phylogenetic diversity of trentepohlialean algae associated

J. Phycol. 47, 282–290 (2011)
2011 Phycological Society of America
DOI: 10.1111/j.1529-8817.2011.00962.x
PHYLOGENETIC DIVERSITY OF TRENTEPOHLIALEAN ALGAE ASSOCIATED WITH
LICHEN-FORMING FUNGI 1
Matthew P. Nelsen2
Committee on Evolutionary Biology, University of Chicago, 1025 E. 57th Street, Chicago, Illinois 60637, USA
Department of Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605-2496, USA
Eimy Rivas Plata
Department of Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605-2496, USA
Department of Biological Sciences, University of Illinois-Chicago, 845 West Taylor Street (MC 066), Chicago, Illinois 60607, USA
Carrie J. Andrew
Department of Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605-2496, USA
Department of Biology, Northeastern Illinois University, 5500 North St. Louis Ave., Chicago, Illinois 60625, USA
Robert Lücking and H. Thorsten Lumbsch
Department of Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605-2496, USA
of known species within the family vary from 70
(López-Bautista et al. 2007) to 100 (Guiry and
Guiry 2010), but the genetic diversity within currently accepted taxa (Rindi et al. 2009) and studies
in underexplored tropical regions (Rindi and LópezBautista 2007, 2008) suggest a much greater number. All Trentepohliaceae have filamentous growth
forms and often contain large amounts of carotenoid pigments (ß-carotene and hematochrome), causing the algae to appear yellow orange in color
(Thompson and Wujek 1997, López-Bautista et al.
2002). With the exception of an early study by Zechman et al. (1990), the phylogenetic diversity of trentepohlialean algae has only recently begun to be
explored (López-Bautista et al. 2002, 2003, 2006,
López-Bautista and Chapman 2003, Rindi et al.
2009, Suutari et al. 2010). These studies and others
(Lewis and McCourt 2004, Pröschold and Leliaert
2007) have confirmed their placement in Ulvophyceae and revealed them to be closely related to Bryopsidales, Cladophorales, and Dasycladales, with this
clade (Trentepohliales ⁄ Cladophorales ⁄ Bryopsidales ⁄
Dasycladales) sister to a group of more basal lineages
such as Ulvales and Ulotrichales. The phylogenetic
placement of Trentepohliales among numerous
groups of primarily marine algae suggests that Trentepohliales have transitioned from aquatic to terrestrial environments (López-Bautista et al. 2002,
López-Bautista and Chapman 2003). Additionally,
the work of Rindi et al. (2009) demonstrated that
traditional generic delimitations, based on morphological, cellular, and reproductive features, as well as
ecological characters, are in need of revision.
Trentepohlialean algae occur free living as well as
in association with lichen-forming fungi. Ahmadjian
(1993) estimated that 31% of lichen-forming fungal
Nearly one-fourth of the lichen-forming fungi associate with trentepohlialean algae, yet their genetic
diversity remains unknown. Recent work focusing on
free-living trentepohlialean algae has provided a phylogenetic context within which questions regarding
the lichenization of these algae can be asked. Here,
we concentrated our sampling on trentepohlialean
algae from lichens producing a diversity of growth
forms (fruticose and crustose) over a broad geographic substratum, ecological, and phylogenetic
range. We have demonstrated that there is no evidence for a single clade of strictly lichenized algae;
rather, a wide range demonstrated the ability to
associate with lichenized fungi. Variation was also
observed among trentepohlialean algae in lichens
from a single geographic area and tree, suggesting
that fungi in close proximity can associate with different trentepohlialean algae, consistent with the findings of trebouxiophycean algae and cyanobacteria.
Key index words: lichen; phylogeny; rbcL; symbiosis; Trentepohliales; Ulvophyceae
Abbreviation: rbcL, ribulose-bisphosphate carboxylase
Trentepohlialean algae are subaerial green algae
in the order Trentepohliales (class Ulvophyceae),
represented by the single family Trentepohliaceae,
which contains five genera: Trentepohlia Martius,
Printzina R. H. Thompson et Wujek, Phycopeltis Millardet, Cephaleuros Kunze ex E. M. Fries, and Stomatochroon Palm (López-Bautista et al. 2002). Estimates
1
Received 3 March 2010. Accepted 13 September 2010.
Author for correspondence: e-mail [email protected].
2
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TRENTEPOHLIALEAN ALGAE IN LICHENS
species associate with trentepohlialean algae. However, we estimate that a slightly lower proportion of
lichen-forming fungal species associate with these
algae ( 23%; to obtain our coarse approximation,
we summed the estimated number of described species in Ostropales [1,700], Arthoniomycetes [1,500],
Trypetheliales [200], Pyrenulales [200], Strigulaceae
[100], Arthopyreniaceae [100], and Monoblastiaceae
[200], all lineages that primarily associate with Trentepohliales algae, and divided by an estimated 17,500
described species of lichen-forming fungi). These
algae are more diverse and abundant in tropical ⁄ subtropical regions (Chapman 1984, Thompson and
Wujek 1997, Chapman and Waters 2002, LópezBautista et al. 2002, 2007, Rindi et al. 2010), a trend
reflected in their associations with chiefly tropical
lichen-forming fungi. For instance, Tucker et al.
(1991) estimated that 38% of the lichen-forming
fungal species in subtropical Louisiana (USA) associate with trentepohlialean algae. Similarly, Ahmadjian
(1967) suggested that 45% of the lichen-forming
fungal species in the tropics associate with trentepohlialean algae. In contrast, the proportion of lichens
containing trentepohlialean species drops dramatically in temperate areas, where 9% of the lichenforming fungal species are thought to associate with
trentepohlialean algae (Santesson 1952, Ahmadjian
1967). In extreme habitats, this number decreases
even further. For example, in Antarctica only a single
species having a trentepohlialean photobiont is
known (Øvstedal and Smith 2001). Interestingly,
some have also suggested that lichens with trentepohlialean photobionts are increasing in temperate
regions due to climate change; for instance, increases
in a number of lichens with trentepohlialean algae
have been attributed to increasing temperatures
(Aptroot and van Herk 2007) and ⁄ or increasing precipitation (van Herk 2009).
Fungi associating with trentepohlialean algae are
not monophyletic and instead occur in four classes
within Ascomycota (Lumbsch and Huhndorf 2007):
Arthoniomycetes, Dothideomycetes, Eurotiomycetes,
and Lecanoromycetes, plus the isolated Eremithallales (Lücking et al. 2008). These algae are especially
frequent as photobionts in foliicolous lichens:
40% of the foliicolous species associate with trentepohlialean algae (Santesson 1952, Lücking 2008).
Thalli produced by lichens in association with these
algae are typically crustose, although fruticose thalli
are formed in genera such as Roccella and Dendrographa and filamentous thalli in Coenogonium, Cystocoleus, and Racodium and occur on a variety of
substrata, such as leaves, tree bark, and rock.
Sipman and Harris (1989) suggested that fungal
associations with Trentepohlia photobionts are adaptations to moist, shaded tropical conditions, and
indeed, a number of studies have demonstrated the
abundance of lichens with trentepohlialean algae
under these conditions. For instance, Wolseley and
Aguirre-Hudson (1997) reported that in one of their
283
plots in an undisturbed evergreen forest in northern
Thailand, the frequency of lichen thalli with trentepohlialean photobionts reached 53%, and that
lichens containing trentepohlialean algae were more
frequent in evergreen forests than deciduous forests
or disturbed evergreen forests. Similarly, Rivas Plata
et al. (2008) demonstrated that several families and
genera of lichenized fungi associating with trentepohlialean algae preferred undisturbed primary
and old-growth secondary forest, fully shaded or
semiexposed microhabitats, and the bark of mature
tree trunks. Cáceres et al. (2008) observed a high
proportion of lichens containing trentepohlialean
algae in a coastal Atlantic rainforest in Brazil (Mata
Atlântica). Taken together, these studies suggest that
lichens with trentepohlialean algae are most abundant in humid, shaded undisturbed tropical forests.
When investigating the community composition
of free-living trentepohlialean algae, Rindi and
López-Bautista (2008) demonstrated that some trentepohlialean taxa were restricted to shady and
humid conditions in rainforests where they occurred
primarily on bark and leaves, whereas other species
occurred primarily in dry, high-light conditions, and
frequently on artificial substrata. Interestingly, some
trentepohlialean taxa have even been reported from
sloth hair (Suutari et al. 2010). In addition, Rindi
and Guiry (2002) determined that some trentepohlialean algae in Ireland showed a preference for
certain substrata (Trentepohlia aurea and T. iolithus:
cement; T. abietina: bark; T. cf. umbrina: limestone),
while others (Printzina lagenifera) were generalists.
Therefore, it will be of interest for future studies to
determine whether these algal species maintain their
environmental and substratum preferences when
lichenized and if understory lichen-forming fungi
are capable of occurring in exposed situations if in
association with an algal partner that typically occurs
in exposed environments.
Nearly all efforts to molecularly characterize
lichen photobionts have focused on cyanobacterial
symbionts (e.g., Miao et al. 1997, Paulsrud and Lindblad 1998, Fewer et al. 2002, Lücking et al. 2009) or
on eukaryotic trebouxiophycean algae (e.g., Beck
et al. 1998, Kroken and Taylor 2000, PierceyNormore and DePriest 2001, Lohtander et al. 2003,
Zoller and Lutzoni 2003, Nyati et al. 2007, Nelsen
and Gargas 2008, Škaloud and Peksa 2010). The
exceptions to this generalization come from Friedl
and Bhattacharya (2002) and Friedl and Büdel
(2008), who reported 18S sequences from ulvophycean algae (Dilabifilum arthopyreniae and Trentepohlia
sp.) in association with lichen-forming fungi, and a
small number of studies that examined one trentepohlialean isolate from the lichen Pyrenula sp.
(López-Bautista and Chapman 2003) and one from
the lichen Racodium rupestre (López-Bautista et al.
2006, Rindi et al. 2009). In addition, E. Baloch and
M. Grube (unpublished) have also begun to explore
the diversity of lichen-associated trentepohlialean
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MA T T H E W P . N E L S E N E T A L .
algae. Herein, we take a step toward elucidating the
phylogenetic position and breadth of trentepohlialean algae found in association with lichenized fungi
by presenting the first major effort to employ molecular markers to characterize the diversity of these
lichen-forming algae.
MATERIALS AND METHODS
Taxon selection. A broad range of lichens were selected (33
collections from 28 species), representing a variety of growth
forms, geographic locations, substrata, and phylogenetic range
of fungi. The majority of the algae sequenced were found in
association with lichen-forming fungi from the families Trypetheliaceae (Trypetheliales: Dothideomycetes) and Graphidaceae (Ostropales: Lecanoromycetes), though algae associating
with other fungal lineages were also included, which adds to
the breadth of substrata, growth forms, and geographic regions
studied. We also included six samples of closely related fungi
(Trypetheliaceae) from a single tree in Madre de Dios, Peru, to
assess algal variation at a small spatial scale.
Molecular methods. The Sigma REDExtract-N-Amp Plant PCR
Kit (Sigma, St. Louis, MO, USA) was used to isolate DNA from
small thallus fragments (1–4 mm2). No attempt was made to
separate the algae from the fungi. The DNA isolations followed
the manufacturer’s instructions, except only 10 lL of extraction
buffer and 10 lL dilution buffers were used, following Avis et al.
(2003). Dilutions of these extractions were found to perform
well for PCR amplifications, and a 20· DNA dilution was then
used in subsequent PCR reactions. A portion of the algal rbcL
gene was sequenced, using the newly designed primers a-chrbcL-203-5¢-MPN: GAA TCW TCW ACW GGW ACT TGG ACW
AC and a-ch-rbcL-991-3¢-MPN: CCT TCT ART TTA CCW ACA
AC. Primers were designed by aligning rbcL sequences of
selected trentepohlialean and trebouxiophycean algae and
selecting conserved regions 835 bp apart. The primer nomenclature is based on that of Gargas and DePriest (1996), where
the organismal group (a = algae) is followed by the location of
the gene (ch = chloroplast), the gene name (rbcL), the beginning position of the primer, the direction of the primer, and the
initials of the designer. The 10 lL PCR reactions consisted of
5 lM of each PCR primer, 3 mM of each deoxynucleoside
triphosphate (dNTP), 2 lL of 10 mgÆmL)1 100· BSA, 1.5 lL
10· PCR buffer, 0.5 lL Taq, 2 lL diluted DNA, and 2 lL
water. The PCR cycling conditions were as follows: 95C for
5 min, followed by 40 cycles of 95C for 1 min, 50C for 1 min,
and 72C for 1 min, followed by a single 72C final extension for
7 min. Samples were visualized on a 1% ethidium-bromidestained agarose gel under UV light, and bands were gel
extracted, heated at 70C for 5 min, cooled to 45C for
10 min, treated with 1 lL GELase (Epicentre Biotechnologies,
Madison, WI, USA), and incubated at 45C for at least 24 h.
The 10 lL cycle sequencing reactions consisted of 1–1.5 lL
of Big Dye version 3.1 (Applied Biosystems, Foster City, CA,
USA), 2.5–3 lL of Big Dye buffer, 6 lM primer, 0.75–2 lL
Gelased PCR product, and water. Samples were sequenced with
PCR primers and occasionally with two newly designed internal
primers a-ch-rbcL-494-5¢-MPN: CGT GAY AAA HTD AAC AAA
TA and a-ch-rbcL-706-3¢-MPN: TTT ARR TAR TGN CCT TT.
The cycle sequencing conditions were as follows: 96C for
1 min, followed by 25 cycles of 96C for 10 s, 50C for 5 s, and
60C for 4 min. Samples were precipitated and sequenced in
an Applied Biosystems 3730 DNA Analyzer, sequences assembled in Sequencher 4.9 (Gene Codes Corporation, Ann Arbor,
MI, USA), and sequences submitted to GenBank (Table S1, see
supplementary material).
Phylogenetic analyses. Available Trentepohliales algae (including one sequence from the lichen Racodium rupestre) and a
range of outgroup rbcL sequences (which were part of
Ulvophyceae, but outside Trentepohliales) were downloaded
from GenBank and aligned with sequences generated in the
current study. GenBank accession numbers for taxa included,
as well as specimen information for newly reported sequences
can be found in Table S1.
Sequences were manually aligned in Se-Al v.2.0a11 (Rambaut
1996). The alignment was partitioned by codon position and a
partitioned maximum-likelihood (ML) analysis was performed
in RAxML 7.0.4 (Stamatakis 2006), using the general time
reversible (GTR)+I+C (GTRGAMMAI) model with four rate
parameter categories (default) for each partition. In addition, support was estimated by performing 1,000 bootstrap
(Felsenstein 1985) replicates.
A partitioned Bayesian (BI) analysis was also performed in
MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). The model
for each partition was selected by first estimating the likelihood
of the data in PAUP* 4.0b10 (Swofford 2002) under 24
substitution models (with a fixed topology) and then calculating Akaike information criterion (AIC) scores for each model in
MrModeltest 2.2 (Nylander 2004). The model with the best AIC
score was selected for each partition and used in the Bayesian
analysis. Two parallel analyses were run for 10,000,000 generations, using the GTR+I+C substitution model for each codon
position, at a temperature of 0.0245 using four chains and
sampling every 100th tree. The average standard deviation of
split frequencies lower than 0.01 was used to suggest convergence between parallel runs (Ronquist et al. 2005). The initial
25,001 trees (25%) were discarded for burn-in, and posterior
probabilities were estimated by constructing a 50% majorityrule consensus tree of all sampled post burn-in trees.
RESULTS
The final alignment of 81 operational taxonomic
units (OTUs) was 712 bases long. Topologies
obtained with ML and BI did not show any strongly
supported conflicts (ML bootstrap ‡70% and BI posterior probability ‡0.95), and the topology from the
ML analysis is shown in Figure 1. Support values for
the ML tree can be found in Figure S1 (see the supplementary material), and the topology obtained
from the Bayesian analysis (with posterior probabilities) can be found in Figure S2 (see the supplementary material). We recovered the three major clades
identified in Rindi et al. (2009) and designated an
additional lineage as clade 4 (Fig. 1). Clade 4 is recovered as sister to clades 2+3, and unpublished 18S data
(M. P. Nelsen, C. J. Andrew, and R. Lücking) suggest
these algae may form a clade with the Trentepohlia
annulata F. Brand 18S sequence (SAG 20.94;
GenBank accession number DQ399588) from Rindi
et al. (2009). In addition, many unique shallow clades
were recovered, which were fairly divergent from
previously published sequences. Algae associating
with lichenized fungi did not form a monophyletic
group; they were instead scattered across the tree.
Algae from clade 1 occurred on a variety of substrata, including bark, concrete, metal, stone, and
leaves. Rindi et al. (2009) cautiously noted that most
clade 1 strains were temperate but that exceptions
existed and more sampling was needed before
drawing generalizations. Our findings have added a
large number of genetically divergent algae from
the tropics to this clade (Fig. 1). Geographically, this
TRENTEPOHLIALEAN ALGAE IN LICHENS
285
Fig. 1. Maximum-likelihood (ML) phylogeny of Trentepohliales algae based on rbcL sequence data. Acetabularia acetabulum and Bornetella
sphaerica were used to root the tree. Black asterisks above or below branches indicate strong support (ML bootstrap ‡70 and BI posterior
probability ‡0.95) for that clade. Nonlichenized haplotypes are listed in black, while lichenized haplotypes are in gray. Gray asterisks following GenBank accession numbers refer to algal haplotypes from the same tree in Madre de Dios, Peru. Lichen growth form, substratum, and
geographic information are indicated for Trentepohliales algae. Clade names refer to those from Rindi et al. (2009) and the present article.
286
MA T T H E W P . N E L S E N E T A L .
clade was recovered from North America, Central
America, and South America, as well as Europe, Fiji,
and the Azores. At present, this clade is found to
associate with lichen-forming fungi from Arthoniomycetes (Dendrographa and Dichosporidium) and Lecanoromycetes (Porina) and was distributed in
crustose (Porina), byssoid (Dichosporidium), and fruticose (Dendrographa) lichen thalli. In addition, two
algae (with an identical rbcL haplotype) associated
with Porina formed a lineage sister to the majority of
clade 1. At present, it is unclear if this represents an
extension of clade 1 or distinct lineage sister to clade
1; here, we tentatively include it in the clade 1.
A number of Trentepohlia and Printzina species
occurred in clade 2, along with several haplotypes or
clades that were fairly divergent from those previously published. Additionally, clade 2 also occurred
on a wide range of substrata including asbestos, bark,
concrete, wood, and leaves. This clade was recovered
from North America, Central America, and South
America, Europe, Africa, Hawaii, Fiji, Thailand, and
the Philippines. The algae in this clade associate with
Dothideomycetes (Astrothelium, Cryptothelium, Laurera,
Trypethelium, and Racodium), Arthoniomycetes
(Cryptothecia), and Lecanoromycetes (Acanthotrema,
Graphis, Thalloloma, Thelotrema, and Coenogonium) and
occurred in filamentous (Coenogonium linkii and
Racodium rupestre) and crustose (all remaining taxa)
lichens. Interestingly, identical rbcL sequences
were recovered from algae distributed in lichens
in Florida, USA (Astrothelium cf. diplocarpum [134],
A. galbineum [131], and Laurera megasperma [138])
and Peru (Trypethelium aeneum [61C] and T. cf.
aeneum [45]), illustrating that some rbcL haplotypes
are widespread. However, more variable loci must be
screened to determine if these individuals have identical genotypes. Nevertheless, similar algal individuals
were recovered from geographically disparate areas.
In contrast to clades 1, 2, and 4, no lichen symbionts were recovered from clade 3 in the present
study, although Strigula species are believed to associate with Cephaleuros (see Discussion). Clade 3
appeared to be primarily epiphyllous and composed
of the genus Cephaleuros, a well-known plant pathogen. These algae were recovered from Africa, Asia,
and North America. Finally, clade 4 contained algae
found in association with Dothideomycetes (Trypethelium), Eurotiomycetes (Anthracothecium), and Lecanoromycetes (Myriotrema). These algae all occurred in
crustose lichens growing on bark in India, Thailand,
and North America, though unpublished results
including 18S data suggest this clade also contains
free-living Trentepohlia annulata from the Czech
Republic (SAG 20.94; GenBank accession number
DQ399588).
DISCUSSION
Recent work on cyanobacterial symbionts of
lichens has identified a clade of Scytonema-like cyano-
bacteria occurring solely in the lichenized state
(Lücking et al. 2009). In contrast to this, our data
suggest that there is not a single lichenized clade of
Trentepohliales algae. Instead, several lineages associate with lichen-forming fungi, which is consistent
with the findings of other lichen-forming eukaryotic
algae or cyanobacteria that frequently occur free
living or in symbiosis with other organisms. For
instance, Nostoc associates with lichen-forming fungi
(Paulsrud and Lindblad 1998, Paulsrud et al. 1998,
2000, 2001, Rikkinen et al. 2002, Summerfield et al.
2002, Lohtander et al. 2003, Wirtz et al. 2003,
O’Brien et al. 2005, Stenroos et al. 2006, Myllys
et al. 2007, Elvebakk et al. 2008), but also with cycads (Vagnoli et al. 1992), the angiosperm Gunnera
(Nilsson et al. 2000, Svenning et al. 2005), the liverworts Blasia and Cavicularia (Rikkinen and Virtanen
2008), and the hornwort Anthoceros (Vagnoli et al.
1992), among other organisms. Phylogenetic studies
of Nostoc have revealed that isolates associating with
lichens do not form a monophyletic group; instead,
free-living isolates and plant symbionts are intermixed with lichen symbionts (O’Brien et al. 2005,
Rikkinen 2009).
The present study also illustrates that very divergent fungi (from different classes) associate with
closely related algae. For instance, the alga associated with Thalloloma hypoleptum (Lecanoromycetes)
is closely related to the algae from various Trypetheliaceae species (Dothideomycetes). A similar situation is seen in the ‘‘Rhizonema’’ clade of Scytonema
cyanobacteria, which were found to associate with a
very wide range of lichenized fungi from Ascomycota and Basidiomycota, suggesting that these distant lineages of fungi associate with closely related
cyanobacteria, representing a remarkable case of
convergence (Lücking et al. 2009). The same is also
true for trebouxioid algae, which are known to
associate with fungi from many lineages in Lecanoromycetidae and also Gomphillaceae in Ostropomycetidae (Lücking 2008).
Many previous studies have identified the photobionts associated with different fungal species on
the basis of morphological characters (see references in Tschermak-Woess 1988, for instance).
These fungal species were from the same genera we
investigated but were often different species from
those studied here. Nevertheless, the present study
confirms earlier morphological observations at a
coarse taxonomic scale, by demonstrating that various fungal species from Anthracothecium (Singh
1982), Coenogonium (Uyenco 1965, Meier and Chapman 1983, Davis and Rands 1993, Lakatos et al.
2004), Cryptothecia (Thor 1997, Jagadeesh Ram et al.
2009), Dendrographa (Sundin and Tehler 1996),
Dichosporidium (Thor 1990), Graphidaceae (Herisset
1946, Verseghy 1961, Nakano 1988, Matthews et al.
1989, Tucker et al. 1991), Porina (Santesson 1952,
Harris 1975, Sérusiaux 1979, Matthews et al. 1989,
Tucker et al. 1991, McCarthy 2001, Baloch and
TRENTEPOHLIALEAN ALGAE IN LICHENS
Grube 2006), Racodium (Koch 1962, Davis and
Rands 1993), and Trypethelium (Harris 1975, Lambright and Tucker 1980, Matthews et al. 1989,
Tucker et al. 1991) associate with trentepohlialean
algae. Remarkably, in several species of Coenogonium
(Uyenco 1965, Rivas Plata et al. 2006), Cystocoleus,
and Racodium (Muggia et al. 2008), the trentepohlialean algal partner determines the structure of the
resulting thalli.
Morphological identification of algal symbionts
associated with Graphis scripta (Trentepohlia umbrina:
Herisset 1946; T. annulata: Verseghy 1961; T. lagenifera
[Printzina lagenifera]: Nakano 1988; Phycopeltis sp.:
Matthews et al. 1989, Tucker et al. 1991) and Coenogonium interplexum [Trentepohlia abietina, T. arborum
(C. Agardh) Hariot, T. aurea, and T. elongata (Zeller) De Toni: Uyenco 1965,] suggest that the same
fungal species can associate with multiple trentepohlialean species, though, as stated earlier, taxonomic
delimitations of many Trentepohliales are in need
of revision (Rindi et al. 2009). The pattern of a single fungal species associating with multiple photobiont species or clades is also found among
associations with cyanobacteria (Rikkinen et al.
2002, Wirtz et al. 2003, O’Brien et al. 2005, Lücking
et al. 2009) and trebouxiophycean algae (Yahr et al.
2004, Blaha et al. 2006, Guzow-Krzeminska 2006,
Ohmura et al. 2006, Nelsen and Gargas 2009, Wornik and Grube 2010), and we expect that future
molecular data will confirm this trend in lichen
associations with trentepohlialean algae. In the present study, we did find evidence that L. megasperma
associated with multiple algal haplotypes, while
algae from two Porina tetracerae thalli from a single
site had identical rbcL sequences.
Two important additional observations were
made. First, several species of lichen-forming Trypetheliaceae were collected from a single tree in the
Peruvian Amazon, and the associated algae
sequenced. Algae from this tree are shown with an
asterisk in Figure 1 and demonstrate that several
haplotypes were recovered, illustrating that fungal
species living in close proximity do not all associate
with the same trentepohlialean haplotype. This is
consistent with the findings of lichen-forming fungi
associating with cyanobacteria (Paulsrud et al. 2000,
Myllys et al. 2007) and trebouxiophycean algae
(Beck 1999, Beck et al. 2002, Zoller and Lutzoni
2003, Guzow-Krzeminska 2006, Ohmura et al. 2006,
Piercey-Normore 2006, Doering and PierceyNormore 2009, Nelsen and Gargas 2009) and demonstrates genetic variation in trentepohlialean algal
communities.
Second, we found that closely related algal haplotypes were found in lichens occurring on both
leaves (Porina imitatrix, P. nucula, and P. distans)
and bark (P. dolichophora, P. aff. farinosa, and P. aff.
dolichophora), suggesting that closely related algae
(possibly the same species) can occur on different
substrata and that leaf and bark-dwelling lichenized
287
fungi may associate with closely related algae (or
possibly the same species). In this particular case,
the associated lichen fungi belong to groups of
Porina that are known to be able to switch between
substrata.
The present study has added several potentially
new lineages to the existing data of Rindi et al.
(2009). This was expected, as the study by Rindi
et al. (2009) was not intended to be an exhaustive
survey of the phylogenetic diversity of Trentepohliales. Nevertheless, the present study suggests that
there is more phylogenetic diversity to be discovered within the Trentepohliales. Additionally,
future work may recover lichen symbionts from
clade 3 as Cephaleuros has been identified as the
photobiont of a number of lichens (Santesson
1952, Harris 1975, Chapman 1976, Chapman and
Good 1983, Matthews et al. 1989, Lücking 2008,
Suto and Ohtani 2009). Future studies should also
include data from more loci and a greater sample
size to verify the findings reported herein. The
present study represents the first step toward understanding the range of trentepohlialean algae
involved in lichen symbiosis and placing them in a
phylogenetic framework.
K. Feldheim is thanked for discussion, and three anonymous
reviewers are thanked for improving the manuscript. This
research was possible thanks to the following grants to the
Field Museum: ‘‘Phylogeny and Taxonomy of Ostropalean
Fungi, with Emphasis on the Lichen-forming Thelotremataceae,’’ PI Thorsten Lumbsch (DEB 0516116); ‘‘Neotropical
Epiphytic Microlichens – An Innovative Inventory of a Highly
Diverse yet Little Known Group of Symbiotic Organisms,’’ PI
Robert Lücking (DEB 0715660); and ‘‘Systematics of the
Dothideomycetes,’’ PI Conrad Schoch, Co-PIs Thorsten
Lumbsch and Joseph Spatafora (DEB 0717476). In addition,
the Caterpillar company provided funds to study lichens
and their photobionts from Panama. Fieldwork was possible
thanks to the doctoral thesis grant ‘‘Estructura de la comunidad de liquenes crustosos, con enfasis en la familia Thelotremataceae, basada en especificidad de forofitos, microclima y
nivel de disturbio forestal en la Concesión de Conservación
Los Amigos (sureste peruano),’’ PI Eimy Rivas Plata (MOORE
Y7 10330), awarded by the Asociación para la Conservación de
la Cuenca Amazónica (ACCA). All DNA analyses were performed at the Pritzker Laboratory for Molecular Systematics
and Evolution at the Field Museum.
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Supplementary Material
The following supplementary material is available for this article:
Figure S1. Maximum-likelihood phylogeny of
Trentepohliales algae based on rbcL sequence
data with bootstrap support values. Acetabularia
acetabulum and Bornetella sphaerica were used to
root the tree. Nonlichenized haplotypes are listed
in black, while lichenized haplotypes are in gray.
Gray asterisks following GenBank accession numbers refer to algal haplotypes from the same tree
in Madre de Dios, Peru.
Figure S2. Majority-rule consensus tree from
Bayesian analysis of Trentepohliales algae based
on rbcL sequence data with posterior probabilities for individual clades. Acetabularia acetabulum
and Bornetella sphaerica were used to root the tree.
Nonlichenized haplotypes are listed in black
while lichenized haplotypes are in gray. Gray
asterisks following GenBank accession numbers
refer to algal haplotypes from the same tree in
Madre de Dios, Peru.
Table S1. Collection information for samples
used in this study. All lichens are deposited in
(F), except Racodium rupestre.
This material is available as part of the online
article.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any
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be directed to the corresponding author for the
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