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Article

Identification of Auchenorrhyncha Nymphs Using DNA Barcoding and Phylogenetic Analysis of the Most Common Genera Collected in Olive Fields

by
Zoi Thanou
1,
Maria Bouga
1,
Georgios Papadoulis
1 and
Antonios Tsagkarakis
2,*
1
Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
2
Laboratory of Sericulture and Apiculture, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(7), 496; https://doi.org/10.3390/d17070496
Submission received: 31 May 2025 / Revised: 14 July 2025 / Accepted: 17 July 2025 / Published: 19 July 2025
(This article belongs to the Section Phylogeny and Evolution)
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Abstract

Due to the potential role of Auchenorrhyncha in the transmission of the bacterium Xylella fastidiosa in a wide variety of cultivations, during recent years in Europe, many studies have focused on species composition, abundance and seasonal appearance of Auchenorrhyncha. However, females and nymphs are difficult to identify, as species-level identification relies primarily on male genitalia morphology. Sampling was conducted over four years in olive fields in Lesvos Island, in the Northeast Aegean, Greece, using sweep nets and Malaise traps. Both adults and nymphs were collected, with males identified to species level, while females and nymphs were separated on different morphotypes. Representatives from each morphotype and identified adults were sequenced using the mitochondrial cytochrome oxidase subunit I (COI) gene. Using a classical morphological approach, 58 species were identified to species level, and using DNA barcoding, nymph morphotypes and females were successfully identified within the families Cicadellidae, Aphrophoridae, Delphacidae and Issidae. A phylogenetic tree was generated, clustering nymphs together with the corresponding adults. Our results demonstrate the utility of combining morphological and molecular methods for accurate species identification and highlight the importance of enriching online databases with additional species records.

1. Introduction

In recent years, the suborder Auchenorrhyncha (Hemiptera) has attracted increased attention in Europe, following the emergence of the devastating Gram-negative bacterium, Xylella fastidiosa (Wells et al., 1987) This pathogen causes a wide range of diseases in economically important crops, including Pierce’s disease (PD) in grapes, citrus variegated chlorosis (CVC), phony peach disease, and olive quick decline syndrome (OQDS). X. fastidiosa is transmitted exclusively by xylem-feeding Auchenorrhyncha.
In Europe, two primary xylem-feeding species, Philaenus spumarius (L.) and Neophilaenus campestris (Fallén), have been identified as the main vectors of X. fastidiosa. These species have been shown to transmit the pathogen in olive orchards in southern Italy, where the bacterium was first detected on the continent [1,2,3]. Although phloem-feeding Auchenorrhyncha may also acquire the bacterium during feeding, their role in transmission remains less understood, underscoring the need for comprehensive faunistic studies [4,5,6].
Taxonomic identification of Auchenorrhyncha species is traditionally based on external morphological characters and often reaches only the subfamily or genus level. Accurate species-level identification typically requires examination of male genitalia. Consequently, identifying adult females or nymphs poses a considerable challenge. Moreover, even experienced taxonomists may struggle to distinguish between closely related taxa due to subtle morphological differences. These limitations highlight the necessity of molecular techniques, such as DNA barcoding, as an assisting tool in taxonomy [7].
DNA barcoding is a widely accepted molecular method, especially for animal species identification. It typically targets a 648 bp fragment of the mitochondrial DNA cytochrome c oxidase subunit I (COI) gene [8,9]. This method has been extensively applied in numerous arthropod groups, including Lepidoptera [10,11], Coleoptera [12,13], Hymenoptera [14,15,16] and Hemiptera [17]. Within Hemiptera, barcoding studies have focused on true bugs, aphids and, to a lesser extent, Auchenorrhyncha [18,19,20,21].
Beyond aiding in species identification, DNA barcoding can delineate cryptic species, resolve synonymies among morphologically diverse taxa, and facilitate biodiversity monitoring. It is particularly useful for assessing species richness in complex ecosystems and tracking population changes over time [8].
In this study, we applied DNA barcoding to morphologically identified male adults of Auchenorrhyncha, collected from olive groves in Lesvos Island, Greece, to enrich reference libraries with species of restricted distribution. Additionally, nymphs and females underwent barcoding and were compared with the corresponding male sequences to achieve species-level identification. Our findings highlight the effectiveness of integrating DNA barcoding with classical morphological taxonomy and underscore the need to expand online sequence databases for improved species resolution.

2. Materials and Methods

2.1. Specimen Collection

Auchenorrhyncha specimens were collected from olive groves across Lesvos Island, the third largest Greek island, located in the Northeast Aegean. Three white Malaise traps were installed in different olive groves: the first site, Lakerda field, was located in the southeastern region, near the capital city of Lesvos, Mytilene; the second, Kalloni field, was located in the northwest, near the wetlands and the pine forest of Lesvos; the third, Nifida field, was in the southern coastal area. Additional samplings were collected using a sweeping net from herbaceous vegetation in olive fields and pine forests across the island at random locations. Specimens were euthanized and stored in 98% ethanol. Sampling was conducted continuously from 2019 to 2022.

2.2. Morphology Identification

Male adult Auchenorrhyncha specimens were identified using standard taxonomic keys [22,23]. Male genitalia were dissected and cleared in 10% KOH for 2 h, except for Typhlocybinae specimens, which were treated for 30 min. The genitalia were then mounted in glycerol on microscope slides and examined using both stereomicroscopes and compound microscopes.
Taxonomic nomenclature followed [23] along with the TaxonPages website. Female specimens were identified to the genus level, and nymphs were grouped into morphotypes and assigned to subfamilies or genera. Representative specimens from each nymph morphotype and adult genus were selected for DNA extraction. Comparative specimens from other locations on the island and from olive fields in Athens were also included.

2.3. DNA Extraction, Amplification and Sequencing

Genomic DNA was extracted from entire adult and nymph specimens using the DNeasy Blood & Tissue extraction kit (Qiagen, Venlo, The Netherlands), following a modified protocol. Each specimen was manually ground in 180 μL of PBS within a 1.5 mL Eppendorf tube, using a polypropylene pestle (Sigma Aldrich, Burlington, MA, USA). Following homogenization, 200 μL of AL buffer and 20 μL of Proteinase K (Qiagen kit components) were added. Samples were incubated at 70 °C for 10 min. Then, 200 μL of 100% ethanol was added and mixed thoroughly, and the standard protocol of the manufacturer was followed.
A fragment of approximately 680 bp of the COI gene was amplified by polymerase chain reaction, and the primers LCO1490 (5′-ggtcaacaaatcataaagatattgg-3′) and HC02198 (5′-taaacttcagggtgaccaaaaaatca-3′) were used [24]. The PCR [25] amplification was carried out in a total volume of 50 μL, comprising 5 μL of 10× reaction buffer (Invitrogen, Carlsbad, CA, USA), 1 μL of a 10 mM dNTP mix (BioLabs, Ipswich, MA, USA), 1.5 μL of 50 mM MgCl2 (Invitrogen, Carlsbad, CA, USA), 1.25 μL of each primer at a concentration of 10 mM (BioLabs, Ipswich, MA, USA), 1.5 units of Taq polymerase (Invitrogen, Carlsbad, CA, USA), 100–150 ng of template DNA, and sterile water. The PCR reactions were conducted using a Thermocycler (Primus 25 advanced) (PEQLAB, London, UK) under the following cycling conditions: an initial denaturation at 94 °C for 1 min; 6 cycles consisting of denaturation at 94 °C for 1 min, annealing at 45 °C for 1 min and 30 s, and extension at 72 °C for 1 min and 15 s; followed by 36 cycles with denaturation at 94 °C for 1 min, annealing at 51 °C for 1 min and 30 s, and extension at 72 °C for 1 min and 15 s. A final extension step was performed at 72 °C for 5 min [26].
PCR products were visualized on a 2% agarose gel using 0.5× TBE buffer. Amplicons approximately 680 bp in length were purified using the Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. Sequencing of the amplified mitochondrial DNA fragment was carried out by CEMIA S.A. (Larisa, Greece) using the same forward primer applied during amplification. The sequences obtained have been deposited in GenBank with accession numbers PV705467–PV705531 (Table S1).

2.4. Data Analysis

All sequences were verified using the BLAST software, version 2.16.0+ of NCBI to confirm their mitochondrial origin and assess similarity to reference sequences, with several matches showing nearly 100% similarity.
Multiple-sequence alignments were performed with MUSCLE [27], and then they were further adjusted manually. Pairwise genetic distances were calculated using MEGA X 10.2.6 [28], and a phylogenetic tree, based on the COI barcode region, was constructed. A Neighbor-Joining analysis was performed, and node support was evaluated using the bootstrap method with 1000 replicates. Outgroup taxa included Aphis gossypii (accession number: KR017753.1) and the GenBank COI sequence of Exitianus capicola (accession number: LC775130.1). A dataset of 67 COI homologous sequences of 524 bp was assembled, with sequence homology ranging from 82.46 to 100%. Haplotype diversity, variable sites and haplotypes were analyzed using DNA sp v.5.10.00 software [29].
Nymph sequences (nymph1–nymph21) were compared against adult sequences to determine species-level identification using DNA barcoding. Nymphs were identified to the species level if they exhibited significant sequence similarity to identified adults or clustered within the same clade of phylogenetic analyses. The same approach was applied to female sequences.
Species delimitation was further addressed using the Automatic Barcode Gap Discovery (ABGD) method (ABGDpy 0.1 [August 2021]), using relative gap width (X = 1.2) and intraspecific divergence (p) values between 0.001 and 0.100, under the Kimura 2.0 model, with all other settings at default. This method identifies the first significant barcode gap beyond the defined threshold and uses pairwise genetic distances to partition the dataset into candidate species [30].

3. Results

A total of 2003 adult specimens and 38 nymphs were collected using Malaise traps from three olive fields on Lesvos Island, yielding 53 species across five families: Aphrophoridae, Cicadellidae, Issidae, Delphacidae, and Cixiidae. Additionally, sweep net collections yielded 1034 adults and 844 nymphs, with 38 species identified morphologically (Table 1).
From both trapping methods, 19 different nymph morphotypes were selected for sequencing (Table 2). Moreover, sequencing included the most commonly identified male adult species, as well as females from common genera, to enable potential correlation with the nymphs.
A total of 65 high-trace COI sequences (524 bp) were successfully generated in this study out of 102 specimens; the remaining 37 specimens either failed sequencing or had low-quality sequences. These included 21 unidentified nymphs and 18 females identified to the genus level, with the remaining being identified from male adults (Table S1). The sequences belonged to five families: Cicadellidae and Aphrophoridae, from Cicadomorpha, and Delphacidae, Issidae and Cixiidae, from Fulgoromorpha. Congeneric p-distances ranged from 10.30% to 16.98%, while intraspecific p-distance reached 5.53% for Cicadomorpha and remained below 0.38% for Fulgoromorpha.
Out of the 21 nymphs sequenced, 18 were identified at the species level. Seven nymphs were identified as Melillaia desbrochersi, which belonged to three different morphotypes (nn = 6, 8, 14) (Figure 1F,H,O). Despite their different morphotypes, the p-distance from the identified male adult of M. desbrochersi was 0.00% (Table S2), and they shared the same haplotype. Two nymphs (nym = 1, 11) were identified as Euscelidius mundus (Figure 1B) and two others (nym = 6, 15) as Streptanus albanicus, showing a 0.19% genetic distance from the adult. The two nymphs of Streptanus had different coloration intensity, leading to their classification as separate morphotypes (Figure 1J,N), although they shared the same haplotype. One nymph (nym = 10) was identified after scanning the GenBank for sequence similarities via blasting, and the sequence with accession number LC775130.1, identified as Exitianus capicola, had 98.64% identity (Figure 1S). Five nymphs were assigned to the genus Euscelis; three were identified as Euscelis lineolata and two as Euscelis alsia, with p-distance ranging from 0.19 to 5.53% with the corresponding male adults, while their sequences clustered together in the phylogenetic tree (Figure 2). Both Euscelis species shared the same morphotype (nn = 3) (Figure 1C), while nymphs with morphotypes nn = 1 and nn = 12 were identified as E. lineolata and E. alsia, respectively (Figure 1A,M). A single Issidae nymph (nym = 3) was identified as Rhissolepus aspinosus, with a p-distance of 0.19% (Figure 1I).
Nymphs (nym = 12, 18) of morphotype nn = 5 (Figure 1E) could not be resolved to species due to overlapping barcodes. Their sequences showed significant similarity (>98.62%) with both Psammotettix alienus and Psammotettix confinis with accession numbers OQ569809.1 and KR573169.1, respectively [31]. Based on morphological identification of adult males, where 144 of 146 individuals belonged to Ps. alienus, it is likely that both nymphs belong to that species as well. One nymph (nym = 8) (Figure 1L) could not be identified even at the genus level, as its p-distance from all adult sequences exceeded 16.98% (Table S2), and no matches were found in reference databases. Genetic distances between nymphs and corresponding male adults are detailed in Table S2.
Four unidentified Fulgoromorpha females were sequenced, representing the genera Latilica, Mycterodus, Kelisia, and Reptalus. Two of them were identified successfully at the species level; the Latilica female showed intraspecific p-distance between 0.19 and 0.38%, while congeneric species L. maculipes and L. antalyica exhibited distances of 14.31–14.50%, allowing identification as Latilica maculipes. The Kelisia female showed 96.88% identity with Kelisia sabulicola (accession number MZ629882.1) [32], suggesting it likely belongs to this species. Females from Mycterodus and Reptalus could not be identified due to a lack of matching sequences in GenBank and the absence of reference males. Similarly, a female was identified as Hebata decipiens, showing 99.81% identity with OQ381262.1 [33]. Another female, resembling Eohardya or Hardya, could not be matched to any reference sequence, but it is presumed to belong to Eohardya fraudulenta, the only known species from these genera in the area.
The Neighbor-Joining (NJ) phylogenetic tree, constructed with 1000 bootstrap replicates, is shown in Figure 2. The tree successfully clustered nymphs within their respective genera and clearly separated major taxonomic groups, such as Aphrophoridae from Cicadellidae and Fulgoromorpha from Cicadomorpha. Subfamilies Typhlocybinae and Deltocephalinae were also distinctly separated. Nearly all branches received strong bootstrap support. Within Deltocephalinae, most sequences belonged to the tribe Athysanini, though their clades were interspersed with sequences from other tribes, including Paralimnini, Selenocephalini, Cicadulini, and Chiasmini. Only the tribe Fieberiellini (Docotettix and Synophropsis) formed distinct clades. A more comprehensive understanding of tribal relationships within Deltocephalinae may require a larger dataset including additional genera and more sequences.
A total of 45 distinct haplotypes were identified. Overall haplotype diversity (Hd) was 0.9587, with a variance of 0.00026. In Euscelidius mundus, five haplotypes were recorded, with a haplotype diversity of 0.9333. In contrast, a single haplotype was found among all nymphs and the adult of Melillaia desbrochersi. For Neophilaenus campestris, four haplotypes were identified, with a haplotype diversity of 0.3956.
ABGD analysis confirmed most species partitions across all tested p-values. Initial partitions revealed intra-population divergence in Neophilaenus campestris, separating four groups corresponding to the number of detected haplotypes. However, divergence remained within intraspecific thresholds and did not indicate speciation.

4. Discussion

This study evaluated Auchenorrhyncha identification using both morphological and molecular approaches based on DNA barcoding of the COI gene. Our findings confirm DNA barcoding as a reliable tool for species identification of Auchenorrhyncha, especially for life stages such as nymphs or female adults. These are difficult or impossible to identify morphologically due to the absence of male genital structures and the limited taxonomic keys for nymphs [34,35].
A total of 71 adult morphotypes were separated, with 58 identified to species level through morphological approach. Twelve morphotypes were identified to genus level and one to family level, due to the exclusive presence of females in the population. The most abundant species in the olive field environment included Cicadulina (Cicadulina) bipunctata, Euscelidius mundus, Euscelis (Euscelis) lineolata, Psammotettix alienus, Thamnotettix zelleri and Zyginidia pullula. They are all commonly reported in Greek olive groves [36,37,38]. Although many species were recorded in olive fields, neighboring plant composition influenced species presence [38]. For example, all the species of the genus Macrosteles were recorded exclusively in Kalloni’s field, which is near the salt pans of Kalloni, as Macrosteles species are predominantly hydrophilous and typically found near lakes and wetlands [39]. Similarly, species of the genus Kelisia prefer wet sites due to their association with sedges of the genus Carex [39]. Another species, Opsius stactogalus, which feeds on Tamaricaceae trees [39], was collected exclusively at the Nifida coastal field, where Tamarix trees are present.
Notably, two species were recorded for the first time in Greece: Kelisia sabulicola and Jassargus kurdicus, both identified morphologically (Figure 3). K. sabulicola was also verified through DNA barcoding, showing 3.12% divergence from a Finnish record in GenBank [32]. For J. kurdicus, no sequence matches were found in databases, since it is endemic in Iraq and had not been previously barcoded. This insect was barcoded in the present study, under accession number PV705487.
DNA barcoding confirmed morphological identifications for 20 adult morphotypes. Among the barcoded females, a Cixiidae (Reptalus) and an Issidae (Mycterodus) could not be assigned to species level due to the absence of corresponding male specimens and lack of similar sequences in GenBank. Two Mycterodus species, Mycterodus (Semirodus) hioles and Mycterodus (Aegaeum) lesbicum, have been previously recorded on Lesvos Island [40]. Both species are endemic to Greece, specifically the Islands of Chios and Lesvos in the Northeast Aegean [40,41]. It is likely that the unidentified specimen in this study belongs to one of these species. The absence of DNA barcodes for species with restricted distributions in online databases presents a significant challenge for the identification of rare species.
Among the 19 identified nymph morphotypes, four (nn = 7, 15, 17, 18) were identified morphologically based on coloration and host plants, as they were collected alongside adults (Figure 1G,P,Q,R). Two morphotypes (nn = 4, 10) could not be identified due to sequencing failure for all samples (Figure 1D,K). However, morphotype nn = 10 was highly abundant in sweep net samplings (Figure 1K) and morphologically matches with Thamnotettix zelleri, a very common species in olive and vineyard agroecosystems of southern Europe [38,42,43]. On Lesvos Island, adults of T. zelleri were collected abundantly from April to May, approximately one month after the emergence of this nymph morphotype, suggesting strong evidence for their conspecificity. However, successful sequencing of these nymphs would be necessary to confirm this identification, as morphological and ecological data alone are insufficient.
From the 13 remaining morphotypes that were successfully sequenced, 9 species were identified. Euscelis alsia and Euscelis lineolata shared a morphotype (nn = 3) and could not easily be distinguished based on morphology alone. DNA barcoding easily separated the two species, revealing congeneric genetic distances ranging from 13.93 to 16.98%. Moreover, it was found that each species exhibited distinct additional nymph morphotypes (nn = 1 and nn = 12 for E. lineolata and E. alsia, respectively). Both species coexist in Greek olive fields, with E. lineolata being more abundant [36,37,38]. Accurate identification is particularly relevant, as E. lineolata has been reported as a vector (though not a confirmed transmitter) of Xylella fastidiosa [5].
Three morphotypes (nn = 6, 8, 14) were identified as Melillaia desbrochersi, another common species across the olive fields of Greece and Italy, feeding in the low vegetation [38,44]. Initially misidentified as Psammotettix alienus based on morphology, these nymphs were reassigned after DNA barcoding revealed >16.98% divergence from Psammotettix sequences. This highlights the limitations of morphology alone, especially for lesser-known taxa lacking reference sequences in GenBank.
Similarly, Rhissolepus aspinosus was confirmed through clustering of nymph sequences (morphotype nn = 9) with a male adult. This species is restricted to Greece and Turkey and had been previously recorded on Lesvos Island [45,46], yet remains underrepresented in online databases. Such cases underscore the importance of expanding barcode reference libraries with vouchered, expertly identified specimens.
The last two species identified were Euscelidius mundus (nn = 2) and Streptanus albanicus (nn = 13 and 16). Both genera were represented in GenBank, showing similarities ranging from 89.45 to 93.09%, with existing records. Sequences from both species clustered reliably with other counterparts in phylogenetic trees and exhibited p-distances of 0.19%. Species-level identification of nymphs belonging to morphotype nn = 5 (Psammotettix sp.) proved problematic due to high sequence similarity with both Psammotettix alienus (GenBank: OQ569809.1) and Psammotettix confinis (GenBank: KR573169.1). Genetic similarity between these reference sequences was lower than the maximum intraspecific divergence observed in this study, complicating species-level resolution. This unexpectedly high similarity may indicate either cryptic species or incomplete lineage sorting, or possibly misidentification of one or both reference sequences. Supporting this, a previous population-level study [47] successfully distinguished P. alienus and P. confinis as separate clades with significant genetic divergence. To improve resolution, future studies should include more barcoded Psammotettix adults, reliably identified in the field. Expanding high-confidence reference datasets will help resolve potential misidentifications and taxonomic ambiguities in this genus.
Nymph morphotype nn = 11 could not be identified even at the genus level, as no adult sequence or GenBank record showed sufficient for matching. Similar challenges have been reported in other studies. For example, Gopurenko et al. [20] found that 24.5% (26/106) of Australian Auchenorrhyncha nymphs could not be matched to known adults or database sequences. Moreover, Ahrens et al. [48] reported that 21% of coleopteran larvae could not be associated with known barcodes. Many species remain absent from reference libraries or are represented by fewer than five barcode sequences. Expanding high-quality barcode records, particularly for species with restricted distributions, will significantly enhance the accuracy and reliability of DNA barcoding [49].
In the phylogenetic tree, the different families and subfamilies of the dataset were clearly separated, clustering all Fulgoromorpha in one distinct branch. Nymphs clustered together with their corresponding adults, providing visual confirmation of results. Similarly, separation at the tribal level within Deltocephalinae was evident. Tribe Fieberiellini formed a distinct clade, while Athysanini sequences were interspersed with those of other tribes. Athysanini is considered polyphyletic, as it is one of the largest tribes and includes many genera that have recently been reassigned to smaller tribes, such as Hardya, now grouped with Cicadulini [50]. The four Issidae species also separated clearly in the phylogenetic tree, consistent with previous studies [51]. All belonged to the tribe Hysteropterini, with interspecific p-distances ranging from 14.31% to 16.98%, and intraspecific distances remaining below 0.38%.
ABGD species delimitation analysis provided additional insights. At higher sensitivity settings (lower p-values), more putative species were detected. For Neophilaenus campestris, sequences were partitioned into four putative groups, indicating population-level divergence. These different groups likely reflect new haplotypes arising from intraspecific mutations. Olive fields in Lesvos, which account for 28% of the local vegetation, are typical of Mediterranean countries. Despite being an island, gene flow remains, as the most common N. campestris haplotype identified here had also been found in olive fields in Attica, Greece and Corsica, France [52,53]. Dispersal ability, shared habitat and environmental conditions may facilitate gene flow, despite potential isolation [53,54]. Local mutations likely produced the additional haplotypes, which coexist with the common haplotype, showing no evidence of structured population.
For Euscelis, ABGD analysis separated sequences into different species but did not differentiate among haplotypes within each species. Euscelis alsia showed three newly recorded haplotypes, showing maximum similarity below 99.42% compared to GenBank records. Euscelis lineolata showed four novel haplotypes. The genetic distance between Euscelis congeneric species ranged from 13.93% to 16.98%, while the highest intraspecific diversity reached 5.53%. Sequences of Euscelis lineolata showed 88.67% to 89.06% similarity to the only GenBank reference (MK188555.1) from Corsica [52]. The widespread distribution of E. lineolata, combined with ecological and geographical variability, may explain the observed haplotype divergence. Similar thresholds have been reported in other Hemiptera; for example, a COI study of true bugs found that 90% of taxa exhibited less than 2% intraspecific divergence, though this reached 7.22% in some species, while congeneric species showed average divergence of 10.7% [21].

5. Conclusions

DNA barcoding proved to be a reliable tool for biodiversity analysis of Auchenorrhyncha, providing species delimitation for specimens with the same morphotype, which would otherwise remain unrecognized. In total, 90.63% of sequences were successfully identified at the species level, either using existing online records or newly generated reference sequences. One nymph remains completely unknown, having a minimum genetic distance of 17.17% from any sequence in the dataset, exceeding congeneric distances recorded for Deltocephalinae genera in this study. Although in most cases there was congruence between classical and molecular taxonomy of Auchenorrhyncha, species identification depended on the presence of closely related sequences in reference databases.
This study highlights that the effectiveness of DNA barcoding relies heavily on the availability of accurate, vouchered reference sequences. For species such as M. desbrochersi, Eu. mundus, S. albanicus, and R. aspinosus, the absence of reference barcodes has historically hindered their inclusion in biodiversity assessments. For endemic or geographically restricted species, this gap results in underrepresentation or misidentification in DNA-based surveys.
Our results confirm strong congruence between morphological and molecular taxonomy in Auchenorrhyncha, but only when closely related reference sequences are available. Comprehensive barcoding of local faunas by expert taxonomists should remain a priority to establish robust reference frameworks.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17070496/s1, Table S1: All specimens that have successfully been sequenced with the species that were assigned and their Accession Numbers; Table S2: Genetic distances (p-distance) between the nymphs and adults, with variance estimation method: Bootstrap method of 1000 reps using Pairwise deletion.

Author Contributions

Conceptualization, A.T. and Z.T.; methodology, Z.T. and M.B.; validation, M.B. and A.T.; investigation, Z.T.; data curation, Z.T. and M.B.; writing—original draft preparation, Z.T. and A.T.; writing—review and editing, Z.T., M.B., G.P. and A.T.; visualization, Z.T.; supervision, A.T. and G.P.; project administration, A.T. and Z.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to also forming part of an ongoing study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Nymph morphospecies: (A) nn = 1, Euscelis lineolata; (B) nn = 2, Euscelidius mindus; (C) nn = 3, Euscelis lineolata, Euscelis alsia; (D) nn = 4, not identified; (E) nn = 5, Psammotettix sp.; (F) nn = 6, Melillaia desbrochersi; (G) nn = 7, Hecalus glaucescens; (H) nn = 8, Melillaia desbrochersi; (I) nn = 9, Rhissolepus aspinosus; (J) nn = 16, Streptanus albanicus; (K) nn = 10, unidentified; (L) nn = 11, not identified; (M) nn = 12, Euscelis alsia; (N) nn = 13, Streptanus albanicus; (O) nn = 14, Melillaia desbrochersi; (P) nn = 15, Cicadella viridis; (Q) nn = 17, Maiestas schmidtgeni; (R) nn = 18, Opsius stactogalus; (S) nn = 19, Exitianus capicola.
Figure 1. Nymph morphospecies: (A) nn = 1, Euscelis lineolata; (B) nn = 2, Euscelidius mindus; (C) nn = 3, Euscelis lineolata, Euscelis alsia; (D) nn = 4, not identified; (E) nn = 5, Psammotettix sp.; (F) nn = 6, Melillaia desbrochersi; (G) nn = 7, Hecalus glaucescens; (H) nn = 8, Melillaia desbrochersi; (I) nn = 9, Rhissolepus aspinosus; (J) nn = 16, Streptanus albanicus; (K) nn = 10, unidentified; (L) nn = 11, not identified; (M) nn = 12, Euscelis alsia; (N) nn = 13, Streptanus albanicus; (O) nn = 14, Melillaia desbrochersi; (P) nn = 15, Cicadella viridis; (Q) nn = 17, Maiestas schmidtgeni; (R) nn = 18, Opsius stactogalus; (S) nn = 19, Exitianus capicola.
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Diversity 17 00496 g002
Figure 2. Neighbor-Joining phylogenetic tree with bootstrap of 1000, a reference sequence of Exitianus capicola (LC775130.1) and the outgroup Aphis gossypii (KR017753.1). The seven different tribes of Deltocephalinae and the families of the dataset are indicated with vertical lines of different colors left and right, respectively.
Figure 2. Neighbor-Joining phylogenetic tree with bootstrap of 1000, a reference sequence of Exitianus capicola (LC775130.1) and the outgroup Aphis gossypii (KR017753.1). The seven different tribes of Deltocephalinae and the families of the dataset are indicated with vertical lines of different colors left and right, respectively.
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Diversity 17 00496 g003
Figure 3. Adult of J. kurdicus (left); aedeagus and connective (right).
Figure 3. Adult of J. kurdicus (left); aedeagus and connective (right).
Diversity 17 00496 g003
Table 1. Total adult species collected in each field with a Malaise trap (Lak, Lakerda; Kal, Kalloni; Nif, Nifida) and with a sweep net from olive fields around Lesvos Island, Greece.
Table 1. Total adult species collected in each field with a Malaise trap (Lak, Lakerda; Kal, Kalloni; Nif, Nifida) and with a sweep net from olive fields around Lesvos Island, Greece.
Morphotype (na) 1SpeciesLakKalNufSweep Net
1Philaenus signatus (Melichar, 1896)---4
2Philaenus spumarius (Linnaeus, 1758)104466
3Neophilaenus (Neophilaenulus) campestris (Fallén, 1805)181187
4Anoscopus albifrons (Linnaeus, 1758)7---
5Allygus sp.-11-
6Anoplotettix sp.12--
7Anoplotettix fuscovenosus (Ferrari, 1882)1---
8Balclutha sp.-81-
9Balclutha frontalis (Ferrari, 1882)31623
10Balclutha saltuella (Kirschbaum, 1868)-2-1
11Cicadulina (Cicadulina) bipunctata (Melichar, 1904)2119-5
12Docotettix cornutus (Ribaut, 1948)1385-
13Eohardya fraudulenta (Horváth, 1903)-1-9
14Euscelidius mundus (Haupt, 1927)271-54
15Euscelis alsia (Ribaut, 1952)371171
16Euscelis (Euscelis) lineolata (Brullé, 1832)2314311404
17Exitianus capicola (Stål, 1855)146325
18Goniagnathus (Goniozygotes) bolivari (Melichar, 1907)--11
19Goniagnathus (Goniagnathus) brevis (Herrich-Schäffer, 1835)1113
20Jassargus (Pontojargus) kurdicus (Remane & Schulz, 1976)---8
21Macrosteles sp.-2--
22Macrosteles quadripunctulatus (Kirschbaum, 1868)-8-1
23Macrosteles ramosus (Ribaut, 1952)-17--
24Macrosteles sexnotatus (Fallén, 1806)-3--
25Maiestas schmidtgeni (Wagner, 1939)215614
26Melillaia desbrochersi (Lethierry, 1889)1--32
27Mocydiopsis sp.1---
28Neoaliturus (Circulifer) haematoceps (Mulsant & Rey, 1855)15--
29Neoaliturus (Neoaliturus) fenestratus (Herrich-Schäffer, 1834)-7-11
30Opsius stactogalus (Fieber, 1866)--2-
31Orosius orientalis (Matsumura, 1914)-10--
32Phlepsius intricatus (Herrich-Schäffer, 1838)-1031
33Psammotettix sp.-225-
34Psammotettix alienus (Dahlbom, 1850)11181373
35Psammotettix confinis (Dahlbom, 1850)-2--
36Proceps acicularis (Mulsant & Rey, 1855)1--1
37Selenocephalus stenopterus (Signoret, 1880)8151931
38Streptanus (Streptanulus) albanicus (Horváth, 1916)1--54
39Synophropsis lauri (Horváth, 1897)-1--
40Thamnotettix zelleri (Kirschbaum, 1868)7861531
41Anaceratagallia (Anaceratagallia) glabra (Dmitriev, 2020)646715
42Anaceratagallia (Anaceratagallia) ribauti (Ossiannilsson, 1938)-202-
43Austroagallia sinuata (Mulsant & Rey, 1855)469113
44Megopthalmus scabripennis (Edwards, 1915)153-
45Anzygina honiloa (Kirkaldy, 1906)1---
46Arboridia sp.362-
47Assymetrasca decedens (Paoli, 1932)-533-
48Eupteryx (Eupteryx) gyaurdagica (Dlabola, 1957)4563-
49Eupteryx (Eupteryx) insulana (Ribaut, 1948)-3--
50Fruticidia sp.12--
51Hauptidia (Hauptidia) provincialis (Ribaut, 1931)11--
52Hebata sp.131611-
53Hebata (Alboneurasca) decipiens (Paoli, 1930)-216126
54Lindbergina cretica (Asche, 1980)3---
55Ribautiana cruciata (Ribaut, 1931)-72-
56Zygina sp.-11-
57Zygina (Zygina) roseipennis (Tollin, 1851)--2-
58Zyginidia adamczewskii (Dworakowska, 1970)--1-
59Zyginidia pullula (Boheman, 1845)31451010
60Reptalus sp.-11-
61Laodelphax striatellus (Fallén, 1826)-10-11
62Toya (Metadelphax) propinqua (Fieber, 1866)25-36
63Kelisia sp.---9
64Kelisia ribauti (Wagner, 1938)---13
65Kelisia sabulicola (Wagner, 1952)---2
66Phantia subquadrata (Herrich-Schäffer, 1838)1513
67Agalmatium flavescens (Olivier, 1792)1---
68Latilica antalyica (Dlabola, 1986)4---
69Latilica maculipes (Melichar, 1906)---5
70Mycterodus sp.---2
71Rhissolepus aspinosus (Dlabola, 1982)---8
1 na: Adult morphospecies.
Table 2. Nymph morphospecies that were collected with Malaise traps and sweep nets and their species that have been assigned to them.
Table 2. Nymph morphospecies that were collected with Malaise traps and sweep nets and their species that have been assigned to them.
Morphotype (nn) 1GenusSpeciesN. (Malaise)N. (Sweep Net)Name of Sequence
1Euscelislineolata04nymph4
2Euscelidiusmundus016nymph1, nymph11
3Euscelislineolata, alsia25130nymph2, nymph9, nymph20
4--60failed
5Psammotettix-03nymph12, nymph18
6Melillaiadesbrochersi048nymph5, nymph7, nymph17, nymph19, nymph21
7Hecalusglaucescens01-
8Melillaiadesbrochersi32nymph16
9Rhissolepusaspinosus134nymph3
10--3548failed
11--011nymph8
12Euscelisalsia06nymph13
13Streptanusalbanicus022nymph6
14Melillaiadesbrochersi07nymph14
15Cicadellaviridis01-
16Streptanusalbanicus02nymph15
17Maiestasschmidtgeni02-
18Opsiusstactogalus06-
19Exitianuscapicola01nymph10
Total 38844
1 nn: nymph morphospecies.
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Thanou, Z.; Bouga, M.; Papadoulis, G.; Tsagkarakis, A. Identification of Auchenorrhyncha Nymphs Using DNA Barcoding and Phylogenetic Analysis of the Most Common Genera Collected in Olive Fields. Diversity 2025, 17, 496. https://doi.org/10.3390/d17070496

AMA Style

Thanou Z, Bouga M, Papadoulis G, Tsagkarakis A. Identification of Auchenorrhyncha Nymphs Using DNA Barcoding and Phylogenetic Analysis of the Most Common Genera Collected in Olive Fields. Diversity. 2025; 17(7):496. https://doi.org/10.3390/d17070496

Chicago/Turabian Style

Thanou, Zoi, Maria Bouga, Georgios Papadoulis, and Antonios Tsagkarakis. 2025. "Identification of Auchenorrhyncha Nymphs Using DNA Barcoding and Phylogenetic Analysis of the Most Common Genera Collected in Olive Fields" Diversity 17, no. 7: 496. https://doi.org/10.3390/d17070496

APA Style

Thanou, Z., Bouga, M., Papadoulis, G., & Tsagkarakis, A. (2025). Identification of Auchenorrhyncha Nymphs Using DNA Barcoding and Phylogenetic Analysis of the Most Common Genera Collected in Olive Fields. Diversity, 17(7), 496. https://doi.org/10.3390/d17070496

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