Acta Tropica 134 (2014) 4351

Contents lists available at ScienceDirect

Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Deep intraspecic divergences in the medically relevant fat-tailed

scorpions (Androctonus, Scorpiones)
P. Coelho a , P. Sousa a,b , D.J. Harris a,b , A. van der Meijden a,
a
b

CIBIO, InBio, Centro de Investigaco em Biodiversidade e Recursos Genticos, Universidade do Porto, Campus Agrrio de Vairo, Vairo 4485-661, Portugal
Departamento de Biologia, Faculdade de Cincias, Rua do Campo Alegre, Porto FC4 4169-007, Portugal

a r t i c l e

i n f o

Article history:
Received 9 October 2013
Received in revised form 28 January 2014
Accepted 1 February 2014
Available online 11 February 2014
Keywords:
Scorpionism
Androctonus
Scorpions
Phylogeny
Biogeography
Cryptic diversity

a b s t r a c t
The genus Androctonus, commonly known as fat-tailed scorpions, contains 22 species distributed from
Togo and Mauritania in the west, North Africa, through the Middle East and to as far east as India. With
13 species, a substantial amount of this genus diversity occurs in North Africa, which is a major hotspot
of scorpion sting incidents. Androctonus are among the most medically relevant animals in North Africa.
Since venom composition within species is known to vary regionally, the improvement of therapeutic
management depends on a correct assessment of the existing regional specic and sub-specic variation.
In this study, we assessed the phylogeographical patterns in six species of Androctonus scorpions from
North Africa using mitochondrial DNA markers. We sequenced COX1, 12S, 16S and ND1 genes from
110 individuals. Despite lacking basal resolution in the tree, we found taxonomical and geographically
coherent clades. We discovered deep intraspecic variation in the widespread Androctonus amoreuxi
and Androctonus australis, which consisted of several well-supported clades. Genetic distances between
some of these clades are as high as those found between species. North African A. australis have a deep
split in Tunisia around the Chott el-Djerid salt-lake. A novel split between A. amoreuxi scorpions was
found in Morocco. We also found deep divergences in Androctonus mauritanicus, corresponding to areas
attributed to invalidated subspecies. In addition we uncovered a clade of specimens from coastal south
Morocco, which could not be ascribed to any know species using morphological characters. Based on
these ndings we recommend a reassessment of venom potency and anti-venom efcacy between these
deep intraspecic divergent clades.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Worldwide, 1.2 million people are stung by scorpions every year.
Scorpionism, dened as the severe to lethal incident as a consequence of a scorpion sting (Lourenco and Cuellar, 1995) may be
responsible for 3250 global annual mortalities which are mostly
concentrated in a few high-risk areas (Chippaux and Goyffon,
2008). North Africa in particular is considered a high-risk area for
scorpionism (Chippaux and Goyffon, 2008), with the genera Leiurus Ehrenberg, 1828 and Androctonus Ehrenberg, 1828 being the
foremost cause of serious envenomation in this area (Goyffon and
Guette, 2005; Graham, 2011; Habermehl, 1994). Five Androctonus
species are considered as dangerous to man, particularly Androctonus mauritanicus (Pocock, 1902) and thewidespread Androctonus

Corresponding author. Tel.: +351 916712100.

E-mail address: mail@arievandermeijden.nl (A. van der Meijden).
http://dx.doi.org/10.1016/j.actatropica.2014.02.002
0001-706X/ 2014 Elsevier B.V. All rights reserved.

australis (Linnaeus, 1758), which are the most dangerous Androctonus in the Maghreb region (Morocco, Algeria, Tunisia) (Goyffon
and Guette, 2005). A. australis is known for envenomating humans
and possessing a high toxicity (LD50 = 0.32 mg/kg in mice; Watt
and Simard, 1984). For this reason, A. australis was one of the rst
species of scorpions to have its venom puried for neurotoxin characterization (Miranda et al., 1966). As in snakes (Daltry et al., 1996;
Prasad et al., 1999), scorpion venom is known to have considerable intraspecic regional variation in composition (Devaux et al.,
2004; El Ayeb and Rochat, 1985; Newton et al., 2007; Smertenko
et al., 2001), and thus a different response to antivenom treatment
(Omran and McVean, 2000). Furthermore, other species such as
Androctonus amoreuxi (Audouin, 1826) may also cause more cases
of scorpionism than currently thought (Goyffon et al., 2012). It
is therefore important to study the phylogeographical patterns of
Androctonus over a great part of their distribution as it may have
direct applications in therapeutic management.
Androctonus is present in deserts and semi-arid regions from
Togo to Morocco in the Atlantic coast of Africa (Lourenco and

44

P. Coelho et al. / Acta Tropica 134 (2014) 4351

Qi, 2007; Lourenco, 2008) to the Maghreb countries and Egypt

where they are also present in relatively elevated areas like the
Atlas Mountains and the Sinai Peninsula mountain range (Vachon,
1952), the Middle East (Levy and Amitai, 1980), reaching across
Afghanistan (Vachon, 1958) to India (Tikader and Bastawade, 1983).
Little is known about the biogeography of Androctonus, although
the radiation of buthids has been associated with the aridication
of the Palearctic Region (Fet et al., 2003, 1998).
In this work, we assess ve species of the Maghrebian countries
except for Libya, plus Egypt and the Sinai Peninsula. A. mauritanicus occurs in Morocco. This country shares a further three species
with Algeria, Tunisia and/or Egypt (Androctonus liouvillei, A. australis and A. amoreuxi), while Androctonus bicolor occurs in Algeria
and Tunisia. A. amoreuxi is known to occur mostly in sandy deserts
while A. australis and A. mauritanicus can be found in anthropogenic
environments (Stockmann and Ythier, 2010). The country in our
area of study with the most species of Androctonus, Morocco, is also
the most orographically diverse. The mountain chains that subdivide Morocco, North to south the Rif Mountains, Middle Atlas, High
Atlas and Anti-Atlas Mountains, are known to be important in the
diversication of the scorpion genus Buthus (Husemann et al., 2012;
Sousa et al., 2012; Pedroso et al., 2013). Similarly, the Tell Atlas
Mountains and Aurs Mountains of Algeria and Tunisia are associated with Buthus (Pedroso et al., 2013). Although Androctonus are
generally not as orophilic as Buthus, these mountain chains may
form a barrier for dispersal of the lowland species. However, at least
one Maghreb Androctonus species is known to be associated with
the Hoggar mountains in Southern Algeria; Androctonus hoggarensis
(Pallary, 1929)
The scorpion genus Androctonus was rst described by Ehrenberg in 1828. Vachon (1952) stabilized the genus taxonomy,
transforming it into a morphological and geographical coherent
group with seven species known in North Africa. Lourenco (2005)
produced an important taxonomical revision of the genus: the subspecies of A. australis and A. mauritanicus were no longer considered
valid, Androctonus crassicauda gonneti Vachon, 1948 was raised to
the status of species, Androctonus aeneas C. L. Koch, 1839 was placed
in the synonymy of A. bicolor Ehrenberg, 1828 and A. liouvillei (Pallary, 1924) was raised to the species level.
Recently, molecular tools have been used to assess the phylogeny of Androctonus in Tunisia. Ben Ali et al. (2000), using nuclear
DNA ITS regions (ITS-rDNA), found paraphyletic clades in three

well-accepted taxa (A. bicolor, A. australis and A. amoreuxi). Ben

Othmen et al. (2004), using allozymic differentiation, found little
support for the monophyly of A. australis and A. amoreuxi individually. However three well-supported monophyletic lineages using
16S-rDNA, each corresponding to a species were recovered in a subsequent study, thus demonstrating the usefulness of this gene as
a barcoding marker (Ben Othmen et al., 2009). Furthermore, they
found a phylogeographic pattern of A. australis in Tunisia, where
each of its two lineages are distributed to the north or south of
the Chott el-Djerid salt lake, situated in central Tunisia. However
their molecular dating makes it unlikely that the salt lake formation has generated a vicariant evolution of Tunisian A. australis. In
recent years, molecular studies using the mitochondrial COX1 gene
already uncovered considerable cryptic diversity in other scorpion
genera in the Maghreb region (Gantenbein and Largiadr, 2003;
Sousa et al., 2012, 2011, 2010). However, a molecular phylogenetic
study of the medically relevant scorpions of the genus Androctonus
across the Maghreb region has thus far not been performed.
In this study, we assess the patterns of diversity estimated
from COX1, 16S and 12S sequence data across the Maghreb region
and Egypt. A separate reduced dataset was also made from ND1
sequences. We here provide the rst molecular phylogeny for
Androctonus scorpions in Morocco, Algeria and Egypt.
2. Materials and methods
2.1. Taxon sampling
We collected samples in the eld in three countries: Morocco,
Tunisia and Egypt (Fig. 1). Algerian samples were donated by Dr.
Said Larbes. Additional specimens were purchased through the
pet-trade for which no locality data were available other than the
country of origin. Sampling sites are illustrated in Fig. 1 and further details are provided in Table 1. Scorpions were captured with
long forceps and preserved in 96% ethanol. To minimize the impact
on scorpion populations, non-lethal sampling methods were used
when possible, and consisted of removing the distal part of one
of the second leg pairs. The scorpion was subsequently placed
in the sand, facilitating the clotting process and thus minimizing haemolymph loss. Scorpions can recover partially amputated
appendages after a few molts and it is common for scorpions to be
active and functional even when missing appendages (pers. obs.).

Fig. 1. Map representing the sampling locations across North Africa of Androctonus scorpions. Inset shows Egyptian samples. Pet trade acquired samples were without locality
data, and are not shown. Symbols correspond to the phylogenetic clades (see Fig. 2).

P. Coelho et al. / Acta Tropica 134 (2014) 4351

45

Table 1
Geographical referencing of the sampled specimens, their voucher identiers and respective countries of origin. Coordinates are in the WGS84 datum, in decimal degrees.
M.D. indicates missing data.
Taxon

A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. amoreuxi
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. australis
A. bicolor
A. bicolor
A. bicolor

Country

Morocco
Algeria
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Algeria
Algeria
Morocco
Morocco
Morocco
Tunisia
Tunisia
Morocco
Morocco
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Algeria
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Tunisia
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Egypt
Algeria
Algeria
Tunisia

Clade

AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM1
AM2
AM2
AM2
AM2
AM2
AM2
AM2
AM2
AM2
AM3
AM3
AM3
AM3
AM3
AM3
AU1
AU1
AU1
AU1
AU1
AU1
AU1
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU2
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU3
AU4
AU4
AU4
AU4
AU4
AU4
AU4
AU4
B
B
B

Latitude

30.176
30.907
31.143
28.250
28.446
28.773
28.606
29.046
29.148
29.060
29.727
29.630
29.680
32.440
32.440
32.476
32.505
31.143
33.943
33.943
32.476
33.892
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
32.440
32.440
32.440
32.440
32.440
32.440
32.440
35.115
35.170
35.368
35.368
34.334
34.334
34.334
33.943
33.943
33.943
33.943
33.943
33.943
33.527
33.527
33.527
33.536
33.533
33.533
33.533
32.785
32.785
32.785
33.650
33.650
33.650
M.D.
31.279
31.279
28.809
28.822
28.822
M.D.
M.D.
35.208
35.208
33.846

Longitude

6.875
3.997
4.387
9.333
9.373
9.459
9.430
8.777
8.605
8.852
7.975
8.010
7.982
3.740
3.740
1.721
1.502
4.022
8.034
8.034
1.721
2.019
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
3.740
3.740
3.740
3.740
3.740
3.740
3.740
5.182
2.217
2.055
2.055
8.579
8.579
8.579
8.034
8.034
8.034
8.034
8.034
8.034
8.790
8.790
8.790
9.522
9.991
9.991
9.991
10.373
10.373
10.373
10.317
10.317
10.317
M.D.
27.055
27.055
34.228
34.182
34.182
M.D.
M.D.
1.541
1.541
10.128

Sc code

304
450
808
1472
1479
1485
1487
1489
1491
1493
1503
1507
1508
423
424
791
794
807
913
920
1056
1403
627
671
770
1165
1375
1413
416
417
418
419
420
421
422
378
382
385
386
903
904
905
914
915
916
917
918
919
922
923
924
928
932
933
934
938
939
940
948
950
951
626
954
957
981
992
993
1374
1414
377
383
947

Accession numbersa
12S

16S

COI

ND1

KJ538271
KJ538273
KJ538277
KJ538280
KJ538284
KJ538288
KJ538292
KJ538295
KJ538299
KJ538303
KJ538307
KJ538311
KJ538315
KJ538421
KJ538425
KJ538428
KJ538431
KJ538434
KJ538437
KJ538441
KJ538445
KJ538448
KJ538476
KJ538479
KJ538481
KJ538484
KJ538487
KJ538490
KJ538337
KJ538340
M.D.
KJ538345
KJ538348
M.D.
KJ538354
KJ538385
KJ538387
KJ538390
KJ538393
KJ538395
KJ538398
M.D.
KF824968
KJ538406
KJ538409
KJ538412
KJ538415
KJ538418
KJ538150
KJ538153
KJ538156
KJ538158
KJ538160
KJ538163
KJ538166
KJ538169
KJ538172
KJ538175
KJ538178
KJ538182
KJ538185
KJ538451
M.D.
KJ538457
KJ538459
KF548101
KJ538464
KJ538468
KJ538472
KF548106
KJ538321
KJ538323

KJ538272
KJ538274
KJ538278
KJ538281
KJ538285
KJ538289
KJ538293
KJ538296
KJ538300
KJ538304
KJ538308
KJ538312
KJ538316
KJ538422
KJ538426
M.D.
KJ538432
KJ538435
KJ538438
KJ538442
KJ538446
KJ538449
KJ538477
M.D.
KJ538482
KJ538485
KJ538488
KJ538491
KJ538338
KJ538341
KJ538343
KJ538346
KJ538349
KJ538352
KJ538355
M.D.
KJ538388
M.D.
KJ538394
KJ538396
KJ538399
KJ538402
KF825083
KJ538407
KJ538410
KJ538413
KJ538416
KJ538419
KJ538151
KJ538154
M.D.
KJ538159
KJ538161
KJ538164
KJ538167
KJ538170
KJ538173
KJ538176
KJ538179
KJ538183
KJ538186
M.D.
KJ538454
M.D.
M.D.
KJ538461
KJ538465
KJ538469
KJ538473
KJ538319
KJ538322
KJ538324

M.D.
KJ538275
KJ538279
KJ538282
KJ538286
KJ538290
KJ538294
KJ538297
KJ538301
KJ538305
KJ538309
KJ538313
KJ538317
KJ538423
KJ538427
KJ538429
KJ538433
KJ538436
KJ538439
KJ538443
KJ538447
KJ538450
KJ538478
KJ538480
KJ538483
KJ538486
KJ538489
KJ538492
KJ538339
KJ538342
KJ538344
KJ538347
KJ538350
KJ538353
KJ538356
KJ538386
KJ538389
KJ538391
M.D.
KJ538397
KJ538400
KJ538403
KF825024
KJ538408
KJ538411
KJ538414
KJ538417
KJ538420
KJ538152
KJ538155
KJ538157
M.D.
KJ538162
KJ538165
KJ538168
KJ538171
KJ538174
KJ538177
KJ538180
KJ538184
KJ538187
KJ538452
KJ538455
KJ538458
KJ538460
KJ538462
KJ538466
KJ538470
KJ538474
KJ538320
M.D.
KJ538325

M.D.
KJ538276
M.D.
KJ538283
KJ538287
KJ538291
M.D.
KJ538298
KJ538302
KJ538306
KJ538310
KJ538314
KJ538318
KJ538424
M.D.
KJ538430
M.D.
M.D.
KJ538440
KJ538444
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
KJ538351
M.D.
M.D.
M.D.
M.D.
KJ538392
M.D.
M.D.
KJ538401
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
KJ538181
M.D.
M.D.
KJ538453
KJ538456
M.D.
M.D.
KJ538463
KJ538467
KJ538471
KJ538475
M.D.
M.D.
KJ538326

46

P. Coelho et al. / Acta Tropica 134 (2014) 4351

Table 1 (Continued )
Taxon

Country

Clade

Latitude

Longitude

Sc code

A. bicolor
A. bicolor
A. bicolor
A. cf. gonneti
A. cf. Gonneti
A. cf. Gonneti
A. cf. Gonneti
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. liouvillei
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus
A. mauritanicus

Tunisia
Egypt
Egypt
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Morocco
Algeria
Morocco
Morocco
Morocco

B
B
B
G
G
G
G
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
M1
M1
M1
M1
M1
M1
M2
M2
M2
M2
M2
M2
M3
M3
M3
M3

33.650
M.D.
M.D.
28.479
28.479
28.479
28.479
30.668
34.286
34.286
33.892
33.213
32.087
32.571
32.571
33.289
33.508
33.892
32.087
33.213
33.892
33.892
32.225
32.526
32.661
32.661
32.661
M.D.
30.183
30.183
30.183
30.159
30.059
30.098
29.068
29.087
29.087
29.087

10.317
M.D.
M.D.
11.212
11.212
11.212
11.212
6.380
3.169
3.169
2.019
2.019
1.241
2.015
2.015
3.780
3.528
2.019
1.241
2.019
2.019
2.019
8.166
7.863
7.793
7.793
7.793
M.D.
9.580
9.580
9.580
8.481
9.084
8.938
10.248
9.898
9.898
9.898

949
1093
1371
1453
1454
1455
1456
210
780
781
786
787
793
796
797
816
817
830
839
1055
1207
1394
15
287
290
291
292
625
1467
1468
1469
1549
1558
1589
438
1443
1444
1445

Voucher specimens were collected at almost all sites. All specimens

are deposited in the collection of CIBIO, Centro de Investigaco
em Biodiversidade e Recursos Genticos, Universidade do Porto,
Vairo, Vila do Conde, Portugal.
Some key species that could not be sampled in the eld,
e.g. Egyptian A. bicolor, were obtained through reputable animal
traders. Morphological identication was done in the lab using
keys by Vachon (1952) and Lourenco (2005). A single specimen of
Opisthacanthus asper (Peters, 1861) (Scorpiones: Liochelidae) was
used as outgroup (details provided in Table 1).

2.2. DNA extraction and PCR conditions

Fresh or preserved leg muscle tissue (or metasoma muscle
for smaller specimens) was used for DNA extraction. Dissection
occurred, preferentially, from the third leg in order to minimize the
loss of important taxonomical characters. Total DNA was extracted
using proteinase K digestion (10 mg/ml concentration) followed by
a standard salt extraction protocol (Bruford et al., 1992).
Four mitochondrial fragments were amplied: 16S ribosomal
RNA (16S rRNA), 12S ribosomal RNA (12S rRNA), cytochrome C
oxidase subunit 1 (COX1) and NADH dehydrogenase 1 (ND1) (see
Table 2). Polymerase chain reactions were performed in a nal volume of 25 L and using 1.0 L each of 10 pmol primer, 12.5 L
REDTaq ReadyMix PCR Reaction Mix with MgCl2 (SigmaAldrich,
St. Louis, USA), 1.0 L of the puried DNA and 9.5 L of water. Difcult PCR reactions were amplied with a touchdown PCR approach.
Puried PCR templates were unidirectionally sequenced using

Accession numbersa
12S

16S

COI

ND1

KJ538327
KJ538331
KJ538335
KJ538372
KJ538375
KJ538379
KJ538383
KJ538188
KJ538190
KJ538193
KJ538196
KJ538199
KJ538202
KJ538206
KJ538209
KJ538213
KJ538216
KJ538218
KJ538221
KJ538224
KJ538227
KJ538230
KJ538253
KJ538257
KJ538260
KJ538263
KJ538267
KJ538269
KJ538233
KJ538237
KJ538241
KJ538244
KJ538247
KJ538251
KJ538357
KJ538360
KJ538364
KJ538368

KJ538328
KJ538332
KJ538336
KJ538373
KJ538376
KJ538380
KJ538384
KJ538189
M.D.
KJ538194
KJ538197
KJ538200
KJ538203
KJ538207
KJ538210
KJ538214
KJ538217
KJ538219
KJ538222
KJ538225
KJ538228
KJ538231
KJ538254
KF825084
KJ538261
KJ538264
KJ538268
M.D.
KJ538234
KJ538238
KJ538242
KJ538245
KJ538248
KJ538252
KJ538358
KJ538361
KJ538365
KJ538369

KJ538329
KJ538333
M.D.
KJ538374
KJ538377
KJ538381
M.D.
M.D.
KJ538191
KJ538195
KJ538198
KJ538201
KJ538204
KJ538208
KJ538211
KJ538215
M.D.
KJ538220
KJ538223
KJ538226
KJ538229
KJ538232
KJ538255
KJ538258
KJ538262
KJ538265
M.D.
KJ538270
KJ538235
KJ538239
KJ538243
M.D.
KJ538249
M.D.
KJ538359
KJ538362
KJ538366
KJ538370

KJ538330
KJ538334
M.D.
M.D.
KJ538378
KJ538382
M.D.
M.D.
KJ538192
M.D.
M.D.
M.D.
KJ538205
M.D.
KJ538212
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
M.D.
KJ538256
KJ538259
M.D.
KJ538266
M.D.
M.D.
KJ538236
KJ538240
M.D.
KJ538246
KJ538250
M.D.
M.D.
KJ538363
KJ538367
KJ538371

dye-labeled dideoxy terminator cycle sequencing on an ABI 3730XL

at Macrogen Inc. The sequencing primers were the same as those
used in the PCRs.

2.3. Data analysis

Chromatographs were checked manually for sequencing errors
using FinchTV 1.4.0 (Geospiza, Inc.; Seattle, USA). Sequences were
edited using MEGA 5 (Tamura et al., 2011) and aligned using default
settings of MUSCLE (Edgar, 2004) for non-coding sequences and
ClustalW (Larkin et al., 2007) for COX1 and ND1.
Phylogeny reconstruction was performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. A homogeneity
partition test executed in Paup* 4.0 rejected homogeneity of the
different genes. We therefore carried out both a partitioned and
a combined analysis. The best tting models of sequence evolution were determined by the AIC criterion in JModeltest 2.1.2
(Darriba et al., 2012) both for the separate genes and the combined dataset. ML tree searches were performed using PhyML,
version 3.0 (Guindon et al., 2010). Bootstrap branch support values
were calculated with 1000 replicates. The BI analysis was conducted with MrBayes 3.2.2, with 5,000,000 generations, sampling
trees every 10th generation (and calculating a consensus tree after
omitting the rst 12,500 trees). Mean genetic distances between
and within clades were calculated with MEGA5 based on COX1
sequences. Variance was estimated with 4000 bootstrap replicates,
with uncorrected p-distances (Table 3). GenBank accession numbers are given in Table 1.

P. Coelho et al. / Acta Tropica 134 (2014) 4351

47

Table 2
List of primers and PCR conditions used for molecular analyses. PCR conditions start with temperature ( C) of each step followed by the time in seconds in brackets.
Gene

Primer name

Sequence (5 3 )

Source

PCR conditions

16S rRNA

18-mer (forward)
20-mer (reverse)

CGATTTGAACTCAGATCA
GTGCAAAGGTAGCATAAT

Simon et al. (1994)

Gantenbein et al. (2000)

94(180), [94(30), 50(45), 72(60)]

35, 72(300), 12()

12S rRNA

12S-F AvdM
12S-R AvdM

AGAG-TGACGGGCAATATGTG
CAGCGGCTGCGGTTATAC

Van der Meijden et al. (2012)

94(180), [94(30), 52(45), 72(60)]

35, 72(300), 12()

COI

LCO1490 (forward)
HCO219 (reverse)
COI avdm F
COI avdm R

GGTCAACAAATCATCATAAAGATATTGG
TAAACTTCAGGGTGACCAAAAAATCA
WTYCTACIAATCAYAARGATATTGG
TAMACYTCIGGGTGWCCAAAAAAYCA

ND1-LR-N-12945 (forward)
ND1-N1-J-12261 (reverse)

CGACCTCGATGTTGAATTAA
TCGTAAGAAATTATTTGAGC

ND1

In addition we downloaded all 16S rDNA sequences in Ben

Othmen et al. (2009) from GenBank and we combined these with
our 16S dataset. Phylogeny reconstruction of this extended dataset
was performed with ML (tree not shown).

3. Results
3.1. Sequence data
We sequenced 110 Androctonus specimens and one outgroup (O.
asper) (Table 1). The combined dataset consists of an alignment of
1457 basepairs (bp) (606 bp of COX1, 374 bp of 16S rRNA and 477 bp
of 12S rRNA). The dataset included 777 variable sites (53.3% of the
total nucleotide positions), 680 bp were constant (46.7%) and 511
positions were parsimony informative (35.1%). Due to difculties
in amplifying ND1 only 41 sequences were available, and thus this
gene was not included in the combined analysis.

3.2. Overall phylogeny

Four of the six species were recovered as monophyletic (Fig. 2).
We identied 13 clades that correspond to biogeographical and/or
taxonomical units. Most of the deeper branches present low values
of bootstrap support (ML) and posterior probability (BI). Consequently, the relationships between species could not always
be resolved. ML and BI trees using a dataset that included ND1
sequences (not shown) did not differ in the topology of wellsupported clades. Trees derived from the individual genes did not
contradict the groupings recovered from the concatenated dataset.

Folmer et al. (1994)

Van der Meijden et al. (2012)

94(180), [94(30), 48(45), 72(60)]

35, 72(300), 12()
94(180), [94(30), 49(45), 72(60)]
35, 72(300), 12()
94(180), [94(30), 47(45), 72(60)]
35, 72(300), 12()

Hedin (1997)

3.3. Species-level clades

A. amoreuxi, is divided into three clades: clades AM1 and
AM2 contain specimens from Morocco, clade AM2 also contains
specimens from Tunisia and Algeria and clade AM3 consists of individuals from Egypt. Clades AM1 and AM2 are placed as sister groups
with a high support (BI = 1.00; ML = 87) and the AM3 clade has no
well-supported afnities but may be sister taxon to A. australis. The
western part of the range, from Morocco to Tunisia, is divided into
two clades; a northeastern (AM2) and a southwestern clade (AM1);
the Southern Moroccan amoreuxi (AM1) clade extends South, from
the regions of Assa and Zag in the South, to the southern area of
the Anti-Atlas Mountains and North toward Taouz, in the Erfoud
area, close to the MoroccoAlgerian border (Fig. 1). The A. amoreuxi
clade AM2 groups specimens from three countries: in Morocco it
is distributed in the Erfoud area and also to the areas surrounding
Bouarfa and Ain-Beni-Mathar (High Plateau in Northeast Morocco);
In Algeria it is represented in one location, Ghardaia (between the
Great Ergs); in Tunisia it is represented in one location, close to
Tozeur (between Chott el-Gharsa and Chott el-Djerid salt lakes).
The third clade, AM3 groups specimens that were acquired via the
pet-trade from Egypt, none of which are georeferenced. Pairwise
genetic distances between these clades are in a range between
7.5% and 9.2% (Table 3). Downloaded 16S sequences of A. amoreuxi
are split between clades AM1 and AM2 with a sequence from Ben
Othmen et al. (2009) (andr05) placed in the AM2 clade whereas the
sequence of Fet et al. (2003) is placed within our AM1 clade (tree
not shown).
Clade L (the liouvillei clade) unites all Moroccan A. liouvillei
scorpions. A total of 15 specimens were collected, ranging from
the High-Atlas Mountains, to the Northeastern range of the

Table 3
Genetic distances between and within groups. Numbers below the diagonal are mean p-distances calculated between clades whereas the corresponding variances, based on
4000 bootstrap replicates, are shown above the diagonal. Mean p-distances within clades are shown to the right.
Distance between clades
AM1
AM2
AM3
AU1
AU2
AU3
AU4
B1
B2
B3
L
M1
M2
M3
G

0.010
0.075
0.092
0.090
0.087
0.090
0.094
0.088
0.077
0.094
0.087
0.084
0.110
0.103
0.101
AM1

0.089
0.102
0.102
0.098
0.093
0.086
0.082
0.100
0.081
0.098
0.103
0.104
0.087
AM2

Within clades

0.011 0.011
0.011 0.012
0.012
0.090
0.093 0.039
0.097 0.061
0.089 0.080
0.096 0.093
0.100 0.093
0.099 0.106
0.096 0.099
0.095 0.087
0.115 0.109
0.097 0.107
0.111 0.100
AM3

AU1

0.011
0.011
0.011
0.007
0.069
0.080
0.090
0.087
0.099
0.099
0.087
0.108
0.102
0.104
AU2

0.011
0.012
0.011
0.009
0.010

0.011
0.012
0.011
0.011
0.010
0.009

0.011
0.010
0.012
0.012
0.012
0.012
0.013

0.010
0.011
0.012
0.011
0.011
0.011
0.011
0.008

0.011
0.011
0.011
0.012
0.012
0.011
0.012
0.011
0.010

0.010
0.010
0.011
0.012
0.011
0.012
0.012
0.011
0.011
0.011

0.010
0.011
0.011
0.011
0.011
0.010
0.011
0.012
0.011
0.010
0.011

0.011
0.011
0.012
0.012
0.011
0.011
0.012
0.011
0.011
0.011
0.011
0.011

0.011
0.011
0.011
0.012
0.011
0.011
0.012
0.012
0.011
0.011
0.011
0.011
0.010

0.056
0.097
0.087
0.098
0.108
0.081
0.099
0.100
0.096

0.108
0.096
0.107
0.108
0.096
0.116
0.107
0.102

0.042
0.079
0.087
0.085
0.096
0.109
0.109

0.074
0.085
0.080
0.099
0.104
0.109

0.083
0.090
0.094
0.095
0.106

0.096
0.099
0.098
0.085

0.105
0.093
0.111

0.090
0.104

0.088

AU3

AU4

B1

B2

B3

M1

M2

M3

0.012
0.011
0.013
0.012
0.012
0.012
0.012
0.012
0.013
0.012
0.011
0.012
0.011
0.011
G

0.016
0.013
0.003
0.004
0.012
0.009
0.004
n/c
0.010
n/c
0.008
0.012
0.023
0.014
0.004

0.002
0.003
0.001
0.002
0.003
0.002
0.001
n/c
0.004
n/c
0.002
0.003
0.004
0.003
0.002

p-Distance

St. err.

A1
A2
A3
AU1
AU2
AU3
AU4
B1
B2
B3
L
M1
M2
M3
G

48

P. Coelho et al. / Acta Tropica 134 (2014) 4351

Fig. 2. Bayesian estimate of phylogenetic relationships of Androctonus (outgroup not shown). Posterior probabilities values and bootstrap support values are shown above
and below nodes respectively. These samples are marked with the same colors and shapes in all gures.

P. Coelho et al. / Acta Tropica 134 (2014) 4351

Middle-Atlas and to the High Plateau along the Algerian borderland.

Average genetic distance within the clade is of 0.8%. It appears to
be sister taxon to A. bicolor (ML 85% bootstrap support, BI posterior
probability 0.96).
A. mauritanicus, is presented in three clades; M2 and M3 are
sister clades (BI = 0.60; ML = 80) whereas M1 was unrelated to these.
Specimens from the M1 clade are located in the northern range of
the Marrakech-Tensif-El Haouz administrative region. The specimens from the M2 clade occur in a horizontal stretch from the
peaks of the Anti-Atlas along the Souss valley with the westernmost
sample being 6 km from the seacoast. The M3 clade is comprised of
specimens from two sampling locations, both from the southwest
slope of the Anti-Atlas Mountains (see Fig. 1). Genetic distances
between clades range from 9.0% to 10.5% while the highest genetic
distance within these clades, in clade M2, is 2.3% (Table 3).
A group of four dark-colored Androctonus, which morphologically resemble Androctonus gonneti most closely, are designated
here as A. cf. gonneti (see Section 4). These specimens (clade G) were
found in one sampling point in the southern limit of the Anti-Atlas
Mountains near the mouth of the Oued Draa.
A. australis forms a monophyletic unit, divided into four clades.
The AU3 (east Tunisian australis) and AU4 (Egyptian australis) clades
are sister groups in the Bayesian tree (BI = 0.80), which is not the
case in the ML analysis. Clades AU1 (Algerian australis) and AU2
(northern Algeria and west Tunisian australis) are sister groups in
both trees (BI = 0.95; ML = 99). Starting from the East, the AU4 clade
is exclusive to Egypt. Some of the specimens present in this clade
are not geographically referenced, as they were obtained through
the pet-trade. The AU3 clade (East Tunisian australis) is distributed
solely in Tunisia: specimens range Southeast of the Chott el-Djerid
salt lake, north to the Chot el-Fejaj salt lake and as far south as
the Tataouine desert region (Fig. 1). The AU2 clade is distributed
from Algeria in the east, and westward to the Chott el-Djerid and
Chot el-Gharsa salt lakes in Tunisia. The AU1 clade is comprised of
specimens from a single location between the Great Ergs of Algeria (Fig. 1). Pairwise genetic distances between these clades range
from 3.9% to 8.0%. The AU1 and AU4 clades present the lowest
values of within group genetic distance (0.4%). Among A. australis,
the highest within group distance was found in clade AU2 (mean
p-distance = 1.2%). Our samples from the Sinai Peninsula conrm
former reports on this species occurrence in the Sinai (Levy and
Amitai, 1980). The extended 16S dataset shows that Ben Othmen
et al. (2009)s northern and southern lineage sequences are placed
throughout the AU2 and AU3 clades respectively (tree not shown).
The B clade (bicolor clade) unites all A. bicolor specimens.
Notably, this clade has longer branches within it than any of the
other clades in this study. For this reason we divided this clade
into three subclades (B1, B2 and B3) and quantied the genetic distances between them. A. bicolor is present in the Tell Atlas in Algeria,
central Tunisia (Fig. 1) and Egypt (no GPS point available). Genetic
distances between these subclades range from 4.2% to 7.9%. These
subclades were too small for mean p-distance within clades to be
calculated.

4. Discussion
Genetic methods, using one or very few mitochondrial genes,
have proven very successful in uncovering cryptic diversity in North
African scorpions (Froufe et al., 2008; Sousa et al., 2012, 2011).
Despite using three genes with a high number of informative sites,
the dataset did not provide sufcient resolution above the species
level. The relationships among these species therefore could not
be resolved. Species-level clades, however, were well-resolved and
received high support, and we resolved several novel and biogeographically coherent clades within the species.

49

For the past 20 M.a. the paleoclimatic history of North Africa

has been characterized by drastic and oscillating events. The
Mediterranean basin experienced extreme changes from the closing of the Mediterranean Seas eastern end (1519 M.a.) to its
isolation from the Atlantic Ocean leading to the Messinian salinity crisis (5.965.33 M.a.) producing signicant sea-level changes.
Afterwards, the violent re-ooding of the Mediterranean basin
submerged large areas of North Africa and created islands in current Morocco and Algeria (Krijgsman et al., 1999; Steininger and
Rogl, 1984). Furthermore, during the last 10 M.a. glacial cycles produced climatic oscillations between wetter and drier conditions in
North Africa. Consequently, the Saharan desert experienced contractions and expansions depending on the formation of heavily
vegetated landscapes or hydrographic systems (Douady et al., 2003;
Schuster et al., 2006). Fragmentation during these climatic oscillations of more xeric habitats (Drake et al., 2011; Le Hourou, 1997),
such as those favored by Androctonus species, may have also contributed to the isolation and subsequent diversication in these
scorpions.
The Atlas Mountains, formed in the mid-late Miocene, are a
major biogeographical barrier, and are thought to be the cause of
diversication in several taxa (e.g. Fonseca et al., 2009; Goncalves
et al., 2012; Habel et al., 2012), and seem to have also played a role in
Moroccan Androctonus. The distribution of the clades of A. mauritanicus suggests that the mountain ranges could act as barriers to gene
ow. A. mauritanicus is found in median and low altitudes (e.g. clade
M2 and M3). The High-Atlas is situated between M1 and M2 clades
and the Anti-Atlas between M2 and M3 clades. It seems from this
pattern that orography might be a probable cause for differentiation
in this species, as is the case in Buthus scorpions (Habel et al., 2012).
Alternatively, the rivers associated with these mountains may have
also formed northsouth barriers to dispersion, thus allowing differentiation within A. mauritanicus. Vachon (1952) described two
subspecies in Morocco: A. m. mauritanicus and A. m. bourdoni. The
distribution of clade M1 largely coincides with the distribution of
the former subspecies, while the distribution of clade M2 coincides
with the distribution of the latter. Although Lourenco (2005) did
not nd evidence to support these two subspecies, the genetic distances between them are at a level of those found between full
species of Androctonus (Table 3). In addition, clades M2 and M3
are also separated by a similar species-level genetic distance. An
exploratory morphological analysis of the specimens of clade M3
found a very granulated prosoma and tergites, which does not t the
description of A. mauritanicus. These morphological characters also
distinguish them from the specimens in clade M2, showing these
scorpions are distinct both genetically and morphologically. However, a more detailed morphological assessment is clearly needed
prior to a taxonomic revision. A. mauritanicus is, together with A.
australis, among the most dangerous scorpions in the Maghreb not
only due to their venom toxicity but also because these species
occurrence often overlaps regions with relatively high human population densities (Chippaux and Goyffon, 2008). Between 1996 and
2006 in Morocco, 53% of deaths that resulted from sting cases
come from incidents with A. mauritanicus (Touloun et al., 2012).
The genetically highly divergent subpopulations of A. mauritanicus
may, similar to A. australis (Devaux et al., 2004) and other scorpion
species (El Ayeb and Rochat, 1985; Newton et al., 2007; Smertenko
et al., 2001), display differences in venom composition. This should
be taken into consideration in both the development of antisera
and the treatment of scorpion stings.
A. liouvillei, although distributed in at least as large an area as
A. mauritanicus, is not as deeply split into separate clades. This
may be due to the less orographically complex area it inhabits. It
is mainly found on the high plateau east of the Middle Atlas, and
eastward into Algeria. Although the A. liouvillei clade is sister to
the A. bicolor clade, genetic distances between them are near the

50

P. Coelho et al. / Acta Tropica 134 (2014) 4351

species-level, corroborating Lourencos (2005) morphology-based

assessment that A. liouvillei is a distinct species from A. bicolor.
A. australis is one of the most widespread scorpions in North
Africa and one of the leading causes of scorpionism in North Africa
(Chippaux and Goyffon, 2008). There is a deep split between clades
AU1 and AU2 on one side, and clades AU3 and AU4 on the other. The
clades in Tunisia (clades AU2 and AU3) are therefore more closely
related to clades distributed in countries as far east as Egypt (AU4)
and as far west as Algeria (AU1) than to each other. Substantial polymorphism for three toxins in the venom of A. australis was reported
in Tunisia (Devaux et al., 2004). Differences were found between
the venoms of specimens from localities corresponding to clades
AU2 or AU3. This shows that it is highly likely that phylogenetically
divergent groups may have different venom compositions in A. australis. The genetic distance also reects a higher distance between
them than between the pairs AU1AU2 and AU3AU4. This suggests either a strong biogeographic barrier between these clades,
or that their current proximity is an area of potential secondary contact. There is a large salt lake, Chott el-Djerid, between the regions
where clades AU2 and AU3 are in closest proximity. Salt lakes as
big as Chott el-Djerid might act as physical barriers for scorpion dispersion (Ben Othmen et al., 2009) and other taxa (Kornilios et al.,
2010). Although Ben Othmen et al. (2009) dealt only with Tunisian
Androctonus, had fewer samples, and used only 16S rRNA data,
they also found a deep divergence between the north-western and
south-eastern Tunisian A. australis. Their northern and southern lineages are genetically and geographically congruent with
our AU2 and AU3 clades respectively. They explored the possibility of the lakes formation causing the vicariant event. However
the relatively recent dates for the lakes formation (90150 ka) do
not coincide with their divergence time estimation (3.439.90 Ma),
and they suggest that the recently formed salt lake acts only as a
barrier to secondary contact, rather than having caused vicariance
between these two populations. Their divergence time estimate
was not based on calibration points, but rather on a constant mutation rate, and should therefore be regarded with caution. Pedroso
et al. (2013) have found a similar high divergence between populations of Buthus scorpions to the North-West and South-East of Chott
el-Djerid. Due to a lack of dependable internal calibration points for
molecular dating, we have refrained from calculating divergence
time estimates in our study, and placing the divergences between
clades and species in a paleogeographic or paleoclimatic context
therefore remains tentative.
Surprising was the nd of two distinct clades of A. amoreuxi
in Morocco. The distribution of clades AM1 and AM2 meet in the
Erfoud area. Although there are no obvious geographical formations
separating them, the genetic distance between clades is 7.5%, which
is similar to the distance recovered between the A. australis clades.
As currently recognized the species appears to be paraphyletic, with
clade AM3 sister to A. australis although with very weak support levels. Although some A. amoreuxi specimens were separated by the
largest geographic distance of any species in this study, we could
not nd morphological differences between them in taxonomically
relevant characters (Lourenco, 2005; Vachon, 1952). The fact that
the genetic distance between clades AM2 and AM3 is relatively high
(8.9%) corroborates the division of A. amoreuxi into two subspecies:
A. a. amoreuxi in the Maghreb and A. a. levyi Fet, 1997 in the Sinai
Peninsula. However, to what taxonomic level these groups should
be ascribed requires further morphological study of the intervening
populations.
Near Tan Tan Plage, a coastal town near the mouth of the Draa
River, we found four specimens morphologically different from
A. mauritanicus and Androctonus sergenti Vachon, 1948 (also, dark
colored species). Morphologically, they seem to be closer related
with A. gonneti. The 4 adult male specimens we captured have the
same pectinal teeth count (33) as reported by Vachon (1952) for A.

gonnetti. A. liouvillei and A. sergenti are reported to have a smaller

pectinal teeth count (Lourenco, 2005; Vachon, 1952). As no other
species are known for that region, we designated the G clade specimens as A. cf gonneti, a species known from this region. Our estimate
of relationships based on mtDNA clearly indicates that these are
distinct from A. liouvillei (Fig. 2). Further sampling will be necessary to study the relationships between this population and other
species from Morocco, but it appears to be A. gonneti, a variant of
this species, or an undescribed new species.
Summarizing, we were able to identify cryptic variation in A.
australis, A. mauritanicus and A. amoreuxi. Some cases were simpler, in which we could corroborate the validity of a distinct
group, such as A. liouvillei. Further work should aim at sequencing nuclear genes in order to resolve deeper relationships within
the genus. It would also be very interesting to obtain samples of
other species like Androctonus aleksandrplotkini, Androctonus hoggarensis, Androctonus maroccanus Lourenco et al., 2009, A. sergenti
and A. gonneti. Those, together with a more extensive sampling of
Algeria, Libya and Mauritania could provide a prime perspective
on the patterns and processes shaping Androctonus evolution and
current distribution in North Africa.

5. Conclusions
We here show that A. australis, A. mauritanicus and A. amoreuxi
have deeply divergent subclades. Such variation can be reected in
the venom these animals produce, as seen in other scorpion species
(Borges et al., 2010; Newton et al., 2007; Omran and McVean, 2000;
Smertenko et al., 2001). We therefore suggest that the venom of A.
australis, A. mauritanicus and A. amoreuxi should be studied with
these deep divergences in mind. Antivenom developed for scorpions in one region should be tested for efcacy against venoms
from regions (Fatani et al., 2010) where scorpions correspond to
different clades as identied here.
Furthermore despite lack of basal resolution, our results have
clear bearing on the taxonomic status of some of the (sub)species
included in our study. In addition, we identied a population of A.
gonneti-like specimens near Tan-Tan (clade G) which merits further
study as a potential new species.

Acknowledgements
We are indebted to Dr. Said Larbes of the Dpartement de Biologie,
Facult des Sciences Biologiques et Agronomiques, Universit M. Mammeri, Tizi-Ouzou, Algeria for supplying important samples from
Algeria. We thank Abdullah M. Nagy of the St. Catherine wildlife
preserve, Srgio Henriques, Diana Pedroso and our colleagues at the
CIBIO institute for assistance in the eld. Thanks to Arendo Flipse
for supplying some of the Egyptian scorpions. This work was funded
by Fundaco para a Cincia e Tecnologia, BIABDE/74349/2006
(to D.J.H.), SFRH/BD/74934/2010 (to P.S.), SFRH/BPD/48042/2008
(A.v.d.M.) and a FCT I&D project (PTDC/BIA-EVF/2687/2012) to
A.v.d.M. DJH is supported by Genomics and evolutionary biology
co-nanced by North Portugal Regional Operational Programme
2007/2013 (ON.2 O Novo Norte), under the NSRF, through the
European Regional Development Fund (ERDF).

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