Songklanakarin J. Sci. Technol. 42 (3), 504-514, May - Jun. 2020 Original Article Molecular systematics and species distribution of foliose lichens in the Gulf of Thailand mangroves with emphasis on Dirinaria picta species complex Achariya Rangsiruji1, Sanya Meesim2, Kawinnat Buaruang2, Kansri Boonpragob2, Pachara Mongkolsuk2, Sutheewan Binchai1, Onanong Pringsulaka3, and Sittiporn Parnmen4* 1 Department of Biology, Faculty of Science, Srinakharinwirot University, Wattana, Bangkok, 10110 Thailand 2 Lichen Research Unit, Department of Biology, Faculty of Science, Ramkhamhaeng University, Bang Kapi, Bangkok, 10240 Thailand 3 Department of Microbiology, Faculty of Science, Srinakharinwirot University, Vadhana, Bangkok, 10110 Thailand 4 Toxicology Center, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Mueang, Nonthaburi, 11000 Thailand Received: 11 October 2018; Revised: 25 December 2018; Accepted: 1 February 2019 Abstract Extensive surveys of mangrove foliose lichens in Caliciaceae and Physciaceae on the Gulf of Thailand revealed eight species of the genera Dirinaria, Physcia, and Pyxine. Species density was highest in the mid-intertidal zone (46%), followed by the landward and seaward zones (31% and 23%, respectively). Fifty-one new internal transcribed spacer sequences were generated and the resulting phylogenies based on maximum likelihood and Bayesian approaches yielded monophyletic lineages of all three genera. However, within the Dirinaria clade formation of polyphyletic assemblages among D. aegialita, D. applanata, and D. picta indicated the presence of homoplasies in certain morphological traits used to characterize them. To address species boundaries of these lichens in Dirinaria picta species complex, methods of DNA barcode-based delineation of putative species were employed. Additional sampling of the Dirinaria species from elsewhere is required to provide further insight into species delimitation of this heterogeneous genus. Keywords: Caliciaceae, Gulf of Thailand, internal transcribed spacer, mangrove foliose lichens, Physciaceae 1. Introduction Mangrove forests consist of different flora and fauna that flourish in estuarine ecosystems subjected to regular tidal flooding. Over 90% of these forests are located within developing countries (Reynolds, Er, Winder, & *Corresponding author Email address: sittiporn.p@dmsc.mail.go.th Blanchon, 2017). Mangroves have been categorized into three major zones that include the landward, the mid-intertidal Rhizophora, and the seaward Avicennia-Sonneratia zones based on dominant plant communities present (Waycott et al., 2011). They provide safe wildlife habitats as well as a variety of socio-economic services to people (Panda et al., 2017) and hence, are recognized as one of the world’s most productive tropical ecosystems (Logesh, Upreti, Kalaiselvam, Nayaka, & Kathiresan, 2012). Despite the well accepted biodiversity value of mangroves across the globe, there has been a A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 continued decline of the mangrove forests due to human influences (Reynolds et al., 2017). Mangrove ecosystems are found in all 23 coastal provinces of Thailand covering areas of approximately 240,000 hectares (Aksornkoae, 2012). However, over the last three decades more than 50% of the total mangrove areas, especially along the Gulf of Thailand, have been depleted mainly due to shrimp farming, tourism, and industrial activities (Giri et al., 2008). Although under threatening conditions, these forests are hosts of manglicolous lichens commonly found as bark-dwelling species. Currently, an increasing number of lichen species has been recorded from Thailand (Buaruang et al., 2017), but there is still a lack of systematic studies of the manglicolous lichens. Therefore, this study focused on extensive surveys of foliose lichens, particularly those belonging to two closely related families, Caliciaceae and Physciaceae, present in the Gulf of Thailand mangroves. Firstly, it aimed to investigate phylogenetic placements of these lichens using internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Furthermore, a recent ITS-based phylogeny of the genus Dirinaria (Caliciaceae) revealed unresolved relationships among the species from South Korea as well as other countries (Jayalal et al., 2013). Thus, the present study also involved in elucidation of species boundaries within Dirinaria using ITS sequence data. 2. Materials and Methods 2.1 Specimens and phenotypic studies The collecting localities were in the mangrove forests of several provinces along the Gulf of Thailand. These included Trat Province (Koh Chang Island: 11°59'N, 102°23'E and Koh Kood Island: 11°37'N, 102°32'E), Rayong Province (12°42'N, 101°41'E), Chachoengsao Province (13°32'N, 101° 00'E), and Chumphon Province (10°21'N, 99°13'E). The foliose lichens were randomly sampled from stem barks, branch barks, and aerial parts of ten host plants located in each type of the mangrove zonation. Forty-eight specimens were obtained and identified on the basis of their morphological, anatomical, and chemical characteristics, and vouchers were deposited in RAMK. Three additional specimens of Dirinaria picta (RAMK 030405-030407) were obtained from secondary forests in Khao Yai National Park (14°24'N, 101°22'E) in Nakhon Ratchasima Province. Morphologically, the specimens were examined for the presence or absence of vegetative propagules (dactyls and soredia) as well as colors and patterns of lobes using a low magnification stereomicroscope (Olympus SZ30). Free-hand sections were performed for anatomical studies of certain characteristics such as textures of the medulla and colors of the asci and ascospores using an Olympus BX51 compound microscope. Stained wet mounts and amyloidity tests were carried out using 10% potassium hydroxide and Lugol’s iodine solution following White & James (1985). Photos were taken using a Canon PowerShot G12. In addition, chemical constituents were identified using thin layer chromatography with two solvent systems: A (Toluene [180 mL], dioxin [60 mL], and acetic acid [8 mL]) and G (Toluene [139 mL], ethyl acetate [83 mL], and formic acid [8 mL) (Elix, 2014). 505 2.2 DNA extraction, PCR amplification and DNA sequencing Small thallus fragments (2–15 mg) excised from freshly collected specimens were ground in liquid nitrogen. Total genomic DNA was extracted using the DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instructions. The DNA obtained was used for PCR amplification of the ITS regions using the primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990). PCR reactions were performed in a thermal cycler (Eppendorf Mastercycler Gradient) and thermal cycling parameters followed Rangsiruji et al. (2016). Amplification products were cleaned using the QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer’s protocol. The purified PCR products were sequenced using the amplification primers. 2.3 Taxon sampling and phylogenetic analyses Voucher information and GenBank accession numbers of the ITS sequences for the specimens obtained from the Gulf of Thailand coastline are shown in Table 1. This also included reference collection details and the GenBank accession numbers of other ITS-derived sequence data from elsewhere. An aligned data matrix of the ITS sequences was obtained using Geneious R8 (http://www.geneious.com). The removal of ambiguous regions was carried out using Gblocks with the less stringent setting (Castresana, 2000) and subsequently, the alignment was subjected to analyses under maximum likelihood and Bayesian approaches. A maximum likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.10) of the CIPRES Science Gateway server (Miller, Pfeiffer, & Schwartz, 2010) based on the GTRGAMMA model (Sta matakis, 2014). Bootstrap values for recovered nodes were estimated from the analysis of 1,000 pseudoreplicate datasets. Only clades that received bootstrap support (BS) ≥75% were considered strongly supported. The Bayesian analysis was accomplished using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). The Markov Chain Monte Carlo (MCMC) algorithm for 10,000,000 generations was used for sampling trees from the distribution of posterior probabilities. These posterior probabilities of the phylogeny were determined by constructing a 50% majorityrule consensus of the remaining trees. Internodes with the posterior probabilities (PP) ≥0.95 were considered statistically significant. The resulting ML and Bayesian consensus trees were visualized with FigTree 1.3.1 software (http://tree. bio.ed.ac.uk/). 2.4 Phylogenies of selected Dirinaria species The ITS sequence alignment of three Dirinaria species (36 specimens) was conducted using Geneious R8. Phylogenetic trees were reconstructed based on the ML and Bayesian analyses under the best-fit mode estimated above. Pyxine asiatica was defined as the outgroup. Two methods of sequence-based species delimitation, including the Automatic Barcode Gap Discovery (ABGD) and Bayesian Poisson Tree Processes (bPTP) were employed. 506 A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 Table 1. Specimens obtained from this study were deposited in RAMK with GenBank accession numbers in bold. Other ITS sequences from elsewhere were retrieved from GenBank with their reference collection details. Species Calicium adspersum Calicium lecideinum Calicium nobile Calicium notarisii Calicium salicinum Calicium trachylioides Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria aegialita Dirinaria applanata Dirinaria applanata Dirinaria applanata Dirinaria applanata Dirinaria applanata Dirinaria applanata Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Dirinaria picta Physcia adscendens Physcia aipolia Physcia alnophila Physcia atrostriata Physcia atrostriata Physcia atrostriata Physcia austrostellaris Physcia caesia Physcia dubia Physcia leptalea Physcia stellaris Physcia subalbinea Physcia tenella Physcia tropica Family Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Physciaceae Voucher information (country of origin) Prieto 3037 Prieto Tibell 21968 Prieto 3007 Prieto Nordin 4002 Kawasaki id9 (Cambodia) RAMK 031278 (Thailand) RAMK 032083 (Thailand) RAMK 032084 (Thailand) RAMK 032085 (Thailand) RAMK 032086 (Thailand) RAMK 032087 (Thailand) RAMK 032088 (Thailand) RAMK 032089 (Thailand) MAF 9854 (Australia) Anonymous (India) Sipman 46067 (Singapore) Hur 041637 (South Korea) RAMK 032090 (Thailand) RAMK 032091 (Thailand) Sipman 45628 (Singapore) Hur 050536 (South Korea) RAMK 032092 (Thailand) RAMK 032093 (Thailand) RAMK 032094 (Thailand) RAMK 032095 (Thailand) RAMK 032096 (Thailand) RAMK 032097 (Thailand) RAMK 032098 (Thailand) RAMK 032099 (Thailand) RAMK 032100 (Thailand) RAMK 032101 (Thailand) RAMK 032102 (Thailand) RAMK 031273 (Thailand) RAMK 031274 (Thailand) RAMK 031275 (Thailand) RAMK 031276 (Thailand) RAMK 031277 (Thailand) RAMK 031279 (Thailand) RAMK 031280 (Thailand) RAMK 031281 (Thailand) RAMK 031282 (Thailand) RAMK 031283 (Thailand) RAMK 031284 (Thailand) RAMK 030405 (Thailand) RAMK 030406 (Thailand) RAMK 030407 (Thailand) RAMK 030408 (Thailand) Moberg 12260 Hernandez & Sicilia XII.2002 Ahti 64008 RAMK 031285 (Thailand) RAMK 031286 (Thailand) RAMK 031287 (Thailand) CBG:Elix 38829 (Australia) Urbanavichus C-01566 BCN-Lich 17042 BCN-Lich 16792 Moberg 12012 Pykälä 9712 Odelvik & Hellström 0827 Elix 36320 (CANB) (Australia) GenBank accession number KX512907 KX512911 KX512913 KX512915 KX512919 KX512933 AB764068 MK028196 MK028167 MK028168 MK028169 MK028170 MK028171 MK028172 MK028173 AY449727 EU722342 AF540512 EU670217 MK028175 MK028176 AF540514 EU670223 MK028177 MK028178 MK028179 MK028180 MK028181 MK028182 MK028183 MK028184 MK028185 MK028186 MK028187 MK028174 MK028192 MK028193 MK028194 MK028195 MK028197 MK028198 MK028199 MK028200 MK028201 MK028202 MK028188 MK028189 MK028190 MK028191 EU682184 EU682185 EU682210 MK028203 MK028204 MK028205 GU074409 EU682197 GU247179 GU247176 EU682183 EU682186 KX512932 FJ822890 A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 Table 1. 507 Continued. Species Physcia tropica Physcia undulata Physcia undulata Pyxine albovirens Pyxine asiatica Pyxine asiatica Pyxine coccifera Pyxine coccoes Pyxine retirugella Pyxine retirugella Pyxine retirugella Pyxine retirugella Pyxine retirugella Pyxine retirugella Pyxine retirugella Pyxine sorediata Pyxine subcinerea Xanthoria elegans Family Voucher information (country of origin) Physciaceae Physciaceae Physciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Caliciaceae Teloschistaceae The ABGD method aims to discover the existence of the DNA barcode gaps and estimate the number of species (Puillandre, Lambert, Brouillet, & Achaz, 2012). The analysis was performed on the ABGD website (http://wwwabi.snv. jussieu.fr/public/abgd/abgdweb.html) with a prior p range of 0.005–0.01, and a relative gap width of 1.5, with the Kimura 2-parameter model. The bPTP model is used for delimiting species boundaries on a rooted phylogenetic tree (Zhang, Kapli, Pavlidis, & Stamatakis, 2013). In this study, a rooted input tree obtained from the Bayesian approach was used and run on a web server (https://species.h-its.org/ptp/) with 500,000 MCMC generations, excluding the outgroup, following the remaining default settings. 3. Results and Discussion 3.1 Species distribution The collected foliose lichens inhabited the mangroves in both the eastern and western parts of the Gulf of Thailand. The sampling localities encompassed four provinces, namely Chachoengsao, Rayong, Trat, and Chumphon. Eight foliose taxa of Caliciaceae and Physciaceae (Figure 1), belonging to three genera, viz. Dirinaria (D. aegialita, D. applanata, D. picta), Physcia (P. atrostriata, P. undulata) and Pyxine (P. asiatica, P. coccifera, P. retirugella) were discovered following in-depth investigations of their morphological, anatomical, and chemical characteristics (Table 2). From these eight species of lichens (48 specimens), the species density was determined among the different types of mangrove zonation. The highest species density was shown in the mid-intertidal zone (46%), followed by the landward and seaward zones (31% and 23%, respectively) (Figure 2A). Within the family Caliciaceae (Figure 2B), D. picta was most abundant as it occurred in all zones, whereas D. aegialita and P. asiatica were restricted to the mid-intertidal Rhizophora zone. On the other hand, D. applanata appeared to inhabit two different life zones of the landward as well as Elix 37727 (CANB) (Australia) RAMK 031298 (Thailand) RAMK 031299 (Thailand) Sipman 25/99-1 (Guayana) RAMK 031288 (Thailand) RAMK 031289 (Thailand) RAMK 031290 (Thailand) Prieto RAMK 031291 (Thailand) RAMK 031292 (Thailand) RAMK 031293 (Thailand) RAMK 031294 (Thailand) RAMK 031295 (Thailand) RAMK 031296 (Thailand) RAMK 031297 (Thailand) Wetmore 91254 AFTOL-ID 686 Odelyik 04532 GenBank accession number FJ822889 MK028206 MK028207 AY143412 MK028208 MK028209 MK028210 KX512936 MK028211 MK028212 MK028213 MK028214 MK028215 MK028216 MK028217 KX512937 HQ650705 KX512947 the mid-intertidal Rhizophora zones. P. retirugella was distributed from the mid-intertidal Rhizophora to the seaward Avicennia-Sonneratia zones and P. coccifera was found in terrestrial forests. Studies by Nayaka, Upreti, and Ingle (2012) and Sethy, Pandit, and Sharma (2012) also revealed that both Dirinaria and Pyxine were among the most common foliose lichens in the mangrove forests of India. In the family of Physciaceae (Figure 2B), P. undulata was widely distributed in the landward zone. In contrast, P. atrostriata occupied the mangrove zonation extending from the mid-intertidal Rhizophora to the seaward Avicennia-Sonneratia zones. Previously, the specimens of P. undulata obtained from Koh Chang Island were recorded as Physcia crispa var. mollescens by Vainio (1909). However, in the present study a voucher specimen was delivered from the University of Turku Herbarium (TUR, Finland) for a thorough re-examination. Based on the detailed morphological and anatomical studies as well as a chemical analysis of this specimen it was re-identified as P. undulata (Figure 3). This finding also supported Buaruang et al. (2017). The presence of lichens in the mangrove ecosystems indicates their tolerance to harsh environmental conditions, including solar radiation, desiccation, salinity, and human interruptions (Delmail et al., 2013; Nayaka, Upreti, & Ingle, 2012). However, in Thailand the knowledge of lichen diversity in such ecosystems is still inadequate. Our previous study showed that the cyanolichen species (blue-green algae photobionts) were most abundant in the seaward zone (Rangsiruji et al., 2016). The present study revealed the existence of dense populations of another group of lichenized fungi with green algae phycobionts in the mid-intertidal zone. Moreover, three halotolerant lichen species, namely D. picta, P. atrostriata, and P. retirugella, were also discovered in the seaward zone. These species produced a major secondary metabolite, atranorin, as a photoprotective pigment to ensure efficient photosynthesis of the phycobionts. Furthermore, it has been shown that these phycobionts were able to produce polyols to enhance the desiccation tolerance of their mycobiont partners (Delmail et al., 2013). 508 A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 Figure 1. Table 2. Habit photographs of foliose lichens in Caliciaceae and Physciaceae present in mangroves of Thailand. (A) Dirinaria aegialita, (B) D. applanata, (C) D. picta, (D) Physcia atrostriata, (E) Physcia undulata, (F) Pyxine asiatica, (G) Pyxine coccifera, and (H) Pyxine retirugella. Blue circle and red circles indicate dactyls and soredia, respectively. Scale = 1 cm. Species collected and their distinctive morphological, anatomical and chemical characteristics. List of species Dirinaria aegialita Dirinaria applanata Dirinaria picta Physcia atrostriata Physcia undulata Pyxine asiatica Pyxine coccifera Pyxine retirugella Morphological and anatomical characteristics Lobes with flabellate apices, thalline dactyls Lobes with flabellate apices, farinose soredia Lobes with discrete apices, farinose soredia Pruinose upper surface and brown-black lower surface, soredia Pruinose upper surface and white to pale brown lower surface, soredia Thallus adnate to tightly, soredia, white medulla Thallus adnate to loosely, red-pigmented soredia Thallus adnate, soredia, white or cream medulla Chemical constituents Atranorin, divaricatic acid Atranorin, divaricatic acid Atranorin, divaricatic acid Atranorin, zeorin Atranorin, zeorin Atranorin, norstictic acid Atranorin, norstictic acid Atranorin, norstictic acid A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 509 Figure 2. (A) Species density of foliose lichens compared among three types of mangrove zonation on the Gulf of Thailand. (B) Distribution of foliose lichen species in the landward, mid-intertidal, and seaward zones. Figure 3. Specimen of Physcia crispa var. mollescens from Koh Chang Island listed by Vainio (1909) was re-identified in this study as P. undulata. (A) Thallus orbicular with frosted-pruinose and (B) soralia marginal (arrow). Scale = 0.2 cm. 3.2 Phylogenies of Caliciaceae and Physciaceae Several groups of lichenized fungi are primarily distinguished on the basis of different growth forms, vegetative propagules, and secondary metabolites. However, classifications based on single vegetative characters have been shown to create non-monophyletic assemblages (Luangsupha bool et al., 2016; Parnmen, Lücking, & Lumbsch, 2012; Parnmen et al., 2012; Rangsiruji et al., 2016; Wedin, Döring, Nordin, & Tibell, 2000). Previous phylogenetic studies showed that members of the mazaedia-producing family Caliciaceae were nested within the genera Dirinaria, Pyxine, and Physcia which were circumscribed in the family Physciaceae. Therefore, some authors treated all Caliciaceae and Physciaceae as one family, and the name Physciaceae was proposed for conservation (Wedin & Grube, 2002; Wedin, Baloch, & Grube, 2002; Wedin, Döring, Nordin, & Tibell, 2000). Recently, however, a two-family concept of Caliciaceae and Physciaceae was adopted and preferred (Gaya et al. 2012). In this study, fifty-one new ITS sequences were generated and aligned with other sequences obtained from GenBank (Table 1). A matrix of 563 unambiguously aligned nucleotide position characters was analyzed. Xanthoria elegans was used as the outgroup. The ML tree had a likelihood of lnL = −6,930.534, while the Bayesian tree possessed a mean likelihood of lnL = −6,755.973 (±0.08). Both trees displayed similar topologies with two major clades. Thus, only the ML tree is shown here (Figure 4) with current phylogenetic placements of the specimens in agreement with those of Wedin et al. (2000, 2002). Clade I (BS=70/PP=0.90) contains the monophyly of Caliciaceae, including Dirinaria, Pyxine, and Calicium. Two subclades consisting of the genera Dirinaria (100/1.00) and Calicium (89/0.99) were strongly supported, whereas the other subclade of the genus Pyxine was lacking support. Morphologically, this clade is characterized by the presence of Bacidia-type asci and ascospores without distinct wall thickenings and hypothecium pigmentation. A close-knit relationship between Dirinaria and Pyxine was observed and this was also revealed by Helms, Friedl, and Rambold (2003). Both genera possessed Dirinaria-type ascospores. They were differentiated based on excipulum types as well as secondary metabolite combinations. The presence of a thalline excipulum and a combination of atranorin and divaricatic acid were typical for Dirinaria, whereas the existence of a proper excipulum and a combination of atranorin and norstictic acid were common for Pyxine. Furthermore, this study demonstrated non-monophyletic lineages of Dirinaria species, 510 A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 Figure 4. ML tree depicting relationships within Caliciaceae-Physciaceae based on ITS sequence data. Only ML bootstrap values ≥75% are reported above the branches and posterior probabilities ≥0.95 are indicated as bold branches. despite their apparent spatial distribution pattern according to geographical origins. Clade II (100/1.00) comprises members of the genus Physcia, representing the family Physciaceae. This clade is characterized by the presence of a pseudoparenchymatous upper cortex, Physcia-type ascospores, and cortical substances such as atranorin and zeorin. This study showed that P. atrostriata and P. undulata from Thailand form a distinct subclade with other species that originated in the southern hemisphere. Both of them belong to palaeotropical taxa in which most members contain soredia, and thus are rapidly dispersed (Galloway & Moberg, 2014). A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 3.3 Dirinaria phylogenies The genus Dirinaria includes approximately 36 species with D. picta as the type species. They occur in pantropical and subtropical regions, as well as oceanic regions. Australia is the center of species diversity where 13 taxa of Dirinaria were recognized (Elix, 2009). In Thailand, eight species were reported to exist mostly in tropical and montane forests (Buaruang et al., 2017). Traditionally, the genus is characterized by the presence of vegetative propagules (soredia/dactyls) and patterns of lobe apices (flabellate/discrete) as well as a combination of secondary 511 metabolites (atranorin, divaricatic acid, sekikaic acid, and xanthones) (Elix, 2009). In this study, three species of Dirinaria, namely D. aegialita, D. applanata, and D. picta, were discovered. Taxonomically, these species are very similar. Dirinaria aegialita can be distinguished from the other two species mainly by the presence of dactyls and absence of orbicular soralia. On the other hand, D. applanata and D. picta are differentiated merely by the presence of flabellate and discrete apices, respectively (Elix, 2009). The morphological attributes of the three species are depicted in Figure 5. Figure 5. ML tree showing relationships within Dirinaria picta species complex based on ITS sequences. Only ML bootstrap values ≥75% are denoted above the branches and posterior probabilities ≥0.95 are demonstrated as bold branches. Phenotypic characters and species delimitation scenarios obtained from different methods are indicated in columns to the right. The proposed putative species are highlighted with different colors and corresponding numbers. 512 A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 The present study revealed the ITS-based phylogenies at the infrageneric level of the genus Dirinaria. The phylogenetic estimates obtained from both the ML and Bayesian approaches were congruent. Thus, only the ML tree is illustrated and it revealed a large group of intermixed species of Dirinaria, giving rise to Dirinaria picta species complex. The presence of putative species (molecular operational taxonomic units) was considered based on the ABGD and bPTP analyses as well as the ML bootstrap values and PP support (Figure 5). The ABGD analysis identified a barcode gap with a prior intra-specific divergence at 0.04 (Figures 6A & 6B). In addition, it revealed 10 putative species in all recursive partitions with prior intra-specific genetic distance thresholds between 0.50 and 0.73% (Figure 6C). On the contrary, the bPTP analysis demonstrated 9 putative species. Eight putative species (1–3 & 5–9) were recognized by both methods. The ABGD method however, further divided the putative species 4 into two more putative species (4a & 4b). Although four putative species, namely 1, 2, 3, and 6, were strongly supported by the ML and PP values, the resulting posterior probabilities based on the bPTP analysis of putative species 1, 3, and 6 were rather low (0.20, 0.56, and 0.65, respectively). Clusters of seven putative species (2–5 & 7–9) were apparently associated with the apex structures of the thallus lobes. Other traits such as soredia and dactyls appeared to be homoplasious and thus, were not reliable for the species delimitation. Therefore, more phenotypically diagnostic characters possessing true synapomorphies are required to reinforce the existence of the proposed delineated species within the current Dirinaria picta species complex. 4. Conclusions Our study showed that the foliose lichens in Caliciaceae and Physciaceae were present in different types of mangrove zonation along the Gulf of Thailand. Three genera Figure 6. Automatic Barcode Gap Discovery (ABGD) outputs based on ITS sequences of Dirinaria. (A) Histogram showing distribution of genetic distances with an arrow indicating the threshold selected to separate between intra- and inter-specific divergences. (B) Ranked genetic distances with a dotted line showing approximate position of gap center. (C) Number of groups inside the partition as a function of the prior limit between intra- and inter-specific divergences. A. Rangsiruji et al. / Songklanakarin J. Sci. Technol. 42 (3), 504-514, 2020 were obtained that included Dirinaria, Physcia, and Pyxine to form monophyletic groups based on the molecular phylogenetic analyses. Phenotypically, the morphological, anatomical, and chemical characteristics of Dirinaria under study were in line with those described by Elix (2009). However, within the Dirinaria clade several species are clearly dispersed, yielding polyphyletic assemblages of taxa. Thus, the ABGD and bPTP methods were employed as the DNA barcode-based delineation of the putative species within the Dirinaria picta species complex. The results confirmed that some vegetative characters were homoplastic synapomorphies and should be avoided in taxonomy. Additional sampling of the Dirinaria species from elsewhere is required to provide more valuable diagnostic traits for a better understanding of the nature of this species complex. Acknowledgements The authors would like to express their appreciation to Prof. Dr. Klaus Kalb from the University of Regensburg, Germany, for his kindness and consideration to reassure the specimen identification of Physcia undulata from Koh Chang Island obtained by Vainio in 1909. We also wish to thank Dr. Thorsten Lumbsch from the Field Museum, U.S.A., for his useful suggestions to improve our manuscript. This research was financially supported by the National Research Council of Thailand. References Aksornkoae, S. (2012). Mangroves … coastal treasure of Thailand. The Journal of the Royal Institute of Thailand, 4, 59-77. Buaruang, K., Boonpragob, K., Mongkolsuk, P., Sangvichien, E., Vongshewarat, K., Polyiam, W., & Lumbsch, H. T. (2017). A new checklist of lichenized fungi occurring in Thailand. MycoKeys, 23, 1-91. doi:10. 3897/mycokeys.23.12666 Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. 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