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Barnett, R et al 2016 Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810), Resolve its Position within the
Panthera
Cats.
Open Quaternary
,
2: 4, pp. 1–11, DOI: http://dx.doi.org/10.5334/oq.24
* Centre for GeoGenetics, Natural History Museum of
Denmark, University of Copenhagen, Øster Voldgade 5-7,
Copenhagen, Denmark
† Department of Ecology and Evolutionary Biology,
University of California Santa Cruz, Santa Cruz,
CA 95064, USA.
‡ School of Life and Environmental Sciences,
University of Sydney, Sydney NSW 2006,
Australia
§ Department of Tourism and Culture, Government of Yukon,
PO Box 2703, Whitehorse, Yukon, Canada
ǁ Department of Biological and Environmental Sciences, Qatar
University, Doha, Qatar
¶ Ice Age Museum, National Alliance of Shidlovskiy “Ice Age”,
119 bld, Mira pr, Moscow, 129223, Russia
** Palaeogenomics and Bio-Archaeology Research Network,
Research Laboratory for Archaeology, Dyson Perrins Building,
South Parks Road, Oxford, OX1 3QY, UK
†† Trace and Environmental DNA Laboratory, Dept of
Environment and Agriculture, Curtin University,
Perth, WA 6102, Australia
Corresponding author: Ross Barnett (drrossbarnett@gmail.com)
RESEARCH PAPER
Mitogenomics of the Extinct Cave Lion,
Panthera
spelaea
(Goldfuss, 1810), Resolve its Position within the
Panthera
Cats
Ross Barnett*
, Marie Lisandra Zepeda Mendoza*
, André Elias Rodrigues Soares†,
Simon Y. W. Ho‡, Grant Zazula§, Nobuyuki Yamaguchiǁ, Beth Shapiro†, Irina V. Kirillova¶,
Greger Larson** and M. Thomas P. Gilbert*,††
The extinct cave lion (
Panthera spelaea
) was an apex predator of the Pleistocene, and one of the
largest felid species ever to exist. We report the rst mitochondrial genome sequences for this species,
derived from two Beringian specimens, one of which has been radiocarbon dated to 29,860 ± 210 14C
a BP. Phylogenetic analysis conrms the placement of the cave lion as the sister taxon to populations of
the modern lion (
P. leo
). Using newly recovered stem pantherine fossils to calibrate a molecular clock, we
estimate that
P. spelaea
and
P. leo
diverged about 1.89 million years ago (95% credibility interval:
1.23–2.93 million years), highlighting the likely position of this extinct carnivore as a distinct species.
Keywords: Cave lion;
Panthera leo spelaea
; Mitochondrial genome; Numt
Background
The extinct cave lion (Panthera spelaea) was an integral
component of the late Pleistocene Holarctic ecosystem,
occupying the position of apex predator (Barnett et al.,
2009, Antón et al., 2005, Yamaguchi et al., 2004, Bocherens
et al., 2011) alongside the scimitar cat (Homotherium sp.)
(Barnett, 2014). The cave lion is known from plentiful
remains preserved in the karstic cave systems of Europe
and the permafrost sediments of Beringia (present day
Siberia, Alaska and the Yukon). Despite this, P. spelaea
has had an interesting history of taxonomic revision,
having been variously considered a highly divergent
population of the modern lion (Dawkins et al., 1866), a
subspecies of the modern lion (Kurtén, 1985), and a full
species in its own right (Sotnikova and Nikolskiy, 2006).
There is currently no consensus on the taxonomic status
of the cave lion.
Cave lions were significantly larger than their modern
lion counterparts, and exhibit a unique cranial morphology
that has a mosaic of characters present in the lion and
tiger (Sotnikova and Nikolskiy, 2006, Hemmer, 2011,
Vereshchagin, 1971, Gromova, 1932). Evidence from
Pleistocene art demonstrates that the cave lion differed in
external morphology from modern lions (Packer and Clotte,
2000). Male cave lions did not possess a mane, a notable
secondary sexual character in the modern species, and this
is likely to have had considerable effect on the ethology
of the species (Yamaguchi et al., 2004, Nagel et al., 2003).
Previous work has also demonstrated that the cave lion
sensu stricto had an enormous range (Barnett et al., 2009),
from western Europe to eastern Beringia, and were coeval
with lion populations in southern North America (Panthera
atrox; Montellano-Ballesteros and Carbot-Chanona, 2009)
and Africa (Panthera leo; Bougariane et al., 2010). Despite
dominating the Holarctic mammoth steppe for most of the
late Pleistocene, the cave lion went extinct nearly simul-
taneously across its range, with terminal dates in Europe,
Siberia, and Alaska all close to 14,000 cal BP. Panthera atrox
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 2of11
disappeared only slightly later in southern North America
(Stuart and Lister, 2010).
Ancient DNA (aDNA) studies based on partial fragments
of the mitochondrial control region, ATP8, and cytochrome
b genes from P. spelaea have not fully resolved the degree
of separation between the various lineages of lion-like
cats (Burger et al., 2004, Barnett et al., 2009, Ersmark et
al., 2015). In particular, although prior analyses recovered
spelaea and leo as sister taxa, these studies have relied on
the Middle Pleistocene appearance of the ancestral cave
lion (Panthera [leo] fossilis) to calibrate the age of the split
for molecular dating analyses, without estimating the
timing of the split directly. Here, we present the complete
mitochondrial genomes of two cave lion specimens and,
using a Bayesian phylogenetic approach, we analyse these
sequences in combination with a strictly vetted set of
published mitochondrial genomes. We use these to infer
the evolutionary timescale of lion-like cats, calibrated
using recently discovered pantherine fossils, and discuss
the species status of P. spelaea.
Materials and Methods
Sample Discovery
Sample YG 401.410, a humerus (Figure 1, Table 1), was
recovered from the Quartz Creek site in Yukon, Canada
(Figure 2), on 24 July 2010. It has subsequently been
stored in the Government of Yukon’s Palaeontology Pro-
gram collection in Whitehorse. The specimen was sub-
sampled in June 2013 by GZ and sent to the Centre for
GeoGenetics (University of Copenhagen, Denmark) for
processing. Hair sample F-2678/70 was found at the right
bank of Malyi Anyui river (Chukotka, Russia) during the
summer of 2008 (Kirillova et al., 2015). A small bundle of
hair was subsampled by IVK and sent to the UCSC Paleog-
enomics Lab (Santa Cruz, USA) for processing.
Radiocarbon Dating
A section of bone from sample YG 401.410 was delivered
to Stafford Research LLC (University of Aarhus, Denmark).
Samples of crushed bone were decalcified and washed,
treated with 0.05 N NaOH overnight to remove humics,
soaked in 0.1 N HCl, gelatinized at 60°C at pH 2, and
ultrafiltered at 30 kDa. The purified collagen was then
graphitised and analysed at the W.M. Keck Carbon Cycle
Accelerator Mass Spectrometry (AMS) Laboratory,
University of California Irvine, according to standard
protocol (Stafford et al., 1988, Waters et al., 2015, Beaumont
et al., 2010).
Extraction and DNA Amplication
Sample YG 401.410
Samples of cortical bone were taken (approx. 1 cm3)
using a Dremel powertool and reduced to powder in
a Mikrodismembrator. DNA extraction was performed
as described by Orlando et al. (2013) in a dedicated
ancient DNA laboratory at the Centre for GeoGenetics in
Copenhagen, in parallel with negative extraction controls.
The DNA extract and negative control were then built
into genomic libraries using the NEB E6070 kit (New
England Biolabs), following a protocol slightly modified
from that by Vilstrup et al. (2013). Briefly, extract (30 µL)
was end-repaired and then passed through a MinElute
column (Qiagen). The collected flow-through was then
adapter-ligated and passed through a QiaQuick column
(Qiagen). Adapter fill-in reaction was then performed on
the flowthrough, before final incubation at 37°C (30 min)
followed by inactivation overnight at −20°C.
We then amplified the DNA in a 50 µL reaction, using
25 µL of library for 12 cycles under the following reaction
conditions. Final concentrations were 1.25 U AccuPrime™
Pfx DNA Polymerase (Invitrogen), 1× AccuPrime™ Pfx
reaction mix (Invitrogen), 0.4 mg/mL BSA, 120 nM primer
in PE, and 120 nM of a multiplexing indexing primer con-
taining a unique 6-nucleotide index code (Illumina).
PCR cycling conditions consisted of an initial denatura-
tion step at 95°C for 2 min, followed by 12 cycles of 95°C
denaturation for 15 s, 60°C annealing for 30 s, and 68°C
extension for 30 s. A final extension step at 68°C for 7 min
was also included. Amplified libraries were first checked
for presence of DNA on a 2% agarose gel before purifi-
cation using the QIAquick column system (Qiagen) and
quantification on an Agilent 2100 BioAnalyzer. Quantified
libraries were communally pooled in equimolar
ratios and sequenced as single-end reads (100 bp) on
an Illumina HiSeq2000 platform at the Danish National
High-Throughput Sequencing Centre.
Sample F-2678/70
DNA extraction from hair sample F-2678/70 was per-
formed in a dedicated, sterile, facility at UC Santa Cruz,
using standard protocols for ancient DNA (Cooper and
Poinar, 2000). The extraction followed the protocol
described by Dabney et al (2013), with the modifications
suggested by Campos and Gilbert (2012). The Illumina DNA
sequencing library was built following Meyer and Kircher
(2010). Between each step, the libraries were cleaned
using Sera-Mag SPRI SpeedBeads (ThermoScientific) in
Specimen Side Minimum
breadth of
diaphysis (mm)
Maximum
depth of
diaphysis (mm)
Maximum
breadth of
distal end (mm)
Minimum anteroposterior
diameter of articulating
surface for ulna (mm)
Notes
YG 401.410 Left 28.6 52.1 86.1 29.1 Missing
proximal end
from pectoral
ridge
Table 1: Metric data from humerus specimen YG 401.410. Measurements to nearest 0.1 mm taken using digital
calipers.
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 3of11
Figure 1: A) Four views of a humerus of cave lion (Panthera spelaea), specimen YG 401.410. This specimen was recovered
from Quartz Creek in the Yukon Territory, Canada. The scale bar is 5 cm. B) Four views of cave lion (Panthera spelaea)
hair bolus, sample F-2678/70, contains guard hair but is mainly represented by thick underfur of tightly packed, wavy
fur hairs. This specimen was recovered from Malyi Anyui river in Chukotka, Russia.
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 4of11
Figure 2: A map of the approximate distributions of late Pleistocene lions and the provenance of samples used in this
study. Red indicates the maximal range of Panthera spelaea; blue the maximal range of Panthera atrox; and green the
maximal range of Panthera leo leo/Panthera leo persica. Stars show approximate locations of lion samples. The insets
show details of the modern boundaries of Yukon Territory, Canada, and Chukotka, Russia, along with regional settle-
ments. Sample YG 401.410 was found at Quartz Creek (Yukon Territory, Canada), 63.49N, 139.27W. Sample F-2678/70
was found at the right bank of Malyi Anyui River (Chukotka, Russia), 68.18N, 161.44E.
18% PEG-8000. The resulting libraries were sequenced in
a MiSeq Illumina sequencer with v3 kits at UCSC Paleog-
enomics Lab.
Sequence Processing
Sample YG401.410
The dataset consisted of 3,444,248 single-end sequences
that were cleaned of adapter and low-quality sequences
using AdapterRemoval v1.2-GG1 (Lindgreen, 2012). In
order to obtain the mitochondrial sequence, we mapped
the resulting 3,429,186 cleaned reads against a published
mitochondrial genome from Panthera leo persica (Gen-
Bank accession JQ904290.1) using bwa (Li and Durbin,
2009). In order to take into account the circularity of the
mitochondrial genome and to recover the reads mapping
at the edges of the extremes of the reference sequence,
we added the first 100 bases to the end. Clonality was
removed with picard-tools v1.92 (Picard, 2013). The map-
ping was analysed for damage at the extremes with Map-
Damage v2.0 (Jónsson et al., 2013) and the quality of the
damaged bases was rescaled with MapDamage. Realign-
ment was performed with GenomeAnalysisTK (McKenna
et al., 2010) on the rescaled mapping file in order to call
SNPs with samtools v0.1.18 and bcftools (Li et al., 2009)
with a minimum coverage of 8 and minimum quality
of 30.
Nuclear copies of mitochondrial genes (numts) are
known to occur in multiple members of the cat family
Felidae. Some phylogenetic studies of felids have been
compromised by the inclusion of numt sequences in
alignment (Davis et al., 2010). Numts were anticipated
to be a particular problem in genomes reassembled from
high-throughput sequencing technologies, which involve
short fragment lengths and are unable to preferentially
target cytoplasmic copies (e.g. by PCR primer design).
In order to obtain a robust, non-chimaeric consensus
sequence with GenomeAnalysisTK, we only used those
SNPs supported by at least 2/3 of the reads mapping to
that position. We used the resulting consensus sequence
as a reference for a second mapping round, using the
same mapping and consensus strategy as before. This
was repeated three more times to make a total of
four mapping rounds. The final consensus sequence
has an average depth of 9.53× covering 89.5% of the
mitogenome.
F-2678/70
The dataset consisted of 6,683,556 paired-end reads.
Initial processing of these reads consisted of using Adap-
terRemoval v1.2-GG1 (Lindgreen, 2012) for the cleaning
step, followed by merging reads including a minimum
overlap of 10 base-pairs between forward and reverse reads
using SeqPrep (http://github.com/jstjohn/SeqPrep).
A total of 6,123,696 merged reads were obtained.
These merged reads were mapped against the Panthera
leo persica mitochondrial genome (GenBank accession
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 5of11
NC_018053.1) using MIA (http://github.com/udo-stenzel/
mapping-iterative-assembler), a reference based, iterative,
short-fragment assembler that accepts circular genomes
as reference sequences. A total of 9,582 reads mapped
to the reference mitochondrial genome. The consensus
sequence was called from the resulting output file, with
each base having a minimum depth of coverage of 3× and
2/3 base agreement. The final assembly had an average
depth of 28.16× covering 99.62% of the mitochondrial
genome.
Phylogenetic Analysis
Complete mitochondrial genomes of lion (Panthera leo),
leopard (Panthera pardus), jaguar (Panthera onca), snow
leopard (Panthera uncia), tiger (Panthera tigris), clouded
leopard (Neofelis nebulosa), and domestic cat (Felis
sylvestris catus) were downloaded from Genbank (Table 2).
Sequences of all 37 mitochondrial genes were extracted
from these genomes.
We estimated the evolutionary relationships among
pantherine cats using maximum likelihood in RAxML
v8.0.14 (Stamatakis, 2014). The data set was partitioned
into five subsets: the three codon positions of the
13 protein-coding genes; the 2 rRNA genes, and the
22 tRNA genes. A separate GTR+G model of nucleotide
substitution was assigned to each subset of the data. The
maximum-likelihood tree was estimated using 10 random
starts. Node support was estimated using 1000 bootstrap
replicates.
To co-estimate the phylogenetic relationships and evolu-
tionary timescale, we analysed the mitochondrial genome
sequences using a Bayesian phylogenetic approach in
BEAST 1.8.2 (Drummond et al., 2012). The Bayesian
information criterion was used to select the best-fitting
model of nucleotide substitution for each data subset.
We compared two tree priors (Yule and birth-death)
using Bayes factors, based on marginal likelihoods
calculated using the stepping-stone estimator (Xie
et al., 2011). To account for the potential presence of rate
variation across lineages, we also used Bayes factors to com-
pare the strict clock against the uncorrelated lognormal
relaxed clock (Drummond et al., 2006). We included relative-
rate parameters to allow each subset of the data to have a
different evolutionary rate.
To calibrate our phylogenetic estimates of divergence
times, we included age constraints based on the fossil
record. Our calibrations were based on the stem snow
leopard Panthera blytheae (Tseng et al., 2014), stem
tiger Panthera zdanskyi (Mazak et al., 2011), and stem
pantherine Panthera paleosinensis (Mazak, 2010). These
fossils were used to specify uniform priors on the ages
of corresponding nodes in the tree (Ho and Phillips,
2009). Previous estimates of the evolutionary timescale
of cave lions were calibrated using fossil evidence from
P. fossilis (Burger et al. 2004, Barnett et al. 2009), but
the exact placement of this taxon is unclear. For example,
incorrect assignment of a stem taxon to a crown
clade can lead to overestimation of divergence times.
Alternatively, divergence times can be underestimated
when a taxon belonging to the crown group is erro-
neously assigned to the stem lineage. By using other
fossil calibrations, we were able to test whether the
split between cave lion and modern lion coincided with
the existence of P. fossilis.
Posterior distributions of parameters, including the
tree and divergence times, were estimated using Markov
Taxon Common name Genbank accession Reference
Panthera leo persica Asian lion NC018053 (Bagatharia et al., 2013)
Panthera leo leo African lion KF776494 (Ma and Wang, 2014)
Panthera leo Lion KP202262 Unpublished, GenBank
Panthera pardus Leopard NC010641 (Wei et al., 2011)
Panthera pardus japonensis North Chinese leopard KJ866876 (Dou et al., 2014)
Panthera onca Jaguar NC022842 Unpublished, Genbank
Panthera tigris sumatrae Sumatran tiger JF357970 (Kitpipit and Linacre, 2012)
Panthera tigris tigris Bengal tiger JF357968 (Kitpipit and Linacre, 2012)
Panthera uncia Snow leopard NC010638 (Wei et al., 2011, Wei et al., 2009)
Neofelis nebulosa Clouded leopard NC008450 (Wu et al., 2007)
Felis sylvestris catus Domestic cat FCU20753 (Lopez et al., 1996)
Felis sylvestris catus NUMT Nuclear pseudogene FCU20754 (Lopez et al., 1994, Lopez et al., 1996)
Panthera tigris NUMT Nuclear pseudogene DQ151551 (Kim et al., 2006)
Panthera spelaea YG401.410 Cave lion KX258451 This study
Panthera spelaea
F-2678/70
Cave lion KX258452 This study
Table 2: Mitochondrial genomes of pantherine cats analysed in this study.
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 6of11
chain Monte Carlo sampling. Samples were drawn every
2000 steps over a total of 20,000,000 steps. Convergence
was checked using two independent chains, and the
resulting samples were combined. Sufficient sampling
was confirmed by inspection of effective sample sizes of
parameters, which were all greater than 200.
Results
Radiocarbon dating
Sample YG401.410
We obtained two separate dating results for sample
YG401.410. The dates were within three hundred 14C years
of each other, with overlapping 95% confidence intervals.
Collagen yield was substantial (Table 3), with δ13C and
δ15N values appropriate for the trophic level of the spe-
cies (Bocherens et al., 2011). Interestingly, the radiocarbon
date for this specimen falls within a noticeable chronolog-
ical gap for Eastern Beringian lions, suggesting that some
of the absences observed by Stuart and Lister (2010) may
be resolved with further sampling.
Sample F-2678/70
The hair sample has previously been radiocarbon dated
by Kirillova et al. (2015). The reported uncalibrated
AMS date of 28,690 ± 130 was much younger than
dates associated with bone and claw from the same
individual, which could have resulted from incomplete
removal of modern carbon. For a full discussion see
Kirillova et al. (2015).
Phylogeny and divergence times
Our initial estimates of the phylogeny gave unexpected
placements for some of the taxa (data not shown). Inclu-
sion of the mitochondrial genomes from the African lion
(Ma and Wang, 2014) and Asian lion (Bagatharia et al.,
2013) produced a tree with P. spelaea as the sister taxon
of P. l. persica, in contradiction with previous studies
(Barnett et al., 2009, Burger et al., 2004, Ersmark et al.,
2015). Analysis of the ATP8 gene, which has been charac-
terised for both cytoplasmic and nuclear origin (Barnett
et al., 2009) in Panthera cats, revealed the presence of
a numt sequence within the mitochondrial genome
published by Ma and Wang (2014). Given that this
mitochondrial genome sequence was assembled from
published nuclear genome data (Cho et al., 2013), it
is likely to include a significant proportion of nuclear-
derived sequence. Therefore, this mitochondrial genome
was excluded from further analyses.
The evolutionary relationships estimated using maxi-
mum-likelihood and Bayesian methods were congruent
(Figure 3), with all nodes being strongly supported. The
two samples of P. spelaea group together as the sister lin-
eage to P. leo. Our estimates of divergence times largely
overlap with previous estimates (Johnson et al., 2006,
Barnett et al., 2005). However, the estimated split between
P. leo and P. spelaea at 1.89 Ma (95% CI: 1.23–2.93 Ma) is
considerably older than the first appearance of Panthera
fossilis, which has been used as a calibrating node in previ-
ous studies (Burger et al., 2004, Barnett et al., 2009).
Discussion
Evolutionary history of the lion-like cats
Despite their global range and continued dominance of
ecosystems in Africa, and until recently in Asia, the lion-
like cats have left a confusing fossil trail. Remains of pan-
therine felids have been found in fossil beds dating to
3.46 million years ago at Laetoli in Tanzania (Barry, 1987),
with recognisably leonine fossils known from Olduvai II
at 1.4–1.2 million years ago (Hemmer, 2011). Lion fos-
sils only become relatively abundant during the Middle
Pleistocene, with the appearance of Panthera fossilis. This
taxon is considered an ancestral form of Panthera spelaea
and provides a minimum age for the separation between
the spelaea and leo lineages. P. fossilis is known from MIS
17-15 (680-600 ka) from European sites such as Mosbach
(Germany), Pakefield (UK) and Isernia (Italy) (Hemmer,
2011, Turner and Antón, 1997, Lewis et al., 2010, Sabol,
2011). Given our divergence-time estimates (Figure 3), it
would appear that P. fossilis must be already on the branch
leading towards P. spelaea rather than close to the split. In
UCIAMS UCIAMS-142833 UCIAMS-143525
Fraction Modern 0.0243 ± 0.0006 0.0234 ± 0.0006
D14C (‰) −975.7 ± 0.6 −976.6 ± 0.6
14C Age BP 29,860 ± 210 30,160 ± 220
>30 kD Collagen yield(%) 8.1 N/A
δ15N (‰) 8 N/A
δ13C (‰) −18.2 N/A
%N 16.3 N/A
%C 44.7 N/A
C/N (wt%/wt%) 2.75 N/A
C/N (atomic) 3.21 N/A
Table 3: Results of two AMS radiocarbon analyses of sample YG401.410 with associated stable isotope and chemical
analysis values.
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 7of11
view of this finding, we recommend that P. fossilis should
only be used to provide a minimum age constraint for the
split between the leo and spelaea lineages.
Dating the divergence between spelaea and leo
Much of the discussion of the taxonomic position of the
cave lion has revolved around its degree of separation
from modern lion populations. Although some authors
have aligned the spelaea and atrox lineages with the tiger
(Groiss, 1996, Herrington, 1986) and with the jaguar
(Simpson, 1941, Christiansen and Harris, 2009), most have
realised its close connection to the modern lion (Goldfuß,
1810, Dawkins et al., 1866, Vereshchagin, 1971, Turner,
1984, Kurtén, 1985, Sotnikova and Nikolskiy, 2006).
Previous genetic studies (Burger et al., 2004, Barnett
et al., 2014) have used the first appearance of the
ancestral cave lion (Panthera fossilis) (Sotnikova and
Foronova, 2014, Peretto et al., 2015) to calibrate
estimates of the pantherine evolutionary timescale.
This study represents the first attempt to identify the
divergence bounds between spelaea and leo without
recourse to P. fossilis as a calibration. The identifi-
cation of this split within the Early Pleistocene at
1.89 Ma, rather than the Middle Pleistocene, shows that
the modern lion and cave lion lineages represent substan-
tially distinct taxa. A caveat to this is that the estimates
rely strongly on the fossil calibrations used. If reanalysis
later shows that these fossils have been ascribed to
the wrong lineages or are re-dated to different periods
then their utility in the analysis will be compromised.
Comparison with other recent pantherines (Figure 3)
demonstrates that the degree of mitochondrial diver-
gence is considerably greater than that found between
well-defined subspecies in modern lion (P. leo) (Barnett
et al., 2014), leopard (P. pardus) (Uphyrkina et al., 2001),
or tiger (P. tigris) (Luo et al., 2004). The estimated
divergence time between P. leo and P. spelaea is also
greater than that between the two newly recognised
species of clouded leopard, Neofelis nebulosa and
N. diardi, which has been estimated at 1.41 Ma (Buckley-
Beason et al., 2006).
Mitochondrial data and the considerable morphologi-
cal differences (Sotnikova and Nikolskiy, 2006) support
the recognition of the cave lion as a full species: Panthera
spelaea. Data from the nuclear genome will allow further
testing of this proposal.
Conclusions
Our analyses of mitochondrial genome sequences reveal
that the Middle Pleistocene Panthera fossilis is likely
to represent a form already on the spelaea lineage. Its
appearance in the fossil record demonstrates the ini-
tial spread of the ancestral cave lion form into Eurasia.
Furthermore, our study has provided an estimate of
1.89 Ma (95% CI: 1.23–2.93 Ma) for the split between the
lineages leading to the cave lion and modern lion. This
molecular estimate appreciably antedates the appearance
of P. fossilis, and provides further evidence that the cave
lion was distinct enough to be considered a species in its
own right.
Figure 3: Bayesian estimate of the pantherine phylogeny based on mitochondrial genomes. The tree is drawn to a time-
scale, given in millions of years. Bars at nodes give 95% credibility intervals of node-age estimates. Values at nodes
denote posterior probabilities and likelihood bootstrap support. The position of the root was inferred by using the
domestic cat (Felis sylvestris catus) as the outgroup.
Barnett et al: Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810),
Resolve its Position within the
Panthera
Cats
Art. 4,page 8of11
Availability of Supporting Data
Sequence data produced for this study have been
uploaded to GenBank, with accession numbers KX258451
and KX258452. Raw data have been uploaded to the
Sequence Read Archive at NCBI under study accession
number SRP075782.
Acknowledgements
We thank the laboratory technicians of the Centre for Geo-
Genetics and the staff of the Danish National High-Through-
put DNA Sequencing Centre for technical assistance. We
thank Tom Stafford Jr and Stafford Research LLC for radio-
carbon dating and discussion. Thanks to the field crew that
recovered bone YG401.410: Matthias Stiller, Duane Froese,
and Tyler Kuhn. We greatly appreciate work of Elizabeth
Hall, Susan Hewitson and Greer Vanderbyl on the Yukon
Palaeontology fossil collections. Thanks to Fedor Shidlovs-
kiy of the Ice Age Museum, Moscow, Russia, for access to
sample F-2678/70. All map outlines downloaded from
d-maps.com. This project received funding from the Euro-
pean Union’s Seventh Framework Programme for research,
technological development and demonstration under grant
agreement no. FP7-PEOPLE-2011-IEF-298820, as well as
from the Lundbeck Foundation grant number R52-A5062.
Additional support was received from the Gordon and Betty
Moore Foundation.
Competing Interests
The authors declare that they have no competing interests.
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How to cite this article: Barnett, R, Mendoza, M L Z, Soares, A E R, Ho, S Y W, Zazula, G, Yamaguchi, N, Shapiro, B, Kirillova, I V,
Larson , G and Gilbert, M T P 2016 Mitogenomics of the Extinct Cave Lion,
Panthera spelaea
(Goldfuss, 1810), Resolve its
Position within the
Panthera
Cats.
Open Quaternary
, 2: 4, pp. 1–11, DOI: http://dx.doi.org/10.5334/oq.24
Submitted: 12 April 2016 Accepted: 18 May 2016 Published: 23 June 2016
Copyright: © 2016 The Author(s). This is an open-access article distributed under the terms of the Creative Commons
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