Ten years of barcoding at CBGP: outputs and prospects. Jean-Claude Streito, Guénaëlle Genson, Eric Pierre, Astrid Cruaud, Jean Yves Rasplus

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Jean-Claude Streito, Guénaëlle Genson, Eric Pierre, Astrid Cruaud, Jean Yves Rasplus. Ten years of Heteroptera barcoding at CBGP: outputs and prospects.. 6. Quadrennial IHS Meeting, Dec 2018, La Plata, Argentina. ￿hal-02788356￿

HAL Id: hal-02788356 https://hal.inrae.fr/hal-02788356 Submitted on 5 Jun 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Ten years of Heteroptera barcoding at CBGP : outputs and prospects Streito Jean-Claude; Genson Guénaëlle; Pierre Éric; Cruaud Astrid & Rasplus Jean-Yves

[email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 Barcoding at CBGP : 10 years

My lab : INRA-CBGP (Centre for Biology and Management of Populations)

Where : Montpellier, France

My job : Produce tools for identification of

Morphology and molecular (barcoding)

.02 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018

Barcoding at CBGP : 10 years

We are interested in Arthropods of agronomic interest

Pests:

Beneficial :

Invasive :

.03 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018

Barcoding at CBGP : 10 years

We have increased true bugs barcoding in recent years

Many true bug species have become major pests in Europe in recent years, Pests: especially on fruit and vegetable crops Lygus spp., Nezara viridula, acuteangulatus ...

Several predatory true bugs are used as biocontrol agents, especially Mirid and Beneficial: Anthocorid species

Invasive : Several alien species have become invasive, including Halyomorpha halys, Corythucha arcuata…

.04 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018

_01 Material and Methods

.05 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 Barcoding at CBGP : methods

Standard Barcode: COI 656 pb (Herbert et al., 2003)

First: Classical PCR and Sanger sequencing

For 3 years: two-step PCR and Miseq sequencing

.06 IHS VI meeting La Plata 3-7 dec 2018 Barcoding at CBGP : methods

We switched to the production of barcodes by NGS for many reasons: - lower cost for massive sample runs - a better understanding of artifacts (pseudogenes, heteroplasmy...) - the ability to detect parasitism

Sanger: only one sequence obtained for each bug (the barcode of the bug or that of its potential parasitoid). NGS: several thousand sequences produced for each bug (the barcode of the bug but also that of its potential parasitoids, its endosymbionts etc.)

.07 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 Barcoding at CBGP : methods

International databases provide barcodes: BOLD Systems. Editor BOLD Canada. DDBJ - DNA Data Bank of Japan. Editor : DDBJ Center. GenBank - NCBI. Editor : NCBI, National Center for Biotechnology Information.

But none of them fully meet our needs:

- many of the we are interested in have not yet been sequenced - international DB (especially GenBank) contain too many misidentifications to allow reliable assignation

To design our identification tools: - we fill the gaps and contribute to barcoding of life - our constant concern is to avoid sequences incorrectly identified (contaminations, misidentifications, pseudogenes…)

.08 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 Species to be Sampling Identification Databases included conditioning Web portal albidipennis storage Orius horwathi Orius laevigatus Orius laticollis Orius lindbergi Orius majusculus Orius minutus Orius niger Orius pallidicornis Orius vicinus Orius insidiosus Orius navaishae Orius tantillus

DNA extraction and Collections storage ADN

PCR amplification COI + nuDNA

Sequencing 2nd quality control

Phylogenetic Sequence correction analysis 1st quality control

Protocol used at CBGP for the production and validation of sequences .09 Barcoding at CBGP : methods One important step: a non-destructive DNA extraction that allows vouchering and morphological re-examination by a taxonomist

Extraction

Dissection

Vouchers

Observation

.010 Barcoding at CBGP : methods

Last quality control : validation of sequences by phylogenetic inference

.011 [email protected] : INRA-CBGP Montpellier _02 Results

.012 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Number of sequences produced :

All database: 21 169 COI sequences

Heteroptera: 2 123 COI sequences http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home Famillies: 35 Genus: 202 Species: 363

A database (Arthemis DB@se) focusing on pests and their natural enemies is continually implemented.

.013 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Representation of biogeographical regions:

5% 75%

5%

11%

3% 1%

.014 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

% Number of sequences produced: sequences Heteroptera: 2 123 COI sequences

Pests 38%

Beneficials 28%

Invasive 12%

Other bugs 22%

.015 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results Number of sequences by families

Species COI COI Sub-order Infra-orders Super-famillies Famillies world Bold CBGP ENICOCEPHALOMORPHA 20 1 0 (425 espèces) 405 2 0 52 2 2 51 2 2 HYPSIPTERYGIDAE 4 - 0 (395 espèces) 287 2 0 STEMMOCRYPTIDAE 1 - 0 751 65 3 HERMATOBATIDAE 9 - 0 962 29 1 HEBRIDAE 221 5 0 (2 120 espèces) HEBROIDEA 3 0 0 PARAPHRYNOVELIIDAE 2 1 0 MESOVELIOIDEA 46 14 0 HETEROPTERA HYDROMETROIDEA 126 6 1 (45 318 espèces) AEPOPHILIDAE 1 - 0 SALDOIDEA 335 42 3 (381 espèces) LEPTOPODIDAE 39 1 0 LEPTOPOIDEA OMANIIDAE 6 - 0 78 2 0 NAUCOROIDEA 391 7 1 POTAMOCORIDAE 8 4 0 160 28 0 NEPOIDEA 268 19 0 CORIXOIDEA 607 89 1 (2 309 espèces) 180 2 0 NOTONECTOIDEA 400 30 2 38 5 0 111 5 0 OCHTEROIDEA 68 2 0

.016 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Number of sequences by families:

Species COI COI Sous-ordres Infra-ordres Superfamilles Familles World Bold CBGP

ANTHOCORIDAE 507 93 41

CIMICIDAE 110 18 1

CIMICOIDEA 27 6 0

PLOKIOPHILIDAE 13 0 0

POLYCTENIDAE 32 1 0

CURALIIDAE 1 0 0 VELOCIPEDOIDEA VELOCIPEDIDAE 31 1 0

HETEROPTERA JOPPEICOIDEA JOPPEICIDAE 1 1 0 (45 318 espèces) (21 277 espèces) MEDOCOSTIDAE 1 - 0 NABOIDEA 386 42 12

MICROPHYSOIDEA 25 8 0

MIRIDAE 11 101 879 107

MIROIDEA THAUMASTOCORIDAE 19 1 1

TINGIDAE 2 124 89 33

PACHYNOMIDAE 21 0 0 REDUVIOIDEA 6 878 236 2

.017 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Number of validated sequences by families:

Species COI COI Sous-ordres Infra-ordres Superfamilles Familles World Bold CBGP 1 931 87 0 9 1 0 359 26 0 3 178 125 9 3 1 0 295 46 12 STENOCEPHALIDAE 16 9 1 IDIOSTOLOIDEA IDIOSTOLIDAE 4 0 0 20 2 0 174 20 2 436 10 3 84 1 0 HETEROPTERA 4 - 0 (45 318 espèces) (18 411 espèces) 54 9 1 280 16 5 HENICOCORIDAE 1 - 0 100 3 0 968 90 26 29 - 0 MESCHIIDAE 5 - 0 13 1 0 150 17 4 78 5 0 44 5 2 1 850 127 15

.018 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Number of validated sequences by families:

Nombre COI COI Sous-ordres Infra-ordres Superfamilles Familles d’espèces Bold CBGP 317 47 1 APHYLIDAE 3 - 0 CANOPIDAE 16 1 0 765 25 2 CYRTOCORIDAE 11 1 0 110 9 0 LESTONIIDAE 2 1 0 MEGARIDIDAE 18 - 0 4 0 0 HETEROPTERA PENTATOMOMORPHA 4 841 438 56 (45 318 espèces) (18 411 espèces) 4 1 0 PLATASPIDIDAE 604 8 1 531 34 5 250 6 1 THAUMASTELLIDAE 3 2 0 220 9 0 UROSTYLIDAE 181 6 0 106 14 0 340 34 2

Total 45 318 2977 363

.019 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Several similar studies has been conducted (Jung et al, 2011; Park et al., 2011; Raupach et al., 2014 etc.) we found quite similar results: - standard COI bacode is a good gene to explore true bugs diversity - for about 10% of genus problems appears (cryptic species and non discrimination of some species)

Some Genus with more taxa than expected Alpagut (cryptic taxa): Notonecta Graphosoma Nezara 11 genus (5,7% of genus studies) Eurydema Nysius Macrolophus Adelphocoris Cardiastethus Elatophilus Orius

.020 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 More species as expected

Macrolophus pygmaeus

Genus Macrolophus

Macrolophus pygmaeus like

Macrolophus pygmaeus like

Probably two new undescribed species

.021 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Several similar studies has been conducted (Jung et al, 2011; Park et al., 2011; Raupach et al., 2014 etc.) we found quite similar results: - standard COI bacode is a good gene to explore true bugs diversity - for about 10% of genus problems appears (cryptic species and non discrimination of species)

Genus for witch COI is non resolutive: Aelia Arocatus Ischnodemus 6 genus (3,1 % of genus studied) Physatocheila Nabis Lygus

.022 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 COI non resolutive

N. rugosus

Genus Nabis N. ferus N. brevis N. pseudoferus

N. provencalis N. ferus

N. punctatus N. pseudoferus

Some misidentifations to be corrected after morphological review of vouchers Morever COI is not fully resolutive to separate all European species

.023 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 10 years Barcoding CBGP : Results

Arthropoda Heteroptera 21 169 2 123

http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home

For the moment we have produced many sequences. Most of them are not accessible because they have not yet been validated and published.

Sq validated We now expect to validate and publish as many (avalaible for blast) sequences as possible to make them accessible. 570 Heteroptera 2 123 Sq published (fully avalaible) 264

.024 [email protected] : INRA-CBGP Montpellier IHS VI meeting La Plata 3-7 dec 2018 MERCI

.025 [email protected] : INRA-CBGP Montpellier