of the requirements for the degree of. Master ... requirements for a master's degree at Montana State University, I ..... and mineral oil added to the top of the tube.
Genetic relationships between white-tailed deer, mule deer and other large mammals inferred from mitochondrial DNA analysis by Matthew Anthony Cronin A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Montana State University © Copyright by Matthew Anthony Cronin (1986) Abstract: Restriction enzyme analyses of mitochondrial DNA (mtDNA) of six artiodactyl species and two bear species were employed to estimate genetic divergence between groups. Estimates of base substitutions per nucleotide were 0.000-0.008 for intra-species, 0.058-0.085 for intra-family, inter-species and 0.113-0.198 for inter-family comparisons. From these, estimates of divergence time between taxa were made which were generally consistent with estimates from the fossil record for species which diverged less than 5 million years ago. My estimates for species with older divergence times are probably underestimates. Comparisons of white-tailed deer and mule deer in Montana revealed species specificity of serum albumin and mtDNA and a probable low level of inter-species hybridization. Despite the species specificity of mtDNA, genetic divergence estimates between the species in Montana ate of the same magnitude as intra-species comparisons of other groups suggesting introgression of white-tailed deer mtDNA into mule deer populations in the past.
GENETIC RELATIONSHIPS BETWEEN WHITE-TAILED DEER, MULE DEER AND OTHER LARGE MAMMALS INFERRED FROM MITOCHONDRIAL DNA ANALYSIS
by M atthew A n th o n y C r o n i n
A t h e s i s subm itted in p a r t i a l f u lf il lm e n t o f th e requirem ents f o r th e degree of Master of Science in B io lo g ical Sciences
MONTANA STATE UNIVERSITY Bozeman, Montana May 1986
//Z T t C t> V
O fIi
APPROVAL
o f t h i s t h e s i s s u b m i t t e d by
Matthew Anthony C r o n i n
Approved f o r t h e M ajo r De p a rtm en t
Zt/
, /9 f ^
Date
Hea d, M ajo r D e p a r tm e n t
Approved f o r t h e C o l l e g e o f G r a d u a t e S t u d i e s
Date
“
ill
STATEMENT OF PERMISSION TO USE
In
p resen tin g
th is
th esis
in
p artial
fu lfillm en t
r e q u i r e m e n t s f o r a m a s t e r ' s d e g r e e a t M ontana S t a t e agree th a t
the
U n iv ersity ,
I
t h e L i b r a r y s h a l l make i t a v a i l a b l e t o b o r r o w e r s u n d e r
r u le s of the L ib ra ry . without
of
special
B r i e f q u o t a t i o n s f ro m t h i s t h e s i s a r e a l l o w a b l e
perm ission,
provided
that
a c c u r a t e a c k now ed gm en t o f
s o u r c e i s made. P e r m i s s i o n f o r e x t e n s i v e q u o t a t i o n f ro m o r r e p r o d u c t i o n o f t h i s t h e s i s may b e g r a n t e d b y my m a j o r p r o f e s s o r , o r i n h i s / h e r a b s e n c e , by t h e D i r e c t o r o f L i b r a r i e s when, i n t h e o p i n i o n o f e i t h e r , t h e p r o p o s e d use of the m a te ria l i s
fo r sc h o la rly purposes.
Any c o p y i n g o r u s e o f
t h e m a t e r i a l i n t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t be a llo w e d w i t h o u t my w r i t t e n p e r m i s s i o n .
Signature Date
iv
ACKNOWLEDGEMENTS
Thanks a r e e x t e n d e d t o Dr s. Dave Cameron and E r n i e Vyse f o r t h e i r support
and
P alm isciano, H am lin,
S.
W ild life
frien d sh ip L.
E llig ,
R iley,
and
and
Parks
d u rin g D.
Pac,
others
assisted
th is R.
M u le ^
w ith in
stu d y . R.
the
Dr.
R.
B ucsis,
M ontana
o b taining
M ackie , G.
Dusek,
D ept,
sam ples.
of
D. K.
Fish,
In a d d itio n
a s s i s t a n c e w a s g i v e n by D. J o n e s a n d J . B a t e s , U t a h D i v . o f Game, C. G ates,
N orthw est T err.
C anadian W i l d l i f e
D e p t , o f R e n e w a b l e R e s o u r c e s , H. R e y n o l d s ,
Service,
C. B l y t h a n d t h e s t a f f a t
Elk Is.
N atl.
P a r k , L. R e n e c k e r , The U. o f A l b e r t a , E. R o l l e r , The U. o f G e o r g i a , S. T a y l o r a n d H. R a n d y , M i n e r A g r i c u l t u r a l I n s t i t u t e a n d
D. W o r l e y , A.
Wood, B. Compton, S. J a c k s o n and S. Denson, Montana S t a t e U. Many h u n t e r s c o n t r i b u t e d t i s s u e s a m p l e s i n c l u d i n g D. W i l l i a m s , E. A r n e t t , R. K a h l e n b e c k , S. A t h e r t o n , R. W h i t e , W. N i e h o f f , J . R a t y , R. Fox,
W. S c h r a d e r ,
Parker,
S. F r e y ,
M. E a r n h a r d t , D. a n d S. C o l l i n s ,
Hogan,
J.
V ore,
J.
D ouglas,
C. K a y a , L. G o l d y , D. W h i t e , D.
H a n s e n , W. Romek, R. S i r o k y a n d D. D e s p a i n .
J . C h a v e z , S. W a t e r s , T.
B l a k e , N. B l a k e a n d S. Ch a o p r o v i d e d t e c h n i c a l a s s i s t a n c e . C arr provided d a ta A lb erta deer.
J.
Steve
f o r T e x a s d e e r a n d V. G e i s t a n d W. W i s h a r t f o r
D r . M. W i l s o n a n d J .
H o r n e r g a v e e n c o u r a g e m e n t and
useful suggestions. I'd Cronin,
especially
lik e
to
t h a n k my p a r e n t s , T h o m a s a n d C o r i n n e
a n d my w i f e V i c k i e a n d d a u g h t e r
encouragement.
C olleen
for
prayers
and
V
TABLE OF CONTENTS
Page ACKNOWLEDGEMENT S ...............................................................................................................
iv
LIST OF TABLES................................................
1±
LIST OF FIGURES.......................................................................... ABSTRACt...........................................
v lli i
INTRODUCTION....................
^
MATERIALS AND METHODS........................................
g
Sample c o l l e c t i o n ..........................
g
Albumin e l e c t r o p h o r e s i s .................................... .............................................
g
M i t o c h o n d r i a l DNA p u r i f i c a t i o n .................................................................
10
D i g e s t i o n an d i d e n t i f i c a t i o n o f mtDNA f r a g m e n t s ........................
H
E s t i m a t i o n o f g e n e t i c d i s t a n c e and d i v e r g e n c e t i m e .................
13
Sam ple s a n a l y s e d .........................................................
14
I n t e r - s p e c i e s c o m p a r i s o n s .....................
14
Mule d e e r —w h i t e - : t a i l e d d e e r c o m p a r i s o n s ................................
14
RESULTS................ C om pa ris on o f a l l s p e c i e s .............................................
15 15
Frag ment p a t t e r n s ...........................................
15
D i v e r g e n c e e s t i m a t e s - ...........................................
21
M u l e - d e e r - w h i t e - t a i l e d d e e r c o m p a r i s o n s . ..........................................
24
Serum a l b u m i n p h e n o t y p i n g ...............................................
24
M i t o c h o n d r i a l DNA a n a l y s i s . . . . .....................
26
vi Page DISCUSSION........................................
30
I n t r a - s p e c i f i c c o m p a r i s o n s .......................
31
I n t r a - f a m i l y , i n t e r - s p e c i e s c o m p a r i s o n s ....................................
32
Mule d e e r - w h i t e - t a i l e d d e e r c o m p a r i s o n s .................................
33
C O N C L U S I O N S ................................
38
LITERATURE
40
C I T E D ....................................
A P P E N D I X .....................................................................................................................
47
vii
LIST OF TABLES
Table
Pa g e s a m p l e d ......... ..................
10
2.
M i t o c h o n d r i a l DNA r e s t r i c t i o n f r a g m e n t p a t t e r n s ........................
17
3.
Number o f b i s o n from v a r i o u s h e r d s a n a l y s e d w i t h d i f f e r e n t r e s t r i c t i o n e n z y m e s ....................................................................
19
4.
V a r i a b l e mtDNA d i g e s t i o n p a t t e r n s i n d e e r . .................................. ..
20
5.
C om p os ite mtDNA p h e n o t y p e s i n d e e r ........................................................
20
6.
Estim ated g e n e tic d is ta n c e i n base s u b s t i t u t i o n s per n u c l e o t i d e ( p ) f o r mtDNA.............................................................................,
21
D i v e r g e n c e t i m e s c a l c u l a t e d from p v a l u e s and e s t i m a t e d f ro m t h e f o s s i l r e c o r d ...................................................
23
Numbers o f m o r p h o l o g i c a l a n d s e r u m a l b u m i n t y p e s o f d e e r from a l l s a m p l e d l o c a t i o n s . ....................................................
24
M o r p h o l o g i c a l , se rum a l b u m i n and mtDNA t y p e s f o r d e e r . . ; . .
28
1.
7.
8.
9.
S pecies,
s u b s p e c i e s and l o c a t i o n s
viii
LIST OF FIGURES
Figure
Page.
1.
P h o t o g r a p h o f Sac I and Cla I d i g e s t i o n p a t t e r n s ......................
16
2.
P h o t o g r a p h o f serum a l b u m i n p h e n o t y p e s f o r d e e r ..................
25
3.
Com po sit e mtDNA d i g e s t i o n p r o f i l e s f o r d e e r .......................
27
4.
Map sh owing g e o g r a p h i c d i s t r i b u t i o n o f mtDNA t y p e s ...................
29
I
ix
ABSTRACT
R e s t r i c t i o n enzyme a n a l y s e s o f m i t o c h o n d r i a l DNA (mtDNA) o f s i x a r t i o d a c t y l s p e c i e s and two b e a r s p e c i e s w e re em p lo y ed t o e s t i m a t e g e n e tic d iv e rg e n c e betw een groups. E stim a te s of base s u b s t i t u t i o n s p e r n u c l e o t i d e w ere 0.000-0.008 f o r i n t r a - s p e c i e s , 0.058-0.085 f o r i n t r a - f a m i l y , i n t e r - s p e c i e s and 0 .113-0.198 fo r in te r - f a m i ly comparisons. From t h d s e , e s t i m a t e s o f d i v e r g e n c e t i m e b e t w e e n t a x a w e r e made w h i c h w e r e g e n e r a l l y c o n s i s t e n t w i t h e s t i m a t e s fro m t h e f o s s i l re c o rd f o r s p e c ie s w hich d iv e rg e d l e s s th an 5 m i l l i o n y e a rs ago. My e s t i m a t e s f o r s p e c i e s w i t h o l d e r d i v e r g e n c e t i m e s a r e p r o b a b l y underestim ates. C o m p a ris o n s o f w h i t e - t a i l e d d e e r and m u le d e e r i n M ontana r e v e a l e d s p e c i e s s p e c i f i c i t y o f se rum a l b u m i n and mtDNA and a p r o b a b l e low l e v e l o f i n t e r - s p e c i e s h y b r i d i z a t i o n . D e s p ite the s p e c ie s s p e c i f i c i t y o f mtDNA, g e n e t i c d i v e r g e n c e e s t i m a t e s b e t w e e n t h e s p e c i e s i n Montana a t e o f t h e same m a g n i t u d e a s i n t r a - s p e c i e s c o m p a r i s o n s o f o t h e r g r o u p s s u g g e s t i n g i n t r o g r e s s i o n o f w h i t e - t a i l e d d e e r mtDNA i n t o m u le d e e r p o p u l a t i o n s i n t h e p a s t .
I
INTRODUCTION
B efore th e developm ent of m o le c u la r g e n e tic te c h n iq u e s in th e 1950's
the assessm en t
p o p u latio n s
felled
of v a r i a b i l i t y
w ithin
and
m a i n l y on t h e d e s c r i p t i o n
m orphological c h a ra c te r s.
M orphological t r a i t s
betw een n a tu r a l
and m e a s u r e m e n t o f are
usually re lia b le
f o r i n d e n t i f c a t i o n o f s p e c i e s and a r e t r a d i t i o n a l l y u s e d a t a l l l e v e l s of taxonom ic c l a s s i f i c a t i o n .
For some a n i m a l s , n o t a b l y D r o s o p h i l a
r o d e n t s and d o m e s t i c l i v e s t o c k ,
sPPm
c o n t r o l l e d b r e e d i n g s t u d i e s ha v e
allow ed e s tim a tio n of the h e r i t a b i l i t i e s of v a rio u s m o rp hological traits
such a s c o a t c o l o r o r body s i z e
(F alconer,
1964; L e w o n t i n ,
1974). To. a c c u r a t e l y a s s e s s
t h e g e n e t i c d i f f e r e n c e s w i t h in o r between
p o p u la tio n s knowledge of a l l e l e f r e q u e n c i e s o r n u c l e o t i d e m ust be a v a i l a b l e . pea
flo w er
co lo rs,
F o r some m o r p h o l o g i c a l t r a i t s , s u c h a s M e n d e l 's sin g le
a lle lic
d ifferen ces
re c o g n iz e a b le phenotypic d if f e r e n c e s . phenotypes i s
under the in flu e n c e of
determ inations are not possible. may f u r t h e r c o m p l i c a t e a n a l y s i s . effect
of
sequences
en v iro n m en t
on
correspond
to
H o w e v er, much o f o b s e r v a b l e polygenes
so s i m p l e
allelic
P l e i o t r o p h i c e f f e c t s o f s i n g l e genes A lso, in n a t u r a l p o p u la tio n s , the
p h en o ty p e
i n d i s t i n g u i s h a b l e fro m g e n e t i c e f f e c t s .
may
be
sig n ific a n t
and
F or ex a m p le Mayr (1970) n o t e s
t h a t B r i t i s h r e d d e e r ( C e r v u s e l a p h u s s c o t i c u s ) t r a n s p l a n t e d t o New Zealand a c q u ir e d th e phenotype of a d i f f e r e n t gen eratio n s.
This
general
lack
of a
Isl
race
w i t h i n one o r two
correspondence
betw een
2
g e n o t y p e a n d p h e n o t y p e p r e v e n t s e n u m e r a t i o n o f g e n o t y p e s and a l l e l e f r e q u e n c ie s f o r m ost n a t u r a l traits
are
p o p ulations
w he n o n l y m o r p h o l o g i c a l
studied.
For many e x t a n t and f o s s i l l a r g e mammals,
s p e c i e s and s u b s p e c i e s
d e s i g n a t i o n s h a v e b e e n b a s e d on v a r i a t i o n s i n t r a i t s co lo ratio n
or
horn
and
an tler
m orphology.
For
such as s i z e ,
exam ple
th irty
s u b s p e c i e s o f w h i t e - t a i l e d d e e r (O d o c o i l e u s v i r g i n i a n u s ) ( B a k e r , 1984) and e i g h t s u b s p e c i e s o f m u le d e e r ( 0 . h e m i o n u s ) ( W a lIm d , 1981) a r e recognized.
Knowledge o f i n t r a - s p e c i e s
m orphological v a r ia tio n
is
u s e f u l f o r s p e c u l a t i o n on t h e l o c a l a d a p t i v e n e s s o f t r a i t s o r g e n e flow
betw een
environm ental
areas
but
u su ally
a
q u a n tita tiv e
and g e n e t i c c o n t r i b u t i o n s
assessm ent
of
to observed v a r i a t i o n i s
not
possible. The u s e o f m o l e c u l a r g e n e t i c , t e c h n i q u e s
allow s q u a n tita tiv e
a n a l y s i s of a c t u a l g e n e ti c d i f f e r e n c e s between o rg an ism s, although n o t n ecessarily observed.
assessm ent
of
the
ad ap tiv e
value
of
the
v ariatio n
M or ph o lo gy , p h y s i o l o g y and e c o l o g y m u st a l s o be s t u d i e d
to
g a in i n s i g h t i n t o a d a p ti v e d i f f e r e n c e s between organism s. P r o t e i n e l e c t r o p h o r e s i s can d e t e c t d i f f e r e n c e s i n p r o t e i n s i z e o r c h a r g e c a u s e d by d i f f e r e n c e s i n a m i n o a c i d s e q u e n c e . sequence
is
determ ined
by n u c l e o t i d e
sequence,
S i n c e ami no a c i d these
p ro tein
v a r i a t i o n s c a n be i n t e r p r e t e d a s a l l e l i c d i f f e r e n c e s a t s p e c i f i c l o c i . Since i t s
first
L ew ontin
and
ap p licatio n
Hubby,
1966)
to p o p u la tio n g e n e tic s p ro tein
i n c r e a s e d our knowledge of g e n e t i c E xam ples i n c l u d e i d e n t i f i c a t i o n
(H arris,
electro p h o resis
variation in
natural
has
1966;
g reatly
populations.
o f population su b d iv isio n in w hite
3
t a i l e d d e e r ( S m i t h e t a I ., 1 9 8 4 ) a n d m o o s e ( A l c e s a l c e s ) ( C h e s s e r e t a l . , 1 982), and g e n e t i c d i f f e r e n c e s b e tw e e n s u b s p e c i e s o f red d e e r (Cervus
elap h u s)
(G y llen sten . et
m a c ro c h iru s ) (Avise e t a l . , to
id en tify
1984).
sp ecies-sp ecific
al.,
1983)
and
sunfish
(L e p o r o i s
E l e c t r o p h o r e s i s h a s a l s o be e n u s e d
form s
of various
p ro tein s
in bears
(W o lf e , 1983) and c e r v i d s (M cClymont e t a l . , 1 9 8 2 ), and to e s t i m a t e t h e g e n e t i c d i s t a n c e b e t w e e n s e v e r a l a r t i o d a c t y l s p e c i e s ( Baccus e t al.,
1983). R e c e n tly th e a n a ly s i s of n u c le ic a c id s has been a p p lie d to th e
s t u d y o f p o p u l a t i o n g e n e t i c s and taxonom y. For e x a m p le , A hlquist
(1983,
1984),
using
DNA-DNA h y b r i d i z a t i o n ,
S i b l e y and
have re v is e d
the
t axo no m y o f b i r d s and p r i m a t e s , e s t i m a t i n g r e l a t e d n e s s o f g r o u p s from s i m i l a r i t y o f c h ro m o s o m a l DNA s e q u e n c e . I n t h e l a s t t e n y e a r s v a r i a t i o n s i n m i t o c h o n d r i a l DNA (mtDNA) have
been
stu d ied
populations m olecule
to
assess
and s p e c i e s .
about
16-17
r e l a t i v e l y sm all s iz e ,
d ifferen ces
Mammalian mtDNA i s kilobases
mtDNA i s
(kb)
in
betw een
in d iv id u als,
a supercoiled
length.
Because
circular of
its
e a s i l y i s o l a t e d w i t h o u t b r e a k a g e and
t h e e n t i r e m o l e c u l e c a n be s u b j e c t e d t o a n a l y s e s .
Mammalian mtDNA i s
s t r u c t u a l l y an e x t r e m e l y c o n s e r v a t i v e m o l e c u l e co mp ar ed t o n u c l e a r DNA which has long non-coding o r d u p l i c a t i v e sequences.
Almost a l l of th e
m a m m a l i a n mtDNA m o l e c u l e c o n s i s t s o f f u n c t i o n a l c o d i n g s e q u e n c e s (A nderson e t a l . , 1981). There
is
substantial
evidence
that
mtDNA i s
homogeneous w i t h i n
i n d i v i d u a l s and m a t e r n a l l y i n h e r i t e d w i t h o u t m e i o t i c r e c o m b i n a t i o n v i a oocyte cytoplasm
(Avise e t a l.,
1979a,
1979b).
P o s s ib le exceptions to
4
th e s e te n e t s have been re p o rte d .
Coote e t a l . (1979) found s l i g h t
d i f f e r e n c e s i n mtDNA f r o m t h e l i v e r a n d b r a i n o f t h e s a m e ox ( B o s taurus).
H a u s w i r t h and L a i p i s
independently a r i s i n g
five
( 19 82 ) found a v a r i a n t mtDNA g e n o t y p e
tim es
Thes e a u t h o r s s u g g e s t t h a t
i n one m a t e r n a l l i n e a g e
of c a t t l e .
t h e two g e n o t y p e s i n one m a t e r n a l l i n e a g e
r e s u l t from i n t r a - i n d i v i d u a l h e t e r o g e n e i t y p o s s i b l y due t o p a t e r n a l in h eritan ce, segregation (1985)
m aternal
n u clear
gene
effects
on mtDNA o r
of m u l t i p l e genotypes w i t h i n an oocyte.
noted
h eterogeneity
in
individual
crickets
individual
h o m o g e n e i t y and m a t e r n a l
(Lansman e t a l . ,
(G r y l lu s ,) .
the
size
of
sim ply
H arrison
et
al.
mtDNA m o l e c u l e s
in
In m am m a lia n s t u d i e s , inheritance
however,
seem
to
in tra
be t h e r u l e
1981).
M i t o c h o n d r i a l DNA f r o m h u m a n s ( A n d e r s o n e t a l . , (Anderson e t a l . ,
1982) an d m ic e ( Mus) (B ib b e t a l . ,
1981), c a t t l e 1981) has been
c o m p le te ly se q u e n c e d , a l l o w i n g co m p a riso n s of e n ti r e m itochondrial genomes.
S e q u e n c i n g i s t i m e c o n s u m i n g and e x p e n s i v e , h o w e v e r , and n o t
necessary fo r ro u tin e population g enetic an aly sis.
Population stu d ie s
i n v o l v i n g mtDNA u s u a l l y i n v o l v e t h e i s o l a t i o n o f mtDNA f o l l o w e d b y treatm ent w ith r e s t r i c ti o n
endonucleases.
Thes e e n z ym e s r e c o g n i z e a
s p e c i f i c s e q u e n c e o f 4, 5 , o r 6 n u c l e o t i d e s a n d c l e a v e t h e m o l e c u l e each tim e th e sequence i s enco u n tered . v a r y i n num ber and s i z e cleavage s i t e s .
depending
Th e r e s u l t i n g DNA f r a g m e n t s
on t h e
number and
lo catio n
The DNA f r a g m e n t s a r e s e p a r a t e d on t h e b a s i s o f s i z e
by e l e c t r o p h o r e s i s and v i s u a l i z e d by s t a i n i n g . o r a u t o r a d i o g r a p h y . size
of
the
of
DNA f r a g m e n t s
can be e s t i m a t e d
by c o m p a r i s o n s
The w ith
s t a n d a r d s o f known s i z e and p a t t e r n s o f d i f f e r e n t o r g a n i s m s c o mp ar ed .
5.
A c o m p l e t e r e v i e w o f t h e t e c h n i q u e i s g i v e n by Lansman e t a l . The
type
of a n a ly s is
of d ig estio n
p attern s
p o p u l a t i o n s u r v e y s ( U p h o l t , 1977; Nei and L i , 1981) i n v o l v e s ,
the in d iv id u a ls
c o mp ar ed .
recommended
for
1979; Lansman e t a l . ,
f o r e a c h d i f f e r e n t enzyme d i g e s t i o n ,
f r a g m e n t s common t o
(1981).
i d e n t i f i c a t i o n of
The common f r a g m e n t s
a r e a s s u m e d t o be h o m o lo g o u s ( g e n e r a t e d by c u t s a t c l e a v a g e s i t e s s h a r e d by d e s c e n t ) . and from
th is
The p r o p o r t i o n o f s h a r e d f r a g m e n t s
is
calculated
th e number of base s u b s t i t u t i o n s p e r n u c le o t id e i s
e s t i m a t e d as an i n d i c a t i o n of g e n e ti c d i s t a n c e (Upholt,
1977).
Brown
e t a l . ( 1 9 7 9 ) a nd F e r r i s e t a l . ( 1 9 8 3 a , 1 9 8 3 b ) e s t i m a t e d t h e r a t e o f n u c l e o t i d e s u b s t i t u t i o n i n mtDNA t o be 2-4% p e r m i l l i o n y e a r s . this
rate,
Usi ng
d i v e r g e n c e t i m e b e t w e e n two m i t o c h o n d r i a l genomes c a n be
estim ated. A n a l y s e s o f mtDNA i n p o p u l a t i o n s o f m i c e (P e r o m y s c u s m a n i c u l a t u s , P. p o l i o n o t u s ) a n d p o c k e t g o p h e r s ( Georoys p i n e t i s ) ( A v i s e e t
al.,
1 9 7 9 a , 1 9 7 9 b ) h a v e r e v e a l e d l o w l e v e l s o f mtDNA d i v e r g e n c e i n l o c a l p o p u l a t i o n s and i n c r e a s i n g l e v e l s o f d i v e r g e n c e i n g e o g r a p h i c a l l y s e p a r a t e d p o p u l a t i o n s and b e t w e e n s p e c i e s .
Evidence o f i n t r o g r e s s i o n
o f mtDNA f r o m one s p e c i e s o r s u b s p e c i e s i n t o a n o t h e r h a s b e e n found i n m ic e ( Mus) ( F e r r i s e t a l . , 1 9 8 3 a ), d e e r (O d o c o i l e u s ) ( C a r r , p e r s o n a l c o m m u n i c a t i o n ) , s u n f i s h (L e p o m i s ) ( A v i s e e t a l . , 1 9 8 4 ) , f r o g s ( R a n a ) ( S p o l s k y and U z z e l l , 1986) and D r o s o p h i l a ( P o w e l l , 1983). al.
(1983) u s e d mtDNA a n a l y s i s t o i n f e r t h a t t h e l i z a r d ,
W right e t
C n e m id o p ho r u s
g u l a r i s , was t h e m a t e r n a l p a r e n t a l s p e c i e s in v o lv e d i n t h e c r e a t i o n of C. I a r e d o e n s i s by h y b r i d i z a t i o n w i t h C. s e x l i n e a t u s . B r o a d e r t a x o n o m i c s u r v e y s u s i n g mtDNA i n c l u d e a n a l y s e s o f g e n e t i c
6
d i v e r g e n c e s o f t h e mtDNA o f h u m a n a n d n o n - h u m a n p r i m a t e s ( B r o w n e t al.,
1 979), s h e e p ( O v i s ) and g o a t s ( C a p r a ) (U p h o lt and Daw id, 1 9 7 7 ),
s p e c i e s o f r a t s (R a t t u s ) (Brown and S im ps o n , et
a l.,
l9 83 fe ) a n d s e v e r a l
1985).
These
restrictio n
stu d ies
breeds
have
1981), m i c e ( Mus) ( F e r r i s
of p i g s ( S u s ) ( W atanabe e t
shown
that
e n d o n u c le a s e s can be u s e d
an aly sis
to
of
estim ate
a l.,
mtDNA w i t h
the
amount o f
n u c l e o t i d e s e q u e n c e d i v e r g e n c e b e t w e e n i n d i v i d u a l s , p o p u l a t i o n s and species. The
fo ssil
in d icatio n s
record,
m orphology
and
p ro tein
p henotypes
give
of g e n e t i c d i s t a n c e b etw e en d i f f e r e n t N orth A m erican
a rtio d A cty ls
and
u rsid s.
The
fam ilies
C ervidae,
Bovidae and
A n t i l o c a p r i d a e a r e b e l i e v e d t o h a v e s p l i t i n t h e Miocen e 2 0 - 3 0 m i l l i o n y e a r s b e f o r e t h e p r e s e n t ( m y b p ) ( R o m e r , 1966.).
The m o r p h o l o g i c a l l y
d i s t i n c t c e r v i d s u b f a m i l i e s C e r v i n a e a n d O d o c o i l i n a e may a l s o h a v e diverged in the Many
M iocene (O sborn,
1910;
e x ta n t sp e c ie s in the c e rv id ,
S co tt,
1937;
b o v i d and u r s i d
G e i s t , 1981). fam ilies
are
t h o u g h t t o h a v e o r i g i n a t e d i n t h e P l i o c e n e o r P l e i s t o c e n e ( K u r te"6 , 1968;
K urtdii and A n d e rso n , 1980).
artio d acty ls relativ e
from
p ro tein
G enetic d is ta n c e e s tim a te s fo r
electro p h o resis
g en erally
d i v e r g e n c e a s i n d i c a t e d by m or p h o l o g y and t h e
( Baccus e t a l.,
agree fossil
w ith record
1983).
M ule d e e r and w h i t e - t a i l e d d e e r a r e s y m p a t r i c i n p a r t s o f w e s t e r n N orth A m erica. 1980),
Each s p e c i e s h as d i s t i n c t i v e m o rp h o lo g y ( W i s h a r t ,
behavior (G eist,
1981),
s e ru m a l b u m i n ( McClymont e t a l . ,
h a b i t a t p r e f e re n c e s (Mackie, 1982).
1981) and
H y b rid iz a tio n betw een n a t u r a l
p o p u l a t i o n s o f t h e s e two d e e r s p e c i e s h a s b e e n r e p o r t e d i n A l b e r t a
7
(W ishart,
1980) and o t h e r a r e a s (K ram er,. 1973).
W lshart (p erso n al
communication) n oted m o rp h o lo g ical c h a r a c t e r s , p a r t i c u l a r l y m e t a t a r s a l gland le n g th , co n tro lled
a s i n t e r m e d i a t e t o e i t h e r p a r e n t a l t y p e i n h y b r i d s fro m
breeding
experim ents.
polyacrylam ide g el e le c tr o p h o r e s is ,
McClymont
et
al.
(1982) u s i n g
i d e n t i f i e d s p e c i e s - s p e c i f i c se rum
a lb u m in p a t t e r n s f o r th e two d e e r s p e c i e s .
S uspected h y b rid s w ith
i n t e r m e d i a t e m o r p h o l o g i e s d i s p l a y e d t h e h e t e r o z y g o t e p a t t e r n o f two album in bands, that
one c h a r a c t e r i s t i c o f e a c h s p e c i e s .
h y b rid izatio n
m echanism s.
G eist
in
natu re
(personal
c o u r t s h i p b e h a v i o r s o f t h e two
w ould
be
K r a m e r (1973) f e l t
m inim ized
com m unication)
has
by noted
behavioral that
s p e c i e s a r e v e r y d i f f e r e n t and f e e l s
t h a t o n ly m ale w h i t e t a i l x fe m a le m ule d e e r c r o s s e s a r e l i k e l y occur.
Hybrids
the
f ro m i n t e r - s p e c i f i c
to
crosses in c a p tiv ity are f e r t i l e
(K ram er, 1973; W allm o, 1981), b u t W i s h a r t ( p e r s o n a l c o m m u n ic a tio n ) reported i n f e r t i l i t y ,
low sperm m o t i l i t y and d e fo r m e d sperm i n F - I
hybrid males w hile F - I hybrid fem ales a re f e r t i l e .
To o u r kn o w le d g e
t h e r e i s no docum entation of h y b r id s bree d in g i n th e w ild . W h i t e - t a i l e d d e e r and m ule d e e r a r e
fou nd t h r o u g h o u t Montana and
a re o fte n considered as h a b ita t s e p a ra te d . o c c u r and i t
is
not
u n c om m o n t o
s p e c i e s can be c o n s id e re d
to
see
O verlap of ranges does
both
species
together.
The
be s y m p a t r i c o r p a r a p a t r i c o v e r m o s t o f
M ontana and i n t e r b r e e d ! n g ^ is^ n o _ t_ _ n ec e ssarily p r e v e n t e d by s p a t i a l s e p a r a t i o n ( M a c k i e , p e r s o n a l communc a t i o n ) .
\
T h i s s t u d y was c o n d u c t e d t o t e s t t h e h y p o t h e s i s t h a t m u le d e e r and
w h ite-tailed
deer
are
g en etically
d istin ct,
i s o l a t e d s p e c i e s and t o c h a r a c t e r i z e i n t e r - s p e c i f i c
rep ro d u ctiv e!y gene flow i f
it
8
occurs.
In a d d i t i o n ,
I applied
restrictio n
endonuclease a n a ly s is of
mtDNA t o s e v e r a l c e r v i d ( e l k , Ce rv u s e l a p h u s ,
w h i t e - t a i l e d d e e r , mule
d e e r) , bovid ( b is o n . Bison b is o n , c a t t l e ) , a n t i l o c a p r id (p ronghorn, A n tilo cap ra
a m e r i c a n a ) and u r s i d
(black
bear,
Ursus
am ericanus,
g r i z z l y b e a r , U. a t c t o s ) s p e c i e s t o e s t i m a t e t h e d e g r e e o f s e q u e n c e d i v e r g e n c e and t i m e o f d i v e r g e n c e o f t h e t a x a .
9
MATERIALS AND METHODS
Sample c o l l e c t i o n
T i s s u e s (50-100 gram s (g) of l i v e r , muscle)
were c o l l e c t e d
o perations
d uring
w hite-tailed hunters.
deer,
k id n ey , b r a i n , or s k e l e t a l
f ro m a n i m a l s k i l l e d
1984-85 and f r o z e n a t species
Table I l i s t s
identification
by h u n t e r s o r i n c u l l i n g -20C.
For m ule d e e r and
was made by b i o l o g i s t s
or
t h e s p e c i e s and l o c a t i o n s s a m p l e d .
Albumin e l e c t r o p h o r e s i s
M u l e d e e r , b l a c k - t a i l e d d e e r ( 0. h . s i t k e h s i s ) a n d w h i t e - t a i l e d d e e r w e r e a n a l y s e d e l e c t r o p h o r e t l e a l I y f o r serum a l b u m i n p h e n o t y p e i w i t h m e t h o d s a d a p t e d fro m McClymbnt^ e t a l . (1982). F or e a c h d e e r 0.5 g t i s s u e , u s u a l l y s k e l e t a l m u s c le , was ground i n I m i l l i l i t e r (m l) d i s t i l l e d w a t e r , c e n t r i f u g e d t e n m i n . a t 1 2 , 0 0 0 x g r a v i t y ( g ) a n d 50 m i c r o l i t e r s ( u l ) o f t h e s u p e r n a t a n t m ixed w i t h 5 u l bro m p h e n o l b lu e a nd o ne d r o p o f g l y c e r o l b e f o r e l o a d i n g 5 - 3 0 u l o n t o t h e g e l . w e r e 6-15% l i n e a r g r a d i e n t p o l y a c r y l a m i d e w i t h a 5% s t a c k i n g g e l . g e l b u f f e r was 1.5 M t r i s - H C l , M tris,
1.5 M g l y c i n e - H C l ,
stain ed
pH 8.3.
during e le c tr o p h o r e s is in
0.1% C o o m a s s i e
(w t/v o l/v o l). in itially ,
D estaining
The
pH 8.8 and t h e e l e c t r o d e b u f f e r was 0.2
a t 100-200 v o l t s f o r 5 - 1 2 h o u r s . plates
G els
V e r t i c a l e l e c t r o p h o r e s i s was done Ice packs were l a i d
to p re v e n t o v e rh e a tin g .
blue, was
a g a in st the gel
in
G els w ere
10% a c e t i c
acid ,
45%
10% a c e t i c
acid ,
45% m e t h a n o l
f o l l o w e d by s e v e r a l w a s h e s i n 7% a c e t i c a c i d .
m ethanol
10
T a b l e I.
Species,
s u b s p e c i e s and l o c a t i o n s s a m p l e d .
Species
Location
W hite-tailed deer (0. v. v irginianus)
"
