Parmelia Barrenoae , a New Lichen Species Related to Parmelia Sulcata
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The Lichenologist 37(1): 37–46 (2005) 2005 The British Lichen Society DOI: 10.1017/S0024282904014641 Printed in the United Kingdom Parmelia barrenoae, a new lichen species related to Parmelia sulcata (Parmeliaceae) based on molecular and morphological data Pradeep K. DIVAKAR, M. Carmen MOLINA, H. Thorsten LUMBSCH and Ana CRESPO Abstract: Parmelia barrenoae is described as new to science in the P. sulcata complex on the basis of morphological and molecular data. The new species is superficially similar to P. sulcata but differs in having simple rhizines whereas the other species of the complex have squarrose rhizines. Nuclear ITS rDNA and partial -tubulin gene sequences have been used as molecular markers. In the phylogenetic analysis, P. sulcata falls into four well supported clades, one of them corresponds to the morphotype that is described here as a new taxon. Six samples of the new taxon from different locations on the Iberian Peninsula form a strongly supported monophyletic group. Key words: Ascomycota, Bayesian inference, lichens, Parmelia, Parmelia barrenoae, Parmeliaceae, rhizines Introduction Parmelia sulcata is a very well known lichen species (with squarrose rhizines and Phylogenetic studies of some widely distrib- sorediate upper surface), reported from all uted lichen species in the Parmeliaceae have continents, including Antarctica. It occurs in shown that in many cases the morphological numerous ecotypes and is one of the most species concept does not coincide with common species in temperate Europe. In a monophyletic unit. Some traditionally Europe it is one of the principal lichens circumscribed species have been shown to recolonizing cities where atmospheric pollu- include several genetically isolated clades, tion has recently decreased (Hawksworth i.e. phylogenetic species (Kroken & Taylor & McManus 1989; Crespo et al. 1999). 2001; Thell et al. 2002; Crespo et al. 2002; Preliminary studies on a worldwide basis Högnabba & Wedin 2003; Molina et al. 2004). These monophyletic groups either using molecular markers have reported an have been proposed to represent cryptic unexpectedly high genetic variability within species or have merely been described as the species (Crespo et al. 1997). distinct populations within a variable The molecular variability detected is species. accompanied by an unusual heterogeneity in rhizine morphology among samples of P. sulcata. Hale (1987: 48) described rhizine P. K. Divikar & A. Crespo (corresponding author): morphology as one of the key characters Departamento de Biología Vegetal II, Facultad de circumscribing P. sulcata: ‘‘The most im- Farmacia, Universidad Complutense, Madrid 28040, Spain. portant diagnostic characters of P. sulcata M. C. Molina: Departamento de Matemáticas y Física are the well–developed laminal soralia Aplicadas y Ciencias Naturales, Escuela Superior de and richly squarrosely branched rhizines.’’ Ciencias Experimentales y Tecnología, Universidad However, other descriptions of this species Rey Juan Carlos, Madrid 28933, Spain. H. T. Lumbsch: Department of Botany, The Field indicate that it has simple, furcate or squar- Museum, 1400 S. Lake Shore Drive, Chicago, IL rose rhizines (e.g. Purvis et al. 1992). This 60605, USA. may be interpreted as individual variation 38 THE LICHENOLOGIST Vol. 37 due to age or ecological conditions. How- sequence of the protein coding -tubulin gene. Fungal ever, it has been shown that thalli with nuITS rDNA was amplified using the primers ITS1F (Gardes & Bruns 1993), ITS4 (White et al. 1990), and rhizines of the squarrose type show a perpen- the protein coding -tubulin gene was amplified using dicular secondary ramification from early the primers Bt3-LM and Bt10-LM (Myllys et al. 2001). stages of thallus development (Molina et al. Amplifications were performed in 50 l volumes con- 2004). On the other hand, the furcate taining a reaction mixture of 5 lof10DNA polym- type of ramification does not form perpen- erase buffer (Biotools) (containing MgCl2 2 mM, dicular secondary branches, suggesting that 10 mM Tris-HCl, pH 8·0, 50 mM KCl, 1 mM EDTA, different rhizine-types are involved. 0·1% Triton X-100), 1 l of dinucleotide triphosphate (dNTPs), containing 10 mM of each base, 2·5 lof Contrary to their utility in other each primer (10 M), 1·25 l of DNA polymerase 1 parmelioid genera, rhizines have not been (1 U l ) and 27.5 ldH2O. Finally, 40 lofthis used as an important taxonomic character in mixture was added to 10 l of DNA of each sample. Parmelia, with the exception of recent com- The amplifications for ITS rDNA and the -tubulin bined molecular and morphological studies gene were carried out in an automatic thermocycler (Molina et al. 2004). Although the type of (Techne Progene) and performed using the following programmes: initial denaturation at 94(C for 5 min, rhizines may vary within monophyletic and 30 cycles of: 94(Cfor1min,54(C (ITS rDNA) or groups of this genus, it may still be taxo- 55–58(C(-tubulin) for 1 min, 72(C for 1·5 min, and nomically informative at specific level. The a final extension at 72(C for 5 min. objective of this work is to test the hypothesis The PCR products were subsequently purified using that rhizine morphology is a useful diagnos- the Bioclean Columns kit (Biotools) according to the tic character at the species level in Parmelia, manufacturer’s instructions. The purified PCR prod- ucts were sequenced using the same amplification and in particular to examine whether primers. The ABI Prism Dye Terminator Cycle samples of P. sulcata with simple rhizines Sequencing Ready reaction kit (Applied Biosystems) form a phylogenetically distinct group. Fresh was used and the following settings were carried out: material collected from different regions of denaturation for 3 min at 94(C and 25 cycles at: 96(C the Iberian Peninsula and other areas has for 10 sec, 50(C for 5 sec and 60( for 4 min. Sequenc- been studied using two molecular markers ing reactions were electrophoresed on a 3730 DNA (ITS and -tubulin sequences). analyzer (Applied Biosystems). Partial SSU rDNA, sometimes including an intron at the end of the 3# (SSU) were removed before the alignment. Sequence fragments obtained were assembled with SeqMan 4.03 (DNAStar) and manually adjusted. Material and Methods Taxon sampling Sequence alignments Thirty-one specimens representing 16 species of An alignment procedure employing a linear Hidden Parmelia s. str., were used in this study and sequence Markov Model (HMM) for the alignment, as imple- data from a part of the protein coding -tubulin mented in the software SAM (Karplus et al. 1998), has gene and nuITS rDNA were collected. Details of the been used. Sequences of 31 specimens (Table 1) were material, area of collection, and location of voucher aligned separately for the two genes. Regions that could specimens, are presented in Table 1. Specimens were not be aligned with statistical confidence were excluded air-dried at room temperature. Parmelia laevior, P. from the phylogenetic analysis. pseudolaevior and P. signifera were used as out-group. The out-group selection was based on the previous Phylogenetic analysis work of Molina et al. (2004). The alignment was analysed using the programs DNA extraction and PCR amplification PAUP* 4.0b10 (Swofford 2003) and MrBAYES 3.0 (Huelsenbeck & Ronquist 2001). Character polarity Small samples prepared from freshly collected and was assessed with outgroup comparison, using Parmelia frozen herbarium specimens were ground with sterile laevior, P. pseudolaevior and P. signifera as outgroup. glass pestles. Total genomic DNA was extracted using The data were analysed using a Bayesian approach the DNeasy Plant Mini Kit (Qiagen) according to the (Larget & Simon 1999; Huelsenbeck et al. 2000). manufacturer’s instructions with the slight modifica- Posterior probabilities were approximated by sampling tions described in Crespo et al. (2001). Dilutions of the trees using a Markov Chain Monte Carlo (MCMC) total DNA were used for PCR amplifications of the method. The posterior probabilities of each branch genes coding for the nuclear ITS rRNA and partial were calculated by counting the frequency of trees 2005 T 1. Specimens of Parmelia used in this study, with location, reference collection detail and GenBank accession numbers GenBank no. No. Species Locality and collector Voucher specimens ITS -tubulin 1 P. squarrosa Forge Creek, USA, 628 m, Cregler MAF 7293 AY036977* AY580308 2 P. squarrosa Parson, USA, 826 m, Cregler MAF 7270 AY036979* AY580309 3 P. sulcata Oerkened, Sweden Thell-Marth AF410840* AF410844* SK-9921 4 P. sulcata Ventorrillo (Madrid), Spain, 1500 m, Divakar MAF 9899 AY580313 AY580310 5 P. sulcata S. Gredos 1 (Ávila), Spain, 1300 m, Crespo MAF 9904 AY579445 AY579460 Parmelia barrenoae 6 P. sulcata Ventorrillo (Madrid), Spain, 1500 m, Divakar MAF 9901 AY579447 AY579462 7 P. sulcata Herbeset (Castellón), Spain, 1140 m, Crespo, Barreno & Divakar MAF 10153 AY579452 AY579466 8 P. sulcata Düsseldorf (Germany), Crespo MAF 10159 AY579454 AY579468 9 P. sulcata Düsseldorf (Germany), Crespo MAF 10155 AY579453 AY579467 10 P. barrenoae La Barranca (Madrid), Spain, 1400 m, Crespo MAF 9750 AY295103* AY295111* 11 P. barrenoae La Barranca (Madrid), Spain, 1400 m, Crespo & Divakar MAF 10151 AY579444 AY579459 12 P. barrenoae S. Gredos 2 (Ávila), Spain, 1300 m, Crespo MAF 9906 AY579446 AY579461 13 P. barrenoae Las Médulas (León), Spain, 850 m, Divakar MAF 9905 AY579448 AY579463 14 P. barrenoae Marvào (Sao Mamede), Portugal, 900 m, Crespo MAF 9900 AY579450 AY579464 — 15 P. barrenoae Herbeset (Castellón), Spain, 1140 m, Barreno, Crespo & Divakar MAF 10154 AY579451 AY579465 Divakar 16 P. sulcata Herbeset (Castellón), Spain, 1140 m, Barreno, Crespo & Divakar MAF 10152 AY579455 AY579469 17 P. sulcata Herbes (Castellón), Spain, 1000 m, Barreno, Crespo & Divakar MAF 10157 AY579456 AY579470 18 P. sulcata Caceres, Spain, 280 m, Crespo et al. MAF 9902 AY579449 — 19 P. fertilis Hokkaido (Tokyo), Japan, 700 m, Hamada MAF 7282 AY036982* AF391143* et al. 39 20 P. cochleata Hokkaido (Tokyo), Japan, 700 m, Harada MAF 7280 AY036985* — 21 P. pinnatifida Kola Peninsula, Russia, Schlensog MAF 7274 AY036987* AF391134* 22 P.