Journal of Apicultural Research
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Southern limit of Africanized honey bees in Argentina inferred by mtDNA and wing geometric morphometric analysis
Leonardo Pablo Porrini, Silvina Quintana, Constanza Brasesco, Martín Pablo Porrini, Paula Melisa Garrido, Martin Javier Eguaras, Fernando Müller & Pedro Fernandez Iriarte
To cite this article: Leonardo Pablo Porrini, Silvina Quintana, Constanza Brasesco, Martín Pablo Porrini, Paula Melisa Garrido, Martin Javier Eguaras, Fernando Müller & Pedro Fernandez Iriarte (2019): Southern limit of Africanized honey bees in Argentina inferred by mtDNA and wing geometric morphometric analysis, Journal of Apicultural Research, DOI: 10.1080/00218839.2019.1681116 To link to this article: https://doi.org/10.1080/00218839.2019.1681116
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ORIGINAL RESEARCH ARTICLE Southern limit of Africanized honey bees in Argentina inferred by mtDNA and wing geometric morphometric analysis Leonardo Pablo Porrinia,b , Silvina Quintanaa,b,c , Constanza Brasescoa,b , Mart ın Pablo Porrinia,b , Paula Melisa Garridoa,b , Martin Javier Eguarasa,b , Fernando Muller€ d and Pedro Fernandez Iriarteb,e aLaboratorio de artr opodos, Centro de Investigaci on en Abejas Sociales (CIAS), Universidad Nacional de Mar del Plata (UNMdP), Buenos Aires, Argentina; bConsejo Nacional de Investigaciones Cient ıficas y T ecnicas (CONICET), Buenos Aires, Argentina; cArea Biolog ıa Molecular Instituto de An alisis Fares Taie, Mar del Plata, Argentina; dCentro de Cr ıa y Mejoramiento Ap ıcola E.K.F de Capiov ı (PROCAyPA), Misiones, Argentina; eLaboratorio de Gen etica, Dto. Biolog ıa, Universidad Nacional de Mar del Plata (UNMdP), Buenos Aires, Argentina
(Received 26 January 2018; accepted 1 April 2019)
African honey bee subspecies Apis mellifera scutellata began to spread in the American continent from southern Brazil in 1956. The process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honey bees. In Argentina, Africanized honey bees dominate in the northern semitropi- cal regions and a hybrid area is defined between (ca.32 –34 latitude). Although previous analysis has been carried out in Buenos Aires province, no analysis has been made south of this latitude. We used mitochondrial DNA (mtDNA) assays and wing geometric morphometrics to determine the prevalence of Africanized honey bees in managed populations. African mitotype origin and subspecies identity, were determined at three regions from Argentina. In our study, we analyzed 480 samples by wing morphometry and 157 by cytochrome-b assays, col- lected during 2013–2016. Our results show that North of 35 N latitude, honey bees with African mitotype were quite common (88.64%) finding high similarity with A. m. scutellata. South of that latitude, in temperate regions, we found African mitotype in only 18 of 133 colonies analyzed and a mixture of both European (A. m. ligustica, A. m. c arnica, A. m. mellifera) and African subspecies, most likely derived from North African honey bee (A. m. inter- missa). Our results confirmed the existence of Africanized honey bee populations with a gradual cline from north to south, as a result from recent A. m. scutellata-derived bee expansion. Beyond the transition area between the 30 –35 S parallels bees carrying the European mitotype were fairly common. Keywords: Africanized honey bee; mtDNA; geometric morphometrics; hybridization; Apis mellifera; Argentina
Introduction ecological conditions (Diniz, Soares, Sheppard, & Del Numerous subspecies of Apis mellifera occur in a wide Lama, 2003; Sheppard, Rinderer, Garnery, & range of distribution and differ from one another in Shimanuki, 1999). The Africanized honey bee distribu- many behavioral and morphological features as a tion is known to be strongly limited by climatic varia- result of the evolutionary process (Le Conte & bles such as temperature and precipitation (Harrison, Navajas, 2008; Ruttner, 1988; Whitfield et al., 2006). Fewell, Anderson, & Loper, 2006). Although In 1956, African honey bee subspecies A. m. scutellata Africanized honey bees have less advantageous adap- wasintroducedtosouthernBrazilinaneffortto tation when moved into more temperate conditions, establish honey bee populations better adapted to some predictive models show a large suitable area in tropical conditions (Scott Schneider, DeGrandi- southern Argentina (Vital, Hepburn, Radloff, & Fuchs, Hoffman, & Smith, 2004). After the accidental escape 2012). The variability of honey bee life-history traits, of 26 African queens (Kerr, 1967)theirdescendants as regards temperature and environment, indicate subsequently spread to South, Central, and North that the species keep such plasticity and genetic vari- America (Gonc¸alves, 1974,Taylor,1988;Winston, ability that could give rise to the selection of develop- 1992). The process of Africanization involved both ment cycles suited to new environmental conditions maternal and paternal bidirectional gene flow between (Le Conte & Navajas, 2008). Besides, it has been sug- European and Africanized honey bees and it is consid- gested that climate itself does not directly impose a ered as one of the most spectacular biological inva- distribution limit; this would be mostly determined by sions that are documented (Pinto, Rubink, Patton, food and nesting site availability (Dietz, Krell, & Coulson, & Johnston, 2005). Populations of Eischen, 1985). Genetic pool is evolving continually in Africanized honey bee, expressing scutellata-like response to natural selection, with bees adapting not reproductive, foraging, and defensive behavior, spread only to changes in their environment but also in rapidly due to their high adaptability to tropical response to human apicultural practices. In areas Corresponding Author. Email: [email protected]
ß 2019 International Bee Research Association 2 L. P. Porrini et al. where their ranges overlap, African and European- based on specimens collected from 2013 to 2016 and derived honey bee interbreed, causing “hybrid zones.” compared with A. m. scutellata and other five subspe- In Argentina, Africanized honey bee dominates in the cies (A. m. carnica, A. m. mellifera, A. m. iberiensis, A. m. northern semitropical regions whereas European intermissa, A. m. ligustica) present in the New World. honey bees dominate in the southern temperate areas. It is also possible to identify a hybrid zone that Materials and methods extends between 32 –34 S latitude where honey bees have varying degrees of African or European traits Samples (Abrahamovich, Atela, De la Rua, & Gali an, 2007; A total of 486 samples from managed honey bee colo- Sheppard, Rinderer, Mazzoli, Stelzer, & Shimanuki, nies were collected from about 100 apiaries in 21 1991; Whitfield et al., 2006). This border area is Argentine provinces during 2013–2016 (Table 1). slightly further south than the one originally suggested Almost 300 specimens per hive, in five colonies from by Kerr, Leon Del Rio, and Barrionuevo (1982). each apiary, were preserved in 96% ethanol and remit- Nevertheless, Africanized honey bees were found ted to the C.I.A.S. (Centro de Investigation en Abejas south of this latitude (39 S) by testing defensive Sociales) Arthropod Laboratory, UNMdP Universidad behavior traits (Dietz et al., 1985), and up to now no Nacional de Mar del Plata for further analysis. analysis have been carried out on honey bee popula- tions to determine the African mitotype or morpho- Molecular diagnosis type. The underlying mechanism for dispersal and the Total genomic DNA was extracted from individual honey genetic composition of Africanized honey bee popula- bee thoraces from 157 colonies using the High Pure PCR tions have been intensively studied in the American Template Preparation kit (Roche Diagnostics) for mito- tropics (Clarke, Oldroyd, Javier, Quezada-Euan, & type identification. Samples were subjected to separate Rinderer, 2001; Del Lama, Figueiredo, Soares, & Del qPCRs to amplify a region of the cytochrome b mitochon- Lama, 1988; Hall, 1990;Hall&Muralidharan,1989; drial gene using the primers Apis-F: Lobo, Del Lama, & Mestriner, 1989;Smith,Taylor,& (50-TATGTACTACCATGAGGA-CAAATATC-30) and Brown, 1989) and to a lesser degree in more temper- Apis-R: (50-ATTA-CACCTCCTAATTTATTAGGAAT- ate regions of South (Diniz et al., 2003;Quezada- 30) (Crozier, Koulianos, & Crozier, 1991), which generate Euan, Perez-Castro, & May-Itza, 2003; Sheppard et al., a control amplicon of 485 bp (Melting temperature (Tm): 1991) and North America (Kono & Kohn, 2015; 80 ± 0.2 C) for both African and European-derived honey Loper, Fewell, Smith, Sheppard, & Schiff, 1999;Pinto bee. Then a second amplification reaction was done with et al. 2005). Studies of feral populations suggest that a combination of primers Apis-R and AHB-F: Africanized honey bees expanded by maternal migra- (CATTACTCTGAGGTGGGTTC-30) (Szalanski & tion (Hall & Muralidharan, 1989;Hall&Smith,1991; McKern, 2007) which generates a 385 bp amplicon (Tm: Smith et al., 1989;Taylor,1988) and have intro- 79 ± 0.2 C) unique of African-derived honey bee. To val- gressed unequal nuclear and mitochondrial propor- idate the modifications made on methodology previously tions of European genes (Clarke et al. 2001; Hall, used by Szalanski and McKern, positive controls were 1990;Hall&McMichael,2001; Sheppard et al., 1991). used for both European (EHB) and Africanized (AHB) Variation in honey bee mtDNA has been detected bees to corroborate products size and melting curves. using a variety of molecular methods, ranging from Additionally partial sequences of cytochrome b mitochon- Restriction Fragment Length Polymorphism’s(RFLPs), drial gene were obtained and maximum likelihood (ml) to PCR-RFLP’s and direct sequencing (Collet, tree was reconstructed from previously uploaded sequen- Ferreira, Arias, Soares, & Del Lama, 2006;DeLaRua, ces (GenBank accession numb. EF016643-48) using Hernandez-Garcia, Jim enez, Gali an, & Serrano, 2005; MEGA7 (Kumar, Stecher, & Tamura, 2015) with 1000 Franck et al., 2001;Garnery,Cornuet,&Solignac, bootstrap replicates (Supplementary material). The 1992; Meixner et al., 2013;Pinto,Munoz,~ Ch avez- F81 þ i model was identified by jmodeltest2 (Darriba, Galarza, & De La Rua, 2012). Likewise the new meth- Taboada, Doallo, & Posada, 2012) as the best fitting sub- ods of automated measures have been used to distin- stitution model as determined by the lowest AICc. Thus guish Africanized honey bees from African and cytochrome b assay discriminates the mitochondrial European subspecies (Baylac, Garnery, Tharavy, African lineage from the mitochondrial western Pedraza-Acosta, & Rortais, 2008;Francoy,Prado, European, eastern Mediterranean, and Middle Eastern lin- Gonc¸alves,DaFontouraCosta,&DeJong,2006, eages. PCR amplifications were performed in a 20 ml total € Francoy et al., 2008;Kandemir,Ozkan, & Fuchs, 2011; volume containing 10 ml of 2X MyTaq PCR mix (Bioline, Miguel et al., 2011) with a high degree of consistency London, UK) master mix, 1 mM of each primer and 1 mlof between wing morphometry and molecular informa- DNA template. The selected cycling program for Apis-F/ tion. Using both wing geometric morphometrics and Apis-R consisted of an initial denaturation of 2 minutes at mtDNA approaches, we examined the current 95 C, and 40 cycles of 94 C2000,52 C3000,72 C3000. African-derived honey bee distribution in Argentina, The thermal cycling conditions of AHB-F/Apis-R were as Southern limit of Africanized honey bees in Argentina 3
Table 1. Localities (city and state) geo-referenced and number of honey bee colonies sampled in Argentina. Locality, Prov.Code Latitude Longitude Region N Yala, Jj 24 47019.5700S65 24044.8000O North 20 San Antonio, Jj 24 15040.1800S61 14024.7900O North 10 San Salvador, Jj 25 40016.4100S60 55058.0400O North 10 Salta Capital, St 25 6055.9600S58 15025.4700O North 5 Laguna Yema, Fr 26 23059.7100S54 37036.5200O North 5 Miraflores, Cc 26 55059.9900S55 3059.9800O North 5 Laguna Blanca, Fr 26 32022.8800S59 20031.1000O North 5 El Dorado, Ms 27 14039.1100S55 32023.4300O North 5 Capiovi, Ms 27 3507.5100S55 806.6100O North 15 San Martin, Cc 27 52027.8500S55 8015.7100O North 5 San Ignacio, Ms 27 54055.9500S55 45011.7200O North 5 F.Ameghino, Ms 27 2301.5600S54 44026.2300O North 5 San Javier, Ms 27 39036.7700S65 45046.1100O North 5 Apostoles, Ms 27 35010.6000S65 37026.8300O North 5 25 de Mayo, Ms 27 30040.7200S58 33024.2400O North 5 La Concha, Tc 30 15031.4200S57 3809.3800O North 5 J. B. Alberdi, Tc 27 5708.9900S58 48011.4900O North 5 S. L. del Palmar, Cr 27 22017.4500S58 30039.4700O North 5 Monte Caseros, Cr 27 35058.4900S66 18057.0900O North 2 Empedrado, Cr 27 46033.8500S64 14017.6400O North 5 San Cosme, Cr 28 15045.2700 65 38013.6000O North 5 Andalgal a, Ct 28 51011.7200S66 1408.7700O North 5 Sgo. Capital, SE 28 44038.5200S65 5703.0900O North 5 Amadores, Ct 28 1800.7800S65 21059.7400O North 5 Chumbicha, Ct 28 4805.2200S66 5700.6400O North 5 Capay an, Ct 28 5003.4400S66 5404.0200O North 5 El alto, Ct 29 1104.5500S58 4024.8400O North 5 Anillaco, LR 29 10022.5200S56 39012.9400O North 5 Aminga, LR 29 9057.0600S67 3001.5100O North 5 Mercedes, Cr 30 1806.3400S66 42050.7900O North 2 Pje. Bacaray, Cr 29 8046.9200S59 38035.5000O North 2 Chilecito, LR 30 5906.1300S57 55011.4300O North 5 Dto. A.Penazola,~ LR 31 9042.2200S58 11013.0200O North 5 Reconquista, SF 31 3002.6000S66 42046.4200O Central 5 Federacion, ER 31 1309.8600S61 43030.0000O Central 5 Concordia, ER 31 32045.8900S68 33023.1500O Central 5 Dto. Rosario, SF 32 56055.0700S60 3901.9400O Central 5 San Antonio, SF 33 40040.8000S65 27028.9300O Central 5 San Juan, SJ 33 9026.6100S64 2106.9500O Central 5 Rosario, SF 34 37059.0600S68 20025.1100O Central 5 Villa Mercedes, SL 34 7012.2700S58 35057.1100O Central 10 R ıo Cuarto, Cd 37 50047.2200S58 15019.3600O Central 5 San Rafael, Md 37 4607.3700S57 41047.2900O Central 10 Bajo Delta, BA 38 101.3000S57 4000.7200O Central 5 Balcarce, BA 38 9048.3900S58 46054.0700O Central 5 Col. Barrag an, BA 38 1007.9700S57 3808.6300O Central 5 Bat an, BA 38 4305.9400S62 15058.8500O Central 5 Lober ıa, BA 38 3301.4800S58 41047.0800O Central 5 Mar del Plata, BA 38 12018.4800S61 46010.9800O Central 15 Bahia Blanca, BA 38 505.9800S63 25050.7900O Central 5 Necochea, BA 40 48041.4500S62 59044.1800O Central 15 Saldungaray, BA 38 59027.0600S64 5038.0600O Central 6 Jacinto Ar auz, LP 38 49050.1900S68 7018.9800O Central 7 Viedma, RN 39 18052.5400S65 45047.8600O Central 10 Rio Colorado, RN 39 17018.4500S65 39054.0500O South 25 Centenario, Nq 40 47055.9300S62 58049.4300O South 10 Luis Beltr an, RN 40 608.4700S64 27029.6200O South 15 Choele Choel, RN 41 57053.7200S71 3207.2000O South 15 C. Patagones, BA 42 2054.0700S71 30056.0400O South 10 Gral. Conesa, RN 42 2047.2800S71 35044.4900O South 25 El Bolson, RN 42 13042.8400S71 20032.8500O South 16 El Hoyo, Ch 46 32055.0600S71 37055.1200O South 19 Lago Puelo, Ch 24 47019.5700S65 24044.8000O South 8 Epuy en, Ch 24 15040.1800S61 14024.7900O South 10 Los Antiguos, SC 25 40016.4100S60 55058.0400O South 4 4 L. P. Porrini et al.
Results To perform matrilineal classification qPCR amplification melting curves were analyzed to check the specific PCR product between AHB-F and Apis-R which shows a peak at the dissociation temperature of 79 C in the African mitotypes (Figure 2A) and Ct values (Cycle threshold) below 25 (Figure 2B). Of the 157 samples Figure 1. Location of 19 landmarks used to characterize sub- collected for mtDNA analysis, 57 were classified as species by geometric Morphometric variation of vein junctions on 10 forewings per colony. African origin and 100 as European. African mitotype was not uniformly distributed throughout the studied area (Figure 3, Table 2). North of 35 latitude, honey follows: an initial denaturation of 2 minutes at 95 C, and bees with African mitochondria were quite common 00 00 00 40 cycles of 94 C20 ,50 C30 ,72 C30 . After amplifi- (88, 64%), having been found in 39 from 44 colonies cation, a melting curve analysis to confirm specific amplifi- located mainly in subtropical provinces. South of that cation of the PCR product was performed. All qPCR latitude, on central and south regions we found African reactions were carried out in a thermocycler Rotor-Gene mitotype in only 18 of 113 analyzed colonies (11, 46%). (Qiagen, Hilden, Germany) in a final volume of 20 ml using The southernmost African mitotype was detected EvaGreen as fluorescent intercalating dye (KAPA Fast, approximately 8 km south of El Hoyo, Chubut Biosystems, Woburn, E.E.U.U.). (42 6019.2600S; 71 27023.0800O). Even though the analysis of A. mellifera populations Geometric morphometrics using the wing morphometry showed consistent results matrilineal origin analysis (Table 2), the distribution limit From the 486 samples obtained, the left forewing of 10 of hybrids with similarity to A. m. scutellata seems to be workers per colony (4860 wings), from 157 apiaries dis- more towards the north than the southern African tributed throughout the country were dissected mitotype found. From the 486 colonies, Mahalanobis (Tofilsky, 2008). The wings were mounted in glass distance was calculated by province to reflect the photographic frames and scanned with a Plustek optic degree of similarity with A. m. scutellata (Figure 3). film 8100 (7200 dpi). For every wing image, the coordi- Likewise, Canonical variate analysis (CVA) of subspe- nates of 19 homologous landmarks (Francoy et al., cies divergence were carried out for the three different 2008) were manually plotted at the wing vein (Figure 1) Regions (north, central and south). To facilitate geomet- using the software tpsDIG, v2.16 and tpsUtil v1.4 ric interpretation of the data, similarities between (Rohlf, 2010). Additionally, for each pure subspecies (A. groups were identified in two-dimensional scatterplots m. carnica, A. m. mellifera, A. m. iberiensis, A. m. intermissa, in which each axis represented a canonical variable. A. m. ligustica and A. m. scutellata obtained from the There were considerable differences between the Morphometric Bee Data Bank in Oberursel, Germany) North, Central and Southern regions. In most of the wing images, representing 50 different colonies were colonies analyzed from the north region (Figure 4A), a included in the analysis. The landmark coordinates high similarity with A. m. scutellata was found and in a obtained were taken to MORPHOJ package lesser degree with A. m. intermissa. In the central region (Klingenberg, 2011). Alignment was performed using (Figure 4B), hybrids were found with fairly balanced Procrustes fit (translation, proportion, and rotation; proportions of the different subspecies, being the ones Dryden & Mardia, 2016). Generalized adjustment of of greater similarity to A. m. ligustica, A. m. intermissa Procrustes method (Viscosi & Cardini, 2011), was per- and in a much smaller proportion to A. m. scutellata, formed. Procrustes and Mahalanobis distance was calcu- compared with the northern region. In the south region lated (Klingenberg & Monteiro, 2005) allowing to reflect the proportion of A. m. ligustica and A. m. carnica the degree of separation between groups. Based on the resulted to be greater than in the central region, and a spectral decomposition of covariance, Principal compo- decreasing proportion of hybrids showed similarity to A. nent analysis (PCA) was performed with all measure- m. scutellata (Figure 4C, Table 2). ments from each locality. Multivariate statistical analysis of the data included the Canonical Variate Analysis (CVA) using the averages of the populations obtained in Discussion the morphometrical analysis in order to verify the dif- The results obtained with geometric morphometric ana- ferences among the populations collected in the three lysis indicate that forewings keep necessary information to regions of the country (north, central, and south distinguish honey bee morphotype from different biogeo- regions). Additionally, a cross validation test was made graphic regions. Likewise, mitotype origin analysis in associ- to check the accuracy of the equations in identifying the ation with these was found to be very informative to colonies and permutation test P values for all the pair- characterize A. mellifera populations. Our results showed a wise tests were also carried out. strong, consistent correlation between A. m. scutellata Southern limit of Africanized honey bees in Argentina 5
Figure 2. (A) Melting analysis used for checking the specific amplification of AHB-F and Apis-R PCR product. In the vertical axis is rep- resented the rate of change in relative fluorescence units (RFU) with time (T) ( d (RFU)/dT) versus temperature ( C) in the horizon- tal axis. (B) Real time amplification with primers AHB-F and Apis-R presented in a graphic format of increase in fluorescence (delta Rn) plotted against number of cycles. 6 L. P. Porrini et al.
Figure 3. Distribution of mtDNA mitotype in honey bee populations sampled in Argentina. Black triangles denotes haplotypes having African origin, while white hexagon denotes haplotypes having European origin. On the lower right margin in a red scale, Mahalanobis distance was calculated by province to reflect the similarity with A. m. scutellata. Southern limit of Africanized honey bees in Argentina 7
Table 2. Mitotype and Morphotype characterization from different regions indicating the number of colonies analyzed. At the top of the table mitochondrial DNA analysis and proportions of African matrilineal origin; at the bottom, identifications of A.mellifera subspecies based on geometric morphometric analysis of forewings are shown. North region Central region South region Mitotype African European African European African European N Matrilineal origin 39 5 11 44 7 51 157 % lineage (A) by region 88.64% 20% 12% Morphotype Analyzed colonies 191 138 157 486 Mahalanobis dist. Apis mellifera carnica 5,2206 4,5944 3,1467 Apis mellifera mellifera 4,1526 4,3156 4,1343 Apis mellifera iberiensis 5,1845 5,7549 5,4583 Apis mellifera intermissa 3,3583 3,5249 3,4996 Apis mellifera ligustica 4,6137 3,5024 3,1581 Apis mellifera scutellata 2,2859 4,9104 5,6508
Figure 4. Canonical variate analysis (CVA) of subspecies divergence carried out in 21 provinces pooled in three regions including 486 colonies analyzed. morphotype and African mitotype throughout the north- localities belonging to central and south regions, sustaining ern region, indicating successful spreading of Africanized the hypothesis that this area represents a zone of hybrid- honey bee since last A. m. scutellata expansion from Brazil ization between Africanized and European honey bees in 1956 (Francoy et al., 2009). However, its geographic dis- (Whitfield et al., 2006). tribution was very heterogeneous if we look together at In Buenos Aires province, molecular diversity of the the three analyzed regions. Our mitotype characterization honey bees has been previously analyzed through showed colonies with both African and European origin in mtDNA resulting in a homogeneous European genetic 8 L. P. Porrini et al. profile with the presence in some colonies of North that needs to be further investigated; first by COI–COII African origin (Abrahamovich et al., 2007). Although intergenic region sequence analysis or by high-density there are populations between the 35 and 42 of lati- assays using single nucleotide polymorphism markers, tude with an African mitotype, the amplified cyto- but also by estimating the degree of adaptations to the chrome b region does not contain polymorphisms that local climates, vegetation and beekeeping. discriminate A. m. scutellata from A. m. intermissa and from some A. m. iberiensis (Pinto et al., 2003). Likewise, Acknowledgements based on geometric morphometric analysis, the honey We thank the beekeepers who collected and forwarded sam- bees at this region were characterized like a mixture of ples from different localities as well as Dr. Tiago Mauricio both European and African subspecies, most likely Francoy, who provided us with the wings images from pure derived from North African honey bee, sustaining the subspecies used in geometric morphometric analysis. This assumption of an Iberian origin for some of the African research was supported by the CIAS and UNMdP. We appre- mitotypes present in Argentina (Sheppard et al., 1999). ciate the contribution of the anonymous reviewers and editor- ial team who helped improve this manuscript. In any case, we must consider that in this analysis, unlike the one conducted by Sheppard, only managed colonies of A. mellifera were analyzed. Therefore, we Disclosure statement believe that these results reflect an earlier expansion of No potential conflict of interest was reported by the authors. African genes since A. m. intermissa and probably A. m. iberiensis were introduced in this region before the Supplementary material arrival of A. m. scutellata to South America. Supplementary material is available for this article at: https:// Subspecies behavior and reproduction traits vary doi.org/10.1080/00218839.2019.1681116. extensively reflecting adaptations to different climatic and ecological conditions (Scott Schneider et al., 2004). It would seem that Africanized honey bees may have ORCID reached a limit to their spread at about the 35 S lati- Leonardo Pablo Porrini http://orcid.org/0000-0002-2480-9635 tude, based in part on their inability to survive extended Silvina Quintana http://orcid.org/0000-0003-1845-7677 Constanza Brasesco http://orcid.org/0000-0001-6207-2825 cold periods. South of this limit, morphotype analysis Mart ın Pablo Porrini http://orcid.org/0000-0002-6070-1664 would make us suppose that African mitotypes found Paula Melisa Garrido http://orcid.org/0000-0001-5989-4596 about the 42 S latitude, are derived from an ancient Martin Javier Eguaras http://orcid.org/0000-0001-6061-7924 expansion to A. m. scutellata introduction in South Pedro Fernandez Iriarte http://orcid.org/0000-0003- 0353-8554 America. We also found evidence of an extensive range of European-African hybrids. This may be due to move- ments of colonies by beekeepers from central to south- References ern region or alternatively, and it may reflect the ability Abrahamovich, A. H., Atela, O., De la Rua, P., & Gali an, J. of African-derived honey bee to survive in temperate (2007). Assessment of the mitochondrial origin of honey areas (Dom ınguez-Ayala, Moo-Valle, May-Itz a, Medina- bees from Argentina. Journal of Apicultural Research, 46(3), Peralta, & Quezada-Eu an, 2016). In our knowledge, 191–194. introgressive hybridization between African and Baylac, M., Garnery, L., Tharavy, D., Pedraza-Acosta, J., & European-derived honey bee’ best explains the current Rortais, A. (2008). ApiClass, an Automatic Wing Morphometric Expert System for Honeybee Identification. patterns of mitotype distribution across the different Retrieved from http://apiclass.mnhn.fr regions analyzed (De La Rua, Hernandez-Garcia, Clarke, K. E., Oldroyd, B. P., Javier, J., Quezada-Euan, G., & Jim enez, Gali an, & Serrano, 2005; Kono & Kohn, 2015; Rinderer, T. E. (2001). Origin of honeybees (Apis mellifera Quezada-Eu an et al., 2003). Finally, phenotypic differen- L.) from the Yucatan peninsula inferred from mitochondrial – ces between them probably result from restrictions to DNA analysis. Molecular Ecology, 10(6), 1347 1355. doi:10. 1046/j.1365-294X.2001.01274.x gene flow between their respective populations (Diniz Collet, T., Ferreira, K. M., Arias, M. C., Soares, A. E. 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