Molecular data support placement of Cameronia in Ostropomycetidae (Lecanoromycetes, Ascomycota)

The phylogenetic position of the Tasmanian endemic genus Cameronia Kantvilas is studied using partial sequences of nuclear LSU and mitochondrial SSU ribosomal DNA. Monophyly of the genus is supported, as is its placement in Ostropomycetidae, although its position within this subclass remains uncertain. Given the lack of close relatives to Cameronia and its morphological differences compared to other families with perithecioid ascomata in Ostropomycetidae, the new family Cameroniaceae Kantvilas & Lumbsch is proposed.


Introduction
The lichen flora of Tasmania has a remarkable number of unique species, as well as several genera that are unknown or very rarely found in other regions.Examples include the genera Jarmania Kantvilas (Kantvilas 1996), Meridianelia Kantvilas & Lumbsch (Kantvilas and Lumbsch 2009), Siphulella Kantvilas, Elix & P. James (Kantvilas et al. 1992), Tasmidella Kantvilas, Hafellner & Elix (Kantvilas et al. 1999), and several species of Cladia (Kantvilas andElix 1987,1999) and thelotremoid Graphidaceae (Kantvilas and Vezda 2000;Mangold et al. 2009).In general, endemism can be either the result of survival of relict taxa (palaeoendemism) or recent speciation events (neoendemism) (Brandley et al. 2010;Brooks et al. 2006;Goldberg et al. 2005;Jans-sen et al. 2008;Kier et al. 2009;Kraft et al. 2010;Lamoreux et al. 2006;Olson et al. 2001;Qian 2001).The reasons for the relatively large amount of endemic taxa in Tasmania are not well understood.In the genus Cladia, for example, molecular data are consistent with a recent speciation and suggest neoendemism (Lumbsch et al. 2010;Parnmen 2011), but for most endemic taxa there are currently insufficient data available to test whether they represent relict lineages or are the product of recent speciation events.In some cases, however, lichens that were believed to be endemic to Tasmania, were subsequently also discovered in New Zealand, e.g.Bunodophoron flaccidum (Wedin 1993;Wedin 2001).
Lichen taxa unique to Tasmania include the genus Cameronia (Kantvilas 2012), which was recently described with an unclear systematic position and placed tentatively in Ostropomycetidae.The genus includes two species that occur on siliceous rocks at high elevations.Although its thallus is superficially similar to that of a species of Lecanora or Pertusaria, the genus is readily distinguished by the presence of eumuriform ascospores in thick-walled, broadly obovate, hemiamyloid asci with a non-amyloid tholus, formed in a hamathecium consisting of richly branched, anastomosing paraphysoids.The ascomata are perithecioid.Secondary metabolites present in the genus include the 9-O-methylpannaric acid chemosyndrome and an unknown triphenyl.
Thick-walled asci having a hemiamyloid wall and non-amyloid tholus, anastomosing paraphysoids and muriform ascospores are all characters reminiscent of Arthoniales (Ertz and Tehler 2011;Grube 1998;Tehler 1990), but the perithecioid ascomata, chlorococcoid photobiont, and morphological details of the ascus differ from this order (Kantvilas 2012).Perithecioid ascomata and thick-walled asci in a hamathecium consisting of anastomosing paraphysoids are characteristic for Protothelenellaceae and Thelenellaceae in Ostropomycetidae (Fryday and Coppins 2004;Mayrhofer 1987a,b;Mayrhofer and Poelt 1985;Schmitt et al. 2005).However, these families differ in having cylindrical asci and, furthermore, Thelenellaceae lacks any amyloid reactions of the asci, whereas Protothelenellaceae have an amyloid tholus.Because phenotypic characters do not place Cameronia in any group unambiguously and the placement in Ostropomycetidae was tentative, we used freshly collected material of the two species of Cameronia to generate DNA sequences of two loci (mtSSU and nuLSU rDNA) to test the monophyly of Cameronia and its placement of Cameronia in Ostropomycetidae, and to identify the closest relatives of the genus and place it in a family.

Taxon sampling and molecular methods
The study is based on fresh material collected by GK and deposited in the Tasmanian Herbarium (HO) and the Field Museum of Natural History (F), and on DNA sequences downloaded from Genbank.Sequences of Umbilicariaceae were included as outgroup since this family has been shown previously to be sister to Lecanoromycetida table 1.Sequences obtained from Genbank for the study.Family or generic group as in figure 1, largely following (Lumbsch and Huhndorf 2010).Newly obtained sequences are indicated in bold.

Sequence alignments and phylogenetic analysis
We assembled partial sequences using Geneious Pro 5.4.3 (Drummond et al. 2011) and edited conflicts manually.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 per- formed 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 as in Lumbsch et al. (2007b).Bootstrapping (Felsenstein 1985) was performed based on 2000 replicates.The B/MCMC analysis was conducted on the concatenated data set using Mr-Bayes 3.1.2(Huelsenbeck and Ronquist 2001), with the same substitution model as in the ML analysis.The dataset was partitioned into two (mtSSU, nuLSU) and each part was allowed to have its 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.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 split 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 depicted using the program FigTree 1.3.1 (Rambaut 2009).

Results and discussion
Eight new sequences were generated for this study and aligned with sequences downloaded from Genbank (Table 1).The single gene locus trees did not show any conflicts and hence the concatenated data set was analyzed.Our combined data set included 1313 unambiguously aligned positions, 370 of which were constant.The ML tree had a likelihood value of -26318.540and in the B/MCMC analysis of the combined data set, the likelihood parameters in the sample had the following mean (Variance): LnL = -27045.138(0.35).The ML tree and the tree from the B/MCMC tree sampling were almost identical, with no differences in well-supported clades.Furthermore, taxon sampling was very similar to that of previous studies focusing on the phylogeny of Ostropomycetidae (Baloch et al. 2010;Lumbsch et al. 2007a;Lumbsch et al. 2007b;Wedin et al. 2005).Thus, only a simplified ML tree, with samples of well-supported families, genera or generic groups collapsed, is shown here (Fig. 1).Individual OTUs are shown only for the species of Cameronia and its sister groups.In our analysis, the four samples of the two Cameronia species form a strongly supported, monophyletic group within the well-supported Ostropomycetidae, confirming the monophyly of the genus and its placement in Ostropomycetidae.The genus Cameronia is another example of a group of lichenized ascomycetes with perithecioid ascomata in this subclass, with others being Porinaceae (Baloch and Grube 2006;Grube et al. 2004), Protothelenellaceae and Thelenellaceae (Schmitt et al. 2005).There are additional families Phylogenetic placement of Cameronia as inferred from a concatenated alignment of mtSSU and nuLSU DNA sequences.This is a simplified cartoon of the optimal tree under maximum likelihood with well supported families and species groups collapsed that were shown in previous studies (Baloch et al. 2010;Lumbsch et al. 2007a;Lumbsch et al. 2007b;Wedin et al. 2005).Asterisks indicate branches with likelihood bootstrap support values above 70% and posterior probabilities equal or above 0.95. in this subclass that also include taxa with more or less perithecioid ascomata, such as Coccotremataceae, Gyalectaceae, Pertusariaceae and Graphidaceae (Baloch et al. 2010;Lumbsch and Schmitt 2002;Lumbsch et al. 2001;Rivas-Plata et al. 2012;Rovas-Plata and Lumbsch 2011;Schmitt et al. 2010;Schmitt and Lumbsch 2004).The diversity of ascomatal morphologies in this subclass has been linked to the hemiangiocarpous type of ascoma development in the group as a whole (Schmitt et al. 2009).
The backbone of the Ostropomycetidae tree largely lacks support and the relationships of Cameronia within Ostropomycetidae remain unclear.Cameronia is the sistergroup of Baeomycetaceae (Ainoa, Baeomyces, Phyllobaeis) but this relationship lacks support.This clade forms a sister-group to a well-supported clade that includes Anzina and Protothelenellaceae, but again, this relationship lacks support.
Although the molecular data support the placement of Cameronia in Ostropomycetidae, they fail to identify any close relatives of the genus, which is also reflected in the similarities of Blast searches of the newly generated sequences (maximal identity -nuLSU: 94%, mtSSU: 93%).Cameronia is distinguished by several characters that are generally used to characterize families, as shown in Table 2 where salient features of Cameronia and other families of Ostropomycetidae with perithecioid ascomata (Porinaceae, Protothelenellaceae, Thelenellaceae) are compared.The ascus type is very different from any of the other perithecioid Ostropomycetidae and also different from the apotheciate Baeomycetaceae, which have cylindrical asci (Gierl and Kalb 1993).Nor is the rudimentary exciple seen in Cameronia found in any of the other perithecioid families.Morphologically, the most similar family in Ostropomycetidae is Protothelenellaceae, with which Cameronia shares a hamathecium of richly branched paraphysoids and a lack of periphyses.However, Prothelenellaceae have a different exciple, different asci with an amyloid apical apparatus in the tholus and an ocular chamber, and halonate ascospores.Furthermore, Protothelenellaceae form a well-supported clade with Anzina (Fig. 1) and are only distantly related to Cameronia.The isolated position of Cameronia is consistent with the hypothesis that this genus is a case of paleoendemism.It will be an exciting project to test this hypothesis at a later stage when more sequence data from Ostropomycetidae become available.
Given the dissimilarity in morphological characters and the lack of close relatives in the phylogenetic tree, we propose a new family Cameroniaceae below to accommodate the genus Cameronia.The new family is placed in Ostropomycetidae with unclear ordinal position.Description.Thallus crustose, photobiont a coccoid green alga.Ascomata perithecioid, immersed in the thallus, proper exciple rudimentary, hamathecium consisting of richly branched, anastomosing paraphysoids, inspersed with oil droplets, containing hymenial algae, periphyses absent.Asci broadly obovate, with outer wall hemiamyloid and with a well-developed, non-amyloid tholus; ocular chamber lacking.Ascospores hyaline, non-halonate, eumuriform.Conidiomata immersed in the thallus, forming baciliform to bone-shaped conidia.