An integrative approach reveals a new species of flightless leaf beetle (Chrysomelidae: Suinzona) from South Korea

The leaf beetle genus Suinzona, consisting of over 20 species, is mainly known for species from Southwest China, and its closely related genus Potaninia, with only two species, is widely distributed in South China and upper Southeast Asia. Despite recent morphological studies, the status of these taxa has long been controversial. Here, we investigated the taxonomic status and phylogenetic relationships of the genera Suinzona and Potaninia within Chrysomelinae using molecular, morphological and ecological data. Molecular phylogenetic analysis supported that they should be regarded as distinct genera, which is consistent with morphological evidence, e.g., well-developed/reduced hind wings. Based on combined evidence from examination of larval and adult morphology, host plants and mitochondrial genomes, we demonstrate that P. cyrtonoides should be placed in the genus Suinzona and that specimens from South Korea represent a new species. Suinzona borowieci sp. nov., occurring in narrow strips of habitat, shows high levels of genetic divergence and distantly related host plants between populations. The population differentiation seems to be correlated with its non-functional wings causing reduced dispersal ability and genetic isolation. Several populations have declined dramatically over the last few decades due to loss of habitat and thus are in need of protection as conservation units.


Results
Diagnosis. Suinzona borowieci sp. nov. is almost identical to S. cyrtonoides in the shape of the flagellum of the aedeagus. However, it can be distinguished by its larger body size (5.5-7.0 mm vs. 4.8-6.0 mm), denser punctures on elytra (less dense punctures in S. cyrtonoides), larger and broader aedeagus with the distal tips of the flagellum quadrifurcated and slightly curved, arising from two sclerotized tubes (with a smaller and narrower aedeagus with distal tips of the flagellum quadrifurcated and almost straight, arising from a sclerotized tube in S. cyrtonoides).
Etymology. Dedicated to the first author's mentor Prof. dr hab. Lech Borowiec (University of Wrocław, Poland), the world's leading specialist in tortoise beetles.
Biological notes. Detailed descriptions of larvae and pupae and the life cycle have been published by Kimoto 16 and Kimoto and Takizawa 11 . Its life cycle is similar to that of S. borowieci sp. nov., but they feed on different host plants.
Remarks. The apical part of the aedeagus is highly variable, narrow to broad, apex narrowly to widely rounded or weakly truncate, mainly with two weak or strong denticles on the apicolateral margin. The aedeagus of the type specimen is narrowly rounded without apicolateral denticles (Fig. 2e). However, we were not able to find an obvious tendency in the morphological variation of the aedeagus at the intrapopulation or interpopulation level. Chrysomela cyrtonoides Jacoby, 1885 was described from Japan. Later, it was transferred to the genus Potaninia by Chûjô and Kimoto 17 and then accepted by various authors until now. However, we found that all materials of P. cyrtonoides have reduced hind wings (Fig. 1d), which are the key diagnostic features of the genus Suinzona, and molecular analysis also suggests its placement in Suinzona. Therefore, Suinzona cyrtonoides (Jacoby, 1885) comb. nov. is proposed. Jacoby 18 gave 'Konose' as the type locality and used at least two specimens collected by G. Lewis for the description. A male specimen (BMNH) from 'Yuyama' , designated by Ge et al. 3 as a lectotype, did not belong to the type series of S. cyrtonoides and thus lost its lectotype status (ICZN: Article 74.2). Indeed, a female specimen (BMNH) was mislabelled as a lectotype. We were able to find four specimens collected from Japan that might belong to the type series of S. cyrtonoides in the G. Lewis collection (BMNH, MCZC), but more precise locality data were not available. Therefore, we regard them as syntypes and defer selection of a lectotype.

Molecular phylogenetic analyses.
It is evident from the clarified phylogenetic inference based on mitogenomes that the genus Suinzona differs from the genus Potaninia, S. borowieci sp. nov. as the sister species of S. cyrtonoides (Fig. 7a). The phylogenetic inferences included a total of 20 mitogenomes of Chrysomelinae and outgroups of Galerucinae (Supplementary Table S1). The complete mitogenomes of the four Suinzona species and one Potaninia species (incomplete) were newly sequenced in the present study. Each mitogenome contains a typical set of mitochondrial genes (13 PCGs, 22 tRNAs and two rRNAs) and a control region. Phylogenetic trees based on ML and BI inferences revealed the presence of two well-supported clades (Chrysomelini and Doryphorini + Entomoscelini + Gonioctenini), placing the genus Suinzona as the sister group of the genus Potaninia. This result matched the morphological character of the hind wing. The COI haplotype network of the genus Suinzona complex (Fig. 7b) confirms the previous results and shows that the currently known single species is well distinguished as a species. Two independent networks were completely separated without any connection due to the existence of the mutation (62 steps) exceeding the 95% parsimony limits between them. 3. Larger, body length 5.5-7.0 mm; elytra more densely punctate (Fig. 1a); aedeagus larger and broader (Fig. 2c) -Smaller, body length 4.8-6.0 mm; elytra less densely punctate (Fig. 1d); aedeagus smaller and narrower (Fig. 2e)

Discussion
Members of Suinzona exhibit high similarity in their external morphology and colouration, often in the shape of the aedeagus; therefore, the flagellum of the aedeagus was used as the most significant diagnostic character between closely related species. Each species has a specific and complex structure of the flagellum, from bifurcated to concentric circles (see Ge et al. 3 ). S. borowieci sp. nov. and S. cyrtonoides comb. nov. show strong similarity in the unique structure of 'the quadrifurcated tips of the flagellum' , despite the slight difference in the microstructure of the flagellum and the substantial difference in the shape of the aedeagus (Fig. 1f,g). The mature larva of S. borowieci sp. nov. is very similar to that of S. cyrtonoides comb. nov. in body shape, colouration and tubercular pattern but differs in the dense setose body and several secondary tubercles between Dae and DLai on the meso-and metathorax. The immature stages and biology of other Suinzona species endemic to China are currently unknown. Based on the larval morphology and biology of Korean and Japanese species, the genus Suinzona belongs to the generic group Potaninia proposed by Kimoto 19 with the genus Entomoscelis Chevrolat and is characterized by the three rows of dorsal tubercles and separated tubercles between Dpe and DLpi on the meso-and metathorax in fourth instar larvae. However, Kimoto 19 examined only one species, S. cyrtonoides comb. nov. (as P. cyrtonoides), for his classification, and hence the generic group name Potaninia needs to be reconsidered. S. cyrtonoides comb. nov. and S. borowieci sp. nov. present a disjunct distribution, with other Suinzona species occurring only in Sichuan and Yunnan (Fig. 3a). The former species is endemic to the major  www.nature.com/scientificreports/ described as having a humeral callus and well-developed hind wings. In addition, Kimoto 20 and Kimoto and Takizawa 11 wrongly described it as apterous, whereas Suzuki et al. 21 stated that it has reduced hind wings, a socalled stenopterous, which is in accordance with the type specimens. The flightless leaf beetle S. borowieci sp. nov. shows high levels of genetic divergence and different host plants at the population level. Each population of this species is small in size and restricted to an extremely narrow habitat area. These effects may lead to allopatric speciation after a long period of time as a result of the increased chance of isolation from other populations. It was demonstrated that the flightless species of carrion beetles retained higher genetic differentiation among the populations than the flying species, and species richness may result from the loss of flight 22 . Unfortunately, several populations of S. borowieci sp. nov. have declined precipitously over the past few decades, especially in Taebaek and Gunwi. Habitat destruction has occurred due to the creation of new hiking trails or the expansion of agricultural land, which is an important cause of local extinction. The morphological, molecular and ecological analysis in this study reveals a new species with generic placement from South Korea. The reduced non-functional hind wing of the genus Suinzona is the most important generic character that distinguishes it from the genus Potaninia and may have promoted population differentiation and species diversification by the reduction in dispersal ability. However, nothing is known about the mitochondrial genome, immature stages and biology of most species (23 species) endemic to southwestern China. Therefore, further studies that include more species are required to obtain a better understanding of the evolution, phylogeny, and biology of the genus Suinzona and the closely related genus Potaninia. All larval specimens used in the study were preserved in 70% ethanol. To examine the morphological characters, some larvae were dissected, cleared in 10% sodium hydroxide solution, rinsed with distilled water, and mounted on slides with glycerine and Swan's liquid (20 g distilled water, 15 g gum arabic, 60 g chlorhydrate, 3 g glucose, and 2 g glacial acetic acid). Genitalia were dissected from adult specimens softened in plastic containers with wet tissue paper for 12-24 h. The aedeagus was softened in 10% sodium hydroxide solution for 2-6 h and placed in distilled water. The careful insertion of a sharp-pointed thick nose hair and injection of 5% ethanol into the foramen of the aedeagus was repeated until the flagellum and internal sac were fully everted. After washing with absolute ethanol, the genitalia were preserved in a microvial with glycerine and pinned to the specimen. Descriptions and illustrations were prepared using Nikon SMZ800 and Nikon Eclipse E600 microscopes, each equipped with a drawing tube. Photographs were taken by a Nikon D850 digital camera attached to a Leica M165C microscope and were edited in Helicon Focus 7.6.4 and Adobe Photoshop 2020. Line drawings were made in Adobe Photoshop 2020 with a Wacom Intuos4 graphics tablet from photographs. In the larval description, the letters L, S, and M after Arabic numerals within parentheses signify long, short, and minute setae on the tubercle, respectively. The terminology used on the larval tubercle is as described by Kimoto 19 . DNA extraction and sequencing. Total genomic DNA was extracted from three species of Suinzona and Potaninia samples using a DNeasy Blood & Tissue Kit (Qiagen Co., Germany) following the manufacturer's protocol. PCR conditions and primers for the mitochondrial COI gene (LCO1490 + eCOI-2H) following Oba et al. 23 . The obtained sequences were deposited in GenBank (Supplementary Table S1). Mitochondrial genomes were sequenced using shotgun sequencing on the HiSeq 2000 platform using libraries with an insert size of 200 bp and paired-end sequencing of 100 bp. Assembly and annotation of genes were performed as described by Nie et al. 24 , using Entomoscelis adonis (GenBank: KX943493) as a reference. The obtained mitochondrial genome sequences were deposited in GenBank (Supplementary Table S1).

Methods
Phylogenetic analyses. All the PCG and rRNA genes were aligned individually using Muscle 3.8.425 25 .
The aligned data from each mitochondrial gene were concatenated with Geneious V.2021.0.3 26 . Phylogenetic inferences were performed using MrBayes 3.2.7 27 and raxmlGUI 2.0 28 . For the BI and ML analyses, the bestfit models of nucleotide substitution and partition schemes were selected using PartitionFinder 2 29 . BI analyses were run for 20 million generations initiated with program-generated trees, two independent runs of four Markov chain Monte Carlo (MCMC) chains and sampling every 100 generations. The first 25% of trees were discarded as burn-in and then visualized using FigTree v1.4.4 30 . For the ML analysis, the GTR-CAT model was chosen for the bootstrapping phase, conducted with initial tree searches, followed by 10,000 ultrafast bootstrap replicates. Sequences of the COI gene were also constructed in unrooted parsimonious networks using TCS 1.21 31  www.nature.com/scientificreports/ that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information can be viewed through any standard web browser by appending the LSID to the prefix "http:// zooba nk. org/". The LSID for this publication is urn:lsid:zoobank. org:act:5DC70067-DB70-4DE3-A4E6-AD287247D19F. The electronic edition of this paper was published in a journal with an ISSN, and it has been archived and is available from PubMed Central.

Data availability
The type series of the new species is deposited in the Národní Muzeum, Prague, Czech Republic [NMPC] and H.-W. Cho's private collection [HCC]. The molecular sequences obtained in this study are available in GenBank. The sequence accession numbers and collection locality for each specimen are presented in Supplementary  Table S1.