Nectar sampling for prairie and oak savanna butterfly restoration.

PREMISE OF THE STUDY: Understanding floral resources is vital for restoring pollinators in habitats affected by anthropogenic development and climate change. As the primary adult food, nectar can limit butterfly longevity and reproduction. For pollinator restoration, it would therefore be useful to examine nectar resources. However, because many flowers preferred by butterflies are too small for microcapillary sampling and the potential for nectar contamination can make accurate measurement difficult, we developed a modified centrifugation method to extract and separate nectar and pollen. METHODS: We sampled nectar from 19 forbs using a glass wool filter to exclude potentially contaminating pollen during centrifugation. To minimize costs, we measured sugar concentration by refractometry and simple ninhydrin tests for amino acids and improved test accuracy by subsequent image analysis. Artificial nectars were used to verify the new techniques. RESULTS: This method eliminated pollen from samples, while only slightly increasing sugar concentrations. Some amino acids were lost during centrifugation, but only samples with high concentrations exhibited substantial loss. We found significant differences in nectar quality among species, as well as an unexpected inverse relationship between amino acid and sugar concentrations. DISCUSSION: This modified centrifugation technique is an efficient, less damaging, inexpensive approach for collecting nectar from small flowers while eliminating pollen contamination, and will facilitate restoration of declining pollinators and thereby the plants they service.

When it pays to cheat: Examining how generalized food deception increases male and female fitness in a terrestrial orchid.

BACKGROUND: Experimental manipulations of floral nectar in food deceptive species can reveal insights into the evolutionary consequences of the deceptive strategy. When coupled to pollen tracking, the effects of the deceptive pollination syndrome on both male and female reproductive success may be quantified. Attraction of pollinators in deceit-pollinated species often relies on producing a conspicuous floral display which may increase visibility to pollinators, but in-turn may increase within plant selfing. METHODOLOGY: To understand the role of deception in Orchidaceae reproduction we studied Cypripedium candidum. All species of the Cypripedium genus employ a generalized food deceptive pollination strategy and have been suggested as a model system for the study of pollinator deception. We conducted a nectar addition experiment that randomly assigned the four plants closest to a transect point to receive one of four histochemical dyes. Two individuals selected for nectar addition in each of altogether 25 blocks received 2µl of 25% sucrose solution in the labellum of each flower, while two others received no artificial nectar. Number of fruits produced, fruit mass and fruit abortion were scored at the end of the four-month experiment. RESULTS: Nectar addition increased (p<0.0001) self-pollination and pollen discounting by nearly 3x, while plants not receiving nectar had greater (p<0.0001) numbers of non-self pollinia deposited and lower rates of pollen discounting. There was a non-significant (p = 0.0645) trend for deceptive plants to set more fruit, while presence of nectar did not affect pollen export. CONCLUSIONS: This study demonstrates the adaptive advantages of food deception by showing a concurrent reduction in particular male and female functions when a food reward is restored to a deceptive flower. We found generalized food deception to not only decrease inbreeding depression in the system, but concurrently have no effect on pollinator attraction and fruit set when compared with rewarding flowers.

Effects of pollination limitation and seed predation on female reproductive success of a deceptive orchid.

For many species of conservation significance, multiple factors limit reproduction. This research examines the contributions of plant height, number of flowers, number of stems, pollen limitation and seed predation to female reproductive success in the deceit-pollinated orchid, Cypripedium candidum. The deceptive pollination strategy employed by many orchids often results in high levels of pollen limitation. While increased floral display size may attract pollinators, C. candidum's multiple, synchronously flowering stems could promote selfing and also increase attack by weevil seed predators. To understand the joint impacts of mutualists and antagonists, we examined pollen limitation, seed predation and the effects of pollen source over two flowering seasons (2009 and 2011) in Ohio. In 2009, 36 pairs of plants size-matched by flower number, receiving either supplemental hand or open pollination, were scored for fruit maturation, mass of seeds and seed predation. Pollen supplementation increased proportion of flowers maturing into fruit, with 87 % fruit set when hand pollinated compared with 46 % for naturally pollinated flowers. Inflorescence height had a strong effect, as taller inflorescences had higher initial fruit set, while shorter stems had higher predation. Seed predation was seen in 73 % of all fruits. A parallel 2011 experiment that included a self-pollination treatment and excluded seed predators found initial and final fruit set were higher in the self and outcross pollination treatments than in the open-pollinated treatment. However, seed mass was higher in both open pollinated and outcross pollination treatments compared with hand self-pollinated. We found greater female reproductive success for taller flowering stems that simultaneously benefited from increased pollination and reduced seed predation. These studies suggest that this species is under strong reinforcing selection to increase allocation to flowering stem height. Our results may help explain the factors limiting seed production in other Cypripedium and further emphasize the importance of management in orchid conservation.

PHYLOGENETIC ANALYSIS OF CHLOROPLAST DNA RESTRICTION SITE DATA AT HIGHER TAXONOMIC LEVELS: AN EXAMPLE FROM THE ASTERACEAE.

Chloroplast DNA variation was examined among 57 genera of Asteraceae representing 15 currently recognized tribes. Complete cleavage maps were constructed for 11 six-base pair restriction enzymes, and a total of 927 cleavage site differences was detected, 328 of which are phylogenetically informative. The data were used to construct phylogenetic trees using both Wagner and Dollo parsimony and the resulting monophyletic groups were evaluated statistically using the bootstrap method. The level of homoplasy in the restriction site data is 54-56% (excluding autapomorphies), most of which is due to parallel site losses. The most parsimonious trees generated by both parsimony methods have nearly identical topologies at lower taxonomic levels, but differ in subfamilial circumscriptions and tribal groupings. Dollo parsimony provides support for the monophyly of two subfamilies, the Lactucoideae (excluding the Barnadesiinae) and Asteroideae, but Wagner parsimony supports the monophyly of the Asteroideae only. This incongruence is due to different assumptions of the two parsimony methods about relative rates of parallel site gains and losses. After eliminating the six most rapidly changing restriction sites or performing successive approximation, Wagner parsimony produces trees with the same subfamilial groupings as the Dollo trees. We conclude that the Dollo tree with two monophyletic subfamilies is the best estimate of phylogenetic relationships in the Asteraceae because this method more accurately reflects the evolution of restriction sites. We also demonstrate that in spite of high levels of homoplasy in chloroplast DNA restriction site data at these higher taxonomic levels, it is possible to make statistically supported estimates of phylogenetic relationships.