Introduction

The genus Parajapyx was erected by Silvestri (1903) with type species Parajapyx isabellae (Grassi, 1886). It is characterized by the mandible with five teeth and four denticles, absence of labial palpus, maxilla with first lobe slender and others pectinate, two pairs of spiracles on meso- and metanotum, four placoid sensilla on the terminal segment of antenna, subcoxal organ on urosternite I, eversible vesicles on urosternites II and III, claw with single medial unguis, and symmetrical cerci with 4-5 inner teeth (Pagés 1952, Xie and Yang 1992).

Later, Parajapyx was divided into two subgenera (Grassjapyx and Parajapyx) according to the shape of cerci (inner margin of cerci straight, tooth 1 not separated from others by a sinus in Grassjapyx vs. teeth 2-5 or 3-5 on a convexity of the internal margin, tooth 1 is separated from others by a sinus in Parajapyx) (Pagés 1952). So far, there are 31 species (16 subspecies) described in subgenus Grassjapyx, and 24 species (7 subspecies) in subgenus Parajapyx (Sendra 2006, Luan et al. 2007) in the world. Five species of genus Parajapyx were reported in China (Xie and Yang 1992, Luan et al. 2007).

In April 2011, during the research of the diversity of basal hexapods in littoral of Asia-Pacific coast, seven specimens of Parajapyx were collected from intertidal zone of several beaches of Hainan Island, South China. Those specimens were identified as Parajapyx pauliani Pagés, 1959, which was firstly described based on only specimen from intertidal zone of Nosy Be, Madagascar Island, and Pagés doubted about the habitat where the species was collected (Pagés 1959).

In this study, we provided a detailed redescription of this species based on our specimens, and more discussion on its littoral habitat. We analyzed the DNA barcoding sequences (Hebert et al. 2003) of Parajapyx pauliani, as well as other four Parajapyx species living in soil, in order to confirm the validity of species, and provide a useful reference for the identification of Parajapyx species.

Materials and methods Samples collection

With flotation method, the specimensof Parajapyx pauliani were collected directly from the water surface in Hainan, China, and stored in 80% ethanol. Specimens of other species were extracted by the Tullgren funnels from soil samples (Table 1). For Parajapyx isabellae, two individuals of its synonym Parajapyx paucidentis identified from the morphology were also sampled.

Taxonomy of Parajapyx pauliani

Seven specimens of Parajapyx pauliani were collected: four of which were mounted in Hoyer’s solution for identification, two were morphological identified in the alcohol first and then used for DNA extraction, and one was reserved in pure alcohol. Measurements and photos were taken by the help of a phase contrast microscope NIKON E600. The species was identified by the comparison of characters of all known species of the genus. For the name of chaetotaxy, we used the nomenclature proposed by Pagés (1952, 1996), and made some minor modifications following García-Gómez (2009). Microsetae on the body and the sensilla on the antenna were studied in detail for this species. Each pro-, meso- and metasternum was divided to three areas to designate setae.

Abbreviations. Ant. I-XXI= antenna segments I-XXI; BS= baculiform sensillum; M = macroseta; the position on dorsal of body as: ma = medial anterior, la= lateral anterior, mp = medial posterior, lp = lateral posterior; ms= microsensillum; m = microseta, n* = normal seta; s=sensillum; t1-t5= teeth of cercus.

* including all “s” setae named by Pagés (1952) and all supplemental setae inserted between M.

Molecular experiments

Eighteen individuals from five Parajapyx species were used for DNA barcoding analyses (Table 1), and two dipluran specimens from Japygidae and Campodeidae were used as the outgroups. All specimens were morphological identified in the alcohol first and then used for DNA extraction. We followed the experimental procedure for Collembola described in Potapov et al. (2010). Genomic DNA was extracted from one individual using the Wizard SV Genomic DNA Purification System (# 2361). The mitochondrial COI gene sequence was amplified (658 bp) by primer pair LCO (5’ - GGTCAACAAATCATAAAGATATTGG-3’) / HCO (5’- TAAACTTCAGGGTGACCAAAAAATCA- 3’) (Folmer et al. 1994). PCR products were purified and then sequenced directly using both of the amplification primers.

Sequences analysis

DNA sequences were analyzed with the software DNASTAR (Burland 2000). The genetic divergences (p-distance) were analyzed using MEGA 4.0 (Tamura et al. 2007). The phylogenetic tree was constructed by PAUP 4.0 beta 10 (Swofford 2002) with Neighbour-joining method and 1000 bootstrap replicates.

Results Discussion and conclusion Littoral records of Parajapyx

This is the first record of littoral dipluran in China. When Parajapyx pauliani was first found in intertidal zone in 1959, Pagés supposed that it is “purely fortuitous, and the single specimen collected was, in fact, might be pulled far away from its normal habitat by runoff” (Pagés 1959). Our records confirm the habitat of the species where it can live in narrow passages between sand particles due to slender and long body.

Numerous normal setae on body of Parajapyx pauliani are shared with Parajapyx botosaneanui Pagés, 1975, described from intertidal zone of Caribbean coast of Cuba (Pagés 1975). The two species can be readly distinguished by the number of the segments of antenna (21 in Parajapyx pauliani vs. 19 in Parajapyx botosaneanui). More dense setaceous covering probably protects the littoral species of Parajapyx against the periodical contact with salt water. Three other Parajapyx species Parajapyx gerlachi, Parajapyx isabellae, and Parajapyx (G.) brasilianus were also recorded in intertidal localities (Pagés 1967).

Barcoding analysis

The DNA barcodes have been widely used in identification of microarthropod species, for instance, collembolans (Hebert et al. 2003, Hogg and Hebert 2004). To our knowledge, this is the first report on DNA barcodes of Diplura, which proved to be useful for dipluran identification. Our analyses confirmed the synonymy of Parajapyx paucidentis and Parajapyx isabellae proposed by Pagés (1998) and Luan et al. (2007). These species differed only by teeth in cerci, absent vs. present. The genetic divergence between Parajapyx paucidentis and Parajapyx isabellae is 1.7% in average (with span 0.8–2.6%), which is exactly in the span of the divergence between individuals of the same species.

The formal morphological difference in second problematic couple, Parajapyx yangi and Parajapyx hwashanensis, is the number of teeth on the cerci: the former species has four teeth, while the latter has five. Our DNA barcoding data showed only one nucleotide difference between examined individuals of Parajapyx yangi and Parajapyx hwashanensis. All individuals, identified formally by us as Parajapyx yangi were, however, immature that indicated the possible age nature of this differences. The type materials of the two species call for study to make the final conclusions.