The ecological structure of communities is determined by a combination of abiotic and biotic factors (Polis, 1994; Stokstad, 2009). Complex relationships between consumers and resources affect food web dynamics both “top-down” and “bottom-up” (Polis and Strong, 1996; Leibold et al., 1997). Interspecific interactions between and within trophic levels structure ecological communities (Diamond, 1975) either directly between species (Lawton and Hassell, 1981) or indirectly (Strauss, 1991) if pairwise interactions are influenced by a third species (Bonsall and Hassell, 1997). According to the competitive exclusion principle, a pair of species with identical ecological demands cannot coexist (Hardin, 1960). Even if there is no universal limit to the similarity of competing species (Abrams, 1983), congeneric species should be engaged in intense competition (Sfenthourakis et al., 2006) and their coexistence in taxonomic assemblages (sensu Pianka, 1973) requires explanation.

Several mechanisms can stabilize the coexistence of closely related species with similar ecological demands (Chesson, 2000b), with the following being most relevant to our study. First, heterogeneity of the environment and differential responses of species to this variability facilitates coexistence (Levin, 1992). Competitive superiority of a species in certain habitat types may lead to local exclusion of inferior competitors, but coexistence can still be favored on a regional scale if spatio-temporal heterogeneity provides the outcompeted species with refuges (Amarasekare, 2003; López-Gómez and Molina-Meyer, 2006). Second, indirect interactions in species assemblages may weaken the intensity of direct interactions such as competition, and thus stabilize coexistence (Glasser, 1979; Holt, 1984; Ashehoug and Callaway, 2015).

Predation-risk may influence the decision of animals to avoid high-risk areas, which may have benefits for other community members (Willems and Hill, 2009; Bidner, 2014). If a predator favors the competitively superior species, either via specific preference or density dependence (Holt and Lawton, 1994; Chesson, 2000b), it reduces the relative abundance of the preferred prey (Sundell et al., 2003). This may indirectly benefit the competitively inferior prey species and prevent interspecific exclusion in assemblages of resource-limited consumers (Paine, 1966; Caswell, 1978; McPeek, 2014). Such changes of the competitive landscape (“keystone predation”: Paine, 1969) can have strong consequences for species richness (Walsh, 2013; Bidner, 2014). Shared natural enemies may lead to competition among prey for enemy-free space, generating spatial patterns resembling those of direct competition (“apparent competition”: Holt, 1977; Bonsall and Holt, 2003). Two species with dietary niche overlap and shared predation risk may outcompete each other in different tasks and spatially segregate into most productive habitats and predator-free space (Chesson, 2000a,b). Finally, if predator and prey belong to the same trophic guild, they may simultaneously affect each other by both direct competition and predation (“intraguild predation”: Polis et al., 1989; Polis and Holt, 1992; Holt and Polis, 1997).

Global patterns of species co-occurrence suggest that primate communities are not randomly structured (Bourlière, 1985; Kamilar, 2009). Primary productivity and interspecific competition have been the major foci in primate community ecology (Schreier et al., 2009; Kamilar and Ledogar, 2011), whereas the effects of predation remain largely neglected (Bidner, 2014). Positive interactions among other terrestrial vertebrates have long been recognized (Terborgh, 1990; Dickman, 1992; Stensland et al., 2003) but remain underacknowledged as determinants of species co-occurrence (Stachowicz, 2001; Bruno et al., 2003; Denis et al., 2019). Studies on costs and benefits of mixed-species associations in primates are numerous, but they largely focused on large-bodied, diurnal, and gregarious haplorrhines (e.g., Heymann and Buchanan-Smith, 2000; Buzzard, 2010; Singh et al., 2011). Thus, additional studies from different primate lineages, in particular strepsirrhines, are needed to test the above-mentioned predictions about community composition and stability.

Lemurs make up about 20% of the global primate species richness (Estrada et al., 2017) and represent more than 60% of mammalian genera in Madagascar (Ganzhorn, 1999). Interspecific competition in lemurs is therefore likely strong (Kamilar and Guidi, 2010; Beaudrot and Marshall, 2011). The non-random checkerboard distribution of frugivore-insectivore (omnivore) lemur species in western Madagascar indeed suggests high levels of interspecific competition for food (Kamilar and Ledogar, 2011). However, based on differences in lemur body size and dietary niches, Schreier et al. (2009) challenged the assumption of a high number of competing species pairs in Madagascar and called for studies comparing simulated distribution patterns with observed community structure to highlight species competing within assemblages. A majority of studies that have investigated habitat utilization and feeding habits of co-occurring lemur species to date were conducted on single study sites (e.g., Rendigs et al., 2003; Schwab and Ganzhorn, 2004; Dammhahn and Kappeler, 2008a,b, 2014; Rakotondravony and Radespiel, 2009; Rakotondranary and Ganzhorn, 2012). We therefore aim at examining the interspecific spatial distribution in a community of nocturnal lemurs throughout the entire area of coexistence.

A taxonomic assemblage of four closely related lemur species of the cheirogaleid family (Lemuriformes: Cheirogaleidae) affords opportunity to study spatial consequences of interspecific interactions: the mouse lemurs Microcebus berthae and Microcebus murinus, the closely related giant mouse lemur Mirza coquereli and dwarf lemur Cheirogaleus medius are nocturnal, arboreal, and relatively small primates endemic to Madagascar (average adult masses of 31 g in M. berthae and 60 g in M. murinus: Schmid and Kappeler, 1994; Rasoloarison et al., 2000, 120 g in C. medius: Fietz, 2003, and 250 g in M. coquereli: Kappeler, 2003). These cheirogaleid species occur sympatrically in the dry forest of the central Menabe region in western Madagascar that is subject to both, spatial and temporal heterogeneities.

The restricted biogeographic range of M. berthae is confined to Menabe Central (Rasoloarison et al., 2000; Schwab and Ganzhorn, 2004), and its low disturbance tolerance indicate ecological specialization (Schäffler, 2012; Schäffler and Kappeler, 2014a). Its sympatric congener M. murinus, in contrast, is found throughout western and southern Madagascar and regionally co-occurs with several other mouse lemur species (Weisrock et al., 2010). The wide biogeographic distribution of M. murinus was explained by its high competitive potential, seasonal plasticity in feeding habits, efficient energy-saving strategies, and behavioral flexibility in habitat selection (Radespiel, 2016). In western Madagascar, M. murinus inhabits degraded forest parts and even village environments (Ganzhorn, 1987; Dammhahn and Kappeler, 2008a; Schäffler, 2012; Schäffler et al., 2015). Mouse lemurs are omnivorous (or more specifically fauni-frugivorous: Steffens and Lehman, 2016) but the feeding niche of M. berthae is narrower than that of M. murinus (Dammhahn and Kappeler, 2008a, 2010). To cope with seasonal fluctuations in food supply (Schwab and Ganzhorn, 2004) mouse lemurs rely on sugary secretions of homopteran larvae (genus Flatidia) as a key fallback food (Dammhahn and Kappeler, 2008a). Increased home range sizes of females in areas of co-occurrence indicated interspecific competition on a local scale (Dammhahn and Kappeler, 2009) and negative interspecific association patterns of populations were also observed on a regional scale (Schäffler et al., 2015).

The mouse lemurs are likely also engaged in interspecific interactions with the other two sympatric cheirogaleids (Hladik et al., 1980; Lahann, 2008). Stable isotope analyses of the cheirogaleid species in Menabe Central suggested that interspecific competition determined ecological structure in this species assemblage (Dammhahn and Kappeler, 2014). In Menabe Central, M. coquereli occurs in relatively low population densities and is heterogeneously distributed whereas C. medius was found in higher numbers in intact habitat than in degraded forest parts (Schäffler and Kappeler, 2014b). Opportunistic (intraguild) predation by M. coquereli was observed on M. murinus (Rakotonirainy, 2003; Schliehe-Diecks et al., 2010) but remains un-reported on M. berthae. Corresponding negative associations of M. coquereli and M. murinus and spatial overlaps between M. coquereli and M. berthae were documented at the population level (Schäffler et al., 2015). While C. medius hibernates throughout the dry season (Dausmann et al., 2004), M. murinus females enter daily and/or prolonged torpor to save energy (Schmid, 1998, 2000, 2001). Microcebus murinus and C. medius compete for tree holes used for resting and breeding (Ganzhorn and Schmid, 1998; Kappeler and Rasoloarison, 2003) and there is evidence for feeding niche overlap in south-eastern Madagascar (Lahann, 2007). In contrast, M. berthae is active throughout the dry season (Schmid and Kappeler, 2005) and rests in open vegetation (Dammhahn and Kappeler, 2005). In a small-scale study, C. medius was found to partially displace M. murinus, whereas positive spatial associations with M. berthae indicated relaxed competition (Schwab and Ganzhorn, 2004).

Ecological patterns emerge from biological mechanisms that operate at different scales: the structure of communities may be imposed by large scale constraints but decisions to spatially avoid or associate with a co-occurring species are made by individuals responding to variations in intra- and interspecific densities (Levin, 1992). In lemurs, competitor and predator recognition is based on acoustic, visual, and olfactory cues: far-reaching acoustic signals and long-lasting odors enable recognition of other individuals over greater distances and longer timespans than visual cues, and olfactory signals are of particular importance to nocturnal arboreal lemurs under poor visibility (Klopfer, 1977). Thus, mouse lemur communication by acoustic and olfactory signals (Braune et al., 2005) should facilitate interspecific recognition. They can also be assumed to recognize other cheirogaleids not only in their immediate vicinity: M. coquereli is easily detectable by a variety of vocalizations, and olfactory signals likely facilitating its recognition as a potential predator (Kappeler, 2003). Microcebus spp. were shown to respond to olfactory cues of mammalian predators, presumably based on metabolites of meat digestion (Deppe et al., 2007; Sündermann et al., 2008; Kappel et al., 2011; Deppe and Kushnick, 2020). Cheirogaleus medius can be recognized by mouse lemurs by its frequent olfactory markings of territorial borders (Fietz, 1999b).

In this study, we aimed at further elucidating determinants of ecological structure in this cheirogaleid species assemblage by linking patterns observed at the population level (Schäffler et al., 2015) to the distribution of individuals. The hypothesized coexistence-stabilizing mechanism operates on the local scale (Huston, 1999), so the interspecific distribution of individuals should reflect competitive or predator-prey interactions. We investigated the distribution of cheirogaleid individuals across spatio-temporal heterogeneities to scrutinize if and under which constraints co-occurring dwarf and giant mouse lemurs may stabilize competitive mouse lemur coexistence. Against this background, we examine the following questions and hypotheses:

1. Do interspecific spatial distributions of individuals reflect competition between the two mouse lemur species? If there is intense competition between M. murinus and M. berthae, as indicated by previous small-scale studies (Schwab and Ganzhorn, 2004; Dammhahn and Kappeler, 2008a,b, 2009, 2010), we expect to observe strong spatial exclusion between these two species (H1a). Interspecific distribution patterns of individuals do not allow for conclusions about the direction of spatial displacement, but evidence from different regions in Madagascar suggests that M. murinus has a higher competitive potential than its sympatric sister species (Dammhahn and Kappeler, 2008b; Rakotondranary et al., 2011; Thorén et al., 2011a). If competition is for food, interspecific avoidance should be most pronounced under resource scarcity, i.e., during the dry season (H1b) and in degraded habitat (H1c).

2. Does intraguild predation by M. coquereli on M. murinus stabilize competitive coexistence between the two mouse lemur species? If (intraguild) predation creates refuges for M. berthae from competition with its congener as interpreted based on the interspecific distribution of cheirogaleid populations observed in non-degraded habitat during the dry season (Schäffler et al., 2015), spatial avoidance of M. coquereli by M. murinus should be reflected in the distribution of individuals. As giant mouse lemur predation on mouse lemurs has only been reported on M. murinus (Schliehe-Diecks et al., 2010), we expect strong spatial exclusion between this species and M. coquereli (H2a), but not between M. berthae and M. coquereli (H1b). Negative spatial association of M. coquereli and M. murinus individuals should be particularly pronounced during the dry season when resource scarcity ought to favor opportunistic predation (H2c).

3. Does C. medius stabilize competitive mouse lemur coexistence by spatial exclusion of M. murinus? Partial displacement of M. murinus, but not M. berthae, suggested that M. berthae would develop large subpopulations where C. medius reduces habitat suitability for M. murinus (Schwab and Ganzhorn, 2004). This hypothesis was not supported by the distribution of mouse lemur populations in relation to the occurrence of C. medius (Schäffler et al., 2015) but may still be reflected by the distribution individuals across spatio-temporal heterogeneities. Therefore, we predict a negative association between C. medius and M. murinus (H3a) but not between C. medius and M. berthae (H3b). Spatial consequences of competition between M. murinus and C. medius should be strongest when resources are scarce (H3c). With respect to food, spatial avoidance would be expected predominately in degraded habitat. Competition for tree holes should be more pronounced in non-degraded habitat with more old-growth trees and high C. medius population densities.

We observed a total of 794 lemurs over the entire survey period. The most common species in both seasons was M. murinus, with 473 individuals. This is much greater than for M. berthae with 91 or M. coquereli with 67 total observations. In the wet season, C. medius was the second most common species with 163 observations. In non-degraded forest parts of Menabe Central, trees grew taller and in higher densities with a more closed canopy cover than in degraded habitat (Schäffler et al., 2015). More lemurs were observed in non-degraded habitats (n = 421) than in degraded habitats (n = 373). Numbers of detected individuals per species did vary with spatio-temporal heterogeneities in that more mouse lemurs were observed in degraded habitat during the dry season (Table 2A), whereas there was no difference between early and late rainy season counts in any species (Table 2B). In most cases, the number of inter- and intraspecific pairs of individuals within 150 m was higher in degraded habitat during the dry season, whereas more pairs were observed within recognition distance in non-degraded habitat during the rainy season (Table 3).

To test the first set of hypotheses (H1), we investigated whether there is evidence of avoidance between M. murinus and M. berthae. Results in Table 4A and Figure 1 (see also Supplementary Figure 1 in Online Resource 2) clearly showed a tendency for mutual exclusion, supporting Hypothesis 1a, as nine of the ten indices (ρ and T) are negative, although only three of these values were statistically significant. The pooled data showed significant evidence of segregation at both scales. At the local scale, there was segregation during the dry season in non-degraded habitat supporting Hypothesis 1b, but no evidence of segregation in the degraded habitats and thus no support for Hypothesis 1c.

Hypothesis 2a predicted segregation between M. murinus and M. coquereli, which was observed at the regional scale in the pooled data (Figure 2A) and in all scenarios but in degraded habitat during the dry season (Table 4B and Supplementary Figure 2A). In contrast, no segregation was observed between M. berthae and M. coquereli (Table 4B and Figure 2B), complying with Hypothesis 2b. On the regional scale, those two species were even positively associated in degraded habitat during the dry season (Supplementary Figure 2B). Negative spatial association of M. murinus and M. coquereli within transects was limited to the dry season with food scarcity (Supplementary Figure 2A) and thus conforms with Hypothesis 2c.

Segregation patterns found between M. murinus and C. medius complied with Hypothesis 3a (Table 4C and Figure 3A). Lack of avoidance between M. berthae and C. medius on the regional scale (Table 4C and Figure 3B) and attraction within transects in non-degraded habitats (Supplementary Figure 3B) supported Hypothesis 3b. Segregation of M. murinus and C. medius was detected at the regional level in non-degraded habitat (Supplementary Figure 3A) and within transects in degraded habitat, which indicates both competition for tree holes and food according to Hypothesis 3c.

This study extends the insights of previous single-site investigations of this cheirogaleid assemblage (Schwab and Ganzhorn, 2004; Dammhahn and Kappeler, 2008a,b) to a wider range of spatial scales, including the local scale, and links them to observed regional-scale patterns (Schäffler et al., 2015). To measure inter-specific interactions within transects, we developed an “Inter-Specific Index of Attraction” (ISIA) which we tested for statistical significance against a null model based on a combination of randomization and bootstrapping, which controls for intra-specific clustering.

The distribution of mouse lemurs indicated habitat partitioning at different levels. First, we detected a higher number of individuals in intact habitats during the wet season and in degraded habitat during the dry season. Our local scale analyses thus reconfirmed that both mouse lemur species crowded in most suitable forest areas during the wet season and tracked scarce resources in degraded habitats during the dry season, corresponding to the pattern described on the population-level (Schäffler et al., 2015). Interspecific competition between individuals within transects was indicated during the dry season predominately in non-degraded habitat, whereas spatial segregation between M. berthae by M. murinus populations was limited to degraded habitat during the dry season (Schäffler et al., 2015). Increased use of degraded habitats when resources are scarce may therefore result from competitive exclusion between individuals in intact forests. Analyses incorporating dwarf and giant mouse lemurs with novel methods expanded knowledge on the interactions with third agents (Schäffler et al., 2015). The interspecific distribution of individuals confirmed that C. medius and M. coquereli mitigate competitive pressure on M. berthae and consequently stabilize mouse lemur coexistence. Our individual-level analyses revealed that the displacement of M. murinus by intraguild predator M. coquereli and the positive association between M. berthae and M. coquereli are not limited to non-degraded habitat during the dry season as previously suggested by the population-level distribution (Schäffler et al., 2015). Moreover, avoidance of superior competitor C. medius by M. murinus individuals and simultaneous local scale co-occurrence of M. berthae and C. medius during the wet season was not evidenced to date (Schwab and Ganzhorn, 2004; Schäffler et al., 2015). Given the variation in cheirogaleid distribution across spatio-temporal heterogeneities, the spatial competition hypothesis (Tilman, 1994) appears best suited to reconcile individual-level interspecific exclusion and population-level coexistence of the two mouse lemur species.

The complex ecological structure in the cheirogaleid assemblage of Menabe Central essentially depends on habitat quality and heterogeneity that provide cheirogaleids with refuges from unfavorable interspecific interactions and stabilizes competitive mouse lemur coexistence. Microcebus berthae was categorized as “Critically Endangered” in the recently updated IUCN Red List of Threatened Species (Markolf et al., 2020). Extensive habitat loss and degradation due to slash and burn agriculture results in continuing decline in the area of occupancy and extent of occurrence. A population decrease by more than 80% over 10 years is not only expected as a direct consequence of ongoing habitat loss, but also as interspecific interactions with dwarf and giant mouse lemurs that stabilize competitive coexistence of mouse lemurs depend on intact habitat. Contrasting association patterns of mouse lemurs between continuous forest sites (Rakotondravony and Radespiel, 2009) and isolated patches (Steffens and Lehman, 2016) in north-western Madagascar demonstrate the impact of habitat fragmentation on interaction regimes.

Species that are subject to the same disturbances do not always react in a similar manner, even if phylogenetically closely related (Lehman et al., 2006a,b,c; Irwin et al., 2010; Herrera et al., 2011; Lehman, 2016). It appears surprising that the massive landscape-level disturbances of recent times have not resulted in more lemur extinctions (Lehman et al., 2006c). However, species extinction in patchy environments may be time-delayed, and particularly threaten species that are abundant in intact habitat due to an interspecific trade-off between colonization and competitive abilities (Tilman et al., 1994). The loss of any species in ecological communities will have indirect effects on multiple levels (Strauss, 1991) and may trigger an extinction cascade with dramatic consequences for system stability (Allesina and Levine, 2011).

Genetic studies revealed that M. coquereli compensates for extensive population fluctuations (Kappeler, 2003) by immigration from adjacent populations (Markolf et al., 2008). Continuing habitat fragmentation may permanently extirpate the species from forest patches if isolation prevents recolonization (Irwin et al., 2009). Metapopulation models for the eight species comprising a lemur assemblage in north-western Madagascar revealed differences in the ability to cope with habitat fragmentation: while the occurrence of all lemurs depended on patch size, C. medius was particularly susceptible to reduction of area and isolation of poorly connected habitat fragments (Steffens and Lehman, 2018). Further loss, degradation and fragmentation of dry forests in Menabe Central will likely cause a reduction of population size in M. coquereli and C. medius and release M. murinus from intraguild predation and resource competition. Increasing interspecific competition between the mouse lemurs would then further reduce the suitability of remaining habitat for M. berthae. In these scenarios, one mouse lemur species might drive the other one to extinction (Schwab and Ganzhorn, 2004). Protection of remaining intact forest parts as well as restoration of degraded habitat and connectivity in the central Menabe region of western Madagascar is therefore critical to preserve M. berthae and coexisting species in this global biodiversity hotspot.

In this paper we tested interspecies associations involving mouse lemurs. Primarily, we were looking for negative associations that might be caused by competitive or (intraguild) predatory interactions. To do this we developed the “Inter-Species Index of Attraction” (ISIA) for local interactions on transects. We used a null model distinct from the ideal gas model because intraspecific clustering is in effect for all species and can obscure the effect of interspecies associations. To control for this, we used a null model based on a combination of randomization and bootstrapping to determine the significance of observed interactions. Our simulation approach comparing the neutral with the observed individual-level distribution of co-occurring species allowed for investigating interspecific interactions on a local scale and link it to observed regional scale patterns rather than examining only spatial overlap of populations (Schäffler et al., 2015). Moreover, our data collected from the cheirogaleid assemblage’s entire area of occupancy considered spatial heterogeneities and thus generated knowledge beyond the insights of previous studies at single sites (e.g. Schwab and Ganzhorn, 2004; Dammhahn and Kappeler, 2008a,b).

The statistical tests used by us in both regional and local analyses are non-parametric. One of the reasons for this is to avoid errors that might arise due to non-normality and zero-inflation in statistical tests. Another advantage of non-parametric statistics is that they are more robust against the effects of spatial autocorrelation. Some degree of spatial autocorrelation arising from external factors (Lennon, 2000) is likely to be present. One of the important external factors is habitat quality, which we controlled for by the fundamental structure of our data: dividing scenarios between degraded versus non-degraded. In comparisons between transect walks, the relatively large distances between transects (>1 km) also reduces the effect of spatial autocorrelation. Another spatial issue arises from the fact that transect distribution was not strictly random, owing to the logistical difficulties of sampling a large number of transects in areas of dense forest.

The raw data supporting the conclusions of this article will be made available by the corresponding author upon request.

The animal study was reviewed and approved by Bundesamt für Naturschutz (BfN) in Germany; Ministère de l’Environment et des Eaux et Forêts (MINEEF), CAFF/CORE committee and the National Association for the Management of Protected Areas (ANGAP) in Madagascar.

LS originally formulated the idea, developed the methodology, conducted fieldwork, and wrote the manuscript. PK provided the research facility and supported project implementation. LS and JH processed the data and performed analyses. JH developed the models and ran statistical analyses. All authors contributed to the article, approved the submitted version and agreed to be accountable for all aspects of the work.

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