Diversity and Ecology of Lobophora Species Associated with Coral Reef Systems in the Western Gulf of Thailand, including the Description of Two New Species

The brown macroalgal genus Lobophora plays important ecological roles in many marine ecosystems. This group has received much attention over the past decade, and a considerable number of new species have been identified globally. However, our knowledge of the genus diversity and ecology along south-east Asian coasts are still limited. Given the growing body of research that uses a combination of molecular and morphological data to identify cryptic species, this study investigates the diversity of Lobophora in the western Gulf of Thailand using morphological and molecular data, as well as their interactions with scleractinian corals. A total of 36 Lobophora specimens were collected from 15 sites in the western Gulf of Thailand and used for molecular and morphological analyses. One mitochondrial (cox3) and two chloroplast (psbA and rbcL) genes were amplified and sequenced for molecular phylogenetic analyses. Based primarily on phylogenetic evidence, two new species were formally described, L. chumphonensis sp. nov. and L. thailandensis sp. nov. Additionally, L. lamourouxii was newly recorded from Thailand. Two new lineages of Lobophora obscura were identified, L. obscura12 and L. obscura13. Among the Lobophora species identified, three were found in interaction with corals, the most notable of which was the massive coral Porites. Lobophora chumphonensis sp. nov. only interacted with Porites by growing on bare coral skeleton between Porites colonies. Furthermore, L. obscura13 was observed under the branching coral Pocillopora. Our findings revealed that Lobophora presented both effects and absence of effects on coral. A thorough understanding of Lobophora diversity and ecology is essential for ongoing and future research on coral–macroalgal ecological relationships.


Introduction
The brown algal genus Lobophora J. Agardh is naturally associated with coral reefs in tropical and subtropical seas [1]. Lobophora species are characterized by a relatively small thalli with a simple morphology, ranging from erect to crustose morphotypes [1]. A more detailed perspective of the species diversity and distribution of Lobophora has been obtained in recent years [2][3][4][5][6][7][8]. Currently, a total of 71 species are taxonomically accepted according to the online database for algae, AlgaeBase [9]. Moreover, the diversity of Lobophora was estimated to surpass 100 species worldwide [5].
Prior to the use of molecular tools for this genus, L. variegata has been widely reported in Thailand, including in the Gulf of Thailand [29][30][31][32]. More recent studies that have made use of molecular tools revealed the presence of three putative species of Lobophora (L. sp.24, L. sp.49, and L. sp.56) in the Southern Andaman Sea, Thailand [4,33], but not the genuine L. variegata. A recent study on the genus diversity from the Western Indian Ocean unveiled an important diversity in this region, with no less than 43 species, which stands in contrast with the only 3 species recorded to date in the Gulf of Thailand adjacent to the Indian Ocean [34]. There is a clear gap in knowledge in this part of the world for the diversity of the genus Lobophora species. Comparatively, our knowledge of Lobophora ecology in this region is very limited. No ecological studies have been conducted in Thailand, including on Lobophora-coral interactions. This study aimed to document Lobophora species diversity and ecology in coral reef areas in the western Gulf of Thailand and interactions with scleractinian corals on coral reef systems in the western Gulf of Thailand.

Taxonomic Results
A total of 36 algal specimens were successfully sequenced with cox3, and 16 algal genomic DNA from representatives of each species were sequenced with psbA and rcbL genes. The molecular analyses based on the individual and concatenated alignments of cox3, rbcL, and psbA sequences revealed five/six Lobophora lineages from coral reefs in the western Gulf of Thailand (Figure 1), including one previously described species, L. lamourouxii, Payri and C.W. Vieira in [8]; one previously identified lineage part of the L. pachyventera complex, L. pachyventera4 from Malaysia ( Figure S1); one lineage related to L. abscondita, C.W.Vieira, Payri, and De Clerck, and L. sp82; and finally two to three lineages part of the L. obscura complex. Based on cox3 and concatenated trees, the L. obscura lineages presently identified from Thailand are closely related to L. obscura7, L. obscura9 and L. astrolabeae, C.W.Vieira and Payri [8], whereas resolution of relationships among these species is less clear in the chloroplast trees (psbA and rbcL) ( Figures S2 and S3). Based on molecular and morphological data, the lineage related to L. abscondita and the one corresponding to L. pachyventera4 are herein formally described as two new species: Lobophora chumphonensis sp. nov. and Lobophora thailandensis sp. nov.  Table 1) Description: Large orbicular thallus, up to 40 cm in diameter, thin, strongly adherent to the substratum across the whole of the ventral surface by abundant rhizoids, dark brown in color. Thallus composed of single-cell-layered medulla, two to six-and two to four-celllayered cortex on the dorsal and ventral side, respectively. The thallus was 75-105 µm thick and composed of 5-11-cell layers. The sporangium was 36-48 µm in height and 60-78 µm in width.
Type locality: Chumphon archipelago, Ang Thong archipelago. Distribution: Thailand. Holotype: THNHM-P-2020-0258, collected 5 March 2020, deposited in the Herbarium of Natural History Museum of the National Science Museum in Thailand.
Habitat: This species is commonly grown on hard substratum between free-space Porites colonies or grown on the upper dead space of Porites colonies in various habitats including fringing reefs and shallow reef flats.  Description: Large orbicular thallus, up to 40 cm in diameter, thin, strongly adherent to the substratum across the whole of the ventral surface by abundant rhizoids, dark brown in color. Thallus composed of single-cell-layered medulla, two to six-and two to four-cell-layered cortex on the dorsal and ventral side, respectively. The thallus was 75-105 μm thick and composed of 5-11-cell layers. The sporangium was 36-48 μm in height and 60-78 μm in width.    Description: Flabellate thallus, up to 5 cm wide and 4 cm tall, thin, adherent to the substratum by rhizoids on the margin, dark brown to orange brown on dorsal in color, and orange brown to orange in color on ventral side. Thallus composed of single-to double-celllayered medulla, two to seven-and two to four-cell-layered cortex on the dorsal and ventral side, respectively. The thallus was 87-110 µm thick and composed of 5-12-cell layers.
Distribution: Thailand, Malaysia. Holotype: THNHM-P-2020-0287, collected 12 March 2020, deposited in the Herbarium of Natural History Museum of the National Science Museum in Thailand.
Habitat: This species is commonly grown on hard substratum, mostly dead corals and large rubbles, and can encrust on live corals in various habitats including fringing reefs and shallow reef flats.
Etymology: the specific epithet refers to Thailand, which is the locality where the materials were collected.

Morphological, Anatomical, and ECOLOGICAL Characteristics
Lobophora obscura exhibited two morphotypes corresponding to two to three different lineages that are part of the Lobophora obscura complex, one (hereafter identified as L. obscura12) closely related to L. obscura7 and the other one/two (hereafter identified as L. obscura13) to L. obscura9 and L. astrolabeae, based on the cox3 and concatenated trees. The anatomical characteristics of the L. obscura lineages, L. chumphonensis sp. nov., and L. thailandensis sp. nov. exhibited intraspecific variation, except that of L. lamourouxii, which exhibited identical characteristics within species in this study (Table 1 and Figure 2). A hierarchical clustering of Lobophora species by using morphology, thallus thickness, height, and width as predictors revealed that the two L. obscura lineages were nearby species by their morphology, as well as Lobophora lamourouxii and L. thailandensis sp. nov., which were also contiguous species. In contrast, L. chumphonensis sp. nov. distinguished itself from both lineages by its morphology. Hierarchical cluster analysis of morphological traits (thallus thickness, height, width, and morphology) revealed two clear groupings of species in different colors ( Figure 3). Thallus height (R 2 = 0.99, p < 0.001) and growth forms (R 2 = 0.36, p < 0.05) contributed to the clustering of Lobophora species, whereas thallus thickness (R 2 = 0.09, p > 0.05) and thallus width (R 2 = 0.09, p > 0.05) did not significantly. In the study areas, one parrotfish (Scarus rivulatus Valenciennes, 1840) and three rabbitfishes (Siganus canaliculatus Park, 1797, S. guttatus Bloch, 1787, and S. virgatus Valenciennes, 1835) were recorded. Lobophora lamourouxii and L. obscura12 were found only on Taen Island at depths of approximately 3.5 m, with low herbivory pressure, and L. chumphonensis sp. nov. was found in only three locations at depths of >3 m, with moderate herbivory pressure. On the  the study areas, one parrotfish (Scarus rivulatus) and three rabbitfishes (Siganus canaliculatus, S. guttatus, and S. virgatus) were recorded. Lobophora lamourouxii and L. obscura12 were found only on Taen Island at depths of approximately 3.5 m, with low herbivory pressure, and L. chumphonensis sp. nov. was found in only three locations at depths of >3 m, with moderate herbivory pressure. On the other hand, L. obscura13 and L. thailandensis sp. nov. are both common in the western Gulf of Thailand and can be found at depths of 1-7 m with moderate herbivory pressure. Figure 3. Hierarchical cluster analysis of Lobophora species by using morphology, thallus thickness, height, length, and width as predictors. Reef types, depth, herbivorous fish pressure, and sites found were not used to analyze hierarchical cluster. Reefs and depth presented where the Lobophora can be found. Herbivorous fish pressure categorized by the abundance of herbivorous fish occurs in areas. The number of sites found is shown. Figure 3. Hierarchical cluster analysis of Lobophora species by using morphology, thallus thickness, height, length, and width as predictors. Reef types, depth, herbivorous fish pressure, and sites found were not used to analyze hierarchical cluster. Reefs and depth presented where the Lobophora can be found. Herbivorous fish pressure categorized by the abundance of herbivorous fish occurs in areas. The number of sites found is shown.

Interactions between Lobophora and Corals
Lobophora-coral interactions in this study focused on Lobophora growing on, at the base, or at the vicinity of live corals. Three species of Lobophora (L. chumphonensis sp. nov., L. obscura13, and L. thailandensis sp. nov.) were in direct physical contact with live corals, whereas L. obcura12 and L. lamourouxii were not found near corals (Table S1). Lobophora chumphonensis sp. nov. interacted only with colonies of the massive coral Porites, by growing on bare space between Porites colonies. Signs of pink line syndrome were systematically observed on the edge L. chumphonensis sp. nov. thalli attached to Porites lutea ( Figure S4e,f). Lobophora obscura13 was the second most important species found in close interaction with the massive coral Porites. Some individuals of L. obscura13 were found underneath the branching coral Pocillopora acuta. Moreover, pink line syndrome was occurred, minor bleaching of Porites lutea was observed, and overgrowth by L. obscura13 with crustose habit covered unhealthy Favites colonies at Kula Island ( Figure S4a-d). Lobophora thailandensis sp. nov. mostly interacted with the massive coral Porites, and occurrences of pink line syndrome was documented as well ( Figure S4g,h).

Discussion
This research is the first taxonomic revision of the brown alga genus Lobophora using both morphological and molecular data in the Gulf of Thailand and reports of Lobophora effects on scleractinian corals. Moreover, two new species are officially described as L. chumphonensis sp. nov. and L. thailandensis sp. nov.
Lobophora species are typically found in association with corals in the western Gulf of Thailand, particularly on Tao and Taen Islands [35,36]. Lobophora were mostly found at depths of less than 3 m in the western Gulf of Thailand, which is consistent with our findings. Lobophora lamourouxii is a new species record in Thailand, and it was recently discovered in Singaporean waters [37,38]. Our results revealed that Lobophora species mostly interact with the massive coral Porites lutea. Porites is susceptible to macroalgae competition, with wide reports of coral tissue damages and bleaching when adjacent to Lobophora or directly in contact with Lobophora [11,16,18].
Diaz-Pulido and McCook [39] demonstrated that Lobophora propagules could not settle on healthy tissue of coral species, but only on bare skeleton next to healthy tissue. In this study, we discovered that L. chumphonensis sp. nov. appears to settle on bare skeleton between Porites lutea colonies. According to Puk et al. [40], Lobophora sp.82, which is related to L. chumphonensis sp. nov., grows at the base of Porites cylindrica colonies, and Jompa and McCook [11] concluded that the coral P. cylindrica provides a refuge for Lobophora from herbivory.
Interestingly, our results show that L. obscura13 with an encrusting form grows under the branching coral Pocillopora acuta, which is the first report of this effect with the branching coral genus Pocillopora. Some Lobophora species grow close to corals, under and among coral branches, where they are protected from herbivores [22,41].
High percentages of algal cover have been reported in Kula Island coral reefs [42], consisting of L. obscura13 and L. thailandensis sp. nov., which both occupy large areas and compete mutually for space. Overall, Lobophora species do not represent a specific threat to corals when the coverage of macroalgae is controlled by coral defenses and herbivores [8], with only one case of aggressive coral overgrowth documented in healthy coral reefs by the species L. hederacea on the coral Seriatopora caliendrum [10]. However, Lobophora overgrowth is most likely caused by a severe event, such as a storm, thermal stress, or mass bleaching [8]. The high sediment load from the mainland may have caused the overgrowth of Lobophora on Kula Island, as the water was relatively turbid [43]. Furthermore, because the depth of the study sites is very shallow, studies suggest that the abundance of herbivorous fishes is inversely related to water clarity. As a result, declining water quality can negatively affect grazing pressure on algal communities in coastal areas. [44,45].
In the Gulf of Thailand, Müller et al.'s [37] results suggest that the rabbitfish and parrotfish species observed in the Gulf of Thailand avoided feeding on Lobophora spp.; however, these observations come from an experimental set-up in a narrow study area, and Lobophora can produce a chemical defense to protect themselves against herbivory [22]. The importance of grazing on Lobophora in natural environments and on a broader scale remains undocumented. In addition, other publications reported active grazing from Scarus spp. and Siganus spp. in adults and recruit of Lobophora [40,46], indicating that in natural conditions species from both genera might control the growth and spread of this alga in Thailand's reefs. Previously, contrasting reports were made on the relationship between Lobophora spp. growth forms, production of secondary metabolites with defensive roles, and their susceptibility to herbivory [10,47,48]; with [22] pointing toward the primary role of ecological habit (e.g., associational defense and spatial refug-es) as defense strategies against herbivory over chemical or morphological defenses [22].
Our results provide baseline data for the Lobophora species diversity and ecology in the western Gulf of Thailand that can be useful for coral management strategies. Further studies are needed to understand the diversity and distribution of Lobophora, including their interaction in other parts of the Gulf of Thailand. The allelopathic effects of L. obscura13 and L. thailandensis sp. nov. and bioactive substances from Lobophora species in this study will be further studied.

Sampling Sites
A total of 36 Lobophora specimens were collected from 15 sites within coral reefs at Chumphon and Ang Thong Archipelagoes, Prachuap Khiri Khan and Samui islands, the western Gulf of Thailand, from January to March in 2020 ( Figure S5). Details of algal specimens are provided in Table S2 in the Supporting Information. Sampling was carried out at 1-7 m depth by snorkeling or SCUBA diving. Algal specimen fragments were preserved in silica gel for molecular analyses, and remains of the specimens were processed as herbarium specimens, later deposited at the Natural History Museum at Klong Ha, Khlong Luang District, Pathum Thani Province, for morphological and anatomical analyses.

DNA Extraction and Phylogenetic Analyses
The DNeasy Plant minikit (Qiagen, Hiden, Germany) was used to extract algal specimens' genomic DNA, following the manufacturer's instruction. Two chloroplast genes (rbcL, psbA) and one mitochondrial gene (cox3) were selected for molecular analyses due to their importance in recent Lobophora phylogenetic studies, e.g., [2,4,7,49]. Information on primers, PCR, and sequencing conditions are listed in Table S3. Phylogenetic trees were reconstructed using a maximum likelihood approach in PhyML v.3.0 [50] based on individual and concatenated alignments of the cox3 (724 bp), psbA (1030 bp), and rbcL (1348 bp) sequences. Genbank accession data used in this study are listed in Table S4.

Morphological and Anatomical Characteristics
Morphological analyses consisted of in situ observations of the thallus color and growth forms of Lobophora specimens following [2]. Dried thalli from the herbarium were rehydrated in seawater, and both transverse and longitudinal sections of the middle part of the plant were made manually using a razor blade and then mounted on a glass slide for microscopic observations. The number and size of both dorsal and ventral cortical cells, including the size of the medulla layer, were measured following [2].

Ecological Studies
Ecological analyses consisted in the identification of the main substratum, categorized as follows: (1) growing on live corals, (2) growing at the base of live coral or vicinity of live coral including dead coral, (3) coral rubbles and bedrock, and (4) growing on seaweed bed. Interactions between Lobophora species and corals were observed using three permanent belt transects at each study site; the effects of Lobophora on corals were conducted from the photo belt transects. Photo belt transects were used as described by [51], consisting of a 30 m transect laid across reef-dominated systems on each site. Photographs were directly taken within 50 cm on each side of belt transect, using a digital camera (Olympus Tough TG-6). Coral species were identified with the genus levels according to [52]. Both negative and positive effects of Lobophora towards corals in the western Gulf of Thailand were recorded. The number of thallus of each species were recorded from the photo belt transect and categorized into three mains: (1) growing on live coral, (2) growing at vicinity of live coral, (3) non-coral substrates to analyze quantification of interaction between Lobophora and corals and expressed in percentage (100% for each Lobophora species). In addition, fish species were identified and counted in situ, and herbivory pressure was estimated. An underwater visual census consisted of three 30 m long and 2 m wide transect per site, performed by Felipe M.G. Mattos (Academia Sinica, Taiwan). Herbivore pressure was categorized from the average abundance of herbivorous fish from where Lobophora species were found as follows: (1) low pressure: 0-50 ind./100 m 2 , (2) moderate pressure: 51-100 ind./100 m 2 , and (3) high pressure: >100 ind./100 m 2 . In addition, reef types and depths where samples were collected were recorded.

Statistical Analyses
To compare the similarities between the characteristics of algal species, Tukey's HSD was used to perform from the package "agricolae" in R. Finally, coefficients of variation for each characteristic were calculated in R to compare results for particular characteristics.
Similarities among species were analyzed in R by using morphological traits (growth forms, thallus thickness, thallus height, and thallus width) as predictors to test for morphological differences among species and create clusters based on algal characteristics. A distance matrix was built by the Euclidean method in the R package "cluster" (Table S5) to examine the distance between species and later build a hierarchical cluster. The linear relationships between two predictors Lobophora species were also analyzed ( Figure S6). A hierarchical cluster was built with "hclust" in the R package "cluster" with "ward.D2" agglomeration to analyze the similarities between the morphological traits in Lobophora species [53].

Data Availability Statement:
The partial genomic sequences used in this study will be available on October 31st at The National Center for Biotechnology Information (NCBI) database, or by prior request from the first author. The raw data used in this study are available upon request from the first author.

Conflicts of Interest:
The authors declare no conflict of interest.