Encyonema equalis L. Tan & B.H. Kim, 2020

Tan, Ling, Wang, Pengbin, Cho, In-Hwan, Hwang, Eun-A, Lee, Hyuk & Kim, Baik-Ho, 2020, Morphology and phylogenetic position of three new raphid diatoms (Bacillariophyceae) from Hangang River, South Korea, Phytotaxa 442 (3), pp. 153-182 : 170-176

Encyonema equalis L. Tan & B.H. Kim , sp. nov. ( Figs 50–63 , LM; Figs 64–69 , SEM)

Description: —Cells solitary. Valves asymmetrical and dorsiventral, ventral margin straight to slightly convex and dorsal margin strongly convex, very slightly protracted to rounded ends ( Figs 51–67 ). Valve length 14–15 µm, width 5–6 µm, 11–12 striae in 10 µm, 33–35 areolae in 10 µm. Striae uniseriate with the ventral striae almost parallel and dorsal striae parallel to slightly radiate near ends. One slightly shortened dorsal stria in the central area ( Figs 64, 65 ). Width of striae equal from central area to margin area ( Figs 67, 69 ). Areolae always elongated externally and internally, except at apices, where the areolae become rounded ( Fig. 66 ). Areolae separated by siliceous struts; a pair of short projections protrude from the opposite position of the adjacent virgae located on the siliceous strut internally ( Figs 67, 69 ). Distance between pair of projections equal near either the central area or the margin area. Rings between pair of projections rarely found ( Fig. 67 ). Raphe positioned near the ventral margin; raphe sternum wide. Distal raphe fissures strongly hooked towards ventral side externally and slightly deflected to ventral side end with small helictoglossae internally, distant from the valve apices ( Figs 66, 67 ). Proximal raphe fissures deflected to dorsal sides externally and hooked towards the dorsal side at right angles internally ( Figs 68, 69 ). Axial area slightly wide. No stigma present.

Type: — Republic of Korea. Gyeonggi-do Province: Byeogundong Valley, 37°41′11.7′′N, 127°04′13.0′′E, river epilithon, L. Tan & M. Z. Miao, 11 December 2017 (Holotype SR!, population on slide SRB11 B 40317, illustrated here in LM as Figs –. Paratype SR!, population on slide SRB11 B 40317a, illustrated here in SEM as Figs –. Isotype HYU!, population on slide HYU-D038) The strain was deposited at the Freshwater Bioresources Culture Research Bureau, Nakdonggang National Institute of Biological Resources with deposition number is ‘ FBCC 210017D’.

Etymology: —The specific epithet refers to the distance between a pair of projections, which are always equidistant on a stria.

Ecology: —The type population of Encyonema equalis was epilithic in Byeogundong Valley, South Korea. Water conditions were: alkaline pH (8.1), low specific conductance (230 μS/cm), low turbidity (7.3 NTU) and high dissolved oxygen (13.5 mg /L). Relatively common species recorded in the same location were: Achnanthidium minutissimum , Gomphonema lagenula , and Achnanthes convergens .

Molecular characterization: —The nucleotide sequences of the SSU rRNA and rbcL genes of the strain were deposited in GenBank (NCBI), under the accession numbers MK567894 and MK576041.

Remarks: —Comparison of morphological characteristics of Encyonema equalis and other closely related species was shown in Table 9. When observed using a LM, Encyonema equalis is similar to E. minutum with the same dimensions. However, it can be easily distinguished from E. equalis by its denser striae, rounded external areolae adjacent to the proximal raphe ends, and narrow axial area. Encyonema quails is morphologically and molecularly closely related to E. silesiacum and E. minutum var. pseudogracilis (Cholnoky) D.B. Czarnecki (1994: 157) . Encyonema silesiacum (Bleisch) D.G. Mann in Round, R.M. Crawford & D.G. Mann (1990: 667) has a very similar valve shape but it differs from E. equalis by the presence of fewer areolae. Encyonema minutum var. pseudogracilis differs from E. equalis in terms of length, width, number of areolae, and valve apices; in addition, it can be easily differentiated from E. equalis because its ventral margin expands at the centre and its ventral striae vary in density and length. Encyonema silesiacum and Encyonema minutum var. pseudogracilis have a stigma in the central dorsal stria, whereas E. equalis lacks this stigma. The three aforementioned species significantly differ from E. equalis in the distance between a pair of projections; in E. equalis , this distance is almost always equal, but it becomes wider in the striae adjacent to the margin. For instance, the size ranges of E. prostratum and E. muelleri are quite different those of E. equalis , and the ventral striae of E. prostratum are radiate to convergent at the ends.

Taxon Encyonema equalis Encyonema silesiacum Encyonema minutum E. minutum var. Encyonema prostratum Encyonema muelleri pseudogracilis

Length (μm) 14-15 14–44 7–23 42–60 48–60 20–60

Width (μm) 5-6 5.9–9.6 4.2–6.9 12.3–13.0 18–21 8–12

Striae/10 μm 11-12 11–14 15–18 7–13 7–8 9–15

(b 1 +b 2)/(a 1 +a 2) 0.73-0.8 0.8–0.94 0.82 n.d. n.d. n.d.

Areolae/10 μm 33-35 28–32 34–38 21– 24 n. d. n.d.

Dorsal margin Convex Convex Convex Broadly rounded Highly arched Strongly convex

Ventral margin Slightly convex Straight/Slightly convex in the Straight Expand at the center Slightly convex Tumid at the center center

Axial area Slightly wide Narrow Narrow Narrow near the apices, Expanded at the apices, Narrow, broadening to widens toward mid- circular to oval in central form an indistinct central valve area area

Apex Slightly protracted, No protracted/Slightly Rounded/Slightly Rounded/Slightly Slightly produced, broadly Rounded/Slightly rounded protracted, narrowly rounded protracted apiculate rounded protracted

External Dorsal Slightly radiate Slightly radiate Slightly radiate Radiate Radiate n.d.

striae Ventral Almost parallel Slightly radiate in central Slightly radiate in Radiate to convergent Radiate to convergent at n.d.

section central section the ends

Parallel/Slightly convergent Parallel/Slightly towards the poles convergent towards the poles

Areolae Almost elongate Elongate apically, round near Elongate apically, round n.d. n.d. n.d.

the raphe near the raphe

Areolae in LM Invisible Visible Visible Visible Visible Visible

Ring between pair of Absent Existent Existent n.d. n.d. n.d.

projections

The distance between Consistent Wider close to the margin Consistent n.d. n.d. n.d.

two projections

Stigma Absent Existent Existent Existent n.d. Absent

Reference This study Krammer (1997a, 1997b); Krammer (1997a, Cholnoky (1958); Krammer (1997b) KÜTzing (1849); Hofmann et al. (2011) 1997b); Hofmann et al. Czarnecki (1994) Krammer (1997a) (2011)

We obtained 18 aligned sequences of SSU rRNA and 17 aligned sequences of rbcL sequences ( Table 13) from GenBank to perform the phylogenetic analyses. Encyonema equalis adjoin the Encyonema clades in the SSU rRNA ( Fig. 70 ) and positioned in the rbcL ( Fig. 71 ) phylogenetic trees. In rbcL phylogenetic tree, Encyonema equalis is closed to the Encyonema muelleri , Encyonema caespitosum and Encyonema macedonicum formed group, which is not show the similar in the SSU tree as Encyonema equalis is out of Encyonema formed clades, though all of Encyonema spp. are closed than other genus species. In phylogenetic analyses, Encyonema equalis were separated to Encyonema muelleri , Encyonema caespitosum and Encyonema macedonicum , with 64% support in the rbcL tree. Both SSU rRNA and rbcL trees show Encyonema equalis is far away to the E. minutum complex. The highest similarity scores (0.990 and 0.963) and lowest genetic distances (0.008 and 0.036) were found between E. equalis and E. minutum in the SSU rRNA and rbcL gene analyses, respectively. Species with similarity scores under or equal to 0.990 ( Table 10) formed a separate branch but were also located in the Encyonema clades. The highest similarity score using the SSU rRNA data (0.990) was between E. equalis and E. minutum , but the similarity score of the rbcL gene between these species was only 0.963 ( Table 11). E. equalis and E. minutum should be two different species according to the phylogeny and genetic distance analysis. Thus, the morphologic and molecular analyses indicated the existence of several differences between E. equalis and other related species of the genus, showing that it is indeed a new species.

Ecology of sampling sites: —In our study, twenty five diatom taxa were identified ( Table 12), this includes 11 orders, 14 families, 15 genera, 25 species.

In S1, we isolated epilithic diatoms of the following seven species: Fistulifera saprophila (Lange-Bertalot & Bonik) Lange-Bertalot (1997: 73) , Gomphonema affine Kützing (1844: 86) , Nitzschia palea (Kützing) W. Smith (1856: 89) , Navicula minima Grunow in Van Heurck (1880: pl. XIV [14]) Sellaphora seminulum (Grunow) D.G. Mann (1989: 2) , Sellaphora pupula (Kützing) Mereschkovsky (1902: 187) , and Conticribra weissflogii (Grunow) Stachura-Suchoples & D.M. Williams (2009: 482) . The relative abundance of Navicula minima was of 3.6%; this species is commonly found in eutrophic and human-impacted polluted environments, besides being associated with heavy metal pollution and pesticides ( Lange-Bertalot & Bonik 1976, Weber & McFarland, 1981, Dayner & Johansen, 1991, Delgado & Pardo, 2014). The relative abundance of the other species was of less than 1%.

Achnanthes convergens was the dominant species in S2. We also isolated Ulnaria ulna from S2, but its relative abundance was less than 1%. This site is a mountainous terrain with high forest coverage. Sits 3 is at the same mountain stream, and the dominant species was Achnanthidium minutissimum (53% of species relative abundance). Gomphonema parvulum (Kützing) Kützing (1849: 65) , Eunotia perpusilla Grunow in Van Heurck (1881: pl. 34), Fragilaria capucina , Fragilaria vaucheriae , and Tabellaria flocculosa (Roth) Kützing (1844: 127) were isolated in S3. Among them, Gomphonema parvulum had a 9.1% of relative abundance and it is pollution-tolerant; it is present in eutrophic waters with a neutral pH value ( Kobayasi & Mayama, 1982).

Sites 4, 5 and 6 had similar water quality parameters, except for turbidity. Achnanthidium minutissimum was the most abundant species in S4 (77.3%) and S5 (42.9%), and we isolated it from S4. Gomphonema parvulum and Cyclotella meneghiniana Kützing (1844: 50) were isolated from site 5. In site 6, we got Nitzschia amphibia , but there were 2 predominant diatom taxa different from the former two sites, that were Diatoma vulgaris (25.6%) and Encyonema minutum (23.2%).

The pH value of S7, S10 and S11 more than S9, but the dissolved oxygen was very low. Achnanthidium alteragracillimum Round & Bukhtiyarova (1996: 349) has high abundance in all three locations. Fragilaria mesolepta Rabenhorst (1861 : no. 1041), Surirella librile (Ehrenberg) Ehrenberg (1845: 139) , Encyonema silesiacum (6.3% of species relative abundance) and Navicula cryptotenelloides Lange-Bertalot (1993: 105) were isolated from there.

Finally, the environmental parameters of sites 8 and 9 very similar despite each them being dominated by a different diatom species. Achnanthidium minutissimum had a very high species relative abundance (39.6%) in S8, we judged the ecological condition of this site to be good by its subdominant species like Hannaea arcus var. recta (Cleve) M. Idei (nom. nud.) and Achnanthes convergens . We isolated Fragilaria capucina and Navicula cryptotenella Lange-Bertalot in Krammer & Lange-Bertalot (1985: 62) from S8. Hannaea arcus var. recta dominated S9 and we obtained Gomphonema affine and Humidophila schmassmanii (Hustedt) Buczkó & Wojtal (2015: 28) .

In the process of investigating diatom species diversity in the Hangang River, three species that could not be identified were investigated using morphological and molecular methods. Thus, we described them as new species in the genera Fragilaria , Nitzschia and Encyonema . Due to each new species were only found in one location, there lack of more environmental information on these three species at different sites. Thus, further study on the ecology of these three species is needed.

Figures

FIGURE 70. Molecular phylogenetic analysis by maximum likelihood (ML) method based on the SSU rRNA showing the phylogenetic position of Encyonema equalis sp. nov. and other species. The tree with the highest log likelihood (-3457.14) is shown. The analysis involved 18 nucleotide sequences.All positions with less than 95% site coverage were eliminated. There were a total of 1312 positions in the final dataset. Species analyzed in this study are overstriking. Only bootstrap values over 50% are shown on tree. Cymbella affinis was chosen as outgroup.

FIGURE 71. Molecular phylogenetic analysis by maximum likelihood (ML) method based on the rbcL gene showing the phylogenetic position of Encyonema equalis sp. nov. and other species. The tree with the highest log likelihood (-2191.15) is shown. The analysis involved 17 nucleotide sequences.All positions with less than 95% site coverage were eliminated. There were a total of 685 positions in the final dataset. Species analyzed in this study are overstriking. Only bootstrap values over 50% are shown on tree. Amphora caribaea was chosen as outgroup.

FIGURES 50–63. Light microscope images of Encyonema equalis sp. nov.. 50. Girdle view. 51–63. Valve view. Scale bar = 10 μm.

FIGURES 64–69. Scanning electron microscope images of Encyonema equalis sp. nov.. 64, 65. External and internal view of valve. 66, 67. External and internal view of valve apices on one side, showing pars of struts (S) between areolae. 68, 69. External and internal view of the central area, showing the dorsally bent proximal raphe fissures. Scale bars: 64, 65 = 2 μm; 66–69 = 1 μm.