Mol Ecol Resour. 2020;00:1–9. wileyonlinelibrary.com/journal/men
1© 2019 John Wiley & Sons Ltd
1 | INTRODUCTION
Natural history museums are biomolecular biobanks. Museum col-
lections maintain fossil and biomolecular archives of the evolution-
ary history of life, including species that are rare, threatened with
extinction, or already extinct (Johnson et al., 2011). Museum col-
lections can be used, for example, to reconstruct evolutionary rela-
tionships and geographic distribution of species (Foote et al., 2013;
White, Mitchell, & Austin, 2018). Historically, specimens archived in
museums have been identified based on their morphological char-
acters. Recently, however, advances in biomolecular techniques
including ancient DNA (aDNA), RNA, and protein sequencing have
provided other sources of information that can be recovered from
museum specimens (Cappellini et al., 2019; Keller et al., 2017; Meyer
et al., 2016). These approaches have expanded the potential utility
of museum collections by making it possible to provide taxonomic
assignments to fragmentary and otherwise difficult to identify spec-
imens (Brown et al., 2016). In some instances, ancient DNA data have
contradicted morphological identifications, leading to both correc-
tion of accidental misidentifications and discover y of unknown taxo-
nomic groups (Heintzman et al., 2017; Krause et al., 2010).
Among the most widely studied taxonomic groups using mu-
seum-preserved remains is the family Equidae. Equids, whose liv-
ing members includes horses, zebras, donkeys, and asses, were
among the first taxonomic groups for which evolutionary history
was inferred through examination of archived fossils and bones
Received: 18 Septemb er 2019
Revised: 25 November 2019
Accepted: 10 December 2019
The case of an arctic wild ass highlights the utility of ancient
DNA for validating problematic identifications in museum
Alisa O. Vershinina1 | Joshua D. Kapp1 | Gennady F. Baryshnikov2 | Beth Shapiro1,3
1Department of Ecology and Evolutionary
Biolog y, Universi ty of California Santa Cr uz,
Santa Cruz, CA , USA
2Laboratory of Theriology, Zoological
Instit ute of the Russian Aca demy of
Science s, St. Petersbur g, Russia
3Howard Hu ghes Medical Institute,
University of California Santa C ruz, Santa
Cruz, C A, USA
Alisa O. Vershinina, Depar tment of Ecology
and Evolutionar y Biolog y, University of
Califo rnia Santa Cruz, Santa Cr uz, CA US A.
Russian Ac ademy of S ciences Presidium
and the Russian Ministry of Education and
Science, Grant/Award Number: "Evolution
of the organic worl d"; National Scien ce
Foundation, Grant/Award Number:
1417036; Institute of Museum and Libr ary
Services, Gr ant/Award Number: M G-30-17-
Museum collections are essential for reconstructing and understanding past bio-
diversity. Many museum specimens are, however, challenging to identify. Museum
samples may be incomplete, have an unusual morphology, or represent juvenile in-
dividuals, all of which complicate accurate identification. In some cases, inaccurate
identification can lead to false biogeographic reconstructions with cascading impacts
on paleontological and paleoecological research. Here, we analyzed an unusual Equid
mandible found in the Far North of the Taymyr peninsula that was identified morpho-
logically as Equus hemionus, an ancestor of present-day Asiatic wild asses. If correct,
this identification represents the only finding of a putative Late Pleistocene hemi-
one in the Arctic region, and is therefore critical to understanding wild ass evolution
and paleoecology. To confirm the accuracy of this specimen's taxonomic assignment,
we used ancient DNA and mitochondrial hybridization capture to identify and place
this specimen in the larger equid phylogeny. We find that the specimen is actually a
member of E. caballus, the ancestor of domestic horses. Our study demonstrates the
utility of ancient DNA to validate morphological identification, in particular of incom-
plete, otherwise problematic, or taxonomically unusual museum specimens.
ancient DNA, Asiatic wild ass, Equus caballus, Equus ferus, Equus hemionus
VERSHININA Et A l.
(MacFadden, 1992). Thanks in part to their abundance and history
of living in colder climates, horse evolution has also been a common
theme in paleogenomics research. In 1984, the first ancient DNA se-
quences were recovered from the skin of a museum-preser ved sub-
species of zebra, the quagga (Higuchi, Bowman, Freiberger, Ryder,
& Wilson, 1984), and the oldest genome yet sequenced is from an
early Middle Pleistocene horse from Canada's Yukon (Orlando et al.,
2013). The abundance and diversity of equid fossils has also made
their taxonomy contentious, with genetic and morphological anal-
yses often leading to taxonomic reassignments and redesignations
(Barron-Ortiz et al., 2019). For example, Przewalski's horse, E. ferus
przewalskii, was considered for many decades to be the only remain-
ing truly wild horse (Der Sarkissian et al., 2015), but has recently
been linked to the lineage of Botai horses that were tamed and
herded five thousand years ago (Gaunitz et al., 2018). In another ex-
ample, DNA from Nor th American horse remains dating to the Late
Pleistocene was recently used to name a new genus, Haringtonhippus
francisci, the New World stilt-legged horse (Heintzman et al., 2017).
While this lineage was known, its phylogenetic placement within
the equids was uncertain, with different authors assigning it to at
least five different species (Eisenmann, Howe, & Pichardo, 20 08;
Heintzman et al., 2017; Weinstock et al., 2005).
Living horses can be broadly subdivided into two major lineages
that diverged 4–4.5 million years ago (Orlando et al., 2013): caballine
horses, which includes domestic horses (most often referred to as
either E. ferus caballus or E. caballus), Przewalski's horses (E. f. prze-
walski or E. c. przewalski); and noncaballine horses, which includes
hemiones (Asiatic wild asses E. hemionus and kiangs E. kiang), zebras
(E. zebra), and donkeys (E. asinus). While caballine horses have been
well characterized using ancient DNA (Fages et al., 2019; Gaunitz
et al., 2018; Heintzman et al., 2017; Orlando et al., 2013), less is
known about the geographic distribution and evolutionary relation-
ships among extinct and extant hemiones. Morphologically, hemi-
ones have both caballine horse features, such as gracile and slender
bodies, and features similar to donkeys, such as a relatively small
body size and a large head. Today, kiangs are found across Tibetan
plateau (St-Louis & Côté, 2009) and Asiatic wild asses are found in
the deserts and arid steppes of southern Mongolia, Kazakhstan,
Iran, and India (Kaczensky, Lkhagvasuren, Pereladova, Hemami, &
Bouskila, 2015). While fossils of kiang-like wild asses are known from
Pleistocene deposits in Alaska (Harington, 1980), the Pleistocene
range of kiangs remains unknown. Wild asses during the Pleistocene
spanned from present-day France, where they were known as the
European wild ass (E. hydrintius), to China (Figure 1a) in regions south
of 50˚N (Bennett et al., 2017).
During the 1980s, a complete mandible, sample ZIN-35608 (the
Zoological Institute of St. Petersburg, Russia), was discovered on the
Begichev Islands in the Laptev sea to the east of the Taymyr penin-
sula, Russia (Figure 1b). The mandible, which was identified as be-
longing to an equid, was small, with both the length of the mandible
bone and the lower tooth row 5 cm smaller than what is typical of
extinct caballine horses. Although the other measurements of the
mandible were inconclusive, it had a curved lower jaw ridge and a
V-shaped linguaflexid of the lower teeth, both of which are char-
acteristics of hemionid horses (Cucchi et al., 2017). Based on these
morphological characters, the mandible was assigned to E. hemionus,
or Asiatic wild ass (Kuzmina, 1997). The discovery of a wild ass in
the Taymyr peninsula was paleontologically significant, as it expands
the range of wild asses far to the nor th and adds them to the list of
arctic Siberian fauna that lived contemporaneously with mammoths
(Figure 1a) (Markova, Smirnov, Kozharinov, Kazantseva, & Kitaev,
There are several reasons to suspect that the taxonomic identifi-
cation of sample ZIN-35608 as a wild ass may have been inaccurate.
First, the location where the specimen was recovered is far outside
the known range of Late Pleistocene hemiones. Although wild asses
were widespread during the Late Pleistocene, no other wild ass sam-
ples are known from northerly regions of Eurasia. To date, the only
equid known from the Pleistocene of Siberian Far North is the cabal-
line horse, thus assigning the specimen in question to caballines is
an alternative hypothesis. Second, the identification of equid fossils
is challenging, given their extensive morphological variation both
within and between taxonomic groups (Bennett, 1980; Forsten,
1998; Geigl & Grange, 2012; Orlando et al., 2009; Twiss et al., 2017).
Taxonomic identification is particularly complicated if the specimen
is a subadult, as suggested for ZIN-35608 based on its small size, as
diagnostic features may have not yet formed.
To confirm the identification of a Pleistocene wild ass in the
Russian Far Nor th, we extracted and captured ancient mitochondrial
DNA and placed sample ZIN-35608 in a phylogenetic tree of the
Equidae. Our analyses revealed the specimen not to be a wild ass,
but instead a caballine horse, the ancestor of the domestic horse.
The unusually small size of the animal, combined with the standard
challenges of morphological identification in equids, probably led to
its misinterpretation as a member of the smaller species, E. hemionus.
Our results underscore the utility of ancient DNA as a paleontolog-
ical tool, in particular when specimens are challenging to identify
morphologically, and highlight the important role that museum
collections play in understanding evolutionary and biogeographic
2 | MATERIALS AND METHODS
ZIN-35608 is a partial mandible found on the Begichev Islands,
Russia, during the 1980s and currently held in the collec tion of the
Zoological Institute of St. Petersburg (Figure 1b). To estimate the age
of the individual at time of death, we followed the protocol outlined
in Hillson (2005). We then collected ~ 1 g of bone surrounding the
M2 tooth socket of the ZIN-35608 mandible, which we subdivided
for radiocarbon dating at the Keck Radiocarbon facility at UC Irvine
and ancient DNA analysis in the purpose-built, sterile, ancient DNA
facilit y at the University of California Santa Cruz Paleogenomics
Laboratory. We performed DNA extraction and subsequent pro-
cessing following standard protocols for working with degraded
DNA (Fulton & Shapiro, 2019). Briefly, the sterile laboratory is
VERSHIN INA Et Al .
located in the building isolated from other molecular research labo-
ratories. Laboratory personnel are instructed to not enter any areas
of campus that have a risk of PCR contamination prior to entering
the aDNA facility. Once inside, they wear coverall suits with hoods,
face masks, and double layer of gloves. All equipment and surfaces
are washed with bleach and ethanol.
We extrac ted DNA following the Dabney et al. (2013) pro-
tocol with modifications for the recovery of short molecules
(Campos et al., 2012), and a sodium hypochlorite pretreatment
(Boessenkool et al., 2017) to reduce the amount of contaminat-
ing DNA potentially adhered to the surface of the bone. Following
extraction, we prepared the extract into Illumina DNA sequenc-
ing libraries following Meyer and Kircher (2010), using Sera-Mag
SPRI SpeedBeads ( ThermoScientific) in 18% PEG-8 000 between
each step for librar y clean-up. To enrich libraries for mitochon-
drial DNA , we performed in-solution hybridization capture using
MyBaits Mito E. caballus RNA bait set (Arbor Biosciences, Ann
Arbor, MI), following the manufacturer's protocol version 3.01. We
incubated the hybridization reactions for 36 hr at 65°C, and then
isolated DNA from the probes using Dynabeads magnetic strepta-
vidin-coated beads. We amplified captured libraries with K APA
HiFi 2X master mix using IS5 and IS6 primers, and purified the
enriched libraries with SPRI beads as above. We then pooled and
sequenced pre- and post-capture libraries on two Illumina MiSeq
runs (v3 chemistry, 75 bp paired end reads).
We used in-house scripts to process the recovered data, map
reads to the reference genomes, and assemble a mitochondrial ge-
nome (https ://github.com/Paleo genom ics/DNA-Post-Proce ssing/
blob/maste r/mito_assem bly_pipel ine.sh). We removed adapters
and merged reads with a minimum overlap of 15 bp and minimum
length of 27 bp using SeqPrep (http://github.com/jstjo hn/SeqPrep).
After verifying the presence of ancient DNA damage at the end
of the reads using MapDamage2 (Jónsson, Ginolhac, Schuber t,
Johnson, & Orlando, 2013), we mapped merged reads to previously
published genomes of E. caballus (EquCab3, GeneBank accession
GCF_002863925, mitochondrial NC_001640), E. asinus (nuclear
GCA_003033725), and E. hemionus (mitochondrial NC_016061). For
alignments to nuclear genomes, we used bwa aln with seed disabled
(Li & Durbin, 2009). To assemble the mitochondrial genome, we used
mia (https ://github.com/mpiev a/mappi ng-itera tive-assem bler), call-
ing bases with at least 10X independent read coverage and > 90%
consensus among reads, so as to avoid false nucleotide calls due to
ancient DNA damage. We imported the BAM file of the mitochon-
drial alignment into Geneious R11 (Biomatters, NZ) to create a con-
sensus nucleotide sequence, which we deposited in GenBank as
accession number MN503280.
To create a data set for comparison to ZIN-35608, we added its
mitochondrial genome to a previously published alignment of 30
equid mitochondrial genomes (table) (Heintzman et al., 2017). We
aligned the assembled mitochondrial genome to this data set using
MAFFT v.7 (Nakamura, Yamada, Tomii, & Katoh, 2018), and manually
checked the alignment for discrepancies. Using the annotation of E.
caballus mitochondrial genome (GenBank ID JN398421), we subdi-
vided the mitochondrial alignment into six partitions (first, second,
and third codon positions for protein coding genes, concatenated
tRNAs, rRNA genes, and the control region). We estimated the ap-
propriate evolutionary models for each partition using jModeltest2
(Darriba, Taboada, Doallo, & Posada, 2012).
FIGURE 1 (a) A map of Eurasia
highlighting the distribution of extinct
European (E. hydrintius) and present-
day Asiatic (E. hemionus) wild asses.
The black dot shows the location of the
Begichev Islands where ZIN-35608 was
found. (b) A mandible found on Begichev
Islands (ZIN-35608) and identified as E.
VERSHININA Et A l.
We estimated phylogenetic trees describing the relation-
ships among the 31 equids in our data set using both a Maximum
Likelihood (ML) and a Bayesian approach. For the ML reconstruc-
tion, we specified the Malasyian tapir, Tapirus indicus as the outgroup
lineage (GenBank ID NC_023838) and ran three instances of RAxML
v.8.2.4 (Stamatakis, 2014) with the GTRGAMMAI nucleotide model
on each par tition. We then selected the best ML tree and estimated
branch supports using 1,000 rapid bootstrap iterations. Bayesian in-
ference does not require an outgroup, therefore we did not include
the tapir sequence in this analysis. Following Heint zman et al. (2017),
we excluded third position of the codon and the control region as to
account for possible convergent mutations in rapidly evolving posi-
tions of the mitochondrial genome. For the first, and second codons,
and the rRNA partitions, we specified the TN93 + I+G nucleotide
model, and for the tRNA partition we specified HKY + I. We ran t wo
instances of BEAST 1.8.4 for 100 million iterations per run, sampling
model parameters and trees every 1,000 iterations (Drummond &
Rambaut, 2007). We assumed the uncorrelated relaxed clock model
and calibrated the molecular clock using the radiocarbon dates of
ancient samples as priors, with ages of the present-day samples set
to 0, and a divergence of the crown caballine group of 4–4.5 Ma
(normal prior, mean 4.25 M, SD: 1.5 M; Orlando et al., 2013). We cal-
ibrated radiocarbon ages of the Late Pleistocene samples reported
in Table 1 using the IntCal13 curve (Reimer et al., 2013) and OxCal
v4.2 (Ramsey, 2009), and assigned the median calibrated age of each
sample as prior information. We used the birth-death process with
serial samples as a tree prior (Drummond, Ho, Phillips, & Rambaut,
2006; Stadler, 2010). We discarded the first 25% of MCMC itera-
tions from each run as burnin, and analyzed parameters for conver-
gence in Tracer (Rambaut, Drummond, Xie, Baele, & Suchard, 2018).
All parameters reached an ef fective sample size > 1,00 0. We then
combined trees from the two BEAST runs in logCombiner and cal-
culated the maximum clade credibility (MCC) tree in Tree Annotator
(Rambaut & Drummond, 2010). We visualized the BEAST MCC tree
and the estimated R AxML phylogeny in Figtree v1.4.2 (Rambaut,
3 | RESULTS
DNA extraction and mitochondrial enrichment were both successful
for specimen ZIN-35608. We sequenced the unenriched library to
a depth of 841,010 reads. When these reads were mapped to the
nuclear reference genomes of E. caballus and E. asinus, 68,276 (8.1%)
of recovered reads mapped to the E. caballus reference nuclear ge-
nome, and 63,916 (7.6%) of reads mapped to E. asinus. Although this
number of reads is small and is not indicative of a final taxonomic as-
signment, it suggests a closer relationship between ZIN-35608 and
caballi ne horses than do nkeys. The median le ngth of aligned DN A se-
quences was 55 bp, and we observed an elevated frequency of G > A
and C > T substitutions at the ends of molecules, consistent with
degraded DNA. After mitochondrial capture, we sequenced the cap-
tured library to a depth of 249,469 reads. Of these, 35,271 (14.1%)
unique reads mapped to the E. hemionus mitochondrial genome, and
44,270 (17.8%) unique reads mapped to E. caballus, resulting in mito-
chondrial genomes with an average coverage of 206x using E. hemio-
nus as the starting reference for mapping and 217x when assembling
on E. caballus. The final assemblies were identical except for three
sections of the control region that were not assembled with E. he-
mionus as the starting reference. We therefore chose the assembly
seeded with E. caballus for phylogenetic reconstruction.
Radiocarbon dating, which was performed at the Keck facility at
UC Irvine, provided an uncalibrated age of 19,470 ± 70 years before
present (UCIAMS-199226). To incorporate this age into the Bayesian
analysis described above, we calibrated it using the IntCal13 radio-
carbon curve as implemented in OxCal 4.3 (Ramsey, 2009; Reimer
et al., 2013). This provided a median age of 23,457 calibrated years
before present (Cal BP 23697–23138, 95.4% probability range).
Based on the state of tooth eruption and microwear analysis (Hillson,
2005), we estimate the age of the individual to be 4–4.5 years at the
time of death.
The mitochondrial phylogenies reconstructed with Maximum
Likelihood and Bayesian approaches were topologically concor-
dant with respect to the major clades within the Equidae family,
although branching order slightly differed within the noncaballine
clade (Figure 2). The major difference is that in the ML phylogeny, E.
ovodovi falls outside of the diversity of zebras, donkeys, and asses,
whereas in the Bayesian phylogeny it is sister to E. asinus, although
with low statistical support. In both ML and BEAST analysis, ZIN-
35608 falls within the clade of caballine horses with strong statis-
tical support (Figure 2b). Based on these results, we conclude that
ZIN-35608 is a caballine horse.
4 | DISCUSSION
Our ancient mitochondrial DNA data indicate that ZIN-35608 is a
member of the caballine clade of ancient horses, rather than a hemi-
one as assigned based on morphology. Although we generated too
few nuclear reads for a confident taxonomic assignment based on
nuclear genomic data, a greater proportion of reads from the shot-
gun library mapped to the E. caballus genome than to the E. asinus
genome, suggesting that the former is a closer evolutionary match.
Both ML and BEAST phylogenies reconstructed with complete high
coverage mitochondrial genomes place ZIN-35608 confidently
within the past and present diversity of c aballine horses (Figure 2).
Our results do not support the proposed expansion of wild
asses to the North of Eastern Siberia, but instead indicate that ca-
balline horses were present in the Begichev Islands during the L ate
Pleistocene (Figure 1a). With a calibrated age of 23,457 years ago,
ZIN-35608 lived during the peak cold interval of the Last Glacial
Maximum. At that time, the sea level was significantly lower than
today, which would have allowed the mainland horse population
to expand to what is today the Begichev Islands. Adjacent to the
Barents-Kara Ice Sheet, the region was at the arctic edge of the dry
Mammoth steppe, dominated by graminoid vegetation (Binney et
VERSHIN INA Et Al .
al., 2017; Mangerud et al., 2004; Tarasov et al., 2000). Such habi-
tats were widely populated by caballine horses at that time (Zimov,
Zimov, Tikhonov, & Chapin, 2012).
The erroneous taxonomic assignment of ZIN-35608 highlights
the challenge of generating an accurate taxonomic identification
from some paleontological remains, which can be problematic when,
GenBank ID Species Age (uncalibrated)
Calendar date BP
NC016 061 Equus hemionus Present day 0
NC018782 E. hemionus Present day 0
JX312732 E. kiang Present day 0
KM881681 E. asinus somalicus Present day 0
NC0 0178 8 E. asinus Present day 0
NC018780 E. zebra har tmannae Present day 0
NC020476 E. zebra Present day 0
NC020432 E. grev yi Present day 0
JX312729 E. burchellii chapmani Present day 0
KM881680 E. burchellii quagga Present day 0
Extinct noncaballine equids
NC018783 E. ovodovi 45,000* 45,000*
MN503280 E. caballus
19,470 ± 70 23,457
KT 75 774 9 E. caballus 28,80 0 ± 1,100 32,918
KT757757 E. caballus 34,460 ± 240 38 ,961
KT757759 E. caballus 13,940 ± 55 16,908
KT 75 7761 E. caballus Pre sent day 0
NC0 0164 0 E. caballus Present day 0
KT 75 776 3 E. scotti 560,000–780,000* 650,000*
KT168318 E. lambei 33,760 ± 400 38,138
KT168322 E. lambei 21,420 ± 80 25,749
New World Stilt Legged horses
28,740 ± 570 32,767
KT168326 H. francisci 46,500 ± 1900 47,770
KT168332 H. francisci 33,400 ± 430 37,6 6 4
KT168333 H. francisci 33,560 ± 440 37, 858
KT168335 H. francisci 14,450 ± 90 17, 5 98
KT168336 H. francisci 28,390 ± 240 32,311
Extinct South American horses
KM8 81671 Hippidion saldiasi 13,990 ± 150 16,980
KM8 81672 H. saldiasi 11,900 ± 60 13,709
KM8 81673 H. saldiasi 13,890 ± 60 16,83 0
KM8 81675 H. saldiasi 10,680 ± 40 12,653
KM881677 H. sp 13,275 ± 30 15,961
Note: The star (*) marks specimen ages estimated by stratigraphic dating. All other nonpresent day
specimens are radiocarbon dated.
TABLE 1 Samples used in the current
study, their NCBI numbers, taxonomic
position, uncalibrated radiocarbon age,
and calendar dates used for BEAST
VERSHININA Et A l.
for example, specimens are fragmentary or come from animals that
have not reached full adult size. In this and other cases, ancient
DNA has proven to be a useful tool for resolving such questions.
Barbanera, Moretti, Guerrini, Al-Sheikhly, and Forcina (2016), for
example, amplified the mitochondrial cytochrome b gene from spec-
imens stored in three museum collections that had been identified as
smooth-coated otters (Lutrogale perspicillata). Ancient DNA revealed
these to belong to three different species, none of which were L. per-
spicillata (Barbanera et al., 2016). Similarly, Cappellini et al. (2014)
used a combination of ancient DNA and proteomics to correct the
taxonomic assignment of the original type material of the Asian el-
ephant Elephas maximus. The biomolecular data revealed that this
sample, a complete ethanol-preserved fetus originally described by
Linnaeus, was in fact an African elephant, Loxodonta sp. (Cappellini
et al., 2014).
In addition to resolving incorrect taxonomic identifications, pa-
leogenomic data can augment the contribution of museum collec-
tions to our understanding of evolutionary history, paleoecology,
and conservation. Samples from organisms that lived before envi-
ronmental shifts or periods of population decline can be used to es-
timate evolutionary changes that occur as a consequence of those
events. For example, ancient DNA from museum preserved speci-
mens collected within the last few centuries has revealed a dramatic
reduction in genetic diversity of the critically endangered Western
Australian woylie and Eastern gorilla, both of which are associated
with anthropogenic habitat changes (Pacioni et al., 2015; van der
Valk et al., 2018). Ancient DNA from much older museum specimens,
coupled with radiocarbon dating and stable isotope analysis, has
also been used to reconstruct ecological changes during the early
Holocene megafaunal mass extinction event (Lorenzen et al., 2011;
Shapiro et al., 200 4). Finally, museum-preserved specimens have tre-
mendous potential value for conservation. Recently, museum spec-
imens of Eurasian beaver, Castor fiber, from the last 10,000 years
helped to identify potential source populations for this species’ rein-
troduction to Britain (Marr, Brace, Schreve, & Barnes, 2018). In the
European beaver study, museum specimens allowed the reconstruc-
tion of past diversity and ancient dispersal events across geographic
locations, which is essential for finding a suitable source population
for controlled genetic rescue (Dietl & Flessa, 2011; Leonard, 2008).
Although the preservation of ZIN-35608 is poor, future advances in
DNA recover y efficiency may allow a complete genome sequence to
be isolated from this specimen, which would help to reveal whether
FIGURE 2 Molecular phylogenies of 31 mitochondrial genomes of various Equid groups sampled worldwide. Colour coding corresponds
to different continents. (a) Maximum clade credibility tree reconstructed with BEAST. Each node has a bar corresponding to a 95% HPD
height interval. (b) Maximum likelihood phylogeny estimated using tapir as an outgroup (the outgroup is not shown). Posterior probabilities
and bootstrap supports higher than 0.95 and 95, respectively are indicated with numbers above and below branches
VERSHIN INA Et Al .
the Begichev populations were genetically isolated from the main-
land Yakutiya and how horse population structure changed with
the separation of the Begichev Islands from the continent. While
the present study of ZIN-35608 is a single example, it highlight s the
potential power of museum specimens, combined with increasingly
sophisticated biomolecular approaches, to reveal the pat tern and
process of biodiversity change over time.
While not all museum specimens retain DNA, advances in paleog-
enomic approaches continue to expand the range of material from
which DNA can be recovered, increasing the value of museum speci-
mens. New methods have been developed to extract DNA from sam-
ples fixed in formalin (Hykin, Bi, & McGuire, 2015; Ruane & Austin,
2017) and ethanol (McGuire et al., 2018) and to decontaminate sam-
ples with sodium hypochlorite prior to extraction (Korlević et al., 2015)
thereby increasing the fraction of useful DNA recovered. In the current
study, we used in-solution hybridization capture, which is an approach
developed to efficiently recover short fragments of a targeted region
of the genome. This method allows recovery of sufficient quantities
of data for population genetic analyses even when samples are poorly
preserved. Such data can be generated US$50–$100 per sample, al-
though costs vary depending on sample preservation, experimental
approach to DNA extraction and library preparation, and local costs of
consumables and sequencing. Other strategies to reduce cost and/or
generate new biological information from ancient specimens include
DNA barcoding and metabarcoding of bulk-extracted bone fragments,
which enables taxonomic identification of a corecovered community
of organisms even when remains are too fragmentary to attempt mor-
phological analysis (Grealy et al., 2015).
Finally, while ancient DNA recovered from museum specimens
has broad utility in ecological and evolutionary analyses, this ap-
proach is most powerful when used in combination with other meth-
ods, including morphological analysis, radiocarbon dating, stable
isotope analysis, and other techniques. Many of these approaches
are inherently destructive, and it is therefore important to consider
the long-term impact on the collections when deciding what ap-
proaches are best for any particular sample.
In summar y, our results reveal that a museum specimen recov-
ered in the Russian Far Nor th and identified based on morphological
characters as a wild ass is actually a caballine horse. This incorrect
identification is probably a consequence of unusually small size of
the horse combined with problematic teeth characteristics. Our
finding therefore disputes the hypothesis that Pleistocene wild
asses expanded to the North of Eurasia during the Pleistocene. Our
study demonstrates how ancient DNA can be used to validate the
taxonomic identity of problematic museum specimens. The growing
diversit y of approaches that can be used to analyze the preserved
remains of organisms highlights the important role of museum col-
lections in advancing our understanding of evolutionary history, pa-
leoecology, and conservation.
This work was funded by National Science Foundation grant
NSF ANS 1417036 and Institute of Museum and Library Services
grant MG-30-17-0045-17. GB is supported by the Program of the
Russian Academy of Sciences Presidium and the Russian Ministry of
Education and Science "Evolution of the organic world. The role and
significance of planetary processes” (2019).
G.B., A .V., and B.S. conceived the study; A.V. obtained the sample;
J.K. ex tracted DNA, prepared sequencing library and performed hy-
bridization capture; A.V. conducted analyses of the sequencing data;
A.V., and B.S. wrote the manuscript. All authors discussed the results
and contributed to the final version of manuscript.
DATA AVAIL ABI LIT Y S TATEM ENT
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How to cite this article: Vershinina AO, Kapp JD, Baryshnikov
GF, Shapiro B. The case of an arctic wild ass highlights the
utility of ancient DNA for validating problematic
identifications in museum collections. Mol Ecol Resour.
2020;00:1–9. ht tps ://doi .org/10.1111/1755- 09 98.1313 0