2 Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou), Guangzhou 511458, China
Polysiphonia Greville is the largest genus of red algae, in which 705 species names are present and about 193 are currently recognized species (Guiry, 2020). These species are common and widely distributed on virtually all coasts of the world (Womersley, 1979). The members in this genus have a wide range of morphological variability that has led to much debate as to how species should be defined and classified, and the generic circumscription has been in an almost constant state of flux since its original description (Mamoozadeh and Freshwater, 2012; Díaz-Tapia et al., 2017a; Huisman et al., 2017).
Recently, based on integrative molecular and morphological studies, the tribal and generic classification of Polysiphonieae was reviewed, and Polysiphonieae had been segmented into Polysiphonieae and Streblocladieae (Díaz-Tapia et al., 2017a). As the tribal classification had been changed, the generic circumscription of Polysiphonia also had been improved, for example, the Polysiphonia species with multinucleate trichoblast cells have been combined into Vertebrata and the Polysiphonia species with plastids lying exclusively on radial walls of pericentral cells have been combined into Melanothamnus (Díaz-Tapia et al., 2017b).
In China, 3 genera and 34 species of Polysiphonieae and Streblocladieae were described based on morphological characteristics (Xiang, 2004; Xia, 2011; Xia and Wang, 2012); nevertheless, molecular analyses have never been performed, and their phylogenetic relationships remain unknown. Considering the recent taxonomic rearrangements in the Polysiphonieae (Díaz-Tapia et al., 2017a, b), the tribal and generic assignments require revision.
Polysiphonia hainanensis Xia et Wang were previously reported from China based on morphological characteristics (Xia and Wang, 2012), but has not been listed in the AlgaeBase (Guiry, 2020). It is unusual in having rhizoids in open connection with pericentral cells and 10(-12) pericentral cells. The rhizoids character is typical of the tribe Polysiphonieae and is unknown in Streblocladieae, and most species with more than four pericentral cells are in the Streblocladieae (Díaz-Tapia et al., 2017a, b, 2018). Therefore, it is uncertain on the relationship of P. hainanensis and its related taxa.
During our sampling surveys in Guangdong and Hainan provinces, we found numerous specimens with characters that matched the main features of P. hainanensis. This study aims to conduct a taxonomic reassessment on P. hainanensis and clearify the phylogenetic position among the related taxa based on molecular and morphological analyses. Furthermore, the morphological observation of sporophyte, male and female gametophytes was conducted.2 MATERIAL AND METHOD
Specimens collected from Nan'ao Island, Shantou City (23°24.32′N, 117°3.42′E), Honghai Bay, Shanwei City (22°39.32′N, 115°34.21′E), Yinggehai, Ledong City (18°28.43′N, 108°46.50′E), Boao, Qionghai City (19°9.27′N, 110°35.11′E) (Fig. 1). They were fixed in 5% formalin in seawater, and voucher specimens were deposited in the herbarium at the Laboratory of Marine Algae, Shantou University, China. Sections for microscopic examination were cut into slices and stained with 1% aceto carmine. Mounted slides were made with fresh or stained fragments. Photomicrographs were taken using a digital Olympus DP20 camera (Olympus, Tokyo, Japan) attached on the microscope Olympus BX53.
DNA was extracted from silica-gel samples using a DNeasy Plant Kit (TIANGEN Biotech, Beijing, China). Partial sequences of the rbcL gene were amplified using one pair primers designed by ourselves: TanF72, 5′-AAAAATGGGATACTGGGATC-3′ and TanR1144, 5′-CCACAATGGATACCACCTGA-3′. Polymerase chain reaction (PCR) cycle was amplified: 94 ℃ for 6 min, 30 cycles at 94 ℃ for 45 s, 52 ℃ for 1 min, 72 ℃ for 1 min, and a final extension at 72 ℃ for 10 min, with a Mycylcer thermal cycler (Bio-Rad, USA). Each 20 μL PCR solution consisted of 10-μL 2×Taq PCR Master Mix, 1-μL template DNA, 1 μL of each primer (10 mmol/L), and 7-μL ddH2O. The sequencing was performed by the BGI Biotech Co. Ltd. (Shenzhen, China). All sequences generated in the present study have been accessioned in GenBank (MN709123–MN709126).
Four rbcL sequences generated in the present study and others 45 sequences obtained from GenBank were aligned with ClustalX v2.1 (Larkin et al., 2007) and corrected manually using MEGA v5 (Tamura et al., 2011). Model parameters were estimated in Modeltest v3.7 (Posada and Crandall, 1998). GTR+I+G model was selected for sequences analyses used in the construction of both Bayesian Inference (BI) and maximum likelihood (ML) trees. Bayesian inference was performed MrBayes v3.2 (Ronquist et al., 2012). The MCMC runs were carried out for 3 million generations each with one cold chain and three heated chains, with sampling and printing every 100 generations. Summary trees were generated using a burnin value of 7500. The ML tree was constructed using PhyML 3.0 software (Guindon and Gascuel, 2003) with 3 000 bootstrap replicates. Pairwise distance estimation used the Kimura two-parameter model. Two species of Pterosiphonia were selected as the out-group based on our phylogenomic analyses of the major lineages of the Rhodomelaceae (Díaz-Tapia et al., 2018).3 RESULT 3.1 Molecular analysis
Four rbcL sequences were generated, and one of them was from the specimen collected in the type locality. Our sequences were compared to those related ones from GenBank and the phylogenetic trees recovered P. hainanensis as a member of the Polysiphonieae (Fig. 2). Within the Polysiphonieae, three clades were resolved: 1) Bryocladia/Falkenbergiella; 2) Polysiphonia sensu stricto; and 3) Lophosiphonia and Epizonaria. P. hainanensis was nested in Bryocladia/Falkenbergiella with high bootstrap value. Sequences from the four specimens of P. hainanensis were essentially identical (0.07% divergence) and formed a stable clade with Bryocladia thyrsigera (J. Agardh) F. Schmitz from Brazil (MF094040) with 100% bootstrap support. The genetic divergence between P. hainanensis and B. thyrsigera was 0.74%–0.81%. On the other hand, Polysiphonia was not monophyletic, and P. hainanensis was distant from the generic type species Polysiphonia stricta (MF101428) with about 8% genetic divergence.3.2 Species description
Bryocladia thyrsigera (J. Agardh) F. Schmitz in Falkenberg, 1901, p 169.
Polysiphonia thyrsigera J. Agardh, 1847 p. 17.
Polysiphonia hainanensis Xia et Wang, 2012, p. 963–965.
Type locality: La Guayra, South America.
Thalli were brownish red or brownish black, saxicolous, 2–7 cm high, and grew in tufts with an entangled prostrate system and erect axes (Fig. 3a). Erect axes had a distinct main axes arising from prostrate axes (Fig. 3a). Main axes were percurrent and borne lateral indeterminate branches in 1/4 spirally arrangement every 1–7 segments, and the axes of lateral indeterminate branches mostly borne determinate branches in 1/4 spirally arrangement every 0–4 segments (Fig. 3b). Main axes and indeterminate branches grew via a dome-shaped apical cell that divided slightly obliquely, whereas determinate branches grew via a spinous apical cell that divided almost transversely (Fig. 3c).
Axes were ecorticate, consisting of a central axial cell and 8–10(-12) pericentral cells (Fig. 3d–f). Plastids were discoid, lying all along the cell walls of pericentral cells (Fig. 3g). Prostrate axes were 100– 180 μm in diameter, and segments were wider than long, 0.2–0.3 L/D (Fig. 3h). Unicellular rhizoids which were open connection with pericentral cells, were 15–40 μm in diameter, with or without a discoid haptera at the terminal part (Fig. 3h). Erect axes grew from an apical cell 3–51 μm in diameter with segments 0.2–0.5 L/D, increasing in diameter to 280–350 (-450) μm in mid and basal parts, with segments 0.2–0.3 L/D (Fig. 3i). Trichoblasts were formed by uninucleate cells, 1–3 times dichotomously divided, and only found in mature female plants (Fig. 3j).
Procarps were formed on a fertile trichoblast (Fig. 4a). Cystocarps were globose, and 250–300 μm in diameter (Fig. 4b). Carpospores are clavate, 60– 100 μm long (Fig. 4c). Spermatangial branches were cylindrical, colorless, 150–180 μm long, 30–50 μm in diameter, without a sterile tip, developed from the entire trichoblast primordium in clusters on short axis with 1/4 spirally arrangement (Fig. 4d–f). Tetrasporangia were arranged in straight series and composed of one per fertile segment of determinate branches, and their stichidia were in clusters on short axis (Fig. 4g & h). The mature ones are divided tetrahedrally, 40–50 μm in diameter, and surround by two presporangial cover cell (Fig. 4h & i).
Habitat: Thalli grow in the intertidal zone attached on rocks.
Domestic distribution: Guangdong, Hainan.
Foreign distribution: America, Africa, and Western Atlantic (Guiry, 2020).
Specimens examined: STU20130405017 (Ⅰ–XXXVI) were collected from Hainan on November 2013 by Tan Huaqiang; STU20140103015 (Ⅰ–Ⅻ) were collected from Guangdong on April 2014 by Tan Huaqiang; STU20150402012 (Ⅰ–Ⅵ) were collected from Guangdong on February 2015 by Tan Huaqiang.4 DISCUSSION
The rbcL sequences from our specimens collected from four sides were essentially identical (0.07% divergence), and formed a clade with Bryocladia thyrsigera from Brazil with strong bootstrap support (100%), among which the divergence was 0.74%– 0.81%. The previous studies of rbcL sequence data in Polysiphonia sensu lato detected comparatively low intraspecific, with values ranging 0–1.3% (McIvor et al., 2001; Kim et al., 2004; Kim and Yang, 2005). Therefore, it suggests that the Chinese and Brazilian specimens should be the same species. Because the Bryocladia/Falkenbergiella clade was distant from the type species P. stricta of the genus Polisiphonia sensu lato, Huisman et al. (2017) had suggested that a separate genus might be appropriate for the Bryocladia/Falkenbergiella. Since molecular information of the type species of this genus, B. cervicornis (Kützing) F. Schmitz, is unavailable, Bryocladia should still be valid.
The genus Bryocladia have the following key morphological characteristics (Schmitz and Falkenberg, 1897; De Toni, 1903; Dawes and Mathieson, 2008; Suárez et al., 2015): 1) thallus comprised of prostrate branches, and erect branches, having a percurrent axis; 2) all branches ecorticate, densely clothed with short, determinate, spine-like, exogenous polysiphonous branchlets, spirally arranged, and with up to 12 pericentral cells; 3) tetrasporangia single in a segment, in a straight series, stichidia in clusters on short axis; and 4) spermatangial capitula replace trichoblasts, conoidal, with or without sterile tip, in clusters on short axis. By comparison, we found that morphological characteristics of Chinese specimens marched the genus Bryocladia (Table 1). Furthermore, morphological comparison (Table 2) suggested B. thyrsigera, P. hainanensis and newly collected specimens from China are conspecific.
The number of pericentral cells was commonly used to distinguish species of Polysiphonia sensu lato (Stuercke and Freshwater, 2008). Xia and Wang (2012) mentioned their new species was closely related to Polysiphonia howei and Vertebrata hendryi var. luxurians based mainly on the morphological character of more than ten pericentral cells. However, in our phylogenic tree, we found B. thyrsigera was placed in the Bryocladia/Falkenbergiella clade, P. howei was near to the Epizonaria clade, and V. hendryi was placed in the family of Streblocladieae. Futher more, in this study, the other eight species (Bryocladia cuspidate, Lampisiphonia iberica, Leptosiphonia schousboei, Polysiphonia adamsiae, and three Vertebrata species) of Polysiphonia sensu lato, all having more than four pericentral cells were also not closely related and scattered in Polysiphonieae and Streblocladieae. It suggests the number of pericentral cells may originate based on parallel evolution, and is not applied well to determine the relationship of species among Polysiphonia sensu lato.5 DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Sequence data that support the findings of this study have been deposited in GenBank under the accession numbers MN709123– MN709126.
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