Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v60i3.289-294Sociobiology 60(3): 289-294 (2013) Melittopalynology and Trophic Niche Analysis of Apis cerana and Apis mellifera in Yunnan Province of Southwest China YJ Liu1, 2, TR Zhao1, XW Zhang3, C Liang3, FY Zhao1 Introduction Honey can be produced from the nectar of plants (nec- tar honey), from secretions of living parts of plants or from the excretion of plant sucking insects (honeydew honey). Ho- wever, honey analysis was focus on nectar honeys and iden- tified the nectar plants in this region through the analysis of pollen of nectar honeys (Caccavari, M. et al. 2010). This re- search method is generally recognized (Behm et al., 1996). Bees are generally regarded as beneficial insects for their role in pollination (Goulson, 2003). So the species of honeybee Apis mellifera Linnaeus was introduced into China 100 years ago for their good production of honey. Apis cerana Fabricius is the most widely distributed native bee species before Apis mellifera been introduced. However, its colonies become less and less in China now. Apis cerana and Apis mellifera are two different species; they have many differen- ces in their body structure and habits. The average length of worker bees body of Apis mellifera is 12.0 ~ 14.0 mm, and Abstract Qualitative melittopalynological data of 34 honey samples obtained from Apis cerana and Apis mellifera between 2011 and 2012 in Mengzi (Yunnan, China) are analyzed. And trophic niche of the two bee species on pollen food resources are also investiga- ted in the present study. Results show that a diverse spectrum of 17 pollen types was identified into 14 families, and the Asteraceae and Myrtaceae are the most frequent at non-nectar flow and the Lythraceae are most frequent at main nectar flow of pomegra- nate in our study area. As for trophic niche analysis of Apis cerana and Apis mellifera, the niche breadth value is 0.65 and 0.57 at non-nectar flow and 0.41 and 0.24 at nectar flow, respectively. Meanwhile, the value of trophic niche overlap index at nectar flow (0.68) is greater than that of non-nectar flow (0.61). And the value of interspecific com- petition index at nectar flow (0.92) is greater than non-nectar flow (0.77), too. These results may promote the development of local beekeeping and provide some reference for scholars to assess the influence of introducing exotic bees on Chinese native bees. Sociobiology An international journal on social insects 1 - Kunming University of Science and Technology, Kunming, PR China. 2 - South China Agricultural University, Guangzhou, PR China. 3 - Yunnan Academy of Agricultural Sciences, Mengzi, PR China. Article History Edited by Kleber Del-Claro, UFU, Brazil Received 21 June 2013 Initial acceptance 18 July 2013 Final acceptance 07 August 2013 Key words Melittopalynology, Apis cerana, Apis mellifera, Trophic niche analysis Corresponding author Feng-Yun Zhao College of Chemistry and Engineering Kunming University of Science and Technology, Kunming 650500 People’s Republic of China E-Mail: zhaofy@kmust.edu.cn that of Apis cerana is 9.5 ~ 13.0 mm. They normally display different strategies in cooling hive temperature (Yang et al., 2010) and guarding against invading bee viruses (Sharm & Dharam, 2005; Ai et al., 2012) or parasitic mites (Peng et al., 1987). Different bee species show their preference for different plants (Ramírez-Arriaga & Navarro-Calvo, 2011), resulting in a variety of pollen types and special proportions of them in honeys. Bees play a very important role in balancing the local ecosystem, especially for the pollination and reproduction of many plant groups (Larkin et al., 2008). Pollen analysis can get an effective assessment on ecological impacts of invasive bee species on native bees (Stout & Morales, 2009). The use of trophic niche analysis methods can effectively assess the impact of bees on the local ecological environment (Santos & Absy, 2010). In this research, we study the pollen types and propor- tion in samples of honey from two bee species, Apis cerana and Apis mellifera, at non-nectar flow and nectar flow two RESEARCh ARTICLE - BEES YJ Liu, TR Zhao, XW Zhang, C Liang, FY Zhao - Melittopalynology and Trophic niche analysis290 periods, to assess the impact of introduced bees on native bees and compare the pollen date and analyze their trophic niche as well. Materials and methods Study area Honey samples were collected from different colo- nies of Apis cerana (n=17) and Apis mellifera (n=17) be- tween 2011 and 2012. Different colonies were placed at two different apiaries (~300 m apart) around the city of Mengzi (23°31′N;103°25′E). The region is located in the Southeast of Yunnan Province, China. The climate is Subtropical plateau monsoon, with an annual rainfall of 857 mm and an annual temperatures of 18.6. The altitude of apiariy is about 1288 m and the sunshine is sufficient with an annual 2234 hours. Honey preparation and melissopalynological analysis Honey samples produced by bees in different months and different main nectar flow were provided by beekeepers and centrifuged at first. The collecting time of honey samples is shown in Table 1. Qualitative melissopalynological analysis were con- ducted by the methods reported by Louveaux et al. (1978) with slightly modification. In brief, 20 g of honey samples were dissolved in 40 mL distilled water and centrifuged (10 minutes, 4000 r/min). The supernatant was disposed and the residue washed again with 20 mL water. Pollen sediment was mounted in glycerine-gelatine and sealed with paraffin to de- termine frequency classes by microscope. Pollen types were Trophic niche analysis Niche is a functional relationship between a spe- cies and other population at the same time and space in the ecosystem. In a biome, niche overlap is a phenomenon of multiple species to feed on the same food, and the resulting competition between different species and increasing compe- tition while having scarce food. In this paper, set up a framework to think about and estimate the difference between Apis cerana and Apis melli- fera in terms of ecological niche. The ecological parameters were calculated using a niche breadth, trophic niche overlap index and interspecific competition index. Niche Breadth (B) were calculated by the equation (Levins, 1968): (1) Where B is niche breadth, S is the level of resource, Pi is the proportion of the species take advantage of i-th level resources accounted in total resources. Trophic niche overlap index (C) were calculated by the equation (Colwell & Futuyma, 1971): (2) Where Cij is the trophic niche overlap index and Cij = Cji, Pjh and Pjh are the proportion of the species i and species j take advantage of h resource sequence accounted in total resources, S is the grade of the resource sequence. Interspecific competition index (a) were calculated by the equation (Southwood, 1978): (3) Where a is the interspecific competition index of spe- cies i and species j in a same resource. Pjh and Pjh are the proportion of the species i and species j in each resource se- quence. Interspecific competition is not fierce in the resource utilization when α≥1 and competition is fierce when α<1. Statistical analysis For descriptive purposes, simple statistical analysis, was applied into our study using SPSS 17.0. The arithmetic means and standard deviations were obtained for variables measured in the present study. The pollen types number of honey samples from two kinds of bee species were compa- red by one-way analysis of variance (ANOVA) and LSD for multiple comparison tests (p>0.05). Data were assessed, such that statistical significance was based on p<0.05. Table 1 - Serial number of honey samples from Apis cerana and Apis mellifera at different main nectar flow. identified by comparing them with a reference collection that was obtained from the plants grown in the area surrounding the beehives. Pollen atlases of China Woody Plants Pollen Photo using Scanning Electron Microscopy (2011) and Nec- tariferous Plant of China (1992) were also consulted. The Pollen types were classified, according to fre- quency, into four categories: predominant pollen (≥ 45%), secondary pollen (16–45%), important minor pollen (3–15%) and minor pollen (≤3%) (Louveaux et al., 1978). The fre- quency occurrence of pollen, expressed as a percentage, was calculated per melliferous area. Jul/2011 (non-necta r flow) Apr/2012 (necta r flow) A.cerana No.1-9 No.19-26 A. mellifera No.10-18 No.27-34 Sociobiology 60(3): 289-294 (2013) 291 Results Honeys were sampled on July 2011(n=18) and April 2012(n=16). At July, there is not any nectar flow of plant in study area. April is nectar flow of Punica granatum and bee activity for honey production. Thus two different periods, co- vering the periods of non-nectar flow and main nectar flow of one plant, were representative of the two different species in trophic niche analysis of the study area. Honey produced by Apis cerana and Apis mellifera in non-nectar flow showed no significant difference (p>0.05). Honey from Apis cerana and Apis mellifera showed a sig- nificant difference in nectar flow of pomegranate flowers (p<0.05), and contained 8.13±1.46 and 6.00±1.77 (mean value ± standard deviation) pollen types, respectively. The number of pollen types are show in Table 2. ta of Myrtaceae is the most represented by Apis cerana, the next is Bidens pilosa. In nectar flow, the Punica granatum of Lythraceae is the main pollen in all samples from Apis melli- fera and Apis cerana, the next is Bidens pilosa (Apis cerana) and Eucalyptus robusta (Apis mellifera). As for the percentage of honey samples produced by Apis cerana and Apis mellifera in different months (Fig 1), the distribution of various pollen types percentage is balan- ced at non-nectar flow, the range of percentage (except minor pollen) is from 11.83% to 18.34% by Apis cerana and from 5.61% to 35.91% by Apis mellifera. In study areas, as soon as the pomegranate began flowering at the end of March, bees started to visit pomegranate flowers, thereby producing the pomegranate honey. At the nectar flow of Punica gra- natum, the percentage of Punica granatum pollen becomes important.However, there is still some other pollen types, like Bidens pilosa (14.59%) and Pisum sativum (12.43%) of Fabaceae sampled by Apis cerana, like Eucalyptus robusta (10.08%) and Rudbeckia laciniata (8.77%) of Asteraceae sampled by Apis mellifera. The range of percentage are from 3.24% to 40.32% by Apis cerana and from 4.05% to 66.82% by Apis mellifera. The distribution of pollen percentage is polarized (Fig. 2). non-nectar flow nectar flow Mean Range S.D. Mean Range S.D. A. cerana 7.44a 5-10 1.42 8.13a 6-11 1.46 A.s mellifera 7.56a 4-8 1.42 6b 4-9 1.77 Table 2 - The number of pollen types in honey samples by Apis cerana and Apis mellifera in different main nectar flow. Note: S.D.-standard deviation. Different letters in the same line in- dicates significant difference (p<0.05) A total of 17 pollen types from 34 honey samples were identified to belong to 14 families (Table 3). It is apparent that none of the pollen types in all samples. The most impor- tant families of pollen were Lythraceae, Asteraceae, Fabace- ae and Myrtaceae according to their percentage frequency. In non-nectar flow, the Bidens pilosa of Asteraceae is the most represented in the pollen of honey by Apis mellifera, the next is Trifolium repens of Fabaceae. And the Eucalyptus robus- Fig 1. Pollen type spectra in honey samples by Apis cerana and Apis melli- fera in different main nectar flow. Fig 2. Frequency of appearance of pollen types in samples by Apis cerana and Apis mellifera. The pollen types were classified into four cate- gories. The percentage of one same category is diffe- rent between Apis cerana and Apis mellifera through data analysis of all honey samples. Important minor pollen is the largest proportion in honey by Apis ce- rana (63%) and Apis mellifera (58%). Comparing the honey produced by Apis cerana and Apis mellifera in other three pollen categories, the percentage is a wide difference. The percentages of four categories are show in Fig 2. Through statistical analysis of all pollen infor- mation and trophic niche analysis, the Niche Breadth, Trophic niche overlap index and Interspecific compe- tition index were calculated to show the relationship of the two kinds of bees in ecology. The proportion of each resource sequence and calculated value are show in Table 4. The Niche breadth compared the different YJ Liu, TR Zhao, XW Zhang, C Liang, FY Zhao - Melittopalynology and Trophic niche analysis292 bee species within the same period. At non-nectar flow, the Niche breadth is 0.65 and 0.57, respectively. At nectar flow, the Niche breadth is 0.41 and 0.24, respectively. The Niche breadth of Apis cerana is greater than Apis mellifera at two different periods. The trophic niche overlap index and In- terspecific competition index compared the bees within two periods of non-nectar flow and nectar flow. The value of Tro- phic niche overlap index at nectar flow (0.68) is greater than that of non-nectar flow (0.61) and the value of Interspecific competition index at nectar flow (0.92) is also greater than non-nectar flow (0.77). Through the two values described above, the two species show stronger interspecific competi- tion at nectar flow. Table 4. Frequency of pollen types from samples of Apis cerana and Apis mellifera and trophic niche analysis. Discussion The present study provides new insights of the eco- logical relationship between two bee species by pollen com- position analysis. The honey samples used in this study were collected for drawing pollen atlases and knowing the main nectar plants at the study area. These studies will help the local beekeeping. The honey from the different bee species of non-nectar flow and nectar flow has their own characteristic. At non-nec- tar flow, they show 8 pollen types, but Eucalyptus robust is the maximum frequency in samples from Apis cerana, follo- wed by Bidens pilosa, Ligustrum lucidum, Trifolium repens and Zea mays. Bidens pilosa is the maximum frequency in samples from Apis mellifera, followed by Trifolium repens, Agave Americana and Leonurus japonicas. At nectar flow, they also show 8 pollen types and they have Punica grana- tum pollen as predominant pollen. The next is Bidens pilosa (Apis ceran) and Eucalyptus robusta (Apis mellifera), respec- tively. Different bee species show their preference of plants, the reasons for which may be that pheromone attract produced by plant to two bee species is different (Jennifer et al., 2009). As for the varying degrees, the number of pollen types in honey and the niche breadth of Apis cerana is greater than Apis mellifera. Whether nectar flow or not, the Trophic niche overlap index and Interspecific competition index all are sho-Ta bl e 3. P ol le n ty pe s id en tifi ed in 3 4 ho ne ys a na ly se d an d th ei r fr eq ue nc y cl as se s– r ef er ri ng to T ab le 1 . Fa m ily Po lle n ty pe 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 A ga va ce ae A ga ve A m er ic an a L. I M - - - M - - I I I S I M I I I - - - - - - - - - - - - - - - - - A st er ac ea e B id en s pi lo sa L. I I I S S S S S I P P S S S I S S S - I I S I I S S I I I I - I I - R ud be ck ia la ci ni at a L. - - - - - - - - - - - - - - - - - - I I I - I I I - I I - I I I I I Ta ra xa cu m m on go lic um h an d. -M az z. - - - - - - - - - - - - - - - - - - - - - - - - - - M - - M - - - - Fa ba ce ae Tr if ol iu m re pe ns L. I I S I I I I I I I I S - S P S I - I I - I I - - I M M - M M - - I Pi su m sa ti vu m L. - - - - - - - - - - - - - - - - - - I I S I S I I I M I I I I I I I G er an ia ce ae Pe la rg on iu m ho rt or um B ai le y M I - - - - I - - - - - - - - - - - - - - - - - - - - - - - - - - - La bi at ae El sh ol tz ia de ns a B en th . - - - - - - - - - - - - - - - - - - I I I S I I I I - - - - - - - - La m ia ce ae Le on ur us ar te m is ia (L au r.) S . Y. h u - - - - - - - - - I I I I I S I S - - - - - - - - - - - - - - - - - La ur ac ea e Ci nn am om um gl an du lif er um (W al l.) N ee s I I - - I - - I - - - - - - - M - - - - - - - - - - - - - - - - - - Le gu m in os ae M el ilo tu s al ba M ed ic . I S S S I I I I I I M I I I S I I S I M M - - - - I - - - - - - - - Ly th ra ce ae Pu ni ca gr an at um L. - - - - - - - - - - - - - - - - - - S S S S S S S P P P P P I P P P M al va ce ae M al va si ne ns is Ca v. - - - - - - - - - - - - - - - - - - M - M - - M - - M M - - - - - M M yr ta ce ae Eu ca ly pt us ro bu st a Sm it h S S - S S S S S S I I I I I I M I - I I I I I I I I I I I I I I I I O le ac ea e Li gu st ru m lu ci du m A it . I I S S I S S I S I M M I I I S I I - - - - - - - - - - - - - - - - Po ac ea e Ze a m ay s L. S I I I I I I I I M M M I M I I S S M I - M - I - I M - - M - - - - Ve rb en ac ea e Ve rb en a of fi ci na lis L. - I - I - - I - - - - - - - - - - - M - - - M - - - - - - - - - - - Family Pi (non-nectar flow) Pi (nectar flow) Apis cerana Apis mellifera Apis cerana Apis mellifera Agavaceae Agave americana L. 1.92 9.60 — — Asteraceae Bidens pilosa L. 18.34 35.91 14.59 4.05 Rudbeckia laciniata L. — — 4.65 8.77 Taraxacum mongolicum hand.-Mazz. — — — 0.28 Fabaceae Trifolium repens L. 11.83 15.71 3.24 1.60 Pisum sativum L. — — 12.43 6.97 Geraniaceae Pelargonium hortorum Bailey 0.89 — — — Labiatae Elsholtzia densa Benth. — — 10.38 — Lamiaceae Leonurus artemisia (Laur.) S. Y. hu — 9.60 — — Lauraceae Cinnamomum glanduliferum (Wall.) Nees 2.37 0.25 — — Leguminosae Melilotus alba Medic. 12.87 9.10 1.19 — Lythraceae Punica granatum L. — — 40.32 66.82 Malvaceae Malva sinensis Cav. — — 0.43 0.75 Myrtaceae Eucalyptus robusta Smith 21.89 8.10 9.62 10.08 Oleaceae Ligustrum lucidum Ait. 16.12 6.11 — — Poaceae Zea mays L. 11.83 5.61 2.92 0.66 Verbenaceae Verbena officinalis L. 1.92 — 0.22 — Niche Breadth 0.65 0.57 0.41 0.24 Trophic niche overlap index 0.61 0.68 Interspecific competition index 0.77 0.92 Sociobiology 60(3): 289-294 (2013) 293 wed that two bee species exist in strong interspecific compe- tition from each other. Bees pollinate most of the world's wild plant species and provide economically valuable pollination services to crops. The western honeybee, Apis mellifera, has been introduced to many parts of the world and sometimes purported to be detrimental to native bees because it reduces their food base(Forup & Memmott, 2005). Except the com- petition in food, there are some more direct competition, for example, Apis mellifera may stolen the honey from hives of Apis cerana sometimes, and Apis mellifera drone chase and attempt to mate with Apis cerana virgin queen caused mating interferes to drone and virgin queen of Apis cerana (Wang et al., 2003). Certainly, we have to face the fact that some plants which mainly depend on Apis cerana for pollination is becoming less with the reduction of Apis cerana. Some researchers have started to pay attention to as- sess the ecological impact of alien bee species on native bee by building various niche models (Villanueva and Roubik, 2004; Franco et al., 2009; Villemant et al., 2011). The alien invasive species, some are not the hopes of the people but some are introduced because of its high production capacity. Apis mellifera, which is really good at production of honey, have been introduced to China. 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