The Relationship Between Microhabitat Use, Allometry and Functional Variation in the Eyes of Hawaiian Megalagrion Damselﬂies

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Functional Ecology 2015 doi: 10.1111/1365-2435.12479 The relationship between microhabitat use, allometry and functional variation in the eyes of Hawaiian Megalagrion damselﬂies

Jeffrey A. Scales*,1,2 and Marguerite A. Butler2

1Department of Integrative Biology, Univeristy of South Florida, Tampa, FL 33620, USA; and 2Department of Biology, University of Hawaii at Manoa, Honolulu, HI 96822, USA

Summary 1. The evolution of visual systems is guided by visual requirements imposed by the environment, the size of the animal’s eyes, and physical limitations imposed by the resolution-sensitivity trade-off. Given a particular eye surface area, resolution and sensitivity cannot be simultaneously maximized: gains in resolution, the ability of the eye to detect detail, will come at the cost of sensitivity, the ability to capture photons, and vice versa, without an increase to eye size. How this constraint interacts with ecology and whether it allows the fine-tuning of the visual system to smaller scale habitat heterogeneity remains an understudied question in visual ecology. 2. Here, we use closely-related species of damselflies in the Hawaiian genus Megalagrion which differ in ecology to test whether variation in the resolution-sensitivity trade-off is the evolutionary result of scaling or differences in microhabitat use. We use regression analyses and phylogenetic comparative methods to examine the effects of size and microhabitat use on traits related to light sensitivity and visual resolution. 3. We find that eye size is tightly associated with body size in these damselflies, but other visual morphology traits related to light sensitivity and resolution are associated with microhabitat type. Furthermore, size and morphology relationships vary across microhabitats, and performance related to resolution tends to be more conserved than to variation in light sensitivity. 4. Additionally, smaller species in visually challenging microhabitats have more regionalized eyes than species with larger eyes in open, well-lit areas. Thus, regionalization of the eye allows a decoupling of size and morphology/performance so that even small insect species can exploit visually challenging habitats. 5. These results suggest that variation in visual performance results from changes in eye geometry as well as size. These morphological changes are likely adaptive to differences in microhabitat, indicating that variation in microhabitat use, even at small scales, can play an important role in the evolution of visual systems.

Key-words: adaptation, allometry, damselﬂy, compound eyes, microhabitat, ommatidia, visual ecology, vision, visual morphology

sensitivity is the ability to capture light. Resolution and Introduction sensitivity cannot be simultaneously maximized due to The eyes, ‘windows to the world’, have long-inspired evo- their opposing dependence on surface area and, as such, lutionary functional study (Walls 1942), uniting a wonder- represent key constraints that guide the evolution of visual fully complex functional system with strong selective systems (Stavenga 1979; Land 1989; Land & Nilsson pressures imposed by environmental variation. Spatial res- 2002). Furthermore, smaller animals are expected to be olution is the ability to distinguish objects, whereas light closer to the functional limit for both parameters. Appo- site to this trade-oﬀ is the notion that eye size is constrained by physiological costs (Laughlin et al. 1998). *Correspondence author. E-mail: [email protected]

Indeed, eye size follows strong allometric scaling in many (D/), two parameters which contribute substantially to taxa (Brooke, Hanley & Laughlin 1999; Kiltie 2000; Hall visual performance (Sherk 1978). The genus Megalagrion 2007; Hall & Ross 2007). Therefore, variation in the resolu- is also exceptional in habitat breadth, covering the full tion-sensitivity trade-off and its relationship to size are range of possible damselfly breeding habitats from streams expected to evolve in response to differences in lifestyle or to waterfall seeps to phytotelmata (pockets of water held habitat. These design principles have received some com- by plants) to terrestrial leaf litter (Polhemus & Asquith parative study, (but often not in a phylogenetic context, 1996). Importantly, these vary in light levels (from full sun Jander & Jander 2002; Kapustjanskij et al. 2007; to very dark) and structural complexity (highly cluttered Rutowski, Gislen & Warrant 2009), or only with respect to to open). All Megalagrion have territorial mating systems the most extreme light habitat transitions (e.g. diurnal vs. and apparently lack female mate choice (Polhemus & nocturnal or crepuscular shifts; Thomas et al. 2006; Hall Asquith 1996), presenting a more direct evolutionary con- 2007; Hall & Ross 2007; Schmitz & Wainwright 2011). nection between sensory system characteristics, male trait Therefore, it is not known whether allometric constraints properties and signalling behaviour. or adaptation dominates in the evolutionary diversification How resolution and sensitivity are potentially tuned to of visual systems in complex habitats. ecological variation is particularly convenient to study in Visual demands can vary widely across environments the compound eyes of insects, which have external retinas (Endler , 1990, 1993; Thery 2001; Warrant 2004). Accord- allowing easy characterization of visual geometry. Apposi- ingly, visual system adaptations are well known from tion compound eyes have a modular design comprised of animals which inhabit extreme visual environments such as ommatidia or individual sensory units that contribute sin- the dim, blue-shifted light of the deep sea (reviewed in: gle ‘pixels’ to the viewed image (Fig. 1). Increasing light Warrant & Locket 2004) or animals active during the low- sensitivity therefore requires an increase in the surface area light periods at dusk or night (Endler 1991; McIntyre & of the lens of each ommatidium, or facet diameter (D). If Caveney 1998; Warrant 2004; Rosenthal 2007). Land- eye size does not correspondingly change, the increases in scape-level differences in terrestrial habitats such as forests (D) result in fewer ommatidia per eye surface area and vs. grasslands that differ in light regimes can likewise pro- therefore reduced resolution capabilities (which is mea- duce visual adaptations (Bauer et al. 1998; Veilleux & sured by interommatidial angle D/, the spatial sampling of Lewis 2011). However, the smaller scale differences offered the eye). Many insects alleviate this trade-off by possessing by heterogeneities within a single habitat or ‘microhabi- a specialized region called an acute zone (analogous in tats’ have received far less study (Marchetti 1993; Endler function to the fovea of vertebrates), where interommati- & Thery 1996; Marshall 2000; Endler & Mielke 2005). Sig- dial angle is decreased, while the lens facet diameter is naling animals are known to choose optimal locations simultaneously increased, resulting in better visual perfor- within a habitat to improve the transmission of their visual mance at the expense of performance elsewhere in the eye signals (Mallet & Gilbert 1995; Thery & Vehrencamp (Stavenga 1979; Land 1989). Specifically, we test whether 1995; Long & Rosenqvist 1998; Gomez & Thery 2004), variation in eye size, D and D/ is explained by allometry, but few examples are known of signal receivers doing the by microhabitat effects, or their interaction (for alternative same, or of visual systems tuned to these smaller differ- hypotheses see Fig. 2). Here, we define allometry as a scal- ences in light quality (Endler 1992; McDonald 1995; Leal ing relationship with body size, with isometry as a special & Fleishman 2002). Yet, specialization to microhabitats case. Isometry is observed when there is a one-to-one may provide ample ecological opportunity for the adaptive increase in length between the different body parts being evolution of animal eyes. compared. Here, we examine how overall body size and variation in microhabitat influence eye morphology and visual per- Materials and methods formance in the endemic Hawaiian damselfly genus Megalagrion. Megalagrion damselflies are ideal for study- STUDY SPECIES ing visual system design, as it contains 16 abundant and extant species, which have entered new breeding habitats Adult male individuals of 13 species of Megalagrion damselflies (and associated light environments) at least five times. In were collected from the wild using hand nets from the islands of Molokai, Maui, Oahu and Kauai (Table 1) from 2010 to October addition, they show nearly twofold variation in body size of 2012. All individuals within a species were collected from the and have evolved bright body coloration numerous times same island. We focused on adult males in this study for two rea- (Polhemus & Asquith 1996; Polhemus 1997). Damselflies sons. First, females are difficult to collect (often located only when are aerial predators and have large eyes which appear to in tandem with a male). Secondly, male odonates use visual signal- have reached a point of physical limitation, occupying the ling in establishing territories and finding mates (Corodoba- Aguilar 2008) which may result in a tighter link between visual majority of the head’s surface (Sherk 1978). Odonates characteristics and microhabitat. Only for M. koelense were adult (damselflies and dragonflies) have the highest visual resolu- females included in eye morphology data (the two males and two tion of any insect group, but also show unusually high females did not show any differences). All morphological measure- variation in facet diameter (D) and interommatidial angle ments were conducted within 3 days of capture.

Facet

Crystalline Increased

diameter cone (FD) } light sensitivity Interommatidial angle Δφ

Rhabdom (b)

Increased Fig. 1. Variation in the design of com- resolution } Ommatidia pound eyes. (a) Light enters the ommatidia; individual sensory units that make up a (a) Smaller compound eye, through a facet lens and is focused on a light receptor rhabdom. (b) Apposition Large facet diameters increase light sensi- compound eye Δφ tivity by creating a larger aperture for incoming light, but this will reduce resolution by increasing interommatidial angle (c) (D/). (c) A ﬁne-grained image requires numerous ommatidia to sample the viewed Regionalization scene. Small D/ indicate that the ommatidia are arranged nearly in parallel. (d) Regional variation of the eye can produce an area of high light sensitivity and resolution. Large ommatidia and small D/ in the Smallest high-resolution zone are achieved by a Δφ localized ﬂattening of the eye, but requires greater curvature (and lower resolution) elsewhere. (e) Eye photograph of Megalag- rion nigrohamatum nigrolineatum, showing the hexagonal array of lenses and large var- (e) (d) iation in D.

appear to be similar across islands however, terrestrial species are MICROHABITAT TYPES found only on Oahu. The five microhabitat types used in this study (stream, pool, seep, plant and terrestrial) are those recognized by field workers and MORPHOLOGICAL MEASUREMENTS taxonomists (Polhemus & Asquith 1996, Table 1). Males of this genus are generally territorial, guarding preferred oviposition sites Gross morphology measurements were comprised of two measure- at specific structural microhabitats where visual performance may ments: Thorax length (THORAXL), the proxy for body size, was be important. We use ‘microhabitat’ here to indicate small loca- the distance from the junction of the second leg and the thorax to tions within a larger habitat that are preferred by a particular spe- the base of the hindwing. Eye height (EYEHT), the proxy for eye cies. Microhabitats may define the ‘niche’ of species within a size, was the diameter of the eye along its vertical axis and community, not only in terms of where it is found, but also corre- measured at the tallest part of the eye. All measurements were per- lating with strategies for feeding, locomotion or other aspects of formed with ImageJ (Rasband 2012) on lateral photographs of functional biology (Elton 1927; Gause 1934; Hutchinson 1957; individual damselflies including a size standard (taken with a MacArthur 1958; Williams 1983). Canon EOS 5D digital camera, Canon, Melville, NY, USA). Each ‘Stream’ species are those that commonly use open areas in morphological variable was checked for deviations from normality large, swift streams. ‘Pool’ species frequently occur along stream using qq-plots in the R computing environment (R Core Team margins, pools and ponds. Pool species use water that is slower 2013). We log-transformed body size and eye size to facilitate moving and often associated with vegetation. ‘Seep’ species are interpretation of scaling relationships. As the data were statisti- associated with the wet vertical surfaces of stream valleys and bed- cally well-behaved (met the assumptions for parametric statistical rock walls in deep forests, as well as bogs. The naiads of seep spe- tests), the remaining variables were analysed on their natural cies are not found in streams, but under wet moss covering the scales (i.e. untransformed) for ease of interpretation. walls with permanent water seepage. ‘Plant’ species breed in water pockets in the leaf axils of plants with a bromeliad growth form and are found in forests or bogs, typically away from streams. INTEROMMATIDIAL ANGLE AND FACET DIAMETER Finally, the single ‘terrestrial’ species breeds in the leaf litter of the MEASUREMENTS native Uluhe fern and can be found in the forest, along trails and ridge tops (Williams 1939). Megalagrion oahuense is one of the We mapped variation in facet diameter and interommatidial angle very few damselfly species in the world known to be fully terres- across the eye as correlates of visual performance. Whereas spa- trial. These five microhabitats are present on all islands and tial resolution is determined by interommatidial angle, several

parameters contribute to light sensitivity (Land & Nilsson 2002) utor to variation in light sensitivity will be differences in facet which can vary between species. Along with facet diameter, differ- diameter (with the caveat that other traits contribute to light sensi- ences in focal length, rhabdom diameter and the ratio between the tivity and to the extent that they vary may also influence visual two may all play a significant role in determining light sensitivity. abilities), an assumption made by previous comparative studies of However, because the species included in our study are very clo- compound eyes (Jander & Jander 2002; Rutowski, Gislen & sely related (separated by between 05–10 Myr Jordan, Simon & Warrant 2009). Polhemus 2003), it is reasonable to assume that a primary contrib- We measured facet diameters (D) and interommatidial angles (D/) using the pseudopupil technique (Horridge 1978; Stavenga 1979; Land 1997; Rutowski & Warrant 2002). Heads were detached from the body and placed on a goniometer stage under a (a) (b) dissecting microscope (Zeiss Stemi DV4 Spot, Zeiss, Jena, Ger- Hyper- Isometric many). The eye was viewed from the same angle as the incident allometry Isometry microhabitat light using orthodromic illumination (i.e. with the light source effect emanating through the lens). The black area apparent from this view defines the ‘pseudo pupil’ (e.g. Fig. 1e). The ommatidia in line with the view absorb all incident light giving these ommatidia a black appearance. Heads were positioned face upward on the Hypo- goniometer stage at the centre of rotation of one eye so that the allometry pseudopupil moved vertically or medially across the eye. Anatomi- cal references were set for the 0 longitude by centring both pupils of the eyes so that they were equidistant from the centre of the (c) (d) Eye size Eye clypeus. The 0 latitude reference was set to the point where the Non-allometric No relationship centre of the pseudopupil was in line with the top of the clypeus microhabitat with the face perpendicular to the scope (Fig. 1). The eye was effect dusted with chalk powder to allow identification of individual facets across photographs. We obtained a 180 dorsal–ventral transect by photographing the head at 10 steps from positive 90 (top eye) to negative 90 (bottom eye) using a Canon Powershot A650 IS digital camera mounted to the ocular of the microscope. Visual inspection of the eye revealed a single (Movie S1, Supporting information), frontally located acute zone. Thus, we focused on Body size the front dorsoventral transect because this transect encompassed the greatest variation in the traits of interest (Fig. S1). Addition- Fig. 2. Hypotheses for scaling relationships between eye morphol- ally, behavioural observations indicated that male Megalagrion ogy and size. We use eye size as an example, but similar allometric damselflies orient themselves to maximize visual cues in the for- relationships could apply to D or D/. (a) Morphological variables ward direction when perching or flying to explore objects may follow one of several simple scaling relationships with body (R. Schroeder & M. A. Butler, unpublished data), suggesting that size. (b) Alternatively, morphology may be a function of scaling the forward visual field is especially important in these damselflies. and microhabitat effects so that eye morphology scales with size Pseudopupil measurements began with importing the photo- across species, but species cluster by microhabitat. (c) Visual graphs into Image J and drawing a circle around the perimeter of demands of different microhabitats take precedence over size effects the pseudopupil to identify its vertical centre in each photograph. such that species occupying similar microhabitats have similar mor- As the eye is rotated, the pseudopupil moves across the eye. The phology, with distinct differences between microhabitats. (d) No average D/ was measured as 10 (the angular rotation between relationship between visual morphology and size or microhabitat pictures) divided by the number of facets crossed by the centre of use. the pseudopupil along the diagonal frontoventral facet rows.

Table 1. Megalagrion species means, standard errors and sample sizes (N) for body size (thorax length), eye size (eye height) and eye mor- D/ phology variables: maximum facet diameter (Dmax), minimum interommatidial angle ( min) and regionalization in facet diameter (regD), and interommatidial angle (regD/). Regionalization variables are reported as fraction of the eye based on a species mean. Island and breeding microhabitats also given

Body size Eye size ðl D/ ðÞ D/ Species (mm) N (mm) NDmax m) N min N regD reg Island Microhabitat

blackburni 625 005 25 243 003 20 327 098 5 0627 0044 5 032 036 Molokai Stream heterogamias 605 005 26 231 002 26 314 085 5 0734 0016 5 027 041 Kauai Stream oceanicum 554 011 3 206 006 3 315 031 3 0704 0048 3 026 034 Oahu Stream hawaiiense 482 004 33 181 001 29 288 083 5 0783 0043 6 025 028 Molokai Seep koelense 456 009 7 177 002 6 286 050 4 0782 048 4 022 035 Molokai Plant vagabundum 449 004 53 178 001 48 338 032 6 0762 0055 6 028 027 Kauai Seep oahuense 446 010 6 189 004 6 367 035 4 0932 0030 4 026 037 Oahu Terrestrial n. nigrohamatum 439 002 21 180 001 20 351 031 6 0574 0022 6 014 026 Maui Pool calliphya 401 003 69 154 001 54 329 090 6 0765 0045 6 018 029 Molokai Pool n. nigrolineatum 392 006 25 167 001 25 342 087 6 0658 0030 6 016 027 Oahu Pool xanthomelas 389 003 32 15 0016 32 342 042 5 0823 0036 5 017 028 Oahu Pool leptodemas 376 006 6 148 003 6 323 035 6 0798 0039 6 018 027 Oahu Pool oresitrophum 343 006 34 150 001 31 324 049 6 0830 0022 6 017 027 Kauai Pool

Facet diameter measurements were based on the average width We also examined whether eye morphology was explained by of three adjacent facets in a row within each pseudopupil. The scaling or by association with ecological variation using ANOVA. hexagonal geometry of the lens facets results in three possible axes For eye size, we tested models including body size, microhabitat for ommatidial rows (roughly at 60 angles to one another). As type and their interaction. For each functional variable (Dmax, D/ D/ each of these directions results in a similar, but slightly different min, regD and reg ), we tested models with eye size, micro- facet diameter, we took the mean D for each of the three axes habitat type and their interaction as dependent variables, dropping through a central ommatidia and averaged these together. Facet non-significant interactions. All statistical analyses were performed diameters were measured every 5 between 40 and 40 and in the R statistical computing environment (R Core Team 2013). every 10 thereafter to 90 along the same transect as D/ were measured. The raw data were smoothed using a cubic spline in PHYLOGENETIC ANALYSES the R computing environment (R Core Team 2013). The mini- Because species share evolutionary history, they are not indepen- mum interommatidial angle (D/ ) and maximum facet diam- min dent data points for standard statistical analyses (Felsenstein eter (D ) were obtained from the smoothed data. We chose max 1985). To account for phylogeny in our analyses, we used a to analyse minimum D/ and maximum D because they model-based approach to analyse the evolution of quantitative should represent maximum visual abilities. If the visual traits, comparing the fit of Brownian motion and Hansen models. demands of microhabitats are limiting to species distribu- Brownian motion models describe the evolution of traits as a tions, we expect peak abilities at the most sensitive part of the purely stochastic process (Eq. 6; Martins & Hansen 1997; Butler eye to be more closely related to habitat use than average & King 2004). Written as a stochastic differential equation, the abilities. Brownian motion model describes evolutionary change in a phenotypic trait (X) through time (t) with dB(t) an increment of a EYE REGIONALIZATION standard Brownian motion (BM) process. One parameter is fit in this model, (r) which is proportional to the strength of stochastic As there is no established standard measure of eye regionaliza- changes or macroevolutionary ‘drift’: tion, we used the following index for the degree to which D dXðtÞ¼rdbðtÞ eqn 5 and D/ vary regionally across the eye. We first calculated mean eye transects for each species. We defined the mid-point D size Hansen models are Ornstein–Uhlenbeck processes developed as halfway between the maximum and minimum D obtained for phylogenetic comparative analysis (Eq. 6; Martins & Hansen for each species mean transect (Fig. 5a). Regionalization in D 1997; Butler & King 2004), which describe the evolution of quanti- was therefore the proportion of the transect with facet diame- tative traits under a model of stabilizing selection. It is important ters (regD) above the mid-point D size for the species. Essen- to note that these are used here to represent macroevolutionary tially, regD is the width of the transect at half-max, so that models that describe evolution over very long time-scales (millions highly regionalized eyes will be low in regD. of years) and cannot be extrapolated down to natural selection Using the transects of D as in Fig. 5a, regD, is calculated as within populations, which occur on very short time-scales. Written follows. Let R be a subrange within the degree range (0, 180). as a stochastic differential equation, the Hansen model describes Then, RD is the degree range of the eye with facet diameters that evolutionary change in a phenotypic trait (X) through time (t) are greater than half-max and is defined as follows: with dB(t) an increment of a standard Brownian motion (BM) process, with additional terms for stabilizing selection (a), drift maxðDÞminðDÞ R ¼ RjD [ eqn 1 (r) and an optimal trait value (h): D 2 dXðtÞ¼a½hðtÞXðtÞdt þ rdbðtÞ eqn 6 Then: Importantly, Hansen models allow for the specification of dif- RD regD ¼ eqn 2 ferent optima on different branches of a phylogeny to indicate 180 transitions to different selective regimes, as when species rapidly A similar procedure was used to obtain the index of regionali- evolve in morphology in response to a new microhabitat type. zation in interommatidial angle, regD/: We used the Megalagrion phylogeny (topology and branch lengths) published by Jordan, Simon & Polhemus (2003, Fig. 3). maxðD/ÞminðD/Þ We pruned the Jordan, Simon & Polhemus (2003) tree to include RD/ ¼ RjD/\ eqn 3 2 only the species for which we have phenotypic data using the ‘ape’ and: package in R (Paradis, Claude & Strimmer 2004), and scaled the time depth of the tree to 1. All evolutionary analyses were con- RD/ ducted on log-transformed variables. regD/ ¼ eqn 4 180

Therefore, regD and regD/ can range in value from nearly 0 to Selective regimes nearly 1, with small values indicating greater regionalization. We constructed three hypotheses for the evolution of body size

and eye morphology (Dmax, Dumin, and regionalization of each). STATISTICAL ANALYSES The first was an adaptive model describing adaption to microhabitat type. The microhabitat model contains 5 optima based on the Linear regressions of log-eye size with log-body size were microhabitat use of each species, and hypothesizes that at least used to test hypotheses of allometry. Isometry implies that each some of the 5 microhabitat types: stream, seep, plant, terrestrial, linear measurement of morphology will scale one to one with or pool, place different selective pressures on the species that are body length. Therefore, if morphological variation scales isomet- associated with them (Fig. 3). The second model described stabi- rically with size, we expect that a log–log regression will result lizing selection towards a single global optimum, hypothesizing in a slope of 10 (Fig. 2a). A slope greater than one indicates that all Megalagrion damselflies experience the same adaptive hyperallometry, and slope < 10 indicates hypoallometry. regime. The final model was Brownian motion, which explains

heterogamias set was used to create 2000 simulated data sets used for parametric blackburni bootstrap assessment of confidence in model selection and param- oceanicum eter estimation (Burnham & Anderson 2002; Scales, King & Butler kauaiense 2009). Each simulated data set was fit to each model to assess con- koelense fidence in model selection. Models were selected as the best if they hawaiiense performed 2 or more BIC units better than all other models for nesiotes each bootstrap replicate. Confidence intervals for parameter esti- oahuense mates for the best-fitting model were also based on 2000 paramet- mauka ric bootstrap replicates. paludicola vagabundum adytum Results eudytum oresitrophum SIZE AND SCALING calliphya leptodemas Means with standard errors and sample sizes for all mea- orobates surements are given in Table 1. We found that body size in n. nigrohamatum Megalagrion varies over twofold in length and is strongly n. nigrolineatum pacificum associated with microhabitat type (Fig. 4a, Table S1). xanthomelas Stream species are significantly larger than seep (P = 001, Tukey HSD test), plant (P = 003), terrestrial (P = 002) Fig. 3. The Megalagrion phylogeny used in this study, with branch and pool species (P < 0001). We found support for lengths drawn proportionally to time (Jordan, Simon & Polhemus hypoallometry (i.e. a trend of reduced eye size with increas- 2003). There were thirteen taxa included in this study, indicated ing body size), along with an allometric microhabitat effect by solid lines. Dashed lines indicate extant species not included in this study as some of these are very rare, endangered or may be (Figs 2b and 4b, Table S1). extinct (e.g. M. nesiotes). Breeding microhabitats (Polhemus & Asquith 1996) are mapped onto the phylogeny represented by different line colours: red – stream, black – plant, green – seep, light FUNCTIONAL MORPHOLOGY OF THE EYES blue – terrestrial, dark blue – pool. These colour codes are used We found extensive variation in facet diameter, interom- throughout the manuscript. matidial angle and their regionalization, and strong associations with microhabitat type (Fig. 5, Table 2). Despite phenotypic evolution by random drift, making no specific assump- being the smallest in size, pool species have some of tions about selection or adaptation. Each evolutionary model was fit to the data and phylogeny the largest maximum facet diameters (light sensitivity, using the OUCH software package (Butler & King 2004; King & Fig. 5a,c) and smallest interommatidal angle (resolution Butler 2009) in the R statistical computing environment (R Core capabilities, Fig. 5b,d) among Megalagrion damselflies. Team 2013). We performed analyses of power and bias for three Dmax and D/ are able to be simultaneously maximized commonly used information criteria and the Bayesian information min via a high degree of regionalization across the eye so that criteria (BIC) performed the best (Appendix S1). Thus, fits of each D/ model were compared using the BIC, which was used to measure the largest D and smallest occur jointly in a small acute the strength of evidence in support of each competing model zone (Fig. 5a,b). This arrangement has allowed small spe-

(Burnham & Anderson 2002). The best-ﬁtting model for each data cies to have larger Dmax than species which are much larger

(a) (b) Eye size (mm) size Eye Body size (mm) Body size Slope = 0·83 R2 = 0·95 Habitat*** Size*** 3·5 4·0 4·5 5·0 5·5 6·0 1·4 1·6 1·8 2·0 2·2 2·4 Plant Pool Seep Stream Terr 3·5 4·0 4·5 5·0 5·5 6·0 Body size (mm)

Fig. 4. The relationships between microhabitat, body size and eye size in Megalagrion damselﬂies.(a) Body size was strongly associated with habitat (see Table S1 for ANOVA results). Box plots represent medians with upper and lower quartiles for each ecotype (there are no boxes for terrestrial and plant ecotypes as only one species is represented). (b) Eye size scaled hypoallometrically with body size as a log–log regression of EYEHT on THORAXL revealed a slope of less than one: slope = 083 0063 SE, P < 0.001. Colours and shapes represent microhabitat use in all ﬁgures: red circle – stream, black triangle – plant, green square – seep, inverted light blue triangle – terrestrial, dark blue diamond – pool.

Light Sensitivity Variation down eye Max capabilities vs. size Regionalization of eye Terrestrial (a) Terrestrial (c) (e) shady Pool Pool Seep variable shady Seep ) m Stream max Stream reg D bright/large D D ( μ

30 32 34 36 Eye size * Plant 20 25 30 35 Plant Habitat *** Habitat ** bright? Eye size X habitat ** Eye size X habitat * 0·15 0·20 0·25 0·30

(b) (d) (f) Δφ min (degrees) Δφ reg

Δφ Stream bright/large Pool Eye size * Eye size n.s. shady 0·60 0·70 0·80 0·90 Habitat ** 01234 Habitat ** 0·25 0·30 0·35 0·40 −50 0 50 1·6 1·8 2·0 2·2 2·4 1·6 1·8 2·0 2·2 2·4 Eye angle (degrees) Eye size (mm) Eye size (mm) Resolution

Fig. 5. Functional morphology of the eye varies more strongly with microhabitat type than with size in Hawaiian damselflies. The top row displays variation in light sensitivity (D), the bottom row, resolution (D/). Panels (a) D and (b) D/ show examples of variation along a vertical transect down the front of the eye for representative species associated with each microhabitat type (representative species: plant = M. koelense, stream = M. heterogamias, seep = M. vagabundum, pool = M. n. nigrolineatum, terrestrial = M. oahuense; see Figs – D/ – S2 S5 for plots of each species with error bars). From these transects, Dmax and min were obtained for statistical analysis. Panels (c f) show species means with solid lines indicating the fitted regression slope for the pool species (regression analyses were conducted as contrasts against pool species; i.e. the eye size slope in Table 3). Dotted lines indicate significant interaction effects with eye size (slopes that are significantly different than the main effect slope). Habitat effects (Table 3) can be seen as differences in intercept among the ecomorph D/ categories. (c) Peak light sensitivity (Dmax) varies strongly with habitat, eye size and their interaction, but (d) peak resolution ( min) has weaker effects that lack an interaction. (e) Regional variation in sensitivity (regD) depends on microhabitat and an eye size–habitat interaction, but (f) regional variation in resolution (regD/) is related to microhabitat and not size (see Table 2 for ANOVA results). *P < 005, **P < 001, ***P < 0001.

in size (sharp high peak in D for pool, compared to lower very poor sensitivity (comparatively small Dmax, Table 1, peak for stream Fig. 5a; also compare Dmax in Fig. 5c). Fig. 5a,b). In contrast, the large stream species have less regionalized eyes. While not best in either sensitivity or resolution com- ECOLOGY OR ALLOMETRY pared to other Megalagrion, they had relatively high reso- D/ lution (small D/) and sensitivity (large Dmax) capabilities For three of the four parameters (Dmax, regD and reg ), over more of their eye when compared to species in other ecology explained more variation than allometry among microhabitats (note broader trough in D/ Fig. 5b and in species (compare P-values for the ANOVA effects in D/ Figs S2–S5). Table 2). Only for min was eye size a stronger organiz- Not all microhabitat types maximized both capabilities, ing principle than microhabitat type (eye size effect: as the terrestrial species had the best light sensitivity over- P < 002, habitat effect: P < 004; Table 2). all (highest Dmax, Fig. 5c) but the worst resolution capabil- D/ ities (largest min, Fig. 5d). In general, resolution Light sensitivity capabilities were more conserved, with greater variation in light sensitivity among microhabitat types. For example, For Dmax, in general, larger eyes had larger Dmax (positive D/ regression slope for eye size Table 3), but there is also evi- the plant and seep species had average resolution ( min) among Megalagrion damselflies, but the plant species had dence for strong ecological effects with highly significant

Table 2. ANOVA tables for maximum facet diameter (Dmax), D/ minimum interommatidial angle ( min), regionalization in facet 17 065* 3 diameter (regD) and regionalization in interommatidal angle 0 (regD/). Each variable was tested in separate ANOVAs as dependent variables explained by variation in eye size, microhabitat type and 84 5 236

their interaction as independent variables. Non-significant interac- 0 tions were dropped from the models. P values in bold indicate significant effects in the model. All models without interactions report type II and models with interaction report type III sums of squares 2** 623 30 Dependent Independent Sum Sq d.f. FP( > F) 0 contained main effects only with no

6 Dmax Eye size 5 40 1 15 09 0 0116 D/

Habitat 4886 4 3417 00008 883 188 0 Eyeht : habitat 1476 2 2065 00038

Residuals 179 5 and reg

D/ min min Eye size 0 034 1 9 31 0 0185

Habitat 0065 4 447 00415 D/ Residuals 0025 7 regD Eye size 00008 1 536 0068 93 028* 08** Habitat 00074 4 1211 0009 019*** 0 0 0 0 Eye size : habitat 0 0024 2 7 83 0 029 Residuals 00008 5 53 D/ 097 337 0 reg Eye size 0 0000 1 0 01 0 908 122 Habitat 00091 4 503 0031 0 Residuals 00032 7 effects for microhabitat type and its interaction with size D/ 041 12* 0 23 129* 0 001. 0 0 6 (see ANOVA effects in Table 2). In particular, seep species 0 0 have a different slope between Dmax and eye size from all ≤ and reg D 70

other microhabitat types, as well as plant species with *** 276 089 5 348 0 0 much smaller Dmax than pool species after controlling for 0 , reg 01, 0

the other eﬀects (Table 3, Fig. 5). min ≤

Regionalization in facet diameter exhibited a dichoto- D/ ** , mous pattern relative to ecology. Variation among the max 05, D large stream species showed that the fraction of the eye 0 2** ≤ 021 06 12 * 54 covered by relatively large D increases with larger eye size. 0 1 0 The opposite was true for the pool species, where the frac- 4 tion of the eye with relatively large D actually decreased 70 003 130 0 with larger eye size (compare slopes for regD/ regressions 0 with eye size and eye size:stream interaction in Table 3, Fig. 5). 76*** 334 016** 025 1 Resolution 07 0 0 0 0 D/ Overall, the interspeciﬁc trend in min indicates better

= 50 6 075 076 resolution with increasing eye size (slope 0 05, Fig. 5, 127 0 0 Table 3), but with different intercepts for the different microhabitat types. Therefore, pool, seep and plant species can be distinguished as having better resolution at smaller eye size in comparison with stream and terrestrial species contained the full model with eye size and microhabitat type as main effects and their interaction. The models for 23* 056* 0 046 (Table 2, Fig. 5). 16* 2 0 0 0 D Pool and seep species have the most regionalized eyes in terms of resolution, whereas plant and terrestrial species 67 8 007 107 488 and reg 0 0 are significantly less regionalized, and stream species are 0 D marginally less regionalized (compare intercepts for micro- Eye size Plant Seep Stream Terrestrial Eye size : seep Eye size : stream habitat effects in regD/ regressions Table 3). For exam- Parameter estimates for the regression slopes and intercepts (effect sizes) of ple, about 27% of the eye has above-average resolution D/

( smaller than the mid-point between the maximum and min D IO D/ max reg D D/ reg Regressions for interaction. The microhabitat termtype. was Standard parameterized errors as are contrasts given against and signiﬁcance the levels pool are microhabitat indicated types, by so asterisks: that the intercept for all other microhab itat types refers to values above that of the pool minimum ( ) in pool and seep species and about 37% of Table 3.

D/ Table 4. Model fit statistics for eye size, Dmax, min, regD and sistent with a scenario where microhabitat use provides regD/. The model with the best fit (Bayesian Information crite- strong selection on body size, with correlated variation in D rion) is listed as 0, with BIC values listed for all other models. eye size (compare Fig. 4b and Fig. 2b). Bootstrap model selection frequencies based on 2000 bootstrap replicates are included in parentheses. The microhabitat model For light capture ability, the microhabitat model pro- D/ best explained variation in all traits except for min vides a different ordering of ecomorph types for optima in Dmax, where terrestrial species have the best light capture Microhabitat (%) BM (%) Global optimum (%) ability with the largest Dmax by several lm (Table 5). Surprisingly, the smaller pool species have the second Size 0 (99) 72106 largest D , overlapping only slightly with the terrestrial Dmax 0 (932) 4541 max D/ min 4 0 (13) 0 (41) 1 0 (6) species (Table 5). Stream and seep species are evolving regD 0 (98) 6 6110 towards similar, intermediate Dmax optima, while the plant regD/ 0 (100) 130149 species have very small Dmax. These evolutionary results are again consistent with a non-allometric microhabitat

effect for Dmax, where the optima represent offsets from a the eye for plant, terrestrial and stream species (Fig. 5). common regression line (i.e. different intercepts, Fig. 2c). There is no size effect for the degree of regionalization in The evolutionary optima recovered for regionalization resolution. in light capture abilities showed the large-bodied stream species with the lowest regD, with seep and terrestrial species overlapping considerably with the stream optima SELECTION AND MICROHABITAT TYPE (Table 5). Pool species are the most regionalized by far, Evolutionary analyses confirmed that many features of showing no overlap in regD with any of the other micro- the Hawaiian damselflies studied here were best explained habitats (Table 5). by a model of divergent selection between microhabitats For regionalization of resolution capabilities, the terres- D/ (body size, Dmax, regD and regD/). For each of these trial, stream and plant species evolving towards low reg features, an adaptive model with microhabitat as the with substantial overlap in their optimal values (Table 5). selective factor was the best-fitting, strongly supported On the other hand, the seep and pool species are distinctly model (Table 4), over the alternative model of neutral more regionalized than the other three microhabitats evolution by drift or evolution towards a single global (Table 5). Thus, microhabitat has a strong evolutionary optimum. Parameters for the selection strength were influence on eye regionalization, with the pool species hav- much stronger than drift, and estimated optima that are ing the most regionalized eyes overall in both light sensitiv- consistent with the observed data (Table 5). In contrast, ity and resolution capabilities. we found no evidence of adaptive evolution in maximal Finally, in contrast to the other three functional parame- D/ resolution, min, with no models outperforming any of ters, microhabitat-based selection did not seem to influence D/ the others (Table 4). min. Instead, maximal resolution was equally well When we fit evolutionary models to our data, the follow- explained by a model of neutral evolution (Table 4). ing combinations of parameters (particularly the positions of optima for the various microhabitat types) predict how Discussion phenotypic variation may have arisen to produce the variation in species we observe today. In terms of body size, our The relationship between size, visual performance and best OUCH model was a microhabitat model with a large ecology is complex in the eyes of Hawaiian Megalagrion optima for stream species, intermediate optima for seep, damselflies. We found strong scaling in only one trait: that plant and terrestrial species, and a small body size optimum of eye size. The strong hypoallometric scaling relationship for pool species (Table 5). This evolutionary finding is con- we found (with an R2 of 095) is not unusual within the

Table 5. Parameters estimated for the microhabitat model for body size and eye morphology. Strength of selection, a, and noise, r, are shown for all morphological variables for which the microhabitat model performed best. Estimated optimal values (h) for each microhabi- l D/ D/ r D/ tat are shown for body size (mm), Dmax ( m), min (degrees), regD and reg . Only is displayed for min; it was not best explained by a selection-based model. Bootstrap 95% conﬁdence intervals for parameter estimates are in parentheses

D/ D/ Size Dmax min regD reg a 100 (953, 152) 77(23, 103) 305 (302, 386) 13 (27, 101) r 12 (295, 167) 2e-5 (4e-6, 3e-4) 002 (0.01, 003) 015 (003, 022) 001 (000, 005) hstream 60(57, 62) 318 (303, 333) 281 (265, 299) 367 (350, 387) hseep 46(43, 50) 313 (294, 330) 264 (242, 286) 277 (235, 299) hplant 46(41, 50) 283 (249, 310) 222 (192, 252) 350 (315, 381) hterr 45(40, 50) 372 (342, 422) 256 (227, 286) 374 (342, 426) hpool 39(37, 41) 336 (326, 346) 166 (154, 177) 273 (261, 285)

© 2015 The Authors. Functional Ecology © 2015 British Ecological Society, Functional Ecology 10 J.A. Scales & M.A. Butler animal kingdom (Brooke, Hanley & Laughlin 1999; Kiltie ter is purely stochastic or highly constrained. We found D/ 2000; Jander & Jander 2002; Werner & Seifan 2006; Hall that across all species min varied by only 0 36 with the & Ross 2007; Hall 2007; Rutowski, Gislen & Warrant majority of species ranging between 070 and 083. This 2009). However, for most of the other functional parame- suggests that maximum resolution is highly conserved, ters, we found that ecology, and not scaling, is a much which would be expected if the tasks of object discrimina- stronger predictor. Even more intriguing is the fact that we tion are similar across all species (and operating at roughly did not find a general trade-off between sensitivity and res- the same distance). Damselflies and all odonates are aerial olution. Instead, many species excel at both resolution predators that use visual cues to locate and capture prey and sensitivity. Smaller-eyed species can circumvent this items (Labhart & Nilsson 1995; Olberg, Worthington & physical limitation by evolving highly regionalized eyes. Venator 2000; Olberg et al. 2005). Thus, the need for excel- Finally, we found strong conservatism in interommatidal lent resolution regardless of size or habitat provides a rea- D/ angle, the trait most related to resolution ability. Below, sonable explanation for the low variation in min we discuss the functional consequences of these complex observed in Hawaiian damselflies as well as the relatively D/ patterns and their potential evolutionary explanations. small min found in odonates in general (Land 1997).

FUNCTIONAL MORPHOLOGY OF THE EYES POTENTIAL SELECTIVE PRESSURES ON VISUAL MORPHOLOGY The dominance of ecology in shaping eye geometry results in the general conclusion that bigger is not always better. In this study, we have taken the first step in trying to under- Indeed, the pool species which are the smallest in size have stand variation in the visual morphology in a radiation of the best visual performance in both sensitivity and resolu- closely related species. Our evolutionary analyses show that tion, whereas the largest species have merely moderate per- facet diameter, regionalization and body size are evolving formance. Among insects, this is a rather unusual pattern. adaptively in response to microhabitat type. What selective In general, larger-eyed species have improved performance pressures may be operative to explain these patterns? The (Jander & Jander 2002; Rutowski, Gislen & Warrant different microhabitats occupied by these damselflies may 2009). Furthermore, all of these species studied here are place different demands on the visual system for a variety of closely related, diurnal, with modest differences in ecology. reasons. The amount of clutter varies greatly across the mi- None have evolved extreme adaptations for nocturnal or crohabitats examined here. For example, the ‘pool’ micro- crepuscular lifestyles, for example. Rather, they are mem- habitat is often heavily vegetated, making it difficult to bers of an adaptive radiation that share the same forest, distinguish prey or mates from the background. Whereas and therefore, these morphological variations may provide this should primarily be a problem of resolution; light sensi- sufficient performance advantages that allow for niche tivity, colour, motion and the neural processing required to partitioning (Arnold 1983). distinguish ‘object of interest’ from background will all This unusual pattern in performance is achieved by come into play. Conversely, the ‘stream’ and some ‘plant’ expanding the dimensionality of visual performance. Rather microhabitats are open so that prey and mate detection than scaling all ommatidia equally with body size, the small- should be relatively easy against a brighter and potentially est species take advantage of high regionalization, concen- more visually uniform background. trating performance at an acute zone. The largest species do The open vs. cluttered axis of habitat variation should not have the best maximal performance, but have larger D also have a strong influence on body size and flight require- and small D/ more evenly distributed over the eye compared ments. The wide-open ‘stream’ habitats, in particular, to the other Megalagrion species (Figs S2–S5). This more expose flying insects to strong wind currents, requiring even distribution should confer improved sensitivity and res- much stronger flying abilities. Indeed, the large-bodied olution over a larger portion of the eye and visual field. ‘stream’ species are much faster fliers, and are almost never Regionalization is well known to be associated with prey found among clutter, as compared to the smaller, slower-fly- capture or mate acquisition in insects (reviewed in Land & ing species (pers. obs; Polhemus & Asquith 1996). It is inter- Nilsson 2002). However, Hawaiian damselflies all share esting to note that the evolutionary optima suggested for similar predatory and mating behaviours (Polhemus & our body size model indicate three distinct groups of body Asquith 1996). Instead, regionalization here is strongly size: large for the stream species; medium for the seep, plant associated with microhabitat specialization. Thus, as evi- and terrestrial species; and small for the pool species. As the denced by the smallest species having the best light-capture three medium-sized microhabitats are much more variable abilities, regionalization may provide a mechanism for alle- in the degree of clutter they contain, this ranking is in gen- viating the resolution-sensitivity trade-off, allowing greater eral concordance with the degree of clutter that these species flexibility in evolutionary adaptation to light levels. experience. We note also that flight speed is known to influ- In contrast to the adaptive patterns above, we found that ence eye geometry in insects, as flight speed should be maximum resolution is more conserved across species, with matched to eye morphology so as to prevent blurring by the no evidence for the influence of ecology on visual resolu- flow field that the animal experiences (Howard & Snyder tion. We therefore conclude that evolution in this parame- 1983; Land 1997). We have no quantitative data on flight

© 2015 The Authors. Functional Ecology © 2015 British Ecological Society, Functional Ecology Microhabitat use and damselfly vision 11 speeds in these damselflies to evaluate this hypothesis; thus, the adaptive significance behind the ecology–morphology it remains a potential contributing factor to the pressures link we have found, future studies should explore variation which may limit stream species in particular from evolving in microhabitat-scale differences in light levels, degree of large ommatidia, especially away from the acute zone. clutter, visual performance and locomotor requirements. Another possibility is that variation in light levels may As adaptation to microhabitat is a common organizing fac- be the selective pressure driving visual system evolution. tor in other functional systems such as locomotion and Several studies have suggested that light levels can be limit- thermoregulation (e.g. Pounds 1988; Adolph 1990; Irschick ing in insects (Land 1997; Kapustjanskij et al. 2007; & Garland 2001; Calvao et al. 2013), this raises the intrigu- Doring & Spaethe 2009), and we found that the strongest ing possibility that microhabitat variation in light levels evolutionary responses to microhabitat type are changes in may be an important factor influencing the design of visual light sensitivity and regionalization of the eye. In these systems (Endler & Thery 1996; Endler & Mielke 2005).

Hawaiian damselflies, Dmax varies widely with microhabitat, generally in a manner consistent with the apparent Acknowledgements light levels of the habitat. For example, pool-dwelling species often occur in heavily shaded microhabitats that may The authors thank D. Preston and the staff of the Bernice Pauahi Bishop Museum for providing locations for study sites and help with species identifi- place a premium on light sensitivity. Our evolutionary cation and data and animal collection. R. Schroeder and J. Walguarnery pro- analyses show that Dmax evolves under an adaptive model vided valuable help in the development of the methods. We also thank K. with strong selection, and pool damselflies have large D Arakaki, S. Evers, R.E.U.K. Higa, C. Kokami, M.A. Pena and J. Rivera for assistance with fieldwork and data collection. We thank C. King and Hawaii for their eye size. When light is not limiting, for example, DLNR for invertebrate collecting permits, R. Kallstrom and the Nature Con- over open, well-lighted streams, no additional functional servancy for collection and land use permission, and the Hawaii Divisions of advantage is gained by increasing D any further. State Parks, and Forestry and Wildlife for land use permits. We thank C. max Cressler for help with mathematical formulations. Helpful comments from K. Rather, greater functional benefit may be gained by Asao, Y. Chang, E. Henry and J. Rivera improved this manuscript. increasing spatial or temporal resolution for tracking aerial prey or mates. Interestingly, ‘stream’ species have among Data accessibility the smallest optima for Dmax, despite being the largest bodied damselflies (Table 5). 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Appendix S1. Competing evolutionary models and power analyses. Supporting Information Table S1.ANOVA table showing a strong association between body Additional Supporting information may be found in the online size and microhabitat (upper table). version of this article: Movie S1. Vertical transect of the damselﬂy eye based on photos taken every 10 degrees as the eye is rotated (M. n. nigrohamatum). Fig. S1. Variation in (a) D and (b) D/ along a lateral transect of Movie S2. Lateral transect of the damselﬂy eye based on photos the eye. taken every 10 degrees as the eye is rotated (M. n. nigrohamatum). Figs S2–S5. Species by species plots of dorsoventral transects D and D/.