New Records of Two unknown Micro-filamentous Endophytic Green Algae in Korea: Phaeophila dendroides and Dilabifilum arthropyreniae

Ⅰ. Introduction

Microfilamentous green algae grow on a variety of solid substrata. They also occur as epiphytes or endophytes of larger algae and seagrasses (Gunnarsson & Nielsen, 2016). A little more than 100 species of microfilamentous ulvophycean taxa are reported in AlgaeBase (Guiry & Guiry, 2016), but the true number may be 200 or more species. This difference in number occurs because these algae are difficult to find and identify due to similarities in their appearances and a lack of information.

In Korea, only several microfilamentous endophytic green algae have been reported (Lee et al., 2013; Kim et al., 2014; Ogandaga et al., 2016). The presence of endophytic the Ulvella species was recorded in Chondrus ocellatus fronds in Korea by Lee et al. (2013), although epiphytic U. viridis was first reported on the fronds of Griffithsia japonica (Lee et al., 1998, Lee & Kang, 2002). Later, Kim et al. (2014) reported three endophytic filamentous algae (Ulvella leptochaete, Blastophysa rhizopus, and Bolbocoleon piliferum). Recently, Korean green endophyte Ulvella ramosa and Korean brown endophyte Mikrosyphar zosterae were added by Choi et al. (2015) and Ogandaga et al. (2016), respectively.

However, until now, we have very little information about Korean endophytes. We now have the opportunity to isolate endophytic algae from Grateloupia lanceolata and Bryopsis sp., which have been collected from the shore of Jeju Island and cultured in the laboratory. These species are newly recorded, and are based on morphological and molecular data in the present study.

Ⅱ. Materials and Methods

The two host species, Grateloupia lanceolata and Bryopsis sp., were collected from Gujwa, Jeju Island, on July, 2015. Samples were stored in a cool box for transport to the laboratory, and then unialgal cultures were prepared by incubating specimens containing the target species in sections of host material. These were incubated in culture dishes with 150 mL of pasteurized seawater containing PES medium in 20℃, 100 μmolm-2s-1, 33 psu, and 16:8h LD cycle.

Taxonomic data were obtained from unialgal culture specimens. Measurements are given as length and diameter. Photographs were taken with a CCD camera (MicroPublisher 5.0, Qimaging, Canada) and a digital camera (TG-4, Olympus, Japan) attached to a microscope (DMLB, Leica, Germany). All specimens examined in this study are now deposited in the herbariums of the Department of Marine Biotechnology, Kunsan National University, Kunsan and of the National Institute of Biological Resources, Incheon, Korea.

Genomic DNA was extracted (Qiagen DNeasy Plant Mini Kit), and the plastid-encoded elongation factor tufA (Fama et al., 2002) and ITS (White et al., 1990) were amplified using published primers. Sequences were determined using an ABI 3130xl Genetic Analyzer (Applied Biosystems, USA), and were assembled in DNASIS^®Max 3.0 (MiraiBio, USA). Phylogenetic analyses were performed using the maximum likelihood estimation method. Bootstrap values were calculated with 1,000 replications. The tufA and ITS sequences of other species were obtained from GenBank. Ulothrixzonata and Desmochloris halophila were used as outgroups.

Ⅲ. Results and Discussion

Phaeophila dendroides (P. Crouan & H. Crouan) Batters: 1902: 13.

Lectotype: CO (Brodie et al. 2007: 74).

Type locality: Rade de Brest, Finistère, France.

Korean name: Ggo-in-teol-sog-sal-i-mal nom. nov. (신칭: 꼬인털속살이말).

Specimens examined: NIBRCL0000112299 (Gujwa, Jeju: 13.v.2015), KSNU000010001-000010003 (Gujwa, Jeju: 13.v.2015).

Habitat: Epi/endophytic on other algae.

Morphology: Plants are endophytic in Grateloupialanceolata. They are green color and composed of uniseriate branched filaments with long setae. These plants formed cylindrical cells of the filaments, measured 5‒19 μm in width, and were 8‒10 times as long. Each cell had several pyrenoids. Undulate or twisted Phaeophila-type hair developed from vegetative cells ([Fig. 1]).

[Fig. 1]

Host Grateloupia lanceolata fronds (A-B), and details of Phaeophila dendroides culture specimens (C-G). A, Host species, G. lanceolata B, An infected G. lanceolata by P. dendrodes (arrows); C, Parts of host vegetative plant; D, Chloroplast with several pyrenoids (arrows); E, Twisted Phaeophila-type hairs; F, Mature sporangium (arrow); G, Empty sporangium.

Dilabifilum arthropyreniae (Vischer & Klement) Tschermak-Woess 1971: 452, 453

Type: ?

Type locality: Macao.

Korean name: Sog-sal-i-ma-di-mal nom. nov. (신칭: 속살이마디말).

Specimens examined: NIBRCL0000112302 (Gujwa, Jeju: 13.v.2015), KSNU000010004-000010005 (Gujwa, Jeju: 13.v.2015).

Habitat: Epi/endophytic on other algae.

Morphology: Plants are endophytic in Bryopsis sp. The frond of this species consists of frequently irregular branching uniseriate filaments. The central cells were rounded (4‒9 μm) and branched filaments were cylindrical cells (1.5‒5 μm in width, and 3‒5 times as long). Each cell had a single pyrenoid without hairs. Sporangia were not observed. The end of the filaments were curved ([Fig. 2]).

[Fig. 2]

Host Bryopsis sp. frond (A), and details of Dilabifilum arthropyreniae culture specimens (B-E). A, Host species, Bryopsis sp.; B, Parts of vegetative plant; C, Curved filaments; D, Chloroplast with single pyrenoid (arrows); E, Distal branches (arrows). Note the shape of growth.

Phaeophila dendroides (Phaeophilaceae) was initially described as Ochlochaete dendroides by Crouan & Crouan (1852). Later this species was transferred to genus Phaeophila by Batters (1902). P. dendroides is a cosmopolitan species (Oliveira Filho & Ugadim, 1976; Kitayama & Garrigue, 1998; Brodie et al., 2007; Dawes & Mathieson, 2008). Moreover, this species has been reported from several Asian countries, including Japan (Kitayama & Yoshida, 1998) and China (Tseng, 2009; Phang et al., 2016). However, its occurrence has not been reported in Korea. According to studies of many researchers (Oliveira Filho & Ugadim, 1976; Kitayama & Yoshida, 1992; Kitayama & Garrigue, 1998), this species has a chaetophoraleous appearance, in that is has characteristically twisted setae. Several traits (form of cells and twisted setae) of our specimens fit well with previous other description of this species <Table 1>. In a molecular analysis based on the tufA sequence ([Fig. 3]), this Korean alga nests in the same clade as Phaeophila dendroides from plants on dead leaves of Zostera marina of France (O’Kelly et al., 2004). The genetic distance between both sequences within the clade was calculated as 0.4 %. This species is here newly recorded in the Korean algal flora.

<Table 1>

Comparison of some traits of several Phaeophila dendroides species

[Fig. 3]

Phylogenetic relationships of the green endophyte, Phaeophila dendroides found in this study among other endophytic species. The tree was obtained with a maximum likelihood analysis of a tufA dataset, using a species of Ulothrix zonata as an outgroup. Numbers above branches reflect bootstrap support based on 1,000 replications.

Dilabifilum was originally considered a member of Chaetophoraceae (Tshemark-Woess, 1971). A reappraisal of Dilabifilum Tschermak-Woess (Johnson & John, 1990) reviews the complicated taxonomic history of the genus (Broady & Ingerfeld, 1993). The systematic position of Dilabifilum, is uncertain and referred to “Ulvalesincertae sedis” by Guiry & Guiry (2016). However, Gunnarsson & Nielsen (2016) suggested recently that it is referred to the family Kornmanniaceae, based on morphological similarity with Psedendoclonium and the molecular data obtained by Thüs et al. (2011). Several traits (form of cells and width) of our specimen closely corresponded to a previously described species, Dilabifilumarthopyreniae <Table 2>. However, we did not observe sporangia. In a molecular analysis based on the ITS sequence ([Fig. 4]), this Korean alga nests in the same clade as from plants of the original isolate (467-2) obtained from the Culture Collection of Algae (SAG), University of Göttingen, Germany. Most D.arthopyreniae have been reported in Europe (Vischer, 1953; Ettl & Gärtner, 1995; Gunnarsson & Nielsen, 2016). In Asia, this species has been reported in Japan (Ihda et al., 1996). This species is here newly recorded in the Korean algal flora.

<Table 2>

Comparison of some traits of several Dilabifilum arthropyreniae species

[Fig. 4]

Phylogenetic relationships of the green endophyte, Dilabifilum arthropyreniae found in this study among other endophytic species. The tree was obtained with a maximum likelihood analysis of an ITS dataset, using a species of Desmochloris halophila as an outgroup. Numbers above branches reflect bootstrap support based on 1,000 replications.

Acknowledgments

※ This work was supported by a grant from National Institute of Biology Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201501204), and by a grant from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries of the Korean Government.

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