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)
"
<0^
"
(&.
Mule d e e r
C l a r k e Co.
Georgia
V .
borealis)
Montgomery Co. I l l .
V .
dakotensis)
Montana ( e a s t e r n )
V1
ochrourus)
Montana ( w e s t e r n )
COju h • he m io nu s )
B l a c k - t a i l e d d e e r (0 ^ h . s i t k e i i s i s )
Montana/Wyoming
Kodiak I s . Ak.
Elk
(C1 e
P r o n g h o rn
(A1. a m e r i c a n a )
Montana
P la in s bison
(B 1 B1 b i s o n )
various herds
see t a b l e 3
Wood b i s o n
(B 1 B1 a t h a b a s c a e )
.
nelson!)
Presumptive
p . bison-w .bison
hybrids
Montana
, Elk I s . Nat. Park,
A lberta
Northwest T e r r.
Canada
C attle
various breeds
(Bos t a u r u s )
Bozeman, Mt.
Black b e a r
(U1 a m e r i c a n u s )
Montana
G rizzly bear
(U1 a r c t o s )
Montana
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
For mtDNA a n a l y s i s
other tis s u e s
brain tissu e
was fou nd t o be f a r s u p e r i o r t o
f o r e a s e o f h o m o g e n i z a t i o n and d i g e s t i o n o f DNA.
Liver
and k i d n e y g a v e good y i e l d s o f mtDNA w h i c h was s o m e t i m e s d i f f i c u l t t o
digest.
S k e l e t a l m u s c l e y i e l d e d l o w e r b u t a d e q u a t e a m o u n t s o f mtDNA.
11
F r e s h t i s s u e was b e s t , b u t f r o z e n s a m p l e s a l s o g a v e good r e s u l t s i f
s t o r e d l e s s t h a n two m o nt h s .
S t a n d a r d p r o c e d u r e s f o r e x t r a c t i o n o f mtDNA w e r e a d a p t e d f r o m
P o w e l l a n d Zu^figa ( 1 9 8 3 ) a n d D a v i s e t a l . ( 1 9 8 0 ) a n d a r e d e s c r i b e d i n
A p p e n d i x A.
For
some
of
the
liv er
and k i d n e y s a m p l e s
sto red
frozen
for
e x t e n d e d p e r i o d s p u r i f i c a t i o n i n c e s i u m c h l o r i d e (CsGl) g r a d i e n t s
was
n e c e s s a ry to a llo w d ig e s t io n w ith endonucleases.
I s o l a t i o n and l y s i s
o f m i t o c h o n d r i a w e r e done a s i n s t e p s 1-10 o f t h e s t a n d a r d p r o c e d u r e s
in
A p p e n d i x A.
Th e
p r o c e d u r e s o f Lansman e t a l .
g r a d i e n t c e n t r i f u g a t i o n were then fo llo w e d .
( 1 981) f o r
CsCl
G r a n u l a r CsCl (1.1 g) was
a d d e d t o e a c h m l o f l y s a t e a l o n g w i t h 0.2 ml o f a 10 m g / m l e t h i d i u m
bromide s o lu t i o n .
T h i s m i x t u r e was p u t i n t o an
and m i n e r a l o i l added t o t h e t o p o f t h e t u b e .
u ltr a c e n trifu g e tube
A fter balancing paired
t u b e s t o 0.01 g s a m p l e s w e r e c e n t r i f u g e d a t 1 6 0 , 0 0 0 x g f o r 48 h r s .
A fter c e n trifu g a tio n
tube
t h e v i s i b l e band o f mtDNA was remo ve d from t h e
w ith a hypoderm ic n eed le.
e x tra ctio n w ith w ater-satu rated
t h e s a m p l e v o lu m e i n c r e a s e d
E thidium
brom ide
was removed by
I -b u tan o l (M aniatis e t a l.,
1.5 t i m e s by a d d i n g w a t e r .
1982) and
P recipitation
and r e s u s p e n s i o n o f mtDNA was done a s i n s t e p s 15- 19 o f t h e s t a n d a r d
p u rific a tio n procedures.
D i g e s t i o n and 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
Six to e ig h te e n u l of s o l u t i o n
p u rified
mtDNA w e r e
reactio n buffer,
I ul
put
in to
a
co ntaining approxim ately
1.5 ml E p p e n d o r f
R N a s e - A (1 0 m g / m l s t o c k ) ,
tube w ith
0.3 ug
2 ul
I ul re s tric tio n
12
enzyme and s t e r i l e
w a t e r t o a v o lu m e o f 20 u l .
The r e a c t i o n b u f f e r s
used were t e n t im e s t h e r e q u i r e d c o n c e n t r a t i o n ,
so t h a t 2 u l added t o
a
20
ul
m ixture
resu lted
D ifferent r e s tr ic tio n
in
the
from
B iochem icals.
hrs.
New
buffer
co n cen tratio n .
enzy mes r e q u i r e d i f f e r e n t b u f f e r s ,
the m anufacturer or M aniatis e t a l.
obtained
proper
E ngland
(1982).
B iolabs
s p e c i f i e d by
R e s t r i c t i o n enzymes w er e
and
B o eh rin g er
M annheim
D i g e s t i o n r e a c t i o n s w e r e c a r r i e d o u t a t 37C f o r 4 - 1 4
The en z y m e s u s e d a r e l i s t e d i n T a b l e 2 on p a g e 17.
E i g h t t o t w e l v e u l o f t h e d i g e s t i o n m i x t u r e was a p p l i e d
t o 0.65%
( 6 - b a s e e n z y m e d i g e s t s ) o f 1.2% ( 4 - b a s e e n z y m e d i g e s t s ) h o r i z o n t a l
agarose g e ls .
G e l a n d e l e c t r o d e b u f f e r w a s 89 mM t r i s ,
a c i d , 2 mM EDTA, pH 8 . 0 ( M a n i a t i s e t a l . , 1 9 8 2 ) .
done
at
20-50
v for
5-16
hrs.
G els
were
89 mM b o r i c
E l e c t r o p h o r e s i s was
stain ed
in
0.5
ug/m l
e t h i d i u m b r o m i d e i n e l e c t r o d e b u f f e r f o r 5 - 30 m in., v i s u a l i z e d u n d e r
s h o r t w a v e u l t r a v i o l e t l i g h t a n d p h o t o g r a p h e d w i t h P o l a r o i d t y p e 55
f i l m ;through a red g e l a t i n f i l t e r .
For e a c h g e l ,
f r a g m e n t s i z e s w e r e e s t i m a t e d by c o m p a r i s o n w i t h
s t a n d a r d s o f k n o w n s i z e (L a mb d a v i r u s . DNA d i g e s t e d w i t h H i n d I I I o r
Bam HI).
The m i g r a t i o n d i s t a n c e s o f t h e s t a n d a r d s w e r e m e a s u r e d
and
p l o t t e d on s e m i l o g g r a p h p a p e r w i t h m i g r a t i o n d i s t a n c e on t h e x a x i s
a n d f r a g m e n t s i z e ( b p ) on t h e l o g a r i t h m i c y a x i s .
m olecules
trav el
proportional
to
through
g els
at
rates
agarose
g els
(M aniatis e t a l.,
approxim ately
inversely
t h e Iog-^Q o f t h e i r m o l e c u l a r w e i g h t s so t h a t t h e l o g
m o l e c u l a r w e i g h t ( o r l o g number b a s e p a i r s )
in
L i n e a r d u p l e x DNA
is
approxim ately
1982).
an
versus m igration d istan ce
inverse
lin ear
The m i g r a t i o n d i s t a n c e s
for
relatio n sh ip
the fragm ents
13
of d ig e s te d
mtDNA w er e t h e n m e a s u r e d and t h e i r s i z e s
estim ated
f ro m
th e graph.
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
F r a g m e n t s we re j u d g e d t o be h o m ol o go u s b e t w e e n tw o o r g a n i s m s i f
m i g r a t i o n d i s t a n c e s a p p e a r e d i d e n t i c a l i n s i d e by s i d e c o m p a r i s o n s on
a g el.
The
calculated
p ro p o rtio n
of
shared
(hom ologous)
fragm ents
was
f o r e a c h p a i r - w i s e c o m p a r i s o n u s i n g e q u a t i o n 21 o f Nei and
L i (1979):
F =
2Nx y /Nx+Ny
w h e r e Nx a n d Ny a r e t h e n u m b e r s o f f r a g m e n t s f r o m a l l d i g e s t i o n s i n
o r g a n i s m s x a n d y r e s p e c t i v e l y a n d Nx y i s t h e n u m b e r o f f r a g m e n t s
s h a r e d by t h e t w o o r g a n i s m s .
nucleotide,
The n u m b e r o f b a s e s u b s t i t u t i o n s p e r .
p , was e s t i m a t e d u s i n g e q u a t i o n 6 b o f U p h o l t (1977):
r
-F+ ( F 2 + 8 F ) 1 / 2
w h e r e n e q u a l s t h e n um ber o f b a s e p a i r s r e c o g n i z e d p e r c l e a v a g e s i t e .
F o r t h e c o m p a r i s o n s b e tw e e n d e e r g ro u p s and b e tw e e n b i s o n and
c a t t l e , p wa s c a l c u l a t e d s e p a r a t e l y f o r t h e 6 ( n = 6 ) a n d 4 ( n = 4 ) b a s e
enzyme a s s a y s ,
and a w e ig h te d a v e r a g e was used a s a f i n a l p v a lu e
u sin g the equation:
P =
w h e r e p^ and N
PiN 1 + P9N9
Nt
ar e the p values
and num ber o f f r a g m e n t s f o r 6 - b a s e
14
enzyme a s s a y s r e s p e c t i v e l y and pg and Ng a r e s i m i l a r v a l u e s f o r 4 - b a s e
enzyme a s s a y s .
is
the
to tal
n um ber o f f r a g m e n t s f o r a l l d i g e s t s .
F o r some o f t h e s p e c i e s , 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 ( t i m e s i n c e
two o r g a n i s m s
last
shared a fem ale a n c e sto r)
w e r e m ad e u s i n g
an
e s t i m a t e d mtDNA s e q u e n c e d i v e r g e n c e r a t e o f 2-4% p e r m i l l i o n y e a r s
(Brown e t a l . ,
1979;
F e rris et al.,
d iv e r g e n c e time =
1983a,
1983b) and t h e e q u a t i o n :
.
p
'.02 ( o r . 0 4 ) X IO -6
Samples a n a l y s e d
I n t e r - s p e c i e s comparisons
At l e a s t one i n d i v i d u a l
of each s p e c ie s (pronghorn,
elk,
w hite­
t a i l e d d e e r , mu le d e e r , b i s o n , and c a t t l e ) was a n a l y s e d w i t h a b a t t e r y
o f t e n 6 - b a s e enz yme s.
A l s o some c a t t l e ,
bison,
m u le d e e r and 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 w i t h two f o u r - b a s e e n zy me s.
One b l a c k b e a r
and one g r i z z l y b e a r w e re s c r e e n e d w i t h s i x s i x - b a s e enzym es.
The
s a m p l e s a n a l y s e d a r e g i v e n i n T a b l e s 2 and 3 on p a g e s 17 a n d 19.
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
A ll d e e r sam pled were s u b je c t to album in e l e c t r o p h o r e s i s .
addition
In
a t o t a l o f 15 Montana m u le d e e r , one Wyoming m u l e d e e r , one
A laska b l a c k - t a i l e d d e e r,
19 M o n t a n a w h i t e - t a i l e d
d e e r, 5 G eorgia
w h i t e —t a i l e d d e e r and 4 I l l i n o i s 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 w i t h
two e n z y m e s , Eco R I , and Hae I I I w h i c h gav e p o l y m o r p h i c p r o f i l e s among
deer.
15
RESULTS
Co m pa ris on o f a l l s p e c i e s
Fragment P a t t e r n s
Table
2
restriction
Figure
I,
shows
the
mtDNA f r a g m e n t
enzyme d i g e s t i o n s .
contains
pattern s
A representative
the fragm ent p a tt e r n s
resu ltin g
from
ele c tro p h o retic gel.
resulting
f ro m d i g e s t i o n o f
d e e r a n d e l k mtDNA w i t h t h e e n z y m e s C l a I a n d Sa c I .
For s e v e r a l of
t h e e n z y m e s t h e p a t t e r n s a r e s i m i l a r t o t h o s e r e p o r t e d by L a i p i s e t
a l . (1979) f o r c a t t l e and 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 ) f o r t h e two
deer species.
Pronghorn,
w h ite-tailed
in tra-sp ecific v ariab ility .
d e e r and m u le d e e r showed
Only d i g e s t s
w i t h Bgl I I
v a r i a b l e p a t t e r n s i n p r o n g h o r n , shown i n T a b l e 2.
observed
in
bison
digestion p attern s
from
several
herds
(Table
f o r d e e r a r e shown i n T a b l e 4,
resu lted
in
No v a r i a b i l i t y wa s
3).
The
v ariab le
and c o m p o s i t e d e e r
mtDNA p h e n o t y p e s a r e g i v e n i n T a b l e 5.
From T a b l e 5 i t c a n be s e e n t h a t t h e c o m p o s i t e p h e n o t y p e s c o n s i s t
of com binations of the v a ria b le d ig e s tio n p a tte rn s
For exam ple,
p a tte rn I,
c o m p o site type A c o n s i s t s
shown i n T a b l e 4.
o f Eco RI p a t t e r n
Cl a I p a t t e r n I and Hae I I I p a t t e r n 2.
I,
Xba I
A l l enzymes e x c e p t
t h o s e l i s t e d i n T a b l e s 4 a n d 5 r e s u l t e d i n i n v a r i a n t p a t t e r n s am o n g
b o t h m u l e d e e r and w h i t e - t a i l e d d e e r , shown i n T a b l e 2-
16
F i g u r e I . P h o t o g r a p h o f Sac I a n d C l a I d i g e s t i o n p a t t e r n s on a 0.65%
a g a ro se g e l s t a i n e d w ith e th ld iu m bromide.
Lane s A-D, F - I , w h i t e - t a i l e d d e e r , Cla I ,
(Lane B i n c o m p l e t e d i g e s t i o n )
Lane E, mule d e e r , Cla I
Lane L, e l k . Sac I
Lane M-P, w h i t e - t a i l e d d e e r .
Sac I
Lane Q, m u le d e e r . Sac I
Lane J ,
III
size standard,
AB
CD
E F G H l
Lambda v i r u s DNA d i g e s t e d w i t h Hind
J K L M N O
pO
17
T a b l e 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
Enzyme
A.
fragment p a t t e r n s .1
Species
S i x - b a s e <anzymes
De e r
Elk
Prong­
horn
Bison
C attle
Black
Bear
4-8(1) 16.6(1)
3.6
3.2
Eco
RI
8.9(18)
3.1(26)
1.9
1.7
12. 0 (6)
3.1
8.4(4)
7.6
7.32
4.6(9)
4.2
7.3(8)
4.6
4.2
Bam
HI
7.5(10)
5.3(19)
3.8
9.3(4)
5.3
2.4
7.4(4)
4.5
3.8
5 . 92
5.2(6)
3.7
11.0(4)
3.4
Hind 1 0 . 5 ( 1 0 )
III
3.9(17)
1,6
.8
XbaI
7.6(4)
5.2(12)
3 .2
11.5(6) 16.6(4)
4.2
1.6
1 0.5(4) 13.0(3)
3.2
3.2
2.6
6.52
3.7(3)
3.0
2.6
2.0
9.4(1)
7.0
2.0
1 5 .02
10.5(7)
1.6(7) 4.6
1.6
15.0(1)
Grizzly
Bear
4.8(4)
3.2
3.0
2.5
7.5(1)
2.7
1.9
1.7
1.5
3.2(1)
2.7
5.6(1)
3.4
3,2
5.6(1)
3.4
3.2
2.0
1.9
1.5
1.8
Bgl
II
16.6(2)
(8 )
11.5(3)
Pvu
II
16.6(7)
(16)
13.0(4)
3.5
2.6
1.2
9.8(1)
6.6
or
16.6(3)
9.8(1)
6.6
9.8(4)
6.6
9.0(1)
7.2
10.5(1)
5.9
6.1(3)
4.6
3.1
1 4 .02
2.7(4)
14.0(4)
2.7
8 . 6(1)
6. 2
10: 0( 1 )
6.6
2.8
ClaI
9.0(6)
7.1(14)
9.0(3)
7.1
7.2(3)
6.0
1.9
1.5
7.2(2) 16.6(2)
6.4
1.6
18
Table 2. c o n tin u ed
B ell
8.0(4)
3.6(7)
3.0
.9
Elk
Pronghorn
4.8(1)
4.6
3.5
2.7
4.0(1)
3.0
2.5 .
Bison
C attle
2.0
1.8 1
1.2
16 .6 (4 ) 16.6(3)
16.6(3)
1 1 . 02
2.8(4)
2.3
P s t I No ( 3 )
cuts(3)
16.6(1) 16.6(3)
No ( I )
cuts
9.4(4)
6.3
Bison
C attle
Hae
III
Sau
961
F o u r - b a s e enzymes
Deer
4.0(18)
2.6(26)
G rizzly
Bear
12. 0(2 ) 11. 0 (2 )
4.0
6. 0
SacI 10. 0 (6 )
3.1(14)
2.7
B.
Black
Bear
2. 0 (2)
1.5
1.2
1. 1
2 . 0 (2 )
1.5
1.3
1.1
.8
3.5(6)
3.2(7)
5.6(2)
3.5
4.0(2)
2.9
2.0
1.5
1.2
.8
1.6
2.2
1.8
1.6
1.0
1.0
.8
Num bers r e f e r t o s i z e o f f r a g m e n t s ( k i l o b a s e s ) g e n e r a t e d by
enzyme d i g e s t i o n .
Djumbers i n p a r e n t h e s e s r e f e r t o n u m b e r o f a n i m a l s
yielding a p a rtic u la r p attern .
F o r ■d e e r t h e u p p e r n u m b e r i n
p a r e n t h e s e s i s m u le d e e r , t h e l o w e r w h i t e - t a i l e d d e e r . Note t h e r e a r e
tw o p a t t e r n s f o r p r o n g h o r n mtDNA d i g e s t e d w i t h BGL I I . Only t h e m o s t
common p a t t e r n s f o r b o t h d e e r s p e c i e s a r e g i v e n h e r e ,
v ariab le
p a t t e r n s and c o m p o s i t e mtDNA p h e n o t y p e s f o r p o p u l a t i o n s and s p e c i e s o f
d e e r a r e g i v e n i n t a b l e 4.
2
I n d i c a t e s Sa m p le i n c l u d e s wood b i s o n .
19
T a b l e 3.
Numbers o f b i s o n fro m 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 enzymes.^
Herd
Enzyme
EINP 2
NWT2
YNP2
HM2
NB2
EcoRI
I
I
3
4
BamHI
I
I
3
I
HindIII
I
2
2
I
I
XbaI
I
I
I
I
2
ClaI
I
B ell
2
SacI
.
I
BglII
PvuII
.
.
I
I
I
2
I
I
PstI
H aeIII
2
Sau96l
2
F r a g m e n t p a t t e r n s f o r a l l d i g e s t i o n s a r e a s i n t a b l e 2, t h e r e i s
no I n t e r - h e r d v a r i a t i o n .
^EINP = E l k I s l a n d N a t i o n a l P a r k , A l b e r t a ( w o o d b i s o n ) , NWT =
N o r t h w e s t T e r r i t o r i e s , C a n a d a , ( p r e s u m e d wqod b i s o n - p l a i n s b i s o n
h y b r i d s ) , YNP = Y e l l o w s t o n e N a t i o n a l P a r k ( p l a i n s b i s o n ) , HM = H e n r y
M o u n t a i n s , U t a h ( p l a i n s b i s o n ) and NB = N i e l s o n h e r d , A l b e r t a ( p l a i n s
bison).
B l a n k s p a c e s i n d i c a t e no a n a l y s e s done.
20
T a b l e 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 . I
EcoRI
1
8.9
3.1
1.9
1.7
2
3
8.9
3.6
3.1
Enzyme
ClaI
1
2
XbaI
1
2
7.0
5.0
4.7
7.6
5.2
3.2
13.1
3.2
1
16.6
9.0
7.1
2
H aeIII
3
4.0
4.0
2.6
1.2
1.1
1.1
1.0
4
4.0
1.3
3.0
1.5
1.1
1. 0
1.2
^Numbers i n d i c a t e f r a g m e n t s i z e i n kb.
T a b l e 5.
Population
MtMD2
MtMD^
GaWT2
MtWT2
IlWT 2
MtMD2
GaWT2
GaWT2
AkBT2
C o m p o s i t e mtDNA p h e n o t y p e s i n d e e r .
,
EcoRI
2
2
3
4
2
I'
I
I
I
■
I
2
2
I
I
I
2
I
3
Enzyme
Xb aI
ClaI
2
I
I
I
I
I
I
I
I
2
I
I
I
. I
I
I
H aeIII
2
' ’
I
I
I
I
3
I
I
4
C o m p o s ite
Pattern
A
B
B
C .
C
D
F
G
E
■'■The n u m b e r s r e f e r t o t h e d i g e s t i o n p a t t e r n s i n T a b l e 4. B l a n k s
i n t h e t a b l e i n d i c a t e no a n a l y s e s done.
^N=number o f a n i m a l s ,
M t=M ontana, G a= G eorgia, I l = I l l i n o i s ,
A k = M a s k a , MD=mule d e e r , W , T = w h i t e - t a i l e d d e e r , B T = b l a c k - t a i l e d d e e r .
21
Divergence e s tim a te s
Table
6 shows
estim ates
of
genetic
distance
e x p r e s s e d a s t h e n um ber o f b a s e s u b s t i t u t i o n
my a n a l y s i s
and t h o s e
reported
elsew here.
betw een
taxa,
p e r n u c l e o t i d e (p) from
T a b l e 7 sh ows d i v e r g e n c e
t i m e o f s e v e r a l t a x a e s t i m a t e d from p v a l u e s and t h e f o s s i l r e c o r d .
I
Table 6 .
E stim ated g en etic d ista n c e
n u c l e o t i d e (p) f o r mtDNA.
Orga nis m s
No.
Enzymes
p
in
base
su b stitu tu tio n s
Standard5
Deviation
per
Sour ce
Intra-species:
MtWTxIIWT1
MtWTxGaWT1
GaWTxGaWT1
MtMDxMtMD1
Pronghorn
WBxPB1
Humans
Pero myscu s
spp.
Lepomis
m acrochiris
12
12
12
12
10
6
7
6
.0000
.0025- .00533
. 0 0 2 6 4- . ' 0 0 8 4 3 » 4
• 00 664
.00284
. 0 0 3 6 - .0 051
.0037- .0063
.0057
.0 0 3 8
.0000
.0036
. 00504- . 0 1 5 0
(Brown, 1980)
( Avise e t a l . ,
1979a)
(Avise e t a l . ,
1984)
.0 870
13
I n t r a - f a m i l y , i n t e r - ■species
MtWTxMtMD1
MtMDxGaWT1
CaMDxSCWT1
12
12
10
. 0 0 2 5 - . 00403
. 0 0 0 0 - . 00953
. 07 20
. 0 0 3 6 - .0045
.0 0 6 8
(Carr, personal
c o m m u n ic a t io n )
BBxGB1 .
Per omy sc us
m aniculatus
P. p o lio n o tu s
6
6
.0 580
.1 3 0 0 - .1700
.0 190
CattlexBison
Elkxdeer^
G oa tx She ep
10
10
2
.0614
. 08 50
.0141
.0170
. 0 6 0 0 - .1100
(Avise e t a l . ,
1979a)
( U p h o l t and
Dawid, 1977)
22
Table 6 continued
O rg an ism s
No.
Enzymes
P
Standard^
D eviation
Source
In tra -o rd e r, in ter-fam ily
DeerxPH^» ^
ElkxPH
BisonxPH
Cat t l exPH
Deerxbison^
D eerxcattle^
Elkxbison
E lkxcattle
Humanxbaboon
10
10
10
10
10
10
10
10
11
.1130.1300.1130.1130-
. 1330*3
. 1 3 I O3
. 15583
. 15583
. 19 80
.1287
.1531
.1274
. 2 3 0 0 - .2 9 0 0
.0189. 0200 .0188.0188-
. 01 98
.01 9 9
.02 1 4
.02 1 4
.0235
.0202
.0 217
.0 203
(Brown e t a l
1979)
P H = p r o n g h o r n , PB=wood b i s o n , WB=wood b i s o n , B B = b l a c k b e a r ,
G B = grizzly b e a r,
C a= C alifo rn ia,
SC =South C a r o l i n a .
O ther
a b b r e v i a t i o n s a s i n T a b l e 5.
‘‘F o r i n t e r - s p e c i e s c o m p a r i s o n s w i t h d e e r t h e m o s t common 6 - b a s e
p a t t e r n s ( T a b le 2) w e r e u s e d .
3
R an g es o f v a l u e s a r e due to i n t r a - g r o u p v a r i a t i o n and r e f l e c t
d i f f e r e n c e s b e tw e e n c o m p o s i te t y p e s i n f a b l e 5 f o r d e e r and t h e two
B g lII p a t t e r n s f o r pronghorn.
^These v a lu e s a re f o r co m p ariso n s w ith in a l i m i t e d g eo g rap h ic
area.
C
S tan d ard d e v i a t i o n s c a l c u l a t e d a s i n Upholt (1 9 7 7 ).
!
23
T a b l e 7.
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 r o m
the f o s s i l record.
Taxa
D i v e r g e n c e t i m e (MYBP)
derived
from p i
f o s s i l record
Source
CervidaeA ntilocapridae
2.83-6.65
20—25 Miocene
(Romer, 1966)
BovidaeA ntilocapridae
2.83-7.79
1 5 - 2 0 Miocene
(Romer, 1966)
( K u r t e n , 1972)
BovidaeCervidae
3.19-9.90
2 0 - 2 5 Miocene
(Romer, 1966)
CervusOdocoileus
2 . 1 3- 4 .2 5
1 5 - 2 5 M io ce ne
( S c o t t , 1937)
( O s b o r n , 1910)
( G e i s t , 1981)
Bos-Bison
1.54-3.07
1 -3
PliocenePleistocene
(McDonald, 1981;
K u r t e n , 1968)
Ursus a r c t o s U. a m e r i c a n u s
1.45-2.90
1 -2
Pleistocene
( H e r r e r o , 1972;
K u r t e n and
A n d e r s o n , 1980)
Odocoileus
virginianusO. hemionus
(C alifornia)
1.80-3.60
0.5-2 P le isto ce n e
(C arr, personal
c o m m u n ic a t io n )
( K u r t e n and
A n d e r s o n , 1980;
G e i s t , 1981)
0 . virginianusO. hemionus
(Mont an a)
0.48-0.60
0.5-2
^Assuming a r a t e o f mtDNA d i v e r g e n c e o f 2-4% p e r m i l l i o n y e a r s .
24
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
I
Serum a l b u m i n p h e n o t y p i n g
The r e s u l t s
Except
for
displayed
et al.
fiv e
o f a l b u m i n e l e c t r o p h o r e s i s a r e p r e s e n t e d i n T a b l e 8.
h eterozygotes
(2%) a l l
w h itetails
and
m ule d e e r
t h e s p e c i e s - s p e c i f i c a l b u m i n band a s r e p o r t e d b y McClymont
(1982).
B l a c k - t a i l e d d e e r had th e m ule d e e r - t y p i c a l band.
i
.
'
F igure 2 i s a photo o f th e d i f f e r e n t p a t t e r n s .
T a b l e 8.
Numbers o f m o r p h o l o g i c a l and serum a lb u m i n t y p e s o f d e e r
fro m a l l s a m p l e d l o c a t i o n s .
M orphological type
Album in
genotype
Total
FF1
Mule
deer
W hite-tailed
deer
B lack-tailed
deer
Total
93
O
3
96
SS
O
162
O
162
FS2
2
3
O
5
95
165
3
263
1F = f a s t , S = s lo w a l l e l e .
^ A l l H e t e r o z y g o t e s were from Mon tana .
i
25
F i g u r e 2.
P h o t o g r a p h o f p o l y a c r y l a m i d e g e l sh owing serum a l b u m i n ban ds
f o r m u l e d e e r ( l a n e s A, B, C, E, H, I ) , w h i t e - t a i l e d d e e r
( l a n e s D, K) and h e t e r o z y g o t e s ( l a n e s F, G). L a n e J s h o w s
a lb u m in from
a deer w ith in te r m e d ia te m orphological
c h a r a c t e r s and t h e w h i t e t a i I - t y p i c a l a l b u m i n ba n d ( s e e
discu ssio n ).
Mitochondrial DNA analysis
Two e n z y m e s , E c o RI a n d Hae I I I ,
w hich v a r i e d
betw een i n d iv id u a l d e e r
provided d ig e s tio n p a tt e r n s
in
M ontana.
The d i g e s t i o n
p r o f i l e s f o r th e te n o th e r enzymes were v i r t u a l l y i d e n t i c a l i n a l l
d e e r o f b o t h s p e c i e s f r o m a l l l o c a t i o n s . B e s i d e s Eco R I a n d Hae I I I
the
only v a r i a b l e
p ro files
in d eer r e s u lte d
d i g e s t i o n s i n Georgia w h i t e t a i l s
Figure
3
is
a
from
C l a I a n d Xba I
( T a b l e 4).
rep resen tatio n
of
the
polym orphic
p r o f i l e s in th e c o m b in a tio n s found i n i n d i v i d u a l d e e r.
d igestion
Note t h a t
t y p e s A-D c o n s i s t o f t w o Eco RI a n d t h r e e Hae I I I p a t t e r n s f o u n d i n
four com binations.
Type E, o b s e r v e d o n l y i n
c o n s i s t s o f two u n i q u e p a t t e r n s .
the
S itka b l a c k t a i l ,
T h e s e t y p e s , A-E, r e p r e s e n t t h e same
t y p e s i d e n t i f i e d i n T a b le 5 e x c e p t o n l y two of th e v a r i a b l e enzymes
w er e u s e d h e r e .
from
the
S i n c e mtDNA i s p r e s u m a b l y i n h e r i t e d a s a n i n t a c t u n i t
m other,
each in d iv id u a l's
in d ic a tiv e of m aternal ancestry.
p ro file
co n stitu tes
a "type",
T a b l e 9 shows t h e d i s t r i b u t i o n and
l o c a t i o n s o f mtDNA, a l b u m i n a n d m o r p h o l g i c a l t y p e s w h i l e F i g u r e 4
shows t h e g e o g r a p h i c l o c a t i o n o f t y p e s i n Montana.
S e v e ra l t h i n g s a r e a p p a re n t from t h e s e d a ta .
t h e m u le d e e r a r e
re la tiv e ly variable,
A,
only
B or D
w hile
type
C is
exhibiting
seen in
F irst,
three
w h itetails.
i n Montana
mtDNA t y p e s ,
Second,
in
M ont an a d e e r t h e r e i s s p e c i e s - s p e c i f i c i t y i n m o r p h o l o g y , serum a l b u m i n
( e x c e p t h e t e r o z y g o t e s ) and mtDNA.
Third,
t h e S i t k a b l a c k t a i l mtDNA i s
v e r y d i f f e r e n t f ro m t h a t o f Montana m u l e d e e r and w h i t e t a i l s .
27
D espite
the
f o r two e n z y m e s ,
distinctions
between th e s e
mtDNA d i g e s t i o n
profiles
t h e o v e r a l l s i m i l a r i t y o f p r o f i l e s f o r mule d e e r and
w h i t e t a i l s fo r the e n ti r e
12 enzyme b a t t e r y i s s t r i k i n g . '
From T a b l e 6
i t c a n be s e e n t h a t t h e p v a l u e f o r c o m p a r i s o n s o f M ontana m ule and
w hite-tailed
d e e r i s a s lo w a s t h o s e f o r 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 th e r t axa.
F i g u r e 3. R e p r e s e n t a t i o n o f a g a r o s e g e l s c o n t a i n i n g c o m p o s i t e mtDNA
d i g e s t i o n p r o f i l e s f o r EcoRI and H a e I I I f o r d e e r .
Fragments
l e s s t h a n 1 000 b a s e p a i r s c o u l d n o t b e c o n s i s t e n t l y
i d e n t i f i e d And a r e n o t i n c l u d e d .
Fragment s i z e s ( k ilo b a s e s )
a r e l i s t e d below each p a t t e r n .
mtDNA COMPOSITE TYPE
A
ECO
X DNA
B
H A E III
Rl
I
1
ECO
C
H A E III
ECO
2
Rl
I
R.I
2
D
H A E III
2
ECO
E
H A E III
ECO
3
Rl
3
Rl
I
H IN D III
H A E III
FR.
S IZ E
Kb
P A IR S
4
O rig in
—
iJ
6.6
——
4 .4
—
23
2. 0-
—-
FRAGM ENT
S IZ E S
8 .9
3.1
1 .9
1 .7
4 .0
8.9
4 .0
8 .9
4 .0
3.6
3.1
2 .6
3.1
2.6
8 .9
3.1
4 .0
1.3
7 .0
5 .0
3 .0
1.1
1 .0
1.2
1.9
1.2
1 .9
1.1
4.7
1.2
1.1
1-7
1.1
1 .7
1 .0
1.5
23.1
28
T a b l e 9.
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 .
morph ol og y
s p e ci m en No.
a lb u m in
type
mtDNA t y p e
location
mule d e e r
n o . 1—6
FF
A
Montana
mule d e e r
no. 7
FS
A
Montana
mule d e e r ■
n o. 8-13
FF
B
Montana
mule d e e r
n o . 14-16
FF
D
B ighorn/Pryor M ts.
M on tan a, Wyoming
w h itetail
n o . 1-17
SS
C
Montana
W hitehall
n o . 18- 19
FS
C
Montana
w hitetail
n o . 20- 23
SS
B
C l a r k e Co. Ga.
w h itetail
n o . 24
SS
C
C l a r k e Co. Ga.
w hitetail
no. 25-28
SS
C
Montgomery Co. 111.
blackball
no. I
FF
E
Kod ia k I s . Ak.
F i g u r e 4. G e o g r a p h i l c d i s t r i b u t i o n o f mtDNA t y p e s f o r i n d i v i d u a l d e e r .
Two l e t t e r a b b r e v i a t i o n s r e f e r t o m o r p h o l o g i c a l t y p e ,
md= m u l e d e e r , wt = w h i t e t a i I , f o l l o w e d b y t h e l e t t e r
d e s i g n a t i o n o f t h e mtDNA t y p e from F i g u r e 3. An a s t e r i s k i n
f r o n t o f th e l e t t e r s i n d i c a t e s t h a t a n im a l i s an a lb u m in
heterozygote.
The two a s t e r i s k s w i t h o u t l e t t e r s r e p r e s e n t
h e t e r o z y g o t e s n o t a n a l y s e d f o r mtDNA.
-',MD1
M O N T A N A
W Y O M I N G
30
DISCUSSION
The d a t a p r e s e n t e d
i n Tables
6 an^ 7 show t h a t d i v e r g e n c e among
t a x a ( a s i n d i c a t e d by t r a d i t i o n a l t a x o n o m i c g r o u p i n g s o r d i v e r g e n c e
tim es
from
the
fo ssil
record)
d iv e rg e n c e (as in d ic a te d
nucleotide,
p).
is
by t h e
accom panied
estim ated
base
b y mtDNA s e q u e n c e
su b stitu tio n s
per
I n t e r - f a m i l y comparisons y i e l d the h ig h e s t e s tim a te s
o f mtDNA s e q u e n c e d i v e r g e n c e ,
f o l l o w e d by i n t r a - f a m i l y - i n t e r - s p e c i e s
and i n t r a - s p e c i e s c o m p a r i s o n s .
T h is r e l a t i v e o r d e r i n g o f d i v e r g e n c e v a l u e s i s e x p e c t e d i f one
a s s u m e s a c o n s t a n t r a t e o f random n u c l e o t i d e s u b s t i t u t i o n by m u t a t i o n .
However,
estim ates
of
genetic
divergence
using
com parisons
of
f r a g m e n t s f r o m mtDNA d i g e s t i o n p r o f i l e s h a v e b e e n f o u n d t o b e m o s t
accurate
when a t
least
20% o f t h e f r a g m e n t s a r e h o m o l o g o u s (F i s
g r e a t e r t h a n 0 .2 0 ( U p h o l t , 1 9 7 7 ) ) o r w h e n p i s l e s s t h a n 0.05 ( F e r r i s
et al.,
scoring
1983b).
W it h d e c r e a s i n g v a l u e s o f F t h e c h a n c e o f i n c o r r e c t l y
fragm ents
p ro p o rtio n al
as
hom ologous
und erestim ate
in creases,
o f p (Brown e t a l . ,
w hich
1979).
c l e a v a g e s i t e s seem t o be h i g h l y c o n s e r v e d i n e v o l u t i o n ,
not
a ll
sites
on
m u t a t i o n a l c h a n g e.
the
mtDNA m o l e c u l e
are
eq u ally
lead s
to
a
A l s o , some
im plying t h a t
su scep tab le
to
The p r e s e n c e o f c o n s e r v e d s i t e s w i l l c a u s e f u r t h u r
u n d e r e s t i m a t i o n o f p (Brown e t a l . ,
1979).
These a u t h o r s s u g g e s t t h a t
p v a lu e s a r e m ost a c c u r a t e f o r l i n e a g e s s e p a r a te d w i t h i n th e l a s t 5
m i l l i o n y e a r s w h i l e t h o s e f o r l i n e s s e p a r a t e d by 25 m i l l i o n y e a r s a r e
u n d e r e s t i m a t e d by a b o u t 50%.
31
Another f a c t o r
apparent
w h i c h may c a u s e u n d e r e s t i m a t i o n
ho mo log y b e t w e e n r e s t r i c t i o n
from c o n v e rg e n t g a in o r
1983).
sites
(or
of divergence i s
fragm ents)
resulting
l o s s o f s i t e s j n o t common a n c e s t r y ( T e m p l e t o n
Given t h e s e c o n s i d e r a t i o n s ,
the u n d erestim atio n of divergence
t i m e s u s i n g p v a l u e s f o r i n t e r - f a m i l y c o m p a r i s o n s ( T a b l e 7) i s n o t
surprising.
O t h e r t e c h n i q u e s , s u c h a s DNA-DNA h y b r i d i z a t i o n
(S ibley
and A h l q u i s t , 1983, 1984) may b e more s u i t a b l e f o r o b t a i n i n g e s t i m a t e s
of n u c l e o t i d e sequence d iv erg e n ce f o r such d i s t a n t l y r e l a t e d taxa.
I n t r a - s p e c i f i c comparisons
The e s t i m a t e d g e n e t i c d i s t a n c e b e t w e e n c o n s p e c i f i c s i n T a b l e 6
range
from
0.0000
in trasp ecific
to
0.0870.
com parisons,
E lk and
displayed
cattle,
not
listed
n o ratDNA v a r i a t i o n
in
the
betw een
c o n s p e c i f i c s (p=0).
The l o w e r o f t h e t w o p v a l u e s f o r i n t r a s p e c i f i c c o m p a r i s o n s o f
P e r o m y s c u s sp p . i n T a b l e 6 was d e r i v e d fro m c o m p a r i s o n s o f i n d i v i d u a l
m i c e w i t h i n l o c a l p o p u l a t i o n s and t h e h i g h e r v a l u e fro m c o m p a r i s o n s o f
geographically
separated
p o p u la tio n s (Avise e t a l . ,
197 9a ).
Av is e e t
a l . (1979b) o b t a i n e d s i m i l a r lo w p v a l u e s f o r l o c a l and h i g h p v a l u e s
for g eo g rap h ically
humans,
separated
p was d e r i v e d
d ifferent
races
p o p ulations
o f Georoys p i n e t i s .
from c o m p a r is o n s of s e v e r a l i n d i v i d u a l s of
(Brown,
1980)
and
for
Lepom is
m acrochirus
c o m p a r i s o n s o f two d i s t i n c t s u b s p e c i e s ( A v i s e e t a l . ,
Intraspecific
to
be
sim ilar
appears,
in
however,
variatio n
a ll
For
w ithin
species
in
lim ited
Table
t h a t l a r g e mammals ( d e e r ,
1984).
geographic a re a s
6
from
appears
(p=0.0026-0.0084).
It
b i s o n and hum ans ) e x h i b i t
32
less
in tra-sp ecific
v ariatio n
in
s u b s p e c i e s and g e o g r a p h i c a r e a s
The a n a l y s e s
of
deer
and
mtDNA ( p = 0 . 0 0 0 0 - 0 . 0 0 5 3 )
betw een
t h a n r o d e n t s and f i s h ( p = . 01 5-0.087).
b ison
mtDNA i l l u s t r a t e
th is
p o in t.
Wood b i s o n a n d p l a i n s b i s o n a r e c o n s i d e r e d m o r p h o l o g i c a l l y d i s t i n c t
s u b s p e c i e s ( G e i s t and K a f s t e n ,
mtDNA and o t h e r w or k w i t h
1977;
McDonald,
1981).
My a n a l y s i s
b l o o d g r o u p s (P ed en and K r a a y ,
of
1979), b l o o d
p r o t e i n s (D. Y a r d l e y , p e r s o n a l c o m m u n i c a t i o n ) , a n d k a r y o t y p e s ( Y i n g
and P e de n,
1977) f a i l e d
to d e te c t d i f f e r e n c e s between th e su b sp ecies.
As W i t h b i s o n , d e e r s h o w e d l i t t l e
o r ho s u b - s p e c i f i c v a r i a t i o n
mt.DNA ( T a b l e s
co n serv atio n
4,
5 and 6 ).
T his
of
mtDNA s e q u e n c e
between g e o g ra p h ic a lly s e p a ra te d p o p u l a t i o n s o f a r t i o d a c t y l s
due t o e x t e n s i v e m i t o c h o n d r i a l ge n e f l o w ,
in
may b e
s e l e c t i o n f o r c e r t a i n mtDNA
sequences or r e l a t i v e l y r e c e n t f o u n d in g e v e n t s and s h o r t d i v e r g e n c e
tim es.
The s i t u a t i o n
is
apparently d iff e re n t
in the geographically
v a r i a b l e r o d e n t s and f i s h .
I n t r a - f a m i l y , i n t e r - s p e c i e s comparisons
E x c e p t f o r c o m p a r i s o n s o f M ontana m ule d e e r w i t h w h i t e - t a i l e d
d e e r th e p v a lu e s 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 com parisons in Table
6 are
interm ediate
to
the
intra-species
and i n t e r - f a m i l y
g e n e r a l l y c o n s i s t e n t w i t h d a ta from o t h e r s t u d i e s .
v a l u e s and
The e s t i m a t e d
d i v e r g e n c e t i m e s f o r t h e s e g r o u p s ( T a b l e 7) a r e l i k e w i s e i n g e n e r a l
a g re e m e n t , w i t h t h e f o s s i l
r e c o rd e x c e p t f o r th e d e e r - e l k comparison-
Osborn (1 91 0) ,
S c o t t (1937) and G e i s t (1981) n o t e t h a t t h e s u b f a m i l i e s
C ervinae
O d o co ilin ae,
and
represented
by C e r v u s
and
O docoileus
r e s p e c t i v e l y , may h a v e b e e n s e p a r a t e l i n e a g e s s i n c e t h e M i d c e n e o r
33
earlier.
Baccus e t a l .
r e la tiv e gen etic
(1983) u s i n g p r o t e i n
e le c tro p h o re s is obtained
d i s t a n c e v a l u e s b e t w e e n c e r v i d s i n d i c a t i v e Of t h i s
su b fam ilial rela tio n sh ip .
T h is would i n d i c a t e
our e s tim a te s of
d i v e r g e n c e , p , and d i v e r g e n c e tim e a r e u n d e r e s t i m a t e s a s w ith th e
i n t e r - f a m i l y com parisons.
N on eth eless, our p value in d ic a te s th a t
t h e s e two d e e r s p e c i e s a r e more c l o s e l y r e l a t e d t o e a c h o t h e r t h a n t o
m em bers
of
the
other
fam ilies.
Since
the
fo ssil
record
is
not
d e f i n i t i v e regarding the o r ig in of these su b fa m ilie s fu rth u r a n aly sis
o f n u c l e a r and m i t o c h o n d r i a l DNA may c l a r i f y t h e d e g r e e o f d i v e r g e n c e
of th ese groups.
Storm ont e t a l .
same genus
as
( 1 961) p r o p o s e d t h a t b i s o n be i n c l u d e d i n th e
cattle,
B o s , due
Baccus e t a l . (1983), r e p o r t i n g
electro p h o resis,
in th is
agreed
to
sim ila ritie s
of
blood
groups.
s i m i l a r i t y , o f p r o t e i n s d e t e c t e d by
with th is
suggestion.
The p v a l u e o b t a i n e d
s t u d y f o r t h e s e two s p e c i e s i s c o m p a r a b l e t o t h o s e from o t h e r
c o n g e n e r i c c o m p a r i s o n s ( T a b l e 6) b u t more i n t e r - g e n u s c o m p a r i s o n s ( i n
a d d itio n
to
d eer-elk )
are
needed
to
estab lish
c rite ria
for
classificatio n .
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
A lack of q u a n t i t a t i v e measurement o f m o r p h o l o g i c a l c h a r a c t e r s
used
to d i s t i n g u i s h
c o l l e c t o r ’s j u d g e m e n t .
reported
to
have
the
species
led
us to c a t e g o r i z e
d e e r by t h e
None o f t h e f i v e a l b u m i n h e t e r o z y g o t e s
in term ed iate
m orphology.
For
some
other
were
deer
i n t e r m e d i a t e c h a r a c t e r s were r e p o r t e d (eg. a w h i t e t a i l w ith l a r g e
34
e a r s , or a m ule d e e r w i t h w h i t e t a i l ty p e a n t l e r s ) .
In a l l c a se s but
one t h e s e a n i m a l s g a v e a l b u m i n p a t t e r n s i n a g r e e m e n t w i t h t h e o v e r a l l
m orphological sp e c ie s d e sig n a tio n .
The o n e e x c e p t i o n
was a m a l e
d e s c r i b e d a s h a v in g W h i t e h a l l a n t l e r s , head and r u n n i n g g a i t , m u le
d e e r rump and t a i l and was a s s o c i a t e d
U nfortunately,
w ith a group of w h i t e t a i l does.
no mtDNA a n a l y s i s was p o s s i b l e b u t he g a v e t h e t y p i c a l
W h i t e h a l l a l b u m i n p a t t e r n . T h i s d e e r was e x c l u d e d f ro m t h e a l b u m i n
d a t a i n T a b le I s i n c e he d id n o t f i t e i t h e r m o r p h o l o g i c a l c a t e g o r y .
Deer w i t h i n t e r m e d i a t e m o r p h o l o g y a r e p e r i o d i c a l l y
w ild life
b io lo g ists
communication).
a l b u m i n and
and
Thes e d e e r ,
hunters
in
M ontana
reported
(M ackie,
by
personal
t h e a p p a r e n t s p e c i e s - s p e c i f i c i t y o f serum
o c c u rr e n c e of r a r e heterozygotes suggests th at lim ited
h y b r id iz a tio n occurs.
Since t h e r e
was no i n d i c a t i o n
of interm ediate
m o rp h o lo g y i n th e a lb u m in h e t e r o z y g o t e s i t c a n n o t be d e te r m i n e d i f
they r e s u l te d
f ro m r e c e n t h y b r i d i z a t i o n o r i n t r o g r e s s i o n o f t h e r a r e
a l l e l e i n t o e a ch s p e c i e s i n th e more d i s t a n t p a s t .
S i n c e mtDNA i s
m a t e r n a l l y i n h e r i t e d , h e t e r o z y g o t e m u l e d e e r n o . 7, w i t h m u l e d e e r
s p e c i f i c t y p e A mtDNA, h a s p r o b a b l e m u l e d e e r f e m a l e a n c e s t r y a n d
w h ite ta il p a te rn al in flu en ce, w h ile w h i t e t a i l h e te r o z y g o t e s nos.
and 19,
w ith w h ite ta il
sp ecific
t y p e C mtDNA, a r e i n a w h i t e t a i l
fe m a le l i n e a g e w i t h m ule d e e r p a t e r n i t y i n t h e i r p a s t .
th a t hybridization
between th e s e
18
It appears
two s p e c i e s may i n v o l v e e i t h e r s e x .
The mtDNA d a t a i n d i c a t e t h a t M o n t a n a m u l e d e e r c o n s i s t o f a t
least
three
m aternal
populational v a ria n ts
fro m
other
sampled
lin es,
while
types
type
populations.
A and
B appear
D may r e p r e s e n t
to
be
a lineage
Montana w h i t e t a i l s
in traisolated
are in a fourth
35
distinct
lineage.
fem ales breed a t
These d a t a
all,
suggest
they w i l l
that
at
present,
if
F - I hybrid
breed back to the m other's s p e c ie s ,
keeping th e m a te rn a l lin e a g e s p e c i e s - s p e c i f i c .
D espite these d i s t i n c t i o n s
t h e o v e r a l l s i m i l a r i t y o f mtDNA t y p e s
b e tw e e n th e d e e r s p e c i e s i n M o n ta n a , and th e d i s s i m i l a r i t y b e tw e en
M ontana m u le d e e r and S i t k a b l a c k t a i l s '
mtDNA
re q u ire explanation.
B l a c k t a i l s and m ule d e e r , b o th s u b s p e c i e s of
m ore
g en etically
sim ilar
to
each
o th er
h e m i o n u s s h o u l d be
than
eith er
is
to
0.
virginianus.
The d a t a f o r se rum a l b u m i n s u p p o r t t h i s
d a t a do n o t .
C arr (p e rs o n a l communication) r e p o r t s d i g e s t i o n p r o f i l e s
o f T exas m ule d e e r and
w h itetails
sim ilar
from
to
my s a m p l e s
and
M ontana,
South
w h i l e t h e mtDNA.
C arolina
Illin o is
and
w h itetails
G eorgia
deer.
C a l i f o r n i a m ule d e e r a r e q u i t e d i f f e r e n t f o r a t e n enzyme b a t t e r y ,
w i t h a n Eco RI p a t t e r n s i m i l a r t o my S i t k a b l a c k t a i l .
I f t h e mtDNA o f
t h e S i t k a b l a c k t a i l and C a l i f o r n i a m u le d e e r r e p r e s e n t s t h e a n c e s t r a l
0 . hem ionus
type
and
that
a n c e s t r a l 0. v i r g i n i a n u s t y p e ,
in e a ste rn
w h itetails
A p la u sib le ex p lan atio n for th is
s u g g e s t e d by 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 ) i s
inter-subspecies
et al.
introgression
o f mtDNA
Other examples of p o s s ib l e i n t e r - s p e c i e s
t r a n s f e r o f mtDNA i n a n i m a l s a r e
( 1 983a), P o w ell (1983), A vise e t a l.
U z z e l l (1986).
the
t h e n t h e Montana (and T e x a s ) mule d e e r
a c t u a l l y have th e w h i t e t a i l type.
follow ing h y b rid izatio n .
represents
or
r e p o r t e d by F e r r i s
( 1 984) and S p o ls k y and
I f i n t r o g r e s s i o n o f w h i t e t a i l mtDNA i n t o m u le d e e r h a s
o c c u r r e d i n t h e p a s t o u r o b s e r v a t i o n s s u g g e s t t h a t i n M o n tan a some
d i v e r g e n c e h a s o c c u r r e d b e tw e en th e s p e c i e s ' m i t o c h o n d r i a l genomes
since the i n i t i a l
h y b rid iz atio n event(s).
36
The d a t a
seem
to
support
the
sym patric
theory
of
o rig in
of
i s o l a t i n g m e c h a n i s m s ( Mayr, 1970) w h ic h p o s t u l a t e s t h a t f o l l o w i n g
geographic
freely .
sp eciatio n ,
in cip ien t
species w ill at
first
hybridize
T h i s i s f o l l o w e d by r e d u c e d l e v e l s o f h y b r i d i z a t i o n a f t e r
s e le c tio n a g a in s t hybrids r e s u l t s
mechanisms.
in e ffe c tiv e reproductive iso la tin g
h e m i o n u s and Ol v i r g i n i a n u s
P l e i s t o c e n e (K urte'n and A n d e r s o n ,
probably sp e c ia te d
in
the
1980) p e rh a p s d u r i n g g e o g r a p h ic
s e p a r a t i o n due to c o n t i n e n t a l g l a c i e r s .
A r e l a t i v e l y high le v e l of
h y b r i d i z a t i o n b e t w e e n t h e s p e c i e s a f t e r c o n t a c t was r e - e s t a b l i s h e d
could have
resu lted
w h itetails
in to
in
the a p p aren t
m ule d e e r.
i n t r ogress ion
of
mtDNA f r o m
Subsequent c h a ra c te r d isp lacem en t of
behavior,
m orphology and h a b i t a t
requirem ents,
th e divergence of
nucleotide
s e q u e n c e s and r e d u c e d l e v e l s o f h y b r i d i z a t i o n
between the
s p e c i e s may a c c o u n t f o r t h e s p e c i e s - s p e c i f i c i t y o f t h e s e t r a i t s i n
e x t a n t p o p u l a t i o n s i n Montana.
P erhaps m ule d e e r p o p u l a t io n s w ere
i n i t i a l l y e s t a b l i s h e d by
m ales d i s p e r s i n g i n t o unoccupied h a b i t a t and b r e e d i n g w i t h r e s i d e n t
w hitetail
back to
fem ales.
The F - I h y b r i d f e m a l e s may h a v e s u c c e s s f u l l y b r e d
m ule d e e r m a l e s e s t a b l i s h i n g a p o p u l a t i o n w i t h m ule d e e r
m orphology
and
w h itetail
mtDNA.
The
apparent
fix atio n
of
the
w h i t e t a i l t y p e mtDNA i n m u l e d e e r p o p u l a t i o n s m ay b e d u e t o r a n d o m
d r i f t i n s m a ll founding p o p u la tio n s ,
t h i s phenomenon
m ito ch o n d rial
allo p atric
but the widespread occurrence of
may demand a n o t h e r e x p l a n a t i o n .
and
n u clear
populations,
genomes,
behavior
F u r t h u r study, of
of
sym patric
and
c o n t r o l l e d b r e e d i n g and r e c o n s t r u c t i o n o f t h e
37
species'
phylogenies
from
the
fo ssil
record
e x p l a i n t h e d i s t r i b u t i o n o f mtDNA r e p o r t e d h e r e .
are
needed
to
fu lly
38
CONCLUSIONS
The r e s u l t s f r o m i n t r a -
and i n t e r - s p e c i e s c o m p a r i o n s i n d i c a t e
that:
I.
mtDNA
restric tio n
fragm ent
an aly sis
may
resu lt
in
u n d e re s tim a te s of div erg en ce fo r i n t e r - f a m i l y comparisons o f
artiodactyls.
Q)
E stim ates
o f bash
su b stitu tio n s
per n u cleo tid e,
i n t r a - s p e c i e s com parisons of a r t i o d a c t y l s
0 .000 t o 0.008 w h i l e i n t r a - f a m i l y ,
p,
for
are g en erally
i n t e r - s p e c i e s comparisons
r e s u l t i n p v a l u e s o f 0.05 t o 0 .0 9 .
X'
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 s from p v a l u e s and t h e f o s s i l
record a re s im i la r fo r sp e c ie s (black b e a r - g r iz z ly b e a r ,
cattle-bison)
The r e s u l t s
indicate
1.
s e p a r a t e d I t o 3 mybp.
from
co m parisons
of
w h ite-tailed
and
mule d e e r
th at:
L im ited
species
h y b rid izatio n
in
M ontana,
probably
both
sexes
occurs
of
betw een
both
the
species
deer
may b e
p articipating.
2.
mtDNA,
serum a l b u m i n and m o r p h o l o g y a p p e a r d i s t i n c t b e t w e e n
Montana m u le and w h i t e - t a i l e d
deer,
suggesting th at
s p e c i e s gene flow i s n o t e x t e n s i v e a t p r e s e n t .
species - sp e c ific ity
of
mtDNA s u g g e s t s
that
in ter­
The a p p a r e n t
F-I
f e m a le s b reed back to th e m o th e rs' s p e c i e s i f a t a l l .
hybrid
39
3.
There i s
apparently lim ite d
m ito c h o n d r ia l g e n e .flo w between
m u le d e e r from t h e B i g h o r n / P r y o r M o u n ta i n s a r e a and o t h e r
sampled l o c a t i o n s .
4.
mtDNA i n b o t h d e e r s p e c i e s i n M o n t a n a a n d Wyo ming i s v e r y
s i m i l a r to t h a t of e a s t e r n w h i t e t a i l s , and d i f f e r e n t from
Sitka
blacktails
type i n t o
suggesting in tr o g re s s io n of the W hitehall
m u le d e e r p o p u l a t i o n s
some t i m e i n t h e p a s t .
follow ing
hybridization
at
40
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47
APPENDIX
Standard procedures f o r e x tr a c t i o n
o f mtDNA f r o m a n i m a l t i s s u e s
a d a p t e d f r o m P o w e l l a n d Z u r fi g a ( 1 9 8 4 ) a n d D a v i s e t
g iv e n below .
al.
(1980) a r e
K e e p i n g t i s s u e s a n d t u b e s on i c e a t a l l t i m e s u n l e s s
o th erw ise noted:
1.
M ince 10-25 g t i s s u e from e a c h a n im a l w i t h a s c a l p e l .
electric
blender at
l o w s p e e d may b e u s e f u l
for
An
m uscle
sam plesi
2.
Hand h o m o g e n i z e i n a n e q u a l v o l u m e o f h o m o g e n i z i n g b u f f e r
(0.2 M s u c r o s e ,
0.015%
(v /v )
I mM EDTA, 2.5 mM C a C l 2 » 0 . 0 3 M t r i s - H C l ,
T rito n -X
homogenizer u n t i l t i s s u e
3.
100,
is
fin a l
pH 7 . 5 )
in
a dounce
liquified.
C e n t r i f u g e 1 00 0 X g f o r t e n m i n . a t 4C t w i c e f o r b r a i n o r
m uscle,
three
tim es
for liv e r
or kidney.
This
removes
n u c l e i and c e l l u l a r d e b r i s .
4.
C e n t r i f u g e t h e s u p e r n a t a n t f ro m s t e p 3 a t
14 ,000 X g 40 min
a t 4C t o p e l l e t m i t o c h o n d r i a .
5.
S u s p e n d p e l l e t i n a b o u t 5 ml 0 . 2 5 M s u c r o s e ,
0. 0 5 M M g C ^
f o r a t o t a l v ol u m e o f t e n ml.
6.
Add I ml DNase I ( 1 0 m g / m l s t o c k ) p e r 1 0 ml s o l u t i o n a n d
i c u b a t e 30 m i n . a t 37C.
especially
tissu es
T h i s r e m o v e s n u c l e a r DNA a n d i s
i m p o r t a n t when n u c l e a r l y s i s
w hich
have
been
te m p e ra tu re s post-m ortem .
frozen
or
is
extensive as in
exposed
to
warm
48
7.
Add I ml 0 . 5 M EDTA p e r 10 ml s o l u t i o n t o q u e n c h t h e DNase
reaction.
8.
C entrifuge
at
14,000
X g 20
m in
at
4C
to
rep e lle t
mitoch o n d ria.
9.
1 0.
Suspend p e l l e t i n 5 - 1 0 ml I M EDTA,
0.2 M T r i s - H C l pH 8.5
Add I m l 10% SDS ( so d iu m d o d e c y l s u l f a t e ) p e r 10 ml s o l u t i o n
to ly se m ito ch o n d ria.
11.
Add 100 u l
proteinase-K
(lOmg/ml s t o c k )
p e r 10 ml s o l u t i o n
and i n c u b a t e a t 6 OC f o r two h o u r s .
12.
Add 4 ml
5 M potassium
acetate
per
10
ml
so lu tio n
to
p r e c i p i t a t e SDS a n d p r o t e i n s a n d l e a v e on i c e a t l e a s t 30
m in .
13.
C e n t r i f u g e a t l e a s t 9000 X g 45 min. a t 4C.
14.
C a r e f u l l y remo ve c l e a r s u p e r n a t a n t c o n t a i n i n g n u c l e i c
w ithout d istu rb in g
15 .
acids
the SD S-protein p e l l e t .
P r e c i p i t a t e n u c le ic a c id s w ith th e a d d itio n of 2-3 tim es th e
s o l u t i o n ' s v o l u m e o f 95% e t h a n o l and k e e p a t —2 OC 30-90 min.
maximum.
16.
C e n t r i f u g e a t l e a s t 9000 X g f o r 3 0 - 6 0 min.
a t 4C t o p e l l e t
nucleic a c id s .
17 .
Wash p e l l e t i n 70% e t h a n o l t o rem o v e s a l t s , and r e c e n t r i f u g e
a s i n s t e p 16 f o r 20 m i n .
18 .
D i s c a r d e t h a n o l b e i n g c a r e f u l n o t t o p o u r o u t t h e p e l l e t and
a llo w th e p e l l e t to a i r dry.
19.
Sus pend p e l l e t i n 100—200 u l 0.01 M T r i s ,
5 mM EDTA, ph 7.8.
for
The f i n a l
s o l u t i o n w i l l be c l e a r f o r b r a i n and m u s c l e and a m b e r
liv er
kidney.
or
This
yields
about
2-5
ug
mtDNA.
B rain
p r e p a r a t i o n s a r e a l m o s t a l w a y s d i g e s t a b l e w i t h r e s t r i c t i o n en zymes.
O th e r t i s s u e s a r e u s u a l l y d i g e s t a b l e i f p r o c e s s e d w i t h i n a m onth of
d e a t h b u t may r e q u i r e u s e o f e x c e s s enzyme d u r i n g d i g e s t i o n o r f u r t h u r
p u r i f i c a t i o n by p h e n o l e x t r a c t i o n o r c e s i u m c h l o r i d e
( M a n i a t i s e t a l . , 1982; L ansm ah e t a l . , 1981).
cen trifu g atio n
M ONTANA ST A T E U N IV E R SIT Y L IB R A R IE S
stks N378.C881
Genetic relationships between white-tail
3 1762 00512051 2