Paracerella Imadaté in China: the description of a new species and the analysis of genetic differences between populations (Protura, Acerentomata, Nipponentomidae)

Abstract The genus Paracerella Imadaté, 1980 is recorded from China for the first time, with the description of a new species, Paracerella sinensis sp. n. Paracerella sinensis is characterized by four pairs of A-setae on tergite I, the presence of setae Pc and P3a on tergite VII, eight A-setae on tergite VIII, the presence of seta Pc on both sternites VI and VII, and 4/2 setae on sternite VIII, which are different from all other members of the genus. The key to the four species of the genus is updated. In addition, DNA barcodes of four populations are sequenced and their genetic differences are analyzed.


Introduction
The genus Paracerella Imadaté, 1980 is separated from Verrucoentomon Rusek, 1974 by the parallel position of the foretarsal sensilla d and a' to t2. It is characterized by having a distinct calyx with racemose appendices on its surface, three pairs of A-setae on mesoand metanota, filiform foretarsal sensillum t1, three A-setae on sternites I-VII, posterior position of setae P3 on tergites II-VI, well-developed labial palps, two subequal setae on abdominal legs II and III and well-developed striate band on segment VIII.
During field work in Inner Mongolia and Heilongjiang Provinces, northeast China, plenty of proturan specimens of Paracerella were found. They were identified as a new species and are described in the present paper, and an updated key to the genus was also provided. In addition, the DNA barcodes of the new species from four localities were sequenced and analyzed, the morphological identification was confirmed, and the genetic differences between different populations were revealed.

Materials and methods
Specimens were collected by Tullgren funnels. All specimens were mounted on slides in Hoyer's medium and dried at 60 °C. Specimens were identified and drawn with the aid of a NIKON E600 phase contrast microscope. The photos were taken by digital camera Nikon DXM1200. Type specimens are deposited in the Shanghai Entomological Museum (SEM), Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Natural History Museum (SNHM).
Abbreviations used in the text follow the paper of Bu and Yin (2007). Head setae and pores are marked according to Rusek et al. (2012). Body setae are marked following Imadaté (1974) and Yin (1999). Terminology of body porotaxy follows Szeptycki (1988) and Shrubovych (2014). Arrangements of the taxa follow the system proposed by Yin (1999).
For the analysis of genetic differences, genomic DNA was extracted from each individual separately by means of a non-destructive method (Gao and Bu 2014). After the DNA extraction, the cuticles of proturans were retrieved and mounted on the slides as voucher specimens. DNA barcoding sequences of mitochondrial COI gene were amplified and sequenced by primer pair LCO/HCO (Folmer et al. 1994). The barcoding sequences are deposited in GenBank. The nucleotide composition and the genetic divergence based on the Kimura-2-parameter (K2P) model were calculated using MEGA 6 (Tamura et al. 2013). Diagnosis. Paracerella sinensis sp. n. is characterized by four pairs of A-setae on tergite I, the presence of seta Pc and P3a on tergite VII, 8 A-setae on tergite VIII, the presence of seta Pc on sternites VI and VII, 4/2 setae on sternite VIII, which are different to any other members of the genus, foretarsal sensillum a extremely long, surpassing base of sensillum e, sensilla d and a' located in subequal level with t2, acrostyli of female squama genitalis each with two fine flaps.
Tergites I-III and VII with pores psm and al, IV-VI with pores psm, al and psl, VIII with pores psm only, IX-XI without pores, XII with single medial pore. Pores psm on tergite VII close to seta P1 (Fig. 3A). Sternites I-V without pores (Fig. 3C, D, E), VI and VII each with single medial pore spm, on VI located close to Pc and on VII located in central position (Fig. 3F, G). Sternites VIII-XI without pores, XII with 1+1 sal pores.
Etymology. The species is named after the Latin name of China, the place where the species was found.
Distribution. Inner Mongolia and Heilongjiang, China.

Remarks.
The new species is placed in the genus Paracerella because of the three pairs of A-setae on both meso-and metanota, filiform sensillum t1 on foretarsus, sensilla d and a' located in subequal level with t2, and well-developed striate band. Paracerella sinensis sp. n. can be easily distinguished from the other three species of the genus by the chaetotaxy of tergites I, IV and VIII, sternites VI-VIII, as well as the length of foretarsal sensillum a.
Among 24 adults of P. sinensis observed, the length of sensillum a is variable between individuals: in most specimens it can surpass base of e (holotype and most of paratypes) (Fig. 1F), in some specimens it is a little shorter, only surpassing base of d (nos. LM6-10, LM6-14D) (Fig. 1H), in some specimens it is extremely long as reaching or surpassing base of f (nos. LM6-13D, HH2-4D, WHS4-6-1), even reaching base of g (no. WHS4-2D). The four species of Paracerella can be distinguished by the following key.   (Imadaté, 1964); Japan (Hokkaido) The K2P genetic divergences of nucleotides for barcode sequences are 0-3.78% between individuals within the same population, and 0.46%-12.54% between individuals from different populations. The numbers of different coded amino acids for this sequence are 0-3 between individuals within the same populations, and 1-4 between individuals from different populations. Except that the COI gene sequence of WHS4-2D is more similar to COI of HH1-2D than to COI of WHS5-2D and WHS6-2D, our data show low genetic variation within populations (LM, WHS, and DZH), but reveal high genetic differentiation among four geographic populations (Table 2).

Discussion
The intraspecific distances of most insects are very low. Virgilio et al. (2010) studied the 15,948 DNA barcodes involving 1,995 insect species across six insect orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera and Orthoptera), and found 95% of all intraspecific K2P distances ranging from 0 to 7.64%. However, the intraspecific genetic distances of P. sinensis sp. n. are very high (up to 12.54%), which is in accord with the previous studies on some other proturan species: up to 21.3% in eight individuals of Ionescuellum haybachae from two Austria populations (Resch et al. 2014), and up to 31.98% separating 21 representatives of Acerentomon italicum in three Italian populations from an Austrian population (Galli et al. 2015). The similar situation  was also found in another basal hexapod group-Collembola: six collembolan species sampled from various locations worldwide with high intraspecific variation for COI from 11.33% to 21.47% (Porco et al. 2012). Compared with insects, basal hexapods are more ancient, and probably accumulated more random genetic mutations. Another possible reason is the lack of gene flow due to the low dispersal ability of basal hexapods. Anyway, we need more data to compare the difference between intra-and interspecific divergence, for the evaluation of the standard DNA barcoding efficacy in Protura.