1. Spatio-temporal distribution and genetic characterization of some marine macroalgae of the Republic of Mauritius. MPhil/PhD Research MPhil/PhD Research By Mrs Persand Jayshree (BSc Hons Biology with Environmental Sciences) Supervisors:Dr Bhagooli & Dr Taleb-Hossenkhan

2.  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,

3. Statement of problem: Statement of problem: Correct identification at molecular level absolute pre-requisite. Wrong identification  misleading for research work Correct identification at molecular level absolute pre-requisite. Wrong identification  misleading for research work Only short-term studies on species distribution and abundance reported Only short-term studies on species distribution and abundance reported

4. 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. 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.

5. M orphotypes 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

6. 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 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. 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.

7. 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

8. Sites of study 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 SiteCharacteristics

9. Jagtap (1993) documented  127 species of macroalgae  confined to intertidal and lagoonal zones.  4 economically important genera: 1. Sargassum (9 species), 2. Gracillaria, (4 species), 3. Ulva (3 species) 4. Enteromorpha (4 species). very high commercial value worldwide and are being harvested in millions of tonnes annualy

10. 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

11. Methodology Genetic analysis of macroalgae Collection of preservation of selected species Collection of preservation of selected species DNA Extraction DNA Extraction PCR amplification of selected regions PCR amplification of selected regions Purification of PCR products, cloning & sequencing Purification of PCR products, cloning & sequencing Comparison of sequences to those already present in DNA databases for identification purposes Comparison of sequences to those already present in DNA databases for identification purposes

12. 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)

13. 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 30-50 individuals tagged (diameter and length)

14.  Water Analysis 2/yr  water samples to be collected for nitrate & phosphates analyses ex situ (cadmium reduction method & ascorbic acid method) 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 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 4 equidistant transects perpendicular to shore evenly spaced macroalgae distribution and seasonal change monitored Wet weight: 3 random samples in 25cm  25cm quadrat/transect Wet weight: 3 random samples in 25cm  25cm quadrat/transect Dry weight: same samples dried at 80 0 c Dry weight: same samples dried at 80 0 c

15. 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 ??? 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 Comparison of re-colonization success of specific macroalgae species in the different regions assessed

16. Work carried out – Primer Design Primers already designed for Ulva and Gracilaria genera for the purpose of genetic characterization 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 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 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

17. 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] 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

18. Thank you for your consideration