The Lichenologist 44(1): 137–146 (2012) © British Society, 2011 doi:10.1017/S0024282911000648

A preliminary survey of lichen associated using pyrosequencing

Scott T. BATES, Donna BERG-LYONS, Christian L. LAUBER, William A. WALTERS, Rob KNIGHT and Noah FIERER

Abstract: Although various eukaryotic organisms, such as , endolichenic/lichenicolous fungi, and , have been isolated from , the diversity and structure of eukaryotic communities associated with lichen thalli has not been well studied. In addressing this knowledge gap, we used bar-coded pyrosequencing of 18S rRNA genes to survey eukaryotes associated with thalli of three different lichen species. In addition to revealing an expected high abundance of lichen biont- related 18S genes, sequences recovered in our survey showed non-biont fungi from the also have a substantial presence in these thalli. Our samples additionally harboured fungi representing phyla (, ) that have not been isolated previously from lichens; however, their very low abundance indicates an incidental presence. The recovery of Alveolata, Metazoa, and Rhizaria sequences, along with recent work revealing the considerable bacterial diversity in these same samples, suggests lichens function as minute ecosystems in addition to being symbiotic organisms. Key words: Ascomycota, , endolichenic/lichenicolous fungi, invertebrates

Introduction ever, debate still continues as to the true The ‘dual’ symbiotic nature of lichens was nature of this relationship, including whether first proposed in the late 19th century by it represents a controlled parasitism or mutu- Simon Schwendener (1868). However, the alism. A bacterial contribution to lichen nu- -fixation) has also been validity of his hypothesis was contested, often trition (e.g., via N2 considered for quite some time (Henckel fervently, for almost another hundred years & Yuzhakova 1936), and contemporary by those who saw lichens as whole organisms culture-independent studies are increasing rather than a symbiosis between fungi and our understanding of diverse populations of ‘algae’ ( or ) bacteria associated with lichens and their po- (Honegger 2000). The concept of lichens as tential functional roles within the symbiosis a mycobiont/photobiont symbiosis is now (Cardinale . 2008; Grube . 2009; universally accepted (see Nash 2008); how- et al et al Hodkinson & Lutzoni 2009; Bates et al. 2011; Hodkinson et al. in press; reviewed in S. T. Bates, D. Berg-Lyons, C. L. Lauber and N. Fierer: Grube & Berg 2009). Cooperative Institute for Research in Environmental In addition to algal and lichenized fungal Sciences, University of Colorado, Boulder, Colorado symbionts, numerous other eukaryotic 80309, USA. Email: [email protected] organisms are known to associate with W. A. Walters: Department of Cellular, Molecular, lichens, which they use for food or shelter and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA. (Gerson 1973). Some moth species, for R. Knight: Department of Chemistry and Biochemistry, example, preferentially lay their eggs under University of Colorado, Boulder, Colorado 80309 and the protective covering of lichens (Thomson Howard Hughes Medical Institute, Boulder, Colorado 1958), and oribatid mites are known to 80309, USA. N. Fierer: Department of Ecology and Evolutionary burrow into lichens on which they feed Biology, University of Colorado, Boulder, Colorado (Grandjean 1950). Members of diverse 80309, USA. invertebrate groups including arthropods, 138 THE LICHENOLOGIST Vol. 44 , nematodes, and have all Surface sterilization and isolation of community been isolated from lichen thalli where they DNA presumably inhabit internal areas (Stubbs Each lichen thallus was surface sterilized using the 1989; Bartels & Nelson 2007). Lichens also most thorough protocol outlined by Arnold et al. (2009), host numerous fungal species, in addition to to avoid amplification of DNA originating from eukary- otic organisms that may have only come into contact the mycobiont, such as lichenicolous (lichen- with external surfaces of our specimens. The steriliza- associated) fungi (Lawrey & Diederich 2003; tion procedure was as follows: samples were washed for Lawrey et al. 2007) and endolichenic fungi w30 s in sterile ultrapure laboratory-grade (Milli-Q) (which grow within the interior of lichens) water to remove debris from outer surfaces; they were then immersed and agitated separately in 96% ethanol (Girlanda et al. 1997; Suryanarayanan et al. for 10 s, followed by 0·5% NaOCl (bleach) for 2 min, 2005; U’Ren et al. 2010). Interestingly, and 70% ethanol for 4 min. A small piece of the lichen recent phylogenetic studies suggest lichen thallus with the approximate dimensions of 2 cm2 was thalli themselves may be ‘cradles’ of fungal removed from the sample immediately after surface diversification in the Ascomycota (Arnold sterilization for use in genomic DNA extraction. We used the commercially available PowerSoil DNA isola- et al. 2009). tion kit (MoBio Laboratories, Carlsbad, CA) to extract Despite a growing body of literature on genomic DNA from thalli after an initial treatment to organisms associated with lichens, we still enhance DNA yield. This treatment included separately have limited knowledge of the extent of grinding each sample with a sterile pestle and mortar under liquid N2, placing the macerated samples into eukaryotic diversity that may be associated individual 2-ml bead-beating tubes with kit buffer, and with individual lichen thalli. Here we carried immersion in a 65°C water bath for 10 min. After out a preliminary survey of eukaryotes associ- heating, extractions proceeded according to the kit ated with surface-sterilized foliose lichen protocol. thalli, using high-throughput pyrosequenc- PCR amplification of 18S rRNA genes and ing with eukaryotic-specific primers targeting bar-coded pyrosequencing the 18S rRNA gene. As an extensive diversity of bacteria has been shown to be associated Extracted genomic DNA representing communities of lichen-associated organisms were prepared for pyro- with internal surfaces of lichens (Cardinale sequencing following the protocol outlined by Fierer et al. 2008; Grube et al. 2009; Bates et al. et al. (2008), using only eukaryotic-specific primers. 2011; B. P. Hodkinson et al. in press), we Briefly, the method includes targeted PCR amplification also compared our eukaryotic diversity re- of a portion (up to w600 bp) of the 18S small subunit rRNA gene, triplicate PCR product pooling (per sults with previously published analyses of sample) to mitigate reaction-level PCR biases, and pyro- bacterial diversity recovered from the same sequencing using the eukaryotic-specific primer set specimens. F515 (5#-GTGCCAGCMGCCGCGGTAA-3#) and R1119 (5#-GGTGCCCTTCCGTCA-3#). We have demonstrated in silico that this primer set should am- plify 18S rRNA genes from a broad range of eukaryotic groups with few biases (see Supplementary Fig. S1). The F515 primer included a Roche FLX+ pyrosequenc- Materials and Methods ing adapter (Roche Applied Science, Indianapolis, IN) and a 2-bp linker sequence (GT), and R1119 incorpor- ated 12-bp bar-coded sequences (each unique to an Sampling individual specimen), an AG linker, and a FLX+ se- quencing adapter (Roche). The PCR was carried out in To minimize the influence of eukaryotic organisms 25 !l reaction mixtures, containing 1 !l (5 !M [each]) of haphazardly found on rock surfaces in our survey, we forward and reverse primers, 10 !lof5PrimeHotmaster selected thalli of three umbilicate (i.e., attached to the mix (Eppendorf-5Prime, Gaithersburg, MD), and 12 !l substratum only at a single, central point), foliose green MoBio PCR water. Each reaction mix received 1 !lof algal lichen species. chrysoleuca, Umbilicaria genomic community DNA as a template, and the follow- americana, and Umbilicaria phaea, were collected at a site ing cycling parameters were used: 35 cycles (94°C for in northern Colorado (40·01°N, 105·47°W) from rock 45 s, 45°C for 30 sec, and 72°C for 1·5 min) were outcrops. Each thallus was removed from the rock sub- performed after an initial denaturation at 94°C for stratum using a sterile knife and placed into individual 3 min. Triplicate reaction mixtures per specimen were sterile plastic collection bags. The specimens were then combined and purified using an UltraClean PCR transported back to the laboratory on ice and processed cleanup kit (MoBio), followed by quantification using immediately. PicoGreen dsDNA (Invitrogen, Carlsbad, CA). The 2012 Lichen eukaryotes—Bates et al. 139 bar-coded PCR products from all samples were normal- Results and Discussion ized in equimolar amounts in a pooled sample, and sent for sequencing at Roche Applied Science. Eukaryotic survey For pyrosequencing, 1 !lofPCRproductwas checked for quality with a Bioanalyzer 2100 DNA 1000 On average, 1008 eukaryotic sequences chip (Agilent Technologies, Santa Clara, CA), quanti- (from 862, 1021, to 1141) were recovered fied using the Quant-iT PicoGreen assay (Invitrogen), 7 from our lichen samples. In addition to phy- and then diluted to 1 × 10 molecules per !l. The PCR lotypes representing the photo- (all 99·5% products were clonally amplified using the GS FLX Titanium LV emPCR Kit (Lib-A) following a modified matches with Trebouxia) and mycobionts version of the manufacturer’s instructions (emPCR (>99% matches with Rhizoplaca or Umbili- Amplification Method Manual: Lib-L LV, GS FLX+ caria) of each lichen species, a total of 50 Series: XL+). Briefly, amplicons were immobilized onto other distinct eukaryotic phylotypes were DNA-capture beads, micro-reactors were formed of a single DNA-containing bead in an emulsion oil sphere, recovered from the 2904 high-quality and emulsified samples were subjected to PCR amplifi- sequences generated by pyrosequencing of cation on 96-well plates. After amplification, emulsions 18S rRNA genes associated with our speci- were chemically broken and beads carrying the amplified mens. These phylotypes represented diverse DNA library were recovered and washed, and then eukaryotic taxa from nine phyla within DNA-positive beads were purified using a biotinylated primer/streptavidin-coated magnetic beads complex. four major clades of Eukarya: Alveolata Recovered library beads were melted from magnetic (Ciliophora), Fungi (Ascomycota, Basidi- beads yielding a population of bead-bound single- omycota, Blastocladiomycota, Chytridiomy- stranded DNA templates. After sequencing primer cota), Metazoa (Rotifera, Tardigrada), annealing, beads were counted with a Mulitsizer 3 (Beckman Coulter, Brea, CA), two million enriched Rhizaria (), and Viridiplantae beads from each of two amplicon pools were loaded into (Chlorophyta). one of two regions in a 70 × 75 mm PicoTiterPlate The relative abundance of each eukaryotic device, and then subjected to 400 cycles of modified phylotype varied considerably within each pyrophosphate-based DNA sequencing on a GS FLX+ specimen, with relative abundances charac- automated sequencer following the manufacturer’spro- tocol (Sequencing Method Manual, GS FLX+ Series: terized by an ‘L-shape’ curve (Fig. 1) that is XL+ Kit, Roche). In this study, amplicons were typical for communities of micro-organisms sequenced from a single direction; however, the ‘A’ (Fuhrman 2009) and indicates that the ma- Adaptor (GS FLX Titanium LV emPCR Kit: Lib-A) jority of phylotypes were relatively rare. is commonly employed for bi-directional amplicon sequencing. Our primer selection combined with These phylotypes essentially represented GS FLX+ technology provided read lengths (up to three levels of abundance: high (representing w600 bp) at a more than sufficient resolution for the between 19% and 70% of the sequences accurate taxonomic classification of micro-organismal recovered from each sample), medium sequences (Liu et al. 2007). (between 7% and 0·5%), and low (less than 0·5%). Invariably the high abundance group Sequence processing comprised two phylotypes representing the The QIIME software pipeline (Caporaso et al.2010) photo- and mycobionts. Three phylotypes was used to process raw sequence data, perform quality were recovered in each lichen species that control, sample grouping (via the unique 12 bp bar- represented the mid-level group, and all codes), phylotype binning, and taxonomic assignment of sequence data. for all eukaryotic phylotypes but one (a 99·7% match to a taxon in (sequences sharing R 97% similarity) recovered from Tardigrada) of these phylotypes corre- our lichen specimens was assigned in QIIME using sponded to fungal taxa within the Asco- BLAST (Altschul et al. 1997), based on sequences from mycota. The numerous low abundance the SILVA comprehensive ribosomal RNA database phylotypes, many of these being ‘singletons’, (http://www.arb-silva.de/). Sequences representing the phylotypes were further screened for chimeras, which represented only 1–4% of all sequences re- were removed from the dataset, and phylotypes assigned covered from each lichen sample. Although as ‘environmental samples’ in the SILVA database were many of these phylotypes matched taxa that subjected to BLASTn (Altschul et al. 1997) searches in are known to be associated with lichens (e.g., GenBank (http://www.ncbi.nlm.nih.gov/genbank/) to determine their taxonomic affiliation. Subsequent diver- lichenicolous fungi, protozoa, rotifers and sity analyses were performed using R statistical software tardigrades), others represented taxa that are (http://www.r-project.org/). not (e.g., chytrids and hypogeous fungi). 140 THE LICHENOLOGIST Vol. 44

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Number of Individual Phylotypes F. 1. Rank abundance graph of unique eukaryotic phylotypes recovered from each lichen species. The number of individual sequences of a given phylotype is shown on the y-axis (log scale) and the x-axis represents the ranked of the phylotypes obtained from the lichens sampled (·····R. chrysoleuca, —— U. Americana,-----U. phaea).

Dominant phylotypes mycetes and Orbiliomycetes) that have been isolated from lichen thalli previously (Pfister Table 1 shows the abundances for all phy- & Liftik 1995; Lawrey & Diederich 2003; lotypes recovered in the survey indicating Arnold et al. 2009), suggesting that this high, medium and low level abundance group corresponds to actual lichenicolous or groups. Considering the large amount of endolichenic fungi inhabiting the thalli of our photo- and mycobiont biomass in these lichen samples. The low abundance group samples, biont phylotype representation in was the most diverse, representing all phyla the high abundance group is consistent with as well as 82% of the unique phylotypes their corresponding 18S rRNA gene copies recovered in this study. Although some of inherent in the sample DNA pool. Although these phylotypes may represent authentic the number of mid-level phylotype group lichen-associated organisms, others are sequences recovered was considerably lower probably only haphazardly present in our than that of the high-level group (see Fig. 2), specimens. For example, some may be fungal they do represent quantities of biomass that remnants in the digestive tract of mycopha- were significant enough to be detected over gous invertebrates, such as tardigrades and the biont 18S signature. Tardigrades of the bdelloid rotifers (Meininger & Spatt 1988; order Echiniscoidea are known to inhabit Wilson & Sherman 2010), which were lichens (Bartels & Nelson 2007); therefore, also among the low abundance phylotypes the recovery of a phylotype corresponding to recovered from our samples. this taxon among our mid-level group is not surprising, as the organism or its eggs would constitute ample biomass to be detected in Comparison of eukaryotic and bacterial our relatively small (2 cm2) lichen sample. diversity The other mid-level phylotypes represent The high relative abundance of biont 18S several classes of ascomycetous fungi copies in our lichen samples is also apparent (, , Leotio- in Figure 2, as is the fraction of non-biont 02Lce eukaryotes— Lichen 2012 T 1. Relative abundance (% of all 18S rRNA gene sequences in each sample) of phylotypes (* = bionts) recovered from the lichens species Rhizoplaca chrysoleuca (R.c.), Umbilicaria americana (U.a.), and Umbilicaria phaea, (U.p.), and their percent identity matches with eukaryotic taxa. High, medium, and low level abundance groups are indicated (darkest to lightest greys, respectively).

2C 5A 5P -ATCH +INGDOM 0HYLUM #LASS /RDER 'ENUS q    &UNGI !SCOMYCOTA ,ECANOROMYCETES ,ECANORALES 2HIZOPLACA q q q q &UNGI !SCOMYCOTA ,ECANOROMYCETES 5MBILICARIALES 5MBILICARIA q q q q 6IRIDIPLANTAE #HLOROPHYTA 4REBOUXIOPHYCAEA -ICROTHAMNIALES 4REBOUXIA q q q q &UNGI !SCOMYCOTA $OTHIDEOMYCETES 0LEOSPORALES !NGUILLOSPORA q q q q &UNGI !SCOMYCOTA $OTHIDEOMYCETES 0LEOSPORALES &ENESTELLA q  q q -ETAZOA 4ARDIGRADA (ETEROTARDIGRADA %CHINISCOIDEA %CHINISCUS  q q q &UNGI !SCOMYCOTA $OTHIDEOMYCETES #APNODIALES 2AMICHLORIDIUM q q q q &UNGI !SCOMYCOTA /RBILIOMYCETES /RBILIALES $ACTYLELLINA q    &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES 3ARCINOMYCES q  q q &UNGI !SCOMYCOTA ,EOTIOMYCETES (ELOTIALES #ROCICREAS  q  q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES 2HINOCLADIELLA q q q q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES #ONIOSPORIUM

 q q q &UNGI !SCOMYCOTA ,ECANOROMYCETES 5MBILICARIALES ,ASALLIA Bates q q   &UNGI !SCOMYCOTA 3ORDARIOMYCETES -AGNAPORTHALES 0HIALOPHORA

 q   &UNGI "ASIDIOMYCOTA !GARICOMYCETES "OLETALES 141 2HIZOPOGON al. et q q  q &UNGI #HYTRIDIOMYCOTA #HYTRIDIOMYCETES 3PIZELLOMYCETALES 2HIZOPHLYCTIS  q  q &UNGI !SCOMYCOTA $OTHIDEOMYCETES #APNODIALES UNKNOWN SP q   q &UNGI !SCOMYCOTA ,ECANOROMYCETES !CAROSPORALES !CAROSPORA  q  q &UNGI !SCOMYCOTA ,ICHINOMYCETES ,ICHINALES %UOPSIS q   q &UNGI "ASIDIOMYCOTA !GARICOMYCETES #ANTHARELLALES -ARCHANDIOBASIDIUM  q  q 2HIZARIA #ERCOZOA 3ILICOFILOSEA 4HAUMATOMONADIDA 0ROTASPIS q    !LVEOLATA #ILIOPHORA )NTRAMACRONUCLEATA 3PIROTRICHEA !MPHISIELLA q    !LVEOLATA #ILIOPHORA )NTRAMACRONUCLEATA 3PIROTRICHEA +AHLIELLA q   q &UNGI !SCOMYCOTA $OTHIDEOMYCETES #APNODIALES UNKNOWN SP q   q &UNGI !SCOMYCOTA $OTHIDEOMYCETES $OTHIDEALES $OTHIDEA 4 H IHNLGS o.44 Vol. LICHENOLOGIST THE 142

2 C 5 A 5 P -ATCH +INGDOM 0HYLUM #LASS /RDER 'ENUS  q   &UNGI !SCOMYCOTA $OTHIDEOMYCETES $OTHIDEALES $OTHIDEA  q   &UNGI !SCOMYCOTA $OTHIDEOMYCETES (YSTERIALES UNKNOWN SP  q  q &UNGI !SCOMYCOTA $OTHIDEOMYCETES )NCERTAE SEDIS 0HAEOSCLERA q   q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES #APRONIA q   q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES UNKNOWN SP  q  q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES UNKNOWN SP  q  q &UNGI !SCOMYCOTA %UROTIOMYCETES #HAETOTHYRIALES UNKNOWN SP  q  q &UNGI !SCOMYCOTA ,ECANOROMYCETES ,ECANORALES ,ECIDEA   q  &UNGI !SCOMYCOTA ,ECANOROMYCETES ,ECANORALES 2HIZOCARPON  q  q &UNGI !SCOMYCOTA ,EOTIOMYCETES (ELOTIALES 3CLEROTINIA  q  q &UNGI !SCOMYCOTA ,EOTIOMYCETES 2HYTISMATALES 0HACIDIOPYCNIS q   q &UNGI !SCOMYCOTA 0EZIZOMYCETES 0EZIZALES ,ECOPHAGUS  q  q &UNGI "ASIDIOMYCOTA !GARICOMYCETES !THELIALES 0ILODERMA q   q &UNGI "ASIDIOMYCOTA !GARICOMYCETES (YMENOCHAETALES 0ORODAEDALEA  q  q &UNGI "ASIDIOMYCOTA !GARICOMYCETES 0OLYPORALES 4RAMETES  q  q &UNGI "ASIDIOMYCOTA !GARICOSTILBOMYCETES !GARICOSTILBALES !GARICOSTILBUM  q  q &UNGI "LASTOCLADIOMYCOTA "LASTOCLADIOMYCETES "LASTOCLADIALES #ATENOMYCES  q  q &UNGI #HYTRIDIOMYCOTA #HYTRIDIOMYCETES 3PIZELLOMYCETALES 2HIZOPHLYCTIS  q  q -ETAZOA 2OTIFERA $IGONONTA "DELLOIDEA 2OTARIA q   q -ETAZOA 2OTIFERA $IGONONTA "DELLOIDEA -NIOBIA q   q -ETAZOA 4ARDIGRADA %UTARDIGRADA 0ARACHAELA 2AMAZZOTTIUS q   q -ETAZOA 4ARDIGRADA (ETEROTARDIGRADA %CHINISCOIDEA %CHINISCUS  q  q 2HIZARIA #ERCOZOA )NCERTAE SEDIS )NCERTAE SEDIS UNKNOWN SP q   q 2HIZARIA #ERCOZOA 3ARCOMONADEA #ERCOMONADIDA "ODOMORPHA  q  q 2HIZARIA #ERCOZOA 3ARCOMONADEA #ERCOMONADIDA #ERCOMONAS  q  q 2HIZARIA #ERCOZOA 3ARCOMONADEA #ERCOMONADIDA UNKNOWN SP  q  q 6IRIDIPLANTAE #HLOROPHYTA 4REBOUXIOPHYCAEA -ICROTHAMNIALES $ICTYOCHLOROPSIS 2012 Lichen eukaryotes—Bates et al. 143

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0 R. chrysoleuca U. americana U. phaea

Fungi, Ascomycota (mycobiont) Fungi, Ascomycota (other) Fungi, other phyla Viridiplantae, Chlorophyta (photobiont) Other Eukaryota

F. 2. Relative abundances of various major eukaryotic lineages recovered from each lichen species. Relative abundance was calculated as the percentage of sequences belonging to a particular lineage of all 18S rRNA gene sequences recovered from the lichens sampled. ascomycetous taxa that probably represents eukaryotes and bacteria (see Bates et al. many true lichen-inhabiting fungi. Overall, 2011) fail to asymptote. With the same sam- just two primarily biont-related phyla (Asco- pling effort, however, eukaryotic diversity mycota and Chlorophyta) dominated our associated with lichens is more restricted lichens, while the remaining phyla (Cilio- than that of bacteria from the same lichen phora, , Blastocladiomycota, specimens. This may be attributed to the Chytridiomycota, Rotifera, Tardigrada and smaller size of bacteria compared to the Cercozoa) had very low representation. In eukaryotic organisms that our surveys sug- contrast, more higher order bacterial taxa, gest inhabit these samples, which may some with members probably having distinct allow for more relative niche space for functional roles in the lichen symbiosis bacteria to occupy. Diversity values for our (Grube & Berg 2009; Hodkinson & Lutzoni lichen samples were generally higher than 2009; Bates et al. 2011; B. P. Hodkinson et those of previous DNA-based studies of al. in press), were noticeably present in these endolichenic fungi (Arnold et al. 2009; same lichen samples (Bates et al. 2011; see U’Ren et al. 2010), which may be attribu- fig. 2 in that publication). table to overestimation by our pyrosequenc- Rarefactionscurves (Fig. 3) highlight the ing approach (Engelbrektson et al. 2010). considerable amount of micro-organismal However, methodological differences make diversity associated with even small frag- such comparisons difficult, and we were con- ments of lichen thalli, as plots for both servative in our method of phylotype binning 144 THE LICHENOLOGIST Vol. 44

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Number of Individual Sample Sequences F. 3. Rarefaction curves depicting the richness (y-axis, as the number of unique phylotypes recovered) determined at an equal sampling effort (x-axis, the number of individual sequences recovered in each sample) of (A) eukaryotic and (B) bacterial (see Bates et al. 2011) phylotypes associated with each lichen species (·····R. chrysoleuca,——U. americana,-----U. phaea). in order to minimize inflation of diversity 3A) also held the lowest level of bacterial estimates. It is also interesting to note that diversity (Fig. 3B) and vice versa for U. the specimen with the highest level of eukary- phaea. This observation raises the question of otic diversity (Umbilicaria americana; Fig. possible competitive interactions between 2012 Lichen eukaryotes—Bates et al. 145 the eukaryotic (principally fungi) and bacte- fragments of lichen thalli. This finding, rial populations that inhabit lichens; however along with the results of previous surveys of the small number of lichens sampled here other lichen-associated micro-organisms, does not allow us to test this hypothesis. reinforces the concept that, in addition to Conversely, the higher levels of diversity for being symbiotic systems where numerous U. americana may simply be the result of symbiotic partners may interact, lichens physical features of this species, such as the can also be considered minute ecosystems dense covering rhizomorphs on the lower (Farrar 1976; Grube et al. 2009). Modern surface of the thallus, which may provide molecular tools are now allowing us to ex- excellent habitat for lichen associated eu- plore the intricacies of these systems in karyotes. ways that were probably inconceivable to Schwendener and his contemporaries.

Members of the Fierer group helped in the laboratory Conclusions and with the analyses. Garrett Cropsey assisted in sample collecting and processing. Laura Wegener Recent DNA-based studies continue to elu- Parfrey provided useful discussion and suggestions. We cidate the extent of fungal (Lawrey et al. thank Mike Robeson and Bill Birky for assistance in 2007; Arnold et al. 2009; U’Ren et al. 2010) locating eukaryotic SEM images, which were graciously and bacterial (Cardinale et al. 2008; Grube provided by Byron Adams, Diego Fontaneto and Robert Roberson. Chinnappa Kodira of Roche Applied Science et al. 2009; Hodkinson & Lutzoni 2009; supervised the pyrosequencing. This research was Bates et al. 2011; B. P. Hodkinson et al. in funded by grants to RK and NF from the National press; reviewed in Grube & Berg 2009) Institutes of Health, the National Science Foundation, diversity that is present in lichen species. and the US Department of Agriculture. Although many invertebrate are known to associate with lichens (e.g., see R Gerson 1973), this is the first culture- Altschul, S. F., Madden, T. J., Schäffer, A. A., Zhang, J., independent survey to document members Zhang, Z., Miller, W. & Lipman, D. J. (1997) of the Alveolata, Metazoa, and Rhizaria Gapped BLAST and PSI-BLAST: a new gener- inhabiting internal surfaces of lichens. We ation of protein database search programs. Nucleic have also contributed to the understanding of Acids Research 25: 3389–3402. Arnold, A. E., Mia˛dlikowska, J., Higgins, K. L., Sarvate, lichen-associated fungal diversity, recover- S. D., Gugger, P., Way, A., Hofstetter, V., Kauff, F. ing phylotypes corresponding to phyla & Lutzoni, F. (2009) A phylogenetic estimation (Blastocladiomycota and Chytridiomycota) of trophic transition networks for ascomycetous of fungi not isolated previously from fungi: are lichens cradles of symbiotrophic fungal lichens. The presence of these fungal phylo- diversification? Systematic Biology 58: 283–297. Bartels, P. J. & Nelson, D. R. (2007) An evaluation of types, as well as those matching other fungal species richness estimators for tardigrades in the taxa that were relatively rare, serves as a Great Smoky Mountains National Park. Journal of caution for culture-dependent studies: Limnology 66 (Supplement 1): 104–110. lichen thalli may contain numerous potential Bates, S. T., Cropsey, G. W. G., Caporaso, J. G., Knight, R. & Fierer, N. (2011) Bacterial communi- fungal propagules that do not represent ties associated with the lichen symbiosis. Applied sizable biomass portions in lichens and are and Environmental Microbiology 77: 1309–1314. perhaps not true lichenicolous or endo- Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, lichenic fungi. K., Bushman, F. D., Costello, E. K., Fierer, N., Although the overwhelming abundance of Gonzalez Peña, A., Goodrich, J. K., Gordon, J. I. et al. (2010) QIIME allows integration and analysis biont related sequences in our survey may of high-throughput community sequencing data. have limited our ability to resolve the full Nature Methods 7: 335–336. extent of eukaryotic diversity in our speci- Cardinale, M., Vieira de Castro, J., Jr., Müller, H., Berg, mens, the sequences recovered in our G. & Grube, M. (2008) In situ analysis of the bacterial community associated with the reindeer pyrosequencing survey revealed diverse lichen Cladonia arbuscula reveals predominance of assemblages of organisms from several major Alphaproteobacteria. FEMS Microbiology Ecology 66: eukaryotic clades which inhabit even small 63–71. 146 THE LICHENOLOGIST Vol. 44

Engelbrektson, A., Kunin, V., Wrighton, K. C., Honegger, R. (2000) Great discoveries in bryology Zvenigorodsky, N., Chen, F., Ochman, H. & and lichenology: Simon Schwendener (1829–1919) Hugenholtz, P. (2010) Experimental factors affect- and the dual hypothesis in lichens. Bryologist 103: ing PCR-based estimates of microbial species 307–713. richness and evenness. ISME Journal 4: 642–647. Lawrey, J. D. & Diederich, P. (2003) Lichenicolous Farrar, J. F. (1976) The lichen as an ecosystem: obser- fungi: interactions, evolution, and biodiversity. vation and experiment. In Lichenology: Progress and Bryologist 106: 80–120. Problems (D. H. Brown, D. L. Hawksworth & R.H. Lawrey, J. D., Binder, M., Diederich, P., Molina, M. C., Bailey, eds): 385–406. London: Academic Press. Sikaroodi, M. & Ertz, D. (2007) Phylogenetic di- Fierer, N., Hamady, M., Lauber, C. L. & Knight, R. versity of lichen-associated homobasidiomycetes. (2008) The influence of sex, handedness, and wash- Molecular Phylogenetics and Evolution 44: 778–789. ing on the diversity of hand surface bacteria. Pro- ceedings of the National Academy of Sciences 105: Liu, Z., Lozupone, C., Hamady, M., Bushman, F. D. & 17994–17999. Knight, R. (2007) Short pyrosequencing reads Fuhrman, J. A. (2009) Microbial community struc- suffice for accurate microbial community analysis. ture and its functional implications. Nature 459: Nucleic Acids Research 35: e120. 193–199. Meininger, C. A. & Spatt, P. D. (1988) Variations of Gerson, U. (1973) Lichen- associations. assemblages in dust-impacted Arctic Lichenologist 5: 434–443. . Artic and Alpine Research 20: 24–30. Girlanda, M., Isocrono, D., Bianco, C. & Luppi-Mosca, Nash, T. H., III (ed.). (2008) Lichen Biology,2ndedn. A. M. (1997) Two foliose lichens as microfungal Cambridge: Cambridge University Press. ecological niches. Mycologia 89: 531–536. Pfister, D.H&Liftik, M. E. (1995) Arthrobotrys Grandjean, F. (1950) Sur deux espèces du genre anamorphs from Orbilia auricolor. Mycologia 87: Dometorina n. g. et les mœurs de D. plantivaga 684–688. (Berl.) (Acariens, Oribates). Bulletin de la Société Zoologique de France 75: 224–242. Schwendener, S. (1868) Ueber die Beziehungen Grube, M. & Berg, G. (2009) Microbial consortia zwischen Algen und Flechtengonidien. Botanische of bacteria and fungi with focus on the lichen Zeitung 26: 289–292. symbiosis. Fungal Biology Reviews 23: 72–85. Stubbs, C. S. (1989) Patterns of distribution and abun- Grube, M., Cardinale, M., Vieira de Castro, J., Jr., dance of corticolous lichens and their invertebrate Müller, H. & Berg, G. (2009) Species-specific associates on Quercus rubra in Maine. Bryologist 92: structural and functional diversity of bacterial com- 453–460. munities in lichen symbioses. ISME Journal 3: Suryanarayanan, T. S., Thirunavukkarasu, N., 1105–1115. Hariharan, G. N. & Balaji, P. (2005) Occurrence of Henckel, P. A. & Yuzhakova, L. A. (1936) On the role non-obligate microfungi inside lichen thalli. of Azotobacter in the lichen symbiosis. Izvestiia Sydowia 57: 120–130. Permskogo Biologicheskogo Nauchno-Issle- Thompson, M. G. (1958) Egg sampling for the western dovatel’skogo Instituta 10: 9–10. hemlock looper. Forestry Chronicle 34: 248–256. Hodkinson, B. & Lutzoni, F. (2009) A microbiotic U’Ren, J., Lutzoni, F., Mia˛dlikowska, J. & Arnold, A. E. survey of lichen-associated bacteria reveals a new (2010) Community analysis reveals close affinities lineage from the Rhizobiales. Symbiosis 49: 163–180. between endophytic and endolichenic fungi in Hodkinson, B. P., Gottel, N. R., Schadt, C. W. & mosses and lichens. Microbial Ecology 60: 340–353. Lutzoni, F. (in press) Photoautotrophic symbiont Wilson, C. G. & Sherman, P.W. (2010) Anciently and geography are major factors affecting highly asexual bdelloid rotifers escape lethal fungal para- structured and diverse bacterial communities in the sites by drying up and blowing away. Science 327: lichen microbiome. Environmental Microbiology. 574–576.

Accepted for publication 22 July 2011