Grateloupia tenuis Wang et Luan sp. nov. (Halymeniaceae, Rhodophyta): A New Species from South China Sea Based on Morphological Observation and rbcL Gene Sequences Analysis

Grateloupia tenuis Wang et Luan sp. nov. is a new species described from Lingshui, Hainan Province, South China Sea. Based on the external form and internal structure, combined with rbcL gene sequence analysis, Grateloupia tenuis is distinct from other Grateloupia species as follows: (1) thalli is slippery and cartilaginous in texture; possess fewer branches, relatively slight main axes, and two or three dichotomous branches; (2) cortex is 5-6 layers; medulla is solid when young, but hollow in old branches; reproductive structures are dispersed in main axes of thalli and lower portions of branchlets; exhibits Grateloupia-type auxiliary cell ampullae; (3) the four studied G. tenuis sequences were positioned in a large Grateloupia clade of Halymeniaceae, which included sister group generitype G. filicina with 68 bp differences; G. tenuis was determined to be a sister taxon to the G. catenata, G. ramosissima, G. orientalis, and G. filiformis subclade. The pairwise distances between G. tenuis and these species were 39 to 50 bp. The sequences of G. tenuis differed by 81–108 bp from the sequences of other samples in Grateloupia; there are 114–133 bp changes between G. tenuis and other genera of Halymeniaceae. In final analysis, we considered Grateloupia tenuis Wang et Luan sp. nov. to be a new species of genus Grateloupia.


Introduction
Red algae Grateloupia C. Agardh is a species-rich genus in the family Halymeniaceae, which not only exhibits highly diverse external morphology, but also is one of the genera in which species discrimination is more difficult. This genus is widely distributed in tropical and temperate coastal areas and includes more than 80 species worldwide and a total of 35 species in China [1][2][3][4][5][6][7][8][9][10][11][12]. The genus Grateloupia was originally built on the foundation of three species, namely, G. ornata, G. hystrix, and G. filicina, with G. filicina identified as the genus generitype [13].
Species morphology is significantly varied in Grateloupia, determining difficulties in species identification and controversy in species classification between algae taxonomists. Yendo [14] described Grateloupia with lomentaceous features as "catenate" and proposed G. catenata as a new species; however, Howe [15] regarded Grateloupia with lomentaceous characteristics as G. filicina (Lamouroux) C. Agardh var. lomentaria Howe. Okamura [16] opined that G. filicina var. lomentaria was a mature stage of G. filicina var. porracea, and integrated them into G. filicina var. porracea f. lomentaria (Howe) Okamura, with G. catenata being a synonym. Wang et al. [1] inspected type material of G. catenata Yendo and proposed to reinstate the Yendo name. G. ramosissima Okamura was discovered by Lin et al. [9], and its morphology resembles the algae researched in the present study. G. orientalis was described based on studies of cystocarp development and rbcL gene sequence analysis [9]. Zhang et al. [12] confirmed a new record of G. orientalis in the Hainan province of China through morphological observations, development of reproductive structures and molecular phylogenetic studies.
In our current research, the above species (G. filicina, G. catenata, G. ramosissima and G. orientalis) showed close relationships with G. tenuis; however, due to the particularly large differences in external and internal morphologies between G. tenuis and other known algae, this species has not been mentioned in previous reports. Based on these differences and analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene sequences, we recommend that this alga is a new species of Grateloupia and should be defined as Grateloupia tenuis Wang et Luan sp. nov. Morphological observations were made on algal specimens preserved in 10% seawater or pressed on herbarium sheets, and molecular analysis was conducted on samples desiccated in silica gel. Photographs of the holotype specimen were taken with a Canon EOS 650D (Canon, Japan). Hand sections were made by cryostat microtome, stained with 0.5% (w/v) cotton blue and discolored with 45% acetic acid. Photomicrographs were taken on an Olympus BH2 digital camera (Olympus Beijing Co. Ltd., China) mounted on a Nikon microscope (Nikon Corporation, Japan).

DNA Extraction and Phylogenetic Tree Construction.
DNA samples of LNU20092087, LNU20092088, LNU20092089, and LNU20092090 were extracted using the DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, Beijing). The procedures for PCR amplification and sequencing were executed as described previously [1]. The rbcL gene sequences of 32 extra relevant Halymeniaceae species and two additional related family (Gelidiaceae and Gracilariaceae) species were selected from GenBank for analysis and were involved in the alignment (Table 1). Gelidiella ligulata Dawson and Gracilaria tenuistipitata Chang et Xia were treated as outgroups.
The rbcL sequences were aligned and compiled with Clustal X version 1.83 [21] for phylogenetic analysis. Phylogenetic tree construction and nucleotide differences analyses were conducted using MEGA 5.0. Maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) were adopted to construct the phylogenetic tree. The MP analysis used heuristic searches for evaluating tree likelihoods, which was carried out with 1000 replicates, employed random addition sequence of taxa and used tree-bisectionreconnection (TBR) branch swapping [22]. The NJ analysis used the ratio test for estimation to seek optimal settings and ensure data dependability and was performed with Modeltest version 3.06 [23]. For ML analysis, a variety of cumulatively complex models of molecular evolution were assessed, as summarized by Litaker et al. [24] and Moncalvo et al. [21]. Bootstrap support values were calculated using 1000 samplings of the dataset [25] to estimate statistical reliability for MP, NJ, and ML methods.

Taxonomic Descriptions.
Thallus simplex, linear, purplish red in color, cartilaginous and slippery in texture, 4-7 cm in height, main axes about 1 mm in width, branches about 0.2-0.6 mm in width, with fewer branches than other thalli ( Figure 1). Main axes of thalli and branches relatively slight, erect axes cylindrical or subcylindrical and bearing alternation or second branches, twice or thrice dichotomously branched (Figures 2(a) and 2(b)). Cortex 5-6 layers, cells elliptic or polygonal and arranged densely; medulla solid with relatively intensive filaments when young, but hollow in the center in old branches. Gametophytes dioecious with reproductive structures dispersed in main axes of thalli and lower portion of branches, and cystocarps of main axes significantly fewer than branches (Figures 2(a)-2(c)). Carposporangium developed from gonimoblasts cells, profoundly immersed inside medulla and revolved by branched ampullar filaments. Mature cystocarps 90-110 m in diameter. Spermatangium shaped from outermost cortex cells of male gametophytes. Mature tetrasporangium cruciately partitioned, 10-16 m long, 6-8 m in diameter, and inserted in outer cortical cells.
Consider the following.
Etymology; "tenuis" refers to the morphology of the thallus.
Holotype; appointed here is a female specimen (LNU20092088 Figure 1). Habitat and seasonality; plant collections were seasonal from January to March, and were attached on rocky reefs or stony marsh of coastal intertidal zone.

Vegetative and Reproductive Structures.
Cross-section of the thallus showed a densely arranged cortex and solid   medulla with dense filaments when young, but hollow in the center in old branches (Figures 3(a) and 3(b)). The 5-6 layers of the cortex consisted of oval outer layers cells arranged in neat rows and three to four inner layers of elliptic or rounded cells; the medulla was composed of slender medullary filaments 11-17 m long and 1-2 m wide, which exhibited irregular distribution (Figure 3(c)). The thallus had Grateloupia-type auxiliary cell ampullae. Auxiliary cells (ac) and carpogonial branches were produced in independent ampulla initiated from inner cortical cells. Two-celled carpogonial branches existed in each carpogonial branch ampulla and contained a terminal carpogonium and hypogynous cell (Figure 3(d)). The auxiliary cell ampullae were narrowly bottle-shaped and comprised of two or three secondary filaments. The mature auxiliary cell (ac) was elliptical in shape and obviously larger than other ampullary cells, and was located at the basal part of the ampulla (Figure 3(e)). Successive stages of the cystocarp development are displayed in Figures 3(f)-3(i). Terminal cells of the gonimoblast filaments gradually matured (Figure 3(f)). Maturing carposporangia developed from gonimoblasts cells, and were surrounded by branched ampullar filaments (Figure 3(g)).  [12] As cystocarps proceeded with development, they became increasingly larger and more deeply embedded in the medulla (Figure 3(h)). Mature cystocarps were spherical or ellipsoidal and 90-110 m in diameter (Figure 3(i)). The mature cystocarp released carpospores (Figure 3(j)). Spermatangia of the male gametophytes were shaped from the outermost cortex cells (Figure 3(k)). Mature tetrasporangia formed from the sporophyte cortical cells were cruciately partitioned, 10-16 m long, and 6-8 m in diameter (Figure 3(l)).

Molecular Analysis.
In the phylogenetic tree, we compared four G. tenuis rbcL gene sequences with a total of 34 extra rbcL sequences from taxa comprising 28 species of Grateloupia, with four species from other genera (Halymenia C. Agardh and Polyopes J. Agardh) in Halymeniaceae and two species from Gelidiaceae and Gracilariaceae treated as outgroups, which were selected from GenBank for analyses ( Table 1). The rbcL sequences of the four Lingshui samples were uniform. The rbcL sequence alignment consisted of 1322 base pairs (bp), but since many rbcL sequences were incomplete at the 5 and 3 ends, the first 67 bp and last 15 bp were excluded from analyses. The maximum likelihood phylogenetic tree (Figure 4) was obtained through the NJ, MP, and ML analysis. The species aggregate of the ML phylogenetic tree was divided into three main clades with high bootstrap support values and included a large Grateloupia clade; a Halymenia/Polyopes clade, which were all within Halymeniaceae; and a Gelidiella/Gracilaria clade, which were in Gelidiaceae and Gracilariaceae, respectively. The four G. tenuis samples and the 28 kinds of Grateloupia genera formed together into one large clade. The Grateloupia clade was divided into a small and large clade, and G. tenuis formed a monophyletic group within the small clade.

Discussion
The family Halymeniaceae exhibits high species richness, especially within Grateloupia, which makes taxonomic species identification difficult. Nevertheless, the formation of auxiliary cell ampullae is a remarkable characteristic for distinguishing genera within Halymeniaceae [2,3,6,26].
The newly described G. tenuis species is morphologically similar to some species, especially G. catenata. Table 2 shows a comparison in morphological features among G. tenuis and the closely related species G. catenata, G. ramosissima, G. orientalis, G. filicina. It was easy to differentiate G. tenuis from G. catenata by its significantly smaller size (4-7 cm high compared to 35 cm high, resp.) and by its dispersed reproductive structures in the main axes of thalli and lower portions of branchlets compared to the scattered reproductive structures over the whole thallus in G. catenata. It was possible to separate G. tenuis from generitype G. filicina by its slippery and cartilaginous texture compared with the mucilaginous and hard texture of G. filicina, and by its dichotomous branches compared with pinnate branchlets. Distinction with G. ramosissima and G. orientalis showed that they had abundant branches. G. tenuis also had representative Grateloupiatype auxiliary cell ampullae, which demonstrated that it was a new species of the family Halymeniaceae and pertained to the genus Grateloupia.
The rbcL sequence data also strongly supported G. tenuis as a new species. In the ML phylogenetic tree (Figure 4)  and generitype G. filicina from Italy. The G. catenata/G. ramosissima subclade was the most closely related sister taxon to G. tenuis and the most similar in appearance. In addition, G. tenuis produced a high bootstrap support value with the G. catenata/G. ramosissima subclade. The generitype species, G. filicina, belonged to the sister position of G.
tenuis, which strongly supported that G. tenuis phylogenetically approached G. filicina. Moreover, G. orientalis, and G. filiformis formed a sister clade with G. tenuis. All Grateloupia species were phylogenetically different to the two clades, especially Gelidiella ligulata and Gracilaria tenuistipitata serving as outgroups.

Conclusions
From morphological observation and rbcL gene sequence analysis, we concluded that the studied specimen was a new species of genus Grateloupia, defined as Grateloupia tenuis Wang et Luan sp. nov. Currently, due to algae species diversity, traditional taxonomic methods are not the most effective way to identify species. With the rapid development of molecular biology and gene sequencing technology; however, taxonomists are increasingly using combined morphological observation with DNA barcoding for algae classification.
In recent years, human impact on marine ecosystems has increased the urgent need to conserve aquatic resources, and accurate species identification is a basic prerequisite in helping protect marine algae.