Next-generation sequencing reveals sterile crustose lichen phylogeny

I recently had a paper published that showed the utility of next-generation sequencing and a microbiome-based approach for basic lichen-forming fungal systematics. DNA sequences derived from the species known as Lepraria moroziana had for many years either been too messy to read or had seemingly been derived from disparate fungi, presumably because the species contains many endo-lichenic associates and/or it often grows in close proximity to other lichen-forming fungi. However, when using 454 sequencing to examine the whole pool of LSU (nuclear ribosomal large sub-unit) sequences from ten samples, it became clear that the dominant fungus in each was from the class Arthoniomycetes. This provided a breakthrough for the group, which was previously classified in the class Lecanoromycetes. After further investigations, it became clear that the two species Lepraria moroziana and Lepraria obtusatica belonged in their own genus, Andreiomyces, which itself we put in its own family, Andreiomycetaceae.

Relative abundances of the various putative classes represented by LSU sequences in each of the ten samples from the species of interest.

Interestingly, there were some sequences found in some of the amplicon pools derived from the "real" genus Lepraria (Lepraria s. str. in the class Lecanoromycetes). Had we gotten these sequences through standard Sanger sequencing, we may have been led astray, making it more difficult to discern the fact that the main lichen-forming fungus belongs to a previously unknown lineage in the class Arthoniomycetes!

I anticipate that this type of methodology will be advantageous for a lot of problematic fungi, especially those that remain uncultured. Here is the full abstract for the associated paper:

"The rapid phylogenetic placement and molecular barcoding of fungi is often hampered in organisms that cannot easily be grown in axenic culture or manually teased apart from their associated microbial communities. A high-throughput procedure is outlined here for this purpose, and its effectiveness is demonstrated on a representative species from an especially problematic group of fungi, the sterile crustose lichens. Sequence data of the LSU and ITS regions were generated from samples of a sterile crustose lichen species, Lepraria moroziana, using next-generation sequencing. DNA fragments most likely to represent the primary lichen-forming fungus were bioinformatically teased out using a specialized data processing pipeline. Phylogenetic analyses of the LSU region revealed that the lichen-forming fungus L. moroziana was previously placed in the incorrect class of fungi (Lecanoromycetes), and actually belongs to the class Arthoniomycetes, in the order Arthoniales. It is here treated as a member of a new family (Andreiomycetaceae Hodkinson & Lendemer fam. nov.) and genus (Andreiomyces Hodkinson & Lendemer gen. nov.). Additionally, Lepraria obtusatica Tønsberg is placed in the newly-defined genus based on its morphological, chemical, and ITS-based molecular similarity to L. moroziana. The procedure outlined here is projected to be especially useful for resolving the dispositions of diverse problematic fungi that remain unnamed, incertae sedis, or have taxonomic positions that are not expected to reflect their true phylogeny."

- Brendan

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Reference

Hodkinson, B. P., and J. C. Lendemer. 2013. Next-generation sequencing reveals sterile crustose lichen phylogeny. Mycosphere 4(6): 1028-1039.
Download publication (PDF file)
Download data and sequence-processing scripts (ZIP archive)
Download Ascomycota LSU alignment and analysis files (ZIP archive)
Download Arthoniales LSU alignment and analysis files (ZIP archive)

Leprocaulales

This year I published a paper with James Lendemer (Lendemer & Hodkinson 2013) in which we established a new order of fungi, Leprocaulales, for a group of sterile crustose lichens.  We first found that the species in the genera Lepraria and Leprocaulon were all shuffled up (they got this way due to their similar appearance), but we were able to sort them properly using molecular data.  It then became apparent, after further analyses, that the Leprocaulon clade is actually quite distantly related to other known groups of fungi (and is not even close to Lepraria), so we gave it a new family (Leprocaulaceae) and new order (Leprocaulales).  One really interesting aspect of this work is that it revealed to us that both groups (Lepraria and Leprocaulon) had crustose and fruticose growth forms within them.  This shows that growth form is more plastic than most of us had probably suspected, and that an entirely new growth form can evolve in fungi on a very short time scale!

Figure 1 from Lendemer & Hodkinson (2013): This tree shows the placement of Lepraria s.l. species (including the species of Leprocaulon) in multiple groups within four families of Ascomycetes. Newly generated sequences of Lepraria s.l. are mapped to the topology of the Schmull et al. (2011) Lecanoromycetidae phylogeny. All taxa with a "leprarioid" growth form are in red.

- Brendan

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Reference:

Lendemer, J. C., and B. P. Hodkinson. 2013. A radical shift in the taxonomy of Lepraria s.l.: molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification. Mycologia 105: 994-1018.
Download publication (PDF file)
View data and analysis file web-portal (website)

Science Lichen Spoof

Recently Science magazine spoofed a bunch of other journals to test their peer-review procedures. It revealed what one might call a very porous filtration system on the part of many journals. A fake study with some pretty obvious problems was accepted for publication in quite a number of journals. You can read more about it here. One thing that caught my attention was that the fake article was on lichens and their potential anti-cancer properties. It is worth noting that there have been several recent articles that were not part of the spoof that show some anti-cancer properties of Usnic Acid; however, it's always important to carefully evaluate the legitimacy of any particular study and remain skeptical until results have been widely validated!

- Brendan

Lichen-Inspired Technology

This article shows how lichens are inspiring new technological advancements:
http://www.ncbi.nlm.nih.gov/pubmed/23881167

Here is one excerpt from the article:

"Inspired by crustose lichens, one of the most common lichens that can grow within rocks, with a root-like fungal structure growing within the rock itself and only a crust-like fruiting body above the surface, we designed a novel integrated counter electrode architecture, in which a porous carbon plate (PCP) was used as a conductive substrate and continuous, vertically oriented ordered mesoporous carbon (OMC) monolithic films were rooted in the porous substrate to serve as catalytic layers (Scheme 1). Morphologically, the main body of crustose lichens (thallus) is made up of a few distinct layers. The lowermost layer consists of densely packed fungal hyphae with a root-like structure (rhizines) that can attach the thallus to various substrates such as rocks, plants, buildings, and even coral reefs. The root-like structure not only enables the lichen to adhere strongly to the substrate, thus preventing it from being peeled off by the wind or waves, but it also ensures the symbiont a much larger active area for mineral and water uptake, and photosynthesis. Herein, instead of a condensed flat conductive substrate such as FTO or metal foil, a porous carbon plate with both high conductivity and high mechanical strength was used as the CE substrate. A root-like integrated OMC–C composite structure formed by a precursor inflation pyrolysis process can harvest the electrons that arrive at the counter electrode and transfer them to the catalytic layer/electrolyte interface efficiently."

Pretty cool stuff!

-Brendan

Copper-Infused Socks for Diabetic Foot Care

I just came across an article about a new type of therapy for diabetic foot ulcers.  It caught my attention especially because it is aimed at treating infections that are fungal in nature.  In short, the product is a new type of sock infused with copper, which is shown to help stop the growth of certain problematic fungi.  This is extremely relevant to the ongoing diabetic foot ulcer microbiome work that we're doing in the Grice Lab.  Hopefully this product will provide some relief and reduce complications for many diabetes patients suffering from infections that may be primarily fungal!

- Brendan

Against the naming of fungi

Here's an interesting opinion piece published in Fungal Biology entitled "Against the naming of fungi:"
http://www.sciencedirect.com/science/article/pii/S1878614613000871
It highlights problems with the Linnean taxonomic system, and proposes using a system of molecularly-based operational taxonomic units (OTUs) that are named in a standardized way.  While this is an interesting and appealing idea, it comes with its own problems.  I think that it is important for everyone to recognize the problems with the current system, but I cannot say I'm on board with any particular alternative.  We'll see how much traction this gets in years to come!

-Brendan

Nelsenium

In a recent paper on the genus Lepraria (Lendemer & Hodkinson 2013), my co-author and I established a new genus named 'Nelsenium' for Lepraria usnica, a corticolous, Paleotropical species (Sipman 2003) that had previously been misclassified. The name honors Matt Nelsen, a fellow North American lichenologist who was the first to sequence Lepraria usnica and conducted molecular phylogenetic analyses to determine its familial affinities within the Lecanoromycetes. When Nelsen et al. (2008) used mtSSU sequence data to infer the higher level systematic placement of Lepraria usnica, they recovered the species as a member of the Pilocarpaceae (which is not the family of Lepraria). In our recent paper we affirmed this based on an analysis of 11 mtSSU sequences of Pilocarpaceae including L. usnica. Nelsen et al., however, did not formally describe a new genus to accommodate this species, so we established the genus Nelsenium to place L. usnica in its proper context.

We used a molecular diagnosis for this genus, naming the particular nucleotide positions that were unique in the mtSSU region.
"Diagnosis: A genus of the Pilocarpaceae, with a sterile, leprose crustose thallus whose mtSSU sequence (AY300894) differs from those of Fellhanera bouteillei (AY567787) and F. subtilis (AY567786) at these nucleotide sites: adenine at positions 96, 106, 232, 355, 689, 760, 806 and 813; cystine at positions 146 and 741; guanine at positions 44, 101 and 292; and thymine at positions 83, 233, 254 and 909."
I think this type of approach will become more and more common, especially in fungal taxonomy, where synapomorphies are often very difficult to find (especially for higher-level taxa) if one does not resort to examining the primary structure of nucleic acids or proteins.

To see how the new genus looks, feel free to peruse these photos of the type specimen of the type species, Nelsenium usnicum (Sipman) Lendemer & Hodkinson:
http://www.bgbm.org/digitalimages/Singa/k-pictures/P1010776k.jpg
http://www.bgbm.org/digitalimages/Singa/k-pictures/P1010777k.jpg

- Brendan

---------------------
References

Lendemer, J.C., and B.P. Hodkinson. 2013. A radical shift in the taxonomy of Lepraria s.l.: molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification. Mycologia 105: 994-1018.
Download publication (PDF file)

Nelsen, M.P., H.T. Lumbsch, R. Lucking, and J.A. Elix. 2008. Further evidence for the polyphyly of Lepraria (Lecanorales: Stereocaulaceae). Nova Hedwigia 87: 361-371.

Sipman, H.J.M. 2003. New species of Cryptothecia, Lepraria, and Ocellularia (lichenized Ascomycetes) from Singapore. Bibliotheca Lichenologica 86: 177-184.

Katydid Lichen Mimic

Here's a cool new species of katydid that looks like a lichen!
http://www.isidewith.com/article/new-to-nature-no-112-lichenagraecia-cataphracta

-Brendan

Beatrix Potter

Today is the 147th birthday of Beatrix Potter.  Although she is best know for her books geared toward children, she was also an amateur mycologist/lichenologist and produced many drawings of fungi.  Here is one lichen drawing that she did.  She was also a big proponent of the dual nature of lichens (i.e., that they represent a symbiosis of fungi and algae), which was still controversial in her day.  You can read more about her scientific pursuits here!

-Brendan

Lichens: A Cure for Cancer?

As the current overseer of recent lichen literature, I have noticed a number of articles that have come out in the past year or so demonstrating the anticancer effects of lichen compounds, specifically usnic acid.

Here are a few recent titles with links to articles:

Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines

Usnic acid inhibits breast tumor angiogenesis and growth by suppressing VEGFR2-mediated AKT and ERK1/2 signaling pathways

Usnic acid inhibits growth and induces cell cycle arrest and apoptosis in human lung carcinoma a549 cells

It will be fascinating to see where this might lead!

- Brendan

mothur MiSeq SOP

As someone who uses the MiSeq platform for high-throughput DNA amplicon sequencing, I was excited to see a new paper in Applied & Environmental Microbiology that presents a pipeline for processing these data. Users of the program "mothur" were sent an email announcing the publication:

"Hello mothur users...
I know many of you have been interested in our protocols and the ideas behind our new MiSeq SOP. We have some great news - the manuscript that we've been working on has been accepted to Applied and Environmental Microbiology. As of today, it is available online ahead of publication. You can find the manuscript here:
http://aem.asm.org/content/early/2013/06/17/AEM.01043-13.full.pdf+html
Until it's published, if you would like the supplement please let me know and I'll send it your way.
Pat Schloss"

So mothur now may become the program of choice for those studying microbial diversity using the MiSeq. Currently, I am using a hybrid approach, with PANDAseq for read assembly, mothur for screening, and QIIME for everything else. However, it is difficult to argue with the notion that it's always easier to use a single program!

- Brendan

Press Release - New Sterile Lichen Taxa

Today I am featured in "Penn News Today"! You can read the full official press release here. It discusses a few of the papers that I published recently describing new lichen taxa. I've already found a number of news sources picking it up. I'll be interested to see how far and wide it'll go!

- Brendan

Reduced PhiX

I recently commented on a post on the 'Next-Gen Seq' blog. The post mentioned the need to use a ~50% spike-in of PhiX DNA to run the MiSeq. Some others started talking about the fact that the software has now been upgraded so that it is not necessary to use such a large spike-in (~1-5% is now recommended). Here is my comment:

"We just did a MiSeq 2x150 amplicon run with the new software and an extremely small PhiX spike-in and it has effectively doubled our yield! It's great to see the technology continually improving; we're now getting almost 10 times as many reads from each 2x150 MiSeq run as we did just a few months back."

As it stands right now, Illumina seems to be doing a really good job of continually improving their technology and staying ahead of the competition on most fronts. We'll see how it goes as the dominant sequencing platforms continue to duke it out in the coming years!

- Brendan

Katydid

Here's a great photo of a katydid that wishes it were a lichen:
http://www.projectnoah.org/spottings/21755032/fullscreen

...and here's a video of one in action, hanging out with Usnea thalli!
http://www.youtube.com/watch?v=bHeiNTe6N9M

- Brendan

When Lichens Meet Water

Here's a pretty cool short video showing what happens to foliose lichens when they get wet:

Thanks to Roger Rosentreter for alerting me to it!

- Brendan

Grice Lab Website

The Grice lab has a new website!  It just went live, so feel free to check it out:

http://www.med.upenn.edu/gricelab/

- Brendan

Public Repositories

I was recently asked by a Library Science graduate student to take a survey regarding my data archiving practices. For part of the survey I was asked to comment on the different databases that I use for archiving data and state why I chose them. Here are my comments on the public repositories for molecular data that I use:

GenBank/NCBI: It is difficult to deposit large data sets here (although it has been improving somewhat in usability), but it is the standard database for molecular biology. A major advantage is that the data are integrated into a larger framework and all sorts of NCBI tools and programs can be used for future researchers to find and analyze the data alongside the data from nearly all other projects in the field of molecular biology.

Data Dryad: It is extremely flexible in the data formats allowed (making my work more reproducible for other scientists) and it's easy to deposit any type of biological data.

MG-RAST: This database is good for metagenomic data sets, but it can be moderately difficult and time-consuming to complete a submission and make it public. However, once the data are public, this repository allows researchers to see various aspects of large molecular biological sequence data sets through a set of tools, and some comparisons can be made between data sets.

TreeBASE: Phylogenetic trees and DNA/protein sequence alignments can be deposited here in a very strictly-regulated way, although there is really no integration of data sets (e.g., comparison or combination of data sets). It is often required by journals that files for evolutionary biology studies be deposited here, but it is usually difficult because of the formatting requirements.

Overall, I think it's best to use multiple databases. I prefer to put all data and analysis files in Dryad, and then use the other databases when appropriate for the specific types of data. The more places the data are, the more likely people are to find them!

- Brendan

Disclaimer: These comments are the opinions of the author based on personal experiences and do not represent the views of any affiliated institutions or funding agencies.

PandaSeq to QIIME

Recently I have been working with some paired-end amplicon MiSeq data. One important step with paired-end amplicon data is assembling each pair of reads to make composite sequences. PandaSeq is a good program for doing this, but when assembling two reads, it seems to use only the portion of the identifier shared between the two reads as the identifier for the assembled read. This becomes problematic for me when performing downstream analyses with QIIME. Therefore, I wrote the following simple Perl script to make the identifiers of the PandaSeq assembly file jive with the identifiers in the corresponding indexing reads file generated after the MiSeq run (so they can both be processed together using QIIME):


#!/usr/bin/perl

my $filename = <$ARGV[0]>;
chomp $filename;
open (FASTQ, $filename);
{
if ($filename =~ /(.*)\.[^.]*/)
{
open OUT, ">$1.fixed.fastq";
}
}

while ()
{
if ($_ =~ /^\@(\w*\-\w*\:\d*\:\w*\-\w*\:\d*\:\d*\:\d*\:\d*)\:/)
{
print OUT "\@$1 2:N:0:\n";
}
else
{
print OUT $_;
}
}


The above text can be copied into a file (to make the Perl script) and then invoked with the following:
perl script.pl assembly.fastq

Note that these instructions presuppose that you have three .fastq files from your paired-end MiSeq run:
01 - Reads from one end of each amplicon
02 - Index reads
03 - Reads from the other end of each amplicon
These files are generated by default when making .fastq files with some Illumina software, but sometimes making all three files (notably the indexing read file) requires specifying certain parameters. As of version 1.6.0 of QIIME, the group of indexing reads must be entered as a separate file if these data are to be properly integrated into the QIIME workflow.

One final issue that arises once reads have been assembled is that there are now fewer reads in the assembly file than there are in the index file. This can be remedied by making a barcode file with only the entries associated with sequences in the PandaSeq assembled data set. I use the following two commands (after running the above Perl script) to take care of this issue:
sed -n '1~4'p assembly.fixed.fastq | sed 's/^@//g' > defs_in_assembly.txt
filter_fasta.py -f SampleID_NoIndex_L001_R2_001.fastq -o index_reads_filtered.fastq -s defs_in_assembly.txt

I then do a final check to see if the entries in the index file and the assembly file are truly the same:
sed -n '1~4'p index_reads_filtered.fastq | sed 's/^@//g' > index_defs_filtered.txt
diff -s index_defs_filtered.txt defs_in_assembly.txt

If those two files are the same, then the .fastq files should be ready to run through the split_libraries_fastq.py script to start the QIIME workflow.

Please let me know if you use the above Perl script or if you run into issues with any of this!

- Brendan


[Update - I just got back a data set from another facility in which the first part of the identifier for each sequence (the section before the first colon) was written in a slightly different format. To deal with this, the above 'if' line that comes after 'while' should read as follows:
if ($_ =~ /^\@(\w*\:\d*\:\w*\-\w*\:\d*\:\d*\:\d*\:\d*)\:/)
If you are not sure which format your identifiers take, it may be best to try the script as written above and then try it with this modification.]

Lichens of GSMNP

A book just came out last week entitled "The Lichens and Allied Fungi of Great Smoky Mountains National Park." It documents the lichen diversity of GSMNP in a far more comprehensive way than has ever been done before. Here is the official summary from Amazon:

"Like the Great Smoky Mountains themselves, much about the lichens of the Smokies has remained shrouded in mystery. This book sheds considerable light on the diversity of these intriguing organisms in the Smokies, a diversity that is unmatched in any other American national park. Written by three of this country's foremost lichen specialists and based on their extensive field and herbarium studies, this book is a comprehensive summary of current knowledge of the lichen biota of Great Smoky Mountains National Park. Included in this treatment: revised and annotated checklist; comprehensive keys to all 804 known species of lichenized, lichenicolous, and allied fungi; extensive ecological notes on noteworthy discoveries; discussion of records for new and interesting taxa; formal description of 2 genera and 12 species new to science; color micrographs illustrating all new genera, and species distribution maps for selected species."

In this book, I co-authored a number of new taxonomic combinations based on morphological and molecular research that I have conducted. The book also includes several species that I have described in past works. This publication will serve as a great resource for researchers studying lichen diversity in eastern North America, since the southern Appalachians (as far as we have documented so far) seem to house more lichen diversity in a smaller area than any other part of the region.

- Brendan

Chirleja buckii, a new genus and species

Recently I co-authored a paper describing a new genus and species from Tierra del Fuego in southern South America. The new genus is Chirleja, named after the local word for lichen/moss in the particular part of the world where it was found. The species is C. buckii, named after Bill Buck, who found it on an NSF-funded expedition. It was published in the New Zealand Journal of Botany, the premiere journal for botany in the southern hemisphere.

Chirleja buckii (scale = 0.5 mm)

We used molecular data from the mtSSU locus to infer the placement of the species in the family Icmadophilaceae, and we could also tell from these analyses that it did not fit within any of the described genera in the family. Many other sterile crustose lichens like this one represent new lineages of fungi that have not previously been described. Our research into crustose lichens is therefore helping to fill in unknown parts of the fungal tree of life and better illuminating the evolutionary history of the fungi.

- Brendan

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Reference

Lendemer, J. C., and B. P. Hodkinson. 2012. Chirleja buckii, a new genus and species of lichenized fungi from Tierra del Fuego, southern South America. New Zealand Journal of Botany 50(4): 449-456.
Download publication (PDF file)
View data and analysis files (website)

Caloplaca reptans, an enigmatic sterile lichen

I recently published a paper in Systematic Botany detailing a case in which DNA and bioinformatics finally made it possible to describe an enigmatic sterile lichen species known from the Appalachian Mountains.

Since sexual characteristics are the primary way that fungi are classified, this sterile species could not be put into our current classification based on how it looks.  The small grayish-greenish species described in the paper (Caloplaca reptans) was known for years in the Appalachian Mountains because it is so distinctive.  However, it was never described formally because no one could figure out what its closest relatives were (and since the genus was uncertain, it could not be given a binomial). We used our DNA-based approach to infer its phylogeny and found that it is closely related to members of the genus Caloplaca, a genus in which most of the species are bright orange or similarly colored.  We can now say that its placement makes sense based on some of its other characteristics, but no one would have guessed that it was just an odd member of that group, one that has apparently lost its ability to make the brightly-colored pigments.

A small, isolated thallus of Caloplaca reptans on rock (scale = 0.5 mm)

I am continuing to conduct research using molecular sequences and bioinformatics to discover and describe new lifeforms so that we can better understand the planet's biodiversity.  Be on the lookout for more papers in the future along these lines!

- Brendan

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Reference

Hodkinson, B. P. and J. C. Lendemer. 2012. Phylogeny and taxonomy of an enigmatic sterile lichen. Systematic Botany 37(4): 835-844.
Download publication (PDF file)
View data and analysis file webportal (website)