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McGuire Raines

BIO 201 A – Botany

15 September 2022

Research Assignment #1: Summary

This dioecious shrub is found in southwestern Appalachian Mountains. It is rare and

often isolated, but conservation is hard since information on its life history and reproduction is

limited. This study seeks to explore the reproductive capacity of this plant to take further steps

in conservation. Previous studies indicate potential for sexual reproduction, but to assume that

the presence of fruits means there is sexual reproduction would be wrong. There is the

possibility for nonsexual embryogenesis and asexual, vegetative reproduction. If this were the

case, then the lack of genetic diversity through sexual reproduction would be concerning for the

health of the species. Thus, this study’s goal is to examine any restraints on the sexual

reproduction of this shrub. In the process, they sought the specific goals of identifying floral

visitors, examining the potential for pollen transfer, deposition, and tube growth, investigating

embryo viability and seed germination, and documenting any seed predation. The site chosen for

this study was Poor Mountain because it is one of the densest populations of the remaining 17

remaining. The piratebush is the common name because of it is hemi-parasitic to trees. The

population is dominantly male, with the male plants flowering in mid-April, a week before

females ending its flowering season in late May, a week before females.

To identify potential pollinators, they observed and even captured pollinators through

various methods, like nets, aspirators, paint brushes, and kill jars. This was done day and night.

These captured organisms were sent to an entomologist for identification. They also used data

from a previous study of piratebush pollinators that had not been published. To track pollen
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transfer, they placed radiant fluorescent pigment dust on the anthers of flowers. This would

hopefully make its way to the stigma of a plant, which would be identified by using a UV light to

reveal the blue dust that had made its way there. Pollen rain trap slides were also set up. To

examine pollen deposition and tube growth, fruits were gathered and prepped to look at the early

stages of development. Each fruit was examined for pollen grains on the stigma as well the

germination of pollen tubes. A controlled test was run on pistillate flowers in which some were

allowed open pollination, some had hand pollination, and others had no pollination. To look at

embryo viability, the embryos of fruits were stained red by a TTC solution, which indicates

respiration. The germination trials included control fruits, fruits bathed in commercial bleach for

10 minutes, fruits bathed in commercial bleach for 60 minutes, and scarified fruits. Each

category contained seeds that were stratified and those that were unstratified.

There was only one nocturnal flower visitor, which was a moth. In the day, 19 were

captured. 47% of the captured visitors were ants, click beetles made up 26%, and sweet bees

made up 11%. These results were compared to a previous study, in which the 2nd most common

visitor was click beetles. Though ants and beetles are not considered effective pollinators, the

result may be due to the lack of different pollinators. It is thought that the sweet-musky odor is

what attracts these visitors. Using the UV light, they discovered the dust to still be on the

stamens, which indicated no pollen transfer. Suggestions were made to retest this in different

seasons. The pollen rain trap slides did not have any pollen, which showed that wind is not a

way that piratebushes spread pollen. The developing fruits had a mean of about 18 + 1.14 (SE)

pollen grains on the stigma, with results ranging from 2 to 58. The results of pollen tube growth

supported the potential for fertilization. Combining these two results helped them determine that

the sexual reproduction of the shrub is not limited by the amount of pollen deposited. Some of
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the fruits would have remained unfertilized due to the lack of pollen tube growth. With the

controlled pollination part of the experiment, the no pollination group had 58% of persisting

flowers and 0% fruit development. The open pollination group had 86% of persisting flowers

and 86% fruit development. The hand pollination group had 81% of persisting flowers and 65%

fruit development. This eliminated the possibility of agamospermy. At the end of September,

the fruit bags were checked to find that 92% of the fruits were missing. This led to the

conclusion of seed predation since the seeds of a piratebush are relatively large. Eastern

chipmunks and gray squirrels were charged with the offense. Thus, they called for further study

into this ecological relationship that restrains the sexual reproduction of this shrub. Embryos had

a 73% high to moderate degree of viability, while the other 23% was reduced. The germination

trials showed that only the stratified seeds had the chance of germination. The mean percent

germination of the control was 70.9%, 72.6% for the 10 minutes in bleach, 27.2% for scarified,

and 54.2% for the 60 minutes in bleach. These results point to the need for a winterization

period. The fact that the control group thrived and the scarified did not do as well, points to the

evidence of rodents that eat the seeds. The study offers the transportation of fruits to

greenhouses or in situ planting as a possible solution to the problem of seed germination. They

recognize that this method would require further research. In conclusion, the study has linked

the poor sexual reproductive health of the piratebush to seed predation. Thus, conservations

efforts should be taken accordingly.

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Literature Cited

Huish, Ryan, Amy Faivre, Melissa Manow, and Conley K. McMullen. 2019. Investigations into

the reproductive biology of the southern Appalachian endemic Piratebush (Buckleya

distichophylla): Pollination biology, fruit development, and seed germination. Castanea.

84(1): 70-80.