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INTRODUCTION
Emus are iconic ratite birds endemic to Australia. Today a
single extant species, known simply as the Emu Dromaius
novaehollandiae (Latham, 1790), is widely distributed
across open habitats, whereas several populations
became extinct on islands surrounding mainland
Australia: Tasmania, Kangaroo Island and King Island.
Each insular population of emu has been recognized as
a distinct taxon, mostly because of its distinct small size,
at the species or subspecies level. The description of the
Kangaroo Island Emu Dromaius baudinianus Parker,
1984 was based on bones collected in a cave in 1926
(Parker, 1984). The diagnosis includes differences of the
length and shape of the tibiotarsus and tarsometatarsus.
The Kangaroo Island Emu is intermediate in size between
the smaller King Island Emu Dromaius minor Spencer,
1906 [previously D. ater Vieillot, 1817, see Dickinson &
Remsen (2013)] and the slightly larger Tasmanian Emu
Dromaius diemenensis Le Souëf, 1907.
Located 110 km southwest of Adelaide, Kangaroo Island
is the third largest Australian island after Tasmania and
Melville Island. It is separated from the mainland by the
13 km wide Backstairs Passage, which is currently 40 m
deep. The island is 145 km long east-west and 54 km
long at its widest north-south section, covering 4405 km2.
Its climate is Mediterranean, having mild winters and dry
summers. Compared to mainland South Australia, the
island has been relatively spared from anthropogenic
degradation with almost 40% of the island still being
covered by native vegetation (Robinson & Armstrong,
1999). The island’s earliest human occupation dates from
ca. 16,000 before present (BP; Lampert, 1981). At the
beginning of the 18th century, when European expeditions
reached the island (see below), evidence suggests that
Aboriginal people were not permanently settled on the
island but that they used the land occasionally (Draper,
2015). In 1802-1803, a British expedition under Matthew
Flinders and a French naval expedition commanded by
Nicolas Baudin each sailed close to South Australia.
Both expeditions landed on Kangaroo Island to explore
and replenish supplies. The French Expedition captured
two living emus there on 31st January 1803 (see details
in Jansen, 2014, 2018). One died en route to Europe
and the other was kept captive in Paris until its death
Revue suisse de Zoologie (September 2019) 126(2): 209-217
Genetic and radiographic insights into the only known mounted specimen
of Kangaroo Island Emu
Alice Cibois1, Laurent Vallotton1, Per G. P. Ericson2, Mozes P. K. Blom2,3, Martin Irestedt2
1 Muséum d’histoire naturelle de Genève, C.P. 6434, CH-1211 Geneva 6, Switzerland
2 Department of Biodiversity Informatics and Genetics, Swedish Museum of Natural History, SE-10405 Stockholm,
Sweden
3 Museum für Naturkunde, Leibniz Institut für Evolutions- und Biodiversitätsforschung, D-10115 Berlin, Germany
* Corresponding author: alice.cibois@ville-ge.ch
Abstract: The Natural History Museum of Geneva holds a mounted specimen of a dwarf emu, which is believed to be
the only preserved skin of the extinct Kangaroo Island Emu, Dromaius baudinianus. We obtained new radiographs that
show the absence of remaining bones in the preparation, conrming previous statements found in the museum’s archives.
Moreover, we sequenced the complete mitochondrial genome of this specimen and we compared it to all available
emu sequences. The mitogenome of the specimen held in Geneva is very close to that of Common Emus Dromaius
novaehollandiae. Overall, the genetic results on insular emus support a shallow divergence between the mainland
population and the – now extinct – populations from King Island, Kangaroo Island and Tasmania. Based on these results,
we agree with previous molecular studies that the insular emu taxa should be treated as three different subspecies of the
Common Emu.
Keywords: Dromaius baudinianus - Casuariidae - Natural History Museum of Geneva - X-ray - complete mitochondrial
genome - ancient DNA.
Manuscript accepted 04.06.2019
DOI:
210 A. Cibois et al.
associated with the specimen. The specimen was
exhibited until 1925 and then removed from its socle.
It was restored in 1955 and mounted again in February
1958. The specimen, which is still on exhibition in
Geneva, was not examined by S. Parker (Parker, 1984).
Jouanin (1959) concluded that it was possible that this
specimen corresponded to the skeleton preserved in
Paris [contra Hume (2017) who misinterpreted Jouanin’s
text], in particular because the measurements of the two
specimens matched perfectly. The taxidermists in 1955
indicated that the specimen has no bones at all, and
that the bill was articial. This was conrmed later by
a radiography commanded by François Poplin (François
Baud in litt.), which was unfortunately not preserved in
the archives of the MHNG. Balouet & Jouanin (1990)
pointed out that a bone could still remain in one claw
of the Geneva specimen, but the evidence for this claim
was uncertain. In the same article, Balouet & Jouanin
(1990) conrmed the identication of the skeleton in
Paris as D. baudinianus, according to measurements and
bone details found in Parker (1984). They considered
that the mounted Geneva specimen was very likely the
skin of the mounted Paris skeleton. They also identied
as “D. ater”= D. minor two other specimens; one other
mounted specimen held in Paris (MNHN-ZO-2012-610)
and another skeleton in Museo di Storia Naturale di
Firenze, Italy (C.G.U. 9588; Barbagli & Violani, 2010).
Worthy et al. (2014) analyzed the skeletal characters of
D. novaehollandiae and D. baudinianus in comparison
to an Oligo–Miocene fossil taxon Emuarius gidju. They
concluded that, apart from their difference in size, the two
Dromaius taxa did not present major qualitative skeletal
differences and that subspecic rank should apply to
D. baudinianus. Two genetic studies have also been
conducted recently on the insular dwarf emus. Heupink
et al. (2011) investigated the phylogenetic relationships
between King Island and mainland emus. They sequenced
two partial mitochondrial DNA (mtDNA) regions
(Control Region and Cytochrome c oxidase subunit 1)
and a small region of a nuclear gene (Melanocortin 1
receptor) for ve bone remains from King Island. They
found that for these genetic markers King Island emus
fall within the diversity of modern samples of mainland
emus. Thomson et al. (2018) sequenced more samples
for mtDNA (Control Region gene only) including ancient
bones from the extinct Tasmanian emus (ve bones) and
Kangaroo Island emus (11 bones). Their conclusions
were similar and they suggested that all insular taxa are
subspecies of D. novaehollandiae.
The rst goal of this study was to re-valuate the
phylogenetic placement of the specimen held in Geneva
relative to all emus previously sequenced, sampled
from Kangaroo Island, the other islands, and mainland
Australia. To achieve this, we used High-Throughput
Sequencing to reconstruct the complete mitochondrial
genome for this specimen and compared it to all
sequences available for emus. Our second objective was
in 1822. The skeleton of the latter was retained at the
Muséum National d’Histoire Naturelle of Paris (MNHN)
(registration number MNHN-ZO-AC-A3525), and it was
believed that its skin corresponds to the specimen from
the Muséum d’histoire naturelle of Geneva (MHNG)
(registration number MHNG-OIS-629.041; Fig. 1).
A report from the archives of the Natural History
Museum of Geneva from 26th January 1828 states that
the mounted specimen was bought in Paris for 150 Swiss
francs in December 1827 by M.-E. Moricand, who was
then the administrator of the “Musée Académique de
Genève”. Jouanin (1959) found in the registers in Paris
that “a Cassowary from New Holland (without beak)”,
of a value of 60 francs, was indeed “given” (“donné”)
to Moricand in December 1827. The specimen was
examined by F. de Schaeck (assistant curator at the
MHNG), and registered, apparently for the rst time,
in September 1892 during curatorial activities at the
collection. A torn piece of paper, which seemed to
read “Ile Decrès”(=Kangaroo Island), was then found
Fig. 1. The Geneva mounted specimen of Kangaroo Island
Emu (MHNG–OIS–629.041). Photo P. Wagneur/
MHNG.
Kangaroo Island Emu 211
to shed light on the origin of the Geneva specimen
in relation with the skeleton held in Paris Museum.
Unfortunately, we were not able to sample this skeleton
for genetic analyses. However, the main uncertainty
in the chronicle regarding the Geneva specimen is the
potential presence of bones within the mount that would
disqualify it from being the same individual as the Paris
skeleton. Therefore, we obtained new radiographs to test
whether bones were left inside the mounted specimen
during the taxidermy preparation.
METHODS
DNA Extraction, Library preparation, sequencing,
assembly and bioinformatics
DNA was extracted from a toe-pad sample taken from
specimen MHNG-OIS-629.041 following the protocol
described in Irestedt et al. (2006) for historical specimens.
Genomic libraries were prepared using the Meyer &
Kircher (2010) protocol, with slight modications as
detailed in Johansson et al. (2018), and included four
unique index libraries to reduce PCR duplicates. All
four libraries were pooled together with an additional
avian sample (Paradise Parrot Psephotellus pulcherrimus
– not part of the current study) and sequenced on a
single Illumina Hiseq X lane at SciLifeLab Stockholm.
Following sequencing, each library was individually
processed using a custom designed workow (available at
https://github.com/mozesblom/NGSdata_tools/clean_up
_raw_reads.py), which removes adapter contamination,
PCR duplicates, merges overlapping read-pairs and
excludes low-complexity reads and low-quality bases.
To avoid any putative bias in mitogenome reconstruction,
an iterative baiting and mapping strategy was employed
to reconstruct the mitogenome for the Geneva specimen.
Using a subsample of polished reads (15 million) and the
extant emu as a seed reference (Genbank - AF338711),
we used MITObim (Hahn et al., 2013) for mitogenome
reconstruction and subsequently corrected the resultant
sequence by mapping the complete dataset against
this initial reference. Each library was mapped using
BWA – mem (Li, 2013), the four corresponding BAM
les being merged with Picard (https://broadinstitute.
github.io/picard/) and variants called using FreeBayes
(Garrison & Marth, 2012). Moreover, we masked sites
with coverage above or below 3X the mean coverage
to avoid the inclusion of putative NUMT calls and low
coverage regions. Finally, uncalled sites were manually
edited using Geneious R10 (https://www.geneious.com)
by lowering the consensus threshold.
Phylogenetic analysis
We compared the complete mitochondrial genome
(hereafter DromGE) of the Geneva specimen to the
three available complete or near complete mitochondrial
genomes of extant continental emus (for clarity hereafter
called Common Emu): AF338711 and NC_002784 (both
16,711 bp; origin of the samples unknown) (Haddrath &
Baker, 2001), and AY016014 (12,280 bp; captive bird)
(Cooper et al., 2001). We also included DromGE to
previously published data sets that included both extant
and extinct emus for two mitochondrial genes: COI
(Heupink et al., 2011) and Control Region (Heupink et
al., 2011; Thomson et al., 2018). The COI and Control
Region sequences from the complete mitogenomes of
Common Emus were added to these data sets.
Divergence time
We used the complete mitogenomes to infer the most
recent time of divergence of the Kangaroo Island Emu
from the Common Emus. We rst applied Lerner et al.’s
(2011) average rate of sequence divergence of 1.8% per
million years for the complete mtDNA genome (0.009
s/s/myr). However it has been suggested that the rates
of mitochondrial genes vary among bird species and
correlate with life history traits, such as body mass
and generation time (Eo & DeWoody, 2010; Nabholz
et al., 2009; Pereira & Baker, 2006). The Common
Emu is a large and long-lived species, sexual maturity
is usually achieved at 2-3 years and longevity is 10-16
years in captivity (Flower, 1938; Del Hoyo et al., 1992).
Size varies from 150 to 190 cm, weight from 30 to
55 kg, males being smaller than females (Folch et al.,
2018). The life history of the Kangaroo Island Emu is
unknown but, although a “dwarf” emu, it was a rather
large and probably long-lived bird. We assume that
DromGE was a full-grown adult, based on its 19 years
in captivity (see introduction). Nabholz et al. (2016)
suggested that body mass could be used as a proxy to
estimate corrected molecular rates for molecular dating
studies. We calculated a mass-corrected molecular rate
of mitochondrial coding genes following the procedure
described in Nabholz et al. (2016), using an extrapolated
weight for DromGE (23 kg) based on the height of the
mounted specimen (116 cm) and on the value for the
smallest Common Emus (30 kg for 150 cm). We also
used the equation proposed by Nabholz et al. (2016) for
the mitochondrial coding genes (10,869 bp), based on all
codon positions and the two sets of calibrations proposed
in this study (“calibrations 2 and 4”, see the original
article for details).
Radiography
The specimen MHNG-OIS-629.041 was X-rayed using a
portable GIERTH TR 90/20 X-ray unit (OR Technology),
equipped with a Toshiba tube D-0814/0.8 mm. The unit
was connected to a Canon Digital Radiography System
CXDI-80C. The images were visualized using a Canon
Dicompacs Acquisition Software. Because of its large
size, only selected sections of the mounted specimen
were examined: the feet, legs, head and back.
212 A. Cibois et al.
RESULTS
Mitogenomes
The complete mitogenome of the Geneva specimen
(DromGe, 16,713 bp after nal editing) was deposited
in GenBank, accession number MK625178. On
average DromGE differs from the three Common Emu
mitogenomes by 0.090% ± 0.008. Lerner’s et al. (2011)
rate applied to emus suggested that DromGE diverged
ca. 100,000 years ago from this set of three Common
Emus (mean 93,074 ± 23,029 years). The rates for
emus corrected for size, used as a proxy for generation
time, varied from 0.004455221 s/s/myr (calibration 2)
to 0.007377136 s/s/myr (calibration 4). Applied on the
coding genes of the mitochondrial genomes, it suggested
that DromGE and the three Common Emus diverged
between 206,510 years ago (calibration 2) and 124,716
years ago (calibration 4).
H1
DromGE
H15
H7
H6
H13
H9
H14
H17
H4 H10
H16
H11
H2
H5
H18
H12
H3
Kangaroo Is.
King Is.
Tasmania
Mainland Australia (modern)
Mainland Australia (ancient)
DE
DromGE
GH
A
I
J
A
B
Fig. 2. Haplotype networks. (A) Control Region (563 bp), modied from Thomson et al. (2018). (B) COI (1,544 bp), modied from
Heupink et al. (2011). Circles are proportional to the number of individuals but the scales are different for the two genes (see
details in the original studies). The black circles represent intermediate or unsampled haplotypes. “DromGE “indicates the
specimen from Geneva Museum.
Kangaroo Island Emu 213
Control region and COI data sets
We added DromGE’s sequence to the genetic haplotype
network provided by Thomson et al. (2018) for the Control
Region (partial gene, 563 bp) (Fig. 2A). This individual
has a new haplotype, different by one substitution
to haplotypes H7 (predominantly found in Common
Emus) and H1 (Common Emus, including AF338711
and NC_002784; AY016014 was not sequenced for
this gene). It differs by two to three substitutions to the
three haplotypes found in bones from Kangaroo Island:
H6, also found in mainland samples; H2, found also in
mainland and Tasmanian samples; and H5, found in a
single Kangaroo Island bone.
Regarding the COI gene (Fig. 2B), DromGE shares
Haplotype A with Common Emus. It differs by two
mutations from the haplotype GH found in King Island
Emus. Bones from Kangaroo Island Emus were not
sequenced for this gene. Common Emus also share three
other haplotypes, DE (including AF338711, NC_002784
and AY016014), I and J.
Radiography
Images were taken at different sections of the specimen:
all showed that no bones were conserved in the
preparation, in particular in the toes (Fig. 3). No elements
of the skull were conserved either, and the bill is articial
and supported by wires.
DISCUSSION
The origin of the Geneva specimen
The genetic data do not provide denitive elements
regarding the origin of the Geneva specimen, DromGE.
Because no unique haplotypes exist for Kangaroo Island
or King Island populations, the putative geographic
origin of Kangaroo Island for this specimen can be
neither conrmed nor refuted. The new radiographs
show the absence of remaining bones in the preparation,
conrming previous statements found in the museum’s
archives. Balouet & Jouanin’s (1990) claim of a remaining
bone in a toe of the Geneva specimen might have been
based on the fact that a bone was missing from the Paris
skeleton (left foot, digit II, proximal phalanx; A. Cibois
pers. obs.). It is common practice to keep the bones in the
legs and feet of birds, even in large birds such as ratites
(Davie, 1894; Larsen, 1945). However, skin and skeleton
for an individual large bird could also have been sold
separately, resulting in boneless mounted specimens. For
instance, we checked the foot of another old mounted
specimen in Geneva (a Common Emu acquired in 1926;
MHNG 837.071) and no bones were preserved in that one
either. Thus, the lack of bones in the Geneva specimen
is not absolute proof that it is the same individual as
the Paris skeleton. Nonetheless, we conclude that after
considering the historical evidence and correspondence
of the measurements of both specimens, the hypothesis
of Kangaroo Island provenance for DromGE remains the
most likely.
Divergence time and isolation of the Kangaroo
Island Emu
Previous studies on the extinct emus showed that the island
populations had a subset of the mitochondrial genetic
diversity found in Common Emus. The numbers of extant
emus on mainland Australia have increased ca. tenfold
since colonization by Europeans, with some uctuation
in numbers, as they beneted from increased water
supplies and the erection of fences to exclude predators
like dingos (Canis lupus dingo) (Folch et al., 2018; Pople
et al., 2000). Their numbers are currently considered as
stable, and no recent bottleneck has led to the erosion
of genetic diversity. The mitogenome of the specimen
held in Geneva is very weakly divergent from that of
Common Emus. Taken together, all the mitochondrial
results on island populations of emus support a shallow
divergence between the extant mainland population and
the extinct populations from King Island, Kangaroo
Island and Tasmania. An alternative explanation could
be that these results are biased by introgression events
that led to the capture of the mainland mitogenome (the
most important population in number) by that of the three
island populations. However, the topology of the largest
haplotype network (for the Control Region, Fig. 2A)
based on 134 individuals, is consistent with incomplete
lineage sorting, the island taxa having both shared and
unique haplotypes. Based on these results, we agree
with Heupink et al. (2011) that all emu taxa should be
considered as subspecies of Dromaius novaehollandiae,
D. n. diemenensis (Tasmania), D. n. minor (King Island)
and D. n. baudinianus (Kangaroo Island).
Heupink et al. (2011) and Thomson et al. (2018) discussed
the morphological differences between the insular dwarf
populations and the mainland emus. They based their
analyses on the hypothesis of a very recent isolation of
these islands during the Holocene, when sea-level rose
after the Last Glacial Maximum and ooded the straits
connecting the islands: 10,000 years ago for Kangaroo
Island and the mainland, 12,000 years ago for King
Island and Tasmania, and 14,000 years ago for Tasmania
and the mainland (Lambeck et al., 2014). These short
time frames would imply that the signicant reduction
of size occurred very rapidly for such long-lived birds.
Our divergence time analysis, although based on a single
insular individual and on Common Emus of unknown
origin, suggests on the other hand that the isolation
of the emus of Kangaroo Island, or some population
structure on nearby parts of mainland Australia, took
place during the Pleistocene, at least ca. 100,000 years
ago. Probably the most parsimonious scenario implies
the mid-Pleistocene episodes of sea-level variations that
could have triggered the isolation of emus on Kangaroo
Island. During the glacial Marine Isotope Stage 6 (which
began 190,000 years ago), sea-level dropped ca. 100 m
below present sea level, permitting a land connection
214 A. Cibois et al.
Fig. 3. Radiographs of the specimen MHNG-OIS-629.041. (A) Left foot. (B) Right foot. (C) Head and neck. (D) Back. (E) Thighs and
upper legs.
Kangaroo Island Emu 215
Because some pristine habitats are still present on Kan-
garoo Island, the island acted as a recent refugium for
species endangered or extirpated from mainland Austra-
lia. For instance, the last population of an endangered
subspecies of the Glossy Black-Cockatoo (Calyptorhyn-
chus lathami halmaturinus) is now restricted to Kanga-
roo Island. The diet of this bird is specialized to the seeds
of sheoaks (Allocasuarina verticillata). Extensive tracts
of this tree and the bird have both disappeared from near-
by parts of mainland Australia, the bird last having been
recorded in the late 1970s (Joseph, 1989; Berris et al.,
2018; Schodde et al., 1993). Species on Kangaroo Island
have also been preserved from alien predators or com-
petitors that have not reached the island. It is the case for
the Bush Rat (Rattus fuscipes greyii), which populations
on Kangaroo Island present higher genetic diversity than
those on the mainland (Hinten et al., 2003).
The entomofauna of Kangaroo Island is poorly known,
but a recent discovery brought to light an endemic
moth species. This new species, called the “enigma
moth”, exhibits a unique combination of morphological
characters and it was placed in a new family Aenig-
matineidae (Kristensen et al., 2015). A molecular
analysis showed that it was basal in the phylogenetic
tree of the moth families, suggesting an old origin. The
current restricted distribution of such an ancient family
suggests that the Kangaroo Island moth could be either
a paleo-endemic that had a wider distribution in the past
and disappeared from most of its range due to climate
modications – or a pseudo-endemic (i.e. a species with
a large distribution anthropogenically reduced by habitat
loss). These alternative hypotheses, which might also
apply to several endemic plants, both support the idea
that Kangaroo Island acted more as a museum than as a
cradle for biodiversity [sensu Stebbins (1974)]. Most of
its diversity did not evolve in situ, but the island, by its
ancient and recent history, has been a refugium for many
plants and animals.
ACKNOWLEDGEMENTS
We thank Michel Dahn, Equine Veterinary, for conducting
the X-ray examination in the public displays of the
Museum, and Philippe Wagneur for the photographic
coverage. We are grateful to Tom Gilbert, Luca
Fumagalli and Dario Zuccon for some preliminary works
and discussions on the specimens of dwarf emus held in
several European museums. The authors acknowledge
support from the National Genomics Infrastructure in
Stockholm funded by Science for Life Laboratory, the
Knut and Alice Wallenberg Foundation and the Swedish
Research Council, and SNIC/Uppsala Multidisciplinary
Center for Advanced Computational Science for
assistance with massively parallel sequencing and access
to the UPPMAX computational infrastructure. Justin
Jansen and Leo Joseph provided helpful comments on
the manuscript.
between Kangaroo Island and Australia (Eldereld
et al., 2012). During the Last Interglacial Maximum
(Marine Isotope Stage 5e, which began 130,000 years
ago and ended about 115,000), sea level was up to 6 m
or 9 m above present sea surface in the Pacic Ocean
(Dickinson, 2001; Hearty et al., 2007), inundating the
40 m deep Backstairs Passage between Kangaroo Island
and Australia and leading to the isolation of the former’s
emu population. This period resulted in the inundation of
many land surfaces in the Pacic Ocean, and in particular
on low atolls, leading for instance to the extirpation of
landbird populations in the Eastern Pacic (Cibois et al.,
2010). Our new estimation of the divergence time for
the Kangaroo Island Emu, based on a molecular clock,
suggests then that the morphological differentiation (i.e.
dwarng) of this insular population, due to selection or
genetic drift, took place over a longer period of time than
proposed by previous studies.
Kangaroo Island, a biodiversity refugium in past and
recent times
Kangaroo Island is one of the six major biodiversity
hotspots identied across the state of South Australia
for plants (Guerin et al., 2016), with 45 endemic species
being inventoried by Kinnear et al. (1999). The island
also includes three recently described species: two
lichens (Kantvilas & Kondratyuk, 2013) and one fungus
(Catcheside et al., 2015). Along with other parts of
southern Australia, Kangaroo Island is thought to have
acted as a refugium for Mediterranean and semi-arid
plants during colder and drier periods (Byrne, 2008).
However, such a high level of endemism has not been
found for animals. The only endemic mammal, the
Kangaroo Island Dunnart (Sminthopsis aitkeni), presents
shallow morphological and genetic differentiation
from closely related mainland taxa and it has recently
been reclassied as a subspecies of the Sooty Dunnart
(Sminthopsis fuliginosus) by Kemper et al. (2012). The
situation is similar for birds, in which the only endemic
species, the Kangaroo Island Emu, has been reevaluated
as warranting only subspecic rank based on its shallow
genetic difference from mainland populations (Thomson
et al., 2018; this study). In fact, the majority of organisms
for which genetic studies have been conducted show a
weak differentiation between Kangaroo Island and
mainland Australia: Western Grey Kangaroos (Macropus
fuliginosus) (Neaves et al., 2009), Tawny Dragon
Lizard (Ctenophorus decresii) (McLean et al., 2014),
Labiosimplex australis (a parasitic nematode) (Chilton
et al., 2009), Narrow-leaf Hopbush (Dodonaea viscosa
angustissima) (Christmas et al., 2017). A more complex
genetic pattern was found in the Crimson Rosella
complex (Platycercus elegans), in which the subspecies
endemic to Kangaroo Island showed introgression from
nearby mainland populations (Joseph et al., 2008). 17
subspecies of birds are endemic to Kangaroo Island
(Schodde & Mason, 1999) but most of them have not
been subjected to molecular analyses.
216 A. Cibois et al.
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