Introduction

Bats are unique among mammals in their capacity for powered flight. Flight is a form of locomotion that enables foraging over large areas and in areas of difficult access, and also allows migration over large distances [1]. Nevertheless, this type of locomotion requires high-energy costs and, therefore, a strong selection must take place on wings as a way to minimize such costs [2]. As such, selection can favor optimal design of wings for locomotion with less effort in accordance with ecological and physiological characteristics of each species [1]–[5].

Size and shape of structures in organisms are features well used to verify differentiation of species [6]–[8], population variations [9]–[11] and make ecological inferences [3], [4], [6], [12]. The employment of techniques that aim at the maximization and the detection of morphological differentiation of bat wings has been shown to be useful as a way to comprehend the dynamics of flight [4], segregation and coexistence in space [13], [14]. To this end, morphometric parameters that take into consideration the mechanics and aerodynamics were proposed to verify the flight performance of bat species that explore habitats differently and have different flight styles [4], [15]–[17]. For example, insectivorous bats of open habitats have long and narrow wings [4], [16], [17]. As these animals pursue agile and flying preys, their wing shape generates a constant and fast flight, but with limited maneuverability. On the other hand, frugivorous and insectivorous bats from complex and closed habitats (i.e. forest environments) require high maneuverability to explore these areas. To such purpose, they usually have short and/or broad wings [4].

Bat wings can be divided in different anatomical features: dactylopatagium, propatagium, plagiopatagium and uropatagium; and each of these parts have different roles in the flight. While dactylopatagium is related to power generation and propulsion of the bat in the air, plagiopatagium is related to the maintenance of this generated force. On the other hand, propatagium and uropatagium are related to the adjustment of flight height, and for some insectivorous bats, uropatagium is used for insect capture during flight [18].

Despite efforts employed to verify morphological differences in bat wings according to ecological characteristics, intraspecific level differences have not been studied in detail. The sex of animals is one of the factors that may influence ecological and morphological characteristics. In this aspect, insectivorous bats were the most studied up until now [19]–[23].

Sexual dimorphism in bats related to body size, skull morphology and, especially, to the forearm length was found in several studies (see ref. [24] for a list of studies). Among the main selective pressures on secondary sexual dimorphism are: competition for resources among females (big mother hypothesis), reduction in competition for resources among males and females, and competition among males for partners (sexual selection) [24]. However, it has previously been proposed that females may present larger body size (including wings), or only larger wings, as a way to compensate the additional weight of carrying a fetus or newborn bat [19], [20], [23]. With the increase of wing area comes a decrease in wing loading (grams per square centimeter of wing area) [1] and therefore, it does not compromise the dynamics of flight during the reproductive period.

Although morphometric parameters related to size have been tested to verify sexual dimorphism in bats [19]–[23], shape analysis, with attention to variation of anatomical landmarks in the wing, was only explored in four species of vespertilionids [25]. Analysis using anatomical landmarks to evaluate the shape of structures can be highly relevant. Area measurements and length of bones, commonly used on morphometry of bats [19], [20], [26], [27], do not fully describe differences in shape and neglect covariation of measurements or specific points in different regions of the wing [25].

In this study we verified possible intersexual differences in the wings of Sturnira lilium using geometric morphometrics, wing area measurements and classical morphometrics to test the hypothesis that females have a different wing to compensate the extra weight caused by fetus mass during pregnancy and breastfeeding of newborns. Newborns of species in the Phyllostomidae family can have 25% to 38% of the mother’s body weight [28]. Thus, we expect sexual dimorphism to be related to wing characteristics that can provide reduction in wing loading of females [20]. More specifically, we expect that this lower wing loading in females will be a result of larger wing and/or different wing shapes for a more efficient flight. Additionally, since no sexual dimorphisms were found in S. lilium by means of classical morphometrics [9], we expect geometric morphometrics to have greater capability to detect these differences between sexes.

Methods

Geometric Morphometric Analysis

We performed morphometric analyses from information collected on photos of bat wings. To photograph specimens the left wing of each individual was extended in the center of a styrofoam board covered in cork and photographed with a digital camera (Canon EOS DIGITAL REBEL XT - Canon EF-S 18–55 lens), which was mounted to a tripod. These images were taken with the camera adjusted to the same height and with no zoom effect. For the standardization of wing position, we have considered the fifth finger parallel to the body of the animal and the largest possible stretching of major (digits IV and V) and medius (digits III and IV) dactylopatagium membranes (Figure 1a). Additionally, we considered the maximum angulation between the humerus and the radio/ulna (Figure 1a). The arm extension of bats is related to the stretch capacity of the propatagium membrane. Therefore, if there was any resistance in the extension of the bat’s arm, to avoid injury, we have considered this as the maximum angulation between humerus and radio/ulna. Wing images were taken of non-pregnant females and males as we assumed this procedure to be too stressful for pregnant females. Fourteen anatomical landmarks were used along the wings of animals with the support of the software TpsDig v.1.18 [31]. Two types of anatomical landmarks were used as a way to sample homologous portions of the wing: maximum curvature points (anatomical landmark 10) and tissue joints (other anatomical landmarks - Figure 1b). These anatomical landmarks provide realistic representations of different wing regions and can be identified in any specimen.

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Figure 1. Wing of Sturnira lilium revealing the main structures evaluated in this morphometric study.

In A) it is possible to observe digits I to V and distinct regions of the wing: (a) propatagium, (b) plagiopatagium, (c) dactylopatagium major, (d) dactylopatagium medius and (e) dactylopatagium minus. In B) it is possible to see the 14 anatomical landmarks used in analyses of partial warps and uniform components of the wing. Anatomical landmarks definition: 1) Articulation between the humerus and radius/ulna; 2) Tissue junction between the propatagium membrane and digit I; 3) Center of the carpus; 4) Articulation between metacarpus and proximal phalange of digit V; 5) Articulation between proximal and distal phalanges of digit V; 6) Tissue junction between distal phalange of digit V and propatagium membrane; 7) Articulation junction between metacarpus and proximal phalange of digit IV; 8) Articulation between proximal and distal phalanges of digit IV; 9) Tissue junction between distal phalange of digit IV and dactylopatagium major membrane; 10) Maximum curvature point of dactylopatagium minus; 11) Articulation between metacarpus and proximal phalange of digit III; 12) Articulation between proximal and intermediate phalanges of digit III; 13) Articulation between intermediate and distal phalanges of digit III; 14) Tissue junction between distal phalange of digit III and dactylopatagium medius membrane.

https://doi.org/10.1371/journal.pone.0049734.g001

During our analysis, we did not use any anatomical landmark that could limit propatagium or plagiopatagium (Figure 1a). This happened because of the consequent difficulty in real delimitation of these structures, because hairs (propatagium insertion onto the shoulder) or arbitrary position of legs (plagiopatagium insertion onto the calcaneus) can generate a high variation in the relative position of such structures. By this means we assumed a dashed straight line from landmark 2 (Figure 1b) towards the body of the animal to estimate the shape of the propatagium, and in the anatomical landmark 6 (Figure 1b) towards the body to estimate the plagiopatagium shape to establish comparisons. For this, we standardized the length of these dashed straight lines taking into account the relative position of the anatomical landmark 1 (Figure 1b). Even though we have not fully sampled the propatagium and plagiopatagium, we were able to verify if the shape of these sampled wing portions differed between males and females by analyzing the relative position of the digit V and the forearm, which retrains the propatagium and plagiopatagium (Figure 1).

To test the amount of error variance that could possibly be related to the standardization method used to extend the bat wings before obtaining the images, we checked the repeatability of all anatomical landmarks in 28 individuals (13 males and 15 females) recaptured which had their wings photographed in other occasions during the study. For this, we used the intraclass correlation coefficient from an analysis of variance on the x and y coordinates of each anatomical landmark, considering the image derived from the first capture as ‘sample 1′ and the image derived from the recapture as ‘sample 2′. From this analysis we were able to verify the error in locating the anatomical landmark position and the differences between individuals. Once all anatomical landmarks presented excellent repeatabilities [32] across samples, ranging from of 0.91 to 0.99, we assumed that the method adopted to extend the wings was standardized throughout the study.

The wing shape variables were obtained from the superimposition of anatomical landmarks (procrustes algorithm) using the software TpsRelW v.1.18 [33]. This method involves the centralization and minimization of distances between anatomical landmarks and the standardization of anatomical landmarks configuration from the Centroid Size (CS) [34], [35]. The CS is a multivariate measurement of size of the structure analyzed. This value is obtained by the square root of the sum of the square distance of each anatomical landmark to the mass center of each configuration (centroid) [35]. This isometric estimator of variation of wing sizes was also obtained from the software TpsRelw 1.18.

Results

We were able to identify significant differences between the body mass of males (N = 30), pregnant females (N = 14) and non-pregnant females (N = 42) (F_2,83 = 7.161; P = 0.001). According to Student’s post-hoc t-test (P<0.05), pregnant females (Mean ± Standard Deviation/Standard Error; 24.7 g ±4.4 g/1.18 g) have greater body weight than non-pregnant females (21.2 g ±3.2 g/0.49 g) (P = 0.001) and males (22.2 g ±1.8 g/0.33 g) (P = 0.001), while non-pregnant females do not differ from males (P = 0.132).

The wing shape of 30 males and 42 females of S. lilium were analyzed. Our analyses of anatomical landmarks resulted in 24 wing shapes variables. The Hotelling’s T² test with these variables revealed differences between males and females (T²_24,47 = 4.098; P<0.0001). At performing LDA with wing shape variables and CS (Wilk's λ = 0.323; F_24,47 = 4.103; P<0.0001), we obtained 80% of correct reclassifications (leave-one-out cross validation) of specimens, but the CS did not contribute to such classifications (stepwise classification −91% of correct reclassifications). The frequency of males and females according to scores generated in LDA can be checked in Figure 2.

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Figure 2. Histogram of scores generated by the Linear Discriminant Analysis (LDA) using wing shape variables of males and females of Sturnira lilium.

The number in parentheses indicates the number of individuals of each sex analyzed.

https://doi.org/10.1371/journal.pone.0049734.g002

Major wing modifications represented by the CV regressed onto shape variables (Figure 3) showed that while females have a displacement of the anatomical landmarks 7, 8 and 9 (Figure 1b) towards the dactylopatagium medius (Figure 1a), males have a displacement of these anatomical landmarks towards the plagiopatagium (Figure 1a). Additionally, we were able to observe a displacement of the anatomical landmarks 4, 5 and 6 towards the plagiopatagium for males, while females have these anatomical landmarks displaced towards the dactylopatagium medius. We observed a similar pattern in the dactylopatagium medius (Figure 1a). The relative position of anatomical landmarks 12, 13 and 14 (Figure 1b) revealed a wingtip more triangular for females and rounded for males (Figure 3). The relative position of the anatomical landmark 10 and 11, which represents the dactylopatagium minus (Figure 1b), also contributed to a less triangular shape in females and resulted in a wider shape when compared to males (Figure 3).

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Figure 3. Canonical Variate (CV) analysis of wing shape variables (partial warps and uniform components) of males and females of Sturnira lilium.

Major modifications in wing shape were obtained with the extreme of the CV from multivariate regression of shape variables onto CV scores.

https://doi.org/10.1371/journal.pone.0049734.g003

Overall differences between males and females of S. lilium were observed in wing length and area measurements as well as body length (T²_12,59 = 2.160; P = 0.027). However, the pair-wise t-test comparisons revealed that the plagiopatatagium and the dactylopatagium minus, medius and major of females have larger areas compared to males, but wing length measurements, body length and propatagium area did not differ between sexes (Table 1). We also found differences in the total wing area between males (Mean ± Standard Deviation/Standard Error; 49.0±4.5/0.8) and females (53.7±4.7/0.7) (t₁,₇₀ = 3.203; P = 0.002), but CS differences between sexes were not significant (t_1,70 = 0.349; P = 0.733).

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Table 1. Results for pairwise comparisons of wing length measurements, body length and wing area measurements between 30 males (M) and 42 females (F) of Sturnira lilium, according to Student’s post-hoc t-test (Hotelling T² test).

https://doi.org/10.1371/journal.pone.0049734.t001

We found no allometric effects between the variables of size (CS, relative wing length and body length) and the CV derived from the wing shape, wing area measurements and wing length measurements (F_3,68 = 1.044; R² = 0.031; P = 0.290).

Discussion

Even though we found differences in the wing shape of Sturnira lilium related to sex, our data indicated that there is no dimorphism related to animal size (i.e. body or wings). Our analyses also confirm that the CS of males and females’ wings do not differ statistically and did not contribute to the correct classification in the LDA. Therefore, our results suggest that the difference in the wing shape of males and females is not related to characteristics regarding the size of wings or body measurements, but it is related to a different distribution of area in distinct portions of the wings. This is supported by our results of wing areas (Table 1), which indicated larger areas in portions of the wing of females where we found major differences in shape (plagiopatagium and dactylopatagium major, medius and minus - Figure 3), while no differences in shape and area were found in the propatagium. Additionally, the lack of allometric effects also supports this idea, indicating important differences in the wing morphology regardless of the size of the body or wings.

Frugivorous bats, such as S. lilium, naturally exhibit a maneuverable flight as they normally inhabit complex environments with several obstacles [4]. Moreover, they usually carry fruits to perches before feeding themselves [42] and, therefore, wing loading for these bats should be smaller than open-area species that do not feed on fruits (i.e. insectivorous bats) [4]. The addition of an extra weight caused by the fetus or infant might compromise some aspects of the animal’s flight dynamics [20]. In our study, we found that pregnant females have a significant increase in body mass (about 16.5%) when compared to non-pregnant females. Taking into consideration that the fetus were still not fully developed, this increase of body mass in females should be grater at birth time. An increase in wing loading increases minimum flight speed, lowering the maneuverability [43] and, therefore, females would likely have a faster flight and greater energy expenditure as a way to maintain flight. Nevertheless, our results suggest different flight patterns between males and females. The increase in the area of specific portions of the wing found in females, responsible for generating and maintaining the thrust range for flight, should increase the efficiency in the use of generated forces and reduce energy expenditure. However, further studies considering experimental measurements of flight performance could confirm these conclusions.

Our limitation in analyzing the entire plagiopatagium could result in omission of features related to wing loading and flight performance in both sexes. However, our results revealed wider shapes (Figure 3) and larger areas (Table 1) in the portion analyzed of plagiopatagium and in the dactylopatagium major (Figure 1a) in females compared to males. The additional load allocated in the proximal portion of the wing, caused by fetus mass, may have great influence on the efficiency and dynamics of flight in females [20]. A larger area allocated in the plagiopatagium and in the dactylopatagium major could generate a smaller wing loading in these regions when females are pregnant or carrying newborns.

Additionally, triangular wingtips in females (Figure 3) are probably related to the need to increase the flight speed due to the additional weight caused by fetal mass or newborn carried during breastfeeding. Wings with pointed tips generate less friction with the air and, consequently, increase flight speed [44] without needing to increase wing flapping rate. Even though this type of flight can compromise the maneuverability of the animal, the larger area found in this portion of the wing (Table 1 and Figure 3) can probably minimize this flight aspect and at the same time contribute for the decreasing of wing loading.

The triangular wingtip in insects, birds and bats represents an evolutionary outcome that increases energy efficiency during the migratory flight [2], [45]. Bowlin and Wikelski [46] observed a positive relation between the heartbeat rate of the migratory bird Catharus ustulatus in function of their rounded wingtips. Therefore, it is possible that females have an increased efficiency in energy expenditure due to their more pointed wingtips in opposition to the more rounded wingtips of males. Additionally, the displacement of anatomical landmarks 10 and 11 (Figure 1b) towards the dactylopatagium medius (Figure 1a) in females can also contribute to a less rounded shape in this wing portion, thus, reducing air friction, which results in a faster flight with an increased efficiency in energy expenditure. Furthermore, the shape of the dactylopatagium minus clearly resulted in a larger area allocated in this portion of the wing (Figure 3), which was confirmed by our area measurements (Table 1).

An alternative hypothesis for a different wing shape between sexes would state that females have different wing shapes to accommodate foraging in different habitats as a resource partitioning pressure. Our results lead to an interpretation of a faster and yet maneuverable flight for females compared to males, which could indicate adaptations for pursuing insects between the canopies [5]. However, previous studies revealed a primarily or entirely frugivorous diet for this bat [46]–[48] with an increase of consumption of insects only when fruits are less abundant [47], and no evidence of intersexual differences in the diet of Sturnira lilium [46], [47]. In addition to this, once our results indicate that the shape is the main determinant for differences in wing between sexes and that there is no dimorphism related to size in S. lilium, the proposed hypotheses on secondary sexual dimorphism [24], at least for this species, are not supported.

This is the first study that uses superimposition of anatomical landmarks in a species of Phyllostomidae. Our results on the wing shape of S. lilium are particularly interesting because, up until now, no secondary sexual dimorphism was confirmed for this species. According to ref. [9], this species has no dimorphism related to any of the 12 cranial and 10 external characteristics of individuals sampled in the Caatinga (xeromorphic biome) and in the Cerrado at the Northeast region of Brazil. This is probably because of the methods used, which were insufficient to detect more subtle morphological variations. The lack of differences between sexes in our results of wing length measurements (Table 1), which were also used in ref. [9], confirms this. Therefore, classical approaches to check morphometric differences between males and females might mask important differences in wings and that have significant impact in interpretations related to lifestyle and ecology of these animals.

In accordance to ref. [25], the method for analyzing the variation in distinct points of the animal’s wing from partial and relative warps seems to have a greater power to detect differences among groups, as well as a greater graphic capability to detect and interpret such differences. But more interestingly is that our study provided evidence of morphological differentiation where traditional morphometrics have failed (in this study and ref. [9]), indicating possible missing features in other bat species. This method can be particularly useful for evaluation and re-evaluation of ecological inferences or flight dynamics in species of bats that have never been studied and those that have already been studied.

Thus, in conclusion, the enlargement or redistribution of wing areas, in both dactylopatagium and plagiopatagium (at least when taking into account the portion analyzed of the plagiopatagium), suggests greater thrust range to maintain flight, leading to a better use of this generated force in maintenance of flight by means of wing shape modifications. Therefore, differences in the distribution of specific areas in the wing seem to reflect an adjustment to pressures related to a higher energy requirement during gestation of females. Moreover, these morphologic features may be related to the transport of newborns during lactation which may also increase wing loading, decrease maneuverability and foraging efficiency [5], [23], and increase energy expenditure [49]. Further studies taking into consideration experimental measurements and analysis of flight performance of S. lilium could confirm our conclusions.

Acknowledgments

We are grateful to the staff of the ecological station of Águas Emendadas, Embrapa Cerrados, IBGE, Brasília National Park, and the private farm owners for authorization and support during fieldwork. We also thank Willian R. F. de Camargo and Thiago F. de Oliveira for fieldwork assistance and Emerson M. Vieira, Rodrigo Gurgel-Gonçalves and the referees for their valuable comments and suggestions on an earlier draft of this manuscript.