A new genus and a new species in the subfamily Polyzosteriinae (Blattodea, Blattidae) from China

Abstract Laevifaciesquadrialatagen. et sp. nov. is described from Hainan Province, China based on morphological data. COI data (DNA barcodes) is utilized to confirm the sexual dimorphism occurring in Laevifaciesquadrialatagen. et sp. nov.Melanozosterianitida Brunner von Wattenwyl, 1865, is reported from Guangxi Province, China. A key to the Chinese Polyzosteriinae is provided.

The genus Melanozosteria was established with Polyzosteria nitida Brunner von Wattenwyl, 1865 as type species (Stål 1874). After that, Mackerras (1967b) did not agree with Melanozosteria as a synonym of Platyzosteria owing to the misidentification of one species of Platyzosteria by Shelford (1909), and placed Melanozosteria and Leptozosteria in Platyzosteria as subgenera. Roth (2003) Walker, 1868. Then Shelford (1909 proposed that Periplaneta polita is a synonym of Cutilia nitida Brunner von Wattenwyl, 1865. Until now, they have both been considered synonyms of Melanozosteria nitida. In the catalogue, Princis (1966) recorded Melanozosteria nitida from Taiwan, China, but he questioned its distribution on Mainland China. The other species, Melanozosteria soror, is mainly distributed in Australia and the Pacific Islands. Walker (1868) firstly recorded this species from Taiwan, China (it was originally described as Periplaneta philpotti, but later synonymized under Melanozosteria soror in Princis (1957)). Then Shiraki (1931) recorded this species from Hainan, but no further information was provided.
DNA barcodes have been proven to be a helpful method to identify species and to successfully match male and female. Barcoding has been applied to resolve the problems of sexual dimorphism and even to identify nymphs in cockroaches (Evangelista et al. 2013;Qiu et al. 2017;Che et al. 2017;Wang et al. 2018). To date, members of the Polyzosteriinae have been identified primarily on the basis of morphological characters (Mackerras 1965a(Mackerras , 1965b(Mackerras , 1965c(Mackerras , 1966a(Mackerras , 1966b(Mackerras , 1967a(Mackerras , 1967b(Mackerras , 1968a(Mackerras , 1968bRentz 2014) and DNA Barcoding has not been employed to investigate the diversity of Polyzosteriinae. In this paper, Laevifacies quadrialata gen. et sp. nov. is described from China and the sexual dimorphism is revealed via DNA barcoding. We also record a specimen from Guangxi, thus proving that Melanozosteria nitida is also distributed in Mainland China. A key to the known Polyzosteriinae species from China is provided.

Morphological study
Morphological terminology used in this paper mainly follows McKittrick (1964), Mackerras (1965a) and Roth (2003). Measurements are based on specimens examined. Genital segments of the examined specimens were macerated in 10% NaOH for 20 minutes and rinsed with distilled water, observed in glycerin jelly using a MOTIC K400 stereomicroscope. Photographs of the specimens were taken using a Canon® 50D plus a Canon® EF 100mm f/2.8L IS USM Macro lens combined with Helicon Focus® software. Photos of other characters were taken using a Leica® M205A stereomicroscope. All photographs mentioned above were modified in Adobe Photoshop® CS6. The type materials are deposited in the Institute of Entomology, College of Plant Protection, Southwest University, Chongqing, China.

DNA extraction, PCR, and sequencing
We used two cockroach specimens for COI sequencing in this study in order to resolve the sexual dimorphism. Both sequences are deposited in GenBank with the accession numbers: MK798103, MK798104 (Table 1). The extraction procedure was according to the Hipure Tissue DNA Mini Kit (Magen Biotech, Guangzhou). Fragments of COI were amplified using PCR. Primers for the amplifications are LCO1490 (5'-GGTCAACAAATCATAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer 1994). The amplification conditions were: initial denaturation at 94 °C for 3 min, followed by 35 cycles for 30 s at 94 °C, 30 s at 49 °C, and 1 min at 72 °C, with a final extension of 10 min at 72 °C.

Sequence processing and phylogenetic analyses
A total of ten COI sequences were analyzed (two sequences of Laevifacies species from our study, six sequences of Blattidae, and two sequences of a mantid outgroup downloaded from GenBank) ( Table 1). All COI sequences were aligned using MEGA 7.0 and adjusted visually after translation into amino acid sequences. Finally, for the phylogenetic analysis we acquired COI sequences whose lengths were 658 bp, except for Angustonicus lifou whose sequence was only 650 bp. The genetic divergence value was quantified based on the Kimura 2-parameter (K2P) distance model (Kimura 1980), using MEGA 7 (Kumar et al. 2016) with 1000 bootstrap replicates. Maximum Likelihood (ML) analysis was implemented in RAxML 7.3.0 (Stamatakis et al. 2008) using GTRGAMMA model with 1000 bootstrap replicates.

Phylogenetic analysis based on COI
In this study, we acquired two COI sequences, whose length, excluding primers, was 658 bp each. The genetic divergence value between male and female of Laevifacies quadrialata sp. nov. is 0.9%; however, the interspecific K2P genetic divergence among Laevifacies quadrialata sp. nov. and other species ranged from 10.4 to 13.1%.
The ML phylogenetic tree ( Figure 1) revealed that male and female of Laevifacies quadrialata sp. nov. grouped together with a high support value (MLB = 100).

Subfamily Polyzosteriinae Tepper, 1893
Polyzosteriinae Tepper, 1893: 32;Princis 1950: 170;Princis 1960: 447;Princis 1966: 561;McKittrick 1964: 66;Mackerras 1965a: 841;Rentz 2014: 121. Generic diagnosis. Body small to medium, thinner in male, thorax slightly broader than abdomen. Surface smooth and shining. Pronotum slightly semicircular, vertex barely exposed. Male with vestigial tegmina and hind wings on mesonotum and metanotum respectively, both nearly triangular; female only with vestigial tegmina, its shape similar to that of male, without hind wings. Legs strong but short, coxae with  punctation, front femora Type A 2 . Mid and hind metatarsus with strong spines, claws symmetrical. Cerci strong, short and symmetrical. Styli long and symmetrical. Supraanal plate in male short, triangular; subgenital plate broad and short, slightly quadrilateral and symmetrical. L1 divided into two parts, L3 bifurcated, one branch short, the other one long, R1 nearly claw-like and R2 large, hooked. Etymology. The name Laevifacies is derived from two Latin words laevis and facies, referring to the smooth and shining surface of terga. The gender of Laevifacies is feminine.

Key to Species of Polyzosteriinae in China
Remarks. Based on former studies (Gutiérrez 2013(Gutiérrez , 2014Mackerras 1965aMackerras , 1965bMackerras , 1965cMackerras , 1966aMackerras , 1966bMackerras , 1967aMackerras , 1967bMackerras , 1968aMackerras , 1968bRehn and Hebard 1927), the Polyzosteriinae is characterized as follows: species having semicircular pronotum, lobiform vestigial tegmina, angles of T2-T7 produced, tarsi usually short, bare or with hind and sometimes mid metatarsi spiny (Laevifacies with mid and hind metatarsi spiny, while in Mackerras (1968b), Australian species of Blattinae and Polyzosteriinae from other Blattidae with all metatarsi spiny), large pulvilli and arolia, cerci strong, short and symmetrical, L1 with hollow finger-like projection and sclerotized projection and R1 claw-like and margin with projection; thus, Laevifacies is placed in the subfamily Polyzosteriinae. Laevifacies has common features with Melanozosteria, Eurycotis, Leptozosteria, and Platyzosteria, such as body small to large, and shining, usually with vestigial tegmina, angles of T5-T6 acute, T6-T7 with punctation and hind metatarsus usually spiny (Gutiérrez 2013(Gutiérrez , 2014Mackerras 1965cMackerras , 1968b. Laevifacies is similar to the Melanozosteria and Eurycotis in general appearance, but it can be distinguished from Melanozosteria by the following characters: 1) body thin and small in male (Figure 2A, B), while in Melanozosteria, it is broad and large ( Figure 4A, B, F, G); 2) the surface of terga smooth (Figure 2A Figure 4K, M); and it can be distinguished from Eurycotis by the following characters: 1) tibiae not specialized, while in Eurycotis, one group of which species have smooth surface, uniform black body and lateral tegmina, with highly specialized caudal tibiae; 2) R2 is hook-like, while in Eurycotis R2 is pincer-like. In addition, Eurycotis is restricted to South and North America and Cuba, while Laevifacies gen. nov. is found in East Asia. Laevifacies is similar to the Methana in the following genitalia characteristics, the margin of L2d smooth, R1 as a strongly claw-like sclerotized process, both of L1 have two structures, L1 of Methana has strong finger-like sclerotization and a membranous lobe, while Laevifacies has a finger-like membrane and a strongly sclerotized lobe ( Figure 3A, B).
Size small to medium, female larger than male. Body oval, vertex nearly unexposed (Figure 2A, B, G, H). Ocelli present, small and round ( Figure 2D). Pronotum nearly semicircular, anterior margin arc-shaped, posterior margin nearly straight, posterior angles blunt ( Figure 2C). Small, vestigial tegmina and hind wings present in male, both extending to notal hind margin, only vestigial tegmina in female (Figure 2A, G); angles of T2-T7 sharp and protruded, sterna smooth and shining (Figure 2A, B, G, H). Legs strong, fore coxae with punctation; front femora Type A 2 ( Figure 2E); mid and hind metatarsus with a row of spines; hind metatarsus fairly long with pulvillus which occupies nearly one-quarter of its length, remainder of surface with hair, claws moderately symmetrical and unspecialized ( Figure 2E, F, I, K). Male: supra-anal plate short, triangular, divided into two round lobes ( Figure 3E); subgenital plate broad and short, posterior margin round; styli long and symmetrical ( Figure 3D). Cerci symmetrical and strong, with indistinct segmentation, ends sharp ( Figure 3E). Female: supra-anal plate with higher sclerotization ( Figure 3G).
Male genitalia. Left phallomere consisting of three parts: L1, L2, and L3. L1 with two parts L1a and L1b, L1a with membranous finger-like projection; L1b with sclerotized projection. L2 consisting of L2d and L2v, L2d strongly sclerotized in anterior part, the posterior part with finger-like and with more small spines; L2va simple and broad, L2vb sclerotized and the posterior with a spinous projection. L3 with a simple hook, elongate to the right and bifurcated ( Figure 3A, B). Right phallomere consisting of R1, R2, and R3. R1 large, claw-like, right margin with a prominent spine; R2 large, curved hook-like, the base strong and gradually becoming thinner, bent to the right; R3 large and cucullate, highly sclerotized ( Figure 3C). Etymology. The species epithet comes from the Latin word quadrialata in reference to the male having four triangular vestigial wings.
Remarks. In our study the interspecific K2P genetic divergence among L. quadrialata sp. nov. and other cockroach species ranged from 10.4 to 13.1%. But the genetic divergence value between male and female of L. quadrialata sp. nov. is only 0.9%, so we pair them based on their similar morphology combined with this COI data. Sexual dimorphism occurs in L. quadrialata sp. nov.: 1) females without hind wings, but males with vestigial hind wings (Figure 2A, 2G); 2) male with narrower body, while female with broader body (Figure 2A, B, G, H).
Remarks. We compared the lectotype of M. nitida (from Ternate, Indonesia) with the specimen from Guangxi and found there are minor differences between them: the styli are straight in the Guangxi individual ( Figure 4I), but in the lectotype of M. nitida, slightly bent ( Figure 4F, G). We also compared the genitalia between the Guangxi individual and the illustration in Mackerras (1968a); they share the typical characters of L1b spinous projection and serration along the margin of L2d, but they are also different in the following characteristics: 1) the terminal of L3 divided into two small forks, which resemble an elephant's nose in the Guangxi individual ( Figure  4J), while in the Mackerras (1968a) individual, L3 has one blunt hook ( Figure 4M); 2) L2v broad and sclerotized, and posterior of L3 membranous in the Guangxi individual ( Figure 4J), while L2v thin, long and with sharp sclerotized terminus in the Mackerras (1968a) individual ( Figure 4M). And the variation of supra-anal plate between samples from Queensland and New Guinea were treated as intraspecific differences in different locations (Mackerras 1968a). Considering Mackerras (1968a) also recorded that the M. nitida is a widely distributed tropical species from Taiwan, Malaya, Moluccas, and Philippines, and due to our specimens being inadequate, the minor difference in the Guangxi individual and the lectotype of M. nitida are temporarily considered as the intraspecific differences of different populations.
Geographical distribution. Australia, Philippines, Malaysia, New Guinea, New Caledonia, New Zealand, China, Thailand.

Discussion
Almost all members in the Polyzosteriinae are brachypterous or apterous (excepting the tribe Methanini), and display high developmental stochasticity (Rentz 2014). The Australian Polyzosteriinae exhibit the best examples of aposematic coloration. They are often being metallically colored, or spotted and barred with bright orange, red, or yellow markings (Rentz 1996;Roach and Rentz 1998). When disturbed, they may first display a warning signal before resorting to defensive measures (Bell et al. 2007). However, Laevifacies quadrialata sp. nov. did not attract our attention due to their bland appearance and life in a hidden habitat (usually hidden in bushes, Lu Qiu, pers. obs.), even with sexual dimorphism. Sexual dimorphism is very common in cockroaches, some of which beinng so extreme that it is a challenge for taxonomists to match the two sexes (Roth 1992). In this study, sexual dimorphism is revealed for the first time in Polyzosteriinae on the basis of COI data, and exhibits mainly in the body size and the vestigial hind wings.