Hidden Genetic Variability, Can the Olive Moth Prays Oleae (Lepidoptera: Yponomeutidae Or Praydidae?) Be a Species’ Complex?
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insects Article Hidden Genetic Variability, Can the Olive Moth Prays oleae (Lepidoptera: Yponomeutidae or Praydidae?) be a Species’ Complex? Marlon Pazian 1 ,Tânia Nobre 1,* , Imen Blibech 2 and Fernando T Rei 1 1 MED—Mediterranean Institute for Agriculture, Environment and Development, Instituto de Investigação e Formação Avançada, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; [email protected] (M.P.); [email protected] (F.T.R.) 2 Laboratory of research LR: Genetic Resources of the Olive Tree: Characterization, Promotion and Protection, Olive Tree Institute, Sousse 4061, Tunisia; [email protected] * Correspondence: [email protected]; Tel.: +351-266-760-885 Received: 28 February 2020; Accepted: 16 March 2020; Published: 26 March 2020 Abstract: Prays oleae is the second most important pest in Mediterranean olive groves, causing substantial damage on olive production. We used mitochondrial [cytochrome c oxidase subunit I(COI), and NADH dehydrogenase subunit 5 (nad5)] and nuclear [ribosomal protein S5 (RpS5)] amplicons to assess the population variability in five main olive producing regions from Tunisia, to support or dismiss the existence of two non-monophyletic groups within the species, as found within Portugal. Our phylogenetic analysis with cytochrome c oxidase subunit I (COI) indeed displayed two distinct and well-supported clades of P. oleae, which were corroborated by the haplotype network reconstructed with both mitochondrial and nuclear amplicons. We were also able to dismiss the hypothesis that one of the clades would not develop on olive fruits. No correlation was observed between clades differentiation and geographic distribution. The existence of cryptic species can impact on the management of agroecosystems and on the perception of how these moths responds to environmental changes. Keywords: Prays oleae; olive moth; cryptic species; phylogenetic; population structure 1. Introduction Global crop losses by insects are estimated to be 13% per annum despite the usage of multiple pesticides [1]. The insect order Lepidoptera includes many crop pests [2]; generally larval stages are crop destroyers that include defoliators, shoot/root borers, and seed predators causing significant agricultural losses. Some Lepidoptera species are known to cause damage to the olive trees, the major agro-ecosystem in the Mediterranean Basin, being the most significant in terms of impact the olive moth, Prays oleae (Bernard, 1978) (Lepidoptera, Yponomeutidae or Praydidae) [3,4]. The population dynamics of this moth is intrinsically dependent on the host-plant characteristics and development as its three yearly larval generations depend on the olive tree: (i) the phylophagous generation feeds on leaves; (ii) the anthophagous generation feeds on olive tree flowers and develops during the plant blooming; and (iii) the carpophagous generation feeds on olive fruits. The dietary preferences of adults are, however, poorly known but likely they feed on floral nectar and on a variety of other liquids similarly to most Lepidopteran adults [5]. Such a close and intricate connection between the olive moth and the olive tree should be reflected in the olive moth population structure and its co-evolutionary history. This co-history is already known and accepted for Bactrocera oleae (Diptera, Tephritidae) [6] whose larvae are monophagous, feeding exclusively on the tissue of olive fruits. Insects 2020, 11, 204; doi:10.3390/insects11040204 www.mdpi.com/journal/insects Insects 2020, 11, 204 2 of 9 Insects 2020, 11, x FOR PEER REVIEW 2 of 10 Recently, NobreNobre and and co-workers co-workers [4] have[4] have questioned questioned the species the statusspecies of P.oleaestatus asof the P. reconstructedoleae as the phylogenyreconstructed based phylogeny on the available based on data the resolves available this data species resolves as non-monophyletic. this species as Moreover,non-monophyletic. the same Moreover,study suggested the same the co-existencestudy suggested of two the sympatric co-existence evolutionary of two sympatric lineages of evolutionary morphologically linea crypticges of morphologicallyolive moth populations. cryptic olive These moth two lineagespopulations. overlapped These two geographically lineages overlapped throughout geographically the extensive throughoutsampling in Portugalthe extensive [4]. Given sampling this scenario,in Portugal a local [4]. diversificationGiven this scenario, could bea local hypothesized, diversification particularly could bebecause hypothesized, the Iberian particularly Peninsula because is known the as Iberian one of Peni the mostnsula importantis known as Pleistocene one of the glacial most important refugia in PleistoceneEurope. This glacial claim isrefugia well supported in Europe. by This several claim lines is well of evidence, supported also by for several Lepidoptera lines of species evidence, (e.g., also two forgenetic Lepidoptera lineages ofspeciesAglaope (e.g. infausta two withgenetic a likely lineages differentiation of Aglaope center infausta in Iberia with [a7 ];likely the phylogeography differentiation centof Melitaeaer in Iberia cinxia [7];shows the phylogeography the importance of of the Melitaea Iberian cinxia refugia shows in current the importance structure of [8 ],the several Iberian refugia refugia in inthe current Iberian structure Peninsula [8], have several been refugia inferred in for the the Iberian protected Peninsula species haveGraellsia been isabellaeinferredand for itsthe recognized protected plantspecies host Graellsia [9]). However, isabellae and the currentlyits recognized available plant data hos ont [9]).Prays However, oleae COI t collectedhe currently outside available Portugal data (one on Praysfrom Spainoleae COI and collected three from outside Tunisia) Portugal suggest (one a similar from Spain pattern and on three the otherfrom sideTunisia) of the suggest Mediterranean a similar patternSea [4]. on the other side of the Mediterranean Sea [4]. For BactroceraBactrocera oleae oleae, Segura, Segura and co-workersand co-workers [10] found [10] that found the mostthat southerlythe most of thesoutherly Mediterranean of the populationsMediterranean sampled populations (Tunisia) sampled differed (Tunisia) significantly differed from significantly the remaining from populations. the remaining However, populations. Nardi However,and co-workers Nardi [ 6and] suggested co-workers that [6] those suggested divergent that Tunisian those divergent samples mightTunisian in factsamples belong might to a clusterin fact belongof olive to fruit a cluster flies in of Central olive fruit/Western flies in Mediterranean Central/Western area. Mediterranean More recently, area Iberian. More and recently, Italian IberianB. oleae populationsand Italian B. were oleae shownpopulations to clearly were split,shown giving to clearly rise tosplit, the giving existence rise ofto atthe least existence three of well at least separated three wellMediterranean separated Mediterranean Basin populations: Basin thepopulations: Western Mediterranean,the Western Mediter the Italianranean, (including the Italian Greece (including and WesternGreece and Turkey) Western and Turkey) the Eastern and Mediterraneanthe Eastern Mediterranean clusters [11]. clusters [11]. These findings findings triggered the present work [4,6,11]: [4,6,11]: we we have have sampled sampled P.P. oleae in fivefive important olive grove regionsregions inin TunisiaTunisia and and proceed proceed similarly similarly with with the the previous previous approach approach done done in Portugalin Portugal [4], [4],to search to search for populationfor population diversity diversity and and co-presence co-presence of the of previouslythe previously identified identified lineages. lineages. 2. Materials Materials and and Methods 2.1. Specimen Specimen Collection Collection SeventySeventy-nine-nine P.P. oleae specimens from Tunisia Tunisia were sampled at at five five locali localitiesties (Bouficha, (Bouficha, Chaffar, Chaffar, Hajeb, Sidi Bouali, and Zarzis) and three specimens of of P.P. oleae from Greece ( (thatthat have emerged from olives collectedcollected in in Crete Crete island island in 2019) in 2019 (Figure) (Figure1). Tunisian 1). Tunisian specimens specimens were sampled were using sampled commercial using commercialtraps with specific traps with pheromones specific pheromones (Biosani) during (Biosani) the year during 2017. the year 2017. Figure 1. MainMain sampling sampling regions regions of Tunisian and Greek Prays oleae utilized in this study study.. Red = Zazis,Zazis, green = Chaffar,Chaffar, blue = HaHajeb,jeb, white white == SidiSidi Bouali, Bouali, yellow yellow == BoufichaBouficha and and black black == Crete.Crete. 2.2. DNA DNA Extraction, Extraction, PCR and Sequencing Adult specimens were stored at 20 C in 96% ethanol until DNA extraction. DNA extraction was Adult specimens were stored at− −20◦ °C in 96% ethanol until DNA extraction. DNA extraction performedwas performed from from whole whole specimen specimen body body following following extraction extraction protocol protocol described described in [4]. DNAin [4]. wasDNA eluted was in 50 µL of sterile ultra-pure water and stored at 20 C for posteriorly utilization in PCR reactions. eluted in 50 µl of sterile ultra-pure water and stored− ◦ at −20 °C for posteriorly utilization in PCR reactionPartial cytochromes. Partial cytochrome c oxidase subunitc oxidase I (subunitCOI), NADH I (COI dehydrogenase), NADH dehydrogenase subunit 5 (subunitnad5) and 5 (