Journal of Apicultural Research

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Markers for the spatial and temporal

differentiation of bee pollen harvested by Apis
mellifera L. in the Eastern Andes of Colombia

Luis Carlos Casas Restrepo, Ilver Eduardo Gutierrez Alabat, Guillermo

Salamanca Grosso & Francisco de Assis Ribeiro dos Santos

To cite this article: Luis Carlos Casas Restrepo, Ilver Eduardo Gutierrez Alabat, Guillermo
Salamanca Grosso & Francisco de Assis Ribeiro dos Santos (2021): Markers for the spatial
and temporal differentiation of bee pollen harvested by Apis�mellifera L. in the Eastern Andes of
Colombia, Journal of Apicultural Research, DOI: 10.1080/00218839.2021.1916188

To link to this article: https://doi.org/10.1080/00218839.2021.1916188

Published online: 18 May 2021.

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Journal of Apicultural Research, 2021
https://doi.org/10.1080/00218839.2021.1916188

ORIGINAL RESEARCH ARTICLE

Markers for the spatial and temporal differentiation of bee pollen harvested by Apis
mellifera L. in the Eastern Andes of Colombia
Luis Carlos Casas Restrepoa
, Ilver Eduardo Gutierrez Alabatb, Guillermo Salamanca Grossoc and Francisco de
Assis Ribeiro dos Santosa,b
a
Programa de P
os-graduaç~ao em Bot^anica, Universidade Estadual de Feira de Santana, Feira de Santana, Bahia, Brazil; bPrograma de
P
os-graduaç~ao em Ecologia e Evoluç~ao, Universidade Estadual de Feira de Santana, Feira de Santana, Bahia, Brazil; cDepartamento de
Qu
ımica, Laboratorio de Propiedades Fisicoqu
ımicas de Alimentos, Universidad del Tolima, Ibagu
e, Tolima, Colombia
(Received 24 February 2020; accepted 12 December 2020)

The plant diversity present in Andean ecosystems is fundamental for the preservation of pollinator species, particularly
for bees, due to their direct dependence on the supply of pollen and nectar. Palynology is an important tool in studying
the interactions between bees and the flora of a region. This work aimed to determine the botanical composition of
bee pollen harvested by Apis mellifera L. in the Altiplano Cundiboyacense over an altitudinal gradient to establish pollen
markers that allow the geographic differentiation of the origin of bee products from the Eastern Andes of Colombia.
Bee pollen samples were gathered in 2015 during both dry and rainy periods. The samples were acetolyzed and ana-
lyzed using a minimum count of 500 pollen grains per sample. Diversity and evenness were analyzed using the Shannon-
Wiener and Pielou indexes respectively. Eighty-nine pollen types belonging to 43 botanical families were identified. The
Asteraceae family had the greatest pollen richness (23 types), followed by Leguminosae (eight types). Statistical signifi-
cant difference between climatic periods was revealed. The rainy period had higher richness and more exclusive pollen
types than the dry period, while lower altitudinal zones had greater diversity and evenness than higher zones. The
results demonstrate the importance of both native and introduced species as nutritional sources in the diet of honey
bees and allow the establishment of pollen types that act as markers useful for recognizing the origin of apicultural
products produced in the Eastern Andes of Colombia.
Keywords: Apis mellifera; bee pollen; botanical origin; Eastern Andes; melissopalynology

Introduction a consequence of changes in environmental and climatic

Bee pollen is an agglomeration of pollen grains collected
by honey bees, which is subsequently transported and
stored in the hive where it is mixed with nectar and sal-
ivary substances (Vit, 2009). Considering its protein,
lipid, vitamin, and mineral content, bee pollen plays a
key role in the nutrition and development of the bee
hive as well as in the honey bee longevity (Corby-Harris
et al., 2019; Keller et al., 2005; Wright et al., 2018).
Besides, the presence of bioactive compounds, such as
flavonoids, carotenoids, steroids, and terpenes, has
increased the demand for bee pollen in the international
market due to its benefits to human health and nutri-
tion (Ares et al., 2018; Sattler et al., 2015).
In the case of Apis mellifera L., its nutritional and
energy requirements lead it to visit and collect pollen
and nectar from a large number of flowers of several
botanical species. For that reason, the resulting bee pol-
len is thus of diverse botanical composition that is influ-
enced by the feeding preferences of the honey bees,
geographical conditions, and the diversity and blooming
period of the plant species that comprise the landscape
of the region (Bradbear, 2009). However, the availability
of floral resources changes, temporally and spatially, as

Corresponding author. Email: lccasasr@gmail.com
conditions, with precipitation being the most influential
factor of flowering processes in tropical regions
(Morellato et al., 2016).
The combination of different geographical and climatic
characteristics of the Altiplano Cundiboyacense such as
variation in the floral supply, heterogeneous vegetation
and landscapes, altitudinal gradient, and different rainfall
levels every three months has provided optimal condi-
tions for the implementation of a beekeeping chain as an
alternative and sustainable economic activity, as well as
its development in the recent decades. Furthermore,
Andean and high-Andean forests belonging to Altiplano
Cundiboyacense have the ideal environmental conditions
that allow high annual yields (36 bee pollen kg/hive)
when compared to other South American countries that
have a more developed beekeeping chain such as
Argentina, Brazil, Chile, and Peru (S
anchez et al., 2013).
Nevertheless, despite the importance and potential of
beekeeping in Colombia, it is still considered a secondary
economic activity as its local practice lacks technical
norms, regulations, and research (Mart
ınez, 2006;
S
anchez et al., 2013).

ß 2021 International Bee Research Association

2 L. C. Casas Restrepo et al.

Table 1. Summary of the location of honey bee-collected pollen sampling.

Groups Apiaries (samples) Locations Altitude (m) Geographic coordinates
Altitudinal zone I Punta larga Nobsa 2528 5 460 30.400 N–72 580 03.900 W
Salitre I Santa Rosa 2546 5 490 17.200 N–72 590 26.400 W
Salitre II Santa Rosa 2560 5 490 56.100 N–72 590 07.300 W
Estaci
on Ap
ıcola Paipa 2564 5 450 38.500 N–73 040 40.900 W
Altamira Firavitoba 2569 5 410 12.300 N–73 010 13.100 W
Altitudinal zone II El para
ıso Duitama 2600 5 460 12.100 N–73 040 38.800 W
San Lorenzo Duitama 2600 5 460 24.400 N–73 040 00.600 W
Ca~nos III Paipa 2650 5 450 50.800 N–73 050 04.100 W
Ca~nos II Paipa 2697 5 450 50.800 N–73 050 02.800 W
Altitudinal zone III Frontera Duitama 2715 5 510 22.300 N–73 010 09.900 W
La laguna Santa Rosa 2721 5 500 24.100 N–73 000 24.100 W
Tutuhaba Firavitoba 2729 5 410 12.300 N–73 010 13.100 W
Espartal Firavitoba 2821 5 390 41.200 N–73 020 44.600 W
Altitudinal zone IV Santa Rosita I Paipa 2937 5 470 41.200 N–73 090 09.500 W
El Carrizal AB2 Sotaquir
a 2998 5 490 04.700 N–73 100 24.200 W
Altitudinal zone V Santa Rosita II Paipa 3006 5 480 04.900 N–73 090 10.100 W
Jamaica III Paipa 3010 5 480 47.500 N–73 100 29.700 W
El Carrizal AB1 Sotaquir
a 3013 5 490 11.400 N–73 100 31.700 W
Jamaica I Paipa 3066 5 480 52.300 N–73 090 31.000 W
El Carrizal I Sotaquir
a 3075 5 480 57.100 N–73 100 37.300 W
Jamaica II Paipa 3087 5 480 42.200 N–73 090 43.300 W

Identifying and understanding honey bee foraging pat-
terns at a spatial and temporal scale improves our
knowledge of how management and conservation prac-
tices can be implemented in areas where native vegeta-
tion is predominant (Hower, 1953; Lau et al., 2019).
Likewise, this information allows a better understanding
of ecosystem services like crop pollination, where
honey bees play a fundamental role in synergy with wild
bees (Garibaldi et al. 2013; Montoya-Pfeiffer et al.,
2016). Nonetheless, studies focused on expanding
knowledge of the botanical origin of honey bee prod-
ucts are scarce in Colombia, and available information is
still insufficient for several areas of the country. In add-
ition, there are only a few works that have studied
honey bee pollen produced in Andean forests, with
honey being the main studied beekeeping product
(Chamorro et al., 2013, 2017; Montoya-Pfeiffer, 2011;
Nates-Parra et al., 2013; Ortiz de Boada et al., 1987;
Ortiz de Boada & Nates-Parra, 1987; Salamanca et al.,
2011, 2014).
The objective of the present work was to determine
the botanical composition of bee pollen harvested by
Apis mellifera L. in the Altiplano Cundiboyacense by sam-
pling over an altitudinal gradient and to establish pollen
markers to spatially and temporally differentiate the ori-
gin of bee pollen in the Eastern Andes of Colombia.
Materials and methods
Study area
The northern region of the Andes is one of the main
biodiversity hotspots in the world. The Altiplano
Cundiboyacense is located in the Eastern Andes of cen-
tral Colombia in the departments of Cundinamarca and
Boyac
a with a mean altitude between 2000 and 3000
masl. The region has a mean annual temperature of
14  C, with a bimodal precipitation regime characterized by
dry and rainy periods every three months and annual pre-
cipitation ranging from 580 to 1000 mm (Baptiste, 2004).
The region is characterized by Andean and high-
Andean ecosystems with a highly diverse plant compos-
ition and a high degree of variation in the dominant flora
as a function of the altitudinal gradient (Brehm et al.,
2008; Myers et al., 2000). Thus, under natural conditions,
it is possible to recognize three main sections: 1) Andean
forests: Located between 2500–2800 meters above sea
level (masl) with heterogeneous vegetation and three
layers compound by several species of trees, shrubs, epi-
phytes, and herbs. 2) High-Andean forest: It is located
between 2800–3300 masl with only one vegetal stratum
compound by low trees which does not exceed 10
meters high, as well as big herbs and a high diversity of
mosses and lichens.3) Sub-P
aramo: It is a transitional zone
between high-Andean forest and P
aramo located between
3300–3600 masl, although in some occasions its vegetation
is able to expand under these limits and colonize high-
Andean forests areas that have been destroyed by
anthropic activity. This zone is characterized by the pres-
ence of semi-open vegetal formations and great variability
in its floristic composition and coverage. For this reason,
it shows a high heterogeneity, richness, and diversity of
vegetal species (Le
on et al., 2015).
Nevertheless, the anthropic intervention has mark-
edly reduced native forest areas, replacing them pro-
gressively with new areas with a predominance of
species of agricultural or logging interest (Baptiste,
2004). The region has areas where agriculture is prac-
ticed and diverse timber species are cultivated such as
Eucalyptus globulus Labill. (Myrtaceae) and Pinus sp.
(Pinaceae). In general, grasslands represent about
66.13% of the total area, where it is possible to find
areas that are dedicated to agriculture and ranching,

Figure 1. Study area. (A) Location of Colombia in South America; (B) Location of Boyac
a; (C) Highlighted study area in Boyac
a. (D)
Distribution of apiaries on altitudinal zones.
while timber species represent 7.69% of the total land-
scape. Besides, urban and suburban areas represent
about 6.18% (Baptiste, 2004).
On the other hand, native forested areas have been
diminished in the last decades and currently represent
20% of their original cover. It is important to distinguish
between bushy areas and remains of native forest, the
former are succession forests which cover about
16.37% of the landscape, while the last represents only
3.63% of the total area. In a native forest, arboreal and
shrubby species, such as Quercus humboldtii Bonpl.
(Fagaceae), Weinmannia tomentosa L.f. (Cunoniaceae),
Clethra fimbriata Kunth. (Clethraceae), Dodonaea viscosa
(L.) Jacq. (Sapindaceae), and Morella parvifolia (Benth.)
Parra-Os. (Myricaceae) are important to vegetation
structure. However, the loss of extensive areas of nat-
ural forest due to human activity has been increasing
progressively, generating changes in the landscape and
vegetal structure (Baptiste, 2004; Van der Hammen,
1998; Young, 2006).
Pollen sampling
Between December 2014 and May 2015, with a dry
period from December to February, and a rainy period
from March to May, 84 samples of bee pollen (20 g/sam-
ple) were gathered from 21 apiaries (two samples/
Melissopalynology in the Eastern Andes of Colombia 3
apiary/season) located in six municipalities of the
department of Boyac
a. Samples were collected directly
from pollen traps placed at the hive entrances and from
the same hive in each season. Considering the hetero-
geneous vegetation of the Altiplano Cundiboyacense
(Baptiste, 2004; Van der Hammen, 1998), previously
described in the study area section, and for a better
understanding of differences in bee pollen composition
throughout the altitudinal gradient, the apiaries were
selected according to their availability in the study area
and their altitudinal location to encompass five altitud-
inal zones: Zone I ¼ 2500–2600 masl; Zone II ¼
2600–2700 masl; Zone III ¼ 2700–2850 masl; Zone IV
¼ 2850–3000 masl, and Zone V > 3000 masl (Table 1,
Figure 1).
Pollen analysis
All laboratory work was carried out in the Plant
Micromorphology Laboratory (LAMIV) of the Feira de
Santana State University (UEFS) in Feira de Santana,
Bahia State, Brazil. Samples were prepared according to
the protocol developed by Alvarado and Delgado
(1985) with adaptations proposed by Novais et al.
(2009): a) pollen loads dehydration in a drying oven at
40  C until they attained a stable weight; b) two grams
of pollen from each sample were re-hydrated in warm
4 L. C. Casas Restrepo et al.
distilled water (20 ml) for at least two hours while being
automatically stirred; c) a 1 ml sub-sample was taken
from each sample and centrifuged (2500 RCF, 10 min)
to concentrate the pollen grains, which were then dehy-
drated with glacial acetic acid (10 ml) for 60 min and
subsequently centrifuged;d) pollen sediment was aceto-
lyzed following Erdtman (1960); e) five permanent slides
were mounted for each sample and 500 pollen grains
were counted per sample to establish the relative abun-
dance [RA ¼ (number of pollen grains of pollen type i/
total pollen grains) x 100] of each recognized pollen
type for each altitudinal zone (Bucher et al., 2004).To
understand honey bees foraging patterns in each hive
individually, pollen types were classified according to
their RA into predominant (>45%), secondary
(>15–45%), important–isolated (3–15%), and isolated
(<3%), following Zander (in Louveaux et al., 1978).
Our study is based on the use of morphological
characteristics of each pollen type to identify the botan-
ical origin of bee pollen samples to a specific taxon
(e.g., a species, a group of species, a genus, or a family)
(De Klerk & Joosten, 2007; Joosten & De Klerk, 2002).
Thus, to identify the pollen grains, we used the pollen
library at LAMIV and databases, as well as comparisons
with the following pollen catalogues: Moncada and Salas
(1983), Barth (1989), Mart
ınez-Hern
andez et al. (1993),
Roubik and Moreno (1991), Vel
asquez (1999), Bogot
a
(2002), Giraldo et al. (2011), and Mercado-G
omez et al.
(2011, 2013). Additionally, information related to the
growing habit (arboreal, Shrubby, subshrubby, herb-
aceous, or climbing) of the plant species related to each
pollen type was obtained from Gonz
alez and L
opez-
Camacho (2012) and Id
arraga et al. (2011).
Relative Frequency of Occurrence [RFO ¼ (the
number of samples with occurrence/total number of
samples) x 100] was estimated for each pollen type for
each climatic period in order to analyze bee pollen
composition and its variation through altitudinal zones.
Based on RFO values, pollen types were classified
according to the classes proposed by Jones and Bryant
Jr. (1996) as very frequent (>50%), frequent (21–50%),
infrequent (10–20%), and rare (<10%). Only plant spe-
cies associated with pollen types with an RFO value
greater than 10% were considered as significant to
honey bee foraging.
Statistical analysis
Statistical analysis was performed using R software (R
Core Team, 2017). To a better understanding of the
results, the Shannon-Wiener diversity index (H’) (1948)
and the evenness index of Pielou (J’) (1977) were used to
measure the level of diversity and evenness, respectively,
of floral resources used by Apis mellifera L. (Apidae) for
each altitudinal zone and climatic period. Statistical differ-
ence in the Shannon-Wiener diversity index value of each
climatic period and the altitudinal zone was estimated
using a t-test modified by Hutcheson (1970) with a
significance level of 0.05. Precipitation data was obtained
from Instituto de Hidrolog
ıa, Meteorolog
ıa y Estudios
Ambientales de Colombia (IDEAM).
Results
A total of 89 pollen types belonging to 43 botanical fami-
lies were identified. Some main pollen types are shown in
Figure 2. The Asteraceae family had the greatest pollen
richness with 23 pollen types, followed by Leguminosae
with eight; Myrtaceae, Apiaceae, Rubiaceae, Rosaceae, and
Solanaceae with three types each; and Adoxaceae,
Araliaceae, Brassicaceae, Ericaceae, Euphorbiaceae,
Melastomataceae, and Polygonaceae with two types each;
each of the remaining 29 families were represented by
only a single pollen type. Five pollen types were classified
as indeterminate since no botanical affinity could be deter-
mined (Table 2).
Fifty-eight pollen types were identified during the dry
period (14.7 mm/month–66.6 mm/month), among which
Hypochaeris radicata and Trifolium pratense were present
in 100% of the samples analyzed. The pollen types
Eucalyptus globulus, Brassicaceae-type, Trifolium repens,
Viburnum, Weinmannia, Rumex, Helianthus, Cardamine
bonariensis, and Myrcia had RFO values above 70%. Five
other pollen types were classified as very frequent,
while 11 were classified as frequent and infrequent and
18 as rare. Fourteen pollen types were exclusive to the
pollen spectrum of the dry period (Table 3).
Analysis over the altitudinal gradient during the dry
period revealed significant (RFO > 10%) pollen types
for each altitudinal zone. Hypochaeris radicata was infre-
quent in zone I; E. globulus and Brassicaceae-type were
infrequent in zone I and frequent in zone II; and T. pra-
tense was infrequent in zones II and III. On the other
hand, Weinmannia was infrequent in Zone I (17%) and
very frequent in zones IV and V with RFO values of
73.7% and 65.46%, respectively (Figure 3).
Eighty pollen types were identified during the rainy
period (14.8 mm/month–181.9 mm/month), with 18 being
classified as very frequent, 15 as frequent, 12 as infrequent,
and 35 as rare. Besides, 36 pollen types were unique in this
period. Despite having greater richness, few pollen types
stood out as significant (RFO >10%) for any of the altitud-
inal zones. Thus, it is possible to highlight the importance
of Brassicaceae-type in all of the zones, being classified as
frequent in zones I and II and as infrequent in zones III, IV,
and V. Additionally, E. globulus was infrequent in zones I
and III, and frequent in zone II. On the other hand, Clethra
and Rumex stood out in zones III and IV being classified as
infrequent and frequent, respectively, whereas Weinmannia
was infrequent in zone III and frequent in zone V
(Figure 3).
From the statistical point of view, several species were
significantly represented (RFO > 10%) throughout the pol-
len spectrum and each climatic period, among them some
introduced species are found: Sambucus nigra L.
(Adoxaceae), Brassica rapa L. (Brassicaceae), Raphanus
 Melissopalynology in the Eastern Andes of Colombia 5

Figure 2. Main pollen types identified in samples of bee pollen harvested by Apis mellifera in Altiplano Cundiboyacense. Colombia. (A)
Adoxaceae: Viburnum; (B) Brassicaceae type; (C–F) Asteraceae: (C) Helianthus; (D) Hypochaeris radicata; (E) Mikania; (F) Vernonia;
(G) Cunoniaceae: Weinmannia tomentosa; (H, I) Leguminosae; (H) Trifolium pratense; (I) Vicia; (J) Myricaceae: Morella parvifolia;
(K) Myrtaceae: Eucalyptus globulus; (L) Solanaceae: Brugmansia. Scale: 10 mm.
6 L. C. Casas Restrepo et al.

Table 2. Relative abundance (RA) (I–V), relative frequency of occurrence (RFO), and frequency classes (FC) of pollen types regis-
tered in honey bee-collected pollen samples collected during dry and rainy season and through an altitudinal gradient in the
Altiplano Cundiboyacense—Colombia.
Dry season Rainy season
Pollen types I II III IV V RFO FC I II III IV V RFO FC
Adoxaceae
Sambucus nigra 2.66 0.07 0.97 0.35 0.1 38.10 F 1.82 0.02 0.82 0.7 33.33 F
Viburnum 0.34 1.57 6.32 0.55 0.35 76.19 VF 2.2 3.2 0.47 1.8 7.63 90.48 VF
Amaranthaceae
Amaranthus 0.02 4.76 R
Anacardiaceae
Anacardiaceae type 0.7 0.1 2.65 23.81 F
Apiaceae
Coriandrum sativum 0.18 0.45 0.08 28.57 F 0.56 0.15 2.1 33.33 F
Eryngium 0.22 4.76 R
Neonelsonia 0.24 1.72 33.33 F
Aquifoliaceae
Ilex kunthiana 0.01 4.76 R 0.76 0.42 0.45 0.01 57.14 VF
Araliaceae
Oreopanax 0.01 4.76 R
Schefflera 0.01 4.76 R
Arecaeae
Arecaceae type 0.02 4.76 R 0.24 0.32 19.05 LF
Asteraceae
Ambrosia arborescens 0.08 0.32 14.29 LF
Aster 0.24 0.07 0.21 14.29 LF 0.02 0.15 4.8 6.18 47.62 F
Austroepatorium inulaefolium 9.36 1.85 4.87 2.3 1.18 90.48 VF
Baccharis 0.1 0.01 14.29 LF 0.16 0.37 0.25 0.03 28.57 F
Barnadesia spinosa 0.07 4.76 R
Eirmocephala brachiata 0.07 4.76 R 0.08 0.05 9.52 R
Espeletia 0.16 0.05 14.29 LF
Eupatorium 1.28 0.15 0.3 0.06 23.81 F 0.22 0.2 1.27 0.35 2.46 61.90 VF
Gnaphalium 0.2 4.76 R
Gynoxys 0.36 0.15 0.1 38.10 F
Helianthus 1.48 0.32 2.32 1.1 0.98 71.43 VF 4.64 0.65 4.52 0.5 0.25 85.71 VF
Hypochaeris radicata 19.34 9.47 27 2.7 3.95 100.00 VF 2.92 6.42 7.12 6.75 11.4 100.00 VF
Lessingianthus yariguiensis 2.3 0.57 0.9 0.05 57.14 VF 0.28 0.15 0.12 0.05 0.08 57.14 VF
Mikania 1.84 0.95 0.95 0.28 52.38 VF 0.88 1.15 33.33 F
Pentacalia 0.02 4.76 R
Senecio formosus 0.11 4.76 R 0.05 0.01 9.52 R
Sonchus 0.14 0.25 14.29 F
Steiractinia 0.05 4.76 R
Taraxacum campylodes 0.6 0.4 28.57 F
Verbesina 0.1 0.22 9.52 R
Vernonia 1.34 0.05 0.02 23.81 F
Asteraceae type1 3.5 0.4 19.05 LF
Asteraceae type 2 0.32 0.35 0.06 14.29 LF
Betulaceae
Alnus acuminata 0.12 0.45 14.29 LF 0.05 4.76 R
Bignoniaceae
Bignoniaceae type 0.6 0.28 19.05 LF
Boraginaceae
Cordia 0.01 4.76 R
Brassicaceae
Cardamine bonariensis 2.1 2.82 1.27 0.8 0.48 71.43 VF 3.56 2.6 0.57 2.8 3.15 80.95 VF
Brassicaceae type 16.84 30.67 3.9 0.35 4.46 90.48 VF 27.56 23.16 12.65 10.65 18.81 100.00 VF
Bromeliaceae
Puya 0.02 0.05 0.075 23.81 F 0.46 0.12 0.3 33.33 F
Campanulaceae
Centropogom 0.36 14.29 LF
Caprifoliaceae
Lonicera 0.01 4.76 R
Clethraceae
Clethra 9.58 0.4 11.42 57.14 VF
Cloranthaceae
Hedyosmun 0.3 14.29 LF
(Continued)
 Melissopalynology in the Eastern Andes of Colombia 7

Table 2. (Continued).
Dry season Rainy season
Pollen types I II III IV V RFO FC I II III IV V RFO FC
Cucurbitaceae
Cucurbita pepo 0.02 4.76 R
Cunoniaceae
Weinmannia 17.52 1.45 2.62 73.7 65.46 76.19 VF 0.68 0.125 14.2 9.95 29.45 71.43 VF
Cyperaceae
Cyperaceae type 0.4 4.76 R
Ericaceae
Bejaria resinosa 0.02 4.76 R
Ericaceae 2 0.05 4.76 R
Eriocaulaceae
Paepalanthus 0.04 0.15 0.03 14.29 LF 2.21 19.05 LF
Euphorbiaceae
Croton 0.06 9.52 R
Euphorbia 0.075 4.76 R 0.05 9.52 R
Fagaceae
Quercus humboldtii 0.3 4.76 R
Hypericaceae
Hypericum 0.38 0.7 2.7 8.51 52.38 VF 1.8 0.27 0.37 0.28 52.38 VF
Lamiaceae
Salvia 0.04 0.05 9.52 R
Leguminosae
Acacia decurrens 0.1 0.52 0.1 0.06 28.57 F 0.1 0.02 0.06 23.81 F
Erythrina 0.12 0.05 14.29 LF 0.02 0.02 9.52 R
Senna 0.18 0.07 0.43 28.57 F 0.36 0.27 1.2 1.15 0.45 80.95 VF
Trifolium pratense 3.44 11.07 14.82 1.5 3.46 100.00 VF 3.66 3.22 5.45 1.9 0.48 90.48 VF
Trifolium repens 2.3 1.22 1.2 6.4 2.36 85.71 VF 3.1 3.8 0.85 2.7 0.96 100.00 VF
Ulex europaeus 2 0.32 1.57 0.25 0.2 42.86 F
Vicia 0.02 0.02 9.52 R
Leguminosae type 0.38 0.15 9.52 R 0.07 9.52 R
Loranthaceae
Gaiadendron punctatum 0.02 0.22 0.3 2.05 47.62 F 0.03 4.76 R
Melastomataceae
Miconia 0.38 0.05 0.47 28.57 F
Melastomataceae type 0.02 4.76 R 0.12 0.3 9.52 R
Myricaceae
Morella parvifolia 0.56 1.5 4.65 28.57 F 0.02 2.42 0.17 23.81 F
Myrtaceae
Eucalyptus globulus 16.28 23.67 8.12 7.45 2.88 95.24 VF 17.46 43.72 12.55 3.15 3.56 95.24 VF
Myrcia 1.4 4 3.7 0.35 0.93 71.43 VF 1.52 3.2 0.92 0.5 0.15 85.71 VF
Myrtaceae type 0.2 4.76 R 0.02 4.76 R
Oleaceae
Fraxinus chinensis 4.74 0.52 9 0.25 0.16 52.38 VF
Passifloraceae
Passiflora 0.02 0.12 14.29 LF 0.02 0.03 14.29 LF
Phyllantaceae
Phyllanthus salviaefolium 0.3 9.52 R
Plantaginaceae
Plantago 0.16 9.52 R
Poaceae
Poaceae type 0.1 0.12 28.57 F 0.02 0.07 0.03 14.29 LF
Polygonaceae
Polygonum segetum 0.06 0.02 14.29 LF 0.02 4.76 R
Rumex 1.32 6.4 5.47 0.85 1.25 76.19 VF 0.68 2.17 2.32 43.35 8.56 71.43 VF
Rosaceae
Hesperomeles 0.05 4 0.2 0.1 38.10 F
Prunus 0.75 4.76 R
Rubus 0.77 0.05 0.06 14.29 LF 0.22 0.45 3.3 0.31 33.33 F
Rubiaceae
Psycotria 0.02 4.76 R 0.02 0.12 0.05 14.29 LF
Spermacoce verticillata 0.05 4.76 R
Rubiaceae type 0.12 4.76 R
Sapindaceae
Dodonaea viscosa 0.3 0.4 1 0.25 0.51 52.38 VF
(Continued)
8 L. C. Casas Restrepo et al.

Table 2. (Continued).
Dry season Rainy season
Pollen types I II III IV V RFO FC I II III IV V RFO FC
Solanaceae
Brugmansia 0.18 9.52 R
Cestrum 0.05 4.76 R
Solanum 0.08 4.76 R 0.04 0.1 9.52 R
Violaceae
Viola 0.14 0.02 0.13 14.29 LF 0.12 9.52 R
Vitaceae
Vitis 0.02 0.05 9.52 R 0.1 4.76 R
Indeterminate types
Indeterminate 1 0.02 0.05 9.52 R 0.05 4.76 R
Indeterminate 2 0.02 4.76 R
Indeterminate 3 0.1 4.76 R
Indeterminate 4 0.15 14.29 LF
Indeterminate 5 0.18 9.52 R
Total pollen grains 5000 4000 4000 2000 6000 21,000 5000 4000 4000 2000 6000 21,000
Pollen types richness 43 33 35 19 32 58 46 38 46 31 40 80
Shanon–Wienner 2.44 2.16 2.52 1.15 1.50 2.42 2.55 1.93 2.85 2.17 2.27 2.78
Pielou 0.64 0.61 0.70 0.38 0.43 0.59 0.66 0.52 0.74 0.62 0.61 0.63
Frequency data in %.
I: 2500–2600 masl; II: 2600–2700 masl; III: 2700–2850 masl; IV: 2850–3000 masl.eV: >3000 masl.RA: Relative abundance; RFO: Relative frequency
of occurrence; FC: Frequency class; VF: Very frequent; F: Frequent; LF: Low frequent; R: Rare.

Figure 3. Relative frequency of occurrence (%) of the main pollen types (RFO > 10%) recorded in honey bee-collected pollen from
Altiplano Cundiboyacense—Colombia during a dry and rainy season. Pollen types in bold were exclusive of a climatic season.

raphanistrum L. (Brassicaceae), Coriandrum sativum L.
(Apiaceae), Eucalyptus globulus Labill.(Myrtaceae), Hypochaeris
radicata L. (Asteraceae), Taraxacum campylodes G.E.
Haglund. (Asteraceae), Fraxinus chinensis Roxb. (Oleaceae),
Rumex acetosella L. (Polygonaceae), Trifolium pratense L.
(Leguminosae), and T. repens L. (Leguminosae).
Additionally, it is important to highlight other pollen
types recorded through the altitudinal zones I, II, and III
such as Anacardiaceae-type, Ambrosia arborescens,
Coriandrum sativum, Sonchus sp., Taraxacum campylodes,
Puya sp., Passiflora sp., Poaceae-type, Brugmansia sp., and
Vitis sp. for the dry season and Amaranthus sp.,
Arecaceae-type, Eirmocephala brachiata, Salvia sp., and
Clethra sp. for the rainy season. Some exclusive pollen
types are presented in Table 3.
Endemic species of Colombia, such as Baccharis boya-
censis Cuatrec (Asteraceae) and B. bogotensis Kunth.
(Asteraceae), as well as species distributed exclusively

Table 3. Exclusive pollen types registered in honey bee-collected pollen samples from different altitudinal zones
of Altiplano Cundiboyacense, Colombia.
Altitudinal zone Dry season
I Croton, Brugmansia
II – Barnadesia spinosa, Spermacoce verticillata
III Cucurbita pepo
IV Quercus humboldtii
V Cyperaceae type, Oreopanax
Figure 4. Richness and diversity of pollen types through an alti-
tudinal gradient and a dry and rainy season in honey bee-col-
lected pollen samples from Altiplano Cundiboyacense—
Colombia. H
¼ Shannon-Wienner Index.
in the Eastern Andes, such as Weinmannia tomentosa L.f.
(Cunoniaceae) and Myrcianthes leucoxyla (Ortega)
McVaugh. (Myrtaceae), were represented in the pollen
samples analyzed. Other native species distributed in
Colombia were represented in the pollen spectrum,
particularly Ambrosia arborescens Mill. (Asteraceae),
Austroepatorium inulaefolium (Kunth) R.M. King. and H.
Rob.(Asteraceae), Baccharis inamoena Gardner.
(Asteraceae), B. macranta Kunth. (Asteraceae), W. pin-
nata L. (Cunoniaceae), Viburnum triphyllum Benth.
(Adoxaceae), V. tinoides L.f. (Adoxaceae), Ilex kunthiana
Triana. (Aquifoliaceae), Oreopanax incisus (Willd. ex
Schult.) Decne. and Planch. (Araliaceae), Alnus acuminata
Kunth. (Betulaceae), Gaiadendron punctatum (Ruiz and
Pav.) G. Don. (Loranthaceae), Morella parvifolia (Benth.)
Parra-Os. (Myricaceae), Polygonum segetum Kunth.
(Polygonaceae), Rubus floribundus Kunth. (Rosaceae), and
Spermacoce verticillata L. (Rubiaceae).
In general, the use of the floral resources by the
honey bees was heterogeneous since various pollen
types related to plant species from several types of
vegetation were registered. Additionally, although a
greater number of pollen types of native species were
recorded, the greatest amount of pollen in most
Melissopalynology in the Eastern Andes of Colombia 9
Exclusive pollen types
Rainy season
Amaranthus, Pentacalia, Bejaria resinosa,
Rubiaceae 1
Eryngium, Gnaphalium
Prunus, Cestrum
Schefflera, Steiractinia, Cordia, Centropogon,
Hedyosmun, Ericaceae 2, Phyllanthus
salviaefolium, Plantago
samples was from introduced plant species, dominating
the pollen spectrum (Table 2). Regarding growth habit
of the plants related to the pollen types registered, the
diet of A. mellifera is sustained mainly by herbs (28), fol-
lowed by shrubs (21) and trees (21), while lianas (5)
and subshrubs (3) were the least represented in the
pollen spectrum.
The Hutcheson t-test revealed significant differences
between diversity index values in all of the altitudinal
zones when compared the dry and rainy period(-
p < 0.001 in all cases; zone I:t ¼ 4195 df ¼ 9955; zone
II: t ¼ 7825, df ¼ 7883; zone III: t ¼ 13,321 df ¼ 7992;
zone IV: t ¼ 22,315 df ¼ 3974; zone V: t ¼ 29,807 df ¼
10,973). In addition, it is important to point out that, in
general, the samples collected in the lower altitudinal
zones (I, II, and III) were more diverse and had greater
evenness than the higher zones (IV and V), with altitud-
inal zone III being the one with the highest degree of
diversity and evenness both in the dry and rainy periods
(H’¼ 2.52 and 2.85; J’¼ 0.70 and 0.74, respectively)
(Table 2, Figure 4).
Discussion
The structural heterogeneity of the vegetation of the
Altiplano Cundiboyacense landscape associated with the
apiaries has a direct effect on differences in the com-
position of the pollen loads of honey bees. Although
the various species associated with native forest were
visited considerably by honey bees, the pollen types
with the greatest RFO were of taxa that were highly
available due to their proximity to hives and associated
with areas modified by human activity such as pastures,
agricultural areas, and areas of timber interest, which
represent about 64% of the total area of the Altiplano
Cundiboyacense (Baptiste, 2004). It has been reported
that bee pollen diversity increases in complex land-
scapes such as those with different land use areas
where honey bees are able to collect pollen from sev-
eral species distributed in native forest areas and mainly
in anthropic ecosystems (Montoya-Pfeiffer & Nates-
Parra, 2020; Ogilvie & Forrest, 2017).
The diversity of pollen types observed in the ana-
lyzed samples evidences the generalist habit of A. melli-
fera, which has been widely reported for most eusocial
10 L. C. Casas Restrepo et al.
bees (Eickwort & Ginsberg, 1980) and has been previ-
ously documented by several authors in the Andean
ecosystems of Colombia (Chamorro et al., 2013, 2017;
Montoya-Pfeiffer, 2011; Nates-Parra et al., 2013; Ortiz
de Boada et al., 1987; Ortiz de Boada & Nates-Parra,
1987; Salamanca et al., 2011, 2014), Peru (Saavedra
et al., 2013; Sayas Rivera & Huam
an, 2009), Chile
(Montenegro et al., 2013; Ram
ırez & Montenegro,
2004), Argentina (Andrada & Teller
ıa, 2005; Faye et al.,
2002), and Venezuela (Barth et al., 2011). According to
Schmidt (1984), honey bees have a preference for a diet
based on pollen material composed of pollen grains of
various species. By combining pollen from several sour-
ces, some of which may have adverse textural (dry or
gummy), nutritional (low protein level), and chemical
properties (feeding deterrents or toxins), the effects of
any one particular pollen species are reduced.
Furthermore, it has been reported that a polyfloral
diets increase worker immunocompetence and colony
tolerances to pathogens (Alaux et al., 2010).
The importance of the Asteraceae family in honey
bee diet is evidenced by the considerable number of
pollen types identified in the samples. Similar results
have been reported by several studies carried out in
Andean areas of Colombia (Chamorro et al., 2013,
2017; Montoya-Pfeiffer, 2011; Vivas et al., 2008). Floral
characteristics, such as a supply of nectar, an abundant
amount of pollen per floral unit, and easy access and
the manipulation of the anthers, directly influence the
selection of these species by honey bees (Chamorro
et al., 2017; Percival 1955; Pernal & Currie, 2002). In
addition, the extensive distribution of Asteraceae spe-
cies in the Altiplano Cundiboyacense contributes
remarkably to the abundance and richness of pollen
types (Gonz
alez & L
opez-Camacho, 2012; Sarmiento &
Cano, 1997). The family Asteraceae has been reported
as the dominant group in several Andean and dry eco-
systems in Colombia (Gonz
alez & L
opez-Camacho,
2012; Sarmiento & Cano, 1997), Venezuela (Hern
andez
et al., 2003), Peru (Arakaki & Cano, 2003), and Chile
(Rodr
ıguez et al., 2008).
The pollen spectrum analysis revealed the import-
ance of introduced species on the diet of A. mellifera, as
well as considerable differences in the flora visited along
the sampled altitudinal gradient. The botanical compos-
ition of bee pollen from altitudinal zones I, II, and III
was diverse during the dry season and rainy season as it
was evidenced by diversity index value (see Figure 4),
possibly by the presence of broad floral resources as a
consequence of a variable landscape and several types
of vegetation (Baptiste, 2004). In these zones, the pollen
spectrum was marked by the dominance of Hypochaeris
radicata, Trifolium pratense, Trifolium repens, Eucalyptus
globulus, and Brassicaceae-type (Brassica sp. and
Raphanus sp.) during both climatic periods, constituting
the main source of nutrition for long periods. These
results support previous studies carried out in
Colombian Andes (Chamorro et al., 2013, 2017), as
well as other researches that have reported the import-
ance of genera Brassica and Trifolium for honey bees
(B€ansch et al., 2020; Lau et al., 2019; Wintermantel
et al., 2019). Most of these pollen types are associated
with introduced species that are widely distributed in
areas modified by anthropic activities, such as pastures
and agricultural areas (Baptiste, 2004), and which supply
large amounts of pollen per floral unit or have flowering
plants with numerous stamens (Montoya-Pfeiffer, 2011).
Additionally, the species Eucalyptus globulus has been
previously noted for its importance in beekeeping in
several regions, and in the study area is widely distrib-
uted as it is considered a timber species, which
increases notably its importance for honey bees diet
(Basilio, 2000; Fag
undez & Caccavari, 2006; Nates-Parra
et al., 2013).
On the other hand, native flora was mostly repre-
sented by pollen types belonging to species of Viburnum,
Myrcia, and Hypericum and, to a lesser extent, those of
Morella, Ilex, Alnus, and Clethra. Several authors have
reported the importance of these genera, whose spe-
cies are prominent floristic elements of the native for-
ests of the region (Galindo et al., 2003; Gonz
alez &
L
opez-Camacho, 2012; Moncada, 2010), as well as their
beekeeping potential (Chamorro et al., 2013, 2017;
Montoya-Pfeiffer, 2011; Nates-Parra et al., 2013;
Salamanca et al. 2011, 2014). The interaction between
the honey bee and the native flora has a positive effect
on the reproduction of Andean species and contributes
significantly to the processes of succession and conser-
vation of the Andean and high-Andean forest.
Furthermore, the knowledge about the ecological
impact of honey bees on the reproduction of the native
flora allows beekeeping to be recognized as a potential
activity to be integrated into the management plans of
Andean ecosystems(Chamorro et al., 2013).
The pollen spectrum of the altitudinal zones IV and
V was represented mainly by Weinmannia pollen type
during the dry and rainy seasons. Its presence and dom-
inance, possibly associated with the species W. tomen-
tosa L.f. and W. pinnata L., evidenced the importance of
this floral source to honey bees, which is characterized
by an abundant supply of nectar and a considerable
number of flowers per inflorescences. Thus, the high
Weinmannia RA and RFO values can possibly be attrib-
uted to the honey bee foraging pattern, as well as it
was mentioned previously, honey bees usually focus
their effort in making use of those species that give
them the best rewards (Nicholls & Hempel de Ibarra,
2017). Salamanca et al. (2011, 2014) reported
Weinmannia as a genus with high potential for the pro-
duction of beekeeping products, such as honey and pol-
len of a monofloral character, given to the dominance
of this genus in the pollen spectrum when the floral
supply exists.

The Hutcheson t-test revealed a statistically signifi-
cant difference in diversity index value between the dry
and rainy periods in each altitudinal zone. This result
can be attributed to the variation in flowering phen-
ology of the species that are the source of pollen for
honey bees which is mainly influenced by variations in
precipitation level in tropical ecosystems (Morellato
et al., 2013). Our results agree with those reported by
Montoya-Pfeiffer and Nates-Parra (2020) which suggest
that, although the pollen amount collected by honey
bees is not influenced by climatic variation, its pollen
composition varies. Nonetheless, as it is normally
expected, richness was not negatively influenced by pre-
cipitation, which is probably in consequence of the het-
erogeneous landscape and diverse vegetation that
provide a more diverse source of pollen for bees
(Escobedo-Kenefic et al., 2020).
Furthermore, it was possible to identify changes in
the qualitative composition of the samples, which
allowed for the identification of pollen markers for each
of the periods and zones. Thus, the combination of pol-
len types such as Eucalyptus globulus, Hypochaeris radi-
cata, Trifolium pratense, Brassicaceae-type, and Trifolium
repens are, generally, indicators of samples produced
between 2500 and 2800 masl, which coincides with
areas with greater anthropic influence, whereas
Weinmannia-type differentiates samples produced in
areas near and above 3000 masl, where fragments of
conserved forest still predominate (Baptiste, 2004; Le
on
et al., 2015; Van der Hammen, 1998). The pollen types
reported in Table 3, allow better differentiation of each
altitudinal zone. In addition, the presence of the pollen
types Gaiadendron punctatum, Fraxinus chinensis, and
Dodonaea viscosa in bee pollen indicates its production
during the dry season, while Ilex kunthiana, Ulex euro-
paeus, and genera such as Vernonia, Clethra,
Hesperomeles, and Vicia are indicators of visits during
the rainy period.
Biodiversity and its pollen offer are fundamental as a
nutritional aspect of the feeding patterns of honey bees
through rainy and dry seasons. Honey bees and other
pollinators are threatened due to agricultural intensifica-
tion, use of insecticides and herbicides, fragmentation of
natural habitats, and homogeneous cropping systems
that reduce the availability of the resources and contrib-
ute to the decreasing in population dynamics of honey
bees (Potts et al., 2016; Kov
acs-Hosty
anszki et al.
2017). For that reason, the availability of a broad range
of floral resources in the landscape is indispensable to
guarantee the honey bees pollen requirements, mainly
during the food supply depletion period, due to honey
bees must multiply their efforts by visiting a greater
number of plant species, as it was evidenced in this
study and others researches carried on in areas where
agricultural and natural landscapes are mixed (B€ansch
et al., 2020; Lau et al., 2019; Montoya-Pfeiffer, 2011;
Requier et al., 2015; Wintermantel et al., 2019).
Melissopalynology in the Eastern Andes of Colombia 11
Conservation of natural forests and restoration of
areas that have been degraded by human activity are fun-
damental to the preservation of honey bees and native
pollinators (Potts et al., 2016; Wratten et al., 2012). Since
beekeeping is associated with suburban areas and crop-
lands, the implementation of organic farming practices
could represent a strategy to increase honey bee colony
performance and survival, as well as enhances ecosystem
services like crop pollination and ecological restoration, as
it was evidenced by previous studies (Wintermantel et al.,
2019). Nevertheless, the effect of organic farming on
honey bee performance and survival has not been tested
in the Altiplano Cundiboyacense and can be the topic of
further researches.
Conclusions
Our results provide important information about the
foraging behavior of A. mellifera in the Eastern Andes of
Colombia and contribute to maintenance of beekeeping
in this region. The species diversity present in the pol-
len spectrum of Altiplano Cundiboyacense demonstrates
its potential for the beekeeping industry and the
importance of the resources such as Hypochaeris,
Eucalyptus, Trifolium, Weinmannia, and the family
Brassicaceae as the main food source for A. mellifera.
The importance of biodiversity is evidenced as honey
bees visited a large number of plant species from nat-
ural forest areas, croplands, and grasslands as a comple-
mentary source of pollen in the region. Our results
suggest that variation in precipitation level is an import-
ant factor that influences variations in honey bee pollen
composition. Thus, the exclusive presence of some pol-
len types among zones of the altitudinal gradient, as
well as during climatic periods, allows pollen types to
be recognized as useful pollen markers in geographically
and temporally differentiating the origin of bee pollen in
the Colombian Andes.
Acknowledgments
The authors express special thanks to APIMUNDO S.A.S,
Efra
ın Mu~noz, Mauricio Mu~noz Barrera, Myriam Barrera and
Carlos Mu~noz for donating the samples for the present study
and their important contribution to Colombian beekeeping.
Special thanks to Oriana Rojas Gonz
alez for her help in the
development of this article. Thanks to CAPES for the scholar-
ship of Masters for the first author and CNPq for a scientific
grant to FARS. Thanks to the Postgraduate Program in Botany
and Plant Micromorphology Laboratory of State University of
Feira de Santana, as well as the Physicochemical Properties of
Foods Laboratory of the University of Tolima, for allowing the
use of their facilities for this research.
Disclosure statement
No potential conflict of interest was reported by the authors.
12 L. C. Casas Restrepo et al.
Funding
This study was financed in part by the Coordenaç~ao de
Aperfeiçoamento de Pessoal de N
ıvel Superior—Brasil
(CAPES)—Finance Code 001.
ORCID
Luis Carlos Casas Restrepo http://orcid.org/0000-0001-
9710-3230
Francisco de Assis Ribeiro dos Santos http://orcid.org/0000-
0002-9246-3146
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