3. History
1985 - a major effluent pipeline was about to begin
discharging industrial effluent over a shallow
continental shelf area in Richards Bay
1985 - surface plankton samples were collected, over
several years, to assess the diversity of fish species
spawning in the area, and the intensity and seasonality
of spawning.
1986 – a second study in Park Rynie was started in
order to collect alive specimens.
1987 – cataloguing of eggs and hatched larvae started
2004 – DNA Barcoding was added to the procedure
(including sampling of adults for reference library)
4. Work flow
• By collecting both offshore (5km) and inshore (0.5km) a reasonable
assessment of location of spawning was obtained for all the common
eggs in the study area.
• A simple “key” based on the physical features of pelagic fish eggs,
was used to separate eggs into basic groups.
• eggs were hatched and both eggs and larvae were photographed.
• once larvae had fully pigmented eyes, they
were anaesthetised with MS222, prior to
fixing in 98% alcohol for DNA Barcoding.
• other larvae were reared to the point
where fin counts and juvenile features
aided in identification.
5. Work flow
• DNA extraction was done using standard protocols at the CCDB.
• A reduced elution volume was used.
• PCR used Fish Cocktail (Ivanova 2007).
• Sequences were queried against BOLD using its Identification
Engine (only 100% were considered).
• reared larvae of the same batch were fixed in formalin and serve as
„para-vouchers‟
6. Today
• some 2100 larvae have been barcoded since 2005
• the local adult reference library (assembled in parallel) contains
some 900 species
• some 1500 species of marine fishes from South Africa are barcoded
• 9000 fish species have been barcoded world-wide
7. Are we done?
40
35
Phylogenetic Diversity (PD)
30
25
20
• PD calculated using Conserve based on
15 NJ trees generated in MEGA 4.0
• Sample size progressively increased by 10
10 random sequences
5
0
0 500 1000 1500 2000
# barcodes
8. Results
• 1638 specimens (78%) could be identified using BOLD
• they represent 280 known species
• 10 of those are new records for South Africa
• the remaining 22% could not be matched to any barcode sequence
on BOLD or GenBank.
11. Trends
• large rainfall causing mud to be washed out from rivers
• the high nutrient load of such a deluge caused massive increase in egg
numbers
• three most prolific pelagic egg spawners: Sardinops sagax, Etrumeus
teres, and Scomber japonicus