Amazonian Rainforest 2005
1

Why French Guiana?
This investigation is targeting the microchlorophytan biodiversity in subaerial habitats from French Guiana (FG) rainforests. We planned exhaustive surveys including coastal, piedmont, riparian, and the high plateau or Guiana Shield.
Factors for a potential high biodiversity in subaerial algae:
• FG is located on the NW shore of SA
• FG with coastal strip and a dense, mostly inaccessible rainforest that covers about 80% of the territory
• The Intertropical Zone of Convergence IZC
• Climate is warm and humid all year
• Temperature ranges between 18 and 36 ° C
• Annual rainfall between 1700-4000 mm/year.
Intertropical Zone of Convergence in
French Guiana (Barret 2004)

Floramazone Lodge at Kaw Mountain
May rainfall in French Guiana
(Barret 2004)

Expeditionary work
• Two summer expeditions, 16 days each
• June and July, end of rainy season
• Areas to be explored: coastal plains and the
Massif Central
• The operation centre is FloraAmazon
• First 3-4 days at the station
• Coastal regions from St-Georges (bordering with Brazil) to St-Laurent-du-Maroni
(bordering with Surinam) for 2-3 days.
• Boat up the river Maroni to Maripasoula
• Aircraft to flight over the high plateau to arrive in Säul (5-6 days).
• Return from Säul to Cayenne for curation of samples (ca. 2 days). d c e a b
Vegetation types in French Guiana (Barret 2004) and proposed target areas for microchlorophytan collection (red)

Algal Sampling
• GPS data is recorded
• Green, yellow, orange, or red patches
• Sampling between 40–50 sites each expedition
• Averaging 3-4 sites per day
• between 10-15 samples per site
• Sterile plastic scrapers
• Samplings 1-2 sq in
• Open plastic bags
• Sealed bags for transportation
• FG collection and export permits
• USA import permit USDA

• 28 taxa recorded
• 2 new species described
• 10 taxa not identified at the species level; some may represent undescribed entities
• Many taxa, especially Phycopeltis and Printzina, associated with shaded rainforest habitats
• 6 new records for the Americas

New records for the Americas
Trentepohlia
dusenii Hariot
Trentepohlia
diffracta var.
colorata Cribb
Printzina bosseae
(De Wildeman)
Thompson &
Wujek
Trentepohlia
cucullata De
Wildeman

Trentepohlia chapmanii Rindi & López-Bautista

Trentepohlia infestans Rindi & López-Bautista

2 new species; 2 new records for the Americas;
4-5 other taxa possibly undescribed
Overall, 93 species of Trentepohliales currently accepted
28 taxa recognized for French Guiana represent 30% of the total
Comparable figures for other tropical regions:
Area of Bogor, Java, Indonesia: 30 taxa
India: 27 taxa
Queensland, Australia: 31 taxa
FRENCH GUIANA IS A BIODIVERSITY HOTSPOT FOR THE TRENTEPOHLIALES

PUBLICATIONS:
Lopez-Bautista, J., Rindi, F. and M. Guiry. 2006. Molecular Systematics of the subearial green algal order Trentepohliales: an assessment based on morphological and molecular data. International
Journal of Systematic and Evolutionary Microbiology 56:1709-1715.
Lopez-Bautista, J., Rindi, F. and D. Casamatta. 2007. The Systematics of Subaerial Algae. In:
Seckbach, J. (Ed.) Extremophilic Algae, Cyanobacteria and Non-Photosynthetic Protists: From
Prokaryotes to Astrobiology, Series: Cellular Origin, Life in Extreme Habitats and Astrobiology,
Volume 11, Springer, pp. 599-617.
Rindi, F. and J.M. Lopez-Bautista 2007. New and interesting records of Trentepohlia
(Trentepohliales, Chlorophyta) from French Guiana, including the description of two new species.
Phycologia 46:698-708.
Rindi, F. and J.M. Lopez-Bautista 2008. Diversity and ecology of the Trentepohliales (Ulvophyceae,
Chlorophyta) in French Guiana. Cryptogamie algologie 29:13-43.
Rindi, F., Guiry, M. D. and J. Lopez-Bautista. 2008. Distribution, morphology and phylogeny of
Klebsormidium (Klebsormidiales, Charophyta) in urban environments in Europe. Journal of
Phycology, IN PRESS.
Rindi, F., Lopez-Bautista, and D. W. Lam. Molecular phylogeny of Trentepohlia and Printzina
(Trentepohliales, ULvophyceae). 2008. To be submitted to Molecular Phylogenetics and Evolution.

• 26 taxa recorded
• 12 taxa not identified at the species level, some probably representing unidentified species
• 1 new record for the Americas; several species newly recorded in French
Guiana also found in Panama
• Extreme richness of Trentepohliales on artificial substrata (concrete, metal, plastic sheeting, curtains of windows in dormitories)

Remarks on the biodiversity of subaerial algae
 Overall, biodiversity studies in Amazonian and Central American rainforests are providing with surprising new records and new species to science.
 Biotic surveys based on algae are useful to unravel biodiversity hot spots in our planet.
 Even short term studies provide with important tools to implement conservation strategies in geographic regions facing extinction

1.
Plan ahead, plan ahead, and plan ahead
2.
Define goals of the collection
3.
Correspondence between goals and fieldwork
4.
Selection of location, people, and logistics

• Camera
• Dissecting trays, forceps and needles
• Portable microscope
• Microscope slides and coverslips
• Ruler
• Proper clothing
• Field (transit) notebook
• Headlamp
• Hand magnifier (10X-15X)
• Insect repellent
• Pencils and permanent markers
• Ziploc bags
• Scrappers
• Field knife
• Labels
• Silica gel
• Enviro-meter
• GPS

Subaerial algae are collected by hand with the aid of scrappers or knives and placed in plastic bags
• Label each and all samples with permanent markers
• Make reference of your collection/plastic bag to your field book
• Minimal data to be included:
• Locality : Place, County, State, if possible use GPS
• Date
• Collector’s name , and…..

• Habitat: tropical rainforest, urban, cemetery, etc
• Color, consistency, and relative abundance
• Substrate type
• Rocks: Epilithic or endolithic
• Animals: Epizoic
• Artificial substrate (road signs, concrete walls)
• Soil: Edaphic
• Lichenized
• Light conditions: shaded or open microhabitat
• On bark (epixylic) and bark roughness
• On leaves (epiphyllous) or inside (endophyllous)

For morphological studies subaerial algae are preserved air-dried in an open plastic bag until their transfer to lab. Protect from direct sunlight. Before returning to lab, close (zip) plastic bags. Upon arrival to the lab keep plastic bags open. and sort and identify ASAP. Most subaerial algae can survive for a couple of weeks after their collection.
For DNA extraction subaerial algae are preserved using a desiccant, silica gel crystals with humidity indicator (blue/pink crystals). Divide each original sample in two: one for morphological/culture studies (see above) and the second portion to be fixed with silica gel. The sample is placed in zip lock bags with enough silica gel. Seal plastic bags, if blue crystals turn pink then add more silica gel. Silica gel can be reused if dehydrated in a microwave or oven.

Vouchers for scientific documentation should be preserved as herbarium specimens
Semi-permanent slides can be made using the glycerin-gel technique:
1.After fixation, stain with 1% aniline blue or lugol
2.Transfer to slide
3.Add warm glycerin-jelly (5 gr gelatin, 30 ml distilled water, 35 ml glycerin)
4.Store flat for one or two days
5.Seal edges with nail polish
6.Label microscope slide
7.Keep flat and away from direct sunlight

• Upon arrival at the laboratory, living samples are sorted out for isolation and culture
• The samples are distributed in liquid and/or agarized Bold Basal Medium (MBB)
• Culture conditions: 16:8 L:D, 20-30 μmol photons m –2 s –1 , at 22-25 ° C
• Tubes and plates are placed inside plant growth chambers

• To obtain unialgal cultures two basic procedures are followed:
• Liquid cultures showing algal growth are selected and samples are transferred using a bacteriological loop to agarized plates
• Field samples are selected with forceps and transferred to agarized plates
• Regular inspections of the culture material are made to isolate colonies by transferring algae into new agar plates
• Colonies showing unialgal features are harvested for molecular/LM and TEM work

• Morphology is documented with a digital camera attached to both, a Microscope with Nomarski optics and a Stereoscope, and fire-wired to a computer for digital imaging processing
• Strain’s morphology is analyzed in LM for:
1. coccoid and colonial forms, cell size, shape, cell wall ornamentation, chloroplast shape, pyrenoids
2. filament, trichome, branching, and cell dimensions
3. type of cell division, degree of constriction at the crosswalls, and fragmentation characteristics
4. apical cell morphology
5. motility
6. color
7. sheath characteristics
8. presence and nature of cellular inclusions
9. reproductive structures

• Ultrastructure as visible in TEM. This work reveals:
1. chloroplast and thylakoid structures
2. cell wall and sheath structure
3. cellular inclusions
4. apical cell features.

Molecular characterization of eukaryotic algae
DNA EXTRACTION
• DNA is extracted from culture strains and some herbarium specimens
• DNA is performed using QIAGEN DNEasy plant mini kit
• DNA samples are stored at -20 ° C
GENE AMPLIFICATION
• Gene amplification by PCR
• Selection of Primers and PCR conditions
• Run PCR product in agarose gels
• Band excision and cleaning with QIAGEN min elute gel extraction kit
• DNA quantification
SEQUENCING
• Gene sequencing
• Selection of primers
• Cyclesequence reaction in thermal cycler
• Purification in Sephadex columns
• Automated sequencing in ABI 3100
• Capture electropherograms

Molecular characterization of eukaryotic algae
PHYLOGENETIC ANALYSES
Goal: To provide with robust phylogenetic hypotheses applicable to the taxonomy and systematics of the subaerial algae at both generic and species levels
• Nuclear-encoded 18S rDNA and chloroplast-encoded rbcL
• They allow us to make comparisons
• Universal primers and new ones with OLIGO
• Sequence data is edited and aligned using Sequencher™
• Adjust manually using MacClade
• Software PAUP* 4.0, MRBAYES 3.0, and ModelTest 3.7
• Phylogenetic reconstruction by distance, parsimony, maximum likelihood, and Bayesian inference
• Bootstrap analyses to assess stability/support of nodes
• Modeltest to estimate a model of evolution
• We assume that if trees are congruent then the same fundamental phylogenetic information is being reconstructed
• It is important to recognize that all methods for phylogenetic reconstruction have limitations, pros and cons

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• Increases the # of taxa in biodiversity surveys
• Fast (1 or 2 weeks)
• Achieve multiple research goals
• Cheaper
• Widely accessible
• Less taxonomically inclined (even novice phycologists can perform these techniques)

• Taxonomically challenged (can’t describe new taxa)
• Might underestimate biodiversity
• Overestimate biodiversity
• Universality of primers
• Small GenBank database
• Primers are never universal
• PCR artifacts: Chimera sequences

Suriname
Gabon

• 432 plastid containing taxa from GenBank
• Sequences from GenBank and our “Env” samples
• 552 characters (including gaps)
• Alignment based on a 2ary structure model from
R.
Gutell (UT)
• Performed ML analysis; GTR+I+G model
• Trees were rooted by the longest branch (midpoint rooting)

Cryptophytes
Haptophytes
Glaucophytes
Cyanophytes
Euglenophytes
Rhodophytes
Heterokonts
Apicomplexa
Streptophyta
Chlororachniophytes
Chlorophytes

Liverworts

Mosses

• Over 50 unique sequences each have been found from Africa and
South America
• Our sequences were represented in several major lineages:
Cyanobacteria, Chlorophyta, Streptophyta (vascular and nonvascular), Heterokontophyta, and an unknown lineage
• 23S rDNA marker is a valuable and powerful environmental sampling tool to study algal biodiversity
• 23S rDNA seems to provide decent phylogenetic signal at this broad level of analysis and with rapid results
• Environmental sampling could be key to unraveling algal biodiversity in the subaerial environments

Remarks on the biodiversity of subaerial algae
• Although economically and ecologically important, subaerial algae have been largely understudied compared to marine and freshwater algae
• For historical reasons, most studies on diversity and ecology of subaerial algae have been carried out in Europe
• Few investigations, limited to Europe, have examined the subaerial algal flora of a certain geographical area
• The large majority of these morphology-based investigations were conducted between
1800 and 1960
• In recent decades limited work has usually focused on a few, specific groups often involving biodeterioration
• Amazingly, the few studies of subaerial algae in North America have yielded a wealth of new taxa to science
• Despite this body of information, the knowledge of subaerial algae is fragmentary
• Even an estimate of their biodiversity is impossible at the present time

Remarks on the biodiversity of subaerial algae
• In the last 20-25 years algal systematics has changed dramatically due to PCR and DNA sequencing techniques
• Molecular data available for subaerial algae is focused primarily on algal phylogeny at the class or order level.
• However, the taxonomic coverage of the molecular data is still uneven; some taxa are represented by large datasets while for others nothing or very little is available
• For example, to date no sequences have been deposited in GenBank for Apatococcus
lobatus, one of the most common subaerial green algae in the world
• For many subaerial algae a correct taxonomic circumscription and an understanding of their diversity would be achieved only through a combined approach, in which morphology is combined with datasets of molecular information
• As the diversity of many groups of terrestrial algae is still poorly understood, the availability of new collections, inventories of their biodiversity, and molecular characterization is urgently needed