Molecular biogeography of the Mediterranean Buthus species complex (Scorpiones: Buthidae) at its southern Palaearctic margin
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Biological Journal of the Linnean Society, 2021, XX, 1–13. With 3 figures.
Molecular biogeography of the Mediterranean Buthus
species complex (Scorpiones: Buthidae) at its southern
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Palaearctic margin
ROBERT KLESSER1,2, MARTIN HUSEMANN2, THOMAS SCHMITT3,4, PEDRO SOUSA5,
ABDELHAMID MOUSSI6 and JAN CHRISTIAN HABEL7,*,
1
Department of Invertebrates, Natural History Museum Leipzig, D-04105 Leipzig, Germany
2
Department of Entomology, Centrum für Naturkunde, Universität Hamburg, D-20146 Hamburg,
Germany
3
Senckenberg German Entomological Institute, D-15374 Müncheberg, Germany
4
Zoology, Institute of Biology, Faculty Natural Sciences I, Martin Luther University Halle-Wittenberg,
D-06099 Halle (Saale), Germany
5
CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto,
Campus Agrário de Vairão, 4485-661 Vairão, Portugal
6
Laboratory Valorization and Conservation of Natural Resources, University of Biskra, Algeria
7
Evolutionary Zoology, Department of Biosciences, University of Salzburg, A-5020 Salzburg, Austria
Received 28 October 2020; revised 21 January 2021; accepted for publication 23 January 2021
Neogene orogenesis and climatic cycles strongly influenced inter- and intraspecific differentiation and variability of taxa.
In this study, we focused on the southern margin of the western Palaearctic, known to be a geographically complex region.
We performed mitochondrial DNA analyses of Buthus scorpions from the Moroccan Atlas Mountains, from the Hoggar
Mountains in Algeria and from Tunisia, Iberia and Israel. Molecular species delimitation suggests the existence of ≥ 24
molecular operational taxonomic units. The data confirm complex differentiation patterns across the Atlas Mountains
of Morocco, but structures in Iberia, Algeria and Tunisia have considerably lower complexity. This identifies the Atlas
Mountain region as the most important differentiation centre of Buthus scorpions. Samples from the Hoggar Mountains
(southern Algeria) cluster with those from the southernmost parts of Morocco in the middle and upper parts of the Draa
Valley. This reinforces a recent connection of these regions. Samples from Israel are genetically similar to individuals
from eastern Algeria and Tunisia. This suggests a widespread group across major parts of North Africa. Divergence time
estimates indicate that differentiation in the genus began during the late Miocene, a period characterized by strong
tectonic activities in this region. Further differentiation could be linked to subsequent climatic changes that have occurred
since the end of the Miocene, with an increasing aridification of the Moroccan area. This also produced many microrefugia
in the mountains of the area during the Pleistocene climatic fluctuations.
ADDITIONAL KEYWORDS: Atlas Mountains – barcoding – genetic differentiation – Green Sahara –
Mediterranean Basin – micro-allopatry – Miocene.
INTRODUCTION shifts, because these structures might act as dispersal
barriers (Hewitt, 1996). In the Mediterranean region,
Over geological time scales, the formation of mountain
the heterogeneous topography and past climatic shifts
ranges and climatic changes are well-known drivers of
(especially during the Pleistocene) together induced
vicariance events (Rosen, 1978; Wiley, 1988; Zink et al.,
a ‘species pump’ mechanism (Schoville et al., 2012),
2000). Mountains can become the prerequisite for
leading to high biodiversity and many range-restricted
range fragmentation driven by climate-induced range
and endemic taxa (Hewitt, 2011). Major parts of this
region, such as the Atlas Mountains, represent highly
*Corresponding author: E-mail: janchristian.habel@sbg.ac.at complex orographic structures, which evolved mainly
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13 1
2 R. KLESSER ET AL.
during the Neogene (Beauchamp et al., 1999). Further from preliminary pieces of work on arthropods of low
complexity is added by a diverse system of large and vagility, such as Buthus scorpions (Gantenbein &
small peninsulas and islands in the entire region. This Keightley, 2004; Habel et al., 2012; Husemann et al.,
geographical setting provides an interesting system in 2012; Pedroso et al., 2013) and Pimelia darkling
which to analyse small-scale inter- and intraspecific beetles (Rangel López et al., 2018). Furthermore,
differentiation, stimulated further by the severe considerably less is known about the relationships
climatic cycles during the Pleistocene (Schmitt, 2007). of Atlas populations to populations in other parts of
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Within the Mediterranean Basin, the biogeography North Africa, despite its vast extent and interesting
of North Africa is particularly complex (Husemann history. For example, trans-Saharan biogeographical
et al., 2014). The region is classified as part of the patterns are still little investigated; yet, considering
Atlanto-Mediterranean refugium (together with that the Sahara Desert went through several more
Iberia), in addition to the Mauritanian refugium south humid periods in the recent past and thus was more
of the Atlas main ridge (de Lattin, 1948). It comprises permeable for many organisms (see Watrin et al., 2009;
particularly high orographic diversity, promoting a Rangel López et al., 2018), these patterns deserve more
high number of endemic plant and animal species attention.
(Cuttelod et al., 2008). Intraspecific fine-grained genetic To strengthen our knowledge on the biogeography of
lineages evolved frequently across the high mountains North Africa, we studied differentiation patterns of the
of North Africa, which, in many cases, represent the scorpion genus Buthus to detect potential connections
centre of origin of such taxa (Gantenbein, 2004; Harris across larger parts of North Africa, including regions
et al., 2004; Fonseca et al., 2008; Husemann et al., to the east and south of the Moroccan Atlas Mountains.
2014; Rangel López et al., 2018). These lineages often We sampled Buthus specimens across North Africa
did not expand across larger parts of the western (Morocco, Algeria and Tunisia) and Israel and
Palaearctic and thus today create hotspots of inter- sequenced the mitochondrial COI gene for these. Based
and intraspecific diversity. However, North Africa on these data, we tested the following hypotheses:
seems to be of great importance as a source for Iberian
(1) The Moroccan Atlas Mountains are the area of
biota, with the Messinian Salinity Crisis (MSC) at the
origin and differentiation centre of the scorpion
Mio-Pliocene transition (Thiede, 1978; Giraudi, 2004,
genus Buthus.
Roveri et al., 2014) representing an important window
(2) Morocco is the primary source for the colonization
for biotic exchange, but not the exclusive phase for
of adjoining North African and European regions.
such interchange (Husemann et al., 2014). Despite
(3) More humid periods allowed the dispersal of
its high relevance for the evolution of species and
Buthus scorpions across areas that are presently
its importance for European biogeographical history,
hostile, such as the Sahara Desert.
North Africa has frequently been neglected in many
studies, which often focus exclusively on the three
large Mediterranean peninsulas, i.e. Iberia, Italy and
the Balkans (Husemann et al., 2014).
Studies incorporating North Africa have shown little MATERIAL AND METHODS
support for the classical assumption of a separation
of North Africa along the Atlas main ridge, with Sampling
one exception being the Mediterranean pond turtle, Sampling was mostly performed by the authors at 82
Mauremys leprosa Schweigger, 1812, but without sites in the Atlas and Rif Mountains of Morocco, in the
further substructuring (Fritz et al., 2006). Instead, Hoggar Mountains in Algeria, and in Tunisia (Fig. 1).
genetic discontinuities are frequently detected across Most specimens were collected during daylight
the Atlas and in the border region between Morocco under rocks. Overnight collections were facilitated
and Algeria (examples are given by Husemann et al., with black light, because all scorpions fluoresce in
2014). Further, more fine-grained structures have ultraviolet light owing to a specific protein in their
been detected along the valleys of the Atlas Mountains exoskeleton (Anglade et al., 1990). Specimens were
for arthropods (Habel et al., 2012; Husemann et al., stored immediately in absolute ethanol until DNA
2012; Pedroso et al., 2013; Rangel López et al., 2018) extraction. Further details on sampling sites are
and reptiles (Perera & Harris, 2010). Although these given in the Supporting Information (Table S1). The
studies provide some evidence for several refugia taxonomy of Buthus scorpions is far from clarified
within the North African refugium (i.e. refugia (Sousa et al., 2017); the genus currently includes 59
within refugium, see Gómez & Lunt, 2007; Abellán & species. However, the status of many species remains
Svenning, 2014), the patterns and underlying processes doubtful, especially for the taxa of North Africa. We
are not yet well understood. The best data supporting used molecular species delimitation to estimate the
the strong substructure within the region so far come number of molecular operational taxonomic units
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13
BIOGEOGRAPHY OF BUTHUS SCORPIONS 3
(mOTUs) and match them with described species, and 1 min elongation at 72 °C. Cycling was terminated
where possible. This might provide a baseline for by a final extension step at 72 °C for 10 min. The PCR
future taxonomic revisions. conditions for high-salt and Chelex extracts were as
follows: activation at 95 °C for 1 min, followed by 35
cycles of 15 s denaturation at 95 °C, 15 s annealing at
Molecular analyses 50 °C and 10 s elongation at 72 °C. Success of the PCR
DNA was isolated from one leg, or from muscle tissue was checked by gel electrophoresis, and successful
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from a caudal segment or the telson, using the Qiagen products were purified with an enzyme mix consisting
DNeasy kit (Qiagen, Hilden, Germany), the high-salt of exonuclease I and shrimp alkaline phosphatase
method (Paxton et al., 1996) or Chelex (Walsh et al., (ExoSap). Amplicons were sequenced by Macrogen
1991). A fragment of COI was amplified using standard (Amsterdam, The Netherlands), GATC Biotech AG
polyerase chain reaction (PCR) procedures with the (Konstanz, Germany) or the Genomics Service Unit of
primers LCO-1490 and HCO-2198 (Folmer et al., the Ludwig Maximilian University of Munich (Munich,
1994). For Qiagen extracts, the PCR was performed Germany). All sequences are deposited at the NCBI
in 20 µL volumes consisting of 10 µL Mastermix GenBank (Supporting Information, Table S2).
(Thermozyme), 0.2 µL of each primer (1 µM), 4.6 µL
PCR-grade water and 5 µL DNA template. For high-
salt and Chelex extracts, the PCR was performed Data analyses
with the following set-up: 5.8 µL PCR-grade water, All chromatograms were checked, trimmed and
2 µL 5× buffer, 0.4 µL of each primer, 0.2 µL dNTPs, proofread in GENEIOUS v.9 (Kearse et al., 2012).
0.1 µL MyTaq polymerase and 1 µL template. The M U S C L E ( E d g a r, 2 0 0 4 ) , a s i m p l e m e n t e d i n
PCR conditions for Qiagen extracts were as follows: GENEIOUS, was used to align all sequences. The
activation step at 94 °C for 4 min, followed by 40 cycles resulting alignments were trimmed to similar length.
of 30 s denaturation at 94 °C, 30 s annealing at 45 °C Overall, we submitted 76 new sequences to GenBank
Figure 1. A, positions and names of mountain ranges. B, map showing the sampling locations of Buthus scorpion species
and the distribution of the 24 molecular operational taxonomic units (mOTUs) derived from Automatic Barcode Gap
Discovery (ABGD) analysis. Known type locations are displayed as circles with dots. The species names of the type numbers
and the metadata of mOTUs can be found in the Supporting Information (Tables S1 and S2). Several sampling locations
match or are close to type locations, making an assignment of mOTUs to species feasible, for the first time. Symbols with
different shapes were used to avoid loss of contrast between too many colours.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13
4 R. KLESSER ET AL.
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Figure 1. Continued.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13BIOGEOGRAPHY OF BUTHUS SCORPIONS 5
(Supporting Information, Table S1). Twenty-seven et al., 2012). We ran the command line version with all
sequences from NCBI GenBank from different given site models (K80, JC69 and Simple Distance),
locations in Portugal, Spain and Algeria were added because the GTR model is not implemented in this
to the alignment (Supporting Information, Table S2). software. Furthermore, we ran all analyses for all gap
We also added some sequences from other genera widths between 0.5 and 1.5, with 50 steps. We then
of Buthidae as outgroups (Mesobuthus caucasicus, compared the results to find the most conservative
HM567334; Mesobuthus gibbosus, KF997876; solution (lowest number of inferred mOTUs). Default
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Mesobuthus martensii, JF700145; and Odontobuthus priors were used [Pmin = 0.001, Pmax = 0.1, Nb bins
sp., KF701326). One hundred and twelve sequences (for distance distribution) = 20]. We also used the
were included from former publications by the authors, Kimura (K80) model and a relative gap width of 1.1,
which are already published on GenBank (Habel et al., because this produced the lowest number of mOTUs
2012; Husemann et al., 2012). The final alignment and led to a good overlap of initial and recursive
included a total of 219 sequences. Nucleotide and partitions. As a second method for defining mOTUs, we
haplotype diversity were estimated with D na SP 6 used GMYC (Pons et al., 2006), a species delimitation
(Rozas et al., 2017). model based on the topology of time-calibrated
We generated phylogenetic trees using Bayesian trees, which was run with Microsoft R mRAN v.3.4
inference, as implemented in BEAST v.2.6.2 (Bouckaert (Microsoft, 2017) in RStudio v.1.0.143 (R Studio Team,
et al., 2014). The best substitution model, HKY+G+I, 2020) and the packages Ape (Paradis et al., 2004),
was determined using the R package PHANGORN Splits (Ezard et al., 2009), Paran (Dinno, 2008) and
(Schliep et al., 2017) in mRAN v.3.4 (Microsoft, 2017) Mass (Venables & Ripley, 2002). We decided to use the
with RStudio v.1.0.143 (R Studio Team, 2020). The single-threshold solution, because the multi-threshold
best model was determined to be HKY+G+I. Input algorithm seemed to generate massive over-splitting.
files for BEAST were prepared with BEAU ti v.2 Third, we used bGMYC, a Bayesian implementation
(Bouckaert et al., 2014). Analyses were also performed of the GMYC model available as an R package (Reid
using the Yule speciation prior and a relaxed log- & Carstens, 2012). We used 50 000 iterations and a
normal clock, with a rate of 2.3% substitutions per burn-in of 40 000, with a thinning of 100. To create an
million years (Brower, 1994), which were published adequate input file, we performed a re-thinning with
for the mitochondrial DNA (mtDNA) of arthropods. LogCombiner v.2.6 (Bouckaert et al., 2014) to create a
Additionally, we ran the analyses with the rates file including only 100 trees instead of 10 000. Fourth,
proposed by Russo et al. (1995) [which is equal to the we used the Poisson tree processes (PTP) algorithm
rate of Gantenbein et al. (2005) for Mesobuthus] and (Zhang et al., 2013) and its Bayesian implementation,
Papadopoulou et al. (2010). We compared the results bPTP (Zhang et al., 2013). These algorithms are based
of the analyses with different rate parameters using on tree topologies and the number of substitutions. We
the summary statistics in TRACER v.1.7 (Rambaut used PTP and bPTP as standalone versions and ran
et al., 2018). We finally chose the rate from the the published python packages under Linux Ubuntu
work of Brower (1994) because this yielded the best 16. In PTP, we processed the best single tree derived
likelihood parameters for final analyses. We performed from BEAST analysis. For bPTP, we processed our
two independent runs in BEAST with the final set of re-thinned multiple tree file from BEAST, again with
parameters. The analyses were run for 100 million a Markov chain Monte Carlo of 50 000 generations, a
generations, sampling every 10 000 iterations. We thinning of 500 and a burn-in of 10%. Convergence of
performed at least three runs for each clock model. bPTP analysis was checked using the trace file output.
Resulting log-files were checked with TRACER to For all species delimitation methods, we removed all
confirm sufficient Effective sample size (ESS > 200) outgroups. We used the most conservative solution
and convergence of the analyses. In a final step, the (lowest number of mOTUs) as our final estimate of
maximum clade credibility (MCC) tree was annotated mOTU diversity and plotted resulting mOTUs on a
with T ree A nnotator v.2 (Bouckaert et al., 2014) map with QGIS v.3.10.2 (QGIS.org, 2021) to visualize
after excluding a burn-in of 10%. The final tree was the geographical distribution.
visualized in FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/ We a l s o p e r f o r m e d D i s p e r s a l - E x t i r p a t i o n -
software/figtree/). Cladogenesis (DEC) (Ree et al., 2005) analyses with
Owing to the unclear taxonomy and distribution RASP v.4.2 (Yu et al., 2015) to understand the origin
of many Buthus species (Sousa et al., 2017), we used of the genus. We again used the consenstree derived
statistical species delimitation to define mOTUs. For with the Brower rate as input together with a Geo-
this, we used four different methods. First, we used the File, wherein we defined nine areas: A, Tell Atlas;
Automatic Barcode Gap Discovery (ABGD) method to B, Aures Mountains; C, Anti-Atlas; D, High Atlas;
search for so-called barcode gaps in the distribution of E, Rif; F, Middle Atlas; G, Hoggar; H, Iberia; and
pairwise differences in a barcoding dataset (Puillandre I, Israel.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–136 R. KLESSER ET AL.
Furthermore, we generated a GenGIS (Parks et al., Species delimitation analyses revealed very different
2013) plot to link phylogenetic and geographical data. numbers, ranging from > 120 mOTUs derived from
We used the tree from our BEAST analysis with the bPTP down to 43 from GMYC, 31 from bGMYC and
clock rate of Brower (1994) and the GPS data of all 24 from ABGD (Fig. 3). Given that these 24 mOTUs
samples to plot both on a map. The outgroups and the were split further in other analyses, we used this as
subtree of our two Israelian samples were collapsed. the most conservative estimate for subsequent analyses
For the two samples from Israel, no precise coordinates and discussion. The mOTUs were well supported in
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were available, although we know that they were the phylogeny, with most posterior probabilities being
collected within a radius of 50 km around Tel Aviv. > 0.95 (Fig. 2). Most mOTUs were found exclusively
Thus, we assumed coordinates nearby Tel Aviv but in Morocco (N = 12, Sc_2–Sc_9, Sc_14, Sc_17, Sc_23
did not include these samples in our geographical and Sc_24); two groups were distributed in Morocco
analyses. and Algeria (mOTUs Sc_15 and Sc_18, Supporting
Finally, we created a GIS map displaying the Information, Table S1), one of which ranged from the
distribution of our mOTUs and the known type Atlas to the Hoggar Mountains (Sc_15, Fig. 1; Fig. 3).
locations of the North African Buthus species, derived The mOTU from Israel (Sc_16) was recovered as a
from the original descriptions when available (see sister group to a clade in Tunisia and Algeria, including
Sousa et al., 2017 for details; Lourenço, 2017; Kovařík exclusive mOTUs from Tunisia (Sc_11) and Algeria
et al., 2020). (Sc_10). Furthermore, we recovered three Iberian
groups and one clade shared between Algeria and Iberia
(more specifically, one Algerian sequence clustered
within an otherwise pure Iberian group). The Iberian
RESULTS lineages were recovered as diversifying out of a larger
We generated an alignment of 215 sequences of group of samples from the Rif Mountains in Morocco.
Buthus specimens from 82 locations (Fig. 1 and 2) and We also generated a G en GIS plot to test our
four outgroups with a trimmed length of 443 bp. The biogeographical hypotheses (Fig. 2) and to visualize
number of invariable (monomorphic) sites was 208, of the relationships between locations. The plot showed
polymorphic (segregating) sites 169, and of polymorphic a high density of mOTUs in the Atlas Mountains and
(segregating) sites with more than two variants 69. a concentration of mOTUs splitting from the others
The alignment contained 152 haplotypes, leading to a rather early in this region. Furthermore, most mOTUs
haplotype diversity of 0.996 and a nucleotide diversity in mountain ranges were strictly limited to small areas,
(per site) of 0.09118. whereas mOTUs distributed in low-elevation areas (e.g.
Figure 2. Graphical visualization of genetic lineages in a BEAST tree (COI, Brower rate) of Buthus scorpion species in
geographical space generated with GenGIS.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13BIOGEOGRAPHY OF BUTHUS SCORPIONS 7
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Figure 3. Dated phylogenetic tree of Buthus scorpion species based on the rate determined by Brower (1994). Black bars
indicate molecular operational taxonomic units (mOTUs) derived from five different models. Green dots indicate posterior
probabilities > 0.90. Species names are given for clearly identified mOTUs. Values next to notes show dates (in millions of
years ago). Below bars, the numbers of mOTUs for each model are given.
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–138 R. KLESSER ET AL.
Portugal or Algeria) had wider distributions. An exception Morocco as a highly important differentiation centre
was the genetic link between Hoggar and the High and and add new information for other parts of North
Anti-Atlas Mountains (separated by > 1500 km). We Africa. In contrast to the high differentiation within the
could also see a close connection between North Africa genus Buthus across the Atlas Mountains in Morocco,
(in particular, the Rif Mountains) and Iberia. the other parts of North Africa, but also Iberia, show
We performed molecular clock analyses with comparably little divergence. The oldest splits between
different rates. Finally, we used the Brower model, lineages are dated at ~8 Mya, i.e. to the upper Miocene
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because all models did not show statistically different (based on the Brower rate). According to the faster
likelihood results (Brower rate, 5758.065; Gantenbein rate from Papadopoulou et al. (2010) and the slower
rate, 5758.218; Papadopoulou rate, 5758.267; all Rate from Gantenbein et al. (2005), this split ranges,
ESS values > 200), and the Brower model led to in different clock models, from 5 to 12 Mya. Our data
an age for the split between the Iberian and North underline that high-elevation mountains with high
Moroccan lineages in line with previous studies and ridges and peaks, in addition to steep valleys, are
geological events (i.e. Messinian Salinity Crisis). In driving differentiation in this group. This indicates
the ages of focused splits, we found large differences the high evolutionary importance of the orographic
depending on the value of the rate; the higher the heterogeneity of the Moroccan Atlas Mountains and
rate, the younger the date of the splits. The oldest suggests an additional strong influence of climatic
splits with the Brower rate were dated at 8 Mya, events for lineage divergence. Our results also show
whereas the Gantenbein and Papadopoulou models the strong connectivity across lowland areas, despite
dated those splits at 12 and 5 Mya, respectively. The the currently harsh desert climate in North Africa.
first split between Iberia and Northern Africa was
dated at ~5.5 (Brower), 9.4 (Gantenbein) and 3.7 Mya
(Papadopoulou). The second split between both Differentiation centre of Buthus scorpions
regions was dated at ~3.8 (Brower), 6.3 (Gantenbein) across Morocco
and 2.5 Mya (Papadopoulou). Overall, the Gantenbein The topology of our COI phylogeny and DEC analyses
rate dated most of the nodes 65–80% older and the suggest that the Moroccan Atlas Mountains (specifically,
Papadopoulou rate dated them 30–40% younger. the High Atlas and the Anti-Atlas) represent the
The DEC analyses suggested that the ancestral area origin and the major differentiation centre for Buthus
of the group was in the Anti-Atlas and the High Atlas scorpions. This is supported by the following findings:
(Supporting Information, Fig. S1), but also suggested (1) some lineages emerging from the oldest splits are
that the Aures Mountains had an important role in endemic to the Moroccan Atlas Mountains; (2) Buthus
the evolution of the group, including populations from scorpions sampled there have high genetic diversity; and
Iberia and the Rif. Furthermore, the analyses suggested (3) the other North African lineages are directly derived
the highest numbers of dispersal events out of the Anti- and nested within lineages from the Moroccan Atlas.
Atlas and the largest number of speciation evens within Molecular clock estimates (independent of the
the Anti-Atlas (112 of 205 total events). DEC suggested rate used) suggest that genetic differentiation
20 dispersal and 14 vicariance events overall. across the Moroccan high mountain systems started
Our GIS plot including the known type locations of during the upper Miocene, a period characterized
North African Buthus species showed that two of our by strong uplift of the Atlas Mountains (Beauchamp
sampling locations matched type locations or were in et al., 1999). The fact that the current topology of
their close proximity (< 5 km): hence, mOTU18 and the Moroccan Atlas Mountains is reflected in the
mOTU09 might represent Buthus boumalenii Touloun genetic structures of Buthus suggests that the entire
& Boumezzough, 2011 and Buthus lienhardi Lourenço, process of formation of these mountains in the upper
2003, respectively. We also sampled in close proximity to Neogene was an important driver of the radiation of
the type localities of Buthus aures Lourenço & Sadine, the taxon and further differentiation in the region,
2016, Buthus draa Lourenço & Slimani, 2004 and Buthus later complemented by range shifts triggered by the
elmoutaouakili Lourenço & Qi, 2006. Several other type Pleistocene climatic oscillations. Previous studies
locations were far outside of our sampling range or at the on the Buthus species complex in the same region
border of two different lineages (e.g. Buthus confluens show similar patterns and differentiation processes
Lourenço, Touloun & Boumezzough, 2012; Fig. 1). starting during the upper Miocene (Gantenbein &
Largiadièr, 2003; Habel et al., 2012; Husemann et al.,
2014). Studies on reptiles, such as the agamid lizard
Agama impalearis Boettger, 1874, also suggest an
DISCUSSION
onset of diversification at 8.5–9.4 Mya, coinciding with
Our phylogeographical data on Buthus scorpions the start of the main period of orogenic uplift of the
provide further evidence for the Atlas Mountains in Atlas Mountains (Brown et al., 2002). Thus, Mio- and
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–13BIOGEOGRAPHY OF BUTHUS SCORPIONS 9
Pliocene vicariance mediated by orographic activities back-colonization to the western Rif Mountains of
resulting in the extant high elevations of the Atlas northern Morocco.
Mountains provides a more general explanation of Although the geographical distances among the
intra- and interspecific biogeographical patterns in sampling locations in Israel, Algeria and Tunisia
this region, as also confirmed by our results. are large, they exhibit only a low level of genetic
differentiation. The close relationships of these
specimens and diversification out of a major Moroccan
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Out of the Atlas Mountains complex of lineages support the idea of a distribution
The observed diversity patterns suggest that for route from the Atlas and Anti-Atlas regions in the
Buthus scorpions, the Atlas Mountains represent the eastern direction, in parallel to the southern shore of
primary source for the colonization of other parts of the Mediterranean Sea, beginning in the Pliocene.
North Africa, Mediterranean regions across south-
western Europe, and the Middle East. The populations
sampled beyond the Atlas Mountains are, for the most Across the Saharan Desert and southern
part, genetically nested within mOTUs otherwise exclaves
found in the Atlas. This suggests that the Moroccan The presence of the same genetic lineage in the upper
Atlas Mountains represent the centre of origin, from Draa valley (located south of the High Atlas, between
which different waves of colonization have taken place the eastern Anti-Atlas Mountains and the Jebel Sarhro)
(and not the Atlantic area of Morocco, as suggested and in the Hoggar Mountains (in the centre of the
by Sousa et al., 2012), mainly during the Pliocene, Sahara Desert), both today separated by > 1500 km of
but also during the Pleistocene. An initial northward (often extreme) desert (e.g. McColl et al., 2005), is most
expansion to Iberia might have occurred during the likely to be the result of dispersal during some periods
Messinian Salinity Crisis (5.96–5.33 Mya), when the of the Pleistocene, or even Holocene, when the climate
Mediterranean Sea desiccated (Giraudi, 2004) and was less arid in this region. During such periods, major
thus established a land bridge for species exchange parts of the western Sahara Desert were transformed
between North Africa and Europe (Carranza et al., into semi-deserts or even savannah ecosystems, i.e. the
2006). However, the phylogeographical structure period of the ‘Green Sahara’ (De Noblet-Ducoudré &
within Iberian Buthus scorpions cannot be explained Prentice, 2000; Prentice et al., 2000; Kuper & Kröpelin,
exclusively by this dispersal event alone. It is more 2006). The ecological changes resulting from the more
likely that Iberia was either colonized repeatedly from humid climate (maybe further supported by palaeo-
Morocco, as also suggested by Habel et al. (2012), or, rivers draining from the High Atlas Mountains to
alternatively, that at least one backward colonization what is currently the Algerian central Sahara) are
from Iberia to North Africa has taken place, resulting assumed to have facilitated migrations of Buthus
in the Moroccan Rif Mountain clade splitting from the scorpions from the upper Draa river valley to the
Iberian clade containing the southern and eastern Hoggar Mountains. When performing these dispersal
samples of the peninsula (Fig. 3); this was also events, these animals must have crossed regions that
suggested by Sousa et al. (2012). are extremely arid today and that, as they are today,
Our data also suggest that eastward expansion are very hostile for these scorpions. The lack of a major
occurred multiple times, with starting points in genetic split in these scorpions between one of their
different subcentres of dispersal. Thus, one expansion south-easternmost population groups in Morocco
originated from south of the High Atlas in southern and the Hoggar Mountains (where the taxon is able
Morocco, with the split being dated at a similar to survive today owing to a higher level of humidity
time to the first expansion to Iberia. However, the within these mountains) also corroborates the former
hereon evolving major lineage found in Algeria high permeability of the western Sahara Desert (i.e.
subdifferentiated only in the Pleistocene, and thus during the period of the Green Sahara) for a variety of
considerably later than the one in Iberia, possibly other taxa showing similar phylogeographical patterns
underlining the high importance of the Pleistocene (Carranza et al., 2004; Rangel López et al., 2018).
climatic oscillations (see Ruddiman et al., 1986; Hewitt
2004) for the differentiation of Buthus in Algeria.
Additionally, it is most likely that two expansions Taxonomic consequences
to northern Algeria originated in the Rif region of By sequencing specimens from type locations (or
northern Morocco. These expansions should have a very close to these), we could match sequence data
Pleistocene age; hence, they might also be a result of with species names, assuming that Buthus species
the Pleistocene climatic fluctuations. Alternatively, normally do not co-occur at the same locality and that
these Algerian populations might be the result of one their ranges do not overlap or overlap only slightly
older expansion, in the Pliocene, with Pleistocene (Habel et al., 2012). As such, it is likely that mOTU18
© 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, XX, 1–1310 R. KLESSER ET AL.
represents B. boumalenii Touloun & Boumezzough, in all, these results call for a critical assessment and
2011 (hence greatly expanding the known distribution revision of the genus Buthus in the future.
for this species) and that mOTU09 coincides with
B. lienhardi.
The mOTU15 contains specimens separated
ACKNOWLEDGEMENTS
by > 1500 km (see above) that were assigned
morphologically to B. draa (samples from southern We thank Thorsten Assmann, Marc Meyer, Frank
Downloaded from https://academic.oup.com/biolinnean/advance-article/doi/10.1093/biolinnean/blab014/6169371 by guest on 15 March 2021
Morocco) and Buthus tassili Lourenço, 2002 (samples E. Zachos and Arie van der Meijden for help in the
from southern Algeria). The type locality of B. draa field and for providing samples. We thank Robert
is geographically close to two of our sample sites, Paxton for access to the molecular laboratory at the
and several more of our sample sites fit well with Martin-Luther-University Halle-Wittenberg. This
the known distribution of the species (Lourenço & research was funded by the Fonds National de la
Slimani, 2004). Our samples of B. tassili coincide Recherche Luxembourg (FNR). P.S. was funded by
with the distribution of the species in the Hoggar project PORBIOTA, Portuguese E-Infrastructure for
Mountains as deduced from the studies by Vachon Information and Research on Biodiversity (POCI-
(1952) and Gysin (1969), although the species is also 01-0145-FEDER-022127), supported by Operational
found further east, in the Tassili n’Ajjer Mountains. Thematic Program for Competitiveness and
Both taxa share the diagnostic feature of a darkened Internationalization (POCI), under the PORTUGAL
fifth metasomal segment, a feature not very common 2020 Partnership Agreement, through the European
in Buthus scorpions, but also shared with another Regional Development Fund (FEDER). We thank
species distributed in the western Sahara region, i.e. in two anonymous referees for sharing their helpful
geographical proximity to these two taxa. Our finding comments on previous versions of the paper.
of a common genetic lineage for these morphologically
similar taxa allows us to argue about a possible
conspecificity with B. draa, potentially representing a
junior synonym of B. tassili. However, a final decision REFERENCES
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Table S1. Overview of Buthus samples collected.
Table S2. Overview of types.
Figure S1. Output graphic of DEC analysis from RASP from BEAST tree (COI).
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