Absence of Premating Barriers in Ploidy-Mixed Populations of Gymnadenia Conopsea S

Supplement 1. Photographs of Gymnadenia cytotypes and their pollinators: (A) tetraploid Gymnadenia conopsea, (B) octoploid G. conopsea, (C) tetraploid G. densiflora, (D) a noctuid, Autographa gamma and (E) a hawkmoth, Deilephila porcellus.

Supplement 2. Primer sequences of the nuclear ribosomal DNA (nrDNA) markers and the chloroplast DNA (cpDNA) markers used in the study.

Type of marker

/

Primer name

/

Primer sequence (5’-3’)

nrDNA

/

ITS 1P

/

CTT TAT CAT TTA GAG GAA GGA AG

nrDNA

/

ITS 4

/

TCC TCC GCT TAT TGA TAT GC

cpDNA

/

ndhJ

/

ATG CCY GAA AGT TGG ATA GG

cpDNA

/

TabE

/

GGT TCA AGT CCC TCT ATC CC

cpDNA

/

psbA

/

GTT ATG CAT GAA CGT AAT GCT C

cpDNA

/

trnH

/

CGC GCA TGG ATT CAC AAT CC

cpDNA

/

sak23F

/

GGA RTA TGA GTG TGT GAC TTG

cpDNA

/

sak24R

/

ATR TGA GAN TTT CAT CTC GTAC

Supplement 3. Occurrence (number of samples containing a specific compound) and percentage amount of compounds found in the Gymnadenia scent samples (tetraploid G. conopsea (4xGc), octoploid G. conopsea (8xGc) and tetraploid G. densiflora (4xGd)). Compounds eliciting antennal responses in the most important flower visitors are printed in italics. Within chemical classes the compounds are ordered according to the Kovats retention index (RI). Within chemical classes, unknowns were pooled, and in the column Occurrence the number of compounds pooled (superscript) and the median number of samples, in which these compounds occurred, is given. Tr.: The percentage amount was less than 0.005. *: Identification based on authentic standards.

4x Gc (26 samples) / 8x Gc (26 samples) / 4x Gd (12 samples)
Compound / Relative Amount / Occurrence / Relative Amount / Occurrence / Relative Amount / Occurrence
RI / Median / Quartiles / Min-Max / Median / Quartiles / Min-Max / Median / Quartiles / Min-Max
Benzenoids
Benzaldehyde* / 980 / 0 / - / 0-0.01 / 3 / 0 / - / 0-0.03 / 1 / 0.14 / 0.07-0.31 / 0.04-0.97 / 12
Benzyl alcohol* / 1045 / 0 / - / 0-0.06 / 2 / 0 / - / 0-Tr. / 1 / 1.79 / 1.31-6.53 / 1.15-10.51 / 12
p-Cresol* / 1080 / 0 / 0-0.01 / 0-0.03 / 12 / Tr. / 0-0.01 / 0-0.03 / 15 / 0.18 / 0.1-0.37 / 0.05-0.99 / 12
Methyl benzoate* / 1106 / 0 / - / 0-0.06 / 1 / - / - / - / - / 0.02 / 0.01-0.05 / 0-0.12 / 10
m/z: 77,133,92,105,79,78 / 1159 / - / - / - / - / - / - / - / - / 0 / 0-0.01 / 0-0.02 / 3
Benzyl acetate* / 1170 / 0.06 / 0-0.07 / 0-0.44 / 19 / 0.02 / 0.01-0.03 / 0-0.11 / 23 / 8.24 / 2.75-10.75 / 0.14-17.71 / 12
1,4-Dimethoxybenzene* / 1172 / 0 / 0-0.06 / 0-0.19 / 10 / 0 / 0-0.01 / 0-0.13 / 10 / - / - / - / -
Dimethoxy toluene / 1241 / 0 / - / 0-0.08 / 2 / 0 / - / 0-0.01 / 4 / - / - / - / -
Benzyl propionate / 1263 / - / - / - / - / - / - / - / - / 0.05 / 0.03-0.07 / 0.02-0.12 / 12
Ethylacetophenone / 1295 / 0.01 / 0-0.16 / 0-1.79 / 13 / 0 / 0-0.02 / 0-0.19 / 7 / 0 / - / 0-0.01 / 1
Benzyl isobutyrate / 1304 / - / - / - / - / - / - / - / - / 0.02 / 0.01-0.03 / Tr.-0.09 / 12
Benzyl butyrate / 1353 / - / - / - / - / - / - / - / - / 0.22 / 0.17-0.27 / 0.07-0.33 / 12
Benzyl isovalerate / 1400 / - / - / - / - / - / - / - / - / 0.04 / 0.01-0.07 / 0-0.17 / 11
Vanillin* / 1412 / 0.13 / 0-0.66 / 0-3.04 / 17 / 0.03 / 0-0.15 / 0-1.36 / 15 / 0.03 / 0-0.1 / 0-0.37 / 8
Methylvanillin / 1486 / 0.19 / 0-0.51 / 0-1.73 / 19 / 0 / 0-0.04 / 0-0.67 / 12 / 0 / 0-0.01 / 0-0.11 / 3
Benzyl tiglate / 1507 / - / - / - / - / - / - / - / - / 0.01 / 0.01-0.02 / 0-0.05 / 10
Vannilylmethylketone / 1543 / 0.04 / 0-0.25 / 0-1.21 / 16 / 0.03 / 0-0.14 / 0-0.76 / 17 / 0.01 / 0-0.03 / 0-0.06 / 7
Phenylmethyl hexanoate / 1551 / - / - / - / - / - / - / - / - / 0.04 / 0.02-0.04 / 0.01-0.08 / 12
3,4,5-Trimethoxybenzaldeyde / 1610 / 1.45 / 0-3 / 0-6.82 / 17 / 0 / - / 0-0.82 / 4 / 0.02 / 0-0.1 / 0-0.53 / 7
Benzyl benzoate* / 1787 / - / - / - / - / - / - / - / - / 25.54 / 18.86-31.58 / 14.47-47.82 / 12
Phenylpropanoids
(Z)-Cinnamyl aldehyde / 1232 / - / - / - / - / - / - / - / - / 0.01 / 0-0.01 / 0-0.02 / 8
Benzenepropanol / 1239 / - / - / - / - / - / - / - / - / 0.23 / 0.2-0.43 / 0.02-0.54 / 12
(Z)-Cinnamyl alcohol / 1269 / - / - / - / - / - / - / - / - / 0 / 0-0.03 / 0-0.15 / 5
(E)-Cinnamyl aldehyde* / 1284 / - / - / - / - / - / - / - / - / 0.47 / 0.42-0.84 / 0.11-1.13 / 12
(E)-Cinnamyl alcohol* / 1315 / - / - / - / - / - / - / - / - / 1.72 / 0.98-2.59 / 0.43-3.65 / 12
Cinnamyl formate / 1362 / - / - / - / - / - / - / - / - / 0.01 / 0-0.03 / 0-0.04 / 7
Eugenol* / 1369 / 1.85 / 0.39-20.14 / 0-62.27 / 23 / 36.01 / 19.3-49.08 / 4.08-63.17 / 26 / 41.24 / 27.43-52.99 / 17.82-65.59 / 12
Phenylpropyl acetate / 1377 / 0.12 / 0-0.21 / 0-1.04 / 19 / 0.03 / 0-0.09 / 0-0.78 / 18 / 0.44 / 0.27-0.79 / 0.03-1.59 / 12
Methyleugenol / 1406 / 0.91 / 0-7.7 / 0-22.43 / 13 / 8.09 / 5.69-12.38 / 0-22.49 / 25 / 5.89 / 1-13.32 / 0.1-34.57 / 12
(Z)-Isoeugenol* / 1419 / 0 / - / 0-0.26 / 5 / 0.03 / 0-0.07 / 0-0.33 / 16 / 0.03 / 0.01-0.09 / 0-0.28 / 9
(E)-Cinnamyl acetate / 1451 / 0 / - / 0-0.01 / 3 / - / - / - / - / 0.67 / 0.46-0.75 / 0.29-1.19 / 12
(Z)-Methylisoeugenol / 1459 / 0 / - / 0-1.27 / 5 / - / - / - / - / - / - / - / -
(E)-Isoeugenol* / 1460 / 0.47 / 0.18-1.08 / 0-24.24 / 21 / 0.49 / 0.26-1.75 / 0.13-17.99 / 26 / 0.28 / 0.17-1.44 / 0.12-4.45 / 12
m/z: 137,164,149,77,103,91 / 1471 / - / - / - / - / - / - / - / - / 0.01 / 0-0.02 / 0-0.04 / 8
(E)-Methylisoeugenol / 1501 / 2.39 / 0.84-4.76 / 0.14-22.66 / 26 / 0.47 / 0.21-1.45 / 0.02-7.8 / 26 / 0.19 / 0.02-0.4 / 0-2.26 / 11
Methoxyeugenol / 1514 / 0.01 / 0-0.3 / 0-0.52 / 13 / 0.34 / 0.19-0.57 / 0.14-0.82 / 26 / 0.05 / 0.01-0.08 / 0-0.21 / 11
Acetyleugenol / 1531 / 0 / - / 0-0.44 / 5 / 0.02 / 0-1.09 / 0-3.04 / 15 / 0.04 / 0.01-0.54 / 0-2.04 / 10
Elemicin / 1557 / 2.06 / 1.19-12.13 / 0.13-41.03 / 26 / 13.16 / 7.91-16.54 / 1.04-47.77 / 26 / 3.30 / 2.35-5.27 / 0.06-7.36 / 12
(E)-Isoeugenyl acetate / 1620 / - / - / - / - / 0 / - / 0-0.1 / 3 / - / - / - / -
(E)-Isoelemicin / 1654 / 4.73 / 1.12-7.43 / 0.11-21.39 / 26 / 0.34 / 0.12-1.33 / 0.08-8.13 / 26 / 0.14 / 0.04-0.22 / 0.01-0.89 / 12

Fatty Acid Derivates

(Z)-3-Hexen-1-ol* / 876 / 0 / - / 0-Tr. / 1 / - / - / - / - / 0 / 0-0.02 / 0-0.11 / 4
(E)-2-Hexen-1-ol / 887 / 0 / - / 0-0.01 / 2 / - / - / - / - / 0.02 / 0.01-0.08 / 0-0.12 / 10
Heptanal / 918 / 0 / 0-0.08 / 0-0.25 / 12 / 0.01 / 0-0.03 / 0-0.15 / 16 / - / - / - / -
Amyl acetate / 929 / 0.03 / 0-0.12 / 0-0.73 / 17 / 0.02 / 0.01-0.08 / 0-0.34 / 24 / - / - / - / -
(Z)-3-Hexenyl acetate* / 1013 / 0.04 / 0-0.2 / 0-1.44 / 17 / 0.04 / 0.02-0.12 / 0-0.33 / 24 / 0.01 / 0-0.02 / 0-0.03 / 7
Hexyl acetate / 1019 / 0.24 / 0.05-0.49 / Tr.-1.49 / 26 / 0.07 / 0.04-0.21 / 0.01-0.74 / 26 / 0 / 0-0.01 / 0-0.02 / 6
Nonanal* / 1103 / 0 / - / 0-0.07 / 2 / - / - / - / - / 0.03 / 0.02-0.04 / 0-0.12 / 10
Heptyl acetate / 1113 / 0.18 / 0.06-0.58 / 0-1.11 / 25 / 0.09 / 0.05-0.2 / 0-0.94 / 25 / - / - / - / -
Nonyl acetate / 1208 / 0.09 / 0-0.32 / 0-1.71 / 16 / 0.22 / 0.1-0.4 / 0-0.79 / 24 / - / - / - / -
(Z)-3-Decenyl acetate* / 1388 / 0.14 / 0-0.27 / 0-0.7 / 19 / 0.08 / 0.04-0.15 / 0-1.16 / 24 / - / - / - / -
(Z)-5-Decenyl acetate* / 1395 / 2.22 / 0.38-5.6 / 0-10.35 / 23 / 2.04 / 1.68-3.79 / 0.07-5.95 / 26 / - / - / - / -
Decyl acetate* / 1405 / 1.23 / 0-4.04 / 0-7.17 / 13 / 0 / - / 0-5.84 / 1 / - / - / - / -
Dodecanal / 1409 / 0.13 / 0-0.87 / 0-2.94 / 18 / Tr. / 0-0.09 / 0-0.41 / 13 / - / - / - / -
(Z)-5-Dodecenyl acetate* / 1587 / 3.72 / 1.3-11.17 / 0-20.79 / 25 / 2.94 / 1.42-4.59 / 0.61-10.85 / 26 / - / - / - / -
(Z)-7-Dodecenyl acetate* / 1592 / 5.56 / 1.55-8.38 / 0-18.19 / 25 / 5.15 / 3.74-8.62 / 0.24-16.73 / 26 / - / - / - / -
Dodecyl acetate* / 1602 / 5.31 / 2.65-6.97 / 0.27-17.7 / 26 / 3.21 / 2.58-5.21 / 1.07-14.4 / 26 / - / - / - / -
Tetradecanal / 1611 / 0 / 0-0.07 / 0-1.64 / 7 / 0.20 / 0-0.33 / 0-0.93 / 18 / - / - / - / -
Tridecenyl acetate I / 1683 / - / - / - / - / 0 / - / 0-0.03 / 4 / - / - / - / -
Tridecenyl acetate II / 1688 / - / - / - / - / 0 / 0-0.01 / 0-0.05 / 7 / - / - / - / -
Tridecyl acetate / 1702 / - / - / - / - / 0 / - / 0-0.04 / 6 / - / - / - / -
Pentadecanal / 1712 / 0 / - / 0-0.02 / 2 / 0 / - / 0-0.02 / 1 / - / - / - / -
Tetradecadienyl acetate I / 1775 / 0.18 / 0-0.66 / 0-2.32 / 18 / 0.30 / 0.2-0.59 / 0.06-1.74 / 26 / - / - / - / -
Tetradecadienyl acetate II / 1782 / 0 / - / 0-0.35 / 3 / 0 / - / 0-0.5 / 4 / - / - / - / -
(Z)-7-Tetradecenyl acetate* / 1784 / 0.39 / 0.01-1.24 / 0-6.39 / 21 / 0.65 / 0.35-1.12 / 0-2.1 / 24 / - / - / - / -
(Z)-9-Tetradecenyl acetate* / 1793 / 0.13 / 0-0.62 / 0-1.59 / 18 / 0.32 / 0.18-0.55 / 0-1.71 / 25 / - / - / - / -
Tetradecyl acetate* / 1801 / 0.99 / 0.53-2.15 / 0-5.1 / 24 / 0.63 / 0.24-0.98 / 0-3.24 / 24 / - / - / - / -
Hexadecanal / 1805 / 0 / 0-0.06 / 0-2.77 / 7 / 0 / - / 0-0.63 / 6 / - / - / - / -
(Z)-11-Hexadecenyl acetate / 1991 / 0.29 / 0.16-0.56 / 0-2.59 / 22 / 0.08 / 0-0.33 / 0-1.52 / 19 / - / - / - / -
Hexadecyl acetate / 1999 / 0.19 / 0.03-0.52 / 0-2.45 / 20 / 0.07 / 0-0.2 / 0-1.41 / 19 / - / - / - / -
Unknowns / 0.36 / 0.08-0.63 / 0-7.62 / 918 / 0.33 / 0.14-2.02 / 0.05-4.28 / 1218 / 0 / 0-0.01 / 0-0.02 / 31
Monoterpenoids
Sabinene* / 988 / - / - / - / - / - / - / - / - / 0 / - / 0-0.14 / 2
ß-Pinene* / 1000 / 0 / 0-Tr. / 0-0.29 / 9 / 0 / - / 0-0.1 / 3 / - / - / - / -
(E)-ß-Ocimene* / 1056 / 0.01 / 0-0.04 / 0-0.32 / 16 / 0 / - / 0-0.11 / 4 / - / - / - / -
Linalool* / 1105 / 0.03 / Tr.-0.09 / 0-0.35 / 21 / Tr. / Tr. -0.01 / 0-0.07 / 22 / - / - / - / -
Unknowns / 0 / 0-0.06 / 0-0.20 / 72 / 0 / - / 0-0.01 / 41
Sesquiterpenoids
(E)-a-Bergamotene / 1448 / 0 / - / 0-0.03 / 2 / 0 / - / 0-0.02 / 1 / - / - / - / -
(E,E)-a-Farnesene* / 1509 / 0.36 / 0.17-0.78 / 0-2.32 / 21 / 0.20 / 0.12-0.34 / Tr. -0.91 / 26 / - / - / - / -
Unknowns / 0.01 / 0-1.55 / 0-1.78 / 63 / 0.02 / 0-0.27 / 0-43 / 33
N-bearing compounds
2-Aminobenzaldehyde* / 1230 / 2.95 / 0.72-9.14 / 0-22.36 / 23 / 1.65 / 0.35-4.07 / 0.1-10.62 / 26 / 0 / 0-0.01 / 0-0.02 / 5
Indole* / 1307 / 19.32 / 10.43-27.19 / 1.28-47.14 / 26 / 7.71 / 6.86-10.07 / 4.25-29.39 / 26 / 0 / 0-0.02 / 0-0.07 / 3
Methylquinoline / 1328 / 0.03 / 0-0.18 / 0-1.2 / 18 / 0 / 0-0.01 / 0-0.22 / 10 / - / - / - / -
Unknown compounds
unknowns / 0.36 / 0-0.57 / 0-8.35 / 149 / 0.2 / 0-0.70 / 0-3.11 / 59 / 0 / 0-Tr. / 0-0.02 / 31