1 INTRODUCTION

The brown alga Lobophora (Dictyotales, Phaeophyceae) occurs in tropical and subtropical seas around the world. The genus is characterized by a marginal row of meristematic cells and a single-layered large central medulla in the thallus (Womersley, 1967, 1987). It is difficult to identify species based on morphological criteria alone, and the recent molecular phylogenetic analyses suggest that the species level divergence is considerably underestimated; as consequence, a dozen new species have been described (Sun et al., 2012; Vieira et al., 2014, 2016, 2019; Schultz et al., 2015; Camacho et al., 2019). So far, 45 specific epithets of Lobophora are listed in AlgaeBase (Guiry and Guiry, 2020), and it is estimated that more than 100 species are present in this genus (Vieira et al., 2017).

Previously, a single species of Lobophora, i.e. L. variegata (J. V. Lamouroux) Womersley ex Oliveira, was reported from Bach Long Vy (Bailongwei) Island, a small island located in the center of the Beibu Gulf (Gulf of Tonkin) (Dam, 1997). This species with erect fan-shaped from Vietnam was identified as L. variegata using gross morphological characters (Pham, 1969; Nguyen et al., 1993; Dam, 1997, 2004; Van Nguyen et al., 2013); however, L. variegata is not geographically distributed in the Pacific (Schultz et al., 2015; Vieira et al., 2016). In recent years, some new species have been reported from Vietnam and the neighbor waters (Sun et al., 2012; Phang et al., 2016; Vieira et al., 2016), and therefore in the present study, we conducted a taxonomic study of Lobophora found in Gulf of Tonkin, Vietnam, using morphological and molecular data.

2 MATERIAL AND METHOD 2.1 Sampling and morphological analysis

New specimens were collected by snorkeling or Self-Contained Underwater Breathing Apparatus (SCUBA) diving from three sites (site 1: 20°08'15"N, 107°43'36"E; site 2: 20°08'01"N, 107°44'06"E; site 3: 20°07'39"N, 107°44'03"E) around Bach Long Vy (Bailongwei) Island (Fig. 1). Specimens for molecular studies were desiccated immediately in silica gel. Other specimens were dried and mounted on herbarium sheets. All the herbaria were deposited in the Marine Biological Museum of the Institute of Marine Environment and Resources (MBMIMER), Haiphong, Vietnam. For morphological observations, thalli were sectioned manually using a razor blade and mounted on glass slides in Karo Syrup/seawater. Photographs were taken with a Motic BA300 microscope (Taiwan, China) equipped with a ZEISS A-Plan 40X/0, 65 N. A. Phase contrast 2 objective (Zeiss, Germany) and an Olympus TG 5 digital camera (Japan).

2.2 DNA sequencing and phylogenetic analysis

Genomic DNA was extracted from the silica gel-dried specimens and herbarium specimens (morphology analyzed later) using a DNeasy^® Plant Mini Kit (Qiagen, Hiden, Germany), according to the manufacturer's instructions. The rbcL and cox3 genes were PCR amplified with TaKaRa Ex Taq enzyme in 25-μL reaction column (TaKaRa, Japan). Primers and PCR conditions were as those described by Sun et al. (2012). The DNA sequencing was performed by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China).

Eight specimens from Bach Long Vy Island were newly sequenced. The sequences of specimens from Hainan Island (Sun et al., 2012) were also added to analyze. A concatenated data set consisting of 24 rbcL and 24 cox3 sequences from GenBank, respectively corresponding to 21 specimens, included 15 species of Lobophora and two species of Zonaria (outgroup). A second cox3 data set consisting of sequences from 47 specimens of L. rosacea, 6 specimens of Lobophora sp. 43 (Vieira et al., 2017, 2019), and 3 specimens of the new species in this study.

The concatenated nucleotide matrices were generated with PhyloSuite (Zhang et al., 2020). The new sequences and the previously published ones for phylogenetic analyses were aligned with MAFFT v7.313 (Katoh and Standley, 2013) and then manually adjusted. The maximum likelihood (ML) and Bayesian inference (BI) analysis were carried out to construct phylogenetic trees by using the software of IQ-TREE v1.6 (Nguyen et al., 2015) and MrBayes v3.2.6 (Ronquist et al., 2012), respectively. The best substitution model for each analysis was evaluated by Partitionfinder 2.0 (Lanfear et al., 2017). For BI analysis, two independent analyses were run with four chains each for 100 million generations, with the first 25% of the resulting trees was discarded as burn-in. FigTree v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the trees.

3 RESULT 3.1 Molecular phylogenetic analysis

Although numerous specimens were obtained from the three sites of Bach Long Vy Island and from two sides of Hainan Island (Changjiang in the west coast, Changpo in the east coast), a single identical sequence was obtained for the rbcL gene (1 348 bp) and the cox3 gene (690 bp) respectively. The specimens from Bach Long Vy Island, Vietnam were 100% identical to those from Hainan Island, China, but differed from Lobophora rosacea Vieira, Payri et De Clerck from New Caledonia. In the phylogenic tree based on concatenated rbcL and cox3 sequences data set (Fig. 2), the two species of Zonaria were designated the outgroup, and the remaining 13 species plus several unnamed Lobophora spp. formed a large Lobophora clade. An Atlantic species L. dispersa (LAF06738, LAF06786) Camacho, Freshwater et Fredericq showed the closest relation with the two taxa of Lobophora from Vietnam and China mentioned above.

In the phylogenic tree based on cox3 sequences data set (Fig. 3), most specimens of Lobophora rosacea shared the same sequence, including the type specimens IRD10213 and one specimen from Kenya (ODC1571). In contrast, the specimens from Hainan and Bach Long Vy Islands had a long genetic distance from other ones and showed a closer relation to those identified as Lobophora sp. 43 from West Australia.

3.2 Morphological analysis

Fan-shaped thalli grew on the substrate in the subtidal zone, attached with a basal holdfast. Plants frequently overlapped each other and formed a dense rosette (Fig. 4a). Larger thalli were predominantly erect, spirally arranged, and up to 9-cm wide and 8-cm high. Fresh specimens were yellow to light brown in color but became dark brown when dried (Fig. 4b). Anatomically, the mid-region of the thallus was commonly composed of nine layers, including a single layer of large medullary cells and four layers of small cortical cells on dorsal and ventral sides (Fig. 4c & d). Sporangial sori were scattered on ventral surface of the mature thallus (Fig. 4e), and sporangia were sessile and ovate without paraphyses in section view (Fig. 4f). Male and female reproductive structures were undetected. Some mature specimens with sporangia had seven layers, the medullary layer surrounded by dorsal and ventral layers of cortical cells with three layers (Fig. 4f).

3.3 Taxonomy

Lobophora tsengii D. Tien et Z. Sun sp. nov.

=Lobophora sp. 67 Vieira et al. (2016)

=Lobophora rosacea Sun et al. (2017)

Description:

Large fan-shaped thallus, up to 9-cm wide and 8-cm high, strongly attaching to the substrate by a basal holdfast. Surface smooth, yellow to brown in color. Thallus 90-190-μm thick, composed of a single layer of large medullary cells and three to four layers of dorsal and ventral cortical cells. The gene sequences (MT779811 and MT779812) for the rbcL and cox3 genes from the type specimen (IMER19036) were unique.

Type locality: Bach Long Vy (Bailongwei) Island, Haiphong, Vietnam.

Holotype designated here: IMER19036, a dried herbarium specimen, deposited in the Marine Biological Museum of the Institute of Marine Environment and Resources (MBMIMER), Haiphong, Vietnam.

Habitat: Growing on hard substrates in subtidal zone 2?-m deep, commonly growing with other macroalgae.

Etymology: Named after the Chinese phycologist and taxonomist Cheng Kwei Tseng.

Distribution: Haiphong, Vietnam; Guangdong, Hainan, China.

Specimens examined: Bach Long Vy Island, Haiphong, Vietnam, Dam Duc Tien, 28 July 1993 (HIO93023, HIO93041); Bach Long Vy Island, Haiphong, Vietnam, Dam Duc Tien, 16 May 2003 (HIO03216, HIO03244, HIO03310); Bach Long Vy Island, Haiphong, Vietnam, Dam Duc Tien and Dao Minh Dong, 14 July 2019 (IMER19036-10942).

Remark: Lobophora tsengii mostly closely resembles L. rosacea but differs from the later in having a thicker thallus caused by the incrassation of one or two cortical layers, and the distinctive DNA sequences MT779811 and MT779812.

4 DISCUSSION

The specimens of Lobophora from Beibu (Tonkin) Gulf and Hainan Island are morphologically similar to those from New Caledonia, in having a basal mound of rhizoids, a predominantly erect and fan-shaped thallus that is arranged into a dense rosette (Table 1). Vieira et al. (2014) documented two morphotypes of L. rosacea from New Caledonia, including the thinner type with five to seven cell layers, and the thicker type with five to eight cell layers. Although some thalli from Beibu Gulf and Hainan Island, such as those with sporangial sori, tend to be thinner, our specimens always have more than seven layers (commonly nine layers, with one medullary layer, four dorsal cortical layers, and four ventral cortical layers), and five layers are never present. Consequently, our new species are morphologically different from L. rosacea due to the increase of one or two cortical layers.

In the concatenated rbcL and cox3 phylogenetic tree, the Vietnamese and Chinese specimens formed a clade that separated from other Lobophora species (Fig. 2). Unfortunately, only a few available rbcL sequences of L. rosacea and its closely related taxa were present in GenBank, which were able to correspond to their cox3 sequences. The rbcL sequence of the type specimen (IRD10213) of L. rosacea was probably mistaken, because it differed from those of other conspecific taxa in more than 8% base substitution. The specimens of L. obscura Vieira, De Clerck et Payri (NVT093) and L. asiatica Sun, Tanaka et Kawai (NVT052), which were also present in the phylogenetic tree.

Additionally, the phylogenetic tree based on all published cox3 sequences closely related to L. rosacea, L. tsengii, and Lobophora sp. 43 (Fig. 3), demonstrated that L. tsengii was separated from the related taxa. Vieira et al.(2016, 2017) had conducted species delimitation analyses with cox3 sequences of Lobophora and treated the specimen (MBM616) from Hainan Island as a separate species (Lobophora sp. 67), instead of merging it into L. rosacea. Almost simultaneously, Sun et al. (2017) reported the same specimens from Hainan Island as a population of L. rosacea without the references of Vieira et al.(2016, 2017). Subsequently, another closely related Lobophora sp. 43 was detected from Coral Bay, Western Australia based on cox3 sequences, without morphological description (Vieira et al., 2017, 2019).

Vieira et al. (2017) suggested that most Lobophora species have small ranges limited to marine realms. It seems L. rosacea is endemic in New Caledonia, expect one specimen (ODC1571) collected from Kenya, probably due to the equator warm current in the Indian Ocean. Lobophora sp. 43, which showed a closer genetic distance to L. tsengii than L. rosacea, may be endemic in West Australia. L. tsengii is distributed around Hainan Island, rare in the south coast and abundant in the west coast and Beibu Gulf. Since the morphological and molecular characters and geographic distribution of our specimens are different from the previously described species, it is reasonable to identify them as a separate species. Considering Bach Long Vy Island where this species grows abundantly, we took this offshore Island as the type locality.

Dam (1997) had misidentified Lobophora tsengii as L. variegata based on the specimens collected from Bach Long Vy Island. Luan et al. (2013) also misidentified this species from Hainan Island as L. variegata. However, L. variegata is not distributed in this region and geographically restricted to the Caribbean Sea (Schultz et al., 2015; Vieira et al., 2016). The misidentification may be due to the morphological resemblance in the erect fan-shaped thallus. As to the species with prostrate thallus, Lobophora sp. reported from Nha Trang, Vietnam (Tsutsui et al., 2005) should be identified as L. obscura (=L. crassa Sun, Lim et Kawai in Sun et al., 2012), based on the distinguished morphological feature. Vieira et al. (2016) detected two prostate species, L. obscura and L. asiatica from Vietnam based on molecular phylogenic analyses. To date, three species of Lobophora were reported from Vietnam, including L. asiatica, L. obscura, and L. tsengii (=Lobophora sp. 67 in Vieira et al., 2016; =L. rosacea in Sun et al., 2017).

In our recent survey on the algal flora in Vietnam, several Lobophora-like taxa were detected, and a further investigation should be carried out based on morphological and molecular phylogenic analyses in the future.

5 CONCLUSION

A new species of Lobophora tsengii was described from Bach Long Vy Island, which had been mistakenly identified as L. variegata and L. rosacea in China and Vietnam. Our study shows that L. tsengii is different from the related L. rosacea based on both morphological and molecular data, and the later should be endemic to the Southern Hemisphere.

6 DATA AVAILABILITY STATEMENT

All datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.