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org/ on June 23, 2018
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Research
Cite this article: Gutiérrez-Garcı́a TA,
Vázquez-Domı́nguez E, Arroyo-Cabrales J, Kuch
M, Enk J, King C, Poinar HN. 2014 Ancient DNA
and the tropics: a rodent’s tale. Biol. Lett. 10:
20140224.
http://dx.doi.org/10.1098/rsbl.2014.0224
Received: 13 March 2014
Accepted: 4 May 2014
Subject Areas:
evolution, taxonomy and systematics,
molecular biology
Keywords:
Cricetidae, Loltún cave, Mexico, Ototylomys
phyllotis, Quaternary, Yucatan peninsula
Author for correspondence:
Tania A. Gutiérrez-Garcı́a
e-mail: taniagutierrezgarcia@gmail.com
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2014.0224 or
via http://rsbl.royalsocietypublishing.org.
Evolutionary biology
Ancient DNA and the tropics:
a rodent’s tale
Tania A. Gutiérrez-Garcı́a1, Ella Vázquez-Domı́nguez1, Joaquı́n Arroyo-
Cabrales2, Melanie Kuch3, Jacob Enk3, Christine King3 and Hendrik N. Poinar3
1
Departamento de Ecologı́a de la Biodiversidad, Instituto de Ecologı́a, Universidad Nacional Autónoma de
México, Ap. Postal 70-275, Ciudad Universitaria 04510, México
2
Laboratorio de Arqueozoologı́a, Instituto Nacional de Antropologı́a e Historia, Moneda no. 16, Centro 06060,
Distrito Federal, México
3
McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, 1280 Main Street West,
Hamilton, Ontario, Canada L8S 4L8
Most genetic studies of Holocene fauna have been performed with ancient
samples from dry and cold regions, in which preservation of fossils is facili-
tated and molecular damage is reduced. Ancient DNA work from tropical
regions has been precluded owing to factors that limit DNA preservation
(e.g. temperature, hydrolytic damage). We analysed ancient DNA from
rodent jawbones identified as Ototylomys phyllotis, found in Holocene and
Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave
located in the Yucatan peninsula. We extracted DNA and amplified six
short overlapping fragments of the cytochrome b gene, totalling 666 bp,
which represents an unprecedented success considering tropical ancient
DNA samples. We performed genetic, phylogenetic and divergence time
analyses, combining sequences from ancient and modern O. phyllotis, in
order to assess the ancestry of the Loltún samples. Results show that all
ancient samples fall into a unique clade that diverged prior to the divergence
of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the
Ototylomys genus. Hence, this rodent’s tale suggests that the sister group to
modern O. phyllotis arose during the Miocene –Pliocene, diversified during
the Pleistocene and went extinct in the Holocene.
1. Introduction
The Loltún cave is a geological formation located over a karst region on the
Yucatan peninsula (figure 1a), which consists of a series of interconnected lime-
stone chambers and tunnels and primarily karst terrains that allow the
concentration of vertebrate remains (see the electronic supplementary material
for a description). The quantity and quality of bone remains from Quaternary
fauna preserved in this neotropical cave make it an extraordinary fossil reser-
voir. It includes approximately 4000 fossil and subfossils, predominantly
mammalian, with neartic and neotropical affinities, and a faunal succession
spanning from the Late Pleistocene to the Holocene [1,2]. Nearly 11% of the
mammalian fossils from Loltún belong to the order Rodentia, including the
monotypic species Ototylomys phyllotis. Identification was carried out by com-
paring the ancient molars and mandibles with those of extant rodents from
the region [2]. A recent phylogeographic study shows that O. phyllotis has
three divergent lineages throughout its geographical distribution, with haplo-
types from Central America, Chiapas and Guatemala highlands and Yucatan
peninsula (figure 1a), and that the origin of the genus was likely earlier than
3.35 Ma [3]. Although the historical affinity of the fossil samples preserved in
Loltún is unknown, based on the extant O. phyllotis phylogeography, a plausible
hypothesis is that they are related to the Yucatan peninsula lineage.
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
 Downloaded from http://rsbl.royalsocietypublishing.org/ on June 23, 2018

(c) H1 2
(a) YP
Loltún cave

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CGH H2 (3)
H5 (2)
CAN YP
H4
CR stratigraphy
H6 (4)
0.93/0.97
Ototylomys
0.80/0.86 phyllotis 1
12.27–5.58 H3
CGH
2
0.99/0.99
3
0.39/0.68
14.49–7.70 4

Biol. Lett. 10: 20140224

CAN
Loltún cave 5
16.46–9.54 CR (haplotype/sampleID/layer)
0.98/0.91 6
CAN H5/10,12/3,4
0.06/0.56 13.39–5.87 H4/2/1 7
H6/4,6,17,21/1,2,5,6 8
outgroups 1/1 H3/7/2
H2/5,11,19/1,3,6
4.07–0.53 H1/1/3
(d) 0 1m
(b) 20 15 10 5 0
Ma

Figure 1. Results for cytochrome b sequence analyses of ancient and modern O. phyllotis samples. (a) Major lineages [3], where YP, Yucatan peninsula; CGH,
Chiapas and Guatemala highlands; CAN, Central America nucleus and CR, Costa Rica. (b) BEAST tree showing the haplotype groups recovered: three major lineages
(as in a) and the LC. Numbers above the diagonal are posterior probabilities/aLRT-SH (values . 0.70); below are range of time of divergence in million years.
(c) Haplotype network: circle size is proportional to haplotype frequency (numbers in parenthesis), short lines are mutational steps and diagram within circles
corresponds to stratigraphic layers. (d ) Stratigraphic schematic of El Túnel ([2]; figure modified from R. Velazquez), layers are organized from recent to oldest
(1 – 8); m, metres. (Online version in colour.)

Comparing ancient and modern DNA sequences using
molecular techniques is a framework that has been used to
decipher the population origin of species or lineages [4,5].
However, the DNA of most ancient samples is heavily
degraded and its preservation depends deeply on different
processes that take place after cellular death, including avail-
ability of water and changes to the environmental conditions
in which bone or soft tissue preservation occurs [6]. The high
diversity and abundance of faunal remains from Loltún
suggest that no drastic environmental changes have occurred
since the Late Pleistocene. Historical humidity fluctuations
have changed the microclimate around and inside the cave
(e.g. turning tropical deciduous forest into grassland) suffi-
ciently to allow for the presence of the noted diverse taxa
[1]. Thus, while humidity has limited DNA preservation in
tropical localities [7], the exceptional preservation of fossils
inside Loltún was likely facilitated by the microclimate
within the cave and by the nature of the karst terrains.
Based on the preservation of our fossils and the value of
ancient DNA to compare historical patterns of diversification
[8], we aimed to address two objectives (i) to confirm the
authenticity of ancient DNA in this tropical setting, and
(ii) to assess their phylogenetic ancestry and relation with
the known phylogeographic history of the species.
2. Material and methods
We selected 28 O. phyllotis hemimandibles and processed them
following strict standards for ancient DNA recovery. These
samples were found in different stratigraphic layers of two exca-
vation units inside Loltún: El Túnel (layers 1 – 7) and El Toro
(12 and 13). The jawbones were broken into three parts as
described in the electronic supplementary material, figure S1,
and approximately 30 – 70 mg were used for DNA extraction.
DNA recovery was performed following Schwarz et al. [9]. We
designed species-specific primers and included those used
by [3] to amplify six overlapping fragments of 100 bp each
(electronic supplementary material, table S1). We included
sequences of modern Ototylomys and two Tylomyini sister
species (Nyctomys and Otonyctomys) used as outgroups to recon-
struct a phylogeny with Bayesian and Maximum-likelihood
methods, using BEAST v. 1.7.5 [10] and PHYML v. 3.1 [11], respect-
ively. Time of divergence for major clades was estimated with
BEAST. We also estimated statistics of genetic diversity and demo-
graphic history, constructed a haplotype network and performed
a Bayesian skyline analysis [12] to infer past population size
changes. All details for ancient DNA extraction, primers, PCR
amplification, methods and parameters for genetic, phylogenetic
and demographic analyses are described in detail in the
electronic supplementary material.
3. Results
Cytochrome b amplicons were successfully obtained for 16 jaw-
bones (of the 28 selected samples from Loltún, an amplification
success rate of 57%; electronic supplementary material, tables
S4 and S5). A total of 666 bp was reconstructed for 12 samples,
based on at least two of three independent amplicons for each
100 bp fragment (see the electronic supplementary material for
details). This DNA fragment represents an unprecedented
success for an ancient DNA sample from a tropical cave [7],
considering comparable sequences come from temperate
regions [13]. The Bayesian and ML trees (figure 1b and the elec-
tronic supplementary material, figure S2, respectively) showed
a concordant topology, with high support for the O. phyllotis
major clades and for a unique clade that includes all ancient
samples (Loltún clade, LC) that differentiated from the other
lineages (posterior probability ¼ 1, aLRT-SH ¼ 1). Surpris-
ingly, LC diverged long before the modern O. phyllotis and
after the Tylomyini sister species (16.46–9.54 Ma), whereas
the divergence within the LC haplotypes occurred much later
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(1.9 Ma). We obtained six ancient haplotypes (figure 1c; acces- amplified [14]. Faunal records from Loltún include species 3
sion numbers KJ751487–KJ751498). The entire ancient dataset with neartic and neotropical affinity in its older layers (Pleis-

did not deviate from neutrality ( p . 0.10; Tajima’s
D ¼ 20.839; Fu and Li’s D ¼ 20.947; F ¼ 21.044), including
656 invariable and 10 polymorphic sites, four parsimony-
informative and six singleton; it showed high haplotype
(h ¼ 0.848 + 0.074) and low nucleotide diversity ( p ¼
0.0039 + 0.007). Genetic divergence and distance values
between LC, O. phyllotis and the Tylomyini sister species were
unexpectedly high (30%; electronic supplementary material,
table S2), considering that modern O. phyllotis phylogroups
have less than 7% divergence between them [3]. On the
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tocene, i.e. Canis dirus, Desmodus cf. D. draculae) [1], which
suggests that the Yucatan peninsula experienced a dry and
cold period; subsequently, during the Holocene, the penin-
sula experienced several droughts that gradually gave way
to the present-day humid conditions [15], all of which may
have played a role in facilitating DNA preservation.
Taking into consideration factors regarding interpretation of
results when working with ancient DNA (e.g. post-mortem
damage and high substitution rate effects [16,17]), our study
shows that the jawbones from Loltún, identified as O. phyllotis,

Biol. Lett. 10: 20140224

other hand, genetic differentiation between layers was low
(0.1–0.5%; electronic supplementary material, table S3), indicat-
ing a continuity between samples within the LC, irrespective of
layer. The latter is also evident in the haplotype network that has
a star-like shape and no more than three mutational steps, which
indicates a recent diversification (figure 1c). Demographic stat-
istics are in agreement with a recent population expansion
signal: negative Fu’s Fs index (Fs ¼ 20.620) and low R 2 and
raggedness indices (R 2 ¼ 0.12; r ¼ 0.20). The latter is supported
by the Bayesian skyline plot, which shows a constant growth
from 1.99 Ma and an effective population size (Ne) of less
than 100 that decreases towards the present (electronic
supplementary material, figure S3).
4. Discussion
Reed et al. [7] and studies cited therein suggest that tropical
humid caves that are located within 308 of the Equator, at
low elevations, or with high temperature and humidity, are
unlikely to preserve DNA over millennia. The fact that we
have successfully extracted and authenticated ancient DNA
(amplicons of 100 bp fragments—totalling 666 bp—of the
cytochrome b gene) from jawbones preserved in Loltún
cave, reiterates the fact that general rules on DNA preser-
vation are limited in use. Understanding this preservation
requires a more systematic review of the environmental con-
text at this site. First, it has been recognized that dry and cold
places, together with a rapid mineralization, allows DNA to
persist longer and in fragments with adequate size to be
are in fact a genetically distinctive clade that is basal to all
O. phyllotis lineages. Despite molar similarities, Alvarez [2]
reported a slight increase in size of these jaws, which became
more pronounced in the deeper stratigraphic layers (12 and
13), and suggested that they could belong to a Pleistocene
form of the Ototylomys genus. Our results support the latter: gen-
etic distances estimated between the cave samples and modern
O. phyllotis are extremely high, even falling in the range of
genus-level distances. Ancient DNA studies have shown that
the Holocene was characterized by cladogenesis, large-scale
extinctions, migrations and changes in population size and con-
nectivity, resulting in the formation of new species and cryptic
populations and also in the subsequent and historically more
recent rapid loss of faunal diversity [18–20]. Our results are con-
gruent with these, where this rodent’s tale suggests that the sister
group of modern O. phyllotis likely arose during the Miocene–
Pliocene, diversified during the Pleistocene and went extinct in
the Holocene; the demographic signal observed showing a con-
stant growth (2–0.5 Ma) followed by a population decrease near
the present, highlights the extinction of this Pleistocene rodent,
whose remains were fortunately preserved in the Loltún cave.
Acknowledgements. We gratefully acknowledge the Consejo de Arqueo-
logia, INAH for granting permission for destructive analysis, project
support and fossil samples, and three anonymous reviewers.
Funding statement. T.A.G.G. acknowledges scholarships from CONA-
CyT (175434) and travel support from PAEP, UNAM, and thanks
McMaster University’s support while conducting scientific visits.
E.V.D. is grateful for financial support from CONACyT (101861).

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Correction
Cite this article: Gutiérrez-Garcı́a TA,
Vázquez-Domı́nguez E, Arroyo-Cabrales J, Kuch
M, Enk J, King C, Poinar HN. 2015 Correction to
‘Ancient DNA and the tropics: a rodent’s tale’.
Biol. Lett. 11: 20150111.
http://dx.doi.org/10.1098/rsbl.2015.0111
Correction to ‘Ancient DNA and the
tropics: a rodent’s tale’
Tania A. Gutiérrez-Garcı́a, Ella Vázquez-Domı́nguez, Joaquı́n Arroyo-Cabrales,
Melanie Kuch, Jacob Enk, Christine King and Hendrik N. Poinar
Biol. Lett. 10, 20140224. (2014; Published online 4 June 2014) (doi:10.1098/rsbl.
2014.0224)
The present erratum is in regards to our article entitled ‘Ancient DNA and the
tropics: a rodent’s tale’. We were made aware of problems with some of the
ancient sequences submitted to GenBank and conducted a systematic review
of all the files used in our study. We discovered that, unfortunately, an incorrect
file was sent to GenBank and was also used in some of our downstream ana-
lyses. We immediately contacted GenBank, explained the situation and
corrected the file. We have redone some analyses with the correct file and
describe these changes below.
The following analyses were performed with the same parameters as
described in the article. The new estimated best-fit substitution model under
the Akaike Information Criterion (AIC) was HKY þ I þ G (A ¼ 0.3298, C ¼
0.3080, G ¼ 0.0981 and T ¼ 0.2641: I ¼ 0.2970 and g ¼ 0.7880). The ML (data
not shown) and Bayesian trees (figure 1a) showed a concordant topology,
with high support for the O. phyllotis major clades. All ancient samples were
grouped within the Yucatan peninsula lineage ( posterior probability ¼ 0.82,
aLRT-SH ¼ 97). Divergence times remain the same for the complete phylogeny
(figure 1a); the new results show that the divergence of the ancient haplotype
basal to the Yucatan lineage occurred within the Pliocene (from 5 Ma onwards)
and that of the rest of the ancient haplotypes occurred much later, coinciding
with the diversification of modern haplotypes (1.9 –0.33 Ma).
We obtained nine ancient haplotypes (figure 1b; accession numbers
KJ751487–KJ751498). The entire ancient dataset did not deviate from neutrality
( p . 0.10) (Tajima’s D ¼ 21.388; Fu and Li’s D ¼ 21.341; F ¼ 21.542) and
showed high haplotype (h ¼ 0.939 + 0.058) and low nucleotide diversity ( p ¼
0.0093 + 0.0018). Genetic differentiation between stratigraphic layers was low
(0.1– 0.5%), indicating continuity between samples within the Loltún ancient
samples irrespective of layer. The haplotype network was constructed for the
Yucatan peninsula lineage in accord with the estimated phylogenetic trees,
given that the ancient set is no longer a unique clade and the ancient sequences
are within this lineage. The resulting haplotype network shows that, with one
exception (5AH), the ancient samples are not dispersed throughout the modern
haplotypes. Instead, they are joined in a central star-like shape, with no more
than nine mutational steps between them. The ancient set connects with
other haplotypes from the Yucatan peninsula that are mostly located basal to
subclades with recent diversifications (figure 1a,b).
To infer past population size changes, demographic indices and a Bayesian
Skyline Plot were calculated for the Yucatan peninsula lineage. The skyline plot
results remained consistent (relatively constant growth from 1.99 Ma and an Ne
of less than 100 that decreases towards the present). Demographic statistics are
in agreement with a recent population expansion signal: negative Fu’s Fs index
(230.083) and low R 2 and raggedness indices (R 2 ¼ 0.0321; r ¼ 0.0059). This
historical demographic pattern was also confirmed by the neutrality test results,
in which this lineage shows a signal of recent expansion indicated by significant
( p , 0.02) and negative values of Tajima’s D and Fu’s D and F (D ¼ 22.267,
D ¼ 25.466 and F ¼ 24.949, respectively).
Importantly, despite the corrections to our sequences as well as the new ana-
lyses we performed, two of our three main results did not change: (i) the DNA
signal is indeed real and tropical caves can be a good source of ancient DNA
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
 (a) (b) 2

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5AH
9AH

3AH

4AH
0.97/0.82
1AH 7AH
7.7–3.1
0.88/0.97 6AH 8AH

0.88/0.79 10.8–4.7 2AH

12.2–5.5 0.88/1

Biol. Lett. 11: 20150111

1/1 (c)
haplotype frequency
14.5–7.4 haplotype - sample ID stratigraphic layers
1AH - 1 Loltún 4 8
2AH - 2 modern
3AH - 4,6,17 1
12.1–5.2 4AH - 5 2
3
7
outgroups 5AH - 7
6AH - 10,12
3
4 2
7AH - 11 5
8AH - 19 1 5
6
9AH - 21 ancient
15 10 5 0
Ma
Figure 1. Results for cytochrome b sequence analyses of ancient and modern O. phyllotis samples. (a) BEAST tree showing the haplotype groups recovered: three
major lineages (as in the manuscript), where blue (bottom), pink (middle) and green (top) bars are Central America Nucleus, Chiapas-Guatemala Highlands and
Yucatan peninsula lineages, respectively; black lines indicate the nine ancient haplotypes. Numbers above the diagonal are aLRT-SH/posterior probabilities (values .
0.80); below are range of time of divergence in million years. (b) Haplotype network for the Yucatan peninsula lineage: circle size is proportional to haplotype
frequency (numbers in circles), ancient haplotypes are indicated in red (shadowed areas). (c) Stratigraphic schematics of El Túnel; layers are organized from recent to
oldest (1 – 6). (Online version in colour.)

regardless of theoretical predictions for these environmental
conditions. We have successfully extracted DNA and amplified
a fragment significantly longer than other reported from Qua-
ternary rodents (666 bp for 12 samples). (ii) Genetic
information obtained from unique fossil samples can reveal
relationships that could not be assessed by morphology due
to the limited availability of complete fossil samples. We were
able to assess the phylogenetic ancestry of the ancient samples,
in which our study shows that the jawbones from Loltún, ident-
ified as O. phyllotis, belong in fact to this species.
Our conclusion in the paper that the ancient samples
formed a distinctive ancestral clade to modern O. phyllotis
lineages is no longer valid. The corrected results show that
the ancient samples fall within the lineage from the Yucatan
peninsula. This result is consistent with our original hypo-
thesis, which stated that the ancient samples were likely to
be related to the Yucatan peninsula lineage. Our results
remain congruent with the main processes of the Holocene,
in that ancient and modern examples of O. phyllotis arose
during the Miocene –Pliocene and diversified during
the Pleistocene. To date we have not found any ancient
haplotypes within extant populations, suggesting drift
(extinction of ancient haplotypes) during the Holocene. This
argues for remarkable genetic continuity within the Yucatan
lineage over at least the last 500 000 years. Further genetic
analyses on the well preserved remains from Loltún cave
will allow for a more refined demographic analysis in the
near future.