Systematics, biogeography and evolution of Asaccus gallagheri (Squamata, Phyllodactylidae) with the description of a new endemic species from Oman

Abstract

The Hajar Mountains are the highest mountain range in eastern Arabia. Despite being classified as a mountain desert, it is considered one of the top biodiversity hotspots of Arabia. As a result of its relatively old geological origin, complex topography, environmental heterogeneity and geographic isolation from other mountain ranges, its fauna and flora have diversified significantly producing high levels of endemicity, particularly amongst reptiles. Several genetic studies indicate that this diversity may still be underestimated, especially within some groups containing morphologically similar species like the nocturnal geckos of the genus Asaccus. These have radiated extensively on both sides of the Gulf of Oman, in the Hajar Mountains and the Zagros Mountains of south-west Asia, and are a good example of the faunal affinities between these two mountain ranges. In the present work, we analyse A. gallagheri, the smallest species of the Arabian radiation, using an unprecedented sampling across its entire distribution range and an integrative approach combining morphological, macroecological and multilocus molecular data with the objective of clarifying its systematics and phylogeography. The results support the presence of two allopatric species within A. gallagheri that split approximately 6 Ma. The newly discovered species is endemic to the Eastern Hajars and is described herein mainly on the basis of its smaller size and high genetic divergence from A. gallagheri. The molecular analyses also uncovered remarkable levels of genetic diversity within both species. The present study highlights the diversity of the genus Asaccus in south-east Arabia and stresses its relevance from a conservation point of view.
http://www.zoobank.org/urn:lsid:zoobank.org:pub:62EB3146-9F79-4857-8CC6-36FE235D84D4

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Systematics and Biodiversity
ISSN: 1477-2000 (Print) 1478-0933 (Online) Journal homepage: http://www.tandfonline.com/loi/tsab20
Systematics, biogeography and evolution of
Asaccus gallagheri (Squamata, Phyllodactylidae)
with the description of a new endemic species
from Oman
Marc SimÓ-Riudalbas, Pedro Tarroso, Theodore Papenfuss, Thuraya Al-Sariri
& Salvador Carranza
To cite this article: Marc SimÓ-Riudalbas, Pedro Tarroso, Theodore Papenfuss, Thuraya
Al-Sariri & Salvador Carranza (2017): Systematics, biogeography and evolution of Asaccus
gallagheri (Squamata, Phyllodactylidae) with the description of a new endemic species from Oman,
Systematics and Biodiversity, DOI: 10.1080/14772000.2017.1403496
To link to this article: https://doi.org/10.1080/14772000.2017.1403496
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Research Article
Systematics, biogeography and evolution of Asaccus gallagheri
(Squamata, Phyllodactylidae) with the description of a new endemic
species from Oman
MARC SIM

O-RIUDALBAS
1
, PEDRO TARROSO
1,2
, THEODORE PAPENFUSS
3
, THURAYA AL-SARIRI
4
& SALVADOR CARRANZA
1
1
Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Mar
ıtim de la Barceloneta 37-49, 08003 Barcelona, Spain
2
CIBIO/InBIO, Research Centre in Biodiversity and Genetic Resources, Universidade do Porto, Campus Agr
ario de Vair~
ao, Rua Padre
Armando Quintas, 4485-661 Vair~
ao, Vila do Conde, Portugal
3
Department of Integrative Biology, Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720, USA
4
Ministry of Environment and Climate Affairs, Thaqafah Street, 100, Muscat, Oman
(Received 20 May 2017; accepted 1 November 2017; published online 15 December 2017)
The Hajar Mountains are the highest mountain range in eastern Arabia. Despite being classified as a mountain desert, it is
considered one of the top biodiversity hotspots of Arabia. As a result of its relatively old geological origin, complex
topography, environmental heterogeneity and geographic isolation from other mountain ranges, its fauna and flora have
diversified significantly producing high levels of endemicity, particularly amongst reptiles. Several genetic studies indicate
that this diversity may still be underestimated, especially within some groups containing morphologically similar species
like the nocturnal geckos of the genus Asaccus. These have radiated extensively on both sides of the Gulf of Oman, in the
Hajar Mountains and the Zagros Mountains of south-west Asia, and are a good example of the faunal affinities between
these two mountain ranges. In the present work, we analyse A. gallagheri, the smallest species of the Arabian radiation,
using an unprecedented sampling across its entire distribution range and an integrative approach combining morphological,
macroecological and multilocus molecular data with the objective of clarifying its systematics and phylogeography. The
results support the presence of two allopatric species within A. gallagheri that split approximately 6 Ma. The newly
discovered species is endemic to the Eastern Hajars and is described herein mainly on the basis of its smaller size and high
genetic divergence from A. gallagheri. The molecular analyses also uncovered remarkable levels of genetic diversity
within both species. The present study highlights the diversity of the genus Asaccus in south-east Arabia and stresses its
relevance from a conservation point of view.
http://www.zoobank.org/urn:lsid:zoobank.org:pub:62EB3146-9F79-4857-8CC6-36FE235D84D4
Key words: Arabia, biogeography, endemicity, geckos, Hajar Mountains, hypervolumes, species delimitation, taxonomy
Introduction
The Hajar Mountains from south-eastern Arabia form a con-
tinuous range that runs for about 650 km alongside the coast
of the Gulf of Oman; from the Musandam Peninsula and
Ruus al Jibal in the north to the Jebel Qahwan in the south-
east. Most of the range is within the Sultanate of Oman but
a small area, just south of the Musandam Governorate,
belongs to the United Arab Emirates, UAE (Fig. 1.1). Cut
by deep canyons or wadis, these arid mountains have a com-
plex topography and can be divided into three distinct areas,
the Western Hajars, the Jebel Akhdar and the Eastern Hajars
(see Fig. 1.1). With a maximum elevation of 3,018 m above
sea level (a.s.l.), the Jebel Shams in the Jebel Akhdar is the
highest peak of the Hajar Mountains, although high peaks
also occur in the Western (2,087 m a.s.l. at Jebel Harim) and
Eastern (2,200 m a.s.l. at Jebel Khadar) Hajars. Despite
being the only area in eastern Arabia with habitats above
2,000 m in elevation and relatively low annual mean tem-
peratures, average precipitation estimates are below
300 mm over much of its range, evapotranspiration is high
and vegetation is very scarce. As a result of that, the Hajar
Mountains are often considered a mountain desert (Edgell,
2006; Mandaville, 1977). Similar to other mountain ranges
Correspondence to: Salvador Carranza. Email: salvador.
carranza@ibe.upf-csic.es
ISSN 1477-2000 print/ 1478-0933 online
ÓThe Trustees of the Natural History Museum, London 2017. All Rights Reserved.
http://dx.doi.org/10.1080/14772000.2017.1403496
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2 M. Sim
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in the Arabian Peninsula such as the Western Mountains of
Yemen and Saudi Arabia, orogeny in the Hajar Mountains
was triggered by the opening of the Gulf of Aden in the Oli-
gocene and continued well into the Miocene, when the latest
phase of plate tectonics affected Oman (Bosworth, Huchon,
&McClay,2005; Glennie, 2006). As a result of its relatively
old geological origin, complex topography, diversity of hab-
itats with local microclimates and geographic isolation from
other mountain areas in Arabia, the Hajar Mountain range is
one of the most biodiverse areas of the whole Arabian Pen-
insula, with high levels of endemicity in reptiles as well as
in other animal and plant groups (Brinkmann, Patzelt, Dick-
hoefer, Schlecht, & Buerkert, 2009; Gardner, 2013; Ghazan-
far, 1991; Mandaville, 1977). Since the first biodiversity
surveys carried out in the mountains in the 1970s, reptiles
have received increasing attention as a result of their abun-
dance and unexpected diversity. With the description of sev-
eral new species, they became, by far, the vertebrate group
with the highest number of endemic species in the Hajar
Mountains (Arnold, 1972,1977,1986; Arnold & Gallagher,
1977; Arnold & Gardner, 1994;Gardner,1994,1999; Gas-
peretti, 1988). More recently, molecular studies have shown
that reptile diversity in the Hajar Mountains was still under-
estimated, with some gecko groups of the genera Hemidac-
tylus (Carranza & Arnold 2012), Pristurus (Badiane et al.,
2014; Garcia-Porta, Sim
o-Riudalbas, Robinson, & Carra-
nza, 2017), Ptyodactylus (Metallinou et al., 2015;Sim

o-Riu-
dalbas et al., 2017), Trachydactylus (de Pous et al., 2016a)
and Asaccus (Carranza, Sim
o-Riudalbas, Jayasinghe,
Wilms, & Els, 2016; Papenfuss et al., 2010) harbouring sev-
eral cryptic species and deep lineages, some of them with
restricted distributions of just a few kilometres. From all the
reptile genera of the Hajar Mountains, Asaccus Dixon &
Anderson, 1973 is the one with the highest number of
endemics (see below) and a good example of the faunal
affinities between the Hajar Mountains in Arabia and the
Zagros Mountains in Iran (Carranza et al., 2016;Papenfuss
et al., 2010; Torki, Ahmadzadeh, Ilgaz, Avcı, & Kumluta¸s,
2011a; Uetz, Goll, & Hallerman, 2017). In a similar way,
closely related taxa and populations from various reptile
groups are present on both sides of the Gulf of Oman and
provide an excellent scenario for reconstructing their
evolutionary relationships and to explore the biogeography
of the region (Dakhteh, Kami, & Anderson, 2007; de Pous
et al., 2016b; Kapli et al., 2008,2015; Krause, Ahmadzadeh,
Moazeni, Wagner, & Wilms, 2013; Metallinou et al., 2012,
2014; Yousofi, Rastegar-Pouyani, & Hojati, 2015).
Commonly known as South-west Asian leaf-toed geckos,
the genus Asaccus (previously part of Phyllodactylus Grey,
1828) is one of the least known genera of the family Phyllo-
dactylidae. All of them are small to medium size, slender,
nocturnal, rock climbing geckos with paired terminal scan-
sors in the digits without lamellae and with no femoral or
preanal pores, cloacal sacs, postanal bones and left oviduct.
By now, 18 species are recognized within the genus, most
of them described in the last decade. A total of 12 species
are found in south-east Anatolia, east Syria, east Iraq and
western Iran (west Zagros Mountains), while the other six
species occur in south-east Arabia, along the Hajar Moun-
tains: Asaccus gallagheri (Arnold, 1972), A.montanus
Gardner, 1994,A. platyrhynchus Arnold & Gardner, 1994,
A. caudivolvulus Arnold & Gardner, 1994 and the two
recently described A. gardneri Carranza et al., 2016 and A.
margaritae Carranza et al., 2016.
Asaccus gallagheri is a small, delicately built gecko
found in stony substrates in wadis and on small cliffs and
boulders on open hillsides. It is currently distributed across
the Hajar Mountains, from sea level up to 1,900 m a.s.l. in
the Jebel Akhdar (pers. obs.). Asaccus gallagheri was
described by Arnold (1972) based on a single juvenile
specimen from Masafi (UAE; locality 14 in Fig. 1.1).
Given the lack of material in the original description, the
species was later revised including more material collected
within 30 km of the type locality (Arnold, 1977). The first
phylogenetic relationships carried out by Arnold and Gard-
ner (1994) using 16 morphological characters suggested a
sister taxa relationship between A. gallagheri and A. platyr-
hynchus, supported mainly by the absence of cloacal
tubercles, by the similar dorsal colour pattern and the pres-
ence of sexually dimorphic tail colouration. A recent
molecular phylogenetic analysis by Papenfuss, Jackman,
Bauer, Stuart, Robinson, and Parham (2010)alsorecovered
the clade formed by A. gallagheri and A. platyrhynchus
and revealed a high level of genetic variability between
Fig. 1. Geographic distribution and phylogenetic relationships.
(1.1) Map of the Hajar Mountains showing the localities of the examined material (see Table S1, see supplemental material online). Type
locality of Asaccus arnoldi sp. nov. indicated by a star. Localities are coloured corresponding to the five mitochondrial lineages identi-
fied (see Fig. 1.2). Maps were drawn using QGIS v.2.8 (available at http://www.qgis.org; digital elevation model freely available at
http://earthobservatory.nasa.gov/); (1.2) Bayesian inference tree of 10 Asaccus species based on the concatenated sequences of two mito-
chondrial (12S and cytb) and two nuclear (c-mos and MC1R) genes. Black dots indicate posterior probability values 0.95 and bootstrap
values 70% are shown next to the nodes (see Fig. S1, see supplemental material online). Age estimates are in italics below the nodes
and include the mean and the HPD 95% confidence interval in brackets; (1.3) Bayesian inference tree of 78 Asaccus based on the same
concatenated genes. Black dots indicate posterior probability values 0.95 and bootstrap values 70% are shown next to the nodes.
Each sequence is labelled with the specimen code followed by the locality code in square brackets (see Fig. 1.1). Colour bars correspond
to the five mitochondrial deep lineages. Detailed information on the samples included in both phylogenetic trees is given in Table S1
(see supplemental material online).
J
Systematics of Asaccus gallagheri 3
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specimens of A. gallagheri from Nizwa in the Jebel Akhdar
and Khasab in the Musandam Peninsula (Fig. 1.1), suggest-
ing that A. gallagheri might include more than one species.
However, the lack of material from the type locality of A.
gallagheri in the UAE as well as other localities across the
distribution range, as for instance the Eastern Hajar Moun-
tains, precluded any taxonomic conclusions.
The objectives of the present work were: (1) to obtain
samples across the whole distribution range of Asaccus
gallagheri in the Hajar Mountains of south-eastern Ara-
bia; (2) to use phylogenetic tools together with species
delimitation algorithms to cluster the samples into line-
ages and to reconstruct their evolutionary relationships
and phylogeography; (3) to contrast the molecular differ-
entiation with measured overlap in morphology and niche;
and (4) to use the information from all these different lines
of evidence to revise the taxonomy of Asaccus gallagheri.
Materials and methods
DNA extraction and sequencing
The molecular study included a total of 86 specimens of
Asaccus and two specimens of the genus Haemodracon,
endemic to the Socotra Archipelago. A list of all individu-
als with their taxonomic identification, sample and
voucher codes, geographic distribution and GenBank
accession numbers is presented in Table S1 (see online
supplemental material, which is available from the
article’s Taylor & Francis Online page at https://doi.org/
10.1080/14772000.2017.1403496). Total genomic DNA
was isolated from ethanol preserved tissue samples using
the SpeedTools Tissue DNA Extraction kit (Biotools,
Madrid, Spain) following the manufacturer’s protocol. All
specimens were sequenced for both strands for two mito-
chondrial gene fragments: the ribosomal 12S rDNA (12S)
and the cytochrome b(cytb), plus two nuclear gene frag-
ments: the oocyte maturation factor MOS (c-mos) and the
melanocortin 1 receptor (MC1R). Primers and PCR condi-
tions used for the amplification of all fragments are shown
in Table S2 (see supplemental material online).
Geneious Pro v. 9.0.5 (Biomatters Ltd) was used for
assembling and editing the chromatographs manually. All
coding gene fragments started by the first codon position
were translated into amino acids to validate the correct read-
ing frame. For the nuclear coding gene fragments, heterozy-
gous positions were identified and coded in both alleles
according to IUPAC ambiguity codes. Multiple sequence
alignments were performed with the online application of
MAFFT v.7 (Katoh & Standley, 2013) with default parame-
ters (Auto strategy, Gap opening penalty: 1.53, Offset value:
0.0). For the 12S ribosomal fragment the Q-INS-i strategy
was applied, in which the secondary structure of the RNA
was considered. SeqPHASE (Flot, 2010) was used to con-
vert the input files and the software PHASE v. 2.1.1 was
used to reconstruct the gametic haplotypes (Stephens,
Smith, & Donnelly, 2001) using default settings except for
the phase probability threshold that was set to 0.7 (see Har-
rigan, Mazza, & Sorenson, 2008). Phased sequences of the
nuclear genes were used for the allele network reconstruc-
tion (see below). Inter- and intra-specific uncorrected p-dis-
tances with pairwise deletion were estimated independently
for both mitochondrial gene fragments using MEGA v.7
(Kumar, Stecher, & Tamura, 2016).
Phylogenetic analyses and ancestral area
reconstruction
A total of three datasets were used in the phylogenetic anal-
yses. Dataset 1 was assembled to infer the phylogenetic
relationships and divergence times amongst Asaccus species
and consisted of 12 terminals including one representative
of A. gallagheri, one from the new species described herein
plus all species of Asaccus available in GenBank: five from
the Hajar Mountains of Arabia (A. caudivolvulus,A. monta-
nus,A. platyrhynchus,A. margaritae and A. gardneri)and
three from the Zagros Mountains (A. griseonotus,A. elisae
and A. nasrullahi). Moreover, one Haemodracon riebeckii
and one H. trachyrhinus were used as outgroups based on
published evidence (Gamble, Bauer, Greenbaum, & Jack-
man, 2008, Gamble, et al., 2011; Gamble, Greenbaum,
Jackman, Russell, & Bauer, 2012; Gamble, Greenbaum,
Jackman, & Bauer, 2015; Garcia-Porta, Morales, G
omez-
D

ıaz, Sindaco, & Carranza, 2016; Garcia-Porta, Sim
o-
Riudalbas, Robinson, & Carranza, 2017). The dataset was
missing nine species from the Zagros Mountains described
based only on morphological evidence: A. andersoni,A.
barani,A. granularis,A. iranicus,A. kermanshahensis,A.
kurdistanensis,A. saffinae,A. tangestanensis and A. zagro-
sicus. Dataset 2 was assembled with the aim of studying in
detail the phylogeographic relationships between the popu-
lations previously described as Asaccus gallagheri. This
dataset consisted of 78 specimens collected from 32 locali-
ties distributed across the Hajar Mountains of Oman and the
UAE (63 A. gallagheri and 15 specimens of the new species
described herein) plus one A. platyrhynchus used as out-
group in the ML analyses. Dataset 1 and 2 included two
concatenated alignments of 1,880 and 1,872 bp (base pair)
respectively; 401 and 393 bp of 12S;399bpofcytb; 414 bp
of c-mos and 666 of MC1R. Dataset 3 was used for prelimi-
nary species delimitation and included 26 unique mitochon-
drial haplotypes from dataset 2 (only 12S sequences).
Datasets 1 and 2 were analysed with maximum likeli-
hood (ML) and Bayesian inference (BI) methods, whereas
dataset 3 was only analysed with BI. The best-fit partition-
ing scheme and models of molecular evolution for all
datasets were selected with PartitionFinder v.2. (Lanfear,
Frandsen, Wright, Senfeld, & Calcott, 2016) with the fol-
lowing settings: branch lengths linked, only models
4 M. Sim
o-Riudalbas et al.
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available in BEAST evaluated, initial partitions by gene,
BIC model selection criterion applied and all partition
schemes analysed. The partition scheme and models of
sequence evolution selected were 12S Ccytb, GTR CIC
G; c-mos, HKI CI; MC1R, HKY CI for dataset 1, 12S,
HKY CG; cytb, HKY CG; c-mos CMC1R, TrNCICG
for dataset 2 and 12S Ccytb, GTR CG for dataset 3. ML
analyses of datasets 1 and 2 were performed in RAxML
v.7.4.2 (Stamatakis, 2006) as implemented in raxmlGUI
(Silvestro & Michalak, 2012) with 100 tree searches,
using the GTRCG model of sequence evolution and inde-
pendent model parameters for the three partitions (see
above). Reliability of the ML tree was assessed by boot-
strap analysis (Felsenstein, 1985) including 1,000 repli-
cates. The software BEAST v.1.8.0 (Drummond, Suchard,
Xie, & Rambaut, 2012) was used for BI and dating analy-
ses. Two individual runs of 5 £10
7
generations were car-
ried out for datasets 1–3, sampling at intervals of 10,000
generations. The following models and prior specifica-
tions were applied, otherwise by default: models of
sequence evolution for the different partitions as selected
by PartitionFinder (see above); Speciation Yule (dataset
1) and Coalescent Constant Size (datasets 2 and 3) tree
prior; uncorrelated lognormal clock for mitochondrial
genes and strict clock for nuclear ones; random starting
tree; base substitution prior Uniform (0,100); alpha prior
Uniform (0,10). Substitution and clock models were
unlinked and the xml file was manually modified to set
Ambiguities D‘‘true’’ for the nuclear gene partitions in
order to account for variability in the heterozygous posi-
tions, instead of treating them as missing data. Posterior
trace plots and effective sample sizes (ESS) of the runs
were monitored in Tracer v1.6 (Rambaut, Suchard, Xie,
& Drummond, 2014) to ensure convergence. The results
of the individual runs were combined in LogCombiner
discarding 10% of the samples and the maximum clade
credibility (MCC) ultrametric tree was produced with
TreeAnnotator (both provided with the BEAST package).
Absolute divergence times were estimated from dataset 1
using BEAST with models and prior specifications as
above and applying previously calculated mean rates of
molecular evolution for the two mitochondrial markers
12S (mean: 0.00755, S.D.: 0.00247) and cytb (mean:
0.0228, S.D.: 0.00806) (Carranza & Arnold, 2012).
Despite the problems associated with using evolutionary
rates from other organisms for time tree calibration, the
rates inferred by Carranza and Arnold (2012) and applied
herein correspond with the rates obtained in other inde-
pendent studies that used different calibration points and
different taxa (Garcia-Porta et al., 2017; Metallinou et al.,
2012; Sindaco et al., 2012). Indeed, the rates by Carranza
and Arnold (2012) have been applied to several different
studies for which reliable internal calibration points based
on biogeographic events or fossil evidence do not exist
(Carranza et al., 2016; de Pous et al., 2016a;G

omez-D
ıaz,
Sindaco, Pupin, Fasola, & Carranza, 2012; Hawlitschek &
Glaw, 2013; Metallinou & Carranza, 2013; Metallinou
et al., 2015; Mil
a, Surget-Groba, Heulin, Gos
a, & Fitze,
2013; Sim
o-Riudalbas et al., 2017;

Sm
ıd et al., 2013;
Tamar et al., 2016a; Vasconcelos & Carranza, 2014).
Tree nodes were considered strongly supported if they
received ML bootstrap values 70% and posterior proba-
bility (pp) support values 0.95 (Huelsenbeck & Rannala,
2004; Wilcox, Zwickl, Heath, & Hillis, 2002).
With the aim of reconstructing the phylogeographic
history and inferring the ancestral origin of Asaccus galla-
gheri and the new species described herein, we used the
Bayesian Stochastic Search Variable Selection (BSSVS;
Lemey, Rambaut, Drummond, & Suchard, 2009) of the
discrete phylogeographic model as implemented in
BEAST v.1.8.0. To match the best tree topology obtained
from the phylogenetic analyses we used dataset 2, which
included all specimens collected across the Hajar Moun-
tains (see above). We established the phylogeographic
traits according to three discrete topographic discontinu-
ities of the Hajar Mountains: the Western Hajars, the Jebel
Akhdar and the Eastern Hajars (Fig. 1.1). Models, prior
settings and parameters were the same ones used for the
BEAST analysis of dataset 2 (see above).
Species delimitation and haplotype networks
Divergent mitochondrial lineages within populations pre-
viously described as Asaccus gallagheri were objectively
identified using the latest version of the general mixed
Yule-coalescent model (GMYC) (Fujisawa & Barra-
clough, 2013; Pons et al., 2006). The single-threshold
approach was tested on the 12S ultrametric tree (dataset 3)
and the analysis was performed using the package ‘splits’
(Ezard, Fujisawa, & Barraclough, 2009) in R (R Develop-
ment Core Team, 2016). This approach essentially detects
the most likely tree depth at which the pattern of tree
branching shifts between a Yule process (reflecting inter-
specific phylogenetic structure) to a coalescent process
(reflecting intra-specific phylogenetic structure). Because
inference of lineages relies on point estimates of the topol-
ogy and branch lengths, the associated phylogenetic error
could decrease the accuracy of the delimitation results.
Therefore, uncertainty in the phylogenetic tree estimation
and model parameters were assessed with a Bayesian
implementation of the GMYC model (bGMYC 1.0; Reid
& Carstens, 2012), which integrates these potential sour-
ces of error via MCMC simulation (Reid & Carstens,
2012). The R package ‘bGMYC’ was used to calculate
marginal posterior probabilities of lineage limits from the
posterior distribution of ultrametric trees reconstructed
with BEAST. A post-burn-in sample of 250 trees
resampled from that posterior was used to calculate the
posterior distribution of the GMYC model, running the
bGMYC analysis for 100,000 generations with a burn-in
of 10,000 generations.
Systematics of Asaccus gallagheri 5
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With the aim of exploring patterns of intra-specific
genetic diversity and nuclear allele sharing within popula-
tions previously classified as Asaccus gallagheri, statisti-
cal parsimony networks on the phased nuclear genes were
constructed independently with the program TCS v.1.21
(Clement, Posada, & Crandall, 2000) using default
settings.
Morphological samples, characters examined
and univariate analyses
A total of 54 alcohol-preserved adult specimens of
Asaccus from across its distribution range in the Hajar
Mountains of Oman and the UAE were examined and
included in the morphological analyses. All voucher
specimens were obtained from S. Carranza’s field
series housed at the Institute of Evolutionary Biology
(IBE), Barcelona, Spain; the Natural History Museum,
London, UK (NHMUK) and the Oman Natural History
Museum, Muscat, Oman (ONHM) (Tables S1 and S4,
see supplemental material online). Variables for the
morphological analyses were selected based on previ-
ous taxonomic studies of Asaccus (Afrasiab & Moha-
mad, 2009; Arnold, 1972; Arnold & Gardner, 1994;
Carranza et al., 2016; Dixon & Anderson, 1973;Gard-
ner, 1994; Rastegar-Pouyani, 1996; Rastegar-Pouyani,
Nilson, & Faizi, 2006; Torki, 2010; Torki et al.,
2011a;Torki, Fathinia, Rostami, Gharzi, & Nazari-
Serenjeh, 2011b;Werner,2006). Specimens were
sexed looking at two external characters: presence of
hemipenal bulges and colour of original tails (yellow
in males). The following measurements were taken
twiceontherightsideofeachspecimenbythesame
person (MSR) using a digital calliper with accuracy to
the nearest 0.1 mm: snout-vent length (SVL), distance
from the tip of the snout to the cloaca; trunk length
(TrL), distance between the fore and hind limb inser-
tion points; head length (HL), taken axially from the
tip of the snout to the anterior ear border; head height
(HH), taken laterally at the anterior ear border; head
width (HW), taken at the anterior ear border; snout
length (SL), from the snout to the anterior eye border;
snout width (SW), taken dorsally at the anterior eye
border; eye diameter (ED), maximal longitudinal
length of the eye; humerus length (LHu), from the
elbow to the insertion of the fore limb on the anterior
part of body; ulna length (LUn), from the wrist to the
elbow; femur length (LFe), from the knee to the inser-
tion of the hind limb on the posterior side of body and
tibia length (LTb), from the ankle to the knee. Tail
length was not measured because many individuals
had a regenerated tail or had lost it. In addition to
these morphometric variables, two pholidotic and one
categorical character were collected using a dissecting
microscope. Pholidotic characters: number of upper
labial scales (ULS) and number of lower labial scales
(LLS); categorical character: postmentals (PM) 1: in
contact, 0: not in contact. All specimens were
Table 1. Descriptive statistics for all characters examined for males and females of A. arnoldi sp. nov. and A. gallagheri.Mean §
Standard Deviation (S.D.) and range (Min-Max) are given in millimetres except for the categorical character (PM; percentage of
individuals with the first pair of postmentals in contact). Abbreviations of characters as explained in the material and methods.
A. arnoldi sp. nov. A. gallagheri
Males (nD6) Females (nD7) Males (nD15) Females (nD26)
Variable Mean§S.D. (Min-Max) Mean§S.D. (Min-Max) Mean§S.D. (Min-Max) Mean§S.D. (Min-Max)
SVL 29.9§2.5 (27.4-33.6) 30.7§1.7 (28.9-33.3) 33.8§2.2 (30-37.3) 32.8§3 (26.5-37.3)
TrL 11.5§1 (10.5-13.2) 12.1§0.7 (11.4-13.3) 13.5§1.3 (11.3-15.4) 13.5§1.6 (9.8-16.8)
HL 7.8§0.6 (7.1-8.7) 7.9§0.4 (7.4-8.5) 8.9§0.5 (8-9.7) 8.5§0.6 (6.9-9.4)
HW 5.1§0.3 (4.6-5.4) 5§0.2 (4.7-5.3) 5.6§0.4 (4.6-6.2) 5.5§0.5 (4.4-6.1)
HH 2.9§0.3 (2.6-3.2) 2.9§0.2 (2.7-3.1) 3.4§0.2 (3.1-3.8) 3.2§0.3 (2.6-3.6)
SL 3.5§0.3 (3.2-3.9) 3.6§0.2 (3.4-4) 4§0.3 (3.7-4.5) 3.8§0.3 (3.1-4.2)
SW 2.6§0.3 (2.3-2.9) 2.7§0.1 (2.5-2.9) 2.9§0.2 (2.5-3.3) 2.7§0.3 (2.1-3.2)
ED 1.8§0.2 (1.6-2) 1.9§0.2 (1.7-2.1) 2.2§0.2 (1.8-2.5) 2.1§0.2 (1.6-2.4)
LUn 4.5§0.5 (4.1-5.1) 4.6§0.2 (4.3-4.7) 5.1§0.3 (4.5-5.6) 4.8§0.4 (3.7-5.5)
LHu 4.3§0.4 (3.9-5.2) 4.4§0.5 (3.9-5) 4.9§0.5 (4.2-6) 4.7§0.6 (3.6-5.6)
LTb 5.8§0.6 (5.3-6.8) 5.8§0.4 (5.2-6.2) 6.8§0.7 (5.6-7.8) 6.2§0.6 (4.8-7.5)
LFe 6.2§0.5 (5.6-7) 6.5§0.3 (6.1-7) 7.1§0.8 (6-8.5) 6.8§0.8 (5-8.2)
ULS 11§0.8 (10-12) 11§0.8 (10-12) 11§0.8 (10-12) 12§0.8 (10-13)
LLS 9§0.8 (8-10) 8§0.5 (8-9) 9§0.9 (8-11) 9§0.8 (8-11)
PM (%) 17 71 87 69
6 M. Sim
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photographed using a Nikon 300 camera with a 60 mm
macro-lens, in order to make all the data easily avail-
able to the scientific community. The complete collec-
tion of 110 high-resolution photographs has been
deposited in MorphoBank (http://morphobank.org/per
malink/?P2755).
The final dataset included 41 specimens corresponding
to A. gallagheri (15 males and 26 females) and 13 (six
males and seven females) to the new species. Summary
descriptive statistics (mean,maximum,minimumand
standard deviation) for males and females independently
(Table 1) and together (Table S4, see supplemental mate-
rial online) were calculated for all the specimens
included in the present study. The 12 morphometric, the
twomeristicandtheonlycategorical variable were ana-
lysedindependentlyandusedinthedescriptionofthe
new species. All variables were log-transformed to
increase the homogeneity of variances. To avoid the
effect of strong correlation between size and the other
morphometric variables, a linear regression between
each variable and the snout-vent length (SVL) as predic-
tor was performed and the residuals were used as a proxy
of shape in the PCA in order to estimate the n-dimen-
sional hypervolumes (see below). Regarding body size,
differences between both species were tested using a
one-way ANOVA on the log-transformed values of
SVL. In addition, all pholidotic characters were tested
using a one-way ANOVA for each variable for taxo-
nomic purposes (see taxonomic account). Sexual dimor-
phism was checked for each variable using one-way
ANOVAs. As a result of the lower number of available
vouchers of the new species described herein, sexual
dimorphismwasonlytestedwithinAsaccus gallagheri.
All morphological analyses were performed in R.
Environmental and presence data
The full environmental dataset included 19 bioclimatic
variables downloaded from WorldClim (http://www.
worldclim.org; Hijmans, Cameron, Parra, Jones, & Jarvis,
2005), plus aridity index and potential evapotranspiration
obtained from the Consortium for Spatial Information
(CGIAR-CSI; http://www.cgiar-csi.org/). The data were
downloaded at 30 00 of degree spatial resolution and
clipped to the extent of the study area (55.94E, 59.50E,
22.02N, 26.30N). The variables were projected to a world
equidistant cylindrical projection centred in the centroid
of the sampled presence data (56.88E, 24.57N) with a spa-
tial resolution of 1 km. The Normalized Difference Vege-
tation Index (NDVI) dataset was obtained from the
MODIS MOD13Q1 (Version 5) product, for the period
between January 2004 and December 2014, with 16 days
frequency and 250 m spatial resolution, resulting in 132
grid images. The data were projected to the study
projection and clipped as above. The final dataset was
summarized by the maximum value obtained per pixel,
allowing a maximum of 34% of missing temporal data for
each pixel. A principal component analysis (PCA) was
done with all the above variables to summarize the envi-
ronmental variation of the study area. Prior to the PCA,
all variables were standardized to Z-scores due to the dif-
ferent units and magnitudes. Environmental variables
were processed in R with ‘rgdal’ library and PCA per-
formed with function ‘princomp’.
Presence data were gathered for A. gallagheri (nD86)
and the new species described herein (nD18) in the field
using a GPS handheld device. Coordinates were stored in
the WGS84 coordinate system and re-projected using the
study projection. The presence dataset for spatial analyses
was processed to remove duplicates within the same
pixel at the spatial resolution of the study (30 arc seconds
1 km), resulting in a reduced dataset with 44 records
belonging to A. gallagheri and 11 to the new species
described herein.
Niche and morphological hypervolume
overlap
The overlap between both species in the environmental
and morphological space was quantified using a three-
dimensional hypervolume. The hypervolume allows cal-
culation of the size, overlap (or intersection) and unique
parts of each species’ niche in a multidimensional space
(Blonder, Lamanna, Violle, & Enquist, 2014). This
method uses a kernel density estimation using the presen-
ces and was shown to produce accurate measurements of
the niche hypervolume, even with low sample size
(Blonder et al., 2014). Presence data were used to extract
the environmental data for each species (see above) and
the three most important components from each of the
environmental and morphological PCAs were used to esti-
mate the hypervolumes (Table S5, see supplemental mate-
rial online). The measurement of both hypervolumes was
used to study the level of differentiation between A. galla-
gheri and the new species described herein at the ecologi-
cal and morphological levels. All analyses were
performed in R with the ‘hypervolume’ package.
Although the hypervolume method may produce results
with any combination of sample size and dimensionality,
low sample size increases the sensitivity of the method to
the bandwidth value (Blonder et al., 2014). We followed
the package guidelines using the maximum of three varia-
bles based on the logarithm of the smallest sample size.
Hypervolumes were built using a Silverman bandwidth
estimator, a quantile threshold of 0% that includes the
total probability density, maximizing the niche overlap
between species, and a set of 1,000 random points to sam-
ple the kernel density.
Systematics of Asaccus gallagheri 7
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In order to quantify the overlap in environmental niches
and morphospaces between species, we used the Sørenson
index (Ahmadzadeh et al., 2016) and the overlap index
(OI) as the ratio of the intersection of the hypervolumes to
the size of the smallest hypervolume. The OI is related to
the Sørenson index (see Appendix 1, see supplemental
material online) and is particularly informative in the
cases of niches with very different sizes. Both indices
range between zero and one, from no overlap to the maxi-
mum possible overlap, respectively. To test the signifi-
cance of the overlap, we followed a randomization
procedure similar to Warren, Glor, and Turelli (2008).
The hypothesis being tested is if the niche of a pair of spe-
cies is more different than the niche drawn from both spe-
cies. For the niche hypervolume we merged the presences
of both species and randomly derived a new set of presen-
ces with the same number of observations as the originals
and measured the Sørenson index and OI. This was per-
formed 999 times in order to generate the null distribution
and a P-value of the observed overlap was calculated in
the R environment. The same procedure was applied to
the morphological data.
Species concept
In this manuscript, we have adopted the General Lineage
Species Concept (de Queiroz, 1998). This unified species
concept considers species as separately evolving metapo-
pulation lineages and treats this property as the single req-
uisite for delimiting species. Other properties, such as
phenetic distinguishability, reciprocal monophyly, and
pre- and postzygotic reproductive isolation, are not part of
the species concept but serve as important lines of evi-
dence relevant to assess the separation of lineages and
therefore to species delimitation (de Queiroz, 2007).
Results
Molecular analyses
The results of the phylogenetic analyses of dataset 1 using
BI and ML analyses produced similar trees, with most
nodes being well supported (Figs 1.2 and S1, see supple-
mental material online) and with the same topology
obtained by Carranza et al. (2016). Asaccus montanus
branches as a sister taxon to all the other Asaccus species
included in the analysis. The other five species from the
Hajar Mountains (A. caudivolvulus,A. margaritae,A.
gardneri,A. platyrhynchus,A. gallagheri and the new
species described herein) form a well-supported clade in
the BI analyses and, within it, the latter three species are
recovered as a highly supported monophyletic group in
both analyses. Asaccus gallagheri and the new species
described herein are sister species in all the analyses (Figs
1.2 and S1, see supplemental material online).
Diversification in the genus Asaccus started at least 28 Ma
(95% HPD D17.1–44.1). The clade formed by A. platyr-
hynchus,A. gallagheri and the new species described
herein started diversifying 9.1 Ma (95% HPD D5.4–14.5)
and, within it, divergence between the two species for-
merly classified as A. gallagheri occurred 6.2 Ma (95%
HPD D3.3–10). The results of the phylogenetic analyses
of dataset 2 show the phylogeographic relationships of all
the specimens previously classified as Asaccus gallagheri
(Fig. 1.3). As a result of the high level of genetic variabil-
ity, we performed a species delimitation approach to
objectively identify the number of distinct lineages. The
results of the GMYC and bGMYC analyses using dataset
3 congruently support five divergent mitochondrial line-
ages: three within A. gallagheri and two within the new
species described herein (Figs 1.3 and Table S1, see sup-
plemental material online).
The examined populations are unequally distributed
throughout the Hajar Mountains and adjacent lowlands,
from sea level to 1,887 m a.s.l., showing an allopatric pat-
tern for all detected lineages (Fig. 1.1,1.3 and Table S1,
see supplemental material online). Within Asaccus galla-
gheri, lineage 1 is widely distributed in the mountains of
northern Oman and eastern UAE (from the Musandam
Peninsula to Wadi Fazah), while lineages 2 and 3 are
restricted to the Jebel Akhdar, separated 165 km on a
straight line from the nearest locality of lineage 1. Despite
very good sampling and thorough exploration across the
northern Hajar Mountains (Garcia-Porta et al., 2017), not
a single specimen was found in this intervening area, in
which only one unvouchered record from Jebel Lahqin
has ever been reported (see Gardner, 2013). The new spe-
cies described herein (including lineages 4 and 5) is dis-
tributed across the Eastern Hajars. It is physically isolated
from lineages 2 and 3 (the Jebel Akhdar populations of A.
gallagheri) by the Semail Valley (see Fig. 1.1). The
results of the discrete phylogeographic analysis carried
out with dataset 2 using the BSSVS model suggest that
speciation between Asaccus gallagheri and the new spe-
cies described herein likely started in the Eastern Hajars
(49% of probability in the deepest node of the phyloge-
netic tree), where the new species differentiated into two
distinct lineages (98% of probability). The origin of A.
gallagheri is inferred to have been in the Jebel Akhdar
(55% of probability), where lineages 2 and 3 split (97% of
probability). The widespread lineage 1 diversified in the
Western Hajars (99% of probability), an area that was
most probably colonised from the Jebel Akhdar (Fig. S2,
see supplemental material online).
Genetic distances between intraspecific lineages (line-
ages 1–3 and 4–5; see Fig. 1) are considerably high, vary-
ing between 6%–8% for the 12S and 8%–13% for the cytb
(Table 2). Genetic distances between Asaccus gallagheri
and the new species described herein are very high: 12.7§
1.5% for the 12S and 20.8§1.7% for the cytb; similar to
8 M. Sim
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the genetic distances calculated between other Asaccus
species (Table 2 and Fig. S3, see supplemental material
online). The level of intraspecific genetic variability for A.
gallagheri is 1.7 §0.3 for the 12S and 2.7 §0.4 for the
cytb, and for the new species described herein 3.8 §0.6
for the 12S and 7.1 §0.8 for the cytb.
The results of the haplotype network analyses show that
A. gallagheri and the new species described herein do not
share a single haplotype (all haplotypes of each species
are private) in both nuclear genes analysed (Fig. S3, see
supplemental material online). Within each species, allele
sharing between all mitochondrial lineages is very low.
Regarding lineages 1, 2 and 3 (Asaccus gallagheri), they
share two haplotypes in the c-mos from a total of 17 hap-
lotypes and do not share any of the 19 haplotypes of the
MC1R gene. Similarly, the two lineages belonging to the
new species described herein (lineages 4 and 5) share two
of the eight haplotypes in the c-mos gene and none of the
eight haplotypes in the MC1R gene. The different haplo-
types of A. gallagheri and the new species described
herein do not present any geographic structure within
them, being distributed evenly over their specific sam-
pling sites (see Fig. 1.1, Fig. S3 and Table S1, see supple-
mental material online).
Morphological differentiation
Descriptive statistics for all 15 morphological traits are
shown in Tables 1 and S4 (see supplemental material
online). Sexual dimorphism between species was not
detectable, so both sexes were pooled together in all pos-
terior analyses (results not shown). Size difference
between species (SVL) was highly significant. The new
species described herein is significantly smaller than A.
gallagheri (F D12.464; d.f. D1; PD0.001). Shape dif-
ferentiation was assessed with a three-dimensional hyper-
volume including the two species (see Fig. 2.1). Nearly
80% of the total variation in the morphological dataset
was explained by the first three PCs used to calculate the
hypervolumes (Table S5.1, see supplemental material
online). The hypervolume of A. gallagheri is 2.6 times
broader than the hypervolume of the new species
described herein. The species’ morphospaces overlap to a
large extent that is »90% of the total morphospace of the
new species and 34% of the morphospace of A. gallagheri
(Fig. S4.1, see supplemental material online). This large
overlap is also suggested by the Sørenson index, although
it is significantly lower than expected under the hypothe-
sis of morphospace overlap (KD0.48; P<0.05). The OI
is high (OI D0.88) suggesting that the morphospace of A.
gallagheri mostly includes the new species described
herein (Fig. S5.1, see supplemental material online) and,
therefore, that we cannot differentiate each other with the
current morphometric data. The significant Sørenson
score is just describing the differences of the morphologi-
cal hypervolume from A. gallagheri to the smaller subset
that represents its sister species described herein.
Niche divergence
The niche overlap between both species was quantified
using three-dimensional hypervolumes (see Fig. 2.2).
Nearly 90% of the total variation in the environmental
dataset was explained by the first three PCs used to calcu-
late the hypervolumes (Table S5.2, see supplemental
material online). The entire hypervolume of A. gallagheri
is 3.7 times broader than the hypervolume of the newly
described species. The species’ niches overlap to a small
extent with an intersection that is »48% of the total niche
of the new species described herein and 13% of the niche
of A. gallagheri (Fig. S4.2, see supplemental material
online). The Sørenson score and OI are low (KD0.19,
OI D0.46) and significantly lower than expected under
the null hypothesis of niche equivalence (Fig. S5.2, see
supplemental material online), suggesting that both spe-
cies have different niches despite a considerable
Table 2. Genetic distances between all Asaccus species included in the molecular phylogenetic analyses. Uncorrected p-distances
(%) for 12S mitochondrial gene (lower-left) and for cytb mitochondrial gene (upper-right). All five mitochondrial lineages identified
within A. gallagheri and A. arnoldi sp. nov. are included. Genetic distances between A. gallagheri and A. arnoldi sp. nov. in bold.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1. A. caudivolvulus 12.8 26.3 27.8 27.3 26.8 25.3 27.3 25.6 30.1
2. A. gardneri 7 25.1 29.7 26.1 26.6 23.2 25.3 25.3 30.6
3. A. margaritae 16 14.5 26.2 22.3 23.1 23.5 26.5 26.1 27.6
4. A. gallagheri (1) 18.1 16.4 16.4 13.6 13.6 21.1 20.6 27.1 31.6
5. A. gallagheri (2) 18 16.7 17 7.9 8.1 19.6 18.4 27.1 29.3
6. A. gallagheri (3) 17.4 16.4 15.8 8.2 6 19.8 19.1 27.1 28.8
7. A. arnoldi sp. nov. (4) 17.4 15.7 15.3 13.3 12.8 11.6 12.5 21.9 29.3
8. A. arnoldi sp. nov. (5) 17.9 16 15.2 12.4 11.8 11.4 6.5 23.2 30.6
9. A. platyrhynchus 18.2 17.4 13.7 13.5 15.8 14.7 12.4 13.1 30.8
10. A. montanus 23.2 21.9 22 20.6 20.2 20.8 17.5 17.1 19.7
Systematics of Asaccus gallagheri 9
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intersection between them. The new species described
herein occupies a smaller niche that, although partly con-
tained within the other species, is not fully overlapping
and has a unique component that differentiates it from A.
gallagheri. The hypervolume overlap shows that the sec-
ond component is where most differentiation between
both species resides (Fig. 2.2). In the comparison with the
respective PCA loadings (Table S5.2, see supplemental
material online), variables with most influence are temper-
ature annual range (BIO7), mean diurnal range (BIO2),
precipitation seasonality (BIO15), temperature seasonality
(BIO4) and precipitation of the wettest month (BIO13).
The unique component of the new species’ niche is, thus,
mostly related to a smaller range of precipitation and
temperature.
Taxonomic account
Despite the high level of crypsis between the populations
from the Eastern Hajars and the rest of the distribution
range of A. gallagheri across all size corrected morpho-
metric characters analysed here (Fig. 2.1), the results of
the four gene fragments analysed clearly show that these
two evolutionary units have been evolving independently
for a long time (Figs 1.2,1.3 and S3, see supplemental
material online). Moreover, this differentiation is also
supported by the macroecological analyses (Fig. 2.2) and
a few morphological traits including size (see diagnosis
below, Tables 1 and S4, see supplemental material
online). Therefore, we describe the unnamed populations
from the Eastern Hajars as a new species endemic to
Oman. Data for the comparison with other Asaccus spe-
cies were obtained from our own morphological dataset
(Table 1 and Fig. S4, see supplemental material online)
and also from morphological information available from
the original descriptions of all 18 species of Asaccus
(Afrasiab & Mohamad, 2009; Arnold, 1972; Arnold &
Gardner, 1994; Carranza et al., 2016; Dixon & Anderson,
1973; Gardner, 1994; Rastegar-Pouyani, 1996; Rastegar-
Pouyani et al., 2006; Torki, 2010; Torki et al., 2011a,
2011b; Werner, 2006).
Family Phyllodactylidae
Asaccus Dixon and Anderson, 1973
Asaccus arnoldi sp. nov.
(Figs 1–3, Figs S1–S3, see supplemental material online;
Tables 1–2, Tables S1 and S3–S4, see supplemental mate-
rial online)
http://www.zoobank.org/urn:lsid:zoobank.org:act:
BF77A117-EA38-4D9C-AF44-9E700348F092
Fig. 2. Estimated three dimensional morphological (2.1) and environmental (2.2) hypervolumes for Asaccus gallagheri (green) and A.
arnoldi sp. nov. (purple). The intersection (dashed blue) between the hypervolumes is also shown.
The three dimensional hypervolumes were calculated with the first three components of the PCA (Table S5.1 and S5.2, see supplemental
material online). Coloured dots correspond to PCA-derived observations. Inset pictures show different specimens of A. arnoldi sp. nov.
from Oman.
10 M. Sim
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Asaccus gallagheri. Arnold & Gardner, 1994: 427
(part.); van der Kooij, 2000: 108 (part.); Sindaco & Jerem-
cenko, 2008: 99 (part.); Gardner, 2013: 95 (part.).
Holotype. BMNH2008.961, adult male, from Wadi Bani
Khalid (Oman), 22.6161N 59.0937E WGS84, elevation
647 m a.s.l. (locality 28 in Fig. 1.1; Table S1), collected
by S. Carranza, F. Amat, E. G
omez-D
ıaz on 3 May 2011,
tissue code S7555.
Paratypes. BMNH2008.962 and ONHM4234, two adult
females and IBES7576, adult male, same data as holotype,
tissue codes S7554, S7252 and S7556, respectively.
Other material examined. Nine specimens used for
genetic and morphological analyses and two specimens
used only for genetic analyses (no voucher available,
juvenile or damaged specimen); all listed in Table S1 and
S4 (see supplemental material online).
Etymology. The species epithet ’arnoldi’ is a genitive
Latin noun to honour the British herpetologist, Dr E.
Nicholas Arnold, for his life-long dedication and
contribution to Arabian herpetology, including the
description of the little-known gecko Asaccus gallagheri
45 years ago.
Diagnosis. A new species of Asaccus from the Eastern
Hajar Mountains of Oman characterized by the combina-
tion of the following morphological characters: (1) small
size with maximum SVL 33.6 mm; (2) first pair of post-
mentals in contact in less than half of the studied speci-
mens; (3) scales across supraorbital region fine; (4) dorsal
tubercles absent on back, occiput, upper arm and else-
where; (5) small subtibial scales; (6) paired terminal scan-
sors on digits not extending markedly beyond claws; (7)
cloacal tubercle minute or absent; (8) tail tip not laterally
compressed or vertically expanded; (9) absence of
enlarged tubercles on tail; (10) subcaudal series of
expanded scales do not reach the vent area anteriorly; (11)
dorsum with a pattern of narrow dark transverse bars; (12)
tail colour sexually dimorphic in non-regenerated tails,
being white barred black in females and yellow in males
(see Fig. 3.3); (13) dorsal dark bars on the tail of females
extend ventrally; (14) tail not coiled and waved in life.
Comparison with other Asaccus species. Asaccus
arnoldi sp. nov. differs from its sister taxon A. gallagheri
mainly in its smaller size (SVL max. 33.6 mm, compared
with max 37.3 mm) and in having less proportion of indi-
viduals with the first pair of postmentals in contact (46%
vs. 76%). It further differs in having fewer upper and
lower labial scales (ANOVA comparison of ULS and
LLS significant, P<0.001), by a genetic distance of
12.7% and 20.8% in the mitochondrial 12S and cytb
genes, respectively (Tables 2 and S3), and by the absence
of allele sharing in the c-mos and MC1R nuclear gene
regions analysed here (Fig. S3). It can be clearly differen-
tiated from all the other species of Asaccus described to
date (A. andersoni,A. elisae,A. caudivolvulus,A. gard-
neri,A. granularis,A. griseonotus,A. iranicus,A. ker-
manshahensis,A. kurdistanensis,A. margaritae,A.
montanus,A. nasrullahi,A. platyrhynchus,A. saffinae,A.
tangestanensis,A. zagrosicus) by its smaller size (SVL
max. 33.6 mm vs. 39.4–71) and by the absence of
enlarged dorsal tubercles on back, occiput, upper arm and
elsewhere.
Description of the holotype. BMNH2008.961 (Fig. 3.1).
Specimen with the tip of the tongue missing (used for
DNA extraction) and the tail partially broken. Data on all
15 morphological traits are provided in Table S4 (see sup-
plemental material online). Adult male, SVL 31.43 mm,
depressed head and body, well-marked neck, limbs and
tail slender. Head length 26% of SVL and head width
66% of head length. Tail not regenerated, 1.3 times the
SVL. Rostral scale twice as wide as high, entire but with a
slight medial depression. Internasals in contact behind
Fig. 3. View of the type locality and general appearance in life
of Asaccus arnoldi sp. nov.
(3.1) Holotype of A. arnoldi sp. nov. (male; voucher code
BMNH.2008.961); (3.2) Rocky sides of Wadi Bani Khalid in
2016 (locality 28 in Fig. 1.1; Table S1, see supplemental mate-
rial online); (3.3) Female (above) and male (below) A. arnoldi
sp. nov. with the characteristic dimorphic tail colouration. All
photographs taken by Salvador Carranza.
Systematics of Asaccus gallagheri 11
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rostral, each one bordering nostril together with two post-
nasal, first labial and rostral scales. Two distinct depres-
sions, one in the anterior loreal region and the other
medially, just anterior to the eyes. Upper scales in the pos-
terior loreal region enlarged (over twice the interorbitals)
and occiput and supraorbital areas covered by homoge-
neous juxtaposed scales, rather smaller than those on the
snout. Eye large, with a diameter of about 24% of the
head length and half of the snout length. Palpebral fold
anteriorly edged with large scales decreasing in size pos-
teriorly, where a row of small ciliate scales projects from
below and two rows of small granules separate the palpe-
bral fold from the supraorbital region. Ear opening almost
twice as long as wide with no denticulation on the border.
Eleven upper labials on both sides and 8 lower labials on
the right side and 9 on the left side. Mental scale large and
triangular, extending backwards to the level of the sutures
between the second and third lower labials. Two pairs of
elongated postmental scales. The first pair is larger, touch-
ing the first lower labials anteriorly and running along the
posterior edge of the mental scale without contacting each
other medially, split by one small rounded scale. The sec-
ond pair is bordered by inner postmentals and slightly sep-
arated from the second lower labials by two granular
scales. Gular scales small and granular. Dorsum covered
with uniform, rounded non-imbricated granules. Ventral
scales slightly larger than dorsals and generally similar in
shape, not flat and overlapping, distinctly larger in the
interfemoral region. Fore and hind limbs covered with
small granular scales. Digits with a series of enlarged
scales beneath that become distally smaller and divided to
form transverse series of two or three scales. Two terminal
pads truncated and surrounding an exposed claw. Tail
with rounded section, not vertically expanded and divided
externally into segments covered above by small juxta-
posed scales (equal size to dorsal body-scales) and with
some irregular extended tubercles situated on the lateral
margin of the segment. Ventral scales of tail enlarged and
overlapping on the tail-base, becoming distally smaller
and interspersed by smaller scales until the tip of the tail.
Colouration faded after fixation, pale whitish-yellow,
and translucent underneath so that viscera are discernible.
A dark stripe from the posterior border of the orbit until
the neck, passing through the upper margin of the ear, and
a small dark mark on the upper loreal region. Dorsum
with five dark crossbands (one on neck, three between
pairs of limbs and one on sacrum). Tail with seven dark
transverse bands not extending onto ventral surface and
decreasing in intensity distally, only the first three ones
clearly visible. Colour in life much richer than in the pre-
served specimen (Fig. 3.1), pale pink with more evident
pattern of dark brownish marks. Yellow shading encir-
cling the eyes and surrounding all the above-described
stripes present on the head area. Iris golden with dark
venations and tail substantially yellow.
Variation. Data on all 15 morphological traits for the three
paratypes (see above) are provided in Table S4 (see supple-
mental material online). Specimens BMNH2008.962 and
IBES7576 with missing tail and specimen ONHM4234 with
broken tail. In all three specimens, the tip of the tongue was
cut and used for DNA extraction. All the specimens are
very similar to each other, with more marked transverse
bands on the body compared with the holotype and darker
stripes on the head joining each other on the neck with the
first crossbands.
Distribution and ecology. As a result of the intensive
sampling across the Hajar Mountain range carried out
between 2005 and 2016, Asaccus arnoldi sp. nov. has
been found from latitude 23.219N in the Wadi Sareen
Nature Reserve, close to Qurayyat (50 km south-east of
Muscat, Oman), to latitude 22.107N in the Ash Sharqiyah
South, around the Jebel Qahwan massif (see Fig. 1.1;
Table S1, see supplemental material online). Thus, it can
be considered an endemic species to the Eastern Hajars,
isolated from its sister taxon A. gallagheri by the Semail
Valley (the minimum distance between them is 100 km
by air). Asaccus arnoldi sp. nov. has been found from sea
level up to 1,683 m a.s.l. (localities 29 and 25, respec-
tively), moving swiftly amongst the rocky terrain in
mountains and coastal wadis, small cliffs on open hillsides
and hiding in caves and fissures (Fig. 3.2). Strictly noctur-
nal, all specimens were captured during the night and
avoided the beam of the flashlight, hiding cautiously in
crevices and holes. This species is not very common and,
even in places where it occurs, sometimes one researcher
needs several hours to find one or two specimens.
Conservation status. Not evaluated.
Proposed common name. English: Arnolds’ Leaf-toed
Gecko;
Arabic:
Discussion
With several species of reptile not found anywhere else in
the world, the Hajar Mountains represent one of the top
biodiversity hotspots in Arabia and an important refuge
for montane species, some of them with clear affinities
with the fauna of south-western Iran and neighbouring ter-
ritories (Arnold, 1986; Balletto, Cherchi, & Gasperetti,
1985; Dakhteh et al., 2007; de Pous et al., 2016a; Garcia-
Porta et al., 2017; Gasperetti, 1988; Gasperetti, Stimson,
Miller, Ross, & Gasperetti, 1993; Kapli et al., 2008,2015;
Krause et al., 2013; Mandaville, 1977; Metallinou et al.,
2012,2014; Sim
o-Riudalbas et al., 2017; Tamar et al.,
2016b; Yousofi et al., 2015). As shown in previous molec-
ular studies of the genus Asaccus (Carranza et al., 2016;
12 M. Sim
o-Riudalbas et al.
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Papenfuss et al., 2010), the Arabian species are not mono-
phyletic, with the Jebel Akhdar endemic Asaccus monta-
nus branching as sister taxon to all other species included
in the phylogenetic analyses. Pending the inclusion of the
remaining species of Asaccus, these results suggest a pro-
visional Arabian origin for the genus, which has under-
gone a substantial radiation across the Zagros and the
Hajar Mountains (Fig. 1.2). This research also provided
the first evidence for the existence of high genetic differ-
entiation within populations of Asaccus from the north-
ernmost section of the Hajar Mountains, revealing the
existence of two new species of the A. caudivolvulus spe-
cies complex living across very short distances (Carranza
et al., 2016). In the present study, we have uncovered a
new speciation event which occurred more than 6 Ma on
the opposite extreme of the Hajar Mountains, involving
populations previously described as A. gallagheri.
Asaccus arnoldi sp. nov. is the smallest species of the
genus, measuring less than 33.6 mm from snout to vent.
Similar to its sister taxon, A. gallagheri, it is characterized
by the absence of enlarged tubercles throughout its body
and the presence of sexual dimorphism in tail colouration,
with yellow tails in males and banded black and white
tails in females. In fact, both Asaccus species from the
Hajar Mountains are morphologically similar, presenting
very conserved proportions in terms of body shape.
Despite the apparent morphological stasis, the genetic dis-
tances inferred from the mitochondrial genes are of simi-
lar magnitude to other interspecific distances within the
genus Asaccus (Table 2 and Table S3, see supplemental
material online). Moreover, the lack of shared haplotypes
in the two nuclear markers analysed further supports the
conclusion that the two species have been genetically iso-
lated for a long time without gene flow (Fig. S3, see sup-
plemental material online). The first-step species
delimitation approach included in this work also reveals
great levels of intraspecific genetic variability, identifying
up to five deep lineages living in allopatry: three within A.
gallagheri and two within A. arnoldi sp. nov. (Fig. 1.1,
1.3 and Table S1, see supplemental material online).
However, we prefer to apply a conservative approach until
more material is available and have decided not to
describe them as independent taxa. The main reasons are
the presence of allele sharing in at least one of the two
nuclear genes and lack of any morphological diagnostic
characters between the divergent mitochondrial lineages.
The ancestral area reconstruction suggests that the first
speciation event occurred in the Eastern Hajars, where lin-
eages 4 and 5 of A. arnoldi sp. nov. are currently distrib-
uted. Afterwards, range expansion progressed northwards,
from the Jebel Akhdar to the Musandam Peninsula, giving
rise to the three lineages detected within A. gallagheri
(Fig. S2, see supplemental material online). A similar
phylogeographic pattern has already been reported for
other two endemic geckos from the Hajar Mountains:
Trachydactylus hajarensis (de Pous et al., 2016a) and
Pristurus rupestris rupestris (Garcia-Porta et al., 2017).
Regarding habitat occupation, A.arnoldi sp. nov. has a
smaller environmental niche with a unique component
that differentiates it from A. gallagheri and that is likely
to be related to the supported variation of temperature and
precipitation. These results highlight the climatic distinc-
tiveness of the only known area inhabited by A. arnoldi
sp. nov. With a total area of 10,436 km
2
, the Eastern
Hajars constitute almost half of the total area of the Hajar
Mountains. Extending from Jebel Qahwan to the north-
west, the Eastern Hajars are isolated from the Jebel Akh-
dar by the Semail Valley and limits with the Sharqiyah
Sands to the south-east (Fig. 1.1). The discovery of these
distinct populations reinforces the importance of this rela-
tively poorly studied area of the Hajar Mountains as a cen-
tre of diversification for this and other reptile groups such
as Hemidactylus (Carranza & Arnold, 2012), Trachydac-
tylus (de Pous et al., 2016a) and Pristurus (Garcia-Porta
et al., 2017). Despite these recent findings, the knowledge
of its fauna and flora is still deficient, especially if one
compares it with the much better-studied areas of Oman,
such as the Jebel Akhdar (Harrison, 1976,1977). In con-
trast to the Western Hajars that do not present a single
protected area, the Eastern Hajars contain five protected
areas in the mountains and surrounding areas (Al Sareen,
Ras al Shajer, Al Saleel, Jebel Qahwan and the Turtle
reserve) with a total area of 1382.26 km
2
. Interestingly,
the two divergent lineages detected within A. arnoldi sp.
nov. have already been found in two protected areas. The
specimen collected in the Al Sareen Nature Reserve
(locality 25, Fig. 1.1, Table S1, see supplemental material
online) belongs to lineage 4, while two other localities
found within the Jebel Qahwan Protected Area belong to
lineage 5 (localities 30 and 31; Fig. 1.1, Table S1, see sup-
plemental material online). Al Sareen, a Protected Area of
»785 km
2
created primarily for the conservation of the
Arabian Tahr, is also home to a new isolated species
within the P. r. rupestris species complex (candidate spe-
cies 16; Garcia-Porta et al., 2017). These results exem-
plify how some emblematic species such as the Arabian
Tahr can act as ’umbrella species’ and contribute to the
protection of other, less visible, but equally relevant spe-
cies. The relatively high level of genetic variability
between lineages 4 and 5 of A. arnoldi sp. nov. and the six
candidate species endemic to the Eastern Hajars found
within the diversification of the P. r. rupestris species
complex (Garcia-Porta et al., 2017), suggest that the East-
ern Hajars are still poorly explored despite their interest
from a biodiversity point of view.
Our findings present the advantage of combining
molecular, morphological and macroecological data to
investigate the existence of morphologically very simi-
lar species, an integrative approach that has already
been done to uncover hidden diversity in Arabia,
Systematics of Asaccus gallagheri 13
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resulting in several taxonomic changes and new spe-
cies descriptions (see for example, Badiane et al.,
2014; Busais & Joger, 2011a,2011b;Carranzaetal.,
2016;Carranza&Arnold,2012;dePousetal.,2016b;
Garcia-Porta et al., 2017; Metallinou & Carranza,
2013; Papenfuss et al., 2010;Sim

o-Riudalbas et al.,
2017;

Sm
ıd et al., 2013,2015,2016; Tamar,

Sm
ıd,
G€
o¸cmen, Meiri, & Carranza, 2016c; Vasconcelos &
Carranza, 2014). The present study also highlights the
diversity of the genus Asaccus in south-east Arabia,
with up to seven endemic species occurring in the
Hajar Mountains and stresses its relevance from a con-
servation point of view. Additional taxonomic work
combining carefully planned fieldwork from geographi-
cally intervening areas together with molecular, mor-
phological and ecological analyses should be applied
to clarify species boundaries for the lineages identified
within A. gallagheri and A. arnoldi sp. nov.
Acknowledgements
We wish to thank Ali Alghafri, Sultan Khalifa, Hamed Al
Furkani, Johnnes Els, Margarita Metallinou, Thomas
Wilms, Jiri Sm
ıd, Raquel Vasconcelos, Roberto Sindaco,
Philip de Pous and F
elix Amat for assisting in sample col-
lection in the field. Special thanks are due to Saleh Al
Saadi, Ahmed Said Al Shukaili, Mohammed Al Shariani,
Ali Al Kiyumi, Mohammed Abdullah Al Maharmi and
the other members of the Nature Conservation Depart-
ment of the Ministry of Environment and Climate, Sultan-
ate of Oman for their help and support. We also wish to
thank the following people for their help with the ship-
ment of tissue samples: J. Vindum from the California
Academy of Sciences (CAS), USA; C. Spencer from the
Museum of Vertebrate Zoology (MVZ), University of
California, Berkeley, USA. Thanks also to Hessa Saif Al
Shamsi for the Arabic translation. Specimens were col-
lected and manipulated with the authorization and under
control and permission of the governments of Oman (Min-
istry of Environment and Climate Affairs, MECA) and the
United Arab Emirates (Environment and Protected Areas
Authority, Government of Sharjah). Specimens were cap-
tured and processed following the guidelines and proto-
cols stated in the collecting permits and agreements
obtained from the competent authorities (see references
below). Members of the government supervised collecting
activities. All efforts were made to minimize animal suf-
fering. All the necessary collecting and export permits for
this study in Oman were issued by the Nature Conserva-
tion Department of the Ministry of Environment and Cli-
mate Affairs, Oman (Refs: 08/2005; 16/2008; 38/2010;
12/2011; 13/2013; 21/2013) and the research in the United
Arab Emirates was done under the supervision and per-
mission of the Environment and Protected Areas
Authority, Government of Sharjah. This work was sup-
ported by the Ministerio de Econom
ıa y Competitividad,
Spain (co-funded by FEDER) under grant numbers
CGL2012-36970 and CGL2015-70390-P; Ministry of
Environment and Climate Affairs under Grant number
22412027; Secretaria d’Universitats i Recerca del Depar-
tament d’Economia i Coneixement de la Generalitat de
Catalunya under grant number 2014-SGR-1532; MSR is
funded by a FPI grant from the Ministerio de Econom
ıa y
Competitividad, Spain (BES-2013-064248); PT is funded
with a grant from Funda¸c~
ao para a Ci^
encia e Tecnologia
with reference SFRH/BPD/93473/2013.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Ministerio de Econom
ıa y
Competitividad, Spain (co-funded by FEDER) under
[grant numbers CGL2012-36970], [grant numbers
CGL2015-70390-P]; Ministry of Environment and Cli-
mate Affairs under [grant number 22412027]; Secretaria
d’Universitats i Recerca del Departament d’Economia i
Coneixement de la Generalitat de Catalunya under [grant
number 2014-SGR-1532]; MSR is funded by a FPI grant
from the Ministerio de Econom
ıa y Competitividad, Spain
[grant number BES-2013-064248]; PT is funded with a
grant from Funda¸c~
ao para a Ci^
encia e Tecnologia with
reference [grant number SFRH/BPD/93473/2013].
Supplemental data
Supplemental data for this article can be accessed here: https://
doi.org/10.1080/14772000.2017.1403496.
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