Two new species of Lecanora sensu stricto ( Lecanoraceae , Ascomycota ) from east Africa

The new sorediate species Lecanora kenyana from Mount Kenya and L. orientoafricana from the Rift Valley in Kenya are described. L. kenyana has red-brown apothecia with a constricted base, a melacarpella–type amphithecium, pulicaris–type epihymenium, a hyaline hypothecium, and contains usnic acid as major constituent. L. orientoafricana is characterized by having a dark hypothecium, pulicaris-type amphithecium, chlarotera-epihymenium, and contains atranorin and gangaleoidin. A phylogenetic analysis using maximum likelihood and a Bayesian approach based on DNA sequence data of mtSSU and ITS rDNA support that both new species belong to Lecanora sensu stricto and cluster with species containing usnic acid or having a dark hypothecium, respectively.


Introduction
Lecanora is the major genus of Lecanoraceae (Lumbsch and Huhndorf 2010) and includes crustose (incl.placodioid) lichens with hyaline, usually non-septate ascospores, Lecanora-type asci and mostly lecanorine apothecia.The morphological and chemical diversity is large in this heterogeneous genus and molecular data have supported that the genus as currently circumscribed is not monophyletic (Arup and Grube 1998;2000;Grube et al. 2004;Lumbsch 2002).The core group of Lecanora sensu stricto is characterized by the presence of calcium oxalate crystals in the amphithecium, filiform conidia, and the presence of atranorin and/or usnic acid.This agrees with an extend-ed circumscription of the Lecanora subfusca group to include taxa containing usnic acid and a dark hypothecium (Guderley 1999;Lumbsch 1995;Lumbsch et al. 1995;Lumbsch et al. 1996;Lumbsch et al. 2003;Papong et al. in press).African species of Lecanora sensu stricto are poorly known but our recent studies resulted in the description of a new species and new records of Lecanora species for Kenya (Kirika et al. in press;Lumbsch et al. 2011).Among the collections from the Mount Kenya area and the Rift Valley we found two corticolous species, one sorediate taxon with usnic acid and a morphologically somewhat similar species with a dark hypothecium.The two taxa do not agree with known species (Lumbsch 1995;Lumbsch et al. 1995;Lumbsch et al. 1996;Papong et al. 2011;Papong and Lumbsch 2011) and consequently are described as new.To confirm the placement of the new species in Lecanora sensu stricto, we also generated DNA sequence data of the internal transcribed spacer region (ITS) and partial sequences of the small subunit of the mitochondrial ribosomal DNA (mtSSU) and performed a phylogenetic analysis with sequences available in Genbank.

Taxon sampling and molecular methods
The study is based on material deposited in EA and F and DNA sequences downloaded from Genbank.Sequences of five Ramboldia spp.were included as outgroup since the genus has been shown previously to be related to Lecanora (Kalb et al. 2008).Sequence data of the two new species were assembled with sequences of the mitochondrial small subunit (mtSSU) and nuclear ITS rDNA downloaded from Genbank (Table 1).Sample preparation, DNA isolation, PCR and direct sequencing were performed as described previously (Mangold et al. 2008;Wirtz et al. 2012).Primers for amplification were: mr SSU1 (Zoller et al. 1999) and MSU7 (Zhou and Stanosz 2001) for mtSSU and ITS1F and ITS4 (Gardes and Bruns 1993) for ITS rDNA.Sequence fragments obtained were assembled with SeqMan 4.03 (DNASTAR) and manually adjusted.

Sequence alignments and phylogenetic analysis
Alignments were done using Clustal W (Thompson et al. 1994).Ambiguously aligned regions were removed manually.The single locus and concatenated alignments were analyzed by maximum likelihood (ML) and a Bayesian approach (B/MCMC).To test for potential conflict, ML bootstrap analyses were performed on the individual data sets, and 75% bootstrap consensus trees were examined for conflict (Lutzoni et al. 2004).Maximum likelihood analyses were performed using the program GARLI (Zwickl 2006), employing the general time reversible model of nucleotide substitution (Rodriguez et al. 1990), including estimation of invariant sites, and assuming a discrete gamma distribution with six rate categories.Bootstrapping (Felsenstein 1985) was performed based on 2000 replicates.The B/MCMC analysis was conducted on the concatenated data set using MrBayes 3.1.2(Huelsenbeck and Ronquist 2001), with the same substitution model as in the ML analysis.The dataset was portioned into the two parts (mtSSU, ITS) and each partition was allowed to have own parameters (Nylander et al. 2004).A run with 20,000,000 generations, starting with a random tree and employing 4 simultaneous chains, was executed.Every 100 th tree was saved into a file.The first 500,000 generations (i.e. the first 5000 trees) were deleted as the "burn in" of the chain.We used AWTY (Nylander et al. 2007) to compare splits frequencies in the different runs and to plot cumulative split frequencies to ensure that equilibrium was reached.Of the remaining trees a majority rule consensus tree with average branch lengths was calculated using the sumt option of MrBayes.Posterior probabilities were obtained for each clade.Only clades that received bootstrap support equal or above 70% under ML and posterior probabilities ≥ 0.95 were considered as strongly supported.Phylogenetic trees were visualized using the program Treeview (Page 1996).

Anatomical and chemical studies
Anatomical studies were conducted using standard light microscopy on hand-cut sections mounted in water.Secondary lichen substances were identified by high performance thinlayer chromatography (HPTLC) according to the standard methods (Arup et al. 1993).

Data resources
The data underpinning the analyses reported in this paper are deposited in the Dryad Data Repository at doi: 10.10.5061/dryad.b1068.
Etymology.The new species is named after the country Kenya where the new species has been found.
Etymology.The new species is named after the area East Africa where it has been collected.
Notes.Lecanora orientoafricana is characterized by the presence of granular soredia, sparsely pruinose, brown apothecia, a pulicaris-type amphithecium, chlarotera-type epihymenium, dark hypothecium, broadly ellipsoid ascospores, and the presence of atranorin and gangaleoidin.Soredia are rare among Lecanora sensu stricto species with a dark hypothecium.Some specimens of L. coronulans are sorediate, but this species is readily distinguished by epruinose apothecial discs, an egranulose epihymenium, and the presence of protoconstipatic acid and zeorin and major constituents in addition to atranorin (Lumbsch et al. 1996).Similar esorediate species include L. egranulosa and L. phaeocardia.The latter differs from L. orientoafricana in having epruinose apothecial discs, a thinner amphithecial cortex, and alternative chemistry.Lecanora egranulosa is readily distinguished by darker, epruinose apothecial discs, an indistinct, thin amphithecial cortex, small crystals in the epihymenium, shorter ascospores, and the presence of zeorin (Lumbsch et al. 1996).
Ecology and distribution.This new species is currently only known from the type locality in the Rift Valley province of Kenya, where it was found growing on bark in a degraded montane forest dominated by Podocarpus falcatus, Rapanea melanophloes and Faurea saligna at an altitude of 3240m.Associated species included Sphaerophorus melanocarpus, Pannaria cf.rubiginosa, and Ramalina spp.

Phylogenetic study
Four new sequences were generated for this study and aligned with sequences downloaded from Genbank (Table 1).The single gene locus did not show any conflicts and hence the concatenated data set was analyzed.Our combined data set included 820 unambiguously aligned positions, 174 of which were constant.The ML tree had a likelihood value of -3718.083and in the B/MCMC analysis of the combined data set, the likelihood parameters in the sample had the following mean (Variance): LnL = -3794.172(0.21).The ML tree and the tree from the B/MCMC tree sampling were almost identical with no differences in well-supported clades.Thus, only the ML tree is shown here (Fig. 3).In our analysis, species of the genus Lecanora form a strongly supported monophyletic group as in a previously published study (Papong et al. in press).Since Papong et al. (in press) discussed the relationships of the different Lecanora groups in detail, these discussions are not reiterated here, but we focus only on the relationships of the two newly described species here.Lecanora kenyana clusters strongly supported with L. ulrikii and L. wilsonii, two species which also contain usnic acid (Lumbsch et al. 1995;Papong et al. 2011;Papong et al. in press;Papong and Lumbsch 2011).Lecanora orientoafricana is sister to L. flavoviridis, which also has a dark hypothecium (Lumbsch et al. 1996;Papong et al. in press).The molecular data support the placement of the new species in Lecanora sensu stricto.However, given the few sequences available from tropical Lecanora in Genbank, the molecular data cannot be used to confirm that the species have indeed not been described previously.We conclude that they are new based on our database of known Lecanora species and our examinations of type material of Lecanora spp.over more than 24 years.

Figure 3 .
Figure 3. Phylogenetic placement of the two new Lecanora species as inferred from a concatenated alignment of mtSSU and ITS DNA sequences.This is the optimal tree under maximum likelihood.Branches in bold received likelihood bootstrap support values above 70%, and posterior probabilities equal or above 0.95