1 Introduction

Leafhoppers (Family Cicadellidae), a diverse group of Hemipteran insects comprising approximately 22,000 species worldwide (Dietrich 2004), exhibit notable traits such as varied colouration, jumping behaviour, variation in life cycle and host plant adaptability, making them valuable bioindicators. Moreover, many species are significant agricultural pests and vectors of phytopathogenic viruses, posing substantial economic threats (Ishii et al. 2013). Particularly in fruit crops, the damage caused by leafhoppers is recognised as a global issue. For example, Zonocyba pomaria (McAtee 1926) (white apple leafhopper) is the most common serious pest in various fruit trees, particularly apple trees, and its damage has been monitored since 1938 (Steiner 1938; EPPO 2024). It feeds on the undersides of leaves, causing whitish spots on the leaf surfaces. Scaphoideus titanus Ball 1932 (American grapevine leafhopper) is another significant pest of vineyards in North America and Europe, primarily due to its role in transmitting Flavescence dorée, a serious phytoplasma disease. The pest damages vines by feeding on sap and spreading this pathogen, which leads to symptoms such as delayed bud break, yellowing or reddening of leaves, reduced photosynthesis and up to a 50% reduction in grape yields (Ripamonti et al. 2022). Early detection and control of nymphs are crucial to prevent the spread of the disease, as adult leafhoppers are highly effective vectors.

Species identification of leafhoppers has historically been challenging due to their small size, morphological ambiguities in nymphs, seasonal phenotypic plasticity and the need for detailed examination of male genitalia (Zhang, Zhang, and Duan 2019; EFSA et al. 2022). To overcome these challenges, DNA barcoding using the COI gene has become essential (Bluemel et al. 2011; Kamitani 2011; Foottit, Maw, and Hebert 2014; Zhang, Zhang, and Duan 2019). Initial efforts by Kamitani (2011) and Bluemel et al. (2011) provided foundational DNA barcodes, while Foottit, Maw, and Hebert (2014) expanded this dataset to include 471 Hemipteran species. Recent studies by Zhang, Zhang, and Duan (2019) and Emam et al. (2020) have further enriched global DNA barcode databases, and Lu et al. (2021) conducted a comprehensive phylogenetic analysis of Typhlocybinae, focusing on COI barcodes.

Nonetheless, previous surveys primarily focused on adult or museum specimens, with limited coverage of nymphs, particularly from the subfamily Typhlocybinae. Early developmental stages of leafhoppers, including nymphs, are equally as damaging to crops as adults (Hower and Flinn 1986; Jarrell et al. 2020). Furthermore, colour variations observed in adult leafhoppers (Ramaiah, Meshram, and Dey 2023) complicate accurate identification of live specimens.

In the Korean Peninsula, about one hundred leafhopper species across thirty-five genera have been recorded in the subfamily Typhlocybinae. Since 2005, severe outbreaks of leafhoppers have been reported in fruit trees and vines, affecting crops such as Vitis vinifera Linnaeus (1753) (grapes), Prunus spp. Linnaeus (1753) (apricot, peach and plum) and Diospyros kaki Thunberg (1784) (persimmon) (Ahn et al. 2005). Notably, the genus Arboridia is distributed in Palaearctic and Oriental regions, with 69 valid species, and ten species have been recorded in Korea (Song and Li 2013; Oh, Choe, and Jung 2015). Arboridia spp. are prominent pests of grapevines, particularly V. vinifera. However, difficulties in identifying Arboridia species, especially at the nymph stage, have hindered early management efforts.

The genus Singapora, primarily distributed across Central Asia and Oriental regions, includes 18 recorded species (Cao, Yang, and Zhang 2014). It is known to infest plants of the Rosaceae family. Singapora shinshana (Matsumura 1932) is the only species from this genus recorded in Korea, where it attacks various orchard trees, particularly Prunus species (Kim et al. 2021). Due to the diversity of host plants within the Prunus genus, genetic studies are needed to clarify its population structure based on host plant associations.

Diospyros kaki (persimmon), originally a fruit tree from China, is now widely cultivated in Korea and Japan. No reports exist of leafhopper species affecting this crop in China or Japan. However, since 2008, Zorka sp. has been reported in Korea as damaging persimmon leaves, causing serious harm, and it continues to affect persimmon crops (Hwang et al. 2009). The genus Zorka is known from the Oriental region, with species primarily recorded in Vietnam and southern China (Huang and Zhang 2013). In Korea, only Zorka sp. has been recorded from this genus.

In this study, we monitored orchard farms in South Korea for leafhopper pests, focusing on both nymphs and adults to facilitate early control. Our approach evaluates the utility of DNA barcoding for matching nymphs and adults, includes photographs of live specimens, reports host plant associations and provides the first DNA barcode sequences for these economically important Korean leafhopper species.

2 Materials and Methods

3 Results

A total of 40 barcode sequences were generated for leafhopper species, and 30 individuals of Korean leafhopper species were successfully amplified and sequenced using universal COI primers. Among these, four leafhopper species from three genera within the subfamily Typhlocybinae were identified: Arboridia kakogawana, A. maculifrons, Singapora shinshana and Zorka sp. The sequences were deposited in NCBI (USA) under Accession Numbers MN972661-MN972679.

Partial neighbour-joining trees, based on Kimura 2-parameter genetic distances, are presented in Figure 1. The trees delineate the leafhoppers into four main clusters with robust bootstrap support (Figure 1). Genetic distances, both intraspecific and interspecific, are provided in Tables 2 and 3. Intraspecific genetic distances among the four species ranged from 0.00% to 2.10%, while pairwise mean genetic distances between species ranged from 15.90% to 26.00%. These values indicate a significant barcoding gap and align with previously reported values for the family Cicadellidae (Foottit, Maw, and Hebert 2014; Zhang, Zhang, and Duan 2019). In prior barcoding studies on Hemiptera, intraspecific distances generally fell below 2%, while interspecific distances exceeded 3% (Hebert et al. 2003; Park et al. 2011). Our results largely support these findings, with the exception of a slightly higher intraspecific genetic distance (0.00%–2.10%). However, Zhang, Zhang, and Duan (2019) reported greater intraspecific divergence within the species of Deltocephalus (Cicadellidae), ranging from 0.6% to 6.3%, indicating that genetic distance may vary depending on the species.

The number of haplotypes identified for each species was 5, 8, 1 and 2 for A. maculifrons, A. kakogawana, Singapora shinshana and Zorka sp., respectively. Haplotype diversity was calculated as 0.857, 0.972, 0.000 and 0.667 for A. maculifrons, A. kakogawana, Singapora shinshana and Zorka sp., respectively.

4 Discussion

The genus Arboridia Zachvatkin 1946, belongs to the leafhopper subfamily Typhlocybinae (Hemiptera: Cicadellidae), comprising 84 species recorded across the Palaearctic and Oriental regions, including several recognised as global pests (Han et al. 2024). Arboridia kakogawana, a significant pest of grapevine (Vitis vinifera), is listed on the EPPO (European and Mediterranean Plant Protection Organization) alert list (EPPO 2024) and has been associated with documented damage in Korea since 2005 (Ahn et al. 2005). Both A. kakogawana and A. maculifrons are prominent pests of cultivated grapevines in Korea, found in both nymph and adult forms (Ahn et al. 2005). A. kakogawana exhibits notable colour variation and intraspecific diversity, complicating accurate identification (EFSA et al. 2022).

Adult A. kakogawana typically display pale yellow bodies with orange or brown venation on the forewings and two black spots at the upper angles of the scutellum (Šćiban, Mirić, and Kosovac 2021). However, these venational patterns and spots vary with maturity, leading to potential misidentification. For example, adult form 1 in Figure 2 lacks venation patterns and black scutellum spots, while form 2 displays the spots but not the venation. Adult form 3 exhibits both features, resembling A. maculifrons. Taxonomic differentiation between A. kakogawana and A. maculifrons primarily relies on the length of male genitalia.

Our neighbour-joining (NJ) tree (Figure 1) distinctly clusters nymph and adult specimens of both species, facilitating accurate identification. Although A. kakogawana and A. maculifrons belong to the same genus, the interspecific genetic distance between them was 15.9% (Table 2), contrasting with the 4%–10% interspecific distances typically observed in closely related congeneric species (Park et al. 2011; Barman et al. 2017). Intraspecific genetic distances also differed between the two species: for A. kakogawana, distances ranged from 0.00% to 1.30%, with a mean of 0.50%, while A. maculifrons showed a range of 0.00%–2.10% and a mean of 0.80% (Table 3), indicating deeper genetic divergence in A. maculifrons.

Arboridia kakogawana exhibited the highest number of haplotypes and the greatest haplotype diversity among the four species studied (Table 3), likely due to sample collection from multiple regions (CB, GG, GW), suggesting that regional differences have influenced its haplotype diversity. Furthermore, more than 15% genetic divergence exists within a single genus, Arboridia, suggesting the possibility of cryptic species (Hebert et al. 2004). This highlights the need for further studies, such as securing additional Arboridia species and conducting a comprehensive phylogenetic study of the genus Arboridia.

The genus Zorka Dworakowska, 1970, also in the subfamily Typhlocybinae, includes eight recognised species worldwide (Huang and Zhang 2013). Zorka sp., a newly discovered species in Korea, was found on persimmon trees (Hwang et al. 2009) and exhibits host plant specificity, not co-occurring with other leafhopper species. This Korean species differs from existing Zorka species by the pattern of white spots on the crown, pronotum, scutellum and forewings.

Both nymph and adult forms of Zorka sp. cluster together in the NJ tree (Figure 1 I, J), clearly separated from other leafhopper species. Zorka sp. exhibited the greatest pairwise mean genetic distance with A. maculifrons, at 26.0% (Table 2). Its intraspecific genetic distance ranged from 0.00% to 0.60%, with a mean of 0.40%, which falls within the typical range (< 2%) (Table 3). The number of haplotypes and haplotype diversity were 2 and 0.667, respectively. These results suggest that regional differences, as indicated by samples collected from CN and GW, have influenced haplotype diversity (Table 3).

In contrast to Arboridia spp. and Zorka sp., Singapora shinshana inhabits various fruit trees in the genus Prunus (e.g., P. armeniaca, P. salicina, P. persica) in Korea (Table 1). Samples from different host plants clustered together in the NJ tree (Figure 1). The pairwise mean genetic distance between Singapora shinshana and Zorka sp. was 22.6% (Table 2). The intraspecific genetic distance for S. shinshana was 0.00% (Table 3), the lowest among the species studied. Moreover, only a single haplotype was observed in S. shinshana in Korea, suggesting low genetic diversity, despite its occurrence on multiple Prunus hosts (Table 3).

5 Conclusions

This study presents newly reported DNA barcodes for Korean leafhopper species: Arboridia kakogawana, A. maculifrons, Singapora shinshana and Zorka sp. The findings confirm that DNA barcoding is an effective tool for identifying leafhoppers across their life stages. Further research is recommended to expand COI data coverage for more precise species identification.

Early detection and accurate identification of nymphs could improve pest control and monitoring efforts, particularly in the early stages, thereby supporting increased crop production.

Author Contributions

Hwalran Choi: conceptualization, writing – original draft, formal analysis, writing – review and editing. Sun-Kook Kim: investigation, resources. Duane D. McKenna: conceptualization, writing – review and editing, writing – original draft. Seunghwan Lee: funding acquisition, writing – original draft, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.