Living specimens were collected at night by visual inspection using a head torch or under light traps. External genitalia were dissected in 10% potassium hydroxide (KOH) solution, clarified with pure water and subsequently stored in 70% ethanol in Eppendorf tubes for further research. Photographs were taken with a Nikon D7100 digital camera. All examined specimens are deposited in the collections of China Agricultural University, Beijing, China (CAU); the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS); the collection of Chao Wu in Beijing, China (CWC); and the collection of Bowen Ye in Shandong, China (CBW).

The classification system follows Schwarz and Roy (2019). Morphological terminology for adults and external male genitalia follows Brannoch et al. (2017) and Schwarz and Roy (2019).

We used the pronotum of 25 male specimens from six species within Phyllothelys werneri species group as representatives to quantitatively assess and visualise the morphological differences between P. qingjinum sp. nov. and others (Table 1). The species P. stigmosum (Zhou & Zhou, 2004) was not considered in analysis due to the unavailability of the type specimen for examination and the possibility that it represents a synonym of Phyllothelys werneri, based on certain aspects of the original description (Wu and Liu 2021). This species is also unlikely to be confused with P. qingjinum sp. nov. due to their distant distributions and significant differences in the pronotum (Zhou and Zhou 2004).

We employed tpsUtil 1.46 (Rohlf 2010) and tpsDig2 v.2.16 (Rohlf 2008) to extract and digitise eight homologous landmarks from the left margin of pronotum in each specimen (Suppl. material 1) (Brannoch et al. 2017). These landmark configurations were then scaled, translated and rotated against a consensus configuration using the Procrustes superimposition method (Bookstein 1991) in MorphoJ 1.06d (Klingenberg 2011). Finally, Principal Component Analysis (PCA) was performed on the reconfigured landmarks in MorphoJ 1.06d to assess variability in the shape space.

The partial Cytochrome Oxidase I (COI) sequence was selected as the molecular marker to conduct the phylogenetic analyses and genetic distance calculation. A total of 19 individuals were sampled for the analyses, covering three species groups in Phyllothelys (P. werneri species group, P. sinense species group and P. westwoodi species group), in which 17 of them are newly sequenced in this study and two are obtained from GenBank databases (Table 2).

For the newly-sequenced specimens, their genomic DNA was extracted from the thoracic and coxal muscle tissues by using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and amplified using Ex Taq (TaKaRa, Tokyo, Japan). Primers for amplification are LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). The PCR protocols implemented using a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) were as follows: an initial 3-min denaturing step at 94°C; 35 cycles of 20 s at 94°C, 20 s at 50°C and 30 s at 72°C; with a final 5-min extension at 72°C. Sequences were bidirectionally sequenced using the same PCR primer pairs. Sequences alignment was carried out using ClustalW and edited using Geneious 10.1.3 (Kearse et al. 2012), generating a matrix with 620 nucleotide characters.

We employed both Maximum Likelihood (ML) and Bayesian Inference (BI) based on the alignment generated above. For the ML analyses, the best substitution model was determined as ‘TIM2+F+G4’ according to BIC (Bayesian information criterion scores) in ModelFinder (Kalyaanamoorthy et al. 2017) and the phylogenetic tree was constructed under the IQ-TREE v.1.6.12 (Trifinopoulos et al. 2016) using an ultrafast bootstrap approximation approach with 1000 bootstrap replicates. For the BI analyses, the best substitution model was determined in PartitionFinder2 v.2.1.1 (Lanfear et al. 2017) as ‘GTR+G+F’. Two simultaneous runs of two million generations were executed using MrBayes v.3.2.7 (Ronquist et al. 2012) for the dataset and trees were sampled every 1000 generations, with the first 25% discarded as burn-in. Stationarity was considered to be reached when the average standard deviation of split frequencies was below 0.01.

We used the same sequences alignment to calculate the genetic distances within Phyllothelys werneri species group. The analysis was conducted in MEGA7 (Kumar et al. 2016) under the Kimura two-parameter (K2P) model and the result visualised in ggplot (package in R v.4.3.0 in RStudio for Windows).

Based on the morphometrics of the pronotum, we compared five previously-described species to P. qingjinum sp. nov. in the P. werneri species group (Fig. 5A). The contribution of the first principal (PC1) component accounted for 62.21% of the total variation, whereas the second principal (PC2) account for 13.92%. In the factorial map, the points representing P. qingjinum sp. nov. were far away from the others. The points of P. chuangtsei and P. werneri were also each distinctly separated from other species. In contrast, the points representing P. jiazhii, P. jianfenglingense and P. dulongense were closer to each other, with their 80%-equal frequency ellipses showing considerable overlap.

Figure 5.  

Morphological and molecular divergence of P. qingjinum sp. nov. relative to other species in the P. werneri group. A. Eight morphological landmarks on the pronotum used for Principal Component Analysis (PCA) (the left) and the resulting pronotum shape differences amongst species shown on the right, with circles are 80%-equal frequency ellipses of each species. B. Phylogenetic relationships within the P. werneri group, constructed using IQ-TREE and MrBayes, feature a cladogram-based framework from IQ-TREE, with branches of multiple samples from the same species collapsed into coloured triangles. Node support values are noted directly beside each node, formatted as Bootstrap value/Bayesian posterior probability (%). C. Heatmap illustrates pairwise genetic distances between species under K2P model, where the intensity of the colour reflects the degree of genetic distance, with deeper reds indicating greater genetic divergence.

Phylogenetic analysis using MrBayes and IQ-TREE revealed identical topologies and generally high support values (Fig. 5B). The results supported the monophyly of all sampled species and species groups and indicated that the P. westwoodi species group is the sister group to the clade (P. sinense species group + P. werneri species group). The phylogenetic position of P. qingjinum sp. nov. is recovered to be within the P. werneri species group, as the sister to the clade (P. werneri + P. dulongense).

The results of the genetic distance analysis showed that the average genetic distance between species in the genus Phyllothelys was 0.11 (Fig. 5C). Specifically, the average genetic distances between P. qingjinum sp. nov. and the P. westwoodi species group, P. sinense species group and other species within the P. werneri species group were 0.14, 0.12 and 0.08, respectively. Notably, the genetic distance between P. qingjinum sp. nov. and P. jianfenglingense was the smallest amongst all comparisons, at 0.07, which is still significantly lower than the average interspecific distance within the genus (0.11).

The posteroventral femoral spines of P. qingjinum sp. nov. are elongate, with their length nearly equal to the width of femora and apex slightly bending backwards (Fig. 1). In the interior surface of prothoracic femora, the basal and middle dark spots merge on the basal half (Fig. 2D). These characteristics align with the key diagnostic features of the P. werneri species group as described by Wu and Liu (2021), while they are different from the other three species groups. In terms of molecular evidence, P. qingjinum sp. nov. and species within the P. werneri species group exhibit relatively low genetic differentiation (average 0.08) compared to the P. sinense species group (average 0.12) and the P. westwoodi species group (average 0.14). Phylogenetic analyses using different methods consistently nest P. qingjinum sp. nov. within the P. werneri species group. Therefore, based on the morphological characteristics of the posteroventral femoral spines and the DNA barcoding evidence, we infer that P. qingjinum sp. nov. should be included in the P. werneri species group.

P. qingjinum sp. nov. can be easily distinguished from other species in the genus Phyllothelys by the morphology and coloured patches of the prothoracic legs (Fig. 2D). The densely arranged large colour patches in the discoidal area of the forewings of P. qingjinum sp. nov. resemble those of P. jianfenglingense and P. dulongense, both of which also belong to the P. werneri species group (Fig. 2E). However, P. qingjinum sp. nov. present a comparatively prominent supracoxal dilation with obvious lateral pronotal expansion, which sets it apart from these two species (Fig. 2B; Suppl. material 1). Within the P. werneri species group, only P. chuangtsei, aside from the new species described here, exhibits a developed lateral pronotal expansion around supracoxa dilation. However, P. qingjinum sp. nov. can be further distinguished from P. chuangtsei by its larger size and the presence of numerous longer, sharp spines on the sdp and afa of the male external genitalia (Fig. 3B).