DNA BARCODING CONTRIBUTES TO THE TAXONOMY OF...

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

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

IDENTIFICATION CHALLENGES

MORPHOLOGY

-  Identification keys: -  Identification keys available for only 60% species of the known fauna; -  Larger the genus, less reliable the identification.


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.

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


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.


AIMS

1)  Construct a DNA barcoding library for Afrotropical hover flies;

AIMS


2)  Compare amplification success obtained with pinned and ethanol-preserved specimens;

AIMS


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.

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

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

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.




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)

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


•  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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Frequency histogram of haplotypes / species

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Nhaplotype per species: 1 to 19 Mean Nhaplotype per species: 3.82 è No haplotype shared between species


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(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.


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


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


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


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


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


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


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


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


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.


Threshold-base method: optimal threshold to separate intra from interspecific K2P distances was defined at 6% for the whole dataset.


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%


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).

Dorsal (top) and lateral (bottom) view of an adult female Toxomerus floralis.

Localities were Toxomerus floralis was found in Africa.

INTRODUCED NEW WORLD SPECIES

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.

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;

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.

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).

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

DNA BARCODING CONTRIBUTES TO THE TAXONOMY OF...

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