INTRODUCTION

The filamentous brown algal genus, Feldmannia, was described by G. Hamel (1939) based on F. lebelii. It is characterized by having basal meristematic zones in axial filaments, branches below meristematic zones and plurilocular sporangia developing from below meristems (Kim and Lee 1994;Abbott and Huisman 2004;Kim 2010). Currently, 28 species of Feldmannia have been known around the world (Guiry and Guiry 2019). Of them, six species have been reported in Korea (Kim 2010): F. lebelii (Areschoug ex Crouan and Crouan) Hamel, F. globifera (Montagne) Hamel 1939, F. irregularis (Kützing) Hamel, F. rhizoidea Hollenberg and Abbott, F. indica (Sonder) Womersley and Bailey, and F. mitchelliae (Harvey) Kim.

Feldmannia chitonicola (De A. Saunders) Levring was originally described as Ectocarpus chitonicolus De A. Saunders from California based on a small specimen growing on the back of a chiton (Saunders 1898). It is characterized by having small size (1-2 mm), epizoic habitat, mostly un-branched erect filaments, plurilocular sporangia produced laterally on basal part of erect filaments and occasionally on prostrate filaments, sessile or short pedicellate plurilocular sporangia, and 2 types (big lanceolate and small ovate) of plurilocular sporangia (Saunders 1898;Setchell and Gardner 1925;Abbott and Hollenberg 1976). Feldmannia chitonicola has been reported only from eastern Pacific, in California (Abbott and Hollenberg 1976), Chile (Ramirez and Santelices 1991) and Peru (Acleto 1980).

We collected unidentified tiny filamentous brown algae from west and east coasts of Korea and cultured them. We observed their detailed morphology and developmental pattern in laboratory cultures. We also analysed molecular data based on rbcL and cox1 genes for their phylogenetic relationships with others in their respective genera and a few selected species within the Ectocarpales. We identify them as Feldmannia chitonicola and add it to the Korean marine algal inventory, representing the first report of this species in western Pacific.

MATERIALS AND METHODS

1. Culture and Morphological studies

Two unialgal isolates of filamentous brown algae were established from samples collected from west and east coasts of Korea in November, 2017 and February, 2019. Young germlines were inoculated in Petri dishes (60 mm diameter×15 mm depth) containing PES medium (Provasoli 1968). Cultures were kept at 10°C, 16°C and 20°C under 20-30 μmol photons m^-2 s^-1 white fluorescent light and 14 : 10-h light/dark photoperiod. Medium was renewed weekly.

After culture process, thalli were sorted into voucher herbarium specimens, silica gel samples, and formalin samples (4-5% formalin/seawater). Fresh cultured materials were used for morphological analysis. Photomicrographs were taken using an Olympus BX51TRF microscope (Olympus, Tokyo, Japan) and an Olympus DP71 camera. At least 25 individuals were selected for the determination of quantitative characters and their means and standard deviations were calculated. For developmental study, a Leica DMi8 inverted microscope (Leica, Wetzlar, Germany) equipped with a Leica DFC450C camera was used. The percentage of fertile plants and different morphologies of sporangia at different temperatures were calculated based on at least 50 plants or sporangia, respectively. Permanent slide specimens were mounted by 70% karo syrup and the examined representative specimens were deposited in the herbarium of Chosun University (CUK) and National Institute of Biological Resources (NIBR), Korea.

2. Molecular study

Genomic DNA was manually extracted from silica-gel samples using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). The extracted DNA was stored at - 20°C and used to amplify rbcL and cox1. Polymerase chain reaction (PCR) was carried out with a Veriti 96-well Termal cycler (Applied Biosystem). The rbcL gene was amplified using the primer combinations NDrbcL2-DRL1R and DRL2F-R3A (Kogame et al. 1999;Hwang et al. 2005) with HelixAmp Ready-2x-Go Series (NanoHelix Co., Ltd., Daejeon, Korea). The GAZF2-GAZR2 combination of primers was used for cox1 (Saunders 2005;Lane et al. 2007). PCR products were purified using a PCRquickspinTM PCR product purification kit (iNtRON Biotechnology, Inc, Seongnam, Korea). Determination of the nucleotide sequence or sequencing was performed by Macrogen Inc., Seoul, South Korea. DNA sequence data (rbcL and cox1) were compiled by the present study and obtained from GenBank and aligned with ClustalW (Thompson et al. 1994). New sequences obtained from Feldmannia chitonicola have been deposited in EMBL/GenBank under the accession numbers MN092346 (CUK18833) and MN092347 (CUK19719) for rbcL, and MN092344 (CUK 18833) and MN092345 (CUK19719) for cox1.

Phylogenetic analyses were conducted using MEGA version 6.06 (Tamura et al. 2013). Maximum likelihood analyses were conducted using the GTR+G+I model, with 1,000 bootstrap replicates. A bayesian inference was performed using MrBayes 3.2.6 (Huelsenbeck and Ronguist 2001;Ronguist and Huelsenbeck 2003). Markov chain Monte Carlo runs were conducted for 2 million generations, each with one cold chain and three heated chains using the GTR+Γ+I evolutionary model and sampling and printing every 1,000 generations. Summary trees were generated using a burn-in value of 800.

RESULTS

Feldmannia chitonicola (De A. Saunders) Levring, 1960

전복솜털 (신칭) (Figs. 1-3)

Type locality: Pacific Grove, California, USA

Habitat: Epiphytic on Sargassum thunbergii.

Material examined: NIBROR0000001611 & CUK188 33 (= MBRB0100TC18833), Chaesokgang, Byeonsanmyeon, Buan-gun, Jeollabuk-do, Korea (35°37ʹ27.04″N, 126°27ʹ56.88″E), November 17, 2017, T. O. Cho and B. Y. Won, at 1 m depth by hand; CUK19719 (=MBRB 0100TC19719), Guryong Pohang, Guryong Pohang-eup, Pohang-si, Gyeongsangbuk-do, Korea (35°58ʹ48.53″N, 129°34ʹ17.33″E), February 02, 2019, T. O. Cho and B. Y. Won, at 1 m depth by hand.

World distribution: Korea, USA, Chile, Peru (Guiry and Guiry 2019).

Culture studies

Culture strains produced plurilocular sporangia after 10-12 days in culture. Spores were settled without showing any signs of sexuality (Fig. 1A). They developed a “germ tube” (Fig. 1B) which septated by cross-wall and later became rhizoid (Fig. 1C). The first erect filament emerged on the opposite side of the rhizoid, forming a heterotrichous thallus after 7 days in culture (Fig. 1D). Additional erect and rhizoidal filaments arose from the prostrate part during the next days (Fig. 1E). The plants produced plurilocular sporangia in the prostrate part after 10 days in culture (Fig. 1F), and then below the meristem near the base of the erect filament after 12 days in culture. The upper filaments did not show branches in older thalli, but they developed long pseudohairs and some of them produced secondary meristems on terminal portion with small plurilocular sporangia and rhizoids (Fig. 1G).

The developmental pattern of thalli was not affected according to different temperatures but the production of plurilocular sporangia was slower at 10°C than in 16°C and 20°C (Table 1). Few irregular morphologies of plurilocular sporangia were observed at 20°C and 16°C (Fig. 1H, I). Direct- monophasic life history by plurilocular sporangia was repeated during four generations in our culture. Unilocular sporangia were not found in all the conditions tested.

Morphological observations

Plants are forming small tufts, 1.2-2.1 mm tall (Fig. 2A), with numerous uniseriate prostrate filaments, and irregularly branched. (Fig. 2C). Uniseriate erect filaments are mostly unbranched or sometimes sparingly branched near base, and slightly attenuated upward or forming long pseudohairs (Fig. 2B). Erect filaments are composed of rectangular cells with 19.3±3.1 μm in width and with numerous discoid chloroplasts and not constricted at the septum (Fig. 2D). Meristematic zones are composed of small-sized cells on erect filaments (Fig. 2E). There are various in the size of cells according to the position: 3.3±0.8 longer than broad in apical and medium parts, 2.1±0.5 longer than broad in basal part, and 1.1±0.3 longer than broad in meristematic zone. Plurilocular sporangia are produced as sessile and on one (or two)-celled pedicels both on erect and prostrate filaments. Plurilocular sporangia are developed below meristematic zone of erect filament (Fig. 2B). There are 2 types of plurilocular sporangia: large lanceolate (Fig. 2H) and small ovate shapes (Fig. 2F, G). Plurilocular sporangia are (42)84-192 μm×26-58 μm in size. Unilocular sporangia not observed.

Phylogenetic analysis

The 1326-nucleotide portion of rbcL and 613-nucleotide portion of cox1 was aligned for Feldmannia chitonicola. The phylogenetic trees were obtained from the alignment of the rbcL sequences newly generated and downloaded from GenBank. Asterocladon rhodochortonoides and A. interjectum tree were selected as outgroups. Our samples were nested within a clade of Feldmannia (Fig. 3). Gene sequence divergence differed from F. irregularis by 2.3-2.4%, while from Acinetospora filamentosa and A. crinita by 3.9- 4.0%.

DISCUSSION

Our brown algal collections from Korea matched with the diagnosis of Feldmannia chitonicola described from the type locality in thallus size, the lanceolate and ovate shapes of plurilocular sporangia, and the development of plurilocular sporangia on both prostrate and basal part of erect filaments. These samples are nested within a clade of Feldmannia in phylogenetic trees based on rbcL and cox1 genes (Fig. 3). We add F. chitonicola in the list of Korean macroalgal flora. This is also the first record of F. chitonicola in western Pacific.

Although Feldmannia chitonicola closely resembles to F. irregularis, in some morphological features as the size of thalli, unbranched erect filament, and terminal hyaline pseudo-hairs, F. chitonicola always develops plurilocular sporangia first on the prostrate filaments while F. irregularis only on the erect filaments in both wild and culture materials (Kim and Lee 1994). Feldmannia chitonicola may be distinguished from F. irregularis by the presence of reproductive organs in prostrate filaments, sporangia shape, and molecular data.

The secondary meristems are the upper part regions producing plurilocular sporangia and rhizoids in erect filaments. The presence of a secondary meristem has been known only from culture materials of F. globifera and F. irregularis. The upper filaments of Feldmannia chitonicola in older thalli developed the secondary meristems with small plurilocular sporangia and rhizoids. These secondary meristems may be attributed to the contact of the filaments with the culture dish, which seems to stimulate the development of new meristems or rhizoids (Clayton 1974;Kim and Lee 1994).

The temperature has been reported as one of important culture conditions for the production of plurilocular sporangia in filamentous brown algae (Clayton 1974). In this study, the production of plurilocular sporangia was increased under higher temperatures (Table 1) and this matched with the Clayton’s result. Also, our result shows that temperature can determine the morphological types of plurilocular sporangia: ovate plurilocular sporangia in lower temperatures and elongated plurilocular sporangia in higher temperatures (Table 1). These effects of different temperatures on the morphologies of plurilocular sporangia have not been reported in Feldmannia species.