Discovering The Genetic Similarities Between and Within The Yucca Species
Naquisha L. Knights, Research Scholar
Faculty Mentor: Jim Leebens Mack
The Pennsylvania State University
Abstract
A total of six individuals from two different Yucca species (Y.carnerosana &
Y.schidigera) were used investigate the genetic diversity and phylogenetic relationship
between and within the Yucca species using Amplified Fragment Length Polymorphism
(AFLP). Five primer combinations produced a total of 318 fragments, but only a193
highly reproducible bands were selected. The phylogenetic relationships were estimated
using the Neighbor Joining (NJ) method in PAUP 4.0 software. The phylogeny tree
generated from the NJ method showed the clustering of different species with high and
low bootstrap values. The bootstrap values were used to determine if the phylogenetic
relationships between and within the Yucca species were supported. Due to technical and
biological problems with reproducibilty and varaiation among individuals the AFLP
analysis may be not be the best method of finding the phyloegenetic relationships
between these two species. Even though these problems have arisen possible solutions
were found to solve these two problems.
Introduction
The Yucca plant plays an important role in the Native American culture. This
plant has been used to make cords, cloths, baskets, and sandals due the plants strong
fibers (Ramsay et al., 1992). The Yucca plant contains three main parts, the flower, dried
pods and root, which were used for various reasons. The raw flowers were eaten in salads,
dried pods were ground into flour and roots were used to make soap, treat headaches,
arthritis and gonorrhea (Ramsay et al., 1992). Recently, a species of the Yucca genus
called Yucca schidigera has been used to treat pain and inflammation. It was also used to
reduce joint inflammation (Ramsay et al., 1992).
Native to the Southwestern region of the United States and Mexico the genus
Yucca includes about 40 species that have evolved as successful semi-desert plants
(W.P.Armstrong, 1992). The Yucca species is apart of the Agavaceae family (Pellmyr &
Leebens-Mack, 2003). There are three major sections in the Yucca genus, which are
spongy fruited, Clistocarpa, fleshy-fruited, Sarcocarpa, and capusular-fruited,
Chaenocarpa (Pellmyr & Leebens-Mack, 2003). The fleshy fruites species is what was
focused on in this project. The Sarcocarpa is one the two largest sections of the Yucca
species. The yucca moth belongs to the Prodoxidae family, which has 78 described
species and 15 undescribed species (Pellmyr & Leebens-Mack, 2003). The
Parategeticula and Tegeticula pollinating moths pollinated the yucca plants for over 41
million years (Pellmyr & Leebens-Mack, 2003). Recent studies have found the genetic
relationships between the different speices of the yucca moths (Pellmyr &Leebens-Mack,
1998). Evolutionist have become involved in finding out the speciation of yucca moth
has effected the speciation of the yucca plants and vice versa.
The relationship between the yucca plant and the yucca moth was first discovered
by entomolistst, C.V. Riley in 1872 (Pellmyr & Leebens-Mack, 2003). This genus is
different from other flowering plants due to its unique method of pollination. The yucca
plant is only pollinated by the yucca moth. The female moth pollinates these plants by
using her mouthpart called the tentacles to collect pollen from the flower anthers
(Pellmyr & Leebens-Mack, 2003). She creates a ball of sticky pollen and stuffs it into the
stigma of flowers she visits (Vos et al., 1995). The moth backs up the flower base and
inserts her ovipositor to lay an egg into the carpel with about 10-20 bobbing movements
(Pelllmyr & Leebens-Mack, 2003). The pollination of the Yucca plant process will allow
the yucca larvae to develop into adults, while feeding on the seeds produced by the plant.
Without this method of pollination both species would become extinct due their
mutualistic relationship. Since, the yucca moth and the yucca are involved in obligate
mutualism they are able to benefit each other in two main ways. The yucca moth can not
produce the next generation of moths without the yucca plant because they depend on the
plant for seeds. Without the yucca moth the yucca plant can not disperse itself or
reproduce seeds.
The yucca plant and the yucca moth depend on each other for survival.
According to Ramsay, author of The Yucca Plant and the Yucca Moth, “If two organisms
become totally dependent upon another, they have locked their future survival together—
they coevolved.” Coevolution is the evolutionary change of one species due to the
changes in another species (Zimmer, 2001). The interactions between the yucca moth
and the yucca plant have lead many evolutionist and ecologist into taking a deeper look at
the genetic similarities between the two organisms. The special interest in the
interactions between these two organisms was how were they able to coevolve over such
a long period of time without killing each other because most plants will produce a
chemical that fends off herbivores when over expolited.
To asses the genetic relationships between many species scientists have been
using a new technique called Amplified Fragment Length Polymorphism (AFLP). This
method combines the replicability of restriction fragment analysis with the power of the
Polymerase Chain Restriction (PCR). AFLP generates hundreds of high-resolution
markers from DNA of any organism (Mueller & Wolfenbarger, 1999). This process
involved three major steps (Vos et al., 1995): (1) restriction digestion of the genomic
DNA and ligation of oligonucleotide adaptors to the ends of the DNA fragments (2)
preselective amplification by primers with a single nucleotide extension and (3) selective
amplification by primers with three selective nucleotide extensions (Figure 1). Other
researchers have found the AFLP method to be successful in studying purple coneflowers
(Kim et al., 2003), rye (Saal & Wricke, 2002), banana ,Musa (Ude et al., 2002; Loh et al.,
2000). Compared to other genetic fingerprinting programs, AFLP was found to be more
reliable, cost efficient, have a high level of repeatability, and produce a larger number of
polymorphisms (Loh et al., 2000; Ude et al., 2002).
The objectives of this study are to (1) provide information on the genetic diversity
and phylogenetic relationship between and within the Yucca species using AFLP analysis,
(2) use the Neighbor Joining (NJ) method to infer relationships and bootstrap analysis to
asses the support for inferred relationships between and within the Yucca species and (3)
asses the sustainability of AFLP technique.
Methodology
We took 2 indpenedent samples from each of the 6 individuals, two each from the
Yucca schidigera and Yucca carnerosana. Therefore, a total of 12 samples were
genetically fingerprinted to determine the degree of genetic similarities between and
within Yucca species. DNA was extracted from the each sample and was digested with
restriction enzymes. To determine if these species were genetically related, Amplified
Fragment Length Polymorphism (AFLP) was used (Vos et al., 1995; Mueller &
Wolfenbarger, 1999). To improve the efficiency of the AFLP technique fluorescent dye
was added to each primer in order to detect the fragments of DNA on the CEQ 8000
automated DNA sequencer (Xu & Mei, 2001). Finally, the phylogenetic relationships
were estimated by the Neighbor Joining method and the Bootstrap analysis using PAUP
4.0 software to determine the similarities within the 6 individuals and between the 2
different species.
AFLP ANALYSIS
Genomic DNA was extracted from the leaf tissue of each sample following the
manufactured protocol with minor modifications (QIAGEN, Venice, CA). The
extraction step was run overnight. Extracted DNA's were run on a 1% agarose gel with
ethidium brominde (EtBr) and visualized on a UV light table. DNA purity was measured
by spectrophotometer by assesing the ratio of absorbances at 260 and 280 nm. Only
samples with 260:280 ratios of 1.8-1.9 were used for AFLP analysis.
Amplified Fragment Length Polymorphism was done following the protocol of
Vos et al. (1995) with minor modifications. Primers used in the AFLP analysis contained
phosphoramidite dye (blue, black and green) that showed differences in signal intensity.
Subsequently, all EcoRI primers contained the blue dye, which gave the highest signal
strength.
The genomic DNA (200 ng) was incubated overnight at 25ºC with 0.2µl of T4
DNA Ligase, 1µl of Ligase Buffer, 1µl of NaCl, 0.5µl BSA, 1µl of Mse adaptor, 1µl of
EcoRI adaptor, 0.05 of µl Mse I enzyme, and 0.05µl of EcoRI enzyme. After ligation,
the reaction was diluted with EB Buffer (10mM Tris·Cl, pH 8.5) and stored at -20ºC.
Two pre-selective primers with a single selective nucleotide were used to amplify
fragments of the DNA template. The single selective nucleotide goes in one base after
the adaptors to amplify the DNA (Figure 1). The PCR reaction mix consisted of 2.5 µl of
10X Titanium Buffer, 2 µl dNTP (2 mM), 0.5 µl Mse I primer, 0.5 µl Eco RI primer, 0.5
µl Titanium Taq Polymerase and 15 µl deionized water (ddH2O) per reaction. The total
volume of the reaction was 20µl containing 3µl of the DNA template. The PCR
conditions were as follows: denature 94ºC (20 sec.), anneal 56ºC (30 sec.), extension
72ºC (30 sec.), final extension 68ºC (30 min.) for 25 cycles. The PCR products were
diluted with 50µl of EB Buffer (10mM Tris·Cl, pH 8.5) and ran on a 1% agarose gel
consisting of ethidium bromide (EtBr) for 40 minutes to determine if the DNA plates
were digested and amplified correctly.
A subset of 5 primer combinations that had three nucleotides was selected for the
Yucca taxa (Table 2). Selective PCR reactions contained 7.5 µl of Core Mix provided by
ABI Bioscience, 0.5 µl of Mse I primer, 0.5 µl of Eco RI primer with a fluorescent dye
and 1.5 µl of diluted preselective amplification DNA. After selective amplification, 1µl
DNA was added to 38.5 µl of deionized formamide and 0.5µl of a 600 bp size standard
(Beckman-Coulter Inc., Fullerton, CA). The samples were analyzed and sequenced using
the CEQ 8000 Fragment Analysis software (Beckman-Coulter Inc., Fullerton, CA).
Data Analysis
All 12 samples, which included the 6 replicates, were all used in the AFLP
analysis. Only those amplifed fragments with high reproducibility were scored. We
manually went into the traces generated with each primer combination and deleted the
loci with low reproducibility for each locus. Each sample was scored for present (1) and
absent (0) by the CEQ Cluster Fragment Analysis (Beckman-Coulter Inc., Fullerton, CA).
To estimate the genetic similarities between each sample a genetic distance matrix (Nei
& Lei, 1979) was used in the PAUP 4.0 software (Swafford, 2003). A phylogeny tree
was constructed using the Neighbor-Joining (NJ) method. This program was used to
group individuals that are genetically related to each other based on the genetic distance
matrix. To support the phyologenetic relationships between and within the species a
bootstrap analysis was done using the PAUP 4.0 software (Swafford, 2003). The higher
the bootstrap values the more likely that the relatioships in the NJ tree are true. The
lower the bootstrap values suggets that we can have less confidence in the relatiosnhips
reflected in the NJ tree.
Results
The total number of fragments produced by each ample was 318 (not shown).
Only 193 fragments of the total 318 were highly reproducible. There was 80%
repeatability after manually selecting the most reproducible fragments. The total number
of fragments in each primer set varied from 9 to 73 (Table 3). Primer Combination 1
produced the highest amount of fragments and Primer Combination 5 produced the least
amount of fragments. The average number of fragments per plant in each primer
combination ranged from 3 to 11. Primer Combination 1 produced the highest amount of
fragments and PC5 produced the least amount of fragments.
The phylogenetic tree indicates that the Y. schidigera species (Red) from San
Diego, California is more likely to be genetically related to a different species the
Y.carnerosana (Blue) than to its own species. This is shown in the top clade in Figure 2.
The Y.carnerosana species from Coah, Mexico is also shown to be more genetically
related to the Y.schidigera species than to its own species, which is shown in the second
clade in Figure 2.
The bootstrap values located in Figure 2 do not lend strong support for many of
the relationships on the phylogeny tree. The bootstrap value of 64% for the Y.schidigera
sample from San Diego, CA being genetically related to the Y.canerosana from
Guadalcazar, Mexico is not very strong. On the otherhand Y.carnerosana from Coah,
Mexico does seem to be genetically related to the Y.schidigera sample from Jacumba,
California. Surprisingly, the bootstrap value was very high for the clustering of these two
different species in the second clade.
Discussion
We were only able to generate 193 highly reproducible fragments from the five
primer combinations in the AFLP analysis. This number is not as high as other studies
have found. Seefelder et al. whom studied the phylogenetic relationships and genetic
diversity of Humulus lupulus (hop) using AFLP found that out of 151 polymorphic
fragments a 130 were highly reproducible (2000). In comparison to the hop study our
data did not generate a high proportion of frgaments out of the 318 fragments. Also, in
this study the choice of primer combinations could have had a large influence on the
amount of fragments recovered (Robinson et al., 1999). The number of fragments
produced by each primer set indicated whether that primer combination should be used in
further experiments. PC5 (E-AGT/M-CAG) did not produce a high amount of fragments,
which indicated that we should use this combination for further studies, but PC1 (EACA/M-CAG) did produce a high amount of fragments. Also, the average number of
fragments was very low also, which indicated that the AFLP did not work properly.
Table 3 showed values ranging from 3 to 11. In the study of hop they were able to
produce 8 to 32 fragments per primer combination (Seefelder et al., 2000). Evidently, the
number of fragments was low, but we were still able to produce a fairly accurate
phylogeny tree.
Recent studies have found that individuals within the same species cluster
together because they are more genetically similar. In the study of banana, it was found
that bananas within the same varieties were more genetically related to each other than to
bananas of a different variety (Ude et al., 2002). Subsequently, in the purple coneflower
study they found that members within the same species were more genetically related to
each other than to members of a different species (Kim et al., 2003). In Figure 2, the
phylogeny tree showed the clustering of individuals from different species in the same
clade. This is completely opposite of what was suppose to happen. The low bootstrap
value for one the clusters in the first clade indicated that the relationship between these
two different is not certainly true.
Although, many other studies have found individuals of the same species to be
more related to each other the second clade in Figure 2 suggest that this is not the caese
for yucca. The high bootstrap value of 89 strongly supports the relationship between
these samples from two different species. This was totally unexpected since the
Y.schidigera and Y.carnerosana live in completely different areas, are pollinated by
different moths and are phenotypically different from one another. Figure 3 indicates the
different areas that these two different species live, which is predominantly in California
and Mexico. The red area is where Y.schidigera species is located and the blue area is
where the Y.carnerosana is located. The distance between these two species is far that
one would not expect these two species to genetically similar. A possible reason for these
different species clustering together could be due to the glaciation period that happened
10,000 years ago, which led to gene flow between the species. Species of the Yucca
genus may have exchanged genes during the glaciation period when many species have
have been living in symaptry. But after the glaciation period the Y.schidigera and
Y.carneosana species could have stopped exhanging genes, leading to the divergence of
the two species. Leebens-Mack et al. (1999) discussed hybridization among Yucca
species, which found Yucca species exchanging genes in symaptry, but were being
pollinated by different moths.
Conclusion
This study found unexpected results and possible problems with the Amplified
Fragment Length Polymorphism (AFLP) Analyses. Two technical problems were
reproduciblity and variation between indiviuals. The reproducubility of frgaments was
low for each sample, which could be solved by runnung replicates of each sample and
only including reproducible loci. There was also variation between individuals, but little
consistent variation between species and few species specific bands. This could be
because recent mutations within the species obliterate mutations tha would give species
specific bands. A biological explanation for our results could be the hybridization
between species. There could have a recent split between the species where each of the
two species was able to hold on to their ancestral variation or long ago the species may
have diverged. The solution to solving the technical problem would be to select species
that have recently evloved by picking species that are located in the same area and are
phenotypically similar and selecting a large population size to asses how much gene flow
has occured. Also, using less rapidly evolving markers so the species specific bands
would not be masked by new mutations.
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Figuure 1. Amplified Fragment Length Polymorphism Technique
Restriction Digest & Ligation of
adaptors
TAA
5'
3'
AAT
AATT
TTAA
MseI
EcoRI
MseI
EcoRI
Denature (95ºC)
GXX
XXT
Cool to allow primers to bind (50ºC)
MseI
EcoRI
MseI
EcoRI
DNA synthesis (72ºC)
Note: The MseI and EcoRI primers bind to allow the Taq polymerase to bind to the amplified DNA
to make complementary
strands of
whenYucca
the temperature
lowered. This process is done for
Table 1. Plant Material
ofDNA
the genus
used in thisisstudy
another 25 cycles creating many copies of the new DNA.
Species
Abrv.
No.
Site
Yucca canerosana
CARN
139
E San Luis Potosi, Mexico (SLP,SLP)
Yucca canerosana
CARN
263
Guadalcàzar, Mexico (GU,SLP)
Yucca canerosana
CARN
260
Saltillo, Mexico (SA,CO)
Yucca schidigera
SCHI
----
San Diego, California (SD,CA)
Yucca schidigera
SCHI
320
N Catavina, Mexico (CAT,BC)
Yucca schidigera
SCHI
379
Jacumba, California (JA,CA)
Table 2. Oligonucleotide adaptors and primers used for AFLP analysis of Yucca
Primers/adapters
AFLP primers
EcoRI+1
MseI+1
EcoRI+3
Sequences
GACTGCGTACCAATTCA
GATGAGTCCTGAGTAAC
GACTGCGTACCAATTCACA
GACTGCGTACCAATTCAGT
MseI+3
GATGAGTCCTGAGTAACAG
GATGAGTCCTGAGTAACTG
GATGAGTCCTGAGTAACTC
Selective Primer Combinations
PC1
E-ACA/M-CAG
PC2
E-ACA/M-CTG
PC3
E-AGT/M-CTG
PC4
E-AGT/M-CTC
PC5
E-AGT/M-CAG
Table 3. Number of fragments generated by five primer combinations in the AFLP
analysis of Yucca.
Primer Combinations
PC1
PC2
PC3
PC4
PC5
Total
Total fragments
73
40
32
39
9
193
Average no. of fragments
per plants
11
7
10
5
3
36
No. of fragments present
in all plants
1
0
2
0
1
5
1
1
The total number of fragments from all plants for each primer combination
Figure 2. Estimated Phylogenetic Relationships between and the Yucca species. The
values on nodes represent the bootstrap values for each cluster.
SLP,SLP_CARN139 1
97
SLP,SLP_CARN139 2
GU, SLP_CARN263 1
97
GU, SLP_CARN263 2
64
100
SD,CA_SCHI 1
SD,CA_SCHI 2
92
SA,COA_CARN260 2
89
99
64
SA,COA_CARN260 1
JA,CA_SCHI379 1
JA,CA_SCHI379 2
99
CAT,BC_SCHI320 1
CAT,BC_SCHI320 2
Figure 3. Species Ranges for Y.schidigera and the Y.carnerosana in the United States
and Mexico