruiz-García
mruizgar@yahoo.es
1 Laboratorio de Genética de Poblaciones Molecular-Biología
Evolutiva, Departamento de Biología, Facultad de Ciencias,
Pontificia Universidad Javeriana, Cra 7A, No. 43-82,
BogotáDC, Colombia
2 Grupo de Investigación en Ecología y Conservación de Fauna
Silvestre, Universidad Nacional de Colombia, SedeMedellín,
Colombia
3 Math, Science andTechnology Department, University
ofMinnesota Crookston, 2900 University Ave, Crookston,
MN56716, USA
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Introduction
The Carnivora Order encloses 16 families divided into
two monophyletic super-families, Caniformia and Feli-
formia (Eisenberg 1989; Wozencraft 2005; Wyss and
Flynn 1993). The first super-family encloses the families
Canidae, Ursidae, Mephitidae, Ailuridae, Procyonidae,
Mustelidae, Otaridae, Odobenidae, and Phocidae, while
the second super-family contains Felidae, Nandiniidae,
Prionodontidae, Eupleridae, Herpestidae, Hyaenidae, and
Viverridae (Wilson and Mittermeier 2009).
The majority of molecular studies including those of
Procyonidae were undertaken to clarify the phylogenetic
relationships among Carnivores (Yu etal. 2004). Koepfli
etal. (2007) were the first to try to resolve the phylogenetic
relationships among the genera within Procyonidae using
nuclear and mitochondrial genes. However, they did not
include sequences of the mountain coati (Nasuella oli-
vacea). This work was important because it was the first
effort to estimate the temporal splits among and within
the Procyonidae genera as well as to analyze possible phe-
nomena which affected the colonization of this family in
Central and South America.
Only a small number of molecular studies have focused
at the level of species for Procyonidae in the Neotropics
(McFadden 2004 for Nasua and Procyon; Helgen etal.
2009, 2013 for Nasuella and Bassarycion; Tsuchiya-
Jerep (2009) for Nasua; Neves-Chaves (2011) for Nasua;
Ruiz-García etal. 2013, 2019 for Potos; Nascimento etal.
2017 for Potos; Silva Caballero etal. 2017 for Nasua; and
Nigenda-Morales etal. 2019 for Nasua).
There are only two publications (Helgen etal. 2009;
Nigenda-Morales etal. 2019) including N. olivacea, the
species covered in the current study. The first study only
included four specimens of Nasuella, one from the Western
Colombian Andes (Cauca Department), two from the north-
ern Ecuadorian Andes, and one from the Venezuelan Merida
Cordillera. Additionally, they studied two N. nasua (Bolivia
and Brazil) and two Nasua narica (Panama and southern
USA). They sequenced and analyzed only a small fraction
of the mitochondrial (mt) Cyt-b gene (366 base pairs, bp).
However, even with these scarce data, they reached the con-
clusion, that the unique sample from Venezuela was a differ-
ent species (Nasuella meridensis). It was differentiated from
the specimens from the Western Colombian Andean Cordil-
lera and northern Ecuador, which belonged to N. olivacea
(100%, bootstrap percentage, and 84%, Bayesian posterior
probability). Overall, this work was too preliminary to com-
pletely elucidate the genetic structure and phylogeography
of the mountain coati.
The second work, with three mtDNA genes and with 11
microsatellites, found a high degree of genetic structure
and divergence in N. narica that conformed to five evolu-
tionarily significant units. The phylogeographic patterns
found within N. narica were associated with geographic
barriers and habitat shifts caused by Pliocene–Pleisto-
cene climate oscillations. Additionally, their findings
suggested the dispersal of N. narica was from southern
Central America to north beginning in the Pliocene, and
not from north to south direction during the Pleistocene
as suggested by the fossil record.
In the current work, we analyzed three mitochondrial
genes (mt genes) of 42 mountain coatis and 101 Nasua
specimens (total 143 specimens). We elected to focus on mt
genes as a first step to understand the phylogeography and
the possible existence of full species within N. olivacea.
Mitochondrial genes are interesting markers for phyloge-
netic tasks because they include a rapid accumulation of
mutations, rapid coalescence time, lack introns, have a neg-
ligible recombination rate and haploid inheritance (Avise
etal. 1987). They also have a high number of copies per cell
which makes mitogenome data easy to obtain and sequence
especially in low-quality samples, such as hair, teeth or small
pieces of skin (Mason etal. 2011; Guschanski etal. 2013).
Despite representing a single linked locus, selection pres-
sures and evolutionary rates are highly heterogeneous across
the mtDNA (Galtier etal. 2006; Nabholz etal. 2012). For all
of these reasons, mitochondrial gene trees are more precise
in reconstructing the divergence history within species, or
among very related species, than other molecular markers
(Moore 1995).
Extreme care should be taken when using mitochondrial
genes for resolving taxonomic problems because gene trees
do not necessarily correspond well with species trees. Spe-
cies can diverge simultaneously with a pair of mitochon-
drial haplotypes or they can diverge after a pair of haplo-
types diverge. However, it is possible for a new haplotype
to develop some time after a population divides. A migrant
could carry the new haplotype to another population and,
both it and the ancestral haplotypes could eventually become
lost from their respective populations. Therefore, if we use
the gene tree to estimate genetic heterogeneity and the diver-
gence time for the species tree, the species will appear to
have diverged more recently than they really did (Freeman
and Herron 1998). Furthermore, mitochondrial data only
show the evolution of the female lineages, and this could
miss hybridization events between close species when males
are the gene flow vectors (‘mitochondrial capture’; Burrell
etal. 2009). To fully resolve the issue about the complete
understanding of the evolutionary biology and number of
taxa in N. olivacea, there needs to be supporting evidence
from nuclear data (microsatellites or SNPs), not just mtDNA
data. Conscious of this two-pronged approach, we offer our
analysis of mitochondrial DNA as the first step towards
resolving this question. In fact, mtDNA have been extremely
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useful in detecting new and previously unnoticed taxa (see
Derenko etal. 2012; Krause etal. 2010; Sawyer etal. 2015).
The mtDNA results should be instrumental in helping to
determine conservation strategies based in the systematic
classification of the differentiated populations below the spe-
cies level. The uncertainty about these conservation units
can lead to confusion in the establishment of management
plans and errors in setting priorities (O’Brien 1994). An
essential second step in the future will be to analyze nuclear
markers to detect possible hybridization or gene flow events
among different lineages.
Thus, the main aims of the current work were: (1) To
determine the number of significantly different groups
within N. olivacea and to determine if any are full species;
(2) To determine if the presumed “new” endemic Venezue-
lan N. meridiensis is a “real” full species; (3) To determine
the kind of phylogenetic relationship of N. olivacea with N.
nasua and N. narica; (4) To determine possible significant
groups within N. nasua and N. narica; (5) To estimate the
levels of genetic diversity in Nasuella and Nasua; (6) To
analyze possible demographic changes in these taxa; and
(7) To determine the possible spatial genetic structure in
N. olivacea.
Materials andmethods
We analyzed a set of 143 individuals, all analyzed at the mt
genes ND5, Cyt-b, and D-loop. Forty-two were N. oliva-
cea, 50 were N. narica, and 51 were N. nasua. Additionally,
as outgroups, we included three specimens of Bassarycion
medius (Ecuador), two specimens of Bassarycion neblina
(Colombia), and 11 specimens of Bassarycion alleni (three
from Ecuador, four from Peru, and four from Bolivia) (see
Table1 and Fig.1).
DNA was obtained from hair, teeth, muscle and blood
from living and dead animals in diverse Indian, colono, or
mestizo communities. We requested permission to collect
biological materials from either carcasses or live animals
that were already present in the community. We sampled
small pieces of muscle, blood drops, or teeth from hunted
animals that were discarded during the cooking process, or
hairs with bulbs plucked from live pets. Communities were
visited only once, all sample donations were voluntary, and
no financial or other inducement was offered for supplying
specimens for analysis. During the sampling process (pets
and hunted animals), we interviewed the indigenous hunt-
ers who claimed that the hunted and captured specimens
came from within 10–15km of their respective communi-
ties. For more information about sample permissions, see the
Acknowledgment section. All the samples used were directly
obtained by the authors for the current work.
Table 1 Sources of the coatis collected and analyzed at three mito-
chondrial genes (ND5, Cyt-b, and D-loop) (42 Nasuella olivacea, 50
Nasua narica, and 51 Nasua nasua)
Species and location Number of
samples
Nasuella olivacea (n = 42)
Colombia (n = 37)
Boyacá Department
(Chita, Cocuy, Iguaqué, Paipa, Villa de Leyva) 5
Caldas Department 2
Cauca Department (Versalles, Puracé) 2
Chocó Department
(Alto Galápagos, San José del Palmar) 2
Cundinamarca Department
(Guasca, Ubaté, Chingaza NP) 12
Nariño Department
(Cumbal) 1
Norte de Santander Department
(Tamá NP) 6
Risaralda Department
(Lago Otún, Santa Rosa, Santa Cecilia) 4
Seized in Bogotá 2
Tolima Department
(Los Nevados NP) 1
Ecuador (n = 5)
Carchi Province
(Tulcán) 1
Cotopaxi Province
(Llanganates NP) 1
Morona-Santiago Province
(Sangay NP) 1
Pichincha Province
(Chiriboya, Guajacito) 2
Nasua narica (n = 50)
Mexico (n = 19)
Campeche State 9
Chiapas State
(San Cristobal de las Casas) 2
Quintana Roo State
(Cozumel Island) 1
Tabasco State 7
Guatemala (n = 13)
Alta Verapaz Department
(Cobán) 1
Izabal Department
(Livingstone, Puerto Barrios) 2
Peten Department
(Tikal NP, Uaxactun) 10
Belize (n = 3)
Rio Bravo Conservation Area 3
Honduras (n = 7)
Olancho Department
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Molecular methods
We extracted DNA from the skin, muscle and blood sam-
ples with a modified phenol–chloroform procedure (Sam-
broock etal. 1989). DNA from hair/follicles and teeth were
extracted using 10–20% Chelex 100 resin (Bio-Rad, USA),
with several modifications from Walsh etal. (1991).
We amplified three mitochondrial genes with primers
from the following papers: (1) 407 base pairs (bp) of mtCyt-
b (Irwin etal. 1991), (2) 1,800bp of mtND5 (Trigo etal.
2008), and (3) 306bp of mtD-loop (Hoelzel etal. 1994).
The total length was 2513bp.
We used a PCR reaction with 2μl of MgCl2 1mM, 1μl
of dNTPs 0.2mM, 1μl of 0.1μM of each primer, 1 unit of
Taq Polymerase, 100–200ng of DNA (2–4μl of DNA), 2μl
of Buffer 10X and 13.5μl of H2O. The PCR temperatures
were 95° for 5min followed by 40 cycles of 1min at 94°C,
1min at 52–56°C (depending on the primers used) and
1min at 72°C, and one final extension of 10min at 72°C.
All amplifications, including positive and negative controls,
were checked in 2% agarose gels. Those samples that ampli-
fied were purified using membrane-binding spin columns
(QIAquick PCR Purification Kit; Qiagen). The PCR prod-
ucts were sequenced in both directions using the Big Dye™
kit in an ABI 377A automated DNA sequencer. A consensus
of the forward and reverse sequences was determined using
the Sequencher software (Gene Codes Corporation).
We translated the mtCyt-b and mtND5 gene sequences
into amino acids to exclude the possibility of nuclear mito-
chondrial DNA segments (NUMTs) in the dataset analyzed
(Lopez etal. 1994). We note that all amino acid transla-
tions of the obtained sequences showed initial start and
terminal stop codons and the absence of premature stop
codons. All the mutations we observed were synonymous
changes. This agrees quite well with the fact that there
were no NUMTs in our sequence data. Moreover, one
Table 1 (continued)
Species and location Number of
samples
(Juticalpa) 5
Roatán Island 2
El Salvador (n = 3)
La Libertad Department
(Jayaque) 3
Nicaragua (n = 1)
Nueva Segovia Department
(Ocotal) 1
Costa Rica (n = 3)
Alajuela Province
(El Arenal) 2
Puntaarenas Province
(Golfo Dulce RF) 1
Panama (n = 1)
Colón Province
(Nombre de Dios) 1
Nasua nasua (n = 51)
Colombia (n = 10)
Amazonas Department
(Leticia, Macedonia, Amacayacu NP) 5
Caquetá Department
(Cuemani River) 1
Guaviare Department
(El Raudal) 2
Meta Department
(La Macarena, Pinacita) 2
Ecuador (n = 2)
Napo Province
(Tena) 1
Sucumbios Province
(Lago Agrio) 1
Peru (n = 13)
Cuzco Department
(Quincemil, Manu NP) 5
Loreto Department
(Nanay River, Napo River) 3
Madre de Dios Department
(Hermosa Grande) 2
Ucayali Department
(Yarinacocha) 3
Bolivia (n = 5)
Beni Department
(Moxos, Mamoré River) 3
La Paz Department 1
Santa Cruz Department 1
Brazil (n = 12)
Amazonas State
(Tabatinga, Javarí River, Manaus) 5
Goias State
Table 1 (continued)
Species and location Number of
samples
(Isla do Banal) 1
Parana State
(Iguazú) 6
Paraguay (n = 5)
Alto Paraná Department
(Pubio, Hernandarias) 5
Uruguay (n = 4)
Tucuarembó Department
(Laureles) 3
Artigas Department 1
The number of specimens analyzed at three mitochondrial genes in
each country is in bold
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Fig. 1 Map of Latin America where specimens of Nasuella oliva-
cea, Nasua nasua, and Nasua narica were sampled. These speci-
mens were sequenced for three mitochondrial genes (ND5, Cyt-b, and
D-loop). A total of 42 Nasuella olivacea, 50 Nasua narica, and 51
Nasua nasua were sampled
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would expect to see a signal relating to NUMTs in the
DNA chromatogram. When the ratio of nuclear to mito-
chondrial genomes per cell is around 1/1000 (Takamatsu
etal. 2002), the proportion of NUMTs and real mitochon-
drial sequences is expected to be amplified. If the potential
amplified NUMTs were highly differentiated from the real
mitochondrial gene or included some insertions or dele-
tions, many double peaks are expected in the chromato-
gram. We did not observe this in our chromatograms.
The GenBank accession numbers of the coati specimens
analyzed are from MT587713 to MT587855.
Phylogenetic studies
MrModeltest v2.3 (Nylander 2004) and MEGA 6.05 soft-
ware (Tamura etal. 2013) were applied to determine the
best evolutionary mutation model for the sequences ana-
lyzed for each individual gene, for different partitions and
for all the concatenated sequences. Akaike information
criterion (AIC; Akaike 1974; Posada and Buckley 2004)
and the Bayesian information criterion (BIC; Schwarz
1978) were used to determine the best evolutionary nucle-
otide model.
We constructed a Maximum Likelihood phylogenetic tree
(MLT) using RAxML v.7.2.6 software (Stamatakis 2006).
To select the best fitting model, 50 independent iterations
were run using three data partitions (codons 1, 2, and 3). For
each analysis, the GTR + G + I model (General Time Revers-
ible model, Tavaré 1986, + gamma-distributed rate variation
among sites + proportion of invariable sites, Yang 1994) was
used to search for the MLT and topological support was
estimated with 500 bootstrap replicates (Stamatakis 2006).
To estimate possible divergence times among the hap-
lotypes found in N. olivacea, N. nasua, and N. narica, we
relied on a Median Joining Network (MJN) (Bandelt etal.
1999) using Network 4.6.0.1 software (Fluxus Technol-
ogy Ltd). Additionally, the ρ statistic (Morral etal. 1994)
and its standard deviation (Saillard etal. 2000) were esti-
mated and transformed into years. The ρ statistic is unbi-
ased and highly independent of past demographic events.
This approach is named “borrowed molecular clocks” and
uses direct nucleotide substitution rates inferred from other
taxa (Pennington and Dick 2010). We used an evolutionary
rate of 1.75% per one MY, which represented one mutation
every 22,742years for the 2513bp analyzed for the dataset
analyzed. One advantage of the MJN procedures compared
to traditional trees is that they explicitly allow for the co-
existence of ancestral and descendant haplotypes, whereas
traditional trees treat all sequences as terminal taxa (Posada
and Crandall 2001). This allows us to observe which current
haplotypes began to evolve first and also to identify the more
recently derived haplotypes.
Genetic heterogeneity
The statistical procedures HST, KST, KST*, γST, NST and FST
(Hudson etal. 1992) were applied to determine the over-
all genetic heterogeneity among the different taxa of Nas-
uella and Nasua detected in the phylogenetic analyses. We
obtained indirect gene flow estimates for all the taxa, assum-
ing an infinite island model (Wright 1965). Significance was
estimated with permutation tests using 10,000 replicates.
These analyses were applied to the three coati species, as
well to the four groups detected within Nasuella olivacea. In
addition, genetic heterogeneity and gene flow statistics were
calculated by taxa pairs. We used FST tests with Markov
chains, 10,000 dememorizations parameters, 20 batches,
and 5,000 iterations per batch. All of these statistics were
calculated with DNAsp 5.1 (Librado and Rozas 2009) and
Arlequin 3.5.1.2 programs (Excoffier and Lischer 2010).
The Kimura 2P genetic distance (Kimura 1980) was
applied to determine the percentage of genetic differences
among the different groups detected in Nasuella and Nasua.
The Kimura 2P genetic distance is a standard measurement
for barcoding tasks (Hebert etal. 2003, 2004). Kartavtsev
(2011) analyzed sequences of mtCOI from 20,731 vertebrate
and invertebrate animal species and obtained 0.89% ± 0.16%
for populations within species, 3.78% ± 1.18% for subspe-
cies or semispecies, and 11.06% ± 0.53% for species within
a genus. At mtCOII, Collins and Dubach (2000), Ascunce
etal. (2003), and Ruiz-García etal. (2014) reported an aver-
age genetic distance of around 8% among species within a
genus, and around 2–5% for subspecies. Bradley and Baker
(2001) claimed, for mtCytb, that values < 2% would equal
intra-specific variation, values between 2 and 13% would
merit additional study, and values > 13% would be indica-
tive of specific recognition. Therefore, for the three mt genes
employed, we consider values around 3–5% for possible sub-
species and values around 12–13% for different species of
the same genus. For species of different genera, this value
should be around 16–18% or higher (Kartavtsev 2011).
Genetic diversity anddemographic changes
We used the following statistics to determine the genetic
diversity for the overall sample of N. olivacea, and for the
two main haplogroups of N. olivacea, and for N. nasua, and
N. narica: number of haplotypes, haplotype diversity (Hd),
nucleotide diversity (π), and θ statistic by sequence. These
genetic diversity statistics were calculated with DNAsp 5.1
software (Librado and Rozas 2009).
We relied on three procedures to detect possible histori-
cal population changes in the overall sample of N. oliva-
cea, the two main haplogroups of N. olivacea, N. nasua,
and N. narica. (1) We used the Fu and Li D* and F* tests
(Fu and Li 1993), the Fu FS statistic (Fu 1997), the Tajima
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D test (Tajima 1989) and the R2 statistic (Ramos-Onsins
and Rozas 2002). A 95% confidence interval and probabili-
ties were obtained with 10,000 coalescence permutations.
(2) The mismatch distribution (pairwise sequence differ-
ences) was obtained following the method of Rogers and
Harpending (1992) and Rogers etal. (1996). We used the
raggedness rg statistic to determine the similarity between
the observed and the theoretical curves. All these previous
demographic analyses were carried out with the DNAsp 5.1
(Librado and Rozas 2009) and Arlequin 3.5.1.2 programs
(Excoffier and Lischer 2010). (3) A Bayesian skyline plot
(BSP) was obtained by means of the BEAST v. 1.8.1 (Drum-
mond etal. 2012) and Tracer v1.6 (Rambaut etal. 2013)
software. The Coalescent-Bayesian skyline option in the tree
priors was selected with four steps and a piecewise-constant
skyline model with 30,000,000 generations (the first 3 mil-
lion discarded as burn-in), kappa with log Normal [1, 1.25]
and Skyline population size with uniform [0, infinite; initial
value 80]. Marginal densities of temporal splits were ana-
lyzed and the Bayesian Skyline reconstruction option was
selected for the trees log file using Tracer v1.6. We selected
a stepwise (constant) Bayesian skyline variant with maxi-
mum time as the upper 95% high posterior density (HPD)
and the trace of the root height as the treeModel.rootHeight.
We considered the last few million years as part of this anal-
ysis. Nevertheless, it is important to recall that all of these
demographic procedures have several caveats. Selection has
effects on effective population sizes reducing the effective
numbers for a time and increasing the coalescence rate later
(Schrider etal. 2016). The same occurs with little changes in
the mutation rates (μ), which can greatly affect the effective
numbers and which in turn affect estimated divergence times
(Sheehan etal. 2013).
Spatial genetic structure inN. olivacea
For all the spatial analyses carried out, we used the lati-
tude and longitude recorded for each specimen analyzed.
No populations or groups of individuals were employed for
these analyses.
A Mantel’s test (Mantel 1967) was used to detect possible
overall relationships between a genetic matrix among speci-
mens of N. olivacea (Kimura 2P genetic distance) and the
geographic distance matrix among the specimens analyzed.
In this study, Mantel’s statistic was normalized according to
Smouse etal. (1986). This procedure transforms the statistic
into a correlation coefficient.
The spatial autocorrelation analysis utilized the Ay sta-
tistic (Miller 2005) for each distance class (DC), where
Ay = Σi = 1, n Σj > i, n (Dijwyij)/Σi = 1, n Σj > i, n wyij, where
n is the number of individuals in the data set, and Dij is the
genetic distance between observations i and j. Elements of a
binary matrix, Wyij, take on values of 1 if the geographical
distance between observation i and j fall within the bounda-
ries specified for a specified DC and are 0 otherwise. Ay can
be interpreted as the average genetic distance between pairs
of individuals that fall within a specified DC. Ay takes on
a value of 0 when all individuals within a DC are geneti-
cally identical and takes on a value of 1 when all individu-
als within a DC are completely dissimilar. The probability
for each DC are obtained using 1,000 randomizations. For
this analysis, there were six defined DCs for the three-gene
datasets (0–50km; 50–150km; 150–210km; 210–270km;
270–440km; 440–800km) each of different size but with an
equal number of individual comparisons. This analysis was
carried out with AIS software (Miller 2005).
Another spatial analysis was carried out with Monmo-
nier’s Algorithm (Monmonier 1973; MMDA) using AIS
software (Miller 2005). This geographical regionalization
procedure is used to detect the locations of putative barriers
to gene flow by iteratively identifying sets of contiguous,
large genetic distances along with connectivity networks
(Doupanloup etal. 2002; Manel etal. 2003; Manni etal.
2004). A Delaunay triangulation (Watson 1992; Brouns
etal. 2003) was used to generate the connectivity network
among sampling points. A graphical representation of puta-
tive “barriers” inferred by the algorithm is superimposed
over the connectivity network to detect rapid identification
of important geographical features reflected by the genetic
dataset. In this case, we used this procedure to detect the
five most important geographical barriers contained in the
dataset for N. olivacea.
Results
Phylogenetic analyses
The nucleotide substitution models which were the best
for the three mt genes taken together were TN93 + G for
BIC (20,268.155) and GTR + G + I for AIC (13,234.043),
respectively.
The MLT (Fig.2) showed that the first Nasua-Nasuella
clade to diverge (76%) consisted of two specimens of N.
olivacea (Risaralda-Colombia, and Pichincha-Ecuador; we
named this group FGO-N) and 14 specimens of N. nasua
from the Colombian, Ecuadorian and northern Peruvian
Amazon, Colombian Eastern Llanos and one specimen
from Santa Cruz-Bolivia (bootstrap support, BS = 97%).
The second clade to diverge had four specimens of N. oliva-
cea (BS = 82%; we named this group SGO-N), all from the
trans-Andean area of Colombia (Chocó, Cauca, and Nariño
Departments) and Ecuador (Pichincha Province). The third
clade to diverge was completely integrated by 37 specimens
of N. nasua. Within this clade, there were two specimens
from the western-central Brazilian Amazon, which presented
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Fig. 2 Maximum likelihood tree
(ML) for 42 Nasuella olivacea,
50 Nasua narica, and 51 Nasua
nasua specimens sampled
throughout the Neotropics
sequenced at three mitochon-
drial genes (ND5, Cyt-b, and
D-loop). Nodes are labelled
with bootstrap percentages
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a low bootstrap. However, the remaining specimens of this
clade yielded an elevated bootstrap (BS = 84%). This clade
was characterized by three large sub-clades from precise
geographical areas: throughout the entire Peruvian Ama-
zon (Loreto, Ucayali, and Manu National Park, Cusco;
BS = 78%), part of the southern Peruvian Amazon and
Bolivia (BS = 86%), and southern South America and the
Brazilian state of Goias (Paraguay, Uruguay, and Iguazú
National Park in southern Brazil; BS = 95%). A few speci-
mens from the western and central Brazilian Amazon were
present among these three geographical sub-clades. The
fourth large clade to diverge consisted of the major frac-
tion of specimens studied of N. olivacea (BS = 84%). Within
this clade, we detected two significant sub-clades. One
composed of nine specimens from the Central Colombian
Andean Cordillera, and a few from the Western Colombian
Fig. 2 (continued)
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Andean Cordillera, plus some specimens from the trans-
Andean northern area of Ecuador (BS = 81%; we named
this group GCENTRAL-NO). Another contained 27 speci-
mens (BS = 80%; we named this group GEASTERN-NO),
all from the Eastern Colombian Andean Cordillera (Boy-
acá, Cundinamarca, and Norte de Santander Department),
and curiously, also a specimen from the Ecuadorian Sangay
National Park in Eastern Ecuador. There was also a mys-
terious small sub-clade consisting of two N. narica from
Panama and southern Costa Rica, but with mitochondrial
DNA from N. olivacea (BS = 90%). The fifth and last large
clade to diverge was composed of all the N. narica, except
the two from southern Central America with the DNA of
N. olivacea (BS = 99%). There were no sub-clades with
high bootstrap percentages within this clade. However, the
specimens sampled in southern Mexico and few of north-
ern Guatemala tend to clump together, whereas the speci-
mens from central and northern Guatemala and Belize tend
to create another cluster, and the specimens from southern
Guatemala, Honduras, El Salvador, Nicaragua, and north-
ern Costa Rica form another cluster. One specimen from
Cozumel Island (N. narica nelsoni or N. nelsoni for some
authors) had mitochondrial sequences that were undifferenti-
ated from the other Mexican coati specimens.
Therefore, the MLT showed the existence of four groups
of specimens with the typical morphology and within the
geographical distribution of N. olivacea. Two small groups
were integrated by a few specimens more related with
mtDNA of N. nasua (these specimens are always from Cen-
tral and Western Colombian Andean Cordilleras and trans-
Andean Ecuador) and the main group of N. olivacea. This,
in turn, showed two differentiated groups, one in the Eastern
Colombian Andean Cordillera and another in Central and
Western Colombian Andean Cordillera and northern trans-
Andean Ecuador. Two specimens with the phenotype of N.
narica and within the southern Central American range of
this species had mitochondrial DNA related with the group
of N. olivacea from the Eastern Colombian Andean Cordil-
lera. It is rather remarkable that the main group of N. oliva-
cea was more related with N. narica than this last with N.
nasua. This provides evidence against Nasuella as a different
genus from Nasua.
The MJN (Fig.3) showed the following picture. If we
assumed that the oldest haplotypes are closest to the out-
groups (three species of Bassarycion), then haplotypes 56
and 79 (two specimens of Nasuella, FGO-N), and haplotype
3 (and related) of N. nasua, the last one widely distributed in
the Amazon and Eastern Colombian Llanos, were the first to
appear within Nasua-Nasuella. Next to diverge (from haplo-
types 56 and 79), was a group of haplotypes from specimens
phenotypically similar to N. olivacea (SGO-N). These haplo-
types are a “bridge” between the first (and oldest) haplotypes
of Nasuella and N. nasua and the remaining haplotypes of
N. olivacea. GEASTERN-NO was the first to appear within
the main body of haplotypes of N. olivacea. GCENTRAL-
NO derived from this first group. Within GEASTERN-NO,
two haplotypes (81 and 82) belonged to specimens of N.
narica (Panama and southern Costa Rica), similar to what
we identified with the MLT. Finally, the haplotypes of all the
Central American coatis, N. narica, derived from GCEN-
TRAL-NO. Based on these data, haplotypes of N. narica
were the youngest to appear.
There were striking temporal splits between and within
the different taxa and groups detected in Nasua-Nasuella.
(1) We estimated a temporal split between the haplotypes
of Bassarycion. neblina-medius and the most original hap-
lotype of N. nasua (H3) of around 17.7 ± 0.4 MYA; (2).
The temporal splits between the GEASTERN-NO and N.
nasua was estimated to have occurred 2.835 ± 0.051 MYA,
whereas the temporal divergence between the GCENTRAL-
NO and N. nasua was dated to around 3.763 ± 0.051 MYA.
Between both main groups of N. olivacea, the temporal
splits were dated around 3.612 ± 0.785 MYA. The temporal
splits between N. nasua and N. narica were estimated to
have occurred around 5.415 ± 0.236 MYA. (3) The inter-
nal temporal split in N. nasua (6.145 ± 1.293 MYA) and in
N. narica (1.089 ± 0.230 MYA) were other time splits. For
additional time splits, see Table2.
Taking into account the diversification within each taxon,
it seems that the oldest haplotypes were that of N. nasua, fol-
lowed by the haplotypes of both main groups of N. olivacea,
with the haplotypes of N. narica as the youngest ones.
Genetic heterogeneity andgenetic distances
The overall genetic heterogeneity tests of the three species
revealed very significant differences (Table3). All the gene
flow statistics were considerably lower than 1 (NmγST = 0.34;
NmNST = 0.22; NmFST = 0.23), which supports that these taxa
are three different species. However, we also analyzed the
overall genetic heterogeneity of the four groups of N. oliva-
cea that were detected in the phylogenetic analyses. In all the
cases, the genetic heterogeneity was highly significant, even
more than in the previous case where the different groups
of N. olivacea were not considered. This means that these
four groups of N. olivacea—detected with the phylogenetic
procedures—are genetically, highly differentiated (Table4).
Again, the gene flow estimates were considerably lower than
1 (three-gene dataset only for N. olivacea: NmγST = 0.30;
NmNST = 0.20; NmFST = 0.22), which not only ratifies the
distinctness of the species but also the differentiation of the
four groups detected within N. olivacea.
The genetic heterogeneity analysis by taxa pairs (the four
groups of N. olivacea, N. nasua, and N. narica) was esti-
mated by the FST statistic (Table5a). This statistic showed
that all the taxa pair comparisons were significant (with the
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Fig. 3 Median Joining Network on three mitochondrial genes (ND5,
Cyt-b, and D-loop) of 42 Nasuella olivacea, 50 Nasua narica, and
51 Nasua nasua specimens from the Neotropics. Taxa analyzed are
showed with different colors for their haplotypes: grey circles = Bas-
sarycion alleni (outgroup); orange circles = Bassarycion neblina
(outgroup); pink circles = Bassarycion medius medius (outgroup);
lilac circles = some specimens phenotypically Nasuella olivacea
(Risaralda Department, Colombia; Carchi Province, Ecuador; FGO-
N); brown circles = some specimens phenotypically Nasuella oli-
vacea (Chocó, Cauca, Nariño Departments, Colombia; Pichincha
Province, Ecuador; SGO-N); yellow circles = Nasua nasua; green
circles = Nasuella olivacea; blue circles = Nasua narica. Red cir-
cles (with mv) indicate missing intermediate haplotypes. Numbers
between haplotypes = number of mutations that differentiated the hap-
lotypes
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Bonferroni’s correction α = 0.0033), with the exception of
FGO-N with SGO-N (p = 0.074), and FGO-N with GEAST-
ERN-NO (p = 0.018). The gene flow estimates for the taxa
pair comparisons (Table5b) showed low levels of gene flow
(lower than 1) for all the comparisons. The value nearest to 1
was that between N. nasua and FGO-N (Nm = 0.94).
The Kimura 2P genetic distances (Table6) showed the
highest range of values among the different taxa of Nasua-
Nasuella and the outgroups (three species of Bassarycion;
between N. narica and Bassarycion neblina, 47.5%, and
FGO-N and Bassarycion neblina, 34.9%). These genetic
distances are in the range of well differentiated genera. The
major part of the genetic distances among the seven Nasua-
Nasuella groups considered were above the value considered
for different species of the same genus (11–12%; see intro-
duction). The lowest values, below this limit were the cases
of N. nasua vs. SGO-N (9.9%), FGO-N vs. SGO-N (9.5%),
GEASTERN-NO vs. GCENTRAL-NO (8.9%), GCEN-
TRAL-NO vs. the two N. narica with mtDNA of N. oli-
vacea (6.8%), and GEASTERN-NO vs. the two N. narica
with mtDNA of N. olivacea (6.5%). Henceforth, values less
than the average genetic distance values of full species were
between N. nasua and one of the small and differentiated
groups of N. olivacea. In addition, they were among some of
the groups of N. olivacea, and among the two main groups
of N. olivacea and the two N. narica with mtDNA of N.
olivacea. Additionally, the magnitude of genetic differentia-
tion between N. narica and the groups of N. olivacea was
Table 2 Some estimated temporal splits among and within the two
species of Nasua (N. nasua and N. narica) and Nasuella olivacea
(four groups: FGO-N, SGO-N, GEASTERN-NO, and GCENTRAL-
NO; for geographical origins of these four groups, see text), and Bas-
sarycion alleni analyzed at three mitochondrial genes (ND5, Cyt-b,
and D-loop), by means of the ρ statistic applied on the median joining
network (assuming 1 mutation every 22,742years)
SD = Standard deviation; MYA = Millions of years ago
Species and groups Estimated temporal splits among and
within species and groups ± SD in MYA
Between N. nasua and N. olivacea (FGO-N) 0.618 ± 0.053
Between N. nasua and N. olivacea (SGO-N) 1.598 ± 0.103
Between N. narica and N. olivacea (GEASTERN-NO) 4.227 ± 0.472
Between N. narica and N. olivacea (GCENTRAL-NO) 3.557 ± 0.708
Within N. olivacea (GEASTERN-NO) 3.331 ± 0.843
Within N. olivacea (Gcentral-NO) 1.900 ± 0.459
Within Bassarycion alleni 0.253 ± 0.128
Table 3 Genetic heterogeneity and gene flow statistics across the
specimens of Nasuella olivacea, Nasua narica, and Nasua nasua
analyzed in this study for three mitochondrial genes (ND5, Cyt-b, and
D-loop); ** p < 0.01, df = degrees of freedom
Nm gene flow statistics, Nm1 Gene flow estimated from γST, Nm2
Gene flow estimated from NST, Nm3 Gene flow estimated from FST
Genetic heterogeneity and
gene flow statistics
Values Probabilities
χ2272.000 df = 160 0.00001**
HST 0.0039 0.00001**
KST 0.5908 0.00001**
KST* 0.3057 0.00001**
ZS1594.771 0.00001**
ZS* 7.0623 0.00001**
Snn 1.0000 0.00001**
γST 0.5969 0.00001**
NST 0.6980 0.00001**
FST 0.6814 0.00001**
Nm10.34
Nm20.22
Nm30.23
Table 4 The genetic heterogeneity and gene flow statistics across the
specimens for the four groups of Nasuella olivacea detected in the
phylogenetic analyses at three mitochondrial genes (ND5, Cyt-b, and
D-loop) *p < 0.05; **p < 0.01, df = degrees of freedom
Nm gene flow statistics, Nm1 Gene flow estimated from γST, Nm2
Gene flow estimated from NST, Nm3 Gene flow estimated from FST
Genetic heterogeneity and
gene flow statistics
Values Probabilities
χ2126.000 df = 93 0.01291*
HST 0.0371 0.0035**
KST 0.5798 0.00001**
KST* 0.3031 0.00001**
ZS216.0235 0.00001**
ZS* 5.1281 0.00001**
Snn 1.0000 0.00001**
γST 0.6223 0.00001**
NST 0.7114 0.00001**
FST 0.6928 0.00001**
Nm10.30
Nm20.20
Nm30.22
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similar, or even lower, than the values between N. narica
and N. nasua.
Genetic diversity levels anddemographic
trajectories
The genetic diversity analyses are shown in Table7 (total
sample of N. olivacea; GEASTERN-NO, GCENTRAL-NO,
N. nasua, and N. narica). In general, the data showed that
the levels of genetic diversity were very high for all the lin-
eages studied. The number of haplotypes and the Hd were
elevated and similar in all five lineages. These statistics are
influenced by the sample sizes and therefore a comparative
discussion is not very explanatory. In contrast, the compari-
son of the statistics π and θ per sequence is more interest-
ing because they are not affected by sample size. The N.
nasua sample presented the highest levels of genetic diver-
sity (π = 0.0647 ± 0.012 and θ = 16.447 ± 4.846), followed
Table 5 A FST pair statistics (below main diagonal; significance
upper main diagonal) and B gene flow estimates among six groups of
coatis, four groups of Nasuella olivacea (FGO-N, SGO-N, GEAST-
ERN-NO, GCENTRAL-NO), Nasua narica, and Nasua nasua ana-
lyzed by means of three mitochondrial genes (ND5, Cyt-b, and
D-loop)
*P< 0.0033 (Bonferroni’s correction)
A FST pair statistics
Taxa N. olivacea FGO-N N. olivacea SGO-N N. olivacea
GEASTERN-
NO
N. olivacea
GCENTRAL-
NO
N. narica N. nasua
N
. olivacea FGO-N
**
*
N. olivacea SGO-N 0.479****
N. olivacea GEASTERN-NO 0.856 0.787*
**
N. olivacea GCENTRAL-NO 0.785 0.740 0.69
6*
*
N. narica 0.855 0.836 0.846 0.79
9*
N. nasua 0.346 0.526 0.708 0.657 0.735
B gene flow
Taxa N. olivacea FGO-N N. olivacea SGO-N N. olivacea
GEAST-
ERN-NO
N. olivacea
GCEN-
TRAL-NO
N. narica N. nasua
N. olivacea FGO-N
N. olivacea SGO-N 0.544
N. olivacea GEASTERN-NO 0.084 0.135
N. olivacea GCENTRAL-NO 0.138 0.175 0.219
N. narica 0.085 0.098 0.091 0.125
N. nasua 0.943 0.450 0.206 0.261 0.180
Table 6 Kimura 2P genetic distance (Kimura 1980) in percent-
ages (%) among different taxa of Nasuella olivacea (four groups;
1 = FGO-N, 2 = SFGO-N, 3 = GEASTERN-NO, 4 = GCENTRAL-
NO), 5 = Nasua narica, 6 = southern Central American Nasua narica
sharing mtDNA of N. olivacea, 7 = Nasua nasua, 8 = Bassarycion
neblina, 9 = Bassarycion medius, 10 = Bassarycion alleni (below
main diagonal) and standard deviations in percentages (%) (above
main diagonal) estimated by means of three mitochondrial genes
(ND5, Cyt-b, and D-loop)
Taxa 12345678910
1 1.5 2.0 2.1 1.9 2.3 1.9 4.8 3.8 4.6
2 9.5 2.7 2.4 2.1 2.6 1.3 4.8 4.2 4.7
3 13.6 17.4 1.4 2.2 1.3 2.5 5.1 4.4 4.8
4 15.6 16.8 8.9 2.0 1.2 2.4 5.0 3.9 4.4
5 14.5 14.6 13.8 11.3 2.6 2.3 5.5 4.2 5.0
6 16.0 17.6 6.5 6.8 12.8 2.5 4.9 4.0 4.7
7 14.1 9.9 18.1 18.0 17.2 18 4.8 4.4 5.0
8 43.3 42.6 43.8 47.3 47.5 44.6 43.0 2.6 2.0
9 36.4 34.9 39.0 39.0 36.9 38.6 37.9 15.6 3.0
10 39.6 40.0 40.2 41.0 43.2 41.7 41.7 11.9 19.1
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by the total sample of N. olivacea (π = 0.0503 ± 0.007 and
θ = 17.008 ± 5.319), GCENTRAL-NO (π = 0.0339 ± 0.0045
and θ = 9.566 ± 4.312) and GEASTERN-NO
(π = 0.0230 ± 0.0049 and θ = 11.156 ± 3.842). The taxa
with the lowest genetic diversity analyses was N. narica
(π = 0.0195 ± 0.0016 and π = 6.985 ± 2.274). These results
agree quite well with the phylogenetic tree and MJN pro-
cedures. The haplotypes of N. nasua seems to be the oldest
with the highest degree of differentiation among populations
classified within this species. The haplotypes of N. olivacea
appeared later and then N. narica, with the youngest of all
the haplotypes analyzed.
The demographic analyses showed the following picture.
For the analyses with diverse statistics and a mismatch dis-
tribution, the overall sample of N. olivacea only showed one
out of six analyses as significant (FS = − 6.713, p = 0.025),
thus indicating population expansion. This was a rather
weak support of a possible population expansion for this
species. For GCENTRAL-NO, there was little evidence of
demographic changes. Only one statistic was significantly
in agreement with a population expansion (FS = − 3.089,
p = 0.034). In contrast, GEASTERN-NO yielded strong
evidence of population expansion. The five statistics
were significant (D = − 1.888, p = 0.014; D* = − 2.308,
p = 0.029; F* = − 2.558, p = 0.026; FS = − 4.963, p = 0.024;
R2 = 0.074, p = 0.042) as well as for the mismatch distribu-
tion (rg = 0.0173, p = 0.048). N. narica also presented strong
evidence of population expansion (D* = -2.666, p = 0.019;
F* = − 2.449, p = 0.023; FS = − 5.014, p = 0.044). No demo-
graphic changes were detected for the last species, N. nasua.
For the BSP analyses (Fig.4), the overall sample of N.
olivacea showed a considerable female population increase
starting 500,000 YA and a very strong population decrease
in the last 20,000 Y. The first temporal estimate was rela-
tively similar to that obtained with the mismatch distribu-
tion, assuming a generation time for the coatis of 1–2years
(τ = 17.129; 251,100–502,200 YA). For GCENTRAL-NO,
the demographic size remained constant during the last 0.9
MYA until the last 20,000 Y when there was a very strong
population decrease. For GEASTERN-NO, there was a
strong population expansion in the last 250,000 Y and a
strong population decrease in the last 20,000 Y. With the
mismatch distribution, the initial expansion was estimated to
have occurred 70,800–141,600 YA (τ = 4.829). For N. nasua,
there was very little clear evidence of population increase.
Maybe, there was a very slight increase from 70,000 YA
to 20,000 YA, but there was a clear decrease in the last
20,000 Y. Finally, N. narica presented evidence of popula-
tion expansion in the last 120,000 Y, with a strong popula-
tion decrease in the last 20,000 Y. With the mismatch dis-
tribution, the initial expansion was very similar to the BSP
estimate. It was dated to 63,400–126,800 YA (τ = 4.322).
Therefore, there was suggestive population expansion for the
overall sample of N. olivacea, for GEASTERN-NO, and for
N. narica. However, there was no clear evidence of popula-
tion increase for GCENTRAL-NO or for N. nasua. Based
on the BSP analysis of all five taxa, there was an extremely
similar strong population decrease in the last 20,000 Y.
Spatial genetic structure ofNasuella olivacea
The application of Mantel’s test offered very similar results
in the detection of global spatial structure. The Mantel’s
test with the three-gene dataset (Fig.5) showed a signifi-
cant relationship between geographical and genetic distances
(r = 0.3713; p = 0.000099). This means that the geographic
distance significantly explained around 13.79% of the
genetic distances.
The spatial autocorrelation analysis using 6 DCs showed
a significant overall correlogram (V = 0.0237; p = 0.0001)
(Fig.6). The first two DCs showed significant positive
autocorrelation (1 DC: 0–50km, p = 0.000001; 2 DC:
50–150km, p = 0.0320). The third DC presented nega-
tive autocorrelation without reaching statistical signifi-
cance. The fourth DC was also significantly positive (4 DC:
210–270km, p = 0.0001). The two last DCs were signifi-
cantly negative (5 DC: 270–440km, p = 0.000001; 6 DC:
440–800km, p = 0.0131). Henceforth, the overall corre-
logram showed a significant spatial pattern with regional
Table 7 Genetic diversity statistics (and ± standard deviation) in the
total sample of Nasuella olivacea, in the N. olivacea from Eastern
Colombian Andean Cordillera (GEASTERN-NO), in the N. olivacea
from Western and Central Colombian and Ecuadorian Andean Cor-
dilleras (GCENTRAL-NO), in Nasua narica, and Nasua nasua ana-
lyzed at three mitochondrial (mt) genes (ND5, Cyt-b, and D-loop)
N Number of haplotypes, Hd Haplotype diversity; π Nucleotide diversity; θ N, Ne effective female population size, μ mutation rate per genera-
tion
Taxa N Hdπ θ
Overall sample of N. olivacea 27 0.943 ± 0.032 0.0503 ± 0.007 17.008 ± 5.319
GEASTERN-NO 17 0.892 ± 0.055 0.0230 ± 0.0049 11.156 ± 3.842
GCENTRAL-NO 9 1.000 ± 0.052 0.0339 ± 0.0045 9.566 ± 4.312
N. narica 20 0.916 ± 0.024 0.0195 ± 0.0016 6.985 ± 2.274
N. nasua 34 0.969 ± 0.012 0.0647 ± 0.0033 16.447 ± 4.846
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patches in the first 200km and isolation by distance from
around 200 to 800km.
The MMDA procedure for the three-gene dataset is
shown in Fig.7. Five possible geographical barriers were
analyzed. The first barrier (blue in Figure) delimited the
geographical area from the Risaralda Department (Colom-
bian Central Andean Cordillera) to the Pichincha Province
(northern Ecuadorian Western Andean Cordillera). This
area agrees quite well with the FGO-N detected in the
MLT and in the MJN. The second barrier (green) discrim-
inated a geographical area from the Chocó Department
(Colombian Western Andean Cordillera) to the Carchi
and Pichincha Provinces (northern Ecuadorian Western
Andean Cordillera). This coincides with the SGO-N and
the major part of the specimens from GCENTRAL-NO
detected in the MLT and in the MJN. The third barrier
(green bluish) was located in a geographical area within
Ecuador (Pichincha and Cotopaxi Provinces), which
Fig. 4 Bayesian skyline plot analyses (BSP) to determine possible
demographic changes across the natural history of different taxa of
Nasuella and Nasua for three mitochondrial genes (ND5, Cyt-b, and
D-loop) in the last 0.7–1 million of years. a Nasua narica; b Nasua
nasua; c Overall sample of Nasuella olivacea; d Sample of Nasuella
olivacea from Western and Central Andean Colombian and Ecuado-
rian Cordilleras (GCENTRAL-NO); e Sample of Nasuella olivacea
from Eastern Andean Colombian Cordillera (GEASTERN-NO)
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agreed quite well with an Ecuadorian sub-group detected
within GCENTRAL-NO in the MLT. The fourth and fifth
barriers (brown and lilac, respectively) delimited two
geographical areas which corresponded to GEASTERN-
NO. The first one contained specimens sampled in the
Departments of Cundinamarca and a fraction of Boyacá,
whereas the second one delimited another fraction of the
Boyacá Department as well as the Norte de Santander
Department. Therefore, there was good correspondence
between the MMDA and the MLT. Maybe the unique dif-
ferences were that the MMDA differentiated less SGO-N
from GCENTRAL-NO than did the MLT. However, it
more remarkably differentiated the Ecuadorian sub-group
within GCENTRAL-NO and distinguished more sharply
two sub-groups within GEASTERN-NO.
Fig. 5 Mantel test between
the geographic and genetic
distances for specimens of
Nasuella olivacea sequenced
for three mitochondrial genes
(ND5, Cyt-b, and D-loop)
Fig. 6 Correlograms with the
Ay statistic and six distance
classes from a spatial autocor-
relation analysis for specimens
of Nasuella olivacea sequenced
for three mitochondrial genes
(ND5, Cyt-b, and D-loop)
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Discussion
The systematics ofNasuella
This work shows the most extensive molecular analysis to
date for N. olivacea trying to establish how many differenti-
ated taxa are within the mountain coati. Aforementioned,
Helgen etal. (2009) only studied a small fragment of mtCyt-
b from an extremely small sample and still determined that
the unique sample of the mountain coati from the Merida
Fig. 7 Monmonier’s algorithm analysis (MMAA) to detect the five
most important geographical barriers for the specimens of Nasuella
olivacea sampled in Colombia and Ecuador for three mitochondrial
genes (ND5, Cyt-b, and D-loop). First barrier (blue) = Geographi-
cal area from the Risaralda Department (Colombian Central Andean
Cordillera) to the Pichincha Province (northern Ecuadorian Western
Andean Cordillera); second barrier (green point) = Geographical area
from the Chocó Department (Colombian Western Andean Cordillera)
to the Carchi and Pichincha Provinces (northern Ecuadorian West-
ern Andean Cordillera); third barrier (green-bluish) = Geographical
area within Ecuador (Pichincha and Cotopaxi Provinces); fourth bar-
rier (brown) = Geographical area, which enclosed the Cundinamarca
Department and a fraction of the Boyacá Department in Colombia;
and five barrier (lilac) = Geographical area, which enclosed a fraction
of the Boyacá Department as well as the Norte de Santander Depart-
ment
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Cordillera in Venezuela was a “new” species, N. meridensis.
Since then, the majority of authors consider the existence
of two mountain coati species: the Eastern mountain coati
(N. meridensis, Thomas 1901) and the Western mountain
coati (N. olivacea, Gray 1865). The type locality for the first
was Monte de Culata, Merida State, Venezuela (Cabrera
1958), while the type locality for the second was Santa
Fé de Bogotá, Colombia (“Bogotá, what should be inter-
preted as the mountains near the capital” claimed Cabrera
1958). Allen (1913) had defined another taxa, N. lagunetae,
also with the type locality in Bogotá and which is usually
consider as a synonym of N. olivacea. Additionally, Hel-
gen etal. (2009) considered that within N. olivacea, two
subspecies should be proposed, although they did not show
any molecular result in favor of this: N. o. olivacea (Gray
1865), distributed throughout the Colombian Andes with a
paler pelage (brown) and dark tail rings, and N. o. quitensis
(Lönnberg 1913; type locality—the southern slope of the
Pichincha volcano), distributed in the Ecuadorian Andes and
slightly smaller than the previous one with a darker pelage
(more blackish) and tail rings less visible than in the previ-
ous subspecies (darker tail).
However, our results from the analysis of three mt genes
from 42 specimens of the traditional N. olivacea (37 from
the three Colombian Andean Cordilleras, and five from the
two Ecuadorian Andean Cordilleras) showed a very differ-
ent perspective. We detected four differentiated molecular
groups within N. olivacea. The first group of specimens with
the morphology and distribution of N. olivacea were com-
posed by two specimens that originated in the middle of the
Central Colombian Andean Cordillera (Risaralda Depart-
ment) and in the Ecuadorian Pichincha Province (Western
Ecuadorian Andes). The haplotypes of these specimens are
more related to the haplotypes of the oldest haplogroup of
N. nasua we detected (Western Amazon Basin and Colom-
bian Eastern Llanos) than to the majority of the haplotypes
detected in the remainder N. olivacea specimens. The same
occurred with a second small group of N. olivacea composed
of four specimens distributed in the Western Colombian
Andean Cordillera (Chocó, Cauca, and Nariño Departments)
as well as in the Western Ecuadorian Andes (Pichincha Prov-
ince). The group’s haplotypes were more related to those of
N. nasua than to the majority of N. olivacea studied. The
MJN analysis showed that the haplotypes of these two small
groups were “a bridge” between the haplotypes of N. nasua
and those from main groups of N. olivacea, although more
related to N. nasua, such as it was found with the MLT. The
haplotypes of these two small groups, although more related
to N. nasua than to the remainder haplotypes of N. olivacea
(in the MLT, not in the genetic heterogeneity analyses), are
sufficiently different from those of N. nasua to form highly
distinctive branches that are significantly different in many
analyses (both phylogenetic and genetic heterogeneity ones).
We discard the notion of very recent or current hybridiza-
tion between N. nasua and specimens of N. olivacea as an
explanation of these results because their haplotypes were
significantly differentiable from the current haplotypes of
the most related N. nasua. However, we offer three hypoth-
eses as an explanation. (a) These two small groups were the
product of two independent events of old introgression of
N. nasua (Western Amazon) into N. olivacea, which came
about 0.6–1.6 MYA, respectively. (b) These two small
groups represent transitional forms from N. nasua haplo-
types to the most derived haplotypes of N. olivacea from
the GEASTERN-NO and GCENTRAL-NO haplogroups.
Figure3 and genetic heterogeneity analyses (see Table5)
agree quite well with this hypothesis. It is clear that the
haplotypes of FGO-N were derived from haplotypes of N.
nasua and, in turn, a haplotype of FGO-N gave origin to the
haplotypes of SGO-N, which, also in turn, gave origin to the
first haplotype of GEASTERN-NO. This hypothesis, which
we consider the most probable, however, is very problematic
from a systematic point of view. Transitional mtDNA, in
some analyses more related to N. nasua (example, MLT) but
with bodies fully of N. olivacea. If so, we should consider
these two groups as the most ancient N. olivacea. The results
of Ruiz-García etal. (2020b), with mitogenomes, reinforced
this hypothesis. For this reason, we have enclosed these two
small groups inside of all the analysis of N. olivacea that we
carried out. (c) The FGO-N and SGO-N haplotypes would
represent N. nasua haplotypes but the specimens with these
haplotypes morphologically evolved by convergent adaption
to very similar morphotype to that shown by N. olivacea
by living in the same Andean biome, but they are really N.
nasua. Only an analysis of nuclear DNA markers should
help to test the three hypotheses.
The other two groups of N. olivaceas haplotypes were
considerably more represented in the specimens analyzed.
The first to appear seemed to be composed by all the speci-
mens sampled in the Eastern Colombian Andean Cordillera
from the Cundinamarca, Boyacá, and Norte de Santander
Departments. Although this group was basically found in
this cited area, we also detected within it, one specimen from
the Caldas Department (Central Colombian Andean Cordil-
lera) and one specimen from Eastern Ecuador (Sangay NP).
Therefore, after this group formed, some migrations could
have occurred relatively recently from the Eastern Colom-
bian Andean cordillera towards the West and South because
the haplotypes of these two specimens are very similar.
Within this group, we found two specimens of N. narica
from southern Central America (Panama, and southern
Costa Rica). Their morphotypes, as well as their geographic
origins, were undoubtedly specimens of N. narica. Some-
thing similar was found by Nigenda-Morales etal. (2019) as
we will discuss in brief. Such as in the previous case, they
cannot represent events of recent hybridization between N.
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olivacea and N. narica. Although the differences between
the haplotypes of southern Central American N. narica and
GEASTERN-NO were not high, as we observed between
the haplotypes of the two first small groups of N. olivacea
and those from N. nasua, they were significantly different.
This could be interpreted as a case of genetic introgression
of the N. olivacea from GEASTERN-NO into the southern
Central American distribution of N. narica. However, this
introgression event occurred more recently than the two pos-
sible introgression events of N. nasua in N. olivacea that we
previously described.
GEASTERN-NO gave origin to another large haplogroup
of N. olivacea, GCENTRAL-NO. Therefore, there was con-
siderably more genetic heterogeneity among the specimens
of N. olivacea sampled within the Western and Central
Colombian Andean Cordilleras (where we found speci-
mens belonging to four different groups, FGO-N, SGO-N,
GCENTRAL-NO, and one specimen from Caldas classified
in GEASTERN-NO), and within Ecuador (where we also
found specimens belonging to these four groups), than in
the Eastern Colombian Andean Cordillera, where only one
gene pool was discovered.
These results radically change the systematics of Nasuella
proposed by Helgen etal. (2009). First, these authors did not
include samples of Nasuella from the Eastern Colombian
Andean in their analyses. We compared the unique sequence
they studied of the proposed new N. meridensis with some
mtCyt-b gene sequences of the Nasua and Nasuella we
studied with a maximum likelihood tree and it was un-dif-
ferentiable from the sequences of the Nasuella that we ana-
lyzed from the Eastern Colombian Andean Cordillera (see
Fig.8). This tree is less robust than that we show in Fig.2,
because only a fraction of the specimens were analyzed (110
specimens vs. 143 specimens) and the length of the mtDNA
sequence was of 366bp vs. 2513bp. This introduces certain
inconsistences in this tree with only a small fraction of the
mtCyt-b gen in reference to the three of Fig.2. This also
shows the importance to obtain large sample sizes represent-
ing the broader geographical distribution as possible of a
species given. However, this tree only wants to show as the
Venezuelan specimen considered a “new species” by Helgen
etal. (2009) is undifferentiated from the N. olivacea from
the Eastern Colombian Andean Cordillera. Therefore, from a
genetic point of view, N. olivacea olivacea (Gray 1865) and
N. meridiensis (Thomas 1901) are synonyms. This means
that the mountain coatis from Eastern Colombian Andean
Cordillera and those from the Venezuelan Merida Cordillera
should be named as N. o. olivacea (Gray 1865). As with the
other two Colombian Cordilleras and in Ecuador, at least
three other haplogroups were detected. If we consider that
the two small groups were caused by genetic introgression
from N. nasua from the Western Amazon (hypothesis 1),
perhaps they should have no systematic value and should be
related to GCENTRAL-NO. If so, all of these specimens of
N. olivacea from Western and Central Colombian Andean
Cordilleras and those from the Western and Central Ecuado-
rian Andean Cordilleras should be named as N. o. quitensis
(Lönnberg 1913). In contrast, if the two small groups were
the first N. olivacea derived from N. nasua, which, in turn,
gave origin to the two main groups of N. olivacea (hypoth-
esis 2, which we believe the most probable), they should be
subspecifically named. Thus, three subspecific names are
required, and only one is available (N. o. quitensis), but we
have no idea which of the three groups is correlated with N.
o. quitensis. Confirmation would require a DNA analysis of
the holotype of N. o. quitensis. In whatever case, two new
subspecific names should be required for two of these three
groups of N. olivacea detected in the Western and Central
Colombian Andean Cordilleras and those from the Western
and Central Ecuadorian Andean Cordilleras. We consider
that the two main Nasuella groups to be subspecies more
than full species as Helgen etal. (2009) considered. If we
take the break for full species values of genetic distances of
11–12%, the relationships of these two groups of N. oliva-
cea were below this amount. For example, GEASTERN-NO
vs. GCENTRAL-NO showed 8.9%. Until there is extensive
work of possible reproductive isolation among these taxa
or important karyotypic differences (which appear to have
no existence, or they are minimal; Jaramillo etal. 2020),
we prefer to treat those taxa as subspecies of N. olivacea.
Finally, if these two small groups were N. nasua, which
evolved by convergent adaption to the Andean biome with a
similar body of N. olivacea, they have not any significance
for the systematics of N. olivacea.
The phylogenetic relationships between Nasuella
olivacea and thetwo species ofNasua andsome
systematic notes for N. nasua and N. narica
Helgen etal. (2009) concluded that Nasuella (Hollis-
ter 1915) and Nasua (Storr 1780) should be unique gen-
era because Nasuella was more related to N. narica than
this last taxon with N. nasua. In fact, Gray (1843, 1865)
described the mountain coati as Nasua olivacea. Later, Hol-
lister (1915) proposed the genus Nasuella for the mountain
coati, and this was retained by Goodwin (1953) and is still
used today.
Our results agree quite well with Helgen etal. (2009)
because we also detected a close relationship between the
two main haplogroups of N. olivacea and N. narica more
so than between N. narica and N. nasua. This was true for
all of the analyses we carried out. For instance, the genetic
distances between the two main groups of N. olivacea
(GEASTERN-NO, and GCENTRAL-NO) vs. N. narica
were 13.8% and 11.3%, respectively, whilst N. nasua and
N. narica offered a value of 17.2%. Nigenda-Morales etal.
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(2019) found three haplotypes from 13 samples derived from
several locations in Panama, which constituted the earliest
branching lineage within the mitochondrial gene tree they
obtained; these haplotypes were highly divergent from the
remaining haplotypes of N. narica that they found in other
areas of Central and North America (9.92–10.78%). Now,
thanks to the current work and Ruiz-García etal. (2020b),
we know that the Panamanian N. narica specimens were
introgressed with the mtDNA from N. olivacea and that
they are not a possible new species. This is the motif why,
both Nigenda-Morales etal. (2019) and the present work,
found high genetic distances (very similar in both works)
between the Panamanian N. narica specimens and the
remainder N. narica specimens. Nigenda-Morales etal.
(2019), throughout the ML and BI analyses that incorporated
mtCyt-b sequences of two N. olivacea specimens, obtained
trees showing that these two specimens were placed inside
the N. narica clade. Now, we know that the Panamanian
N. narica (as well as some specimens from southern Costa
Rica and northern Colombia; Ruiz-García etal. 2020b)
Fig. 8 Maximum likelihood tree
(ML) for 1 Nasuella meridien-
sis, 15 Nasuella olivacea, 41
Nasua narica, and 53 Nasua
nasua specimens sampled
throughout the Neotropics
sequenced for the mtCyt-b gene.
Potos flavus was employed as
out-group. Nodes are labelled
with bootstrap percentages
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were nested inside one of the haplogroups of N. olivacea
(GEASTERN-NO).
The ancestors of GEASTERN-NO, and GCENTRAL-
NO diverged from the ancestor of N. narica around 3.6–4.2
MYA (and the split between these two haplogroups was
dated around 3.6 MYA; Late Pliocene). Nigenda-Morales
etal. (2019), employing two different analytical schemes
that differed in the calibration priors employed, estimated
the temporal divergence between the haplotypes of the
Panamanian N. narica (introgressed with mtDNA of N. oli-
vacea) and the remainder N. narica haplotypes around 4
MYA (95% highest posterior density, HPD = 2.0–6.7 MYA
and 2.6–5.1 MYA for the two different analytical schemes
employed, respectively). Therefore, these temporal diver-
gence estimates were very similar in both works.
All the phylogenetic analyses showed that the haplotypes
of N. narica were the youngest. This means that the evolu-
tion of the current mitochondrial haplotypes of the coatis
Fig. 8 (continued)
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began in South America (around 6.1 MYA in N. nasua; Late
Miocene; and split between the ancestors of N. nasua and N.
narica, around 5.4 MYA). This result totally concords with
that found by Nigenda-Morales etal. (2019). These authors
estimated divergence times with the two different analytical
schemes they employed and they estimated a split between
the ancestors of N. nasua and N. narica, around 6 MYA,
very similar to the estimates herein reported. Ruiz-García
etal. (2020b), in an extensive work with N. nasua, estimated
that the original haplotypes of the ancestor of the current
coatis were dated around 13 MYA. This agrees quite well
with the model M2 of the three DEC models employed by
Nigenda-Morales etal. (2019), which suggested that disper-
sion of the ancestor of Nasua across the Isthmus of Panama
likely occurred before 9.5 MYA.
More recently, N. narica derived from South America
and became distributed in Central America (1.1 MYA;
Pleistocene). Nigenda-Morales etal. (2019) estimated the
divergence time for the four clades of N. narica they found,
excluding the Panamanian group, in around 1.3 MYA (95%
HPD = 0.59–2.1 MYA). Thus, both works also showed simi-
lar temporal divergence splits.
The traditional paleontological view maintained that
the procyonids dispersed from North America into South
America two separate times, being one of the first groups of
North American mammals to colonize South America. The
first dispersion event took place around 7.3–5 MYA (Late
Miocene) and the fossil genus Cyonasua in South America
is an evidence, long before the closure of the Isthmus of Pan-
ama and the GABI, which approximately occurred around
2.4 to 2.8 MYA according to Marshall etal. (1979, 1982),
Webb (2006), Woodburne (2010). All descendants from
that first colonization apparently went extinct by the end
of the Middle Pleistocene (Marshall 1985; Soibelzon and
Prevosti 2013). The second dispersion of procyonids into
South America was, following this hypothesis, the one made
by the ancestors of the extant genera in the last 0.125 MYA
(Woodburne 2010) in the Late Pleistocene. Therefore, the
current procyonid genera are not considered to be descend-
ants of the procyonids that originally invaded South America
(Baskin 2004; Soibelzon and Prevosti 2013; Forasiepi etal.
2014). This traditional view is based on that the complete
emergence of the Isthmus of Panama was around 3.0–3.5
MYA in the Middle Pliocene, resulting in the closing of the
Central American Seaway, CAS (Coates and Obando 1996;
O’Dea etal. 2016). After this event, the fossil record indi-
cates that the mammalian lineages predominantly migrated
from North America to South America around 2.4–2.8 MYA
(Simpson 1980; Woodburne 2010).
More recently, a second possible hypothesis has been
established. This hypothesis affirms that the appearance
of a land bridge and the closure of the CAS was around
13–15 MYA, during the Middle Miocene (Farris etal. 2011;
Montes etal. 2012, 2015; Carrillo etal. 2015). Indeed, the
Isthmus of Panama formation began earlier and seems to be
associated with the northern Andean uplift, around 24 MYA
(Farris etal. 2011). Some recent studies proposed that the
most significant periods of migration of mammals occurred
during 20 and 6 MYA, with similar migration rates between
North and South America, and that asymmetric migration
emerged after 6 MYA, with higher migration from South to
North America (Bacon etal. 2015; Marko etal. 2015). In
fact, Carrillo etal. (2015) showed that many faunal coloniza-
tion events associated with GABI began around 10 MYA.
Meanwhile, in the first hypothesis, geological events might
be responsible for preventing, or not, fauna colonization
events, in this second hypothesis, environmental processes
are the causes of promoting, or not, the faunal dispersion
(Bacon etal. 2015; Montes etal. 2015). For example, moist
and warm climate occurring in northern South America and
Central America before 3.5 MYA favored tropical environ-
ments preventing faunal interchange of some North Ameri-
can mammal species that do not thrive in densely forested
environments (Molnar 2008; Leigh etal. 2014). Neverthe-
less, N. narica is well adapted to forested habitats (Gomp-
per 1995) and likely would have easily colonized different
areas through tropical forests before dry savanna-like habi-
tats evolved in the Middle Pliocene (3.0–3.5 MYA; Webb
2006; Molnar 2008).
Our results with the coatis, as well as the results of
Nigenda-Morales etal. (2019), agree better with the second
hypothesis. Our results also agree with the other results with
molecular data found that the diversification within some
extant procyonid genera, as Procyon and Potos, occurred
in the Middle to Late Miocene, temporally coincident with
the diversification of the extinct genera in South America
(Koepfli etal. 2007; Ruiz-García etal. 2019). Addition-
ally, the results of Nigenda-Morales etal. (2019), and the
current ones, correlated well with the fact that the diversi-
fication of the South American extinct species Cyonasua
spp. and Chapalmalania spp. and of extant Nasua, Potos,
and Procyon species, 13–5 MYA, may have been part of
a temporally concordant diversification event predating the
GABI (Koepfli etal. 2007; Eizirik etal. 2010; Forasiepi
etal. 2014; Carrillo etal. 2015; Ruiz-García etal. 2019).
Moreover, Nasua and Procyon fossils dated around 1.5–3
MYA from Venezuela, showed the presence of these genera
in South America during the time of the full emergence of
the Panamanian isthmus (Ruiz-Ramoni etal. 2018).
This hypothesis aligns well with the origin of the cur-
rent coatis “in situ” in South America. It is correlated with
the results of the evolutionary histories of other Neotropical
mammals. The ancestors of some today’s South American
mammalian species colonized South America before the
complete closure of the Panamanian land bridge. Examples
of this, for example, come from data on Cebus capucinus
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(Ruiz-García etal. 2012a), Tapirus bairdii (Ruiz-García
etal. 2012b, 2016), Eira barbara (Ruiz-García etal. 2017),
and Potos flavus (Ruiz-García etal. 2019). The mitochon-
drial diversification of the Nasua-Nasuella complex within
South America during the Late Miocene-Pliocene aligns
with the fossils of some mammalian species collected from
the Panama Canal region and their close similarities to
taxa in North America. This suggests a broad connection
between Central and North America during the Miocene as
it was advanced by Whitmore and Stewart (1965). Indeed,
some current mammal species, which traditionally were
thought to have been radiated from North America during
the GABI and which they colonized South America in the
Pleistocene, we now know that the original radiation of the
current genetic lineages of these species occurred “in situ”
in South America. They were the cases of the jaguar (Pan-
thera onca; Ruiz-García etal. 2013), the puma (Puma con-
color; Culver etal. 2000), or the Andean bear (Tremarctos
ornatus; Ruiz-García etal. 2020a). Some of the results of
Nigenda-Morales etal. (2019) were related to this fact. They
found that the numbers of migrants between the Panama-
nian N. narica population and all other populations were
consistently asymmetric, with migration from Panama into
northern populations usually greater than in the opposite
direction (migration north to south). The S-DIVA and BBM
biogeographic analyses, carried out by Nigenda-Morales
etal. (2019), identified South America as an area of distri-
bution for the most recent common ancestor of Nasua and
Bassaricyon.
This work together with that of Nigenda-Morales etal.
(2019) showed that the initial diversification of the Nasua
species may have been in the northern Andes (current
Colombian and Ecuadorian Andes). The rapid uplift of the
northern Andes during the last 5–10 MYA (Hoorn etal.
2010; Mora etal. 2010) coincides with the temporal coati
splits found.
In contrast to the slight genetic differences among the
three sub-groups of N. narica found, the genetic differences
among the four detected groups within N. nasua were very
elevated. In fact, the mitochondrial divergence process
within N. nasua was estimated to begin around 6 MYA (Late
Miocene). The genetic distances among these four groups
were also noteworthy. These groups were placed in (1) the
Western Amazon (Colombia, Ecuador, and northern Peru-
vian Amazon) and Eastern Colombian Llanos, (2) through-
out a large area of the Peruvian Amazon, (3) southern Peru
and Bolivia, and in (4) Paraguay, Uruguay, and southern
Brazil frontier with Argentina. Traditionally, 10 subspecies
of N. nasua have been established (Cabrera 1958; Hershko-
vitz 1959; Decker 1991; Gompper and Decker 1998). The
four groups we detected could be in agreement with four of
these putative subspecies: (1) N. n. dorsalis (Western Ama-
zon and Eastern Colombian Llanos); (2) N. n. montana (in
the majority of the Peruvian Amazon); (3) N. n. boliviensis
(southern Peru and majority of Bolivia); and (4) N. n. spa-
dicea (Paraguay, Uruguay, and southern Brazil). The genetic
distances among these N. nasua groups (shown elsewhere)
are very high and the temporal splits among them were
considerable (between dorsalis and spadicea: 4.6 MYA;
between spadicea and boliviensis: 2.9 MYA; dorsalis and
boliviensis: 6.4 MYA). Many of these temporal splits were
similar, or even higher, to the time splits between N. nasua
and N. narica, or between N. nasua and different groups
of N. olivacea. Henceforth, these groups should be cryp-
tic species affected by morphological stasis (Eldredge etal.
2005; Gould 2007). This was also discovered in another pro-
cyonid, Potos flavus (Ruiz-García etal. 2019). Coatis are
extremely well adapted to many different environments and
it could be responsible for morphological stasis and cryptic
morphological species in N. nasua. There is considerable
molecular differentiation among these cryptic species. Obvi-
ously, nuclear DNA, karyotypic, ecological, and reproduc-
tive behavioral studies are needed to confirm that these N.
nasua taxon groups are completely different species. If so,
these four groups should be named as N. dorsalis, N. mon-
tana, N. boliviensis, and N. spadicea, respectively.
Genetic diversity, demographic changes, andspatial
structure
The levels of genetic diversity (especially nucleotide diver-
sity) were highly correlated with the time splits within each
one of the taxa analyzed. N. nasua showed the highest lev-
els of genetic diversity, followed by N. olivacea from the
Eastern Colombian Andes Cordillera, and then N. olivacea
from the Western and Central Colombian Andes Cordillera.
Finally, N. narica is the last and least genetically diverse
taxon, which also supports it as the youngest.
N. nasua did not show any evidence of population
expansion. This could be related to two events. First, if
N. nasua evolved the first and oldest haplotypes within
coatis, it would also be more challenging to detect possible
demographic changes. Second, within N. nasua, we found
robust genetic heterogeneity, to such an extent that maybe
each group should be considered as a cryptic species. This
strong genetic heterogeneity can make it difficult to detect
possible demographic changes in N. nasua. However, the
overall sample of N. olivacea showed a population expan-
sion which began in the last 0.5 MY, although this sam-
ple contained several different groups, but with a lower
genetic heterogeneity than found among the groups of N.
nasua. GEASTERN-NO also showed clear evidence of
population expansion in the last 0.2 MY. Nevertheless, the
other main group of N. olivacea, GCENTRAL-NO, did not
show clear evidences of demographic changes. This was
probably due to the relatively small sample size of this
Author's personal copy
Mammalian Biology
1 3
group. In contrast, N. narica showed a clear population
expansion in the last 0.12 MY, nicely correlating with it
as the youngest taxon. One common feature among the
four taxa is that they all had strong population declines in
the last 20,000years. This aligns with the Upper Plenigla-
cial period, around 22,000–14,000 YA, concomitant with
major cold and dry periods (20,000–18,000 YA) of the
fourth large Pleistocene glaciation (Climap 1976; Brown
1982; Haffer 1997, 2008; Van der Hammen etal. 1991).
Clark (2002) showed that the rain level and the humid-
ity in the Amazon basin at that time were extremely low.
Even the Atlantic Ocean along the Brazilian coast had
its temperature lowered to almost 6°C. Metivier (1998)
showed that the central and northern Andes had an ice
surface area totaling around 371,306 km2, 18,000 YA. This
means an ice cover nearly 100 times greater than today.
The last glacial advance in the Andes occurred during that
epoch (Younger Dryas or III Dryas; Clapperton 1993).
This last glacial advance is reported to have happened in
different parts of the Andean cordilleras (Wright 1983),
such as the Manachaque Valley (Cordillera Blanca), the
Upismayo-Jalacocha (Cordillera de Vilcanota), and Puna
de Junin in Peru, Choque-Yapu mountain in Bolivia and
the Chimborazo volcano in Ecuador. Indeed, Rodbell and
Seltzer (2000) showed that the glacial limits in the Cor-
dillera Blanca (San Martin Department in Peru), around
12,000 YA, were around 3,170 and 3,827m above sea level
(masl). Compare this to today’s limit of around 4,600 masl.
Maslin and Burns (2000) showed that the Amazon River
reached its maximum dry peak around 16,000–15,000 YA
and this situation continued until 12,000 YA. The analysis
with O18 isotopes revealed that the mouth of the Ama-
zon River only had 40% of the water compared to today.
This climate change might have produced the last massive
extinction event, which eliminated around 80% of the large
vertebrates of North America. For example, this elimi-
nated the pumas in North America (Culver etal. 2000) as
well as 40 other species including Smilodon, lions, chee-
tahs, as well as the giant ground sloths and glyptodonts
(Lessa etal. 1997; Lyons etal. 2004).
The existence of a very significant spatial structure for N.
olivacea is not surprising due to the significant differences
within the Western and Central Colombian and Ecuado-
rian Andean Cordilleras and, especially, by the differences
between these last cordilleras and the Eastern Colombian
Andean Cordillera.
The current mitochondrial genetic analysis is only a first
step to understand the molecular evolution of the coatis. It
should be complemented, in the future, with nuclear DNA
markers, especially to analyze gene flow among the four
groups of N. olivacea and determine whether there are sub-
species within N. olivacea or full species. It would also be
helpful to analyze nuclear DNA to determine the possible
level of historical introgression or current hybridization
among N. olivacea, N. nasua, and N. narica.
Acknowledgements Thanks to Dr. Diana Alvarez, Pablo Escobar-
Armel, Nicolás Lichilín, Luisa Fernanda Castellanos-Mora, Dr. Clara
Saldamando, Armando Castellanos, and Jorge Brito for their respective
help in obtaining Nasua and Nasuella during the last 20years. This
work was financed by Project 6839 (Pontificia Universidad Javeriana).
Thanks to the Ministerio del Ambiente Ecuatoriano (MAE) in Santo
Domingo de Tsáchilas and in Coca, to the Instituto von Humboldt
(Colombia), to the Peruvian Ministry of Environment, PRODUCE
(Dirección Nacional de Extracción y Procesamiento Pesquero), Con-
sejo Nacional del Ambiente and the Instituto Nacional de Recursos
Naturales (INRENA) from Peru, to the Colección Boliviana de Fauna
(Dr. Julieta Vargas), and to CITES Bolivia for their role in facilitating
the obtainment of the collection permits in Ecuador, Colombia, Peru
and Bolivia. The first author also thanks the many people of diverse
Indian tribes in Ecuador (Kichwa, Huaorani, Shuar and Achuar), in
Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri
and Yucuna), in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua,
Angoteros, Orejón, Cocama, Kishuarana and Alamas), Bolivia (Siri-
onó, Canichana, Cayubaba and Chacobo), and multiple Mayan com-
munities in Central America for their assistance in obtaining samples
of Nasua and Nasuella.
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... A species that meets these characteristics is the whitenosed coati (Nasua narica Linnaeus 1766, Order Carnivora, Family Procyonidae) since it has a wide distribution from North America to northern South America (González-Maya et al. 2011). The evolutionary and biogeographical history of procionids is controversial from the molecular and paleontological perspectives (Soibelzon and Prevosti 2013;Nigenda-Morales et al. 2019;Ruiz-García et al. 2019b, 2020a. In fact, procionids are one of the taxonomic groups that can provide surprising insights into how and when GIBA occurred (Koepfli et al. 2007;Forasiepi et al. 2014). ...
... These evolutionary rates were reported for the family Canidae ). In the present work, this methodology was used to estimate divergence times as previous studies (Ruiz-García et al. 2020a, 2021a have used Bayesian inference methods to investigate the divergence between the species of Nasua, Nasuella, and Bassaricyon. However, the use of the MJN is preferable because the present study preferably analyzed the divergence times within N. narica, and there is a scarce fossil record for coatis. ...
... However, the use of the MJN is preferable because the present study preferably analyzed the divergence times within N. narica, and there is a scarce fossil record for coatis. In fact, there are no fossil remains attributable to either N. narica or Nasuella, and those attributable to N. nasua do not exceed 0.125 MYA (Woodburne 2010), which significantly underestimates all studies of divergence times within procionids (Koepfli et al. 2007;Nigenda-Morales et al. 2019;Ruiz-García et al. 2019b, 2020a, 2021a. ...
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... This increase of crops by Spaniards and Portuguese, mainly, enhanced the quantity of food and, very likely, increased the size of the Dasyprocta populations in different areas of the Neotropics in the last few centuries. Similar demographic patterns have been observed in other Neotropical mammals (spectacled bear, Tremarctos ornatus; Ruiz-García et al. 2020a,b; coatis, Nasua nasua, Nasua narica, and Nasuella olivacea; Ruíz-García et al. 2020c, 2021aTamandua sp., and Myrmecophaga tridactyla;Ruiz-García et al. 2021b). However, this recent population increase might be an artifact of population structure and therefore caution should be exercised with this last population increase (Heller et al. 2013;Grant 2015). ...
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... Nuestros resultados también muestran que el segundo linaje podría corresponder a N. o. quitensis, aunque no tenemos total certeza que esta denominación no pudiera corresponder también a alguno de los tres linajes moleculares de distribución geográfica más restringida encontrados en las cordilleras occidentales y central de Colombia y Ecuador. Además, ponemos en duda que Nasuella sea un género realmente diferenciado de Nasua, ya que encontramos mayores distancias genéticas entre N. nasua y N. narica que entre esta última especie y N. olivacea (RuIz-GARcíA et al., 2020a). 9. Árbol de Máxima Verosimilitud obtenido para el cánido (Carnivora) Cerdocyon thous. ...
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... This increase of crops by Spaniards and Portuguese, mainly, enhanced the quantity of food and, very likely, increased the size of the Dasyprocta populations in different areas of the Neotropics in the last few centuries. Similar demographic patterns have been observed in other Neotropical mammals (spectacled bear, Tremarctos ornatus; Ruiz-García et al. 2020a,b; coatis, Nasua nasua, Nasua narica, and Nasuella olivacea; Ruíz-García et al. 2020c, 2021aTamandua sp., and Myrmecophaga tridactyla;Ruiz-García et al. 2021b). However, this recent population increase might be an artifact of population structure and therefore caution should be exercised with this last population increase (Heller et al. 2013;Grant 2015). ...
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Geographical assignment of individuals, or tissues, seized from illegal traffic and hunting is relevant for the conservation of many species. For this, the real number of genetically differentiated groups within a species should be determined to know from where the specimens were illegally extracted or to know where the seized and rehabilitated specimens should be liberated. This determination is also crucial to study the evolutionary history of the species. In the current work, we show, by means of three examples, that sample size is more important than the number of genes or markers studied in determining the total number of well-differentiated genetic groups. The examples were related to the number of groups detected for the white-fronted capuchins (Cebus albifrons) in Ecuador, and for the number of well-differentiated groups throughout Latin America for the kinkajou (Potos flavus), and for the different species of coatis (Nasua and Nasuella). In all cases, larger sample sizes with fewer genes detected more genetically different groups than did smaller-sized with entire mitogenomes. Therefore, in regards to the geographical assignment of seized specimens from illegal traffic it is better to obtain larger sample sizes, which cover the most extensive geographical range possible even if they have just one or few mitochondrial genes rather than to rely on smaller sample sizes with entire mitogenome. Furthermore, we take into consideration that analyses of entire mitogenomes are more costly and require a higher DNA quality than a few mitochondrial genes.
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Knowledge of how a species is divided into different genetic units, and the structure among these units, is fundamental to the protection of biodiversity. Procyonidae was one of the families in the Order Carnivora with more success in the colonization of South America. The most divergent species in this family is the kinkajou (Potos flavus). However, knowledge of the genetics and evolution of this species is scarce. We analyzed five mitochondrial genes within 129 individuals of P. flavus from seven Neotropical countries (Mexico, Guatemala, Honduras, Colombia, Ecuador, Peru, and Bolivia). We detected eight different populations or haplogroups, although only three had highly significant bootstrap values (southern Mexico and Central America; northern Peruvian, Ecuadorian, and Colombian Amazon; and north-central Andes and the southern Amazon in Peru). Some analyses showed that the ancestor of the southern Mexico–Central America haplogroup was the first to appear. The youngest haplogroups were those at the most southern area analyzed in Peru and Bolivia. A “borrowed molecular clock” estimated the initial diversification to have occurred around 9.6 million years ago (MYA). All the spatial genetic analyses detected a very strong spatial structure with significant genetic patches (average diameter around 400–500 km) and a clinal isolation by distance among them. The overall sample and all of the haplogroups we detected had elevated levels of genetic diversity, which strongly indicates their long existence. A Bayesian Skyline Plot detected, for the overall sample and for the three most significant haplogroups, a decrease in the number of females within the last 30,000–50,000 years, with a strong decrease in the last 10,000–20,000 years. Our data supported an alignment of some but not all haplogroups with putative morphological subspecies. We have not discounted the possibility of a cryptic kinkajou species.
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We sequenced mitochondrial genes ND5, 12s rRNA, and COI in 302 Andean or spectacled bears (Ursidae) from Venezuela, Colombia, Ecuador, Peru, and Bolivia. Of this total, 294 of the bears were from the wild whereas the remaining eight were from zoos in Mexico, Argentina, France, and Switzerland. A subset of 127 individuals, representing the above five South American countries, was genotyped at seven nuclear microsatellites (GID, G10B, G10C, G10L, G10M, G10P, and G10X). Our results support the following: (1) There are two evolutionary significant units (ESUs), following the definition of Moritz (1994). The first (Northern Andean Clade, NAC) comprises all the bears from Venezuela, Colombia, Ecuador, and northcentral Peru, whereas the second (Southern Andean Clade, SAC) comprises the bears from southern Peru and the northern and central Bolivian Andes. The temporal split between the ESUs was estimated to have occurred around 500,000 years ago (YA). Additionally, in Bolivia, a few of the sampled Andean bears in the Santa Cruz Department were more related to NAC than to SAC; (2) The eight captive bears belonged to the NAC, and thus, individuals from the SAC could be underrepresented in international zoos; 3) Different historical demographic analyses showed signatures of significant population expansions for the species as a whole and in each one of the ESUs found. These population expansions began between 690,000 and 450,000 YA. Nevertheless, one procedure detected a population decrease in the last few hundred to few thousand years for the species as a whole and in each one of the ESUs.
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In this work, fossil material of Nasua and Procyon from the Marplatan of El Breal of Orocual, Venezuela, is reported as the oldest record of these procyonids in South America. This gives evidence of an earlier origin of these genera in the continent than was previously thought. © 2018, Sociedade Brasileira de Paleontologia. All rights reserved.
Article
The coatis are social carnivores from the Procyonidae family distributed by the Neotropics (from Arizona, US, to northern Argentina and Uruguay). Traditionally, three species of coatis are placed in two different genera [Nasua (Nasua nasua distributed in South America; Nasua narica, distributed in Central America) and Nasuella (Nasuella olivacea, distributed in the Andean Cordilleras of Venezuela, Colombia, and Ecuador)]. Recently, a supposed new species of Nasuella has been reported (N. meridensis) in the Venezuelan Andean Cordillera. However, the systematics and the evolutionary history of the coatis are extremely confusing. Herein, we analyzed a dataset, which was composed by 345 coati specimens sampled from southern Mexico to Uruguay sequenced at eight mitochondrial gene (ND5, ND4,Cytb, D-loop, COI, COII, ATP6, and 12s rRNA). The phylogenetic analyses detected 19 main haplogroups (10 haplogroups in N. nasua, five haplogroups in N. olivacea, and four haplogroups in N. narica) and eight-10 sub-haplogroups (five-seven in N. nasua, and three in N. narica). The oldest and original haplotypes were from Colombian and Ecuadorian Andean Cordillera N. nasua specimens, followed by some haplotypes of specimens with the phenotype of N. olivacea also in the Colombian and Ecuadorian Andes, but with DNA more resemblant to that of N. nasua. Thus, the Northern Andes seems to be the original point where the mitochondrial DNA coati diversification began around 13 millions of years ago (Late Miocene). This totally disagrees with the traditional paleontological view, which considers that coati appeared in North America and later migrated into South America in the Pleistocene. The present molecular results agree better with a unique genus (Nasua) than the traditional two genera. If we will apply the Phylogenetic Species Concept (PSC), at least, 19 species of coatis should be described. However, the evolutionary history of the coatis is complex and we agree better with one-three species. Two (or three) N. olivacea haplogroups were more related to some N. nasua haplogroups to other N. oivacea haplogroups. This means that N. olivacea is polyphyletic or there were some Andean N. nasua groups that have evolutionary converged to similar phenotypes to those of the “true” N. olivacea. In fact, we detected the presence of two N. olivacea haplogroups in the Peruvian and Bolivian Andes. One of them was expanded by Peru and Bolivia (phenotype of N. olivacea but with similar mtDNA to a N. nasua haplogroup); the other corresponded to a “true” N. olivacea haplogroup distributed for the Cuzco Department in southern Peru. Thus, we detected molecularly this taxon for first time in Peru. Furthermore, we detected N. nasua specimens with haplotypes of transition very related with those of the original N. narica haplogroup. The first N. narica haplogroup was detected in the trans-Andean and Pacific Ecuador. This haplogroup later generated the Central American N. narica. In Central America, we clearly detected three haplogroups, two of them highly related (northern Costa Rica, Nicaragua, El Salvador, Honduras, Guatemala, and southern Mexico). However, the fourth N. narica haplogroup presented in southern Costa Rica, Panama, and northern Colombia were mitochondrially introgressed by one N. olivacea haplogroup. Therefore, N, narica was originated in South-America and later migrated to Central America, but still two N. narica haplogroups are living within South America together with N. nasua.
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Modern glaciers in the cordillera west and east of the plain of Laguna Junín northeast of Lima are confined to small summits and west-facing cirques. Snowline today is at about 4900 m on the west and 4800 m on the east. That these small glaciers were larger in the recent past is indicated by small moraines and by areas nearly bare of vegetation peripheral to the present glacier limits. In the Pleistocene a mountain glacier complex covered most of the cordillera and spread to all but the center of the plain, thus damming Laguna Junín. Two phases of glaciation can be distinguished. The younger is marked by sharp moraines dotted with small depressions. The older has smooth landforms, no undrained depressions, and locally a thin cover of loess. Retreat from the younger moraines may not have commenced until about 12,000 years ago. Maximum late-Pleistocene snowline depression is calculated at about 300 m in the western cordillera and 500 m in the eastern, on the basis of the elevation of small cirque lakes. Snowline depression of 300 m could be caused by a depression in mean annual temperature of only 2°C. Greater snowline depression on the east may reflect the southward shift of the tropical rain zone in the Amazonian lowlands, as a secondary effect of the vast Laurentide ice sheet of North America.
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An accurately resolved gene tree may not be congruent with the species tree because of lineage sorting of ancestral polymorphisms. DNA sequences from the mitochondrially encoded genes (mtDNA) are attractive sources of characters for estimating the phylogenies of recently evolved taxa because mtDNA evolves rapidly, but its utility is limited because the mitochondrial genes are inherited as a single linkage group (haplotype) and provide only one independent estimate of the species tree. In contrast, a set of nuclear genes can be selected from distinct chromosomes, such that each gene tree provides an independent estimate of the species tree. Another aspect of the gene-tree versus species-tree problem, however, favors the use of mtDNA for inferring species trees. For a three-species segment of a phylogeny, the branching order of a gene tree will correspond to that of the species tree if coalescence of the alleles or haplotypes occurred in the internode between the first and second bifurcation. From neutral theory, it is apparent that the probability of coalescence increases as effective population size decreases. Because the mitochondrial genome is maternally inherited and effectively haploid, its effective population size is one-fourth that of a nuclear-autosomal gene. Thus, the mitochondrial-haplotype tree has a substantially higher probability of accurately tracking a short internode than does a nuclear-autosomal-gene tree. When an internode is sufficiently long that the probability that the mitochondrial-haplotype tree will be congruent with the species tree is 0.95, the probability that a nuclear-autosomalgene tree will be congruent is only 0.62. If each of k independently sampled nuclear-gene trees has a probability of congruence with the species tree of 0.62, then a sample of 16 such trees would be required to be as confident of the inference based on the mitochondrial-haplotype tree. A survey of mtDNA-haplotype diversity in 34 species of birds indicates that coalescence is generally very recent, which suggests that coalescence times are typically much shorter than internodal branch lengths of the species tree, and that sorting of mtDNA lineages is not likely to confound the species tree. Hybridization resulting in transfer of mtDNA haplotypes among branches could also result in a haplotype tree that is incongruent with the species tree; if undetected, this could confound the species tree. However, hybridization is usually easy to detect and should be incorporated in the historical narrative of the group, because reticulation, as well as cladistic events, contributed to the evolution of the group.