Variation of fore wing shape in Melipona mandacaia Smith , 1863 ( Hymenoptera , Meliponini ) along its geographic range

Melipona mandacaia is a stingless bee species responsible for the pollination of many native plants in Brazil, South America. In spite of its ecological and economic importance, natural populations of M. mandacaia have been depleted because of deforestation. In order to evaluate the interpopulation morphometric structure of remaining populations, we carried out geometric morphometric studies based on fore wing shape in this native bee species. The grouping analysis by UPGMA revealed three distinct clusters and significant differences in fore wing size were observed (p<0.001) among populations. The three groups were also reflected in the first two principal components explaining about 60% of the total variation. These results indicate differentiation among populations, which can be regarded as unique management units. Therefore, efforts should be directed to the conservation of local populations of M. mandacaia to avoid the negative impacts of loss in pollination over plant species and environmental services. JHR 48: 85–94 (2016) doi: 10.3897/JHR.48.6619 http://jhr.pensoft.net Copyright Arlete Prado-Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Arlete Prado-Silva et al. / Journal of Hymenoptera Research 48: 85–94 (2016) 86


Variation of fore wing shape in Melipona mandacaia
Smith, 1863 (Hymenoptera, Meliponini) along its geographic range

Introduction
Stingless bees play a key role in natural and agricultural systems (Eardley et al. 2006) as they account for about 30% to 90% of pollination in native flora (Kerr 1997).Besides, these bees produce high-quality honey, propolis, pollen and wax used as food sources and/or as pharmaceutical products.However, human activities, such as deforestation and habitat loss, are a potential threat to these insects, leading to population declines (Fortuna and Bascompte 2006).
In Brazil, the stingless bees of the tribe Meliponini encompass most of native social bee taxa, in which Melipona is the species-richest genus (Michener 2007, Camargo andPedro 2013).Melipona mandacaia Smith, 1863 is an endemic bee species from semiarid regions in northeastern Brazil, including one of the poorest Brazilian regions, named the "drought polygon".Being typical of the caatinga (dry bushland) biome, M. mandacaia is well adapted to high temperatures and reduced rainfall conditions (Carvalho et al. 2003, Batalha-Filho et al. 2011).The nests of this species are built in tree holes (Camara et al. 2004) and some populations have been raised in boxes by local farmers for honey production, representing a major portion of economy and food in traditional agriculture (Carvalho et al. 2003).
In fact, morphometric analyses based on landmarks allow evaluating homologous morphological features that are independent of size (Francoy and Fonseca 2010).Furthermore, the geometric morphometrics of wings have been useful to establish the geographic origin of individuals of a single species (Bischoff et al. 2009, Nunes et al. 2012).
In spite of being a precise and simple technique to assess population diversity (Adams et al. 2004), no geometric morphometrics reports are available in M. mandacaia.Therefore, we focused on analyzing morphogenetic traits in populations of M. mandacaia in order to examine how populations are structured throughout their range.

Material and methods
The samples were collected in 15 localities (3-26 colonies per collection site) along the semiarid regions of the state of Bahia, northeastern Brazil.Right after collection, the specimens were stored in absolute ethanol for further morphometric analyses.The right fore wings were removed from 9 to 13 adult workers per colony, totaling 127 colonies and 1199 specimens (Table 1).Afterwards, they were placed onto glass slides and photographed using a stereomicroscope equipped with an image capture system (Leica Application Suite version 3.4.1).
Based on high-resolution images, two dimensional landmarks were digitized with the software TPSDIG2 (Rohlf 2006).A total of 12 landmarks were recorded in each fore wing for morphometric analyses (Figure 1).
Based on Cartesian coordinates, Procrustes superimposition and Principal Component Analysis (PCA) were performed using the software MORPHOJ version 2.0 (Klingenberg 2011) based on mean values of colonies.Subsequently, a cluster analysis using UPGMA (Unweighted Pair-Group Method with Arithmetic Average) was carried out with the software Past (Paleontological Statistics) version 1.81 (Hamer 2008).The cophenetic correlation coefficient was also calculated using Past.
A variance analysis (ANOVA) followed by a Tukey test was performed to evaluate centroid size.Shape and size of fore wings from each population were compared to altitude and geographic distance of colonies via a Mantel test in the software NTSYS version 2.02 (Rohlf 1993).

Results
The first two principal components explained about 60% of the variation in fore wing shape.Three groups were distinguished according to collection site in PCA, as follows: 1 -Pilão Arcado, Remanso and Casa Nova; 2 -Juazeiro, Macururé and Paulo Afonso; and 3 -Ourolândia, Morro do Chapéu, Serra do Ramalho, São Gabriel, Morpará, Itaguaçu da Bahia, Muquém de São Francisco, Uibaí and Hidrolândia (Figure 2).Similarly, the dendrogram based on UPGMA (Figure 3) revealed three distinct clusters.It should be pointed out that the third group is composed of colonies from both sides of the São Francisco river (Serra do Ramalho and Muquém de São Francisco are located on the left margin while Morro do Chapéu, São Gabriel, Morpará, Itaguaçu da Bahia, Ourolândia, Uibaí and Hidrolândia are located on right margin of São Francisco River) with a cophenetic correlation coefficient of 84% (Figure 4).
Fore wing size was significantly different among colonies from distinct collection sites (p<0.001)(Figure 5) by the Tukey test.However, the fore wing shape was more informative in discriminating the groups than size, thus demonstrating the efficiency of geometric morphometrics.The Mantel test revealed no correlation between both size x altitude and shape x altitude (p>0.05)(Table 2).On the other hand, both shape (p<0.05) and size (p<0.001)were weakly but significantly correlated to geographic distance in M. mandacaia.

Discussion
The present analyses indicated gene flow among individuals from the three morphogenetic groups of M. mandacaia (Figure 2) inasmuch as populations are weakly structured.This result suggests that São Francisco River should not act as an effective geographic barrier to the dispersal of individuals from both river margins.
Similarly, molecular markers revealed a moderate genetic structuring (ΦST = 0.2961) among these populations (Miranda et al. 2012)  reservoir (about 320 km of extension and a water surface of 4.214 km²) seems to disrupt gene flow between groups 1 and 2, as identified in this study (Figure 4).Thus, the construction of dams related to human disturbance seems to be responsible for the population structure associated with decreased genetic variation and high inbreeding in colonies of M. mandacaia.A similar result was reported in Apis mellifera L. (Miguel et al. 2007) and rodents (Calomys expulsus Lund, 1841) (Nascimento et al. 2011).Additional support to this hypothesis is that the Sobradinho dam was built about 40 years ago.This relatively long period is compatible with, at least, 10 generations of queen bees that could potentially lead to both fixation and losses of alleles (i.e.reduced genetic variation).Moreover, previous reports have shown that fore wing size and shape of some Melipona species are influenced by both geographic distance and altitude, like that observed in M. scutellaris Latreille (Nunes et al. 2007), M. quadrifasciata anthidioides Lepeletier (Nunes et al. 2013) and M. subnitida Duke (Lima et al. 2014).Nonetheless, no significant morphometric differences were observed in M. mandacaia in relation to altitude (p>0.05),but both fore wing size and shape were correlated to geographic distance (p<0.001 and p<0.05, respectively).This isolation-by-distance model in M. mandacaia can be explained by the eusocial behavior of these bees and their short flight range (about 2,000 m) (Araújo et al. 2004), causing restricted gene flow among populations.
Therefore, the morphometric analysis based on fore wingsis useful to evaluate the interpopulation genetic divergence of M. mandacaia, once they seem to be less susceptible to environmental influence.In this sense, studies of geometric morphometrics are useful to the identification of variation in populations and species of bees (Oleksa and Tofilski 2015) that are essential to conservation plans.

Figure 2 .Figure 3 .
Figure 2. Analysis of Principal Components and Minimum Spanning Tree of fore wings of Melipona mandacaia.

Figure 4 .
Figure 4. Map of Bahia, in Brazil, with sampling sites of Melipona mandacaia.

Figure 5 .
Figure 5. Boxplot of fore wing centroid size of Melipona mandacaia and comparison of mean values by a Tukey test (same letters represent no significant statistical differences).
Sampling sites of Melipona mandacaia with altitude, geographic coordinates and number of samples colonies and specimens. Table1.

Table 2 .
Comparison between matrixes of geographic distances, altitude and shape and size of fore wings using Mantel's test with 5000 permutations.
. Only the Sobradinho