Two New Benthic Diatoms of the Genus Achnanthidium (Bacillariophyceae) from the Hangang River, Korea

: Two new benthic freshwater species belonging to the genus Achnanthidium were found in Korea. Achnanthidium ovale sp. nov. and A. cavitatum sp. nov. are described as new species based on light and scanning electron microscopy observations and molecular analyses. Both species are compared with the type material of morphologically similar taxa. Achnanthidium ovale di ﬀ ers from other species belonging to the A. pyrenaicum complex in outline, striation pattern, raphe central endings, and freestanding areolae at the apices. Achnanthidium cavitatum di ﬀ ers from other species in the A. minutissimum complex in outline, broad axial central area in the raphel ess valve, and slit-like areolae near the axial central area. We assessed their molecular characteristics by analyzing nuclear small subunit (SSU) rRNA and chloroplast-encoded rbcL gene sequences. Both the morphological comparison and the SSU and rbcL sequence analyses provide strong evidence to support the recognition of A . ovale and A . cavitatum as new species.

The genus Achnanthidium currently includes freshwater monoraphid species with the following characteristics: (1) linear-lanceolate to lanceolate elliptic cells with length and width less than 30 µm and 5 µm, respectively, (2) concave raphe valve, uniseriate striae, and a wide central area; (3) a well-developed raphe that can be straight or turned to one side [4]. Because of their small size and inadequate morphological features, Achnanthidium species can be complicated to identify. Currently, species in the genus Achnanthidium can be divided into three major groups: (1) the A. minutissimum complex, which includes species with straight raphe fissures on the apical area;

Sample Collection, Isolation, and Culture
To collect diatoms, two or three pebbles were collected from the littoral zones (0.1 m depth) of rivers. The sampling points at which diatoms were collected are shown in Table 1 and located in the Hangang River, Republic of Korea ( Figure 1). Epilithon was collected from the surfaces of the stones using a toothbrush. Single diatom cells were isolated using a Pasteur pipette (Hilgenberg GmbH, Germany) and the capillary method [37] under an Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan). Cells were isolated and cultured in 96-well cell plates, and each well contained 160 μL of Diatom Medium (DM) [38]. After 10-14 days of isolation, diatoms reached the exponential growth stage [39]. The cells that grew and had a healthy aspect were transferred into 24-well cell plates with 1 mL of DM. Again, after 10-14 days, the cells that were in good condition were transferred to 50 cm 3 culture flasks with 20 mL of DM. To maintain healthy cells, each strain was sub-cultured at 40-day intervals. All the strains were cultured at 20 °C, with an irradiance of c. 50 μmol quanta m −2 s −1 , and a 12:12 h light: dark cycle with cool white fluorescent light. Two new diatom cultures were eventually established; of these, one culture was used for this study. The other two cultures were preserved at a lower temperature (<10 °C) and light intensity (<20 μmol m −2 s −1 ) for growth limitation.

Light Microscopy (LM)
For LM, both natural and cultured cells were fixed with Lugol solution. To remove organic compounds, HNO 3 and H 2 SO 4 (1:3) were added to the samples, which were then boiled at 100 • C for 2-3 min. To remove the acid from the oxidized cultures, the samples were washed four times with distilled water, following one day of sedimentation. Morphological characteristics were observed using an upright microscope (Nikon E600, Nikon, Tokyo, Japan). Slides of the washed frustules were mounted using Wako Mountmedia (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Light micrographs were collected at 1000× magnification using an MSC-C5.0 microscope digital camera (SONY, Tokyo, Japan). Measurements of length and width of frustules were obtained from at least 50 diatom cells.

Scanning Electron Microscopy (SEM)
For SEM, the washed samples were gently filtered through a 0.2-µm pore-sized GTTP Millipore filter membrane (Millipore Filter Corporation, Cork, Ireland) using gravity. The membrane was then stuck to the SEM stubs with carbon tape (Shintron Enterprise CO., Ltd., Kaohsiung, Taiwan). Mounted specimens were dried for at least 12 h at room temperature. Finally, the specimens were coated for 120 s with platinum and examined using field emission SEM (Nova Nano SEM 450, FEI Inc., Hillsboro, OR, USA).

DNA Extraction, PCR Amplification, and Sequencing
Clonal cultures (10 mL) were prepared in the mid-logarithmic growth phase and centrifuged in a conical tube at 4000× g for 10 min. A DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) was used for genomic DNA extraction. PCR reactions were performed in 40 µL reaction mixtures, and the primers used in the PCR amplification of SSU and rbcL genes are shown in Table 2. Each reaction mixture contained 23.8 µL of distilled water, 4 µL of 10× Ex PCR Buffer (TaKaRa, Tokyo, Japan), 4 µL of dNTP (deoxyribonucleotide triphosphate) (TaKaRa), 0.2 µL of ExTaq polymerase (TaKaRa), 2 µL of each primer, and 4 µL of DNA template. PCR amplification was carried out in a Bio-Rad iCycler (Bio-Rad, Hercules, CA, USA) using the following conditions: pre-denaturation at 94 • C for 4 min; 37 cycles at 94 • C for 20 s, 56 • C for 30 s, and 72 • C for 50 s; and a final extension at 72 • C for 5 min. The PCR products were separated by electrophoresis in 1% agarose gel with a staining solution (Genetics, Dueren, Germany) and then sent to the company BIONICS (ISO: 9001, Seoul, Korea) for SSU rRNA and rbcL gene sequencing. Table 2. Primers used to amplify and sequence the SSU rRNA and rbcL genes.

Phylogenetic Analyses
Sequences were viewed and assembled in ContigExpress (Vector NTI version 1.6, Invitrogen, Grand Island, NY, USA). The SSU rRNA and rbcL sequences from this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank (Table 3). Multiple sequence alignment between the sequences generated in this study and those obtained from the NCBI database was performed using ClustalW [41] in MEGA version 7.0 [42]. The alignments were manually edited, and ambiguously aligned characters were excluded using MEGA version 7.0 [42]. MEGA 7.0 was also used to calculate the genetic distance (p-distance) by means of a bootstrap method with 1000 replicates Diversity 2020, 12, 285 5 of 20 and a Kimura 2-parameter model [42]. The final alignment of the SSU rDNA dataset contained 39 taxa and 1628 characters (including gaps introduced for alignment), and the rbcL dataset contained 52 taxa and 1628 characters (1390 bp). Sequences of Aulacoseira granulata were used as outgroups for the SSU and rbcL phylogenetic trees. Phylogenetic trees for the sequence alignments (SSU and rbcL) were inferred from maximum likelihood (ML) analyses (using RaxML version 8 [43]) and Bayesian inference (using MrBayes version 3.2: [44]). The general time-reversible model with parameters accounting for γ-distributed rate variation across sites was used in all analyses, taking into account a 6-class gamma. Bootstrap analyses for both datasets were carried out for ML with 1000 replicates to evaluate statistical reliability. The Markov chain Monte Carlo method was used with four runs for 10 million generations, sampling every 100 generations. A majority-rule consensus tree was created to examine the posterior probabilities of each clade. The final trees were visualized with MEGA version 7.0.   replicates and a Kimura 2-parameter model [42]. The final alignment of the SSU rDNA dataset contained 39 taxa and 1628 characters (including gaps introduced for alignment), and the rbcL dataset contained 52 taxa and 1628 characters (1390 bp). Sequences of Aulacoseira granulata were used as outgroups for the SSU and rbcL phylogenetic trees. Phylogenetic trees for the sequence alignments (SSU and rbcL) were inferred from maximum likelihood (ML) analyses (using RaxML version 8 [43]) and Bayesian inference (using MrBayes version 3.2: [44]). The general time-reversible model with parameters accounting for γ-distributed rate variation across sites was used in all analyses, taking into account a 6-class gamma. Bootstrap analyses for both datasets were carried out for ML with 1000 replicates to evaluate statistical reliability. The Markov chain Monte Carlo method was used with four runs for 10 million generations, sampling every 100 generations. A majority-rule consensus tree was created to examine the posterior probabilities of each clade. The final trees were visualized with MEGA version 7.0.   Description: Cells are elliptical, 6.3-7.7 µm long, and 3.8-4.1 µm wide. Striae density varies by location; 30-35 in 10 µm in the center, and up to 55 near the apices of the raphe valve. The number of striae on the primary side is higher than on the secondary side of the rapheless and raphe valves. Therefore, a "T" pattern can be seen in the LM images ( Figure 2B-O).

Species Description
At the external part of the raphe valve, the striae are parallel but radiate very slightly and curve near the apices. The shorter striae in the central part consist of 4-6 areolae. The terminal fissures of the raphe are hooked toward the same side (arrow in Figure 3A). Central raphe endings are laterally expanded ( Figure 3C). The sternum is narrow and slightly broader in the central area ( Figure 3A).        The rapheless valve is convex. The axial area is below the valve plane, and it forms a shallow V (arrow in Figure 4A). A row of slit-like areolae is present on the mantles of the raphe and rapheless valves ( Figure 4A). In the oblique view, the helictoglossae are raised internally ( Figure 4B, arrow). Areolae are occluded by hymens, which are connected in adjacent areolae. Pairs of unconnected vimines are present above the adjacent areolae ( Figure 4B); however, the areolae near the margin area are separate from the neighboring areolae. The structure of the areolae from the inner parts of both valves is formed by hymenes with marginal slits; the thickness of the central disk differs from that of the marginal area ( Figure 4C). Hymenes have perforations of the parallel array type (solid arrow) and the centric array type (dotted arrow) ( Figure 4D).
Holotype: A slide of the isolate 180409KCB8B40511, illustrated in Figure 2A-N, was deposited at the Freshwater Bioresources Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210015D).
Isotype: A slide of the isolate 180409KCB8B40511, illustrated in Figure 2O, was deposited at the Freshwater Bioresources Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210015D).
Molecular characterization: Nucleotide sequences of the SSU rRNA and rbcL genes of strain 180409KCB8B40511 were deposited in GenBank (NCBI; accession numbers MK578710 and MK639354, respectively).
Locality  Diversity 2020, 12, x; doi: FOR PEER REVIEW www.mdpi.com/journal/diversity striae on the primary side is higher than on the secondary side of the rapheless and raphe valves. Therefore, a "T" pattern can be seen in the LM images ( Figure 2B-O). At the external part of the raphe valve, the striae are parallel but radiate very slightly and curve near the apices. The shorter striae in the central part consist of 4-6 areolae. The terminal fissures of the raphe are hooked toward the same side (arrow in Figure 3A). Central raphe endings are laterally expanded ( Figure 3C). The sternum is narrow and slightly broader in the central area ( Figure 3A).
The rapheless valve is convex. The axial area is below the valve plane, and it forms a shallow V (arrow in Figure 4A). A row of slit-like areolae is present on the mantles of the raphe and rapheless valves ( Figure 4A). In the oblique view, the helictoglossae are raised internally ( Figure 4B, arrow). Areolae are occluded by hymens, which are connected in adjacent areolae. Pairs of unconnected vimines are present above the adjacent areolae ( Figure 4B); however, the areolae near the margin area are separate from the neighboring areolae. The structure of the areolae from the inner parts of both valves is formed by hymenes with marginal slits; the thickness of the central disk differs from that of the marginal area ( Figure 4C). Hymenes have perforations of the parallel array type (solid arrow) and the centric array type (dotted arrow) ( Figure 4D).
Holotype: A slide of the isolate 180409KCB8B40511, illustrated in Figure 2A-N, was deposited at the Freshwater Bioresources Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210015D).
Isotype: A slide of the isolate 180409KCB8B40511, illustrated in Figure 2O, was deposited at the Freshwater Bioresources Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210015D).         Figure 6A,B). Slit-like areolae are more numerous on the rapheless valve than on the raphe valve ( Figure 6A,B). Striae are more numerous on the primary side of the raphe and rapheless valves than on the secondary side ( Figure 6A,B). On the internal side of the raphe valve, the central raphe endings gently curve in opposite directions ( Figure 6C). The central area of rapheless valves is broadly lanceolate to linear and narrow ( Figure 6D).
The raphe valves are concave, and the rapheless valves are convex ( Figure 7A,B). The axial area is below the valve plane in the rapheless valves ( Figure 7B). Areolae on the valve mantle are elongated to slit-like on both raphe and rapheless valves ( Figure 7A,B). Internally, there are two types of hymenes, including the valve mantle, in the raphe and rapheless valves ( Figure 7C,D, solid arrow). The external valve of the cell can be seen through the broken hymen, which has slit-like openings. Areolae with slit-like openings are loculate ( Figure 7D, solid arrow). On the other side, externally, two shapes of areolae can be seen in the raphe and rapheless valves: slit-like (arrow S) to elongate or round (arrow E) ( Figure 7E,F). There are two types of areola occlusions: (1) round or elongate-round opening, with hymenes between the external and internal valves; and (2) slit-like opening on the external valve, covered by hymenes on the internal valve plate, different from the round or elongate areolae.
Holotype: A slide of the isolate 180419HG03C4C30524, illustrated in Figure 5A,C-AB, was deposited at the Freshwater Bioresources Culture Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210016D).
Isotype: A slide of the isolate 180419HG03C4C30524, illustrated in Figure 5B, was deposited at the Freshwater Bioresources Culture Research Bureau, Nakdonggang National Institute of Biological Resources (slide number FBCC210016D).
Molecular characterization: Nucleotide sequences of the SSU rRNA and rbcL genes of strain 180419HG03C4C30524 were deposited in GenBank (NCBI; accession numbers MK578711 and MK639355, respectively). Etymology: The epithet cavitata refers to the specimens' broad axial central area on the rapheless valve.
Habitat: This species is an epilithon diatom and lives in flowing freshwater. The environmental variables of its habitat are shown in Table 1

Molecular Phylogeny
The phylogenetic positions of Achnanthidium ovale sp. nov. (HYU-D036) and Achnanthidium cavitatum sp. nov. (HYU-D037) were inferred using SSU rRNA and rbcL gene sequences (Figures 2G and 8). ML and Bayesian analyses generated four similar trees that differed in only a few topological features. The results of the SSU-generated phylogenetic trees show that sequences of Achnanthidium species formed a monophyletic group with high statistical support (100% ML bootstrap support and 1.00 Bayesian posterior probability [PP]) ( Figure 8). The phylogenetic positions of A. ovale (HYU-D036) and A. cavitatum (HYU-D037) were clearly different from those of other Achnanthidium species. The similarity scores based on the SSU rRNA data are shown in Table 4. The highest similarity score and lowest p-distance of A. ovale were found in the comparisons to A. reimeri (Arei2) (0.988) and A. anastasiae (Ros1) (0.006), respectively. The highest similarity score and lowest p-distance of A. cavitatum were found in the comparisons to A. catenatum (TCC849) (similarity score = 0.990; p-distance = 0.004).
Diversity 2020, 12, x FOR PEER REVIEW 10 of 19 Diversity 2020, 12, x; doi: FOR PEER REVIEW www.mdpi.com/journal/diversity D036) and A. cavitatum (HYU-D037) were clearly different from those of other Achnanthidium species. The similarity scores based on the SSU rRNA data are shown in Table 4. The highest similarity score and lowest p-distance of A. ovale were found in the comparisons to A. reimeri (Arei2) (0.988) and A. anastasiae (Ros1) (0.006), respectively. The highest similarity score and lowest p-distance of A. cavitatum were found in the comparisons to A. catenatum (TCC849) (similarity score = 0.990; p-distance = 0.004).   The rbcL-generated phylogenetic tree also shows that the position of A. ovale (HYU-D036) is distinct from those of other Achnanthidium species (Figure 9). A. cavitatum (HYU-D037) and A. straubianum (TCC831) form a single cluster with strong support (93% ML bootstrap and 1.00 Bayesian PP). The similarity scores based on rbcL gene sequences are shown in Table 5. Achnanthidium ovale had the highest similarity score (0.956) and lowest genetic distance (0.045) compared with A. anastasiae (Ros1), and A. cavitatum had the highest similarity score (0.971) and lowest genetic distance (0.028) compared with A. straubianum (TCC831).   Table 5. Similarity scores and genetic distances of Achnanthidium species sequences based on 594 bp of chloroplast-encoded rbcL gene sequences. GenBank accession and strain numbers follow the taxon names.

Achnanthidium ovale as a New Species
Achnanthidium ovale sp. nov. has terminal raphe endings that turn to the same side. This characteristic is typical of species belonging to the A. pyrenaicum complex. Table 6 shows a detailed comparison between A. ovale and similar species from the A. pyrenaicum complex taxa: A. rivulare Potapova & Ponader [10], A. pyrenaicum (Hustedt) Kobayasi (Karthick et al. [13]), and A. convergens Kobayasi [45]. Although the smaller A. rivulare is similar to A. ovale in valve outline, these species differ in (1) areola openings, (2) striation pattern on the raphe valve, and (3) internal raphe endings. The areola openings in A. ovale are mostly elongated or sometimes small and rounded, unlike the areolae in A. rivulare, which are mostly rounded. The striae on the apical area of the raphe valve in A. ovale are radiate, unlike the convergent striae of A. rivulare. Internally, A. ovale has deflected raphe central endings, whereas A. rivulare has hooked raphe central endings. In A. rivulare, the number of mantle areolae at the valve ends that do not correspond to the areolae on the valve face varies between 1 and 4 but is usually 2 or 3 ([10]; Figure 5E-K,M,S); however, in A. ovale, the number of areolae that do not have corresponding areolae on the valve face is as high as 5 ( Figure 3A). According to Kobayasi [5], the number of freestanding areolae is a species-specific characteristic in Achnanthidium. Potapova & Ponader [10] considered that the number of areolae varied not only among species, but also within a single valve. Achnanthidium ovale differs from A. pyrenaicum in outline, as the latter has slightly drawn-out ends. Moreover, the outline and striation pattern of A. ovale differ from those of A. convergens. In both SSU rRNA and rbcL phylogenetic trees, A. ovale has a unique phylogenetic position, as appropriate to establish a new species. In addition, it is slightly related to A. daonense and A. anastasiae in the A. minutissimum complex, with less support.
Achnanthidium cavitatum has two conspicuous characteristics that are typically observed in species from the A. minutissimum complex: (a) axial central area broadly lanceolate to linear and narrow, which can be observed under LM ( Figure 5R-AB), and (b) slit-like areolae, mostly near the axial central area, which can be observed using SEM ( Figures 6B and 7B,F). On the other hand, A. cavitatum differs from A. minutissimum in outline and A. saprophilum in outline and number of areolae per stria. Moreover, A. cavitatum differs from A. eutrophilum in outline and central area of the raphe valve and from A. duriense in outline and areola arrangement on internal view [16].
The SSU rRNA and rbcL gene phylogenetic trees indicate that A. cavitatum is part of the clade containing Achnanthidium strains (Figures 2G and 8). Therefore, based on its morphological characteristics and molecular data, it is correct to classify A. cavitatum in the genus Achnanthidium. The axial central area of A. cavitatum differs from those of other Achnanthidium species. In addition, the molecular data show that A. cavitatum has low genetic distance and similarity scores compared to sequences from the NCBI database ( Table 4; Table 5). Therefore, we propose A. cavitatum as a new Achnanthidium species based on morphological and molecular analyses.

Areolae Occlusions and Openings
Loculate areolae are markedly constricted at one surface and occluded [19] by a velum (cribrum, rota, vola) or a hymen at the other. Yana & Mayama [23] described A. pseudoconspicuum var. yomensis to have loculate type areolae and incomplete vimines through lost hymenes. In the present study, we found a similar arrangement in A. ovale: the ultrastructure of the loculate areolae can be seen from the broken valve. Moreover, most A. ovale vimines are incomplete, and the hymens have different thicknesses from the margin area to the central area. However, A. cavitatum also has a different ultrastructure of the valve on the internal view. In the external view, the areola openings are slit-like or round and elongate on both the valve face and mantle ( Figure 7A,B). On the internal view, slit-like and elongate areola openings can be seen through broken hymenes, and vimines are complete between areolae. Around the rapheless valve, the slit-like areola openings occluded by hymenes differ from the hymenes occluded by elongate areolae openings, depending on the depth of the valve ( Figure 7D). Thomas [46] recognized the areola type of A. minutissimum as poroid. However, using SEM, we observed that the areolae of Achnanthidium are loculated and internally covered by hymenes.

Ecological Characteristics of Two Achnanthidium Species
Achnanthidium ovale sp. nov. and A. cavitatum sp. nov. were recorded in the Gye Stream and the Yeongpyeong Stream (Korea), respectively. The summary of environmental data is shown in Table 1. Previous studies on Achnanthidium stated that species of this genus live in alkaline to acidic environments [14]; this is supported by the results of the present study. Achnanthidium ovale and A. minutissimum were collected from an alkaline environment, whereas A. cavitatum was collected from an acidic environment. The water velocity in the location of the two species was 0-80 cm/s ( Table 1). Dissolved oxygen (5.93-9.86 mg/L), water temperature (4.10-11.71 • C), conductivity (57-148 µS/Cm), and turbidity (0.0-5.4 NTU) in the two locations differed widely. Studies have shown that water-quality assessment methods can be based on genus-level identifications because species within a genus can live in different ecological conditions [15,[47][48][49]. The two species in our study, although they are from the same genus, live in different pH, dissolved oxygen, water temperature, velocity, conductivity, and turbidity conditions; therefore, our results support previous conclusions about the genus Achnanthidium.
Achnanthidium ovale was collected from a stone in Gye Stream, which has a fast flow and low conductivity and turbidity. Land use and cover conditions within a 1 km radius of this area are forest (50%) and agriculture (50%) [50]. The dominant species here is A. minutissimum (77.31%), a widespread species found in low abundance in polluted rivers [15].
Achnanthidium cavitatum was collected from a stone in the Yeongpyeong Stream, which is a slightly acidic environment with low conductivity and turbidity. Land use and cover conditions of this area are forest (80%) and urban (20%) [50]. The stream is fast-flowing and has a stony substratum. The dominant species in this area is Hannaea arcus var. recta Idei (41.33%), which is a saproxenous species [51]. The subdominant species is Achnanthidium minutissimum (22.45%).