DPR for the Plan projects 2014-15
Centre for Wildlife Studies (Plan)
Head of Account: 205-2225
Project Title
:
DNA fingerprinting and phylogenetics of the endemic
and threatened biodiversity of Western Ghats and their
conservation
Principal Investigator
:
Dr. P.O. Nameer
Assoc. Professor and Head
Department of Wildlife Science
College of Forestry, KAU, Vellanikkara. 680656.
Associates / Collaborators
:
Dr. D Girija
Professor & Head
Department of Microbiology
College of Horticulture, KAU, Vellanikkara. 680656.
Centre
:
College of Forestry
Kerala Agricultural University
Vellanikkara. 680656.
Thrissur District, Kerala.
Budget Outlay
:
Rs. 103.6376 Lakhs
Type of scheme
:
Centre for Wildlife Studies (Plan)
Head of Account: 205-2225
if it is spill over give all details
:
NA
Period / Phrase
:
2014-15
Background / rationale
:
The Western Ghats hill range in South India contains
spectacular landscapes and an incredible array of wild species,
many found nowhere else in the world. One among the
world’s 34 most biologically diverse “hotspots”, the region has
representation of a wide variety of natural ecosystems from
grasslands and dry forests to rainforests, rivers, and streams,
threatened by a multitude of human activities such as
industrialisation, agriculture, grazing, hunting, deforestation,
fragmentation, and degradation. The hotspots are chosen for
their species richness, endemism, taxonomic uniqueness,
unusual ecological or evolutionary phenomena, and global
rarity. Today, rainforests in the Western Ghats occur largely as
fragments within a landscape matrix dominated by commercial
plantations of tea, coffee, and other cash crops. With an
annual deforestation rate of 1.2%, the southern Western
Ghats is losing about 500 square kilometers of forest every
year.
Biodiversity documentation of the Western Ghats has been
grossly inadequate. This is particularly true on the case of
lower formers of vertebrate fauna such as chiropteria,
rodentia, insectivore, and herpetaufauna. These organisms
play extremely important functions in the ecosystem
functioning and thus important for the mankind including the
farming community. They perform functions such as
pollination, seed dispersal, biological control of the various
pests and vermins etc.
Biodiversity is the variation of life forms within a given
ecosystem, biome or for the entire Earth. Biodiversity is often
used as a measure of the health of biological systems. Since
1986 the terms and the concept have achieved widespread
use among biologists, environmentalists, political leaders, and
concerned citizens worldwide. It is generally used to equate to
a concern for the natural environment and nature
conservation. This use has coincided with the expansion of
concern over extinction observed in the last decades of the
20th century.
Measurement of Biodiversity: Biodiversity is a broad concept,
so a variety of objective measures have been created in order
to empirically measure biodiversity. Each measure of
biodiversity relates to a particular use of the data. For practical
conservationists, this measure should quantify a value that is
broadly shared among locally affected people. For others, a
more economically defensible definition should allow the
ensuring of continued possibilities for both adaptation and
future use by people, assuring environmental sustainability.
Biodiversity is usually plotted as taxonomic richness of a
geographic area, with some reference to a temporal scale.
Whittaker described three common metrics used to measure
species-level biodiversity, encompassing attention to species
richness or species evenness: Species richness, Simpson index
and Shannon index.
The state of the art technique in documenting the biological
diversity is the molecular techniques combined with
phylogenitic studies. Thus it is proposed to take up a study on
the “DNA fingerprinting and phylogenetics of the endemic and
threatened biodiversity of Western Ghats and their
conservation”, with following objectives.
Objective
:
a. To DNA fingerprint the faunal biodiversity of the
Western Ghats, with particular emphasis on the
endemic and threatened species
b. To undertake phylogentic analysis of the molecular
assessment
c. To develop a database on the vertebrate biodiversity of
the Western Ghats
d. To find out the economic significance of these
biodiversity elements on the mankind in general and the
farming community in particular
Technical Programme
:
A: FIELD STUDIES:
Camera trap studies:
Camera traps were used to survey the small carnivores in the
study site in both the habitats. Since most of the small
carnivores are nocturnal animals, camera trapping is one of
the best methods to study them. Digital scout cameras having
passive infra-red sensors for heat and motion detection
(Wildview Xtreme 4 model no. STC-TGL4M) were used for this
survey (Plate. 2). Total 270 trap-nights (nine cameras X three
days X ten months) were carried out in the sanctuary during
the study period. Trap stations were selected randomly with
200m distance between the stations. Cameras were active for
12 hours (1800h to 0600h) in three consecutive days for ten
months and the locations were changed in every month.
Line-transect studies for both direct and indirect evidences:
Line transects of one kilometer length were selected randomly
in each habitat. Total 240 km (120 km in each habitat)
transect walk was carried out in the sanctuary ie., four
kilometer X five days X 12 months. Transects were surveyed
from 0700h to 1000h in the morning and 1500h to 1800h in
the evening. The direct sightings of the animals as well as
other indirect evidences which give the presence of the
animals were recorded in the prescribed datasheets
(Appendix I and II) during the transect walk.
Night spotlight studies
For some of the nocturnal mammals which are strictly arborial,
the day transects and camera trapping are ineffectual. Night
spotlight survey is an effective method for these animals. The
same transect used for direct and indirect evidence in day
time was used for spotlight survey also. A total of 120 km
(two kilometer X five days X 12 months) was surveyed as
night transect. It was carried out from 2000h to 2130h using
High Beam LED torch. Animals directly sighted and calls heard
were recorded.
Sherman traps for rodents:
Most of the rodents are nocturnal and some are burrowing
animals. Live trapping is the only method for studying these
animals. Sherman traps were used for live trapping of rodents.
Traps were set in 7 X 7 grids with 10m between the trap
stations. Each station had a single Sherman trap 23cm X 9cm
X 8cm in dimension (Plate 4), placed on ground giving a total
of 49 traps per site covering an area of 0.49 ha (Fig.3).
Additional ten traps were set on trees at five meter height
from the ground. Trapping was carried out for five days
consecutively in every month. This gave approaximately 5000
trap-nights (50 traps X five days X 10 months in both the
semi-evergreen and moist deciduous habitats) during the
study period. Traps were baited using peanut butter with fried
coconut kernel, and checked and rebaited in every morning at
0700h. The rodents captured were removed immediately and
placed in a cone (Plate 5), identified, measured and released.
The morphological measurements such as the head to body
length (HBL), tail length (TL) and weight (W) were measured.
In addition to this, other behavioural and habitat observations
of the individuals captured were also recorded.
Mistnet studies for bats
Mist nets are used most commonly for the small, volant
mammals, because they are easily deployable and suitable in
a variety of situations (Greenhall and Paradiso, 1968;
Nagorson and Peterson, 1980; Kunz and Kurta, 1988). Mist
nets made of monofilament nylon with a mesh size of 36 mm
and an overall size of 10 x 1.5 m were used to capture bats
during the study. The net was erected about half an hour
before dusk and was kept open for two to four hours after
dusk. Total 80 hours of mist-netting was carried out in the
sanctuary during the study period. Nets were watched
continuously. The bats, which were trapped in the mist net
were removed immediately with gloved hands and placed in
cloth bags, measured and released. Measurements such as
forearm length (FL), ear length (EL), tail length (TL), were
taken using digital calliper.
B. LABORATORY STUDIES
Extraction and purification of DNA from tissue samples:
Obtaining good quality DNA is the major difficulty while
working with forensic animal samples. The possible tissue
samples may include cooked meat, blood smear, hair, dry
skin, bones and so on. The problem is aggravated further, as
in most cases such samples are poorly handled/collected and
thus are presented at different stages of degradation.
Moreover, in most instances, the forensic samples available for
analysis are in very small amounts, making extraction of DNA
for forensic analysis even more difficult.
Therefore,
developing efficient methods for successful isolation of DNA
useful for molecular/DNA analysis (the most important step)
would be the major focus of the proposed project. For the
purpose, a suite of DNA isolation protocols ranging from the
conventional methods (developed for isolation of animal DNA
as per Sambrook et al. 1989), to more recent ones (such as,
based on solid-surface binding/purification, resin based
separation, column based separation etc.) would be tested
using known reference as well as forensic samples. This would
be followed by standardization of quantity, quality of the
source tissue for isolation of reasonably good quality DNA
amenable for subsequent PCR-amplification/DNA analysis.
The method which yields good quality DNA from small sample
will be selected for further work
Amplification of specific DNA sequences using PCR: The DNA
isolated from the forensic/control samples will be used for PCR
amplification/sequencing of taxonomically informative domains
to develop species-specific DNA signatures. Few such domains
of interest, which we plan to analyze, are part sequences of
cytochrome C-oxidase subunit 1 (COI), cytochrome-b, and dloop control region. The amplifications would be carried out
using primers already available in the literature/ NCBI
database.
Sequencing: Amplified product will be separated by agarose
gel electrophoresis and eluted from the gel. The amplicons will
be cloned directly in pGEMT and commercially sequenced.
Parallel efforts would be made to standardize PCR
conditions/purification protocols enabling direct sequencing of
amplified products without the need for cloning.
Create database on sequences of the threatened and endemic
biodiversity of Kerala: The sequence data will be generated for
each species under study and will be analyzed with the
sequences available in the public domain/NCBI database. In
this way, unique species-specific DNA sequences based on
mitochondrial cytochrome c oxidase subunit 1, cytochrome b
genes, and d-loop region would be developed.
Phylogenetics studies: The phylogenetic analysis would be
done through neighbour joining method (Saitou and Nei,
1987) using the software MEGA 5.0 (Tamura et al., 2011).
Confidence values for internal lineages were assessed with the
bootstrapping option (Felestein, 1985). Pairwise distances
between all sequences were calculated using the Kimura two
parameter model (Kimura, 1980) in MEGA 5.0 (Tamura et al.,
2011). This model corrects for multiple hits, taking into
account transitional and transversional substitution rates,
whilst assuming that the four nucleotide frequencies are the
same and that rates of substitution do not vary among sites
(Nei and Kumar, 2000). This model was used as it can
provide direct comparison with distance measures reported by
Bradley and Baker (2001).
Timeframe work
:
2013-14
Outcome
:
The biodiversity documentation of the faunal elements of the
biodiversity hotspot of Western Ghats would be done, giving
emphasis on the threatened and endemic species in Western
Ghats. This would help further in the conservation
prioritisation of these biological wealth of the Western Ghats,
which we are losing at an alarming pace.
Manpower requirement if any
:
Research Associates - 3 nos.
Skilled assistants
- 3 nos.
Equipments if any
:
Camera traps
Digital SLR camera and accessories
Radio telemetry equipments
Microscope with photo attachment
Mist net
Sherman traps
Real time PCR
Gel documentation system
Incubator shaker
Digital caliper etc
Infrastructure requirement if any
Budget
:
:
Renovation of existing lab
Subheads
Rs. In lakhs
A. Recurring contingencies
Pay and allowances
9.216
TA & vehicle hire charges
3.00
Operational expenses
7.00
Sub Total A
19.216
B. Non Recurring Contingencies
Equipments/ machineries
50.00
Works
25.00
Sub Total B
75.00
Total (A+B)
94.216
Institutional charges@10%
9.4216
Grand Total
103.6376