Cladonia verticillata ( Cladoniaceae , Ascomycota ) , new record to Iberian Peninsula

Pino-Bodas, R.; Martín, M. P.; Stenroos, S. & Burgaz, A. R. 2013. Cladonia verticillata (Cladoniaceae, Ascomycota), new record to Iberian Peninsula. Bot. Complut. 37: 21-25. The identity of a putative collection of Cladonia verticillata is assessed by means of ITS rDNA region. The phylogenetic analyses confirmed that this specimen belongs to C. verticillata, and this species is reported as new to Iberian Peninsula.


INTRODUCTION
Cladonia verticillata (Hoffm.)Schaer. is a broadly distributed species, found in Europe, Asia, America and Australasia (Ahti 2007).It is characterized by its scyphose, corticate podetia that present proliferations growing from the scyphus center.Not long ago it was commonly considered as a subspecies of C. cervicornis (Ach.)Flot.However, van Herk & Aptroot (2003) deemed that the morphological differences between them were enough to raise C. verticillata to the species rank.This taxonomical change has been accepted by most of the authors (Ahti 2007, Burgaz & Ahti 2009).
Though C. verticillata had been previously reported from the Iberian Peninsula (Navás 1904, Tavares 1950, Seaward 1983, Valcárcel et al. 1993), the recent studies on Cladoniaceae in this area (Burgaz & Ahti 1998, 2009) excluded the species from the area, indicating that all the samples identified as C. verticillata were in fact specimens of C. cervicornis.During our studies of Cladonia in the Iberian Peninsula, we collected a sample morphologically resembling C. verticillata.The aim of this study was to clarify the identity of this sample of Cladonia by means of ITS rDNA region.
Molecular analyses.The DNeasy Plant Mini Kit (Quiagen) was used to extract DNA, according to the manufacturer's instructions.The primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) were used to amplify the ITS rDNA region.PCRs were carried out with Ready-to-Go-PCR Beads (GE Healthcare Life Sciences, UK).The volume of reaction was 25 Ìl for each tube, with 0.4 mM final concentration of primers.The amplification program was 94 ºC for 5 min; 5 cycles of 94 ºC for 30 s, 54 ºC for 30 s and 72 ºC for 1 min; and 33 cycles of 94 ºC for 30 s, 48 ºC for 30 s and 72 ºC for 1 min; with a final extension of 72 ºC for 10 min.PCR products were purified with ExoSAP-IT (USB Corporation, OH, USA) and sequencing was carried out in Macrogen (South Korea) service (www.macrogen.com).The sequences were edited using Sequencher™ (Gene Codes, Ann Arbor, MI, USA).The alignment was done manually, using SE-AL v2.0 (Rambaut 2002) program.Maximun Parsimony (MP) analysis was done in PAUP 4.0b10 (Swofford 2003), using heuristic searches with 100 random taxon-addition replicates with TBR branch swapping and MulTrees option in effect, equally weighted characters and gaps treated as missing data.Bootstrap analysis was used with 1000 replicates and the heuristic option.The ML analyses were implemented in RAxML 7.04 (Stamatakis 2006), assuming the GTRGAMMA model.The nucleotide substitution model was choosen using MrModeltest (Nylander 2004).SYM+G was the best-fitting evolutionary model according to AIC criterion.The Bayesian analysis was carried out using MrBayes 3.1.2(Huelsenbeck & Ronquist 2001).The posterior probabilities were approximated by sampling trees using Markov Chain Monte Carlo (MCMC).Two simultaneous runs with 10.000.000generations, each starting with a random tree and employing 4 simultaneous chains were executed.Every 1,000th tree was saved into a file.Tracer v. 1.0 (http://evolve.zoo.ox.ac.uk/ software.html?id=tracer) was used to determine when the chains reached the stationary stage and to assess the number of generations that should be discarded as burn-in.The 50% majority-rule consensus tree was calculated using MrBayes "sumt" command, deleting the first 2.000.000generations.
The hypothesis that the sample identified as C. verticillata forms a monophyletic group with C. cervicornis samples was tested with Shimodaira-Hasegawa (SH) (Shimodaira and Hasegawa 1999) and the expected likelihood weight (ELW) tests (Strimmer & Rambaut 2002), implemented in Tree-PUZZLE 5.2 (Schmidt et al. 2002).

RESULTS AND DISCUSSION
In this study, four new sequences of ITS rDNA region have been generated (Table 1).The ITS alignment contained 565 unambiguous characters, 84 of which were parsimony-informative and 413 were constant.The MP analysis generated 81 equally parsimonious trees, 240 steps long, with CI = 0.7167 and RC = 0.5515.ML analysis generated a tree with likelihook value of LnL = -2126.72,while Bayesian analysis likelihook was LnL = -2158.97.The phylogenetic trees yielded in the three analyses had the same topology and only the 50% majority-rule consensus tree of the Bayesian analysis is shown (Fig. 1).
The specimen identified as C. verticillata from Spain gathers together with other samples of C. verticillata from Europe and North America, in a well supported clade.All the samples of C. verticillata except one from Canada joined in this clade.The hypothesis tests rejected that the putative specimen of C. verticillata from Spain belongs to C. cervicornis (SH, P-value = 0.01; ELW, P-value = 0.0013).The phylogenetic analyses give evidence that C. verticillata is present in the Iberian Peninsula.This species is morphologically closely related to C. cervicornis.In fact, it is difficult to tell them apart (Ahti 1980), particularly in Europe.Even their secondary metabolites are the same, viz. the fumarprotocetraric acid complex.Traditionally, the main characters used to differentiate them were that C. verticillata has slenderer podetia and more proliferatiing tiers than C. cervicornis (Poelt & Vûzda 1977, Wirth 1995, Clauzade & Roux 1985).However, the study by van Herk & Aptroot (2003) revealed that the podetia of both taxa are often indistinguishable in the European populations, though they can be separated by the primary thallus characters.Cladonia verticillata has small squamules with white lower surface, while C. cervicornis has big and deeply incised squamules with grey to pink lower surface.The specimen assessed here has squamules 3.5-6 mm long, with white lower surface (Fig. 2), and podetia 16-40 mm tall, with up to three successive proliferations.All these characters are consistent with those given by van Herk & Aptroot (2003).In the Mediterranean region, C. verticillata s. str.was probably first reported in Italy (Nimis 1993), where it is more common in the North, in the Alps, but it was found in some localities in the south of Italy (Puglia, Basilicata and Sicilia) as well.
Other well supported clades appeared on the phylogenetic tree, such as those of C. cervicornis, C. pulvinata (Sandst.)Herk & Aptroot, and two specimens of C. rappii A. Evans (not monophyletic).These results are similar to those given by Stenroos et al. (2002) and Pino-Bodas et al. (2010).Ahti (2007) indicated that the status of several species in the C. verticillata group is uncertain and a deep worldwide study is necessary to clarify the limits among the species.The core species of this group, C. verticillata, probably hides more than one species.In fact, in our analyses one sample of this species did not form a group with the other samples.Further genetic studies will be necessary to verify the genetic homogeneity, or lack of it, in the populations of C. verticillata.

Table 1
List of taxa included in this study with the respective sampling localities, collections numbers and GenBank accession numbers.In bold, the sequences generated in this study Fig. 1-Phylogeny of the Cladonia verticillata group.50% Majority Rule Bayesian tree based on ITS rDNA region.Branches supported with posterior probability ≥ 0.95 and bootstrap > 70% are indicated in bold.Bootstrap for MP/Bootstrap for ML/posterior probability for Bayesian analysis at branches.Botanica Complutensis 37: 21-25.2013