Contribution to a modern treatment of Graphidaceae biodiversity in South Africa: genera of tribe Graphideae with hyaline ascospores

Abstract

Additions and corrections are provided for the South African species of Graphidaceae tribe Graphideae with hyaline ascospores. Allographa oldayana I. Medeiros sp. nov. is described as new to science based on morphological, chemical and molecular data. The new species is characterized by lirellae with striate labia and a complete thalline margin, a completely carbonized excipulum, large, muriform ascospores, and the presence of hirtifructic acid. Allographa consanguinea (Müll. Arg.) Lücking, A. leptospora (Vain.) Lücking & Kalb, Diorygma aff. minisporum Kalb et al., Graphis crebra Vain., Gr. dupaxana Vain., Gr. furcata Fée, Gr. handelii Zahlbr., Gr. longula Kremp., Gr. pinicola Zahlbr., Gr. proserpens Vain, Gr. subhiascens (Müll. Arg.) Lücking and Platythecium sp. are reported as new records for South Africa. Allographa striatula (Ach.) Lücking & Kalb, Graphis analoga Nyl. and Gr. scripta (L.) Ach. are shown to be misapplied names that should be removed from the South African checklist. The new combination Mangoldia bylii (Vain.) I. Medeiros comb. nov. (bas. Graphis bylii Vain) is made; this represents an earlier name for M. atronitens (A. W. Archer) Lücking et al. Taxonomic notes are provided for Graphis bylii var. lividula Vain. and Gr. denudans Vain., species that are known only from their South African holotypes. Phylogenetic analyses that include new DNA sequence data from the nrLSU, mtSSU and RPB2 loci confirm the generic placements of several species for which molecular data were lacking: Allographa consanguinea, Glyphis atrofusca (Müll. Arg.) Lücking, Graphis crebra and Gr. subhiascens.

Introduction

The diverse topography, geology, climate and vegetation of South Africa contribute to a rich lichen biota estimated to contain at least 2500–3000 species (Fryday 2015). Approximately 1750 species of lichenized and allied fungi, many of which are endemic to southern Africa, are presently recorded from the country (Crous et al. 2006; Fryday 2015; Ahti et al. 2016). The majority of the at least 750–1250 undocumented or undiscovered species are expected to be crustose lichens, which have not yet received the taxonomic attention given in South Africa to macrolichen genera such as Xanthoparmelia (Fryday 2015).

Graphidaceae is the largest family of crustose lichens worldwide (Staiger 2002; Frisch 2006; Lücking et al. 2014, 2017). Research on this family in South Africa has been piecemeal, beginning with the description of Thelotrema henatomma Ach. by Acharius (1804). Nylander (1868) published the first taxonomic paper that described new lirelliform Graphidaceae from South Africa, based on specimens collected near Pietermaritzburg by Olivia Armstrong and William Mackenzie (Medeiros 2019). His article described one new species in the family and reported several records of previously described species. Additional new species were described by Müller (1886, 1887, 1895), Vainio (1926) and Zahlbruckner (1926, 1932). More recently, Egea & Torrente (1996) described the new saxicolous species Gymnographopsis latispora Egea & Torrente from fresh material and Kalb et al. (2009) described the new species Acanthothecis dialeucoides Kalb & Staiger from a historical specimen. Several species described from South African type material have been revised by modern authors as part of global revisions (Frisch 2006; Lücking et al. 2009). The most substantial recent work on Graphidaceae in South Africa is a country-level revision of the genus Diploschistes (Guderley & Lumbsch 1996), which has an unusual ecology within the family. Whereas most species of Graphidaceae occur on bark in shrubby or forest vegetation, Diploschistes species occur on rock or soil, often in arid or semi-arid regions (Staiger 2002; Frisch 2006; Lücking et al. 2014). Moreover, the only molecular data available for South African Graphidaceae is a single nuclear large subunit ribosomal RNA (nrLSU) sequence from Diploschistes actinostoma (Ach.) Zahlbr. (Rivas Plata et al. 2013).

Throughout this literature, species descriptions and records have accumulated without any synthesis of the systematics of lirelliform Graphidaceae in southern Africa. The only attempts at summarizing these scattered data have been the lichen checklists compiled by Doidge (1950) and Fryday (2015), which dealt with all lichens and did not make new taxonomic conclusions. Furthermore, no recent publications have dealt with new collections of lirelliform Graphidaceae on bark, where we expect a large portion of this family's South African biodiversity. Lücking et al. (2014) estimated that the lichen biota of Namibia, Botswana and South Africa includes over 220 species of Graphidaceae, with more than 160 species either unreported or undescribed. The method Lücking et al. (2014) used to estimate Graphidaceae biodiversity excluded strictly extratropical species, but subtropical biodiversity hotspots in South Africa (Mucina & Rutherford 2006) could support substantial biodiversity in this family. The present study is an initial contribution to a modern treatment of Graphidaceae biodiversity in South Africa, focusing on the species of the tribe Graphideae that have hyaline ascospores, namely species of the lirelliform genera Allographa, Diorygma, Glyphis, Graphis, Mangoldia and Platythecium (Rivas Plata et al. 2013; Lumbsch et al. 2014a). Species of Graphideae with pigmented ascospores (i.e. Phaeographis, Platygramme and related genera) will be treated in a subsequent publication.

Materials and Methods

Specimens were collected in February 2016 and May–June 2019 at forest and savannah sites in Mpumalanga, KwaZulu-Natal and Western Cape provinces (Figs 1 & 2). The forested areas were classified as Northern Mistbelt Forest, Southern Mistbelt Forest, and Southern Afrotemperate Forest (Mucina & Rutherford 2006). Additional relevant specimens were obtained from B, BOL, BM, DBN, LD, NU, PRE, SBBG, TRH, TUR, UPS, US and WIS. These included a large set of Graphidaceae from KwaZulu-Natal, collected by Ove Arbo Høeg in the 1920s, that has not previously been studied or discussed in any published work. Approximately 50 new collections and 110 herbarium specimens from South Africa, including four type specimens, were examined for this study.

Fig. 1. Map of South Africa showing collection localities for specimens in the present study. Open circles with letters A–D refer to localities depicted in Fig. 2. Filled black circles indicate other collection localities, including historical specimens. Extent of the forest biome in South Africa, Eswatini and Lesotho indicated in green (in colour) or mid grey (South African National Biodiversity Institute 2012). In colour online.

Fig. 2. Example collection sites of South African Graphidaceae. A, Northern Mistbelt Forest in a ravine at Buffelskloof Private Nature Reserve. Note planted Pinus on former natural grassland in the background. Photograph by József Geml. B, savannah in Kruger National Park. Zebras for scale. Photograph by Shuzo Oita. C, Diorygma aff. minisporum in situ on a tree trunk in Southern Mistbelt Forest in the Karkloof Nature Reserve. Photograph by Ian D. Medeiros. D, Southern Afrotemperate Forest on the coast at Nature's Valley, Garden Route National Park. Photograph by Betsy Arnold. See Fig. 1 for locations of these sites. In colour online.

Morphology and chemistry

Specimens were observed under Leica M60, Olympus SZ61, or Leica MZ6 dissecting microscopes. Photographs were taken with a Canon Rebel XSi camera attached to a Leica MZ125 dissecting microscope or with a Nikon D3200 camera with the VariMag DSLR modular imaging system attached to an Olympus SZ61 dissecting microscope. Hand-cut thin sections of apothecia were mounted in tap water for observation under either a Leica DM1000, Leica DMLB, or Olympus CH30 compound microscope. Morphological characters were documented following Lücking (2009). Recently published keys (Staiger 2002; Lücking et al. 2009; Barcenas Peña et al. 2014; Joshi et al. 2016) and species descriptions from the primary literature were used to assess whether collections represented previously described species. Genera and species have been designated as first reports for South Africa if they do not appear in the South African lichen checklist (Fryday 2015; Ahti et al. 2016).

Secondary chemistry was assessed with thin-layer chromatography (TLC) using standard methods (Culberson & Kristinsson 1970; Orange et al. 2010). Plates were run in solvent systems C (170 toluene: 30 glacial acetic acid) for standard analyses and G (139 toluene: 83 ethyl acetate: 8 formic acid) when necessary to separate stictic acid satellite compounds. Elix (2018) was consulted as an additional reference for identifying TLC spots.

Molecular phylogenetics

All new molecular data are from South African specimens except for one specimen of Platygramme pachnodes (Fée) Fée from Florida, USA (LaGreca et al. 2021). DNA was isolated either from fresh specimens or from specimens stored at −20 °C in silica gel. Small fragments (c. 1–2 mm²) of thallus or ascomata were excised with a sterile needle. DNA extraction followed the procedure outlined in Hughes et al. (2020) using laboratory-made extraction and dilution buffers, except that thallus fragments were frozen in the extraction buffer for 1 h at −20 °C and thawed to room temperature prior to the heating step.

The internal transcribed spacer (ITS) was amplified with primers ITS1F and LR3 (Vilgalys & Hester 1990; Gardes & Bruns 1993). ITS sequences are not widely available for Graphidaceae (Cáceres et al. 2020) and the few ITS sequences we obtained were not used in subsequent phylogenetic analyses. The nuclear large subunit (nrLSU) was amplified with primer pair AL2R and LR6 (Vilgalys & Hester 1990; Mangold et al. 2008), while the mitochondrial small subunit (mtSSU) was amplified with primer pair mrSSU1 and mrSSU3R (Zoller et al. 1999). The RNA polymerase II second largest subunit (RPB2) was amplified with a nested approach, first using the fungal primers fRPB2-7cF and fRPB2-11aR (Liu et al. 1999) and then the internal primers GD-RPB2-7cF and GC-RPB2-11aR, which were designed specifically for Graphidaceae (Kraichak et al. 2015). Thermal cycler conditions followed Kraichak et al. (2015). PCR products were checked on a 1% agarose gel and cleaned with exonuclease I and shrimp alkaline phosphatase (ThermoFisher Scientific, Waltham, MA, USA). Sanger sequencing was performed by Eurofins Genomics (Louisville, KY, USA) using the PCR primers. Forward and reverse reads were assembled and checked for errors in Geneious Prime v. 2022.0.1. GenBank Accession numbers for newly generated sequences are provided in Table 1 (for nrLSU, mtSSU and RPB2) or in the remarks under individual species (for ITS). We generated two new ITS sequences, 14 new nrLSU sequences, 21 new mtSSU sequences and 19 new RPB2 sequences from a total of 21 specimens.

Table 1. Voucher information and GenBank Accession numbers for new sequences of Graphidaceae generated for this study (GenBank Accession numbers in bold) and reference taxa used in the phylogenetic analysis. Dashes indicate missing data. Note that the new data include one specimen from outside South Africa (Platygramme pachnodes from Florida, USA).

The phylogenetic analyses were based on nrLSU, mtSSU and RPB2. We prepared a dataset of reference sequences (Table 1) generated in previous studies of Graphidaceae and other fungi (Kalb et al. 2004; Staiger et al. 2006; Mangold et al. 2008; Rivas Plata et al. 2011, 2013; Cáceres et al. 2012; Nelsen et al. 2012; McDonald et al. 2013; Lumbsch et al. 2014b; Fazio et al. 2018; Vu et al. 2019). Species of Acanthothecis, Diploschistes and Fissurina, representing three different Graphidaceae clades outside tribe Graphideae (Lumbsch et al. 2014a), were used for the outgroup. The nrLSU and mtSSU sequences were initially aligned in MAFFT with the G-INS-1 option (Katoh et al. 2019), while the RPB2 sequences were aligned by translated amino acids in Mesquite (Maddison & Maddison 2021). Alignments were subsequently corrected and ambiguously aligned regions were delimited in Mesquite. Ambiguous regions were excluded from subsequent analyses. Complete alignments with delimited ambiguous regions are available at FigShare (DOI: 10.6084/m9.figshare.c.6007096).

Model selection and maximum likelihood tree inference were performed using IQ-TREE v. 2.1.2 (Nguyen et al. 2015; Chernomor et al. 2016) run on the CIPRES server (Miller et al. 2010). We first inferred separate trees for each locus. We performed 5000 ultrafast bootstrap pseudoreplicates to calculate bipartition support (Hoang et al. 2018) and examined the trees for well-supported conflicts (i.e. ≥ 95% ultrafast bootstrap). There were no well-supported conflicts, so the nrLSU, mtSSU and RPB2 alignments were concatenated and used as input for a partitioned analysis in IQ-TREE. Our concatenated alignment included 78 specimens, including 15 South African representatives from Graphidaceae tribe Graphideae. ModelFinder (Kalyaanamoorthy et al. 2017) was used to optimize the partitioning scheme and determine the best substitution models (Table 2). We performed 5000 ultrafast bootstrap pseudoreplicates to calculate bipartition support for the concatenated tree topology. The alignments and three-locus phylogeny from this paper have been made available on T-BAS (Carbone et al. 2017, 2019) to facilitate access to these alignments and placement of new sequence data from this clade.

Table 2. Alignment statistics and substitution models. The full concatenated alignment included 78 specimens and 2404 sites. RPB2 was partitioned by codon position. PI = parsimony-informative sites; Inv = invariant sites.

Results

The phylogenetic analysis based on the concatenated dataset recovered hyaline-spored samples from South Africa in four highly supported clades, corresponding to the genera Allographa, Diorygma, Glyphis and Graphis s. str. (Fig. 3). We also recovered a highly supported clade containing species of the brown-spored genera Leiorreuma, Phaeographis, Pallidogramme, Platygramme and Thecaria, including three South African specimens. The monophyly of Graphideae was highly supported but relationships among genera were not, with the exception that Mangoldia was supported as sister to Allographa (Fig. 3).

Fig. 3. Maximum likelihood phylogeny of Graphidaceae tribe Graphideae based on concatenated analysis of nrLSU, mtSSU and RPB2. Specimens in bold are from South Africa. Ultrafast bootstrap (UFBoot2; Hoang et al., 2018) values ≥ 95 are indicated with thickened branches. Scale indicates substitutions per site. Loci included in the alignment are indicated for each specimen, and their GenBank Accession numbers are shown in Table 1. For information on how Graphideae is situated in a wider phylogenetic context within Graphidaceae, see Lumbsch et al. (2014a).

Morphological study of herbarium material and fresh collections yielded one species of Allographa new to science, one species based on a South African type that needed to be transferred to Mangoldia, and 12 new records for South Africa, all described below. The herbarium material also included a substantial number of Opegrapha and Enterographa specimens misidentified as Graphidaceae; these have been annotated but a complete listing of examined, non-Graphidaceae specimens is beyond the scope of this study.

Taxonomy

Note that taxonomic synonyms are provided only when they concern names described from South African material.

Allographa consanguinea (Müll. Arg.) Lücking & Kalb

Herzogia 31, 549 (2018).—Graphina consanguinea Müll. Arg., Nuov. Giorn. Bot. Ital. 21, 362 (1889).—Graphis consanguinea (Müll. Arg.) Lücking, in Lücking et al., Fieldiana, Bot. 46, 67 (2008); type: Brazil, Glaziou s. n. (G—holotype, not seen).

(Fig. 4A)

Fig. 4. Images of South African Graphidaceae. A, Allographa consanguinea (TRH L-17909). B, A. leptospora (Medeiros L449). C, A. oldayana (Medeiros L506). D, A. oldayana (Medeiros 2078—holotype). E, ascospore of A. oldayana (Medeiros L507). F, Diorygma aff. minisporum (Medeiros 2106). Scales: A–D & F = 1 mm; E = 50 μm. Images by IDM (A–E) and Thomas Barlow (F). In colour online.

Remarks

Allographa consanguinea has not previously been recorded for South Africa; it was reported from Kenya by Kirika et al. (2012) and is otherwise known only from the Neotropics (Lücking et al. 2009). We provide the first molecular data for A. consanguinea and confirm its placement in Allographa (Table 1, Fig. 3). This corticolous species co-occurs and shares morphological similarities with Allographa oldayana, described below; see the remarks under the new species for characters that separate them. Records of Allographa acharii (Fée) Lücking & Kalb from South Africa (Nylander 1868; Doidge 1950; Almborn 1988) may represent A. consanguinea or A. oldayana. Allographa acharii has prominent lirellae and 2–6 large, muriform spores per ascus versus erumpent lirellae and one spore per ascus in A. consanguinea (Lücking et al. 2009).

Specimens examined

South Africa: Eastern Cape: ‘Grahamstown, natural forest just above Fern Kloof’, iii 1961, H. B. Johnston s. n. (LD 1948852). KwaZulu-Natal: ‘distr. Eshowe, on Eucalyptus along main road’, 26 viii 1919, O. A. Høeg s. n. (TRH L-17858, L-17909); ‘distr. Eshowe, S of Solheim Miss. St.’, 7 ix 1929, O. A. Høeg s. n. (TRH L-17921); uMgungundlovu District Municipality, Karkloof Nature Reserve, 29°17ʹ52ʺS, 30°13ʹ40ʺE, 2019, Medeiros 2095 (BOL). Limpopo: De Hoeck Forest, west of Tzaneen, 1953, R. Kräusel 4b [with Allographa oldayana] (B).

Allographa leptospora (Vain.) Lücking & Kalb

In Kalb et al., Phytotaxa 377, 19 (2018).—Graphis leptospora Vain., Ann. Bot. Soc. Zool.-Bot. Fenn. Vanamo 1(no. 3), 53 (1921); type: Thailand, Hosseus s. n. (TUR-V—holotype, not seen).

(Fig. 4B)

Remarks

This is the first report of this corticolous species for South Africa. It differs from the other species with a striate, completely carbonized excipulum and a complete thalline margin (A. consanguinea and A. oldayana) in having transversely septate ascospores.

Specimen examined

South Africa: Mpumalanga: Buffelskloof Private Nature Reserve, 25°15ʹ59ʺS, 30°31ʹ6ʺE, 1725 m, 2016, Medeiros L449 (BOL).

Allographa oldayana I. Medeiros sp. nov.

MycoBank No.: MB 843785

Differing from Allographa cerradensis (Marcelli et al.) Lücking & Kalb in the clear hymenium, larger ascospores, and presence of hirtifructic acid.

Type: South Africa, Mpumalanga, Buffelskloof Private Nature Preserve, 2019, Medeiros 2078 & Flakus (BOL—holotype; DUKE, PRE—isotypes).

(Fig. 4C–E)

Thallus corticolous, epiperidermal, 30–120 μm thick, continuous; surface smooth, pale green; prothallus absent. Thallus in section with prosoplectenchymatous cortex 12–30 μm thick, algal layer (Trentepohlia) 20–110 μm thick, and irregular clusters of calcium oxalate crystals.

Apothecia lirelliform, unbranched, erumpent to prominent, with thin to thick complete thalline margin, 1.0–7.5 mm long, 0.5–0.6 mm wide, 0.29–0.37 mm high; disc concealed; proper margin thick, labia striate, black. Excipulum striate, completely carbonized, 60–105 μm wide; hypothecium prosoplectenchymatous, 20–30 μm high, colourless to pale yellow; hymenium 100–180 μm high, colourless, clear; paraphyses unbranched, smooth; epithecium granulose, 12–25 μm high, dark olive-brown. Asci fusiform, 100–180 × 19–32 μm. Ascospores 1–4 per ascus, ellipsoid, muriform, transversely 20–30-septate, longitudinally 2–4-septate, 100–150(–175) × (16–)18–24(–27) μm, 4.5–7 times as long as wide, hyaline, I+ violet.

Conidiomata not seen.

Chemistry

Hirtifructic acid (major), cf. conhirtifructic acid (minor).

Etymology

The specific epithet honours Professor Fred C. Olday (College of the Atlantic, Bar Harbor, Maine, USA), who introduced the first author to the study of lichens.

Distribution and ecology

This species occurs in forested areas of eastern South Africa (e.g. Fig. 2A). Although fresh collections were made only in Northern and Southern Mistbelt Forest at the Buffelskloof and Karkloof reserves, respectively (Mucina & Geldenhuys 2006), one herbarium specimen from near Eshowe suggests that this species occurred in more coastal forest, at least historically (Mucina et al. 2006).

Remarks

Molecular data confirm the placement of this species in Allographa (Fig. 3). Allographa elixii (A. W. Archer) Lücking & Kalb, the only other species in the genus with hirtifructic acid, differs from the new species in having an inspersed hymenium and ascospores which are only terminally muriform (Archer 2001a). Allographa cerradensis differs in the inspersed hymenium, the presence of stictic acid instead of hirtifructic acid, and the smaller (80–100 × 15–20 μm) ascospores (Lumbsch et al. 2011). The North American Appalachian species Allographa sterlingiana (E. A. Tripp & Lendemer) Lücking & Kalb lacks lichen substances and has an inspersed hymenium (Lendemer et al. 2013).

The new species and A. consanguinea occur in the same habitats and both have a complete thalline margin, striate labia and large, muriform ascospores. They differ in secondary chemistry (no secondary metabolites in A. consanguinea), the more prominent lirellae in A. oldayana, and ascospore number (A. consanguinea has one spore per ascus). Records of Allographa acharii from South Africa (Nylander 1868; Steiner 1907; Doidge 1950; Almborn 1988) may represent A. oldayana or A. consanguinea. Allographa acharii has ascospores of similar size and number to the new species but lacks secondary compounds (Lücking et al. 2009).

Hirtifructic acid is an uncommon secondary compound in Graphidaceae, known from a handful of species in Allographa and Ocellularia (Lücking et al. 2009; Rivas Plata et al. 2012). This substance is rare in Lecanoromycetes more broadly and its chemical structure is unknown, although it is believed to be related to echinocarpic acid (Elix 2018). In addition to Graphidaceae, it is also known in Parmeliaceae, where it is found in several species of Relicina and one species of Hypotrachyna (Elix 1996; Sipman et al. 2009). Elix (1996, 2018) does not report an R _f value for this substance in solvent G; we found the R _f value to be 59. Elix (2018) reported that there were no spot test reactions for hirtifructic acid but the new species reacts weakly K+ yellow, best observed when the secondary compounds are eluted with acetone onto filter paper. This is not diagnostic, however, as many Graphidaceae species react K+ yellow for more common substances (Lücking et al. 2009). TLC is thus required to confirm the presence of hirtifructic acid.

Additional specimens examined (paratypes)

South Africa: KwaZulu-Natal: ‘distr. Eshowe, on the trees in open forest along the small stream at Solheim’, 3 ix 1929, O. A. Høeg s. n. (TRH L-17871); uMgungundlovu District Municipality, Karkloof Nature Reserve, 29°17ʹ52ʺS, 30°13ʹ40ʺE, 2019, Medeiros 2102 (DUKE, BOL). Mpumalanga: Buffelskloof Private Nature Preserve, 25°15ʹ59ʺS, 30°31ʹ6ʺE, 1725 m, in ravine forest, on bark, 2016, Medeiros L448 (PRE); ibid., 25°17ʹ31ʺS, 30°30ʹ15ʺE, 1380 m, wet forest near bottom of ravine, 2016, Medeiros L506, L507 (PRE). Limpopo: De Hoeck Forest, west of Tzaneen, 1953, R. Kräusel 4b [with Allographa consanguinea] (B).

Allographa sp. 1

Remarks

Molecular data place the specimen cited below in Allographa without a close match to other species for which sequence data are available (Fig. 3). The specimen has a lateral thalline margin, an excipulum that is striate and apically to peripherally carbonized, and a clear hymenium. No ascospores could be found, precluding a species determination.

Specimen examined

South Africa: KwaZulu-Natal: uMgungundlovu District Municipality, Karkloof Nature Reserve, 2019, Medeiros 2099 (BOL).

Diorygma aff. minisporum Kalb, Staiger & Elix

Symb. Bot. Upsal. 34, 161 (2004); type: Guatemala, Kalb & Plöbst s. n. (WIS, hb. Kalb—holotype, not seen).

(Figs 2C, 4F)

Remarks

Diorygma minisporum was described from Guatemala (Kalb et al. 2004) and occurs widely in South America (Medina et al. 2012; Aptroot & Cáceres 2018; Pereira et al. 2018). Kirika et al. (2012) reported this corticolous species from Kenya and cited this as the first record from outside the Americas. Although the specimen cited below fits Diorygma minisporum morphologically, the molecular data suggest that our collection may not be conspecific with the neotropical material (Fig. 3).

Specimen examined

South Africa: KwaZulu-Natal: uMgungundlovu District Municipality, Karkloof Nature Reserve, 29°17ʹ52ʺS, 30°13ʹ40ʺE, on bark at base of large tree, 2019, Medeiros 2106 (BOL, DUKE).

Glyphis atrofusca (Müll. Arg.) Lücking

In Archer, Fl. Australia 57, 651 (2009).—Graphina atrofusca Müll. Arg., Flora 70, 74 (1887).—Graphis atrofusca (Müll. Arg.) Stizenb., Ber. Tätigk. St. Gallischen Naturwiss. Ges. 1889-1890, 186 (1891); type: South Africa, F. Wilms 70 (G—lectotype, not seen, designated by Lücking in A. W. Archer, Fl. Australia 57, 651 (2009)).

Graphina polycarpa Müll. Arg., Flora 70, 63 (1887); Graphis polycarpa (Müll. Arg.) Stizenb., Ber. Tät. St. Gallisch. Naturw. Gesellsch. 1889–1890, 184 (1891); type: South Africa, F. Wilms 48 (G—holotype).

(Fig. 5A)

Fig. 5. Images of South African Graphidaceae. A, Glyphis atrofusca (Medeiros 2089a). B, Gl. cicatricosa (Medeiros 2100). C, Graphis crebra (Medeiros 2088a). D, Gr. denudans (van der Bijl 126—holotype). E, Gr. dupaxana (Høeg s. n.). F, Gr. furcata (Høeg s. n.). Scales = 1 mm. Images by Willow Torrey (D) and IDM (A–C, E & F). In colour online.

Remarks

We provide molecular data (ITS, ON507259; for other loci see Table 1) for a topotype of Glyphis atrofusca, a corticolous species originally described from the general vicinity of modern-day Kruger National Park (Müller 1887) and subsequently reported from Australia and North America (Archer 2001b, 2009; Staiger & Kalb 2004; Lücking & Kalb 2018; Guzmán-Guillermo et al. 2021). Lücking & Kalb (2018) noted that this species can be mistaken for a species of Graphis (its immersed, more or less elongate lirellae can suggest that genus if the presence of brown pruina is missed or indistinct) and that molecular data had not yet confirmed its placement in Glyphis. We show that Gl. atrofusca falls in Glyphis with high support (Fig. 3).

Staiger (2002) established Glyphis subgenus Pallidoglyphis for two species with a non-carbonized hypothecium: Glyphis substriatula (Nyl.) Staiger and Gl. atrofusca, the latter under its synonym Gl. montoensis (A. W. Archer) Staiger. Recently, Kalb (2020) described a third species from this group, Gl. frischiana Kalb. Prior to our study, only Gl. substriatula had molecular data. In our phylogenetic analysis, relationships within Glyphis were not well supported, and we can neither confirm nor refute the subgeneric classification adopted by Staiger (2002).

We have only seen South African specimens of this species from savannah vegetation (Fig. 2B). It has also been found in dry, well-lit scrubland in other parts of its range (Archer 2001b; Staiger & Kalb 2004). The collection by Wilms cited below (which is from the type locality) is the basis for the record of Graphis sophistica Nyl. in Stizenberger (1891) and therefore is also one of the records cited for Gr. platycarpa (Eschw.) Zahlbr. by Doidge (1950). There are other historical records of Gr. sophistica not from the type locality of Glyphis atrofusca (Doidge 1950), so for the present we refrain from excluding Gr. platycarpa from the South African checklist as a misapplied name.

Specimens examined

South Africa: Mpumalanga: Kruger National Park, on bark of Spirostachys africana, 2019, Medeiros 2089a (BOL, DUKE); ‘Corticolam prope urbem Lydenburg’, s. d., F. Wilms s. n., Lichenotheca Universalis 90 (UPS L-203515).

Glyphis cicatricosa Ach.

Syn. Meth. Lich. (Lund), 107 (1814); type: Guinea, s. col. (H-ACH—holotype, not seen).

(Fig. 5B)

Remarks

This distinctive pantropical species has numerous records from KwaZulu-Natal (Nylander 1868; van der Byl 1931; Doidge 1950). There is substantial morphological variation within this species (Staiger 2002) and previous phylogenetic analyses have failed to recover all sequenced individuals in a single, highly supported clade (Rivas Plata et al. 2013). In our phylogenetic analysis, specimens from South Africa, Kenya and El Salvador clustered together with poor support (Fig. 3). Glyphis dictyospora Staiger, described from Kenya, is externally similar to Gl. cicatricosa but has submuriform ascospores (Staiger 2002). It has not been found in South Africa.

Specimens examined

South Africa: Eastern Cape: ‘Alexandria Div., Alexandria State Forest’, on Pinus radiata, 60 m, G. Degelius SA-417 (UPS L-53775, with Phaeographis sp.). KwaZulu-Natal: ‘distr. Eshowe, Solheim near Eshowe’, on loquat trees, 5 ix 1929, O. A. Høeg s. n. (TRH L-17867); uMgungundlovu District Municipality, Karkloof Nature Reserve, 29°17ʹ52ʺS, 30°13ʹ40ʺE, on bark, 2019, Medeiros 2100 & Flakus (BOL); Karkloof Region, 1868, Armstrong & Mackenzie s. n. (DBN); ‘Dist. Stanger, Hill north of Umgeni River’, 1920, N. S. Pillans 10071 (BOL 216555); ‘Dist. Nongoma, Umzinene’, 18 viii 1929, O. A. Høeg s. n. (TRH L-17901).

Graphis bylii var. lividula Vain.

Ann. Univ., Fenn. Aboënsis, Ser. A 2(no. 3), 27 (1926); type: South Africa, Western Cape, Knysna, on Ficus elastica, s. d., P. A. van der Bijl 277 (TUR-V—holotype!).

Remarks

Graphis bylii var. lividula, known only from the South African type collection (Vainio 1926), is not closely related to Gr. bylii Vain. s. str. (see below) and is morphologically indistinguishable from the northern European species Gr. inustuloides Lücking. Both are corticolous species characterized by open, pruinose discs; an apically carbonized, entire excipulum; a clear hymenium; broadly ellipsoid, muriform spores; and the absence of secondary metabolites. The spores of the type of Gr. bylii var. lividula are 37–49 × 16–22 μm with 9–10 × 4–5 locules, while Lücking & McCune (2012) give measurements of 35–50 × 15–20 μm for Gr. inustuloides.

If these are indeed conspecific, their disjunction would be comparable to that of the closely related Gr. pergracilis, which is known from South Africa, the Solomon Islands, and the north-western United States (Archer 2007; Lücking et al. 2009; Lücking & McCune 2012). Graphis bylii var. lividula, Gr. pergracilis and Gr. inustuloides, together with the Australian species Gr. coenensis A. W. Archer and the neotropical Gr. dimidiata Vain., form a species complex that requires further study (Lücking & McCune 2012). We have also seen additional specimens from this group that vary slightly from the described species. For example, a South African specimen at PRE (E. Retief 551) differs from typical Gr. pergracilis in having shorter lirellae, discs that may be partially exposed, and a laterally carbonized excipulum (in some sections carbonized only in the upper two-thirds). Molecular data are not currently available for any species from this group, but such data will be essential for understanding species boundaries in what may represent a complex of cryptic species (Lücking & McCune 2012; van der Pluijm 2014). A precedent for such a pattern can be found in the Graphis scripta complex (Neuwirth & Aptroot 2011; Kraichak et al. 2015).

Graphis crebra Vain.

Hedwigia 38, 256 (1899); type: Guadeloupe, Duss 541 (TUR-V 27617, not seen).

(Fig. 5C)

Remarks

Graphis crebra has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species for South Africa, and we provide the first molecular data for the species (Table 1, Fig. 3). Graphis crebra and Gr. handelii (newly reported from South Africa below) both have open discs, an inspersed hymenium, hyaline, transversely septate ascospores c. 25 μm in length, and contain norstictic acid. The hyaline ascospores separate these two taxa from species of Phaeographis or Platygramme with open discs, whereas these two Graphis species can be distinguished from one another by the discs that are white pruinose in Gr. crebra and epruinose in Gr. handelii. Glyphis atrofusca was found at the same savannah site as Gr. crebra (Fig. 1B) and also has open discs, but can be readily distinguished by the brown pruina on the discs, clear hymenium, and muriform ascospores. Graphis pyrrhocheiloides Zahlbr., which was reported from South Africa by Lücking et al. (2009) as an accessory species on the type of Gr. pergracilis, is similar to Gr. crebra in having open, pruinose discs, a laterally carbonized excipulum, transversely septate ascospores and norstictic acid, but differs in the clear hymenium.

Specimen examined

South Africa: Mpumalanga: Kruger National Park, on bark of Euclea divinorum, 2019, Medeiros 2088a (BOL, DUKE).

Graphis denudans Vain.

Ann. Univ., Fenn. Aboënsis, Ser. A 2(no. 3), 27 (1926); type: South Africa, KwaZulu-Natal, near Durban, s. d., P. A. van der Bijl 126 (TUR-V—holotype!).

(Fig. 5D)

Remarks

This corticolous species is characterized by very short (< 1 mm), unbranched to sparsely branched, erumpent lirellae with lateral thalline margin and entire (or weakly striate?) labia, discs that are often partially exposed, a completely carbonized excipulum (sometimes weakly at base) and a clear hymenium; ascospores 8 per ascus, hyaline, transversely 5–6-septate, 17–22(–25) × 7–8 μm; and the presence of norstictic acid. Graphis denudans is known only from the holotype and has not been reported since its original description by Vainio (1926). It may be related to G. schiffneri Zahlbr., an eastern palaeotropical species that differs in having longer lirellae, obscurely striate labia and slightly longer ascospores (Lücking et al. 2009); additional material might show the two to be conspecific.

Graphis dupaxana Vain.

Ann. Acad. Sci. Fenn., Ser. A 15(no. 6), 241 (1921); type: Philippines, McGregor 14313 (TUR-V—lectotype, not seen).

(Fig. 5E)

Remarks

Graphis dupaxana has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species for South Africa. It differs from other species of Graphis in South Africa by having a completely carbonized, striate excipulum, a combination of characters more associated with Allographa (Lücking & Kalb 2018). It differs from South African species of Allographa in lacking a thalline margin and having a distinctly white (not greenish) thallus.

Specimen examined

South Africa: KwaZulu-Natal: ‘distr. Eshowe, in a small krantz at Inyezane R., between the Mtunzeni Road and S. Siding’, very dense vegetation of small trees, 31 viii 1929, O. A. Høeg s. n. (TRH L-17906).

Graphis furcata Fée

Essai Crypt. Exot. (Paris), 40 (1825); type: South America, s. col. (G—holotype, not seen).

(Fig. 5F)

Remarks

Graphis furcata has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species for South Africa. See remarks under Gr. pinicola for how to differentiate these two species. Graphis furcata is known only from a single historical collection and its current status in South Africa is uncertain.

Specimen examined

South Africa: KwaZulu-Natal: ‘distr. Durban, Salisbury Island’, small trees in rather dense vegetation outside the mangrove, 20 ix 1929, O. A. Høeg s. n. (TRH L-17915).

Graphis handelii Zahlbr.

Symb. Sinic. 3, 44 (1930); type: China, Handel-Mazzetti 12788 (W—lectotype, not seen).

(Fig. 6A)

Fig. 6. Images of South African Graphidaceae. A, Graphis handelii (Killick 535). B, Gr. librata (Garside s. n.). C, Gr. longula (Medeiros L497). D, Gr. pinicola (Sipman 20.181). E, Gr. proserpens (Medeiros L444). F, Gr. subhiascens (Medeiros 2088b). Scales = 1 mm. All Images by IDM. In colour online.

Remarks

Graphis handelii has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species for South Africa. It is very close to Gr. crebra and in the field could also be mistaken for a species of Phaeographis; characters to separate these taxa are provided above in the remarks for Gr. crebra. We have seen Graphis handelii only in historical specimens from KwaZulu-Natal, where it was found in both coastal and inland forest vegetation. We lack recent collections from the Indian Ocean Coastal Belt vegetation of this province, a threatened vegetation type (Jewitt 2018), so the current status of this species in South Africa is uncertain.

Specimens examined

South Africa: KwaZulu-Natal: ‘distr. Durban, summit of the Bluff, on trees in dense vegetation’, 22 ix 1929, O. A. Høeg s. n. (TRH L-17922); ‘distr. Umgeni, at Petermaritzburg, near Town Bush Road. On small tree (Solanum) along small stream’, 29 ix 1929, O. A. Høeg s. n. (TRH L-17908); ‘distr. Eshowe, Solheim near Eshowe’, on branches of loquat trees, 5 ix 1929, O. A. Høeg s. n. (TRH L-17899, mixed collection with other Graphis spp.); ‘Pietermaritzburg Division, Table Mountain, epiphloeodal on Croton sylvaticus growing along forest margin, 2000 ft’, 1948, D. J. B. Killick 535 (BOL 207564).

Graphis librata C. Knight

Trans. Proc. New Zeal. Inst. 16, 404 (1884); type: New Zealand, Knight 67:23 (WELT—lectotype, not seen, designated by Hayward, New Zealand J. Bot. 15, 571 (1977)).

Graphis diaphoroides Müll. Arg., Flora (Regensburg) 69(20), 316 (1886); type: South Africa, Mpumalanga, near Lydenburg, 1884, Wilms 11 [Lojka Lichenothecum Universalis 91] (G—holotype, not seen; BM, M, MICH, NY—isotypes, not seen; US—isotype!).

(Fig. 6B)

Remarks

Lücking et al. (2009) synonymized Graphis diaphoroides, a corticolous species described from South African material (Müller 1886), with G. librata. As currently circumscribed, this species is pantropical; it is one of the most common and widely distributed species of Graphis in South Africa. We obtained molecular data from one specimen of G. librata. In our phylogenetic analysis, this specimen was recovered distant to G. librata from El Salvador but without strong support (Fig. 3). Molecular data for Graphis librata from other locations, especially the type locality in New Zealand, will be necessary to understand the delimitation of this species.

Specimens examined

South Africa: KwaZulu-Natal: Karkloof, on bark, 2019, Medeiros 2112 (BOL). Western Cape: Kirstenbosch, on Brabejum, ii 1946, S. Garside s. n. (BOL 207569); Kirstenbosch, on bark, 2019, Medeiros 2145 (BOL); Table Mountain National Park, Silver Mine pond, on bark of tree at base of dam, 2019, Medeiros 2143 (BOL); ‘S. Cape, Zitzikamma forest near Coldstream, on twigs of fruit trees’, vii 1944, J. Pont s. n. (PRE 766676); Nature's Valley, on vining Euphorbiaceae, 2019, Medeiros 2134 (BOL).

Graphis longula Kremp.

Flora 59, 414 (1876); type: Brazil, Glaziou 5497 (M—lectotype, not seen).

(Fig. 6C)

Remarks

Graphis longula has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species from South Africa.

Specimen examined

South Africa: Mpumalanga: Buffelskloof Private Nature Preserve, 25°17ʹ31ʺS, 30°30ʹ15ʺE, 1380 m, wet forest near bottom of ravine, 2016, Medeiros L497 (BOL).

Graphis pinicola Zahlbr.

In Handel-Mazzetti, Symb. Sinic. 3, 43 (1930); type: China, Handel-Mazzetti 2829 (W—holotype, not seen; US—isotype, not seen).

(Fig. 6D)

Remarks

Graphis pinicola has a pantropical distribution (Lücking et al. 2009). This is the first report of this corticolous species from South Africa, although it is a widespread species with numerous historical and modern collections. Graphis pinicola is similar to Gr. furcata; however, the former has a corticate thallus and well-defined thalline margin, while the latter is ecorticate and the thalline margin slopes gradually into the thallus. Graphis librata differs from Gr. pinicola in producing norstictic acid.

Specimens examined

South Africa: KwaZulu-Natal: ‘distr. Eshowe, S. of Solheim’, on trees, 7 ix 1929, O. A. Høeg s. n. (TRH L-17911); ‘Solheim near Eshowe’, on branches of loquat trees, 5 ix 1929, O. A. Høeg s. n. (TRH L-17899, mixed collection with other Graphis spp.); Karkloof region, Armstrong & Mackenzie s. n., 1866 (DBN). Western Cape: Cape Town, east slope of Table Mtn at Kirstenbosch Botanical Garden, 400 m, 1986, H. Sipman 20.181 (B); Garden Route National Park, Nature's Valley, 2019, Medeiros 2135a (with Phaeographis sp.) (BOL).

Graphis proserpens Vain.

Botanisk Tidsskrift 29, 132 (1909).—Graphis disserpens Vain., Acta Soc. Fauna Flora Fenn. 7(2), 123 (1890), nom. illeg. (non Graphis disserpens Nyl.); type: Brazil, Vainio s. n. (TUR-V—holotype, not seen).

(Fig. 6E)

Remarks

Graphis proserpens has not previously been reported from South Africa, although this corticolous species is probably pantropical (Lücking et al. 2009). Historical references to Gr. striatula Ach. (Nylander 1868) probably represent this species (see discussion of misapplied names, below). Diederich et al. (2017) recently reported this species as new to the Seychelles. In our phylogenetic analysis, Gr. proserpens from South Africa was recovered as sister to Gr. proserpens from the Philippines (Fig. 3). The GenBank Accession number for the ITS sequence of Medeiros 2105 is ON507258.

Specimens examined

South Africa: KwaZulu-Natal: Karkloof, on bark, 2019, Medeiros 2105 (BOL, DUKE). Mpumalanga: Buffelskloof Private Nature Preserve, 25°15ʹ59ʺS, 30°31ʹ6ʺE, 1725 m, 2016, Medeiros L444 (PRE), L447 (BOL); 25°17ʹ31ʺS, 30°30ʹ15ʺE, 1380 m, wet forest near bottom of ravine, 2016, Medeiros L498 (PRE).

Graphis subhiascens (Müll. Arg.) Lücking

In Lücking et al., Fieldiana, Bot. 46, 111 (2008).—Graphina subhiascens Müll. Arg., Bot. Jahrb. 20, 2811 (1894); type: Tanzania, Holst 696 (G—holotype, not seen).

(Fig. 6F)

Remarks

Graphis subhiascens has not previously been reported from South Africa, although this corticolous species is pantropical (Lücking et al. 2009). We provide the first molecular data for this species (Table 1, Fig. 3). It has medium-sized, muriform ascospores and a complete thalline margin, and can be distinguished from Allographa consanguinea and A. oldayana on the basis of the larger ascospores and striate, completely carbonized excipulum in those species. Other South African species with an entire, laterally carbonized excipulum (Gr. crebra, Gr. furcata, Gr. handelii and Gr. pinicola) have transversely septate ascospores.

Specimen examined

South Africa: Mpumalanga: Kruger National Park, on bark of Euclea divinorum, 2019, Medeiros 2088b (BOL).

Mangoldia bylii (Vain.) I. Medeiros comb. nov.

MycoBank No.: MB 843786

Graphis bylii Vain., Ann. Univ., Fenn. Aboënsis, Ser. A 2(no. 3), 27 (1926).—Graphina bylii (Vain.) Zahlbr., Cat. Lich. Univers. 8, 604 (1932); type: South Africa, Western Cape, Knysna, on bark of Ocotea bullata, 1922, P. A. van der Byl 265 (TUR-V—holotype!).

Syn. nov.: Graphina atronitens A. W. Archer, Mycotaxon 77, 162 (2001).—Thalloloma atronitens (A. W. Archer) A. W. Archer, Telopea 11, 77 (2005).—Mangoldia atronitens (A. W. Archer) Lücking et al., Phytotaxa 69, 4 (2012); type: Australia, New South Wales, Black Rock, c. 7 km S of Brunswick Heads, 5 November 1998, A. W. Archer G292 (NSW—holotype, not seen).

(Fig. 7A)

Fig. 7. Images of South African Graphidaceae. A, Mangoldia bylii (van der Bijl 265—holotype). B, Platythecium sp. (Høeg s. n.). Scales = 1 mm. Images by Willow Torrey (A) and IDM (B). In colour online.

Remarks

Lücking et al. (2012) established the genus Mangoldia for two Australian species with Phaeographis-like ascomata but Graphis-like ascospores. Mangoldia is closely related to Allographa (Fig. 3). The holotype of Graphis bylii closely matches the description of Graphina atronitens from Archer (2001b); the ascospores of G. bylii are slightly larger (41–51 × 12–18 μm versus 30–45 × 10–15 μm) but the difference is not sufficient to treat them as separate species. The previously unrecognized presence of this genus in South Africa highlights biogeographical connections between southern Africa and Australasia (Almborn 1988; Galley & Linder 2006; McCarthy 2006). We have not seen any specimens other than the holotype, and the present status of this species in South Africa is therefore unknown.

Platythecium sp.

(Fig. 7B)

Remarks

The only specimen of Platythecium we have seen from South Africa could not be determined to species because mature spores were not seen and none of the TLC spots could be positively identified; secondary chemistry is important for species determination in this genus (Neuwirth et al. 2017). We include it here because this genus has not otherwise been reported from South Africa. In South Africa, Platythecium is probably restricted to the vegetation of the Indian Ocean Coastal Belt, which is a threatened vegetation type (Jewitt 2018) that lacks recent lichen sampling.

Specimen examined

South Africa: KwaZulu-Natal: ‘distr. Eshowe, rather dense and dark situation in the indigenous forest’, 26 viii 1929, O. A. Høeg s. n. (TRH L-17910).

Misapplied names

Allographa striatula (Ach.) Lücking & Kalb

In Kalb et al., Phytotaxa 377, 26 (2018).—Opegrapha striatula Ach., Syn. Meth. Lich. (Lund), 74 (1814).—Graphis striatula (Ach.) Spreng., Syst. Veg., Edn 16 4, 250 (1827); type: Guinea, s. col. (H-ACH 629—holotype, not seen).

Remarks

This species is known for South Africa only from Nylander (1868). Several specimens examined by Nylander are in the Armstrong collection at DBN. A small number lack spores and are undeterminable, while several others represent Graphis proserpens.

Graphis analoga Nyl.

Annls Sci. Nat., Bot., Sér. 4 11, 244 (1859); type: Tahiti, Viellard & Planchet G13:8 (H-NYL 7432—holotype, not seen).

Remarks

This species is known for South Africa only from Nylander (1868). The single specimen in the Armstrong collection at DBN is in very poor condition, but the excipulum is striate and completely carbonized. We could not find ascospores, although Nylander's notes on the specimen give spore dimensions of 46–80 μm × 18–30 μm. The excipulum characters and ascospore size contradict the description of this species provided by Lücking et al. (2009).

Graphis scripta (L.) Ach.

K. Vetensk-Acad. Nya Handl. 30, 145 (1809).—Lichen scriptus L., Spec. Plant. 2, 1140 (1753); type: Sweden, Malme s. n. [Lich. Suec. Exs. 47] (UPS—epitype, not seen).

Remarks

This is primarily a temperate, Northern Hemisphere species (Kraichak et al. 2015), although there are a small number of confirmed records from the Southern Hemisphere (Neuwirth & Aptroot 2011). Frequent records of this species in older literature (see Doidge (1950) and citations therein) date to an era when Graphidaceae biodiversity was poorly known. All South African specimens we have seen identified as Graphis scripta are misidentifications of other taxa.

Discussion

The species outlined above consist mostly of pantropical taxa, especially in Glyphis and Graphis s. str. Conversely, Allographa oldayana is a candidate southern African endemic, and Mangoldia bylii represents a biogeographical connection between South Africa and Australia. Several species known only from their type specimens (e.g. Graphis bylii var. lividula and Gr. denudans) will require additional study to resolve their taxonomy and biogeography.

The forest habitats preferred by many Graphidaceae species are a small proportion of the total land cover in South Africa (Fig. 1; Mucina & Geldenhuys 2006), and their natural patchiness might contribute to both the evolution of endemic species and the susceptibility to disturbance. More than one third of the species we treat in this paper are known from South Africa only from pre-1950s collections. Their current status in the country is uncertain given high levels of forest habitat loss, especially in the Indian Ocean Coastal Belt (Mucina & Geldenhuys 2006; Jewitt 2018).

In addition to the new species and records listed in this paper, we have seen many additional specimens that cannot clearly be assigned to a known species and several putative new species known only from a single specimen. For the time being, we refrain from describing these new species pending the availability of additional specimens and molecular data. The present paper is only a first step towards a modern understanding of lirelliform Graphidaceae in South Africa, and we hope it will be a starting point for more researchers to investigate taxonomic problems such as the Graphis pergracilis complex and the description of additional new species.

The separation of Allographa and Graphis s. str. (Lücking & Kalb 2018) resolved one of the broader systematic issues in Graphideae: the polyphyly of Graphis s. lat. (Berger et al. 2011; Rivas Plata et al. 2011). Two major problems remain. First, Thalloloma is a junior synonym of Diorygma, but the two genera have not yet been formally merged. When these genera were resurrected in the modern understanding of Graphidaceae (Staiger 2002; Kalb et al. 2004), they were recognized as close relatives. Molecular data now strongly support a single genus for this group (Fig. 3) but, to avoid excessive name changes, more molecular data from atypical species of Thalloloma are necessary before the genera are formally synonymized. Second, and a greater problem, phylogenetic relationships and generic boundaries among the genera with pigmented ascospores (Leiorreuma, Pallidogramme, Phaeographis, Platygramme, Sarcographa and Thecaria) are unresolved or not well supported (Fig. 3). This group has a complicated nomenclatural and taxonomic history (Staiger 2002; Lücking et al. 2007). Phaeographis has long been known to be polyphyletic (Rivas Plata et al. 2013), and our new data on Platygramme pachnodes suggests that Platygramme is also polyphyletic (Fig. 3). Genera in this clade have probably been oversplit; for example, there is much more sequence variation within Graphis or Allographa than there is among Leiorreuma, Pallidogramme and Thecaria (Fig. 3). We will address some of these taxonomic and nomenclatural issues in a subsequent paper on the South African species of Graphidaceae tribe Graphideae with pigmented ascospores, which will also include a complete key to Graphideae in South Africa.