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'

THESIS

QCO\

 

lIBRARY
M Michigan State 3

L... University ......

 

 

This is to certify that the
thesis entitled
ARE HYBRIDS MORE FIT THAN THEIR PARENTAL
TYPES? A TEST USING TWO TIGER SWALLOWTAIL

BUTTERFLY SPECIES. PAPILIO GLAUCUS AND

P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)
presentedby

Jennifer Laura Donovan

has been accepted towards fulfillment
of the requirements for

Master ' 3 degree inmmy

[fl Via/w

 

Major professor

Date

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

DATE DUE DATE DUE

 

DATE DUE

 

OCT 0 2 2004

:IC/

Ar
h.

 

 

C“!
CD

1x

LR)

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRCIDateDue.p65-p.15

 

 

APE HYBRII

PAP/£10 GL

ARE HYBRIDS MORE FIT THAN THEIR PARENTAL TYPES? A TEST USING
Two TIGER SWALLOWTAIL BUTTERFLY SPECIES
PAPILIO GLA UCUS AND P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)
By

Jennifer Laura Donovan

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Entomology

2001

 

 

 

ARE HYBRl

PAM/0(1)

The rangCS ot
boreal and “’7
between 41: :1
adults 0f P- «‘3‘.‘
cross or F3 0

and the field tl

 

tion (Deering 1
postzygotie bat
tors of fitness t
reciprocal cros
type and recip
perature (153C
loides. Przmus
rates or small:
required to co

less than at le

did hybrids eV

results indicate

ABSTRACT
ARE HYBRIDS MORE FIT THAN THEIR PARENTAL TYPES? A TEST USING
TWO TIGER SWALLOWTAIL BUTTERFLY SPECIES
PAPILIO. GLA UCUS AND P. CANADENSIS (LEPIDOPTERA: PAPILIONIDAE)
By

Jennifer Laura Donovan

The ranges of P. canadensis and P. glaucus overlap and form a hybrid zone at the

boreal and temperate forest transition zone in the eastern half of the United States,
between 41° and 44° north latitude. It has been documented that laboratory hybrid
adults of P. glaucus and P. canadensis are fertile, produce normal gametes, and back
cross or F-2 offspring (Hagen et al. 1991). It has also been shown in the labora‘ofi
and the field that prezygotic barriers are weakly effective at preventing cross fere‘yflfi’ {
tion (Deering 1998, Stump 2000). This study was designed to examine the Sven?” O
postzygotic barriers at the larval stage by simultaneously examining multiple igdica-
tors of fitness (growth rate, pupal weight, larval duration and survival to pupat ion) in
reciprocal crosses hybrid and parental types of P. canadensis and P. glaucus. P arenta]
type and reciprocal hybrid larvae were randomly assigned to a combination of t e
perature (15°C, 23°C, or 31°C) and host plant (Liriodendron tulipifizra, Populus ire”, ~
Ioides, Prunus serotina). Hybrids showed patterns of significantly slower 81‘th

rates or smaller pupal weights than their parental types. The number of degree days
required to complete hybrid larval development to pupation was always equal to 0r

less than at least one of the parent types. Furthermore, in no treatment combination

did hybrids ever perform less well than both parental types for any fitness trait. These

results indicate that postzygotic barriers at the larval stage could potentially be weak,

 

 

 

l \Vt‘Ul

 

guidance. Th.

DI. Rufus lsr‘.

   

(DEB-Will fit I.
Thank

for profession;

 

Thank
this research:
Janice Bossan

Finally

WOllld l'taVe m

ACKNOWLEDGEMENTS

I would like to thank my major advisor, Dr. Mark Scriber for his support and
guidance. Thanks to my committee, Dr. James Miller, Dr. Deborah McCullough, and
Dr. Rufus Isaacs. Research was supported by National Science Foundation Grant
(DEB-9981608) and the MAES (Project #1644)

Thank you to Dr. Thomas Emmel for his advice and providing opportunities
for professional development.

Thank you to the people that have played a vital role in helping me complae
this research: Gabriel Ording, Sara Sanders, Michelle Oberlin, Jenny Muehfi‘afis’
Janice Bossart, Jim Maudsley, and Mark Deering.
Einally, thank you to Aram Stump: without your support, advice and mgiflgx

would have never finished.

iii

 

 

 

 

 

 

 

 

 

ABSTKAC T. .

LlST 0T TATI
1

LIST OF FlGl

LlTERATl'Ri

 

 

 

INTRODECT
METHODS...
RESULTS...
Drscrrssros

APPENDIX 1
RECORD OT

APPENDIX
VOUCHER:

FAMILY 1 5

APPENDU
“RAGE

‘AEPPENDU
NUMBER

LITERATr

TABLE OF CONTENTS

ABSTRACT..- .................................................................................... ii

LIST OF TABLES ................................................................................ v
LIST OF FIGURES ............................................................................. vii
LITERATURE REVIEW ........................................................................ 1
INTRODUCTION ................................................ ‘ ............................... 11
METHODS ....................................................................................... 14
RESULTS ......................................................................................... 22
DISCUSSION .................................................................................... 47
APPENDIX 1:

RECORD OF DEPOSITION OF VOUCHER SPECIMENS .............................. 54
APPENDIX 1.1:
VOUCHER SPECIMEN DATA ............................................................... 56
APPENDIX 2:
FAMILY 15116 .................................................................................. 63
APPENDIX 3:
ALLOZYME ELECTROPHORSIS ANNAYLSIS OF PARENTS ...................... 65
APPENDIX 4
A VERAGE NUMBER OF DAYS FROM HATCH To PUPATION .................... 70

APPENDIX 5

NUMBER OF LARVAE THAT SURVIVED To PUPATION ........................... 72

LITERATURE CITED .......................................................................... 74

iv

 

 

 

 

Table 1: Diff!

( Adapted fror

Table 2: F-\;|
The model \1
and hostplant
Block’Tempc

Table 3: F-va
The model we
and hostplant
Block‘Tempc

 

Table 4: Ave
error. ”N 1

until pupation

Table 5; F_
P<0.05, The

genotype and
lure BlOthT

Table 6: l:
P<0_05. Tl]:
genon-pe am
ture Block-4]

Table 7: A‘
pupation. n,

Table 8: R.

e mOdel .
and hogtpla
lOthTem'

Table 9. F-

e model
and hOStha
Block‘Tem

Table 10:
pupallOn. n

LIST OF TABLES

Table 1: Differing Heritable Characteristics of Papilio glaucus and Papilio canadensis
( Adapted from Hagen et a1 1991) ............................................................. 10

Table 2: F-values (Type 111 SS) from ANOVA of 1999 degree day analysis. P<0.05.
The model was 3 Split plot design with temperature as the whole plot and genotype
and hostplant as the split plots. ‘ To obtain the correct F - value for temperature
Block*Temperature is used as the error term ................................................ 23

Table 3: F-values (Type III SS) from ANOVA of 2000 degree day analysis. P<0.05.
The model was a Split plot design with temperature as the whole plot and genotype
and hostplant as the split plots. To obtain the correct F - value for temperature

Block*Temperature is used as the error term ................................................ 23

Table 4: Average Number of Degree Days From Hatch to Pupation with a: 1 standard
error. **** Denotes no survival until pupation. n=number of larvae that survived

until pupation .................................................................................... 27

Table 5: F-values (Type III SS) from ANOVA of 1999 pupal weight analysis.
P<0.05. The model was a Split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F - value for tempera-
ture Block*Temperature is used as the error term. .......................................... 30

Table 6: F-values (Type III SS) from ANOVA of 2000 pupal weight analysis.
P<0-05. The model was a Split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F- value for tempera-
ture Block*Temperature is used as the error term ........................................... 30

Table 7: Average pupal weight 3: 1 standard error. **** Denotes no survival until
Pup ation. n=number of pupae .................................................................. 34

Table 8: F -values (Type 111 SS) from ANOVA of 1999 growth rate analysis. P<0.05.
The model was a Split plot design with temperature as the whole plot and genotype
and hostplant as the split plots. To obtain the correct F- value for temperature
Block*Temperat11re is used as the error term ................................................ 36

Table 9: F-values (Type III SS) from ANOVA of 2000 growth rate analysis. P<0.05.
The model was a Split plot design with temperature as the whole plot and genotype
and hostplant as the split plots. To obtain the correct F- value for temperature
BIOCk * Temperature is used as the error term ................................................ 36

Table 1 0: Average growth rate i 1 standard error. **** Denotes no survival until
pupati on. n=number of pupae .................................................................. 41

 

 

Table 11: A\
survival until

Table 12: l
P. canadens1WI

resolution \x'a.

Table 13: Pa
males were car
were hand pa;
used in 1999
allozyme wen.

Table 14: A“
m‘ Denotes

Table 15: Tm
531111) at the st

 

 

Table 11: Average Percent Survival to PUpation :t 1 standard error. **** Denotes no
survival until pupation. n=number of pupae ................................................ 46

Table 12: Diagnostic allozymes ran to confirm genotypic identity of putative
P. canadensis female mother 15116 and her offspring. Question marks indicate that
resolution was difficult but best guesses were made. ..................................... 64

Table 13: Parental allozyme and geographical origin. Wild males denotes that fe-
males were captured in the field and mates are unknown wild males. All other females
were hand pair with the male indicated. Numbers beginning with 15 are butterflies
used in 1999 and numbers beginning with 16 are from 2000. *** Indicates that
allozyme were run but were not able to be visualized ....................................... 67

Table 14: AVerage number of days from hatch to pupation i 1 standard error.
**** Denotes no survival until pupation. n=number of pupae ............................ 71

Table 15: The number of larvae that survived to pupation/the total number of larvae
set up at the start of the experiment ....................................................... i ..... 73

vi

 

 

Figure 1: R311

Figure 2: Col
cleft P. gluzrr'zr.

 

Ficure 3: Flo

Figure 4: Ave
pansons are n1
a treatment grr
means from 21
loner case lett
s.e. Bars not S

Figure 5: Ave
Parisons are m
a treatment grcl
means from 21’,
lower case lettt
5-6- Bars not 5

Figure 6; AVG}
pal-150118 are m
a treatment th
means from 21
lower Case 1m
s.e. Bars not 5

Figure 7: FOP
mem group ar.
9r SignlfiCam
\‘alues are ex]
mficamb' dir‘n

Figure 8: F

r 31‘.
epresem mea‘
{or Slgm'fiCam

alues Eire e\1

Tllllcaml}, (11 ff,-

LIST OF FIGURES

Figure 1: Ranges and hybrid zone of Papilio canadensis and Papilio glaucus .......... 9

Figure 2: Collection sites for P. canadensis and P. glaucus. P. canadensis (solid cir-
cle) P. glaucus (open circle) .................................................................... 16

Figure 3: Florida collection site for P. glaucus ............................................. 17

Figure 4: Average number of degree days from hatch to pupation at 15°C. All com-
parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means from 2000. Capital letters for Significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are :tl
s.e.‘ Bars not sharing a letter are significantly different from each other at P<0.05. . . .24

Figure 5: Average number of degree days from hatch to pupation at 23°C. All com-
parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means from 2000. Capital letters for Significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are $1
s.e. Bars not sharing a letter are significantly different from each other at P<0.05....25

Figure 6: Average number of degree days from hatch to pupation at 31°C. All com-
parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means from 2000. Capital letters for significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are :tl
s.e. Bars not sharing a letter are significantly different from each other at P<0.05. . ..26

Figure 7: For average pupal weight at 15°C. All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars
represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are 21:1 s.e. Bars not Sharing a letter are sig-

nificantly different from each other at P<0.05 .............................................. 31

I Figure 8: For average pupal weight at 23°C. All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars
represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are 21:1 s.e. Bars not sharing a letter are sig-

nificantly different from each other at P< 0.05 .............................................. 32

vii

 

Figure 9: Forl
ment group :1
represent me;
for significan
Values are e.\=
niticantly dil':

 

 

   
  
   

Figure 10: F0
ment group or
represent men
for significant
Values are ex
niticantly dit‘t

Figure 11; F1
ment grourp :1
represent me.-
for significar
Values are e:
nificantly d1:

Fierrre 12; 1
them grout)
rePresent m
for SignllICZ
Values are .
nificantly (1

Figure 13:
group and
Sem Percei
James are
ETEnceS l I
PTCSSed as
ter are Sig

Figure 14
group anc
Sim Defer
Values 31’
er‘enCeS i
preSSEd a
181‘ are SIS

Figure 9: For average pupal weight at31°C. All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars '
represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are i1 8. e. Bars not sharing a letter are sig-

nificantly different from each other at P<O. 05 ............................................. 33

Figure 10: For average growth rate at 15°C. All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars
represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are :tl s.e. Bars not sharing a letter are sig-

nificantly different from each other at P<0.05 ............................................... 38

Figure 11: For average growth rate at 23°C . All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars

represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are :1 s.e. Bars not sharing a letter are sig-

nificantly different from each other at P<0.05 ............................................... 39

Figure 12: For average growth rate at 31°C. All comparisons are made within a treat-
ment group and within a year. Each graph represents a treatment group. White bars

represent means from 1999 and black bars represent means from 2000. Capital letters
for significant differences indicate 1999 data and lower case letters indicate 2000 data.
Values are expressed as means, error bars are i=1 s.e. Bars not sharing a letter are sig-

nificantly different from each other at P<0.05 ............................................... 40

Figure 13: Percent survival at 15°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant dif-
ferences indicate 1999 data and lower case letters indicate 2000 data. Values are ex-
pressed as an average of the family means, error bars are $1 s.e. Bars not sharing a let-
ter are significantly different from each other at P<0.05 .................................. 43

Figure 14: Percent survival at 23°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant dif-
ferences indicate 1999 data and lower case letters indicate 2000 data. Values are ex-
pressed as an average of the family means, error bars are $1 s.e. Bars not sharing a let-
ter are significantly different from each other at P<0.05 ................................... 44

viii

 

 

Figure 15 Pu
group and \1’1'
sent pace at s
Values at t ex
t‘erencesi id'n
pressed 3 . an
ter are 511 niti

 

 

Figure 15: Percent survival at 31°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant dif-
ferences indicate 1999 data and lower case letters indicate 2000 data. Values are ex-
pressed as an average of the family means, error bars are 21:] s.e. Bars not Sharing a let-
ter are significantly different from each other at P<0.05 ................................... 45

ix

 

Since
been constant
end of the spc
hybrids. in g:~
1963). This it
combinations
to spread into
adapted in an}
Hodges (1095
3110131385 111:1
lineages."

This (1:
flature. SPCCI
cause the ind
1997). Damn
in a Perfect Co

H}bn d5, 0n IL.

 

 

'°-\\

LITERATURE REVIEW

Since Charles Darwin published On the Origin of Species in 1859, there has
been constant debate over the role of hybridization in the evolutionary process. At one
end of the spectrum is the view that it should play little or no role in evolution because
hybrids, in general, have been considered unfit relative to their progenitors (Mayr
1963). This is illustrated by Dobzhansky (1970): “To be sure, a minority of the gene
combinations formed by the hybridization of species might be fit, perhaps fit enough
to spread into as yet unoccupied adaptive peaks. A majority would be relatively ill
adapted in any environment.” At the other end is the View championed by Arnold and
Hodges (1995): “hybridization may often lead to the production of relatively fit hybrid
genotypes that possess novel genetic variation and the founding of new evolutionary
lineages.”

This debate has been evident in the many proposed species concepts in the lit-
erature. Species concepts typically take the View that hybridization is maladaptive be-
cause the individuals involved produce fewer and/or less fertile progeny (Arnold
1997). Darwin wrote (1859) “Pure species have of course their organs of reproduction
in a perfect condition, yet when intercrossed they produce either few or no offspring.
Hybrids, on the other hand, have their reproductive organs functionally impotent....”

More recently Mayr (1963) reinforced this viewpoint by saying “. . .The majority of . . .
hybrids are totally sterile . . . Even those hybrids producing normal gametes in one or
both sexes are nevertheless unsuccessful in most cases and do not participate in repro-

duction . . . when they do backcross to the parental species, they normally produce

 

genotypes 01.
hybridization
The fr
Cohesion. anC
sentative Of a1
cies concepts
pairings have
the fitness of
considered “b;
(Arnold l997)|
Biological Sp
(1937) earlier
ing natural
grOUpg“ (Mayr
Cies' Mayr de:
heme hybrids
SDefies Or S em
«The majority

"lgor." Even t”-

 

 

theless unstrccc
the)” do backcrl
Inability that a}

d
oes SUCCGszu;

 

genotypes of inferior viability that are eliminated by natural selection. Successful
hybridization is indeed a rare phenomenon among animals.”

The four species concepts that will be discussed are: Biological, Recognition.
Cohesion, and Phylogenetic. They were chosen because they are considered to be repre-
sentative of all other Species concepts (Arnold, 1997). They also exemplify that all Spe-
cies concepts consider hybridization to be “bad” because the offspring of heterospecific
pairings have lower levels of fertility and/or viability and therefore effectively reduce
the fitness of the parental types to zero (Arnold, 1997). Natural hybridization is also
considered “bad” when it is Viewed as a violation of the process of divergent evolution
(Arnold 1997).

Biological Species Concept: Mayr, basing his definition of species on Dobzhansky’s
(1937) earlier work, defined species as “. . .groups of actually or potentially interbreed-
ing natural populations, which are reproductively isolated from other such
groups” (Mayr, 1942). This strict definition does not allow for the hybridization of Spe-
cies. Mayr dealt with this problem in two ways. First in 1942, he argued that if viable,
fertile hybrids were produced, then one should consider the hybridizing forms to be sub-
species or semispecies. Secondly, in 1963, he stated that if hybridization does occur,
“The majority of such hybrids are totally sterile, even where they display “hybrid
vigor.” Even those hybrids that produce normal gametes in one or both sexes are never-
theless unsuccessful in most cases and do not participate in reproduction. Finally, when
they do backcross to the parental species, they normally produce genotypes of inferior
Viability that are eliminated by natural selection.” Mayr concludes that if hybridization

does successfully occur it will most likely lead to evolutionary dead ends. Mayr (1963)

 

states. "In “3"
The failure 01'
already 3 gm:
species."

The Recognir
most inclusive
fertilization Sj
byproduct of
Systems“ (Par

selection. Thi

subset that aft

1

Species Concc
Characters of
life" (Paterson

Strict a
reticulation Car

1995). ReCOg]

et'olutjcmarV ir
SPEClfiQ K’Iale

 

states, “In natural populations there is usually severe selection against introgression.
The failure of most zones of conspecific hybridization to broaden Shows that there is
already a great deal of genetic unbalance between differentiated populations within a
species.”

The Recognition Species Concept: Under this theory, Species are defined as “that
most inclusive population of individual biparental organisms which share a common
fertilization system” (Paterson, 1985). Heterospecific reproductive isolation is a
byproduct of adaptive evolution. The development of “Specific Mate Recognition
Systems” (Paterson, 1985) occurs when an allopatric population undergoes directional
selection. This selection acts upon a variety of aspects of the organism, including a
subset that affects mate recognition (Paterson, 1985). Speciation withthe Recognition
Species Concept is therefore “an incidental effect resulting from the adaptation of the
characters of the fertilization system, among others, to a new habitat, or way-of-
life” (Paterson, 1985).

Strict adherence to the Recognition Species Concept leads one to conclude that
reticulation can only occur between forms that are not yet species (Arnold and Hodges,
1995). Recognition Species Concept views natural hybridization between species as
defmitionally impossible. The Recognition Species Concept denies that these taxa are
evolutionary independent because gene exchange between them indicates a common
Specific Mate Recognition System (Paterson, 1985). However, this View conflicts
with observations of crosses in nature that involve organisms clearly belonging to
differentiated species (Templeton, 1989; Arnold, 1992).

The Cohesion Species Concept: A. R. Templeton originally put the Cohesion

 

Species Concr
having the pt
19$».Tennfl:
Concept did 1
syngameons (.
account all 0:
lAnkal997

tion:

“The (
diffe re;
exchan
0f Sper
Lhflike

COHCEP

 

I'eduCe
heremS
Species
COhesi
Placed
Species.
degTEe

,ethOSe r

Species Concept forth. He defines species as “the most inclusive group of organisms
having the potential for genetic and/or demographic exchangeability” (Templeton,
1989). Templeton felt that the Biological Species Concept and the Recognition Species
Concept did not apply to either asexual organisms or individuals that belonged to
syngameons (Arnold, 1997). The Cohesion Species Concept was designed to take into
account all of the microevolutionary processes thought to contribute to Speciation
(Arnold, 1997).
Arnold (1997) best describes how the Cohesion Species Concept addresses hybridiza-
tion:
“The Cohesion Species Concept accepts that there are species that show
differences in their boundaries defined by either genetic or demographic
exchangeability (Templeton, 1989). For example, syngameons are a result
of species having greater genetic than demographic exchangeability.
Unlike the Biological Species Concept and the Recognition Species
Concept, the Cohesion Species Concept does not require that these taxa be
reduced to subspecific categories and thus allows for the process of
heterospecific natural hybridization. Furthemore, the production of hybrid
species from such heterospecific hybridization is also possible under the
Cohesion Species Concept. It is important to be aware of that Templeton
placed those taxa belonging to a syngameon into a category called “bad
Species.” These species are “bad” because they demonstrate an elevated
degree of genetic exchangeability. “Good species,” on the other hand are

“those that are well defined both by genetic and demographic exchange-

"F‘

 

ability”
The P101039
species as “at?
such clusters.
.
descent” (C rat
phylogenetic r
identification 1
of branching

boundaries of

ships among SI

 

Both 11
heterospecifrc '
Phyletic. Crac
be demonstrab
error.“ Henni
species im’olx-
Times of One S
Events are Con

Specie:

for study. Frc

ability” (Templeton, 1989).”

The Phylogenetic Species Concept: The Phylogenetic Species Concept defines
species as “an irreducible (basal) cluster of organisms, diagnosably distinct from other
such clusters, and within which there is a parental pattern of ancestry and
descent” (Cracraft, 1989). The application of this concept to determine patterns of
phylogenetic relationships, define species and the process of Speciation depends on the
identification of the ancestral and derived states of particular characters. The pattern
of branching within these cladograms is the basis for determining not only the
boundaries of species (i.e. irreducible clusters; Carcraft, 1989), but also the relation-
ships among species.

Both Hennig (1966) and Cracraft (1989) argue that species cannot arise from
heterospecific hybridization because species must be of monophyletic origin not poly-
phyletic. Cracraft (1989) wrote: “in the majority of cases, phylogenetic species will
be demonstrably monophylectic; they will never be nonmonophyletic, except through
error.” Hennig argues that if new species arose from polyphyletic origin that the
species involved were so closely related that they could just as well be considered
races of one species (Hennig, 1966). Consequently fertile offspring of hybridization
events are considered evidence that the parental types are the same species.

Species concepts influence the paradigm from which researchers develop ideas
for study. From the above described paradigms one would conclude that all hybrids
are unfit and therefore of little evolutionary importance or that the process of hybridi-
zation is maladaptive because it is an impediment to divergent evolution (Arnold and

Hodges, 1995; Arnold, 1997; Arnold, 1999) .

 

 

There
However. Ari
studies and fr.
"ey'olutionaril;
may reinforct
(prezygotic is:
isolating mecl:

Hybrid
may create a 1
gene combinar

for on the ham

 

 

 

been demonstr

h}'brid P0pu1at

Definltlo n5: 1

“ith u-

use HamSOn~S

There are many examples of hybrids having lower fitness than their parents.
However, Arnold and Hodges (1995) have recently reanalyzed data from several
studies and found that not all hybridization is “bad”. Hybridization can be seen as
“evolutionarily important” even in cases when the hybrids are unfit. For example, it
may reinforce reproductive isolation of separate Species by assortative mating
(prezygotic isolating mechanisms), through selection against the hybrids (postzygotic
isolating mechanisms) (Dowling and Moore 1985).

Hybridization can also be seen as important in cases where hybrids are fit. It
may create a bridge between species and allow the passage of unique and beneficial
gene combinations. Novel and superior adaptive gene combinations could be selected
for on the basis of increased genetic variability due to introgression. This notion has
been demonstrated in laboratory cultures of Drosophila (Nagle and Mettler 1969) and

hybrid populations of Hyalophora moths (Collins 1984).

Definitions: Hybrid and Hybrid Zone
With the problems inherent with Species definitions in relation to hybrids, I will

use Harrison’s (1993) definition for hybrid and Amold’s (1997) for hybrid zone.
“Hybridization is the interbreeding of individuals from two populations, or
groups of populations, which are distinguishable of the basis of one or
more heritable characters.”

The term hybrid will be applied to F1 individuals and individuals of mixed ances-

try. F 1 will be specified when appropriate. Using this definition has three advan-

tages (Harrison, 1993): first, its use does not depend on reaching agreement on a

 

single Species
to particular 1
require judgmr
types in “adap

A hyb:
individuals th.
overlap Spatial

offspring" ( A r:

The Papilio g

C0mplex

 

 

Hybnar
216 ofthe 557
the 216 SPecies
reliable dOCUm
group. 11 is tho:

a gene reCEiVeC

also suspeCted

EndangEred Sp
Tare interSPEC if

It has 1

bpecies (Haven
TGIF-f
llC, perUCE

single species concept. Second, it does not require the assignment of populations
to particular taxonomic categories (e.g. races or subspecies). Third, it does not
require judgments about relative fitness of hybrids or differences between parental
types in “adaptive norms.”

A hybrid zone will be defined simply as a place “where two populations of
individuals that are distinguishable on the basis of one or more heritable characters,
overlap spatially and temporally and cross to form viable and at least partially fertile

offspring” (Arnold, 1997).

.The Papilio glaucus and Papilio canadensis (Lepidoptera: Papilionidae) Hybrid
Complex

Hybridization is relatively widespread in the genus Papilio. Papilio comprises
216 of the 557 species in the family Papilionidae and it is estimated that up to 15% of
the 216 species form natural hybrids (Sperling, 1990). One of the most extensive and
reliable documentations of hybridization in the genus Papilio is within the machaon
group. It is thought that the dark morph of P. zeIicaon and P. machaon is expressed by
a gene received through introgression from P. polyxenes asterius (Sperling 1990). It is
also suspected that Ornithoptera allottei, a Convention on International Trade in
Endangered Species of Wild Fauna and Flora (CITES), protected species, is actually a
rare interspecific hybrid (Sperling 1990).

It has been documented that P. glaucus and P. canadensis are hybridizing
species (Hagen et a1. 1991). Laboratory hybrids of P. glaucus and P. canadensis are

fertile, produce normal gametes, and participate in reproduction (Hagen et a1. 1991).

 

 

I

These two SP“
from New Eng,
1).

The fat
are they consit
(1991) convint
on several dif

suitabilities be

 

tree and high 5
quaking aspen
The P.
toxic Balms p}
lation. This dc
sex linked sup]
There a

l-Hk-t). Lactate c

Pupalc

em"

These two species form a hybrid zone between 41° and 44° North latitude and extends

from New England through the Great Lakes region (Hagen and Scriber 1991) (Figure
1).

The fact that these two species can hybridize so easily begs the question why
are they considered different species? It has been generally accepted that Hagen et al.,
(1991) convincingly described P. glaucus and P. canadensis as separate species based
on several differing heritable characteristics (Table 1). There are differential host
suitabilities between the parental types. P. canadensis has low larval survival on tulip
tree and high survival on quaking aspen. Conversely, P. glaucus has low survival on
quaking aspen and high survival on tulip tree.

The P. glaucus female is polymorphic. Where P. glaucus in sympatric with the
toxic Battus philenor swallowtail a P. glaucus dark morph is present within the popu-
lation. This dark morph is absent in P. canadensis females. It is thought that there is a
sex linked suppressor for the dark morph carried by P. canadensis males.

There are also fixed differences in three diagnostic allozyme loci: Hexokinase
(Hk), Lactate dehydrogenase (th), 6-Phosphogluconate dehydrogenase (Pgd).

Pupal diapause determination is also different for the two species. Diapause is
environmentally determined for P. glaucus and obligate for P. canadensis.

This evidence also meets the criteria of Arnold’s (1997) definition of a hybrid
zone: “where two populations of individuals that are distinguishable on the basis of
one or more heritable characters, overlap spatially and temporally and cross to form

viable and at least partially fertile offspring”.

 

 

F1Stlre

 

      

as

P. canadensis

 

 

 

Figure 1: Ranges and hybrid zone of Papilio canadensis and Papilio glaucus.

 

Table 1: Diffs
( Adapted fror‘

Character

Physiological”:
Environmental d
pupal diapause

Lower lethal tem
diapausing pUpas
Larval survival 0
Lanal survival 0
Larval survival 0
Pohmorphism ft
Suppression of rr

Biochemical
Hexokinase (Hkl
Lactate dehydros
6-Phosphoglucoi
(Pgd’) alleles

l. Hagen and Sc:
H988); 6. Scrib
(.1996); 10 Scrib

 

Table 1: Differing Heritable Characteristics of Papilio glaucus and Papilio canadensis

( Adapted from Hagen et al 1991).

Character

Physiological/Developmental
Environmental determination of
pupal diapause

Lower lethal temperature for
diapausing pupae

Larval survival on aspen foliage
Larval survival on birch foliage
Larval survival on tulip tree foliage
Polymorphism for female color
Suppression of melanic color

Biochemical

Hexokinase (Hk) alleles

Lactate dehydrogenase (th) alleles
6-Phosphogluconate dehydrogenase
(Pgd) alleles

P. glaucus

Yes

~23°C
Very low
Low
High
Present
Absent

£61009,
“100”

(6100,99 £65099

P. canadensis

No

-23°C to -27°C
High

High

Low

Absent

Present

“ l I 0”
6680,39 664099

£6125,’9 6‘80,,9
£6150”

Inheritance

X-linked

9

Polygenic
Polygenic
Polygenic
Y-linked
X-linked

Autosomal
X-linked

X-linked

Ref.

1,2

3
45,6
5,7
4,5
8.9
1,9, 10

ll

1,7,1]

1,7,1]

1. Hagen and Scriber (1989); 2. Rockey et a1. (1987); 3. Scriber (1994); 4. Scriber (1986); 5. Scriber
(1988); 6. Scriber et a1. (1989); 7. Hagen (1990); 8. Clarke and Sheppard (1962); 9. J. M. Scriber, et al.
(1996); 10 Scriber et a1. (1987); l 1. Hagen et al. (1991).

10

'7‘

 

Conve
1997): First. _
isms. Second-
flow. with the
to the process
thought to be sf

Arnolt.

of hyb
ferent 1
ml con
Arnold
hybrids are fit
44 Cases of by
all lower fitne;
that the genera
the hybrids an

 

INRODUCTION

Conventionally, research using hybrids has had one of three emphases (Arnold,
1997): First, hybrids are used to study the systematics of a particular group of organ-
isms. Secondly, they have been used to understand the mechanisms that limit gene
flow, with the assumption that the development of barriers to gene flow is equivalent
to the process of Speciation . Thirdly, individual examples of natural hybridization are
thought to be evolutionarily important and therefore worthy of study.

Arnold (1992) suggests:

“The most informative approach for estimating the adaptive effects

of hybridization and introgression is to measure the relative fitness of dif-

ferent hybrid and parental genotypes under identical experimental or natu-

ral conditions.”

Arnold and Hodges took a careful look at the question of whether or not
hybrids are fit or unfit relative to their parents. They reviewed research that looked at
44 cases of hybridization and found that only 13 hybrid classes demonstrated an over-
all lower fitness than their parental types. (Arnold and Hodges, 1995). They concluded
that the general pattern of hybrid fitness is not that hybrids are uniformly unfit but that
the hybrids are as fit as the two parental taxa or demonstrate a higher level of fitness
than at least one the parents (Arnold and Hodges, 1995).

In this paper Arnold and Hodges (1995) suggest that “simultaneous
measurements of fitness components for hybrid and parental individuals allow direct

comparisons of fitness among parental and hybrid classes and among different hybrid

11

£73

or:
[Wm

indkan

t’ . commas
l’. conudcn
\‘aiwgb

its

”hat

4

“8hr

classes.”

With these ideas in mind I conducted an experiment that simultaneously
measured multiple components of fitness in the larval stage of the Tiger Swallowtail
butterflies, P. glaucus and P. canadensis and their reciprocal hybrids. The fitness
indicators that I measured or calculated were: average growth rate, average pupal
weights, average number of degree days from hatch to pupation and larval survival to
pupation.

These fitness indicators were measured on larvae from four genotypic classes:
P. canadensis, P. glaucus, P. canadensis x P. glaucus and P. glaucus and
P. canadensis. The two types of hybrids, P. glaucus x P. canadensis and
P. canadensis x P. glaucus were chosen because they are easily made, through hand-
pairing, in the laboratory. Three host plants were chosen: tulip tree, black cherry and
quaking aspen. The host plants were included in the study because they all are present
within the hybrid zone and in some part of the two parental types ranges. Tulip tree is
a preferred host of P. glaucus and P. canadensis has low survival on it (Scriber, 1988).
Quaking aspen is a preferred host of P. canadensis and P. glaucus has low survival on

1't(Scriber, 1988). Black cherry is mutually suitable for both parental types.
Three temperature treatments were used: 15°C, 23°C and 31°C. These
temperatures approximate the lower and upper limits of survivorship of both parental
SPecies.
There were four main objectives of this study:
Objective 1: To determine if hybrid larvae of P. glaucus and P. canadensis will need

Significmtly different number of degree days from hatch to pupation.

12

W.

{/v.
0

(1'_

01
Mina

Obiect.

cools cause \‘
he occupv‘

mm“

Objective 2: To determine if hybrid pupae of P. glaucus and P. canadensis have
significantly different weights.

Objective 3: To determine if hybrid larvae of P. glaucus and P. canadensis have sig—
nificantly different growth rates from hatch to pupation than parental type larvae.
Objective 4: determine if hybrid larvae of P. glaucus and P. canadensis have signifi-
cantly different survival rates to pupation than parental type larvae.

Determining the fitness of hybrids relative to their parents will allow us to
make predictions about what to expect from hybrids if they are naturally occurring and
begin to asses their evolutionary importance. If we find that hybrids are unfit, this
could cause the reinforcement of reproductive isolation. If they are fit, hybrids might
be occupying adaptive peaks or allowing the passage of novel beneficial gene

combinations from one parental species to another.

13

 

[my

hybrids.

11...... ...ww

f or em
mm co\\

\\m \{\s
has

for

2000. f
Pupae v

ilOm Si

iltlfll
Tonal:

Were re

Wilt Sll

METHODS

Research was conducted that measured multiple components of fitness of the
larval stage of Tiger Swallowtail butterflies, P. glaucus and P. canadensis, and their
hybrids. Larvae of P. glaucus, P. can'adensis and their reciprocal hybrids were
randomly assigned to host plant/temperature treatments (3 temperatures: 3 hostplants)

and then reared in growth chambers.

Sources of Broods
Parental types: Parental types were obtained from the offspring of wild
females collected from outside the hybrid zone. P. canadensis females were collected
from several counties in Michigan: Charlevoix County, Emmet County, Cheboygan
County, and Isabella County. P. canadensis were also collected from Bennington
County Vermont and Clark County Wisconsin.
In 1999, P. glaucus females were collected from Warren County Missouri. In
2000, P. glaucus females were bought as pupae from a breeder in Pennsylvania.
Pupae: were allowed to emerge and were then handpaired with wild P. glaucus males
from St. Joseph County, Michigan.
Hybrids: In 1998, wild females were collected for laboratory stock, from
Several sites in Michigan: Cheboygan County, Dickinson County and Isle Royale.
Females were placed in oviposition arenas and eggs were harvested daily. Caterpillars

Were reared under diapause inducing conditions: 12:12 light/dark and at 25°C. Pupae

Were stored in 24 hour dark and 4°C conditions until spring of 1999. In May of 1999,

14

 

 

flax/m

Romacl

I. ...l

mid P. curtuu

\mkafi

pupae were brought out of diapause conditions and were allowed to emerge in
cylindrical screen cages and then handpaired with wild P. glaucus males. In 2000,
P. canadensis pupae were supplied by Howard Romack. He raised larvae from
Bennington County, Vermont on Black Cherry (Prunus serotina). Pupae received from
Romack were allowed to emerge and then handpaired with wild P. glaucus males.

In 1999, offspring of P. glaucus females that were collected in Highland
County, Florida in April of that year, were handpaired to wild P. canadensis males. In
2000, P. glaucus pupae from Southeast Pennsylvania were bought as pupae from a
breeder from Pennsylvania. Adult females emerged and then were handpaired with

wild P. canadensis males.

Handpairing
All pairings were initiated by hand, after methods described in Clarke &
Sheppard (1956), using laboratory-reared, virgin females. All males that were used for
pairings were wild caught. After being handpaired, coupled pairs were put into a
Cylindrical screen cage and monitored for separation. If pairs uncoupled with in 5
minutes they were paired again or discarded. Matings were considered successful if

they lasted longer than 30 minutes (Stump, 2000).

Oviposition
A1 1 females that were wild caught and handpaired were placed individually in a
r ound plastic oviposition arena, 10 cm in height and 27 cm in diameter, with a 3 choice

hOSt plant arrangement: Liriodendron tulipifera, Populus tremuloides and

15

 

.7 '
‘ I
I
I \ II
I
f) \
/ , \ \
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I \
l v
I “,A y .. / lo- \’.\ \
,Il- ‘ I ‘ H7 "
~’ _~ - I /’
i \K, ‘-~..-:p

 

 

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-~- 2‘ / * ,1 ~41 s .... 73's...
L -. " T ' »‘ 5 ‘ ‘
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i , ' /
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i——»—-—..

1

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“ykfiaeér

. ‘f ‘ A { I”
N If/ ,\ ‘ . ,_Il"_] \
‘ f I } , K; i l 1“.) If
i O ‘./‘\ \ & If); ‘1 1’3 A '
t I ‘ z "I f i \‘
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“’J :-_~__. -..—v ._._- .... bl" —- 4 ‘— 4.__. v A
- -—— — —~ W’- .... \ 7
l ‘ ' T
I 1" ”j H I 1 kg;
| l .. \ .r .l " — ‘
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' . ,1 ,r' --.~ NV " /c
: -""“_"“1’“h" » a :7 ‘ ”a;
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l '1 1 /

 

Figure 2: Collection sites for P. canadensis and P. glaucus.
P. canadensis (solid circle)

P. glaucus (open circle)

16

 

 

fig

 

 

 

Figure 3: Florida collection site for P. glaucus.

17

 

.1

LII-rm' '-.'l

 

 

 

Mm

Wit? 0!}

Larval Exact
hitc
\sgm \ais

so

Prunus serotina. The arenas were placed on a rotating platform (10 times per hour),
0.7 — 1.0 meter from 6-8 100-watt incandescent lights on a 6 hour light: 6 hour dark:
6 hour light: 6 hour dark cycle (Scriber, 1993). The 6 hours light alternating with
6 hours of dark is to maximize the number of eggs laid by the female. If the females
were on a 24 hour light cycle they would over heat and die in the oviposition arenas.
The females were fed daily with a 20% honey/80% water solution. Females were
allowed to oviposit until death. Eggs were collected and counted daily. Thirty-six

larvae from each brood were used in the experiment.

Larval Experiments
After hatching, 36 larvae from each brood of P. glaucus, P. canadensis and
their reciprocal hybrids, P. canadensis x P. glaucus and P. glaucus x P. canadensis
(PCPG and PGPC), were randomly assigned to a host plant/temperature treatments.
In 1999, there were 5 (180 larvae) broods of P. canadensis, 6 (216 larvae)
broods of P. canadensis x P. glaucus, 4 (144 larvae) broods of P. glaucus x
P. canadensis and 2 (72 larvae) broods of P. glaucus. In 2000, there were 5 (180
larvae) broods of P. canadensis, 4 (144 larvae) broods of P. canadensis x P. glaucus,
4 ( 144 larvae) broods of P. glaucus x P. canadensis and 5 (180 larvae) broods of

P. glaucus.

Rearing

The survival of P. glaucus, P. canadensis and their reciprocal crosses was

StUdied 0.11 three host plants: Liriodendron tulipifera, Populus tremuloides and Prunus

18

 

 

 

.er

ll'l
llz’fc’ /

placed

Source oil

\

.3“

12d.

Adult 1

The pr
tool p
31 da}
ale to

Seltd g

serotina and under three different temperature regimes (15°C, 23°C, 31°C).

Using a randomized complete split block design with temperature being the
whole plot and genotype and host plant being the split plots, 36 larvae from each brood
were randomly assigned to a temperature and a host plant treatment. Each larva was
placed in a Petri dish that was lined with paper towel and contained the appropriate
host plant. To maintain turgidity, the stems of the leaves were inserted into a water
filled aquapicTM. Leaves were changed every other day or more frequently if required.

Hatch date, weights every 10 days, pupation date, pupal weight, emergence

date, sex, and death date, were recorded.

Source of Leaves

Pesticide free host plant foliage was collected from the same East Lansing sites
in both years. Branches were cut and transported to the laboratory. To maintain
turgidity branches were placed in 10 gallon buckets filled with water. To ensure fresh-

ness of the leaves, all leaves for larval feeding were used within 24 hours of being cut.

Adult Emergence
After pupation a pupae were put into a cylindrical screen cage to emergence.
The pupae were weighed and then placed back in the chamber where larval rearing
took place- Pupae were allowed to stay in these conditions for emergence for at least
3 1 days, If they hadn’t emerged after 31 days they were placed in overwintering stor-

age conditions (3°-5°C/total darkness). When emergence occurred, each adult was

sexed and then frozen at -80° C for possible future allozyme work.

19

 

 

 

[tr/4%" .

among

masts of \"

Moat a u

Statistics and Calculations

To determine if there were significant differences between pupal weight,
growth rate. larval duration and survival until pupation between parental types and by-
brids a standard split plot design. Temperature as whole-plot with two split plots:
genotype (P. glaucus, P. canadensis, P. glaucus x P. canadensis, and P. canadensis x
P. glaucus) and host plant (black cherry, quaking aspen and tulip tree). The
experiment was blocked by temperature. Each one of these indices was measured or
calculated for every individual of parental type and reciprocal hybrids.

Pupal weight, growth rate and larval duration data were analyzed using an
analysis of variance (Proc glm: SAS Institute Inc. 1990). Treatment means were
compared using a least squares means comparison.

Time from hatch to pupation, expressed in number of degree-days (to see
number of days from hatch to pupation refer to appendix 4), was calculated using the
following formula: {(average daily temperature — threshold temperature) x number of
days from hatch to pupation} using a threshold temperature of 10°C (Ritland and
Scriber, 1985). Growth rate was calculated by dividing pupal weight by the number of

days from hatch to pupation.
Percent survival to pupation was analyzed using a nonparametric chi-square
analysis (Proc npariway wilconxon anova: SAS Institute Inc. 1990).
The experiment was run twice over two consecutive summers: 1999 and 2000.
S.tatistical 1y a full model was ran and reported so that ultimately all effects and
interactions could be compared but for the purposes of this thesis comparisons were

Only made within a year and treatment combination . .

20

RESULTS

All comparisons. were made within a treatment group and within a year. For
example, comparisons were made between the hybrids and parental types on black

cherry at 15°C in 1999. No comparisons were made between years, across tempera-

tures or between host plants. Sample sizes and data used in the graphs are reported in

a table at the end of each section.

Objective 1: To determine if hybrid larvae of P. glaucus and P. canadensis will
need significantly different number of degree days from hatch to pupation.

In 1999 there was a significant effect of temperature at P<0.01. In 1999 and
2000 there were significant effects of genotype at P<0.0001/ P<0.0001 and host plant
at P<0.0001 and P<0.0001 on the time from hatch to pupation (Tables 2 and 3).

In both years, when Pch larvae were fed black cherry, at all temperatures (15°
C, 23°C and 31°C), they needed significantly fewer degree days to reach pupation than
P. canadensis (Figure 4, 5 and 6). When Pch larvae were compared to P. glaucus
there was no difference (Figure 4, 5, and 6). Except in two cases (Pch larave in 1999
at 15°C and 31°C needed significantly fewer degree days to reach pupation than
P. canadensis), Pch larvae did not differ from either parental types (Figure 4, 5 and
6).

In both years, when fed quaking aspen at 15°C, 23°C and 31°C (with the ex-
ception of Pch at 23°C in 2000) , the surviving larvae did not differ from each other

(Figure 4, 5, and 6). In 2000 Pch larvae needed at least 43 more degree days to reach

21

pupation on quaking aspen than P. canadensis larvae (Table 4).

On tulip tree at 15 °C and 23°C, in both years the hybrid larvae needed as
many or fewer degree days to reach pupation as their parental types. For example, in
1999 and 2000 at 23°C, Pch larvae needed at least 55 and 27 respectively fewer
degree days to reach pupation than P. canadensis(table'4). At 31°C in both years the
surviving larvae did not differ statistically from each other in the number degree days
needed to reach pupation (Figure 6).

Over all hybrids at each host plant and temperature treatment combination and
in both years, needed the same or few number of degree days to reach pupation at the

parental types.

22

Table 2: F-values (Type 111 SS) from ANOVA of 1999 degree day analysis.
P<0.05. The model was a Split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F- value for tem-
perature Block*Temper'ature is used as the error term.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

plant

Sources of Variation DF SS MS F Pr<F
Block 1 25.94 25.94 0.01 0.92
Temperature 2 85695.27 42847.64 91.44 0.01
Block * Temperature 2 ”937.19 468.60 0.17 0.84
Genotype 3 "nu-76272.36 25424.12 9.32 0.0001
LHostplant .__ M I T 2 233002.64 116504.32 42.73 0.0001
“e;n;”t;pe"¥"ii;;“{p1;‘n‘ ' 5 37556.85 7511.37 2.75 i 0.019”
Temperature * Genotype T “aw-"6T- 29214.71 4869.12 1.79 "”136...
Temperature * Hostplant 4 20623.37 5155.84 1.89 0.1 1
”Temperature * Genotype* Host- 8 12708.69 1588.56 0.58 0.79

 

Table 3: F -values (Type 111 SS) from ANOVA of 2000 degree day analysis.
P<0.05. The model was a Split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F- value for tem-
perature Block*Temperature is used as the error term.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1L1...

23

Sources of Variation DF SS MS F Pr<F
Block 1 3087.39 -‘ 3087.39 1.25 0.2646
Temperature -2 134884.45 67442.23 1.14 0.47
Block * Temperature 2 118035.70 59017.85 23.96 0.0001
Genotype 3 90914.81 30304.95 12.30 0.0001
Hostplant 2 230076.29 115038.14 * 46.71 0.0007".
Genotype * Hostplant—MW 5“ 72928.56 14585.71 5.92 0.0001
Temperature * Genotype 6 22695.06 3782.51 1.54 0.17
Temperature *3 Hostplant 4 2675.38 , 668.85 .27 0.90
TT-amperature * Genotype* Host- 8 12070.34 1508.79 .61 ”0.7—7M

 

 

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a

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500.

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(Degree Days)

 

 

 

 

 

 

 

 

 

 

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Figure 4: Average number of degree days from hatch to pupation at 15°C. All com-
parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means from 2000. Capital letters for significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are 4:]
s.e. Bars not sharing a letter are significantly different from each other at P<0.05.

24

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600 - a .

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parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means from 2000. Capital letters for significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are 21:]
s.e. Bars not sharing a letter are significantly different from each other at P<0.05.

25

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(Degree Days)

 

 

 

 

 

 

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Black Cherry 31°C
b AB

    

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Figure 6: Average number of degree days from hatch to pupation at 31°C. All com-
parisons are made within a treatment group and with in a year. Each graph represents
a treatment group. White bars represent means from 1999 and black bars represent
means fi'om 2000. Capital letters for significant differences indicate 1999 data and
lower case letters indicate 2000 data. Values are expressed as means, error bars are $1
s.e. Bars not sharing a letter are significantly different from each other at P<0.05.

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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27

Objective 2: To determine if hybrid pupae of P. glaucus and P. canadensis have
significantly different weights.

In 1999 and 2000 there was a significant effect of genotype on pupal weight at
P<0.0001/P<0.0001 (tables 5 and 6). In 1999 there was a significant effect of host
plant (P<0.0001) on pupal weight (Table 5).

In general, when larvae were fed black cherry, the hybrids produced pupae that
were as large as the parental types. The only exception was in 2000 at 23°C and 31°
C , Pch had significantly small pupae than P. glaucus (Figure 7, 8, and 9).

On quaking aspen, across all temperature treatments (15°C, 23°C and 31°C),
there was only one case were a hybrid produced pupae that were significantly smaller
than the surviving parental type (Figure 7, 8, and 9). Pch’s pupae, in 2000, were at
least .109g small than P. canadensis pupae (Table 7).

By and large, when larvae were fed tulip tree they produced pupae that were
statistically similar is size to at least one of the parental types. Pch, in 1999 at 15°C,
and in 2000 at 23° and 31°C, produced pupae that were significantly smaller than
P. glaucus (Figure 7, 8 and 9). In all other cases, (except one, Pch pupae in 1999 at
31°C were significantly larger than the surviving parental type: P. glaucus) there were
no differences in pupal weights of the hybrids compared to the parental types.

Even though there were three cases where in one year hybrids produced pupae
that were significantly smaller than the surviving parental type (tulip tree 15°C, tulip
tree 31°C and quaking aspen 23° ) this did not occur in both years and the other paren-
tal type did not have any larvae that survived at those treatment levels. In all other

cases hybrids produced pupae that were statistically the same or larger as at least one

28

of the parental types.

29

Table 5: F-values (Type 111 SS) from ANOVA of 1999 pupal weight analysis.

P<.05. The model was a split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F- value for tem-
perature Block*Temperature is used as the error term

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sources of Variation DF SS MS F Pr<F
Block 1 .001 _ .0001 0.06 .8058
Temperature 2 _____ 0.54 0.27 8.40 .1063
one. * Temperature ...... 2 M“0.306%“-W0.03 1.38 M0725“
Genotype "W W T 3 ‘ 16605523670000]
”115.51.; FT “W“ T _ 2 _ ”“3758" 0.29 12.35 00001 T
Genotype * Hostplant ”I __._ T's—T 0.35 0.07 2.99 0.01
Temperature * Genotype” 6 ..,, "“3"”-“0.05—2mm" 0.008 0.37m"”0.90"Tm"
Temperatures Hoopla}? " 4' M amt—3.17m“---u"01'04'WW'1387*"a 6MB -
Temperature; Genotype * 8 0.14 0.02 0.76 0.64m
Hostplant

 

 

Table 6: F-values (Type III SS) from ANOVA of 2000 pupal weight analysis.

P<.05. The model was a split plot design with temperature as the whole plot and
genotype and hostplant as the split plots. To obtain the correct F - value for tem-
perature Block*Temperature is used as the error term

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 
  
  

 

 

 

    
   
 

 

 

 

 

 

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Sources of Variation DF SS MS F Pr<F
£1355 _ 1 0.002 0.001 0.09 011—1
Temperature 2 1.89 0.94 645.79 0.002
3163;:Te‘mfiperatgure ., 2 0.002 0.001 0.07 0794mi
”Genotype ,_. 3 _. 0.51 0.16 7.54 30.30001“
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Témpuature * Hostplant mam—W; 0.09 30.02 1.00 . “041‘ T
. impugn“ * Genotype *WMJTT8 T m 011WMWWO.01 0.6 0.77% I

 

30

 

 

 

 

 

 

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1,60 _ Black Cherry 15 C

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160 . Quaking Aspen 15°C

1.40 -
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1.00 -
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0.40 _
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(Grams)

 

 

 

Average Pupal Weight

1.60
1.40 l
120.
1.00 -
0.80 -
0.60 -
0.40 .
0.20 .
0.00

 

 

 

 

 

 

 

 

 

PC PCPG PGPC pa

Figure 7: For average pupal weight at 15°C. All comparisons are made within a

treatment group and within a year. Each graph represents a treatment group. White

bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data- Values are expressed as means, error bars are :1 s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

31

 

I 60 Black Cherry 23°C

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1.20 -

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1.60 1

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(Grams)

 

 

   

 

 

 

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Average Pu pal Weight

Tulip Tree 23°C
1.60 .

1.40 _
1.20 e
1.00 .
0.80 .
0.60 'i
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0.20 -

abA

   

 

 

 

 

 

 

PC PC PC PGPC PG

Figure 8: For average pupal weight at 23°C. All comparisons are made within a

treatment group and within a year. Each graph represents a treatment group. White

bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data. Values are expressed as means, error bars are i] s.e. Bars not sharing a
letter are significantly different from each other at P< 0.05.

32

1 60 Black Cherry 31°C

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Average Pupal Weight

° 0
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Figure 9: For average pupal weight at 31°C. All comparisons are made within a

treatment group and within a year. Each graph represents a treatment group. White

bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data. Values are expressed as means, error bars are :1 s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

33

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

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34

Objective 3: To determine if hybrid larvae of P. glaucus and P. canadensis have
significantly different growth rates from hatch to pupation than parental type
larvae.

In 1999 and 2000 there was a significant effect of temperature (P<0.0046/
P<0.013I), genotype (P<0.0001/P<0.0006) and hostplant (P<0.0001/P<0.0001) on
growth rate (Tables 8 and 9).

In 1999 and 2000, of the larvae that survived to pupation at 15°C, there were
no significant differences in growth rate, at any level of the host plant treatments
(black cherry, quaking aspen and tulip tree), between the hybrids and the parental
types (Figure 10).

In general, at 23°C they hybrid’s growth rates, across all host plant treatments,
Were the same or faster than the surviving parental type larvae (in 1999 and 2000
P. glaucus did not survive to pupation on quaking aspen) (Figure 11). However, in
2000 on black cherry and in 2000 on quaking aspen and tulip tree, Pch’s growth rates
were significantly slower than one of the parental types (Figure 11).

In both years on black cherry at 31°C Pch did not differ statistically from
P. glaucus and in 2000 it did not differ from P. canadensis (Figure 12). In 1999, Pch
grew an average of at least 0.012 g/day faster than P. canadensis (Table 10). In both
years Pch had a significantly slower growth rate than P. glaucus but grew at the same
rate or faster than P. canadensis.

In both years, on quaking aspen at 31°C, the surviving hybrid larvae did not
differ statistically in growth rate from P. canadensis.

Even though in 2000, on tulip tree at 31°C, Pch and Pch had significantly

35

Table 8: F -values (Type 111 SS) from ANOVA of 1999 growth rate analysis.
P<0.05. The model was a split plot design with temperature as the whole plot
and genotype and hostplant as the split plots. To obtain the correct F- value for
temperature Block*Temperature is used as the error term

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sources of Variation DF SS MS F - Pr<F
Block 1 0.000001 0.000002 .05 0.83
re;;;;.};};m' -...-- 2 0.02 00.01 217.67 0.004%
BIockilWrempeer—oremwiW"m 2 ”_0—.0001W070001 1.28 ___ 028*“
_ Genotype " 3 0.001 0.001 12.87 0.0001 _
”1423;157:131“ MW" W“ 2 W 0004 ""'0.'002“'—""-"'1.5707”m M00001 W
"Gaotype * Hostplant-WMWw5” M01001“ 0.0002 4.65m“ii—M00005“ *—
Temperature * Genotype 6 A 0.001 0.0001 2.48 0.02—m
Temperature * Hostplant 4 0.001 “0.0004 8.45 ___ 0.0001
”Temperature * GZIOtype * "MM—"MWS “a- —0‘0004 00000] D 126 027
Hostplant

 

 

Table 9: F-values (Type III SS) from ANOVA of 2000 growth rate analysis.
P<0.05. The model was a split plot design with temperature as the whole plot
and genotype and hostplant as the split plots.. To obtain the correct F- value for
temperature Block*Temperature is used as the error term

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sources of Variation DF SS MS F Pr<F
Block 1 0.0001 0.0001 1.37 0.24
Temperature 2 0.03 0.02 75. I 2 0.01
Block * Temperature-“MW F I 2 0.0004» 0.0002 3.36 N 0.04
Genotype h -____3.__. 0.001 0.0004 ------Eh-.- ' 0.0006
Hostplant ‘2” 0.003 0.001 20.98 0.0001
Genotype * Hostplant ‘5“ ._ 0.001 0.0003 4.29 0.001
Temperature * Genotype 6 0.001 I J ' 0.0002 3.56 0.003
Te;;,;r;ture * Hostplaiit W“ .4 0.001 Vin—00002.“— E40 0.01
\_.— - m m... W--- ..-..._ ---... __________._-.. -..-
Temperature * Genotype * 8 0.0003 0.00003 0.60 0.78
Hostplant

 

 

   

36

slower growth rates than P. glaucus, in 1999, they had a statistically identical or faster
(Pch had a faster growth rate than P. glaucus of at least 0.01g/day) growth rates than
the parental types.

When hybrids are compared to their parental type they did not show any reduc-
tion in growth rate. Furthermore, across all treatment levels of host plant and tempera-

ture, the hybrids had a growth rate that was as faSt or faster than at least on of the

parental types.

37

 

0 10 Black Cherry 15°C

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0.09 A
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Average Growth Rate
(Grams/Day)

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Figure 10: For average growth rate at 15°C. All comparisons are made within a

treatment group and within a year. Each graph represents a treatment group. White

bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data- Values are expressed as means, error bars are :tl s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

38

Average Growth Rate

(Grams/Day)

 

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Figure 11: For average growth rate at 23°C. All comparisons are made within a
treatment group and within a year. Each graph represents a treatment group. White
bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data. Values are expressed as means, error bars are :21 s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

39

 

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PC PCPG PGPC PG

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Figure 12: For average growth rate at 31°C. All comparisons are made within a
treatment group and within a year. Each graph represents a treatment group. White
bars represent means from 1999 and black bars represent means from 2000. Capital
letters for significant differences indicate 1999 data and lower case letters indicate
2000 data. Values are expressed as means, error bars are i1 s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

40

 

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Objective 4
I
nificantly d

Peru
Krusl'al-llu
1990}-

We

 

 

 

 

sun'ired as
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ferences we
in W99; 7}
at 31°C (7.:
ln 1
P- glaucus
3000.016 '
313ClFigu
On
than the h
tree sun“
Ever}- treat

Objective 4: determine if hybrid larvae of P. glaucus and P. canadensis have sig-

nificantly different survival rates to pupation than parental type larvae.

Percent survival until pupation data was tested for significant differences with a
Kruskal-Wallis Chi-Square test (Proc npariway wilconxon anova: SAS Institute Inc.
1990).

When fed to mutually-acceptable host plant black cherry, both hybrid types
survived as well as both parental types in all temperature treatments. Except for lower
survival of P. canadensis at 23°C in 1999 ()8, P>0.02) (Figure 14 ) no significant dif-
ferences were observed between the four genotypes of butterflies at 15°C ()8, P>O.66
in 1999; x2 , P> 0.28 in 2000, Figure 13) , at 23°C (17-, P>0.08 in 2000, Figure 14) nor
at 31°C ()8 , P> 0.63 in 1999; x2 , P> 0.1 in 2000, Figure 15).

In general, on quaking aspen, hybrids survived as well as P. canadensis and
P. glaucus at all temperatures in both years (Figures 13, 14 and 15). However, in
2000, the Pch hybrid larvae had a significantly lower survival than P. canadensis at
31°C(Figure 15).

On tulip tree at 15°C and 23°C, P. canadensis survival was significantly lower
than the hybrids in 1999 but not at significant levels in 2000. Hybrid larvae on tulip
tree survived at numerically higher levels than both P. canadensis and P. glaucus in
every treatment (15, 23, 31°C) but in no case did these levels of hybrid survival reach

statistical significance (Figures 13, 14, 15; table 11)

42

 

 

Percent Survival to Pupation

 

Figure 13: {

 

Black Cherry 15°C

100%

 

PC PCPG PC PC PC

Quaking Aspen 15°C

100%
00%
80%
70%
60%
50%
. 40% .
30%
20%
10%
0%

 

PC PCPG PGPC PG

Percent Survival to Pupation

 

PC PC PC PC PC PC

Figure 13: Percent survival at 15°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant
differences indicate 1999 data and lower case letters indicate 2000 data. Values are
expressed as an average of the family means, error bars are :tl s.e. Bars not sharing
a letter are significantly different from each other at P<0.05.

43

.Perccnt Survival to Pupation

l

gIOuP and \K

 

 

 

I00°/o 1
90% l
“”6-
70'/o 4
6070 -l
flné-
4070 -

. 3070 -
20%»-
1095+

 

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lMHfi-
9070 -
8070 -
WR6J
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“”64
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10934

070 4

 

Percent Survival to Pupation

lMHfi-
“”6-
80%»«
una-
“H6-
5H6
4W%
MH6«
2W%~
1W%u

L4

 

096«

Figure 14: Percent survival at 23°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant
differences indicate 1999 data and lower case letters indicate 2000 data. Values are
expressed as an average of the family means, error bars are i] s.e. Bars not sharing

 

 

PC

 

 

PCPG

PCPG

 

PC

PCPG

 

Black Cherry 23°C

a l

ab

  

 

PGPC ' PG

Quaking Aspen 23°C

 

 

 

a C
rope I PG
Tulip Tree 23°C

 

 

a letter are significantly different from each other at P<0.05.

44

' tion
’upa
ival to l
' rv
t Su
l’crccn

Figure 15: pl
121011;) and 11:
Sam Percent __
yallles are fl
”ferences 1'!
expressed as
me’ are si

7
9i
k

 

 

 

Black Cherry 31°C

100% .
90% . A
80% .
70%
60%
50% .
40%» <
30% .
20%
10% .

0% l

 

 

 

 

 

PCPG PGPC PG

Quaking Aspen 31°C

100% -
90% «
80% <
70% <
60%

L

 

 

 

 

Percent Survival to Pupation

 

50% .
40% l
30% l
20% .
10% 1 B a B
070 -l 1
PC PCPG PGPC PG
Tulip Tree 31°C
10070 -
90% .
8070 «l
70% .
60% .
50% .
40V. -
80%1
20% . A
10% .
0% a CF" .

 

 

Figure 15: Percent survival at 31°C. All comparisons are made within a treatment
group and within a year. Each graph represents a treatment group. White bars repre-
sent percent survival from 1999 and black bars represent percent survival from 2000.
Values are expresses as percent survival to pupation. Capital letters for significant
differences indicate 1999 data and lower case letters indicate 2000 data. Values are
expressed as average of the family means, error bars are $1 s.e. Bars not sharing a
letter are significantly different from each other at P<0.05.

45

 

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Cant reduction

Consequ‘mm

Three K

 

 
   
  

toms: 3) fusi

 

DISCUSSION

This study was an attempt to compare the fitness of parental type and hybrid
larvae of P. glaucus and P. canadensis Swallowtail butterflies under different thermal
and host plant regimes. I looked at four indicators of fitness: number of degree days
from hatch to pupation, pupal weight, growth rate and percent survival until pupation.
Over the two year period of this study, I did not observe any evidence for hybrid larvae
performing more poorly than both their parental types. In fact, hybrids of both types
performed at least as well as one parent and sometimes better than both parents.

It is clear that pairings between P. canadensis and P. glaucus parental types
produced healthy and viable larvae. Hybrids were able to survive on host plants that
their parental types did poorly on. P. canadensis larvae have low survival when fed
tulip tree (Liriodendron tulipfera) and P. glaucus have low survival on quaking aspen
(Populus tremuloides) (Hagen et al., 1991). Hybrids consistently produced pupae that
were well within the normal size range for both species and their growth rates also fell
well within the range of their parental types. Finally, hybrids don not show a signifi-

cant reduction in survival of larvae to pupation.

Consequences of Hybridization

Three commonly described consequences of hybridization are : l) reinforce-
ment of Speciation due to the evolution of pre-mating barriers to gene exchange in re-
sponse to selection against the hybrids; 2) extinction of one or the other parental

forms; 3) fusion of the species (Harrison 1993, Rhymer and Simerloff 1996, Arnold

47

P_ gz’J.
517011460
P, curzuc
94.2% 01
ot‘interac
Sn
looked at 1
tion durati<
shoxm that '
3000). when
dance for co
females mate
found that SO]
Others Used 3F
mates (Stump‘
The fix

the Parental I}.

 

lilil'OdUCed (RE

  
  

and/9

‘ C'anaa’L

(figure 1).

Inc0m ,

1997)

To date evidence of complete prezygotic barriers in the P. canadensis and
P. glaucus hybrid complex have not been found (Deering, 1998). In 1998, Deering
showed, through field tethering studies, that wild males of both P. glaucus and
P. canadensis prefer females of P. glaucus. P. glaucus preferred conspecific females
94.2% of interactions and P. canadensis males preferred heterospecific females 82.3%
of interactions (Deering, 1998).

Stump (2000) found no evidence for postpairing prezygotic. barriers. He
looked at four indices of success for heterospecific and conspecific matings: copula-
tion duration, spermatophore deposition, oviposition and egg hatchability. It was
shown that their was no reduction in any of the four factors of pairing success (Stump,
2000), when females were mated once. He went on to show that that their was no evi-
dence for consistent conspecific sperm precedence in P. canadensis or P. glaucus in
females mated to both a conspecific and a heterospecific male (Stump, 2000). It was
found that some females continued to exclusively use sperm from the original mating
others used sperm from the second mate exclusively and some used sperm from both
mates (Stump, 2000).

The extinction of one of the parental types usually occurs in cases where one of
the parental types is rarer than the other and the and the species that is “taking over” is
introduced (Rhymer and Simberloff 1996). This in not the case with the P. glaucus
and P. canadensis system. Both are native abundant species and have vast ranges

(figure 1).

incomplete prezygotic barriers and postzygotic barriers through the larval stage

48

may resuli
away as tl‘
press. Ryn‘.
It is

P. glaucus
P. multicau
“proto” P. (2’
continuous r
they may hat
the Beringial
It is knovm.
”EMU/01.4153
The i

Fears B.P_) “
ability to de
1938) to be s
qufiking 35m

P' glaucus. I

 

What are no“
With
0” Iul

1p tree (

Seem ‘0 be r

 

3001b, Scn'b

 

may result in the two taxa diffusing into each other with their unique characters fading
away as their trait clines “decay” and become broader or less steep (Scriber 2001a in
press, Rymer and Simberloff 1996; Porter et al., 1997).

It is thought that the North American P. glaucus group, which is comprised of
P. glaucus, P. canadensis, P. alexiares, P. eurymedon, P. rutulus, and
P. multicaudatus, are descendent from one common ancestor (Scriber 1.996). This
“proto” P. glaucus type, before the Pleistocene glaciations, was believed to have had a
continuous range through out North America. As the glaciers expanded southward
they may have spilt the proto P. glaucus type into two populations, one to the North in
the Beringial refugia (Alaska) and one into a refuge South of the Laurentide ice sheet.
It is known, though fossil records that populations of Populus balsamifera and Populus
tremuloides persisted in the Beringial refugia during the glaciation, 36,000 B.P.

The isolation of the Beringial population for 25,000 years (36,000 to 9,500
years B.P.) with only Salicaceae as an host plant would have been enough time for the
ability to detoxify allelochemicals produced by the Salicaceae host plant (Scriber
1988) to be selected for. This would explain the ability of P. canadensis to survive on
quaking aspen and the loss of the ability to survive on the current southern host of
P. glaucus, tulip tree. With the retreat of the glaciers we see a secondary contact of
what are now two species of butterflies: P. glaucus and P. canadensis.

With the increased frequency of P. canadensis near the hybrid zone surviving
on tulip tree over the last three years, other morphological and physiological traits that
seem to be moving northward, possibly as a result of global warming (Scriber et al

2001b, Scriber et a1 2001d) and the lack of strong prezygotic and postzygotic barriers

49

 

W5
8811‘

ends Ii
561161115
an Cm
{Rhymer

near this i
ments in e
Birch give
for Aa’apIal
expanding ll
parent that ti.
has not been

against the h)

each other’s g

P. canadensi.8‘ .

As a ca

tions in the hi

 

the parental IV

   
 
 

hat four nearl‘

Wing by din

we could be seeing the beginning of the two species fusing together.

Benefits of Hybridization

- Traditional wisdom has generally emphasized hybrids as evolutionary dead
ends for animal species e.g. Mayr (1963). However, it is important to recognize the
benefits that hybridization can play. Hybridization can also allow for rapid evolution-
ary change in- populations by creating novel and beneficial gene combinations
(Rhymer and Simberloff 1996, Lewontin and Birch 1966). Botanists have recognized
that this phenomenon may lead to increased fitness and adaptation to new environ-
ments in existing taxa (Rhymer and Simberloff 1966, Arnold 1997). Lewontin and
Birch give an insect example in their paper “Hybridization as a Source of Variations
for Adaptation to New Environments”. They cite the example of Dacus tryoni
expanding its range by hybridizing with the closely related D. neohumeralis. It is ap-
parent that the two species exchange genes through hybridization. The gene exchange
has not been great enough to merge the species, presumable because of selection
against the hybrids, but it has been enough to incorporate foreign species genes into
each other's gene pool (Lewontin and Birch 1966). This could be the case with
P. canadensis larvae being able to survive on tulip tree.

As a catalyst for the evolutionary process by the creating novel gene combina-
tions in the hybrids, hybrids can occupy and thrive in novel (disturbed) habitats were
the parental types were unable to survive. Anderson and Stebbins (1954) proposed
that four nearly concurrent events during the Cretaceous period: retreat of seas, over-

grazing by dinosaurs, the diversification modern like birds which transported seeds

50

long (115!
the diver
ated com
ing togetl
have actet
sperms.

It i'
sects. Wit

could also I

Future Res

Even
at the larva]
Funher int'es
determine “h
35 tit 35 their 1
105‘ setting,

the role of

    

lGregor}, and I
depend on exr:
might reveal I}:
not rEpleable I.

“76 Dot
I

 

long distances, and rise of flower pollinating bees and other insects all contributed to
the diversification and dominance of angiosperms. They propose that these events cre-
ated conditions favorable for hybridization. i.e. new habitat for colonization and bring-
ing together of species that were previously isolated. In this case, hybridization may
have acted as one of the fuels of evolution that led to the diversification of the angio-
sperms.

It is known that with the radiation of the angiosperms came the radiation of in-
sects. With these conditions fostering an ideal environment for plant hybridization, it

could also have been an ideal environment for insect hybridization.

Future Research

Even though this study did not show any reduction in fitness, in the laboratory,
at the larval stage, hybrids may show lower fitness at any life stage in the field.
Further investigation in the P. canadensis-and P. glaucus hybrid complex is needed to
determine what forces are at work in this system and whether or not hybrids are truly
as fit as their parental types. Research in this area has been primarily done in a labora-
tory setting. Laboratory studies have been useful in analyzing fitness of hybrids and
the role of prezygotic and postzygotic barriers in isolating closely related taxa
(Gregory and Howard 1993). However, they cannot look at components of fitness that
depend on external effects (Gregory and Howard 1993). Further research in the field
might reveal that the hybrids are being selected against by some factor that is
not replicable in the laboratory setting.

The possibility of hybrids being unfit at the pupal and adult stage needs investi-

51

gallor
P513114
:QQlC
acting
that 111

3 (am.

needs I‘
P, canaa
has im'eS
It
and backer
a cohort :11
within and
of all Classes
genetically a
several field 5
hybrids emerg

are the h't'hrid;

 
  

selected as m;

being selected

 

gation. Preliminary laboratory work has shown that hybrid pupal survival at high tem-
peratures (30°C, 33°C, 36°C) is greater for hybrids than parental types (Scriber et al
2001c). Even with this being true, reduction of fitness could manifest itself as hybrids
acting as a parasitoid/predator sink (West and Hazel 1982). It also has been shown
that there is a slight Haldane effect in the slightly higher mortality of P. glaucus x
P. canadensis hybrid female pupae (Hagen and Scriber 1995).

Whether or not hybrids are being selected as mates by either male parental type
needs to be looked at. An intriguing study by Deering (1998) showed that
P. canadensis males prefer P. glaucus females 82.3% of the time but no study to date
has investigated male selection of hybrids as mates.

It has been determined, through electrOphoretic analysis that natural hybrids
and backcrosses do exist but at unknown natural frequency and fitness. I suggest that
a cohort analysis, similar to the Howard et a1 (1993) study, of Tiger Swallowtails
within and through the hybrid zone be undertaken to begin to characterize the fitness
of all classes of hybrids in a field setting. This would involve repeatedly sampling and
genetically analyzing different populations within the hybrid zone over the span of
several field seasons. A cohort analysis would begin to answer such questions as: Are
hybrids emerging synchronously with their parental counterparts? At what frequency
are the hybrids present? What kinds of genetic classes are present? Are hybrids being
selected as mates by heterospecifics and conspecifics or other hybrids? Are hybrids

being selected against by unknown forces?

52

APPENDICES

53

APPENDIX 1

RECORD OF DEPOSITION OF VOUCHER SPECIMENS

54

The speci

seumts) a

Voucher
fluid-pres

Voucher I

Title of th

 

 

Are Hybr‘
A Test L':
Papilio g
("LCpidop
Museum

E

(

* REfe
AmEr

DCDC

S\a

Appendix 1
Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in the named mu-
seum(s) as samples of those species or other taxa, which were used in this research.
Voucher recognition labels bearing the Voucher No. have been attached or included in
fluid-preserved specimens.

Voucher No.: 2001-04

 

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

Are Hybrids More Fit Than Their Parental Types?

A Test Using Two Tiger Swallowtail Butterfly Species

Papilio glaucus and P. canadensis

(Lepidoptera: Papilionidae)

Museum(s) where deposited and abbreviations for table on following sheets:
Entomology Museum, Michigan State University (MSU)
Other Museums:

Investigator’s Name(s) (typed)

Jennifer Donovan

 

 

 

Date 7/23/2001

 

*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.

55

 

APPENDIX 1 .1

VOUCHER SPECIMENT DATA

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58

Appendix 1.1

Voucher Specimen Data

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Appendix 1.1
Voucher Specimen Data

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Voucher Specimen Data

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61

Appendix 1.1

Voucher Specimen Data

 

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APPENDIX 2

FAMILY 15116

63

In 1999 one P. canadensis female was caught in Charlevoix county, MI. Her
offspring were used as one of the P. canadensis families in the multiple indicators of
fitness study presented in this thesis. Her off spring demonstrated high survival on Tu-
lip Tree, a known poor host of Canadensis larvae and direct development. P. canaden-
sis are obligate diapausers. It was suspected that her offspring were actually hybrid
type not parental type. To confirm suspicions, diagnostic allozymes were rtm on the
mother and her offspring. The results confirmed that female 15116 was a pure paren-
tal type P. canadensis and her offspring were hybrids. This has led us to believe that
this is evidence of field mating between a heterospecific species. i..e. P. canadensis
female was mated to a P. glaucus male in the field (Donovan et al 2001).

Consequently, P. canadensis female 15116’s offspring were eliminated from
the analysis that compared multiple indicators of fitness.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INDIVIDUALS RUN HK PGD LDH
15116 male offspring 110 ? -125/-100 80/100
15116 male offspring l 10? ~125/-100 80/100
15116 female offspring l 10? -100 100
15116 female offspring 100/1 10? - 100 100
15116 female offspring 1 10? -100 100
P. glaucus control 100 -100/-50 100
P. glaucus control 100 - l 00 100
P. canadensis control 110 -125 80
15116 female mother 110 -125 80?
15116 female offspring 100/ 1 10? -100 100
15116 female offspring 100/1 10? - 100 100
15116 female offspring 100/1 10? - l 00 100

 

 

 

 

 

Table 12: Diagnostic allozymes ran to confirm genotypic
identity of putative P. canadensis female mother 15116 and
her offspring. Question marks indicate that resolution was
difficult but best guesses were made.

64

APPENDIX 3

ALLOZYME ELECTROPHORSIS ANNAYLSIS OF PARENTS

65

 

Individual parents of all families used in these studies were characterized
through analysis of two diagnostic enzyme loci: 6-phosphogluconate dehydrogenase
(PGD) and lactate dehydrogenase (LDH). In cases where the allozymes could be visu-
alized parental origin was as expected based on morphological characters (Table 3).

All parental butterflies and subsequent offspring were frozen at —80°C. Al-
lozyme electrophoresis protocols followed Hagen and Scriber (1991). Specimens were
prepared by grinding the proximallhalf of the abdomen or part of the thorax for fe-
males (using the proximal half of the abdomen or part of the thorax avoided including
spermatophore proteins from male mates) or distal half of the abdomen of males in
IOOuL buffer (0.1M tris, 1.07M EDTA, 0.15mN NAD, .l3mM NADP, 35.75mM 2-
mercaptoethanol, pH 7.0). After grinding, the specimens were centrifuged for 10 min-
utes at 12,000 x g. Allozymes were separated by electrophoresis on thin layer cellu-
lose acetate plates. We then stained for the enzymes LDH and PGD.

Formulae for enzyme stains followed Richardson et a1. (1986). Gel scoring
protocols were after Hagen and Scriber (1991). Allozymes were scored by assigning
the origin (where samples were applied) a score of ‘0’ and the most common allozyme
a score of “100”. All other allozymes were assigned a score relative to their location
in relation to the origin and the most common allozyme. .P glaucus’ PGD allozyme
scores a -100 with a rarer allozyme at —50 and P. canadensis scores at —125 and a rarer
allozyme at —80 (Hagen and Scriber 1991). The LDH allozyme scores at 100 for P.
glaucus. P. canadensis scores at 80 and a rarer allozyme at 40. All specimens were
run twice to confirm allozyme types. Every plate was run with controls: a previously

scored P. glaucus and P. canadensis.

66

 

 

 

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69

APPENDIX 4

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71

APPENDIX 5

NUMBER OF LARVAE THAT SURVIVED TO PUPATION

 

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73

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74

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Pupation Site in Swallowtail Butterflies. Evolution. 36(1): 152-159.

79

IIIII

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177 42