I’ve recently completed migration of this blog from wordpress.com to photobiontdiversity.org
This move will allow the creation of a number of new features, the first of which is described in this post.
Unfortunately, I’ve not been able to migrate subscriptions to the new site, so you’ll have to resubscribe if you want to keep receiving updates about new posts.
Up to this point, I have been focusing on the rbcX locus for all investigations of cyanobacterial photobionts because it is probably the most extensively sampled locus and it is more variable than 16S rDNA. However, it is limited because some groups of symbiotic cyanobacteria do not have rbcX sequences in the database. These include symbionts of the water fern Azolla which has traditionally been called Anabaena azolae and the photobionts of a variety of primarily tropical lichens that have traditionally been classified within the genus Scytonema. 16S sequences are available for both of these groups, as are sequences from a variety of other related genera of cyanobacteria. Furthermore, it is useful to compare the patterns revealed from analyses of rbcX to those based on an independent locus, often sampled from independent specimens. Continue reading
In the three months or so that I’ve been working on this blog there has been some evolution in the methods I’m using. I though it would be worthwhile to revisit the first group I looked at to see if these changes in the methods affect my results. There have also been some additional sequences released since I started…
While most work has focused on coccoid green algae and/or cyanobacteria, a largely overlooked lineage of lichen photobionts is the Trentepoliales, a group of filamentous, carotenoid producing green algae. Trentepohlian algae are associated with one fifth of lichen species world wide and are particularly common as a photobiont of eipiphytic lichens in the tropics. Trenepohlian algae are also very commonly found as free-living colonies on tree bark, leaves and rocks. Like most of the groups discussed here, the taxonomy of the Trentepholiales has not held up to scrutiny with molecular phylogenetic methods and the various genus names do not appear to be meaningful.
The only published study that I am aware of on the diversity of Trenetpohlian photobionts is Nelsen et al. 2011 (J. Phycol. 47, 282–290). They found that lichenized strain are mixed with free-living ones throughout the phylogeny and that lichen fungi from different classes can associate with very similar photobionts. Since the publication of this study, a large number of additional sequences from Trentepohlian photobionts have been deposited in the databases. These includes sequences of both ITS, which is the marker that has been used the most widely in studies of Trebouxiophycean photobionts and rbcL, a chloroplast gene that is probably the most extensively used phyologenetic marker in plants. Since rbcL was used in the study mentioned above, that is what I used for the analysis presented here.
Methods are the same as those described previously. Because the rbcL is so highly conserved, long sequences from more distantly related algae had higher Evalues than shorter sequences from Trenetepohlian algae. This means that I had to manually add missed sequences with accession numbers that were bracketed by the ones that were found. It also meant that I had to exclude sequences from other algae that formed a large clade sister to the Trentepohliales in preliminary analyses. The detailed steps of this analysis are here. Datasets can be found here.
This is what the tree looks like:
Trenetpohliales rbcL phylogeny color-coded by host class (green: Lecanoromycetes, orange: Arthoniomycetes, blue: Dothideomycetes, red: Eurotiomycetes, grey: free-living). Sequences recovered from multiple genera are in black. Black circles indicate aLRT support >= 0.9. Clades correspond to those of Nelsen et al. 2011. Tree is rooted by Chloromonas sp. U80809
This phylogeny includes the four major clades discussed by Nelsen, but there are also a large number of lichenized strains that branch near the base of clades 2 and 3. The new sequences also greatly expand clade 4 (from 3 to 14 photobionts) and add a lichenized strain to clade 3 (a Strigula photobiont, as predicted by Nelsen et al.). Non-lichenized strains are distributed throughout the tree, and there are two case where a free-living and a lichen photobiont have identical rbcL sequences. There are a few subclades that appear to be specific to a single lichen genus (Roccella photobionts in clade 1, Coenogonium photrobionts in clade 2), but the most remarkable thing about this tree is the phylogenetic breadth of lichens that share the same photobionts. Clades 2 and 4 both include photobionts of four different classes of Ascomycetes: Lecanoromycetes, Arthoniomycetes, Eurotiomycetes and Dothideomycetes. Indeed, there are two cases of photobionts of Pyrenula (Eurotiomycetes) and Graphis (Lecanoromycetes) having identical rbcL sequences.
This is clearly a group that is worthy of a lot more study. There appear to be a broad range of specificities, from species that can switch among two or more classes of fungi (in addition to living independently), to ones that are specialised on a single host genus. Additional sampling of the Eurotiomycetious lichens in particular would be helpful to determine the phylogentic breadth of hosts for many of these lineages. Extensive sampling within species would also be helpful to range of suitable photobionts for individual species.
Heath OBrien (2013). Green Algal Photobionts: Trentepolia PhotobiontDiversity.wordpress.com : http://dx.doi.org/10.6084/m9.figshare.750445
After a few weeks of for a vacation, it’s time to get back to green algal photobionts. In addition to Trebouxia and Asterochloris, there are several other genera of Trebouxiophycean algae that act as photobionts for various groups of lichens. The most significant of these is probably Coccomyxa, which is the photobiont of many mushroom-forming basidiolichens and is also the green algal component of tripartite lichens in the Peltigerales.
There hasn’t been much work on Coccomyxa, but there were two papers in 2003 that each sequenced ITS from 20-25 specimens. One study found that photobionts of basidiolichens, photobionts of Peltigeralian lichens and free-living strains each formed a distinct lineage (Zoller et al. 2003), while the other found that photobionts of two species each of Nephroma and Peltigera were nearly identical (variable at a single position), which a single Peltigera britannica photobiont was found to be significantly different (Lohtander et al. 2003). Coccomyxa and Pseudococcomyxa have also been reported as symbionts of protists including Paramecium and Stentor.
Seventy-seven ITS sequences were obtained and analysed as described previously. Representative ITS sequences from Lohtander et al. were expanded based on the data in Table 2 of their paper. Details of the analyses are here. Datasets can be found here. The resulting tree looks like this:
Coccomyxa ITS phylogeny color-coded according to the clades identified by Zoller et al, 2003: blue, L/O, red, L/P, green, F. Black circles indicate aLRT support >= 0.9. Tree is mid-point rooted
The clade coloured blue in the figure corresponds to the basidiolichen clade mentioned above. It also includes the outlier sequence from P. britannica found by Lohtander et al. The clade in red corresponds to the Peltigeralean photobiont clade above and includes all other Peltigera and Nephroma sequences obtained by Lohtander et al. The clade in green corresponds to the free-living clade. This free-living clade also includes two symbionts of Paramecium bursaria while two other P. bursaria symbionts and one from Stentor amethystinus fall outside of these three main clades. There are also a large number of additional sequences from free-living strains, which are scattered throughout the tree. Many of these in the basidiolichen clade are described as “mucilaginous overgrowth on rotting wood”, which is similar to the growth habit and habitat of many basidiolichens, so these may actually represent lichenized strains, but the study that these sequences are from is unpublished so I don’t know the details. This basidiolichen clade also includes unpublished sequences from photobionts of three Solorina specimens, which are Peltigeralean lichens.
Strains identified as Pseudococcomyxa, Paradoxia and Choricystis are nested within Coccomyxa, so it appears that all of these strains represent the same genus, with different species specialised on rotting wood (either in association with basidiolichen fungi or possibly free-living), Peltigeralean lichen fungi, and fresh water (often in association with Paramecium). However, this specificity is not absolute, at least for the two lichenized species. There are also one or more additional species that grow free-living and in association with with protists, but that do not appear to be able to lichenize, though additional sampling will be required to confirm this.
References:
Katileena Lohtander, Ilona Oksanen, & Jouko Rikkinen (2003). Genetic diversity of green algal and cyanobacterial photobionts in Nephroma (Peltigerales) Lichenologist DOI: 10.1016/S0024-2829(03)00051-3
Zoller S, & Lutzoni F (2003). Slow algae, fast fungi: exceptionally high nucleotide substitution rate differences between lichenized fungi Omphalina and their symbiotic green algae Coccomyxa. Molecular phylogenetics and evolution, 29 (3), 629-40 PMID: 14615198
Heath OBrien (2013). Green Algal Photobionts: Coccomyxa PhotobiontDiversity.wordpress.com : http://dx.doi.org/10.6084/m9.figshare.743673
I have been kicking around the idea of setting up an online catalog of lichen photobionts for years before I started doing this. The main impetus to finally start was this paper:
Fernández-Martínez, M., de los Ríos, A., Sancho, L., & Pérez-Ortega, S. (2013). Diversity of Endosymbiotic Nostoc in Gunnera magellanica (L) from Tierra del Fuego, Chile Microbial Ecology DOI: 10.1007/s00248-013-0223-2
I have been interested in the relationships between lichenized Nostoc and those that form symbioses with ferns, cycads, liverworts and the flowering plant Gunnera for a long time, but my efforts to address the issue were hampered by inadequate sampling and others who had better sampling used a genetic marker with a complex history that made such comparisons difficult. Finally, here was a paper with extensive sampling across the range of a plant host of Nostoc who used the same genetic marker that has been widely adopted in work on lichen photobionts. Unfortunately, while the paper has a lot of interesting things to say about the Nostoc-Gunnera symbiosis (genetically monomorphic within individuals, lots of variability among individuals, reduced symbiont diversity in recently deglaciated areas, etc), they included very few lichen photobionts in their analyses, so I wasn’t able to get the answers to the questions I’m interested in from the paper.
Now that I’ve developed a decent phylogentic framework for symbiotic Nostoc, it should now be possible to address these questions. Here is how the G. magellanica symbionts fit in:
Nostoc rbcX phylogeny with Gunnera magellanica symbiont hilighted, coloured by type of association (purple: lichen photobionts, green: plant symbionts, blue: free-living, red: fungal endosymbiont). Names in black indicate genotypes found in more than one group. Circles on internal nodes indicate aLRT ≥0.9.
A few things about this tree are interesting. For one, G. magellanica symbionts form two well-supported lineages to exclusion of all other strains. This contradicts the results from the paper, where two G. magellanica symbiont haplotypes did not group with the others and where one lichen photobiont was nested within one of the G. magellanica symbiont clusters, though resolution and support were low for these nodes in their tree. Secondly, the G. magellanica symbionts do not group with any other plant symbionts, including the other Gunnera symbiont. Thirdly, the G. magellanica symbionts are on relatively long branches, suggesting that the evolutionary rate is higher in this lineage.
One major note of caution are in order when interpreting this tree, however: all of these G. magellanica symbionts were collected in the southern tip of South America, while the vast majority of the sampling of other lineages, including many of the plant symbionts, is from the northern hemisphere. Indeed, several of the symbionts of tropical plants were isolated from botanic gardens in Europe, well outside of the native range of the plants. It is certainly possible that lichens and other plant hosts from South America would associate with some of the same Nostoc strains isolated from G. magellanica.
So, in conclusion, it’s fair to say that these data support the notion of frequent host shifts between lichens and plants in the evolutionary history of Nostoc, but that there is no evidence that the same strains of Nostoc routinely form symbionses with lichens and with Gunnera. However, more geographically appropriate sampling may provide this evidence in the future.
Methods:
Sequences KF142679 to KF142710 were downloaded from genbank and added to the Nostoc rbcX dataset. These 32 haplotype sequences represent 110 specimens, but it is not clear how many specimens each haplotype represents. Details of the analysis can be found here. Data files are here.
Heath OBrien (2013). Gunnera symbionts do not cluster with lichen photobionts PhotobiontDiversity.wordpress.com : http://dx.doi.org/10.6084/m9.figshare.726140
In this post I will be taking a look at the diversity of the junior partner to Trebouxia: Asterochloris. Originally described by Elisabeth Tschermak-Woess in 1980, Asterochloris was subsequently merged with Trebouxia before being split out again on the basis of sequence data in the late 1990s. For the most part, Asterochloris is thought to be restricted to associations with lichens in two closely related families, Cladoniaceae and Stereocaulaceae. However, this includes Cladonia, one of the more charismatic (and ecologically important) lichen groups, so Asterochloris has been extensively sampled.
Sequences were obtained and analysed as described previously, except that I decided to use a command-line application to remove redundant sequences instead of the GUI-based program MetaPIGA. In addition to being easier to automate, this has the advantage of being much better documented and WAY faster:
usearch -cluster_fast Asterochloris_ITS.fa -id 1 -centroids Asterochloris_ITS_nr.fa -uc Asterochloris_ITS_groups.txt
This works on unaligned sequences and produces a file of non-redundant sequences in addition to the list of groups, which can be used directly for alignment and phylogenetic inference. The detailed steps of this analysis are here. Datasets can be found here.
The resulting phylogeny, colour-coded by algal species names looks like this:
Asterochloris ITS phylogeny color-coded by species (light blue: A. irregularis, red: A. glomerata, dark blue: A. phycobiontica, dark green: A. magna, ornage: A. excentrica, purple: A. italiana, light green: A. erici, grey: A. sp.). Sequences recovered from multiple names species are in black. Black circles indicate aLRT support >= 0.9
The structure of the tree looks quite similar to that of Trebouxia, and this paper argues that many of these clusters represent distinct species. However, the sequence divergence within the genus is much, much lower than it is for Trebouxia:
Trebouxia and Asterochloris ITS trees with branch lengths drawn to the same scale
This has more to do with the huge amount of sequence divergence within Trebouxia than it does any lack of diversity in Asterochloris. Most of Asterochloris clusters above have at least 2% sequence divergence from one another, so it’s not unreasonable to consider them different species.
The named representatives of three species (A. phycobiontica (dark blue), A. erici (light green) and A. excentrica (light blue) ) form coherent clusters in the tree. Strains identified as A. glomerata (red), A. magna (dark green) and A. irregularis (orange) each fall into two clusters, though it is difficult to see for the latter two species because some of the sequences are identical to those from representatives of other species and are thus labeled black. Two additional species, A. italiana and A. pyriformis are only represented by sequences that are identical to sequences from other species.
As for the host association patterns, the vast majority of isolates are from members of the genera Cladonia (blue), Lepraria (red) and Stereocaulon (purple):
Asterochloris ITS phylogeny color-coded by host genus ( dark blue: Cladonia, red: Lepraria, purple: Stereocaulon, light blue: Pilophorus, dark green: Anzina, light green: Varicellaria, orange: Diploschistes, grey: unknown/other). Sequences recovered from multiple names species are in black. Black circles indicate aLRT support >= 0.9
There are also photobionts of Cladia, Pilophorus, Pycnothelia, Anzina, Diploschistes, Ochrolechia, Varicellaria and Xanthoria. The first three of these are closely related to Cladonia, while the others are a diverse assemblage of lichens. With the exception of Xanthoria, none of these genera have been found to associate with Trebouxia. Xanthoria is listed as the host for A. italiana in GenBank, but the authors make no mention of this in their paper and the host is not listed in the culture collection info, so it should probably be taken with a grain of salt.
Interestingly, there are also a number of Asterochloris sequences that were obtained from environmental sampling (one from limestone rock and two from forest soil and ten from a glacier forefield). It is certainly possible that these were derived from lichen fragments or vegetative propagules, but there was only a single Trebouxia sequence that was not from a lichen thallus despite almost three times as much sampling, so these results suggest that Asterochloris may be a facultative lichen photobiont while Trebouxia is obligately lichenized.
Other than the A. phycobiontica cluster which is associated with a diverse array of lichen genera, Lepraria tends to be associated with distinct lineages compared to Cladonia and Stereocaulon, while the latter genera overlap in their photobiont preferences. There is also a lot of interesting host association patterns at the species level for this group, a topic that I hope to explore in the future.
Heath OBrien (2013). Green Algal Photobionts: Asterochloris PhotobiontDiversity.wordpress.com : http://dx.doi.org/10.6084/m9.figshare.717196
Having gone through the steps to build a phylogeny of the most common lichen photobiont, Trebouxia in my last post, I will now go on to discussing the host association patterns that it reveals. Here is the Trebouxia ITS tree generated previously:
Trebouxia ITS phylogeny. Major clades are differentilly coloured and named according to authentic strains
I’ve coloured all of the taxa within clades according to the colours of the named strains and I’ve assigned unique colours to each clade that does not contain named strains. I have not attempted to break up T. jamesii or T. impressa into sub-clades, though doing so would probably be justified. This will be a topic for a future post. I should also point out that T. jamesii is referred to as T. simplex is some papers.
In contrast to Nostoc photobionts where the fasta headers were consistently labeled with the host information, these sequences are …not. I used a bioperl wrapper to NCBI’s Eutils interface to download genbank format sequences and parsed them to extract host association information from the “host”, “note” and “isolation source” annotions. I also extracted information about the author of each sequence and where it was published while I was at it:
../Scripts/GetGB.pl Trebouxia_ITS_acc.txt heath.obrien-at-gmail-dot-com > Trebouxia_ITS.gbk
../Scripts/ParseHost.pl Trebouxia_ITS.gbk > host_info.txt
../Scripts/GetRef.py Trebouxia_ITS.gbk >ref_info.txt
This information was added to Trebouxia_ITS_metadata.txt in Excel and missing values were filled in manually where possible. I also added culture collection numbers where available and started to fill in locality information, but I haven’t gotten very far with the latter.
Next, I added information about which clade each sequence fell into to the Trebouxia metadata file. The ETE python toolkit was invaluable for this step and was my main proximate motivation for switching from perl to python for my scripting, but I was also really, REALLY tired of having to keep my ‘\@’s and ‘%{$’s straight:
../Script/GetClades.py -t Trebouxia_ITS.nwk -m Trebouxia_ITS_metadata.txt >temp
mv temp Trebouxia_ITS_metadata.txt
In this case, the information was added to the metadata file automatically.
Lastly, I wrote a script to count the number of times that Trebouxia from each clade was associated with each lichen genus that has been sampled:
../Scripts/CountAssociations.py -m Trebouxia_ITS_metadata.txt > AssocaitionCounts.txt
After some fiddling with conditional formatting in Excel, these analyses produced this:
Counts of associations between Trebouxia clades and lichen genera. Colour coding matches the phylogeny
Before discussing the patterns, I should point out that these counts are of how many sequence records have been deposited in genbank, not the number os specimens that have been sampled as many authors deposit representative haplotype sequences rather than all of their data. Incorporating this information will change the counts dramatically in some cases.
The most extensively sampled genus is Letharia, which is exclusively associated with Trebouxia jamesii. In fact, there appears to be strong reciprocal specificity acting between species of Letharia and subclades within T. jamesii, a topic I would like to explore further in the future.
Eight other genera in the Parmeliaceae are also exclusively associated with T. jamesii photobionts, including Cetraria (76 sequences), Evernia (19 sequences), Flavocetraria (17 sequences), Hypogymnia (12 sequences) and Pseudevernia (10 sequences). However, Parmelia (10 sequences), Flavoparmelia (6 sequences), and 4 other genera associate with photobionts in the T. impressa/T. gelatinosa clade (among others) and Parmotrema is highly specific for T. corticola photobionts, with 135 P. tinctorum photobionts grouping with T. corticola (the Trebouxia sp. 3 photobiont is from a different Parmotrema species). The P. tinctorum / T. corticola association is another case of reciprocal specificity as 135 of 141 T. corticola sequences are from P. tinctorum photoboints.
All photobionts from Lasallia (28 sequences) and most from Umbilicaria (105 of 131 sequences) also grouped with T. jamesii, with most of the exceptions being specimens collected in the Antarctic (see also this paper). T. jamesii was also the predominant photobiont of Thamnolia (22 out of 28 sequences) and Chaenotheca (7 of 8 sequences). T. jamesii was also a common photobiont of Lecanora and Lecidea, but photobiont diversity in both of these genera, and the Lecaonraceae in general, is extremely high, with Lecanora photobionts falling into 11 different species and Lecidea photobionts falling into 8. Indeed, 6 species of photobiont have been recovered from L. rupicola alone. Lichens in the Lecanoraceae are the hosts for the vast majority of T. asymmetrica, T. incrustata, T. showmanii and T. sp. 1 photobiots that have been sampled.
Similar to the Parmeliaceae, most of the genera in the Physciaceae are specific for the same Trebouxia lineage, while other genera do not associate with it at all. Physconia (40 sequences) and Phaeophyscia (7 sequences) are exclusively associated with T. impressa, as are 29 out of 36 Physcia sequences while only 2 of 5 Rinodina photobionts and none from Anaptychia (8 sequences) group with T. impressa.
All but 9 of 133 Xanthoria photobionts belong to T. decolorans or T. arboricola, which are sister species. These photobionts also predominate in most of the other genera in the Teloschistaceae, including 53 of 65 Caloplaca photobionts, 4 of 4 Huea photobionts, and 34 of 68 Tephromela photobionts, a genus that is also frequently associated with Trebouxia sp. 2 photobionts.
Specificity is also high for Ramalina, with 139 of 150 photobiont sequences restricted to two Trebouxia clades. Boreoplaca is associated with one of the same photobionts (Trebouxia sp. 2), but not with the most common Ramalina photobiont (T. decolorans; 116 of 150 sequences).
Thus, there is a wide range of association patterns, from extreme reciprocal specificity (135 of 141 T. corticola sequences associated with Parmotrema and all P. tinctorum specimens associated with T. corticola) to generalism (Lecanora rupicola associating with 6 different Trebouxia species). There is some evidence of phylogenetic inertia, as lichen genera from the same family are more likely to share similar photobiont association patterns than unrelated lichens, but there is also a lot of plasticity. There are a lot of ideas out there about the ecological and life history factors that could be causing these differences, but given the complexity of the patterns and our lack of knowledge of things like lichen demography and dispersal mechanisms, it will probably be some time before definitive explanations can be provided.
Heath O’Brien (2013). A preliminary look at host association patterns in Trebouxia PhotobiontDiversity.wordpress.com : http://dx.doi.org/10.6084/m9.figshare.711786
Having beaten the phylogeny of symbiotic cyanobacteria into submission in my previous post, I am now tackling the green algae. My plan was to start with a big-picture analysis of 18S ribosomal RNA sequences, but my initial blast search returned over 10,00o 454 reads from metagenomic projects which was a lot more “environmental isolate XXX” than I felt like dealing with. Besides, I don’t know that I could add much to this recent overview. Therefore, I am going to focus on the most important lineage of lichenized algae: Trebouxia. There have been a large number of studies that have obtained photobiont ITS sequences from a variety of Trebouxia associated lichens, so these are the data that I looked at.
The methods are the same as the ones that I described in detail previously for Nostoc ITS sequences. Briefly, I used two ITS sequences (T. impressa JN204819 and T. arboricola JQ993781) as queries to identify all homologous (E-value <= 1e-100) sequences in the nt database. Sequences were aligned with MAFFT, duplicate sequences were removed with MetaPIGA, alignment positions corresponding to gaps in the references sequence (T. arboricola JQ993758) were removed with trimal, and phylogenetic relationships were inferred with PhyML.
This procedure produced a tree with 794 taxa representing 1840 Trebouxia ITS sequences. The actual number of Trebouxia associated lichens that have been sequenced is much higher than this because many authors only deposit representative sequences of each haplotype that they obtained. At some point I will dig into the papers where this has been done to extract the real numbers, but I have not done so yet.
For now, I am going to focus on the taxonomy of the algae. I will leave a discussion of the host-association patterns for a future post. Here is the Trebouxia ITS phylogeny color-coded by species (tree file can be found here):
Trebouxia ITS phylogeny color-coded by species (dark green: T. jamesii, yellow: T. corticola, light green: T. incrustata, brown: T. asymmetrica, ornage: T. gigantea, purple: T. gelatinosa, dark blue: T. impressa, light red: T. arboricola, light blue: T. decolorans, dark red: other, grey: T. sp.). Sequences recovered from multiple named species are in black. Black circles indicate aLRT support > 0.9
With a few exceptions, sequences from named algae tend to cluster very well. T. gelatinosa (purple) is nested within T. impressa (dark blue), though given the long branch separating these two species from all of the others, I don’t entirely trust the rooting of this clade. T. jamesii (dark green) is a very heterogeneous group as has been recognised previously. A number of photobionts that group with T. decolorans (light blue) have been identified as T. arboricola (light red). Three major lineages have no named members (except for some presumably misidentified T. decolorans sequences).
In addition to the differentially coloured species, there are several additional species names that are represented by a small number of sequences, all of which are colored dark red in the tree. T. australis, T. brindabellae, T. showmanii and T. usneae are each found in distinct clusters and are likely to represent additional good species. T. australis and T. brindabellae are both in clusters near the base of one of the T. jamesii clades (dark green). Two T. showmanii sequences form the sister group to T. incrustata (light green). T. usneae forms a distint lineage with a misidentified T. corticola sequence sister to the T. corticola lineage (yellow). All other rare species are deeply nested within other common species and appear not to be distinct. These include T. potteri which is nested within T. impressa (dark blue), T. aggregata and T. crenulata which are nested within T. arboricola (light red) and T. simplex, which includes six sequences that are identical to T. jamesii (black) and two other sequences that are nested within one of the T. jamesii clades (dark green). T. flava is identical to a T. impressa sequence and is coloured black in the tree.
In conclusion, >1840 Trebouxia ITS sequences that have been obtained from lichens cluster into about 24 distinct species, 13 of which appear to have suitable named representatives in the database. Two of the T. jamesii clusters have been given the provisional names T. “vulpinea” and T. “letharii” but it looks like at least three additional names are needed for this group.
That’s it for now. In my next post I will map host information onto this tree.
**Post has been updated with some corrections to the host information in the first phylogeny**
Today I am finally going to take a detailed look at the Nostoc phylogeny that I have been working on. But before I can begin, I have to figure out a way to highlight interesting taxa in an automated way. To do this, I wrote a script that adds html color tags after taxon names according to various classifications. While I was at it, I converted the branch support values to a binary system (≥0.9 vs. <0.9), which I can display as black circles on significantly supported branches. Note that this script requires that the tree be in NEXUS format rather than the plain Newick that is produced by PhyML. Opening the tree file in FigTree and saving it converts it to NEXUS, or the conversion could be scripted using Bioperl.
First, lets compare lichen photobionts to other free-living and symbiotic Nostoc strains:
../Scripts/ColourTree.pl ../Nostoc_rbcX_metadata.txt ../Nostoc_rbcX_host.nwk host >host_tree.nwk
Nostoc rbcX phylogeny, coloured by type of association (purple: lichen photobionts, green: plant symbionts, blue: free-living, red: fungal endosymbiont). Names in black indicate genotypes found in more than one group. Circles on internal nodes indicate aLRT ≥0.9.
As mentioned last time, the earliest branching taxa are free-living Nostoc isolates, along with a culture isolated from Peltigera, which I suspect may not be a true photobiont. There are also other free-living strains throughout the rest of the phylogeny that have been identified as N. edaphicum, N. calcicola, N. commune, N. muscorum and N. flagelliforme. Cyanobacterial taxonomy is a mess, but that is a topic for another day. There are also symbionts from a variety of plant groups throughout the main crown group including Cycads (Cycas, Macrozamia and Encephalartos), Bryophytes (Blassia and Anthoceros) and the angiosperm Gunnera. There are two cases where lichen photobionts are identical to plant symbionts (coloured black in the tree). Finally, there are two symbionts from Geosiphon pyriforme, a weird unicellular primative fungus, that hosts intracellular symbionts in sac-like, multinucleate cells (coloured red). There is some debate as to whether this symbiosis should be classified as a lichen or not.
Next, we can look at photobionts of different lichen families (the taxonomy of the lichen is based on that of the fungal partner):
../Scripts/ColourTree.pl ../Nostoc_rbcX_metadata.txt ../Nostoc_rbcX_host.nwk family >family_tree.nwk
Nostoc rbcX phylogeny, coloured by host family (purple: Stereocaulaceae, green: Lobariaceae, blue: Peltigerales, red: Collemetaceae, yellow: Nephromataceae, brown: Pannariaceae). Names in black indicate genotypes found in more than one group or photobionts of lichens with uncertain taxonomic position (Massalongia). Names in grey indicate non-lichenized strains. Circles on internal nodes indicate aLRT ≥0.9.
At the deepest nodes in the tree, there is clearly a lot of host switching between different lichen families, but there is a lot of clustering of photobionts from the same lichen family at the tips of the tree. Photobionts of lichens in the Lobariaceae, Nephromataceae and Pannariaceae are all mixed up, which has been noted previously and has been proposed to reflect the ecological similarities of the hosts. There also appears to be a lot of historic photobiont sharing between lichens in the Peltigeraceae and the Collmenataceae, but such sharing is not ongoing as in all cases there are long branches separating photobionts of these families. Stereocaulon is the only species represented in the tree that is not part of the Peltigerales, an order of lichens that are universally associated with Nostoc, either as the sole photosynthetic partner or as a secondary photobiont. It would be interesting to see if other non-Peltigeralean lichens also associate with such divergent Nostoc genotypes.
Lastly, let’s take a look at species-level patterns. There is a lot of host switching among members of the same genus, but there do appear to be some species that are highly specialised:
../Scripts/ColourTree.pl ../Nostoc_rbcX_metadata.txt ../Nostoc_rbcX_host.nwk specialists >specialist_tree.nwk
Nostoc rbcX phylogeny, coloured by host species Names in grey indicate non-specialist hosts. Circles on internal nodes indicate aLRT ≥0.9.
With the current sampling, it is possible to identify four species of Leptogium and one each of Collema, Peltigera and Sticta that exclusively associate with a single cluster of photobionts, which is, in turn, exclusively associated with that lichen species (reciprocal specificity). Note that there is one P. malacea photobiont that falls out of the P. malacea cluster, but there are about four times as many specimens of this species as there are for any of the other specialists. As noted previously, these specialists predominate in the basal symbiotic Nostoc lineage.
There is a lot more that could be said about this tree, but I think I’ll leave it there for now. See this paper for a more detailed analysis of the complex photobiont specialisation patterns in Peltigera, including geographic patterns. On to the green algal photobionts in my next post…
PhotobiontDiversity 