Biodiversity Data Journal :
Research Article
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Corresponding author: Badrul Munir Md-Zain (abgbadd1966@yahoo.com)
Academic editor: Chloe Robinson
Received: 30 Jun 2022 | Accepted: 19 Sep 2022 | Published: 20 Oct 2022
© 2022 Nor Hafisa Syafina Mohd-Radzi, Kayal Vizi Karuppannan, Nurfatiha Akmal Fawwazah Abdullah-Fauzi, Abd Rahman Mohd-Ridwan, Nursyuhada Othman, Abdul-Latiff Muhammad Abu Bakar, Millawati Gani, Mohd Firdaus Ariff Abdul-Razak, Badrul Munir Md-Zain
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mohd-Radzi NHS, Karuppannan KV, Abdullah-Fauzi NAF, Mohd-Ridwan AR, Othman N, Muhammad Abu Bakar A-L, Gani M, Abdul-Razak MFA, Md-Zain BM (2022) Determining the diet of wild Asian elephants (Elephas maximus) at human–elephant conflict areas in Peninsular Malaysia using DNA metabarcoding. Biodiversity Data Journal 10: e89752. https://doi.org/10.3897/BDJ.10.e89752
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Human–elephant conflict (HEC) contributes to the increasing death of Asian elephants due to road accidents, retaliatory killings and fatal infections from being trapped in snares. Understanding the diet of elephants throughout Peninsular Malaysia remains crucial to improve their habitat quality and reduce scenarios of HEC. DNA metabarcoding allows investigating the diet of animals without direct observation, especially in risky conflict areas. The aim of this study was to determine: i) the diet of wild Asian elephants from HEC areas in Peninsular Malaysia using DNA metabarcoding and ii) the influence of distinct environmental parameters at HEC locations on their feeding patterns. DNA was extracted from 39 faecal samples and pooled into 12 groups representing the different sample locations: Kuala Koh, Kenyir, Ulu Muda, Sira Batu, Kupang-Grik, Bumbun Tahan, Belum-Temengor, Grik, Kampung Pagi, Kampung Kuala Balah, Aring 10 and the National Elephant Conservation Centre, which served as a positive control for this study. DNA amplification and sequencing targeted the ribulose-bisphosphate carboxylase gene using the next-generation sequencing Illumina iSeq100 platform. Overall, we identified 35 orders, 88 families, 196 genera and 237 species of plants in the diet of the Asian elephants at HEC hotspots. Ficus (Moraceae), Curcuma (Zingiberaceae), Phoenix (Arecaceae), Maackia (Fabaceae), Garcinia (Clusiaceae) and Dichapetalum (Dichapetalaceae) were the highly abundant dietary plants. The plants successfully identified in this study could be used by the Department of Wildlife and National Parks (PERHILITAN) to create buffer zones by planting the recommended dietary plants around HEC locations and trails of elephants within Central Forest Spine (CFS) landscape.
Asian elephant, diet, rbcL, DNA metabarcoding, next-generation sequencing
Asian elephants (Elephas maximus) are charismatic animals that have been categorised as an endangered species by the International Union for Conservation of Nature Red List (
E. maximus are primarily threatened by the loss, fragmentation and degradation of habitat and poaching for ivory, skin, meat and leather (
Asian elephants are “mega-gardeners” of the Malaysian tropical rainforests (
Using genomics tools, the diet of elephants can be studied from the perspective of conservation. Metagenomics is the combination of next-generation sequencing (NGS) and DNA barcoding (
With HEC incidents increasing yearly (
1) Feacal Sampling
Feacal samples of E. maximus were provided by the Wildlife Genetic Resource Bank (WGRB) of PERHILITAN, who collected them from various localities in Peninsular Malaysia based on HEC complaints that were lodged by the public (Fig.
Map of Peninsular Malaysia with a close-up of various locations, where this study took place (1 = National Elephant Conservation Centre, 2 = Bumbun Tahan, 3 = Aring 10, 4 = Kg. Kuala Balah, 5 = Kg. Pagi, 6 = Kuala Koh, 7 = Kenyir, 8 = Kupang-Grik, 9 = Belum-Temenggor, 10 = Grik, 11 = Ulu Muda, 12 = Sira Batu).
2) DNA extraction and amplification
Laboratory work was performed at NWFL, PERHILITAN. Approximately 150 mg of the feacal sample was subjected to DNA extraction using the Qiagen QIAamp Fast DNA Stool mini kit (Qiagen, Germany). The extracted DNA was quantified spectrophotometrically on an Implen Nanophotometer. In this study, the 39 samples were pooled into 11 different DNA extracts corresponding to the different HEC localities with distinct environmental parameters, with one additional pooled sample representing the positive control (captivity) (
Polymerase chain reaction (PCR) for Illumina sequencing was performed twice. The first PCR was to amplify the targeted region of the ribulose-bisphosphate carboxylase (rbcL) gene; the second PCR was to index the purified PCR products. The gene was amplified using the forward primer rbcLZ1: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGTCACCACAAACAGAGACTAAAGCAAGT-3’ and the reverse primer rbcL19b: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTCTTCAGGTGGAACTCCAG-3’ (
3) Library construction for NGS
The first PCR products were sent to GeneSeq Sdn. Bhd for library preparation and sequencing. They were purified using solid phase reversible immobilisation beads (
4) Bioinformatics and metabarcoding analysis
The quality filtering and demultiplexing of sequences were performed using the CLC Genomic Workbench software (CLC) (Qiagen, USA) at the Evolutionary and Conservation Genetic Laboratory of the Department of Technology and Natural Resources, Universiti Tun Hussein Onn Malaysia. Quality scores were initially assessed across the Illumina data using FASTQ files. The operational taxonomical units (OTUs) were clustered at 97% similarity and represented by a single sequence. Rarefaction curves were plotted with the number of OTUs observed at a given sequencing depth using CLC. The plant genus classification of the OTUs was performed against an rbcL plant database with a confidence threshold of 97%. Using PAST 4.02 software, the alpha diversity indices of Shannon and Chao-1 index estimators measured the plant species richness in the elephants’ diet. The relationship between the samples was established using principal coordinate analysis (PCoA) in PAST 4.02. Paired t-test and analysis of variance were conducted to measure the significance of beta diversity at P < 0.05. To evaluate dietary diversity relationships amongst HEC areas, a heatmap was constructed using 1000 bootstrap replications of Bray–Curtis measurements. A Venn diagram was created to determine the shared and unique OTUs between distinct environmental parameters of HEC areas at 97% similarity.
Faecal samples of E. maximus were provided by the Wildlife Genetic Resource Bank (WGRB) of PERHILITAN, who collected them from various localities in Peninsular Malaysia. All samples from Kuala Koh, Kenyir, Ulu Muda and Belum-Temenggor were collected by the Management and Ecology of Malaysian Elephants, University of Nottingham Malaysia. Samples were also obtained from HEC locations, such as logged forests, non-logged forests, highways, human settlements, near human settlements etc. Captive samples from the National Elephant Conservation Centre in Kuala Gandah, Pahang served as positive controls and baseline data for the diet of E. maximus. A total of 39 feacal samples were utilised, of which 33 were retrieved from 11 HEC areas and six were from captivity (Table
No |
Sample ID |
Origin |
Pooled samples |
Environmental parameters |
1 |
EM274 |
Kuala Koh, Kelantan |
KK |
Near human settlement |
2 |
EM276 |
Kuala Koh, Kelantan |
KK |
Near human settlement |
3 |
EM283 |
Kuala Koh, Kelantan |
KK |
Near human settlement |
4 |
EM152 |
Grik, Perak |
G |
Human settlement |
5 |
EM155 |
Grik, Perak |
G |
Human settlement |
6 |
EM159 |
Grik, Perak |
G |
Human settlement |
7 |
EM364 |
Kupang-Grik, Perak |
KG |
Highway |
8 |
EM365 |
Kupang-Grik, Perak |
KG |
Highway |
9 |
EM366 |
Kupang-Grik, Perak |
KG |
Highway |
10 |
EM367 |
Kupang-Grik, Perak |
KG |
Highway |
11 |
EM368 |
Kupang-Grik, Perak |
KG |
Highway |
12 |
EM425 |
Belum-Temenggor, Perak |
BT |
Lake side |
13 |
EM426 |
Belum-Temenggor, Perak |
BT |
Lake side |
14 |
EM427 |
Belum-Temenggor, Perak |
BT |
Lake side |
15 |
EM1363 |
Ulu Muda, Kedah |
UM |
Logged forest |
16 |
EM1365 |
Ulu Muda, Kedah |
UM |
Logged forest |
17 |
EM1370 |
Ulu Muda, Kedah |
UM |
Logged forest |
18 |
EM752 |
Kg. Pagi, Pahang |
KP |
Human trail |
19 |
EM755 |
Kg. Pagi, Pahang |
KP |
Human trail |
20 |
EM759 |
Kg. Pagi, Pahang |
KP |
Human trail |
21 |
EM739 |
Kg. Kuala Balah, Pahang |
KKB |
Human trail |
22 |
EM740 |
Kg. Kuala Balah, Pahang |
KKB |
Human trail |
23 |
EM668 |
Aring 10, Pahang |
A10 |
Human trail |
24 |
EM669 |
Aring 10, Pahang |
A10 |
Human trail |
25 |
EM679 |
Aring 10, Pahang |
A10 |
Animal trail |
26 |
EM781 |
Kenyir, Terengganu |
K |
Non-logged forest |
27 |
EM831 |
Kenyir, Terengganu |
K |
Non-logged forest |
28 |
EM832 |
Kenyir, Terengganu |
K |
Non-logged forest |
29 |
EM148 |
Bumbun Tahan. Pahang |
BB |
Non-logged forest |
30 |
EM149 |
Bumbun Tahan. Pahang |
BB |
Non-logged forest |
31 |
EM2167 |
Sira Batu, Kedah |
SB |
Non-logged forest |
32 |
EM2168 |
Sira Batu, Kedah |
SB |
Non-logged forest |
33 |
EM2169 |
Sira Batu, Kedah |
SB |
Non-logged forest |
34 |
EM1524 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
35 |
EM1531 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
36 |
EM1548 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
37 |
EM1541 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
38 |
EM1527 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
39 |
EM1537 |
National Elephant Conservation Centre (NECC), Pahang |
C |
Captivity |
All next-generation sequence data were deposited into National Center of Biotechnology Information (NCBI), under Sequence Read Archive (SRA) accession numbers; SRR19811599, SRR19806293, SRR19806081, SRR19806065, SRR19805810, SRR19805808, SRR19805784, SRR19805749, SRR19805748, SRR19804224, SRR19801341.
NGS data analysis
The concentration of the DNA extracted ranged from 3.2 ng/µl to 385.7 ng/µl. The quantity of DNA measured by the quality check assay was between 3.75 pM to 7.1 pM. High-throughput DNA metabarcoding was used to assess the specific plants consumed from different HEC locations. Illumina NGS successfully produced 379,580 reads, ranging from 4,865 to 99,383 sequences, which were filtered to exclude low-quality sequence reads and chimeras. Subsequently, the OTUs were clustered and 5,385 known OTUs were identified at the 97% similarity cut-off, with the highest in captivity (980) followed by Belum-Temenggor (923) and Kampung Pagi (835) (Table
Number of sequences, OTUs and unique OTUs of plants consumed by E. maximus.
Samples |
Sequences |
OTUs |
Unique OTUs |
KK |
7,769 |
71 |
17 |
KG |
7,448 |
272 |
55 |
K |
9,574 |
294 |
48 |
KP |
46,757 |
835 |
176 |
KKB |
34,625 |
648 |
175 |
G |
6,967 |
78 |
19 |
BT |
70,022 |
923 |
360 |
BB |
4,865 |
60 |
12 |
UM |
41,396 |
605 |
152 |
SB |
15,248 |
76 |
19 |
A10 |
35,526 |
543 |
85 |
C |
99,383 |
980 |
445 |
Total |
379,580 |
5385 |
1563 |
List of pooled samples according to distinct environmental parameters of all HEC areas.
No |
Pooled Samples |
Environmental Parameters |
1 |
Kuala Koh, Kelantan (KK) |
Human Settlements (HS) |
2 |
Grik, Perak (G) |
|
3 |
Belum-Temenggor, Perak (BT) |
Logged Forests (LF) |
4 |
Kupang-Grik, Perak (KG) |
|
5 |
Ulu Muda, Kedah (UM) |
|
6 |
Bumbun Tahan, Pahang (BB) |
Non-logged Forests (NLF) |
7 |
Kenyir, Terengganu (K) |
|
8 |
Sira Batu, Kedah (SB) |
|
9 |
Aring 10, Pahang (A10) |
Human Trails (HT) |
10 |
Kg. Kuala Balah, Pahang (KKB) |
|
11 |
Kg. Pagi, Pahang (KP) |
Number of sequences, OTUs and unique OTUs of plants eaten by elephants at different environmental parameters of HEC areas (HS = human settlement; LF = logged forest; NLF = non-logged forest; HT = human trail).
Samples |
Sequences |
OTUs |
Unique OTUs |
HS |
14,736 |
144 |
46 |
LF |
118,866 |
1,356 |
679 |
NLF |
29,687 |
412 |
83 |
HT |
116,908 |
1,414 |
792 |
Total |
280,197 |
3,326 |
1,600 |
Plant species identification
The plants consumed by all E. maximus sampled in this study were taxonomically classified into 35 orders, 88 families, 196 genera and 237 species (Table
Taxonomic classification of plants consumed by E. maximus according to rbcL gene analysis.
Taxonomic level |
Total number |
Order |
35 |
Family |
88 |
Genus |
196 |
Species |
237 |
Distribution (%) of plants consumed by E. maximus at the genus level (20 most abundant genera). (KK = Kuala Koh; KG = Kupang-Grik; K = Kenyir; KP = Kg. Pagi; KKB = Kg. Kuala Balah; G = Grik; BT = Belum-Temenggor; BB = Bumbun Tahan; UM = Ulu Muda; A10 = Aring 10; SB = Sira Batu; C = Captive; N/A = Not Available).
Alpha diversity indices, rarefaction curve, heatmap and Venn diagram
The alpha diversity (Shannon and Chao-1 indices) indicated that the diet of Asian elephants varied depending on the HEC localities (Table
Samples |
Shannon_H |
Chao-1 |
KK |
1.112 |
141 |
KG |
2.763 |
326.3 |
K |
2.58 |
411.8 |
KP |
2.983 |
1,212 |
KKB |
2.267 |
968.9 |
G |
1.806 |
303.6 |
BT |
2.702 |
1,056 |
BB |
1.162 |
93.83 |
UM |
2.730 |
913.4 |
SB |
0.746 |
193.2 |
A10 |
1.998 |
828.3 |
C |
2.811 |
1,045 |
Alpha diversity indices of Shannon and Chao-1 values at different environmental parameters in HEC areas.
Samples |
Shannon_H |
Chao-1 |
HS |
2.020 |
427.6 |
LF |
3.033 |
1,551 |
NLF |
2.400 |
614.8 |
HT |
3.006 |
1,870 |
Paired t-test diversity statistical analysis of plant diets in different environmental parameters, based on Shannon indices (HS = human settlement; LF = logged forest; NLF = non-logged forest; HT = human trail).
Pairing |
t |
df |
p-value |
LF–HS |
-69.212 |
23029 |
0 |
LF–NLF |
-48.916 |
52632 |
0 |
LF–HT |
-2.9376 |
2.34E+05 |
0.003 |
HT–NLF |
-47.93 |
48619 |
0 |
HT–HS |
-68.583 |
21555 |
0 |
HS–NLF |
-22.167 |
34775 |
4.00E-108 |
Percentage of plants relative abundance consumed by Asian elephants at studied HEC areas at family level (N/A = not available) (> 0.1% abundance).
No |
Family |
BT |
BB |
A10 |
C |
G |
K |
KG |
KK |
KKB |
KP |
SB |
UM |
Total |
1 |
N/A |
6.21 |
2.52 |
3.97 |
45.84 |
3.61 |
3.68 |
3.82 |
4.03 |
5.45 |
8.89 |
7.91 |
4.07 |
50.77 |
2 |
Moraceae |
46.39 |
0.00 |
0.02 |
0.10 |
0.00 |
3.61 |
0.00 |
0.00 |
29.19 |
1.98 |
0.00 |
18.71 |
17.52 |
3 |
Zingiberaceae |
0.23 |
0.00 |
62.36 |
11.01 |
0.00 |
0.00 |
0.01 |
0.00 |
7.45 |
18.92 |
0.00 |
0.02 |
11.41 |
4 |
Arecaceae |
18.95 |
0.00 |
0.14 |
1.34 |
0.00 |
0.14 |
0.03 |
0.00 |
2.83 |
46.62 |
0.00 |
29.95 |
9.25 |
5 |
Fabaceae |
72.27 |
0.03 |
0.02 |
18.54 |
0.00 |
0.05 |
0.02 |
0.00 |
0.09 |
2.75 |
0.00 |
6.22 |
3.40 |
6 |
Clusiaceae |
3.25 |
0.00 |
0.03 |
0.03 |
0.00 |
0.00 |
0.00 |
0.00 |
0.03 |
0.00 |
0.00 |
96.67 |
0.93 |
7 |
Dichapetalaceae |
76.96 |
0.00 |
0.03 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.03 |
0.00 |
0.00 |
22.98 |
0.89 |
8 |
Nelumbonaceae |
14.15 |
0.00 |
0.00 |
32.16 |
0.00 |
0.00 |
0.00 |
0.00 |
4.65 |
2.72 |
0.00 |
46.31 |
0.53 |
9 |
Bromeliaceae |
1.03 |
0.00 |
0.05 |
0.59 |
0.00 |
0.00 |
0.00 |
0.00 |
6.81 |
2.22 |
0.00 |
89.30 |
0.49 |
10 |
Myristicaceae |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.06 |
0.00 |
0.00 |
99.94 |
0.47 |
11 |
Hypoxidaceae |
0.17 |
0.00 |
0.62 |
0.11 |
0.00 |
0.00 |
0.00 |
0.00 |
0.17 |
98.70 |
0.00 |
0.23 |
0.46 |
12 |
Poaceae |
23.10 |
0.00 |
43.13 |
30.06 |
0.00 |
0.00 |
0.00 |
0.00 |
1.31 |
2.17 |
0.00 |
0.23 |
0.46 |
13 |
Asteraceae |
32.01 |
0.00 |
0.27 |
44.34 |
0.00 |
0.07 |
0.00 |
0.00 |
0.20 |
22.64 |
0.00 |
0.47 |
0.39 |
14 |
Datiscaceae |
98.32 |
0.00 |
0.00 |
0.23 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.46 |
0.00 |
1.00 |
0.34 |
15 |
Musaceae |
38.80 |
0.00 |
2.97 |
8.27 |
0.00 |
0.08 |
0.00 |
0.00 |
8.11 |
37.83 |
0.00 |
3.94 |
0.33 |
16 |
Convolvulaceae |
0.62 |
0.00 |
0.00 |
98.75 |
0.00 |
0.18 |
0.00 |
0.00 |
0.09 |
0.09 |
0.00 |
0.27 |
0.30 |
17 |
Celastraceae |
99.80 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.10 |
0.00 |
0.00 |
0.10 |
0.26 |
18 |
Solanaceae |
100.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.25 |
19 |
Metteniusaceae |
66.50 |
0.12 |
0.12 |
14.96 |
0.00 |
0.49 |
0.49 |
0.00 |
0.62 |
16.56 |
0.00 |
0.12 |
0.21 |
20 |
Theaceae |
99.87 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.13 |
0.20 |
21 |
Nothofagaceae |
28.53 |
0.00 |
0.00 |
3.57 |
0.00 |
0.16 |
0.00 |
0.00 |
6.05 |
8.37 |
0.00 |
53.33 |
0.17 |
22 |
Pandaceae |
66.75 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
33.25 |
0.11 |
Percentage of plants relative abundance consumed by Asian elephants at studied HEC areas at genus level (N/A = not available) (> 0.1% abundance).
No. |
Genus |
BT |
BB |
A10 |
C |
G |
K |
KG |
KK |
KKB |
KP |
SB |
UM |
Total |
1 |
N/A |
6.21 |
2.52 |
3.97 |
45.84 |
3.61 |
3.68 |
3.82 |
4.03 |
5.45 |
8.89 |
7.91 |
4.07 |
50.77 |
2 |
Ficus |
46.39 |
0.00 |
0.02 |
0.10 |
0.00 |
3.64 |
0.00 |
0.00 |
29.21 |
1.96 |
0.00 |
18.68 |
17.39 |
3 |
Curcuma |
0.21 |
0.00 |
62.39 |
11.02 |
0.00 |
0.00 |
0.01 |
0.00 |
7.45 |
18.90 |
0.00 |
0.02 |
11.39 |
4 |
Phoenix |
18.97 |
0.00 |
0.13 |
1.32 |
0.00 |
0.14 |
0.02 |
0.00 |
2.62 |
46.58 |
0.00 |
30.21 |
8.98 |
5 |
Maackia |
90.40 |
0.04 |
0.03 |
0.44 |
0.00 |
0.07 |
0.02 |
0.00 |
0.05 |
2.91 |
0.00 |
6.03 |
2.52 |
6 |
Garcinia |
3.25 |
0.00 |
0.03 |
0.03 |
0.00 |
0.00 |
0.00 |
0.00 |
0.03 |
0.00 |
0.00 |
96.66 |
0.93 |
7 |
Dichapetalum |
76.96 |
0.00 |
0.03 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.03 |
0.00 |
0.00 |
22.98 |
0.89 |
8 |
Wisteria |
15.46 |
0.00 |
0.00 |
76.25 |
0.00 |
0.00 |
0.00 |
0.00 |
0.23 |
1.59 |
0.00 |
6.47 |
0.81 |
9 |
Nelumbo |
14.15 |
0.00 |
0.00 |
32.16 |
0.00 |
0.00 |
0.00 |
0.00 |
4.65 |
2.72 |
0.00 |
46.31 |
0.53 |
10 |
Myristica |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.06 |
0.00 |
0.00 |
99.94 |
0.47 |
11 |
Curculigo |
0.00 |
0.00 |
0.63 |
0.11 |
0.00 |
0.00 |
0.00 |
0.00 |
0.17 |
99.09 |
0.00 |
0.00 |
0.46 |
12 |
Borrichia |
31.81 |
0.00 |
0.27 |
44.66 |
0.00 |
0.00 |
0.00 |
0.00 |
0.21 |
22.71 |
0.00 |
0.34 |
0.39 |
13 |
Datisca |
98.32 |
0.00 |
0.00 |
0.23 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.46 |
0.00 |
1.00 |
0.34 |
14 |
Ensete |
39.98 |
0.00 |
1.08 |
8.36 |
0.00 |
0.08 |
0.00 |
0.00 |
7.78 |
38.66 |
0.00 |
4.06 |
0.32 |
15 |
Camonea |
0.00 |
0.00 |
0.00 |
100.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.29 |
16 |
Guzmania |
0.09 |
0.00 |
0.09 |
0.37 |
0.00 |
0.00 |
0.00 |
0.00 |
9.59 |
0.00 |
0.00 |
89.86 |
0.29 |
17 |
Loeseneriella |
99.79 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.10 |
0.00 |
0.00 |
0.10 |
0.26 |
18 |
Nicotiana |
100.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.24 |
19 |
Rhaphiostylis |
66.50 |
0.12 |
0.12 |
14.96 |
0.00 |
0.49 |
0.49 |
0.00 |
0.62 |
16.56 |
0.00 |
0.12 |
0.21 |
20 |
Ottochloa |
0.51 |
0.00 |
93.13 |
4.96 |
0.00 |
0.00 |
0.00 |
0.00 |
0.64 |
0.25 |
0.00 |
0.51 |
0.21 |
21 |
Rhapis |
20.52 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
3.12 |
53.25 |
0.00 |
23.12 |
0.20 |
22 |
Franklinia |
99.87 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.13 |
0.20 |
23 |
Fuscospora |
28.31 |
0.00 |
0.00 |
3.60 |
0.00 |
0.16 |
0.00 |
0.00 |
5.56 |
7.86 |
0.00 |
54.50 |
0.16 |
24 |
Werauhia |
2.18 |
0.00 |
0.00 |
0.54 |
0.00 |
0.00 |
0.00 |
0.00 |
0.91 |
5.44 |
0.00 |
90.93 |
0.15 |
25 |
Morus |
45.73 |
0.00 |
0.00 |
0.21 |
0.00 |
0.00 |
0.00 |
0.00 |
26.07 |
4.70 |
0.00 |
23.29 |
0.12 |
26 |
Echinochloa |
91.76 |
0.00 |
0.23 |
7.55 |
0.00 |
0.00 |
0.00 |
0.00 |
0.23 |
0.23 |
0.00 |
0.00 |
0.12 |
27 |
Microdesmis |
66.58 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
33.42 |
0.11 |
28 |
Cylicomorpha |
64.64 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
21.64 |
0.00 |
13.72 |
0.10 |
29 |
Ventilago |
100.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.10 |
OTU and percentage of plants plants eaten by elephants at different environmental parameters of HEC areas at top 30 genus level (N/A = not available) (HS = human settlement; LF = logged forest; NLF = non- logged forest; HT = human trail).
No. |
Genus |
DM |
% |
HP |
% |
HTP |
% |
PM |
% |
|||
1 |
N/A |
35 285 |
30.42 |
27 180 |
23.27 |
27 187 |
91.67 |
14 721 |
100.00 |
|||
2 |
Ficus |
20 588 |
17.75 |
42 955 |
36.78 |
2402 |
8.10 |
0 |
0.00 |
|||
3 |
Curcuma |
38 371 |
33.08 |
103 |
0.09 |
2 |
0.01 |
0 |
0.00 |
|||
4 |
Phoenix |
16 816 |
14.50 |
16 773 |
14.36 |
50 |
0.17 |
0 |
0.00 |
|||
5 |
Maackia |
286 |
0.25 |
9211 |
7.89 |
11 |
0.04 |
0 |
0.00 |
|||
6 |
Garcinia |
2 |
0.00 |
3532 |
3.02 |
0 |
0.00 |
0 |
0.00 |
|||
7 |
Dichapetalum |
2 |
0.00 |
3392 |
2.90 |
0 |
0.00 |
0 |
0.00 |
|||
8 |
Myristica |
1 |
0.00 |
1781 |
1.52 |
0 |
0.00 |
0 |
0.00 |
|||
9 |
Curculigo |
1749 |
1.51 |
0 |
0.00 |
0 |
0.00 |
0 |
0.00 |
|||
10 |
Nelumbo |
149 |
0.13 |
1222 |
1.05 |
0 |
0.00 |
0 |
0.00 |
|||
11 |
Datisca |
6 |
0.01 |
1297 |
1.11 |
0 |
0.00 |
0 |
0.00 |
|||
12 |
Ensete |
574 |
0.49 |
532 |
0.46 |
1 |
0.00 |
0 |
0.00 |
|||
13 |
Guzmania |
105 |
0.09 |
976 |
0.84 |
0 |
0.00 |
0 |
0.00 |
|||
14 |
Loeseneriella |
1 |
0.00 |
967 |
0.83 |
0 |
0.00 |
0 |
0.00 |
|||
15 |
Nicotiana |
0 |
0.00 |
924 |
0.79 |
0 |
0.00 |
0 |
0.00 |
|||
16 |
Borrichia |
339 |
0.29 |
470 |
0.40 |
0 |
0.00 |
0 |
0.00 |
|||
17 |
Rhapis |
434 |
0.37 |
336 |
0.29 |
0 |
0.00 |
0 |
0.00 |
|||
18 |
Franklinia |
0 |
0.00 |
768 |
0.66 |
0 |
0.00 |
0 |
0.00 |
|||
19 |
Ottochloa |
739 |
0.64 |
8 |
0.01 |
0 |
0.00 |
0 |
0.00 |
|||
20 |
Wisteria |
56 |
0.05 |
675 |
0.58 |
0 |
0.00 |
0 |
0.00 |
|||
21 |
Rhaphiostylis |
140 |
0.12 |
543 |
0.46 |
5 |
0.02 |
0 |
0.00 |
|||
22 |
Fuscospora |
82 |
0.07 |
506 |
0.43 |
1 |
0.00 |
0 |
0.00 |
|||
23 |
Werauhia |
35 |
0.03 |
513 |
0.44 |
0 |
0.00 |
0 |
0.00 |
|||
24 |
Morus |
144 |
0.12 |
323 |
0.28 |
0 |
0.00 |
0 |
0.00 |
|||
25 |
Microdesmis |
0 |
0.00 |
407 |
0.35 |
0 |
0.00 |
0 |
0.00 |
|||
26 |
Echinochloa |
3 |
0.00 |
401 |
0.34 |
0 |
0.00 |
0 |
0.00 |
|||
27 |
Cylicomorpha |
82 |
0.07 |
297 |
0.25 |
0 |
0.00 |
0 |
0.00 |
|||
28 |
Ventilago |
0 |
0.00 |
377 |
0.32 |
0 |
0.00 |
0 |
0.00 |
|||
29 |
Sinningia |
0 |
0.00 |
332 |
0.28 |
0 |
0.00 |
0 |
0.00 |
|||
30 |
Camonea |
0 |
0.00 |
0 |
0.00 |
0 |
0.00 |
0 |
0.00 |
Figs
Heatmap with dendrogram showing dietary plant abundance at the genus level for E. maximus in different HEC localities. Gradient of the heatmap shows the 20 most abundant genera. KK = Kuala Koh; KG = Kupang-Grik; K = Kenyir; KP = Kg. Pagi; KKB = Kg. Kuala Balah; G = Grik; BT = Belum-Temenggor; BB = Bumbun Tahan; UM = Ulu Muda; A10 = Aring 10; SB = Sira Batu; C = Captive; N/A = Not Available.
Heatmap with dendrogram showing dietary plant abundance at the genus level for E. maximus in different environmental parameters. Gradient of the heatmap shows the 30 most abundant genera. HS = human settlement; LF = logged forest; NLF = non-logged forest; HT = human trail; N/A = Not Available.
The Venn diagram in Fig.
Principal coordinate analysis (PCoA) between Asian elephant samples from different HEC areas, based on Bray–Curtis distances. KK = Kuala Koh; KG = Kupang-Grik; K = Kenyir; KP = Kg. Pagi; KKB = Kg. Kuala Balah; G = Grik; BT = Belum-Temenggor; BB = Bumbun Tahan; UM = Ulu Muda; A10 = Aring 10; SB = Sira Batu; C = Captive.
Metabarcoding analysis proved that areas affected by HEC are very attractive to wild Asian elephants. In this study, we examined the diet of wild E. maximus from various HEC locations throughout Peninsular Malaysia, without any direct observation of the plants consumed. Previous studies have relied on indirect observations of feeding, including elephant footprints, fresh dung piles near browsed foliage and typical plant damage caused by elephant browsing, such as debarkation, branch breaking and uprooting (
According to our findings, the diversity and richness of the dietary plant taxa are correlated to the quality of an elephant’s habitat (F = 2.159, P = 0.014). Alpha diversity analysis demonstrated that KP presented the highest plant diversity, with a Shannon index value of 2.983 and a significantly superior species richness, with a Chao-1 value of 1,212 (Table
The most dominant plant genus Ficus (family Moraceae) is the elephant’s main diet preference at BT, UM and KKB. It is a good source of nutrition for fruit-eating animals like E. maximus in tropical areas. Figs are rich in fibres, trace minerals, antioxidant polyphenols, proteins, sugars, organic acids, cholesterol-free and contain high number of amino acids (
The genus Curcuma (family Zingiberaceae) is the second highest abundant plant genus consumed by wild Asian elephants in the HT of A10, KP and KKB at TNNP. It consists of rhizomatous herbs, such as ginger and turmeric, which are distributed in the tropical and subtropical regions of Southeast Asia, Papua New Guinea and northern Australia (
Secondary forests and areas with disturbed vegetation have been shown to attract wild Asian elephants and cause HEC (
On the other hand, KG, KK and G contained only a small percentage of the top 20 abundant plant genera. Besides grasses, samples retrieved from KG consisted of palm plants, like Elaeis oleifera and Burretiokentia hapala, which indicates the presence of palm plants near the highway.
Metabarcoding analysis followed by t-test and analysis of variance revealed significant differences in the diets of wild Asian elephants according to environmental parameters (F = 3.002, P = 0.029). Elephants were attracted to the diversity of plants in disturbed vegetation locations, such as LF (H = 3.033). In Peninsular Malaysia, secondary forests are areas that are highly suitable for elephant habitats (
Feacal samples were collected from HT of three HEC sites in the primary rainforests of TNNP, namely KP, KKB and A10. HT presented the highest species richness (Chao-1 = 1,870) and a significant diversity index (H = 3.006). Vegetation in these regions showed the maximum number of OTUs (1,414) and unique OTUs (792) compared with other environmental parameters studied. The dominant plant genera in HT were Curcuma, Ottochloa and Curculigo (Fig.
Feacal samples from NLF and HS mostly comprised unidentified plants (Fig.
Even though 50% of the plant families and genera could not be identified by the rbcL metabarcoding database, this study managed to list up to 237 plant species. The most abundant among them were: fig, Ficus sp. (17.4%); date palm, Phoenix dactylifera (9.0%); black ginger, Curcuma aeruginosa (8.0%); white turmeric, Curcuma zedoaria (2.9%); flowering plants, like Maackia floribunda (2.52%), Garcinia hopii (0.93%), Dichapetalum crassifolium (0.89%) and Wisteria floribunda (0.81%); and turmeric, Curcuma longa (0.51%). Most of the flowering plant species found are not native to Malaysia as the rbcL database tends to identify plants native to African, American and Asian countries. Therefore, the plant species recognised could be close relatives to those in the tropical rainforests of Malaysia (
As this was a pilot study, the number of feacal samples examined and HEC areas covered were limited due to the limited availability of stored E. maximus feces at WGRB. This study utilised the feces collected by PERHILITAN during opportunistic sampling at HEC areas between 2011 and 2021. Although the poor condition of certain feacal samples may have affected the quality of the extracted DNA or of the sequencing, the rbcL primer intron sequence of all 39 samples was successfully amplified. Prior studies have established that fresh feacal samples lead to high DNA quality and high percentage of sequencing reads (
This study examined plant diversity and richness in the diet of wild E. maximus at HEC localities throughout Peninsular Malaysia. DNA metabarcoding using NGS enabled us to identify dietary plant taxa at HEC locations up to the species level. The plant metabarcoding database can be used by PERHILITAN in building buffer zones with plant genera detected in HEC areas through habitat development programs. Future studies with increased periodic sampling at HEC localities are essential to completely understand the dietary diversity of the wild E. maximus. Future studies should also include sample of plants damaged by elephants browsing activity in the areas of fecal sample collection to confirm the identification of plant species with voucher samples in Herbarium at Forest Research Institute Malaysia (FRIM). Additional information on fecal freshness level, environmental conditions and surrounding vegetation at sampling sites could improve the quality of findings on metabarcoding diet of wild Asian elephants as well as the habitat enrichment programs within CFS landscape. Conservational efforts to improve the habitat of elephants may mitigate HEC cases and maintain the population of endangered E. maximus in Peninsular Malaysia. Metabarcoding using NGS is a useful tool to elucidate the dietary patterns of other significant herbivores to reduce human-wildlife conflicts all around the world.
We would like to take this opportunity to thank the Director General of the Department of Wildlife and National Parks (PERHILITAN), Peninsular Malaysia, YBhg. Dato' Abdul Kadir bin Abu Hashim and all the PERHILITAN staff involved. We are grateful to National Wildlife Forensic Laboratory (NWFL) and Universiti Kebangsaan Malaysia (UKM) for their great laboratory facilities. We would like to express our gratitude to the collaborators; Mr. Kaviarasu Munian from Forest Research Institute Malaysia (FRIM), Dr. Wong Ee Phin from University of Nottingham Malaysia, as well as Ms. Hidayah Haris and Ms. Hartini Suriyati from Universiti Tun Hussein Onn Malaysia (UTHM) for their enormous assistance throughout this project. This study was fully funded by Fundamental Research Grant Scheme (FRGS/1/2019/WAB13/NRE//1) and UKM grant ST-2020-002.
Fundamental Research Grant Scheme, Dana Luar UKM
Fundamental Research Grant Scheme (FRGS/1/2019/WAB13/NRE//1), UKM grant ST-2020-002 and GUP-2019-037
Department of Wildlife and National Parks (PERHILITAN), Peninsular Malaysia and Universiti Kebangsaan Malaysia
Research methods, reported in this manuscript, adhered to the legal requirements of Malaysia and were approved by Department of Wildlife and National Parks (PERHILITAN), Peninsular Malaysia, KM10 Jalan Cheras, Kuala Lumpur, Malaysia under research permit (JPHL&TN(IP):100-34/1.24 Jld 16 (18).
N.H.S.M.R, K.V.K. and B.M.M.Z. designed the project. N.H.S.M.R., K.V.K., N.A.F.A.F., M.G. and M.F.A.A.R were involved in field sample collection, collected, processed and analysed the samples. N.H.S.M.R. and N.A.F.A.F. performed the laboratory work. N.O and A.R.M.R. performed data analyses. N.H.S.M.R drafted the manuscript. M.A.B.A.L., A.R.M.R., K.V.K. and B.M.M.Z. reviewed and edited the manuscript. M.A.B.A.L., K.V.K. and B.M.M.Z. provided resources, project administration and funding acquisition. All authors read and approved the final manuscript.
The authors declare that they have no conflict of interests.