dirk steinke - vertebrates plenary
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Marine fish eggs and larvae from theEast coast of South Africa
Dirk Steinke, Allan Connell, Tyler Zemlak, Paul Hebert
History
Allan Connell
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)
History
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, theywere 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.
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’
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
0 500 1000 1500 20000
5
10
15
20
25
30
35
40
Are we done?
Phy
loge
netic
Div
ersi
ty (
PD
)
# barcodes
• PD calculated using Conserve based on NJ trees generated in MEGA 4.0
• Sample size progressively increased by 10 random sequences
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.
0
10
20
30
40
50
60
70
80
90
100
% Offshore
% Inshore
Monodactylus falciformis Cubiceps pauciradiatusPomadasys olivaceus
Some observations
Mean monthly eggs per sample, averaged over 24 years
Trends
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
www.fisheggsandlarvae.com
Acknowledgements:
Erin CorstorphineTyler ZemlakPhilip Heemstra
Biodiversity Institute of Ontario
Thank you!
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