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.  Also, 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/