SupplementaryFig.S1Mitochondrial rRNA large subunit gene (mtLSU) region containing two exons and up to three introns (positions 1, 2, 3) of haplotypes A, B, I-XI, XIII-XXXIII (R. irregularisand its close relatives; see Supplementary Fig.S2) and haplotype XII (R. intraradices) as defined by Börstler et al.(2008; 2010)in 5′–3′ orientation. The corresponding clones used as representatives for the haplotypes are shown in Fig.2, and Supplementary Table S1. Exons are shaded in grey. Different introns can be recognized by the type code defined by Börstler et al.(2008; 2010). Intron regions identical to those found in the haplotypes A and B are shown in the same color. Minor sequence differences of four single base pairsubstitutions compared to the corresponding intron region of haplotype Bare highlighted in dark orange for haplotype XVII. RNL-primers (2c, 16, 38, 85, 87, 89, 142, 145) used for the specific detection of the haplotypes A and B are shown as arrows at the respective annealing sites. Drawn to scale.

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SupplementaryFig.S2Phylogenetic tree of Rhizophagus irregularisand its close relatives,R. intraradices,R. proliferus and R. clarus, the latter was used as outgroup. The phylogeny was obtained using 929 characters of mtLSU exon sequences by a heuristic search under the maximum likelihood criterion. Neighbor-joining and Maximum parsimonybootstrap percentages from 1000 replicates are shown above branches. Sequences of both haplotypes of R. irregularis BEG140 are shown in boldface. R. irregularis,its relativesand R. intraradices sequenced by Börstler et al. (2008; 2010) are labeled with their accession numbers and roman numerals indicating the corresponding mtLSU haplotype.

SupplementaryFig.S3Verification of the specificity of the PCR approach developed for haplotype A, using the nested primer pairs RNL-16/RNL-89(a) and RNL-2c/RNL-145(b): DNA extracts of the mtLSU haplotypes X (isolate NB102c), XXIV (field root sample), I (isolate JJ291), II (isolate CC-4), IV (isolate SW205), III (field root sample) and VIII (isolate JA202) were tested for potential amplifications. The PCR products of the first reaction (a) were used as template (diluted 1:100 in distilled water) for the second PCR (b). For the origin of isolates and field root samples, see Börstler et al. (2008; 2010). A 100 bp ladder was used (#SM0242; Fermentas Life Science).

SupplementaryTableS1Results of in silico amplifications for all PCR reactions developed for the specific detection of the mtLSU haplotypes A and B of the Rhizophagus irregularis isolate BEG140.

1. reaction/
haplotype A / 2. reaction/
haplotype A / 1. reaction/
haplotype B / 2. reaction/
haplotype B
Accession
number / Haplotype / RNL-16/
RNL-89 / RNL-2c/
RNL-145 / RNL-85/
RNL-38 / RNL-87/
RNL-142
FN678790 / A / yes / yes, 403 bp / (yes) / no
FN678789 / B / yes / no / yes▲ / yes, 484 bp▲
FN377598 / XXXIII / yes / no / no / no
FN377597 / XXXII / yes / no / no / no
FN377595 / XXXI / no / no / no / (yes, 1423 bp)
FN377594 / XXX / no / no / no / no
FN377593 / XXIX / yes / no / no / no
FN377592 / XXVIII / yes / no / no / no
FN377590 / XXVII / yes / no / no / no
FN377589 / XXVI / yes / no / no / no
FN377588 / XXV / (yes)▲ / no / no / (yes, 1601 bp)▲
FN377586 / XXIV / yes / no / (yes) / no
FN377585 / XXIII / yes / no / (yes) / no
FN377584 / XXII / no / no / no / no
FN377580 / XXI / yes / no / (yes) / no
FN377579 / XX / yes / no / (yes) / no
FN377578 / XIX / no / no / no / no
FN377576 / XVIII / yes / no / no / no
AJ841288 / XVII / no / no / (yes) / yes, 484 bp▲
AM950227 / XVI / yes / no / no / (yes, 359 bp)
AM950226 / XV / yes / no / no / no
AM950222 / XIV / no / no / no / no
AM950221 / XIII / yes / no / no / no
AM950220 / XII / no / no / no / no
AM950218 / XI / yes / no / no / no
AM950217 / X / yes / no / (yes) / (yes, 358 bp)*
AM950216 / IX / no / no / no / no
AM950215 / VIII / no / no / no / no
AM950214 / VII / yes / no / (yes) / no
AM950213 / VI / yes / no / no / no
AM950210 / V / no / no / no / (yes, 359 bp)
AM950208 / IV / yes / (yes, 952 bp)* / no / (yes, 525 bp)▲
AJ938171 / III / no / yes, 445 bp / no / no
AM950204 / II / yes / yes, 445 bp / (yes) / no
AJ973192 / I / no / no / no / (yes, 358 bp)
AM040980 / R. proliferus / no / no / no / no
FN377601 / R. clarus / no / no / no / (yes, 360 bp)

[1]

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[1]The program Amplify (version 3, Bill Engels, University of Wisconsin 2005) was used. In addition to the haplotypes A and B, one representative of all mtLSU haplotypes sequenced so far between the priming sites RNL-29/RNL-30 were tested for successful amplification based on all available sequences: haplotypes I-XI, XIII-XXXIII (R. irregularis and its close relatives; see Supplementary Fig.S2), haplotype XII (R. intraradices) as defined by Börstler et al. (2008; 2010), R. proliferus (Raab et al. 2005) and R. clarus (Thiéry et al. 2010). Possible products caused by primer mismatches are shown in brackets. For all results of the second PCR reactions, the size of the amplicons is shown. These results are marked by an asterisk if amplification would most probably fail in a nested PCR reaction, because of missing priming sites in the products of the first PCR reaction. In addition, products (including those of primer mismatches) are shaded when they are part of a complete nested PCR. Some reactions revealed additional misamplifications (▲). In these cases, only the most likely product is shown.