Mitochondrial DNA Part A

DNA Mapping, Sequencing, and Analysis

ISSN: 2470-1394 (Print) 2470-1408 (Online) Journal homepage: http://www.tandfonline.com/loi/imdn21

Mitogenomics phylogenetic relationships of the

current sloth’s genera and species (Bradypodidae
and Megalonychidae)

Manuel Ruiz-García, Diego Chacón, Tinka Plese, Ingrid Schuler & Joseph
Mark Shostell

To cite this article: Manuel Ruiz-García, Diego Chacón, Tinka Plese, Ingrid Schuler & Joseph
Mark Shostell (2018) Mitogenomics phylogenetic relationships of the current sloth’s genera and
species (Bradypodidae and Megalonychidae), Mitochondrial DNA Part A, 29:2, 281-299, DOI:
10.1080/24701394.2016.1275602

To link to this article: https://doi.org/10.1080/24701394.2016.1275602

Published online: 27 Jan 2017.

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 MITOCHONDRIAL DNA PART A, 2018
VOL. 29, NO. 2, 281–299
http://dx.doi.org/10.1080/24701394.2016.1275602

RESEARCH ARTICLE

Mitogenomics phylogenetic relationships of the current sloth’s genera and

species (Bradypodidae and Megalonychidae)

na, Tinka Pleseb, Ingrid Schulera and Joseph Mark Shostellc
Manuel Ruiz-Garc
ıaa, Diego Chaco
a
Departamento de Biolog
ıa, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogot
a, DC, Colombia; bFundaci

on AIUNAU, Medell
ın,
Colombia; cMath, Science and Technology Department, University of Minnesota Crookston, Crookston, MN, USA

ABSTRACT ARTICLE HISTORY

We sequenced the complete mitogenome of 39 sloths (19 Bradypus variegatus, 4 B. tridactylus, 1 B. Received 21 September 2016
pygmaeus, 1 B. torquatus, 4 Choloepus didactylus, and 10 C. hoffmanni). A Bayesian tree (BI) indicated Accepted 19 December 2016
a temporal split between Bradypus and Choloepus around 31 million years ago (MYA, Oligocene) and
the other major splits within each genera during the Miocene and Pliocene. A haplotype network (MJN)
KEYWORDS
estimated a lower temporal split between the sloth genera (around 23.5 MYA). Both methods detected Bradypus; Choloepus;
Downloaded by [Manuel Ruiz-Garcia] at 18:13 03 January 2018

the ancestor of B. torquatus as the first to diverge within Bradypus (21 for BI and 19 MJN), followed trans-Andean B. variegatus;
by that of the ancestor of B. tridactylus. The split of B. pygmaeus from the common ancestor with B. mitogenomics; mt Cyt-b
variegatus was around 12 MYA (BI) or 4.3 MYA (MJN). The splits among the previous populations of
B. variegatus began around 8 MYA (BI) or 3.6 MYA (MJN). The trans-Andean population was the first
to diverge from the remaining cis-Andean populations of B. variegatus. The genetic differentiation
of the trans-Andean B. variegatus population relative to the cis-Andean B. variegatus is similar to
that found for different species of sloths. The mitogenomic analysis resolved the differentiation of C.
hoffmanni from the C. didactylus individuals of the Guiana Shield. However, one C. didactylus from the
Colombian Amazon specimen was inside the C. hoffmanni clade. This could be the first example of pos-
sible natural hybridization in the Amazon of both Choloepus taxa or the existence of un-differentiable
phenotypes of these two species in some Amazonian areas.

The sloths are classified within of the supraorder Xenarthra
(anteaters, armadillos and the sloths), one of the most basal
mammalian clades in Placentalia (Murphy et al. 2001a,b;
Delsuc et al. 2004, 2012; Gibb et al. 2016). The morphology
of their reproductive system shows that they represent one
of the earliest divergent clades among mammals. They are
also the sister group to all other eutherians (Engelmann 1985;
McKenna & Bell 1997).
Ho€ss et al. (1996) determined that the divergences among
anteaters, armadillos, and the sloths date back 80 million
years (range of 73–88 MYA). This is in agreement with
immunological comparisons between albumins from sloths,
anteaters, and armadillos (Sarich 1985). The time frame
extends further according to Delsuc et al. (2004). They
detected the divergence between Xenarthra and the remain-
ing main placental clades to have occurred at the end of the
Early Cretaceous, around 103 MYA (95% confidence interval:
94–113 MYA). This is compatible with the fossil record in that
armadillo scutes appear in Brazil during the Upper Paleocene
around 58–60 MYA (Carroll 1988).
Xenarthra subsequently underwent an explosive tertiary
radiation with multiple fossil forms (Patterson & Pascual
1972). McKenna and Bell (1997) recognized 218 genera within
this unique clade of mammals that evolved exclusively in
the New World. Thus, it is the quintessential taxon of
South American mammals. Among the xenarthrans, sloths
constitute the most diverse group, with almost 100 fossil
genera recorded (McKenna & Bell 1997). Sloths are arranged
in the suborder Folivora (¼ Phyllopahaga), which enclose
eight different families (Rathymotheriidae, Orophodontidae,
Scelidotheriidae, Mylodontidae, Bradypodidae, Megatheriidae,
Nothrotheriidae, and Megalonychidae). Megalonychidae con-
tains Choloepus (current two-toed sloths) and Bradypodidae
contains Bradypus (the current three-toed sloths) (MacKenna
et al. 2006). Both current sloth genera have been placed into
distinct families to reflect their numerous morphological dif-
ferences and probable diphyletic origin from two different
fossil groups (Webb 1985).
Gaudin (2004) analyzed 286 osteological characters of all
extant and fossil sloth forms, showing Bradypus as the sister
taxon of other sloths (Eutardigrada). The author also showed
that similarity between Megatheriidae and Bradypus was the
product of evolutionary convergence. Furthermore, he was
the first to run a deep phylogenetic analysis where Choloepus
was with the extinct members of the Megalonychidae family.
According to the author, the temporal split between the
two extant sloth genera could have occurred around 40
MYA during the Middle Eocene. This event could be related
to the Incaic tectonic phase, when great volcanic
activity occurred before the Andes uplift (Noble et al. 1979;

CONTACT Manuel Ruiz-Garc
ıa mruizgar@yahoo.es, mruiz@javeriana.edu.co Departamento de Biolog
ıa, Facultad de Ciencias, Pontificia Universidad
Javeriana, Bogot
a, DC, Colombia
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
 282 M. RUIZ-GARC
IA ET AL.

Sempere et al. 1994). The oldest known sloths come from the
Eocene of Patagonia and Antarctica but they cannot be pre-
cisely assigned to any of the recognized lineages (Vizca
ıno &
Scillato-Yan
e 1995).
Both genera have a quadrupedal suspensory posture and
locomotion, and due to their phylogenetic distance and the
fact that no extinct species appear to have possessed this
trait, many authors accept that these provide a case of
extreme convergent evolution (Greenwood et al. 2001;
Gaudin 2004; Delsuc & Douzery 2008; Billet et al. 2011;
Nyakatura 2012).
Many of the extinct sloths were large and ground-dwelling
such as the Mylondontidae that were at least the size of a
black bear. The families contained several large species, some
the size of elephants. They also lived in a variety of ecological
conditions. For example, Thalassocnus genus was a semi-
aquatic sloth in South America (Canto et al. 2008).
The arrival of North American immigrants (around 3 MYA
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morphological variation. In Central America, it almost always
has a tan face. Many specimens from Southwestern
Colombia, Western Ecuador, and Northcentral Brazil are char-
acterized by strikingly golden faces, although the base of
each facial hair is usually dark brown. Populations of the
Tapajos River in Brazil also show a strong golden frosting on
the throat. This sloth taxon also varies widely in the blotchi-
ness of its dorsal coloration. However, the coat characteristics
could be highly variable within a population without having
any clearly differentiated and transitional forms (Anderson &
Handley 2001). Wetzel (1982) lists nine subspecies of B. varie-
gatus, but the classification of Gardner (2005, 2008) recog-
nizes seven subspecies as the most accepted (see Table 1A).
Bradypus tridactylus, the pale-throated sloth, inhabits parts
of Venezuela, Guyana, Suriname, French Guiana, and
Northeastern Brazil from the delta of the Orinoco River to the
Amazon River and from the Negro River to its mouth in
the Amazon River (Wetzel & Avila-Pires 1980; Wetzel 1982).

or around 12 MYA, depending on the hypotheses of the ori-
gin of the Isthmus of Panama; GABI, Marshall et al. 1982;
Coates et al. 2004; Coates & Stallard 2013; Bagley & Johnson
2014) seems to have not affected the xenarthrans. This
included giant terrestrial forms, because they became suc-
cessful southern invaders of Central and North America
(Patterson & Pascual 1972; Webb 1976). However, this abun-
dant continental sloth fauna became extinct in the Americas
during the Pleistocene around 10,000 YA (Long & Martin
1974; Cartelle 1994), although Steadman et al. (2005) showed
that the Caribbean island sloths lasted until 4000 YA. Only six
species belong to the two quoted genera (Bradypus and
Choloepus) arrived until the present.
The brown-throated three-toed sloth (B. variegatus) is the
sloth species with the widest geographical distribution. It’s
range is from Honduras south into Ecuador, Colombia,
Venezuela (with the exception of the Orinoco River delta and
the Guiana highlands), Peru, and also includes Bolivia to
Northern Argentina and a major part of Brazil (except Amap
a
in Northern Brazil and Paran
a and Rio Grande do Sul in
Southern Brazil; Wetzel & Avila-Pires 1980). This taxon is the
only species of the genus that displays notable geographic
The actual distribution of B. tridactylus seems to be narrower
than previously inferred (Gardner 2008). Anderson and
Handley (2001) pointed out that the distribution of B. tridac-
tylus probably does not extend southwest of the Negro River
nor as far south as the Amazon River. No morphological sub-
species have been reported for this taxon.
Bradypus torquatus, the maned three-toed sloth, shows a
disjunct distribution in the Atlantic coastal forests of
Southeastern Brazil (Lara-Ruiz et al. 2008). One population is
located in the Sergipe, Bahia, and part of Minas Gerais State.
Another population is in the states of Minas Gerais, Espiritu
Santo, and Rio de Janeiro. The historic distribution extended
further north along the coast (from Rio Grande do Norte to
Rio de Janeiro) and was not disjunct (Gardner 2008). No mor-
phological subspecies have been reported for this species.
Bradypus pygmaeus, the pygmy sloth, is only found on the
4.3 km2 Isla of Escudo de Veraguas (islands of Bocas del
Toro), 17.6 km off the Northern Caribbean coast of Panama. It
was recently described by Anderson and Handley (2001,
2002). These authors showed significant morphological and
morphometric data of this taxon relative to the other
Bradypus species recognized. They claimed that these islands

Table 1. (A) Different B. variegatus subspecies recognized by Gardner (2005, 2008) and (B) Different C. hoffmanni subspecies recog-
nized by Gardner and Naples (2007).
Author who
Subspecies described the taxon Geographical place where it was described (Type locality)
A.
B. v. boliviensis Gray (1871) ‘Bolivia’ restricted to Buena Vista, Santa Cruz Department in Bolivia by Cabrera (1958)
B. v. brasiliensis Blainville (1840) ‘Brazil’ restricted to Rio Janeiro by Thomas (1917)
B. v. ephippiger Philippi (1870) ‘Republic Ecuador and Northern Peru’ but restricted to N. W. Colombia by Thomas (1917)
and further restricted to the Atrato River by Cabrera (1958)
B. v. gorgon Thomas (1926) Gorgona Island, off the coast of Colombia
B. v. infuscatus Wagler (1831) ‘Brasilia versus Peru restricted to the confluence of the Solimoes and the Ica rivers in the
razilian Amazon by Cabrera (1958)
B. v. trivittatus Cornalia (1849) prope Gurupe ad Amazonum ripas in Par
a, Brazil’
B. v. variegatus Schinz (1825) ‘Sudamerika’ restricted to Bahia (Brazil) by Mertens (1925)
B.
C. h. agustinus Allen (1913) San Agustin, Huila Department, Colombia
C. h. capitalis Allen (1913) Barbacoas, Colombia
C. h. hoffmanni Peters (1858) ‘Costa Rica’ corrected to ‘Heredia, Volc
an Barba’ by Wetzel and Avila-Pires (1980)
C. h. juruanus Lonnberg (1942) Santo Antonio, Eastern of Eiru River, South of Jurua River, Brazil
C. h. pallescens Lonnberg (1928) Calavera, a place near Roque on the way to Moyabamba, San Mart
ın Department, Peru

were separated from the adjacent Panamanian mainland by
rising sea levels during the past 10,000 years (Holocene).
They further claimed that this provoked at least four inde-
pendent events, with sloths on five of the islands evolving
smaller sizes following insularization. Sloths on the younger
islands remained conspecific with mainland Panamanian pop-
ulations of B. variegatus. However, on the oldest and the
most separated island of this archipelago, the three-toed
sloth was extremely differentiated and quickly formed a new
species, B. pygmaeus.
Two-toed sloths are grouped in two species: C. hoffmanni
(Hoffman’s two-toed sloth) and C. didactylus (Linnaeus’s two-
toed sloth).
Choloepus hoffmanni has two disjoint populations; the
northern most population extends from Nicaragua south into
western Venezuela and the trans-Andean areas of Colombia
and Ecuador. The southern one is found from north-central
Peru through the extreme southwestern part of Brazil to
Central Bolivia (Meritt 2008). Five morphological subspecies
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have been defined for this species (Gardner & Naples 2007)
(see Table 1B).
Choloepus didactylus is found throughout Venezuela and
the Colombian Eastern Llanos (Meta Department), Guyana,
Suriname, French Guiana, and the Maranhao State in Brazil
south along the Brazilian Amazon (Amazon River) and west
into the upper Amazon Basin of Colombia, Peru, and Ecuador
(Wetzel 1982, 1985; Meritt 2008). No subspecies have been
described but Wetzel (1982) proposed three names for pos-
sible subspecies: C. d. columbianus, C. d. florenciae, and C. d.
napensis.
Many mitogenomic analyses have proved to be extremely
useful in determining phylogenetic relationships and in esti-
mating divergence times in many different groups of organ-
isms. Examples of these groups are fishes (Miya et al. 2003;
Inoue et al. 2010), amphibians (Zhang et al. 2005; Zhang &
Wake 2009), birds (Pereira & Baker 2006; Slack et al. 2007),
and different groups of mammals (Arnason et al. 2002, 2004,
2008; Hodgson et al. 2009; Matsui et al. 2007; Chiou et al.
2011; Finstermeier et al. 2013; Pozzi et al. 2014; Ruiz-Garc
ıa
et al. 2016a,b). Mitochondrial gene trees are more precise in
reconstructing the divergence history among species than
other molecular markers (Moore 1995). Cummings et al.
(1995) showed that mitochondrial genomes have higher
information content per base than nuclear DNA. However, we
must take into account that lineage specific variation in evo-
lutionary rates within individual mitochondrial DNA has been
previously determined (Gissi et al. 2000). This variation has
been linked to differences in mutation rates and agrees quite
well with longevity in mammals for mt Cyt-b, for instance
(Nabholz et al. 2008). It is for this reason that the mammalian
mitochondrial DNA clock for individual genes can be particu-
larly erratic (Nabholz et al. 2009). Also, by using relaxed clock
models, we can account for substitution rate variations
among lineages (Thorne et al. 1998). The analyses of com-
plete mitochondrial genomes could overcome this problem.
Taking into consideration all that has been commented
upon, we focus, in this work, on the reconstruction of the
phylogeny of the six extant sloth species using
mitogenomics.
MITOCHONDRIAL DNA PART A 283
Materials and methods
The origins of the 39 we sampled are listed in Figure 1. We
sequenced and analyzed the entire mitogenome of each sam-
ple save for the mt control region. There were 19 B. variega-
tus from Colombia, Venezuela, Peru, and Brazil and four B.
tridactylus from Guyana, Surinam, and French Guiana. There
was also a B. pygmaeus from Veraguas, Panama, and a B. tor-
quatus from Rio de Janeiro State, Brazil. The sample also
included four C. didactylus from Colombia, Venezuela, and
Surinam as well as 10 C. hoffmanni from Costa Rica, Panama,
and Colombia. For an outgroup, we sequenced the complete
mitochondrial genome of two Tamandua tetradactyla from
Peru and one T. mexicana from Panama.
DNA was extracted and isolated from either hair or muscle
samples using the QIAamp DNA Micro Kit (Qiagen, Inc.,
Valencia, CA) following the protocol provided by the manu-
facturer. Muscle extractions followed the protocol ‘DNA
Purification from Tissues.’ Mitochondrial genomes were
sequenced by long-template PCR, which minimizes the
chance of amplifying mitochondrial pseudogenes from the
nuclear genome (numts) (Thalmann et al. 2004; Raaum et al.
2005). PCR amplification of mitochondrial DNA was carried
out using a LongRange PCR Kit (Qiagen, Inc.), with a reaction
volume of 25 ll and a reaction mix consisting of 2.5 ll of 10
LongRange PCR Buffer, 500 lM of each dNTP, 0.6 lM of each
primer, 1 unit of Long-Range PCR Enzyme, and 50–250 ng of
template DNA. Cycling conditions were as follows: 94  C for
5 min, followed by 45 cycles denaturing at 94  C for 30 s, pri-
mer annealing at 50–57  C (depending on primer set) for 30 s,
and extension at 72  C for 8 min, followed by 30 cycles of
denaturing at 93  C for 30 s, annealing at 45–52  C (depend-
ing on primer set) for 30 s, and extension at 72  C for 5 min,
with a final extension at 72  C for 8 min. To minimize the pos-
sibility of amplifying nuclear mitochondrial pseudogenes
(numts), four sets of primers were used to generate overlap-
ping amplicons from 3687 to 4051 bp in length, thereby ena-
bling a quality test for genome circularity (Bensasson et al.
2001; Thalmann et al. 2004). Additional information about
these primers and amplified genes is provided by Ruiz-Garc
ıa
et al. (2016a). The control region was not included in the
mitogenomic analysis because of the difficulty of aligning this
region among the Bradypus, Choloepus, and Tamandua sam-
ples. This difficulty is due to the high quantity of indels in
the mitochondrial DNA fragment. Both mt DNA strands were
sequenced directly using BigDye Terminator v3.1 (Applied
Biosystems, Inc., Foster City, CA). Sequencing products were
analyzed on an ABI 3730 DNA Analyzer system (Applied
Biosystems, Inc.). Sequences were then assembled and edited
using Sequencher 4.7 Software (Gene Codes, Corp., Ann
Arbor, MI). Overlapping regions were examined for irregular-
ities such as frameshift mutations and premature stop
codons. A lack of such irregularities indicates an absence of
contaminating numt sequences.
We used approximately 15,473 bp (although numerous but
minor indels were detected within and between Bradypus
and Choloepus mitochondrial genomes). The alignments with
all the genes were concatenated after removing problematic
regions using Gblocks 0.91 (Talavera & Castresana 2007)
 284 M. RUIZ-GARC
IA ET AL.
Downloaded by [Manuel Ruiz-Garcia] at 18:13 03 January 2018
Figure 1. Map with the geographical origins of the Bradypus and Choloepus specimens sequenced for all the mitogenomes.
under a relaxed approach. This Software removes all poorly
aligned regions and it has been shown to be particularly
effective in phylogenetic studies including very divergent
sequences (Castresana 2000; Talavera & Castresana 2007).
The individual alignments were then concatenated by means
of the SequenceMatrix v1.7.6 Software (Vaidya et al. 2011) to
create a master alignment.
Data analyses
Phylogenetics procedures
The MrModeltest v2.3 Software (Nylander 2004) and the
Mega 6.05 Software (Tamura et al. 2013) were applied to
determine the best evolutionary mutation model for the
sequences analyzed for each individual gene, for different
partitions and for all the concatenated sequences. Akaike
information criterion (AIC; Akaike 1974; Posada & Buckley
2004) and the Bayesian information criterion (BIC; Schwarz
1978) were used to determine the best evolutionary nucleo-
tide model.
Phylogenetic trees were constructed by using two proce-
dures: Maximum-Likelihood tree (MLT) and Bayesian Inference
(BI). 1- A MLT was obtained using RAxML v.7.2.6 Software
(Stamatakis 2006). To select the best fitting model, 50 inde-
pendent iterations were run using three data partitions
(codon 1, codon 2, codon 3). Additionally, 50 iterations were
run using two data partitions (codons 1 þ 2 combined, codon
3). For each analysis, the HKY þ G þ I model (Hasegawa et al.
1985) was used to search for the MLT and topologic support
was estimated with 500 bootstrap replicates using the
HKYGAMMA (Stamatakis 2006). Maximum-likelihood bootstrap
proportions (MLBS) higher than 70% were considered strong
support (Hillis & Bull 1993; Wilcox et al. 2002). 2- BI was per-
formed using a HYK þ G þ I model with the gamma distrib-
uted rate varying among sites, because it was determined to
be the better model using MrModeltest v2.3 Software. BI was
completed with the BEAST v. 1.8.1 program (Drummond et al.
2012). Four independent iterations were run using three data
partitions (codon 1, codon 2, codon 3) with six MCMC chains
sampled every 10,000 generations for 30 million generations
after a burn-in period of 3 million generations. We checked
for convergence using Tracer v1.6 (Rambaut et al. 2013). We
plotted the likelihood versus generation and estimated the
effective sample size (ESS >200) of all parameters across the
four independent analyses. Additionally, AWTY (Are We There
Yet?) Software (Wilgenbusch et al. 2004; Nylander et al. 2008)
was used to plot pairwise split frequencies for the four inde-
pendent MCMC runs and to check the posterior probabilities
of clades for nonoverlapping trees in the sample using
the compare and slide commands. The results from different
runs were combined using LogCombiner v1.8.0 Software

(Rambaut & Drummond 2013a) and TreeAnnotator v1.8.0
(Rambaut & Drummond 2013b). A Yule speciation model and
a relaxed molecular clock with an uncorrelated log-normal
rate of distribution (Drummond et al. 2006) were used.
Posterior probability values provide an assessment of the
degree of support of each node on the tree. Values higher
than 0.95 were considered strong support for monophyletic
clades (Erixon et al. 2003; Huelsenbeck & Rannala 2004). We
estimated the lower and upper 95% highest posterior den-
sities (HPD), the means, geometric means, medians, marginal
densities, and traces with Tracer v1.6 Software. Trees were
visualized with FigTree v. 1.4 Software (Rambaut 2012). This
program was run to estimate the time to most recent com-
mon ancestor (TMRCA) for different nodes of BI. For the six
sloth species with mitogenomics BI, we employed two priors.
One was around 58.4 ± 2 MYA for the split between the
ancestors of the anteaters and the sloths. The second was
around 30 ± 2 MYA for the split of the ancestors of Bradypus
and Choloepus. Both priors were taken from Delsuc et al.
Downloaded by [Manuel Ruiz-Garcia] at 18:13 03 January 2018
(2012) and Gibb et al. (2016).
We constructed a Median Joining Network (MJN) (Bandelt
et al. 1999) using Network 5.0.0.0 Software (Fluxus
Technology Ltd) to estimate possible divergence times (using
mitogenomics) among the haplotypes found in the six extant
sloth species. Additionally, the q statistic (Morral et al. 1994)
and its standard deviation (Saillard et al. 2000) were esti-
mated and transformed into years. The q statistic is unbiased
and highly independent of past demographic events. For the
overall mitogenome, we employed a mutation rate of
1  106 (an average from the studies of Wayne et al. 1997;
Michaux et al. 2002; Benton & Donoghue 2007; Nabholz et al.
2008, 2009), which corresponds to one mutation each
206,000 years.
Some phylogenetics analyses, as BI and MJN, were
also carried out using only the mtCyt gene and including
some sequences of two extinct sloths Mylodon darwinii
(Mylodontidae) and Nothrotheriops shastensis
(Nothrotheriidae) generated by Greenwood et al. (2001).
We also determined the differences among the specimens
analyzed for mitogenomics by means of a factorial analysis
with the Kimura-two-parameter genetic distance (Kimura
1980) and gap blocks correction using DARwin v. 6.0
Software (Perrier & Jacquemoud-Collet 2006).
Genetic heterogeneity and demographic evolution for
Bradypus and Choloepus with mitogenomics
The genetic heterogeneity among the extant sloth species
was estimated with a FST population comparison pair analysis
with exact tests by means of Markov chains with the follow-
ing parameters: 10,000 dememorizations parameters, 20
batches, and 5000 iterations per batch.
A Bayesian skyline plot (BSP) was obtained for the con-
catenated mitochondrial sequences by means of the BEAST v.
1.8.1 and Tracer v1.6 software. The Coalescent-Bayesian sky-
line option in the tree priors was selected with four steps
and a piecewise-constant skyline model with 30,000,000 gen-
erations (the first 3,000,000 discarded as burn-in), kappa with
log Normal [1, 1.25], and Skyline population size with uniform
MITOCHONDRIAL DNA PART A 285
[0, infinite; initial value 80]. We used Tracer v1.6 to analyze
the marginal densities of temporal splits. The Bayesian
Skyline reconstruction option was selected for the trees log
file. A stepwise (constant) Bayesian skyline variant was
selected with the maximum time as the upper 95% high pos-
terior density (HPD) and the trace of the root height as the
treeModel.rootHeigh. To determine the time ranges for pos-
sible demographic changes for B. variegatus and C. hoffmanni
with mitogenomics, we considered that the evolution of
these extant sloth taxa began during the Miocene (7-11
MYA).
Results
Mitogenome phylogenetics for the sloths
The MLT and the BI basically showed the same results for the
mitogenomics analyses (Figure 2(A,B)). Here, we only com-
ment on the BI. The first split was between the ancestor of
the anteaters (Tamandua) and the sloths (p ¼ 1, 55.74 MYA;
95% HPD ¼ 53.52–61.31 MYA). The split between the two spe-
cies of Tamandua (T. mexicana and T. tetradactyla), following
this procedure, was around 11.4 MYA (95% HPD ¼ 2.87–18.06
MYA; p ¼ 1). The split between the ancestors of the two
sloths (Bradypus and Choloepus) was around 30.93 MYA (95%
HPD ¼ 28.52–36.25 MYA; p ¼ 1). The mitochondrial diversifica-
tion seems to begin earlier within Bradypus than in
Choloepus. The first split within the first genus was that of
the ancestor of B. torquatus and the remaining Bradypus taxa
(p ¼ 1, 21.28 MYA; 95% HPD ¼ 19.14–31.37 MYA). The next
split within Bradypus was that of the ancestor of B. tridactylus
(p ¼ 1, 14.15 MYA; 95% HPD ¼ 11.7–23.45 MYA). Within B. tri-
dactylus, the mitochondrial diversification began around 4.57
MYA (95% HPD ¼ 1.41–9.79 MYA; p ¼ 1). It was followed by
the separation of the ancestor of B. pygmaeus (p ¼ 0.67, 12.27
MYA; 95% HPD ¼ 9.28–20.18 MYA). The mitochondrial diversi-
fication within B. variegatus began around 8.07 MYA (95%
HPD ¼ 6.21–16.21 MYA; p ¼ 1) with the separation of the
ancestor of the trans-Andean haplogroup. In turn, the diversi-
fication within this haplogroup began around 1.42 MYA (95%
HPD ¼ 0.45–4.49 MYA; p ¼ 1). The ancestor of the cis-Andean
B. variegatus split into two well differentiated populations
(p ¼ 0.79, 8.01 MYA; 95% HPD ¼ 4.44–12.48 MYA). On one
hand, a Western Amazon clade (p ¼ 1, 3.15 MYA; 95%
HPD ¼ 0.81–5.66 MYA) and on the other hand, a clade consti-
tuted by individuals from the Negro River, Tocantins River,
and Brazilian Eastern area (p ¼ 0.74, 3.44 MYA; 95%
HPD ¼ 2.98–10.77 MYA).
The first split within Choloepus separated the ancestors of
C. didactylus and C. hoffmanni around 18.68 MYA (95%
HPD ¼ 8.66–23.57 MYA; p ¼ 1). Within C. didactylus, the mito-
chondrial diversification began around 0.51 MYA (95%
HPD ¼ 0.01–2.56 MYA; p ¼ 1). The mitochondrial diversifica-
tion was within C. hoffmanni older (12.58 MYA; 95%
HPD ¼ 5.66–16.49 MYA, p ¼ 0.78). The first haplotype to
diverge within C. hoffmanii was from Jerico
(Antioquia
Department, Colombia), followed by a clade with two Central
American individuals (Panama and Costa Rica), whose ances-
tor diverged around 10.43 MYA. These two Central American
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Figure 2. (A) Maximum-likelihood tree with the mitogenomes of 19 B. variegatus (BV), one B. pygmaeus (BP), four B. tridactylus (BTr), one B. torquatus (BTo), four C.
didactylus (CD), 10 C. hoffmanni (CH), one T. mexicana and two T. tetradactyla. In the nodes, bootstrap percentages. (B) Bayesian tree with mitogenomes analyzed for
19 B. variegatus (BV), one B. pygmaeus (BP), four B. tridactylus (BTr), one B. torquatus (BTo), four C. didactylus (CD), 10 C. hoffmanni (CH), one T. mexicana, and two T.
tetradactyla. Thicker tree branches correspond to a posterior probability of 1. Moderately thick tree branches correspond to posterior probabilities higher .5 and
lower than .99. The thin tree branches correspond to posterior probabilities than .5. In nodes, the mean temporal splits in millions of years ago and into parentheses
the temporal split interval for the 95% HPD.

haplotypes diverged around 3.49 MYA (95% HPD ¼ 0.35–6.5
MYA; p ¼ 1). Thus, in Northern Colombia there are live haplo-
types that precede the split between other Northern
Colombian and Central American haplotypes in C. hoffmanni.
A very interesting fact is the inclusion of the haplotype of
one individual of C. didactylus sampled in the Colombian
Amazon with a typical phenotype of this species but within
the haplotypes of C. hoffmanni.
The MJN procedure in Figure 3 shows on the right side
of the figure the relationship among the haplotypes of both
Tamandua species. The temporal split between both
Tamandua species was 1,600,000 ± 244,949 YA, around nine
times lower than the corresponding BI temporal estimate.
The time split between the Tamandua and both sloth’s spe-
cies was 30,353,846 ± 2,143,559 YA, considerably lower than
the BI estimate (around 55 MYA). The next haplotypes to
diverge were those of Choloepus. The separation of the hap-
lotypes of C. didactylus and C. hoffmanii is not clear. One C.
hoffmanii’s haplotype (21; Jerico
, Antioquia Department,
Colombia) was more related to the unique haplotype found
in the three C. didactylus specimens we sampled in the
Guiana Shield. However, the unique Amazonian C. didactylus
individual we sampled shared its haplotype with a C.
hoffmanni individual sampled in the Santander Department

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Figure 2. Continued.
(Colombia). The temporal split between both Choloepus spe-
cies was 5,757,143 ± 869,553 YA, which is considerably lower
than 19 MYA estimated with BI. The temporal split between
Choloepus and Bradypus was 23,450,000 ± 1,910,871 YA,
which is also minor compared to 31 MYA estimated with BI.
The B. torquatus’ haplotype diverged from all the other
Bradypus species’s haplotypes 19,056,000 ± 1,770,134 MYA
similar to the 21 MYA estimate obtained with BI. In this
case, B. torquatus was more related to B. tridactylus than to
other species of Bradypus. The temporal split between B. tri-
dactylus and B. variegatus was estimated at
6,566,667 ± 961,769 YA, which is lower than the date of 14
MYA detected by BI. As in the previous analysis, the trans-
Andean B. variegatus was the first to diverge for this species
and this haplogroup is more related to B. tridactylus than
any other B. variegatus’s haplogroup. The temporal split
between the trans-Andean and all the other cis-Andean
MITOCHONDRIAL DNA PART A 287
haplotypes was 3,600,000 ± 582,204 YA (against eight MYA
for BI). The splits between the Western Amazon haplogroup
and the Brazilian Atlantic forest þ Tocantins, and the Negro
River haplogroups were 1,500,000 ± 100,000 YA and
2,100,000 ± 244,949 YA, respectively, which were also lower
than the BI estimate (3.4 MYA). Finally, B. pygmaeus was
derived from an undetermined haplotype placed between
those from the Western Amazon and the Negro River hap-
logroups. The temporal split between these B. variegatus
haplotypes and B. pygmaeus was estimated at
4,300,000 ± 616,441 YA, which is substantially less than the
12 MYA value obtained by BI.
The factorial analysis for the anteaters and sloths for the
complete mitochondrial genome (Figure 4) showed that the
most differentiated individuals belonged to the individuals of
Tamandua. The individuals of Bradypus and Choloepus were
perfectly differentiated, the first on the right side of the
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Figure 3. Median Joining Network (MJN) for the haplotypes detected in the different species of Bradypus, Choloepus, and Tamandua. T. mexicana (H28), T. tetradac-
tyla (H26-H27), C. didactylus (H18-H24), C. hoffmanii (H17 and H22), B. torquatus (H14), B. tridactylus (H12, H13 and H16), trans-Andean B. variegatus (H3 and H4),
Western Amazon B. variegatus (H1, H10 and H11), Brazilian Eastern Atlantic B. variegatus H5, H6, H8, H9 and H10), Negro River B. variegatus (H2), B. pygmaeus (H15).
All mv circles are extinct or not found haplotypes.

Figure 4. Factorial analysis for Tamandua, Choloepus, and Bradypus species sequenced for complete mitogenomes.

figure and the second on the left side. The individuals of
B. torquatus are extremely differentiated (at the centre of the
figure) from the individuals of the other three species of
Bradypus. For the other three species of Bradypus, from left to
right, we observe the individuals of B. tridactylus, B. pygmaeus
(in middle), and B. variegatus. Within B. variegatus in the
upper sector, we observed two groups of individuals, one
with trans-Andean individuals and the other with Western
Amazon individuals. Within the lower sector, there was a mix-
ture of individuals from the Negro River, Tocantins, and
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Figure 5. Bayesian tree (which is identical to the ML, Maximum Parsimony and NJ trees) of the extant sloth genera (Bradypus; Choloepus) and two fossil sloths,
Mylodon darwinii (Mylodontidae) and Nothrotheriops shastensis (Nothrotheriidae) by means of the mt Cyt-b gene. All nodes showed 1 (posterior probability) or 100%
bootstrap percentages (order ML, Maximum Parsimony and NJ). The unique exceptions were between Mylodon darwinii (Mylodontidae) and Choloepus (0.67 for the
Bayesian tree and 89% for the ML tree) and for a clade within the cis-Andean B. variegatus population (0.82 for the Bayesian tree). As outgroups, mt Cyt-b gene
sequences of Dasypus novemcinctus and T. tetradactyla. In the x-axis, times in millions of years.

Brazilian Eastern populations. The individuals of C. didactylus
from the Guiana Shield were clearly differentiated from C.
hoffmanni, but the C. didactylus individual from the
Colombian Amazon was undifferentiated from the C. hoff-
manni individuals. One individual of this last species was
highly differentiated from the other specimens (Cesar
Department in Colombia).
The first analysis only at the mtCyt-b gene with BI
(although we also obtained ML and NJ trees; Figure 5)
showed very high posterior probabilities and bootstraps for
all the clades obtained (p ¼ 1 and bootstraps 100%).
The clade of Mylodon darwini þ Choloepus had the lowest
posterior probability (0.67) and the lowest bootstrap (89%).
The BI indicated that Mylodon darwini þ Choloepus and
Nothrotheriops shastensis þ Bradypus formed two groups
which diverged around 39 ± 9 MYA. The split between
Bradypus and Nothrotheriops was estimated to have occurred
around 31.5 ± 5 MYA, and that of Mylodon and Choloepus to
have occurred around 31 ± 5 MYA. Therefore, it was practic-
ally a synchronous split. In the same manner, the split
between the Guiana Shield C. didactylus and the C. hoffmanni
(including one Amazonian C. didactylus) and the split
between the trans- and the cis-Andean B. variegatus were
synchronous around 13 ± 6 MYA (Miocene).
The second analysis with the MJN procedure (Figure 6),
the temporal splits were considerably minor: 23.49 MYA for
the split of the two current sloth genera, 12.1 MYA for
Bradypus-Nothrotheriops and 16.12 MYA for Choloepus-
Mylodon. The split between C. didactylus-C. hoffmanni and
that between the trans- and cis-Andean B. variegatus popu-
lations were synchronous around 7 MYA. These splits seem
to be independent of the different temporal split estimates
obtained by BI and MJN and seem to be correlated with a
global temperature decrease (Figure 6).
Mitogenomics population genetic heterogeneity of the
sloths
A comparison of the genetic heterogeneity among the differ-
ent sloth taxa we analyzed for mitogenomics indicated
extremely high and significant levels of differentiation. All the
genetic differentiation statistics were highly significant
(Table 2). Some traits are very interesting in the comparison
pairwise analysis (Table 3). The lowest degree of genetic dif-
ferentiation was between the Western Amazon B. variegatus
and the Brazilian Eastern B. variegatus populations
(FST ¼ 0.686). Nevertheless, the trans-Andean B. variegatus
population showed levels of genetic differentiation relative
to the Western Amazon and the Brazilian Atlantic Eastern
populations (FST ¼ 0.889 and 0.869, respectively). These are
similar to those found among different species pairs such
as for example the Western Amazonian B. variegatus vs. B.
tridactylus (FST ¼ 0.890) and the Brazilian Eastern B. variegatus
vs. B. tridactylus (FST ¼ 0.840). Thus, the genetic differentiation
of the trans-Andean B. variegatus population in regard to the
other cis-Andean B. variegatus populations is comparable to
the genetic differentiation among other defined sloth species.
The BSP with mitogenomics showed the exact same
trends for B. variegatus and for C. hoffmanni (Figure 7) with
constant or very slight increases during the Pliocene and a
clear decrease in the last 0.5 MY (Pleistocene).
Discussion
Temporal divergences in the origins of the Xenartharns,
between the two extant arboreal sloth genera and
within Tamandua
We determined a temporal split of around 65 MYA for the
ancestor of the armadillos (Dasypus) and the remaining
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Figure 6. Double tree, one obtained from an MJN procedure (top; lower times) and other obtained from a Bayesian tree (below; higher times) for the extant sloth
genera (Bradypus; Choloepus) and two fossil sloths, Mylodon darwinii (Mylodontidae) and Nothrotheriops shastensis (Nothrotheriidae) by means of the mt Cyt-b gene.
In blueish grey, the moments of three relevant tectonic events in the Andes. In the below area of the tree, it is shown the overall ocean cooling throughout the
time by means of the quantification of d18O isotopes (Zachos et al. 2001). The dotted line shows the different formation of glacial layers in the time considered
(Zachos et al. 2001).

Table 2. Genetic heterogeneity and indirect gene flow (Nm) statistics among Table 3. Genetic differentiation by means of the FST statistic for B. variegatus
different B. variegatus populations (trans-Andean, Amazonian, and Brazilian (three populations: 1 ¼ trans-Andean, 2 ¼ Amazonian, and 3 ¼ Brazilian Eastern
Eastern Atlantic ones), B. tridactylus, C. didactylus, and C. hoffmanni. Atlantic ones), 4 ¼ B. tridactylus, 5 ¼ C. didactylus, and 6 ¼ C. hoffmanni pairs
Genetic differentiation estimated p Gene flow throughout the results of mitogenomes.
v2 ¼ 180 df ¼ 105 .0000 Taxa 1 2 3 4 5 6
HST ¼ 0.2333 .0000 cST ¼ 0.8874 Nm ¼ 0.06 1 –
KST ¼ 0.8737 .0000 NST ¼ 0.9176 Nm ¼ 0.04 2 0.889 –
KST ¼ 0.5857 .0000 FST ¼ 0.9105 Nm ¼ 0.05 3 0.869 0.691 –
ZS ¼ 61.0785 .0000 4 0.943 0.863 0.840 –
ZS ¼ 3.8725 .0000 5 0.991 0.968 0.955 0.971 –
Significant probability (p < .001). 6 0.924 0.897 0.873 0.880 0.809 –
p < .01;
p < .0001, significant heterogeneity.

xenarthrans (Figures 5 and 6). This agrees quite well with

that reported by Delsuc et al. (2001) with the nuclear VWF
gene and mitochondrial 12S rRNA and 16S rRNA genes. They
estimated an interval of around 59–76 MYA for the process
of diversification of the current Xenarthra. Other studies indi-
cated similar findings (67.8 MYA [61.3–74.7 MYA], Delsuc
et al. 2012; 67.7 MYA [60.4–71.6 MYA], Gibb et al. 2016). We
also estimated a temporal split with BI of 56 MYA (53–61
MYA) between the ancestors of Tamandua and the sloths.
This estimate agrees quite well with that determined by
Delsuc et al. (2004) [55.2 MYA (46–65 MYA)], Delsuc et al.
(2012) [60.1 MYA (53.1–67.2 MYA)] and Gibb et al. (2016)
[58.4 MYA (48.6–64.7 MYA)]. Our MJN value clearly seems to
be an underestimate of that age (30.4 MYA). However, Barros
et al. (2008) estimated the split between sloths and anteaters
to have occurred at about 37 MYA, relatively similar to the
last estimation.
We have obtained three temporal estimations for the split-
ting between the ancestors of the two extant sloth genera
(Bradypus and Choloepus): 31 MYA (for BI with mitogenomes),
23.5 MYA (for MJN with mitogenomes), and 39 MYA (for BI
with mt Cyt-b gene). It is interesting to note the discrepancy
between the BI (fossil-calibrated DNA phylogeny) and

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Figure 7. Bayesian skyline plot analysis (BSP) to determine possible demographic changes across the natural history of (A) B. variegatus and (B) C. hoffmanni On the
x-axis, time in millions of years; on the y-axis, log effective population size of females.
borrowed molecular clock (MJN). This is due to two facts.
First, we do not know if we are using precise fossil data for
BI and second we don’t know the exact mutation rates for
entire mitogenomes. In fact, age estimates from DNA sequen-
ces show some difficulties related to the variation of evolu-
tionary rates both over time and among lineages. It is
challenging to overcome these problems because there is no
unambiguous separation of evolutionary rates and time
(Smith & Peterson 2002).
These three temporal estimates could generate three
hypotheses for the split of the ancestors of the two current
sloth genera:
(1) The lowest estimate (23.5 MYA) is relatively similar to
those estimates obtained by other authors. Delsuc et al.
(2001) estimated this split to have occurred around 18 MYA.
Delsuc et al. (2004) studied three nuclear genes (5130 bp)
and estimated it to have occurred 21 MYA (15–27.8 MYA)
during the uplift of the Central and Northern Andes in the
Quechua I phase (23–17 MYA). At this time (Early Miocene)
MITOCHONDRIAL DNA PART A 291
the temperature decreased and the ocean level changed. This
coincides with the end of the first major Bolivian Andean cri-
sis. This diastrophic event was an intense deformational and
magmatic episode widespread along the Andes (Sempere
et al. 1994). The intensity of this Andean change is seen as a
turning point in Andean tectonics because the Andes created
a rain shadow that significantly influenced South American
climates (Marshall & Sempere 1993). Together with the
Bolivian Andean crisis, it was correlated with a thermal opti-
mum followed by sharp marine regressions (Patterson &
Pascual 1972; Haq et al. 1987) and by a brief but deep glacial
maximum (Zachos et al. 2001). This could be correlated with
the split of the two extant sloth genera as well as with the
beginning of the Miocene radiation of ground sloths
(Patterson & Pascual 1972).
(2) Our highest estimate (39 MYA) agrees with that
claimed by Gaudin (2004), who suggested that the split
between the two extant genera is an ancient event that
occurred at about 40 MYA. Also Katzourakis et al. (2009)
 292 M. RUIZ-GARC
IA ET AL.
concluded that the sloth endogenous foamy virus invaded
the genome of a sloth ancestor in the late Middle Eocene
around 39 MYA. This hypothesis could be related with the
Incaic Tectonic Phase (42–35 MYA) and the uplifting of the
Central Andes and strong volcanic activity (Noble et al. 1979;
Sempere et al. 1994). During this epoch there was also a
cooling sea process (Mackensen & Ehrmann 1992) and a con-
comitant increase of the Antarctic ice sheet and an expansion
of the Notophagus forests. This indicates a trend of general
cooling of the continental zones (Barreda & Palazzesi 2007).
Related with this, Gibb et al. (2016) estimated a divergence of
42 MYA between two lineages of armadillos that they
claimed as two different families, Dasypodidae (dasypodines),
and Chlamyphoridae (euphractines, chlamyphorines, and
tolypeutines).
(3) Between these two extreme estimates, there is our
own intermediate estimate of 31 MYA. Sarich (1985), with
immunological data, suggested that both sloth genera
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diverged 35 MYA. Thus, our estimate, is strongly in agree-
ment with that obtained by other molecular studies such as
by Delsuc et al. (2012) (28.3 MYA [22–35.2 MYA]) and Gibb
et al. (2016) (29.9 MYA [16.5–39.6 MYA]). This could be sup-
ported by a megalonychid fossil from Puerto Rico (30 MYA)
and other equally old megalonychids from Southern South
America (MacPhee & Iturralde Vinent 1995). This agrees that
the oldest, reasonably well preserved fossils assigned to
Folivora are Oligocene (Carlini & Scillato-Yan
e 2004; Pujos &
De Iuliis 2007). Similarly, Slater et al. (2016), using the
Deseadan fossil taxa and fall them within crown families (as
suggested by morphological data), obtained an estimation of
29 MYA. However, Slater et al. (2016), treated these fossils as
either stem taxa or allowed them to be crown taxa that fell
outside of recognized families, and estimated the initial diver-
sification in the sloths to have occurred around 21.5 MYA.
This coincided with the first hypothesis.
Thus, following the two last hypotheses, major sloth
groups probably diverged during a short period of time in
the Late Eocene and Oligocene. However, much of the diver-
sification within both extant sloth genera could have
occurred in the Miocene or early Pliocene, which agrees with
Delsuc et al. (2002, 2004) and Gibb et al. (2016).
Episodes of tectonic uplift since the Cretaceous period and
throughout the Cenozoic (Paleocene until Pliocene) led to an
increase in elevation of the Andes and subsequent modifica-
tions in atmospheric circulation. This in turn led to major
changes in habitats and climatic conditions, thus shaping
faunal and floral assemblages (Pascual & Ortiz Jaureguizar
1990; Marshall & Sempere 1993; Hoorn et al. 2010). These
uplifts occurred 125–112 MYA (Peruvian Tectonic phase),
42–35 MYA (Incaic Tectonic phase), 23–17 MYA (Quechua I
Tectonic phase), 9.5–7 MYA (Quechua II Tectonic phase) and
6–2.7 MYA (Quechua III Tectonic phase) and they went from
the South to the North. The uplift of the Andes could gener-
ate important barriers for sloths and other species (Lessa
et al. 2003; Miller et al. 2008; Hoorn et al. 2010; Rull 2011).
The problem with the three hypotheses is that the Andean
uplift’s influence on the origins of the current sloth genera
could happen at any time because of the continuous uplift of
the Andes. What seems certain is the synchronicity of
speciation phenomena within the two current sloth genera or
within the two main sloth groups. For the BI estimate with
mitogenomics, the process of speciation in Bradypus was
around 21.3 MYA and in Choloepus around 18.7 MYA. The
diversification of the armadillo subfamily Tolypeutinae
(Priodontes, Cabassous, and Tolypeutes) occurred within the
same epoch around 21 MYA (15–28 MYA) (Gibb et al. 2016),
in the Early Miocene. The BI estimates (using mt Cyt-b) of the
split of Choloepus-Mylodon (31 MYA) and Bradypus-
Nothrotheriops (31.5 MYA) were almost the same. This tem-
poral split is in line with the first offshoot within
Myrmecophagidae around 31.8 MYA (Cyclopes; Barros et al.
2008). Our temporal splits for Bradypus and Nothrotheriidae,
31 MYA for BI and 12 MYA for MJN, overlapped with the tem-
poral estimates provided by Slater et al. (2016) (23.5–17.5
MYA). Also, the BI estimate with mt Cyt-b offered the same
time (around 13 MYA) for the diversification process within B.
variegatus (trans- and cis-Andean populations) as well as for
the trans-Andean C. hoffmanni and the cis-Andean C. didacty-
lus populations. This also agrees with the split between
Myrmecophaga and Tamandua at 12.9 MYA (Barros et al.
2008). This temporal estimate was lower when it was
obtained with the MJN procedure. But, it was identical to
that for the Bradypus and Choloepus taxa (around 7 MYA).
The time agrees quite well with the diversification of extant
Euphractine armadillos and the split among the long-nosed
armadillos (Dasypus) in the late Miocene (7 MYA) following
Delsuc et al. (2004).
Similalrly to what was commented by Gibb et al. (2016),
and in consideration of the model of Condamine et al. (2013),
a temperature-dependent model showed that the speciation
rate over the entire xenarthran temporal splits (in our case,
sloth temporal splits) was negatively correlated with tempera-
ture. The periods of intense cooling (Zachos et al. 2001) since
the Late Eocene, Oligocene, and the Middle Miocene, fol-
lowed by the presence of the circum Antarctic current and
the continuous uplift of the Andes (Garzione et al. 2008),
could have caused the aridification of South America. These
events could have also caused and the formation of dry bio-
mes (Caatinga, Cerrado, Chaco, Pampas; Simon et al. 2009;
Hoorn et al. 2010) and the formation of forest refugia. During
the dry periods rainforest communities split into isolated refu-
gia separated by savannah or arid pampas (Vanzolini &
Williams 1970; Whithmore & Prance 1987; Haffer 1969, 1997,
2008). These forest refugia could be the centers of evolution
and differentiation of the different sloth taxa.
Our mitogenomics results with BI and MJN for the two
species of Tamandua are extremely different. BI offered a split
of 11.4 MYA (Miocene), whilst MJN showed an estimate of 1.6
MYA (Pleistocene). We believe that the second estimate could
be more realistic than the first one for two reasons. The first
one is related with the mitogenomics results of Gibb et al.
(2016). They found a small genetic differentiation between
the trans-Andean T. mexicana and the cis-Andean T. tetradac-
tyla (2.8% for all the mitogenome and 2.1% for mt COI, virtu-
ally identical to our estimates) and a temporal split of 1 ± 0.4
MYA. The two tamanduas are considered two distinct species
based on differences in coat colouration, body size, skull
characters, and number of caudal vertebrae that can be quite

variable within populations (Wetzel 1985). If our second esti-
mate is right, then we can question if these Tamandua taxa
are really different species. Only the analyses of nuclear
molecular markers, chromosomal differences, and the positive
or negative existence of post- and/or pre-zygote barriers
could shed light on the question of one versus two species
within Tamandua. Indeed, the number of chromosomes
(2n ¼ 54) and the fundamental numbers (FN ¼ 104) are the
same in both taxa (Hayssen 2011). This could be correlated to
the fact that both taxa could be a unique species. The second
one is related with the fossil record. Although anteaters are
known to have been around since the Santacrucian South
American Land Mammal Age (around 16 MYA; Delsuc et al.
2004), the fossil record indicates that Tamandua has only
been around since the Pleistocene and the Holocene
(McKenna & Bell 1997; Simpson 1945; McDonald et al. 2008).
Some coprolites have been found in Brazil (Ferreira et al.
1989). Thus, it seems that the diversification process in
Tamandua could be more related with Pleistocene events
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than with Miocene ones.
Temporal splits within Bradypus and Choloepus
Our mitogenomics split estimates for the ancestor of B. tor-
quatus from the other Bradypus taxa were 21.3 MYA for BI
and 19 MYA for MJN. These estimates agree quite well with
that of Gibb et al. (2016), who obtained a value of 19 ± 4.7
MYA. All these estimates are clearly higher than the estimate
from Barros et al. (2008) who stated that the split occurred
approximately 7.7 MYA. They concluded that the split of the
ancestor of B. torquatus from the ancestors of other Bradypus
species agrees quite well with the separation of two monkey
genera (Brachyteles and Lagothrix; around eight MYA). The
geographic distributions of these monkey genera in the
Amazon and Brazilian Atlantic forest are similar to that of
Bradypus. Moraes-Barros et al. (2011) estimated a divergence
of 12 MYA for the ancestor of B. torquatus and the ancestor
of B. tridactylus þ B. variegatus. Clearly, our estimates for B.
torquatus are much older than estimates obtained by some
authors for other congeneric endemic species of these two
biomes (Amazon vs. Brazilian Atlantic forest). For example,
consider the curassows (Pereira & Baker 2006), monkeys
(Meireles et al. 1999; Cort
es-Ortiz et al. 2003; Bloch et al.
2016), and vesper mice (Salazar-Bravo et al. 2001). The esti-
mate of around 20 MYA for the separation of B. torquatus
preceded, for instance, the split of Tamandua and
Myrmecophaga around 12.9 MYA (Barros et al. 2003). For this
reason, we agree with other authors that B. torquatus merits
the status of a different genus (Scaeopus; S. torquatus) follow-
ing Barros et al. (2008), Paula-Couto (1979) and Santos (1977).
Our temporal estimates for the split of the ancestor of B.
tridactylus from the remaining Bradypus species were 14.1
MYA for BI and 6.6 MYA for MJN. The estimate carried out by
Gibb et al. (2016) was around 5.7 MYA (3.6–12.6 MYA) and
that of Moraes-Barros et al. (2011) was around 6–4.8 MYA.
Therefore, our MJN estimate is more similar to those of these
two authors. Also, our BI could be an overestimation of the
reality. In the wild, sometimes it is difficult to separate mor-
photypes of B. trivirgatus from those of B. variegatus.
MITOCHONDRIAL DNA PART A 293
This could be the reason why Barros et al. (2003), who ana-
lyzed the mt 16s RNA gene, estimated the divergence of the
taxa to have occurred 0.4 MYA. They probably estimated the
temporal split between two B. variegatus specimens.
However, B. tridactylus showed the presence of a pair of fora-
mina at the anterodorsal nasopharynx. This pair has been
used as reference for a stable diagnostic character to help dif-
ferentiate B. variegatus from B. tridactylus (Wetzel & Avila-
Pires 1980; Wetzel 1985; Anderson & Handley 2001).
Our temporal split estimates between the ancestors of B.
pygmaeus and B. variegatus were 12.3 MYA with BI and 4.3
MYA with MJN. A unique comparative data set comes from
Gibb et al. (2016), who estimated a split around 7.7 MYA
(3.6–12.6 MYA), which is within the range of our two esti-
mates. Thus, our data are in agreement with the findings of
Anderson and Handley (2001, 2002), as well Gibb et al.
(2016). The pygmy form of the three-toed sloths from Isla
Escudo in Panama is a completely different species from B.
variegatus. The morphological differences observed by
Anderson and Handley (2001, 2002) for B. pygmaeus, there-
fore, have a phylogenetical signal (absent or minute external
carotid foramen; tiny stylomastoid foramen at the posterior
external base of the auditory bulla; large external auditory
meatus; ventral edge of stylohyoid usually smoothly concave;
coronoid process of the mandible slender, and strongly fal-
cate). The sloths, having evolved on islands, are extremely
interesting from an evolutionary point of view. For example,
B. variegatus on Gorgona Island (Pacific area of Colombia), is
at a greater distant from the Colombian shore than Isla
Escudo is from the Panama mainland. This taxon seems to
have similar morphological traits to the mainland trans-
Andean B. variegatus population but we ignore the molecular
genetics characteristics of this island population.
The diversification within B. variegatus has been intensively
studied in Moraes-Barros et al. (2007), Moraes-Barros and
Arteaga (2015) and Ruiz-Garc
ıa et al. (2016a). Mitogenomic
and mt control region studies have both shown the trans-
Andean B. variegatus population (Ruiz-Garc
ıa et al. 2016a) to
clearly be monophyletic and well differentiated relative to
other B. variegatus populations. Ruiz-Garc
ıa et al. (2016a) con-
cluded that it should be defined as a new species of three-
toed sloth: Bradypus ephippiger following the Phylogenetic
Species Concept (PSC; Cracraft 1983). However, we are reluc-
tant to define this population as a new species until new
data indicate strong karyotype differences and/or pre or post-
zygote reproductive barriers (Biological Species Concept, BSC;
Mayr 1942) between the trans and the cis-Andean popula-
tions. The temporal split estimates between the ancestors of
the trans and the cis-Andean B. variegatus were 8 MYA for BI
and 3.6 MYA for MJN.
Similarly, the Western Amazon population is well differen-
tiated according to an analysis of the mt control region (Ruiz-
Garc
ıa et al. 2016a). Also, like in several mt control region
analyses, the Negro River’s haplotype was more related to
the Brazilian Eastern haplogroup than to the Western
Amazon one. But, in contrast to that found with the mt con-
trol region (Ruiz-Garc
ıa et al. 2016a), the Tocantins River’s
haplotypes were more related with the Brazilian Eastern hap-
logroup by using mitogenomics. However, the mt control
 294 M. RUIZ-GARC
IA ET AL.
region showed a more intense relationship between the
Tocantins River and Western Amazon haplogroups (Ruiz-
Garc
ıa et al. 2016a). The mitogenomics probably recon-
structed better relationships among taxa above the species
level. In contrast the mt control region is more efficient in
reconstructing the relationships below the species level. For
example, Tchaicka et al. (2007) described a similarity between
the southern region of the Atlantic forest and the eastern
region of the Amazonian forest for Cerdocyon thous
(Canidae). Our temporal splits between the Western Amazon
haplogroup and the other one (Negro River þ Tocantins
River þ Brazilian Eastern Atlantic forest) were 3.4 MYA for BI
and 1.5 MYA for MJN.
This mitogenomics study also detected a Northern and a
Southern Brazilian Eastern Atlantic haplogroups which split
around 1.26 MYA, following BI. This estimate is somewhat
lower than that detected between these two areas of the
Eastern Brazilian Atlantic area for other species. Silva et al.
Downloaded by [Manuel Ruiz-Garcia] at 18:13 03 January 2018
(2012) determined divergence estimates around 7–2 MYA
between these two areas for many other vertebrate species.
The lack of a complete separation between these Northern
and Southern lineages (more clear in Ruiz-Garc
ıa et al. 2016a)
could be the result of events during the Holocene when ever-
green forests and the open savannas became denser than in
previous epochs (Turcq et al. 1997; Ledru et al. 1998; Auler &
Smart 2001). This favoured the interchange of sloths between
the two areas. For instance, following Rothlisberger (1987),
around 6300 YA, there was a significant increase in tempera-
ture especially in Southern South America as well as in the
Central Andes, evidenced by O18 levels in Huascaran snow.
The situation could be more complex when more individuals
of the Brazilian Eastern Atlantic area are studied. Indeed, Silva
et al. (2012) analyzed different studies of Brazilian Atlantic for-
est vertebrates and described three large regions. They were
subdivided into nine sub-groups which showed latitudinal
and longitudinal patterns. These authors established a com-
plex history for the organisms of the Brazilian Atlantic forest
and temporal diversification events within the Brazilian
Atlantic forest, which were not restricted to the Pleistocene.
Our temporal split estimates between the ancestors of the
two species of Choloepus were 18.7 MYA with BI and 5.8 with
MJN. Gibb et al. (2016) provided an estimate of 9.2 MYA
(3.5–16.7 MYA) which is within the range of our estimates.
Slater et al. (2016) recently estimated the split between C.
hoffmanni and C. didactylus to have occurred about 6–7 MYA,
which is relatively similar to our lower estimate with MJN.
Both time estimates are possible according to the literature.
For example, some data of the fossil record could agree with
an estimate of 18.7 MYA. Choloepus has been nested within
the endemic Antillean megalonychid radiation as a close rela-
tive of Acratocnus (Gaudin 1995; White & MacPhee 2001;
Gaudin 2004; McDonald et al. 2013). The earliest firm evi-
dence of an Antillean megalonychid is that of Imagocnus
zazae from the Early Miocene (Burdigalian, 16.1 – 21.5 MYA in
Cuba; MacPhee et al. 2003). If so, the ancestral Choloepus
(re)colonized the mainland from the islands in the Late
Miocene. A lower estimate of 9–6 MYA would be more agree-
able to what has been claimed by other authors (Pujos et al.
2007). Choloepus is not highly related to the Antillean
megalonychid radiation. Moraes-Barros and Arteaga (2015)
used the mt COI gene to estimate a divergence to have
occurred around 7 MYA between Central and South
American populations of C. hoffmanni. Indeed, our mitoge-
nomic BI estimate enlarges this temporal split to 12.6 MYA.
These finding suggest that Choloepus’s disjunct distribution
today was at least partly a result of an uplift of the Northern
Andes and supports the hypothesis of a long history of
Choloepus on the mainland. However, this does not negate
the possibility that the Antillean and Central American areas
could be of extreme importance to the origins of the current
Choloepus in South America. Indeed, this study, Ruiz-Garc
ıa
et al. (2016a) and Gibb et al. (2016) show that the origins of
B. pygmaeus precede that of B. variegatus or that of B.
variegatus þ B. tridactylus. Therefore, the origins of the ances-
tors of these species are more likely the Caribbean Islands or
Central America than South America. In contrast to C. hoff-
manni, the diversification within C. didactylus seems to be
very recent (0.51 MYA), although we analyze a very limited
number of specimens of this species. Its distribution along
the Caribbean South America coasts (Venezuela, Guyana,
Surinam, French Guiana) could be related to its Caribbean
island origins.
Independent of when the ancestors of both species of
Choloepus split, they present some distinctive osteological dif-
ferences, although their external differences are sometimes
difficult to recognize. The morphological differences between
C. hoffmanni and C. didactylus include the number of cervical
and caudal vertebrate (Wetzel 1985; Maslin et al. 2007;
Hautier et al. 2010; Asher et al. 2011). According to Wetzel
(1985), the two Choloepus species also have a different num-
ber of foramina in the anterior half of the basipharyngeal
canal. There are two pairs of foramina in the basypharyngeal
canal of C. hoffmanni, whereas there is only one pair in C.
didactylus. Hautier et al. (2014) discovered some differences
between the two species relative to the length, width, and
height of the skull. The two species are well discriminated on
a third principal component (9.69% of the explained vari-
ance). On this principal component, the C. didactylus skull
appeared narrower with a short snout and long zygomatic
arches whereas the C. hoffmanni skull appeared wider with a
longer snout and shorter zygomatic arches. However, the
interspecific differences in the skulls of both Choloepus spe-
cies are not apparent on the first and the second principal
components, which explain 18.71% and 9.93% of the vari-
ance, respectively. Also, a weak interspecific differentiation
was detected on the mandibular morphology. The first princi-
pal component (30.56% of total shape variation) weakly dis-
criminated C. didactylus from C. hoffmanni.
It is quite notable to have detected the mitogenome of a
specimen of C. didactylus by morphology and by geograph-
ical distribution within the clade of C. hoffmanni. We ignore if
this was a case of incomplete lineage sorting, genetic intro-
gression, recent hybridization in the wild (there are hybrids in
captivity; Jorge et al. 1978; Steiner et al. 2011) or that the
geographical distribution of the Choloepus species is relatively
unknown. Maybe the incomplete lineage sorting could be
discarded because this phenomenon occurs in very recent
differentiated species, whereas the two Choloepus species

(18.7–5.8 MYA) may not have recently differentiated. Maybe it
represents a case of recent hybridization. Steiner et al. (2011)
identified one animal as a hybrid using the nuclear gene
Enam. It had alleles derived from both Choloepus species. In
whatever case, the taxonomic status of both Choloepus spe-
cies and possible subspecies within them are completely
unknown from a molecular point of view. More extensive
sampling and additional molecular data would be helpful
especially from C. hoffmani along the southern distribution
where the species is sympatric with C. didactylus The situation
is complex because within C. dydactylus there are different
chromosomal numbers. Jorge et al. (1985), Adam (1999), and
Steiner et al. (2011) showed a low chromosomal number vari-
ation in C. hoffmanni (2n ¼ 49, 50 and 51; FN ¼ 61 and vari-
able sex chromosomes; Corin-Frederic 1969; Jorge et al. 1978;
Jorge & Pereira 2008). In contrast, C. didactylus shows several
karyotypes consisting of highly polymorphic chromosomes,
often unpaired (Jorge et al. 1985) with 2n ¼ 51, 52, 53, 59, 64,
Downloaded by [Manuel Ruiz-Garcia] at 18:13 03 January 2018
65, 66, 67 (Svartman 2012). Jorge et al. (1985) proposed that
Choloepus chromosomes vary geographically. They identified
three geographic clusters: one that includes individuals from
Central America, Peru, and Colombia with diploid numbers of
49 and 50 (C. hoffmanni); a second group composed of
female two-toed sloths from Belem to Central Brazil with dip-
loid numbers of 51 and 53 (C. didactylus), and a third group
with one individual from Manaus with a diploid number of
65 (C. didactylus). Variation in chromosome number might
suggest the existence of different geographic groups or sub-
species in the Amazon Basin for C. didactylus (Kovacs et al.
2001) or even new different species if chromosomal speci-
ation (stasipatric, parapatric or peripatric; Lewis 1966; White
1968, 1978; Reig 1980) could be demonstrated. This occurs in
the night monkeys (Aotus; Ruiz-Garc
ıa et al. 2016c). However,
no molecular studies have been carried out to validate this
possibility.
More molecular results are especially needed to resolve
the relationships among populations of trans-Andean B. varie-
gatus (or B. ephippiger) in Central America and Northern
Colombia. Additional results are also needed to help us deter-
mine the relationship of the B. variegatus population on
Gorgona Island with other B. variegatus populations. It would
also be beneficial for future studies to determine the genetic
structure within B. tridactylus, C. hoffmanni, and in C. didacty-
lus and the possibility of hybridization between the two last
taxa.
Ackowledgements
Thanks to Dr Diana Alvarez, Pablo Escobar-Armel, Nicol
as Lichil
ın, Luisa
Castellanos-Mora, Kelly Luengas, and Alan Velarde for their respective
help in obtaining sloth samples during the last 20 years. Thanks to the
Instituto von Humboldt (Villa de Leyva in Colombia; Janeth Mun ~oz), the
Peruvian Ministry of Environment, PRODUCE (Direccio
n Nacional de
Extraccio
n y Procesamiento Pesquero), Consejo Nacional del Ambiente
and the Instituto Nacional de Recursos Naturales from Peru, to the
Coleccio
n Boliviana de Fauna (Dr Julieta Vargas) and to CITES Bolivia.
Also, thanks to the National Environmental authority from Panama, and
the Brazilian IBAMA for their role in facilitating the obtainment of collec-
tion permits in Colombia, Peru, Bolivia, Panama, and Brazil. We also thank
the many people of diverse Indian tribes in Peru (Bora, Shipigo-Comibo,
Kishuarana, and Alamas), Bolivia (Siriono
, Canichana, and Chacobo), and
MITOCHONDRIAL DNA PART A 295
Colombia (Tucano, Nonuya, Yuri, and Yucuna) and many people in
Panama and Brazil for their support in obtaining samples of Bradypus
and Choloepus.
Disclosure statement
The authors report no conflicts of interest. The authors alone are respon-
sible for the content and writing of this article.
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