Transcript Investigating spatio-temporal distribution, genetic

Spatio-temporal distribution and
genetic characterization of some
marine macroalgae of the Republic
of Mauritius.
MPhil/PhD Research
By Mrs Persand Jayshree
(BSc Hons Biology with Environmental Sciences)
Supervisors:Dr Bhagooli & Dr Taleb-Hossenkhan
Studies on macroalgae distribution based on
morphological characteristics:
Dickie, 1875, data collected during 1860’s
Boergesen 1940-1957, data collected during late 1920’s
& 1930’s
Jagtap, 1993, data collected in 1987
No DNA analyses done
Relatively sparse data, dating back to 21 years,
Statement of problem:
Correct identification at molecular level
absolute pre-requisite.
Wrong identification  misleading for
research work
Only short-term studies on species
distribution and abundance reported
Rationale of study
Identification of macroalgae heavily reliant on
cell detail
and cell arrangement,
gross morphology,
Morphology of a single species vary in response to
environmental conditions, for example low salinity
and salinity shocks creating morphotypes.
Molecular genetic tools  to identify morphotypes.
Morphotypes
low salinity and salinity shocks can induce branching in
Ulva intestinalis
(U. intestinalis being unbranched) creating morphotypes
similar to Ulva compressa.
U. intestinalis and U. compressa are two distinct,
genetically divergent and reproductively isolated
species
Rationale of study
Mschigeni (1985) highlighted
level of misidentification of specimens in some areas in the
Indian Ocean may have an adverse effect on other studies and
the commercial application of these specimens
This study proposes a re-evaluation of macroalgae
identification, abundance and distribution in the Mauritian
lagoons and help identify possible effects on macroalage in our
lagoons of the recent development that have occurred along
the coastal shoreline in the recent years in Mauritius.
Objectives of the study:
1. Monitor spatio-temporal changes in population dynamics
of macroalgae
3 consecutive years.
2. Determine seasonal changes in wet weight and biomass
of selected macroalage
3. Genetically characterize different species of macroalgae
using well-established molecular genetic protocols.
4. Monitor the photo-physiological status of macroalgae
over time in the field
5. Measure physico-chemical parameters of water
Sites of study
Site
Characteristics
Flic en Flac
Possible water current that cause enough flushing of
lagoon
Palmar/ Belle Mare
Site of onion plantation along coast hence input of
fertilizers through runoff
Trou aux biches
Site of intense boating activities and freshwater seepage
Pointe aux Biches
Site whereby there is drastic temperature fluctuations as
macroalgae beds emerge out of water during low tides
Balaclava
Undisturbed site (control) with low nutrients levels (AFRC,
2004)
Poste La Fayette
Constant exchange of water (control) from off lagoon
Jagtap (1993) documented

127 species of macroalgae


1.
2.
3.
4.
confined to intertidal and lagoonal zones.
4 economically important genera:
Sargassum (9 species),
Gracillaria, (4 species),
Ulva (3 species)
Enteromorpha (4 species).
very high commercial value
worldwide and are being
harvested in millions of tonnes
annualy
Genetic characterisation
Well-established genetic tools and markers  to
identify macroalgal species
E.G.

utility of rDNA internal transcribed spacer
(ITS) sequences
gene encoding the large subunit of RUBISCO,
small subunit rDNA
Methodology
Genetic analysis of macroalgae
• Collection of preservation of selected species
• DNA Extraction
• PCR amplification of selected regions
• Purification of PCR products, cloning & sequencing
• Comparison of sequences to those already present in DNA
databases for identification purposes
Photo-physiology of macroalgae
Chl a fluorescence
determined each season using Pulse
Amplitude Modulated (PAM) fluorometer
Measurement of:
 minimum fluorescence (Fo)
 maximum fluorescence (Fm)
 maximum quantum yield of photosystem II (PSII) (Fv/Fm)
 photosynthetic electron transport rate (ETR)
 non-photochemical quenching (NPQ) at PSII derived from rapid
light curves (RLCs)
Population dynamics
% cover & density
monitored
(recording the time taken for algal re-colonization
in cleared area)
Specific growth rates of macroalgal species
monitored
length / mass
tagged
and length)
30-50 individuals
(diameter
 Water Analysis
2/yr  water samples to be collected for nitrate & phosphates
analyses ex situ (cadmium reduction method & ascorbic acid
method)
water physico-chemical parameters including temperature, pH,
salinity, turbidity & dissolved oxygen monitored in situ
 Macroalgae distribution
4 equidistant transects perpendicular to shore evenly spaced
macroalgae distribution and seasonal change monitored
Wet weight: 3 random samples in 25cm  25cm quadrat/transect
Dry weight: same samples dried at 800c
Expected output
 Macroalgae species distribution assessed on a spatio-temporal
scales in the selected lagoons of Mauritius.
 Anthropogenic stressors
v/s
macroalgae species
distribution/abundance
suitability of certain macroalgae as bioindicators ???
 Verification of identity of morphologically characterized
selected macroalgae using molecular genetic tools.
• Comparison of re-colonization success of specific macroalgae
species in the different regions assessed
Work carried out – Primer Design
•
Primers already designed for Ulva and Gracilaria genera
for the purpose of genetic characterization
•
DNA sequences corresponding to the 18S, ITS1, 5.8S, ITS2,
28S rRNA genes and RuBisCo gene from Ulva and Gracilaria
genera - retrieved from GenBank.
•
DNA sequences belonging to several different species in each
genus retrieved so that sequences could be compared and
primers designed in the regions showing polymorphism only
Multiple Sequence Alignments (MSAs) deriving from different
Ulva spp. and of the rubisco gene cluster for Gracilaria spp. were
then produced using the MultAlin Software
Primers, specific for the amplification of the polymorphic regions,
were then designed using Primer3WWW software
Figure 1: Organisation of one rDNA array. Single repeat units (arrows) are
tandemly organised. Each of them consists of the rRNA genes: 18S, 5.8S and
28S. Spacers separate these genes, namely the external transcribed spacer
(ETS), the internal transcribed spacers (ITS 1 and ITS 2) and the intergenic
spacer
(IGS).
[http://webdoc.sub.gwdg.de/ebook/y/1999/whichmarker/index.htm]
Thank you
for your consideration