Perciformes: Pomacentridae) of the Eastern Pacific

Home , Abudefduf, Abudefduf notatus, Abudefduf troschelii, Azurina, Chrominae, Chromis punctipinnis

LINNE AN .«ito/ BIOLOGICAL “W s o c í e T Y JournalLirmean Society

Biological Journal of the Linnean Society, 2011, 102, 593-613. With 9 figures

Patterns of morphological evolution of the cephalic region in damselfishes (Perciformes: Pomacentridae) of the Eastern Pacific

ROSALÍA AGUILAR-MEDRANO1*, BRUNO FRÉDÉRICH2, EFRAÍN DE LUNA 3 and EDUARDO F. BALART1 laboratorio de Necton y Ecología de Arrecifes, y Colección Ictiológica, Centro de Investigaciones Biológicas del Noroeste, La Paz, B.C.S. 23090 México 2Laboratoire de Morphologie fonctionnelle et évolutive, Institut de Chimie (B6c), Université de Liège, B-4000 Liège, Belgium 3Departamento de Biodiversidad y Sistemática, Instituto de Ecología, AC, Xalapa, Veracruz 91000 México

Received 20 May 2010; revised 21 September 2010; accepted for publication 22 September 2010

Pomacentridae are one of the most abundant fish families inhabiting reefs of tropical and temperate regions. This family, comprising 29 genera, shows a remarkable diversity of habitat preferences, feeding, and behaviours. Twenty-four species belonging to seven genera have been reported in the Eastern Pacific region. The present study focuses on the relationship between the diet and the cephalic profile in the 24 endemic damselfishes of this region. Feeding habits were determined by means of underwater observations and the gathering of bibliographic data. Variations in cephalic profile were analyzed by means of geometric morphometries and phylogenetic methods. The present study shows that the 24 species can be grouped into three main trophic guilds: zooplanktivores, algivores, and an intermediate group feeding on small pelagic and benthic preys. Shape variations were low within each genus except for Abudefduf. Phylogenetically adjusted regression reveals that head shape can be explained by differences in feeding habits. The morphometric phylogeny recovered the subfamily Stegastinae and the relation ship between Abudefduf troschelii and Chromis species. The cephalic profile of damselfishes contains a clear and strong phylogenetic signal. © 2011 The Linnean Society of London,Biological Journal of the Linnean Society, 2011, 102, 593-613.

ADDITIONAL KEYWORDS: Gulf of California - head shape - phylogenetic morphometries - reef fishes - trophic niche.

INTRODUCTION (California, USA) in the north to the south of Chile, including all the oceanic islands of this region (Fig. 1). The damselfishes (Pomacentridae) are a species-rich In this part of the Pacific, Pomacentridae is one (approximately 360), worldwide distributed family of of the most abundant families, having radiated in marine fishes that inhabit tropical and temperate coral reefs, rocky reefs, and kelp forests. Twenty-four waters (Allen, 1991). Most inhabit tropical coral reefs, species belonging to seven genera (i.e. Stegastes, although a number of species live in rocky reefs of Microspathodon, Hypsypops, Nexilosus, Chromis, cooler temperate waters. They have been present Azurina, and Abudefduf) have been reported in the within these ecosystems for 50 million years (Bell- Eastern Pacific and all are endemic to this region wood, 1996; Bellwood & Sorbini, 1996). In the Eastern (Robertson & Allen, 2008). The genera Azurina, Pacific, their range extends from Monterey Bay Hypsypops, and Nexilosus are solely present in this part of the Pacific, whereas the genus Microspath “■“Corresponding author. E-mail:[email protected] odon contains two species in the Eastern Pacific and

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F ig u r e 1. Distribution of damselfish species in the coast and islands of the Eastern Pacific Ocean. Left: localities of underwater observations in the Gulf of California: National Marine Park of Loreto, La Paz Bay, and National Marine Park of Cabo Pulmo. two others in the Atlantic Ocean. On the other hand, Pomacentrinae the genera A budefduf, Chrom is, and Stegastes are A budefduf Abudefdufinae distributed worldwide (Allen, 1991). All genera rep resented in this region belong to the basal groups Chromis Chrom inae of the family Pomacentridae (i.e. the subfamilies Ste- Pomacentridae Lepidoziginae gastinae, Chrominae and Abudefdufinae; as defined by Cooper, Smith & Westneat, 2009) (Fig. 2). The Microspathodon members of the most derived subfamily Pomacentri Hypsypops nae have radiated in the Indo-West Pacific region only Nexilosus (Cooper et al., 2009). Despite the use of numerous Stegastinae molecular data, the phylogenetic relationships within Stegastes the family Pomacentridae are not fully resolved Plectroglyphydodon

(Tang, 2001; Quenouille, Bermingham & Planes, Parma 2004; Cooperet al., 2009). Similiparma The damselfishes of the Eastern Pacific display a remarkable diversity with regards to habitat prefer O utgroup ences, feeding habits, and behaviours. Their colora F ig u r e 2. Phylogeny of Pomacentridae family sensu tion is highly variable, ranging from drab hues of Cooper etal. (2009). Dotted lines refer to groups not brown, grey, and black to brilliant combinations of represented in the Eastern Pacific region. orange, yellow, and neon blue (Robertson & Allen, 2008). Generally speaking, the damselfishes living in coral reefs of the Indo-West Pacific region feed of the brightly patterned species (e.g. members mainly on filamentous algae as well as small plank of the genus Chromis) obtain their nourish tonic and benthic invertebrates (Allen, 1991; Kuo ment from the current-borne plankton (Allen, 1991). & Shao, 1991; Frédérich et a l ., 2009). The drab- To our knowledge, the trophic ecology of only two coloured species feed mainly on algae, whereas most damselfish species from the Eastern Pacific has

© 2011 The Linnean Society of London,Biological Journal of the Linnean Society, 2011, 102, 593-613 HEAD SHAPE DIVERSITY IN DAMSELFISHES 595 been studied in detail: Stegastes rectifraenum and and/or phylogeny. In particular, we investigate the Microspathodon dorsalis (Montgomery, 1980). ecomorphological hypothesis that cephalic shape Ecomorphological studies attempt to understand variation is associated with dietary differences in the relationships between the morphological varia damselfishes of the Eastern Pacific. On the basis tion among species and their corresponding ecolo of previous analyses in damselfishes (Emery, 1973; gical variation (Norton, Luczkovich & Motta, 1995; Gluckmann & Vandewalle, 1998; Frédérich et al., Wainwright, 1996; Costa & Cataudella, 2007). For 2008; Cooper & Westneat, 2009), we predict that diet example, head morphology is subject to various con is correlated to shape variations. We also test the straints dealing with the strategy of feeding and the presence of phylogenetic signal (i.e. the expectation type of ingested food (Liem, 1979, 1993; Wainwright that the phylogenetic relatedness is associated with & Richard, 1995). The relationship between cephalic shape similarity) (Cardini & Elton, 2008) in the morphology and diet has been broadly studied in cephalic region of damselfishes. We have no a priori freshwater fishes such as perches (Svanbäck & Eklöv, hypothesis regarding this relationship because such 2002), centrarchids (Collar, Near & Wainwright, studies are limited in fishes (Guill, Heins & Hood, 2005), and cichlids (Barel, 1983; Albertson, Streelman 2003). & Kocher, 2003), as well as marine fishes such as sparids (Costa & Cataudella, 2007) and labrids (West- neat, 1995; Streelman & Karl, 1997; Wainwright MATERIAL AND METHODS et al., 2004). The first ecomorphological studies in Pomacentridae (Emery, 1973; Gluckmann & Vande- T r o p h ic d a ta walle, 1998) suggested that a detailed study of cepha Underwater observations (UO) were carried out in lic morphology could reveal different trophic groups. three areas in the Gulf of California, Mexico (Fig. 1): Using geometric morphometries (Rohlf & Marcus, (1) N ational M arine P ark of Loreto Bay; (2) La Paz 1993; Lawing & Polly, 2009), Frédérich et al. (2008) Bay; and (3) National Marine Park of Cabo Pulmo. highlighted strong size and shape variations in four The National Marine Park of Loreto Bay is located at skeletal units of the damselfish skull among species 26°07' to 25°43'N and 111°21' to 111°13'W Coronado, living in the Indo-West Pacific region and belonging to Carmen and Danzante islands delimit this area different trophic groups. Recently, Cooper & Westneat where the substrate composition varies from fine sand (2009) investigated the evolution of skull shape to cobbles, boulders, and rocks (Campos-Dávila et al., within Pomacentridae and showed that planktivory 2005). La Paz Bay is located at 24°07' to 24°21'N and has involved important changes in damselfish head 110°17' to 110°40'W, the reef presents fine sand, big morphology. In this latter study, the authors used (30 m) and small (12 m) boulders, big walls (15 m), only one species per genus to infer morphological and rocky reef (Aburto-Oropeza & Balart, 2001). The evolution. Consequently, the inference of intra-generic National Marine Park of Cabo Pulmo is the north variation in trophic morphology was not possible. ernmost coral reef in the Eastern Pacific and is Recently, Barneche et al. (2009) confirmed the appli located near the entrance of the Gulf of California in cability of Bergmann’s rule to the family Pomacen a transitional zone between the tropical and tem tridae, stating that the larger species are found at perate Pacific at 23°50'N, 109°25'W (Alvarez-Filip, higher latitudes. The damselfishes of the Eastern Reyes-Bonilla & Calderón-Aguilera, 2006). This park Pacific are distributed along a wide range of latitudes presents a barrier reef and a lagoon composed of (-33°N to 35°S; Fig. 1) and show a great variation of small boulders with sandy areas. The UO were body size among species (standard length in the range carried out during January, May, and August 2008, 11-36 cm; Table 1). Moreover, the genera Chromis, and January and March 2009, by scuba diving and Hypsypops, Nexilosus and, Microspathodon contain snorkeling. Observations of territorial species were the largest damselfishes (Allen, 1991). This axis of carried out for 2 min per organism; for fishes in body size variation could have promoted morphologi constant movement (e.g. planktivorous), a maximum cal variation within this group of endemic species. of 10 min were devoted per organism. Observations In the present study, we collect trophic data about were conducted on 20-100 individuals per species, all endemic damselfish species of the Eastern Pacific according to the availability. During these observa region and apply geometric morphometric techniques tions, we mainly focused on two main components of and phylogenetic methods to the cephalic region of their trophic ecology: (1) social behaviour: territorial these damselfishes. The main goal of the study is to and solitary species versus species forming groups provide an overview of the trophic diversity of dam and (2) feeding habit: feeding areas (especially for selfishes in the Eastern Pacific and to determine territorial, algivorous species), feeding strategy (i.e. whether variations in cephalic shape can be explained biting, grazing or feeding in the water column), and by size variation (i.e. allometry), feeding behaviour, type of prey. Fourteen damselfish species were

Table 1. List of studied damselfish species m m - ,-P HP PQ Ph u cd o ¡3 Cd pq £ m PQ O cd A

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Sites of underwater observation in the Gulf of California, Mexico. 21, 0, 593-613 102, 2011, , N, number of specimens used for the geometric morphometric analysis;N u, number of specimens from museum collections;N cw, number of specimens collected in the wild; SL, standard length; SL A, average SL of the fishes used in the present study; SL B, maximum SL of the fishes used in present study; SL C, maximum SL reported by Allen (1991). HEAD SHAPE DIVERSITY IN DAMSELFISHES 597 directly observed and studied (Table 1). Consequently, (La Paz, BCS, México), CICIMAR (La Paz, BCS, the diet of all Eastern Pacific species was described by México), SIO (San Diego, CA, USA), LACM (Los means of our UO and completed by a review of results Angeles, CA, USA), and USNM (Washington, DC, from previous general studies. Using all these data, USA) (Table 1). The list of museum specimens used in an alimentary item matrix of the 24 endemic dam this study is available upon request from the first selfishes of the Eastern Pacific was built (Table 2). It author (R.A.-M.). was analyzed for the redundancy of the 16 alimentary The specimens were photographed in lateral view items in all the species using the Kendall correlation with a camera (Kodak x 4 optical and 4 mega pixels) index for presence-absence data; this analysis was and the x- and y-coordinates of 18 homologous land conducted to group the alimentary items and reduce marks (Fig. 3) were digitized from the left side of each the main matrix. All alimentary items with a corre individual using TPSDIG, version 2.05. Superimposi lation higher than 0.80 were grouped (Table 2). tion of landmark data was achieved using a general Additionally, the data of diet composition were also ized procrustes analysis (Rohlf & Slice, 1990) which used for the estimation of the trophic level of each aligned landmark configurations such that the sum of damselfish species. The TROPH index, initially used squared distances between corresponding landmarks for Mediterranean fishes (Stergiou & Karpouzi, 2002), was minimized by scaling, translating, and rotating expresses the position of organisms within the food specimens with respect to a mean consensus con webs that largely define aquatic ecosystems. Real figuration. The consensus configuration (‘the grand consumers do not usually have TROPHs with integer mean’) was obtained and used as the reference. values and the definition of TROPH for any consumer Partial warp scores (PWs) including both uniform and species (1) is: non-uniform components were calculated and used as descriptors of shape variation (Bookstein, 1991; Rohlf, G 1993). TROPH = 1 + ^ D C , • troph. A principal components analyses (PCA) was used j =i to find hypothetical variables (components) that w here G is the total number of prey species, DCy account for as much of the variance in the morpho represents the fraction of y in the diet ofi and trophy logical data (Davis, 1986). Subsequently, two kinds is the fractional trophic level of prey y. The TROPH of discriminant analyses were carried out. Differ value was calculated from the dataset using TRO- ences among species were tested by means of analy PHLAB (Pauly et al., 2000), which is a stand-alone ses of variance (ANOVA), the Tukey-Kramer test application for estimating TROPH and its standard and multivariate analyses of variance (MANOVA). error using qualitative information from the list of Then, a canonical variance analysis (CVA) was per items known to occur in the diet of each species. If such formed to compare cephalic profile among groups. trophic levels are missing, TROPHLAB uses default Indeed, CV axes allow us to maximize the differ troph values for various prey (based on data in FISH- ences in shape among groups relative to within BASE; Froese & Pauly, 2000). TROPH values were group variance. MANOVA and CVA were computed used as feeding habit data, allowing the study of the using all shape variables (PWs). Deformation grids relationships between head shape of damselfishes and using the thin-plate spline (TPS) algorithm were their trophic ecology in a quantitative way, and giving used to visualize the patterns of shape varia an ecomorphological meaning to shape differences (see tions along PC axes and CV axes (Thompson, 1917; methods below) (Costa & Cataudella, 2007). Bookstein, 1991; Rohlf, 1993). To determine whether shape data are hierarchically clustered, we applied phenetic and phylogenetic G e o m e t r ic morphometrics a n d parsimony methods for grouping. Phenetic distance PHYLOGENETIC ANALYSIS methods are based upon a different conceptual frame The sample comprised 509 specimens belonging to all work of grouping than parsimony. We included a 24 damselfishes of the Eastern Pacific (Table 1). Addi phenogram to determine whether there was evidence tionally, the embiotocid Zalembius rosaceus (Jordan & for the assertion that similarity in morphometric data Gilbert, 1880) (family Embiotocidae) was used as an is not the result of a phylogenetic signal. Phenetic ‘outgroup’. The Embiotocidae are now recognized as relationships were summarized using a cluster analy the sister group of Pomacentridae (Streelman & Karl, sis on the matrix of mean shape. Phenogram of the 25 1997; Mabuchi et al., 2007), Zalembius rosaceus is a species was calculated using the unweighted pair- small benthic carnivorous fish distributed inside the group method algorithm, and the Procrustes distance same area of the studied pomacentrids. Some speci as measure of the similarity. The goodness of fit of the mens were speared during the field studies and the cluster analysis was measured by the coefficient of others came from the museum collections of CIBNOR cophenetic correlation (Cardini & Elton, 2008). The

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Benthic diet: A, sponges; B, macroalgae, microalgae, crustaceans, and sessile worms; C, sessile mollusks; D, mobile crustaceans; E, mobile worms; F, anem ones; G, gastropods; H, fish eggs. Pelagic diet: I, Zooplankton; J, crustaceans; K, fish larvae. Other: L, detritus; M, ectoparasites. UO, Underwater observations. HEAD SHAPE DIVERSITY IN DAMSELFISHES 599

The molecular phylogenetic hypothesis by Cooper et al. (2009) and our morphological phylogeny were used for phylogenetic adjusted regressions of the shape variables on the TROPH index, centroid size (CS), and standard length (SL) using the module PDAP in MESQUITE (Midford, Garland & Maddison, 2009). Our morphological and molecular phylogenies were used to obtain an estimate of character distri bution and correlation along a phylogeny (Garland et al., 1993). Phylogenetic regression analyses to test interspecific allometry were performed using the mean values of CS and SL versus the 32 shape variables. Similarly, we performed phylogenetic regression analysis using the mean values of the 32 shape variables and the TROPH index to test the relationship between feeding habit and cephalic shape. Additionally, we compared these results with conventional nonphylogenetic regression analyses performed in TPSREGRES, version 1.37. Geometric morphometric analyses were performed using computer programs from the TPS series (TpsDig, TpsRegres) written by F. J. Rohlf (http://life. bio.sunysb.edu/morph/), and the IMP series (PCAGen, CVAGen), created by H. D. Sheets ( http://www2. canisius.edu/~sheets/morphsoft.html). Multivariate analyses (correlation analyses, ANOVA, PCA, CVA, MANOVA) were computed with the statistical pack ages: PAST, version 1.74 (Hammer, Harper & Ryan, 2001; http://folk.uio.no/ahammer/past), Statistica, version 8.0 http://www.StartSoft.com ( ) and JMP, version 8.0 (SAS Institute Inc.). Phylogenetic analysis was computed in TNT, version 1.1 (Goloboff, Farris & Nixon, 2008; http://www.zmuc.dk/public/phylogeny ). Figure 3. Anatomical landmarks used in geometric mor- Phylogenetic regressions were performed in the phometric analyses: (1) tip of the premaxilla; (2) nostril; PDAP package (Midford, Garland & Maddison, 2010; (3-6) inferior, anterior, superior, and posterior margin of http://mesquiteproject.org/pdap_mesquite/) for M ES the eye; (7) center of the eye; (8) superior tip of the QUITE, version 2.73 (Maddison & Maddison, 2010; preopercular; (9) superior tip of the operculum; (10) pos http://mesquiteproject.org/mesquite/mesquite.html). terior tip of the operculum; (11, 12) superior and inferior insertion of the pectoral fin; (13) insertion of the pelvic fin; (14) insertion of the operculum on the body profile; (15) RESULTS posterio-ventral corner of the preopercular; (16, 17) ante T r o p h ic a n d behavioural d a ta rior and posterior extremity of the dentary; (18) posterior Chromis limbaughi, Chromis punctipinnis, Hypsy extremity of the premaxilla. pops rubicundus, and Stegastes redemptus were only observed at one locality (Table 1), whereas the trophic morphological phylogenetic analysis was executed behaviours were always consistent among sites in the using the 32 shape variables (RWs) as characters for other species. Unfortunately,Chromis alta was never all studied species. For each species, we scored either observed at any location, maybe as a result of its RW intervals, RW means or both. Phylogenetic analy depth preferences (down to 150 m; Allen, 1991). Gen ses were performed using new algorithms for the erally, the underwater observations corroborate data direct optimization of continuous characters in TNT obtained from the literature related to diet of each (Goloboff, M attoni & Quinteros, 2006). We used a studied species. However, some new trophic behav combination of ratchet, sectorial search, tree drifting, iours were also observed. For example, the mainly and tree fusing search algorithms for the selection of algivorousS. rectifraenum was observed recurrently optimal trees and for the estimation of Jackknife picking feces from Zooplankton feeders (e.g. A bu values as a measure of support. defduf troschelii) directly in the water column.

The trophic behaviour appeared relatively constant G e o m e t r ic morphometrics within all genera, except within the genus A budefduf (Table 2). Abudefduf troschelii mainly fed on plank The main shape variations across species can be tonic preys in the water column, whereasA budefduf examined by a distribution of specimens in the PC concolor and Abudefduf declivifrons mainly grazed figure defined by the axis PCI and axis PC2 (Fig. 4). algae or fed on some small sessile animals attached to The first two PCs account for 70% of the total shape the rocks. variance (PCI = 58.41% and PC2 = 10.32%). Shape The analysis of alimentary items shows a strong variation was relatively low within each genus except relationship (r > 0.80) between four items: macroal for A budefduf. This genus could be clearly divided gae, microalgae, sessile crustaceans, and worms. into two groups, one comprisingA. concolor and A. Consequently, these prey items were grouped in declivifrons and the other comprising A. troschelii. Table 2. Thus, a mainly algivorous species such as PCI allowed a clear distinction between mainly all Stegastes species, Microspathodon species, N ex benthic feeders (low PCI values) and mainly Zoop ilosus latifrons, and H. rubicundus may be con lankton feeders (high PCI values). The main morpho sidered as a benthic feeder grazing on algae and logical variation along axis PCI is related to the snout feeding on small benthic invertebrates. On the basis length, the cephalic depth, and the eye size and of a review of the published results and our UO, position. The benthic feeders, especially Microspath four alimentary items are species-specific (Table 2): odon species, were characterized by a very short sponges (A): H. rubicundus; detritus (L): S. rectifrae snout and small eyes; the pelagic feeders (Azurina n u m ; gastropods (G): Z. rosaceus; and ectoparasites species, Chromis species, and A. troschelii) were char (M): A. troschelii. Abudefduf troschelii showed the acterized by relatively larger eyes and longer cephalic largest diet of the damselfish investigated in the profile. Along axis PC2, the main shape variation was present study, feeding in benthic and pelagic areas. related to the position of the mouth and the pectoral All seven species of Chromis and the two species fin, and explains the variation within some genera of A zurina mainly feed on Zooplankton. Zalem bius such as Stegastes and Chromis. rosaceus is considered as a benthic carnivorous When species were used as grouping factor, species, feeding only on mobile benthic crustaceans the MANOVA revealed significant differences among

(shrimps, small crabs), worms, gastropods, and them (V w i l k s = 2.36 x 1CT5, E = 42.02, d.f.i = 256, bivalves (Table 2). d.f.2 = 3665.2; P< 0.001) and pairwise comparisons As for the diet, the social behaviour was con supported that all species showed significant dif served within genera. Hypsypops rubicundus and ferences in mean shape (P < 0.05). MANOVA was Stegastes species are solitary, protecting small ter repeated using genus as grouping factor, and the ritories, forming couples only during the repro results also show significant differences among all ductive season. Hypsypops rubicundus showed a genera; however, A budefduf genus show a strong preference for small caves around big rocks and patter of segregation into two groups. According to the all Stegastes species prefer small rocks. Both trophic data, the feeding habit is consistant within Microspathodon species are solitary or live in pairs genera except for the genus Abudefduf. The segrega and protect big rocks with high vertical walls. N ex tion pattern of Abudefduf was tested by exploratory ilosus latifrons was not observed in the wild, analyses (ANOVA, PCA, and CVA). Because the although some authors (Grove & Lavenberg, 1997; pattern was repeated in all analyses, the final Angel & Ojeda, 2001; Robertson & Allen, 2008) MANOVA was performed using genera as grouping reported that it is a grazing species living close to factor, with the genus A budefduf split into two the rocks in small groups (up to ten individuals). All groups: (1) A. concolor and A. declivifrons and (2) A. Abudefduf, Azurina, and Chromis species live in troschelii. The results of MANOVA show significant groups (i.e. schooling species) and are territorial differences among all groups (X w i l k s = 0.0000263, only during the reproductive season. The main dif F = 41.23, d.f. = 265, P < 0.001), and all pairwise ference between these species is the number of inte comparisons based on Mahalanobis distances show grants in the groups (Abudefduf species: 5-30 significant differences (P < 0.05). individuals versus Chromis species: 10-60 indivi Discrimination among groups can be also inter duals), although it proved complicated to achieve preted by examining the ordination of specimens a good description of these numbers because they in the morphospace defined by the CV axes (Fig. 5). changed according to the study area and the The first three CV axes accounted for 84% of the habitat. Neither A zurina species were observed in total shape variation in the dataset and allow the wild; thus, we do not have any estimation of the discrimination of five main groups. The axis CV1 number of fish per group. Specimens ofA. declivi distinguishes three groups (Table 3): (A) Micros frons and A. concolor could also be found solitary. pathodon species; (B) A. concolor, A. declivifrons, H.

+PC 2

(N Ü CL

-PC 2 m m m

-PC 1 +pc 1

Figure 4. Scatterplot of principal components (PC) 1 and 2. Cross,A budefduf, equis, Azurina', square, C hrom is; triangle, H ypsypops; circle, Microspathodon; asterisk, N exilosus; black circle, Stegastes ; black square, Zalem bius. Thin plate spline deformation grids for the extreme points of each axis are shown; these are superimposed on the shapes predicted when the average landmark configuration of all specimens is deformed into that of a hypothetical specimen positioned at the extreme point of an ordination axis. rubicundus, N. latifrons and Stegastes species; (C) (+CV2). The eye of the two A zurina species and Z. A zurina species, Chromis species, A. troschelii and Z. rosaceus was in a more forward and lower position, rosaceus. This last group (group C) could be subdi and the snout region is also longer than in all vided into three groups in the morphospace defined by Chromis species and A. troschelii (CV3; Fig. 5). The CV1 and CV3: Z. rosaceus in the extreme high values, phenogram shows a segregation of four morphological A zurina species in the middle values, and A. troschelii groups (Fig. 6): (1) all species of the genusStegastes, and Chromis species in the extreme low values A. concolor, and A. declivifrons, which are mainly (Fig. 5, Table 3). algal feeders; (2) all species of the genus Chromis and In general, the CVA strengthened the profile and A. troschelii, which are mainly Zooplankton feeders; the position of the eye and the mouth. Having the (3) both Microspathodon species are algal feeders, lowest scores of CV1, the two speciesoí Microspath with an extremely flat cephalic shape; and (4) both odon showed a higher and flatter cephalic profile; A zurina species, which are zooplanktivorous, andZ. Chromis species, A zurina species, A. troschelii, and Z. rosaceus, which is carnivore benthic feeder, form a rosaceus present a lengthened cephalic profile with group having a highly sharp cephalic profile. The big eyes; H. rubicundus, N. latifrons, Stegastes coefficient of cophenetic correlation is relatively high species, A. concolor, and A. declivifrons showed an (7 ' = 0.76). intermediate shape. Along CV2, the main shape variation was related to the position of the mouth and of the pectoral fin. The twoMicrospathodon species P hylogenetic a n a l y s is showed a more horizontally oriented pectoral fin, the The morphological phylogenetic hypothesis (Fig. 6) cephalic profile is high and the mouth is small shows a grouping pattern with a high degree of

+CV2

-CV 2

+CV3

-CV 3 CV 1 (61%)

-CV1 +CV1

Figure 5. Scatterplot of canonical variâtes (CV): CVl versus CV2 and CV1 versus CV3. Black cross,Abudefduf concolor and A. declivifrons; grey cross, Abudefduf troschelii; equis, Azurina', white square, Chromis alta, C. crusma, C. intercrusma, C. limbaughi, C. meridian and C. punctipinnis', grey square, Chromis atrilobata', triangle, Hypsypops', circle, Microspathodon', asterisk, Nexilosus', black circle, Stegastes', black square, Zalem bius. For both plots, thin plate spline deformation grids for the extreme points of each axis are shown; these are superimposed on the shapes predicted when the average landmark configuration of all specimens is deformed into that of a hypothetical specimen positioned at the extreme point of an ordination axis.

correlation between head morphology and feeding A budefduf that belong to the Abudefdufinae sub habits. The morphological phylogeny is not totally family according to the molecular phylogeny. Within congruent with the molecular phylogeny, except this group, the clade of two species ofA budefduf is in that the subfamily Stegastinae is recovered highly supported (Jackknife = 96). The subfamilies with the genus Stegastes, Nexilosus, Hypsypops, Chrominae and Abudefdufinae are not recovered as and Microspathodon. This clade presents mode monophyletic groups in our morphometric phylogeny. rate support values (Jackknife = 63). However, our Rather, the Chrominae is a partially resolved grade morphometric phylogeny includes two species of that also includes A. troschelii.

Table 3. Analysis of variance and Tukey-Kramer test of the three first axes of canonical variance analysis

A B c D E F G H I

CV 1 A 5.694 7.324 7.605 0.743 8.480 0.143 0.118 4.959 B < 0.0001 1.630 1.910 6.437 14.175 5.837 5.576 0.735 C < 0.0001 < 0.0001 0.280 8.067 15.805 7.467 7.206 2.365 D < 0.0001 < 0.0001 0.876 8.348 16.085 7.747 7.487 2.646 E 0.246 < 0.0001 < 0.0001 < 0.0001 7.737 0.600 0.861 5.702 F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 8.338 8.598 13.439 G 1.000 < 0.0001 < 0.0001 < 0.0001 0.631 < 0.0001 0.261 5.102 H 1.000 < 0.0001 < 0.0001 < 0.0001 0.002 < 0.0001 0.986 4.841 I < 0.0001 0.083 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 CV 2 A 0.443 2.710 1.015 1.418 4.645 1.408 2.889 1.692 B 0.799 3.153 1.458 0.975 5.088 0.965 2.446 1.249 C < 0.0001 < 0.0001 1.695 4.127 1.935 4.118 5.599 4.402 D 0.001 < 0.0001 < 0.0001 2.432 3.630 2.423 3.904 2.707 E < 0.0001 0.005 < 0.0001 < 0.0001 6.063 0.010 1.471 0.275 F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 6.053 7.534 6.337 G < 0.0001 0.033 < 0.0001 < 0.0001 1.000 < 0.0001 1.481 0.284 H < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 1.197 I < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.987 < 0.0001 0.993 < 0.0001 CV 3 A 1.33 2.82 1.20 1.13 0.11 0.54 0.35 6.72 B < 0.0001 4.15 0.13 0.20 1.44 1.87 0.98 8.05 C < 0.0001 < 0.0001 4.02 3.95 2.71 2.28 3.17 3.90 D < 0.0001 1.00 < 0.0001 0.07 1.31 1.74 0.85 7.92 E 0.01 1.00 < 0.0001 1.00 1.24 1.67 0.78 7.85 F 1.00 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.43 0.46 6.62 G 0.80 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.86 0.89 6.18 H 0.86 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.14 0.02 7.07 I < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.01 < 0.0001 < 0.0001 < 0.0001

A: Abudefduf concolor, Abudefduf declivifrons', B: Abudefduf troschelii', C: Azurina spp.; D: Chromis spp.; E: Hypsypops rubicundus', F: Microspathodon spp.; G: Nexilosus latifrons', H: Stegastes spp.; I: Zalembius rosaceus. Above the diagonal: difference degree of Tukey-Kramer; below the diagonal: P-value.

R elationship b e t w e e n c e p h a l ic s h a p e a n d siz e the main shape variation among the studied dam Interspecific allometry was tested using linear regres selfishes is related to their feeding habit (Fig. 7). sions analyses. The relationship between shape and Cephalic shapes are more related to trophic levels size (CS and SL) is low (r2 < 0.4; Table 4). The lowest than to the phylogenetic relationships (Fig. 7, 7'2-values were found with the nonphylogenetic Table 4). Trophic data group the 24 damselfishes in regression analysis and the highest ones with the three main groups: mainly algal feeders, mainly phylogenetic regression analyses (Table 4). However, Zooplankton feeders, and an intermediate group shape always showed a stronger relationship with CS feeding on small pelagic and benthic preys. CVA and than with SL. cluster analysis allow the discrimination of the three main trophic groups and also show a clear degree of variation between two extremes of cephalic shape R elationship b e t w e e n c e p h a l ic (i.e. Zooplankton feeders, A zurina species, with SHAPE AND TROPHIC DATA highly sharpness cephalic shape and algal feeders, Both nonphylogenetic regression analyses and the Microspathodon species, with highly flattened cepha equivalent with phylogenetically independent con lic shape). On the other hand, the phylogenetic trasts show a significant positive relationship hypothesis constructed with shape data produces between the TROPH index and shape variables two monophyletic groups: (1) subfamily Stegastinae, (Table 4). Values of the coefficient of determination which group mainly algal feeders and (2) a group (r2) were lower in the phylogenetic regression analy composed ofA. troschelii, Chromis meridiana, and sis than in the nonphylogenetic analysis. These tests C. lim baughi, which are mainly zooplanktivorous, and mirror trees analyses clearly demonstrate that feeding also on fish eggs.

Main trophic groups

M. b aird ii M. d o r s a lis ^ ^ 9 0 M. d o rs a lis M, b aird ii A. concolor A. c o n c o lo r ^ ^ 9 6 | A. d ec liv ifro n s A. declivifrons — R rubicundus Microspathodon S. acapulcoensis N. latifro n s Mainly S. rectifraerum S. acapulcoensis algal 5. arcifrons a rc ifro n s feeder 5 . H a l l i s red e m p tu s H. r u b ic u n d u s ' b e e b e i H. la tifro n s S. flavilatus S. redemptus le u c o ru s S . b e e b e i S3| Steg ast inae b ald w in i S. leucorus' S. rectifraenum S. baldwini A, tro s c h e lii A troschelii C, m erid ia n a C. meridiana C. a lta C. limbaughi C, a trilo b a ta C a l l a » chromis Mainly C. lim b au g h i Zooplankton C. crusma ■ C . c ru sm a feeder C. pinctipinnis - C. intercrusma C. a trilo b a ta ■ 100 C. pinctipinnis C intercrusma ■ A, e u p a la m a A h i r u n d o » A. hirundo A. e u p a l a m a » ¿ r o s a c e u s Carnivore I rosaceus » benthic 0.30 0.25 0.20 0.15 0.10 0.00 feeder

Procrustes distance Coph. corr. = 0.757

Figure 6. Comparison of two hierarchical models. Phenogram (A) and morphometric phylogeny (B) compared to main trophic groups of damselfish. Images of some species are added to help to visualize the pattern of cephalic shape variation in relation to each trophic group. In the phylogeny, the number on branches are support values (jackknife, 1000 replicates, cut = 50, jackknifing P = 36). Coph. Corr., coefficient of cophenetic correlation.

Table 4. Phylogenetic and nonphylogenetic regression analysis for testing interespecific allometry and the relationship between morphology and trophic data

Least square regression Variables r2 F d.f. P

Phylogenetic CS versus shape variables (molecular and morphological phylogeny) 0.41 16.32 23 0.0005 SL versus shape variables (molecular and morphological phylogeny) 0.17 4.64 23 0.042 TROPH index versus shape variables (molecular phylogeny) 0.51 23.16 23 < 0.0001 TROPH index versus shape variables (morphological phylogeny) 0.51 23.16 23 < 0.0001 Nonphylogenetic CS versus shape variables 0.20 6.22 32-736 < 0.0001 SL versus shape variables 0.09 2.44 32-736 < 0.0001 TROPH index versus shape variables 0.36 13.17 32-736 < 0.0001

CS, centroid size; SL, Standard length.

The optimization of the TROPH index and morphol although some shape variation can be observed ogy in the molecular phylogeny revealed that all among all Stegastes species, N. latifrons, and the species of the genus Stegastes and N. latifrons are two Microspathodon species. Morphological phylog highly similar with respect to morphology and feeding eny highlighted a pattern of convergence with two habits. The subfamily Stegastinae show a high cor members of A budefduf genus (A. concolor and A. relation between morphology and feeding habits, declivifrons). Although A. concolor and A. declivifrons

Character: TROPH Index Character: Morphology Parsimony reconstruction M. bairdii Parsimony reconstruction Squared length: 0.1650 Squared length: 7.6695 M. dorsalis I 2.6 to 2.74 I t A. concolor -12.396 to -10.607 A. declivifrons Í! I 2.74 to 2.88 I I -10.607 t o -8.819 S. acapulcoensis □ 2.88 to 3.02 S. rectifraenum □ -8.819 to -7.030 □ 3.02 to 3.16 <1> S. arcifrons [ I -7.030 to-5.242 CO ] 3.16 to 3.3 S. flavilatus n -5.242 to -3.453 (/) H. rubicundus [ I 3.3 to 3.44 co □ -3.453 to 1.665 O) N. latifrons

Abudefdufinae A. concolor A. declivifrons A. troschelii A. eupalama A. hirundo C. crusma C. intercrusma C. alta Chrominae C. punctipinnis C. limbaughi C. atrilobata C. meridiana M. bairdii M. dorsalis H. rubicundus N. latifrons S. acapulcoensis S. arcifrons S. baldwini S. beebei OJ S. flavilatus S. leucorus S. rectifraenum S. redem ptus B Z. rosaceus

Figure 7. Mirror tree optimization of the TROPH index (left) and morphology (right). A, morphological hypothesis; B. molecular hypothesis.

© 2011 The Linnean Society of London,Biological Journal of the Linnean Society, 2011, 102, 593-613 606 R. AGUILAR-MEDRANO ETAL. are close according to the morphological phylogeny, with the trophic data (Table 2), the cephalic profile of the feeding habit of A. declivifrons is different and A. troschelii is more similar to that ofChromis and more similar to that of members of the Chrominae Azurina, suggesting that this species should be con subfamily. Both Microspathodon species and H. rubi sidered as an omnivorous species mainly feeding on cundus are close according to their feeding habits. Zooplankton. On the other hand,A. declivifrons and The cephalic shape is highly conserved in the sub A. concolor are more similar toStegastes, Hypsypops, family Stegastinae through both phylogenies. Accord and Nexilosus, showing that they should be consid ing to the categories of the TROPH index (Fig. 7), the ered as omnivorous species and mainly benthic Chrominae subfamily shows five categories distrib feeders. uted between medium and high values of the TROPH To our knowledge, H. rubicundus is the only index. On the other hand, the range of morphological damselfish feeding on sponges. No damselfish of the variation of this group is low (i.e. only two extreme Indo-West Pacific is known to be a common con morphological categories of colour code; Fig. 7). sumer of such kind of prey (Allen, 1991; Frédérich According to the morphological phylogeny,A. trosche et al., 2009). The angelfishes (Pomacanthidae) is lii is closely related to the Chrominae. The con specialized in such prey catching in coral reef envi vergence pattern ofA. troschelii introduces a new ronments (Konow & Bellwood, 2005). According morphology within this group. The Abudefdufinae to Robertson & Allen (2008), one Pomacanthidae, subfamily is highly diverse. Two morphological cat Pomacanthus zonipectus, is distributed in the cooler egories were found in this subfam ily (Fig. 7); (1) temperate regions and lives sympatrically with A. troschelii is more similar in shape to the outgroup H. rubicundus. However, this angelfish achieves its (Z. rosaceus) and (2) A. concolor and A. declivifrons maximum population density in the Tropical Eastern show similar values to the Stegastinae subfamily Pacific and is scarce in cooler temperate regions. members. Each A budefduf species has a different Consequently, a lower competition level could permit colour code for its trophic index (Fig. 7). an extension of the trophic width of damselfishes such as H. rubicundus in temperate regions.

DISCUSSION E comorphology a n d m o r p h o -f u n c t io n a l T r o p h ic d iv e r s it y o f damselfishes IMPLICATIONS The trophic data obtained in the present study The bucco-pharyngeal cavity of a fish has been mod confirm the division of damselfishes of the Eastern elled as a truncate eone, whose small base comprises Pacific into three main trophic groups, as reported for the circular opening of the mouth and whose large species living in the Indo-West Pacific region (Allen, base is located behind the branchial basket on the 1991; Kuo & Shao, 1991; Frédérichet al., 2009): (1) level of the opercles (Alexander, 1967; Lauder, 1980; the pelagic feeders mainly sucking zooplanktonic Lauder & Lanyon, 1980; Liem, 1993). The efficiency of preys (e.g. Chromis spp., A zurina spp.); (2) the the eone depends on various factors such as the benthic feeders mainly grazing filamentous algae and morphology of the skull and particularly the bucco picking small invertebrates (e.g. Stegastes spp., N ex pharyngeal cavity (Liem, 1990). There are three basic ilosus latifrons, Hypsypops rubicundus, Microspathmodes of feeding according to the degree of truncation odon spp.); and (3) an intermediate group gathering of the eone (Liem, 1980, 1993): suction feeding, ram species feeding both on small pelagic and benthic feeding, and biting. However, a mode of prey capture preys (e.g. A budefduf spp.). is not exclusive; many teleosts are able to modulate The social behaviour of damselfishes is constant their feeding mode and to move from one category in the whole Indo-Pacific, Western Atlantic, and to another (Liem, 1980, 1993; Ferry-Grahamet ál., Eastern Pacific region (Emery, 1973; Frédérich et al., 2002). If the head morphology of damselfishes 2009; present study): species mainly feeding on prompts the consideration that they are good suction benthic prey are solitary, whereas zooplanktivorous feeders (Emery, 1973; Frédérich et al., 2008; Cooper species live in groups. However,N. latifrons is rela & Westneat, 2009; present study), geometric mor tively atypical because it is a grazing species living phometric analyses allow a deeper understanding of in small groups (up to ten individuals) (Grove & the different ways of feeding and reveal functional Lavenberg, 1997; Angel & Ojeda, 2001; Robertson differences among species. & Allen, 2008). The main difference between morphological groups In general, the diet is consistent within damselfish is the degree of sharpness of the cephalic shape, genera. However, our ecomorphological approach indi which goes from a long angular cephalic profile as in cated that the three A b u d efd u f species should be Z. rosaceus, both A zurina species, A. troschelii, and grouped into two different trophic guilds. Consistent all Chromis species; followed by angular but shorter

feeding in that they attack the individual planktonic A preys they select visually. The possession of relatively large eyes (Fig. 5) should increase their ability to find and target planktonic preys, as exemplified in cichlids (Barel, 1983). Their elongated head profile facilitates the capture of these organisms using ram-suction feeding (Coughlin & Strickler, 1990), although further functional studies should aim to test whether the differences in cephalic profile between Chromis and B A zurina species (Fig. 5) could be related to differences in feeding strategy and performance. For example, the contribution of ram (i.e. the predator movement towards the prey) and suction (i.e. the prey movement towards the predator as a result of aspiration) during feeding may differ between both genera (Wainwright et al., 2001). Abudefduf troschelii is an omnivorous species C feeding mainly on Zooplankton (Groveet al., 1986; present study). Robertson & Allen (2008) considered this species to comprise two feeding groups: omnivo rous and planktivorous.A budefduf troschelii shows a very similar cephalic profile to the almost exclusive zooplanktivorousChromis and A zurina genera. Simi larly, A. troschelii mainly occurs along rocky shores or coral reefs, in shallow waters, foraging on Zooplank ton in aggregations. By contrast, A. concolor, A. Figure 8. Difference in the mouth orientation when it is declivifrons, H. rubicundus, N. latifrons, all Stegastes open in (A) Zalembius rosaceus, (B) Azurina hirundo, and species and both Microspathodon species mainly (C) Chromis atrilobata. graze filamentous algae growing on rocks. Within this trophic group, the two Microspathodon species present a highly different morphology. Our underwa cephalic profiles as in A. concolor, A. declivifrons, H. ter observations revealed that the way of feeding in rubicundus, N. latifrons, and all Stegastes species; both Microspathodon species differ from that of the and, finally, to an almost flat cephalic profile as in others. Indeed, all Stegastes species grazed algae or both Microspathodon species (Fig. 6). The benthic car picked up small invertebrates on small, mainly hori nivorousZ. rosaceus feeds mainly on gastropods, zontal rocks or rubble, whereas the twoMicrospath mobile worms, and crustaceans (Table 2). As observed odon species scraped on big rocks with high vertical in some cichlids (Liem, 1993), a long angular cephalic walls. This type of feeding is probably facilitated by profile may facilitate the catching of these items. an almost flat cephalic profile in Microspathodon Despite a similar angular cephalic profile as in both species. Moreover, the premaxillary bones on their A zurina species and Chromis species, Z. rosaceus anterior region reveal a loose connective tissue where shows a great morpho-functional difference compared teeth are continuously produced (Ciardelli, 1967). to these zooplanktivorous species, which was not indi When the fish scrapes the rocky wall, the teeth are cated by our geometric morphometric analyses. continuously eroded and, consequently, need to be Indeed, this difference is solely observed when the produced constantly (Trapani, 2001). Furthermore, mouth is extended (i.e. during mouth protrusion) this connective tissue that supports and nurtures the (Fig. 8). During feeding, the mouth is oriented more teeth could act as a buffer, supporting the movement ventrally in Z. rosaceus, optimizing the capture of of the teeth and the premaxillary bones on the benthic animal preys, whereas the mouth is directed rock. Although, when the food items are similar, the rostrally in A zurina and Chromis species, facilitating morphological pattern could diverge if the methods of prey capture in the water column. Further morpho- prey catching differs. functional studies should precisely address the differ The results of the present study indicate that dam ences in the degree of protrusion of the premaxillary selfishes from the Eastern Pacific show strong differ bones during feeding among these species. Zooplank ences with respect to their cephalic profile. These tivorous damselfishes such as the A zurina and differences are mainly related to the degree of sharp Chromis species can be described as being particulate ness and the position of the eye and the mouth. A

© 2011 The Linnean Society of London,Biological Journal of the Linnean Society, 2011, 102, 593-613 608 R. AGUILAR-MEDRANO ETAL. pattern of relationship between head morphology and to two different feeding habits. In A. troschelii, the diet was found, as in previous analysis (Frédérich, mouth is superior (omnivorous, feeding mainly on Parmentier & Vandewalle, 2006; Frédérich et al., Zooplankton); this shape is very similar to that ofC. 2008; Cooper & Westneat, 2009). However, the rela m eridiana and C. limbaughi (Fig. 6). By contrast, the tionship between the cephalic profile and feeding m outh ofA. concolor and A. declivifrons is inferior habit found in damselfishes extends beyond this. In (omnivorous, feeding mainly on benthic preys); thus, the present study, head morphology was observed to the shape is similar to Microspathodon bairdii and be related to the way that the food resource is Microspathodon dorsalis (Fig. 6). The m olecular phy extracted: two species can use the same food resource logenetic analysis of Quenouilleet al. (2004) consi (,Stegastes species and Microspathodon species) and dered 16 A b u d efd u fspecies, and found three main present a different head morphology, and these clades within this genus: A. declivifrons, A. concolor, respond to the way that each species extracts the and Abudefduf taurus are the sister group to the resources of the environment. two principal groups.Abudefduf taurus is a mainly herbivorous fish (Randall, 1967; Allen, 1991), with similar behaviour toA. concolor and A. declivifrons, E volutionary considerations showing strong preference for limestone shorelines The shape data of the present study confirm a and tide pools in regions with surf (Allen, 1991; morphological group composed byZ. rosaceus, our R. Aguilar-Medrano, pers. observ.). The second group chosen outgroup (Figs 4, 5). The family Embiotocidae comprises Abudefduf sordidus, Abudefduf septemfas includes mainly carnivorous (zoobenthos) species with ciatus, and Abudefduf notatus, which are also mainly a pointed cephalic profile. Interestingly, Z. rosaceus algal feeders (Allen, 1991). All A budefduf species of shows a cephalic profile more similar to the most these two groups possess a dark body with clear derived zooplanktivorous damselfishes (i.e.Chromis vertical bars and have a strong preference for very and Azurina) (Fig. 2). The sim ilarity among these shallow areas (0-4 m) with surf (Allen, 1991; R. A.-M. three genera is a convergence in the cephalic profile. & B. F. pers. observ.). Their mouth is placed in a lower The present study confirms the results reported by position than the others. The third cluster of Que Frédérich et al. (2008), who stated that shape varia nouille et al. (2004) includes A. troschelii, these group tion is not correlated with size variation in damself present species which show an overall lighter body ishes. Interspecific allometry is low and size does not with dark vertical bars and are laterally compressed; predict convergent shapes. Consequently, variations all are omnivorous, feeding mainly on Zooplankton in size and shape may be viewed as two independent (Emery, 1973; Frédérich et al., 2009; present study). evolutionary factors explaining the diversification of They are gregarious, their mouth is in a superior the family Pomacentridae. When the relationships position, their habitat distributions are in a water between CS, SL, and shape variables are analyzed depth in the range 1-15 m, and they are associated through nonphylogenetic analyses, 7'2-values are with rocky and coral reefs (Emery, 1973; Allen, 1991; lower than when they are analyzed through phyloge Frédérich et al., 2009; present study). netic analyses, indicating that size is related to the Previous molecular phylogenetic analyses found phylogeny. th a t Microspathodon, Hypsypops, Nexilosus, and S te The morphometric results of the present study gastes are closely related genera belonging to the show A zurina to be very similar in cephalic shape to subfamily Stegastinae (Tang, 2001; Quenouille et al., Chromis. In a Euclidean plane, the slender A zurina 2004; Cooperet al., 2009), although relationships are species lie at the extreme point of the Chromis dis not fully resolved (Fig. 2). The trophic data of the tribution along an axis of cephalic length (Figs 4, 5). present study revealed that all these species are According to the morphological phylogeny,A zurina benthic feeders, mainly grazing filamentous algae. species are closely related to Chromis species (Fig. 6); Our geometric morphometric analyses and pheno this relationship is also reported by Cooper et al. gram clearly distinguish the two Microspathodon (2009), who proposed synonymizing the genus species from the other species belonging to this A zurina w ith Chromis and re-classifying the two trophic group (i.e.H. rubicundus, N. latifrons, and all species of Azurina. Their proposal was based on a studied Stegastes species) based on their highly flat strong phylogenetic relationship between A zurina tened cephalic profile. Hypsypops rubicundus and N. hirundo from the Eastern Pacific and Chromis mul latifrons are predominantly found in temperate tilineata from the Atlantic. waters, in the extreme north and south, respectively, The genus A b u d efd u fhad been traditionally segre of the Eastern Pacific damselfish distribution. Their gated into two groups. According to our mophometric cephalic profiles are relatively similar to those of analyses, the main difference between these groups is Stegastes species, although geometric data show the position of the mouth, which clearly corresponds some differences among these three genera. In the

© 2011 The Linnean Society of London,Biological Journal of the Linnean Society, 2011, 102, 593-613 HEAD SHAPE DIVERSITY IN DAMSELFISHES 609 morphospace,H. rubicundus and N. latifrons species are placed peripheral of the Stegastes morphological distribution (Fig. 5) and a schematic representation of A the observed differences within this group is illus trated in Figure 9. If we hypothesize that Nexilosus, Hypsypops, and Microspathodon are derived from the genus Stegastes (Cooper et al., 2009), the results of the present study demonstrate that the main shape variation during the evolutionary process was related to the form of the cephalic profile (pointed versus flattened), the size of the eye, and the position of the eye and mouth. From a Stegastes ancestor, a first evolutionary step could produced two new forms (Fig. 9): (1) H. rubicundus with an pointed profile, a B long horizontal distance between the mouth and the eye, and a short vertical distance between the mouth and the eye, and (2) N. latifrons with a more rounded profile, a short horizontal distance between the mouth and the eye, and a long vertical distance between the mouth and the eye. The possible next evolutionary step from the Nexilosus brand produced a strongly specialized cephalic profile: both Microspathodon species present a highly flattened profile, no horizon tal space between the eye and the mouth and high vertical distance between the mouth and the eye (Fig. 9). C The molecular data reported by Tang (2001) showed th a t Hypsypops is closer toParm a (Subfamily Stegas tinae) than to Stegastes. Indeed, when the genus Parm a was first described, H. rubicundus was included as one of its members (Tang, 2001). These two genera are predominantly distributed in temper ate cool water and rocky reefs; furthermore,Parma includes some species that can be found in kelp forests, such asH. rubicundus (Allen, 1991; Buckle & Booth, 2009). Future geometric studies should include Parm a species to obtain a good overview of the phe notypic differences or similarities among Parma, Hypsypops, and Stegastes. The phylogenetic position D of N. latifrons is unknown (Cooper et al., 2009), although the data of the present study strongly suggest that N. latifrons is closely related to Stegastes species in morphology and ecology, even though the former lives in small groups. According to Robertson & Allen (2008), the main differences between this genus and Stegastes are that the margins of the preopercle and infraorbital bones are smooth inN ex ilosus and serrated in Stegastes. Both genera present a single row of teeth and the number of dorsal fin spines of Nexilosus (XIII) is included in the range of F igure 9. Schematization of the variation related to Stegastes (XII to XIV) (Allen, 1991). Similar to the the position of the eye and mouth in the cephalic profile question of synonymy between the genusA zurina and of (A) Hypsypops rubicundus, (B) Stegastes flavilatus, Chromis, the close relationship between Nexilosus, (C) Nexilosus latifrons, and (D) Microspathodon dorsalis. Stegastes, and Hypsypops allows us to question w hether Nexilosus and Hypsypops (both monospecific genera: N. latifrons and H. rubicundus, respectively)

should be considered as valid genera. Further exhaus Noroeste, México (CIBNOR) and CT001 (CONABIO). tive phylogenetic studies are needed to better under Finally, we thank Mark Webster and two anonymous stand the phyletic relationships among M icrospa reviewers for their enlightening comments. thodon, Hypsypops, Nexilosus, Stegastes, and Parma. Our morphometric and phylogenetic analyses of the cephalic region show that the subfamilies Stegastinae REFERENCES and Chrominae present a specific morphological Aburto-Oropeza O, Balart EF. 2001. Community structure pattern, but not Abudefdufinae (Fig. 7). The mor of reef fish in several habitats of a rocky reef in the Gulf of phological average appeared twice throughout the California. Marine Ecology 22: 283-305. m olecular phylogeny (Fig. 7B): (1) Stegastinae Albertson RC, Streelman JT, Kocher TD. 2003. subfamily, in Stegastes genus, N. latifrons, and H. 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