﻿Three cryptic Anaplecta (Blattodea, Blattoidea, Anaplectidae) species revealed by female genitalia, plus seven new species from China

﻿Abstract Morphological characteristics, including male and female genitalia, combined with DNA barcodes were used to identify 470 Anaplecta specimens sampled from China. Ten Anaplecta species are new to science, including three cryptic species: A.paraomei Zhu & Che, sp. nov., A.condensa Zhu & Che, sp. nov., and A.longihamata Zhu & Che, sp. nov., which are distinguished mainly by their female genitalia. The other seven new species are as follows: A.bicruris Zhu & Che, sp. nov., A.spinosa Zhu & Che, sp. nov., A.ungulata Zhu & Che, sp. nov., A.anomala Zhu & Che, sp. nov., A.serrata Zhu & Che, sp. nov., A.bombycina Zhu & Che, sp. nov., and A.truncatula Zhu & Che, sp. nov. This study illustrates that differences in female genitalia can be used to distinguish among species of Anaplecta. The female genitalia of 19 Chinese Anaplecta species are described and illustrated in this paper.


PCR amplification and sequencing
A total of 38 specimens was used for COI sequencing in this study. Total DNA was extracted from the muscles of the thorax and legs according to the Hipure Tissue DNA MiniKit. Primers for polymerase chain reaction (PCR) were COI-F3 (5'-CAACYAATCATAAAGANATTGGAAC-3') and COI-R3 (5'-TAAACTTCTG-GRTGACCAAARAATCA-3'). The thermal cycling conditions were as follows: initial denaturation 2 min at 98 °C, followed by 35 cycles of 10 s at 98 °C, 10 s, annealing at 49-50 °C, 15 s extension at 72 °C, and a final extension of 2 min at 72 °C; the samples were then held at 8 °C. The PCR products were sequenced by Tsingke (Beijing, China). All sequences were deposited in GenBank with the following accession numbers OL790028-OL790065 (Table 1).

Species delimitation and distance analyses
A total of 58 COI sequences was analyzed: 38 sequences of Anaplecta species in this study, 17 published sequences of Anaplecta, 3 sequences of Periplaneta Burmeister, 1838 (as outgroup) downloaded from GenBank (Table 1). All COI sequences were aligned using MEGA 7.0 and adjusted visually after translation into amino acid sequences. Genetic divergence values were quantified based on the Kimura 2-parameter (K2P) distance model (Kimura, 1980). Maximum Likelihood (ML) method was implemented in IQ-TREE (Nguyen et al. 2015) with the GTR+I+G model selected by PartitionFinder v.2.1.1 according to the corrected Akaike Information Criterion (AICc) (Lanfear et al. 2017), and nodal support values were estimated using 1000 bootstrap replicates. We then performed the Automatic Barcode Gap Discovery (ABGD; Puillandre et al. 2012) molecular species delimitation method to provide auxiliary evidence for distinguishing species. As a simple, quick, and efficient method, ABGD is available as a web interface (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) and was used with default settings, using the Jukes-Cantor (JC69) and p distance model with relative gap width (X = 1.0).

Phylogenetic analysis based on COI and MOTUs estimations
In this study, we acquired 38 COI sequences of Anaplecta species. The ML phylogenetic tree showed that males and females of the same morphospecies form monophyletic groups (Fig. 2). Most specific clades have 100 bootstrap values, except A. strigata (B = 86), A. omei (B = 94), and A. corneola (B = 87), indicating that the same morphospecies we identified were well clustered. The relatively low bootstrap values may be caused by the large geographical distances and lack of transitional population. In addition, ABGD analysis produced 20 MOTUs with prior intraspecific divergence (P) = 0.004642, 0.007743, 0.012915, 0.021544, and 0.035938, 17 morphospecies were detected as a single MOTU, but GZ2, GZ5, GZ6, DS4_2, formed one branch, SP4 and ZWLS2 formed a second, and GZ4, GX8, and GZ10 formed a third branch; all were distinct from A. omei but more closely related than the other species. The K2P genetic distance between the 38 individuals ranged from 0 to 27.4% (Suppl. material 1: Table S1).

Establishment of ten new species based on molecular and morphological data
The results of species delimitation by ABGD were nearly consistent with those by morphological identification (Fig. 2), except 13 samples, which were initially identified as A. omei based on external morphological characters and male genitalia, that were divided into 4 MOTUs. But it was insufficient and challenging to distinguish the 13 samples based on the characteristics of male genitalia. Therefore, we examined the females of all Anaplecta species from China carefully (except A. furcata, A. malayensis, A. simplex, and A. arisanica, Figure 2. Maximum Likelihood tree derived from COI sequences analyzed with a GTR+I+G model and with 1000 bootstrap replicates. The numbers at nodes are bootstrap values, the sex of the specimens is given in brackets (we checked the voucher specimens of the published sequences to determine whether they were males or females), the red bar indicated morphospecies, the blue bar indicated MOTUs in ABGD (P = 0.0046).
for which no female specimen was available), and found there were significant differences among their female genitalia. The sample from Mt. Wuliang (ZWLS2) has a robust and long first valvifer arm, while the first valvifer arm of others (CQ5, GZ10, DS4_2) are short and curled. The sample from Libo (GZ10) has a small and filamentous intercalary sclerite, while the intercalary sclerite of others (ZWLS2, CQ5, DS4_2) are strip-shaped or sheet-like. The anterior arch of the sample from Dushan (DS4_2) has a hip-shaped posterior margin, while that from Mt. Wuliang (ZWLS2) has two transverse finger-like protrusions, and that of CQ5 and GZ10 are smooth. The shape of the basivalvulae are also varied (Fig. 16). Ultimately, we discerned 20 Anaplecta species among our 470 samples, including ten new species, using both morphological characteristics and molecular data. The ten new species are Anaplecta bicruris sp. nov., A. spinosa sp. nov., A. ungulata sp. nov., A. anomala sp. nov., A. serrata sp. nov., A. bombycina sp. nov., A. truncatula sp. nov., A. longihamata sp. nov., A. paraomei sp. nov., and A. condensa sp. nov.

Diagnosis of the genus
The characteristics of the external structure and male genitalia are given in Deng et al. (2020) and are therefore not repeated here. Female genitalia: paratergites connected to crosspiece by membrane. First valvifer arm usually short, fused with crosspiece. Anterior margin of anterior arch with weakly sclerotized protrusions, and the shape of basivalvula is always irregular. Spermathecal plate almost merged with basivalvula. Subgenital plate symmetrical. Intersternal fold always simple, sheet-like.

Key to species of Anaplecta in China
Head and thorax. The distance between antennal sockets slightly narrower than interocular space. Fifth maxillary palpus nearly oval, slightly thicker and wider than others (Fig. 3D). Pronotum nearly sub-elliptical, posterior margin slightly straight (Fig. 3C). Tegmina with slightly indistinct veins; radius posterior veins of hind wings slightly indistinct, without transverse veins between M and CuA (Fig. 3E, F). Front femur Type B 2 (Fig. 3B). Pulvilli absent, tarsal claws symmetrical.
Distribution. China (Hainan). Diagnosis. This species is slightly similar to A. anncajanoae Lucañas, 2016, but can be distinguished from the latter by the spines on the left phallomere. It is also similar to A. cruciata Deng & Che, 2020 in body color and size, but can be distinguished as follows: 1) sclerites of the left phallomere spinous in A. spinosa sp. nov., while spineless in A. cruciata; 2) one of R2 with dense punctuations in A. spinosa sp. nov., while A. cruciata without; 3) anterior margin of anterior arch with a long horn-shaped protrusion in A. spinosa sp. nov., that of A. cruciata blunter and rounder; and 4) basivalvula nearly triangular in A. spinosa sp. nov., while nearly rectangular in A. cruciata.

Anaplecta spinosa
Etymology. The specific epithet is derived from the Latin word spinosus, referring to the left phallomere that is spiny.
Head and thorax. The distance between antennal sockets slightly narrower than interocular space. Fifth maxillary palpus nearly oval, slightly thicker and wider than others (Fig. 4D). Pronotum nearly sub-parabolic, anterior and posterior margins straight (Fig. 4C). Tegmina with slightly indistinct veins; radius posterior veins of hind wings slightly indistinct, with one discontinuous or no transverse veins between M and CuA (Fig. 4E, F). Front femur Type B 2 . Pulvilli absent, tarsal claws symmetrical.
Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, not extending to the posterior margin of supra-anal plate. Intercalary sclerite strip-shaped, slightly curved. First valve robust, with inward protrusions. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch slightly sclerotized, with a long horn-shaped protrusion, lateral area with dense tiny punctuations ( Fig. 13D, E). Basivalvula broad, the right lateral deeply concave, lateral area with dense punctuations (Fig. 13D). Spermatheca slightly sclerotized at base. Laterosternal shelf slightly sclerotized, lateral margin slightly curved, with dense spinules at base (Fig. 13F).
Distribution. China (Hainan). Diagnosis. This species is similar to A. cruciata Deng & Che, 2020 in body color and size, but can be distinguished as follows: 1) R2 serrated in A. serrata sp. nov., while that of A. cruciata without serration; 2) anterior margin of anterior arch with a sheet-like protrusion in A. serrata sp. nov.; while the protrusions of A. cruciata nearly Y-shaped; and 3) basivalvula extremely curled in A. serrata sp. nov., while slightly in A. cruciata.

Anaplecta serrata
Etymology. The specific epithet is derived from the Latin word serratus, in reference to the serrated lateral edges of R2.
Distribution. China (Yunnan).  Diagnosis. This species can be easily separated from other species by its hoof-shaped right phallomere, and the vestibular sclerite with two serrated and curved long spines.

Anaplecta ungulata
Etymology. The specific epithet is derived from the Latin word ungulatus, referring to the apex of R2 shaped like a pig or horse hoof.
Head and thorax. The distance between antennal sockets slightly narrower than interocular space. Fifth maxillary palpus nearly triangular, slightly thicker and wider than others (Fig. 6D). Pronotum sub-elliptical, anterior margin slightly curved and posterior margin straight (Fig. 6C). Tegmina with slightly indistinct veins; the radius posterior veins of hind wings slightly indistinct, with one or two transverse veins between M and CuA (Fig. 6E, F). Front femur Type B 2 . Pulvilli absent, tarsal claws symmetrical.
Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, extending to the posterior margin of supra-anal plate. Intercalary sclerite stripshaped. First valve tubular, with inward protrusions. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch protrudes in the shape of two triangles. Irregularly shaped basivalvula with dense punctuations, posterior margin curled. The base of vestibular sclerite nearly hyaline, posterior margin bifurcated into two highly sclerotized spines (Fig. 14A, B). Laterosternal shelf nearly hyaline (Fig. 14C).
Distribution. China (Yunnan). Diagnosis. This species is slightly similar to A. falcifer Hebard, 1925 but differs in the coloration of pronotum and tegmina. It is also similar to A. strigata Deng & Che, 2020 in body color and pronotum, but can be distinguished as follows: 1) the base of the tegmina almost black, while A. strigata mostly uniform dark yellowish brown; 2) L2d nearly rectangular in A. anomala sp. nov., while slightly bent in A. strigata; and 3) anterior margin of anterior arch with a finger-like protrusion, while the protrusion of A. strigata nearly wavy.

Anaplecta anomala
Etymology. The specific epithet is derived from the Latin word anomalus, referring to the left phallomere being different from other species.
Distribution. China (Yunnan). Diagnosis. This species can be easily separated from other species by dark brown tegmina and the extremely slender filamentous structure in the male genitalia.

Anaplecta bombycina
Etymology. The specific epithet is derived from the Latin word bombycinus, referring to the slender filamentous structure with which L1 is connected.
Distribution. China (Yunnan). Diagnosis. This species is similar to A. japonica Asahina, 1977 in body color and tegmina marking, but may be distinguished from the latter by the straight interstylar margin, Since A. japonica was described by external structures lacking genitalia, a comparison of this part is impossible. It is also similar to A. nigra Deng & Che, 2020, but can be distinguished as follows: 1) subgenital plate sub-rectangular in A. truncatula sp. nov., while A. nigra fan-shaped; 2) R1 needle-shaped in A. truncatula sp. nov., while arc-shaped in A. nigra; 3) anterior margin of anterior arch with a strip-shaped protrusion in A. truncatula sp. nov., while the protrusion of A. nigra triangular; and 4) vestibular sclerite with two long spines in A. nigra, A. truncatula sp. nov. without.

Anaplecta truncatula
Etymology. The specific epithet is derived from the Latin word truncatulus, referring to the truncated end of the bifurcation of the paraprocts.
Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, not extending to the posterior margin of supra-anal plate. Intercalary sclerite short, nearly spindle-shaped. Right first valvifer finger-like. First valve robust. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch slightly sclerotized, with a bifurcated strip-shaped protrusion (Fig. 15A, B). Basivalvula irregular, posterior margin and center with dense punctuations, the left of anterior margin extending back, connecting to crosspiece by membrane (Fig.  15A). Laterosternal shelf slightly sclerotized, lateral margin slightly curved, with dense spinules at lateral base (Fig. 15C).
Diagnosis. This species is similar to A. omei Bey-Bienko, 1958, but can be distinguished as follows: 1) right paraproct long hooked in A. longihamata sp. nov., while sheet-like in A. omei; 2) R1 bifurcated in A. omei, while unbranched in A. longihamata sp. nov.;3) anterior arch with two transversely finger-like protrusions in A. longihamata sp. nov., while A. omei without; and 4) first valvifer arm lateral edges folded up in A. longihamata sp. nov., while not folded in A. omei .
Etymology. The specific epithet is derived from the Latin words longi and hamatus, referring to the right paraproct extended backward in a long hook shape.
Distribution. China (Yunnan). Diagnosis. This species is very similar to A. omei, but can be distinguished as follows: 1) the paraprocts not extending backward in A. paraomei sp. nov., while left paraproct extending backward in A. omei; 2) the apex of R1 nearly symmetrical in A. paraomei, while asymmetrical in A. omei; 3) intercalary sclerite nearly strip-shaped in A. paraomei, while spindle-shaped in A. omei; and 4) posterior margin of anterior arch hip-shaped in A. paraomei sp. nov., while smooth in A. omei.

Anaplecta paraomei
Etymology. The Latin word para means similar, referring to its close resemblance to A. omei.
Distribution. China (Guizhou). Diagnosis. This species is very similar to A. omei, but can be distinguished as follows: 1) paraprocts both extending backward in A. condensa sp. nov., while only the left extending backward in A. omei; 2) R1 needle-shaped in A. condensa sp. nov., while bifurcated in A. omei; and 3) intercalary sclerite of A. condensa sp. nov. very small, filamentous, while that of A. omei is spindle-shaped.

Anaplecta condensa
Etymology. The specific epithet is derived from the Latin word condensus, referring to the paraprocts with dense spines on curly posterior margin.

Anaplecta nigra Deng & Che, 2020
Distribution. China (Xizang). Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, extending to the posterior margin of supra-anal plate. Intercalary sclerite nearly stripshaped, tapering to inside. First valve robust, finger-like protrusions on the inner edge with dense spines. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch protrudes forward in a flaky shape, slightly sclerotized, with an angular protrusion. Basivalvula highly irregular, most areas of the basivalvula with dense punctuations, the rest part curled (Fig. 15J, K). Vestibular sclerite sheet-like. Laterosternal shelf broad, slightly sclerotized, lateral margin straight (Fig. 15L).

Anaplecta bicolor
Distribution. China (Yunnan).   Male genitalia. On the basis of careful observation, we suspect that the L2d mentioned by Deng et al. (2020) may be the degraded right phallomere.

Anaplecta omei
Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, not extending to the posterior margin of supra-anal plate. Intercalary sclerite slender. First valve robust. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch slightly sclerotized, extending forward in a flaky shape with a deep concave in the middle. Basivalvula nearly elliptic with dense punctuations. Vestibular sclerite sheet-like (Fig. 17D, E). Laterosternal shelf slightly sclerotized, lateral margin slightly curved (Fig. 17F).
Distribution. China (Hainan). Female genitalia. Supra-anal plate nearly symmetrical. Paraprocts broad, not extending to the posterior margin of supra-anal plate. Intercalary sclerite nearly stripshaped, tapering to insides. First valve robust. Second valve small, basally fused. Third valve broad. The anterior margin of anterior arch slightly sclerotized, extending forward in a heart shape, with a nodular protrusion at apex (Fig. 17G, H). Basivalvula irregular, the left anterior margin extending posteriorly to first valvifer arm, deep depression in the center, posterior margin broad with dense punctuations (Fig. 17G). Laterosternal shelf slightly sclerotized, lateral margin slightly curved (Fig. 17I).

Discussion
In recent years, male genitalia were the main characteristics in the species delimitation of Anaplecta (Lucañas, 2016;Deng et al. 2020) but DNA barcodes can also help to delimit and distinguish species . During examination of samples of Anaplecta omei, we found some subtle morphological differences between samples collected in Libo, Dushan, Mt. Wuliang, and other regions. This included color, paraprocts, subgenital plates, and phallomeres. Although these specimens were recovered as four MOTUs in ABGD, these subtle differences in morphology were insufficient to determine whether they reflect intraspecific variation or interspecific differences. Therefore, we turned to the female genitalia for more evidence. Surprisingly, we found the shapes of first valvifer arm, intercalary sclerite, anterior arch, and basivalvula were stable within these four MOTUs and differed between MOTUs. Khalifa (1950) mentioned that when a pair of Blattella germanica mated, the hooked left phallomere (L3) extended and secured the first valve allowing the male to physically attach to the female during copulation. Therefore, we hypothesize that the long and robust hook of male genitalia of SP4 is to match the robust first valvifer arm of its female. Graves (1969) speculated that when transferring the spermatophore, the soft outer layer of the spermatophore hardens and would be against the female genital sclerites in order to ensure the openings of the sperm sacs aligned directly with the female spermathecal opening. Thus, we infer that the anterior arch and basivalvula might be related to this process of transferring the spermatophore. Taking all this evidence together, we can consider these MOTUs as different species: A. longihamata sp. nov., A. paraomei sp. nov., and A. condensa sp. nov. Similarly, we also found significant differences in other species in the anterior arch and basivalvula, indicating that the variation in female genitalia can be applied to identify the species of Anaplecta. However, this has often been neglected in the past study of Anaplecta, with the exception of McKittrick (1964), who described the female genitalia in detail. Only the valvular subgenital plate was involved in other studies (Roth, 1990;Deng et al. 2020). In our study, the characteristics of the female genitalia played an important role in detecting these three cryptic species; hence, researchers should pay more attention to female genitalia in future studies.