Unveiling community patterns and trophic niches of tropical and temperate ants using an integrative framework of field data, stable isotopes and fatty acids

Background The use and partitioning of trophic resources is a central aspect of community function. On the ground of tropical forests, dozens of ant species may be found together and ecological mechanisms should act to allow such coexistence. One hypothesis states that niche specialization is higher in the tropics, compared to temperate regions. However, trophic niches of most species are virtually unknown. Several techniques might be combined to study trophic niche, such as field observations, fatty acid analysis (FAA) and stable isotope analysis (SIA). In this work, we combine these three techniques to unveil partitioning of trophic resources in a tropical and a temperate community. We describe patterns of resource use, compare them between communities, and test correlation and complementarity of methods to unveil both community patterns and species’ niches. Methods Resource use was assessed with seven kinds of bait representing natural resources available to ants. Neutral lipid fatty acid (NLFA) profiles, and δ15N and δ13C isotope signatures of the species were also obtained. Community patterns and comparisons were analyzed with clustering, correlations, multivariate analyses and interaction networks. Results Resource use structure was similar in both communities. Niche breadths (H′) and network metrics (Q and H2′) indicated similar levels of generalization between communities. A few species presented more specialized niches, such as Wasmannia auropunctata and Lasius fuliginosus. Stable isotope signatures and NLFA profiles also indicated high generalization, although the latter differed between communities, with temperate species having higher amounts of fat and proportions of C18:1n9. Bait use and NLFA profile similarities were correlated, as well as species’ specialization indices (d′) for the two methods. Similarities in δ15N and bait use, and in δ13C and NLFA profiles, were also correlated. Discussion Our results agree with the recent view that specialization levels do not change with latitude or species richness. Partition of trophic resources alone does not explain species coexistence in these communities, and might act together with behavioral and environmental mechanisms. Temperate species presented NLFA patterns distinct from tropical ones, which may be related to environmental factors. All methods corresponded in their characterization of species’ niches to some extent, and were robust enough to detect differences even in highly generalized communities. However, their combination provides a more comprehensive picture of resource use, and it is particularly important to understand individual niches of species. FAA was applied here for the first time in ant ecology, and proved to be a valuable tool due to its combination of specificity and temporal representativeness. We propose that a framework combining field observations with chemical analysis is valuable to understand resource use in ant communities.

226 in different sample points were considered independent, and chemical results represent the 227 average of different samples from that colony.

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To analyze resource use, we used clustering and network analysis. UPGMA clustering 229 was used for species, to show functional groups based on similar use of resources, and for baits, 240 is an extension of d' and shows how specialized the network is overall. H 2 ' = 0 would mean that 241 all species used resources in the same proportions, and H 2 ' = 1 that each species has its exclusive 242 pattern of resource use.

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Specialization indices were also used to analyze species × fatty acids contingency tables.
244 In this case, they indicate how exclusively NLFAs are distributed across species (Brückner & 245 Heethoff, 2017). H 2 ' = 0 would mean that all compounds occur in the same proportion in all 246 species, and H 2 ' = 1 that each species has its exclusive compounds. Correspondingly, relatively 247 high d' represents NLFAs that occur more exclusively in certain species, or species with more 248 exclusive proportions of certain NLFAs. Low d' means a compound that is widespread among Manuscript to be reviewed 249 species, or species with similarly generalized profiles. Additionally, we tested whether the two 250 communities differed in their overall NLFA composition with PERMANOVA, using site as a 251 fixed factor (Anderson, 2001). Homogeneity of multivariate dispersion was tested a priori with 252 PERMDISP (Anderson & Walsh, 2013). To detect which NLFAs contributed to differences, we 253 used SIMPER (Clarke, 1993). These tests were performed using package "vegan" (Oksanen et 254 al., 2017).

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To test whether niche breadths and NLFA profile diversity were different between 256 communities at species level, we calculated Shannon diversity indices for each ant species as H'  Figure 1, Table 2).

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In Germany, only Lasius fuliginosus showed a relatively high specialization level and 303 clustered separately, due to its almost exclusive use of animal resources (living prey and dead 304 arthropods). Other species showed low specialization and dissimilarity ( Figure 1, Table 2).

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The main reason for this difference was the predominant role of C18:1n9 in temperate 315 species (SIMPER, dissimilarity contribution = 47%, p < 0.01, Figure 2  Manuscript to be reviewed 317 p < 0.01), although amounts were similar. C16:0 was proportionally the most abundant NLFA in 318 Brazil and the difference from Germany was marginally significant (contribution = 20%, p = 319 0.06), although amounts again were higher in temperate species. A few other NLFAs had 320 statistically significant, but very small contributions to the difference (Table S2).

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In samples from Germany, there was no correlation between total amount of fat and
334 Notice that the first two Principal Components explained only 60% of the variance and linear 335 regressions were not strong. In Germany, most variation was along the sugar-protein axis. C18:0 336 and C17:0 (margaric acid) were strongly correlated with PC axes (r 2 = 0.84, p = 0.05 and r 2 = 337 0.78, p = 0.04, respectively). Both were higher in species that used more sugars, and C17:0 was 338 also related to use of feces, although its relative abundance was very low in all species (< 0.5%, 339 345 to -27 ( Figure 5, Table 4).

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In Germany, δ 15 N was lower overall, ranging from 3.6 (Lasius niger) to -1.1 (Lasius   (Table 5). While 355 δ 15 N similarities were correlated with similarities in bait use, but not with NLFAs, the opposite 356 was found for δ 13 C. That is, similar use of resources among species was reflected in similar body 357 fat composition, and both were related to their long-term trophic position, albeit in different ways.
358 In Germany, no such correlations were found between datasets (although it was marginally 359 significant for NLFAs and δ 15 N).  The hypothesis proposed by MacArthur (1972) suggested that specialization is higher in 374 tropical communities because the environmental stability allows species to adapt to more 375 specialized niches without increasing extinction risk, thus allowing more species to coexist. The correlations we observed between methods are interesting from both the 537 methodological and the biological perspective. From a methodological viewpoint, for terrestrial 538 animals, this is the first time an empirical relationship is shown between patterns of resource use 539 and composition of stored fat in natural conditions, and that both relate to their long-term trophic 540 position. Although differences between species were small, these relationships were robust 541 enough to be detected by different methods. From a biological viewpoint, it highlights several 542 physiological mechanisms involved in such relationships. We will discuss in the following some Manuscript to be reviewed 543 of these mechanisms, as well as caveats that are often cited for these methods. They probably 544 still influence our results and correlations, but did not completely override the patterns.

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A commonly cited caveat for using baits is that ants could be attracted to the most limited 546 resources, instead of the ones they use more often. Evidence for this comes mainly from 547 nitrogen-deprived arboreal ants (Kaspari & Yanoviak, 2001), and some cases are discussed 548 below (see Method Complementarity). However, this effect might be less pronounced in epigeic 549 species, and our results suggest that there is convergence between bait attendance, and medium-550 and long-term use of resources. We also found correlations with stable isotopes. They were weaker than between baits 566 and NLFAs, and different for each isotope. For δ 15 N, it shows that patterns of resource use are 567 more correlated with trophic level. Protein amino acids must be obtained from diet or 568 synthesized from other nitrogenated compounds, so the signal relative to nitrogen sources should 569 be more preserved. This also fits to the idea that δ 15 N reflects larval diet, because it is in this 570 stage that ants grow and build most of their biomass . On the other 571 hand, it makes sense that the signal relative to carbon sources is related to NLFA composition.
572 We should note that we removed the gaster of the ants used in SIA, so we observed only the 573 signal of carbon incorporated in the other body parts. This is related to dietary carbon, but a 574 stronger signal could be expected if the fat body is included.

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The low source-specificity of stable isotope signatures might also lead to relatively weak

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In Germany, no correlation was observed between methods. This is probably a 581 consequence of the low number of species available in the community The relationships found in  wasmau  linein  odonch  campze  pachst  pheiap  wasmaf  gnamst  phei07  sole03  sole04  sole08  sole01  sole02  phei04  pheisa  phei02  linemi  pheilu  nyla01  pheine  sole06  pheisi