A regional study of the genus Phyllopsora (Ramalinaceae) in Asia and Melanesia

Abstract Phyllopsora is a crustose to squamulose lichen genus inhabiting the bark of trees in moist tropical forests and rainforests. Species identification is generally challenging and is mainly based on ascospore morphology, thallus morphology and anatomy, vegetative dispersal units, and on secondary chemistry. While regional treatments of the genus have been conducted for Africa, South America and Australia, there exists no study focusing on the Asian and Melanesian species. Previously, 24 species of Phyllopsora s. str. have been reported from major national studies and checklists representing 13 countries. We have studied herbarium material of 625 Phyllopsora specimens from 18 countries using morphology, anatomy, secondary chemistry, and molecular data to investigate the diversity of Phyllopsora species in Asia and Melanesia. We report the occurrence of 28 species of Phyllopsora including the following three species described as new to science: P.sabahana from Malaysia, P.siamensis from Thailand and P.pseudocorallina from Asia and Africa. Eight species are reported as new to Asia. A key to the Asian and Melanesian species of Phyllopsora is provided.


Introduction
The genus Phyllopsora Müll. Arg. consists of 54 crustose or squamulose species (Kistenich et al. in press). They grow mostly on bark of trees in (sub-)tropical rainforests or moist woodlands. The genus was described in 1894 from New Zealand (Müller 1894), but the first modern revision of the pantropical genus was conducted 87 years later by Swinscow and Krog (1981) focusing on the East African species. Ten years later, Brako (1991) monographed the Neotropical species, while Elix (2009) summarized the Australian species and their occurrence. Additional reports and regional studies of the genus and its species, distribution can be found from Eastern Africa (Timdal and Krog 2001), Peru (Timdal 2008b) and the West Indies (Timdal 2011). From Asia, however, only a few reports exist for selected countries. Upreti et al. (2002) listed five Phyllopsora species from India. Later, Mishra et al. (2011) described two species and one variety from India as new to science. Recently, Kondratyuk et al. (2016) described a new species from South Korea. Phyllopsoroid specimens have been reported in additional checklists and geographical studies from, for example, Bangladesh (Aptroot and Iqbal 2011), Northeast India (Logesh et al. 2017), Sri Lanka (Weerakoon and Aptroot 2014), South Korea (Joshi et al. 2011) and Thailand (Aptroot et al. 2007). A general Asian, transnational study focusing on Phyllopsora has to date not been published. So far, 24 of the 54 accepted Phyllopsora species have been reported to occur in Asia and Melanesia (Table 1). An additional nine species reported from Asia represent either synonyms or have recently been excluded from the genus (Kistenich et al. in press;2018b; Table 1).
Species of Phyllopsora are generally challenging to identify by morphology only. In a molecular phylogeny of the lichen family Ramalinaceae C. Agardh, Kistenich et al. (2018b) showed the genus Phyllopsora to be polyphyletic. Consequently, they excluded ten species from the genus. Three of the excluded species most likely belong in the family Malmideaceae Kalb, Rivas Plata & Lumbsch. An additional three of the excluded species were transferred to the new genus Parallopsora Kistenich, Timdal & Bendiksby. The species of Parallopsora grouped together in a poorly resolved clade with a number of tropical genera, such as Eschatogonia Trevis., Krogia Timdal and Physcidia Tuck. (Kistenich et al. 2018b). Little is known about these genera in Asia, which are generally very similar to Phyllopsora in their macromorphology. They often differ, however, from Phyllopsora in ascospore size and arrangement, presence of prothallus, thallus construction and chemistry (Kalb and Elix 1995;Kistenich et al. 2018b;Timdal 2008a;. Recently, Kistenich et al. (2018a) described three new species of Krogia from Asia and Oceania, which were all tentatively identified as Phyllopsora sp., indicating the morphological similarity between these two genera.
The scope of the present study is to revise Asian and Melanesian Phyllopsora specimens mainly collected between 1990 and 2017 by the authors. Herein, we provide an overview of the species of Phyllopsora occurring in the Asian countries with an updated taxonomy based on multiple sources of evidence, including DNA sequence data. We describe three new species and provide a key to the Asian and Melanesian species of Phyllopsora.

The specimens
We investigated material from 18 different countries in Asia and Melanesia (Table 1) based on herbarium collections made mainly between 1990 and 2017. Older material of Phyllopsora is generally not suitable for DNA sequencing (Kistenich et al. in press). In addition to material from our own herbaria directly available to us (BM, BORH, O, PDA), we received loans from the institutional herbaria B, E, H, TNS, and UPS, as well as from the private herbarium of P. Diederich. In total, we investigated 908 specimens of Phyllopsora and related genera. Author names for the studied species are provided in Tables 1 and 2.
The definition of Melanesia follows the United Nations geoscheme for Oceania as devised by the United Nations Statistics Division based on the M49 coding classification (https://unstats.un.org/unsd/methodology/m49/). Accordingly, it includes the five countries Fiji, New Caledonia, Papua New Guinea, Solomon Islands, and Vanuatu.

Morphology and secondary chemistry
All specimens were studied morphologically and when necessary, also anatomically. Microscope sections were prepared using a freezing microtome and mounted in water, 10% KOH (K), lactophenol cotton blue, and a modified Lugol's solution in which water was replaced by 50% lactic acid. The types of upper cortex referred to in this paper (types 1 and 2) are those described by Swinscow and Krog (1981). Amyloid reactions in the apothecium were observed in the modified Lugol's solution after pretreatment in K, and crystals of lichen substances were observed using polarized light. Ascospore measurements are given as X ± 1.5 × SD rounded to 0.5 µm, where X is the arithmetic mean and SD the standard deviation.
We performed thin-layer chromatography (TLC) as routine investigation for identification of lichen substances in accordance with the methods of Culberson (1972), modified by Menlove (1974) and Culberson and Johnson (1982). Generally, we examined the acetone-extracts in solvent system B'; fatty acids were not examined. In difficult cases, we additionally used solvent systems A and C for lichen substance identification.

Molecular methods and phylogenetic analysis
For DNA extraction, PCR amplification and DNA sequencing of the mitochondrial ribosomal small subunit (mtSSU) and the nuclear ribosomal internal transcribed spacer region (ITS: ITS1, 5.8S, ITS2), we followed the protocols outlined in Kistenich et al. (2018a). For sequence assembly and preliminary alignment, we used Geneious R9 (Kearse et al. 2012). As many of the specimens, from which we generated sequences, had not been previously identified, we needed to find out, which specimens belonged in Phyllopsora s. str. and consequently, which sequences to use in the final phylogenetic analyses. Hence, we phylogenetically analysed a combined alignment of our Ramalinaceae dataset (Kistenich et al. 2018b) and the newly generated sequences using standard RAxML (i.e., applying the GTR substitution model for each pre-defined partition [mtSSU,ITS1,5.8S and ITS2] with 100 rapid bootstrap inferences and the GAMMA model for evaluating and optimizing the likelihood of the final tree; Stamatakis 2014). Based on these RAxML trees, we selected those specimens falling into Phyllopsora s. str. and incorporated them into our Phyllopsora dataset (Kistenich et al. in press). This dataset was analysed phylogenetically in more detail (see below) to provide evidence for undescribed species.
Each marker was aligned separately using MAFFT v.7.408 (Katoh and Standley 2013) with the E-INS-i algorithm and the nucleotide scoring matrix set to 1PAM / κ=2. We trimmed the ends of the ITS alignment to comprise only the ITS-region and deleted the residual 18S and 28S sequence information. Each dataset was initially analysed by IQ-TREE v.1.6.7 (Nguyen et al. 2015) to infer a maximum likelihood tree using 1000 ultrafast bootstrap repetitions (Hoang et al. 2018). We checked for genetree incongruence using compat.py (Kauff and Lutzoni 2002) with a cut-off of 90.
As we did not find any strongly supported incongruences, which would affect the circumscription of the new species, we concatenated the mtSSU and ITS alignments. We ran a detailed IQ-TREE analysis to find the best-fitting nucleotide substitution models and partitioning schemes (Chernomor et al. 2016;Kalyaanamoorthy et al. 2017) among models implemented in MrBayes (i.e., 1-, 2-, and 6-rate models) and to infer a maximum likelihood tree using 1000 standard non-parametric bootstrap repetitions (BS). We defined four subsets, one for mtSSU and three for ITS corresponding to the ITS1, 5.8S and ITS2 regions, and analysed those with the TESTMERGE function resembling PartitionFinder2. In addition, we analysed the dataset with MrBayes v.3.2.6 ( Altekar et al. 2004;Ronquist and Huelsenbeck 2003) as described in Kistenich et al. (2018b). The temperature increment parameter was set to 0.05. We projected the BS values from the IQ-TREE analysis onto the MrBayes consensus tree with posterior probabilities (PP) and collapsed branches with BS < 50 and PP < 0.7. The resulting trees were edited in TreeGraph2 (Stöver and Müller 2010)

Morphology and secondary chemistry
Morphological identification of many specimens was challenging, but with data obtained by TLC, many specimens could be identified to species level. Of the 908 studied specimens, we found 625 specimens to belong in Phyllopsora, while 283 specimens were found to belong in other genera of the Malmideaceae and Ramalinaceae (not treated in this study). Of the 625 Phyllopsora specimens, 480 were identified to species level in Phyllopsora (Table 2, Suppl. material 2: Table S1), while 141 specimens (23%) were left unidentified (not included in Suppl. material 2: Table S1), most of which were not sequenced and did not contain lichen substances. The morphology and anatomy of the Phyllopsora species have been described in detail by Swinscow and Krog (1981) and Brako (1991), and are not repeated here. We often found the distinction between cortex type 1 and type 2 useful for species identification; however, in many species the cortex type is intermediate (type 1-2). The chemistry of the 54 accepted Phyllopsora species is summarized in Kistenich et al. (in press).

Molecular data and phylogenetic analysis
We obtained sequences for 140 phyllopsoroid specimens with 132 mtSSU and 106 ITS sequences (Tables 2, 3). Based on the initial RAxML analyses (not shown), 93 specimens were found to belong in Phyllopsora s. str. (Table 2) and were used in the subse-  (Table  3) and are referred to at the family level only due to many problems of generic affiliation. The concatenated alignment had a length of 1,825 bp with 264 accessions including one specimen of Biatora beckhausii (Körb.) Tuck. and one of B. vacciniicola (Tønsberg) Printzen for rooting of the phylogenetic trees. The alignment contained ca. 20% missing data and is available from TreeBase (study no. 23881).
The software IQ-TREE suggested the following substitution models for four subsets: GTR+I+Γ for mtSSU and SYM+I+Γ for ITS1, 5.8S and ITS2. Bayesian phylogenetic analysis halted automatically after 40×10 6 generations, when the ASDSF in the last 50% of each run had fallen below 0.01. Following a burnin of 50%, we used 80,004 trees for the final Bayesian majority-rule consensus tree. The phylogenetic results generated by IQ-TREE vs. MrBayes showed no incongruences. The extended majorityrule consensus tree ( Fig. 1; see Suppl. material 1: Fig. S1 for the uncollapsed version of the tree), based on the Bayesian topology with all compatible groups (BS ≥ 50 and/or  PP ≥ 0.7), shows an overall good resolution of Phyllopsora species. Seventeen unidentified specimens did not associate with any known species in the phylogenetic tree (Fig.  1). Four unidentified specimens (1017, 7227, 7230, and 7231) were resolved on long branches, while the remaining 13 specimens grouped into three distinct, strongly supported clades (Fig. 1A-C). Clade A is resolved as sister to a clade consisting of P. cinchonarum and P. concinna, clade B is found in a clade with P. castaneocincta, P. foliata and P. neofoliata among others, and clade C is resolved as sister to P. neotinica.

Discussion
In this study, we present the first revision of the genus Phyllopsora for Asia and Melanesia based on the integrative study of morphology, chemistry and DNA sequence data.
We investigated 625 specimens of Phyllopsora collected from 18 countries and found the material to comprise at least 28 species of Phyllopsora s. str. (Figs 2-10) including three supported clades that we describe as species new to science. With this study, the genus Phyllopsora comprises 57 species.
Among the 28 species of Phyllopsora, eight are reported as new for Asia and Melanesia ( Table 1). One of these new species is P. africana ( Fig. 2A). This species has recently been found to be morphologically and chemically heterogeneous, comprising three chemotypes (Kistenich et al. in press). In addition to the known isidiate morph, a lacinulate morph was detected among P. africana material by Kistenich et al. (in press). Moreover, they described two new chemotypes. The lacinulate morph occurred in specimens of chemotype 1 and 3, but has so far never been found in those of chemotype 2. Specimens of chemotype 2, however, were shown to be morphologically cryptic to the sister species P. swinscowii (Kistenich et al. in press). In this study, we added twelve specimens of P. africana to our phylogeny (mainly lacinulate specimens of chemotype 3), but are not able to disentangle the difficult nature of this species complex. While we found most specimens of P. africana to roughly group according to chemotype in the phylogenetic tree (Suppl. material 1: Fig. S1), one specimen of P. africana chemotype 1 (7224) was more closely related to P. swinscowii (Suppl. material 1: Fig. S1). This raises the question of whether the two species should be synonymized based on their morphological and chemical similarity in combination with the short branches in the phylogenetic tree (Suppl. material 1: Fig. S1). We refrain from synonymizing them here, awaiting more data.
The two species P. cuyabensis (Fig. 4B) and P. longiuscula (Fig. 7B) are reported as new for the Asian continent. Specimens of both species are morphologically congruent with their Neotropical representatives. In the phylogenetic tree (Suppl. material 1: Fig. S1), however, the respective Asian accessions sit on rather long branches, clearly distinct from the Neotropical specimens. In these cases, there seem to exist genetically different populations for Neotropical and Asian specimens and more specimens should be collected to investigate the extent of genetic variation.
The genus Phyllopsora was recently shown to be polyphyletic by Kistenich et al. (2018b). The typical growth form, which characterizes this genus, has evolved multiple times independently in the family Ramalinaceae. These findings corroborate the morphological co-evolution in tropical lichens already indicated by Lakatos et al. (2006). Hence, molecular methods are often the only means of reliably assigning specimens to Phyllopsora or rather to its morphologically similar relatives (e.g., Bacidia De Not., Bacidina Vězda, Eschatogonia, Parallopsora). It is thus not surprising that several of our sequenced specimens (Table 3) were extraneous to Phyllopsora s. str. We did not assign those specimens to genus level, but all but one belong in the Ramalinaceae. The non-Ramalinaceae specimen appears to belong in the Malmideaceae. This indicates that correct taxonomic assignment even at family level using morphology may prove chal- lenging in certain cases. Furthermore, about a quarter of the total material investigated could not be identified, partly because many of those unidentified specimens were sterile and deficient in lichen substances. Unfortunately, we were not able to generate sequences of all unidentified specimens in the course of this study.

New species
The three new species, P. pseudocorallina, P. sabahana and P. siamensis, fall into distinct and well-supported clades in the phylogeny ( Fig. 1A-C). They were originally assumed to comprise Asian populations of the species P. porphyromelaena, P. corallina and P. imshaugii, respectively, based on morphology and/or chemistry. Their sequence data, however, revealed them as separate species clearly distinct from their look-alikes (Fig. 1). Phyllopsora pseudocorallina (Fig. 9B) is distinguished from its namesake, i.e. P. corallina, by forming a partly more rosulate thallus. Poorly developed specimens, however, might be difficult to assign to the correct species. Specimens of P. sabahana (Fig. 9C) are challenging to identify based on morphology only. The species is morphologically and chemically almost identical to P. porphyromelaena chemotype 1. It differs only in forming slightly smaller ascospores. Thus, sterile specimens cannot be identified without DNA sequence data. Phyllopsora siamensis (Fig. 10B) is described from material collected in Thailand and we have not been able to detect this species in collections from other countries. The specimens resemble P. imshaugii in morphology and chemistry, but may be readily distinguished by forming larger ascospores. See also the remarks in the Taxonomy section. In addition, we found sequences of the four unidentified specimens with extraction numbers 1017, 7227, 7230, and 7231 to be resolved on rather long branches (Fig. 1,  Suppl. material 1: Fig. S1). Hence, we could not assign them to any other Phyllopsora species, for which DNA sequences of the mtSSU or ITS region were available, based on molecular data, either. It is possible that these specimens represent several new species. In this study, however, we refrain from describing them as new species pending the collection of more material. Even though specimens 1017 and 7230 are clustered together in a clade with short branches (Suppl. material 1: Fig. S1), they are morphologically quite distinct and more specimens are needed to support the hypothesis that they belong to the same species.

Unconfirmed species records
Despite investigating about 600 phyllopsoroid specimens, we were not able to find in our material any specimens belonging to seven species (i.e., P. chlorophaea, P. corallina, P. isidiotyla, P. mauritiana, P. nemoralis, P. pyxinoides, and P. swinscowii) previously reported from India, South Korea, Sri Lanka, Taiwan, Thailand, and Vietnam (Table 1), respectively. We have only investigated a few collections from especially India, South Korea, Taiwan, and Vietnam (Suppl. material 2: Table S1), though. Also for the other countries, collections are limited to certain areas and we cannot exclude the species' occurrence in other parts of the respective countries. About 23% of the investigated material could not be identified to species level and it is possible that some of these unidentified specimens represent a poorly developed individual of any of these seven species. Regarding P. corallina, for instance, we found two candidate specimens from Papua New Guinea, but DNA sequence data is necessary to resolve their species status unambiguously. Alternatively, some of these species records might be based on misidentifications. In the case of P. swinscowii, we have shown the species to be morphologically identical to the isidiate morph of P. africana, a very widespread species. It is therefore possible that the records of P. swinscowii indeed represent P. africana. In general, we have repeatedly experienced difficulties in correctly identifying species of Phyllopsora based on morphology only. For many of the species records, it remains unclear whether anatomical studies and/or chemical investigations were performed as part of the identification process or not. Especially P. chlorophaea, P. corallina and P. isidiotyla may be difficult to identify without TLC or even sequence data.

Taxonomy
This taxonomy section is a result of the integrative species delimitation process primarily based on the conclusions from the statistically inferred species delimitation analyses combined with morphological and chemical evaluations as performed in the global Phyllopsora study by Kistenich et al. (in press). The additional material of the present study complements the global dataset for the phylogenetic analysis (Fig. 1, Suppl.  material 1: Fig. S1) and revealed three new species, which were mainly delimited by forming separate clades on long branches compared to their neighboring clades.
Distribution references for Asia and Melanesia are cited in Table 1; for all other distributions, references are cited below. zeorin. Chemotype 5 is described here for the first time, and contains an unknown compound with R f values similar to argopsin in solvent system B' but with a distinct blue UV 366 fluorescence (not quenching) on the chromatograms after development. Chemotype 2 (pannarin, phyllopsorin, zeorin) is Neotropical and chemotype 4 (argopsin, norargopsin, zeorin) is known from the Norfolk Islands. We were unable to sequence specimens of chemotype 4 and 5, but specimens of chemotype 1-3 are resolved in a clade with chemotype 1 and 2 of P. porphyromelaena (Fig. 1, Suppl. material 1: Fig.  S1). The species is new to China, Japan, and Sri Lanka.
Remarks. This is one of the most common species in our material (Suppl. material 2: Table S1). It is usually easily recognized by the well-developed squamulose thallus on a reddish brown prothallus and by containing furfuraceic acid (Fig. 3A), but care is needed as about 10% of the examined specimens were actually deficient in lichen substances. It is new to Cambodia, Malaysia, Nepal, New Caledonia, Papua New Guinea, The Solomon Islands, Taiwan, and Thailand.
Remarks. This species resembles P. porphyromelaena and P. sabahana, to which it is closely related in the phylogenetic tree (Fig. 1), but differs in the presence of xanthones and the absence of argopsin and norargopsin. Kistenich et al. (in press) showed that probably all Neotropical records of this species (e.g., by Timdal 2008bby Timdal , 2011 belong in another species, P. neotinica. Phyllopsora chodatinica (Fig. 3B) is new to Malaysia, New Caledonia, and Vanuatu.
Remarks. The species is recognized by the squamulose thallus on a white prothallus, long isidia, and the presence of lobaric acid (Fig. 3C). Several additional com- longer ascospores and in containing additional substances (methyl furfuraceiate and methyl homofurfuraceiate). Judging from the number of examined specimens (Suppl. material 2: Table S1), P. dolichospora seems to be more common than P. furfuracea in Asia, although the number of reports (Table 1) suggests the opposite. This, however, might be a result of morphological misidentifications when TLC has not been run. The species is new to Japan and Papua New Guinea.
Remarks. This rarely reported Australian species (Fig. 5A) is here confirmed from Japan and Sri Lanka mainly based on our DNA sequences (both mtSSU and ITS), which were compared with sequences obtained from Australian material ( Table 2). It is new to Japan.
Distribution. Pantropical (Brako 1991;Timdal and Krog 2001;Elix 2009). Remarks. Despite widespread reports in the literature, we were able to confirm the presence of this species (Fig. 5B) in Papua New Guinea, Sri Lanka, and Thailand, only. In the phylogenetic tree, P. furfuracea forms a clade with P. dolichospora and P. foliatella (Fig. 1).
Distribution. Apparently pantropical. Remarks. The species (Fig. 5C) was included in the genus Crocynia until recently (Kistenich et al. 2018b), and not originally a part of our taxon sampling; hence the few specimens examined. The accession from Sri Lanka (7201) clusters together with specimens of C. molliuscula (Suppl. material 1: Fig. S1), from which it is morphologically and chemically different. Further specimens need to be investigated to inform about its relationship to C. molliuscula. The species is the phylogenetic sister to P. imshaugii (Fig. 1).

Phyllopsora halei (Tuck.) Zahlbr.
Description. Swinscow and Krog (1981, as P. pannosa), Timdal and Krog (2001). Distribution. North America (Brako 1991), Africa (Timdal and Krog 2001), Asia. Remarks. This species (Fig. 6A) was previously known from the type collection from North America (Louisiana), East Africa (Ethiopia, Kenya, Tanzania), and a few reports from Asia (Table 1). We here confirm its presence in Asia, based on DNA sequences from material from Sri Lanka compared with sequences from Kenya and Tanzania (Suppl. material 1: Fig. S1). Three chemotypes of this species are known (Timdal and Krog 2001), differing in terpenoid patterns and presence of an unknown compound. Our two specimens from Sri Lanka belong in chemotype 3 of Timdal and Krog (2001). The species is the phylogenetic sister to P. amazonica (Fig. 1). It is new to Sri Lanka.
Remarks. The species was not studied by us due to lack of response from LWG to our repeated loan requests.
Distribution. Pantropical, also occurring in temperate Asia and North America (Kistenich et al. in press).
Remarks. The species (Fig. 6C) was reported from India by Mishra et al (2011), and we confirm its presence in Asia by DNA sequences (mtSSU and ITS; 456) from material from Thailand. The species is sister to a clade comprising P. byssiseda and P. fendleri (Fig. 1). It is new to Thailand.
Distribution. Asia. Remarks. The species (Fig. 7A) was recently described from South Korea by Kondratyuk et al. (2016), and we report it as new to Japan and Nepal. Our sequences were compared to unpublished sequences of the holo-and isotype kindly provided to us by Sergey Kondratyuk. Our accessions form a strongly supported clade together with accessions of P. confusa (Fig. 1), from which it is difficult to distinguish. See also remarks for P. confusa.
Remarks. In the concept of Brako (1991) and Timdal (2011), this species is lacinulate. Kistenich et al. (in press), however, extend the concept to include the isidiate species P. intermediella, which they synonymize. The Asian material we have examined is lacinulate. The species (Fig. 7B) is the phylogenetic sister to P. thaleriza (Fig. 1). It is new to Asia (Sri Lanka, Thailand, and Vietnam).
Distribution. Africa (Timdal and Krog 2001), Asia. Remarks. The species (Fig. 7C) was previously known from East Africa and the Mascarenes (Timdal and Krog 2001). Although not sequenced, we here report it as new to Asia based on a specimen (Moberg 2750, UPS) from Sri Lanka (Suppl. material 2: Table S1). The species is the phylogenetic sister to P. parvifolia (Fig. 1).
Remarks. This originally Australian species is reported as new to Africa (Kenya) by Kistenich et al. (in press) and here as new to Asia (Sri Lanka; Fig. 8A). Both the African and Sri Lankan specimens were sequenced (mtSSU and ITS) and found to conform with sequences of an isotype (O L-1319). The species is generally identified by the squamulose, lacinulate thallus containing furfuraceic acid.

Distribution.
Pantropical, but mainly Neotropical, extending into the temperate zones in North and South America and in Europe (Brako 1991, as P. parvifolia var. parvifolia;Kistenich et al. in press).
Remarks. Despite several reports from Asia and Melanesia (Table 1), we have seen only a single specimen of this species (Sri Lanka, Kistenich & Weerakoon SK1-661, PDA, not sequenced; Fig. 8B) from the area. The species is the phylogenetic sister to P. mediocris (Fig. 1). It is new to Sri Lanka.
Remarks. This squamulose, isidiate species contains atranorin and parvifoliellin (Fig.  8C); characters it shares with P. concinna and P. rappiana. The molecular phylogeny ( Fig.  1) shows that the three species are not closely related, though; rather P. parvifoliella belongs in a clade together with P. africana, P. ochroxantha, and P. swinscowii. We have sequenced material from Indonesia and Thailand, and here report the species as new to Asia and Melanesia, i.e. from Indonesia, Papua New Guinea, The Philippines, and Thailand.
In the phylogenetic tree (Fig. 1, Suppl. material 1: Fig. S1), accessions of chemotypes 1 and 2 group into a weakly supported clade with P. buettneri, while accessions of chemotype 3 form a clade with P. chodatinica and the P. buettneri/P. porphyromelaena clade. Additional specimens of chemotype 3 should be sequenced to find out whether it indeed represents a chemical strain of P. porphyromelaena or rather a distinct species.
The species is morphologically very similar to P. sabahana; see that species for discussion. It is possible that some specimens listed as P. porphyromelaena chemotype 1 in Suppl. material 2: Table S1, especially those from Malaysia, represent P. sabahana. It is new to Fiji, Indonesia, Japan, Malaysia, New Caledonia, Papua New Guinea, South Korea, Sri Lanka, and Thailand. Description. Thallus effuse or forming irregular rosettes up to 1 cm diam., squamulose; squamules medium sized, up to 1 mm wide, adnate to ascending, elongate, contiguous or partly imbricate, crenulate to incised, plane to weakly convex, medium green, glabrous on the upper side, faintly pubescent along the margin; isidia common, attached marginally to the squamules, cylindrical, simple or slightly branched, up to 0.1 mm wide and 0.6 mm long; upper cortex formed by thick-walled hyphae with rounded lumina (type 2), 20-30 µm thick; cortex and medulla not containing crystals (PD-, K-); prothallus indistinct to partly well developed, white.
Chemistry. No lichen substances. Distribution. Cambodia, Malaysia, Papua New Guinea, The Seychelles. Etymology. The specific epithet refers to its morphological and chemical similarity to P. corallina.
Remarks. The species is morphologically, anatomically, and chemically very similar to P. corallina. There is, however, a tendency of P. pseudocorallina being more rosulate, i.e., composed of more radiating and elongated marginal lobes. The phylogenetic tree (Fig. 1), however, shows the two species not to be closely related: P. corallina is resolved in a clade with P. glaucella and P. rappiana as sister to P. martinii, while P. pseudocorallina appears in a clade with P. castaneocincta and P. neofoliata among others (Fig.  1). The new species is widely distributed in Asia and also found on The Seychelles. It is unclear whether P. corallina occurs in Asia at all or if previously reported specimens of P. corallina rather represent specimens of P. pseudocorallina. Description. Thallus effuse, squamulose; squamules medium sized, up to 0.8 mm wide, ascending, elongated, often imbricate, incised to deeply divided, plane to weakly convex; upper side pale green to medium green, glabrous, epruinose; margin concolorous with upper side, often finely pubescent; lacinules common, developing from lobe-tips; upper cortex formed by thick-walled hyphae with cylindrical lumina (type 1), 30-40 µm thick, containing crystals dissolving in K (PD+ orange, K-); medulla containing crystals partly dissolving in K (PD+ orange, K-); prothallus well developed, reddish brown.
Distribution. Malaysia (Borneo). Etymology. The specific epithet refers to its occurrence in Sabah, Malaysia.
Remarks. The species is morphologically and chemically very similar to P. porphyromelaena chemotype 1, and is close to be regarded as a morphologically cryptic species. It may, however, be distinguished in forming smaller ascospores (6-8 × 2-2.5 vs. 8-13 × 2-4 µm). Apothecia are not common in neither species, however, and the measurements are based on only 20 spores from each species (the holotype of P. sabahana and two specimens of P. porphyromelaena from La Réunion). In the phylogenetic tree ( Fig. 1), the five accessions of P. sabahana form a strongly supported clade as sister to the Neotropical species P. neotinica, from which it may readily be distinguished in its composition of lichen substances (P. neotinica contains xanthones). So far, P. sabahana is only known from Borneo.
Remarks. The species was previously reported from Japan, Papua New Guinea, and The Philippines (Table 1), and is here reported from four localities in Thailand (Fig. 10A). We were unable to produce DNA sequences from our material, and the identification is based on typical morphology and presence of argopsin (major) and noragopsin (minor). New to Thailand. Description. Thallus effuse, crustose to squamulose; squamules small, up to 0.4 mm wide, adnate, isodiametrical, more or less scattered when young, later contiguous or fusing, more or less crenulate, plane to weakly convex; upper side medium green, somewhat shiny, epruinose, glabrous; margin concolorous with upper side, often pubescent; isidia common, attached marginally to the squamules, cylindrical, simple or slightly branched, up to 0.15 mm wide and 1.5 mm long; upper cortex formed by thick-walled hyphae with rounded lumina (type 2), 15-30 µm thick, containing a few scattered crystals dissolving in K; medulla containing crystals dissolving in K and recrystallizing by forming acicular, red crystals, PD+ yellow, K+ red; prothallus well developed, thick, white.

Phyllopsora siamensis
Apothecia seen in the holotype only, up to 1.5 mm diam., more or less plane when young, soon becoming weakly to moderately convex, medium brown, rounded to irregular, simple, when young with a rather thick, paler, weakly pubescent margin, later becoming more or less immarginate; excipulum pale brown in the rim, darker brown in inner part; hypothecium dark brown, K-; crystals present in inner part of exciple and in hypothecium, dissolving in K and recrystallizing by forming acicular, red crystals; epithecium pale brown to colourless, K-; ascospores narrowly ellipsoid or fusiform to bacilliform, simple, 15-22 × 3.5-4.5 µm (n=20). Conidiomata not seen.
Distribution. Thailand. Etymology. The specific epithet refers to its occurrence in Thailand.
Remarks. The species is morphologically and chemically very similar to P. imshaugii. Phyllopsora siamensis, however, may be distinguished by forming slightly larger squamules and longer ascospores (15-22 × 3.5-4.5 vs 10.5-14.5 × 3-4 µm; the latter measurements are based on 40 spores in the type material from Jamaica) than P. imshaugii. So far, P. imshaugii is only known to occur in the Neotropics, while P. siamensis is solely known from Thailand. In the phylogenetic tree (Fig. 1), the four accessions of P. siamensis cluster in a strongly supported clade as sister to a clade comprising P. cinchonarum and P. concinna, from which the new species is readily distinguished by its chemistry. Phyllopsora imshaugii and P. siamensis are the only Phyllopsora species known to contain norstictic acid; the major compound of the two other species are lobaric acid and parvifoliellin, respectively.
Key to the phyllopsoroid genera in Asia and Melanesia