The family Gracilariaceae Nageli belongs to Gracilariales, the order of marine red algae described by Fredericq and Hommersand (1989). The gracilarioid algae include some of the most valuable marine plants. They have been extensively investigated over the last 30 years, and many studies have provided comprehensive information on their life history, cultivation, taxonomy, and utilization (Bellorin et al. 2002, Rueness 2005). Studies on the structure of their reproductive organs and the phylogenetic relationships among species inferred from
DNA sequence analysis is the most widely used molecular technique for inferring phylogenetic relationships at the species level within the Gracilariaceae (Iyer et al. 2005, Gargiulo et al. 2006, Bellorin et al. 2008, Kim et al. 2008
In China, there are several papers on the morphological description of Gracilariaceae with 14 novel species (Chang and Xia 1976, Zhang and Xia 1988, 1992, 1994), particularly Chang and Xia (1976) dealt with 24 species of the genus
This study was performed as part of survey on the gracilarian species diversity in Asia-Pacific region and focused on the molecular phylogeny of Gracilariaceae on Hainan Island for the first time. We conducted molecular and morphological analyses to clarify their taxonomic identities and better understand their distributions. As a result, we reported the phylogenetic relationships of five species:
Gracilariaceae specimens were collected from intertidal substrates in Sanya, Hainan, China: G058 (JN605794), January 29, 2010, Matou; G056 (JN605796), January 30, 2010, Xiadonghai Bay; G052 (JN605793), January 31, 2010, Hongsha River; G057 (JQ026028), January 31, 2010, Yulin Harbor; G054 (JN605795), January 29, 2010, Qingshui Bay. Specimens were air-dried and stored in silica gel desiccant for DNA extraction. Voucher specimens are housed at the herbarium of Jeju National University (JNUB), Jeju, Korea. Specimen slides were stained with aniline blue solution and examined for morphological diagnostic features. Photographs were taken using a μ-Tough-8000 digital camera (Olympus, Tokyo, Japan), and QIMAGING 1394 (QImaging, Surrey, BC, Canada) with a BX50 microscope (Olympus). After obtaining the digital images, plates were edited using Photoshop 7.0.1 (Adobe, San Jose, CA, USA).
Total genomic DNA was extracted from dried thalli ground in liquid nitrogen using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. For amplification and sequencing of the
To confirm the taxonomic position of Gracilariaceae from Hainan, maximum likelihood (ML) and Bayesian analysis (BA) were performed using PAUP 4.0 (Swofford 2002) and MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003). Five
Gracilaria salicornia (C. Agardh) Dawson (
Description. Thallus ranges from being erect to prostrate, 2-4 cm tall, forming a loose tuft-like aggregation from the discoid holdfast (Fig. 1B & C). The texture is car-
[Fig. 1.] Gracilaria salicornia (C. Agardh) Dawson. (A) Holotype photo cited from C. Agardh 1820. (B) Specimen collected from Xiadonghai bay, Sanya, Hainan in January 30, 2010. (C) Photograph showing the habitat of specimens growing on the rock. (D) Shape of the apical part of branches showing the constricted segments. (E & F) Transverse section of branches showing gradual transition of cells from medulla to cortex with thin-wall and small-celled cortex. Scale bars represent: B & C, 2 cm; D, 1 cm; E, 200 μm; F, 50 μm.
tilaginous and succulent, and it is light brown to orange in color. Cylindrical branches are distinctly constricted at the base with two branchlets at each node and smooth margins 2-2.5 mm in diameter (Fig. 1D). Branchlets are elongated, club-shaped, and generally di- to trichotomously arranged. The medullary cells gradually increase in diameter toward the center (Fig. 1E). The medullary cell is 200-400 μm in diameter, with walls approximately 10-15 μm thick. The cortical layer consists of 2-3 small cells that are 7-10 μm in diameter (Fig. 1F).
Type locality. Manila, the Philippines.
Distribution. Asia (Xia 1985, Ohno et al. 1999), Pacific Islands (Meneses and Abbott 1987), Australia (Witherell et al. 1994, Millar and Xia 1997), Indian Ocean (Silva et al. 1996), Africa (Silva et al. 1996).
Xia (1986) recognized that the degree of constriction at the branch base was not distinguishable among similar species, such as
Gracilaria tenuistipitata C. F. Chang et B. M. Xia (
Description. Plants are 10-20 cm long, slender, with 2-3 orders of branching and easily detached from the substratum because of the small holdfast and slender stipe (Fig. 2A). Main axes have densely and irregularly arranged branches that bear progressively shorter, delicate, and numerous branchlets of 1.0-1.5 mm in diameter (Fig. 2B). The medulla consists of 4-5 cells that increase markedly in size towards the center and reaches up to 130-200 μm in diameter, with thick walls of 10-25 μm in diameter (Fig. 2D). The cortex consists of 1-2 layers of rounded cells of 14-22 μm in diameter (Fig. 2E).
Type locality. (as
Distribution. Asia (Ohno et al. 1999, Terada et al. 2000).
Remarks. Its distribution is restricted to China, Malaysia, Thailand, and Vietnam. Two varieties,
Description. Plants are solitary, up to 15 cm in length, and erect axes rise from small discoid holdfasts (Fig. 3A). Branches are terete and always constricted at the base (Fig. 3B), tapering gradually towards acute apices (Fig. 3C). The cortex consists of globular cells with a dense cytoplasm, 9 μm high and 7 μm wide in size. The medulla consists of 7-8 cells with thin-walled (15 μm), unpigmented, spherical or isodiametric cells. Medullary cells increase abruptly in size toward the center, reaching up to 600 μm in diameter (Fig. 3D). Cystocarps prominently protrude from the entire surface, except the basal and apical parts (Fig. 3E). They are globose, 900 μm high and 1,100 μm wide, slightly constricted at the base, and have tubular nutritive cells connecting gonimoblasts to the pericarp (Fig. 3E). Pericarp tissue is 200-230 μm thick, and carposporangia is globose to obovoid and 17-20 μm in diameter. Only cystocarpic plants were collected at Matou, Sanya, on January 29, 2010, growing on sand in the intertidal zone.
[Fig. 2.] Gracilaria tenuistipitata C. F. Chang et B. M. Xia. (A) Herbarium specimen collected from Hongsha river, Hainan in January 31, 2010. (B) A part of the main axis bearing many terminal branchlets. (C) Habitat of specimens growing in the muddy. (D & E) Transverse section of the branch showing the abrupt transition in medullary cell to cortical cell size with thick-wall and small-celled cortex. Scale bars represent: A, 2 cm; B, 1 cm; C, 15 cm; D, 200 μm; E, 50 μm.
[Fig. 3.] Gracilaria sp. (A) Herbarium specimen collected from Matou, Sanya, Hainan in January 29, 2010. (B) Shape of markedly constricted branches bearing many cystocarps. (C) The apical part of the branches tapering gradually toward apex. (D) Transverse section of the main axis showing the abrupt transition in medullary cell size and small-celled cortex. (E) Vertical section of cystocarp showing two nutritive filaments (arrows). Scale bars represent: A, 3 cm; B & C, 1 cm; D & E, 200 μm.
analysis (Fig. 6,
Gracilariopsis bailiniae J. Zhang et B. M. Xia (
Description. Plants are 15-20 cm long, solitary or caespitose, arising from a small discoid holdfast (Fig. 4A). The thallus is cylindrical, 1.4-1.6 mm in diameter with
[Fig. 4.] Gracilariopsis bailiniae J. Zhang et B. M. Xia. (A) Herbarium specimen collected from Yulin harbour, Sanya, Hainan in January 31, 2010. (B) Shape of branches and terminal branchlets showing non-constricted them at the base. (C & D) Transverse section of the branch showing the abrupt transition in medullary cell size and small cortical cells. (E) Transverse section of the branchlet showing a cruciately divided tetrasporangia. Scale bars represent: A, 5 cm; B, 2 cm; C, 300 μm; D, 50 μm; E, 30 μm.
2-4 orders of fleshy, brittle, and easily broken branches. Branches are 0.2-0.4 mm in diameter, gradually tapering toward the apex and are non-constricted at the base (Fig. 4B). The transverse section of the thallus consists of a large medullary cell 400-650 μm in diameter and with walls 10-15-μm-thick, surrounded by 2 to 3 layers of small roundish cortical cells with 4-6 μm in diameter (Fig. 4D). The transition of cell size is abrupt from medulla to cor-tex (Fig. 4C). Tetrasporangia are scattered over the surface of branches, ranging from 25-30 × 30-35 μm in diameter, and cruciately divided (Fig. 4E).
Type locality. Yinggehai, Hainan Island, Guangdong Province, China.
Distribution. Asia (Ohno et al. 1999).
The specimen of
Hydropuntia edulis (S. G. Gmelin) Gurgel et Fredericq (
Description. Plants are 5-8 cm tall and terete throughout, and grow in tufts from a discoid holdfast (Fig. 5A & B). Branches are fastigiated, wiry, arranged as either dichotomous or trichotomous, and 0.9-1.0 mm in diameter. Several branches arise from the base of the plant with 4 to 6 orders. Ultimate branches are short and spine-like (Fig. 5C). The medulla consists of 7-9 roundish thin-walled cells, 100-250 μm in diameter, and 1 to 2 small cortical cell of 15-20 μm in diameter (Fig. 5E). The diameter is gradually decreased from medulla to cortex (Fig. 5D).
Type locality. Indonesia.
Distribution. Asia (Ohno et al. 1999), Pacific Islands (Tsuda 1985), Australia (Millar and Xia 1997), India (Rao 1972), Africa (Silva et al. 1996).
[Fig. 5.] Hydropuntia edulis (S. G. Gmeli) Gurgel et Fredericq. (A & B) Living and dried specimen collected from Qingshui bay, Sanya, Hainan in January 29, 2010. (C) Shape of the branches with acute apex. (D & E) Transverse section of the branch showing the gradual transition in medullary to cortex cell size. Scale bars represent: A, 3 cm; B, 5 cm; C, 1 cm; D, 200 μm; E, 30 μm.
Among the 1,171 base pairs analyzed, 688 were constant, 483 were variable, and only 414 were parsimony-informative. No insertion or deletion mutations were found in the
[Fig. 6.] Maximum likelihood phylogenetic tree of the family Gracilariaceae estimated using rbcL sequence data. Numbers above each clade represent maximum likelihood bootstrap values and Bayesian posterior probabilities, respectively. Species name of the boldface type are shown the specimens from Hainan Island, China. Scale bar represents: substitutions / site.
in the genus
This study represents the first molecular contribution to the taxonomy of gracilarioid algae from Hainan Island. The