The Licheniad

The Licheniad

by Sean Q. Beeching

Canto I

A lichen, one may theorize,
When on the future casts his eyes,
His dear descendants he descries.

Eternal life, so it appears,
And a youth that lasts a thousand years,
The lichen spurns as cause for tears.

He dreams, or she, perhaps I’ll say,
Of numerous, happy, progeny,
With whom it would, perforce, parté.

The truth to which one must attest,
Is that our lives may not be blest,
And nuclear winter may end the fest.

In which event the lichen too,
Will perish along with me and you,
Unless a lifeboat he can constue.

But how to effect the goal sublime?
To reproduce two souls entwined,
Involves a course most labyrinthine.

Too sadly he must bid ado,
To sex, I’m sorry, but it’s true,
The ordinary method won’t work for two.

By sex what here we represent,
Does not demand adult consent,
Simple meiosis is all that’s meant.

These poor dears, for aught we know,
Lack the genders and hence forgo,
What here to say would not be apropos.

And had they genders, why they’d be four,
Two for the fungus, for the alga two more,
As state of affairs one would deplore.

It would lead to confusion,
To mishap and exclusion,
And not, in fine, to the hoped for diffusion.

One half, if the better, I’ll not say,
May be engendered in the usual way,
What results, I’m afraid, is a lichen manqué.

Oft tales are told of the sailing spores,
Which travel the heavens beyond our shores
And by means unknown the lichen restores.

Yet how the bionts reunite,
On what rare moonlit starry night;
It remains conjecture, that secret rite.

Yet how the lichen reunites,
By what fantastic arcane rites
Remains unknown, they’re unseen sights.

(Betwixt the preceding tercets twain
Neither of the other could the advantage gain,
I’ve left them both as a short refrain.)

Though it may happen, it seems farfetched,
Belief must needs be sorely stretched,
To credit the procedure I have thus far sketched.

Instead the lichen puts his trust,
In structures far removed from lust;
They seem to us no more than dust.

Within the thallus, all unseen,
Assembles the lichen his breeding machine,
As in an ant hill he were the queen.

At length the surface of its skin,
Reveals the tumult deep within,
It writhes and wrinkles and waxes thin.

Upon its face begin to vent,
Depending on the creature’s bent,
Features odd but of small extent.

Their smallness is indeed a test
Of our student’s eyesight. They protest,
And think our labels a cruel jest.

They’re each quite different, we declare,
Which drives them all to black despair,
As at the mocking plants they stare.

And truth to tell not even we,
Are always sure which one we see,
Isidia, soredia, which could it be?

Regardless which these fly aloft,
Both symbionts combined but soft,
They fall to earth, not seldom but oft.

Back on the earth, behold, it sprouts,
It lives, it breathes, convention flouts,
And grows to manhood, or thereabouts.

Thus, dear readers, have you now heard,
A tale as marvelous but far less blurred,
Than that of the logos, the living word.

Should that comparison seem extreme;
By it am I seen to blaspheme:
It’s nothing, I assure you, but blown off steam.

Fast UniFrac for barcoded 454 16S amplicons

When analyzing large 16S sequence data sets from multiple samples to infer community patterns, one of the best analytical methods available is Fast UniFrac.  Barcoded 454 sequencing (a type of 'pyrosequencing') is quickly becoming the method of choice for generating the data for these types of analyses, and Fast UniFrac has been built especially to handle the large data sets generated using this method.  However, there are simple data management issues that can keep researchers from using this methodology.  One major hindrance with Fast UniFrac is that, if one wants to take advantage of the 16S reference tree that is built in to the program (something that becomes almost obligatory with sufficiently large data sets), it is necessary to put all of the sequence names in a specific format that reveals the sample of origin while preserving the individual sequence identifiers.  Below I describe two distinct procedures that I developed for assembling the input file for the first step of the Fast UniFrac pyrosequencing analysis procedure.  I have tested the first procedure on a data set of ~120,000 sequences (this required sending an email to request a higher Fast UniFrac quota, which is typically capped at 100,000 sequences) of ~500bp in length and the second procedure on a data set of ~40,000 sequences of the same approximate length.

Files needed:
Sequence files ('.fna'+'.qual' or '.sff' or '.fasta'+'.groups') with sequence names that are all the same length (standard for 454 data)
Oligos file with primers and barcodes for each sample (for use with Mothur) [http://www.mothur.org/wiki/Trim.seqs]

Special programs needed:
Mothur
A text editor that can perform search and replace on returns (e.g., TextWrangler for Macintosh or TextPad for Windows)
Microsoft Excel 2008 (previous versions max out at ~65000 rows and may do other unexpected things to large data sets)
BLAST (local)
PyCogent
Enthought Python Distribution (I used v6.3) (alternatively, you can download Python and Numpy, but the EPD has versions of these that play well together and should hopefully work with PyCogent as long as they are first in your path... getting Python, Numpy, and PyCogent to talk can be more complicated than it would seem)

Method #1 (no UNIX scripting required):

1) Take the '.fna' (fasta) and '.qual' (quality) files (or alternatively the '.sff' file) plus the manually-produced '.oligos' file for your data set and process them with Mothur using the 'trim.seqs' function [http://www.mothur.org/wiki/Trim.seqs].  This will produce a '.fasta' file with your sequences and a '.groups' file that shows which sequences come from which samples ('environments' in the language of UniFrac).  I recommend using the many functions of Mothur to further cull your data set; however, the amazing power of Mothur will be the subject of future posts.  If you do decide to change the composition of the '.fasta' file in any way (this is always necessary at least to some degree), you can use the Mothur 'list.seqs' function (performed on the finalized '.fasta' file) to generate an '.accnos' file, then follow that by the 'get.seqs' function (performed on the '.accnos' file and the original '.groups' file) to generate a finalized '.groups' file that correlates with the finalized '.fasta' file.

2) Open the '.groups' file in Microsoft Excel and edit the file to create a four-column spreadsheet: (A) '>' on every row; (B) sample names; (C) delimiter (I use '#') on every row, and (D) sequence names.  Highlight all four columns and sort ascending according to column D (sequence names).  This is done by going to 'Data' > 'Sort' > 'Column D', 'ascending'.  Save as 'file1.csv' (comma-delimited text).

3) Open the new 'file1.csv' file in a text editor and remove all commas (this can be easily done in TextWrangler for Mac by opening the file and going to 'Search' > 'Find', putting ',' in the 'Find:' box, typing nothing in the 'Replace:' box, and clicking 'Replace All').  Save as 'file2.txt' (plain text).

4) Open the '.fasta' file with a text editor (e.g., TextWrangler).  Remove all returns (e.g., Find: '\r', Replace: '' in TextWrangler with the 'Grep' box checked on the Find/Replace screen).  Replace all instances of '>' with a return and '>' (e.g., '\r>'; remember that 'Grep' must be checked if you are using TextWrangler).  Now the first line is empty; manually delete that line.  Save as 'file3.txt' (plain text).

5) Import 'file2.txt' into the first column of Microsoft Excel (column A).  On the same spreadsheet, import the names and sequences from file3.txt to the second and third columns (columns B and C) by clicking on cell B1 (column B, row 1) and importing 'file3.txt' as a text file with 'fixed width'; set the field width for the column break manually at the interface between the sequence name and the beginning of the nucleotide sequence (for my data, it was around the 15th place; if this doesn't work with the text import wizard, fixed-width delimitation can be performed under 'Text to Columns...' in the 'Data' menu).  Highlight columns B and C *only* (if you highlight column A as well, this will not work).  Go to 'Data' > 'Sort' > 'Column B', 'ascending'.  At this point, you should have rows that look something like: '>CLSt#GR5DBVW03HJKND' '>GR5DBVW03HJKND' 'TACGATCGATCGATCAGCATCGATCA...' where the columns are correlated with one another and reading across a row should show the same identifier in column A as in column B.  Be sure that this is the case throughout the spreadsheet.  Now delete column B (the one with the identifiers from the imported fasta file).  Save as 'file4.csv' (comma-delimited text).

6) Open 'file4.csv' with a text editor.  Replace all instances of ',,' with a single return (e.g., find ',,' and replace with '\r' in TextWrangler... remember to have the 'Grep' box checked).  Save as 'file5.fasta'.

7) Follow the instructions for "The BLAST to GreenGenes protocol" and 1-7 of the "Steps" in the Fast UniFrac tutorial (download raw pairwise distance matrices for further analyses), all found here:
http://bmf2.colorado.edu/fastunifrac/tutorial.psp

Method #2 (simpler overall, but requires assembling and running a customized UNIX shell script):

1) Take the '.fna' (fasta) and '.qual' (quality) files (or alternatively the '.sff' file) plus the manually-produced '.oligos' file for your data set and process them with Mothur using the 'trim.seqs' function [http://www.mothur.org/wiki/Trim.seqs].  This will produce a '.fasta' file with your sequences and a '.groups' file that shows which sequences come from which samples ('environments' in the language of UniFrac).  I recommend using the many functions of Mothur to further cull your data set; however, the amazing power of Mothur will be the subject of future posts.  If you do decide to change the composition of the '.fasta' file in any way (this is always necessary at least to some degree), you can use the Mothur 'list.seqs' function (performed on the finalized '.fasta' file) to generate an '.accnos' file, then follow that by the 'get.seqs' function (performed on the '.accnos' file and the original '.groups' file) to generate a finalized '.groups' file that correlates with the finalized '.fasta' file.

2) Create and run a shell script on the '.fasta' file that looks something like this:

#!/bin/bash
#$ -S /bin/bash 
#$ -cwd
#$ -o search_replace.log -j y


sed -i s/GMQ03P202B3SVL/MID08#GMQ03P202B3SVL/g file_name.fasta
sed -i s/GMQ03P202BTOMD/MID08#GMQ03P202BTOMD/g file_name.fasta
sed -i s/GMQ03P202CGYZW/MID08#GMQ03P202CGYZW/g file_name.fasta
sed -i s/GMQ03P202BT4E9/MID08#GMQ03P202BT4E9/g file_name.fasta
sed -i s/GMQ03P202BXELV/MID08#GMQ03P202BXELV/g file_name.fasta

It will have to contain a row for every sequence.  This sort of thing can be assembled in Microsoft Excel:
Column A: 'sed -i s/' all the way down the column
Column B: sequence IDs (from a Mothur '.groups' file)
Column C: backslashes all the way down the column
Column D: sample identifiers (correlated with the sequence IDs... easily done if the sequence IDs and sample identifiers are both taken from a Mothur '.groups' file and the order is preserved)
Column E: delimiter (e.g., #) all the way down the column
Column F: identical to column B
Column G: '/g file_name.fasta' all the way down the column
After that is put together, save it as comma-delimited text, open it with a text editor, remove all commas, add the first few lines manually to make it a working script, and run.

3) Follow the instructions for "The BLAST to GreenGenes protocol" and 1-7 of the "Steps" in the Fast UniFrac tutorial (download raw pairwise distance matrices for further analyses), all found here: http://bmf2.colorado.edu/fastunifrac/tutorial.psp


Once you have the raw pairwise distance matrices, they can be analyzed in R or another statistical package/program.  This will be the subject of a later post.  As part of the Fast UniFrac protocol (when using the GreenGeenes core backbone tree), it will be necessary to use PyCogent.  If you have trouble with PyCogent itself (or the command-line in general), please see my earlier post with some tricks and tips on getting that part of the Fast UniFrac 'BLAST-to-GreenGenes' protocol to work.  Hopefully the current post can help someone in constructing the initial input file for the first part of the Fast UniFrac procedure!

- Brendan

P.S. This procedure places '#' in the sequence names as the delimiter. This information will be needed during the PyCogent-dependent portion of the Fast UniFrac protocol. Here is an example of what I type in the command line once I have navigated to the folder where I have placed the 'create_unifrac_env_file_BLAST.py' python script and the 'blast_output...' file (the confusing thing with my nomenclature is that the Python input is named 'output' because it is the BLAST output, while the Python output is named 'input' because it becomes the Fast UniFrac input; the logic behind this is that the essence of this step is the transformation of the BLAST output file into the Fast UniFrac input file):

python create_unifrac_env_file_BLAST.py blast_output_allreplaced_ready4python.txt fastunifrac_input_file.txt #

Update: These instructions are now published as part of my dissertation (Hodkinson 2011) and an article in Environmental Microbiology (Hodkinson et al. 2012a); the supporting data/analysis/instruction files for the latter are available from the Dryad data repository (Hodkinson et al. 2012b). 

----------------------------------------------

References

The above instructions are published in the following sources:

Hodkinson, B. P. 2011. A phylogenetic, ecological, and functional characterization of non-photoautotrophic bacteria in the lichen microbiome. Doctoral Dissertation, Duke University, Durham, NC.
Download Dissertation (PDF file)

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2012a. Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Environmental Microbiology 14(1): 147-161.

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2012b. Data from: Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Dryad Digital Repository doi:10.5061/dryad.t99b1.

----------------------------------------------

The development of the above protocols was supported in part by NSF (DEB-1011504) and the US Department of Energy.

PyCogent for Fast UniFrac

As someone studying the composition of lichen-associated bacterial communities, I have generated several data sets of 16S rRNA gene sequences from bacteria that live in this specialized niche. Beyond the simple question of "who lives there?" we can start to use phylogenetic inferences to examine the ecology of this niche by comparing sets of 16S sequences from different communities and taking into account where the different members fall in a phylogeny. UniFrac is a tool that allows the integration of phylogenetic information into ecological comparative community analyses, and its hip new cousin Fast UniFrac is all the rage these days. But, alas, fully utilizing the special features of Fast UniFrac (such as mapping pyrosequencing reads to a reference phylogeny) requires PyCogent, the installation of which has given me much grief recently. 

PyCogent is a great Python-based toolkit that can be used for conducting a number of analyses on biological sequence data (DNA, RNA, proteins); it is billed as "making sense from sequence" (Knight et al. 2007).  There is a good guide to PyCogent known as the PyCogent Cookbook.  Some programs/packages/pipelines that depend on PyCogent include QIIME and Fast UniFrac (for the latter, PyCogent is required only if you have a large 16S data set that requires a guide tree).

I have had trouble getting the different versions of Python, NumPy, and PyCogent to communicate with one another through UNIX (on both CentOS and MacOSX... although all of the various versions of the different dependencies may have been an issue, since I do not own the machines and I run several versions of Python myself locally), but I ran through the simple 2-step protocol listed below on Windows XP and Windows 7 and it worked very well for running the Python script associated with the Fast UniFrac 'BLAST-to-GreenGenes' protocol. This is a little odd since it is written that installation of PyCogent by itself is not supported for Windows... and the procedure that I outline below seems to be a pretty simple way to get it installed.

Installing and running PyCogent requires using the command line. If you would like to do this on a Windows machine and you are unfamiliar with the Windows command line, you can google tutorials on "MS-DOS" and/or "command prompt".  There is a decent introductory guide here. The instructions below are written in a broad, inclusive way so that they should work with a UNIX-based system as well (including Macintosh; if you are a Mac user and are unfamiliar with the command line, you can google something like "Mac OSX Terminal" or find a good beginners' tutorial here).  

Whatever type of system it is, the PATH variables must be set correctly so that the programs can find one another.  As long as you do not have previous versions of Python, NumPy, or PyCogent installed, Windows should automatically set the environmental variables so that this protocol will work without a hitch (Macintosh most likely will not set the variables automatically because it usually comes with a pre-installed Python that it will always want to use).  Click here to see a post that further addresses one of the issues with the wrong version of Python/NumPy getting in the way. 

Here is my simplistic protocol for getting PyCogent moving enough to run the Python script mentioned above (I should note that this protocol is not approved by the makers of PyCogent, since it may not produce a fully-functional package, but it does allow me to run the script):

1) Installing Python, NumPy, etc.:
Install the most recent version of the Enthought Python Distribution package (free for academics).

2) Installing PyCogent:

Download the most recent version of PyCogent ('.tgz' file).
Unzip the folder (using, e.g., WinRAR, WinZip, or 7-Zip; an automatic partial unzip might leave it as '.gz' but one of the previously mentioned programs will allow you to unzip it fully and you can drag the folder to your desktop if necessary).

In the command line, navigate to the PyCogent directory.
Type in the command line:

python setup.py install

There are some further notes on installation here and in the README, but please note that it was the fact that these instructions didn't quite get me to where I was going that inspired me to write this post. Still, they are likely to provide exactly what is needed for most situations.

Depending on the sort of jobs you need to run using PyCogent, a single computer may or may not have enough computing power.  I have an interest in PyCogent because I need it to run the aforementioned script that makes the Fast UniFrac '.env' input file (see the Fast UniFrac tutorial for more details on how this fits into the overall Fast UniFrac protocol).  A single computer processor has more than enough computing power to handle this job, but some of the more advanced QIIME functions will certainly require greater power for sufficiently large data sets.

Hopefully the notes here can make Fast UniFrac more accessible to more people (specifically, when the mapping of pyrosequencing reads to a reference tree is required), since the various errors that may occur with PyCogent, NumPy, Python, etc. can be difficult. If you wish to use PyCogent directly, you will probably have to be somewhat familiar with the Python programming language, although the cookbook has enough examples that one may be able to stumble through it naively (not that I would recommend it). If you're like me, and only use PyCogent so that you can map sequences to a reference tree for Fast UniFrac, then everything else you'll need to know can probably be found in the excellent Fast UniFrac tutorial. The Fast UniFrac 'BLAST-to-GreenGenes' procedure also requires a local installation of BLAST (installation instructions for PC, Mac, Linux, etc.). Making the initial input file for this specific type of Fast UniFrac analysis can require some creative thinking, and will be the subject of a future post.

- Brendan

Poemules

Those involved in Southeastern U.S. licheneering have probably met Sean Q. Beeching on a foray. His knowledge of the local lichen flora of Georgia and neighboring states surpasses that of most lichenologists for any region. He even has a species named after him: Megalaria beechingii (Lendemer 2007). He has also authored a book of essays... I highly recommend the fascinatingly-titled: "'I Like You But What Can You Do, Can You Be a Bird?': Adventures In The Lichen Trade." Page 3 of this bulletin provides a review of the book that gives some clues as to what kinds of essays you will find between the covers.

I recently received a message from Sean regarding the video of James Lendemer on the NPR website and some of the thoughts that it inspired in him:

"
I sent James's lichen video, by way of your blog, to a friend of mine, the botanist Lisa Kruse, and she, being sharper than me, noticed that the narrator says that the lichen fungus eats the algae. She then asked me if that is true because her understanding, and mine, was that the fungus appropriates from the algae, in one way or another, its nutrients without actually consuming the little green fellows. Instead of answering the question I sent her these three poems, or perhaps, poemules, would more accurately describe them. And thus I send them to you in the hope that you find them amusing.... on the other hand, if recent investigations have determined that the fungus does indeed devour the algae, I would like to know about that.

Three rhymes on the lichen symbiosis occasioned by a lichen video’s narrator having said that the lichen fungus eats the lichen algae.

Were I to venture to explain
How doth the fungus entertain,
I’d not have said he eats his guests
But rather shares in their bequests.

If to me the burden fell,
To illustrate the lichen, well,
More like the cow-maid, I would hold,
The fungus cultivates her fold,
She slaughters not her gentle beasts,
But rather milks them by their teats.

How doth the lichen food obtain?
The fungus, I would ascertain,
The alga probes with fingers shrewd.
His ticklish damsels something lewd,
And makes the ladies to surrender,
Unto its hands the purloined provender.
"

My only thoughts on the issue of whether the fungus 'eats' the algae are that [1] the fungi eat the products made by the algae (and they often even seem to enhance the production of food by the algae) and [2] the fungi presumably must devour the carcasses of the dead algae. Whether or not the fungi may, under certain conditions, 'kill' some of the algae or speed along their ultimate demise is not something that I think is known, although I would welcome additional commentary on the matter!

- Brendan

---

Works Cited:
Lendemer, J.C. 2007. Megalaria beechingii, a new species from the southern Appalachian Mountains of eastern North America. Opuscula Philolichenum 4: 41-44.
Download publication (PDF file)

PICS-Ord

Just this week I had a 'methodology' article published in BMC Bioinformatics.  Robert Lücking of the Field Museum was the first author, and we worked with Alexis Stamatakis (of RAxML fame) and Reed Cartwright (creator of Ngila and Dawg).  The paper is entitled "PICS-Ord: Unlimited Coding of Ambiguous Regions by Pairwise Identity and Cost Scores Ordination" and it presents a method for encoding data found in ambiguously-aligned regions of multiple sequence alignments in a way that makes it possible to integrate such data into standard molecular phylogenetic analyses.  Most researchers simply exclude data found in ambiguously-aligned regions of nucleotide or amino-acid alignments when conducting phylogenetic inferences.  While such practices are perfectly sound, a large amount of potentially informative data is subsequently left out of downstream analyses.  However, using a method to recode these regions and integrate the data into phylogenetic analyses allows one to consider all of the data present in the larger molecular regions being analyzed.

Until PICS-Ord, no method had been devised for properly integrating this type of data into likelihood-based analyses (e.g., ML, Bayesian).  INAASE (Lutzoni et al. 2000) is a program that recodes ambiguously-aligned regions, but since the distances between different sequence types are encoded as cost matrices, its utility is limited to parsimony-based analyses.  It also has a finite number of symbols, making it impractical for large data sets.  For each ambiguously-aligned region, PICS-Ord uses ordination of scores (which are based on pairwise alignments between the sequences for each taxon) to create a series of axes that are converted to discreet characters which can be appended to a multiple sequence alignment.  The matrix of the sequence alignment plus the recoded characters can then be analyzed phylogenetically based on any number of criteria, including maximum likelihood (ML) and Bayesian inference.

PICS-Ord is available here as an R-based program.  As academic software goes, it's pretty friendly, but please let us know if you run into any troubles.  The publication of this method along with a program for implementation represents a great leap forward in phylogenetics, with the ability to finally integrate data from ambiguously-aligned regions into likelihood-based analyses!

-Brendan

P.S. Find out more about PICS-Ord here on Reed Cartwright's blog:
http://pandasthumb.org/archives/2011/01/pics-ord-unlimi.html

---

Works Cited:

Lücking, R., B. P. Hodkinson, A. Stamatakis, and R. A. Cartwright. 2011. PICS-Ord: Unlimited Coding of Ambiguous Regions by Pairwise Identity and Cost Scores Ordination. BMC Bioinformatics 12: 10.
Download publication (PDF file)
Download R-based PICS-Ord program (zipped program package)
View program wiki (website)

Lutzoni, F., P. Wagner, V. Reeb, and S. Zoller. 2000. Integrating ambiguously aligned regions of DNA sequences in phylogenetic analyses without violating positional homology. Systematic Biology 49: 628-651.
Download publication (PDF file)
Download Java-based INAASE program (zipped program package)

Snow Camp Lichens

A few years back I stopped into a little town called Snow Camp, NC, and saw some amazing rooftops with wooden shingles filled with Cladonia lichens!  I had never seen colonies so large in this region before.  Recently, I realized that I would be passing by the town again and decided to take some photographs of this phenomenon.  I had to do some acrobatics to get close-ups of them.  You can click on the photos to see greater detail!

 

Standing on the back of a car to photograph the lowest of the Cladonia-filled rooftops.

 One half of a roof almost entirely covered with lichens.

 

A roof corner with Cladonia cristatella and other species.

A close-up of just one shingle.

Happy New Year!

- Brendan

Our Lady of Lichens

This statue of Mary sits in a graveyard near our home.  Notice all of the beautiful orange lichens that cover her!

 

Have a merry Christmas!

- Brendan

Lichens in the Media

Recently, National Public Radio (NPR) interviewed James Lendemer of the New York Botanical Garden for a portion of the "Science Friday" program about lichens. The primary product that resulted from this interview was a video that they posted on their website:
http://www.sciencefriday.com/videos/watch/10336
My favorite part of the video occurs when they show James hiking through the forest hunting for lichens and there is a voice-over of James saying "I think of myself as a bounty hunter." On the weekly national broadcast they did a five-minute piece in which they briefly discussed lichens and heavily referenced the above video. Online there is a transcript and a link to an mp3 of the broadcast. There are also some lichen photos that viewers/listeners sent in to NPR after hearing the program:
http://www.sciencefriday.com/newsbriefs/read/203

Lepraria hodkinsoniana (photo taken by an NPR listener)

It's great to see a piece like this promoting lichenology in the media!

-Brendan

USA Science

Last month I volunteered to take part in the USA Science and Engineering Festival Expo in our nation's capital, Washington D.C.! There were all kinds of exhibits hosted by agencies, professional science organizations, and businesses like NASA, the American Society for Microbiology, and Lockheed Martin.

"
Mission:
Our mission is to re-invigorate the interest of our nation’s youth in science, technology, engineering and math (STEM) by producing and presenting the most compelling, exciting, educational and entertaining science gatherings in the United States.
"

If the powers that be decide to throw this festival a second time, it would be great to have a representation of the country's leading lichenological, bryological, and mycological societies. I'll keep an eye on future plans!

- Brendan

Beneficial Microbes

Recently I attended the American Society for Microbiology's 3rd Conference on Beneficial Microbes, which was held in Miami, FL. It was great to meet so many people using techniques similar to the ones that I am using for my research into the bacteria of the lichen microbiome. I was especially excited to go to the session entitled 'Ecology and evolution of microbial communities' since this is one of the major topics that interests me. There were talks by some of the stars of microbial community ecology (e.g., Dr. Rob Knight and Dr. Ruth Ley) and other great talks as well. There were certainly other sessions with interesting talks, too, including one by Dr. Giles Oldroyd on 'Reprogramming Plant Cells for Endosymbiosis.'

Here is a short description of the conference:
"Biologists are becoming increasingly aware that animals and plants have coevolved with diverse assemblages of microorganisms that are required for normal health and development. Not surprisingly, the activity of these symbiont communities is also likely to profoundly affect all aspects of the host’s physiology. An understanding of these complex interactions requires contributions from a diverse, multidisciplinary group of researchers, including microbial ecologists and geneticists. The goal of this conference is to bring together a wide array of scientific expertise to foster the development of this rapidly expanding field of biology."

I returned excited to continue my research on the lichen microbiome, and I look forward to speaking again with some of the people that I met!

- Brendan

Tennessee Journey

Over the past couple of weeks I've been in Tennessee working at the Oak Ridge National Laboratory and attending a class on High Performance Computing for Phylogenetics at the University of Tennessee.  My family came along, and since we were staying in Knoxville, which is only an hour from the Great Smoky Mountains, we spent one Saturday visiting the G.S.M. National Park.  We happened to be there right in the peak of fall color season and had a great time looking at the foliage and exploring the amazing diversity of lichens in and around the park!

 

An official park stamp with Cladonia apodocarpa.

 

Traveling companions.

Checking out the lichen diversity!

-Brendan

NC Science Festival

A little over a week ago, I participated in a Science Expo that was one of the culminating events of the North Carolina Science Festival.  It was great fun to bring my traveling lichen show, complete with real lichens, microscopes, and plenty of amazing lichen photos.  I was also sure to engage passersby and answer any questions that they might have about lichen biology and the science of lichenology.  While my table was one of the only 'natural history' type displays, there were all sorts of science demos, hands-on activities, lab tours and talks.  It was a great time!

- Brendan

Bayesian reversible-jump MCMC: deflating inflated support values

Recently, on the Botanical Society of America Student ListServ, commonly-used phylogenetic methods were being discussed, and a specific problem was raised with regard to Bayesian phylogenetic inference: it has been documented that, within a Bayesian framework, support values (in the form of posterior probabilities) can become inflated or skewed.  This is an issue of special interest to me, since I commonly use Bayesian inference alongside other phylogenetic methods in my research (e.g., Hodkinson & Lendemer 2010, Hodkinson & Lutzoni 2009, Miadlikowska et al. 2006).  I decided to contribute to the discussion by sending the following message to the ListServ:

"
I have found that Bayesian phylogenetic inference methods can do amazing things (like finding the same topology with one gene that only emerges with two or three genes in an analysis based on maximum parsimony (MP) or maximum likelihood (ML)).  However, this problem of Bayesian methods inflating support (especially at short internodes) makes me feel very suspicious of any given Bayesian posterior probability value. Therefore, if I want to evaluate support, I always look at MP- and/or ML-bootstrap proportions.  The Bayesian problem that we're talking about has been discussed by Lewis et al. (2005), and they state that the solution is to use reversible-jump MCMC (rjMCMC).  However, I have wondered myself how I could actually implement this.

I recently found an article that clearly stated the fact that MrBayes and BEAST cannot perform rjMCMC (Kodandaramaiah et al. 2010): http://dx.doi.org/10.1016/j.ympev.2009.08.012
In this article, they used Phycas for their rjMCMC analyses.

So I went to the Phycas manual (Lewis et al. 2010), and found that it does give the nitty-gritty of how to implement this (see section 2.3 on 'Polytomy Priors'):
http://phylo.bio.ku.edu/phycas/manual.pdf
I have not yet done it myself, but I am very excited to try it out!  Perhaps now I will have more faith in my posterior probabilities!
"

The Phycas manual gives the best, most concise summary of the issue that I have seen anywhere:

"
A solution to the 'Star Tree Paradox' problem was proposed by Lewis, Holder, and Holsinger (2005). Their solution was to use reversible-jump MCMC to allow unresolved tree topologies to be sampled during the course of a Bayesian phylogenetic analysis in addition to fully-resolved tree topologies. If the time between speciation events is so short (or the substitution rate so low) that no substitutions occurred along a particular internal edge in the true tree, then use of the polytomy prior proposed by Lewis, Holder, and Holsinger (2005) can improve inference by giving the Bayesian model a 'way out.' That is, it is not required to find a fully resolved tree, but is allowed to place a lot of posterior probability mass on a less-than-fully-resolved topology. Please refer to the Lewis, Holder, and Holsinger (2005) paper for details.
"

Please post comments here if you have performed these types of analyses and have any additional insights!

-Brendan

P.S. Please see my more recent posts for the details of using Phycas:
http://squamules.blogspot.com/2011/06/installing-and-running-phycas.html
http://squamules.blogspot.com/2011/06/writing-phycas-script.html

---

Works Cited:

Hodkinson, B. P., and J. C. Lendemer. 2011. Molecular analyses reveal semi-cryptic species in Xanthoparmelia tasmanica. Bibliotheca Lichenologica 106: 115-126.
Download draft (PDF file)
Download alignment (NEXUS file)

Hodkinson, B. P., and F. Lutzoni. 2009. A microbiotic survey of lichen-associated bacteria reveals a new lineage from the Rhizobiales. Symbiosis 49: 163-180.
Download publication (PDF file)
Download alignment (NEXUS file)

Kodandaramaiah U., C. Pena, M. F. Braby, R. Grund, C. J. Muller, S. Nylin, and N. Wahlberg. 2010. Phylogenetics of Coenonymphina (Nymphalidae: Satyrinae) and the problem of rooting rapid radiations. Molecular Phylogenetics and Evolution 54(2): 386-394.
View Publication (webpage)

Lewis, P. O., M. T. Holder, and K. E. Holsinger. 2005. Polytomies and Bayesian phylogenetic inference. Systematic Biology 54(2): 241-253
View Publication (webpage)

Lewis, P. O., M. T. Holder, and D. L. Swofford. 2010. Phycas User Manual, Version 1.2.0.
View Manual (PDF file)

Miadlikowska, J., F. Kauff, V. Hofstetter, E. Fraker, M. Grube, J. Hafellner, V. Reeb, B. P. Hodkinson, M. Kukwa, R. Lücking, G. Hestmark, M. Garcia Otalora, A. Rauhut, B. Büdel, C. Scheidegger, E. Timdal, S. Stenroos, I. Brodo, G. Perlmutter, D. Ertz, P. Diederich, J. C. Lendemer, P. May, C. L. Schoch, A. E. Arnold, C. Gueidan, E. Tripp, R. Yahr, C. Robertson, and F. Lutzoni. 2006. New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two protein-coding genes. Mycologia 98: 1088-1103.
Download publication (PDF file)
Download supplement (PDF file)
Download alignment (zipped NEXUS file)

A recipe with fungi and algae!

I have never actually eaten a lichen, but I was thinking the other day about how I do like to eat both fungi and algae, especially together.  Several years ago, my wife and I took a Korean cooking class, where we learned some great recipes.  The one that we enjoy cooking the most is Bee-bim Bop, a dish with all kinds of veggies served over rice.  We usually serve it like a salad bar, where people can put whatever items they want on their rice then mix it themselves.  In addition to the veggies, both fungi (mushrooms) and algae (seaweed) are a traditional part of the dish, and help to form a flavor symbiosis of sorts.

Here's our recipe for Bee-bim Bop:

Ingredients (you can vary the quantities of each vegetable depending on your preference)
Rice (cook ~1/2 cup per person)
Shiitake mushrooms
Bean Sprouts (~1 cup)
Eggs (to be whipped, fried, and sliced; we use 3 eggs)
Carrots (to be cut into matchsticks and fried; 1 big carrot should do it)
Onion (to be chopped and fried)
Cucumber (to be cut into matchsticks)
Lettuce (1 small head or less)
Tofu (firm, cubed; ~ 1 cup)
green onion tops for garnish
Seasonings: salt, garlic, sesame oil, sesame seeds, sugar, soy sauce, and cayenne pepper

Now there is a lot of vegetable chopping to be done!

Directions:
1. Soak dried mushrooms at least 30 minutes
2. Wash and get rice cooking.
3. Prepare marinade sauce for mushrooms and tofu: 1t salt, 1/2t garlic, and 1t sesame oil (make as much as you need using those proportions).  Put the tofu (raw) in a bowl with some of the sauce to marinate while you do the next few steps.
3. Wash the bean sprouts and put them in a pot with a little water (not quite covering them) and a pinch of salt. Cook them, covered, about 15 minutes on medium-high heat, then drain and place in a bowl.
4. Heat some sesame oil in a frying pan for the eggs. Mix the eggs and a dash of salt vigorously with a fork and then pour them into the hot pan. Let it spread out as much as possible. Flip it after a couple minutes, and when the "pancake" is cooked through, take it out of the frying pan and slice it into long, thin strips.
5. Cut the carrots and onions into thin pieces and fry them (with salt) in the hot oil until the carrots are tender.
6. Drain the re-hydrated mushrooms and squeeze excess water out of them.  Mix them with some of the sauce from step 3 and fry.
7. Fry the tofu in hot oil with black pepper.
8. Drain the cooked bean sprouts and season them with a little bit of salt, garlic, sesame seeds, and sesame oil.
9. Cut the cucumber and lettuce into long thin strips. 
10. Prepare Goo Choo Chang sauce: mix 1/2t cayenne pepper, 1/2t garlic, 1t sesame seeds, 1T soy sauce,  1T sugar, and 1T sesame oil.  You'll want to put this in a little container for pouring at the dinner table.

When you have it all ready, you should have on the table a big bowl of rice and small bowls with the mushrooms, sprouts, egg strips, carrots & onions, tofu, cucumber, and lettuce. 

To Serve:
Put cooked rice into individual serving bowls.
Put vegetables, eggs, tofu, and mushrooms on top of the rice.
Pour Go Choo Chang sauce over bowl.
Mix thoroughly, and add more sesame oil to the mixture depending on your preference.
Serve with Kim Chee and Laver seaweed (thin and crispy... not sushi nori).
 

The ingredients on display (with a small one who is quite excited about the algae)!

The final product, with algae around the edges and fungi in the mix with the rest.

All that's left at this point is to give your hands a thorough washing and dig in; we typically eat this by using the little sheets of laver as our utensils!

Enjoy!

-Brendan

Virginia Lichen Checklist

Back in 2004 I began assembling the first checklist of Virginia lichen taxa. The first draft of this work consisted of a literature review plus an inventory of Virginia specimens held by the DUKE Cryptogamic Herbarium (DUKE) and various herbaria with publicly-accessible databases. However, I decided not to publish any of that work, instead favoring an approach that would produce a list of expert-verified taxa for the state. I did this because I was in a unique position to correct some of the taxonomic problems that have arisen over the years, and a literature review with an herbarium inventory would only perpetuate previous errors.

Beginning in 2006, I worked primarily with Dr. Richard C. Harris ('Dick') of the New York Botanical Garden to assemble the lichen checklist for the state, and ensured that Dick verified at least one specimen for each taxon in the list. This resulted in the first checklist of Virginia lichens, lichenicolous fungi, and allied taxa, published last year in Evansia (Hodkinson et al. 2009). However, taxonomy is ever-evolving, and new species are constantly being discovered. To address this issue, I have designed a website that can be updated at any time to reflect changes in our understanding of the Virginia lichen flora:
http://www.duke.edu/~bph8/VirginiaLichens/checklist.html

Virginia’s physiographic provinces

The most recent addition to the list, from just this week, is the lichenicolous fungus Skyttea radiatilis (Tuck.) R.Sant., Etayo & Diederich, identified by James Lendemer. The species was described just within the past decade; it seemingly grows only on a single sterile crustose lichen species, and is probably not rare, even though it is seldom collected and identified. In order to understand why it is so seldom seen, one must simply ask: 'How many people are collecting and carefully examining sick-looking sterile crustose lichens?' (Hint: not many). Additions like this have brought the total number of verified taxa for the state to just over 600, and additional collecting work that I have recently done in the outer Coastal Plain of Virginia has revealed many more (keep an eye out for this work, to be published sometime next year).

I welcome any comments or corrections to the Virginia Lichen Checklist. Let us not forget that, since so much remains to be discovered about the diversity of lichenized fungi, regional inventories like this one are still crucial for the advancement of the field!

-Brendan

---

References:

Hodkinson, B. P., R. C. Harris, and M. A. Case. 2009. A Checklist of Virginia Lichens. Evansia 26(2): 64-88.
Download publication (PDF file)

Hodkinson, B. P., R. C. Harris, and M. A. Case. 2010. A Checklist of Virginia Lichens. http://www.duke.edu/~bph8/VirginiaLichens/checklist.html [updated: 25 August 2010].
View authors' updated checklist (website)

Hodkinson, B. P., and M. A. Case. 2008. A lichen survey of Williamsburg, Virginia. Banisteria 31: 24-30.
Download publication (PDF file)
Download supplement (Excel file)

Hodkinson, B. P. 2010. A First Assessment of Lichen Diversity for One of North America's 'Biodiversity Hotspots' in the Southern Appalachians of Virginia. Castanea 75(1): 126-133.
Download publication (PDF file)

Parmelia barrenoae

Recently I published an article in the journal North American Fungi on the distribution and morphology of the foliose lichen Parmelia barrenoae (Hodkinson et al. 2010). One significant aspect of this paper is that it highlights the importance of good herbarium collections. Bill and Chicita Culberson traveled to Morocco in 1971, and to Calfornia in 1972; on both trips, they collected generally, and happened to pick up specimens of P. barrenoae without knowing it (since the species was not yet described). However, when I went into the DUKE herbarium to look for specimens, I found two continental records (for both Africa and North America) just sitting there!  

The species was described from Spain a few years back (Divakar et al. 2005), but the emphasis of the originial paper was on the molecular phylogeny and the fact that this distinctive species was differentiated by having simple rhizines (as opposed to the squarrose rhizines seen in the closely-related Parmelia sulcata). An examination of the herbarium material from a broader geographic range allowed us to additionally highlight the fact that the species has distinct soralia that are erose, instead of the more erumpent soralia seen in Parmelia sulcata. 

Figure 1. Parmelia barrenoae (A-E; all from Lendemer 19720) and comparison of rhizines with P. sulcata (F, from McGarrity s.n.). A, lobe morphology (scale = 2.0 mm). B, detail of lobe tip (scale = 0.5 mm). C, young soralium (scale = 0.5 mm). D, soralia (scale = 1.0 mm). E-F, comparison of rhizines in P. barrenoae (E) and P. sulcata (F) (scale = 0.2 and 0.5 mm respectively). 

Several lichenologists (e.g., Trevor Goward and Ernie Brodo) had recognized this entity in western North America as a species not on the continental checklist (N.A. lichen checklist), but Ted Esslinger recognized it as P. barrenoae and James Lendemer generated the ITS rDNA sequence data confirming it. Additional ITS rDNA sequences from this species recently deposited in GenBank from Morocco and Turkey (previously deposited sequences were from Spain, and ours from California) give molecular barcode sequence data from throughout the species's known worldwide range.  Now there is at least one ITS sequence (the more-or-less 'official' fungal barcode) from each of the continents from which the species has been reported, giving a good sampling of the sequence diversity found across its range. 

For another nice set of photos of this species, check out these ones on MushroomObserver.com!

-Brendan

---

Works Cited:

Divakar, P. K., M. C. Molina, H. T. Lumbsch, and A. Crespo. 2005. Parmelia barrenoae, a new lichen species related to Parmelia sulcata (Parmeliaceae) based on molecular and morphological data. Lichenologist 37: 37-46.

Hodkinson, B. P., J. C. Lendemer, and T. L. Esslinger. 2010. Parmelia barrenoae, a macrolichen new to North America and Africa. North American Fungi 5(3): 1-5.
Download publication (PDF file)
View journal volume (website) 
View sequence data (NCBI website)

---

After posting this, I was alerted to the following article in Spanish, which gives some additional discussion of morphology and ecology, with some nice micrographs:
Barreno, E., and M. A. Herrera-Campos. 2009. Parmelia barrenoae Divakar, MC. Molina & A. Crespo un liquen nuevo para la flora asturiana. Bol. Cien. Nat. R.I.D.E.A. 50: 333-341.
Download publication (PDF file)

New Lichen Web Resources

The New York Botanical Garden has recently made available a set of excellent web resources for the study of lichens in North America.  Here is the announcement sent out by James Lendemer:

"

We are pleased to announce that two new lichen websites have been posted that can be accessed via the NYBG Virtual Herbarium (http://sciweb.nybg.org/science2/VirtualHerbarium.asp). These websites treat the lichen biotas of two large biogeographic regions of North America: the Ozarks and the southeastern Coastal Plain.

1) Lichens of the Ozarks - http://sweetgum.nybg.org/ozarklichens/

This website presents the results of Richard Harris and Doug Ladd's research on the lichen biota of the Ozark Ecoregion in central North America. Highlights of the website include a dynamic specimen-based checklist, dynamic literature listings including linked pdfs when applicable, and a pdf of the keys to Ozark lichens produced for the Tuckerman Workshop in Eureka Springs, Arkansas.

2) Lichens of the Southeastern Coastal Plain - http://sweetgum.nybg.org/southeastlichens/index.php

This website is an ongoing effort intended to present the results of our continuing research on the lichen biota of the Coastal Plain of southeastern North America.

We welcome all comments, corrections, and suggestions. These should be sent to jlendemer@nybg.org

"

The southeastern Coastal Plain project builds on the research and collecting done by Richard Harris for the classic works Some Florida Lichens (Harris 1990) and More Florida Lichens (Harris 1995).  Currently, we are actively working further north in the Coastal Plain, conducting inventories (e.g., Hodkinson & Case 2008, Hodkinson et al. 2009, Lendemer & Hodkinson in prep), elucidating distributional patterns (Lendemer & Hodkinson 2009, 2009a), and describing new species that occur in the region (e.g., Lendemer & Hodkinson 2010, in prep).  

Those who are attending the Botany 2010 conference in Rhode Island will have an opportunity to see a talk by James Lendemer on the progress of the work so far in the southeastern Coastal Plain (Lendemer et al. 2010).  At the upcoming conference, I will be co-leading a workshop on an integrated approach to lichen systematics (Lendemer et al. 2010a), presenting a poster on semi-cryptic species (Hodkinson & Lendemer 2010, 2010a), and giving a talk on 'rhizobes' associated with lichens (Hodkinson & Lutzoni 2010).  Hopefully, I will see some of you there!

- Brendan

[Research on the lichens of the Ozarks was supported by NSF Grant DEB-0206023.]

---

References:

Harris, R. C. 1990. Some Florida Lichens. Published by the author. Bronx, NY. 109 pp.

Harris, R. C. 1995. More Florida Lichens, including the 10 cent tour of the pyrenolichens. Published by the author. Bronx, NY. 192 pp.

Download publication (PDF file)

Harris, R. C., and D. Ladd. 2005. Ozark lichens; Enumerating the lichens of the Ozark Highlands of Arkansas, Kansas, Illinois, Missouri, and Oklahoma. Published by the authors. Bronx, NY. 249 pp.

Download publication (PDF fie)

Hodkinson, B. P., and M. A. Case. 2008. A lichen survey of Williamsburg, Virginia. Banisteria 31: 24-30.
Download publication (PDF file)

Hodkinson, B. P., R. C. Harris, and M. A. Case. 2009. A Checklist of Virginia Lichens. Evansia 26(2): 64-88.
Download publication (PDF file)
View authors' updated checklist (website)

Hodkinson, B. P., and J. C. Lendemer. 2010. Molecular analyses reveal semi-cryptic species in Xanthoparmelia tasmanica. Bibliotheca Lichenologica: in press.
Download draft (PDF file)
Download alignment (NEXUS file)

Hodkinson, B. P., and J. C. Lendemer. 2010a. How do you solve a problem like Xanthoparmelia? Molecular analyses reveal semi-cryptic species in an Australasian-American 'disjunct' taxon. Botany 2010, abs. 355.
View abstract (website)

Hodkinson, B. P., and F. Lutzoni. 2010. Do lichens harbor their own 'rhizobia'? A large-scale phylogenetic survey of lichen-associated bacteria from the order Rhizobiales. Botany 2010, abs. 347.
View abstract (website)

Lendemer, J. C., and B. P. Hodkinson. 2009. The Wisdom of Fools: new molecular and morphological insights into the North American apodetiate species of Cladonia. Opuscula Philolichenum 7: 79-100.
Download publication (PDF file)
Download alignment (NEXUS file)
 
Lendemer, J. C., and B. P. Hodkinson. 2009a. Stretching the boundaries: A range extension for Buellia wheeleri R.C. Harris. Evansia 26(4): 172-176.
Download publication (PDF file)
View dynamic range map of Buellia wheeleri (google map)
 

Lendemer, J. C., and B. P. Hodkinson. 2010. A new perspective on Punctelia subrudecta in North America: previously-rejected morphological characters corroborate molecular phylogenetic evidence and provide insight into an old problem. Lichenologist 42(4): 405-421.
Download publication (PDF file)
Download alignment (NEXUS file)

Lendemer, J. C., R. C. Harris, and B. P. Hodkinson. 2010. Connecting the dots: progress and problems in assessing lichen biodiversity and biogeography in the coastal plain of southeastern North America. Botany 2010, abs. 30.
View abstract (website) 

Lendemer, J. C., B. P. Hodkinson, and M. Piercey-Normore. 2010. Cladonia Systematics: What can we infer from morphology and molecules? Botany 2010, abs. 1052.
View abstract (website)

Lichens of Nome, Alaska

Lichens of Alaska, Part III: Nome

Pannaria pezizoides surrounded by Stereocaulon sp.

Candelariella aurella with Lecanora sp. and unknown Collemataceae.

Bryocaulon divergens with Alectoria ochroleuca  and Cladonia spp.

Lichen-covered igloo mounds on the tundra.

Ochrolechia upsaliensis with Flavocetraria spp. and Cladonia sp.

Xanthoria-covered rocks overhanging clear waters.

Thanks for all the interest in my photos.  A nice field photo accompanied by a good specimen can have great scientific and educational value, so I encourage everyone to snap and collect (with permission, of course)!

-Brendan

[Funding provided by NSF Award DEB-0640956.]