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

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

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

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

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)

RLL 2012

The final installment of the 2012 portion of the Recent Literature on Lichens series has been published in the December issue of The Bryologist. This year I have been the first author on all of the installments and have gotten a chance to see the field of lichenology really moving forward! The count for the RLL series reaches nearly 1,000 citations each year. Having a resource like this for the field of lichenology does seem to help to keep works from slipping into obscurity: it provides at least one formal citation for each work and allows the citations to be further integrated into numerous online digital databases. I look forward to continuing the compilation of these annotated bibliographies into the new year and beyond!

- Brendan

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

References

Hodkinson, B. P. and S. Z. Hodkinson. 2012. Recent literature on lichens—227. The Bryologist 115(4): 626-632.

View publication

Hodkinson, B. P. and S. Z. Hodkinson. 2012. Recent literature on lichens—226. The Bryologist 115(3): 465-473.

View publication

Hodkinson, B. P. and S. Z. Hodkinson. 2012. Recent literature on lichens—225. The Bryologist 115(2): 365-374.

View publication

Hodkinson, B. P. and S. Z. Hodkinson. 2012. Recent literature on lichens—224. The Bryologist 115(1): 183-193.

View publication

Some South Carolina Lichens

This year I co-authored a paper on the findings of the 19th Tuckerman Lichen Workshop, which took place in 2010 near August, Georgia. While sites in both Georgia and South Carolina were visited, the paper focused on the collections from South Carolina, since that state is more neglected lichenologically. Here's the abstract, which gives more of the exciting details:

"The Tuckerman Lichen Workshop is an annual event where professional and amateur lichenologists convene to practice their skills in lichen identification while exploring the lichen diversity of an area in eastern North America. The 19th workshop in this series was held in the Augusta-Aiken area of Georgia and South Carolina from 11-15 March 2010. While we visited sites on both sides of the state line, this report presents checklists from three sites visited in South Carolina. Habitats visited represent the Piedmont and Southeastern Plains ecoregions and contain forested and rocky habitats, including granitic outcrops and bluffs, mesic and bluff forest, and sandhill oak-pine forest. We found a combined diversity of 255 taxa with 93, 125 and 148 taxa per site. Site lichen biotas were found to have low diversity similarities with Jaccard results each = 0.25, and were found to differ in terms of habit (growth form) and substrate. Noteworthy finds include three recently described species: Caloplaca yuchiorum, Lepraria hodkinsoniana, and Ramboldia blochiana. Also newly reported for South Carolina are Graphis oxyclada and G. pinicola, both recently reported as new to North America."

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

Reference:

Perlmutter, G. B., J. C. Lendemer, J. C. Guccion, R. C. Harris, B. P. Hodkinson, W. P. Kubilius, E. Lay, and H. P. Schaefer. 2012. A provisional survey of lichen diversity in south-central South Carolina, U.S.A., from the 19th Tuckerman Lichen Workshop. Opuscula Philolichenum 11: 104-119.
Download publication (PDF file)

Vertiliquens

Here's an interesting blog I came across: Lichens & Climbing. If you speak Catalan, you might get more out of it than me! It's got a lot of great photos on lichens and the scenery surrounding them.

- Brendan

QIIME vs. mothur

When conducting 16S-based microbial community analyses for my dissertation (Hodkinson 2011; Hodkinson et al. 2012a, 2012b), I decided to process most of my next-generation sequencing data (454 at that time) using the program mothur (Schloss et al. 2009). This program worked well, as it was easy to install and extremely flexible. At the time, I knew about the program QIIME (Caporaso et al. 2010), which also seemed to be a good option for these types of analyses, but many of its dependencies were in conflict with system installations on the computer cluster I was using at the time, which made it extremely difficult to install. The QIIME virtual box was meant to get around this problem, but the size of the data sets it could handle were too small for it to be useful for my purposes. I had colleagues who were in favor of QIIME and colleagues who were in favor of mothur, but some of my collaborators on the project were already accustomed to using mothur. So mothur it was, and I was very happy with its performance, especially once I learned enough programming in R to supplement my mothur pipeline with additional statistical analyses in R.

I am now working with 16S sequence data from microbial communities generated through paired-end Illumina instead of 454. Since barcodes are sequenced in a fundamentally different way in the Illumina system, Illumina sequence data are not really very compatible with the current version of mothur [update: please see more recent comments below regarding the mothur MiSeq SOP], at least at the beginning of an analysis pipeline. Of course, complex Perl scripts could be written to manipulate the Illumina output so that mothur could use it; however, QIIME already has specific scripts for processing Illumina data. Since I am now in a lab that uses QIIME regularly (and, therefore, it has been expertly installed on our main Linux server), there are no longer roadblocks in place for using this program. So I am now using QIIME for all of my 16S data processing, and it's great! I really like its visual outputs and its to-the-point scripts that can perform complex functions with a single command.

So my question for everyone who conducts microbial community analyses is: mothur or QIIME? ...or do you prefer something else? ...perhaps a combination of different programs?

I suppose at this point I prefer QIIME for very standard 16S-based community analyses, but if I want to do something creative and complicated with amplicon data (as I am doing for one upcoming paper), a pipeline built based on mothur commands still seems best due to that program's extreme flexibility.

-Brendan

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

References

Caporaso, J.G., J. Kuczynski, J. Stombaugh, K. Bittinger, F.D. Bushman, E.K. Costello, N. Fierer, A.G. Pena, J.K. Goodrich, J.I. Gordon, G.A. Huttley, S.T. Kelley, D. Knights, J.E. Koenig, R.E. Ley, C.A. Lozupone, D. McDonald, B. D Muegge, M. Pirrung, J. Reeder, J.R. Sevinsky, P.J. Turnbaugh, W.A. Walters, J. Widmann, T. Yatsunenko, J. Zaneveld and R. Knight. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7: 335-336.
View publication (website)

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. 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
View data and analysis file web-portal (website)
Download data and analysis file archive (ZIP file)

Hodkinson, B.P., N.R. Gottel, C.W. Schadt and F. Lutzoni. 2012b. Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Environmental Microbiology 14(1): 147-161. doi:10.1111/j.1462-2920.2011.02560.x
Download publication (PDF file)
Download supplementary phylogeny (PDF file)

Schloss, P.D., S.L. Westcott, T. Ryabin, J.R. Hall, M. Hartmann, E.B. Hollister, R.A. Lesniewski, B.B. Oakley, D.H. Parks, C.J. Robinson, J.W. Sahl, B. Stres, G.G. Thallinger, D.J. Van Horn and C.F. Weber. 2009. Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Applied and Environmental Microbiology 75(23): 7537-7541.
View publication (website)

Cryptogamic Invaders

Over the weekend, I spent most of my time up on the roof of my new home. My main objective was to clean it, which meant physically scraping the lichen and moss from the shingles. Here are some photos:

Standing at the apex of the garage roof

Scraping a particularly cryptogamically-encrusted roof portion

For those worried about the scientific implications, we did take some samples to document the property's cryptogamic flora!

- Brendan

Relocating to Philadelphia

Last week I relocated to the University of Pennsylvania in Philadelphia.  I have started a new position as a Postdoctoral Research Fellow in the lab of Dr. Elizabeth Grice in the Department of Dermatology (School of Medicine).  The lab that I have joined is focused primarily on the skin microbiome, the collection of organisms that live on the surfaces of our bodies.  This position will give me a perfect opportunity to conduct research that integrates my main interests, specifically biodiversity, bioinformatics, ecology, evolution, and genomics, in a system that is new to me.  In this position, I will be able to maximally utilize my computational skills and continue to keep pace with the ever-changing next-generation sequencing technology.  I've already started on a few projects, and have encountered some exciting bioinformatics challenges.  As I continue my blog, I will surely place a greater emphasis on bioinformatics of next-generation sequencing data in my posts, so be prepared!

Thanks to all for your support!

- Brendan

Platismatia wheeleri

Last week I had an article published in North American Fungi on the recently described species Platismatia wheeleri. The first author is Jessi Allen, currently a graduate student at NYBG who is partially funded by our NSF grant to study the lichens of the Mid-Atlantic Coastal Plain. The study was inspired by her recent collection of the species, which was described just last year. After carefully examining a number of reference specimens, it became clear that the species, described from intermountain western North America (southern British Columbia, Idaho, Montana, Oregon and Washington), was collected from both the southwestern-most corner of the United States (southern California) and the Tatra Mountains of Slovakia. We were very surprised by the huge disjunction in Europe based on a historical specimen, and we're interested to see if any additional collections turn up from that region! Hopefully this article will alert researchers to the potential presence of P. wheeleri in different regions of the world so we can better understand its historical and current distribution and abundance.

Current distribution of Platismatia wheeleri worldwide (A) and in western North America (B). Red dots are specimens newly reported by us, green dots are from the original description, and yellow dots are from McCune (2012).

A lobe of Platismatia wheeleri (scale = 1 mm)

-Brendan

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

Reference:

Allen, J. L., B. P. Hodkinson, and C. R. Björk. 2012. A major range expansion for Platismatia wheeleri. North American Fungi 7(10): 1-12.
View publication (PDF file)
View data file webportal (website)

Clearwater Valley

A call-to-action has come out over the lichens-l listserve and I thought it would be good to highlight it here:

"Dear lichenologists

A local forestry company is gearing up to log a critical portion of the Clearwater Valley near Wells Gray Park.

Because this valley supports one of the world's richest macro- and mesolichen floras - more than 430 species recorded so far - I'm asking the lichenological community will help us put a stop to this.

There are three ways you can help:

(1) Link to the following website: http://wellsgrayworldheritage.ca/.
Doing this will make clear to the B.C. government that this is more than a local issue.

(2) Share this link with your Facebook friends, with a request that they pass it along.

(3) Write a letter of support. If you've ever visited the Clearwater Valley and would like to help us out in this time of crisis, please drop me a line (trevor.goward@botany.ubc.ca) and I'll send you the details.

Many thanks to all

Trevor Goward"

RLL Q&A

Some people have asked me questions regarding the Recent Literature on Lichens (RLL), and I thought it would be helpful to post some answers here.

Q: What is 'recent'?

A: If a work was published more than 5 years ago, it will not be included in a 'recent literature' list.

Q: What should be done with literature that is more than 5 years old and never made it to a published list?

A: Since there is a database associated with RLL that is routinely used by lichenologist to find literature, it is important to make sure certain older works do not fall by the wayside. If a particular work is never cited in a list as part of the RLL series, it can still be added to the database as part of the 'RLL supplement.' Anyone is allowed to add anything to the supplement at any time through this web-form. Please direct any questions regarding the supplement to Einar Timdal.

Q: How should literature/citations be sent for inclusion in the published RLL lists?

A: Although I am happy to receive reprints of articles, the best way to send along citations for inclusion is by using a modified RIS format, as follows:

TY - JOUR 
T1 - Lichens are cool 
JF - Journal of Awesome Stuff 
VL - 223 
IS - 3 
SP - 123 
EP - 124 
PY - 2012 
AU - Rindalo, F. D. 
AU - L. V. Marisco 
AU - C. Denado 
AB - This article is about why lichens are the coolest. A new species of <i>Parmotrema</i> is described. 
KW - Lichens 
KW - Hipness 
UR - http://dx.doi.org/10.1007/s127-011-0-2 
PN - In Old Norse; new species: <i>Parmotrema redentenii</i> F. D. Rindalo 
ER -

This can simply be typed or pasted into a text file and sent to me. Feel free to look up more information here on RIS format. The modifications used for RLL are (1) the use of html tags for designating italics, (2) a "PN" field for published notes [new taxa and language of article (when not English) are always reported], and (3) the placement of first initials after the last name for the first author, but before the last name for all subsequent authors. So the rule of thumb for literature not yet in the database should be: send it to me if it's less than 5 years old (preferably in RIS format), but if it's older than that, add it to the supplement.

Q: Why do the newer RLL lists have fewer pages than the older ones?

A: There was an editorial request from the staff of The Bryologist to cut down on the length of RLL lists to conserve space. Therefore, summaries of articles are not provided in the most recent lists (only the language of the article and new taxa are given as notes). However, full abstracts are given in the database for the articles that have abstracts freely available electronically.

- Brendan

Recent Literature on Lichens - A New Approach

Recently, I took over as the lead author of the Recent Literature on Lichens (RLL) publication series in The Bryologist.  I want to thank Bob Egan for all of his work over the past many years putting together these lists!  I also thank Sarah Hodkinson, my wife and Master of Library Science, for taking over in the interim while I was finishing up my dissertation work and for staying on as a co-author.  Although RLL may seem pretty much the same on the surface, there have been many changes over the past year going on behind the scenes.  It's finally reached the point where there is a stable set of protocols, thanks to a lot of help from Einar Timdal, who has been working to reconfigure the long-standing RLL database so that it can be updated instantaneously, with citations of recent literature appearing online even before the literature lists are submitted for publication.

The main motivations for changing the RLL protocols were (1) to make it possible to add citations to the list from any computer connected to the internet, (2) to open up the possibility of having multiple authors, and (3) to ensure that citations could become available immediately to the community.  Previously, RLL lists were assembled using the proprietary software EndNote, which is an excellent tool for citation management.  However, this meant that the library of citations typically had to be kept as a single document on a single computer that had the software installed.  Also, the citations needed to be exported in batches and uploaded to the public RLL database every so often.  The procedure now involves adding citations to the online database immediately, either through manual web-form entry or RIS-formatted citation imports.  This means that if one searches the database at http://nhm2.uio.no/botanisk/lav/RLL/RLL.HTM and keeps the "Work file" box checked, it will be possible to see the most recent literature citations that have just been entered into the database.  When it is time to send in the publication, I can export the citations in a format appropriate for The Bryologist, and paste them into the newest manuscript.

Although the workflow seems relatively simple and straightforward, a great deal of 'curation' is still necessary.  However, the procedure meets the goals of portability, flexibility, and accessibility... and it has worked extremely well for the first few lists!

- Brendan

Summer Conferences

It has been a busy month so far! I have attended two conferences this month, giving a talk and presenting a poster at each. The first was the annual Botany conference ("Botany 2012: The Next Generation") in Columbus, Ohio. There were some great talks and a lot of buzz about "next-gen" sequencing technologies (in keeping with the conference's subtitle).  I gave a talk about using next-gen sequencing for elucidating the phylogeny and resolving the taxonomy of sterile crustose lichens. I also discussed the utility of our data sets for examining fungal communities associated with lichens. The poster that I presented put my work in the broader context of my current NSF grant, in which we integrate ecology, phylogeny, taxonomy, and floristics both to expand our understanding of biodiversity and to achieve specific conservation goals. At the Mycological Society of America conference at Yale University, I organized a symposium on phylogenetic, ecological, and functional diversity of fungi. It was a great symposium, with an award-winning student talk given by Kathryn Picard! I also had a great time playing music with other conference attendees. I can't wait to see everyone again next year!

- Brendan

Lichen Roll

I recently came up with another dish featuring both fungi and algae. Inspired by traditional Japanese cuisine, I call my creation the 'Lichen Roll.'  It's basically a standard sushi roll (wrapped in seaweed, the algae), but stuffed with Shiitake mushrooms (the fungi).  

After putting down the rice on the seaweed, I added the mushrooms.

After also adding crab (optional), I rolled it all up tightly.

The roll was sliced and served with ginger, wasabi, and soy sauce on the side.

Let me know if you try the Lichen Roll any time soon... and don't forget to savor the flavor of fungal/algal symbiosis in your mouth!

-Brendan

New taxa from NYBG

A new press release recently came out from the New York Botanical Garden (NYBG) highlighting discoveries of new taxa during the year 2011. Specifically mentioned in the release is the description of the orders Trapeliales and Sarrameanales from my Phytologia paper published last year. Additional taxa that I described (either published or in press in 2011) include Punctelia eganii and Lepidostroma vilgalysii. This release is now being picked up by other websites, such as Science Daily.

- Brendan

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

References

Hodkinson, B. P., and J. C. Lendemer. 2011. Punctelia eganii, a new species in the P. rudecta group with a novel secondary compound for the genus. Opuscula Philolichenum 9: 35-38.
Download publication (PDF file)

Hodkinson, B. P., and J. C. Lendemer. 2011. The orders of Ostropomycetidae (Lecanoromycetes, Ascomycota): Recognition of Sarrameanales and Trapeliales with a request to retain Pertusariales over Agyriales. Phytologia 93(3): 407-412.
Download publication (PDF file)

Hodkinson, B. P., J. K. Uehling, and M. E. Smith. 2012. Lepidostroma vilgalysii, a new basidiolichen from the New World. Mycological Progress 11(3): 827-833. doi:10.1007/s11557-011-0800-z.
View publication (website)
View data and analysis file webportal (website)

Crespoa

Recently, I co-authored a paper establishing the new genus Crespoa (Lendemer & Hodkinson 2012). Here is the abstract:

"Recent molecular phylogenetic analyses of the lichen family Parmeliaceae have revealed that the members of the Parmelia crozalsiana group form a sister clade to one containing members of the genus Parmotrema. The four species in this group were classified first in Parmelia, then Pseudoparmelia, and later Canoparmelia. Recently, the classification of this group was resolved by placing the species in the newly-described Parmotrema subg. Crespoa. This placement was justified by an absence of characters from the fungal reproductive structures distinguishing members of the group from those classified in Parmotrema subg. Parmotrema. As this classification obfuscates a morphologically and phylogenetically discrete group of foliose macrolichens that has always been recognized as distinct from Parmotrema s. str., we here recognize the group as the genus Crespoa. A discussion of taxonomic rank assignment based on character-types that are preconceived as diagnostic is also provided."

This work led to four new combinations:
Crespoa carneopruinata (Zahlbr.) Lendemer & Hodkinson
Crespoa crozalsiana (B. de Lesd. ex Harm.) Lendemer & Hodkinson
Crespoa inhaminensis (C.W. Dodge) Lendemer & Hodkinson
Crespoa schelpei (Hale) Lendemer & Hodkinson

Crespoa crozalsiana is a common inhabitant of the eastern United States, including the study region on which I am primarily focused these days!

- Brendan

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

Reference

Lendemer, J. C., and B. P. Hodkinson. 2012. Recognition of the Parmelia crozalsiana group as the genus Crespoa. North American Fungi 7(2): 1-5.
Download publication (PDF file)

Chincoteague

Last week we collected lichens at the Chincoteague National Wildlife Refuge. Here are some photos showing off the fauna of the refuge:

The ponies of Chincoteague.

The Delmarva Fox-Squirrel, a protected species.

It was a great place for lichens and we collected at several sites throughout the refuge. During the week we collected at several other places along the Delmarva Peninsula. Here's a small portion of the collections that I set out to dry along the top bunk in my cabin:

- Brendan

Smithsonian Botanical Symposium

Last weekend I attended the Smithsonian Botanical Symposium. I greatly enjoyed the talks and meeting the people organizing and attending the event. This year's theme was "Transforming 21st Century Comparative Biology using Evolutionary Trees," which made the conference particularly relevant to my interests. There was a good mix of talks, from ones that were almost purely methodological to ones that were focused on the evolutionary history of a particular group of organisms.

It was also great to be at the National Museum of Natural History in Washington, D.C. Having been born in D.C. and raised in the area, I spent my childhood visiting the NMNH. I even recall a class trip downtown when we had to break into two groups, one of which would go to the Air and Space Museum and the other of which would go to the NMNH; I was the only one who chose the latter (I was never one to conform). It holds a special place in my heart, and it was great to attend a symposium there!

The NMNH mascot.

- Brendan

Turtles of the Mid-Atlantic Coastal Plain

Over the course of the fieldwork for this grant, we have (so far) rescued four turtles from the road.  Amazingly, they all seemed to be different species.  Here is a photograph of me rescuing a snapping turtle, which was easily the most ferocious of all the turtles that we encountered.  Since they are often aggressive and have long necks that can reach ~3/4 of the way across their backs, the only safe way to move a snapping turtle is to pick it up by its tail.  Their tails are very tough and leathery, so there is little risk of hurting the turtle that way.  It's just very important to keep the turtle a sufficient distance from your body when transporting it!

- Brendan

Dismal Swamp

This week marks the first major field expedition under the recent NSF award entitled "Lichen biodiversity in the threatened Middle Atlantic Coastal Plain of North America: Improving classification, conservation, and communication" [DEB-1145511]. I am collecting lichens in Virginia and North Carolina with James Lendemer, Jessi Allen, and Dick Harris, all of the New York Botanical Garden. Today we went to the Great Dismal Swamp. We had two separate bear encounters, which were very exciting! We are also finding a lot of great lichens, and despite being almost all wetlands, the Great Dismal Swamp has proven to be an extremely diverse and heterogeneous area!

-Brendan

MSA Symposium on Fungal Diversity

As I noted in my last post, I will be attending the Mycological Society of America meeting at Yale this summer. At the meeting, I will be chairing a symposium that I put together, entitled Phylogenetic, Ecological, and Functional Diversity of Fungi. Financial support for the symposium will be provided by 454 Life Sciences, a Roche company. The symposium will have an introduction from a representative of 454, one of the major companies providing solutions for large-scale studies of diversity through metagenomics and related fields. After that, there will then be five talks on a variety of topics relating to fungal diversity. Abstracts for all of the talks in the symposium can be found here: http://msa2012.net/media/pdf/diversity.pdf.

Here is the brief formal summary of the event:
"How do we best characterize fungal diversity? The speakers in this symposium will explore fungi in the environment from varied perspectives, examining the relationships and interactions between organisms. This symposium links various disciplines (e.g., systematics, ecology, genetics, metagenomics, metaproteomics) around the theme of studying environmental fungal diversity."

My own talk at the symposium will focus on a new pyrosequencing-based approach that I am using to obtain sequence data for studying the systematics of lichen-forming fungi. Here is the abstract:
"Lichen-forming fungi represent one of the most readily visible and publicly accessible groups of organisms studied by mycologists. Remarkably, despite their 'high profile' nature, many aspects of lichen biology and taxonomy remain poorly understood and critically understudied. These two factors converge noticeably in a highly speciose assemblage of lichens that are referred to as sterile asexually-reproducing crustose lichens, or more simply, 'sterile crusts.' These taxa do not represent a monophyletic group, but rather are treated together because they have all evolved to reproduce primarily through the dispersal of lichenized diaspores (specialized dispersal units that include the major constituents of the lichen microbiome). Species that employ this mode of reproduction have evolved in nearly all of the diverse lineages that comprise lichen-forming fungi. In an effort to overcome the taxonomic impasse in this group, we have developed a set of high-throughput procedures to test for correlations between molecular sequence data and non-molecular character data to phylogenetically place and phenotypically define a large number of species so that they can be formally described and subsequently integrated into conservation and management plans. This work includes using slower-evolving loci (e.g., mtSSU and nucLSU) to place many of the 'incertae sedis' lichen-forming fungi into a higher-level framework and testing species boundaries using the ITS fungal 'barcode' region."

Thanks to all of the speakers for agreeing to participate in the symposium and to all of the people at MSA and 454 who helped me to get this symposium together!

- Brendan

A Modern, Integrated, High-Throughput Biodiversity Research Workflow

This summer I will be attending the Mycological Society of America (MSA) meeting at Yale.  I will be presenting a poster there that details the overall workflow for the research I am conducting at the New York Botanical Garden. You can find the abstract online here:
http://msa2012.net/registration/abstract_status.php?abstract_id=105

A modern, high-throughput workflow for biodiversity research integrating floristics, taxonomy, phylogeny, ecology and conservation

Brendan P Hodkinson* and James C Lendemer

Abstract: The field of Systematic Biology has increasingly become fragmented in recent years, with many scientists focusing in on a small number of methods or approaches for studying biodiversity and its origins. Here we present an integrated workflow for studying the many facets of biodiversity and systematics. This workflow is currently being used to conduct a large-scale inventory of lichens in the Mid-Atlantic Coastal Plain of the United States that includes the collection and analysis of ~35,000 new specimens. Using floristic habitat sampling (FHS), specimens representing the organismal group of interest are collected from individual sites throughout the study region, and are subsequently analyzed anatomically and chemically. Specimens that cannot be identified taxonomically with anatomical/chemical analyses alone are sequenced using a multi-locus 454-based approach. Sequences from the genes of interest (in our case, mtSSU, nucLSU and nucITS) are compared with publicly-available reference sequences to determine the phylogenetic affinities of species that remain either unnamed or 'incertae sedis.' All collections are databased rapidly using modular databasing software (KE EMu). Large-scale ecological analyses are performed on database-generated taxon lists by hijacking data management software originally designed for organizing and analyzing large molecular sequence data sets. Overall results are compiled and made available on the web in the form of, e.g., checklists (for regions, sub-regions and sites), detailed taxonomic treatments, molecular sequence alignments, phylogenetic trees, and specimen-based ecological data sets. Both through these online resources and workshops/forays for the public, information is disseminated for integration into conservation and management plans to preserve biodiversity for the future.

Please contact me if you'd like more specific information on this type of work.  I'm looking forward to putting together the poster and discussing it at the conference!

-Brendan

Lichens of the Mid-Atlantic Coastal Plain

The National Science Foundation (NSF) recently granted me an award to study the lichen biodiversity of the Mid-Atlantic Coastal Plain (MACP) of North America. Here is a link to the official public abstract for the award:
http://www.nsf.gov/awardsearch/showAward.do?AwardNumber=1145511
[DEB-1145511; "Lichen biodiversity in the threatened Middle Atlantic Coastal Plain of North America: Improving classification, conservation, and communication;" PI: Brendan P. Hodkinson, Co-PI: James C. Lendemer, Co-PI: Richard C. Harris]

Although the focus of the project is a lichen diversity survey of the MACP, the project has many additional facets, including ecological hypothesis-testing (to obtain information relevant to pressing conservation issues), molecular phylogenetic work (to resolve species boundaries and place incertae sedis taxa), outreach (to grade-schoolers, undergraduates, graduate students, land managers, etc.), and the production of two major book-length taxonomic works. The taxonomic, floristic, and ecological research will be informed by the collections housed at the New York Botanical Garden's herbarium (NY; totaling ~1/4 of a million specimens) as well as 35,000 new specimens that we will collect from the study area as part of this project.

Here is the full text of the award abstract:

"Lichens are fungi with a unique lifestyle involving a semi-parasitic relationship with an alga and/or cyanobacterium. Although lichens are critical to maintaining healthy ecosystems on land, they remain chronically understudied. This project centers on a survey of lichen diversity in the environmentally threatened Middle Atlantic Coastal Plain of the United States and represents an efficient, comprehensive means of addressing a critical information gap for one of the least known and most environmentally sensitive organismal groups. The survey data will be used to resolve numerous problems related to conservation and ecology through a unique system that integrates newly written computer scripts and programs.

"The project will set conservation priorities in the region and will result in two book-length works on lichens. While preparing these treatments, the authors will depart from traditional approaches by using dynamic online resources to incorporate the insights of professionals and amateurs alike. The investigators will teach a series of courses, mentor a diverse cohort of high school and undergraduate students, and train a PhD student. Extensive interactions and collaborations with regional land managers and naturalists, including a three-day workshop led by the investigators, will ensure that this project has the maximum impact on conservation efforts."

As we make progress in the later stages of the project, we will update the NYBG Southeastern Coastal Plain Lichens website:
http://sweetgum.nybg.org/southeastlichens/index.php
If you have any interest in Mid-Atlantic Coastal Plain lichens, please let me know and we can stay in touch!

-Brendan

Darwin Day

Today my family and I attended a celebration of Darwin's 203rd birthday hosted by the Metropolitan Society of Natural Historians! As part of the birthday party, there was a cake contest.

My wife's entry (which won first place!) was a series of cupcakes illustrating the evolution of mankind, beginning with an ancestral primate akin to a shy, nocturnal aye-aye, moving through our common ancestors with the monkeys and great apes, and culminating in Darwin himself:

The Descent of Man

My entry was a bit more cryptic, but got to the heart of one of the most important aspects of evolution: the fact that individuals within a species vary. Without this, evolution would not be possible. I think that many of the misunderstandings about what evolution is, and how it works, among the general public seem to stem from the under-appreciation of this fact. So I created a group of cupcakes that showed the variation within one species:

Variation Within Species

Happy Darwin Day!

- Brendan

Lepidostroma vilgalysii

I recently had a paper published in Mycological Progress with Jessie Uehling and Matt Smith in which we described a new species of lichen.  This is a special lichen because it is one of the few species in the Basidiomycetes that forms an intimate association with an alga (note that >98% of lichen-forming fungi belong to the Ascomycetes).  The species was first collected by Rytas Vilgalys of Duke University, so naturally we named it Lepidostroma vilgalysii.  There is a "DukeTODAY" post about the species, which was followed up by a podcast.  There was even a Q&A with Dr. Rytas Vilgalys, and an announcement was posted in The Herald-Sun.

Here's a shot of the species in the field:

Notice the yellow clubs (these are the fungal reproductive structures) and the tiny light-green white-rimmed squamules on the soil (these are the lichenized part with the sterile fungus and the algae).

- Brendan

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

References

Hodkinson, B. P., J. K. Uehling, and M. E. Smith. 2012. Lepidostroma vilgalysii, a new basidiolichen from the New World. Mycological Progress doi:10.1007/s11557-011-0800-z.
View publication (website)

Hodkinson, B. P., J. K. Uehling, and M. E. Smith. 2012. Data from: Lepidostroma vilgalysii, a new basidiolichen from the New World. Dryad Digital Repository doi:10.5061/dryad.j1g5dh23.
View data and analysis file webportal (website)

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

P.S. I found this interesting blog post that gives the Lepidostroma vilgalysii article as an example for citation of data packages associated with articles. The public archiving of all data and analysis files associated with published results is extremely important (but often neglected), and is analogous to the archiving of physical resources such as specimens and cultures.

NYBG Lichenology - Year in Review

Recently, Bill Buck of the New York Botanical Garden wrote up a summary of the past year in cryptogamic botany at NYBG.  A portion of this was specifically on lichenology, which I have reproduced below:

"This was a monumental year at New York for lichenology. Several things all came together. First, our lichenologists, led by graduate student James Lendemer, have prepared a manuscript on the lichens of the Great Smoky Mountains National Park, based on their own recent collections and increasing the lichens known from the park by over 60%! This will be published in the Garden’s Memoir series. Next, we received a National Science Foundation grant to digitize our North American bryophytes and lichens. As part of this project we brought to New York Dr. Brendan Hodkinson, who just finished his Ph.D. at Duke University looking at the role that bacteria play in the lichen symbiosis. He is organizing the project and coordinating with other centers of the project. Next, we heard from Dr. Jonathan Dey, at Illinois Wesleyan University, that he would donate his private lichen herbarium to NYBG. Since his thesis was on the lichens of the higher elevations of the Appalachian Mountains, this was a wonderful addition to our holdings of lichens from the Great Smoky Mountains National Park. Although we are still processing this amazing gift, it appears to be about 30,000 specimens! Next, Brendan Hodkinson and James Lendemer applied to the National Science Foundation to inventory the lichens of the mid-Atlantic coastal plain (southern New Jersey to southern Georgia), as postdoctoral students of our lichenologist, Dr. Richard Harris. Much to our delight, this project was fully funded and will begin later this month. In addition to funding the two postdocs, the grant will also cover the expenses of a new graduate student. So, we will continue to have lichenology here at New York for some time to come. Brendan will move off of his administrative role in the one grant and onto the other."

So within the coming weeks, I will officially become the PI on this new grant, entitled "Lichen biodiversity of the threatened North American Middle Atlantic Coastal Plain: Improving classification, conservation, and communication."  I can't wait to get started!

- Brendan

Lichenology Conference in Thailand

I recently attended the 7th International Association for Lichenology Symposium in Bangkok, Thailand.  It was a great conference with many exciting presentations about the most cutting-edge lichen research!  Besides networking, presenting, and listening to talks, I also took a few hours to do some sightseeing around the city.  Here are some photos:

 

See more photos from the conference here!

- Brendan

Lichen Taxonomy

This week a new issue of the journal Opuscula Philolichenum came out. It contains two pieces to which I contributed. The first is an announcement of the new International Committee for the Nomenclature of Lichens and Allied Fungi (ICNLAF), which was formed only very recently (Lendemer et al. 2012). The second is an annotated phylogenetically based summary of lichen taxonomy that I have put together based on the system in use at the New York Botanical Garden (NYBG) (Hodkinson 2012). This will be up on the internet in a format that can be continually updated as our understanding of the organisms evolves. Hopefully it can serve as a useful guide for lichen herbarium/data managers wishing to arrange taxa within a higher-level framework.

Here is the official announcement from the editor:
"The latest issue (volume 11, number 1) of Opuscula Philolichenum is now available online at http://sweetgum.nybg.org/philolichenum/ free of charge as is tradition. This is the first volume to be published in the new age of electronic publication as sanctioned in Melbourne last year. As was noted in the previous issue, the journal has ceased its print run (although one copy is still deposited in the library at NYBG). Volumes will also now consist of multiple numbers, published throughout the year.

"The current issue comprises five contributions covering a diverse array of topics. This includes a higher level taxonomic scheme for lichen-forming fungi that synthesizes current literature. The system may be particularly useful to those wishing to organize herbaria using a phylogenetic system as is common practice in vascular plants. Also included are contributions to our understanding of Hypogymnia in eastern Asia, Parmeliella in South America, and Acarospora in South America. Those who have been following the changes in generic concepts in Acarosporaceae, particularly pertaining to Silobia, will be interested in the discovery by Linda in Arcadia and Kerry Knudsen that Myriospora is actually an earlier available name for Silobia. The authors make the appropriate new combinations and place the taxa previously assigned to Myriospora in a new genus.

"In addition to the above contributions the issue contains the notice of the formation of an International Committee for the Nomenclature of Lichens and Allied Fungi. A group that that aims to work with IAL and various bodies (ICNF, NCF) in the event that matters of nomenclatural governance are formally delegated to special committees by the IBC/IAPT."

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

References

Hodkinson, B. P. 2012. An evolving phylogenetically based taxonomy of lichens and allied fungi. Opuscula Philolichenum 11: 4-10.
Download publication (PDF file)

Lendemer, J. C., M. N. Benatti, T. L. Esslinger, J. Hafellner, B. P. Hodkinson, K. Knudsen, and J. Kocourková. 2012. Notice of the formation of the International Committee for the Nomenclature of Lichens and Allied Fungi (ICNLAF). Opuscula Philolichenum 11: 1-3.
Download publication (PDF file)

Environmental Microbiology

Just yesterday I had an article come out in print in Environmental Microbiology (Hodkinson et al. 2012a) that represents the central chapter of my doctoral dissertation (Hodkinson 2011). It has been published as part of a special issue on 'Omics' and their utility in environmental microbiology and microbial ecology. The study uses 16S rRNA gene sequence data (generated both through molecular cloning and pyrosequencing) to illuminate the bacterial diversity found in lichens. There are a number of interesting discoveries presented, e.g.:
- lichens harbor complex, diverse bacterial communities
- mycobiont, photobiont, and geography are all significant factors in determining bacterial community composition in lichens
- the most taxonomically diverse group is the order Rhizobiales (which contains many symbiotic nitrogen fixers... and many of the groups found within lichens have members that are symbiotic nitrogen fixers)
- the LAR1 (Lichen-Associated Rhizobiales #1; Hodkinson & Lutzoni 2009) lineage is one of the most abundant lineages and seems to be nearly exclusive to lichens
- Acidobacteria is also extremely common, which could potentially be a result of the abundant acidic secondary compounds produced by lichens.

In addition to the above findings (along with the exciting future directions that they point to!), the paper presents many scripts and protocols for managing and analyzing large 16S rRNA gene sequence data sets (Hodkinson et al. 2011). Many of the scripts and protocols that I have posted elsewhere on this blog were derived from the studies that led to this publication. For much of my future research I plan to tweak these scripts and re-purpose them for all sorts of exciting and interesting new applications!

I'll be giving a talk next Monday on this work at the International Association for Lichenology conference (IAL-7) in Bangkok, Thailand (Hodkinson et al. 2012b). I hope to see some of you there!

- Brendan

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

References

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., and F. Lutzoni. 2009. A microbiotic survey of lichen-associated bacteria reveals a new lineage from the Rhizobiales. Symbiosis 49(3): 163-180.
Download publication (PDF file)
Download nucleotide alignment (NEXUS file)
View Dryad data package (website)

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2011. 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

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. [doi:10.1111/j.1462-2920.2011.02560.x]
Download publication (PDF file)
Download supplementary phylogeny (PDF file)
View data and analysis file webportal (website)
Download data and analysis file archive (ZIP file)

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2012b. Pyrosequencing reveals previously unknown phylogenetic, metabolic and ecological complexity within the lichen microbiome. In: IAL-7, International Association for Lichenology, Bangkok, Thailand, in press.

Using R for community analyses

For the central chapter of my doctoral dissertation (published in Environmental Microbiology), I wanted to visualize similarities and differences between lichen-associated bacterial communities, find which factors were most strongly correlated with community differences, and determine the significance of these correlations.  When communities are characterized using large sets of DNA sequence data, this can be achieved by using both genetic measures (e.g., UniFrac distance) and taxon-based measures (e.g., Bray-Curtis OTU-based dissimilarity).

This post gives an example of a script (or a series of commands) to be run with the R statistical package (R Development Core Team 2010) for performing ANOSIM and NMDS analyses of (1) an OTU-based relative abundance matrix derived from a set of clone libraries and (2) a UniFrac distance matrix derived from a 454 amplicon library (see example files at the bottom of this post).  These examples are derived from Hodkinson et al. (2012), although the scripts and example files shown here are abbreviated in order to save space.  Full example files can be downloaded from the Dryad data package associated with this publication [Hodkinson et al. 2011; http://dx.doi.org/10.5061/dryad.t99b1].  The matrices were obtained using the Fast UniFrac program (see instructions here) and Mothur (using the get.relabund command; for the full pipeline that I authored to process these data, see the Dryad data package).  Some minor modifications were required to get the matrices into the precise format needed for R (edits were performed in Microsoft Excel and files were saved in .csv format... note that all of the spaces/tabs in the examples below would actually be commas in the raw text versions of the files).

Running the script as it is presented below (with hash marks in front of the 'plot' commands) will allow one to obtain an output file with the numerical results of all analyses.  However, in order to visualize the NMDS plots, one can run each of the commands line-by-line in the R-GUI (of course, all 'anosim' commands can be ignored if the only goal is NMDS), removing the hash marks in front of the plot commands (note that R has many fancy options for visualization using 'plot'; these may be worth investigating further for your own purposes).  Here is a link to the version of R that I used for these analyses: http://cran.r-project.org/bin/windows/base/old/2.12.0/.  Once R is installed, you can call the script with a command like this one:
C:\\"Program Files"\R\R-2.12.0\bin\i386\Rterm.exe --verbose --no-restore --file=C:\\ANOSIM_NMDS_Clon_454_win.R > C:\\ANOSIM_NMDS_Clon_454_win.out

Here is the script, named 'ANOSIM_NMDS_Clon_454_win.R' (abbreviated example script; full file available at http://dx.doi.org/10.5061/dryad.t99b1):

# load the necessary libraries
library(ecodist)
library(vegan)
# set the output file
sink("ANOSIM_nmds.out", append=TRUE, split=TRUE)
# load the clone data set
samplesClon<-read.csv("C:\\SamplesClon.csv", header=TRUE)
samplesClon
# load the Bray-Curtis OTU-based matrix
OTUsClon<-read.csv("C:\\HodkinsonClon16S_OTUs_1per_171.csv", head = FALSE, row.names = 1)
# transform the OTU matrix into a Bray-Curtis matrix
OTUsClon.dist<-vegdist(OTUsClon, "bray")
# run Bray-Curtis OTU-based ANOSIM
anosim(dis = OTUsClon.dist, grouping = samplesClon$Photobiont, strata = samplesClon$Site)
anosim(dis = OTUsClon.dist, grouping = samplesClon$Mycobiont, strata = samplesClon$Site)
anosim(dis = OTUsClon.dist, grouping = samplesClon$Mycobiont, strata = samplesClon$Photobiont)
anosim(dis = OTUsClon.dist, grouping = samplesClon$Site, strata = samplesClon$Photobiont)
anosim(dis = OTUsClon.dist, grouping = samplesClon$Site, strata = samplesClon$Mycobiont)
# run Bray-Curtis OTU-based NMDS
nmds <- nmds(as.dist(OTUsClon.dist))
nmin <- nmds.min(nmds)
nmin
#plot(nmin, pch=as.numeric(samplesClon$Photobiont))
#plot(nmin, pch=as.numeric(samplesClon$Mycobiont))
#plot(nmin, pch=as.numeric(samplesClon$Site))
# load the 454 data set
samples454<-read.csv("C:\\Samples454.csv", header=TRUE)
samples454
# load the normalized weighted UniFrac matrix
unifracnw454<-read.csv("C:\\DistUFnw454.csv", head = FALSE, row.names = 1)
# run normalized weighted UniFrac ANOSIM
anosim(dat = as.dist(unifracnw454), grouping = samples454$Photobiont, strata = samples454$Site)
anosim(dat = as.dist(unifracnw454), grouping = samples454$Site, strata = samples454$Mycobiont)
anosim(dat = as.dist(unifracnw454), grouping = samples454$Site, strata = samples454$Photobiont)
anosim(dat = as.dist(unifracnw454), grouping = samples454$Mycobiont, strata = samples454$Photobiont)
anosim(dat = as.dist(unifracnw454), grouping = samples454$Mycobiont, strata = samples454$Site)
# run normalized weighted UniFrac NMDS
nmds <- nmds(as.dist(unifracnw454))
nmin <- nmds.min(nmds)
nmin
#plot(nmin, pch=as.numeric(samples454$Photobiont))
#plot(nmin, pch=as.numeric(samples454$Mycobiont))
#plot(nmin, pch=as.numeric(samples454$Site))
# close the output file
sink()
# unload the libraries
detach("package:ecodist")
detach("package:vegan")


Samples454.csv (referenced in the above script)

SampleID	Photobiont	Site	Mycobiont
CLCl	Ch	C	Cl
CLDi	Cy	C	Di
CLLe	Cy	C	Le
CLPe	Cy	C	Pe
CLSt	Cy	C	St
CLUs	Ch	C	Us
ELCl	Ch	E	Cl
ELFl	Ch	E	Fl
ELOp	Ch	E	Op
ELPe	Cy	E	Pe
ELUm	Ch	E	Um
HLCl	Ch	H	Cl
HLLe	Cy	H	Le
HLPe	Cy	H	Pe
HLSt	Cy	H	St
HLUs	Ch	H	Us
NLCl	Ch	N	Cl
NLFl	Ch	N	Fl
NLOp	Ch	N	Op
NLPe	Cy	N	Pe
NLUm	Ch	N	Um

DistUFnw454.csv (referenced in the above script; full file available at http://dx.doi.org/10.5061/dryad.t99b1)

CLCl		0.16	0.17	0.11	0.08	0.12	...
CLDi	0.16		0.24	0.18	0.13	0.18	...
CLLe	0.17	0.24		0.11	0.16	0.20	...
CLPe	0.11	0.18	0.11		0.10	0.18	...
CLSt	0.08	0.13	0.16	0.10		0.13	...
CLUs	0.12	0.18	0.20	0.18	0.13		...
ELCl	0.20	0.22	0.30	0.25	0.19	0.17
ELFl	0.14	0.18	0.24	0.19	0.14	0.15	...
ELOp	0.33	0.35	0.40	0.37	0.34	0.31	...
ELPe	0.16	0.21	0.24	0.19	0.15	0.19	...
ELUm	0.22	0.24	0.32	0.27	0.22	0.22	...
HLCl	0.19	0.18	0.27	0.24	0.17	0.15	...
HLLe	0.13	0.16	0.20	0.15	0.11	0.12	...
HLPe	0.09	0.17	0.15	0.11	0.11	0.16	...
HLSt	0.14	0.16	0.21	0.18	0.12	0.13	...
HLUs	0.10	0.20	0.18	0.15	0.14	0.11	...
NLCl	0.19	0.21	0.28	0.25	0.19	0.15	...
NLFl	0.18	0.21	0.27	0.24	0.19	0.15	...
NLOp	0.24	0.27	0.32	0.29	0.25	0.25	...
NLPe	0.10	0.18	0.13	0.09	0.10	0.14	...
NLUm	0.09	0.18	0.21	0.15	0.13	0.16	...

SamplesClon.csv (referenced in the above script)

SampleID	Photobiont	Site	Mycobiont
CL01	Cy	C	Peltigera
CL02	Ch	C	Alectoria
CL03	Ch	C	Cladonia
CL04	Tr	C	Placopsis
CL05	Cy	C	Erioderma
CL06	Cy	C	Sticta
CL07	Cy	C	Dictyonema
CL08	Cy	C	Leptogium
CL09	Tr	C	Stereocaulon
CL10	Ch	C	Usnea
EL01	Cy	E	Peltigera
EL01t	Tr	E	Peltigera
EL02	Ch	E	Alectoria
EL03	Ch	E	Cladonia
EL04	Ch	E	Rhizocarpon
EL05	Ch	E	Ophioparma
EL06	Ch	E	Flavocetraria
EL07	Ch	E	Arctoparmelia
EL08	Ch	E	Umbilicaria
EL09	Ch	E	Masonhalea
EL10	Ch	E	Dactylina
HL01	Cy	H	Peltigera
HL02	Ch	H	Platismatia
HL03	Ch	H	Cladonia
HL04	Ch	H	Parmotrema
HL05	Ch	H	Flavoparmelia
HL06	Cy	H	Sticta
HL07	Ch	H	Xanthoparmelia
HL08	Cy	H	Leptogium
HL09	Tr	H	Stereocaulon
HL10	Ch	H	Usnea
NL01	Cy	N	Peltigera
NL01t	Tr	N	Peltigera
NL03	Ch	N	Cladonia
NL04	Tr	N	Amygdalaria
NL05	Ch	N	Ophioparma
NL06	Ch	N	Flavocetraria
NL07	Ch	N	Parmelia
NL08	Ch	N	Umbilicaria
NL09	Tr	N	Stereocaulon
NL10	Ch	N	Cetraria

HodkinsonClon16S_OTUS_1per_171.csv (referenced in the above script; full file available at http://dx.doi.org/10.5061/dryad.t99b1)

CL01	0.00	0.00	0.00	0.00	0.00	0.00	...
CL02	0.00	0.00	0.00	0.00	0.00	0.00	...
CL03	0.00	0.25	0.00	0.00	0.00	0.00	...
CL04	0.11	0.00	0.00	0.00	0.00	0.00	...
CL05	0.00	0.03	0.34	0.05	0.00	0.00	...
CL06	0.00	0.10	0.02	0.03	0.00	0.02	...
CL07	0.00	0.56	0.00	0.00	0.00	0.19	...
CL08	0.00	0.03	0.00	0.00	0.00	0.00	...
CL09	0.02	0.13	0.06	0.15	0.00	0.00	...
CL10	0.00	0.00	0.00	0.00	0.00	0.00	...
EL01	0.00	0.00	0.00	0.00	0.00	0.17	...
EL01t	0.00	0.00	0.00	0.00	0.00	0.31	...
EL02	0.00	0.26	0.00	0.00	0.00	0.00	...
EL03	0.00	0.03	0.00	0.00	0.00	0.00	...
EL04	0.00	0.10	0.00	0.00	0.00	0.00	...
EL05	0.00	0.04	0.00	0.00	0.00	0.00	...
EL06	0.00	0.07	0.00	0.00	0.00	0.00	...
EL07	0.00	0.00	0.00	0.00	0.00	0.00	...
EL08	0.00	0.34	0.00	0.00	0.00	0.00	...
EL09	0.00	0.21	0.00	0.00	0.00	0.00	...
EL10	0.00	0.21	0.00	0.00	0.04	0.00	...
HL01	0.00	0.00	0.00	0.00	0.00	0.00	...
HL02	0.13	0.00	0.00	0.00	0.00	0.00	...
HL03	0.00	0.00	0.00	0.00	0.00	0.00	...
HL04	0.06	0.00	0.00	0.00	0.00	0.00	...
HL05	0.00	0.03	0.00	0.00	0.00	0.00	...
HL06	0.00	0.00	0.00	0.06	0.00	0.00	...
HL07	0.00	0.14	0.00	0.00	0.00	0.00	...
HL08	0.00	0.00	0.00	0.00	0.00	0.00	...
HL09	0.00	0.00	0.00	0.00	0.00	0.00	...
HL10	0.00	0.02	0.00	0.00	0.00	0.00	...
NL01	0.00	0.15	0.00	0.00	0.00	0.00	...
NL01t	0.00	0.00	0.00	0.00	0.00	0.15	...
NL03	0.00	0.00	0.00	0.00	0.00	0.00	...
NL04	0.00	0.15	0.00	0.00	0.00	0.00	...
NL05	0.00	0.17	0.00	0.00	0.00	0.00	...
NL06	0.00	0.02	0.00	0.00	0.00	0.00	...
NL07	0.00	0.33	0.00	0.00	0.00	0.00	...
NL08	0.00	0.80	0.00	0.00	0.00	0.00	...
NL09	0.00	0.00	0.00	0.00	0.00	0.00	...
NL10	0.00	0.05	0.00	0.00	0.00	0.00	...

Here are some examples of non-metric multidimensional scaling plots produced from OTU-based Bray-Curtis dissimilarities between lichen-associated bacterial communities (Hodkinson et al. 2012). Photobiont is indicated by color (light green = green algae; dark blue = Cyanobacteria; black = Tripartite), while the site is indicated with symbols (• = Eagle Summit, AK; + = Nome, AK; x = Highlands, NC, and * = Cerro de la Muerte, CR). Numbers indicate the identity of different mycobiont types (see associated publication for details). Continuous lines act as visual aids to delimit communities associated with the two major photobiont-types, whereas dashed lines delimit communities associated with chlorolichens from northern versus southern sites.

Plot A shows results obtained from clone library data of 16S sequences from the order Rhizobiales.



Plot B was produced from 454 barcoded 16S amplicon data (representing approximately half the number of samples as the clone library set but ~100 times as many sequences from a much wider range of bacterial diversity; since this plot was produced through OTU-based methods it does not precisely correlate with what would be produced through the abbreviated script above, which uses UniFrac distances to generate NMDS plots based on 454 data).

For additional information and documentation, including results of ANOSIM analyses, please see the references below!

- Brendan

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

References

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. 2012. Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Environmental Microbiology 14(1): 147-161. [doi:10.1111/j.1462-2920.2011.02560.x]
Download publication (PDF file)
Download supplementary phylogeny (PDF file)
View data and analysis file webportal (website)
Download data and analysis file archive (ZIP file)

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2011. 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

R Development Core Team. 2010. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. http://www.r-project.org/