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presented by
M. VirgilioRoyal Museum for Central Africa, Tervuren, Belgium
Royal Belgian Institute of Natural Sciences, Brussels, Belgium
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Tephritid Barcoding Initiative
and barcoding of agricultural pest
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Why DNA Barcode Fruit Flies?
• large number of species
• many economically relevant species
• difficult identification of larvae.
• need for standardized diagnostic methods
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
The Tephritid Barcode Initiative (TBI)
TBI Chair:Bruce McPheron, Penn State
TBI Coordinators:Allen Norrbom, USDA, USAMarc De Meyer, RMCA, Belgium
CBOL obtained funding from the Sloan Foundation to support a “Demonstrator System”
Steering Committee formed in April, 2006, in Belgium
Steering Committee Members:Karen Armstrong, New ZealandNorman Barr, USAAmnon Freidberg, IsraelHo-Yeon Han, South KoreaGeorge Roderick, USAIan White, UK
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI proposal: Goals
Generate barcode database for 2,000 species
– 5 individuals/species (10,000 specimens)– 100% of economically important (EI) species– >75% of EI congeners– 1 species per genus in subtribes containing EI species– 1 species per genus in other higher taxa– representative tephritoid families
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI proposal: Beneficial Outcomes
1) Establish a globally-available DNA database of barcodes
2) Establish a globally-available DNA repository
3) Generate a collection of identified vouchers for future
systematic work
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Who is generating fruit fly barcodes?
1. Penn State University, USA: Bruce McPheron, Md. Sajedul Islam
2. Lincoln University, New Zealand: Karen Armstrong
3. Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio
4. Yonsei University, Korea: Ho-Yeon Han
5. California Department of Agriculture, USA: Peter Kerr
6. Smithsonian National Museum of Natural History, USA: Allen Norrbom
7. APHIS-PPQ Mission lab, USA: Norman Barr
8. University of Guelph
9. Biodiversity Institute of Ontario
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Financial support
Penn State University, USA: technical support
APHIS: collecting & sequencing
Belgian Federal Government: sequencing, pilot study mini-barcodes
California Citrus Board: general expenditure
Korean Government: sequencing
(Dutch Government: collecting)
Various contributions through staff time and general core funding of institutions or other projects
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
What needs to be provided in BOLD for TBI
Euleia fratria (Trypetinae)TEPH101 (from BOLD)
1. identification of specimen by an expert
taxonomist
2. voucher specimen
3. collection information (collection date
and location)
4. other infos (GPS, elevation,
photodocumentation) not mandatory but
strongly encouraged
5. barcode: at least 500bp with less than
1% missing data.
6. trace files stored in BOLD.
Other COI records (e.g., Genbank submissions) are integrated into the BOLD database but kept separate.
Deposited in :
Tephritidae {family}
Subfamilies (5)Dacinae [572] Phytalmiinae [7] Tachiniscinae [1] Tephritinae [219] Trypetinae [243]
Lineage: Arthropoda: Insecta: Diptera
Specimen Record: 1047
Specimens with barcodes: 828
Public Sequences: 240
Search performed on October 20, 2008
http://www.barcodinglife.org
Barcodes : Species :
213
73
533
95
240
44 34
119
19 37
0
200
400
600
An
astr
eph
a
Rh
ago
leti
s
Bac
tro
cera
Cer
atit
is
Dac
us
all species EI species
Five Genera of Highest Economic Importance
0
20
40
60
80
100
An
astr
eph
a
Rh
ago
leti
s
Bac
tro
cera
Cer
atit
is
Dac
us
number of species % of species barcoded
insights into
DNA barcoding
of tephritid fruitflies
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
0%
4%
8%
12%
16%
20%
Bactrocera Ceratitis Dacus
between species
within species
intra- and inter-specific genetic differentiation in 3 genera of tephritid fruitflies
p-distances
12.6 6.3 6.6
ratio inter/intra specific p-dist
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
DNA barcoding of tephritid fruitflies:
where it works nicely....
44 barcodes of Bactrocera cucurbitae
from 11 countries:
• Bangladesh• Cambodia• China• Hawaii• India• Kenya• La Reunion• Malaysia• Philippines• Sudan• Tanzania
K2p = 0.02% (S.E.= 0.01%)
average genetic distance
within species
Ceratitis fasciventris
C. anonae
C. rosa
... and where not:
the FAR complex
0.00
0.01
0.02
withinspecies
betweenspeciesK
2P
average genetic distances
ratio = 1.2
other potentially “problematic”
tephritid species groups and complexes:
B. dorsalis complex
C. cosyra complex
C. capitata / C. caetrata
T. occipitale / T. quadrimaculatum
...
Armstrong and Ball (2005) Phil. Trans. R. Soc. B 360: 1813-1823. (B. dorsalis complex)
Virgilio et al. (2008) Molecular Phylogenetic and Evolution 48: 270-280 (FAR complex, Ceratitis)
Dacus:
from identification issues
to classification issues
as expected low genetic differentiation
in taxonomically closer species...
D. chiwira - D. famona, p-dist.= 0.3% (subgenus Dacus)
D. apostata - D. triater, p-dist.= 0.0% (subgenus Psilodacus)
COINJT-K2P
DACUS
DIDACUS
LOPHODACUS
LEPTOXYDA
NEODACUS
PSILODACUS
...but also discrepancies between morphological and molecular taxonomy
subgeneric classification
Apocynaceae
Cucurbitaceae
Passifloraceaeunknown
COI+16S+perBayesian
DNA barcoding may provide clues for an improved morphological classification
host plant choice
DNA barcoding may reveal cryptic speciation:
Virgilio M., De Meyer M, White I.M., Backeljau T. (submitted) Phylogenetic relationships among African Dacus species (Diptera: Tephritidae) as inferred from mitochondrial and nuclear DNA.
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
methodological problems
in the barcoding of tephritid fruitflies
from museum collections
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
DNA Extraction Procedures:Several pilot studies performed to assess the success of an invasive,
nondestructive DNA extraction method for museum specimens.
Study 1: L. Weigt and A. Driskell (Laboratory of Analytical Biology, National
Museum of Natural History, Smithsonian Institution) -16S and COI
Study 2: N. Barr (USDA) and R. Ruiz (Penn State) -16S
Study 3. M. Virgilio (Royal Museum Central Africa, BE) – 16S, COI
Study 4. Md. Sajedul Islam (Penn State) - COI (ongoing study)
Qiagen DNeasy kit: ok for DNA from recent dry and alcohol specimens not crushed during the extraction process.
methodological problems
in the barcoding of museum specimens
age of specimens vs barcoding success
% specimens amplified
% specimens sequenced
methodological problems
in the barcoding of museum specimens
0
20
40
60
80
100
2007
2006
2005
2004
2003
2002
2001
2000 90
s
80s
< 1
980
(n=394)
0
20
40
60
80
100
2007
2006
2005
2004
2003
2002
2001
2000
90s
80s
< 1
980
from EtOH specimens
from pinned specimens
% of succesfully sequenced specimens (n=394)
methodological problems
in the barcoding of museum specimens
pinned vs EtOH preserved specimens
>200041%
90s4%
80s3%
70s1%
60s2%
50s13%
40s4%
<194032%Ceratitis, Bactrocera and Dacus
in the collections of the RMCA:(n=1804)
methodological problems
in the barcoding of museum specimens
DNA extraction protocols tested:
• Qiagen DNeasy• Qiagen Biosprint• Bio101 Ancient DNA kit • Autogen • Machery-Nagel Filterservice kit • Phenol-Chloroform• DNAzol• E.Z.N.A. kits for forensics and insects • chargeswitch magnetic beads• etc.
methodological problems
in the barcoding of museum specimens
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
problem:• the quality of DNA rapidly decreases in time (shearing)• the amount of barcodes obtained from older museum specimens
is not significantly affected by • the extraction method used• the use of genus- and species-specific primers
objective:
develop internal primers to improve the collection of barcodes from
sheared DNA
methodological problems
in the barcoding of museum specimens
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
the BARFLY projectshort term project funded by the Belgian Science Policy (BELSPO)
Joint Experimental Molecular Unit
of RMCA and RBINS (www.jemu.be)
Jeroen Van Houdt, Floris Breman
• development of internal primers for the barcoding of Tephritids• collection of new barcodes from museum specimens
methodological problems
in the barcoding of museum specimens
LCO 1490 HCO 2198
full barcode - c. 670 bp
frag. 1 - 343bp
frag. 2 - 269bp
frag. 3 - 227bp
a new set of internal primers for the barcoding of tephritids
VanHoudt J., Breman F. C., Virgillio M., De Meyer M. (in prep.)A protocol for DNA barcoding of African tephritid fruitflies from museum collections using mini barcodes.
% of pcr products
obtained
% of barcode sequences
obtained (>500bp)
(n=229)
0
20
40
60
80
100
>2000 90s 80s 70s 60s 50s 40s <1940
fragment 1
fragment 2
fragment 3
.
(n=229)
>2000 90s 80s 70s 60s 50s 40s <1940
0
20
40
60
80
100
higher performances
compared to the standard primers
0
20
40
60
80
100
>2000 90s 80s <1980
standard primers
internal primers
(n=229)
+7% +32% +6% +7%
a new set of internal primers for the barcoding of tephritids
% of barcodes obtained (>500bp)
RMCA:
internal primers as a standard protocol
for the barcoding of museum specimens
collected before 2000
a new set of internal primers for the barcoding of tephritids
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
work in progress:
could ‘minibarcodes’ represent a
temporary alternative to
‘full’ barcodes?
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Dacus armatus
400 bp
600 bp
500 bp
400 bp
400 bp
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
300 bp
400 bp
500 bp
Bactrocera oleae
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
TBI current challenges
– Coordination / tracking specimens: BOLD
– Older specimens: mini barcodes / internal primers
– Fresh material: collecting activities
– Taxon coverage: non EI genera
RMCA current challenges
RMCA is collecting barcodes as a “complementary activity” to the currently
ongoing research lines:
- phylogeny and population genetics of African Dacus
- molecular taxonomy of species complexes in the genus Ceratitis
- phylogeny of African and Australasian Bactrocera
- population genetics and phylogeography of B. cucurbitae
Marc De Meyer: [email protected]
Massimiliano Virgilio: [email protected]:
RMCA current challenges
RMCA is opened to collaborations with African Institutions aiming to:
- identify tephritid samples through morphological and
molecular characters
- prepare species inventories from African countries
- collect / rear tephritid fruit flies
- investigate population genetics and host races evolution of
tephritid species
Marc De Meyer: [email protected]
Massimiliano Virgilio: [email protected]:
Royal Museum for Central Africa – Royal Belgian Institute of Natural SciencesRoyal Museum for Central Africa – Royal Belgian Institute of Natural Sciences
Acknowledgments
Karen Armstrong
Abdelaziz Babikir
Thierry Backeljau
Norman Barr
Marc DeMeyer
Sajedul Islam
Bruce McPheron
researches at the RMCA are currently funded by the
Belgian Science Policy Action 1 (project MO/37/017)
and benefit from practical support by the
Joint Experimental Molecular Unit (JEMU)