POTENTIAL IMPACT OF FOREST DISTURBANCE ON
GENETIC DIVERSITY OF AMPHIBIAN POPULATIONS
by
RASHIDAH HALIMAH FARID
A THESIS
Submitted in partial fulfillment of the requirements
for the degree of Masters of Science
in the Department of Biological and Environmental Sciences
in the School of Graduate Studies
Alabama A&M University
Normal, Alabama 35762
May 2014
Submitted by RASHIDAH FARID in partial fulfillment of the requirements for
the degree of MASTER OF SCIENCE specializing in PLANT AND SOIL SCIENCE.
Accepted on behalf of the Faculty of the Graduate School by the Thesis
Committee:
_____________________________
_____________________________
_____________________________ Major Advisor
_____________________________
_____________________________ Dean of the Graduate School
_____________________________ Date
ii
Copyright by
RASHIDAH HALIMAH FARID
2014
iii
The thesis is dedicated to my sisters Samaiyah, A’ishah, Sabaah, and Janet and to
my Grandmothers Rose and Effie who gave me breath of life when I had none. For
without their strength and courage I am nothing.
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POTENTIAL IMPACT OF FOREST DISTURBANCE ON GENETIC DIVERSITY OF
AMPHIBIAN POPULATIONS
Farid, Rashidah, M.S., Alabama A&M University, 2014. 84 pp.
Thesis Advisor: Dr. Khairy Soliman, Ph.D
Populations of many amphibian species have declined because of habitat destruction,
fragmentation, and alteration. In a forest community that has experienced dynamic
changes in habitat structure and composition, it is expected that amphibian populations’
genetic variations could be affected due to declined success of breeding and survivorship,
which might lead to the bottleneck effect over multiple generations. A study was initiated
at Bankhead National Forest in Alabama to examine how past forest management
practices have affected genetic structures of pool-breeding amphibian species. Molecular
markers simple sequence repeats (SSR) were used to assess the level of heterozygosity
among and within individual populations and species in the area of study. Five vernal
pools of different size and disturbance history were selected and tissue samples were
collected from two targeted species: Ambystoma maculatum and Notophthalmus
viridescens viridescens. Heterozygosity of Ambystoma maculatum populations ranges
from 35-55%, with inbreed coefficient not exceeding 55%. However, homozygosity was
highly prevalent in all Notophthalmus viridescens viridescens populations. All three
populations of Eastern Red Newt exhibited bottleneck events; allele frequency (0-0.22)
was lowest in the first distribution class. Two populations of Spotted Salamander
v
exhibited bottleneck events; allele frequencies were 0 and 0.21. The results provided a
glimpse into the landscape’s genetic connectivity and created a baseline for future genetic
monitoring studies of these species.
KEY WORDS: salamanders, gene drift, habitat fragmentation, bottleneck
vi
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ........................................................................................................... xi
LIST OF ABBREVIATIONS ........................................................................................... xii
BNK - William Bankhead National Forest ....................................................................... xii
ACKNOWLEDGMENTS ............................................................................................... xiii
CHAPTER 1 ....................................................................................................................... 1
INTRODUCTION .............................................................................................................. 1
1.2 Statement of the Problem ................................................................................... 3
1.3 Objectives of the Study ...................................................................................... 4
CHAPTER 2 ....................................................................................................................... 5
LITERATURE REVIEW ................................................................................................... 5
2.1 Amphibian Decline ............................................................................................ 5
2.2 Bottleneck Theory.............................................................................................. 6
2.3 Mutation-Drift Equilibrium ............................................................................... 6
2.4 Application of Microsatellites............................................................................ 7
2.5 Impacts of Timber Harvest ................................................................................ 8
2.6 Spotted Salamander (Ambystoma maculatum) Ecology .................................... 9
2.7 Red-Spotted Newt (Notophthalmus viridescens viridescens) Ecology ........... 10
CHAPTER 3 ..................................................................................................................... 12
MATERIALS AND METHODOLOGY .......................................................................... 12
3.1. Sampling ......................................................................................................... 12
3.2 Genomic Deoxyribonucleic Acid Purification................................................. 15
3.3 Random Amplified Polymorphic DNA: SSRs Identification & PCR ............ 17
3.4. Bacteria Transformation and Cloning ............................................................. 19
3.5. Sequencing of Plasmid DNA .......................................................................... 26
3.6. Population Genetic Analysis with Microsatellites .......................................... 29
CHAPTER 4 ..................................................................................................................... 34
RESULTS ......................................................................................................................... 34
4.1. Genomic Deoxyribonucleic Acid Quantification ........................................... 34
4.2. Random Amplified Polymorphic DNA .......................................................... 38
vii
4.3. Bacteria Transformation and Cloning ............................................................. 42
4.3.2. Plasmid DNA Extracted from Positive Clones ............................................ 44
4.4. Sequenced Plasmid DNA ................................................................................ 45
4.5. Fragment Analysis .......................................................................................... 46
4.6. Bottleneck Analysis ........................................................................................ 52
CHAPTER 5 ..................................................................................................................... 56
DISCUSSION ................................................................................................................... 56
REFERENCES ................................................................................................................. 64
VITA .....................................................................................................................................
viii
LIST OF TABLES
Table 1. SSR Spotted Salamander Primer Set. ................................................................. 31
Table 2. SSR Eastern Red Newt Primer Set. .................................................................... 31
Table 3. One hundred twenty-two DNA samples with 260/280 ratios between 1.7 and
1.8...................................................................................................................................... 34
Table 4. Eastern Red Newt DNA samples used for RAPD analysis are highlighted. ...... 39
Table 5. : Spotted Salamander DNA samples used for RAPD analysis are highlighted. . 39
Table 6. Random Amplified Polymorphic DNA Kit, US Biological .............................. 40
Table 7. Amplified Spotted Salamanders Polymorphic Regions...................................... 42
Table 8. Amplified Eastern Red Newt Polymorphic Regions .......................................... 42
Table 9. Selected Clones for Sequencing from Bacteria PCR. ......................................... 45
Table 10. Eastern Red Newt heterozygosity and inbreeding coefficient per locus within
BaPo 2 population. ............................................................................................................ 46
Table 11. Eastern Red Newt heterozygosity and inbreeding coefficient per locus within
BaPo 3 population. ............................................................................................................ 46
Table 12. Eastern Red Newt heterozygosity and inbreeding coefficient per locus within
BaPo 8 population. ............................................................................................................ 47
Table 13. Observed Heterozygosity of Eastern Red Newt Populations by Locus. .......... 47
Table 14. ANOVA of Loci Heterozygosity (Eastern Red Newt) Across and Within
Populations........................................................................................................................ 48
Table 15. Spotted Salamander heterozygosity and inbreeding coefficient per locus within
BaPo 6 population. ............................................................................................................ 48
Table 16. Spotted Salamander heterozygosity and inbreeding coefficient per locus within
BaPo 4 population. ............................................................................................................ 49
Table 17. Spotted Salamander heterozygosity and inbreeding coefficient per locus within
BaPo 3 population. ............................................................................................................ 49
ix
Table 18. Spotted Salamander heterozygosity and inbreeding coefficient per locus
within BaPo 2 population.
50
Table 19. Observed Heterozygosity of Spotted Salamander Populations by Locus......... 51
Table 20. ANOVA of Loci Heterozygosity (Spotted Salamander) Across and Within
Populations........................................................................................................................ 51
Table 21. Wilcoxon Test for Heterozygosity for Eastern Red Newt Populations. ........... 53
Table 23. Wilcoxon Test for Heterozygosity for Spotted Salamander Populations. ........ 54
x
LIST OF FIGURES
Figure 1. Map of pCR 4-TOPO. The sequence and restrictions sites are labeled to indicate
actual cleavage sites (Life Technologies, Inc.) ................................................................. 21
Figure 2. Flow chart of experimental steps of cloning (Life Technologies, USA). ......... 24
Figure 3. Life Technology visual depiction of the incorporation of terminating fluorescent
nucleotides. ....................................................................................................................... 26
Figure 4. Chromatogram of successfully sequencing of short PCR products using BigDye
Terminator chemistry; sample run of ABI 3100 Genetic Analyzer using POP-6 Polymer
(Life Technologies USA). ................................................................................................. 27
Figure 5. Gel electrophoresis of seventy one genomic DNA samples on a ethidium
bromide stained 1% agarose 1 x TBE gel; 80 voltages for 2.5 hours. Samples represented
are from study species; in numerical order. ...................................................................... 38
Figure 6. Transformed Bacteria Plates.............................................................................. 43
Figure 7. Allele Frequency Distribution for Eastern Red Newt Populations. .................. 53
Figure 8. Allele Frequency Distribution for Spotted Salamander Populations. ............... 55
Figure 9. BaPo 8 Stand First Year. ................................................................................... 57
Figure 10. Location of Sites BaPo 2 and BaPo 6. ............................................................. 59
Figure 11. Bankhead National Forest Study Sites Locations. .......................................... 60
xi
LIST OF ABBREVIATIONS
BNK - William Bankhead National Forest
DNA - Deoxyribonucleic Acid
dNTP - 2’-deoxyribonucleotide triphosphate
NSF - National Science Foundation
PCR - Polymerase Chain Reaction
RNA - Ribonucleic Acid
SSR - Simple Sequence Repeats
USDA - United States Department of Agriculture
CDC - Charge-capture Digital Camera
HWE - Hardy-Weinberg Expectations
S.O.C. Medium - Super Optimal with Catabolism repression
TAE Buffer - Tris-Acetate-EDTA Buffer
TBE Buffer - Tris-Borate-EDTA Buffer
EDTA Buffer - Ethylenediaminetetraacetic Acid
TE Buffer - Tris-EDTA Buffer
xii
ACKNOWLEDGMENTS
I would like to thank my advisors, Dr. Khairy Soliman and Dr. Yong Wang, for
their guidance. I am sincerely grateful to my advisory committee members, Dr. William
Stone and Dr. Luben Dimov, for their expertise and time. My research would not have
been possible without the financial support of the CREST Program and the National
Science Foundation (NSF) and tissue collection permission from the US Forest Service
and the Alabama Department of Conservation and Natural Resources. I am thankful to
my colleagues for their moral support, vast knowledge, and expertise in the molecular
field: Dr. Govind Sharma, Dr. Ramesh Kantety (late), Safira Sutton, Abreeotta Williams,
Fetun Desta, Angelica Durrah, Dr. Venkateswara Sripathi, Dr. Seloame Nyaku, Timley
Watkins, and Sarah Cseke. Thank you Mrs. M. Saintjones for all of your help. For their
time and dedication, I would also like to thank my students: Rogericus Denish, Meseret
Sima, and Calvin Means. Finally, I would like to thank the support staff of the
Agricultural Research Center and CREST Program: Penny Stone, Mila Sangalang, and
Lisa Gardner.
xiii
CHAPTER 1
INTRODUCTION
Although many amphibian adults are faithful to their breeding pools across
breeding seasons, they are increasingly being forced to move to more suitable pools for
breeding to increase their probability of breeding success (Gibbs, 1998; Chan-Mcleod,
2003). The combination of fewer breeding adults and reduced pool suitability could result
in fewer breeding events and egg masses at these breeding pools. These demographic
changes may lead to increasingly smaller population sizes of some pool breeding
amphibians in the areas being impacted by forest management practices (Renken et al.,
2004; Patrick et al., 2006). With smaller populations or more isolation, gene frequencies
can drift dramatically by chance effect. A reduction in population that is maintained at a
smaller size for several generations will have different genetic characteristics than it had
prior to the reduction in size. This reduction will change the populations genetically
owing to the loss of connectivity between subpopulations and genetic drift and increases
in the possibilities of inbreeding (Andersen et al., 2004). Genetic drift and inbreeding
could result in predicted low levels of genetic variation as detected by significant
deviations from Hardy-Weinberg expectations (HWEs) (Frankham et al., 2002).
1
Reductions in population size can also be changes in forest composition. Tree
composition of the canopy directly determines the type and abundance of organic matter
and therefore indirectly regulates microbial community and subsequently the macro
invertebrate community. Dietary requirements vary considerably between species and life
stages. For those species dependent of macroinvertebrates, species presence is directly
correlated to sediment load, chemical concentrations, and humus. Therefore in a forest
community that has experienced dynamic changes in tree composition, amphibian
populations’ genetic variations would reflect periods of resources limitation or abundance
throughout multiple generations. An understanding of genetic variation in amphibian
populations and how these variations limit species abundance could have significant
impacts on the way forest and mitigate wetlands are managed.
For this study, three species were initially sampled to assess population effective
size number and species comparison feasibility to include: the Spotted Salamander
(Ambystoma maculatum), Red-Spotted Newt (Notophthalmus viridescens viridescens),
and Southern Leopard Frog (Lithobates sphenocephalus utricularius). Additionally,
tissue from the following species was included when time permitted: Marble Salamander
(Ambystoma opacum), Green Frog (Lithobates clamitans melanota), and Eastern
Spadefoot (Scaphiopus holbrookii). To study amphibian genetic variation in response to
changes in forest management practices, The William Bankhead National Forest (BNF)
was chosen because of its management history.
Originally a mixed hardwood forest, the BNF was cut and converted into
agricultural fields and pastures prior to 1914. As small agricultural use gradually declined
(USDAFSBNF 2003), the USDA Forest Service (FS) purchased the land and reforested it
2
with loblolly pine, a fast-growing species that is excellent for erosion control and quality
timber. With the older pine stands dilapidated, due to severe inter-specific competition
and southern pine beetle infestations, sites quickly began regenerating with native
hardwood species such as oaks, red maple, black gum, and yellow poplar. Presently, a
thriving amphibian population is well established within its forest boundaries (USDAFS
BNF 2003). The management practices and changes in forest community provide a
unique opportunity to study forest restoration effects (Wang et al., 2010) on long term
amphibian population vitality.
1.2 Statement of the Problem
The effect of forest disturbances specifically prescribed as thinning and burning,
on animal communities with a focus on herpetofauna was conducted. In recent years,
several research studies examined the effect of these treatments and their interactions on
species richness and abundance and how the mechanisms (microclimate, vegetation
structure, food availability, density of competitors, vernal pool hydrology, and
metapopulations dynamics) are responsible for the changes in population demographics.
In this study, the main focus was to examine such effects by experimentally determining
the specific habitat features, landscape level changes, and metapopulation genetic
dynamics and structures (Wang et al., 2010). This research will address the specific
research hypotheses that silvicultural practices have had an evident effect on the genetic
diversity of pool breeding amphibians.
3
1.3 Objectives of the Study
The overall objectives of this research project are to (1) evaluate if genetic
bottleneck events are present and (2) determine the current level of genetic variation per
species. The specific objectives of this study were to: (1) calculate species effective
population size per species, (2) determine expected allele/ heterozygosity ratios, (3)
analyze data for environmental correlations between species abundance and
environmental factors, (4) conduct DNA extraction and amplification, and (5) per
species, evaluate presence of bottle neck in each population.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Amphibian Decline
Loss and degradation of habitat are considered the foremost causes of amphibian
populations decline worldwide (Collins and Storfer, 2003). Amphibians have relatively
low dispersal abilities and are often philopatric, leading to distinct populations that can
represent unique genetic entities despite geographic proximity (Kimberling et al., 1996;
Waldmann & Tocher, 1997; Driscoll, 1999; Scribner et al., 2001). With life cycles based
both in water and on land, amphibians are dramatically affected by changes in canopy
cover and demographics, hydrology, climate change and tonicity levels. Amphibians are
well suited to address questions at the level of metapopulations (Hanski, 1997), and have
in recent years become a focus for studies on the effect of landscapes and landscape
alterations to wildlife (Halley et al., 1996; Vos et al., 2001). The restoration of habitat has
shown to have a direct effect on the recovery of a species population numbers, as the
correlation between environmental variation and fitness components are widely accepted
(Bruce and Stiven, 1988). It has been debated that the success of the population is
dependent on the extent and prolonged degradation of the habitat as well as the associated
recovery time laps following the habitat degradation. It is difficult to distinguish real
5
long-term downward trends from natural population oscillations (Beebee and Rowe,
2001). In many cases, statistical analysis of demographic information is often the only
way of determining population declines (Reed and Blaustein, 1995).
2.2 Bottleneck Theory
Bottlenecks, long standing reductions in effective population size, were
effectively determined by measuring genotype frequencies at multiple polymorphic loci
at a single point in time (Beebee and Rowe, 2001). Cornuet and Luikart (1996) developed
an approach to identify recent population bottlenecks by genetic analysis. The test
requires the determination of genotype frequencies at multiple polymorphic loci at a
single point in time, based on the expectation that a bottlenecked population will
demonstrate an excess of heterozygosity over the expected, under mutation-drift
equilibrium. The test also assumes that such loci evolved according to the Infinite Allele
Model (IAM) and Stepwise Mutation Model (SMM); and the relationship between excess
heterozygosity and observed number of alleles is a function of time elapsed since the
beginning of the bottleneck (Cornuet and Luikart, 1996). Testing for bottleneck events
has become increasingly more accessible by the application of routine sequencing and
Polymerase Chain Reactions; particularly, with the application of DNA-based markers
such as microsatellite loci (Jehle and Arntzen, 2002).
2.3 Mutation-Drift Equilibrium
Bottleneck calculations assume mutation-drift equilibrium to compute the
distribution of gene diversity expected from the observed number of alleles.
6
Microsatellite genetic diversity can be used to calculate estimates of evolutionarily
effective population size (Ne) given particular assumptions about mutation patterns and
rates. The expected average heterozygosity is calculated through simulations of Infinite
Allele and Stepwise Mutations Models (Knaepkens et al., 2004). Under Infinite Allele
Model (IAM) each mutation that arises is unique. Mismatch at a given allele is observed
as an occurrence of a single mutation. At equilibrium, the heterozygosity under the
infinite allele model (IAM) is expressed as:
, which leads to
(Crow and Kimura, 1970), which µ is the mutation rate
(Knaepkens et al., 2004). In Stepwise Mutation Model (SMM), actual mutations are
observed and scored on a frequency spectrum at a given loci; no mismatch (0), (1) one
mismatch, (2) two mismatch, etc. The simplest form of SMM is a one-step symmetric
model where it is assumes that each mutation is a single step with equal probability of
increasing (+1) or decreasing (-1) (Walsh, 2001). Under SMM at equilibrium, the
heterozygosity is equal to:
), which yields:
(Ohta and Kimura, 1973). Populations without a recent change in size will be in
mutation-drift equilibrium where the expected heterozygosity (HEQ) will equal the
Hardy–Weinberg equilibrium heterozygosity (HE) (Knaepkens et al., 2004).
2.4 Application of Microsatellites
Microsatellites (SSRs) consist of tandem repetitive units of DNA, typically less
than five base pairs in length. The number of alleles segregating over a panel of
microsatellite loci enables the genetic recognition of individuals without physical
marking (Luikart, 1999). On average, microsatellites exhibit mutation rates between 10-3
7
and 10-5 (Goldstein and Schlötterer, 1999) and mostly mutate through the addition or
deletion of one repeat unit, following a stepwise mutation model or alternatively, by a
certain number of repeats simultaneously (Goldstein and Schlötterer, 1999). Beebee and
Rowe (2001) found genetic analysis using microsatellite loci was effective in
distinguishing between short-term fluctuation and long-term trend in accurately assessing
amphibian declines.
Seven microsatellite loci were used in examine the relationship
between the current six of bullhead fish and genetic diversity in the 2004 study by
Knaepkens. Though the loci number was below the recommended 10-15 (Cornuet and
Luikart, 1996), Knaepkens et al. (2004) were able to observe a positive correlation
between genetic variability and the size of the population implying that concerns about
loss of genetic diversity in small populations are indeed warranted.
2.5 Impacts of Timber Harvest
Prior to the 1960s, selective timber harvest, leaving a few remaining large trees
was the preferred method of cutting. Since the 1960’s, clear cutting has almost
completely replaced selective cutting as the preferred method of timber harvesting by the
U.S. Forest Service in the southern Appalachians (Petranka et al., 1993). However since
FEMAT (1993) percent shelter wood, or partly removal of canopy, harvest has dominated
as the preferred method on USFS lands. Amphibians’ skin must be kept moist to facilitate
gas exchange. Consequently, adults generally are restrict their activity to moist forestfloor microhabitats in the day and are active on open surface ground only at night when
humidity is high, to reduce the chance of dehydration. Although limited studies have
been conducted on clear cutting effects on amphibians, studies collectivity suggest that
8
timber harvesting is detrimental to amphibians, especially salamanders, in eastern forests
(Bury and Corn, 1988; Raphael, 1988; Welsh, 1990). Clear cutting degrades forest-floor
microhabitats for salamanders by eliminating shading, reducing leaf litter, increasing soil
surface temperature and reducing soil-surface moisture (Bury, 1983; Ash, 1988; Raphael,
1988; Welsch, 1990). Petranka et al. (1993) concluded that most animals died, estimated
loss of 75-80%, from physiological stress following the removal of trees from sites.
Increased sedimentation and general deterioration of stream quality may also have
contributed to the decline of species with aquatic larval stages (Bury and Corn, 1988).
Despite their ecological importance, amphibians (especially salamanders) are often
neglected in forest management studies (Petranka et al., 1993).
2.6 Spotted Salamander (Ambystoma maculatum) Ecology
Spotted salamanders are large, metamorphs 50 mm and adults 228 mm, dark body
salamanders with yellow and/or orange spots on the tail, body, and head. However, spots
do not appear until several months after metamorphosis. Occurring throughout the
southeast and along the east coast into Canada, spotted salamanders prefer primarily
hardwood and mixed deciduous forests. Breeding is restricted to vernal or ephemeral
wetlands with mature upland terrestrial habitats (Mitchell and Gibbons, 2010). Adults
breed in the winter and early spring; often following rainy or foggy nights. Breeding in
Alabama has been documented in late December and early February (Mount, 1975).
Males normally arrive earlier than females and are 1.5 times more numerous (Flageole
and Leclair, 1992). Adults are fateful to specific breeding ranges, often by pass other
closer ponds and navigated to their original home range (Downs, 1989).
9
Olfaction has been suggested to guide migration routes and facilitate entry and
exit near the same point yearly (Petranka, 1998). During mating, males deposits
spermatophores on substrate near a courted female. Females absorbs multiple, 15-20,
spermatophores with her cloacal lips before the close of mating season (Petranka, 1998).
Females can deposit between one-four clutches of eggs, each up to 250 eggs, within a
single season. However, embryonic mortality can be as high as 75%, depending on depth
of water, time of season, and temperature at time of deposit. Clutch incubation period
normal lasts between 4-7 weeks. After hatching, larvae feed on a variety of zooplankton
and macroinvertebrates. Transformation into metamorphs occurs within 2-4 weeks.
Metamorphs disperse into nearby forest within weeks of their transformation and will not
return to breeding pond until sexually mature 2-5 years. Adults feed on forest floor
invertebrates and live an average of 6-8 years and a maximum of approximately 20 years
(Petranka, 1998).
2.7 Red-Spotted Newt (Notophthalmus viridescens viridescens) Ecology
N. virdescens, with exception populations, has four complex and distinct life
history stages: the egg, aquatic larva, terrestrial red eft (juvenile stage), and aquatic adult.
Two stages, red eft and aquatic adult are distinctively identifiable. The juvenile stage, red
eft, migrates to land and remains for several years, approximately seven, until sexual
maturity. The in the red eft stage, individuals are gill less with lungs; the larvae losses gill
function prior to existing the aquatic environment as an eft. Red efts bright red coloration
is visible within two weeks after metamorphosis (Chadwick, 1950). A second
transformation occurs at maturity were terrestrial red efts metamorph into a gill-less,
aquatic adult and remain aquatic for extent of their lives. The morphological changes
10
include a dorsal tail fin and development of non granular skin. Aquatic adults measure
approximately 90- 165 mm of total length. Eastern Newts (N. virdescens) are the second
widest distributed salamander in North American; their range extending as west as Texas,
south to Florida and north into southern Canada (Petranka, 1998).
11
CHAPTER 3
MATERIALS AND METHODOLOGY
3.1. Sampling
Sampling was carried out in multiple steps due to the nature of amphibian
breeding and migratory patterns. Amphibian populations are highest during the breeding
seasons, and can vary depending on specific species. Breeding season may begin in early
spring and extend throughout the fall. Sampling was conducted monthly during the
breeding months of each species and sampled randomly during periods of high rainfall.
Sampling occurred in the fall and spring seasons or when populations were most
abundant; throughout the study period of approximately two years. Minnow net/traps
were used for surveying tadpoles, hatchlings and larva. A minimum quantity of tissue
was collected, to minimize stress and reduce morality, from each of the 5 research
breeding pools; BaPo 2, BaPo 3, BaPo 4, BaPo 6, and BaPo 8. BaPos 2, 3, 4, 6 and 8
were sampled at higher priority level due to their uniformity of two are more species
presences:
Spotted
Salamander
(Ambystoma
maculatum),
Red-Spotted
Newt
(Notophthalmus viridescens viridescens), and/or Southern Leopard Frog (Lithobates
sphenocephalus). Tissue samples from the tail and/or toe were taken of no greater than
10% of total body mass of any specimen to minimize stress. Toe clippings were taken
12
from adults. Tail clippings were collected from metamorphs with a total body mass equal
or greater than 2 grams. For metamorphs and larva less than 2 grams or eggs, the whole
specimen was sacrificed; not exceeding more than ten individuals or 1% of a given
species per site within a sampling season. Whole specimens were euthanized in a
benzocaine hydrochlorid bath (CCAC, 2006). Samples were kept in separate, labeled test
tubes and stored with dry ice in the field. Samples were stored long term at -80oC.
3.1.2 Sample Size
Recent BNF amphibian population estimates are currently undetermined.
However, a sample size between 20-30 individuals per species/site is considered adequate
in assessing several estimates of genetic diversity (number of alleles per locus, average
observed heterozygosity, and average expected heterozygosity) for both within and
between, species specific, pool populations (Pruett and Winker, 2007). Therefore, ponds
of high estimated population densities, a minimum of 20 samples were collected for
analysis. Additionally, more tissue samples were collected, to account for potential loss
due to degradation during handling. Sampling varied with species and site resulting in N
values form 5-20. Estimates of alleles per locus at small sample sizes can be greatly
biased, especially when compared to populations from which a larger sample size is
obtained (Pruett & Winker, 2007; Petit et al., 1998). Therefore to account for allele
diversity, we used the rarefaction method (smallest N value 5) of to normalize standard
error across sample sizes. Rarefaction is commonly used to compare allelic diversity
across unequal sample sizes (Petit et al., 1998, Leberg, 2002).
13
3.1.3 Live Capture Precautions
Minnow nets were positioned to ensure the air-water interface is maintained to
avoid drowning or asphyxiation. Due to amphibian sensitivity to temperature and
dehydration, care was taken to handle specimens quickly. Contact was limited by
examining individuals on plexiglass, to avoid the transfer of body heat. Handling time
was minimized in compliance with CCAC protocol. Trap deaths from exposure to
unfavorable temperatures, drowning, shock, predation and desiccation was minimized by
checking traps daily and not sampling during unfavorable weather conditions (CCAC,
2006). Sampled individuals were released at individual prospective capture sites.
3.1.4 Protocol for Pathogens (Chytrid Fungus)
Due to the risk the potential persistence of amphibian pathogens in aquatic
environments and the risk of transporting them, the Southeast Partner’s in Amphibian and
Reptile Conservation protocol for disinfecting equipment was followed. This protocol is
sufficient in eliminating the transport of chytrid fungus between wetlands or aquatic sites.
Once sampling at an aquatic site was completed and before moving to a new site or
returning from the field, all field equipment (e.g., nets, buckets, and water quality meters)
and personal gear (e.g., boots and waders) were rinsed with water, and all debris and mud
removed. If the tires of a vehicle contacted water with amphibians, they will be cleaned.
A 10% bleach disinfectant solution or equivalent was used to kill pathogens. The
disinfectant remained in contact with equipment or personal gear for at least 2-5 minutes
to ensure complete inactivation pathogens. Equipment and footwear were rinsed with
municipal water after the minimum disinfecting time to remove residual chemical, which
14
can be toxic to aquatic life. Disinfectant solutions were discarded and replaced after 5
days (Miller and Gray, 2009).
3.2 Genomic Deoxyribonucleic Acid Purification
3.2.1 Genomic DNA Extraction
Deoxyribonucleic acid (DNA) was purified from a 5-20 mg sample of frozen toe,
tail, skin, or other tissue using Qiagen, Gentra Puregene Tissue Kit. Tissue was prepared
using liquid nitrogen and either mortar and pestle or the TissueLyser and 8 mm steal
beads. The TissueLyser and steal beads were used for skin and tail samples. However it
was not efficient in homogenizing hard tissues from toe cuttings; therefore, mortar and
pestle was used. Following homogenization, chemical treatment was the same. Generally,
cells were lysed with an anionic detergent in the presence of a DNA stabilizer. Tissue
homogenates were digested at 55°C with proteinase K (0.2mg/mL) in the presence 0.5–
1% SDS before extraction with phenol, phenol–chloroform (1:1 v/v), and chloroform –
isoamyl alcohol (25:1 v/v). Ribonucleic acid (RNA) was removed by treatment with
RNase at 37°C; followed by salt precipitation of proteins and contaminants. Finally,
purified DNA was recovered by supernatant with isopropanol; then washed the 70%
ethanol and dissolved in hydration solution (1 mM EDTA, 10 mM Tris-Cl pH 7.5) for 1
hour at 65°C. Genomic DNA was stored at -20ºC long term use.
3.2.2 Spectrophotometric Analysis
A spectrophotometer (NanoDrop 1000, Thermo Fisher) was used to determine the
concentration of nucleic acid (ng/µl) and purity of the each purified sample. The
15
spectrophotometer is design to distinguish between protein, RNA and DNA by the
molecular weight at a given absorbency range (at wavelength of 260/280 nm). The
absorbance analysis is conducted at a baseline established from the sample suspension
buffer. A liquid column (1 mm) is formed from the sample between a upper and lower
optical base. Light is pass through the sample, quantifying a molecule concentration
within a know path length (l mm). A second measurement is taken at an adjust height of
0.2 mm to provide a reliable concentration range and estimate of sample purity
(NanoDrop Technologies, USA).
3.2.3 NanoDrop Procedure
The instrument was calibrated and initialized using 2 µl of RNase and DNase free
water; following by blanking with 2 µl DNA hydration solution (1 mM EDTA, 10 mM
Tris-Cl pH 7.5). DNA samples were loaded (2 µl) individual on the optical base, labeled
and measured. Each sample was assigned a unique name in which the first letter and
number represent site name, middle letters indicate species, and latter numbers indicated
sequential sample extracted per site/species.
3.2.2 Gel Electrophoresis
Gel electrophoresis was used to visualize the physical quality and condition of the
nucleic acid. The electrophoresis apparatus uses a DNA stabilizing and conductive buffer
to create an electrical current. Moving from negative and positive electrodes, electricity
passes through a porous gel of polyacrimanide or agarose at a set voltage and prescribed
time. Ethidium bromine was used to stain the DNA for florescence. Because, DNA is
16
negative, larger fragments move relatively short distances versus small fragments, when
compared to a commercially produced DNA ladder of predetermined fragment sizes.
Therefore, sample fragment length, containments presences and shearing are visualized
under UV light.
3.3 Random Amplified Polymorphic DNA: SSRs Identification & PCR
Random Amplified Polymorphic DNA (RAPD) is fragments of high GC content
(Allison, 2007) between 50-65% designed to amplify genomic region of high
polymorphism. SSRs are found within regions of high polymorphic variation. A
preliminary screening was performed with DNA from several individuals from the
studied populations to allow for the identification of RAPD primers that yielded distinct,
well-separated and reproducible bands. These bands were subsequently chosen for final
analyses. Band repeatability for each primer was confirmed by duplicate PCRs with DNA
from a subset of each population sampled. Forty RAPDs were used to target regions that
may contain SSRs. Repeating PCR product size was desired from multiple samples
before presiding to cloning and sequencing.
3.3.1 Introduction: Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) (Allison, 2007) is a technique to directly
amplify target regions of a genome. A primer anneals and flanks the desired region
followed by repeated polymerase enzymatic cycles. These repetitive cycles produce
(amplification) multiply copies of the desired gene. The PCR product of desire gene can
17
be concentrated and quantified using gel electrophoresis for downstream application such
as cloning.
3.3.2 RAPDs: PCR Protocol
PCR was carried out conducted using a MJ Research PTC 200 or a Bio-Rad
Tetrad 2 peltier thermal cycle. Each 12.5µl reaction consisted of 100-200 ng of DNA,
6.5 µl of Qiagen Taq PCR Master Mix (2.5 units of Taq DNA Polymerase, 1 X Qiagen
PCR buffer, 200 µM of each dNTP, 1.5 µM of mM MgCl2), 2 µl of each primer (0.2
µM) and 1-3 µl of DNase free water. The following PCR program was conducted:
initialized at 95°C for 2-5 minute, 30-35 cycles of denatured for 30 seconds at 95°C,
annealing at either 39.5°C or 43.6°C, extension at 72°C for 1 minute and final extension
for 10 minutes at 72°C and storage at 4°C. The follow samples were tested.
3.3.3 RAPDs: Electrophoresis and Band Extraction
Identified products were subjected to electrophoresis on a 2% agarose, 1x TAE
(Tris-Acetate Electrophoresis) buffer gel for 90 minutes at 80 volts. DNA bands were
stained with ethidium bromide and visualized under UV light. One-hundred base-pair
marker was use to determine base size. Desired products were cut from the gel using a
dark light eliminator and DNA was extracted use a PureLink Quick DNA Gel Extraction
Kit (Life Technologies, USA). The gel bands were dissolved in solvent release DNA
fragments. The solution was added to a silica spin column. DNA was adhered to the spin
column, washed to remove containments, and eluted in a TE buffer. Selected products
were cloned via chemically competent E.coli transformation.
18
DNA Gel Extraction Procedure
Gel bands were excised from gel using a scapula, minimizing excess agarose.
Band was when weighed to ensure a 3:1 ratio of Gel Solubilization Buffer to DNA band
in a 1.6 ml polypropylene microcentrifuge tube. Gel was incubated at 50°C for 10
minutes and remained in a dry block for additional 5 minutes. After dissolving, content
was transferred to extraction column/tube apparatus and centrifuged for 1 minute at
>12000 x g. Filter column was washed with 500 µl of wash buffer, containing ethanol,
and centrifuged again. Residual wash buffer was removed by centrifuging at >1200 x g
for an addition 1-2 minutes. DNA was eluted through incubation at room temperature for
1 min with 50 µl of Elution Buffer; followed by recovery centrifugation at > 12000 x g
for 1 min. Expected DNA recovery was between 85% - 95% of original concentration.
3.4. Bacteria Transformation and Cloning
3.4.1 Cloning Introduction
The TOPO TA Cloning Kit (Life Technologies, USA) was used for direct insert
of PCR products into pCR 4-TOPO plasmid vector. Plasmid used has a single 3’
deoxthymidine (T) residues allowing for efficient bind to the 5’ poly- A tails added with
use of Taq polymerase.
TOPO cloning kit exploits the enzymatic reaction of
topoisomerase I from Vaccinia virus. The DNA phosphodiester backbone is cleaved after
5’ –CCCTT by topoisomerase I in one strand; energy release is conserved through the
formation of covalent bond between 3’ phosphate and tyrosyl residual of Topisomerase I.
Five prime hydroxyl of the original stand ultimately attaches to the phopho-tyrosyl bond
reversing the reaction and releasing the topoisomerase (Life Technologies, USA). Vector
19
pCR 4-TOPO also contains the lethal ccdB gene allowing for direct positive screening.
The ccdB gene fuses directly to the c-terminus of the LacZα fragment. Only the ligation
of a PCR fragment disrupts the expression of the lacZα -ccdB gene permitting only
positive recombinants to be transformed into chemically competent E. coli cells (Life
Technologies, USA). Figure 1 depicts plasmid structure and recognition sites.
20
Figure 1. Map of pCR 4-TOPO. The sequence and restrictions sites are labeled to
indicate actual cleavage sites (Life Technologies, Inc.)
________________________________________________________________________
__________________________________________________________________
21
3.4.2 LB + Ampicillin Media Plates
Nutrient rich LB+ ampicillin plates were prepared before 2 days before
transformation to allow for any contaminated plates to be eliminated. Standard LB broth
was prepared with 10 grams of bactro- tryptone, 5 grams of bactro- yeast extract and 10
grams of NaCl. Contents were heat dissolved in 750 ml of ddH2O, pH adjusted to 7.5
with sufficient 1 M NaOH. Final solution volume was brought to 1 liter. Lastly, 15 grams
of bacro- agar was added. LB broth was autoclaved at 121°C for sterilization for
minimum of 35 minutes. After sterilization, broth was allowed to cool to 50°C before
added 100 mg of ampicillin dissolved in 2 ml of ddH2O; stock ampicillin concentration of
50 mg/ml. Plates were poured immediately in a sterilized hood; after solidification, plates
were sealed, bagged and stored at 4°C for 48 hours or until the night before use. Plates
were heated at 37°C overnight before use to ensure (1) no contaminate growth occurs and
(2) pre-warm the plates to encourage colony growth prior to spreading transformed E.
coli.
3.4.3 TOPO Cloning Reaction Procedure
Fresh PCR product (4 µl) was gently mix with 1 µl of salt solution (1.2 m NaCl;
0.06 M MgCl2) and 1 µl of TOPO vector; incubated for 5 minutes at room temperature.
The reaction was placed on ice for 5-30 minutes until ready of transformation into
competent cells.
22
3.4.4 Chemical Transformation Procedure
In each vial of One Shot chemically competent E coli, 2 µl of cloning reaction
was added, mix gently, and incubate for 5 – 30 minutes on ice. Cells were heat shocked
for 30 seconds at 42°C using a dry heat block; then immediate transfer back onto ice.
While remaining on ice, 250 µl of room temperature S.O.C. medium was added to each
vial. Vials were shaken (200 rpm) horizontally at 37°C for one hour. Two volumes, 20 µl
and 50 µl, of the each transformation were plated on the pre-warm ampicillin treated LB
plates and allowed to incubate for 48 hours. Positive clones were selected for bacteria
PCR analyzes. Figure 2 describes a complete layout of cloning procedure.
23
Figure 2. Flow chart of experimental steps of cloning (Life Technologies, USA).
_______________________________________________________________________
3.4.5. Analysis of Transformants by Bacteria PCR
Positive single colonies were selected for bacteria PCR analyzer. Sterile
toothpicks were gently dabbed into a colony and mixed into 0.2 µl tube with 6.25 µl of
Taq master mix, 1 µl of appropriate RAPD primer, and 5.25 µl DNA grade water. The
reactions were incubate at 94°C for 10 minutes to lysed the cells; followed by
amplification for 20-30 cycles and final extension for 10 minutes at 72°C. PCR products
were quantified using the Tape Station. Positive clones with the target gene were selected
for plasmid DNA extraction and sequencing.
24
3.4.6. Plasmid DNA Extraction form E. coli
Each selected clone was grew overnight in Innova 44 Incubator Shaker. Five
milliliters of ampicillin treated of sterile LB broth was placed in a 50 mL conical tube
along with the selected colony; colonies were incubated at 37°C and shaken at 200 rpm to
encourage growth. Cells were harvested by centrifuging 1 mL of the overnight LB culture
in a 1.6 µl micro-centrifugation tube. Gathered pellet was processed using the PureLink
Quick Plasmid Miniprep Kit (Life Technologies, USA).
Harvested pellet was resuspended with 250 µl of buffer R3 ( 50mM Tris- HCL,
pH 8.0; 10 mM EDTA) with RNase A (20 mg/mL in R3 buffer); mixed by pipeting until
homogeneous. Lysis buffer (250 µl; 200mMNaOH, 1% w/v SDS) was added add gently
mix by inverting; incubated at room temperature for 5 minutes. Proteins were precipitated
with 250 µl of Precipitation Buffer, added to each tube; mix vigorously by inversion and
centrifuge at > 12,000 x g for 10 minutes. Supernatant was then loaded onto a spin
column / wash tube apparatus and centrifuged at 12,000 x g for l minute. Plasmid DNA
bound to the spin column was then washed with 700 µl of 70% ethanol washing buffer
and centrifuge at 12,000 x g for 1 minute; washing was repeated and followed by a dry
centrifugation of 12,000 x g for l minute to remove residual ethanol. Plasmid DNA was
finally eluted from the spin column into a clean 1.6 mL tube with 50-75 µl of preheated
TE buffer. TE buffer was incubated at the center of the spin column at room temperature
for 1 minute and centrifuge at 12,000 x g for 2 minutes. Plasmid DNA recovered was
stored at -20°C to 4°C until sequencing.
25
3.5. Sequencing of Plasmid DNA
3.5.1. Sequencing Platform and Chemistry Introduction
Sequence was conducted on the ABI 3100 Genetic Analyzer. Samples were
prepared with the Big Dye Terminator v3.1 Standard kit. Big Dye chemistry works by
incorporating signature fluorescent (A, T, C, G) nucleotides into the amplification of the
target gene’s PCR products. As a fluorescent nucleotide is incorporated, the extension of
the primer stops; allowing for a single terminal nucleotide per fragment. This
incorporated continues until the target gene sequence is represented by a multiple of
single fluorescent nucleotide fragments.
________________________________________________________________________
________________________________________________________________________
Figure 3. Life Technology visual depiction of the incorporation of terminating
fluorescent nucleotides.
Nucleotides fluoresce during capillary (16 capillary/ 50 cm) electrophoresis at a
distinct wavelength. This flexible chemistry allows for reads up to 850 bps. The result is
visualization of the DNA sequencing in a chromatogram. See (Figure 4).
26
_______________________________________________________________________
Figure 4. Chromatogram of successfully sequencing of short PCR products using
BigDye Terminator chemistry; sample run of ABI 3100 Genetic Analyzer using
POP-6 Polymer (Life Technologies USA).
3.5.2. Sequencing PCR Reaction
Reaction volumes of 20 µl consisted of 8 µl of master mix, 3 ng of plasmid DNA,
3.2 pmol of M13 primer, and de-ionized water. Cycle sequencing occurred using a
thermal cycler. After mixing, reactions were denatured at 95°C for 5 minutes; followed
by 50-70 cycles of 95°C for 30 sec, annealing at 50-55 °C for 10 seconds, and extension
for 4 minutes at 60°C. PCR products were held at 4°C until purification (Life
Technologies USA).
3.5.3. PCR Purification
The ethanol-EDTA purification method was used to precipitate PCR products
within a 96 well reaction plate. EDTA (125 µl, 125mM) and 60 µl of 100% were added
to the bottom of each well. The reaction plate was sealed with aluminum tape and mixed
thoroughly by inverting approximately four times. After incubated at room temperature
for 15 minutes, the plate was centrifuged at 3000 x g for 30 minutes. The reaction plate
was immediately inverted and centrifuged at 185 x g for 1 minute to remove all buffer.
27
Reactions were washed in 60 µl of 70% ethanol and spun at 4°C and 1600 x g for 15
minutes. Plate was immediately inverted again and spun at 185 x g for 1 min. Purified
reactions were re-suspended in 10 µl of formamide, cover with aluminum foil to protect
from light and stored at 4°C (Life Technologies USA).
3.5.4. Sequencing Parameters
The ABI 3100 Analyzer was standardized with BigDye Terminator v3.1 Matrix
Standard kit. A multi-component matrix was required to normalize the data collection
software to the four different colored fluorescent dyes labeling DNA fragments in a
single capillary (Life Technologies USA). Matrix Standard (5 µl) was mixed with 195 µl
formamide; 1:40 dilution. Reaction denature at 95 °C for 2 minutes. Reaction mixture
was used to run a spectral calibration for the dye set Z; A-green, C-blue, G-yellow and Tred. A spatial analysis was also conducted for each capillary array. Spatial test checks for
even light penetration and absorbency through each of the 16 capillaries (Life
Technologies USA).
The Run 3100 Data Collection v2.0 software allows for the labeling and sequence
run parameters to be set using the Plate Manger application. Samples identification was
paralleled with well position, dye set, sequence protocol, and data storage location. After
designing plate parameters, reaction plate was linked to run manger until sequencing was
completed. Data was formatted, viewed and analyzed using Sequencing Analysis 5.1.1
software. Sequences results were BLAST to determine whether or not they were from
know origins or presence unknown and potentially new microsatellites.
28
3.6. Population Genetic Analysis with Microsatellites
3.6.1. Fragment Analysis Platform and Chemistry Introduction
Fragment Analysis chemistry works by comparing the known signature standard
dye marker (ROX) fragments peaks size to the amplified target gene’s PCR products.
This comparison allows for the determination of fragment size and genetic variation of
alleles. Target loci are distinguished by fluorescent dyes colors: 6-FAM is blue, HEX is
green, and NED is yellow. Labels fluoresce during capillary (16 capillary/ 50 cm)
electrophoresis, as they pass in front of a laser. The fluorescence is captured by the
charge-capture digital camera (CDC). The result is visualized on an electropherogram.
For each fragment a single peak represents the relative dye concentration, used as a label,
against time of exposure. Resolution, Rs, of two peaks in an electropherogram is defined
as: Rs =
(Life Technologies, 2010).
Fragment analysis was conducted on the ABI 3100 Genetic Analyzer. PCR
products were prepared with the ROX 400 or 500 Dye Standard; 50 µl of Standard with
1000 µl of formamide.
ROX Dye Standards contain florescent fragments of sizes
ranging from 40- 400 or 500 bps. Unlike sequencing, fluorescent primers are used to tag
desired loci. The diluted PCR product (1 µl) was then added to the dye standard; heated
at 95°C for 5 minutes, then immediately returned to ice for at least 5 minutes. The dye
standard serves as a genetic marker within each well, determining the base pair size for
each fragment. Each analysis reaction contained 1 µl of dilution PCR product and 6-8 µl
of dye standard solution.
29
3.6.2. Fluorescent Primers and PCR
A total of ten microsatellite primers were using for Spotted Salamanders
(Ambystoma maculatum) isolated by Wieczorek et al. (2002). The seven polymorphic
microsatellites isolated by Croshaw and Glenn 2003 for Red Newts (Notophthalmus
viridescens) were also used. Primers were synthesized by Operon, Inc. with either a HEX
or 6-FAM labeled forward primer 5’ end.
Sample size ranged from five to eight
depending on pool site and species tissue availability. Each reaction contained 20-100 ng
of DNA, 6.25 µl of Taq Qiagen Master Mix, 2 µl of 100 mM primer and DNA grade
water. Thermal cycles were conducted according to the authors’ recommendations using
Bio-Rad Tetrad 2 Cycler. Wieczorek et al. (2002) loci were amplified under the
following conditions: 5 minutes of denaturation at 95°C; 35 cycles of 30 seconds at 95°C,
30 seconds at specific annealing temperature, 1 min at 72°C; and final extension for 10
minutes at 72°C (Table 1). Croshaw & Glenn (2003) loci for red newts were amplified
under a touchdown program with two annealing temperature groups (either 60-50°C or
55-45°C); additional modifications of a denaturation and final extension period were
added, program as follows: 3 minutes of denaturation at 95°C; 5 cycles of 96°C for 20
seconds, 30 seconds at the highest annealing temperature (either 60° or 55°C), and 72 °C
for 1 minute; 21 cycles of 96 °C for 30 seconds, highest annealing temperature (either
60° or 55°C), minus 0.5 °C each cycle for 30 seconds, and 72 °C for 1 minute; lastly 10
cycles of 96°C for 30 seconds, the lower annealing temperature (either 50° or 45°C), for
30 seconds, and 72°C for 1 minutes; and final extension of 72°C for 3 minutes. PCR
product purification is not required for fragment analysis. However, products must be
diluted in DNA grade water to minimize background under analysis. PCR products were
30
diluted to a 1:20 ratio; 1 µl of product mix with 20 µl of DNA grade water for each
reaction.
Table 1. SSR Spotted Salamander Primer Set.
Wieczorek et al. (2002).
Table 2. SSR Eastern Red Newt Primer Set.
Primer
Primer Sequence
5' - 3'
Dye
GenBank
Number
Touchdown
Temp.
Nvi2F
AGC CAC TTG
TAA GAA TTG T
CCA TCA CAC
ACG TTA TTT
TGC CTT GCT
GTG ATT C
GGA CAT TCA
AGC TCA CAT
ACT
GGG AGA GAG
GAA TAG AC
ATG GTA TTG
TGA TTA CTC
TAT
HEX
AY29145
2
60
Nvi2R
Nvi7 F
Nvi7 R
Nvi11 F
Nvi11 R
6-FAM
HEX
AY29145
4
AY29145
5
31
60
60
Size of
Range
(bps)
176194
127157
178214
No. of
Alleles
HO
HE
6
0.38
0.33
0.88
0.77
0.5
0.62
0.63
0.75
0.88
0.85
0.75
0.81
8
8
(Continued). Table 2. SSR Eastern Red Newt Primer Set.
Primer
Primer Sequence
5' - 3'
Nvi14 F
Nvi14 R
Nvi18 F
Nvi18 R
Nvi19 F
Nvi19 R
Nvi24 F
Nvi24 R
AAG GTC ATC
TAA CAA AAG
AGT
ACA GCA TGG
CAC AGT AT
TAT GGA GTC
CTT TGT ATT
TTT TTC AGG CTT
CAT C
TGT CAC CCA
CTT CAG TA
GTG GCG ACT
TGT ATG T
CCT CCA TGT
TCT CTC ATA
CTC ATT CCA
ACA CTT AAC
TAT
Dye
6FAM
HEX
6FAM
HEX
GenBank
Number
Touchdown
Temp.
Size of
Range
(bps)
No. of
Alleles
HO
HE
AY291456
60
284-308
6
0.63
0.78
0.38
0.76
0.5
0.87
0.38
0.88
0.75
0.83
0.88
0.86
0.38
0.81
0.25
0.9
AY291457
AY291458
AY291459
60
60
60
146-190
163-201
100-163
10
8
10
Croshaw & Glenn (2003).
3.6.2. Fragment Analysis Parameters
The ABI 3100 Analyzer was standardized with Multi-Capillary DS-30 Matrix
Standard Kit. A multi-component matrix was required to normalize the data collection
software to the four different colored fluorescent dyes (6-FAM, Hex, NED and ROX
dyes) labeling DNA fragments in a single capillary. Matrix Standard (5 µl) was mixed
with 195 µl formamide; 1:40 dilution. Reaction denature at 95 °C for 5 minutes. Reaction
mixture was used to run a spectral calibration for the DS-30 dye set D; green, blue, red
(ROX standard), and yellow. A spatial analysis was also conducted for each capillary
32
array. Spatial test checks for even light penetration and absorbency through each of the
16 capillaries.
The Run 3100 Data Collection v2.0 Software allows for the labeling and fragment
analysis run parameters to be set using the Plate Manger application. Samples’
identification was paralleled with well position, dye set, sequence protocol, and data
storage location. After designing plate parameters, reaction plate was linked to run
manger until sequencing was completed.
3.6.3. Fragment Analysis Parameters
Data were formatted and analyzed using the GeneMapper Software v3.5.
GeneMapper Software is designed to analyze the data generated using several fragment
analysis chemistry kits on the ABI PRISM® 3100 Genetic Analyzer. Run results were
exported in a .csv format and later viewed with Excel for data cleaning and formatting.
CONVERT software (Glaubitz, 2004) was used to convert the cleaned data into a useable
format for heterozygosity analysis using GENEPOP (Raymond & Rousset, 1995).
CONVERT program is designed to transfer codominant, diploid genotypic data outputs
form common genetic software packages. Excel files of clean codominant marker data
were used as an input file for conversion into GENEPOP format (Glaubitz, 2004).
GENEPOP software package computes exact tests: for Hardy-Weinberg equilibrium,
population differentiation, classical Fis, allele frequencies and allele size-based statistics
(Raymond & Rousset, 1995). To determine bottleneck presence in each population,
BOTTLENECK program was used. Bottleneck is a program for detecting recent effective
population size reductions from allele data frequencies (Piry et al., 1999).
33
CHAPTER 4
RESULTS
4.1. Genomic Deoxyribonucleic Acid Quantification
Approximately 230 tissue samples were collected; of which, total genomic DNA was
extracted from 107 samples. DNA samples were allocated towards SSR identification and
isolation. Following isolation, DNA samples were quantified and qualified for downstream
application using the NanoDrop Spectrophotometer and assigned a distinctive code. The
following table displaces all genomic DNA samples with a 260/280 value of between 1.7- 2.0,
indicating purity of sample.
Table 3. One hundred twenty-two DNA samples with 260/280 ratios between 1.7
and 1.8.
Sample
ID
B10SS1
B10SS2
B1ES3
B1ES4
B1ES5
B1ES7
B1ES8
B1ES9
B1SS2
B2GF1
Date
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
8/28/2012
Time
6:59 PM
6:37 PM
6:52 PM
6:53 PM
6:54 PM
6:56 PM
6:58 PM
6:36 PM
6:46 PM
10:22 AM
ng/ul
284.04
108.32
47.78
48.1
61.26
38.46
32.05
25.42
43.68
68.29
34
A260
5.681
2.166
0.956
0.962
1.225
0.769
0.641
0.508
0.874
1.366
A280
3.027
1.159
0.522
0.562
0.684
0.403
0.34
0.27
0.474
0.703
260/280
1.88
1.87
1.83
1.71
1.79
1.91
1.89
1.88
1.84
1.94
(Continued) Table 3. One hundred
between 1.7 and 1.8.
Sample
ID
Date
Time
B2GF2
8/28/2012
10:24 AM
B2LF15 8/28/2012
4:50 PM
B2LF15 8/28/2012
4:51 PM
B2LF22 8/28/2012
5:00 PM
B2LF24 8/28/2012
5:51 PM
B2LF29 8/28/2012
5:57 PM
B2LF30 8/28/2012
5:58 PM
B2LF31 8/28/2012
6:10 PM
B2RN10 8/28/2012
10:39 AM
B2RN11 8/28/2012
10:40 AM
B2RN20 8/28/2012
4:57 PM
B2RN21 8/28/2012
4:58 PM
B2RN32 8/28/2012
6:11 PM
B2RN4
8/28/2012
10:27 AM
B2RN6
8/28/2012
10:33 AM
B2RN7
8/28/2012
10:34 AM
B2RN8
8/28/2012
10:35 AM
B2SS14 8/28/2012
4:48 PM
B2SS16 8/28/2012
4:52 PM
B2SS17 8/28/2012
4:53 PM
B2SS18 8/28/2012
4:54 PM
B2SS28 8/28/2012
5:56 PM
B2SS34 8/28/2012
6:39 PM
B3MB10 8/28/2012
6:28 PM
B3MB5 8/28/2012
6:19 PM
B3MB5 8/28/2012
6:21 PM
B3MB8 8/28/2012
6:25 PM
B3MB9 8/28/2012
6:26 PM
B3RN1
8/28/2012
6:15 PM
B3RN1
8/28/2012
6:14 PM
B3RN2
8/28/2012
6:16 PM
B3RN3
8/28/2012
6:17 PM
B3RN7
8/28/2012
6:23 PM
B4SS11 8/28/2012
11:04 AM
B4SS12 8/28/2012
4:41 PM
twenty-two DNA samples with 260/280 ratios
ng/ul
65.51
21.51
20.95
12.71
22.41
65.81
25.6
301.51
43.47
53.22
50.22
109.33
110.66
95.52
114.18
90.13
251.06
344.57
6.01
430.21
201.27
46.77
114.22
29.05
1556.63
688.66
49.13
21.06
157.63
146.4
208.66
56.94
214.72
53.53
35.5
35
A260
1.31
0.43
0.419
0.254
0.448
1.316
0.512
6.03
0.869
1.064
1.004
2.187
2.213
1.91
2.284
1.803
5.021
6.891
0.12
8.604
4.025
0.935
2.284
0.581
31.133
13.773
0.983
0.421
3.153
2.928
4.173
1.139
4.294
1.071
0.71
A280
0.679
0.222
0.224
0.13
0.251
0.688
0.278
3.227
0.446
0.559
0.53
1.131
1.255
0.965
1.178
0.927
2.615
3.754
0.062
4.775
2.207
0.514
1.186
0.332
16.872
7.438
0.54
0.246
1.809
1.722
2.284
0.646
2.293
0.568
0.367
260/280
1.93
1.93
1.87
1.95
1.78
1.91
1.84
1.87
1.95
1.9
1.89
1.93
1.76
1.98
1.94
1.95
1.92
1.84
1.93
1.8
1.82
1.82
1.93
1.75
1.85
1.85
1.82
1.72
1.74
1.7
1.83
1.76
1.87
1.88
1.93
(Continued) Table 3. One hundred
between 1.7 and 1.8.
Sample
ID
Date
Time
B4SS15 8/28/2012
4:46 PM
B4SS16 8/28/2012
5:02 PM
B4SS18 8/28/2012
5:04 PM
B4SS30 8/28/2012
6:00 PM
B4SS31 8/28/2012
6:02 PM
B4SS33 8/28/2012
6:05 PM
B4SS35 8/28/2012
6:07 PM
B4SS5
8/28/2012
10:55 AM
B6MB2 8/28/2012
5:05 PM
B6SS1
8/28/2012
4:42 PM
B6SS10 8/28/2012
5:15 PM
B6SS11 8/28/2012
5:17 PM
B6SS12 8/28/2012
5:18 PM
B6SS13 8/28/2012
5:21 PM
B6SS14 8/28/2012
5:22 PM
B6SS15 8/28/2012
5:25 PM
B6SS16 8/28/2012
5:29 PM
B6SS17 8/28/2012
5:31 PM
B6SS18 8/28/2012
5:32 PM
B6SS19 8/28/2012
5:33 PM
B6SS20 8/28/2012
5:35 PM
B6SS21 8/28/2012
5:36 PM
B6SS22 8/28/2012
5:38 PM
B6SS23 8/28/2012
5:39 PM
B6SS24 8/28/2012
5:41 PM
B6SS25 8/28/2012
5:43 PM
B6SS26 8/28/2012
5:44 PM
B6SS27 8/28/2012
5:45 PM
B6SS28 8/28/2012
5:47 PM
B6SS29 8/28/2012
5:48 PM
B6SS30 8/28/2012
7:00 PM
twenty-two DNA samples with 260/280 ratios
ng/ul
11.25
12.41
22.52
13.55
22.83
27.78
27.26
9.23
38.97
15.79
13.63
37.81
123.12
95.97
266.05
55.7
195.79
50.37
79.74
339.59
315.3
171.44
242.4
262.87
347.5
146.98
289.22
349.33
276.28
244.12
360.63
36
A260
0.225
0.248
0.45
0.271
0.457
0.556
0.545
0.185
0.779
0.316
0.273
0.756
2.462
1.919
5.321
1.114
3.916
1.007
1.595
6.792
6.306
3.429
4.848
5.257
6.95
2.94
5.784
6.987
5.526
4.882
7.213
A280
0.12
0.142
0.235
0.142
0.238
0.29
0.289
0.094
0.439
0.177
0.146
0.43
1.419
0.999
3.035
0.632
2.145
0.539
0.891
3.648
3.655
1.826
2.609
2.857
3.763
1.554
3.157
3.851
2.937
2.614
3.878
260/280
1.87
1.75
1.91
1.91
1.92
1.92
1.89
1.96
1.78
1.79
1.87
1.76
1.73
1.92
1.75
1.76
1.83
1.87
1.79
1.86
1.73
1.88
1.86
1.84
1.85
1.89
1.83
1.81
1.88
1.87
1.86
(Continued) Table 3. One hundred twenty-two DNA samples with 260/280 ratios
between 1.7 and 1.8.
Sample
ID
Date
Time
ng/ul
A260
A280
260/280
B6SS4
8/28/2012
5:08 PM
24.38
0.488
0.279
1.75
B6SS5
8/28/2012
5:09 PM
133.96 2.679
1.441
1.86
B6SS6
8/28/2012
5:10 PM
38
0.76
0.411
1.85
B6SS7
8/28/2012
5:11 PM
162.72 3.254
1.873
1.74
B6SS8
8/28/2012
5:13 PM
137.4
2.748
1.486
1.85
B6SS9
8/28/2012
5:14 PM
303.09 6.062
3.225
1.88
B8SS1
8/28/2012
6:41 PM
153.39 3.068
1.669
1.84
B8SS2
8/28/2012
6:42 PM
183.22 3.664
1.925
1.9
B9SS1
8/28/2012
6:38 PM
29.93
0.599
0.315
1.9
___________________________________________________________________
In each label: first two spaces represent site name, middle two letters represent
common name species, last digits are order of quantification. Note the tables include
all samples and species extracted; however three species were targeted for SSR
primer development.
Purity and quality of genomic DNA were verified by gel electrophoresis. Samples
were run on a 1 x TBE 1% agarose gel. Below in figure 5, 60 genomic samples were
visualized. A one kb genetic marker was used to visualize the percentage of genomic
DNA above 2,000 bps. Shearing of DNA was also visible in some samples as a sign of
contamination and degradation. Samples with contamination or significant shearing were
removed from the study.
37
________________________________________________________________________
Figure 5. Gel electrophoresis of seventy one genomic DNA samples on a ethidium
bromide stained 1% agarose 1 x TBE gel; 80 voltages for 2.5 hours. Samples
represented are from study species; in numerical order.
4.2. Random Amplified Polymorphic DNA
Random Amplified Polymorphic DNA (RAPDs) primers are being used to isolate highly
polymorphic fragments of the conservative regions of the genome, pre species. Initial PCR was
conducted on twelve DNA samples for each of the 40 RAPD primers. PCR trials produced
adjusted annealing temperatures and primer concentrations that will be use for the remaining
DNA samples. In Table (7 & 8) below, highlighted samples were amplified with RAPD
primers. After gel electrophoresis, products proceeded to cloning.
38
Table 4. Eastern Red Newt DNA samples used for RAPD analysis are highlighted.
Sample ID
B2RN13
B2RN12
B2RN4
B2RN10
B2RN7
B2RN6
B2RN21
B2RN8
ng/ul
46.87
40.4
95.52
43.47
90.13
114.18
109.33
251.06
260/280
2.23
2.12
1.98
1.95
1.95
1.94
1.93
1.92
Sample ID
B2RN11
B2RN20
B3RN7
B3RN2
B2RN32
B3RN3
B3RN1
B3RN1
ng/ul
53.22
50.22
214.72
208.66
110.66
56.94
157.63
146.4
Table 5. : Spotted Salamander DNA samples used for RAPD analysis are
highlighted.
Sample ID
B10SS1
B10SS2
B2SS14
B2SS17
B2SS18
B2SS34
B4SS14
B6SS12
B6SS13
B6SS13
B6SS14
B6SS16
B6SS19
B6SS20
B6SS21
ng/ul
284.04
108.32
344.57
430.21
201.27
114.22
95.54
123.12
91.22
95.97
266.05
195.79
339.59
315.3
171.44
260/280
1.88
1.87
1.84
1.8
1.82
1.93
2.01
1.73
2.03
1.92
1.75
1.83
1.86
1.73
1.88
Sample ID
B6SS22
B6SS23
B6SS24
B6SS25
B6SS26
B6SS27
B6SS28
B6SS29
B6SS30
B6SS5
B6SS7
B6SS8
B6SS9
B8SS1
B8SS2
ng/ul
242.4
262.87
347.5
146.98
289.22
349.33
276.28
244.12
360.63
133.96
162.72
137.4
303.09
153.39
183.22
260/280
1.86
1.84
1.85
1.89
1.83
1.81
1.88
1.87
1.86
1.86
1.74
1.85
1.88
1.84
1.9
A number of primers produced distinctive polymorphic regions, visualize on a 2%
agarose 1X TBE buffer gel under UV light. Six primers yielded tight reproducible bands
for Red-Spotted Newt, 9 primers for Spotted Salamander. The results for Spotted
Salamanders and Red Newts are listed in Table 7 and 8 prospectively. In these tables
39
RAPD primers are listed by their working identification name. The specific sequence and
catalog numbers corresponding to each working name are listed in Table 9 below.
Table 6. Random Amplified Polymorphic DNA Kit, US Biological
Random Amplified Polymorphic DNA Kit, BioAssay: Lot No. L10041901 &
L12102655
GC
Content
TA: 39.5°C Primers
Catalog no.
Sequence
%
TM
Primer Working Name
1
R1125-01-3
AGT CAG CCA C 60%
39.5 C
2
R1125-01-4
AAT CGG GCT G 60%
39.5 C
3
R1125-01-5
AGG GCT CTT G 60%
39.5 C
4
R1125-01-7
GAA ACG GGT G 60%
39.5 C
5
R1125-01-8
GTG ACG TAG G 60%
39.5 C
6
R1125-01-10
GTG ATC GCA G 60%
39.5 C
7
R1125-01-11
CAA TCG CCG T 60%
39.5 C
8
R1125-01-12
TCG GCG ATA G 60%
39.5 C
9
R1125-01-14
TCT GTG CTG G
60%
39.5 C
10
R1125-01-15
TTC CGA ACC C
60%
39.5 C
11
R1125-01-16
AGC CAG CGA A 60%
39.5 C
12
R1125-01-17
GAC CGC TTG T
60%
39.5 C
13
R1125-01-18
AGG TGA CCG T 60%
39.5 C
14
R1125-01-19
CAA ACG TCG G 60%
39.5 C
15
R1125-01-20
GTT GCG ATC C
60%
39.5 C
16
R1125-02-01
GTT TCG CTC C
60%
39.5 C
17
R1125-02-02
TGA TCC CTG G
60%
39.5 C
18
R1125-02-04
GGA CTG GAG T 60%
39.5 C
40
(continued) Table 6. Random Amplified Polymorphic DNA Kit, US Biological
Random Amplified Polymorphic DNA Kit, BioAssay: Lot No. L10041901 &
L12102655
GC
Content
TA: 39.5°C Primers Catalog no.
Sequence
%
TM
Primer Working Name
19
R1125-02-11
GTA GAC CCG T
60%
39.5 C
20
R1125-02-12
CCT TGA CGC A
60%
39.5 C
21
R1125-02-15
GGA GGG TGT T
60%
39.5 C
22
R1125-02-16
TTT GCC CGG A
60%
39.5 C
23
R1125-02-17
AGG GAA CGA G 60%
39.5 C
24
R1125-02-18
CCA CAG CAG T
60%
39.5 C
25
R1125-02-20
GGA CCC TTA C
60%
39.5 C
GC
Content
TA: 42°C Primers Cat. #
Sequence
%
TM
26
R1125-01-01
CAG GCC CTT C
70%
43.6 C
27
R1125-01-02
TGC CGA GCT G
70%
43.6 C
28
R1125-01-06
GGT CCC TGA C
70%
43.6 C
29
R1125-01-09
GGG TAA CGC C 70%
43.6 C
30
R1125-01-13
CAG CAC CCA C
70%
43.6 C
31
R1125-02-03
CAT CCC CCT G
70%
43.6 C
32
R1125-02-05
TGC GCC CTT C
70%
43.6 C
33
R1125-02-06
TGC TCT GCC C
70%
43.6 C
34
R1125-02-07
GGT GAC GCA G 70%
43.6 C
35
R1125-02-08
GTC CAC ACG G
70%
43.6 C
36
R1125-02-09
TGG GGG ACT C
70%
43.6 C
37
R1125-02-10
CTG CTG GGA C
70%
43.6 C
38
R1125-02-13
TTC CCC CGC T
70%
43.6 C
39
R1125-02-14
TCC GCT CTG G
70%
43.6 C
40
R1125-02-19
ACC CCC GAA G 70%
43.6 C
41
Table 7. Amplified Spotted Salamanders Polymorphic Regions
Primer # bands
Band Size I
Size
Size
Size Comments
1
3
490
194
110
3
2
955
534
5
3
927
737
269
2 CLEAR BANDS
27
3
793
556
281
31
4
1116
796
320
95
32
33
35
38
2
7
4
3
660
739
705
1003
1080
525
565
673
307
391
304
106
366
Table 8. Amplified Eastern Red Newt Polymorphic Regions
Primer
# bands
Band Size
Size
1
3
350
600
6
2
700
800
7
2
495
600
8
2
512
800
10
1
750
19
2
350
675
21
2
21
75
26
1
166
27
1
175
118
39
1
213
VERY CLEAR
660
VERY
CLEAR
5 CLEAR BANDS
VERY CLEAR
Size
820
4.3. Bacteria Transformation and Cloning
RAPD PCR products bands were exorcized from agarose gels; then dissolved and
DNA was extracted. Fragments were inserted into vectors and transformed into e. coli; in
the presence of ampicillin for selection. The growth of white colonies indicates
successful transformation of plasmid into bacteria. A number of positive clones are
visible in Figure 6. Of the white colonies, single isolates were selected for bacteria PCR
42
to determine which fragments were inserted. Clones within fifty base pair range of the
original isolated product from RAPD PCR were selected for sequencing.
Figure 6. Transformed Bacteria Plates.
43
(continued) Figure 6. Transformed Bacteria Plates.
4.3.2. Plasmid DNA Extracted from Positive Clones
For the study species, ten individual transformation plates were selected for
sequencing. However, twenty other plates of positive colonies were sequences what were
not from the study species. Colonies were replicated overnight in 5 mL of LB broth in
the presence of ampicillin. Selected colonies and fragment insert sizes are listed below in
Table 10.
44
Table 9. Selected Clones for Sequencing from Bacteria PCR.
Final Selected Clones for Sequencing by Fragment Size
Clone ID
Colony 1
Colony 2
Colony 3
Colony 4
1SS33
2SS33
198/364
86/852
137/461
3SS5
710
57
172
787
4SS27
163/626
84
630
61/539/632
5SS5
375
605
6SS27
101
X
X
7SS27
8SS33
45
X
X
X
9SS31
10SS31
11SS3
12SS27
13SS35
14SS33
15SS33
16SS5
535
247/450
20RN27
425
427
21RN27
418
425
427
22RN6
310
563
23RN6
24RN39
4.4. Sequenced Plasmid DNA
From thirty sequenced plasmids, only 3 produced unknown sequence. However,
these sequences for another species not included in the remainder of study. All positive
clones were replicated overnight in LB broth in the presence of ampicillin; shaken at 200
rpm for aeration, over night at 37°C. A total of 2 mL of LB broth with bacteria was
suspended in sterile glycerol and stored at -80°C for later sequencing.
45
4.5. Fragment Analysis
Eastern Red Newt
Table 10. Eastern Red Newt heterozygosity and inbreeding coefficient per locus
within BaPo 2 population.
Bapo 2 Eastern Red Newt
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
Nvi11
0.25
0.9167
0.7273
Nvi14
0
1
1
Nvi18
0
0
Nvi19
0
0.6667
1
Nvi24
0.3333
0.8333
0.6
Nvi7
0
0.9048
1
Nvi2
0
0
POP(wt.)
0.087
0.7174
0.8788
The eastern red newt populations at site BaPo 2 had an average observed
heterozygosity of 8.7 %; the expected heterozygosity for this population was 71.7 %.
This population was found to be more than 90% homozygosis and 87.8 % (Fis) inbred.
Eight individuals were analyzed and 5 loci amplified.
Table 11. Eastern Red Newt heterozygosity and inbreeding coefficient per locus
within BaPo 3 population.
Bapo 3 Eastern Red Newt
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
Nvi11
0.6667
0.9833
0.322
Nvi14
0.6
0.95
0.3684
Nvi18
0
0.6667
1
Nvi19
0.3333
1
0.6667
Nvi24
0
0.8333
1
Nvi7
0
0.6667
1
Nvi2
POP(wt.)
0.32
0.866
0.6305
46
The eastern red newt populations at site BaPo 3 had an average observed
heterozygosity of 32 %; the expected heterozygosity for this population was 86.6 %.
This population was found to be more than 65% homozygosis and 63 % (Fis) inbred.
Seven individuals were analyzed and 6 loci amplified.
Table 12. Eastern Red Newt heterozygosity and inbreeding coefficient per locus
within BaPo 8 population.
BaPo 8 Eastern Red Newt
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
Nvi11
0.6
0.65
0.0769
Nvi14
Nvi18
0
1
1
Nvi19
0.2
0.5
0.6
Nvi24
0.3333
0.8333
0.6
Nvi7
Nvi2
POP(wt.)
0.3333
0.6833
0.5122
The eastern red newt populations at site BaPo 8 had an average observed
heterozygosity of 33.3 %; the expected heterozygosity for this population was 68.3 %.
This population was found to be more than 67% homozygosis and 51 % (Fis) inbred. Five
individuals were analyzed and 4 loci amplified.
Table 13. Observed Heterozygosity of Eastern Red Newt Populations by Locus.
Eastern Red Newts
Observed Heterozygosity per Population by Locus
Locus
Nvi11
Nvi14
Nvi19
Nvi24
Nvi18
Nvi7
Nvi2
B2
0.25
0
0
0.3333
0
0
0
B3
0.6667
0.6
0.3333
0
0
0
B8
0.6
0.2
0.3333
0
47
MEAN
0.50557
0.3
0.17777
0.2222
0
0
0
SD
0.22383
0.42426
0.16776
0.19243
0
0
0
Table 13 summarizes observed heterozygosity across all populations of Eastern
Red Newt by locus. Of the seven loci, only one (Nvi11) was heterozygous in all three
populations. Three loci, (Nvi18, Nvi7, Nvi2), were homozygous in each population. All
loci without a value failed to amplify within a given population.
Table 14. ANOVA of Loci Heterozygosity (Eastern Red Newt) Across and Within
Populations.
ANOVA
Source of
Variation
SS
Between
Groups
0.189729
Within
Population 0.410541
Total
df
MS
F
P-value
F crit
3 0.063243 1.078338 0.418128 4.346831
7 0.058649
0.600271
10
No significant difference (p = 0.41) was observed of heterozygosity between
populations or within populations when grouped by loci; p < 0.05. The variation from
loci is not contributing significantly to the heterozygosity variations between populations.
Spotted Salamanders
Table 15. Spotted Salamander heterozygosity and inbreeding coefficient per locus
within BaPo 6 population.
BaPo 6 Spotted Salamander
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
AMA07
0.5
0.875
0.4286
AMA112B
0.1429
0.8333
0.8286
AMA127
0
0
AMA2C2
0.25
1
0.75
AMA33
0.875
0.9643
0.0926
AMA34
0.3333
1
0.6667
AMA410
AMA51
0
0
AMA61
0.2857
0.7024
0.5932
AMA94
0.4
0.9
0.5556
POP(wt)
0.4286
0.8801
0.513
48
The spotted salamander population at site BaPo 6 had an average observed
heterozygosity of 42.9 %; the expected heterozygosity for this population was 88 %.
This population was found to be more than 57.1% homozygosis and 51.3 % (Fis) inbred.
Eight individuals were analyzed and 7 loci amplified.
Table 16. Spotted Salamander heterozygosity and inbreeding coefficient per locus
within BaPo 4 population.
BaPo 4 Spotted Salamander
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
AMA07
0.1429
0.1429
0
AMA112B
0.5
0.6964
0.2821
AMA127
0.4286
0.7143
0.4
AMA2C2
0.7143
0.8571
0.1667
AMA33
0.7143
0.8571
0.1667
AMA34
0.875
0.8214
-0.0652
AMA410
0.4286
0.7857
0.4545
AMA51
0
0.4
1
AMA61
0.75
0.7589
0.0118
AMA94
0.75
0.9464
0.2075
POP(wt)
0.5556
0.7123
0.2201
The spotted salamander population at site BaPo 4 had an average observed
heterozygosity of 55.6 %; the expected heterozygosity for this population was 71.2 %.
This population was found to be more than 44.4% homozygosis and 22.0 % (Fis) inbred.
Eight individuals were analyzed and 9 loci amplified.
Table 17. Spotted Salamander heterozygosity and inbreeding coefficient per locus
within BaPo 3 population.
BaPo 3 Spotted Salamander
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
AMA07
0.25
0.5179
0.5172
AMA112B
1
0.5
-1
AMA127
0
0.25
1
AMA2C2
0.375
0.4464
0.16
49
(Continued). Table 15. Spotted Salamander heterozygosity and inbreeding
coefficient per locus within BaPo 3 population.
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
AMA33
0.625
0.875
0.2857
AMA34
0.2857
0.5476
0.4783
AMA410
0
0.5714
1
AMA61
0.75
0.7589
0.0118
AMA94
0.75
0.9464
0.2075
POP(wt)
0.5556
0.7123
0.2201
The spotted salamander population at site BaPo 3 had an average observed
heterozygosity of 35.5 %; the expected heterozygosity for this population was 57.8 %.
This population was found to be 64.5% homozygosis and 38.7 % (Fis) inbred. Eight
individuals were analyzed and 9 loci amplified.
Table 18. Spotted Salamander heterozygosity and inbreeding coefficient per locus
within BaPo 2 population.
BaPo 2 Spotted Salamander
Locus
Ho (1-Qintra)
He (1-Qinter)
Fis (per locus)
AMA07
0.3333
0.6667
0.5
AMA112B
0.5
1
0.5
AMA127
AMA2C2
0.5
0.9583
0.4783
AMA33
0.4
1
0.6
AMA34
AMA410
0
0
AMA51
0
0
AMA61
0.25
1
0.75
AMA94
0.5
0.5
0
POP(wt)
0.36
0.7933
0.5462
The spotted salamander population at site BaPo 2 had an average observed
heterozygosity of 36 %; the expected heterozygosity for this population was 79.3 %.
50
This population was found to be 64 % homozygosis and 54.6 % (Fis) inbred. Five
individuals were analyzed and 8 loci amplified.
Table 19. Observed Heterozygosity of Spotted Salamander Populations by Locus.
Locus
AMA07
AMA112B
AMA2C2
AMA33
AMA34
AMA61
AMA94
AMA51*
AMA127
AMA410
Mean Pop.
Spotted Salamanders
Observed Heterozygosity per Population by Locus
BaPo 6
BaPo 4
BaPo 3
BaPo 2
0.5
0.1429
0.25
0.3333
0.1429
0.5
1
0.5
0.25
0.7143
0.375
0.5
0.875
0.7143
0.625
0.4
0.3333
0.875
0.2857
0.2857
0.75
0.875
0.25
0.4
0.75
0.4286
0.5
0
0
0
0
0.4286
0
0.4286
0
0
0.3097
0.5304
0.4266
0.3104
Mean
0.30655
0.53573
0.45983
0.65358
0.498
0.54018
0.51965
0
0.14287
0.14287
SD
0.15068
0.35233
0.19798
0.19819
0.32736
0.3189
0.15922
0
0.24745
0.24745
Table 19 summarizes observed heterozygosity across all populations of Spotted
Salamanders by locus. Of the ten loci, one locus (AMA51) was homozygous in all
populations of Spotted Salamander. All other loci, except AMA127 and AMA 410, were
heterozygous in at least three populations. All loci without a value failed to amplify
within a given population.
Table 20. ANOVA of Loci Heterozygosity (Spotted Salamander) Across and Within
Populations.
ANOVA
Source of
Variation
Between Groups
Within Groups
SS
0.89691
1.516364
Total
2.413274
df
MS
F
P-value
F crit
8 0.112114 1.774463 0.1322638 2.355081495
24 0.063182
32
51
No significant difference (p = 0.13) was observed of heterozygosity between
populations or within populations when grouped by loci; p < 0.05. The variation from
different loci is not contributing significantly to the heterozygosity variations between
populations.
4.6. Bottleneck Analysis
Summary of Heterozygosity and Fis Correlation
Heterozygosity is the individual of population level parameter measuring gene
diversity. Heterozygosity ranges from 0-1.0 and reflects the proportion of loci expected to
be heterozyotes. Observed heterozygosity is the calculated as the number of
heterozygotes of the total loci. Expected heterozygosity is calculated as 1 minus the sum
of squared gene frequencies. Fis, inbreeding coefficient, is the proportion of the variance
in the sub population contained in an individual. Bottleneck calculations assume
mutation-drift equilibrium to compute the distribution of gene diversity expected from
the observed number of alleles; i.e. the various molecular weights of allele. Expected
heterozygosity is computed from Hardy- Weinberg equilibrium (HWE) (He=
1) (Knaepkens et al., 2004). Populations without a recent change in size will be in
mutation-drift equilibrium where the expected heterozygosity (HEQ) will equal the
Hardy–Weinberg equilibrium heterozygosity (HE) (Knaepkens et al., 2004). A normal Lshaped distribution of allele frequency across distribution classes is expected in a normal
population. The loss of heterozygosity in the first distribution class (0-0.1) represents
gene drift and a bottleneck event (Piry et al., 1999).
52
Eastern Red Newt
Table 21. Wilcoxon Test for Heterozygosity for Eastern Red Newt Populations.
Wilcoxon Test (Loci Heterozygosity)
BaPo 2
BaPo 3
One Tail Excess
0.01563
0.01563
Two Tail Ex or Def.
0.03125
0.03125
One Tail Deficiency
1
0.99219
BaPo 8
0.03125
0.0625
1
Excess heterozygosity was significant, where p < 0.5, in all populations of Eastern
Red Newt p- values ranging from (0.01- 0.03).
Figure 7. Allele Frequency Distribution for Eastern Red Newt Populations.
In all populations of Eastern Red Newt, allele frequency (0-0.22) was lowest in
the first generation class (0-0.1) and highest in the second distribution class (0.101-0.2) in
53
BaPo 2 and BaPo 3. In the BaPo 3 population, allele frequency was the highest in the first
class and resembles an L-shape distribution. In BaPo 8, allele frequency was highest in
distribution class 0.5-0.6 and least resembles an L-shape distribution.
Table 22. Wilcoxon Test for Heterozygosity for Spotted Salamander Populations.
Wilcoxon Test
One tail He Deficiency One Tail He Excess
Two tail Ex. or Def.
BaPo 2
0.98438
0.02344
0.04688
BaPo 4
0.34766
0.6875
0.69531
BaPo 6
0.53125
0.53125
1
BaPo 3 *
Raw data error
Excess heterozygosity was significant, where p < 0.5, in only the BaPo 2
population of Spotted Salamanders; p- value 0.02. BaPo 4 and Bapo 6 p-values were not
significantly different from a normal population variation. Raw data formatting error
prevented Wilcoxon analysis for the BaPo 3 population.
54
Figure 8. Allele Frequency Distribution for Spotted Salamander Populations.
Normal L- shape distribtion of allele frequency was observed in populations BaPo
4 and BaPo 6; allele frequency was highest in the first distruction class (0-0.1). Bapo 3
allele frequency (0.207) was lower in the first class than the second distrubtion class
(0.241). BaPo 2 allele frequency (0) was lowest in the first distrubution class.
55
CHAPTER 5
DISCUSSION
In all populations of eastern red newts, observed heterozygosity was less than
expected heterozygosity. Further the results were supported by Fis values indicating
inbreeding was highly present. Excess heterozygosity was significant, where p < 0.5, in
all populations of Eastern Red Newt p-values ranging from (0.01- 0.03). These results
were consistent with the allele frequency distribution results. In all populations of Eastern
Red Newt, allele frequency (0-0.22) was lowest in the first generation class. All three
populations exhibited bottleneck events with gene drift occurring most recently in BaPo
3. In the BaPo 3 population, allele frequency was the higher (0.222) in the first class and
resembled an L-shape distribution. It is possible BaPo 3 population is recovering from a
recent bottleneck which would be further substantiated with additional study of the
migration rate of the population. Site locations are mapped below in Figure 8. In BaPo 8,
allele frequency was highest in distribution class 0.5-0.6 and least resembles an L-shape
distribution; indicating a bottleneck event occurred with a recovery time much slower
than other populations. Bapo 8 is located within meters of a dirt road and within a FS
stand with forest disturbance dating, by first year, to approximately 1960 (USFS-CISC
database). See Figure 7. What portion of the canopy cover removed and by what means
56
the disturbance was caused requires further research. Little to no migration is expected
for this site without of nearby pond sites. The semi-aquatic adults’ movement is limited;
only the eft juveniles’ dispersal movement is a migration factor in the metapopulation
genetic variation (Williams, 2009).
Figure 9. BaPo 8 Stand First Year.
The observed heterozygosity levels differ from the results of Croshaw & Glenn
(2003). However, considering sample size, these results may not be representative of the
whole populations for two reasons. The markers used were developed from another
population of the same species but not the same sub species. Additionally, the markers
57
used were designed to amplify consistently in populations of average to high expected
heterozygosity. High homozygosity was present in these populations which could
account for the over estimation of inbreeding.
Spotted Salamander
Heterozygosity of spotted salamander populations ranges from 35-55%, with
inbreed coefficient not exceeding 55%. Collectively these populations exhibited normal
range of heterozygosity. Population BaPo 4 had the highest level of heterozygosity at
55.6% and the lowest lever of inbreeding (22%) presences. However, populations BaPo 3
(0.3548) and BaPo 2 (0.36) heterozygosity levels did not exceed 40%; both populations
Fis also exceeded 50 percent. Populations BaPo 6 (0.4286) and BaPo 4 (0.5556)
heterozygosity levels were highest; allele frequency distributions were also normal Lshape. Bapo 3 allele frequency (0.207) was lower in the first class than the second
distrubtion class (0.241); representative of a bottleneck. BaPo 2 allele frequency (0) was
lowest in the first distrubution class; representatiove of the most recent bottleneck of all
populations of Spotted Salamanders.
The occurrence of a bottlenck in Spotted Salamander population is BaPo 2 is
unexpected. BaPo 2 and BaPo 6 are within migrational range of for the species. However,
BaPo 2 site is a permanent pond, located near a clearing cultivated as a wildlife food plot.
Spotted Salamanders are known to prefer vernal pools and avoid clearings during
migration. The FS stand in which BaPo 2 is located is dated, by first year, to
approximately 1993 (USFU- CISC database) compared to BaPo 6, by first year, Stand
date of approximately 1935 (USFU- CISC database). See Figures 8 and 9. Again, what
portion of the canopy cover removed and by what means the disturbance was caused
58
requires further research. It is also worth noting after AL State Highway 33 is location
between these sites.
Figure 10. Location of Sites BaPo 2 and BaPo 6.
59
Figure 11. Bankhead National Forest Study Sites Locations.
60
CHAPTER 6
CONCLUSION
This research has contributed to establishing a long-term genetic study of
amphibian populations at Bankhead NF (1) by establishing a base line of heterozygosity
to study gene drift in established populations and (2) by determined expected allele/
heterozygosity ratios. The project sample size was greatly limited because of (1) DNA
and tissue samples lost, and (2) the availability of chemicals for fragment analysis. A
considerable about of samples were lost due to power outages cause by weather.
Additional samples were lost when the sequencing platform was changed from Ion
Torrent Touch to ABI 3100; samples prepared for the Ion Torrent could not be used for
the ABI 3100 fragment analysis process. Final since ABI 3100, was not the intended
platform for analysis, this project was preformed with remaining reagents from another
project.
However, both objectives were accomplished for this project. This research has
provided better insight into distinguishing short and long term population declines; as
well as contributed to understanding why genetic diversity is important for longer
population fitness- and ultimately the survival of a species. Without specific data on the
types of forest disturbance that have occurred surrounding these study sites, no
management recommendations can be made.
61
The study concluded:
1. Establishing a base line of heterozygosity to study gene drift in established
populations.
2. In all populations of eastern red newts, observed heterozygosity was less than
expected heterozygosity.
3. Excess heterozygosity was significant, where p < 0.5, in all populations of
Eastern Red Newt p-values ranging from (0.01- 0.03).
4. Fis values indicating inbreeding was highly present in all populations of
Eastern Red Newts (RN).
5. High homozygosity was presence in RN populations could account for the
over estimation of inbreeding.
6. In all populations of Eastern Red Newt, allele frequency (0-0.22) was lowest
in the first distribution class.
7. All three populations of RN exhibited bottleneck events with gene drift
occurring most recently in BaPo 3.
8. In RN BaPo 3 population, allele frequency was the higher (0.222) in the first
class and resembled an L-shape distribution.
9. In RN population BaPo 8, allele frequency was highest in distribution class
0.5-0.6 and least resembles an L-shape distribution.
10. Heterozygosity of spotted salamander populations ranges from 35-55%, with
inbreed coefficient not exceeding 55%.
11. Collectively Spotted Salamander (SS) populations were with normal range of
heterozygosity.
62
12. SS population BaPo 4 had the highest level of heterozygosity at 55.6% and
the lowest lever of inbreeding (22%) presences.
13. SS Populations’ BaPo 3 (0.3548) and BaPo 2 (0.36) heterozygosity levels did
not exceed 40%; both populations Fis also exceeded 50 percent.
14. In Spotted Salamanders populations’ BaPo 6 (0.4286) and BaPo 4 (0.5556)
heterozygosity levels were highest; allele frequency distributions were also
normal L- shape.
15. Spotted Salamander population Bapo 3 allele frequency (0.207) was lower in
the first class than the second distrubtion class (0.241); representative of a
bottleneck.
16. Spotted Salamander population BaPo 2 allele frequency (0) was lowest in the
first distrubution class; representative of the most recent bottleneck of all
populations of Spotted Salamanders.
63
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69
VITA
Rashidah Halimah Farid, the daughter of Wali Farid and Dr. Ashanty Farid, was
born on April 10, 1985 in Abbeville, Alabama. She received her Bachelor of Science
degree in Animal Science from Tuskegee University in May 2008. She later returned to
graduate school at Alabama Agricultural and Mechanical University in May of 2011 to
begin the Master of Science degree program in conservation genetics. Miss. Farid
received her Master of Science degree in May 2014 from the department of Biological
and Environmental Sciences with a concentration in Plant and Molecular Biology.