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Molecular evolution
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Research
Cite this article: Der Sarkissian C et al. 2015
Mitochondrial genomes reveal the extinct
Hippidion as an outgroup to all living equids.
Biol. Lett. 11: 20141058.
http://dx.doi.org/10.1098/rsbl.2014.1058
Received: 15 December 2014
Accepted: 11 February 2015
Subject Areas:
evolution, molecular biology
Keywords:
ancient DNA, Hippidion, mitochondrial
genomes, Great American Biotic Interchange
Author for correspondence:
Ludovic Orlando
e-mail: [email protected]
Mitochondrial genomes reveal the extinct
Hippidion as an outgroup to all living
equids
Clio Der Sarkissian1, Julia T. Vilstrup1, Mikkel Schubert1,
Andaine Seguin-Orlando1,2, David Eme3, Jacobo Weinstock4,
Maria Teresa Alberdi5, Fabiana Martin6, Patricio M. Lopez7, Jose L. Prado8,
Alfredo Prieto9, Christophe J. Douady3, Tom W. Stafford1, Eske Willerslev1
and Ludovic Orlando1,10
1
Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5 –7,
1350 Copenhagen K, Denmark
2
Danish National High-throughput DNA Sequencing Centre, Øster Farimagsgade 2D, 1353 Copenhagen K, Denmark
3
Universite´ de Lyon, UMR5023 Ecologie des Hydrosyste`mes Naturels et Anthropise´s, Universite´ Claude Bernard
Lyon 1, ENTPE, CNRS, 6 rue Raphae¨l Dubois, 69622 Villeurbanne, France
4
Faculty of Humanities, University of Southampton, Avenue Campus, Highfield, Southampton SO17 1BF, UK
5
Departamento de Paleobiologı´a, Museo Nacional de Ciencias Naturales, CSIC, Jose´ Gutie´rrez Abascal 2,
28006 Madrid, Spain
6
Centro de Estudios del Hombre Austral, Universidad de Magallanes, Instituto de la Patagonia, Av. Bulnes 01890,
Punta Arenas CP 6200000, Chile
7
Instituto de Investigaciones Arqueolo´gicas y Museo Gustavo Le Paige, Universidad Cato´lica del Norte,
Calle Gustavo Le Paige No. 380, San Pedro de Atacama, Chile
8
INCUAPA, CONICET-UNICEN, Del Valle 5737, Olavarrı´a B7400JWI, Argentina
9
Campus Puerto Natales, Km 1.5 Norte, Universidad de Magallanes, Puerto Natales, Chile
10
Laboratoire AMIS, Universite´ Paul Sabatier 3, Universite´ de Toulouse, UMR CNRS 5288, 37 Alle´es Jules Guesde,
31073 Toulouse cedex 3, France
CDS, 0000-0003-4312-9085; JTV, 0000-0001-6534-8452; MS, 0000-0003-2401-9921
Hippidions were equids with very distinctive anatomical features. They lived in
South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion
morphospecies is still disputed. Based on palaeontological data, Hippidion
could have diverged from the lineage leading to modern equids before 10 Ma.
In contrast, a much later divergence date, with Hippidion nesting within
modern equids, was indicated by partial ancient mitochondrial DNA sequences.
Here, we characterized eight Hippidion complete mitochondrial genomes at
3.4–386.3-fold coverage using target-enrichment capture and next-generation
sequencing. Our dataset revealsthat the two morphospecies sequenced (H. saldiasi
and H. principale) formed a monophyletic clade, basal to extant and extinct Equus
lineages. This contrasts with previous genetic analyses and supports Hippidion as
a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6–6.5 Ma, suggesting an early divergence in North
America prior to the colonization of South America, after the formation of the
Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange.
1. Background
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2014.1058 or
via http://rsbl.royalsocietypublishing.org.
All contemporary equids belong to a single genus, Equus [1]. Their most recent
common ancestor (MRCA) lived in North America approximately 4.0–4.5
million years ago (Ma) [2], before dispersing into other continents and radiating
into a wide range of extant and now-extinct forms. The ancestors of South
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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Table 1. Sample and sequencing information. cal. BP, calibrated years before present; #reads (#hits), total number of reads (high-quality hits); M, million; cov,
average depth-of-coverage. #hits and cov are estimated following mapping against the sample’s mitogenome; LP, Late Pleistocene.
location
age (cal. BP)
#reads (M)
#hits
cov (fold)
AE57-17
CDM4
H. saldiasi
H. saldiasi
Cueva Nordenskjo¨ld, Magallanes, Chile
Cueva del Milodo´n, Magallanes, Chile
16 809– 17 400
13 575– 13 810
34.6
18.6
57 640
37 151
293.1
186.6
CE99208-31
CM66
H. saldiasi
H. saldiasi
Cueva Escondida, Magallanes, Chile
Cueva del Medio, Magallanes, Chile
16 870– 17 020
LP
26.5
36.1
11 718
75 624
51.5
386.3
CM-A37
H. saldiasi
Cueva del Medio, Magallanes, Chile
12 570– 12 640
37.4
16 441
69.8
EQB
EQF
H. sp.
H. sp.
Quebrada Opache, Calama, Chile
Cueva Ban˜o Nuevo-1, Chile
LP
15 602– 16 724
5.5
7.7
138
18 522
0.6
78.7
JW251
OH
H. principale
H. saldiasi
Provincia de Buenos Aires, Argentina
Cueva del Milodo´n, Magallanes, Chile
LP
13 197– 13 468
47.1
29.7
1028
2286
3.4
7.1
American equids are believed to have left North America
through intercontinental migrations after the formation of the
Panamanian Isthmus 3.0–3.7 Ma [3,4], which promoted
the Great American Biotic Interchange. No American equid
survived the megafaunal extinction approximately ten
thousand years ago (kya) [2].
Two main equine lineages are found in the South American
Pleistocene fossil record [5]. The first defines a subgenus of its
own, Equus (Amerhippus; but see [6]), which appeared approximately 1 Ma and showed horse-like anatomical features and
genetic affinities [6]. Hippidiforms represent the second lineage, which appeared approximately 2.5 Ma and includes two
genetically similar generalists (Hippidion principale, H. saldiasi)
[7] and one genetically distinct high-altitude specialist
(H. devillei) [7–9].
Like Amerhippus, hippidiforms were stocky, but displayed
very long nasal bones and extremely deep nasoincisival notches
[5]. Palaeontological data support affinities with the northern
American Pliohippus [3,8,10], which lived 6–14.5 Ma. This considerably predates the time when hippidiforms first entered
South America [3], implying an early divergence from the
Equus lineage. However, recent molecular studies based on
short mitochondrial sequences from 20 fossil specimens have
suggested clustering of hippidiforms within Equus [6,7,11,12],
or possible affinities with Dinohippus [12], a North American
lineage that lived 5–8 Ma [13]. Therefore, the evolutionary
origin of Hippidion remains unclear.
Mitochondrial genomes (mitogenomes) have recently
resolved the evolutionary tree underlying the main radiation
of equids [1], which was confirmed by whole-exomes [14].
This established mitogenomes as reliable phylogenetic markers
for equids. We therefore used target-enrichment capture and
next-generation sequencing to characterize mitogenomes of
Hippidion and resolve its evolutionary origin.
2. Methods
(a) Samples
We analysed nine Hippidion samples excavated in northern Chile,
southern Chile and Argentina (table 1 and the electronic supplementary material, table S1). The specimens represent two
morphospecies from the Late Pleistocene (H. principale and
H. saldiasi) with six samples radiocarbon-dated to 12.6–17.4
calibrated kya.
(b) Molecular analyses
We drilled samples (94–485 mg powder), built DNA libraries and
set up PCR amplification in ancient DNA facilities following
published methods (electronic supplementary material, table S1).
We first shotgun-sequenced one/two amplified libraries per
sample on Illumina HiSeq 2000 platforms, except for libraries from
EQB and EQF, which were directly processed for target-enrichment
capture. As sequencing revealed a limited number of mitochondrial
sequences, we performed target-enrichment capture for equine
mitogenomes for all but JW251 and OH samples (electronic supplementary material, table S2). We followed [15] and built probes
from mitochondrial PCR amplicons from living equids (electronic
supplementary material). Enriched libraries were amplified for an
additional 12 cycles post-capture before being quantified on an Agilent 2100 Bioanalyser, pooled and sequenced on an Illumina HiSeq
2000 platform. DNA contamination was monitored using mock controls during DNA extraction, library construction, PCR amplification
and target enrichment. Contamination levels were estimated from
sequence reads using the maximum-likelihood (ML) method from
Fu et al. [16] and a database of 245 equine mitogenomes (electronic
supplementary material, table S3).
(c) Sequence analyses
DNA read processing (adapter trimming), mapping (read alignment, PCR duplicate removal, indel realignment) and damage
analyses were performed using the PALEOMIX pipeline [17].
Seeding was disabled for mapping, a relaxed edit distance
(2n 0.03) was used, and reads showing mapping qualities less
than 25 were excluded. To ensure that all equine-like reads could
be identified in the absence of a mitochondrial reference for Hippidion, we first recovered, for each sample, high-quality hits mapping
uniquely against at least one equine mitogenome and/or partial
Hippidion sequence (electronic supplementary material, table S4).
We then used a majority rule and a minimum depth-of-coverage
of 2 to call a preliminary consensus sequence for each sample,
which was used for read mapping with default parameters.
A new and final consensus was then called using a majority rule,
a minimum depth-of-coverage of 3 and filtering for base scores
less than 30. Alignment statistics and DNA damage parameters
were calculated based on a final mapping against each individual
mitochondrial sequence.
(d) Phylogenetic analyses
We reconstructed the phylogenetic tree of equid complete mitogenomes using both ML (PHYML v. 3.0; [18]) and Bayesian
algorithms (BEAST v. 1.8.0; [19]). We constructed a 40-taxon mitochondrial dataset by aligning Hippidion mitochondrial sequences
Biol. Lett. 11: 20141058
species
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2
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(b)
(a)
H. sp. (EQB KM881676)
87
H. saldiasi (AE57-17 KM881671)
99
100
H. saldiasi ACAD1652 (EU030679)
B
H. sp. CLS144887 JW245 (DQ007561)
90
H. sp. CLS142563 JW260 (DQ007566)
E. caballus (JN398398)
94
99
85
H. saldiasi E.1.20.103 (AY152862)
H. saldiasi ACAD3613 (GQ324596)
E. caballus (JN398378)
E. przewalski (AP013095)
97
H. saldiasi CM-A37 (KM881675)
96
A
E. caballus (JN398420)
E. caballus (JN398431)
H. principale JW251 (KM881678)
H. saldiasi OH (KM881679)
E. caballus (JN398450)
100
99
99
E. caballus (JN398437)
E. caballus (JN398456)
D
H. saldiasi CE99208-31 (KM881673)
New World Stilt Legged horse (JX312727)
H. sp. CDM28/6c-780 JW24 (DQ007560)
H. sp. EQF (KM881677)
H. saldiasi CDM4 (KM881672)
E. ovodovi (JX312734)
86
100
99
C
H. saldiasi ACAD3612 (GQ324595)
89
100
H. saldiasi. AE57-17 (KM881671)
E. h. onager (JX312730)
100
81
E. h. kulan (JX312728)
E
E. asinus (X97337)
H. saldiasi ACAD3609 (GQ324594)
F
E. a. somaliensis (KM881681)
100
H. saldiasi ACAD3601 (GQ324593)
100
H. devillei ACAD3625 (GQ324599)
H. devillei ACAD3615 (GQ324598)
91
E. q. boehmi (KM881680)
100
83
E. b. chapmani (JX312729)
H. devillei ACAD3627 (GQ324600)
82
100
E. b. quagga (Extinct quagga JX312733)
100
E. grevyi (JX312725)
H. devillei ACAD3629 (GQ324602)
Equus
99
E. grevyi (JX312723)
100
96 E. grevyi (JX312722)
E. zebra (JX312724)
E. zebra (JX312717)
100
99
100
0.01 substitutions/site
E. a. somaliensis (AP012271)
E. b. chapmani (JX312721)
99
H. devillei ACAD3628 (GQ324601)
C
E. kiang (JX312732)
H. saldiasi CM66 (KM881674)
H. sp. EQB (KM881676)
99
E. kiang (HM118851)
E. kiang (JX312731)
E. zebra (JX312718)
E. zebra (JX312719)
0.05 substitutions per site
Figure 1. Maximum-likelihood phylogenetic reconstructions. (a) Control region. (b) Mitochondrial genome (rooted using rhinoceroses as outgroups, not shown). The
Hippidion devillei clade is represented in brown. The other Hippidion clade including H. saldiasi, H. principale and H. sp., is shown in red. In bold, Hippidion sequences
generated in this study. Node supports (aLRT SH-like) ,0.80 are not shown. See table 2 for node supports and Bayesian divergence estimates. New World Stilt
Legged Horse ¼ extinct Pleistocene North American equid [12].
against equine orthologous sequences, adding the white and black
rhinoceroses as outgroups (electronic supplementary material,
table S5). As BEAST allows the application of different models of substitution to partitions in datasets, we divided the alignment into six
partitions (one per codon position, rRNA, tRNA, control region
CR). For PHYML analyses, we merged these partitions into one
alignment, as PHYML does not handle multiple partitions. The
best substitution model for each partition/merged dataset (with
and without outgroups) was determined with MODELGENERATOR v.
0.85 [20] on the basis of its Bayesian information criterion, and
applied to the corresponding partition/dataset in BEAST (unlinked
substitution models) and PHYML analyses (electronic supplementary material, tables S6–S7). BEAST analyses were performed
assuming a birth–death serially sampled tree model, and divergence dates were estimated using a log-uncorrelated molecular
clock model together with tip-sampling (electronic supplementary material, table S8) and assuming a time for the MRCA at
4.0–4.5 Ma for Equus [2].
To further support tree topology, we performed approximate
unbiased (AU) and Shimodeira – Hasegawa (SH) topological tests
in CONSEL [21] using five trees differing in their Hippidion
placement and site-wise-likelihood values determined by PHYML.
Finally, in order to compare our Hippidion CR sequences with
those characterized previously, we ran ML analyses under a
GTR þ I þ G8 model (GTR, general time reversible model; I, invariant sites; G8, 8-class gamma distribution; electronic supplementary
material, table S9).
3. Results and discussion
We characterized eight Hippidion mitogenomes at 3.4–386.3fold coverage (table 1 and electronic supplementary material,
table S2). Compared with shotgun sequencing, target-enriched
libraries showed a 3.0–147.8-fold increase (average 42.4-fold;
electronic supplementary material, table S2) in the number of
high-quality hits mapping uniquely against the Hippidion mitogenome. An additional sample (EQB) showed poor DNA
preservation and resulted in limited mitogenome coverage
(0.6-fold). Several lines of evidence support our data as authentic: (i) presence of typical nucleotide mis-incorporation
and ancient DNA fragmentation patterns [22], i.e. increasing C ! T (G ! A) substitutions toward read starts (ends),
preferential read starts (ends) at genomic positions following
purines ( pyrimidines; electronic supplementary material,
figures S1–S9), (ii) 6.9–32.5-fold higher cytosine deamination
rates at overhangs than in double-stranded DNA (electronic
supplementary material, table S10), (iii) contamination levels
less than or equal to 4.5% ( per library) and 1.4% (per sample;
electronic supplementary material, table S11), (iv) ML phylogenetic clustering with all H. saldiasi and H. principale CR
sequences previously characterized (figure 1a). In addition,
albeit only partially characterized owing to limited coverage,
sample JW251 CR sequence clustered with the sequence
previously published for this specimen [12].
ML analyses (figure 1b) and Bayesian phylogenetic inference rooted on rhinoceros outgroups revealed Hippidion as
a monophyletic assemblage basal to all Equus. Similar Bayesian phylogenetic placement was found when excluding
rhinoceros outgroups (data not shown). We furthermore
used AU and SH topological tests to show that this topology
provided a significantly better alternative than four other
topologies where Hippidion was forced to nest within
Biol. Lett. 11: 20141058
H. sp. CLV JW256 (DQ007563)
89
H. saldiasi (CM-A37 KM881675)
H. saldiasi (CE99208-31 KM881673)
H. saldiasi ACAD5559 (GQ324597)
H. sp. MLP6-272 JW251 (DQ007562)
H. sp. (EQF KM881677)
H. saldiasi (OH KM881679)
H. principale (JW251 KM881678)
98
H. sp. TA14001 JW257 (DQ007564)
92
H. saldiasi (CDM4 KM881672)
H. saldiasi 03.20.104-b (AY152861)
3
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94
H. saldiasi 01.20.104 (AY152863)
87
H. saldiasi (CM66 KM881674)
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Table 2. Clade phylogenetic support and divergence dates. See figure 1 for nodes A – F. CR, control region; MG, mitochondrial genome; O, outgroups; n.a., not
applicable. Node ages correspond to the median of Bayesian posterior distributions [5% and 95% quantiles].
A
Bayesian inference
supportb
support
CR
n.a.
0.891
MG 1 O
MG – Oc
MG 1 O
MG– O
1.000
n.a.
1.000
1.000
6.502
5.596
1.000
[5.551 – 7.608]
0.377
[4.599 – 6.629]
0.322
[0.276 – 0.494]
(4.000– 4.500)a
[0.247 – 0.425]
(4.000 – 4.500)a
1.000
0.915
1.000
MG 1 O
MG – O
C
0.994
0.860
1.000
1.000
1.000
D
0.888
0.999
0.996
1.000
1.000
3.126
[2.648 – 3.603]
2.833
[2.314 – 3.331]
1.000
1.000
1.000
1.000
2.965
2.650
n.a.
n.a.
n.a.
n.a.
[2.600 – 3.340]
n.a.
[2.290 – 3.017]
n.a.
E
—
F
0.895
a
Calibration point.
aLRT SH-like.
c
Manually rooted according to the MG þ O tree.
b
different Equus clades ( p-values 0.001 and 0.044; electronic supplementary material, figure S10), following all
possible alternative topologies highlighted in reference [7].
Our analyses therefore contrast with previous results based
on partial mitochondrial sequences [6,7,11,12] and support
palaeontological models, which place Hippidion outside the
range of variation of all extinct and extant Equus species.
Additionally, we found that H. devillei clusters outside a paraphyletic assemblage consisting of H. principale and H. saldiasi,
as shown in reference [7]. Whether the latter should still
be described as two distinct morphospecies remains to be
determined with nuclear data.
We estimated the divergence between Hippidion and
Equus at approximately 5.6–6.5 (confidence range ¼ 4.6–
7.6 Ma; table 2), before Hippidion entered South America
following the formation of the Panamanian Isthmus 3.0–
3.7 Ma [5]. This is supported by fossils found in southern
North America, which exhibit a more primitive nasal notch
than South American hippidions, and could represent earlier
hippidiforms [23].
4. Conclusion and perspectives
In this study, we sequenced for the first time complete mitogenomes of Hippidion saldiasi and H. principale. We reject
previous claims that Hippidion is nested within Equus, and estimate an early split between Hippidion and Equus 5.6–6.5 Ma.
Although the Equidae family flourished during the Miocene,
all present-day equids originate from an MRCA that lived
4.0–4.5 Ma [2]. With closest living relatives diverging 55 Ma,
the early evolutionary stages of the genus Equus are difficult
to reconstruct based on present-day genetic information
alone. The survival of Hippidion until 10 kya and the possibility
to sequence whole genomes from extinct species [24] offer a
unique opportunity to access a closer phylogenetic outgroup
for equids, and thereby reveal the genetic foundation of Equus.
Data accessibility. Hippidion mitogenome sequences can be accessed from
GenBank (KM881671 –KM881679).
Acknowledgements. We thank the Danish National High-throughput
DNA Sequencing Centre; Tina Brand and Pernille Selmer-Olsen for
laboratory assistance; Philip Johnson for sharing R-scripts estimating
contamination levels.
Authors’ contributions. L.O. conceived and designed the study. C.D.S., J.T.V.,
A.S.O. and L.O. performed ancient DNA extraction, DNA library construction and amplification. C.D.S. performed target-enrichment
capture experiments. M.S. and L.O. performed sequence analyses. D.E.,
J.W., M.T.A., F.M., P.M.L., J.L.P., A.P., C.J.D., T.W.S., E.W. and L.O. contributed samples, reagents and methods. All authors participated to the
interpretation of the data. C.D.S., J.T.V. and L.O. wrote the article.
Funding statement. This research was supported by the Marie Curie CareerIntegration Grant (293845), the Danish Council for Independent
Research (Natural Sciences), the Danish National Research Foundation
(DNRF94), the “Chaires d’Attractivite´ 2014” IDEX programme (University of Toulouse), and the Lundbeck Foundation (R52-A5062). F.M. was
supported by the FONDECYT 1100822 and CD MAG0901 grants.
Competing interests. We have no competing interests.
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