Reconstructing the Phylogeny of Capsosiphon fulvescens (Ulotrichales, Chlorophyta) from Korea Based on rbcL and 18S rDNA Sequences

Capsosiphon fulvescens is a filamentous green algae in the class Ulvophyceae. It has been consumed as food with unique flavor and soft texture to treat stomach disorders and hangovers, and its economic value justifies studying its nutritional and potential therapeutic effects. In contrast to these applications, only a few taxonomic studies have been conducted on C. fulvescens. In particular, classification and phylogenetic relationships of the C. fulvescens below the order level are controversial. To determine its phylogenetic position in the class, we used rbcL and 18S rDNA sequences as molecular markers to construct phylogenetic trees. The amplified rbcL and 18S rDNA sequences from 4 C. fulvescens isolates (Jindo, Jangheung, Wando, and Koheung, Korea) were used for phylogenetic analysis by employing three different phylogenetic methods: neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The rbcL phylogenetic tree showed that all taxa in the order Ulvales were clustered as a monophyletic group and resolved the phylogenetic position of C. fulvescens in the order Ulotrichales. The significance of our study is that the 18S rDNA phylogenetic tree shows the detailed taxonomic position of C. fulvescens. In our result, C. fulvescens is inferred as a member of Ulotrichaceae, along with Urospora and Acrosiphonia.


Introduction
Capsosiphon fulvescens (C. Agardh) Setchell and N. L. Gardner, filamentous chlorophycean seaweed, is found in the North Atlantic [1,2] and in the Northern Pacific, including Korea [3] and Japan [4]. Its natural habitat is the upper intertidal regions of coastal sediments and rocky shores which it shares with the common edible seaweed Ulva prolifera Mueller. Capsosiphon fulvescens, a filamentous green algae, reproduces by biflagellated isogametes released from bisexual gametophytes [5]. This seaweed is known to be a contaminant in Porphyra cultivation [6]. However, in the Southwestern province of Korea, it has been consumed as food with unique flavor and soft texture to treat stomach disorders and hangovers [7], and its economic value justifies studying its nutritional and potential therapeutic effects. Several physiological studies conducted in vitro and in vivo have suggested that extracts of C. fulvescens had an inhibitory effect on melanogenesis in B16 cells [8], induced apoptosis in AGS gastric cancer cells [9], and reduced cholesterol levels in hypercholesterolemic rats [10]. The potential economic interest in C. fulvescens could justify its large-scale cultivation in both the laboratory and the field [11].
In contrast to attention to its applications, only a few taxonomical studies have been conducted regarding C. fulvescens. In particular, classification and phylogenetic relationships of the C. fulvescens below the order level are controversial. The reasons for discrepancies among classification schemes include disagreements regarding the evaluation of morphological characters. For example, C. fulvescens produces gametes and zoospores like those of Ulothrix and Urospora [12] while it was also considered to be closely related to Monostroma by Migita [5] because of discontinuous reproductive patches near the thallus apex as well as similarity of gametes. However, Chihara [4] considered that it was closely related to Percuriaria and Ulva because fronds produce zygotes that germinate directly without formation of a thickwalled zygote [4]. It is often difficult to identify symplesiomorphies characters because similar characters can be derived from convergent or parallel evolution of Ulotrichales.
Molecular systematics in seaweeds has progressed rapidly with the use of PCR coupled with sequencing methods. This molecular approach has been effective in addressing many phylogenetic questions that had not been solved using phenotypic characters. The gene for the large subunit of ribulose-bisphosphate carboxylase (rbcL) located in the chloroplast genome and the 18S rDNA in the nuclear genome have been extensively used for the inference of phylogenetic relationships at higher taxonomic levels because of their slow synonymous nucleotide substitution rates and strong functional constraints of rbcL sequence that reduced the evolutionary rate of nonsynonymous substitutions. The first report to mention the classification of C. fulvescens collected from North Atlantic with a molecular marker (cf. 18S rDNA) was presented by Hayden and Waaland [13]. They suggested that C. fulvescens was in the order Ulotrichales, which was consistent with Nagata's report [12]. In 2008, Hanic and Lindstrom [14] also used 18S rDNA sequence to prove that C. fulvescens and Pseudothrix borealis (entity formerly called C. groenlandicus) do not belong in the same genus Capsosiphon.
In this study, for the first time, C. fulvescens have been examined using rbcL gene and 18S rDNA sequences of newly collected material from different provinces in South Korea and these sequences have been employed to understand the phylogenetic position of Korean Capsosiphon fulvescens in the Ulvophyceae.

Sample Collection and
Culture. C. fulvescens thalli were collected from five different seaweed farms located in Jindo, Koheung, Wando, and Janghuyn, South Korea, during December 2011 to February 2012. They were washed several times in clean cold seawater and kept on ice until being returned to the laboratory. Seawater samples were collected from discrete depths using 10 Niskin bottles arranged on a conductivity, temperature, and depth (CTD) rosette. The entire contents of the bottles were gravity filtered onto a 47 mm Poretics membrane filter (GE Osmonics, Fairfield, CT, USA), with a pore size of 5 m, held within a Millipore Swinnex filter holder (Millipore, Bedford, MA, USA). The filtration time varied between 30 min and 2 h; if the Niskin bottle was not completely filtered at the end of 2 h, the filter was processed, noting the amount of seawater filtered. Freshly collected plants were grown in seawater filtered medium in glass culture vessels at 8 ∘ C under 30-50 mol photons m −2 s −1 , 14 : 10 h LD cycle. The species was identified microscopically, manually separated from other algae, and washed with tap water and distilled water. Authentic standard compounds were purchased from Tokyo Kasei Kogyo Co., Ltd. (Japan), and Supelco Inc. (Bellefonte, USA).

DNA Extraction.
Whole cultured biomass of Capsosiphon fulvescens was freeze-dried, and genomic DNA from freeze dried materials was extracted using a modified hexadecyltrimethylammonium bromide (CTAB) method [15], in which samples (∼0.5 g) were ground in 1 mL CTAB using a sterile mortar and pestle. DNA extracts were cleaned with a Wizard PCR purification system (Promega, Madison, WI, USA), according to the manufacturer's instructions.  (Table 1) with the following conditions. The reactions were conducted using an initial denaturation at 94 ∘ C for 3 min, followed by 35 cycles each of 1 min at 94 ∘ C, 1 min at 50 ∘ C, and 1 min at 72 ∘ C, followed by final extension for 10 min at 72 ∘ C. The PCR products were analyzed by 1.2% (w/v) agarose gel electrophoresis and purified with a Wizard PCR purification system (Promega, Madison, WI, USA). The cleaned PCR products were sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Foster City, CA, USA) following the manufacturer's instructions. Sequences were generated in both the forward and reverse directions using an automatic sequencer ABI PRISM 310 Genetic Analyzer (Applied Biosystems Foster City, CA, USA) (see Supplementary Materials available online at http://dx.doi.org/10.1155/2016/1462916).

Phylogenetic Analyses.
Assemblies of the newly created DNA sequences in this study were carried out using the DNASTAR program (DNASTAR, Inc., Madison, WI, USA). The coverage of sequences determined was 2x. Multiple sequence alignments were performed using the ClustalX 2.1 with default parameters and manually edited. Other sequences were obtained from GenBank using BLASTN search with the identified sequences as queries. Twentyfive rbcL sequences belonging to Ulvophyceae were used to construct a phylogenetic tree with 2 outgroup sequences. Thirty-three 18S rDNAs were used to construct phylogenetic tree. For phylogenetic analyses, 2 different methods were applied: neighbor joining (NJ) and maximum parsimony (MP) using software Mega v6 [18] with complete deletion of gaps. In MP analyses, nucleotide positions and character state changes were weighted equally after removing uninformative characters. ML analysis was carried out using RAxML v8

Discussion
After Hayden and Waaland [13] reported that Ulvales and Ulotrichales sensu Floyd and O'Kelly [20] are monophyletic sister orders, the systematics in Ulvales has been well supported by molecular marker as 18S rDNA [21,22] and shows 3 main families: Ulvaceae, Kornmanniaceae, and Ulvellaceae. However, classification and phylogenetic relationships in Ulotrichales are complicated. Despite several studies of the beneficial value of C. fulvescens to health, the position of C. fulvescens in the order Ulotrichales is still unclear. Phylogenetic trees were constructed on the basis of rbcL, 18S rDNA, and combined sequences. This information has been further used for phylogenetic analysis and classification of Ulvophyceae and other related taxa.
Phylogenetic trees based on rbcL and 18S rDNA sequences exhibit topological differences that are due to the different rates at which these genes evolve. To make any statement from our phylogenetic trees, our study only selected the tree topology supported by >50% in at least 2 different phylogenetic trees. The rbcL tree (Figure 1) recovered Ulvales as a monophyletic group, which was consistent with the previous studies and showed that C. fulvescens was weakly supported in Ulotrichales because all phylogenetic trees have showed a corresponding topology with at least 50% of bootstrap value.  The 18S rDNA phylogenetic tree ( Figure 2) provided more information about the phylogenetic systematics of Ulotrichales and the position of C. fulvescens. Capsosiphon fulvescens appeared on its own branch of the Ulotrichales in the NJ and ML analysis; in the MP analysis, it appeared on its own branch at the base of the Ulotrichaceae. In all analyses, it clearly belonged to the Ulotrichales since the subtending branch, separating the Ulotrichales from members of the Ulvales, had 70% bootstrap support.
Our phylogenetic tree shows that Ulotrichales appears to be paraphyletic. Two clearly different lineages in Ulotrichaceae (Acrosiphonia and Urospora) were also successfully recovered with >80% of bootstrap numbers in all 3 phylogenetic methods (ML/MP/NJ = 81/83/85), which was also consistent with the SSU rDNA tree [14,21]. The key result here is that the tree topology in Ulotrichaceae shows 4 distinct lineages: Gloeotilopsis, Acrosiphonia, Capsosiphon, and Urospora + Protomonostroma ( Figure 2). This tree topology [Acrosiphonia, and (Capsosiphon, Urospora + Protomonostroma)] was supported by >50% of bootstraps in all 3 different phylogenetic trees, suggesting that Capsosiphon is more closely related to Urospora + Protomonostroma. In both MP and NJ analyses with the 18S rDNA data set, 3 well-supported groups were recovered: (1) Gomontiaceae, (2) Monostromataceae, and (3) Ulotrichaceae. The relationship between Ulotrichaceae and other genera is not well supported in either NJ or MP tree. In NJ tree, the proximal outgroup Ulotrichaceae and Gomontiaceae + Monostromataceae are similar to the phylogram including 5 representatives of Kornmanniaceae [22].
The present study showed that both phylogenetic analysis based on 18S rDNA and rbcL sequences analysis resolved  the phylogenetic position of C. fulvescens in Ulotrichales (Figures 1 and 2). All of 4 C. fulvescens were clustered with Urospora wormskioldii and Acrosiphonia arcta in the family Ulotrichaceae, Ulotrichales, with high bootstrap number in all phylogenetic trees (Figure 2), suggesting that Capsosiphon is a genus in the order Ulotrichales, although additional information is needed to support this hypothesis.
We will further study the vegetative morphology of this genus to compare it with that of known genera in the Ulotrichales.