Redescription of arenicolous dipluran Parajapyx pauliani (Diplura, Parajapygidae) and DNA barcoding analyses of Parajapyx from China

Abstract Littoral dipluran Parajapyx pauliani Pagés, 1959 was redescribed based on the specimens collected in Hainan Island, South China. The littoral habitat was confirmed for the species, as the first report of arenicolous dipluran in China. DNA barcoding fragment was sequenced for five Parajapyx species (18 individuals) from China, and this is the first report on DNA barcodes used for dipluran identification. The mean intra- and interspecific divergencesare 1.9% and 19.1% respectively. Synonymy of Parajapyx paucidentis and Parajapyx isabellae was confirmed.


Introduction
The genus Parajapyx was erected by Silvestri (1903) with type species P. 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 andYang 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 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 P. 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.

Samples collection
With flotation method, the specimens of P. 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 P. isabellae, two individuals of its synonym P. paucidentis identified from the morphology were also sampled.

Taxonomy of P. pauliani
Seven specimens of P. 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 (1952Pagés ( , 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.

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'-TAAACTTCAGGGT-GACCAAAAAATCA-3') (Folmer et al. 1994). PCR products were purified and then sequenced directly using both of the amplification primers.
Mouthparts. Lacinia composed by five lobes, the first lobe (distal) is very acute and smooth, and the following four larger and pectinate. Mandible with five teeth and three denticles between them. Maxillary palpus with 10 n and 2 m setae.
Distribution. So far, the species is known only from two localities: Hainan, China and Madagascar.
Remarks. Parajapyx pauliani is characterized by the antenna with 21 segments, nota each with 5+5 M setae and numerous normal setae, urotergites II-VII each with 8+8 M setae and numerous normal setae, and prescutum of urotergites II-V each with 2+2 normal setae. It has more normal setae than in other congeners. The numbers of M and m setae are relatively stable, but the numbers of normal setae are quite variable in different individuals.

Littoral habitat of Parajapyx pauliani
Three intertidal locations where P. pauliani was found are shown in Figs 18-20. All habitats are positioned lower than supralittoral, devoid of halophytes, and are directly influenced by sea water. The animal lives in shingly or sand beaches (Figs 18-20), between particles of different size: from 9 mm (with the whole variation from 5 to 16 mm, n=100) to 1.5 mm (1.0-2.3 mm, n=150) in diameter. P. pauliani appears to be a dipluran member of a genuine littoral community and is often associated with collembolan species like Yuukianura sp., Isotogastrura trichaetosa Potapov et al. 2011, Thalassaphorura sp., Oudemansia sp., Acherontiella sp., Archisotoma sp.

The DNA barcoding
The DNA barcoding of 18 individuals from five Parajapyx species from China were sequenced, and deposited in GenBank (the accession numbers showed in Table 1). The genetic divergence between individuals of the same species is 1.9% in average, with span 1.5-5.3%, and it is 19.1% in average, with span 16.3-21.3% between different Parajapyx species. The Neighbour-joining tree was constructed based on the barcoding sequences (Fig. 21). P. pauliani is clustered with P. isabellae. P. isabellae and P. emeryanus are valid species respectively well supported by barcoding analyses. Two individuals of P. isabellae (Syn. P. paucidentis) (teeth absent on the cerci) clustered together with five individuals of P. isabellae (teeth present on the cerci). The genetic divergence between P. isabellae (Syn. P. paucidentis) and P. isabellae is only 1.7% in average (with span 0.8-2.6%). In addition, individuals of P. yangi and P. hwashanensis clustered together with high support value, and the genetic divergence between them is low (0.2%).

Littoral records of Parajapyx
This is the first record of littoral dipluran in China. When P. 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 P. pauliani are shared with P. 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 P. pauliani vs. 19 in P. botosaneanui). More dense setaceous covering probably protects the littoral species of Parajapyx against the periodical contact with salt water. Three other Parajapyx species P. gerlachi, P. isabellae, and P. (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 andHebert 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 P. paucidentis and P. 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 P. paucidentis and P. 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, P. yangi and P. 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 P. yangi and P. hwashanensis. All individuals, identified formally by us as P. 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.