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Systematics and Biodiversity
ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20
The revenant: rediscovery of Margaritifera
homsensis from Orontes drainage with remarks
on its taxonomic status and conservation (Bivalvia:
Margaritiferidae)
Ilya V. Vikhrev, Ivan N. Bolotov , Ayhan Altun, Mikhail Y. Gofarov, Gennady A.
Dvoryankin, Alexander V. Kondakov, Tahir Ozcan & Gulnaz Ozcan
To cite this article: Ilya V. Vikhrev, Ivan N. Bolotov , Ayhan Altun, Mikhail Y. Gofarov, Gennady
A. Dvoryankin, Alexander V. Kondakov, Tahir Ozcan & Gulnaz Ozcan (2017): The revenant:
rediscovery of Margaritifera homsensis from Orontes drainage with remarks on its taxonomic
status and conservation (Bivalvia: Margaritiferidae), Systematics and Biodiversity, DOI:
10.1080/14772000.2017.1343876
To link to this article: http://dx.doi.org/10.1080/14772000.2017.1343876
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Published online: 17 Jul 2017.
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Research Article
The revenant: rediscovery of Margaritifera homsensis from Orontes
drainage with remarks on its taxonomic status and conservation
(Bivalvia: Margaritiferidae)
ILYA V. VIKHREV
1,2
, IVAN N. BOLOTOV
1,2
, AYHAN ALTUN
3
, MIKHAIL Y. GOFAROV
1,2
,
GENNADY A. DVORYANKIN
4
, ALEXANDER V. KONDAKOV
1,2
, TAHIR OZCAN
3
& GULNAZ OZCAN
3
1
IBIGER – Institute of Biogeography and Genetic Resources, Federal Center for Integrated Arctic Research, Russian Academy of
Sciences, Severnaya Dvina Emb. 23, Arkhangelsk 163000, Russian Federation
2
Scientific Department, Northern (Arctic) Federal University, Severnaya Dvina Emb. 17, Arkhangelsk 163002, Russian Federation
3
Faculty of Marine Sciences and Technology, Iskenderun Technical University, Iskenderun 31200, Hatay, Turkey
4
SevPINRO – Northern Branch of the Polar Scientific-Research Institute of Fish Husbandry and Oceanography, Uritskogo 17,
Arkhangelsk 163000, Russian Federation
(Received 4 July 2016; accepted 17 May 2017)
Since Margaritifera marocana (Pallary, 1918) and M. laosensis (Lea, 1863) were rediscovered, M. homsensis (Lea, 1865)
remains the only pearl mussel species known solely based on old shell samples from natural history museums. This is also
the last pearl mussel species, which is absent in a phylogeny of the family. Here, we aimed to provide an integrative
revision of the taxonomic status of M. homsensis from the Orontes Basin. Using a newly collected specimen from the River
Karasu, Hatay Province, southern Turkey, five gene partitions were sequenced, the cytochrome c oxidase subunit I (COI),
large ribosomal subunit rRNA (16S), large ribosomal subunit rDNA (28S) and its D3 expansion segment (D3), and small
ribosomal subunit rDNA (18S). The multi-gene phylogeny indicates that M. homsensis is a sister taxon of M. auricularia,
but both these species are closely related to M. marocana by nuclear genes. The main conchological features, i.e., the shell
shape, teeth morphology, and mantle attachment scars, as well as Fourier shell shape analysis have not shown principal
differences between M. homsensis and M. auricularia. Based on these data, we concluded that M. homsensis is a valid
species that is most closely related to M. auricularia. Special conservation efforts for a population of M. homsensis
discovered in Turkey, including the formation of a nature reserve, might contribute to the conservation of the species.
Finally, an extensive search for surviving populations in Orontes drainage (southern Turkey, Lebanon, and Syria) and the
Nahr-el-Kabir River (Lebanon and Syria) remains necessary to develop a transboundary conservation strategy for this
unique taxon.
Key words: freshwater endemism, freshwater pearl mussel, Mediterranean, River Orontes, Turkey
Introduction
The Mediterranean region is considered to be one of the
most important freshwater biodiversity hotspots in the
world, reflecting exceptional species richness and high
level of endemism (Myers, Mittermeier, Mittermeier,
da Fonseca, & Kent, 2000). The circum-Mediterranean
freshwater mollusc fauna is much more diverse than some
larger regions, such as Africa and Europe (Seddon,
Kebap¸cı, Lopes-Lima, Damme, & Smith, 2014). Four
‘classic refugia’, i.e., Iberia, Maghreb, South-western
Greece and Southern Turkey, were reported for the Medi-
terranean region (Froufe et al., 2016b). Southern Turkey
has been less studied with respect to Unionoida.
The family Margaritiferidae is one of the smallest, but
widely distributed, groups within freshwater mussels with
only 12 species spread over Eurasia, North America and
north-western Africa (Bolotov et al., 2015; Haas, 1969;
Huff et al., 2004; Smith, 1983,2001). Despite increasing
studies and low numbers of species in the genus, some
significant gaps were only recently closed. Since Margari-
tifera marocana (Pallary, 1918) and M. laosensis (Lea,
1863) were rediscovered (Ara
ujo, Toledo, Van Damme,
Ghamizi, & Machordom, 2009; Bolotov et al., 2014;
Correspondence to: Ilya V. Vikhrev. E-mail: vikhrevilja@gmail.
com
ISSN 1477-2000 print/ 1478-0933 online
ÓThe Trustees of the Natural History Museum, London 2017. All Rights Reserved.
http://dx.doi.org/10.1080/14772000.2017.1343876
Systematics and Biodiversity (2017), 1–12
Kottelat, 2009; Sousa et al., 2016), M. homsensis (Lea,
1865) remains the only pearl mussel species known solely
based on old shell samples from natural history museums.
Margaritifera homsensis is one of the three pearl mus-
sel species distributed throughout the Mediterranean
region. Two other species, M. auricularia (Spengler,
1793) and M. marocana (Pallary, 1918), are distributed
throughout southern Europe and Morocco, respectively
(Ara
ujo et al., 2009). The range of M. homsensis is
restricted by the four river basins, i.e., the Orontes, Nahr-
el-Kabir, Nahr-el-Harun (Drouet, 1893; Sch€
utt, 1983,
1987; Smith, 2001) and, apparently, Litani River
(Tristam, 1865).
Margaritifera homsensis was initially described as
Unio species (Lea, 1865), but during subsequent years the
species was stated as Unio,Psilunio,Potomida,Pseudu-
nio,Margaritana or Margaritifera (reviews: Haas, 1940,
1969; Smith, 2001). Based on the general appearance of
the shell morphology, Haas (1940) concluded that three
nominal taxa described from Orontes Basin, i.e.,Unio
episcopalis Tristram, 1865,Unio barroisi Drouet, 1893
and Margaritana syriaca Pallary, 1929, are synonyms of
Potomida littoralis homsensis (DM. homsensis). Further
studies have accepted that these names are synonyms of
M. homsensis. Based on conchological features, Sch€
utt
(1983) considered that M. homsensis is a subspecies of
Potomida littoralis. In contrast, Kinzelbach and Roth
(1984) considered M. homsensis to be a valid species with
respect to Modell (1964) and noted that it is closely
related to M. auricularia. However, Sch€
utt (1987) sug-
gested that M. homsensis is either a sibling species or a
subspecies of M. auricularia and listed this species as M.
sinuatus homsensis. Falkner (1994) transferred this taxon
to the genus Pseudunio. Smith (2001) stated that M. hom-
sensis is a valid species that is distinct from M. auricu-
laria based on contrasting conchological characters, i.e.,
the external morphology such as shell shape and folds at
dorsal-posterior flanks. At the same time, Smith (2001)
mentioned that presence of folds depends on shell age. He
also noted similarities between M. homsensis and M.
auricularia, and M. marocana,M. hembeli (Conrad,
1838) and M. marrianae R.I. Johnson, 1983, and placed
these taxa within the genus Pseudunio using conchologi-
cal data. Additionally, Smith (2001) specifically noted
that discovery and study of a living population of M. hom-
sensis is of great importance to enhance the current under-
standing of such a poorly known species.
Such a complicated taxonomic history is common for
freshwater bivalves, reflecting the high phenotypic plas-
ticity of the shell features (Ara
ujo et al., 2009;
Lopes-Lima et al., 2016; Prie, Puillandre, & Bouchet,
2012), leading to the restricted applicability of traditional
conchological characters for the delimitation of taxo-
nomic units (Ortmann, 1920; Zieritz & Aldridge, 2011;
Zieritz, Hoffman, Amos, & Aldridge, 2010).
The aim of the present study was to provide an integra-
tive revision of the taxonomic status of M. homsensis
from the Orontes Basin. For this purpose we combined
molecular, morphological and morphometric analyses to
evaluate the phylogenetic position and taxonomic status
of M. homsensis within Margaritiferidae. We used newly
obtained sequences of five mitochondrial and nuclear
genes and applied an elliptic Fourier analysis to generate
the first separation of M. homsensis from other
Margaritiferidae.
Materials and methods
Data sampling
The main channel as well as riversides of the Orontes
River and its six tributaries (three irrigation channels, Small
Karachay, Big Karachay, and Karasu river) were surveyed
during a field trip in Hatay Province, Turkey (Fig. 1,
Appendix 1, see online supplemental material, which is
available from the article’s Taylor & Francis Online page
at https://doi.org/10.1080/14772000.2017.1343876). Stream
observations were performed by wading or snorkelling;
however, snorkelling was inappropriate in most cases as a
result of the muddy water. A small sample of foot tissue
was obtained from one specimen and preserved in 96%
ethanol for molecular analyses. Fragmented empty shells
were collected from the shores and riverbed. These recently
collected materials were deposited in the collection of the
Russian Museum of Biodiversity Hotspots, Institute of Bio-
geography and Genetic Resources, Federal Centre for Inte-
grated Arctic Research, Arkhangelsk, Russia (RMBH). We
also examined M. auricularia (nD11), M. homsensis (nD
25), and M. marocana (nD3) shell samples from collec-
tions at the Natural History Museum, London, UK
(NHMUK); Senckenberg Research Institute and Natural
History Museum, Frankfurt, Germany (SMF); National
Museum of Natural History, Paris, France (MNHN); and
National Museum of Natural History, Madrid, Spain
(MNCN) (Appendices 2, 3, and 4, see supplemental mate-
rial online). Additionally, 10 field photographs of shells of
M. auricularia and 21 field photographs of shells of M.
marocana were provided for shape analysis by Dr Keiko
Nakamura and Dr Manuel Lopes-Lima, respectively.
Molecular analyses
To examine the phylogenetic affinities of M. homsensis,
we sampled representatives of 11 margaritiferid species,
including previously published sequences from NCBI’s
GenBank (Araujo, Schneider, Roe, Erpenbeck, &
Machordom, 2016; Bolotov et al., 2016; Breton et al.,
2011; Giribet et al., 2006; Giribet & Wheeler, 2002; Gon-
zalez & Giribet, 2015; Graf, 2002; Graf & Foighil, 2000;
Hoeh et al., 1998; Huff et al., 2004; Sharma et al., 2013;
2 I.V. Vikhrev et al.
Takeuchi, Okada, & Kakino, 2015; Appendix 5, see sup-
plemental material online). Four outgroup bivalve taxa
from two families were used (Appendix 5, see supplemen-
tal material online). In two cases, we were forced to use
chimaeric combined sequences, collected using gene frag-
ments from different specimens; however, these sequen-
ces were avoided when possible (Graf, Jones, Geneva,
Pfeiffer, & Klunzinger, 2015). Five molecular markers
were used to reconstruct the phylogeny, i.e., the cyto-
chrome c oxidase subunit I (COI, 656 bp), large ribosomal
subunit rRNA (16S rRNA, 498 bp), large ribosomal sub-
unit rDNA (28S rDNA, 797 bp), and its D3 expansion seg-
ment (D3, 305–308 bp), and the small ribosomal subunit
rDNA (18S rDNA, 1770 bp). The tissue samples were
stored in 95% ethanol and extracted using the Nucle-
oSpinÒTissue Kit (Macherey-Nagel GmbH & Co. KG,
Germany). New sequences were amplified using standard
PCR protocols (Bolotov et al., 2015: COI; Huff et al.,
2004: 18S and D3; Prie & Puillandre, 2014: 28S and 16S)
and primers Folmer, Black, Hoeh, Lutz, & Vrijenhoek,
1994; Giribet, Carranza, Baguna, Riutort, & Ribera, 1996;
Graf & Foighil, 2000; Jovelin & Justine, 2001; Simon et
al., 1994; Palumbi, 1996; Whiting, Carpenter, Wheeler, &
Wheeler, 1997; Appendix 6, see supplemental material
online). Forward and reverse sequence reactions were per-
formed directly on purified PCR products using the ABI
PRISMÒBigDyeTM Terminator v. 3.1 reagents kit and
run on an ABI PRISMÒ3730 DNA (Thermo Fisher Scien-
tific Inc., Waltham, MA, USA). The resulting sequences
were assessed using a sequence alignment editor (BioEdit
version 7.2.5, Hall, 1999,2013).
Sequence alignment and phylogenetic conflict
testing
The sequence alignment was performed for each gene
separately using the Muscle algorithm implemented in
MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar,
2013). The aligned sequence datasets were assessed using
GBlocks v. 0.91b (Castresana, 2000; Talavera & Castre-
sana, 2007) to exclude hypervariable fragments from the
sequence alignments with options for less stringent selec-
tion, enabling gap positions, smaller final blocks and less
strict flanking positions. The resulting lengths of the
sequence alignments are listed in Appendix 7 (see supple-
mental material online). A partition homogeneity test was
applied in PAUP v. 4b10 to confirm the lack of incongru-
ence of phylogenetic signals amongst all of the sequence
datasets (Farris, K€
allersj€
o, Kluge, & Bult, 1995; Swofford,
2002). The alignment datasets were joined in the com-
bined nucleotide sequence alignment (COI C16S C18S
C28S & D3) and collapsed into unique haplotypes
(Appendix 5) using an online FASTA sequence toolbox
(FaBox 1.41, Villesen, 2007). The absent sites were
treated as missing data. For phylogenetic analyses, we
used the resulting combined dataset with unique haplo-
types. In all variants of the analyses, the base fragment of
the 28S rRNA and its D3 expansion fragment were treated
as a uniform partition because these sequences are parts of
a single gene and do not reveal any differences in the phy-
logenetic signal (partition homogeneity test, pD1.00).
Phylogenetic and barcoding gap analyses
Maximum likelihood (ML) analyses of the combined data-
set (six partitions: 3 codons of COI C16S C18S C28S
& D3) were conducted by using RAxML v. 8.2.6 HPC
Black Box (Stamatakis, 2006) at the San Diego Supercom-
puter Centre through the CIPRES Science Gateway
(Miller, Pfeiffer, & Schwartz, 2010). A unique GTR model
was applied for each partition, with corrections for gamma
Fig. 1. Range of Margaritifera homsensis. The red circle shows
the location of the live population in the Karasu River; yellow
and green circles indicate collection localities of studied
museum specimens and published records, respectively (Appen-
dices 2 and 12, see supplemental material online). Additionally,
white circles indicate sites in which the species was not recently
recorded (see Appendix 1). The former Amik Lake in Hatay,
artificially drained for agricultural purposes, is indicated with
diagonal hatching.
The revenant: rediscovery of Margaritifera homsensis from Orontes drainage 3
distribution. Nodal support values were estimated using an
automatic rapid bootstrapping algorithm in accordance
with developer’s recommendations (Stamatakis, Hoover, &
Rougemont, 2008), and majority-rule consensus tree was
constructed from independent searches.
Bayesian inference (BI) analyses of the same dataset
were performed using MrBayes v. 3.2.6 (Ronquist et al.,
2012) at the San Diego Supercomputer Centre through the
CIPRES Science Gateway (Miller et al., 2010). The parti-
tion combinations were similar under the ML model. The
best models of sequence evolution for each partition as
suggested based on corrected Akaike Information Crite-
rion (AICc) of MEGA6 (Tamura et al., 2013) are pre-
sented in Appendix 8 (see supplemental material online).
Four runs, each with three heated (temperature D0.1) and
one cold Markov chain, were conducted for 10 million
generations. The trees were sampled every 1000th genera-
tion. After the completion of the Markov Chain Monte
Carlo (MCMC) analysis, the first 10% of trees were dis-
carded as burn-in, and the majority-rule consensus trees
were calculated from the remaining trees. The conver-
gence of the MCMC chains to a stationary distribution
was visually assessed based on the plotted posterior esti-
mates using a MCMC trace analysis tool (Tracer v. 1.6;
Rambaut, Suchard, & Drummond, 2013). The effective
sample size (ESS) value for each parameter sampled from
the MCMC analysis was always recorded as >1000. The
combined set of trees showed a smooth frequency plot.
To examine the genetic divergences between M. hom-
sensis and other taxa of Margaritiferidae, we estimated
the number of nucleotide substitutions per site for each
gene partition and determined the number of amino acid
differences per site for the protein-coding COI gene. The
divergences were calculated using a pairwise uncorrected
p-distance of MEGA6 (Tamura et al., 2013). Thresholds
between intraspecific and interspecific genetic distances
were examined using barcoding gap analysis (Pri
e&
Puillandre, 2014).
Morphological study and morphometric
analysis
Conchological studies were conducted on shells, paying
special attention to the characters used to describe pearl
mussel morphology (Ara
ujo et al., 2009; Bolotov et al.,
2015; Kondo & Kobayashi, 2005; Smith, 2001). To ana-
lyse the intra- and interspecific variability of the sagittal
shell shape, we conducted a Fourier shape analysis using
the SHAPE ver. 1.3 software package as described by
Iwata and Ukai (2002). The outline of the shell was
extracted from a photograph and digitized using Adobe
Photoshop CC 2015. Full colour bmp images with 20-mm
scale bar were analysed using the ChainCoder program
which describes the geometrical information concerning
the contours in numbers ranging from 0 to 7. After that
Elliptic Fourier Descriptors (EFDs) were calculated using
the Chc2Nef programme. We used normalization based
on the longest radius. Thanks to this mode we performed
manual normalization during Chc2Nef processing and all
of the contours were orientated in the same position. The
contour shapes were described in the first 20 Fourier har-
monics. Principal component analysis (PCA) was per-
formed on the normalized EFD coefficients using the
PrinComp program from the SHAPE software package.
The number of principal components (PC) to be retained
was determined using the broken stick model of the scree
plot. The shape variations explained by each PC were
visualized using the PrinPrint program by drawing syn-
thetic outlines of extreme (§2SD) shell shapes. This
method was also successfully applied to examine and
describe the bivalve shell shape variability, but using dif-
ferent software (Froufe et al., 2016a; Zieritz & Aldridge,
2011; Zieritz et al., 2010). To test whether the sagittal
shell shape significantly differed between species, the
Kruskal–Wallis test as well as Mann–Whitney pairwise
test with Bonferroni corrected p-values were performed.
Statistical analysis was performed using PAST v. 3.01
(Hammer, Harper, & Ryan, 2001).
Results
Recent record of living M. homsensis with
notes on its habitat
We examined 10 river sites within the Orontes Basin in
the territory of Hatay Province of Turkey (Fig. 1 and
Appendix 1, see supplemental material online). Amongst
the sites surveyed, we observed conclusive evidence of a
living population of M. homsensis only in an upstream
section of the Karasu River in which a small-sized live
specimen (shell length of 71.4 mm) in the river and
numerous shell fragments on the shores were collected.
Fig. 2. Habitat of Margaritifera homsensis, a section of River
Karasu, lower Orontes Basin, Turkey.
4 I.V. Vikhrev et al.
The live M. homsensis specimen was buried into the silty-
sand substrate near the bridge (Fig. 2) together with five
other unionoid species, i.e.,Potomida semirugata
(Lamarck, 1819), Leguminaia wheatleyi (Lea, 1862), L.
cf. saulcyi (Bourguignat, 1852), Unio aff. caffer Krauss,
1848, and U. aff. crassus Retzius, 1788 (our unpublished
data based on DNA barcoding identification). The river
section, where pearl mussel was found, is quite different
from the downstream parts of the river and other surveyed
streams based on the well-preserved natural channel, riv-
erbed substrate, and rich vegetation alongside the river
(Fig. 2 and Appendix 1, see supplemental material
online). The fine gravel and sand, as the main substrate
types in the central part of the river channel, converted to
silty-sand substrate near the shore.
Phylogenetic affinities of M. homsensis
The partition homogeneity test revealed the congruence of
phylogenetic signals amongst almost all of the sequence
datasets, excluding those between the 28S CD3 fragment
and the 18S gene (Appendix 9, see supplemental material
online). However, we considered that the conflict between
these datasets did not affect the phylogenetic position of
M. homsensis as this conflict might reflect the lack of 28S
sequences for some taxa. The congruence between the D3
fragment and 18S gene partitions supports this suggestion.
Searches using RAxML and MrBayes based on the com-
bined dataset (six partitions: 3 codons of COI C16S C
18S C28S & D3) resulted in congruent topologies.
The resulting phylogenetic tree of Margaritiferidae is pre-
sented in Fig. 3. This phylogenetic analysis indicates that
M. homsensis is a close relative of M. auricularia (Bayes-
ian posterior probability and bootstrap support D100%).
These two species together with M. marocana belong to a
well-supported clade (Bayesian posterior probability and
bootstrap support D100%).
DNA sequence divergences and barcoding
gap analyses
The mean interspecific and intraspecific divergences
within different Margaritifera taxa are presented in
Appendices 10 and 11 (see supplemental material online).
The p-distance between M. homsensis and M. auricularia
based on nucleotide substitutions in the COI gene is com-
parable with the divergence within M. monodonta. How-
ever, there is a lack of intraspecific amino acid differences
with respect to the COI gene in almost all Margaritifera
species, excluding M. auricularia,M. middendorffi, and
M. marrianae. The p-distance between M. homsensis and
M. auricularia based on the amino-acid structure is identi-
cal to that between the two most distant haplotypes of
M. auricularia (both from River Ebro, Spain). The p-dis-
tance between M. homsensis and M. auricularia based on
the 16S rRNA gene corresponded well with the typical
intraspecific difference within Margaritifera species and
two-fold lower than that between two haplotypes of M.
falcata. In contrast, with respect to the nuclear genes, the
p-distance between M. homsensis and other species,
including M. marocana and M. auricularia, is clearly
higher than that of the intraspecific differences.
The results of the barcoding gap analyses were also
controversial (Fig. 4). According to nucleotide substitu-
tions in mtDNA genes, the p-distances between
M. homsensis and M. auricularia, with mean values of
1.5% (COI) and 0.5% (16S rRNA), fell within those of
intraspecific divergences, situated below the barcoding
gap observed in each gene (2–4% and 1.5–2.0% in COI
and 16S rRNA genes, respectively) (Figs 4.1 and 4.3). In
contrast, p-distances between M. homsensis and M. maro-
cana are much higher and correspond well to interspecific
differences (9.3% and 6.9% in COI and 16S rRNA genes,
respectively). A barcoding gap in the amino acid differen-
ces based on the COI gene is lacking (Fig. 4.2). The p-dis-
tance between M. homsensis and the haplotype AUR-2 of
M. auricularia clearly corresponded to intraspecific amino
Fig. 3. Majority rule consensus phylogenetic tree of the Margari-
tiferidae recovered from BI/ML analyses of the multi-gene
sequence dataset (six partitions: 3 codons of COI C16S C18S C
28S & D3). Numbers near branches are the BI posterior probabil-
ity/ML bootstrap support values. The outgroup taxa are omitted.
The red filling represents Margaritifera homsensis and its close
relatives: M. auricularia and M. marocana. An asterisk indicates
BPP and BS values of 95%. BPP and BS values of <50% are
omitted.
The revenant: rediscovery of Margaritifera homsensis from Orontes drainage 5
acid differences that were typical for other Margaritifera
taxa. In contrast, the p-distance between M. homsensis
and the haplotype AUR-1 of M. auricularia by the amino
acid differences is higher than that between distant spe-
cies, e.g., M. margaritifera and M. dahurica,M. laevis
and M. middendorffi,orM. margaritifera and M. falcata.
With respect to nuclear genes, the p-distances between
M. homsensis and M. auricularia were low but well above
the barcoding gap observed in 18S rDNA and the D3
expansion segment of 28S rDNA genes (Figs 4.4,4.5 and
4.6; Appendix 10, see supplemental material online). The
p-distances between M. homsensis and M. marocana also
fell within those of interspecific divergences. Sequences
of the base fragment of the 28S rDNA gene of M.
marocana and M. auricularia were not available.
Morphology and shell shape variations
The morphology of our specimens from Hatay is similar
to the morphology of M. homsensis,M. auricularia, and
M. marocana (Fig. 5). The shell is oval-elongate in shape,
with a roundish and shortened anterior part and an elon-
gated and rounded posterior margin. The dorsal margin is
straight, forming a visible ridge with the anterior margin.
The ventral margin is slightly curved. The curvature of
the shell appeared during the last few years, as growth
lines demonstrate. Umbos are well projected. A deep ser-
rated triangular cardinal tooth and well-pronounced ser-
rated lateral tooth were observed in the right valve. Two
serrated cardinal teeth were observed in the left valve.
The anterior one was smaller and pointed; the posterior
one was larger with a rotund top. Two well-developed lat-
eral teeth were also observed in the left valve. Mantle
attachment scars are weak and few in number.
Fig. 4. Barcoding gap analyses for Margaritiferidae taxa (uncor-
rected p-distance, %). 4.1 – COI (nucleotide substitutions). 4.2 –
COI (amino acid differences). 4.3 – 16S rRNA. 4.4 – 18S rDNA.
4.5 – D3 expansion fragment of 28S rDNA. 4.6 – Base fragment
of 28S rDNA. The red arrow indicates barcoding gap (the gap
between intraspecific and interspecific distances for all taxa
except Margaritifera homsensis). The barcoding gap in the fre-
quency distribution of p-distances from between amino acid
sequences of the COI gene is not detected. The single asterisk
reveals an interval containing the distance between M. homsensis
and M. auricularia. The double asterisk indicates an interval
containing the distance between M. homsensis and M. marocana.
Sequences of base fragment of 28S rDNA for M. auricularia and
M. marocana were not available.
Fig. 5. Shell morphology of Margaritifera homsensis,M. auricularia,andM. marocana.5.1 M. auricularia from the La Garonne River,
France (MNCN 15.07/178). 5.2 M. homsensis from the Orontes River, Syria (NHMUK 1936.3.10.3). 5.3 M. marocana from the Darna
River, Taghzirt, Morocco (SMF 83158/2) 5.4 M. auricularia, Europe (MNCN 15.07/1437). 5.5 M. homsensis from the Karasu River, a trib-
utary of lower Orontes, Hatay Province, Turkey (RMBH biv0176). 5.6 M. marocana from the Laabid river, Morocco. Scale bar – 2 cm.
6 I.V. Vikhrev et al.
Two significant principal components were obtained
using PCA with 20 EFDs. PC1 and PC2 explained
63.87% and 18.95% of the total variance of sagittal shell
shape, respectively. Significant differences between ana-
lysed species were observed for both PCs according to
Kruskal–Wallis test (PC1: x
2
D31.47, nD72, P<
0.0001; PC2: x
2
D7.90, nD72, P<0.01). Paired Mann–
Whitney test with Bonferroni corrected P-values revealed
that significant differences within pairs M. auricularia/M.
homsensis and M. auricularia/M. marocana for PC2 are
lacking. The two morphotypes for the overall shell shape
(trapezoid and ovate) were determined, as the synthetic
outlines of the extreme shell forms illustrate (Fig. 6). First
and second components reflect trends from oblique dorso-
posterior margin to elevated umbonal ridge and slightly
curved ventral margin to straight ventral margin. In addi-
tion, PC1 is associated with a trend from a high trapezoi-
dal shell to a relatively narrow ovate shell, while PC2,
conversely, is associated with trend from a narrow trape-
zoidal shell to a relatively high ovate shell.
The clustering results, showing M. auricularia versus M.
homsensis versus M. marocana in the scatter plot (Fig. 6),
revealed that both morphotypes and transitional forms
were observed amongst species. Moreover, the second
component, explaining near to 20% of the shell shape vari-
ability, did not indicate significant differences between
M. auricularia and two other taxa. The only difference
between species in the scatter plot was that shells of
M. marocana are associated, in general, with the morpho-
type with well-developed umbonal ridge. The live speci-
men of M. homsensis from the Karasu River belongs to
ovate morphotype (Fig. 5.5) characterized by an elevated
umbonal ridge, straight ventral margin and rounded poste-
rior and anterior parts. An example of a M. homsensis indi-
vidual, with a shell shape belonging to the trapezoid
morphotype, with oblique upper part of the posterior edge,
a practically disappearing umbonal ridge, a curved ventral
margin and a pointed anterior part is presented in Fig. 6.2.
The museum specimens of M. auricularia also correspond
to these two primary morphotypes (Figs 5.1 and 5.4)while
the trapezoid morphotype is prevailing amongst small and
large individuals of M. marocana (Figs 5.3 and 5.6).
Discussion
Taxonomic implications
The mtDNA phylogeny clearly indicated that M. homsen-
sis is an intraspecific form of M. auricularia, which is
partly consistent with the hypotheses of Kinzelbach and
Roth (1984), Nesemann (1993), Sch€
utt (1987), and Smith
(2001). In contrast, we observed M. homsensis as a close
relative of both M. auricularia and M. marocana based on
the two nuclear genes, which agrees with suggestions of
Pallary (1927,1928), who found that it resembled North
African margaritiferids. The results of phylogenetic analy-
ses were confirmed by using a barcoding gap approach.
Based on these data, we concluded that M. homsensis is a
valid species that is most closely related to M. auricularia,
but both of these species are closely related to M. maro-
cana by nuclear genes. These three species are representa-
tives of the subgenus Pseudunio Haas (1910), as Bolotov
et al. (2016) already suggested.
The low mtDNA divergence between these taxa, despite
the relatively high distance within nuclear genes, is a pecu-
liar feature compared with the majority of other animals in
which mtDNA typically evolves more rapidly than nuclear
DNA (Hellberg, 2006; Shearer, Van Oppen, Romano, &
W€
orheide, 2002). In contrast, anthozoans (corals, sea fans,
and their relatives) are the only known examples within the
animal realm with mtDNA that evolves slower than the
nuclear genome (Hellberg, 2006), consistent with the pres-
ent findings inferred from the M. homsensis and M. auricu-
laria species pair. To our knowledge, this is the first case
of a mismatch in the divergence level of mitochondrial and
nuclear genes in Mollusca recorded to date. This finding is
of particular importance in the context of applying the
results of DNA barcoding using the COI gene to a taxo-
nomic revision of molluscs. Within the framework of an
integrative taxonomic approach, nuclear markers should be
used together with mtDNA data, as the direct application
of divergence values inferred only from mitochondrial
genes into taxonomy might lead to inaccurate taxonomic
solutions on the status of certain taxa.
Another possible explanation of this mismatch is an
ancient introgressive hybridization event between the two
Fig. 6. Principal component scores for the first two PC axes
obtained by using PCA on the Fourier coefficients of the shell
shape of Margaritifera homsensis,M. auricularia, and M. maro-
cana. Synthetic shell outlines of ‘extreme’ morphotypes are dis-
played with the posterior margin facing to the right and the
dorsal margin to the top of the page. The black dots indicate M.
homsensis, red dots indicate M. auricularia and light green dots
indicate M. marocana. A 95% confidence ellipse is shown for
each species.
The revenant: rediscovery of Margaritifera homsensis from Orontes drainage 7
different Margaritifera species. Introgressive hybrids
were discovered in certain marine mussel taxa, e.g., Myti-
lus (Gosset & Bierne, 2013; Doherty, Brophy, & Gosling,
2009; Fra€
ısse, Belkhir, Welch, & Bierne, 2016; Gosset &
Roux et al., 2014), albeit interspecific hybridization is
almost unknown in unionoideans (Ferguson, 2009; Hoeh,
Stewart, & Guttman, 2002).
Such a close phylogenetic relationship of M. homsensis
with two other species, M. auricularia and M. marocana,
is reflected in similar conchological features amongst taxa.
There are no clear, reliable shell characteristics for distin-
guishing M. homsensis from M. auricularia.Smith(2001)
separated these species based on shell shape, but this fea-
ture might overlap, as PCA reveals (Fig. 6). The PCA scat-
ter plot on PC1 vs. PC2 (Fig. 6) shows that the shells of M.
homsensis and M. auricularia are highly variable, ranging
from trapezoid to ovate morphotypes and belong to the
same cluster. Distinguishing M. homsensis from M. maro-
cana could be based on shell form, as the former is primar-
ily associated with the trapezoid morphotype, but not in all
cases (Fig. 6). Interestingly, PC2 explains near to 20% of
the variation in shell shape, but only M. homsensis and M.
marocana show significant differences based on this com-
ponent. We assumed high age-dependent variability in
shell shape from an ovate morphotype in small individuals
to a trapezoidal morphotype in large specimens. Neverthe-
less, this trend is more common for shell shapes of M.
auricularia and M. homsensis, whereas the ovate morpho-
type with well-developed umbonal ridge is spread amongst
M. marocana’s shells of different size.
In general, these findings indicate that shell shape partially
supports species delimitation but cannot be used for reliable
species identification, as the features overlap. Moreover, this
feature could not be considered to be reliable, reflecting the
observed high variability. Multiple studies have justified that
shell shape is highly dependent on environment, sex, ontoge-
netic growth, and other variables (Froufe et al., 2016a;Ort-
man, 1920; Rufino et al., 2013; Watters, 1994; Widarto,
2007; Zieritz & Aldridge, 2011; Zieritz et al., 2010). More-
over, the Mediterranean pearl mussel taxa, i.e.,M. homsen-
sis,M. auricularia,andM. marocana, could be considered
to be cryptic species as the former two species also have no
clear conchological differences (Ara
ujo et al., 2009).
Distribution range and ecology
With respect to the results obtained in the present study,
M. homsensis is a true endemic taxon of the Orontes Basin
and probably some of the nearby smaller river systems,
particularly the Nahr-el-Kabir River and, apparently, the
Litani River (Fig. 1). In the description of Unio episcopa-
lis Tristram, 1865, a synonym of M. homsensis, Tristram
(1865: 544) stated “This, the prince of Oriental Unioni-
dae, is not uncommon in the Orontes. I found a dead valve
by the Leontes [old name of the Litani River], but did not
meet with it in the Lake of Galilee”, clearly indicating the
occurrence of the species in the Litani River, southern
Lebanon. A Unio episcopalis specimen from Tristram’s
collection is stored in NHMUK (no. 1936-3-10-3),
labelled “River Orontes”, but it is most likely another
specimen collected from the Orontes Basin, as this species
possesses a shell with both valves. Additionally, Tristram
(1865) clearly separated M. homsensis from P. semirugata
because the latter taxon was described as Unio simonis
Tristram, 1865. However, further studies are needed to
confirm the distribution of M. homsensis in the Litani
River, as there are no other records from that basin. The
majority of the collected specimens of M. homsensis origi-
nated from the Nahr-el-Kabir River on the Lebanon-
Syrian border (Appendix 1, see supplemental material
online) and from middle Orontes near Homs, Syria
(Sch€
utt, 1983,1987). Sch€
utt (1983,1987) reported only
two available records for lower Orontes ( DAsi) in Tur-
key, one of which is the Karasu River.
Kinzelbach and Roth (1984) assumed that M. homsensis
and M. auricularia originated from a common ancestor
from Paleo-Danube Basin, distributed from the lower
Danube through the Vardar depression and the Egean
lakes into the Orontes system during the Pliocene (Fig. 7).
This assumption is consistent with palaeontological data
because M. flabellata was the most abundant naiad species
in the Paleo-Danube system during the Neogene
(Nesemann, 1993). With respect to the close phylogenetic
relationships between these taxa and to the data on low
evolutionary rates in Margaritiferidae (Bolotov et al.,
2016), such a scenario is probably correct but needs to be
confirmed using a molecular clock approach.
According to published data, M. marocana and M.
auricularia prefer gravel-sandy substrates (Ara
ujo et al.,
2009;G
omez & Araujo, 2008; Sousa et al., 2016). How-
ever, M. auricularia might occasionally survive in mud
sediments, as reported for adult specimens in Canal Impe-
rial, Ebro Basin, Spain (Gomez & Araujo, 2008). We
observed a M. homsensis specimen at the small sand-silty
site of the Karasu River. This patch of the main channel is
most likely the appropriate place for mussels inhabiting
this section of the river. Numerous fragments of empty
shells indicate that the colony of M. homsensis is poten-
tially located upstream, but the upstream section of the
river is under military control and impossible to survey, as
it is situated along the Syria border.
The host fishes of M. auricularia glochidia are stur-
geons and blennies, but the hosts of other Mediterranean
pearl mussel taxa are unknown (Araujo, Bragado, &
Ramos, 2001). The river blenny (Salaria fluviatilis)isa
circum-Mediterranean species (Almada et al., 2009),
which also inhabits tributaries of the Orontes River (Okur
& Yal¸cın-
€
Ozdilek, 2008) and appears to be the first candi-
date as the host of M. homsensis glochidia.
8 I.V. Vikhrev et al.
Conservation notes
The occurrence of a population of endemic M. homsensis
in the Karasu River supports the high value of southern
Turkey as one of the main four ‘classic refugia’ for fresh-
water fauna in the Mediterranean region (Froufe et al.,
2016b). To our knowledge, Karasu is the only known river
that supports a living population of M. homsensis. This
stream flows through this area which is under strong
anthropogenic impact, reflecting intensive agriculture and
deficient water resources, while the regular irrigation
affects the water level, which dramatically decreases dur-
ing the summer season (Dr T. Ozcan, pers. comm.). The
middle course of the Karasu River, upstream of the
recorded population of M. homsensis, defines the Turkey–
Syrian border, which is a closed area as a result of the dif-
ficult social and political situation in this transboundary
region. Such an indirect preservation of the natural vege-
tation and freshwater habitats along this watercourse
could facilitate the survival of M. homsensis. However,
special conservation efforts for the discovered population
of M. homsensis in Turkey, including the formation of a
nature reserve, might contribute to the conservation of
this species.
Many open questions remain unanswered, including an
evaluation of the abundance of M. homsensis in Turkey
and within Orontes Basin as a whole, existence of this spe-
cies in smaller river drainages, estimation of the genetic
diversity of this species and studies of the biological
features, e.g., the spawning time and period of glochidia
metamorphosis. The host fish identification is one of the
primary goals of future investigations of M. homsensis,as
this information is urgently needed for the conservation
management of this local endemic species. Moreover, an
extensive search for the surviving populations in Orontes
drainage (southern Turkey, Lebanon and Syria) is needed
to develop a transboundary conservation strategy for this
unique taxon. The middle Orontes (e.g., Lake Homs) and
the Nahr-el-Kabir River should be considered the highest
priority watercourses for further studies.
Acknowledgements
The fieldwork was performed under the Agreement on
Scientific Cooperation between Iskenderun Technical
University (Turkey) and Federal Centre for Integrated
Arctic Research of Russian Academy of Sciences
(Russian Federation). We are grateful to Dr R. Janssen
and Dr K.-O. Nagel (SNM, Frankfurt, Germany),
Dr J. Ablett (NHMUK, London, UK), Dr P. Bouchet and
Dr V. H
eros (MNHN, Paris, France), and Dr R. Araujo
(MNCN, Madrid, Spain) for their generous assistance in
morphological studies of discussed taxa. Our special
thanks go to Dr M. Lopes-Lima and Dr K. Nakamura who
provided photographs of shells of M. marocana and M.
auricularia for this paper.
40°E
30°E
30°E
20°E
20°E
10°E
10°E
0°
0°10°W
40°N
40°N
30°N
0300 600 900150
Kilometers
1
2
3
4
Fig. 7. Past and modern ranges of Margaritifera spp. in the Mediterranean region. The distribution of each recent species is illustrated
based on the corresponding river drainages. 1 – M. auricularia (range data: Lopes-Lima et al., 2016); 2 – M. homsensis (range data:
Fig. 1); 3 – M. marocana (range data: Ara
ujo et al., 2009; Sousa et al., 2016); 4 – Danube Basin, which harbours abundant populations
of M. flabellata, a potential ancestral lineage of M. auricularia and M. homsensis, during the Neogene (Nesemann, 1993).
The revenant: rediscovery of Margaritifera homsensis from Orontes drainage 9
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Ministry of Education and
Science of the Russian Federation [grant number
6.2343.2017], Russian Federal Agency for Scientific
Organizations [grant number 0409-2016-0022,0410-2014-
0028], Grant Council of the President of Russia [grant
number MD-7660.2016.5] and Russian Foundation for
Basic Research [grant number 16-34-60152 mol_a_dk].
Supplemental data
Supplemental data for this article can be accessed here: https://
doi.org/10.1080/14772000.2017.1343876.
ORCID
Ivan N. Bolotov http://orcid.org/0000-0002-3878-4192
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Associate Editor: Joanne Porter
12 I.V. Vikhrev et al.
