Neoprotoparmelia gen. nov. and Maronina (Lecanorales, Protoparmelioideae): species description and generic delimitation using DNA barcodes and phenotypical characters

Abstract Multilocus phylogenetic studies revealed a high level of cryptic diversity within the lichen-forming fungal genus Maronina (Protoparmelioideae, Parmeliaceae). Coalescent-based species delimitation suggested that most of the cryptic molecular lineages warranted recognition as separate species. Here we study the morphology and chemistry of these taxa and formally describe eight new species based on phenotypical and molecular characters. Further, we evaluate the use of ITS rDNA as a DNA barcode for identifying species in this genus. For the first time, we obtained an ITS sequence of Maroninaaustraliensis, the type species of the genus and showed that it is phylogenetically not closely related to species currently placed in Maronina or Protoparmelia. We assembled a dataset of 66 ITS sequences to assess the interspecies genetic distances amongst the twelve Maronina species using ITS as DNA barcode. We found that Maronina and Protoparmelia form a supported monophyletic group whereas M.australiensis is sister to both. We therefore propose a new genus Neoprotoparmelia to accommodate the tropical-subtropical species within Protoparmelioideae, with Neoprotoparmeliacorallifera as the type, N.amerisidiata, N.australisidiata, N.brasilisidiata, N.capensis, N.crassa, N.pauli, N.plurisporibadia and N.siamisidiata as new species and N.capitata, N.isidiata, N.multifera, N.orientalis and N.pulchra as new proposed combinations. We provide a key to Neoprotoparmelia and confirm the use of ITS for accurately identifying species in this group.


Introduction
The taxonomic status of the genus Maronina and its phylogenetic relationships have been a matter of debate. Maronina was formally described in 1990 (Hafellner and Rogers 1990) for two species with multispored asci, the type species Maronina australiensis and M. multifera. The authors suggested a close relationship of Maronina and Protoparmelia based on ascus characters and considered Maronina a multispored derivative of Protoparmelia. Later, molecular data confirmed the phylogenetic relationship between Maronina with Protoparmelia and Maronina was merged with Protoparmelia (Papong et al. 2011). Recently, Kraichak et al. (2017) suggested the use of a temporal banding approach for a consistent grouping of taxa at higher taxonomic levels, i.e. at family and genus level, for lichen-forming fungi. This approach identifies a divergence time of ~102-112 Ma for families and 29-33 Ma for genera ). Based on this approach, the genus Maronina has been resurrected (Maronina-Protoparmelia split ~70 Ma; Divakar et al. 2017, Singh et al. 2018) and both genera, Protoparmelia and Maronina, have been placed together in the subfamily Protoparmelioideae (Parmeliaceae). Currently, the genus Protoparmelia comprises arctic, boreal, temperate and Mediterranean species, whereas the genus Maronina comprises subtropical and tropical species.
Presently Maronina includes 11 species (Aptroot 2002, Aptroot et al. 1997a, 1997b, 2007 Barbero et al. 2006, Elix 2007, Hafellner and Rogers 1990, Kantvilas and Elix 2007, Lendemer and Lumbsch 2008, Papong et al. 2011. Molecular data are available for six species (Maronina capitata, M. corallifera, M. multifera, M. orientalis, M. isidiata and M. pulchra). A recent study aimed at molecular identification of species in Maronina and Protoparmelia, based on a multilocus dataset and species delimitation analysis, included these six species and two putatively novel species . Molecular analysis confirmed the presence of the abovementioned species in Maronina and suggested seven additional species: M. isidiata A, M. isidiata B (Brazil), M. isidiata C (Thailand), M. isidiata D (Australia), M. isidiata E (Australia), M. ZA (South Africa) and M. KE (Kenya). These candidate species were strongly supported by species delimitation approaches BP&P and speDeSTEM , but not formally proposed at the time. In the present study, we describe the seven novel Maronina species sensu Singh et al. (2015) and a further new saxicolous species from Brazil based on phenotypical and molecular evidence. In addition, we include the type specimen M. australiensis, which has not been sequenced before.

Materials and methods
We included 66 ITS rDNA sequences of Protoparmelia and Maronina in this study. Out of these, 61 ITS sequences are from Singh et al. (2015) and five sequences are new, representing two additional specimens of M. capitata, two sequences of a new taxon M. plurisporibadia and a sequence of the type species of the genus Maronina, M. australiensis (Table 1).

Molecular methods
For DNA extraction, amplification and sequencing, we followed the protocols from Singh et al. (2015). We used the Protoparmelia specific primers (Suppl. material 1:Table S1) and Ex Taq polymerase (Takara Bio Europe, France) for the PCRs. Generating an ITS sequence from the 32-year-old M. australiensis sample required a PCR cloning approach. The amplified products were cloned into the pJET1.2 / blunt cloning vector using the Thermo Scientific CloneJET PCR cloning kit and transformed into E. coli XL1-Blue cells (for details see: https://www.chem-agilent.com/pdf/strata/200249. pdf ). The cloned PCR products were analysed using the "colony PCR". For the PCR reactions and sequencing, we used the pJET1.2 Forward Sequencing Primer and the pJET1.2 Reverse Sequencing Primer. We performed a BLAST search using the M. australiensis ITS sequence to infer the phylogenetic affinities of M. australiensis.

Phylogenetic analyses
We aligned the sequences using MAFFT v5 with Geneious version 5.6.5 (Katoh et al. 2005, Drummond et al. 2011. To infer the phylogenetic position of M. australiensis within Protoparmelioideae, we produced an alignment using the ITS sequences of M. australiensis, Protoparmelia and Maronina species. Using this alignment, we generated a maximum likelihood tree using the ITS sequences from Protoparmelia (9 species, 20 sequences) and Maronina (13 species including the type species, 44 sequences; Fig. 1), with GTR + G as the substitution model. This dataset contains overall 66 sequences, including 2 sequences of the outgroup (Gypsoplacaceae). The maximum likelihood search was performed using the RAxML-HPC BlackBox v8.1.11 on the Cipres Scientific gateway (Miller et al. 2010, Stamatakis 2014.

Analysis of sequence variation in the ITS barcode marker
To infer intra-and interspecific ITS sequence variation within and amongst putative lineages of Neoprotoparmelia (Maronina s.l.), we calculated pairwise distances amongst Neoprotoparmelia species (Maronina s.l. species, 43 sequences from 12 species, excluding  Singh et al. (2015) and the newly generated sequences, including the voucher information, were submitted to the BOLD database, under the project name 'Neoprotoparmelia species description'. M. isidiata E and M. plurisporibadia (in Singh et al. 2015), morphological examination was performed with an Olympus SZX7 and pictures were taken with Nikon Coolpix 995. Hand-made sections of ascomata and thallus were studied in water, 5% KOH (K) and/or Lugol's reagent (1% I 2 ) after pre-treatment with KOH (IKI). Microscopic photographs were prepared using an Olympus BX50 with Nomarski interference contrast and Nikon Coolpix 995.

For the samples Maronina isidiata
For the samples Maronina ZA and M. KE, morphological examination was performed under a Nikon SMZ-1500 stereomicroscope and Nikon Eclipse-80i microscope, with bright field and DIC. Photographs were taken with a Nikon DS-Ri2 coupled to the microscope and stereomicroscope. Observations and measurements of ascospores and conidia were made in water. When possible, for each species, at least 30 spores and conidia from different specimens were measured and length width (l:b) were calculated. In the description of the new species, n (number of spores and conidia measured) are given in parentheses. Spot tests (K, C, I and Pd) and thin-layer chromatography (TLC) were carried out following Orange et al. (2010). We used TLC solvent system C (200 ml toluene / 30 ml acetic acid), with concentrated acetone extracts at 50 °C spotted on to silica gel 60 F254 aluminium sheets (Merck, Darmstadt).

Results and discussion
In the ML phylogenetic tree of Protoparmelioideae, both Protoparmelia and Maronina s.l. form supported monophyletic clades (Fig. 1). Protoparmelia and Maronina s.l. are supported as sister groups, whereas Maronina australiensis is sister to the Protoparmelia-Maronina s.l. clade. This suggests that Maronina, as currently circumscribed, is polyphyletic. The heterogeneous nature of Maronina has already been indicated by Kantvilas and Elix (2007), based on ascomatal characters. Originally, Hafellner and Rogers (1990) Singh et al. 2015, 2017 based on a 6-locus phylogeny). Based on molecular and phenotypical evidence, we thus propose to restrict the genus Maronina s.str. to M. australiensis, the type species of the genus and M. hesperia. In its restricted circumscription, the genus Maronina is currently only known from Australia. To accommodate the Maronina s.l. taxa, sister of Protoparmelia, we propose the new genus Neoprotoparmelia with N. corallifera, as the type species. The following species are here recognised in Neoprotoparmelia: N. capitata, N. isidiata, N. multifera, N. orientalis and N. pulchra and  Table 1; New Neoprotoparmelia species are marked in red and bold.  . amerisidiata, N. australisidiata, N. brasilisidiata, N. capensis, N. crassa, N. pauli, N. plurisporibadia and N. siamisidiata. All Neoprotoparmelia species are well supported in the ML tree inferred from the ITS sequences ( Fig.  1). The genus occurs throughout the tropics. The presently available data do not allow us to infer the exact phylogenetic position of Maronina s.str. (M. australiensis and M. hesperia). The first 30 BLAST hits of the M. australiensis ITS fragment suggest close affinity of M. australiensis to Lecanora species.

Distance summary
The mean intra-and inter-specific divergence was 0.56% (SE = 0.01) and 19.94 (SE = 0.01), respectively (Table 2). Our results thus show that, within species, divergence was much lower than inter-species divergence for all Neoprotoparmelia species (Table 2, Fig. 2). The maximum sequence divergence amongst individuals of a species was, in all cases, lower than the minimum interspecies sequence divergence, which supports the barcode-based taxonomic assignments of Neoprotoparmelia species (Table 2, Fig. 2). The maximum intraspecific genetic variation did not overlap with the nearest neighbour and a barcode gap was present amongst all neighbouring species (Fig. 2). Hence, we conclude that ITS is a suitable barcode marker to identify Neoprotoparmelia species.   only, as was suggested by Kantvilas and Elix (2007)) and Kantvilas et al. (2010). The genus Maronina contains depsides instead of depsidones as found in Neoprotoparmelia. Etymology. Derived from the Greek neos (=new) and its close relationship to Protoparmelia.
Distribution and ecology. The taxa of this genus occur in open habitats, mostly on bark, with only a few species growing on siliceous rock. This genus has a Pantropical distribution and is currently known from Australia, Brazil, Kenya, Papua New Guinea, South Africa, Thailand and south-eastern USA. Remarks. The new genus is morphologically similar to Maronina but can be distinguished by containing depsidones instead of depsides as found in Maronina and branched paraphyses. The genus is morphologically similar to Protoparmelia but was recognised as "tropical Protoparmelia clade" in Singh et al. (2015). The asci are essentially variations of the Lecanora-type sensu Hafellner (1984), and mainly coincides with those well studied by Kantvilas and Elix (2007) and Kantvilas et al. (2010). A detailed illustration of the ascus of N. pulchra is given in Aptroot et al. (1997a: 148, fig . 101a); it is similar to the ascus illustrated of Protoparmelia badia by Hafellner (1984: 393, fig.40).  Diagnosis. Similar to Neoprotoparmelia brasilisidiata, but differing by the thicker, 0.07-0.11 mm wide, isidia.

Neoprotoparmelia amerisidiata
Etymology. Named after its distribution in North America and the presence of isidia. Description. Thallus up to ca. 0.05 mm thick, shiny, pale olive-green to olive-grey, continuous, delimiting marginal prothallus line (brown, thin or absent). Isidia always numerous, initially widely dispersed or somewhat clustered, eventually covering much of the thallus, up to 1.5 mm long, persistently 0.07-0.11 mm wide over their whole length, cylindrical, usually irregularly repeatedly branched and somewhat nodulose, glossy, pale olivegreen to olive-grey, tips distinctly brown and dull. Apothecia and pycnidia not observed.
Distribution and ecology. On tree bark in forest. Known only from the southeastern USA (North Carolina, Alabama, Georgia, Mississippi and Florida).
Remarks. This species comprises the specimens recovered within 'P. isidiata A' in 'Protoparmelia tropical clade' in Singh et al. (2015). It is morphologically most similar to N. brasilisidiata which only differs by the generally thinner isidia. Some specimens have been reported before as Protoparmelia isidiata (Lendemer and Lumbsch 2008).   Diagnosis. Similar to Neoprotoparmelia isidiata, but differing by the larger number of isidia per thallus areole.
Etymology. Named after Australia and the presence of isidia. Description. Thallus consisting of almost contiguous, flat to convex areoles with irregular shape, of up to ca. 0.1 mm thick and 0.7 mm wide, somewhat shiny, pale brown to dark brown or pale olive-green to olive-grey, marginal prothallus black, thin or absent. Isidia usually in groups on almost each thallus areole, up to 0.9 mm long, persistently 0.07-0.1 mm wide over their whole length, cylindrical, usually rather irregularly once or more rarely repeatedly branched and somewhat nodulose, somewhat shiny, pale to dark brown or pale olive-green to olive-grey, of thallus colour, tips not darkened or somewhat brown. Apothecia (only young ones observed) sessile, round, 0.4-0.6 mm diam., disc concave to flat, smooth, glossy, orange brown. Margin glossy, ca. 0.05 mm wide, glossy brown at the outside, slightly higher than the disc. Hymenium hyaline, not inspersed with oil droplets, up to 50 μm high; epihymenium fuscous brown, pigment in K becoming soluble and paler; hypothecium hyaline, up to 90 μm thick including subhymenium; excipulum hyaline throughout, with a 5-12 μm thick layer of cortex, without crystals, with algae, extending below the hypothecium (cupulate). Paraphyses branched, ca. 2.5 μm wide, not thickened at the tips. Mature asci and ascospores not observed. Pycnidia not observed.
Chemistry. Spot tests: medulla of thallus and isidia C-, P-, K-, KC+ pink, UV+ greenish-white. TLC: alectoronic acid (major), dehydroalectoronic acid (minor or trace) and β-alectoronic acid (trace). Remarks. This species comprises the specimens recovered within 'P. isidiata E' in 'Protoparmelia tropical clade' in Singh et al. (2015) and referred to as Maronina in Divakar et al. (2017) and Singh et al. (2018). Coalescent-based species delimitation inferred from the six-locus dataset supports these taxa as distinct lineage from the other isidiate samples collected from the geographically distant populations. This species is morphologically very similar to Neoprotoparmelia isidiata, but has larger and contiguous thallus areoles, usually bearing more isidia. Members of this species may differ considerably in colour and the abundance and maximum length of the isidia.  Diagnosis. Very similar to Neoprotoparmelia amerisidiata, but differing by having thinner, 0.04-0.08 mm wide, isidia.
Distribution and ecology. On tree bark in parks, open areas, Cerrado and Atlantic rain forests. Neotropical -known from Costa Rica, El Salvador and Brazil, where it is widespread and known from the following states: Sergipe, Matto Grosso, Rio de Janeiro, São Paulo, Maranhão, Tocantins, Minas Geraes and Rio Grande do Sul.
Remarks. This species comprises specimens recovered within 'P. isidiata B' in 'Protoparmelia tropical clade' in Singh et al. (2015). It is similar to N. amerisidiata, but, however, differs in having slightly thinner isidia. It is a common species on exposed bark in the neotropics and can easily be recognised in the field, from other isidiate crusts even when sterile, due to the strong UV-reaction visible with a portable UV-torch and thus can be distinguished from other isidiate crusts, even when sterile.   -Lich. 19584;isotypes: MAF-Lich. 19624, 19625, 19626 and 19628).
Diagnosis. Morphologically similar to the northern hemispheric Protoparmelia montagnei (Fr.) Poelt & Nimis, but mainly differing from it by the presence of alectoronic acid as major secondary metabolite in the medulla. The two species, P. montagnei and N. capensis, are also genetically not closely-related and belong to different genera.
Etymology. The specific epithet refers to its occurrence in Cape Province of South Africa.
Remarks. This comprises the specimens recovered within 'P. sp. ZA' in 'Protoparmelia tropical clade' in Singh et al. (2015). Neoprotoparmelia capensis is morphologically similar to the Protoparmelia montagnei complex, in the sister genus Protoparmelia, but differs from the latter in its chemistry and distribution. The major secondary metabolite found in N. capensis is alectoronic acid whereas, in P. montagnei, it is lobaric and/ or gyrophoric acids or fatty acids. Protoparmelia montagnei is distributed in Eurasia on acid rocks, with mainly a broad Mediterranean distribution, from Turkey to The Canary Islands and from Ireland to Morocco (Coppins andChambers 2009, Barbero et al. 2006). In contrast, N. capensis grows on sandstone in the Cape Region. Molecular data also clearly supports N. capensis and P. montagnei as distantly related, evolutionary independently lineages . Details on the morphology and chemistry of the similar P. montagnei species complex can be found in Coppins and Chambers (2009) and Barbero et al. (2006). The grey to brown thalli, 8-spored asci, α-collatolic acid absence, distribution and/or molecular data, supports N. capensis as an evolutionary independent lineage from the other two saxicolous Neoprotoparmelia species here described.
The analysed material of Neoprotoparmelia capensis was rich in lichenicolous ascomycetes, some of which make its characterisation confusing. Portions of the studied specimens serve as host to species of Phacographa and Sphaerellothecium similar to those living on taxa of the Protoparmelia badia complex (Hafellner 2009 andTriebel 1989, respectively), causing visible symptoms. A Phoma-type fungus, with hyaline pycnidia and conidia, frequently infected the hymenium of N. capensis. Moreover, in some adult apothecia of N. capensis, an endohymenial Arthonia species develops its asci, together with those of the host. The latter two taxa lacked visible symptoms on the host. These four lichenicolous fungi are currently under further investigation and the results will be published in a subsequent study.   Diagnosis. Similar to Neoprotoparmelia isidiata, but differs from it in having shorter isidia and a thicker thallus.
Etymology. Derived from crassus (Lat. = fat) indicating that the thallus is thicker than that of the other isidiate species.
Description. Thallus consisting of contiguous to centrally fusing, flat to rather convex areoles with irregular shape, of up to ca. 0.1 mm thick and 0.3 mm wide, somewhat shiny, pale brown to dark brown, marginal prothallus absent. Isidia covering most of the thallus except the outer margins, globose to ellipsoid, up to 0.15 mm long, persistently 0.07-0.1 mm wide, unbranched, of thallus colour, tips not darkened or somewhat brown. Apothecia and pycnidia not observed. Remarks. This comprises the specimens recovered within 'P. isidiata D' in 'Protoparmelia tropical clade' in Singh et al. (2015). Similar to Neoprotoparmelia isidiata but differing in having a thicker thallus and shorter isidia.  Description. Thallus consisting of isolated convex areoles of up to ca. 0.1 mm thick and 0.2 mm wide, somewhat shiny, pale brown to dark brown or mottled whitish-grey, on a fully immersed hyaline hypothallus, marginal prothallus black, thin or absent. Isidia usually solitary on almost each thallus areole, up to 0.5 mm long, persistently 0.07-0.1 mm wide over their whole length, cylindrical, usually rather irregularly once or more rarely repeatedly branched and somewhat nodulose, glossy, pale to dark brown, tips dark brown to almost black. Apothecia sessile, initially round, older ones usually with wavy outline, 0.6-3.5 mm diam., disc flat, smooth, glossy, dark brown to orange brown. Margin glossy, ca. 0.25 mm wide, glossy brown at the outside, not or only slightly higher than the disc. Hymenium hyaline, not inspersed with oil droplets, up to 70 μm high; epihymenium fuscous brown, pigment in K becoming soluble and paler; hypothecium hyaline, up to 120 μm thick including subhymenium; excipulum hyaline throughout, with a 20-30 μm thick layer of cortex, without crystals, with algae, extending below the hypothecium (cupulate). Paraphyses branched, ca. 2.5 μm wide, not thickened at the tips. Asci cylindrico-clavate, up to 35 × 9 μm, with 8 mostly biseriate ascospores. Ascospores hyaline, simple, narrowly ellipsoid, not constricted, (9-)11-13(-17) × 2-3 μm, without appendages. Pycnidia not observed. Chemistry. Spot tests: medulla of thallus and isidia UV++ greenish-white, C-, P-, K-, KC+ pink. TLC: alectoronic acid (major), dehydroalectoronic acid (minor or trace) and β-alectoronic acid (trace).
Distribution and ecology. On bark of trees in forests. Known from Papua New Guinea only.
Remarks. This species differs from the other species by having a thallus consisting of tiny areoles, generally bearing just one isidium each and by large apothecia.
Additional  Diagnosis. Similar to Neoprotoparmelia capensis but differs from it by having a reduced, olive tinged thallus and smaller apothecia. Moreover, the major secondary metabolite produced by Neoprotoparmelia pauli is α-collatolic acid, absent in N. capensis.
Etymology. The new species is named after our colleague, the Kenyan lichenologist, Paul M. Kirika, who was one of the collectors of the type material.
Distribution and ecology. Only known from the type locality in Kenya, covered with upland dry forest ecosystems (Wass 1995), growing on exposed sandstones.
Remarks. Consists of specimens recovered within 'P. sp. KE' in 'Protoparmelia tropical clade' in Singh et al. (2015), supported as an evolutionary independent lineage based on the coalescent-based species delimitation analysis. The thalli of the type material were poorly developed, immature apothecia and only a few mature spores were found. This hindered us in providing detailed morphological features (especially ascomatal) and thus future collections may slightly change the morphological description. Its olive-brown thalli, 8-spored asci, α-collatolic acid presence, distribution and/ or molecular data supports it as an evolutionary independent lineage from the other two saxicolous Neoprotoparmelia species.  Diagnosis. Differing from the morphologically similar Protoparmelia badia (Ach.) M. Choisy by the presence of multispored asci and different chemistry and distribution.

Neoprotoparmelia plurisporibadia
Etymology. Named after pluri = many, spores and badia = dark brown. Description. Thallus consisting of areoles with wavy border of up to ca. 1.3 mm thick and 2.0 mm wide (but mostly much smaller) that are tightly packed together and occasionally become almost lobe-like, somewhat shiny, pale brown to dark brown, marginal prothallus black, thin or absent. Isidia absent. Apothecia immersed in areoles to erumpent, usually up to one per areole, initially round, later usually compressed and with wavy elongated shape, 0.4-1.3 mm diam., disc concave to flat, smooth, glossy, dark brown. Margin dull, ca. 0.3 mm wide, indistinguishable from the thallus, not or only slightly higher than the disc. Hymenium hyaline, not inspersed with oil droplets, up to 100 μm high; epihymenium fuscous brown, pigment in KOH becoming soluble and paler; hypothecium hyaline, not distinguishable from the thallus medulla and thus extending to over 1 mm; excipulum hyaline throughout, with a 10-15 μm thick layer of pseudocortex without crystals, with algae, not extending below the hypothecium. Paraphyses simple to somewhat branched, ca. 2.5 μm wide, not thickened at the tips. Asci cylindrico-clavate, blue, up to 95 × 15 μm, with ca. 50 ascospores. Ascospores hyaline, simple or occasionally with a pseudoseptum, narrowly ellipsoid, not constricted, 7.0-8.0 × 2.5-3.5 μm, wall ca. 0.5 μm thick, without appendages. Pycnidia abundant, immersed, dark brown; surrounding areole usually slightly raised. Conidia hyaline, linear to slightly clavate, 5-7.5 × 0.9-1.1 μm.
Distribution and ecology.  Diagnosis. Similar to Neoprotoparmelia brasilisidiata, but mainly differs from it by the presence of 16-spored asci.
Etymology. Named after the place of discovery, Siam (Thailand) and the presence of isidia. Description. Thallus consisting of slightly convex areoles of up to ca. 0.1 mm thick and 0.3 mm wide which are mostly coalescent to form a rimose thallus, somewhat shiny, pale brown to dark brown or mottled whitish-grey, on a fully immersed dark hypothallus, marginal prothallus black, thin or absent. Isidia always numerous, initially widely dispersed or somewhat clustered, eventually covering much of the thallus, up to 1.5 mm long, persistently 0.05-0.07 mm wide over their whole length, cylindrical, usually rather irregularly once or repeatedly branched and somewhat nodulose, glossy, pale to dark brown, tips generally dark brown. Apothecia sessile, initially round, older ones usually with wavy boundaries, 0.6-1.5 mm diam., disc flat, smooth, glossy, dark brown to orange brown. Margin glossy, ca. 0.25 mm wide, glossy brown at the outside, not or only slightly higher than the disc. Hymenium hyaline, not inspersed with oil droplets, up to 90 μm high; epihymenium fuscous brown, pigment in KOH becoming soluble and paler; hypothecium hyaline, up to 120 μm thick including subhymenium; excipulum hyaline throughout, with a 20-30 μm thick layer of cortex, without crystals, with algae, extending below the hypothecium (cupulate). Paraphyses branched, ca. 2.5 μm wide, not thickened at the tips. Asci cylindrico-clavate, blue, up to 35 × 9 μm, with 16 mostly biseriate ascospores. Ascospores hyaline, simple, broadly ellipsoid, not constricted, 9-11 × 6.5-8 μm, without appendages. Pycnidia not observed.
Distribution and ecology. On tree bark in a Park. Known only from Thailand (Chiang Mai).
Remarks. This comprises the specimens recovered within 'P. isidiata C' in 'Protoparmelia tropical clade' in Singh et al. (2015). It is similar to the other four isidiate Neoprotoparmelia species but can be distinguished from them by the presence of 16-spored asci. For additional specimens from Thailand, see Aptroot et al. (2007, as Protoparmelia isidiata). It can be distinguished from Neoprotoparmelia corallifera only by presence of 8-spored asci (Aproot et al. 1997a) and by using molecular data.