Lack of macro- and microscopic characters in fungi severely hampers species identification. This is particularly true for the inconspicuous species complex known as Sebacina vermifera (Oberw.) P. Roberts (family Sebacinaceae, order Sebacinales, informally assigned as subgroup B [Weiss et al., 2004]). These fungi are not known to form basidiocarp or hymenial layers (Oberwinkler et al., 2013). However, DNA sequencing of samples taken from soil or root samples indicates that there is extensive diversity within this species complex (Weiss et al., 2004). Thus, it is likely that there are many species yet to be determined in this group. Australian terrestrial orchids within the genera Caladenia R. Br., Glossodia R. Br., Elythranthera (Endl.) A. S. George, and Eriochilus R. Br. form mycorrhizal associations with fungi of the S. vermifera subgroup B species complex. However, the actual number of Sebacina species associated with these orchids is presently unknown. Two commonly used markers for fungal species identification, the ribosomal internal transcribed spacer (ITS) and mitochondrial large subunit (mLSU), both suggest that species-level differences exist among orchid mycorrhizal isolates extracted from these orchid genera (Weiss et al., 2004; Wright et al., 2010). Here we report the development of additional loci to aid accurate species identification of orchid mycorrhizal fungi allied to S. vermifera.
METHODS AND RESULTS
Partial genome sequencing for genetic marker development—A Sebacina isolate from C. huegelii Rchb. f. and Eriochilus pulchellus Hopper & A. P. Br., as well as three isolates from Eriochilus dilatatus Lindl., were grown in liquid culture and DNA was extracted as described previously (Roche et al., 2010). For the C. huegelii isolate, sequences were generated with a 3-kb mate-pair library on the GS FLX 454 platform with GS XL70 sequencing chemistry (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). CLC Genomics Workbench software (CLC bio, Aarhus, Denmark) was used to separately assemble the C. huegelii sequences using the default assembly parameters. Sequences for the isolates from Eriochilus were produced with a HiSeq2500 (Illumina, San Diego, California, USA) using paired-end libraries with 350-bp fragments. Reads were quality filtered, and the adapters were removed using libngs ( https://github.com/sylvainforet/libngs) using a minimum quality of 25 bp and a minimum read size of 150 bp. The genomic assemblies were carried out with SPAdes (Bankevich et al., 2012) using the following options: “-k 21,33,55,77,99,127–careful -t 16 -m 64”.
To design phylogenetic markers that are broadly applicable, we focused primer design on common regions of high sequence homology. Initially, we compared the four Sebacina isolates from Eriochilus against the C. huegelii isolate within CLC Genomics Workbench software. This allowed us to target the high homology sequence regions shared across the five isolates for positioning the primers. Out of the 58,295 successful reads of Sebacina from C. huegelii, there were 1251 shared reads with E. pulchellus (average length 285 bp) and 950 to 1605 shared reads with E. dilatatus isolates (average length 375 and 588 bp). GenBank BLAST searches ( http://www.ncbi.nlm.nih.gov/) were conducted on 159 of C. huegelii sequences with high homology with at least two Eriochilus isolates. A total of 31 sequences (out of the 159) were identified as being associated with annotated or predicted genes from Basidiomycola R. T. Moore or Ascomycota (Berk.) Caval.-Sm. fungi. Priming sites were selected for 19 consensus sequences using Primer3 (Rozen and Skaletsky, 2000); selection was based on product length, ease of primer design, and gene designation.
Characteristics of phylogenetic primers developed for Sebacina isolates in this study.
Genetic marker amplification success and sequence diversity—Successful fungal isolation, following Roche et al. (2010), was obtained for several Sebacina isolates from host species within the Australian orchid genera Caladenia (14 spp.), Glossodia (2 spp.), Eriochilus (3 spp.), and Elythranthera (1 sp.). A total of 31 isolates were evaluated for DNA sequence diversity with the new fungal primers described below (Tables 1 and 2, Appendix 1). This set represented one to four isolates each from the 13 most strongly supported phylogenetic clades as revealed by a preliminary screen with ITS (bootstrap support >0.78; data not shown) of Sebacina isolates associated with several Australian orchids.
PCR reactions were performed in 30-µL reactions containing 2 µL of 20–100 ng of template DNA, 14.75 µL of H2O, 6 µL MangoTaq 5× PCR buffer (Bioline, Sydney, Australia), 1 µL dNTPs (2.5 mM), 1.5 µL of MgCl2 (50 mM), 2 µL bovine serum albumin (BSA; 10 mg/mL), 1.5 µL of each primer (10 µM), and 1 unit of MangoTaq polymerase (Bioline). A touchdown thermal profile was used consisting of a 3-min denaturation at 94°C; followed by 12 touchdown cycles at 94°C (30 s) with the first annealing temperature at 66°C (40 s) (−3°C/second cycle) and a primer extension at 72°C (1 min); then 30 cycles at 94°C (30 s), 48°C (40 s), 72°C (1 min); with a final extension at 72°C for 20 min. Products were sequenced bidirectionally with ABI PRISM BigDye Terminator version 3.1 sequencing kit on an ABI 3100 automated sequencer (Applied Biosystems, Carlsbad, California, USA). Sequences were edited using the program Sequencher version 4.7 (Gene Codes Corporation, Ann Arbor, Michigan, USA) and aligned in Geneious Pro version 6.1.6 (Drummond et al., 2011). Consistent and high-quality amplification of all tested Sebacina isolates occurred for eight primer sets (Table 2). Estimates of variability across the alignment of up to 31 sequences were performed within MEGA version 5.2 (Tamura et al., 2011). The eight markers revealed 15–10% nucleotide diversity and 11–31% parsimony informative sites across these sequences (Table 2).
Characteristics of markers in Sebacina mycorrhizal fungi from Australian orchids.
Phylogenetic analyses—A multiple sequence alignment was constructed using the alignment tool in Geneious Pro version 6.1.6 (Drummond et al., 2011) before performing manual checks and minor adjustments. Phytogenies of individual and concatenated loci were estimated with a maximum likelihood (ML) analysis using RAxML 7.0.3 (Stamatakis et al., 2008). Support for nodes was assessed for ML trees using 1000 pseudoreplicates of nonparametric bootstrapping in RAxML. A GTR+G substitution model was used for all analyses as all other models are nested inside this model. Trees were visualized using FigTree version 1.3.1 ( http://tree.bio.ed.ac.uk/software/figtree/) and midpoint rooted. Among the 31 isolates, eight to 12 well-supported clades were identified with the newly developed loci (Fig. 1, Appendices S1–S7 (appsd1400015_appendixs1-7.pdf)).
We successfully designed polymorphic markers for eight putative gene coding loci that successfully amplified across the mycorrhizal fungi species complex S. vermifera. These new markers will allow investigations of the species diversity, phylogenetic relationships, and the specificity of orchid mycorrhizal associations for a wide range of Australian terrestrial orchids.