Journal of Threatened
Taxa | www.threatenedtaxa.org | 26 July 2026 | 18(7): 29196–29211
ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.10450.18.7.29196-29211
#10450 | Received 09 February 2026 | Final received 01 July 2026 |
Finally accepted 01 July 2026
Metabarcoding-based diet
characterization of the Kolar Roundleaf Bat Hipposideros hypophyllus Kock
& Bhat, 1994 (Chiroptera: Hipposideridae) and its peninsular Indian
congeners
Bhargavi Srinivasulu 1 , Aditya Srinivasulu 2 & Chelmala Srinivasulu 3
1,3 Centre for Biodiversity and
Conservation Studies, Osmania University, Hyderabad, Telangana 500007, India.
1,2 Systematics, Ecology &
Conservation Laboratory, Zoo Outreach Organisation, Saravanampatti, Coimbatore,
Tamil Nadu 641035, India.
3 Wildlife Biology & Taxonomy
Lab, Department of Zoology, University College of Science, Osmania University,
Hyderabad, Telangana 500007, India.
1 bharisrini@gmail.com, 2 a.chelmala1@gmail.com
(corresponding author), 3 chelmala.srinivasulu@osmania.ac.in
Editor: H. Raghuram, Sri. S. Ramasamy Naidu Memorial
College, Virudhunagar, India. Date of publication: 26 July
2026 (online & print)
Citation:
Srinivasulu, B., A. Srinivasulu & C. Srinivasulu (2026).
Metabarcoding-based diet characterization of the Kolar Roundleaf Bat Hipposideros
hypophyllus Kock & Bhat, 1994 (Chiroptera: Hipposideridae) and its
peninsular Indian congeners. Journal of
Threatened Taxa 18(7):
29196–29211. https://doi.org/10.11609/jott.10450.18.7.29196-29211
Copyright: © Srinivasulu et al. 2026. Creative Commons Attribution 4.0 International License.
JoTT allows unrestricted use, reproduction, and distribution of this article in
any medium by providing adequate credit to the author(s) and the source of
publication.
Funding: This study was funded by Bat Conservation International (Texas, USA) under the Endangered Species Grant (Award number:
210427-51000). CS acknowledges a research grant from DST-SERB (New Delhi,
India), and partial funding and research facilities provided by MoE-RUSA 2.0
(Government of India). AS acknowledges doctoral funding during the time of
the study from the University of Reading (Reading, UK) International PhD
Studentship (ref. GS21-026).
Competing interests: The authors declare no competing interests.
Author details: Bhargavi Srinivasulu is a senior scientist at the
Deccan Regional Station of Zoo Outreach Organisation, India. Her research
focuses on bat systematics, phylogenetics, and conservation, and herpetofauna
macroecology. Aditya Srinivasulu is a research affiliate at the
Deccan Regional Station of Zoo Outreach
Organisation, India. He is interested in understanding factors driving habitat
suitability, extinction risk, and biodiversity loss in the Global South. His
work explores conservation knowledge shortfalls, bat and herpetofauna macroecology, extinction risk, and the climate and land-use futures of
South Asian vertebrates. Chelmala Srinivasulu is a professor of Zoology at
Osmania University, Hyderabad, where he heads the Wildlife Biology and Taxonomy
Lab and directs the Centre for Biodiversity and Conservation
Studies. He researches biodiversity conservation, systematics, and taxonomy of
mammals, reptiles, and birds, as well as climate-change modelling.
Author contributions: BS and CS led the conceptualisation, methodology, investigation,
formal analysis, data curation, visualisation, funding acquisition, and initial
writing for the study. AS contributed to investigation, formal analysis,
visualisation, and final writing.
Acknowledgements: We thank Bat Conservation International (Texas, USA) for funding this
study under the Endangered Species Grant (Award number: 210427-51000), and Dr.
Jonathan Flanders for his interest in this study. CS thanks DST-SERB, New Delhi
for the research grant. We thank the principal chief
conservator of forests (wildlife) and chief wildlife warden, Karnataka Forest
Department, Government of Karnataka for the necessary permissions to carry out
the study; the deputy conservator of forests, Kolar Forest Department; the range forest officers; and staff of Mulbagal and Kolar forest
departments Mr. Bharat, Mr. Zameer, and Mr. Venkatappa for their help during
the study period. AS thanks the University of Reading for funding during the
study period under the University of Reading International PhD
Studentship (ref GS21-026). We also thank Dr. G. Devender, Ms. N. Mohini
Sastry, Ms. B. Jyothi, and Ms. Vyshnavi Sneha for assistance in the fieldwork,
the head, Department of Zoology, Osmania University for encouragement and facilities, and MoE-RUSA 2.0 for facilities and partial funding.
Abstract: We used faecal DNA metabarcoding
to analyse the diets of the Kolar Roundleaf Bat Hipposideros hypophyllus,
and its three congeners (H. durgadasi, H. fulvus, & H.
speoris) cohabiting a roost in Kolar District (Karnataka, India). We
collected faecal pellets of these bats returning to the roost after the first
feeding bout over three seasons between 2021 and 2022. The whole diet of all
these species consisted of 7,309 Operational Taxonomic Units (OTU) belonging to
phylum Arthropoda. Although no significant difference among the whole diets of
the four species was found, H. hypophyllus consumed the most amount of
insect taxa (4,114 species) compared to others. The core diets consisted of 48
taxa belonging to 10 orders, 37 families, and 46 genera. A significant
difference was found between the core diets, with H. hypophyllus and H.
speoris feeding on more diverse insects; diets did not overlap, hinting
that the study species are diet specialists. Prey species belonging to orders
Lepidoptera and Diptera were abundant in the diet of all four species of bats.
The core diet of the Kolar Roundleaf Bat varied significantly between the
seasons, with ≥70% intake of lepidopterans in the monsoon and winter seasons.
All four species fed on medically and agriculturally significant insect pests
as well, indicating their role as bio-controllers in this ecosystem. This
study, the first of its kind in India, is an initial exploration of the use of
non-invasive diet characterisation for enhancing our knowledge of the
ecological interactions of endemic and threatened bat species in sensitive and
diverse ecosystems.
Keywords: Biocontrollers, core diet,
endemic and threatened, faecal analysis, Hipposideros durgadasi,
Hipposideros fulvus, Hipposideros hypophyllus, Hipposideros
speoris, insect pest, leaf-nosed bat, whole diet.
Introduction
Comprising more than 1,500
species worldwide, bats constitute 22% of global mammalian diversity
(Upham et al. 2024). The majority
of bat species are insectivorous, consuming invertebrates up to 70–80% of their
body weight during their numerous feeding bouts each night (Kurta et al. 1989;
Kalka et al. 2008; Kunz et al. 2011). Much research has highlighted the
beneficial effects of bats and their ecosystem services, especially as
biocontrollers of pest insects (Leelapaibul et al. 2005; Cleveland et al. 2006; Boyles et al. 2011; Kunz
et al. 2011; Wanger et al. 2014; Puig-Montserrat et al. 2015; Taylor et al.
2018; Kemp et al. 2019; Linden et al. 2019). Despite this diversity and
ecological significance, there exists a shortfall in the knowledge of diets of
most insectivorous bats (Adams & Sandbrook 2013; Tuneu-Corral et al. 2023).
Effective conservation prerequires
an understanding of the ecological interactions of any species (Adams &
Sandbrook 2013), and the diet of an animal is a vital interaction to understand
(Whitaker Jr. 1988; Racey et al. 2003; Clare 2014; Groom et al. 2017; Cravens
et al. 2018) as the quality and quantity of food resources available
for an animal are crucial both for individual- and population-level survival
(Eeva et al. 1997; Vickery et al. 2001; Taylor & Schultz 2008; Sorensen et
al. 2009; Johnsen et al. 2017; Serrano-Davies & Sanz 2017). Due to their
diverse diets and varied foraging ranges, bats consume diverse insect species,
and the ecosystem services that they provide cannot be compensated by predators
such as birds; the loss of such services leads to increased insect pest
abundance, reduced crop production, increased use of pesticides, and
deleterious effects on the health of the habitat, ecosystem, and humans (Maas
et al. 2013; Maslo et al. 2017; Scholz & Voigt 2022). Additionally, bats
provide value to the ecosystems as primary, secondary, and tertiary consumers
sustaining both unmodified natural and human-dominated landscapes (Kunz et al.
2011). They help maintain diversity of the forests by seed dispersal, introduce
novel plant species, maintain genetic diversity of flowering plants (especially
those that bloom exclusively at night), and redistribute nutrients through their
guano (Kunz et al. 2011).
However, identifying and analysing
the diet of bats is complicated due to their highly mobile nature (Painter et
al. 2009). The most widely used approach to assessing bat diets is through the
analysis of faeces, either through ‘classical’ methods such as manual
dissection of faeces, or more modern approaches including molecular analysis of
DNA: bat faeces contain the DNA of the species that produced them as well as
the remains (and consequently the DNA) of prey insects (Reed et al. 1997). As
classical methods usually result in relatively coarse family- or even
order-level identification of remains (with many unidentifiable fragments),
DNA-based methods are increasingly used (Zeale et al. 2011; Clare et al. 2014;
Krüger et al. 2014; Foo et al. 2017; Krauel et al. 2018; Scholz & Voigt
2022) mainly due to the higher taxonomic resolution and detection rates of rare
or overlooked prey taxa (Clare et al. 2009, 2011; Ashrafi et al. 2011; Razgour
et al. 2011; Krüger et al. 2012; Clare 2014; Vesterinen et al. 2013; Vesterinen
et al. 2016; De Sousa et al. 2019; Gordon et al. 2019; Hayes et al. 2019).
Of the 153 species of bats extant in
South Asia (Srinivasulu et al. 2025), only the Kolar Roundleaf Bat Hipposideros
hypophyllus has been assessed to be ‘Critically Endangered’ by the IUCN Red
List (Chakravarty et al. 2015). Its only known roost is a single subterranean
cave on a granite hill in the vicinity of Hanumanhalli Village in the Kolar
District of Karnataka, southern India, and the population has been estimated to
be around 200 individuals (Srinivasulu et al. 2014). Since its ‘rediscovery’ in
2014, significant steps have been taken for its conservation, including the
designation of the roost and its environs as a conservation reserve, and the
inclusion of the species in the Indian Wildlife (Protection) Amendment Act,
2022, granting it the highest legal protection by the Government of India. It
shares its roost with Durga Das’ Roundleaf Bat Hipposideros durgadasi (VU;
Mishra & Dookia 2016), the Fulvous Roundleaf Bat Hipposideros fulvus
(LC; Srinivasulu & Srinivasulu 2018), and Schneider’s Roundleaf Bat Hipposideros
speoris (LC; Srinivasulu & Srinivasulu 2019). This study explores the
diets and variations therein of the highly threatened Kolar Roundleaf Bat and
congeners in its only known roost. It is an initial exploration of the
potential dietary niche of this sensitive and endangered species alongside its
congeners, aiming to provide a vital foundation for future conservation
management and policymaking to ensure the continued survival of this species
and the health of this multispecies colony and its surrounding ecosystem.
Materials
and Methods
Study area
The study site, Hanumanahalli Betta
(near Hanumanhalli Village, Kolar District, Karnataka, India; 13.16° N, 78.29°
E) is a monolithic granite hill with a single narrow subterranean cave housing
the only known population of the ‘Critically Endangered’ Kolar Roundleaf Bat Hipposideros
hypophyllus (Srinivasulu et al. 2014). The general habitat surrounding the
hill is comprised of scrublands with scattered boulders and hills, agricultural
lands, and patches of reserve forest dominated by tropical dry deciduous and
tropical thorny forest vegetation (Champion & Seth 1968;
Srinivasulu et al. 2014), with small rain-fed waterbodies located just beside
the hill and scattered in the study area.
Fecal sample collection
Between April 2021 and March 2022,
individuals of each of the four study species (Hipposideros hypophyllus (Image
1), H. durgadasi (Image 2), H. fulvus (Image 3), & H.
speoris) (Image 4) were captured from the cave using mist-nets and were
handled strictly according to the ethical and responsible handling practices
outlined in the American Society of Mammalogists guidelines (Sikes
& American Society of Mammalogists 2016 – importantly including
double-layered gloves and masks) to ensure minimal stress and
exposure. All collections and surveys were conducted under express permission
from the Karnataka State Forest Department (No. PCCF(WL)/E2/CR/2016-17, dated
19.i.2022 vide Government Order No. FEE 404 FWL 2014, dated 20.i.2015),
Government of Karnataka. The experimental protocols and procedures were
reviewed and approved (59A/CBCS/OU/2021-22) by the Research Committee of Centre
for Biodiversity and Conservation Studies, Osmania University, Hyderabad. The
study is reported in accordance with ARRIVE guidelines.
Mist-nets were erected after the
bats had completed their emergence and were returning from their feeding bouts.
Bat emergence times were initially determined based on Srinivasulu et al.
(2014) and confirmed based on active echolocation call analysis. Echo Meter
Touch 2 Pro (Wildlife Acoustics Inc., USA) and u384 USB
ultrasound microphone (Pettersson Elektronik, AB, Uppsala, Sweden) ultrasound
detectors were used at the entrance/exit points to determine bat species
identity based on call frequencies, corroborated by published data (Srinivasulu
et al. 2016). We captured four individuals of each species, which were safely
held in clean cloth bags for 30 minutes or until they voided 3–5 faecal pellets each, and were
then released back near the roosting site. The pellets were pooled by species
(and by season for Hipposideros hypophyllus) and placed in vials filled
with RNAlater stabilization solution (Invitrogen) for DNA extraction.
DNA extraction
DNA was extracted from bat faecal
samples using Qiagen QIAamp fast DNA stool mini kit. The method of DNA
extraction involved bead-beating, use of QIAamp fast DNA Stool Mini Kit and
RNase treatment. From the collected faecal samples, an aliquot of the stool
sample (500 μl) was pipetted into a micro-centrifuge tube using a cut tip.
About 3.1 ml of InhibitEX buffer was added to each stool sample and vortexed
continuously for one minute until thoroughly homogenized and sample was
incubated at 70°C for 10 minutes. The samples were treated with 200 μl of
lysozyme (10 mg/ml) and incubated at 37°C for 30 minutes. Around 4–5 stainless
steel beads were added, and bead-beating was performed for five cycles (Vortex
at maximum speed for one minute, resting time 5 minutes). The tubes were
allowed to cool on ice for 2–3 minutes and centrifuged at 16,000 xg for 5
minutes at 4°C. The supernatant was transferred to a fresh 2 ml Eppendorf tube.
To the cell pellet 200 μl of lysozyme was added, followed by incubation at 37°C
for 30 minutes. The bead-beating step was repeated for three cycles in the
following scheme: vortex at maximum speed for one minute, resting time five
minutes. The Eppendorf tube was centrifuged at 16,000 xg for 5 minutes at 4°C
and the supernatant was transferred to a fresh 2 ml Eppendorf tube. The
supernatant obtained from the earlier steps was pooled, and 20 μl proteinase K
and 200 μl buffer AL was added, the tubes vortexed for 15 seconds and incubated
at 56°C for 2 h. The supernatant was treated with 30 μl of RNase A and
incubated at 65°C for 30 minutes. The lysate was thoroughly mixed with half
volume of absolute ethanol and loaded into a QIAamp spin column placed in a 2
ml collection tube. The collection tube was then centrifuged at ≥ 6,000 xg for
one minute and the flow-through was discarded. To the column, 500 μl Buffer AW1
was added and centrifuged for one minute at ≥ 6,000 xg and the flow-through was
discarded. 500 μl of Buffer AW2 was added and centrifuged for one minute at
20,000 xg and the flow-through was discarded. This step was repeated twice. The
QIAamp spin column was placed in a new 2 ml collection tube, and a dry spin was
performed by centrifuging at 20,000 xg for one minute. The QIAamp spin column
was then transferred into a new labelled 1.5 ml microcentrifuge tube, and the
DNA was eluted by adding pre-warmed 30-50 μl 10 mM Tris HCl (pH 8.0), directly
on the QIAamp membrane and incubated at room temperature (25°C) for 5 min. The
microcentrifuge tube was centrifuged at 20,000 xg for one minute, and the DNA
pellet was retained. The concentration and purity of genomic DNA was quantified
using a nanodrop spectrophotometer. The integrity of DNA in the sample was
confirmed using gel electrophoresis and the DNA concentration was quantified
using a Qubit dsDNA HS assay kit.
Library Preparation
Genomic DNA was amplified for COI
gene using ANML LCO1490 and COI-CFMRa primers with KAPA HiFi HotStart PCR
Master Mix (KAPA Biosystems, USA). The forward and reverse primer
concentrations were kept at 5 μm each. A 25 μl PCR was setup using 12.5 μl of
the 2X KAPA HiFi HotStart Ready mix, 10.5 μl of the genomic DNA and
nuclease-free water and one μl each of the forward and reverse primers using a
two-step protocol: 94oC for one minute for initial denaturation,
followed by 94oC for one minute, 45oC for one minute 45
seconds and 72oC for one minute and 15 seconds for five cycles; 94oC
for one minute, 51oC for 30 seconds, 72oC for one minute
and 15 seconds for 35 cycles followed by a final extension step of 72oC
for 5 minutes. The amplicons were visualized on 1.2% agarose gel and purified
using Sera-MagTM Select beads (Cytiva). Sequencing libraries were prepared with
Illumina-compatible NEXTflex Rapid DNA sequencing Bundle (BIOO Scientific, USA)
at Genotypic Technology (Bangalore, India). 100 ng of Qubit quantified purified
amplicons were end-repaired, adenylated, and ligated to Illumina multiplex
barcode adaptors as per NEXTflex Rapid DNA sequencing bundle kit protocol.
Illumina universal adapters used in the study were:5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
and index adapter: 5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC [INDEX]
ATCTCGTATGCCGTCTTCTGCTTG-3’.
Adapter-ligated fragments were
purified using Sera-MagTM select beads (Cytiva). Resultant fragments
were amplified for six cycles of PCR using Illumina-compatible primers provided
in the NEXTflex Rapid DNA sequencing Bundle. Final PCR products (sequencing
library) were purified with Cytiva beads followed by library quality control.
Sequencing libraries were quantified by qubit fluorometer (ThermoFisher
Scientific, USA) and the fragment size distribution was analysed on Agilent
2200 TapeStation. The libraries were paired-end sequenced on Illumina HiSeq X
Ten sequencer (Illumina, USA) for 150 cycles following the manufacturer’s
instructions. The raw sequencing data was demultiplexed using bcl2fastq
software v2.20 (Illumina, USA). The sequencing quality (base call accuracy and
adapter contamination) was assessed using FastQC v0.11.8 software (Andrews
2010). The adapter sequences were trimmed using TrimGalore (Felix 2015) and
bases above Q30 were considered for downstream analysis. The high-quality
processed reads were stitched using Fastq-join (Aronesty 2013).
The centrifuge tool (Kim et al.
2016) was used to align the trimmed reads against centrifuge-NT database. This
database is designed to facilitate the classification and identification of
sequences in metagenomic and microbiome datasets. It contains reference sequences
from a wide range of organisms, including bacteria, archaea, viruses, and
eukaryotes, classifying from kingdom to higher taxonomic ranks. Centrifuge is a
very rapid and memory-efficient system which uses a novel indexing scheme based
on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index,
optimized specifically for the metagenomic classification. Pavian (Breitwieser
& Salzberg 2020) was later used to estimate and interactively explore the
taxonomic content. Only matches with a similarity level of 95% or higher were
considered significant and retained during the alignment process. The sequences
generated in this study are deposited in NCBI database (BioProject accession
number PRJNA1281322).
From the classified report, OTU
IDs were assigned using kraken-biom Python tools. kraken-biom parses the
k-reports, counts for each operational taxonomic unit (OTU), and includes
lineage information. The extracted OTU from the kraken-report is then stored in
a BIOM (Biological Observation Matrix) table, linking each count to the
respective sample and OTU. The final obtained OTU-specific read abundance was
used for further analysis. The frequency of occurrence of each OTU (or taxa) in
the diet of each bat species studied was calculated as percent relative
abundance (i.e., each OTU’s read abundance divided by total read abundance of
each bat species multiplied by 100).
Niche overlap and niche breadth
Pianka’s niche overlap and
Levins’ niche breadth were calculated using spaa
package in R (Zhang & Ma 2014). The values of the niche breadth obtained
were standardised to get values between zero and one using the Levins’ index
equation (BA= B-1/n-1; (Levins 2020)). We performed a non-parametric
analysis of variance of relative prey-item read abundance (number of reads for
a prey species divided by total number of reads) grouped by bat species (for
both whole and core diets) to investigate if the dietary composition differed
between the four species of bats, and diversity indices (for both whole and
core diets) to investigate the diet breadth of each species in PAST statistical
software (Hammer et al. 2001). We used RAWGraphs 2.0 (Mauri et al.
2017) to visualize the whole diet and the core diets of the four species of
hipposiderid bats across three seasons (summer, monsoon, & winter).
Results
Of the 18,483,216 raw reads
generated, only those with a taxonomic identity match percentage of > 95%
were retained, resulting in 17,360,540 reads post-processing. The final
processed reads were further checked against the records available from India
on the public data portal of the BOLD and GenBank databases. We obtained 17,179
records corresponding to 4,177 species (combined data from BOLD and GenBank
databases), which were further filtered to obtain a final 7,309 OTUs
(Supplementary Tables 1,2).
Dietary analysis
The final dataset of whole diets
of the four hipposiderid species revealed that our samples of Hipposideros
hypophyllus comprised the most insect diversity (4,114 species), followed
by H. durgadasi (2,410 species), H. fulvus (1,945 species), and H.
speoris (1,160 species). No significant difference among the diets of the
four species of bats was found (Kruskal-Wallis H[2] =
5.407; p > 0.05).
From the whole diet dataset, we
filtered out frequently consumed prey-items (≥ 1% frequency of occurrence);
this was classified as the core diets of the four species of hipposiderid bats.
In the core diets of the four species, we identified 48 taxa belonging to 10
orders, 37 families, 46 genera, and 48 species. A significant difference was
found among the core diets of the four hipposiderid species (Kruskal-Wallis H[2] = 22.81; p < 0.05). Variation between the core diets
of the four study species accounted for 22.8% of the
total variation, while the rest (77.2%) was due to within-group variance. Hipposideros
hypophyllus and H. speoris consumed 17 taxa each, while H.
durgadasi consumed eight taxa, and H. fulvus consumed six taxa of
insects. Pairwise comparisons using Dunn’s test highlighted a significant
difference (p < 0.05) between the diet of Hipposideros speoris and
that of H. hypophyllus and H. fulvus. There was no overlap in the
whole and core diets between the four study species
(Table 1), and all four species showed a high degree of specialization in the
dietary items they consumed in both whole and core diets (Table 2).
Core diets
In the core diets of all four
hipposiderid bat species, prey belonging to orders Lepidoptera and Diptera were
consumed to a greater extent in comparison to other insect orders which is common
for any bat species (Ingala et al. 2021). The core diet of Hipposideros
hypophyllus consisted of four insect orders with lepidopterans figuring
majorly in its diet with six families followed by the three other insect orders
(Image 5). The core diet of Hipposideros durgadasi consisted of taxa
exclusively belonging to the orders Lepidoptera (4 families) and Diptera (1
family) (Image 6). H. fulvus fed on five insect orders including two
families of lepidopterans and the other insect orders in equal measure (Image
7). While the core diet of H. speoris was very diverse and comprised
eight insect orders lepidopterans were fed upon more in comparison to the rest
of the insect orders (Image 8).
Amongst the lepidopteran families
consumed by these four species of hipposiderid bats, the most consumed was the
family Noctuidae, followed by Pyralidae, Gelechiidae, Geometridae,
Papilionidae, and Elachistidae. The least consumed families were Tineidae and
Nolidae (Image 9). Hipposideros hypophyllus preferred prey items
belonging to the family Noctuidae (30.90%), followed by Papilionidae (7.89%),
Elachistidae (6.70%), Pyralidae (5.03%). Prey items belonging to families
Geometridae and Nolidae combined contributed to 3.59% of the diet of this
species. H. durgadasi consumed prey items belonging to the family
Pyralidae (43.9%), followed by Noctuidae (25.85%), Geometridae (10.58%), and
Tineidae (1.65%). H. fulvus consumed prey items belonging to the family
Gelechiidae (16.95%), followed by Noctuidae (2.67%). The least consumption of
lepidopteran prey was by H. speoris with the family Pyralidae (3.39%),
Gelechiidae (1.78%), and Noctuidae and an unclassified family (combined
amounting to 2.05%).
Among the dipteran families,
these hipposiderid bat species consumed families Chironomidae, Muscidae,
Culicidae, Asilidae, Calliphoridae, Sciaridae, and Tabanidae (Image 10). Hipposideros
hypophyllus consumed the family Chironomidae (16.20%), followed by Asilidae
(2.88%), and Sciaridae (1.35%). H. durgadasi consumed the least number
of dipterans and fed only on the family Calliphoridae (2.30%). Similarly, H.
fulvus fed only on the family Muscidae (19.68%), while H. speoris
fed on Culicidae (5.86%), Chironomidae (3.94%), and Tabanidae (1.10%).
Diversity indices
The degree to which a species
influences other species in the community (or makes up most of the biomass) is
quantified by the dominance index (Di), where Di ≤ 0.50 indicates no species
dominates, 0.50–0.75 indicates moderate dominance of a given species, and
0.75–1 indicates high dominance. None of the study species were found to be
dominant in their whole diet [Di (H. fulvus) = 0.31; (H. durgadasi)
= 0.23; (H. speoris) = 0.16; (H. hypophyllus) = 0.09]. Diet
species diversity is quantified by Simpson’s diversity index, where an index of
≤ 0.50 indicates low diversity, between 0.50–0.75 indicates moderate diversity,
and between 0.75–1 indicates high diversity. Simpson’s diversity index for the
whole diet showed a high diversity of diet in Hipposideros hypophyllus
(0.91), followed by H. speoris (0.84), and H. durgadasi (0.76).
However, the diet of H. fulvus was relatively less diverse (0.69).
Shannon’s H index of species diversity ranges between 1.5 and 3.5 and rarely
exceeds 4.5; the Shannon index of the prey species in the four
study species’ whole diets shows that Hipposideros hypophyllus (H
= 3.05) fed on highly diverse dietary items, followed by H. speoris (H =
2.75), H. durgadasi (H = 2.38), and H. fulvus (H = 1.75).
In the core diets of the four study species, the dominance index (Di) showed no
dominant species [Di (H. fulvus) = 0.37; (H. durgadasi) = 0.33; (H.
speoris) = 0.21; (H. hypophyllus) = 0.12]. Simpson’s diversity index
showed a high diversity in the core diets of H. hypophyllus (0.87) and H.
speoris (0.79), and moderate diversity in H. durgadasi (0.67) and H.
fulvus (0.63). Shannon’s H index showed that H. hypophyllus (H =
2.39) and H. speoris (H = 2.05) had a diverse diet, while H.
durgadasi (H = 1.44) and H. fulvus (H = 1.22) had a relatively low
diversity of insect species in their core diets. The core diets of H.
hypophyllus and H. speoris included six species of pest insects
each, while that of H. durgadasi and H. fulvus included three
insect pest species each (Supplementary Table 3).
Seasonal variations in
Hipposideros hypophyllus diet
The whole diet of our sampled H.
hypophyllus individuals (N = 4) over summer, monsoon, and winter seasons
showed no significant difference (Welch F-test: F = 2.79, p >
0.05). In the summer season, the diet was dominated by lepidopteran prey
species (35.81%), followed by the orders Diptera (31.80%), Blattodea (20.72%),
Coleoptera (2.96%), Mantodea (2.52%), followed by other insect orders. The
monsoon diet was dominated by Lepidoptera (92.80%), followed by Diptera (1.5%)
and other insect orders. The winter diet was similarly dominated by the order
Lepidoptera (85.82%), followed by Diptera (12.86%) and other insect orders
(Image 11). The core diet of H. hypophyllus over the three seasons
showed a significant difference (Welch F-test: F = 3.99, p =
0.05). The summer diet of H. hypophyllus sampled in the study period was
dominated by the order Lepidoptera (32.70%), followed by Diptera (29.37%),
Blattodea (20.72%), and Coleoptera (2.12%). In the monsoon, the diet was
comprised mostly by lepidopteran prey (74%), while in the winter it consisted
of orders Lepidoptera (82.68%) and Diptera (12.58%) (Image 12).
Discussion
Despite the diversity of
insectivorous bats in southern Asia (Srinivasulu et al. 2025), studies on
insectivorous bat diets are sporadic and scattered, often employing either gut
content analysis (Advani 1981) or faecal pellet dissection (Whitaker
Jr. 1988), which is more common due to its non-invasive nature (Kunz &
Whitaker Jr. 1983; Dickman & Huang 1988; Whitaker Jr. 1988). Studies
conducted on the faecal pellets or echolocation of some roundleaf bats in
southern Asia have provided a broad understanding of their diets. For instance,
studies based on the flight behaviour and echolocation calls of H. fulvus
mentioned that it may prefer feeding on cockroaches and beetles, and on insects
of intermediate size to avoid competition with other sympatric species such as H.
ater and H. speoris (Madhavan et al. 1978; Jones et al. 1994).
Khajuria (1980) states that the diet of H. durgadasi is similar to H.
fulvus, and they likely feed on small insects such as beetles and small
crickets. H. speoris was observed to feed on mosquitoes and flies
(Brosset 1962), and beetles and other low-flying insects, especially termites
(Phillips 1980). Jones et al. (1994) predicted it to feed on large-bodied
insects based on its echolocation. There have been no studies yet on the diet of
the Kolar Roundleaf Bat, however, Kock & Bhat (1994) mention that they
observed fat deposits at the base of the tail of the species between the months
of November and February.
The number of identifiable insect
taxa is limited in a microscopic examination of faecal pellets due to the level
of fragmentation of insect parts, making it rarely possible to identify insect
remnants beyond order and, at times, family level (Hubancheva et al. 2023). DNA
metabarcoding of bat guano provides higher-resolution appraisal of diet, often
up to species level (Hubancheva et al. 2023; Liu et al. 2023; Ling et al.
2025). This has resulted in faecal DNA analysis becoming increasingly common in
ecological, dietary, spatial, and phylogenetic studies (Thuo et al. 2019; Ando
et al. 2020; Guan et al. 2020; Ingala et al. 2021; Hubancheva et al. 2023; Liu
et al. 2023; Treloar et al. 2023; Ling et al. 2025), emphasising its
effectiveness in helping to make informed conservation decisions. However,
metabarcoding studies are not without limitations. Metabarcoding analyses of
diet are still in the nascent stages in southern Asia, and none yet have been
conducted on Indian bats. This technique is limited by resource and equipment
costs, human resource expenditure, and computational limitations due to the
need to effectively analyse many gigabytes of data – these costs are
significantly lower in physical faecal dissection-based diet analyses. However,
with the advent of more accessible and portable technologies (e.g., nanopore
sequencing), this suite of techniques may grow more methodologically and
economically feasible in southern Asia in the near future.
Our exploration of the diets of
some individuals of the Kolar Roundleaf Bat Hipposideros hypophyllus and
its congeners, the Fulvous Roundleaf Bat H. fulvus, Khajuria’s Roundleaf
Bat H. durgadasi, and Schneider’s Roundleaf Bat H. speoris
revealed highly similar whole diets, likely due to sharing a colony and
therefore a resource space, but significant differences between the core diets
(i.e., the most frequent prey items) in H. hypophyllus & H.
speoris and H. speoris & H. fulvus. Further, no species
dominated in terms of prey abundance, and Simpson’s and Shannon’s diversity
indices of prey species in core diets indicated that the Kolar Roundleaf Bat
likely preys on a wide diversity of invertebrate species. The core diet of the
Kolar Roundleaf Bat also varied significantly between the three seasons
(Summer, monsoon, & winter) sampled across one year, but in all seasons was
dominated by lepidopteran prey.
Interestingly, there were some
medically and agriculturally significant pests seen in the prey diversity of
the four study species. For instance, all species were
seen to feed on mosquitoes of Aedes, Anopheles, and Culex
genera, known to carry various significant diseases (Achee et al. 2015; Choo &
Blackwood 2017; Wilkman et al. 2023; Lim et al. 2025). Hipposideros
durgadasi was the biggest suppressor of Tetranychus urticae
(Trombidiformes: Tetranychidae), a mite that infests many vegetable crops and Withania
somnifera, a medicinal plant (Mason 2001). Cockroaches, vectors of many
viral and bacterial diseases (Donkor 2020), were majorly fed upon by H.
hypophyllus and H. fulvus. Hishimonus phycitis (Hemiptera:
Cicadellidae), a major hemipteran pest of citrus and brinjal (EFSA
Panel on Plant Health et al. 2017), was suppressed mostly by H. speoris.
Finally, the tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae),
a highly invasive tomato pest (Biondi et al. 2018), was majorly predated upon
by H. fulvus and H. hypophyllus. However, natural predators of
pests were also fed upon by our study species, including predatory mosquitoes Lutzia
fuscana (Diptera: Culicidae; Surendran et al. 2013; Mudi et al. 2025),
hatchet wasps Prosevania sp. (Hymenoptera: Evaniidae) which are
predators and parasites of cockroaches (Tee & Lee 2017), Ichneumonid wasps
of the genus Leptobatopsis (Hymenoptera: Ichneumonidae; Quicke et al.
2023), and Megastigmus viggianii (Hymenoptera: Megastigmidae), a natural
suppressor of the Eucalyptus gall wasp Leptocybe invasa (Mendel et al.
2017). However, the bats sampled were seen to suppress pests much more
effectively than their insect predators.
It is important to note that this
study is only an initial exploration of the diets of these four species,
limited to a small sample of four individuals per species per season, and three
seasons over one cycle in one site – such a small sample size inherently leads
to potential biases in prey species detectability through primer bias, and the
diversity reported in our study may not represent the entire dietary breadth of
our study species. Given the nascent state of DNA metabarcoding as a method for
non-invasive assessment of biodiversity in southern Asia, a limited sample size
also hampers the generalisability of this study, which insofar may only serve
as a foundational piece of research on bat diets using genetic techniques.
Further research using DNA metabarcoding is needed to clarify and confirm prey
diversity patterns and seasonal variations in these and other bat species, and
replicated metabarcoding studies are required across peninsular India and
southern Asia as a whole to establish standard practices for these techniques.
Our study explores the diets of these four roundleaf bats, identifying their
importance as suppressors of medical and agricultural pests, and emphasising
the effectiveness and importance of non-invasive assessment of bat diets in
helping to guide future conservation research and planning. In the case of such
sensitive roosts as Hanumanhalli Betta and threatened species such as the Kolar
Roundleaf Bat and Durga Das’ Roundleaf Bat, a non-invasive approach towards
understanding their ecological interactions, appraising their ecosystem
services (especially pest suppression in the case of insectivorous bats), and
assessing these against the threats they face is vital for effective
conservation, ensuring the long-term survival of these irreplaceable species.
Data availability statement
All relevant data are within the
manuscript and its Supporting Information files. The BioProject accession
number is PRJNA1281322.
Table 1.
Niche overlap (Pianka’s overlap index – 0 indicates no overlap, 1 indicates
full overlap; top right values represent the whole diet; bottom left values
represent the core diet) of the four Hipposideros bat species in the
study.
|
Species |
H. hypophyllus |
H. durgadasi |
H. fulvus |
H. speoris |
|
H. hypophyllus |
- |
0.03183 |
0.02088 |
0.00086 |
|
H. durgadasi |
0.02586 |
- |
0.00012 |
0.00110 |
|
H. fulvus |
0.01580 |
0.0 |
- |
0.00116 |
|
H. speoris |
0.0 |
0.0 |
0.0 |
- |
Table 2.
Niche breadth (Levins’ index – 0 indicates specialism, 1 indicates generalism;
top row represents the whole diet, bottom row represents the core diet) of the
four Hipposideros bat species in the study.
|
Species |
H. hypophyllus |
H. durgadasi |
H. fulvus |
H. speoris |
|
Whole diet |
0.00237 |
0.00134 |
0.00115 |
0.00448 |
|
Core diet |
0.42540 |
0.25349 |
0.28695 |
0.21752 |
For
images - - click here for full PDF
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