(Teleostei, Cyprinodontidae, Orestias) from Lake Titicaca

Journal of Fish Biology (2009) 74, 502–520 doi:10.1111/j.1095-8649.2008.02140.x, available online at http://www.blackwell-synergy.com

Morphology–diet relationships in four killiﬁshes (Teleostei, Cyprinodontidae, Orestias) from Lake Titicaca

E. MALDONADO*†‡, N. HUBERT§, P. SAGNES AND k B. DE ME´ RONA* k *U.R. 131 Institut de Recherche pour le Developpement (IRD), 43 Bd du 11 novembre 1918, 69622 Villeurbanne, France, †Instituto de Limnologıa,´ Universidad Mayor de San Andre`s, La Paz, Bolivia, §U.R. 175 Institut de Recherche pour le Developpement (IRD), GAMET, BP 5095, 361 rue JF Breton, 34196 Montpellier Cedex 05, France and UMR CNRS 5023 Ecologie des hydrosyste`mes ﬂuviaux, Universite Claude Bernard Lyonk1, 43 Bd du 11 novembre 1918, 69622 Villeurbanne, France

(Received 21 January 2008, Accepted 22 October 2008)

This study explores the relationship between morphology and diet in four Andean killiﬁshes (Orestias) from Lake Titicaca that are known to differ in habitat use. Species that fed preferentially on amphipods (Orestias albus) or molluscs (Orestias luteus) separated in multivariate space from other species that feed on cladocera and algae (Orestias agassii and Orestias jussiei). Generally, specimens feeding on cladocera were characterized by a short, blunt nose with a small mouth; whereas, specimens feeding on amphipods exhibited a long snout with a large mouth. Specimens including molluscs in their diet tended to have a larger posterior part of the head and the larger opercles than others; while the occurrence of substratum in gut content was generally related to a short but deep head. The present analysis suggests that the littoral O. jussiei has an intermediate phenotype and diet between the pelagic (O. agassii) and benthic (O. albus and O. luteus) species. Results suggest that resource partitioning was occurring and that several morphological traits relate to characteristics of the diet, and it is inferred that the benthic, the pelagic and the littoral zones in the lake host different prey communities constituting distinct adaptive landscapes. # 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles

Key words: ecomorphology; functional morphology; gut content; habitat partitioning; head morphometry.

INTRODUCTION The origin of phenotypic divergence and the inﬂuence of environment in shap- ing phenotypes are a central question in the evolutionary biology (Barel, 1983; Via & Lande, 1985; Winemiller, 1991; Smith & Skulason,´ 1996; Schluter, 2000, 2001; West-Eberhard, 2003, 2005). As ecosystems are dynamic by nature, individuals may experience a vast array of ecological conditions throughout the

‡Author to whom correspondence should be addressed: Tel.: 33 472 43 1642; fax: 33 472 43 2892; þ þ email: [email protected] 502

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 503 species range distribution leading to local adaptation and ‘phenotypic novelties’ (Schluter & Grant, 1984; Meyer, 1993; Schluter, 1993, 1996; Losos et al., 1998; Kocher, 2004). Hence, it has been suggested that natural selection may be driv- ing species occupying the same ecological niche to be similar in some key morphological features as a response to similar selective pressures (Wainwright, 1991, 1996; Hugueny & Pouilly, 1999; Pouilly et al., 2003; de Merona et al., 2008). The major goal of ecomorphology is to understand the morphological response of organisms to ecological conditions by comparing patterns of variation in ecological characteristics (e.g. diet) with patterns of variation in morphological characteristics measured across populations, species, higher-order taxa and communities (Motta et al.,1995; Norton et al., 1995). In this context, foraging strategies and associated morphological adaptations constitute an important part of the phenotype–environment relationship (Grant, 1986; Wainwright, 1991; Winemiller, 1991; Douglas & Matthews, 1992; Motta et al., 1995; Skulason´ & Smith, 1995; Winemiller et al., 1995; Svanback¨ & Eklo¨ v, 2002; Langerhans et al., 2003; Higham et al., 2006; Parsons & Robinson, 2006, 2007). Several studies have provided evidence for associations between foraging and feeding strategies and particular morphological adaptations (Pouilly et al., 2003) that may reflect convergent response to ecological pressures. Other studies, however, have found little or no relationship between ecological and morphological characters (Motta et al., 1995). So, it cannot always be assumed that environment constrains morphology and ecology in a parallel fashion. This study examines the relationship between diet and morphology in a genus of freshwater killifish as a first step in understanding the high degree of ecological and the phenotypic diversity exhibited in the group. The freshwater killifish genus Orestias (Teleostei, Cyprinodontidae) encom- passes 43 species endemic to the Andes (Parenti, 1984; Dejoux & Iltis, 1991). The genus has been hypothesized to have its origin 5 million years ago with an important diversification stage during the last 1 million years (Parenti, 1981, 1984; Lussen et al., 2003). There is great ecological and phenotypic diversity within the genus across the wide range of habitats in which it is found in the Andes (Dejoux & Iltis, 1991). Currently, 15 species occur in the Titicaca Lake where they occupy different niches and constitute important components of the trophic network (Lauzanne, 1982; Loubens et al., 1984; Loubens & Sarmiento, 1985). They are found either in the pelagic, the benthic or the littoral zones, and resource partitioning has been suggested to occur among the species (Lauzanne, 1982; Dejoux & Iltis, 1991). Currently, four of the largest and abundant Orestias species from Lake Titicaca illustrate the ecological diversification in the genus: (1) Orestias jussiei Valenciennes, reaches 130 mm standard length (LS) and is found almost exclusively in the littoral zone from Lake Titicaca and neighbouring water- sheds (Lauzanne, 1982; Froese & Pauly, 2000); (2) Orestias agassii Valenciennes, reaches 150 mm LS and is mainly found in the pelagic zone. This species occurs throughout the Altiplano in both lakes and rivers (Lussen, 2003); (3) Orestias albus Valenciennes, reaches 130 mm LS, lives in the benthic zone and is one of the few Ore- stias known to include fish in its diet. This species in tightly restricted to the Lago Menor and the southern part of Lago Mayor in Lake Titicaca (Lauzanne, 1982; Froese & Pauly, 2000) and (4) Orestias luteus Valenciennes, reaches 115 mm LS and lives in the benthic zone (Lauzanne, 1982; Dejoux & Iltis, 1991). This species

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 504 E . M A L D O N A D O E T AL . is found throughout Lake Titicaca from Lago Arapa north to Lago Menor south as well as outside the lake in the Laguna Umayo (Lussen, 2003). Previous studies provided evidence for dietary specialization in these four species (Lauzanne, 1982), and suggested that the genus may have diversiﬁed through an adaptive radiation (Vill- wock, 1986). In this context, phenotypic diversity in the genus may be related to divergent selection in contrasted habitats (Wainwright, 1991, 1996; Hugueny & Pouilly, 1999; Pouilly et al., 2003). Nevertheless, in the genus Orestias the extent to which morphological divergence is related to foraging and feeding remains poorly assessed, and potential mechanisms underlying the origin of the ecological diversity in the genus are still unknown. This study quantiﬁes the relationships between some head characters, considered as functionally important during the foraging activity and diet using a multivariate morphometric approach.

MATERIALS AND METHODS

STUDY SYSTEM Lake Titicaca is the world’s highest lake standing at an altitude of 3810 m and cov- ering 8 300 km2 with a length of 195 km and a width of 50 km (Dejoux & Iltis, 1991). The lake is currently divided into two orogenetic basins (Fig. 1). The Lago Mayor is the largest and deepest with a mean and maximum depth of 150 and 285 m, respectively. By contrast, Lago Menor reaches only 40 m in depth (mean depth 10 m) and covers <1000 km2. This tropical lake experiences almost constant light and temperature conditions throughout the year and high salinity content (Dejoux & Iltis, 1991). Three major types of habitat are found: (1) the pelagic zone (i.e. open water of the lake) rich in Cladocera; (2) the benthic zone (i.e. bottom area) rich in molluscs and amphipods;

FIG. 1. Study area and location. All ﬁshes were captured in Lago Menor, and samples were obtained from the Copacabana market between June and November 2003.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 505

(3) the littoral zone (i.e. weedy habitat of the shores) rich in macrophytes and amphipods (Dejoux & Iltis, 1991). The four species of Orestias studied are of economic interest in the Bolivian and Peruvian Andes, and commonly captured by Amerindian communities in the vicinity of the cities of Copacabana and Puno (Fig. 1). All specimens analysed were captured with gillnets of small mesh-size in the Lago Menor by local ﬁshermen and brought back by them to the Copacabana market, where they were purchased for this study between June and November 2003. If specimens were still alive when purchased, they were euthanized with a clove oil solution. Specimens were frozen immediately to prevent deg- radation of the gut content. Ontogenetic changes in diet and morphology occurring during growth may affect interspeciﬁc comparison of both morphology and diet (Machado-Allison & Garcıa,´ 1986; Winemiller, 1989). Hence, only specimens close to the maximal LS were retained for subsequent analysis.

DIET Specimens with an empty stomach (only 5% of the specimens analysed) were dis- carded from the data sets for subsequent analyses of both diet and morphology. In order to characterize the diet of the four species, the relative abundance of 10 trophic items were considered: t1, cladocerns; t2, amphipods; t3, substratum; t4, algae; t5, arthropods; t6, macrophytes; t7, fish eggs; t8, fishes; t9, molluscs and t10, bryozoans. For each specimen, the gut was opened and each of the 10 dietary item types was iso- lated. Stomach contents were measured using a surface area covered by graduated paper. The aggregate mass of each prey type for each individual fish was laid over the graduated paper in a Petri dish and measured. Although the substratum is not prey, this item may indicate the location of foraging activity (more substratum should be ingested during benthic activities) and was treated as a diet item in subsequent analyses.

MORPHOLOGY Functional morphological traits selected were those related to foraging activity and prey capture and included many morphometric traits in the head, since head morphology is strongly related to foraging. Hence, morphometric variables in the anterior part of the body were considered to assess the variability of functionally important morphological characters among the four species (Gatz, 1979; Hugueny & Pouilly, 1999; Pouilly et al., 2003). A total of 13 homologous points were identiﬁed on the head (Fig. 2). Most of the distances between these points were measured following a ‘truss’ as described by Strauss & Bookstein (1982). Digital pictures from lateral and dorsal views were used 1 (118 pixels cmÀ or 300 dpi), and measurements to the nearest 0 1 mm were obtained through the software ImageJ 1.3 (freeware available at http://rsbÁ.info.nih.gov/ij/). The LS was measured using a digital calliper. A total of 25 measurements were obtained for each of the 106 specimens (Table I).

STATISTICAL METHODS Does diet differ between species? Temporal heterogeneity in prey abundance and habitat characteristics may affect diet during the annual cycle (Winemiller, 1989). Ecological conditions and prey abundance, however, are relatively homogeneous during the annual cycle in Lake Titicaca (Dejoux & Iltis, 1991). Hence, specimens collected during the whole sampling period were pooled for diet analyses. The relative abundance of each item was estimated as the per cent of an item from the overall amount of food recovered from the gut of an individual ﬁsh. The mean

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 506 E . M A L D O N A D O E T AL .

FIG. 2. Location of the 13 homologous points identified on the head of Orestias species, delimiting 22 measurements and three angles used for the estimation of morphological variability (see Table I). relative abundance for each item within a species was obtained by averaging the relative abundances from all the specimens of a species. The per cent frequency of occurrence for a species was computed as the per cent of specimens with the item compared to the total number of specimens analysed for this species (non-empty stomachs only). The relative abundances of trophic items for the four species were analysed using the between groups principal component analysis (bPCA; Doledec & Chessel, 1987) following the procedure implemented in ADE-4 (Thioulouse et al., 1997). The bPCA is a clas- sical principal component analysis using a matrix of species means for the variables at hand, with individuals projected back into this space. Hence, the principal components (PC), namely the axis for projections, are established using the mean individual fish represented only by the means of the variables. This procedure allows projections to be constructed by taking only into account differences among groups, and allows a distinc- tion to be made between the proportion of variance explained by differences among groups and the variance explained by differences among individuals within groups. In the present study, the bPCA was used to estimate the effective trophic differentiation among species by removing the intraspecific variability. In this way, it was possible to buffer potential residual ontogenetic effects on diet coming from the smallest specimens, even if specimens were chosen as close as possible to the maximum LS of each species. Differences in relative abundance among species were focused on in order to identify the items that differentiate the diet of the four species. The significance of the differences among groups in the bPCA was tested by a permutation test. Individuals were permuted among groups, and n new permuted matrices were constituted. A bPCA was then performed for each of the n permuted matrices, and the proportion of variance explained by differences among groups was compared to one obtained from the real data set. If the variance explained by differences among groups was higher in the original matrix in 95% of the cases, groups were considered to be significantly different at the 5% level (Dol edec & Chessel, 1987). The significance of the differences in diet among species across the principal component of the bPCA was further assessed. For each axis of the bPCA, the effect of species differences in the individual projections onto each PC was assessed with ANOVA performed with Statgraphics (Statistical Graphics Corp., www.statisticalgraphics.blog.com). Then, the

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 507

TABLE I. List and description of the 25 morphometric variables used in the present study

Abbreviation Description Homologous points d1 Dentary length 1–2 d2 Maxillary length 2–3 d3 Diagonal 1 of cheek 2–7 d4 Ventral length of cheek 2–8 d5 Forehead length 3–5 d6 Dorsal length of cheek 3–7 d7 Diagonal 2 of cheek 3–8 d8 Head length 3–9 d9 Snout length 3–12 d10 Head height 4–5 d11 Height of upper and 6–7 posterior part of head d12 Height of anterior 7–8 part of opercle d13 Dorsal length of opercle 7–9 d14 Diagonal 1 of opercle 7–10 d15 Diagonal 2 of opercle 8–9 d16 Ventral length of opercle 8–10 d17 Height of posterior 9–10 part of opercle d18 Height of forehead 5–13 above orbit d19 Eye diameter 11–12 d20 Mouth width 3 of one side to 3 of the other side, dorsal view d21 Snout width 12 of one side to 12 of the other side, dorsal view d22 Width between eyes 5 of one side to 5 of the other side, dorsal view d23 Forehead orientation Angle between d5 (3–5) and d6 (3–7) d24 Mouth orientation Angle between d2 (2–3) and d6 (3–7) d25 Opercle orientation Angle between d16 (8–10) and d17 (10–9)

significance of the species pair differences in the individual projections onto the PC was assessed with a Tukey HSD test for unequal sample size using Statistica (Statsoft Inc.; www.statsoft.com). Significant correlations between diet items and PC onto the bPCA were checked by testing for the significance of a linear relationship between individual co-ordinates in the PC and each diet items in order to detect the variables that significantly contribute to the differentiation of the species. Tests of correlation were performed using an ANOVA as implemented in Statgraphics (Statistical Graphics Corp.). This procedure tests the fit of a linear regression model between two variables and assess if a continuous explanatory variable (i.e. co-ordinates of individuals projections onto the PC) accounts for a significant amount of variance in a dependant variable (i.e. diet items). Finally, a potential artifact due to sampling of individuals belonging to different ontogenetic stages was tested for analysing the fit of a linear regression model between specimen co-ordinates in the PC of the bPCA and LS in order to detect a potential correlation. Since the aim was to detect bias related to ontogeny, the LS was transformed to a per cent of the maximal LS for the species in order to have

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 508 E . M A L D O N A D O E T AL . a homogeneous surrogate of ontogeny in the data. As mentioned above, the correlation was tested using the ANOVA procedure implemented in Statgraphics (Statistical Graphics Corp.). Throughout the analyses made in the present study, significance levels were corrected for multiple comparisons by using the sequential Bonferroni procedure (Rice, 1989). If n multiple comparisons were made, the significance level of a test a was corrected 1 for multiple comparisons and divided by the number of comparison made a’ anÀ . Then, if k comparisons were significant, a new significance level was calculated¼ 1 a’ a (n k)À . ¼ À

Does morphology differ between species? Measurements were transformed into the ratio of the trait measurement to LS, and angles expressed in decimals. Allometric bias in the use of ratios has been debated intensively, especially in the case of the comparison of species with indeterminate growth (Reist, 1985). As specimens belonged to the same ontogenetic stage in this study (i.e. only mature individuals close to the maximal LS were used), allometric bias in the use of ratio was reduced. Moreover, morphological variability was explored using the same statistical procedure as described above, and potential residual effects of allometric changes were buffered by removing the effect of variability among individuals within groups. Differences among groups as implemented in the bPCA were focused on. The signiﬁcance of species differences in morphology was tested with the procedure aforementioned in the analysis of diet.

Is morphology related to diet? The co-variation of morphometric variables and trophic items was analysed with a multivariate approach (Blows, 2007) as implemented in the co-inertia analysis (Doledec & Chessel, 1994) in ADE-4. This analysis searches for composite axes maximizing simultaneously the variance explained in two distinct matrices. Projections of individuals based on trophic items and morphometric variables can be made on the same axis. Afterward, an index of similarity (IS) between the two matrices was computed and tested by permutation of individuals between groups. This analysis presents several ad- vantages when compared with others that have a higher sensitivity to the shape of the matrix, namely the relative number of columns and rows (Doledec & Chessel, 1994; Dray et al., 2003). Finally, correlations between morphological characters and diet items were tested with a Pearson correlation test.

RESULTS

DIET VARIABILITY Cladocerans and amphipods were the most common items found (Table II). Except for O. albus, they occurred in the gut contents of >80% of the specimens analysed and represented 14–77% of ingested food. Likewise, algae, other arthropods and macrophytes were common in terms of their frequency of occurrence which ranged between 27 and 76% of the specimens of the species. The exception was O. albus, where arthropods were not recorded. The relative abundance of these items, however, was low ranging from 0 6 to 13%. Substra- tum and bryozoans were found only in O. albus and O. agassiiÁ but were more abundant in the latter. Fish eggs were found in all the species except O. agassii, but they represented only a small fraction of the ingested food even if they occurred in up to 40% of the specimens. Molluscs were observed in O. albus

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 509

TABLE II. Abundance of 10 trophic items (t1–t10) in the gut of Orestias agassii, Orestias jussiei, Orestias albus and Orestias luteus

O. agassii O. jussiei O. albus O. luteus n 30 25 29 22 L range (mm) 102–145 88 5–127 102–150 76 4–114 S Á Á L mean S Æ 122 1 10 3 112 1 10 7 116 7 10 1 102 1 10 1 S.D. (mm) Á Æ Á Á Æ Á Á Æ Á Á Æ Á %O %R %O %R %O %R %O %R t , Cladocerans 91 9 30 0 23 0 100 37 0 16 0 43 3 0 8 1 5 95 5 38 0 23 0 1 Á Á Á Á Á Á Á Á Á Á Á t , Amphipods 97 3 43 0 Æ 23 0 96 26 0 Æ 13 0 96 7 77 0 Æ 20 0 81 8 14 0 Æ 11 0 2 Á Á Á Á Á Á Á Á Á Á Á t , Substratum 18 9 1 4 Æ 4 1 0 Æ0 6 7 0 4 Æ 1 8 0 Æ0 3 Á Á Á Á Á Á t , Algae 51 4 9 2 Æ 16 68 3 2 5 7 43 3 0 6 Æ 1 2 54 6 1 9 3 2 4 Á Á Á Á Á Á Á Á Á Á t , Arthropods 54 1 6 2 Æ 7 1 60 5 6 Æ 9 4 0 Æ0 36 4 2 8 Æ 4 9 5 Á Á Á Á Á Á Á Á t , Macrophytes 27 3 9 Æ 9 7 76 13 0 Æ 13 0 76 7 3 4 3 6 54 6 1 4 Æ 1 8 6 Á Á Á Á Á Á Á Á Á Á t , Fish eggs 0 Æ0 12 2 2 Æ 10 0 40 0 2 7 Æ 6 3 13 6 0 3 Æ 0 8 7 Á Á Á Á Á Á Á Á t , Fishes 0 0 0 Æ0 6 7 0 9 Æ 3 4 0 Æ0 8 Á Á Á t , Molluscs 0 0 0 0 23 3 0 1 Æ 0 2 72 7 18 0 28 0 9 Á Á Á Á Á Á t , Bryozoans 16 2 0 2 0 5 0 0 3 3 0 01 Æ 0 3 0 Æ0 10 Á Á Æ Á Á Á Æ Á

LS, standard length; n, number of specimens analysed; %O, per cent frequency of occurrence; %R, mean S.D. relative abundance. Æ and O. luteus but were common in the latter only. Finally, fishes were found only in the gut contents of O. albus. The bPCA indicated that 80% of the overall variance in the relative abundance of the 10 items could be attributed to the variation among individuals within species, while 20% could be attributed to the variation among species. Although the proportion of variance explained by differences among species was low, species differentiation was highly significant (permutation test, P < 0 001). Projection of the individuals along the two first axes of the bPCA ac- coÁ unted for 85 9% of the overall variance with 66 5 and 19 4% for PC 1 and PC 2, respectiveÁly [Fig. 3(a)]. A test was made for aÁcorrelatioÁn between individual projections in PC1 and PC2 and LS (i.e. per cent of the maximal LS observed for each species) in order to check a possible ontogenetic effect in the present analysis. Neither PC1 (ANOVA, r 0 18, P >0 05) nor PC2 (ANOVA, r Á Á 0 07, P >0 05) were significantly r¼elated by a linear regression model to th¼e À Á Á LS. The analysis of variance indicated that the variance distribution was significantly related to species differences for both PC1 and PC2 (ANOVA; Table III). The PC1 clearly differentiated O. albus, O. agassii, O. luteus and O. jussiei that only partially overlapped and significantly differed from each other [Fig. 3(a); Tukey HSD test, Table III]. In PC1, cladocerans (t1), amphipods (t2) and molluscs (t9) harboured the highest absolute contributions, and all three were significantly correlated to PC1 with t1 and t9 loading positively and t2 loading negatively [Fig. 3(b) and Table IV]. The PC2 significantly differentiated O. luteus and O. albus from O. agassii and O. jussiei (Table III) due to the contributions of the molluscs (t9), substratum (t3), algae (t4), arthropods (t5) and macrophytes (t6) which were all significantly correlated to PC2 and loaded positively, except for t9 which loaded negatively [Fig. 3(b) and Table IV). Overall, the bPCA separated

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 510 E . M A L D O N A D O E T AL .

FIG. 3. Morphology and diet variability in Orestias agassii, Orestias albus, Orestias jussiei and Orestias luteus. (a) Projection of specimens onto the first two principal components (PC1 and PC2) of the between groups principal component analysis (bPCA) based on relative abundance of trophic items. (b) Correlation circle of the 10 trophic items (see Table II) for the first two principal components (PC1 and PC2) of the bPCA. (c) Projection of specimens onto the first two principal components (PC1 and PC2) of the bPCA based on the 25 morphometric variables. (d) Correlation circle of the 25 morphometric variables (see Table I and Fig. 2) for the first two principal components (PC1 and PC2) of the bPCA. species that feed preferentially on amphipods (O. albus) or molluscs (O. luteus) from the others that feed on cladocerans but include a substantial amount of algae in their diet (O. agassii and O. jussiei).

MORPHOLOGICAL VARIABILITY Species differences accounted for 32% of the overall morphological variability, while 68% could be attributed to differences between individuals within species. The partition of the specimens into four species contributed

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TABLE III. Results of the ANOVA and Tukey HSD test of Orestias species’ differences in individual projections onto the principle component (PC) of the between groups principal component analysis (bPCA) diet items and morphometric data

Diet Morphometry

PC1 PC2 PC1 PC2

ANOVA F 116 9 11 2 103 8 47 6 Á Á Á Á P <0 01* <0 01* <0 01* <0 01* Á Á Á Á Tukey HSD test P (Orestias agassii Orestias albus) <0 01* <0 01* <0 01* <0 01* Á Á NS Á Á P (O. agassii OrestiasÂ jussiei) <0 01* 0 44 <0 01* <0 01* Á Á Á Á P (O. agassii Â Orestias luteus) <0 01* <0 01* <0 01* 0 02* Á Á NS Á Á P (O. albus ÂO. jussiei) <0 01* 0 09 0 04* <0 01* Á Á NS Á Á P (O. albus Â O. luteus) <0 01* 0 66 0 02* <0 01* Á Á Á Á NS P (O. jussieiÂ O. luteus) <0 01* <0 01* <0 04* 0 14 Â Á Á Á Á F, ratio of the variance explained by differences between groups to the variance explained by differences between individuals within groups; NS, non-significant; P, probability of the test; *, significant after sequential Bonferroni correction. significantly to the overall variance in the data (permutation test, P < 0 001). The projection of the individuals across the two first axes accountedÁ for 98% of the overall variance with 80 and 18% in axis 1 and 2, respectively [Fig. 3(c)]. A test was made for a correlation between projections of individuals in PC1 and PC2, and LS transformed in per cent of the maximal LS observed for each species in order to check a possible size effect. Neither PC1 (ANOVA; r 0 15; P > 0 05) nor PC2 (ANOVA; r 0 16; P > 0 05) was significantly ¼ À Á Á ¼ À Á Á related by a linear regression model to LS. ANOVA indicated that the variance distribution was significantly related to species differences for both PC1 and PC2, with the exception of one comparison between O. jussiei and O. luteus for PC2 [ANOVA; Table III and Fig. 3(c)]. The PC1 differentiated all four species [Fig. 3(c); Tukey HSD test; Table III]. In PC1, all the variables except d2, d14, d17 and d18 were significantly correlated, but forehead length (d5), dorsal length of cheek (d6), diagonal 2 of cheek (d7), head length (d8), snout length (d9), head height (d10) and dorsal length of opercle (d13) showed the highest absolute contributions [Fig. 3(d) and Table IV]. PC2 significantly differentiated O. luteus and O. jussiei from O. albus and O. agassii (Table III). Forehead length (d5), snout length (d9), head height (d10), height of upper and posterior part of head (d11), height of forehead above the orbit (d18), mouth width (d20), forehead orientation (d23) and opercle orientation (d25) loaded positively while dentary length (d1), maxillary length (d2) and mouth width (d20) loaded negatively. All these variables significantly contributed to PC2 and harboured the highest absolute contributions [Fig. 3(d) and Table IV]. Overall, the bPCA described variations from short, deep and wide heads (O. albus and O. jussiei) to long, shallow, narrow heads (O. agassi) in PC1 (O. luteus being intermediary), and PC2 described variations from deep,

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 512 E . M A L D O N A D O E T AL .

TABLE IV. Contributions of diet items and morphometric variables to the principal components (PC) of the between group principal component analysis (bPCA) performed on each data set separately. The absolute contribution (AC) represents the contribution of each variable to the construction of each PC of the bPCA. The relative contribution (RC) represents the amount of variance in the data explained by each PC

PC1 PC2

Diet AC RC r P AC RC r P

NS t1, Cladocera 3754 9814 0 70 <0 01* 89 68 0 05 0 66 Á Á Á Á NS t2, Amphipods 4257 9885 0 75 <0 01* 99 67 0 06 0 06 À Á Á NS À Á Á t3, Substratum 33 311 0 07 0 59 2017 5482 0 28 <0 01* À Á Á NS Á Á t4, Algae 51 554 0 08 0 36 2206 6910 0 29 <0 01* Á Á NS Á Á t5, Arthropods 226 6254 0 17 0 02 460 3718 0 22 0 003* Á Á NS Á Á t6, Macrophytes 20 166 0 05 0 24 833 2003 0 18 0 001* À Á Á NS Á Á NS t7, Fish eggs 316 6274 0 20 0 02 283 1639 0 10 0 13 Á Á NS Á Á NS t , Fishes 247 8075 À0 18 0 20 199 1900 À0 16 0 12 8 À Á Á À Á Á t9, Molluscs 984 4285 0 36 <0 01* 3675 4670 0 36 <0 01* Á Á NS Á Á NS t , Bryozoa 107 4921 0 18 0 04 134 1806 À0 12 0 04 10 Á Á Á Á Morphometry À d1 425 7304 0 52 <0 01* 655 2536 0 31 <0 01* Á Á NS Á Á d2 37 511 À0 16 0 02 3112 9488 À0 67 <0 01* Á Á Á Á NS d3 484 9421 À0 55 <0 01* 129 565 À0 14 0 12 Á Á Á Á NS d4 487 9313 À0 56 <0 01* 41 178 À0 08 0 11 Á Á Á Á d5 724 8856 À0 68 <0 01* 380 1047 0 23 <0 01* Á Á Á Á NS d6 706 9725 À0 67 <0 01* 82 256 0 11 0 30 Á Á Á Á NS d7 648 9158 À0 64 <0 01* 224 713 À0 18 0 01 Á Á Á Á NS d8 907 9869 À0 76 <0 01* 50 123 À0 09 0 97 Á Á Á Á NS d9 827 9653 0 72 <0 01* 83 219 À0 11 0 13 Á Á Á Á d10 817 8581 À0 72 <0 01* 563 1333 À0 28 <0 01* Á Á Á Á d11 229 6092 À0 38 <0 01* 639 3824 0 30 <0 01* Á Á Á Á NS d12 219 9357 À0 38 <0 01* 62 596 0 09 0 90 Á Á Á Á NS d13 670 9535 À0 65 <0 01* 144 464 0 15 0 27 Á Á NS Á Á NS d14 50 8577 0 18 0 05 27 1061 0 06 0 36 Á Á Á Á NS d15 316 9278 À0 45 <0 01* 2 14 0 02 0 88 Á Á Á Á NS d16 480 9556 0 55 <0 01* 98 443 À0 12 0 14 Á Á NS Á Á NS d17 59 6068 0 19 0 02 2 53 À0 02 0 90 Á Á NS Á Á d18 26 2138 À0 13 0 02 135 2468 0 14 <0 01* Á Á Á Á NS d19 250 9703 À0 40 <0 01* 0 4 0 01 0 30 Á Á Á Á d20 300 7093 À0 44 <0 01* 442 2355 0 25 <0 01* Á Á Á Á NS d21 334 9535 À0 46 <0 01* 6 38 À0 03 0 40 Á Á Á Á NS d22 436 8910 À0 53 <0 01* 8 41 À0 04 0 76 Á Á Á Á d23 180 2458 À0 34 <0 01* 2434 7490 0 59 <0 01* Á Á Á Á NS d24 216 9358 À0 37 <0 01* 48 470 0 08 0 03 Á Á Á Á d25 162 5339 0 32 <0 01* 622 4611 0 30 <0 01* Á Á Á Á NS, non-significant; P, probability of the analysis of variance (ANOVA) with d.f. of 1 (model) and 104 (residual); r, correlation coefficient to the PC of the bPCA; *, significant after sequential Bonferroni correction.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 513 arched heads with large mouth (O. albus) to shallow, ﬂat heads with small mouth (O. jussiei and O. luteus with O. agassii being intermediary).

RELATING MORPHOLOGY AND DIET The co-inertia analysis provided evidence for a relationship between morphology and diet. Similarity between the two data sets was low (RV 0 14); Á however, the co-structure was highly significant (permutation test,¼ P < 0 001). The correlation between morphology and diet reached 0 49 and 0 51 alongÁ the axes 1 (CI1) and 2 (CI2), respectively (Fig. 4). The twoÁ first axesÁ of the projection accounted for 80% of the overall variance in the data. The proportion of variance in the separate analyses explained by CI1 and CI2 in the co-analysis reached 89 and 88% for diet and 67 and 98% for morphology (i.e. the co-analysis displayed most of the variance of each data set). It is worth noting that variability was higher in morphology than diet as the variance displayed by the two first axes was three times higher in morphology than diet. Overall, the projection of individuals onto the two first axes was very similar to the projections obtained from separate analyses of diet and morphology. CI1 separated species according to cladocerans (t1), amphipods (t2), substratum (t3), algae (t4), molluscs (t9) and bryozoans (t10) for the diet, and all the variables but d11, d14, d17, d18 and d23 for morphology (Table V). Nevertheless, length of dentary (d1), height of anterior part of the opercle (d12) and orientation of the forehead (d23) harboured the highest absolute contribution and best contributed to the differentiation of the four species in CI1 (Table V). Clado- cerans (t1), amphipods (t2), substratum (t3) and algae (t4), and morphological variables d3–d13, d15 and d16, d18 and d19, d21–d23 significantly contributed to CI2 and to the differentiation of the four species (Table V). Cladocerans (t1) and amphipods (t2) for diet, and the length of the maxillary (d2), head height (d1) and orientation of the forehead (d23) for morphology, however, showed the highest absolute contributions in the differentiation of the four species in CI2 (Fig. 4 and Table V). Several morphological variables and diet items were significantly correlated (Table VI). The occurrence of cladocerans (t1) in the diet was negatively correlated with the length of the maxillary (d2), but positively correlated with the orientation of the forehead (d23) indicating that species feeding on cladocerans have a short, blunt nose with a small mouth. By contrast, a positive correlation between the occurrence of amphipods (t2) and the length of the maxillary (d2) and a negative correlation between the amphipods and the orientation of the forehead (d23) indicated that individuals feeding on amphipods were characterized by a long snout with a large mouth. Length of dentary (d1), diagonal 2 of cheek (d7) and head height (d10) were positively correlated to the occurrence of substratum (t3), while head length (d8) and mouth orientation (d24) were negatively correlated to the occurrence of substratum indicating that specimens with substratum in their gut contents generally exhibited a short but deep head (Table V). Finally, a positive correlation was found between molluscs (t9) and head length (d8). A negative but not significant correlation was found between the length of the snout (d9) and the occurrence of molluscs, providing weak

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# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 515 evidence that species including molluscs in their diet have a larger posterior part of the head and the larger opercles than others.

DISCUSSION Predation has three general components: prey search, capture and processing. Prey search may be expected to influence overall body shape to optimize mobil- ity and energetic cost of swimming in a given habitat (Webb, 1984; Parsons & Robinson, 2007). By contrast, capture and processing are tightly related to head and jaw morphology. The results of the study on four species of Orestias are in agreement with theoretical expectations of morphological adaptation to diet. Prey capture in Orestias involves ‘suction’ feeding during which prey are drawn into the mouth through a water flow generated by the opercles (Higham et al., 2006; Parsons & Robinson, 2007). A trade-off exists, however, between maximizing water velocity and water volume moved, that should translate into jaw morphology. Indeed, optimal head shape for large and evasive prey should involve a large oral region that maximizes the volume of water; whereas feeding on small and non-evasive prey should translate into a small mouth that allows quick jaw movement at a higher frequency. The positive correlation found between the maxillary length (d2) and the relative abundance of amphipods (t2) was consistent with this hypothesis given that large and evasive prey were more abundant in the diet of species with the larger mouth (i.e. O. albus). By contrast, as a pelagic species, O. agassii includes pelagic prey, such as cladocerans, in its diet, a foraging behaviour that may be reflected in a smaller maxillary. Molluscs are large hard-bodied prey that should be actively extracted from the substratum (Wainwright et al., 1991). In this study, species including molluscs in their diet, namely O. albus and O. luteus, are both benthic and both were characterized by a larger opercle and a longer head than the two other species (i.e. pelagic and littoral). This relationship was of interest, since these characters confer the ability to move a large flow of water (Higham et al., 2006). Together these results suggest that feeding on benthic prey leads to similar constraints in feeding morphology in the two benthic species of Orestias, since both shared morphological characters of functional importance in the flow of water during prey capture. Cladocerans are abundant in the littoral zone (Dejoux & Iltis, 1991), and O. jussiei harboured a small mouth that may reflect the dominance of cladocerans in its diet. Orestias jussiei, however, also occasionally consumed fish eggs and macrophytes, a usual trend in bottom-feeding species, which was also observed in the benthic species O. albus and O. luteus. This result may be related to the mixed influence of the prey communities from the benthic and the pelagic zones in the littoral habitat where both benthic and pelagic prey may be found jointly. The present analysis indicated that O. jussiei has an intermediate phenotype and diet between the pelagic (O. agassii) and benthic

FIG. 4. Results of the co-inertia analysis: projections of specimens onto the two first axes according to diet (origin of the arrow) and morphology (end of the arrow) (a) Orestias agassii, (b) Orestia albus, (c) Orestias jussiei and (d) Orestias luteus (e) trophic items (see codes in Table II) to the two first axes and (f) correlation circle of the 25 morphometric variables (see codes Table I) for the two first axes.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 516 E . M A L D O N A D O E T AL .

TABLE V. Contributions of diet items and morphometric variables (see Tables I and II) to the axes of the co-inertia analysis (CI) performed on both data sets simultaneously. The absolute contribution (AC) represents the contribution of each variable to the construction of each CI. The relative contribution (RC) represents the amount of variance in the data explained by each CI

CI1 CI2

Diet AC RC r P AC RC r P t , Cladocerans 1657 3792 0 62 <0 01* 3361 5457 0 75 <0 01* 1 Á Á Á Á t , Amphipods 1362 4186 0 56 <0 01* 2386 5204 -0 63 <0 01* 2 Á Á Á Á t , Substratum 1772 7056 À-0 64 <0 01* 721 2039 0 35 <0 01* 3 Á Á Á Á t4, Algae 3123 7360 0 85 <0 01* 1334 2232 0 47 <0 01* À Á Á NS Á Á NS t5, Arthropods 25 261 0 08 0 85 530 3904 0 30 0 01 Á Á NS Á Á NS t6, Macrophytes 31 521 0 09 0 44 414 4901 0 26 0 75 Á Á NS À Á Á NS t7, Fish eggs 42 296 0 10 0 68 3 18 0 03 0 01 Á Á NS Á Á NS t8, Fishes 350 5772 0 29 0 74 31 372 0 08 0 02 À Á Á Á Á NS t9, Molluscs 1588 6532 0 61 <0 01* 699 2043 0 34 0 13 Á Á Á Á NS t , Bryozoans 46 423 0 10 <0 01* 515 3354 À0 30 0 81 10 Á Á Á Á Morphometry À À d1 1740 9443 0 64 <0 01* 1 5 0 02 0 80NS Á Á Á Á NS d2 1570 6450 À0 61 <0 01* 1157 3373 0 44 0 02 Á Á Á Á d3 241 7436 À0 24 <0 01* 0 0 À0 01 <0 01* Á Á Á Á d4 99 2656 À0 15 <0 01* 176 3341 À0 17 <0 01* Á Á Á Á d5 18 276 À0 07 <0 01* 768 8225 0 36 <0 01* Á Á Á Á d6 550 8681 À0 36 <0 01* 1 17 0 02 <0 01* Á Á Á Á d7 1346 9055 À0 56 <0 01* 12 58 0 05 <0 01* Á Á Á Á d8 608 4406 À0 38 <0 01* 935 4808 0 39 <0 01* Á Á Á Á d9 423 7150 0 32 <0 01* 13 164 À0 05 <0 01* Á Á Á Á d10 93 738 À0 15 <0 01* 1629 9129 0 52 <0 01* Á Á NS Á Á d11 2 29 À0 02 0 01 902 8952 0 39 <0 01* Á Á Á Á d12 128 2297 0 17 <0 01* 280 3558 0 22 <0 01* Á Á Á Á d13 595 8524 À0 38 <0 01* 1 20 0 02 <0 01* Á Á NS Á Á NS d14 34 964 0 09 0 02 161 3236 0 17 0 06 Á Á Á Á d15 185 3165 À0 21 <0 01* 58 705 0 10 <0 01* Á Á Á Á d16 14 401 0 06 <0 01* 315 6060 À0 23 <0 01* Á Á NS Á Á NS d17 29 464 0 08 0 10 45 500 À0 08 0 02 Á Á NS Á Á d18 26 626 0 08 0 80 98 1643 0 13 <0 01* Á Á Á Á d19 382 5404 0 30 <0 01* 255 2563 0 21 <0 01* Á Á Á Á NS d20 364 4887 À0 30 <0 01* 73 700 0 11 0 01 Á Á Á Á d21 144 3416 À0 18 <0 01* 24 417 À0 06 <0 01* Á Á Á Á d22 55 1427 À0 11 <0 01* 163 2970 0 17 <0 01* Á Á NS Á Á d23 217 963 À0 23 0 19 2786 8742 0 68 <0 01* Á Á Á Á NS d24 650 7503 0 39 <0 01* 17 144 0 05 0 04 Á Á Á Á NS d25 474 6143 0 33 <0 01* 116 1069 À0 14 0 48 Á Á Á Á NS, non-significant; P, probability of the ANOVA with d.f. of 1 (model) and 104 (residual); r, correlation coefficient; *, significant after sequential Bonferroni correction.

(O. albus and O. luteus) species. Orestias jussiei feeds on pelagic cladocerans but is able to forage on the substratum. This result was of particular interest since O. jussiei seems to be more abundant in this ecotone where both the benthic

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TABLE VI. Results of the Pearson test of multiple correlations

t1 t2 t3 t4 t9 t10 d1 r 0 26 r 0 19 r 0 45 r 0 26 r 0 17 r 0 13 Á NS Á NS Á Á NS Á NS Á NS P¼ À0 01 P¼ 0 05 P¼< 0 01* P¼ 0 01 P¼ À0 08 P¼ 0 18 Á Á Á Á Á Á d2 r ¼ 0 48 r ¼0 45 r 0 22 r ¼0 07 r ¼ 0 14 r ¼0 09 Á Á Á NS Á NS Á NS Á NS P¼<À0 01* P¼< 0 01* P¼ 0 02 P¼ 0 50 P¼ À0 17 P¼ 0 35 Á Á Á Á Á Á d3 r 0 06 r 0 14 r ¼0 10 r ¼0 08 r ¼ 0 12 r ¼0 07 Á NS Á NS Á NS Á NS Á NS Á NS P¼ À0 56 P¼ 0 15 P¼ 0 29 P¼ 0 41 P¼ À0 20 P¼ 0 48 Á Á Á Á Á Á d4 r ¼0 01 r ¼ 0 01 r ¼0 08 r ¼0 13 r ¼ 0 12 r ¼0 10 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 96 P¼ À0 95 P¼ 0 42 P¼ 0 18 P¼ À0 22 P¼ 0 32 Á Á Á Á Á Á d5 r ¼0 22 r ¼ 0 10 r ¼0 16 r ¼0 11 r ¼ 0 15 r ¼0 02 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 02 P¼ À0 32 P¼ 0 11 P¼ 0 27 P¼ À0 12 P¼ 0 88 Á Á Á Á Á Á d6 r ¼ 0 16 r ¼0 14 r ¼0 18 r ¼0 19 r ¼ 0 15 r ¼0 02 Á NS Á NS Á NS Á NS Á NS Á NS P¼ À0 10 P¼ 0 15 P¼ 0 06 P¼ 0 05 P¼ À0 13 P¼ 0 82 Á Á Á Á Á Á d7 r ¼ 0 26 r ¼0 14 r ¼0 33 r ¼0 26 r ¼ 0 19 r ¼0 17 Á NS Á NS Á Á NS Á NS Á NS P¼ À0 01 P¼ 0 16 P¼< 0 01* P¼ 0 01 P¼ À0 06 P¼ 0 09 Á Á Á Á Á Á d8 r ¼ 0 11 r ¼0 03 r 0 28 r ¼ 0 26 r ¼0 35 r ¼ 0 10 Á NS Á NS Á Á NS Á Á NS P¼ À0 25 P¼ 0 75 P¼<À0 01* P¼ À0 01 P¼< 0 01* P¼ À0 30 Á Á Á Á Á Á d9 r ¼ 0 11 r ¼0 14 r 0 12 r ¼0 17 r 0 20 r ¼0 08 Á NS Á NS Á NS Á NS Á NS Á NS P¼ À0 26 P¼ 0 17 P¼ 0 23 P¼ 0 08 P¼ À0 04 P¼ 0 40 Á Á Á Á Á Á d10 r ¼0 24 r ¼ 0 21 r ¼0 29 r ¼0 19 r ¼ 0 18 r ¼0 10 Á NS Á NS Á Á NS Á NS Á NS P¼ 0 01 P¼ À0 03 P¼< 0 01* P¼ 0 05 P¼ À0 07 P¼ 0 32 Á Á Á Á Á Á d11 r ¼0 22 r ¼ 0 21 r 0 19 r ¼0 09 r ¼ 0 06 r ¼ 0 05 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 02 P¼ À0 03 P¼ 0 05 P¼ 0 35 P¼ À0 56 P¼ À0 61 Á Á Á Á Á Á d12 r ¼0 07 r ¼ 0 07 r ¼0 19 r ¼0 11 r ¼ 0 08 r ¼0 14 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 46 P¼ À0 48 P¼ 0 05 P¼ 0 28 P¼ À0 40 P¼ 0 16 Á Á Á Á Á Á d13 r ¼0 20 r ¼ 0 14 r ¼ 0 19 r ¼ 0 20 r ¼0 13 r ¼ 0 04 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 04 P¼ À0 17 P¼ À0 05 P¼ À0 04 P¼ 0 20 P¼ À0 65 Á Á Á Á Á Á d15 r ¼0 12 r ¼ 0 02 r ¼ 0 12 r ¼ 0 16 r ¼0 12 r ¼0 01 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 22 P¼ À0 80 P¼ À0 21 P¼ À0 11 P¼ 0 24 P¼ 0 93 Á Á Á Á Á Á d16 r ¼ 0 11 r ¼0 02 r ¼ 0 08 r ¼ 0 09 r ¼0 08 r ¼ 0 03 Á NS Á NS Á NS Á NS Á NS Á NS P¼ À0 26 P¼ 0 82 P¼ À0 39 P¼ À0 36 P¼ 0 42 P¼ À0 78 Á Á Á Á Á Á d18 r ¼0 10 r ¼ 0 07 r ¼0 02 r ¼ 0 11 r ¼ 0 14 r ¼ 0 02 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 31 P¼ À0 51 P¼ 0 88 P¼ À0 26 P¼ À0 16 P¼ À0 84 Á Á Á Á Á Á d19 r ¼0 01 r ¼ 0 02 r ¼0 26 r ¼0 26 r ¼ 0 12 r ¼ 0 05 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 92 P¼ À0 86 P¼ 0 01 P¼ 0 01 P¼ À0 20 P¼ À0 64 Á Á Á Á Á Á d20 r ¼ 0 10 r ¼0 17 r ¼0 16 r ¼0 03 r ¼ 0 10 r ¼0 09 Á NS Á NS Á NS Á NS Á NS Á NS P¼ À0 32 P¼ 0 08 P¼ 0 10 P¼ 0 73 P¼ À0 32 P¼ 0 37 Á Á Á Á Á Á d21 r ¼0 06 r ¼0 09 r ¼0 12 r ¼0 05 r ¼ 0 14 r ¼0 05 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 55 P¼ 0 34 P¼ 0 20 P¼ 0 65 P¼ À0 16 P¼ 0 64 Á Á Á Á Á Á d22 r ¼0 14 r ¼0 01 r ¼0 11 r ¼0 07 r ¼ 0 13 r ¼0 05 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 16 P¼ 0 95 P¼ 0 27 P¼ 0 49 P¼ À0 18 P¼ 0 62 Á Á Á Á Á Á d23 r ¼0 45 r ¼ 0 46 r ¼0 18 r ¼0 07 r ¼ 0 07 r ¼ 0 03 Á Á Á NS Á NS Á NS Á NS P¼< 0 01* P¼<À0 01* P¼ 0 07 P¼ 0 49 P¼ À0 47 P¼ À0 79 Á Á Á Á Á Á d24 r 0 21 r 0 05 r ¼ 0 27 r ¼ 0 21 r ¼0 12 r ¼ 0 05 Á NS Á NS Á Á NS Á NS Á NS P¼ 0 03 P¼ À0 62 P¼<À0 01* P¼ À0 03 P¼ 0 21 P¼ À0 58 Á Á Á Á Á Á d25 r ¼0 16 r ¼ 0 24 r 0 10 r ¼ 0 05 r ¼0 25 r ¼0 03 Á NS Á NS Á NS Á NS Á NS Á NS P¼ 0 09 P¼ À0 01 P¼ À0 32 P¼ À0 60 P¼ 0 01 P¼ 0 79 ¼ Á ¼ Á ¼ Á ¼ Á ¼ Á ¼ Á NS, non-significant; P, probability of the test; r, correlation coefficient; *, significant after sequential Bonferroni correction.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 518 E . M A L D O N A D O E T AL . feeding species O. albus and O. luteus and the pelagic feeding species O. agassii are less abundant (Lauzanne, 1982). Although data about distribution and ecology are still scarce for the genus, this trend suggests that, in ecotones, gen- eralist species are more abundant than ecologically specialized species. The relationships emphasized in this study between morphology and diet are consistent with the expectations of the adaptive radiation hypothesis, namely that a lineage diversifying through ecological specialization produces species with phenotypes related to habitat (Schluter, 2000). The adaptive radiation hypothesis requires that habitats with distinct characteristics lead to distinct adaptive landscapes, whereby competition tends to favour specialist phenotypes. The relationship depicted here between some morphological characters and diet suggests that benthic, pelagic and littoral habitats constitute distinct adaptive landscapes leading to distinct morphological adaptations (Schluter, 1993; Sibbing et al., 1998; Lu & Bernatchez, 1999; Parsons & Robinson, 2007). The species studied here, however, are not sister species within a monophyletic group, and it remains unclear whether adaptive divergence may have promoted limited gene flow between diverging populations. What is clear is that contrasting habitats with distinct trophic resources and environmental conditions may be found in Lake Titicaca, and that this ecological heterogeneity is reflected in ecomorphological variation among the four species of Orestias that suggests axes of niche segregation. Future studies that integrate phylogeny and reproductive isolation will be important in determining the mechanism underlying the observed interspecific variation and the speciation event.

This work was a part of the Master project of E.M. in the Master E2M2 from the Universite Claude Bernard Lyon 1. The project was supported by funds from the French Institut de Recherche pour le Developpement, Unite de Recherche 131 Bio- diversite des grands cours d’eau. Many thanks go to M. Pouilly, V. Iniguez, R. Marin, J. Pinto and M. Baudoin for their logistic and academic support in La Paz, Bolivia. We thank D. Pontier and D. Pont for their support in the Master E2M2 and UMR CNRS 5023, respectively. We acknowledge L. Chapman and the anonymous referees for their helpful comments on the manuscript.

References Barel, C. D. N. (1983). Toward a constructional morphology of cichlid ﬁshes (Teleostei, Perciformes). Netherlands Journal of Zoology 33, 357–424. Blows, M. W. (2007). A tale of two matrices: multivariate approaches in evolutionary biology. Journal of Evolutionary Biology 20, 1–8. Dejoux, C. & Iltis, A. (1991). El Lago Titicaca. La Paz, Bolivia: HISBOL. Doledec, S. & Chessel, D. (1987). Rythmes saisonniers et composantes stationnelles en milieu aquatique I- Description d’un plan d’observations complet par projection de variables. Acta Oecologica, Oecologia Generalis 8, 403–426. Doledec, S. & Chessel, D. (1994). Co-inertia analysis: an alternative method for studying species-environment relationships. Freshwater Biology 31, 277–294. Douglas, M. E. & Matthews, W. J. (1992). Does morphology predict ecology? Hypothesis-testing within a freshwater stream ﬁsh assemblage. Oikos 65, 213–224. Dray, S., Chessel, D. & Thioulouse, J. (2003). Co-inertia analysis and the linking of ecological tables. Ecology 84, 3078–3089. Froese, R. & Pauly, D. (2000). FishBase 2000: Concepts, Design and Data Sources. Los Ban˜ os, CA: ICLARM.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 E C O M O R P H O L O G Y O F OR E ST I A S F I S H E S 519

Gatz, A. J. (1979). Ecological morphology of freshwater stream fishes. Tulane Studies in Zoology and Botany 21, 91–124. Grant, P. R. (1986). Ecology and Evolution of Darwin’s Finches. Princeton, NJ: Princeton University Press. Higham, T. E., Day, S. W. & Wainwright, P. C. (2006). Multidimensional analysis of suction feeding performance in fishes: fluid speed, acceleration, strike accuracy and the ingested volume of water. Journal of Experimental Biology 209, 2713–2725. Hugueny, B. & Pouilly, M. (1999). Morphological correlates of diet in an assemblage of West African freshwater fishes. Journal Fish Biology 54, 1310–1325. Kocher, T. D. (2004). Adaptive evolution and explosive speciation: the cichlid fish model. Nature Reviews Genetics 5, 288–298. Langerhans, R. B., Layman, C. A., Langerhans, A. K. & Dewitt, T. J. (2003). Habitat- associated morphological divergence in two Neotropical fish species. Biological Journal of the Linnean Society 80, 689–698. Lauzanne, L. (1982). Les Orestias (Pisces, Cyprinodontidae) du petit lac Titicaca. Revue d’Hydrobiologie Tropicale 15, 39–70. Losos, J. B., Jackman, T. R., Larson, A., De Queiroz, K. & Rodrıguez-Schettino,´ J. (1998). Contingency and determinism in replicated adaptive radiations of island lizards. Science 279, 2115–2118. Loubens, G. & Sarmiento, J. (1985). Observations sur les poissons de la partie Bolivienne du lac Titicaca. Revue d’Hydrobiologie Tropicale 18, 159–171. Loubens, G., Osorio, F. & Sarmiento, J. (1984). Observations sur les poissons de la partie bolivienne du lac Titicaca. I. Milieux et peuplements. Revue d’Hydrobiologie Tropicale 17, 153–161. Lu, G. & Bernatchez, L. (1999). Correlated trophic specialization and genetic divergence in sympatric lake whitefish ecotypes (Coregonus clupeaformis): support for the ecological speciation hypothesis. Evolution 53, 1491–1505. Lussen, A. (2003). Zur Systematic, Phylogenie und Biogeographie chilenischer Arten der Gattung Orestias Valenciennes, 1839 (Teleostei, Cyprinodontidae): morphologi- sche, biochemische und molekularbiologische Befunde. PhD Thesis, Hamburg University, Germany. Lussen, A., Falk, T. M. & Villwock, W. (2003). Phylogenetic patterns in populations of Chilean species of the genus Orestias (Teleostei: Cyprinodontidae): results of mitochondrial DNA analysis. Molecular Phylogenetics and Evolution 29, 60–151. Machado-Allison, A. & Garcıa,´ C. (1986). Food habits and morphological changes during ontogeny in three Serrasalmins fish species of the Venezuelan floodplains. Copeia 1986, 193–196. de Merona, B., Hugueny, B., Tejerina-Garro, F. L. & Gautheret, E. (2008). Diet-morphology relationship in a fish assemblage from a medium sized river of French Guiana. The effect of species taxonomic proximity. Aquatic Living Resources 21, 171–184. Meyer, A. (1993). Phylogenetic relationships and evolutionary processes in East African Cichlid fishes. Trends in Ecology and Evolution 8, 279–284. Motta, P. J., Norton, S. F. & Luczkovich, J. (1995). Perspectives on the ecomorphology of bony fishes. Environmental Biology of Fishes 44, 11–20. Norton, F., Luczkovich, J. & Motta, P. J. (1995). The role of ecomorphological studies in the comparative biology of fishes. Environmental Biology of Fishes 44, 287–304. Parenti, L. (1981). A phylogenetic and biogeographic analyses of cyprinodontifom fishes (Teleostei, Atherinomorpha). Bulletin of the American Museun of Natural History 168, 335–557. Parenti, L. (1984). A taxonomic revision of the Andean Killifish genus Orestias (Cyprinodontiformes, Cyprinodontidae). Bulletin of the American Museum of Natural History 178, 107–214. Parsons, K. J. & Robinson, B. W. (2006). Replicated evolution of integrated plastic responses during early adaptive divergence. Evolution 60, 801–813. Parsons, K. J. & Robinson, B. W. (2007). Foraging performance of diet-induced morphotypes in pumpkinseed sunfish (Lepomis gibbosus) favours resource poly- morphism. Journal of Evolutionary Biology 20, 673–684.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520 520 E . M A L D O N A D O E T AL .

Pouilly, M., Lino, F., Bretenoux, J. G. & Rosales, C. (2003). Dietary-morphological relationships in a fish assemblage of the Bolivian Amazonian floodplain. Journal of Fish Biology 62, 1137–1158. Reist, J. D. (1985). An empirical evaluation of several univariate methods that adjust for size variation in morphometric data. Canadian Journal of Zoology 63, 1429–1439. Rice, W. R. (1989). Analysing tables of statistical tests. Evolution 43, 223–225. Schluter, D. (1993). Adaptive radiation in sticklebacks: size, shape, and habitat use efficiency. Ecology 74, 699–709. Schluter, D. (1996). Ecological causes of adaptive radiation. American Naturalist 148, 40–64. Schluter, D. (2000). The Ecology of Adaptive Radiation. New York, NY: Oxford University Press. Schluter, D. (2001). Ecology and the origin of species. Trends in Ecology and Evolution 16, 372–380. Schluter, D. & Grant, P. R. (1984). Determinants of morphological patterns in communities of Darwin’s finches. American Naturalist 123, 175–196. Sibbing, F. A., Nagelkerke, L. A. J., Stet, R. J. M. & Osse, J. W. M. (1998). Speciation of lake Tana barbs (Cyprinidae, Ethiopia) driven by trophic resource partitioning: a molecular and ecomorphological approach. Aquatic Ecology 32, 217–227. Skulason,´ S. & Smith, T. B. (1995). Resource polymorphisms in vertebrates. Trends in Ecology and Evolution 10, 366–370. Smith, T. B. & Skulason,´ S. (1996). Evolutionary significance of resource polymorphisms in fishes, amphibians, and birds. Annual Review of Ecology and Systematics 27, 111–133. Strauss, R. E. & Bookstein, F. L. (1982). The truss: body form reconstructions in morphometrics. Systematic Zoology 31, 113–135. Svanback,¨ R. & Eklo¨ v, P. (2002). Effects of habitat and food resources on morphology and ontogenetic growth trajectories in perch. Oecologia 131, 61–70. Thioulouse, J., Chessel, D., Doledec, S. & Olivier, J. M. (1997). ADE-4: a multivariate analysis and graphical display software. Statistics and Computing 7, 75–83. Via, S. & Lande, R. (1985). Genotype-environment interaction and the evolution of phenotypic plasticity. Evolution 39, 502–522. Villwock, W. (1986). Speciation and adaptive radiation in Andean Orestias fishes. In High Altitude Tropical Biogeography (Vuilleumier, F. & Monasterio, M. M., eds), pp. 387–403. Oxford: Oxford University Press. Wainwright, P. C. (1991). Ecomorphology: experimental functional anatomy for ecological problems. American Zoologist 31, 680–693. Wainwright, P. C. (1996). Ecological explanation through functional morphology: the feeding biology of sunfishes. Ecology 77, 1336–1343. Wainwright, P. C., Osenberg, S. W. & Mittlebach, G. G. (1991). Trophic polymorphims in the pumpkinseed sunfish (Lepomis gibbosus Linnaeus): effects of environment on ontogeny. Functional Ecology 5, 40–55. Webb, P. W. (1984). Body form, locomotion and foraging in aquatic vertebrates. American Zoologist 24, 107–120. West-Eberhard, M. J. (2003). Developmental Plasticity and Evolution. New York, NY: Oxford University Press. West-Eberhard, M. J. (2005). Developmental plasticity and the origin of species differences. Proceedings of the National Academy of Sciences of the United States of America 102, 6543–6546. Winemiller, K. O. (1989). Ontogenetic diet shifts and resource partitioning among piscivorous fishes in the Venezuelan llanos. Environmental Biology of Fishes 26, 177–199. Winemiller, K. O. (1991). Ecomorphological diversification in lowland freshwater fish assemblages from five biotic regions. Ecological Monographs 61, 343–365. Winemiller, K. O., Kelso-Winemiller, L. & Brenkert, A. L. (1995). Ecomorphological diversification and convergence in fluvial cichlid fishes. Environmental Biology of Fishes 44, 235–261.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 502–520