Modular Diversification of the Locomotor System inDamselfishes (Pomacentridae)

Rosal�ıa Aguilar-Medrano,1,2* Bruno Fr�ed�erich,3 and Paul H. Barber1

1Department of Ecology and Evolutionary Biology, University of California, 621 Charles E. Young Dr. South,Los Angeles, California, 90095, USA2Instituto De Ecolog�ıa Aplicada, Universidad Aut�onoma De Tamaulipas, 356 Divisi�on Del Golfo, Col. Libertad,Ciudad Victoria, Tamaulipas, 87029, M�exico3Laboratoire De Morphologie Fonctionnelle Et Evolutive, AFFISH Research Center, Universit�e De Liege,Liege, Belgium

ABSTRACT As fish move and interact with theiraquatic environment by swimming, small morphologicalvariations of the locomotor system can have profoundimplications on fitness. Damselfishes (Pomacentridae)have inhabited coral reef ecosystems for more than 50million years. As such, habitat preferences and behaviorcould significantly constrain the morphology and evolv-ability of the locomotor system. To test this hypothesis,we used phylogenetic comparative methods on morpho-metric, ecological and behavioral data. While body elon-gation represented the primary source of variation in thelocomotor system of damselfishes, results also showed adiverse suite of morphological combinations betweenextreme morphologies. Results show clear associationsbetween behavior, habitat preferences, and morphology,suggesting ecological constraints on shape diversifica-tion of the locomotor system. In addition, results indicatethat the three modules of the locomotor system areweakly correlated, resulting in versatile and independ-ent characters. These results suggest that Pomacentri-dae is shape may result from the interaction between (1)integrated parts of morphological variation that main-tain overall swimming ability and (2) relatively inde-pendent parts of the morphology that facilitateadaptation and diversification. J. Morphol. 000:000–000,2016. VC 2016 Wiley Periodicals, Inc.

KEY WORDS: modularity; integration; locomotion; geo-metric morphometrics; reef fish; ecomorphology; evolu-tionary morphology

INTRODUCTION

Considerable morpho-functional diversity infishes is seen in the structures of the locomotorsystem because density-related inertial forces inaquatic systems have high impacts on swimmingperformance (Videler, 1993). Because swimming isthe primary way that fish interact and movethrough their environment (Fulton, 2010), swim-ming performance is a major determinant of (1)individual survival, (2) growth, and (3) lifetimereproductive fitness (Webb, 1994; Plaut, 2001;Blake, 2004). As such, small variations in swim-ming abilities within and among species can have

profound impacts on these three key life historyparameters (Koch and Wieser, 1983; Boisclair andSirois, 1993), making swimming performance aprimary target for natural selection (Huey andStevenson, 1979; Hertz et al., 1988).

Many studies have examined the role of body andfin shape on swimming performance in teleosts(Webb, 1982; Bushnell and Moore, 1991; Lighthill,1993; Gibb et al., 1994; Gibb et al., 1999; Druckerand Lauder, 2001, 2005; Wainwright et al., 2002;Fulton et al., 2005; Fulton, 2007; Standen, 2008;Aguilar-Medrano et al., 2013). Fewer studies haveexamined the coordination of the locomotor andfeeding system (Rice and Westneat, 2006; Higham,2007), and only one study has explicitly tested thecorrelated evolution of teleost feeding and locomotorsystems within a single clade (i.e., labrids; Collaret al., 2008). As such, it is unclear how morphologi-cal variation within a single taxonomic group maybe correlated to niche partitioning via swimmingperformance during evolution.

Pomacentridae (damselfishes) is a speciose fam-ily of 394 species of marine fish (Eschmeyer, 2014)that has inhabited reef ecosystems for more than50 million years (Bellwood, 1996; Bellwood andSorbini, 1996). The vast majority of damselfishinhabit tropical coral reefs, although some specieslive in rocky reefs or kelp forests in temperatecoastal waters. Due to their wide range of

Additional Supporting Information may be found in the onlineversion of this article.

*Correspondence to: Rosal�ıa Aguilar Medrano, Department ofEcology and Evolutionary Biology, University of California, 621Charles E. Young Dr. South, Los Angeles, CA 90095.E-mail: liabiol@gmail.com.

Received 30 September 2015; Revised 24 January 2016;Accepted 28 January 2016.

Published online 00 Month 2016 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20523

VC 2016 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 00:00–00 (2016)

behaviors and habitat preferences in structurallycomplex environments, damselfishes are an excel-lent model to test the role of the locomotor systemshape on niche partitioning and diversification.

The majority of ecomorphological studies on dam-selfishes have focused on the relationship betweenmorphology and diet (Emery, 1973; Fr�ed�erich et al.,2008; Cooper and Westneat, 2009; Aguilar-Medranoet al., 2011; Fr�ed�erich et al., 2013). Only two keystudies analyzed the ecomorphological variation ofswimming performance (Fulton, 2007; Aguilar-Medrano et al., 2013). In this study, we analyze themorphology of the locomotor system of damselfishesfrom the perspective of modularity. Modularityrefers to semiautonomous units (modules) that com-prise higher organisms (Simon, 1962; Wagner andAltenberg, 1996). Modules are assemblages of partsthat are integrated internally by strong interac-tions, but are relatively independent of one anotherby few or weak interactions (Wagner and Altenberg,1996; Klingenberg, 2005). We focus on modulesbecause of their possible role as “building blocks” ofphenotypic adaptation (Wagner, 1995).

Functional modularity is an important determi-nant of evolutionary diversification because it pro-vides a link between the modular structure ofmorphological traits and selection on performancein organismal functions (Klingenberg, 2008). Hence,in an evolutionary context, a module is a set of mor-phological characters that (1) collectively serve acommon functional role, (2) are tightly integrated,and (3) are relatively independent from other mod-ules (Mitteroecker and Bookstein, 2007).

Here, we focus on three morphological modulesthat play different roles in swimming fitness: mod-ule 1, cephalic region, associated with drag reduc-tion and lift forces (Bushnell and Moore, 1991;Lighthill, 1993; Larouche et al., 2015); module 2,trunk, dorsal, pelvic, and anal fins, associated withstability and thrust (Harris, 1938; Drucker andLauder, 2001, 2005; Fulton, 2007; Standen, 2008);and module 3, caudal peduncle, associated withacceleration and thrust (Webb, 1982; Gibb et al.,1999; Fig. 1). We study the variation along thesemorpho-functional axes in an evolutionary context,focusing on the relationship between habitat prefer-ences and behavior to address three questions: (1)how the morphological modules vary within Poma-centridae, (2) do these morpho-functional modulesevolve individually or as an integrated system?, and(3) do behavior and habitat preferences impact themorphological variation of each module?

MATERIAL AND METHODSMorphological Data

We conducted morphometric analyses on 739 lateral-viewphotographs of adult damselfish, representing 205 species fromthe time-calibrated phylogeny of Fr�ed�erich et al. (2013). Thesesamples represent 27 of 29 genera; however, the two missinggenera, Amblypomacentrus and Altrichthys, only contain two

species, are very similar in shape to Pomacentrus and Chrysip-tera (Allen, 1991) and are recently diverged from these genera(Fr�ed�erich et al., 2013). As such, the absence of these generashould not impact the results of the study. Photographs werelargely obtained directly from accessioned museum specimens,with additional photographs from online resources such asMorphBank (www.morphbank.net), FishBase (Froese andPauly, 2014) and John E. Randall’s Fish Photos (pbs.bishopmu-seum.org/images/JER). The complete list of species included inthis study, their sources and accession numbers are availableas supplementary online material 1.

We conducted landmark (LM)-based geometric morphometricanalyses (Bookstein, 1991; Rohlf and Marcus, 1993; Rohlf,1999; Zelditch et al., 2004), recording x, y coordinates of LMsand semilandmarks (SLMs) using TpsDig, version 2.17 (Rohlf,2014). SLMs were identified using a slider file made in TpsUtil(Rohlf, 2014). First, we studied the whole body shape variationusing a configuration made of 17 LMs and 28 SLMs. Then, theoverall body was divided into three modules based on func-tional analyzes: module 1: cephalic region delimited by eightLMs and 10 SLMs; module 2: trunk delimited by seven LMsand 14 SLMs, and module 3: caudal peduncle delimited by twoLMs and six SLMs (Fig. 1). For every data set, superimpositionof LM configurations was achieved using Generalized Pro-crustes Analysis (Rohlf and Slice, 1990). We calculated PartialWarp scores, and we performed a Principal Components Analy-ses (PCA) of shape variables (also called Relative Warps analy-sis, RWs) using TpsRelw, version 1.54 (Rohlf, 2014). PCA wasused to find hypothetical variables (components) that accountfor much of the variance in the morphological data (Davis,1986). Principal components scores were used as descriptors ofshape variation (Bookstein, 1991; Rohlf, 1993). Deformationgrids using the thin-plate spline algorithm were used to visual-ize the patterns of shape variations along PC axes (Bookstein,1991; Rohlf, 1993).

We tested the relevance of modules defined a priori based onfunctional considerations using Escoufier’s RV-coefficient andthe multi-set RV-coefficient (Klingenberg, 2009; Larouche et al.,2015). RV-coefficient analysis is a robust method to testwhether a priori defined modules fit observed covariance matri-ces better than a distribution of randomly generated modules ofequivalent size and number (Goswami and Polly, 2010a). TheRV coefficient is analogous to a correlation coefficient, butinstead of measuring the association between two individualvariables, it measures the association between two sets of vari-ables. It ranges from zero to one, with zero representing a case

Fig. 1. Landmaks (LMs, red dots) and semilandmarks (SLMs,blue dots) used to analyze the locomotor system of damselfishes.A: module 1, cephalic region (CR; LMs & SLMs 1–18); module 2,trunk (LMs & SLMs 19 to 25 and 34 to 45); module 3, caudalpeduncle (CP; LMs & SLMs 26 to 33).

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of perfect modularity, in which there is no covariance betweenthe sets. Thus a lower value in a priori defined modules thanin randomly generated modules indicate that none of the ran-domly generated modules are better than the a priori definedmodules. The validation of this modular configuration was doneusing the function compare modular partitions in geomorphV.20 (Adams et al., 2014) for R.

Allometric Variation

We tested the relationship between shape and size acrossspecies using a multivariate regression of shape variables(RWs) onto the centroid size (CS) (Bookstein, 1991; Monteiro,1999) using TpsRegr, version 1.41 (Rohlf, 2014).

Ecological and Behavioral Diversity

We collected ecological and behavioral data from publishedliterature for each species, focusing on four major characters.First, we recorded wave exposure, binning this variable into:(1) zero to low, (2) moderate, and (3) high wave exposure. Sec-ond, we characterized position in the water column in relationto the substratum as: (1) on the substratum (e.g., territorials)/close to the substratum, (2) midwater/close to the surface, or (3)both. Finally we characterized behavior as: (1) perennial terri-torials, solitary, or couples, (2) schooling non-perennial territori-als, or (3) solitary, pairs, or small groups non-perennialterritorials (Supporting Information 1).

Phylogenetic Comparative Methods

We examined the evolution of morphological and ecologicaltraits using the time calibrated phylogeny of Fr�ed�erich et al.(2013). We hypothesized that evolution should favor integrationwithin the swimming system. So, we used a phylogenetic gener-alized least square method (PGLS) that incorporated Brownianmotion (BM) to test for a correlation between the three func-tional modules.

To test for a correlation between ecology and morphology, weused PGLS methods that incorporated two models of trait evo-lution, including BM, in which disparity increases as a functionof time (Harmon et al., 2003), and the Ornstein-Uhlenbeck(OU) model that incorporates one or more optimum trait valuehn and strength of selection a (Beaulieu et al., 2012). In otherwords, BM model is a completely stochastic model, while theOU-model incorporates both stochastic and stabilizing compo-nents (Butler and King, 2004). To identify the pattern of mor-phological diversification, we compared the fit of a BM-modeland OU-model for each one of the three ecological variables: (a)wave exposure; (b) position in the water column in relation tothe substratum; and (c) behavior. If these ecological variablesshaped the evolution of damselfish morphology, we expectedthat the OU-model would show better fit than the BM model.To evaluate the best model for our data, we compared the fit ofboth models using Akaike’s information criterion (AIC), whichconsiders the trade-off between the fit and the complexity ofthe model (Akaike, 1973).

For PGLS analysis, we used packages in the R environmentversion 3.0.3 (The R Foundation for Statistical Computing2014): NLME (Pinheiro et al., 2014), GEIGER (Harmon et al.,2008), APE (Paradis et al., 2004), and CAPER (Orme et al.,2011).

RESULTSMain Axes of Morphological Variation

For each module, we considered the PCs thatexplained at least 70% of the shape variation (Can-gelosi and Goriely, 2007). For the cephalic region,four PCs accounted for 71% of shape variation(PC1 5 30%, PC2 5 22%, PC3 5 11%, PC4 5 8%). In

the trunk, three PCs accounted for 75% of the shapevariation (PC1 5 48%, PC2 5 17%, PC3 5 10). Thecaudal peduncle was the least complex module withtwo PCs accounting for 81% of shape variation(PC1 5 68%, PC2 5 13%). For each module, the firstaxis of shape variation (PC1) was related to an elon-gation along the rostro-caudal axis of the body. Foranalyses of the whole body, the first four PCsaccounted for 71% of the total variation (PC1 5 41%;PC2 5 12%; PC3 5 10%; PC4 5 8%; SupportingInformation 2).

Within the cephalic region (module 1) PC1shows that species with horizontally lengthenedcephalic profiles, typically have a snout at thesame height or higher than the eyes, big eyes, aninferior region of the cephalic profile that is elon-gated downwards and backwards, a supraoccipitalcrest (SLMs 17 and 18) that is angled backwardsand a more angular opercular margin (e.g., Azur-ina and Lepidozygus; Fig. 2B, PC1-). Conversely,species with horizontally short and vertically longcephalic profiles present the opposite characterstates (e.g., Dascyllus; Fig. 2A, PC11). The secondaxis of variation shows that in species with bigeyes close to the snout, the frontal area of thecephalic region is biggest and an elongated supra-occipital crest (SLMs 17 and 18; e.g., Azurina andsome Chromis species; Fig. 2A,B, PC21). In con-trast, species with smaller eyes located below thesnout present the opposite character states (e.g.,Amphiprion; Fig. 2A, PC22). The third axis of var-iation shows that in species with big eyes close tothe snout, the frontal area is higher, producing amore rounded cephalic profile (e.g., some Pomacen-trus, Chrysiptera Chromis, and Neopomacentrusspecies; Fig. 2C, PC32), while species with smallereyes located below the snout present the oppositecharacter (e.g., some Abudefduf and Microspatho-don species; Figs. 2C and 3D, PC31). Finally, PC4segregates species with big eyes and almost roundcephalic profiles (e.g., some Chromis, Dascyllus,and Pomachromis species; Figs. 2C and 3D,PC41) from medium sized eyes with and deep-narrow cephalic profiles (e.g., some Abudefduf andLepidozygus species; Figs. 2C and 3D, PC42).

The first axis of variation of the trunk (module2) is related to an elongated profile of the trunkregion resulting from a dorsal/ventral compressionof the trunk, with greater compression in the cen-ter and anterior edges and less in the posterior.This morphology is associated with an elongatedbase of the pectoral fin, which is positioned ante-rior to the pelvic fin and the origin of the dorsalfin (e.g., some Lepidozygus and Azurina species;Fig. 3B, PC11). The opposite morphology, shorterprofile of the trunk region, is associated with thePC12 axis (e.g., Dascyllus; Fig. 3A, PC12). Thesecond axis of variation shows that in species withpectoral fins positioned anterior to the pelvic finand origin of the dorsal fin, the dorsal and anal

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fins are typically short resulting in a longer ventraldistance between the pelvic and anal fin (e.g., someAbudefduf and Azurina species; Fig. 3A,B, PC22).The opposite morphology, more elongated dorsaland anal fin, is associated with the PC21 axis (e.g.,some Amphiprion, Chromis, and Premnas species;Fig. 3A,B, PC21). Finally, PC3 show that in specieswith pectoral fin positioned approximately at thesame high of the pelvic fin, the anal fin is typicallylong and the dorsal fin short (e.g., some Lepidozygusspecies; Fig. 3D, PC31). The opposite morphology,pectoral fins positioned anterior of the pelvic fin,more elongated dorsal fin and shorter anal fin, isassociated with the PC32 axis (e.g., some Stegastes,Chrysiptera, and Neophomacentrus species; Fig. 3C,PC32).

The main axis of variation of the caudal peduncle(module 3) is elongation and degree of curvature.The first axis of variation shows that long caudalpeduncles are more curved (e.g., Azurina; Fig. 4B,PC12) than the short ones (e.g., some Abudefduf;Fig. 4A, PC11). The second axis of variation showsthat species with an elongated ventral edge presenta less curved posterior edge (e.g., some Chromis spe-cies; Fig. 4A, PC21), while species with a shorterventral edge present a more curved posterior edge(e.g., Amphiprion; Fig. 4A, PC21).

For the analyses of whole body shape, the firstaxis of variation showed that in elongated specieswhere the eye and the snout are at a similar height,the pectoral fin is anterior to the pelvic fin and thedorsal fin origin. There is a general elongation ofthe cephalic region and caudal peduncle in a verti-

cal axis, in relation to the trunk (e.g., Azurina andLepidozygus; Fig. 5B, PC11). The opposite charac-ters, deep-bodied species, are associated with PC12

axis (e.g., Dascyllus; Fig. 5A, PC12). The PC2showed a segregation among species with relativelyelongated cephalic shape, less curved caudalpeduncle, shorter anal and dorsal fins, and pectoralfins in a less horizontal angle of insertion (e.g., someAbudefduf, Chromis, and Azurina species; Fig.5A,B, PC22). The PC21 present the contrary char-acters, elongated anal and dorsal fin (e.g., Premnasand Pristotis; Fig. 5B, PC21).

Inter-Specific Allometry

Results showed CS was a significant predictor ofshape variation (P< 0.001), but only for a low per-centage of shape variation. The trunk shape wasmost strongly correlated to size (r2 5 0.3; df 5 38–27740; F 5 350), such that elongated trunks tendto be smaller than deep-trunks. A smaller propor-tion of shape variation was explained by size vari-ation in the caudal peduncle (r2 5 20.08; df 5 12–8760; F 5 61) and the cephalic region (r2 5 0.02;df 5 30–21900; F 5 12). Larger caudal pedunclestend to be more curved than the smaller ones, andlarger cephalic regions are generally more verti-cally elongated than smaller ones (Fig. 6).

Test of Modular ConfigurationDefined A Priori

The RV-coefficient value obtained for prioridefined modules (RV 5 0.57; P 5 0.01; Supporting

Fig. 2. Principal components analysis of the cephalic region. To highlight genera with few species the same graph is presentedtwice. Graph A includes all genera with more than five species while graph B only includes genera with less than five species. PC,principal component. Thin plate spline deformation grids represent the deformation of the most extreme specimen in each directionof the PC axes versus the grand mean.

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Information 3) is lower that any randomly gener-ated modules (RV 5 0.69–1) indicating that ourpriori defined modules are better option of mod-ules segregation than any randomly generatedpossibility.

Modular Evolution

PGLS analysis revealed limited relationshipamong modules, supporting a modular evolution ofthe body parts (Table 1). In total, only 16 of 36comparisons revealed a significant correlationbetween axis of variation in the three modules andphylogenetic relationship. Although each module

is significantly related to at least one other module(P� 0.05), the correlation values were always verylow (r2 value �0.02, Table 1).

Ecological Driving Forces

Models of trait evolution showed strong supportfor the OU-model mainly in the second, third, andfourth axes of variation (PC2, 3, and 4; Table 2)across all modules and all ecological variables.Behavior and position in the water column arerelated to cephalic region and caudal peduncle inaxes representing a high percentage of the totalvariation (behavior, module 1, PC2 5 23% and

Fig. 3. Principal components analysis of the trunk. To highlight genera with few species the same graph is presented twice. GraphA includes all genera with more than five species while graph B only includes genera with less than five species. PC, principal com-ponent. Thin plate spline deformation grids represent the deformation of the most extreme specimen in each direction of the PC axesversus the grand mean. Red dots in the grids represent fin insertions.

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module 3, PC2 5 13%; position in the water col-umn, module 1, PC3 5 11% and module 3,PC2 5 13%). Conversely, wave exposure is relatedto all modules, in axes representing a lower per-centage of the total variation (module 1,PC3 5 11% and PC4 5 7%, module 2, PC3 5 10%and module 3, PC2 5 13%; see the morphologicalvariation related to each axis on Results; Mainaxes of morphological variation). Thus, our resultsclearly indicate that the ecological variables ana-lyzed here influenced the shape of major anatomi-cal modules during the evolutionary processes ofthe Pomacentridae family.

DISCUSSION

Although organisms are commonly viewed asintegrated morphological units, they can also beviewed as a collection of semi-autonomous moduleswith unique positions and functions. Therefore,

understanding the adaptation, evolution, and diver-gence within groups of organisms requires under-standing how the underlying morphological andfunctional interactions act on these units (modules)and how changes in one unit may impact other unitsand functions across phylogenies (Zelditch and Car-michael, 1989; Marroig and Cheverud, 2001; Youngand Hallgr�ımsson, 2005; Goswami, 2006a,b; Klin-genberg, 2008; Singh et al., 2012).

By examining morphometric and ecological datafrom 204 species of damselfishes (including 27 of29 genera) in a comparative phylogenetic frame-work, this study found evidence for modular evolu-tion in the locomotor system of damselfishes.Specifically, results show that the modules of thedamselfish locomotor system are constrained bybehavior and habitat preferences. The integrationof these modules produces internal constraints onthe overall body morphology, resulting in distinc-tive morphologies. However, the integration among

Fig. 4. Principal components analysis of the caudal peduncle. To highlight genera with few species the same graph is presentedtwice. Graph A includes all genera with more than five species while graph B only includes genera with less than five species. PC,principal component. Thin plate spline deformation grids represent the deformation of the most extreme specimen in each directionof the PC axes versus the grand mean.

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locomotory modules is low (Table 1), potentiallyproviding flexibility in generating morphologicaldiversity that could contribute to species diversity.

Modularity and Ecological Constraints onLocomotor System Shape of Damselfishes

There are different ways to conceptualize mor-phological modules, including origins during devel-opment (Monteiro et al., 2005), integration amongLMs with similar patterns of variation (Goswami,2006a,b), and interactions of traits that performone or more coordinated functions (Klingenberg,2008). The latter “functional modality” definition(Mitteroecker and Bookstein, 2007) is more appro-priate for characterizing the damselfish locomotorysystem because the cephalic region, trunk, andcaudal peduncle collectively serve a common func-tional role, are integrated, and relatively inde-pendent from other modules.

Each module was described according to its ownfunctions; however they all serve the same pur-pose, locomotion. For example, the trunk, dorsal,

pelvic and anal fins are associated with stabilityand thrust (Harris, 1938; Drucker and Lauder,2001, 2005; Fulton, 2007; Standen, 2008), whilethe caudal peduncle is associated with accelerationand thrust (Webb, 1982; Gibb et al., 1999), how-ever, both trunk and caudal peduncle functiontogether to increase and keep speeds during loco-motion. Thus, even if our results show that ana-tomically nearby modules are more related amongit, these results are related to share functions.

To our knowledge, only few studies have ana-lyzed morphological modularity of fishes consider-ing the whole body. Those analyses resulted insomewhat diverging results. Mabee et al. (2002)considered the anal and dorsal fins as a singlemodule and the pelvic and pectoral fins separately.Larouche et al. (2015) just two modules in thewhole body, the cephalic region as one module,and trunk-caudal peduncle, including all fins asthe second module. Our results partially agreedwith both suggestions. We found a strong relationamong the caudal and dorsal region, and a rela-tion among the pectoral and pelvic fins (Fig. 3).

Fig. 5. Principal components analysis of the whole body. To highlight genera with few species the same graph is presented twice.Graph A includes all genera with more than five species while graph B only includes genera with less than five species. PC, principalcomponent. Thin plate spline deformation grids represent the deformation of the most extreme specimen in each direction of the PCaxes versus the grand mean. Red dots in the grids represent the fins.

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Also when we found a level of integration amongthe trunk and the caudal peduncle, our result stillsupport the presence of three modules in thewhole body.

The largest source of morphological variationobserved in the damselfish locomotor system wasrelated to the rostro-caudal elongation of the fishbody, a major trend in shape diversity in reeffishes (Ward and Mehta, 2010; Aguilar-Medranoet al., 2011; Aguilar-Medrano, 2013; Claverie andWainwright, 2014). Although, it is interesting tonote that the three studied ecological variables donot show significant relationships with the firstaxis of variation but instead with the secondaryaxes. This might indicate that the rostro-caudalelongation of the body presents a strong phyloge-netic signal in this fish group, while all secondarycharacters have being constrained by ecology.

Most elongated damselfish were largely school-ing pelagic feeders, inhabiting the midwater col-umn or close to the surface (SupportingInformation 1). These species are generally small,and their elongated body tended to be associatedwith angular cephalic regions, creating a morestreamlined profile that decreases drag andincreases swimming speeds (Bushnell and Moore,1991; Lighthill, 1993). This morphology was alsoassociated with big eyes that are level with thesnout, a common characteristic of predatory fishesthat improves the efficiency of feeding (Griffinet al., 2001; Karplus and Algom, 1981). Lastly,elongated body forms were associated with anelongated caudal peduncle that is curved on thehorizontal edge and posterior edge, creating alarge area of thrust that promotes rapid accelera-tion (Webb, 1984; Webb and Weihs, 1986; Lauder,2000). The combination of these morphologicaltraits results in species that are fast and efficientswimmers, capable of rapid accelerations (e.g.,Azurina and Lepidozygus), characteristics requiredto successfully exploit midwater habitats as wellas those close to the surface.

In contrast, most deep-bodied damselfishes areterritorial, benthic feeders, and inhabit environ-ments close to the substratum (Fr�ed�erich et al.,2008; Cooper and Westneat, 2009; Aguilar-Medrano et al., 2011), and are larger than

Fig. 6. Morphological variation related to size in the threemodules of the locomotor system of damselfishes. Numbers rep-resent the centroid size.

TABLE 1. Results from phylogenetic generalized least squares analyses using a Brownian motion model for testing correlated evolu-tion between morphological modules: Module 1, cephalic region; Module 2, trunk; and Module 3, caudal peduncle

Cephalic region Trunk Caudal peduncle

PC1 PC2 PC3 PC4 PC1 PC2 PC3 PC1

Cephalic regi�on PC1PC2 nsPC3 ns nsPC4 ns ns 0.01*

Trunk PC1 0.02* 0.02* 0.02* nsPC2 20.01* 20.01* 20.01* ns 20.01*PC3 ns 20.02* 20.02* ns ns 20.02*

Caudal peduncle PC1 20.01* 20.01* 20.01* ns 20.01* 20.01* nsPC2 ns Ns ns ns ns ns ns ns

Significant correlations are indicated by an asterisk, while ns indicate no significant results. PCs refer to each principalcomponent.

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schooling damselfishes. Results showed that deep-bodied species generally have deep cephalic profileswith lower positioned snouts that allow them tograze on algae, or prey on benthic invertebrateswhile maintaining visual awareness of predators(Bellwood et al., 2014). In addition, these speciestypically have short caudal peduncles that facilitatestrong initial swimming bursts rather than sus-tained fast swimming required of midwater species.Furthermore, these species have deep-trunks thatfavor stability at slow swimming speeds, and arelikely advantageous for negotiating structurallycomplex habitats (e.g., Dascyllus aruanus living inbranching corals; Webb, 1982; Fulton, 2007).

Despite this generality, numerous taxa deviatedfrom this pattern. For example Amphiprion pre-sented a rounded cephalic profile, a trait com-monly observed in benthic species, but its trunkand caudal peduncle were relatively long and simi-lar to species living in the upper and mid-watercolumn. In contrast, Hypsypops exhibited an angu-lar cephalic profile, characteristic of species fromthe water column, but also had a high trunk andshort caudal peduncle, traits generally associatedwith benthic habitats. Thus, while the evolution ofpomacentrids is biased in specific phenotypic direc-tions due to general rules that link the evolution

of modules of the locomotor system (Klingenberg,2005), these links are flexible. The low associationamong modules allows morphological diversity toincrease by producing unexpected combinations.In turn, this variation may facilitate shifts intonovel niche spaces, contributing to lineage diversi-fication in pomacentrids.

Previous studies have demonstrated the relation-ship between cephalic shape and diet in damsel-fishes (Emery, 1973; Gluckmann and Vandewalle,1998; Aguilar-Medrano et al., 2011; Fr�ed�erich et al.,2013). While the ecological variables studied in ouranalysis are highly related to locomotion, they alsoinfluence feeding behavior. For example, deep-bodies species with short caudal peduncles weretypically associated with slower, more maneuver-able swimmers (Bartol et al., 2002; Fulton, 2007;Aguilar-Medrano, 2013). In turn, these characteris-tics were associated with being territorial and her-bivorous (Fr�ed�erich et al., 2008; Cooper andWestneat, 2009; Aguilar-Medrano et al., 2011). Sim-ilarly, results showed that elongated species withlonger caudal peduncles were associated withfaster, more efficient swimming (Videler, 1993; Ful-ton, 2007; Aguilar-Medrano, 2013); these specieswere typically schooling and mostly zooplanktivo-rous (Fr�ed�erich et al., 2008; Cooper and Westneat,

TABLE 2. Results of phylogenetic generalized least squares analyses testing correlated evolution between morphology and ecology

Wave exposurePosition in the water

column Behavior

AIC Corr. AIC Corr. AIC Corr.

Whole body PC1 BM 2812.08 0.004* 2806.45 0.000 2809.17 20.001OU 2687.34 0.003 2686.84 0.003 2690.27 0.009

PC2 BM 2831.40 0.000 2846.50 0.005* 2833.32 0.002OU 2936.07 20.006* 2930.93 0.002 2931.29 20.001

PC3 BM 2831.28 0.002 2836.26 0.003* 2831.96 0.002OU 21,047.55 20.001 21,046.14 0.000 21,049.23 20.003

PC4 BM 21,018.83 0.000 21,019.09 0.001 21,023.00 20.005OU 21,059.78 0.003 21,057.73 20.002 21,079.59 20.009*

Module 1 Cephalic regi�on PC1 BM 2605.10 0.008* 2606.13 0.006* 2601.40 0.001OU 2552.53 0.000 2553.62 0.006 2570.03 20.029*

PC2 BM 2649.38 0.003 2664.71 20.008* 2651.80 0.009OU 2610.05 20.001 2610.89 20.005 2655.71 0.040*

PC3 BM 2623.04 20.012* 2616.40 0.006* 2611.70 20.002OU 2800.40 20.007* 2804.24 0.007* 2796.20 20.001

PC4 BM 2642.36 20.004 2699.05 0.014* 2643.41 20.006OU 2794.05 20.007* 2788.80 0.000 2790.81 20.004

Module 2 trunk PC1 BM 2726.34 20.009* 2712.94 20.001 2715.48 20.002OU 2599.56 20.005 2598.19 0.002 2603.65 20.014*

PC2 BM 2774.47 0.005* 2770.41 20.001 2772.61 20.004OU 2833.52 0.006 2829.78 0.002 2831.17 20.003

PC3 BM 2863.82 0.000 2863.90 0.001 2868.08 20.007OU 2952.95 0.005* 2947.45 20.001 2950.08 20.003

Module 3 Caudal peduncle PC1 BM 2497.20 0.006 2496.62 0.004 2497.55 0.003OU 2458.71 20.006 2468.17 0.018* 2458.81 20.005

PC2 BM 2636.52 0.005 2636.36 0.004 2635.89 20.001OU 2790.93 0.009* 2793.38 20.007* 2799.06 0.015*

Two models were used, BM (Brownian motion) and OU (Ornstein-Uhlenbeck) and compared using Akaike information criterionscores (AIC). Corr.: correlation values. Bold values indicate the best fitted model. Asterisk indicated significant correlation (P5<0.05). Dashed circled values indicated significant correlation on BM as best fitted model and solid circled values indicated sig-nificant correlation on OU as best fitted model.

9MODULAR DIVERSIFICATION OF THE LOCOMOTOR SYSTEM IN DAMSELFISHES

Journal of Morphology

2009; Aguilar-Medrano et al., 2011). The strong con-nections observed between locomotion and feedingin damselfishes (see above) is similarly seen in otherfishes, such as the Labridae (Collar et al., 2008) sug-gesting that constraints of swimming and feeding inan aquatic medium can have similar effects acrossdistantly related taxa.

Morphological Evolution of the DamselfishLocomotor System

Our study system highlights the balance betweenevolvability and morphological constraints. Evolv-ability is the potential of populations to respond tonatural selection and evolve (Wagner and Altenberg,1996; Klingenberg, 2005; Singh et al., 2012) rising tonovel phenotypes, while the morphological con-straints imposed by integration of morphology withinlocomotor modules reduces the potential to varywithin a population, conserving aspects across evolu-tionary time (Klingenberg, 2005; Singh et al., 2012).

Evolutionary analysis of damselfish morphologyilluminates a limited range of morphological varia-tion and a tendency to repeat shapes through clades(Cooper and Westneat, 2009; Fr�ed�erich et al., 2013).In particular, the integration among the cephalicregion, trunk, and caudal peduncle suggest theaction of morphological constraints because theintegration of modules potentially limits the varia-tion of each module (Martin et al., 2005). As such,evolutionary change is biased in specific phenotypicdirections (Klingenberg, 2005), potentially slowingthe rate of evolution and/or constraining morpholog-ical evolution to a smaller range of possible varia-tion (Goswami and Polly, 2010b). Thus, theintegration among modules may explain the limitedrange of morphological variation in pomacentrids(giving this group their distinctive shape) and thetendency to reiterate patterns throughout the evo-lutionary history of this group.

While results suggest integration among modules,a high percentage of the morphological variationwithin damselfishes shows little integration. Thefreedom to evolve more independently from themain body pattern may result in greater morpholog-ical variation and species diversity in some lineagesof pomacentrids. For example, a higher level of mod-ularity has probably operated for the evolution ofAmphiprion. This genus combines rounded cephalicprofile with elongated trunk and caudal peduncle,and this atypical combination of shapes may be con-sidered as a novelty in damselfishes. In addition tophysiological adaptations, this modular noveltycould be related to the colonization of untappedniches such as sea anemones and could explain thehigh rate of diversification observed in the clade ofclownfishes (Litsios et al., 2012). Such “independent”characters are likely critical for evolvability byincreasing the potential for morphological variation(Goswami and Polly, 2010b).

As expected, behavior and habitat preferencesimpact the morphological variation of each moduleof the fish body during the evolution. Thus, selec-tion pressure and integration of damselfish loco-motory modules should result in a relatively lownumber of morphological possibilities and corre-spondingly limited morphological diversity. How-ever, evolution has produced lineages ofdamselfish that do not seem to follow these con-straints. Hence the results suggest that the evolu-tionary success of Pomacentridae may lie in thedelicately balanced interaction between integratedparts of the morphology that produce a functionalbody design and weakly integrated or relativelyindependent morphological aspects that facilitateadaptation and diversification.

ACKNOWLEDGMENTS

Consejo Nacional de Ciencia y Tecnolog�ıa (CON-ACYT), Mexico and University of California, Insti-tute for Mexico and the United States (UCMEXUS), USA funded the Postdoctoral research ofR. Aguilar-Medrano. B. Fr�ed�erich is a Postdoctoralresearcher of the F.R.S.-FNRS (Belgium).

CONFLICT OF INTEREST

The authors declare that they have no conflict ofinterest.

ETHICAL APPROVAL

None of the organisms used in this analysiswere collected in the field. Most photographs wereobtained from accessioned museum specimens,with additional photographs obtained from onlineresources such as MorphBank (www.morphbank.net), FishBase (Froese and Pauly 2014), and JohnE. Randall’s Fish Photos (pbs.bishopmuseum.org/images/JER).

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