doi: 10.1111/jeb.12133

Effects of ornamentation and phylogeny on the evolutionof wing shape in stalk-eyed flies (Diopsidae)
J. F. HUSAK*, G. RIBAK†, R. H. BAKER‡, G. RIVERA§, G. S. WILKINSON¶& J. G. SWALLOW*1*Department of Biology, University of South Dakota, Vermillion, SD, USA†Department of Aerospace Engineering, Technion Autonomous Systems Program, Technion Israel Institute of Technology, Haifa, Israel‡Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, NY, USA§Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA, USA¶Department of Biology, University of Maryland, College Park, MD, USA

Keywords:

Abstract

compensatory traits;functional convergence;
many-to-one mapping;ornament costs;
stalk-eyed flies;
wing shape.

Exaggerated male ornaments are predicted to be costly to their bearers, butthese negative effects may be offset by the correlated evolution of compensatory traits. However, when locomotor systems, such as wings in flying species, evolve to decrease such costs, it remains unclear whether functionalchanges across related species are achieved via the same morphological routeor via alternate changes that have similar function. We conducted a comparative analysis of wing shape in relation to eye-stalk elongation across 24 species of stalk-eyed flies, using geometric morphometrics to determine how
species with increased eye span, a sexually selected trait, have modified wing
morphology as a compensatory mechanism. Using traditional and phylogenetically informed multivariate analyses of shape in combination with phenotypic trajectory analysis, we found a strong phylogenetic signal in wing
shape. However, dimorphic species possessed shifted wing veins with theresult of lengthening and narrowing wings compared to monomorphic species. Dimorphic species also had changes that seem unrelated to wing size,
but instead may govern wing flexion. Nevertheless, the lack of a uniform,
compensatory pattern suggests that stalk-eyed flies used alternative modifications in wing structure to increase wing area and aspect ratio, thus taking
divergent morphological routes to compensate for exaggerated eye stalks.

Introduction
Locomotion is of key importance to fitness in manytaxa, and locomotor ability is expected to exhibit correlated evolution with other aspects of the phenotype
that do not constitute the primary locomotion system.
Locomotor ability of individuals in a population is thus
likely to represent a balance between natural and sexual selection acting on locomotion directly, as well as
indirectly due to selection on other aspects of theCorrespondence: Jerry Husak, Department of Biology, University of St.
Thomas, 2115 Summit Ave., St. Paul, MN 55105, USA.
Tel.: +651 962 5223; fax: +651 962 5201;
e-mail: jerry.husak@stthomas.edu
1
Present address: Department of Integrative Biology, University ofColorado Denver, Denver, CO, USA

phenotype that may influence locomotion (Dickinson
et al., 2000; Irschick & Garland, 2001; Husak & Fox,
2008; Irschick et al., 2008). Typically, locomotion, andthe lower level traits that underlie it, is thought of interms of how natural selection selects against thoseindividuals too slow to escape predators or acquire food(Irschick et al., 2008). However, recent studies suggestthat better locomotor performance may also directly
increase mating success (Husak et al., 2006) and be
under the direct influence of sexual selection (Lailvaux& Irschick, 2006; Husak & Fox, 2008; Byers et al.,2010). On the other hand, sexual selection may haveindirect negative effects on locomotion, via the direct
detrimental effects of sexually selected traits on locomotor abilities (reviewed in Oufiero & Garland, 2007;
Husak & Swallow, 2011). Indeed, negative effectsª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
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J. F. HUSAK ET AL.

associated with bearing sexually selected signals, ornaments and weapons are required in many theoreticalmodels of sexual selection (Zahavi, 1975; Grafen, 1990;
Kotiaho, 2001; but see review by Szamado, 2011), andthis burden has often been hypothesized to take theform of reduced locomotor performance (reviewed in
Kotiaho, 2001; Oufiero & Garland, 2007) because of its
presumed link to individual fitness. Nevertheless, selection should also favour, within some constraints, traits
that mitigate the detrimental performance effects ofsexually selected traits (reviewed in Oufiero & Garland,
2007; Husak & Swallow, 2011). Despite many decadesof theoretical and empirical studies on sexually selectedtraits, however, many key questions about how theircosts may be ameliorated remain unresolved. Whatmorphological changes allow functional changes inlocomotion to compensate for the detrimental effects ofsexually selected traits? Does phylogenetic history influence the evolution of compensatory traits, thus limitingor constraining morphological change? Does evolution
proceed through different combinations of morphological changes that arrive at similar functional compensation? A more general way to phrase these questions in
a comparative framework is to ask whether related taxa
facing similar selective pressures (i.e. reduced locomotor
ability due to similar sexually selected traits) evolve afunctional solution via similar morphological changes
or via different morphological routes to the same functional end.
As the last question suggests, studying the evolutionof morphological traits in a comparative analysis can becomplicated by the presence of multiple morphological‘solutions’ to a given functional ‘problem’. Such manyto-one mappings of form to function have been documented in multiple functional systems for numerous
taxa (Wainwright et al., 2005; Wainwright, 2007; Losos,
2011). For example, the 4-bar linkage system in theoral jaws of labrid fishes allows for the evolution ofmultiple 4-bar shapes that produce similar jaw movement and function (e.g. Alfaro et al., 2004, 2005; Wainwright et al., 2005). Sprint speed of Anolis lizard species
is determined, in general, by hindlimb length and muscle mass, but similar sprint speeds can be obtained indifferent species with various combinations of limb
segment dimensions and muscle mass (Vanhooydonck
et al., 2006). Similarly, Drosophila subobscura populations
along a geographical gradient evolved convergent wing
size, but this convergence in size, which functions toincrease force production, was accomplished via
changes in different parts of the wing (Huey et al.,2000). Specifically, North American and European flies
both increased wing length over evolutionary time withincreasing latitude, but they did so by increasing different segments of the wing. These examples emphasizethat when similar selective pressures favour a specificfunctional capacity, evolutionary change may take avariety of routes. Conversely, there are numerous cases

where similar selective pressures have resulted inconvergent changes in morphology. For example, convergence in wing shape has been shown in previous
studies to result from similarity in migration distance(Marchetti et al., 1995; Voelker, 2001; Johansson et al.,2009), foraging strategies (Bullen & McKenzie, 2007;
Kaboli et al., 2007) and mating displays (Hedenstr€om &Møller, 1992). Because much of this work has been
conducted on bird species, generalization to the most
species-rich group of flying animals (insects) remains
unclear (Dudley, 2000; Johansson et al., 2009). Also,
little remains known about how selective pressuresresulting from bearing an ornament affect flight performance and morphological components of the flightapparatus (reviewed in Husak & Swallow, 2011). Multiple studies have shown that tail ornament length inbirds is positively correlated with wing size across species (Andersson & Andersson, 1994; Balmford et al.,1994), but whether wing size is increased via lengthening of the same morphological features in all species
remains unknown.
Stalk-eyed flies (Diopsidae) represent an ideal systemwith which to test hypotheses about morphological andfunctional adaptations to compensate for ornaments(Ribak et al., 2009; Husak et al., 2011a,b). Stalk-eyedflies have their eyes displaced laterally from the sides of
their heads on stalks, and the males of many species
have eye stalks that far exceed that of females, with themale eye span of some species greatly exceeding bodylength (Wilkinson & Dodson, 1997). The family Diopsidae is a species-rich lineage of flies with 200–300
described species within 10–14 genera (Feijen, 1989)
and marked variation in the degree of hypercephaly(Wilkinson & Dodson, 1997; Baker & Wilkinson, 2001).
This exaggerated male ornament is under both intersexual selection via female choice (Burkhardt & de laMotte, 1988; Wilkinson et al., 1998; Hingle et al., 2001;
Cotton et al., 2006) and intra-sexual selection during
male–male competition (Burkhardt & de la Motte,
1985; Panhuis & Wilkinson, 1999; Small et al., 2009).
Even though the significantly increased moment ofinertia of the head of males (Ribak & Swallow, 2007) is
predicted to decrease aerial turning performance compared with females, flight performance trials have failed
to consistently detect flight performance decrements inmales (Swallow et al., 2000; Ribak & Swallow, 2007).
The best supported hypothesis for the apparent lack ofa flight performance cost in males is that they have
evolved compensatory mechanisms to offset such costs.
In eye-span dimorphic species, male stalk-eyed flies
have larger wings than females (Ribak et al., 2009;
Husak et al., 2011a). Looking only at males, those withrelatively longer eye stalks have relatively larger wings,
both within (Husak et al., 2011a) and among dimorphic
species (Ribak et al., 2009). Finally, as dimorphism ineye span increases across species, so too does dimorphism in wing length and area, as well as aspect ratioª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYStalk-eyed fly wing shape(Ribak et al., 2009; Husak et al., 2011b). Since wings do
not appear to be under direct sexual selection in malesof dimorphic stalk-eyed fly species (Burkhardt & de laMotte, 1985; Panhuis & Wilkinson, 1999; Egge et al.,2011), the increase in wing size that is associated withincreased eye span is unlikely to be due to direct sexual
selection on wing size or function. The finding that the
aspect ratio of wings may be affected by male ornamentation suggests that shape differences between males
and females exist. Aspect ratio is a shape property(non-dimensional) affecting the aerodynamic performance (i.e. function) of a wing with a given area. However, it only describes the ratio between wing lengthand average width (chord), thereby providing only acoarse measure of wing shape. Since increasing wingarea and/or aspect ratio can be accomplished in manydifferent morphological ways, we used geometric morphometric techniques to examine the finer details ofchange in wing shape across stalk-eyed fly species todetermine how dimorphism in ornamentation specifically alters wing shape.
Our objective was to determine if stalk-eyed fly species increase wing size and aspect ratio in association
with eye-span dimorphism via similar or divergent
morphological changes in wing shape. We studied
wing shape in 24 species of stalk-eyed flies in four
genera, using phylogenetic comparative methods todetermine which aspects of wing shape, if any, areevolutionarily correlated with dimorphism in eye span
and whether there are phylogenetic patterns in wing
shape unrelated to dimorphism in eye span. If species
have taken similar morphological routes to increase
wing size, then we predicted that there would be astrong relationship between dimorphism in eye spanand dimorphism in wing shape. Conversely, if there
are divergent morphological solutions to increase wing
size, then we predicted that there would be weak or
no evolutionary relationships across species between
dimorphism in eye span and dimorphism in shapevariables. Finally, we examined changes in shapewithin genera to determine if there is concordance or

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discordance in morphological changes associated withdimorphism in eye span.

Materials and methods
We studied 24 species of stalk-eyed flies, obtaining dataon average male and female eye span and body lengthfrom Baker & Wilkinson (2001) and Ribak et al. (2009).
We obtained wing images from subsets of individuals
used in previous studies: Diasemopsis aethiopica, D. dubia,
D. meigenii, D. signata, Diopsis apicalis, Sphyracephala beccarii, Teleopsis dalmanni, T. quinqueguttatta and T. thaii wereobtained from flies used in Ribak et al. (2009); Diasemopsis species’ wings were from flies used in Baker & Wilkinson (2003); T. quadriguttata are from flies used inWilkinson & Taper (1999); and the remaining species
were from flies used in Baker & Wilkinson (2001). We
refer readers to those papers for details of laboratory rearing of flies and specimen preparation. Previous work has
shown considerable variation in eye-stalk dimorphism(Baker & Wilkinson, 2001, 2003), and we follow thosestudies of eye-stalk allometry and their classifications ofdimorphic and monomorphic species for analyses.
We used tpsDig v. 2.1 (Rohlf, 2006) to obtain coordinates for 14 landmarks (Fig. 1) on each wing (n = 1031
individuals; sample sizes provided in Fig. 2). With one
exception, landmarks chosen represent the intersectionsof wing veins and are comparable to landmarks used inother studies of fly wing shape (e.g. Pelabon et al.,2010). Landmark 3 was defined as the intersection of
the trailing edge of the wing with the straight line
defined by landmarks 4 and 5. Vein intersections thatwere more proximal to the wing hinge than landmarks
1 and 2 were not reliably present in many individuals.
Although some methodological details in preparing
wings for photographs differed, the various techniques
did not affect obtaining reliable or comparable landmark data from the images (see Supporting Information
available online).
We used a custom-written (Bush et al., 2002) program for SAS (9.1, SAS Institute Inc., Cary, NC, USA)

Fig. 1 Landmarks used in analysis of wing shape. Image is of a Teleopsis dalmanni wing.
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
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J. F. HUSAK ET AL.

Diasemopsis fasciata (1.18; 24, 24)
Diasemopsis signata (0.96; 19, 14)Diasemopsis obstans (1.31; 30, 22)Diasemopsis dubia (1.33; 16, 16)Diasemopsis silvatica (1.36; 28, 24)
Diasemopsis albifascies (0.99; 33, 27)
Diasemopsis meigenii (1.16; 21, 19)Diasemopsis conjuncta (1.20; 25, 21)Diasemopsis nebulosa (1.16; 27, 24)
Diasemopsis aethiopica (1.14; 15, 29)Diasemopsis elongata (1.27; 18, 8)
Diasemopsis longipedunculata (1.36; 22, 19)Diasemopsis hirsuta (1.11; 28, 22)
Diopsis apicalis (1.16; 23, 26)
Teleopsis quinqueguttata (1.00; 13, 21)
Teleopsis breviscopium (1.80; 19, 10)
Teleopsis rubicunda (1.07; 21, 8)
Teleopsis quadriguttata (0.99; 32, 32)
Teleopsis thaii (1.66; 13, 19)
Teleopsis whitei (1.71; 27, 29 )Teleopsis dalmanni (1.44; 22, 26)
Sphyracephala brevicornis (0.95; 14, 22)
Sphyracephala munroi (0.99; 21, 15)Sphyracephala beccarii (0.98; 23, 20)
0.04

Fig. 2 Phylogenetic relationships among the stalk-eyed fly species studied. Dimorphism indices and male and female sample sizes,
respectively, are given in parentheses. The phylogeny is from Baker et al. (2001), with the addition of T. thaii. See Materials and methods
for details on tree construction.

to perform a generalized least-squares Procrustes analysis (GPA) on landmark coordinates. GPA rotates, translates and scales coordinates to remove positioning andisometric size effects in landmark data (Rohlf & Slice,
1990; Bookstein, 1991; Marcus et al., 1996). Orthogonally projected tangent coordinates were obtained for
all individuals (Slice, 2001), and these coordinates wereentered into a principal components analysis (PCA).
We then analysed wing shape and dimorphism of shapeusing two different approaches, each of which answers
different questions. First, we examined differencesamong species and between the sexes, using multivariate analysis of variance (MANOVA), as well as by examining dimorphism indices of wing shape (see below).
Second, we analysed the differences in magnitude anddirection (i.e. angles) of phenotypic trajectories (Collyer& Adams, 2007; Adams & Collyer, 2009). Although thefirst approach examines how specific segments of thewing differ among species, the second considers how
sexual shape dimorphism varied among species in multidimensional morphospace (see below). We also visually examined changes in shape with thin-plate splines(using tpsSpline v. 1.20; Rohlf, 2004) within genera,
qualitatively comparing deformation grids of average

male consensus shapes of species monomorphic for eyespan to those dimorphic for eye span within Diasemopsis, Sphyracephala and Teleopsis.
We conducted a two-way ANOVA to examine theeffects of sex and species on centroid size (Gidaszewski
et al., 2009). We then conducted a two-way multivariate analysis of variance (MANOVA) with principal component scores as response variables to examine the effects
of sex and species on wing shape. This allowed us to
examine specific differences among species considering
individual variation within species and analysing thesexes simultaneously. We complimented this analysis
with phylogenetic PCA (Revell, 2009; see below),which considers species as data points.
We then calculated dimorphism indices for each variable of interest. For eye-span dimorphism, we used species averages from Baker & Wilkinson (2001) andRibak et al. (2009); for centroid size, we used values
obtained from GPA. The dimorphism index for eye spanand centroid size was the male average divided by thefemale average, which is desirable for this variable dueto the marked variation in body size among the stalkeyed fly species studied (Baker & Wilkinson, 2001). To
examine dimorphism in shape, we computed theª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
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Euclidean distance in tangent space (hereafter, ‘shapedimorphism’) between the mean shapes of the males
and females of each species (Dryden & Mardia, 1998;
Gidaszewski et al., 2009). We note that this measure ofshape dimorphism is equivalent to the magnitude ofshape change in the phenotypic trajectory analysis
described below.
We used phenotypic trajectory analysis (Collyer &Adams, 2007; Adams & Collyer, 2009) to more fully
understand effects of the interaction between species
and sex on wing shape. Briefly, this method examines
phenotypic evolution as phenotypic change vectors
between two evolutionary units (the two sexes in ourcase), comparing the vectors across pairs of taxa. The
magnitude of each vector represents the level of phenotypic change (or difference) between the two units,
whereas the direction (or orientation) of the vector
represents the way in which the phenotype differs
between the two units (Collyer & Adams, 2007;
Adams & Collyer, 2009). When comparing the vectorsof two species, differences between the magnitudes
indicate differences in dimorphism, whereas differencesin directions (i.e. the angle between vectors) describe
the difference in the evolutionary trajectories between
the units across the taxa compared. For magnitudes
and directions, the smaller the differences between
taxa, the more similar are the levels of sexual dimorphism and evolutionary trajectories respectively. Thismethod allowed us to examine whether sex-based differences in wing shape are concordant across monomorphic and dimorphic species, how patterns ofsexual shape dimorphism differ within and between
genera and whether species differ predominantly inthe direction of change or the magnitude of change.
Specifically, we calculated a magnitude and directionof change between the least-squares means of maleand female individuals for each species to generate theendpoints of the species vector. We then tested forconcordance across species vectors by calculating pairwise differences in magnitude and direction (i.e.
angles) between vectors and testing for statistical significance using a residual randomization procedure(9999 iterations)(ter Braak, 1992; Gonzalez & Manly,1998). To adjust for Type I error rates, sequential Bonferroni corrections (Holm, 1979; Rice, 1989) wereapplied to all pairwise tests. In addition, we assessed
the relationship between phenotypic trajectories andphylogenetic relatedness. As vector direction in multidimensional space is only interpretable as the angle
between two vectors, we used Mantel correlations todetermine the relationship between the angle between
two species’ vectors and the patristic distance between
the two taxa. Mantel correlations were also used tocompare magnitude and patristic distance, and for acomparison of magnitudes and angles between taxa.
These statistical analyses were conducted using R (RDevelopment Core Team, 2011).

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Because common ancestry makes species averages
nonindependent of each other (e.g. Felsenstein,1985), we tested for phylogenetic signal in our dataand performed correlation analyses with phylogeny
taken into account. The phylogeny used for our analyses was generated from the nucleotide matrix consisting of three mitochondrial genes (CO II, 12S and
16S), and three nuclear genes (Elongation factor-1alpha,wingless and white) that was constructed in Baker
et al. (2001). Twelve taxa for which we had no morphological measurements were pruned from thematrix and DNA sequence for 4 genes (CO II, 16S,
wingless and white) from a new species, T. thaii
(F€
oldv
ari et al., 2007), was added to the matrix. A partitioned maximum likelihood (ML) search, with 100
bootstrap replicates, using six distinct models withjoint branch length optimization and an estimatedGamma model of rate heterogeneity, was conductedin RAxML (Stamatakis et al., 2005). The ML tree wasconverted to ultrametric in Tree Edit (http://tree.bio.
ed.ac.uk/software/treeedit/) using the nonparametricrate smoothing option.
We conducted phylogenetic PCA (Revell, 2009) on
males and females separately using the ‘phytools’ package (Revell, 2012) of R (R Development Core Team,
2011). Phylogenetic PCA uses species averages as data
points input into a PCA that takes phylogeny into
account when computing eigenvalues, eigenvectors,
component loadings and component scores (Revell,2009). We obtained principal component scores formales and females, separately, for each species. This
analysis does not give scores that are ‘phylogeneticallycorrected,’ but they are adjusted to reduce type I error
rates, compared with nonphylogenetic procedures,
when further analysed. We then conducted ANOVA oneach of the first four principal components (eigenvalues  3) for males and females separately, testingfor differences among genera. Diopsis was included in
the phylogenetic PCA, but not in the subsequent ANOVAs, because we only had data for one species of Diopsis.
When ANOVAs were significant, we used Tukey Honestly
Significant Difference post hoc tests to determine which
genera differed from each other statistically in each of
the principal components. These analyses allowed us totest whether genera differed in wing morphology using
a technique that gives us more confidence in our
hypothesis tests.
We tested for phylogenetic signal in all dimorphism
variables using a randomization test implemented inthe ‘picante’ package (Kembel et al., 2010) of R. The
K-statistic gauges the amount of phylogenetic signal relative to the amount expected for a character undergoing Brownian motion evolution along the specified
topology and branch lengths (Blomberg et al., 2003),
and a significant K-value means there is statistically significant phylogenetic signal for the variable being
tested.

ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
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We then used the PDAP:PDTREE module (Garland
et al., 1999; Midford et al., 2005) in Mesquite v. 2.72
(Maddison & Maddison, 2009) to calculate standardized
phylogenetically independent contrasts (Felsenstein,1985) for species and sex averages of dimorphism ineye span, centroid size and wing shape. None of thedimorphism indices showed significant correlations
between the absolute value of the contrast and thestandard deviation of the contrast (eye-span dimorphism, 2-tailed P = 0.22; centroid dimorphism, 2-tailed
P = 0.33; shape dimorphism, 2-tailed P = 0.67), so contrast analyses should perform equally well to alternative
methods of analysis (Garland et al., 1992; Revell, 2010).
To examine how increases in eye span might influencechanges in wing size and shape, we used independent
contrasts in Pearson correlation analyses, testing forcorrelations between eye-span dimorphism and dimorphism in both centroid size and wing shape.
To examine how species monomorphic and dimorphic for eye span differ in wing size and shape, we usedthe data set of 24 species to conduct both a standard(nonphylogenetic) and phylogenetic analysis of variance (ANOVA), following Garland et al. (1993), with species averages as data points, centroid size, wing shapeand dimorphism indices as dependent variables, andeye-span dimorphism/monomorphism (following Baker& Wilkinson, 2001; Ribak et al., 2009; Husak et al.,2011b) as the main effect. This offers a compliment toour analysis of independent contrasts by considering
eye-span dimorphism as a categorical variable insteadof as a continuous variable, and allows us to test for
average differences in wing size and shape between
monomorphic and dimorphic species. Our phylogenetic
ANOVA used 1000 simulations with a Brownian motion
model to produce the null distribution of F-statistics
against which we tested the empirical F-statistic (Garland et al., 1993). Males and females were analysedboth separately and combined for centroid size, since
we were interested in how the sexes individually may
be affected by the evolution of eye-span dimorphism.
Although the standard and phylogenetic ANOVAs havedifferent assumptions, for completeness we present theresults from both. ANOVAs were conducted in the ‘geiger’(Harmon et al., 2008) package of R.

Results
Wing shape and dimorphism
The first four principal components from the PCA
described 83% of the variation in shape, while subsequent component axes each explained less than 5% of
the variation. The ANOVA including all individuals of all
species revealed a significant species*sex interaction(F23,983 = 4.55, P < 0.001) for centroid size (species
effect: F23,983 = 148.24, P < 0.001; sex effect: F1,983 =
0.22, P = 0.646). The MANOVA with PC scores that

0.12

0.08

0.04

PC 2

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0

–0.04

–0.08
–0.1

–0.08

–0.06

–0.04

–0.02

0

0.02

0.04

0.06

PC 1

Fig. 3 Species averages of principal component (PC) 2 scoresplotted against PC 1 scores for female (open symbols) and male(filled symbols) stalk-eyed flies from a principal components
analysis of tangent coordinates from a procrustes analysis of wing
landmarks (see Fig. 1). Polygons are drawn around the genera
included in this study.

included all individuals of all species revealed a significant
species*sex
interaction(F552,22586 = 2.88,
P < 0.001), in addition to significant species (F552,22586 =
41.70, P < 0.001) and sex (F24,960 = 28.74, P < 0.001)
main effects, for wing shape. These differences can beseen in a plot of the first two principal components(Fig. 3), which explain the most variation in wing shape(67.4%). Genera are distinctly separated from each otherin the first two dimensions of morphospace.
The phylogenetic PCA showed similar results as thenonphylogenetic PCA. For females, ANOVA on each of
the first 4 principal components (eigenvalues = 8.20,
4.77, 3.46, 3.21 respectively), revealed that genera differed from each other along all four principal component axes, with Teleopsis being most different from theother genera (Table 1; Fig. 4). For males, ANOVA on eachof the first 4 principal components (eigenvalues = 9.99,
4.37, 3.51, 2.96 respectively), revealed that genera differed from each other along the first three, but not the
fourth, principal component axes, with Teleopsis being
most different from the other genera (Table 1; Fig. 4).
Wing centroid size had significant phylogenetic signal
whether males and females were examined separately(Males: K = 0.625, P = 0.001; Females: K = 0.786,
P = 0.001) or together (K = 0.716, P = 0.001). Centroid
dimorphism did not have significant phylogenetic signal(K = 0.297, P = 0.34), nor did shape dimorphism(K = 0.312, P = 0.19), but eye-span dimorphism
approached significance (K = 0.409, P = 0.088). The
standard and phylogenetic ANOVAs revealed that malesof eye-span dimorphic species had larger wings thanmales of monomorphic species, but this was not thecase for females (Table 1). Species dimorphic for eyespan significantly differed in eye-span dimorphism andcentroid dimorphism; however, species dimorphic foreye span did not differ from species monomorphic forª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
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Table 1 Results of standard(nonphylogenetic) ANOVAs testing fordifferences among genera in the first four
principal components from a phylogenetic
PCA (see text for details). For significant
ANOVAs, results from Tukey’s Honestly
Significant Difference tests are shown,comparing specific genera. Males andfemales were analysed separately, and thegenus Diopsis was not included, because it
was represented by only one species.
Significant differences are bolded.

10

F2,20

P

Females
PC 1
PC 2
PC 3
PC 4

7.96
9.60
36.86
40.23

0.003
0.001
< 0.001< 0.001

Males
PC 1
PC 2
PC 3
PC 4

12.25
43.18
27.14
1.33

< 0.001< 0.001< 0.001
0.29

Diasemopsis– Teleopsis

Sphyracephala– Teleopsis

0.004
0.12
0.16
0.04

0.86
0.001
< 0.001< 0.001

0.003
0.60
0.002
< 0.001< 0.001
0.53
0.33
–

0.99
< 0.001< 0.001–

0.001< 0.001< 0.001–

Table 2 Results of standard (nonphylogenetic) and phylogenetic
ANOVAs testing for differences in wing centroid size and dimorphism
indices (see text for details) between species of stalk-eyed flies
monomorphic and dimorphic for eye span. Males and females
were analysed separately for centroid size. Significant differences
are bolded.

(a)Teleopsis

5

Diasemopsis– Sphyracephala

Sphyracephala

PC2

0
–5

Diopsis–10

Diasemopsis
–15
–20

–15

–10

–5

0

5

10

15

PC1
20

Centroid size
Males
Females
Eye-span dimorphism
Centroid dimorphism
Shape dimorphism

F1,22 = 8.08
F1,22 = 2.33
F1,22 = 8.37
F1,22 = 8.96
F1,22 = 1.08

Standard P

Phylogenetic P

0.009
0.14
0.008
0.007
0.28

0.008
0.16
0.014
0.019
0.43

(b)
Teleopsis

15
10

PC2

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5

Sphyracephala0

Diopsis–5–10

Diasemopsis
–15
–15

–10

–5

0

5

10

15

PC1

Fig. 4 Species averages of phylogenetic principal component (PC)
2 scores plotted against phylogenetic PC 1 scores for female (opensymbols) and male (filled symbols) stalk-eyed flies from aphylogenetic principal components analysis of tangent coordinatesfrom a procrustes analysis of wing landmarks (see Fig. 1).
Polygons are drawn around the genera included in this study.

eye span in the magnitude of wing shape dimorphism(Table 2).
When analysing phylogenetically independent contrasts of dimorphism indices, we found that eye-spandimorphism was positively correlated across species
with centroid dimorphism (r = 0.73, P < 0.001; Fig. 5a)and shape dimorphism (r = 0.52, P = 0.012; Fig. 5b).

Shape dimorphism was not significantly correlated withcentroid dimorphism (r = 0.39, P = 0.07).
In our qualitative assessment of how eye-span dimorphism is associated with wing shape, deformation gridsthat compared consensus (i.e. average) shapes of species
monomorphic for eye span and those dimorphic for eyespan within each genus showed dissimilar patterns ofmorphological differences among genera when monomorphic and dimorphic males were compared (Fig. 6).
Whereas in both Diasemopsis and Teleopsis dimorphic species appear to increase wing size with longer, narrower
wings, the specific sections of the wing that change differ between these two genera (Fig. 6).
Phenotypic trajectory analysis
When examining the magnitude and direction (angle)of change between the least-squares means of male andfemale individuals for each species vector, we foundthat sexual shape dimorphism differed in magnitude
among species more often than in direction (Table 3).
The lowest percentage of significant differences withinor among genera occurred between species monomorphic for eye span, and this was true for both the magnitude and direction of change (Table 3). Whenconsidering only differences in magnitude, the largest
percentage of significant differences occurred betweenª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1288

J. F. HUSAK ET AL.

species dimorphic for eye span, and differences weremore often significant when comparing species within agenus than between genera (Table 3). However, whencomparing species dimorphic for eye span to thosemonomorphic for eye span, differences were more often
significant when comparing species between genera
than within a genus (Table 3). When considering thedirection (angle) of change, differences between species
dimorphic for eye span were more often significant
within a given genus than between genera, and this was
also true when comparing species dimorphic for eye(a)Table 3 Summary of phenotypic trajectory analysis of sexual
shape dimorphism, showing the proportion of significant tests (andafter correction with sequential Bonferroni adjustment) whencomparing the magnitude and direction (angle) of change between
the least-squares means of male and female individuals for eachspecies vector. Comparisons were made between species dimorphic
for eye span (DM vs. DM, N = 153 comparisons), species
monomorphic for eye span (MM vs. MM, N = 15 comparisons),
and between species dimorphic for eye span and monomorphic foreye span (DM vs. MM, N = 108 comparisons), as well as within(congeneric, N = 102 comparisons) and between genera(intergeneric, N = 174 comparisons).

(b)

Fig. 5 Relationships between (a) wing centroid size dimorphismand eye-span dimorphism and (b) wing shape dimorphism andeye-span dimorphism across species of stalk-eyed flies (Diopsidae)
taking phylogenetic relationships into account with independent
contrasts. Shape dimorphism is the tangent distance between themean shapes of the males and females for each species (see text
for details). Least-squares regression lines are shown for visualindication of statistical significance.

DM vs. DM

MM vs. MM

DM vs. MM

All

Magnitude
All
CongenericIntergeneric49.7 (16.3)
50.8 (18.5)
48.9 (14.8)

13.3 (0.0)
0.0 (0.0)
16.7 (0.0)

38.0 (6.5)
32.4 (2.9)
40.5 (8.1)

43.1 (11.6)
43.1 (12.7)
43.1 (10.9)

Direction (angle)
All
CongenericIntergeneric

4.6 (0.7)
7.7 (1.5)
2.3 (0.0)

0.0 (0.0)
0.0 (0.0)
0.0 (0.0)

3.7 (1.9)
5.9 (2.9)
2.7 (1.4)

4.0 (1.1)
6.9 (2.0)
2.3 (0.6)

Fig. 6 Thin-plate spline deformation
grids of males within each genus,
showing comparison of wing shape
between species monomorphic versus
dimorphic for eye span. Deformation
grids have been accentuated by a factorof three to enhance visualinterpretation.
ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYStalk-eyed fly wing shapespan to those monomorphic for eye span (Table 3).
Taken together, species differed predominantly in themagnitude of shape change, and less so in the direction.
Most of the differences resulted from the comparisons
between species from the same genera that are dimorphic for eye span. For example, 18% of the of the 55
species comparisons of eye-span dimorphic Diasemopsis
species differed in the magnitude of shape dimorphism,
and one (1.8%) of the comparisons was significant forthe direction of shape dimorphism. Similarly, 20% of
the of the 10 species comparisons of eye-span dimorphic
Teleopsis species differed in the magnitude of shapedimorphism, and none of the comparisons were significant for the direction of shape dimorphism. On the other
hand, only 22% of the 55 species comparisons between
Teleopsis and Diasemopsis species dimorphic for eye spandiffered in the magnitude of shape dimorphism, andnone of the comparisons were significant for the direction (angle) of shape dimorphism (see also Supporting
Information available on-line). None of the comparisons
between monomorphic species in magnitude or direction of change were significant.
Mantel correlations to assess the relationship between
phenotypic trajectories and phylogenetic relatedness
revealed a significant relationship between patristic distance and difference in angle between vectors (r = 0.22,
P = 0.006), but there was no such relationship detected
between patristic distance and difference in magnitude
of vectors (r = 0.10, P = 0.09). There was also no correlation between the difference in angle between vectors
and the difference in magnitude of vectors (r = 0.01,
P = 0.54).

Discussion
Stalk-eyed flies illustrate how morphological traits that
control locomotion (wing shape and size) can display
correlated evolution with a sexually selected ornament(eye span). Males of species dimorphic in eye span havelarger wings compared to females, and wing shape differs among species in association with eye-span dimorphism (Ribak et al., 2009; Husak et al., 2011b; thisstudy). Our finding of a positive correlation between
eye-span dimorphism and two measures of wing dimorphism (centroid size and wing shape) is consistent withthese previous findings. Thus, our analyses of dimorphism indices and phenotypic vectors indicate thateye-span dimorphism may have affected wing shapeevolution in at least some consistent ways across all
species examined. Moreover, we show that wing shapeis also affected by phylogeny; much of the variation inwing shape (i.e. the first two principal components)grouped members according to genus (Fig. 3), evenwhen phylogeny had been accounted for (Fig. 4), andthe direction of the sex-difference in shape dimorphism
increased with phylogenetic distance between taxa.
Thus, our results reveal a complicated picture for the1289

evolution of wing size and shape in stalk-eyed flies. It
appears that compensatory increases in wing size are acommon feature of all dimorphic males, but there aresome differences among genera in the way wing shape
has changed between monomorphic and dimorphic
species. Our results provide some evidence that compensatory increases in wing size have occurred via
alternative morphological routes consisting of different
structural wing changes among the genera. The factthat we found dimorphism effects overlaid on the phylogenetic component of wing shape further corroborates
that stalk-eyed fly wings are under strong indirect sexual selection pressure from the presence of a sexually
selected ornament (Husak & Swallow, 2011) that mayotherwise reduce flight performance if not for the compensatory adjustment of the wings.
Examination of deformation grids comparing averageconsensus shapes of species monomorphic for eye span
and those dimorphic for eye span within each genus
revealed that morphological differences between malesof monomorphic and dimorphic species are dissimilar
among genera (Fig. 6; see also Figs. 3–4). This was supported by the phenotypic trajectory analysis, which
revealed that the greatest number of significant differences between eye-span dimorphic and eye-spanmonomorphic species was between rather than within
genera. In both Diasemopsis and Teleopsis, dimorphic species appear to increase wing size with longer, narrower
wings. However, the specific sections of the wing thatchange differ between these genera (Fig. 6). In Diasemopsis, the distal section of the wing is narrowed, withthe trailing distal edge extended. Conversely, in Teleopsis, the proximal section of the wing is narrowed, withthe leading distal edge extended. Dimorphic Sphyracephala species exhibit a completely different pattern ofshape change compared to the other genera, withincreases in wing width along much of the wing length.
The phylogenetic PCA supported these findings, as species within a genus grouped in morphospace. In thephenotypic trajectory analysis, 18% of the comparisons
between eye-span dimorphic Sphyracephala and Diasemopsis were significantly different for direction of change,and 20% comparing Sphyracephala to Teleopsis (thoughnone remained significant after Bonferroni correction).
Numerous studies have now shown that eye-stalk elongation is evolutionarily associated with increases inwing area and changes in some measures of wing shape(Ribak et al., 2009; Husak et al., 2011a,b), but theresults of our current study suggest that the generalinterspecies trend is in fact comprised of genera-specificchanges in wing architecture converging into a similarfunctional means of compensation, thus representing
another example of many-to-one mapping of form tofunction (Wainwright et al., 2005; Wainwright, 2007).
Some care should be taken when interpreting thephylogenetic ANOVA results since most of the dimorphic
species are Diasemopsis and Teleopsis species, whereasª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY1290

J. F. HUSAK ET AL.

two of the three Sphyracephala species are monomorphic(and S. beccarii is only moderately dimorphic, with
substantially smaller eye stalks than most Diasemopsis or
Teleopsis species). Thus, our phylogenetic ANOVA may still
have been somewhat influenced by phylogeny despite
taking it into account, because two of the five monomorphic species came from one genus (Sphyracephala),
and there were only three species from that genus in
the analysis. Regardless, the lack of a significant difference in the phylogenetic ANOVA between monomorphic
and dimorphic species in wing shape dimorphism did
not match with our correlation analysis of wing sizeand shape dimorphism indices relative to eye-spandimorphism. Although phylogenetic effects may partly
explain this disparity, another reason for our somewhat
discordant results is that classifying species as ‘monomorphic’ or ‘dimorphic’ in the phylogenetic ANOVA does
not completely capture the large range of variation
across species in eye-span dimorphism (Baker & Wilkinson, 2001) as well as the correlation analysis did.
Nevertheless, the lack of a general difference between‘monomorphic’ and ‘dimorphic’ species with phylogenetic ANOVAs reinforces the notion that the genera have,to some degree, attained wing size and shape dimorphism via different morphological routes. Moreover, we
did find significant differences in eye-span dimorphism(as would be expected), as well as centroid dimorphism.
These results, taken together, provide support to thenotion that species dimorphic for eye span have larger
wings, but have done so via different shape changes.
One surprising finding in our data was that many ofthe differences among genera when comparing monomorphic and dimorphic species seem related to internal
movement of wing vein intersections and due less tochanges in landmarks that would signify differences inwing length, area, or aspect ratio (see Fig. 6). This may
be due to phylogenetic constraints coupled with isometric increases in wing size within a species, instead of dramatic shape changes between the sexes to compensate
for eye-span elongation (Ribak et al., 2009). With thedata in hand we can only postulate on the functional significance of evolutionary changes in wing morphology
across stalk-eyed fly species. Many of the differencesamong genera appear related to positioning of wing veins(see Fig. 1) that are either proximal-anterior (landmarks
5 and 6) or define cross-veins that connect longitudinalveins (landmarks 7–10). Movement of landmarks 9 and
10 affects the size and shape of the basal medial – distal
medial cell. Similarly, movement of landmarks 7 and 8
affects the size and shape of the basal-radial cell in thewing. Note that these landmarks (5–10) form a diagonalband traversing from the leading edge at approximately
1/3 of the wing length (measured from the hinge) to thetrailing edge at approximately 2/3 the wing length(Fig. 1). Since these landmarks represent the most distalintersections of the wing veins (excluding the wing tip)
their specific positions should have important roles instructurally stiffening and controlling deformation of thewing area. When aerodynamic load and wing inertia act
on the flapping wing they will tend to bend it, and, since
the bending moment is highest at the hinge, the proximal section of the wing is reinforced with thicker veinsthat are closer to one another. The thickness and positioning of wing veins along the leading edge determines
wing stiffness along the wing span during flight (Combes& Daniel, 2003). The trailing edge has a reduced numberof veins and these are much thinner (e.g. see Fig. 1). As
a result, the trailing edge is less rigid and has a higher
tendency to elastically deform during flapping. We postulate that the diagonal line of landmarks 5–11 can serve
as a reinforcement of the wing structure for limiting wing
deflection beyond the distal 1/3 of the wing and controlling wing camber. If this presumption is correct, theneven small changes in the positions of landmarks 6–10
can have an effect on the elastic deformation of the wing
during flapping (Ennos, 1989; Wootton, 1992). Ennos(1989) showed that ventral flexion of dipteran wings canallow force vectors to be changed (and hence maneuvers
to be performed) with little to no body angle changes
and that such flexion reduces stress on the wing articulation (Ennos, 1988, 1989; Wootton, 1992). Obviously, thefunctional implications of the wing vein intersectionchanges found in our study, and why different patternsof wing shape are associated with certain lineages,
require confirmation from empirical studies. We hopethat our findings and functional interpretations catalyse
future work on the subtleties of wing veins and how theyaffect flight mechanics.
The phenotypic trajectory analysis was in generalagreement with our other analyses in that it emphasizedthat most differences in wing shape dimorphism among
species are due to the magnitude of shape change
between the sexes and not the direction of shape change.
As in our other analyses, there was a significant phylogenetic effect, with greater differences in the direction ofshape change occurring between more distantly related
species. Despite this phylogenetic effect on wing shape,
we still detected numerous shape dimorphism differencesamong species, and even within genera. This finding suggests that many species within and among genera have
similarities in wing shape, but that there are also strong
differences, likely related to dimorphism in eye span as
indicated by the significant relationship between eyespan dimorphism and shape dimorphism (Fig. 5). The
variety of morphological solutions across species to compensate for exaggerated eye stalks is consistent with eyespan dimorphism and monomorphism having evolved
multiple times in stalk-eyed flies (Baker & Wilkinson,
2001). Unlike the other analyses we used, the phenotypic
trajectory analysis allowed us to divide dimorphism into
magnitude and direction. Doing so revealed that patternsof dimorphism are dominated by differences in magnitude, not direction. Further, Mantel correlations revealed
phylogenetic effects on direction of change, but notª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGYStalk-eyed fly wing shapemagnitude. We found no phylogenetic signal in wing
shape dimorphism indices using the K-statistic, but thoseindices indicate only the magnitude of dimorphism.
Taken together, these results suggest that, not only isthere phylogenetic signal in wing shape but also there is
phylogenetic signal in the direction of morphologicalchange between the sexes. We would not have detected
this effect if we had not examined phenotypic trajectories.
Although considerable research has been conducted
on insect wing form and function (Wootton, 1992; Grodnitsky, 1999; Stark et al., 1999; Dudley, 2000; Combes &Daniel, 2003), even specifically within flies (Ennos,
1989), most of this work has examined functional differences among widely disparate taxa with highly divergent
wing morphology and venation patterns. Although wehave speculated on some of the potential functional consequences of smaller scale changes in wing shape, actual
studies that examine functional differences at the magnitude of morphological divergence that we observed
within diopsid flies are greatly needed. Such studies
could be done with correlational biomechanical analysesof closely related species or with robotics (e.g. Shanget al., 2009). Nevertheless, our comparative geometricmorphometric analysis of wing shape allows for an
examination of specific wing form changes that might be
expected at the microevolutionary scale in response to
selection. Our study also emphasizes the strong indirect
effects that sexual selection may have on morphology, in
addition to the direct effects on ornaments and othertraits that directly enhance mating success. Testingwhether the detrimental effects of sexually selectedtraits, in general, are ameliorated by the evolution of
convergent form and function or convergent function
via divergent form (i.e. many-to-one mapping) will be a
fruitful area of research in the future. Such studies willallow a better understanding of how selection on theentire phenotype acts to shape locomotor systems in
addition to direct selection on the locomotor apparatus
and its performance.

Acknowledgments
Funding was provided by the National Science Foundation to GSW (DEB 9409369 and 0952260), JGS (IOB
0448060), RHB (DEB 0951816) and G. Rivera (DBI
0905898). Thanks to S. Lailvaux (University of New
Orleans) for help with phylogenetic statistics. G. Ribak
was supported in part at the Technion by a Fine fellowship and by the Center for Absorption in Science, Ministry of Immigrant Absorption, State of Israel.

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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Data S1 Wing Mounting Techniques.
Table S1 Results from phenotypic trajectory analysis,
showing differences in magnitude (upper diagonal) andangle (lower diagonal) of change between the leastsquares means of males and females for each species
vector.
Figure S1 Plot of 14 landmarks shown in Figure 1
from stalk-eyed fly wings using three different techniques.
Received 20 September 2012; revised 9 January 2013; accepted 10
January 2013ª 2013 THE AUTHORS. J. EVOL. BIOL. 26 (2013) 1281–1293
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2013 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY