Living Together but Apart: Spatial and trophic niche segregation of two termite species sharing the same nest

Living Together but Apart: Spatial and trophic niche segregation of two termite species sharing the same nest | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Living Together but Apart: Spatial and trophic niche segregation of two termite species sharing the same nest Johanne Timmermans, Nicolas Fontaine, Gilles Lepoint, Yves Roisin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6323213/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2025 Read the published version in Insectes Sociaux → Version 1 posted 5 You are reading this latest preprint version Abstract Resource differentiation and segregation is widely recognized as a key factor enabling species coexistence. However, patterns of niche segregation remain poorly understood in faunal assemblages confined by physical barriers, such as species cohabiting in the same nest. In termite-termite symbiosis where a species (inquiline) is hosted in the nest built by another species (host), resource partitioning within the nest appears critical for species coexistence. Here we aim at disentangling the habitat and trophic niche segregation between Constrictotermes cavifrons and its inquiline, Inquilinitermes inquilinus. We assess how spatial segregation contributes to reducing competition by analyzing where the inquiline constructs its galleries within the host nest. Using an isotopic niche approach, we also examine whether I. inquilinus imposes costs on its host by depleting shared food resources or mitigates conflict through niche differentiation, by exploiting distinct dietary resources. Our findings suggest that the inquiline's persistence within the nest is linked to spatial segregation, with the inquiline occupying zones rich in dark organic material, while the host inhabits clay-rich, friable galleries constructed by itself. Isotopic analyses further revealed dietary segregation between the two species, likely reducing competition and facilitating coexistence. The actual food used by the inquiline is most probably the dark mineral-organic material found in the bottom of the host nest. These observations support a commensal symbiosis, wherein the inquiline imposes no significant cost upon the host. Termitidae Constrictotermes Inquilinitermes behavioral ecology inquilinism stable isotopes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Termites (Blattodea), exhibit diverse foraging and nesting strategies (Abe, 1987 ; Donovan et al., 2001 ; Eggleton & Tayasu, 2001 ). Their diets encompass various stages of decomposing organic matter, such as dead wood, leaf litter, dry grass, fungi, lichens, and soil (Donovan et al., 2001 ; Lavelle et al., 1993 ; Miura & Matsumoto, 1997 ). Termite nests, which can be subterranean, epigeal, or arboreal, are complex structures of interconnected galleries and tunnels made from soil, feces, or a mixture of both. These nests provide physical protection and stable microclimates, suitable for colony survival and functioning (Noirot & Darlington, 2000 ). A particular phenomenon, called inquilinism, occurs when one termite species (inquiline) inhabits the nest built and maintained by another termite species (host) (Shellman-Reeve, 1997 ). This strategy benefits the inquiline by providing shelter, protection from predators, and reduced costs of nest construction and maintenance (Florencio et al., 2013 ; Jirošová et al., 2016 ; Shellman-Reeve, 1997 ). Inquilinism may be either facultative, when inquilines colonize nests not necessarily inhabited by their builder (Jirošová et al., 2016 ; Shellman-Reeve, 1997 ), or obligatory, when inquilines colonize host-inhabited nests and are specialized in their association with the host species (Calaby, 1956 ; Gay, 1966 ). The latter category includes the inquiline genus Inquilinitermes , always encountered in nests of Constrictotermes species (Constantino, 1992 ; Florencio et al., 2013 ; Shellman-Reeve, 1997 ). Unlike social parasites, which exploit the social systems of their hosts, inquiline termites employ avoidance strategies (Stuart, 2002 ). They often inhabit spatially segregated parts of the nest and use chemical cues to evade detection or conflict with their hosts (Jirošová et al., 2016 ). For instance, Inquilinitermes microcerus recognizes and avoids the trail and alarm signals of its host, Constrictotermes cyphergaster , while soldiers of Inquilinitermes inquilinus produce repellent substances to deter Constrictotermes cavifrons (Cristaldo et al., 2014 , 2016 ; Jirošová et al., 2016 ). Furthermore, many inquiline species consume food sources distinct from those of their hosts, which reduces direct competition. For example, I. microcerus and I. fur are thought to feed on material excreted by their hosts, a dark substance containing a high quantity of lignin relative to cellulose (Barbosa-Silva et al., 2016 , 2019 ; Constantino, 2005 ; Mathews, 1977 ). However, the nature of the host–inquiline relationships —ranging from mutualism to parasitism— varies and still remains poorly understood (Cristaldo et al., 2016 ; Shellman-Reeve, 1997 ). For instance, while inquilines benefit from the nest's defenses and stable microclimate, they may impose costs by consuming stored food or damaging nest structures, requiring constant repair by the host. In some other cases, the host may benefit from inquilines as they could contribute to nest maintenance by eating the refuse of the host. In this study we aimed at disentangling the relationship between Constrictotermes cavifrons (Holmgren, 1910) and Inquilinitermes inquilinus , an obligatory inquiline (Emerson, 1925), and to place it into the parasitism–mutualism continuum. Constrictotermes cavifrons builds elongated arboreal nests up to 10 meters above ground. Large nests often contain I. inquilinus , either as a single mature colony or in the form of multiple incipient colonies (Martius et al., 2000 ; Timmermans et al., 2024 ). Workers of C. cavifrons use a mix of soil, feces and saliva for construction, giving the nest a light brown to reddish color (indicating high clay content). Known for processional foraging, C. cavifrons creates exposed trails to forage irregularly by day or by night on nearby trees and litter, feeding on microepiphytes, and more precisely on lichens, bryophytes, algae and fungi growing on bark or leaves (Martius et al., 2000 ; Timmermans et al., submitted). A notable feature of these nests is the presence of dark material accumulated at the bottom, which is thought to consist of either stored food or segregated excrement (Barbosa-Silva et al., 2016 ; Emerson, 1938 ; Mathews, 1977 ). Numerous organisms live inside this matter, including many insect larvae (tineid caterpillars, acanthocerid, elaterid beetles, larvae of Sciara flies) (Emerson, 1938 ). Moreover, the inquiline could use this dark material to dig its galleries inside (Emerson, 1938 ; Timmermans et al., submitted). Inquilinitermes inquilinus appears to live totally enclosed within the host nest, as individuals have never been observed outside it, except for alates. If the two species encounter each other, hostile interactions may occur (Emerson, 1938 ; Noirot, 1970 ). The nature of the food source of I. inquilinus remains uncertain but they may feed on the host nest and more precisely on the dark material accumulated at the bottom of the nest (Barbosa-Silva et al., 2019 ; Emerson, 1938 ; Timmermans et al., submitted). The origin and specific role of this dark material remain subjects of investigation (Mathews, 1997; Rezende, 2012 ; Souza Santos et al., 2022 , Timmermans et al., submitted). The success of inquilines within a host nest often depends on their ability to reduce direct competition and conflict with their hosts. Here we aim to analyze the spatial and dietary segregation between C. cavifrons and I. inquilinus to better understand the ecological and evolutionary underpinnings of their coexistence. Specifically, we seek to determine whether I. inquilinus imposes costs on its host by depleting shared food resources or minimizes conflict through niche differentiation, for instance by consuming distinct dietary components. We also investigate how inquiline termites alter the structural integrity of the host nest and if there is a spatial segregation between the two species by understanding where the inquiline constructs its galleries inside the host nest. MATERIAL AND METHODS Study site and nests collection Five expeditions in French Guiana were realized between 2021 and 2023. For this study, 24 nests were sampled in the rainforest at four different localities: Paul Isnard road (n = 1), Petit Saut dam area (n = 14), Kaw mountain (n = 5) and Saül (n = 4) (Figure S1 ). Collection details and composition of samples are referenced in Table S1 . Each C. cavifrons nest (length range: 0.61‒3.56 m) was carefully dissected in 10-cm sections, starting from the bottom to the top of the nest. Each section was measured (width and length) immediately before being isolated in a ziplock bag. Sections were carefully sorted at the base camp to extract host and inquiline (when present) individuals of each caste (Figs. 1 a and 1 b). Samples were stored in 100% ethanol and conserved at -20°C. During the sorting process several material types were collected: galleries of C. cavifrons (gcav), galleries of I. inquilinus (ginq), dark material (dm: black material accumulated inside the C. cavifrons galleries often at the bottom of the nest), exterior galleries (gext: galleries outside of the nest connecting the bottom nest entrance to the ground for pellet transportation), pellets (p: pellets of soil used for nest construction), topsoil layer (soil) taken below the nest and foraging material (fm: material on which C. cavifrons was observed feeding in the field, such as lichen, mosses, fungi and algae growing on bark or leaves). The term galleries is here referring to all the living space of the species inside the nest, comprising chambers and galleries. Additional measurements were taken on the nest: length, maximum width, maximum circumference (starting from the merging point of the nest on the tree to the other side of the nest) and height from the ground (from lowest part of the nest to the ground level) (Table S1 ). C. cavifrons nests were separated in four categories: nests inhabited only by the host (n = 9), nests inhabited by the host and a mature inquiline colony (n = 8), nests inhabited by the host and an incipient inquiline colony (n = 4) and nests abandoned by the host but inhabited by a mature inquiline colony (n = 3). Nest characterization We described qualitatively and quantitatively the galleries of I. inquilinus as well as their location inside the host nest. For one nest (N3), we recorded the width, length and height of galleries selected randomly in the first three 10-cm sections of the nest (0–30 cm), as well as the population density of workers, soldiers, nymphs and alates in the galleries, and the possible presence of primary reproductives. For nests N1 to N5 we photographed and measured every section during the fieldwork. We used the pictures of N4 to analyze the horizontal spatial segregation by marking the center of each I. inquilinus galleries on every section (0–130 cm, pictures of the upper part of the sections). We then checked if more galleries were present (1) in the center of the nest rather than near the periphery, (2) closer to the tree (= interior) rather than near the exterior wall (Figure S2 a). For each of these analyses, we divided the sections in similar areas of center/periphery and interior/exterior. To obtain the surface of the sections, we first outlined on the pictures the border of each section and used the software ImageJ to calculate the area (Schneider et al., 2012 ). We scaled each section independently with its length measured during the fieldwork. The volume of each section was also obtained by multiplying the area with the section height (usually 10 cm). To obtain a “center” part that is 50% of the surface of the section, we reduced a copy of it by a factor of \(\:\sqrt{0.5}=0.707\) and centered it on the original section (Figure S2 b). For the interior/exterior analysis we divided the section in two with a line (Figure S2 c). Resulting surfaces of the divided sections were checked with ImageJ and the number of galleries found in each part was normalized with the surface (Table S2 ). We also described the vertical spatial segregation between the two species and noted the differences in nest composition with the height inside the nest (N1–N5). For each section of 10 cm, we approximated the percentage of dark material, inquiline galleries and host galleries as well as the friability of the nest material. We calculated the sections’ volumes with the method described above. The density of I. inquilinus was obtained by counting the individuals of each caste, and approximated for larvae and eggs. For C. cavifrons the population size was always estimated due to the high population density of this species. We used a scale from 0 to 5: for neuters 0 = no individual, 1 = 0 to 50, 2 = 50 to 500, 3 = 500 to 1.000, 4 = 1.000 to 2.500 and 5 = 2.500 to 5.000 individuals; for other castes 0 = no individual, 1 = very few, 2 = few, 3 = average, 4 = high, 5 = very high. We compared our estimation of the neuter population size with the model proposed by Vasconcellos et al. ( 2007 ) for the closely related species C. cyphergaster : \(\:Population=7113.7+\left(1667.9\text{*}\text{N}\text{e}\text{s}\text{t}\:\text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\right)\) . Isotopic and elemental composition Termite tissues of C. cavifrons (n = 46 individuals and n = 19 colonies) and I. inquilinus (n = 30 individuals and n = 11 colonies), nest material of different types, i.e. , C. cavifrons galleries (n = 58 samples and n = 21 colonies), I. inquilinus galleries (n = 15 samples and n = 8 colonies), dark material (n = 33 samples and n = 18 colonies) and exterior galleries (n = 5 samples and n = 5 colonies), pellets (n = 6 samples and n = 5 colonies), topsoil layer (n = 23 samples and n = 23 colonies) and foraging material (n = 5 samples and n = 1 colonies) were subjected to carbon and nitrogen isotopic analyses (Table S3). Samples of termites were stored at -20°C in 100% ethanol to preserve tissues for the isotopic composition analyses (Florencio et al., 2011 ). Only the heads of termites were kept for the analysis to avoid contamination from gut content. We dissected 10 worker heads per section (with sometimes several replicates) and directly dried the tissues as well as the nest, soil, foraging material and pellet samples at 60°C for 24h in a stove before weighing. To prevent bias from carbonates in the final isotopic composition, we tested their absence by reaction with 10% HCl in the dried topsoil and nest samples. None of the samples presented signs of carbonate presence. Samples were weighed using a Mettler AT261 DeltaRanger (Mettler Toledo) precision balance (0.1 mg) in tin capsules. For dried tissues of termites, the samples ranged from 0.9 to 1.1 mg and for nest, soil, foraging material and pellets samples ranged from 4.5 to 5.5 mg. Measurements were performed using an elemental analyzer (Vario Microcube, Elementar Analysensysteme GMBH, Germany) coupled to an isotope ratio mass spectrometer (PrecisION, Elementar Analysensysteme GMBH, Germany). Isotopic ratios were expressed following the δ notation (‰) (Coplen, 2011 ) based on international standards: Vienna Pee Dee Belemnite for carbon, atmospheric air for nitrogen. Glycine, and replicates were interspersed every 15 samples for elemental calibration and secondary isotopic CRM. The standard deviation for δ 15 N and δ 13 C were 0.3 and 0.1‰ for termite replicates ( I. inquilinus ), 0.4 and 0.1‰ for nest material replicates, 0.2 and 0.1‰ for foraging galleries and 0.3 and 0.3‰ for glycine. Raw data are given in the Online Resource ESM_2.xlsx. For certified reference materials (CRM), we used sucrose (IAEA-C6; mean ± SD: δ 13 C = − 10.8 ± 0.5‰) and ammonium sulphate (IAEA-N2; δ 15 N = 20.3 ± 0.2‰). Elemental data are expressed as a percentage of dry weight over weight (W:W). Average, standard deviation and confidence interval at 95% for δ 15 N, δ 13 C, N%, C% and C:N were calculated for each material type (Table S4). All the analyses were conducted in the R environment version 4.3.1 (R Core Team, 2023 ). As none of the data distribution were normal, we used the non-parametric Kruskal-Wallis test (function: kruskal.test ) to evaluate the significance of differences between materials averages (Hollander et al., 2014 ). A pairwise Wilcoxon test with a Benjamini and Hochberg correction for multiple testing was then used to identify significantly different pairs using the function pairwise.wilcox.test (Benjamini & Hochberg, 1995 ; Hollander et al., 2014 ) (Tables S5). One biplot of δ 15 N and δ 13 C per nest was obtained with all the material types represented (Figure S3). To compare the isotopic niche of the different material types, SIBER package in R was used (Jackson et al., 2011 ). Bayesian standard ellipses were inferred with the function plotSiberObject from the δ 15 N and δ 13 C biplot. Overlap between ellipses were obtained using the function sea.overlap.comm1 and Layman metrics were calculated with function groupMetricsML (Jackson et al., 2011 ). The overlap between two ellipses in percent was calculated using this formula: \(\:100\left.\left(\:\frac{overlap}{SEAc\:A\:+\:SEAc\:B\:-\:overlap}\right.\right)\) . We then proceeded to a correlation analysis between each group of minimum six colonies, using Python 3.12.5 to obtain Pearson coefficient and R 2 with the scikit-learn and SciPy package (Freedman et al., 2007 ; Van Rossum & Drake, 1995 ; Virtanen et al., 2020 ). Biplots of the linear regression were also obtained with Python 3.12.5 using the Seaborn packages, and confidence intervals were calculated with a bootstrap method (Waskom, 2021 ) (Figure S4). Correlation between materials, nest size and height were also tested (Table S6). Another analysis was conducted on the nest sections to observe whether there is an effect of the location inside the nest on the composition of the material studied. Pearson coefficient and R 2 were calculated for each material type according to the nest section and between materials within a nest section (Freedman et al., 2007 ). A Benjamini and Hochberg correction for multiple testing was applied to avoid statistical overinterpretation (Benjamini & Hochberg, 1995 ) (Table S7). RESULTS Nest characterization Three clearly different material types are found inside the C. cavifrons nest. The first one is the host galleries, made of soil pellets collected from the ground underneath the nest and mixed with feces (Timmermans et al., submitted) (Fig. 1 g). The color is light-brown or reddish depending on the soil composition, and the material is way more friable than the other two. The galleries are all connected to each other, none being enclosed inside inquiline galleries. Another interesting point is that we found a royal chamber of C. cavifrons adjacent to I. inquilinus galleries (in section 80–90cm on a 172cm nest length, N4) (Fig. 1 h). The second type of material is the dark mineral-organic material accumulated at the bottom of the nest, filling the galleries of the host (Fig. 1 c). This material can also be observed in the upper part of the nest and was sometimes found in smaller quantity in the uppermost section. The material is often hard to break and not really friable. Note that this material is always present inside C. cavifrons nests, even in the absence of inquiline, provided they have reached a sufficient size. The horizontal location of the inquiline galleries inside the host nest (performed on 13 sections of one nest) shows no difference between the center of the nest and the periphery (respectively: 50.7% and 49.3% of the galleries) (Table S2 ). The interior/exterior analysis gives similar results with respectively 48.4% and 51.6%. The relative abundance of the three material types is clearly influenced by the vertical position in the nest (Fig. 2 a and Figure S5a). In nests without inquilines (N1, N2 and N5) the dark material is the most abundant material in the lowest part of the nest; it decreases with the height and is null above 30 to 60 cm. In the nest with inquilines (N4), I. inquilinus galleries are the most common material in the lowest part of the nest, decreasing with height in opposition with C. cavifrons galleries. Dark material is also less present in this nest than in nests where I. inquilinus is absent (Table S9). In general, the friability of the nest increases with the proportion of C. cavifrons galleries. Globally, the proportion of C. cavifrons galleries is always higher than dark material (respectively; N1: 96.88% and 3.13%, N2: 85.30% and 14.70%, N4: 69.76% and 6.12% and N5: 83.43% and 16.57%). In the presence of inquilines, the proportion of inquiline galleries (24.12%) is higher than dark material (6.12%) (Table S9). Density curves of the neuter population of C. cavifrons show an almost complete absence in the lowest section of the nest, as in nest N5 were no individual was seen in the lowest 40 cm of the nest (Fig. 2 b and S5b). The peak of density is around the two-thirds of the nest, above which the density decreases, being low at the highest section of the nest. The queen and/or king were often found near the peak of density of neuters. The density curve of I. inquilinus in the N4 nest indicates a pattern opposite to C. cavifrons , with the highest density in the lowest section, decreasing slowly until the first third of the nest (Fig. 2 b). The queen and king of I. inquilinus were found higher in the nest than the peak of highest neuter density (king: 30–40 cm, queen: 40–50 cm). The C. cavifrons queen was found in the 80–90 cm section, where the inquiline was still present (until section 130–140 cm). Analysis of the caste distribution of I. inquilinus inside nest N4 shows the highest density of neuters in the lowest sections of the nest, reproductive individuals and juveniles being almost totally absent (Figure S6a). Neuter density decreases with nest height while reproductives increase up to 40–50 cm, corresponding to the presence of the queen. After the peak, all castes decrease slowly and are absent above section 130–140 cm. In C. cavifrons the density of each caste is quite similar: they increase slowly until the highest peak near the middle of the nest (where the queen is found) and then decrease (Figure S6b). Isotopic and elemental analyses Constrictotermes cavifrons tissues have a mean values of 2.0‰ for δ 15 N (95% CI 1.7‰ to 2.3‰, n = 19), and − 29.5‰ for δ 13 C (95% CI -29.8‰ to -29.3‰, n = 19). Tissues of I. inquilinus show mean values of 5.5‰ (95% CI 4.5‰ to 6.5‰, n = 11) for δ 15 N and − 28.5‰ (95% CI -28.8‰ to -28.2‰, n = 11) for δ 13 C (Table S4). Wilcoxon tests revealed a significant difference between the two species in the isotopic composition of carbon and nitrogen and in the C:N ratio (p-value < 0.05) (Table S5). There is no overlap between the two isotopic niches and the difference between standard ellipse areas (SEAc) is significant ( C. cavifrons : 1.06‰² and I. inquilinus : 2.66‰², p-value = 0.008) (Fig. 3 a and Table S10). For nest materials we compared the galleries of C. cavifrons (mean δ 15 N: 4.6‰ and δ 13 C: -30.2‰), the galleries of I. inquilinus (mean δ 15 N: 4.9‰ and δ 13 C: -30.9‰), the dark material (mean δ 15 N: 4.5‰ and δ 13 C: -31.2‰) and the soil (mean δ 15 N: 5.1‰ and δ 13 C: -29.2‰). Wilcoxon tests show no significant difference between these materials for nitrogen isotopes, but a clear significant difference for carbon isotopes except between the dark material and I. inquilinus galleries. For the isotopic niches, we observe 9.7% of overlap between the galleries of the host and the inquiline, 6.4% between C. cavifrons galleries and the dark material, 12.8% between the soil and the host galleries, 39.7% between I. inquilinus galleries and dark material, and no overlap between other groups (Fig. 3 b). A significant correlation between host and inquiline tissues is observed in the isotopic composition of nitrogen (R 2 : 0.898, p-value < 0.001, n = 8) but not of carbon (R 2 : 0.450, p-value: 0.069, n = 8) (Fig. 4). Correlations between inquiline galleries, dark material and inquiline tissues are significant for both isotopic ratios. For nitrogen there is a significant correlation between soil and dark material, soil and host galleries, host tissues and dark material as well as host tissues and inquiline galleries. For carbon isotopic ratios, only C. cavifrons and dark material are significantly correlated. Figure 4 Correlation matrix between each material type (n ≥ 6) for carbon and nitrogen isotope. R 2 are indicated for each correlation with p-value and sample size. Graphs are generated with Python 3.12.5. If we compare the different colony types (host alone, presence of a mature I. inquilinus colony and presence of an incipient I. inquilinus colony), we observe differences in the isotopic composition of C. cavifrons tissues between colonies where I. inquilinus is present versus absent (Figure S7a). These colony types also differ by the mean size of the nest (respectively: 1.12 m, 2.05 m and 1.96 m). The SIBER plot of the C. cavifrons nests arranged by size classes also shows a relationship with the isotopic composition, without overlap between small and large nests (Figure S7b). Standard ellipse areas (SEAc) are larger for small nests than for middle and large nests (respectively: 1.48, 0.92 and 0.54‰²). However, no significant difference between these groups is observed and correlations of isotopes between C. cavifrons tissues and nest size were not significant. There is only a significant correlation between nest size and C. cavifrons galleries for δ 15 N (R 2 : 0.36, p-value: 0.01), and between nest ground height and C. cavifrons tissues for δ 13 C (R 2 : 0.22, p-value: 0.04, data in Table S6). The nest size and the ground height are also significantly correlated (Spearman ρ: 0.47, p-value: 0.001, shown in Figure S7c). We observe a relation between the proportion of carbon and nitrogen throughout a gradient of the different material types (Fig. 5 ). Pellets have the lowest C and N content, followed by soil and exterior galleries, host galleries, inquiline galleries, dark material and foraging material. Dark material shows a high heterogeneity in the global composition (C% mean: 28.33, σ: 11.47 and N% mean: 1.29, σ: 0.50) and has significantly higher nitrogen and carbon proportions than pellets, soil, exterior galleries and C. cavifrons galleries (p-value < 0.002). For I. inquilinus galleries the proportion of carbon is significantly smaller than in dark material (p-value: 0.007) but the proportion of nitrogen is not clearly different (p-value: 0.057). Galleries of the inquiline show a higher C% and N% than host galleries (p-value < 0.002). Foraging material has a greater proportion of carbon and nitrogen than every other material, only its proportion of nitrogen is not clearly different from that of dark material (p-value: 0.07). Analysis by nest interval shows no significant correlation between isotopic ratios of materials from the same interval. Only two correlations between isotopic ratios and height inside nest reach p-values below 0.05: C. cavifrons galleries for δ 13 C (‰) and I. inquilinus tissues for δ 15 N (‰) in nest N4 (p-value: 0.04) (Table S7). DISCUSSION Segregation in resource use is recognized as a major factor enabling species to coexist, with evidence across plant (Silvertown, 2004 ), invertebrates (Sarty et al., 2007 ) or vertebrates (Lejeune et al., 2018 ; Mason et al., 2008 ). In termites, it is known that diet but also habitat influences species coexistence (Bourguignon et al., 2009 , 2011 ; Korb & Linsenmair, 2001 ). However, niche segregation patterns remain largely unexplored in assemblages confined by physical barriers, as when species are sharing the same nest. In termite-termite symbiosis such as inquilinism, resource partitioning inside the nest through spatial and diet segregation appears to be essential to understand their cohabitation and potential negative or positive effect of this particular symbiosis. Spatial niche segregation We observe no horizontal spatial segregation in the nest between C. cavifrons and I. inquilinus . The inquiline shows no preference for the central versus peripheral zone or the interior versus exterior zone. Interestingly, in C. cyphergaster nests, I. fur and I. microcerus tend to be restricted to the central region where the dark organic matter is concentrated (Cunha et al., 2003 ). A hypothesis to explain this difference between related species is that the dark material does not display any specific horizontal distribution pattern in C. cavifrons nests. As to the vertical spatial segregation, the inquiline is restricted to the zone rich in dark material, in which it seems to build its galleries. This dark organic-rich material is located principally at the bottom of the nest and decreases with nest height until approximately the bottom one-third of the nest. However, some nests contain dark material in upper zones, such as nests N4 (80–90 cm on a 172 cm nest) and N5, where we observed it in the top section of the nest (130–145 cm). In the presence of the inquiline, the proportion of dark material is lower in the lowest section of the nest than in its absence. The proportion of inquiline galleries is way higher than the dark material in this section (respectively 0–10 cm: 85% and 5%). It suggests that when the inquiline is present, the dark organic material is replaced by inquiline galleries. In summary, as a general observation, the presence of dark material, probably produced by host species, highly determines the location of inquiline species. We found individuals of I. inquilinus and their galleries until section 120–130 cm on a 172 cm nest. This is way higher than the one-third of the nest occupancy expected for termite inquiline species (Cruz et al., 2023 ). Moreover, we found inquiline galleries totally enclosed inside host galleries and some of them adjacent to the host royal chamber. One hypothesis to explain this observation is that the inquiline species only inhabits zone of dark material and never ventures into host galleries. Additionally, we never found the host in zones of dark material. The two species thus coexist closely using different spaces in the nest. To ensure this segregation, is it possible that the inquiline uses chemical cues. It is known that I. inquilinus soldiers repel C. cavifrons by producing chemical substances (Jirošová et al., 2016 ), and the feces of the related species I. microcerus seem to be repellent to its host C. cyphergaster (Hugo et al., 2020 ). Moreover, according to Cristaldo et al. ( 2014 , 2016 ), I. microcerus can recognize the trail and alarm signals of its host and avoid them. Therefore, the cost imposed on the host by the inquiline on the spatial niche is surely minimized by the population size (probably five-fold lower than the host’s) and the difference in occupancy of the nest (i.e. dark organic material for inquiline versus host galleries for C. cavifrons ). In this termite-termite close relationship, the habitat niche of involved species are actually segregated at the microhabitat scale, despite the fact that the two species share the same nest. Trophic niche segregation There is no overlap between the isotopic niches of I. inquilinus and C. cavifrons (Fig. 3 a). The isotopic niche being used as a proxy of trophic niche, it suggests a clear trophic segregation through the utilization of distinct food sources. Such results are consistent with other studies realized on inquiline-host relationships in termites (Florencio et al., 2013 ; Hellemans et al., 2019 ). The standard ellipse aeras (SEAc) are significantly higher for I. inquilinus than for C. cavifrons ( C. cavifrons : 1.06 vs. I. inquilinus : 2.66‰², p = 0.008). These findings indicate a higher isotopic heterogeneity in the diet of the inquiline species. The correlation between the two species is significant for δ¹⁵N (R² = 0.898, p < 0.001). It appears less conclusive for δ¹³C (R² = 0.450, p = 0.069), but this ratio shows very low intercolonial variations. This suggests a clear connection between the two species, with at least one species being dependent on the other. It is therefore likely that the inquiline feeds, at least partially, on material excreted by the host. The organic-rich dark material is highly variable in terms of elemental and isotopic composition, and is probably produced by the host species. However, the presence of high amounts of mineral crystals inside the dark material, while such crystals are very scarce in the gut of the microepiphyte-feeding C. cavifrons (Timmermans et al., submitted), shows that this matter is not solely composed of segregated excrement. One possible explanation for the presence of mineral crystals is that the host incorporates soil pellets into its feces when depositing them at the bottom of the nest, resulting in the formation of this dark, mineral-organic material. In this study, we aimed to resolve the true nature of the dark organic material by examining the two possible explanations presented in the literature: segregated excrement or stored food (Barbosa-Silva et al., 2016 ; Emerson, 1938 ; Mathews, 1977 ). The isotopic composition of the dark material does not align with sampled food sources of host, which refutes the “food storage” hypothesis. More likely, dark material is reprocessed material (i.e. host feces, undigested food remnants), undergoing significant isotopic and elemental modifications compared to host food sources. Dark material and I. inquilinus galleries constitute the only pair of materials that are not significantly different in carbon isotope ratios. This is supporting the idea that I. inquilinus digs its galleries out of the dark organic mass accumulated at the bottom of the host nest (Emerson, 1938 ; Mathews, 1977 ). The high correlation across nests between dark material and I. inquilinus galleries further support this hypothesis (correlation for δ 15 N (‰): 0.865 and δ 13 C (‰): 0.935, p-value = 0). In contrast, if I. inquilinus built its galleries within the host galleries, the isotopic composition of both materials would be more similar. Furthermore, the significant differences in carbon and nitrogen percentages between these two materials highlight a clear distinction in the composition of the structures built by the inquiline or by the host. As found in a previous study (Bourguignon et al., 2011 ), the δ 15 N values of the inquiline is significantly higher than the host’s. I. inquilinus is thus feeding on material more reprocessed than its host, which agrees with the confinement of the inquiline inside the host nest. It is assumed that Inquilinitermes species feed on matter excreted by their host, allegedly the dark organic-rich substance accumulated inside the host galleries (Barbosa-Silva et al., 2019 ; Emerson, 1938 ; Mathews, 1977 ). Here, the strong correlations observed for δ 15 N and δ 13 C values between I. inquilinus tissues, galleries, and dark matter (Fig. 4) provide further evidence for this hypothesis, with the restriction that the dark matter is not solely composed of host feces. However, Cruz et al., ( 2023 ) showed that the inquiline species I. fur and I. microcerus can live in absence of dark material in nests of C. cyphergaster . This means that some inquiline species can have different trophic habits. We also examined the isotopic ratios among the different types of C. cavifrons colonies: nests inhabited by the host only, nests inhabited by both the host and a mature inquiline colony, and nests inhabited by the host and an incipient inquiline colony. The analysis of the isotopic niches reveals no overlap between the isotopic composition of colonies inhabited by the host only and by the dyad (Figure S7a). As it is known that C. cavifrons nests are colonized by I. inquilinus only if they are sufficiently large (Cristaldo et al., 2012 ), a possible explanation for the isotopic difference between host-only and host inquiline colonies is that young C. cavifrons colonies (small nests) do not feed on the same resources as older colonies (large nests). This hypothesis is supported by the observation that smaller nests had higher δ 15 N and lower δ 13 C than larger nests, independently of their colonization status (Figure S7b). Moreover, smaller nests are typically found closer to the ground as shown by the correlation between nest size and ground height we found in this study. Additionally, foragers from small nests have been observed feeding on the forest floor litter, while those from larger nests seem to feed higher up in the trees, particularly on tree bark. This difference in foraging behavior depending on the host colony age could explain the distinct trophic niches observed for these nests. Conclusion For species being partitioned inside the same termitarium with physical barriers, it is important to have different ecological niches to avoid competition and potential threats (i.e. aggressive behavior). Here we show that the inquiline permanence within the nest seems to be related to spatial and trophic segregation between the cohabiting species. Spatially, the inquiline occupies a zone where dark organic-rich material is concentrated, mostly at the bottom of the nest, while the host inhabits the light-colored friable galleries they build out of clay pellets. Based on isotopic analyses, we also observe a clear dietary segregation between the two species. This mechanism likely benefits the inquiline by reducing competition with its host. Since the two species make different uses of the food and spatial niches, it seems logical to consider their relationship as a commensalism, in which the inquiline species imposes no cost on its host. However, we cannot totally exclude that I. inquilinus may feed directly on the host galleries and thus impose some cost of maintenance and construction on the host. Declarations Author contributions Johanne Timmermans and Yves Roisin designed the study. Funding was secured by Johanne Timmermans and Yves Roisin. Collection of the samples was assured by Johanne Timmermans, Nicolas Fontaine and Yves Roisin. Laboratory work was performed by Johanne Timmermans and Gilles Lepoint. Johanne Timmermans conducted the analyses with help from Gilles Lepoint. Johanne Timmermans wrote the first draft of the manuscript. All authors contributed to the final version and approved the submitted manuscript. Acknowledgments We acknowledge support from the Fonds de la Recherche Scientifique—FNRS and FRIA (Formation à la Recherche dans l'Industrie et dans l'Agriculture). G.L. is Senior Researcher at FRS-FNRS. We also thank Loane Wu, Paule Vanessa Fopa Diffo, Matsvei Tsishyn, Esméralda Rodriguez Palacio, Sarah Gravier et Maxime Lenaerts for their help in the field collecting samples and sorting the nests. Funding information Funding was provided by the Fonds de la Recherche Scientifique—FNRS (JT, YR), through Grant CDR J.0180.20 (to YR), and FRIA (Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture) PhD fellowship (to JT). Conflict of interest statement The authors declare no conflict of interest. Financial interest statement The authors declare they have no financial interests. Data availability All data are accessible in the Online Resource ESM_1.pdf and Online Resource ESM_2.xlsx. References Abe, T. (1987). Evolution of life types in termites. In: Evolution and coadaptation in biotic communities. (Eds S. Kawano, J. H. Connell & T. Hidaka) pp. 126–148. University of Tokyo Press , Tokyo. 83–112. Barbosa-Silva, A. M., Farias, M. A. A., Mello, A. P., Souza, A. E. F., Garcia, H. H. M., & Bezerra-Gusmão, M. A. (2016). 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(2021). seaborn: Statistical data visualization. Journal of Open Source Software , 6(60), 3021. https://doi.org/10.21105/joss.03021 Supplementary Files ESM1.docx ESM2.xlsx Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2025 Read the published version in Insectes Sociaux → Version 1 posted Editorial decision: Minor Revisions Needed 19 May, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers invited by journal 03 Apr, 2025 Editor assigned by journal 01 Apr, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6323213","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437852323,"identity":"b76e2a08-c026-4639-a5ab-7be45be2c3f5","order_by":0,"name":"Johanne Timmermans","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYBACxgYeKIsZLsZ8gOFhA0MCQS08CC1sCQyJeLQA1aJQYKYBXi3M7b0HPxfusGGwZ+dO3fBxzzZ5c/4z3yQSdzDk8eNyWM+5ZOmZZ9KADuPddnPGs9uGO2fkbpNIPMNQLNmAQ8uMHANp3rbDYC23eQ7cZtxwgxeopY0hccMBnFqMf/O2/Ydo+XPgtv2G82eegbXsx63FDGjLAYgWhgO3gYbnsEFswemXM2bWvG3JPDyHgX7pOXA7ecONNGOLxDMSxRI4bDFs7zG+zdtmJ8fef3bbjR8HbttuOH/44Y2PO2zy+HF43xAqzoMuIYHDWQwM8jhlRsEoGAWjYBTAAAANmGCIjzdCOQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-7883-2081","institution":"Universite Libre Bruxelles Faculte des Sciences","correspondingAuthor":true,"prefix":"","firstName":"Johanne","middleName":"","lastName":"Timmermans","suffix":""},{"id":437852324,"identity":"4c658d31-fad1-4551-a830-ab0e7c892dcd","order_by":1,"name":"Nicolas Fontaine","email":"","orcid":"","institution":"Université Libre de Bruxelles: Universite Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Fontaine","suffix":""},{"id":437852325,"identity":"eed8eeac-28fa-4e35-9ed7-5379b5c5804f","order_by":2,"name":"Gilles Lepoint","email":"","orcid":"","institution":"Université de Liège: Universite de Liege","correspondingAuthor":false,"prefix":"","firstName":"Gilles","middleName":"","lastName":"Lepoint","suffix":""},{"id":437852326,"identity":"d70387a1-d4f0-4f72-8dc0-457fc02ae706","order_by":3,"name":"Yves Roisin","email":"","orcid":"","institution":"Universite Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Yves","middleName":"","lastName":"Roisin","suffix":""}],"badges":[],"createdAt":"2025-03-27 20:59:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6323213/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6323213/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00040-025-01061-x","type":"published","date":"2025-09-09T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81284038,"identity":"bc6c106b-cb06-46a7-bf70-5561c1d919b0","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":819978,"visible":true,"origin":"","legend":"\u003cp\u003ePictures of\u003cstrong\u003e a.\u003c/strong\u003e castes in \u003cem\u003eI. inquilinus \u003c/em\u003e1. Worker. 2. Soldiers. 3. Nymph, 4\u003csup\u003eth\u003c/sup\u003e instar. 4. Nymph, 5\u003csup\u003eth\u003c/sup\u003e instar. 5. alates 6. King. 7. Queen. \u003cstrong\u003eb. \u003c/strong\u003ecastes in \u003cem\u003eC. cavifrons \u003c/em\u003e1. Larvae. 2. Soldier. 3. Worker. 4. Nymph,\u0026nbsp; 4\u003csup\u003eth\u003c/sup\u003e instar.\u0026nbsp; 5. Queen. \u003cstrong\u003ec.\u003c/strong\u003e dark material accumulated inside the host nest \u003cstrong\u003ed.\u003c/strong\u003e \u003cem\u003eI. inquilinus \u003c/em\u003egalleries \u003cstrong\u003ee. \u003c/strong\u003enodules found inside \u003cem\u003eI. inquilinus \u003c/em\u003egalleries\u003cstrong\u003e f. \u003c/strong\u003econnecting hole from inquiline galleries \u003cstrong\u003eg.\u003c/strong\u003e \u003cem\u003eC. cavifrons \u003c/em\u003egalleries \u003cstrong\u003eh.\u003c/strong\u003e \u003cem\u003eC. cavifrons \u003c/em\u003eroyal chamber surrounded by inquiline galleries.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/7f812f4ce470b3e19b18fae9.png"},{"id":81284036,"identity":"06bb7c2f-3f8d-42d7-929e-8dbd07e30866","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169181,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs for N4 nest of \u003cstrong\u003ea.\u003c/strong\u003epercentage of the three different materials observed inside \u003cem\u003eC. cavifrons\u003c/em\u003enest with the nest height. And \u003cstrong\u003eb.\u003c/strong\u003e density of neuters of \u003cem\u003eC. cavifrons\u003c/em\u003eand \u003cem\u003eI. inquilinus \u003c/em\u003ewith height inside the nest. The sections where the queen or king was found is indicated by a symbol.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/6e5e717bda9792e8803cefa9.png"},{"id":81284039,"identity":"41838989-05d7-4338-8400-67ec02fbbd6b","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171604,"visible":true,"origin":"","legend":"\u003cp\u003eStable isotope ratios of carbon (δ\u003csup\u003e13\u003c/sup\u003eC) and nitrogen (δ\u003csup\u003e15\u003c/sup\u003eN) of \u003cstrong\u003eA.\u003c/strong\u003e tissues of host species (\u003cem\u003eC. cavifrons\u003c/em\u003e) and inquiline species (\u003cem\u003eI. inquilinus\u003c/em\u003e). \u003cstrong\u003eB.\u003c/strong\u003e nest materials. Bayesian ellipses were generated with SIBER using R.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/2813c8f0010dfbbed780ea5f.png"},{"id":81284040,"identity":"ee63a9b4-3962-4c97-94a3-93449cff9aaf","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":254028,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation matrix between each material type (n ≥ 6) for carbon and nitrogen isotope. R\u003csup\u003e2\u003c/sup\u003e are indicated for each correlation with p-value and sample size. Graphs are generated with Python 3.12.5.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/ba6a8aa44fb2b8f0c37a6ad8.png"},{"id":81284037,"identity":"1faf1930-909e-44c9-99da-a683a4ec0abe","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92791,"visible":true,"origin":"","legend":"\u003cp\u003eBiplot of the proportion of nitrogen and carbon for all material types. Graph is generated with R v4.3.1.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/96740f13388c476d30430b97.png"},{"id":91359019,"identity":"9bf0e9d9-c545-426b-ae69-2679f4c60a48","added_by":"auto","created_at":"2025-09-15 16:04:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2083572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/b64716aa-7cd3-49c0-bec4-725260c90ca5.pdf"},{"id":81284056,"identity":"487d4a79-a40c-4d98-84a8-32bd53f6e79e","added_by":"auto","created_at":"2025-04-24 10:37:03","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5695037,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/fa814702d7f876231d06b6a8.docx"},{"id":81284047,"identity":"e8cb46b6-8711-4a4a-8cf7-0b67f2a3db0e","added_by":"auto","created_at":"2025-04-24 10:37:02","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":33278,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6323213/v1/28cf353fbb26d93c4a054a60.xlsx"}],"financialInterests":"","formattedTitle":"Living Together but Apart: Spatial and trophic niche segregation of two termite species sharing the same nest","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTermites (Blattodea), exhibit diverse foraging and nesting strategies (Abe, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Donovan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Eggleton \u0026amp; Tayasu, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Their diets encompass various stages of decomposing organic matter, such as dead wood, leaf litter, dry grass, fungi, lichens, and soil (Donovan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Lavelle et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Miura \u0026amp; Matsumoto, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Termite nests, which can be subterranean, epigeal, or arboreal, are complex structures of interconnected galleries and tunnels made from soil, feces, or a mixture of both. These nests provide physical protection and stable microclimates, suitable for colony survival and functioning (Noirot \u0026amp; Darlington, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA particular phenomenon, called inquilinism, occurs when one termite species (inquiline) inhabits the nest built and maintained by another termite species (host) (Shellman-Reeve, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This strategy benefits the inquiline by providing shelter, protection from predators, and reduced costs of nest construction and maintenance (Florencio et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jirošov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shellman-Reeve, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Inquilinism may be either facultative, when inquilines colonize nests not necessarily inhabited by their builder (Jirošov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shellman-Reeve, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), or obligatory, when inquilines colonize host-inhabited nests and are specialized in their association with the host species (Calaby, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1956\u003c/span\u003e; Gay, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). The latter category includes the inquiline genus \u003cem\u003eInquilinitermes\u003c/em\u003e, always encountered in nests of \u003cem\u003eConstrictotermes\u003c/em\u003e species (Constantino, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Florencio et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shellman-Reeve, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Unlike social parasites, which exploit the social systems of their hosts, inquiline termites employ avoidance strategies (Stuart, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). They often inhabit spatially segregated parts of the nest and use chemical cues to evade detection or conflict with their hosts (Jirošov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For instance, \u003cem\u003eInquilinitermes microcerus\u003c/em\u003e recognizes and avoids the trail and alarm signals of its host, \u003cem\u003eConstrictotermes cyphergaster\u003c/em\u003e, while soldiers of \u003cem\u003eInquilinitermes inquilinus\u003c/em\u003e produce repellent substances to deter \u003cem\u003eConstrictotermes cavifrons\u003c/em\u003e (Cristaldo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jirošov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, many inquiline species consume food sources distinct from those of their hosts, which reduces direct competition. For example, \u003cem\u003eI. microcerus\u003c/em\u003e and \u003cem\u003eI. fur\u003c/em\u003e are thought to feed on material excreted by their hosts, a dark substance containing a high quantity of lignin relative to cellulose (Barbosa-Silva et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Constantino, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mathews, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). However, the nature of the host\u0026ndash;inquiline relationships \u0026mdash;ranging from mutualism to parasitism\u0026mdash; varies and still remains poorly understood (Cristaldo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shellman-Reeve, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). For instance, while inquilines benefit from the nest's defenses and stable microclimate, they may impose costs by consuming stored food or damaging nest structures, requiring constant repair by the host. In some other cases, the host may benefit from inquilines as they could contribute to nest maintenance by eating the refuse of the host.\u003c/p\u003e \u003cp\u003eIn this study we aimed at disentangling the relationship between \u003cem\u003eConstrictotermes cavifrons\u003c/em\u003e (Holmgren, 1910) and \u003cem\u003eInquilinitermes inquilinus\u003c/em\u003e, an obligatory inquiline (Emerson, 1925), and to place it into the parasitism\u0026ndash;mutualism continuum. \u003cem\u003eConstrictotermes cavifrons\u003c/em\u003e builds elongated arboreal nests up to 10 meters above ground. Large nests often contain \u003cem\u003eI. inquilinus\u003c/em\u003e, either as a single mature colony or in the form of multiple incipient colonies (Martius et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Timmermans et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Workers of \u003cem\u003eC. cavifrons\u003c/em\u003e use a mix of soil, feces and saliva for construction, giving the nest a light brown to reddish color (indicating high clay content). Known for processional foraging, \u003cem\u003eC. cavifrons\u003c/em\u003e creates exposed trails to forage irregularly by day or by night on nearby trees and litter, feeding on microepiphytes, and more precisely on lichens, bryophytes, algae and fungi growing on bark or leaves (Martius et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Timmermans et al., submitted). A notable feature of these nests is the presence of dark material accumulated at the bottom, which is thought to consist of either stored food or segregated excrement (Barbosa-Silva et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Mathews, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Numerous organisms live inside this matter, including many insect larvae (tineid caterpillars, acanthocerid, elaterid beetles, larvae of \u003cem\u003eSciara\u003c/em\u003e flies) (Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e). Moreover, the inquiline could use this dark material to dig its galleries inside (Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Timmermans et al., submitted). \u003cem\u003eInquilinitermes inquilinus\u003c/em\u003e appears to live totally enclosed within the host nest, as individuals have never been observed outside it, except for alates. If the two species encounter each other, hostile interactions may occur (Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Noirot, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). The nature of the food source of \u003cem\u003eI. inquilinus\u003c/em\u003e remains uncertain but they may feed on the host nest and more precisely on the dark material accumulated at the bottom of the nest (Barbosa-Silva et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Timmermans et al., submitted). The origin and specific role of this dark material remain subjects of investigation (Mathews, 1997; Rezende, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Souza Santos et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Timmermans et al., submitted).\u003c/p\u003e \u003cp\u003eThe success of inquilines within a host nest often depends on their ability to reduce direct competition and conflict with their hosts. Here we aim to analyze the spatial and dietary segregation between \u003cem\u003eC. cavifrons\u003c/em\u003e and \u003cem\u003eI. inquilinus\u003c/em\u003e to better understand the ecological and evolutionary underpinnings of their coexistence. Specifically, we seek to determine whether \u003cem\u003eI. inquilinus\u003c/em\u003e imposes costs on its host by depleting shared food resources or minimizes conflict through niche differentiation, for instance by consuming distinct dietary components. We also investigate how inquiline termites alter the structural integrity of the host nest and if there is a spatial segregation between the two species by understanding where the inquiline constructs its galleries inside the host nest.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eStudy site and nests collection\u003c/p\u003e \u003cp\u003eFive expeditions in French Guiana were realized between 2021 and 2023. For this study, 24 nests were sampled in the rainforest at four different localities: Paul Isnard road (n\u0026thinsp;=\u0026thinsp;1), Petit Saut dam area (n\u0026thinsp;=\u0026thinsp;14), Kaw mountain (n\u0026thinsp;=\u0026thinsp;5) and Sa\u0026uuml;l (n\u0026thinsp;=\u0026thinsp;4) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Collection details and composition of samples are referenced in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Each \u003cem\u003eC. cavifrons\u003c/em\u003e nest (length range: 0.61‒3.56 m) was carefully dissected in 10-cm sections, starting from the bottom to the top of the nest. Each section was measured (width and length) immediately before being isolated in a ziplock bag. Sections were carefully sorted at the base camp to extract host and inquiline (when present) individuals of each caste (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Samples were stored in 100% ethanol and conserved at -20\u0026deg;C. During the sorting process several material types were collected: galleries of \u003cem\u003eC. cavifrons\u003c/em\u003e (gcav), galleries of \u003cem\u003eI. inquilinus\u003c/em\u003e (ginq), dark material (dm: black material accumulated inside the \u003cem\u003eC. cavifrons\u003c/em\u003e galleries often at the bottom of the nest), exterior galleries (gext: galleries outside of the nest connecting the bottom nest entrance to the ground for pellet transportation), pellets (p: pellets of soil used for nest construction), topsoil layer (soil) taken below the nest and foraging material (fm: material on which \u003cem\u003eC. cavifrons\u003c/em\u003e was observed feeding in the field, such as lichen, mosses, fungi and algae growing on bark or leaves). The term galleries is here referring to all the living space of the species inside the nest, comprising chambers and galleries. Additional measurements were taken on the nest: length, maximum width, maximum circumference (starting from the merging point of the nest on the tree to the other side of the nest) and height from the ground (from lowest part of the nest to the ground level) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. cavifrons\u003c/em\u003e nests were separated in four categories: nests inhabited only by the host (n\u0026thinsp;=\u0026thinsp;9), nests inhabited by the host and a mature inquiline colony (n\u0026thinsp;=\u0026thinsp;8), nests inhabited by the host and an incipient inquiline colony (n\u0026thinsp;=\u0026thinsp;4) and nests abandoned by the host but inhabited by a mature inquiline colony (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003eNest characterization\u003c/p\u003e \u003cp\u003eWe described qualitatively and quantitatively the galleries of \u003cem\u003eI. inquilinus\u003c/em\u003e as well as their location inside the host nest. For one nest (N3), we recorded the width, length and height of galleries selected randomly in the first three 10-cm sections of the nest (0\u0026ndash;30 cm), as well as the population density of workers, soldiers, nymphs and alates in the galleries, and the possible presence of primary reproductives. For nests N1 to N5 we photographed and measured every section during the fieldwork. We used the pictures of N4 to analyze the horizontal spatial segregation by marking the center of each \u003cem\u003eI. inquilinus\u003c/em\u003e galleries on every section (0\u0026ndash;130 cm, pictures of the upper part of the sections). We then checked if more galleries were present (1) in the center of the nest rather than near the periphery, (2) closer to the tree (=\u0026thinsp;interior) rather than near the exterior wall (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). For each of these analyses, we divided the sections in similar areas of center/periphery and interior/exterior. To obtain the surface of the sections, we first outlined on the pictures the border of each section and used the software ImageJ to calculate the area (Schneider et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We scaled each section independently with its length measured during the fieldwork. The volume of each section was also obtained by multiplying the area with the section height (usually 10 cm). To obtain a \u0026ldquo;center\u0026rdquo; part that is 50% of the surface of the section, we reduced a copy of it by a factor of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sqrt{0.5}=0.707\\)\u003c/span\u003e\u003c/span\u003e and centered it on the original section (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). For the interior/exterior analysis we divided the section in two with a line (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec). Resulting surfaces of the divided sections were checked with ImageJ and the number of galleries found in each part was normalized with the surface (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also described the vertical spatial segregation between the two species and noted the differences in nest composition with the height inside the nest (N1\u0026ndash;N5). For each section of 10 cm, we approximated the percentage of dark material, inquiline galleries and host galleries as well as the friability of the nest material. We calculated the sections\u0026rsquo; volumes with the method described above. The density of \u003cem\u003eI. inquilinus\u003c/em\u003e was obtained by counting the individuals of each caste, and approximated for larvae and eggs. For \u003cem\u003eC. cavifrons\u003c/em\u003e the population size was always estimated due to the high population density of this species. We used a scale from 0 to 5: for neuters 0\u0026thinsp;=\u0026thinsp;no individual, 1\u0026thinsp;=\u0026thinsp;0 to 50, 2\u0026thinsp;=\u0026thinsp;50 to 500, 3\u0026thinsp;=\u0026thinsp;500 to 1.000, 4\u0026thinsp;=\u0026thinsp;1.000 to 2.500 and 5\u0026thinsp;=\u0026thinsp;2.500 to 5.000 individuals; for other castes 0\u0026thinsp;=\u0026thinsp;no individual, 1\u0026thinsp;=\u0026thinsp;very few, 2\u0026thinsp;=\u0026thinsp;few, 3\u0026thinsp;=\u0026thinsp;average, 4\u0026thinsp;=\u0026thinsp;high, 5\u0026thinsp;=\u0026thinsp;very high. We compared our estimation of the neuter population size with the model proposed by Vasconcellos et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) for the closely related species \u003cem\u003eC. cyphergaster\u003c/em\u003e: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Population=7113.7+\\left(1667.9\\text{*}\\text{N}\\text{e}\\text{s}\\text{t}\\:\\text{v}\\text{o}\\text{l}\\text{u}\\text{m}\\text{e}\\right)\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIsotopic and elemental composition\u003c/p\u003e \u003cp\u003eTermite tissues of \u003cem\u003eC. cavifrons\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;46 individuals and n\u0026thinsp;=\u0026thinsp;19 colonies) and \u003cem\u003eI. inquilinus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;30 individuals and n\u0026thinsp;=\u0026thinsp;11 colonies), nest material of different types, \u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003eC. cavifrons\u003c/em\u003e galleries (n\u0026thinsp;=\u0026thinsp;58 samples and n\u0026thinsp;=\u0026thinsp;21 colonies), \u003cem\u003eI. inquilinus\u003c/em\u003e galleries (n\u0026thinsp;=\u0026thinsp;15 samples and n\u0026thinsp;=\u0026thinsp;8 colonies), dark material (n\u0026thinsp;=\u0026thinsp;33 samples and n\u0026thinsp;=\u0026thinsp;18 colonies) and exterior galleries (n\u0026thinsp;=\u0026thinsp;5 samples and n\u0026thinsp;=\u0026thinsp;5 colonies), pellets (n\u0026thinsp;=\u0026thinsp;6 samples and n\u0026thinsp;=\u0026thinsp;5 colonies), topsoil layer (n\u0026thinsp;=\u0026thinsp;23 samples and n\u0026thinsp;=\u0026thinsp;23 colonies) and foraging material (n\u0026thinsp;=\u0026thinsp;5 samples and n\u0026thinsp;=\u0026thinsp;1 colonies) were subjected to carbon and nitrogen isotopic analyses (Table S3).\u003c/p\u003e \u003cp\u003eSamples of termites were stored at -20\u0026deg;C in 100% ethanol to preserve tissues for the isotopic composition analyses (Florencio et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Only the heads of termites were kept for the analysis to avoid contamination from gut content. We dissected 10 worker heads per section (with sometimes several replicates) and directly dried the tissues as well as the nest, soil, foraging material and pellet samples at 60\u0026deg;C for 24h in a stove before weighing. To prevent bias from carbonates in the final isotopic composition, we tested their absence by reaction with 10% HCl in the dried topsoil and nest samples. None of the samples presented signs of carbonate presence. Samples were weighed using a Mettler AT261 DeltaRanger (Mettler Toledo) precision balance (0.1 mg) in tin capsules. For dried tissues of termites, the samples ranged from 0.9 to 1.1 mg and for nest, soil, foraging material and pellets samples ranged from 4.5 to 5.5 mg.\u003c/p\u003e \u003cp\u003eMeasurements were performed using an elemental analyzer (Vario Microcube, Elementar Analysensysteme GMBH, Germany) coupled to an isotope ratio mass spectrometer (PrecisION, Elementar Analysensysteme GMBH, Germany). Isotopic ratios were expressed following the δ notation (\u0026permil;) (Coplen, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) based on international standards: Vienna Pee Dee Belemnite for carbon, atmospheric air for nitrogen. Glycine, and replicates were interspersed every 15 samples for elemental calibration and secondary isotopic CRM. The standard deviation for δ\u003csup\u003e15\u003c/sup\u003eN and δ\u003csup\u003e13\u003c/sup\u003eC were 0.3 and 0.1\u0026permil; for termite replicates (\u003cem\u003eI. inquilinus\u003c/em\u003e), 0.4 and 0.1\u0026permil; for nest material replicates, 0.2 and 0.1\u0026permil; for foraging galleries and 0.3 and 0.3\u0026permil; for glycine. Raw data are given in the Online Resource ESM_2.xlsx. For certified reference materials (CRM), we used sucrose (IAEA-C6; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD: δ\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026permil;) and ammonium sulphate (IAEA-N2; δ\u003csup\u003e15\u003c/sup\u003eN = 20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026permil;). Elemental data are expressed as a percentage of dry weight over weight (W:W). Average, standard deviation and confidence interval at 95% for δ\u003csup\u003e15\u003c/sup\u003eN, δ\u003csup\u003e13\u003c/sup\u003eC, N%, C% and C:N were calculated for each material type (Table S4). All the analyses were conducted in the R environment version 4.3.1 (R Core Team, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs none of the data distribution were normal, we used the non-parametric Kruskal-Wallis test (function: \u003cem\u003ekruskal.test\u003c/em\u003e) to evaluate the significance of differences between materials averages (Hollander et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A pairwise Wilcoxon test with a Benjamini and Hochberg correction for multiple testing was then used to identify significantly different pairs using the function \u003cem\u003epairwise.wilcox.test\u003c/em\u003e (Benjamini \u0026amp; Hochberg, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Hollander et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (Tables S5). One biplot of δ\u003csup\u003e15\u003c/sup\u003eN and δ\u003csup\u003e13\u003c/sup\u003eC per nest was obtained with all the material types represented (Figure S3). To compare the isotopic niche of the different material types, SIBER package in R was used (Jackson et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Bayesian standard ellipses were inferred with the function \u003cem\u003eplotSiberObject\u003c/em\u003e from the δ\u003csup\u003e15\u003c/sup\u003eN and δ\u003csup\u003e13\u003c/sup\u003eC biplot. Overlap between ellipses were obtained using the function \u003cem\u003esea.overlap.comm1\u003c/em\u003e and Layman metrics were calculated with function \u003cem\u003egroupMetricsML\u003c/em\u003e (Jackson et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The overlap between two ellipses in percent was calculated using this formula: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:100\\left.\\left(\\:\\frac{overlap}{SEAc\\:A\\:+\\:SEAc\\:B\\:-\\:overlap}\\right.\\right)\\)\u003c/span\u003e\u003c/span\u003e. We then proceeded to a correlation analysis between each group of minimum six colonies, using Python 3.12.5 to obtain Pearson coefficient and R\u003csup\u003e2\u003c/sup\u003e with the scikit-learn and SciPy package (Freedman et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Van Rossum \u0026amp; Drake, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Virtanen et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Biplots of the linear regression were also obtained with Python 3.12.5 using the Seaborn packages, and confidence intervals were calculated with a bootstrap method (Waskom, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Figure S4). Correlation between materials, nest size and height were also tested (Table S6).\u003c/p\u003e \u003cp\u003eAnother analysis was conducted on the nest sections to observe whether there is an effect of the location inside the nest on the composition of the material studied. Pearson coefficient and R\u003csup\u003e2\u003c/sup\u003e were calculated for each material type according to the nest section and between materials within a nest section (Freedman et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). A Benjamini and Hochberg correction for multiple testing was applied to avoid statistical overinterpretation (Benjamini \u0026amp; Hochberg, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) (Table S7).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eNest characterization\u003c/p\u003e \u003cp\u003eThree clearly different material types are found inside the \u003cem\u003eC. cavifrons\u003c/em\u003e nest. The first one is the host galleries, made of soil pellets collected from the ground underneath the nest and mixed with feces (Timmermans et al., submitted) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). The color is light-brown or reddish depending on the soil composition, and the material is way more friable than the other two. The galleries are all connected to each other, none being enclosed inside inquiline galleries. Another interesting point is that we found a royal chamber of \u003cem\u003eC. cavifrons\u003c/em\u003e adjacent to \u003cem\u003eI. inquilinus\u003c/em\u003e galleries (in section 80\u0026ndash;90cm on a 172cm nest length, N4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eThe second type of material is the dark mineral-organic material accumulated at the bottom of the nest, filling the galleries of the host (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This material can also be observed in the upper part of the nest and was sometimes found in smaller quantity in the uppermost section. The material is often hard to break and not really friable. Note that this material is always present inside \u003cem\u003eC. cavifrons\u003c/em\u003e nests, even in the absence of inquiline, provided they have reached a sufficient size.\u003c/p\u003e \u003cp\u003eThe horizontal location of the inquiline galleries inside the host nest (performed on 13 sections of one nest) shows no difference between the center of the nest and the periphery (respectively: 50.7% and 49.3% of the galleries) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The interior/exterior analysis gives similar results with respectively 48.4% and 51.6%. The relative abundance of the three material types is clearly influenced by the vertical position in the nest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Figure S5a). In nests without inquilines (N1, N2 and N5) the dark material is the most abundant material in the lowest part of\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ethe nest; it decreases with the height and is null above 30 to 60 cm. In the nest with inquilines (N4), \u003cem\u003eI. inquilinus\u003c/em\u003e galleries are the most common material in the lowest part of the nest, decreasing with height in opposition with \u003cem\u003eC. cavifrons\u003c/em\u003e galleries. Dark material is also less present in this nest than in nests where \u003cem\u003eI. inquilinus\u003c/em\u003e is absent (Table S9). In general, the friability of the nest increases with the proportion of \u003cem\u003eC. cavifrons\u003c/em\u003e galleries. Globally, the proportion of \u003cem\u003eC. cavifrons\u003c/em\u003e galleries is always higher than dark material (respectively; N1: 96.88% and 3.13%, N2: 85.30% and 14.70%, N4: 69.76% and 6.12% and N5: 83.43% and 16.57%). In the presence of inquilines, the proportion of inquiline galleries (24.12%) is higher than dark material (6.12%) (Table S9).\u003c/p\u003e \u003cp\u003e Density curves of the neuter population of \u003cem\u003eC. cavifrons\u003c/em\u003e show an almost complete absence in the lowest section of the nest, as in nest N5 were no individual was seen in the lowest 40 cm of the nest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and S5b). The peak of density is around the two-thirds of the nest, above which the density decreases, being low at the highest section of the nest. The queen and/or king were often found near the peak of density of neuters. The density curve of \u003cem\u003eI. inquilinus\u003c/em\u003e in the N4 nest indicates a pattern opposite to \u003cem\u003eC. cavifrons\u003c/em\u003e, with the highest density in the lowest section, decreasing slowly until the first third of the nest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The queen and king of \u003cem\u003eI. inquilinus\u003c/em\u003e were found higher in the nest than the peak of highest neuter density (king: 30\u0026ndash;40 cm, queen: 40\u0026ndash;50 cm). The \u003cem\u003eC. cavifrons\u003c/em\u003e queen was found in the 80\u0026ndash;90 cm section, where the inquiline was still present (until section 130\u0026ndash;140 cm).\u003c/p\u003e \u003cp\u003eAnalysis of the caste distribution of \u003cem\u003eI. inquilinus\u003c/em\u003e inside nest N4 shows the highest density of neuters in the lowest sections of the nest, reproductive individuals and juveniles being almost totally absent (Figure S6a). Neuter density decreases with nest height while reproductives increase up to 40\u0026ndash;50 cm, corresponding to the presence of the queen. After the peak, all castes decrease slowly and are absent above section 130\u0026ndash;140 cm. In \u003cem\u003eC. cavifrons\u003c/em\u003e the density of each caste is quite similar: they increase slowly until the highest peak near the middle of the nest (where the queen is found) and then decrease (Figure S6b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIsotopic and elemental analyses\u003c/p\u003e \u003cp\u003e \u003cem\u003eConstrictotermes cavifrons\u003c/em\u003e tissues have a mean values of 2.0\u0026permil; for δ\u003csup\u003e15\u003c/sup\u003eN (95% CI 1.7\u0026permil; to 2.3\u0026permil;, n\u0026thinsp;=\u0026thinsp;19), and \u0026minus;\u0026thinsp;29.5\u0026permil; for δ\u003csup\u003e13\u003c/sup\u003eC (95% CI -29.8\u0026permil; to -29.3\u0026permil;, n\u0026thinsp;=\u0026thinsp;19). Tissues of \u003cem\u003eI. inquilinus\u003c/em\u003e show mean values of 5.5\u0026permil; (95% CI 4.5\u0026permil; to 6.5\u0026permil;, n\u0026thinsp;=\u0026thinsp;11) for δ\u003csup\u003e15\u003c/sup\u003eN and \u0026minus;\u0026thinsp;28.5\u0026permil; (95% CI -28.8\u0026permil; to -28.2\u0026permil;, n\u0026thinsp;=\u0026thinsp;11) for δ\u003csup\u003e13\u003c/sup\u003eC (Table S4). Wilcoxon tests revealed a significant difference between the two species in the isotopic composition of carbon and nitrogen and in the C:N ratio (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table S5). There is no overlap between the two isotopic niches and the difference between standard ellipse areas (SEAc) is significant (\u003cem\u003eC. cavifrons\u003c/em\u003e: 1.06\u0026permil;\u0026sup2; and \u003cem\u003eI. inquilinus\u003c/em\u003e: 2.66\u0026permil;\u0026sup2;, p-value\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Table S10). For nest materials we compared the galleries of \u003cem\u003eC. cavifrons\u003c/em\u003e (mean δ\u003csup\u003e15\u003c/sup\u003eN: 4.6\u0026permil; and δ\u003csup\u003e13\u003c/sup\u003eC: -30.2\u0026permil;), the galleries of \u003cem\u003eI. inquilinus\u003c/em\u003e (mean δ\u003csup\u003e15\u003c/sup\u003eN: 4.9\u0026permil; and δ\u003csup\u003e13\u003c/sup\u003eC: -30.9\u0026permil;), the dark material (mean δ\u003csup\u003e15\u003c/sup\u003eN: 4.5\u0026permil; and δ\u003csup\u003e13\u003c/sup\u003eC: -31.2\u0026permil;) and the soil (mean δ\u003csup\u003e15\u003c/sup\u003eN: 5.1\u0026permil; and δ\u003csup\u003e13\u003c/sup\u003eC: -29.2\u0026permil;). Wilcoxon tests show no significant difference between these materials for nitrogen isotopes, but a clear significant difference for carbon isotopes except between the dark material and \u003cem\u003eI. inquilinus\u003c/em\u003e galleries. For the isotopic niches, we observe 9.7% of overlap between the galleries of the host and the inquiline, 6.4% between \u003cem\u003eC. cavifrons\u003c/em\u003e galleries and the dark material, 12.8% between the soil and the host galleries, 39.7% between \u003cem\u003eI. inquilinus\u003c/em\u003e galleries and dark material, and no overlap between other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA significant correlation between host and inquiline tissues is observed in the isotopic composition of nitrogen (R\u003csup\u003e2\u003c/sup\u003e: 0.898, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8) but not of carbon (R\u003csup\u003e2\u003c/sup\u003e: 0.450, p-value: 0.069, n\u0026thinsp;=\u0026thinsp;8) (Fig.\u0026nbsp;4). Correlations between inquiline galleries, dark material and inquiline tissues are significant for both isotopic ratios. For nitrogen there is a significant correlation between soil and dark material, soil and host galleries, host tissues and dark material as well as host tissues and inquiline galleries. For carbon isotopic ratios, only \u003cem\u003eC. cavifrons\u003c/em\u003e and dark material are significantly correlated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4\u003c/b\u003e Correlation matrix between each material type (n\u0026thinsp;\u0026ge;\u0026thinsp;6) for carbon and nitrogen isotope. R\u003csup\u003e2\u003c/sup\u003e are indicated for each correlation with p-value and sample size. Graphs are generated with Python 3.12.5.\u003c/p\u003e \u003cp\u003eIf we compare the different colony types (host alone, presence of a mature \u003cem\u003eI. inquilinus\u003c/em\u003e colony and presence of an incipient \u003cem\u003eI. inquilinus\u003c/em\u003e colony), we observe differences in the isotopic composition of \u003cem\u003eC. cavifrons\u003c/em\u003e tissues between colonies where \u003cem\u003eI. inquilinus\u003c/em\u003e is present versus absent (Figure S7a). These colony types also differ by the mean size of the nest (respectively: 1.12 m, 2.05 m and 1.96 m). The SIBER plot of the \u003cem\u003eC. cavifrons\u003c/em\u003e nests arranged by size classes also shows a relationship with the isotopic composition, without overlap between small and large nests (Figure S7b). Standard ellipse areas (SEAc) are larger for small nests than for middle and large nests (respectively: 1.48, 0.92 and 0.54\u0026permil;\u0026sup2;). However, no significant difference between these groups is observed and correlations of isotopes between \u003cem\u003eC. cavifrons\u003c/em\u003e tissues and nest size were not significant. There is only a significant correlation between nest size and \u003cem\u003eC. cavifrons\u003c/em\u003e galleries for δ\u003csup\u003e15\u003c/sup\u003eN (R\u003csup\u003e2\u003c/sup\u003e: 0.36, p-value: 0.01), and between nest ground height and \u003cem\u003eC. cavifrons\u003c/em\u003e tissues for δ\u003csup\u003e13\u003c/sup\u003eC (R\u003csup\u003e2\u003c/sup\u003e: 0.22, p-value: 0.04, data in Table S6). The nest size and the ground height are also significantly correlated (Spearman ρ: 0.47, p-value: 0.001, shown in Figure S7c).\u003c/p\u003e \u003cp\u003eWe observe a relation between the proportion of carbon and nitrogen throughout a gradient of the different material types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Pellets have the lowest C and N content, followed by soil and exterior galleries, host galleries, inquiline galleries, dark material and foraging material. Dark material shows a high heterogeneity in the global composition (C% mean: 28.33, σ: 11.47 and N% mean: 1.29, σ: 0.50) and has significantly higher nitrogen and carbon proportions than pellets, soil, exterior galleries and \u003cem\u003eC. cavifrons\u003c/em\u003e galleries (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.002). For \u003cem\u003eI. inquilinus\u003c/em\u003e galleries the proportion of carbon is significantly smaller than in dark material (p-value: 0.007) but the proportion of nitrogen is not clearly different (p-value: 0.057). Galleries of the inquiline show a higher C% and N% than host galleries (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.002). Foraging material has a greater proportion of carbon and nitrogen than every other material, only its proportion of nitrogen is not clearly different from that of dark material (p-value: 0.07).\u003c/p\u003e \u003cp\u003eAnalysis by nest interval shows no significant correlation between isotopic ratios of materials from the same interval. Only two correlations between isotopic ratios and height inside nest reach p-values below 0.05: \u003cem\u003eC. cavifrons\u003c/em\u003e galleries for δ\u003csup\u003e13\u003c/sup\u003eC (\u0026permil;) and \u003cem\u003eI. inquilinus\u003c/em\u003e tissues for δ\u003csup\u003e15\u003c/sup\u003eN (\u0026permil;) in nest N4 (p-value: 0.04) (Table S7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSegregation in resource use is recognized as a major factor enabling species to coexist, with evidence across plant (Silvertown, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), invertebrates (Sarty et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) or vertebrates (Lejeune et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mason et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In termites, it is known that diet but also habitat influences species coexistence (Bourguignon et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Korb \u0026amp; Linsenmair, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, niche segregation patterns remain largely unexplored in assemblages confined by physical barriers, as when species are sharing the same nest. In termite-termite symbiosis such as inquilinism, resource partitioning inside the nest through spatial and diet segregation appears to be essential to understand their cohabitation and potential negative or positive effect of this particular symbiosis.\u003c/p\u003e \u003cp\u003eSpatial niche segregation\u003c/p\u003e \u003cp\u003eWe observe no horizontal spatial segregation in the nest between \u003cem\u003eC. cavifrons\u003c/em\u003e and \u003cem\u003eI. inquilinus\u003c/em\u003e. The inquiline shows no preference for the central versus peripheral zone or the interior versus exterior zone. Interestingly, in \u003cem\u003eC. cyphergaster\u003c/em\u003e nests, \u003cem\u003eI. fur\u003c/em\u003e and \u003cem\u003eI. microcerus\u003c/em\u003e tend to be restricted to the central region where the dark organic matter is concentrated (Cunha et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A hypothesis to explain this difference between related species is that the dark material does not display any specific horizontal distribution pattern in \u003cem\u003eC. cavifrons\u003c/em\u003e nests.\u003c/p\u003e \u003cp\u003eAs to the vertical spatial segregation, the inquiline is restricted to the zone rich in dark material, in which it seems to build its galleries. This dark organic-rich material is located principally at the bottom of the nest and decreases with nest height until approximately the bottom one-third of the nest. However, some nests contain dark material in upper zones, such as nests N4 (80\u0026ndash;90 cm on a 172 cm nest) and N5, where we observed it in the top section of the nest (130\u0026ndash;145 cm). In the presence of the inquiline, the proportion of dark material is lower in the lowest section of the nest than in its absence. The proportion of inquiline galleries is way higher than the dark material in this section (respectively 0\u0026ndash;10 cm: 85% and 5%). It suggests that when the inquiline is present, the dark organic material is replaced by inquiline galleries. In summary, as a general observation, the presence of dark material, probably produced by host species, highly determines the location of inquiline species.\u003c/p\u003e \u003cp\u003eWe found individuals of \u003cem\u003eI. inquilinus\u003c/em\u003e and their galleries until section 120\u0026ndash;130 cm on a 172 cm nest. This is way higher than the one-third of the nest occupancy expected for termite inquiline species (Cruz et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, we found inquiline galleries totally enclosed inside host galleries and some of them adjacent to the host royal chamber. One hypothesis to explain this observation is that the inquiline species only inhabits zone of dark material and never ventures into host galleries. Additionally, we never found the host in zones of dark material. The two species thus coexist closely using different spaces in the nest. To ensure this segregation, is it possible that the inquiline uses chemical cues. It is known that \u003cem\u003eI. inquilinus\u003c/em\u003e soldiers repel \u003cem\u003eC. cavifrons\u003c/em\u003e by producing chemical substances (Jirošov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and the feces of the related species \u003cem\u003eI. microcerus\u003c/em\u003e seem to be repellent to its host \u003cem\u003eC. cyphergaster\u003c/em\u003e (Hugo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, according to Cristaldo et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), I. \u003cem\u003emicrocerus\u003c/em\u003e can recognize the trail and alarm signals of its host and avoid them. Therefore, the cost imposed on the host by the inquiline on the spatial niche is surely minimized by the population size (probably five-fold lower than the host\u0026rsquo;s) and the difference in occupancy of the nest (i.e. dark organic material for inquiline versus host galleries for \u003cem\u003eC. cavifrons\u003c/em\u003e). In this termite-termite close relationship, the habitat niche of involved species are actually segregated at the microhabitat scale, despite the fact that the two species share the same nest.\u003c/p\u003e \u003cp\u003eTrophic niche segregation\u003c/p\u003e \u003cp\u003eThere is no overlap between the isotopic niches of \u003cem\u003eI. inquilinus\u003c/em\u003e and \u003cem\u003eC. cavifrons\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The isotopic niche being used as a proxy of trophic niche, it suggests a clear trophic segregation through the utilization of distinct food sources. Such results are consistent with other studies realized on inquiline-host relationships in termites (Florencio et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hellemans et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The standard ellipse aeras (SEAc) are significantly higher for \u003cem\u003eI. inquilinus\u003c/em\u003e than for \u003cem\u003eC. cavifrons\u003c/em\u003e (\u003cem\u003eC. cavifrons\u003c/em\u003e: 1.06 vs. \u003cem\u003eI. inquilinus\u003c/em\u003e: 2.66\u0026permil;\u0026sup2;, p\u0026thinsp;=\u0026thinsp;0.008). These findings indicate a higher isotopic heterogeneity in the diet of the inquiline species. The correlation between the two species is significant for δ\u0026sup1;⁵N (R\u0026sup2; = 0.898, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). It appears less conclusive for δ\u0026sup1;\u0026sup3;C (R\u0026sup2; = 0.450, p\u0026thinsp;=\u0026thinsp;0.069), but this ratio shows very low intercolonial variations. This suggests a clear connection between the two species, with at least one species being dependent on the other. It is therefore likely that the inquiline feeds, at least partially, on material excreted by the host.\u003c/p\u003e \u003cp\u003eThe organic-rich dark material is highly variable in terms of elemental and isotopic composition, and is probably produced by the host species. However, the presence of high amounts of mineral crystals inside the dark material, while such crystals are very scarce in the gut of the microepiphyte-feeding \u003cem\u003eC. cavifrons\u003c/em\u003e (Timmermans et al., submitted), shows that this matter is not solely composed of segregated excrement. One possible explanation for the presence of mineral crystals is that the host incorporates soil pellets into its feces when depositing them at the bottom of the nest, resulting in the formation of this dark, mineral-organic material. In this study, we aimed to resolve the true nature of the dark organic material by examining the two possible explanations presented in the literature: segregated excrement or stored food (Barbosa-Silva et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Mathews, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). The isotopic composition of the dark material does not align with sampled food sources of host, which refutes the \u0026ldquo;food storage\u0026rdquo; hypothesis. More likely, dark material is reprocessed material (i.e. host feces, undigested food remnants), undergoing significant isotopic and elemental modifications compared to host food sources.\u003c/p\u003e \u003cp\u003eDark material and \u003cem\u003eI. inquilinus\u003c/em\u003e galleries constitute the only pair of materials that are not significantly different in carbon isotope ratios. This is supporting the idea that \u003cem\u003eI. inquilinus\u003c/em\u003e digs its galleries out of the dark organic mass accumulated at the bottom of the host nest (Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Mathews, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). The high correlation across nests between dark material and \u003cem\u003eI. inquilinus\u003c/em\u003e galleries further support this hypothesis (correlation for δ\u003csup\u003e15\u003c/sup\u003eN (\u0026permil;): 0.865 and δ\u003csup\u003e13\u003c/sup\u003eC (\u0026permil;): 0.935, p-value\u0026thinsp;=\u0026thinsp;0). In contrast, if \u003cem\u003eI. inquilinus\u003c/em\u003e built its galleries within the host galleries, the isotopic composition of both materials would be more similar. Furthermore, the significant differences in carbon and nitrogen percentages between these two materials highlight a clear distinction in the composition of the structures built by the inquiline or by the host.\u003c/p\u003e \u003cp\u003eAs found in a previous study (Bourguignon et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the δ\u003csup\u003e15\u003c/sup\u003eN values of the inquiline is significantly higher than the host\u0026rsquo;s. \u003cem\u003eI. inquilinus\u003c/em\u003e is thus feeding on material more reprocessed than its host, which agrees with the confinement of the inquiline inside the host nest. It is assumed that \u003cem\u003eInquilinitermes\u003c/em\u003e species feed on matter excreted by their host, allegedly the dark organic-rich substance accumulated inside the host galleries (Barbosa-Silva et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Emerson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Mathews, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Here, the strong correlations observed for δ\u003csup\u003e15\u003c/sup\u003eN and δ\u003csup\u003e13\u003c/sup\u003eC values between \u003cem\u003eI. inquilinus\u003c/em\u003e tissues, galleries, and dark matter (Fig.\u0026nbsp;4) provide further evidence for this hypothesis, with the restriction that the dark matter is not solely composed of host feces. However, Cruz et al., (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed that the inquiline species \u003cem\u003eI. fur\u003c/em\u003e and \u003cem\u003eI. microcerus\u003c/em\u003e can live in absence of dark material in nests of \u003cem\u003eC. cyphergaster\u003c/em\u003e. This means that some inquiline species can have different trophic habits.\u003c/p\u003e \u003cp\u003eWe also examined the isotopic ratios among the different types of \u003cem\u003eC. cavifrons\u003c/em\u003e colonies: nests inhabited by the host only, nests inhabited by both the host and a mature inquiline colony, and nests inhabited by the host and an incipient inquiline colony. The analysis of the isotopic niches reveals no overlap between the isotopic composition of colonies inhabited by the host only and by the dyad (Figure S7a). As it is known that \u003cem\u003eC. cavifrons\u003c/em\u003e nests are colonized by \u003cem\u003eI. inquilinus\u003c/em\u003e only if they are sufficiently large (Cristaldo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), a possible explanation for the isotopic difference between host-only and host inquiline colonies is that young \u003cem\u003eC. cavifrons\u003c/em\u003e colonies (small nests) do not feed on the same resources as older colonies (large nests). This hypothesis is supported by the observation that smaller nests had higher δ\u003csup\u003e15\u003c/sup\u003eN and lower δ\u003csup\u003e13\u003c/sup\u003eC than larger nests, independently of their colonization status (Figure S7b). Moreover, smaller nests are typically found closer to the ground as shown by the correlation between nest size and ground height we found in this study. Additionally, foragers from small nests have been observed feeding on the forest floor litter, while those from larger nests seem to feed higher up in the trees, particularly on tree bark. This difference in foraging behavior depending on the host colony age could explain the distinct trophic niches observed for these nests.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFor species being partitioned inside the same termitarium with physical barriers, it is important to have different ecological niches to avoid competition and potential threats (i.e. aggressive behavior). Here we show that the inquiline permanence within the nest seems to be related to spatial and trophic segregation between the cohabiting species. Spatially, the inquiline occupies a zone where dark organic-rich material is concentrated, mostly at the bottom of the nest, while the host inhabits the light-colored friable galleries they build out of clay pellets. Based on isotopic analyses, we also observe a clear dietary segregation between the two species. This mechanism likely benefits the inquiline by reducing competition with its host. Since the two species make different uses of the food and spatial niches, it seems logical to consider their relationship as a commensalism, in which the inquiline species imposes no cost on its host. However, we cannot totally exclude that \u003cem\u003eI. inquilinus\u003c/em\u003e may feed directly on the host galleries and thus impose some cost of maintenance and construction on the host.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eJohanne Timmermans and Yves Roisin designed the study. Funding was secured by Johanne Timmermans and Yves Roisin. Collection of the samples was assured by Johanne Timmermans, Nicolas Fontaine and Yves Roisin. Laboratory work was performed by Johanne Timmermans and Gilles Lepoint. Johanne Timmermans conducted the analyses with help from Gilles Lepoint. Johanne Timmermans wrote the first draft of the manuscript. All authors contributed to the final version and approved the submitted manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe acknowledge support from the Fonds de la Recherche Scientifique\u0026mdash;FNRS and FRIA (Formation \u0026agrave; la Recherche dans l\u0026apos;Industrie et dans l\u0026apos;Agriculture).\u0026nbsp;G.L. is Senior Researcher at FRS-FNRS. We also thank Loane Wu, Paule Vanessa Fopa Diffo, Matsvei Tsishyn, Esm\u0026eacute;ralda Rodriguez Palacio, Sarah Gravier et Maxime Lenaerts for their help in the field collecting samples and sorting the nests.\u003c/p\u003e\n\u003cp\u003eFunding information\u003c/p\u003e\n\u003cp\u003eFunding was provided by the Fonds de la Recherche Scientifique\u0026mdash;FNRS (JT, YR), through Grant CDR J.0180.20 (to YR), and FRIA (Fonds pour la Formation \u0026agrave; la Recherche dans l\u0026apos;Industrie et l\u0026apos;Agriculture) PhD fellowship (to JT).\u003c/p\u003e\n\u003cp\u003eConflict of interest statement\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eFinancial interest statement\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no financial interests.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data are accessible in the Online Resource ESM_1.pdf and Online Resource ESM_2.xlsx.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbe, T. 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(2021). seaborn: Statistical data visualization. \u003cem\u003eJournal of Open Source Software\u003c/em\u003e, 6(60), 3021. https://doi.org/10.21105/joss.03021\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Termitidae, Constrictotermes, Inquilinitermes, behavioral ecology, inquilinism, stable isotopes","lastPublishedDoi":"10.21203/rs.3.rs-6323213/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6323213/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResource differentiation and segregation is widely recognized as a key factor enabling species coexistence. However, patterns of niche segregation remain poorly understood in faunal assemblages confined by physical barriers, such as species cohabiting in the same nest. In termite-termite symbiosis where a species (inquiline) is hosted in the nest built by another species (host), resource partitioning within the nest appears critical for species coexistence. Here we aim at disentangling the habitat and trophic niche segregation between \u003cem\u003eConstrictotermes cavifrons\u003c/em\u003e and its inquiline, \u003cem\u003eInquilinitermes inquilinus.\u003c/em\u003e We assess how spatial segregation contributes to reducing competition by analyzing where the inquiline constructs its galleries within the host nest. Using an isotopic niche approach, we also examine whether \u003cem\u003eI. inquilinus\u003c/em\u003e imposes costs on its host by depleting shared food resources or mitigates conflict through niche differentiation, by exploiting distinct dietary resources. Our findings suggest that the inquiline's persistence within the nest is linked to spatial segregation, with the inquiline occupying zones rich in dark organic material, while the host inhabits clay-rich, friable galleries constructed by itself. Isotopic analyses further revealed dietary segregation between the two species, likely reducing competition and facilitating coexistence. The actual food used by the inquiline is most probably the dark mineral-organic material found in the bottom of the host nest. These observations support a commensal symbiosis, wherein the inquiline imposes no significant cost upon the host.\u003c/p\u003e","manuscriptTitle":"Living Together but Apart: Spatial and trophic niche segregation of two termite species sharing the same nest","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 10:36:57","doi":"10.21203/rs.3.rs-6323213/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revisions Needed","date":"2025-05-19T12:15:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-06T01:32:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T08:52:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T12:47:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2025-03-28T06:27:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8f0e842-965a-4401-a1bd-c88a21eadf83","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T16:00:18+00:00","versionOfRecord":{"articleIdentity":"rs-6323213","link":"https://doi.org/10.1007/s00040-025-01061-x","journal":{"identity":"insectes-sociaux","isVorOnly":false,"title":"Insectes Sociaux"},"publishedOn":"2025-09-09 15:57:01","publishedOnDateReadable":"September 9th, 2025"},"versionCreatedAt":"2025-04-24 10:36:57","video":"","vorDoi":"10.1007/s00040-025-01061-x","vorDoiUrl":"https://doi.org/10.1007/s00040-025-01061-x","workflowStages":[]},"version":"v1","identity":"rs-6323213","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6323213","identity":"rs-6323213","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}