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Morphology and molecular phylogeny of Pleurosira nanjiensis sp. nov., a new marine benthic diatom from the Nanji Islands, China
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Yuhang LI1, Tamotsu NAGUMO3, Kuidong XU1, 2, 4, *
Acta Oceanologica Sinica | 2018, 37(10) : 33 - 39
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Acta Oceanologica Sinica | 2018, 37(10): 33-39
• Articles •
Morphology and molecular phylogeny of Pleurosira nanjiensis sp. nov., a new marine benthic diatom from the Nanji Islands, China
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Yuhang LI1, Tamotsu NAGUMO3, Kuidong XU1, 2, 4, *
Affiliations
  • 1 Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
  • 2 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
  • 3 Department of Biology, School of Life Dentistry at Tokyo, The Nippon Dental University, Tokyo 102-8159, Japan
  • 4 University of Chinese Academy of Sciences, Beijing 100049, China
Published: 2018-10-25 doi: 10.1007/s13131-018-1298-x
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A new marine benthic diatom, Pleurosira nanjiensis sp. nov., is described from the rocky intertidal zone of the Xiaochaiyu Island of the Nanji Islands in China. Its morphology was examined with light and scanning electron microscopy. Molecular phylogeny was reconstructed based on SSU rRNA and rbcL gene sequences. Pleurosira nanjiensis differs from congeners in possession of a combination of morphological features including the domed valve with broadly lanceolate, elliptical or circular valve outline, two elevated marginal ocelli, two (rarely three) rimportulae, and radiate striae.

Pleurosira nanjiensis  /  rocky intertidal area  /  marine diatom  /  Nanji Islands  /  new species
Yuhang LI, Tamotsu NAGUMO, Kuidong XU. Morphology and molecular phylogeny of Pleurosira nanjiensis sp. nov., a new marine benthic diatom from the Nanji Islands, China[J]. Acta Oceanologica Sinica, 2018 , 37 (10) : 33 -39 . DOI: 10.1007/s13131-018-1298-x
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1 Introduction
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Trevisan (1848) erected the genus Pleurosira Meneghini for members of a subgenus in Melosira Agardh, which has cylinder cells connected by mucilage. Based on the redefinition of Compère (1982), Pleurosira is characterized by a cylindrical frustule, circular to broadly elliptical valve with clear separation between the usually flat valve face and a vertical mantle, the possession of poroid areolae, 2–4 marginal ocelli and 2–15 labiate processes located about midway between the valve centre and margin.
To date, the genus Pleurosira contains five species including four varieties and one forma (Compère, 1982; Guiry and Guiry, 2017). Among these, two species were reported only from freshwater environments. Pleurosira laevis f. laevis (Ehrenberg) Compère inhabits both freshwater and brackish environments and P. laevis f. polymorpha Compère dwells in both brackish and marine environments (Compère, 1982), while P. inusitata (Hohn and Hellerman) Desianti and Potapova and P. socotrensis var. bengalensis Compère have been reported only from brackish waters (Compère, 1982; Desianti et al., 2015).
In terms of distribution, P. indica Karthick and Kociolek, P. socotrensis (Kitton) Compère and three varieties of P. socotrensis were found from tropical Asia (Compère, 1982; Karthick and Kociolek, 2011). Pleurosira inusitata and P. minor were described from North and South America, respectively (Metzeltin et al., 2005; Desianti et al., 2015). Only P. laevis have been reported worldwide. Among these, P. laevis, P. socotrensis and P. minor were also reported in China (Pei et al., 2008; Liu et al., 2011). In the present study, we describe a new species of Pleurosira from a rocky intertidal zone in the East China Sea and its phylogenetic position is investigated with DNA sequencing. The morphological delimitation of the genus Pleurosira is also discussed.
2 Materials and methods
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2.1 Sample collection, cultivation and morphological observation
Samples of benthic diatoms were collected from the lower rocky intertidal zone of the Xiaochaoyu Island (27°25.348′N, 121°05.459′E) of the Nanji Islands on Chinese coast of the East China Sea on 15 May 2015. Single cells were isolated from the samples and transferred to F/2 medium. Clonal cultures were established and maintained at 20–23°C, with 20–30 μmol photons m–2 s–1 from cool-white fluorescent tubes. The photoperiod was 14:10 light:dark (L:D).
Cleaned frustules were prepared by the bleach solution method (Nagumo and Kobayasi, 1990) and were mounted on a glass slide with Mountmedia (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Nikon Eclipse 80i light microscopes (LM) equipped with differential interference contrast (DIC) were used for LM observation. For scanning electron microscope (SEM) observation, vegetative cells were fixed with 2.5% glutaraldehyde before cleaning. Cleaned frustules were air-dried and coated with osmium for SEM observation. A Hitachi S-3400 was used for SEM observation.
Terminology follows Anonymous (1975), Ross and Sims (1971), and Compère (1982).
2.2 DNA extraction, sequencing and phylogenetic analysis
Diatom pellets obtained by centrifuging the liquid cultures for 5 min at 1 000 g. Total DNA was extracted using Plant Genomic DNA Kit (Tiangen Biotech Co., China). Partial fragments of 18S small subunit rDNA (SSU rDNA) sequence and the chloroplast encoded large subunit of RUBISCO (rbcL) gene were amplified by polymerase chain reaction (PCR). The volume of each PCR reaction was 25 μL, containing 2.0 μL template DNA, 12.5 μL of 2× EasyTaq PCR SuperMix polymerase (TransGen Biotech, China), 0.5 μL of each primer (10 mmol/L) and sterile distilled H2O. Primers SSU1 and ITS1DR were used to amplify SSU rDNA (Medlin et al., 1988; Edgar and Theriot, 2004). Primers rbcL 66+ and rbcL 1444– were used to amplify rbcL gene (Alverson et al., 2007; Ruck and Theriot, 2011). The PCR cycles for the two markers followed Alverson et al. (2007). PCR products were purified using the TIANgel Midi Purification Kit (Tiangen Biotech Co., China) and sequenced by an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
The sequences were aligned using MAFFT v. 7 and further modified manually in Mesquite v. 3.2 (Katoh and Standley, 2013; Maddison and Maddison, 2017). Highly variable regions in which the alignment could not be determined unambiguously were excluded before phylogenetic analysis. The final alignment of a concatenated alignment of SSU rDNA and rbcL gene sequences included 3 036 positions. The concatenated alignment of SSU rDNA and rbcL gene sequences partitioned by different gene, and in the case of rbcL, by codon position. The GTR+G+I model was selected under the AICc criterion by Partitionfinder 2 for all partitions except the GTR+G model for the second condon of rbcL (Lanfear et al., 2017)
Maximum likelihood (ML) analyses were performed with RAxML v8.0.0 (Stamatakis, 2014). The reliability of internal branches was assessed using a non-parametric bootstrap method with 1 000 replicates. Gaps were treated as missing data. Bayesian inference (BI) analyses were carried out with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The programs ran for 107 generations with trees sampled every 1 000 generations and the first 25% of trees were discarded as burn-in. Convergence was judged based on the average standard deviation of split frequencies (all less than 0.01) and the ESS values (more than 4 000) analyzed in the R Package RWTY (Warren et al., 2017). The remaining trees were used to generate a consensus tree and calculate the posterior probabilities of all branches using a majority-rule consensus approach. FigTree v1.4.2 and Adobe illustrator CS6 were used to view and edit trees for publication.
3 Results
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3.1 Morphological description
Pleurosira nanjiensis Yuhang Li, Nagumo and Kuidong Xu sp. nov.
Diagnosis: Valve domed with broadly lanceolate, elliptical or circular outline. Valve length 15.5–46.8 μm, width 13.0–31.1 µm, pervalvar height 13.5–15.2 μm. Valve with external spins. Two ocelli, ocellate elevation blunt widely rounded. Poroid areolae with domed cribra. Two (rarely three) rimoportula about midway between centre and margin. Six bands. Striae radiated 15–21 per 10 μm, 14–18 pores per 10 μm on bands.
Holotype: Holotype slide MBM285985 has been deposited in the Marine Biological Museum, Chinese Academy of Sciences (MBMCAS) at Qingdao, China
Type locality: Xiaochaiyu Island, Nanji Islands, Wenzhou City, Zhejiang Province, China.
Etymology: Named after the type locality Nanji Islands.
Distribution and ecology: The species is currently known only from the type locality, where it inhabited the rock surface in the lower intertidal zone.
Gene sequences: The SSU rDNA and rbcL gene sequences of Pleurosira nanjiensis have been deposited in GenBank with the accession number MF578764 and MF578765, respectively.
Description: Cells form zig-zag colony (Figs 1a, b). Plastids are discoid. Valves are domed (rarely flat in culture, Fig. 2b arrow) with broadly lanceolate, elliptical or circular outline (Figs 1c–h and 2a–f). Valve is 15.5–46.8 μm long, 13.0–31.1 µm wide, and 13.5–15.2 μm high (Figs 1c–k). Two conspicuous blunt and robust ocellate elevations are present at two poles (Fig. 2c arrows). Each ocellus is surrounded by a thin hyaline area (Fig. 2g). On the external valve surface, spins are randomly distributed (Fig. 2g arrow). Poroid areolae are occluded by domed cribra externally (Fig. 2e arrow) and round internal foramina (Fig. 3b) in different size. Striae are radiated, 15–21 per 10 μm (Figs 1c–h and 3a–b). Usually two, rarely three small rimoportulae are positioned on both sides of the axis passing through the ocelli, about midway between the centre and the margin (Figs 1d, g–h, 2d and 3a–b arrows). The valve constricts near the margin (Figs 1j and 2h arrow). The cingulum is composed of six porous open bands in mature individuals, 14–18 per 10 μm (Figs 2b and 3b). The valvocopula has a dentate margin (Fig. 3c).
3.2 Phylogenetic analysis
The combined SSU rDNA and rbcL gene analysis showed that Pleurosira nanjiensis sp. nov. formed a robust clade with P. laevis and P. laevis f. polymorpha, the only two Pleurosira species have sequences information in GenBank, with strong nodal support (ML bootsrap=100%, Posterior probability=1.00). The genus Odontella Agardh is polyphyletic (Fig. 4). The Pleurosira clade is clustered with Odontella aurita (Lyngbye) Agardh, the generitype of Odontella, but with relatively lower support (ML bootstrap=79%, Posterior probability=0.94). Odontella obtusa Kützing, which resembles P. nanjiensis to some extent, is sister to Triceratium dubium Brightwell and T. dictyotum Sims and Ross.
4 Discussion
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In Compère’s definition of Pleurosira, the valve face is usually flat and distinctly separated from the vertical valve mantle, and the ocelli are not elevated from the general valve face. Pleurosira nanjiensis sp. nov. possesses a domed valve and ocellate elevations and thus are deviated from the generic diagnosis. Nonetheless, Compère (1982) ever described P. laevis f. polymorpha with a domed valve and elevated ocelli, a form that can tolerate higher salinity than P. laevis f. laevis. Likewise, a domed valve and elevated ocelli were reported in P. inusitata and P. socotrensis var. benalensis, both of which were found from brackish environments (Compère, 1982; Desianti et al., 2015). Moreover, these characters also occurred in the freshwater species P. socotrensis, when it was cultured in enriched seawater medium (Li and Chiang, 1979). Thus, both the ocellate elevation and the domed valve shape are likely associated with the saline environments these species inhabit, and should not be used to define the genus Pleurosira.
Based on a phylogenetic study of Biddulphiaceae and Eupodiscaceae, Ashworth et al. (2013) suggested that the morphological features such as the valve perforation and the position of rimoportulae and ocelli are important for discriminating the eupodiscacean diatoms (see Fig. 6 in Ashworth et al., 2013). Like congeners, P. nanjiensis has the rimoportulae present about the midway between the valve centre and margin on both side of the valve, and the simple valve perforation which is open in round forma internally. These important features together with the result of the molecular phylogenetic analysis support the assignment of P. nanjiensis to the genus Pleurosira.
Pleurosira nanjiensis differs from congeners by a combination of morphological features, including the domed valve with broadly lanceolate, elliptical or circular valve outline, two elevated marginal ocelli, two (rarely three) rimportulae, and radiate striae (Table 1). As mentioned above, P. nanjiensis resembles all the Pleurosira species that inhabits brackish or marine environment, namely P. laevis f. polymorpha, P. inusitata and P. socotrensis var. benalensis, in possessing a domed valve or elevated ocelli. However, P. nanjiensis has 2 or 3 rimoportulae, whereas there are 6–15 in P. socotrensis var. benalensis. Pleurosira nanjiensis differ from P. inusitata in the radiate striae (vs. irregular areolae in P. inusitata), and differ from P. laevis f. polymorpha in the broadly lanceolate valve outline. Furthermore, the two species can also be separated by the DNA sequences dissimilarity (Desianti et al., 2015). In addition, Metzeltin et al. (2005) described P. minor with a more or less broadly lanceolate outline, but the species has no domed valve and elevated ocelli.
Pleurosira nanjiensis is also similar to the generitype of Odontella, O. aurita, in the presence of a domed cribra. However, O. aurita has centrally located rimoportulae with externally spine-like tubes. Pleurosira nanjiensis is also similar to Odontella obtusa in the presence of 2 or 3 rimoportulae, 2 ocelli, and with no clear separation between the valve face and mantle. However, the ocellate elevations in P. nanjiensis are bluntly rounded and the cingulum is composed of six bands, while in O. obtusa the ocellate elevations are acute and there are only five bands. Moreover, P. nanjiensis has no network of ridge surrounding the areolae on the external valve surface as in O. obtusa (Lavigne et al., 2015). The phylogenetic tree also suggested O. obtusa is closed related to Triceatium rather than P. nanjiensis (Fig. 4). Nonetheless, the species status of O. obtusa needs to be confirmed because the original description and illustrations by Kützing (1844) are rather simple and many subsequent descriptions of the species are inconsistent.
Acknowledgements
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The authors thank Cai Houcai, Chen Wandong and all the staff of the Nanji Islands National Marine Natural Reserve Research Institute for their help in sample collection.
Funding
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  • The Basic Work of Science and Technology Project of China under contract No. 2013FY111100-03; the Nanji Islands National Marine Natural Reserve Postdoctoral Research Funding under contract No. NJKJ-2015-005.
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Article Info
doi: 10.1007/s13131-018-1298-x
  • Receive Date：2017-08-14
  • Online Date：2026-04-14
  • Published：2018-10-25
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  • Received：2017-08-14
  • Accepted：2017-11-30
Funding
The Basic Work of Science and Technology Project of China under contract No. 2013FY111100-03; the Nanji Islands National Marine Natural Reserve Postdoctoral Research Funding under contract No. NJKJ-2015-005.
Affiliations
    1 Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
    2 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
    3 Department of Biology, School of Life Dentistry at Tokyo, The Nippon Dental University, Tokyo 102-8159, Japan
    4 University of Chinese Academy of Sciences, Beijing 100049, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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