OUTLINE BASED GEOMETRIC MORPHOMETRIC ANALYSIS OF

WING SHAPE AND SIZE IN SELECTED SPECIES OF BEES

(Apis sp. and Bombus sp.)

Ray Vincent E. Araña

rayvincentarana@gmail.com

INTRODUCTION

The complex shape of an organism cannot easily be summarized by using linear

measurements as in traditional morphometrics (Pavlinov 2001). Because these measurements are

highly correlated with size, much effort was spent for size correction (Zelditch et al, 2004). But

there is no consensus on different size correction methods and several difficulties remain. For

instance, the homologies of linear distances are difficult to assess and the same set of distance

measures can be obtained from totally different shapes (Zelditch et al, 2004). In general, it

is not feasible to generate graphical representations of shapes from the linear distances (Adams

et al, 2004).

Morphological features are an important source of information for many areas of

biological investigation, including systematics and taxonomy. Most studies in these areas are

done using meristic and morphometric characteristics. Meristic characteristics are generally

countable and informative for species, genera and higher taxonomic levels. However, in

interspecific and populational studies, these measurements are not informative and it is therefore

necessary to obtain information on morphometric characteristics (Bookstein et al, 1985). These

characteristics are generally quantitative phenotypic values obtained from continuous

measurements and ratios in which classes are often defined based on means and standard

deviation (Prado et al, 2006). On the other hand, morphometric characteristics normally use

general morphometric analysis based on measurements such as distances or angles (Bitner-

Mathe & Klaczko, 1999; Bookstein et al, 1985; Klaczko & Bitner-Mathe, 1990; Moraes et al,

2004; O’Higgins, 2000; Stock & Kaya, 1996; Timm & Genoways, 2004; Ventura et al, 2000;

cited in Prado et al, 2006) These values are obtained from the distance between homologous

points or landmarks of a particular structure (Rohlf, 1993). The values generated can then be

used to quantify morphological variation. The efficiency of this approach depends on the

existence and precise definition of landmarks for these structures and on the association of these

homologous points with morphological variation. Since landmarks are not always easy to find,

alternative descriptive algorithms that allow the analysis of morphometric variation without

defining homologous points are being increasingly used. Such algorithms include methods based

on outline data in which the form (shape + size) of the structure is obtained from its contour

(Rohlf & Marcus, 1993).

The studies conducted by Cesar Jr. and Costa (1996) and Costa and Cesar Jr. (2000)

proposed the use of continuous curvature as an alternative method for analyzing morphometric

characteristics, without the need for landmarks. Continuous curvature allows an analysis of the

shape of the object and the length of its arc, both of which are related to the size of the object.

Curvature is a particularly important geometric measurement that expresses the rate of change of

the angle between the tangent to the curve and the x-axis (Rohlf & Marcus, 1993. This

measurement can be used to characterize a curve, with low values of curvature along a portion of

the curve indicating a straight region whereas abrupt variation in the curvature corresponds to a

vertex. In addition, concavity along the curve can be determined by the sine of the curvature.

Two important additional properties of curvature as a geometric measurement are that the

original curve can be reconstructed from the curvature values and are invariant for translation

and rotation (Prado et al, 2006).

The morphometric analysis of insect wings, principally in the Diptera, has been used to

define relationships between closely related taxa (Brown and Shipp, 1978; Rohlf and Archie,

1984; cited by Moraes et al, 2004). Investigations of insect wing architecture can reveal patterns

in both flight performance and evolutionary history (Wootton, 1992, cited by Maglinte, 2006).

Despite their structural simplicity, fly wings are an excellent system for studying morphological

variation (Klingenberg, 2002, cited by Moraes et al, 2004). Because wings are solid or rigidly

articulated structures they have become very useful tools for geometric morphometric studies

(Pavlinov, 2001).

In this work, the variation in wing shape and size of selected species of bees (Apis sp. and

Bombus sp.) was described and explored. Sexual dimorphism based on shape and size was also

examined with both left and right forewings and hind wings as characters.

METHODOLOGY

Samples

One hundred fifteen (115) bee species; fifty-six Apis sp. (40 females & 16 males), fifty-

nine Bombus sp. (33 females & 26 males) were used in this study. The wings were mounted on

slides and scanned images (1200 dpi) were obtained (Figs. 1 and 2). One hundred-twenty outline

points were then digitized on each of the wings of the two bee species. Wings from male and

female bees of each species were analyzed separately.

Data Analysis

The software PAST version 1.89 was used as platform for the analysis (Hammer et al,

2001). The relative warp scores and centroid size were obtained to verify the shape and size

variations respectively, within sexes. Differences in size among the two bee species were

determined using Kruskal-Wallis test performed on the PC scores and visualized using box plots.

Scatter-plot was used in the determination of the differences in shape.

Figure 1A. Left and right forewing of Apis sp.

Figure 1B. Left and right hind wing of Apis sp.

Figure 2A. Left and right forewing of Bombus sp.

Figure 2B. Left and right hind wing of Bombus sp.

RESULTS AND DISCUSSION

There are one hundred-fifteen (115) species of bees, fifty-six Apis sp. (40 females & 16

males) and fifty-nine Bombus p. (33 females & 26 males) were examined in this study.

The coordinate data gathered from the samples collected was subjected to different computer

software and programs for analysis. In this case, Kruskal-Wallis test and scatter plot were used in

determining the variation in size and shape of selected species of bees respectively, using an

outline method.

Case Study 1: Within Species Morphological Variation of Bees Based on Wing Shapes

Shape as defined in mathematics is a set of all features of a configuration of landmarks

excluding size, position, and orientation relative to an original coordinate system. Even if body

size, including wing size, is highly variable among individuals of the same species, wing shape

appears more stable. The shape is generally believed to be more stable, submitted to strong

developmental and phylogenetic constraints, so that significant variations are expected mostly

among species, and is often used as basis for taxonomic classification (Sturtevant, 1942). In

opposition to this hypothesis, recent empirical observations have shown that variations in shapes

within species, has a genetic basis (Huey, 2000; Gilchrist and Partridge, 2001).

The advent of geometric morphometrics opened an arena to study shape variations in

organisms. Most studies using geometric morphometrics characterize shape given a set of

landmark data – X and Y coordinated of points that can be located unambiguously on a

specimen.

A. Apis sp.

The wing shape diversity can be measured based on how dispersed the fitted outline

points are. Mean shapes of each sex are shown in Figure 3 for both left and right forewing and

hind wing. Visual inspection of the scatter plots in Figure 4 revealed less wing shape diversity

among individuals for both sexes. There is an overlapping between male and female individuals

based on their forewings and hind wings suggesting minimal variation and absence of sex

dimorphism.

Figure 3. Mean shapes of both sexes of Apis sp. (a) left and right fore/front wings (b) left and

right hind wings

Figure 4. Scatter plot diagrams of both sexes of Apis sp. (females are labelled red, males are

labelled blue) (a) left forewing, (b) right forewing, (c) left hind wing, (d) right hind wing

Table 1 shows the results for PCA-number of significant components and their

corresponding percent contribution to the shape variation on the forewings and hind wings of

both sexes of Apis sp. Fifteen (15) significant components contributed to the variation of the left

and right forewing and hind wing of both sexes of Apis sp.

Table 1. Proportion of variation associated with the significant components using the Procrustes-

fitted coeeficients on the forewings and hind wings of both sexes of Apis sp.

B. Bombus sp.

Individuals of Bombus sp. differ less with respect to shape as seen on the mean shapes

(Figure 5) and scatter plot diagrams (Figure 6). Overlapping of individuals is visible on both

wings in both sexes. Majority of the plot shows overlapping of individuals which indicates that

there is absence of dimorphism in Bombus sp.

Significant components of each wing in both sexes that contribute to variation are shown

in table 2. Each of the four wings has component 1 with the highest value of % variance and

Eigenvalue. There are 23 components in the left forewing while 25 components each are present

in the right forewing, left hind wing, and right hind wing. These numerous significant

components indicate that variations are contributed by many outline points.

Figure 5. Mean shapes of both sexes of Bombus sp. (a) left and right fore/front wings (b) left and

right hind wings

Figure 6. Scatter plot diagrams of both sexes of Bombus sp. (females are labelled red, males are

labelled blue) (a) left forewing, (b) right forewing, (c) left hind wing, (d) right hind wing

Table 2. Proportion of variation associated with the significant components using the Procrustes-

fitted coeeficients on the forewings and hind wings of both sexes of Bombus sp.

Case Study 2: Within Species Morphological Variation of Bees Based on Centroid Sizes of

the Wings.

There is a simplistic view, not uncommon in behavioral ecology that bigger is always

better. This statement is supported by Sokolovska in 2000 that there is general fitness benefit to

larger size in odonates. Anderson (1994) found that large size was advantageous to male fitness

in 51 cases and to female fitness in 27 cases. Absent notably, however, were quantitative studies

indicating a small size advantage or those demonstrating stabilizing selection with respect to

size. Though Anderson admitted that in some cases sexual selection may be self-limiting,

stabilizing selection on size in relation to fitness has rarely been considered (Thornbill and

Aloeck, 1993; Arnqvist et al, 1996; Sokolovska et al, 2000). In fact, stabilizing selection on body

size with respect to measure of lifetime mating success has been demonstrated only for three

species of insect, all of which are odonates.

Though constructing separate variables for general size and for shape variation, the

centroid size was used as a measure of wing size and was extracted from the set of coordinates.

Each individual centroid size was calculated and was used as a variable to various statistical test.

Box plots of centroid sizes of the wings were constructed to detect wing size differences.

A. Apis sp.

Shown on Figure 7 are the box plots of the male and female sexes of Apis sp. There were

a number of 16 males and 40 females tested. Based on the obtained results, size maybe regarded

as a basis for sex determination and that wing size is relatively a sexual dimorphic trait among

Apis species. The size of the left and right forewing of male individuals is larger than female

individuals. There is only slight difference in size of the left and right hind wings of female

and male Apis sp.

Another test was considered to further justify the results achieved in the box plots.

Kruskal-Wallis test (Table 3) was carried out to provide a basis on how significant the results are

and how identical the means of the two groups are. F

_LF

W

M_LF

W

F_R

FW

M_R

FW

1050

1075

1100

1125

1150

1175

Centr

oid

Siz

e

F_LH

W

M_LH

W

F_R

HW

M_R

HW

690

720

750

780

810

840

Centr

oid

Siz

e

(a) (b)

Figure 7. Box plot showing the centroid size differences between males and females in

(a) forewings and (b) hind wings of Apis sp.

Table 3. Kruskal-Wallis Test

(a) Forewings (b) Hind wings

B. Bombus sp.

A number of 33 females and 26 males of Bombus sp. are tested to determine wing size

variation. Results obtained are parallel with the Apis sp., as shown in Figure 7, that male

individuals have larger forewings compared to females. Slight variation was observed for both

sexes in terms of hind wings, which indicates that both individuals exhibit common size

characteristic and absence of dimorphism. Thus, size maybe considered too as a basis for sex

determination and that wing size is somewhat a sexual dimorphic trait among Bombus species.

Kruskal-Wallist test (Table 4) was done to further prove the result attained in the box

plots.

F_LF

W

M_LF

W

F_R

FW

M_R

FW

1600

1700

1800

1900

2000

2100

Centr

oid

Siz

e

F_LH

W

M_LH

W

F_R

HW

M_R

HW

1020

1080

1140

1200

1260

1320

1380

Centr

oid

Siz

e

(a) (b)

Figure 7. Box plot showing the centroid size differences between males and females in

(a) forewings and (b) hind wings of Bombus sp.

Table 4. Kruskal-Wallis Test

(a) Forewings (b) Hind wings

The description of patterns of variations in morphological shape and size within and

among populations is fundamental for defining the boundaries of independent evolutionary units

in nature, and an important step in the recognition of such evolutionary units is the identification

of groups of populations that share morphological features of shape and geographic continuity

over geographic space. In this study, the methodology of morphometrics were used against a

background of the philosophy and practical application of various methods, and evaluate the

competence of these tools to interpret the morphological complexities that are the product of

biological processes. Outline-based geometric morphometrics were used to quantify size and

shape variation, to assess wing variation of bees that is used to investigate the occurrence of

sexual dimorphism.

The observed variations as shown in the results are not enough to conclude that

dimorphism is present. There is major overlapping of individuals as depicted in the scatter plot,

which means that the variations examined has minimal contribution on the wing variation of

individuals and that commonness of male and female individual for both bee species is

preserved.

The trend and extent of sexual differences in body size vary greatly among different

animal taxa (Andersson, 1994; Nylin and Wedell, 1994). The body size of insects is a general

trait that can be related to ecological, behavioural, or evolutionary patterns. Commonly a

dimorphism in body size between males and females is exhibited in insects, with the females

having a larger body size in most species. This pattern is mainly attributed to egg production in

the females. The fecundity advantage hypothesis (Andersson, 1994) suggests that large females

have a higher fecundity (Arnqvist et al., 1997; Bateman, 1998). Yet, as a deviation from this

general rule, larger males or males with prolonged organs (e.g. antennae, extensions of the head

or pronotum) occur in some species (Hanks et al., 1996; Preziosi and Fairbairn, 1996; Kawano,

1997; Panhuis and Wilkinson, 1999). However in this study, wherein the occurrence of sexual

dimorphism in shape and size based on wing variation, it was found out those wings of bees,

Apis sp. and Bombus sp., were not sexually dimorphic in shape and size, be it forewing or hind

wing after subjected to various geometric morphometric analyses. This may be due to the fact

that sexual dimorphism usually occurs on sexual characters of an insect (e.g. abdomen and eyes)

and that wings are non-sexual character. The sexual trait thus function directly in males sexual

competition, and are probable targets of sexual selection in both species, whereas the non-sexual

trait do not function directly in male sexual competition, and are probably not subject to strong

or direct selection (Bonduriansky and Rowe, 2003). Body size strongly influences animal flight

performance (Elington, 1991; Norberg, 1990; Dudley, 2000) and in this study, bees that are

being examined have minimal difference in their body size, and as we know wing shape and size

are associated with body size. Hence, the forewings and hind wings of bees are not sexually

dimorphic as observed.

CONCLUSION AND RECOMMENDATIONS

This study was conducted to describe and explore the variation in wing shape and size

within species of Apis sp. and Bombus sp., and to examine the incidence of sexual dimorphism

on shape and size. The disparity of these characters was investigated for both left and right

forewings and hind wings using geometric morphometric analysis.

There is absence of sexual dimorphism within species of Apis sp. and Bombus sp. as

shown on the results wherein there is overlapping of individuals. The species shared

commonness in their structure although there are variations in their wing shape.

The study also distinguished that the possession of a larger forewing over the hind wing

is common in both sexes of Apis sp. and Bombus sp. This wing arrangement is assumed to

provide the insect proper maneuvering and flight. The left and right forewing and hind wing of

these species also exhibit varying patterns of asymmetry. Further studies should be geared

toward understanding variations in other flight morphologies, such as body size, length and

weight. Larger sample size is also recommended for future investigation to have more precise

result in statistical analysis. Developmental stages should also be considered in order to fully

understand the biological concept behind every variation. And lastly, biochemical test should be

carried out in order to justify genetic variability among the species of bees.

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