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dissertation entitled

AN ANALYSIS OF CLIMATE INDUCED HYBRID
SPECIATION IN TIGER SWALLOWTAIL BUTTERFLlES
(PAPILIO)

presented by

Gabriel J. Ording

has been accepted towards fulﬁllment
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AN ANALYSIS OF CLIMATE INDUCED HYBRID SPECIATION IN TIGER
SWALLOWTAIL BUTTERFLIES (PAPIIJO)

By

Gabriel J. Ording

A DISSERTATION
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Entomology

2008

ABSTRACT

AN ANALYSIS OF CLIMATE INDUCED HYBRID SPECIATION IN TIGER
SWALLOWTAIL BUTTERFLIES (PAPILIO)

By

Gabriel J. Ording

North American Papilio canadensis and P. glaucus (Lepidoptera: Papilionodae,
these Papilio = Pterourus), have been described as having allopatric distributions
separated by a narrow hybrid zone. The range of hybridization is directly correlated with
a well-deﬁned thermal landscape. In light of recent climate shifts, potentially associated
with global warming, there have been increased levels of genetic introgression. This
dissertation describes the morphological and genetic status of unique isolated hybrid
swarm populations, on South Manitou Island in Michigan and in the Battenkill River
Valley near the border of New York and Vermont, that have arisen near the range
boundaries of these two closely related species of Tiger Swallowtail butterﬂies. Climate
induced genomic mixing may be responsible for ‘climatic speciation’, as appears to be
occurring in a case of incipient speciation near the border of southern New York and
Vermont. Additionally, it is suggested that the generation of these unique genetic
populations best explains the origins of a recently described new Papilio species
(Pterourus appalachiensis Pavulaan & Wright, 2002) via hybrid speciation.
Furthermore, the signiﬁcance of an identiﬁed X-linked gene complex controlling both
diapause and the expression of the lactate dehydrogenase enzyme is examined in
laboratory crosses and is proposed as a primary mechanism in the historic maintenance of

the hybrid zone and also is likely instrumental in the reported cases of hybrid speciation.

Again, to my family

iii

ACKNOWLEDGMENTS

I would like to thank my co-major advisers, Dr. Mark Scriber for his never ending
supply of enthusiasm, guidance, patience and understanding; Dr. Larry Besaw for many
years of mentorship in the realms of science education. Thank you to the additional members
of my guidance committee, Dr. Kim Scribner and Dr. Jim Smith for their willingness to
participate in my doctoral program, both have put forth an astounding amount of energy
towards helping to improve this research project.

Thanks to the National Park Service, at Sleeping Bear Dunes, for allowing this
research to take place and providing special use and specimen collection permits. Special
thanks to Steve Yancho, the resource manager at Sleeping Bear Dunes National Park.
Thanks to Howard Romack for his extraordinary help in obtaining specimens from Vermont
and New York.

Many thanks to all of the graduate students and lab technicians of the Scriber lab that
have come and gone over the course of this project. A great deal of work was accomplished
as a result of help ﬁ'om a large group of wonderful people. Among this group I would like to
speciﬁcally thank Michelle Oberlin, Martha Smith Caldas, and Matt Lehnert. Also ﬁ'om this
group, I would like to extend an enormous thank you to Rodrigo Mercader for ongoing

support and ﬁ'iendship as we worked through our doctoral programs together.

A very special thanks and a huge hug to my big brother, Dr. Dominic Ording, for

sharing his thoughtful wisdom and loving support.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vii
LIST OF FIGURES .................................................................................. x
CHAPTER 1:

INTRODUCTION ..................................................................................... 1
CHAPTER 2:

A STABLE HYBRID SWARM .................................................................... 35
Introduction ............................................................................................ 35
Materials and Methods ............................................................................... 38
Results .................................................................................................. 46
Discussion .............................................................................................. 51
CHAPTER 3:

CAN INTROGRESSION LEAD TO REPRODUCTIVE ISOLATION? :INCIPIENT
HYBRID SPECIATION BETWEEN PAPILIO GLA UC US AND P. CANADENSIS ....... 53
Introduction ............................................................................................. 53
Materials and Methods ................................................................................ 56
Results ................................................................................................... 63
Discussion ............................................................................................... 80
CHAPTER 4:

TRAIT LINKAGE ON THE X-CI-IROMOSOME OF PAPILIO CANADENSIS AND P.

GLA UCUS, REVEALED BY A HIGHLY INF ORMATIVE BACKCROSS ................. 86
Introduction ............................................................................................. 86
Materials and Methods ................................................................................. 93
Results ................................................................................................... 96
Discussion .............................................................................................. 103
CHAPTER 5:

SUMMARY AND CONCLUSIONS ............................................................... 108
APPENDIX 1:

RECORD OF DEPOSITIN OF VOUCHER SPECIMENS ...................................... 116
APPENDIX 1.1

VOUCHER SPECIMEN DATA ..................................................................... 118
APPENDD( 2:
POPULATION WING MEASUREMENTS AND ALLOZYME DATA ..................... 120

APPENDIX 3:
POPULATION EMERGENCE DATA ............................................................. 138

LITERATRUE CITED ................................................................................ 154

vi

LIST OF TABLES

Table 1.1 — Summary of genetic differences discussed between Papilio glaucus and Papilio
canadensis. (Modiﬁed ﬁom Table I in Scriber 1990) .............................................. 21

Table 2.1. South Manitou Island male P. canadensis specimen collection data ................ 39

Table 2.2. Forewing length measurements for male specimens collected on South Manitou
Island, Leelenau County, Michigan from 1998-2005. Values represented are the average
length in mm +/- s.e. from the distal tip of the wing to the basal thoracic attachment. Values
are based upon the calculated means of the left and right forewing lengths for each specimen
analymd. The years not connected by the same letter are signiﬁcantly different based upon a
comparison for each pair using the Tukey’s HSD (adjusted P-value of signiﬁcance <0.05)..48

Table 2.3. Hind Wing Black Band widths for male specimens collected on South Manitou
Island, Leelenau County, Michigan ﬁ'om 1998-2005. Values represented are the percent of
the hind wing anal cell that is ﬁlled by a dark pigmented band. Values are based upon the
calculated means of the left and right hind wing black bands for each specimen analyzed.
There were no signiﬁcant differences across years based upon analysis using Tukey’s HSD
(adjusted P-value of signiﬁcance <0.05) ............................................................. 48

Table 2.4. Allele ﬁ'equencies for the species diagnostic Pgd allozyme from male specimens
collected on South Manitou Island, Leelenau County, Michigan ﬁom 1998-2005. Allele
frequency values represented are percentages of introgressed glaucus alleles detected through
gel electrophoresis. The years not connected by the same letter are signiﬁcantly different
based upon a determination of both Genic Differentiation and Genotypic Differentiation as
calculated using Genepop Version 3.6 1999 (P-values of <0.05) ................................. 50

Table 3.1. Forewing length measurements for male specimens collected in the Early Flight
and the Late Flights of the Battenkill River Valley of Vermont and New York from 2002-
2004. Values represented are the mean length in mm +/- s.e. from the distal tip of the wing
to the basal thoracic attachment. Values are based upon the calculated means of the left and
right forewing lengths for each specimen analyzed. Sample sizes are presented in
parentheses. Probability values are derived ﬁ'om a nonparametric 2-sample Wilcoxon test
performed using JMP 6.0. Probabilities for comparisons that are signiﬁcantly different are
indicated with an *. All comparisons are signiﬁcantly different with p-values <0.0001......64

Table 3.2. Hind wing black bandwidth measurements for male specimens collected in the
Early Flight and the Late Flights of the Battenkill River Valley of Vermont and New York
from 2002-2004. Values represented are the mean percent +/- s.e. of the hind wing anal cell
that is ﬁlled by a dark pigmented band. Values are based upon the calculated means of the
left and right hind wing black bands for each specimen analyzed. Sample sizes are presented
in parentheses. Probability values are derived from a nonparametric 2-sample Wilcoxon test

vii

performed using JMP 6.0. Probabilities for comparisons that are signiﬁcantly different are
indicated with an *. All comparisons are signiﬁcantly different with P-values <0.0001 . .64

Table 3.3. Pupal weight measurements for ﬁeld reared specimens from both the Early Flight
and the Late Flights of the Battenkill River Valley of Vermont and New York from 2003-
2004. Values represented are the mean pupal weights in milligrams +/- s.e. Sample sizes are
presented in parentheses. Probability values are derived ﬁ'om a nonparametric 2-sarnple
Wilcoxon test performed using JMP 6.0. Probabilities for comparisons that are signiﬁcantly
different are indicated with an *. All comparisons signiﬁcantly different with p-values
<0.0001 .................................................................................................... 65

Table 3.4. Species diagnostic allele frequencies for two X-linked loci (Pgd and th) in wild
captured male specimens from the Early Flight and the Late Flight populations of the
Battenkill River Valley for 2000-2004. Allele frequencies represented are the combinations
of all of the diagnostic alleles for each species lumped together in order to represent the
degree of genetic introgression. The species diagnostic Pgd alleles for P. canadensis are 150,
125 and 80, and for P. glaucus are 100 and 50. The species diagnostic th alleles for P.
canadensis are 80 and 40, and for P. glaucus is 100. The th hybrizyme is the novel th 20
allele that was consistently detected in the Late Flight. The *’s for this allele in the 2000 and
2001 Late Flight populations indicate that the allele was in fact present at high frequencies,
but as a result of being novel, failed to be identiﬁed and had been misinterpreted as faulty gel
results. An estimation of the likely allele frequency of the hybrizyme for those two years is
shown in parentheses .................................................................................... 67

Table 3.5. Wright’s Fst values for the Early and Late Flight populations of the Battenkill
River Valley (2000-2004) based upon allozyme data collected ﬁom wild captured male
specimens. Pairwise values were calculated using Genepop Version 3.6. Values of greater
than 0.15 indicate signiﬁcant population differentiation and suggest little gene ﬂow (F rankarn
et a]. 2002). F st values are presented for both of the individual X-linked loci analyzed (Pgd
and th). An "' indicates that year for which the novel th 20 hybrizyme was potentially
present but unidentiﬁed and therefore not appropriately accounted for. F st values that are
signiﬁcant are underlined ............................................................................... 70

Table 3.6. mtDNA haplotypes and diagnostic X-linked loci (Pgd and th) allozyme
genotypes for fourteen wild captured males ﬁorn 2003 from both the Early and Late ﬂights
of the Battenkill River Valley populations of Papilio. Introgressed glaucus alleles are
presented in bold print .................................................................................. 72

Table 3.7. Comparisons of the days to emergence between Early Flight and Late F light ﬁeld
reared pupae. These comparisons were conducted in two sequential years across multiple
temperatures (2003 at 18 °C, 22 °C, 26 °C and 2004 at 14 °C, 18 °C, 22 °C, 26 °C). Values
represented are the mean number of days +/- s.e. from the time that pupae were removed
from winter like conditions to the day of pupal eclosion Sample sizes are presented in
parentheses. Probability values are derived ﬁ'om a nonparametric 2-sample Wilcoxon test
performed using JMP 6.0. Probabilities for comparisons that are signiﬁcantly different are
indicated with an *. All comparisons are signiﬁcantly different with p-values <0.0001. ....74

viii

Table 3.8. Comparisons of the days to emergence between males and females in both the
Early Flight and Late Flight ﬁeld reared pupae. These comparisons were conducted in two
sequential years across multiple temperatures (2003 at 18 °C, 22 °C, 26 °C and 2004 at 14
°C, 18 °C, 22 °C, 26 °C). Values represented are the mean number of days +/- s.e. from the
time that pupae were removed from winter like conditions to the day of pupal eclosion
Sample sizes are presented in parentheses. Probability values are derived from a
nonparametric 2-sampleWilcoxon test performed using IMP 6.0. Probabilities for
comparisons that are signiﬁcantly different are indicated with an * ............................. 79

Table 4.1. X-chromosome linkage maps for each of the offspring produced in the18006
brood. The linkage maps represented indicated the following loci in sequential order: dark
morph suppressor / Pgd / diapause / th. Color-coding allows for determination of the
parental origin of each chromosome or chromosomal segment. Portions of chromosomes
that could not be determined with conﬁdence are indicated with ??? ........................... 97

Table 4.2. Relative survival probabilities for 18006 backcross females. Survival
probabilities are relative to those of males with the same paternally inherited [db and Pgd
alleles. Probabilities were obtained by dividing the number of females with each genotype
by the number of males with the corresponding paternal haplotype. These numbers are given
in parentheses as (#females / #males) ............................................................... 102

ix

LIST OF FIGURES

Figure 1.1. A diagrammatic illustration of the process by which incompatibilities could arise
in diverging populations. Time is represented as moving forward moving up the diverging
lineages. In each case the lower case letters represent the ancestral allele, the upper case
letters represent the derived characters. Each derived allele has not been “tested” against the
genetic background of the opposite lineage. With each newly derived allele there is an
increasing opportunity and number of possible combinations in which genetic
incompatibilities could occur. The end result of the “snowballing effect” would be complete
reproductive isolation. (Figure modiﬁed from Coyne and Orr 2004.) ........................... 13

Figure 1.2. The geographic ranges of two closely related species of Tiger Swallowtail
Butterﬂies, Papilio canadensis to the north and Papilio glaucus to the south (modiﬁed from
Scriber 1996a). Indicated in the Western United States are the ranges of three largely
sympatric species, P. rutulus, P. eurymedon, and P. multicaudams. Represented in Mexico
is the range of P. alaxiares .............................................................................. 20

Figure 1.3. Proposed linkage map of the X-chromosome loci in Tiger Swallowtail Butterﬂies
(Modiﬁed from Hagen and Scriber 1989). The chromosome represented in white depicts the
canadensis-type chromosome, whereas that represented in black depicts the glaucus-type.

Chapter 4 identiﬁes the likely possibility that the th 100 and od- loci are in fact much more
closely linked that suggested by this linkage map ................................................... 22

Figure 1.4. Geographic representation of the historic hybrid zone as well as the identiﬁed
distribution of the recently described Papilio appalachiensis, and also the location of
incipient speciation in the Battenkill river valley of New York and Vermont .................. 24

Figure 1.5. X-linked allele ﬁequencies (Pgd-100 and th-lOO allozymes) along the thermal
landscape (degree-days above a base of 50°F [°Dp]) across the eastern United States. Note
the sharp decline in th-lOO at 2700on and the nonconcordant but steep increase in Pgd-
100 frequencies between 2300 and 2800on. The 70 different populations (each represented
by 10-125 males) are from Vermont and northern Michigan to southern Ohio and North
Carolina (data from Scriber and Ording 2005; and IMS unpublished data). The bivoltine P.
glaucus are at the right (>2800°Dp), while everything else is univoltine. These other
populations include a range of P. canadensis populations that exhibit differential degrees of
P. glaucus introgression. (Figure modiﬁed ﬁ'om Scriber et a1. 2007) ............................ 32

Figure 2.1. Papilio canadensis and P. glaucus hybrid zone across the Midwest. The shaded
region in Michigan roughly indicates what has historically been considered the hybrid zone
between Papilio glaucus and P. canadensis (Nielsen, 1999). The shaded region across
Wisconsin into Minnesota represents the 50 year average degree-day accumulation and
northern limit allowing for two generations of Papilr'o glaucus. The star indicates the
location of South Manitou Island ...................................................................... 36

Figure 2.2. Forewing length measurements (measurement A) are the distance from the tip of
the forewing to the thoracic wing base attachment (Figure modiﬁed from Leubke et al.
1988) ...................................................................................................... 41

Figure 2.3 Black band width measurements are the percentage of the hindwing anal cell that
is ﬁlled by the dark band labeled A. (Modiﬁed from Luebke et al. 1988) ..................... 42

Figure 2.4. Bivariate linear regression of black band width by forewing length (R2 = 0.004)
for male Papilio canadensis specimens collected on South Manitou Island from 1998-2005.
Analysis was performed using IMP statistical software version 6.0 ............................ 47

Figure 3.1. Degree of P. glaucus introgression into both the Early Flight and the Late Flight
populations of the Battenkill River Valley, for the diagnostic X-linked Pgd locus. The
portion of each graph that is ﬁlled in with white represents the proportion of introgressed
glaucus Pgd alleles in that population for 2000-2004 ............................................. 68

Figure 3.2. Representation of the presence and proportion of the allele frequencies of the
novel X-linked th 20 hybrizyme in both the Early Flight and the Late Flight populations of
the Battenkill River Valley. The portion of each graph that is ﬁlled in with gray represents
the proportion of the hybrizyme th allele in these populations for 2002-2004 ............... 69

Figure 3.5. Histogram comparison of the days to emergence between Early Flight and Late
Flight ﬁeld reared pupae in 2003 at 18 °C, 22 °C, and 26 °C ..................................... 75

Figure 3.6. Histogram comparison of the days to emergence between Early Flight and Late
Flight ﬁeld reared pupae in 2004 at 14 °C, 18 °C, 22 °C, and 26 °C ............................. 77

xi

CHAPTER 1:

INTRODUCTION

An American Entomologist ﬁrst coined the term speciation in 1906. In the words
of Orator F. Cook, “Speciation, to give the process a name, is the origination or
multiplication of species by subdivision, usually, if not always, as a result of
environmental incidents ” (Berlocher 1998). Before and since that time there has been an
increasingly heated debate about the “species concept” and the best deﬁnition to apply
(Hey 2006). Until recently the biological species concept (BSC) championed by Ernst
Mayr (1963) has been used as the primary default in most biological investigations. The
BSC indicates that species are groups of interbreeding populations that are reproductively
isolated from each other (Mayr 1963, 1995). However, there are weaknesses associated
with the BSC that make it difficult to apply to every biological situation. As a result
there have been many attempts at redeﬁning species, but so far no single deﬁnition works
in every situation. Concisely deﬁning the species concept will likely be a never-ending
source of scientiﬁc controversy. A concrete deﬁnition is of great importance in certain
situations, in taxonomy for example. A concise deﬁnition is also important in
conservation efforts, where funding and resources are limiting. For many evolutionary
biologists however, who are more concerned with evolutionary processes, the need for a
single species deﬁnition is less crucial, and accurately identifying the mechanisms by
which differentiated populations of organisms arise is of the utmost importance.
Identiﬁcation of the mechanisms that drive evolution will allow for explanations as to the

“origins” of the vast amount of biodiversity on the planet.

As indicated by Cook, speciation is best described as a process rather than a
singular event, and is the series of events that lead to populations of organisms becoming
genetically differentiated and ultimately reproductively isolated from one another
(Harrison 1993; Coyne and Orr 2004). The relationship between two distinct populations
can be placed along a continuum on a sliding scale between early phases of
differentiation and true species. Central to the BSC is the establishment of reproductive
isolation between populations, such that the genetic differences between unique
populations can become enhanced through further evolutionary processes. As ﬁrst
developed by Mayr, the BSC indicated that reproductive isolation is accomplished
through intervening “isolating mechanisms”. Many evolutionary biologists have opted
for the use of the alternative phrase “isolating barriers” (Coyne and Orr 2004), to describe
the combination of factors that either prevent mating altogether (prezygotic) and / or
those factors that result in infertile or inviable offspring being produced if mating were to
occur (postzygotic). It has been suggested that the presence of only postzygotic barriers
to gene ﬂow indicates that the differentiating populations have only recently begun down
the evolutionary pathway towards complete speciation, and that the presence of
prezygotic barriers is indicative of being further along the evolutionary pathway towards
complete speciation (Presgraves 2002). Reinforcement has been proposed as the process
by which prezygotic barriers to gene ﬂow arise in areas where recently diverged
populations overlap, such as in hybrid zones (Howard 1993). These additional prezygotic
barriers would act to prevent futile attempts at mating that would only result in wasted

energy expenditure.

When originally developed, the BSC relied upon geographic isolation, to allow
for divergence in allopatry (Mayr 1963). The work of Cook indicates that he too meant
that speciation occurred as a result of geographic isolation, when he referred to
“environmental incidents” (Berlocher 1998). Any geographic barrier (a newly arising
mountain range, river, ocean, or glacial advance) that can prevent gene ﬂow between
populations can act as a ready made isolating mechanism. This is a piece of what makes
allopatric speciation so appealing and intuitive is that there is a clear barrier that prevents
any gene ﬂow, thus allowing for an accumulation of genetic changes to occur
independently in both populations. Because of its elegant simplicity, allopatric speciation
has been the dominating view as to how speciation has occurred.

A recognized alternative to allopatry, heavily championed by Guy Bush of
Michigan State University in the early 1960’s, is the concept of sympatric speciation.
Sympatric speciation does not require any geographic distance or barrier to allow
speciation to occur. Typically the mechanism that allows for sympatric speciation
involves disruptive selection leading to a shift in the ecological niche of an organism.
Two of the model examples exploring sympatric speciation have been an alteration in the
gene complexes that determine host plant preferences in Rhagoletis pomonella (Bush
1975, 1993; Bush and Smith 1998) and sexual selection in African cichlids (Higashi et al.
1999; Mendelson and Shaw 2005).

Another potential pathway to allow for speciation has been proposed, allochronic
speciation (Alexander and Bigelow 1960). Allochronic speciation, similar to and perhaps
best described as a subcategory of sympatry (Bush 1975; Tauber and Tauber 1981), does

not require any geographic boundaries to prevent gene ﬂow, but relies upon populations

becoming temporally isolated. If populations are separated by time, during the
reproductive portion of their lifecycle, this has the same effect of preventing mating
Wmmdoﬂmghﬂplmtmfmmterwogﬁﬁmsystemsmmwnmin
chainsandglaciers. quuaﬁlycﬁedexamplesofspeciatiomproposedtohavebeen
driven by allochronic speciation include North American crickets (Alexander and
Bigelow 1960; Harrison 1979), paiodic cicadas (Cooley et al. 2001), and gall-forming
aphids (Abbot and Withgott 2004). The ultimate ecological effect of both sympatric and
allochronic speciation is “niche packing”, allowing unique populations to take advantage
of slightly different and untapped ecological resources, while minimizing or eliminating
niche overlap and competition.

Speciationbeingviewedasaprocesssuggeststhattheremustbeasequenceof
events that initiates the process of divergence, and that an accumulation of genetic
changes must occur before complete reproductive isolation can be achieved. Temporal
isolation may be the ﬁrst step in the process of speciation, but it may also be the only
necessary step to allow for complete reproductive isolation (T auber and Tauber 1981).
This being the case, if the timing of signiﬁcant life-history traits is controlled by a
relatively small number of genes, or even an individual locus, minimal genetic

modiﬁcations could result in striking effects.

Climate Change
Biogeography is the ecological discipline devoted to investigating the interaction of
environmental factors that ultimately determine the distribution and abundance of

organisms. There is a unique and intricate interplay of ecological factors (abiotic and

biotic) for which every species has evolved its own range(s) of tolerance. Global climate
change is providing evolutionary ecologists unique opportunities to investigate factors
that are involved in the maintenance of distinct populations and also the factors that may
contribute to the processes of speciation. Global warming is having a diverse
combination of ecological impacts on natural populations. Climate change is leading to
shifting environmental conditions and selective pressures. Increased average global
temperatures have resulted in a shortening of winter like weather in many locations
(Stenseth and Mysterud 2002; Parmesan and Yohe 2003). One result of this is the
phenology of ﬂowering plants and animals is being drastically altered. Earlier annual
ﬂowering times have been described for many plants (Miller-Rushing et al. 2006) and the
observed ﬂight times of many insects have been altered (Parmesan 2006). Physiological
shifts in the photoperiodic induction of diapause have been reported in response to
climate change (Bradshaw and Holzapfel 2001). These shifts then have resulted in the
disruption of coevolved synchronized insect plant interactions (Parmesan 2006). The
genetic diversity of some insect species has rapidly eroded in response to natural
selection associated with climate change (Rodriguez-Trelles and Rodriguiz 1998).
Ultimately, this loss of genetic variation will reduce these populations ability to cope with
environmental changes. The predicted rates at which global temperatures are expected to
shift will be far too rapid for even robust populations to cope with, let alone populations
that are genetically challenged.

However, climate change may actually lead to increased levels of genetic diversity
and rates of evolution in some insect populations for a combination of reasons. Insect

population growth rates are strongly inﬂuenced by thermodynamics. Increased

temperatures associated with anthropogenic climate change are expected to lead to
increased population growth rates of certain species in many locations (Frazier et al.
2006). This in itself may have tremendous ecological and evolutionary impacts. In
addition, climate-speciation hypotheses have been proposed that suggest that increased
metabolic rates brought about by increased temperatures may result in increased rates of
mutation, and in turn increased rates of evolution (Rohde 1992; Balanya et al. 2006;
Wright et al. 2006). The ranges of many insects, including butterﬂies, in both Europe
and North America have been found to expand and shift towards higher latitudes and
elevations (Karban and Strauss 2004; Parmesan and Yohe 2003). Shifting species ranges
is now allowing for increased contact and increased levels of gene ﬂow between
historically isolated populations (Ording 2001). Additionally, it has been shown that
mutation rates and genome diversity frequently increases in organisms that are under
stressful environmental conditions (Nevo 2001), as would be the case for animals near
range boundaries or during periods of climatic change. When historically stable hybrid
zones between closely related species are destabilized by shifting climates, this often
serves to promote increased levels of introgression. This in turn, promotes enhanced
levels of genetic variability that can allow newly formed genotypes to evolutionarily
shadow environmental changes. This in effect can lead to ‘climatic speciation’ (Masaki
1978) through the recombination of preexisting characters (Dowling and Secor 1997;

Balanya et al. 2006).

Hybridization

For a long time, botanists have viewed the occurrence of hybridization in nature
as an important process in the evolution of plants. Zoologists on the other hand, have
historically viewed animal hybrids to be evolutionary dead ends that have little or no
ecological value, with hybrids expressing reduced ﬁtness (Mayr 1942). Recently
however, more and more animal investigations are identifying potential ecological and
evolutionary value in hybridization as sources of new genetic combinations (Dowling and
Secor 1997). In some situations, these novel genotypes may exhibit hybrid vigor and
levels of increased ﬁtness as compared to either parental genotype (Arnold and Hodges
1995; Harrison 1993). Additionally, it has been suggested that an evolutionary value of
hybrids is that they can potentially be well adapted to new and unique environmental
conditions (Arnold 1997).

A hybrid zone is a geographic region where the ranges of two genetically distinct
populations overlap, and in which a certain amormt of interspeciﬁc mating occurs that
results in some offspring of mixed ancestry (Harrison 1993). Hybrid zones have been
described as unique locations in which to study the processes of evolution in that they act
as “windows into the evolutionary process” or as “nattu'al laboratories” (Barton and
Hewitt 1985). Hybrid zones can be identiﬁed by the presence of clines. A cline is a
geographic location between two genetically distinct populations in which there is a
gradient identiﬁable ﬁ'om one genetic character state to another. Clines between unique
populations allow for unique opportunities to investigate the processes and mechanisms
that are driving the evolution of isolating mechanisms between distinct populations, and

cline theory is of extreme signiﬁcance when discussing hybrid zones.

Closely related, but distinct species, are often times recognizable based upon one
or more diagnostic characters or markers (morphological or genetic). A cline would
represent the geographic location that demarcates the hybrid zone, the geographic
location in which one could expect to ﬁnd specimens of mixed ancestry. Some authors
use the terms cline and hybrid zone interchangeably. Investigations conducted in these
hybrid zones can help to shed light on the various mechanisms involved in evolutionary
processes, such as speciation (Harrison 1993; Arnold 1997; Coyne and Orr 2004).

The maintenance of the width and shape of a cline is largely the result of a
balance between dispersal and selection. There is a direct relationship between the ability
for long-range dispersal and cline width (Harrison 1993). Similarly, the strength of
selective pressures on various characters will help to dictate cline width. If a cline
exhibits a comparable width along its entire length, this might indicate that the strongest
selective pressures acting to maintain distinct populations are the result of negative
endogenous selection through genetic incompatibilities (Barton and Hewitt 1985).
However, if the cline exhibits a mosaic pattern, this could be indicative of a
heterogeneous environment in which exogenous selective pressures are acting more
strongly. Mosaic clines and hybrid zones, transitions from one population to another,
often times coincide with ecotones (distinct boundaries across an environmental
gradient). The varying environmental conditions on either side of these ecotone
boundaries may in fact be the primary selective pressures that initiated population
divergence (Barton and Hewitt 1985).

The slope of a cline can potentially infer the strength of selection (Kirkpatrick and

Barton 1997). A steep cline for any given character would indicate extremely strong

selective pressures. Different diagnostic characters may exhibit differences in the mean
slope. This would indicate that the relative strength of selection on each locus is variable.
If a steep slope indicates strong selection, a subtler slope would be indicative of relaxed
selective pressures. This differential strength of selection allows for asymmetrical gene
ﬂow at different loci. Certain genetic alleles may have an increased ability to introgress
further beyond the center of the cline than can other alleles. A steep cline for one trait
can potentially be the result of strong selection on that locus directly, or perhaps could be
the result of tight genetic linkage with some other loci under strong selective pressures
(Harrison 1993). In this way, clines and hybrid zones can act as semipermeable barriers
to gene ﬂow.

When sampling and identifying the genotypes of specimens within a cline, hybrid
zones can be categorized along a continuum, ranging from unimodal to bimodal, with
unimodal hybrid zones being primarily composed of individuals that are genotypically
intermediate between both parental forms, and bimodal hybrid zones being composed of
individuals whose genotypes resemble one of the two parental form (J iggins and Mallet
2000). Bimodality in a hybrid zone is thought to represent a system in which the parental
forms are more fully diverged and unimodal hybrid zones represent the juncture between
two populations that are less fully reproductively isolation (Kondrashov et al. 1998).

Hybrid zones and cline centers often remain highly stable over the course of time
and do not shift in position. This is often the result of the cline being coincident with an
ecological gradient that maintains the position of the cline center through exogenous
selection (Coyne and Orr 2004). In fact, the ﬁequency of hybrid individuals can some

times be much higher than would be expected in these intermediate environmental

conditions due to bounded hybrid superiority (W oodruff 1989), a condition in which
hybrid offspring express increased ﬁtness to that of either parental population under
unique environmental conditions (Collins 1984). Cline movement, the gradual shifting of
the cline center farther towards one or the other parent population has however been
known to occur. This is most often the case when endogenous selective pressures are
acting to prevent complete mixing of the parental gene pools, but some amount of

introgression and “leaking” is occurring near the population borders (Harrison 1993).

Genetic Incompatibilities

There is an ongoing quest to identify common patterns and mechanisms that can
help to explain the various processes of evolution and speciation in plants and animals.
Among the common patterns, identiﬁed by both botanists and zoologists, that aids to
prevent hybridization is that of hybrid breakdown. The ﬁtness of hybrid offspring is
ﬁequently greatly reduced ﬁ'orn that of either parent population. Without knowing the
molecular mechanisms by which it could arise, Darwin suggested that hybrid sterility was
the result of tmknown differences in the parent populations leading to incompatibilities in
hybrid individuals (Darwin 1859; Presgraves 2007). There are intrinsic forces at work
that help to prevent gene ﬂow between distinct populations and thus the melding of the
respective gene pools.

In the vast majority of taxa studied, hybrid offspring are frequently found to be
sterile or inviable. It has been identiﬁed that the combination of diverged genetic
backgrormds leads to intrinsic genetic incompatibilities (Coyne and Orr 2004). Over
time, populations evolve coadapted gene complexes that have been “tested” and

“sculp ” by selective forces under unique local environmental conditions. These gene

10

combinations have been “crafted”, through natural selection, to work in concert. When
hybridization occurs, incompatibilities arise due to disruptions of these coadapted gene
complexes that result in negative epistatic interactions between alleles that have not
coevolved. These endogenous factors resulting in a reduction of ﬁtness, due to the
crossing of diverged genetic backgrounds, is one mechanism that results in outbreeding
depression.

Among the most widely accepted “rules” that can be applied to the vast majority
of evolutionary systems that helps to in part offer a mechanism that explains outbreeding
depression is Haldane’s Rule, or the Haldane Effect (Orr 1997). Haldane’s Rule suggests
that when hybridization occurs the result is a distinct increase in the occurrence of
sterility or inviability in the hybrid offspring of the heterogarnetic sex. According to
Haldane’s Rule, in a hybrid zone the heterogarnetic sex will be extremely rare or
completely absent. This has been largely supported in the vast majority of animal hybrid
zones investigated. This indicates that there are some seemingly universal processes
driving evolution and speciation. The result of Haldane’s Rule is postzygotic isolation
driven by genetic incompatibilities of loci on sex chromosomes (Presgraves 2002).

There are several hypotheses that have been proposed to explain the
mechanism(s) underlying Haldane’s Rule, for which the Dominance Theory has received
the vast majority of support. The Dominance Theory suggests that complementary alleles
on the X chromosome can lead to sterility or inviability and are partially recessive. A
hybrid individual in a hemizygous state (receiving only one X chromosome) would have

no alternative allele to mask the deleterious recessive (Orr 1997).

ll

The severity of the Haldane Effect has been found to increase as the level of
genetic divergence between the hybridizing taxa increases. This process of enhanced
genetic incompatibility is the result of a “snowballing effect” (Coyne and Orr 2004). Use
of a model put forth by Dobzhansky (1937) and Muller (1942) is very useful in
explaining how this would occur. Consider two populations with common ancestry but
that have diverged over time. Before divergence had occurred, the ﬁxed genotype for the
pepulation at two loci could have been described as aabb. Alter divergence a new allele
arose through mutation in population 1 for the a locus and through drift and/or selection,
populations 1 genotypes had become ﬁxed as AAbb. Similarly, population 2 became
ﬁxed for a mutation at the b locus, leading to the population 2 genotype being aaBB.
These mutations arose independently of each other but in the presence of comparable
genetic backgrounds. However, the new A and B alleles had never been “tested”
together. In hybrid offspring, these are the complementary alleles, in that they are
genetically incompatible (Orr 1997).

Extend this idea to populations that have diverged and have become distinct at
multiple loci. With each new allele arising in each of the populations, there are
increasing opportunities for allele combinations that are incompatible. Figure 1.1
illustrates the process of incompatibilities arising in diverging populations. Time is
represented as moving forward moving up the diverged lineages. In each case the lower
case letters represent the ancestral allele, the upper case letters represent the derived
characters. Each derived allele has not been “tested” against the genetic background of

the opposite lineage. With each new derived allele there is an increasing opportunity and

12

 

Figure 1.1. A diagrammatic illustration of the process by which incompatibilities could
arise in diverging populations. Time is represented as moving forward moving up the
diverging lineages. In each case the lower case letters represent the ancestral allele, the
upper case letters represent the derived characters. Each derived allele has not been
“tested” against the genetic background of the opposite lineage. With each newly
derived allele there is an increasing opportunity and number of possible combinations in
which genetic incompatibilities could occur. The end result of the “snowballing effect”
would be complete reproductive isolation. (Figure modiﬁed from Coyne and Orr 2004.)

13

number of possible combinations in which genetic incompatibilities could occur. The
end result of the “snowballing effect” would be complete isolation.

When Haldane’s Rule is identiﬁed between hybridizing populations, this is an
indication that complete reproductive isolation has not yet been achieved. If genetic
incompatibilities, especially on the X-chromosome, are a major driving force in the
process of speciation, the “snowballing” effect associated with Haldane’s Rule will in
theory ultimawa lead to complete isolation and speciation (Coyne and Orr 2004).

Exogenous factors can also lead to outbreeding depression. Populations of
organisms can become highly adapted to local environmental conditions. The alleles that
form the coadapted gene complexes are suited to speciﬁc ecological conditions.
Ecological constraints can prevent the movement of certain alleles across environmental
gradients for which they are not adapted. Often times, this is one of the mechanisms
found to delineate the range boundaries between closely related species. A widely
recognized example of an ecological gradient constraining the adjacent distribution of
closely related populations is temperature and the effect that it can have on certain
metabolic enzymes (Powers et al. 1986; Dimichele and Powers 1991).

Distinct alleles of certain enzymes have been shown to exhibit structural
variability and temperature dependent differences in thermal stability and function
(Adams et al. 1973). Some alleles perform much better at higher or lower temperatures,
whereas others perform poorly under certain temperature regimes (Angiletta et al. 2003).
Different allelic forms of enzymes having differing thermal stabilities have resulted in a
steep latitudinal cline in marine ﬁshes directly correlating with environmental

temperatures (Crawford and Powers 1989; Dimichele and Powers 1991). Evidence that

14

differing thermal environments impose striking selective pressures on different structural
forms of enzymes has been clearly shown (Eanes 1999). A combination of these genetic
and ecological incompatibilities prevents widespread hybridization and complete mixing
of distinct population genomes. However, where population ranges meet, there can be
opportunities for hybridization and a localized zone of genetic mixing. Hybrid zones are
typically narrow geographic ranges, often times correlated with some distinct ecological
boundary, in which specimens of mixed ancestry can be found (Harrison 1993).
Typically, hybrid zones are narrow geographic regions where extensive genetic
exchange and introgression is prevented through some form of hybrid unﬁtness (Barton
and Hewitt 1985). However, under conditions where two closely related populations
meet and hybridize, localized populations may arise, that under localized environmental
conditions, exhibit no apparent loss of ﬁtness. These populations may be composed of
individuals that exhibit a diverse array of genetically recombined forms. These
populations composed of diverse recombinant types are best described as hybrid swarms
(Harrison 1993). Given that hybrid zones often correlate to environmental gradients, they
represent boundaries for both parental populations that are potentially imposed as a result
of ecological limitations (e.g., thermal landscape). The mixing of two distinct parental
genotypes can result in genetic incompatibilities and a loss of ﬁtness, but alternatively
can offer an opportunity for the production of unique genetic combinations that can
actually result in offspring that have increased ﬁtness compared to either parental form
under the localized environmental conditions. This state of hybrid vigor, where offspring

exhibit higher ﬁtness than either parental form under the extreme environmental

15

conditions associated with an ecotone is described as “bounded hybrid superiority”
(Moore 1977).

Populations near species range bormdaries are often times under stress and experience
relatively increased levels of mutation and recombination (Hoffmann and Hercus 2000).
A common phenomenon has been described in many hybrid zones, the rare allele effect,
where allozymes that are extremely rare or absent ﬁ'orn either of the parental populations
appear at surprisingly high frequencies within hybrid swarm populations (W oodruff
1989). The most ﬁequently proposed mechanism by which these alleles are brought to
high frequencies suggests that it is the result of the strong selection that can occur within
hybrid zones, purging genetic incompatibilities and the alleles that are closely linked to
them. Alleles that had been rare, but associated with genetically viable allelic
combinations, are then brought to high frequencies through genetic “hitchhiking”
(Schilthuizen et al. 1999). As a result of this rare allele phenomenon occurring in
association with hybrid zones, these novel allozymes have been referred to as
“hybrizymes” (W oodruff 1989). Often times, these hybrizymes are completely unique to
the hybrid population, and are completely absent ﬁom either parental population. These
alleles are not the result of recombination of parental genetic material, but instead are
typically the result of single nucleotide substitution in a parental allele (Schilthuizen et al.
2001). These hybrizymes are established local evolutionary novelties that have been
described in systems as neutral alleles, but could also potentially provide adaptive
beneﬁts under local environmental conditions.

Hybridization between distinct populations has historically been viewed as an

evolutionary waste of reproductive energy. More recently however a great deal of

16

research is indicating that the novel genotypes that arise through the crossing of distinct
parental populations, can possess great evolutionary potential. Hybrid offspring are
genetically distinct from either parental population and can potentially exhibit higher
ﬁtness in a novel habitat or under extreme conditions for which neither parental
population is well adapted (Gompert et al. 2006). There are an increasing number of
investigations exploring hybridization as a mechanism that can be involved in the process
of speciation. Hybrid speciation has been proposed in plants (Wolfe et al. 1998; Riesberg
et al. 2003), ﬁsh (Salzburger and Sturmbauer 2002), and insects (Schwarz et al. 2005,
2007), including Lepidoptera (Scriber and Ording 2005; Gompert et al. 2006; Maverez et
al. 2006). The intent of this dissertation is to explore the multiple components that are
thought to be responsible for, in part, the current distribution of the hybrid zone between
two closely related species of Tiger Swallowtail, Papilr'o canadensis and Papilio glaucus,
but more importantly, to investigate the ecological and genetic factors that are thought to

have led to unique cases of hybrid speciation.

Butterflies as Model Organisms for Ecological and Evolutionary Study

Insects in general have served as ideal model systems through which to gain a
tremendous amount of scientiﬁc insight into genetics, associated with both
developmental and evolutionary biology. There have been a great number of signiﬁcant
advances in population genetics and evolutionary theory accomplished through
investigations on Lepidoptera. Classic textbook examples of genetic research conducted
on Lepidoptera include natural selection and industrial melanism in the Peppered Moth

(Biston betularia) originally reported by J.W. Tutt (1896) and later made widely known

17

by B. Kettlewell (1973) (see reviews by Coyne 1998; Grant 1999; Cook 2003; Majerus
2005); the migratory movements of Monarch Butterﬂies (Danaus plexippus) originally
described by F .A. Urquhart (1960) and their associated population genetic structuring
later described by Eanes and Koehn (1978); the idea of coevolution of insects and plants
described by Ehrlich and Raven (1964) was heavily based upon descriptions of butterﬂies
and their associated host plants; and widely cited examples of the variety of processes
that potentially drive speciation have been put forth by Chris J iggins and James Mallet
(Jiggins and Mallet 2000; Jiggins et al. 2001).

Studies on Lepidoptera have also greatly enhanced our understanding of the
dynamic genetic processes associated with hybrid zones (Barton and Hewitt 1985; Mallet
and Barton 1989; Dasmahapatra et al. 2002). The emphasis of hybrid zone research has
primarily been focused on making inferences ﬁom distinct populations as to the
mechanisms that initially drove differentiation and speciation, and then also the
mechanisms that allow for the maintenance of two distinct species across hybrid zone
boundaries (Harrision 1993; Jiggins et al. 1997). Recently however, there have been an
increasing number of investigations identifying the possibility of hybridization as a
mechanism that can ultimately allow for speciation (Ungerer et al. 1998; Salzburger et
al. 2002; Gross and Rieseberg 2005; Scriber and Ording 2005; Schwarz et al. 2005, 2007;
Gompert et al. 2006). The hybrid zone between Papilio glaucus and Papilio canadensis

provides an ideal system with which to investigate this possibility.

l8

Two Species

North America is home to two closely related species of Tiger Swallowtail
butterﬂies, Papilio canadensis and Papilio glaucus. Only recently were these two
species elevated ﬁ'om subspecies making and granted distinct species status (Hagen et al.
1991). These two species are distinguishable on the basis of multiple morphometric,
physiologic, behavioral, and biochemical traits (Table 1.1; Scriber 1994). The
documented range of the northern of the two species, Papilio canadensis, extends all the
way into Alaska, whereas the range of the southern species, Papilio glaucus, extends
southward all the way to the tip of Florida (Figure 1.2; Scriber 1994).

For many species across North America, a Berengial refuge has long been
proposed as an historic isolated geographic location that would have allowed for
allopatric speciation to man: After the glacial retreat, some 15,000 — 25,000 years ago,
diverged populations would have been able to repopulate areas to the south and come into
secondary contact with ancestral populations. It has been suggested that these two
Papilio species arose through genetic differentiation in allopatry during the Pleistocene
ice age. With the spread of the ice sheets over vast portions of North America, a small
relic population, that eventually became P. canadensis, was isolated in the far
northwestern Berengial refuge (Scriber 1988, Scriber et al. 1991). P. glaucus populations
were forced southward by the ice sheets. Glacial retreat then allowed for the ranges of

these two populations to expand and again meet.

19

{I} (KR _- //’?{: ‘36:}.-

     
 
 
  

   

”iii-t-
.a'W::-J.' ' .
2 tiniest» :2
iritrgt’iigétia
t§2p2f’,- <5 5.. :'|'
i"§-r,§i‘f: GREAT LAKES
-.: '. ! gag-El HYBRID ZONE
:' '.' "'5 I": B;
.12.2. -=§s-=t§§:.=;
5'“ ‘ lauc
2: =\\ 9 us
:3\‘.2“\
xx. ., H
\‘x \
\,\

Figure 1.2. The geographic ranges of two closely related species of Tiger Swallowtail
Butterﬂies, Papilio canadensis to the north and Papilio glaucus to the south (modiﬁed
ﬁorn Scriber 1996a). Indicated in the Western United States are the ranges of three
largely sympatric species, P. rutulus, P. eurymedon, and P. multicaudatus. Represented
in Mexico is the range of P. alexiares.

Table 1.1 — Summary of genetic differences discussed between Papilio glaucus and
Papilio canadensis. (Modiﬁed from Table l in Scriber 1990).

 

 

P. glaucus trait P. canadensis trait Mode of inheritance Reference
1. Pgd 100, 50 Pgd 150,125,80 x-linked allozyme Hagen & Scriber
1991
2. th 100 th 80, 40 x-linked allozyme Hagen & Scriber
1991
3. Hk 110 HR 100 autosomal allozyme Hagen & Scriber
1991
4. Tuliptree 2-4 loci? dominant Scriber 1986
detoxiﬁcation autosomal?
Quaking aspen 2-4 loci? dominant Scriber 1986
5. detoxiﬁcation autosomal?
6. Narrow anal Broad anal wing ? Luebke et al. 1988
wing band band
7. Large Forewing Small Forewing polygenic Luebke et al. 1988
length length
8. Prefers tuliptree Prefers aspen ? Scriber 1994
for oviposition for oviposition
(b+) 09-)
Scriber 1994

“Enabler” (s-) SW” (9+)

 

21

 

 

 

 

 

 
 
 
  
 

 

 
     

s Pgd od TpI
L I 7 J
th
#38 I
"'I
Pgd Acp
Pagdh
Oviposition
P ef rence W
r e Pgd
8+ -125 OD+ th 80
l l l 1
I .1 l I’ LJ

 

 

 
   

s- Pgd 00- um 100
77 -100 DIAGNOSTIC
Oviposit. ALL ELES
Preference

 

 

Figure 1.3. Proposed linkage map of the X—chromosome loci in Tiger Swallowtail
Butterﬂies (Modiﬁed ﬁom Hagen and Scriber 1989). The chromosome represented in
white depicts the canadensis-type chromosome, whereas that represented in black depicts
the glaucus-type. Chapter 4 identiﬁes the likely possibility that the th 100 and od- loci
are in fact much more closely linked that suggested by this linkage map.

22

Papilio Hybrid Zone

After the Pleistocene glaciers retreated, the ranges of P. canadensis and P.
glaucus were able to again expand and meet in secondary contact. There is currently a
narrow band that extends across the Midwest into New England at approximately 41 -44
°N where the ranges of these two species overlap and hybridization is able to occur
(Figure 1.4; Remington 1968; Scriber 1996a). This hybrid zone is best characterized as
unimodal in nature (J iggins and Mallet 2000). Towards the East, this range of overlap
becomes more confused and sweeps southward into the Appalachian Mountains
(Pavulaan and Wright 2002; Scriber 1996a). Much of this narrow region of hybridization
has remained stable for more than a century (Scriber and Lederhouse 1992; Scriber et al.
1996). As is commonly reported of hybrid zones (Harrison 1993), this P. canadensis and
P. glaucus hybrid zone, at fast glance, corresponds to the boundary between two
ecotones, the boreal coniferous forest to the north (or high altitudes in mountainous
regions) and the temperate deciduous forest to the south (or lower altitudes). It has been
shown that perhaps a more accurate tool, than latitude, by which to predict the location of
potential hybridization between these two species would be the geographic location that
corresponds to between approximately 2300 and 2700 F degree days of thermal unit
accumulation. A minimum of 2800 F degree-days is the amount of thermal energy
required allowing for the bivoltine physiology of P. glaucus in a single season (Scriber
1994)

Under laboratory conditions, P. canadensis and P. glaucus can be readily hand

paired, resulting in viable offspring. The resulting F1 hybrids are intermediate in many

23

3.2 . .V V

 

 

 

 

 

 

 

 

 

CENTRAL I ,
WISCONSIN
lied
CENTRAL
MIC GAN new YORK ‘ .
- I BArI-ENKILL RIVER
‘ NY/ VERMONT
"P. appalachiensis
(Pavulaan &_ Wright 2002)
Historical Hybrid Zone ' Wrasrvvmo‘wu
Papilio (glaucus/canadensis) ._ {
(before I998-2006 wanting) . 1
1 ; ,z30 N
HABERSHAMJL -, ,
J :_ RABUN Cos ‘.3 '
; GEORGIA

 

 

 

Figure 1.4. Geographic representation of the historic hybrid zone as well as the identiﬁed
distribution of the recently described Papilio appalachiensis, and also the location of
incipient speciation in the Battenkill river valley of New York and Vermont.

24

respects with regards to signiﬁcant life-history traits and morphological characters.
Under growth chamber controlled environmental conditions, it has been shown that pupal
eclosion of male F1 hybrids (P. glaucus X P. canadensis) is intermediate between both
parental types, being delayed compared to P. canadensis and emergence is earlier than
that of P. glaucus. Female F 1 hybrids of this same cross have been found to be delayed
by an average of two weeks (Scriber et al. 2002). The width of the black bands on the
hind wings and the length of the forewings of hybrid adults are intermediate between
both parental types (Scriber 1982). F1 larvae are able to detoxify both tulip tree and
quaking aspen. Survival, growth rates, and pupation of F1 hybrids are as successful as
that of either parental type (Donovan 2001). Lastly, as would be predicted, lab reared

offspring exhibit a mixture of species diagnostic allozymes.

Barriers to Gene Flow?

In nature, barriers to gene ﬂow between natural populations can be categorized as
either prezygotic or postzygotic. Mechanisms under either of these categories ultimately
result in the maintenance of distinct populations and therefore, species reinforcement
(Harrison 1993). Even in the heart of the known hybrid zone, identiﬁcation of a ﬁeld-
captured individual exhibiting a genotype of mixed glaucus and canadensis ancestry has
been rare in occurrence (<10%) (Leubke et al. 1988; Scriber et al. 2003). A great deal of
research has been conducted investigating what mechanisms maintain and shape the
narrow zone of hybridization between P. glaucus and P. canadensis. If hybrids are
viable, what prevents these two populations ﬁom fusing into a single panmictic

population?

25

P. canadensis and P. glaucus have species speciﬁc host plant preferences and
detoxiﬁcation abilities that correspond to some of the available plants in their respective
ranges. In three choice host plant investigations, P. canadensis females consistently
prefer ovipositing on quaking aspen (Populus tremuloides), whereas P. glaucus prefer
ovipositing on tulip tree (Liriodendron tulipifera). Additionally, the larvae of each
species are able to successfully detoxify the secondary chemicals associated with their
respective choice of host plant, but an inability to detoxify the alternative host plant leads
to larval death. However, there are suitable host plants, including black cherry (Prunus
serotina) and white ash (F raxinus americana) that the two species share in cormnon,
which exist throughout their combined species ranges (Scriber 1982; Scriber 2002). Host
plant availability is therefore not likely a sufﬁcient mechanism preventing range
expansion of either species.

It is frequently the case that species reinforcement and reproductive isolation are
accomplished through mechanisms associated with mating systems (Harrison 1993).
Male mate preference has not been shown to be a strong candidate explanation.
Presented with a choice of size-matched tethered virgin females of both species, wild
males in both glaucus and canadensis ranges signiﬁcantly prefer mating with P. glaucus
females (Deering and Scriber 2002). It has also been shown that conspeciﬁc sperm does
not exhibit sperm precedence in multiple mating situations (Stump and Scriber 2006).

These investigations alone do not identify any strong barriers to bi-directional gene ﬂow.

26

Impacts of Temperature

Many life history trait patterns of insects are dramatically inﬂuenced, or are
directly driven, as a result of temperature. It has been hypothesized that temperature is
the strongest selective force dictating the geographic range limits of many polyphagous
insects, including P. glaucus (T esar and Scriber 2000). A great deal of research has
indicated that both P. canadensis and P. glaucus are drastically impacted by, and can
exhibit strong local adaptations to cope with local thermal environments (Ayres and
Scriber 1994; Scriber 1996b, 2002; Scriber and Lederhouse 1992). It is very likely that
temperature, acting directly or indirectly, is the major factor preventing unchecked gene
ﬂow between populations of P. canadensis and P. glaucus.

Consider the farthest northern reaches of the range of P. glaucus. It consistently
correlates with the geographic range that has >2800 degree day thermal unit
accumulation. This makes intuitive sense given the genetically based multivoltine
physiology of P. glaucus. Both P. canadensis and P. glaucus over winter as pupae on the
ground in the leaf litter. Diapause is facultative in nature in P. glaucus. Environmental
cues, primarily photoperiod, either induce diapause (relatively shortened daylight period)
or induce direct development (relatively long daylight period). It would be suicidal for a
second generation of P. glaucus to be attempted in locations without sufﬁcient thermal
units accumulated annually. An absolute minimum of 2800 degree-days is necessary to
allow for the completion of two full generations. Additionally, ﬁeld studies have been
conducted that indicate that P. glaucus pupae are less cold tolerant than are pupae of P.
canadensis. Glaucus pupae have been shown to experience increased mortality under the

thermal environments frequently encountered in the P. canadensis home range (Kukal et

27

al. 1991). In these ways, temperature likely acts to prevent the advancement of P.
glaucus northward.

Alternatively, it appears as though temperature may also play a role in preventing
signiﬁcant movement of P. canadensis southward. Diapause in P. canadensis is obligate
in nature, requiring a cold period (winter) followed by spring like temperatures, in order
to induce eclosion. Physiologically, diapause has likely evolved to help insects avoid
prolonged times of environmental stress. However, while in diapause butterﬂies are
unable to behaviorally modify their immediate environment. It has been shown that the
extreme summer temperatures that frequently occur south of the hybrid zone could
induce heat stress in diapausing canadensis pupae. In fact, experimental results identiﬁed
an inability of nearly 100% of both pure P. canadensis and hybrid glaucus X canadensis
pupae from eclosing after brief exposure to temperatures of 36°C (Scriber et al. 2002).
This kind of strong selection would prevent signiﬁcant amounts of extensive southward

P. canadensis gene ﬂow.

Recent Evidence of Introgressive Hybridization

Likely as a result of thermal constraints acting on a variety of life history traits,
the geographic location of the hybrid zone between P. canadensis and P. glaucus has
. remained relatively stable for more than a century (Scriber and Lederhouse 1992; Scriber
et al. 1996). However, recent global climate changes have led to drastic alterations in the
typical thermal environment across much of North America. It appears as though this has
greatly facilitated increased levels of gene ﬂow between populations of P. canadensis

and P. glaucus. Over the course of the past 8-10 years there has been both direct and

28

indirect evidence of northward gene ﬂow of glaucus genes into various populations of
historically “pure” P. canadensis.

There are two isolated pieces of evidence indicating the possibility for long-range
gene ﬂow of P. glaucus northward into populations of P. canadensis. The ﬁrst direct
observation of potential gene ﬂow was the capture of a dark morph female in the Upper
Peninsula of Northern Michigan in the summer of 1997. This is the farthest northern
collection of a dark morph female on record. The location where it was captured is
nearly 300 kilometers ﬁ'om where a dark morph female would be anticipated. This
isolated incident of a pure dark morph female being captured so far from where it would
be expected was hypothesized to be the result of long-range transport by a strong storm
front that had passed through the day before (Scriber et al. 1998). There is no guarantee
that the arrival of this dark P. glaucus would have resulted in genetic introgression.
Potentially no male P. canadensis would have recognized her as a suitable mate. The
second isolated source of evidence was a wild P. canadensis female captured in northern
Michigan in June of 2000. The eggs produced by this female resulted in a brood of
offspring that in every way appeared to be primary hybrids between P. glaucus and P.
canadensis (Donovan and Scriber 2003). These isolated incidents should highlight that

opportunities do exist for gene ﬂow well beyond species boundaries.

A Newly Described Species
In 2002 a new species of Tiger Swallowtail, Papilio appalachiensis (Lepidoptera:
Papilionidae), was described ﬁom various sites of the Appalachian Mountains in the

Southeastern United States (Figure 1.4). It has been described as a univoltine species

29

sympatric to populations of P. glaucus. The primary differences from P. glaucus initially
justifying it being given unique species status were based on a delayed spring emergence
pattern, an apparent obligate diapause physiology, an absence of dark morph females,
larger size (than typical spring form glaucus in that region), and multiple wing
morphometrics. The wing morphometrics of this new species actually appeared to be
very hybrid like. However, preliminary mtDNA analysis indicated that this unique
species was more closely related to P. glaucus (Pavulaan and Wright 2002).

Since ﬁrst being described, collaborative investigations have indicated that P.
appalachiensis is a hybrid species between P. glaucus and P. canadensis (Scriber and
Ording 2005). This hypothesis is supported by data collections associated with
oviposition preferences, larval survival abilities, and morphometric analysis, each of
which show this new species to be intermediate between both parental types in all
categories, as is the case with lab reared hybrids. The most compelling data indicating
the mixed ancestry of this new species has been the allozyme electrophoresis. Specimens
collected and described as P. appalachiensis by the original authors (Pavulaan and
Wright), that have been analyzed through allozyme gel electrophoresis, for two X-linked
species diagnostic loci, exhibit a striking genetic pattern. Nearly all individuals scored
have glaucus like Pgd and canadensis like th (Scriber and Ording 2005). Highly
noteworthy, is that this new species has been identiﬁed ﬁ'om geographic regions that

correspond to an accumulated 2300-2700 thermal degree-days.

30

There is clearly an intimate link and an interaction between the thermal landscape and
population genetics, that initially allowed for differentiation between P. glaucus and P.
canadensis, has maintained these two groups as unique populations with distinct ranges,
and now allows for differential rates of gene ﬂow and introgression. Figure 1.5 (adapted
from Scriber et al. 2008) represents the allele frequencies of two X-linked species
diagnostic allozymes along the thermal landscape across the Eastern United States.
Those populations represented ﬁom regions in which there are less than 2300 degree
days are univoltine and essentially pure P. canadensis, whereas those populations ﬁom
regions in which there are 2800 degree days or more are bivoltine and essentially pure P.
glaucus. Those populations represented between 2350 and 2750 degree days are found
relatively near species range boundaries or in the midst of the species hybrid zone, and
are therefore prone to varying degrees of genetic introgression. This dissertation will
clearly illustrate how recent shifting thermal landscapes have differentially impacted the
range limits of P. glaucus in certain areas, allowing for variable levels of genetic
introgression into once pure populations of P. canadensis. In those populations in which
the highest levels of genetic introgression have been recorded, there is clear evidence of

genetic recombination and divergence in key species diagnostic traits.

31

 

 

 

 

 

100- O 00 ‘ 0
E8 08 9 8 O

902 O
T. .0- 8
.2 O
2 70-
§ ‘ ’
a 60‘ LF ergo—now
f 50‘ ”W o th-Ioo
2 40’ Vermont P. appalachiensis
3 Populations
“(‘2' 30' ..
2. r.r~_———Ip

20- I4yrs) i f

.09 o

0 O O
2000 2200 28'... so. 3.00 2.00 .2500
r
Univoltine P. canad Univoltine EF/LF/P. app Bivoltine P. glaucus
(Q300°Dp) (2350-2750°Dp) (>2800°Dp)

Figure 1.5. X-linked allele frequencies (Pgd-100 and th-lOO allozymes) along the
thermal landscape (degree-days above a base of 50°F [°Dp]) across the eastern United
States. Note the sharp decline in th-lOO at 2700on and the nonconcordant but steep
increase in Pgd—100 ﬁequencies between 2300 and 2800°Dp. The 70 different
populations (each represented by 10-125 males) are ﬁom Vermont and northern
Michigan to southern Ohio and North Carolina (data from Scriber and Ording 2005; and
JMS unpublished data). The bivoltine P. glaucus are at the right (>2800°Dp), while
everything else is univoltine. These other populations include a range of P. canadensis
populations that exhibit differential degrees of P. glaucus introgression. (Figure
modiﬁed from Scriber et al. 2008).

32

Chapter 2 of this dissertation investigates an isolated hybrid swarm located on South
Manitou Island in Lake Michigan. This population of P. canadensis is unique in that it is
located over 150 km north of the historic hybrid zone across a relatively broad thermal
landscape, and yet levels of P. glaucus introgression there are as high if not higher than
across the heart of the hybrid zone in the State of Michigan. It is thought that the
introgression exhibited on South Manitou Island is the result of historic episodes of gene
flow into the area, and given the isolated nature of this island population, signiﬁcant
levels of introgression are not the result of ongoing gene ﬂow (Ording 2001). The
environmental conditions of this northern location are unique in that the typically severe
northern temperatures are heavily moderated by the signiﬁcant lake effect of this region.
This provides an opportunity to monitor the stability of an introgressed hybrid swarm
population and to investigate how environmental conditions on the “cooler side of the
hybrid zone” exert differential selective pressures on the variety of hybrid like trait

combinations.

Chapter 3 will describe populations what were once pure P. canadensis near the New
York and Vermont border, which have exhibited extremely high levels of genetic
introgression over the course of the past 10 years. The combination of reasons for the
high levels of introgression in these populations includes the recent series of unusually
warm years associated with anthropogenic climate change, allowing for shifting species
boundaries. Additionally, the topography of the region results in an extremely condensed
thermal landscape (compared to that in the State of Michigan), bringing populations of P.

glaucus much closer contact to populations of P. canadensis. The high levels of genetic

33

mixing that have occurred have resulted in unique combinations of hybrid traits being
expressed, including changes in signiﬁcant life history traits (diapause). This has resulted
in rapid divergence and provided a mechanism for temporal isolation. This temporal
isolation is an example of a rapidly developed mechanism that could allow for hybrid
speciation (Scriber and Ording 2005) and could be identical in nature to the processes
that have led to the formation of the newly described P. appalachiensis (Pavulaan and

Wright 2002).

Chapter 4 will highlight the genetic mechanisms that allow for the recombinant
genotypes that exist in the introgressed populations of P. canadensis to arise, through the
use and analysis of laboratory reared hybrids. These laboratory investigations provide
evidence as to the ﬁequency with which recombination likely occurs in nature, and also
provides support for the presence of genetic incompatibilities and a Haldane effect that
has likely played a central role in the historic maintenance of the P. canadensis and P.

glaucus range boundaries.

34

CHAPTER 2:

A STABLE HYBRID SWARM

Introduction

Ongoing research (1998-2007) conducted on and adjacent to the Manitou
Islands in Lake Michigan, has identiﬁed an isolated “hybrid swarm” over 150 km.
north of the historic Papilio canadensis and P. glaucus hybrid zone in Michigan
(Figure 2.1). Specimens collected from this hybrid swarm (1998-2001) exhibited
intermediacy and mixed ancestry for several of the species diagnostic characters
(Ording 2001). The hypothesis being tested is that the levels of P. glaucus
introgression in this hybrid swarm have remained stable and consistent in the years
following the original investigation. The extent to which P. glaucus introgression
continues to be present, and the stability of the hybrid swarm on South Manitou
Island has been investigated through further analysis of those same diagnostic
characters between P. canadensis and P. glaucus. A combination of morphologic,
ecological and biochemical traits have been considered utilizing samples taken over

an eight-year period (1998-2005).

35

 

 

Figure 2.1. Papilio canadensis and P. glaucus hybrid zone across the Midwest. The
shaded region in Michigan roughly indicates what has historically been considered
the hybrid zone between Papilio glaucus and P. canadensis (Nielsen, 1999). The
shaded region across Wisconsin into Minnesota represents the 50 year average
degree-day accumulation and northern limit allowing for two generations of Papilio
glaucus. The star indicates the location of South Manitou Island.

36

Banding patterns on butterﬂy wings is a common method by which species
can be distinguished, as is the case between Papilio glaucus and P. canadensis
(Hagen et al. 1991), or can also be used to discern differences between populations
within a species. The Monarch butterﬂy (Danaus plexippus) has a range that extends
across the Western Hemisphere, from Alaska in the north to Patagonia in the south.
There are differences in wing banding patterns that allow individuals coming ﬁom
one region to be distinguished ﬁorn an individual coming ﬁom another (Williams et
al. 1942).

In P. canadensis and P. glaucus there is a distinct black band in the hind wing
that partially ﬁlls the anal cell. This morphologic character is one used to distinguish
between Papilio canadensr's and P. glaucus (Hagen et al. 1991). This dark band is
signiﬁcantly wider in P. canadensis, on average ﬁlling 70 percent of the anal cell,
whereas in P. glaucus this black band ﬁlls on average only 30 percent of the anal cell
(Hagen et al. 1991). Lab reared hybrids and ﬁeld collected specimens of mixed
ancestry exhibit intermediacy for this trait (Luebke et al. 1988). As a result this black
band character has been heavily relied upon as a reliable morphometric character with
which to identify specimens of mixed ancestry.

Analysis of species diagnostic allozyme allele frequencies is extremely useful
in estimating levels of genetic introgression in a population. P. canadensis specimens
from South Manitou Island have been analyzed for levels of P. glaucus introgression
using the Pgd (6-Phosphogluconate dehydrogenase) allozyme locus. This locus was
chosen as a result of it being highly polymorphic (Hagen & Scriber 1991), relatively

consistent and easy to interpret, and is among the species diagnostic characters.

37

MATERIALS AND METHODS

Specimen Acquisition and Transport

Papilio specimens utilized in this research were live-captured by net, from a
variety of locations on South Manitou Island each year (Table 2.1). Specimen
collections were primarily made during mid-day, between approximately ten o’clock
am. and four o’clock pm. on warm surmy days, these being the predominant hours
for ﬂight activity. Specimens were most frequently captured while puddling, feeding
on available somoes of nectar, and sometimes while in ﬂight. This method of
specimen collection often led to the discovery of desirable puddling locations or high
concentrations of appropriate nectar sources, each often times with high densities of
butterﬂies. These locations could then be returned to multiple times in a day and
within a season for further collections. These collection locations also proved to be
consistently productive in subsequent years.

Upon capture, individual specimens were placed with their wings folded back
into 2 oz. Glassine envelopes, which were labeled with specimen sex, date and
location of capture. Collections were transported alive to the laboratory in
Tupperware® plastic containers in ice coolers, which lowered specimen body
temperatures and slowed metabolism. Upon arrival to Michigan State University in
East Lansing, Michigan, specimens were preserved by freezing them alive in an -80°

C ultra-low biological freezer for later processing.

38

Table 2.1. South Manitou Island male P. canadensis specimen collection data.

Year
1998
l 999
2000
2001
2002
2003
2004
2005

Date(s)
June 17
June 18
June 10
June 11
June 29 and July 8
June 20
June 18
June 25

39

Sample Sizes (n)
32

120

72

100

6 and 3

70

43

25

Total (n)
32
120
72
100
9
70
43
25

Wing Morphometrics

Two wing characteristics were chosen for analysis in order to identify the
presence of Papilio glaucus introgression into the South Manitou Island population of
P. canadensis. Forewing length (Fig. 2.2) and hind wing anal cell black bandwidth
(Fig. 2.3) are morphometric characters of adult butterﬂies, which can be used in the
ﬁeld to help distinguish between P. canadensis and P. glaucus. On the average, P.
glaucus forewings are signiﬁcantly larger than those of P. canadensis. P. glaucus
forewings have been shown to be 8-10 mm longer, ﬁom thoracic attachment to tip,
than P. canadensis forewings (Hagen et a1 1991). The more powerful diagnostic
morphometric wing character is the width of the black band along the anal margin of
the hind wing. For P. glaucus males, this band ﬁlls on average 30 percent of the
width ﬁorn the wing margin to the CuA2 vein; whereas for P. canadensis the width of
the band ﬁlls 70 percent of this anal cell (Hagen et al. 1991). Hybrid individuals
display a black bandwidth intermediate between the two, averaging 50 percent
(Scriber et al. 2001). Intermediacy for genetically based morphometric traits is a good
indicator of a hybrid individuals or populations (Harrison 1993).

After the wings were detached from adult swallowtail specimens during the
allozyme electrophoresis preparatory protocol, they were assayed for the two major
morphological features. F orewing length, from the distal tip of the wing to the basal
thoracic attachment (Fig. 2.2), was measured using a clear plastic metric ruler to the
nearest mm. On the ventral surface of the wings, the width of the anal black band
was assessed as a percentage of the distance ﬁom the wing edge to the CuA2 vein.

This measurement was taken at a line of intersection with the junction of vein CuA2

40

Forewing ‘
f‘\
\
\

 

Figure 2.2. Forewing length measurements (measurement A) are the distance
ﬁom the tip of the forewing to the thoracic wing base attachment (Figure
modiﬁed from Leubke et al. 1988).

41

Hindwing Black band

 

Figure 2.3 Black band width measurements are the percentage of the hindwing
anal cell that is ﬁlled by the dark band labeled A. (Modiﬁed ﬁom Luebke et al.
1988).

42

and the discal cell (Fig. 2.3). This anal black band measurement was taken to the
nearest . 1mm using a dissecting microscope and a WILD glass micrometer slip. For
both of these morphometric characters assayed, measurement values for both wings
were taken when available, and the mean values for each individual have been
utilized for analysis. In cases where wings were damaged and ripped, preventing an
accurate measurement; if one wing was undamaged a single measurement has been
utilized for analysis; if both wings were damaged, the specimen has not been included

in the analyses.

Allozyme Electrophoresis

Allozyme electrophoresis was performed on adult male Tiger Swallowtail
Butterﬂies in this study. Electrophoresis protocol follows that of Hagen and Scriber
1991. Adult specimens were removed from -80° C and processed in 3 4° C cold
room. Using a scalpel or razorblade, wings were dissected ﬁ'orn the thorax at their
place of attachment and returned to Glassine envelopes for morphometric analysis.
Tissue extracts were prepared by grinding one half of the abdomen with 100 pl of
grinding buffer. The lower half of the abdomen was utilized in male specimens. The
remaining abdomen portion, head and thorax, were returned to the —80° C ﬁ'eezer for
future use. The extract was centrifuged for 10 minutes at 14,000 rpm. At this point
the extract could be stored at —80° C until ready to continue the electrophoresis
protocol. Female specimens were not utilized in the electrophoretic portions of this
study for several reasons. First, the sample sizes for females were relatively low for
many of the years of sampling. More importantly however, the allozyme banding
patterns produced by females are ﬁ'equently not as clear as those produced by male

43

specimens and prove to be difﬁcult to interpret. It is possible that the eggs contained
in the abdomen somehow disrupt the normal staining process.

Samples were removed from -80° C and allowed to thaw in 4° cold room for
approximately 10 minutes and were then centrifuged for 5 minutes at 14,000 rpm.
7.5 ul of extract from each sample was applied to thin layer acetate plates (Titan 111
[94 by 76 mm], Helena Laboratories) for electrophoresis. The allozyme locus scored
for this study portion is Pgd (6-Phosphogluconate dehydrogenase). Scoring of gel
banding patterns was accomplished following methods of Hagen and Scriber 1991
using original gels and photographs. Known P. g. and P. c. standards were run
alongside all samples analyzed, to provide for allozyme banding pattern

standardization.

Statistical Analysis

In order to determine whether the wing morphometric characters in the South
Manitou Island hybrid swarm had remained stable, multiple statistical analyses of
both forewing length and black bandwidths were performed using JMP statistical
software version 6.0 by the SAS Institute Inc. An analysis of variance (ANOVA), a
nonparametric Wilcoxon test and a Student’s t-test were performed comparing both
forewing lengths and hind wing black bandwidths across and between years (1998-
2005). To be sure that there was no effect of forewing length on black bandwidth, a
bivariate linear regression was performed to be sure that these characters are in fact
independent of one another.

The stability of the hybrid swarm was also investigated through statistical

analysis of the allele ﬁ'equencies that were identiﬁed through gel electrophoresis.

44

These statistical analyses were performed using Genepop 3.6 (Raymond and Roussett
1995). Tests for both genotypic and genic differentiation were performed comparing

allele frequencies from each year (1998-2005).

45

RESULTS

Wing Morphometrics

The bivariate linear regression indicates that the two characters are in
fact independent of one another (Fig. 2.4). Plotting black bandwidth against forewing
length indicates that there was no effect of forewing length on black bandwidth (R2 =
0.004). This provides validity to the use of these two characters as being independent
of one another. Both the ANOVA and the pairwise statistical analysis comparing the
forewing lengths of specimens from South Manitou Island across each year indicates
that the average forewing length does vary signiﬁcantly between certain pairs of years
(Table 2.2). There are not signiﬁcant pairwise differences however between six of
the eight years analyzed, and these include the earliest year sampled (1998) and the
four most recent years analyzed (2002 — 2005). The same pairwise analysis of the
black bandwidths of these same South Manitou Island specimens across each year
indicates that there is no signiﬁcant difference between any pair of years analyzed

(Table 2.3).

46

 

 

Black Band Width (%)

 

 

 

 

2G I I ' I I I I I I I ‘I I I I I T
40 50
Forewing Length (mm)

 

 

 

Figure 2.4. Bivariate linear regression of black band width by forewing length (R2 =
0.004) for male Papilio canadensis specimens collected on South Manitou Island
from 1998-2005. Analysis was performed using JMP statistical software version
6.0.

47

Table 2.2. Forewing length measurements for male specimens collected on South
Manitou Island, Leelenau County, Michigan ﬁom 1998-2005. Values represented are
the average length in mm +/- s.e. ﬁ'om the distal tip of the wing to the basal thoracic
attachment. Values are based upon the calculated means of the left and right
forewing lengths for each specimen analyzed. The years not connected by the same
letter are signiﬁcantly different based upon a comparison for each pair using the
Tukey’s HSD (adjusted P-value of signiﬁcance <0.05).

Year Similarity Sample Size (n) Mean Forewing Length +l- s.e.

1998 A B 32 46.01 +/- 0.48
1999 A 120 48.58 +/- 0.43
2000 B 72 47.29 +/- 0.23
2001 A B 60 47.68 +/- 0.29
2002 A B 9 46.00 +/- 1.27
2003 A B 70 47.54 +/- 0.26
2004 B 43 46.86 +/- 0.37
2005 A B 25 47.48 +/- 0.39

Table 2.3. Hind Wing Black Band widths for male specimens collected on South
Manitou Island, Leelenau County, Michigan ﬁ'om 1998-2005. Values represented are
the percent of the hind wing anal cell that is ﬁlled by a dark pigmented band. Values
are based upon the calculated means of the left and right hind wing black bands for
each specimen analyzed. There were no signiﬁcant differences across years based
upon analysis using Tukey’s HSD (adjusted P-value of signiﬁcance <0.05).

Year Sample Size (n) Mean Black Band Width +/- s.e.

1998 32 55.20 +/- 1.29
1999 120 56.27 +/- 0.62
2000 72 56.14 +/- 0.96
2001 60 54.91 +/- 0.91
2002 9 55.17 +/- 2.91
2003 70 55.43 +/- 0.75
2004 43 55.45 +/- 1.23
2005 25 53.66 +/- 1.85

48

Allozyme Electrophoresis

Of the nine Pgd alleles that exist in the closely related species groups of
Papilionidae (Hagen and Scriber 1991), six were encountered in these analyses. The
South Manitou Island population allele frequencies are listed in Table 2.4. Using
Genepop Version 3.6 pairwise analysis was conducted to determine the level of Genie
and Genotypic differentiation between each year for which there was allozyme data
available on South Manitou Island (T able 2.4). These analyses indicate that there are
no signiﬁcant genie or genotypic differences between any years, other than the
population in 2001 signiﬁcantly differs from the population in both 1998 and 2000

(P-values of signiﬁcance <0.05).

49

Table 2.4. Allele frequencies for the species diagnostic Pgd allozyme ﬁ'om male
specimens collected on South Manitou Island, Leelenau County, Michigan from
1998-2005 (allozyme data for 2003 and 2004 are unavailable). Allele frequency
values represented are percentages of introgressed glaucus alleles detected through
gel electrophoresis. The years not connected by the same letter are signiﬁcantly
different based upon a determination of both Genic Differentiation and Genotypic
Differentiation as calculated using Genepop Version 3.6 1999 (P-values of <0.05).

Year

1998
1999
2000
2001
2002
2005

Similarity

EEJ’E”

Sample Size (n)

50

32
120
100
100

9

20

Allele Frequencies

.109
.079
.095
.035
.l 11
.025

DISCUSSION

There are two morphometric wing characters that have consistently been
applied in distinguishing between P. glaucus and P. canadensis, forewing length and
hind wing black bandwidth. Intermediacy for these two characters has been applied
towards the identiﬁcation and characterization of hybrid like specimens. The analysis
of data collected over an extended period of time (1998-2005) indicates that these
characters have remained intermediate and stable in the introgressed hybrid swarm
population that exists on South Manitou Island. Additionally, the allele frequencies
of introgressed allozymes in this same population have remained relatively constant
over this same time period, this also indicating that the isolated island hybrid swarm
population has remained relatively stable.

Given the island location and the difficulty of accessing this hybrid swarm,
sampling efforts have until recently been primarily restricted to making the annual
collection in a single day each year. However, other more recently identiﬁed P.
canadensis populations that show indications of P. glaucus introgression exhibit the
possibility that detectable levels of introgression can be dependent on the timing of
collections. It appears in these populations that specimens that possess certain

hybrid-like genotypes exhibit a delayed emergence.

51

Chapter 3 introduces a unique Papilio population in the midst of the P.
glaucus and P. canadensis hybrid zone in the Battenkill River Valley of New York
and Vermont. This population possesses unique traits that appear to be the result of
hybridization. The result of these new genetic combinations appear to have resulted
in the production of an incipient species, reproductively isolated from either parental

population through temporal mechanisms.

52

CHAPTER 3:
CAN INTROGRESSION LEAD TO REPRODUCTIVE ISOLATION? :
IN CIPIENT HYBRID SPECIATION BETWEEN PAPILIO GLA UCUS AND

P. CANADENSIS

Introduction

Hybridization can lead to the production of a vast diversity of genotypes unique
ﬁom that of either parental population. It has been shown that hybrid offspring can
exhibit “bounded hybrid superiority”, or increased ﬁtness compared to that of either
parental population, under certain environmental conditions (Collins 1984; Woodrnff
1989). Bounded hybrid superiority has been implicated in cases of hybrid speciation in
extreme environments (Riesberg et al. 2003; Gompert et al. 2006) and in novel ecological
situations allong for adaptive radiation (Schwarz et al. 2005). In theory, hybrid
speciation would be an unlikely evolutionary phenomenon given that viable hybrid
offspring would be able to mate and exchange genetic material with parental forms. This
would allow for the production of a hybrid swarm, but not speciation. What is required
for hybrid speciation to occur is that the hybrid offspring be afforded some signiﬁcant
genetic modiﬁcation, which would result in reproductive isolation. Hybridization
between Papilio glaucus and P. canadensis is the proposed mechanism that explains the
newly described Papilio appalachiensis species (Scriber and Ording 2005). There are
other locations in which high levels of hybridization are occurring between these closely
related Papilio species. Investigations in these locations are of extreme value in that it is
possible to identify the mechanisms that can ultimately lead to speciation and provide an

opportunity to witness the process while it is occurring.

53

“False Second Generation”

Populations of P. canadensis outside of the Great Lakes have shown evidence of
high levels of P. glaucus introgression. Several New England populations of P.
canadensis including the Battenkill River Valley population of New York and Vermont
have recently (since 1999) exhibited a strikingly uncharacteristic life history trait
phenomenon. These locations have displayed what has been described as a “False
Second” generation (Hagen and Lederhouse 1985). As described earlier, P. canadensis
has a univoltine life cycle. These locations however, have recently begun displaying
what appear to be two distinct ﬂights within a single summer. The ﬁrst ﬂight occurs as
would be expected, in late May through June, and a second ﬂight occurs in mid July.
This second ﬂight was being described as a “false second generation” due to the fact that
it appears far too quickly in the season (mid July) to be a true second ﬂight, derived ﬁ'om
the ﬁrst. This second ﬂight is composed of individuals that appear more “glaucus-like”
than does the ﬁrst ﬂight, being signiﬁcantly larger (based upon forewing length) and
possessing signiﬁcantly narrower hind wing black bands (see Table 1.1). In addition,
individuals ﬁom this second ﬂight appear “glaucus-like” for other species-diagnostic
traits, including oviposition preference and host plant detoxiﬁcation abilities (Scriber and
Ording 2005).

With respect to ﬂight times in the ﬁeld, a variety of morphometric characters, and
host use abilities, the Early Flight (EF) and the Late Flight (LF) Papilio populations from
the Battenkill river valley appear distinct. Many of the apparent character differences in
the LF population appear to be similar to populations of P. canadensis that have been

heavily introgressed by P. glaucus, or similar to lab reared hybrids between the two

54

species. This chapter is devoted to the analysis of data that investigates three primary
questions. First, is there genetic evidence of P. glaucus introgression into either the EF
or the LF populations? Second, are the EF and LF populations genetically unique?
Lastly, are the EF and LF reproductively isolated? This study provides an example of an
individual trait that is greatly modiﬁed as a result of introgressive hybridization, that can
provide strong reproductive isolation between parental populations and hybrid offspring.
In order to determine the presence and degree of genetic introgression of P.
glaucus into the Battenkill Valley population of P. canadensis, a combination of
morphological and molecular techniques were employed, similar to those described in
Chapter 2. In order to determine whether the EF and the LF are genetically unique and
reproductively isolated, a combination of ﬁeld observations, lab controlled emergence

experiments, and additional molecular analyses have been employed.

55

MATERIALS AND METHODS
Specimen Acquisition

Wild captured adults ﬁ'om the Battenkill River Valley populations that have been
used for morphological and molecular analysis were ﬁeld captured by net, from a variety
of locations. Specimen collections were primarily made during mid-day, between
approximately ten o’clock am. and four o’clock pm. on warm sunny days, these being
the predominant hours for ﬂight activity. Specimens were most ﬁequently captured
while feeding on available sources of nectar, puddling, and sometimes while in ﬂight.
Sample sizes of males captured for each of the years surveyed are as follows: Early Flight
(EF) (May — June) 2002 n = 48, 2003 n = 28, 2004 n = 128; Late F light (LF) (Mid July)
2002 n= 15, 2003 n=14, 2004n= 12.

Offspring ﬁom the females of both EF and LF ﬂights were collected as pupae
from ﬁeld-rearing in sleeved tree branches of wild black cherry (Prunus serotina). The
eggs and larvae obtained inside of these sleeved branches were ﬁ'om 25—30 EF females
and 15-18 LF females in 2002, and from 15-20 EF females and 12-15 LF females in
2003. The resulting pupae were collected in mid-September and stored in darkness at 3-5
°C under controlled environmental conditions beginning in October until the

commencement of the emergence experiments in both 2003 and 2004.

Wing Morphometrics
The same two quantitative polygenic wing characteristics (forewing length and
hind wing black band width) that were applied as a component towards the classiﬁcation

of the South Manitou Island population as a hybrid swarm (see details in Chapter 2), were

56

applied in order to compare and contrast the EF and LF ﬂight of the Battenkill River
Valley. Genetic differentiation between two populations is possible through phenotypic
analysis because shared genes would be reﬂected in similar phenotypes (Boag and van
Noordwijk 1987). The characteristics under investigation, forewing length and hind wing
anal cell black bandwidth, again were chosen to apply to this investigation for the same
reasons that they were applied towards the South Manitou Island investigation. Both of
the traits are heritable and polygenic (Luebke et al. 1988), thus conceivably impacted by
mutation, genetic drift, and natural selection under differing niche speciﬁc environmental
conditions. In addition, these two traits have been consistently applied as highly
informative measurements to apply when identifying interspeciﬁc hybridization. (Scriber
et al. 2001; 2003). Female specimens were placed in sleeves on trees in the ﬁeld to allow
for oviposition. In this process, wings became tattered and were not readily usable. For
this reason only male specimens were used for wing morphometric analyses. Sample
sizes for each of the years analyzed are as follows: 2002 EF 11 = 48, LF n = 15; 2003 EF 11

=28, LFn= 14; 2004 EFn= 128, LFn= l2).

Pupal Weights

There is a direct correlation between pupal weight and overall size of adult
Papilio. P. glaucus consistently have larger pupae than do P. canadensis. This correlates
directly with P. glaucus being a signiﬁcantly larger butterﬂy than P. canadensis (Hagen
et al. 1991). Pupal weight, like the wing morphometric characters, is a polygenic trait
that can prove valuable in comparing genetic differences between these two species.

Pupae were removed ﬁom winter diapause chambers and a random subsample were

57

weighed to the nearest milligram using a Mettler ® macroanalytical balance. Sample
sizes for each of the years analyzed were as follows: 2003 BE n = 406 and 399, LF n =
446‘ and 429; 2004 EF 11 = 736‘ and 779, LF n = 786‘ and 6232. Sex ofpupae was
determined post emergence for guaranteed accuracy. Only the pupal weights of those
specimens that successfully emerged were used in analysis. Pupal weight data ﬁom those

pupae that did not successfully emerge were omitted.

Allozyme Electrophoresis

In order to determine and compare the level of P. glaucus introgression
into both the EF and LF populations, and to determine whether the EF and LF
populations were reproductively isolated, allozyme electrophoresis was employed. In
this investigation the two X-linked species diagnostic allozymes were analyzed, Pgd (6-
Phosphogluconate dehydrogenase) and th (lactate dehydrogenase). The same
techniques as were described in Chapter 2 were applied. Of the nine Pgd alleles that exist
in the closely related species groups of Papilionidae (Hagen and Scriber 1991), ﬁve were
encountered in this analysis. For the purposes of determining the degree of glaucus
introgression, the ﬁ'equency of Pgd alleles that have been described as diagnostic
characters associated with glaucus (Pgd 100 and 50) have been grouped together, and the
canadensis diagnostic alleles (Pgd 150, 125, and 80) have also been grouped together for
interpretation. Analysis was performed only on wild male individuals captured in the
ﬁeld. The specimens that were used for the emergence investigations that resulted from
ﬁeld rearing were not used due to the fact that these were many individuals from

relatively few families and would not represent independent samples. Allele ﬁequencies

58

for Pgd are presented for 2000-2004, whereas allele ﬁ'equencies for th are only
presented for 2002-2004. The reason that th allele ﬁequencies were not included for
2000 and 2001 is due to the fact that before 2002 all specimens scored had indicated zero
glaucus introgression and 2002 was the ﬁrst year that a novel hybrizyme allele was
recognized. Sample sizes for the number of male specimens from each year are as
follows: 2000EFn= 116, LFn=33;2001 EFn=0,LFn=51;2002 EFn= ll7,LFn=

13;2003 EFn=29, LFn= 14; 2004 EFn= 158, LFn= 12.

Mitochondrial DNA Analysis

Another technique employed in order to identify the extent of P. glaucus
introgression into both the EF and LF populations was an analysis of mtDNA. Fourteen
EF and the fourteen LF male specimens that were ﬁeld captured during the summer of
2003 were analyzed for mtDNA characters that are known to be diagnostic between P.
glaucus and P. canadensis. DNA extraction and PCR-RFLP analysis techniques follow
those used by Stump et al. (2003), which were modiﬁed from Sperling & Hickey (1995).
For each specimen, two legs were plucked and macerated in 800 pl of Litton buffer
(0.2M sucrose, SOmM EDTA, lOOmM Tris, and 0.5% SDS). Samples were vortexed and
left at room temperature for 30 minutes. Then 100 ul 8M KOAc was added and each
sample was inverted and put on ice for 60 minutes. Samples were centrifuged for 20
minutes and the supernatant was transferred to a new tube. Samples were extracted once
with phenol and once with chloroform/isoamyl alcohol (24:1). DNA was then

precipitated with isopropanol. Samples were centrifuged at 14,000 rpm for 10 minutes.

59

The resulting pellet was washed with 70% ethanol, then dried and resuspended in 200 pl
1X TE buffer.

The PCR primers that were used had sequences 5’ ATA ATT GGA GGA TIT
GGA AAT TG 3’ and 5’ A'IT GTA GTA ATA AAA Tl‘A AT'I‘ GCT CC 3’. These
primers were produced as a result of sequencing work on canadensis and glaucus
mitochondrial C01 and C011 genes (Caterino and Sperling 1999), and were expected to
produce a DNA ﬁ'agment 294 base pairs long. Within this fragment were ﬁve potentially
diagnostic restriction sites. A Tan restriction site was expected to be present in
specimens carrying glaucus mtDNA and absent in those carrying canadensis mtDNA.
An SspI restriction site was expected to be present in specimens carrying canadensis
mtDNA and absent in those carrying glaucus mtDNA.

PCR was carried out using the above primers in a total reaction volume of 100 pl
using AmpliTaq Gold DNA polymerase in a Perkin Elmer GeneAmp 9600 Cycler. PCR
products were veriﬁed by running them out on a 2% agarose gel along with a 100bp
DNA ladder and visualized with ethidium bromide (EtBr) and ultraviolet light All PCR
products were then digested independently by both Tan and SspI restriction enzymes
incubated at 65 °C for 120 minutes. The two alternative diagnostic restriction enzymes
were used on every specimen to provide corroboration. Digested DNA was run on a

1.5% EtBr Agarose gel with a 100bp DNA ladder for comparison.

Emergence Investigations
After pupal weights were determined, specimens were evenly distributed and
randomly assigned to controlled environmental chambers (l 8 °C, 22 °C, and 26 °C in

2003; 14 °C, 18 °C, 22 °C, and 26 °C in 2004; all under a L18:D6 photoperiod) to

60

determine the length of time until adult emergence. Emergence investigations began on
April 15, 2003 and on April 8, 2004. Pupae were either placed alone in a screened Petri
dish or in a screened petri dish in a group of up to 20 pupae. Petri dish placement within
each chamber was split, with EF and LF specimens on each shelf level, so as to avoid the
possibility that the shelf height or the distance to the light sources would act as
conformding variables. Temperatures of each environmental chamber were kept constant
throughout the emergence investigations and were monitored for stability on a daily
basis. Checks were made twice daily (early morning and late aftemoon) to collect freshly

eclosed specimens.

Statistical Analysis

In order to identify whether the EF and LF populations were in fact
morphologically distinct, multiple statistical analyses of data associated with forewing
length, black bandwidths, pupal weights, and emergence timing were performed using
JMP statistical software version 6.0 by the SAS Institute Inc. Both an analysis of
variance (ANOVA) and a nonparametric 2-sample Wilcoxon test was performed
comparing both forewing length and hind wing black bandwidths between wild captured
EF and LF for each individual year (2002-2004). These same analyses were performed
comparing the pupal weights of the ﬁeld-reared pupae and also their respective days to
emergence from EF and LF populations ﬁ'om each individual year (2003 and 2004).

Analysis of the allozyme data allowed for a determination and a comparison of
the amount of P. glaucus introgression in both the EF and the LF populations. These

temporal and genetic data were also valuable in determining whether the EF and the LF

6]

were reproductively isolated. These analyses were performed using Genepop 3.6
(Raymond and Roussett 1995). Tests for both genotypic and genie differentiation were
performed comparing allele frequencies ﬁom both populations for each year (2000-
2004).

Additionally, a classic method by which evolutionary biologists estimate gene
ﬂow between populations is the use of Wright’s Fst (Weir and Cockerharnl984), which is
a comparison of the allele frequencies between populations. Fst is on a scale from 0 to l,
with low values indicating that there is a great deal of gene ﬂow, and high values
inferring that there is very little. F st values were calculated for the BF and LF for each

year (2000-2004) for both individual loci investigated using Genepop 3.6.

62

RESULTS

Wing Morphometrics

For both the ANOVA and the nonparametric 2-sample Wilcoxon analyses
that were conducted, comparing the forewing lengths for each year (2002-2004) between
EF and LF (Table 3.1), the EF forewing lengths were consistently identiﬁed as being
signiﬁcantly smaller than those of the LF (p-values <0.0001). The same analyses applied
towards a comparison of the EF and LF black bandwidths for each year analyzed (2002-
2004) also resulted in signiﬁcant differences between the two populations (Table 3.2),
with the black bands of the EF being signiﬁcantly wider than those in the LF (p-values

<0.0001).

Pupal Weights

Both an analysis of variance (ANOVA) and a nonparametric 2-sample Wilcoxon
analysis were performed comparing the pupal weights of specimens ﬁeld reared for each
year (2003-2004) between BF and LF (Table 3.3). The BF pupal weights were
consistently identiﬁed as being signiﬁcantly smaller than those of the LF (p-values
<0.0001). It should be noted that pupal weights are not independent observations in that
the pupae are derived from only a limited number of mothers for each year and ﬂight

population.

63

Table 3.1 . Forewing length measurements for male specimens collected in the Early
Flight and the Late Flights of the Battenkill River Valley of Vermont and New York ﬁ'om
2002-2004. Values represented are the mean length in mm +/- s.e. from the distal tip of
the wing to the basal thoracic attachment. Values are based upon the calculated means of
the left and right forewing lengths for each specimen analyzed. Sample sizes me
presented in parentheses. Probability values are derived from a nonparametric 2-sample
Wilcoxon test performed using JMP 6.0. Probabilities for comparisons that are
signiﬁcantly different are indicated with an *. All comparisons are signiﬁcantly different
with p-values <0.0001.

‘

Mean Forewing Length +/- s.e.

 

Year BF (n) LP (11) Prob>lll
2002 45.42 +/- 0.34 (48) 50.60 +/. 0.51 (15) <0.0001*
2003 45.18 +/- 0.34 (28) 51.00 +/- 0.49 (14) <0.0001*
2004 47.05 +/- 0.15 (128) 51.42 +/- 0.65 (12) <0.0001*

 

Table 3.2. Hind wing black bandwidth measurements for male specimens collected in
the Early Flight and the Late Flights of the Battenkill River Valley of Vermont and New
York from 2002-2004. Values represented are the mean percent +/- s.e. of the hind wing
anal cell that is ﬁlled by a dark pigmented band. Values are based upon the calculated
means of the left and right hind wing black bands for each specimen analyzed. Sample
sizes are presented in parentheses. Probability values are derived from a nonparametric
2-sample Wilcoxon test performed using JMP 6.0. Probabilities for comparisons that are
signiﬁcantly different are indicated with an *. All comparisons are signiﬁcantly different
with P-values <0.0001.

 

J

Mean Black Bandwidth +/- s.e.

 

Year EF (n) LP (11) Prob>[Z]
2002 67.46 +/. 1.23 (48) 47.97 +/— 2.23 (15) <0.0001*
2003 69.22 +/- 1.60 (29) 50.29 +/- 1.98 (14) <0.0001*
2004 71.02 +/- 0.66 (128) 47.46 +/. 2.09 (12) <0.0001*

 

 

Table 3.3. Pupal weight measurements for ﬁeld reared specimens ﬁ'om both the Early
Flight and the Late Flights of the Battenkill River Valley of Vermont and New York from
2003-2004. Values represented are the mean pupal weights in milligrams +/- s.e.

Sample sizes are presented in parentheses. Probability values are derived ﬁom a
nonparametric 2-sample Wilcoxon test performed using JMP 6.0. Probabilities for
comparisons that are signiﬁcantly different are indicated with an *. All comparisons
signiﬁcantly different with p-values <0.0001.

 

Mean Male Pupal Weight +/- s.e.

 

Year Early Flight (11) Late Flight (11) Prob>[Z]
2003 0.77 +/- 0.01 (40) 0.97 +/- 0.02 (44) <0.0001*
2004 0.74 +/- 0.01 (73) 1.03 +/- 0.10 (78) <0.0001*

 

 

Mean Female Pupal Weight +/- s.e.

 

Year Early Flight (r1) Late Flight (n) Prob>[Z]
2003 0.87 +/- 0.08 (39) 1.04 +/- 0.02 (42) <0.0001*
2004 0.79 +/- 0.01 (77) 1.10 +/- 0.01 (62) <0.0001*

 

65

Allozyme Electrophoresis

Of the nine Pgd alleles that exist in the closely related species groups of
Papilionidae (Hagen and Scriber 1991), ﬁve were encountered in this analysis. For the
purposes of determining the degree of glaucus introgression, the Pgd alleles that have
been described as diagnostic characters associated with glaucus (Pgd 100 and 50) have
been lumped together, and the canadensis diagnostic alleles (Pgd 150, 125, and 80) have
been lumped together for interpretation (Table 3.4; Figure 3.1).

Of the four th alleles that have been described in this species group, three were
encountered in this analysis (th 40 and 80 which are diagnostic for P. canadensis and
th 100 which is diagnostic for P. glaucus). In addition, a novel allele that has never
been described before (here described as th 20 and will be referred to as a hybrizyme)
was encountered and found at high frequencies (ZS-50%) in the late ﬂight. This novel
hybrizyme was not detected in the EF until 2004, but was only present at an extremely
low allele ﬁ'equency from a very large population sampling (1.3% out of 158 specimens
analyzed). Table 3.4 and Figure 3.2 represent the ﬁ'equencies of th diagnostic alleles,
and also the allele frequencies of the novel th 20, in both the EF and LF for 2000-2004.

Fst values were also calculated for the combination of the two loci (Table 3.5).
Typically an F st value of greater than 0.15 is considered to be an indication that
populations are signiﬁcantly differentiated (Frankham et al. 2002). Based upon this
standard, the EF and the LF populations exhibit low levels of gene ﬂow and signiﬁcant
genetic differentiation. This is even true in 2000 when the novel th 20 hybrizyme may
have been present in the LF population at ﬁequencies comparable to that of the years

since its discovery.

66

Table 3.4. Species diagnostic allele frequencies for two X-linked loci (Pgd and th) in
wild captured male specimens ﬁ'om the Early Flight and the Late Flight populations of
the Battenkill River Valley for 2000-2004. Allele ﬁequencies represented are the
combinations of all of the diagnostic alleles for each species lumped together in order to
represent the degree of genetic introgression. The species diagnostic Pgd alleles for P.
canadensis are 150, 125 and 80, and for P. glaucus are 100 and 50. The species
diagnostic th alleles for P. canadensis are 80 and 40, and for P. glaucus is 100. The
th hybrizyme is the novel [Ali 20 allele that was consistently detected in the Late
Flight. The "s for this allele in the 2000 and 2001 Late Flight populations indicate that
the allele was in fact present at high ﬁ'equencies, but as a result of being novel, failed to
be identiﬁed and had been misinterpreted as faulty gel results. An estimation of the
likely allele ﬁ'equency of the hybrizyme for those two years is shown in parentheses.

 

 

Loci Pgd th
Population EF LF EF LF
2000 2000
n=116 n=33 n=1 l6 n=33
canadensis 0.927 0.621 1.000 1.000 (0.850)
glaucus 0.073 0.379 0.000 0.000
hybrizyme *" *" (0.150)
2001 2001
n= n=5 1 n=0 n=5 1
canadensis 0.529 1.000 (0. 745)
glaucus 0.471 0.000
hybrizyme *" (0.255)
2002 2002
n=ll7 n=l3 n=ll7 n=l3
canadensis 0.927 0.539 1.000 0.539
glaucus 0.073 0.462 0.000 0.000
hybrizyme 0.000 0.462
2003 2003
n=29 n=l4 n=29 n=l4
canadensis 0.828 0.500 1.000 0.500
glaucus 0.172 0.500 0.000 0.000
hybrizyme 0.000 0.500
2004 2004
n=158 n=12 n=158 n=12
canadensis 0.930 0.542 0.984 0.750
glaucus 0.070 0.458 0.003 0.000
hybrizyme 0.013 0.250

67

Early Flight Late Flight

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2000
n=1 16 ’*
2001
n=51
r
2002
n=ll7 if Wn=l3
2003
_ ,,J
n=l4
l” 1
2004

   

 

 

 

 

 

hnéTSEW ‘ ‘ n=12

Figure 3.1. Degree of P. glaucus introgression into both the Early Flight and the Late
Flight populations of the Battenkill River Valley, for the diagnostic X-linked Pgd locus.
The portion of each graph that is ﬁlled in with white represents the proportion of
introgressed glaucus Pgd alleles in that population for 2000-2004.

68

Early Flight Late Flight

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2002
n=117 n=l3

2003
JH=14

2004

   

 

 

 

 

 

 

n=158 ’ n=12

Figure 3.2. Representation of the presence and proportion of the allele ﬁ'equencies of the
novel X-linked th 20 hybrizyme in both the Early Flight and the Late Flight populations
of the Battenkill River Valley. The portion of each graph that is ﬁlled in with gray

represents the proportion of the hybrizyme th allele in those populations for 2002-2004.

69

Table 3.5. Wright’s Fst values for the Early and Late Flight populations of the Battenkill
River Valley (2000-2004) based upon allozyme data collected from wild captured male
specimens. Pairwise values were calculated using Genepop Version 3.6. Values of
greater than 0.15 indicate signiﬁcant population differentiation and suggest little gene
ﬂow (F rankarn et al. 2002). F st values are presented for both of the individual X-linked
loci analyzed (Pgd and th). An * indicates that year for which the novel th 20
hybrizyme was potentially present but unidentiﬁed and therefore not appropriately
accounted for. Fst values that are signiﬁcant are underlined.

 

Year Pgd Mb
2000* 0253 0.013*
2002 m m
2003 0. 148 QM
2004 0.381 0.086

 

70

Mitochondrial DNA

All of the specimens analyzed ﬁorn both the EF and the LF populations for PCR-
RF LP were successfully ampliﬁed by PCR, except one specimen (LF 6‘#8-03). The PCR
products for all specimens were slightly shorter than 300bp long, as expected based on
previous work (Sperling & Hickey 1995; Stump et al. 2003). No specimens produced
two PCR fragments. All of the specimens analyzed from both the EF and the LF had
PCR fragments that were both out by SspI, and were not cut by Tan, except one
individual specimen ﬁ'om the EF (EF 6#1-03). The PCR product from this EF specimen
was both out by Tan, and was not cut by SspI (Table 3.6). In addition, for unknown
reasons, one of the LF specimens (LF 6‘#14-03) did not produce any banding pattern
whatsoever after the restriction enzyme digestion.

The corroborated data for all specimens that were successfully analyzed indicates
that the mtDNA carried by all of the LF specimens and all of the EF specimens except
one (EF 6‘#l), is canadensis like. P. glaucus mtDNA was only found in the single
individual from the EF. Table 3.6 provides both the mtDNA haplotypes of all individuals
analyzed as well as the associated genotypes for the two diagnostic X-linked loci (Pgd
and th). EF 6‘#1 possess glaucus mtDNA and also exhibits some X-linked
introgression. That specimen scored as heterozygous for the Pgd locus (one canadensis
allele and one glaucus allele) but scored as carrying two canadensis alleles for th. This

indicates that that specimen was not an F1 hybrid, but is rather a recombinant type.

71

Table 3.6. mtDNA haplotypes and diagnostic X-linked loci (Pgd and th) allozyme
genotypes for fourteen wild captured males from 2003 ﬁom both the Early and Late
ﬂights of the Battenkill River Valley populations of Papilio. Introgressed glaucus alleles

are presented in bold print.

 

 

 

 

 

 

Specimen mtDNA Pgd th

Early Flight 6#1 (+) 125/100 80/80
Early Flight 6#4 (—) 125/100 80/40
Early Flight 6#6 (—) 125/125 80/80
Early Flight 6#7 (—) 125/80 80/80
Early Flight 6#8 (—) 125/125 80/80
Early Flight 6#9 (—) 125/125 80/80
Early Flight 6#10 (—) 100/80 80/80
Early Flight 6#11 (—) 125/125 80/80
Early Flight 6#12 (—) 125/80 80/80
Early Flight 6#13 (—) 125/125 80/80
Early Flight 6#14 (—) 125/80 80/40
Early Flight 6#15 (—) 125/80 80/40
Early Flight 6#16 (—) 125/125 80/80
Early Flight 6#17 (—) 125/125 80/80
Early Flight 6#18 (—) 125/100 80/80
Late Flight 6#1 (—) 125/125 80/20
Late Flight 6#2 (—) 125/100 80/20
Late Flight 6#3 (—) 125/100 80/20
Late Flight 6#4 (-) 125/100 80/20
Late F light 6#5 (—) 100/80 8000
Late Flight 6#6 (—) 100/100 40/20
Late Flight 6#7 (—) 100/80 80/20
Late Flight 6#8 (?) 100/100 40/20
Late Flight 6#9 (—) 125/125 80/20
Late Flight 6#10 (-) 100/100 80/20
Late Flight 6#11 (—) 150/100 80/20
Late Flight 6#12 (—) 125/125 40/20
Late Flight 6#13 (—) 125/100 80/20
Late Flight 6#14 (?) 125/100 80/20

Key

Species mtDNA PE th

canadensis (—) 150, 125, 80 80, 40
glaucus (+) 100 100

 

72

Emergence Investigations

The timing of pupal eclosion and adult emergence of the LF broods, that were
derived from wild captured LF females, was consistently delayed compared to that of the
EF broods for both years assayed under every temperature regime (2003 at 18 °C, 22 °C,
26 °C and 2004 at 14 °C, 18 °C, 22 °C, 26 °C). The mean days to emergence was
signiﬁcantly less for the EF broods than it was for the LF broods in all categories (Table
3.7). And in fact there was no overlap of the timing of any of the emerging broods under
any of the individual temperature conditions, meaning that the last individual had
emerged ﬁom the EF brood before the very ﬁrst individual emerged from the LF brood
with only three individual exceptions to this (see Figs. 3.3 and 3.4). In the 18 °C
chamber in 2003, and in the 14 °C and 26 °C chambers in 2004, a single specimen each
that were among the LF brood specimens (LF 18 °C 6#1-03, LF 14° SB #1-04, and LF
26° 6 #1-04) emerged well ahead of their conspeciﬁcs and in fact emerged directly in the
midst of the EF emergence ﬂush.

In addition to determining that the timing of emergence was distinct between the
EF and LF populations, analysis was conducted to compare the timing of emergence
between males and females under each temperature regime (Table 3.8). For both years
that analysis was performed (2003-2004), and under every temperature regime, the mean
days to emergence was slightly less for males than for females. In all categories except
two, these differences in emergence timing were signiﬁcantly different. The average
emergence timing of females being slightly delayed ﬁorn that of male specimens is
highly adaptive considering how important protandry has been found to be as a

component of reproductive strategies in many animals including Papilio (Wiklund 2003).

73

Table 3.7. Comparisons of the days to emergence between Early Flight and Late Flight
ﬁeld reared pupae. These comparisons were conducted in two sequential years across
multiple temperatures (2003 at 18 °C, 22 °C, 26 °C and 2004 at 14 °C, 18 °C, 22 °C, 26
°C). Values represented are the mean number of days +/- s.e. ﬁom the time that pupae
were removed from winter like conditions to the day of pupal eclosion Sample sizes are
presented in parentheses. Probability values are derived from a nonparametric 2-sample
Wilcoxon test performed using JMP 6.0. Probabilities for comparisons that are
signiﬁcantly different are indicated with an *. All comparisons are signiﬁcantly different
with p-values <0.0001.

 

Mean Days to Emergence +/- s.e.

 

 

Year Temp. EF (n) LF (n) Prob>[Z]
2003 18 °C 26.37 +/- 0.70 (67) 50.29 +/- 1.27 (34) <0.0001*
22 °C 15.78 +/- 0.34 (64) 32.94 +/- 1.31 (36) <0.0001"
26 °C 12.32 +/- 0.27 (65) 25.92 +/- 0.68 (36) <0.0001*
2004 14 °C 50.40 +/- 0.82 (42) 93.97 +/- 2.97 (29) <0.0001*
18 °C 24.71 +/- 0.30 (48) 46.29 +/- 0.83 (35) <0.0001“
22 °C 17.02 +/- 0.28 (47) 32.78 +/- 0.82 (37) <0.0001“
26 °C 11.69 +/- 0.16 (48) 25.28 +/- 0.64 (39) <0.0001“

 

74

2003 Emergence Experiments
Early Flight 18 °C

 

 

#Emergod

 

 

 

O 510152025303540455055606570
DayatoEmergenca

 

 

 

Late Flight 18 °c

 

 

12

I
a
#Emergod

L4
8-2

 

 

 

0 510152025303540455055606570
DayatoEmamenca

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Early Flight 22 °C
--30
no 3
8
~10 ,,
I I11 I I It
0 510152025303540455055606570
DaystoEmeIgence
Late Flight 22 °C
~15
~10 3
8
r—5 2

 

 

 

O 510152025303540455055606570
DaystoEmergance

 

 

 

Figure 3.5. Histogram comparison of the days to emergence between Early Flight and
Late Flight ﬁeld reared pupae in 2003 at 18 °C, 22 °C, and 26 °C.

75

Early Flight 26 °c

 

 

 

 

 

 

 

 

Late Flight 26 °c

 

 

 

 

 

 

 

1

I
0 510152025303540

Daysto Emergence

I I
45

I
50

—30
8
-20 g’
5
—10 *
0510152025303540455055606570
DayatoEmerenca
~10
l-8
—4 Lu
r—2 *

 

Figure 3.5 continued.

76

 

 

2004 Emergence Histograms

 

 

   
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

Early Flight 14 °C
—10 8
— 8’
—6 E
_ 1.1.1
as
I . —2
05152535455565758595110125140
DaystoEmergence
LateFlight 14°C
8
9
o
E
ur
at
05152535455565758595110125140
DaystoEmemence
Early Flig ht 18 °c
8
2'
o
E
In
at
0 510152025303540455055606570
DaystoEmergence
lateFlight18°c
—10
—a 8
-6 g
—4 ul
.—2 ﬁ
0 510152025303540455055606570
DaystoEmeIgenoe

 

 

 

Figure 3.6. Histogram comparison of the days to emergence between Early Flight and
Late Flight ﬁeld reared pupae in 2004 at 14 °C, 18 °C, 22 °C, and 26 °C.

77

Early Flight 22 °c

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

—30
8
—20 2’
0
5
--10 at:
TllllllVllllllllllUlllITTIIIII
0 510152025303540455055606570
DaystoEmergence
Late Flight 22 °C
—8
_. 8
6 8
r—4 E
Lu
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0 510152025303540455055606570
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Early Flight 26 °C
~40
— 8
3° 8
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Lu
_1o %
Tlllllllllllllllllll
0 510152025303540455055606570
DaystoEmergence
Late Flight 26 °C
—10 g
—6 E
_ In
a:
—2
0 510152025303540455055606570
DayatoEmargonce
Figure 3.6 continued.

78

 

 

 

 

Table 3.8. Comparisons of the days to emergence between males and females in both the
Early Flight and Late Flight ﬁeld reared pupae. These comparisons were conducted in
two sequential years across multiple temperatures (2003 at 18 °C, 22 °C, 26 °C and 2004
at 14 °C, 18 °C, 22 °C, 26 °C). Values represented are the mean number of days +/- s.e.
from the time that pupae were removed from winter like conditions to the day of pupal
eclosion Sample sizes are presented in parentheses. Probability values are derived from
a nonparametric 2-sampleWilcoxon test performed using JMP 6.0. Probabilities for
comparisons that are signiﬁcantly different are indicated with an *.

 

Mean Days to Emergence +/- s.e.

 

 

 

 

Year Temp. Males (n) Females (n) Prob>[Z]
Early Flight 18 °C 24.74 +/- 0.88 (38) 28.52 +/- 1.01 (29) 00004"
2003 22 °C 15.54 +/- 0.45 (28) 15.97 +/- 0.50 (36) 0.3560
26 °C 12.03 +/- 0.31 (30) 12.57 +/- 0.42 (35) 0.1440
Late Flight 18 °C 46.41 +/- 1.59 (17) 54.18 +/- 1.50 (17) 0.0008"
2003 22 °C 29.93 +/- 1.00 (15) 35.10 +/- 2.03 (21) 0.0250"
26 °C 24.43 +/- 0.95 (21) 28.00 +/- 0.65 ( 15) 0.0025"
Early Flight 14 °C 48.15 +/- 0.77 (26) 54.06 +/- 1.33 (16) 0.0002*
2004 18 °C 23.24 +/- 0.28 (21) 25.85 +/- 0.36 (27) <0.0001“
22 °C 15.80 +/- 0.19 (25) 18.41 +/- 0.38 (22) <0.0001"
26 °C 11.12 +/- 0.14 (26) 12.36 +/- 0.22 (22) <0.0001*
Late Flight 14 °C 89.41 +/- 2.56 ( 17) 100.42 +/- 5.85 ( 12) 0.0237"
2004 18 °C 44.15 +/- 0.92 (20) 49.13 +/- 1.17 (15) 0.0036"
22 °C 30.18 +/- 0.99 (17) 35.00 +/- 1.03 (20) 0.0029"
26 °C 23.67 +/- 0.76 (24) 27.87 +/- 0.74 (15) 0.0007"

 

79

DISCUSSION

Based upon the morphometric and molecular evidence, both the Early and Late
Flight populations of the Battenkill River Valley can be characterized as hybrid swarms.
Both of these ﬂights exhibit higher levels of P. glaucus introgression than does the stable
South Manitou Island hybrid swarm (see Chapter 2). The level of P. glaucus
introgression exhibited in the Late Flight however, is far greater than that of the Early
Flight.

The LF is likely the product of relatively high rates of glaucus introgression into
the EF. This is made possible as a result of recent warming due to climate change in a
highly condensed thermal landscape of the region near the hybrid zone (refer to Figure
1.4). Certain recombined backcross genotypes apparently result in an alteration of
developmental rates leading to a signiﬁcantly delayed spring emergence. It can be
argued however that the LP is not merely a genetic “sink”, annually being replenished by
backcross offspring. The timing of the emergence of pupae derived ﬁom LF adults
consistently results in their emergence being appropriately delayed, compared to that of
the EF. This indicates that the LF is primarily the result of recurring annual reproductive
activities within the LF. Additionally, the complete absence of the novel th 20
hybrizyme in the EF through 2003 suggests that this novel allele arose as a mutation in
the LF and that there has been little opportunity for genetic mixing between these two
populations.

Based upon ﬁeld observations and sampling success, it is clear that the LF is not
nearly as numerous as is the EF. As a result, the th 20 hybrizyme may have become so

prevalent in the LF due to genetic drift in a small population. Alternatively, there may be

80

some metabolic ﬁtness advantage associated with this hybrizyme in the novel thermal
niche of the LF, favored through natural selection. It is important to note that the range
of this Late Flight occurs in the geographic area that correlates with an accumulated
2300-2700 thermal degree-days. This is nearly the same thermal niche being exploited
by P. appalachiensis (2600-2850 degree days) in the mountains of West Virginia and
Georgia (Scriber et a1. 2007).

An important element to the production of a stable, self-sustaining, reproductively
isolated population is that the emergence of males and females is appropriately timed (i.e.
synchronous), so as to maximize reproductive success. Protandry is a key element to this
in many animals, and widely observed in butterﬂies (W iklund 2003). The degree to
which the timing of LF male emergence is appropriately just prior to the emergence of
LF females (see Table 3.8) would strongly promote successful annual regeneration of a

late ﬂight derived from the LF of the previous year.

Both ﬁeld observations and laboratory investigations suggest that the Late Flight
population may in fact be temporally isolated enough to constrain gene ﬂow between
these two populations. Field observations over the course of the past ﬁve years in the
Battenkill River Valley report the early canadensis like ﬂight occurring primarily in late
May and early June. The Late Flight adults are observed ﬂying in mid to late July
(Romack personal communication). Additionally, ﬁeld reared pupae from both ﬂights
held in cold storage, have been simultaneously placed side by side under different
temperature regimes (l 8, 22, and 26° Celsius) in climate controlled growth chambers,
and monitored for emergence times (see Figures 3.5 and 3.6 and Table 3.7). Two years

worth of collected data indicates that both the males and female from pupae from the

81

Late Flight exhibit a delayed emergence from that of the Early Flight adults. In every
temperature regime, the last adult had emerged ﬁom the Early Flight before the ﬁrst adult
had emerged from the Late Flight, with only 3 individual exceptions. This time lag
between the two broods was enhanced in the colder temperature regimes.

The comparatively premature emergence of the three individual LF specimens
could potentially be explained by an error in labeling, or were in fact the result of
unnoticed larvae that were already on the branch of the tree that had been oviposited by a
free ﬂying female. Either of these scenarios could potentially result in EF specimens
being artiﬁcially grouped with LF specimens. This possibility was investigated by
looking at the pupal weights of these individuals. One of the three specimens (LF 26° 6‘
#1-04) did in fact have a very low pupal weight (0.683 grams) that is much more typical
of those pupae ﬁ'om the EF. The other two specimens however had relatively large pupal
weights (.965 and 1.116 grams), the second of which is larger than any EF pupae
measured. Based upon pupal weight, these individuals would be categorized as LF
specimens. An alternative possibility to explain unusually premature emergence in these
specimens is that they possessed a genotypic backcross combination that resulted in this.

The emergence data is an indication of expected relative ﬂight times but is highly
temperature dependent. It should be noted that these emergence investigations were
conducted at constant temperatures for both years in each temperature regime. This is
not an ideal replication of ﬁeld conditions in that there would be naturally occurring
ﬂuctuations in temperatures. Early spring would be somewhat cooler, and the
temperatures would gradually increase as spring progressed. Additionally, ﬂuctuations

would be experienced between day and nighttime temperatures.

82

Under all of the laboratory maintained temperature regimes, the mean emergence
times did appear to be distinct between these two populations but did not provide for a
signiﬁcant window of time, if any at all, between the ﬂights. For instance in 2003 under
the 26 °C temperature regime, the last EF specimen to emerge in the chamber is a female,
and the very next day, the ﬁrst LF specimen, a male emerges. The BF female would only
have needed to survive a single day to have potentially encountered and mated with this
LF male. This emergence pattern certainly would not afford sufﬁcient temporal isolation
to prevent gene ﬂow. However, the laboratory temperature regime that is more indicative
of what these populations would likely encounter in the wild is the 18 °C chamber.
Considering the 2003 emergence data, there is a 10—day window between the last EF
emergence and the ﬁrst LP emergence under those conditions. Under these conditions,
which more closely resemble ﬁeld conditions, there is increased temporal isolation,
which could greatly limit gene ﬂow. In fact, it is important to remember that based upon
ﬁeld observations in the Battenkill River Valley, the EF in the ﬁeld occurs from early to
mid June and the LF emerges in mid July (Romack personal observations).

This pupal eclosion time lag does not in itself guarantee complete reproductive
isolation however. It is still plausible that a late emerging Early Flight female could
remain active for two weeks in the ﬁeld and cross paths with one of the ﬁrst to emerge
protandrous males from the Late Flight. There is however, additional evidence, which
suggests that this is inﬁequent at most. Allozyme electrophoresis has been employed to
monitor the X-linked canadensis and glaucus like allele frequencies of both Pgd and th
enzymes. As previously stated, the ﬁequency of glaucus like Pgd is much higher in the

Late Flight, and there is zero glaucus like th introgression in either ﬂight. However, a

83

startling number of the Late Flight generation specimens exhibit the novel th 20
hybrizyme allele that had never before been reported. th 20 allele frequencies have
been identiﬁed to be as high as 50% in the Late Flight (Fig. 3.2). This rare allele was
identiﬁed in the Early Flight, but in only the most recent year (2004) and at a very low
frequency (003%).

In the ﬁeld, those rare LF individuals (1.3% of those LF specimens reared in the
sleeve rearing experiment) that were seen to have emerged well in advance of the rest of
the LF brood, and in the midst of the EF emergence pattern, would serve to allow for low
levels of genetic introgression from the LF population into the EF population. In this
way the th 20 hybrizyme could have been successfully transmitted. This would explain
why it appeared in the last year of the specimens analyzed. Overall, the striking contrast
of the allele frequencies for both of the X-linked loci, especially the newly identiﬁed
“hybrizyme” suggests that there is very little genetic exchange between these two

populations.

Incipient Speciation?

Based upon the analyzed data presented, a snapshot in time indicates that EF and
LF are distinct subpopulations that appear to be largely reproductively isolated. This
would indicate that this localized “Late Flight” phenomenon exhibits a combination of
circumstances and mechanisms that could result in allochronic isolation that has resulted
from introgressive hybridization. The timing of diapause and pupal eclosion has been
reported to be a likely mechanism by which to provide an isolating mechanism other

systems. Recent investigations done on the well studied Rhagoletis group has indicated

84

that in fact the host race differences associated with diapause may have been the key trait
that allowed for the initiation of differentiation (F eder et al. 2003a). It has been found
that this character trait actually arose in allopatry F eder et al. 2003b).

Ifthe newly described species, P. appalachiensis, is in fact the result of hybrid
speciation, and the Battenkill River Valley is another location where there are
signiﬁcantly increased levels of genetic introgression due to recent climate shifts, is it
possible to identify and capture climatic speciation in the act? The Late Flight could be
described as an incipient species. The morphological characteristics (forewing length and
hind wing black band width) and the timing of emergence of specimens collected ﬁ'om
this population indicate that they are virtually identical to P. appalachiensis. The major
difference between P. appalachiensis and the Late Flight is that P. appalachiensis
populations have nearly ﬁxed allozyme frequencies (glaucus like Pgd and canadensis
like th), while the Late Flight still exhibits a higher retention of canadensis like Pgd
alleles.

A combination of mechanisms could eventually allow for the Late Flight allele
frequencies to become ﬁxed for the glaucus-like Pgd alleles, thus making the Late Flight
virtually indistinguishable from P. appalachiensr’s. First, the Late Flight would need to
be sufﬁciently temporally, and thus reproductively isolated ﬁ'om the ﬁrst ﬂight of
canadensis to prevent the continued inﬂux of canadensis-like genes. Second, there
would need to be either sufﬁcient selection (intrinsic or extrinsic) against the canadensis-
like Pgd allele to eliminate it (or something closely linked on the X-chromosome), or the
elimination of the canadensis Pgd alleles could be accomplished through random genetic

drift over time. These possibilities are examined in Chapter 4.

85

CHAPTER 4:
TRAIT LINKAGE ON THE X-CHROMOSOME OF PAPILIO CANADENSIS
AND P. GLA UC US, REVEALED BY A HIGHLY INFORMATIVE

BACKCROSS

Introduction

In all of the P. canadensis populations that have begun to exhibit P. glaucus
introgression, including the populations of P. appalachiensrs, there appears to be a
consistent pattern of “glaucus-like” traits, both present and absent. These populations
possess individuals, who score either intermediate or glaucus-like for most of the
species diagnostic traits (forewing length, hind wing black-band width, oviposition
preference, tulip tree detoxiﬁcation ability, and Pgd and I-lk allozymes (Scriber and
Ording 2005)). However, none of the several hundred individuals analyzed ﬁ'om
these populations has possessed the glaucus X—linked th allele (100). Nor have
there been any individuals captured that have demonstrated the ability for direct
development, which is dictated by the X-linked od- gene allowing for facultative
diapause.

The latter of these two traits not being present is not so surprising. As stated
previously, the offspring of any individual possessing the od- gene, that underwent
direct development, would stand no chance of completing development to pupation
given the limited thermal environment. As earlier stated, completion of two full
generations within a single year requires 2800 degree-days, even on the highest

quality host plant species for rapid larval growth (Scriber and Lederhouse 1992;

86

Scriber 1996b). On average, there are simply insufﬁcient annual thermal units in the
locations where these hybrid swarm populations exist. This thermal constraint would
likely act to strongly select against the od- gene.

As described in Table 1.1, studies of diagnostic traits in these two Papilio
species have determined that ﬁve known species-speciﬁc genetic differences exist on
the X—chromosome (Scriber 1994, Hagen and Scriber 1995). The sequence in which
these loci exist along the length of the X-chromosome, as well as an estimated
relative map distance between each locus, is depicted in Figure 1.3.

Individual specimens have been ﬁeld collected ﬁ'om these introgressed
populations that have exhibited traits that suggest that they possess recombinant
genotypes of X-linked diagnostic traits. There are two potential methods by which an
individual could express an apparently non-concordant genotype. One method by
which apparently recombined genotypes could arise is through segments of the X-
chromosome being translocated to an autosome, and in this way passed on to
offspring. Sex chromosome segment translocation has been described in the
Mediterranean ﬂour moth (Marec et al. 2001). A more commonly described method
by which recombinant types could arise in this Papilio group is through chromosomal

crossovers during meiosis in males.

Why is there zero introgression of “glaucus-like” th (100)?
Given the apparent frequency with which chromosomal crossovers can occur
in these hybridizing butterﬂies, why is it then that the “glaucus-like” th 100 allele is

never expressed in individuals sampled from the introgressed populations of P.

87

canadensis? One possible explanation is that the [Ab locus is far more closely linked
to the diapause locus than has been previously suggested (Hagen and Scriber 1984).
A close linkage between the “glaucus-like” facultative diapause and th 100 would
explain why neither trait occurred in these populations. As earlier stated, individuals
possessing the facultative diapause gene would be rapidly eliminated ﬁ'orn
populations anywhere with fewer than 2750 °F degree days in the colder more
northern regions of North America.

An alternative hypothesis explaining the absence of [Ab 100 in introgressed
populations of P. canadensis is that there is direct selection on the th gene.
Allozyrnes are often utilized as genetic markers in population investigations and are
assumed to be neutral genetic markers. However, lactate dehydrogenase is an
important enzyme in the metabolism of carbohydrates. Distinct alleles of this enzyme
have been shown to exhibit structural variability and temperature dependent
differences in thermal stability and ﬁmction (Adams et al. 1973). Some alleles
perform much better at higher or lower temperatures, whereas others perform poorly
under certain temperature regimes (Angiletta et al. 2003). Possession of different
allelic forms of the [db enzyme having differing thermal stabilities has resulted in a
steep latitudinal cline in marine ﬁshes directly correlating with environmental
temperatures (Crawford and Powers 1989; Dimichele and Powers 1991). There is
clear evidence that differing thermal environments impose striking differential
selective pressures on different structural forms of enzymes (Eanes 1999). This has
been shown in several studies to be true speciﬁcally for th (Crawford and Powers

1989; Johns and Somero 2004; Schulte et al. 2000).

88

Furthermore, it has been shown that minor modiﬁcations to the gene that
codes for th can result in signiﬁcant structural changes that in turn result in
differences in thermal stability. Minimal amino acid substitutions have been
identiﬁed in the th enzyme in six different species of barracuda living in different
thermal environments (Holland et al. 1997). It has been shown that there is a single
amino acid substitution in the th enzyme between two distinct species of
damselﬁshes, one native to cold-temperate habitats, while the other is native to
tropical waters (Johns and Somero 2004). This too has been identiﬁed to be the case
in the th enzyme between P. canadensis and P. glaucus. Preliminary results
indicate that a single base pair substitution is responsible for the structlnal differences
between th 100 (glaucus) and Mb 80 (canadensis) (Andolfatto 2004 personal
communication). Collaborative work is now underway to identify DNA sequence

differences between the various th alleles.

Lab rearing organisms under environmentally controlled conditions can be of
great value in that it can help to eliminate the uncertainties and confounding variables
that can arise under ﬁeld conditions. Additionally, there is great value to being able
to produce lab reared crosses in that you can better determine and manipulate the
genetic background of the parents and resulting offspring. When investigating the
mechanisms that prevent widespread gene ﬂow across a hybrid zone, lab reared
crosses between individuals of known genetic material allows controlled
investigations to distinguish between intrinsic and extrinsic barriers to gene ﬂow.

Recent investigations associated with newly described hybrid species have allowed

89

the “recreation” of hybrid species genotypes under laboratory conditions (Riesberg et
al. 2003; Maverez et al. 2006).

A great deal of work has been done with laboratory breeding investigations
conducted with Papilio glaucus and P. canadensis. Most of this work has been
applied towards identifying the mechanisms that are involved in the maintenance of
their narrow hybrid zone and also those factors that were involved in the processes of
their speciation (Luebke et al. 1988; Hagen et a1 1991; Hagen and Scriber 1995;
Scriber et al. 1999). It was determined through genetic analysis of laboratory-reared
crosses and backcrosses between- these two species, that the Haldane effect plays a
signiﬁcant role as a postzygotic isolating mechanism. It was found that an
incompatibility between canadensis X—chromosome genes and the glaucus Y-
chromosome or cytoplasm results in the disruption of female development (Hagen
and Scriber 1995). A laboratory hybridization study conducted by Pavulaan and
Wright (2002), who originally described the new P. appalachiensis, illustrates that
the Haldane effect was also exhibited in a brood produced through hand pairing of an
appalachiensis male and a dark morph P. glaucus female. This cross resulted in 24
eggs and 1St instar larvae but only resulted in 10 successﬁrl pupae. Of those 10 pupae,
only 7 males successfully emerged. The remaining 3 were females that never
successfully eclosed. (Pavulaan and Wright 2002)

There is a good deal of evidence that suggests that chromosomal crossovers
ﬁequently occur in these Papilio species. This is the abundance of individuals ﬁeld
captured from introgressed P. canadensis populations (South Manitou and Vermont),

that when analyzed through allozyme electrophoresis, exhibit the “glaucus-like” Pgd

9O

allele (100) but one of the “canadensis-like” th alleles (40 or 80), as is the ﬁxed
condition of P. appalachiensis (Scriber and Ording 2005). In addition, many lab
reared crosses have produced offspring expressing X-linked trait combinations that
can most readily be explained through chromosomal crossovers (Scriber 1994).

Data gathered through laboratory crosses between P. glaucus and P.
canadensis have also been applied toward the development of the linkage map of the
X-linked loci (Fig. 1.3) for which there are species diagnostic alleles (Hagen and
Scriber 1989). This linkage map provides an indication as to the sequence and an
estimate of the relative distances between major landmark loci along the length of the
X chromosome in this Papilio group. This provides a mechanism by which to predict
the likelihood of certain genetic recombinations occurring through genetic crossovers.

The rate at which genetic recombinations occur in P. glaucus and P.
canadensis has been found to be higher than in other insect groups including
Drosophila, relative to mutation rates (Putnam et a1. 2007). Fixed states of genetic
recombination between two parental species has been an element in characterizing
and a mechanism in helping to explain some cases of hybrid speciation (Riesberg et
al. 2003; Schwarz et al. 2005; Gompert et al. 2006) including the new Papilio
appalachiensis (Scriber and Ording 2005). Comparable genetic recombination
between P. glaucus and P. canadensis appears to explain the origin of the Late Flight
in the Battenkill River Valley of Vermont and New York. Lab-reared backcrosses
could help to “recreate” the genotypes that result in a delayed emergence mimicking
that of the Late Flight. Analysis of individuals of this type would then help to

identify the genetic combinations that result in this shift in such a major life history

91

trait, as is the timing of diapause. This chapter is devoted to the analysis of a unique
lab reared backcross that helps to illustrate the striking frequency with which
recombination can occur, the variety of genetic combinations that can be produced,
and partially determine whether it is intrinsic (genetic incompatibilities) or extrinsic
(environmental) factors that have acted as a mechanism to prevent gene ﬂow between

P. glaucus and P. canadensis.

92

MATERIALS AND METHODS

Laboratory Cross

The most informative type of cross in this Papilio system is to produce a
backcross between a dark morph female and a hybrid male. This provides the most
information regarding X-linked loci. In this investigation, a wild captured dark
morph female P. glaucus produced a series of offspring in the lab. One of the lab—
reared offspring was a virgin dark morph female (€2#18006). A lab-reared male was
produced from a hand—pairing between a dark morph P. g. Q ﬁom Pennsylvania and a
wild caught PC. 6‘ from Oscoda County, Michigan. S2#18006 was then hand paired
to this lab-reared F1 hybrid. After the pairing, S2#l8006 was placed in an oviposition
arena with suitable host plants upon which to deposit eggs. These eggs were
collected and the resulting larvae were reared through to pupation on appropriate host
plants under early to mid summer like photoperiod conditions (L18:D6) with the
intent of inducing direct development in those offspring that possessed the X-linked
facultative diapause gene (Rockey et al. 1987).

The resulting pupae were placed in screened cages and monitored for direct
development. Those specimens that direct-developed (n=16 dark morph S2, n=4
yellow S2, and n=396) were placed in —80 °C freezer for storage until allozyme
electrophoresis was conducted. The resulting pupae that did not direct develop were
collected in mid-September and stored in darkness at 3-5 °C under controlled
environmental diapause conditions until the recommencement of emergence
investigations. Those pupae were brought out of cold-storage in early May and

placed in screened petri dishes in a 26 °C growth chamber under a L18:D6

93

photoperiod. Specimens were monitored and as they emerged, they were placed in —
80 °C storage to await allozyme electrophoresis. Pupae that did not emerge after this
ﬁrst emergence opportunity were again placed in refrigerated cold storage for 12
weeks to mimic a second winter period. They were then again removed from cold

storage and monitored through another emergence period.

Allozyme Electrophoresis

Allozyme electrophoresis was conducted on Q#18006, the parental F 1 hybrid
to which she was hand paired, and all of the offspring that successfully pupated and
direct developed, or emerged the next year. Every specimen was assayed for both of
the X-linked diagnostic loci (Pgd and th). Allozyme electrophoresis was

accomplished following those methods described in chapters 2 and 3.

X-Chromosome Mapping

Based upon the suite of known X-linked diagnostic characters, analysis of the
sex, color, diapause behavior, and allozyme electrophoresis data, it was possible to
map and determine the parental source of each portion of the X-chromosome(s)
carried by each ofthe offspring produced in the 18006 brood. In this way it was
possible to detect which individuals carried X-chromosomes that were the result of a
genetic crossover.

Some assumptions have been made in the process of determining some of the
specimen allelic identities. The identity of the diapause locus has been based purely
on whether or not the specimen entered diapause. If they went into diapause they

were assumed to be carrying the canadensis diapause allele od+. Also, in the case of

94

the X-linked dark morph suppressor gene (s+) it is impossible to say for certain in the
case of male specimens, as they do not carry a Y-chromosome and therefore never
express the dark morph phenotype (b+; Scriber et al. 1996). In these male specimens

the allelic identity was assumed based upon the identity of the Pgd locus.

Analysis for Haldane Effect

A determination as to whether a Haldane effect is exhibited in hybrid
backcrosses was accomplished by determining the relative survival probabilities for
female offspring (heterogametic sex in Papilio) from the 18006 brood. Survival
probabilities were determined relative to those of male offspring from the 18006
brood with the same paternally inherited th and Pgd alleles. These probabilities
were obtained by dividing the number of females with each genotype by the number

of males with the corresponding paternal haplotype.

95

RESULTS

Emergences

Of the pupae that were produced from the 18006 brood, 39 male and 20
female specimens (4 yellow and 16 dark morph) direct developed. Four male and
four dark morph female specimens emerged during the ﬁrst post diapause emergence
period. One additional male and three additional dark morph female specimens

emerged after the second post diapause emergence period.

Allozyme Electrophoresis and X-chromosome Mapping

Table 4.1 illustrates the determined character state for each of the four X-
linked loci for 92#18006, her F1 mate, all of the direct developing offspring (n=16
dark morph E2, n=4 yellow S2, and n=396), the diapausing specimens that emerged
the following year (n= 4 dark morph S2 and n=46), and the diapausing specimens that
emerged after two winter like diapause periods (n=3 dark morph S2 and n=1 6‘).
Images in this dissertation are presented in color. The degree of certainty of the
mapping of the individual X-chromosomes for every individual was possible only due
to the fact that the male specimen to which Q#18006 was paired carried a relatively
rare Pgd allele. From his dark morph Pennsylvania mother he received an X-
chromosome that carried an extremely rare Pgd 50 allele. This allele exists in
relatively low frequencies in glaucus populations (Hagen and Scriber 1991). Had he
carried the far more commonly observed glaucus Pgd 100 allele, this would have

served to mask and prevent a true detection of some chromosomal crossovers.

96

Table 4.1. X-chromosome linkage maps for each of the offspring produced in
the18006 brood. The linkage maps represented indicated the following loci in
sequential order: dark morph suppressor/ Pgd / diapause / th. Images in this
dissertation are presented in color. Color-coding allows for determination of the
parental origin of each chromosome or chromosomal segment. Portions of
chromosomes that could not be determined with conﬁdence are indicated with ???.

 

Mother — Pg. 9 # 18006 Penn. (Dark) s- 100 0d- 100
Y'
Father — Pg. ‘2 Dark Penn. X Re. 6‘ # 17047 Oscoda 8+ 125 od+ 40

s- 50 od- 100

F1 -— Direct Developing 52s

1. s- 50 od- 100 11. 3+ 125 od- 100
8’ ‘Y

2. s- 50 od- 100 12. s- 50 od- 100
8’ 8’

3. s- 50 od- 100 13. s- 50 od- 100
Y' 8’

4.. s- 50 od- 100 14. 5+ 125 od- 100
8’ ‘Y

5. s- 50 od- 100 15. s- 50 od- 100
Y' ‘Y

6. s- 50 od- 100 16. s- 50 od- 100
8’ ‘Y

7. s- 50 od- 100 17. 5+ 125 od- 100
Y' ”Y

8. s— 50 od- 100 18. s- 50 od- 100
8’ ”Y

9. s- 50 0d- 40 19. s- 50 od- 100
Y’ 8’

10. 8+ 125 od- 100 20. s- 50 od- 100
8’ 'Y

97

F 1 - Direct Developing 6‘s

1.

10.

11.

12.

13.

14.

15.

50
100

50
100

50
100

125
100

50
100

50
100

125
100

50
100

50
100

125
100

125
100

50
100

50
100

50
100

125
100

od-
od-

0d-
od-

od-
od-

od-
od-

0d-
od-

od-
od-

od-
od-

???

od-

od-
od-

???

od-

???
od-

od-
od-

0d-
od-

???
od-

od-
od-

100
100

100
100

100
100

100
100

100
100

100
100

100
100
100
100
100
100
???
100

100
100

100
100
100
100
100

98

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

50
100

50
100

125
100

50
100

125
100

50
100

50
100

50
100

125
100

125
100

125
100

50
100

125
100

50
100

125

.100

od+
od-

od-
od-

od-
od-

od-
od-

od+
od-

od-
od-

od+
od-

od+
od-

od-
od-

od-
od-

od+
od-

od+
od-

od+
od-

0d-
od-

od-
od-

40
100

100
100

100
100

100
100

40
100

l ()0
100

40
100

40
100

100
100

100
100

40
100

40
100

40
100

100
100
100
100

31.

32.

33.

34.

35.

125
100
50

100

50
100

50
100

125
100

od+
od-

od-
od-

od+
od-

od-
od-

od+
od-

40
100

100
100

40
100

100
100

40
100

First Emergence Fl Diapausing 82s

21. s- 50 od+ 40
8’

22. s- 50 od- 100
8’

23. s- 50 od- 100
Y'

24. s- 50 od+ 40
8’

Second Emergence Fl Diapausing

£28

25. s- 125 ??? 100
8’

26. s- 125 od+ 4O
8’

27. s- 50 ??? 100

36. 5+ 125 od- 100
s- 100 od- 100
37. 3+ 125 od- 100
s- 100 od- 100
38. ?? ??? ??? ???
s- 100 od- 100
39. 5+ 125 od+ 40
s- 100 od- 100
First Emergence Fl Diapausing 6s
40. s- 50 od+ 40
s- 100 od- 100
41. 5+ 125 od- 100
s- 100 od- 100
42. 5+ 125 od+ 40
s- 100 od- 100
43. 5+ 125 od- 100
s- 100 od- 100

Second Emergence F1 Diapausing 6

44.

5+

S-

125
100

???
od-

100
100

All of the X-linked alleles that were detected in both parents were observed
throughout the offspring. Of the total number of offspring analyzed, 28 out of 71
specimens (39.4%) were determined to possess a recombined X-chromosome. Of the
20 direct developing females, ﬁve of them (25%) carried recombined X-
chromosomes. Of the 39 direct developing males, 15 of them (38.5%) carried
recombined X-chromosomes. Four out of seven diapausing females and four out of
ﬁve diapausing males carried recombined X-chromosomes. Of those crossovers that
were detectable, 90% of them (26 out of 29) were the result of a cross between the
Pgd and diapause loci. Two specimens (diapausing 821726 and Q#27) exhibited a
crossover that was determined to be between the dark morph suppressor (s) and the
Pgd loci. Also, there was only a single specimen (direct developing €2#9) that
exhibited a crossover that was determined to be between the diapause and th loci.

Evidence for multiple crossovers on the same individual X-chromosome
exists in only one female specimen (Q#25). This is a yellow female carrying a Pgd
125 allele and an th 100 allele. This indicates that a crossover must have occurred
between the dark morph suppressor and Pgd loci and also somewhere between the
Pgd and th loci. This observation indicates that it is possible that there were other
specimens in which there were multiple crossovers, some of which were undetectable

based upon the markers available.

Analysis for Haldane Effect
Of the 65 total offspring that were successfully reared to adulthood from the
18006 brood, there were a total of 39 males and only 26 females. The ratios of

female to male specimens that were produced in each of four genotypic categories,

100

these based on the species diagnostic X-linked alleles possessed for both the Pgd and
th loci are contained in Table 4.2. The results indicate that there is a Haldane Effect
represented in this brood. The haplotype for which there is the greatest female
survival are those specimens carrying an apparently in tact glaucus X-chromosome,
and in this category survival probability is actually greater than for males. Females in
each of the alternative haplotype categories, either an X-chromosome that exhibits
mixed canadensis and glaucus elements, but especially those that carry an apparently

pure canadensis X-chromosome, exhibit reduced survival probabilities compared to

their male counterparts.

101

Table 4.2. Relative survival probabilities for 18006 backcross females. Survival
probabilities are relative to those of males with the same paternally inherited th and
Pgd alleles. Probabilities were obtained by dividing the number of females with each
genotype by the number of males with the corresponding paternal haplotype. These
numbers are given in parentheses as (#females / #males).

 

 

Pgd 50 Pgd 125
(glaucus) (canadensis)
th 100 1.29 (18/14) .33 (4/12)
(glaucus)
th 40 .50 (3/6) .14 (1/7)
(canadensis)

 

102

DISCUSSION

The X-chromosome has been found to play a disproportionately large role in
the evolution of isolating mechanisms and the process of speciation in Lepidoptera
(Prowell 1998; J iggins et al. 2001). Butterﬂies (and birds) are somewhat unusual in
that the male sex is homogametic. It has been found that traits carried on the X-
chromosome are primarily responsible for the maintenance of reproductive isolation
between Papilio glaucus and P. canadensis and that there is a detectable Haldane
effect (Hagen and Scriber 1995). The Haldane effect results in decreased viability of
the heterogarnetic sex due to genetic incompatibilities. Analysis of hybrid
backcrosses can help to differentiate between which X-linked loci combinations are
involved in hybrid genetic incompatibilities and those that are not.

This 18006 backcross, and other backcrosses of this nature, provide valuable
information as to the hybrid recombinant genotypes that can be produced in
introgressed hybrid swarms. The ability to identify the relative locations of multiple
loci, both those of value as species diagnostic tools and also those that impact
signiﬁcant life history traits, provides a clear environmental mechanism by which to

explain the sharply contrasting clines that are exhibited across the hybrid zone.

The analysis of the 18006 brood, derived from a laboratory hand-pairing, has
proven extremely valuable. Given that male Papilio are the homogarnetic sex, any X-
chromosome crossovers that might occur could only have taken place during the
process of sperm production. One thing that is obvious and striking about the genetic

analysis of the 18006 brood, is the high ﬁequency with which recombination can

103

occur. In this single brood, over 37% of the offspring produced were recombinant
types, possessing individual X-chromosomes that carry alleles that are diagnostic for
two different species.

The next observation that can be made is the location at which the crossovers
have been determined to occur, and with what ﬁequencies. Out of the high number
of individuals possessing a recombinant X-chromosome, 93% of them exhibit a
crossover that is between the Pgd and diapause loci, and only a single specimen
exhibits a crossover between the diapause and th loci. This is striking considering
the relative distances between these loci as represented on the linkage map previously
developed (see Fig. 1.3) by Hagen and Scriber (1989). That linkage map suggests
that the distance between the Pgd and diapause loci is relatively small, and in
comparison the distance between the diapause and th loci is large. The results ﬁrm
the 18006 analysis indicates that the likelihood of a crossover occurring between the
Pgd and diapause loci is extremely high, and that the likelihood of a crossover
between the diapause and th loci is surprisingly low. This suggests that there must
be a great deal more distance between the Pgd and diapause loci than between the
diapause and the th loci.

The glaucus th 100 allele has previously never been reported in ﬁeld captured
specimens collected in the hybrid zone or in any identiﬁed hybrid swarm population.
This could be explained through extrinsic (ecological) selection. If in fact the
diapause and th loci are linked much more closely than has historically been
suggested, environmental factors would act strongly to prevent such combinations

from surviving in the ﬁeld. P. glaucus carry a facultative diapause gene (od-)

104

allowmg for a bivoltine life cycle in thermally appropriate conditions. Diapause in P.
glaucus is triggered by the shortening photoperiods associated with late summer. If
this od- allele, tightly linked to the glaucus th 100 allele, were donated to a hybrid
brood, the offspring would likely direct develop in preparation to produce a second
ﬂight. The photoperiod at the locations at which such a cross would occur would
typically induce direct development in the offspring. However, in these northward-
extended latitudes, the continuing warmth of summer would be relatively short,
providing insufﬁcient thermal energy to allow for the second brood to successfully
achieve pupation. These hybrid individuals would die in the ﬁeld, and the [db 100

allele would be strongly selected against.

The Haldane effect has been reported as a mechanism that results in postzygotic
isolation between P. glaucus and P. canadensis (Hagen and Scriber 1995). The
explanation has been that there is some form of genetic incompatibility between the
canadensis X-chromosome and either the glaucus Y—chromosome or cytoplasm.
Often times, backcross females remain in “extend ” or permanent diapause. The
genotypes of these female backcrosses that die as pupae would be reﬂected as a
deﬁcit in the sex ratio of adults carrying each allelic combination. Analysis of the
offspring of the 18006 brood lends support to this. Of the 71 offspring produced only
38% are female. The 18006 brood contains male specimens, both direct developing
and diapausing, that carry what appear to be completely intact X-chromosomes ﬁom
both parental types. However, among the female specimens, there is not a single

individual that possesses a completely intact canadensis X-chromosome, and a strong

105

deﬁcit of females carrying either of the canadensis allozyme alleles (Table 4.2). This
deﬁcit is most obvious in the category in which both allozyme alleles are the
canadensis type. The ratios derived in each of the four allelic “classes” are similar to
the ratios derived in similar crosses performed when the original X-chromosome
linkage map was developed (Hagen and Scriber 1995).

Closer scrutiny of the allelic identities contained in the recombined 18006 females
illustrates that there are a diverse combination of partial canadensis X-chromosomes
associated with the glaucus Y-chromosome. Assuming that there were no crossovers
that were undetected, it would appear as though every single portion of the X-
chromosome has successfully complemented the glaucus Y-chromosome and
cytoplasm. This might suggest that any genetic incompatibilities might be the result
of a combination of loci along the length of the X-chromosome or that the
incompatibilities are not 100% lethal. Another possibility that needs to be considered

is that X-chromosome crossovers occurred in this brood that went undetected.

This 18006 investigation clearly indicates that the ﬁequency with which
chromosomal crossovers occur within this Papilio system is high. Analysis of the
offspring from this brood also provides additional support, identifying the genetic
components that result in the Haldane effect being exhibited in hybrid
backcrosses between P. glaucus and P. canadensis. It also provides evidence for
the ﬁrnctional mechanisms by which genetic crossovers can result in the
genetically compatible backcross genotypes that are exhibited in the Late Flight

hybrid swarm of Vermont and the newly described species, P. appalachiensis. A

106

revised linkage map that accurately depicts the sequence and relative distances
between the species diagnostic loci on the X—chromosome would be extremely
valuable, but alternative techniques might be more efﬁciently applied to this task.
An improved linkage map and alternative techniques of analyzing the X-
chromosome would be invaluable in determining which portions of the glaucus
and canadensis genomes were incompatible. Additional backcross investigations
need to be conducted in an attempt to consistently “recreate” the genotypes that
result in an obligate diapausing brood, which exhibits the delayed emergence, as

is the case for the Late Flight and P. appalachiensis.

107

CHAPTER 5:

SUMMARY AND CONCLUSIONS

Speciation is typically viewed as a process through which populations gradually
evolve mechanisms that confer reproductive isolation (Harrison 1993; Coyne and Orr
2004). Populations that have recently diverged in allopatry may be lacking strong
reinforcement mechanisms (Howard 1993). If these populations are brought back
together in secondary contact, weak isolating mechanisms allow for introgressive
hybridization. Historically hybridization has been viewed as an evolutionary dead end.
More recently however, it has been discovered that hybridization can allow for rapid
recombination and can ultimately act to enhance genetic diversity (Lewontin and Birch
1966; Seehausen 2004). Levels of enhanced genetic diversity can allow for rapid
adaptation to unique environmental conditions. Furthermore increased rates of mutation
near species borders can help to enhance the rate at which these populations can become
adapted to a unique ecological niche (Nevo 2001).

Hybridization between closely related populations with distinct life history traits,
such as timing of diapause and / or patterns of voltinism can potentially lead to offspring
whose expression of these traits are truly distinct from either parental population (Gross
and Riesberg 2005). These distinct patterns can act as a mechanism by which to rapidly
isolate these offspring populations from either parental population (Thomas et al. 2003).
This is especially true when the genetic determination of these life history traits is based

relatively few genes (Henrich and Denlinger 1983) or even a single gene.

108

When comparing closely related species it is often difﬁcult to disentangle whether
traits that differ between the two species were actually involved in the process of
speciation or whether they arose after the two species were already reproductively
isolated. Most species groups are evolutionarily old enough so that it is difﬁcult to
identify the order of genetic changes. For example, the differences that exist between the
host-races of Rhagoletis, for host plant preference, diagnostic enzymes, and differences in
the timing of eclosion (F eder et al. 2003). However, it is uncertain as to the sequence in
which these genetic differences arose and whether each of these had an initial role in

initiating reproductive isolation.

Global climate change has resulted in dramatic changes in the thermal
environment in various regions. Most ecological predictions associated with the impacts
of climate change and global warming disturbingly warn of disastrous events (ecosystem
simpliﬁcation and collapse, decline of genetic diversity within populations, loss of
biodiversity) (Parmesan and Yohe 2003; Thomas et al. 2004). However, a great deal of
recent research is indicating that shifting climate patterns inducing range shifts of closely
related species can provide opportunities for the shufﬂing of co-adapted gene complexes
resulting in climate induced hybrid speciation (Dowling and Secor 1997). This genetic
reshuﬁling would allow evolutionary changes to shadow environmental shifts, adapting

organisms to new and unique niches.

109

It has been thought that Papilio glaucus and Papilio canadensis became initially
differentiated in allopatry and have recently met in secondary contact along an ecological
transition zone, which directly correlates with their range boundaries. The geographic
distribution of P. canadensis and P. glaucus seem to be primarily dictated as a result of
an interaction between genetics and the thermal environment. The univoltine P.
canadensis is limited to the north perhaps due to stress induced mortality resulting from
exposure to high temperatures common to the south of its range. The multivoltine P.
glaucus is likely prevented ﬁeln moving farther north, due to cold induced pupal
mortality but more signiﬁcantly insuﬂicient degree-days to allow for a second generation
to succeed in a single generation. An attempted glaucus second generation would be
suicidal in any geographic range that accumulated <2800 thermal degree-days. In the
absence of strong reinforcement mechanisms a narrow hybrid zone has existed along the
length of this ecological ecotone.

Unique thermal landscapes exist along the hybrid zone between Papilio
canadensis and Papilio glaucus into which climate change has facilitated high levels of
introgressive hybridization. Eight of the ten warmest years on record coincide with the
dramatic increase of introgression of Papilio glaucus alleles into populations of P.
canadensis. Signiﬁcant cline shifts have occurred for many of the species diagnostic
characters, resulting in hybrid swarms at locations near and distant ﬁ'orn the historic
hybrid zone (Ording 2001; Scriber and Ording 2005). The newly identiﬁed species, P.
appalachiensis (Pavulaan and Wright 2002), appears to be the result of hybrid speciation

that was possibly due to climate change (Scriber and Ording 2005; Scriber et al. 2008).

110

This research has analyzed a variety of wild populations that have been
differentially impacted by introgressive hybridization, and has also analyzed a highly
informative lab reared hybrid backcross. The ﬁndings of this research help to elucidate
those factors that can contribute to the process of climate induced hybrid speciation in

this Papilio system.

South Manitou Island maintains a unique hybrid swarm population at a location
over 150 km north of the hybrid zone across a wide thermal landscape. Climate change
resulting in unusually warm years has likely allowed for P. glaucus relatively historic
introgression into the region (Ording 2001). Analysis of a variety of traits in this
population over the course of 10 years indicates that the relative levels of genetic
introgression in this population have been retained and have remained relatively stable.
Out of over 1000 samples taken from this location, no pure glaucus or F1 hybrids have
ever been collected ﬁom this location. However, there are a suite of glaucus—like hybrid
traits present (longer forewing length, narrow hind wing black band width, X-linked Pgd
100 allele). Specimens possessing these hybrid traits have been present across the span
of this research indicating that there doesn’t appear to be any strong selection against

these genetically recombined individuals.

Climate change is also very likely the major contributing factor that has allowed
for increased levels of introgression into the Battenkill River Valley at the Vermont /
New York border. This location, in the Appalachian Mountains of the Eastern United

States, is very different from South Manitou Island in that there is more signiﬁcant

11]

topography, which results in a much more condensed thermal landscape. This has
allowed for signiﬁcantly increased levels of introgressive hybridization. The result has
been the newly described Late Flight (LF). These highly introgressed LF populations are
also best described as hybrid swarms, but exhibit even higher levels of introgression
compared to South Manitou Island. This Late Flight population exhibits unique life
history traits, including ﬂight times and voltinism patterns, from either parental Papilio
species.

It appears as though the LF population reported in the Battenkill River Valley
may represent an ongoing process that over time could lead to incipient species. Each of
these introgressed populations shares the unique feature of retaining the canadensis th
enzyme that is closely associated with the diapause gene on the X—chromosome. It has
been shown that thermal selection is strong on this enzyme in other systems (Crawford
and Powers 1989; Johns and Somero 2004; Schulte et al. 2000) and that only minor
changes to this gene can result in functionally different forms of the enzyme (Holland et
a1. 1997; Johns and Somero 2004), potentially conferring increased ﬁtness in light of
shifting thermal environments.

The Late F light of the Battenkill River Valley provides a unique opportunity in
that it is clear which trait has arisen to initiate isolation. The delayed emergence of the
recently reported Late Flight is a physiological shift that appears to be the result of
hybridization. This new trait provides for a signiﬁcant level of prezygotic isolation. The
LF is now either geographically isolated or temporally isolated from both of the parental
populations. Being temporally isolated could then potentially allow for the accumulation

of further genetic differences.

112

The analysis of the lab-reared backcross (18006) provides strong evidence that
this Papilio system can experience high rates of genetic recombination and X—
chromosome crossovers. The analysis of survival probabilities for the variety of potential
genotypes indicates that there is a Haldane effect, intrinsic genetic incompatibilities that
reduce the frequency with which certain female recombined genotypes survive.
Additionally, the trait analysis and the mapping of the X-chromosome indicate that the
diapause regulatory locus and the th locus are tightly linked. This provides for an
extrinsic explanation as to why the glaucus th 100 allele would never be found in
introgressed populations. Chromosomal crossovers would rarely occur between such
tightly linked loci. Any individual possessing the facultative diapause regulatory gene
(od- allele) would direct develop in a thermal landscape with insufﬁcient time to
complete another round of reproduction, thus eliminating th 100 with the od- allele.
Most importantly, the rapid rates of recombination in nature between these hybridizing
taxa is the mechanism that can result in unique genotypes that are well adapted to unique
thermal niches.

As a result of shifting thermal landscapes, likely caused by climate change,
populations in this Papilio system have exhibited high levels introgression and genetic
recombination. This has resulted in novel trait combinations with delayed post diapause
emergences, and provides an example of hybridization leading to the formation of
populations allochronically isolated ﬁ'om either parental population. The temporal

isolation in the Late Flight of the Battenkill River Valley has been strong enough to allow

113

for identiﬁable genetic differentiation via strong divergent selection on the X-
chromosome. This is a potential ﬁrst step down the avenue towards complete

reproductive isolation and speciation.

114

APPENDICES

115

APPENDIX 1:

RECORD OF DEPOSITION OF VOUCHER SPECIMENS

116

Appendix 1
Record of Deposition of Voucher Specimens'
The specimens listed on the following sheet(s) have been deposited in the named
museum(s) as samples of those species or other taxa, which were used in this research.
Voucher recognition labels beeﬁng the Voucher No. have been attached or included in
ﬂuid-preserved specimens.
Voucher No.: 2008-01

Title of thesis or dissertation (or other research projects):

AN ANALYSIS OF CLIMATE INDUCED HYBRID SPECIATION IN TIGER
SWALLOWTAIL BUTTERFLIES (PAPILIO)

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

investigator's Name(s) (typed)

ngriel J. Ordjﬂg

 

 

 

 

Date 1/10/08

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North

America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix 1 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 1 in copies of thesis or dissertation.
Museum(s) ﬁles.
Research project ﬁles.

This form is available from and the Voucher No. is assigned by the Curator, Michigan State
University Entomology Museum.

117

APPENDIX 1.]:

VOUCHER SPECIMEN DATA

118

Appendix 1.1

Voucher Specimen Data

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119

APPENDIX 2:

POPULATION WING MEASUREMENTS AND ALLOZYME DATA

120

APPENDIX

Table 1. South Manitou island specimen information for each year (1998-2005).
Forewing length, hind wing black bandwidth, and/or allozyme data are given for all
specimens collected. Each specimen allozyme data presented represents two
alleles. If only one allele is indicated, the individual is homozygous for that allele.
If two alleles are indicated the individual is heterozygous for those two alleles.

South Manitou Island Data
1998 1999
Hind Wing Fore Wing Hind Wing Fore Wing
Black Band Width Length Black Band Width Length

6 #1 61.5 49.5 6 #1 51 48
6 #2 55 49.5 6 #2 65 46.5
6 #3 52.5 38.5 6 #3 55 51
6 #4 54 47 6 #4 55 46
6 #5 53 39 6 #5 67 46
6 #6 60.5 46.5 6 #6 55 44.5
6 #7 57 44.5 6 #7 60 40.5
6 #8 53 49 6 #8 56 45
6 #9 50 49 6 #9 36 52.5
6 #10 61 48.5 6 #10 53 45
6 #11 73 50 6 #11 64 46
6 #12 38.5 47 6 #12 52 42.5
6 #13 60.5 48.5 6 #13 62 46
6 #14 62.5 46.5 6 #14 60 50
6 #15 54 49.5 6 #15 51 47
6 #16 49 49.5 6 #16 57 46

6 #17 52 45 6 #17 57
6 #18 43 44 6 #18 66 48
6 #19 38 46.5 6 #19 52 49.5
6 #20 62.5 45 6 #20 66 49
6 #21 53.5 45.5 6 #21 56 46
6 #22 52 48 6 #22 56 44.5
6 #23 60 48 6 #23 52 49.5
6 #24 57 48.5 6 #24 50 51.5
6 #25 57 46.5 6 #25 60 52.5
6 #26 53 46.5 6 #26 64 47.5
6 #27 56.5 48 6 #27 57 49.5
6 #28 53.5 45 6 #28 58 48
6 #29 62 47.5 6 #29 58 48
6 #30 64 49 6 #30 72 47
6 #31 60.5 42.5 6 #31 66 48.5
6 #32 47 46 6 #32 60 50
6 #33 55 47

121

122

6 #34
6 #35
6 #36
6 #37
6 #38
6 #39
6 #40
6 #41
6 #42
6M3
6 #44
6%
6 #46
6M7
6M8
6M9
6 #50
6m
6 #52
6 #53
6 #54
6 #55
6 #56
6 #57
6 #58
6 #59
6 #60
6%
6 #62
6%3
MM
6 #65
am
6 #67
6 #68
6 #69
6 #70
6m
6 #72
6 #73
6 #74
6 #75
6 #76
6 #77
6 #78
6 #79
6 #80
6m

8%88

52

339883883888838883

53
61
49
45

51

59

E883

63

62
67

55
61
59

55
47
62
45

54.5
43.5

50

51
48.5
47.5
47.5

47.5
46.5
46.5
49.5

51
48.5
50.5
46.5

48
48.5
52.5

2000
Hind Wing Fore Wing
Black Band Width Length
6 #1 56 50
6‘ #2 61.5 47.5
6‘ #3 53.5 48

123

6#82
6 #83
6#84
6 #85
6 #86
6 #87
6 #88
6#89
6 #90
6 #91
6 #92
6#93
6 #94
6 #95
6#96
6 #97
6#98
6#99
6#100
6 #101
6 #102
6#103
6#104
6 #105
6#106
6 #107
6 #108
6 #109
6 #110
6 #111
6#112
6 #113
6 #114
6#115
6 #116
6#117
6 #118
6 #119
6 #120

6#1
6#2
6#3

63 50
50 52.5
56 49
54 47
49 46
59 45.5
54 46
5O 50
47 48
50 49
55 45
59 51
56 47.5
43 50.5
58 46
65 47.5
56 49
62 47
58 48.5
53 49
52 49.5
46 50
52 45
50 49.5
63 46
58 51
58 51
53 44.5
59 48
50 54
48 47
46 46
54 49
53 51
49 47.5
56 48
73 5O
39 47.5
59 47
2001
Hind Wing Fore Wing Allozyrnes

Black Band Width Length Pgd

1 25

1 25

125/80

6 #4
6 #5
6 #6
6 #7
6#8
6 #9
6 #10
6 #11
6#12
6 #13
6#14
6 #15
6#16
6 #17
6#18
6#19
6 #20
6#21
6 #22
6 #23
6 #24
6 #25
6#26
6 #27
6 #28
6 #29
6 #30
6 #31
6 #32
6 #33
6 #34
6 #35
6 #36
6 #37
6 #38
6 #39
6 #40
6 #41
6#42
6 #43
6 #44
6#45
6 #46
6#47
6 #48
6 #49
6 #50
6 #51

50.5
57.5
55.5
43.5
45.5

58

63
55.5

58.5

61
58.5
67
41.5
55.5
54.5
64.5
67
52
51.5

60.5

53.5

63.5
53

65.5
57
63.5
65
60.5

65.5

51.5
45.5
62

67
60.5
57
45.5

50.5

48
46.5
47
45
50
47
46.5
47
49
47

688555

49
45

45
47
49
50.5
50.5
46.5
46.5
48.5
47.5

44.5
51
45.5
48
48.5
43.5

83386

48.5

45
48.5
46.5

45.5

124

6 #4
6 #5
6 #6
6 #7
6 #8
6 #9
6 #10
6 #11
6 #12
6 #13
6 #14
6 #15
6 #16
6 #17
6 #18
6 #19
6 #20
6 #21
6 #22
6 #23
6 #24
6 #25
6 #26
6#27
6 #28
6 #29
6 #30
6 #31
6 #32
6 #33
6 #34
6#35
6 #36
6 #37
6 #38
6 #39
6 #40
6 #41
6 #42
6 #43
6 #44
6 #45
6 #46
6 #47
6 #48
6 #49
6 #50
6 #51

50.5
53.5
53
47.5

54.5

47
58.5
59.5
49.5

46
46
50
47
52
49
48
43
47.5

51
47

125
125/80
125
125
125
125
125
125
125
125
125
125
125/100
125/100
125
125
125/100
125
125/80
125
125/80
125/150
125
125
125
125
125
125
125
125/100
125
125
125/100
1501125
125
150I125
125
125
125/80
125
125
125
125
125
125
125
125
125

6 #52
6 #53
6 #54
6 #55
6 #56
6 #57
6 #58
6 #59
6 #60
6 #61
6 #62
6 #63
6 #64
6 #65
6 #66
6 #67
6 #68
6 #69
6 #70
6 #71
6 #72

6#1
6#2
6#3
6#4
6#5
6#6
6#7
6#8
6#9

2002

46.5
61
59
51

55.5

60.5

53.5

53.5

62.5
71
51

51.5

23.5
59
47.5
64.5
51.5
57
52.5
54

Hind Wing

Black Band Width Length Pgd

55
64
55
70
57.5
50.5
49
39.5

48 6 #52
50 6 #53
46.5 6 #54
45.5 6 #55
48 6 #56
46.5 6‘ #57
47 6 #58
49 6 #59
47.5 6 #60
47 6 #61
45.5 6 #62
44 6 #63
45.5 6 #64
43 6 #65
47 6 #66
48 6 #67
47 6 #68
49 6 #69
50 6 #70
44.5 6 #71
44 6 #72
6 #73

6 #74

6 #75

6 #76

6 #77

6 #78

6 #79

6‘ #80

6 #81

6 #82

6 #83

6 #84

6 #85

6 #86

Fore Wing Allozyme 6 #87

6 #88
43 125 6 #89
50 125 6 #90
46 125 6 #91
38 125 6 #92
47125180 6 #93
49125/80 6 #94
48 100 6 #95
49 125 6 #96
44 125 6 #97
6 #98
6 #99

125

54.5
69
41.5

eﬁaggaasaa

67.5

88888818

48.5

888

56.5
43.5

59
63.5
43.5
49.5

59
59.5

8838831928

39
63.5
62
49
61
57
59

47.5
47.5

44.5

50.5
45

47
48.5

45.5
51
47.5
44
49
49
47

632888

46.5

49.5

125
150I125
1501125

125

125

80

125

125

125

125

125/80

125

125

125

125

125

125

125/80
125/80
125/80
125
125/80

125

125

125

125

125/80

125

125

125
125/100

125

125

125

125

125

125/80
125
125

125/80

125/100

125

125

125

125

125

125

125

6 #1
6 #2
6 #3
6 #4
6 #5
6 #6
6 #7
6 #8
6 #9
6 #10
6 #11
6 #12
6 #13
6 #14
6 #15
6 #16
6 #18
6 #19
6 #20
6 #21
6 #22
6 #23
6 #24
6 #25
6 #26
6 #27
6 #28
6 #29
6 #30
6 #31
6 #32
6 #33
6 #34
6 #35
6 #36
6 #37
6 #38
6 #39
6 #40
6 #41
6 #42

2003

Hind Wing

Black Band Width
57.5

51.5

48

48.5

46

40

50.5

53.5
57.5
62.5

57
58.5
62
58.5
64.5
61.5
52.5
47.5
59

55.5
62.5
55.5
57.5

47.5
57
59.5

49
58.5
55

Fore Wing
Length

46

48

49

47

46
49
44
48
47

47
48
47
48
53
51
45
48
47
49
47
47
48
51
44
47
52
45
52
49
45
47
46
51
47
50
49
45
53
48
45
47

126

6 #100

6 #1
6 #2
6 #3
6 #4
6 #5
6 #6
6 #7
6 #8
6 #9
6 #10
6 #11
6 #12
6 #13
6 #14
6 #15
6 #16
6 #17
6 #18
6 #19
6 #20
6 #21
6 #22
6 #23
6 #24
6 #25
6 #26
6 #27
6 #28
6 #29
6 #30
6 #31
6 #32
6 #33
6 #34
6 #35
6 #36
6 #37
6 #38
6 #39
6 #40
6 #41

44

2004

Hind Wing

Black Band Width
61
54.5
46.5
60
52.5
65
60
58.5
69
59.5
65
39.5
51.5
69
61.5
57.5
60
62
55
52
53
50
35
56.5
47.5
55.5
58
63.5
62.5
41
51
46.5

8888

60
45
63
50

50.5

Fore Wing

Length
49

44
49
50
45
41
48
49

49
43

88833888838883

47
51
49
47
49
49

47
45
47
47
50
48
51
43
47
47

125

6 #43
6 #44
6 #45
6 #46
6M7
6M8
6 #49
6 #50
6 #51
6 #52
6 #53
6 #54
6 #55
6 #56
6 #57
6 #58
6 #59
6 #60
6%
6 #62
6 #63
6 #64
6 #65
6 #66
6 #67
6 #68
6 #69
6 #70

59.5
48.5
54.5

56.5
54.5
70
48.5
57.5
51.5
58
62
49

48
50.5
60.5

50
54.5
52.5
48.5

55.5
63

55.5
51.5
54.5

8888

46
45

47

45
49
50
49

8888888

42
47
46
49
48
48

46

127

6#42
6#43

6 #1
6#2
6#3
6 #4
6 #5
6#6
6#7
6 #8
6 #9
6#10
6#11
6#12
6#13
6#14
6#15
6 #16
6#17
6#18
6#19
6#20
6#21
6#22
6 #23
6 #24
6#25

2005

56
44

Hind Wing
Black Band Width Length Pgd

39.5
53.5
46.5
57.5
57.5
40
60.5
56
63.5
62
58.5
59
55.5
57.5
63.5
58.5
67.5
56.5
53.5
29
49
61
38.5
47
50.5

48

Fore Wing Allozyme

48 125
49 125
47 125
48 125/80

46 125
50 125
49 125
50 125
46 125
46 125
49 125/100
49 125
44 125
49 125
47 125
48 125
46 125/80

50 125
46 125
47 125
48

42

49

46

48

Table 2. Wild collected Vermont specimen information for each year (2002-2004).
Forewing length, hind wing black bandwidth, and/or allozyme data are given for
specimens collected. Each specimen allozyme data presented represents two
alleles. If only one allele is indicated, the individual is homozygous for that allele.
If two alleles are indicated the individual is heterozygous for those two alleles.

Vermont 2002
Forewing Hindwing

Wild Early Flight Length Blackband Pgd th
6 #1 *150/80 80
6 #2 125 80
6 #3 125 80
6 #4 125/80 80
6 #5 150I125 40
6 #6 *125 80
6 #7 * 80
6 #8 *150 80
6 #9 *125 80

6 #10 *125 80
6 #11 125/80 80
6 #12 125/80 80
6 #13 125/80 80
6 #14 125 80
6 #15 100 80
6 #16 100/50 80
6 #17 46 60 100 80
6 #18 48 58 100I50 80
6 #19 49 56 125 80
6 #20 44 54.5 80 80
6 #21 44 73 125 80
6 #22 49 76.5 *125 80
6 #23 48 52.5 * 80
6 #24 45 63 * 80
6 #25 47 65 * 80
6 #26 41 68 * 80
6 #27 42 77.5 125 80
6 #28 49 61 125 80
6 #29 44 69.5 125 80
6 #30 47 70.5 125 80
6 #31 48 58 125 80
6 #32 45 60 125 80
6 #33 44 76 125 80
6 #34 44 83.5 125 80
6 #35 45 82 125/80* *

6 #36 39 62 125 *

6 #37 40 69.5 125 80
6 #38 46 73 125 80

128

6 #39
6 #40
6#1
6 #42
6 #43
6 #44
6 #45
6 #46
6 #47
6#8
6#9
6 #50
6 #51
6#2
6 #53
6 #54
6 #55
6 #56
6 #57
6 #58
6 #59
6 #60
6 #61
6 #62
6 #63
6 #64
6%5
6 #66
6 #67
6M8
6 #69
6 #70
6 #71
6 #72
6 #73
6 #74
6 #75
6W6
6 #77
6 #78
6W9
6 #80
6 #81
6 #82
6 #83
6 #84
6 #85
6 #86

88888888

#«5
V0!

3388888888388838

65.5
75
76
70
83

74.5

73.5

59.5

69.5

67.5
79.5
74.5
73.5
66.5
70
58.5
72.5
67.5

52
72.5
71

888

125
125
125

i

1 25
1 25
1 25180
1 25
1 25
1 25

H

H

125
125
125
125
125
1501125
125/80
125
125
125
125
125/100
“1 25180
125180
1 251100
100
”125
”125
12511 00
“1 25180
125
125
125
1 25180
1251100
*100
125180
125
125
125180
125
125
125
125
125

129

80140

61187
61188
6#89
61190
67191
61192
6#93
6#94
6#95
6 #96
6#97
6 #98
6 #99
6#100
6#101
6#102
6#103
6#104
6#105
6#106
6#107
6#108
6#109
614110
611111
6#112
6#113
6#114
6#115
6#116
6#117
6#118
611119
671120
6#121
6#122
6#123
6#124
614125
611126
6#127
6#128
611129
611130
671131
6#132
6#133
6#134

125 80
125 80
125 80
125 80
125/80 80
125/80 80
1501100 *40 or 20
125 80
125/80 80
125/80 *80
125 80
125 40
125 80
125 80
125 '80140
125 40
125 80
1501125 *
125 80
125 *
125 *80/40
125 80
125/80 80
125 80
125 40
125 80
125 40
150I125 *
125 80
125 80
125/100 80
125 80
125 80
1501125 80
125 40
125 80
125 80
125 80
1501125 80
125/80 80
125/80 80
125 80
125 80
125 80
125/100 80
125 80
125 80
125 80

130

6 #135
6 #136
6 #137
6#138
6 #139
6 #140
6 #141
6 #142
6 #143

Wild Late Flight

6711
6#2
6#3
6#4
6#5
6#6
6#7
6#8
6#9
6#10
6#11
6#12
6#13
6#14
6#68

6#1
6#2
6#3
6#4
6#5
6#6
6#7
6#8
6#9
6#10
6#11
6#12

Vermont 2002

Forewing Hindwing

125

1251100
125

125
125

Length Blackband Pgd

50 48
49 52
51 39
54 39.5
49 63.5
52 58.5
51 43.5
47 57.5
54 33.5
48 37.5
50 49
50 48.5
51 57
51 47.5
52 45
Vermont 2003

45
45
49
43
45
46
45
47
48
47
44
46

Wild Early Flight Forewing Hindwing
Length Blackband

72
74
73.5
70
63
59
77
66.5
66.5
68
70
65.5

100180
125
100
100
125

1251100
1251100
125
125
80
100
1251100
100

H

P96

1251100
1251100
125
1251100
125
125
125180
125
125
100180
125
125180

131

80

888888

80

L111!
80120
40120
80120

80120
80120
80120
80120
80120
80
80120
20
80120

th

80

80140
80140

8888888

80

6 #13 44 54.5 125 80
6 #14 44 69 125180 80140
6 #15 45 62 125180 80140
6 #16 45 75.5 125 80
6 #17 46 72 125 80
6 #18 44 69.5 125/100 80
6 #19 46 74 125/100 80
6 #20 43 72.5 125180 80
6 #21 95 125 80
6 #22 46 74.5 125 80
6 #23 43 63.5 125 80
6 #24 43 72.5 100 80
6 #25 47 77 125 80
6 #26 48 73 125/100 80
6 #27 41 44.5 125 80
6 #28 44 70 125 80
6 #29 46 63.5 150/100 80
Vermont 2003

Wild Late Flight Forewing Hindwing Pgd th
Length Blackband

132

6 #1 49 53 125 80l20
6 #2 52 44.5 125/100 *80/20
6 #3 53 53 125/100 *80/20
6 #4 51 46.5 1251100 80120
6 #5 51 54 100I80 80120
6 #6 51 51 100 40120
6 #7 53 41.5 100I80 80120
6 #8 49 41 100 40120
6 #9 50 54.5 125 80120
6 #10 49 59.5 100 *80/20
6 #11 48 53.5 1501100 80120
6 #12 51 61 125 40120
6 #13 53 35.5 1251100 80120
6 #14 54 55.5 125/100 80120
Vermont 2004
Wild Early Flight Forewing Hindwing Pgd th
Length Blackband

6 #1 150/125 80

6 #2 125 80

6 #3 125 80

6 #4
6 #5
6 #6
6 #7
6 #8
6 #9
6 #10
6 #11
6 #12
6 #13
6 #14
6 #15
6 #16
6 #17
6 #18
6 #19
6 #20
6 #21
6 #22
6 #23
6 #24
6 #25
6 #26
6 #27
6 #28
6 #29
6 #30
6 #31
6 #32
6 #33
6 #34
6 #35
6 #36
6 #37
6 #38
6 #39
6 #40

6 #200
6 #201
6 #202
6 #203
6 #204
6 #205
6 #206
6 #207
6 #208
6 #209

125
125
125
125
125
125
1251100
125
125
125
125
125
125
125
125
125
125
125/100
1251100
125
125
125180
125
125
125
125
125
125180
1501125
125
125
1251100
125
125
125
125
125

125
125
125180
125
125180
125180
1251100
125
125
125

133

888888888888888'*88888888888888888888

3888888

888

6mm
6ml
6mm
6M3
6mm
6mm
6mm
6mm
6mm
6mm
6 #220
6 #221
6 #222
6 #223
6 #224
6 #225
6 #226
6 #227
6 #228
6 #229
6 #230
6 #231
6 #232
6 #233
6 #234
6 #235
6 #236
6 #237
6 #238
6 #239
6 #240
6 #241
6 #242
6 #243
6 #244
6 #245
6 #246
6 #247
6 #248
6 #249
6 #250
6 #251
6 #252
6 #253
6 #254
6 #255
6 #256
6 #257

48
47
47

48
53
48
47
49

8888

47
45
48
48

72
76.5
67.5
78.5

68
75.5

80
72.5
51.5
68.5
72.5
68.5
85.5

68

69
73.5
62.5

125
125
125180
125
125
1501125
1251100
125180
125
125
125
125
1251100
1501125
1501125
125
125
1251100
100
1501125
125
125180
125
125
1251100
125
125
125
125
125
125180
125
125
125180
125
125
125
125180
125
125
125
1501125
125
125
125
125
125
125

134

8888388888888888888888888888888888 188888888

8888

(D
O

6 #258
6 #259
6 #260
6 #261
6 #262
6 #263
6 #264
6 #265
6 #266
6 #267
6 #268
6 #269
6 #270
6 #271
6 #272
6 #273
6 #274
6 #275
6 #276
6 #277
6 #278
6 #279
6 #280
6 #281
6 #282
6 #283
6 #284
6 #285
6 #286
6 #287
6 #288
6 #289
6 #290
6 #291
6 #292
6 #293
6 #294
6 #295
6 #296
6 #297
6 #298
6 #299
6 #300
6 #301
6 #302
6 #303
6‘ #304
6 #305

49
50

48
42
48
49
48
49
47
45
48
48
45
47
46
48
49

50

47
47
47
48

48

47
47
45
47
48

45
48
48
45
49
48
48
49
47

49
49
47
49

62.5
49.5
71.5
67.5
65.5
69.5
79
74.5

81
72
62
66.5
73.5
75
68
68
76
87
72
7 I
70
82
79
76
65.5
74
65.5
60.5
80.5
63.5
76
70
73.5
76
69.5
63
74
63.5
77
76
64.5
79.5
53
6| .5
76
75.5
76

1 25
1 25150

1251100

125180
125
125
125

125180

125

1251100

125
125
125180
1 25150
125
1 25
125

125
1 25
1 25
1 25180

1501125

125
125
125
125
125
125
125
125
125

1251100

125
125
125
125
125
100
125
125

1251100
1251100

125
125
125
125

135

888888888888888888888888888883888888888888888

888

6 #306
6 #307
6 #308
6#309
6 #310
6 #311
6 #312
6 #313
6 #314
6#315
6 #316
6#317
6 #318
6 #319
6 #320

6 #344
6 #345
6 #346
6 #347
6 #348
6 #349
6 #350
6 #351
6 #352
6 #353
6 #354
6 #355
6 #356
6 #357
6 #358
6 #359
6 #360
6 #361
6 #362
6 #363
6 #364
6 #365
6 #366
6 #367
6 #368
6 #369
6 #370
6 #371
6 #372
6 #373
6 #374
6 #375

45
42
47
47
47
45
48

-48

43
48
48
47

50
46

48
47
50
47
45
47
45
47
45
48
47

8888

49

47
46
50
49
47
48

85.5
69
70.5
72
77.5
76.5
65
67
83.5
69
77.5
72
70.5
74
57

74.5
76.5
79.5
71
76.5
57.5
78
71
76.5
77.5

75.5
82.5
79
85.5
63.5
74.5
57
44.5
72

72.5

125/100
125
125

1251100
125

1501125
125

80
125
125180
125
125

1501125
125
125

136

40
40
80

80
80

888888

6 #376 47 82.5

6 #377 48 65.5
6 #378 48 67
6 #379 47 67
6 #380 47 74
6 #381 45 68.5
6 #382 46 76
6 #383 46 80
6 #384 47 66.5
6 #385 46 66.5
6 #386 44 74.5
6 #387 47 70
6 #388 44 63
6 #389 47 63.5
6 #390 48 65.5
6 #391 47 58
6 #392 43 75
6 #393 47 69.5
6 #394 45 68
6 #395 49 82
6 #396 45 64.5
6 #397 46 70.5
6 #398 47 77
6 #399 48 74
6 #400 47 68.5
Vermont 2004

Wild Late Flight Forewing Hindwing Pgd th
Length Blackband

6 #1 50 45 125180 80120
6‘ #2 54 48 1251100 80
6 #3 52 44.5 100180 80
6 #4 52 39 1251100 80120
6 #10 52 44 125/100 80
6‘ #1 l 54 60.5 125 40
6 #12 51 46.5 100 80120
6 #13 48 55 100180 80
6 #14 55 46.5 100 80120
6 #15 48 53.5 1501100 80
6 #16 51 34 125 80120
6 #17 50 53 100180 80120

137

APPENDIX 3:

POPULATION EMERGENCE DATA

138

APPENDIX

Table 3. Specimen emergence data for Early and Late Flight Vermont pupae reared
in sleeved branches of black cherry (Prunus serotina). Data represented are the
number of days from the beginning emergence investigation under controlled
laboratory conditions.

Vermont Early Flight 2003 Emergence Data
Pupae placed in emergence chambers 411 5103

ID Pupal Wt. Days to Black Band Fore Wing
Emerge Width (Eye) Length

18° 6 #1 0.866 12 60 43
18° 6 #3 0.724 13 80

18° 6 #4 0.599 14 55 40
18° 6 #6 0.73 15 80 43
18° 6 #7 0.852 16 80 43
18° 6 #8 0.758 19 80 46
18° 6‘ #10 0.623 17 60 42
18° 6 #11 0.7 21 85 45
18° 6 #12 23 80 41
18° 6 #14 0.729 23 70 43
18° 6 #15 0.756 24 75 41
18° 6 #17 0.691 24 75 44
18° 6 #18 25 75 48
18" 6‘ #19 25 60 40
18° 6 #21 26 65 41
18° 6 #22 26 75 47
18° 6 #23 26 70 45
18° 6 #24 26 60 40
18° 6 #25 0.888 26 75 42
18° 6 #26 0.654 26 75 42
18° 6 #27 0.967 26 55 48
18° 6 #28 0.743 26 55 42
18° 6‘ #29 27 65 44
18’ 6 #30 0.822 27 65 45
18’ 6 #32 27 65 42
18° 6 #33 28 70 46
18° 6 #37 0.806 28 75 44
18° 6 #38 0.832 28 70 45
18° 6 #39 28 65 47
18° 6 #40 28 75 42
18° 6 #42 29 65 42
18° 6 #43 29 70 45

139

18° 6#4-4
18° 6#46
18° 6#47
18° 6#52
18° 6#53
18° 6#66

ID

89%
18° 9 #5
18° 9 #9
18° 9 #13
18° 9 #16
18° 9 #20
mwm1
wwn4
18° 9 #35
18° 9 #36
18° 9 #41
wwms
mwM8
wWM9
18° 9 #50
18° 9 #51
18° 9 #54
18° 9 #55
wwws
18° 9 #57
18° 9 #58
18° 9 #59
wwwo
18° 9 #61
18° 9 #62
wwma
www4
wwms
18° 9 #67

0.872

Pupal Wt.
0.913
0.897
0.702
0.923
0.803
0.916

0.844
1.004

0.861

0.796

29
29
29
30
30

Days to
Emerge
1 2
14
1 9
23
24
25
27
28
28
28
29
29
29
29
29
30
31
31
31
31
32
32
33
33

88888

Width (Eye)

140

60
70

8883

Black Band

70
80
90
75
75

85
80
90
75
85
85

65
70
75
70
60
75
75
70
60
60
60
75
70
65
65
75
75
75

8888

43
42

Fore Wing
Length
46
45
32
47

888888

45
47

43
46
45
42

43
45

8888888

41
45

Vermont Late Flight 2003 Emergence Data

ID

18° 6#1
18° 6#2
18° 6#4
18° 6#5
18° 6#6
18° 6#8
18° 6#9
18° 6#10
18° 6#11
18° 6#12
18° 6#14
18° 6#16
18° 6#17
18° 6#20
18° 6#21
18° 6#22
18° 6#24

ID

18° 9 #3
18° S2 #7
18° 9 #13
18° 9 #15
18° 92 #18
18° 92 #19
18° 9 #23
18° SB #25
18° 82 #26
18° 9 #27
18° 92 #28
18° 9 #29
18° 9 #30
18° S? #31
18° 9 #32
18° 9 #33
18" S? #34

Pupal Wt.

1.116
1.092
0.981

0.841

0.986

0.934
1.11

1.087
1.109

0.873
1.229
0.914
0.829
1.089

Pupal Wt.

1.072
1.085
0.888
0.841
1.139
1.099

1.035
1.133
1.291
1.168

0.905

1.15
1.041

Days to
Emerge
23
45
45
45
46
46
46
46
47
47
48
49
49
51
51
52
53

Days to
Emerge
45

46
43
48
50

50
53
53
54
55
55
55
56
61
63
64
65

141

Pupae placed in emergence chambers 4/15/03

Black Band
Width (Eye)

888888888

55
50
45
75
65
70
60
70

Black Band
Width (Eye)
65
70

70
70
60
65
70
70
75

70
65
65
75
55

50
55

Fore Wing
Length
47
51
47

47
45
48
49

53

888888

49
43
48

Fore Wing
Length
50
47

88888888888888

Vermont Early Flight 2003 Emergence Data

ID

22° 6 #1

22° 6 #2

22° 6 #6

22° 6 #12
22° 6 #13
22° 6 #14
22° 6 #15
22° 6 #18
22° 6 #20
22° 6 #21
22° 6 #26
22° 6 #28
22° 6 #30
22° 6 #32
22° 6 #36
22° 6 #37
22° 6 #38
22° 6 #39
22° 6 #40
22° 6 #41
22° 6 #42
22° 6 #43
22° 6 #44
22° 6 #46
22° 6 #49
22° 6 #50
22° 6 #51
22° 6 #59

ID

22° 9 #3
22° 9 #4
22° 9 #5
22° 9 #7
22° 9 #8
22° 9 #9
22° 9 #10
22° 9 #11

Pupal Wt.

0.679
0.645
0.695

0.645
0.904

0.785
0.805
0.742

0.772

Pupal Wt.

0.753
0.903
0.747
0.843
0.754
0.834
0.822
0.839

Days to
Emerge
9
10
11
13
14
14
14
15
15
15
16
16
16
16
16
17
17
17
17
17
17
17
17
17
18
18
18
18

Days to
Emerge
10
10
10
12
11
11
13
13

142

Pupae placed in emergence chambers 411 5103

Black Band
Width (Eye)
65

888388888838888888888888388

Black Band
Width (Eye)
75
60
90
60
70
75
70
60

Fore Wing
Length
40
42
41

88888888888888888

£88882

#«h
NOD

Fore Wing
Length

49
43
47
43
45
43
46

22° 9 #16
22° 9 #17
22° 9 #19
nwuz
22° 9 #23
22° 9 #24
22° 9 #25
22° 9 #27
awns
22° 9 #31
nwma
22° 9 #34
22° 9 #35
uwms
22° 9 #47
an8
22° 9 #52
nwwa
RW%4
22° 9 #55
22° 9 #56
22° 9 #57
nmms
22° 9 #60
22° 9 #61
22° 9 #62
22° 9 #63
22° 9 #64

ID

22° 9 #1
22° 82 #8
22° S2 #9
22° 9 #12
22° 9 #13
22° SB #14
22° 92 #16
22° 9 #18
22° SB #19
22° 9 #20
22° 9 #22
22° S2 #25
22° 9 #26
22° 9 #27
22° 92 #28

0.829

0.996

1 .044
0.842
0.898

1.0007
0.856

Pupal Wt
0.581

1.081
1.238
0.901
1.033

1.168
1.008
1.177
0.916
1.19

0.979

15
15
15
15
16
16
16
16
16
16
16
16
16
18
18
18
19
19
19
18
18
18
18
19
19
20
20
20

Days to
Emerge
21
29

8888888888888

143

70

45

75
75

75
70
75
70

70

8888888888888

Black Band
Width (Eye)

8888388888

8888

42

47

42
47
47
49
47

888888888888888888

49

Fore Wing
Length

88.888888888819588

22° 9 #31
22° 9 #32
22° 9 #33
22° 9 #34
22° 9 #35
22° 32 #36

1.149
1.232
1.286
0.929
1.021
1.103

38
39

40
41
70

50

45
45
45
65

50
53
50
47

Vermont Late Flight 2003 Emergence Data

ID

22° 6 #2
22° 6 #3
22° 6 #4
22° 6 #5
22° 6 #6
22° 6 #7
22° 6 #10
22° 6 #11
22° 6 #15
22° 6 #17
22° 6 #21
22° 6 #23
22° 6 #24
22° 6 #29
22° 6 #30

Pupal Wt.

1.073
0.779
0.833
1.051
0.805
0.951
0.756
0.972

0.939
0.945

1.027
0.973

Days to
Emerge
25
25
25
27
27
27
30
30
31
32
32
32
33
36
37

Pupae placed in emergence chambers 4/1 5103

Black Band
Width (Eye)
35

88888888888888

Fore Wing
Length
49

8888888188818

45
49

Vermont Early Flight 2003 Emergence Data

ID

26° 6 #1
26° 6 #2
26° 6 #3
26° 6 #10

Pupal Wt.

0.835

0.804

0.704
0.73

Days to
Emerge
9

9
9
9

144

Pupae placed in emergence chambers 4/15/03

Black Band
Width (Eye)
70
65
70
55

Fore Wing
Length
42
44
45
44

26° 6 #13
26° 6 #14
26’ 6 #16
26° 6 #18
26° 6 #19
26° 6 #20
26° 6 #21
26° 6 #22
26° 6 #23
26° 6 #27
26° 6 #29
26° 6 #30
26° 6 #31
26° 6 #32
26° 6 #34
26° 6 #36
26° 6 #37
26° 6 #38
26° 6 #42
26° 6 #44
26° 6 #50
26° 6 #51
26° 6 #52
26° 6 #53
26° 6 #55
26° 6 #57

ID

26° 9 #4
26° 9 #5
26° 9 #6
26° 9 #7
26° 9 #8
26° 9 #9
26° 9 #11
26° 9 #12
26° 9 #15
26° 9 #17
26° 9 #24
26° 9 #25
26° 9 #26
26° 9 #28
26° 9 #33
26° 9 #35
26° 9 #39
26° 9 #40
26° 9 #41

0.718

0.785
0.737
0.857

0.847

0.893

0.801
0.837

Pupal Wt.

0.878
0.833
0.807
0.836
0.754
0.891
0.948
0.771

0.921

0.885
0.9

9
10
11
12
12
12
12
12
12
11
13
13
13
13
13
13
13
13
11
13
14
14
14
14
14
14

Days to
Emerge

145

88888888888888888838838883

Black Band
Width (Eye)
70

883

88888388888383

88888888888888888888888888

Fore Wing
L009”!
43
42
43
43

46
43
42
50
50
47

45
47
46
51

47
44
48
45

26° 9 #43
26° 9 #45
26° 9 #46
26° S? #47
26° 9 #48
26° S? #49
26° S? #54
26° S? #56
26° 52 #58
26° 9 #59
26° S2 #60
26° 9 #61
26° S2 #62
26° S? #63
26° £2 #64
26° S2 #65

0.844
0.83
1.026

13
14
14
14
14
14
14
14
14
15
15
15
15
16
16
17

55

50

70

88888

55

65
70
80
60

46
47
49
50

49

88888888

47
47

Vermont Late Flight 2003 Emergence Data

ID

26° 6 #1

26° 6 #2

26° 6 #3

26° 6 #4

26° 6 #5

26° 6 #6
26° 6 #8
26° 6 #9
26° 6 #10
26° 6 #11
26° 6 #12
26° 6 #13
26° 6 #14
26° 6 #16
26° 6 #17
26° 6 #18
26° 6 #19
26° 6 #24
26° 6 #32
26° 6 #35
26° 6 #36

Pupal Wt.

1.16
0.985
0.99
0.968
1.086
0.925
1.09
0.826
0.855

0.628
1.046
0.896
0.891
0.932

1 .076
0.949
0.955
1.056

Days to
Emerge
18
18
20
21
21
22
23
23
23
24
24
24
24
25
25
26
26

27
31
34
34

146

Pupae placed in emergence chambers 411 5103

Black Band
Width (Eye)
60
45

45
60
50
55
50

45
45
50
50
35
45
45
60

888888

Fore Wing
Length
51

88888888888888888888

ID

26° 92 #7
26° 9 #15
26° S? #20
26° 32 #21
26° 9 #22
26° 92 #23
26° 92 #25
26° 9 #26
26° 2 #27
26° S2 #28
26° 9 #29
26° 9 #30
26° 82 #31
26" S2 #33
26° S? #34

Pupal Wt.

0.884
0.926
1.131

1.072
1.18

0.997

0.846
0.896
0.86
1.072
1.222
0.848

Days to
Emerge
22
25
26
27
27
27
28
28
29
29
29
30
30
31
32

147

Black Band
Width (Eye)
55
75
50

75
40
50
60
50

65
60
55
55
75
70

Fore Wing
Length
47
46
50

50
54
52
50
50
48

50
45
50
43
45

Vermont Early Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

ID Pupal Wt. Days to Fore Wing ID Pupal Wt. Days to Fore Wing
Emerge Length Emerge Length

14° 6‘ #1 0.7082 42 43 14° 52 #1 49 41
14° 6 #2 0.6633 42 42 14° S2 #2 0.8209 49 49
14° 6 #3 0.6288 44 40 14° 9 #3 0.7219 49 44
14° 6 #4 0.7929 44 45 14° 92 #4 0.733 49 46
14° 6 #5 0.7194 45 44 14° 9 #5 0.6102 51 41
14° 6 #6 0.6078 46 39 14° 9 #6 0.6859 52 38
14° 6 #7 0.7098 46 42 14° 9 #7 0.8296 52 45
14° 6 #8 0.7755 46 43 14° 9 #8 0.7352 52 44
14° 6 #9 0.7081 46 14° S2 #9 0.8312 52 43
14° 6 #10 0.7658 46 43 14° S2 #10 0.7451 54 45
14° 6 #11 0.7835 47 44 14° S? #11 56 43
14° 6 #12 0.6802 47 42 14° 9 #12 56
14° 6 #13 0.7922 47 44 14° S? #13 0.7556 57 47
14° 6 #14 0.7397 47 44 14° S? #14 0.7377 57 44
14° 6 #15 0.8514 47 46 14° 9 #15 0.9398 61 47
14° 6 #16 0.7219 47 42 14° S? #16 0.8187 69 44
14° 6 #17 0.7468 49 44
14° 6 #18 50 43
14° 6 #19 50 38
14° 6 #20 52 43
14° 6 #21 0.8341 51 44
14° 6 #22 0.7631 51
14° 6 #23 0.8016 52 43
14° 6 #24 56 42
14° 6 #25 0.6972 56
14° 6 #26 0.6519 56 40

Vermont Early Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

148

ID Pupal Wt. Days to Fore Wing ID Pupal Wt. Days to Fore Wing
Emerge Length Emerge Length

18° 6 #1 21 41 18° S2 #1 0.8936 21 47

18° 6 #2 0.6597 22 41 18° 9 #2 0.7848 23 46

18° 6 #3 0.7469 21 44 18° 9 #3 0.6932 24 44

18° 6 #4 0.8441 21 46 18° S2 #4 0.6873 24 45

18° 6#5
18° 6#6
18° 6#7
18° 6#8
18° 6#9
18° 6#10
18° 6#11
18’ 6#12
18° 6#13
18° 6#14
18° 6#15
18° 6#16
18° 6#17
18° 6#18
18° 6#19
18° 6#20
18° 6#21

0.6866
0.8687
0.6463
0.8251
0.8173

0.697

0.7388
0.81 16
0.7578

0.7274
0.7979

23
23
23
23
23
23
23
23
23
24
24
24
24
25
25
25
25

:88

88888888888888

18° 9 #5
18° 82 #6
18’ SB #7
18° 9 #8
18° 9 #9
18° 9 #10
18° 9 #11
18° 9 #12
18° 9. #13
18° S2 #14
18° 9 #15
18" Q #16
18° 9 #17
18° 9 #18
18° 9 #19
18° 9 #20
18° S? #21
18° 9 #22
18° SB #23
18° 9 #24
18" S? #25
18" Q #26
18° 52 #27

0.6269
0.7958
0.7936
0.8618
0.7166
0.8091
0.8182
0.8194
0.9585
0.6287

0.678
0.9076

0.8164
0.8177
0.7426
0.7704
0.8132
0.8105

0.8531
0.7448
0.7773

24
24
25
25
25
25

3338

26
26
26
27
27
27
27
27
27
28
28
29
30

888888:

47

88888888888888

Vermont Early Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

149

ID Pupal Wt Days to Fore Wing ID Pupal Wt Days to Fore Wing
Emerge Length Emerge Length

22° 6 #1 0.7952 14 45 22° 9 #1 0.8315 15 45
22° 6 #2 0.7089 15 45 22° 9 #2 0.8502 16 48
22° 6 #3 0.7122 15 42 22° 92 #3 0.6795 17 44
22° 6 #4 15 44 22° 9 #4 0.7478 17 42
22° 6 #5 0.6756 15 41 22° 92 #5 0.7169 17 46
22° 6 #6 0.808 15 45 22° 9 #6 0.7821 17 45
22° 6 #7 0.7166 15 44 22° 9 #7 0.7664 17
22° 6 #8 0.7361 15 44 22° 9 #8 0.8242 18 45
22° 6 #9 0.658 15 41 22° 9 #9 0.8933 18 47
22° 6 #10 0.5528 16 42 22° S? #10 0.6964 18 42
22° 6 #11 0.7653 16 46 22° 9. #11 0.7329 18 42
22’ 6 #12 15 42 22° S2 #12 0.8497 18 47
22° 6 #13 16 42 22" S2 #13 0.7485 19 46
22° 6 #14 16 43 22° 9 #14 0.7402 19 45
22° 6 #15 16 43 22° 9 #15 0.8601 19 48
22° 6 #16 16 44 22° 92 #16 0.79 19 45
22° 6 #17 16 22" $2 #17 1.0246 19 51

22° 6 #18
22° 6 #19
22° 6 #20
22° 6 #21
22° 6 #22
22° 6 #23
22° 6 #24
22° 6 #25

0.7038
0.7124
0.6738
0.7676

0.7249
0.8256
0.6613

16
16
16
16
17
17
18
18

42
43
43
45

45
44
43

22° S2 #18
22" 9 #19
22° SB #20
22° 9 #21
22° 9 #22

0.6982

0.7567
0.7766
0.8079

19
20
21
21
23

Vermont Early Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

ID

26° 6 #1

26° 6 #2

26° 6 #3

26° 6 #4
26° 6 #5

26° 6 #6

26° 6 #7

26° 6 #8

26° 6 #9
26° 6 #10
26° 6 #11
26° 6 #12
26° 6 #13
26° 6 #14
26° 6 #15
26° 6 #16
26° 6 #17
26° 6 #18
26° 6 #19
26° 6 #20
26° 6 #21
26° 6 #22
26° 6 #23
26° 6 #24
26° 6 #25
26" 6 #26

ID

0.6623
0.6991
0.706

0.7802
0.7873
0.8738
0.6173
0.7047
0.7451
0.6825
0.6704

0.829
0.8224
0.7084

0.786

0.8002
0.7381
0.6328
0.7469
0.8703

10
10
10
10
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
12
12
12
12
12
13

45
42

888888888888888888888

42
45

Pupal Wt Days to Fore Wing ID
Emerge Length

26° S? #1
26" SB #2
26° S2 #3
26° 9 #4
26° 9 #5
26° 9 #6
26° 9 #7
26° 92 #8
26° 9 #9
26° 9 #10
26° SB #11
26° S2 #12
26° 9 #13
26° 9 #14
26° 9 #15
26° 9 #16
26° 9 #17
26° S? #18
26° S2 #19
26° 52 #20
26° 9 #21
26° 9 #22

0.8187
0.9599
0.9187

0.7358
0.7573
0.8493
0.7318
0.7133
0.6901
0.8221
0.8363

0.838
0.7049
0.8784
0.8546
0.8063
0.8508

0.8335

11
11
11
11
12
12
12
12
12
12
12
12
12
12
13
13
13
13
13
14
14
15

Vermont Late Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

Pupal Wt Days to Fore Wing ID

150

88888

Pupal Wt. Days to Fore Wing
Emerge Length

8888888888888888888888

Pupal Wt. Days to Fore Wing

14° 6 #1
14° 6#2
14° 6#3
14° 6 #4
14° 6 #5
14° 6 #6
14° 6#7
14° 6#8
14° 6 #9
14° 6 #10
14° 6 #11
14° 6 #12
14° 6 #13
14° 6 #14
14° 6 #15
14° 6 #16
14° 6 #17

ID

18° 6#1
18° 6#2
18° 6#3
18" 6#4
18° 6#5
18° 6#6
18° 6#7
18° 6#8
18° 6#9
18° 6#10
18° 6#11
18° 6#12
18° 6#13
18° 6#14
18° 6#15
18° 6#16
18° 6#17
18° 6#18
18° 6#19
18° 61120

0.8936
1.0219
1.0303
1.0224
0.9916
0.9891
0.9538
0.9083
1.1649
1.0353
1.1038
1.1506
1.0923
1.0086
1.1023
1.2209

1.108

Emerge Length

75
75
75
78
81
82

87
91

95

97
97

102
110

88888888888888888

14° 9 #1
14° 9 #2
14° 9 #3
14° 9 #4
14° 9 #5
14° 9 #6
14° 9 #7
14° 9 #8
14° 9 #9
14° 9 #10
14° 9 #11
14° 9 #12

0.9654
1 .0707
0.9808
1 .1026
1 .0659
1 .1 122
0.9525
1 .1722
1 .1 144
1 .0167
0.9724
1 .1 149

Emerge Length

102
104
111
122
138

Vermont Late Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

1.1192
1.1706
1.0548
1.1164
1.0317
1.0502
1.1943
1.0163
1.0607
0.8168
1.0897
1.1589
1.1449
1.0989
1.0322
0.9906
1.0776
1.1129
1.1571
1.0002

2888888888888

88888

51

888888888888

51
49

Pupal Wt Days to Fore Wing ID
Emerge Length

18° 9 #1
18’ 9 #2
18° 9 #3
18° 9 #4
18° 9 #5
18° 9 #6
18° 9 #7
18° 9 #8
18° 9 #9
18° 9 #10
18° 9 #11
18° 9 #12
18° 9 #13
18° 9 #14
18° 9 #15

151

0.936

1.223
0.8084
1.0102

0.985
1.2856
1.0223
1.2892
1.0409
1.1991
0.9674
1.1661
1.3188
1.1231
1.2497

43
45
45
45
46
47
47

888892888

47
49
49
49

88888

45
47

Pupal Wt Days to Fore Wing
Emerge Length

50
51
41
49
48
51
49
52
47
53
43
52
52
52
55

ID

22° 6 #1
22° 6 #2
22° 6 #3
22° 6 #4
22° 6 #5
22° 6 #6
22° 6 #7
22° 6 #8
22° 6 #9
22° 6 #10
22° 6 #11
22° 6 #12
22° 6 #13
22° 6 #14
22° 6 #15
22° 6 #16
22° 6 #17

ID

26° 6 #1
26° 6 #2
26° 6 #3
26° 6 #4
26° 6 #5
26° 6 #6
26° 6 #7
26° 6 #8
26° 6 #9
26° 6 #10
26° 6 #11

Vermont Late Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

Pupal Wt.

1.0099
0.9229
0.8256
0.9927
0.9874
1.1853
0.8833
1.0132
1 .1293

0.974
1.0869

0.992
1.0794
0.9945
1.0172
0.9105
1.0926

Days to Fore Wing ID

Emerge Length

24
25
26
27
28
28
29
29

29
29
30
32
33
34
35

37
38

48
47
47
49

53
43
50
50
43
50
50
51
51
51

51

22° 9 #1
22° 9 #2
22° 9 #3
22° 9 #4
22° 9 #5
22° 9 #6
22° 9 #7
22° 9 #8
22° 9 #9
22° 9 #10
22° 9 #11
22° 9 #12
22° 9 #13
22° 9 #14
22° 9 #15
22° 9 #16
22° 9 #17
22° 9 #18
22° 9 #19
22° 9 #20

Pupal Wt.

1 .0206
1.1 161
1.1909
1.00323
0.9683
1.0187
1 .0828
1.3412
1 .0228
1 .0678
1 .3561
0.9329
1 .0048
1.1676
1.1612
0.9678
1.2123
1 .2428
1 .0902
1.1223

Days to Fore Wing

Emerge Length

28
29
30
31
31

8888888888

39
40
41
41
45

Vermont Late Flight 2004 Emergence Data
Pupae placed in emergence chambers 418104

Pupal Wt.

0.6829
1.1102
1.0456
0.9519
0.8109
1.0692
0.9964
0.8775
0.9538
1.0049
0.9492

Days to Fore Wing ID

Emerge Length

12
20
21
21
21

88888

23

43
49
50
50
43
50
51

48
50
50
49

26° S2 #1
26° S? #2
26° S2 #3
26° 92 #4
26° 9 #5
26° 92 #6
26° 9 #7
26° 9 #8
26° 32 #9
26° SB #10
26° 92 #11

152

Pupal Wt.

1.0896
1.1348
0.9876

1.208
1.0086
1.2064
1.1013
1.1969
1.0888
1.1769
1.1694

51
53
51
52
43
51
51
53
51
51

Days to Fore Wing

Emerge Length

24
24
24
25
26
26
27
29
30
30
30

50
50
43
52
50
52
53
54
49
54
51

26° 6 #12
26° 6 #13
26° 6 #14
26° 6 #15
26° 6 #16
26° 6 #17
26° 6 #18
26° 6 #19
26° 6 #20
26° 6 #21
26° 6 #22
26° 6 #23
26° 6 #24

0.9458
1.0314
0.8649
1 .0587
0.9894
1.1722
1.0106
1.1 1 16
1.0163
1.1347
1.0072

0.883
1.1 198

23
24
25
25
25
25
25
25
26
27
29
29
31

49
50

50
51
54
45

3.8888

52

26° 9 #12
26° £2 #13
26° 9 #14
26° 9 #15

153

1.1673
1.1883
0.9874
1.1146

8888

888

52

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