FUSE 2014 – Houser
Taxonomic classification of an endemic Bermudian fern, using molecular and morphological data
Description of Project
Diplazium is a genus in the Athyriaceae family of ferns. One specimen of Diplazium Laffanianum was
collected in Bermuda in 1905, and this species is believed to be endemic to Bermuda. Because few
individuals remain, it is not very well studied. In this study, I will use morphological and molecular
methodology to determine the exact taxonomic placement of this species.
Background:
Diplazium is an immense genus containing roughly 400 species of ferns (Wood, 2006). Ferns in this
genus are mostly terrestrial, developing in humid wetlands to steep mountain woodlands and sometimes
on limestone rocks (Wei et al., 2013). The name Diplazium originates from two words, “di” meaning two
and “plasion” meaning oblong and also refers to the double row of spore-producing sori (clusters of
spore-containing sacs) underneath the fern fronds (leaves). The Diplazium genus has been found in the
United States, China, India, Thailand, and Cambodia.
It is very important to study Diplazium Laffanianum (Figure 1),
commonly called Governor Laffan’s Fern, because it is rare,
endangered, and believed to be endemic to the islands of Bermuda.
It needs to be classified and named so that it can be properly
protected. Beyond determination of the exact species, there is a
question as to whether this fern is properly placed in the genus
Diplazium or if it is even part of the family Athyriaceae. Diplazium
may be part of the genus Asplenium in the Aspleniaceae family,
but evidence also supports the placement of this fern in the
Woodsiaceae family (Smith et al., 2006). A recent publication on
the phylogeny and classification of Diplazium, did not mention
Diplazium Laffanianum (Wei et al., 2013).
Diplazium Laffanianum is listed in the Flora of Bermuda published
in 1918 (Britton, 1918); however, no representatives are currently
living in Bermuda (the species is extinct in the wild) (Bermuda
Conservation). The last individual was seen in the wild in 1905.
The Plant Conservation Research Laboratory at Henry Doorly Zoo in
Figure 1 - Diplazium Laffanianum
Omaha, Nebraska has some of these ferns. They have been cultivating from Bermuda, Bermuda
them and growing them from spores for many years, and they recently Department of Conservation
returned some of these to Bermuda in hopes that they can return them to the wild. So far, they have not
been successful. Henry Doorly Zoo has twelve mature plants and hundreds of immature specimens
growing in sealed containers in various nutrient solutions.
The government of Bermuda wants to know if this species is a distinct species that is endemic to
Bermuda or is it the same as a currently recognized species. If so, which species is it?
In this study, I will collect and compare morphological and molecular data from the zoo and borrowed
specimens to address the following questions:
a. Is the rare Bermudian fern a distinct (and possibly unrecognized) species?
b. Is the rare Bermudian fern related to ferns that are related to North America or deciduous ones in
the tropics?
FUSE 2014 – Houser
Activities and Methodology:
I will collect leaf fronds for DNA extraction. In the lab, I will extract total genomic DNA from the leaf
tissue and then send the DNA samples to UNMC for Illumina sequencing. I will compare the DNA
sequences between the fern individuals from Bermuda and those of close relatives (available on GenBank
database). This information will be used to determine to which other species my sample may be closely
related.
With assistance from UNO’s herbarium curator, Dr. David Sutherland, we will borrow samples of
closely-related species from other herbaria in the United States. With these samples, I will then be able to
investigate the morphological characteristics of the individuals housed at the Henry Doorly Zoo in
Omaha, Nebraska. At the zoo, I will compare individual plants, fronds, and reproductive structure
characteristics with the samples borrowed. I will develop a character matrix that includes a wide range of
morphological characteristics along with descriptors such as color, mass, size, and other metrics.
Determining the correct taxonomic placement of the aforementioned Diplazium fern is my fundamental
goal for this project. To meet my goal, I will commit to several hours of work, seek advice as needed and
prepare my discoveries in a timely manner. Once I have correctly placed the Diplazium fern in its family,
it can be properly protected.
Project Timeline:
Spring
2014
Summer
2014
Fall
2014
Spring
2015
Collect leaf tissue for extraction of genomic DNA and send samples to UNMC for
Illumina sequencing. Begin investigating the morphological differences within the
Athyriaceae, Woodsiaceae and Aspleniaceae families.
With the genomic DNA sequence that I get from my sample and the sequences I have
downloaded from GenBank, I will estimate a phylogeny. I will examine my phylogeny
for where it has placed my sample. This will give me a favorable opinion about close
relatives of the Diplazium fern. Then, I will borrow these close relatives from other
herbaria in the United States with the help of Dr. David Sutherland, UNO’s herbarium
coordinator.
I will investigate the morphological characteristics of the individual ferns which are
housed at Omaha Zoo’s Plant Laboratory with help from their scientists, and compare
these to the borrowed herbarium specimens.
Present results at the UNO’s 2015 Student Research and Creative Activity Fair
Student and Mentor Roles:
Dr. P. Roxanne Kellar will be mentoring this project. I, the student, will be performing the work for this
experiment. I will commit to working 20 hours per week at $10 per hour. I will consult with Dr. Kellar
with any questions or concerns I may have. Weekly group and lab meetings will be held and I will be
expected to discuss my progress, results and will be given feedback. Additionally, I will be reporting my
findings, not only to Dr. Kellar, but also to fellow Kellar lab members. When I am in need of samples
from other herbaria in the United States, I will consult with Dr. Kellar and Dr. Sutherland on which
samples to borrow from other herbaria in the United States. I will actively participate in the FUSE
program activities such as their orientation sessions and FUSE Speaker Series. I will also take
responsibility for completing the project by preparing my results for presentation in UNO’s 2015 Student
Research and Creative Activity Fair.
FUSE 2014 – Houser
Budget Justification:
The proposed budget will cover multiple trips to the Omaha Henry Doorly Zoo to study morphology and
DNA extraction and sequencing.
6 trips to the Plant Conservation Research Laboratory at Omaha’s Henry Doorly Zoo:
 Gas (6 trips * 12 miles round-trip * $0.56 per mile) -------------------------------------------------- $41
Lab Work:
 DNA extraction supplies (1 sample * $4.00/sample * 4 rounds of extraction) -------------------- $16
 Illumina sequencing ($400 each) ---------------------------------------------------------------------- $400
Total Materials and Supplies ---------------------------------------------------------------------------------- $457
Student stipend (200 hours) ----------------------------------------------------------------------------------- $2000
Total Requested ------------------------------------------------------------------------------------------------- $2457
Note: These expenses represent only a portion of those needed to complete the entire project. Dr. Kellar
and the Plant Research Laboratory at Omaha’s Henry Doorly Zoo will cover additional costs.
FUSE 2014 – Houser
References:
Bermuda Conservation - Governor Laffan's Fern. (n.d.). Bermuda Conservation - Governor Laffan's
Fern. Accessed January 29, 2014, from http://www.conservation.bm/governor-laffans-fern
Britton, N. L. (1918). Diplazium Sw. In Flora of Bermuda: (illustrated) (p. 423). New York: Scribner's.
Diplazium. (n.d.). Diplazium. Accessed January 29, 2014, from
http://hardyfernlibrary.com/ferns/listSpecies.cfm?Genus=Diplazium
Smith, A. R., Pryer, K. M., Schuettpelz, E., Korall, P., Schneider, H., & Wolf, P. G. (2006). A
classification for extant ferns. Taxon, 55(3), 705-731.
Wei, R., Schneider, H., & Zhang, X. (2013). Toward a new circumscription of the twinsorous-fern genus
Diplazium (Athyriaceae): A molecular phylogeny with morphological implications and infrageneric
taxonomy. Taxon, 62(3), 441-457
Wood, K. R. (2006). Conservation status of the Hawaiian endemic fern Diplazium molokaiense
(Athyriaceae) in Honomanu, East Maui, Hawai'i. Endangered Species Research, 2, 1-5.
COLLEGE OF ARTS AND SCIENCES
Department of Biology
3 February 2014
Dear FUSE Review panel:
Subject: Letter of mentor support for Dilys Houser
I am pleased to support the undergraduate research project by Dilys Houser. I have known Dilys
for one semester, and I have every confidence in her determination and ability to conduct the
proposed research. She was enrolled in my Flora of the Great Plains course in the fall of 2013
and was one of the top performers in the class. She has the desire and drive to excel in
everything she does. Dilys’ educational background has prepared her to take on this project.
Research in my lab focuses on plant taxonomy, phylogenetics, and biodiversity on various plant
groups. Dilys’ project uses similar methods and techniques as others in my lab, but her project
focuses on a taxonomic group that is different from other current research projects. This is a
very important study that may lead to the protection of this endangered species. It is also a great
opportunity for her to work with the world-class plant researchers at Henry Doorly Zoo. I have
read Dilys' proposal, and it accurately describes the overall importance of the research, project
goals, and methods, and it is achievable by the student within the stated timeframe.
Additionally, I assert that the student was the primary author of this proposal (except for the
budget section). She received editorial assistance from me throughout the writing process.
I appreciate and support the FUSE program, and I am delighted to have a responsible and
enthusiastic student applying for one of the grants. We appreciate your consideration.
Sincerely,
P. Roxanne Kellar, Ph.D.
Assistant Professor
402-554-2840; [email protected]
Department of Biology / 114 Allwine Hall / Omaha, NE 68182-0040
(402) 554-2641 / FAX (402) 554-3532 / www.unomaha.edu/biology