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Research Article
Phylogeny, morphology and chemistry reveal two new multispored species in the Lecanora subfusca group (Lecanoraceae, Ascomycota)
expand article infoLijuan Li‡§, Yanyun Zhang|, Christian Printzen§
‡ Goethe University Frankfurt, Frankfurt am Main, Germany
§ Senckenberg Research Institute and Natural History Museum, Frankfurt am Main, Germany
| Anhui Normal University, Wuhu, China

Corresponding author: Lijuan Li ( lijuan.li@senckenberg.de )

Academic editor: Thorsten Lumbsch
© 2023 Lijuan Li, Yanyun Zhang, Christian Printzen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Li L, Zhang Y, Printzen C (2023) Phylogeny, morphology and chemistry reveal two new multispored species in the Lecanora subfusca group (Lecanoraceae, Ascomycota). MycoKeys 99: 25-43. https://doi.org/10.3897/mycokeys.99.108462
Open Access

Abstract

Two new multispored species from China, Lecanora anhuiensis Li J. Li & Printzen, sp. nov. and Lecanora pseudojaponica Li J. Li & Printzen, sp. nov. are described and illustrated here, based on morphological, chemical and molecular evidence. Lecanora anhuiensis is characterised by an epruinose, yellowish-brown to deep brown apothecial disc, an epihymenium with fine crystals, an amphithecium with small crystals, 16-spored asci and the presence of zeorin, in addition to atranorin. Lecanora pseudojaponica is characterised by an epruinose, red-brown apothecial disc, an epihymenium without crystals, an amphithecium with small crystals, 8 or 16- spored asci and the presence of zeorin and the stictic acid complex, in addition to atranorin. Phylogenetic reconstructions, based on mtSSU, nrITS and nrLSU suggest that these two species are members of the Lecanora subfusca group. They are compared with morphologically similar and phylogenetically related species, based on a nrITS dataset. Phylogenetic results show that the multispored taxa of Lecanora are polyphyletic. The number of ascospores per ascus appears to be a taxonomic character of minor importance. Detailed descriptions, discussions and figures for the two new species from China and a key for the multispored species of Lecanora worldwide are provided.

Key words

Ascospores, China, identification key, Lecanora, lichen, taxonomy

Introduction

Lecanora Ach. is one of the largest genera of lichens, including species with lecanorine apothecia, Lecanora-type asci and simple, hyaline ascospores. The majority of species produce eight ascospores per ascus. Multispored species with more than eight spores per ascus are relatively rare amongst Lecanora. To date, only 14 species have been reported worldwide: L. cateilea (Ach.) A. Massal., L. bruneri Imshaug & Brodo, L. loekoesii L. Lü, Y. Joshi & Hur, L. moniliformis L. Qiu & L. Lü, L. pleospora Müll. Arg., L. praesistens Nyl., L. weii L.F. Han & S.Y. Guo, L. japonica Müll. Arg., L. subjaponica L. Lü & H.Y. Wang, L. subpraesistens Nayaka, Upreti & Lumbsch, L. shangrilaensis Z.T. Zhao & L. Lü, L. strobilinoides Giralt & Gómez-Bolea, L. polysphaeridia Alstrup and L. sambuci (Pers.) Nyl. [= Polyozosia sambuci (Pers.) S.Y.Kondr., Lőkös & Farkas] (Miyawaki 1988; Alstrup 1993; Guderley and Lumbsch 1999; Nayaka et al. 2006; Han et al. 2009; Lü et al. 2011, 2012; Lü and Zhao 2017; Brodo et al. 2019; Qiu and Lü 2022).

As a result of systematic revisions and phylogenetic studies, several genera or species groups have been segregated from or within Lecanora sensu lato (s.lat.) (Kalb 1991; Rodriguez-Flakus and Printzen 2014; Zhao et al. 2016; Kondratyuk et al. 2019; Davydov et al. 2021). In this context, the attribution of multispored species to these groups has often been discussed, indicating that the multispored species are polyphyletic within Lecanora s.lat. However, a more comprehensive overview of the species is lacking so far.

Lecanora cateilea and L. bruneri were assigned to the L. albella group in the broad sense since they have pruinose apothecial discs and a poorly-developed amphithecium cortex (Lumbsch et al. 1997; Brodo et al. 2019). Lecanora polysphaeridia belongs to the L. fuscescens group, based on its Biatora-type ascus and globose ascospores (Alstrup 1993; Øvstedal et al. 2020). ‘Lecanora’ sambuci was combined into Polyozosia A. Massal. (= Myriolecis Clem., Lecanora dispersa group), because it conforms with the general circumscription of the genus having a more or less immersed thallus, small apothecia with brown discs and pale margins and no lichen substances (Laundon 2003a; Śliwa 2007; Zhao et.al 2016; Kondratyuk et al. 2019). Lecanora strobilinoides was identified as a member of the Lecanora varia group on the basis of phenotypic characters and closely related to L. strobilina (Giralt and Gómez-Bolea 1991; Laundon and Rodney 2003). Laundon and Rodney (2003), on the other hand, considered L. strobilinoides to be a geographical race of L. strobilina and suggested a new combination L. strobilina subsp. strobilinoides (Giralt & Gómez-Bolea) J.R.Laundon, since they only differed by ascospore size and number. Subsequently Pérez-Ortega and Kantvilas (2018) confirmed the species status of the two taxa and their position within the L. symmicta group by phylogenetic analysis. Lecanora weii and L. shangrilaensis are only known from China so far and their phylogenetic position within Lecanora is not mentioned in the original descriptions. It might be assumed that L. weii is a member of the L. albella group since it has heavily pruinose apothecial discs and produces atranorin (Han et al. 2009), while L. shangrilaensis might belong to the L. varia group, because of the presence of usnic acid instead of atranorin, the yellowish apothecia with prominent margin and its preferred substrate, pine cones (Laundon 2003a; Laundon and Rodney 2003; Lü and Zhao 2017).

As they produce atranorin and large calcium oxalate crystals in the amphithecium, L. pleospora, L. praesistens and L. subpraesistens have been identified as typical members of the L. subfusca group (Brodo 1984; Lumbsch 1994). These species mainly differ by the presence or absence of crystals in the epihymenium and chemical traits (Guderley and Lumbsch 1999; Nayaka et al. 2006). In addition, L. japonica, L. loekoesii, L. moniliformis and L. subjaponica have been described as members of the L. subfusca group, since they all have small oxalate crystals in the amphithecium and also produce atranorin (Miyawaki 1988; Guderley and Lumbsch 1999; Wang et al. 2007; Lü et al. 2011; Lü and Zhao 2017; Qiu and Lü 2022).

These systematic attributions were almost all based on phenotypical characters because genetic data are lacking for most of the species. In addition to discussing the phylogenetic position of the multispored species, it is also interesting to study whether the number of ascospores per ascus is a useful character for species delimitation. Since Lecanora japonica contains 8-spored and multispored asci, Guderley and Lumbsch (1999) considered that the number of ascospores is of minor taxonomic significance in the L. subfusca group (Miyawaki 1988). The same is true for L. cateilea, containing both 8-spored and multispored asci and several other taxa with ‘(8–)12 –16’-spored asci (Guderley and Lumbsch 1999; Nayaka et al. 2006; Brodo et al. 2019; Qiu and Lü 2022).

While studying the species diversity of the L. subfusca group in China, within the ongoing project ‘Lecanomics’ (https://lecanomics.org), two multispored taxa, consistent with the general circumscription of the L. subfusca group, came to our attention. One of them contains both 8-spored and multispored asci. A phylogenetic analysis, based on molecular data from multiple collections, indicated that both taxa are so far undescribed and we describe them below in detail. By including all available molecular data of multispored taxa in Lecanora s.lat., we attempted to confirm the phylogenetic affinity of these and the newly-described species to genera or species groups within Lecanoraceae. In addition, we also tested whether the number of ascospores may be considered a distinguishing feature for species (or genera) within Lecanoraceae.

Materials and methods

Phenotypic studies

The specimens in this study are deposited in Anhui Normal University (AHUB), Herbarium Mycologicum Academiae Sinicae-Lichenes (HMAS-L) and Lichen Herbarium Kunming Institute of Botany (KUN-L).

We took macrophotographs using a Zeiss Axio Zoom V16. External morphological characters were studied on air-dried material under a stereomicroscope (Zeiss Stemi SV11). Anatomical features were studied using a light microscope (Zeiss Axioskop 2 plus) on transverse sections of apothecia and thalli, cut with a freezing microtome (Zeiss HYRAX KS 34) to 16–20 µm thickness and mounted in water or lactophenol cotton blue. Spore measurements are presented in the following way: (minimum–) x̄ - SD – x̄ – x̄ + SD (–maximum), where x̄ is the arithmetic mean and SD is the standard deviation (values were rounded to the nearest 0.5 µm), followed by the number of measurements (n). Crystals in apothecia were observed in polarized light (POL), their solubility was studied in 20% nitric acid (HNO3) (N) and 10% potassium hydroxide (KOH) (K), N-sol/K-sol means crystals dissolved, N-insol/K-insol means they did not dissolve.

One of the goals of this study was to investigate whether samples with 8-spored and multispored asci or species that produce both kinds of asci belonged to different species. Therefore, chemical and molecular data were generated from apothecia after verifying the number of ascospores on hand-cut sections.

Spot tests were conducted using K and a saturated aqueous solution of sodium hypochlorite (NaClO) (C). High-performance thin layer chromatography (HPTLC) was performed in solvents A, B′ and C to identify lichen chemical compounds, following standardised methods (Culberson and Kristinsson 1970; Arup et al. 1993).

DNA extraction, PCR and sequencing

Apothecia were cleaned with acetone before DNA extraction. DNA was extracted using the GeneOn Plant DNA Extraction Kit (GeneOn BioTech, China) by the magnetic bead method or the Chelex® 100 Resin (Bio-Rad, USA) method following Ferencová et al. (2017). The fungal internal transcribed spacer (ITS) region of the rDNA was amplified via polymerase chain reaction (PCR) using the primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). The large subunit of the nuclear ribosomal DNA (LSU) was amplified using the primers AL2R (Mangold et al. 2008) and LR6 (Vilgalys and Hester 1990) and the mitochondrial small subunit (mtSSU) of ribosomal RNA using the primers 16F and 972R (Li et al. 2023). PCR amplifications were performed in 25 µl volumes Ready-To-Go PCR Beads (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) containing 5 µl of DNA extract and 1 µl of each primer. Cycling conditions included initial denaturation at 94 °C for 5 min, followed by 4 cycles of 94 °C for 30 s, 54 °C (53 °C for mtSSU) for 45 s and 72 °C for 60 s, 30 cycles of 94 °C for 30 s, 48 °C for 30 s and 72 °C for 60 s and a final extension at 72 °C for 10 min. The PCR products were visualised on 1% agarose gels and sequenced by Macrogen Europe (Amsterdam, The Netherlands) with the same primers as the original PCR amplifications.

Phylogenetic analyses

A mtSSU-nrITS-nrLSU concatenated dataset and an nrITS dataset with Protoparmelia badia (Hoffm.) Hafellner and P. picea (Hoffm.) Hafellner as outgroup were used for this study, respectively (Zhao et al. 2016). First, each locus was aligned and analysed separately. Sequences were assembled and edited in Geneious Prime 2021.0.3 (https://www.geneious.com/). Each gene dataset was aligned using the MAFFT v.7 online service (https://mafft.cbrc.jp/alignment/server/index.html) and GUIDANCE2 web server (http://guidance.tau.ac.il/) to remove poorly- or ambiguously-aligned regions with the default parameter settings. Before concatenating the single-gene datasets, these were tested for potential incongruencies using the online version of IQ-TREE (Trifinopoulos et al. 2016, http://iqtree.cibiv.univie.ac.at/) with 1000 ultrafast bootstrap replicates. No well-supported conflict was detected.

A Maximum Likelihood (ML) phylogenetic tree with simultaneous inference of the optimal partitioning scheme and substitution models for each data partition was inferred using IQ-TREE, suggesting five initial partitions (mtSSU, ITS1, 5.8S, ITS2, nrLSU). The best-fit model for each partition was selected according to the Bayesian Information Criterion (BIC) as follows: TPM2u+F+R3 for mtSSU, TIMe+G4 for ITS1, TNe+G4 for 5.8S and ITS2 and TNe+I+G for nrLSU. The Branch support was assessed using both ultrafast bootstrap approximation (UFBoot) (Minh et al. 2013) with 10000 replicates and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010) with 1000 replicates. Nodes with support values of both UFBoot ≥ 95% and SH-aLRT ≥ 80% were considered well-supported (Minh et al. 2013). Bayesian reconstructions of phylogenies were performed with MrBayes 3.2.6 (Ronquist et al. 2012) to infer phylogenetic trees applying the models inferred by IQ-TREE and slightly simplified as: HKY+F+I+G4 for mtSSU, SYM+G4 for ITS1, K2P+G4 for 5.8S and ITS2, K2P+I+G for nrLSU. All model parameters were unlinked amongst partitions and we used the default distributions for priors. Two parallel runs of four Markov chains each were run for 4 million generations, sampling every 1000th generation and the first 25% discarded as burn-in. The average standard deviation of split frequencies had fallen below 0.01 at the end of the analysis. Tracer v.1.7 (Rambaut et al. 2018) was used to assess chain convergence by checking the effective sampling size (ESS > 200) for all model parameters. Bayesian posterior probabilities ≥ 0.95, UFBoot ≥ 95% and SH-aLRT ≥ 80% were visualised on the ML tree.

The nrITS dataset was analysed based on Maximum Likelihood (ML) using IQ-TREE with automated substitution model selection with three partitions (ITS1, 5.8S and ITS2). The best-fit models were selected as TIMe+G4 for ITS1, TIM2e+I+G4 for 5.8S and TNe+G4 for ITS2. Branch support was assessed using both ultrafast bootstrap approximation (UFBoot) with 10000 replicates and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) with 1000 replicates. UFBoot ≥ 95% and SH-aLRT ≥ 80% are given on the tree and the editing and annotation are in Microsoft PowerPoint.

Results and discussion

The mtSSU-nrITS-nrLSU concatenated alignment comprised 61 terminals (Table 1) and included all multispored species with available sequences, as well as species to which they were attributed in previous publications. It also comprised several other major genera and species groups within Lecanoraceae, to figure out the phylogenetic positions of the multispored taxa. The nrITS alignment comprised 34 terminals, including all sequences from the L. subfusca group and outgroup shown in Fig. 1, as well as the newly-generated sequences (Fig. 2). Phenotypical characters are mapped on to the ML tree to highlight the significance of the different numbers of ascospores and other distinguishing characters.

Figure 1. 

Phylogenetic tree generated from Maximum Likelihood (ML) analysis, based on combined mtSSU, nrITS and nrLSU sequences. SH-aLRT support (%) ≥ 80 / ultrafast bootstrap support (%) ≥ 95 / Bayesian posterior probabilities (PPs) ≥ 0.95 are given above the nodes. Newly-generated sequences are indicated in bold. Multispored species are indicated by black triangles.

Figure 2. 

Phylogenetic tree generated from Maximum Likelihood (ML) analysis of the Lecanora subfusca group, emphasising multispored taxa, based on nrITS sequences. SH-aLRT support (%) ≥ 80 / ultrafast bootstrap support (%) ≥ 95 are given above the nodes. Newly-generated sequences are indicated in bold. Phenotypical characters are mapped next to the tree, solid grey rectangles indicate the presence of corresponding features: 1, multispored asci; 2, apothecial amphithecium with large crystals; 3, amphithecium with small crystals; 4, epihymenium without crystals; 5, epihymenium with fine crystals; 6, epihymenium with coarse crystals; 7, thallus with soredia; 8, atranorin; 9, usnic acid; 10, zeorin; 11, stictic acid; 12, norstictic acid.

Table 1.
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Specimens used for the phylogenetic analyses with the corresponding voucher information and GenBank Accession numbers for mtSSU, nrITS and nrLSU sequences. Newly-obtained sequences in this study are in bold. Species names are followed by their Lecanomics IDs, referring to the project of “Lecanomics” (https://lecanomics.org). “na” indicates that there is no sequence available.

Species name Voucher details Country GenBank Accession number
mtSSU nrITS nrLSU
Adelolecia pilati Ekman 3373 (BG) Austria AY567713 MG925949 AY300826
Bryonora castanea Westberg PAD321 (UPS) Sweden OM417201 OM423658 OM423613
B. castaneoides Svensson 3156 (UPS) Sweden OM417202 OM423659 OM423614
Carbonea vitellinaria Svensson 3963 Sweden MZ468129 MZ474888 na
C. vorticosa Tuerk 43031 Antarctica na JN873869 na
Frutidella caesioatra Andersen 91 (BG) Norway AY567765 na AY756349
F. furfuracea Vondrák 26120 (PRA) Czech Republic OQ682951 OQ717391 na
Japewia subaurifera Spribille & Wagner s.n. (GZU) USA na JN009716 KR017230
J. tornoensis Printzen s.n. (hb. BG) Canada HQ660559 HQ650656 na
Lecanora albella Malíček 7336 (hb. JM) Czech Republic KY502423 KY548048 na
L. allophana Malíček 9626 (hb. JM) Russia KY502421 KY548050 na
L. anhuiensis L1454 Ren 20200748 (HMAS-L) China OR096240 OR098679 OR096274
L. anuiensis L1456 Ren 20200731 (HMAS-L) China OR096242 OR098678 OR096275
L. pseudojaponica L1424 Yao 20200919 (HMAS-L) China OR096248 OR098686 OR096277
L. pseudojaponica L1464 Yao 20200932 (HMAS-L) China OR096246 OR098687 OR096276
L. argopholis Printzen 12558 (FR) Austria MH520108 MH512978 MW257122
L. baekdudaeganensis B.G.Lee 2019-000065 (BDNA) South Korea MN879871 MN879847 na
L. baekdudaeganensis L1461 Zhang 20200762 (HMAS-L) China OR096239 OR098693 OR096273
L. caesiorubella Lumbsch 19094a (F) USA JQ782666 JN943722 JN939506
L. campestris Arup & Grube 2000 (hb. Arup) Sweden na AF159930 na
L. carpinea Kondratyuk 21337 (KW) Ukraine MK693683 MK672827 na
L. cateilea Goward & Poelt (GZU) Canada na AY541250 na
L. conizaeoides Palice 21292 (PRA) Czech Republic MT939177 MT938947 na
L. excludens Palice 21929 (PRA) Norway MK541649 MK541647 na
L. glabrata Arup L011003 (LD) Sweden DQ787360 na DQ787359
L. hybocarpa Lumbsch s.n. (F) Spain EF105417 EF105412 EF105421
L. imshaugii Lumbsch 19273b (F) USA JQ782681 JQ782717 na
L. intricata Flakus 29565b (KRAM) Bolivia OL604112 OL604030 OL663890
L. intumescens Malíček 8480 (hb. JM) Czech Republic KY502441 KY548040 na
L. layana 0 Lendemer 37519 (NY) USA KR094857 KR094859 na
L. layana 1 Lendemer 38131 (NY) USA KR094858 KR094860 na
L. loekoesii L1476 Wei et al. HLJ201400311 (HMAS-L) China OR096237 OR098694 na
L. paramerae Lumbsch s.n. (F) Spain EF105418 EF105413 EF105422
L. polytropa Flakus 29524 (KRAM) Bolivia OL604125 OL604045 OL663904
L. praesistens LIFU083-16 (WSL) Switzerland na KX132991 na
L. pulicaris Malíček 10262 (hb. JM) Russia MK778539 MK778611 na
L. rupicola Flakus 29527(KRAM) Bolivia OL604094 OL604012 OL663876
L. saligna Palice 21284 (PRA) Czech Republic MT939225 MT938996 na
L. strobilinoides Garrido-Benavent s.n. (MA) Spain na MG973238 na
L. subintricata Printzen 15562 (FR) Japan MT939239 MT939010 na
L. symmicta Davydov 18083 (hb. Davydov) Russia ON553202 ON553209 na
L. varia Kondratyuk 21325 (KW) Ukraine MK693694 MK672852 na
Lecidella patavina ZX 20140501-2 China KT453845 KT453767 KT453799
L. stigmatea ZX 20141254 China KT453852 KT453758 KT453808
Miriquidica gyrizans Fryday 10175 (MSC) USA MN508282 MN483126 MN460217
M. leucophaea Kossowska 1354 (hb. Kossowska) Thailand KP822516 KP822311 KP796397
Palicella_ filamentosa Hauck s. n. (FH) Germany HQ660568 HQ650663 HQ660543
P. glaucopa Flakus 2539 (FR) Argentina KJ152471 KJ152486 KJ152460
Polyozosia altunica Xahidin 20071910 (XJU) China MH698407 MH698406 MH698407
P. contractula Brodo 31501(DUKE) USA DQ986898 HQ650604 DQ986746
P. sambuci BIOUG24047-E06 Canada na KT695378 na
Protoparmelia badia Fryday 8575 USA KY012807 KY066254 KY066280
P. picea Haugan 9612 (O) Norway na KF562194 KF562186
Protoparmeliopsis garovaglii 0 Wang et al. 19-63467 (KUN-L) China ON807176 ON807160 na
P. garovaglii 1 Leavitt 089 (BRY-C) USA KT453818 KT453728 KT453775
Pulvinora pringlei McCune 36799 (OSC & ALTB) USA MW257153 MW257114 MW257114
P. stereothallina Davydov 14817 (LE & ALTB) Russia MW257159 MW257118 MW257118
Pyrrhospora petraeoides Elix 36816 (F) EU075531 EU075531 EU075545 EU075521
P. sanguinolenta Elix 28835 (F) Australia EU075534 EU075548 EU075523
Rhizoplaca chrysoleuca BRY 55000 USA KT453856 HM577233 KT453812
R. pachyphylla Wang et al. 18-59561 (KUN-L) China MN192154 MK778050 na

The three-loci phylogenetic tree (Fig. 1) shows that the species of the L. subfusca group form a well-supported monophyletic clade (SH-aLRT = 96.5%, UFBoot = 95%, PP = 0.99), within which two well-supported branches correspond to the two new multispored species, L. anhuiensis (SH-aLRT = 99.7%, UFBoot = 100%, PP = 1.00) and L. pseudojaponica (SH-aLRT = 98.3%, UFBoot = 100%, PP = 1.00). The two new species show a sister group relationship (SH-aLRT = 84.5%, UFBoot = 99%), both sharing the characteristics of multispored asci, small POL+ crystals in the amphithecium, the major production of atranorin and zeorin, characters in agreement with typical members of the L. subfusca group. Lecanora imshaugii with 8-spored asci, reported from Eastern Asian and eastern North America (Miyawaki 1988, 1994), is the most closely-related species (SH-aLRT = 96.7%, UFBoot = 100%, PP = 1.00). Another multispored species, L. loekoesii, is closely related to the sorediate L. layana. These two species form a group with L. baekdudaeganensis reported from South Korea. All these species form a strongly supported clade (SH-aLRT = 100%, UFBoot = 100%, PP = 1.00) nested within the L. subfusca group. The multispored L. praesistens also belongs to the L. subfusca group and appears to be closely related to L. pulicaris (SH-aLRT = 97.5%, UFBoot = 100%, PP = 1.00), with which it shares red-brown apothecial discs and large crystals in the amphithecium, but differs by its multispored asci and an epihymenium with coarse crystals (Guderley and Lumbsch 1999). Lecanora cateilea and L. intumescens form a strongly supported clade (SH-aLRT = 97%, UFBoot = 100%, PP = 1.00). Together with the closely-related L. excludens, they produce zeorin as a constant compound in addition to atranorin. The multispored L. strobilinoides clusters with L. symmicta (SH-aLRT = 100%, UFBoot = 100%, PP = 1.00), in accordance with previous results showing it to be a member of the L. symmicta group with (12–)16(–32)-spored asci (Pérez-Ortega and Kantvilas 2018).

Another major clade (UFBoot = 99.9%, SH-aLRT = 100%, PP = 0.99) combines species belonging to Carbonea, the L. polytropa-, L. saligna- and L. varia groups, Polyozosia, Protoparmeliopsis and Rhizoplaca. These genera and species groups conform largely to the ‘MPRPS’ clade (Medeiros et al. 2021) which, however, also comprised Bryonora, Carbonea and the L. varia group, but were not included in the analysis of Medeiros et al. (2021). The multispored Polyozosia sambuci forms a clade with P. altunica and P. contractula (SH-aLRT = 100%, UFBoot = 100%, PP = 1.00).

The majority of multispored Lecanora species has previously been classified into different genera or species groups, based on phenotypic characters. Our results provide phylogenetic evidence for these assignments. The affinities of multispored taxa in Lecanora s.lat. are primarily supported by other phenotypical characters, such as epihymenium and amphithecium characteristics, as well as chemical compounds, rather than the numbers of ascospores.

In the nrITS tree (Fig. 2), the multiple sequences of the two new species, L. anhuiensis and L. pseudojaponica, formed well-supported clades, respectively. The overall topology of other related species in the tree is consistent with our concatenated tree. Significant phenotypical characters for the species are depicted next to the phylogenetic tree. In the new species L. pseudojaponica, we observed the occurrence of apothecia containing both 8-spored and multispored asci growing intermixed on the same thallus (samples L1424 and L1425). We sequenced these apothecia separately (labelled as P1 = multispored and P2 = 8-spored). The results revealed that both types of apothecia had identical sequences where they overlapped (616 bp). This is further support that the number of ascospores is of minor taxonomic significance within the L. subfusca group (Guderley and Lumbsch 1999).

Taxonomy

Lecanora anhuiensis Lijuan Li & Printzen, sp. nov.

MycoBank No: 849250
Fig. 3A–E

Diagnosis

Distinguished from other multispored species of Lecanora by brown apothecial discs, fine crystals in the epihymenium, small crystals in the amphithecium and the presence of atranorin and zeorin.

Type

China. Anhui Prov.: Lu’an Ci., Jinzhai Co., The main peak of the Tiantangzhai, Ta-pieh Mountain, 31°06′20″N, 115°46′15″E, alt. 1720 m, on bark, 12 Oct 2020, Ren Qiang 20200731, HMAS-L-0147383—holotype.

Description

Thallus corticolous, continuous to rimose to verrucose areolate, thin and tightly attached to the substrate, surface dirty grey to greenish, epruinose, lacking soredia, prothallus not visible.

Apothecia lecanorine, numerous, rounded or deformed by mutual pressure, dispersed to aggregated, sessile to adnate, 0.4–1 mm in diameter; disc plane to slightly concave or convex, yellowish-brown to deep brown, epruinose, margin persistent and prominent, entire or slightly flexuous, cream-white; amphithecium with numerous algal cells, containing small crystals (POL+, K-insol, N-sol); cortex indistinct, interspersed with fine crystals (POL+, K-sol, N-insol); parathecium colourless, 15–25 (–40) µm wide, with fine crystals (POL+, K-sol, N-insol) mostly in the uppermost part; epihymenium with fine crystals (POL+, K-sol, N-insol) on the surface and interspersed to upper part of paraphyses and amongst the apical cells, with deep orangish-brown to deep brown amorphous pigmentation, becoming faint dull brown or dissolving in K; hymenium colourless, 80–110 µm high; paraphyses simple to somewhat branched, ca. 1.5–2 µm thick, tips expanded up to 4 µm; hypothecium colorless, composed of anastomosing hyphae; asci clavate, Lecanora-type, 55–70 × 15–25 µm, 16-spored; ascospores simple, hyaline, narrowly ellipsoid to ellipsoid or ovoid, occasionally subglobose, (9.0–)11.0–12.0–13.5(–15.0) × (5.0–)5.5–6.0–7.0(–8.0) µm (n = 74), wall ca. 0.5 µm. Pycnidia not found.

Figure 3. 

The new species Lecanora anhuiensis A lichen thallus and apothecia, habit B vertical sections of apothecia in normal light C vertical sections of apothecia in polarized light D 16-spored ascus E ascospores. Scale bars: 1 mm (A); 20 µm (B, C); 10 µm (D, E).

Chemistry

Thallus K+ yellow, C-; containing atranorin and zeorin.

Distribution

This species occurs on bark and is known from Anhui Prov., in the south-eastern part of the Ta-pieh Mountains at elevations between 850 and 1720 m. The Ta-pieh Mountains are located at the junction between Anhui, Hubei and Henan Provinces in China.

Etymology

The species is named after its locality in Anhui Province, China.

Notes

Lecanora loekoesii is similar to L. anhuiensis in having somewhat yellowish-brown apothecial discs, a granulose epihymenium and small crystals in the amphithcium, but differs in having relatively larger ascospores (12.1–)12.6–15.3(–16.2) × (7–)7.5–8.5(–9) µm in size and producing usnic and norstictic acid in addition to atranorin and zeorin [according to Lü et al. (2011)]. In the species delimitation of the L. subfusca group, the type of epihymenial crystals is one of the most important diagnostic characters, as illustrated by Brodo (1984). The original description mentioned that the epihymeniun of L. loekoesii contains fine crystals. Subsequently, Wang et al. (2013) examined the holotype and 68 Chinese specimens, suggesting that the crystals are, in fact, coarse. Both types can be distinguished by examining their solubility in N: fine crystals are insoluble in N, while coarse crystals dissolve in N (Brodo 1984). We found that the epihymenial crystals are insoluble in N, indicating the presence of fine crystals, consistent with Lü et al. (2011) and Qiu and Lü (2022).

Lecanora shangrilaensis, with yellow to yellowish-brown apothecial discs, a granulose epihymenium and small crystals in the amphithecium, might also be confused with L. anhuiensis. However, it can be easily distinguished by the presence of coarse epihymenial crystals, K-soluble crystals in the amphithecium and the production of usnic acid instead of atranorin. Lecanora weii is also similar to L. anhuiensis in forming a granulose epihymenium, an amphithecium with K-insoluble small crystals and the presence of atranorin and zeorin, but differs in having heavily pruinose apothecial discs, an epihymenium with coarse crystals (K-sol, N-sol) and 12–16-spored asci (Han et al. 2009).

Additional specimens examined

China: Anhui Prov.: Lu’an Ci., Jinzhai Co., The main peak of the Tiantangzhai, Ta-pieh Mountain, 31°6′20″N, 115°46′15″E, alt. 1720 m, on bark, 12 Oct 2020, Ren Qiang 20200748a (HMAS-L-0147384); Anqing Ci., Yuexi Co., Yangtianwo, Yaoluoping National Nature Reserve, 31°58′11″N, 116°04′10″E, alt. 1160 m, on bark, 15 Oct 2020, Yao Zongting 20200911b (HMAS-L-0147400); Lu′an Ci., Jinzhai Co., JiangjunYan of the TiantangZhai, 31°12′26″N, 115°76′61″E, alt. 1501 m, on bark of Rhododendron, 19 Sep 2022, Zhang Yanyun 22-956 (AHUB-00810); Anqing Ci., Yuexi Co., Yangtianwo, Luchai River, 31°03′94″N, 116°11′38″E, alt. 850 m, on the bark of Pine, 20 Sep 2022, Zhang Yanyun 22-985 (AHUB-00839); Anqing Ci., Qianshan Co., Xianren Cave, Tianzhu Mountain World Geopark, 30°74′50″N, 116°45′30″E, alt. 1377 m, on bark, 21 Sep 2022, Zhang Yanyun 22-1026 (AHUB-00880); Anqing Ci., Qianshan Co., Qixing Chi, Tianzhu Mountain World Geopark, 30°74′50″N, 116°46′07″E, alt. 1250 m, on the bark of an oak tree, 21 Sep 2022, Zhang Yanyun 22-1040 (AHUB-00894).

Lecanora pseudojaponica Lijuan Li & Printzen, sp. nov.

MycoBank No: 849251
Fig. 4A–G

Diagnosis

Distinguished from other species of Lecanora by the red brown apothecial discs, the 16- and 8-spored asci, an epihymenium without crystals, small crystals in the amphithecium and the presence of atranorin, zeorin and the stictic acid complex.

Type

China, Anhui Prov.: Yuexi Co., Yangtianwo, Yaoluoping National Nature Reserve, 31°58′11″N, 116°4′10″E, alt. 1160 m, on bark, 15 Oct 2020, Yao Zongting 20200919, HMAS-L-0147402—holotype.

Description

Thallus corticolous, continuous to rimose, thin and tightly attached to the substrate, surface pale green to dull greenish-grey, epruinose, lacking soredia, prothallus black or not visible.

Apothecia lecanorine, numerous, rounded, dispersed to aggregated, sessile to adnate, 0.3–1 mm in diameter; disc plane or moderately concave, yellowish brown to reddish-brown, weakly shiny, epruinose, margin persistent and prominent, entire, cream white or greyish-white; amphithecium with numerous algal cells, small crystals (POL+, K-insol, N-sol); cortex indistinct, interspersed with fine crystals (POL+, K-sol, N-insol); parathecium colourless, 15–20 µm thick, without crystals (POL-); epihymenium without crystals (POL-), with orangish-brown to deep brown amorphous pigmentation, 10–20 µm high, not altered by K (sometimes becoming slightly more dark brown), orange intensifying in N, occasionally topped by a layer of hyaline gel ca. 5 µm thick; hymenium colourless, 60–100 µm high; paraphyses with few anastomoses, weakly branched, ca. 1.5 µm thick, tips expanded to 4 µm with an orangish-brown cap; hypothecium colourless, composed of anastomosing hyphae; asci clavate to narrowly clavate, Lecanora-type, 50–65 × 20–25 µm, 8- and 16-spored; ascospores simple, hyaline, ellipsoid to ovoid, (11.0–)13.0–14.5–16.5(–18) × (6.0–)5.5–6.5–8.0(–9.0) µm (n = 153), wall ca. 0.5 µm. Pycnidia not found.

Figure 4. 

The new species Lecanora pseudojaponica A lichen thallus and apothecia, habit B vertical sections of apothecia in polarized light C vertical sections of apothecia mounted in lactophenol cotton blue in normal light D 16-spored ascus E 8-spored ascus F ascospores in 8-spored asci G ascospores in 16-spored asci. Scale bars: 1 mm (A); 20 µm (B, C); 10 µm (D, E, F, G).

Chemistry

Thallus K+ yellow, C-; containing atranorin, zeorin and the stictic acid complex.

Distribution

This species occurs on bark at similar localities as L. anhuiensis in Anhui Province at elevations between 1160 and 1720 m.

Etymology

The specific epithet refers to the similar species L. japonica.

Notes

In our collections, we observed the presence of apothecia containing 8-spored asci as well as others containing 16-spored asci, growing mixed and distributed randomly on the same thallus. On rare occasions, 8-spored asci have been also been observed in the apothecia containing 16-spored asci. In order to rule out the possibility that we were actually observing two species growing intermixed, we sequenced both types of apothecia separately. The phylogeny (Fig. 2) showed no genetic differences between these apothecia. The only phenotypic difference seems to be that ascospores in 8-spored asci are, on average, larger than those in 16-spored asci, although both have a similar size range. It might be confused with other species of the L. subfusca group, especially if only 8-spored asci are found in the hymenium, but it is readily distinguished by its unique chemistry.

Three multispored species from the L. subfusca group previously reported from China, L. japonica, L. subjaponica and L. moniliformis, are morphologically similar to L. pseudojaponica in having red-brown apothecia, an epihymenium without crystals and small crystals in the amphithcium. Lecanora japonica differs by the lack of lichen substances other than atranorin (Miyawaki 1988), L. subjaponica contains (16–) 32-spored asci and lacks stictic acid (Lü et al. 2012) and L. moniliformis has crenate apothecial margins and produces psoromic acid (Qiu and Lü 2022). Lecanora subpraesistens is another multispored species with an egranulose epihymenium, but it can be distinguished from L. pseudojaponica by large crystals in the amphithecium, slightly larger apothecia (0.5–1.5 mm) and the absence of stictic acid (Nayaka et al. 2006).

Additional specimens examined

China: Anhui Prov.: Anqing Ci., Yuexi Co., Yangtianwo, Yaoluoping National Nature Reserve, 31°58′11″N, 116°4′10″E, alt. 1160 m, on bark, 15 Oct 2020, Yao Zongting 20200915 (HMAS-L-0147401), Yao Zongting 20200911 (HMAS-L-0147400), Yao Zongting 20200932 (HMAS-L-0147405); Lu’an Ci., Jinzhai Co., the main peak of the Tiantangzhai Scenic Area, Da-pie Mountain, 31°06′20″N, 115°46′15″E, alt. 1720 m, on bark, 12 Oct 2020, Ren Qiang 20200751 (HMAS-L-0147385); Lu′an Ci., Jinzhai Co., Waterfalls area of the Tiantangzhai Scenic Area, 31°12′26″N, 115°76′67″E, alt. 1492 m, on bark, 19 Sep 2022, Zhang Yanyun 22-959 (AHUB-00813); Lu′an Ci., Jinzhai Co., Waterfalls area of the Tiantangzhai Scenic Area, 31°12′27″N, 115°76′69″E, alt. 1490 m, on oak bark, 19 Sep 2022, Zhang Yanyun 22-961 (AHUB-00815); Lu′an Ci., Huoshan Co., Baimajian in the Main Scenic Area of Ta-pieh Mountain, 31°11′45″N, 116°17′95″E, alt. 1459 m, on bark, 09 Sep 2021, Zhang Yanyun 21-124 (AHUB-01027).

Material of additional species examined

Lecanora baekdudaeganensis: China. Anhui Prov.: Anqing Ci., Yuexi Co., 31°10′16″N, 115°35′35″E, alt. 770 m, on bark, 13 Oct 2020, Zhang Jiarong 20200766 L1452 (HMAS-L-0147386), Zhang Jiarong 20200762 (HMAS-L-0147387).

Lecanora cateilea: China. Yunnan Prov.: Diqing Tibetan Autonomous Prefecture, Baima Snow Mt., 27°24′00″N, 98°56′99″E, alt. 4100 m, on stump, 23 Oct 2003, Wang Lisong et al. 03-22910 (KUN-L).

Lecanora loekoesii: China. Shaanxi Prov.: Baoji Ci., Taibai Mt., 33°54′20″N, 107°47′99″E, alt. 2200 m, on Betula bark, 2014, Wang Lisong et al. 14-45264 (KUN-L-47212); Heilongjiang Prov.: Heihe Ci., Sunwu Co., 49°38′99″N, 127°17′59″E, alt. 335 m, on bark, 24 Aug 2014, Wei Xinli et al. HLJ201400311 (HMAS-L-0131277); Yichun Ci., Hongxing Co., 49°48′01″N, 127°25′34″E, alt. 280 m, on bark, 26 Aug 2014, Wei Xinli et al. HLJ201400640 (HMAS-L-0131305); Yichun Ci., Fenglin Co., 49°46′01″N, 127°25′20″E, alt. 262 m, on bark, 27 Aug 2014, Wei Xinli et al. HLJ201400853 (HMAS-L-0131321).

Lecanora subjaponica: China. Yunnan Prov.: Diqing Tibetan Autonomous Prefecture, Baima Snow Mt., 27°24′00″N, 98°56′99″E, alt. 4100 m, on bark, 23 Oct 2003, Wang Lisong et al. 03-22905 (KUN-L); Xizang Prov.: Lizhi Ci., 29°43′99″N, 94°43′99″E, alt. 3131 m, on bark, 19 Aug 2004, Huang Manrong 1662 (HMAS-L-0148671).

Key to the multispored species of Lecanora and similar genera with lecanorine apothecia and multispored asci

1 Asci Fuscidea-type, 32–200-spored Maronea (Fuscideaceae)
– Asci Lecanora-type or Biatora-type 2
2 Asci Biatora-type, ascospores globose, 4.5–5.5 µm diam., asci 24–32-spored, only known from the type locality in Greenland at an elevation of 20 m Lecanora polysphaeridia
– Asci Lecanora-type 3
3 Ascospores narrowly ellipsoid to fusiform to elongate, asci 8–100-spored 4
– Ascospores ellipsoid, asci 8–32-spored 5
4 Paraphyses branched and anastomosing, asci 8–64-spored, containing depsidones Neoprotoparmelia (Parmeliaceae)
– Paraphyses slender and mostly simple, asci 32–100-spored, containing depsides Maronina (Parmeliaceae)
5 Apothecial discs epruinose or occasionally slightly pruinose 6
– Apothecial discs pruinose 17
6 Thallus K+ yellow 7
– Thallus K- 15
7 Amphithecium with large crystals 8
– Amphithecium with small crystals 9
8 Epihymenium without crystals (POL-), with red-brown pigmentation not altered by K, asci 12–16-spored, only known from the type locality in northern India, at elevations between 2500 and 2800 m Lecanora subpraesistens
– Epihymenium with crystals (POL+, K-sol) 10
9 Epihymenium with fine crystals (POL+, K-sol, N-insol) 11
– Epihymenium without crystals (POL-) 12
10 Apothecia sessile, 0.3–0.7 mm diam., discs red-orange, epihymenium yellowish-brown, asci 8–(16)-spored, only known from the type locality in Kenya at elevations between 1500 and 2000 m Lecanora pleospora
– Apothecia sessile to subimmersed, 0.5–1.6 mm diam., discs red-brown to blackish-orange, epihymenium reddish-brown to yellowish-brown, asci (8–)12–16-spored, known from different parts of the Alps and Ukraine at elevations between 900 and 2000 m Lecanora praesistens
11 Apothecial discs yellowish-brown, epruinose or slightly pruinose, amphithecium with small crystals (K-sol), asci 16-spored, producing atranorin, zeorin, usnic and norstictic acid, known from China, South Korea and the Russian Far East, at elevations between 150 and 2900 m Lecanora loekoesii
– Apothecial discs yellowish-brown to deep brown, amphithecium with small crystals (K-insol), asci 16-spored, producing atranorin and zeorin, known from the east of China at elevations between 850 and 1720 m Lecanora anhuiensis
12 Asci (16–)32-spored, ascospores 7.5–12.5 × 4.0–6.0 μm, apothecia 0.5–1.6 mm diam., discs shiny, apothecial margin entire, producing zeorin, only known from China at elevations between 2400 and 3800 m Lecanora subjaponica
– Asci 8–16 spored, apothecia usually smaller than 1 mm diam 13
13 Apothecial discs plane to convex, margin crenate, asci (8–)12–16-spored, producing atranorin and psoromic acid, only known from China at elevations between 1300 and 1700 m Lecanora moniliformis
– Apothecial discs plane to concave, margin entire or slightly flexuous, asci 8- and 16-spored 14
14 Apothecia crowded, only with atranorin, known from Japan and China at elevations between 70 and 2700 m Lecanora japonica
– Apothecia dispersed to aggregated, with zeorin and stictic acid complex in addition to atranorin, known from China at elevations between 1300 and 1700 m Lecanora pseudojaponica
15 Apothecia disc red brown to black brown, asci 16(–32)-spored, no lichen products, known from Europe and North America Polyozosia sambuci
– Apothecia disc yellow brown or brown, asci 12–16(–32)-spored, containing usnic acid 16
16 Apothecia 0.2–0.5 mm diam., disc yellowish, epruinose, asci 12–16-spored, ascospores simple, with fumarprotocetraric acid besides usnic acid, only known from Yunnan Province in south-western China at elevations of 3500 m Lecanora shangrilaensis
– Apothecia 0.5–1 mm diam., disc brown, usually slightly pruinose, asci (12–)16(–32)-spored, ascospores simple or 1-septate, with zeorin besides usnic acid, known from north-eastern Spain at elevations between 25 and 700 m Lecanora strobilinoides
17 Apothecial section P-, discs red brown to dark brown, with slightly to heavily pruinose, asci 12–16-spored, only known from the type locality in north-eastern China at the elevation between 350 and 400 m Lecanora weii
– Apothecial section P+ yellow 18
18 Apothecia densely clustered, discs yellowish-brown to orange-brown, with heavily whitish-grey pruina, asci (8–)12(–14)-spored, known from Northern Hemisphere Lecanora cateilea
– Apothecia scattered, discs red brown, with heavily bluish-grey pruina, asci 12–16-spored, known from the type locality in Mexico and China Lecanora bruneri

Acknowledgements

We thank the Herbaria AHUB, HAM-L and KUN-L for the loan of specimens, Heike Kappes from the Grunelius-Möllgaard-Lab of the Senckenberg Research Institute for technical support.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work benefited from the sharing of expertise within the DFG priority programme SPP 1991 ‘Taxon-Omics’, as well as financial support from DFG grant PR 567/19–1 to CP, the Anhui Provincial Education Department (No. 2022AH050207) to YZ and the China Scholarship Council to LL.

Author contributions

LL conducted the lab work, analyzed the data, and wrote the draft. YZ provided partial data and samples, and revised the manuscript. CP supervised the research, revised the manuscript, and provided funding.

Author ORCIDs

Lijuan Li https://orcid.org/0000-0003-1048-1971

Yanyun Zhang https://orcid.org/0000-0002-0902-5066

Christian Printzen https://orcid.org/0000-0002-0871-0803

Data availability

All of the data that support the findings of this study are available in the main text.

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