# Distinct barcodes for the Cereal leaf beetles Oulema melanopus and Oulema duftschmidi (Coleoptera: Chrysomelidae), two syntopical sibling species

Julie Leroy, Jean-David Chapelin-Viscardi, Guénaëlle Genson, Julien Haran,
Eric Pierre, Jean-Claude Streito

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Julie Leroy, Jean-David Chapelin-Viscardi, Guénaëlle Genson, Julien Haran, Eric Pierre, et al. Distinct barcodes for the Cereal leaf beetles Oulema melanopus and Oulema duftschmidi (Coleoptera: Chrysomelidae), two syntopical sibling species. European Journal of Entomology, 2020, 117, pp.490-503. 10.14411/eje.2020.052. hal-03276037

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# Distinct barcodes for the Cereal leaf beetles *Oulema melanopus* and *Oulema duftschmidi* (Coleoptera: Chrysomelidae), two syntopical sibling species

JULIE LEROY<fnref n="1" />, JEAN-DAVID CHAPELIN-VISCARDI<fnref n="1" />, GUÉNAËLLE GENSON<fnref n="2" />, JULIEN HARAN<fnref n="2" />, ÉRIC PIERRE<fnref n="2" />
and JEAN-CLAUDE STREITO<fnref n="2" />

**Key words.** Coleoptera, Chrysomelidae, *Oulema*, cereal leaf beetles, DNA barcode, COI, identification, crop pests, Europe

**Abstract.** *Oulema melanopus* (Linnaeus, 1758) and *Oulema duftschmidi* (Redtenbacher, 1874) (Coleoptera: Chrysomelidae) are two native West Palaearctic species developing on various cultivated and wild grasses. Along with *O. obscura* they are considered to be secondary pests of cereal crops. However, local outbreaks have been recorded recently and their status as secondary pests may evolve, especially as the use of broad-spectrum insecticides is now greatly reduced. *Oulema melanopus* and *O. duftschmidi* are considered to be sibling species. They are morphologically very close and difficult to distinguish from each other, which makes it difficult to study them. We tested the reliability of the standard barcode fragment (COI) for distinguishing between these species. A total of 92 samples of the two species, covering the majority of their natural range, was sequenced for the barcode fragment and inter- and intraspecific genetic distances were estimated. Our results confirm those of Bezděk & Baselga (2015, *Acta Entomol. Mus. Nat. Prag.* **55**: 273-304) in that this marker cannot differentiate between all the species of the *Oulema melanopus* complex, which in the Mediterranean basin contains several described and possibly some undescribed cryptic species. However, this marker may be useful in an agricultural context in areas where only *O. melanopus* and *O. duftschmidi* occur (such as in cereal crops in France) where it can be used to reliably and rapidly separate all stages of these two taxa and can therefore help in studying their ecology and dynamics.

## INTRODUCTION

Cereal leaf beetles belong to Coleoptera of the subfamily Criocerinae within the family Chrysomelidae and their larvae feed and develop on various cultivated or wild grasses (Venturi, 1942; Jolivet, 1997). Nine species are recognized in France, one belonging to the genus *Lema* [*Lema cyanela* (Linnaeus, 1758)] and 8 to the genus *Oulema* [*Oulema duftschmidi* (Redtenbacher, 1874), *O. erichsonii* (Suffrian, 1841), *O. obscura* (Stephens, 1831), *O. hoffmannseggii* (Lacordaire, 1845), *O. melanopus* (Linnaeus, 1758), *O. rufocyanea* (Suffrian, 1847), *O. septentrionis* (Weise, 1880) and *O. tristis* (Herbst, 1786)]. The genus *Oulema* comprises about 130 species worldwide, of which 19 are Nearctic and 21 Palearctic. Currently, 11 species are known from Europe (Bezděk & Schmitt, 2017; Rilet et al., 2003), among which two new species were recently described from Italy (*Oulema mauroi* Bezděk & Baselga, 2015) and Spain (*Oulema verae* Bezděk & Baselga, 2015). Only two species of *Oulema* are frequently cited as cereal pests in the Palearctic region: *O. melanopus* and *O. obscura* (Bala-

chowsky & Mesnil, 1935; Bonnemaison, 1962; Labeyrie, 1963; Chambon et al., 1983; ACTA, 2016). The history of the taxonomy of *O. obscura* is very confusing: early works mention *O. obscura* as *Lema lichenis* Weise, 1882 or *Lema lichenis* Voet, 1806 (an invalid name according to White, 1981), then as *O. gallaeciana* (Heyden, 1870), before it was synonymized with *O. obscura* (Stephens, 1831) (Cox, 2000; Bezděk & Schmitt, 2017). Labeyrie (1963) further cites *O. tristis* as a pest, as does Feytaud (1924). With the exception of *O. tristis*, which has not been reported on crops since then, three species of *Oulema* are regularly observed in crops and are likely to damage them in France and Europe: *O. obscura*, *O. melanopus*, but also *O. duftschmidi*, which was confused with the previous species until 1989 and is still not included in plant protection manuals. *O. melanopus* and *O. duftschmidi* are considered to be sibling species, as they are very similar both in their internal (genitalia) and external morphology. Their identification requires the dissection of the flagellum located in the penis of the males (Berti, 1989; Bezděk & Baselga,

<footnote n="1">¹ Laboratoire d'Éco-Entomologie, 5 rue Antoine Mariotte, 45000 Orléans, France;
e-mails: leroy@laboratoireecentomologie.com, chapelinviscardi@laboratoireecentomologie.com</footnote>

<footnote n="2">² CBGP, INRAE, CIRAD, IRD, Institut Agro, Univ Montpellier, Montpellier, France; e-mails: guenaelle.genson@inrae.fr, julien.haran@cirad.fr, eric.pierre@inrae.fr, jean-claude.streito@inrae.fr</footnote>
2015; Chapelin-Viscardi & Maillet-Mezeray, 2015; Leroy & Chapelin-Viscardi, 2018), which makes their study particularly difficult for non-specialists, especially as it is not possible to identify females and the immature stages. The existence of both species in France was reported by Berti (1989), who provides reliable morphological criteria for identifying them (Fig. 2) and reports the existence of specimens of *O. duftschmidi* identified as *O. melanopus* in the collections of the French National Museum of Natural History (MNHN, Paris). Berti (1989) also states that both species are sympatric and widely distributed in France. More recently, Bezděk & Baselga (2015) revised the *Oulema melanopus* species complex in Western Europe. They recognize five species, including two new ones and review the taxonomy of the group.

*Oulema melanopus* and *O. duftschmidi* can damage crops, especially the larvae that feed on leaves of cereal plants (Philips et al., 2011). These sibling species could potentially harm cereal crops in France (Bonnemaison, 1962; Labeyrie, 1963; Anglade et al., 1976; Chambon et al., 1983; ACTA, 2016 etc.) and other European countries (Labeyrie, 1963): Romania (Knechtel & Monolache, 1936), Hungary (Sajó, 1893), Spain (Urquijo, 1940), Greece (Pélécassis, 1951) and Italy (Bechini et al., 2013). Most of these publications refer only to *O. melanopus* whereas *O. duftschmidi* may be also involved (Chapelin-Viscardi & Maillet-Mezeray, 2015). Recently, high population densities of larvae of cereal leaf beetles were recorded in various parts of mainland France, such as the Ille-et-Vilaine, Loiret and Allier departments (pers. obs.). In addition, extreme climatic events, which are becoming increasingly frequent, are conducive to pest outbreaks, particularly of species that are highly dependent on the climate, such as cereal leaf beetles (Guppy & Harcourt, 1978; Olfert & Weiss, 2006; Bechini et al., 2013). The greatly reduced use of neonicotinoid insecticides, and more generally that of pesticides, along with the increase in organic farming in Europe, could also provide suitable conditions for future outbreaks of these pests. Their status could shift from secondary to major pests, as has occurred in the United States and Asia (Philips et al., 2011).

Due to the impossibility of identifying the larvae of these species little is known about the life history traits and relative abundance of these two species. Preliminary agricultural monitoring indicates that *O. duftschmidi* is the more common in several French agricultural landscapes (Chapelin-Viscardi & Maillet-Mezeray, 2015). These surveys also reveal that the flight activity of the adults of both these species is synchronous, which indicates simultaneous larval development. A more recent biogeographical study provides clear evidence that both species are sympatric and coexist throughout France (Leroy & Chapelin-Viscardi, 2018).

In order to better understand the structure of the *O. melanopus/O. duftschmidi* species complex it is crucial to have a reliable and routine method for identifying all the development stages of the species. Kubisz et al. (2012) used the standard barcode fragment of the mitochondrial

COI gene (Hebert et al., 2003a) to successfully distinguish between and identify several species of Criocerinae in the genus *Crioceris*. Similarly, Bezděk & Baselga (2015) use this DNA fragment for identifying European species of *Oulema*, but their results, based on a small number of specimens, indicate that the COI barcode is not appropriate for the molecular identification of these species as there are discrepancies between the species boundaries revealed by morphology and DNA barcodes. In this study the effectiveness of this gene for identifying the species of *Oulema* feeding on cereals in Europe is re-evaluated using a larger number of specimens.

# MATERIALS AND METHODS

## Sampling and morphological identification

Specimens were collected between 2005 and 2017 in France, Portugal (Madeira), Greece (Crete), Spain and Italy (Table 1 and Fig. 1). At each site sampled, one to three adults or larvae were collected and killed directly in 96.5% ethanol. The adult specimens were identified to species, based on external morphological characters (Warchałowski, 2003; Bezděk & Mlejnek, 2016), except for specimens belonging to the *Oulema melanopus/duftschmidi* species pair (Fig. 2) for which the identification was based on the structure of dissected male genitalia as only by examination of the flagellum can the two species in this complex be reliably separated (Chapelin-Viscardi & Maillet-Mezeray, 2015). *O. duftschmidi* has a thin, elongated flagellum (Fig. 2d, f) whereas *O. melanopus* has a short, stocky flagellum (Fig. 2e, g) (Bukejs & Ferenca, 2010; Bezděk & Baselga, 2015). For this reason, only males were used to validate our DNA sequences for *O. melanopus* and *O. duftschmidi*. Three species of *Oulema* were sequenced, including 92 male specimens belonging to the *O. melanopus/duftschmidi* complex (44 *O. melanopus* ♂, 48 *O. duftschmidi* ♂) and 43 specimens of both sexes of *O. obscura*. To account for these species in an evolutionary context and validate our means of identification, 25 additional specimens of 8 different species belonging to the subfamily Criocerinae were also sampled. The species *Epitrix pubescens* (Chrysomelidae: Galerucinae) was used as an outgroup to root the phylogenetic analysis reported below. After validation of the barcoding method, we tested the molecular identification of 17 females and 7 larvae of the *O. melanopus/duftschmidi* complex. As a result, the sample for molecular analyses included a total of 185 specimens (Table 1).

## DNA sequencing and analysis of sequences

The extraction and amplification protocol was that used by Streito et al. (2018): extraction of the total genomic DNA was carried out in a non-destructive manner, on whole specimens, using the DNeasy 96 Blood & Tissue extraction kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The standard barcode fragment (Hebert et al., 2003a) was amplified using a mixture of tailed primers (based on Cruaud et al., 2010; Germain et al., 2013 and Ivanova et al., 2007) (Table 2). PCRs were carried out in 25 µl of reagents with 2 µl of matrix DNA, 0.07 mM of each primer, 2.5 mM of MgCl₂, 0.05 mM of dNTPs and 0.025 U/µl Dreamtaq DNA Polymerase (Thermo Scientific, Waltham, USA). The PCR conditions used were 94°C for 3 min followed by 5 cycles at 94°C for 30 s, 45°C for 40 s, 72°C for 60 s, then 35 cycles at 94°C for 30 s, 51°C for 40 s, 72°C for 60 s, with a final extension phase at 72°C for 10 min. PCR products were purified, then sequenced directly by Eurofins MWG Operon according to their protocol using M13 sequencing primers (M13F and M13R). Forward and reverse overlapping strands were assembled
<table><caption>**Table 1.** List of sequenced specimens and the sequence accession numbers available in Bold and GenBank. For presentation purposes, COI sequences of specimens highlighted in grey were not included in the phylogenetic trees (Figs 3 and S1).</caption><thead><tr><th>Species</th><th>Specimens</th><th>Phylogenetic tree codes</th><th>Life stage (sex)</th><th>Country</th><th>Locality</th><th>Geographic coordinates Lon./Lat., decimal degr.</th><th>Sampling date</th><th>BOLD ID</th><th>GenBank Access no.</th></tr></thead><tbody><tr><td rowspan="5">Crioceris asparagi (L., 1758)</td><td>CCOC09462_0101</td><td>–</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.238952/0.185079</td><td>15/07/2010</td><td>BCELB001-19</td><td>MT456383</td></tr><tr><td>CCOC09463_0101</td><td>–</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.238952/0.185079</td><td>15/07/2010</td><td>BCELB002-19</td><td>MT456384</td></tr><tr><td>CCOC09464_0101</td><td>–</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.238952/0.185079</td><td>15/07/2010</td><td>BCELB003-19</td><td>MT456382</td></tr><tr><td>CCOC00752_0101</td><td>C007520101</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.249645/0.210105</td><td>10/07/2005</td><td>BCELB004-19</td><td>MT456385</td></tr><tr><td>CCOC02373_0101</td><td>C023730101</td><td>adult</td><td>France</td><td>Vernou-sur-Brenne</td><td>47.400450/0.863481</td><td>19/05/2007</td><td>BCELB005-19</td><td>MT456386</td></tr><tr><td rowspan="2">Crioceris bicruciata (Sahlberg, 1823)</td><td>JSTR00373_0101</td><td>R003730101</td><td>adult</td><td>Greece</td><td>Askinou (Crete)</td><td>35.291880/24.178090</td><td>25/05/2013</td><td>BCELB006-19</td><td>MT456388</td></tr><tr><td>JSTR00373_0103</td><td>R003730103</td><td>adult</td><td>Greece</td><td>Askinou (Crete)</td><td>35.291880/24.178090</td><td>25/05/2013</td><td>BCELB007-19</td><td>MT456387</td></tr><tr><td rowspan="5">Crioceris duodecimpunctata (L., 1758)</td><td>CCOC00757_0101</td><td>–</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.249645/0.210105</td><td>13/07/2005</td><td>BCELB008-19</td><td>MT456390</td></tr><tr><td>CCOC02404_0101</td><td>–</td><td>adult</td><td>France</td><td>La Chapelle-sur-Loire</td><td>47.248332/0.194144</td><td>13/05/2007</td><td>BCELB009-19</td><td>MT456391</td></tr><tr><td>JSTR01304_0101</td><td>–</td><td>adult</td><td>France</td><td>Villandry</td><td>47.340936/0.500805</td><td>18/06/2012</td><td>BCELB010-19</td><td>MT456392</td></tr><tr><td>CCOC00555_0101</td><td>C005550101</td><td>adult</td><td>France</td><td>Montauroux</td><td>43.617652/6.807396</td><td>18/09/2005</td><td>BCELB011-19</td><td>MT456389</td></tr><tr><td>CCOC01914_0101</td><td>C019140101</td><td>adult</td><td>France</td><td>Saint-Martin-en-Ré</td><td>46.198628/-1.344658</td><td>12/08/2007</td><td>BCELB012-19</td><td>MT456393</td></tr><tr><td rowspan="3">Crioceris paracenthesis (L., 1767)</td><td>JSTR01291_0102</td><td>–</td><td>adult</td><td>France</td><td>Saint-Raphaël</td><td>43.417100/6.859020</td><td>10/07/2005</td><td>BCELB013-19</td><td>MT456395</td></tr><tr><td>JSTR01296_0101</td><td>–</td><td>adult</td><td>France</td><td>Salte-Croix-de-Quintillargue</td><td>43.775419/3.907606</td><td>07/05/2005</td><td>BCELB014-19</td><td>MT456396</td></tr><tr><td>JSTR01291_0101</td><td>R012910101</td><td>adult</td><td>France</td><td>Saint-Raphaël</td><td>43.417100/6.859020</td><td>10/07/2005</td><td>BCELB015-19</td><td>MT456394</td></tr><tr><td rowspan="2">Lilioceris lili (Scopoli, 1763)</td><td>JSTR01294_0101</td><td>R012940101</td><td>adult</td><td>France</td><td>Mireval</td><td>43.531030/3.802800</td><td>30/04/2005</td><td>BCELB016-19</td><td>MT456397</td></tr><tr><td>JSTR01305_0101</td><td>R013050101</td><td>adult</td><td>France</td><td>Montlouis-sur-Loire</td><td>47.370624/0.837422</td><td>26/04/2012</td><td>BCELB024-19</td><td>MT456406</td></tr><tr><td>JSTR01305_0102</td><td>R013050102</td><td>adult</td><td>France</td><td>Montlouis-sur-Loire</td><td>47.370624/0.837422</td><td>26/04/2012</td><td>BCELB025-19</td><td>MT456407</td></tr><tr><td rowspan="3">Lema aenea (Lacordaire, 1845)</td><td>JSTR01223_0101</td><td>–</td><td>adult</td><td>France</td><td>Salazie (Reunion Island)</td><td>–21.052480/55.525760</td><td>29/03/2014</td><td>BCELB017-19</td><td>MT456399</td></tr><tr><td>JSTR00629_0101</td><td>R006290101</td><td>adult</td><td>France</td><td>Saint-Benoît (Reunion Island)</td><td>–21.096460/55.653440</td><td>25/03/2014</td><td>BCELB018-19</td><td>MT456400</td></tr><tr><td>JSTR00629_0102</td><td>R006290102</td><td>adult</td><td>France</td><td>Saint-Benoît (Reunion Island)</td><td>–21.096460/55.653440</td><td>25/03/2014</td><td>BCELB019-19</td><td>MT456401</td></tr><tr><td rowspan="3">Lema borboniae (Jolivet, 1979)</td><td>JSTR01222_0101</td><td>–</td><td>adult</td><td>France</td><td>Salazie (Reunion Island)</td><td>–21.052480/55.525760</td><td>29/03/2014</td><td>BCELB020-19</td><td>MT456402</td></tr><tr><td>JSTR00629_1301</td><td>R006291301</td><td>adult</td><td>France</td><td>Saint-Benoît (Reunion Island)</td><td>–21.096460/55.653440</td><td>25/03/2014</td><td>BCELB021-19</td><td>MT456403</td></tr><tr><td>JSTR00629_1302</td><td>R006291302</td><td>adult</td><td>France</td><td>Saint-Benoît (Reunion Island)</td><td>–21.096460/55.653440</td><td>25/03/2014</td><td>BCELB022-19</td><td>MT456404</td></tr><tr><td rowspan="2">Lema cyanella (L., 1758)</td><td>JSTR04552_0201</td><td>R045520201</td><td>adult (♂)</td><td>Spain</td><td>Villafranca de Ebro</td><td>41.540200/-0.565540</td><td>6/8/2017</td><td>BCELB023-19</td><td>MT456405</td></tr><tr><td>JCH00002_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB026-19</td><td>MT456451</td></tr><tr><td rowspan="21">Oulema duftschmidi (Retdenbacher, 1874)</td><td>JCH00003_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB027-19</td><td>MT456452</td></tr><tr><td>JCH00003_0303</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB028-19</td><td>MT456453</td></tr><tr><td>JCH00004_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Houville-la-Branche</td><td>48.447991/1.625547</td><td>18/07/2014</td><td>BCELB029-19</td><td>MT456454</td></tr><tr><td>JCH00005_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB030-19</td><td>MT456455</td></tr><tr><td>JCH00006_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB031-19</td><td>MT456456</td></tr><tr><td>JCH00009_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB032-19</td><td>MT456457</td></tr><tr><td>JCH00010_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Thiverval-Grignon</td><td>48.846643/1.951940</td><td>01/06/2014</td><td>BCELB033-19</td><td>MT456458</td></tr><tr><td>JCH00015_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Orléans</td><td>–</td><td>15/06/2013</td><td>BCELB034-19</td><td>MT456459</td></tr><tr><td>JCH00018_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277301/2.806932</td><td>15/05/2014</td><td>BCELB035-19</td><td>MT456460</td></tr><tr><td>JCH00018_0303</td><td>–</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277301/2.806932</td><td>15/05/2014</td><td>BCELB036-19</td><td>MT456461</td></tr><tr><td>JCH00020_0302</td><td>–</td><td>adult (♂)</td><td>Portugal</td><td>Porto Santo Island (Madeira)</td><td>33.083000/-16.334400</td><td>04/04/2015</td><td>BCELB038-19</td><td>MT456463</td></tr><tr><td>JCH00021_0301</td><td>–</td><td>adult (♂)</td><td>Portugal</td><td>Porto Santo Island (Madeira)</td><td>33.083000/-16.334400</td><td>04/04/2015</td><td>BCELB039-19</td><td>MT456464</td></tr><tr><td>JCH00025_0302</td><td>–</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB040-19</td><td>MT456465</td></tr><tr><td>JCH00026_0302</td><td>–</td><td>adult (♂)</td><td>Greece</td><td>Chania (Crete)</td><td>35.476613/23.933068</td><td>31/05/2016</td><td>BCELB041-19</td><td>MT456466</td></tr><tr><td>JSTR01261_0101</td><td>–</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.525900/2.809390</td><td>15/05/2014</td><td>BCELB037-19</td><td>MT456462</td></tr><tr><td>JSTR03883_0102</td><td>–</td><td>adult (♂)</td><td>Italy</td><td>Collemeto</td><td>40.203419/18.093920</td><td>16/11/2016</td><td>BCELB042-19</td><td>MT456467</td></tr><tr><td>JCH00002_0301</td><td>H000020301</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB043-19</td><td>MT456468</td></tr><tr><td>JCH00003_0301</td><td>H000030301</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB044-19</td><td>MT456469</td></tr><tr><td>JCH00004_0301</td><td>H000040301</td><td>adult (♂)</td><td>France</td><td>Houville-la-Branche</td><td>48.447991/1.625547</td><td>18/07/2014</td><td>BCELB045-19</td><td>MT456470</td></tr><tr><td>JCH00005_0301</td><td>H000050301</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB046-19</td><td>MT456471</td></tr><tr><td>JCH00006_0301</td><td>H000060301</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB047-19</td><td>MT456472</td></tr><tr><td>JCH00007_0301</td><td>H000070301</td><td>adult (♂)</td><td>France</td><td>Cossé-le-Vivien</td><td>47.947795/-0.921883</td><td>15/05/2014</td><td>BCELB048-19</td><td>MT456473</td></tr><tr><td>JCH00008_0301</td><td>H000080301</td><td>adult (♂)</td><td>France</td><td>Beugnâtre</td><td>50.129035/2.874760</td><td>14/05/2014</td><td>BCELB049-19</td><td>MT456474</td></tr><tr><td>JCH00009_0301</td><td>H000090301</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB050-19</td><td>MT456475</td></tr><tr><td>JCH00010_0301</td><td>H000100301</td><td>adult (♂)</td><td>France</td><td>Thiverval-Grignon</td><td>48.846643/1.951940</td><td>01/06/2014</td><td>BCELB051-19</td><td>MT456476</td></tr><tr><td>JCH00012_0301</td><td>H000120301</td><td>adult (♂)</td><td>France</td><td>Bruxerolles</td><td>46.611001/0.386810</td><td>18/05/2014</td><td>BCELB052-19</td><td>MT456477</td></tr><tr><td>JCH00013_0301</td><td>H000130301</td><td>adult (♂)</td><td>France</td><td>Mouzeuil-Saint-Martin</td><td>46.465235/-0.983798</td><td>15/06/2014</td><td>BCELB053-19</td><td>MT456478</td></tr><tr><td>JCH00014_0301</td><td>H000140301</td><td>adult (♂)</td><td>France</td><td>Saint-Priest</td><td>45.687722/4.967944</td><td>30/06/2014</td><td>BCELB054-19</td><td>MT456479</td></tr><tr><td>JCH00015_0301</td><td>H000150301</td><td>adult (♂)</td><td>France</td><td>Orléans</td><td>–</td><td>15/06/2013</td><td>BCELB055-19</td><td>MT456432</td></tr><tr><td>JCH00016_0301</td><td>H000160301</td><td>adult (♂)</td><td>France</td><td>Parcieux</td><td>45.917306/4.835083</td><td>07/07/2014</td><td>BCELB056-19</td><td>MT456433</td></tr><tr><td>JCH00017_0302</td><td>H000170302</td><td>adult (♂)</td><td>France</td><td>Plaisir</td><td>48.793955/1.942767</td><td>03/05/2014</td><td>BCELB057-19</td><td>MT456434</td></tr><tr><td>JCH00018_0301</td><td>H000180301</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277301/2.806932</td><td>15/05/2014</td><td>BCELB058-19</td><td>MT456435</td></tr><tr><td>JCH00019_0301</td><td>H000190301</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.525900/2.809400</td><td>23/07/2014</td><td>BCELB059-19</td><td>MT456436</td></tr><tr><td>JCH00020_0301</td><td>H000200301</td><td>adult (♂)</td><td>Portugal</td><td>Porto Santo Island (Madeira)</td><td>33.083000/-16.334400</td><td>04/04/2015</td><td>BCELB064-19</td><td>MT456441</td></tr><tr><td>JCH00021_0302</td><td>H000210302</td><td>adult (♂)</td><td>Portugal</td><td>Porto Santo Island (Madeira)</td><td>33.083000/-16.334400</td><td>04/04/2015</td><td>BCELB065-19</td><td>MT456450</td></tr><tr><td>JCH00022_0301</td><td>H000220301</td><td>adult (♂)</td><td>France</td><td>Lectoure</td><td>43.933604/0.623621</td><td>12/05/2015</td><td>BCELB066-19</td><td>MT456449</td></tr><tr><td>JCH00024_0301</td><td>H000240301</td><td>adult (♂)</td><td>France</td><td>Ladon</td><td>48.004543/2.533953</td><td>05/07/2015</td><td>BCELB067-19</td><td>MT456448</td></tr><tr><td>JCH00025_0301</td><td>H000250301</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB068-19</td><td>MT456447</td></tr><tr><td>JCH00026_0301</td><td>H000260301</td><td>adult (♂)</td><td>Greece</td><td>Chania (Crete)</td><td>35.476613/23.933068</td><td>31/05/2016</td><td>BCELB069-19</td><td>MT456446</td></tr><tr><td>JCH00026_0303</td><td>H000260303</td><td>adult (♂)</td><td>Greece</td><td>Chania (Crete)</td><td>35.476613/23.933068</td><td>31/05/2016</td><td>BCELB070-19</td><td>MT456445</td></tr><tr><td>JSTR00666_0102</td><td>R006660102</td><td>adult (♂)</td><td>France</td><td>Arles</td><td>43.510278/4.781944</td><td>27/04/2014</td><td>BCELB060-19</td><td>MT456437</td></tr><tr><td>JSTR01258_0101</td><td>R012580101</td><td>adult (♂)</td><td>France</td><td>Lorgies</td><td>50.554260/2.793420</td><td>15/05/2014</td><td>BCELB061-19</td><td>MT456438</td></tr><tr><td>JSTR01297_0102</td><td>R012970102</td><td>adult (♂)</td><td>France</td><td>Sainte-Croix-de-Quintillargue</td><td>43.775419/3.907606</td><td>23/04/2006</td><td>BCELB062-19</td><td>MT456439</td></tr><tr><td>JSTR01302_0101</td><td>R013020101</td><td>adult (♂)</td><td>France</td><td>Assay</td><td>47.062201/0.270064</td><td>12/05/2006</td><td>BCELB063-19</td><td>MT456440</td></tr><tr><td>JSTR03882_0101</td><td>R038820101</td><td>adult (♂)</td><td>Italy</td><td>Tricase</td><td>39.945573/18.352039</td><td>17/11/2016</td><td>BCELB071-19</td><td>MT456444</td></tr><tr><td>JSTR03888_0101</td><td>R038880101</td><td>adult (♂)</td><td>Italy</td><td>Cascina Litta</td><td>40.091173/18.167204</td><td>16/11/2016</td><td>BCELB072-19</td><td>MT456443</td></tr><tr><td>JSTR04562_0101</td><td>R045620101</td><td>adult (♂)</td><td>Spain</td><td>Aguillar-de-Segarra</td><td>41.738710/1.610790</td><td>6/9/2017</td><td>BCELB073-19</td><td>MT456442</td></tr></tbody></table>
<table><caption>Table 1 (continued).</caption><thead><tr><th>Species</th><th>Specimens</th><th>Phylogenetic tree codes</th><th>Life stage (sex)</th><th>Country</th><th>Locality</th><th>Geographic coordinates Lon./Lat., decimal degr.</th><th>Sampling date</th><th>BOLD ID</th><th>GenBank Access. no.</th></tr></thead><tbody><tr><td rowspan="24">Oulema melanopus (L., 1758)</td><td>JDCH00001_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Berstett</td><td>48.682758/7.649488</td><td>07/07/2014</td><td>BCELB141-19</td><td>MT456518</td></tr><tr><td>JDCH00002_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB142-19</td><td>MT456517</td></tr><tr><td>JDCH00003_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB143-19</td><td>MT456516</td></tr><tr><td>JDCH00003_0203</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB144-19</td><td>MT456515</td></tr><tr><td>JDCH00005_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB145-19</td><td>MT456514</td></tr><tr><td>JDCH00006_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB146-19</td><td>MT456513</td></tr><tr><td>JDCH00007_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Cossé-le-Vivien</td><td>47.947795/-0.921883</td><td>15/05/2014</td><td>BCELB147-19</td><td>MT456512</td></tr><tr><td>JDCH00012_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Bruxerolles</td><td>46.611001/0.386810</td><td>18/05/2014</td><td>BCELB148-19</td><td>MT456511</td></tr><tr><td>JDCH00013_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Mouzeuil-Saint-Martin</td><td>46.465235/-0.983798</td><td>15/06/2014</td><td>BCELB149-19</td><td>MT456510</td></tr><tr><td>JDCH00014_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Saint-Priest</td><td>45.687722/4.967944</td><td>30/06/2014</td><td>BCELB150-19</td><td>MT456509</td></tr><tr><td>JDCH00015_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Orléans</td><td>–</td><td>15/06/2013</td><td>BCELB151-19</td><td>MT456508</td></tr><tr><td>JDCH00016_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Parcieux</td><td>45.917306/4.835083</td><td>07/07/2014</td><td>BCELB152-19</td><td>MT456507</td></tr><tr><td>JDCH00019_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809400</td><td>23/07/2014</td><td>BCELB153-19</td><td>MT456506</td></tr><tr><td>JDCH00019_0203</td><td>–</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809400</td><td>23/07/2014</td><td>BCELB154-19</td><td>MT456505</td></tr><tr><td>JDCH00020_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Verchain-Maugré</td><td>50.266552/3.474370</td><td>19/04/2015</td><td>BCELB156-19</td><td>MT456503</td></tr><tr><td>JDCH00024_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Ladon</td><td>48.004543/2.533953</td><td>05/07/2015</td><td>BCELB157-19</td><td>MT456502</td></tr><tr><td>JDCH00025_0202</td><td>–</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB158-19</td><td>MT456501</td></tr><tr><td>JDCH00025_0203</td><td>–</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB159-19</td><td>MT456520</td></tr><tr><td>JSTR00657_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Clapiers</td><td>43.650652/3.873110</td><td>16/04/2014</td><td>BCELB155-19</td><td>MT456504</td></tr><tr><td>JDCH00001_0201</td><td>H000010201</td><td>adult (♂)</td><td>France</td><td>Berstett</td><td>48.682758/7.649488</td><td>07/07/2014</td><td>BCELB160-19</td><td>MT456499</td></tr><tr><td>JDCH00002_0201</td><td>H000020201</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB161-19</td><td>MT456500</td></tr><tr><td>JDCH00003_0201</td><td>H000030201</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB162-19</td><td>MT456519</td></tr><tr><td>JDCH00004_0201</td><td>H000040201</td><td>adult (♂)</td><td>France</td><td>Houville-la-Branche</td><td>48.447991/1.625547</td><td>18/07/2014</td><td>BCELB163-19</td><td>MT456521</td></tr><tr><td>JDCH00005_0201</td><td>H000050201</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB164-19</td><td>MT456522</td></tr><tr><td>JDCH00006_0201</td><td>H000060201</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB165-19</td><td>MT456480</td></tr><tr><td>JDCH00007_0201</td><td>H000070201</td><td>adult (♂)</td><td>France</td><td>Cossé-le-Vivien</td><td>47.947795/-0.921883</td><td>15/05/2014</td><td>BCELB166-19</td><td>MT456523</td></tr><tr><td>JDCH00009_0201</td><td>H000090201</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB167-19</td><td>MT456481</td></tr><tr><td>JDCH00012_0201</td><td>H000120201</td><td>adult (♂)</td><td>France</td><td>Bruxerolles</td><td>46.611001/0.386810</td><td>18/05/2014</td><td>BCELB168-19</td><td>MT456482</td></tr><tr><td>JDCH00013_0201</td><td>H000130201</td><td>adult (♂)</td><td>France</td><td>Mouzeuil-Saint-Martin</td><td>46.465235/-0.983798</td><td>15/06/2014</td><td>BCELB169-19</td><td>MT456483</td></tr><tr><td>JDCH00014_0201</td><td>H000140201</td><td>adult (♂)</td><td>France</td><td>Saint-Priest</td><td>45.687722/4.967944</td><td>30/06/2014</td><td>BCELB170-19</td><td>MT456484</td></tr><tr><td>JDCH00015_0201</td><td>H000150201</td><td>adult (♂)</td><td>France</td><td>Orléans</td><td>–</td><td>15/06/2013</td><td>BCELB171-19</td><td>MT456485</td></tr><tr><td>JDCH00016_0201</td><td>H000160201</td><td>adult (♂)</td><td>France</td><td>Parcieux</td><td>45.917306/4.835083</td><td>07/07/2014</td><td>BCELB172-19</td><td>MT456486</td></tr><tr><td>JDCH00018_0201</td><td>H000180201</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277301/2.806932</td><td>15/05/2014</td><td>BCELB173-19</td><td>MT456487</td></tr><tr><td>JDCH00019_0201</td><td>H000190201</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809400</td><td>23/07/2014</td><td>BCELB174-19</td><td>MT456488</td></tr><tr><td>JDCH00020_0201</td><td>H000200201</td><td>adult (♂)</td><td>France</td><td>Verchain-Maugré</td><td>50.266552/3.474370</td><td>19/04/2015</td><td>BCELB179-19</td><td>MT456493</td></tr><tr><td>JDCH00024_0201</td><td>H000240201</td><td>adult (♂)</td><td>France</td><td>Ladon</td><td>48.004543/2.533953</td><td>05/07/2015</td><td>BCELB180-19</td><td>MT456494</td></tr><tr><td>JDCH00025_0201</td><td>H000250201</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB181-19</td><td>MT456495</td></tr><tr><td>JSTR00656_0101</td><td>R006560101</td><td>adult (♂)</td><td>France</td><td>Clapiers</td><td>43.658835/3.867831</td><td>16/04/2014</td><td>BCELB175-19</td><td>MT456489</td></tr><tr><td>JSTR00666_0101</td><td>R006660101</td><td>adult (♂)</td><td>France</td><td>Arles</td><td>43.510278/4.781944</td><td>27/04/2014</td><td>BCELB176-19</td><td>MT456490</td></tr><tr><td>JSTR00769_0101</td><td>R007690101</td><td>adult (♂)</td><td>France</td><td>Le-Bouchet-Saint-Nicolas</td><td>44.900330/3.748820</td><td>08/08/2014</td><td>BCELB177-19</td><td>MT456491</td></tr><tr><td>JSTR01303_0101</td><td>R013030101</td><td>adult (♂)</td><td>France</td><td>La-Roche-Clermault</td><td>47.139333/0.228444</td><td>08/07/2005</td><td>BCELB178-19</td><td>MT456492</td></tr><tr><td>JSTR04448_0101</td><td>R044480101</td><td>adult (♂)</td><td>Spain</td><td>Maçanet de la Selva</td><td>41.762700/2.758420</td><td>5/28/2017</td><td>BCELB182-19</td><td>MT456496</td></tr><tr><td>JSTR04454_0101</td><td>R044540101</td><td>adult (♂)</td><td>Spain</td><td>Tordera</td><td>41.712840/2.686380</td><td>5/28/2017</td><td>BCELB183-19</td><td>MT456497</td></tr><tr><td>JSTR04552_0101</td><td>R045520101</td><td>adult (♂)</td><td>Spain</td><td>Villafranca de Ebro</td><td>41.540200/-0.565540</td><td>6/8/2017</td><td>BCELB184-19</td><td>MT456498</td></tr><tr><td rowspan="24">Oulema obscura (Stephens, 1831)</td><td>JDCH00001_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Berstett</td><td>48.682758/7.649488</td><td>07/07/2014</td><td>BCELB074-19</td><td>MT456542</td></tr><tr><td>JDCH00001_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Berstett</td><td>48.682758/7.649488</td><td>07/07/2014</td><td>BCELB075-19</td><td>MT456543</td></tr><tr><td>JDCH00002_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB076-19</td><td>MT456544</td></tr><tr><td>JDCH00002_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB077-19</td><td>MT456545</td></tr><tr><td>JDCH00003_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB078-19</td><td>MT456536</td></tr><tr><td>JDCH00003_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB079-19</td><td>MT456535</td></tr><tr><td>JDCH00004_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Houville-la-Branche</td><td>48.447991/1.625547</td><td>18/07/2014</td><td>BCELB080-19</td><td>MT456534</td></tr><tr><td>JDCH00005_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB081-19</td><td>MT456533</td></tr><tr><td>JDCH00005_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB082-19</td><td>MT456546</td></tr><tr><td>JDCH00006_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB083-19</td><td>MT456547</td></tr><tr><td>JDCH00007_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Cossé-le-Vivien</td><td>47.947795/-0.921883</td><td>15/05/2014</td><td>BCELB084-19</td><td>MT456548</td></tr><tr><td>JDCH00009_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB085-19</td><td>MT456549</td></tr><tr><td>JDCH00009_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB086-19</td><td>MT456550</td></tr><tr><td>JDCH00010_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Thiverval-Grignon</td><td>48.846643/1.951940</td><td>01/06/2014</td><td>BCELB087-19</td><td>MT456551</td></tr><tr><td>JDCH00010_0103</td><td>–</td><td>adult (♂)</td><td>France</td><td>Thiverval-Grignon</td><td>48.846643/1.951940</td><td>01/06/2014</td><td>BCELB088-19</td><td>MT456552</td></tr><tr><td>JDCH00012_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Bruxerolles</td><td>46.611001/0.386810</td><td>18/05/2014</td><td>BCELB089-19</td><td>MT456553</td></tr><tr><td>JDCH00020_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Verchain-Maugré</td><td>50.266552/3.474370</td><td>19/04/2015</td><td>BCELB095-19</td><td>MT456559</td></tr><tr><td>JDCH00024_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Ladon</td><td>48.004543/2.533953</td><td>05/05/2015</td><td>BCELB096-19</td><td>MT456560</td></tr><tr><td>JSTR01259_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Lorgies</td><td>50.554260/2.793420</td><td>15/05/2014</td><td>BCELB090-19</td><td>MT456554</td></tr><tr><td>JSTR01260_0101</td><td>–</td><td>adult (♂)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809390</td><td>15/05/2014</td><td>BCELB091-19</td><td>MT456555</td></tr><tr><td>JSTR01265_0101</td><td>–</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.283450/2.813050</td><td>15/05/2014</td><td>BCELB092-19</td><td>MT456556</td></tr><tr><td>JSTR01265_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.283450/2.813050</td><td>15/05/2014</td><td>BCELB093-19</td><td>MT456557</td></tr><tr><td>JSTR01272_0102</td><td>–</td><td>adult (♂)</td><td>France</td><td>Saint-Laurent-de-Lin</td><td>47.511404/0.271791</td><td>22/07/2014</td><td>BCELB094-19</td><td>MT456558</td></tr><tr><td>JDCH00001_0101</td><td>H000010101</td><td>adult (♂)</td><td>France</td><td>Berstett</td><td>48.682758/7.649488</td><td>07/07/2014</td><td>BCELB097-19</td><td>MT456561</td></tr><tr><td>JDCH00002_0101</td><td>H000020101</td><td>adult (♂)</td><td>France</td><td>Mirecourt</td><td>48.286220/6.106403</td><td>25/03/2014</td><td>BCELB098-19</td><td>MT456562</td></tr><tr><td>JDCH00003_0101</td><td>H000030101</td><td>adult (♂)</td><td>France</td><td>Gannat</td><td>46.100167/3.199103</td><td>24/04/2014</td><td>BCELB099-19</td><td>MT456563</td></tr><tr><td>JDCH00004_0101</td><td>H000040101</td><td>adult (♂)</td><td>France</td><td>Houville-la-Branche</td><td>48.447991/1.625547</td><td>18/07/2014</td><td>BCELB100-19</td><td>MT456564</td></tr><tr><td>JDCH00005_0101</td><td>H000050101</td><td>adult (♂)</td><td>France</td><td>Treffendel</td><td>48.037544/-1.983165</td><td>06/06/2014</td><td>BCELB101-19</td><td>MT456565</td></tr><tr><td>JDCH00006_0101</td><td>H000060101</td><td>adult (♂)</td><td>France</td><td>Saint-Aubin-du-Désert</td><td>48.322195/-0.203475</td><td>15/05/2014</td><td>BCELB102-19</td><td>MT456566</td></tr><tr><td>JDCH00007_0101</td><td>H000070101</td><td>adult (♂)</td><td>France</td><td>Cossé-le-Vivien</td><td>47.947795/-0.921883</td><td>15/05/2014</td><td>BCELB103-19</td><td>MT456524</td></tr><tr><td>JDCH00008_0102</td><td>H000080102</td><td>adult (♂)</td><td>France</td><td>Beugnâtre</td><td>50.129035/2.874760</td><td>14/05/2014</td><td>BCELB104-19</td><td>MT456525</td></tr></tbody></table>
<table><caption>Table 1 (continued).</caption><thead><tr><th>Species</th><th>Specimens</th><th>Phylogenetic tree codes</th><th>Life stage (sex)</th><th>Country</th><th>Locality</th><th>Geographic coordinates Lon./Lat., decimal degr.</th><th>Sampling date</th><th>BOLD ID</th><th>GenBank Access no.</th></tr></thead><tbody><tr><td rowspan="23">"Oulema group melanopus"</td><td>JDCH00009_0101</td><td>H000090101</td><td>adult (♂)</td><td>France</td><td>Querrieu</td><td>49.945034/2.424406</td><td>25/07/2014</td><td>BCELB105-19</td><td>MT456526</td></tr><tr><td>JDCH00010_0101</td><td>H000100101</td><td>adult (♂)</td><td>France</td><td>Thiverval-Grignon</td><td>48.846643/1.951940</td><td>01/06/2014</td><td>BCELB106-19</td><td>MT456527</td></tr><tr><td>JDCH00011_0101</td><td>H000110101</td><td>adult (♂)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277301/2.806932</td><td>15/05/2014</td><td>BCELB107-19</td><td>MT456528</td></tr><tr><td>JDCH00012_0101</td><td>H000120101</td><td>adult (♀)</td><td>France</td><td>Bruxerolles</td><td>46.611001/0.386810</td><td>18/05/2014</td><td>BCELB108-19</td><td>MT456529</td></tr><tr><td>JDCH00020_0101</td><td>H000200101</td><td>adult (♀)</td><td>France</td><td>Verchain-Maugré</td><td>50.266552/3.474370</td><td>19/04/2015</td><td>BCELB114-19</td><td>MT456539</td></tr><tr><td>JDCH00024_0101</td><td>H000240101</td><td>adult (♂)</td><td>France</td><td>Ladon</td><td>48.004543/2.533953</td><td>05/05/2015</td><td>BCELB115-19</td><td>MT456540</td></tr><tr><td>JDCH00025_0101</td><td>H000250101</td><td>adult (♂)</td><td>France</td><td>Montargis</td><td>47.995572/2.730073</td><td>12/07/2015</td><td>BCELB116-19</td><td>MT456541</td></tr><tr><td>JSTR01259_0101</td><td>R012590101</td><td>adult (♂)</td><td>France</td><td>Lorgies</td><td>50.554260/2.793420</td><td>15/05/2014</td><td>BCELB109-19</td><td>MT456530</td></tr><tr><td>JSTR01260_0102</td><td>R012600102</td><td>adult (♀)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809390</td><td>15/05/2014</td><td>BCELB110-19</td><td>MT456531</td></tr><tr><td>JSTR01262_0102</td><td>R012620102</td><td>adult (♀)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277510/2.799190</td><td>15/05/2014</td><td>BCELB111-19</td><td>MT456532</td></tr><tr><td>JSTR01272_0101</td><td>R012720101</td><td>adult (♂)</td><td>France</td><td>Saint-Laurent-de-Lin</td><td>47.511404/0.271791</td><td>22/07/2014</td><td>BCELB112-19</td><td>MT456537</td></tr><tr><td>JSTR01299_0101</td><td>R012990101</td><td>adult (♀)</td><td>France</td><td>Chambray-lès-Tour</td><td>47.339980/0.722099</td><td>16/05/2006</td><td>BCELB113-19</td><td>MT456538</td></tr><tr><td>EPIE00642_0103</td><td>–</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB122-19</td><td>MT456426</td></tr><tr><td>EPIE00642_0104</td><td>–</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB123-19</td><td>MT456425</td></tr><tr><td>EPIE00642_0105</td><td>–</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB124-19</td><td>MT456424</td></tr><tr><td>EPIE00642_0106</td><td>–</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB125-19</td><td>MT456423</td></tr><tr><td>JSTR00656_0103</td><td>–</td><td>adult (♀)</td><td>France</td><td>Clapiers</td><td>43.658835/3.867831</td><td>16/04/2014</td><td>BCELB117-19</td><td>MT456431</td></tr><tr><td>JSTR00657_0101</td><td>–</td><td>adult (♀)</td><td>France</td><td>Clapiers</td><td>43.650652/3.873110</td><td>16/04/2014</td><td>BCELB118-19</td><td>MT456430</td></tr><tr><td>JSTR00657_0103</td><td>–</td><td>adult (♀)</td><td>France</td><td>Clapiers</td><td>43.650652/3.873110</td><td>16/04/2014</td><td>BCELB119-19</td><td>MT456429</td></tr><tr><td>JSTR01263_0101</td><td>–</td><td>adult (♀)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.277510/2.799190</td><td>15/05/2014</td><td>BCELB120-19</td><td>MT456428</td></tr><tr><td>JSTR01264_0101</td><td>–</td><td>adult (♀)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.283450/2.813050</td><td>15/05/2014</td><td>BCELB121-19</td><td>MT456427</td></tr><tr><td>JSTR03884_0101</td><td>–</td><td>adult (♀)</td><td>Italy</td><td>Cascina Litta</td><td>40.091173/18.167204</td><td>16/11/2016</td><td>BCELB126-19</td><td>MT456422</td></tr><tr><td>CCOC02368_0101</td><td>C023680101</td><td>larva</td><td>France</td><td>Mer</td><td>47.721926/1.500711</td><td>15/05/2007</td><td>BCELB127-19</td><td>MT456421</td></tr><tr><td>EPIE00642_0101</td><td>E006420101</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB136-19</td><td>MT456412</td></tr><tr><td>EPIE00642_0102</td><td>E006420102</td><td>larva</td><td>France</td><td>Beyssenac</td><td>45.411704/1.324250</td><td>07/06/2008</td><td>BCELB137-19</td><td>MT456411</td></tr><tr><td>JSTR00656_0102</td><td>R006560102</td><td>adult (♀)</td><td>France</td><td>Clapiers</td><td>43.658835/3.867831</td><td>16/04/2014</td><td>BCELB129-19</td><td>MT456419</td></tr><tr><td>JSTR00666_0103</td><td>R006660103</td><td>adult (♀)</td><td>France</td><td>Arles</td><td>43.510278/4.781944</td><td>27/04/2014</td><td>BCELB130-19</td><td>MT456418</td></tr><tr><td>JSTR00669_0801</td><td>R006690801</td><td>adult (♀)</td><td>France</td><td>Combaillaux</td><td>43.666567/3.785286</td><td>18/05/2014</td><td>BCELB128-19</td><td>MT456420</td></tr><tr><td>JSTR01258_0102</td><td>R012580102</td><td>adult (♀)</td><td>France</td><td>Lorgies</td><td>50.554260/2.793420</td><td>15/05/2014</td><td>BCELB131-19</td><td>MT456417</td></tr><tr><td>JSTR01261_0102</td><td>R012610102</td><td>adult (♀)</td><td>France</td><td>Neuville-Vitasse</td><td>50.255900/2.809390</td><td>15/05/2014</td><td>BCELB132-19</td><td>MT456416</td></tr><tr><td>JSTR01264_0102</td><td>R012640102</td><td>adult (♀)</td><td>France</td><td>Tilloy-lès-Mofflaines</td><td>50.283450/2.813050</td><td>15/05/2014</td><td>BCELB133-19</td><td>MT456415</td></tr><tr><td>JSTR01297_0101</td><td>R012970101</td><td>adult (♀)</td><td>France</td><td>Sainte-Croix-de-Quintillargue</td><td>43.775419/3.907606</td><td>23/04/2006</td><td>BCELB134-19</td><td>MT456414</td></tr><tr><td>JSTR01301_0101</td><td>R013010101</td><td>adult (♀)</td><td>france</td><td>Preuilly-sur-Claise</td><td>46.854377/0.928917</td><td>17/05/2006</td><td>BCELB135-19</td><td>MT456413</td></tr><tr><td>JSTR03883_0101</td><td>R038830101</td><td>adult (♀)</td><td>Italy</td><td>Collemeto</td><td>40.203419/18.093920</td><td>16/11/2016</td><td>BCELB138-19</td><td>MT456410</td></tr><tr><td>JSTR03885_0101</td><td>R038850101</td><td>adult (♀)</td><td>Italy</td><td>Galugnano</td><td>40.249590/18.229184</td><td>16/11/2016</td><td>BCELB139-19</td><td>MT456408</td></tr><tr><td>JSTR03886_0101</td><td>R038860101</td><td>adult (♀)</td><td>Italy</td><td>Collemeto</td><td>40.203419/18.093920</td><td>11/16/2016</td><td>BCELB140-19</td><td>MT456409</td></tr><tr><td>Epitrix pubescens (Koch, 1803)</td><td>CCOC11818_0101</td><td>C118180101</td><td>adult</td><td>La Ville-aux-Dames</td><td>47.394035/0.770874</td><td>6/4/2011</td><td>BCELB185-19</td><td>MT456398</td></tr></tbody></table>

using Geneious v4.6.2 (Drummond et al., 2010). Sequences were aligned with default ClustalW parameters (1.81) (Thompson et al., 1997). Consensus sequences were translated to amino acids using MEGA 7 software (Kumar et al., 2016) to detect frame-shift mutations and premature stop-codons, which may have indicated the presence of pseudogenes or contaminations. Voucher specimens and associated DNA are preserved in the INRAE collections of the CBGP (Centre de Biologie pour la Gestion des Populations, Montferrier-sur-Lez, France). All sequences were deposited in Barcode of Life Data System (BOLD; Ratnasingham & Hebert, 2007; www.boldsystems.org) and GenBank (NCBI). Our sequence dataset is available in BOLD (through the DOI dx.doi.org/10.5883/DS-BCOUL) and NCBI, accession numbers are provided in Table 1.

Pairwise nucleotide sequence divergences were calculated using the Kimura 2-parameter model of substitution (Kimura, 1980) in MEGA 7 software (Kumar et al., 2016) and the “pairwise-deletion” option. A preliminary phylogenetic tree of the genus *Oulema*, including all the specimens sequenced, was reconstructed based on the *COI* sequences. For a clearer view of the inferred tree, only the sequences of one or two specimens per locality are presented in Fig. 3 (specimens not highlighted in Table 1). We reconstructed a tree (Fig. 3) including the standard barcode *COI* solely for specimens reliably identified to species (only dissected males for the *melanopus/duftschmidi* complex) and the sequences published by Bezdkěk & Baselga (2015). Phylogenetic analyses were conducted using the Maximum Likelihood (ML) method and the SMS model (Smart Model Selection)

(Lefort et al., 2017) in Phylm 3.0 software (Guindon et al., 2010). The aLRT approximation (approximate likelihood ratio test) was used to calculate the bootstrapping values for each node (Anisimova, 2006) (5000 replicates). The resulting phylogenetic trees were edited using iTOL software (Letunic & Bork, 2016).

## RESULTS

### Analysis of newly obtained sequences

The barcode fragment was successfully amplified for all specimens, regardless of sex or stage. In total, 185 specimens belonging to 12 species were sequenced (Table 1). The observed minimum interspecific genetic distance between species of Chrysomelidae in the neighbouring genera of *Oulema* based on *COI* ranged from 10.2 to 19.3% (Table 3). In the genus *Oulema*, *O. obscura* showed a minimum divergence of 18.5% with *Oulema melanopus* and *Oulema duftschmidi*. *O. melanopus* showed a minimum divergence of 3.1% with *O. duftschmidi*, its closest relative. Between genera of the Criocerinae, the minimum divergence varied from 16.5% between *Lema* and *Oulema* to 21.8% between *Crioceris* and *Oulema*. *Epitrix* had a minimum distance from the Criocerinae species tested ranging from 22.2 to 26.2%.

The intraspecific divergence levels ranged from 0 to 0.9% (mean 0.19% ± 0.06%) for *O. melanopus*, from 0 to
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Map showing the locations of the sites where males of *Oulema melanopus* (44 specimens) and *O. duftschmidi* (48 specimens) were collected. J. Leroy Design [QGIS Software version 2.18.12 (QGIS, 2016), Mapping Holdings Association: UE (Burke, 2012) and GREAT (UMS 2414 RIMATE, 2018)].</figcaption></figure>

<table><caption>**Table 2.** Mixture of PCR primers used in this study (based on Cruaud et al., 2010 and Germain et al., 2013). M13 tails (Ivanova et al., 2007) were used.</caption><thead><tr><th>Name of the primer</th><th>Sequence 5'–3' of the primer</th></tr></thead><tbody><tr><td>Forward</td><td>TGTAAAACGACGGCCAGTTTTCAACWAATCATAAAGATATTGG</td></tr><tr><td>LCO1490puc_t1</td><td>TGTAAAACGACGGCCAGTTTTCAACTAAYCATAARGATATYGG</td></tr><tr><td>LCO1490Hem1_t1</td><td></td></tr><tr><td>Reverse</td><td>CAGGAAACAGCTATGACTAAACTTCWGGRGWCCAAARAATCA</td></tr><tr><td>HCO2198puc_t1</td><td>CAGGAAACAGCTATGACTAAACYTCAGGATGACCAAAAAAYCA</td></tr><tr><td>HCO2198Hem2_t1</td><td></td></tr><tr><td>HCO2198Hem1_t1</td><td>CAGGAAACAGCTATGACTAAACYTCDDGATGBCCAAARAATCA</td></tr></tbody></table>

1.7% (mean 0.57% ± 0.14%) for *O. duftschmidi* and from 0 to 0.5% (mean 0.09% ± 0.05%) for *O. obscura* (Table 4).

## Phylogenetic reconstruction

The substitution models selected by PhyML 3.0 as the most appropriate were the GTR + G + I model for the tree in Fig. 3 (AIC = 10403,93488). That tree is based on both our sequences and those published by Bezděk & Baselga (2015). The genus *Oulema* is monophyletic (support 83.26%) with two sister clades. One (support 91.40%) contains *O. obscura* and *O. hoffmannseggii* from Spain (as in the phylogenetic tree of Bezděk & Baselga, 2015, in which, however, the latter species is paraphyletic); *O. obscura* contains two sister groups, one comprising specimens from the Iberian Peninsula and the other from France

and the Czech Republic. The other clade containing the *O. melanopus* complex (including the 5 species recognized by Bezděk & Baselga, 2015) is highly supported (99.42%), but its internal relationships are problematic. *O. duftschmidi* forms a well-supported clade* (91.36%) which, however,

<footnote>* Among the dissected males, only one mismatched specimen (JSTR02905_0101 from Alentejo, Portugal, morphologically clearly belonging to *O. duftschmidi*) was nested with specimens of *O. melanopus*. This could be a labeling error or contamination (see Discussion); therefore the specimen was removed from the final analyses, from the trees in Figs 3 and S1 and consequently from sequences deposited in Bold (Table 1).</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Habitus and male genitalia of *Oulema* spp. A – *O. obscura* (specimen JSTR1259_0101). B – *O. duftschmidi* (specimen JSTR0666_0102). C – *O. melanopus* (specimen JSTR00769_0101). D – *O. duftschmidi* male aedeagus in lateral view with flagellum extracted. E – *O. melanopus*, idem. F – *O. duftschmidi* flagellum (specimen JSTR01302_0101). G – *O. melanopus*, idem (specimen CCOC11910_010, not sequenced). Photographs: J.-C. Streito, except D and E, which are from Bukejs & Ferenca, 2010.</figcaption></figure>

includes also the single available sequence of *O. mauroi* and one specimen from Morocco identified as *O. melano-pus* (again similar to the tree in Bezděk & Baselga, 2015), whereas the other specimens (*O. rufocyanea*, *O. verae* and all remaining *O. melanopus*) form a markedly paraphyletic cluster and the latter two species (represented by more than one specimen) are both polyphyletic. In particular, five specimens from Galicia (NW Spain) identified as *O. mel-anopus* stand out and form a sister clade to *O. rufocyanea* (as in Bezděk & Baselga, 2015).

If we exclude the sequences of Bezděk & Baselga (2015), the resulting tree (not shown because it was essentially similar to the present Fig. S1) is congruent with morphological identifications and all species are monophyletic. The three cereal pest species had support of 100% for *O. obscura*, 65.94% for *O. melanopus* and 95.72% for *O. duftschmidi*.

When we included the unidentified specimens of the *O. melanopus* complex from France and Italy (see Table 1) in the above analysis, they were clearly placed in one or the other species (Fig. S1).

## DISCUSSION

During this study, we sequenced 184 specimens of 11 species of Criocerinae in four genera (*Crioceris*, *Lema*, *Lilioceris* and *Oulema*) with a view to testing the possibility of routinely using the standard barcode *COI* for high-throughput and reliable identification of the three species of *Oulema* of agronomic interest, *O. obscura*, *O. melano-pus* and *O. duftschmidi*. We included European specimens (*O. duftschmidi* was also available from Madeira) of 9 out of 28 species and subspecies of Criocerinae known to occur in Europe, and added specimens of 2 more *Lema* species from the Mascarenes. Combining our sequences with
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Phylogenetic tree constructed using the Maximum Likelihood method (ML) and the COI gene sequences of our identified specimens of *Oulema melanopus* (25), *Oulema duftschmidi* (31), *Oulema obscura* (20) and 10 other species of Chrysomelidae (15), including *Epitrix pubescens* (in bold) used to root the tree, and the 30 sequences (also in bold) published by Bezděk & Baselga (2015), giving a total of 122 sequences. Each colour corresponds to a morphologically identified species. The sequence code, identity and geographical origin are given for each specimen. The bootstrap values located at branch nodes are percentages and only those greater than 64% are presented (over 5,000 replicates). The country name is coded according to ISO 3166-1 alpha-2 (ISO 3166, 2016).</figcaption></figure>
<table><caption>**Table 3.** Kimura two-parameter pairwise distance values between species (interspecific divergence). In each box the first line gives the average value and the second the minimum value. The estimated standard errors (SE) are indicated in red above the diagonal. The blue boxes show the values of distances between genera and the yellow boxes the values within the genus. 161 sequences of *O. duftschmidi* (48) and *O. melanopus* (44) males and other species of Chrysomelidae (69) were included in this analysis. Sequences from Bezdźěk & Baselga (2015) are not included.</caption><thead><tr><th></th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th><th>I</th><th>J</th><th>K</th><th>L</th></tr></thead><tbody><tr><td>A Lilioceris lilii</td><td></td><td>0.020</td><td>0.019</td><td>0.018</td><td>0.021</td><td>0.019</td><td>0.019</td><td>0.020</td><td>0.020</td><td>0.020</td><td>0.021</td><td>0.020</td></tr><tr><td>B Crioceris asparagi</td><td>0.218<br>[0,217]</td><td></td><td>0.018</td><td>0.018</td><td>0.017</td><td>0.019</td><td>0.020</td><td>0.019</td><td>0.021</td><td>0.021</td><td>0.021</td><td>0.023</td></tr><tr><td>C Crioceris bicruciata</td><td>0.202<br>[0,202]</td><td>0.180<br>[0,176]</td><td></td><td>0.013</td><td>0.018</td><td>0.020</td><td>0.020</td><td>0.020</td><td>0.021</td><td>0.019</td><td>0.020</td><td>0.022</td></tr><tr><td>D Crioceris paracentesis</td><td>0.189<br>[0,186]</td><td>0.191<br>[0,186]</td><td>0.104<br>[0,102]</td><td></td><td>0.019</td><td>0.022</td><td>0.021</td><td>0.020</td><td>0.021</td><td>0.019</td><td>0.020</td><td>0.021</td></tr><tr><td>E Crioceris<br>duodecimpunctata</td><td>0.237<br>[0,224]</td><td>0.184<br>[0,179]</td><td>0.209<br>[0,184]</td><td>0.207<br>[0,193]</td><td></td><td>0.019</td><td>0.020</td><td>0.021</td><td>0.020</td><td>0.020</td><td>0.021</td><td>0.021</td></tr><tr><td>F Lema aenea</td><td>0.204<br>[0,204]</td><td>0.197<br>[0,193]</td><td>0.218<br>[0,217]</td><td>0.242<br>[0,198]</td><td></td><td>0.015</td><td>0.018</td><td>0.017</td><td>0.018</td><td>0.018</td><td>0.021</td><td>0.021</td></tr><tr><td>G Lema borboniae</td><td>0.208<br>[0,208]</td><td>0.226<br>[0,223]</td><td>0.219<br>[0,219]</td><td>0.230<br>[0,226]</td><td>0.229</td><td>0.136</td><td></td><td>0.017</td><td>0.018</td><td>0.019</td><td>0.019</td><td>0.020</td></tr><tr><td>H Lema cyanella</td><td>0.214<br>[0,214]</td><td>0.211<br>[0,205]</td><td>0.231<br>[0,230]</td><td>0.227<br>[0,223]</td><td>0.228</td><td>0.169</td><td>0.159</td><td></td><td>0.019</td><td>0.020</td><td>0.021</td><td>0.020</td></tr><tr><td>I Oulema obscura</td><td>0.206<br>[0,204]</td><td>0.240<br>[0,232]</td><td>0.242<br>[0,240]</td><td>0.229<br>[0,226]</td><td>0.222</td><td>0.165</td><td>0.166</td><td>0.190</td><td></td><td>0.020</td><td>0.019</td><td>0.022</td></tr><tr><td>J Oulema melanopus</td><td>0.214<br>[0,208]</td><td>0.225<br>[0,217]</td><td>0.220<br>[0,215]</td><td>0.218<br>[0,211]</td><td>0.223</td><td>0.175</td><td>0.190</td><td>0.197</td><td>0.193</td><td></td><td>0.007</td><td>0.022</td></tr><tr><td>K Oulema duftschmidi</td><td>0.222<br>[0,218]</td><td>0.232<br>[0,224]</td><td>0.226<br>[0,220]</td><td>0.235<br>[0,230]</td><td>0.237</td><td>0.176</td><td>0.191</td><td>0.206</td><td>0.192</td><td>0.037</td><td></td><td>0.021</td></tr><tr><td>L Epitrix pubescens<br>(Galerucinae) – outgroup</td><td>0.235<br>[0,235]</td><td>0.263<br>[0,262]</td><td>0.260<br>[0,260]</td><td>0.252<br>[0,250]</td><td>0.240</td><td>0.222</td><td>0.234</td><td>0.224</td><td>0.245</td><td>0.233</td><td>0.234</td><td></td></tr></tbody></table>

those of Bezdźěk & Baselga (2015), 7 European species of *Oulema* (from the 11 known in Europe) were documented. Our results confirm those of Kubisz et al. (2012) and show that the standard DNA barcode can reliably differentiate between most European species of Criocerinae, but confirm also the results of Bezdźěk & Baselga (2015) that this marker cannot differentiate between all species of the genus *Oulema*. A complex of species occurs in the Mediterranean basin including at least *O. melanopus*, *O. verae*, *O. duftschmidi*, *O. mauroi* and possibly some undescribed cryptic species, which cannot be reliably distinguished by the standard barcode. In this study, sampling and marker selection were used to address agronomic questions. The lack of material coming from non-cultivated Mediterranean ecosystems prevented us addressing the problem of the species occurring around the Mediterranean basin where

much more extensive sampling and the use of other markers and other methods would be needed to clarify the taxonomy of *Oulema*.

## Distinguishing *Oulema obscura* from the *O. melanopus* complex

While adult specimens of *O. obscura* can be easily distinguished from those of the *melanopus* group on the basis of their general coloration (body entirely blue versus red pronotum and legs, respectively) (Fig. 2a versus 2b, c), the use of DNA barcodes for species identification can also be used to reliably identify the immature stages of these species. The sequence of the COI gene tested makes it possible to distinguish this species from the entire *melanopus* complex which includes the other two *Oulema* cereal pests (*O. melanopus* and *O. duftschmidi*). The minimum divergence of 18.5% between *O. obscura* and *O. melanopus/O. duftschmidi* is rather high and comparable to distances recorded between species in different genera in the same subfamily, such as *Crioceris* and *Lema* (Table 3).

## Distinguishing between *O. melanopus* and *O. duftschmidi*

The minimum interspecific divergence of 3.1% between specimens of *O. melanopus* and *O. duftschmidi* (Table 3) means that these sibling species are more closely related than all the other species studied, which is consistent with their similar morphology and biology. In addition, such a value is congruent with what is reported for other sibling species in the family Chrysomelidae (Cognato, 2006). However, we recorded lower genetic distances between specimens of *O. duftschmidi* from very distant populations (North of France and the Italian province of Puglia) or isolated populations (such as those in Madeira and Crete), than between specimens of *O. melanopus* and *O.*

<table><caption>**Table 4.** Kimura two-parameter pairwise average distance values within the species studied (intraspecific divergence) (d – average; max – maximum). The estimated standard errors (SE) are given. 161 sequences of *O. duftschmidi* (48) and *O. melanopus* (44) males and other species of Chrysomelidae (69) were included in this analysis. Distances could not be estimated for species with only one individual (*Lema cyanella* and *Epitrix pubescens*). Sequences from Bezdźěk & Baselga (2015) are not included.</caption><thead><tr><th></th><th>d</th><th>SE</th><th>max</th></tr></thead><tbody><tr><td>Oulema melanopus</td><td>0.0019</td><td>0.0006</td><td>0.009</td></tr><tr><td>Oulema duftschmidi</td><td>0.0057</td><td>0.0014</td><td>0.017</td></tr><tr><td>Oulema obscura</td><td>0.0009</td><td>0.0005</td><td>0.005</td></tr><tr><td>Crioceris duodecimpunctata</td><td>0.0183</td><td>0.0042</td><td>0.035</td></tr><tr><td>Crioceris asparagi</td><td>0.0037</td><td>0.0015</td><td>0.009</td></tr><tr><td>Crioceris bicruciata</td><td>0.0015</td><td>0.0015</td><td>0.002</td></tr><tr><td>Crioceris paracentesis</td><td>0.0038</td><td>0.0016</td><td>0.008</td></tr><tr><td>Lema aenea</td><td>0</td><td>0</td><td>n/c</td></tr><tr><td>Lema borboniae</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Lilioceris lilii</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
*duftschmidi* from France, despite being collected together,
in the same place and at the same time (Tables 3 and 4).

Lastly, the interspecific percentage divergences (Table 3)
were well above the maximum percentages of intraspecific
divergence recorded (Table 4) and there was no overlap
between the intra- and interspecific distances of O. mela-
nopus and O. duftschmidi. Consequently, this argues in fa-
vour of a clear genetic differentiation of the sibling species
O. duftschmidi and O. melanopus, which is supported by
the phylogenetic trees (Figs 3 and S1). Of the 92 males
studied, only one (JSTR02905_0101, a male specimen of
O. duftschmidi from Alentejo, Portugal), was placed in a
cluster that does not correspond with the species identifica-
tion based on the morphology of its genitalia. A posteriori
examination of the preserved adult and its dissected geni-
talia definitively excluded misidentification. However, we
cannot exclude an error in tube labelling during handling
or contamination. Wherever possible, we deliberately se-
lected specimens of the two species for our dataset that
were collected on the same day at the same location (see
Table 1), to maximize the chances of recording potential
hybridisation. Apart from this specimen, for which it was
not possible to rule out a handling error, no other individual
was incorrectly assigned in our data set. A second case of
a mismatch between molecular and morphological identifi-
cation was that of the Moroccan specimen (PK406715.1),
which was genetically assigned to O. duftschmidi, whereas
it was identified as O. melanopus by Bezděk & Baselga
(2015). These authors (pers. com.) suspected that there was
an undescribed cryptic species in Morocco to which this
specimen belonged. Indeed, they noted differences between
the genitalia of this specimen and typical specimens of O.
melanopus with which it was tentatively identified. The
method we used (extraction, amplification and sequencing
of a gene) enabled correct assignment of males previously
identified on the basis of dissected genitalia. The uniden-
tified females and larvae that we tested were also unam-
biguously assigned to one of the two taxonomic groups. It
would be interesting to test the method on a larger number
of specimens in order to check whether introgression has
occurred and assess its percentage of occurrence. Breeding
tests would also be required to test this hypothesis.

Distinguishing other species in the *melanopus*
complex and their intraspecific differences

The intraspecific diversity of specimens from the Medi-
terranean basin, especially those from the Iberian Peninsu-
la, was greater than that of the French and Czech specimens
(Fig. 3). This increase in genetic diversity with increase
in the geographical coverage is documented (Bergsten et
al., 2012) and due to the presence of Mediterranean glacial
refugia and their associated biological diversity (Hewitt,
2001). Currently the data from the Mediterranean areas is
limited and more extensive sampling could provide addi-
tional insights into the biological or biogeographical pro-
cesses that resulted in the present diversity.

The other issue is distinguishing between the cereal pests
and the rarer or more localized species that are described

in the genus *Oulema*. Phylogenetic relationships between
the five species of the *melanopus* group have been stud-
ied by Bezděk & Baselga (2015) based on the *COI* gene.
They conclude that the relationships between the different
species in this group were not well resolved on the basis
of this gene. The results we obtained by combining their
work with ours are slightly more optimistic at least in the
possibility of distinguishing *O. duftschmidi* from any Eu-
ropean specimens currently being morphologically identi-
fied as *O. melanopus* and support the idea that these two
groups are genetically well separated. In Fig. 3, *O. mauroi*
and the Moroccan specimen KF406717.1 presently identi-
fied as *O. melanopus* are nested within *O. duftschmidi*, and
the sequences of *O. verae* are intermixed with the remain-
ing specimens identified as *O. melanopus*. We did not un-
dertake a morphological study of *O. mauroi* and *O. verae*,
which are rare in collections, but according to Bezděk &
Baselga (2015), the morphological differences between
*O. verae*/*O. melanopus* and *O. mauroi*/*O. duftschmidi* are
much more marked than the differences between *O. mela-
nopus* and *O. duftschmidi*. *O. rufocyanea*, also a member
of the *O. melanopus* complex, is clustered with a subgroup
of Spanish specimens from Galicia (Bold BIN ACJ0414)
morphologically identified as *O. melanopus*. The popula-
tion from Morocco remains to be studied. It is possible that
the standard *COI* barcode is not suitable for discriminating
between species in the Mediterranean area. The possibility
of introgressions having occurred will have to be explored
along with increased sampling and use of more relevant
molecular markers, especially nuclear markers.

Other European species of *Oulema*

*Oulema obscura* and *O. hoffmannseggii* form a sister
group to the *O. melanopus* complex (as in Bezděk & Ba-
selga, 2015). A more comprehensive sampling of the un-
der-represented species and the addition of the 4 remaining
species (*O. erichsonii*, *O. septentrionis*, *O. tristis* and *O.
magistrettiorum*) should further improve our understand-
ing of this genus. The fact that the specimens of *O. mela-
nopus* from Galicia differed from the others also indicates
that there may be additional cryptic diversity in the genus
*Oulema*, warranting further studies using an integrated ap-
proach.

*Oulema* sequences available in BOLD and reliability
of the barcoding identification of European species

BOLD system (Ratnasingham & Hebert, 2007) currently
(November 2020) contains 436 *Oulema* sequences forming
15 Barcode Index Numbers (BINs). Three BINs contain
most of the sequences:

AAK5928: 178 sequences of which 155 are identified as *O. melanopus*, two as *O. erichsonii*, and the remaining sequences are not identified to species level. This BIN includes 44 sequences from the present study identified as *O. melanopus*.

AAO0694: 107 sequences of which 83 are identified as *O. duftschmidi*, one as *O. mauroi* and two as *O. melanopus*. This BIN includes 48 sequences from the present study, all identified as *O. duftschmidi*.
AAN1559: 77 sequences of which 76 are identified as *O. obscura* (or its synonym *O. gallaeciana*) and one is unidentified.

Surprisingly, *O. verae* is not included in BOLD while *O. mauroi* from the same study (Bezděk & Baselga, 2015) is included.

The remaining 12 BINs are represented by a limited number of sequences (one to ten). Several species names are associated with several BINs: *O. erichsonii* (four different BINs among which is AAK5928); *O. duftschmidi* (AAO0694 and ADK1309 for one Indian sequence); *O. obscura* under the name *O. gallaeciana* (AAN1559 and ABW1444 for the 7 sequences from Spain, Galicia, see Fig. 3); *O. hoffmannseggii* (ABV0207 and ADU7791 for two sequences from Spain); *O. melanopus* (AAK5928 and four other BINs for sequences from different European and non-European countries, of which ABW1460 contains among other the 5 sequences from Spain, Galicia, sister to *O. rufocyanea* in our Fig. 3). That sequence of *O. rufocyanea* (KP406722.1) is associated with a unique BIN (ACJ0414). 44 sequences were not identified to species and 46 were not associated to a BIN due to their poor quality, insufficient length, etc.

BOLD provides the state-of-the-art barcoding information of the genus *Oulema* and highlights the need of clarification of the taxonomy in this group. The association of one species with several BINs and conversely several species within the same BIN may not be only due to misidentification. We cannot exclude some cryptic species such as the 7 specimens identified as *O. gallaeciana* from Spain that form a separate BIN. At the present state of knowledge, the use of the database for routine identification of *Oulema* can only be considered in a limited geographic context, keeping in mind possible misidentifications and the partly unresolved taxonomy. For that reason we chose to compare our results only to sequences resulting from a taxonomic study (Bezděk & Baselga, 2015).

## CONCLUSION

Bergsten et al. (2012) highlighted that limited sampling, and thus a restricted set of sequences reflecting local biodiversity, improves the identification by barcoding. This is supported by our results. Depending on the geographical context and the agronomic versus natural context, the identification of *Oulema* species by barcoding may be more or less efficient.

This study showed that the standard *COI* barcode can be used to distinguish between some *Oulema* species, including *O. melanopus* and *O. duftschmidi*, but cannot distinguish some other species in the *melanopus* complex, suggesting that further analyses might be needed to validate their taxonomic status.

Very extensive sampling has been carried out recently in agricultural regions in France (Chapelin-Viscardi & Maillet-Mezeray, 2015; Leroy & Chapelin-Viscardi, 2018). Several thousand specimens were identified based on the shape of the male flagellum. Given the relatively clear morphological criteria that characterise *O. mauroi*

and *O. verae* (Bezděk & Baselga, 2015), it is unlikely that they would have been confused with *O. melanopus* and *O. duftschmidi* in those studies. Identification based on male genitalia, and especially flagella, tested by specialists, was validated by our study, which also confirmed the quality of the morphological identifications. These surveys provide evidence that only three species of *Oulema* are present in cereal crops in France: *O. obscura*, *O. melanopus* and *O. duftschmidi*. In the absence of the other species of the *melanopus* complex, the results obtained show that DNA barcoding is a good method for differentiating between species of the genus *Oulema* in cereal crops in France, regardless of the developmental stage or sex of the specimens. In order to meet the needs of plant protection professionals, the method must provide both unambiguous and reliable results. To achieve this, it will be necessary first of all to associate the reference sequences present in the database (on which the identification of sequences will be carried out) to a given geographical area and context, in our case cereal fields in mainland France.

**ACKNOWLEDGEMENTS.** We thank the many contributors who collected samples in the field: C. Schaub (Alsace Chamber of Agriculture), D. Marcolet (INRA Mirecourt), J. Dourlens, A. Roncier, S. Boucher (Bayer CropScience), J.-C. Lebreton (Mayenne Chamber of Agriculture), F. Couturier-Boiton (Aube Chamber of Agriculture), A. Gardarin (INRA Grignon), P. Cantot (Société Entomologique de France), D. Dentinger (Vendée Chamber of Agriculture), B. Cailleret (FREDON) and C. Cocquempot (INRA Montpellier). We are grateful to B. Gauthier and J. Fleury for assisting us in laboratory analyses at the Laboratoire d'Eco-Entomologie. Thanks to A. Bukejs for allowing us to use the photographs of *Oulema* aedeagi. Many thanks to P. Biggins (Cirad) who reviewed the English language of this manuscript.

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Received March 24, 2020; revised and accepted July 14, 2020
Published online December 21, 2020

Supplementary Fig. S1 follows on next page.
<figure><img src="image_7.png" /><figcaption>**Fig. S1.** Phylogenetic tree constructed using the Maximum Likelihood method (ML) and the COI gene sequences of identified specimens from Table 1 plus unidentified specimens (in bold, 11 females and 3 larvae), belonging to the complex *Oulema melano-pus/duftschmidi. Epitrix pubescens* (in bold) was used to root the tree (106 sequences in total). Each colour corresponds to a morphologically identified species. The sequence code, identity and geographical origin are given for each specimen. The bootstrap values at the branch nodes are percentages and only those greater than 64% are presented (over 5,000 replicates). The country name is coded according to ISO 3166-1 alpha-2 (ISO 3166, 2016).</figcaption></figure>