Understanding the mechanisms of adaptive population differentiation requires that both the functional and adaptive significance of divergent traits are characterized in contrasting environments. Here, we (a) determined the effects of floral spur length on pollen removal and receipt using plants with artificial spurs representing the species-wide variation in length, and (b) quantified pollinator-mediated selection on spur length and three traits contributing to floral display in two populations each of the short-spurred and the long-spurred ecotype of the orchid Platanthera bifolia. Both pollen receipt and removal reached a maximum at 28-29 mm long spurs in a short-spurred population visited by short-tongued moths. In contrast, pollen receipt increased linearly across the tested range (4-52 mm) and pollen removal was unrelated to spur length in a long-spurred population predominantly visited by a long-tongued moth.  The experimentally documented effects on pollen transfer were not reflected in pollinator-mediated selection through female fitness or pollen removal indicating that the natural within-population variation in spur length was insufficient to result in detectable variation in pollen limitation. Our study illustrates how combining trait manipulation with analysis of causes and strength of phenotypic selection can illuminate the functional and adaptive significance of trait expression when trait variation is limited.

The excel files provide data on: 

(1) Pollination success (pollen removal and pollen receipt) collected in a field experiment conducted in a short-spurred grassland and in a long-spurred woodland population of the orchid Platanhera bifolia on the island Öland in southeastern Sweden in 2017. In this experiment, artificial nectar spurs were used to vary the spur length of experimental plants.

(2) Data on sugar concentration in nectar produced by  flowers in a short-spurred grassland and in a long-spurred woodland population. This information was used to decide the sugar concentration to be used in "nectar" added to artificial spurs.

(3) Pollen removal and receipt in flowers with intact nectar spurs and in flowers with artificial nectar spurs in plants included in the artificial-spur experiment. These data were used to check that pollination success was similar in intact and manipulated flowers within the natural range of spur lengths in each of the two study populations.

(4) Data on pollen removal, female fitness, and floral traits in two grassland and in two woodland populations of P. bifolia on the island Öland in southeastern Sweden in 2016. These data were used to quantify phenotypic selection among open-pollinated control plants and among plants receiving supplemental hand-pollination, and from these results estimate pollnator-mediated selection, non-pollinator mediated selection, and net selection on spur length, plant height, number of flowers, and flower size.