﻿Redescription of Periplanetaarabica (Bey-Bienko, 1938) (Blattodea, Blattidae), with a comparative analysis of three species of Periplaneta Burmeister, 1838 (sensu stricto)

﻿Abstract The blattid cockroach Periplanetaarabica (Bey-Bienko, 1938) has been poorly understood since its original description. In this study, male and female (including nymph) of P.arabica are paired using DNA barcoding, and their morphological characters (including both external characteristics and genitalia) are described. A detailed comparative morphological study of this species and the closely related Periplanetaamericana (Linnaeus, 1758) and Periplanetalateralis Walker, 1868 was carried out to explore phylogenetically relevant characters.


Introduction
According to Beccaloni (2014), the Blattinae genus Shelfordella Adelung, 1910 comprised three species before it was synonymized with Periplaneta (Deng et al. 2023). The taxonomic status of Shelfordella remains unclear even though several revisions were carried out by Princis (1954) and Bohn (1985) based on the external morphological characters. In addition, many molecular phylogenetic studies (Legendre et al. 2015;Bourguignon et al. 2018;Arab et al. 2020;Liao et al. 2021;Djernaes and Murienne 2022;Li et al. 2022;Deng et al. 2023) have shown that Periplaneta americana (Linnaeus, 1758), the type species of Periplaneta Burmeister, 1838, is the sister species to Sh. lateralis (Walker, 1868). Considering both molecular data and morphological characters of male genitalia of P. americana and Sh. lateralis, Shelfordella was considered as a synonym of Periplaneta (Deng et al. 2023), resulting in the restoration of Periplaneta lateralis Walker, 1868 and Periplaneta monochroma Walker, 1871, and the transference of Shelfordella arabica Bey-Bienko, 1938 to Periplaneta. Periplaneta arabica was originally described with a female specimen as its type, and the male has not been described.
DNA barcoding has been confirmed to be a helpful tool in discovery of new species, matching nymphs with adults, and revealing sexual dimorphism and cryptic species in cockroaches (Evangelista et al. 2013;Che et al. 2017;Yang et al. 2019;Li et al. 2022;Zhu et al. 2022). Herein, we use DNA barcoding to pair male, female and nymphs of P. arabica, allowing a comprehensive redescription of this species. We also take the opportunity to compare the morphological characters of P. arabica, P. americana and P. lateralis, to show the structural complexity and diversity of species of Periplaneta s.s., as well as to provide detailed information useful for future phylogenetic studies on the genus.

Morphological study
Specimens (stored in absolute ethanol at -20 °C) examined are deposited in the Institute of Entomology, College of Plant Protection, Southwest University, Chongqing, China (SWU). Abdominal segments were soaked in 10% NaOH solution at 70 °C for 10 minutes. They were cleaned in distilled water, dissected in glycerol under a Motic K400 stereomicroscope, then stored in glycerol. Photographs were taken using a Canon M5 plus a Laowa 65 mm F2.8 CA-Dreamer Macro 2X Macro lens attached to a Leica M205A stereomicroscope. All figures were modified in Adobe Photoshop CC 2019. The morphological terminology used in this paper mainly follows Roth (2003). The terminology of veins follows Li et al. (2018), and those of the sclerites of male and female genitalia mainly follows Klass (1997) and McKittrick (1964) sclerites of the right phallomere

Molecular analysis
In this study, we used six COI sequences of P. arabica, five COI sequences of P. americana and six COI sequences of P. lateralis. All new sequences were deposited in GenBank with accession numbers OP727638 to OP727652. Intraspecific COI genetic divergence (K2P) of P. arabica and P. lateralis is 0%, but for P. americana, the intraspecific COI genetic divergence ranged from 0.00% to 2.30%. Interspecific COI genetic divergence ranged from 9.9% (P. arabica and P. americana) to 13.1% (P. americana and P. lateralis). In our ML analyses, samples including adults and nymphs from the same morphospecies are clustered together with high support values ( Fig. 1). Periplaneta arabica was recovered as the sister to P. americana on the basis of COI data but with a rather low support (bootstrap support (BS) = 79). These three species (i.e., P. arabica, P. lateralis and P. americana) formed a monophyletic group with Blatta orientalis as the sister (BS = 79 and 60, respectively). Diagnosis. Combining the following characteristics, this species is easily distinguished from its congeners: 1) interocular space slightly wider than the interocellar space and less than interantennal space in male, interocular space wider than interantennal space in female; 2) tegmina of female reduced and nearly square; 3) legs slender, pulvilli and arolia absent; 4) hind margin not extending outward and slightly concave in the middle, forming an obtuse angle in supra-anal plate of male; 5) caudal part of L2 with a well sclerotized spine; 6) hlap weakly developed, but larger than that of the other two species; 7) distal part of R1H with two long spines and no serration.
Coloration. Body brown or reddish brown, eyes black, ocelli white; tegmina and wings yellowish brown.
Male (Fig. 2). Head and thorax. Vertex exposed. Interocular space slightly wider than the interocellar space, less than interantennal space. Antenna longer than the body (Fig. 2C). Pronotum subelliptical, with surface sparsely pubescent, the central part of anterior margin depressed, and hind margin slightly convex, the widest point approximately in the middle (Fig. 2D). Tegmina and wings well developed, exceeding the end of abdomen by about 5.3-7.7 mm. Tegmina with ScP strong, the distal part fusing with anterior branches of R; anterior branches of R with 2-4 bifurcations, posterior branches reaching the outer margin; the base of M distinct with 2-4 bifurcations; CuA slender with a few branches; V indistinct (Fig. 2J). Wings with ScP slender, the distal part of RA indistinct, RP slightly strong and distinct; M with 2-3 bifurcations at the end; CuA strong; V distinct (Fig. 2K). Legs (Fig. 2E-I) slender. Front femur type A 2 (Fig. 2E). Hind metatarsus longer than the remaining segments combined (Fig. 2H). Pulvilli and arolia reduced; claws symmetrical (Fig. 2I). Abdomen. First tergite unspecialized. Supra-anal plate transversely broad, the lateral margins curved, and the hind margin slightly concave in the middle; the distal part less sclerotized and hyaline (Fig.  2L). Paraprocts (pp.) long strip-shaped and symmetrical. Cerci long, apically tapering. Subgenital plate nearly square, the hind margin slightly convex (Fig. 2M). Styli long, slender. Genitalia (Fig. 2N, O). L1 weakly sclerotized with pubescence. L4C with microspines on the lateral margin; the distal part expanded, hind margin nearly truncated. L2 curved and extended to left, the caudal part with a long spine toward right. L4D small (Fig. 2O). L4E flat. L3 unciform and well sclerotized; the base wide, downwardly tapering; the distal part bifurcated, hlap weakly developed. L4G elliptic with the basal part constricted. R1H flaky, with two long spines at the apex. The basal part of R1G broad, the distal with a long and curved narrow process toward right. R1F irregular and its outer margin thickened. R2 with a ridge-like projection in dorsal view. R3 located at the upper right, triangular and weakly sclerotized. Female (Fig. 3). Head and thorax. Interocular space wider than interantennal space (Fig. 3B). Pronotum campaniform; anterior margin straight and hind margin convex, the widest point after the middle (Fig. 3A). Tegmina square, reduced and not reaching the first tergite of abdomen; lateral margins of tegmina truncated, forming nearly right angle with the anterior margin; R parallel to the anterior margin (Fig. 3I). Hind wings small lobe-like (Fig. 3J). Abdomen (Fig. 3K, L). Hind margin of tergum X (TX) with median invagination, and with a membranous line inside. Paraprocts (pp.) wide, nearly triangular. Subgenital plate divided; median with intersternal fold (inst.f.). Genitalia (Fig. 3K, L). First valve (v.I) sclerotized with dense punctures; the distal part hyaline, and the base fused with first valvifer (vlf.I). First valvifer short, parallel to paratergites (pt.) and laterosternite IX (ltst.IX). Paratergites slender and laterosternite IX irregular. Valvifer II (p.l.) annular. Second valve (v.II) small and flaky, the base fused, connecting with third valve (v.III) by membrane. Third valve (v.III) large and less sclerotized. Anterior arch (a.a.) wide and its central part with two symmetrical foot-shaped projections, surface with microtrichia. Spermathecal plate (sp.pl) well sclerotized and nearly crescent-shaped. Spermathecal opening (sp.o.) located at anterior margin of spermathecal plate. Spermatheca (sp.) divided into two branches, one branch with the distal part enlarged. Basivalvulae (bsv.) subelliptical with punctures. Postero-lateral angle of laterosternal shelf (ltst.sh.) extended towards outer margin. Vestibular sclerite (vst.s.) strip-shaped.
Nymph. Early instars are yellowish brown with ocelli and eyes small; in older nymphs, the body turns brown or reddish brown (Fig. 3E-H).

Distribution. Saudi Arabia (Mecca); Yemen; United Arab Emirates; Oman; Iran (Ilam Province; new country record).
Remarks. Bey-Bienko (1938) first documented and described this species based on a female specimen from Mecca, Saudi Arabia. Bohn (2007) provided some morphological characteristics of the male in the key to genera and species occurring in the United Arab Emirates. After checking the original description by Bey-Bienko (1938) and Bohn (2007) and the images of the type specimens, we consider P. arabica to be characterized by: 1) interocular space slightly wider than the interocellar space in female; 2) pronotum anterior margin straight and hind margin convex in female; 3) tegmina nearly square in female; 4) hind margin slightly concave in the middle to form an obtuse angle in supra-anal plate of male; these characteristics are present in our specimens as well. Therefore, we concluded that our material collected from western Iran should belong to P. arabica. Matching of individuals of different sexes and life stages was possible with DNA barcoding.
Comparative morphology of P. americana, P. arabica and P. lateralis A detailed morphological comparison of P. americana, P. arabica and P. lateralis was performed in this study. The following intraspecific variations were found in all three species: 1) the number of veins branches of wings; 2) the marks on disc of pronotum in male and female of P. americana; and 3) the color of the pronotum and abdominal tergite of female of P. lateralis.

External morphological characters
The external morphological characteristics of P. americana, P. arabica and P. lateralis (Fig. 4) are compared in Table 2. Males of the three species have similar shapes of pronotum, wings, and supra-anal and subgenital plates, and lack tergite gland. Interocular space and interantennal space of females were both wider than the single eye spacing. The main differences among these three species are as follows: body size (i.e., P. americana > P. arabica > P. lateralis), tegmina and wings of females, and the presence or absence of arolia and pulvilli.

Genitalia of male and female
As depicted in Fig. 5, the genitalia of P. americana, P. arabica and P. lateralis are highly similar in appearance but differ in the degree of development of the sclerites. In males (see P. arabica for detailed description), the results ranked in descending order are as follows: P. lateralis > P. arabica > P. americana for the pubescence density in L1, P. arabica > P. americana > P. lateralis for the sclerotization degree of spine in L2, and P. arabica > P. lateralis > P. americana for the development degree of the hlap in L3. In addition, there are certain differences in other aspects, for example, the basal margin of L4C in P. americana and P. arabica bears a row of microspines that is absent in P. lateralis, and a row of serration at the margin of R1H is present in P. americana but absent in P. arabica and P. lateralis. In females (see P. arabica for detailed description), the degree of development of some sclerites (i.e., valvifer II, laterosternite IX, basivalvulae and laterosternal shelf ) is ranked as P. americana > P. arabica > P. lateralis. Periplaneta americana differs from P. lateralis and P. arabica in the following characters: hind margin of basivalvulae (bsv.) with two symmetrical protrusions in the former, which is lacking in the latter two; furthermore, the enlargement of spermathecae (sp.) in P. americana is longer and curved (the degree of curvature varies among samples), but usually irregular in P. arabica and subelliptical in P. lateralis.

Discussion
In recent years, molecular phylogenetic analyses have shown that P. americana has phylogenetic affinity with P. lateralis (Legendre et al. 2015;Bourguignon et al. 2018;Arab et al. 2020;Liao et al. 2021;Djernaes and Murienne 2022;Li et al. 2022;Deng et al. 2023), whereas P. australasiae+P. fuliginosa+P. brunnea would be the sister group to Homalosilpha (Liao et al. 2021;Djernaes and Murienne 2022;Deng et al. 2023). Deng et al. (2023) also included P. japonica and P. karnyi, neither of which clustered with P. americana. This inevitably raised doubts about the characteristics used in the past to distinguish Periplaneta and Shelfordella. Until recently, the development of tegmina and wings, pulvilli and arolia were usually considered the main diagnostic characters between these two genera (Adelung 1910;Bey-Bienko 1938;Bohn 1985). But, based on the phylogenetic results and some genital characteristics, Deng et al. (2023) considered Shelfordella as a synonym of Periplaneta. Considering the results of the current study, we also confirmed that P. americana differs significantly from P. arabica and P. lateralis in these characteristics. Our DNA-based analyses provided favorable evidence in the matching of females and males in all three species, as well as the pairing of adults and nymphs in P. arabica. Therefore, we had the possibility to compare males of these species and found that genitalia of both sexes of these three species were extremely similar, with differences in the developmental degree of sclerites. After a comparative morphological study on the genitalia of Blaberidae, Roth (1970Roth ( , 1972Roth ( , 1973 concluded that genital characters could be used as diagnostic characters for tribes, genera and groups. Until now, no such detailed genital comparison has been done in Blattidae, and our study might be helpful to solve the polyphyly of Periplaneta (Djernaes and Murienne 2022;Deng et al. 2023). In addition, considering the close relationship of P. americana and P. lateralis and the fact that both P. arabica and P. lateralis originated from West Asia (Beccaloni 2014), we speculate that P. americana might have originated in this region as well, and later dispersed naturally or was introduced by humans to other parts of the world, before gradually becoming a notorious indoor pest. Before the extensive usage of molecular data in cockroach systematics, most genera of Blattinae were established mainly on the basis of external morphological characters. As a matter of fact, the wings, pulvilli and arolia of cockroaches are heavily influenced by the environment and lifestyle (Arnold 1974;Bell et al. 2007). In deserts, a cave-dwelling lifestyle is a survival strategy for cockroaches (Roth and Willis 1960). Material of P. arabica reported in this study were sampled from a natural cave in western Iran (Fig. 6), which has a subtropical desert climate (Burstyn et al. 2019). Morphologically, slender antennae and legs, absent pulvilli and arolia, lighter body and very small ocelli of early instars are consistent with the convergent evolution of cave-dwelling species (Bell et al. 2007;Lucañas and Lit 2016). In contrast, P. americana has well-developed tegmina and wings, and developed pulvilli and arolia in both sexes, which could be favorable to facilitate its dispersal and climbing ability (Clemente and Federle 2008), and also beneficial for this species to colonize other environments (e.g., human settlements, tree trunks in the wild, landfills, and shallow caves with abundant guano; Lucañas et al. 2022) in search for food. Therefore, influenced by their environment and lifestyle, these three species have maintained a high similarity in genitalia, but greatly diverged in external morphology.