Results
We analysed a total of 138 dropping samples for Myotis escaleraiand 90 for Myotis crypticus from 49 locations, 26 of which were
in the broad-scale allopatric regions and 23 in sympatric regions.
Within the sympatric regions (La Rioja and southern Cantabria), 91
samples were classified as locally allopatric and 28 as locally
sympatric (Fig. 1, Table 1; Supplementary Table S1). We recovered a
total of 2,859,300 reads (Supplementary Table S3 for details) from the
228 dropping samples for the four combinations of PCR replicates and
primers (1,403,636 from ANML1 and 1,455,664 from ZBJ). These reads were
associated into 1461 different BINs. Based on the BINs present in
sequencing blanks, we removed for the ANML primer 6 BINs from the first
run of 56 dropping samples, and 2 BINs for the second run of 10 samples.
For the ZBJ primer, we removed 5 BINs from the first run of 10 samples
and 23 BINs from the second run of 88 samples. Based on BINs present in
extraction blanks, we removed a total of 39 BINs from 16 dropping
samples
Characterising the diet of M.
escalerai and M.
crypticus
A total 19 arthropod orders were obtained based on the 1461 BINs
(Supplementary Data file S1 for list of prey items obtained for each bat
species). The diets of M. escalerai and M. crypticus were
characterised by high arthropod diversity, and were composed mostly of
the orders Lepidoptera (M. escalerai = 26.6 %; M.
crypticus = 23.7%), Diptera (24.8%; 33.2%), Araneae (20.7%;
17.2%), but also included Hemiptera (11.8%; 6.2%), Coleoptera (4.8%;
5.1%), and Orthoptera (4.3%; 4.8%), among others (<5%)
(Fig. 2a-b; Supplementary Fig. S2 for diet composition based on POO and
RRA measures). Diet composition at the prey order level was very similar
between bat species (OJK = 0.98, above 1000 null
models). However there were differences in the number of BINs per sample
of Diptera, which was lower in M. escalerai (5.27 versus 6.75)
(Negative binomial GLM: z1,226=-2.03, P = 0.042), and
Hemiptera, which was higher in M. escalerai (2.09 versus 1.68)
(Negative binomial GLM: z1,226=2.85, P = 0.004
Supplementary Fig. S3).
At the prey species (BIN) level, Levins’ niche breadth was similar for
both species, BA = 0.17 for M. escalerai and
BA = 0.19 for M. crypticus . Niche overlap between
species was higher than expected by chance (OJK = 0.71,
above 95% of 1000 null models). The samples from the two bat species
showed some differences in prey item composition in NMDS ordination
space (Fig. 3a, Stress: 0.25, k=3, non-metric fit
R2=0.934, Linear fit, R2= 0.532). An
analysis of similarity confirms that distance in prey item composition
among samples is greater between species than within species (ANOSIM R
statistic: 0.10, P= 0.001).
Trophic partitioning in sympatric versus allopatric
locations
At the arthropod order level, there is no clear pattern of shift from
high similarity in order composition between species to differential use
in sympatry at any of both spatial scales (OJK regional
allopatry =0.88, OJK regional sympatry = 0.96,
>1000 null models, Fig 2c-d; OJK local
allopatry =0.95, OJK local sympatry = 0.98,
>1000 null models, Fig 2e-f). When examining the number of
BINs of the main arthropod orders per sample, there were differences
between bat species between the allopatric regions for Araneae and
Hemiptera, which were both higher in M. escalerai (M.
escalerai = 4.00, 1.65, M. crypticus = 2.16, 0.55 respectively),
and for Lepidoptera, which was higher in M. crypticus (4.6,
11.94) (Negative binomial GLM: df=1,98, P<0.05). In the
sympatric region, the higher number of Hemiptera in M. escaleraiholds (M. escalerai = 2.50, M. crypticus 1.50), and in
Lepidoptera there is a shift whereby is M. escalerai the one that
consumes a higher number (M. escalerai = 6.78, M. cryptius= 4.09, Negative binomial GLM: P<0.05). At the fine-scale,
within the sympatric region, the only difference found between the bat
species was the higher number of BINs per sample of Hemiptera (2.67,
1.49) (Negative binomial GLM: z1,89= -2.68, P=0.007) and
Lepidoptera (6.70, 3.64) in M. escalerai in allopatric locations
(Negative binomial GLM: z1,89= -2.92, P=0.004). There
were no differences in arthropod orders consumed between the bat species
in locally sympatric locations (Negative binomial GLM:
P>0.05; Supplementary Fig. S4).
At the prey species (BIN) level, at the broad-scale, trophic niche
similarity between species was lower in allopatric than in sympatric
regions (OJK allopatric = 0.35, OJKsympatric= 0.62). Conversely, at the fine-scale, within the sympatric
region, trophic niche overlap between species was higher in locally
allopatric locations (OJK= 0.56) than in locally
sympatric locations (OJK= 0.37). Despite the low values
of overlap in regionally allopatric and locally sympatric locations, in
all the four cases, observed niche overlap was higher than 1000 null
models. When measuring trophic niche overlap between species using pairs
of locations, we observed the same pattern. At the broad scale we found
higher diet overlap in sympatric than allopatric locations
(OJK sympatric= 0.107±0.056, OJKallopatric= 0.050±0.04; Gausian hurdle model: binomial GLM:
z1,316=4.76, P<0.05; Gaussian GLM:
t1,265=8.26, P<0.05). In contrast, at the
fine-scale, niche overlap was lower among pairs of locally sympatric
than among locally allopatric locations (OJK sympatric=
0.099±0.065, OJK allopatric= 0.126±0.057; Linear model:
F1,73=6.34, P=0.014; Fig 3b).
Functional diet analysis
Both species had a similar high percentage of non-volant (M.
escalerai =21.4 %, M. crypticus =19.5%) and not
actively-volant (44.6%, 45.8%) prey items in the diet. Only 34.0% and
34.7% of weighted percent of occurrence (wPOO) was composed of
arthropods classified as nocturnally volant (Fig. 4a). There were no
differences in the overall percentage of not nocturnally volant prey
taxa (BINs) per sample between bat species (66% ±20%, 66% ±21%,
Linear model: F1,217 <0.001, P= 0.990; Fig.
4b). When analysing functional diet differences separately in allopatric
versus sympatric regions, we found differences between species in
allopatric regions, whereby M. crypticus consumed lower
percentage of prey that were not nocturnally volant (allopatric regions:M. escalerai = 66% ±19%, M. crypticus =48% ±25%;
F1,98=11.72, P<0.05; Fig. 4c; sympatric
regions: 65% ±22%, 71% ±17%; F1,117=2.3, P=0.13;
Fig. 4d). At the fine-scale, there were no differences among bats in
locally allopatric locations (M. escalerai = 67% ±22%, M.
crypticus =72% ±16%, F1,89=1.325, P=0.250; Fig. 4e)
while in locally sympatric locations the percent of prey that were not
nocturnally volant was borderline lower in the diet of M.
escalerai (52% ±17%) than M. crypticus (68% ±18%;
F1,26=4.03, P=0.055; Fig. 4f).
Prey consumption relative to
availability
In nearly all cases, we could not detect over- or under-selection of
arthropod orders by the bats relative to their availability in sweeping
samples. The distribution of prey order selection values between the
1st and 3rd quartiles overlapped
with zero in all cases, except in the case of M. escalerai and
Lepidoptera, where positive selection values could indicate
over-selection (1st-3rd quartile:
+0.43 — +18.7; Supplementary Fig. S5).
Metabarcoding and primer
performance
There were compositional differences in the prey orders that each primer
recovered. A large proportion of the BINs identified in dropping samples
were only recovered by one of the primers (Supplementary Fig. S6).
Neuroptera, Orthoptera, Coleoptera were more frequently recovered by
ZBJ, while Plecoptera and Thysanoptera, Dermaptera Mantodea where more
frequently recovered by ANML (Supplementary Fig. S6). Supplementary
Figure S7a-b shows composition for a subset of dropping samples
comparing each primer.
In sweeping samples, 7065 insects were identified morphologically to
order level, with an average of 174.3 individuals per sample (range
7-624). Using molecular tools, we recovered 899,853 reads (Supplementary
Table S2), and identified 813 different BIN items. Some of the rarer
orders were under-represented in the molecular analysis. Specifically,
Opiliones, Dermaptera, and
Archaeognatha, appeared in more than 10 sweeping samples each identified
morphologically, but were rarely recovered in the molecular approach,
despite being present in the reference databases (Supplementary Fig.
S8).