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Patterns of morphological evolution of the cephalicregion in damselfishes (Perciformes: Pomacentridae)of the Eastern Pacific
ROSALÍA AGUILAR-MEDRANO1*, BRUNO FRÉDÉRICH2, EFRAÍN DE LUNA3 andEDUARDO F. BALART1
1Laboratorio de Necton y Ecología de Arrecifes, y Colección Ictiológica, Centro de InvestigacionesBiológicas del Noroeste, La Paz, B.C.S. 23090 México2Laboratoire de Morphologie fonctionnelle et évolutive, Institut de Chimie (B6c), Université de Liège,B-4000 Liège, Belgium3Departamento de Biodiversidad y Sistemática, Instituto de Ecología, AC, Xalapa, Veracruz 91000México
Received 20 May 2010; revised 21 September 2010; accepted for publication 22 September 2010bij_1586 593..613
ADDITIONAL KEYWORDS: Gulf of California – head shape – phylogenetic morphometrics – reef fishes –trophic niche.
INTRODUCTION
The damselfishes (Pomacentridae) are a species-rich(approximately 360), worldwide distributed family ofmarine fishes that inhabit tropical and temperatewaters (Allen, 1991). Most inhabit tropical coral reefs,although a number of species live in rocky reefs ofcooler temperate waters. They have been presentwithin these ecosystems for 50 million years (Bell-wood, 1996; Bellwood & Sorbini, 1996). In the EasternPacific, their range extends from Monterey Bay
(California, USA) in the north to the south of Chile,including all the oceanic islands of this region (Fig. 1).In this part of the Pacific, Pomacentridae is oneof the most abundant families, having radiated incoral reefs, rocky reefs, and kelp forests. Twenty-fourspecies belonging to seven genera (i.e. Stegastes,Microspathodon, Hypsypops, Nexilosus, Chromis,Azurina, and Abudefduf) have been reported in theEastern Pacific and all are endemic to this region(Robertson & Allen, 2008). The genera Azurina,Hypsypops, and Nexilosus are solely present in thispart of the Pacific, whereas the genus Microspath-odon contains two species in the Eastern Pacific and*Corresponding author. E-mail: [email protected]
Biological Journal of the Linnean Society, 2011, 102, 593–613. With 9 figures
two others in the Atlantic Ocean. On the other hand,the genera Abudefduf, Chromis, and Stegastes aredistributed worldwide (Allen, 1991). All genera rep-resented in this region belong to the basal groupsof the family Pomacentridae (i.e. the subfamilies Ste-gastinae, Chrominae and Abudefdufinae; as definedby Cooper, Smith & Westneat, 2009) (Fig. 2). Themembers of the most derived subfamily Pomacentri-nae have radiated in the Indo-West Pacific region only(Cooper et al., 2009). Despite the use of numerousmolecular data, the phylogenetic relationships withinthe family Pomacentridae are not fully resolved(Tang, 2001; Quenouille, Bermingham & Planes,2004; Cooper et al., 2009).
The damselfishes of the Eastern Pacific display aremarkable diversity with regards to habitat prefer-ences, feeding habits, and behaviours. Their colora-tion is highly variable, ranging from drab hues ofbrown, grey, and black to brilliant combinations oforange, yellow, and neon blue (Robertson & Allen,2008). Generally speaking, the damselfishes livingin coral reefs of the Indo-West Pacific region feedmainly on filamentous algae as well as small plank-tonic and benthic invertebrates (Allen, 1991; Kuo& Shao, 1991; Frédérich et al., 2009). The drab-coloured species feed mainly on algae, whereas most
of the brightly patterned species (e.g. membersof the genus Chromis) obtain their nourish-ment from the current-borne plankton (Allen, 1991).To our knowledge, the trophic ecology of only twodamselfish species from the Eastern Pacific has
Figure 1. Distribution of damselfish species in the coast and islands of the Eastern Pacific Ocean. Left: localities ofunderwater observations in the Gulf of California: National Marine Park of Loreto, La Paz Bay, and National Marine Parkof Cabo Pulmo.
Figure 2. Phylogeny of Pomacentridae family sensuCooper et al. (2009). Dotted lines refer to groups notrepresented in the Eastern Pacific region.
been studied in detail: Stegastes rectifraenum andMicrospathodon dorsalis (Montgomery, 1980).
Ecomorphological studies attempt to understandthe relationships between the morphological varia-tion among species and their corresponding ecolo-gical variation (Norton, Luczkovich & Motta, 1995;Wainwright, 1996; Costa & Cataudella, 2007). Forexample, head morphology is subject to various con-straints dealing with the strategy of feeding and thetype of ingested food (Liem, 1979, 1993; Wainwright& Richard, 1995). The relationship between cephalicmorphology and diet has been broadly studied infreshwater fishes such as perches (Svanbäck & Eklöv,2002), centrarchids (Collar, Near & Wainwright,2005), and cichlids (Barel, 1983; Albertson, Streelman& Kocher, 2003), as well as marine fishes such assparids (Costa & Cataudella, 2007) and labrids (West-neat, 1995; Streelman & Karl, 1997; Wainwrightet al., 2004). The first ecomorphological studies inPomacentridae (Emery, 1973; Gluckmann & Vande-walle, 1998) suggested that a detailed study of cepha-lic morphology could reveal different trophic groups.Using geometric morphometrics (Rohlf & Marcus,1993; Lawing & Polly, 2009), Frédérich et al. (2008)highlighted strong size and shape variations in fourskeletal units of the damselfish skull among speciesliving in the Indo-West Pacific region and belonging todifferent trophic groups. Recently, Cooper & Westneat(2009) investigated the evolution of skull shapewithin Pomacentridae and showed that planktivoryhas involved important changes in damselfish headmorphology. In this latter study, the authors usedonly one species per genus to infer morphologicalevolution. Consequently, the inference of intra-genericvariation in trophic morphology was not possible.Recently, Barneche et al. (2009) confirmed the appli-cability of Bergmann’s rule to the family Pomacen-tridae, stating that the larger species are found athigher latitudes. The damselfishes of the EasternPacific are distributed along a wide range of latitudes(–33°N to 35°S; Fig. 1) and show a great variation ofbody size among species (standard length in the range11–36 cm; Table 1). Moreover, the genera Chromis,Hypsypops, Nexilosus and, Microspathodon containthe largest damselfishes (Allen, 1991). This axis ofbody size variation could have promoted morphologi-cal variation within this group of endemic species.
In the present study, we collect trophic data aboutall endemic damselfish species of the Eastern Pacificregion and apply geometric morphometric techniquesand phylogenetic methods to the cephalic region ofthese damselfishes. The main goal of the study is toprovide an overview of the trophic diversity of dam-selfishes in the Eastern Pacific and to determinewhether variations in cephalic shape can be explainedby size variation (i.e. allometry), feeding behaviour,
and/or phylogeny. In particular, we investigate theecomorphological hypothesis that cephalic shapevariation is associated with dietary differences indamselfishes of the Eastern Pacific. On the basisof previous analyses in damselfishes (Emery, 1973;Gluckmann & Vandewalle, 1998; Frédérich et al.,2008; Cooper & Westneat, 2009), we predict that dietis correlated to shape variations. We also test thepresence of phylogenetic signal (i.e. the expectationthat the phylogenetic relatedness is associated withshape similarity) (Cardini & Elton, 2008) in thecephalic region of damselfishes. We have no a priorihypothesis regarding this relationship because suchstudies are limited in fishes (Guill, Heins & Hood,2003).
MATERIAL AND METHODSTROPHIC DATA
Underwater observations (UO) were carried out inthree areas in the Gulf of California, Mexico (Fig. 1):(1) National Marine Park of Loreto Bay; (2) La PazBay; and (3) National Marine Park of Cabo Pulmo.The National Marine Park of Loreto Bay is located at26°07′ to 25°43′N and 111°21′ to 111°13′W. Coronado,Carmen and Danzante islands delimit this areawhere the substrate composition varies from fine sandto cobbles, boulders, and rocks (Campos-Dávila et al.,2005). La Paz Bay is located at 24°07′ to 24°21′N and110°17′ to 110°40′W, the reef presents fine sand, big(30 m) and small (12 m) boulders, big walls (15 m),and rocky reef (Aburto-Oropeza & Balart, 2001). TheNational Marine Park of Cabo Pulmo is the north-ernmost coral reef in the Eastern Pacific and islocated near the entrance of the Gulf of California ina transitional zone between the tropical and tem-perate Pacific at 23°50′N, 109°25′W (Alvarez-Filip,Reyes-Bonilla & Calderon-Aguilera, 2006). This parkpresents a barrier reef and a lagoon composed ofsmall boulders with sandy areas. The UO werecarried out during January, May, and August 2008,and January and March 2009, by scuba diving andsnorkeling. Observations of territorial species werecarried out for 2 min per organism; for fishes inconstant movement (e.g. planktivorous), a maximumof 10 min were devoted per organism. Observationswere conducted on 20–100 individuals per species,according to the availability. During these observa-tions, we mainly focused on two main components oftheir trophic ecology: (1) social behaviour: territorialand solitary species versus species forming groupsand (2) feeding habit: feeding areas (especially forterritorial, algivorous species), feeding strategy (i.e.biting, grazing or feeding in the water column), andtype of prey. Fourteen damselfish species were
directly observed and studied (Table 1). Consequently,the diet of all Eastern Pacific species was described bymeans of our UO and completed by a review of resultsfrom previous general studies. Using all these data,an alimentary item matrix of the 24 endemic dam-selfishes of the Eastern Pacific was built (Table 2). Itwas analyzed for the redundancy of the 16 alimentaryitems in all the species using the Kendall correlationindex for presence–absence data; this analysis wasconducted to group the alimentary items and reducethe main matrix. All alimentary items with a corre-lation higher than 0.80 were grouped (Table 2).
Additionally, the data of diet composition were alsoused for the estimation of the trophic level of eachdamselfish species. The TROPH index, initially usedfor Mediterranean fishes (Stergiou & Karpouzi, 2002),expresses the position of organisms within the foodwebs that largely define aquatic ecosystems. Realconsumers do not usually have TROPHs with integervalues and the definition of TROPH for any consumerspecies (1) is:
TROPH DC troph= + ⋅=
∑11
ij j
j
G
where G is the total number of prey species, DCij
represents the fraction of j in the diet of i and troph jis the fractional trophic level of prey j. The TROPHvalue was calculated from the dataset using TRO-PHLAB (Pauly et al., 2000), which is a stand-aloneapplication for estimating TROPH and its standarderror using qualitative information from the list ofitems known to occur in the diet of each species. If suchtrophic levels are missing, TROPHLAB uses defaulttroph values for various prey (based on data in FISH-BASE; Froese & Pauly, 2000). TROPH values wereused as feeding habit data, allowing the study of therelationships between head shape of damselfishes andtheir trophic ecology in a quantitative way, and givingan ecomorphological meaning to shape differences (seemethods below) (Costa & Cataudella, 2007).
GEOMETRIC MORPHOMETRICS AND
PHYLOGENETIC ANALYSIS
The sample comprised 509 specimens belonging to all24 damselfishes of the Eastern Pacific (Table 1). Addi-tionally, the embiotocid Zalembius rosaceus (Jordan &Gilbert, 1880) (family Embiotocidae) was used as an‘outgroup’. The Embiotocidae are now recognized asthe sister group of Pomacentridae (Streelman & Karl,1997; Mabuchi et al., 2007), Zalembius rosaceus is asmall benthic carnivorous fish distributed inside thesame area of the studied pomacentrids. Some speci-mens were speared during the field studies and theothers came from the museum collections of CIBNOR
(La Paz, BCS, México), CICIMAR (La Paz, BCS,México), SIO (San Diego, CA, USA), LACM (LosAngeles, CA, USA), and USNM (Washington, DC,USA) (Table 1). The list of museum specimens used inthis study is available upon request from the firstauthor (R.A.-M.).
The specimens were photographed in lateral viewwith a camera (Kodak ¥ 4 optical and 4 mega pixels)and the x- and y-coordinates of 18 homologous land-marks (Fig. 3) were digitized from the left side of eachindividual using TPSDIG, version 2.05. Superimposi-tion of landmark data was achieved using a general-ized procrustes analysis (Rohlf & Slice, 1990) whichaligned landmark configurations such that the sum ofsquared distances between corresponding landmarkswas minimized by scaling, translating, and rotatingspecimens with respect to a mean consensus con-figuration. The consensus configuration (‘the grandmean’) was obtained and used as the reference.Partial warp scores (PWs) including both uniform andnon-uniform components were calculated and used asdescriptors of shape variation (Bookstein, 1991; Rohlf,1993).
A principal components analyses (PCA) was usedto find hypothetical variables (components) thataccount for as much of the variance in the morpho-logical data (Davis, 1986). Subsequently, two kindsof discriminant analyses were carried out. Differ-ences among species were tested by means of analy-ses of variance (ANOVA), the Tukey–Kramer testand multivariate analyses of variance (MANOVA).Then, a canonical variance analysis (CVA) was per-formed to compare cephalic profile among groups.Indeed, CV axes allow us to maximize the differ-ences in shape among groups relative to withingroup variance. MANOVA and CVA were computedusing all shape variables (PWs). Deformation gridsusing the thin-plate spline (TPS) algorithm wereused to visualize the patterns of shape varia-tions along PC axes and CV axes (Thompson, 1917;Bookstein, 1991; Rohlf, 1993).
To determine whether shape data are hierarchicallyclustered, we applied phenetic and phylogeneticparsimony methods for grouping. Phenetic distancemethods are based upon a different conceptual frame-work of grouping than parsimony. We included aphenogram to determine whether there was evidencefor the assertion that similarity in morphometric datais not the result of a phylogenetic signal. Pheneticrelationships were summarized using a cluster analy-sis on the matrix of mean shape. Phenogram of the 25species was calculated using the unweighted pair-group method algorithm, and the Procrustes distanceas measure of the similarity. The goodness of fit of thecluster analysis was measured by the coefficient ofcophenetic correlation (Cardini & Elton, 2008). The
morphological phylogenetic analysis was executedusing the 32 shape variables (RWs) as characters forall studied species. For each species, we scored eitherRW intervals, RW means or both. Phylogenetic analy-ses were performed using new algorithms for thedirect optimization of continuous characters in TNT(Goloboff, Mattoni & Quinteros, 2006). We used acombination of ratchet, sectorial search, tree drifting,and tree fusing search algorithms for the selection ofoptimal trees and for the estimation of Jackknifevalues as a measure of support.
The molecular phylogenetic hypothesis by Cooperet al. (2009) and our morphological phylogeny wereused for phylogenetic adjusted regressions of theshape variables on the TROPH index, centroid size(CS), and standard length (SL) using the modulePDAP in MESQUITE (Midford, Garland & Maddison,2009). Our morphological and molecular phylogenieswere used to obtain an estimate of character distri-bution and correlation along a phylogeny (Garlandet al., 1993). Phylogenetic regression analyses totest interspecific allometry were performed usingthe mean values of CS and SL versus the 32shape variables. Similarly, we performed phylogeneticregression analysis using the mean values of the 32shape variables and the TROPH index to test therelationship between feeding habit and cephalicshape. Additionally, we compared these results withconventional nonphylogenetic regression analysesperformed in TPSREGRES, version 1.37.
Geometric morphometric analyses were performedusing 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). Multivariateanalyses (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 analysiswas computed in TNT, version 1.1 (Goloboff, Farris &Nixon, 2008; http://www.zmuc.dk/public/phylogeny).Phylogenetic regressions were performed in thePDAP package (Midford, Garland & Maddison, 2010;http://mesquiteproject.org/pdap_mesquite/) for MES-QUITE, version 2.73 (Maddison & Maddison, 2010;http://mesquiteproject.org/mesquite/mesquite.html).
RESULTSTROPHIC AND BEHAVIOURAL DATA
Chromis limbaughi, Chromis punctipinnis, Hypsy-pops rubicundus, and Stegastes redemptus were onlyobserved at one locality (Table 1), whereas the trophicbehaviours were always consistent among sites in theother species. Unfortunately, Chromis alta was neverobserved at any location, maybe as a result of itsdepth preferences (down to 150 m; Allen, 1991). Gen-erally, the underwater observations corroborate dataobtained from the literature related to diet of eachstudied species. However, some new trophic behav-iours were also observed. For example, the mainlyalgivorous S. rectifraenum was observed recurrentlypicking feces from zooplankton feeders (e.g. Abu-defduf troschelii) directly in the water column.
Figure 3. Anatomical landmarks used in geometric mor-phometric analyses: (1) tip of the premaxilla; (2) nostril;(3–6) inferior, anterior, superior, and posterior margin ofthe eye; (7) center of the eye; (8) superior tip of thepreopercular; (9) superior tip of the operculum; (10) pos-terior tip of the operculum; (11, 12) superior and inferiorinsertion of the pectoral fin; (13) insertion of the pelvic fin;(14) insertion of the operculum on the body profile; (15)posterio-ventral corner of the preopercular; (16, 17) ante-rior and posterior extremity of the dentary; (18) posteriorextremity of the premaxilla.
The trophic behaviour appeared relatively constantwithin all genera, except within the genus Abudefduf(Table 2). Abudefduf troschelii mainly fed on plank-tonic preys in the water column, whereas Abudefdufconcolor and Abudefduf declivifrons mainly grazedalgae or fed on some small sessile animals attached tothe rocks.
The analysis of alimentary items shows a strongrelationship (r > 0.80) between four items: macroal-gae, microalgae, sessile crustaceans, and worms.Consequently, these prey items were grouped inTable 2. Thus, a mainly algivorous species such asall Stegastes species, Microspathodon species, Nex-ilosus latifrons, and H. rubicundus may be con-sidered as a benthic feeder grazing on algae andfeeding on small benthic invertebrates. On the basisof a review of the published results and our UO,four alimentary items are species-specific (Table 2):sponges (A): H. rubicundus; detritus (L): S. rectifrae-num; gastropods (G): Z. rosaceus; and ectoparasites(M): A. troschelii. Abudefduf troschelii showed thelargest diet of the damselfish investigated in thepresent study, feeding in benthic and pelagic areas.All seven species of Chromis and the two speciesof Azurina mainly feed on zooplankton. Zalembiusrosaceus is considered as a benthic carnivorousspecies, feeding only on mobile benthic crustaceans(shrimps, small crabs), worms, gastropods, andbivalves (Table 2).
As for the diet, the social behaviour was con-served within genera. Hypsypops rubicundus andStegastes species are solitary, protecting small ter-ritories, forming couples only during the repro-ductive season. Hypsypops rubicundus showed apreference for small caves around big rocks andall Stegastes species prefer small rocks. BothMicrospathodon species are solitary or live in pairsand protect big rocks with high vertical walls. Nex-ilosus latifrons was not observed in the wild,although some authors (Grove & Lavenberg, 1997;Angel & Ojeda, 2001; Robertson & Allen, 2008)reported that it is a grazing species living close tothe rocks in small groups (up to ten individuals). AllAbudefduf, Azurina, and Chromis species live ingroups (i.e. schooling species) and are territorialonly during the reproductive season. The main dif-ference between these species is the number of inte-grants in the groups (Abudefduf species: 5–30individuals versus Chromis species: 10–60 indivi-duals), although it proved complicated to achievea good description of these numbers because theychanged according to the study area and thehabitat. Neither Azurina species were observed inwild; thus, we do not have any estimation of thenumber of fish per group. Specimens of A. declivi-frons and A. concolor could also be found solitary.
GEOMETRIC MORPHOMETRICS
The main shape variations across species can beexamined by a distribution of specimens in the PCfigure defined by the axis PC1 and axis PC2 (Fig. 4).The first two PCs account for 70% of the total shapevariance (PC1 = 58.41% and PC2 = 10.32%). Shapevariation was relatively low within each genus exceptfor Abudefduf. This genus could be clearly dividedinto two groups, one comprising A. concolor and A.declivifrons and the other comprising A. troschelii.PC1 allowed a clear distinction between mainlybenthic feeders (low PC1 values) and mainly zoop-lankton feeders (high PC1 values). The main morpho-logical variation along axis PC1 is related to the snoutlength, the cephalic depth, and the eye size andposition. The benthic feeders, especially Microspath-odon species, were characterized by a very shortsnout and small eyes; the pelagic feeders (Azurinaspecies, Chromis species, and A. troschelii) were char-acterized by relatively larger eyes and longer cephalicprofile. Along axis PC2, the main shape variation wasrelated to the position of the mouth and the pectoralfin, and explains the variation within some generasuch as Stegastes and Chromis.
When species were used as grouping factor,the MANOVA revealed significant differences amongthem (lWILKS = 2.36 ¥ 10–5, F = 42.02, d.f.1 = 256,d.f.2 = 3665.2; P < 0.001) and pairwise comparisonssupported that all species showed significant dif-ferences in mean shape (P < 0.05). MANOVA wasrepeated using genus as grouping factor, and theresults also show significant differences among allgenera; however, Abudefduf genus show a strongpatter of segregation into two groups. According to thetrophic data, the feeding habit is consistant withingenera except for the genus Abudefduf. The segrega-tion pattern of Abudefduf was tested by exploratoryanalyses (ANOVA, PCA, and CVA). Because thepattern was repeated in all analyses, the finalMANOVA was performed using genera as groupingfactor, with the genus Abudefduf split into twogroups: (1) A. concolor and A. declivifrons and (2) A.troschelii. The results of MANOVA show significantdifferences among all groups (lWILKS = 0.0000263,F = 41.23, d.f. = 265, P < 0.001), and all pairwisecomparisons based on Mahalanobis distances showsignificant differences (P < 0.05).
Discrimination among groups can be also inter-preted by examining the ordination of specimensin the morphospace defined by the CV axes (Fig. 5).The first three CV axes accounted for 84% of thetotal shape variation in the dataset and allow thediscrimination of five main groups. The axis CV1distinguishes three groups (Table 3): (A) Micros-pathodon species; (B) A. concolor, A. declivifrons, H.
rubicundus, N. latifrons and Stegastes species; (C)Azurina species, Chromis species, A. troschelii and Z.rosaceus. This last group (group C) could be subdi-vided into three groups in the morphospace defined byCV1 and CV3: Z. rosaceus in the extreme high values,Azurina species in the middle values, and A. troscheliiand Chromis species in the extreme low values(Fig. 5, Table 3).
In general, the CVA strengthened the profile andthe position of the eye and the mouth. Having thelowest scores of CV1, the two species of Microspath-odon showed a higher and flatter cephalic profile;Chromis species, Azurina species, A. troschelii, and Z.rosaceus present a lengthened cephalic profile withbig eyes; H. rubicundus, N. latifrons, Stegastesspecies, A. concolor, and A. declivifrons showed anintermediate shape. Along CV2, the main shapevariation was related to the position of the mouth andof the pectoral fin. The two Microspathodon speciesshowed a more horizontally oriented pectoral fin, thecephalic profile is high and the mouth is small
(+CV2). The eye of the two Azurina species and Z.rosaceus was in a more forward and lower position,and the snout region is also longer than in allChromis species and A. troschelii (CV3; Fig. 5). Thephenogram shows a segregation of four morphologicalgroups (Fig. 6): (1) all species of the genus Stegastes,A. concolor, and A. declivifrons, which are mainlyalgal feeders; (2) all species of the genus Chromis andA. troschelii, which are mainly zooplankton feeders;(3) both Microspathodon species are algal feeders,with an extremely flat cephalic shape; and (4) bothAzurina species, which are zooplanktivorous, and Z.rosaceus, which is carnivore benthic feeder, form agroup having a highly sharp cephalic profile. Thecoefficient of cophenetic correlation is relatively high(r = 0.76).
PHYLOGENETIC ANALYSIS
The morphological phylogenetic hypothesis (Fig. 6)shows a grouping pattern with a high degree of
Figure 4. Scatterplot of principal components (PC) 1 and 2. Cross, Abudefduf; equis, Azurina; square, Chromis; triangle,Hypsypops; circle, Microspathodon; asterisk, Nexilosus; black circle, Stegastes; black square, Zalembius. Thin plate splinedeformation grids for the extreme points of each axis are shown; these are superimposed on the shapes predicted whenthe average landmark configuration of all specimens is deformed into that of a hypothetical specimen positioned at theextreme point of an ordination axis.
correlation between head morphology and feedinghabits. The morphological phylogeny is not totallycongruent with the molecular phylogeny, exceptin that the subfamily Stegastinae is recoveredwith the genus Stegastes, Nexilosus, Hypsypops,and Microspathodon. This clade presents mode-rate support values (Jackknife = 63). However, ourmorphometric phylogeny includes two species of
Abudefduf that belong to the Abudefdufinae sub-family according to the molecular phylogeny. Withinthis group, the clade of two species of Abudefduf ishighly supported (Jackknife = 96). The subfamiliesChrominae and Abudefdufinae are not recovered asmonophyletic groups in our morphometric phylogeny.Rather, the Chrominae is a partially resolved gradethat also includes A. troschelii.
Figure 5. Scatterplot of canonical variates (CV): CV1 versus CV2 and CV1 versus CV3. Black cross, Abudefduf concolorand 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, Zalembius. For both plots, thin plate splinedeformation grids for the extreme points of each axis are shown; these are superimposed on the shapes predicted whenthe average landmark configuration of all specimens is deformed into that of a hypothetical specimen positioned at theextreme point of an ordination axis.
Interspecific allometry was tested using linear regres-sions analyses. The relationship between shape andsize (CS and SL) is low (r2 � 0.4; Table 4). The lowestr2-values were found with the nonphylogeneticregression analysis and the highest ones with thephylogenetic regression analyses (Table 4). However,shape always showed a stronger relationship with CSthan with SL.
RELATIONSHIP BETWEEN CEPHALIC
SHAPE AND TROPHIC DATA
Both nonphylogenetic regression analyses and theequivalent with phylogenetically independent con-trasts show a significant positive relationshipbetween the TROPH index and shape variables(Table 4). Values of the coefficient of determination(r2) were lower in the phylogenetic regression analy-sis than in the nonphylogenetic analysis. These testsand mirror trees analyses clearly demonstrate that
the main shape variation among the studied dam-selfishes is related to their feeding habit (Fig. 7).Cephalic shapes are more related to trophic levelsthan to the phylogenetic relationships (Fig. 7,Table 4). Trophic data group the 24 damselfishes inthree main groups: mainly algal feeders, mainlyzooplankton feeders, and an intermediate groupfeeding on small pelagic and benthic preys. CVA andcluster analysis allow the discrimination of the threemain trophic groups and also show a clear degree ofvariation between two extremes of cephalic shape(i.e. zooplankton feeders, Azurina species, withhighly sharpness cephalic shape and algal feeders,Microspathodon species, with highly flattened cepha-lic shape). On the other hand, the phylogenetichypothesis constructed with shape data producestwo monophyletic groups: (1) subfamily Stegastinae,which group mainly algal feeders and (2) a groupcomposed of A. troschelii, Chromis meridiana, andC. limbaughi, which are mainly zooplanktivorous,feeding also on fish eggs.
Table 3. Analysis of variance and Tukey–Kramer test of the three first axes of canonical variance analysis
The optimization of the TROPH index and morphol-ogy in the molecular phylogeny revealed that allspecies of the genus Stegastes and N. latifrons arehighly similar with respect to morphology and feedinghabits. The subfamily Stegastinae show a high cor-relation between morphology and feeding habits,
although some shape variation can be observedamong all Stegastes species, N. latifrons, and thetwo Microspathodon species. Morphological phylog-eny highlighted a pattern of convergence with twomembers of Abudefduf genus (A. concolor and A.declivifrons). Although A. concolor and A. declivifrons
Figure 6. Comparison of two hierarchical models. Phenogram (A) and morphometric phylogeny (B) compared to maintrophic groups of damselfish. Images of some species are added to help to visualize the pattern of cephalic shape variationin 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 relationshipbetween morphology and trophic data
Least squareregression Variables r2 F d.f. P
Phylogenetic CS versus shape variables (molecular and morphological phylogeny) 0.41 16.32 23 0.0005SL versus shape variables (molecular and morphological phylogeny) 0.17 4.64 23 0.042TROPH index versus shape variables (molecular phylogeny) 0.51 23.16 23 < 0.0001TROPH index versus shape variables (morphological phylogeny) 0.51 23.16 23 < 0.0001
Nonphylogenetic CS versus shape variables 0.20 6.22 32–736 < 0.0001SL versus shape variables 0.09 2.44 32–736 < 0.0001TROPH index versus shape variables 0.36 13.17 32–736 < 0.0001
are close according to the morphological phylogeny,the feeding habit of A. declivifrons is different andmore similar to that of members of the Chrominaesubfamily. Both Microspathodon species and H. rubi-cundus are close according to their feeding habits.The cephalic shape is highly conserved in the sub-family Stegastinae through both phylogenies. Accord-ing to the categories of the TROPH index (Fig. 7), theChrominae subfamily shows five categories distrib-uted between medium and high values of the TROPHindex. On the other hand, the range of morphologicalvariation of this group is low (i.e. only two extrememorphological categories of colour code; Fig. 7).According to the morphological phylogeny, A. trosche-lii is closely related to the Chrominae. The con-vergence pattern of A. troschelii introduces a newmorphology within this group. The Abudefdufinaesubfamily is highly diverse. Two morphological cat-egories were found in this subfamily (Fig. 7); (1)A. troschelii is more similar in shape to the outgroup(Z. rosaceus) and (2) A. concolor and A. declivifronsshow similar values to the Stegastinae subfamilymembers. Each Abudefduf species has a differentcolour code for its trophic index (Fig. 7).
DISCUSSIONTROPHIC DIVERSITY OF DAMSELFISHES
The trophic data obtained in the present studyconfirm the division of damselfishes of the EasternPacific into three main trophic groups, as reported forspecies living in the Indo-West Pacific region (Allen,1991; Kuo & Shao, 1991; Frédérich et al., 2009): (1)the pelagic feeders mainly sucking zooplanktonicpreys (e.g. Chromis spp., Azurina spp.); (2) thebenthic feeders mainly grazing filamentous algae andpicking small invertebrates (e.g. Stegastes spp., Nex-ilosus latifrons, Hypsypops rubicundus, Microspath-odon spp.); and (3) an intermediate group gatheringspecies feeding both on small pelagic and benthicpreys (e.g. Abudefduf spp.).
The social behaviour of damselfishes is constantin the whole Indo-Pacific, Western Atlantic, andEastern Pacific region (Emery, 1973; Frédérich et al.,2009; present study): species mainly feeding onbenthic prey are solitary, whereas zooplanktivorousspecies live in groups. However, N. latifrons is rela-tively atypical because it is a grazing species livingin small groups (up to ten individuals) (Grove &Lavenberg, 1997; Angel & Ojeda, 2001; Robertson& Allen, 2008).
In general, the diet is consistent within damselfishgenera. However, our ecomorphological approach indi-cated that the three Abudefduf species should begrouped into two different trophic guilds. Consistent
with the trophic data (Table 2), the cephalic profile ofA. troschelii is more similar to that of Chromis andAzurina, suggesting that this species should be con-sidered as an omnivorous species mainly feeding onzooplankton. On the other hand, A. declivifrons andA. concolor are more similar to Stegastes, Hypsypops,and Nexilosus, showing that they should be consid-ered as omnivorous species and mainly benthicfeeders.
To our knowledge, H. rubicundus is the onlydamselfish feeding on sponges. No damselfish of theIndo-West Pacific is known to be a common con-sumer of such kind of prey (Allen, 1991; Frédérichet al., 2009). The angelfishes (Pomacanthidae) isspecialized in such prey catching in coral reef envi-ronments (Konow & Bellwood, 2005). Accordingto Robertson & Allen (2008), one Pomacanthidae,Pomacanthus zonipectus, is distributed in the coolertemperate regions and lives sympatrically withH. rubicundus. However, this angelfish achieves itsmaximum population density in the Tropical EasternPacific and is scarce in cooler temperate regions.Consequently, a lower competition level could permitan extension of the trophic width of damselfishessuch as H. rubicundus in temperate regions.
ECOMORPHOLOGY AND MORPHO-FUNCTIONAL
IMPLICATIONS
The bucco-pharyngeal cavity of a fish has been mod-elled as a truncate cone, whose small base comprisesthe circular opening of the mouth and whose largebase is located behind the branchial basket on thelevel of the opercles (Alexander, 1967; Lauder, 1980;Lauder & Lanyon, 1980; Liem, 1993). The efficiency ofthe cone depends on various factors such as themorphology of the skull and particularly the bucco-pharyngeal cavity (Liem, 1990). There are three basicmodes of feeding according to the degree of truncationof the cone (Liem, 1980, 1993): suction feeding, ramfeeding, and biting. However, a mode of prey captureis not exclusive; many teleosts are able to modulatetheir feeding mode and to move from one categoryto another (Liem, 1980, 1993; Ferry-Graham et al.,2002). If the head morphology of damselfishesprompts the consideration that they are good suctionfeeders (Emery, 1973; Frédérich et al., 2008; Cooper& Westneat, 2009; present study), geometric mor-phometric analyses allow a deeper understanding ofthe different ways of feeding and reveal functionaldifferences among species.
The main difference between morphological groupsis the degree of sharpness of the cephalic shape,which goes from a long angular cephalic profile as inZ. rosaceus, both Azurina species, A. troschelii, andall Chromis species; followed by angular but shorter
cephalic profiles as in A. concolor, A. declivifrons, H.rubicundus, N. latifrons, and all Stegastes species;and, finally, to an almost flat cephalic profile as inboth Microspathodon species (Fig. 6). The benthic car-nivorous Z. rosaceus feeds mainly on gastropods,mobile worms, and crustaceans (Table 2). As observedin some cichlids (Liem, 1993), a long angular cephalicprofile may facilitate the catching of these items.Despite a similar angular cephalic profile as in bothAzurina species and Chromis species, Z. rosaceusshows a great morpho-functional difference comparedto these zooplanktivorous species, which was not indi-cated by our geometric morphometric analyses.Indeed, this difference is solely observed when themouth is extended (i.e. during mouth protrusion)(Fig. 8). During feeding, the mouth is oriented moreventrally in Z. rosaceus, optimizing the capture ofbenthic animal preys, whereas the mouth is directedrostrally in Azurina and Chromis species, facilitatingprey capture in the water column. Further morpho-functional studies should precisely address the differ-ences in the degree of protrusion of the premaxillarybones during feeding among these species. Zooplank-tivorous damselfishes such as the Azurina andChromis species can be described as being particulate
feeding in that they attack the individual planktonicpreys they select visually. The possession of relativelylarge eyes (Fig. 5) should increase their ability to findand target planktonic preys, as exemplified in cichlids(Barel, 1983). Their elongated head profile facilitatesthe capture of these organisms using ram-suctionfeeding (Coughlin & Strickler, 1990), although furtherfunctional studies should aim to test whether thedifferences in cephalic profile between Chromis andAzurina species (Fig. 5) could be related to differencesin feeding strategy and performance. For example,the contribution of ram (i.e. the predator movementtowards the prey) and suction (i.e. the prey movementtowards the predator as a result of aspiration) duringfeeding may differ between both genera (Wainwrightet al., 2001).
Abudefduf troschelii is an omnivorous speciesfeeding mainly on zooplankton (Grove et al., 1986;present study). Robertson & Allen (2008) consideredthis species to comprise two feeding groups: omnivo-rous and planktivorous. Abudefduf troschelii shows avery similar cephalic profile to the almost exclusivezooplanktivorous Chromis and Azurina genera. Simi-larly, A. troschelii mainly occurs along rocky shores orcoral reefs, in shallow waters, foraging on zooplank-ton in aggregations. By contrast, A. concolor, A.declivifrons, H. rubicundus, N. latifrons, all Stegastesspecies and both Microspathodon species mainlygraze filamentous algae growing on rocks. Within thistrophic group, the two Microspathodon speciespresent a highly different morphology. Our underwa-ter observations revealed that the way of feeding inboth Microspathodon species differ from that of theothers. Indeed, all Stegastes species grazed algae orpicked up small invertebrates on small, mainly hori-zontal rocks or rubble, whereas the two Microspath-odon species scraped on big rocks with high verticalwalls. This type of feeding is probably facilitated byan almost flat cephalic profile in Microspathodonspecies. Moreover, the premaxillary bones on theiranterior region reveal a loose connective tissue whereteeth are continuously produced (Ciardelli, 1967).When the fish scrapes the rocky wall, the teeth arecontinuously eroded and, consequently, need to beproduced constantly (Trapani, 2001). Furthermore,this connective tissue that supports and nurtures theteeth could act as a buffer, supporting the movementof the teeth and the premaxillary bones on therock. Although, when the food items are similar, themorphological pattern could diverge if the methods ofprey catching differs.
The results of the present study indicate that dam-selfishes from the Eastern Pacific show strong differ-ences with respect to their cephalic profile. Thesedifferences are mainly related to the degree of sharp-ness and the position of the eye and the mouth. A
Figure 8. Difference in the mouth orientation when it isopen in (A) Zalembius rosaceus, (B) Azurina hirundo, and(C) Chromis atrilobata.
pattern of relationship between head morphology anddiet was found, as in previous analysis (Frédérich,Parmentier & Vandewalle, 2006; Frédérich et al.,2008; Cooper & Westneat, 2009). However, the rela-tionship between the cephalic profile and feedinghabit found in damselfishes extends beyond this. Inthe present study, head morphology was observed tobe related to the way that the food resource isextracted: two species can use the same food resource(Stegastes species and Microspathodon species) andpresent a different head morphology, and theserespond to the way that each species extracts theresources of the environment.
EVOLUTIONARY CONSIDERATIONS
The shape data of the present study confirm amorphological group composed by Z. rosaceus, ourchosen outgroup (Figs 4, 5). The family Embiotocidaeincludes mainly carnivorous (zoobenthos) species witha pointed cephalic profile. Interestingly, Z. rosaceusshows a cephalic profile more similar to the mostderived zooplanktivorous damselfishes (i.e. Chromisand Azurina) (Fig. 2). The similarity among thesethree genera is a convergence in the cephalic profile.The present study confirms the results reported byFrédérich et al. (2008), who stated that shape varia-tion is not correlated with size variation in damself-ishes. Interspecific allometry is low and size does notpredict convergent shapes. Consequently, variationsin size and shape may be viewed as two independentevolutionary factors explaining the diversification ofthe family Pomacentridae. When the relationshipsbetween CS, SL, and shape variables are analyzedthrough nonphylogenetic analyses, r2-values arelower than when they are analyzed through phyloge-netic analyses, indicating that size is related to thephylogeny.
The morphometric results of the present studyshow Azurina to be very similar in cephalic shape toChromis. In a Euclidean plane, the slender Azurinaspecies lie at the extreme point of the Chromis dis-tribution along an axis of cephalic length (Figs 4, 5).According to the morphological phylogeny, Azurinaspecies are closely related to Chromis species (Fig. 6);this relationship is also reported by Cooper et al.(2009), who proposed synonymizing the genusAzurina with Chromis and re-classifying the twospecies of Azurina. Their proposal was based on astrong phylogenetic relationship between Azurinahirundo from the Eastern Pacific and Chromis mul-tilineata from the Atlantic.
The genus Abudefduf had been traditionally segre-gated into two groups. According to our mophometricanalyses, the main difference between these groups isthe position of the mouth, which clearly corresponds
to two different feeding habits. In A. troschelii, themouth is superior (omnivorous, feeding mainly onzooplankton); this shape is very similar to that of C.meridiana and C. limbaughi (Fig. 6). By contrast, themouth of A. concolor and A. declivifrons is inferior(omnivorous, feeding mainly on benthic preys); thus,the shape is similar to Microspathodon bairdii andMicrospathodon dorsalis (Fig. 6). The molecular phy-logenetic analysis of Quenouille et al. (2004) consi-dered 16 Abudefduf species, and found three mainclades within this genus: A. declivifrons, A. concolor,and Abudefduf taurus are the sister group to thetwo principal groups. Abudefduf taurus is a mainlyherbivorous fish (Randall, 1967; Allen, 1991), withsimilar behaviour to A. concolor and A. declivifrons,showing strong preference for limestone shorelinesand tide pools in regions with surf (Allen, 1991;R. Aguilar-Medrano, pers. observ.). The second groupcomprises Abudefduf sordidus, Abudefduf septemfas-ciatus, and Abudefduf notatus, which are also mainlyalgal feeders (Allen, 1991). All Abudefduf species ofthese two groups possess a dark body with clearvertical bars and have a strong preference for veryshallow areas (0–4 m) with surf (Allen, 1991; R. A.-M.& B. F. pers. observ.). Their mouth is placed in a lowerposition than the others. The third cluster of Que-nouille et al. (2004) includes A. troschelii, these grouppresent species which show an overall lighter bodywith dark vertical bars and are laterally compressed;all are omnivorous, feeding mainly on zooplankton(Emery, 1973; Frédérich et al., 2009; present study).They are gregarious, their mouth is in a superiorposition, their habitat distributions are in a waterdepth in the range 1–15 m, and they are associatedwith rocky and coral reefs (Emery, 1973; Allen, 1991;Frédérich et al., 2009; present study).
Previous molecular phylogenetic analyses foundthat Microspathodon, Hypsypops, Nexilosus, and Ste-gastes are closely related genera belonging to thesubfamily Stegastinae (Tang, 2001; Quenouille et al.,2004; Cooper et al., 2009), although relationships arenot fully resolved (Fig. 2). The trophic data of thepresent study revealed that all these species arebenthic feeders, mainly grazing filamentous algae.Our geometric morphometric analyses and pheno-gram clearly distinguish the two Microspathodonspecies from the other species belonging to thistrophic group (i.e. H. rubicundus, N. latifrons, and allstudied Stegastes species) based on their highly flat-tened cephalic profile. Hypsypops rubicundus and N.latifrons are predominantly found in temperatewaters, in the extreme north and south, respectively,of the Eastern Pacific damselfish distribution. Theircephalic profiles are relatively similar to those ofStegastes species, although geometric data showsome differences among these three genera. In the
morphospace, H. rubicundus and N. latifrons speciesare placed peripheral of the Stegastes morphologicaldistribution (Fig. 5) and a schematic representation ofthe observed differences within this group is illus-trated in Figure 9. If we hypothesize that Nexilosus,Hypsypops, and Microspathodon are derived from thegenus Stegastes (Cooper et al., 2009), the results ofthe present study demonstrate that the main shapevariation during the evolutionary process was relatedto the form of the cephalic profile (pointed versusflattened), the size of the eye, and the position of theeye and mouth. From a Stegastes ancestor, a firstevolutionary step could produced two new forms(Fig. 9): (1) H. rubicundus with an pointed profile, along horizontal distance between the mouth and theeye, and a short vertical distance between the mouthand the eye, and (2) N. latifrons with a more roundedprofile, a short horizontal distance between the mouthand the eye, and a long vertical distance between themouth and the eye. The possible next evolutionarystep from the Nexilosus brand produced a stronglyspecialized cephalic profile: both Microspathodonspecies present a highly flattened profile, no horizon-tal space between the eye and the mouth and highvertical distance between the mouth and the eye(Fig. 9).
The molecular data reported by Tang (2001) showedthat Hypsypops is closer to Parma (Subfamily Stegas-tinae) than to Stegastes. Indeed, when the genusParma was first described, H. rubicundus wasincluded as one of its members (Tang, 2001). Thesetwo genera are predominantly distributed in temper-ate cool water and rocky reefs; furthermore, Parmaincludes some species that can be found in kelpforests, such as H. rubicundus (Allen, 1991; Buckle &Booth, 2009). Future geometric studies should includeParma species to obtain a good overview of the phe-notypic differences or similarities among Parma,Hypsypops, and Stegastes. The phylogenetic positionof N. latifrons is unknown (Cooper et al., 2009),although the data of the present study stronglysuggest that N. latifrons is closely related to Stegastesspecies in morphology and ecology, even though theformer lives in small groups. According to Robertson& Allen (2008), the main differences between thisgenus and Stegastes are that the margins of thepreopercle and infraorbital bones are smooth in Nex-ilosus and serrated in Stegastes. Both genera presenta single row of teeth and the number of dorsal finspines of Nexilosus (XIII) is included in the range ofStegastes (XII to XIV) (Allen, 1991). Similar to thequestion of synonymy between the genus Azurina andChromis, the close relationship between Nexilosus,Stegastes, and Hypsypops allows us to questionwhether Nexilosus and Hypsypops (both monospecificgenera: N. latifrons and H. rubicundus, respectively)
Figure 9. Schematization of the variation related tothe position of the eye and mouth in the cephalic profileof (A) Hypsypops rubicundus, (B) Stegastes flavilatus,(C) Nexilosus latifrons, and (D) Microspathodon dorsalis.
should be considered as valid genera. Further exhaus-tive phylogenetic studies are needed to better under-stand the phyletic relationships among Microspa-thodon, Hypsypops, Nexilosus, Stegastes, and Parma.
Our morphometric and phylogenetic analyses of thecephalic region show that the subfamilies Stegastinaeand Chrominae present a specific morphologicalpattern, but not Abudefdufinae (Fig. 7). The mor-phological average appeared twice throughout themolecular phylogeny (Fig. 7B): (1) Stegastinaesubfamily, in Stegastes genus, N. latifrons, and H.rubicundus, and (2) in Abudefdufinae subfamily, inA. concolor and A. declivifrons. In these groups, themean cephalic shape is related to medium-highvalues of the TROPH index. Nevertheless, species ofthe genus Chromis showed the same TROPH values.Consequently, A. concolor and A. declivifrons pre-sent convergent morphological adaptations to themembers of the subfamily Stegastinae in the familyin the Eastern Pacific.
In conclusion, the present study shows that varia-tion in the cephalic shape of Pomacentridae of theEastern Pacific can be clearly explained by differencesin diet and trophic behaviour. Shape and size may beviewed as two independent evolutionary factorsexplaining the diversification of Pomacentridae.Cepahlic shape is a significant predictor of trophichabit. Finally, the morphological groups discovered byour morphometric analyses partially agree with themain clades delimited by molecular phylogenetichypothesis. Consequently, the cephalic profile of dam-selfishes shows a clear and strong historical (phylo-genetic) signal only for the Stegastineae and partiallyfor Chrominae. Members of the subfamily Abudefdu-finae show convergences of cephalic shape. Themainly zooplanktivorous A. troschelii is very similarto Chromis species (Chrominae), and the mainly algalfeeders A. concolor and A. declivifrons are verysimilar to Microspathodon species (Stegastinae).
ACKNOWLEDGEMENTS
We thank the Consejo Nacional de Ciencia y Tec-nología (CONACYT, México) for a doctoral scholar-ship support to A.M.-R. to develop this work. We alsothank Lucia Campos Dávila (CIBNOR), Sandra J.Raredon (USNM), H. J. Walkers (SIO), Philip A.Hastings (SIO), Victor Cota Gómez (CICIMAR), JoséDe La Cruz Agüero (CICIMAR), and Rick Feeney(LACM) for their help with the museum collections.Thanks also to Fernando Aranceta Garza, IsmaelMascareñas Osorio, Juan J. Ramírez Rosas, MarioCota Castro, and Enrique Calvillo (all from CIBNOR)for their enthusiastic assistance on field trips. Thiswork was supported in part by the projects EP2 andEP3 of the Centro de Investigaciones Biológicas del
Noroeste, México (CIBNOR) and CT001 (CONABIO).Finally, we thank Mark Webster and two anonymousreviewers for their enlightening comments.
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