MELISSOPALYNOLOGY AND CLIMATIC VARIATION: CASE OF HONEY ATTRIBUTED TO EUPHORBIA RESINIFERA, REGION OF AZILAL (MOROCCO)

Abderrahim Ihitassen 1,2 , Younes Belrhazi 1 , Ismail Bettar 1 , Fouad Msanda 2 . 1. Laboratory of Applied Geology and Geo-environment, Faculty of Sciences, Ibn Zohr University, Agadir Morocco. 2. Laboratory of Biotechnologies and Valorization of Natural Resources, Faculty of Sciences, Ibn Zohr University, Agadir Morocco. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 12 April 2019 Final Accepted: 14 May 2019 Published: June 2019

This work is an example of the use of honey in the study of the impact of global warming on nectar production and the relationship between plants and pollinators. It is the result of the melissopalynological analysis of 42 honey samples attributed to Euphorbia resinifera Berg, from the region of Azilal (Morocco). Quantitative analysis showed that these honeys are mostly poor in botanical elements. Although beehives were established during the flowering period of Euphorbia resinifera, in areas dominated by it, only 16.7% of honeys harvested showed the monofloral character associated with the species. The others are multifloral or honeydew produced mainly by the holm oak (Quercus ilex). The qualitative analysis of the honeys showed the presence of 55 botanical taxa belonging to 24 families. Pollen from herbaceous species is better represented than pollen from shrubs and trees. Entomophiles are dominant. The evolution of the pollen percentages of E. resinifera in the pollen spectrum has shown that the precocity of its flowering related to the increase of the temperature and the diminution of the precipitations has negatively influenced its nectar production, its attraction for bees, and its contribution to the elaboration of honey.
The present study was carried out during 2014 and 2015 in order to specify the palynological characteristics of monofloral honey of Euphorbia resinifera, an endemic species of Morocco which dominates the flora of certain areas of the Region of Azilal (Morocco).

Sampling areas:
In order to have a good representation of the area of the Euphorbia, we chose 5 areas representing different ecogeographical situations ( Fig. 1):  Ait Attab (840 m), a forest area consisting mainly of holm oak (Quercus ilex), cedar and of two juniper species (Juniperus oxycedrus and J. phoenicia). In this location, Euphorbia resinifera spreads over large areas along the forest;  Foum Jamâa (890m), located in a generally forest environment where Euphorbia resinifera is associated with cedar (Tetraclinis articulata) and red juniper (Juniperus phoenicea) and where cereal crops, almond and olive tree cultivation are dispersed on small fields at the edge of the forests;  Tanant (940 m), the Euphorbia colonizes an important area of a forest consisting mainly of cedar, red juniper and of holm oak;  Azilal (1300 m), a mid-mountain area where oak is associated with cedar, red juniper, lentisk (Pistacia lentiscus) and cistus (Cistus villosus); Euphorbia occupies the slopes;  Ait Mhamed (2000 m), an area of high mountains where the Euphorbia occupies the slopes dominated by holm oak. 704

Analysis methods:
Hives were planted in sites dominated by E. resinifera, at the beginning of its flowering, from mid-April in the lower elevation areas (Ait Attab, Foum Jemâa and Tanant) and for the second week of May in the highest sectors (Azilal and Ait Mhamed). We also established a reference collection of pollen grains from the flora of the region during the study period. The samples were collected at the end of the floraison period of Euphorbia resinifera in July for the two years of study.
The melissopalynological analyzes, quantitative and qualitative, were performed according to the method described by Louveaux et al. (1978), reviewed by Ohe et al. (2004) and summarized in Terrab et al. (2014). Analyzes are done on a 10 grams of honey basis. The determination of the different palynomorphs, their counting and their photographs were done under the Leitz laborlux S microscope.
The identification of pollen grains is based on their morphological criteria. For their botanical attribution, we used our reference collection and the Atlas of literature (Moore et al., 1991;Reille, 1992;Valdés et al., 1987). Some taxa are determined up to the species or genus. When a set of genera of the same family coexisted during the foraging period and have the same pollen type, we assigned the pollen with the same characteristics to the most dominant genus with the indication "type" which follows the name of the genre (i.e: Lotus Type).
For the classification of honeys, we followed the approach proposed by the International Commission for Bee Botany (ICBB) (Louveaux et al., 1978) based on the percentages of the various elements of the melissopalynological residue (pollen grains and elements of honeydew). For each sample, we counted and determined a minimum of 1000 pollen grains which we grouped according to the 4 frequency classes defined by Louveaux et al., (1970Louveaux et al., ( , 1978 designating as : 1. Dominant or predominant pollen (n> 45% of a specific type of pollen); 2. Accompanying or secondary pollen (15% <n <45%); 3. Minor important or tertiary pollen (3% <n <15%); 4. Minor or rare pollen (n<3%).
For the botanical attribution, we used the measurement of electrical conductivity (EC), an important parameter according to the International Honey Commission (IHC) (Bogdanov et al., 1997(Bogdanov et al., , 2004, to differentiate flower honeys from those of honeydew (EC>800 μS/cm for honeydew's honey and EC<800 μS/cm for nectar's honey). The proportions of the different pollen grains of nectariferous taxa allowed us to decide between monofloral and multifloral honeys.

Results:-
The harvested honeys showed the following sensory characteristics: amber colored with a sweet smell and a persistent taste sticking to the throat after swallowing. These characteristics allow beekeepers, resellers and consumers to consider this type of honey as Euphorbia monofloral.
The results of the quantitative analysis (       (Fig.2). Table 2 summarizes the frequency of pollen in the samples studied as well as their class of abundance. 720 For their botanical origin, and according to the electrical conductivity values (Table 1), all honeys harvested in 2014 are nectarous honey whereas in 2015, 12 are attributed to honeydew and 3 are from flowers.
Among the thirty flower honeys, seven are monofloral of E. resinifera (samples 2, 3, 5, 9, 10, 12, and 26) with a percentage of pollen grains from this species varying between 25% and 58% among pollen grains of nectariferous taxa. Euphorbia pollen is among the underrepresented group and a result of 25% of pollen presence is sufficient to qualify a honey as monofloral (Damblon, 1988;Terrab et al., 2014). These honeys have a low botanical content (five belong to Maurizio's class I (1979) and two to class II with an average of = 14027 grains / 10g). Their honeydew index is very low (average = 0.362), a characteristic value of monofloral honeys (Louveaux et al., 1970(Louveaux et al., , 1978Von Der Ohe, 2004). The remaining flower honeys are multifloral, and belong to classes I, II or III, with an average of 26953 grains / 10g and a low honeydew index (average = 0.508).
Honeydew honeys have an electrical conductivity that varies between 1017 and 1456 μS / cm, a honeydew index of less than 3 (average = 2.067) for most samples and an average content of botanical elements with two samples belonging to class I, eight to class II, two to class III and one to class V (average = 196020). The very large number of botanical elements of the last sample results from the fact that this honey was very adherent to the alveoli and we were forced to extract it by pressing (Table 1).

Discussion:-
The study was carried out during 2014 and 2015 characterized by a precocious flowering, a low yield of honey per hive (4 kg/hive for 2014 and 3 kg/hive for 2015) compared to a normal year (11 kg/hive on average) and a small contribution of Euphorbia resinifera to honey production. The melissopalynological characteristics of the studied samples did not reflect the dominance of E. resinifera, nor the sensory characteristics that allowed beekeepers to label honeys from the Azilal region as monofloral. Of the 42 samples analyzed, only seven (16.7%) had characteristics of monofloral honey of Euphorbia resinifera, the others are either honeydew or multifloral.

Early flowering of Euphorbia resinifera:
The  (Fig. 4). For 2014, the cumulative rainfall of the 4 months preceeding the flowering period (January-April) was 220 mm compared to 169 mm for 2015.
Therefore, for 2014, the climatic conditions were relatively favorable (higher rainfall and cooler temperatures) for the production of nectar honeys (Table 1), although the yield remains modest. In the field, despite the "heat wave" that hit the region in mid-May (Fig3), E. resinifera in flowers dominated the nectarine flora in all areas studied until the end of June. The pollen spectra of the honeys sampled show an increase in the pollen percentages of the E. resinifera with altitude. In the highest areas, the percentages are more in line with the situation in the field ( Table 2). Seven out of twenty honeys are monofloral (one in Azilal and six, higher, in Ait Mhamed). The honeys in the lower areas are multifloral and are low in pollen of the species (0 to 4.7% among nectarifers). In these areas, the species was less visited than other taxa. 722 nectar production (Carrión-Tacuriet al., 2012) and increase sugar concentration (Carroll et al., 2001). Domesticated bees prefer average sugar concentrations (Waller, 1972;Roubik and Buchmann, 1984) however low (Bolten and Feinsinger, 1978) or high concentrations of sugar (Adgaba et al., 2017) in nectariferous plants can limit melittophilia. For Mitchell and Waser (1992) and Mitchell (1993), there is a positive relationship between nectar production and frequency of pollinator visits.
In Morocco, over the past few decades, aridity has continuously transgressed from low to high altitudes and from south to north (Ait Brahim et al., 2016), in response to increasing temperatures and decreasing rainfall (Mokssit, 2012).
Thus, for areas of lower altitudes, more affected by temperature rise and drought, the early flowering of E. resinifera, negatively influenced its productivity of nectar, its attractiveness for honey bees, and thus its contribution to honey production. For the mountain areas (Azilal and Ait Mhamed), less affected by climatic variations, the stability of the flowering period of E. resinifera is responsible for a more attractive nectar productivity for bees and a sufficient contribution to producing monofloral honeys.
For 2015, there was no harvest for the sectors of Ait Attab and Foum Jemâa. In the Tanant area of comparable altitude but close to the l'Oued Lakhdar and Azilal, the composition of honeys was different compared to 2014. The samples are rich in honeydew. This is related to the evolution of climatic parameters and their impact on flora. As for 2014, the season started in good conditions. This allowed the hive to flourish and the bees to harvest the nectar. But the heat waves that occurred during the study period (Fig. 4), destroyed flowering and weakened the herd. The bees have consumed their stock. It was only after that they started storing honey again during the honeydew period (something that beekeepers found). Hence, the important part of honeydew in these samples and the low contribution of the nectariferous of which E. resinifera.

Conclusions:-
Honey is the product of a close collaboration between plants, which secrete the raw material (nectar and honeydew), and the bees that produce it. Studying it allows us to trace the evolution of honeybee-plant interaction under the control of natural and/or anthropogenic factors. Thus, using melissopalynology, this study has highlighted, for the first time, the relationship between the early flowering of Euphorbia resinifera, endemic species of Morocco, in response to global warming, on one hand, and its low nectar productivity, its attractiveness for bees and its contribution to honey making, on the other hand.
If the decline in honey yield from hives can be attributed to adverse weather conditions, affecting melliferous plants and bee colonies, several questions, about the relationship between early flowering and the contribution of Euphoribia resinifera in the elaboration of honey, need answers. We have already put forward various hypotheses such as the decrease in the quantity of nectar secreted by the species compared to other melliferous plants or the variation of its sugar concentration and / or the proportions of it. Studies are planned to unmask the origin of this low contribution to honey production, its impact on plant pollination and honey bees welfare, and the interaction between pollinators and entomophilous plants in general.