﻿A taxonomic review of the order Mantodea in Korea based on morphology and DNA barcodes

﻿Abstract A taxonomic study of Korean Mantodea using morphological and molecular characters (COI) is presented. Eight species [Amantisnawai (Shiraki, 1908), Acromantisjaponica Westwood, 1889, Mantisreligiosasinica Bazyluk, 1960, Statiliamaculata (Thunberg, 1784), Tenoderaangustipennis Saussure, 1869, T.sinensis Saussure, 1871, Hierodulachinensis Werner, 1929, H.patellifera (Audinet-Serville, 1838)] belonging to six genera in three families are recognized. Interspecific genetic divergence of COI using uncorrected p-distance ranged from 6.7% to 22.4%, while intraspecific divergence ranged from 0% to 2.2% among eight Korean Mantodea species. All eight species were each strongly supported as a single lineage using COI on both neighbor-joining and parsimony trees. An illustrated key, redescriptions, habitus photographs, and illustrations of diagnostic characters of the species of Korean Mantodea are provided to facilitate identification.


Introduction
The order Mantodea comprise approximately 2,400 species in 460 genera, making it a distinctive group of predatory insects (Ehrmann 2002;Otte et al. 2024).They exhibit remarkable diversity in morphology, hunting strategies, and habitat specialization and are widely distributed in tropical and subtropical regions (Otte et al. 2024).Members of this order are generally characterized by a carnivorous diet, mimetic behavior, triangular head, large compound eyes, raptorial foreleg with elongated forecoxa, forefemur and foretibia with characteristic spines, femoral brush for grooming, elongated pronotum, male subgenital plate (coxosternite IX), asymmetrical male genitalia, female subgenital plate (coxosternite VII), and ootheca covered by a secretion (Klass 2001;Klass and Meier 2006;Yager and Svenson 2008;Wieland 2013;Svenson et al. 2015;Hashimoto et al. 2016;Brannoch et al. 2017).
In this paper, we present a taxonomic reassessment of Korean Mantodea, recognizing eight species belonging to six genera in three families, including A. nawai (Shiraki, 1908), Ac.Japonica Westwood, 1889, M. religiosa sinica Bazyluk, 1960, S. maculata (Thunberg, 1784), T. angustipennis Saussure, 1869, T. sinensis Saussure, 1871, H. chinensis Werner, 1929, and H. patellifera (Audinet-Serville, 1838).Re-descriptions incorporate salient morphological features critical for accurate identification of these species, including the male genitalia.We also used molecular criteria including genetic divergence and gene tree monophyly using a COI barcode region as a multiple lines of evidence approach for the species identification.Our taxonomic review of Korean Mantodea provides redescriptions, habitus photographs, an interactive key, and diagnoses.

Materials and methods
Studied specimens were mostly collected from inland and the islands of the Korean peninsula.Specimens were collected by direct sweeping, scanning, shifting leaf litter and light trapping.If nymphs or oothecae were found, samples were reared until the adult insect emerged.The collected specimens were killed by freezing to prevent discoloration and were moved to a drying chamber for dehydration at 60 °C for 10 days until completely hardened.The subsequent sample preparation followed methods by Brannoch et al. (2017) and Shim et al. (2021b).Briefly, the male genitalia were incubated overnight in 10% potassium hydroxide (KOH), and washed with distilled water, then 75% ethanol, before storage in glycerol.Depository of specimens examined is as follows: National Institute of Agricultural Sciences Insect Collection (NASIC, Wanju, Korea); Kunsan National University (KsNU, Gunsan, Korea); National Institute of Biological Resources (NIBR, Incheon, Korea).
The specimens were examined with a stereomicroscope (MS5, Leica Microsystem, Wetzlar, Germany).Images were obtained using a Canon DSLR (EOS 5D; Tokyo, Japan) with an attached Canon MP-E 65 mm f/2.8 1-5× lens.Several layers of photographs were combined in Helicon Focus 5.3 software (Helicon Soft Ltd, Kharkov, Ukraine) and edited using Adobe Photoshop CC 2020 (Adobe, San Jose, CA, USA).Measurements were recorded in millimeters using digital Vernier calipers (CD-15APX; Mitutoyo, Sakado, Japan).The terminology of taxonomic characters and measurements of specimens mainly followed Wieland (2013) and Brannoch et al. (2017) for external morphology, and Klass (1997) and Schwarz and Roy (2019) for male genitalia.
For the study of molecular characters, we included a total of 74 specimens for DNA extraction in the dataset and the specimens used are listed in Suppl.material 1.DNA extraction, sequencing and alignments follow the methods described by Shim et al. (2021b).The mitochondrial COI was selected.Primers and amplification strategies are provided in Shim et al. (2021b).Data from Gen-Bank for 50 foreign specimens were incorporated into the study, as indicated in Suppl.material 1. Parsimony (PA) analyses were conducted using MEGA X (Kumar et al. 2018) with 1000 bootstrap replications.A neighbor-joining analysis (NJ) was performed in MEGA X (Kumar et al. 2018) using the Kimura-2-Parameter (K2P) model (Kimura 1980).Bootstrap support values for each node were evaluated via MEGA X with 1000 replicates.Intra-and inter-specific distances in the different taxonomic levels were calculated using an uncorrected pairwise distance method (Srivathsan and Meier 2012).
Diagnosis.Very small sized mantises.Body with mottled dark spot pattern.Pronotum short, kite-shaped, its dorsal surface with longitudinal striped pattern.Genicular spurs length as long as Pvfs length.Second Avfs is as long as second Ds, much longer than the neighboring spines.Wings brachypterous or macropterous.
Biological notes.Acromantis japonica is found in the southern islands of the Korean Peninsula (Jeon et al. 1999;An 2011).They occur in leaf litter, under broad leaves and on tree trunks.Adults exhibit positive phototaxis, and oothecae are typically laid under stones and the bark of rotten logs.This species overwinters in the egg stage inside their oothecae (Taniguchi 1987;Befu 1992).They exhibit behaviors such as vibrating their antennae and swinging forelegs.Nymphs fold their abdomens back during rest.The first instar nymphs hatch from the end of May to early June, while adults emerge beginning from the end of August.

Type species. Gryllus (Mantis) religiosa Linnaeus, 1758
Diagnosis.Medium to large sized mantises.Body color mostly bright green, yellow, brown.Male antenna reddish, conspicuously thicker than the female's.Vertex swollen.Forecoxal proximal area with dark spot, ventral surface with numerous yellow spots.Tibial spur groove area with yellow spot.Furcasternite with numerous spots.Metathoracical episternum with a dark lateral spot.Male inter-stylar margin notched.
Distribution.China, Japan, Nepal, South Korea, Taiwan.Invasive in Eastern USA and Russia.
Remarks.Statilia maculata is predominantly found in the eastern Palearctic regions (S. maculata has also been introduced to Eastern USA and Russia), whereas S. nemoralis is described from the Philippines and, erroneously, from various Southeast Asian countries (Ehrmann 2002;Patel and Singh 2016;Schwarz et al. 2018).These two species, with S. nobilis (Brunner de Wattenwyl, 1893), are frequently confused in the taxonomic literature, leading to numerous misidentifications (see details in Schwarz et al. 2018); for example, both S. nobilis and S. maculata have been erroneously reported as S. nemoralis (Wang 1993;Jeon et al. 1999;Oshima 2017;Schwarz et al. 2018;Oshima 2020;Shcherbakov and Govorov 2020).Notably, the green morph is relatively uncommon in female S. maculata (Fig. 54) but prevalent in both sexes of S. nobilis and S. nemoralis (Zhu et al. 2012;Ehrmann and Borer 2015;Schwarz et al. 2018).Jeon et al. (1999) initially reported S. nemoralis in Korea based on two female specimens lacking black spots on the furcasternite.Differences in the male genitalia, specifically the margins of the sdpl and sdpm, are reliable for distinguishing between S. maculata and S. nobilis (Schwarz et al. 2018).On the other hand, the lack of a black spot on the furcasternite is a common feature of the green morph of S. maculata.The occurrence of the Philippine Statilia nemoralis is continental SE Asia needs confirmation.Subsequent examinations revealed that the specimens initially identified by Jeon et al. (1999) as S. nemoralis were, in fact, misidentifications of S. maculata.

Measurements
Biological notes.Tenodera angustipennis occurs throughout the Korean peninsula and both adults and oothecae can be commonly found on trees and shrubs.First instar nymphs hatch from late May to mid-June, and adults emerge in mid-August.
Distribution.China, India, Japan, Java, South Korea.Invasive in NE USA and Hawaii.
Remarks.Tenodera angustipennis is morphologically similar to T. sinensis in Korea but can be distinguished by their more slender bodies, the pronotum length/width ratio, the orange coloration between the forecoxa base (Oshima 2018) (Fig. 81), lack of reddish coloration on the hindwing radius area , and the pointed, perpendicular apex of the aafa (Fig. 86).
Biological notes.Tenodera sinensis occurs throughout the Korean peninsula and has adapted well to urban, suburban, and riverside environments.It spawns ootheca in various locations, such as on stones, tree trunks, and branches.First instar nymphs hatch from mid-April to mid-May, while adult mantises typically emerge in mid-August.
Distribution.China, Nepal, Japan, Russia, Thailand, South Korea.Invasive in Canada and the USA.Remarks.Tenodera sinensis was originally described as a variation of the widely distributed species Tenodera aridifolia (Stoll, 1813), and treated as a subspecies for quite a long time (Saussure 1871b;Rehn 1903;Shiraki 1932;Beier 1932;Tinkham 1937;Bazyluk 1977;Iwasaki 1996;Bruins 1999), but already Giglio-Tos (1927) and Ehrmann (2002) considered T. sinensis as a valid species.Tenodera aridifolia and T. sinensis are extremely close and morphologically very similar (Ehrmann and Borer 2015), but Jensen et al. (2010) provide differences in male genitalia of T. sinensis and T. aridifolia.According to Jensen et al. (2010), it is a distinctly divided sister species pair based on phylogeny using nuclear genes (histone III, wingless gene) and mitochondrial genes (large and small rRNA, cytochrom oxidase II).Tenodera aridifolia is found in tropical and subtropical regions but is replaced by T. sinensis in temperate habitats.Our study used one male West Javan T. aridifolia specimen to comparatively examine and recovered remarkable characters from T. sinensis including the narrow pronotum (length/maximum width = 5.1) and forewing, a smaller head proportional to body size, short and stout aafa, pafa, and sdpl curved at ~ 45° (Mukherjee et al. 1995;Jensen et al. 2010).
Description.See Shim et al. (2021a) for detailed diagnosis and description.Biological notes.Hierodula chinensis is sparsely distributed in the Korean peninsula, primarily inhibiting wooded areas with shrubs and tall trees.It typically lives under tree leaves throughout its lifespan and deposits its oothecae on tree branches.Nymphs have the ability to camouflage themselves by folding their abdomens backwards.The first instar nymphs of H. chinensis hatch in early July, with adult mantises typically emerging at the end of August.
Distribution.China.Invasive in Japan and South Korea.
Remarks.Hierodula chinensis was recently reported in the Korean peninsula by Shim et al. (2021a).This species was previously recorded in China by Werner (1929) and Beier (1932) and was recently reported in Japan (Yamasaki et al. 2022).However, this species has been erroneously confused with H. membranacea Burmeister and H. macrodentata Wang, Zhou & Zhang, 2020 by some authors, including Tinkham (1937), Wang (1993), andZhu et al. (2012).Recent studies by Wang et al. (2020) and Liu et al. (2020) have provided a redescription of H. chinensis, clarifying its taxonomic status.See Shim et al. (2021a) for detailed description.
Remarks.Hierodula patellifera is a widely distributed species (Ehrmann and Borer 2015;Patel and Singh 2016;Shcherbakov and Anisyutkin 2018;Battiston et al. 2019;Moulin 2020).The species exhibits a high degree of morphological variation, which has led to the recognition of numerous synonyms (Audinet-Serville 1838; Wang 1993, Schwarz et al. 2018).Morphological variation, including differences in the number of forecoxal spines, and shapes of forewing stigma and afa, have been observed among H. patellifera populations, including those in Korea.Shim et al. (2021b) discuss the challenges

DNA barcoding of Korean Mantodea
In total, 56 new sequences from seven species in six genera were generated (657 bp of COI).All new sequences were deposited in GenBank under the accession numbers OQ826709-OQ826764 (Suppl.material 1).Table 1 and Fig. 137 present the p-distances of COI regions for specimens at each taxonomic level.
Intraspecific distances from eight species were either identical or very similar (0%-2.2%).The minimum interspecific genetic distance between congeners (6.7%) was ~ 3× higher than the maximum intraspecific genetic distance (2.2%), indicating a significant barcoding gap.All eight species were supported as a single lineage using COI on both NJ and PA trees, respectively (Fig. 138).

Discussion
This study presents the first comprehensive taxonomic review of the Mantodea species in Korea, recognizing eight species based on morphology and DNA barcodes.In contrast to previous studies that primarily focused on the documentation of unrecorded species, our study meticulously examined 494 specimens, encompassing all eight species that have been recorded in Korea.Notably, while the majority of species exhibit a broad distribution across the Korean peninsula , the genera Amantis and Acromantis are confined to the southern islands of Korea (Fig. 139).Furthermore, Hierodula chinensis, initially documented in Jeonbuk Province in 2021 (Shim et al. 2021a), has been found in more locations since (Fig. 142).The morphology of male genitalia is a crucial diagnostic feature for species identification and delineation in mantids, supporting the monophyly of high-er taxa (Schwarz and Roy 2019;Liu et al. 2021).However, it is important to note that misidentification at the species level may occur in some cases due to afa structural variations in male genitalia, which can occur intraspecifically in mantids, even under sympatric conditions (Svenson and Roy 2011;Svenson and Vollmer 2014;Shim et al. 2021b).For example, the Korean populations of Amantis nawai exhibit two morphotypes of afa structural variations at the intraspecific level, which could lead to confusion at the species level (Figs. 16,17).This situation makes it challenging to determine whether the observed variation in male genitalia represents a cryptic species or a morphological variation.However, the genetic divergence between the two morphotypes based on COI barcode data were not significantly different (0.3%, Table 1), and they were also supported as a single lineage (Fig. 138).This is consistent with our findings that the intraspecific genetic divergence ranges from 0% to 2.2%, while interspecific divergence among congeners ranges from 6.7% to 14.6% (Tables 1, 2; Fig. 137).Furthermore, Mantodea species are each formed as distinct lineages on NJ (Fig. 138) and PA trees (not shown).Consequently, we consider all of them as separate and valid species, given that they exhibit morphological differences as discussed above.
Although the diversity of Mantodea in Korea is relatively modest when compared to the high species diversity and endemism observed in neighboring China and Japan (Otte et al. 2024), this study substantially advances our understanding of mantodean diversity within Korea.To further elucidate the diversity of Mantodea across the Korean peninsula, it is imperative that future research initiatives prioritize intensified specimen collection efforts, particularly targeting areas that have been under-sampled to date.
Table 2. Inter-and intraspecific genetic differences among Korean Mantodea species for COI (657 bp) calculated using p-distance.

Figure 137 .
Figure 137.Intra-and interspecific uncorrected distances of partial COI gene sequences for each taxonomic level of Mantodea.

Figure 138 .
Figure 138.Neighbor-joining tree of Korean Mantodea species based on partial COI gene sequences with bootstrap values (left) and Parsimony analysis bootstrap values (right).Scale bar indicates the expected number of substitutions per site.0.02

Table 1 .
Inter-and intraspecific genetic differences in Korean Mantodea species at each taxonomic level for COI (657 bp) calculated using p-distance.