I.

Project Summary

Invasive species and climate change have both proven to be obstacles to the health of our

regional ecosystems (Gian-Reto 2002). In the proposed study, we will be observing both above-

and below-ground plant growth in order to draw a conclusion on whether increased growth of

invasive species due to climate change will negatively affect native species. Invasive species can

act as a fierce, uninhibited competitor to the native species and fight with them for space,

nutrition, and other necessities. In the experimentation process, we will be growing a total of

twenty-eight plants, fourteen Tussilago farfara and fourteen Lonicera maachii. These specific

plants are both considered to be invasive species in the state of West Virginia (Batcher and Stiles

2000, Innes 2011). Seven plants of each species will be grown in an environment that maintains

an average temperature of 21.1 C (or 70 F). The remaining seven plants of each species will be

grown in an average temperature of 26.7 C (or 80 F). By the year 2100, it is expected that there

will be an average temperature increase of 3 C (Boer, 2000). In our study we will be measuring

a difference of 5.6 C because this experiment will be conducted in a short period of time, in

contrast to the period of a century that the projected climate change is occurring over. The effect

on overall plant health will be measured. These factors include root length, root biomass,

stomatal density, amount of photosynthetic pigments, and rate of growth. The association

between increased temperature and the growth of the plant, measured in height and dry mass,

will give us insight on how climate change may affect the survival rate of invasive species in the

region. Therefore, we will be able to predict the fitness of invasive species and use this to draw

conclusions about how they may affect native species in the future.

II. Project Description

 A. Introduction

In this study, two important problems are being addressed, climate change and invasive

species. Over the past one hundred years, the Earth’s average temperature has increased by 0.6

C (Gian-Reto 2002). Although this data may not seem extreme, this is just an average across the

entire Earth. Regional data has proven to be much more useful in determining what effect

climate change has on species, due to the asymmetrical way that warming occurs (Gian-Reto

2002). Climate change is expected to facilitate many changes in biodiversity including changes

in phenology, gene expression, species ranges, and interactions between species (Hellman et al.

2008). While it has been established that climate change will invariably cause resulting changes

in ecosystems, but more research is necessary in order to visualize the long-term effects of

climate change (Gian-Reto 2002).

In this study, we will be applying this information and exploring regional data by using

plants that are invasive species in the state of West Virginia. It is important to provide a

definition of the term “invasive species”, which we will define as a plant that has been

introduced to the area relatively recently and causes discernable harm to some aspect of

ecosystem or human health (Hellman et al. 2008). Many invasive plant species have

characteristics that help them survive- and even thrive- in harsh environments, which is how they

are able to spread so quickly. These characteristics may include low seed mass and quick

germination time (Hellman et al. 2008). The importance behind using invasive species is that the

presence of these species has been linked to widespread extinctions of native plants (Gurevitch

2004). In order to preserve the flora of West Virginia, it is important that ecologists understand

the relationships between invasive and native species. This is an important relationship to be
discerned because both invasive species and climate change may couple to cause devastating

effects on native plant species.

At this point, there have been very few studies completed that have directly identified the

consequences of climate change on invasive species survival in their nonnative habitat. In order

to evaluate how the invasive species survive, we will be taking measurements daily. This will

allow us to develop an understanding of their growth rates. The faster their growth rate is, the

more likely that they will negatively affect native species by heavy competition for space and

other resources. However, some publications have suggested that climate change is likely to

favor invasive species (Hellman et al. 2008). In order for a species to become classified as

invasive, it must have overcome a number of environmental obstacles in its new environment

(Hellman et al. 2008). Due to this fact, it seems increasingly likely that these plants may be able

to continue this process by being successful even in the face of global warming. Since there has

been little to no solid evidence that climate change enhances the growth of invasive species, we

are attempting to construct a new type of study in order to gather new information. We will

attempt to build on past research studies in order to provide a more well-rounded understanding

of how the invasive plant species Tussilago farfara and Lonicera maachii here in West Virginia

react to increasing temperatures related to climate change. In this future, this research may assist

in allowing us to design better plans for management of invasive species.

B. Background

1. Lonicera maachii

Lonicera maachii is an invasive species to the United States and is commonly called bush

honeysuckle. This plant is a deciduous shrub with opposite, simple, entire leaves, brightly

colored, showy petals, and red, fleshy berries (Batcher and Stiles 2000). They can grow
anywhere from two to six meters tall and are upright and multi-stemmed (Batcher and Stiles

2000). As this species becomes increasingly more common in the eastern United States, it has

been linked to decline of native plant communities. This includes reduced richness of ecosystems

of native herbs as well as reduced rates of tree regeneration in successional forests (Batcher and

Stiles 2000). The plant’s high rates of seed production and short-term viability of seeds are major

contributors to its ability to take over established ecosystems (Batcher and Stiles 2000).

Lonicera maachii is native to China, Manchuria, and Korea and was introduced in Europe

around 1887 (Batcher and Stiles 2000). It was introduced to North America soon after, around

1898 and is now reported to be found in 24 states in the eastern and central United States as well

as parts of Canada (Batcher and Stiles 2000). The species’ native habitat is generally in forests

where they live in association with large trees such as oak, elm, fir, and spruce trees (Batcher and

Stiles 2000). Since L. maachii has made its way to North America, where it is an invasive

species, they continue to be found in forests, but are also notably located in lakeside habitats as

well as abandoned agricultural sites (Batcher and Stiles 2000). Additionally, they are aggressive

weedy complexes that have been found to inhibit tree regeneration in forests that have been

disturbed (Batcher and Stiles 2000). The successful restoration of these forests is being treated by

this species, causing us to be able to classify L. maachii as an invasive species according to our

previous definition.

These seeds germinate at around 4.4 C (or 40 F) over a period of three months in nature;

however, in the greenhouse they have been found to germinate in a time period of just 18 days

(Batcher and Stiles 2000). Since this is an invasive species that has proven to grow quickly,

numerous methods have been used in attempt to control their growth. One process is controlled

burning, which is annual burning of the top layer of shrubs and other material on the floor of
forests (Batcher and Stiles 2000). This allows the invasive and damaging species to be killed, and

this process may be repeated annually or even more often than that in order to continue to control

growth (Batcher and Stiles 2000). Another growth control mechanism is mechanical control,

including the grubbing or pulling of either seedlings or mature shrubs, repeatedly (Batcher and

Stiles 2000). This utilizes the same ideas as above in order to keep growth controlled. The last

factor that is known to help control the growth of L. maachii is the use of herbicides (Batcher

and Stiles 2000).

2. Tussilago farfara

This plant is a rhizomatous perennial forb and is commonly known as coltsfoot (Innes

2011). The overall size of this plant ranges from two to twenty inches tall (Innes 2011). The

leaves found on this plant are deciduous, long-petioled, and heart shaped (Innes 2011). There is

flowering on this plant and the flowers mature before the leaves become present on the plant

(Innes 2011). Just as it was seen with the Lonicera maachii, Tussilago farfara has also caused a

decline in native plant species found not only in West Virginia but throughout the United States.

Its quick spread and its ability to cause problems for the area, is found in its ability to

create a large abundance of seeds once the plant has matured. Particularly a single plant can

produce up to 3,500 seeds once the plant fully matures at two years (Innes 2011). The seed

production usually depends on the amount of flower heads and the overall temperature of the

season. As for seed dispersal, the seed is easily spread through wind or water (Innes 2011).

Another reason that contributes to its wide-spread is its ability to easily travel large distances

with the wind, and in some cases a seed can travel as far as eight miles away from its parental

plant (Innes 2011). When the seed itself comes to germination, this type of seed does not show

dormancy and therefore germinate within the season that their seed was produced (Innes 2011).
 In a laboratory setting, T. farfara has a high viability, however that may not be the case in

the wild, as there is a decline in seed germination during cold temperatures or dry, acidic type

soils (Innes 2011). In most cases this plant can germinate in a range of temperatures, amount of

light, and various soil pH and moistures (Innes 2011). Its optimal germinating environment

would be in temperatures ranging from 12.8 °C (or 55 °F) to 25 °C (or 77 °F), moisture heavy

soils, an abundance of light, and a pH that falls between 4.5 and 6.5 (Innes 2011). There were

slower germination rates if the pH was in the lower ranges, and there was no germination at all if

the pH was lower than 3.5 (Innes 2011). It is also to note that germination will be slower if the

plant is not in full light and there is shaded periods of time that the plant goes through, it was

seen that in just 70% of daylight a mean dry weight of the plant was 8,770 mg compared to a

study done in 10% of daylight that lead to a mean dry weight of the plant being 7mg (Innes

2011).

This plant is non-native to West Virginia; however, it is now a concern for the state as it is

an invasive plant species. It is native to Europe, western Asia, and northwestern Africa, it was

said to be brought to the United States in the 1840s for medical purposes (Innes 2011). By the

1920s the plant had spread and taken root within Canada (Innes 2011). In terms of its spread in

the United States, it goes from Minnesota to Tennessee and upwards towards Maine (Innes

2011). This plant can be found in many different habitats, but they are all similar to its native

location. Floodplain forests and woodland areas, especially around riverbanks and ponds, you

can easily find this invasive species, it has even traveled to anthropogenically disturbed areas

such as roadsides and ditches as well (Innes 2011). One common place in West Virginia that T.

farfara can be found is along the New River Gorge National River (Innes 2011). This is a prime
area as it is on the riverbank of a nice woody forest as it is common to be found in various

hardwood forests (Innes 2011).

3. Temperature and Invasive Species

Both the Tussilago farfara and Lonicera maachii plants fall under explosive plants. Their

growth rates are highly influenced by the prevailing environmentalconditions.The T. farfara and

L. maachii plants can spread through wide areas because of their roots ability to traverse in a

faster way. Research shows that during the month of May and June T. farfara seeds germinate

within twenty-four hours (Innes 2011).However, the faster germination and growth only happens

in uncrowded environments that are moist and conducive in all manner (Innes 2011).These

species are mainly spread by humans who are carrying the seeds, whole plants or root pieces

through pets, vehicles and transportation of goods, they are also spread naturally in their

ecosystems by wind and other natural occurrences (Innes 2011). The invasive plants produce

thousands of seeds which can lie dormant for years and germinate whenever the conditions

become preferred. They caused harm to other plant species because of their abilities to reproduce

rapidly, hence competing against the native vegetation(Tennessee Exotic Pest Plant Council

2009).

Temperature specifically, play an important role in enhancing explosiveness among

invasive species. Explosiveness is the ability of the plant to grow rapidly in a way that threatens

other species in the ecosystem. To begin with, T. farfara seemed to spread at a faster rate when

exposed to temperatures which were high. When they are exposed to temperatures that fall

between 26.7 C (or 80 F) to 37.8 C (or 100 F), the roots acquire nutrients from the soil in

large quantities hence expanding at a faster rate. However, low temperatures of 21.1 C (or 70

F) and below limits the spreading of the vegetation (Lee 2002).
 On the other hand, it is very different from the L. maackii species regarding how they

react to high and low temperatures. This is because their roots become explosive specifically

when the temperatures range between 21.1C downwards to -17.8 C (or 0 C). When the

species is exposed to temperatures above 21.1 C, their roots are inhibited and grow at a slower

pace. Therefore the L. maackii species is favored inmoist habitats which have temperatures less

than 21.1 C(Mooney and Cleland 2001). In conclusion these findings depict that extremely high

temperatures help the T. farfara species to spread faster whereas lower temperatures favor the

growth and development of the L. maackii species. Another environmental factor that influences

the explosive nature of the T. farfara and L. maackii plants is the humidity and moisture of the

soil (Mooney and Cleland 2001). With high precipitation and moisture, the roots spread very

fast, and the seeds are also able to remain viable for germination for an extended period of time

(Mooney and Cleland 2001).

C. Objectives, Hypotheses, or Questions

Question 1. How does the rising average temperature from 21.1 °C to 26.7 °C affect the growth

of invasive species in West Virginia, specifically Lonicera maackii and Tussilago farfara?

For this experiment, this is the general question that we are seeking answers for. There

are two sub-questions, listed below, that incorporate more specificity into the experiment. In

order to answer this question, we will be monitoring the growth of each plant on a daily basis in

order to obtain a cumulative review of how they independently developed. Plants will be grown

in two different temperatures, 21.1 °C and 26.7 °C, and then have their features compared. For

this, we will be using both qualitative and quantitative data for our experiment. Qualitative data

will be observations in plant color, stage of life, and relative healthiness. As well as quantitative
data such as root length, root biomass, stomatal density, amount photosynthetic pigments and

overall growth rate.

Question 2. Does an increase in temperature affect different species differently based on their

native habitat or climate?

Monitoring the plants almost daily, we will be able to take measurements throughout the

experiment to watch its overall growth rate. We will be able to compare the growth rates between

the two treatment groups, for the specific plant, to observe if there was either a decline or an

increase in its ability to grow. It is predicted that Lonicera maackii, will not be as successful

growing in the higher temperature, as it has a preferred germination temperature at and around

4.4 °C (or 40 °F) (Batcher and Stiles 2000). Specifically, L. maackii was originally found in

China, Manchuria, and Korea (Batcher and Stiles 2000). These three countries all have an

average climate that fall between 22.5 °C and 25 °C (or 72.5 °F to 77 °F), so they are similar to

West Virginia’s climate (Zhang et al. 2019). When compared to the other plant species we are

observing, Tussilago farfara, its native habitat is found in Europe, western Asia, and Africa

(Innes 2011). These areas have an average climate that fall between 16 °C and 26 °C (or 60.8 °F

and 78.8 °F) which is a much larger range in temperatures, along with a much lower average

temperature that also can reach higher than that of China, Manchuria, and Korea (Jury 2018).

From this data we believe to see better growth with the T. farfara, as it is better adapted to

various temperatures because its native climate has a larger range compared to the habitat that L.

maackii is found in.

Question 3. Does an increase in average temperature affect plant growth differently in above

ground structures, such as height or photosynthetic pigments found in their tissues, and below

ground structures, such as root length?

 By measuring different parts of the plants, mentioned above, we will be able to compare

growth of different areas of plants. We will be measuring these factors in an environment that is

simulated to show possible climate change in the future, as described thoroughly in previous

sections. By measuring above ground height on a weekly basis, we will be able to construct data

showing growth rate of the individual species throughout the length of the experiment. This will

allow us to evaluate not only the final amount of growth experienced by the plants, but the

increments of growth that they have experienced along the way. This will give us the ability to

more accurately compare plant growth between species. Additionally, we will be measuring

stomatal density once at the end of the experimentation process. By doing this we will be able to

visualize the number of stomata the plants have and make observations based on the stomata’s

gas exchange properties and its overall effect on the plant’s growth. Photosynthetic pigments,

specifically chlorophyll a, chlorophyll b, and carotenoids will be observed to make conclusions

on the plant’s ability to absorb various light waves and its ability to affect the plant’s growth.

With increased pigment, this indicated increased fitness since this suggests that the plant is better

suited for the environment and bale to perform more efficient photosynthesis. By measuring the

roots at the end of the experiment, we will be able to compare root growth between environments

to see if temperature affected the plant’s ability to reach various nutrients in the soil. Therefore,

we may be able to demonstrate how global warming may change the growth of plant roots in the

future. As previously discussed, we hypothesize that Tussilago farfara will exhibit higher levels

of growth, making it better suited for a changing environment.

D. Research Plan

1. Experimental Design
 The two factors to be studied over the duration of this experiment are species and soil

temperature. The two temperatures being tested will be 21.1C and 26.7 C, which are the

average summer temperatures in West Virginia, and the average global summer temperature

predicted for the year 2100 respectively (Boer et al. 2000). The two species being studied during

this experiment will be Lonicera maackii and Tussilago farfara, two invasive species that are

found in West Virginia.

2. Methods

The seeds for Lonicera maackii and Tussilago farfara will be obtained from the Life

Sciences Greenhouse here at WVU. The seeds will be planted in 28 4”x4”x5” planting pots with

5 seeds in each pot. There will be seven pots for each treatment combination, so seven replicates

for each combination. Seven pots will have the L. maackii low temperature treatment, seven pots

will have the L. maackii high temperature treatment, seven pots will have the T. farfara low

temperature treatment, seven pots will have T. farfara high temperature treatment. The

temperature treatment will be applied continuously throughout the experiment using heat mats

supplied by the Life Sciences Greenhouse, but the plants will only be watered every other day.

Plant height measurement will be taking every other day during watering so that a growth rate

can be obtained. Root length and root dry mass will be obtained after the plants have been

harvested at the end of the experiment and this will be done with a rule for the length, and paper

bags, the Greenhouse oven, and scale to obtain a root dry mass. In order to get more data

pertaining to plant growth rate, we will also obtain a photosynthetic pigment count using

acetone, a centrifuge, and a spectrophotometer. We will also obtain a stomatal count as more data

for growth rate, and this will be performed using the microscope.

3. Safety Precautions
 A necessary safety precaution for this experiment is to carefully handle the heat mats as

they may be hot enough to cause burns if handled incorrectly. Also, the acetone used in the

procedure necessary to obtain photosynthetic pigment count needs to be handled with care as it

can cause burns to the eyes or skin.

4. Statistical Analysis

To test the effects of temperature and species on invasive plant growth patterns, we will

use a two-way ANOVA to analyze the data obtained from this experiment. We will use Microsoft

Excel and SAS-JMP to analyze our data and get our statistical results from our experiment. We

will be testing to see if there is a significant variation in the root length, root dry mass, growth

rate, photosynthetic pigment count, and stomata count of these plants that varies due to species

or temperature difference. For our statistical significance, an α-value of < 0.05 will be used.

Plant Species

Temperature Tussilago farfara Lonicera maachii

21.1 °C n=7 n=7

(Today’s Average)

26.7 °C n=7 n=7

(Future Average)

Figure 1: 2 x 2 Factorial Design. Two different invasive species, Tussilago farfara and

Lonicera maachii, will be studied at two temperatures, the average climate today, 2019, and the

predicted climate in the future, 2100. Seven replicates will be used for each combination of

factors.

5. Research Schedule

Date Task Completed Group Member(s)

Present
2/11/19 Experiment begins; 50 L. maackii seeds and 50 T. farfara seeds All
planted and placed into their treatment groups
2/11/19 – L. maackii and T. farfara seeds are watered and monitored Alternating
2/25/19 every other day members
2/25/19 Both plants should be successfully germinated Alternating
members
2/25/19 – Plants watered every day (5.5-week duration) Alternating
4/5/19 members
2/25/19 – Plants measured every other day (5.5-week duration) Alternating
4/5/19 members
4/5/19 Final measurements are taken: root length, root biomass, All
growth rate, and amount of chlorophyll.
4/5/19 – Perform statistical analysis on results and interpret data All
4/8/19

E. Expected Significance

As global temperatures continue to rise due to global warming, it is important to test and

make predictions about the effects that this increased temperature will have on plant growth. By

testing these invasive species’ growth patterns in the increased temperature, we expect to obtain

significant results. Due to the fact that the Lonicera maackii plant comes from a milder climate

natively, we expect that the effects of the increased temperature will be more severe on this plant

than they will be on the Tussilago farfara plant. Due to this hypothesis, we expect that they T.

farfara plants will show higher stomatal density, have higher photosynthetic pigment counts, and

a higher calculated growth rate, all indicating a better ability to cope with the increased

temperature. In terms of below ground growth, we expect that T. farfara may be able to grow

deeper roots than L. maackii but that their root dry mass will be relatively the same. In simple

terms, we expect that the growth of the T. farfara plants will be less severely affected by the high

temperature.

By finding significant results with this experiment, we would hopefully be able to apply these

results in the future plans for dealing with invasive species. By knowing how certain plants react

to the increasing global temperatures, we can come up with a better plan for how to stop their

invasive nature. Specifically, if their above ground growth or below ground growth shows
significant differences, this could indicate that targeting a specific area of the plant might be an

effective treatment. In addition to this, the results of this study could be used as a starting off

point to get an idea of how invasive species hailing from all different areas of the world will be

affected in the future by rising global temperatures.