Despite the coexistence of phylogenetically closely related and morphologically similar species, niche theory suggests that a complete niche overlap may evolutionarily be unlikely (e.g., Hutchinson, 1957; Hardin, 1960). Coexisting species evolved varying forms of resource utilization, such as niche partitioning or niche differentiation (Levins, 1968; MacArthur, 1972), which have been widely used to explain coexistence patterns in community assemblages (Schoener, 1974; Giller, 1984; Ross, 1986). Coexistence may occur due to segregation of specific resources (also known as resource partitioning) or through spatiotemporal variation in resource use (Pianka, 1974; Schoener, 1974; Ross, 1986). In tropical forest bird assemblages, partitioning of dietary resources is one of the fundamental mechanisms of niche separation (e.g., Frith, 1984; Symes and Woodborne, 2010; Mansor and Ramli, 2017; Mansor et al., 2022). In line with the competitive exclusion principle (Hardin, 1960) if any species with identical niches or ecological roles compete, one will drive the other to extinction. By implication, multiple species cannot occupy the same exact niche in one habitat and coexist in a stable manner. Consequently, when these species differentiate their niches, they tend to reduce competition, and promote coexistence. This niche differentiation can be achieved through different mechanisms, which includes consuming various dietary items or partitioning of the environment, such as using different vertical strata of the tropical rainforest (Thiel et al., 2021).

The family of tetrakas (Bernieridae) consists of mostly greenish to yellowish songbirds, all of which are endemic to Madagascar. Some resemble typical warblers (Sylviidae), others are bulbul-like (Pycnotidae), and some did not appear to fit in any systematic group (Cibois et al., 1999, 2001). Their systematic relationship was resolved by the genetic studies of Cibois et al. (2010), which referred to them as a separate family, the Bernieridae. Under current taxonomy, they represent an adaptive radiation of 13 species in eight genera (Reddy et al., 2022; Safford et al., 2022). Some show extensive interspecific resemblance but also intraspecific cryptic diversity (Bickford et al., 2007; Block, 2012; Safford and Hawkins, 2013; Block et al., 2015; Reddy et al., 2022).

The adaptive radiation of tetrakas has been at a relatively continuous diversification rate (Block, 2012) since its estimated dispersal to Madagascar between 9–17 MY (Cibois et al., 2001) or 19.2–25.2 MY ago (Beresford et al., 2005). These range estimates are approximately the same as that for the better known adaptive radiation of a Malagasy bird family, the vangas (Vangidae) (Jønsson et al., 2012; Reddy et al., 2012). Contrary to the vangas, the adaptive radiation of the tetrakas does not appear to have resulted in such a large range of morphological variations and feeding behaviors. Present knowledge of the tetrakas describes all of them as almost exclusively insectivorous (Goodman and Parrillo, 1997; Raherilalao and Goodman, 2011; Block, 2012; Hawkins et al., 2015; Faliarivola et al., 2020). Using their slender bills, their feeding behaviors are relatively homogenous, mostly gleaning insects, probably also using flutter-chase or even flush-pursue strategies (Remsen and Robinson, 1990) when participating in mixed-species foraging flocks (Eguchi et al., 1993; Sridhar et al., 2009). Eleven species are understory or even ground-dwelling in relatively pristine or even exclusively in primary forests, while the Rand’s Warbler (Randia pseudozosterops) and Cryptic Warbler (Cryptosylvicola randrianasoloi) can be found in the canopy (Goodman et al., 1996; Raherilalao and Goodman, 2011; Hawkins et al., 2015).

In the eastern tropical rainforests of Madagascar, up to eight species of tetraka can be found living sympatrically. Since they share dense habitats or the high canopy, they can be very difficult to observe. Previously, some information relating to the ecological niche and microhabitats of these species has been described (Raherilalao and Goodman, 2011; Safford and Hawkins, 2013). However, the mechanisms behind the successful sympatry of these phylogenetically closely related species have yet to be explained in detail. A species’ ecological space, which is often linked to diet, substrate use and foraging behavior can be indirectly approximated by the morphological space filled by each species (Jønsson et al., 2012) and may be visible in their isotopic signatures (Procházka et al., 2010; Yohannes and Woog, 2020).

Stable isotopes have been employed effectively as a tool to determine the origin of dietary nutrient sources for various taxonomic groups (Kelly, 2000). The stable isotopes in feathers can reveal the dietary sources of birds by providing information on the habitat use and nutrient intake during feather growth, which occurs during molt. These isotopes can indicate whether the birds consumed plant matter, such as fruits or seeds, or invertebrate-based diets, such as insects or spiders or both. The stable isotope ratios, particularly nitrogen (δ¹⁵N), can provide insight into the trophic level of the prey and thus the bird, as (δ¹⁵N) values tend to increase at higher trophic levels (Inger and Bearhop, 2008). Stable nitrogen isotopes undergo an increase ranging from 2 to 4‰ in heavy isotope enrichment with each trophic level and can therefore serve as a tool to determine dietary shifts and trophic positions (Inger and Bearhop, 2008). Stable carbon isotopes can be used to distinguish between plants that use either C₃, C₄ or CAM modes of photosynthesis, since the C₄ and CAM pathways lead to lower carbon fractionation than C₃ photosynthesis (Karasov and Martínez del Rio, 2007). Carbon isotopes can thus be used to reconstruct habitat use regarding C₃, C₄ and CAM plants and therefore, the diet preferences of the study species (Hobson, 1999). Changes in δ¹³C due to consumer tissue-diet fractionation range from 1 to 2‰. Furthermore, there is a discernible difference in the stable isotope ratios of plants growing in different vertical strata within closed canopy forests, as seen in carbon (δ¹³C) values. This phenomenon is commonly referred to as the canopy effect (van der Merwe and Medina, 1991), whereby vertical gradients in sunlight penetration, humidity, water source and photosynthetic processes regulate the stable isotope values. Consistently, this produces ¹³C-depleted plant values from ground to canopy; the most positive values are usually at the upper vertical portion. Canopy effects on avian studies (understorey or ground-dwelling birds) have been measured as height from the ground, rather than estimating or measuring the vertical distance from the canopy (e.g., Rajaonarivelo et al., 2020, 2021; Lowry et al., 2021).

Compared with carbon and nitrogen isotopes, stable sulfur isotopes have been used less frequently, and their potential as tracers in biochemical and physiological studies are only beginning to be realized. Mechanisms and accompanying minor or null isotope fractionations involved during the uptake and assimilation of sulfur compounds by animals can be utilized as bioindicators of the foraging niche (Richards et al., 2003; Arneson and MacAvoy, 2005; Florin et al., 2011), to distinguish the origin of dietary sources (e.g., Lott et al., 2003) and to track movement and habitat use (e.g., Date et al., 2022). Sulfur plays an essential role in the synthesis of key metabolic intermediates. For example, the metabolism of sulfur-containing amino acids, methionine and cysteine are essential to protein synthesis in birds (Griffith, 1987). For optimal growth and survival of individuals, the diet must provide these two amino acids and sulfur-containing vitamins, such as thiamine and biotin (Brosnan and Brosnan, 2006). Variations in δ³⁴S values on a small geographic scale are described as local soil sulfate variability. It can significantly influence the plants that grow in these soils and is expressed in the animal tissues (e.g., insects, birds) that feed on them. The fractionation of sulfur isotopes in increasing food chains is comparatively low (McCutchan et al., 2003). Consequently, a wide range of feather δ³⁴S values indicate variable input of sulfur-containing amino acids from a range of consumed dietary sources or recycled body proteins during molt.

In tetrakas, only qualitative descriptions of their feeding habits and diets have been published so far, but information on the stable isotopes in their feathers or prey have not yet been assessed. As a first step, to explore their niche differentiation that may be related to their diet or to the strata they use in the forest, we used a multi-elemental approach analyzing feather isotopes combined with morphometric measurements. We aim to link multi-isotope results toward canopy stratigraphy and avian feeding niches in a rainforest ecosystem and to construct isotopic food web relations among the species dwelling in the same ecosystem. To uncover this diversification and niche segregation, we use a combination of morphometry and multi-element stable isotope analyses of body feathers (δ¹³C, δ¹⁵N, and δ³⁴S) that can be linked to a variety of microhabitats and prey. For keratinous tissues that are metabolically inert following synthesis (e.g., feather), their isotopic values are “fixed” specifically representing the time over which the feather was grown (Hobson, 1999). We hypothesize that niche partitioning and dietary resource segregation may explain the sympatry of insectivorous tetrakas in the Maromizaha rainforest in eastern Madagascar. Specifically, we compare five species of Tetraka: the Grey-crowned Tetraka (Xanthomixis cinereiceps), Long-billed Tetraka (Bernieria madagascariensis), Spectacled Tetraka (Xanthomixis zosterops), White-throated Oxylabes (Oxylabes madagascariensis), and Wedge-tailed Tetraka (Hartertula flavoviridis) and explore niche partitioning patterns and the mechanism of coexistence between them. In two sexually dimorphic species (Long-billed Tetraka and Spectacled Tetraka), we investigate the effect of sex on isotopic niches. We examine whether morphological trait-based niche differentiation was evident. The degree of isotopic niche overlap was expected to reflect the extent of interspecific feeding competition between these co-occurring species.

Between 2003 and 2018, a total of 205 tetrakas were caught and measured at five different sites (mountain ridge: n = 13, river valley: n = 99, mountain saddle: n = 67, plantation: n = 25, dry savannah: n = 1). Tetrakas were abundant in almost all habitats, except for the highly degraded dry savannah. All species were most abundant in the river valley and were captured predominantly in the lower pockets of the nets: 0.01–0.5 m (n = 29); 0.51–1.0 m (n = 63); 1.01–1.5 m (n = 57); 1.51–2.0 m (n = 29); 2.01–2.5 m (n = 14); 2.51–3.0 m (n = 4); 3.01–3.5 m (n = 0); 3.51–4.0 m (n = 2); 4.01–4.5 m (n = 5); and 4.51–5.0 m (n = 2) (Figure 2). White-throated Oxylabes and Wedge-tailed Tetrakas were consistently captured at the lower pockets, most often between 0.5 and 1 m (68% of total captures). In contrast, Long-billed Tetrakas were captured at higher net heights (capture heights 5 to 10, above 2 m) more often than the other species (Figure 2). Chi-Square test of independence: χ² = 12.57, df = 4, p = 0.014, for a total of 205 captures; Grey-crowned Tetraka: n = 32, Long-billed Tetraka: n = 65, Spectacled Tetraka: n = 71, Wedge-tailed Tetraka: n = 12, White-throated Oxylabes: n = 25).

The five tetraka species studied herein, all members of a bird family endemic to Madagascar and representing an adaptive radiation, showed considerable differences in morphological characters, exhibited varying degrees of sexual dimorphism, and had low isotopic similarity and limited isotopic niche overlap. The observed trait-based niche differentiation in tetrakas leads to the assumption that at Maromizaha, each of the five species (and individuals within the species) might consume a subset of the resources locally available, a phenomenon which could be described as species diet specialization in relation to morphology. Such differentiation might play a pivotal role in the diversification, adaptation and speciation in tropical birds. It is possible that these species have overlapping diets acquired from different strata of the forest canopy. This could explain why there is dietary variation among these tropical birds, with differences in the utilization of vertical forest strata playing a critical role in establishing unique niches (Frith, 1984).

Long-billed Tetrakas were the most widely distributed, surprisingly even using a dry quarry outside the forest, whereas all other taxa were restricted to native forest with little degradation. Within the forest, most tetraka species were caught in the lower strata below 2 m and very rarely above 2.5 m, with only Long-billed Tetrakas regularly using higher forest strata. Long-billed Tetrakas seem to have a larger ecological niche, as they have been observed to venture beyond the forest and utilize higher strata, which is different from the behavior of other tetraka species investigated in this study. Additionally, the morphological differentiation of this species is more pronounced than anticipated, particularly with regards to their tarsus, wings, and bill length. Each of these measures has been attributed to specialization regarding avian feeding ecology and behavior. For instance, differences in bill morphology point toward how species or sexes within a species specialize in the prey they feed on (Leisler and Winkler, 1985, 1991), shorter wings may facilitate moving through dense undergrowth (Winkler and Leisler, 1985; Forstmeier et al., 2001), whilst more robust tarsi enable birds to have a stronger grip on vegetation and substrate such as branches (Winkler, 1988; Leisler et al., 1989) and may provide insight into the types of perching substrates used by the birds. Males were often larger than females and this was most striking in the Long-billed Tetraka, where males had 7% longer tarsi, 18% longer wings and impressively 28% longer bills than females. The other tetraka species also showed sexual size dimorphism in multiple morphological measures, including the wing, tarsus, and beak, which point toward possible resource partitioning between sexes.

Regarding stable isotopes, there were only slight differences in δ¹⁵N, as expected by a comparison between insectivorous taxa, however, species occurring closer to the ground had higher δ¹⁵N values, indicating that their prey may be higher up in the food chain. Spiders, for example, should have higher δ¹⁵N values as they prey on arthropods and are abundant in leaf litter (Nyffeler, 1999). However, elevated δ¹⁵N values may indicate not only trophic level but also the patterns of nitrogen cycling in the ecosystem. Different habitats can have varying levels of nitrogen availability and cycling rates, which can affect the δ¹⁵N values of organisms living in those habitats (Craine et al., 2009).

Comparison of the δ³⁴S data between the four species shows an overall similar trend, with higher values of δ³⁴S for the ground-dwelling White-throated Oxylabes, but interestingly also for the Long-billed Tetrakas, which uses higher strata more often than the other species. This may indicate differences in protein sources and sulfur-containing amino acids in diet such as cysteine and methionine (Brosnan and Brosnan, 2006), the two principal sulfur-containing building blocks of proteins. However, additional research is necessary to confirm this hypothesis, for example, through the study of protein and amino acid specific stable isotopes found in insect prey.

The canopy effect describes vertical variation in δ¹³C within plants throughout the forest canopy (van der Merwe and Medina, 1991). Long-billed Tetrakas had the highest δ¹³C values, which may be due to their use of higher strata in the canopy. Significant variation in δ¹³C in response to height in combination with light availability and tree species was expected, as leaves in lower strata of the forest tend to exhibit lower δ¹³C values compared to those in higher strata. While this vertical isotopic variation was not tested using plant leaves and insect consumers, the effect was expected to be directly reflected in the δ¹³C of avian insect diet in the forest. Based on the triple elemental dissimilarity in the four tetraka species, we argue that some prey taxa were commonly consumed by one species and not others. Alternatively, the tetraka species might be preying on different taxa at the same trophic level. The correlations between wing and bill length and δ¹³C and δ¹⁵N values provides evidence that individual tetrakas may segregate into different habitat structures based on a combination of their physical characteristics and food preferences. Potential ecological factors that may contribute to this habitat segregation may include differences in microhabitat preference.

Thus, marginal nestedness in isotopic niche space indicates that the species use unique resources, enhancing their ability to avoid competition and the means for niche partitioning. We are aware that assumed dietary segregation was not assessed concurrently with resource abundance, which is likely to influence the degree of partitioning (Holmes et al., 1986). More data is needed on this.

The observation of incomplete niche overlap and distinct isotopic signatures can be interpreted as evidence for the absence of strong competitive interactions between the organisms in question. Yet, the enhanced dissimilarities in isotope values could parsimoniously be explained by species foraging non-opportunistically as specialists, targeting specific prey items. Nevertheless, since there is a lack of detailed information on the diet and insect species involved, this assumption remains uncertain. The presence of differences and dissimilarities in these sympatric species appears to be essential to species coexistence, at least during the molting season. Insofar the presence of resource partitioning is not expected to lead to exclusion in a resource-rich system. Yet, if prey is scarce and resources are depleted, particularly during the dry season, it can skew the resource utilization pattern if molting coincides with this period. Overall, the isotopic distributions are consistent with well-known stable isotope distribution patterns along vertical strata in forest ecosystems (Van der Merwe and Medina, 1991), supporting the prediction that similar endemic species partitioned their resources and habitat use in a complex manner.

Tetrakas varied in their morphology and isotopic niches, which could be described as ecological specializations, presumably necessary for coexistence. Some species showed a certain level of overlap in resource use, with modest resource specialization. The nature of this resource segregation, coupled with the data on morphological traits, provided evidence that birds occupy different niches to avoid dietary competition. In groups with high resource overlap with complementary distinct specializations, it is often understood that coexistence is explained as species being imperfect generalists with “own” resources available to individuals or species (Sánchez-de León et al., 2014). In fact, this might be linked to morphological traits. Elsewhere, this pattern is referred to as Liem’s Paradox, where species show foraging specializations that permit resource access to unique prey items but still exhibit high niche overlap (Liem, 1980; Bandl et al., 2015; Golcher-Benavides and Wagner, 2019). This might well be the case for the Spectacled Tetraka and its potential competitor, the White-throated Oxylabes, since they had an overlap of about 62% in the three dietary elements.

While the capture height data in this study is only qualitative and cannot be statistically linked to the isotope data, it can provide additional insight into the strata that the birds utilized, along with observations from previously published studies on foraging heights. Evidently, tetrakas enhance the likelihood of coexistence by dividing foraging spaces at a microhabitat level and not necessarily resources partitioning per se (e.g., Schoener, 1974). These results complement the concept of species utilizing resources that other competitors could not, presumably due to morphological or behavioral constraints (e.g., Robinson and Wilson, 1998; Sánchez-de León et al., 2014). It could well be expected that this may lead to a coexistence mechanism in the tetraka species, generating sex-based morphometric and isotopic niche differentiation within the larger niche space (Wilson, 2010).

In Madagascar, when not breeding, insectivorous birds of different sizes, taxonomic groups and dietary preferences are known to form mixed-species flocks (Rand, 1936), which are characterized by two or more individuals moving together to forage, profiting (1) from the disturbance created by the other members of the flock, i.e., by flushing up insects and (2) from increased vigilance with increasing flock size to detect predators. Most tetraka species are known to take part in such flocks (Safford and Hawkins, 2013). This highly specialized cooperative behavior, coupled with the dissimilarities in their respective morphometric and isotopic spaces, could be an indication of their shared sympatric coevolutionary history. We assume that the co-adaptation of these species is based on niche differentiation, rather than outcompeting one another. This theory needs to be corroborated by detailed foraging studies and more precise data on the diet and habitat use of the tetrakas, including more species.

Morphological differences between the sexes were also reflected in their isotopic niches. The female isotopic niche was only part of the male niche in at least two species, the Long-billed and the Spectacled Tetraka. Males of at least two species (Long-billed and Spectacled Tetraka) appear to have extended niches, interestingly, away from the isotopic niches of the other taxa, which suggests that this leads to a reduction in competition for food but more data is needed. Long-billed and Spectacled Tetrakas had a wide range of δ³⁴S values implying a diverse sulfur containing dietary origins. Male Long-billed Tetrakas had heavier bills and it is very likely they prey on larger insects relative to females, presumably contributing to trophic niche extension in males. Thus, it could be argued that species dietary selection plays an important role in micro-habitat partitioning (vertical spatial distribution) and toward avoiding competition and therefore modulating the patterns of spatial use and coexistence between the sexes. Moreover, these data complement the assumption that niche overlaps and intraspecific competition might be minimized by increased specialization enforced through sexual dimorphism (e.g., Pyke et al., 1977). These and additional assumptions described here are solely based on morphology and isotope values obtained during the feather growing season. To validate this, repeated and long-term empirical data as well as evidence for the differences in prey items taken by individuals of the same species are essential (Roughgarden, 1972 Bolnick et al., 2002 Bolnik et al., 2007). Furthermore, stable isotope composition of the different species of insect prey needs to be studied in the different forest strata across different seasons and habitats. Morphometric data are crucial for the description of a species’ niche. Detailed assessment of diet and foraging behaviors are required to infer the sources of differences in morphometry. Identifying which ecological and evolutionary factors are the most important drivers of species specialization is pivotal in understanding how these ecological variations are promoted and sustained.

We report considerable variation in isotopic niche positions, niche breadth and interspecific niche overlap in tetraka species that exhibit a close phylogenetic relationship. Morphological traits coupled with potential dietary groups and micro-habitat trends provided explanations of patterns of isotopic niche areas and positions. We conclude that dietary segregation may be based on specialized foraging strata and species “spatial-resource” specialization. The measurable differences found among these related species could be considered stratified resource use along the vertical canopy gradient, indicating habitat-based structural niches in tetrakas that might assist in reducing interspecific competition. These results refine knowledge of the species and their ecology. Even minor differences in the degree of species specialization among related taxa may have important implications for trophic structure. While disentangling these causes and differences is beyond the scope of this study, these results offer insights into the evolution of differential resource utilization through comparison of both phenotype and ecological aspects of endemic species in a tropical rainforest community. Finally, our results emphasize the efficacy of these proposed coexistence mechanisms through spatial segregation and therefore variation in nutrient acquisition. Future studies should examine the role of coexistence as a flexible strategy in complex, multi-species communities with phylogenetically closely related species. Studies that do not focus on a species’ sex groups may miss important ecological relationships, such as differences in resource use between sex groups. More detailed mechanisms can be elucidated by conducting further studies with larger sample sizes per species and sex group in multiple tetraka species across a wider geographic scale. Additionally, information on the isotopic signatures of plants and invertebrates in their habitats would be useful in interpreting the findings further.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

J-LB, EY, and FW conceived and designed this study and prepared and edited the manuscript. FW collected the samples in the field and took all the measurements. EY performed the stable isotope analyses at the Stable Isotope Lab of the University of Konstanz. J-LB performed the laboratory work for molecular sexing at the Natural History Museum Stuttgart. J-LB and EY performed the morphological and stable isotope data analyses. All authors have read and agreed to the published version of the manuscript.

This project was supported by funding by the “Ausschuss für Forschungsfragen” from the University of Konstanz.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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