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Master's Theses and Doctoral Dissertations
Master's Theses, and Doctoral Dissertations, and
Graduate Capstone Projects
11-1-2013
Survivorship of Ploidy-variable Unisexual
Ambystoma Salamanders across Developmental
Stages
Christina Marie Casto
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Recommended Citation
Casto, Christina Marie, "Survivorship of Ploidy-variable Unisexual Ambystoma Salamanders across Developmental Stages" (2013).
Master's Theses and Doctoral Dissertations. Paper 601.
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Survivorship of Ploidy-variable Unisexual Ambystoma Salamanders across Developmental
Stages
by
Christina Casto
Thesis
Submitted to the Department of Biology
Eastern Michigan University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
in
Ecology and Organismal Biology
Thesis Committee:
Katherine Greenwald, Ph.D., Chair
Margaret Hanes, Ph.D.
Steven Francoeur, Ph.D.
November 1, 2013
Ypsilanti, Michigan
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DEDICATION
This thesis is dedicated to my mother, father, and fiancé, Keith Teltser. I would have never been
where I am today without you.
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ACKNOWLEDGMENTS
I would like to acknowledge Dr. Susan Studlar and Dr. Donna Ford-Werntz for renewing
my love of biology (even if it was through botany) and inspiring me to go to graduate school. I
would like to thank my advisor, Dr. Katherine Greenwald, for all her help and patience and for
everything else, from teaching me the basics of DNA extractions to helping me collect samples
when my grandmother was sick to the grammatical critiques on my thesis. I would also like to thank
Dr. Maggie Hanes for her patience as a committee member and for her corrections on my thesis and
proposal. I am also very grateful for Dr. Steven Francoeur for helping me through the statistical
analyses involved and for catching my mistakes, as well as for his involvement on my committee.
I want to thank my lab mates, especially those who helped with DNA extractions and
collections in the last two years (that’s you, Jay Krystyniak, Thomas Nuttall, Kaitlyn Kono,
Olivia Scheffler, Chase Stevens, Danielle Hulvey, Katrina Nicholls, Sarah Sherburne, Justin
Straub, Natalie Colletti, Paul Anderson, and David Clipner). I would also like to thank the
graduate assistants for the cathartic moments at the Corner Brewery (and I suppose I should also
thank the Corner Brewery for allowing us to have our cathartic moments there). My friends and
editors also deserve a special thank you for their patience, time, and expertise.
I gratefully acknowledge the U of M, Edwin S. George Reserve and Dr. Earl Werner for
allowing me to use the facilities there. Mike Benard also deserves a special thank you for the
hours he stayed at the ESGR and checked traps for us when we could not. Financial support was
provided by the Greenwald Lab, as well as the Meta Hellwig and Jon Brown Research awards
given to me by the Department of Biology at Eastern Michigan University.
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Finally, I cannot forget to thank my family for their enduring support. My parents have
fostered my love of biology since I was old enough to flip over rocks in the creek by our house,
and today I get paid to do the same thing. I love you both so much and hope you are as proud of
me as I am of you. My brother, who never fails to make me laugh and cheer me up when it
seems everything I’ve worked for is going wrong. And finally, my fiancé, Keith, deserves
endless thanks because I would certainly not have looked for a graduate school in Michigan if it
hadn’t been for him, and because of that, I have had the most exciting and educational (and
admittedly, frustrating) two years of my life. Thank you, Keith, both for keeping me focused on
my final goal and for being understanding and not complaining too much about the times I made
you go out in the field with me.
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ABSTRACT: Unisexual Ambystoma produce ploidy-variable offspring that differ in
survivorship to adulthood. These populations reproduce through kleptogenesis, persisting by
"stealing" genetic material from males of compatible bisexual Ambystoma species (e.g.,
Jefferson’s Salamander A. jeffersonianum, and the Blue-Spotted Salamander A. laterale).
Kleptogenesis can result in ploidy-variable embryos within an egg mass because the female may
or may not incorporate the male ambystomatid genome. Little is known about the survivorship of
ploidy-variable individuals. In previous studies, triploid individuals are the most abundant class,
suggesting a greater mortality in high-ploidy (tetraploid and pentaploid) individuals. We assessed
the frequency of ploidy levels (determined by microsatellite analysis) across four life stages
within a single year: adults, early larvae, late larvae, and metamorphs. We found that, instead of
an abrupt change due to individuals dying at or during metamorphosis, there was a gradual
decline in tetraploids across all stages as the larvae develop into adults.
Key words: Ambystoma; Caudata; Development; Kleptogenesis; Microsatellite analysis;
Ploidy-variable; Survival; Unisexual
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TABLE OF CONTENTS
Dedication ....................................................................................................................................... ii
Acknowledgements........................................................................................................................ iii
Abstract ............................................................................................................................................v
List of Tables ................................................................................................................................ vii
List of Figures .............................................................................................................................. viii
Introduction......................................................................................................................................1
Methods............................................................................................................................................8
Results............................................................................................................................................15
Discussion ......................................................................................................................................20
Tables.............................................................................................................................................25
Figures............................................................................................................................................32
Literature Cited ..............................................................................................................................41
Appendices.....................................................................................................................................48
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LIST OF TABLES
TABLE 1. —Allele size ranges and diversity at each locus within early, late, and juvenile
stages..............................................................................................................................................26
TABLE 2. —Average ambient temperatures during life stages in 2012 and 2013.........................27
TABLE 3. —Number of individuals in each ploidy level in each life stage...................................28
TABLE 4. —Unique alleles within ponds.......................................................................................29
TABLE 5a. —2012 results from Chi-squared analyses of the independence of ploidy levels
between life stages .........................................................................................................................30
TABLE 5b. —2013 results from Chi-squared analyses of the independence of ploidy levels
between life stages .........................................................................................................................30
TABLE 5c. —2012-2013 comparison of each stage; results from Chi-squared analyses .............30
TABLE 5d. —2012-2013 comparison within individual ponds; results from Chi-squared
analyses ..........................................................................................................................................31
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LIST OF FIGURES
FIG 1. The Edwin S. George Reserve location in Michigan, satellite view, and road map ...........35
FIG 2. An example of Geneious results portraying the three loci and peaks of a triploid
individual .......................................................................................................................................36
FIG 3. Average temperature and rain events during the 2012 and 2013 breeding seasons............37
FIG 4. The percentage of samples that were tetraploid in each life stage between ponds ............38
FIG 5. Percentage of the triploid and tetraploid samples from 2012 .............................................39
FIG 6. Percentage of the triploid and tetraploid samples from 2013 .............................................40
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INTRODUCTION
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IN THE animal kingdom, thirteen families of fish, reptiles, and amphibians undergo
10
unusual reproductive modes including, but not restricted to, parthenogenesis, gynogenesis,
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hybridogenesis, and the development of ploidy-variable individuals (Mittwoch, 1978; Vrijenhoek
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et al., 1989). Parthenogenesis, or virgin birth, is defined as the creation of an embryo from a
13
female without any genetic contribution from a male (Mittwoch, 1978; Vrijenhoek, 1998).
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Gynogenesis is described as parthenogenesis induced by sperm but with the paternal genome
15
absent from the developing embryo (Schlupp, 2005). Finally, hybridogenesis is explained as a
16
maternal genome inherited clonally and an additional genome incorporated sexually to produce a
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viable, hybrid embryo (Stock et al., 2012). All three modes of reproduction may produce
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polyploid offspring: parthenogenic triploid whiptail lizards, Cnemidophorus (Lueck 1985),
19
gynogenic polyploid white sturgeon, Acipenser transmontanus (Van Eenennaam et al., 1996), and
20
the hybridogenic triploid Iberian minnow, Squallius alburnoides (Sousa-Santos et al., 2007).
21
In contrast, typical pond-breeding salamanders reproduce sexually, incorporating both
22
parents’ genetic material into the offspring. In the sexual species, a male salamander, after a brief
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courtship, will deposit several spermatophores (packets of sperm) on the bottom of the pond. If
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the female is interested, she will investigate and may eventually pick up the genetic material.
25
Offspring are produced through internal fertilization and are genetically a combination of the
26
maternal and paternal genomes (Uzzell, 1964).
27
Not all modes of reproduction can be described by variations in parthenogenesis,
28
gynogenesis, hybridogenesis, or sexual reproduction, and occasionally a new mode of
29
reproduction is designated. The entirely female, or unisexual, subpopulation of Ambystoma,
30
known as the Jefferson’s complex or more appropriately the Ambystoma complex, produces
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offspring through a mode not found to date in any other reproductively active organism (Panek,
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1978; Weller and Menzel, 1979; Splosky et al., 1992; Brodman and Krouse, 2007). Unisexual
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Ambystoma salamanders are known to pick up spermatophores produced by males of multiple
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sympatric, compatible, sexually reproducing Ambystoma species. Within a single egg mass, both
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eggs that are unreduced by mitotic division and eggs that are reduced through meiotic mechanisms
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have been found (Bogart, 1982; Bogart et al., 2007). Only inseminated eggs develop, ruling out
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asexuality and therefore parthenogenesis as the reproductive mode (Bogart et al., 1989). In genetic
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studies, it has been shown that genomes are not consistently inherited clonally throughout the
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generations, further excluding both hybridogenesis and gynogenesis as the sole reproductive
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modes (Bogart et al., 2007). In addition, genomes from Ambystoma spp. have been found in
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populations of unisexual salamanders where that bisexual species is not present (e.g. A.
42
jeffersonianum genomes present in a unisexual population outside of the A. jeffersonianum range),
43
meaning the unisexual salamanders are capable of movement to other breeding ponds and
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switching sperm donor species (e.g. using A. laterale sperm instead of A. jeffersonianum sperm to
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reproduce) (Bogart et al., 2007). Bogart et al. (2007) therefore proposed a new reproductive mode
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to represent this unique method of procreation: kleptogenesis.
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For a unisexual ambystomatid to reproduce they must use sperm from a male whose
48
genome and cytoplasm is compatible with the egg (Bogart et al., 2007; Brodman and Krouse,
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2007). Compatible males, or hosts, include five species (Ambystoma laterale, Ambystoma
50
jeffersonianum, Ambystoma barbouri, Ambystoma tigrinum, and Ambystoma texanum), all of
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which are fully sexual species with both male and female members (Bogart et al., 2009; Bi and
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Bogart, 2010). Therefore, any unisexual population is theoretically confined to an area where
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they are sympatric with one or more of these species (Ramsden, 2005). The male sperm may or
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may not be incorporated into the egg. This results in a range of ploidy-variable individuals within
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an egg mass. If the sperm is incorporated, the resulting embryo may either have an extra set of
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chromosomes or genome (ploidy elevation) or may have the paternal genome replace the
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maternal genome, resulting in the same ploidy level as the mother (genome replacement). If the
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sperm is not incorporated, the resulting offspring are produced asexually. However, in ploidy-
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elevated organisms, the offspring are produced through an endomitotic event followed by
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meiosis, resulting in offspring which could be genetically variable (Macgregor and Uzzell, 1964;
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Bogart et al., 2007).
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Positive Effects of Kleptogenesis
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Unisexuality in vertebrates typically results in short-lived populations, but the
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Ambystoma complex has been estimated to be five million years old (Bi and Bogart, 2010). In
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theory, unisexual vertebrate populations should be short lived because of several factors, one of
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which being the cost of producing males. This is the hypothesis that bisexual populations are half
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as efficient at reproducing, because both males and females are required to produce offspring. A
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unisexual population consisting of asexual females could grow at twice the rate of a sexual
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population (Maynard Smith, 1978). The Ambystoma complex overcomes the cost of males by
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using the sperm of other species (Ramsden, 2005; Bogart et al., 2007). Therefore, the population
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growth of these unisexual salamanders could theoretically far outpace that of their bisexual
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congeners under the right conditions.
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The Red Queen hypothesis further suggests there should be an untimely demise of purely
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asexual organisms and one may think that this could apply to the Ambystoma complex; however,
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with small amounts of sex, a species such as those in the Ambystoma complex should be able to
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overcome this obstacle (Van Valen, 1973; Bogart, 2003). The Red Queen hypothesis, in reference
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to Lewis Carroll’s Through the Looking Glass, states that to keep up with the evolutionary arms
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race, a parasite (in the sense of an organism which benefits from another organism), must evolve
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at a rate parallel to the host to survive. Dries (2003) related this hypothesis to the Amazon mollies
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(Poecillia formosa). In this system, gynogenetic females require sperm from sympatric males to
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produce offspring and, in doing so, must first attract a mate (Dries, 2003). Dries (2003) found that
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Amazon mollies are not red queens because the ability to attract mates results from the original
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hybridization event in this system “freezing” qualities males are attracted to. In the case of
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kleptogens, it is possible that these females are also not red queens because they may forego the
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courtship phase entirely and scavenge extra spermatophores (Bogart, 2003). This act would allow
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unisexual Ambystoma to evolve separately from the bisexual Ambystoma spp.
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The Ambystoma complex has been shown to have a broader tolerance to different habitats
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than their bisexual counterparts because they can incorporate the genomes of any compatible
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sympatric species (Bogart, 2003). Unisexual salamanders are typically the first to hatch in spring
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(Licht and Bogart, 1989), are larger at hatching (Panek, 1978), show increased growth rates
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(Licht and Bogart, 1989), have potentially increased stamina (Gerald and Greenwald, personal
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observation), and portray increased aggressiveness leading to niche partitioning (Brodman and
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Krouse, 2007). Therefore, the Ambystoma complex should, in theory, be much more fit than any
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Ambystoma species within the same habitat.
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Negative Effects of Kleptogenesis
There are many negative consequences to ploidy elevation and unisexuality in
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vertebrates. Because sperm is required to trigger reproduction, there are limitations on this
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complex similar to species that reproduce through gynogenesis. Males are rarely produced in this
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complex, so unisexual Ambystoma are forced to live within the range of compatible species
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(Ramsden, 2005). Due to the increased fitness of the Ambystoma complex displacing potential
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sperm donors, this increased fitness may lead to the ultimate demise of the salamander
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population within an area. Some populations seem to be made up entirely of unisexuals;
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however, these populations are presumably not reproducing (K. Greenwald, personal
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observation) or males are elusive but present, such as on North Bass Island where they have not
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been found but sperm has been recovered from female cloaca (Bogart, 2003).
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The high embryonic mortality in unisexual populations (as high as 83% in some
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populations) shows that there are cellular complications in these populations (Bi and Bogart,
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2010; Ramsden, 2005). It was once thought that intergenomic crossover events could result in
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nonviable offspring, but this event was shown to be rare and crossover events seem to occur
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intragenomically (e.g., between A. laterale genomes rather than between A. laterale and A.
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jeffersonianum genomes), therefore the cause of such low hatch rates in these populations is still
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unknown (Bi et al., 2008; Bogart, 2010). It is also possible that increased ploidy levels result in
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decreased cell function, such as the ability to transport oxygen throughout the body (Uzzell,
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1964), and affect the ability to undergo metamorphosis (Lowcock et al., 1991). This decrease in
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cell function could be the mechanism preventing the infinite addition of genomes.
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Rationale
Little research has been published on the frequencies of ploidy levels over time in the wild
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within the same population of these salamanders. Ploidy, or the number of sets of chromosomes
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(genomes) within an organism’s cells, can range from diploid to pentaploid in these populations.
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The triploid salamanders are the most common within the Ambystoma complex, and pentaploid
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organisms are rare (Lowcock et al., 1991; Bogart and Klemens, 2008). Higher ploidy levels are
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more common in larvae than in adults, so it appears that some selection occurs against the higher
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ploidy levels as the population ages (Bogart et al., 2007). In the published literature, it seems that
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frequencies of ploidy variable occurrences are often clumped over life stages (e.g., metamorphs
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and adults; Ramsden, 2005; Bogart et al., 2007). Due to this clumping, it is impossible to
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determine at what point in development selection against the higher ploidy levels is the strongest.
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This study focuses on the frequencies of ploidy levels through four life stages of
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unisexual Ambystoma salamanders (adults, early larvae, late larvae, and juveniles). I hypothesize
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that if cellular complications occur, then organisms will suffer most at metamorphosis and there
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will be a reduction in the frequency of tetraploid individuals from late larvae to juveniles. As a
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whole, these data will provide insight into the significance of ploidy elevation and the possibility
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of heterozygous genome advantage. These data will also identify trends in survival of ploidy-
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variable individuals throughout the Edwin S. George Reserve and indicate at what point
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developmental complications hinder survival.
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METHODS
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Study Site
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The University of Michigan’s Edwin S. George Reserve (ESGR) is near Pinckney,
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Michigan, about 35 miles from Eastern Michigan University (Fig. 1). The ESGR includes a 540-
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hectare area of oak-hickory forest with many wetland areas and vernal ponds (Skelly, 1999). The
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ponds at the ESGR have been sites of ongoing, long-term amphibian research (Skelly, 1999;
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Franker, 2009; Benard and Maher, 2011). We collected samples from three amphibian breeding
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ponds within the ESGR: Ilex Pond, West Woods Big, and Dreadful Swamp (Fig. 1). There are
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three sexually reproducing ambystomatids in these ponds: Blue-Spotted Salamanders
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(Ambystoma laterale), Spotted Salamanders (A. maculatum), and Tiger Salamanders (A.
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tigrinum). However, unisexual females only incorporate genomes from A. laterale males (K.
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Greenwald, personal observation). Ambystoma jeffersonianum genomes are present in all of the
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unisexual salamanders at the Edwin S. George Reserve regardless of bisexual A. jeffersonianum
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not being present at this site. This A. jeffersonianum genome could have been inherited from a
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female ancestor from which the Pinckney, Michigan population is now isolated (Bi et al. 2008).
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Each pond is fully encircled by a drift fence made of aluminum window screen and
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wooden stakes buried to roughly 15 cm. The fences are accompanied by pitfall traps; 1-gallon
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plastic buckets sunk into the ground every 2 m on both sides of the fence. When amphibians
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encounter the fence, they walk along it until they fall into a trap. At the beginning of the breeding
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season, we “opened” traps on the outside edge of the fence by placing partial lids on the buckets.
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Buckets include a small sponge raft so organisms can survive if a bucket floods with rainwater
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and a small branch to allow trapped mammals to escape. While open, we checked traps at least
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twice daily. After the breeding season, we opened gates built into the drift fence, allowing
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animals to pass in and out, and closed pitfall traps with full lids.
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Sampling Techniques
We took tail snips from breeding adults caught in pitfall traps which we checked twice
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daily for the duration of the breeding season (from March 8th to March 17th in 2012 and from
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March 11-12th and March 30th-April 10th in 2013). We sampled every adult female recognized as
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a member of the unisexual complex at each pond. Only female salamanders are sampled, as male
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unisexuals are exceedingly rare (Ramsden, 2005), so all males are assumed to be A. laterale. We
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processed all adult according to current mark and recapture research, which involved
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anesthetizing organisms in Tricaine Methanesulfonate (MS-222), marking with passive
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integrated transponders (PIT tags), taking tail snips for genetic analysis, and measuring mass and
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snout vent length (SVL) (Greenwald, in press). When previously tagged adults were recaptured,
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we only measured mass and SVL.
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Six weeks after the initial sampling during the breeding season (April 28 th to May 5th,
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2012, and May 11th to May 22nd, 2013), we captured early larvae through dip-netting because
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their small size permitted them to escape minnow traps. Dip-netting consisted of scooping the
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first layer of substrate (mostly decaying leaf litter) and checking for larvae. We kept organisms
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identified as members of the Ambystoma complex in a large bucket of pond water in the shade
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until sampling. We returned all other organisms to the pond immediately. We measured total
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length and then excised 3-5 mm of the tail. We kept tail snips and organisms that died in
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sampling (roughly 4%) whole and stored them in 95% ethanol for microsatellite analysis.
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Ambystoma salamanders are known to regenerate lost appendages, so this amount of tissue loss
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has little effect on the individual’s fitness (Grinfeld et al., 1996). After sampling, we returned all
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organisms to the pond where they were captured.
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One month later (June 5th to June 12th, 2012 and June 24th to July 9th, 2013), we caught
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late larval samples through both dip-netting and minnow traps. Minnow traps were 9 x 9 x 18
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inches, and a collapsible mesh design with a funnel on each end leading to a 3-inch diameter
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hole. We did not bait the traps, which we set up with the hole submerged and room above the
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water’s surface, so any trapped organism which required air would not perish. When traps were
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used, we set them up in the evening and left them out overnight. We never left traps unchecked
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for more than 15 hours to reduce trap mortality (Anderson and Giacosie, 1967). We processed
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late larval samples similarly to early larval samples taking no more than 5mm for a tail snip. We
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also recorded total length.
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The field season of 2012 was hot and very dry, so the juveniles were moving out of the
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ponds as the ponds dried up (July 5th-July 9th). We sampled any organisms identified as members
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of the unisexual complex after finding them under cover objects and along the drift fences under
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leaf litter. Again, we measured SVL, and removed roughly 5 mm of the tail for the DNA sample,
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which we stored in 95% ethanol until microsatellite analysis. We released individuals on the
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forest side of the drift fences near cover objects. The field season of 2013 was wet, and
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organisms moved out of the ponds much later (July 17th to July 29th 2013). We sampled
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organisms in 2013 similarly to those collected in 2012.
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We strove to get 30-40 samples from each life stage (early larvae, late larvae, and
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juvenile) from each pond for each year. Therefore, we hoped to achieve a sample size of at least
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270 organisms (not including adult samples) so the sample size would be large enough for
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appropriate statistical analysis. Eastern Michigan University’s Institutional Animal Care and Use
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Committee (IACUC) approved animal use and sampling procedures, reference number 2011-
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049, through the year 2014 (Appendix A). The work was approved by the Michigan Department
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of Natural Resources under a Scientific Collector’s Permit issued to Dr. Katherine Greenwald;
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Dr. Earl Werner (director of the ESGR) and the University of Michigan approved access to the
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ESGR.
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Lab Methods
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We completed DNA extraction and purification using the QIAGEN DNeasy blood and
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tissue kit (QIAGEN Sciences, Germantown, MD) following the manufacturer instructions. We
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used PCR analysis to amplify three microsatellite loci for each individual: AjeD378, AjeD94, and
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AjeD346 (Julian et al., 2003; Ramsden et al., 2006). We fluorescently tagged forward primers so
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the three loci could be multiplexed in PCR reactions: AjeD378 with 6-FAM (blue), AjeD346 with
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NED (yellow, but shows black in the software), and AjeD94 with HEX (green). Both AjeD94 and
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AjeD346 amplify in both Ambystoma jeffersonianum and A. laterale but in different size ranges,
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and AjeD378 amplifies only in the A. jeffersonianum locus (Julian et al., 2003; Ramsden et al.,
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2006). For example, AjeD94 amplifies in the 160-200 bp range for A. jeffersonianum (J) and in the
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250-300 bp range for A. laterale (L). If three peaks occur (e.g., 185-253-293), the individual is a
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triploid and the biotype for that individual is an LLJ salamander, having two genomes from A.
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laterale and one genome from A. jeffersonianum. This is double checked by a second locus,
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AjeD346, which amplifies A. laterale in the 130-160 bp range and A. jeffersonianum in the 220-
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260 bp range. The QIAGEN multiplex kit was used to run all loci at the same time (QIAGEN
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Sciences, Germantown, MD). (Fig. 2)
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The conditions for all PCR programs were 2 min and 45 s of initial denaturation at 94°C,
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annealing for 45 s at 58°C, and extension for 1 min and 30 s at 72°C. The program repeated this
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cycle 34 times with only 45 s for the denaturation of the following repetitions. The PCR program
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ended after a final 5 min at 72°C. We sent PCR products in 96-well plates to the Georgia
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Genomics Facility (GGF, University of Georgia, Athens, GA) for genotyping with the Applied
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Biosystems 3730xl 96 capillary DNA Analyzer. The GGF adds formamide and 500-ROX size
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standard to 2μl PCR products for electrophoresis.
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Genetic Data Analysis
We used the Geneious software package to visualize electropherograms received from
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the GGF in .fsa format (Geneious version 6.0.4 created by Biomatters), and verified allele calls
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similarly to Ramsden (2005). We determined ploidy level by the number of peaks present in each
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individual across all three loci. When the number of locus peaks differed between two loci, the
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locus with the greatest amount of peaks was used to determine the ploidy level. For example, if
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locus AjeD94 shows peaks at 185-145 (diploid) and AjeD346 shows peaks at 175-250-305
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(triploid) for an individual, the individual would be designated as a triploid organism instead of a
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diploid organism, based on the assumption that AjeD94 was homozygous at one locus.
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Size ranges for each genome present in this population are listed in Table 1. Peak
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processing involved deleting peaks caused by “stutter” (excess PCR product) and “pull-up”
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(peaks called in other primer dyes because one peak was so large it caused the other dyes to
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register as peaks as well).
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Statistical Analysis
The independence of observed biotype frequencies was determined using chi-squared
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analyses (Zar, 2010). Chi-squared tests determined if there was a difference between ponds. If no
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difference was found, samples from all ponds were pooled allowing for an increased sample size.
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We then used chi-squared analyses to test the independence of observed biotype frequencies
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between each life stage to determine where, if any, selective pressure occurs on the population as
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it ages. We also used chi-squared analyses to determine if there was a difference in frequencies
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of observed biotypes between years within ponds. We performed t-tests to determine if there was
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a difference in the size of organisms at each life stage between years.
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RESULTS
C. Casto 16
273
Weather Data
274
During the breeding season of 2012 (March 8th- March 17th), there was 2.29 cm of rain
275
total, and the average temperature was 9.4ºC (49ºF; Fig. 3). During a period of nine days at the
276
end of breeding activity (March 16th-March 24th), the average temperature was 17ºC (63ºF).
277
From the beginning of the season to the last day of collection, only 27.86 cm of rain were
278
recorded, precipitation events were brief, and the average temperature was 11.4ºC (52.5ºF). Of
279
the three ponds studied, West Woods Big had no remaining standing water on July 11 th 2012. On
280
the same date, Dreadful Swamp consisted of a small, shallow pool surrounded by deep muck,
281
and Ilex Pond was a dense, mostly dry patch of button bush with water standing only in the
282
deepest sections of the pond.
283
The 2013 field season was cooler and wetter than the 2012 field season. A period of over
284
two weeks where the average temperature was -1.3ºC (29.6ºF) broke up the breeding season, so
285
the breeding activity occurred March 11-12th and March 30th- April 10th. During this time, there
286
was 4.11 cm of rain total, and the average temperature was 4.4ºC (40ºF; Fig. 3). From the
287
beginning of the season to the last day of collection, 42.37 cm of rain was recorded, and the
288
average temperature was 13.3ºC (56ºF). Of the three ponds studied, all had water remaining as
289
the last juvenile sample was collected on July 29th, 2013.
290
On average, Pinckney, Michigan, gets 38.53 cm of rain from March through July and has
291
an average temperature of 12.8ºC (55ºF ) during that time, the average temperature for March
292
alone being 1.1ºC (34ºF; Weatherdb.com). These averages show that 2013 would be considered a
293
standard spring and summer for the Pinckney area. The average egg incubation temperature and
C. Casto 17
294
the average temperature during the life of the larval stages are very similar between 2012 and
295
2013 (Table 2).
296
The Hell, Michigan, weather station, National Weather Service ID HELM4, was used for
297
all weather data analyses. When data from Hell, Michigan, weather station were unavailable, the
298
Howell, MI, Sewage Plant, National Weather Service ID HOWM4 data were used. All weather
299
data were obtained via Weather Source, LLC (Amesbury, MA).
300
301
Sampling and Genotypes
In 2012, we collected 99 early larvae, 84 late larvae, and 83 juveniles in total across the
302
three ponds. In addition, 352 adult samples were collected. In 2013, we collected 111 early
303
larvae, 102 late larvae, and 63 juveniles in total across the three ponds. In addition, 136 adult
304
samples were collected.
305
Of the 581 salamanders we sampled in 2012, only two were diploid organisms and one was
306
a pentaploid organism (Table 3). Of the 412 salamanders sampled in 2013, three were diploid
307
organisms and two were pentaploid organisms (Table 3). The 2013 adults did not produce offspring
308
through genome addition (ploidy elevation), which resulted in very few tetraploid organisms being
309
produced (Fig. 4).
310
Across both years, AjeD346 had the highest allelic diversity, with 26 alleles (Table 1). We
311
found that some alleles were only in one or two of the three ponds (Table 4). Individual sample
312
allele calls and biotypes for early larvae, late larvae, and juvenile samples may be found in
313
Appendix B.
314
C. Casto 18
315
316
Size
In 2012, the average size was 21 mm for early larvae, 38 mm for late larvae average, and 32
317
mm for juvenile (SVL). In 2013, the average size was 31 mm for early larvae, 44 mm for late
318
larvae, and 34 mm for juvenile (SVL). All three life stages were, on average, larger in 2013 than in
319
2012 (Early larvae t = -12.66, p < 0.0001; Late larvae t = -3.72, p = 0.0001; Juvenile t = -3.77, p =
320
0.0002).
321
322
Chi-squared analyses
Within year comparisons.—Because of low sample sizes in both years, we left diploid
323
and pentaploid classifications out of the analysis, and only analyzed the frequencies of triploid
324
and tetraploid individuals across developmental stages. A chi-squared analysis of the frequencies
325
of tetraploid organisms within each pond across life stages did not show any significant
326
differences (2012, χ² = 4.248, p = 0.83; 2013, χ² = 2.016 p = 0.98; Fig. 4). In addition, the ponds
327
are close enough that inter-pond dispersal may occur (Trenham et al., 2001). Therefore, we
328
grouped the three ponds into one population for further analysis. We determined the total sample
329
and the sample size of each life stage prior to data collection; thus, the data were organized into a
330
2x4 contingency table with one fixed margin (Zar, 2010).
331
A chi-squared analysis of the frequencies of tetraploid organisms within each pond across life
332
stages did not show any significant differences for either year (2012: χ² = 4.25, p > 0.8; 2013: χ² =
333
2.02, p > 0.9; Fig. 4). Therefore, we grouped the three ponds into one population for each year for
334
further analysis. The sample size of each life stage was again predetermined so the chi-squared
335
analyses were performed the same way for both years. The frequencies of occurrence differed among
336
life stages in 2012 (χ² = 35.7, p < 0.0001) but only shows a trend toward independence in 2013 (χ² =
C. Casto 19
337
13.7, p = 0.057). Subdividing the 2012 contingency table by removing the adults from the
338
analysis to determine if this result was caused by the larger sample size of this life stage, we
339
found that the frequency of ploidy variability differed significantly among the remaining stages
340
as well (χ² = 7.82, p < 0.05; Zar, 2010). Further subdividing the 2012 contingency table, we
341
found that the frequency of ploidy levels did not differ between early larvae and late larvae (χ² =
342
2.98), late larvae and juveniles (χ² = 3.96), or juveniles and adults (χ² = 0.02) (p > 0.2). However,
343
the frequency of ploidy levels did significantly differ between all remaining combinations (Table
344
5a; Fig. 5). Because the initial 2013 analysis was not statistically significant, subdividing the
345
contingency table further was unnecessary (Zar, 2010). However, the adult and juvenile samples
346
in 2013 were the cause of the trend seen this year as there was a statistically significant
347
difference between these life stages (χ² = 8.005, p = 0.04; Table 5b).
348
Between year comparisons.—The frequency of occurrence of triploid and tetraploid
349
organisms differed between years for the population’s early larvae stage and late larvae stage (χ²
350
= 20.47, p = 0.001 and χ² = 8.76, p = 0.03 respectively; Table 5c). A higher percentage of the
351
breeding adults in 2013 were tetraploid, and very few early larvae produced in 2013 were
352
tetraploid (or the result of ploidy elevation) when compared to 2012. There was no statistically
353
significant difference between years in the occurrence of triploid and tetraploid individuals for
354
adult and juvenile stages (χ² = 5.09, p = 0.1 and χ² = 3.24, p = 0.36 respectively; Table 5c). The
355
ploidy levels at individual ponds did differ between life stages sampled each year. The frequency
356
of occurrence of triploid and tetraploid individuals in the early larval stage for Ilex Pond and
357
West Woods Big did show a statistical difference between years (χ² = 13.35 p = 0.0039; χ² =
358
8.748, p = 0.033 respectively; Table 5d).
359
C. Casto 20
360
361
362
363
364
365
366
367
368
DISCUSSION
C. Casto 21
369
This study focused on the frequencies of ploidy levels through four life stages of
370
unisexual Ambystoma salamanders (breeding adults, early larvae hatchlings, late larvae, and
371
juveniles leaving the pond) to determine survivorship of ploidy-variable individuals. We
372
hypothesized that if cellular complications have the greatest selective consequences, then
373
organisms will suffer most at metamorphosis and the frequency of tetraploid biotypes from late
374
larvae to juveniles would be reduced. The distributions of ploidy levels in each consecutive life
375
stage (with the exception of adult to early larvae) showed no significant differences; however,
376
the remaining combinations of life stages were significantly different (e.g., late larvae and adult).
377
There was a gradual decline in tetraploid individuals as early larvae age to adults at the Edwin S.
378
George Reserve during the summer of 2012. Therefore, the decline in higher ploidy levels does
379
not directly result from the transition between aquatic and terrestrial habitats and instead occurs
380
in the ponds before this transition takes place.
381
There is variation in the ploidy levels of organisms in the Ambystoma complex depending
382
on what host species is used for reproduction; however, triploid individuals are the most common
383
ploidy level in the Ambystoma complex (Panek, 1978; Bogart and Klemens, 2008; Bogart, 2003).
384
Therefore, it is no surprise that in previous sampling, some ponds had samples that were close to
385
100% triploid (Panek, 1978; Bogart et al., 1989; Lowcock and Murphy, 1990; Hedges, 1992). Many
386
studies also show that increased ploidy levels may be more common in the larvae (Bogart et al.,
387
1989; Ramsden, 2005; Bogart et al., 2007). The current study supports these conclusions by adding
388
evidence of ploidy-elevation in early larvae samples in the 2012 field season (Table 2, Fig. 4).
389
The lack of ploidy-elevated offspring in 2013 is likely a result from that year’s cooler
390
breeding season temperatures (4.4ºC) and shows in the early larvae samples from West Woods
391
Big and Ilex Pond. Bogart et al. (1989) determined that at 6ºC, unisexual salamanders were more
C. Casto 22
392
likely to reproduce in captivity through gynogenesis than through hybridization (genome
393
replacement) or ploidy elevation. Bogart et al. (1989) found that at higher temperatures (15ºC),
394
these same organisms produced ploidy-elevated offspring more often. In addition, Bogart et al.
395
(1989) found that even salamanders not compatible with the Ambystoma complex (A.
396
maculatum) could be used as sperm donors in colder temperatures because the sperm was less
397
likely to be incorporated into the offspring.
398
The difference between 2012 and 2013 breeding seasons (9.4ºC vs. 4.4ºC, respectively) not
399
only acts as supporting evidence from the field, but also shows that temperatures as low as 9.4ºC
400
can potentially induce genome addition. The current study is the only study to support this finding
401
in the field and shows that temperature may drastically change the results between field seasons. It
402
is also interesting to note that the differences between years seen in the early larval stages at Ilex
403
Pond and West Woods Big disappear in the late larvae stages, showing that the most detrimental
404
phase for the tetraploid individuals is within the first month after hatching.
405
The mean sizes of the salamanders in 2013 were also larger for early, late, and juvenile
406
life stages than those collected in 2012. Such a result was expected, as aquatic larvae develop
407
faster at warmer temperatures and respond to faster drying ponds by undergoing metamorphosis
408
sooner; both aspects result in smaller body size at warmer temperatures (Ryan, 1941; Anderson,
409
1972; Voss, 1993; Bridges, 2002).
410
In 2013, adult samples from Ilex Pond were not collected. Ilex pond, historically, has
411
been very close to 100% triploid (Greenwald, correspondence). Had these samples been
412
collected, it is likely that there would be no significant differences in the frequency of occurrence
413
in the juvenile and adult samples of that year. In addition, there was a large die-off event at West
C. Casto 23
414
Woods Big between June 7th and June 30th, 2013, resulting in very low numbers for the late
415
larval sample at this pond. This die-off event killed many organisms of different species, most
416
noticeably wood frogs (Rana sylvatica), which we found bloated and floating around the pond.
417
This particular event’s cause is unknown, but previous outbreaks of Ranavirus have been
418
documented at the ESGR with similar consequences.
419
The current study differs from published works when looking at diploid and pentaploid
420
organisms; however there seems to be enormous variation in ploidy composition across the
421
unisexual range. We were surprised to find only five diploid LJ organisms and three pentaploid
422
LLLLJ samples out of 993 individuals (0.5% LJ and 0.3% LLLLJ). Bogart and Licht (1986)
423
found 33 diploid organisms (11%) with Ambystoma laterale - A. texanum nuclear genetics in
424
only 283 samples. Noel et al. (2011) reports finding 100% diploid individuals (N=36) in Quebec
425
where A. jeffersonianum genomes are used. Bogart et al. (2007) also found ten diploid (LJ)
426
larvae from two egg masses (in an analysis of 29 egg masses) where both A. laterale and A.
427
jeffersonianum were used as hosts, and found no pentaploid organisms. In contrast, Ramsden et
428
al. (2006) only found one diploid LJ (0.7%) salamander in a sample of 153 but found 13
429
pentaploid (LJJJJ) terrestrial individuals (8.5%) where the host species was A. jeffersonianum.
430
The range of the Ambystoma complex spans from Kentucky to Ontario. Southern
431
populations are exposed to warmer temperatures earlier in the year than populations farther
432
north. There seems, however, to be no relationship between latitude and genome addition
433
because southern populations simply breed earlier in the year after the first thaw of spring
434
(Bogart, correspondence). Future research is required to determine what other factors are
435
involved with the increased fitness of ploidy-elevated and diploid organisms seen in previous
C. Casto 24
436
studies. At this time, there is no answer to why some populations ploidy elevate more than others
437
and why some ploidy-elevated populations survive longer in some areas than others.
438
This study further supports previous work on the unisexual Ambystoma complex and
439
provides evidence that multiple factors affect the fitness of tetraploid and pentaploid individuals.
440
This study is perhaps the first to note a gradual decline in tetraploid individuals as the population
441
ages. It seems the unisexual population at the Edwin S. George Reserve is unique in the low
442
number of diploid and pentaploid organisms produced. Diploid organisms must be selected
443
against in this population, lending further evidence to the hypothesis of heterozygous genome
444
advantage in triploid organisms. This study is also the first to find evidence in the field of warm
445
temperatures during the breeding season linked to ploidy-elevated offspring. Globally warming
446
temperatures and an increasing amount of heat waves could therefore have severe consequences
447
on the survival of high-ploidy individuals as triploid adults produce more ploidy-elevated
448
offspring that are selected against, possibly resulting in a declining population over time
449
(Carlson, 2008).
450
Many factors about the unisexual Ambystoma complex are still unknown. Future work is
451
required to determine if ploidy-elevated unisexuals show similar declines as the population ages
452
when interacting with different host species. Future work is also required to monitor populations
453
as the climate changes. The only way to further our understanding of this unique system of
454
reproduction is to perform long-term studies as opposed to year-long snapshots to determine
455
population trends.
456
C. Casto 25
457
458
459
460
461
462
463
464
465
466
TABLES
C. Casto 26
467
TABLE 1. —Allele size ranges and diversity at each locus within early, late, and juvenile stages.
Locus
Ambystoma
Ambystoma
jeffersonianum
laterale
AjeD378
210-290 bp
AjeD94
185-250
135-155
Reference
Diversity in alleles at
the ESGR (# of alleles)
Julian et al., 2003
6
Julian et al., 2003;
19
Ramsden et al., 2006
AjeD346
160-200
245-305
Julian et al., 2003;
Ramsden et al., 2006
468
469
26
C. Casto 27
470
TABLE 2. —Average ambient temperatures during life stages in 2012 and 2013.
Average temperature 2012
471
472
Average temperature 2013
Time of season
Fahrenheit
Celsius
Fahrenheit
Celsius
Breeding season
49
9.4
40
4.4
Egg Incubation
49
9.4
51
10.6
Larval life
66
18.9
67
19.4
C. Casto 28
473
TABLE 3. —Number of individuals in each ploidy level in each life stage.
Parentheticals are the numbers found in Dreadful Swamp/Ilex Pond/ West Woods
Big.
Year
Life stage
Diploid
Triploid
Tetraploid
Pentaploid
2012
Adult
0
290
25
0
(27/120/143)
(4/7/14)
2
67
29
1
(1/0/1)
(28/21/18)
(7/11/11)
(0/0/1)
0
68
16
0
(24/23/21)
(6/7/3)
76
7
(30/26/20)
(4/2/1)
114
20
2
(10/0/104)
(2/0/18)
(0/0/2)
104
7
0
(35/40/29)
(4/1/2)
3
94
5
(1/2/0)
(47/38/9)
(2/2/1)
0
62
1
(29/33/0)
(1/0/0)
Early Larvae
Late Larvae
Juveniles
2013
Adult
Early Larvae
Late Larvae
Juveniles
474
475
0
0
0
0
0
0
C. Casto 29
476
TABLE 4. —Unique alleles within ponds.
AjeD378
AjeD94
+
Dreadful Swamp
West Woods Big
Ilex Pond
AjeD346
+
227
262, +286
260,~248,
139, 191, ~203, 215, 259
202, ~238, ~266, 302
~248,
187, 195, 199, ~203, +227
148, 174, 218, ~238, +262,
~
266, +286
+
denotes alleles shared between Dreadful Swamp and Ilex Pond.
~
denotes alleles shared between West Woods Big and Ilex Pond.
Bold alleles are those unique to one pond.
477
C. Casto 30
478
TABLE 5a. —2012 results from Chi-squared analyses of the independence of ploidy levels
between life stages.
Comparison A-E-L-J
E-L-J
E-L
E-J
E-A
L-J
L-A
A-J
χ²
35.72
13.34
2.976
13.14
31.98
3.960
8.880
0.022
p-value
<0.001*
0.020*
0.395
0.004*
<0.001*
0.27
0.031*
0.999
* denotes statistical significance, A=adult, E= early larvae, L= late larvae, J= juvenile.
479
TABLE 5b. —2013 results from Chi-squared analyses of the independence of ploidy levels
between life stages.
Comparison A-E-L-J
E-L-J
E-L
E-J
E-A
L-J
L-A
A-J
χ²
13.68
2.002
0.153
2.040
4.599
1.295
5.796
8.005
p-value
0.057~
0.849
0.984
0.564
0.204
0.730
0.122
0.046*
~ denotes statistical trend, * denotes statistical significance, A=adult, E= early larvae, L=
late larvae, J= juvenile.
480
TABLE 5c. —2012-2013 comparison of each stage; results from Chi-squared analyses.
Comparison
A
J
L
E
χ²
5.092
3.241
8.764
20.47
p-value
0.165
0.356
0.033*
<0.001*
* denotes statistical significance, A=adult, E= early larvae, L= late larvae, J= juvenile
481
482
C. Casto 31
TABLE 5d. —2012-2013 comparison within individual ponds; results from Chi-squared
analyses.
Comparison
Dreadful Swamp
Ilex Pond
West Woods Big
between years
E
L
J
E
L
J
E
L
χ²
1.384
5.181
1.573
13.35
5.143
2.437
8.748
0.0425
p-value
0.244
0.159
0.666
0.004*
0.162
0.487
0.033*
0.998
* denotes statistical significance, A=adult, E= early larvae, L= late larvae, J= juvenile
483
C. Casto 32
484
485
486
487
488
489
490
491
FIGURES
C. Casto 33
492
493
FIG 1. —The Edwin S. George Reserve location in Michigan, satellite view, and road map. Ponds
used in analysis are labeled.
494
495
496
497
FIG 2. —An example of Geneious results portraying the three loci and peaks of a triploid
498
individual; AjeD378-6FAM (blue) shows a single peak here at 238, AjeD94-Hex (green) shows
499
three peaks at 147-151-243, and AjeD346-NED (black) shows three peaks at 185-253-293.
500
501
502
503
504
FIG 3. —Average temperature and rain events during the 2012 and 2013 breeding
seasons. Dotted lines represent average temperatures on days without breeding activity.
505
506
507
508
FIG 4. —The percentage of samples that were tetraploid in each life stage between ponds.
509
Here, there were no statistical differences within years, so the ponds could be clumped into a
510
single population for statistical analysis. Because of a die-off event at West Woods Big, juveniles
511
were not found in 2013. Ilex Pond adults were not sampled 2013.
512
513
514
515
516
FIG 5. —Percentage of the triploid and tetraploid samples from 2012. In 2012, there was
no statistical difference between each consecutive life stage (with the exception of adult-early
C. Casto 34
517
larvae), but every second life stage showed a statistically significant difference in the frequencies
518
of triploids and tetraploids in the populations. Significant differences are denoted when bars do
519
not have corresponding letters. Only triploid and tetraploid samples are shown; stages that had
520
diploid and pentaploid organisms do not add to 100%.
521
522
523
524
FIG 6. —Percentage of the triploid and tetraploid samples from 2013. There was no
525
statistical difference between the life stages in 2013 with the exception of the adult and juvenile
526
stages. Significant differences are denoted when bars do not have corresponding letters. Only
527
triploid and tetraploid samples are shown; stages that had diploid and pentaploid organisms do
528
not add to 100%.
C. Casto 35
529
530
FIG 1. The Edwin S. George Reserve location in Michigan, satellite view, and road map. Ponds used in analysis are labeled.
C. Casto 36
531
147 151
185
Base pairs
238 243 253
293
532
FIG 2. An example of Geneious results portraying the three loci and peaks of a triploid individual; AjeD378-6FAM (blue) shows a single
533
peak here at 238, AjeD94-Hex (green) shows three peaks at 147-151-243, and AjeD346-NED (black) shows three peaks at 185-253-293.
534
535
C. Casto 37
536
537
FIG 3. Average temperature and rain events during the 2012 and 2013 breeding seasons. Dotted lines represent average temperatures
538
on days without breeding activity.
539
C. Casto 38
540
541
FIG 4. The percentage of samples that were tetraploid in each life stage between ponds. Here, there were no statistical differences
542
within years, so the ponds could be clumped into a single population for statistical analysis. Because of a die-off event at West Woods
543
Big, juveniles were not found in 2013. Ilex Pond adults were not sampled 2013.
C. Casto 39
544
545
FIG 5. Percentage of the triploid and tetraploid samples from 2012. In 2012, there was no statistical difference between each
546
consecutive life stage (with the exception of adult-early larvae), but every second life stage showed a statistically significant
547
difference in the frequencies of triploids and tetraploids in the populations. Significant differences are denoted when bars do not have
548
corresponding letters. Only triploid and tetraploid samples are shown; stages that had diploid and pentaploid organisms do not add to
549
100%.
C. Casto 40
550
551
FIG 6. Percentage of the triploid and tetraploid samples from 2013. There was no statistical difference between the life stages in 2013
552
with the exception of the adult and juvenile stages. Significant differences are denoted when bars do not have corresponding letters.
553
Only triploid and tetraploid samples are shown; stages that had diploid and pentaploid organisms do not add to 100%.
C. Casto 41
554
555
556
557
558
559
560
561
LITERATURE CITED
C. Casto 42
562
563
564
565
Anderson, J.D. 1972. Embryonic temperature tolerance and rate of development in some
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Anderson, J.D., and R.V. Giacosie. 1967. Ambystoma laterale in New Jersey. Herpetologica
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566
Benard, M.F., and J.M. Maher. 2011. Consequences of intraspecific niche variation: phenotypic
567
similarity increases competition among recently metamorphosed frogs. Oecologia
568
166:585-592.
569
570
Bi, K., and J.P. Bogart. 2010. Time and time again: unisexual salamanders (genus Ambystoma)
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571
Bi, K., J.P. Bogart, and J. Fu. 2008. Genealogical relationships of southern Ontario polyploid
572
unisexual salamanders (genus Ambystoma) inferred from intergenomic exchanges and
573
major rDNA cytotypes. Chromosome Research 16:275-289.
574
Bogart, J.P. 1982. Ploidy and genetic diversity in Ontario salamanders of the Ambystoma
575
jeffersonianum complex revealed through an electrophoretic examination of larvae.
576
Canadian Journal of Zoology 60:848-855.
577
Bogart, J.P. 2003. Genetics and systematics of hybrid species. Pp. 120-143 in D.E. Sever (Ed),
578
Reproductive Biology and Phylogeny of Urodela. Science Publishers, Enfield.
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Bogart, J.P. Email correspondence with author. Sept. 19th, 2013.
580
Bogart, J.P., and L.E. Licht. 1986. Reproduction and the origin of the polyploids in hybrid
581
salamanders of the genus Ambystoma. Canadian Journal of Genetics and Cytology
582
28:605-617.
C. Casto 43
583
Bogart, J.P., and M.W. Klemens. 2008. Additional distributional records of Ambystoma laterale,
584
A. jeffersonianum (Amphibia: Caudata) and their unisexual Kleptogens in Northeastern
585
North America. American Museum of Natural History 3627:1-58.
586
587
588
Bogart, J.P., R.P. Elinson, and L.E. Licht. 1989. Temperature and sperm incorporation in
polyploid salamanders. Science 246:1032-1034.
Bogart, J.P., K. Bi, J. Fu, D.W.A. Noble, and J. Niedzwiecki. 2007. Unisexual salamanders
589
(genus Ambystoma) present a new reproductive mode for eukaryotes. Genome 50:119-
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136.
591
Bogart, J.P., J. Bartoszek, D.W.A. Noble, and K. Bi. 2009. Sex in unisexual salamanders:
592
discovery of a new sperm donor with ancient affinities. Heredity 103:483-493.
593
Bridges, C.M. 2002. Tadpoles balance foraging and predator avoidance: effects of predation,
594
595
596
597
598
599
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pond drying, and hunger. Journal of Herpetology 36:627-634.
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Franker, M.E. 2009. Predation risk assessment by green frog (Rana clamitans) tadpoles through
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chemical cues produced by multiple prey. Behavioral Ecology and Sociobiology
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tail regeneration in space: effect on the pigmentation of the blastema. Advances in Space
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Hedges, S.B., J.P. Bogart, and L.R. Maxson. 1992. Ancestry of unisexual salamanders. Nature
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Julian, S.E., T.L. King, and W.K. Savage. 2003. Novel Jefferson salamander, Ambystoma
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complexes. Molecular Ecology Notes. 3:95-97.
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Licht, L.W., and J.P. Bogart. 1989. Embryonic development and temperature tolerance in diploid
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Lowcock, L.A., and R.W. Murphy. 1990. Pentaploidy in hybrid salamanders demonstrates
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619
Lowcock, L.A., H. Griffith, and R.W. Murphy. 1991. The Ambystoma laterale-jeffersonianum
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621
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Lueck, B.E. 1985. Comparative social behavior of bisexual and unisexual whiptail lizards
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Macgregor, H.C., and T.M. Uzzell, Jr. 1964. Gynogenesis in salamanders related to Ambystoma
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629
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631
Panek, F.M. 1978. A developmental study of Ambystoma jeffersonianum and A. platineum
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Ambystoma laterale, A. jeffersonianum and sympatric unisexuals. Molecular Ecology
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646
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651
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652
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653
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654
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655
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658
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659
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660
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661
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662
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C. Casto 47
663
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664
Voss, S.R. 1993. Effect of temperature on body size, developmental stage, and timing of
665
hatchling in Ambystoma maculatum. Journal of Herpetology 27:329-333.
666
667
668
669
670
671
672
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C. Casto 48
673
674
675
676
677
678
679
680
681
APPENDICES
C. Casto 49
682
683
684
Appendix A: Approval notification from Eastern Michigan University’s Institutional Animal
Care and Use Committee (IACUC).
C. Casto 50
685
686
Appendix B: Allele calls for early larvae, late larvae, and juvenile samples. D: Dreadful Swamp,
I: Ilex Pond, W: West Woods Big, E: Early larvae, L: Late larvae, M: Juveniles.
Year
ID
BIOTYPE
AjeD378
AjeD94
AjeD94
2012
DE 1.01
LLLJ
240
147
2012
DE 1.02
LLJ
240
2012
DE 1.03
LLLJ
2012
DE 1.04
2012
AjeD94
AjeD94
AjeD346
AjeD346
AjeD346
AjeD346
151
243
186
254
258
294
147
151
255
178
254
240
147
151
243
186
250
LJ
240
147
DE 1.05
LLLJ
240
147
151
2012
DE 1.06
LLLJ
240
147
151
2012
DE 1.07
LLLJ
240
147
2012
DE 1.08
LLJ
240
147
2012
DE 1.09
LLJ
240
147
2012
DE 1.10
LLJ
244
147
2012
DE 1.11
LLJ
240
147
2012
DE 1.12
LLJ
240
2012
DE 1.13
LLJ
2012
DE 1.14
2012
2012
155
294
254
294
282
243
186
250
254
294
247
186
254
270
294
151
251
194
250
290
294
151
251
182
254
294
151
247
186
250
294
231
182
250
298
151
251
182
250
294
147
151
251
178
254
294
240
147
151
247
186
254
294
LLJ
240
147
151
243
186
254
DE 1.15
LLJ
244
147
231
182
250
298
DE 1.16
LLJ
240
147
235
186
254
294
2012
DE 1.17
LLJ
256
147
247
194
242
2012
DE 1.18
LLJ
240
147
151
243
186
254
294
2012
DE 1.19
LLJ
240
147
151
251
186
254
294
2012
DE 1.20
LLJ
244
147
231
182
250
298
2012
DE 1.21
LLJ
240
147
151
243
186
254
294
2012
DE 1.22
LLLJ
240
147
151
243
186
254
2012
DE 1.23
LLJ
240
147
151
251
194
250
294
2012
DE 1.24
LLJ
240
147
151
247
186
254
294
2012
DE 1.25
LLJ
240
147
151
247
190
250
294
2012
DE 1.26
LLJ
240
147
151
235
186
254
294
2012
DE 1.27
LLJ
240
147
151
235
186
254
294
2012
DE 1.28
LLJ
240
147
151
251
186
254
294
2012
DE 1.29
LLJ
240
147
151
243
186
254
294
2012
DE 1.30
LLJ
244
147
227
178
250
298
2012
DE 1.31
LLJ
256
147
227
194
242
2012
DE 1.32
LLJ
240
147
151
247
190
250
2012
DE 1.33
LLLJ
240
147
151
247
186
250
2012
DE 1.34
LLJ
240
147
151
235
190
254
294
2012
DE 1.35
LLJ
240
147
151
247
186
254
294
2012
DE 1.36
LLJ
240
147
151
251
186
254
294
2012
DL 1.01
LLJ
240
147
151
251
178
254
294
2012
DL 1.02
LLLJ
240
147
151
247
186
254
155
274
274
282
294
274
294
278
270
294
294
AjeD346
C. Casto 51
2012
DL 1.03
LLJ
240
147
151
243
186
254
2012
DL 1.04
LLLJ
240
147
151
255
186
250
2012
DL 1.05
LLJ
244
2012
DL 1.06
LLJ
244
147
231
178
250
298
147
231
178
250
298
2012
DL 1.07
LLJ
244
147
251
186
254
294
2012
DL 1.08
LLJ
244
147
231
178
250
298
2012
DL 1.09
LLJ
240
147
151
247
186
254
294
2012
DL 1.10
LLJ
240
147
151
243
186
254
294
2012
DL 1.11
LLLJ
236
147
151
255
186
250
254
2012
DL 1.12
LLJ
256
147
247
194
242
274
2012
2012
DL 1.13
LLJ
240
147
151
235
190
254
DL 1.14
LLLJ
240
147
151
235
190
250
2012
DL 1.15
LLJ
240
147
151
251
186
254
294
2012
DL 1.16
LLJ
240
147
151
239
186
250
294
2012
DL 1.17
LLJ
244
147
231
178
250
298
2012
DL 1.18
LLJ
240
147
151
247
186
250
294
2012
DL 1.19
LLJ
240
147
151
243
186
254
294
2012
DL 1.20
LLJ
240
147
151
251
186
254
294
2012
DL 1.21
LLJ
244
147
151
251
190
254
294
2012
DL 1.22
LLJ
240
147
151
251
186
254
294
2012
DL 1.23
LLJ
240
147
151
243
186
254
294
2012
DL 1.24
LLJ
240
147
151
243
186
254
294
2012
DL 1.25
LLLJ
240
147
151
247
186
254
2012
DL 1.26
LLJ
240
147
151
243
186
254
294
2012
DL 1.27
LLJ
240
147
151
243
186
254
294
2012
DL 1.28
LLLJ
240
147
151
243
182
250
2012
DL 1.29
LLJ
240
147
151
251
186
254
294
2012
DL 1.30
LLJ
240
147
151
251
186
254
294
2012
DM 1.01
LLJ
240
147
151
251
178
254
294
2012
DM 1.02
LLJ
240
147
151
255
186
254
294
2012
DM 1.03
LLJ
256
147
247
194
242
2012
DM 1.04
LLJ
240
147
151
255
186
254
294
2012
DM 1.05
LLJ
240
147
151
251
186
254
294
2012
DM 1.06
LLJ
240
147
151
251
186
254
294
2012
DM 1.07
LLLJ
240
147
151
235
186
254
2012
DM 1.08
LLJ
240
147
151
247
190
250
294
2012
DM 1.09
LLJ
240
147
151
251
186
254
294
2012
DM 1.10
LLJ
240
147
151
243
186
254
2012
DM 1.11
LLLJ
240
147
151
243
190
254
2012
DM 1.12
LLJ
240
147
151
251
186
254
294
2012
DM 1.13
LLJ
240
147
151
247
186
254
294
155
151
155
294
254
294
294
294
254
282
254
294
294
294
274
270
294
294
294
298
C. Casto 52
2012
DM 1.14
LLJ
240
147
151
251
186
254
2012
DM 1.15
LLLJ
240
147
151
251
186
254
2012
DM 1.16
LLJ
240
147
151
251
186
254
294
2012
DM 1.17
LLJ
240
147
151
251
186
254
294
2012
DM 1.18
LLJ
240
147
151
247
186
250
294
2012
DM 1.19
LLJ
240
147
151
251
186
254
294
2012
DM 1.20
LLJ
240
147
151
255
182
254
290
2012
DM 1.21
LLJ
240
147
151
239
186
254
294
2012
DM 1.22
LLJ
240
147
151
243
186
254
294
2012
DM 1.23
LLJ
240
147
151
243
186
254
294
2012
DM 1.24
LLJ
240
147
151
239
186
254
294
2012
DM 1.25
LLJ
240
147
151
251
186
250
294
2012
DM 1.26
LLJ
244
147
231
178
250
298
2012
DM 1.27
LLJ
240
147
151
247
186
254
294
2012
DM 1.28
LLLJ
240
147
151
251
186
254
2012
DM 1.29
LLJ
240
147
151
251
186
254
294
2012
DM 1.30
LLJ
240
147
151
243
186
254
294
2012
DM 1.31
LLJ
240
147
151
235
186
254
294
2012
DM 1.32
LLJ
240
147
151
243
182
254
294
2012
DM 1.33
LLJ
244
147
231
178
250
298
2012
DM 1.34
LLJ
256
147
247
194
242
2012
IE 1.01
LLJ
240
147
151
243
186
258
294
2012
IE 1.02
LLJ
240
147
151
247
182
258
294
2012
IE 1.03
LLJ
240
147
151
231
186
254
294
2012
IE 1.04
LLLJ
244
147
151
235
190
254
2012
IE 1.05
LLJ
240
147
151
247
182
258
294
2012
IE 1.06
LLJ
240
147
151
243
186
258
294
2012
IE 1.07
LLJ
240
147
151
243
182
258
294
2012
IE 1.08
LLJ
236
147
151
251
186
262
294
2012
IE 1.09
LLLJ
240
147
151
251
186
254
2012
IE 1.10
LLLJ
240
147
151
251
186
254
286
294
2012
IE 1.11
LLLJ
240
147
151
243
186
254
278
294
2012
IE 1.12
LLJ
240
147
151
251
186
254
2012
IE 1.13
LLLJ
240
147
151
251
186
254
2012
IE 1.14
LLJ
240
147
151
251
186
254
2012
IE 1.15
LLLJ
240
147
151
243
186
258
2012
IE 1.16
LLJ
240
147
151
243
186
254
294
2012
IE 1.17
LLJ
240
147
151
251
186
254
294
2012
IE 1.18
LLJ
240
151
235
186
254
294
2012
IE 1.19
LLJ
240
147
151
243
186
2012
IE 1.20
LLLJ
240
147
151
243
182
187
294
262
286
294
294
274
270
294
290
294
282
294
294
262
294
294
254
262
294
C. Casto 53
2012
IE 1.21
LLLJ
240
147
151
235
190
254
2012
IE 1.22
LLJ
240
147
151
243
186
254
2012
IE 1.23
LLJ
240
147
151
243
186
254
2012
IE 1.24
LLLJ
236
147
151
243
182
254
2012
IE 1.25
LLJ
240
147
151
247
186
254
294
2012
IE 1.26
LLJ
240
147
151
243
186
254
294
2012
IE 1.27
LLJ
240
147
151
247
182
254
290
2012
IE 1.28
LLLJ
240
147
151
247
186
254
2012
IE 1.29
LLJ
244
147
151
255
190
254
294
2012
IE 1.30
LLJ
240
147
151
235
186
254
294
2012
IE 1.31
LLJ
240
147
151
247
182
254
2012
IE 1.32
LLLJ
240
147
151
247
186
250
2012
IL 1.01
LLJ
240
147
151
235
186
254
294
2012
IL 1.02
LLJ
240
147
151
235
186
254
294
2012
IL 1.03
LLJ
240
147
151
235
186
254
294
2012
IL 1.04
LLJ
240
147
151
251
186
254
294
2012
IL 1.05
LLJ
240
147
151
247
186
254
294
2012
IL 1.06
LLJ
240
147
151
251
186
254
294
2012
IL 1.07
LLJ
240
147
151
235
186
254
294
2012
IL 1.08
LLJ
240
147
151
251
186
254
294
2012
IL 1.09
LLJ
240
147
151
251
186
254
294
2012
IL 1.10
LLJ
240
147
151
235
186
254
294
2012
IL 1.11
LLJ
240
147
151
243
182
254
294
2012
IL 1.12
LLLJ
240
147
151
243
186
254
282
294
2012
IL 1.13
LLLJ
240
147
151
251
186
250
254
294
2012
IL 1.14
LLJ
240
147
151
251
186
254
298
2012
IL 1.15
LLJ
240
147
151
239
190
254
294
2012
IL 1.16
LLLJ
240
147
151
255
190
254
2012
IL 1.17
LLJ
240
147
151
251
186
254
2012
IL 1.18
LLJ
240
147
151
251
186
254
2012
IL 1.19
LLLJ
240
147
151
247
186
254
2012
IL 1.20
LLJ
240
147
151
235
186
254
294
2012
IL 1.21
LLJ
240
147
151
251
186
254
294
2012
IL 1.22
LLJ
240
147
151
243
186
254
294
2012
IL 1.23
LLJ
240
147
151
239
186
254
294
2012
IL 1.24
LLJ
240
186
262
270
2012
IL 1.25
LLLJ
240
147
151
243
186
258
282
2012
IL 1.26
LLJ
240
147
151
247
186
250
294
2012
IL 1.27
LLJ
240
147
151
247
186
250
294
2012
IL 1.28
LLJ
240
147
151
247
186
254
294
2012
IL 1.29
LLLJ
240
147
151
243
186
254
151
155
262
294
294
294
266
278
294
294
290
254
270
294
294
294
294
282
282
294
294
294
C. Casto 54
2012
IL 1.30
LLLJ
240
147
151
243
186
250
2012
IM 1.01
LLJ
240
147
151
251
186
250
294
2012
IM 1.02
LLJ
240
147
151
243
186
258
294
2012
IM 1.03
LLJ
240
147
151
243
186
254
290
2012
IM 1.04
LLJ
240
147
151
243
186
254
294
2012
IM 1.05
LLJ
240
147
151
251
186
254
294
2012
IM 1.06
LLJ
240
147
151
251
186
254
294
2012
IM 1.07
LLJ
240
147
151
243
186
254
294
2012
IM 1.08
LLJ
240
147
151
251
186
254
294
2012
IM 1.09
LLJ
240
147
151
251
186
254
294
2012
IM 1.10
LLJ
240
147
151
243
186
254
294
2012
IM 1.11
LLJ
240
147
151
235
186
254
294
2012
IM 1.12
LLJ
240
147
151
251
186
254
294
2012
IM 1.13
LLJ
240
147
151
251
186
254
298
2012
IM 1.14
LLJ
240
147
151
251
186
254
294
2012
IM 1.15
LLJ
240
147
151
251
186
250
294
2012
IM 1.16
LLJ
240
147
151
243
186
254
294
2012
IM 1.17
LLJ
240
147
151
243
186
254
290
2012
IM 1.18
LLJ
240
147
151
251
190
254
294
2012
IM 1.19
LLJ
240
147
151
243
186
254
294
2012
IM 1.20
LLJ
240
147
151
251
186
254
294
2012
IM 1.21
LLJ
240
147
151
239
186
254
294
2012
IM 1.22
LLJ
240
147
151
243
186
254
294
2012
IM 1.23
LLJ
240
147
151
251
186
254
294
2012
IM 1.24
LLJ
240
147
151
243
186
254
294
2012
IM 1.25
LLJ
240
147
151
235
186
254
294
2012
IM 1.26
LLJ
240
147
151
251
186
254
294
2012
IM 1.27
LLLJ
240
147
151
251
186
250
282
290
2012
IM 1.28
LLLJ
240
147
151
243
182
254
270
294
2012
WE 1.01
LLLJ
244
147
151
239
186
258
270
294
2012
WE 1.02
LLLJ
240
147
151
243
186
254
270
294
2012
WE 1.03
LLJ
240
147
151
251
186
254
294
2012
WE 1.04
LLJ
240
147
151
251
186
254
294
2012
WE 1.05
LLJ
236
147
151
215
194
250
2012
WE 1.06
LJ
240
147
151
2012
WE 1.07
LLLJ
240
147
151
235
190
254
266
294
2012
WE 1.08
LLLJ
240
147
151
255
186
254
266
294
2012
WE 1.09
LLJ
240
147
151
235
186
254
294
2012
WE 1.10
LLJ
240
147
151
235
186
254
294
2012
WE 1.11
LLJ
240
147
151
243
186
254
294
2012
WE 1.12
LLJ
240
147
151
251
186
254
294
155
186
254
294
258
266
C. Casto 55
2012
WE 1.13
LLLJ
240
147
151
235
186
254
2012
WE 1.14
LLJ
240
147
151
239
186
254
2012
WE 1.15
LLJ
240
147
151
239
186
254
2012
WE 1.16
LLLJ
240
147
151
255
186
254
2012
WE 1.17
LLJ
240
147
151
239
190
254
294
2012
WE 1.18
LLJ
240
147
151
247
186
254
294
2012
WE 1.19
LLJ
240
147
151
251
186
254
294
2012
WE 1.20
LLLJ
240
147
151
251
186
250
2012
WE 1.21
LLJ
236
147
151
259
186
254
2012
WE 1.22
LLLJ
240
147
151
243
186
254
2012
WE 1.23
LLJ
240
147
151
235
186
254
2012
WE 1.24
LLLJ
244
147
231
178
186
250
298
2012
WE 1.25
LLLJ
240
147
151
243
186
254
270
294
2012
WE 1.26
LLJ
240
147
151
251
186
254
294
2012
WE 1.27
LLJ
240
147
151
243
186
254
294
2012
WE 1.28
LLLJ
256
147
247
194
238
2012
WE 1.29
LLJ
240
147
151
243
186
254
2012
WE 1.30
LLLLJ
240
147
151
243
186
254
2012
WE 1.31
LLJ
240
147
151
235
186
254
294
2012
WL 1.03
LLJ
240
147
151
251
186
254
294
2012
WL 1.04
LLJ
240
147
151
235
190
254
294
2012
WL 1.06
LLLJ
240
147
151
251
186
254
2012
WL 1.07
LLJ
240
147
151
251
186
254
2012
WL 1.08
LLJ
240
147
191
203
202
270
2012
WL 1.09
LLJ
240
147
151
251
186
254
2012
WL 1.10
LLLJ
240
147
151
235
190
254
2012
WL 1.11
LLJ
240
147
151
251
186
254
2012
WL 1.12
LLJ
260
147
247
194
242
274
2012
WL 1.13
LLLJ
240
147
151
243
186
254
290
2012
WL 1.14
LLJ
236
147
151
251
186
254
294
2012
WL 1.15
LLJ
240
147
151
235
186
254
294
2012
WL 1.18
LLJ
240
139
147
246
254
294
2012
WL 1.19
LLJ
240
147
151
247
182
254
294
2012
WL 1.21
LLJ
244
147
231
182
250
294
2012
WL 1.22
LLJ
240
147
151
243
186
254
294
2012
WL 1.23
LLJ
240
147
151
243
186
254
294
2012
WL 1.24
LLJ
240
147
151
243
186
254
294
2012
WL 1.25
LLJ
240
147
151
235
186
254
294
2012
WL 1.26
LLJ
240
147
151
243
186
254
294
2012
WL 1.27
LLJ
240
147
151
243
186
254
294
2012
WL 1.28
LLJ
240
147
151
239
186
254
294
155
274
294
294
294
270
254
294
302
294
270
294
294
242
274
294
270
266
282
294
294
278
294
266
294
294
294
294
C. Casto 56
2012
WL 1.29
LLJ
240
147
151
251
186
254
294
2012
LLJ
240
147
151
235
186
254
294
LLJ
240
147
151
247
182
254
294
LLJ
240
147
151
251
186
254
294
LLJ
240
147
151
235
186
254
294
LLJ
240
147
151
235
186
254
294
LLJ
240
147
151
247
186
250
294
LLJ
240
147
215
186
270
274
LLJ
236
147
215
194
250
258
LLJ
244
147
231
178
250
298
LLJ
240
147
151
243
186
254
294
LLJ
240
147
151
247
182
254
294
LLJ
240
147
151
251
186
254
294
LLJ
240
147
151
235
190
254
294
LLLJ
240
147
151
243
186
254
LLJ
240
147
151
255
186
254
LLJ
236
147
151
215
194
250
LLJ
240
147
151
247
186
250
294
LLJ
240
147
151
243
182
254
294
LLJ
240
147
243
182
258
294
LLJ
240
147
151
235
186
254
294
LLJ
240
147
151
235
186
254
294
2012
WL 1.30
WM
1.01
WM
1.02
WM
1.03
WM
1.04
WM
1.05
WM
1.06
WM
1.07
WM
1.08
WM
1.09
WM
1.10
WM
1.11
WM
1.12
WM
1.13
WM
1.15
WM
1.16
WM
1.17
WM
1.18
WM
1.19
WM
1.20
WM
1.21
WM
1.22
LLJ
240
147
151
251
186
254
294
2013
DE 2.01
LLLJ
244
147
151
243
186
254
2013
DE 2.02
LLJ
240
147
151
247
186
254
294
2013
DE 2.03
LLJ
240
147
151
251
186
254
294
2013
DE 2.04
LLJ
236
147
151
255
186
258
294
2013
DE 2.05
LLJ
244
147
231
178
250
298
2013
DE 2.07
LLJ
244
147
231
178
250
298
2013
DE 2.08
LLJ
256
147
247
194
242
2013
DE 2.09
LLJ
240
147
151
243
182
258
294
2013
DE 2.10
LLJ
240
147
151
251
186
254
294
2013
DE 2.11
LLJ
244
147
151
251
186
254
294
2013
DE 2.12
LLJ
240
147
151
235
186
254
294
2013
DE 2.13
LLJ
240
147
151
251
186
254
294
2013
DE 2.14
LLJ
240
147
151
251
186
254
294
2013
DE 2.15
LLJ
240
147
151
243
186
254
294
2013
DE 2.16
LLJ
240
147
151
251
190
254
294
2013
DE 2.17
LLJ
240
147
151
243
186
254
294
2013
DE 2.18
LLJ
240
147
151
251
186
254
294
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
2012
151
151
270
294
294
258
282
294
274
C. Casto 57
2013
DE 2.19
LLJ
240
147
151
243
186
254
294
2013
DE 2.20
LLLJ
240
147
151
251
186
254
2013
DE 2.21
LLJ
240
147
151
235
186
254
294
2013
DE 2.22
LLJ
240
147
151
251
186
254
294
2013
DE 2.23
LLJ
240
147
151
247
186
250
294
2013
DE 2.24
LLJ
240
147
151
182
254
294
2013
DE 2.25
LLJ
240
147
151
251
186
254
294
2013
DE 2.26
LLJ
240
147
151
251
186
254
294
2013
DE 2.27
LLJ
240
147
151
255
178
254
294
2013
DE 2.28
LLJ
240
147
151
251
186
254
294
2013
DE 2.29
LLJ
240
147
151
247
186
254
294
2013
DE 2.30
LLJ
240
147
151
231
186
254
294
2013
DE 2.31
LLJ
240
147
151
243
186
254
294
2013
DE 2.32
LLJ
240
147
151
243
186
254
294
2013
DE 2.33
LLJ
240
147
151
255
178
254
294
2013
DE 2.34
LLJ
240
147
151
235
186
254
294
2013
DE 2.35
LLJ
240
147
151
251
186
254
294
2013
DE 2.36
LLJ
240
147
151
255
178
254
294
2013
DE 2.37
LLLJ
244
147
151
227
178
186
250
294
2013
DE 2.38
LLLJ
256
147
151
247
182
194
242
274
2013
DE 2.39
LLJ
240
147
151
239
186
254
2013
DE 2.40
LLJ
240
147
151
243
186
254
2013
DL 2.01
LLJ
240
147
151
251
186
254
294
2013
DL 2.02
LLJ
236
147
151
243
186
254
294
2013
DL 2.03
LLJ
240
147
151
251
186
254
294
2013
DL 2.04
LLJ
240
147
151
251
186
254
294
2013
DL 2.05
LLJ
240
147
151
251
186
254
294
2013
DL 2.06
LLLJ
244
147
231
178
250
270
2013
DL 2.07
LLJ
240
151
190
250
270
2013
DL 2.08
LLJ
244
151
231
182
250
298
2013
DL 2.09
LLJ
236
147
151
255
186
254
294
2013
DL 2.10
LLJ
240
147
151
2013
DL 2.11
LLJ
240
147
151
2013
DL 2.12
LLJ
240
147
2013
DL 2.13
LLJ
240
147
2013
DL 2.14
LLJ
240
147
2013
DL 2.15
LLJ
240
147
2013
DL 2.16
LLJ
240
147
2013
DL 2.17
LLJ
244
147
2013
DL 2.18
LLJ
240
147
2013
DL 2.19
LLJ
240
147
270
294
294
278
298
186
270
251
186
254
243
186
270
243
186
254
186
270
182
254
294
186
282
294
231
178
250
298
151
251
186
250
294
151
247
186
250
294
151
155
151
155
282
294
286
294
282
C. Casto 58
2013
DL 2.20
LLJ
240
147
151
243
186
254
2013
DL 2.21
LLJ
256
147
2013
DL 2.22
LLJ
240
147
2013
DL 2.23
LLJ
240
147
151
2013
DL 2.24
LLJ
240
147
2013
DL 2.25
LLJ
240
2013
DL 2.26
LLJ
2013
DL 2.27
2013
247
194
242
227
182
254
294
247
186
254
294
151
251
186
254
294
147
151
235
186
254
294
240
147
151
243
186
254
294
LLJ
240
147
151
251
186
254
294
DL 2.28
LLJ
244
147
231
178
250
298
2013
DL 2.29
LLJ
256
147
247
194
242
2013
DL 2.30
LLJ
244
147
231
178
250
298
2013
DL 2.31
LLJ
240
147
151
235
190
254
294
2013
DL 2.32
LLJ
240
147
151
243
186
254
294
2013
DL 2.33
LLJ
256
147
247
194
242
2013
DL 2.34
LLJ
244
147
231
182
250
2013
DL 2.35
LLJ
240
147
151
186
270
2013
DL 2.36
LLJ
240
147
151
243
186
250
294
2013
DL 2.37
LLJ
240
147
243
186
270
298
2013
DL 2.38
LLJ
240
147
251
186
254
294
2013
DL 2.39
LLJ
244
147
231
182
250
298
2013
DL 2.40
LLJ
240
147
151
251
186
254
294
2013
DL 2.41
LLJ
240
147
151
243
186
254
294
2013
DL 2.42
LLLJ
240
147
151
247
186
254
2013
DL 2.43
LLJ
244
147
231
178
250
298
2013
DL 2.44
LLJ
240
147
151
251
186
254
294
2013
DL 2.46
LLJ
240
147
151
186
246
282
2013
DL 2.47
LLJ
240
147
151
186
270
278
2013
DL 2.48
LJ
240
147
151
2013
DL 2.49
LLJ
240
147
151
227
178
250
294
2013
DL 2.51
LLJ
240
147
151
247
186
254
294
2013
DL 2.52
LLJ
240
147
151
239
182
254
294
2013
DM 2.01
LLJ
240
147
151
247
186
254
294
2013
DM 2.02
LLJ
240
147
151
251
186
254
294
2013
DM 2.03
LLJ
240
147
151
251
186
254
294
2013
DM 2.04
LLJ
240
147
151
251
186
254
294
2013
DM 2.05
LLJ
240
147
151
251
186
254
294
2013
DM 2.06
LLJ
240
147
151
251
186
254
294
2013
DM 2.07
LLJ
240
147
151
251
190
254
294
2013
DM 2.08
LLJ
240
147
151
251
186
250
294
2013
DM 2.09
LLJ
240
147
151
251
186
254
294
2013
DM 2.10
LLJ
240
147
151
247
186
254
294
151
243
186
294
270
274
274
298
278
282
294
278
C. Casto 59
2013
DM 2.11
LLJ
240
147
151
239
186
254
294
2013
DM 2.12
LLJ
240
147
151
243
186
254
294
2013
DM 2.13
LLJ
240
147
151
243
186
254
294
2013
DM 2.14
LLJ
240
147
151
235
186
254
294
2013
DM 2.15
LLJ
240
147
151
235
186
254
294
2013
DM 2.16
LLJ
240
147
151
247
186
250
294
2013
DM 2.17
LLJ
240
147
151
251
186
254
294
2013
DM 2.18
LLJ
240
147
151
251
186
254
294
2013
DM 2.19
LLJ
240
147
151
251
186
250
294
2013
DM 2.20
LLJ
240
147
151
243
186
254
294
2013
DM 2.21
LLLJ
240
147
151
251
186
254
2013
DM 2.22
LLJ
240
147
151
2013
DM 2.23
LLJ
240
147
151
247
186
250
294
2013
DM 2.24
LLJ
240
147
151
243
186
254
294
2013
DM 2.25
LLJ
240
147
151
235
186
254
294
2013
DM 2.26
LLJ
240
147
151
247
186
254
294
2013
DM 2.27
LLJ
240
147
151
247
186
254
294
2013
DM 2.28
LLJ
240
147
151
235
190
254
294
2013
DM 2.29
LLJ
240
147
151
243
186
254
294
2013
DM 2.30
LLJ
240
147
151
231
178
250
298
2013
IE 2.01
LLJ
240
147
151
251
186
254
294
2013
IE 2.02
LLJ
240
147
151
247
186
254
294
2013
IE 2.03
LLJ
240
147
151
243
186
254
294
2013
IE 2.04
LLJ
240
147
151
251
186
254
290
2013
IE 2.05
LLJ
240
147
151
235
186
254
294
2013
IE 2.06
LLJ
240
147
151
182
266
2013
IE 2.07
LLJ
240
147
151
251
186
254
298
2013
IE 2.08
LLJ
240
147
151
235
186
254
294
2013
IE 2.09
LLJ
240
147
151
251
186
254
294
2013
IE 2.10
LLJ
240
147
151
243
186
254
294
2013
IE 2.11
LLJ
240
147
151
243
186
254
294
2013
IE 2.12
LLJ
240
147
151
251
186
254
294
2013
IE 2.13
LLJ
240
147
151
243
186
254
294
2013
IE 2.14
LLLJ
240
147
151
247
186
254
282
2013
IE 2.15
LLJ
240
147
243
182
266
282
2013
IE 2.16
LLJ
240
147
151
243
182
274
2013
IE 2.17
LLJ
240
147
151
247
186
250
294
2013
IE 2.18
LLJ
240
147
151
243
186
254
294
2013
IE 2.19
LLJ
240
147
151
243
186
254
294
2013
IE 2.20
LLJ
240
147
151
251
190
254
294
2013
IE 2.21
LLJ
244
147
151
231
148
242
298
186
270
294
270
298
278
294
C. Casto 60
2013
IE 2.22
LLJ
256
147
247
194
242
2013
IE 2.24
LLJ
240
147
2013
IE 2.25
LLJ
240
147
2013
IE 2.26
LLJ
240
2013
IE 2.27
LLJ
240
2013
IE 2.28
LLJ
2013
IE 2.29
2013
151
243
186
254
151
251
186
254
151
247
190
254
147
151
247
186
254
294
240
147
151
251
186
254
294
LLJ
240
147
151
243
182
258
294
IE 2.30
LLJ
240
147
151
251
186
254
294
2013
IE 2.31
LLJ
240
147
151
251
186
254
294
2013
IE 2.32
LLJ
240
147
151
243
186
254
294
2013
IE 2.33
LLJ
240
147
151
251
186
254
294
2013
IE 2.34
LLJ
240
147
151
251
186
254
294
2013
IE 2.35
LLJ
240
147
151
243
182
246
282
2013
IE 2.36
LLJ
240
147
186
278
286
2013
IE 2.37
LLJ
236
147
151
243
182
246
290
2013
IE 2.38
LLJ
240
147
151
243
186
254
294
2013
IE 2.39
LLJ
240
147
151
243
186
254
294
2013
IE 2.41
LLJ
240
147
151
231
186
254
294
2013
IE 2.42
LLJ
240
147
151
251
186
254
2013
IE 2.43
LLJ
240
147
151
182
254
2013
IL 2.01
LLJ
240
147
151
247
186
254
2013
IL 2.02
LLJ
240
147
151
251
186
270
282
2013
IL 2.03
LLJ
240
147
151
182
250
270
2013
IL 2.04
LLJ
240
147
151
251
186
254
294
2013
IL 2.05
LLJ
240
147
151
251
186
254
294
2013
IL 2.06
LLJ
244
147
227
174
250
298
2013
IL 2.07
LLJ
240
147
186
254
286
2013
IL 2.08
LLJ
256
147
247
186
242
274
2013
IL 2.09
LLJ
240
147
247
186
270
282
2013
IL 2.10
LLJ
240
147
251
186
254
2013
IL 2.11
LLJ
240
147
251
186
270
278
2013
IL 2.12
LLJ
240
147
243
186
254
278
2013
IL 2.14
LLJ
244
147
235
178
250
2013
IL 2.15
LJ
240
147
243
186
266
2013
IL 2.16
LLJ
240
147
243
186
270
278
2013
IL 2.17
LLJ
240
147
243
186
254
270
2013
IL 2.18
LLJ
240
147
247
186
254
266
2013
IL 2.19
LLJ
240
147
151
186
258
282
2013
IL 2.20
LLJ
240
147
151
186
254
2013
IL 2.21
LLJ
240
147
151
186
258
282
2013
IL 2.22
LLJ
240
147
186
254
266
151
151
255
243
274
294
294
266
274
298
294
298
294
C. Casto 61
2013
IL 2.23
LLJ
240
147
247
186
254
266
2013
IL 2.24
LLJ
240
147
239
182
270
286
2013
IL 2.25
LLJ
240
151
251
186
254
294
2013
IL 2.26
LLJ
240
147
151
251
186
254
294
2013
IL 2.27
LLJ
240
147
239
186
250
290
2013
IL 2.28
LLJ
240
147
247
186
254
294
2013
IL 2.29
LLJ
244
147
231
178
250
298
2013
IL 2.30
LLJ
240
147
151
251
186
250
294
2013
IL 2.31
LLLJ
240
147
151
243
186
254
2013
IL 2.32
LLJ
240
147
151
239
186
254
2013
IL 2.33
LLJ
240
147
151
235
190
254
2013
IL 2.34
LLLJ
248
195
199
251
218
254
2013
IL 2.35
LLJ
240
147
151
251
2013
IL 2.36
LLJ
240
147
151
251
2013
IL 2.37
LLJ
240
147
2013
IL 2.38
LLJ
240
147
151
2013
IL 2.39
LLJ
240
147
2013
IL 2.40
LLJ
240
2013
IL 2.41
LJ
2013
IL 2.42
LLJ
2013
IL 2.43
2013
151
203
266
294
294
294
262
270
254
294
148
254
294
243
186
270
251
186
250
151
186
262
274
147
155
186
270
278
240
147
151
186
262
240
147
151
186
270
286
LLJ
240
147
151
186
278
282
IM 2.01
LLJ
240
147
151
247
186
254
294
2013
IM 2.02
LLJ
240
147
151
243
186
254
294
2013
IM 2.03
LLJ
240
147
151
239
186
254
294
2013
IM 2.04
LLJ
240
147
151
251
190
254
294
2013
IM 2.05
LLJ
240
147
151
243
186
254
2013
IM 2.06
LLJ
240
147
151
243
186
254
294
2013
IM 2.07
LLJ
240
147
151
251
186
254
294
2013
IM 2.08
LLJ
240
151
247
186
254
294
2013
IM 2.09
LLJ
240
147
151
251
186
254
294
2013
IM 2.10
LLJ
240
147
227
178
250
294
2013
IM 2.11
LLJ
240
147
151
243
186
254
294
2013
IM 2.12
LLJ
240
147
151
251
186
254
294
2013
IM 2.13
LLJ
240
147
151
243
186
254
294
2013
IM 2.14
LLJ
240
147
151
251
186
254
294
2013
IM 2.15
LLJ
240
147
151
247
186
254
294
2013
IM 2.16
LLJ
240
147
151
247
186
254
294
2013
IM 2.17
LLJ
240
147
151
251
186
254
294
2013
IM 2.18
LLJ
240
147
151
254
186
254
294
2013
IM 2.19
LLJ
240
147
151
247
186
254
294
2013
IM 2.20
LLJ
240
147
151
243
182
254
294
278
294
C. Casto 62
2013
IM 2.21
LLJ
240
147
151
255
186
254
294
2013
IM 2.22
LLJ
240
147
151
251
186
254
294
2013
IM 2.23
LLJ
240
147
151
243
186
254
294
2013
IM 2.24
LLJ
240
147
151
243
186
254
294
2013
IM 2.25
LLJ
240
147
151
255
186
254
294
2013
IM 2.26
LLJ
240
147
151
251
186
254
294
2013
IM 2.27
LLJ
240
147
151
251
186
254
294
2013
IM 2.28
LLJ
240
147
151
254
186
254
2013
IM 2.29
LLJ
240
147
151
251
186
254
294
2013
IM 2.30
LLJ
240
147
151
251
186
254
294
2013
IM 2.31
LLJ
240
147
151
235
186
254
294
2013
IM 2.32
LLJ
240
147
151
243
186
254
294
2013
IM 2.33
LLJ
256
147
247
194
242
274
2013
WE 2.01
LLJ
240
147
151
251
186
254
282
2013
WE 2.02
LLJ
240
147
151
251
186
254
294
2013
WE 2.03
LLJ
244
147
151
231
182
250
298
2013
WE 2.04
LLLJ
240
147
151
243
182
254
2013
WE 2.05
LLJ
240
147
151
251
186
254
294
2013
WE 2.06
LLJ
244
147
151
231
182
250
298
2013
WE 2.07
LLJ
240
147
151
251
186
254
294
2013
WE 2.08
LLJ
240
147
151
247
186
254
294
2013
WE 2.09
LLJ
236
147
151
255
186
254
294
2013
WE 2.11
LLJ
240
147
151
235
186
254
294
2013
WE 2.12
LLJ
240
147
151
251
186
254
294
2013
WE 2.13
LLJ
260
147
247
190
242
2013
WE 2.14
LLJ
240
147
151
231
190
254
294
2013
WE 2.15
LLJ
240
147
151
251
186
254
294
2013
WE 2.16
LLJ
240
147
151
247
186
254
294
2013
WE 2.25
LLJ
240
147
151
243
182
254
294
2013
WE 2.26
LLJ
240
147
151
251
186
254
294
2013
WE 2.27
LLJ
236
147
151
235
186
254
294
2013
WE 2.28
LLJ
240
147
151
251
186
254
294
2013
WE 2.29
LLJ
240
147
151
255
186
254
294
2013
WE 2.30
LLJ
240
147
151
239
186
254
294
2013
WE 2.31
LLJ
244
147
151
243
190
258
294
2013
WE 2.32
LLJ
240
147
151
247
186
254
294
2013
WE 2.33
LLJ
240
147
151
251
186
254
294
2013
WE 2.34
LLJ
240
147
151
251
186
254
2013
WE 2.35
LLLJ
240
147
151
239
190
254
2013
WE 2.36
LLJ
240
147
151
251
186
254
290
2013
WE 2.37
LLJ
240
147
151
243
186
254
294
282
270
294
278
294
270
294
C. Casto 63
687
2013
WE 2.38
LLJ
240
147
151
186
254
294
2013
WE 2.39
LLJ
240
147
151
251
186
254
294
2013
WE 2.40
LLJ
240
147
2013
WL 2.01
LLJ
256
147
243
186
254
202
270
2013
WL 2.02
LLJ
236
147
186
254
2013
WL 2.03
LLJ
260
147
202
270
2013
WL 2.05
LLJ
240
147
151
251
186
254
294
2013
WL 2.07
LLJ
240
147
151
239
190
254
294
2013
WL 2.11
LLJ
240
147
151
239
186
254
270
2013
WL 2.12
LLLJ
240
147
151
243
182
250
270
2013
WL 2.15
2013
WL 2.16
LLJ
240
147
151
251
186
254
294
LLJ
236
147
151
251
186
254
294
2013
WL 2.18
LLJ
240
147
151
251
186
254
294
191
151
255
191
294
278
294
278
294