Specimens used in this study were collected using a sweep net and are deposited at the Institute of Entomology, Guizhou University, Guiyang, China (GUGC). External morphological features were examined under an Olympus SZ2-ILST stereo microscope. Adult images were captured using a Keyence VHX-6000 imaging system. Genitalia were illustrated using Adobe Illustrator CS6 and Adobe Photoshop CS6. Male genitalia were prepared by boiling in an 8–10% NaOH solution for 1.5–2.5 min, then rinsed with water and stored in glycerol.

Morphological terminology and higher-level classification follow Zhang (1990), Rakitov (1997), and Dietrich (2005). Nomenclature of the new species adheres to the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature [ICZN] 1999).

Total genomic DNA was extracted from the insect abdomen using the DNeasy^® Blood & Tissue Kit (Qiagen, Germany). DNA purity and concentration were assessed using a Nanodrop 2000 spectrophotometer and 1% agarose gel electrophoresis. Extracted DNA was stored at -20 °C.

The complete mitochondrial genome was sequenced using the Illumina HiSeq 6000 platform (Berry Genomic, Beijing, China), generating 150 bp paired-end reads with an average insert size of 350 bp. Approximately 2 GB of clean data was obtained. Raw sequence data were assembled in Geneious R9 (Kearse et al. 2012), using C. hamata (GenBank: MN922303; Xu and Dai 2020) as a reference.

Genome annotation was performed using the MITOS web server (http://mitos2.bioinf.uni-leipzig.de/index.py; Donath et al. 2019). The 13 protein-coding genes (PCGs) were identified using ORF Finder in Geneious Prime with the invertebrate mitochondrial genetic code. The secondary structures of the 22 tRNA genes were predicted using tRNAscan-SE 1.21 and ARWEN 1.2 (Lowe and Eddy 1997; Laslett and Canbäck 2008).

Base composition and relative synonymous codon usage (RSCU) values were calculated using MEGA X (Kumar et al. 2018), which was also used to compute amino acid usage frequency. A graphical map of the mitogenome was generated using the CGView comparison tool (https://proksee.ca; Grant et al. 2023). Strand asymmetry was calculated using the following formulas (Perna and Kocher 1995):

AT skew = (A − T) / (A + T)

GC skew = (G − C) / (G + C)

To clarify the phylogenetic position of C. pala sp. nov., we analyzed its complete mitogenome along with those of 33 additional Deltocephalinae species obtained from GenBank. Two species from the subfamily Iassinae (Krisna rufimarginata and Batracomorphus lateprocessus) were used as outgroups (Table 1).

Phylogenetic reconstruction was based on sequences from 13 PCGs and two ribosomal RNA genes (12S and 16S rRNA). These sequences were extracted using Geneious Prime 2019.2.1 (Kearse et al. 2012). Multiple sequence alignment was conducted using MAFFT v7.313 within PhyloSuite v. 1.2.1. Poorly aligned regions and gaps were removed using Gblocks 0.91b (Talavera and Castresana 2007; Katoh et al. 2019; Zhang et al. 2020).

Gene alignments were concatenated using MEGA X (Kumar et al. 2018). Two datasets were prepared: (i) PCG-rRNA dataset: 13 PCGs + 12S + 16S rRNA (12,105 bp), and (ii) CG-only dataset: 13 PCGs (10,875 bp). Phylogenetic trees were constructed using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods based on these datasets (Huelsenbeck and Ronquist 2001; Nguyen et al. 2015).

The complete mitochondrial genome of C. pala sp. nov. (GenBank accession no. PQ615145) is circular, with a total length of 15,433 bp (Fig. 1). It contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region (CR) rich in A+T nucleotides. Its gene arrangement and composition are consistent with those of other Cicadellidae mitogenomes (Cameron 2014a; Wang et al. 2020; Li et al. 2023; Yang et al. 2023; Lu et al. 2024; Sun et al. 2024). Among the 13 PCGs, four genes (ND1, ND4, ND4L, and ND5) are located on the N-strand, while the remaining genes (ND2, ND3, ND6, COX1, COX2, CYTB, ATP6, and ATP8) are located on the J-strand (Fig. 1), which is typical of Cicadellidae mitogenomes (Wang et al. 2017a, 2017b).

The nucleotide composition of the mitogenome of C. pala sp. nov. and its components (whole genome, PCGs, rRNAs, tRNAs, and CR) is shown in Table 3: A (%): 43.2, 43.2, 43.9, 42.4, 42.5; T (%): 33.5, 32.3, 36.7, 37.5, 35.3; G (%): 9.5, 9.8, 7.5, 9.0, 10.2; and C (%): 13.8, 14.7, 11.9, 11.2, 11.6, respectively. The A+T content of the whole mitogenome is 76.7%, and the A+T content in the PCGs, rRNAs, tRNAs, and CR is 75.5%, 80.6%, 79.9%, and 77.8%, respectively (Table 3), indicating a strong A+T bias, similar to other leafhoppers (Wang et al. 2017a, 2017b, 2018). The AT-skew and GC-skew values for the genome, PCGs, rRNAs, tRNAs, and CR are as follows: AT-skew: 0.13, 0.14, 0.09, 0.06, 0.09; and GC-skew: -0.18, -0.20, -0.23, -0.11, -0.06, respectively.

The start and stop codons of the 13 PCGs, as well as the anticodons of the 22 tRNAs, are listed in Table 2. Most PCGs use typical ATN start codons (ATA, ATT, ATC, or ATG) and terminate with either TAR (TAA or TAG) or an incomplete stop codon (T). Notably, ND1 and ND4 were reported to end with ATT, and ND5 initiates with AAC and terminates with a single A, which may be sequencing artifacts or indicative of RNA editing. Among the 13 PCGs, ND5 is the longest gene (1,660 bp), and ATP8 is the shortest (148 bp), consistent with other insects (Mao et al. 2017; Wang et al. 2018; Du et al. 2019; Xu and Dai 2021).

Figs 2, 3 summarize the relative synonymous codon usage (RSCU) and amino acid frequency. The most frequently used codon is CGA (Arg), followed by UUA (Leu), whereas the least used is UCG (Ser1). The most frequently encoded amino acids are Ser, Leu, Lys, Asn, and Ile. The prevalent use of codons composed entirely of A or T reflects the high A+T content of the genome.

All 22 tRNAs were predicted using tRNAscan-SE 1.21, ARWEN 1.2, and MITOS2. Most tRNAs exhibit the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm and instead forms a simple loop, with is a common feature in many insects (Sheffield et al. 2008; Cao and Du 2014; Wang et al. 2014; Zhang et al. 2014; Wang et al. 2017a, b). Additionally, trnG lacks the TΨC arm (Fig. 4). The lengths of the tRNAs range from 61 bp (tRNA-His) to 69 bp (tRNA-Gln) (Table 2). Several base mismatches were observed in tRNA secondary structures (Table 4), including: A-A mismatches (e.g., in trnE), U-U mismatches (e.g., in trnQ, trnL1, trnL2, trnW, and trnR), and G-U mismatches, which were most frequent, observed in 13 tRNAs (trnA, trnD, trnQ, trnG, trnH, trnL1, trnK, trnF, trnP, trnS1, trnW, trnY, trnV). These findings suggest that further research on tRNA secondary structures is warranted.

As in other insect mitogenomes, C. pala sp. nov. contains two rRNA genes: rrnL (1,181 bp) and rrnS (731 bp) (Table 2). Their locations are conserved: rrnL lies between trnL2 and trnV, and rrnS lies between trnV and the control region (CR) (Table 2, Fig. 1). The CR (also called the A+T-rich region) is the longest non-coding segment and varies greatly among species in both length and sequence composition, which is a key factor in mitogenome diversity among leafhoppers (Wang et al. 2020). In C. pala sp. nov., the CR is 1,134 bp in length, located between rrnS and trnI, with an A+T content of 77.8% (Tables 2, 3).

The phylogenetic relationships of Deltocephalinae were inferred using 34 species based on two datasets: (i) 13 protein-coding genes (PCGs) and two ribosomal RNA genes (12S and 16S rRNA), comprising 12,105 nucleotides (PCG+rRNA); and (ii) PCGs only, comprising 10,875 nucleotides. Four phylogenetic trees were generated using Bayesian Inference (BI) and Maximum Likelihood (ML) methods (BI-PCGs, BI-PCG+rRNA, ML-PCGs, ML-PCG+rRNA), all of which yielded similar topologies.

Previous studies have examined the phylogenetic relationships within Deltocephalinae using either morphological or molecular data (Zahniser and Dietrich 2008, 2010, 2013; Du et al. 2017a, 2017b; Hassan et al. 2023). In our study, mitogenomic data produced well-resolved trees supporting several established relationships. The results indicate that Penthimiini occupies a basal position in the phylogenetic tree (Figs 5, 6; Suppl. material 1: figs S1, S2), which is consistent with previous findings (Zahniser and Dietrich 2013; Wu et al. 2021). C. pala sp. nov. clusters with other Deltocephalinae species and specifically with C. theae and C. hamata within Penthimiini.

Within Deltocephalinae, Scaphoideini forms a distinct clade with Drabescini, with both lineages recovered as sister groups (Figs 5, 6; Suppl. material 1: figs S1, S2), corroborating previous findings by Zahniser and Dietrich (2013), Xu et al. (2020b), and Lu et al. (2023). Stable sister-group relationships were also observed between Deltocephalini and Paralimnini, and between Selenocephalini and Cicadulini. However, the phylogenetic placement of Athysanini remained unresolved and unstable across datasets, echoing earlier studies (Wu et al. 2021; Hassan et al. 2023). In addition, the monophyly of Chiasmini, Deltocephalini, Drabescini, Paralimnini, Penthimiini, and Scaphoideini was strongly supported in all analyses (Figs 5, 6; Suppl. material 1: figs S1, S2).

(males, Fig. 10A–P)