The melissopalynological investigation in the Eastern Dry Zone of Karnataka, India

Melissopalynology, the analysis of pollen grains present in honey, indicates about the pollen and nectar sources in a region utilized by bees, which is used to determine the bee floral resources and botanical origin of the honey. This study investigated the melissopalynological analysis of the honey samples from the Eastern Dry zone of Karnataka. 24 honey samples were examined based on pollen analyses, among them 14 samples were unifloral, rest were multifloral. The unifloral honey had pollens of Callistemon viminalis, Areca catechu, Citrus sp., Mallotus philippensis, Cocos nucifera, Eucalyptus sp., Ocimum sp., Moringa oleifera and Pongamia pinnata. Samples collected in October, November, December, and January were rich in pollens of Eucalyptus sp.. Similarly, samples collected in January, February and March had pollen of tree species viz., Swietenia mahagoni, Canthium parviflorum, Simarouba glauca, Eucalyptus sp., Moringa oleifera, Syzygium cumini, Tabebuia sp., Pongamia pinnata, Acanthaceae, Anacardium occidentale, Cocos nucifera, Areca catechu, Mallotus philippensis, Bauhinia variegata, Psidium guajava, Alangiaceae, Euphorbiaceae, Ulmaceae, Capparis zeylanica, Convolvulaceae. GKVK-11 followed by GKVK-12 sample recorded the highest Shannon diversity and GKVK-9 followed by GKVK-7 sample recorded the least diversity. Based on the similar floral composition samples were classified into four clusters. The PCA revealed that most of the samples grouped into a single cluster, except 7, 19, 20, 21, and 22 which were placed away from the origin. The presence of pollen in the honey of a particular plant species during different months is related to the blooming of that particular plant species from which the bees forage. The flora of honey changes with the season. The diversity of pollen grains in honey varied with location to location. The present study provides scientific knowledge to the beekeepers by indicating important plants for the development of the regional apiculture, through the identification of pollen types.


Introduction
Melissopalynology is a study of pollen grains present in honey. It helps to assess the bee floral availability of the region, where the particular types of honey are produced (Sajwani et al., 2007;Song et al., 2010). It also provides the exact information regarding the floral resource to bees in the vicinity and also becomes useful in the construction of the floral calendar (Sekhar, 2000).
The honey bees depend on the flora for nectar and pollen. The quality of honey varies concerning the flora available in and around them (Ponnuchamy et al., 2014). The Eastern dry zone of Karnataka is one of the important areas for beekeeping since the region comprises of a rich diversity of bee flora. One or the other flora bloom in every month which makes the bees survive throughout the year. The relative abundance of bee plants in the eastern dry zone consisted of trees (46%), herbs (25%), shrubs (18%), trailers (9%), climbers, and palms (2%) (Marc,3 2012). The major bee flora available in and around the eastern dry zone region was Callistemon viminalis Byrnes, Anacardium occidentale L., Antigonon leptopus Hook. & Arn., Simarouba glauca DC., Eucalyptus sp., Tabebuia sp. L., Cocos nucifera L., Mallotus philippensis Lour, Santalum album L., Tamarindus indica L., Trewia nudiflora L., and Bauhinia purpurea L. (Sekhar, 2000).
The urban beekeeping industry is gaining popularity and is accepted as a complementary activity to agriculture in India (Attri, 2010). The analysis of the honey pollen spectrum is extremely useful to detect the contribution of different nectar sources during a different period of the year (Oliviera et al., 2010). It contributes to the development of the beekeeping industry by the way of proper utilization of floral resources and provides the exact information about the floral resources of the bees in the particular region. Melissopalynological studies are least touched in various parts of India, especially in South India (Jhansi et al., 1994).
The studies on the source of pollen have been very few in Asia and with respect to different species of honey bees, the melissopalynological information available is very less (Ramalho et al., 2007 andNovais et al., 2009).
Although honey has been the most widespread bee product, the bee pollen trade is undergoing expressive growth. The quantity and quality of the bee pollen produced have attracted increased attention for Indian Apiculture. Eastern dry zone of Karnataka needs in-depth studies on the bee floras available also to support the interest of the urban beekeepers related to when is the honey flow season, dearth period, when to feed bees, when not to feed bees for managing the colonies.
It also helps in developing the pollen spectrum through pollen combination.
Henceforth, to encourage them and to make them aware of the floral calendar and the flora to which bee visits. This study aimed to identify the botanical origin of pollen loads and the melissopalynological investigation of Apis cerana honey in the Eastern dry zone of Karnataka.

Honey sampling and Melissopalynology
Twenty-one honey samples (Tab.1) of Apis cerana were collected from different regions of the Eastern dry zone (Fig. 1) during 2016-17. A piece of Apis cerana comb completely sealed with honey was collected from different places and brought to the laboratory for extraction. The cut comb was unsealed using the uncapping knife, honey was extracted using a honey extractor, and the obtained honey thus filtered into a beaker using a muslin cloth and was transferred to an airtight container and was labeled concerned to the place, date and time of collection. The samples were stored in a refrigerator at 4ºC for further studies (Method adopted from Saxena et al., 2010). After the complete removal of honey, the frame along with an empty comb was placed back to the same colony and the same position.
Apis cerana honey was analyzed for the presence of pollen using the acetolysis method suggested by Erdtman (1952Erdtman ( , 1966. One gram of honey was diluted in 9ml of water and centrifuged at 4000rpm for 15 min. The pollen so obtained at the bottom was collected, identified, and counted in all the 25 cells of hemocytometer under a microscope (Motic). Based on the percentage of each pollen type present in the honey was classified as unifloral (more than 45% of single pollen) or multifloral (less than 45% of single pollen) (Chaturvedi, 1989).
The grouping of honey is done by taking into account of pollen grains in 10g honey following Maurizio's classes. Group I (20,000 pollen grains per 10 g honey), Group II (20,000-100,000 grains per 10 g honey), Group III (100,000-500,000 grains per 10 g honey) (Louveaux et al., 1978). The images of pollen collected from honey samples were captured by Moticam 2300 3.0M Pixel USB2.0 and the software used was Motica image plus 2.0 ML .
These pollen images were identified by making slides of available flora.
To study the similarity between the pollen sources agglomerative hierarchical clustering method was run using XLSTAT. The dendrogram was constructed using the Jaccard co-efficient and Unweighted pair group average method. PAST 3.24 version was used for principal component analysis (PCA) of the pollen data. Myrtaceae count to 18.52% (Figure 4). The absolute pollen counts per 10 g of honey samples indicated that 50% of honey samples belong to the group I (2,500-15,00 pollen grains), 37.50% of samples belong to group II (25,000-60,000) and 12.5%
The floral calendar of the melisopalynological studies ( Figure 2)  To study the pollen diversity in the honey samples collected from different locations, Shannon diversity was determined. The diversity indices varied between 0 to 2.11 ( Figure 6). Among the 24 different sources, the highest Shannon diversity was recorded in GKVK-11 followed by GKVK-12. The least diversity was recorded in GKVK-9 followed by GKVK-7. At 0.09 similarity co-efficient, The Principal Component analysis revealed that most of the samples grouped into a single cluster except 7, 19, 20, and 21 which were placed away from the origin ( Figure 8). The absolute pollen count and the frequency of each taxon are presented in Figure 9.

Discussion and Conclusion
The presence of pollens in honey symbolized the bee foraging plants. Among several flowering plants bee forage specific plants (Dimou, 2007). The diversity of pollen grains in honey varied with locations and availability of bee flora (Song et al., 2014). The present study provides a vision on the pollen spectrum of the Similarly, in Brazil, Eucalyptus sp. and Citrus sp. were predominant (Barth, 1970: Ramlho et al., 1991. In India, honey from Uttar Pradesh predominated with pollen from Antegonon and Moringa (Nair and Singh, 1974), Rumex sp., Nephelium sp., and members of Myrtaceae, Liliaceae, Rosaceae, and Euphorbiaceae (Sharma and Nair, 1965;Gaur and Nanwani, 1989). Honey from Himachal Pradesh had a preponderance of Brassica, Adathoda, Clematis, Mussenda, and Helianthus sp. (Singh et al., 1994). Honey from Andhra Pradesh revealed that Sapindus, Eucalyptus, Anacardium, and Cleome were major pollen types (Kalpana and Ramanujan, 1997). In Karnataka, honey samples were having pollens from Cocos, Eucalyptus, Schefflera, and Mimosa (Singh and Suryanarayana, 1990). Also, the Samples collected in October, November, December, and January were rich in pollens of Eucalyptus. Many Eucalyptus species offer pollen and nectar to the pollinators (House, 1997). In the many tropical to subtropical regions, where Eucalyptus species have been planted, and often become naturalized, they have become important, or even dominant, nectar sources for beekeeping in southern Asia (Chauhan et al., 2017), in South America (Daners, 1998;Bonilla et al., 2016), in Africa (Carroll, 2006) and other tropical regions (Rasoloarijao, 2014), and many 13 countries within the Mediterranean Basin (Seijo et al., 2003;Terrab et al., 2003;Fea´s et al., 2010). Similarly, in January, February and March had forest pollen species viz., Pongamia, Syzygium, Santalum, Neem, Mahagoni, etc. The presence of pollen in the honey of particular plant species during different months is related to the blooming of that particular plant species from which the bees collected the pollen during foraging activity (Joshi et al., 1998). China (Song et al., 2012). Some of the locations had similar pollen compositions based on which the cluster analysis is plot. From the statistical analysis of cluster analysis, the samples are grouped into five clusters. This result is in accordance with the four clusters formed out of 89 samples which were collected from the Leon and Palenica provinces (Herrero et al., 2001). This is based on the common pollen taxa recorded in different months. Eucalyptus sp. L was predominant in samples of cluster 4. GKVK 1 recorded unique compared to other cluster; this may be due to Sesamum and Citrus, generally, Sesamum isn't grown in the Eastern dry zone of Karnataka and few experimental plots of GKVK campus of UAS, Bangalore, had Sesamum which reflected in GKVK 1 sample. Majority samples in cluster 3 are unifloral and cluster 4 are multifloral. Based on this study, honey samples are grouped according to their botanical origin.
The grouping in PCA might be due to pollen diversity, the season of collection and abundance or dearth period (Sekhar, 2000). This was reflected in PCA plots wherein a few samples (7,19,20,21 ) were distributed away from the origin (Fig.   8). Since most of the tree species in the study location bears flowers in February and March, the honey bees had the opportunity to explore the diverse flora and collect pollens of different tree species. It was also observed that samples that had pollens from more than 5 species might have been placed away from the origin. It can be observed that the time of sampling would have affected the pollen variability in the honey sample (Marc, 2012;Raja, 2012). Among these samples, 75% were multifloral and were sampled during March.
Overall, this study provides useful data regarding the favoured plant and the foraging preference of Apis cerana from the Eastern dry zone of Karnataka, which will help beekeepers to develop the apiary. It provides information regarding the agricultural crop which gets pollinated and can be utilized for pollination purposes.
This study will in-turn contribute to the conservation of bees, apiary development and leads to sustainable honey production. Also, this will improve the socioeconomic status of the farmers and beekeepers.