?? So what is “biodiversity” ??

“Biodiversity refers to the variety and variability among living organisms and the ecological complexes in which they occur …” (US Congress Office of Technology Assessment, OTA 1987)

An abstract concept …..

Different divisions: (3) genetic, species, ecosystem diversities(5) genes, populations, species, assemblages, ecosystem(hierarchical scales)

A measurable entity …..

Although based on the definition of “entities”, the concept embraces “processes”.

Measuring biodiversity enables to rigorously and empirically test the concepts. There is no way to get a unique measure of biodiversity!

The most classical measure of biodiversity combine the # of species, and the evenness or equability of their abundance.

(n / N)2

A social construct … Biodiversity = nature conservation. Out of its neutral scientific concept, biodiversity is perceived as a value.

Species concepts

The world is populated by organisms that form clusters of similar individuals, called species. Speciation is the splitting of populations into different evolutionary independent units. The species is the critical transition level between micro- and macroevolution.

There are many different ways of defining species, none of which is able to cover all known organisms. The 2 most important are:

1) the Biological Species Concept of Dobzhansky (1937) and Mayr (1942) –a group of organisms that can mate with each other and produce viable grand-children-2) the Phylogenetic Species Concept: a monophyletic group composed of the smallest diagnosable cluster of individual organisms within which there is a parental pattern of ancestry and descent. The PSC brings a historical dimension that does not exist in the BSC.

Finlay 2002

For microbes: everything is everywhere …Species-Area curves

(~3000 spp)

Finlay 2002

Molecular tools for the study of marine micro- diversity:

DNAPCR

Sequencing

RFLP, DGGE

Environmental Clone Librairies

FISH

Quantitative PCR

1

DNAMicroarrays

Shotgun Sequencing

Taq Polymerase

Thermophilusaquaticus.

More than 2 million copies in 20 cycles

The Sanger sequencing reaction:

Automatic DNA sequencing using fluorescent dyes:

Electropherogram

Denaturing Gradient Gel Electrophoresis

-fragment: around 500 bp-GC clamp: 39-50 bp

Denaturant: Urea, Formamide,High temperature (TGGE) Formation of heteroduplex!!

Typical DGGE gel:

HorseradishPeroxydase

Basic steps in FISH:

1) Collection of natural samples through filtration

2) Cells are dehydrated with EtOH

3) Cells are stored at –80 degreeC.

4) Probe hybridization

5) Washing of unhybridyzed probe

6) Addition of Fluorescein-Tyramide

7) Unfixed Fluorescein-Tyramide is washed

8) DNA staining with DAPI or propidium iodide

Not necessaryfor monolabelledprobes

Flow cytometers count and analyse individual particles in a fluid. Fast and one by one. The analysis consist of forward and sideward scattered light as well as fluorescence of the cells. The cells are funneled by a sheath fluid in single file through a laser beam focus at typically 1,000 or more per second. The detected optical signals from each passing particle are digitized and listed into correlated data. If a sorter unit is present, individual particles may be sorted physically depending on their optical signals.

PRINCIPLE OF FLOW CYTOMETRIC ANALYSIS

Side scatter

Chl

orop

hyll

fluor

esce

nce

Each dot represents a single measured particle.

Courtesy Glen Tarran, PML

Data are displayed as dual-parameter plots of combinations of light scatterand fluorescence depending on the particles being analysed. Forward light scatter is governed by particle size and sideward scatter is mostly sensitive for small cellular structures. The fluorescence, probed in several emission bands relates to the amount of natural cellular pigment and its composition, and/or artificially introduced pigment. Applications range from simple phytoplankton counting and sizing to more elaborate physiological analysis, and species recognition using artificial neural net algorithms.

- PICO-EUKARYOTES -

One water samples in the Pacific; - 75m35 SSU rDNA sequences

1. Moon Van der Stay et al. 2001

Results:11 already known pico-eukaryotes24 totally new phylotypes, with twonew groups of alveolate.

2. Lopez-Garcia et al. 2001

One site at the Antarctic Polar Front4 depths: 250 / 500 / 2000 / 3000m

24 complete SSU rDNA sequences

2 novel groups of Alveolatesthat account for 65 to 76%of the sequences retrieved

A few new Heterokonts

ALVEOLATES

Sub-surface Deep Sea

Globorotalia truncatulinoides

L R

Single Cell PCR amplification (ITS rDNA) of 40 Globorotalia truncatulinoidescollected along an Indian Ocean transect.Success rate = 90%

After PCR

After RFLP (Sau 96 I)

PCR amplification : 30 centsRPLF : 20 cents

DNA extraction : 4 cents$ 0.5/reaction !!

Type I Type IIIType IV

Foissner 2002

17 bi-monthly samplings from 100m2 area of beech forest soil in Austria.

On a total of … 1000 soil ciliates!

Foissner’s estimation: at least 30 000 species of free-living ciliates, and eventually an order of magnitude higher!!

(Corliss, 2001: 213 000 species of protists)