pis mellifera beekeeping in Colombia began with European lineages brought by the Spanish colonizers of America. Experimental swarms of African lineages that accidentally escaped in Brazil in the 1950s reached Colombia in the 1970s, starting a genetic crossing with local European populations. Today it is assumed that the majority of the Colom- bian beekeeping is of Africanized type, although some beekeepers continue to import European queens, to confer meekness. To determine the prevalence of African mitotypes in bee populations of the country, PCR and sequencing of a fragment from the intergenic region of the cytochrome oxidase I and II (COI–COII) mitochondrial genes, were employed. This study analyzed 645 A. mellifera specimens collected in six regions of Colombia, representing the largest sampling survey of apiculture for a Latin American country. The results indicated that 98.3% of these populations had African mitotypes, represented in 17 classes (A): A1, A1b, A1e, A4, A11, A26, A26a, A26b, A26c, A26d, A29a, A30, A36, A39, A44, A46 and A47. Only 1.7% of beehives had European mitotypes (C), corresponding to ligustica subspecies. These results confirm that beekeeping in the country is mostly of the Africanized type.

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Determination of the Africanized mitotypes in
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Víctor Manuel Tibatá, Edgar Arias, Miguel Corona, Fernando Ariza Botero,
Judith Figueroa-Ramírez & Howard Junca
To cite this article: Víctor Manuel Tibatá, Edgar Arias, Miguel Corona, Fernando Ariza Botero,
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2. ORIGINAL RESEARCH ARTICLE
Determination of the Africanized mitotypes in populations of honey bees
(Apis mellifera L.) of Colombia
Vı´ctor Manuel Tibata´a,b
* , Edgar Ariasa
, Miguel Coronac
, Fernando Ariza Boteroa
, Judith Figueroa-Ramı´reza
and
Howard Juncaa,d
a
Facultad de Medicina Veterinaria y Zootecnia, Grupos de Investigacio´n AYNI – Ciencia y Tecnologı´a Apı´cola y Gene´tica Molecular Animal,
Universidad Nacional de Colombia, Bogota´, Colombia; b
VM LAB Veterinary Molecular Diagnostics and Research, Bogota´, Colombia; c
Bee
Research Lab – United States Department of Agriculture, USDA, Beltsville, MD, USA; d
RG Microbial Ecology, Div. Ecogenomics & Holobionts,
Microbiomas Foundation, Chı´a, Colombia
(Received 28 April 2017; accepted 30 October 2017)
Apis mellifera beekeeping in Colombia began with European lineages brought by the Spanish colonizers of America.
Experimental swarms of African lineages that accidentally escaped in Brazil in the 1950s reached Colombia in the
1970s, starting a genetic crossing with local European populations. Today it is assumed that the majority of the Colom-
bian beekeeping is of Africanized type, although some beekeepers continue to import European queens, to confer
meekness. To determine the prevalence of African mitotypes in bee populations of the country, PCR and sequencing
of a fragment from the intergenic region of the cytochrome oxidase I and II (COI–COII) mitochondrial genes, were
employed. This study analyzed 645 A. mellifera specimens collected in six regions of Colombia, representing the largest
sampling survey of apiculture for a Latin American country. The results indicated that 98.3% of these populations had
African mitotypes, represented in 17 classes (A): A1, A1b, A1e, A4, A11, A26, A26a, A26b, A26c, A26d, A29a, A30,
A36, A39, A44, A46 and A47. Only 1.7% of beehives had European mitotypes (C), corresponding to ligustica subspecies.
These results confirm that beekeeping in the country is mostly of the Africanized type.
Determinacio´n de los mitotipos de africanizacio´n en poblaciones de abejas Apis mellifera L. en Colombia
La apicultura en Colombia ha contado desde sus inicios con linajes europeos, traı´dos por los colonizadores de Ame´r-
ica. Con la llegada al paı´s en 1970 de las abejas africanizadas que escaparon accidentalmente en Brasil en 1950, se inicio´
un cruce gene´tico natural de e´stas con las abejas europeas. Hoy dı´a se asume que la gran mayorı´a de la apicultura
colombiana es de tipo africanizado, aunque algunos apicultores continu´an importando reinas europeas para conferir
mansedumbre. Para determinar la prevalencia de mitotipos africanizados en poblaciones de abejas del paı´s, se
emplearon las te´cnicas de amplificacio´n por PCR y secuenciacio´n de un fragmento de la regio´n interge´nica de los genes
de la citocromo oxidasa I y II (COI y COII) mitocondrial. Este estudio analizo´ 645 especı´menes de Apis mellifera, colec-
tados en seis regiones de Colombia, representando el ma´s grande muestreo de la apicultura para una paı´s latinoameri-
cano. Los resultados indicaron que el 98.3% de estas poblaciones, tuvieron de mitotipos de africanizacio´n,
representados en 17 clases (A): A1, A1b, A1e, A4, A11, A26, A26a, A26b, A26c, A26d, A29a, A30, A36, A39, A44,
A46 y A47. Solamente el 1,7% de las colmenas tuvieron mitotipos europeos (C), correspondientes a la subespecie
europea, ligustica. Estos resultados confirman que la apicultura del paı´s es mayormente de tipo africanizado.
Keywords: bee; Apis mellifera; Africanization; mitotypes; Colombia
Introduction
Apis mellifera, originally native to Europe, Africa and Asia,
is the most used bee worldwide for the production of
honey, pollen, propolis, royal jelly and wax. A. mellifera
includes several sub-species, classified into 5 lineages: C
(Carnica): A. m. carnica and A. m. ligustica; M (Honey
bees of Europe): A. m. mellifera and A.m. iberiensis; A
(African Group): A. m. scutellata, A. m. capensis, A. m.
lamarckii, A. m. litorea, A. m. adansonii, A. m. intermissa
and A. m. unicolor; Y (bees from Ethiopia) and the group
O (Eastern bees including A. m. anatoliaca, A. m. cauca-
sica, A. m. syriaca, A. m. pomonella, and A. m. cypria) (Sza-
lanski & Magnus, 2010).
At genetic level, the 5 lineages of A. mellifera can be
identified on the basis of nuclear and mitochondrial
DNA markers (Arias & Sheppard, 2005; Franck et al.,
2001; Han, Wallberg, & Webster, 2012; Miguel et al.,
2010). Most of the studies published so far determining
africanization from honey bee populations around the
world have used the PCR-RFLP technique or DNA
sequencing of the cytochrome b (cyt b) region of mito-
chondrial DNA (mtDNA). Another approach with
higher resolution is the genotyping by sequencing ampli-
fied fragments of the intergenic regions of cytochrome
oxidase I and II (COI and COII) genes, also harboured
in the mitochondrial DNA, but exhibiting a higher
degree of genetic variation between the different
*Corresponding author. Email: vmtibatar@unal.edu.co
© 2017 International Bee Research Association
Journal of Apicultural Research, 2017
https://doi.org/10.1080/00218839.2017.1409065
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3. lineages and therefore constituting a molecular marker
of superior precision for detecting mitotypes that repre-
sent intraspecific genetic variations. This genetic region
was used on PCR-RFLP analyses of bee populations in
Turkey (Solorzano et al., 2009); Mexico; South America
(Collet, Ferreira, Arias, Soares, & Del Lama, 2006;
Ferreira, e Silva, Arias, & Del Lama, 2009); Africa
(Franck et al., 2001); and Australia (Chapman, Lim, &
Oldroyd, 2008). In the United States of America, a sam-
pling survey of honey bees was analyzed using
sequenced fragments of this intergenic region detecting
a total of 12 different mitotypes of which two (A1 and
A1d of the African lineage) represented 77%, thus
demonstrating the high degree of Africanization of the
populations (Szalanski & Magnus, 2010).
Regarding honey production in Colombia, Amerindi-
ans have been doing so for centuries from several sting-
less bees of the autochthonous Meliponini tribe (Vit,
Pedro, & Roubik, 2013). Honey production with A. mel-
lifera was originated in the country with European lin-
eages brought by the Spaniards colonizing America since
the fifteenth century. In the first part of the twentieth
century, the Colombian Ministry of Agriculture
imported European bees to promote the beekeeping
industry. In the decade of the 1970s, with the arrival to
Colombia of the Africanized bees derived from A. mellif-
era scutellata that escaped accidentally in Brazil, began a
natural genetic crossing of these bees with the managed
European lineages. Given this situation, many of the bee-
keepers abandoned the activity, mainly due to the high
defensiveness of the Africanized bees, thus reducing the
number of hives and therefore the production of honey
(Francoy et al., 2009). Nevertheless, in the following
years and until today, the majority of Colombian bee-
keepers have worked with the resulting bee hybrids, as
they consider the hybrids generate a higher yield and
are believed to be more resistant to diseases, but no
scientific studies in local populations have been reported
confirming or contradicting these claims. Additionally,
despite the high defensiveness of these hybrids, the
majority of beekeepers acquire queens from others local
producers, whom only bred Africanized bees or they
capture feral swarms to increase the number of colo-
nies. However, a few beekeepers continue importing
European queens to confer meekness, thus facilitating
the management of hives and reducing risks of attacks
when manipulating Africanized hybrids. Despite the
introduction of European queens, it is assumed that
most of the beekeeping in the continental area of
Colombia is of the Africanized type, and only in the
northern Caribbean insular area (San Andre´s and Provi-
dence Islands), it is expected that no hybridization has
occurred there yet with European lineages. At present,
the majority of Colombian beekeeping is used mainly
for honey production; only in cold or temperate regions
(Boyaca´), beehives are employed in pollen production.
The use of beekeeping for crop pollination in Colombia
is still minimal (Sa´nchez, Castan˜eda, Mun˜os, & Tellez,
2013).
About previous genetic studies in Colombia, there is
one report based on analysis of restriction patterns
(RFLP) of amplified fragments from the ribosomal 16S
RNA gene and mitochondrial DNA (Prada, Duran, Sala-
manca, & Del Lama, 2009) finding that 87% of the sam-
ples were of the African haplotype and represented in 6
mitotypes: A1, A4, A26, A28, A29 and A30. In the pre-
sent study we determined, by PCR and sequencing anal-
yses, the mitotypes present in a larger number of
samples representative of honey bee populations in
Colombia, aiming to confirm such high level of African-
ization, to expand the knowledge of the mitotype diver-
sity, predominance and relative abundances, and to
provide evidences whether there is an active population
expansion or balancing selection. Since the level of
Africanization may have implications on adaptability, bio-
logical fitness, behavior, pollination, resistance to disease
and honey production, as well as the incidence and
prevalence of diseases in A. mellifera (Hamiduzzaman
et al., 2015; Mendoza et al., 2014), this study aims to
contribute with a such genetic background baseline.
Materials and methods
Sampling
Samples were collected from apiaries belonging to
Colombian associations of beekeepers from six regions
(“departamentos”). These regions are recognized as the
main apicultural producers in the country: (1) Mag-
dalena: Association of Beekeepers Conservationists of
the Sierra Nevada of Santa Marta – APISIERRA. (2)
Sucre: Rural Association of Beekeepers of Sucre –
ARPA. (3) Boyaca´: Association of Beekeepers of Boyaca´
– ASOAPIBOY. (4) Antioquia: Association of Beekeep-
ers of Bethany – ASOAPIBE. (5) Huila: APISRED and (6)
Cundinamarca: Association of Fruit Growers of Sumapaz
(FRUTIPAZ). A total of 645 hives were sampled; the
number of apiaries, municipalities and sampled beehives
are detailed in Table 1. Each hive was randomly selected
within each apiary (Figure 1(A)). An adult bee was col-
lected from each of the 645 selected hives and sacri-
ficed by inhalation with ethyl acetate in a lethal
chamber, according to international standards for the
slaughter of experimental animals (Ma´rquez Luna, 2005).
Bees were stored in containers with 70% ethanol. As
controls of European honey bees, 12 bees were brought
from Israel (four), United States (four) and San Andres
Islands (four); and preserved in 70% ethanol.
DNA extraction
Genomic DNA was obtained from the thorax of each
bee by the phenol chloroform method (Sambrook &
Rusell, 2001). Briefly, each thorax was washed in 1× TE
buffer and then macerated with disposable pistils and
2 V.M. Tibata´ et al.
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4. resuspended in 500 ul of lysis buffer, plus 10 uL of Pro-
teinase K (20 mg/ml), and incubated at 55 ˚C for 12 h.
500 μl of the supernatant was taken and mixed with
500 μl of saturated phenol (pH 8.0), chloroform and
isoamyl alcohol (25:24:1); and centrifuged at 12,000×g
for 5 min. The aqueous phase was transferred to a new
vial and a volume of isopropanol plus 1/10 volume of 3
Molar sodium acetate was added. The solution was cen-
trifuged at 12,000×g for 15 min. The isopropanol was
removed and the pellet washed with 75% ethanol; and
then centrifuged at 12,000×g for 5 min and the remain-
ing ethanol was removed. The DNA obtained was
reconstituted in 1X TE buffer and stored in refrigeration
at 4 ˚C. In order to determine the integrity and quality
of the obtained DNA, aliquots of this genetic material
were quantified by fluorometry (Qubit 2.0 Invitrogen)
and visualized by electrophoresis in agarose gels
(Sambrook & Rusell, 2001).
DNA amplification
Genomic DNA from the 645 bees was used as a tem-
plate to amplify a fragment of the intergenic region of
the cytochrome oxidase I and II genes (COI and COII)
and was amplified by PCR with the primers E2 (5´-GGC
AGA ATAAGT GCA TTG-3´) and H2 (5´-CAA TAT
CAT TGATGA CC-3´) (Garnery, Solignac, Celebrano,
& Cornuet, 1993). The PCR amplification profile con-
sisted of a denaturation step of 94 ˚C for 2 min, fol-
lowed by 35 cycles of 94 ˚C for 45 s, 46 ˚C for 45 s
and 72 ˚C for 45 s; and a final step of 72 ˚C for 5 min.
The size of the amplified fragment was verified by elec-
trophoresis in 2% agarose gel.
Sequencing and phylogenetic analysis
Amplified fragments were sequenced with the forward
primer (H2) used in the PCR protocol. In order to
establish the identity of the obtained sequences with the
A. mellifera mitotypes reported in the GenBank, nucleo-
tide sequences with a continuous quality score of
Phred > 28, were used for the BLASTn (Basic Local
Alignment Search Tool) of the NCBI (National Center
for Biotechnology Information, Bethesda, MD, USA),
with default parameters for nr/nt, with high similarity
sequences (megablast) http://blast.ncbi.nlm.nih.gov/Blast.
CGI. To confirm the Blast results about the affiliation of
the sequences obtained to a certain reference mitotype
sequence reported, a data-set was constructed contain-
ing all the sequences obtained from the specimens col-
lected in Colombia and all the closest reference
sequences reported in the databases as representatives
of mitotypes. A multiple sequence alignment was per-
formed using MUSCLE program with default parameters
for both DNA sequence alignment as implemented in
the MEGA program (Tamura, Stecher, Peterson, Filipski,
& Kumar, 2013). After the initial alignment, a sequence
data-set was extracted consisting of the continuous col-
umns blocks with common information between all the
obtained sequences and the reference sequences. This
data-set was realigned for further phylogenetic analyses.
Trees were calculated by Neighbor-Joining, UPGMA,
Maximum Likelihood, Maximum Parsimony and Mini-
mum Evolution methods with a bootstrap of 1000. The
aligned sequences and the resulting trees can be found
as supplementary online material (SP5-SP9). Program
DNAsp 5.10 (Rozas, Librado, Sanchez-Delbarrio, Messe-
guer, & Rozas, 2009) was used for population genetics
analyses.
Results
Mitotypes amplification and sequencing
Amplifications were obtained from 645 hives sampled.
COI-COII intergenic regions were polymorphic in
sequence and size among the different haplotypes. We
obtained fragments of lengths between 600 and 800
base pairs from the different samples, a finding similar
to the report by Szalanski and Magnus (2010) (supple-
mental online material, SP0). Blast analyses of the 645
sequences against the GenBank nr database allowed to
identify that 98.3% of the samples were of Africanized
lineage (A), represented in 17 mitotypes: A1, A1b, A1e,
A4, A11, A26, A26a, A26b, A26c, A26d, A29a, A30,
A36, A39, A44, A46 and A47. The most prevalent
(93%) were A1e (31.9%), A26a (23.1%), A1 (19.8%), A4
(12.4%), A26d 4% and A26c (2.2%). The remaining 11
Africanized mitotypes comprised 5.3%, with individual
percentages varying between 0.1 and 0.8%.
Concerning to European lineage (C), ligustica mito-
types were found, corresponding to 1.7% (11 samples):
Table 1. Number of apiaries and sampled hives per region.
Region Number of municipalities Number of apiaries Number of sampled hives
Magdalena 6 11 153
Sucre 9 13 164
Boyaca´ 12 16 158
Antioquia 3 6 57
Huila 3 6 54
Cundinamarca 3 6 59
Total 36 58 645
Determination of the Africanized mitotypes in populations of honey bees 3
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5. 7 samples from Huila region (C1a mitotype) and 3 from
Cundinamarca (C1 mitotype) (Figure 1 and supplemen-
tary online material SP2). The European M mitotypes
were not detected in any sample. From the European
bee sample controls, the obtained mitotypes were: C1
(ligustica) from 4 samples from San Andre´s Islands, C11
(carnica) of 4 samples from United States and ligustica
mitotypes from 4 samples from Israel.
Regarding the prevalence of mitotypes from each
region, A1e was the most frequent in 4 of the 6 regions;
while A26a was prevalent in Boyaca´ and Magdalena,
additionally these two regions had more variety of
Figure 1. Frequencies of A. mellifera mitotypes from 6 regions of Colombia, South America. (A) Distribution of mitotypes (percent-
ages) detected on each region. Bars represent the frequency of the sequences grouped according to the closest relative sequence
found in Blast analyses in GenBank nr/nt database: A1e (GU326335), A26a (FJ743640), A1 (EF033649), A4 (EF033650), A26d
(GU326336). LFM stands for Low Frequency Mitotypes, those found in a range of 0.2–2.2% of the samples, including sequences
ascribed to A26c, A26, A44, A47, A30, A26b, A1b, A39, A11, A46, A36, A29a and C1. Number of hives sampled: Magdalena (153),
Sucre (164), Boyaca´ (158), Antioquia (57), Cundinamarca (59) and Huila (54). (B) Cumulative frequencies from each mitotype in a
total of 645 hives sampled in Colombia.
4 V.M. Tibata´ et al.
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6. mitotypes (10 and 12 respectively). As mentioned
above, European mitotypes were found only in three
apiaries in Huila and one in Cundinamarca (18 and 5%,
respectively). In the apiaries from the other 4 regions
(Boyaca´, Magdalena, Sucre and Antioquia), 100% of the
samples corresponded to Africanized lineages (Figure 1
and supplementary online material).
Phylogenetic analysis
The sequences of the amplicons of the COI-COII inter-
genic region, obtained from bees collected in the 6
regions, showed that the great majority of the
sequences were indeed highly related to the inferred
African origin (mitotype A). Within this large cluster of
Africanized mitotypes, variability was identified between
groups in the country. The sequences retrieved from
specimens collected at insular Colombian territories
(San Andre´s Island) clustered as expected with Euro-
pean lineages (Figure 2). Multiple sequence alignment
datasets of the information, are available in supplemen-
tary material SP1 and SP3.
In order to have a more detailed interpretation of the
mitotypes found in Colombia, the pairwise distances of all
the aligned sequences were calculated (sequence data-
set alignment and resulting distance matrix as supplemen-
tary online material SP1 and SP2). The sequences were
clustered in groups of high similarity, and included the ref-
erence mitotype sequences. The overall average distance
among all the sequences was 0.030 (3%), indicating a high
level of relatedness of the sequences compared. Those
results are in agreement with the mitotype frequency affil-
iations assigned, based on Blast searches (Figure 1).
Regarding multiple alignment, the pairwise distance
calculation showed that sequence divergence of 6% or
less, were affiliated to a given reported mitotype. The
exceptions to this criterion, were 45 sequences (repre-
senting 8% of all the 543 sequences included in the anal-
yses). Those sequences exhibited a high similarity to a
reference mitotype sequence in GenBank (above 99%),
when using a Blast local alignment analysis; but in the
global multiple sequence alignment showed total similari-
ties between 84 and 93%. These resulted on being
excluded of the clusters containing the reference mito-
type sequences (A1e or A26d), to which they are clo-
sely related based on Blast results.
The total number of haplotypes calculated (DNAsp
V.5.0) in the data-set of the sequences obtained (exclud-
ing the reference mitotype sequences, supplementary
material SP3 and SP4) are 80, with an Haplotype (gene)
diversity Hd of 0,307 and a Variance of Haplotype diver-
sity of 0.00071, where it is also evidenced and excess of
singletons and a low frequency polymorphims. Consid-
ering that in single gene polymorphism assessments in
populations with a high frequency of new mutations it
could be interpreted as the evidence of population
growth (Alonso & Armour, 2001), the statistical tests
of neutrality (Fu & Li, 1993) were applied on the
nucleotide data-set of of COI-COII intergenic regions
sequences from sampled specimens in honey bee popu-
lations in Colombia. It resulted in Fu and Li’s F test
statistic values of −5.31821 (**, p < 0.02), where such
large negative value indicates an excess of the number
of young mutations and a reduction of the number of
common variants, that can be taken as evidence against
the neutrality of mutations (Fu & Li, 1993); for Tajima’s
D, equaling to zero for neutral variation, the negative
value obtained of −2.72345 (***, p < 0.001) means
excess of mutations in external branches (Fu & Li,
1993), in this case, an excess of low frequency polymor-
phisms; for Fu and Li’s D test statistic, a negative value
was obtained, −6.46381 (**, p < 0.02), indicating an
excess of singletons. Altogether this concurrent rejec-
tion of neutral “null” hypothesis with negative values on
these tests allows the proposal of a population growth,
defined (Waxman, 2012) as the overall rate of loss of
resident polymorphisms having a negative contribution
from population size change and a positive contribution
from random genetic drift, as it may be resulting from
the recent hybridization, expansion and diversification
evidenced in this study.
Discussion
In 2006 it was estimated that Colombia had 2,100 bee-
keepers and 40,000 hives. Today it is calculated that the
number of hives is 80.000, with an annual production of
honey of 2000 tons per year and 150 tons of pollen,
indicating that this agricultural activity is becoming more
important in the country (Sa´nchez et al., 2013). Despite
this remarkable growth, there is no information system
to determine key aspects of bee identification and
health. Africanized bee hybrids have several traits that
are expected to have contributed to the ongoing
growth of Colombian beekeeping. These may include
lower infestation levels of Varroa mites and low preva-
lence of pathogenic virus (Tibata´, Junca, Corona, Ariza-
Botero, & Figueroa-Ramı´rez, 2017). However, the
higher defensiveness of Africanized hybrids have caused
some beekeepers to continue importing European
queens from other countries, or even from the insular
territory of Colombia (San Andres Islands), because this
region is the only one that retains its beekeeping with
European bees (Sa´nchez et al., 2013). Although the
introduction of these European lineages has been pri-
marily aimed to confer meekness, not many beekeepers
use them; the vast majority continues to work with
Africanized hybrids, including the capture of wild
swarms. This handling has made the genetic mix of api-
aries increasingly diverse, but there is no accurate
record of imports or previous characterizations of the
genetic diversity of these populations.
As mentioned above, the only study of mitotypes
characterization in Colombia was reported by Prada
et al. (2009), performing a restriction pattern
analysis (RFLP) of PCR amplicons obtained from the 16S
Determination of the Africanized mitotypes in populations of honey bees 5
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7. ribosomal RNA, COI and COII of mitochondrial DNA
regions; that study revealed 87% of the African haplo-
type from 5 regions, represented in 6 mitotypes: A1,
A4, A26, A28, A29 and A30. These results, compared
to the present study based on sequencing, allowed us to
improve the resolution of such valuable initial observa-
tion, as we detected sequences closely related to 17
additional mitotypes present in Colombia. The smaller
Figure 2. Overview of phylogenetic relationships inferred from COI-COII intergenic mt sequences retrieved from A. mellifera spec-
imens collected in Colombia and reference mitotype sequences representative of lineages A, C, M, and O. Sequences obtained from
Colombian continental samples are labeled in green. Sequences from insular Colombian Caribbean samples (San Andre´s Islands) are
labeled with light blue. Reference mitotypes sequences are labeled in red. Sequences obtained in this study from control samples
from A. mellifera specimens from USA are labeled with dark blue and controls from Israel are labeled with purple. Tree was inferred
using the Maximum Parsimony method. The most parsimonious tree with length = 7314 is shown. The consistency index is
0.285890 (0.266330), the retention index is 0.504741 (0.504741), and the composite index is 0.144300 (0.134427) for all sites and
parsimony-informative using the Subtree-Pruning-Regrafting (SPR) algorithm. Analyses and visualization were performed in software
MEGA7 (Kumar, Stecher, & Tamura, 2016). Phylogenetic trees with different methods (MP, ML, ME, NJ, UPGMA) provided analo-
gous clustering of the majority of sequences obtained from specimens in Colombia in A group in branches with closer node dis-
tances to where A1, A26d, A1e, an A4 reference mitotype sequences are located. Files of complete alignments sequences datasets
and trees are included as Supplementary Online Material (SP1-SP9).
6 V.M. Tibata´ et al.
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8. number of mitotypes detected in the 2009 study, is very
likely due to the RFLP technique employed; however it
must also be taken into account that 8 years have
passed. That study provided a valuable insight about the
very high level of africanization in the country, which is
indeed maintained and expanded. In another work
determining bee mitotypes frequency in the United
States, 12 mitotypes were detected (Szalanski & Magnus,
2010). As this country has been the last to be colonized
in the Americas by the Africanized hybrids (about 1990)
(Szalanski & Magnus, 2010), it would be reasonable to
expect a higher variety of mitotypes in Colombia than
in the U.S.A. These authors suggested that the findings
reported for Colombia may be due to the limited reso-
lution of the PCR-RFLP technique used in the study by
Prada et al. (2009), as well as others performed in Mex-
ico by Kraus et al. (2007) and in Brazil and Uruguay by
Collet et al. (2006), where a relatively low number of
mitotypes were found as well. Additionally in the study
in the United States, of the 12 mitotypes, 5 were not
found in any of the South American countries. Szalanski
and Magnus (2010) argue that it is possibly due to the
use of PCR-RFLP, in particular to the use of the enzyme
DraI, which would not allow the differentiation of mito-
types of A1 and A 29, into sub-types.
In this study we are reporting 98.3% of samples with
Africanized mitotypes that can be assigned to 17 differ-
ent haplotypes: A1b, A1e, A11, A26a, A26b, A26c,
A26d, A29a, A36, A39, A44, A46 and A47. The only
mitotype that was not found in the present work was
the A28. It is important to note that the previous study
was also carried out in 6 regions (departamentos), 5 of
which coincided with 5 of the present work; the differ-
ent region included in the previous study is Valle del
Cauca, while in our case we included Antioquia. One
important difference is that we analyzed 645 samples
versus 391 in the previous study. Regarding the pres-
ence of European haplotypes in the first study, 12.5% of
these samples corresponded to the ligustica subspecies,
whereas here, only 1.7% was classified in this type, rep-
resented in 11 samples. The description of these two
mitotypes, corresponded exactly with what was stated
by the beekeepers who owned these hives, because
they used European queens; these apiaries were located
in Cundinamarca and Huila. Interestingly, this ligustica
mitotype coincided with the origin of this genetic mate-
rial, since they were brought from San Andre´s Islas,
from where also two European bee samples were
obtained for this study, and they also evidenced the
same mitotype.
Regarding the mitotypes found in Colombia, com-
pared with results from United States, we detected 5
more (17 vs. 12), of which only 2 (A1a and A1d) were
present in North America and were not detected in
Colombia. A1d was the most prevalent in the United
States accounting for 53.4% of the 172 samples analyzed
(Szalanski & Magnus, 2010), probably a variant with
higher expansion and/or more adapted to temperate/
seasonal conditions. Ten mitotypes were found in the
two countries: A1, A1e, A4, A26, A26a, A26b, A26c,
A26d, A29a and A30. The 7 mitotypes detected in
Colombia that were not found in the USA were A1b,
A11, A36, A39, A44, A46 and A47. This higher number
of Africanized mitotypes obtained in Colombia com-
pared to the USA, agrees with the idea that there had
been a greater number of colonizations of African
hybrids derived from Apis mellifera scutellata; because
Colombia is closer to Brazil, origin of the Africanized
bees after its accidental escape. Perhaps among the fac-
tors that have allowed the expansion of Africanized
hybrids in Colombia, have been the mild and constant
climatic conditions, compare to more temperate sea-
sonal latitudes, the flora variety and continuous produc-
tion across the year, offering a range of resources and
habitats for their development. Additionally, the fact
that the majority of beekeepers reproduce their hives
with Africanized hybrids, and many of them trap wildlife
swarms, may increase the persistence of more haplo-
types and their wider spread and survival in Colombia.
Another possible determining factor in the expansion of
Africanized hybrids, was the introduction of Varroa
destructor in the Colombia in 1980. It is known that
European lineages are more susceptible to this para-
sitism, contributing to its population decline in the con-
tinent (Hamiduzzaman et al., 2015; Rosenkranz,
Aumeier, & Ziegelmann, 2010). According to Pinto,
Rubink, Coulson, Patton, and Johnston (2004), Pinto,
Rubink, Patton, Coulson, & Johnston (2005), the
replacement of European bee populations in Texas by
Africanized hybrids, coincided with the arrival of Varroa
to that state and with large losses of European beehives.
Varroa is present throughout the Colombian territory,
except in San Andres Islands, where the bee populations
are from European lineages. Our findings suggest that
the presence of Varroa possibly contributed in the selec-
tion of the Africanized populations and the decline of
the European ones.
Additionally to climatic and environmental factors,
the replacement of European populations by Africanized
hybrids in Colombia could be due, as experienced in
other countries, to their higher fitness and adaptability
favoring their expansion. In this regard, Kono and Kohn
(2015) demonstrated that 70% of feral hives in San
Diego California (USA) had the African mitotype, mean-
while only 13% of managed colonies presented this mar-
ker; additionally there was no observed correlation
between morphology and mitotype, suggesting that
Africanized bees are a product of a bidirectional
hybridization. Rangel et al. (2016), studied populations
of feral hives that had been previously examined for
Africanization processes 23 years before, revealing that
hybrid colonies had high African mitotype content and
low levels of European ancestry. Similar results were
found by Nelson, Wallberg, Simo˜es, Lawson, and Web-
ster (2017) by genome comparisons of European and
African bees with Africanized hybrids from Brazil,
Determination of the Africanized mitotypes in populations of honey bees 7
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9. determining that they retain 84% of the African ancestry
and 16% of European genetic; an admixture that seems
to have adaptive advantages.
Statistical estimates from the polymorphisms and the
groups of haplotypes inferred, indicated an active popu-
lation growth in Colombia; considering the distances
found for several sequences, obtained with the closest
defined mitotype reported. This finding is an interesting
path to follow i.e., to pinpoint if such recent diver-
gences, possibly derived from the abundant mitotypes,
could be correlated to minute morphometric differences
in individual specimens.
The large number of samples analyzed and the
sequencing technique used allowed a reliable and up to
date assessment of beekeeping hybridization status in
Colombia. Despite the continued introduction of Euro-
pean queens to Africanized apiaries, our results showed
that the takeover by Africanized hybrids is a highly
selective and a dominant phenomenon in the continental
beekeeping of Colombia. These findings can serve as a
valuable resource to studies aiming to understand and
to evaluate the relationships of Africanization with bee
ecology, expansion, and differential resistance to para-
sites such as Varroa, and the prevalence of viruses and
other pathogens.
Supplementary material
Supplementary material is available for this article at:
https://doi.org/10.1080/00218839.2017.1409065.
Acknowledgements
The authors wish to express their sincere thanks to Andre´s
Sa´nchez, Carlos Baez, Umberto Moreno and Rogelio Rodrı´guez
for assistance in the collection and processing of the samples.
We also thank the associations of beekeepers for providing the
samples. We are grateful with Dr. Jay Evans of Bee Research
Lab USDA (Beltsville MD-USA) for scientific advices.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The Bogota´ Research Division (DIB) of National University of
Colombia funded this work.
ORCID
Vı´ctor Manuel Tibata´ http://orcid.org/0000-0002-3737-5418
Howard Junca http://orcid.org/0000-0003-4546-6229
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