Electronic appendices
Appendix A Preliminary pollination experiments using emasculated flower heads of Aster amellus
Materials and methods: Two groups of flower heads were prepared: unmanipulated pollen donor flower heads and recipient emasculated flower heads (the upper part of the florets was cut at the bud stage with the help of a razor blade to remove self-pollen). In this species, it was difficult to successfully emasculate the flower heads without completely damaging the stigmatic surface, and a series of control groups were included to determine the proportion of pollination due to residual pollen in the emasculated flowers. Recipient flowers were pollinated with donor flower heads by gently rubbing the two flower heads together two to five times over the course of five days. The pollination treatments were the following for each cytotype: emasculated and bagged flowering heads pollinated with emasculated flowering heads from the same ploidy level - control for pollen residues in emasculated florets (C); emasculated and bagged flowering heads self-pollinated with a pollen donor head from the same individual - control for self-incompatibility (A); and emasculated and bagged flowering heads cross pollinated with pollen from the other cytotype (2x × 6x and 6x ×2x). Because the florets within a flowering head were in different developmental stages, the emasculation treatment frequently led to the removal of the stigmatic lobes of some florets, and thus it was impossible to determine the number of florets that were successfully emasculated and pollinated. Therefore, instead of measuring the proportion of developed seeds per flowering head, it was only possible to calculate the proportion of fruiting heads per treatment. Seeds were divided in two groups to assess the ploidy level at the seed and seedling stages using flow cytometry. Ploidy level was assessed in 2-3 seeds per fruiting head and for all the germinated seedlings.
Results and conclusions: The results obtained in this preliminary experiment show that perfect emasculation is very difficult to achieve in A. amellus (Fig. A1). To avoid severely damaging the stigmatic surface, residual pollen grains remain in the florets, influencing the pollination treatments (Fig. A1).
Figure A1. Preliminary pollination experiments using emasculated flower heads of Aster amellus. Proportion of flower heads with developed seeds, proportion of flower heads with germinated seeds and proportion of flower heads with germinated seeds from the ploidy level of the mother are provided for diploid (a) and hexaploid individuals (b). Hand-pollination treatments: C – control, emasculated and bagged flowering heads pollinated with emasculated flowering heads from the same ploidy level; A – autogamy, emasculated and bagged flowering heads self-pollinated with a pollen donor head from the same individual; 2x × 6x and 6x × 2x – emasculated and bagged flowering heads cross pollinated with pollen from the other cytotype. Ploidy levels of the seeds and seedlings after inter-cytotype crosses are also provided for diploid (c) and hexaploid individuals (d)
Appendix B. Pollinators of Aster amellus
Table B1 Pollinator assemblage and number of interactions for three diploid (2x) and three hexaploid (6x) populations; values are given as total number of flower heads visited followed by the proportion of floral interactions (in %), given in parenthesis; empty cells represents zero visits by a given insect to the flower heads of A. amellus
2x / 6xVisitor species / Malíč / Holý vrch / Encovany / Malešov / Sovice / Vrutice sad
Order Diptera
Episyrphus balteatus (Syrphidae) / 12 (1.7) / 9 (5.0) / 6 (1.1) / 24 (3.7) / 11 (1.1)
Eristalis arbustorum (Syrphidae) / 78 (10.8) / 5 (0.9) / 26 (4.0) / 75 (7.6)
Eristalis tenax (Syrphidae) / 191 (26.3) / 22 (4.1) / 2 (0.8) / 54 (8.4) / 6 (0.6)
Eupeodes corolla (Syrphidae) / 1 (0.1) / 9 (5.0) / 5 (0.9) / 19 (7.9) / 47 (7.3) / 15 (1.5)
Helophilus pendulus (Syrphidae) / 62 (8.6) / 6 (1.1) / 4 (0.6)
Scaeva pyrastri (Syrphidae) / 4 (0.6) / 12 (6.7) / 48 (19.8) / 12 (1.9)
Sphaerophoria scripta (Syrphidae) / 226 (31.2) / 65 (36.3) / 93 (17.4) / 85 (35.1) / 103 (15.9) / 51 (5.2)
Musca sp. (Muscidae) / 15 (2.1) / 4 (2.2) / 8 (1.5) / 20 (8.3) / 19 (2.9) / 18 (1.8)
Phora sp. (Phoridae) / 7 (3.9) / 2 (0.4) / 6 (0.6)
Order Hymenoptera
Apis mellifera (Apidae) / 2 (0.3) / 7 (3.9) / 9 (0.9)
Bombus sp. (Apidae) / 29 (4.0) / 4 (2.2) / 29 (5.4) / 18 (7.4) / 1 (0.2) / 28 (2.8)
Dasypoda hirtipes (Melittidae) / 2 (0.8) / 89 (9.0)
Andrena sp. (Andrenidae) / 11 (6.1) / 15 (2.8) / 35 (5.4) / 48 (4.9)
Heriades truncorum (Megachilidae) / 3 (0.4) / 8 (4.5) / 1 (0.2) / 48 (7.4) / 47 (4.8)
Hoplosmia spinulosa (Megachilidae) / 10 (1.4) / 1 (0.6) / 75 (14.0) / 73 (11.3) / 131 (13.3)
Megachile centuncularis (Megachilidae) / 1 (0.1) / 11 (6.1) / 24 (4.5) / 10 (4.1) / 61 (9.4) / 3 (0.3)
Halictus simplex (Halictidae) / 5 (0.7) / 8 (1.2) / 142 (14.4)
Halictus sp. (Halictidae) / 12 (1.7) / 72 (13.5) / 16 (6.6) / 27 (4.2) / 37 (3.8)
Lasioglossum sp. (Halictidae) / 10 (1.4) / 3 (1.7) / 112 (21.0) / 8 (1.2) / 5 (0.5)
Epeolus ariegatus (Anthophoridae) / 19 (2.9)
Nomada fucata (Anthophoridae) / 1 (0.1) / 29 (5.4) / 6 (0.9) / 7 (0.7)
Formica sp. (Formicidae) / 1 (0.6) / 8 (1.2) / 2 (0.2)
Ammophila sabulosa (Sphecidae) / 1 (0.2) / 13 (1.3)
Unknown (Chalcidoidea) / 10 (5.6) / 15 (2.8) / 7 (2.9) / 17 (2.6) / 3 (0.3)
Order Lepidoptera
Aphantopus hyperantus (Nymphalidae) / 1 (0.6) / 2 (0.8) / 82 (8.3)
Maniola jurtina (Nymphalidae) / 27 (3.7) / 1 (0.2) / 1 (0.1)
Vanessa cardui (Nymphalidae) / 6 (3.4) / 3 (1.2) / 5 (0.8)
Polyommatus icarus (Lycaenidae) / 6 (0.8) / 10 (1.0)
Satyrium acaciae (Lycaenidae) / 5 (0.7) / 1 (0.2) / 7 (1.1) / 54 (5.5)
Pieris napi (Pieridae) / 7 (1.0) / 3 (1.7) / 1 (0.2) / 3 (0.5) / 49 (5.0)
Eilema sp. (Arctiidae) / 15 (1.5)
Zygaena cf. ephialtes (Zygaenidae) / 6 (0.8) / 3 (0.6) / 2 (0.8) / 3 (0.5) / 11 (1.1)
Order Coleoptera / 12 (1.7) / 3 (1.7) / 5 (0.9) / 8 (3.3) / 19 (2.9) / 8 (0.8)
Order Heteroptera / 4 (2.2) / 5 (0.9) / 3 (0.5) / 8 (0.8)
Total number of visits / 725 / 179 / 534 / 242 / 646 / 984
Table B2 Visitation rates of each floral visitor to Aster amellus flower heads in three diploid (2x) and three hexaploid (6x) populations; values indicate flower visitation rates (number of flowers visited per 10 min) for each insect species; empty cells represents zero visits by a given insect; the five highest visitation rates in each population are highlighted in bold
Visitor species / Malíč / Holý vrch / Encovany / Malešov / Sovice / Vrutice sad
Order Diptera
Episyrphus balteatus (Syrphidae) / 0.522 / 0.474 / 0.261 / 0.750 / 0.367
Eristalis arbustorum (Syrphidae) / 3.391 / 0.217 / 0.813 / 2.500
Eristalis tenax (Syrphidae) / 8.304 / 0.957 / 0.111 / 1.688 / 0.200
Eupeodes corolla (Syrphidae) / 0.043 / 0.474 / 0.217 / 1.056 / 1.469 / 0.500
Helophilus pendulus (Syrphidae) / 2.696 / 0.261 / 0.125
Scaeva pyrastri (Syrphidae) / 0.174 / 0.632 / 2.667 / 0.375
Sphaerophoria scripta (Syrphidae) / 9.826 / 3.421 / 4.043 / 4.722 / 3.219 / 1.700
Musca sp. (Muscidae) / 0.652 / 0.211 / 0.348 / 1.111 / 0.594 / 0.600
Phora sp. (Phoridae) / 0.368 / 0.087 / 0.125 / 0.200
Order Hymenoptera
Apis mellifera (Apidae) / 0.087 / 0.368 / 0.300
Bombus sp. (Apidae) / 1.261 / 0.211 / 1.261 / 1.000 / 0.031 / 0.933
Dasypoda hirtipes (Melittidae) / 0.111 / 2.967
Andrena sp. (Andrenidae) / 0.579 / 0.652 / 1.094 / 1.600
Heriades truncorum (Megachilidae) / 0.130 / 0.421 / 0.043 / 1.500 / 1.567
Hoplosmia spinulosa (Megachilidae) / 0.435 / 0.053 / 3.261 / 2.281 / 4.367
Megachile centuncularis (Megachilidae) / 0.043 / 0.579 / 1.043 / 0.556 / 1.906 / 0.100
Halictus simplex (Halictidae) / 0.217 / 0.250 / 4.733
Halictus sp. (Halictidae) / 0.522 / 3.130 / 0.889 / 0.844 / 1.233
Lasioglossum sp. (Halictidae) / 0.435 / 0.158 / 4.870 / 0.250 / 0.167
Epeolus ariegatus (Anthophoridae) / 0.594
Nomada fucata (Anthophoridae) / 0.043 / 1.261 / 0.188 / 0.233
Formica sp. (Formicidae) / 0.053 / 0.250 / 0.067
Ammophila sabulosa (Sphecidae) / 0.031 / 0.433
Unknown (Chalcidoidea) / 0.526 / 0.652 / 0.389 / 0.531 / 0.100
Order Lepidoptera
Aphantopus hyperantus (Nymphalidae) / 0.053 / 0.111 / 2.733
Maniola jurtina (Nymphalidae) / 1.174 / 0.031 / 0.033
Vanessa cardui (Nymphalidae) / 0.316 / 0.167 / 0.156
Polyommatus icarus (Lycaenidae) / 0.261 / 0.333
Satyrium acaciae (Lycaenidae) / 0.217 / 0.043 / 0.219 / 1.800
Pieris napi (Pieridae) / 0.304 / 0.158 / 0.043 / 0.094 / 1.633
Eilema sp. (Arctiidae) / 0.500
Zygaena cf. ephialtes (Zygaenidae) / 0.261 / 0.130 / 0.111 / 0.094 / 0.367
Order Coleoptera / 0.522 / 0.158 / 0.217 / 0.444 / 0.594 / 0.267
Order Heteroptera / 0.211 / 0.217 / 0.094 / 0.267
Overall visitation rate / 31.522 / 9.421 / 23.217 / 13.444 / 20.188 / 32.800
Table B3 Frequency of interactions of each floral visitor with Aster amellus flower heads in three diploid (2x) and three hexaploid (6x) populations; values indicate the frequency of interactions for each insect (visitation rates multiplied by the mean no. of insects per 10 min); empty cells represents zero visits by a given insect; the five highest frequencies of interaction in each population are highlighted in bold
Visitor species / Malíč / Holý vrch / Encovany / Malešov / Sovice / Vrutice sad
Order Diptera
Episyrphus balteatus (Syrphidae) / 0.181 / 0.125 / 0.034 / 0.234 / 0.073
Eristalis arbustorum (Syrphidae) / 2.064 / 0.009 / 0.076 / 1.083
Eristalis tenax (Syrphidae) / 11.554 / 0.208 / 0.006 / 0.527 / 0.007
Eupeodes corolla (Syrphidae) / 0.002 / 0.075 / 0.028 / 0.293 / 0.918 / 0.050
Helophilus pendulus (Syrphidae) / 2.344 / 0.023 / 0.016
Scaeva pyrastri (Syrphidae) / 0.023 / 0.166 / 1.333 / 0.082
Sphaerophoria scripta (Syrphidae) / 29.478 / 3.601 / 4.922 / 9.969 / 4.325 / 0.850
Musca sp. (Muscidae) / 0.113 / 0.011 / 0.076 / 0.123 / 0.148 / 0.180
Phora sp. (Phoridae) / 0.136 / 0.008 / 0.016 / 0.013
Order Hymenoptera
Apis mellifera (Apidae) / 0.004 / 0.058 / 0.030
Bombus sp. (Apidae) / 0.658 / 0.022 / 0.384 / 0.444 / 0.001 / 0.311
Dasypoda hirtipes (Melittidae) / 0.012 / 1.681
Andrena sp. (Andrenidae) / 0.091 / 0.113 / 0.171 / 0.480
Heriades truncorum (Megachilidae) / 0.011 / 0.089 / 0.002 / 1.078 / 0.888
Hoplosmia spinulosa (Megachilidae) / 0.057 / 0.003 / 2.694 / 1.497 / 3.493
Megachile centuncularis (Megachilidae) / 0.002 / 0.152 / 0.318 / 0.123 / 0.536 / 0.003
Halictus simplex (Halictidae) / 0.028 / 0.016 / 4.576
Halictus sp. (Halictidae) / 0.091 / 1.497 / 0.198 / 0.316 / 0.658
Lasioglossum sp. (Halictidae) / 0.038 / 0.017 / 5.293 / 0.016 / 0.011
Epeolus ariegatus (Anthophoridae) / 0.130
Nomada fucata (Anthophoridae) / 0.002 / 0.548 / 0.018 / 0.031
Formica sp. (Formicidae) / 0.003 / 0.031 / 0.004
Ammophila sabulosa (Sphecidae) / 0.001 / 0.058
Unknown (Chalcidoidea) / 0.111 / 0.255 / 0.065 / 0.133 / 0.003
Order Lepidoptera
Aphantopus hyperantus (Nymphalidae) / 0.003 / 0.012 / 0.820
Maniola jurtina (Nymphalidae) / 0.715 / 0.001 / 0.001
Vanessa cardui (Nymphalidae) / 0.066 / 0.028 / 0.020
Polyommatus icarus (Lycaenidae) / 0.034 / 0.033
Satyrium acaciae (Lycaenidae) / 0.009 / 0.002 / 0.007 / 0.540
Pieris napi (Pieridae) / 0.040 / 0.017 / 0.002 / 0.006 / 0.653
Eilema sp. (Arctiidae) / 0.083
Zygaena cf. ephialtes (Zygaenidae) / 0.045 / 0.017 / 0.012 / 0.006 / 0.061
Order Coleoptera / 0.091 / 0.025 / 0.019 / 0.074 / 0.186 / 0.044
Order Heteroptera / 0.022 / 0.038 / 0.009 / 0.044
Overall frequency of interaction / 286.4 / 38.7 / 154.4 / 64.2 / 145.1 / 264.6
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