Journal of Heredity 2012:103(2):221–229
doi:10.1093/jhered/esr130
Advance Access publication February 6, 2012
Ó The American Genetic Association. 2012. All rights reserved.
For permissions, please email: [email protected].
Evolution and Radiation in the Scorpion
Buthus elmoutaouakili Lourencxo and Qi
2006 (Scorpiones: Buthidae) at the
Foothills of the Atlas Mountains
(North Africa)
MARTIN HUSEMANN, THOMAS SCHMITT, IASMI STATHI,
AND JAN
CHRISTIAN HABEL
From the Biology Department, Baylor University, Waco, TX 76798 (Husemann); the Department of Biogeography, Tier
University, 54296 Trier, Germany (Schmitt); the Natural History Museum of Crete, University of Crete, 71409 Heraklion,
Crete, Greece (Stathi); and the Natural History Museum Luxembourg, Department of Invertebrate Biology, 25, rue
Münster, L-2160 Luxembourg (Habel).
Address correspondence to Jan Christian Habel at the address above, or e-mail: [email protected].
Abstract
When low dispersal ability of an organism meets geographical barriers, the evolution of inter- and intraspecific
differentiation is often facilitated. In the Atlas massif of North Africa, the genus Buthus splits into several species and
diverges into numerous genetic lineages, often following the orographic structures of mountain systems. Such high
mountain ranges often act as barriers for species with reduced mobility even on small spatial scales. To study the effect of
orographic structures on organisms with low dispersal ability, we collected 61 individuals of the scorpion species Buthus
elmoutaouakili at 18 locations around the southwestern foothills of the High Atlas and Antiatlas and in the Sousse valley
(western Morocco). We analyzed intraspecific differentiation patterns within this geographically restricted area of about
100 50 km using 452 bp of the cytochrome oxidase I mitochondrial gene. We detected 5 distinct genetic lineages. In
a second analysis, we added 61 previously published sequences from Buthus species from Europe and North Africa. Using
a molecular clock approach, we detected old splits (4–5 Ma) separating the samples from 1) the western High Atlas and
north of these mountains, 2) the Sousse valley and adjoining mountain areas, and 3) the southwestern Antiatlas. Further
differentiation happened in the first 2 geographical groups about 3 Ma. Thus, the divergence time estimates based on
a Bayesian approach support the onset of differentiation into these main clades along the Pliocene (5–2.3 Ma) when
climatic oscillations started and a constant global cooling preceded the glacial–interglacial cycles of the Pleistocene. Further
genetic splits into parapatric groups are detectable for the Sousse valley main group in the early Pleistocene. The climatic
oscillations of the Pliocene and early Pleistocene might have caused repeated range shifts, expansions, and retractions
leading to repeated vicariance, hereby producing the hierarchical structure of genetic differentiation in B. elmoutaouakili.
A taxonomic revision, including morphological and molecular data, is needed to assess the status of each of these Buthus
scorpion lineages.
Key words: COI, haplotype diversity, mountain barriers, small-scale radiation, vicariance, microendemism
Genetic diversity is not homogenously distributed in space.
Whereas intraspecific variability declines to the actual
leading edge of the distribution range of a species, the rear
edge (often located in the ice age refuge areas) is often
characterized by high genetic diversity and differentiation
(Hampe and Petit 2005). This phenomenon has been
identified for the refuge areas of the southern European
peninsulas. Several studies even showed strong genetic
substructures within these peninsulas as resulting from
organisms surviving in several ‘‘refugia within refugia’’
(cf. Gómez and Lunt 2007). Strong genetic substructuring
within refugia has been identified in molecular studies
(reviewed in Hewitt 2011).
North Africa served as a further important but often
neglected retreat, differentiation and speciation center of
thermophilic western Palearctic organisms (see Habel et al.
221
Journal of Heredity 2012:103(2)
2011; with references therein). The heterogeneous altitudinal
relief of the Maghreb region with the Atlas massif in
Morocco and the Tell-Atlas in Algeria, the former exceeding
elevations of 4000 m a.s.l., favored the evolution of strong
intraspecific differentiation due to its topographic diversity.
Thus, these extraordinary orographic structures prevent the
exchange of individuals and hence gene flow, ultimately
resulting in a remarkable pattern of allo- and parapatric
genetic lineages. Previous molecular analyses revealed
intraspecific north–south (e.g., Fritz et al. 2006) and east–
west splits (e.g., Cosson et al. 2005; Paulo et al. 2008) or
even show further substructures scattered over these
mountain ranges (Gantenbein et al. 2001; Gantenbein
2004; Harris et al. 2002, 2004).
However, even on smaller geographical scales, philopatric
organisms with low dispersal ability frequently show strong
genetic differentiation. For example, molecular studies
revealed patterns of restricted gene flow and resulting
speciation in darkling beetles of the genus Pimelia even among
individual valleys on the Canary Islands (Emerson 2002; Moya
et al. 2006).
Scorpions display highly restricted home ranges limited
around their burrows, and a maximum dispersal of only
a few meters per year has been estimated (Polis et al. 1985).
This extreme philopatry and limited dispersal ability leads to
low rates of gene flow. Therefore, scorpions have served as
examples of extreme microdiversification, and molecular
analyses using allozymes, nuclear, and mitochondrial DNA
(mtDNA) data revealed high FST estimates even among
local populations of scorpions (Yamashita and Polis 1995;
Gantenbein et al. 2001; Gantenbein 2004). The limited
exchange rates of individuals among populations and hence
limited rates of gene flow might be the prime reason for the
tremendous species richness within the Buthid scorpions, as,
for example, described for the Atlas region (cf. Fet et al.
2000; Stockmann and Ythier 2010).
Combining strong orographic structures (as present in
the Atlas massif) and the low dispersal ability shown for
scorpions, one can predict strong genetic imprints of the
past (e.g., cold and warm phases) over limited geographic
ranges. Here, we focus on the scorpion Buthus elmoutaouakili
(Lourencxo and Qi 2006) as model system to analyze smallscale diversification processes and the impact of past
climatic oscillations in the western Atlas region of North
Africa. We analyzed cytochrome oxidase I (COI) sequence
data of 61 individuals belonging to 18 populations to test for
geographic patterns and the underlying geographic structures
and historic events.
Materials and Methods
Study Species
Buthus scorpions are widely distributed over large parts of
Africa and show a circum-Mediterranean stronghold including the Mediterranean islands (Gantenbein and Keightley
2004), with extraordinary high diversity in North Africa. In
Morocco, 14 species have been recognized (Vachon 1952;
222
Lourenc
xo 2002, 2003; Lourencxo and Slimani 2004; Lourenc
xo
and Geniez 2005; Lourencxo and Qi 2006). Yet, molecular
studies showed that diversity may be even underestimated,
and additional cryptic species may exist (Gantenbein and
Largiadèr 2003). Most of these species are strictly territorial
and show high philopatry and low dispersal abilities resulting
in restricted home ranges around their burrows (Polis et al.
1985; Polis and Sissom 1990).
Sampling and Identification
In total, we sampled 61 individuals of the scorpion species
B. elmoutaouakili of 18 populations during spring 2008 and
2009. An individual of Scorpio maurus (Out) served as outgroup for our phylogenetic analyses. In total, our sampling
covers an area of about 100 50 km at the western High
Atlas and Antiatlas. Most specimens were collected during
daylight under rocks. Collections at night were facilitated by
the use of a blacklight lamp as all scorpions fluoresce in UV
light due to a specific protein in their exoskeleton (Anglade
et al. 1990). Collected specimens were stored in absolute
ethanol until DNA extraction. All sampling sites are shown
in Figure 1 and listed in Table 1, in which further details
including sampling date and location are given.
Species were identified using a key by Vachon (1952),
and individual descriptions of later described Moroccan
Buthus species and therein provided diagnoses (Lourenc
xo
2002, 2003; Lourenc
xo and Slimani 2004; Lourencxo and
Geniez 2005; Lourencxo and Qi 2006). Buthus elmoutaouakili
belongs to a group of closely related species referred to as
the ‘‘B. occitanus complex.’’ Although largely similar in
general morphology, the species can be distinguished from
its close relatives by its relatively larger size (.50 mm),
a lack of dark pigmentation on the fifth metasomal segment
and its carapace, the number of pectinal teeth (males: 30–30,
females: 23–26), the number of denticle rows on the fixed
and movable fingers of the pedipalp (10–11), and the
numbers of lobes of the anal arc (2) (Lourencxo and Qi
2006). All species identifications were performed by I.S.
PCR and DNA Sequencing
For all individuals, DNA was isolated from leg or telson
muscle tissue using the Qiagen DNeasy kit with standard
protocols for tissue samples. A 452-bp fragment of the
mitochondrial COI gene (being typically included in barcoding studies as a global bioidentification system, see Hebert,
Cywska et al. 2003; Hebert, Ratnasingham et al. 2003) was
amplified using standard PCR procedures with the following
primers: forward 5# GGT CAA CAA ATC ATA AAG ATA
TTG G 3#, reverse 5# TAA ACT TCA GGG TGA CCA AAA
AAT CA 3# (Folmer et al. 1994). PCRs were performed in 20
ll volumes: 10 ll Mastermix (Thermozyme), 0.2 ll of each
Primer (1 lM), 4.6 ll PCR grade water, and 5 ll DNA
template. The cycle program comprised an activation step at
94°C for 4 min, followed by 40 cycles of 30-s denaturation at
94°C, 30-s annealing at 45°C, and 1-min elongation at 72°C.
Cycling was terminated by a final extension step at 72°C for 10
min. Amplicons were subsequently sequenced in both
Husemann et al. Diversification in Buthus Scorpions
Figure 1. Geographic distribution of sampled Buthus
elmoutaouakili populations. Genetic clusters are identified by
identical letters also given in Figures 2 and 3.
directions on an automated sequencer (3730xl DNA
Analyzer; Applied Biosystems, Carlsbad, CA) at the University of Kiel. All sequences are deposited at the National Center
for Biotechnology Information GenBank (accession numbers
are given in Table 1).
Statistical Analyses
Sequences were inspected and aligned using GENEIOUS
5.0.3 (Drummond et al. 2006) and BIOEDIT 7.0.9.0 (Hall
1999). Standard measures of genetic diversity and differentiation, including pairwise FST values as well as nucleotide and
haplotype diversities, were calculated with DNASP v5.10
(Librado and Rozas 2009), ARLEQUIN 3.5.1.2 (Excoffier and
Lischer 2010), and MEGA 5 (Tamura et al. 2011). A maximum
parsimony (MP) tree with close neighbor interchange search
algorithm (search level 1) based on 10 replicates of random
addition trees and 1000 bootstrap replicates (support values
lower than 60 are not shown) was calculated using MEGA 5.
The same program was employed to calculate a maximum
likelihood (ML) tree for 2 different data sets. The first data set
was similar to our original data, whereas in the second analysis,
we included 61 additional sequences of North African and
European Buthus species obtained from GenBank. These
additional data were added to test for monophyly of B.
elmoutaouakili. We used the general time reversible (GTR) þ
Gamma þ Invariable substitution model and performed 1000
bootstrap replicates to obtain measures of branch support.
Furthermore, a haplotype minimum spanning network was
calculated with the program TCS1.21 (Clement et al. 2000) using
a connection limit of 95%.
The geographic distance matrix was calculated with
the program GEODIST (Heidenreich A, unpublished data).
A reduced major axis regression to calculate intercept and
slope of genetic distance versus geographic distance were
applied. To test for an isolation by distance pattern, a matrix
correlation between genetic distance (calculated as FST and
PhiST) and geographic distance using a mantel test was
performed in the online software IBDweb 3.16 (http://
ibdws.sdsu.edu/~ibdws/).
In order to estimate divergence times of clades, we used
a Bayesian approach as implemented in BEAST v. 1.6.1
(Drummond and Rambaut 2007). For this, we employed our
widened data set, which also included samples from the
European part of the Mediterranean. We based our
estimates on the split between the European specimens
from a purely Moroccan clade. This split likely coincided
with the salinity crisis dating back 5.33 Ma. This geographic
event is often used to date divergence events that followed
the split between North Africa and Europe. The Strait of
Gibraltar reopened about 5.33 Ma and since then presented
a permanent barrier to migration between the Iberian
Peninsula and the Maghreb (Gantenbein and Largiadèr
2003). Accordingly, we calibrated the molecular clock with
this split between the European and Moroccan clade at 5.33
± 0.1 Ma (Figure 4). This calibration is strongly based on the
assumption that the divergence among the Spanish sister
clade and the B. elmoutaouakili lineage is the result of
vicariance due to the reopening of the Strait of Gibraltar.
Although this has been proved to be a valid assumption in
other taxa (Fromhage et al. 2004; Veith et al. 2006; Albert
et al. 2007), the presented dating estimates should still be
treated with care. We used the BEAUti application v. 1.6.1
to generate an input file for BEAST assuming a GTR
substitution model with estimated base frequencies and
Gamma þ Invariable sites setting for site heterogeneity. We
employed a relaxed clock model with an uncorrelated
lognormal distribution. The tree prior was set to Yule
process as recommended for data sets including multiple
species or distinct clades. The Markov Chain Monte Carlo
length was set to 10 000 000 steps with a sampling interval
of 500.
We used TREEANNOTATOR v. 1.6.1 to discard
a burn-in of 2000 sampled trees as specified by the software
Tracer v. 1.5. FIGTREE v. 1.3.1 was used to display the
obtained dated phylogeny.
Results
We used a total of 61 DNA sequences (plus out-group) of 18
populations with a total length of 452 bp. We detected 101
segregating (22.3%) and 351 monomorphic sites with a total
of 114 mutations. We recovered 36 haplotypes resulting in
a total haplotype diversity (Hd) of 0.97869. The average
number of nucleotide differences (Kt) was 32.97377, the
average nucleotide diversity (PiT) was 0.07295, and the
nucleotide diversity sensu Jukes and Cantor is 0.07802. As
sample sizes ranged from 2 to 5 individuals per population,
further details about genetic diversities are not shown.
The total genetic differentiation among and within
B. elmoutaouakili populations are as follows: genetic differentiations among all populations FST 5 0.93827; genetic
differentiation among the 5 major groups (see below)
FCT 5 0.88725; and genetic differentiation within these
223
Journal of Heredity 2012:103(2)
Table 1
Sampling sites of all studied Buthus elmoutaouakili populations in western Morocco
Cluster
Pop. ID
Population
Locality
GPS coordinates
Date of sampling
N
E
09-79
1
Chichoaua
31,27; 8,47
13-3-2009
4
D
09-78
2
Argane
30,55; 9,03
12-3-2009
4
B
09-77
3
Ameskrout
30,37; 9,20
12-3-2009
5
09-74
4
Agadir
30,28; 9,18
12-3-2009
4
08-10
5
Tiz-n-Test
30,52; 8,23
12-5-2008
3
09-72
6
Tassoumate
30,35; 8,15
11-3-2009
3
09-71
7
Tassoumate
30,35; 8,15
11-3-2009
3
09-73
8
W Tassoumate
30,30; 8,34
11-3-2009
4
09-3
9
Ait Aissa
30,18; 8,31
22-2-2009
3
09-70
10
Taliouine
30,22; 8,09
11-3-2009
3
09-5
11
Tassga
30,10; 8,28
22-2-2009
4
09-4
12
Igherm
30,06; 8,7
22-2-2009
3
09-6
13
Ait Saha
30,06; 9,12
23-2-2009
2
8-6
14
Et Tnine
29,42; 9,16
11-5-2008
3
09-10
15
Tighermi
29,32; 9,20
24-2-2009
4
09-11
16
Izerbi
29,20; 9,04
24-2-2009
3
09-12
17
Onafka
29,24; 9,15
24-2-2009
3
A
C
224
National Center
for Biotechnology
Information
accession numbers
JN832012
JN832013
JN832014
JN832015
JN832008
JN832009
JN832010
JN832011
JN832003
JN832004
JN832005
JN832006
JN832007
JN831999
JN832000
JN832001
JN832002
JN831974
JN831975
JN831976
JN832026
JN832027
JN832028
JN832023
JN832024
JN832025
JN832029
JN832030
JN832031
JN832032
JN832016
JN832017
JN832018
JN831997
JN831998
JN831980
JN831977
JN831978
JN831979
JN831981
JN832019
JN832020
JN832021
JN831995
JN831996
JN831971
JN831972
JN831973
JN831982
JN831983
JN831984
JN831985
JN831986
JN831987
JN831988
JN831989
JN831990
JN831991
Husemann et al. Diversification in Buthus Scorpions
Table 1 Continued
Cluster
Out
Pop. ID
Population
Locality
GPS coordinates
Date of sampling
N
09-13
18
Tiznit
29,18; 9,45
24-2-2009
3
09-68
Scorpio maurus
Taliouine
30,31; 7,53
10-3-2009
1
National Center
for Biotechnology
Information
accession numbers
JN831992
JN831993
JN831994
JN832022
Given are the respective genetic clusters, location name, running population number (coinciding with figures). S. maurus (Out) was defined as out-group.
groups: FSC 5 0.45248 (significance levels for all given
fixation values are P , 0.001). Respective genetic variances
are given in Table 2.
MP and ML analyses of our original data set indicate
several splits among geographic locations in B. elmoutaouakili
(see Figure 2). We found 5 distinct genetic groups (A–E),
mostly well supported by high bootstrap values and
distinguishable by their geographical distributions: northwestern Antiatlas (A), lower Sousse valley (B), southwestern
Antiatlas (C), southwestern High Atlas (D), and northwestern High Atlas (E). Group A shows a further structuring
into rather localized subgroups (A1–A5), which are strongly
supported by our haplotype network (Figure 3).
Our widened data set (121 in-group sequences, 383 bp)
reveals several monophyletic groups with several distinct
geographic lineages. Bootstrap values are generally low partially
due to limited data quality of GenBank sequences reflected by
a high amount of missing data. On the other hand, fairly recent
divergence among most lineages is suggested by molecular
clock analyses. Monophyly of B. elmoutaouakili appears to be
rejected in this analysis. However, considering that B.
elmoutaouakili has been described in 2006 (other Moroccan
species of Buthus have been described in 2003 and 2005) and
that the sequences obtained from GenBank originate from B.
occitanus mardochei identified and analyzed in 2003, these samples
do not provide any further taxonomic evidence to reject the
monophyly of B. elmoutaouakili. However, 4 additional genetic
lineages have been recovered within this large monophyletic
group by these additional individuals. Furthermore, a sister
group relationship to a lineage from Northern Spain and
France is recovered. However, this analysis also suggests that
not all Moroccan species might be monophyletic; yet, support
Table 2 AMOVA (Analyses of Molecular Variance) results
with groups assigned according to phylogenetic lineages of Buthus
elmoutaouakili in western Morocco
Source of
variation
Among groups (Va)
Among populations
within groups (Vb)
Within populations (Vc)
Total
df
SS
VC
%
9
8
907.226
33.304
16.272
0.935
88.73
5.10
43
60
48.683
989.213
1.132
18.340
6.17
df, degree of freedom; SS, sum of squares; VC, variance components.
for these basal branches is weak, and additional data will be
needed to resolve these basal lineages.
Mantel tests show a rather low correlation between
geographic and genetic distance, and thus, geographical
distance per se might have lower impact on differentiation processes than the orographic structures: pairwise
FST (Z 5 15 225.5974, r 5 0.4560, P , 0.05), and PhiST
(Z 5 7364.5738, r 5 0.2820, P , 0.05).
Discussion
Retraction–Expansion Dynamics, Vicariance, and the
Accumulation of Differentiation
As already observed in other Buthus species (Gantenbein and
Largiadèr 2003) and other scorpion genera (e.g., Scorpio),
mtDNA variation is fairly high showing nucleotide divergence rates from 10% to 15% (cf. Froufe et al. 2008), and
strong genetic differentiation over limited geographic ranges
is especially known for Buthus species as shown for mtDNA
and nDNA sequences (Gantenbein and Largiadèr 2003;
Froufe et al. 2008; Sousa et al. 2010) and allozymes
(Gantenbein 2004). The hereby detected ‘‘cryptic species’’
led to the description of several new taxa for the
Mediterranean region, but especially for the Maghreb region
(e.g., Lourencxo and Geniez 2005; Froufe et al. 2008).
The prerequisite for the evolution of such genetic
lineages might be the limited dispersal behavior of
scorpions, the heterogeneous terrain topography, and severe
climatic oscillations during the past 5 My. Organisms react
on changing climatic conditions by range modifications
(Hampe and Petit 2005). Buthus elmoutaouakili was probably
restricted to very small refugia, where the climate was not
too dry (in the lowlands during interglacials) or too cold (in
mountain ranges during glacial periods). The high Moroccan
Mountain ranges most likely acted as effective barriers for
dispersal within this taxon. These repeated expansion–
retraction dynamics caused microallopatry and have produced the highly diverse intraspecific variability of this
species.
In the case of B. elmoutaouakili, the first split happened
during the same time period as the separation from the
Iberian populations with the opening of the Strait of
Gibraltar (about 5.33 Ma). Most probably, a vicariance event
separated the population groups in the High Atlas from the
225
Journal of Heredity 2012:103(2)
Figure 2. (A) ML and (B) MP phylogenies for 18 populations of Buthus elmoutaouakili in western Morocco. Bootstrap values
calculated with 1000 replicates (values lower than 60 hidden). Letters of clades and population numbers coincide with all other
figures and tables.
ones in the Antiatlas. As this period was characterized by
severe droughts in the Mediterranean region, the plain of the
Sousse valley between these 2 mountain areas with their
somewhat higher precipitation rates might have acted as
a strong geographic barrier.
The split between southwestern and northwestern
Antiatlas occurred about 1 My later, maybe due to retraction
B
3
5
E
3
3
5
A
10
9
10
6/7
D
16
2
2
9
6/7
2
1
3
C
15
Lineage Richness: Poor Europe versus Rich Maghreb
16
12
13
17
14
11/12
14
13
18
14
11
8
11
8
17
8
15
15
Figure 3. Haplotype network calculated with maximum
connection steps at 95% 5 9, gaps treated as fifth state, 36
haplotypes for 18 populations of Buthus elmoutaouakili in western
Morocco. Given numbers and letters coincide with all other
figures and tables.
226
to the northern and southern foothills of these mountains.
The final splits between the 5 major groups were 1) in the
High Atlas group in which the beginning oscillations of the
climate cause a restriction to the southern (group D) and
northern (group E) slopes of this high mountain system
and 2) between groups A and B. The substructures in group
A, which should have evolved along the Pleistocene, are
likely the result of the climatic shifts restricting populations
to different valley systems of this geographically highly
complex region.
In contrast to the diverse B. elmoutaouakili exhibiting several
strongly differentiated genetic lineages, the closely related
scorpion B. ibericus and B. occitanus occurring at the Iberian
Peninsula represent only 2 genetic lineages distributed over
major parts of southern Iberia as well as eastern Iberia and
southern France, respectively, and only some accumulation
of genetic diversity and differentiation at its southernmost
distribution edge in south Iberia (Sousa et al. 2010). This
might evidence the existence of distinct refuge areas only in
southern Iberia during glacial phases. Nevertheless, these
Iberian differentiation patterns are low if compared with the
strongly differentiated lineages found for B. elmoutaouakili
over some parts of the Atlas Mountains. We therefore
Husemann et al. Diversification in Buthus Scorpions
assume that the climatic conditions in North Africa were
suitable for Buthus species over major parts of this area
throughout the past 5 My, at least in climatically buffered
pockets scattered all over the area. However, regional
retractions causing repeated vicariance events triggered the
subsequent differentiation process within this region, but
even the cold phases of the Pleistocene did not eradiate Buthus
scorpions from major parts of our study area in western
Morocco, thus enabling the survival of cryptic diversity. This
is in strong contrast to Iberia, where conditions for these
highly thermophilic animals only existed in some restricted
parts of the very south during ice age conditions causing
a much less pronounced genetic differentiation pattern in
this area.
Species Explosion
The molecular studies of the past decade recovered several
distinct lineages within Buthus species (from the Iberian
Peninsula and North Africa), and some of these have even
been recognized as distinct species (Lourencxo and Vachon
2004; Lourencxo and Geniez 2005). However, most of these
are rather cryptic species (i.e., taxa that are genetically
distinct but extremely similar in their morphology and
ecology, Lomolino et al. 2006). Although the allopatric
differentiation processes observed in B. elmoutaouakili have
historically been considered the most frequent, if not the
essential, first step in speciation (sensu Mayr 1942, 1963).
However, results obtained from a single molecular marker
should always be interpreted with caution in order to avoid
species misidentifications and taxonomic inflation (e.g.,
Isaac et al. 2004; Bickford et al. 2006). Future taxonomic
revisions should combine analyses of molecular data with
detailed morphological and morphometrical analyses of
indicative characters such as sculpturation and granulation,
shape of prosoma and telson, the number of pectinal teeth
and denticle rows on the fixed and movable finger of the
pedipalp, and the trichobothria (Vachon 1962).
Funding
Luxembourg Research Fund; German Academic Exchange
Service; Natural History Museum Luxembourg.
Figure 4. ML tree constructed using Buthus elmoutaouakili
sequences generated in this study and including all Buthus
sequences available from National Center for Biotechnology
Information GenBank; black circles represent sequences obtained
from GenBank; the star indicates the split between an Iberian
clade and the monophyletic B. elmoutaouakili clade, which has been
used as calibration point for our molecular clock analysis; vertical
bars represent geographic location of the clade (black, Europe;
gray, North Africa); location and clade IDs correspond to other
figures; numbers at branches are confidence values obtained from
1000 bootstrap replicates (values lower than 70 hidden).
Acknowledgments
We thank Frank E. Zachos for the DNA sequencing service at Kiel
University, Germany.
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Corresponding Editor: Rob DeSalle
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