Intraspecific venom variation in southern African scorpion species of the genera Parabuthus, Uroplectes and Opistophthalmus (Scorpiones: Buthidae, Scorpionidae)

Accepted Manuscript Intraspecific venom variation in southern African scorpion species of the genera Parabuthus, Uroplectes and Opistophthalmus (Scorpiones: Buthidae, Scorpionidae) Stephan Schaffrath, Lorenzo Prendini, Reinhard Predel PII:

S0041-0101(18)30047-3

DOI:

10.1016/j.toxicon.2018.02.004

Reference:

TOXCON 5816

To appear in:

Toxicon

Received Date: 2 November 2017 Revised Date:

7 February 2018

Accepted Date: 11 February 2018

Please cite this article as: Schaffrath, S., Prendini, L., Predel, R., Intraspecific venom variation in southern African scorpion species of the genera Parabuthus, Uroplectes and Opistophthalmus (Scorpiones: Buthidae, Scorpionidae), Toxicon (2018), doi: 10.1016/j.toxicon.2018.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Intraspecific venom variation in southern African scorpion species of the genera Parabu-

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thus, Uroplectes and Opistophthalmus (Scorpiones: Buthidae, Scorpionidae)

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Stephan Schaffratha, Lorenzo Prendinib and Reinhard Predela,*

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a

Institute for Zoology, University of Cologne, D-50674 Cologne, Germany

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b

Division of Invertebrate Zoology, American Museum of Natural History, NY-10024, USA

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*corresponding author: Reinhard Predel, University of Cologne, Institute for Zoology, Zuelpicher

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Straße 47b, D-50674 Cologne, Germany; e-mail: [email protected]

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Keywords: Scorpion, venom mass fingerprinting, intraspecific variation, hierarchical clustering

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Highlights

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- Venom samples of 60 individuals from different populations of four southern African scorpion

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species were analyzed

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- Due to high intraspecific variation, venom fingerprinting by MALDI-TOF MS revealed only a

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few potentially species-specific components

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- Conclusive information about population-specific diversity was obtained by hierarchical clus-

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tering analyses

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- Hierarchical clustering enabled samples to be unambiguously identified to species

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Abstract

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Scorpion venoms comprise cocktails of proteins, peptides, and other molecules used for immobi-

26

lizing prey and deterring predators. The composition and efficacy of scorpion venoms appears to

27

be taxon-specific due to a coevolutionary arms race with prey and predators that adapt at the mo-

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lecular level. The taxon-specific components of scorpion venoms can be used as barcodes for

29

species identification if the amount of intraspecific variation is low and the analytical method is

30

fast, inexpensive and reliable. The present study assessed the extent of intraspecific variation in

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newly regenerated venom collected in the field from geographically separated populations of four

32

southern African scorpion species: three buthids, Parabuthus granulatus (Ehrenberg, 1831),

33

Uroplectes otjimbinguensis (Karsch, 1879), and Uroplectes planimanus (Karsch, 1879), and one

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scorpionid, Opistophthalmus carinatus (Peters, 1861). Although ion signal patterns were general-

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ly similar among venom samples of conspecific individuals from different populations, MALDI-

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TOF mass spectra in the mass range m/z 700–10,000 revealed only a few ion signals that were

37

identical suggesting that species identification based on simple venom mass fingerprints (MFPs)

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will be more reliable if databases contain data from multiple populations. In general, hierarchical

39

cluster analysis (HCA) of the ion signals in mass spectra was more reliable for species identifica-

40

tion than counts of mass-identical substances in MFPs. The statistical approach revealed conclu-

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sive information about intraspecific diversity. In combination with a comprehensive database of

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MALDI-TOF mass spectra in reflectron mode, HCA may offer a method for rapid species identi-

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fication of venom MFPs.

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81 MFPs offer a useful chemotaxonomic tool

53 proteins, peptides, and other molecules 54 used for immobilizing prey and deterring 55 predators

(Casewell

et

al.,

2013;

56 Morgenstern and King, 2013). The compo57 sition and efficacy of scorpion venoms 58 appear to be taxon-specific due to a coevo59 lutionary arms race with prey and predators 60 that adapt at the molecular level (Zhang et

62 specific components of scorpion venoms

63 can be used as barcodes for species identi64 fication if the amount of intraspecific var-

65 iation is low and the analytical method is 66 fast, inexpensive and reliable.

83 information for the differentiation of taxa, 84 i.e., if interspecific variation exceeds intra85 specific variation.

86 However, venom composition, even among 87 genetically identical individuals, may vary 88 depending on the age of animals, time 89 since the previous venom injection, method 90 of venom collection, and other factors. 91 Such variation was described in diverse

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61 al., 2015; Zhijian et al., 2006). The taxon-

82 if mass spectra reveal sufficient diagnostic

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52 Scorpion venoms comprise cocktails of

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51 1. Introduction

92 venomous taxa (Abdel-Rahman et al., 93 2011; Malina et al., 2017), including scor94 pions (Inceoglu et al., 2003; Newton et al., 95 2007; Pimenta et al., 2003; Rodriguez96 Ravelo et al., 2013). Therefore, the repro97 ducibility of mass fingerprints may be 98 more affected by natural variation than by

68 simple

tool for species identification

99 mass spectrometry instrumentation (Fa-

69 (Borges et al., 2006; Debont et al., 1998;

100 vreau et al., 2006). The problem of varia-

70 Dyason et al., 2002; Newton et al., 2007;

101 tion between the venom composition of old

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67 Venom barcoding is a promising and rather

102 vs. newly regenerated venoms (see Pimen-

72 2014; Schwartz et al., 2008). Whereas Or-

103 ta et al., 2003) can be minimized by col-

73 bitrap/ESI-MS analyses of venom samples

104 lecting only newly generated venom after

74 result in unsurpassed information regarding

105 repeated electrical stimulation of the telson

75 number, molecular masses and sequences

106 (Schaffrath and Predel, 2014). This method

76 of venom peptides (Favreau et al., 2006),

107 of venom collection should facilitate the

77 mass fingerprints (MFPs) obtained by ma-

108 reproducibility of mass fingerprints even if

78 trix-assisted

desorption/ionization

109 venom is collected in the field. The present

79 time-of-flight (MALDI-TOF) mass spec-

110 study assessed the extent of intraspecific

80 trometry are cheaper and more efficient.

111 variation in newly regenerated venom col-

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71 Pimenta et al., 2003; Schaffrath and Predel,

laser

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126 Natural History (New York, U.S.A.). The 127 venom of adult scorpions of both sexes 128 was collected in the field by electrical 129 stimulation according to a protocol pre-

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130 sented by (Schaffrath and Predel, 2014). 131 Transcutaneous electrical stimulation was 132 performed with a pulse width of 200–250 133 µs and a pulse rate of 60–130 Hz. The ven-

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134 om of each individual was collected into a Fig. 1. Collection localities of four southern African scorpion species, Opistophthalmus carinatus (Peters, 1861) (circles), Parabuthus granulatus (Ehrenberg, 1831) (triangles), Uroplectes otjimbinguensis (Karsch, 1879) (stars), and Uroplectes planimanus (Karsch, 1879) (diamonds) analyzed in the present study. Colors represent different populations of conspecifics. Codes and corresponding GPS data are listed in Table 1. Topographic map adapted from Global MultiResolution Topography synthesis (Ryan et al., 2009). (color figure in printed version)

135 glass capillary and subsequently trans-

112 lected in the field from geographically sep-

142 was analyzed. The extent of variation in

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136 ferred into a 0.5 ml microtube (Eppendorf, 137 Hamburg, Germany) containing 200 µl 35 138 % ethanol/ 0.1 TFA. Diluted venom sam139 ples were stored at 4 °C. Only newly re140 generated venom, collected 24 h after the 141 first electrical stimulation of the telson,

113 arated populations of four southern African

143 venom samples collected with the same

114 scorpion species: three buthids, Parabu-

144 methods after the first electrical stimula-

115 thus

1831),

145 tion, 24 h later, and seven days later was

(Karsch,

146 tested under laboratory conditions using

granulatus

and

Uroplectes

planimanus

147 Parabuthus villosus (Peters, 1862) from

118 (Karsch, 1879), and one scorpionid, Opis-

148 Uis, Namibia, and U. planimanus, from 30

119 tophthalmus carinatus (Peters, 1861).

149 km NE of Otjiwarongo, Namibia.

120 2. Material and Methods

150 2.2. MALDI-TOF MS analysis

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117 1879),

otjimbinguensis

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116 Uroplectes

(Ehrenberg,

121 2.1. Venom collection and storage

151 0.3 µl of 10 mg/ml 2,5-dihydroxybenzoic

122 Samples of the four scorpion species were

152 acid (Sigma-Aldrich, Steinheim, Germa-

123 collected in Angola, Botswana and Namib-

153 ny), dissolved in 20 % acetonitrile/1 %

124 ia (Fig. 1; Tab. 1). Voucher specimens are

154 formic acid, was used as a matrix and

125 deposited in the American Museum of

155 mixed with the same quantity of stored 4

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173 UltrafleXtreme or ABI 4800 proteomics

157 MALDI-TOF mass spectrometry before air

174 analyzer (Applied Biosystems, Framing-

158 drying at room temperature. MALDI-TOF

175 ham, U.S.A.) in gas-off and gas-on (as-

159 mass spectrometry was performed in re-

176 signment of Leu/Ile ambiguities) mode.

160 flectron positive ion mode on an UltrafleX-

177 Peptide sequences were identified by man-

161 treme

178 ual analysis of fragment ions and subse-

TOF/TOF

162 (Bruker

mass

spectrometer

Daltonik, Bremen, Germany).

179 quent

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156 venom directly on the sample plate for

comparison

of

predicted

180 (http://prospector.ucsf.edu) and experimen-

164 of m/z 700–4,000 and m/z 4,000–10,000.

181 tally-obtained fragment patterns.

165 Intensity and number of laser shots were

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163 Samples were recorded in the mass ranges

182 2.3. Data Analysis

166 adjusted to obtain an optimal signal-to-

183 MFPs were processed with FlexAnalysis

168 shots at a laser frequency of 666 Hz.

184 3.0 (Bruker Daltonik, Bremen, Germany).

169 Bruker peptide and protein standard kits

185 Only ion signals with a minimum intensity

170 were used for calibration. MS/MS experi-

186 of 3 % were manually selected for subse-

171 ments

Bruker

187 quent analysis. Ion signals uniquely ob-

technology implemented in the

188 tained in all individuals from a single pop-

TM

conducted

using

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172 LIFT

were

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167 noise ratio; most data resulted from 3,000

Table 1 Collection localities of four southern African scorpion species, Parabuthus granulatus (Ehrenberg, 1831), Uroplectes otjimbinguensis (Karsch, 1879), Uroplectes planimanus (Karsch, 1879), and Opistophthalmus carinatus (Peters, 1861). Coordinates in decimal degrees for the WGS84 datum.

Code LUC MAM MQT RDF SDN TH BWLL HAL NT SH TL WB

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Locality Lucira Mamué Mariquita Rio dos Flamingo Serra de Neve Tsodilo Hills Brandberg Halali Tirasberge Sophienhöhe Tsumkwe Waterberg

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Country Angola Angola Angola Angola Angola Botswana Namibia Namibia Namibia Namibia Namibia Namibia

Latitude -13.646 -13.799 -14.771 -15.592 -13.695 -18.785 -21.023 -19.039 -26.189 -20.119 -19.444 -20.478

Longitude 12.619 13.118 12.386 12.194 12.922 21.739 14.682 16.470 16.353 16.059 19.756 17.303

Altitude 374 m 639 m 289 m 130 m 462 m 1002 m 464 m 1112 m 1005 m 1330 m 1219 m 1460 m 5

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Fig. 2. Comparison of three venom extractions from Parabuthus villosus (Peters, 1862). (left panel m/z 700– 4,000; right panel m/z 4,000–10,000). Prominent ion signals are labeled. A) Extracted venom from the first electrical stimulation. B) Second venom extraction 24 hours later. C) Third venom extraction seven days later. Despite variation in the relative abundance of some ion signals, the presence of ion signals (yes/no) did not change.

189 ulation or a single species were considered

205 3. Results and Discussion

190 species-specific

population-specific

206 3.1. Comparison of MFPs of venom sam-

191 markers, respectively. The percentage of

207 ples collected from the same individuals

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or

208 immediately after the first electrical stim-

193 for each population. In this context, the

209 ulation, 24 h later, and seven days later

194 average number of ion signals in MFPs

210 Recent analyses have shown that venom

195 represented the initial value of 100%. Ma-

211 composition may differ between freshly

196 trix signals and signals from multiple

212 regenerated venom and venom from the

197 charged ions were excluded. Similarity

213 first

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192 population-specific signals was calculated

extraction (Schaffrath & Predel,

198 analysis of the binary mass list was per-

214 2014). These differences appear to be more

199 formed using Dice’s coefficient. This simi-

215 obvious in the scorpionid taxa with large

200 larity analysis provided the basis for a sin-

216 pedipalps which seem to use their stinger

201 gle

linkage agglomerative hierarchical

217 less regularly than buthid species with ra-

202 clustering analysis (HCA). HCA was per-

218 ther weak pedipalps (S. Schaffrath, un-

203 formed with MATLAB R2017a (Math-

219 published results; see also Casper 1985).

204 Works, MA, U.S.A.).

220 Two individuals of P. villosus and U. 221 planimanus were tested under laboratory 6

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238 ent scorpion species to be clearly identified

223 after the first electrical stimulation is suffi-

239 (Fig. 4). Most of the ion signals observed

224 cient to obtain reproducible venom MFPs

240 were species-specific. Only individuals of

225 in buthid scorpions. Both individuals had

241 the two species of Uroplectes shared sev-

226 their last meal 14 days before the first ven-

242 eral mass-identical substances. These re-

227 om extraction. The presence of ion signals

243 sults were expected, because a separation

228 was very similar in samples from both in-

244 of southern African Parabuthus species

229 dividuals (Figures 2 and 3). All subsequent

245 with MALDI-TOF MFPs was previously

230 venom extractions were performed 24 h

246 reported (Dyason et al., 2002). However,

231 after a first electrical stimulation of the

247 the species-specific venom code for P.

232 telson in the field.

248 granulatus reported by Dyason et al.

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222 conditions to verify that an interval of 24 h

233 3.2. Comparison of venom MFPs from

249 (2002), which covered only three putative

234 single individuals of four southern Afri-

250 Na channel toxins in the mass range of

235 can scorpion species

251 m/z 6,500–7,500, did not match the data

236 Venom compounds collected in the field

252 presented here for the same species. A sin253 gle possible hit may refer to another spe-

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237 from single individuals enabled the differ-

+

Fig. 3. Comparison of venom fingerprints from three venom extractions of Uroplectes planimanus (Karsch, 1879) (left panel m/z 700–4,000; right panel m/z 4,000–10,000). Prominent ion signals are labeled. A) Extracted venom extraction from the first electrical stimulation. B) Second venom extraction 24 hours later. C) Third venom extraction seven days later. Despite variation in the relative abundance of some ion signals, the presence of ion signals (yes/no) did not change.

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Fig. 4. Comparison of MALDI-TOF MFPs of diluted venom samples collected in the field from four southern African scorpion species. MFPs are specific for each sample. Two mass ranges (left panel m/z 700–4,000; right panel m/z 4,000–10,000) were recorded separately. Prominent ion signals are labeled. A) Opistophthalmus carinatus (Peters, 1861), TL; B) Parabuthus granulatus (Ehrenberg, 1831), RDF; C) Uroplectes otjimbinguensis (Karsch, 1879), SH; D) Uroplectes planimanus (Karsch, 1879), HAL.

268 dress this question. Conspecific individuals

255 listed average masses in monoisotopic

269 from different populations were then ana-

256 masses, but this match could be coin-

270 lyzed in the same manner to estimate geo-

257 cidental. Two conclusions can be drawn

271 graphic variation and constancy in the

258 from these results. First, the published

272 MFPs of each scorpion species.

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254 cies, P. kalaharicus, when converting the

259 venom code for Parabuthus species is not

but rather population-

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260 species-specific

261 specific (exact collection localities were

273 3.2. Comparison of venom MFPs ob274 tained from multiple conspecific individu275 als collected at the same localities

262 not given by Dyason et al., 2002). Second, 263 a wider mass range should be evaluated to

276 The MFPs of different individuals collect-

264 search for species-specific components in

277 ed at the same localities (see Fig. 5) were

265 the MFPs. MFPs of several conspecific

278 compared to assess the variation among

266 individuals from the same locality (i.e.,

279 MFPs within a single population; regard-

267 population) were initially compared to ad-

280 less of sex. Previous work reported sex8

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Fig. 5. Comparison of MALDI-TOF MFPs of four specimens of the southern African scorpion, Parabuthus granulatus (Ehrenberg, 1831) from Angolan locality RDF (left panel m/z 700–4,000; right panel m/z 4,000– 10,000). Prominent ion signals are labeled. Ion signal identities decrease in the higher mass range.

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281 related differences as a potential source of

296 for ion signals in a mass range around m/z 297 5000 by De Sousa et al. (2010) and Yamaji

283 of these studies reported variation in a

298 et al. (2004). No significant differences

284 mass range not analyzed in the present

299 were observed among the venom MFPs of

285 study (Abdel-Rahman et al., 2009) or sug-

300 male and female Heterometrus cyaneus

286 gested quantitative variation in the venom

301 (C.L. Koch, 1836) (Scorpionidae), under

287 complements (Miller et al., 2016; Schwartz

302 similar laboratory conditions, however

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282 variation in the venom of scorpions. Some

288 et al., 2008). In the present study, only

303 (Schaffrath, unpublished) nor among the

289 qualitative

pres-

304 MFPs of male and female U. planimanus

290 ence/absence of ion signals were consid-

305 sampled in the field in Botswana and Na-

291 ered. Different ion intensities of otherwise

306 mibia. Variation within a single population

292 mass-identical ion signals were not consid-

307 was observed among four individuals of P.

293 ered to be variation. Qualitative differences

308 granulatus from Angolan locality RDF

294 in venom compounds observed among

309 (Fig. 5). In the complete mass range of m/z

295 males and females were, however, reported

310 700–10,000, an average of 58 distinct ion 9

differences

in

the

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317 Among the P. granulatus individuals from

312 MFPs; 44 of these signals were in the mass

318 locality RDF, 35 ion signals were observed

313 range of m/z 700–4,000. Only ion signals

319 in all MFPs, i.e., 60 % of the average num-

314 occurring in all MFPs were considered

320 ber of ion signals obtained in each of the

315 typical for a population and divergent

321 four MFPs. This does not imply that only

316 MFPs were not eliminated a posteriori.

322 60 % of the observed ion signals represent

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311 signals was recorded among the individual

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Table 2. Number of specimens (n) of four southern African scorpion species, Parabuthus granulatus (Ehrenberg, 1831), Uroplectes otjimbinguensis (Karsch, 1879), Uroplectes planimanus (Karsch, 1879), and Opistophthalmus carinatus (Peters, 1861) analyzed per locality, number of ion signals in MFPs, and percentage of ion signals present in MFPs of all conspecific individuals from each locality. Percentage calculated using average number of ion signals in MFPs of different individuals from each locality. Note decrease of ion signals among conspecific individuals from each population in the higher mass range (m/z 4,000–10,000).

Code

Ion signals

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Species

n

Ion signals

all MFPs

Opistophthalmus carinatus

> 4000

17

67.3

76.5

23.5

3

23–25

19

80.3

73.7

26.3

4

29–40

25

70.9

84.0

16.0

LUC

3

34–54

26

61.9

84.6

15.4

RDF

4

56–65

35

59.3

88.6

11.4

NT

2

45

40

88.9

87.5

12.5

MAM

2

35

30

85.7

90.0

10.0

SDN

4

36–37

19

52.8

94.7

5.3

BWLL

4

33–46

17

44.7

70.5

29.5

SH

5

38–48

28

65.1

89.3

10.7

MAM

4

56–73

40

64.0

75.0

25.0

MQT

3

51–69

23

37.7

100.0

0.0

SDN

4

68–76

38

54.1

78.9

21.1

TH

5

24–41

15

47.2

73.3

26.7

HAL

4

47–59

29

55.8

79.3

20.7

WB

5

46–59

23

44.1

73.9

26.1

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Uroplectes planimanus

< 4000

24–26

TL

Uroplectes otjimbinguensis

%

4

LUC MAM

Parabuthus granulatus

% %

10

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353 the ion signals typical of each species were

324 within the population. It reflects those ions

354 identified from the data compiled for the

325 detected in all venom MFPs from a single

355 different conspecific populations (Supple-

326 population. Variation among the ion sig-

356 mentary Material 2). Only four ion signals

327 nals increased in the higher mass range and

357 (ca. 9 % of the average number of ion sig-

328 included the putative sodium channel

358 nals obtained in single MFPs) were found

329 modulators.

359 in all mass spectra of the individuals from

331 individuals of all populations (n = 60) of 332 the four scorpion species (Table 2; Sup-

361 These signals were detected in the low 362 mass range of m/z 750–900. At least two of

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333 plementary Material 1). The average num-

360 different populations of P. granulatus.

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330 The same approach was repeated using the

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323 compounds common to all individuals

334 ber of ion signals observed in MFPs varied

335 considerably but, in most conspecific 336 populations, 50–70 % of ion signals were

337 observed in MFPs of all individuals ana338 lyzed. In general, the signals common to

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339 all conspecific individuals collected from 340 the same locality (population) decreased in 341 the higher mass range. The list of ion sig-

342 nals shared by all conspecific individuals

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343 from the same population (Supplementary 344 Material 2) was used in subsequent anal-

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345 yses to detect variation among populations. 346 This list also includes potentially species347 specific ion signals. 348 3.3.

Comparison

349 obtained from

of

venom

MFPs

conspecific individuals

350 collected at different localities 351 In order to discriminate among population352 specific and species-specific ion signals,

Fig. 6. MSMS spectra of two related peptides observed in MFPs of all specimens of the southern African scorpion, Parabuthus granulatus (Ehrenberg, 1831) analyzed in the present study. Prominent b- and y- ions are labeled. Mass spectra do not differentiate between Nterminal R or GV. Sequences resemble recently described new-type peptides without disulfide bridges and with GKR processing signal (NPDB21) (Zhong et al., 2017).

11

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represent

peptides

379 cluding putative potassium channel modu-

364 (RFYPSR-NH2, RFIPSR-NH2; Fig.6) and

380 lators, were shared among them. In this

365 exhibit sequence similarity to recently de-

381 species, one of the commonly occurring

366 scribed NPDB21, new-type peptides with-

382 shorter peptides was fragmented, yielding

367 out disulfide bridges, and with a GKR pro-

383 the sequence of an unknown histidine-rich

368 cessing signal (Zhong et al., 2017). The

384 peptide (GDKHHHGPED-NH2).

369 three ion signals mentioned as a species370 specific code in Dyason et al. (2002) were 371 not detected in the MFP from P. granula372 tus venom samples analyzed for the present

385 The potentially species-specific ion signals 386 of the four scorpion species investigated in 387 the present study are summarized in Table 388 3. These signals do not cluster in a mass 389 range typical of venom components like

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373 study. The number of potentially species-

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substances

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363 these

374 specific ion signals does not necessarily 375 decrease as more individuals or popula-

376 tions are analyzed. In the case of U. plani-

377 manus, 25 individuals from six populations

390 antimicrobial, hemolytic, immunemodulat391 ing, antifungal or hormone-like peptides 392 (Lee et al., 2004; Pimenta and De Lima, 393 2005;

Ramirez-Carreto

et

al.,

2015;

378 were compared, and five ion signals, in-

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Table 3. Average number of potentially species-specific ion signals observed in MFPs compared to percentage of ion signals present in MFPs of all individuals of four southern African scorpion species, Parabuthus granulatus (Ehrenberg, 1831), Uroplectes otjimbinguensis (Karsch, 1879), Uroplectes planimanus (Karsch, 1879), and Opistophthalmus carinatus (Peters, 1861).

Species

AC C

EP

Opistophthalmus carinatus

% average ion signals in all conspecific specimens 19 %

Parabuthus granulatus

9%

Uroplectes otjimbinguensis

5%

Uroplectes planimanus

9%

Ion signals in all conspecific specimens 1412.8 1490.9 1528.8 1606.9 2589.5 2601.3 774.5 812.4 824.5 862.4 1143.6 1149.6 1092.58 1127.53 1149.53 3490.46 3619.62 12

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Fig. 7. Hierarchical clustering of venom fingerprint data of 60 individuals of four southern African scorpion species, Parabuthus granulatus (Ehrenberg, 1831), Uroplectes otjimbinguensis (Karsch, 1879), Uroplectes planimanus (Karsch, 1879), and Opistophthalmus carinatus (Peters, 1861), based on similarity analysis with the Dice coefficient. Distances correlate with the obtained pairwise similarities. Individuals of the different species are clearly separated from each other and the topology of the dendrogram is consistent with currently accepted phylogenetic relationships. Note that the Angolan populations of all species (red letters) are always separated from Namibian/ Botswana populations. (color figure in printed version)

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394 Ramirez-Carreto et al., 2012; Zeng et al.,

409 along a transect of nearly 2,000 km and it

395 2005), potassium channel modulators (m/z

410 is likely that MFPs from additional popula-

396 3000-4500

Martin-

411 tions will overlap MFPs from populations

397 Eauclaire, 1995; Pimenta et al., 2001;

412 analyzed in the present study. These results

398 Rodriguez de la Vega and Possani, 2004;

413 suggest that species identification based on

(Lima

and

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414 simple MFPs will be more reliable if data-

400 modulators (m/z 6500-8000; (Batista et al.,

415 bases contain information from multiple

401 2004; Rodriguez de la Vega and Possani,

416 conspecific populations rather than a few

402 2005). Considering the documented varia-

417 species-specific ion signals.

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399 Tytgat et al., 1999) or sodium channel

403 tion among the venom composition of dif404 ferent conspecific populations, it seems 405 possible that the number of species406 specific ion signals will further decrease as 407 more populations are analyzed. However,

418 The percentage of species-specific ion sig419 nals in MFPs of different conspecific popu420 lations varies among the four genera stud421 ied (Table 3). The least congruence was 422 observed among the U. otjimbinguensis

408 the widespread P. granulatus was analyzed 13

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423 populations. One possible reason is the

453 (Prendini and Wheeler, 2005). Opistoph-

424 small size of these scorpions which might

454 thalmus (Scorpionidae) was placed sister to

425 result in a low abundance of several venom

455 the remaining species which belong to

426 components that are present but not clearly

456 Buthidae.

427 detectable in MFPs. Nevertheless, a de-

457 Uroplectes species formed a group, sister

428 tailed analysis of the MFPs among popula-

458 to the species of Parabuthus. Even indi-

429 tions of U. otjimbinguensis and U. plani-

459 viduals of the closely related U. otjimb-

430 manus indicated that populations which are

460 inguensis and U. planimanus, collected at

431 geographically proximate to one other

461 the same localities, always clustered to-

432 share more similarities. In addition, more

462 gether. Although the number of identical

433 congruence was observed among individu-

463 ion signals in MFPs among different popu-

Buthidae,

the

two

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464 lations was rather low, the Angolan popu-

435 (northern

Namibia–northeast Botswana)

465 lations of all species analyzed were dis-

436 than along a South–North transect (north-

466 tinctly separated from the Namibian popu-

437 ern Namibia–southern Angola). These dif-

467 lations, in each case. These results suggest

438 ferences suggest possible vicariance be-

468 that species identification is more reliable

439 tween populations rather than technical

469 with HCA than using counts of mass-

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434 als collected along a West–East transect

440 problems during venom extraction. North441 ern Namibia is separated from southern

470 identical substances in MFPs. 471 4. Conclusions

442 Angola by the Kunene River.

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443 3.4. Hierarchical cluster analysis (HCA) 444 of the venom fingerprints

472 The use of complex venom MFPs for spe473 cies identification is a widely-accepted 474 concept and works effectively with MAL475 DI-TOF mass spectra which are easy to

446 identification of species and even popula-

476 obtain. As demonstrated in the present

447 tions is illustrated by the HCA (Fig. 7).

477 study, these spectra contain sufficient in-

448 Conspecific samples grouped according to

478 formation to consistently identify species.

449 species. Moreover, the arrangement of the

479 The data presented, based on newly regen-

450 four species was consistent with currently

480 erated venom extracted in the field, con-

451 accepted phylogenetic relationships among

481 firmed considerable variation among ven-

452 the

482 om components even within conspecific

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445 The relevance of venom fingerprints for

families and genera in question

14

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512 We acknowledge the support and assis-

484 creased with geographical distance until

513 tance of the National Museum of Namibia,

485 few ion identical signals remained among

514 Windhoek (Benson Muramba), Vollrat von

486 conspecific individuals. HCA was more

515 Krosigk (Windhoek, Namibia) and the

487 reliable for species identification than

516 German Research Foundation (PR766/11-

488 counts of mass-identical substances in

517 1).

490 yses also contain potential information 491 about intraspecific diversity, they might 492 offer a promising taxonomic tool that com-

519 The authors declare no conflict of interest.

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493 plements other methods of species identifi-

518 Conflicts of interest

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489 mass fingerprints. As these statistical anal-

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483 populations. This variation generally in-

494 cation by using ion signal identity. For this 495 to work, multiple mass spectra from differ-

496 ent conspecific populations will need to be

497 included in a reference database that can be 498 queried to reliably assign new venom sam-

500 spectra

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499 ples to species by comparison with mass

from authoritatively identified

501 samples.

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502 Author contributions

503 Stephan Schaffrath performed experiments 504 (including collecting and ‘milking’ scorpi-

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505 ons), and data analysis; Lorenzo Prendini 506 assisted with collecting activities in Angola 507 and determined the species; Reinhard Pre508 del designed the research, organized col509 lecting activities. All authors contributed to 510 the writing of the manuscript. 511 Acknowledgments 15

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