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Systematics and Phytogeography |
Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado 80309-0334 USA
Received for publication November 17, 2006. Accepted for publication May 31, 2007.
ABSTRACT
Many plant species are characterized by a life cycle with a long-lived, subterranean phase that is completely dependent on mycorrhizal fungal symbionts for fixed carbon. This type of life cycle is both phylogenetically and ecologically widespread and is found in diverse vascular plant lineages from the tropics to subalpine meadows. Here we report on the molecular identities of the arbuscular mycorrhizal fungi associated with the autotrophic and underground mycoheterotrophic life cycle phases of the ferns Botrychium crenulatum and B. lanceolatum. We show that the Glomus taxa found in the mycoheterotrophic life cycle phases of B. crenulatum and B. lanceolatum are also found in conspecific and heterospecific photosynthetic neighboring plants. From our DNA sequence data, we infer carbon flow from photosynthetic plants to mycoheterotrophic plants through shared glomalean fungal networks. Finally, our phylogenetic analyses identify a major Glomus clade that forms associations with mycoheterotrophic life cycle stages of B. crenulatum and B. lanceolatum.
Key Words: arbuscular mycorrhizal symbioses • Botrychium • Glomus • mycoheterotrophs
Between 60 and 80% of all land plant species have intracellular mutualistic associations with arbuscular mycorrhizal (AM) fungi in which the photosynthetic plant host gains increased access to mineral nutrients and the fungal symbiont acquires fixed carbon (Smith and Read, 1997
). To date, most AM research has focused on these mutualistic associations in seed plant lineages.
Many lineages of vascular nonseed plants (pteridophytes) are characterized by a life cycle with a long-lived, subterranean gametophytic phase that is completely dependent on its AM fungal symbionts for fixed carbon (Leake, 1993
; Read et al., 2000
). This life history is found in roughly 1000 fern and lycopsid species from the tropics to the subalpine (Bierhorst, 1971
; Kenrick and Crane, 1997
). Although AM fungal symbionts have been documented by microscopy in the underground phases of diverse fern and lycopsid lineages (reviewed in Bower, 1935
; Bierhorst, 1971
; Leake, 1993
, 2004
; Read et al., 2000
; recent reports include Duckett and Ligrone, 1992
; Schmid and Oberwinkler, 1994
, 1996
; Carafa et al., 2003
), nothing is known about the molecular phylogenetic identities of the fungal partners within these life cycles. Moreover, the nature of the AM networks that facilitate carbon flow to these subterranean nonphotosynthetic plants remains a mystery. In essence, the ecology and evolutionary biology of this relatively common pattern of plant–mycorrhizal association has been largely overlooked in the coevolutionary history of land plants and fungi.
We employed a DNA sequenced-based approach to address four fundamental questions: What is the molecular phylogenetic identity of the mycorrhizal symbionts in taxa (specifically Botrychium) with underground life cycle stages? In plant life cycles with autotrophic and mycoheterotrophic stages, are the AM fungal symbionts the same throughout the alternation of generations or do they differ? How do the AM partners in the mycoheterotrophic life cycle stages of plants integrate with the mycorrhizal fungal network in the surrounding autotrophic plant community? What are the evolutionary relationships of Glomus taxa that form associations with autotrophic and mycoheterotrophic life cycle stages of Botrychium?
We chose to investigate these questions in Botrychium Swartz subgenus Botrychium (Ophioglossaceae). The taxa we examined are typical of plant lineages with a long-lived underground mycoheterotrophic phase. Botrychium is a small group of ferns of approximately 25 species, with the majority found only in North America. Botrychium sporophytes are small (5–15 cm) and have only one aboveground leaf with a fertile and sterile segment (Bower, 1935
; Bierhorst, 1971
; Gifford and Foster, 1988
). In the past 20 years, studies have documented the anatomy and morphology of underground phases of Botrychium (including gametophytes, juvenile sporophytes, and sporophyte-produced gemmae [Farrar et al., 1986
; Mason and Farrar, 1989
; Farrar and Johnson-Groh, 1990
; Camacho, 1996
]), their spatial and temporal distribution (Kelly, 1994
; Johnson-Groh and Lee, 2002
; Johnson-Groh et al., 2002
), and their genetic variation (Camacho and Liston, 2001
). Although detailed light and electron microscopy has been used to examine the AM symbionts of Botrychium gametophytes and sporophytes (Campbell, 1911
; Nozu, 1954
; Bierhorst, 1971
; Schmid and Oberwinkler, 1994
; Kovacs et al., 2003
), little is known about the phylogenetic identity of the AM symbionts that are the main source of carbon to these underground life cycle phases of Botrychium.
We characterized the AM symbionts in B. lanceolatum and B. crenulatum at three distinct ontogenetic stages (Fig. 1). The first stage was the subterranean mycoheterotrophic gametophyte that is completely dependent on its mycorrhizal symbiont for a source of fixed carbon (Fig. 2). The second stage was the subterranean mycoheterotrophic sporophyte that initially receives carbon from the gametophyte and is known to establish its own separate AM associations (Campbell, 1911
; Schmid and Oberwinkler, 1994
). These mycoheterotrophic stages in Botrychium collectively can live as long as 10 yr underground, during which time the plants rely on their fungal symbionts for a source of fixed carbon (Johnson-Groh and Lee, 2002
). The third stage was the photosynthetic diploid sporophyte that has AM symbionts (Fig. 3) that are assumed to engage in a mutualistic relationship with the host plant. In addition, we investigated the mycorrhizal partners of neighboring flowering plants of B. crenulatum to more completely characterize the potential network of glomalean partners of the mycoheterotrophic life cycle stages (Fig. 3).
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Specimen collection
Botrychium crenulatum was collected in July 2003 at Silver Meadow in the Uinta National Forest in Utah. Plugs 8.9 cm in diameter by 15.2 cm deep were obtained from four sites in that meadow. Each plug contained neighboring flowering plants and all three life cycle stages of B. crenulatum. The dominant vegetation in the meadow was Caltha palustris (Ranunculaceae) and various grasses. Gametophytes and heterotrophic sporophytes of B. crenulatum were isolated by sieving soil with USA Standard Testing Sieves no. 80 and no. 20 and examining the material retained on the sieves with a dissecting microscope. Eight C. palustris plants, eight autotrophic B. crenulatum sporophytes, four heterotrophic B. crenulatum sporophytes, and four B. crenulatum heterotrophic gametophytes were used for DNA amplification of fungal 18S small subunit (SSU) rDNA (Table 1). Despite numerous attempts, no sequences matching Glomus were amplified from neighboring grass species, and sectioned grass roots did not have evidence of any glomalean symbionts.
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Histology
Tissue was fixed in the laboratory in 4.0% acrolein solution in PIPES buffer pH 6.8. The tissue was dehydrated in an ethanol series to 95% and then infiltrated and embedded in the plastic monomer glycol methacrylate (JB-4 embedding kit, Polysciences, Warrington, Pennsylvania, USA). Serial sections (5 µm thick) of plastic embedded specimens were cut using a Leica rotary microtome (Leica Microsystems, Bannockburn, Illinois, USA) and glass knives. Serial sections were mounted on slides and stained with 0.1% toluidine blue. Sections were digitally photographed using a Zeiss Axiophot microscope equipped with a Zeiss Axiocam digital camera (Zeiss MicroImaging, Thornwood, New York, USA).
Sequence amplification
Plant material was surface sterilized before being frozen and stored for DNA sequence analysis. DNA was extracted from plant material using the standard phenol–chloroform extraction method. Nuclear small subunit ribosomal DNA (18S) was amplified using primers previously used in the amplification of glomalean fungi from plant roots and isolated spores; the primers were VANS1 (Simon et al., 1992
), GEOA2, GEO11 (Schwarzott and Schussler, 2001
), GLOM1311R (Bidartondo et al., 2002
), and SS1492 (Simon et al., 1993
). The PCR program was 95°C for 1.5 min; 35 cycles of 94°C for 45 s, 56.5°C for 55 s, 72°C for 2.5 min + 1 s per cycle; and 72°C for 10 min.
Sequencing
All amplified DNA samples were cloned using TOPO TA cloning kits (Invitrogen, Carlsbad, California, USA). A minimum of 12 and usually 20 colonies were chosen from each reaction for sequencing. Colonies were isolated and grown in TSB broth at 35°C for 18 h. Plasmid DNA was extracted using the Mini Wizard Prep Plasmid Kit (Promega, Madison, Wisconsin, USA). Extracted plasmid DNA was used directly in a cycle sequence reaction using the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, New Jersey, USA). The cycle sequence reaction mixture contained 2 µL of sequencing reagent premix, 2 µL of template, 0.33 µL of primer, 7 µL of water, and 0.75 µL of 5x sequencing buffer. The cycle sequence reaction was 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, repeated 25 times. The cycle sequence reactions were cleaned using the Sephadex Column System (Applied Biosystems, Foster City, California, USA). Samples were run at the University of Colorado Sequencing Facility in the Department of Ecology and Evolutionary Biology on an MJ Research Base Station 51. The 18S sequences (EF109816-EF109888; AY930720-AY930722) were submitted to GenBank (www.ncbi.nlm.nih.gov/Genbank) (Appendix 1).
Phylogenetic analyses
Each sequence was run through a BlastN (http://www.ncbi.nlm.nih.gov/blast/) search on GenBank to confirm it as glomalean. Sequences were aligned and edited in Clustal-X version 1.83 (Thompson et al., 1997
) and Sequencher version 4.2 (Gene Codes Corporation, Ann Arbor, Michigan, USA) (Appendix S1 see Supplemental Data accompanying online version of this article). Regions of the alignment that were ambiguous (base pairs 232–260; 394–410; 997-1011) were realigned with different parameters within ClustalX and tested to determine whether the changes affected the resulting tree. Changing the sequence alignment in these regions or removing these regions from the phylogenetic analyses did not impact the topology of the resulting phylogenetic tree. Neighbor joining and Bayesian analyses were conducted in MrBayes (Huelsenbeck and Ronquist, 2001
) and PAUP* version 4.2.b. (Swofford, 2002
). Bayesian analyses were carried out using models of evolution determined using the program Modeltest (Posada and Crandall, 1998
). Previously isolated complete 18S sequences from nonphotosynthetic angiosperms Arachnitis uniflora, Voyria corymbosa, and Voyriella parviflora (Bidartondo et al., 2002
) were included in our phylogenetic analyses. The published 18S sequences for Glomaceae, Acaulosporaceae, Gigasporaceae, Archaeosporaceae, Geosiphonaceae, and Paraglomaceae used in phylogenetic analyses are from Schwartzott et al. (2001) (Appendix S2, see Supplemental Data accompanying online version of this article).
RESULTS
We chose the small subunit ribosomal DNA (18S) to amplify, clone, and sequence to characterize the glomalean AM partners of mycoheterotrophic gametophytes, mycoheterotrophic sporophytes, and autotrophic sporophytes of B. crenulatum and B. lanceolatum. We also characterized the glomalean endosymbionts of flowering plants growing adjacent to B. crenulatum in the field. Based on BlastN searches, most of the sequences we isolated share identity with glomalean fungi. A small percentage of the isolated sequences share an identity with ascomycetes. We chose not to focus on those sequences because light microscopy of sporophyte roots and gametophytes only revealed typical glomalean AM associations (Figs. 2 and 3), and it has been demonstrated that ascomycete 18S sequences can be found inside healthy glomalean spores and hyphae (Redecker et al., 1999
; Hijri et al., 2002
).
Glomalean phylogenetic diversity
The 18S phylogeny (Fig. 4) based on 1260 characters and 76 unique sequences (39 from B. crenulatum, 22 from B. lanceolatum, 15 from C. palustris) is well resolved. In our analyses, the phylogenetic relationships among representatives of previously reported sequence diversity within Glomeromycota are similar to those formerly published: monophyletic clades of Glomus A, B, and C, as well as Acaulosporaceae, Gigasporaceae, Archaeosporaceae, Geosiphonaceae, and Paraglomaceae (Schussler et al., 2001a
, b; Schwartz and Schussler, 2001; Bitardondo et al., 2002; Vandenkoornhuyse et al., 2002
) were recovered. All 76 isolated sequences are in Glomus group A, as described by Schussler et al. (2001b)
and represent previously unknown glomalean sequence diversity based on both phylogenetic analyses and GenBank searches.
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; Hijri and Sanders, 2005
). Within a single spore, cloned 18S and ITS sequences have been shown to vary between 0.5–3% and 6–18%, respectively (Clapp et al., 1995
; Antoniolli et al., 2000
; Kjoller and Rosendahl, 2001
; Jansa et al., 2002
). In some cases, sequences from the same spore are less closely related to each other than to sequences isolated from other Glomalean species (Lloyd-Macgilp et al., 1996
). As a result, circumscription of glomalean species as discrete evolutionary units, based on molecular data, can be difficult.
The community composition of fungal symbionts within a plant can be characterized phylogenetically via the number of recovered phylotypes: clades of closely related sequences that have high bootstrap statistical support (recent examples include Vandenkoornhuyse et al., 2002
; Opik et al., 2003
; Gollette et al., 2004
; and Rosendahl and Stukenbrock, 2004
). Previous studies have identified glomalean phylotypes with bootstrap values ranging from 77–100%. Sequence identity within these phylotypes ranged from 96.5–100% (Vandenkoornhuyse et al., 2002
; Opik et al., 2003
; Gollette et al., 2004
; Rosendahl and Stukenbrock, 2004
). Our phylogenetic analyses partition the 76 Glomus 18S sequences isolated from the two populations of Botrychium into 11 phylotypes that are supported by greater than 99% bootstrap and posterior probability values. Sequence identity within the clusters ranged from 98.5–99.8%. The 11 phylotypes cannot be further divided into statistically well-supported groupings. The 11 phylotypes can be collapsed into seven phylotypes that are supported by greater than 99% bootstrap values. However, the sequence identity within these phylotypes ranges from 91.2–96.7%, which represents as much as 18 times greater sequence difference than previously reported from sequences isolated from a single spore (Clapp et al., 1995
; Kjoller and Rosendahl, 2001
; Jansa et al., 2002
).
We are not equating the 11 phylotypes with species, but we do view them as statistically robust phylogenetic groupings of genotypes that share an evolutionary history. Consequently, although we do not know how many "species" of Glomus the 11 isolated phylotypes represent, we can characterize the distribution and evolution of the isolated AM phylotypes in terms of the two populations of Botrychium and the known diversity of glomalean fungi.
Arbuscular mycorrhizal associations in B. crenulatum
Four 18S phylotypes were isolated from B. crenulatum mycoheterotrophic gametophytes and juvenile subterranean sporophytes (Fig. 4, Table 1). In all subterranean gametophytes and heterotrophic sporophytes that were sampled (with one exception), each individual harbored a single 18S phylotype of Glomus. Each phylotype isolated from a mycoheterotrophic life cycle stage was also always isolated from a neighboring conspecific or heterospecific photosynthetic plant.
Ten glomalean phylotypes were isolated from photosynthetic sporophytes of B. crenulatum. Each photosynthetic sporophyte of B. crenulatum harbored at least two and as many as four AM phylotypes. One of the phylotypes isolated in each autotrophic sporophyte was always one of the four 18S phylotypes isolated from a nearby mycoheterotrophic plant. Four phylotypes were only recovered from autotrophic B. crenulatum sporophytes; they were not recovered from any mycoheterotrophic plants (Table 1).
Our DNA sequence data further demonstrated that the mycoheterotrophic gametophytes and mycoheterotrophic sporophytes of B. crenulatum share specific 18S AM fungal phylotypes with neighboring flowering plants. Three of the four 18S AM fungal sequences isolated from a mycoheterotrophic life cycle stage were also isolated from neighboring C. palustris plants. In addition, we isolated one 18S fungal phylotype unique to C. palustris plants and one phylotype that C. palustris plants shared with photosynthetic sporophytes of B. crenulatum (Fig. 4, Table 1).
Arbuscular mycorrhizal associations in B. lanceolatum
Three 18S Glomus phylotypes were isolated from both the subterranean mycoheterotrophic gametophyte and aboveground photosynthetic sporophyte life cycle stages of B. lanceolatum collected in Oregon. As with B. crenulatum, only one Glomus phylotype was isolated from any single B. lanceolatum subterranean gametophyte. Each Glomus phylotype isolated from a subterranean gametophyte was also always isolated from neighboring photosynthetic sporophytes of B. lanceolatum. Individual B. lanceolatum sporophytes always harbored two of the three glomalean phylotypes recovered from neighboring underground gametophytes (Fig. 4, Table 1).
Two of the three 18S phylotypes found in the Oregon B. lanceolatum population were also recovered from the population of B. crenulatum from Utah. One of the two shared phylotypes isolated from both the autotrophic and mycoheterotrophic life cycle phases of B. lanceolatum from Oregon was also recovered in the autotrophic and mycoheterotrophic life cycle phases of B. crenulatum from Utah (Fig. 4, Table 1). The other phylotype found in both Botrychium species was isolated from the autotrophic and mycoheterotrophic life cycle phases of B. lanceolatum from Oregon and the autotrophic life cycle phases of B. crenulatum from Utah.
Phylogenetic relationships of isolated phylotypes
Ten of the 11 Glomus phylotypes isolated from B. lanceolatum and B. crenulatum compose a monophyletic lineage that is sister to the glomalean sequences isolated from the mycoheterotrophic angiosperm Arachnitis uniflora (Bidartondo et al., 2002
). Support for the monophyly of these 10 glomalean phylotypes is moderate (70%). However, support for the monophyly of the clade of glomalean fungi isolated from Arachnitis uniflora + 10 closely related phylotypes isolated from Botrychium is high (>95%). Hereafter, this clade will be referred to as the mycoheterotrophic Glomus clade 1 or MH1 clade.
Phylogenetic relationships within the MH1 clade are resolved with statistical support ranging from low to high (54–100%). The two phylotypes isolated only from B. crenulatum from Oregon are more closely related to phylotypes isolated from B. lanceolatum from Utah than to phylotypes isolated from conspecific plants in the same population (bootstrap support greater than 99%). Some Glomus phylotypes isolated from mycoheterotrophic Botrychium life cycle stages are more closely related to phylotypes isolated only from photosynthetic plants than to other mycoheterotrophic phylotypes (Fig. 4).
One 18S phylotype isolated from photosynthetic sporophytes of B. crenulatum falls within a clade of glomalean fungi that includes glomalean sequences isolated from the mycoheterotrophic angiosperm Voyria corymbosa (hereafter referred to as the mycoheterotrophic Glomus clade 2 or MH2 clade) (Fig. 4). Phylogenetic relationships within this clade are unresolved but bootstrap and posterior probability values for the clade are high (100%). Relationships between the MH1 clade, the MH2 clade, and other Glomus taxa in the larger Glomus A clade (sensu lato Schussler et al. [2001b
]) are unresolved (Fig. 4).
We conducted further BlastN searches in GenBank to determine whether additional Glomus sequences fall within the MH1 or MH2 clade. We recovered two unique sequences, containing the same number of base pairs, both of which were isolated from the liverwort Marchantia foliacea (Russell and Bulman, 2005
). Bayesian and neighbor-joining phylogenetic analyses demonstrate that one Glomus sequence isolated from M. foliacea is in the MH1 clade and the other is in the MH2 clade.
DISCUSSION
Roughly 1000 fern and lycopsid species spend a significant portion of their lives (as gametophytes and young sporophytes) underground, deriving fixed carbon from their mycorrhizal fungal partners (Goebel, 1905
; Campbell, 1911
; Bower, 1923
, 1935
; Eames, 1936
; Smith, 1938
; Bierhorst, 1971
; Read et al., 2000
). Virtually unstudied, this type of life cycle is ecologically and phylogenetically widespread. Although the presence of fungal symbionts has been well documented by microscopy in these underground phases, nothing is known of the phylogenetic affinity of the fungal partners or nature of the AM networks that facilitate carbon flow from neighboring autotrophic plants.
Identity and phylogenetic relationships of Glomus symbionts of Botrychium
Using 18S rDNA sequence data, we have characterized the molecular and phylogenetic identity of glomalean AM fungi that form symbiotic associations with mycoheterotrophic and autotrophic stages of Botrychium. We isolated 11 18S phylotypes from field-collected B. crenulatum and B. lanceolatum gametophytes and sporophytes (Fig. 4, Table 1). The phylogenetic relationships of the isolated 18S sequences provide compelling evidence for the discovery of a major clade of Glomus, the MH1 clade, capable of forming mycoheterotrophic AM associations with underground gametophytes and underground sporophytes of B. crenulatum and B. lanceolatum, and with one species of nonphotosynthetic angiosperm.
Within the MH1 clade, four of the phylotypes were only recovered from Botrychium photosynthetic sporophytes and neighboring photosynthetic Caltha plants. It is possible that the AM phylotypes isolated from photosynthetic plants alone also engage in symbioses with mycoheterotrophic plants but were not recovered in our environmental sampling of mycoheterotrophic life cycle stages of B. crenulatum and B. lanceolatum. This could be due to our small sample sizes of mycoheterotrophic plants (they are notoriously difficult to find and isolate) and/or to PCR bias (unequal amplification of certain sequences over others when the template is a mixture homologous genes [Kanagawa, 2003
]). If this is the case, the glomalean fungi in the MH1 clade are all capable of forming mycoheterotrophic AM associations.
To date, studies of mycoheterotrophic plant–fungal interactions (mostly characterized in relationships between ectomycorrhizal fungi and their mycoheterotrophic angiosperm hosts) have demonstrated that mycoheterotrophic plant species appear to specialize on "narrow" clades of fungi (Leake, 1993
, 2004
; Bidartondo et al., 2002
; Trudell et al., 2003
; Leake et al., 2004
; Bidartondo, 2005
). This contrasts with the diverse clades of fungi typically recovered from environmental sampling of ectomycorrhizal and AM symbionts from photosynthetic plant species (recent examples include Vandenkoornhuyse et al., 2002
; Bergemann and Garbelotto, 2006
; reviewed in Johnson et al., 2005
; Opik et al., 2006
). Our analyses recovered phylotypes restricted to a single clade of Glomus (excluding one phylotype recovered only from photosynthetic sporophytes of B. crenulatum) that forms AM associations with the two species of Botrychium. In addition, our broader analysis of relationships among members of the Glomus A clade showed that two previously identified lineages of glomalean fungi that form AM associations with the nonphotosynthetic angiosperms Voyria corymbosa and Voyriella parviflora (Bidartondo et al., 2002
) (Fig. 4) are not closely related to the MH1 clade. Thus, our data, along with those of Bidartondo et al. (2002)
, are congruent with the pattern observed in other mycoheterotrophic plant–fungal associations: AM mycoheterotrophic plants specialize on Glomus fungi that are a subset of the total AM fungal diversity and that are polyphyletic and probably selected to form AM mycoheterotrophic associations with specific nonphotosynthetic plant lineages.
Botrychium mycorrhizal networks
Our DNA sequence data demonstrated that the mycoheterotrophic life cycle phases of B. crenulatum and B. lanceolatum harbor the same glomalean AM phylotypes as neighboring photosynthetic conspecific and heterospecific sporophytes (Fig. 4, Table 1). In nature, Botrychium gametophytes will not grow past the eight-celled stage without a fungal symbiont (Campbell, 1911
). In axenic culture, Botrychium gametophytes will not grow without a source of fixed carbon (Whittier, 1972
, 1984
; Whittier and Thomas, 1993
). Thus, it is reasonable to conclude that Botrychium mycoheterotrophic gametophytes and mycoheterotrophic sporophytes (before their emergence aboveground) acquire fixed carbon through glomalean fungal symbionts that are shared with neighboring photosynthetic plants.
The distribution of Glomus phylotypes among the different life cycle stages of Botrychium and neighboring flowering plants indicates that the ultimate upstream source of fixed carbon that flows to mycoheterotrophic stages of Botrychium could originate in conspecific and/or heterospecific neighboring photosynthetic plants. However, Botrychium sporophytes (often found under dense vegetation and in low light environments), even when aboveground and photosynthesizing, may be partially mycoheterotrophic and dependent upon AM symbionts for a source of fixed carbon (Wagner et al., 1984
, 1985
; Wagner and Wagner, 1986
, 1994
; D. Farrar, Iowa State University, and C. Johnson-Groh, Gustavus Adolphus College, personal communication). Furthermore, individual "photosynthetic" sporophytes of various Botrychium species have been reported to remain underground (perenate) for as many as 2 years (Kelly, 1994
), during which time they too might function as mycoheterotrophs.
Given that all of the phylotypes recovered in mycoheterotrophic stages of B. crenulatum and B. lanceolatum were also isolated in the photosynthetic sporophytic stages of these two species, it may well be that these sporophytes partially depend on fixed carbon derived from neighboring heterospecific photosynthetic plants via shared glomalean networks. If this is the case, then heterospecific neighboring plants are most likely the primary source of fixed carbon to underground, and possibly aboveground, Botrychium life cycle phases through shared glomalean networks. Field-based carbon labeling experiments are needed to determine the direction and extent of carbon flow through the complex networks of shared Glomus phylotypes between the different life cycle phases of Botrychium and neighboring photosynthetic plants.
Conclusions
In summary, we have identified a major clade of Glomus (MH1) that is capable of forming mycoheterotrophic associations with the eusporangiate ferns B. crenulatum and B. lanceolatum and the angiosperm Arachnitis uniflora. Furthermore, our analyses suggest at least three lineages within Glomus A contain taxa that form mycoheterotrophic AM associations with diverse lineages of land plants. With further sampling of Glomus symbionts in the more than 1000 vascular plants with mycoheterotrophic glomalean AM associations (including Lycopodiaceae, basal leptosporangiates [Goebel, 1905
; Campbell, 1911
; Bower, 1923
, 1935
; Eames, 1936
; Smith, 1938
; Bierhorst, 1971
], eudicots, and monocots [Wang and Qiu, 2006
]), there is good reason to believe that researchers will discover many additional Glomus A phylotypes that engage in symbioses with mycoheterotrophic plants. Without question, future integration of field molecular identification of Glomus symbionts and carbon flow dynamics of AM fungal networks in ecosystems with mycoheterotrophic plants should yield a fascinating picture of the complex biological relationships of mycoheterotrophs, their neighboring autotrophs, and the fungi that interconnect them.
APPENDIX
Taxon—GenBank accessions 18S; Source; Voucher specimen.
Botrychium crenulatum—EF109825, EF109827–EF109829, EF109840–EF109846, EF109851–EF109854, EF109864–EF109869, EF109872–EF109882, EF109875–EF109888, AY930720–AY930722; Utah; JW-03–03, CU. Botrychium lanceolatum—EF109816–EF109824, EF109831–EF109834, EF109847–EF109850, EF109856–EF109860; Oregon; JW-03–02, CU. Caltha palustris—EF109826, EF109830, EF109835–EF109839, EF109855, EF109861–EF109863, EF109870, EF109871, EF109883, EF109884; Utah; JW-03–01, CU.
FOOTNOTES
1 The authors thank C. Johnson-Groh and D. Farrar for assistance with the collection of Botrychium crenulatum and B. lanceolatum and D. O'Conner and K. Ryerson for help with DNA sequencing. This work was supported by a NASA Astrobiology grant to W.E.F. 
2 Author for correspondence (ned{at}colorado.edu
)
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99% neighbor-joining bootstrap (values of 10 000 bootstraps) and Bayesian posterior probability (values of 4 million generations) values. Lower bootstrap and posterior probability values are indicated above the branch. Triangles represent a phylotype of glomalean fungus, and sequences within a phylotype share 98.5–99.8% sequence identity. AM fungal isolates (in blue) from mycoheterotrophic angiosperms Voyriella parviflora, Voyria corymbosa, and Arachnitis uniflora (Bidartondo et al., 2002