See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/273766529 Taxonomic study of the genus Anzia (Lecanorales, lichenized Ascomycota) from Hengduan Mountains, China Article in The Lichenologist · March 2015 Impact Factor: 1.45 · DOI: 10.1017/S0024282914000644 READS 60 8 authors, including: Xinyu Wang Chinese Academy of Sciences 41 PUBLICATIONS 131 CITATIONS SEE PROFILE Bernard Goffinet University of Connecticut 155 PUBLICATIONS 2,761 CITATIONS SEE PROFILE Available from: Xinyu Wang Retrieved on: 03 June 2016 The Lichenologist 47(2): 99–115 (2015) r British Lichen Society, 2015 doi:10.1017/S0024282914000644 Taxonomic study of the genus Anzia (Lecanorales, lichenized Ascomycota) from Hengduan Mountains, China Xin Yu WANG, Bernard GOFFINET, Dong LIU, Meng Meng LIANG, Hai Xia SHI, Yan Yun ZHANG, Jun ZHANG and Li Song WANG Abstract: Analyses of morphological, anatomical, chemical and DNA sequences led to the recognition of ten species of Anzia in the Hengduan Mountains, which harbour all species known from China, including A. pseudocolpota sp.nov.andA. hypomelaena comb.&stat.nov.Furthermore,populations similar to A. hypoleucoides but with narrow lobes and a yellow-orange pigmented medulla may be a phylogenetically distinct species tentatively recognized as A. aff. hypoleucoides. The species are primarily distinguished by the presence or absence of a central axis, the colour and shape of the spongy cushion and the nature of the secondary compounds. A key to all known species of Anzia from China is presented. Key words: lichens, molecular phylogeny, Parmeliaceae, taxonomy, Yunnan Province Accepted for publication 7 November 2014 Introduction the asci containing eight spores and the yellow- green upper cortex (Darbishire 1912). Asahina Anzia Stizenb. (Parmeliaceae; Lumbsch & (1935) divided Anzia into three sections Huhndorf 2010; Thell et al. 2012) com- (Simplices, Duplices,andNervosae)basedon prises foliose lichens characterized by a the anatomy of the medulla. Section Nervosae brown-black or pale brown spongiostratum was identified by the presence of a central axis. (spongy cushion) covering the lower Sections Simplices and Duplices were defined by surface (Calvelo 1996), pale greyish white the presence of a single-layered or double- to greyish green narrow lobes, and asci layered medulla, respectively. Yoshimura with numerous crescent-shaped ascospores (1987) observed both of these medulla types (Stizenberger 1861). The genus has a rather within a single species (i.e. A. japonica)and cosmopolitan distribution, and includes c. therefore combined these two sections as 38 species, occurring primarily between section Anziae. 1000 and 4000 m in subtropical or temperate The circumscription of the genus and latitudes of both hemispheres. Anzia may geographical ranges of the species have be closely related to Pannoparmelia (Mull. ¨ been revised for Macaronesia (Haugan Arg.) Darb., which also has a spongy 1992), Asia (Yoshimura & Elix 1993; cushion on the lower surface, but differs by Yoshimura 1995; Yoshimura et al. 1997; Jayalal et al. 2012; Liang et al. 2012) and X. Y. Wang, D. Liu, M. M. Liang, Y. Y. Zhang South America (Calvelo 1996). Eight spe- and L. S. Wang (corresponding author): Key Labora- cies are currently recognized from mainland tory for Plant Diversity and Biogeography of East Asia, China (Wei 1991; Liang et al. 2012), mostly Kunming Institute of Botany, Chinese Academy of from Yunnan Province (Wu & Wang Sciences, Heilongtan, Kunming, Yunnan 650204, China. Email: [email protected] 1992; Wang 1995), but the diversity, varia- B. Goffinet: Department of Ecology and Evolutionary tion and hence boundaries of species have Biology, University of Connecticut, Storrs, CT 06269- not been comprehensively investigated. 3043, USA. Anzia leucobatoides (Nyl.) Zahlbr., for &QJ. H. X. Shi: Yunnan University of Traditional Chinese Medicine, Kunming, Yunnan 650500, China. example, was described based on a single J. Zhang: Yunnan Open University, Kunming, Yunnan collection from Yunnan and has not been 650223, China. collected since. 100 THE LICHENOLOGIST Vol. 47 Here we critically revise the genus Anzia 1000TM), with the following profile: initial denaturation in China based on a morphological and at 948C for 5 min, followed by 30 cycles, each of three chemical study of all collections held in steps (948Cfor1min,568Cfor1min,728Cfor1?5min), and a final extension at 728Cfor7min.ThePCR KUN, and on phylogenetic inferences from products were sanger-sequenced by Sangon Biotech. ITS sequences obtained for a set of exem- plars. We propose to recognize ten species, Phylogenetic analyses including A. pseudocolpota sp.nov.and All raw sequences were assembled and edited using A. hypomelaena comb. & st. nov., based on SeqMan (DNAstar packages). The newly generated ITS A. leucobatoides f. hypomelaena, and highlight sequences were complemented by sequences available in under A. aff. hypoleucoides a set of popula- GenBank. Sequences were aligned using MUSCLE v3.6 tions that are closely related to A. hypoleu- (Edgar 2004), and ambiguous regions were excluded using Gblocks (Talavera & Castresana 2007) with the coides but differ by their narrower lobes and default settings. Phylogenetic relationships were inferred their pigmented medulla, and which may using Bayesian Inference (BI) and under the Maximum constitute an additional species. A key to all Likelihood criterion (ML), with Pannoparmelia angustata Anzia taxa in China is provided. (Pers.) Zahlbr. selected as the outgroup. BI analyses were performed using MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003). The optimal substitution model implemented in MrBayes was deter- Materials and Methods mined based on the Akaike Information Criterion (AIC) using jModelTest 3.7 (Posada 2008). BI analyses were Morphological and chemical studies run for 1 000 000 Markov chain Monte Carlo (MCMC) Approximately 500 specimens were examined in the generations and 4 incrementally heated chains; MCMC Lichen Herbarium of the Kunming Institute of Botany started from a random tree and trees were sampled every (KUN-L), including recent collections from 2013 and 1000 generations, with the first 10% of trees discarded as 2014, made mostly in the Hengduan Mountains. Speci- burn-in. Posterior probabilities (PP) were obtained from mens were examined using standard microscopy techni- the 95% majority-rule consensus tree of all saved trees. ques and hand-cut sections under a NIKON SMZ 745 T ML analyses were performed with an online version dissecting microscope. Anatomical descriptions are based of RAxML v7.2.6 (Stamatakis 2006). All parameters in on observations of these preparations under a NIKON the ML analysis were set to their default setting, and Eclipse 50i microscope and photographs were taken statistical support values were estimated by 1000 non- using a NIKON digital camera head DS-Fi2. Size of the parametric bootstrapping pseudoreplicates. Bootstrap thallus, apothecia and lobes are based on measurements support values (MLBS) were obtained from the 70% for each specimen, and ascospore dimensions reflect ten majority-rule tree of all saved trees, using RAxML. measurements typically from a single apothecium per specimen. Size is in each case represented by the range between the smallest and largest single values. Results Secondary metabolites of all the specimens were Phylogenetic analyses identified using spot test and thin-layer chromatogra- phy as described by Elix et al. (1987), White & James Sixteen new ITS sequences were generated (1985) and Orange et al. (2001). Solvent C (toluene: acetic acid 5 85:15) was used for TLC analysis. and the final matrix included 25 samples of Anzia and the outgroup exemplar (Table 1). The most likely tree is composed of one DNA extraction well-supported (MLBS 5 100; PP 5 1?0) Total DNA was extracted from small fragments and one unsupported clade (Fig. 1). The of fresh thallus tips following the protocol by presence or absence of a central axis does not Ekman (1999) using Axyprep Multisource Genomic define a single clade wherein the character is DNA Miniprep Kit. The nrDNA ITS region conserved. All species producing lobaric (ITS1-5.8S-ITS2) was amplified by PCR using the acid, divaricatic or divaricatic and sekikaic ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) primers. Conditions previously described by acids form respective monophyletic lineages. Arup (2002) were used for PCR: 2 ml of genomic By contrast, species producing anziaic and DNA was added to the following mix: 2 ml of primer divaricatic acids compose a polyphyletic (1 ml for each primer of a 10 mM solution), 12?5 mlof group (Fig. 1). 23Taq PCR Mastermix (Aidlab) (containing Taq DNA Polymerase: 0?1 unit/ml; MgCl2:4mM;dNTPs: 0?4mM) and 8?5 mldH2O for a total volume of 25 ml. The three populations of a putative new Amplifications were carried out in a thermocycler (C species (i.e., A. pseudocolpota), similar to 2015 Anzia from Hengduan Mountains, China—Wang et al. 101 TABLE 1. Specimens used in this study, newly obtained sequences are in bold. Voucher GenBank accession Species name Locality Collector(s) Specimens number Anzia colpota S. Korea SO Oh, JS Hur & KM Lim Hur 041492 DQ068379 A. colpota S. Korea SO Oh, JS Hur & KM Lim Hur 040952 DQ068380 A. colpodes USA: Lumbsch 4.VI.04. UPS DQ980000 Tennessee A. aff. hypoleucoides China: LS Wang, XY Wang & JW Li 13–37933 KJ486586 Yunnan A. aff. hypoleucoides China: LS Wang, XY Wang & JW Li 13–37994 KJ486587 Yunnan A. aff. hypoleucoides China: LS Wang & XY Wang 13–38259 KJ486588 Yunnan A. aff. hypoleucoides China: LS Wang & XY Wang 13–37971 KJ486589 Yunnan A. flavotenuis Sri Lanka U. Jayalal, P. Wolseley et al. BM:1088238 JN873338 A. formosana China: A. Thell et al. TWN 267–666 AY251411 Taiwan A. hypoleucoides China: LS Wang & XY Wang 13–38280 KJ486575 Yunnan A. hypoleucoides S. Korea KM Lim, JS Hur & LS Wang Hur 040112 DQ001273 A. leucobatoides f. China: LS Wang & XY Wang 13–38008 KJ486574 hypomelaena Yunnan A. leucobatoides f. China: LS Wang & XY Wang 13–38273 KJ486576 hypomelaena Yunnan A. leucobatoides China: LS Wang & MM Liang 11–32320 KJ486584 Yunnan A.