Molecular Evidence of Cryptic Species Diversity in the Perinereis Nuntia Species Group (Annelida: Nereididae) with first Records of P
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Plankton Benthos Res 14(4): 287–302, 2019 Plankton & Benthos Research © The Japanese Association of Benthology Molecular evidence of cryptic species diversity in the Perinereis nuntia species group (Annelida: Nereididae) with first records of P. nuntia and P. shikueii in southern Japan 1,2, 2 3 4 HIROAKI TOSUJI *, KIHO NISHINOSONO , HWEY-LIAN HSIEH , CHRISTOPHER J. GLASBY , 5,† 1,5 TAKERU SAKAGUCHI & MASANORI SATO 1 Research Field in Science, Science and Engineering Area, Kagoshima University, Kagoshima 890–0065, Japan 2 Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Kagoshima 890–0065, Japan 3 Research Center for Biodiversity, Academia Sinica, Taipei, Taiwan 115, R.O.C 4 Museum and Art Gallery of the Northern Territory, PO Box 4646, Darwin, Northern Territory 0801, Australia 5 Department of Earth and Environmental Sciences, Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890–0065, Japan † Present address: Kagoshima City Aquarium, Kagoshima 892–0814, Japan Received 17 June 2019; Accepted 16 September 2019 Responsible Editor: Shigeaki Kojima doi: 10.3800/pbr.14.287 Abstract: Taxonomic reexamination of Japanese populations of the Perinereis nuntia species group, which consti- tutes a major polychaete component in intertidal benthic communities, was carried out by analyzing the mitochondrial 16S rDNA and the nuclear ribosomal ITS sequences of 80 specimens, newly collected from 27 sites in Japan, together with 18 museum-preserved specimens collected from southern neighboring countries outside Japan. The Japanese populations of the Perinereis nuntia species group could be divided into four genetically different groups, which cor- responded morphologically to four nominal species (P. mictodonta, P. wilsoni, P. shikueii, and P. nuntia), with some exceptions. Perinereis nuntia and P. shikueii were recorded as new to the Japanese fauna, with their distributions re- stricted to southern Japan. The clade containing P. shikueii was subdivided into two sister clades (forms A and B), indi- cating that cryptic speciation has occurred within this clade. Our results revealed a remarkable variability in the number of bars in area VI of the proboscis in form B of P. shikueii, which appeared to be caused by breakage of the long bar into short bars during growth from a juvenile to an adult. Key words: 16S rDNA, ITS, paragnaths, polychaetes, taxonomy the temperate and tropical Indo-Pacific (Wilson & Glasby Introduction 1993, Glasby & Hsieh 2006). The genus Perinereis Kinberg, 1865 (Annelida: Nerei- Only two species of the Perinereis nuntia species group, didae) includes approximately 66 species in the world, oc- P. mictodonta (Marenzeller, 1879) and P. wilsoni Glasby curring as major components of intertidal benthic com- & Hsieh, 2006, are currently recognized from Japan (Sato munities (Sampértegui et al. 2013). The Perinereis nuntia 2017) and Korea (Park & Kim 2007); whereas, six spe- species group is characterized by the presence of an arc of cies have been recorded from nearby Taiwan (Glasby & bar-shaped paragnaths (or a mixture of bars and cones) on Hsieh 2006). Prior to the taxonomic revision of Glasby area VI of the eversible proboscis. The species group cur- & Hsieh (2006), the Japanese and Korean populations of rently comprises 15 species, recorded from a wide range in P. mictodonta and P. wilsoni were known as P. nuntia var. brevicirris sensu Fauvel, 1932 and P. nuntia var. val- * Corresponding author: Hiroaki Tosuji; E-mail, [email protected] lata sensu Fauvel, 1932, respectively (Imajima 1972; Paik u.ac.jp 1972). Though the two species are morphologically very 288 H. TOSUJI et al. Table 1. Comparison of selected diagnostic characteristics of paragnaths in areas V and VI on the proboscis and of lower neurochaetae in four species of the Perinereis nuntia species group, based on Glasby & Hsieh (2006) and Park & Kim (2007). Area V Area VI Heterogomph spiniger Species (No. of cones and arangement) (No. and length of bars) in lower neurochaetae P. mictodonta 1–5, usually 3 in a triangle 2–10, uneven length present throughout (Marenzeller, 1879) P. wilsoni Glasby & 1–4, in a longitudinal line 3–9, uneven length present throughout Hsieh, 2006 P. shikueii Glasby & 3 or 4, usually 3 in a transverse line or 4–10, short even-length bars present throughout Hsieh, 2006 flat triangle P. nuntia 0–5, at more proximal position to 4–17, short even-length bars absent in anterior parapodia (Savigny, 1818) area VI similar (Glasby & Hsieh 2006), molecular studies using the dana (Ehlers, 1868), P. mictodonta, and P. wilsoni) is much ribosomal internal transcribed spacers (ITS) (Chen et al. higher than intraspecific divergence, and the interspecific 2002) and the mitochondrial cytochrome oxidase I (COI) divergence of ITS 2 is significantly higher than that of ITS gene (Park & Kim 2007) suggested that they are distinct 1. species. In Japan, the two species have been classified ac- In the present study, we examine the molecular phy- cording to the key of Imajima (1972, 1996): P. mictodonta logeny of 80 Japanese specimens of the Perinereis nun- has three paragnaths in a triangle on area V, whereas P. tia species group, together with 18 reference materials of wilsoni has a single paragnath in the same area. However, four foreign species sourced from museum collections. Glasby & Hsieh (2006) and Park & Kim (2007) showed Our analysis is based on molecular markers of the mito- that the paragnath numbers in area V are variable with- chondrial 16S rDNA and the nuclear ribosomal ITS se- in each species and overlap between the two species (P. quences Our results indicate that the Japanese specimens mictodonta: 1–3 in the type locality (Japan), 1–5 in Taiwan of the Perinereis nuntia species group can be divided into and Korea; P. wilsoni: 1–3 in Taiwan, 1–4 in Korea) (Table four genetically different groups, which correspond to four 1). Moreover, these two species are also morphologically nominal species (P. mictodonta, P. wilsoni, P. nutria, and very similar to another two species, P. nuntia (Savigny, P. shikueii) and that the clade of P. shikueii can be clearly 1818) and P. shikueii Glasby & Hsieh, 2006 (Glasby & subdivided into two sister clades, suggesting the existence Hsieh, 2006), which are known from Taiwan. Therefore, of a cryptic species closely related to P. shikueii. it seems that the species diversity of the Perinereis nuntia species group may be underestimated in Japan (especially Materials and Methods in southern Japan) because some tropical and subtropical species could be easily misidentified as P. mictodonta or Collection and preparation of specimens for DNA analy- P. wilsoni by Imajima’s key. ses Molecular approaches by DNA analyses can be of sig- nificant help in taxonomy. The most widely used molecular A total of 80 Perinereis specimens were collected from markers for both intraspecific and interspecific relation- 27 locations of the intertidal zone in Japan from June 1981 ships in polychaetes are the 18S ribosomal RNA genes to April 2019 (Table 2 and Figs. 1–5). We also obtained from nuclear DNA (e.g., Bleidorn et al. 2003), and the an additional 18 reference materials, which were collected COI and the 16S ribosomal RNA genes from mitochon- outside of Japan and had been deposited in the Northern drial DNA (e.g., Jolly et al. 2006, Iannotta et al. 2007). The Territory Museum and Art Gallery, Darwin, Australia nuclear ribosomal DNA (rDNA) of eukaryotes consists of (NTM) and the Research Museum, Research Center for an external transcribed spacer (ETS), small-subunit ribo- Biodiversity, Academia Sinica, Taipei, Taiwan (ASIZW), somal RNA (18S rRNA), first inter-transcribed spacer (ITS including specimens examined in Glasby & Hsieh (2006) 1), 5.8S rRNA, second inter-transcribed spacer (ITS 2), and (Table 3). large-subunit rRNA (28S rRNA) (Gerbi 1986). Ribosomal The specimens, fixed in 80–99% ethanol, were used for ITS sequences are also used for phylogenetic construction DNA isolation, with the reference material specimens be- at and below the species level, because they can provide a ing soaked for 30 min in phosphate-buffered saline (PBS) spectrum of signals for phylogenetic resolution, and their prior to the DNA extraction. Total DNA was extracted degree of polymorphism has been shown to vary with spe- from several millimeters of the middle section of each cies (e.g., Chen et al. 2002). Chen et al. (2002) revealed that worm using two methods: the hexadecyltrimethylammo- the interspecific divergence of ITS among the four spe- nium bromide (CTAB) method from Winnepenninckx et cies of Perinereis (P. aibuhitensis (Grube, 1878), P. flori- al. (1993) and a combination method from Huelsken et al. Perinereis nuntia species complex in Japan 289 Table 2. List of specimens of five species of Perinereis nuntia species group newly collected in Japan in the present study. Site Accession number Species Code Location (latitude and longitude) Sampling date numbers* 16S rDNA ITS P. nuntia 1 Pnun-7 Nagashima Island, Kagoshima Prefecture (32°06.50′N, 130°08.53′E) April 29, 2014 LC482160 2 Pnun-5 Hirakawa, Kagoshima, Kagoshima Prefecture March 20, 2014 LC482160 LC482139 (31°26.77N, 130°31.03′E) 2 Pnun-6 Hirakawa, Kagoshima, Kagoshima Prefecture March 20, 2014 LC482160 LC482140 (31°26.77N, 130°31.03′E) 3 Pnun-8 Tokunoshima Island, Kagoshima Prefecture June 18, 2016 LC482159 (27°52.00′N, 128°53.32′E) 3 Pnun-9 Tokunoshima Island, Kagoshima Prefecture June 18, 2016 LC482159 LC482142 (27°52.00′N, 128°53.32′E) 3 Pnun-10 Tokunoshima