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1 Article Title Genetic div ersity of the endemic honeybee: Apis mellifera

unicolor (Hymenoptera: Apidae) in Madagascar

2 Article Sub- Title 3 Article Copyright

-Year

INRA, DIB and Springer-Verlag France 2015 (This w ill be the copyright line in the final PDF)

4 Journal Name Apidologie

5

Corresponding Author

Family Name Delatte

6 Particle

7 Given Name Hélène

8 Suffix

9 Organization CIRAD, UMR PVBMT

10 Division

11 Address 7, chemin de l’IRAT, Saint Pierre 97410, La

Réunion, France

12 e-mail helene.delatte@cirad.fr

13

Author

Family Name Rasolofoariv ao

14 Particle

15 Given Name Henriette

16 Suffix

17 Organization CIRAD, UMR PVBMT

18 Division

19 Address 7, chemin de l’IRAT, Saint Pierre 97410, La

Réunion, France

20 Organization Université d’Antananarivo

21 Division Département d’Entomologie, Faculté de Sciences

22 Address B.P. 906, Antananarivo 101, Madagascar

23 Organization Université de La Réunion, UMR PVBMT

24 Division

25 Address Saint Denis CEDEX 9 97715, La Réunion, France

26 e-mail

27

Author

Family Name Clémencet

28 Particle

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30 Suffix

31 Organization Université de La Réunion, UMR PVBMT

32 Division

33 Address Saint Denis CEDEX 9 97715, La Réunion, France

34 e-mail

35

Author

Family Name Techer

36 Particle

37 Given Name Maév a Angélique

38 Suffix

39 Organization CIRAD, UMR PVBMT

40 Division

41 Address 7, chemin de l’IRAT, Saint Pierre 97410, La

Réunion, France

42 Organization Université de La Réunion, UMR PVBMT

43 Division

44 Address Saint Denis CEDEX 9 97715, La Réunion, France

45 e-mail

46

Author

Family Name Rav aomanariv o

47 Particle

48 Given Name Lala Hariv elo Rav eloson

49 Suffix

50 Organization Université d’Antananarivo

51 Division Département d’Entomologie, Faculté de Sciences

52 Address B.P. 906, Antananarivo 101, Madagascar

53 e-mail

54

Author

Family Name Reynaud

55 Particle

56 Given Name Bernard

57 Suffix

58 Organization CIRAD, UMR PVBMT

59 Division

60 Address 7, chemin de l’IRAT, Saint Pierre 97410, La

Réunion, France 61 e-mail 62 Schedule Received 1 October 2014 63 Revised 20 February 2015 64 Accepted 9 March 2015

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65 Abstract Apis mellifera unicolor is a tropical honeybee endemic of Madagascar. Comprehensive knowledge about its mitochondrial and nuclear genetic diversity and structuration was our main purpose. Samples of worker bees were collected from 867 colonies in 76 sites in Madagascar and 1 reference population in South Africa. PCR-restriction fragment length polymorphism (RFLP) and sequencing were used to reveal variability in the COI–COII mtDNA region. Seventeen microsatellite loci were used for studying the nuclear diversity. Three PCR-RFLP profiles were observed, among which 99.4 % belonged to A1 haplotype, 0.2 % to a new A

haplotype, and 0.4 % to A4 haplotype. In microsatellite analysis, moderate genetic diversity values were found for Madagascar, together with low mean number of alleles ranging from 2.47 to 3.88 compared to South Africa. Bayesian clustering assignment methods and principal component analysis (PCA) separated populations into two genetic clusters which matched with

geographic areas. Several hypotheses are discussed regarding to the low genetic diversity of A. m. unicolor in its native range.

66 Keywords

separated by ' - '

Apis mellifera unicolor - microsatellite - African lineage - genetic diversity - mtDNA

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67 Foot note information

Manuscript editor: Marina Meixner

Henriette Rasolofoarivao holds an Ir degree, CIRAD, UMR PVBMT. Johanna Clémencet holds a PhD degree, Université de La

Réunion, UMR PVBMT.

Maéva Angélique Techer holds an Ir degree, CIRAD, UMR PVBMT. Lala Harivelo Raveloson Ravaomanarivo holds a PhD degree, Université d’Antananarivo.

Bernard Reynaud holds a PhD degree, CIRAD, UMR PVBMT. Helene Delatte holds a PhD degree, CIRAD, UMR PVBMT.

The online version of this article (doi:10.1007/s13592-015-0362-1) contains supplementary material, which is available to authorized users.

Div ersité génétique d'une abeille endémique de Madagascar: Apis mellifera unicolor (Hymenoptera: Apidae)

Microsatellite / lignée africaine / ADNmt / aire de répartition / haplotype

Genetische Div ersität der endemischen Honigbiene v on Madagaskar, Apis mellifera unicolor (Hymenoptera: Apidae) Apis mellifera unicolor / Mikrosatelliten / Afrikanische Lineage / genetische Div ersität / mtDNA

Electronic supplementary material

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1 Q1

2

Genetic diversity of the endemic honeybee:

Apis mellifera

3

unicolor (Hymenoptera: Apidae) in Madagascar

4 Henriette RASOLOFOARIVAO1,2,3,Johanna CLÉMENCET3,

Q2 Maéva Angélique TECHER1,3,

5 Lala Harivelo Raveloson RAVAOMANARIVO2,Bernard REYNAUD1,Hélène DELATTE1

6 1

Q3 CIRAD, UMR PVBMT, 7, chemin de l’IRAT, 97410, Saint Pierre, La Réunion, France

7 2

Département d’Entomologie, Faculté de Sciences, Université d’Antananarivo, B.P. 906, 101, Antananarivo, Madagascar

8 3

Université de La Réunion, UMR PVBMT, 97715, Saint Denis CEDEX 9, La Réunion, France 9 Received 1 October 2014– Revised 20 February 2015 – Accepted 9 March 2015 10

11 Abstract– Apis mellifera unicolor is a tropical honeybee endemic of Madagascar. Comprehensive knowledge about

12 its mitochondrial and nuclear genetic diversity and structuration was our main purpose. Samples of worker bees were

13 collected from 867 colonies in 76 sites in Madagascar and 1 reference population in South Africa.

Q4 PCR-restriction

14 fragment length polymorphism (RFLP) and sequencing were used to reveal variability in the COI–COII mtDNA

15 region. Seventeen microsatellite loci were used for studying the nuclear diversity. Three PCR-RFLP profiles were

16 observed, among which 99.4 % belonged to A1 haplotype, 0.2 % to a new A haplotype, and 0.4 % to A4 haplotype. In

17 microsatellite analysis, moderate genetic diversity values were found for Madagascar, together with low mean number

18 of alleles ranging from 2.47 to 3.88 compared to South Africa. Bayesian clustering assignment methods and principal

19 component analysis (PCA) separated populations into two genetic clusters which matched with geographic areas.

20 Several hypotheses are discussed regarding to the low genetic diversity of A. m. unicolor in its native range.

21

22 Apis mellifera unicolor / microsatellite / African lineage / genetic diversity / mtDNA

23

24 1. INTRODUCTION

25 Apis mellifera subspecies are the most

econom-26 ically valuable pollinators of crop monocultures

27

worldwide (Klein et al.2007). In addition, honeybee

28

contribution to floral biodiversity and conservation

29

through pollination is estimated to affect 80 % of

30

wild flora (Batra1995; De la Rua et al.2009).

31

Remarkable morpho-geographical

differentia-32

tions are found throughout honeybee distribution

33

areas; 28 subspecies are endemic to Africa,

Eu-34

rope, and Middle East (Ruttner et al. 1978;

35

Ruttner 1988; Sheppard et al. 1997; Sheppard

36

and Meixner 2003). Four evolutionary lineages

37

have been described based on phenotypes and

38

molecular traits (Garnery et al. 1992, 1993;

39

Estoup et al. 1995; Franck et al. 2000, 2001;

40

Alburaki et al.2013).

41

In Africa, 11 subspecies are found from all

42

lineages except from the C lineage (Hepburn and

43

Radloff1998; Meixner et al.2011). The African A

44

lineage is native to Africa and subspecies include

45

Apis mellifera scutellata (Lepeletier 1836), Apis

46

mellifera capensis (Eschscholtz1822), and Apis

47

mellifera unicolor (Latreille1804).

Electronic supplementary material The online version of this article (doi:10.1007/s13592-015-0362-1) contains supplementary material, which is available to authorized users.

Corresponding author: H. Delatte, helene.delatte@cirad.fr Manuscript editor: Marina Meixner

Henriette Rasolofoarivao holds an Ir degree, CIRAD, UMR PVBMT.

Johanna Clémencet holds a PhD degree, Université de La Réunion, UMR PVBMT.

Maéva Angélique Techer holds an Ir degree, CIRAD, UMR PVBMT.

Lala Harivelo Raveloson Ravaomanarivo holds a PhD degree, Université d’Antananarivo.

Bernard Reynaud holds a PhD degree, CIRAD, UMR PVBMT.

Helene Delatte holds a PhD degree, CIRAD, UMR PVBMT.

Apidologie

Original article

* INRA, DIB and Springer-Verlag France, 2015 DOI:10.1007/s13592-015-0362-1

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48 A. m. unicolor is the endemic subspecies of

49 Madagascar, a drifted tropical island, located

50 400 km off the East coast of Africa. Madagascar

51 is among the five richest biodiversity hotspots of

52 the world in terms of endemic plants and vertebrate

53 species (Myers et al. 2000). However, less than

54 10 % of its original habitat still remains (Myers

55 et al.2000). In such an area of endemism, with over

56 80 % of endemic phanerogam (Ganzhorn et al.

57 2001), the role of pollinating insects is vital for

58 the reproduction of these plants. Although the

pol-59 linating role of the honeybee has never been

inves-60 tigated in detail for these floras, A. m. unicolor is

61 thought to play a crucial role in the pollination of

62 endemic phanerogam species (Ruttner 1975;

63

Q5 Ralalaharisoa-Ramamonjisoa et al.1996).

64 After molecular analysis, mitotype A1 was

at-65 tributed to the honeybee of Madagascar, with a

66 surprising absence of mitochondrial polymorphism

67 within this endemic subspecies (Franck et al.

68 2001). The latest molecular study using SNP

anal-69 yses suggested that A. m. unicolor had a specific

70 SNP (Whitfield et al.2006). However, those

as-71 sumptions were based on a low number of samples

72 (<50) taken from regions of the vast territory of

73 Madagascar. In terms of morphological and

behav-74 ioral criteria, two ecotypes were described, the first

75 from the highlands (Hauts Plateaux) and the

sec-76 ond from the coastal area (Ruttner1988).

77 Since the recent arrival of the parasitic mite

78 Varroa destructor documented in Madagascar in

79 2010, severe colony losses have been observed

80 (Rasolofoarivao et al.2013). This situation may

81 dramatically impact the biodiversity of

Madagas-82 car. Therefore, considering the lack of genetic data

83 and the threat to this subspecies, it appears

neces-84 sary to improve our knowledge on A. m. unicolor

85 genetic diversity in its native area.

86 2. MATERIAL AND METHODS

87 2.1. Area of study

88 The land area and relief of Madagascar gives rise to

89 various climatic zones. The year is characterized by two

90 distinct seasons: the austral winter (April to November)

91 and the austral summer (December to March). The coastal

92 zones enjoy a warm climate, and mean annual

tempera-93 tures range between 22 and 25 °C. The upland areas of the

94

island have a more temperate climate with a mean annual

95

temperature of 20 °C. Tropical forests mainly consist of

96

deciduous woodland in Western Madagascar and

xero-97

phytic thorn forests in the southern region (Figure1).

98

2.2. Sampling

99

One adult worker honeybee per colony was sampled

100

between August 2011 and March 2013 from 76 sites

101

(n =867) in Madagascar (Figure1, SD TableI).

Sam-102

pling was performed on managed colonies and 33 wild

103

colonies (collected in Djamajar (S8) n =6, Antsoha (S7)

104

n =9, Rantolava (S24) n =3, and Tsararano (S21) n =

105

15). As a reference population from African lineage,

106

samples were collected from 22 managed colonies in

107

South Africa (one apiary from Cape region) in 2013.

108

Honeybees were preserved in ethanol (96 %) and kept at

109

−20 °C until molecular analysis.

110

2.3. DNA extraction

111

The six worker legs were used for DNA isolation. DNA

112

was extracted from individual honeybees as previously

113

described (Delatte et al.2010). All individuals were

sub-114

jected to both microsatellite and mitochondrial analyses.

115

2.4. Microsatellite amplification

116

and genotyping

117

Microsatellite population studies were carried out using

118

17 loci published in Solignac et al. (2003) and combined

119

into four different mixes (mix 1: A024, A113, Ac306,

120

Ap055, Ap081; mix 2: A (B)124, A028, A029, A088,

121

Ap273, Ap289; mix 3: Ap033, A035, Ap036; mix 4:

122

A014, Ap043, Ap066). PCR reactions were performed

123

in a 10 μL final reaction volume with a primer mix

124

(10 μM) using Type-it Multiplex PCR Master Mix

125

(Qiagen) kits. PCR programs were run with an initial

126

denaturation of 94 °C for 5 min, followed by 35 cycles

127

of denaturation at 94 °C for 30 s; annealing was 55 °C for

128

30 s for mix 1 and 52 °C for mixes 2–4, followed by

129

elongation at 72 °C for 30 to 45 s. A final extension was

130

done at 72 °C for 10 to 20 min.

Figure 1. Madagascar map with ecological zonation and honeybee sampling sites (emplacements of each site are presented in red polygons ). The 76 sites are spread in 6 geographic regions. Each number repre-sents a site with correspondence in TableI.

„

H. Rasolofoarivao et al.

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North: dry deciduous forest, dry tropical climate

West: dry deciduous forest, hot tropical climate during dry season

Center: high land, sub humid forest, altitude tropical climate

East: wetter area of island, lowland forest, sub equatorial climate

South: half desert, spiny thickets, dry tropical climate

South west: succulent woodlands, dry tropical climate

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131 Samples were further genotyped using an automated

132 DNA sequencer (Applied Biosystems 3130XL) with

133 capillaries. Allele sizes were scored using GeneMapper

134 4.0 Software. Individuals with genotype data missing

135 for more than 40 % of all loci were excluded from

136 statistical analysis. Small sample sizes under 10

indi-137 viduals per site were not included in the nuclear genetic

138 analysis at population level.

139 2.5. Microsatellite analysis

140 Observed (Ho), expected (He), and Nei’s 1987

un-141 biased expected (Hn.b) heterozygosity and fixation

in-142 dices (Fis) (

Q6 Weir et al. 1984) were estimated using

143 Genepop 4.2 (Rousset 2008) and Genetix 4.05

144 (Belkhir et al.1996). All pairs of loci were tested for

145 linkage disequilibrium using Genepop 4.2 (Rousset

146 2008). Deviations from Hardy–Weinberg equilibrium

147 (HWE) were tested using a two-tailed Fisher exact test

148 based on Markov chain (Rousset2008). Permutation

149 tests conducted by FSTAT (Goudet2001) determined

150 whether genetic diversity (He, Hn.b, Ho) and Fis

dif-151 fered significantly between geographical regions.

152 FreeNA (

Q7 Chapuis and Estoup 2007) was used to

153 estimate null allele frequencies. Population

differentia-154 tion was quantified by calculating pairwise FSTvalues

155 (Weir and Cockerham1984) and verifying their

signif-156 icance through the permutational test in Genetix 4.05

157 (Belkhir et al.1996). Relationships between genetic and

158 geographic distances at all sites were tested using the

159 Mantel’s test in Genepop 4.2 (Rousset2008). The

sig-160 nificance of the correlation between matrices of

geo-161 graphical and genetic distances among pairs of sites

162 was tested using 1000 permutations of the data. As

163 potential isolation by distance (ibd) patterns may not hold

164 over the entire range (1470 km), because at some point

165 the influence of gene flow is expected to be weak relative

166 to the influence of genetic drift and homoplasy

167 (Hutchison and Templeton1999), ibd patterns were

in-168 vestigated at smaller spatial scales, i.e., in sites from the

169 northern regions (500 km, nsites=7), the central regions

170 (370 km, nsites=15), and southern regions of the island

171 (380 km, nsites=9). We further investigated the

impor-172 tance of scale on spatial genetic structuring by

173 performing a hierarchical F analysis, which estimates

174 the genetic variation found at each hierarchical level. A

175 nested tree-level analysis of molecular variance

176 (AMOVA, Excoffier and Lischer2010) was performed

177 by partitioning the total sum of squares into components

178

representing variation between geographical regions,

179

among sites within regions and among individuals within

180

sites using Arlequin V3.5 (Excoffier and Lischer2010).

181

Levels of population admixture were quantified

182

using a number of Bayesian clustering procedures as

183

implemented in Structure V2.3.4 (Pritchard et al.2000).

184

The number of population clusters was inferred

accord-185

ing to Evanno et al. (2005) and the ad hoc statisticΔK

186

was calculated for K ranging from 1 to 10 for the full

187

dataset comprising the reference population and 1 to 20

188

within the Madagascar dataset (1 million simulations

189

and 100,000 burn-in with 10 iterations for each K ). This

190

ad hoc statistic was processed through the Structure

191

Harvester website (http://taylor0.biology.ucla.edu/

192

structureHarvester/). Clumpp v.1.1.2 (Jakobsson and

193

Rosenberg2007) was used to align the best of the five

194

repetitions of the K . Distruct v.1.1 (Rosenberg 2004)

195

was used to graphically display the results. A principal

196

component analysis (PCA) was performed on the

ge-197

netic data to visualize genetic differentiation among the

198

population groups using adegenet 1.4 (Jombart2008) in

199

R software (Team2005). Adegenet 1.4 was also used to

200

check the dataset structure using an alternative

cluster-201

ing analysis with a discriminant analysis of principal

202

component (DAPC).

203

2.6. Mitochondrial analysis

204

Mitochondrial DNA COI–COII region was

ampli-205

fied using two specific primers, E2 and H2 (Garnery

206

et al. 1992). Amplification and PCR cycles were as

207

described in Garnery et al. (1992). The size of the

208

fragment amplified was visualized using 5μL of the

209

PCR products electrophoresed on 2 % agarose gel. PCR

210

products were then used both for enzymatic restriction

211

and sequencing.

212Q8

Ten microliters of PCR products from Madagascar

213

(n =867) and South Africa (n =22) were enzymatically

214

digested by the DraI (Promega©) enzyme according to

215

manufacturer recommendations. The resulting

frag-216

ments were separated in 4 % agarose gel. Each

enzy-217

matic profile was scored and compared to expected

218

sizes published (Garnery et al. 1992; Franck et al.

219

2001). Each profile with a different band pattern

ob-220

served on gel in Madagascar was sent for sequencing

221

(121 samples of the dominant profile, 2 per other

pro-222

file). PCR products (n =173) were sent to Macrogen©

223

for sequencing. Of these, some were taken from wild

224

colonies (site Tsararano S21 n =10). DNA sequence H. Rasolofoarivao et al.

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225 results were aligned using MEGA 5.04 (Kumar et al.

226 2008) then analyzed by BLAST search on GenBank.

227 Genetic relationships between the different sequences

228 obtained for the A1 haplotypes from Madagascar were

229 investigated by constructing a minimum-spanning

net-230 work of the haplotypes with TCS software (Clement

231 et al.2000).

232 3. RESULTS

233 3.1. Diversity indices

234 The amplification was successful for 710 out of

235 867 Malagasy and for all South African (n =22)

236 individuals (with at least 10 loci amplified per

237 sample). Of the 77 sampled sites, only 33 have

238 more than 10 individuals (SD TableI). Analysis

239 for linkage disequilibrium showed no significant

240 deviation from equilibrium among the 17

micro-241 satellite marker pairs (all P >0.05), except

be-242 tween loci A113 and A24 (P <0.05).

243 The average number of alleles per population

244 and over 17 markers varied between 2.47 and 3.88

245 in Madagascar with an overall average of 7.76

246 alleles/locus , and for the single reference

popula-247 tion of South Africa, the average number was

248 12.57 alleles/locus (TableI).

249 Unbiased estimated heterozygosity (Hn.b)

250 ranged from 0.36 to 0.50 for Madagascar, and

251 for the reference population Hn.b=0.86 (TableI).

252 Across all loci, less than 10 % null allele was

253 observed (Table I). Inbreeding coefficient Fis

254 ranged from heterozygote excess (outbreeding,

255 −0.07) to heterozygote deficiency (inbreeding, 256 0.15) compared with HWE expectations. No

dif-257 ference was detected among geographical regions

258 within Madagascar (SD Table II, Comparisons

259 among groups, all P >0.4).

260 3.2. Genetic structure

261 FSTestimates among pairs of sites ranged from

262 −0.02 to 0.17, with 318 out of 496 genetic differ-263 entiation estimates being significantly different

264 from zero (SD Table III) within Madagascar

265 dataset. FSTestimates between the overall

Mala-266 gasy populations and the reference population of

267 South Africa were highly significant and

268 equal to 0.34.

269

Within each of the northern, central, and

south-270

ern parts of Madagascar (<500 km between sites),

271

nuclear genetic differentiation between sites was

272

positively correlated with the logarithm of

geo-273

graphical distance (Figure2a, b, c). However, at

274

the island scale (north to south 1470 km, nsites=

275

32), the correlation between the two matrices was

276

no longer significant (Mantel test, P = 0.87;

277

Figure 2d). The analysis of molecular variance

278

comprising only Madagascar populations showed

279

that the main contribution to the genetic variance

280

was variation within sites (94 %). Differences

281

among regions (2.3 %) and among sites within

282

regions (3.34 %) were much lower but contributed

283

significantly to the total genetic variation (SD

284

Table IV). When all regions were taken into

ac-285

count (within Madagascar), all hierarchical levels

286

accounted for a significant part of the genetic

287

diversity.

288

Variation in allelic composition was

highlight-289

ed by Structure. The optimal K =2 was the most

290

strongly supported in likelihood using the

refer-291

ence population (Figure 3), with South African

292

individuals being far apart from the Malagasy

293

individuals. Then, looking at the substructure

294

intra-Madagascar populations, the optimal and

295

strongest K population is 2 (SD Fig. 1, SD

296

Fig.2). The DAPC analysis corroborates the same

297

clustering analysis using both datasets. For the full

298

dataset (South Africa and Madagascar), three

ge-299

netic clusters were found (one for South African

300

individuals, and two closer ones within

Madagas-301

car; Figure4).

302

Under the K =2 population clustering

assump-303

tion within Madagascar, a geographic structuring

304

appears with populations from the north,

north-305

west, and south together in cluster 1 as opposed to

306

populations from the west and east (cluster 2). The

307

highland populations were mostly a mix of two

308

genetic clusters (Figures1and4, SD Fig.2).

309

3.3. PCR-restriction fragment length

310

polymorphism patterns and sequences

311

After analysis of the Madagascar samples,

312

three different PCR-restriction fragment length

313

polymorphism (RFLP) profiles were detected.

314

Two restriction profiles were congruent with ones

315

already published. The dominant PCR-RFLP

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316 profile observed was the African A1 haplotype

317 (99.4 %), which exhibited three lengths of

318

fragments (∼47, 108, and 483 bp) (Franck et al.

319 2001). Another PCR-RFLP profile referred to as

t1:1

Q9 Table I. Genetic diversity indices by sites. t1:2 Geographical regions Map codes Sites (S) Number He Hn.b. Ho Fis Nbr An t1:3 Madagascar t1:4 North 2 Namakia 10 0.38 0.40 0.42 −0.07 2.47 0.05 t1:5 Northeast 11 Analamandrorofo 16 0.42 0.44 0.38 0.11a 3.47 0.06 t1:6 13 Analapenja 11 0.35 0.37 0.34 0.08 2.76 0.02 t1:7 15 Antohomarina 22 0.38 0.39 0.34 0.14a 3.29 0.07 t1:8 Northwest 20 Tsararano M. 10 0.38 0.40 0.39 0.01 3.00 0.02 t1:9 21 Tsararano M. 15 0.43 0.44 0.37 0.16a 3.35 0.07 t1:10 22 Tsaramandroso 10 0.42 0.45 0.35 0.21a 3.18 0.07 t1:11 Hauts Plateaux 34 Ambotseheno 19 0.40 0.41 0.34 0.18a 3.24 0.08

t1:12 35 Betoho 16 0.38 0.39 0.35 0.10a 2.94 0.05 t1:13 36 Anjepy 12 0.42 0.44 0.42 0.04 3.24 0.04 t1:14 37 Maharidaza 10 0.38 0.41 0.40 0.02 2.76 0.03 t1:15 38 Mandraka 12 0.40 0.41 0.38 0.09 3.06 0.05 t1:16 40 Ivato 17 0.43 0.45 0.41 0.06 3.18 0.07 t1:17 42 Andramasina 10 0.43 0.46 0.40 0.14a 3.12 0.06 t1:18 43 Toronala 33 0.38 0.39 0.36 0.07a 3.53 0.05 t1:19 47 Ihazolava 12 0.38 0.40 0.36 0.11a 3.06 0.06 t1:20 49 Anativato 11 0.40 0.42 0.43 −0.02 3.00 0.05 t1:21 50 Mahazina 10 0.34 0.36 0.32 0.11 2.94 0.05 t1:22 Hauts Plateaux south 52 Soanirina 11 0.37 0.40 0.40 −0.04 2.56 0.05 t1:23 53 Ambositra 10 0.36 0.39 0.32 0.17a 2.59 0.05 t1:24 54 Tsararano 43 0.38 0.39 0.36 0.07a 3.76 0.05 t1:25 55 Vohimasina 22 0.40 0.41 0.38 0.09a 3.29 0.07 t1:26 West 63 Androvabe 11 0.47 0.50 0.43 0.15a 3.24 0.07 t1:27 Southwest 66 Toliary 30 0.39 0.39 0.33 0.15a 3.88 0.06 t1:28 67 Bezaha 23 0.42 0.43 0.42 0.03 3.47 0.02 t1:29 South 68 Mahatalaky 17 0.44 0.45 0.41 0.10a 3.47 0.06 t1:30 70 Ifarantsy 10 0.35 0.37 0.35 0.07 2.53 0.03 t1:31 71 Ifarantsy 16 0.39 0.40 0.38 0.04 3.24 0.04 t1:32 72 Ampasy N. 10 0.42 0.45 0.39 0.13a 3.12 0.05 t1:33 74 Soanierana 21 0.40 0.41 0.38 0.08a 3.47 0.06 t1:34 75 Ankaramena 13 0.35 0.36 0.36 0.03 2.71 0.05 t1:35 76 Ambovombe 15 0.42 0.44 0.39 0.09 3.35 0.04

t1:36 South Africa 77 Cape 22 0.84 0.86 0.83 0.031 12.57 0.03

For each site, indicated are as follows: their geographical region, number of individuals per site, expected heterozygosity (He),

expected unbiased heterozygosity (Hn.b), observed heterozygosity (Ho), estimate of Wright’s (Weir and Cockerham1984) fixation

index (Fis),mean number of allele (Nbr). Only sites with at least 10 individuals per apiary are considered

An mean null allele frequency

aDeviations from HWE

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320 the A4 haplotype (∼47, 108, 192, and 483 bp) was

321 found in three individuals in our sample, two from

322 S66 and one from S23. Another notable profile

323 was found in two individuals from S22 and S29

324 (∼47, 108, 150, 350 bp). The total sizes of each

325 haplotype ranged from 638 bp for A1, 830 bp for

326 A4, and 655 bp for the new haplotype. Within the

327 South African samples, two different PCR-RFLP

328 profiles were detected: A1 haplotype (n =1) and

329 A4 haplotype (n =21).

330 A total of 173 samples collected in 49 sites

331 were sequenced. A total of 18 different sequences

332 were found with variable sequence sizes

belong-333 ing to the A lineage (SD Table V). The 16

se-334 quences obtained for the A1 restriction profile

335 have never been reported and were named

336 A1_Mad1 to A1_Mad16 (accession numbers

337 KF976992 to KF977009). These 16 sequences

338 were characterized by one unit P0and one unit

339 Q . The most frequent sequence, A1_Mad3, was

340

present in all sites (n =121, 70 %). The sequences

341

of the two individuals exhibiting the new

PCR-342

RFLP haplotype (in S29, S22) cluster within the

343

A. m. unicolor group (it was subsequently named

344

A1_Mad13) and we propose to classify it as a

345

subtype of A1. The two other sequences

346

(A4_Mad1 and A4_Mad2) were genetically close

347

to the A4 haplotypes of A. m. scutellata

(acces-348

sion number FJ 477987 (Franck et al. 2001),

349

similarity=98 %) and found in two different

re-350

gions of the West coast of Madagascar (R4 and

351

R16) (SD TableV, SD Fig.2). The A4 haplotypes

352

were characterized by one unit P0and two units

353

Q sequences.

354

4. DISCUSSION

355

Previous PCR-RFLP analyses performed on 48

356

individuals from Madagascar by several authors

357

(Garnery et al.1992; Franck et al.2001) detected

Figure 2. Relationship between logarithm of geographical distance and nuclear genetic differentiation as estimated as FST/(1−FST), between a sites from the northern region only (nsites=7, P =0.014), b the southern region only

(nsites=9, P =0.0027), c sites from Hauts Plateaux only (nsites=16, 120 combinations, Mantel test P =0.014), and d

sites from all regions sampled in Madagascar (nsites=32, 496 combinations, Mantel test P =0.84).

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358 a single restriction profile (A1). The larger

sam-359 pling scale of this study in Madagascar detected a

360 new A1 restriction profile and the occurrence of

361

an A4 restriction profile. The sequencing

ap-362

proach used in our study revealed mtDNA

vari-363

ability with 16 new sequences. Haplotype

Figure 3. Population structure and Euclidean distances among genetic clusters based on 17 microsatellites loci. Top : Structure bar plots (K =2, 3, and 4) with 22 reference samples from South Africa and 710 individuals of Madagascar organized by sampling sites (S1–76). Each horizontal bar represents one individual, and sites are delimited by black lines . The height of each bar represents the probability of assignment to a genetic cluster (one color ). Bottom : PCAs among individual genotypes assigned to the different clusters (K =2, 3, and 4). Inertia percentage of each axis is indicated (using 232 variables).

H. Rasolofoarivao et al.

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364 frequencies and network analyses suggested that

365 divergences are quite recent (SD Fig.3), with all

366 A1 sequences in Madagascar diverging from the

367 predominant one through one single mutation

368 (except for A1Mad_12). The A1 haplotype is

369 widespread. It was found in this study in South

370 Africa, and it has also been reported in Morocco

371 (De la Rua et al. 2006), Algeria (Chahbar et al.

372 2013), Sudan (El-Niweiri and Moritz2008), and

373 in the Middle East (Alburaki et al.2011). Three

374 samples from A4 were found in the Western

re-375 gion of Madagascar. Two of these samples show

376 high genetic diversity compared to A4 published

377 sequences. A4 haplotypes might probably result

378 from ancestral introductions to the island together

379 with ancestral A1, as both haplotypes (belonging

380 to AIsublineage) are commonly distributed within

381 the different African subspecies populations

382 (Franck et al.2001).

383

The absence of haplotypes belonging to other

384

lineages in our study implies that introductions of

385

foreign queens are rare. This can be explained by

386

the fact that other subspecies i) if imported/

387

introduced were too few to be seen in our sampling,

388

ii) are not well adapted to this environment (climate

389

and specific endemic vegetation), or iii) are not

390

selected by traditional Malagasy beekeepers, A. m.

391

unicolor being easy to handle compared to other

392

much more aggressive subspecies like the

African-393

ized honeybee (Ruttner1988; Winston1992).

394

Madagascar populations were highly different

395

(FST=0.34) to the reference population

(compris-396

ing A1 and A4 haplotypes). Furthermore, A. m.

397

unicolor has a poor allelic diversity in terms of

398

number of alleles for each population marker

399

(Nbr=2.47 to 3.88), in comparison to our

refer-400

ence population (Nbr=12.57) or even with other

401

studies performed on African honeybee

Figure 4. DAPC based on individual genotypes of Madagascar (n =710) and reference population of South Africa (n =22). Dots of different colors indicate honeybee samples from different genetic clusters (K =3). Inertia percent-age of each axis, PCs eigenvalues, and discriminant factors retained are indicated.

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402 populations with allelic diversity ranging from 7.9

403 (A. m. capensis ) to 9 (Apis mellifera intermissa )

404 and even 11 (A. m. scutellata ) (for 7 to 12

micro-405 satellite loci, with most loci being the same in both

406 studies) (Estoup et al.1995).

407 The levels of heterozygosity observed in

Mala-408 gasy populations were also much lower than the

409 reference population used in this study and those

410 reported from African populations. Across

Mada-411 gascar, levels varied from 0.34 to 0.47 (He), while

412 the reference population He=0.84, and the average

413 Heranged between 0.78 and 0.90 (A. m. intermissa

414 to A. m. scutellata) in African populations (Franck

415 et al.2001). In parallel, the lowest levels ranged

416 from 0.26 to 0.66 in west Mediterranean subspecies

417 (Apis mellifera iberiensis and Apis mellifera

418 siciliana , respectively) (Garnery et al.1998).

419 High levels of nuclear polymorphism in African

420 populations have been explained by i) quaternary

421 climate changes that could be responsible for

hon-422 eybee subspecies diversification and expansion in

423 Africa (Franck et al. 2001), ii) larger effective

424 population size (Estoup et al.1995), allowing more

425 alleles to be maintained, and iii) the high migratory

426 behavior of colonies which is typical for African

427 honeybees south of the Sahara (Hepburn and

428 Radloff 1998; Jaffe et al. 2009). Allelic richness

429 within populations can also be increased by

intro-430 gression of foreign genes into zones with other

431 subspecies. Due to the lack of data on the biology

432 of the Malagasy subspecies, it is difficult to

com-433 pare effective population size of A. m. unicolor to

434 other subspecies. However, its insular situation

435 prevents frequent natural introductions and may

436 in part explain the low nuclear polymorphism.

437 Both the significant pairwise FSTvalues observed

438 between neighboring sites (i.e., S34–S35 only

439 10 km apart, SD Table III) and the significant

440 isolation by distance patterns observed among sites

441 500 km apart (Figures1and2) suggest that gene

442 flow is restricted. The larger variance of FSTat

443 longer distances (>500 km, Figure 2d) indicates

444 that at the island scale, the influence of genetic drift

445 is strong relative to gene flow (Hutchison and

446 Templeton1999) and that problems of homoplasy

447 could be more important (Jarne and Lagoda1996).

448 As observed in A. m. capensis from South Africa,

449 A. m. unicolor populations may be less mobile

450 than other African subspecies (Estoup et al.1995)

451

because of the topography of the island (coastal vs.

452

Hauts Plateaux areas) and the climatic variations

453

between regions. Indeed this was underlined with

454

the results of Structure indicating genetic

455

subclustering of the observed populations into at

456

least two major clusters. The observed genetic

457

subclustering did not match the distribution of the

458

two honeybee ecotypes described by Ruttner

459

(1988). Furthermore, we found such an admixture

460

of genetic clusters between populations from

dif-461

ferent regions and the region surrounding the

cap-462

ital (on the Hauts Plateaux) that those ecotypes

463

might have been mixed in the recent past. Indeed,

464

in Madagascar, most goods pass through the

capi-465

tal, central market, and free commercial exchanges,

466

which facilitate honeybee movement. Transport

467

routes around the island are limited but all of them

468

lead to the capital.

469

Wild populations, uninfluenced by beekeeping,

470

exist in many regions of Africa, and honeybees

471

from natural habitats have been shown to have a

472

higher genetic diversity than managed populations

473

(Allsopp2004), so more intensive studies of wild

474

colonies in protected and wild zones of

Madagas-475

car would be interesting to confirm or not our

476

findings on the genetic diversity of A. m. unicolor .

477

Several clues and hypotheses point out the fact

478

that A. m. unicolor might be derived from a recent

479

(in geological time) colonization event of this

480

continental island: i) relatively low mitochondrial

481

and nuclear genetic diversity were found on A. m.

482

unicolor in Madagascar, compared to other

sub-483

species of the A lineage (Estoup et al. 1995;

484

Franck et al. 1998,2001). ii) The hypothesis on

485

molecular data showing A lineage split from other

486

lineages 6 million years ago with A. m. unicolor

487

divergence from other subspecies more recently

488

(1 million years ago) (Han et al. 2012). iii) The

489

prehistoric breakup of the supercontinent

Gond-490

wana which separated Madagascar from mainland

491

Africa is dated much earlier (around 135 million

492

years ago; Rabinowitz et al. 1983) than the first

493

honeybee species.

494

Nevertheless, its morphological (two ecotypes)

495

and behavioral differences (one of the most gentle

496

honeybees in the world (Ruttner1988)) from

oth-497

er African honeybees suggest that, such as the

498

flora of the island, A. m. unicolor seems to have

499

evolved in relative isolation. Furthermore, the low

H. Rasolofoarivao et al.

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500 genetic diversity observed, if confirmed in wild

501 and conserved areas, might also be the result of

502 over 1400 years of high deforestation rates and

503 habitat fragmentation on the island (Campbell

504 1993; Gade1996) which has been increasing over

505 the last 50 years (Harper et al.2007).

506

507 ACKNOWLEDGMENTS

508

509 We thank Ravelomanana A. for mapping sites with

510 GIS and Simiand C. for technical help in the laboratory.

511 We thank the following people for their help with data

512 collection: Rousse P., Porphyre V. (and QualiREG

net-513 working), Borsa C., Mandirola V., Andrianaivoariseta N.,

514 Razafindrazaka D., Cattel J., Chesnais Q., ADEFA, and

515 FENAM. We are grateful to the Malagasy beekeepers who

516 participated in the study. We would like to thank Garnery

517 L. for the fruitful discussions related to those genetic

518 results. This work is part of the Ph.D. of Rasolofoarivao

519 H., recipient of a grant of CIRAD-AIRD-Sud. Field work

520 had been partly funded by CIRAD, the Enlargement and

521 sustainability of the Plant Protection Network supported by

522 the European Union, the French government, and the

523 Région Réunion and the Département of la Réunion. In

524 addition, we would like to thank the editor (M. Meixner)

525 and the anonymous referees for their remarks that greatly

526 improved our manuscript.

527

528 Diversité génétique d'une abeille endémique de Mada-529 gascar: Apis mellifera unicolor (Hymenoptera: 530 Apidae)

531 Microsatellite / lignée africaine / ADNmt / aire de ré-532 partition / haplotype

533 Genetische Diversität der endemischen Honigbiene von 534 Madagaskar, Apis mellifera unicolor (Hymenoptera: 535 Apidae)

536 Apis mellifera unicolor / Mikrosatelliten / Afrikanische 537 Lineage / genetische Diversität / mtDNA

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720 Sheppard, W., Arias, M., Grech, A., Meixner, M.

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729 Team R.D.C. (2005) R: A language and environment for 730 statistical computing. R Foundation for Statistical 731 Computing, Vienna, Austria

732 Weir, B.S., Cockerham, C.C. (1984) Estimating F-statistics

733 for the analysis of population structure. Evolution

734 38 (6), 1358–1370

735 Whitfield, C.W., Behura, S.K., Berlocher, S.H., Clark,

736 A.G., Johnston, J.S., Sheppard, W.S., Smith, D.R.,

737 et al. (2006) Thrice out of Africa: ancient and recent

738 expansions of the honeybee, Apis mellifera . Science

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740 Winston M. (1992) Killer bees. The Africanized honeybee

741 in the Americas. Harvard University Press, 162 p.

742

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