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

CONTACT Manuel Ruiz-Garcıa mruizgar@yahoo.es, mruiz@javeriana.edu.co Departamento de Biologıa, Facultad de Ciencias, Pontificia Universidad
Javeriana, Bogota, DC, Colombia
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
282 M. RUIZ-GARCIA ET AL.

Sempere et al. 1994). The oldest known sloths come from the morphological variation. In Central America, it almost always
Eocene of Patagonia and Antarctica but they cannot be pre- has a tan face. Many specimens from Southwestern
cisely assigned to any of the recognized lineages (Vizcaıno & Colombia, Western Ecuador, and Northcentral Brazil are char-
Scillato-Yane 1995). acterized by strikingly golden faces, although the base of
Both genera have a quadrupedal suspensory posture and each facial hair is usually dark brown. Populations of the
locomotion, and due to their phylogenetic distance and the Tapajos River in Brazil also show a strong golden frosting on
fact that no extinct species appear to have possessed this the throat. This sloth taxon also varies widely in the blotchi-
trait, many authors accept that these provide a case of ness of its dorsal coloration. However, the coat characteristics
extreme convergent evolution (Greenwood et al. 2001; could be highly variable within a population without having
Gaudin 2004; Delsuc & Douzery 2008; Billet et al. 2011; any clearly differentiated and transitional forms (Anderson &
Nyakatura 2012). Handley 2001). Wetzel (1982) lists nine subspecies of B. varie-
Many of the extinct sloths were large and ground-dwelling gatus, but the classification of Gardner (2005, 2008) recog-
such as the Mylondontidae that were at least the size of a nizes seven subspecies as the most accepted (see Table 1A).
black bear. The families contained several large species, some Bradypus tridactylus, the pale-throated sloth, inhabits parts
the size of elephants. They also lived in a variety of ecological of Venezuela, Guyana, Suriname, French Guiana, and
conditions. For example, Thalassocnus genus was a semi- Northeastern Brazil from the delta of the Orinoco River to the
aquatic sloth in South America (Canto et al. 2008). Amazon River and from the Negro River to its mouth in
The arrival of North American immigrants (around 3 MYA the Amazon River (Wetzel & Avila-Pires 1980; Wetzel 1982).
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or around 12 MYA, depending on the hypotheses of the ori- The actual distribution of B. tridactylus seems to be narrower
gin of the Isthmus of Panama; GABI, Marshall et al. 1982; than previously inferred (Gardner 2008). Anderson and
Coates et al. 2004; Coates & Stallard 2013; Bagley & Johnson Handley (2001) pointed out that the distribution of B. tridac-
2014) seems to have not affected the xenarthrans. This tylus probably does not extend southwest of the Negro River
included giant terrestrial forms, because they became suc- nor as far south as the Amazon River. No morphological sub-
cessful southern invaders of Central and North America species have been reported for this taxon.
(Patterson & Pascual 1972; Webb 1976). However, this abun- Bradypus torquatus, the maned three-toed sloth, shows a
dant continental sloth fauna became extinct in the Americas disjunct distribution in the Atlantic coastal forests of
during the Pleistocene around 10,000 YA (Long & Martin Southeastern Brazil (Lara-Ruiz et al. 2008). One population is
1974; Cartelle 1994), although Steadman et al. (2005) showed located in the Sergipe, Bahia, and part of Minas Gerais State.
that the Caribbean island sloths lasted until 4000 YA. Only six Another population is in the states of Minas Gerais, Espiritu
species belong to the two quoted genera (Bradypus and Santo, and Rio de Janeiro. The historic distribution extended
Choloepus) arrived until the present. further north along the coast (from Rio Grande do Norte to
The brown-throated three-toed sloth (B. variegatus) is the Rio de Janeiro) and was not disjunct (Gardner 2008). No mor-
sloth species with the widest geographical distribution. It’s phological subspecies have been reported for this species.
range is from Honduras south into Ecuador, Colombia, Bradypus pygmaeus, the pygmy sloth, is only found on the
Venezuela (with the exception of the Orinoco River delta and 4.3 km2 Isla of Escudo de Veraguas (islands of Bocas del
the Guiana highlands), Peru, and also includes Bolivia to Toro), 17.6 km off the Northern Caribbean coast of Panama. It
Northern Argentina and a major part of Brazil (except Amapa was recently described by Anderson and Handley (2001,
in Northern Brazil and Parana and Rio Grande do Sul in 2002). These authors showed significant morphological and
Southern Brazil; Wetzel & Avila-Pires 1980). This taxon is the morphometric data of this taxon relative to the other
only species of the genus that displays notable geographic 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 Para, 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, Volcan 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 Martın Department, Peru
MITOCHONDRIAL DNA PART A 283

were separated from the adjacent Panamanian mainland by Materials and methods
rising sea levels during the past 10,000 years (Holocene).
They further claimed that this provoked at least four inde- The origins of the 39 we sampled are listed in Figure 1. We
pendent events, with sloths on five of the islands evolving sequenced and analyzed the entire mitogenome of each sam-
smaller sizes following insularization. Sloths on the younger ple save for the mt control region. There were 19 B. variega-
islands remained conspecific with mainland Panamanian pop- tus from Colombia, Venezuela, Peru, and Brazil and four B.
ulations of B. variegatus. However, on the oldest and the tridactylus from Guyana, Surinam, and French Guiana. There
most separated island of this archipelago, the three-toed was also a B. pygmaeus from Veraguas, Panama, and a B. tor-
sloth was extremely differentiated and quickly formed a new quatus from Rio de Janeiro State, Brazil. The sample also
species, B. pygmaeus. included four C. didactylus from Colombia, Venezuela, and
Two-toed sloths are grouped in two species: C. hoffmanni Surinam as well as 10 C. hoffmanni from Costa Rica, Panama,
(Hoffman’s two-toed sloth) and C. didactylus (Linnaeus’s two- and Colombia. For an outgroup, we sequenced the complete
toed sloth). mitochondrial genome of two Tamandua tetradactyla from
Choloepus hoffmanni has two disjoint populations; the Peru and one T. mexicana from Panama.
northern most population extends from Nicaragua south into DNA was extracted and isolated from either hair or muscle
western Venezuela and the trans-Andean areas of Colombia samples using the QIAamp DNA Micro Kit (Qiagen, Inc.,
and Ecuador. The southern one is found from north-central Valencia, CA) following the protocol provided by the manu-
Peru through the extreme southwestern part of Brazil to facturer. Muscle extractions followed the protocol ‘DNA
Central Bolivia (Meritt 2008). Five morphological subspecies Purification from Tissues.’ Mitochondrial genomes were
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have been defined for this species (Gardner & Naples 2007) sequenced by long-template PCR, which minimizes the
(see Table 1B). chance of amplifying mitochondrial pseudogenes from the
Choloepus didactylus is found throughout Venezuela and nuclear genome (numts) (Thalmann et al. 2004; Raaum et al.
the Colombian Eastern Llanos (Meta Department), Guyana, 2005). PCR amplification of mitochondrial DNA was carried
Suriname, French Guiana, and the Maranhao State in Brazil out using a LongRange PCR Kit (Qiagen, Inc.), with a reaction
south along the Brazilian Amazon (Amazon River) and west volume of 25 ll and a reaction mix consisting of 2.5 ll of 10
into the upper Amazon Basin of Colombia, Peru, and Ecuador LongRange PCR Buffer, 500 lM of each dNTP, 0.6 lM of each
(Wetzel 1982, 1985; Meritt 2008). No subspecies have been primer, 1 unit of Long-Range PCR Enzyme, and 50–250 ng of
described but Wetzel (1982) proposed three names for pos- template DNA. Cycling conditions were as follows: 94  C for
sible subspecies: C. d. columbianus, C. d. florenciae, and C. d. 5 min, followed by 45 cycles denaturing at 94  C for 30 s, pri-
napensis. mer annealing at 50–57  C (depending on primer set) for 30 s,
Many mitogenomic analyses have proved to be extremely and extension at 72  C for 8 min, followed by 30 cycles of
useful in determining phylogenetic relationships and in esti- denaturing at 93  C for 30 s, annealing at 45–52  C (depend-
mating divergence times in many different groups of organ- ing on primer set) for 30 s, and extension at 72  C for 5 min,
isms. Examples of these groups are fishes (Miya et al. 2003; with a final extension at 72  C for 8 min. To minimize the pos-
Inoue et al. 2010), amphibians (Zhang et al. 2005; Zhang & sibility of amplifying nuclear mitochondrial pseudogenes
Wake 2009), birds (Pereira & Baker 2006; Slack et al. 2007), (numts), four sets of primers were used to generate overlap-
and different groups of mammals (Arnason et al. 2002, 2004, ping amplicons from 3687 to 4051 bp in length, thereby ena-
2008; Hodgson et al. 2009; Matsui et al. 2007; Chiou et al. bling a quality test for genome circularity (Bensasson et al.
2011; Finstermeier et al. 2013; Pozzi et al. 2014; Ruiz-Garcıa 2001; Thalmann et al. 2004). Additional information about
et al. 2016a,b). Mitochondrial gene trees are more precise in these primers and amplified genes is provided by Ruiz-Garcıa
reconstructing the divergence history among species than et al. (2016a). The control region was not included in the
other molecular markers (Moore 1995). Cummings et al. mitogenomic analysis because of the difficulty of aligning this
(1995) showed that mitochondrial genomes have higher region among the Bradypus, Choloepus, and Tamandua sam-
information content per base than nuclear DNA. However, we ples. This difficulty is due to the high quantity of indels in
must take into account that lineage specific variation in evo- the mitochondrial DNA fragment. Both mt DNA strands were
lutionary rates within individual mitochondrial DNA has been sequenced directly using BigDye Terminator v3.1 (Applied
previously determined (Gissi et al. 2000). This variation has Biosystems, Inc., Foster City, CA). Sequencing products were
been linked to differences in mutation rates and agrees quite analyzed on an ABI 3730 DNA Analyzer system (Applied
well with longevity in mammals for mt Cyt-b, for instance Biosystems, Inc.). Sequences were then assembled and edited
(Nabholz et al. 2008). It is for this reason that the mammalian using Sequencher 4.7 Software (Gene Codes, Corp., Ann
mitochondrial DNA clock for individual genes can be particu- Arbor, MI). Overlapping regions were examined for irregular-
larly erratic (Nabholz et al. 2009). Also, by using relaxed clock ities such as frameshift mutations and premature stop
models, we can account for substitution rate variations codons. A lack of such irregularities indicates an absence of
among lineages (Thorne et al. 1998). The analyses of com- contaminating numt sequences.
plete mitochondrial genomes could overcome this problem. We used approximately 15,473 bp (although numerous but
Taking into consideration all that has been commented minor indels were detected within and between Bradypus
upon, we focus, in this work, on the reconstruction of the and Choloepus mitochondrial genomes). The alignments with
phylogeny of the six extant sloth species using all the genes were concatenated after removing problematic
mitogenomics. regions using Gblocks 0.91 (Talavera & Castresana 2007)
284 M. RUIZ-GARCIA ET AL.
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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 (codon 1, codon 2, codon 3). Additionally, 50 iterations were
aligned regions and it has been shown to be particularly run using two data partitions (codons 1 þ 2 combined, codon
effective in phylogenetic studies including very divergent 3). For each analysis, the HKY þ G þ I model (Hasegawa et al.
sequences (Castresana 2000; Talavera & Castresana 2007). 1985) was used to search for the MLT and topologic support
The individual alignments were then concatenated by means was estimated with 500 bootstrap replicates using the
of the SequenceMatrix v1.7.6 Software (Vaidya et al. 2011) to HKYGAMMA (Stamatakis 2006). Maximum-likelihood bootstrap
create a master alignment. 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-
Data analyses uted rate varying among sites, because it was determined to
Phylogenetics procedures be the better model using MrModeltest v2.3 Software. BI was
completed with the BEAST v. 1.8.1 program (Drummond et al.
The MrModeltest v2.3 Software (Nylander 2004) and the 2012). Four independent iterations were run using three data
Mega 6.05 Software (Tamura et al. 2013) were applied to partitions (codon 1, codon 2, codon 3) with six MCMC chains
determine the best evolutionary mutation model for the sampled every 10,000 generations for 30 million generations
sequences analyzed for each individual gene, for different after a burn-in period of 3 million generations. We checked
partitions and for all the concatenated sequences. Akaike for convergence using Tracer v1.6 (Rambaut et al. 2013). We
information criterion (AIC; Akaike 1974; Posada & Buckley plotted the likelihood versus generation and estimated the
2004) and the Bayesian information criterion (BIC; Schwarz effective sample size (ESS >200) of all parameters across the
1978) were used to determine the best evolutionary nucleo- four independent analyses. Additionally, AWTY (Are We There
tide model. Yet?) Software (Wilgenbusch et al. 2004; Nylander et al. 2008)
Phylogenetic trees were constructed by using two proce- was used to plot pairwise split frequencies for the four inde-
dures: Maximum-Likelihood tree (MLT) and Bayesian Inference pendent MCMC runs and to check the posterior probabilities
(BI). 1- A MLT was obtained using RAxML v.7.2.6 Software of clades for nonoverlapping trees in the sample using
(Stamatakis 2006). To select the best fitting model, 50 inde- the compare and slide commands. The results from different
pendent iterations were run using three data partitions runs were combined using LogCombiner v1.8.0 Software
MITOCHONDRIAL DNA PART A 285

(Rambaut & Drummond 2013a) and TreeAnnotator v1.8.0 [0, infinite; initial value 80]. We used Tracer v1.6 to analyze
(Rambaut & Drummond 2013b). A Yule speciation model and the marginal densities of temporal splits. The Bayesian
a relaxed molecular clock with an uncorrelated log-normal Skyline reconstruction option was selected for the trees log
rate of distribution (Drummond et al. 2006) were used. file. A stepwise (constant) Bayesian skyline variant was
Posterior probability values provide an assessment of the selected with the maximum time as the upper 95% high pos-
degree of support of each node on the tree. Values higher terior density (HPD) and the trace of the root height as the
than 0.95 were considered strong support for monophyletic treeModel.rootHeigh. To determine the time ranges for pos-
clades (Erixon et al. 2003; Huelsenbeck & Rannala 2004). We sible demographic changes for B. variegatus and C. hoffmanni
estimated the lower and upper 95% highest posterior den- with mitogenomics, we considered that the evolution of
sities (HPD), the means, geometric means, medians, marginal these extant sloth taxa began during the Miocene (7-11
densities, and traces with Tracer v1.6 Software. Trees were MYA).
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 Results
sloth species with mitogenomics BI, we employed two priors. Mitogenome phylogenetics for the sloths
One was around 58.4 ± 2 MYA for the split between the
ancestors of the anteaters and the sloths. The second was The MLT and the BI basically showed the same results for the
around 30 ± 2 MYA for the split of the ancestors of Bradypus mitogenomics analyses (Figure 2(A,B)). Here, we only com-
and Choloepus. Both priors were taken from Delsuc et al. ment on the BI. The first split was between the ancestor of
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(2012) and Gibb et al. (2016). the anteaters (Tamandua) and the sloths (p ¼ 1, 55.74 MYA;
We constructed a Median Joining Network (MJN) (Bandelt 95% HPD ¼ 53.52–61.31 MYA). The split between the two spe-
et al. 1999) using Network 5.0.0.0 Software (Fluxus cies of Tamandua (T. mexicana and T. tetradactyla), following
Technology Ltd) to estimate possible divergence times (using this procedure, was around 11.4 MYA (95% HPD ¼ 2.87–18.06
mitogenomics) among the haplotypes found in the six extant MYA; p ¼ 1). The split between the ancestors of the two
sloth species. Additionally, the q statistic (Morral et al. 1994) sloths (Bradypus and Choloepus) was around 30.93 MYA (95%
and its standard deviation (Saillard et al. 2000) were esti- HPD ¼ 28.52–36.25 MYA; p ¼ 1). The mitochondrial diversifica-
mated and transformed into years. The q statistic is unbiased tion seems to begin earlier within Bradypus than in
and highly independent of past demographic events. For the Choloepus. The first split within the first genus was that of
overall mitogenome, we employed a mutation rate of the ancestor of B. torquatus and the remaining Bradypus taxa
1  106 (an average from the studies of Wayne et al. 1997; (p ¼ 1, 21.28 MYA; 95% HPD ¼ 19.14–31.37 MYA). The next
Michaux et al. 2002; Benton & Donoghue 2007; Nabholz et al. split within Bradypus was that of the ancestor of B. tridactylus
2008, 2009), which corresponds to one mutation each (p ¼ 1, 14.15 MYA; 95% HPD ¼ 11.7–23.45 MYA). Within B. tri-
206,000 years. dactylus, the mitochondrial diversification began around 4.57
Some phylogenetics analyses, as BI and MJN, were MYA (95% HPD ¼ 1.41–9.79 MYA; p ¼ 1). It was followed by
also carried out using only the mtCyt gene and including the separation of the ancestor of B. pygmaeus (p ¼ 0.67, 12.27
some sequences of two extinct sloths Mylodon darwinii MYA; 95% HPD ¼ 9.28–20.18 MYA). The mitochondrial diversi-
(Mylodontidae) and Nothrotheriops shastensis fication within B. variegatus began around 8.07 MYA (95%
(Nothrotheriidae) generated by Greenwood et al. (2001). HPD ¼ 6.21–16.21 MYA; p ¼ 1) with the separation of the
We also determined the differences among the specimens ancestor of the trans-Andean haplogroup. In turn, the diversi-
analyzed for mitogenomics by means of a factorial analysis fication within this haplogroup began around 1.42 MYA (95%
with the Kimura-two-parameter genetic distance (Kimura HPD ¼ 0.45–4.49 MYA; p ¼ 1). The ancestor of the cis-Andean
1980) and gap blocks correction using DARwin v. 6.0 B. variegatus split into two well differentiated populations
Software (Perrier & Jacquemoud-Collet 2006). (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-
Genetic heterogeneity and demographic evolution for
tuted by individuals from the Negro River, Tocantins River,
Bradypus and Choloepus with mitogenomics
and Brazilian Eastern area (p ¼ 0.74, 3.44 MYA; 95%
The genetic heterogeneity among the extant sloth species HPD ¼ 2.98–10.77 MYA).
was estimated with a FST population comparison pair analysis The first split within Choloepus separated the ancestors of
with exact tests by means of Markov chains with the follow- C. didactylus and C. hoffmanni around 18.68 MYA (95%
ing parameters: 10,000 dememorizations parameters, 20 HPD ¼ 8.66–23.57 MYA; p ¼ 1). Within C. didactylus, the mito-
batches, and 5000 iterations per batch. chondrial diversification began around 0.51 MYA (95%
A Bayesian skyline plot (BSP) was obtained for the con- HPD ¼ 0.01–2.56 MYA; p ¼ 1). The mitochondrial diversifica-
catenated mitochondrial sequences by means of the BEAST v. tion was within C. hoffmanni older (12.58 MYA; 95%
1.8.1 and Tracer v1.6 software. The Coalescent-Bayesian sky- HPD ¼ 5.66–16.49 MYA, p ¼ 0.78). The first haplotype to
line option in the tree priors was selected with four steps diverge within C. hoffmanii was from Jerico  (Antioquia
and a piecewise-constant skyline model with 30,000,000 gen- Department, Colombia), followed by a clade with two Central
erations (the first 3,000,000 discarded as burn-in), kappa with American individuals (Panama and Costa Rica), whose ances-
log Normal [1, 1.25], and Skyline population size with uniform tor diverged around 10.43 MYA. These two Central American
286 M. RUIZ-GARCIA ET AL.
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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 times lower than the corresponding BI temporal estimate.
MYA; p ¼ 1). Thus, in Northern Colombia there are live haplo- The time split between the Tamandua and both sloth’s spe-
types that precede the split between other Northern cies was 30,353,846 ± 2,143,559 YA, considerably lower than
Colombian and Central American haplotypes in C. hoffmanni. the BI estimate (around 55 MYA). The next haplotypes to
A very interesting fact is the inclusion of the haplotype of diverge were those of Choloepus. The separation of the hap-
one individual of C. didactylus sampled in the Colombian lotypes of C. didactylus and C. hoffmanii is not clear. One C.
Amazon with a typical phenotype of this species but within hoffmanii’s haplotype (21; Jerico , Antioquia Department,
the haplotypes of C. hoffmanni. Colombia) was more related to the unique haplotype found
The MJN procedure in Figure 3 shows on the right side in the three C. didactylus specimens we sampled in the
of the figure the relationship among the haplotypes of both Guiana Shield. However, the unique Amazonian C. didactylus
Tamandua species. The temporal split between both individual we sampled shared its haplotype with a C.
Tamandua species was 1,600,000 ± 244,949 YA, around nine hoffmanni individual sampled in the Santander Department
MITOCHONDRIAL DNA PART A 287
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Figure 2. Continued.

(Colombia). The temporal split between both Choloepus spe- haplotypes was 3,600,000 ± 582,204 YA (against eight MYA
cies was 5,757,143 ± 869,553 YA, which is considerably lower for BI). The splits between the Western Amazon haplogroup
than 19 MYA estimated with BI. The temporal split between and the Brazilian Atlantic forest þ Tocantins, and the Negro
Choloepus and Bradypus was 23,450,000 ± 1,910,871 YA, River haplogroups were 1,500,000 ± 100,000 YA and
which is also minor compared to 31 MYA estimated with BI. 2,100,000 ± 244,949 YA, respectively, which were also lower
The B. torquatus’ haplotype diverged from all the other than the BI estimate (3.4 MYA). Finally, B. pygmaeus was
Bradypus species’s haplotypes 19,056,000 ± 1,770,134 MYA derived from an undetermined haplotype placed between
similar to the 21 MYA estimate obtained with BI. In this those from the Western Amazon and the Negro River hap-
case, B. torquatus was more related to B. tridactylus than to logroups. The temporal split between these B. variegatus
other species of Bradypus. The temporal split between B. tri- haplotypes and B. pygmaeus was estimated at
dactylus and B. variegatus was estimated at 4,300,000 ± 616,441 YA, which is substantially less than the
6,566,667 ± 961,769 YA, which is lower than the date of 14 12 MYA value obtained by BI.
MYA detected by BI. As in the previous analysis, the trans- The factorial analysis for the anteaters and sloths for the
Andean B. variegatus was the first to diverge for this species complete mitochondrial genome (Figure 4) showed that the
and this haplogroup is more related to B. tridactylus than most differentiated individuals belonged to the individuals of
any other B. variegatus’s haplogroup. The temporal split Tamandua. The individuals of Bradypus and Choloepus were
between the trans-Andean and all the other cis-Andean perfectly differentiated, the first on the right side of the
288 M. RUIZ-GARCIA ET AL.
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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 (in middle), and B. variegatus. Within B. variegatus in the
B. torquatus are extremely differentiated (at the centre of the upper sector, we observed two groups of individuals, one
figure) from the individuals of the other three species of with trans-Andean individuals and the other with Western
Bradypus. For the other three species of Bradypus, from left to Amazon individuals. Within the lower sector, there was a mix-
right, we observe the individuals of B. tridactylus, B. pygmaeus ture of individuals from the Negro River, Tocantins, and
MITOCHONDRIAL DNA PART A 289
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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 Mitogenomics population genetic heterogeneity of the
from the Guiana Shield were clearly differentiated from C. sloths
hoffmanni, but the C. didactylus individual from the
Colombian Amazon was undifferentiated from the C. hoff- A comparison of the genetic heterogeneity among the differ-
manni individuals. One individual of this last species was ent sloth taxa we analyzed for mitogenomics indicated
highly differentiated from the other specimens (Cesar extremely high and significant levels of differentiation. All the
Department in Colombia). genetic differentiation statistics were highly significant
The first analysis only at the mtCyt-b gene with BI (Table 2). Some traits are very interesting in the comparison
(although we also obtained ML and NJ trees; Figure 5) pairwise analysis (Table 3). The lowest degree of genetic dif-
showed very high posterior probabilities and bootstraps for ferentiation was between the Western Amazon B. variegatus
all the clades obtained (p ¼ 1 and bootstraps 100%). and the Brazilian Eastern B. variegatus populations
The clade of Mylodon darwini þ Choloepus had the lowest (FST ¼ 0.686). Nevertheless, the trans-Andean B. variegatus
posterior probability (0.67) and the lowest bootstrap (89%). population showed levels of genetic differentiation relative
The BI indicated that Mylodon darwini þ Choloepus and to the Western Amazon and the Brazilian Atlantic Eastern
Nothrotheriops shastensis þ Bradypus formed two groups populations (FST ¼ 0.889 and 0.869, respectively). These are
which diverged around 39 ± 9 MYA. The split between similar to those found among different species pairs such
Bradypus and Nothrotheriops was estimated to have occurred as for example the Western Amazonian B. variegatus vs. B.
around 31.5 ± 5 MYA, and that of Mylodon and Choloepus to tridactylus (FST ¼ 0.890) and the Brazilian Eastern B. variegatus
have occurred around 31 ± 5 MYA. Therefore, it was practic- vs. B. tridactylus (FST ¼ 0.840). Thus, the genetic differentiation
ally a synchronous split. In the same manner, the split of the trans-Andean B. variegatus population in regard to the
between the Guiana Shield C. didactylus and the C. hoffmanni other cis-Andean B. variegatus populations is comparable to
(including one Amazonian C. didactylus) and the split the genetic differentiation among other defined sloth species.
between the trans- and the cis-Andean B. variegatus were The BSP with mitogenomics showed the exact same
synchronous around 13 ± 6 MYA (Miocene). trends for B. variegatus and for C. hoffmanni (Figure 7) with
The second analysis with the MJN procedure (Figure 6), constant or very slight increases during the Pliocene and a
the temporal splits were considerably minor: 23.49 MYA for clear decrease in the last 0.5 MY (Pleistocene).
the split of the two current sloth genera, 12.1 MYA for
Bradypus-Nothrotheriops and 16.12 MYA for Choloepus- Discussion
Mylodon. The split between C. didactylus-C. hoffmanni and
Temporal divergences in the origins of the Xenartharns,
that between the trans- and cis-Andean B. variegatus popu-
between the two extant arboreal sloth genera and
lations were synchronous around 7 MYA. These splits seem
within Tamandua
to be independent of the different temporal split estimates
obtained by BI and MJN and seem to be correlated with a We determined a temporal split of around 65 MYA for the
global temperature decrease (Figure 6). ancestor of the armadillos (Dasypus) and the remaining
290 M. RUIZ-GARCIA ET AL.
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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 [58.4 MYA (48.6–64.7 MYA)]. Our MJN value clearly seems to
gene and mitochondrial 12S rRNA and 16S rRNA genes. They be an underestimate of that age (30.4 MYA). However, Barros
estimated an interval of around 59–76 MYA for the process et al. (2008) estimated the split between sloths and anteaters
of diversification of the current Xenarthra. Other studies indi- to have occurred at about 37 MYA, relatively similar to the
cated similar findings (67.8 MYA [61.3–74.7 MYA], Delsuc last estimation.
et al. 2012; 67.7 MYA [60.4–71.6 MYA], Gibb et al. 2016). We We have obtained three temporal estimations for the split-
also estimated a temporal split with BI of 56 MYA (53–61 ting between the ancestors of the two extant sloth genera
MYA) between the ancestors of Tamandua and the sloths. (Bradypus and Choloepus): 31 MYA (for BI with mitogenomes),
This estimate agrees quite well with that determined by 23.5 MYA (for MJN with mitogenomes), and 39 MYA (for BI
Delsuc et al. (2004) [55.2 MYA (46–65 MYA)], Delsuc et al. with mt Cyt-b gene). It is interesting to note the discrepancy
(2012) [60.1 MYA (53.1–67.2 MYA)] and Gibb et al. (2016) between the BI (fossil-calibrated DNA phylogeny) and
MITOCHONDRIAL DNA PART A 291
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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. the temperature decreased and the ocean level changed. This
First, we do not know if we are using precise fossil data for coincides with the end of the first major Bolivian Andean cri-
BI and second we don’t know the exact mutation rates for sis. This diastrophic event was an intense deformational and
entire mitogenomes. In fact, age estimates from DNA sequen- magmatic episode widespread along the Andes (Sempere
ces show some difficulties related to the variation of evolu- et al. 1994). The intensity of this Andean change is seen as a
tionary rates both over time and among lineages. It is turning point in Andean tectonics because the Andes created
challenging to overcome these problems because there is no a rain shadow that significantly influenced South American
unambiguous separation of evolutionary rates and time climates (Marshall & Sempere 1993). Together with the
(Smith & Peterson 2002). Bolivian Andean crisis, it was correlated with a thermal opti-
These three temporal estimates could generate three mum followed by sharp marine regressions (Patterson &
hypotheses for the split of the ancestors of the two current Pascual 1972; Haq et al. 1987) and by a brief but deep glacial
sloth genera: maximum (Zachos et al. 2001). This could be correlated with
(1) The lowest estimate (23.5 MYA) is relatively similar to the split of the two extant sloth genera as well as with the
those estimates obtained by other authors. Delsuc et al. beginning of the Miocene radiation of ground sloths
(2001) estimated this split to have occurred around 18 MYA. (Patterson & Pascual 1972).
Delsuc et al. (2004) studied three nuclear genes (5130 bp) (2) Our highest estimate (39 MYA) agrees with that
and estimated it to have occurred 21 MYA (15–27.8 MYA) claimed by Gaudin (2004), who suggested that the split
during the uplift of the Central and Northern Andes in the between the two extant genera is an ancient event that
Quechua I phase (23–17 MYA). At this time (Early Miocene) occurred at about 40 MYA. Also Katzourakis et al. (2009)
292 M. RUIZ-GARCIA ET AL.

concluded that the sloth endogenous foamy virus invaded speciation phenomena within the two current sloth genera or
the genome of a sloth ancestor in the late Middle Eocene within the two main sloth groups. For the BI estimate with
around 39 MYA. This hypothesis could be related with the mitogenomics, the process of speciation in Bradypus was
Incaic Tectonic Phase (42–35 MYA) and the uplifting of the around 21.3 MYA and in Choloepus around 18.7 MYA. The
Central Andes and strong volcanic activity (Noble et al. 1979; diversification of the armadillo subfamily Tolypeutinae
Sempere et al. 1994). During this epoch there was also a (Priodontes, Cabassous, and Tolypeutes) occurred within the
cooling sea process (Mackensen & Ehrmann 1992) and a con- same epoch around 21 MYA (15–28 MYA) (Gibb et al. 2016),
comitant increase of the Antarctic ice sheet and an expansion in the Early Miocene. The BI estimates (using mt Cyt-b) of the
of the Notophagus forests. This indicates a trend of general split of Choloepus-Mylodon (31 MYA) and Bradypus-
cooling of the continental zones (Barreda & Palazzesi 2007). Nothrotheriops (31.5 MYA) were almost the same. This tem-
Related with this, Gibb et al. (2016) estimated a divergence of poral split is in line with the first offshoot within
42 MYA between two lineages of armadillos that they Myrmecophagidae around 31.8 MYA (Cyclopes; Barros et al.
claimed as two different families, Dasypodidae (dasypodines), 2008). Our temporal splits for Bradypus and Nothrotheriidae,
and Chlamyphoridae (euphractines, chlamyphorines, and 31 MYA for BI and 12 MYA for MJN, overlapped with the tem-
tolypeutines). poral estimates provided by Slater et al. (2016) (23.5–17.5
(3) Between these two extreme estimates, there is our MYA). Also, the BI estimate with mt Cyt-b offered the same
own intermediate estimate of 31 MYA. Sarich (1985), with time (around 13 MYA) for the diversification process within B.
immunological data, suggested that both sloth genera variegatus (trans- and cis-Andean populations) as well as for
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diverged 35 MYA. Thus, our estimate, is strongly in agree- the trans-Andean C. hoffmanni and the cis-Andean C. didacty-
ment with that obtained by other molecular studies such as lus populations. This also agrees with the split between
by Delsuc et al. (2012) (28.3 MYA [22–35.2 MYA]) and Gibb Myrmecophaga and Tamandua at 12.9 MYA (Barros et al.
et al. (2016) (29.9 MYA [16.5–39.6 MYA]). This could be sup- 2008). This temporal estimate was lower when it was
ported by a megalonychid fossil from Puerto Rico (30 MYA) obtained with the MJN procedure. But, it was identical to
and other equally old megalonychids from Southern South that for the Bradypus and Choloepus taxa (around 7 MYA).
America (MacPhee & Iturralde Vinent 1995). This agrees that The time agrees quite well with the diversification of extant
the oldest, reasonably well preserved fossils assigned to Euphractine armadillos and the split among the long-nosed
Folivora are Oligocene (Carlini & Scillato-Yan e 2004; Pujos & armadillos (Dasypus) in the late Miocene (7 MYA) following
De Iuliis 2007). Similarly, Slater et al. (2016), using the Delsuc et al. (2004).
Deseadan fossil taxa and fall them within crown families (as Similalrly to what was commented by Gibb et al. (2016),
suggested by morphological data), obtained an estimation of and in consideration of the model of Condamine et al. (2013),
29 MYA. However, Slater et al. (2016), treated these fossils as a temperature-dependent model showed that the speciation
either stem taxa or allowed them to be crown taxa that fell rate over the entire xenarthran temporal splits (in our case,
outside of recognized families, and estimated the initial diver- sloth temporal splits) was negatively correlated with tempera-
sification in the sloths to have occurred around 21.5 MYA. ture. The periods of intense cooling (Zachos et al. 2001) since
This coincided with the first hypothesis. the Late Eocene, Oligocene, and the Middle Miocene, fol-
Thus, following the two last hypotheses, major sloth lowed by the presence of the circum Antarctic current and
groups probably diverged during a short period of time in the continuous uplift of the Andes (Garzione et al. 2008),
the Late Eocene and Oligocene. However, much of the diver- could have caused the aridification of South America. These
sification within both extant sloth genera could have events could have also caused and the formation of dry bio-
occurred in the Miocene or early Pliocene, which agrees with mes (Caatinga, Cerrado, Chaco, Pampas; Simon et al. 2009;
Delsuc et al. (2002, 2004) and Gibb et al. (2016). Hoorn et al. 2010) and the formation of forest refugia. During
Episodes of tectonic uplift since the Cretaceous period and the dry periods rainforest communities split into isolated refu-
throughout the Cenozoic (Paleocene until Pliocene) led to an gia separated by savannah or arid pampas (Vanzolini &
increase in elevation of the Andes and subsequent modifica- Williams 1970; Whithmore & Prance 1987; Haffer 1969, 1997,
tions in atmospheric circulation. This in turn led to major 2008). These forest refugia could be the centers of evolution
changes in habitats and climatic conditions, thus shaping and differentiation of the different sloth taxa.
faunal and floral assemblages (Pascual & Ortiz Jaureguizar Our mitogenomics results with BI and MJN for the two
1990; Marshall & Sempere 1993; Hoorn et al. 2010). These species of Tamandua are extremely different. BI offered a split
uplifts occurred 125–112 MYA (Peruvian Tectonic phase), of 11.4 MYA (Miocene), whilst MJN showed an estimate of 1.6
42–35 MYA (Incaic Tectonic phase), 23–17 MYA (Quechua I MYA (Pleistocene). We believe that the second estimate could
Tectonic phase), 9.5–7 MYA (Quechua II Tectonic phase) and be more realistic than the first one for two reasons. The first
6–2.7 MYA (Quechua III Tectonic phase) and they went from one is related with the mitogenomics results of Gibb et al.
the South to the North. The uplift of the Andes could gener- (2016). They found a small genetic differentiation between
ate important barriers for sloths and other species (Lessa the trans-Andean T. mexicana and the cis-Andean T. tetradac-
et al. 2003; Miller et al. 2008; Hoorn et al. 2010; Rull 2011). tyla (2.8% for all the mitogenome and 2.1% for mt COI, virtu-
The problem with the three hypotheses is that the Andean ally identical to our estimates) and a temporal split of 1 ± 0.4
uplift’s influence on the origins of the current sloth genera MYA. The two tamanduas are considered two distinct species
could happen at any time because of the continuous uplift of based on differences in coat colouration, body size, skull
the Andes. What seems certain is the synchronicity of characters, and number of caudal vertebrae that can be quite
MITOCHONDRIAL DNA PART A 293

variable within populations (Wetzel 1985). If our second esti- This could be the reason why Barros et al. (2003), who ana-
mate is right, then we can question if these Tamandua taxa lyzed the mt 16s RNA gene, estimated the divergence of the
are really different species. Only the analyses of nuclear taxa to have occurred 0.4 MYA. They probably estimated the
molecular markers, chromosomal differences, and the positive temporal split between two B. variegatus specimens.
or negative existence of post- and/or pre-zygote barriers However, B. tridactylus showed the presence of a pair of fora-
could shed light on the question of one versus two species mina at the anterodorsal nasopharynx. This pair has been
within Tamandua. Indeed, the number of chromosomes used as reference for a stable diagnostic character to help dif-
(2n ¼ 54) and the fundamental numbers (FN ¼ 104) are the ferentiate B. variegatus from B. tridactylus (Wetzel & Avila-
same in both taxa (Hayssen 2011). This could be correlated to Pires 1980; Wetzel 1985; Anderson & Handley 2001).
the fact that both taxa could be a unique species. The second Our temporal split estimates between the ancestors of B.
one is related with the fossil record. Although anteaters are pygmaeus and B. variegatus were 12.3 MYA with BI and 4.3
known to have been around since the Santacrucian South MYA with MJN. A unique comparative data set comes from
American Land Mammal Age (around 16 MYA; Delsuc et al. Gibb et al. (2016), who estimated a split around 7.7 MYA
2004), the fossil record indicates that Tamandua has only (3.6–12.6 MYA), which is within the range of our two esti-
been around since the Pleistocene and the Holocene mates. Thus, our data are in agreement with the findings of
(McKenna & Bell 1997; Simpson 1945; McDonald et al. 2008). Anderson and Handley (2001, 2002), as well Gibb et al.
Some coprolites have been found in Brazil (Ferreira et al. (2016). The pygmy form of the three-toed sloths from Isla
1989). Thus, it seems that the diversification process in Escudo in Panama is a completely different species from B.
Tamandua could be more related with Pleistocene events
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variegatus. The morphological differences observed by


than with Miocene ones. 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
Temporal splits within Bradypus and Choloepus
external base of the auditory bulla; large external auditory
Our mitogenomics split estimates for the ancestor of B. tor- meatus; ventral edge of stylohyoid usually smoothly concave;
quatus from the other Bradypus taxa were 21.3 MYA for BI coronoid process of the mandible slender, and strongly fal-
and 19 MYA for MJN. These estimates agree quite well with cate). The sloths, having evolved on islands, are extremely
that of Gibb et al. (2016), who obtained a value of 19 ± 4.7 interesting from an evolutionary point of view. For example,
MYA. All these estimates are clearly higher than the estimate B. variegatus on Gorgona Island (Pacific area of Colombia), is
from Barros et al. (2008) who stated that the split occurred at a greater distant from the Colombian shore than Isla
approximately 7.7 MYA. They concluded that the split of the Escudo is from the Panama mainland. This taxon seems to
ancestor of B. torquatus from the ancestors of other Bradypus have similar morphological traits to the mainland trans-
species agrees quite well with the separation of two monkey Andean B. variegatus population but we ignore the molecular
genera (Brachyteles and Lagothrix; around eight MYA). The genetics characteristics of this island population.
geographic distributions of these monkey genera in the The diversification within B. variegatus has been intensively
Amazon and Brazilian Atlantic forest are similar to that of studied in Moraes-Barros et al. (2007), Moraes-Barros and
Bradypus. Moraes-Barros et al. (2011) estimated a divergence Arteaga (2015) and Ruiz-Garcıa et al. (2016a). Mitogenomic
of 12 MYA for the ancestor of B. torquatus and the ancestor and mt control region studies have both shown the trans-
of B. tridactylus þ B. variegatus. Clearly, our estimates for B. Andean B. variegatus population (Ruiz-Garcıa et al. 2016a) to
torquatus are much older than estimates obtained by some clearly be monophyletic and well differentiated relative to
authors for other congeneric endemic species of these two other B. variegatus populations. Ruiz-Garcıa et al. (2016a) con-
biomes (Amazon vs. Brazilian Atlantic forest). For example, cluded that it should be defined as a new species of three-
consider the curassows (Pereira & Baker 2006), monkeys toed sloth: Bradypus ephippiger following the Phylogenetic
(Meireles et al. 1999; Cort es-Ortiz et al. 2003; Bloch et al. Species Concept (PSC; Cracraft 1983). However, we are reluc-
2016), and vesper mice (Salazar-Bravo et al. 2001). The esti- tant to define this population as a new species until new
mate of around 20 MYA for the separation of B. torquatus data indicate strong karyotype differences and/or pre or post-
preceded, for instance, the split of Tamandua and zygote reproductive barriers (Biological Species Concept, BSC;
Myrmecophaga around 12.9 MYA (Barros et al. 2003). For this Mayr 1942) between the trans and the cis-Andean popula-
reason, we agree with other authors that B. torquatus merits tions. The temporal split estimates between the ancestors of
the status of a different genus (Scaeopus; S. torquatus) follow- the trans and the cis-Andean B. variegatus were 8 MYA for BI
ing Barros et al. (2008), Paula-Couto (1979) and Santos (1977). and 3.6 MYA for MJN.
Our temporal estimates for the split of the ancestor of B. Similarly, the Western Amazon population is well differen-
tridactylus from the remaining Bradypus species were 14.1 tiated according to an analysis of the mt control region (Ruiz-
MYA for BI and 6.6 MYA for MJN. The estimate carried out by Garcıa et al. 2016a). Also, like in several mt control region
Gibb et al. (2016) was around 5.7 MYA (3.6–12.6 MYA) and analyses, the Negro River’s haplotype was more related to
that of Moraes-Barros et al. (2011) was around 6–4.8 MYA. the Brazilian Eastern haplogroup than to the Western
Therefore, our MJN estimate is more similar to those of these Amazon one. But, in contrast to that found with the mt con-
two authors. Also, our BI could be an overestimation of the trol region (Ruiz-Garcıa et al. 2016a), the Tocantins River’s
reality. In the wild, sometimes it is difficult to separate mor- haplotypes were more related with the Brazilian Eastern hap-
photypes of B. trivirgatus from those of B. variegatus. logroup by using mitogenomics. However, the mt control
294 M. RUIZ-GARCIA ET AL.

region showed a more intense relationship between the megalonychid radiation. Moraes-Barros and Arteaga (2015)
Tocantins River and Western Amazon haplogroups (Ruiz- used the mt COI gene to estimate a divergence to have
Garcıa et al. 2016a). The mitogenomics probably recon- occurred around 7 MYA between Central and South
structed better relationships among taxa above the species American populations of C. hoffmanni. Indeed, our mitoge-
level. In contrast the mt control region is more efficient in nomic BI estimate enlarges this temporal split to 12.6 MYA.
reconstructing the relationships below the species level. For These finding suggest that Choloepus’s disjunct distribution
example, Tchaicka et al. (2007) described a similarity between today was at least partly a result of an uplift of the Northern
the southern region of the Atlantic forest and the eastern Andes and supports the hypothesis of a long history of
region of the Amazonian forest for Cerdocyon thous Choloepus on the mainland. However, this does not negate
(Canidae). Our temporal splits between the Western Amazon the possibility that the Antillean and Central American areas
haplogroup and the other one (Negro River þ Tocantins could be of extreme importance to the origins of the current
River þ Brazilian Eastern Atlantic forest) were 3.4 MYA for BI Choloepus in South America. Indeed, this study, Ruiz-Garcıa
and 1.5 MYA for MJN. et al. (2016a) and Gibb et al. (2016) show that the origins of
This mitogenomics study also detected a Northern and a B. pygmaeus precede that of B. variegatus or that of B.
Southern Brazilian Eastern Atlantic haplogroups which split variegatus þ B. tridactylus. Therefore, the origins of the ances-
around 1.26 MYA, following BI. This estimate is somewhat tors of these species are more likely the Caribbean Islands or
lower than that detected between these two areas of the Central America than South America. In contrast to C. hoff-
Eastern Brazilian Atlantic area for other species. Silva et al. manni, the diversification within C. didactylus seems to be
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(2012) determined divergence estimates around 7–2 MYA very recent (0.51 MYA), although we analyze a very limited
between these two areas for many other vertebrate species. number of specimens of this species. Its distribution along
The lack of a complete separation between these Northern the Caribbean South America coasts (Venezuela, Guyana,
and Southern lineages (more clear in Ruiz-Garcıa et al. 2016a) Surinam, French Guiana) could be related to its Caribbean
could be the result of events during the Holocene when ever- island origins.
green forests and the open savannas became denser than in Independent of when the ancestors of both species of
previous epochs (Turcq et al. 1997; Ledru et al. 1998; Auler & Choloepus split, they present some distinctive osteological dif-
Smart 2001). This favoured the interchange of sloths between ferences, although their external differences are sometimes
the two areas. For instance, following Rothlisberger (1987), difficult to recognize. The morphological differences between
around 6300 YA, there was a significant increase in tempera- C. hoffmanni and C. didactylus include the number of cervical
ture especially in Southern South America as well as in the and caudal vertebrate (Wetzel 1985; Maslin et al. 2007;
Central Andes, evidenced by O18 levels in Huascaran snow. Hautier et al. 2010; Asher et al. 2011). According to Wetzel
The situation could be more complex when more individuals (1985), the two Choloepus species also have a different num-
of the Brazilian Eastern Atlantic area are studied. Indeed, Silva ber of foramina in the anterior half of the basipharyngeal
et al. (2012) analyzed different studies of Brazilian Atlantic for- canal. There are two pairs of foramina in the basypharyngeal
est vertebrates and described three large regions. They were canal of C. hoffmanni, whereas there is only one pair in C.
subdivided into nine sub-groups which showed latitudinal didactylus. Hautier et al. (2014) discovered some differences
and longitudinal patterns. These authors established a com- between the two species relative to the length, width, and
plex history for the organisms of the Brazilian Atlantic forest height of the skull. The two species are well discriminated on
and temporal diversification events within the Brazilian a third principal component (9.69% of the explained vari-
Atlantic forest, which were not restricted to the Pleistocene. ance). On this principal component, the C. didactylus skull
Our temporal split estimates between the ancestors of the appeared narrower with a short snout and long zygomatic
two species of Choloepus were 18.7 MYA with BI and 5.8 with arches whereas the C. hoffmanni skull appeared wider with a
MJN. Gibb et al. (2016) provided an estimate of 9.2 MYA longer snout and shorter zygomatic arches. However, the
(3.5–16.7 MYA) which is within the range of our estimates. interspecific differences in the skulls of both Choloepus spe-
Slater et al. (2016) recently estimated the split between C. cies are not apparent on the first and the second principal
hoffmanni and C. didactylus to have occurred about 6–7 MYA, components, which explain 18.71% and 9.93% of the vari-
which is relatively similar to our lower estimate with MJN. ance, respectively. Also, a weak interspecific differentiation
Both time estimates are possible according to the literature. was detected on the mandibular morphology. The first princi-
For example, some data of the fossil record could agree with pal component (30.56% of total shape variation) weakly dis-
an estimate of 18.7 MYA. Choloepus has been nested within criminated C. didactylus from C. hoffmanni.
the endemic Antillean megalonychid radiation as a close rela- It is quite notable to have detected the mitogenome of a
tive of Acratocnus (Gaudin 1995; White & MacPhee 2001; specimen of C. didactylus by morphology and by geograph-
Gaudin 2004; McDonald et al. 2013). The earliest firm evi- ical distribution within the clade of C. hoffmanni. We ignore if
dence of an Antillean megalonychid is that of Imagocnus this was a case of incomplete lineage sorting, genetic intro-
zazae from the Early Miocene (Burdigalian, 16.1 – 21.5 MYA in gression, recent hybridization in the wild (there are hybrids in
Cuba; MacPhee et al. 2003). If so, the ancestral Choloepus captivity; Jorge et al. 1978; Steiner et al. 2011) or that the
(re)colonized the mainland from the islands in the Late geographical distribution of the Choloepus species is relatively
Miocene. A lower estimate of 9–6 MYA would be more agree- unknown. Maybe the incomplete lineage sorting could be
able to what has been claimed by other authors (Pujos et al. discarded because this phenomenon occurs in very recent
2007). Choloepus is not highly related to the Antillean differentiated species, whereas the two Choloepus species
MITOCHONDRIAL DNA PART A 295

(18.7–5.8 MYA) may not have recently differentiated. Maybe it Colombia (Tucano, Nonuya, Yuri, and Yucuna) and many people in
represents a case of recent hybridization. Steiner et al. (2011) Panama and Brazil for their support in obtaining samples of Bradypus
and Choloepus.
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- Disclosure statement
cies and possible subspecies within them are completely
The authors report no conflicts of interest. The authors alone are respon-
unknown from a molecular point of view. More extensive
sible for the content and writing of this article.
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 References
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