New
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 Research
 Geographic variation in algal partners of Cladonia
 subtenuis (Cladoniaceae) highlights the dynamic nature of
 a lichen symbiosis
 Rebecca Yahrl,2*, Rytas Vilgalys1 and Paula T. DePriest2
 'Duke University, Department. of Biology, Box 90338, Durham, NC 27708 USA; 2Department of Botany, National Museum of Natural History, Smithsonian,
 Institution, PO Box 37012, Washington, DC 20013-7012 USA and Smithsonian Institution, Museum Conservation Institute, 4210 Silver Hill Road, Suitland,
 MD 20746 USA; *Present address: Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh EH3 5LR, UK.
 Author for correspondence:
 Rebecca Yahr
 Tel: +44 131 248 2957
 Fax: +44 131 248 2901
 Email: r.yahr@rbge.nc.uk
 Received: 28 February 2006
 Accepted: 20 April 2006
 Summary
 * Multiple interacting factors may explain variation present in symbiotic associa-
 tions, including fungal specificity, algal availability, mode of transmission and fungal
 selectivity. To separate these factors, we sampled the lichenized Cladonia subtenuis
 and associated Asterochloris algae across a broad geographic range.
 * We sampled 87 thalli across 11 sites using sequence data to test for fungal spe-
 cificity (phylogenetic range of association) and selectivity (frequency of association),
 fungal reproductive mode, and geographic structure among populations. Permuta-
 tion tests were used to examine symbiont transmission.
 * Four associated algal clades were found. Analysis of molecular variation (AMOVA)
 and partial Mantel tests suggested that the frequency of associated algal genotypes
 was significantly different among sites and habitats, but at random with respect to
 fungal genotype and clade. The apparent specificity for Clade II algae in the fungal
 species as a whole did not scale down to further within-species lineage-dependent
 specificity for particular algae. Fungal genotypes were not structured according to
 site and appeared to be recombining.
 * We suggest that ecological specialization exists for a specific lichen partnership
 and a site, and that this selectivity is dynamic and environment-dependent. We
 present a working model combining algal availability, fungal specificity and selectivity,
 which maintains variation in symbiotic composition across landscapes.
 Key words: symbiotic association, environmental specialization, Cladonia subtenuis
 (Cladoniaceae), lichenized fungus, specificity, selectivity.
 New Phytologist (2006) 171: 847-860
 ? The Authors (2006). Journal compilation ? New Phytologist (2006)
 doi: 10.1111/j.1469-8137.2006.01792.x
 Introduction
 The ecology and selective environment of all organisms include
 biotic interactions. Across these interactions, the processes
 shaping patterns of association among organisms are major
 forces shaping biodiversity. For example, strong specificity
 observed in insect herbivores in tropical forests has been
 suggested to be responsible for the huge diversity of herbivorous
 beetles (Erwin, 1982). Similarly, high estimates of fungal
 diversity (1.5 million species) originate from inferences about
 narrow specialization with plant hosts (Hawksworth, 2001).
 Roughly half the true fungi are known to be symbiotic with
 plants, as mycorrhizae, lichens or pathogens.
 Specialization in symbioses can arise independently through
 direct selection on one or both partners, or via indirect
 selec ion on the interacting partners as a selective unit (the
 holobiont). However, the mechanisms shaping the associa-
 tions in these symbioses are only beginning to be unravelled,
 and the relative contributions of phylogenetic history vs envi-
 ronmental variation are poorly understood in most groups.
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 848 Rsrch' l
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 Lichens are a classic example of symbiosis, and an
 important and conspicuous part of biodiversity, representing
 approximately half the known ascomycetes and totaling more
 than 16 000 fungal species (Eriksson, 2006). The symbiont
 composition and diversity of lichen associations is poorly
 known, however. Although major macroevolutionary patterns
 (such as cospeciation and phylogenetic congruence) have been
 rejected for green algal (Kroken & Taylor, 2000; Piercey-
 Normore & DePriest, 2001) and cyanobacterial lichens
 (Rikkinen etal., 2002; Lohtander etal., 2003; Wirtz et al.,
 2003), many fungal genera are found in nature only with
 specific photobiont genera (DePriest, 2004). In addition, many
 lichen species-level associations appear consistent (Paulsrud
 et al., 1998; Kroken &Taylor, 2000; Paulsrud etal, 2001), even
 across large geographic scales (Paulsrud et al., 2000). Taken
 together, these results clearly demonstrate a role for at least
 some phylogenetically constrained associations in lichen fungi.
 In addition to phylogenetic constraints on symbiotic
 associations, ecological mechanisms may also contribute to
 evolutionarily correlated patterns of association. For example,
 environmental variation (for example in geographic position,
 temperature or light conditions) correlates with temporally
 and spatially variable associations among horizontally trans-
 mitted symbionts in hard corals (Rowan & Knowlton, 1995;
 Rowan et aL, 1997; LaJeunesse & Trench, 2000; van Oppen
 etal., 2001) and fungi (Taylor & Bruns, 1999). In these
 examples, associations with different symbionts may act to
 maintain variation among generalist host populations,
 allowing selective specialization for particular environments
 or conditions by associating with and exploiting symbionts
 differentially. In lichen associations, population-level studies
 indicate that lichen fungi specialize on particular algae despite
 the availability of other species (Beck et al, 1998; Beck et al.,
 2002; Romeike et al., 2002; Yahr et al., 2004), or form
 'ecological guilds' of lichen fungi linked through shared
 photosynthetic partners (Rikkinen, 2003). Few studies have
 examined simultaneously the relative contributions of several
 possible mechanisms responsible for structuring symbioses.
 The elimination of one or more possible mechanisms can be
 achieved by taking an explicitly geographic approach, and by
 varying environmental factors (Brodie et al., 2002).
 Studies of other symbiotic systems provide a set of testable
 expectations for green algal-lichen associations, focusing on
 the roles of both phylogenetic and ecological hypotheses in
 determining the composition of the holobiont in nature. For
 example, at a single site a fungal species may be limited to a
 narrow group of algae by several possible mechanisms. First,
 dispersal limitation or strict vegetative reproduction may lead
 to a lack of, or infrequent, horizontal transmission, as seen in
 bacterial endosymbionts (Saffo, 1992; Moran & Baumann,
 1994; Huigens et al., 2000), grass endophytes (Schardl et al.,
 1997, 2004) or vertebrate parasites (Clayton & Johnson, 2003).
 Second, phylogenetic constraint, or a narrow genetically
 defined compatibility, may limit potential interactions, as
 observed in hard corals (Santos et al., 2004), fish parasites
 (Desdevises etal., 2002) and mycorrhizal fungi (Bruns &
 Read, 2000; Bidartondo & Bruns, 2001, 2002). Third, eco-
 logical dominance, or the increased probability of encounter-
 ing a single compatible type of symbiotic partner because of
 its high local frequency or fitness in a site, may result in a lim-
 ited set of associations, as suggested for some coral communi-
 ties (Rowan et al., 1997; LaJeunesse & Trench, 2000; Toller
 et al., 2001). Fourth, selectivity, or the symbiont's preference
 for a particular host in a particular site or habitat, may allow
 the symbiont to take advantage of variable fitness benefits of
 different hosts (Thompson, 1994; Desdevises et al., 2002).
 Finally, random processes, including drift, lineage sorting and
 historical artifacts, may result in stochastic associations not
 related to any of the above factors. Few of these hypotheses
 have been examined explicitly in green algal lichens.
 In lichens, the mechanism for symbiotic contact and thallus
 formation in nature is only partially understood. Honegger
 (1992) proposed a model for formation of symbiotic lichen
 structures in early developmental stages. In this model, an
 undifferentiated thallus stage exists in which fungal-algal
 contacts are formed in all sexual, and some asexual, lichens.
 Even in the case of asexual reproduction, where both partners
 are codispersed in specialized structures, de-differentiation
 (separation of algal and fungal partners) allows codispersed
 algae to be replaced by others available in the environment, or
 even 'robbed' from other nearby lichen associations (Friedl,
 1987). The frequency of such algal substitution or 'switching'
 (Piercey-Normore & DePriest, 2001) in nature is unknown,
 but this strategy may provide a certain level of flexibility in
 early stages of symbiosis and, importantly, a mechanism for
 optimizing symbiotic composition in a local environment.
 Cladonia species are symbiotic with green algal photobionts
 in the Asterochloris group of the genus Trebouxia (sensu Friedl,
 in Rambold et aL, 1998, hereafter referred to as Asterochloris).
 The lichen fungus Cladonia subtenuis, used as a model in the
 present study, provides the opportunity to test evolutionary
 and ecological mechanisms producing variable patterns of
 specificity and selectivity between symbionts. In Florida
 habitats, C. subtenuis was observed to associate with a single
 lineage of algae in Asterochloris Clade IIa (Yahr et al., 2004),
 appearing to have relatively high specificity for this algal partner.
 This lineage, Clade IIa Asterochloris, has at the present time
 been found only in Florida, although the diversity and distri-
 bution of Asterochloris lineages is in general poorly known.
 Intriguingly, however, C. subtenuis is distributed over much of
 eastern North America, suggesting that this fungus may form
 associations with other algae in different regions.
 In the present study, we investigate the geographic structure
 ofgenotypic variation among algal partners that are associated
 with C. subtenuis, and we compile and analyze data to create
 a conceptual model of factors responsible for structuring
 lichen associations. First, we compare fungal specificity across
 different geographic and taxonomic scales of study, comparing
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 Table 1 Pairwise F s for nrlTS of Asterochloris algal pools among sampling sites
 GA FL-3 FL-1 FL-2 NC-1 SC NJ NC-2 OZ PA VA
 n=6 n=ll n=3 n=21 n=6 n=7 n=2 n=7 n=6 n=6 n=9
 GA 0.000
 FL-3 0.051 0.000
 FL-1 -0.088 0.011 0.000
 FL-2 -0.002 0.143 -0.019 0.000
 NC-1 0.275 0.082 0.175 0.476 0.000
 SC 0.377 0.192 0.239 0.617 -0.206 0.000
 NJ 0.984 0.799 0.987 0.968 0.369 0.042 0.000
 NC-2 0.950 0.814 .0.943 0.953. 0.491.0.281 0.239 0.000
 OZ 0.892 0.801 0.864 0.934. 0.523.0.386.0.216 0.136 0.000
 PA 0.756 0.622 0.697 0.855 0.225 0.003 -0.163 0.039 0.152 0.000
 VA 0.880 0.790 0.859 0.921 0.529 0.383 -0.003 -0.020 -0.075 0.074 0.000
 Significantly positive values are shown in bold (P < 0.05). Comparisons across habitats are shaded. Overall FsT = 0.689, P = 0.00000.
 GA, Georgia; FL, Florida; NC, North Carolina; SC, South Carolina; NJ, New Jersey; OZ, the Ozark region of Missouri and Arkansas.
 the results ofYahr et al. (2004) with this study; second, we test
 hypotheses concerning the roles of phylogenetic constraint,
 clonal reproduction, ecological dominance, selectivity, or random
 processes in explaining observed patterns of association. In
 particular, we (1) examine phylogenetic associations among
 lineages of C. subtenuis and its algal partners; (2) test for clonal
 reproduction and evidence of recombination in C. subtenuis
 and for clonal transmission of algal partners; and (3) investigate
 geographic variability across eastern North America in algal
 associations with C. subtenuis and other Cladonia species to
 seek evidence for ecological dominance of algal lineages.
 Random associations serve as our null hypotheses for each of
 these proposed mechanisms. Our goal was to produce a con-
 ceptual model to integrate the various factors that may explain
 observed patterns of association in C. subtenuis. This model may
 also be applied to other lichens.
 Materials and Methods
 Study species
 Cladonia subtenuis (Abbayes) Mattick is an endemic of the
 West Indies and eastern North America, ranging from Florida
 westward to the Ozarks and north to the upper mid-west and
 Nova Scotia (Ahti, 1984). Populations of C. subtenuis can be
 found from open, acid, sandy soils in the coastal plain to
 granite outcrops and open pine forests over poor soil inland.
 Populations increase in density following fire (Hawkes &
 Menges, 1996) or disturbance. In open situations, it can occur
 with several other terrestrial lichen species, including several
 Cladonia species (Evans, 1952; Yahr, 2000).
 Sampling
 We sampled between three and 10 C. subtenuis thalli from
 each of 11 sites, in two habitats: coastal plain scrub and high pine
 vs inland pine forests (Table Si in Supplementary Material).
 Collections were made between 2001 and 2004, except for
 rece tly collected herbarium material from the Ozarks, SC,
 and NJ (see Table 1). Sites FL-2 and FL-3 (both in Florida) are
 aggregates of five and two nearby lo at ons, respectively, less than
 50 km apart. All thalli were collected along meandering transects
 through each site, taking care to sample from noncon iguous
 th lli (at least 1 m apa t if possible, typically > 5 m). We sampled
 a otal of 87 thalli. Clean, apparently healthy branches of each
 specimen, weighing approx. 10-25 mg, were selected under
 a dissecting microscope and placed in 1.5-ml tubes for
 extraction. Vouchers for each specimen are housed at Duke
 University, except those fr m SC and NJ (housed at PH
 Academy of N tural Sciences, Philadelphia, PA, USA).
 Molecular methods
 DNA extractions were performed using cetyltrimethylam-
 monium bromide (CTAB) and N-tris(hydroxymethyl) methyl-
 2-aminoethanesulfonic acid (TES) extraction buffers, and a
 protocol modified from Grube et al. (1995) and Ivanova et al.
 (1999). DNA was resuspended in 50 iil dH20 and frozen at
 -200C until needed. Dilutions of total genomic extracts were
 used as a PCR template for all amplifications, totaling between
 2 and 5 ng DNA per reaction. For internal transcribed spacers
 (ITS), we used forward primers specific for either fungi
 (1780-5'F: 5'-CTG CGG AAG GAT CAT TAA TGA G-3')
 or algae (1780-5'A: 5'-CTG CGG AAG GAT CAT TGA
 TTC-3') (Piercey-Normore & DePriest, 2001), and ITS4
 (White et al., 1990), at 10 mM each. We also sampled two
 single-copy nuclear genes, translation elongation factor 1
 (EFlac) and RNA polymerase subunit II (RBP2) regions 5-7
 (Liu et al., 1999). For these, we used newly designed primers
 (forward EFlao, CLEF-3F: 5'-GGC AAA GGC TCC TTC
 AAG T-3'; reverse EFla], CLEF-3R: 5'-GCC AAT ACC
 ACC GAT CTT GT-3'; forward RBP2, CLRPB5F: 5'-CTG
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 TTT CGA ACG CTG TTT CA-3'; reverse RBP2,
 CLRPB7R: 5'-CGC ATC CAC GTA TTC AAC AA-3'),
 each encompassing an entire (EFlat) or partial (RPB2) intron.
 Each PCR reaction also included 0.1 mm dNTP and 2.5 units
 Klentaq with the manufacturer's buffer (AB Peptides, St. Louis,
 MO, USA). Fungal ITS was amplified using a 94'C 3-min
 denaturation step followed by 27 cycles of 940 for 1 min, 500C
 for 1 min and 720C for 2 min, with a 3-s autoextension. EFlax
 and RPB2 were amplified using a 940C 3-min denaturation
 step followed by 30 cycles of 950 for 1 min, 550C for 30 s and
 720C for 1.5 min. Algal ITS was amplified using a 94'C 3-min
 denaturation step followed by 35 cycles of 940 for 1 min,
 50'C for 30 s and 720C for 1 min. PCR products were cleaned
 using Qiagen's QiaQuick kit (Qiagen, Valencia, CA, USA).
 Sequencing reactions were performed with PCR primers at
 2 mM using BigDye dye-terminator chemistry (Perkin Elmer,
 Boston, MA, USA) with 10 ng PCR template. Sequences were
 determined on an ABI Prism 3700 (Applied Biosystems,
 Foster City, CA, USA), edited and assembled using SEQUENCHER
 ver. 3.1, and exported into PAuP*4.0bl0 (Swofford, 2000).
 Sequence alignments were produced separately for each
 fungal and algal data set. For the fungal ITS data set, all
 sequences were compared with a database of Cladonia ITS
 sequences to verify field identifications. We used SITES (Hey &
 Wakeley, 1997) to identify identical sequences in both data
 sets, which were removed for phylogenetic analysis. For the
 aligned sequences, likelihood ratio tests were used to identify
 the best-fit model of sequence evolution using MODELTEST ver.
 3.06 (Posada & Crandall, 1998). Bayesian phylogenetic analysis
 was conducted using MRBAYES ver. 3.0b4 (Huelsenbeck &
 Ronquist, 2001). Model parameters were estimated in each
 analysis and begun using random starting trees for 1 000 000
 generations sampled every 100 for four complete runs for the
 fungi, and using 2 000 000 generations sampled every 100
 for the algae. We examined plots of likelihood for each run
 to determine the number of generations required to reach
 stationarity (burn-in). After discarding the trees collected during
 burn-in, we assembled all saved trees to calculate posterior
 probability as branch support. From this set of trees, for each
 of the algal and fungal data sets a tree with the lowest log-
 likelihood was selected for illustration purposes. In addition,
 we assessed branch support by parsimony bootstrapping using
 100 replicate heuristic searches using PAUP*4.0b 10 (Swofford,
 2000) in addition to Bayesian posterior probability.
 Phylogenetic associations
 Several terms have been used to describe patterns of associa-
 tion among interacting species. In this paper we use the term
 'specificity' to refer to the phylogenetic range of compatible
 partners for a given symbiont (Smith & Douglas, 1987).
 Analogous to the concept of host range in parasitology, we use
 'specificity' from the perspective of only one of the symbionts
 - the fungal partner. We adopt this perspective for two
 reasons. (1) In lichen algae the phylogenetic diversity is poorly
 known, and many divergent lineages of the commonest lichen
 algae, Trebouxia and Asterochloris (sensu Friedl, in Rambold et aL,
 1998) lack names (Kroken & Taylor, 2000; Helms et al., 2001;
 Piercey-Normore & DePriest, 2001). (2) Specificity of algae
 seems less restricted than fungal specificity (for example Cladonia
 is restricted to Asterochloris, while Asterochloris associates with
 several fungal genera; Piercey-Normore & DePriest, 2001).
 We used several methods to test for phylogenetic corre-
spondence between fungal and algal ITS variation as a
 description of specificity. First, to group algal genotypes we
 used analysis of molecular variation (AMOVA; Excoffier, 2000)
 with partitions structured according to statistically supported
 clades in the fungal ITS phylogeny. AMOVA partitions variation
 by calculating the proportion of covariance of components
 f om user-defined categories (in this case, fungal clade). We
 tested two fungal clade groupings using either four or five
 clades: three well supported fungal clades plus the remaining
 C. subtenuis genotypes (four clades); or including the three
 supported clades plus the division of the unresolved genotypes
 into the two lineages found in lowest log-likelihood tree (five
c ades). A Mantel test was used to calculate the correlation
 between matrices of algal and fungal pairwise genetic distance.
 Permutation tests using 10 000 replicates were used to test
 significance (Smouse et al., 1986) using ARLEQUIN (Schneider
 et al., 2000).
 Fungal reproductive mode and algal transmission
 To test for recombining fungal population structure and
 reproductive mode, we used several methods. Clonal codis-
 persive reproduction can be dismissed if either partner
 appears to have recombining population structure, or if
 partners appear to recombine with respect to one another.
 First, using SITES and RECOMBINATION DETECTION PROGRAM 2
 (RDP2; Martin et al., 2005), we examined a sample of 33
 EFla sequences plus one outgroup, and eight RPB2 sequences
 plus one outgroup. Alignments were produced manually in
 PAUP. Using each data set alone and by concatenating the
 sample of eight RPB2 sequences with the corresponding EFla
 a d fungal ITS, we used SITES to generate a table of site-by-site
 congruency and a table of the minimum set of recombination
 intervals (Hudson & Kaplan, 1985). As the number of
 sequences (eight for RPB2) or sites (642 bp for EFlax; 763 for
 RPB2) were in the range of values for which highly biased
 estimates of recombination parameters were expected, we did
 not estimate gamma (Hey & Wakeley, 1997) or Hudson's 4Nc.
 RDP (Martin & Rybicki, 2000) and MaxChi (Maynard-
 S ith, 1992; Posada & Crandall, 2001), which both performed
 we l in empirical tests, including situations using sequences with
 low sequence divergence (Posada, 2002), were implemented
 in RDP2 to estimate the number of recombination events.
 In addition, we used FST measures to test for population
 differentiation in the fungal populations. FST values range
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 from 0 to 1, with 0 indicating no population differentiation
 caused by gene flow among populations, and 1 indicating
 complete isolation. FST values that are significantly different
 from zero can therefore demonstrate genetic subdivision and
 reject the null hypothesis of panmixia. In addition to seeking
 evidence for recombination within the fungal genome, we
 also sought evidence of nonclonal transmission of algal
 partners in C. subtenuis. Using known frequencies of fungal
 and algal ITS genotypes, we performed a permutation test for
 random association between these fungal and algal ITS
 genotypes. There were 77 pairs for which both fungal and
 algal genotypes were known in this study. We therefore
 sampled with replacement from the 77 algae for which the
 genotypes were known, and from the 77 fungi; randomly
 associated these sampled algae and fungi; and recorded the
 number of unique genotype pairs. We repeated this process
 10 000 times. We then performed a one-tailed test to ask if the
 observed number of unique combinations was significantly
 lower than the fifth percentile (corresponding to a significance
 level of 0.05) of the distribution of random combinations, as
 would be expected if clonal propagation and codispersal of
 fungi and algae occur.
 Geographic associations
 In contrast with phylogenetically correlated specificity, select-
 ivity describes the frequency of association with any of the
 compatible partners (Rambold et al., 1998), and represents
 the combined effects of phylogenetically determined specificity
 and ecologically determined holobiont fitness. Selectivity, then,
 is a measure of ecological specialization. We used FST tests,
 AMOVA and partial Mantel tests to test geographic patterns of
 variation in symbiotic associations. Sites < 50 km apart were
 tested for differentiation using FST tests, and were combined
 if no significant structuring among sites was observed. Using
 the entire sample of either fungal or algal sequences separately,
 we calculated the overall FST to test for variation corre-
 sponding to geographic population structure. Significant
 values of FST are nonzero, indicating genetic subdivision.
 Exact tests of pairwise FST are calculated by generating
 replicated permuted data sets representing the null hypothesis
 of random association in ARLEQUIN. Pvalues were adjusted for
 multiple tests using a Bonferroni correction to adjust the
 threshold for significance proportional to the number of tests
 performed.
 Where significant population structure was detected in an
 analysis, Mantel tests were used to describe the correlation
 between genetic and geographic distance/habitat, or between
 the genetic distance of algal and fungal partners (Smouse
 et al., 1986), using ARLEQUIN. For the algal and fungal genetic
 distance matrices, we used the pairwise genetic distance calcu-
 lated using the K2P model with y = 0.0167 and y= 0.2215,
 respectively. Geographic distances were calculated based on
 latitude and longitude using a javascript program (http://
 www.wcrl.ars.usda.gov/cec/java/lat-long.htm). For all distance
 measures, we tested untransformed and log-transformed
 distances to ensure the measures used for correlation tests
 fitted the assumptions of linearity, and used untransformed
 geographic and genetic distance. The habitat matrix was con-
 structed by assigning 0 to all coastal plain sites and 1 to all sites
 outside the coastal plain, using the classification of Fenneman
 & Johnson (1946). We use these broad physiographic regions,
 coastal plain vs inland, as proxies for direct habitat measures.
 Partial correlations were calculated using Mantel tests in ARLE-
 QUIN with either algal or fungal pairwise distance matrices as
 the Ymatrix, geographic distance as matrix X, and habitat as
 X2. The null hypothesis tested was whether a random value
 for association between matrix measures was higher than the
 observed association. For significance of all tests, we used 10 000
 permutations.
 Results
 We examined a total of 87 thalli of C. subtenuis from 11 sites
 (Table S1). A total of 55 new algal ITS sequences were
 produced. Aligned sequences were 568 bp with 38 variable
 and 30 parsimony-informative sites. Of the 81 algal ITS
 samples, a total of 24 unique algal genotypes were present, all
 from Asterochloris Clade II (Piercey-Normore & DePriest,
 2001; Yahr et al., 2004). The most abundant algal genotypes
 were found 17 (J) and 12 (E) times, respectively, with another
 three common genotypes found eight, eight and seven times
 (II, AA and Q), respectively. The other 19 algal genotypes
 were observed four or fewer times. The 24 algal genotypes
 were members of three well supported clades: IIa, IIb (those
 identified in Yahr et al., 2004) and IIc, and one genotype of
 uncertain placement (genotype II, Fig. 1, inset), Clade IId.
 We sequenced the fungal ITS of 79 samples: 77 of these
 have corresponding algal sequences. Aligned fungal ITS was
 566 bp, with 112 variable characters of which 58 were
 parsimony-informative. We resolved 32 unique fungal ITS
 genotypes, distributed in three well supported clades plus
 several unresolved genotypes (Fig. 1). Sequences representing
 each fungal and algal ITS genotype in Fig. 1 have been depos-
 ited in GenBank (algal ITS accession nos DQ482671-82;
 fungal ITS accession nos DQ482683-3711).
 In addition, for C. subtenuis we generated 33 EFlao and
 eight RPB2 sequences. EFlca sequences were 643 bp with
 nine variable positions and five parsimony-informative sites
 (GenBank accession nos DQ490091-DQ4900105). These
 were sampled from five populations (FL-2, FL-3, GA, NC-1
 and PA) and from four of the five fungal ITS clades found
 (excluding the clade with only two representative genotypes;
 Table S1). A total of 15 genotypes were detected, all with > 99.7%
 similarity. The RPB2 data set included seven genotypes from
 eight sequences of 763 bp, nine variable positions and two
 parsimony-informative sites, all with > 99.3% similarity
 (GenBank accession nos DQ522282-DQ522289).
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 852 Rsearc
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 Clade I T. glomerata UTEX896
 m . e"u , fuMN7x i N -594:FL-1--------- 0 Tirregularis UTEX2236 u 1252:NJ, VA ----AA BB
 FF 943:FL-2, Ozarks - - . -1126:NC-2-------
 EE - 1112:FL-3 -------
 ED 1259:SC
 H 1240:VA-
 L M I - 579:FL-1- - - 3
 -N 1255
 cae-* 1008:FL-3--------
 _MM 1210:GA --
 00QQ 1136:NC-1 ------
 II
A  L973:FL-2 --------4 3~e I 1258:SC
 T. ericiUTEX910 PA, SC, Ozarks
 -0.001 substitutions/site 1190:NC-1 -
 1086:FL-3, Ozarks '-*L 24:NC2 1159:Ozarks - ------ II
 - 1124:NC-2 -2 ------------- C. subtenuis 1208:GA ---------------
 1231:PA-------------- /PP
 1229:PA-------------
 1234:VA --------------- BB
 11216:GA- -----------
 - 1216:GA 11213:GA ------ ------i
 1215:FL, GA-------- 01E =
 839:FL-2---------------
 1233: NC, PA ------ AA
 1230: PA ------------------- II
 C. conspicua 1341
 C. stygia 2815
 C. rangiferina 1302
 -- 0.001 substitutions/site
 Fig. 1 Phylogenetic relationships of fungal and algal partners and their associations. Each tree shown has the lowest log likelihood from each
 (algal or fungal) set of trees in the posterior distribution. Branch support of over 95% posterior probability is shown as thickened branches. Trees
 were rooted with Cladonia conspicua, Cladonia stygia and Cladonia rangiferina (fungal), or T erici (algal). (a, inset). Estimated internal
 transcribed spacer (ITS) phylogeny of unique Asterochloris algal genotypes. Genotypes of Asterochloris from Cladonia subtenuis are in capital
 letters and grouped by clade. (b) Estimated phylogeny of unique C. subtenuis ITS genotypes (numeric code according to voucher number) with
 their algal genotypes, corresponding to lettered codes in (a). States in which fungal genotypes were found are listed by standard two-letter
 abbreviations (see Table 1 footnote) following voucher numbers. Several sampling sites shown with single fungal voucher numbers indicate that
 the same genotype was found in samples from multiple sites. Asterisk indicates a fungal clade specific to algal Clade Ila.
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 In no case did we find evidence for more than a single copy
 of any sequenced region.
 Phylogenetic associations
 There is no clear association of fungal and algal genotypes
 overall, on the basis of mapping algal genotypes and clades
 onto the estimated fungal ITS phylogeny (Fig. 1). In three of
 four main fungal lineages, fungal relationships do not predict
 algal partners. For example, for eight distinct and unrelated
 fungal genotypes (Fig. 1, fungal genotypes 943, 1126, 973,
 1258, 629, 1124, 1086, 1233), associations were detected
 with at least two, and up to four, separate algal clades. No
 correlation between genetic distance of algae and fungi was
 found with a Mantel test (r = -0.079, P= 0.739; 0.06% of
 variation explained). One fungal clade, however, shows a
 relatively narrow host range, including only Clade IIa algae
 (asterisk in Fig. 1), although the algal partner of one member
 of this clade was not sequenced. This clade is found throughout
 Florida, northward along the coastal plain into New Jersey.
 An additional test of association was performed using an
 AMOVA to examine the genetic structure of algae according
 to fungal clade membership. We tested algal partitioning
 according to either four or five fungal clades. In both tests, only
 2-5% of algal genetic variation was distributed among fungal
 clades, whereas 95-98% of variation was found within
 fungal clades, resulting in an overall algal FST (according to
 fungal clade) not significantly different from zero (5 clade:
 FST = 0.0500, P= 0.110; 4 clade: FST = 0.0188, P= 0.252).
 We therefore found little evidence of multiple cryptic fungal
 lineages specific to narrow but different algal host ranges.
 Fungal reproductive mode and algal transmission
 Two hypotheses can be tested in relation to fungal repro-
 ductive mode: H1, clonal reproduction; and H2, random
 mating. Evidence of recombination can reject the null
 hypothesis of clonality (H1), while evidence against panmixia
 rejects the null hypothesis of random mating (H2). We use
 FST, a measure of gene flow (Cockerham & Weir, 1993), as a
 proxy for random mating. FST values approaching 0 indicate
 unlimited gene flow, whereas FST values approaching 1 indicate
 complete isolation of populations caused by the absence of
 gene flow among sites. Using the EFla data, SITES detected a
 minimum set of one recombination interval, shown between
 positions 114 and 254. No recombination events were detected
 in the RPB2 data. Using the combined data set (EFlIa, RPB2
 and ITS) with 1973 positions, two recombination events were
 detected, one at each of the boundaries between loci, with
 significant linkage disequilibrium detected only within loci.
 However, using RDP2 and MaxChi with single and concaten-
 ated data sets, no recombination events were detected. Fungal
 populations were poorly but significantly differentiated
 according to ITS variation (FsT = 0.091, P= 0.008) rejecting
 the hypothesis of panmixia. Finally, in a permutation test for
 association between fungal and algal genotypes, we found that
 the observed number of unique combinations (n = 56) was
 not significantly different from random (P = 0.360). If clonal
 propagation and codispersal of fungi and algae occurred, we
 would expect significantly fewer combinations than random.
 Algal geographic associations
 Algal ITS genetic variation was significantly structured by site,
 with overall FST = 0.687, P< 0.00001. Sites FL-2 and FL-3
 (both in Florida) are aggregates of five and two nearby loca-
 tions, respectively, < 50 km apart. These locations were tested
 for differentiation using FST tests, and combined as no
 significant differentiation among them was observed. Among
 all sites studied, pairwise comparisons showed especially strong
 differences between southern coastal plain (FL-1, FL-2, GA,
 FL-3) vs inland (NC-2, VA, PA, OZ) sites, with 15 of 16
 comparisons being statistically significant (Table 1, shaded).
 Nine of 12 comparisons between mid-Atlantic coastal plain
 (SC, NC-1, NJ) vs inland sites, and 12 of 21 within coastal
 plain comparisons, also had high FST values (> 0.15), although
 most were not statistically significant. Both geographic position
 and habitat predict variation in algae, with > 40% of the
 genetic variation we detected in algal symbionts explained by
 geographic distance (23.5%) and habitat (19.8%) in partial
 Mantel tests (Table 2). Geographic distance and habitat were
 themselves weakly correlated (r12 = 0.296), and both the
 partial correlations for geographic distance and habitat with
 algal genetic variation were significant (Table 2).
 Figure 2 shows the frequency of algal ITS clades associated
 with C. subtenuis for each sampling site, and highlights the
 unequal distribution of clades across sites. For example, all
 Clade IIa genotypes (dark gray) occur in the southern coastal
 Table 2 Partitioning of internal transcribed
 spacer (ITS) genetic variation in Asterochloris
 algae of Cladonia subtenuis according to
 partial Mantel tests, using pairwise genetic
 distance of algae (K2P + y= 0.0167) vs
 geographic distance (X1) and habitat (coastal
 plain vs inland; X2)
 Correlation Percentage
 Source of variation coefficient variation explained P
 Geographic distance 0.5449 23.5 0.000000
 Habitat 0.5137 19.8 0.01
 Geographic distance by habitat 0.2957 na na
 Geographic distance (habitat fixed) 0.4796 na 0.000000
 Habitat (geographic distance fixed) 0.4402 na 0.019
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 854ReIs
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 Fig. 2 Distribution (stippled) and collection
 sites for Cladonia subtenuis with proportion
 of major algal clades sampled in each site.
 Pie charts for coastal plain sampling sites
 are shown with dashed lines; solid lines
 surround inland sites. Colors of major algal
 clades correspond to Fig. 1: dark gray, Clade
 Ila; black, lIb; light gray, IIc; white, Ild.
 Table 3 Partitioning of internal transcribed
 spacer (ITS) genetic variation in Cladonia
 subtenuis according to partial Mantel tests,
 using pairwise genetic distance of fungal
 ITS (K2P + y = 0.3639) vs log (geographic
 distance) and habitat (coastal plain vs inland)
 Correlation Percentage
 Source of variation coefficient variation explained P
 Log (geographic distance) -0.232 7.0 0.903
 Habitat 0.226 6.7 0.059
 Geographic distance (habitat fixed) -0.300 na < 0.957
 Habitat (geographic distance fixed) 0.296 na 0.020
 plain south of 35' N, and most of Clades IIb (black) and IIc
 (light gray) algal genotypes occur northwards. Sites that har-
 bor different proportions of the same or partially overlapping
 algal clades (e.g. OZ vs NC-2; FL-2 vs FL-3, NC-1 or SC)
 also contain significantly different algal associates according
 to FST tests (Table 1).
 Fungal geographic associations
 We detected high diversity of fungal ITS genotypes within
 sites, with gene diversity (equivalent to heterozygosity estimates
 for haploid organisms) values between 0.44 and 0.78, calcul-
 ated according to Weir (1996). This is also consistent with
 gene diversity of 0.48 reported in an earlier study of C.
 subtenuis at NC-2 (Beard & DePriest, 1996). In comparison
 with the algal partners, C subtenuis has little population genetic
 structure as measured by pairwise FST or corresponding to
 geographic position. Only one of 55 pairwise comparisons
 shows significant structuring as measured by FST: between PA
 vs GA (data not shown). The overall measure of population
 differentiation using FST was 0.092 (P = 0.008). This value
 represents approx. 9% of the total genetic variation across all
 C. su tenuis samples, compared with the average > 90% of
 variat on found within populations: on average, any single
 popu ation of C. subtenuis fungi contains > 90% of that found
 across the entire species. This genetic structure among fungal
ITS genotypes is ot significantly correlated with geographic
 distance (r = -0.232, P= 0.903). Fungal genetic structure is
 marginally significantly associated with habitat differences
 (r = 0.226, P= 0.059; Table 3), explaining approx. 6.7% of
 the variation in fungal ITS distribution. The partial
 correlation of habitat with the fixed effect of geographic distance
 (r = 0.296, P= 0.020) was significant, but the converse was
 not (r = -0.300, P= 0.957).
 Discussion
 The phylogenetic breadth of a species' potential symbiotic
 partners - its specifici y - may be a product of intrinsic or
 extrinsi  factors. The combination of intrinsic, genetically
 determined constraints with extrinsic factors such as
 vailability of partners r fitness in a particular site leads to
 observed pattern  of association among symbionts. Selectivity
 is def ned as the frequency of observed association among
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 compatible partners (Rambold et al., 1998), and may be a
 function of these interacting forces. In the lichenized fungus
 C subtenuis, significant differences among sites in the asso-
 ciations with Asterochloris algae exist in eastern North America
 (Fig. 2). This geographic structure in algal associations
 occurred in the context of limited population structure in the
 fungal partners, and without obvious genetic correlations
 between fungi and algae - without evidence of clonal trans-
 mission of partners or within species clade-level specificity.
 Among sites, a comparison of the distribution of known
 compatible algal genotypes for C subtenuis with the patterns
 of association with these genotypes shows that not all com-
 patible algae present in a particular site are associated. Instead,
 a degree of ecological specialization exists between the part-
 nership of fungus plus alga for a particular site, and this
 specialization appears to depend on the local environment.
 Across the range of C. subtenuis, we found four associated
 algal clades. This algal specificity is much lower than that for
 a smaller sampling area (Yahr et al., 2004) for the same fungal
 species, reinforcing the observation that sampling scale has
 important implications for studies of ecological interactions
 (Thompson, 1997). In addition, despite high algal clade
 diversity within C. subtenuis (Fig. 1), we cannot detect
 specificity of these fungal lineages for particular algal clades.
 Instead, in our sample of C subtenuis, both geographic
 position and habitat are good predictors of algal genotype
 distribution. This result is consistent with the selective asso-
 ciations suggested for algae in Cladonia rangiferina (Piercey-
 Normore, 2004) and for 'Trebouxia sp. 3' in subalpine
 Letharia populations of the Pacific north-west (Kroken &
 Taylor, 2000). We found that algal associations are not
 predicted by fungal lineage, as distribution of fungal genetic
 variation was not, or was only weakly, structured by geographic
 factors, and fungal populations appear panmictic across their
 range.
 Fungal specificity decreases over increasing geographic
 area
 Fungal specificity is the pattern of taxonomically limited
 associations and may indicate genetically determined com-
 patibility, a mechanism that may result in tight associations
 between symbionts. In C. subtenuis, algal associations vary
 across its range, but appear specific for members of Clade II.
 The apparent specificity for Clade II algae in the fungal species
 as a whole does not scale down to further within-species,
 lineage-dependent specificity for particular algae. An example
 ofwithin-species, clade-level specificity is illustrated by the C.
 subtenuis clade indicated by an asterisk in Fig. 1, which
 associated with a single clade of algae (IIa). In contrast, we
 found that most (four of five) lineages within C. subtenuis
 were compatible with several distinct Clade II algae.
 Although almost all samples of C. subtenuis in Florida are
 apparently restricted to Clade IIa algae, this study demonstrates
 that association of C subtenuis with Clade IIa algae in Florida
 cannot be explained solely by phylogenetic correlation or
 specificity below the species level. First, most of the lineages
 of C. subtenuis detected in our sample across eastern North
 America were found in Florida, ruling out the possibility that
 fungal lineages are geographically restricted within the area
 sampled. Second, identical lineages (and genotypes) of fungi
 restricted to Clade IIa algae in Florida are found with different
 Clade II lineages in other parts of the geographic range of
 C. subtenuis (Fig. 1), demonstrating a lack of specificity for
 individual fungal lineages. In the light of these new observa-
 tions, C. subtenuis appears specific for the large group of Clade
 II algae in general, not with a single lineage within that group.
 In addition, these observations exclude the possibility that
 cryptic species that may be currently included in the concept
 of C. subtenuis are specific for different algal lineages. These
 Clade II Asterochloris algae may represent a very disparate
 group, containing several unnamed clades (IIa, IIb, IIc) as well
 as sequences belonging to Trebouxia magna and Trebouxia
 excentrica (Piercey-Normore & DePriest, 2001; Yahr et al.,
 2004).
 Because determination of specificity here is based on
 molecular phylogenetic criteria, failure to resolve fine-scale
 associations between fungal and algal lineages is highly
 dependent on obtaining appropriate phylogenetic resolution,
 in this instance using ITS sequences to estimate phylogenies.
 For some fungal studies, ITS has proved a less variable marker
 than other single-copy nuclear genes such as EFlcl, RPB2 or
 even anonymous DNA markers (Kroken & Taylor, 2001).
 However, screens of EFla and RPB2 have so far revealed less
 variation than in ITS for C. subtenuis (see Results). Although
 we cannot rule out further resolution altering these conclu-
 sions, we believe the associations suggested by fungal ITS are
 conservative. The amount of apparent algal sharing indicated
 by Fig. 1 suggests that, even if additional lineages could be
 resolved, they would also be likely to associate with multiple
 algal genotypes (e.g. fungal genotype 629,, Fig. 1). The reso- lution in our algal phylogeny is similarly restricted to infor-
 mation from the ITS. Although we did not sample any other
 markers, other researchers have used actin-coding regions and
 introns for studies of Trebouxia photobionts; these markers
 have been largely congruent with, and less variable than, ITS
 (Kroken & Taylor, 2000; Piercey-Normore, 2006).
 Clonal propagation
 A simple model of tight associations between symbionts
 predicts that t e partners cannot be separated, or are only
 infrequently separated. In some symbiotic systems, such as
 maternally inherited endosymbionts (Saffo, 1992; Moran &
 Baumann, 1994; H igens et al., 2000) or grass endophytes
 (Clay, 1990; Saikkonen et al., 2002), strong population struc-
 ture and shared phylogenetic history ofsymbionts are expected
 because of the vertical transmission of symbionts (Brem &
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 856 Research
 New
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 Leuchtmann, 2003). In lichens, vegetative fragmentation
 of thalli is likely within populations, producing physically
 separate but genetically identical thalli, or 'symbiotic
 individuals', including genetically identical fungal and algal
 components (Paulsrud et al., 1998). Although C. subtenuis
 has no specialized vegetative propagules, we detected 11 such
 putative clones, unique combinations of fungal and algal
 genotypes, found more than once in a single site or among
 sites (a total of 32 samples), consistent with that expectation.
 The average geographic distance between putative clones was
 approx. 160 km, with only nine of 45 pairs sampled from the
 same site. In some cases, indistinguishable symbiotic indi-
 viduals were found widely distributed among distant sites, for
 example between North Carolina's Coastal Plain and Florida's
 North Gulf Coast, > 550 km apart. This distribution may
 reflect either widespread dispersal of clones; lack of resolution
 in these markers; or, perhaps most likely, chance reassociation
 with identical genotypes.
 Despite the repeated occurrences of symbiotic individuals
 in C. subtenuis, the absence of association between fungal and
 algal genetic variation using both Mantel tests and AMOVA
 illustrates the reassorting of algal and fungal lineages, and sug-
 gests horizontal transmission of symbionts (any transmission
 not between parents and offspring). Furthermore, the number
 of unique symbiotic individuals is well within the number
 expected under random association of fungal and algal geno-
 types (Fig. 2). Finally, the occurrence of seven different algal
 genotypes with a single fungal genotype (629 in Fig. 1)
 further supports the hypothesis of horizontal transmission of
 symbionts. This indication of horizontal algal transmission is
 also consistent with observations suggesting de novo licheni-
 zation in this species, for example frequent observations of
 juvenile recruitment in the field, presumably from spores.
 Although clonal propagation cannot be rejected as contrib-
 uting to the patterns of association between fungal and algal
 symbionts, it does not appear to be a major factor in this
 species. In addition to the evidence above, the sexual strategy
 inferred from the evidence of recombination between sam-
 pled gene regions (and within EF 1 a) using SITES also suggests
 that re-lichenization must occur at each new generation. The
 lack of significant evidence for recombination using the other
 two algorithms implemented in RDP probably indicates
 insufficient statistical power, as the ability to detect recombi-
 nation in empirical studies relies especially on the amount of
 sequence divergence (Posada, 2002). Sexual reproduction has
 been demonstrated in several other species of Cladonia using
 fingerprinting techniques (Seymour et al, 2005).
 Algal availability does not limit lichen associations
 The geographic distribution of algal strains associated with
 lichen fungi is so far incompletely known. Trebouxioid symbionts
 may not require lichenization for all or even part of their life
 cycle, and may frequently occur as free-living (Mukhtar et al.,
 1994). Inferences from recent molecular work also stress the
 likelihood of a pool of free-living green algae in explaining
 observed lichen associations (Beck et al., 1998; Beck, 1999;
 Kroken & Taylor, 2000; Piercey-Normore & DePriest, 2001;
 Romeike et al., 2002). However, even if some algal strains are
 capable of surviving as free-living, they may not be frequent
 enough to be encountered by a Cladonia spore or fragment
 (Courtney & Chew, 1987). Therefore the frequency and
 distribution of available algae in each site may determine algal
 abundance in lichen associations.
 In studies of algal associations, some geographic differences
 in algal genotypic distribution have been detected. Clade II
 algae are widely distributed and genetically diverse, with a
 clade of neotropical genotypes (Piercey-Normore & DePriest,
 2001) and a clade known only from the south-eastern USA,
 Clade IIa (Yahr et al., 2004). Although in Florida C. subtenuis
 associates almost exclusively with Clade IIa algae, Clade IIb
 algae are also known to be compatible (Fig. 1) and abundant
 in Florida, being the most common symbionts of other
 co-occurring Cladonia species: C. leporina, C. pachycladodes,
 C. perforata and C. prostrata (Yahr et al., 2004). Therefore the
 almost exclusive association of C. subtenuiswith Clade IIa algae
 in Florida cannot be explained by either its absence or low
 frequency, at least across the coastal plain in Florida. Likewise,
 in the Ozarks, Clade IIa algae have been found in Cladonia
 caroliniana, although they are absent from C. subtenuis in that
 area (R.Y., unpublished results). Finally, single algal genotypes
 can sometimes be found across several continents (Piercey-
 Normore & DePriest, 2001), indicating that although
 narrow algal distributions are possible, they are clearly not
 solely responsible for observed associations between lichen
 symbionts.
 Random processes
 Historical artifacts such as population bottlenecks are expected
 to produce patterns of low diversity and strong, correlated
 geographic structure. In C. subtenuis, there is no evidence of
 low diversity in subsets of sites or genetically isolated popu-
 lations, as expected with bottlenecks or founder events. Further-
 more, random processes cannot explain the observed geographic
 patterns in algae, as the same patterns of association across
 distant sites (e.g. associations with only Clade IIa across
 multiple sites in the south-eastern Coastal Plain) would not
 be expected. The finding of strong patterns with respect to
 geographic associations suggests active processes beyond those
 with a stochastic explanation (Fig. 2).
 Fungal selectivity
 The variable fitness ofsymbiont associations across a range of
 ecological settings may determine their success, and therefore
 their frequency of association, in a site or habitat (Fox &
 Morrow, 1981). Although the distribution of fungal variation
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 New
 Phytologist  Resep~~~D i
 Fig. 3 A hypothetical model of fungal-algal
 association for lichens, showing the
 interactions between partners and the
 mechanisms that structure them. (a) Algal
 availability determines which algae can be
 contacted. (b) Fungal specificity determines
 the ability to form prethallus stages. (c)
 Fungal selectivity, dependent on ecological
 context, determines the final composition of
 observed lichens.
 ) Algal availability
 yes
 switching peict
 n o yes no
 , ............
 * *: * ' Symbiont selectivity
 no yes
 ................. ....................
 Paaiimorcesu
 is weakly correlated with habitat differences, this factor
 explains only about 7% of the variation observed (Table 3).
 Therefore the strong relationship between geographic position
 and C subtenuis algal associations shown here could be caused by
 either algal factors alone (i.e. fitness or abundance of free-living
 algae), or mechanisms that act on the composite symbiotic
 thallus. These dynamic species interactions may produce
 evolutionarily specialized lineages and coevolution between
 partners (e.g. Clade IIa in Florida sites; Thompson, 1994;
 Thompson, 1999), and empirical studies have shown geographic
 structure in the organization (Taylor & Bruns, 1999) and
 outcomes of other symbiotic interactions (Lively, 1999; Brodie
 et al., 2002). Although the role of genetically structured
 populations is well understood in establishing and maintain-
 ing mosaics of differentially specialized species interactions,
 studies of lichens have so far not investigated this factor in
 detail.
 Two possibilities exist to explain local patterns of speciali-
 zation in C. subtenuis for algae. (1) Given both the presence
 of, and the specificity for, Clade II algae, C. subtenuis may
 associate randomly with compatible Clade II algae it encounters
 at a site. Following initial contacts and establishment, varying
 site-specific selective forces may remove unfit symbiotic com-
 binations, leaving only the subset of successful thalli with an
 apparently specialized symbiotic composition. (2) Alterna-
 tively, the fungus may recognize and 'select' one partner over
 another (for example via more efficient interactions; Schaper
 & Ott, 2003). Such local and variable selection is akin to the
 local specialization of insects for host plants, which is known
 to occur on small spatial scales (Jaenike, 1990). In our study,
 the habitat division tested is broad, but is known to be
 important for the distribution of variation between Cladonia
 polycarpia and Cladoniapolycarpoides, with soil type explain-
 ing distributional differences in sibling species (Park,
 1985). Habitats in the coastal plain are well differentiated
 from inland sites in terms of both geology and vegetation,
 with the former typically characterized by sandy, acidic and
 nutrient-poor soils, supporting open vegetati n types such as
 coastal dune scrubs and sandhills (Fenneman & Johnson,
 1946). The differences in symbiotic associations detected here
 between coastal plain and inland sites may reflect the local
 specialization of the holob ont to selective factors in these
 differ nt habitats. For example, varying forest ight levels have
 been hypothesized to be important for determining mixtures
 of alg l genotypes within thalli optimal fo  photosynthesis
 in a recent study of an epiphytic l chen association (Piercey-
 Normore, 2006).
 A model of lichen associations in nature
 Based on the observations above, we propose the following
 generalized model for factors involved in lichen associations
 (Fig. 3). This model is similar to Combes's 'filter model',
 which emphasizes those mechanisms - 'filters' - determining
 encounter and compatibility between parasites (Combes,
 2001). However, we add an important new ecological filter
 determining fitness of the symbiotic association. Pre-thallus
 stages have not been studied and are not included.
 Algal availability First, algae disperse widely, perhaps globally,
 providing a continuous and diverse source of potential algal
 partners. Studies of wind patterns (Munoz et al., 2004) and
 airborne algae and soredia (Tormo etal., 2001) support this
 idea. These are the algae available for contact by fungal partners,
 although algal frequency and habitat suitability may also play
 a role in what algae are available. Certain habitats may
 prove unsuitable for a portion of this algal 'rain', and these
 genotypes are not available in these sites, or at such low
 abundance as to be undetectable. Clade IIc algae may fit this
 scenario: in a study of eight species of Cladonia in Florida's
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 -58 RU,
 New
 Phytologist
 coastal plain with over 200 samples investigated, no genotypes
 of this clade were detected (Yahr et al., 2004). As shown in this
 study, its absence from such sampling may be caused by those
 algae not being 'selected' by the sampled fungi in these sites.
 An empirical study of algal frequency in environmental (e.g.
 soil, air) samples is needed to address this aspect of the model,
 for example by PCR detection (Walser et al., 2001). None-
 theless, it is possible that certain algae are habitat specialists to
 some extent, with such low fitness in others as to be func-
 tionally absent from the lichen association.
 Fungal specificity Following thinning of available genotypes
 by environmental selection, the inherent and phylogenetically
 determined compatibility of fungal propagules may limit the
 algae with which they can associate. In vitro studies have shown
 that compatibility varies between fungal and algal species,
 with variable efficiency of interactions in terms of timing of
 contact and establishment ofhaustoria (Schaper & Ott, 2003),
 and with variable outcomes for the algae and fungi. For example,
 a fungus equally compatible with two algal strains, but more
 efficient in terms of establishing contacts and conduits for
 symbiotic interactions with one, will probably be more frequent
 in nature with that strain. Likewise, some fungi are able to
 parasitize nonsymbiotic algae temporarily, while in some
 instances lichen symbiosis seems unlikely to establish at all
 (Ahmadjian et aL, 1980; Schaper & Ott, 2003). In this example,
 C. subtenuis may be phylogenetically compatible with any of
 the available Clade II algae, but apparently not with Clade I
 algae, to the extent that this association has not yet been
 detected. Lack of association at this stage may be a product of
 failure to recognize, communicate with, or establish stable
 associations with potential partners.
 Fungal selectivity Lastly, even compatible pairings in one
 habitat may not be optimal in another, leaving only a portion
 of any possible associations detectable at any site. Ecological
 context is known to be an important variable determining the
 evolutionary outcomes (Bronstein, 1994) and distribution of
 interacting species (Fox & Morrow, 1981; Thompson, 1999;
 Desdevises et al., 2002). For example, the importance of
 spatial scale has been shown in several coral symbioses (Rowan
 & Knowlton, 1995; Rowan et al., 1997; Rowan, 1998), where
 strong patterns of variation in algal associations correlate with
 ecological variables (e.g. light, depth, latitude). In the lichen
 model, a compatible - but nonoptimal - photobiont may be
 parasitized by the fungus, or an association of low fitness may
 form, resulting in poor performance and low abundance.
 Such an association may persist, or algal switching may occur
 (dashed line, Fig. 3).
 Therefore the frequency of associations in a site is not
 necessarily determined by the frequency of those partners
 that arrive and survive by themselves, but rather by the
 environmentally determined success of the partnerships that
 form.
 In conclusion, we have provided indirect evidence that
 differential selection maintains variation among populations
 for symbiotic interactions in the field, and that the variation
 in associations among populations can be explained by
 ecological determinants. We suggest that the variation in
 ecological parameters is a frequently ignored, yet important
 factor in determining associations in lichens and should be the
 subject of experimental tests in future studies.
 Acknowledgements
 The authors thank Carl Weekley, Alia Wally, James Lendemer
 (PH), Dick Harris (NY) and Philip May for collections;
 William E Morris for assistance in performing permutation
 tests; and John Willis, Stuart McDaniel, Daniel Henk,
 Thomas Meagher and three anonymous reviewers for helpful
 comments on earlier versions of the manuscript. This study
 was funded by NSF Partnerships for Enhancing Expertise
 in Taxonomy grant number 9712484 to P.T.D., R.V. and
 S. Hammer, Monographic Studies in the Cladoniaceae
 (Lichen-forming Ascomycetes).
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 Supplementary Material
 The following supplementary material is available for this
 article online
 Table S1 Collection information for specimens sampled, with
 algal and fungal internal transcribed spacer (ITS) genotypes
 (vouchers are housed at DUKE unless otherwise indicated)
 This material is available as part of the online article from
 http://www.blackwell-synergy.com
 New Phytologist (2006) 171: 847-860 www.newphytologist.org ? The Authors (2006). Journal compilation ? New Phytologist (2006)
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