﻿Discovery of five new species of Allacta from Yunnan and Hainan, China (Blattodea, Pseudophyllodromiidae)

﻿Abstract We examined new Allacta materials from Yunnan and Hainan Province, China, and discovered new species using both morphological and molecular species delimitation (ABGD) methods. Five new species are described: A.bifolium Li & Wang, sp. nov., A.hemiptera Li & Wang, sp. nov., A.lunulara Li & Wang, sp. nov., A.redacta Li & Wang, sp. nov., and A.unicaudata Li & Wang, sp. nov. All five species are placed under the hamifera species group. An updated key and checklist of Allacta species from China are provided.


Introduction
The genus Allacta Saussure & Zehntner, 1895 belongs to Pseudophyllodromiidae, with 47 known species mainly distributed in the Oriental and Australasian regions (Beccaloni 2014;He et al. 2019;Prabakaran et al. 2019;Senraj et al. 2021).Species of this genus are found inhabiting tree trunk surfaces at night or under the barks in the daytime (He et al. 2019).They are distinguished from other Pseudophyllodromiidae by the tarsal pulvillus only present on the fourth tarsomere.Recently, Prabakaran et al. (2019) and Senraj et al. (2021) described four new Allacta species from India, all with a solidly dark pronotal disk, broadly V-shaped or keel-like male interstylar margin, and all belonging to the hamifera species group (Roth 1993b).
Eight species were recorded from China according to He et al. (2019).However, the specimen depository of Allacta hainanensis (Liu et al. 2017) was not assigned (see Liu et al. 2017).Therefore, this species is invalid according to the International Code of Zoological Nomenclature (see Article 16.4.2) (ICZN 1999).
DNA barcoding has been widely used in cockroach identification in recent years, but is considered more reliable when used in combination with morphological evidence (Evangelista et al. 2013;Che et al. 2017;Yang et al. 2019;He et al. 2021).Although male genital morphology is traditionally used in cockroaches, female genitalia characters have also been shown to be useful in the identification of cockroaches, such as genera Cryptocercus and Anaplecta (Bai et al. 2018;Zhu et al. 2022).
In this study, newly collected Allacta materials from Yunnan and Hainan Provinces were examined, and they were found morphologically different from the known species.Based on morphological characters as well as the ABGD approach, five new species were confirmed, including the establishment of Allacta hemiptera Li & Wang, sp. nov.for the nomen nudum Temnopteryx hainanensis Liu et al., 2017.

Morphological examination
Twenty-six studied specimens of Allacta were collected from Yunnan, Hainan and Xizang Provinces and were deposited in College of Plant Protection, Southwest University, Chongqing, China (SWU) and Shanghai Entomology Museum, Shanghai, China (SEM).Morphological terminology used in this paper follows Roth (2003), McKittrick (1964) and Li et al. (2018) All materials are preserved in absolute ethanol and stored at -20 °C.The genitalia were handled based on a standard procedure in which terminal segments of the abdomen were cut off, heated in tap-water and rinsed with sterile water to remove trace amounts of NaOH, and then immersed in glycerol for further photography, dissection and preservation.Photos were taken with Leica DFC camera through a Leica M205A stereomicroscope; dissection and observation were performed under a Motic K400 stereomicroscope.All photos and images were edited with Adobe Photoshop CC 2019.

DNA sequencing
Total DNA was extracted from hindleg tissues by the Hipure Tissue DNA Mini Kit (Magen Biotech, Guangzhou), and the remaining body parts were stored in absolute ethanol as voucher specimens.Primers for PCR amplification were LCO1490 (5'-GGTCAACAAATCATAAGATATTGG-3') and HCO2198 (5'-TA AACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994).The PCR reactions were carried out in a 25 μL volume.The amplification conditions were: initial denaturation at 98 °C for 2 min, followed by 35 cycles for 15 s at 98 °C, 10 s at 49 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C.All DNA purification and sequencing were carried out by Tsingke Biotech Co., Ltd.(Beijing, China) using the aforementioned primers.

Results
ML analysis clustered females together with morphologically similar males.We identified 11 morphospecies of Allacta on the basis of morphological characters, mainly body color, pronotum pattern, head features, legs, wing venation and male genitalia (Fig. 1A), of which, four new morphological species were identified (four branches with red, yellow, green and blue highlights in Fig. 1).All Allacta species were divided into 12 molecular operational taxonomic units (MOTUs) by ABGD analysis as indicated by the pink bar (Fig. 1B).Taxonomic results were identical between morphological delimitation and ABGD except the branch highlighted with blue color, which is a single morphospecies but divided into two MOTUs by ABGD.

Allacta Saussure & Zehntner, 1895
Diagnosis.The characteristics of the external structure and male genitalia were given in full in Roth (1993b) and He et al. (2019).The following are supplements or adjustments.Tegmina and wings usually fully developed, sometimes reduced (A.bifolium Li & Wang, sp. nov. and A. redacta Li & Wang, sp. nov.).Subgenital plate usually with two styli, a few with one stylus (A.unicaudata Li & Wang, sp. nov.).
Description.Male.Body dark brown (Fig. 2A, B).Face brown with a yellowish-brown transverse crescent band below antennal sockets (Fig. 2C).Lateral portions of thorax yellowish brown, including pronotum, mesonotum and metanotum as well as most of the tegmina (Fig. 2A).Tibiae yellowish brown with base dark brown.Cerci dorsally with basal half blackish brown, terminal parts yellowish brown; each segment ventrally with basal half dark brown and apical half light brown.
Vertex with interocular space greater than the distance between antennal sockets.The third, and fourth maxillary palpi of approximately the same length, slightly longer than the fifth (Fig. 2E).Pronotum subparabolic with hind margin nearly straight.Tegmina greatly reduced (Fig. 2H), slightly surpass mesonotum, without veins.Hind wings absent.Anteroventral margin of front femur Type B 3 (Fig. 2F).Pulvillus only present on the fourth tarsomere (Fig. 2G).Tarsal claws symmetrical and unspecialized, arolia present.
Etymology.The Latin words bi-means pair, double, and folium means leaf, referring to the tegmina being degenerated into small leaf-like structures and hind wings absent.
Distribution.China (Yunnan).Diagnosis.This species can be easily distinguished from its congeners by the wings being reduced and the pronotal disk with a brownish mushroom-shape marking.
Vertex with interocular space greater than distance between antennal sockets.Pronotum subparabolic with hind margins nearly straight (Fig. 3D).The third and fourth maxillary palpi of approximately same length, slightly longer than the fifth (Fig. 3E).Tegmina and hind wings reduced, tegmina reach anterior edge of the second abdominal tergite; hind wings reach nearly two-thirds length of metanotum, width approximately one-tenth of tegmina (Fig. 3H, I).Anteroventral margin of front femur Type B 3 (Fig. 3F).Pulvillus only present on the fourth tarsomere (Fig. 3G).Tarsal claws symmetrical and unspecialized, arolia present.
Etymology.The specific name redacta derived from Latin, refers to both the tegmina and hind wings being reduced, which do not reach half the length of the normal wings of its congeners.
Distribution.China (Yunnan).Diagnosis.This species can be easily distinguished from all congeners by the absence of the left stylus in males, except for Allacta lunulara sp.nov., of which males are unknown (see below for females).This species shares a similar appearance with A. lunulara sp.nov., but it can be differentiated from the latter mainly by the following characters of the female genitalia: 1) third valves asymmetrical and slender rod-shaped, while symmetrical and broad crescent-shaped in A. lunulara; 2) posterior half of basivalvula narrower than the basal half, while basivalvula oval-shaped in A. lunulara; 3) spermatheca plate rounded with a sharp protrusion in the middle of the spermatheca plate, while front margin of spermatheca plate truncated in A. lunulara; and 4) laterosternal shelf asymmetrical, narrow, long and slightly curved, while symmetrical, broad and triangular in A. lunulara.
Vertex with interocular space narrower than distance between antennal sockets.The third and fourth maxillary palpi slightly longer than the fifth (Fig. 4G).Pronotum subparabolic with hind margins truncated (Fig. 4F).Tegmina and hind wings fully developed, both extending beyond the end of abdomen.Tegmina slender, with M and CuA longitudinal, CuA with four branches (Fig. 4J).M of hind wings with two branches, CuA curved with three complete branches (Fig. 4K).Anteroventral margin of front femur Type B 3 (Fig. 4H).Pulvillus only present on the fourth tarsomere.Tarsal claws symmetrical and unspecialized, arolium present.
Female genitalia.Supra-anal plate nearly symmetrical.Paraprocts broad.Intercalary sclerite irregular plate-shaped and translucent.First valves robust, with inward protrusions.Second valves small.Third valves asymmetrical, slender rod-shaped, and the left branch obviously curved outward.The anterior margin of anterior arch slightly sclerotized.First valvifer long and narrow platelike with setae on the inside.Basivalvula spindle-shaped, wide in the middle and tapers at both ends.Laterosternal shelf asymmetrical, narrow, long and slightly curved.Front margin of spermathecal plate rounded with a sharp protrusion in the middle.The spermatheca lobe forked, the end of one spermatheca branch enlarged, and the other long and tubular (Fig. 4R, S).
Remarks.This species resembles A. alba, but it can be differentiated from the latter by the following characters: 1) pronotal disc with an inverted triangular yellowish spot, while with subtrapezoidal symmetrical white markings in A. alba; 2) tegmina and hind wings slightly extending beyond the end of the abdomen, while extending far beyond the end of the abdomen in A. alba; and 3) subgenital plate without left stylus, while with two styli in A. alba.This species is placed in the hamifera species group by having the male interstylar margin broadly V-shaped.
Etymology.The Latin words uni-meaning one, single, and caudata meaning tailed, referring to subgenital plate with only one stylus.
Distribution.China (Yunnan).Diagnosis.This species resembles A. unicaudata, but it can be differentiated from A. unicaudata mainly by the symmetrical and crescent-shaped third valves.
Vertex with interocular space narrower than distance between antennal sockets (Fig. 5C).The third and fourth maxillary palpi of approximately same length, slightly longer than the fifth (Fig. 5E).Pronotum subparabolic with hind margins truncated (Fig. 5D).Tegmina and hind wings fully developed, both extending beyond the end of abdomen.Tegmina slender, with M and CuA longitudinal, CuA with four branches.M of hind wings with three branches, CuA curved with three complete branches (Fig. 5H, I).Anteroventral margin of front femur Type B 3 (Fig. 5F).Pulvilli only present on the fourth tarsomere.Tarsal claws symmetrical and unspecialized, arolium present.
Female genitalia.Supra-anal plate nearly symmetrical.Paraprocts broad, not extending to the posterior margin of supra-anal plate.Intercalary sclerite irregular plate-shaped and translucent.First valves robust, with inward protrusions.Second valves small.Third valves symmetrical and broad crescent-shaped.The anterior margin of anterior arch slightly sclerotized.First valvifer long and narrow plate-like with setae on the inside.Basivalvula oval-shaped.Laterosternal shelf symmetrical, broad and triangle.Front margin of spermathecal plate truncated.The spermatheca lobe forked, the end of one spermatheca branch enlarged, and the other long and tubular (Fig. 5K, L).
Remarks.This species was found to be a cryptic species, very similar to A. unicaudata Li & Wang, sp.nov.but it can be differentiated from A. unicaudata by the female genitalia characters aforementioned.In this study, after comparing the female genitalia in five Allacta species, it is confirmed that the variation in female genitalia can be applied to identify the species of Allacta.The COI divergence between them (6.6%) is significantly larger than the usual intraspecies distance in Allacta (Suppl.material 1).This species is placed in the hamifera species group by having a dark pronotum.
Etymology.The specific epithet is derived from the Latin word lunulara which means moon-like or relating to a crescent shape, referring to its crescent-shaped third valves.
Distribution.China (Yunnan).Diagnosis.This species resembles A. redacta, but it can be differentiated from A. redacta mainly by the pronotal disk with a nib-shaped yellowish spot.
Vertex with interocular space obviously narrower than distance between antennal sockets (Fig. 6D).The third and fourth maxillary palpi of approximately same length, slightly longer than the fifth (Fig. 6E).Pronotum nearly triangle with front margins blunt round and hind margins truncated (Fig. 6C).Tegmina and hind wings reduced.Tegmina nearly quadrilateral, veins not obvious.Hind wings small, thin and transparent, about half the length of tegmina (Fig. 6I,  J).Anteroventral margin of front femur Type B 3 (Fig. 6F).Pulvilli only present on the fourth tarsomere.Tarsal claws symmetrical and unspecialized, arolium present (Fig. 6G).
Female genitalia.Supra-anal plate nearly symmetrical.Paraprocts broad, not extending to the posterior margin of supra-anal plate.Intercalary sclerite irreg- ular plate-shaped and translucent.First valves robust, with inward protrusions.Second valves small.Third valves symmetrical and broad rod-shaped.The anterior margin of anterior arch slightly sclerotized.First valvifer irregular swollen and oval with short setae on the inside.Basivalvula oval-shaped.Laterosternal shelf symmetrical, broad and trapezoid.Front margin of spermathecal plate truncated.The spermatheca lobe forked, the end of one spermatheca branch enlarged, and the other long and tubular (Fig. 6H).
Remarks.According to the International Code of Zoological Nomenclature (Article 16.4.2) (ICZN 1999), Temnopteryx hainanensis Liu et al., 2017is invalid. He et al. (2019) did not realize that and moved Temnopteryx hainanensis to the genus Allacta.We here describe it as a new species based on the new material and the type specimens of Temnopteryx hainanensis Liu et al., 2017.Etymology.The Latin terms hemi-means half, ptera means wing, and hemiptera means that the tegmina is half the normal wing length.

Discussion
External characteristics and male genitalia have been traditionally used to define species of Allacta (Roth 1993b;Wang et al. 2014;He et al. 2019), but identifying species relied too much on male characteristics.For example, the male genitalia of A. lunulara Li & Wang, sp.nov.was not available and therefore could not be used to determine whether it was a new species in this study.As such, we tried to look for morphological divergence in female morphology.We compared the female genitalia of four known species and A. hemiptera sp.nov., and found that there were significant differences mainly in valvifer, first valvifer, basivalvula and laterosternal shelf (Figs 6, 7), indicating that female genitalia could be used for identification in Allacta.
The clade highlighted in blue (Fig. 1) was divided into two MOTUs.We carefully compared the female external genitalia of these two MOTUs and found evidence that they differed in the third valves, basivalvula, spermatheca plate and laterosternal shelf.According to the material sampled here, the maximum intraspecific genetic distances of Allacta for COI was 1.1%, but the minimum divergence of these two clades reaches 6.6% (Suppl.material 1).Female genitalia differences combined with this larger genetic distance indicated that specimen 5015272 was not A. unicaudata Li & Wang, sp.nov.Therefore, we proposed that it is a new species, A. lunulara Li & Wang, sp. nov.In future research, morphology specific to females should be taken into greater consideration, as it played a key role in the discovery of new species in this study.Sometimes female genitalia can even be used to discover cryptic species, for example, Zhu et al. (2022) distinguished three new cryptic species from Anaplecta omei through differences in female genitalia.
In this study, the absence of one stylus in the family Pseudophyllodromiidae was observed for the first time (A. unicaudata Li & Wang, sp. nov.), but this is not a unique case in Blattodea (Kumar and Prinis 1978;Roth 1989Roth , 1993a)), for example, Blattella parilis Walker, 1868, Symplocodes euryloba Zheng et al., 2015 andSymplocodes ridleyi Shelford, 1913 in Blattellidae; Pycnoscelus indicus Fabricius, 1775 and Pycnoscelus nigra Brunner von Wattenwyl, 1865 in Blaberidae.However, the causes and mechanisms for this phenomenon remains to be discovered.

Figure 1 .
Figure 1.Maximum likelihood (ML) tree based on COI sequence.Branch node labels are support values.Colored bars indicate different species delimitation by different methods A morphology (green) B ABGD results (pink).The colored clades (red, yellow, green and blue highlights) on the tree correspond to four new morphological species.

Figure 2 .
Figure 2. Allacta bifolium sp.nov., male holotype A dorsal view B ventral view C head, ventral view D pronotum, dorsal view E maxillary palpi, ventral view F front femur, ventral view G tarsus and tarsal claws of front leg H tegmen, dorsal view I supra-anal plate and paraprocts, ventral view J subgenital plate, dorsal view K left phallomere, dorsal view L median phallomere, dorsal view M hook-like phallomere, dorsal view.Scale bars: 5 mm (A, B); 1 mm (C-M).

Figure 3 .
Figure 3. Allacta redacta sp.nov., male holotype A dorsal view B ventral view C head, ventral view D pronotum, dorsal view E maxillary palpi, ventral view F front femur, ventral view G tarsus and tarsal claws of front leg H tegmen, dorsal view I hind wing, dorsal view J supra-anal plate and paraprocts, ventral view K subgenital plate and median phallomere, dorsal view L left phallomere, dorsal view M hook-like phallomere, dorsal view.Scale bars: 5 mm (A, B); 1 mm (C-M).
. Vein abbreviations in the figures are as follows:

Table 1 .
Samples used in this study.