Trophic specialization and morphological divergence between two sympatric species in Lake Catemaco, Mexico

Abstract The association of morphological divergence with ecological segregation among closely related species could be considered as a signal of divergent selection in ecological speciation processes. Environmental signals such as diet can trigger phenotypic evolution, making polymorphic species valuable systems for studying the evolution of trophic‐related traits. The main goal of this study was to analyze the association between morphological differences in trophic‐related traits and ecological divergence in two sympatric species, Astyanax aeneus and A. caballeroi, inhabiting Lake Catemaco, Mexico. The trophic differences of a total of 70 individuals (35 A. aeneus and 35 A. caballeroi) were examined using stable isotopes and gut content analysis; a subset of the sample was used to characterize six trophic and six ecomorphological variables. In our results, we recovered significant differences between both species in the values of stable isotopes, with higher values of δ15N for A. caballeroi than for A. aeneus. Gut content results were consistent with the stable isotope data, with a higher proportion of invertebrates in A. caballeroi (a consumption of invertebrates ten times higher than that of A. aeneus, which in turn consumed three times more vegetal material than A. caballeroi). Finally, we found significant relationship between ecomorphology and stable isotopes (r = .24, p < .01), hence, head length, preorbital length, eye diameter, and δ15N were all positively correlated; these characteristics correspond to A. caballeroi. While longer gut and gill rakers, deeper bodies, and vegetal material consumption were positively correlated and corresponded to A. aeneus. Our results are consistent with the hypothesis that morphological divergence in trophic‐related traits could be associated with niche partitioning, allowing the coexistence of closely related species and reducing interspecific competition.


| INTRODUC TI ON
Understanding the mechanisms that influence speciation and their relationship with the appearance of new traits is a key question for evolutionary biologists. According to ecological speciation theory, adaptive divergence plays a key role in the formation and perpetuation of barriers between species that inhabit different environments (Schluter, 2001). In this scenario, the evolution of morphological traits could be influenced by adaptive processes, especially in the case of traits subject to divergent selection (Schluter, 2001). Thus, it is important to identify the main triggering elements of diversification related to phenotypic divergence.
In addition to morphological differences, we have proposed that functional modularity plays a role in the body and skull variation between the two species, probably in association with divergent evolution (Ornelas-García et al., 2017). Modular evolution was observed in the preorbital region of the skull of A. caballeroi. In contrast, body shape variation of both species is partitioned into two modules, the head and the rest of the body, affecting both trophic morphology and swimming performance of the fish (Ornelas-García et al., 2017;Webb, 1984).
Despite the high degree of morphological differentiation between the two species inhabiting Lake Catemaco, there is no evidence of trophic segregation between them. Therefore, the main goal of the present work was to assess the relationship between morphological disparity and trophic divergence between two sympatric species, Astyanax aeneus and A. caballeroi. To this end, 12 ecomorphological variables were evaluated between the two species, while trophic divergence was assessed by stable isotopes and gut content analyses. We expected to observe an association between ecomorphological variation and dietary habits, which would suggest a divergence in traits that are ecologically relevant for niche partitioning among sympatric characid species.

| Sample collection and taxonomic determination
A total of five localities in Lake Catemaco (located at Sierra de Los Tuxtlas, Veracruz, Mexico) were sampled; the sites were selected to F I G U R E 1 Map of the sampled localities in Lake Catemaco.  : 5, 6.25, 8,10, 12.5, 15.5, 19.5,24, 29, 35, 43, and 55 mm.
After sampling, the fish were kept alive in aerated coolers until tissue processing (normally <1 hr after the last sampling). Under more sterile conditions, the fish were euthanized by putting them in ice water (around 4°C), and the stomach and muscle biopsies were collected.
All processed specimens were preserved as vouchers in 95% ethanol and deposited in the National Fish Collection, IBUNAM. The fish were assigned to nominal species based on diagnostic morphological characters (Contreras-Balderas & Rivera-Teillery, 1983;Miller et al., 2005).

| Stable isotopes
A total of 70 individuals (35 Astyanax aeneus and 35 Astyanax caballeroi) from Lake Catemaco were examined using stable isotope analysis (Table S1). Muscle tissue samples were collected individually, dried using silica gel and then frozen at −80°C. Samples were dried at 60°C for 48 hrs, ground into a fine powder with a mortar and pestle, and stored in sterile 2-ml tubes. Subsamples of each ground sample were weighed, packaged into tin capsules and sent to the Center of Stable Isotopes at the University of New Mexico (Department of Earth and Planetary Sciences) to measure the stable isotope ratios of carbon ( 13 C/ 12 C) and nitrogen ( 15 N/ 14 N). The isotopic ratios were expressed in standard delta "δ" notation. The mean standard deviation between replicates was 0.04 ‰ for δ 13 C and 0.07 ‰ for δ 15 N.
The analysis of the trophic niche of both species was performed by adapting the community niche analysis using δ 13 Cδ 15 N bi-plots (Layman, Arrington, Montaña, & Post, 2007;Zambrano, Valiente, & Vander Zanden, 2010). Trophic niche analysis evaluates the trophic niche of a single species based on the carbon range (CR), nitrogen range (NR), total niche area (TA), mean distance to centroid (CD), mean nearest neighbor distance (NND), and standard deviation of nearest neighbor distance (SDNND) (Layman et al., 2007). These parameters define the range of food sources consumed by certain species, the capacity of a species to consume organisms from different trophic levels, the trophic niche occupied by each species, and the similarity of food sources among individuals. Student's t-test was performed to compare the F I G U R E 2 Trophic niche analysis of A. aeneus (black circles) and A. caballeroi (gray circles). Polygons encompass the convex hull area of all individuals. CD, mean distance to centroid; CR, carbon range; NR, nitrogen range; NND, mean nearest neighbor distance; SDNND, standard deviation of nearest neighbor distance; TA, total niche area F I G U R E 3 Relative compositions (biomass) of gut contents of A. aeneus and A. caballeroi mean differences between species for each stable isotope ratio; a multivariate analysis of variance (MANOVA) was performed when both isotopes were considered. Differences in carbon and nitrogen between seasons and species were analyzed using an analysis of variance (ANOVA).

| Stomach content
Only the stomach sack was used to compare the stomach content, and the intestines were discarded. Individuals with empty stomachs were omitted from the analysis. Dietary items were categorized into the following groups: detritus, vegetal material, invertebrates, and fish remains (eggs, eyes, scales, bones, teeth, fin rays, gills and fish vertebrae) (Table S2). We used these categories to estimate diet composition, using a standard gravimetric method to account for broad differences in diet habits (Zacharia & Abdurahiman, 2004).
The wet weight of the food items was measured after removing superfluous water. The mean proportions of diet items were compared between species using a chi-square test.

| Morphology and trophic habits
Morphological and trophic data were correlated to characterize the ecomorphological patterns of the two sympatric species.
Multivariate ordination analysis was carried out with dietary, isotopic, and morphological data for a total of 33 individuals (Table S3 and S4), with a similar proportion of each species (A. aeneus, N = 17; A. caballeroi, N = 16). A total of 12 measurements were included; of these, six were morphological, mainly related to locomotion and food acquisition; the other six corresponded to trophic-related variables (Córdova-Tapia, Hernández-Marroquín & Zambrano, 2017; Montaña & Winemiller, 2013;Winemiller, 1991). The morphological variables included the head length (HL), standard length (SL), eye diameter (ED/(HL/SL), the preorbital region (PrOL/HL); the body depth (BD/SL); the gill raker length (GRL, i.e., the longest gill raker/SL); and the gut length (GL, i.e., intestine length/SL) (Table   S4 and S5). Trophic data corresponded to the stomach content (fish remains, vegetal material, detritus and invertebrates) and stable isotope data (δ 13 C and δ 15 N). To equally weight each trait, the data were standardized using a z-transformation; thus, the mean of each trait was equal to 0, and its standard deviation was equal to 1 (Córdova-Tapia et al., 2017). Student's t-test was performed to compare the mean differences between species for each morphological and trophic variable; a multivariate analysis of variance (MANOVA) was performed when the six variables were considered together.

| Stable isotopes and gut content analyses
The bi-plot comparing trophic niche areas revealed a substantial overlap in niche space between the two species in the middle of the trophic structure ( Figure 2). However, significant differences were found between both species with respect to carbon (δ 13 C: t (68) = −3.27, p < .01) and nitrogen (δ 15 N: t (68) = −5.73, p < .001) values, as well as when both isotopes were considered together (δ 13 C + δ 15 N: F (2, 67) = 16.22, p < .001). A. caballeroi had a higher mean F I G U R E 4 Boxplots of the comparisons of relative gut length and relative gill raker length between A. aeneus and A. caballeroi. Both relative gut length and gill raker length were significantly different (p < .05) nitrogen value (δ 15 N: 7.2 ± 0.38) than A. aeneus (δ 15 N: 6.6 ± 0.39).
Regarding the stomach content analysis, detritus was the dominant item in both species (Figure 3), although significant differences were found in the mean proportions of diet items (χ 2 = 16.74 (3), p < .001). A. caballeroi showed a higher consumption of invertebrates (14.8%) and fish debris (24.2%) than A. aeneus, which showed values of 1.29% and 19.55%, respectively. Furthermore, A. aeneus had a higher proportion of vegetal items than A. caballeroi (8.8% vs. 3.3%, respectively, Table S2).
Mantel's test showed a significant relationship between morphology and stable isotopes (r = .24, p < .01). However, no significant correlation was found between gut content and morphology (r = −.11, p = .88), nor between gut content and stable isotopes (r = −.12, p = .89). Spearman's correlation coefficients between variables (Table 1) showed a negative and significant correlation between HL and the next variables: BD, GRL, and GL. Moreover, there was a positive and significant correlation between the next pairs of variables: HL and ED; δ 15 N and δ 13 C; ED and δ 15 N; and GRL and GL. In this respect, A. caballeroi had a longer HL, a slender body (BD), shorter GRL, shorter GL, larger ED, and higher ratios of δ 15 N and δ 13 C. In contrast, the individuals of A. aeneus showed a deeper body (BD), longer gut length (GL) and gill rakers, and lower values of δ 15 N.
The PCA results allowed us to explore morphospace differences between both species; the first and second components accounted for 50.6% of the total variance (36.1% for PC1 and 14.5% for PC2; Table 2, Figure 5). The positive scores of PC1 were associated with HL, ED, δ 15 N, δ 13 C, invertebrates, and fish remains, while the negative scores were associated with GRL, GL, BD, and PrOL.

| D ISCUSS I ON
Ecological opportunity has been proposed as a key element in the colonization of new habitats, and it is believed to contribute to morphological diversification by providing a means to exploit alternative resources (Losos, 2010). The evolution of a trait that enables the exploitation of a new resource has a strong influence in the diversification of species as well as in shifts of trophic and habitat niches (Burress et al., 2016;Losos, 2010). Thus, morphology has been considered a good indicator of ecology and feeding habits (Gatz, 1979).
In characiforms, previous studies have found correlations between ecomorphological traits and trophic ecology, which can help us to understand the factors that enable the coexistence of closely related species (Bonato et al., 2017;Mise et al., 2013). The present study corroborated the occurrence of a significant correlation between ecomorphological traits and trophic habits (i.e., stable isotopes values) of two sympatric species of Astyanax genus. As such, these results provide additional evidence supporting the hypothesis that morphology could reflect the ecology of an organism and could help predict its trophic habits (Bonato et al., 2017).

| Stable isotopes and gut content analyses
In the present study, a significant correlation between stable isotopes and ecomophology was observed, in contrast to the latter and diet content. In this respect, diet analysis provides a snapshot of fish feeding habits and shows temporal variations that could be difficult to approach. Stable isotopes analysis provides a time-integrated indicator of energy resources (Vander Zanden, Casselman, & Rasmussen, 1999).
When combined, these two sources of evidence provide robust analytic tools that can be used to better understand the consumption and assimilation of food by fish (Vander Zanden & Vadeboncoeur, 2002).
Stable carbon and nitrogen isotope ratios have been widely used to provide a time-integrated perspective of feeding relationships (Vander Zanden, Casselman, et al., 1999). The nitrogen stable isotope signature (δ 15 N) increases proportionally to the trophic position in the food web and shows habitat-dependent variation; in contrast, a source of variation in the carbon stable isotope (δ 13 C) could be associated with the base of the food webs among other factors (Vander Zanden, Shuter, Lester, & Rasmussen, 1999;Vander Zanden, Casselman, et al., 1999).
This study confirmed that Astyanax species are an opportunistic group (Mise et al., 2013) with the ability to exploit a wide variety of trophic resources; however, some important differences were found between the two coexisting species studied here. For example, δ 15 N and δ 13 C showed wider variation in A. aeneus (Figure 2), which has been suggested as a characteristic of fish with omnivorous habits in lacustrine systems (Córdova-Tapia, Contreras, & Zambrano, 2015;Jepsen & Winemiller, 2002;Vander Zanden & Vadeboncoeur, 2002). In this respect, the niche area of A. caballeroi was more restricted than that of A. aeneus (TA = 2 and 2.6, respectively), which is characteristic of fish with specialized trophic habits (Jepsen & Winemiller, 2002). The results of our stomach content analysis showed that this interpretation coincides with the trophic habits of A. caballeroi, which showed a much higher consumption of invertebrates than A. aeneus, which in turn showed a higher consumption of vegetal material.

| Morphology and trophic habits
Our results showed that the feeding patterns were correlated with morphological differentiation. Firstly, we observed bimodal distributions of trophic-related traits between species, that is, A. aeneus showed longer gut length and gill raker than did A. caballeroi.  (Bonato et al., 2017).
Furthermore, short oral jaws have been associated with a powerful bite and are considered advantageous for low-mobility or immobile food items (Arbour & López-Fernández, 2014;Cooper et al., 2010;Wainwright & Richard, 1995).
Based on both ecomorphology and diet, we can provide some suggestions regarding prey capture behavior for both species.
A. caballeroi depicts a large head, upwardly facing mouth position, fusiform body, and conical teeth, consistent with the capture of elusive preys and access to floating terrestrial insects (e.g., hymenopterans or coleopterans) (Bonato et al., 2017). In contrast, A. aeneus had a relatively deeper body and shorter snout, which is associated with the ability to move upward and downward in the water column, from the bottom to the surface, as befits an omnivorous species (Gatz, 1979;Mise et al., 2013;Winemiller, 1991).
In conclusion, the Astyanax aeneus and A. caballeroi model system showed a significant correlation between ecomorphological traits and trophic habits (i.e., stable isotopes values), supporting the hypothesis that morphological divergence in trophic-related traits is associated with niche partitioning. This study also provides additional evidence that trophic partitioning could trigger morphological changes and ultimately promote speciation, as seen in other groups of fishes.