Discussion
Morphologically almost identical species are likely to compete for
resources, and therefore offer good case studies to understand processes
that drive species coexistence. We find evidence of reduced trophic
niche overlap in recently separated cryptic bat species in sympatric
locations relative to allopatric ones based on DNA metabarcoding and
high throughput sequencing. The functional analysis suggests that the
subtle trophic shift seen may be driven by differential foraging mode.
Our results support niche theory predictions of the role of biotic
interactions in driving species assemblages (Schoener, 1974). Trophic
resource partitioning was only evident at the fine spatial scale, within
areas of range overlap, suggesting that fine-scale mechanisms of
coexistence could have implications for the maintenance of broad- scale
diversity patterns (Godsoe, Murray, & Plank, 2015).
Trophic ecology of Myotis escalerai and Myotis
crypticus
Our results reveal that the two bat species have a broad generalist
diet, tend to consume prey relative to their availability and use
gleaning to a high extent. We found high similarity in their trophic
ecology in terms of both order and prey species composition. Both bat
species’ diets are mostly composed of Lepidoptera, Diptera and Araneae,
but also include several other prey orders. However, M. escaleraiconsumes a higher percentage of Hemiptera, while M. crypticu s
Diptera. Functionally, the two bat species consume an equally high
proportion of prey items that are not nocturnally volant, which suggests
that both bats predominantly glean prey from vegetation.
The trophic ecology of these two recently described bat species is very
similar to their cryptic sister-species M. nattereri, which also
feeds mostly on Lepidoptera, Diptera and Araneae (Hope et al., 2014;
Swift, 1997; Swift & Racey, 2002; Vaughan, 1997) and is known to catch
a high proportion of its prey through gleaning (Arlettaz, 1996; Hope et
al., 2014; Shiel, McAney, & Fairley, 1991; Swift, 1997; Swift & Racey,
2002). Similarly to our study, Shiel et al., (1991) estimated that 68%
of M. nattereri ’s diet is made up of arthropod families that are
not active at night. The row of hairs in the uropatagium border is a
characteristic trait of members of the M. nattereri species
complex that is thought to be functionally linked with gleaning (Czech,
Klauer, Dehnhardt, & Siemers, 2009). Although the presence of more
developed hairs in M. escalerai is one of the characters which
separates these taxa (Juste et al., 2018), we found no difference in the
extent of gleaning between the two bat species.
Trophic partitioning across spatial
scales
Despite overall high trophic niche similarity between the two bat
species at the prey order level, we detect a signature of trophic shift
at the prey species (BIN) level, whereby diet overlap is lower in
locally sympatric compared to locally allopatric locations at the
fine-scale. This supports the contribution of trophic partitioning to
species coexistence even when overall trophic niche overlap is high. A
similar trend was seen at the functional level, whereby the proportion
of prey items that are not nocturnally volant is borderline different
between bat species only when locally sympatric. This suggests that the
differentiation in diet composition seen at the prey species level when
locally sympatric may be driven by a shift in foraging strategy (e.g.
Krüger et al., 2014), through M. escalerai decreasing its extent
of gleaning. However, our inference is limited by small sample sizes,
which reduced the power of the analysis. At the arthropod order level,
we find differences in the use of some arthropod orders among allopatric
regions, likely due to differences in arthropod availability between the
Mediterranean region, where only M. escalerai is found and the
Atlantic region, where M. crypticus is present.
Several studies have identified trophic niche shifts from allopatry to
sympatry, for instance between morphologically similar fish (Gkenas,
Magalhães, Cucherousset, Orjuela, & Ribeiro, 2019; Schmitt & Coyer,
1983) and reptile species (Huey, Pianka, Egan, & Coons, 1974; Klawinski
et al., 1994). However, in bats, previous coexistence studies looking at
trophic ecology only focused on sympatric populations, and rarely found
evidence of trophic resource partitioning. A few exceptions are the
gleaning bats M. nattereri , Plecotus auritus andMyotis bechsteini in a sympatric population in central Europe
(Andreas, Reiter, & Benda, 2012a), and evidence of low dietary overlap
between sympatric P. auritus and Plecotus macrobullaris(Ashrafi, Beck, Rutishauser, Arlettaz, & Bontadina, 2011).
The observed trophic shift, albeit subtle, suggests that the two bat
species are likely competing for food resources. It has been previously
hypothesised that arthropods are abundant and do not constitute a
limiting resource for bats (Arlettaz, 1999; Krüger et al., 2014).
However, exclusion experiments in both tropical (Kalka, Smith, & Kalko,
2008) and temperate forests (Böhm, Wells, & Kalko, 2011) show that bats
can control the abundance of arthropods, and therefore arthropods could
be a limiting resource to competitors (Salinas‐Ramos et al., 2020).
Our study does not refute the possibility that other coexistence
mechanisms, such as habitat or temporal partitioning (Schoener, 1974),
occur among these two species, or the role of environmental variability
in facilitating coexistence (Chesson & Warner, 1981). Spatial
partitioning is frequently cited as a key mechanism of coexistence in
other bat studies (e.g. Arlettaz, 1999; Emrich, Clare, Symondson,
Koenig, & Fenton, 2014; Kunz, 1973; Russo et al., 2014). Although in
many cases, contrary to our study, spatial partitioning may be driven by
slight differences in bat morphology (e.g. Salsamendi et al., 2008,
2012), which would affect their performance in different habitats
(Norberg, 1994). However, in our study the two species were caught in
the same sampling sites, some of which were forests, where they are
known to forage, suggesting they may share the same foraging sites.
A better understanding of the
spatial scales of species coexistence is an important advance in our
understanding of the maintenance of diversity (Hart et al., 2017). Our
finding that trophic partitioning only occurs at the fine spatial scale
is consistent with other bat (Peixoto et al., 2018), ant (Albrecht &
Gotelli, 2001), parasitoid insects (Harvey, Snaas, Malcicka, Visser, &
Bezemer, 2014) and bobcat (Lewis et al., 2015) studies, showing that
interspecific interactions are more important for shaping community
structure at fine rather than broad spatial scales. However, this
pattern is not universal (e.g Harmáčková, Remešová, & Remeš, 2019).
Fine-scale coexistence mechanisms could prevent competitive effects from
scaling-up (Godsoe et al., 2015), which in our study system could
contribute to enabling broad-scale range overlap across the north of the
Iberian Peninsula.
Prey consumption relative to
availability
The diet of a species is a function of both consumer selection and
trophic resource availability within the foraging habitat (Lawlor,
1980). Therefore, considering resource availability allows for a better
inference of species trophic preferences. Previous studies comparing bat
prey consumption with prey availability pointed to selection of certain
prey orders, such as Coleoptera by Eptesicus fuscus (Agosta,
Morton, & Kuhn, 2003), chironomid flies by Myotis daubentoni(Vesterinen et al., 2016) and certain prey traits like moth size byBarbastella barbastella (Andreas, Reiter, & Benda, 2012b).
Similarly, M. nattereri was found to over-select arachnids,
Opiliones, Coleoptera, and several Diptera families, and under-select
Hemiptera (Swift & Racey, 2002). In this study we do not detect clear
trends of over-selection for specific prey orders matching the
generalist broad trophic niche of the studied bats. However, diet
selection results should be interpreted with caution due to the
difficulty of obtaining a representative estimation of arthropod
availability. Any arthropod sampling technique is biased towards certain
types of arthropods (Cooper & Whitmore, 1990) and the habitats sampled
and their respective sampling effort may not adequately represent where
bats actually forage, especially given that they can use large areas and
arthropod communities change depending on habitat type (Lamarre et al.,
2016) and vertical stratification (Ulyshen, 2011). Our molecular diet
analysis results confirm that the two studied bat species indeed glean
prey from the vegetation, and therefore the arthropod community sampled
using sweep nets likely represents at least part of the prey resources
available to the bats.
Methodological considerations and study
limitations
Primer bias towards certain taxonomic groups is a major issue in
metabarcoding studies (Elbrecht et al., 2019). In this study, prey items
were frequently recovered by only one of the primers, and differences
existed in the recovery of the different arthropod orders. This supports
previous studies that suggest that more than one set of primers should
be used when the expected diet covers a broader taxonomical spectrum
(Alberdi et al., 2018). The inclusion in this study of a set of samples
with known composition based on morphological analysis (albeit only at
the order level) gives us some idea of potential biases in the molecular
identification. Opiliones, in particular, were morphologically
identified in several sweep net samples and are known to be present in
the diet of M. nattereri (Galan et al., 2018; Swift, 1997; Swift
& Racey, 2002), but were absent from the molecularly-characterised
diets of the two bats. Thus, their absence in this study is likely the
result of primer amplification bias.
Parameter choice during bioinformatic analysis can modify the diet
composition recovered (Alberdi et al., 2018). The strong match between
the inferences drawn using the additive and the conservative approach of
dealing with PCR replicates (Fig. S9), show that the results are robust
to that choice, and mirror other studies showing that parameters choice
does not change ecological conclusions (Clare, Chain, Littlefair,
Cristescu, & Deiner, 2016). In the same manner, high similarity in diet
composition based on weighted Percent of Occurrence (wPOO) and Relative
Read abundance (RRA), indicates that the results are also robust to the
measure used.
Because prey development stage cannot be identified using the
metabarcoding approach, some of the prey species (BINs) classified as
nocturnally volant may correspond to non-flying larval stages. This
could be important in Lepidoptera, and could increase the inferred
importance of the gleaned behaviour of both species because larval
stages are known to be consumed by M. nattereri (Hope et al.,
2014). More generally, nocturnal aerial activity is not directly
quantifiable, and therefore our classification is subject to a certain
degree of subjectivity. However, because arthropod orders most difficult
to categorise due their diversity, like Coleoptera, are consumed in
similarly low proportion by both bat species, potential classification
biases are expected to be low and standardised across species.
Nevertheless, due to potential classification biases and low sample
sizes in sympatric locations, interpretations of functional prey shift
should be considered with caution.
Conclusions
In line with niche theory predictions, we show that coexistence among
morphologically identical (cryptic) species can be facilitated through
fine-scale mechanisms of resource partitioning, despite high levels of
trophic similarity at the broad-scale, even in sympatric regions. Hence,
this study highlights the importance of using appropriated spatial
scales when studying impacts of biotic interactions on community
assembly (Viana & Chase, 2019). Our findings that trophic resource
partitioning is only evident at the fine spatial scale, within areas of
range overlap, suggest that fine-scale mechanisms of coexistence could
have implications for the maintenance of broad- scale diversity
patterns. This is the first study to identify a trophic shift between
allopatric and sympatric populations of insectivorous bats, supporting
the role of trophic resource partitioning in enabling species
co-occurrence in the same foraging site. It thereby addresses some of
the key limitations identified in a recent review of interspecific
competition in bats (Salinas‐Ramos et al., 2020). We highlight the
importance of using high taxonomic resolution and allopatric populations
at meaningful spatial scales for identifying patterns of niche shift,
and the utility of using a functional approach that better links
mechanistically with species trophic ecology.
Understanding mechanisms of
coexistence is essential for predicting species vulnerability under
climate change because range shifts will result in new community
assemblages and competitive interactions (HilleRisLambers, Harsch,
Ettinger, Ford, & Theobald, 2013). This is particularly relevant in our
study system as both species are restricted to the Mediterranean region,
where climate change is predicted to be particularly severe (Sala et
al., 2000), and both are predicted to experience range shifts and
changes in range overlap under climate change (Razgour et al., 2019).