﻿Description of three new species of Callyntrura (Japonphysa) (Collembola, Entomobryidae) from China with the aid of DNA barcoding

﻿Abstract Callyntrura(s.l.) Börner, 1906 is the largest genus of the subfamily Salininae and contains 11 subgenera and 98 species from all over the world (mainly Asia), with eight species recorded from China. In the present paper, three new species of Callyntrura(s.l.) are described from China: C. (Japonphysa) xinjianensissp. nov.; C. (J.) tongguensissp. nov. and C. (J.) raoisp. nov. Their differences in colour pattern, chaetotaxy and other characters are slight, however distances of COI mtDNA support their validation as three new distinct species. A key to the Chinese Callyntrura(s.l.) is provided.


Introduction
The genus Callyntrura (s.l.) Börner, 1906 was previously considered a member of the family Paronellidae, but now belongs to the family Entomobryidae (Godeiro et al. 2022).It is mainly characterized by the smooth dens, fusiform scales on body, the presence of frontal spines on the head and more than three teeth on the mucro.Callyntrura was subdivided into 11 subgenera on the base of labral chaetae, antennae, dental spines and other characters (Yoshii 1992).The subgenus Japonphysa was established by Yoshii in 1982 and it contains four species.The subgenus can be separated from the other subgenera of Callyntrura by the absence of modified labral chaetae and the presence of a blunt basal chaeta on the maxillary outer lobe.
Callyntrura (s.l.) specimens are medium-sized and their colour pattern plays a key role in its classification.So far, 97 species of Callyntrura have been described from Southeast and South Asia and one species from Africa and the descriptions of most species were quite simple (Bellinger et al. 1996(Bellinger et al. -2023)).
Prior to this study, eight species belonging to four subgenera were described or reported from China (Ma 2013).Here we describe three new species of Callyntrura (Japonphysa) from China, based on their morphology and molecular data.
A key to Chinese Callyntrura (s.l.) is also provided.

Taxon sampling and specimen examinations
Specimens were collected with an aspirator and stored in 99% alcohol.They were mounted on glass slides in Marc André II solution, and were studied with a Leica DM2500 phase contrast microscope.Photographs were taken under a Leica DFC300 FX digital camera which mounted on the microscope and enhanced with Photoshop CS2 (Adobe Inc.).SEM photographs were taken under a ZEISS Gemini SEM 300 after the specimens were coated with a Leica EM ACE600.Type specimens are deposited in School of Life Sciences, Nantong University, Jiangsu, China.

Molecular analysis
DNA was extracted from one specimen per species by using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) following the manufacturer's standard protocols.Amplification of a 658 bp fragment of the mitochondrial COI gene was carried out using a Prime Thermal Cycler (TECHNE, Bibby Scientific Limited, Stone, Staffordshire, UK) in 25 μl volumes using Premix Taq polymerase system (Takara Bio, Otsu, Shiga, Japan).The primers and PCR progams followed Greenslade et al. (2011).All PCR products were checked on a 1% agarose gel electrophoresis.Successful products were purified and sequenced by Majorbio (Shanghai, China) on an ABI 3730XL DNA Analyser (Applied Biosystem, Foster City, CA, USA).
DNA sequences were assembled using Sequencher 4.5 (Gene Codes Corp), and then deposited in GenBank.Sequences were aligned by ClustalW implemented in MEGA 6 (Tamura et al. 2011) with default settings.Pairwise genetic distances were analyzed in MEGA 6 employing the Kimura 2-parameter (K2-P) model (Kimura 1980).

Results
The distribution in China of the species described in present paper is shown in Fig. 1.Head.Antenna not annulated and 1.37-1.57times length of body.Ratio of Ant.I-IV as 1.00/0.85-1.00/0.49-0.72/1.37-2.00.Distal part of Ant.IV with many sensory chaetae and normal ciliate chaetae, without apical bulb   long as inner edge of unguis; unguis with four inner teeth, most distal tooth very faint, basal pair located at 0.30-0.42distance from base of inner edge of unguis, unpaired teeth at 0.65-0.70 and 0.82-0.90distance from base, respectively; unguiculus lanceolate, with one median inner tooth and outer edge slightly serrate .
Etymology."raoi" (in apposition) refers to Lieutenant General Shoukun Rao, who made immortal achievements in the Chinese People's War of Resistance against Japanese Aggression and the War of Liberation.
Ecology.Found in the leaf litter.Remarks.The three new species are very similar in overall chaetotaxy, colour pattern and other characters.The chaetotaxy of each studied specimen is listed in Table 1, and the differences between these three species are slight.One main difference in chaetotaxy is that Abd.I has 7( 8 (Kinoshita, 1917), C. (J.) oligosetosa Kim, Rojanavongse & Lee, 1999, C. (J.) semilineata Yosii, 1961 and C. (J.) unilineata Yosii, 1961.The differences between the three new species and the four known species are great, especially in chaetotaxy of body (Table 2).

Molecular results
Sequenced individuals in the present study had a mean K2-P distance of COI sequences between 0.190-0.197 (about 19%) 3).Therefore, the interspecific distances of COI between the three new species were more than the accepted barcoding gap recently reported for the species of Entomobryidae (Zhang et al. 2018b) and Tomoceridae (Yu et al. 2018).The molecular distances coincided with the morphological divergences, thus further supporting the distinction of the three species.with a similar colour pattern and well used in classification in some genera of Collembola, such as Coecobrya (Nilsai et al. 2017;Zhang et al. 2018a), Dicranocentrus (Zhang et al. 2018b) and Tomocerus (Yu et al. 2016(Yu et al. , 2017;;Gong et al. 2018).

Figure 1 .
Figure 1.Record locality of all Chinese species of Callyntrura (s.l.) in China.Scale bar: 1000 km.
pigment; medial and posterior margin of Abd.IV with pair of irregular brown patches, respectively; brown pigment scattered on basal Ant.I and distal Ant.IV, legs, anterior part of ventral tube and distal dentes (Figs 2-6).

Table 2 .
Comparison between the new species and all known species of Callynthrura (Japonphysa).anteriorly in C. (J.) xinjianensis sp.nov., absent in C. (J.) tongguensis sp.nov.andpresent almost entirely in C. (J.) raoi sp.nov.The subgenus Japonphysa contains four species at present: C. (J.) japonica present . The shortest interspecific distance was 0.190 between C. tongguensis sp.nov.and C. raoi sp.nov.and the longest was 0.197 between C. tongguensis sp.nov.and C. xinjianensis sp.nov.(Table

Table 3 .
Genetic distances (mean K2-P divergence) of the COI sequences between the new described species.
SpeciesC.(J.) tongguensis sp.nov.C. (J.) raoi sp.nov.C. (J.) xinjianensis sp.nov.Discussion Colour pattern usually plays a very important role in the classification of Collembola and many species were described based on it previously.Although colour pattern is a good character and intraspecific variability is low in Callyntrura taxa, it is sometimes very difficult for taxonomists to distinguish those different species who share similar colouration.DNA barcoding is a good tool to separate species ZooKeys 1187: 237-260 (2023), DOI: 10.3897/zookeys.1187.109608Mei-Dong Jing et al.: Three new species of Callyntrura from China