New record of Cyrtonotula Uvarov, 1939 (Blaberidae, Epilamprinae) from China, with three new species based on morphological and COI data

Abstract The genus Cyrtonotula Uvarov, 1939 (Blaberidae, Epilamprinae) is recorded for the first time from Hainan Island, China. Three new species, Cyrtonotula epunctata Wang & Wang, sp. nov., C. maculosa Wang & Wang, sp. nov., and C. longialata Wang & Wang, sp. nov., are described based on morphological data and a molecular analysis using Automatic Barcode Gap Discovery (ABGD). Additional barcode data of blaberid species, including these three new species, are provided to facilitate future species identification. Morphological photographs and habitat photos of these new species, as well as a key to the known species, are provided.


Introduction
The Epilamprinae genus Cyrtonota was proposed by Hanitsch (1929), with C. lata as type species, on the basis of one single female specimen from Sumatra. It was characterized by its comparatively large pronotum, hind metatarsus length barely equal to the succeeding joints combined, and the reduced tergum with nearly truncate apex. Owing that the name is preoccupied by a genus of spider (Simon 1864), a replacement name, Cyrtonotula, was proposed by Uvarov (1939).
Since then, no more species have been reported from this genus. Roth (2003) removed it from Epilamprinae and treated it as Blaberidae incerate sedis. Recently, Mavropulo et al. (2015) returned it back to Epilamprinae as a result of a phylogenetic analysis based on 28S and the male genital characters; this was verified by Djernaes et al. (2020) using four mitochondrial and three nuclear genes. Mavropulo et al. (2015) described two species from Indonesia and provided a description on the male genitalia of this genus for the first time. Later, a fourth species was described from the Philippines (Lucañas 2017). To date, there are four known Cyrtonotula species worldwide and none from China.
Species of Cyrtonotula are currently identified primarily on the basis of morphological characters, mainly the shortened tegmina and wings, the shape of the pronotum, and male genitalia. DNA barcoding had not been employed to explore the diversity of Cyrtonotula.
DNA barcodes (the standard COI sequence) have been proven to be a useful supplementary method in identifying cockroach species and have been effective in resolving problems, such as sexual dimorphism and the identification of nymphs (Che et al. 2017;Bai et al. 2018;Wang et al. 2018). In DNA barcoding studies of cockroaches, the Automatic Barcode Gap Discovery (ABGD) method of species delineation (Puillandre et al. 2012) is widely used and has proven effective in discerning cockroach species (Bai et al. 2018;Wang et al. 2019;Li et al. 2020). Here, Cyrtonotula is reported from China, and three new species are described, with the aid of DNA barcoding.

Morphological study
Type specimens are deposited in the Institute of Entomology, College of Plant Protection, Southwest University, Chongqing, China (SWU). Male genital segments were processed with 10% NaOH for maceration of the soft tissues, observed in glycerol with a Motic K400 stereomicroscope or a Leica M205A stereomicroscope, and preserved with the remainder of the specimen in ethyl alcohol. Photographs were taken with a Leica DFC digital microscope camera attached to a Leica M205A stereomicroscope. All photos and images were processed with Adobe Photoshop CS6. Species descriptions are based on the holotype male. Measurements are given according to the whole sample studied for the description. Sclerites in male genitalia are named according to Klass (1997). The terminology of venation follows Li et al. (2018). Vein abbreviations in this article are as follows:

DNA extraction, amplification, and sequencing
We used standard methods to sample cytochrome c oxidase subunit I (COI) of four species (Table 1) as follows. Total DNA was extracted using Hipure Tissue DNA Mini Kit from the hind legs of alcohol-preserved specimens according to the standard DNA barcoding methods for the cockroach. The mitochondrial COI gene was amplified by PCR using primer sets of COI-F3 (5'-CAACYAATCATAAAGANATTGGAAC-3') and COI-R3 (5'-TAAACTTCTGGRTGACCAAARAATCA-3') resulting in a fragment length of 658 bp for genetic analysis after trimming the primers from the amplified product. The amplification reaction was performed in a total volume of 25 µL, including 23 µL T3 DNA polymerase, 1 µL of each primer and 1 µL DNA template.
The thermal cycling conditions were as follows: initial denaturation of 2 min at 98 °C followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 53 °C for 10 s, extension at 72 °C for 10 s, and a final extension at 72 °C for 5 min; the samples were then held at 8 °C. The amplified samples were evaluated in 1% agarose gels. Sequencing in both directions was performed by BGI Technology Solutions Co. Ltd (BGI-Tech) (Beijing, China).

Sequence processing and phylogenetic analyses
A total of 11 mitochondrial COI sequences were obtained from four Epilamprinae species, plus one Cyrtonotula sequence, another seven Epilamprinae sequences, and one sequence representing the mantis outgroup were downloaded from NCBI for phylogenetic analyses (Table 1). Sequences were aligned in online MAFFT 7 (https:// mafft.cbrc.jp/alignment/server/) using the Q-INS-i algorithm. The alignment was then manually corrected in MEGA 7 (Kumar et al. 2007). Intraspecific and interspecific genetic distances are quantified based on the Kimura 2-parameter (K2P) distance model (Kimura 1980) in MEGA 7. The neighbor joining (NJ) tree was constructed in MEGA 7 under the Kimura 2 parameter model (K2P). Statistical support was estimated with 1000 bootstrap replicates. To determine putative species in our study, we used the species delimitation approach, Automatic Barcoding Gap Discovery (ABGD), which was performed using the online webserver (http://wwwabi.snv.jussieu.fr/public/ abgd/). The settings were as follows: Pmin = 0.001, Pmax = 0.1, Steps = 10, X (relative gap width) = 1.0, Nb bins = 20, and using Jukes Cantor (JC69) distance.

Species delimitation based on COI and morphological data
In this study, we acquired 11 COI sequences representing three Cyrtonotula and one Opisthoplatia species. All new sequences were deposited in GenBank (accession numbers MW649972 to MW649982 in Table 1). An NJ analysis revealed that clades from the same species, including male and female samples, constituted monophyletic groups with high support values ( Fig. 1). We observed the lowest and highest K2P interspecies genetic distance among these species, 0.1056 for C. maculosa sp. nov. and C. longialata sp. nov., and 0.1367 for C. epunctata sp. nov. and C. maculosa sp. nov. We used the ABGD method to delimit Cyrtonotula species. Three MOTUs were detected in the ABGD analysis, which are completely consistent with the results based on morphological characters ( Fig. 1) for C. epunctata sp. nov., C. maculosa sp. nov., and C. longialata sp. nov.

Cyrtonotula Uvarov, 1939
Cyrtonota Diagnosis. Medium-sized cockroaches. Both sexes similar. Ocular distance slightly narrower than the distance between antennal sockets, greater than ocellar distance. Pronotum broad, anterior margin curved and posterior margin obtusely produced. Tegmina and wings usually brachypterous, not reaching the abdominal apex (except for macropterous C. longialata sp. nov.), their apices somewhat rounded or approximately truncated. Anteroventral margin of front femur Type B; tarsi moderately long; hind metatarsus slender, distinctly longer or nearly equal to the remaining segments combined, armed with two or less equal rows of spines and large apical pulvilli; succeeding tarsomeres armed only with spines surrounding the large pulvilli; the pretarsus with arolium, claws symmetrical and unspecialized. Supraanal plate entire, with a median incision. Cerci elongate. Subgenital plate large, nearly symmetrical or somewhat asymmetrical. Styli cylindrical. Male genitalia. Right phallomere Morphnini-type (Anisyutkin and Yushkova 2017): consisting of sclerites R1T, R2, R3, R4, and R5; R4 irregular plate-like, separated; R3 connected to R5. The shape of apical sclerite of L2D irregular and variable. Sclerite L3 hook apically blunt; the folded structure distinct with bristles (visible at high magnification), sclerite L4U present.
The genus Cyrtonotula differs from Rhabdoblatta, Pseudophoraspis, and Stictolampra principally by its reduction of the tegmina and wings. Additionally, C. longialata sp. nov. is morphologically somewhat similar to some Rhabdoblatta and Stictolampra species but can be distinguished by the presence of glandular specialization on the abdominal tergites, basal portion of sclerite L2D, and the non-punctate pronotum.
The genus Cyrtonotula can be distinguished from Morphna by the structure of hind tarsi: metatarsus distinctly longer or about as long as other segments combined, with relatively numerous tarsal spines (metatarsus slightly shorter or nearly equal to remaining segments combined with larger pulvilli, tarsal spines few or absent). Differential diagnosis. The new species readily differs from all its congeners in the spination of hind tarsi. Cyrtonotula epunctata sp. nov. resembles C. lata Hanitsch, 1929 in testaceous body color and the length of hind metatarsus, but the new species can be distinguished from C. lata by the following characters: the coloration of facial part black, with clypeo-labral area yellowish brown, and vertex without visible lines (vs deep testaceous and vertex with three longitudinal dark lines in C. lata), and tegmina only reaching to the posterior margin of the third abdominal segment (vs reaching over the sixth abdominal tergite in C. lata).
Vertex concealed. Interocular space same as the width between the antennal sockets, slightly greater than ocellar distance. Pronotum nearly semicircular, anterior margin parabolic, posterior margin obtusely angled (Fig. 2E). Tegmina reduced, reaching up the 4 th abdominal tergite only; apex rounded; venation distinct, all main veins (Sc, R, and CuP) present, Sc thickened (easily visible on ventral side of tegmen) ( Fig. 2A,  G). Wings vestigial, only reaching to the posterior margin of the 3 rd abdominal segment, completely covered by tegmina. Front femur Type B 2 (Fig. 2F). Hind metatarsus depressed-cylindrical, nearly equal to the succeeding segments combined, with single complete row of spines along ventral margin and several additional spines on inner side; four proximal tarsomeres with pulvilli terminal, the one on the second tarsomere occupying practically the whole length of the segment; claws symmetrical and simple; arolium present (Fig. 5A). Abdominal tergites unspecialized; knobs along the posterior margin indistinct; weak spiracle-bearing outgrowths of tergite VIII with distinct spiracle. Supra-anal plate with the posterior margin widely rounded and a weak mesal incision. Cerci distinctly segmented, densely covered with bristles. Paraprocts of blaberid type, asymmetrical (Fig. 2H). Subgenital plate rounded, slightly asymmetrical; the base of the inner plate bifurcated. Styli cylindrical, apically rounded (Fig. 2I).
Male genitalia. Right phallomere with caudal part of sclerite R1T rectangular in shape; cranial part of R1T more or less straight; R2 curved; R3 long; R4 irregular platelike; R5 large, fused with sclerite R3 in caudal part (Fig. 2J). "chaetae-bearing membranous area" absent. Sclerite L2D not divided into basal and apical parts, slender and rod-like, with basal end tapering and a bifurcated outgrowth born near the basal end (Fig. 2K). Sclerite L3 hooked, apex slightly rounded, with a small tooth on the inner margin less distinct; folded structure present, with bristles. Sclerite L4U distinct (Fig. 2L). Differential diagnosis. This new species is closely related to C. tertia Mavropulo, Anisyutkin, Zagoskin, Zagoskina, Lukyantsev & Mukha, 2015 in the shape of tegmina and body color, but the former can be distinguished from the latter by the specialized abdominal terga (vs unspecialized) and the shape of sclerite L3 of male genitalia, in which L3 hook is comparatively robust and posteriorly truncate distinctly (vs comparatively slender and rounded apically in C. tertia).
Female. Similar to the male. Body color lighter. Tegmina only reaching the second abdominal tergite, with apex distinctly truncated. Abdominal tergites unspecialized.
Etymology. Derived from the Latin word maculosus, referring to the scattered with dense spots pronotum and tegmen. Differential diagnosis. The new species principally differs from all its congeners, except for C. maculosa sp. nov., in the presence of abdominal tergal glands. From C. maculosa sp. nov., C. longialata sp. nov. differs in having the completely developed tegmina and wings extending beyond the abdominal apex, the shape of tergal glands (see description below).
Female. Similar to the male. Abdominal tergites unspecialized. Remarks. Currently, this is the only species of Cyrtonotula with fully developed tegmina and wings. This species is placed in Cyrtonotula because it closely resembles C. maculosa sp. nov. in having sclerite L3 hooked (apex nearly truncate and inner margin with a distinct point) and in the location of tergal gland.
Etymology. The species epithet is derived from the Latin adjective longialatus, which refers to the well-developed wings.

Discussion
Flightless cockroaches are usually considered to persist in stable habitats, where food, shelter, and mates are easily accessible (Bell et al. 2007). All of previously known Cyrtonotula species are brachypterous, but C. longialata sp. nov. is noteworthy for being the first macropterous in this genus. Brachypterous cockroaches, including C. epunctata sp. nov. and C. maculosa sp. nov., were observed on leaf litter and scree in areas with trickling water ( Fig. 7A-C), while C. longialata sp. nov., maybe a canopy species, was collected in low vegetation (Fig. 7D). We speculate that habitats of C. epunctata sp. nov. and C. maculosa sp. nov. may be different from the habitat of C. longialata sp. nov., and habitat might be one of the determining factors in variations of the wings. This study is also the first to use COI DNA barcode to evaluate diversity of Cyrtonotula species. Our results show that DNA-based species delimitation methods perform well and that individuals were correctly assigned to their corresponding species, although only 10 sequences of Cyrtonotula were included here. Therefore, taking into consideration that there are only seven species of the genus found worldwide, we expect more species of Cyrtonotula, especially macropterous ones, will be discovered and observed with further sampling, so that the knowledge of this genus Cyrtonotula could be comprehensive and explored more deeply.