Morphology and taxonomy of the Aphanizomenon spp. (Cyanophyceae) and related species in the Nakdong River, South Korea

The purpose of this study is to describe the morphological characteristics of the Aphanizomenon spp. and related species from the natural samples collected in the Nakdong River of South Korea. Morphological characteristics in the four species classified into the genera Aphanizomenon Morren ex Bornet et Flahault 1888 and Cuspidothrix Rajaniemi et al. 2005 were observed by light microscopy. The following four taxa were identified: Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault, Aphanizomenon klebahnii Elenkin ex Pechar, Aphanizomenon skujae Komárková-Legnerová et Cronberg, and Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. Aph. flos-aquae and Aph. klebahnii always formed in fascicles; the others only occurred in solitary. Aph. flos-aquae was similar to Aph. klebahnii, whereas these species differed from each other by the size and shape of fascicles, which was macroscopic in Aph. flos-aquae and microscopic in the Aph. klebahnii. One of their characteristics was that trichomes are easily disintegrating during microscopic examination. C. issatschenkoi could be clearly distinguished from other species by hair-shaped terminal cell. Its terminal cell was almost hyaline and markedly pointed. Young populations of the species without heterocytes run a risk of a misidentification. Aph. skujae was characterized by akinete. Morphological variability of akinetes from natural samples collected in the Nakdong River was rather smaller than those reported by previous study. C. issatschenkoi are described for the first time in the Nakdong River. In addition, Aph. klebahnii and Aph. skujae are new to South Korea.


Background
The genus Aphanizomenon Morren ex Bornet et Flahault 1888 (type species: Aph. flos-aquae) belongs to order Nostocales and family Nostocaceae, which has a worldwide distribution (Rajaniemi et al. 2005a). The species of genus Aphanizomenon and several of its members have been described as the cause for harmful bloom (Mcdonald and Lehman 2013;Ma et al. 2015). Some species can produce hepatotoxic and neurotoxic, such as aphantoxin, anatoxin-a, cylindrospermopsin, and saxitoxin, cyanobacterial secondary metabolites which can cause critical problems (Paerl and Huisman 2009;Ballot et al. 2010;Zhang et al. 2015). Therefore, it is very important for the accurate species identification of Aphanizomenon because of water bloom with several toxin-producing species (Guzmán-Guillén et al. 2015).
The classification of genus Aphanizomenon which frequently form blooms is in some cases difficult that is due to lack of the study for morphology and taxonomy in South Korea. The purpose of this study is to describe the morphological characteristics of the Aphanizomenon spp. and related species from the natural samples collected in the Nakdong River, South Korea.

Methods
The cyanobacteria samples were collected on three stations of the Nakdong River where the stations located in Sangju (N 35°27′ 14.69″/E 128°15′ 27.11″), Daegu (N 35°50′ 35.58″/E 128°27′ 33.92″), and Haman (N 35°23′ 40.89″/ E 128°31′ 11.84″), respectively (Fig. 1). The samples were collected from June 2015 to May 2016 with 1-month interval using the plankton net (mesh size 32 μm). It was preserved in 4% Lugol's solution or formaldehyde water and was transferred at the laboratory. The morphology of trichomes, vegetative cells, heterocytes, and akinetes were studied using Nikon ECLIPSE 80i light microscope with a digital camera. NIS-Elements F 3.0 software was used for image analysis. The following parameters were selected to describe the morphology of the studied specimens: length and width of vegetative cell, heterocytes, and akinetes; morphology of terminal cell; distance between heterocytes and distance between a heterocyte and the nearest akinete (counted as the number of cells); presence or absence of terminal heterocytes and gas vesicles; and shape of trichomes and its aggregation in colonies. All measurements were obtained with the preserved materials.

Results and discussion
Within the genus Aphanizomenon, three clusters were distinguished by Komárek and Komárková (2006) and Komárek (2013) for classification. The first cluster (i) included the type species Aph. flos-aquae Ralfs ex Bornet et Flahault 1888 and Aphanizomenon klebahnii Elenkin ex Pechar 2008, together with Aph. yezoense, Aph. paraflexuosum, Aph. flexuosum, Aph. solvenicum, Aph. platense, and Aph. hungaricum. Trichomes of Aph. flos-aquae and Aph. klebahnii taxa always formed macroscopic and microscopic fascicles, and those were able to cause intensive water blooms in eutrophic stagnant water (Hindák 2000). Aph. flos-aquae was common species with Microcystis spp. and Anabeana spp. and the major component of the water bloom in the Nakdong River (Park et al. 2015;Yu et al. 2014). Whereas Aph. klebahnii was described for the first time in the South Korea. Cluster (ii) included species with slightly curved or flexuous trichomes. The terminal cells were narrowed, elongated, and hyaline with sharply pointed. Akinetes were distant to heterocytes. This cluster included Cuspidothrix issatschenkoi (Usačev) , together with C. elenkinii, Aph. tropicalis, Aph. capricorni, and Aph. ussatchevii. C. issatschenkoi was described for the first time in the Nakdong River. Cluster (iii) was comprised of species described as morphotype of Aphanizomenon gracile with straight, solitary trichomes and with narrowed ends, which belong into the vicinity of Dolichospermum according to molecular sequences. Aph. skujae Komárková-Legnerová et Cronberg 1992 belonged to this cluster, together with Aph. gracile, Aph. Schindleri, Aph. manguinii, Aph. chinense, and Aph. sphaericum. Identification of the species is the first report in South Korea. The four investigated species in the Nakdong River were classified in the genus Aphanizomenon (Aph. flos-aquae, Aph. klebahnii, Aph. skujae) and in the genus Cuspidothrix (C. issatschenkoi). Morphological characteristic of trichomes, heterocytes, and akinetes from natural samples collected in the Nakdong River is shown in Figs. 2 and 3 and Table 1 and that investigated from studies cited is shown in Fig. 4.
Ecology: This species is planktonic in eutrophic reservoir (Komárek 2013). It has shown positive growth within a wide range of temperatures (16-25°C) (Preussel et al. 2009) and can grow below 10°C (Üveges et al. 2012). It has a competitive advantage under situations of low light intensities (Mehnert et al. 2010). We collected this specimen in waterbodies of mesotrophic or eutrophic status (range of total phosphorus 0.017-0.040 mg L −1 ).
Ecology: This species is planktonic in eutrophic up to hypertrophic reservoir (Komárek 2013). It is adapted to high water temperatures (Yamamoto and Nakahara 2006). We collected this specimen in waterbodies of mesotrophic or eutrophic status (range of total phosphorus 0.028-0.036 mg L −1 ).
Ecology: This species is planktonic in lakes. It is distributed in northern and colder parts of temperate zone in Eurasia (Komárek 2013). We collected this specimen in waterbodies of oligotrophic status (total phosphorus 0.009 mg L −1 ).
Material examined: Sangju (Jun. 2015) Cuspidothrix issatschenkoi (Usačev)   (Fig. 2d; Fig. 3A-4~C-4) (John et al. 2002, p. 96, pl. 18k;Rajanieimi et al. 2005b, Fig. 7. c;Komárek and Komárková 2006, Fig. 31;Figueiredo et al. 2011, Fig. 1. a-c;Ballot et al. 2010. Fig. 1;Komárek 2013, p. 668, Figs. 822-823) Fig. 6 Comparison of size and shape of fascicles from natural samples collected in the Nakdong River. a Aphanizomenon klebahnii (microscopic size) and b Aphanizomenon flos-aquae (macroscopic size). Scale bar is 10 mm. Shape of fascicles in circles was depicted by Pechar and Kalina from Komárek and Komárková (2006) Synonyms: Aphanizomenon issatschenkoi Usačev 1938 The presence of this species has been reported in freshwaters from many European countries (Kastovsky et al. 2010) and in Asia including China (Wu et al. 2010;Ballot et al. 2010), Japan (Watanabe 1985), and Singapore (Pham et al. 2011). In South Korea, this species has been described only one time until a recent date in the Han River (Park 2004) and it is described for the first time in the Nakdong River. The species was characterized by solitary, bent, or slightly coiled trichomes. The trichomes were isopolar, cylindrical in central part, and continually narrowed or pointed towards ends (developed trichomes). Other morphological features include a not or slightly constricted at the cross-walls and subsymmetric (Fig. 2d). Cells were cylindrical to long cylindrical, usually with scarce aerotopes, 4.4-7.0 × 2.5-3.3 μm; terminal cell was almost hyaline and markedly pointed. The hair-shaped terminal cell was in general narrower than the vegetative cells and continually elongated ( Fig. 3A-4). Heterocytes were solitary, intercalary, 1-2 (rarely 3) on a trichome, cylindrical, and 6.6-8.7 × 3.4-3.7 μm. Akinetes were not found from natural samples collected in the Nakdong River. This species was easily recognized by trichomes with hair-shaped terminal cells. However, young field populations of this species without heterocytes can be easily misidentified as the very similar Rapidiopsis mediterranea Skuja or as the non-heterocystous life stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju (Moustaka-Gouni et al. 2010).
Ecology: This species is sporadically planktonic in mesotrophic and eutrophic reservoirs (Komárek 2013). It has shown positive growth within a moderate range of temperatures (22-28°C) (Dias et al. 2002). It has also been observed thriving in freshwater, as well as in oligohaline and brackish waters (Marshall et al. 2005). We collected this specimen in waterbodies of eutrophic status (range of total phosphorus 0.032-0.043 mg L −1 ).

Conclusions
The four investigated species in the Nakdong River were classified in the genus Aphanizomenon (Aph. flos-aquae, Aph. klebahnii, Aph. skujae) and in the genus Cuspidothrix (C. issatschenkoi). C. issatschenkoi are described for the first time in the Nakdong River. In addition, Aph. klebahnii and Aph. skujae are new to South Korea.