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KURT JORDAENS, GEORG GOERGEN, AUDREY VOKAER, NATHALIE SMITZ, MASSIMILIANO VIRGILIO, GONTRAN SONET, THIERRY BACKELJAU, MARC DE MEYER
DNA BARCODING CONTRIBUTES TO THE TAXONOMY OF AFROTROPICAL HOVER FLIES
(INSECTA: DIPTERA, SYRPHIDAE)
From left to right: Phytomia natalensis, Syritta bulbus, Rhingia cyanoprora, Mesembrius cyanipennis, Ornidia obesa © CNC/BIO Photography Group, Biodiversity Institute of Ontario
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AFROTROPICAL HOVER FLIES
• After Hymenoptera, most important
group of pollinators, including pollinisation of commercial horticultural crops;
• Larvae may be predators of aphids and other pests è useful for biological control;
• 600 species (6000 worldwide);
• 4 subfamilies: • Eristalinae • Syrphinae • Microdontidae • Pipizinae
Eristalinus smaragdinus © Mengual, Ximo
Paragus borbonicus © CNC/BIO Photography Group, Biodiversity Institute of Ontario
Allobaccha eclara © CNC/BIO Photography Group, Biodiversity Institute of Ontario
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IDENTIFICATION CHALLENGES
MORPHOLOGY
- Identification keys: - Identification keys available for only 60% species of the known fauna; - Larger the genus, less reliable the identification.
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IDENTIFICATION CHALLENGES
MORPHOLOGY
- Identification keys: - Identification keys available for only 60% species of the known fauna; - Larger the genus, less reliable the identification.
- Original species description: - Many are too brief for species identification; - Several based on males only; - 40-50% of the species only known from the type locality and
description based on one or few specimens.
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BARCODING:
à helpful to assist morphological identification (target: a 648bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI))
- Currently, more than 2 800 COI barcodes of hover flies on GenBank and BOLD
IDENTIFICATION CHALLENGES
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BARCODING:
à helpful to assist morphological identification (target: a 648bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI))
- Currently, more than 2 800 COI barcodes of hover flies on GenBank and BOLD
è BUT only 6 COI barcodes >550bp from Afrotropics: Difficulties in assignment of Afrotropical hover flies species.
IDENTIFICATION CHALLENGES
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AIMS
1) Construct a DNA barcoding library for Afrotropical hover flies;
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AIMS
1) Construct a DNA barcoding library for Afrotropical hover flies;
2) Compare amplification success obtained with pinned and ethanol-preserved specimens;
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AIMS
1) Construct a DNA barcoding library for Afrotropical hover flies;
2) Compare amplification success obtained with pinned and ethanol-preserved specimens;
3) Test multiple analytic methods of species identification and compare accuracy and shortcomings of the database.
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SAMPLING SITES
Ghana (Nsites= 2) Togo(Nsites= 2)
Benin (Nsites= 21) Nigeria (Nsites= 4)
Total Sites = 29
Mosaic of savannahs, plantations, degraded and natural forest
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SAMPLING SIZE
• 590 specimens collected with sweep net: • From 1994 to 2013: pinned specimen kept dry (N= 276) • From 2013 to 2014: kept in absolute ethanol (N= 314)
• Identification based on external morphology: • 94 morphospecies from 27 genera
• For each specimen: 1 leg was removed for molecular analysis
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ANALYTICAL APPROACHES
1) Genetic Distance method: Kimura 2-parameter pairwise distances
• Construction of frequency distribution histograms of conspecific and heterospecific pairwaise distances
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ANALYTICAL APPROACHES
1) Genetic Distance method: Kimura 2-parameter pairwise distances
• Construction of frequency distribution histograms of conspecific and heterospecific pairwaise distances
2) Threshold-based method: estimation of the proportion of correctly identified specimens (only for Nspecies > 2)
• Best Match: identification based on the best-matching sequence • Best Close Match: identification relying on an optimal distance threshold
value of sequence similarity determined to minimize assignement errors.
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ANALYTICAL APPROACHES
1) Genetic Distance method: Kimura 2-parameter pairwise distances
• Construction of frequency distribution histograms of conspecific and heterospecific pairwaise distances
2) Threshold-based method: estimation of the proportion of correctly identified specimens (only for Nspecies > 2)
• Best Match: identification based on the best-matching sequence • Best Close Match: identification relying on an optimal distance threshold
value of sequence similarity determined to minimize assignement errors.
3) Tree-based method: reconstruct Neighbor-Joining and maximum likelihood trees
• Species clustering • Outgroup: Clistoabdominalis ancylus (Family Pipunculidae)
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RESULTS AND DISCUSSION
• Amplification success rate (>550bp) : • 480 specimens = 87.4% (mean of 5.3 barcodes per species)
• 83 morphospecies of 23 genera • 26 taxa represented by one specimen
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RESULTS AND DISCUSSION
• Amplification success rate (>550bp) : • 480 specimens = 87.4% (mean of 5.3 barcodes per species)
• 83 morphospecies of 23 genera • 26 taxa represented by one specimen
• Pinned vs ethanol-preserved (calculated per 5 consecutive years)
2013-‐2014 2008-‐2012 2003-‐2007 1998-‐2002 1993-‐1997
pinnedethanol
% of sa
mples with
a DN
A barcod
e >55
0 bp
Period2013-‐2014 2008-‐2012 2003-‐2007 1998-‐2002 1993-‐1997
pinnedethanol
% of sa
mples with
a DN
A barcod
e >55
0 bp
Period2013-‐2014 2008-‐2012 2003-‐2007 1998-‐2002 1993-‐1997
pinnedethanol
% of sa
mples with
a DN
A barcod
e >55
0 bp
Period
Absolute ethanol
Pinned specimens
Ethanol-preserved: 98.9% Pinned: all: 64.9% >15 years old: 25%
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RESULTS AND DISCUSSION
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
(26) (2)
(14) (2)
(4) (2)
(7)
(2)
(2)
(2)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
(26) (2)
(14) (2)
(4) (2)
(7)
(2)
(2)
(2)
Nhaplotype per species: 1 to 19 Mean Nhaplotype per species: 3.82 è No haplotype shared between species
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RESULTS AND DISCUSSION
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
(26) (2)
(14) (2)
(4) (2)
(7)
(2)
(2)
(2)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
0 2 4 6 8 10 12 14 16 18
12
34
56
78
910
1112
1314
1516
1718
19
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
A
C
B
Frequency histogram of specimens / species
Number of specimens / species
Num
ber o
f hap
loty
pes
/ spe
cies
Num
ber o
f spe
cies
Frequency histogram of haplotypes / species
0 2 4 6 8 10 12 14 16 18
Number of species (1)
(26) (2)
(14) (2)
(4) (2)
(7)
(2)
(2)
(2)
Nhaplotype per species: 1 to 19 Mean Nhaplotype per species: 3.82 è No haplotype shared between species
Significant correlation between the Nindividuals sampled per species and Nhaplotypes recorded. è BUT mean and maximum intraspecific K2P distances were not affected by sample size.
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RESULTS AND DISCUSSION
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
010
0020
0030
0040
0050
00
0 0.04 0.08 0.12 0.16 0.2 0.24 0.28
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
020
4060
8010
012
0
0 0.03 0.06 0.09 0.12 0.15 0.18
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
050
010
0015
0020
00
0 0.03 0.06 0.09 0.12 0.15 0.18
Numb
er of
pairw
ise co
mpari
sons
K2P distance
MicrodontinaeEristalinae Syrphinae
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
010
0020
0030
0040
0050
00
0 0.04 0.08 0.12 0.16 0.2 0.24 0.28
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
020
4060
8010
012
0
0 0.03 0.06 0.09 0.12 0.15 0.18
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
050
010
0015
0020
00
0 0.03 0.06 0.09 0.12 0.15 0.18
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
010
0020
0030
0040
0050
00
0 0.04 0.08 0.12 0.16 0.2 0.24 0.28
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
020
4060
8010
012
0
0 0.03 0.06 0.09 0.12 0.15 0.18
intraspecificinterspecific
K2P distance
Numb
er of
pairw
ise co
mpar
isons
050
010
0015
0020
00
0 0.03 0.06 0.09 0.12 0.15 0.18
Numb
er of
pairw
ise co
mpari
sons
K2P distance
MicrodontinaeEristalinae Syrphinae
Mean K2P distances: - intraspecific = 0.78% (0-5.63%) - interspecific = 9.29% (0-16.3%)
72 morphospecies were >3% divergent from their nearest neighbor è K2P = good proxy to distinguish intra- from interspecific genetic variability. BUT in Syrphinae and Eristalinae subfamilies, absence of perfect DNA barcoding gap.
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RESULTS AND DISCUSSION
Threshold-base method: optimal threshold to separate intra from interspecific K2P distances was defined at 6% for the whole dataset.
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RESULTS AND DISCUSSION
Threshold-base method: optimal threshold to separate intra from interspecific K2P distances was defined at 6% for the whole dataset. è BUT large differences among subfamilies :
- Eristalinae: 3.5% - BM: 100% identification success - BCM: 98%
- Syrphinae: 5.8% - BM and BCM: 97.1%
- Microdontinae: 0.7-2% (presence of a barcoding gap) - BM and BCM: 100%
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RESULTS AND DISCUSSION
Threshold-base method: optimal threshold to separate intra from interspecific K2P distances was defined at 6% for the whole dataset. è BUT large differences among subfamilies :
- Eristalinae: 3.5% - BM: 100% identification success - BCM: 98%
- Syrphinae: 5.8% - BM and BCM: 97,1%
- Microdontinae: 0.7-2% (presence of a barcoding gap) - BM and BCM: 100%
Tree-based method: 55 morphospecies (90.1%) formed monophyletic groups (high support).
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Dorsal (top) and lateral (bottom) view of an adult female Toxomerus floralis.
Localities were Toxomerus floralis was found in Africa.
INTRODUCED NEW WORLD SPECIES
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CONCLUSION
• Construction of a reference dataset of 480 COI barcodes of the more common species from Ghana, Togo, Benin and Nigeria;
• DNA barcoding is effective for most of the morphospecies examined;
• Different analytical methods give highly congruent results, with high identification success.
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PERSPECTIVES
• Enlarge sampling size and sites: 2/3 of the common
species is not represented yet. A larger geographical area would also allow to better account for intraspecific variation in barcodes;
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PERSPECTIVES
• Enlarge sampling size and sites: 2/3 of the common
species is not represented yet. A larger geographical area would also allow to better account for intraspecific variation in barcodes;
• High intraspecific and low interspecific sequence divergence in some taxa suggests that the taxonomy of several taxa deserves further study:
• More variable markers to increase resolution; • Re-evaluate the morphological characters currently used to
separate morphospecies; • Ex: Allobaccha picta, Asarkina ericetorum, Syritta bulbus, Phytomia
natalensis etc.
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PERSPECTIVES
• Link immature stage and female morphotype with barcoded specimens (for sp. for which it wasn’t done so far);
• Pinned specimen > 15 years old: next-generation DNA barcoding to retrieve DNA barcodes of very old material (ex: holotype).
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ACKNOWLEDGEMENTS
• Belgian Science Policy (BELSPO)
• JEMU and the Molecular DNA labs of RBINS and RMCA
• Belgian Network for DNA barcoding and FWO
• Organizers of the 6th international Barcode of Life Conference
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THANK YOU FOR YOUR ATTENTION
© Mengual, Ximo