Morphological versus genetic diversity

Transcription

Annals of Anatomy 194 (2012) 88–102
Contents lists available at ScienceDirect
Annals of Anatomy
journal homepage: www.elsevier.de/aanat
Eurasian wild asses in time and space: Morphological versus genetic diversity
Eva-Maria Geigl ∗ , Thierry Grange
Institut Jacques Monod, UMR7592 CNRS Université Paris Diderot, 15 Rue Hélène Brion, Paris, France
a r t i c l e
i n f o
Article history:
Received 13 February 2011
Received in revised form 3 June 2011
Accepted 5 June 2011
Keywords:
Equids
Hemione
Palaeogenetics
Phylogeography
Conservation biology
Taxonomy
Speciation
s u m m a r y
The Equidae have a long evolutionary history that has interested palaeontologists for a long time. Their
morphology-based taxonomy, however, is a matter of controversy. Since most equid species are now
extinct, the phylogenetic tree based on genetic data can be established only imperfectly via deduction
of present day genomes and little is known about the past genetic diversity of these species. Recent
studies of ancient DNA preserved in fossil bones have led to a simplification of the phylogenetic tree and
the classification system. The situation is still particularly unclear for the wild asses whose geographical
distribution in the Pleistocene and the early Holocene stretched from Northern Africa to Eurasia before
they became endangered or extinct. Therefore, we performed a phylogeographic study of bone remains
of wild asses covering their former geographic range over the past 100,000 years based on the analysis of
ancient mitochondrial DNA. Here, we will not show but rather discuss our results calling the morphologybased classification into question and indicating that morphological criteria alone can be an unreliable
index in inferring various equid species. Indeed, the diversity of mitochondrial lineages in populations
with similar morphology along with genetic signatures shared between morphologically distinct animals
reveal a significant morphological plasticity among Equus species. The classification of palaeontological
species based on morphological and genetic criteria will be discussed.
© 2011 Elsevier GmbH. All rights reserved.
1. Introduction
The concept of species has evolved from the static view of
Linnaeus that strongly influenced taxonomy, to a dynamic view
integrating evolutionary biology and systematics. Ernest Mayr has
proposed a very influential definition of species as populations
that are reproductively isolated from one another (Mayr, 1963).
This biological definition has gained wide acceptance but has also
been challenged by numerous alternative definitions in the last
40 years that have emphasized various biological properties (for
reviews: de Queiroz, 2005, 2007). One can distinguish biological,
ecological, phenetic, evolutionary and phylogenetic concepts that
are favoured by different subgroups of biologists. De Queiroz has
proposed a unifying, more general concept of species as segments
of separately evolving metapopulation lineages where the various
biological properties proposed in previous definitions are no
longer essential but become contingent (de Queiroz, 2005). In this
concept, the various properties so far used to define species have
solely the status of cumulative lines of evidence that support the
hypothesis that two separating and diverging lineages are distinct
species (de Queiroz, 2007). Thus, the secondary species criteria are
not relevant to species conceptualization but only to species delimitation (de Queiroz, 2007). Indeed, when two lineages diverge,
∗ Corresponding author. Tel.: +33 157278132.
E-mail address: [email protected] (E.-M. Geigl).
0940-9602/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.aanat.2011.06.002
they will progressively acquire distinctive properties. In particular,
they will (i) accumulate specific quantitative and morphological
characteristics that will make them phenetically and anatomically
distinguishable, (ii) occupy specific niches and adaptive zones
that will make them ecologically distinguishable, (iii) accumulate
genetic differences that will separate them into monophyletic
groups in terms of multiple gene trees, (iv) or develop an intrinsic
reproductive barrier due to ethological, anatomical or genetic
reasons (de Queiroz, 2005, 2007). Since they will not acquire all
of these properties simultaneously or in a defined order, different
opinions arise as to whether the two lineages should be considered
as distinct species or not. Furthermore, since fluctuations in the
environment can, over time, modify the geographical and ecological ranges of the diverging lineages, novel opportunities for gene
flow can emerge, thus reversing the divergence underway. The
concept of subspecies is sometimes used to characterize metapopulation lineages that have not accumulated all distinctive properties
of species. What is considered as distinctive properties for species
and subspecies, however, may depend on the school of thought.
In the “general metapopulation lineage concept of species” (de
Queiroz, 2005), there is no space for subspecies since they correspond solely to segments of lineages unified by secondary criteria.
Thus, the term “subspecies” should be considered only as a working
hypothesis to facilitate communication among taxonomists.
Species classification is particularly challenging when analysing
extinct species or lineages based on the morphological analyses of
fossils since mostly only anatomical or phenetical characteristics
E.-M. Geigl, T. Grange / Annals of Anatomy 194 (2012) 88–102
can be used (tentative palaeoecological reconstructions of the
species habitat could be used as well but are rarely used). The definition of new palaeontological species based on minor anatomical
differences is a classical methodological trap because of the underestimation of the range of morphological diversity within a species
population, or among sexes, or among the various growth stages of
a single genus (e.g., Scanella and Horner, 2010). Proper characterization of distinctive features that could be used for classification of
distinct species requires identification of a large number of complete, or almost complete, skeletons. This allows assessment of
the range of morphological diversity within each putative species
population and thus ascertainment that the distinctive features are
accurately differentiating two non-overlapping groups. Depending
on the situation, the distinctive features could result from either
a small or a large number of allelic variations within the genome,
and the lower the number of allelic variations involved, the lower
the discriminative value of the criterion involved. For example,
variations in the expression level of a single dental morphogenic
regulator in the mouse, Ectodysplasin, which is a member of the
tumor necrosis factor family of proteins, can induce multiple
correlated morphological changes of the dentition with simultaneous variations in the number, positions and shapes of cusps,
longitudinal crests form, and number of teeth (Kangas et al., 2004).
Thus, phylogenetic history based on seemingly independent characteristics can be obscured when the characteristics are correlated
to a small number, or even to a single, allelic variation affecting
the expression of a developmental regulator (Kangas et al., 2004).
Finally, deduction of classifications based on morphological similarities and differences with extant species can also be misleading
due to changes in the range of variation of morphological features
within a population lineage over time. This is particularly true
for domesticated animals whose wild ancestor has disappeared.
Indeed, the domesticates cannot be used to infer the variability
of the morphology of the wild population because domestication
is well known to cause extensive changes in the range of morphological features. Such changes can considerably complicate the
task of palaeontologists who rely on comparisons with modern
reference collections for their determination. The accuracy of the
identification of extinct species would thus benefit considerably
from the analyses of other classes of biological properties which are
independent of morphology, just as the identification of modern
species relies on the accumulation of independent lines of evidence.
Palaeogenetic analyses of the genetic information contained in
ancient bones of extinct lineages offer the opportunity to extend the
range of arguments supporting the identification of extinct species.
2. Asiatic wild asses: the present situation
The equid family is a vivid example of a complex lineage
with multiple closely related extant and extinct species that are
sometimes defined based on a limited number of fossils and that
would benefit from clarification of the relationships between lineages using palaeogenetic analyses. The bushy phylogenetic tree
of the Equidae spans 60 million years and comprises a multitude of controversially discussed species and subspecies defined
by morphological criteria. Identification is a matter of considerable
difficulty and debate since the genus Equus comprises many closely
related forms, most of which are extinct or rare in the wild (Payne,
1991). E. caballus is extinct in the wild, Equus hydruntinus is completely extinct; E. hemionus survives in Iran and farther east, but
the Syrian onager (E. hemionus hemippus) has been extinct since the
beginning of the 20th century; African wild asses (Equus africanus)
are almost or already extinct in the wild. Zebras are the only wild
equids that are still widely distributed, even though only in a single continent: Africa. This situation explains the scarcity of reliable
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reference collections to which archaeological bone finds could be
compared.
The Asiatic wild asses, or half-asses, stilt-legged equids of the
desert, now survive in small patches of populations that are distributed over a wide area (see Fig. 1 and Table 1). The natural habitat
of the Asiatic half-asses is the open semi-arid plains and alluvial
valleys characteristic of much of the Middle East from the Mediterranean to the Indus (Meadow and Uerpmann, 1986), Tibet and
Mongolia. There are populations that are in contact and hybridize
at a low level, producing hybrids of reduced fertility or hybridize
at one point of contact but not at another. They can be described
as diverging metapopulation lineages that have not accumulated
all lines of evidence to be considered as distinct species and are
sometimes referred as subspecies, a vague enough concept that
leaves plenty of room for controversy (George, 1869; Groves and
Mazak, 1967; Groves, 1986; Forsten, 1990; Clark and Duncan, 1992;
Denzau and Denzau, 1999; Eisenmann and Mashkour, 1999, 2000).
Groves and Mazak ranked the kiang Equus kiang of the Tibetan
Plateau as a full species separate from the onager E. hemionus
(Groves and Mazak, 1967; Groves, 1986). Indeed, the two are
allopatric (their ranges nowhere meet), their biology and morphology are very different, and the various geographic variants
of kiang and onager are phenotypically much more different from
each other than they are among themselves. Furthermore, Groves
and Mazak (1967) and Groves (1986) divide each group in several
subspecies, thus defining nine geographical taxa: three subspecies
of E. kiang (E. k. kiang in Western Tibet, E. k. polyodon in Southern Tibet, and E. k. holdereri in Eastern Tibet), and six subspecies
of E. hemionus: the extinct Hemippe of Syria, E. h. hemippus; the
Onager of Persia, E. h. onager; the Kulan of Turkmenistan, E. h.
kulan; the Mongolian Kulan, E. h. hemionus in northern Mongolia
and adjacent Transbaikalia and Kazakhstan; and the Gobi Kulan or
Dziggetai of southern Mongolia and adjacent Gansu in China, E. h.
luteus; and finally the Khur of the Rann of Kutch in India, E. h. khur
(see Fig. 1 and Table 1). The hemiones fall into three size groups:
the two large Mongolian lineages (hemionus and luteus), the three
small southerly ones (kulan, onager and khur), and the diminutive
Syrian one (hemippus) (Groves and Mazak, 1967). The two Mongolian lineages are generally considered as a single subspecies and
E. h. luteus is believed to be a localized sample of a unitary Mongolian lineage living in extreme desert conditions (Groves, 1986).
The Mongolian Khulan is the largest remaining hemione population that is currently concentrated mostly in Southern Mongolia
and neighbouring Northern China.
The exact relationships between all these forms are still a matter
of debate. The access to the animals is difficult, some forms have
completely disappeared, and many are endangered and parked in
reserves or zoos (Denzau and Denzau, 1999). According to Groves
(1986), the six subspecies of E. hemionus are quite easy to distinguish by means of their coat colour. Other characteristics, however,
do not follow this classification. Therefore, Groves concludes that
the differences between the six subspecies are of a mosaic nature,
not steps on a unidirectional cline (Groves, 1986). For this author,
the easiest way to reconstruct their differentiation is to visualize
a formerly continuous population sharing a common gene pool
spread over at least the present range, but with differing frequencies of particular characteristics in different parts of the range.
Groves concludes that the most parsimonious explanation for the
generation of this situation is habitat fragmentation that would
have led to well-differentiated isolates within each of which rapid
homogenization would have occurred (Groves, 1986). Indeed, the
range of the species is known to have been more nearly continuous in the past. It remains unclear, however, whether it was
totally continuous and whether the population fragmentation was
the consequence of human extermination of intervening populations (Groves, 1986). Analyses of ancient specimens from localities
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Fig. 1. Current natural distribution range of the Asiatic wild ass and geographical location of the archaeological sites that were sampled in the present study and the
palaeogenetic results yielded: Upper Palaeolithic sites (blue dots), Neolithic sites (green dots); Chalcolithic sites (red dots); Bronze Age sites (orange dots); Iron Age sites
(pink dots). Brown clouds indicate the areas where Asiatic wild asses naturally occur at present. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
where onagers no longer occur are needed to clarify the situation.
Most half-ass populations are presently endangered, some are
already extinct, like E. h. hemippus of Syria, and several of the
few remnant hemione populations may soon become extinct (see
Table 1). As pointed out by Meadow and Uerpmann (1986), the situation at the end of the Pleistocene and beginning of the Holocene
was very different since several wild equid species roamed the Middle East in large numbers. Better characterization of this situation
necessitates the palaeontological analysis of extinct individuals.
3. Palaeontology of the Equidae
In order to evaluate the distribution of equids in the past, we
have to rely on the fossil record. There is an abundant literature
from many authors that is very well covered by the conference
proceedings “Equids in the ancient world”, edited by Meadow and
Uerpmann (1986). As stated by Eisenmann (1986), the discrimination of the osteological remains of the various equid species,
varieties and hybrids is a prerequisite to the understanding of
the geographical and chronological distributions. Table 2 summarizes the main morphological features used to discriminate
between E. caballus, E. hemionus and E. hydruntinus. Discriminative characteristics are mostly found on limb bones and teeth.
Unambiguous species identification seems to be extremely difficult since the different anatomies of modern equids are mosaics of
various combinations of a relatively small number of characteristics (Eisenmann, 1986). All kinds of combinations, however, do not
seem possible. Teeth of asses, for example, do not exhibit caballine
double knots and half-asses do not have robust metapodials
(Eisenmann, 1986). How often, however, are teeth and metapodials
of the same animal found together at an archaeological site? Thus,
even if the whole range of combinations of characteristics in modern species is known, the task remains a challenge when dealing
with incomplete and generally fragmented, fossil material. Indeed,
whole skeletons would be needed in order to determine their taxonomic status with confidence, but these are extremely rare. Most
of the time, palaeontologists and archaeozoologists have to cope
with individual and often fragmentary teeth and bones. Fragmentary bones, however, are difficult to identify (Uerpmann, 1986), and
so is the determination of equids on single finds from archaeological sites (Von den Driesch, 2000). Groves and Willoughby (1981)
conclude that the postcranial skeleton offers the best chances for
discrimination especially if different bones are available (the proportions between different bones being better than the shape of
any individual bone, the metapodials alone excepted), followed by
the cranium, followed by the dentition, which trails badly as a discriminator. This view is not generally shared; others reckon that
teeth and complete metapodials are the most reliable indicators
(Davis, 1987). Form and length of the protocone are considered a
valid distinguishing feature between the various equid species for
some authors (e.g., Bökönyi, 1986) but not for others (e.g., Turnbull,
1986). In her thesis, Mashkour summarized the experience of Eisenmann stating that for complex and systematic questions, a global
approach to the skeletal material is necessary, if possible entire
skulls and, in their absence since they are rare, of metapodials and
first phalanxes but that the discriminative characteristics are not
necessarily on the same bones or teeth for each species (Mashkour,
2001).
One major difficulty in discriminating species stems from poor
knowledge of past intraspecies morphological diversity. Palaeontologists seek to analyse as many fossil samples as possible in order
to understand the variability of the past populations but generally
face the problem that archaeological collections often consist of
Table 1
Classification and distribution range of the Asiatic wild ass.
Common names
Zoological name
Synonymous
zoological names
supposed subspecies
Current distribution
range
Former distribution
range
Status
Palestine to Iraq,
including Syria, Israel,
Jordan, and Arabian
Peninsula
Extinct in the wild and
in captivity (20th
century)
Iran (Touran and
Bahram-e-Goor
Reserves)
Endemic to Iran
Endangered
Protected but isolated
in disconnected natural
reserves; hybrid
population
onager/kulan
introduced in Israel
NW India (Rann of
Kutch); gradually
moving out and
colonizing Greater
Rann of Kutch,
extending into the
neighbouring areas
(State of Rajasthan and
Jalore in Gujarat, India)
Western India, Sindh
and Baluchistan,
Afghanistan,
south-eastern Iran
Endangered
Protected in the Little
Rann of Kutch;
suggestions to
reintroduce a few
individuals into the
Thar desert in
Rajasthan
Turkmenistan,
Uzbekistan,
Kazakhstan, Tajikistan,
Kyrgyzstan
Endangered
Reintroduced to
Uzbekistan and
Kazakhstan;
introduced to Ukraine;
hybrid population
onager/kulan
introduced in Israel
Hemippus
Equus hemionus
hemippus
Onager
Persian onager
Khur
Indian wild ass
Equus hemionus
khur
Turkmenian
Kulan
Kulan
Equus hemionus
kulan
E. onager kulan
Turkmenistan
(Badkhys Nature Park)
Mongolian
wild ass
Mongolian
Khulan
North Mongolian
Dziggetaia
Equus hemionus
hemionusa
E. hemionus
luteus?a
Gobi desert of Southern Formerly possibly
Mongolia and N China distributed over NE
Mongolia and China.
Unclear – possibly
extinct if once existed
Gobi Asiatic
wild ass
Gobi Khulan
Gobi Dziggetai
Equus hemionus
luteus
E. hemionus
hemionus
Gobi region of
Southern Mongolia,
rare in adjacent areas
of China
Most of Mongolia,
small parts of Siberia
and Manchuria, W
Inner Mongolia and N
Xinjiang (China)
Unclear – population
decline likely
Kiang
Tibetan
wild ass
Khyang, Gorkhar
Equus kiang
E. hemionus kiang
90% China: provinces
of Qinghai, Gansu,
Xinjiang, and Tibet;
10%: Nepal and
Sikkem, India (Ladak),
Pakistan and Bhutan
Roughly the same as
current range
Lower risk, but data
insufficient
Persian wild ass
Equus hemionus
onager
E. onager onager
E. k. kiang
E. k. holdereri
E. k. polyodon
E. k. chu
Syrian
wild ass
Conservation measures
China: Kalameili
Reserve
Based on the status report 2007 of the Large Herbivore Foundation; http://www.largeherbivore.org/assets/pdf/status-report-2007-student-version.pdf)
a
Unclear whether N Mongolian subspecies ever existed.
91
According to Gromova (1955), Payne (1972b, 1975), Groves (1986), Davis (1980), Bonifay (1991), Burke et al. (2003), Eisenmann et al. (in press) and V. Eisenmann and S. Davis, personal communications. For pictures and schematic
drawings of the teeth, see Fig. 3.
Both premolars and
molars “V” shaped
As for E. hemionus
3 (smallest)
E. asinus
2 (more gracile)
Slenderer
Absent
Absent
Larger, two lobes of
more-or-less equal size
Both premolars and
Premolars are like E. hemionus but
in the molars there is complete
penetration of the external fold
between the flexids to the extent
that sometimes the external fold
touches the internal fold.
Short
triangular-shaped (like
a slipper)
Absent
Absent
Slenderest
3 (smallest)
E. hydruntinus
3 (most gracile)
Both premolars and
Shallow, no penetration between
the flexids of the external fold in
premolars and molars
2 (smaller)
E. hemionus
2 (more gracile)
Slenderer than
horse
Absent
Absent
Larger, two lobes of
more-or-less equal size
Both premolars and
molars “U” shaped
Less deep than E. hemionus but in
the molars there is slight
penetration of the external fold
between the flexids and none at all
in the premolars
Largest, two lobes with
the posterior lobe
tending to extend some
way back (posteriad)
Present
Present
Robust
1 (robust)
1 (biggest)
E. caballus
Pli caballin
Caballine double
knots
Relative robustness
Relative body size
Relative robustness
index of skeleton
Teeth
Limb bones and
metapodials
Whole body
Table 2
Summary of the most prominent morphological features on which the morphological discrimination between Eurasian equids is based.
Protocone
Linguaflexid –
lower premolars
and molars
Ectoflexid – lower premolars and
molars
92
material that is fragmentary and not very abundant. This difficulty
is particularly exacerbated for subfossil equid remains because the
number of equid bones has generally been rather small in faunal
assemblages. This small sample size limits the statistical reliability of many studies (Bökönyi, 1986; Gilbert, 1991). Furthermore,
proper assignment of fossil bones is complicated by unsatisfactory modern museum reference collections that poorly sample the
morphological variability of the different living wild Equus species.
It is difficult to assess the degree of morphological and metrical
variation to be expected within the various forms and to identify
characteristics that might be restricted to particular populations
given the scarcity of modern skeletons of wild true and half-asses
and hybrids of any sort. Moreover, very often wild equid specimens
in these collections are poorly documented, often come from zoos
rather than from the wild, are often juvenile or senile individuals and postcranial skeletons are often completely missing (Payne,
1991). The fact that distinguishing characteristics must be taken
from the closest available samples when local samples are not available may clearly introduce a bias (Payne, 1991). This situation has
led to contradictory conclusions: Some authors consider true and
half-asses no more closely related than are asses and zebras claiming the existence of six groups (Horse, Ass, Hemione, Mountain
Zebra, Burchell Zebra, Grevy Zebra) that are separated for a long
evolutionary time (Groves and Willoughby, 1981), others found
true and half-asses very similar and distinguishable only by a combination of skull and metapodial characteristics (Eisenmann, 1979,
1980). Thus, the classification of equids based on osteological data
poses serious problems, which are the consequence of uncertainties
of overlapping and variable skeletal criteria (Gilbert, 1991).
On the basis of phenotypical characteristics, six different
hemione populations or subspecies have been recognized since the
end of the Pleistocene, which occupy a geographical area reaching
from China to Syria (Eisenmann, 1992b). This phenotypical distinction is reflected also in the post-cranial proportions of the skeleton
when the metric measurements are transformed in logarithmic
differences with a reference, a method called VSI (Variability Size
Index) (Uerpmann, 1986; Meadow, 1999). Following this method,
Mashkour (2001) compared the measurements (cumulative frequencies) of different hemiones populations, defined on the basis
of their geographical occurrence and their phenotype as discussed
before, and found clear size differences between the extinct E.
h. hemippus, the Khur from India (E. h. khur), the Kulan of Turkmenistan (E. h. kulan), the Dziggetai of Mongolia (E. h. hemionus)
and the Kiang from Tibet (E. h. kiang or E. kiang). These data show
a gradient toward larger body size from the Southwest to the
Northeast, conforming to Bergmann’s hypothesis linking environmental temperature to body size within a genus (Bergmann, 1847).
The Persian Onager and the Turkmenian Kulan, distinguished by
very slight morphological differences, occupied close territories in
the past, although they are now confined to natural reserves in
Touran National Park and Bahramgor Reserve in Iran and in Badkhyz Reserve in Turkmenistan (Schreiber et al., 2000). It is not fully
clear whether the differentiation of these two populations has not
been exacerbated by recent reduction of their habitats.
The existence of half-asses west of the Zagros Mountains is subject to debate (e.g., Ducos, 1986; Uerpmann, 1986). The now extinct
“Syrian Onager”, survived in captivity until the first third of the 20th
century. Several specimens that supposedly belong to E. hemionus
hemippus have been preserved in the Natural History Museums
of Vienna, Paris, London, Harvard-Cambridge and Chicago. Their
exact geographic provenience, however, is uncertain (Ducos, 1986).
The two specimens from the Natural History Museum in Paris, one
of which is mounted in the Gallery of Anatomy, were described
by Isidore Geoffroy St-Hilaire in 1855: “These two wild horses
(“chevaux sauvages”), both females and not yet fully adult, were
sent by the Viceroy of Egypt under the name of onagers to Her
Majesty the Empress. Moreover, he wrote in a note about the origin
of these animals: “The Viceroy of Egypt had received these animals
from Seraskier Izzet-Pacha, governor of Syria, who in turn had taken
them from the Arab chief Atha-Bey. They came originally, we are
assured, from the desert of Syria between Palmyra and Baghdad.
This species had not yet been seen either in Egypt or even in Damascus” (translation by Ducos, 1986). The lack of precision concerning
the provenience of the animals is characteristic of all the specimens that were received as living animals and then preserved in
the Natural History collections around the world after their death
(Ducos, 1986). The fact that their physical appearance resembles
one of the equids depicted in the reliefs of the palace of Assurbanipal at Nineveh demonstrates the existence west of the Zagros of a
small wild equid with short, horse-like ears and a tail partly devoid
of hair, these characteristics being present in the two stuffed and
mounted specimens in Paris and Vienna (Ducos, 1986) as well as in
the specimen described and photographed by Antonius (1929) in
the Schönbrunn Zoo in Vienna. These features point to the Syrian
onager as being a half-ass rather than a true ass (Bökönyi, 1986).
In 1986, Uerpmann stated that the early Holocene specimens
from Syrian sites such as Muraibit and Shams ed-Din could not
be called “hemippus” since they were larger than the subrecent
hemippus, size being the only criterion of separation of the hemippus from the rest of the hemiones in the case of bone remains
(Uerpmann, 1986). Therefore, he attributed the equid bone finds
from these archaeological sites to the hemiones and hypothesized
that the so-called modern hemippus represent remnants that were
dwarfed through isolation and deterioration of its habitat due to
human settlement and agriculture (Uerpmann, 1986). An alternative hypothesis is that the modern hemippus were not wild
but rather hybrid forms (Ducos, 1986), which would imply that
they were not related to the ancient small equids found in Syria
and depicted in historical times at Assurbanipal’s palace in Nineveh. Ducos (1986) suggested that these latter were related to
wild African asses. This argument does not account satisfactorily
for the physical resemblance between the depicted equids and the
museum specimens from the 19th and 20th century.
Furthermore, the difficulties of sorting out, from bone remains,
the presence and distributions of at least three and possibly five
forms of equids in the western part of the Middle East at the end of
the Pleistocene increase in complexity when the remains of domestic equids begin to appear, at the end of the fourth millennium
B.C. In fact, the situation becomes considerably more complicated
because of the possibility of encountering the remains of horse-ass,
horse-hemione, and ass-hemione hybrids. If characteristics useful
for distinguishing horses, asses, and hemiones are hard to work
out even for modern specimens of known taxonomic status, the
situation when faced with hybrids is that much more troublesome.
For example, curious mixed morphological characteristics of some
teeth of the Bronze Age site of Godin Tepe, Western Iran, have
led the authors to mention mixed zebrine/E. hydruntinus appearance (Gilbert, 1991). Did such mixed characteristic patterns really
arise from interbreeding rather than intra- and inter-population
variability?
4. The special case of E. hydruntinus
A small equid, first described by Regàlia in 1907 (Regàlia, 1907)
from the Late Pleistocene site of Grotta di Romanelli (Apulia, Italy)
and named E. (asinus) hydruntinus (Stehlin and Graziosi, 1935),
shows a mixture between characteristics similar to Equus stenonis, an ancestral European horse from the end of the Pliocene,
and hemione characteristics (Eisenmann, 1992b). Its spatiotemporal distribution is still poorly known, as are its phylogenetic
affinities (Eisenmann, 1986). The earliest appearance of this small
93
Fig. 2. (A) Rock engravings of equids in the cave of “Les Trois Frères” (MontesquieuAvantes, Ariège, France) attributed to E. hydruntinus. The left drawing is inspired
from that represented in Nores Quesada and Liesau von Lettow-Vorbeck (1992).
The right drawing was performed by Henri Breuil (1877–1961) and was reproduced
in Clot and Duranthon (1990). (B) “Panneau de l’Hémione” in the cave of Lascaux,
France. This Magdalenian fresco is situated on the bottom of the wall. On the left
side, a depiction of a horse and on the right side, another equid with long ears, a
straight back and a long tail suggest that the depicted animal was a hemionus.
equid is attested in West Europe around 350,000 years ago at the
site of Lunel-Viel (Bonifay, 1991), until the end of the Pleistocene
in the Near East in the Levant (Davis, 1980; Clutton-Brock, 1986;
Eisenmann, 1992b, 1995; Clutton-Brock, 1999) and in Anatolia until
the Neolithic (Payne, 1972a; Uerpmann, 1987; Buitenhuis, 1997;
Russel and Martin, 2000) although it does not appear to be present
in a systematic manner (Uerpmann, 1986). It was not found in Syria
during this period (Meadow, 1986; Vila, 1996), although it seemed
to be present earlier (Reynaud Savioz and Morel, 2005). Its presence
is also attested in Azerbaidjan (Eisenmann and Mashkour, 1999)
and Iran (Mashkour, 2002; Orlando et al., 2006).
In addition to the presence of bone material attributed to this
animal in many Palaeo-, Meso- and Neolithic sites, its presence is
attested to in Europe in Palaeolithic art. Indeed, in the Magdalenian cave of Putois (Haute-Garonne, France), the anterior part of the
body of an equid with long ears and slender front legs is engraved
with fine, precise lines on a bone fragment, presumably from a
reindeer, that has been polished and smoothed (Cleyet-Merle and
Madelaine, 1991). Does this engraving represent a hydruntine?
This is at least the interpretation of the authors who also correlate the reappearance of this animal in the faunal assemblages of
the end of the Middle Magdalenian in the Southwest of France
where it had been absent for thousands of years (Cleyet-Merle
and Madelaine, 1991). Other art objects possibly representing E.
hydruntinus are known from “La Salpétrière” (Pont du Gard, Gard).
Various rock art representations of E. hydruntinus are known from
“Gabillou” (Sourzac, Dordogne, France), “Bernifal” (Meyrals, Dordogne, France), “Les Combarelles I” (Les Eyzies, Dordogne, France),
“Les Cavernes du Volp” including “Les Trois Frères” (MontesquieuAvantes, Ariège, France, Albarracin (Teruel, Spain), and Levanzo
(Egedi, Italy) (Cleyet-Merle and Madelaine, 1991). The most convincing depiction, in our eyes, is the one from “Les Trois Frères”
(Fig. 2A) since it shows a slender skull, slender legs, a fine tail, long
ears, and a moderate convexity of the back. Another interesting
example is the Lascaux cave (Montignac, Dordogne, France), where
a panel presumably depicts a horse on the left and another equid
94
appearing to be a hemionus on the right (Fig. 2B). The long ears, the
straight back and the tail are characteristic hemionus features.
The disappearance of the hydruntine is estimated to be relatively recent, since it was found in numerous Neolithic sites in
Eastern Europe (Spassov and Iliev, 2002; Haimovici and Balasescu,
2006), as well as in some Iron Age sites (Wilms, 1989). Based on
medieval manuscripts, its disappearance in Portugal was suspected
to be very recent (Nores Quesada and Liesau von Lettow-Vorbeck,
1992; Antunes, 2002).
The taxonomic position is subject to debate. The species has
been included in the subgenus asinus (Stehlin and Graziosi, 1935),
hemionus (Azzaroli, 1992), or zebras (Davis, 1980; Bonifay, 1991),
or declared the last representative of the Plio-Pleistocene group of
the stenonian equids (Gromova, 1949; Forsten, 1986, 1990; Forsten
and Ziegler, 1995; Forsten, 1999), or as belonging to a subgenus
hydruntinus (Radulesco and Samson, 1965). The main characteristics of hydruntine are its small body and tooth size, its slender
leg bones, and the patterns of its dental elaborations (Table 2).
Measurement of the length and width of its metapodials allows
us to appreciate its slenderness, whereas the limb segment proportions, in particular the relative length of the 3rd metacarpal and
metatarsal reveal slightly different proportions relative to the similar E. hemionus (Eisenmann et al., in press). Its relative microdonty
compared to various hemiones can be appreciated when relating
the size of its upper premolars and molars to the length of its
metapodial (Eisenmann et al., in press). The pattern of its dental
elaboration is a particular distinctive feature (Fig. 3). Its upper and
lower molars are more similar to zebra’s teeth and can be distinguished from horse, hemione and donkey teeth (Fig. 3A). For the
upper cheek teeth, the characteristic features are the absence of
the pli caballin and a short tri-angular shaped protocone, for the
lower cheek teeth, a deep ectoflexid, in particular on the M1 and
M2 (Fig. 3A and B) (Davis, 1980; Bonifay, 1991; Eisenmann et al., in
press). These features make it similar to Equus stenonis, from which
it differs by its slightly longer protocone and simpler enamel pattern (Gromova, 1955). The primitive nature of the distinguishing
characteristics led some authors to consider it as a survivor of the
Villafranchian E. stenonis, even though common retention of primitive characteristic states does not indicate relationship (Payne,
1972b, 1975). Some of the characteristics, such as the short length of
the upper M3 and the particular evolution of the protoconic index,
seem to be specific to E. hydruntinus and to give this group a taxonomic identity, in particular as they are combined with the more
evolved skeletal characteristics, such as the cursorial proportions
and the gracility (Boulbes, 2009). On the basis of the cranial proportions of two almost complete skulls from the Middle Palaeolithic
kill and butchering site of Kabazi II (Ukraine), E. hydruntinus was
classified as a separate species unrelated to any known Pliocene
or Plio-Pleistocene monodactyl equid, to Stenonids, and to zebras,
but close to the hemiones (Burke et al., 2003). The aforementioned
characteristic dental features allow, however, distinction between
hemiones and hydruntines (Fig. 3B). Discrepancies between the
characteristic features of various bones and teeth all attributed to
E. hydruntinus could be due, however, to the fact that they are not
all truly conspecific (Groves, 1986).
Gradient-like morphological variations have been observed in
this lineage over time and over space. Indeed, its metapodials seem
to get more slender over time, judging from Russian and Caucasian
material (Groves, 1986). A geographical and chronological gradient
for the occurrence of bones attributed to E. hydruntinus has been
described, the first occurring from the West to the East with Eastern
Europe showing the highest density of bone finds, the latter increasing their numbers in ever earlier archaeological sites reaching up to
forty percent of the analysed assemblages, for example from sites
dating to the end of the last glaciations in the Mediterranean regions
of France (Bonifay, 1991).
The closeness with E. hemionus of the dental features, also seen
in Late Pleistocene specimens in Southern France (Boulbes, 2009),
could be the result of convergent evolution of the two species.
Boulbes points out, however, that the enamel structure of all equids
shows a high degree of polymorphism, which makes it difficult
to determine the degree of extension of the variability of these
species and the identification of the limits between them (Boulbes,
2009). Moreover, period-specific size variations are observable in
the Pleistocene, the populations of isotope stage 9 to 5 having
smaller and those of stage 3 to 1 larger crowns (Boulbes, 2009).
Stehlin and Graziosi (1935) argued that, since in the Upper Pleistocene E. hydruntinus was contemporary with hemiones in Asia, it
cannot simply be explained away as a hemione with archaic dental
characteristics. They speculate that it could have been adapted to
a different substrate type rather than to climate. Was it adapted to
a particular ecological niche that allowed it to exist in parallel to E.
caballus and E. hemionus?
E. hydruntinus was a member of the fossil communities that were
associated with milder climatic conditions although it was also
found in colder and dryer periods if they were not too intense (Prat,
1968; Delpech, 1984; Eisenmann, 1984; Azzaroli, 1990; Bonifay,
1991). Therefore, its geographic distribution varied according to
the climatic conditions but it was particularly abundant in the
Northern Mediterranean regions and Southeastern Europe. The
ecological demands of this species seemed to have forced it either
to adapt or to migrate. Population migrations in the second half of
the Upper Pleistocene have been identified in well-dated sites in
the Southwest of France (Delpech, 2003). Climatic constraints can,
when they are tolerated, lead to skeletal adaptations such as size
changes, alterations in proportions of the limbs and, in the particular case of equids, of the metapodials (Bignon and Eisenmann, 2002;
Bignon, 2003). A certain plasticity of the enamel structure of the
teeth has been observed at the classical palaeontological species
and subspecies level as a function of the ecological conditions
(Eisenmann, 1992a). An increase in the length of the protocone and
of the size of the teeth could be an adaptation to a different food
source (Gromova, 1949; Guadelli, 1987; Eisenmann, 1991). Thus,
climate and nutrition could have favoured morphological changes
that might at least partly account for the observed variations.
These ecological variations, however, could have also led to
population fragmentation and independent evolution. Thus, the
populations West of the Alps could have evolved independently
of those of the Adriatic region as a consequence of the withdrawal
of mammal populations to refuge areas during the Ice Age maxima
(Taberlet et al., 1998; Avise, 2000; Hewitt, 2001). Such geographic
isolation could have led to genetic differentiation (Michaux et al.,
2003; Schmitt, 2007; Provan and Benentt, 2008). In the case of
Late Pleistocene horses (Bignon and Eisenmann, 2002; Bignon,
2003) and other taxa (Weinstock, 2000), a regionalization of the
populations has been identified on the basis of morphological characteristics such as the size of the metapodials. The analysis of the
protocones of E. hydruntinus finds in the Balkans and the South
of France suggests a fractionation of the populations that was also
explained by migration and differentiation (Boulbes, 2009). Indeed,
the scarcity of remains of E. hydruntinus has been interpreted as an
indication of small population sizes, which must have undergone
differentiation to survive throughout the glacial periods. Moreover,
interspecies competition with small-sized caballine (Uerpmann,
2005) or asinine equids (Azzaroli, 1979), E. hemionus (Eisenmann
and Mashkour, 1999), and other herbivores (Janis et al., 1994;
Grange and Duncan, 2006) could have shaped the ecological and
morphological evolution of the hydruntine populations. The coexistence of two equid species is presently attested in Africa (Grevy
Zebras and African Wild Ass) and also described for the MiddleUpper Pleistocene site of Binagady (Azerbaidjan) and the Holocene
of Iran (Eisenmann and Mashkour, 1999), where these authors also
95
Fig. 3. Characteristic dental elaboration patterns allowing distinction between equid teeth. (A) Photographs and schematic drawings of the patterns of the enamel folds
of molar teeth allowing distinction between the various equids. The drawings were kindly provided by Davis (1987) and the pictures by Eisenmann et al. (in press). (B)
Comparison of left upper and lower cheek teeth of a representative hemione (Upper: E. h. khur, Lower: E. h. hemionus) and hydruntine (Agios Georgios, SGK 76 and 574,
University of Thessaloniki, Laboratory of Geology and Paleontology). The pictures were kindly provided by Eisenmann et al. (in press).
propose the co-existence of two “subspecies” of E. hemionus and
of E. hydruntinus. The sympatry of two “subspecies”, if correctly
assigned, can only be explained if the subspecies/species occupied
different ecological niches within the same territory (Eisenmann
and Mashkour, 1999). The authors add a cautionary note concerning their taxonomic attribution but consider the possibility that
the morphological characteristics of E. hydruntinus, none of which
is specific, developed several times in different places and within
96
different phylogenetic lines. Indeed, the association of primitive
characteristics, such as the small size, primitive patterns of upper
and lower cheek teeth with evolved characteristics of the skeleton
(such as gracility, cursorial proportion as an adaption to dry environment) and teeth (hypsodonty) is also found in the case of the
South African E. lylei that differs by having more robust limb bones
(Eisenmann and Mashkour, 1999). In contrast, for other authors,
E. hemionus and E. hydruntinus clearly occupy different territories
and ecological niches in neighbouring areas; for example, in the
late Pleistocene in Israel, E. hemionus was restricted to the desertic
south and E. hydruntinus was present in the wetter north (Davis,
1980, 1987). It should be noted that the conclusions of each author
rely on the taxonomic attributions of the bone finds, which depend
on the weights given to various morphological criteria that differ
among palaeontologists and archaeozoologists.
In conclusion, there seems to be general agreement between
many palaeontologists/archaeozoologists that there was a distinctive population of small equids in Europe that could correspond to a
species that they all agree to call E. hydruntinus, which would have
evolved over time and space, raising the possibility that different
equid populations could be associated to the same species by different palaeontologists. Furthermore, there is no general agreement
on its phylogenetic relationship to other equids. Genetic analyses
should allow us to clarify this situation.
5. Population structure of equids based on modern genetic
data
The analysis of the hypervariable region of mitochondrial DNA,
which is the part of this maternally inherited molecule that mutates
at the highest rate, is commonly used to analyse intra- and
interspecific relationships. This approach allows a confrontation
between the taxonomic classification established from comparative anatomy and a genetic classification. In order to infer properly
taxonomic classification from genetic data, it might be necessary
to sample a sufficiently large number of loci and individuals and to
use probability modelling (Knowles and Carstens, 2007). Furthermore, mitochondrial DNA being exclusively maternally derived can
give an erroneous picture when there are sex-specific migration
behaviours (e.g., Portnoy et al., 2010). Mitochondrial DNA information is, however, the most readily accessible and can nevertheless
provide useful information that enlightens a complex situation,
even if it is reasonable to consider it as a starting point that will
require more exhaustive analyses to allow genetic data to fully
enrich the picture.
The Median Joining Network approach is a useful tool to visualize the genetic relationships between DNA sequences (Bandelt
et al., 1999). When applied to the mitochondrial hypervariable
region from equids, one can easily distinguish 6 clades (clearly separated from each other at roughly equal distances that correspond
to the 6 unambiguous equid species, horse, ass, mountain zebra,
plain zebra, Grevy’s zebra and half-asses (Fig. 4). The hemione group
is separated from all other equids by a 28 bp deletion within the
hypervariable region, which certainly resulted from a single evolutionary event, thus precluding homoplasy. This deletion can be
used as a “barcode” for the unambiguous genetic identification of
the half-asses.
In contrast to the horses and the donkeys, there are very few
mitochondrial sequences of zebras and half-asses available in Genbank (Benson et al., 2011). Within the half-asses, a phylogeographic
pattern could not be deduced, only the relative separation of the
Kiangs became apparent (Fig. 4). A similar situation was found
when constructing a maximum likelihood tree of the combined
mitochondrial hypervariable region and 12S rRNA gene sequences
and using the rhinoceros C. simum as an outgroup (Oakenfull et al.,
2000). Here as well, the mitochondrial sequences separated two
kiang individuals from kulans and onagers. The mitochondrial
sequences of two kulans from Turkmenistan clustered with one
onager from Iran, while that of two other kulans from Turkmenistan
clustered in a different group with those of three other onagers from
Iran (Oakenfull et al., 2000). The authors conclude that in these
species the speciation events are difficult to resolve, supposedly
due to one or more rapid radiation events or to extensive secondary contacts of the metapopulation lineages. The fact that kulan
and onager populations share two haplotypes pleads in favour of
the secondary contact hypothesis since it suggests that there was
recent gene flow between them. Gene flow could have occurred
in the ancestral population that only recently split into two populations from which kulans and onagers are derived. Alternatively,
kulans and onagers could have separated earlier and mixed recently
before separating into their current populations (Oakenfull et al.,
2000). A study by Ryder and Chemnick suggested in 1990 that
the chromosome numbers of some kulans and onagers deviated
from the average chromosome number of 55, which is the same
for onagers and kulans (Ryder and Chemnick, 1990). These deviating karyotypes could be the consequence of stable Robertsonian
rearrangements, i.e., partial and complete fusion/fission of chromosome pairs 23 and 24, as shown by G- and C-banding studies
(Ryder, 1978), indicating an initial difference in chromosome number in the two subspecies followed by limited mixing afterwards
(Oakenfull et al., 2000). Alternatively, this situation was explained
more parsimoniously with a founder effect, i.e., the separation from
the original population of a small population in which the proportion of the chromosome ratios was not the same as in the original
population but easily underwent a skew at variance to that of the
population from which they were derived, an explanation that is
in agreement with the scenario proposed for mtDNA haplotypes
(Oakenfull et al., 2000). In this study, DNA from captive animals was
analysed. Thus, these founder effects in both mtDNA haplotype and
chromosome frequencies could have occurred as the captive populations of onagers and kulans were formed. A more recent study
focusing on 31 microsatellite loci and analysing two other captive
kulan and onager populations (7 and 11 individuals, respectively)
could show some separation between them although supported by
low bootstrap values (Kruger et al., 2005). Some individuals in each
population were, however, not clearly differentiated from the other
population. Because different genetic markers and different captive populations were studied, it remains difficult to ascertain the
exact extent of genetic differentiation between these two metapopulation lineages. Should onagers and kulans therefore be classified
as evolutionarily significant populations even though their differences could be due to recent human activities causing the isolation
of the populations?
The skull morphology between these subspecies was shown to
have a large overlap in their variation (Eisenmann and Shah, 1996).
Thus, the differentiation of morphological characteristics has not
been successful in interpreting the evolution of these subspecies.
To shed light on this question, it is necessary to obtain data that
elucidate not only the dispersal of the populations in extent but
also in time. This prompted us to perform a palaeogenetic analysis
of individuals belonging to ancient, now extinct populations.
6. Discussion of our palaeogenetic analysis of ancient
hemione and hydruntine populations
In an attempt to characterize the population structure of
half-asses in the past, we sampled roughly 200 ancient E. hemionus
and E. hydruntinus specimens spanning the area from Western
France to the Caucasus and roughly 100,000 years. We subjected
them to palaeogenetic analyses of the hypervariable region of
97
Fig. 4. Median Joining network (Bandelt et al., 1999) depicting the relationships between extant equids based on 408 bp of the mitochondrial hypervariable region.
mitochondrial DNA. Here we will describe and discuss the results
but the original data will be published elsewhere (Champlot et
al., in preparation). The bone and teeth samples came from wellknown archaeological contexts in Spain, France, Belgium, Italy,
Slovakia, Serbia, Romania, Turkey, Georgia, Dagestan, Armenia,
Syria and Iran, and had been analysed by archaeozoologists and
palaeontologists prior to their palaeogenetic analysis. One Iberian
sample was roughly 7000 years old and one came from an Iberian
Bronze Age context. One sample from France came from a burial
context that belongs to isotope stage 5 (roughly 100,000 years ago),
one from the Middle Pleistocene and one from a Solutrean context.
The Belgian samples belonged to the Middle Pleistocene and one of
them was dated to 34,600 years BP. The Slovakian sample was 14 C
dated to 3000 BP. The Serbian samples were archaeologically dated
to c. 5–4000 BC. The Romanian samples come from a Chalcolithic
context dated to c. 4700 BC. The Turkish samples came from
different archaeological contexts from the PPNA to the early PPNB
(c. 9300–8200 BC), the Late Neolithic (c. 6200–5500 BC), to the
Bronze Age (c. 4200–3000 BP). The samples from the Caucasus
were archaeologically dated to the 5th and 4th millennia. Finally,
the samples from Iran were dated to the period between 6000 and
1000 BC, and those from Syria to the late Bronze Age.
The identification of bones as belonging to E. hydruntinus turned
out to be a difficult issue that often did not lead to a consensus
between eminent archaeozoologists. For all bone and teeth samples that we obtained originally as E. hydruntinus samples and
for which we had obtained the entire skeletal fragment, i.e., for
23 samples, the palaeontological determination was performed by
several palaeontologists/archaeozoologists. Surprisingly, in almost
all cases, there was a divergence in their classification. In the most
spectacular cases, five different palaeontologists/archaeozoologists
each came up with a different determination of the same sample.
Some of them remained cautious and characterized the species only
as “little equids” or did not classify them. Often they included the
supposed period and geographical origin in their determination,
implying that they would have come up with a different taxonomic
status if the sample came from a different archaeological context.
This shows the difficulty of species classification of the equids
based only on morphology and reflects the difficulties discussed
above. We established a reliability factor reaching from 0.125 to
1, which expresses the proportion of palaeontological determination in agreement with the determination as E. hydruntinus. Three
E. hydruntinus samples had a reliability factor of one (all palaeontologists agreed), two of 0.75 (three out of four palaeontological
determinations yielded E. hydruntinus), one of 0.66 (two hydruntinus determinations, one against hydruntinus and one excluding the
horse), three of 0.5 and five of 0.33, both with various combinations
of determinations, eight of 0.25 (one out of four determinations)
and one 0.125. For another 15 samples, we could not establish the
reliability factor for the morphological determination since we only
obtained a small, uncharacteristic portion of the samples. Of these,
13 samples had been determined by only one archaeozoologist.
The bone and tooth specimens were then subject to palaeogenetic analysis in a high-containment laboratory dedicated to
ancient DNA research under rigorous experimental conditions as
described previously (Charruau et al., 2011). The mitochondrial
hypervariable region was analysed using UQPCR that eliminates
99.99% of potential carry-over PCR products (Pruvost et al., 2005).
98
The initial quantity of target DNA molecules was determined via
an internal, homologous reference dilution series as described
(Pruvost et al., 2007). The PCR products of overlapping DNA
fragments were sequenced, aligned and the 73 obtained and
authenticated sequences of various lengths as well as one sequence
from an E. hemippus museum specimen from the 19th century, one
modern E. h. onager and one modern E. h. luteus sequence, three
modern kulan and six modern kiang sequences were analysed by
median joining network analysis (Bandelt et al., 1999).
Two types of sequences were obtained from the bones assigned
as “E. hydruntinus” with various reliability indices, a caballinetype and a hemionus-type. Among the European samples, a
100,000-year-old bone from France that had been determined
unambiguously as E. hydruntinus by several archaeozoologists and
the four Romanian samples all yielded hemionus-type sequences
including the hemionus-characteristic 28 bp-deletion. All the other
14 European samples, however, even those determined by at least
one eminent archaeozoologist as being E. hydruntinus, yielded
caballine sequences. In contrast, most samples (39) from Southwest
Asia, determined as being either E. hydruntinus with various reliability indexes, or as being E. hemionus or “small equids”, yielded
hemionus-type sequences. There were, however, exceptions: (i)
two samples from the Caucasus and one sample from Turkey that
had been determined by eminent archaeozoologists as E. hydruntinus or at least as ass-like types and definitely not caballine-types
yielded typical caballine sequences; (ii) for two other samples, one
from Iran and one from Turkey, the archaeozoological determination was contradictory, but the sequences that were obtained were
clearly of caballine-type.
The 28 ancient caballine sequences all cluster with modern caballine sequences, i.e., they are unambiguously contained
within the mitochondrial diversity of modern horses. Seven of
those came from specimens that were palaeontologically determined as hydruntinus-like and definitely not caballine-like. The two
sequences obtained from the Caucasian E. hydruntinus samples represent caballine haplotypes that are now extinct (or were never
sampled) and so do some of the samples that had been determined
palaeontologically as E. caballus. Since all these sequences were
obtained more than once from PCR products produced under rigorous experimental conditions in a high-containment laboratory
dedicated to ancient DNA research and in addition using UQPCR
that eliminates carry over contamination prior to PCR, we consider
it highly unlikely that they are due to laboratory contamination.
Moreover, the substitutions are not due to chemical transformations during bone diagenesis either and the sequences can therefore
be considered authentic. For one of the Serbian and one of the
Caucasian mitochondrially caballine samples, we also obtained a
characteristic caballine Y chromosome-specific sequence so that
we could exclude the possibility that the corresponding animal was
a hybrid.
The network of the hemione-type sequences clearly shows a
strong population structure of seven well-separated haplogroups
or clades (data not shown, Champlot et al., in preparation). This
population structure became apparent solely through the ancient
sequences, in our study they constitute the majority of available
hemionus sequences to date. Only two haplogroups correspond to
purely modern populations that are geographically distinct and not
covered by our study due to a lack of ancient samples: the kiangs in
Tibet and the Mongolian kulans or dziggetais. Moreover, the ancient
sequences form three more haplogroups that are void of any modern sequence and characterize populations that are extinct today:
there is one haplogroup containing sequences from ancient samples from the Caucasus and Iran, one cluster containing sequences
from ancient samples from Turkey and Romania as well as the
Pleistocene sample from France, and one cluster containing ancient
sequences from E. hemionus samples from Syria and the 19th cen-
tury hemippus. Finally, the three modern kulans cluster in two
different haplogroups, each one together with ancient sequences
from Iran that play a key role in defining the clusters without ambiguity. One of the three modern kulan sequences from Turkmenistan
cluster in one haplogroup together with the modern Iranian onager
and the other two modern Turkmen kulans belong to the other
haplogroup.
The 19th century hemippus from the Natural History Museum in
Paris has the same haplotype as the four prehistoric samples from
Syria. They are well separated from all other haplogroups but distantly linked to one haplogroup constituted by modern Iranian and
Caucasian samples. The ancient Syrian E. hemionus were of larger
size than the hemippe, and thus the very close genetic relationships
between these two groups indicate there was a size reduction in the
Syrian hemiones that preceded their extinction in the 20th century,
confirming the hypothesis of Uerpmann (1986) that the modern
hemippes represent remnants that have been dwarved through isolation and deterioration of their habitat due to human settlement
and agriculture.
One of the haplogroup clusters all samples from Turkey and
Romania for which the archaeozoological determination as E.
hydruntinus had the highest support in the ranking list of reliability (three bone samples were determined as E. hydruntinus with a
reliability factor of 1, two of 0.75, one of 0.66, one of 0.33, one of 0.5
and four of 0.25). This group also contains the French specimen from
isotope stage 5, a perfectly preserved complete 3rd metacarpal that
was unanimously determined as E. hydruntinus. One can therefore
conclude that this clade shows the best correspondence to what
is thought to have been E. hydruntinus. This hemionus-type clade
seems to have been present during the Middle Pleistocene in Western Europe and survived in Southeastern Balkans and in Turkey
until at least the Late Neolithic.
7. Discussion
7.1. Ambiguities, biases and uncertainties in palaeontological
determination of equid bones
The phylogenetic tree of the Equidae based on morphological
criteria established on a rich fossil record comprises a multitude of
species and subspecies that are controversially discussed. Indeed,
as discussed above, identification is a matter of considerable difficulty and debate (Payne, 1991). Recent advances in ancient DNA
technology have made the reanalysis of a number of ancient Equus
samples possible, which has led to proposals for taxonomic revisions at the generic, subgeneric, and species levels (Orlando et al.,
2003, 2006, 2009; Weinstock et al., 2005). In particular, while South
American hippidions were considered to be descendants of the Pliohippines based on their distinct nasal morphology, the mitochondrial DNA of several hippidion specimens from Argentina formed
a tight haplogroup within a larger paraphyletic group of Equus,
suggesting either that hippidions and living Equus belong to the
same genus or that living equids should be split into several genera
(Orlando et al., 2003, 2006, 2009; Weinstock et al., 2005). Similarly,
the South American subgenus Amerhippus that was considered on
morphological grounds to be rather Equus-like (Azzaroli, 1998),
had a mitochondrial haplotype that clustered with the caballine
horses (Orlando et al., 2008). Moreover, the group of the stilt-legged
equids of North America, that were morphologically similar to the
Asiatic wild asses, were shown through mitochondrial aDNA analysis to be endemic to the New World and to harbour fewer species
than deduced from morphological criteria (Weinstock et al., 2005).
Finally, it was proposed recently on the basis of ancient mitochondrial DNA data to synonymize Cape zebras, quaggas and plain
zebras, on the one hand, and E. hydruntinus and E. hemionus, on
the other hand (Orlando et al., 2009). There are thus precedents for
inconsistency between palaeontological determination of species
based on bone morphology and palaeogenetic data.
Although we could clearly establish that the samples that
were determined by most archaeozoologists to be E. hydruntinus
all carry a characteristic hemione-type sequence, in agreement
with the observation of Orlando and colleagues (Orlando et al.,
2006, 2009), we observed a large proportion of samples morphologically determined by some archeozoologists as being E.
hydruntinus that turned out to carry a typical caballine mitochondrial sequence, and in two cases even a typical caballine Y
chromosomal sequence ruling out the possibility of hybrids. This
polyphyly is clearly incompatible with the determination of all
these bones as a unique species. Most of the caballine-type samples
originated from Europe showing that for this geographical area,
there is a tendency to attribute horse bones to E. hydruntinus. The
difficulties in determining ancient bone remains at the species level
are best illustrated by the experimental strategy that we adopted in
order to obtain a reliability index for the determination: we asked
several palaeontologists/archaeozoologists to determine the same
bones. For most of the bones, this procedure led to their attribution to more than one species and for several bones a consensus
could not be reached, i.e., each palaeontologist/archaeozoologist
attributed a given bone to another species. This suggests that several palaeontologists/archeozoologists, including very experienced
and renowned ones, tend to underestimate the morphological
variability of caballine equids in this period and area and to
overuse the ill-defined E. hydruntinus in Europe to determine
bones that seem to be undeterminable. Furthermore, we could
also observe that some of the misattributions could be linked to
erroneous dating of the bones: for two bones, even though the corresponding stratigraphic layer was archaeologically dated to the
Neolithic, 14 C dating showed that the bones were from Late Bronze
Age/Early Iron Age, thus biasing the determination that was based
on expectations about the size range of wild horses during the
Neolithic.
Orlando and colleagues previously performed a mitochondrial
DNA analysis of four ancient bone samples that had been determined palaeontologically to belong to E. hydruntinus and that were
found to carry the 28 bp deletion that is characteristic of the Asiatic
wild asses (kiangs, onagers, kulans) (Orlando et al., 2006, 2009).
Thus, despite the fact that the average relative dimensions of the
metapodials and upper-cheek teeth parameters of E. hydruntinus
differed in the specimens that they analysed, the authors conclude, on the basis of the mitochondrial DNA data obtained, that E.
hydruntinus was a subspecies of E. hemionus (Orlando et al., 2009).
Three other presumed E. hydruntinus specimens from Southwest
Siberia, however, formed a new monophyletic group lacking the
hemione-specific 28 bp deletion and any specific affinity with other
non-caballine horses (Orlando et al., 2009). Therefore, these authors
suggest that these latter constitute an extinct group with no extant
relatives, a proposal that has been supported by a revisited morphological analysis of these bones (Eisenmann, 2010). The morphology
of the lower cheek teeth as well as the size of the third metapodials of these specimens from Proskuryakova and Akhalkalaki were
compared to those of E. altidens from Süssenborn, and E. hydruntinus from Lunel-Viel, Staroselie and Dorog. This analysis showed
only poor affinities between the Siberian specimens and hemiones
and hydruntines but rather strong affinities with the archaic Middle
Pleistocene Süssemiones from Süssenborn, Germany (Eisenmann,
2006; Orlando et al., 2009; Eisenmann, 2010). Based on the age and
the mitochondrial sequences of the analysed samples, the age of
the emergence of this new species was estimated to have occurred
between 0.4 and 2.3 MYA (Orlando et al., 2009). Süssemiones supposedly became extinct well before the beginning of the Late
Pleistocene (Eisenmann, 2006), but the 14 C dates of the specimens
99
analysed by Orlando et al. (2009), indicate that the Siberian species
survived at least until c. 45–50,000. Our identification of the most
reliable E. hydruntinus bones to the hemione-type mitochondrial
groups is in agreement with the proposal that the hydruntine population is closely linked to E. hemionus, and thus would favour their
interpretation that the other mitochondrial lineage identified can
clearly be assigned to another lineage, presumably süssemiones
as proposed (Orlando et al., 2009; Eisenmann, 2010). As discussed
before, the taxonomic position of E. hydruntinus based on morphological criteria was subject to debate and for some authors it
was believed to be the last representative of the Plio-Pleistocene
group of the stenionans (Gromova, 1949; Forsten, 1986, 1990;
Forsten and Ziegler, 1995; Forsten, 1999). Presumably, this attribution was based on bones that belonged to süssemiones rather
than to hydruntines. Thus, our results as well as the results of
Orlando et al. indicate that the erroneous attribution of bones from
another lineage to E. hydruntinus affects at least two equid lineages,
süssemiones and caballines, and these erroneous attributions may
explain the past discrepancies of the phylogenetic relationships
between E. hydruntinus and other equids. Here, the equids may
not only suffer taxonomic oversplitting as proposed (Orlando et al.,
2009), but also taxonomic undersplitting, several lineages being
fused into a single species.
The discrepancies between the phylogenetic trees of equids
based either on mitochondrial DNA data or morphological characteristics require reconsidering the strength of each line of evidence.
We suggest that the morphological variability of ancient equids
is underestimated. In particular, temporal and regional body-size
variations among Late Pleistocene and Holocene equids might
have been more pronounced than generally believed, which could
introduce biases in morphological taxonomy. One should keep
in mind that morphological characteristics are not as independent as they may seem at first glance. They may rather change
when the expression level of a small number of regulatory factors changes due to minor genetic or even epigenetic modifications
(e.g., Kangas et al., 2004). Morphological variability within a species
could be more widespread than currently assumed. Thus, the use
of a few “characteristic” morphological traits to reconstruct phylogeny can be misleading and sometimes provide inconsistencies
with phylogenies based on a few genetic markers not related to
the morphological traits. In contrast, morphological traits that are
based on a large number of genetic variants distributed throughout the genome, as seen for example in the case of size differences
in chicken (Johansson et al., 2010), could provide more reliable
phylogenies. It is probably presently still premature to rely on morphological traits the genetic basis of which is unknown.
Several equid populations might have lived and interacted in
the same area at the same time, possibly thriving in different ecological niches, such as different species of zebras do in Africa today.
Indeed, natural selection can lead to parapatric occupation of different ecological niches through morphologically distinct populations
between which gene flow occurs (Smith et al., 1997; Schneider
et al., 1999). If this phenomenon lasts for periods long enough to
be detectable in the fossil record but not long enough for complete
divergence of the metapopulation lineages, then a situation could
arise such as that concerning E. hydruntinus revealed in the course
of our study.
7.2. E. hydruntinus as a separate species or an E. hemionus
subspecies?
Orlando et al., emphasize that extant kiangs and onagers-kulans
are classified as separate species based on differences in coat colour,
morphology, geographic distribution, and the number of chromosomes although they show only poor mitochondrial differentiation.
Similarly, they allow for the possibility that E. hydruntinus is a
100
separate species, which might have experienced multiple introgression events from different onager or kulan stocks (Orlando
et al., 2009). Our identification of the most reliable E. hydruntinus
bones to the hemione-type mitochondrial groups is in agreement
with the proposal of these authors that the hydruntine population is closely linked to E. hemionus. However, our mitochondrial
DNA analysis involving a larger part of the hypervariable region
and a large sample size of ancient E. hemionus and E. hydruntinus
bones has allowed us to identify substructures within the hemionus
sequences that substantially change the evidence supporting the
taxonomic status of E. hydruntinus. Indeed, we could identify seven
distinct haplogroups within Asiatic wild asses. This in turn allowed
us to identify one haplogroup for which independent palaeontological determinations as E. hydruntinus were established with the
highest consensus among all the bone remains that we studied.
This hydruntine-like clade was present during the first half of the
Holocene in the Southwest of the Balkans and in Turkey. The haplotype of the 100,000-year-old specimen from Southwest France
clusters with the hydruntine-like haplogroup, but it is not found at
a position that connects this haplogroup to the other haplogroups,
but rather at the other end. Thus, this sequence, although belonging to a bone that is more ancient than those from the Balkans
and Turkey, derives from the Balkan hydruntine population. This
suggests that the West European hydruntine corresponded to a
subpopulation that evolved from an ancestral population originating in Eastern Europe and Turkey. The hydruntine haplogroup is
as distant from the other haplogroups as are the kiangs from the
onagers. Thus, if kiangs are considered a distinct species, on the
basis of several lines of evidence (phenotypical and genotypical),
then the level of genetic differentiation between E. hemionus and
E. hydruntinus would be sufficient to consider them as different
species as well.
A counter argument, however, is that such a genetic distance can
also be found separating the two haplogroups that exist within the
extant Turkmen kulan and the Iranian onager populations. These
two haplogroups comprise ancient Iranian hemiones found near
the Caspian Sea from the 6th to the 2nd millennium BC the bone
morphology of which does not allow for classification between
kulans and onagers. A Bayesian analysis of our mitochondrial DNA
sequence data showed that the two groups separated a long time
ago (estimated 59 kyrs), at about the same time as they separated from kiangs (Champlot et al., in preparation). The fact that
these two haplogroups can be found in the extant Turkmen kulan
population, as well as in the Iranian onager population as suggested previously (Oakenfull et al., 2000), indicate that there was
gene flow between these two populations at some point after
their separation. This could have occurred recently, possibly as
a consequence of human-driven habitat disruption and population rearrangements, or could have occurred earlier, with a mixed
hemione population that separated only recently in the current
onager and kulan population, an hypothesis that was favoured by
Oakenfull et al. (2000) because it was the most parsimonious interpretation. Our observation that the two haplogroups had already
overlapped in the same geographical region several thousand years
ago argues in favour of the recent differentiation of a unique population comprising several distinct haplogroups. Thus, this level
of genetic differentiation can accompany a speciation event, as
seen for the Kiangs, but does not convincingly demonstrate speciation.
Finally, a specimen from Portugal dated to the 17th century was
analysed by Orlando et al. (2009) that was reported to be the last
specimen of the so-called zebro, a striped equid described in the
Medieval Iberian literature and supposed to be at the origin of
the name of the African equids, the zebras (Nores Quesada and
Liesau von Lettow-Vorbeck, 1992; Antunes, 2002). Cranial morphology and mitochondrial DNA sequence, however, indicate that
this animal was a donkey (Orlando et al., 2009). This suggests that E.
hydruntinus disappeared earlier than assumed based on the determination of ancient bones, and Orlando and colleagues proposed
pushing back the upper limits of hydruntine survival from the Middle Age to the Iron Age. Our study reveals that all the Western
European bones that were assigned to E. hydruntinus dating from
35,000 to 6000 BP were erroneous attributions of horse bones.
This suggests that the disappearance of E. hydruntinus may have
occurred much earlier than believed, or at least, that there was a
severe reduction in the size of this population long before the Iron
Age.
8. Conclusion
Palaeogenetic studies like the one described here can highlight and help to correct systematic biases of the morphological
characterization of ancient bones and be useful guiding tools to
better define ancient species and their phylogenetic relationships.
One should, however, keep in mind, that the present study, as
well as those that have preceded it (Orlando et al., 2003, 2006,
2008; Weinstock et al., 2005), have analysed only the maternally inherited, non-recombining mitochondrial DNA that does
not take into account certain etiological factors such as territorial and mating behaviour, which is known to be different in
horses and wild asses (Burke, 2002). Mitochondrial DNA is a
characteristic independent of anatomical traits the analysis of
which delivers a certain type of information, different from that
which can be obtained from the analysis of the dentition and
bone morphology, and can usefully complement the anatomist’s
tools. Both the palaeontological and the palaeogenetic and genetic
approach analyse secondary species criteria that should be taken as
lines of evidence helping to define the species boundary between
separately evolving metapopulation lineages. The complexity of
speciation events and the scarcity of data from ancient populations
mean that caution is necessary before assigning metapopulation lineages as species, whatever the methods used to collect
the lines of evidence, whether they are based on genetics or
anatomy.
Acknowledgments
We thank Sophie Champlot and E. Andrew Bennett for the production of the palaeogenetic data and Mathieu Gautier for Bayesian
analysis; Véra Eisenmann, Hans-Peter Uerpmann, and Simon Davis
for help with palaeontological determinations; Véra Eisenmann,
Hans-Peter Uerpmann, Simon Davis, Mietje Germonpré, Marjan
Mashkour, Arturo Morales Muniz and Joris Peters for helpful discussions; Benjamin Arbuckle, Adrian Balasescu, Marie-Françoise
Bonifay, Jean-Philippe Brugal, Jean-Jacques Cleyet-Merle, Lydia
Gamberi, Mietje Germonpré, Christophe Griggo, Stéphane Hinguant, Stéphane Madelaine, Gabriella Mangano, Marjan Mashkour,
Arturo Morales Muniz, Pierre-Elie Moullé, Marylène Patou-Mathis,
Joris Peters, Maryline Rillardon, Jean-François Tournepiche, and
Hans-Peter Uerpmann as well as the Muséum National d’Histoire
Naturelle of Paris for providing samples; Véra Eisenmann for providing the photos and validating Fig. 3, and help with Table 2;
Simon Davis for critical reading of the manuscript, valuable suggestions, correction of Table 2, providing the drawings of Fig. 3
and correction of the English language; an anonymous reviewer
for valuable suggestions; E. Andrew Bennett for critical reading of
the final manuscript;
The palaeogenetic analyses were funded by the French National
Research Centre CNRS.
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Morphological versus genetic diversity

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