PhotobiontDiversity has moved!

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.

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Another perspective on diversity of symbiotic cyanobacteria: 16S

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

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Symbiotic Nostoc Revisited

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…

Continue reading

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Green Algal Photobionts: Trentepohlia

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

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

  • Lichen (apbiology2014mathew.wordpress.com)
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Green Algal Photobionts: Coccomyxa

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

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 PseudococcomyxaParadoxia 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

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Gunnera symbionts do not cluster with lichen photobionts

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.

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

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Green Algal Photobionts: Asterochloris.

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

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

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

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

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