Morphometric Characterization of a Population of Tetrapedia diversipes in Restricted Areas in Bahia, Brazil (Hymenoptera: Apidae)

Tetrapedia species are solitary bees which collect floral oils, being restricted to tropical regions of the Americas. Information on forms of nesting has been little researched in the literature, requiring studies on the diversity and variability of species to obtain better management and conservation strategies for their populations. Morphometry is a efficient technique and has been used to detect variation and for identification of species of bees in order to detect changes in quantitative traits within and among populations of bees. This study aimed to compare the variability of the population of Tetrapedia diversipes in artificial nests located in orchards and their surroundings (other fruit) of acerola in a restricted area of the Reconcavo region of Bahia, Brazil. Right wings were extracted from 155 individuals of the T. diversipes species, to perform the morphometric analysis. In conventional morphometry, 9 variables contributed significantly to the sexual dimorphism in the study areas (α = 0.05). The geometric morphometric analysis revealed low gene flow in populations of T. diversipes demonstrating loss of genetic diversity, requiring proper management of this bee for its conservation and maintenance of the associated flora.


INTRODUCTION
Solitary bees represent approximately 330 species in the Neotropical region (Michener 2000) including the genus Tetrapedia (Tetrapediini) with 18 described species in Brazil (Silveira et al. 2002), presenting different morphological and behavioral characters in their nesting (Cordeiro et al. 2010).
The Tetrapedia species use preexisting cavities as nesting sites which allows the sampling of these bees by means of trap-nests or artificial nests (Camillo 2005), and this solitary behavior is another hallmark of the Tetrapediini tribe characterized by independence of the female in the construction and supply their nest (Alves-dos-Santos et al. 2007).
The study of oil-collecting bees and their importance in the pollination of many plant groups is an important tool to contribute to the knowledge of their ecology, economic importance (Imperatriz-Fonseca 2010) and morphological variation of bee populations in their natural environment (Francoy & Imperatriz-Fonseca 2010). Research for the evaluation of morphological diversity is important in ecological and genetic studies, and one of the techniques used in these studies are morphometric tools (Rohlf 1990).
In morphometric analysis variation is studied through the covariation between pairs of linear measurements, and geometric morphometry is able to more clearly describe and locate the regions of changes and rebuild and reconstruct these differences graphically (Francoy et al. 2011).
Morphometry has been widely applied in many studies with social bees using tools to detect or describe genetic patterns in colonies, species identification, geographical variations and phylogenetic relationships among and within populations of bees (Peruquetti 2003, Mendes et al. 2007Nunes et al. 2008, Souza et al. 2009, Carvalho et al. 2011Ferreira et al. 2011;Francoy et al. 2011, Souza et al. 2010, and variability in shape and size of wings of social bees Quezada-Euán et al. 2007). However, there are few morphometric studies on solitary bees (Bosch 2008), with the exception of a study on the patterns of wing venation and geographical differences between populations of the Centris genus (Ferreira et al. 2011) and one study on the morphology of Tetrapedia diversipes applying linear morphometric analysis to length and width of the wings, head width and cephalic and mouth appendages (Smith et al. 2011).
In this context, considering the importance of morphometric studies in populations of bees, it is necessary to collect data on solitary bees and offer suggestions for future research aimed at the morphological variability within and among populations, diversity and biology of nesting. The study aimed to compare the degree the morphological variability of Tetrapedia diversipes residing in artificial nests located in orchards and their surroundings (other fruit) of acerola in a restricted area in the Bahia Reconcavo region, Brazil.

MATERIAL AND METHODS
The experiment was conducted in Embrapa (Brazilian Agricultural Research Corporation) Cassava and Tropical Fruits in the period between March/2008 and August/2009 in the Municipality of Cruz das Almas (12 ° 40'12 "S, 39 º 06'07" W, 220 m), located in the Reconcavo of Bahia, Brazil. The nests were installed in four areas: Area I -Active Germplasm Bank of Malpighia emarginata DC; Area II -Other Orchards (Citrus spp., Spondias sp., Musa spp. and Mangifera indica); Area III -Transition area (the area between the forest and other orchards) and Area IV -Forest Fragment. The artificial nests were made of colored paper timber of 5 mm in diameter with a length of 15 cm (Machado 2011), and the species collected from the Tetrapediini tribe was Tetrapedia diversipes.
The sealed nests were transferred to the Laboratory of the Center for the Study of Insects (INSECTA) of the Federal University of Bahia Reconcavo (UFRB), and placed in BOD (Biologic Oxygen Demand) chambers at a temperature of 25 ± 1 ° C, humidity 80% ± 1% with a photoperiod of 12 hours. Right wings of T. diversipes were removed and placed between two plates to capture images using the program Motic 2.0 ML with a digital camera coupled to a stereomicroscope, with 7.5 X magnification (Carvalho et al. 2011). The veins and cells wings were classified according to the methodology described by Silveira et al. (2002), and biological material has been identified, and stored in Eppendorf tubes of 1.5 mL and deposited in the collection of the Laboratory of the Center for the Study of Insects (Núcleo de Estudos dos Insetos-INSECTA), at the Center of Agricultural, Environmental and Biological Sciences (Centro de Ciências Agrárias, Ambientais e Biológicas) of the Federal University of Bahia Reconcavo (UFRB).

Geometric Morphometric Analysis
18 landmarks were defined and recorded using intersections of the veins of the forewings of Tetrapedia diversipes specimens ( Fig. 1) using software tpsDig2 version 2.12 (Rohlf 2008a). The alignment of the x and y coordinates of each line of the wings (alx aly) and the centroid size of the wings can be used as a data matrix (matrix W) in order to perform multivariate analysis (Rohlf 2008b).
The coordinates of the landmarks of the wings and centroid size were used in principal component analysis (PCA), the matrix W was used in multivariate analysis of variance (MANOVA) and cluster analysis by UPGMA (Unweighted Pair-Group Method using Arithmetic Average).

Conventional Morphometric Analysis
In this analysis we used 24 linear measurements in the right wings (Figs. 2, 3) of 155 individuals measured by Motic software 2.0 ML. Measurements of the veins and cells were applied according to the methodology of de Souza et al. (2010), which used 17 measures on the forewing (length -C and Width -L of the wing, width and length of 1st, 2nd and 3rd submarginal cells, and 1st and 2nd Medial cells (M), length of the Subcostal veins + Radial (Sc + R) and Anal-1 vein; length of the cells: Marginal (M) and the 2nd cubital (Cu); distance from the intersection of the Anal-1 vein with Cubital-anal (Cu-a) until the intersection of the Cubital vein with 1st Middle-Cubital (m-Cu) and the hindwings with 7 measurements (length and width of the wing; length of the ribs sector Radial (R), Anal-1 and Cubital-anal veins; distance from the intersection of the rib Anal -1 with cubital-anal until the intersection of the median-Radial sector (Rs-m) with the Radial sector (Rs) and Radial (R) cell width. The differences between the sexes of the individuals of the species T. diversipes were analyzed by multivariate variance (MANOVA), canonical variables (AVC), principal components (PCA) and cluster analysis by the UPGMA method.

Conventional Morphometrics
For the multivariate analysis of variance (MANOVA), there was a significant difference between groups of individuals (Wilk's λ = 0.65109, p <0.00001), with only 10 variables contributing significantly to the separation of individuals (α = 0.05), confirming the existence of sexual dimorphism of this species in relation to the size of the wings. This separation is represented graphically by principal component analysis (Fig. 4).
In principal component analysis (PCA) performed to test the existence of sexual dimorphism of T. diversipes the first three variables explained 74.40% of the variation, with the first component explained 63.28%, the second and the third 6.70%, and 4.42% (Table 1), and the variables that contributed most to the first component were length and width of forewings (Table 2). To evaluate the differences between the areas another PCA was performed (Fig. 5), where an overposition between the three areas of the population occurred, demonstrating the existence of low morphometric divergence between individuals of the three areas. This can be explained by the vicinity of the sampled areas, which contributes to gene flow between the population and therefore, maintains a similar phenotypic pattern.
The analysis of canonical variables showed morphometric differences between individuals in Area I and II, and area III formed a group with characteristics that are similar to areas I and II (Fig. 6). Multivariate analysis of variance (MANOVA) for the three areas showed a significant difference between individuals collected in areas I, II and III (Wilk's λ = 0.57864, p <0.00001), where only 9 variables corresponding to the forewings contributed significantly to the separation of individuals in each area (α = 0.05).
Considering the Mahalanobis D 2 distances (Table 3) between the areas, it appears that individuals of the areas I (Active Germplasm Bank of M. emar- ginata) and II (Other Orchards) showed highly significant morphological differences (p <0.00001). However, comparing areas I and III (Transition area: between the forest and other orchards), and II and III there was no significant difference between the total sample, demonstrating morphometric similarity, which can be attributed to the similarity of available resources between these areas.      Ferreira et al. (2011) studied the degree of differentiation of populations in areas of Centris aenea in acerola orchards, where they observed significant morphometric differences between individuals, corroborating the present study. It is very important to consider that there are fluctuations of trophic resources over time, and the phenotypic expression of the bees may vary according to availability of resources.

Geometric Morphometrics
Multivariate analysis of variance (MANOVA) with the matrix W to observe the difference between the areas, also found significant differences (Wilk's λ = 0.459, p <0.0001) between individuals of T. diversipes collected in the three areas, and all variables of the matrix W contributed significantly (α = 0.05) to discriminate between the groups in the areas.
With the triangular matrix of Mahalanobis D 2 distances from the data coming from the matrix W, we found again significant differences between area I and areas I and II. However, among areas I and III, there was no significant difference (Table 4). Ghaderi et al. (1984) found that when several characteristics are analyzed at once, the squared Mahalanobis distances can be used as estimates of genetic diversity among the groups or areas where this variability is the result of morpho-physiological characteristics and ecological differences.
The analysis of canonical variables produced a difference between individuals in areas I, II and III (Fig. 7). The individuals of T. diversipes in areas I and III showed similar morphometrics, so there is probably a greater genetic interaction between the bees in these two areas. This similarity can be attributed to a low level of gene flow, with the assumption of the occurrence of inbreeding. According to Breda et al. (2004) inbreeding occurs among individuals related by descent, or is the union of individuals more closely related than the average population, increasing the homozygosity and phenotypic effect of expressing the recessive genes. It is possible that the fragmentation of the environment caused by anthropogenic activities and is mainly favoring the isolation of the population of T. diversipes, increasing the rate of inbreeding, weakening the genetic diversity and therefore favoring the disappearance of bees in these areas. Similar data were found by Mendes et al. (2007). Analyzing the population Nannotrigona testaceicornis collected in two urban areas and an area of natural vegetation, they found that in urban areas there were more similarities when compared with areas of natural vegetation, affecting gene flow of these bees. However, Gonçalves (2010) noted that the geometric morphometry of wings was not sensitive enough to separate samples of Frisieomelita varies in natural and disturbed areas and diverged from the present work.
The conservation of native flora and reducing human disturbance around natural and agricultural areas can contribute to the maintenance of intrapopulation genetic material of T. diversipes, increasing their genetic variability and ensuring survival, demonstrating the need for further studies on the evolutionary variations of this species.

CONCLUSION
A conventional morphometric analysis revealed the existence of sexual dimorphism of the species Tetrapedia diversipes. However, geometric mor-phometrics indicated the existence of low levels of gene flow, facilitating the isolation and weakening of the genetic diversity in the population. The study of morphometric characterization has provided important data on the degree of intrapopulational differentiation of T. diversipes, facilitating future research on other species of Tetrapedia, supporting strategies for the conservation and maintenance of this bee.