Polyphasic characterization of Stigonema dinghuense, sp. nov. (Cyanophyceae, Nostocophycidae, Stigonemaceae), from Dinghu Mountain, south China Author Song, Gao-Fei Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China. & Graduate University of Chinese Academy of Sciences, Beijing, 100049, P. R. China. Author Xiang, Xian-Fen Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China. Author Wang, Zhong-Jie Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, P. R. China. Author Li, Ren-Hui Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China. reli@ihb.ac.cn text Phytotaxa 2015 2015-06-17 213 3 212 224 http://dx.doi.org/10.11646/phytotaxa.213.3.2 journal article 302105 10.11646/phytotaxa.213.3.2 24a32f66-f68c-4f93-899a-3a592a89f44d 1179-3163 13638055 Stigonema dinghuense Song et Li , sp . nov . ( Fig. 1 ) Thallus macroscopic, caespitose, cushion like, dull-brown to blackish-green. Sheaths thin, firm, colorless, yellow or yellowbrown. Mature filaments polyseriate, heteropolar, 41.3–77.1 μm (=57.8μm) wide, richly branched at the base. Branches Ttype and narrower than the main filament, 14.4–50.2 μm (=34.8 μm) wide, with long, broadly developed lateral branches or with short mammilliform hormogonia. Hormogonia short, uniseriate, arising on both sides, 13.2–27.9 μm (=19.6 μm) wide and 35.2–59.2 μm (=45.8 μm) long. Cells spherical, 8.2–18.3 μm (=12.6cμm) wide and 8.8–18.5 μm (=13.1μm) long, in older parts regularly barrel-shaped. Heterocytes mostly lateral, nearly spherical, 6.2–13.8 μm (=9.2 μm) wide and 6.2–12.9 μm (=8.5μm) long. Akinetes not observed. Type: — CHINA . Guangdong Province : Dinghu Mountain, 23°9’34.19” N , 112°33’23.62” E , 415 m a.s.l., G . F . Song , 22 January 2013 ( holotype CHCB ! DH- 2013012030-1). FIGURE 1. Main characteristics of Stigonema dinghuense . A. Habit of thallus, B. Mature filaments. C. Lateral branches. D. Hormogonia. Scale bars: 200 μm (A), 50 μm (B, C), and 20 μm (D). Etymology: —The name of the species was chosen for the sampling location. Habitat: —Wet rocky walls in the temperate region. Notes: —Several species morphologically similar to S . dinghuense have been described (see a comparison in Table 2 ). This species was found only in unpolluted soil habitats and under mosses on wet rocky walls from the temperate region. These populations are similar to S . mamillosum according to the descriptions in the literature, but there are doubts about their identity outside North Europe and North America. Despite many records of S . mamillosum outside of this area of distribution have been reported, there are few morphological descriptions, indicating that the S . mamillosum requires a revision comparing the tropical, subtropical, and temperate populations with those from subfrigid and frigid zones. Pigment examination: —Observations under fluorescence microscope indicated that S . dinghuense simultaneously contained chlorophyll a , phycocyanin, and phycoerythrin ( Fig. 2 ). The fluorescence intensity of branches was significantly higher than that of the main filaments. Chlorophyll and phycocyanin are widely present in cyanobacteria, and phycoerythrin is a taxonomically useful marker for certain cyanobacterial groups/species. TABLE 2. Several species morphologically similar to Stigonema dinghuense with their main morphological diagnostic characters
S . dinghuense S . mamillosum S . lechangense S . multipartitum S . sinuatum S . leprieurii
Colony Brown or blackish green Dark brown or blackish green Brownish-black or black Bluish-green, brown or Brown -
Sheath Colorless, yellow or Colorless, yellow or yellow- Wide, yellow-brown or black Following the outline of Individual, brown and Wide, hyaline or brown,
yellow-brown, thin and brown, firm, lamellate, brown, scarcely lamellate whorls, wide yellow to lamellate smooth from outside,
firm around cells or cell groups rusty brown homogeneous
there are individual
mucilaginous, stratified
Main Multiseriate, 5-seriate, envelopes Multiseriate, filaments up to Multiseriate, 3–5- seriate, Multiseriate, up to 8- Multiseriate, up to 5-seriate, Rigid, 62–102.5μm wide,
filaments 41.34–57.79–77.11 μm 65(90) μm wide 37–60 μm seriate, 49–160(333) μm 80–100(120) μm wide usually 3–4-seriate
Branches Branches 14.38–50.18 μm Branches ±40–50 μm wide Branches wider than wide Attenuated at bases and Branches only a little Filament with numerous lateral
main filament, pectinate, slightly narrower thinner than main filaments, branches with different lengths.
narrowed to the apex, up 40–60 μm wide Branched perpendiculary to the
to 150×85 μm main filament, 28.7–53.1(90)
Cell shape Spherical, rounded- Spherical or irregularly Cells in groups, enveloped Cells generally rounded- Cells globose or subglobose μm wide, with cylindrical ends Smaller peripheral cells
quadrate, barrel-shaped, spherical by individual lamellated quadrate to subcylindrical. are sunspherical, oblong or
Key or irregularly spherical With long, broadly With numerous lateral sheaths. Hormogonia long, usually Filaments rigid, Filaments blue-green with subquadratic Central cells in main filaments
character developed lateral branches perpendicular short developing unilaterally multiseriate, with up to 8 wavy outline distinctly larger than the
or short mammilliform mammilliform hormogonia from main trichomes cells aside, with principal external ones or in branches
hormogonia (hormocytes) and inflated branched, axes up to 80–120 μm wide
Ecology On wet rocks, In mesotrophic rivers, uniseriate and cylindrical On wet rocks, Water biotope, On wet rocks in Aquatic, attached to substrates
Subtropical region submersed, benthic, subfrigid Subtropical region Pantropical and subtropical mountainous, temperate in stagnant and slowing
and frigid zones regions regions streaming waters, in tropical
regions
TABLE 3. Comparison of 16S rRNA gene sequence similarity among Stigonema and outgroup taxa
Seq-> 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
1. Stigonema dinghuense DHS 0071 -
2. Stigonema dinghuense DHS 0072 99.2 -
3. Stigonema turfaceum CBFS A-031 97.6 98.3 -
4. Stigonema ocellatum CBFS A-032 97.6 98.0 98.0 -
5. Stigonema ocellatum CBFS A-029 96.5 96.4 97.0 96.4 -
6. Stigonema ocellatum SAG 48.90 96.5 96.7 96.6 97.6 96.8 -
7. Stigonema ocellatum SAMA 35 96.2 96.2 96.3 96.6 97.3 97.8 -
8. Stigonema sp. WI53 95.9 96.1 96.5 96.3 96.6 98.6 96.9 -
9. Stigonema panniforme CBFS A-026 96.4 96.5 96.8 96.8 97.7 97.1 98.2 96.5 -
10. Stigonema tomentosum CBFS A-028 96.8 97.2 97.3 97.0 96.4 95.8 95.9 95.9 96.4 -
11. Stigonema informe CBFS A-034 97.7 98.5 98.0 98.0 96.2 96.7 95.9 96.1 96.2 97.6 -
12. Stigonema hormoides CBFS A-030 97.1 96.7 96.3 96.7 95.9 95.8 95.6 95.1 95.7 98.0 97.3 -
13. Stigonema cf. robustum CBFS A-027 97.0 97.4 98.2 97.9 96.7 96.4 96.0 96.4 96.6 98.0 98.7 97.2 -
14. Chlorogloeopsis fritschii PCC 6912 91.3 91.7 91.5 91.8 91.3 92.8 92.1 93.0 91.8 91.7 91.7 91.5 91.5 -
15. Fischerella muscicola HA 7617_LM2 91.8 92.3 92.7 92.4 92.1 93.8 92.8 94.0 92.4 91.5 92.3 91.0 92.2 92.1 -
16. Hapalosiphon delicatulus IAM M 266 92.3 92.4 92.7 92.8 92.5 94.7 93.6 94.4 93.3 92.2 92.6 91.7 92.6 92.3 97.3 -
17. Iphinoe spelaeobios LO 2B1 91.6 92.1 92.2 91.9 91.6 92.9 92.5 93.0 92.4 91.6 92.4 91.1 92.1 92.0 93.1 92.8 -
18. Loriellopsis cavernicola LFB 5 89.7 89.6 89.8 89.9 89.9 90.5 89.9 89.9 90.1 88.9 90.1 89.1 89.8 89.1 88.7 89.7 90.3 -
19. Mastigocladopsis repens MORA 92.9 93.0 93.1 93.2 93.2 93.3 93.8 93.5 93.8 93.3 93.5 93.0 93.6 91.7 91.7 92.4 94.1 90.0 -
20. Nostochopsis lobatus strain 92.1 92.3 92.7 92.6 93.1 92.5 94.4 93.3 94.0 92.6 92.0 93.0 91.8 92.6 92.7 97.6 97.1 92.7 90.0 92.4 -
21. Capsosira lowei HWF 3JJ1 93.6 94.3 94.4 94.0 94.2 94.2 93.8 94.6 94.0 94.4 94.5 93.6 94.6 91.2 90.8 90.9 90.8 88.4 92.4 91.2 -
22. Symphyonema sp. strain 1517 93.0 93.3 93.9 93.5 93.2 93.6 94.1 94.3 93.7 93.6 94.1 93.0 94.3 92.0 92.8 92.6 94.2 90.0 96.4 93.2 92.3 -
23. Westiellopsis prolifica VBCCC 022 91.1 91.5 92.0 91.4 91.9 93.0 92.3 93.1 91.6 91.5 91.7 91.0 91.6 91.8 97.3 97.0 92.3 88.9 91.3 97.0 90.5 92.0 -
24. Nostoc commune HK 02 94.3 94.9 95.1 94.9 94.5 95.4 94.8 95.4 94.7 95.0 95.0 94.3 94.9 91.1 91.8 92.2 92.6 88.8 93.3 92.5 96.4 93.0 91.1
FIGURE 2. Microphotographs by fluorescence microscope of a free living filament. A. No filter. B. Using the channel for chlorophyll a . C. Using the channel for phycocyanobilin. D. Using the channel for phycoerythrobilin. Scale bar = 50 μm. Phylogenetic analysis: —The direct PCR without routine DNA extraction resulted in obtaining two 16S rRNA gene, two rbc LX and one nif H sequences. The two 16S rRNA gene sequences from the population of S . dinghuense showed 99.2% similarity, and these two sequences shared 96.5% and 96.7%, 96.2% and 96.2%, 97.6% and 98.3% similarities with Stigonema ocellatum SAG 48.8, S . ocellatum SAMA 35, and S . turfaceum CBFS A-031, respectively. The S . dinghuense and other Stigonema sequences obtained in GenBank shared relatively high similarities, within the range of 95.9%─98.3% ( Table 3 ). The similarities in nif H and rbc LX sequences among heterocytous cyanobacteria including S . dinghuense examined in this study, are shown in Tables S2 and S 3 . Based on 16S rRNA gene sequences from 81 cyanobacterial taxa, including both S . dinghuense sequences in this study, the phylogenetic trees were constructed using the NJ, ML, and Bayesian methods. The ML tree is shown in Fig. 3 , with support values from the three methods at each node. Phylogenetic analysis indicated that S . dinghuense was in a very deep branch within the heterocytous cyanobacterial group, together with S . turfaceum . The heterocytous cyanobacteria were separated into five clusters (A, B, C, D and E), and S . dinghuense was located within the Stigonemataceae (B1) ( Fig. 3 ). The phylogenetic trees based on nif H and rbc LX sequences are also shown in Figs. 4 and 5 respectively.