Download Invasive Species: The Japanese Hop

Population Distribution and Genetics of the Invasive Plant Species Japanese Hops
(Humulus Japonicus)
By: Samantha Christianson, Advisor – Dr. Ryan Haasl
Abstract
The recent introduction of the invasive plant species Japanese hops to Southwest
Wisconsin has important implications for the future of the waterways of this area. Japanese hops
is a fast growing annual vine that quickly overtakes river banks and pulls down other plants. The
current distribution of the plant, especially in Grant county Wisconsin is not well known. One of
the main focuses of this research was to develop an understanding of the current distribution of
the plant. This object was accomplished and distinct patterns of distribution were noted. The
other focus of this project was to develop genetic markers to determine populations, size of
populations, and the history of invasion of the plant in Wisconsin. The ultimate goal of this
research is to help groups such as the DNR and Southwest Badger focus their eradication efforts
to keep the plant species contained.
Introduction
Invasive species have significant impacts on the areas in which they invade. These
impacts effect a wide variety of disciplines ranging from ecological to economical (Strayer,
2010). For these reasons, it is so imperative that when a new invasive species is found that all
efforts are used to eradicate that species. There are many methods to accomplish this goal, but
before the species can be eradicated, its distribution must be known. One of the main goals of
this project was to find and map the current distribution of Japanese hops in Southwest
Wisconsin where the frontal wave of the infestation is occurring. The frontal wave of the
infestation is a genetically interesting area to investigate. In these pioneer populations genetic
mutations are occurring rapidly and scientists are able to see the plant evolve as it adapts to its
new environment. These mutations to specific regions of the DNA can be compared to other
individuals to determine populations, population sizes, and the history of invasion.
Japanese hops is an herbaceous vine with opposite five to seven palmately lobed leaves.
The leaves of the plant have a rough texture and along the stem hairs are present that irritate the
skin when brushed against. The plant has distinct male and female flowers which bloom in late
summer. The plant is considered an annual in Wisconsin, but grows quickly enough in one
season to cover entire banks and pull down plants it has climbed up. The pollen of the plant is
wind dispersed and the seeds are animal or more commonly water dispersed (Japanese hops,
2014). The plant is closely related to common hops, but has no potential use in brewing (Central
Office, 2013). The plant can be a severe irritant to those suffering from allergies (Hong et al.,
1999). Japanese hops preferentially grows in moist soil along river and stream banks and in full
sun but can tolerate some shade as we found at multiple sites (Japanese hops, 2014).
The history of the plant’s invasion is well known. The plant came to America from
Eastern Asia in the 1890’s as an ornamental. By the 1550’s the plant had spread to the midMissouri area and by the 1990’s had made its way up the Mississippi into Southwest Wisconsin.
It is believed that Blockhouse Creek was the first site of the plant’s invasion. It was reported at
the site after a construction project to a bridge. This leads us to believe that the invasion of
Japanese hops in Wisconsin was human mediated and not a result of natural distribution.
The invasion of Japanese hops will have potentially significant impacts for our state. The
plant prefers to grown in riparian zones which are located alongside rivers and streams. These
areas perform important functions for our ecosystems. Such as providing food and shelter for
native species, preventing erosion of stream banks, and purifying run off water before it enters
the main water way. These critical functions help to keep our ecosystems functioning properly
and keep us healthy. Japanese hops outcompete our native plant species because of its rapid
growth. This loss of native plant species can mean the loss of critical food and shelter for native
animal species. In addition, without the complex network of native plant species roots holding
the soil together it is possible for the banks to erode much more quickly and without these root
the runoff water will not be purified to nearly the same level as before (Kang and Park, 2010). If
a municipality downstream uses this water for its citizens they will have to spend more money
purifying the water which could have trickle down effects on that whole city.
Since the introduction of the plant into Wisconsin, few groups have had either the time or
resources to do much work with the plant. This research project will give important insight into
the current distribution of the plant and its movement into Wisconsin, both of which can be used
by conservation groups to help better manage this invasive plant.
When we searched for distributional data of the plant in Wisconsin we did not find any
that was a comprehensive as we liked. We contacted both the DNR and Southwest Badger for
their current known locations of the plant and used those locations as starting points for our
search of the plant. This again,
Methods
Sampling
In the spring of 2014 we contacted both the Wisconsin DNR and Southwest Badger for
their known sites of Japanese hops. Over the summer of 2014, our group sampled these sites
given to us by the Wisconsin DNR and Southwest Badger. In addition, if Japanese hops was
present at a particular site we would continue sampling both up and down stream of the site to
find the end of the infestation. We sampled 197 sites and collected approximately 300 samples.
Our sites were chosen for ease of accessibility at locations where the water way and the road
crossed. In August of 2014, we spent two days sampling locations in Wisconsin, Minnesota, and
Iowa.
All of our samples were collected and stored under the same conditions. Collected
samples were cut from young leaves from the plant, cut into smaller pieces to increase surface
area, placed into tea bags and then placed into a plastic bag containing silica gel beads. The
plastic bag was marked with both the site number and sample letter. Young leaves were chosen
because they are undergoing the greatest amount of mitosis and as a result will have a greater
amount of DNA located in them then compared to older leaves. The samples were placed in bags
containing silica gel beads to quickly dry the leaves helping to reduce the amount of DNA
degradation caused by DNAses. The samples were stored in the lab at the University of
Wisconsin-Platteville at room temperature until DNA extraction could be performed on the
sample.
DNA Extraction
The DNA was extracted from the samples using the Qiagen DNeasy Plant Mini kit. The
samples were ground using a mortar and pestle before being run through the suggest protocol by
the kit. The extracted DNA was placed in a tube marked with both the site number and sample
letter before being placed in the freezer to await Polymerization Chain Reaction (PCR).
Polymerization Chain Reaction (PCR)
The primers used in this experiment were designed for Common Hops (Humulus
lupulus). These primers were chosen because there are no Japanese hops currently available for
purchase in the regions we were interested in and because there has not been enough molecular
work done on Japanese hops, little of its genome is sequenced to even design our own primers.
As a result, and due to findings in the literature suggesting that the same microsatellite regions in
Common hops also exist in Japanese hops, we elected to use those Common hops primers. This
is possible because of the very close relationship between Common and Japanese hops. The four
primer pairs used were EU094866, EU094885, EX518839, and EX516827. All four of these
primers were found to work and amplify a specific microsatellite region of the Japanese hops
genome (Patzak and Matoušek, 2011) and (Jakse, Luthar, and Javornik, 2008).
The areas we were interested in amplifying through PCR are called microsatellite
regions. Microsatellites are located in regions where the genome undergoes mutation regularly.
These areas are characterized by specific nucleotide repeats where the number of repeats changes
between individuals. The unique number of these repeats for different individual will allow us to
compare different populations to one another because of these differences.
The thermocycler was set to run 30 cycles of PCR which each cycle being composed of
40 seconds at 95°C, 45 seconds at 55°C, and 45 seconds at 72°C. To each individual PCR
reaction, 5µl of 10X Taq buffer, 1 µl 10mM dNTP mix, 2 µl of the forward primer, 2 µl of the
reverse primer, 0.25 µl of Taq DNA Polymerase, 4 µl of sample DNA, and 35.75 µl of pure
water was added.
Gel Electrophoresis and PCR Product Purification
The PCR products were visualized by performing gel electrophoresis on the PCR products. A
0.8 percent Agarose gel containing Ethidium Bromide was used. The resulting bands were
expressed under UV light. The bands of PCR product were cut from the gel and purified used
the gel purification kit.
DNA Sequencing
The purified PCR products will be sent to the University of Wisconsin-Madison for sequencing.
Results
Figure 1. Sites visited during the summer of 2014. Blue circles indicated locations where
Japanese hops is present and orange squares indicate sites where Japanese hops was not found.
Figure 2. Sites visited in the summer of 2014. Visited waterways have been highlighted either
blue or orange to indicate the presence or lack thereof for that particular segment of the water
way. Blue circles indicate sites where Japanese hops was found and orange squares locations
where there is no presence of Japanese hops.
Discussion
Distribution
From the sites we sampled during the summer of 2014 we developed a working
understanding of the current distribution of the species Japanese hops in Southwest Wisconsin
specifically in Grant County. As shown in Figure 1 effected rivers and creeks include the
Pecatonica River, Grant River, Rattlesnake creek, Little Platte, Platte, Blockhouse creek,
Rountree branch, and Kickapoo River.
The presence of Japanese hops on the Kickapoo River is an isolated location in
comparison to the other populations perhaps suggesting that this is another example of human
mediate dispersal. This river is widely used for recreation such as fishing and canoeing, it would
not seem unlikely that Japanese hops could have been brought in on recreational equipment and
spread from there. Only one site was visited on the river so the full extent of the infestation is not
current known. The site we did visit had a very large, well established population of the plant.
In figure 2, we are more clearly able to see the regions of each waterway that are effected
by Japanese hops invasion. As stated above, the distribution of seeds is generally downstream as
upstream movement is more difficult and thus less likely to occur. The majority of the waterways
in figure 2 they follow this trend. After the most upstream location of Japanese hops the invasion
continues downstream until it meets the Mississippi. One of the few rivers that does not follow
this pattern is the Pecatonica River. This could be due to multiple invasion sites along the river
that have not yet grown into each other, conditions that do not favor Japanese hops growth, or
restoration projects.
Molecular
The majority of our samples’ DNA has been extracted and is awaiting PCR. We have
encountered problems getting single clear bands from our PCR products suggesting that we need
to adjust our PCR temperatures to get one clear single band that we know will be the product we
are interested in sequencing. In addition, we have been having issues with getting our gels to
stain properly. The gels were not showing any bands even in the lane containing the standard
DNA ladder which acts as a positive control. As a result of this we have ordered new Ethidium
Bromide and DNA ladder to eliminate those as possible explanation for the gel to not staining
properly. As a result of these setbacks, we have not yet been able to send any samples UWMadison to be sequenced. We want to wait until we have definite desired product to send in
order to reduce cost of sequencing products that do not contain the microsatellite region we are
interested in. Over this coming summer and fall the remaining molecular work will be
conducted.
Conclusion
The current distributional data collected from our summer 2014 sampling give
considerable insight into the populations of Japanese hops present. This is especially true in
Grant County where the sampling was most concentrated. In this area there is a better
understanding of the extent of the invasion as well because more samples were collected in this
area.
The distributional data we collected will have significant implications for conservation
groups who will be able to use this data to better understand the spread of the plant and where to
focus their eradication methods. As more distributional data is collected in subsequent years we
will be able to help predict the movement of this species using a simulation designed by Dr.
Ryan Haasl.
The potential genetic data achieved in this project will continue to give more insight into
the plant’s spread. The information will allow future students to determine the original site,
where the plant moved from there, and where the plant will be moving to next.
Acknowledgements
First, I would like to start by thanking Dr. Haasl for his patience and tireless help on this
project. Without his generosity of time and expertise this project would not have been made
possible. Next, I would like to thank the Undergraduate Research and Creative Endeavors offices
for selecting me at a 2014-2015 Pioneer Undergraduate Research Fellow, funding this project,
and all their work help promote undergraduate research at UW-Platteville. In addition, I would
like to thank the UW-Platteville Biology Department for supporting my research. Lastly, to my
family and friends thank you for all love, help, and support as I completed this project.
Works Cited
Central Office. (2013). Japanese hops impressive but undesirable invasive plant.
http://dnr.wi.gov/news/weekly/article/?id=2663
Hong, C.S., Kim, C. W. Ko, S.H, Jeoung, B.J., & Park, J.W. (1999). Identification and
characterization of the major allergen of the Humulus japonicus pollen. Clinical and
Experimental Allergy, 29, 1080-1086.
Japanese hop. (2014). Invasive Plant Atlas of the United States.
http://www.invasiveplantatlas.org/subject.html?sub=10091
Kang, H. and Park, S. (2010). Impact of invasive plant and environmental conditions on
denitrification potential in urban riparian ecosystems. Chemistry and Ecology. 26. 535360.
Strayer, D.L. (2010). Alien species in fresh waters: ecological effects, interactions with other
stressors, and prospects for the future. Freshwater Biology. 55.152-174.