Morphological and molecular characteristics of Homoeostrichus formosana sp. nov. (Dictyotaceae, Phaeophyceae) from Taiwan

In the marine brown macroalgae, the morphological characters are highly similar between two widely distributed genera, Homoeostrichus and Zonaria (Dictyotaceae), thereby resulting in the difficulty of exploring their hidden biodiversity. Owing to the help of the molecular tools, it is now easy for scientists to objectively describe a new species in nature. In this study, we make a description on the Homoeostrichus formosana sp. nov. from Taiwan, Indo-Pacific Ocean based on the morphological evidence and molecular data. Our morphological observations revealed that this species has marginal row of apical cells responsible for thallus growth and the thallus with four layers of cells except the marginal regions. The cortical cell lies upon each medullary cell in transverse section, and two cortical cells upon each medullary cell in longitudinal section. Tetrasporangium is developed from cortical cell with stalk cell and singly scattered over the thallus surface, and has no indusia and paraphyses. Molecularly, the phylogenetic trees based on SSU, psaA, psbA, and rbcL gene sequences supported that Homoeostrichus species are closely related to Exallosorus species and clearly separated from each others in addition to Zonaria species. Homoeostrichus formosana sp. nov. can now be clearly distinguished from E. harveyanus and Japanese H. flabellatus.

A species of brown alga with external morphology similar to Exallosorus and Zonaria was collected from several collecting sites (Figure 1) in southern Taiwan. The plants of Homoeostrichus formosana Wang, Lin, Lee et Liu sp. nov. have been identified as Z. diesingiana or Z. harveyana in Taiwan, due to short information of their reproductive structures and morphological characteristics, especially no gametangia. It is the first time to describe the characteristics of sporangia of H. formosana sp. nov. in this study. We also described the morphological and phenological characteristics of this species, and determined its phylogeny among the related species based on nuclear-encoded SSU rRNA and plastid encoded rbcL, psaA, and psbA gene sequences.

Survey on morphological characteristics
Collections were made by SCUBA or snorkeling in southern Taiwan ( Figure 1) from 1999 to 2002. Voucher specimens were fixed with 10% formalin/sea water or pressed on herbarium sheets and deposited in the Herbarium of the Department of Biology, National Chunghua University of Education, Taiwan. Microscopic sections were made using a freezing microtome (Leica CM1850), then stained with 0.1% Toluidine Blue O (TBO) and mounted in 50% Karo syrup. Microphotographs were taken on a Pixera digital camera attached to a Carl Zeiss Axioskop 2 microscope with differential interference contrast (DIC) optics.
Other specimens deposited in the following institutions were also examined: the Institute of Oceanography, National Taiwan University, Taipei (IONTU), the National Museum of Natural Science, Taichung, Taiwan (NMNS) and the National Museum of Marine Biology and Aquarium, Hengchun, Taiwan (NMMBA).

Gene sequence analyses
Collections for gene sequencing were made by SCUBA or snorkeling at Kenting, in southern Taiwan on 23 April 2004. Nuclear-encoded SSU rRNA and plastid encoded rbcL gene were selected for elucidating the phylogenetic relationship of Homoeostrichus formosana sp. nov. with other Dictyotaceae. Genomic DNA was extracted from 0.01 g of powder ground in liquid nitrogen using Dneasy Plant Mini Kit™ (Qiagen, Hilden in Germany), according to the manufacturer's instructions. The partial rbcL gene and rbcS, except for short 3′-terminal of rbcL and 5′-terminal region of the rbcS, were amplified and sequenced as two fragments using the primers sets, DRL1F-DRL2R and DRL2F-RU2 (Hwang et al. 2005). The psaA gene sequences were also amplified and sequenced by two 130 F-970R and 870 F-1760R primers sets, psbA gene by one fragment with psbA F-psbA R primers set (Yoon et al. 2002). The partial 18S rRNA gene (SSU) was amplified and sequenced using primers set, SR1-SR7 and SR4-SR12. The amplified DNA was purified using High Pure PCR Product Purification Kit™ (Roche, Indianapolis,USA), in accordance with the manufacturer's instructions. The forward and the reverse sequences were determined for all samples using an ABI PRISM 377 DNA sequencer. The sequences were aligned using PHYDIT (Chun 1995) with final visual confirmation and then submitted to GenBank under the accession numbers (Table 1). The alignment of each coding gene sequence was based on the alignment of inferred amino acid sequences, and reconfirmed by eye. The Padina species were selected as the outgroup species in the phylogenetic analyses.

Species description
Thalli are 5-23 cm in height and (1-)3-7(−10) cm in width, dark brown in color, complanate, flabellate, split to form branches with narrow lower axes and upper flabellate segment, and prostrate at the base arising from a conspicuously rhizoid holdfast to upright blades. Thallus composed of two to four layers of cells throughout, 88-100 μm in thickness. In transverse section medullary cells, 80-157 μm in height, 15-25 μm in width, are overlain by a single cortical cell, 25-50 μm in height, 15-25 μm in width, and then in longitudinal section, two to three cortical cells over lay each medullary cell. Tetrasporangia are spherical, scattered over the both sides of thallus except the margins, 80-100 μm in height by 85-95 μm in diameter, with one basal stalk cell projecting out from the thallus surface, without forming a sorus, indusia and paraphyses absent.

Holotype
The holotype is deposited at Department of Biology, National Changhua University of Education, Changhua (NCUE-CAF91072101) ( Figure 2).
Etymology "formosana" refers to Taiwan, where the alga was collected.

Distribution
Known only from southern Taiwan (Figure 1).

Habitat and phenology
Absence of perennial stipes indicates that this species may be annual. Plants were found all year round, mainly at 2-5 m depth, where they were abundant on coral reefs or on reef rocks.

Habitat and anatomical structures
Thalli are yellow or dark brown in color, composed of upright blades (Figures 2 and 3), and which basal portions are creeping with a conspicuously rhizoid holdfast. They are 5-23 cm in height and (1-)3-7(−10) cm in width (Figures 2 and 3). Thallii are fan-shaped when young and splitting into numerous bladelets when old. The surfaces of thallus are covered with hyaline hairs that are arranged in interrupted concentric bands (Figure 4), and with the blanketing brown rhizoidal filament at the base ( Figure 5). Thallus growth is by a row of marginal meristem cells, which are dark in color ( Figure 6). The apical cell is 120-240 μm in length and 70-78 μm in width ( Figure 12). The blades are polystromatic, two or four cell layers, with 88-100 μm in     8). In longitudinal section of thallus, two or three cortical cells overlay a single medullary cell (Figure 8), whereas a single cortical cell overlays each medullary cell in transverse section (Figures 11 and 13).

Reproductive structures
Sporangia are scattered over the surface on both sides of the blade (Figures 9 and 10). Tetrasporangia are roughly spherical and projected above the surface of the thallus, 80-100 μm in height and 85-95 μm in diameter, with a basal stalk cell which measured 12-26 μm in height by 17-25 μm in diameter, and lacked indusium and paraphyses (Figures 11 and 13). Gametophytes were not observed.

Characteristics of gene sequences
The SSU sequences determined and aligned in this study were 1,814 nucleotides long. The 20 aligned SSU sequences had 106 (5.8%) variable bases and 176 (9.7%) parsimoniously informative sites and 49.4% G+C contents. Transitions occurred more than transversions (Ts/ Tv=1.16). The average of uncorrected pairwise distances (p-distances) was 0.059 from the aligned data set ( Figure 14). The uncorrected pair wise distance (p-distances) between Zonaria species and Homoeostrichus species ranged from 0.057 to 0.077, and between Exallosorus species and Homoeostrichus species from 0.009 to 0.014. We could find 5 nucleotide base pairs differences in the    aligned 1,723 nucleotide base pairs sequences between H. formosana sp. nov. and H. flabellatus from Japan (~0.3%), and 11 nucleotide base pairs differences between H. formosana sp. nov. and H. sinclairii. We determined and aligned 1,351 nucleotides long rbcL sequences in this study. The 28 aligned rbcL sequences had 82 (6.07%) variable bases and 420 (31.08%) parsimoniously informative sites. The G+C content was 38.2% in the aligned sequence data set. Transitions were almost less than transversions (Ts/Tv=0.89). The average of p-distances was 0.122 from the aligned data set (Figure 14). The "p-distance" between Zonaria species and Homoeostrichus species ranged from 0.118 to 0.125, and between Exallosorus species and Homoeostrichus species from 0.098 to 0.119. Sixteen nucleotide differences were found between H. formosana sp. nov. and H. flabellatus in 1,305 nucleotide base aligned sequences (~1.2%).
The psaA sequences determined and aligned in this study were 1,395 nucleotides long. The aligned 24 psaA sequences had 87 (4.49%) variable bases and 496 (35.55%) parsimoniously informative sites and had 35.1% G+C content, and ratio of 0.82 transitions to transversions (Ts/Tv). The "p-distances" was 0.154 from the aligned psaA sequences data set ( Figure 14). The "pdistances" between Zonaria species and Homoeostrichus species ranged from 0.143 to 0.152, and between Exallosorus species and Homoeostrichus species from 0.132 to 0.137. We found 182 nucleotide differences between H. formosana sp. nov. and H. sinclairii in aligned sequences of 1,394 base pairs.
The total 845 base pairs of psbA sequences were determined and aligned in this study. The aligned 25 psbA sequences had 45 (5.33%) variable bases and 213 (25.21%) parsimoniously informative sites with 37.8% G+C content. Transitions occurred more frequently than transversions (Ts/Tv=1.22) and p-distance ranged from 0.030 to 0.134 with average of 0.089 in aligned psbA sequences data set ( Figure 14). The "p-distances" between Zonaria species and Homoeostrichus species ranged from 0.072 to 0.102, and between Exallosorus species and Homoeostrichus species from 0.084 to 0.098.
Overall, the sequence divergence is smallest in SSU, followed by psbA (Figure 14). In contrast, the sequence divergence is much larger in rbcL and psaA (Figure 14). Our observations suggest that SSU is more suitable to resolve the phylogenetic relationship of higher taxonomic level and other plastid genes used in this study are more suitable to tackle the phylogenetic relationship for the lower taxonomic level.

The phylogeny based on gene sequences
The phylogenetic tree based on SSU sequences showed that genera of tribe Zonarieae made four clades with no phylogenetic resolution among them in the ML analyses ( Figure 15). The clade of Homoeostrichus and Exallosorus species is separated from that of Zonaria and Lobophora species. Three Homoeostrichus species made a subclade distinguished from Exallosorus species except for H. canaliculatus. Especially H. formosana sp. nov. made a clade with H. flabellatus with very low supporting value in three analyses.
The topology of phylogenetic tree based on rbcL sequences also show that four clades are distinguished ( Figure 15). The clade comprising Homeostrichus and Exallosorus species figured out as basal sister group in this phylogeny although the results showed pale phylogenetic resolution. Homoeostrichus formosana sp. nov. made a clade with H. flabellatus with very high supporting value, and a sister group of H. sinclairii with low supporting value in three analyses. Exallosorum olsenii also made a sister group to three Homoeostrichus species clade and closely related to H. cnaliculatus. The clade of Zonaria and Lobophora species made a  concrete clade with high supporting value distinguished from others.
The aligned psaA gene sequences data set made the phylogenetic tree with five clades, which have a basal clade of Stypopodium species although with pale phylogenetic resolution ( Figure 15). As in the former trees, the clade of Homeostrichus and Exallosorus species is distinguished as basal sister group in this phylogeny although having pale phylogenetic resolution. Homoeostrichus formosana sp. nov. made a clade with H. flabellatus with very high supporting value, with a sister group of H. sinclairii with low supporting value in three analyses. Exallosorum olsenii also made a sister calde with three Homoeostrichus species and closely related to H. cnaliculatus as in the phylogeny of rbcL. The clade of Zonaria and Lobophora species made a concrete clade with high supporting value distinguished from Homoeostrichus and Exallosorus species.
The phylogenic tree based on psbA gene also show that H. formosana sp. nov. is involved in a clade with H. sinclairii and H. canaliculatus. This phylogenic tree is composed of five clades with very pale phylogenic resolution ( Figure 15). Exallosorus species are closely related to Homoeostrichus species as in the other phylogenic trees.

Discussion
The taxonomy of the Dictyotales is largely based on the comparison of vegetative and reproductive growth and organization (Phillips 1997). Homoeostrichus formosana sp. nov. is mainly characterized by blades composing of two to four layers of cells, single tetrasporangia scattered over both thallus surfaces, sporangia borne on a stalk cell, and lacking indusium and paraphyses. In the erect to recumbent fan-like fronds of Lobophora, unusual large medullary cell and indusiate sporangial sorus, and Padina, rolling margin and concentric arrangement of reproductive structures, which both are conspicuously differed from Homoeostrichus, whatever the habit, texture, anatomical and reproductive structures. Homoeostrichus has been very easily confused with Exallosorus and Zonaria, based on the vegetative and reproductive characters used for separating among them summarized in Table 2. The phylogenic trees based on SSU, psaA, psbA, and rbcL gene sequences supported that Homoeostrichus species are closely related to Exallosorus species but clearly separated from each others in addition to Zonaria species.
The genus Exallosorus is separated from Zonaria and Homoeostrichus in having regularly arranged cells in transverse section, densely placed basally stalked sporangia within sori that possess brown paraphyses and indusium (Phillips 1997) (Table 2). Sporangia of Zonaria lacked basal stalk cells, are surrounded by whitish paraphyses (except in Z. angustata) in the indusiate sori, and released eight spores (Womersley 1987;Phillips 1997) (Table 2). Sporangia of Homoeostrichus are distributed among brown paraphyses in non-indusiate sori, and released four spores (Womersley 1987;; Phillips 1997) ( Table 2). In this study, we also found the sporangia in H. formosana sp. nov. are singly scattered over the surfaces of the thallus without forming a sorus and lacking indusium and paraphyses (Table 3). Classifying the genera of tribe Zonariae based on these morphological and anatomical characteristics is basically agreed to five clades in phylogenic analyses based on gene sequences.
Homoeostrichus formosana sp. nov. is superficially similar to Zonaria diesingiana found from Taiwan in external morphology. However, H. formosana sp. nov. can be distinguished vegetatively and reproductively from Z. diesingiana, especially it makes four cell layers. The thallus of Z. diesingiana is composed of 4-8 layers of cells, in which the one medullary cell is flanked by 2 cortical cells in transverse section, the octosporangia are borne on no stalk cell, and white paraphyses are present in indusiate sori. However, the tetrasporangium of H. formosana sp. nov. is borne on a basal stalk cell and lacks paraphyses and indusium. Homoeostrichus formosana sp. nov. was previously misidentified as E. harveyanus (as Z. harveyana, H. multifidus) in Taiwan (Yamada 1925;Okamura 1936;Shen and Fan 1950;Chiang 1960;Lewis and Norris 1987). The thallus of E. harveyanus is composed of 6 layers of cells, which measured 120-170 μm in thickness, and the sporangia are formed in a dark brown band of an indusiate sorus, whereas the sporangia in H. formosana are singly scattered over the surfaces of the thallus without forming a sorus (see Table 3). Although Yamada (1925) and Okamura (1936) had documented the thallus of "Homoeostrichus multifidus" (as H. formosana sp. nov. in this study) as being composed of four layers of cells, they did not observe reproductive structures, moreover, it is now known that E. harveyanus (as H. multifidus) is only distributed in southern Africa, the type locality (Silva et al. 1996;Phillips 1997). All molecular data also supported that H. formonosa sp. nov. is clearly distinguished from E. harveyanus in the psbA sequences molecular analyses in this study. Another Exallosorus species, E. olsenii, comprised of six cell layers, has sporangia assembled in indusiate sori that are connected with hairs and paraphyses, and with the reproductive structures only occurring on one thallus surface (Womersley 1987, as H. olsenii;Phillips 1997), which is not agreed with H. formosana sp. nov. (Table 3).  Homoeostrichus formosana sp. nov can possibly be confused with other species of Homoeostrichus: H. canaliculatus and H. sinclairii (Womersley 1987;Phillips 1997). However, H. formosana sp. nov. can be distinguished from the other species of Homoeostrichus by its 2-4 layers of cells thallus and sporangial stalk cells opposed to a 6-7 cell layer thallus and by multicellular stalk cells which are found in Homoeostrichus (see Table 3). The phylogentic tree especially based on psbA gene sequences showed that H. canaliculatus is distinguished from other Homeostrichus species and from Exallosorus species. Moreover, H. flabellatus Okamura, another Dictyotaceae species from Taiwan, might also be confused with H. formosana sp. nov. (Taniguti 1976;Lewis and Norris 1987;Wang and Chiang 2001). Okamura (1936) reported the thallus of H. flabellatus was composed of three layers of cells but he did not observe reproductive structures. Womersley (1987) speculated that Japanese H. flabellatus did not belong to the genus Homoeostrichus, and Papenfuss (1944) transferred H. flabellatus to Zonaria flabellata (Okamura) C. However, this combination is not recognized by some phycologists (see Phillips 1997;Phillips and Nelson 1998). The molecular characteristics of SSU show Japanese H. flabellatus is more related to H. formosana sp. nov. in this study. These show that the status of this taxon should be required further study especially examining voucher specimens of H. flabellatus. Furthermore, it is noted that an undescribed Zonaria sp. was recently reported from Chaojing, Keelung, northern Taiwan by Kitayama and Lin (2012). Though they only showed single photo of the thallus of this alga without any anatomical observations, this alga is highly similar to H. formasana in appearance. Considering that H. flabellatus (as Zonaria flabellatus) in Okinawa is biogeographically close to northern Taiwan (Figure 15), it will be interesting to examine the phylogenetic affinity of this undescribed Zonaria sp. from the northern Taiwan to test whether this alga is phylogenetically close to H. flabellatus or H. formasana.

Conclusions
We describes a new species, Homoeostrichus formosana Wang, Lin, Lee et Liu, collected from Taiwan. This species has marginal row of apical cells responsible for thallus growth and the thallus with four layers of cells except the marginal regions. The cortical cell lies upon each medullary cell in transverse section, and two cortical cells upon each medullary cell in longitudinal section. Tetrasporangium is observed for the first time, which is developed from cortical cell with stalk cell and singly scattered over the thallus surface, and has no indusia and paraphyses. The phylogenetic trees based on SSU, psaA, psbA, and rbcL gene sequences supported that Homoeostrichus species are closely related to Exallosorus species but distinctly different from Zonaria species.