Palynotaxonomical study of honeybees ( Apis mellifera var. adansonii ) forage and/or preference plants in South-Western Nigeria

The disputed issues of higher plant taxonomy and phylogeny are greatly impacted by pollen morphology because pollen grains have clear distinctive characteristics that are typically labeled. Palynotaxonomical study of Honeybees preference plants in South-Western Nigeria were conducted between the months of May, 2019 and June 2021 using fresh polleniferous materials which were extracted through acetolysis methods. Results showed that ninety-two (92) plant species belonging to thirty-six (36) plant families of apicultural importance most visited by honeybees ( Apis mellifera var. adansonii ) were documented. At x100 magnification, thorough morphological studies were performed, and the results showed that pollen grains from plant species in the family Asteraceae were spinolous and spherical in shape; those from the Fabaceae sub-families Caesalpinoideae, Mimosoideae, and Papilionoideae, on the other hand, had a great deal of morphological diversity with variations in symmetry, position and distribution. Those of the families Malvaceae was echinate and Myrtaceae were colporate; syncolpate and prolate respectively. However, the variations above were generally reduced within species in the same genera. Tricolpate aperture type was observed in the genus Euphorbia ; giving reason for their classification in the same genera. The genus Combretum was all heterocolpate, circular in shape and heteroporate. Sculpturing was psilate or scabrate and pollen shape class were sub-prolate, oblate-spheroidal or prolate spheroidal. Similar form and symmetry observed in all the species studied were reason for their classification into the same family. The study revealed that the flowering period for most of these honeybees’ forage plants were between the months of October to March. Identification, propagation, cultivation, conservation, and sustainable exploitation of these honeybee ( Apis mellifera var . adansonii ) forage and preference plants would be beneficial in potentially increasing the efficiency of apiculture and commercial cultivation in South-Western Nigeria.


INTRODUCTION
Palynotaxonomy deals with the study of description of variation in organisms, the investigation of causes and consequences of this variation, and the manipulation of the data obtained to produce a system of classification.Taxonomy is defined as a science dealing with the study of nomenclature, description, classification, identification and relationships, with basic idea of determining the rank of the taxa.Several characteristic features of plants are used to determine the rank of the taxa, of which palynological evidence has proven useful at all taxonomic levels, particularly in verifying relationship in established taxonomic groups.On the basis of pollen characters, study of this nature is very useful in systematic considerations.Palynotaxonomy could provide a convenient method of identification and communication.A workable classification having the taxa arranged in hierarchy, detailed and diagnostic descriptions are essential for identification.It could also detect evolution at work: to reconstruct the evolutionary history of the plant kingdom, determining the sequence of the evolutionary change and character modification.For example, Chanda and Ghosh (1979) proposed a genealogical tree which reflects the evolutionary tendencies among the monocots and early dicots in terms of apertural evolution.Chanda et al. (1988) studied the pollen morphology of the order Alismatales (including Butomaceae, Limnocharitaceae and Alismataceae).They reported that the pollen grains of monotypic Butomaceae are monosulcate and boat-shaped, those of Limnocharitaceae have 4 to 10 ill-defined, fimbriate globally distributed pores, and of Alismataceae are pantoporate and spheroidal, or rounded polyhedral.The bee is the most valuable insect on planet earth.This is not because of the value of its direct products as they represent only 0.5% of the total agricultural production, but because of the enormous benefits accruing from the cross pollination of plants.This cross pollination ensures the improved quality and quantity of produce, fruits and seeds, improved species of selfgerminating plants and also maintain the ecobalance on earth (Sivaram, 1995).
The honey bees (Apis mellifera var.adansonii), the pollinators of plants the world over; play a crucial role for wild and cultivated plants, especially in the tropics where insect pollination is vital (Winfree, 2010;Ollerton et al., 2011).Some honey components, such as carbohydrates, water, traces of organic acids, enzymes, amino acids, and pigments, come from bees and plants, while others, such as pollen and wax, appear during honey maturation (Agwu and Okeke, 1997).
The Apis mellifera var.adansonii (African honey bee) is very defensive and unpredictable; darker and smaller; more energetic and aggressive; and also irritable during the hot hours and hates noise.It builds its nest in closed spaces but migrates (swarms) often and abandoned its nest (absconds) when disturbed.It produces more drones (male bees).It gathers food all the year round, produces large quantity of honey yields every year (Breadbear, 2009).Co-evolution and mutualism have been cited as examples of relationships between honeybees and flowering plants.Honeybees and flowering plants are mutually dependent; honeybees need flowering plants for food in the form of pollen and nectar, whereas plants need honeybees for pollination.Honey contains pollen grains which are collected by honeybees while foraging the flowers for nectar (Essien, 2020).Taxonomy, phylogeny, palaeobotany, aeropalynology, and pollen allergy study all rely heavily on pollen morphological characterization.Analysis of pollen (extant and extinct form or taxa) is the most important approach to reconstruction of past flora, vegetation and environment (Faegri and Iversen, 1989).Understanding pollen's functional characteristics, such as pollination biology and pollen-pistil interaction, depends on the knowledge of pollen morphology (Essien and Ige, 2019).
The microscopic analysis of pollen is a standard method and an effective tool to understanding the distribution and abundance of floral nectar sources in any given region (Agwu et al., 2013).Palynological analysis is used to determine the characteristics, types and quality of honey.Microscopical pollen assessment has been used to determine the geographical and floral origins of honeys (Maurizio, 1975;Agwu et al., 2015).The significance of a standardized procedure that minimizes characterization errors from the pollen analysis of honey obtained from various geographical locations was demonstrated by Low et al. (1989) and Lutier and Vaissière (1993).
Due to the fact that honeybees are known to travel more than 3 km in search of their preferred forage sources, studying the pollen content in honey significantly aids in understanding the geographical, ecological, and botanical origins of honey.Knowledge of botanical source of honey is a prerequisite for beekeepers to undertake migratory beekeeping for increasing honey production and pollination.When determining the honey's commercial quality, characterization is crucial because the season of flowering and nectar production for the same species can vary depending on location (Zamarlicki, 1984).Additionally, palynological investigations have been performed to determine single-and multiplefloral honeys (Seijo and Jato, 1998;Valencia-Barrera et al., 1994;2000).Identification of honey sources in an ecological zone is important for commercial beekeeping with the goal of increasing honey production.Knowledge of honeybee plants and time of pollen and nectar flow greatly influence the brood rearing activity and the functioning of honeybee colonies and production of honey as well as other hive products (Sivaram, 1995;Ostrowsha, 1998).
Beekeeping is entirely depending on the types of flowering plants available in any given area.Understanding the relationship between honeybees and plants is important for researching honeybee food preferences and pollination needs.An essential prerequisite for developing apiary is pollen from various plants that could serve as potential sources of nectar and pollen for the honeybees (Kalpana and Ramanujam, 1997).Different plants have pollen with distinctive shapes, sizes, apertures, and ornamentations.Melissopalynology is the study of the pollen that bees intentionally and unintentionally gather and then incorporate into honey.A lot of research has been done using Melissopalynology to identify the floral, geographic, purity and origins of honeys (Waters, 1915;Nair, 1964;Agwu et al., 2013).It is also used to access correlations between in-situ climate parameters, such as temperature and rainfall, which are crucial in the context of outside factors affecting pollinators and pollination networks (Jato et al., 1994;Bilisik et al., 2008;Jens et al., 2008;Thomas et al., 2009).According to Bryant and Jones (2001), a variety of factors, including honeybees' capacity to filter out specific kinds and quantities of pollen from the nectar they gather before returning to the hive, affect the production of honey.They acknowledged that the complexity brought on by these various factors suggests a significant variation in the pollen content of honey produced in the same hives from year to year or season to season.Finding the source of honey in the area can be done using a well-established technique called microscopic analysis of pollen grains (Agwu et al., 2013;Essien et al., 2022).Around the world, numerous studies on pollen morphology have been conducted (Raj, 1969;Sowunmi, 1973;Tomb et. al., 1974;Nair and Kapoor, 1974;Gill and Chinnappa, 1982;Paul et al., 2014).On the basis of their pollen profiles, Kral (1992) conducted a palynological investigation of forest trees in relation to the history of the forest and the natural mixture of tree species.A palynological study of the cultivated plants in Rawalpindi, Pakistan, was conducted by Noor et al. (2004) while in Nigerian cultivated plants with 20 different pollen morphologies have been described by Adekanmbi and Ogundipe (2006).The Moringaceae and Berberidaceae families' pollen studies were carried out by Perveen and Qaiser (2009;2010).

Several
taxonomists use phenotypic characteristics of plants to identify plant species.Therefore, pollen morphological studies can serve as a foundation for identification of plant species.The full applications of pollen morphology in systematics, palaeobotany, and allergy have increased due to interest in these fields, which are all widely acknowledged (Noor et al., 2004).Therefore, the identification of bee plants is a significant application of pollen research.Understanding the main nectar and pollen producing plants and their flowering periods are of great benefit to maximize the effectiveness of the bees according to Jato et al. (2002) and Balasurbramanyam (2011).South-Western Nigeria has a wide variety of flowering plants and a promising future for commercial beekeeping.The floras of some areas in South-Western Nigeria are under threat from increased, unchecked, and indiscriminate destruction as a result of cultural and agricultural practices.The knowledge of the significant forage plants for honeybees in this area may one day result in their legal protection and/or planned propagation for the construction of bee farms.The purpose of this study are to identify the types of plants that honeybees (Apis mellifera var.adansonii) most frequently visited while collecting nectar, the plant that the bees prefer, and whether the study area is a suitable location for beekeeping.

Extraction of pollen grains
With the help of a piercing needle and a pair of forceps, anthers from both flower buds and fully opened flowers from the preserved materials were carefully removed and crushed in an ethanol solution.These were then decanted after being sieved and centrifuged for five minutes at 2000 rpm.Before acetolysis, glacial acetic acid was applied to the pollen sediments, and then they were centrifuged and decanted to remove any remaining water.Acetic anhydride and concentrated sulphuric acid were used to make an acetolysis mixture that was freshly prepared in a 9:1 ratio.Agwu and Akanbi (1985), Paul et al. (2014), and Essien and Ige (2019) adaptations of Erdtman's (1969) methods were used in boiling the sediments in a water bath at 100 0 C for acetolysis.The mixture was stirred for 5 minutes in a water bath set at 100 0 C, after which it was centrifuged for 5 minutes, with the supernatant being decanted.The precipitates were decanted after being centrifuged twice with distilled water and washed once with glacial acetic acid.The recovered precipitates were kept in plastic vials with 2:1 solutions of glycerin and ethanol.

Microscopic examination and pollen analysis
On a 25.4 mm x 76.2 mm (1"×3") slide 1 mm-1.2mm thick, one drop of thoroughly shaken precipitates suspension was mounted and covered with 18mm x 18mm cover slip.To keep the precipitation from drying out, the mount was sealed off at the edges with colorless nail polish.For thorough morphological studies, the prepared slide was then scrutinized under a microscope at x400 and x1000 magnifications.In the Palynology and Environment Research Laboratory, Department of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba-Akoko, Ondo State, Nigeria, pollen samples were examined and analyzed morphologically using reference descriptions, photomicrographs (Bonnefille and Riollet (1980), Agwu and Akanbi (1985), Punt et al. (2007), Essien and Ige (2019), and prepared slides.In documenting the dimensions and other parameters, averages of ten (10) pollen grains were used and this helped in easy description and convenient identification of the pollen studied.Photomicrographs were taken using a Fluorescent PEC Medical Microscope Model No.: OZJ088.

RESULTS AND DISCUSSION
Palynotaxonomical study of Honeybees preference plants in South-Western Nigeria were conducted between the months of May, 2019 and June 2021 using fresh polleniferous materials and results showed that ninety-two (92) plant species belonging to thirty-six (36) plant families of apicultural importance were most visited by honeybees during the course of pollen and nectar collection.The study revealed great similarities and diversity in aperture types, sculpturing pattern, size of the pollen grain, exine thickness, colpi length and width among other diagnostic features (Table 1).Photomicrographs of some selected pollen types examined in the study are presented in Plate 1.The flowering calendar for these honeybees' preference plants and their forage sources were also documented in the study and these is anticipated to provide baseline information to beekeepers in determining major season of honey production in the study area.These would also make it easier to identify the botanical and geographic origins of Nigerian honeys for use in the region's developing, commercially viable honey industry.Similar findings were reported by Nair (1970)
This study is anticipated to be useful to beekeepers in South-Western Nigeria to formulate these seasonal bee management strategies and schedule especially for migration of bee colonies to different floral sources.The pollen morphology of the taxa examined could also provide useful information about their respective families.Study is also anticipated to aid in ascertaining the ecological, geographical as well as the botanical origin of honey (Agwu and Akanbi, 1985;Ige and Apo, 2007;Essien et al., 2022).
This study also showed that most of these honeybees' forage plants taxa flowers during the period of reduced rainfall (dry season).That is, the months of November to March when the sun shines more brightly and the atmospheric humidity is lower.The finding is in line with the report of Jato et al. (2002) who opined that these climatic conditions are most suitable for the flight of insects (for example honeybees) and positive pollination ecology.The information obtained during this phenology study has a great significance because it did not only provide knowledge about the plants flowering calendar but also gives insight into how environmental variables, such as selective pressure on flowering, can affect a species' behavior, demonstrating how food is produced year-round in the study area.Findings conformed favourably with the report of Essien (2020).

CONCLUSION
The pollen taxa described in this work reveal a wide spectrum of plant communities common in the Southwestern Nigeria.In the present study, palynotaxonomy of some selected monocots and dicots foraged by honey bees in Southwestern Nigeria are presented.Modern bee keeping is the art of handling bees and maximizing their benefits such as harvesting of honey, pollen, wax, royal jelly, propolis, beevenom and other bee products.Plants flowering and honeybees have been used as illustrative examples of co-evolution and mutualism.The findings of this study indicated that majority of these foraged plants flower primarily between the months of October and March.
This study revealed that pollen grains of plant species belonging to the family Asteraceae were spinolous and spherical in shape; those of the Fabaceae sub-families Caesalpinoideae, Mimosoideae and Papilionoideae had great morphological diversity with variations in symmetry, position and distribution of apertures, exine structure and sculpture of the pollen wall.The pollen grains of plants belonging to the family Malvaceae were echinate (spinolous) and those of the family Myrtaceae were syncolpate and prolate.However, the variations above were generally reduced within species in the same genera.Tricolpate aperture type was observed in Euphorbia heterophylla L, Euphorbia hirta L and Euphorbia hyssopiifolia L; a possible reason why they are classified in the same genera.Pollen grains of the genus Combretum were all heterocolpate, circular in shape and heteroporate.Sculpturing was psilate or scabrate and pollen shape class were subprolate, oblate-spheroidal or prolate spheroidal.Similar form and symmetry observed in all the species studied were reason for their classification into the same family.The morphological characteristics of pollen grains are manifested in the outermost pollen wall (exine).The stratification of exine along with number, position and character of apertures are useful in classifying pollen grains.Hence the orientation of polarity is an important criterion in the identification and description of pollen grains.On the basis of apertural characters along with shape, size and surface ornamentations of the exine, a comparative study of pollen grains is very useful in systematic consideration.
Palynologically, plant families can be segregated into two groups: Stenopalynous, where the taxa of the family display more or less the same type of pollen grains, for example, Poaceae, etc. and Eurypalynous, where the taxa are characterized by an obvious difference in pollen types, for example, Rubiaceae, Solanaceae, Convolvulaceae and Fabaceae, among others.The knowledge of pollen morphology has been to substantiate many taxonomic revisions sometimes even up to the formation of new taxa.Segregation of Bombacaceae from Malvaceae is an example of the contribution of pollen morphological study to taxonomy.

Table 1 :
Palynotaxonomical characters of some selected honeybees' preference plants inSouth-western Nigeria As such, it is therefore necessary to examine large numbers of pollen types from each family so as to obtain an elaborate, comprehensive, detailed and complete information on different species and genera within that family.