Species delimitation of Margattea cockroaches from China, with seven new species (Blattodea, Ectobiidae, Pseudophyllodromiinae)

Abstract Nearly 450 Margattea specimens were collected from 27 locations in China and their morphology was examined. Then 68 Margattea COI sequences were obtained and used to carry out phylogenetic analyses as well as species delimitation analyses using General Mixed Yule Coalescent (GMYC), Automatic Barcode Gap Discovery (ABGD), and Poisson Tree Processes (bPTP). GMYC analysis resulted in 21 molecular operational taxonomic units (MOTUs) (confidence interval: 20–22), which was completely consistent with the result of the bPTP. There were 15 MOTUs using the ABGD method. The number of MOTUs was slightly different from the assigned morphospecies (16). As to the incongruence between molecular and morphological results, we checked the specimens again and made sure that most morphological differences were determined to be intraspecific differences (except the difference between M. angusta and M. mckittrickae), although a large genetic distance existed. Finally, 16 Margattea species from China were defined in this study, of which, seven new species are established, i.e. Margattea deltodonta J-J He & Z-Q Wang, sp. nov., Margattea cuspidata J-J He & Z-Q Wang, sp. nov., Margattea caudata J-J He & Z-Q Wang, sp. nov., Margattea paratransversa J-J He & Z-Q Wang, sp. nov., Margattea disparilis J-J He & Z-Q Wang, sp. nov., Margattea transversa J-J He & Z-Q Wang, sp. nov., and Margattea bicruris J-J He & Z-Q Wang, sp. nov.


Introduction
Until now, 59 species have been included in the genus Margattea worldwide. Of these, 19 are from China (Wang et al. 2009;Liu et al. 2011;Beccaloni 2014). Margattea is known by the following characters: 1) eighth abdominal tergum unspecialized or specialized with a tuft; 2) median phallomere usually with accessory structure; 3) styli simple, cylindrical; and 4) symmetrical stripes and spots scattered on disc of pronotum, and in some species, the color of stripes and spots is similar to the body color (Roth 1989;Wang et al. 2009;Wang et al. 2014). As with other cockroach species, females of Margattea spp. are difficult to identify and match with males due to their strong resemblance in appearance and given that diagnostic characters are based on male genitalia (Wang et al. 2009(Wang et al. , 2014. DNA barcoding has proven to be a reliable and cost-effective method for identifying species in insect groups (Foster et al. 2004;Rach et al. 2008). General Mixed Yule-Coalescent (GMYC) (Pons et al. 2006), Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012), and Poisson-Tree-Processes (bPTP) (Zhang et al. 2013) have been used for species delimitation based on COI data Bai et al. 2018;Yang et al. 2019;Li et al. 2020).
In this study, we explore the diversity of Margattea species in China using both morphological features and GMYC, ABGD, and bPTP approaches to estimate the number of molecular operational taxonomic units (MOTUs), describe new species, and pair the female specimens with the males.
Measurements are based on observed specimens. The genital segments of the studied specimens were dissected and immersed in 10% NaOH, heated to dissolve the fat, and rinsed with distilled water to make the segments and genitalia observable. They were then stored in glycerin. Genitalia were observed in glycerin using a MOTIC K400 stereomicroscope. All photos were made with a Leica DFC digital microscope camera attached to a Leica M205A stereomicroscope, and were modified with Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA). Type materials are all deposited in the Institute of Entomology, Southwest University, Chongqing, China (SWU).

DNA extraction, PCR and sequencing
DNA was extracted according to the Hipure Tissue DNA Mini Kit (Magen Biotech, Guangzhou). Fragments of COI were amplified using PCR. Primers used for the amplifications are LCO1490 (5'-GGTCAACAAATCATAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994). Each PCR was performed in Analytik Jena Easy Cycler with 25 μl volumes using the aforementioned primers, followed by agarose gel electrophoresis. Amplification conditions were: initial denaturation at 98 °C for 2 min, followed by 35 cycles for 10 s at 98 °C, 10 s at 49 °C, and 1 min at 72 °C, with a final extension of 3 min at 72 °C.

Sequence processing and phylogenetic analyses
A total of 81 COI sequences were used for analysis, of which 68 sequences are newly sequenced and nine sequences were downloaded from GenBank. Four sequences were selected as outgroups from species of four genera (Allacta, Sorineuchora, Balta, and Shelfordina) of the subfamily Pseudophyllodromiinae (Table 1). All sequences were aligned using MEGA 7 and adjusted visually after translation into amino acid sequences, whose lengths were 658 bp. The genetic divergence value was quantified by MEGA 7 based on Kimura 2-parameter (K2P) (Kumar et al. 2016). Maximum likelihood (ML) analysis was implemented in RAxML 7.3.0 (Stamatakis et al. 2008) using a GTR GAMMA model with 1000 bootstrap replicates.
We used three molecular species delimitation methods (GMYC, ABGD, bPTP) to delimit Margattea species based on COI sequences. For GMYC, time-resolved gene trees were estimated in BEAST 1.8.1 (Drummond and Rambaut 2007) with the parameters as follows: the uncorrelated lognormal (UCLN) relaxed clock model, the mean clock rate fixed to 1, the UPGMA starting tree and the tree prior as constant-size coalescent. The single-threshold GMYC method was then applied to generate the ultrametric gene tree using the SPLITS package (Ezard et al. 2009;Team 2013). Ultimately, we compared the groups delimited with the one-species null model using a likelihood ratio test. For ABGD, we used the Jukes-Cantor (JC69) model with a relative gap width X = 1.0, the rest of the parameters are set by default. For bPTP, we uploaded the converted file of the ML tree into the web site (https://species.h-its.org) with the default setting to obtain the results.

Molecular analysis
All Margattea members were clustered together to form a monophyletic group in ML analysis (Fig. 1). Samples of Margattea species each formed monophyletic groups and most of branches with high support values, and females were recovered and grouped together with males (more details in Table 1). GMYC and bPTP analyses established 21 MOTUs as blue and purple bars indicate (Fig. 1); the ABGD analysis established 15 MOTUs (green bars). Compared to the other two molecular divisions, ABGD results were mostly consistent with morphological results (revealed by pink bars) (Fig. 1).

Four methods to identify species
On the basis of morphological characters including male genitalia, we were able to identify 16 morphospecies of Margattea. ML analysis revealed each morphological species of the genus as a robust clade (Fig. 1). There were some similarities and differences in the results of these four methods. Both GMYC and bPTP divided all Margattea species into 21 MOTUs, while ABGD was different from the above two methods in that all species were divided into 15 MOTUs. And there were some disagreements between morphospecies and MOTUs, such as the colored clades on the ML tree. According to the GMYC and bPTP results, M. speciosa (with orange highlight) and M. concava (with lavender highlight) were grouped into two MOTUs. Moreover, M. bisignata (with light blue highlight) was divided into four MOTUs. And for ABGD, most species were consistent with morphospecies, except for M. angusta (with yellow highlight) and M. mckittrickae (with green highlight), which were considered to be one MOTU. As to the incongruence, we checked the specimens of M. speciosa, M. concava, and M. bisignata again and found there were no differences in male genitalia of their different samples (Figs 2, 3), so that the genetic variations among different samples of M. speciosa, M. concava, and M. bisignata were determined to be intraspecific differences despite a relatively large genetic distance existed (2.9% in M. concava, 3.1% in M. speciosa, and 5.9% in M. bisignata) (Suppl. material 1: Table S1). Also, upon examination of specimens of M. angusta and M. mckittrickae, we found some stable differences between the two species (in the former, the interstylar region barely protruding, right phallomere simple and hook-like, and in the latter, the interstylar region strongly produced, hook-like phallomere on the right side with a brush-shaped sclerite), although the genetic distance between them was only about 5%.

Diagnosis of the genus Margattea
Third and fourth palpi both obviously longer than the fifth. Tegmina and wings usually fully developed, beyond end of abdomen, but slightly reduced in a few species, not reaching end of abdomen. Disc of pronotum usually with symmetrical maculae. ScP of tegmina simple, R multi-branched, M with 4-7 complete branches; hind wings of the ScP and RA expanded at base, CuA usually with 4-6 branches. Eighth abdominal tergum unspecialized or specialized with a tuft. Anteroventral margin of front femur type B 2 , or B 3 , rarely C 2 . Tarsal claws symmetrical and usually specialized, inner margin serrated. Styli simple, cylindrical. Hook phallomere on right. Median phallomere with accessory structure.
The genus Margattea Shelford, 1911 is closely related to Balta Tepper, 1893; however, they can be distinguished by the following characteristics: 1) In the former, the front femur is always of type B 2 , or B 3 , rarely C 2 ; in the latter, the front femur always of type C 2 or in a few, type B 3 ; 2) in the former, the tarsal claws are symmetrical and specialized, but in the latter, the tarsal claws are asymmetrical and unspecialized; 3) in the former, the interstylar region is always convex or nearly straight, while in the latter, the interstylar region is always concave.  Diagnosis. This species is similar to M. satsumana (Asahina, 1979) in general appearance, but can be differentiated from the latter by the following characters: 1) median phallomere slender rod with base sharp, and apex expanded with three spines; while in the latter, base slightly expanded, and apex curved with some short spines; 2) subgenital plate not folded; while in the latter, folded inwards.
Female unknown. Etymology. The word "delt" and "odont" from Greek and means triangular, the species name "deltodontus" refers to the posterior margin of subgenital plate with small spines.
Distribution. China (Yunnan).  Wang et al., 2014 in general appearance and male genitalia, but it can be differentiated from the latter by the following characters: 1) interstylar region obviously convex with both sides curved inwards, three spines on each side, while in the latter, two sides curled up with 5-6 small thorns; 2) the left end of the accessory structure with a slender bone; the latter absent.
Description. Male. Coloration: body yellowish-brown (Fig. 5A, B). Face yellowishbrown. Interocular space with a dark brown band. Ocelli spots white, interocelli space with a brown band. Antennae dark linen-colored. Clypeus dark yellowish-brown (Fig.  5C). Maxillary palps yellowish-brown (Fig. 5E). Pronotal disc yellowish-brown with dark brown stripes, and lateral borders light linen-colored (Fig. 5D). Tegmina pale yellow, wings medium brown (Fig. 5F, G). Abdomen pale yellowish-brown. Cerci pale yellowish-brown (Fig. 5K). Styli faint yellow (Fig. 5M). Head: vertex slightly exposed, distance between interocular same length as antennal sockets space (Fig. 5C). Pronotum nearly trapezoidal, broader than long, the widest part after the midpoint, the front and posterior margins nearly straight, and the postero-lateral angle blunt and round; the disc with symmetrical irregular macules (Fig. 5D). The third, fourth palpi of approximately same length, both obviously longer than the fifth, the fifth obviously expanded (Fig. 5E). Tegmina and wings: tegmina and wings fully developed, both extending beyond the end of abdomen (Fig. 5A, B). Tegmina with ScP simple, R multi-branched, M straight with six complete branches. Hind wings with ScP and RA expanded at apex; M straight and simple, without branches; CuA with five complete branches (Fig. 5F, G). Legs: anteroventral margin of front femur type B 3 (Fig. 5H). Pulvilli present on four proximal tarsomeres. Tarsal claws symmetrical and specialized, inner margin serrated, arolia present (Fig. 5I). Abdomen and genitalia: eighth abdominal tergum specialized with a tuft (Fig. 5J). Supra-anal plate transverse, posterior margin protruded. Paraprocts similar, splitting into two pieces, apex with tufts (Fig. 5K). Subgenital plate symmetrical, lateral borders flip inwards with spines and hairs. Styli similar, slender; interstylar region obviously convex, two sides convex and curved inwards, each side with three spines (Fig. 5M). Left phallomere complex, irregular bone-shaped, with a short spine (Fig. 5L). Median phallomere slender rod-shaped, obviously curved, apex with ordered long spines; the accessory structure arched, on at rightmost end with spines, left apex with a slender bone with apex sharp (Fig. 5M). Hook phallomere on the right side, apex curved inwards with a short spine (Fig. 5N).
Female unknown. Etymology. The latin name "cuspidatus" refers to interstylar region obviously convex, two sides convex and curved inwards.
Distribution. China (Guangxi).  Diagnosis. This species is similar to M. flexa Wang et al., 2014 in male genitalia, but it can be differentiated from the latter by the following characters: 1) interstylar region obviously irregularly convex, the left part obviously larger than the right, while in the latter, interstylar region obviously regularly convex; 2) the left part of the accessory structure of median phallomere with a brush, absent in the latter.
Description. Male. Coloration: body yellowish-brown with pale brown (Fig. 7A,  B). Face yellowish-brown. Interocular space with a dark brown band. Ocellar spots white and small, interocellar space with a brown band. Antennae light linen-colored. Clypeus medium yellowish-brown (Fig. 7C). Maxillary palps light yellowish-brown to yellowish-brown (Fig. 7E). Pronotal disc yellowish-brown with brown stripes and two lateral borders light yellow (Fig. 7D). Tegmina light fawn, wings and legs pale brown (Fig.  7F, G). Abdomen light linen with pale yellowish-brown. Cerci pale brown (Fig. 7K). Styli light yellowish-brown (Fig. 7M). Head: vertex slightly exposed, distance between interocular same length antennal sockets space (Fig. 7C). Pronotum nearly trapezoidal, broader than long, the widest part after the midpoint, the front and posterior margins nearly straight, and postero-lateral angle blunt and round; disc with symmetrical irregular macules (Fig. 7D). Third and fourth palpi of approximately the same length, both obviously longer than fifth, fifth palp obviously expanded (Fig. 7E). Tegmina and wings: tegmina and wings developed, both extending the end of abdomen (Fig. 7A-B). Tegmina with ScP simple, R multi-branched, M straight with seven complete branches. Hind wings with ScP and RA expanded at apex; M straight and simple, without branches; CuA with five complete branches (Fig. 7F, G). Legs: anteroventral margin of front femur type B 3 (Fig. 7H). Pulvilli present on four proximal tarsomeres. Tarsal claws symmetrical and specialized, inner margin serrated, arolia present (Fig. 7I). Abdomen and genitalia: eighth abdominal tergum specialized with a tuft (Fig. 7J). Supra-anal plate transverse, posterior margin convex. Paraprocts simple, similar, splitting into two pieces, base with tufts (Fig. 7K). Subgenital plate asymmetrical. Styli similar, slender, distinctly separated; interstylar region obviously irregularly convex, middle part concave, two lateral angles spherical with some short thorns, left angle obviously larger than right (Fig. 7M). Left phallomere complex, irregular bone-shaped, with a long spine (Fig. 7L). Median phallomere slender rod-shaped, base splitting into some long spines; the accessory structure arched, at leftmost end with a brush (Fig. 7M). Hook phallomere on right side, base curved inwards with a short spine (Fig. 7N).
Female unknown. Etymology. The latin name "disparilis" refers to the interstylar region obviously irregular convex.
Distribution. China (Yunnan). Diagnosis. This species is similar to M. nimbata (Shelford, 1907) in male genitalia, but it can be differentiated from the latter by the following characters: 1) median phallomere base with a curved spine, while in the latter, with two curved spines; 2) a long piece of bone extends from the right side of the accessory structure, while absent in the latter; and 3) left phallomere with four long spines; the latter with two long spines.
Female same as male. Etymology. The latin name "transversus" refers to the interocular space having a dark brown transverse band.
Distribution. China (Yunnan). Diagnosis. This species closely resembles Margattea transversa sp. nov., but they can be distinguished by the following characteristics: 1) Left phallomere of the former with three long spines, while the latter with four long spines; 2) In the former, median phallomere apex with a slightly curved spine, while the median phallomere apex of latter with a distinct curved spine. In addition, this species is also similar to M. nimbata (Shelford, 1907) in general appearance, but it can be differentiated from the latter by the following characters: 1) median phallomere base with a curved spine, while in the latter, with two curved spines; 2) A long piece of bone extends from the right side of the accessory structure, while absent in the latter; and 3) left phallomere with three long spines; the latter with two long spines.
Female similar as male. Etymology. The species name "paratransversa" reflects its similarity to M. transversa sp. nov.
Distribution. China (Yunnan). Diagnosis. This species is similar to M. brevialata (Caudell, 1927) in male genitalia, but it can be differentiated from the latter by the following characters: 1) median phallomere slender rod, apex forked without spines; while in the latter, one side of splitting apex with 2 long spines; 2) left phallomere without a spine; the latter with a spine; and 3) supra-anal plate symmetrical, the front and the posterior margin straight; while in the latter, posterior margin convex, the middle part concave.
Female unknown. Etymology. The Latin name "bicruris" refers to the median phallomere having the base forked.

Discussion
The number of Margattea MOTUs (21) recovered from GMYC and bPTP analysis were greater than the number of species (16) determined by morphological characters. Of these, 13 MOTUs totally correspond to 13 species, while the remaining three species were overestimated as eight MOTUs. The ABGD method yielded 15 MOTUs because two morphospecies were considered as one MOTU. After re-examining the specimens, we still adhere to the morphological hypotheses, that is, 16 species. Our results therefore show that ABGD was, for Margattea with the parameters used, more in agreement with the morphological species hypotheses than the other methods tested. DNA-based identification methods were also proven to be useful in Margattea male and female matching. There is no denying that DNA-barcoding methods have performed well in the rapid identification and assessment of species diversity, in finding cryptic species, and in the matching of males and females (Yang et al. 2019;Li et al. 2020). However, when there is a divergence between the morphology and mo- Figure 11. Twenty-seven collecting locations of Margattea species in China. The location corresponding to each number on the map was shown in Suppl. material 2: Table S2. The map originates from https:// www.simplemappr.net/. lecular results, we need to look for morphological evidence to show which approach is best supported. For this group of cockroaches in our study, the intraspecific and interspecific K2P genetic distances (0.0-5.9% and 4.9-25.2%, respectively) were more or less similar to values found for other cockroach groups (Cryptocercus: 0.00-0.61% and 2.18-20.36% (Bai et al. 2018); Ectobiidae: 0.0-7.0% and 4.6-30.8% ). There is an overlap, also known as no barcoding gap, between the intraspecific and interspecific distance according to our results; but this barcoding gap was treated as an artifact of insufficient sampling across lycaenid butterfly taxa by Wiemers et al. (2007). The maximum intraspecific genetic distance (5.9%) existed in M. bisignata samples. Four MOTUs were suggested within this species in the GMYC and bPTP analyses. No obvious variation could be discerned in these different geographical populations (Fig. 11) using morphological characters, including male genitalia, in spite of this larger genetic distance (Fig. 2). Therefore, we speculate that sufficient sampling of M. bisignata locations resulted in greater genetic distance. While the two morphospecies with an interspecific genetic distance of 5% were hypothesized as a single MOTU in ABGD, they did have obvious and stable morphological differentiation characters, which may be the result of insufficient sampling or rapid morphological differentiation.