View
3
Download
0
Category
Preview:
Citation preview
MOLECULAR SYSTEMATICS OF THE AGKISTRODON COMPLEX
by
RALPH ALEXANDER KNIGHT, JR., B.S., M.S.
A DISSERTATION
IN
BIOLOGY
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Apptiqved
Accepted
May, 1991
- LB
/^?/ JV^ ^^?/^ /^n JX^ ACKNOWLEDGMENTS
I thank Dr. Llewellyn D. Densmore for his assistance and
support during the course of my studies at Texas Tech
University. I am very appreciative of Dr. Robert J. Baker
for the invaluable guidance and inspiration he provided.
Dr. Jonathan A. Campbell was unfailing in his assistance,
continuously providing helpful discussions, specimens,
guidance, and encouragement. I thank Dr. Robert D. Owen for
training in data analysis and many helpful discussions.
Dr. John M. Bums deserves a great deal of credit for
creating an atmosphere in the department where graduate
studies flourish. In addition to the above named advisory
committee members, I wish to thank Dr. Francis L. Rose for
his many helpful discussions, reviewing of manuscripts, and
contribution of specimens.
Many individuals contributed specimens or collecting
locality information. Without their assistance and
generosity, this work could not have been accorrplished. They
are: Charles C. Carpenter, Robert Dean, John Greene, David
Kizirian, William W. Lamar, David R. Long, Randall
Montgomery, Louis Porras, Buzz Ross, Thomas Schultz and the
San Diego Zoo, and Robert T. Zappalorti.
Kelly D. Cahill and Lesley Ann Baker assisted with
verification of data. Drs. Marc Allard, James Derr, and
David P. Mindell freely provided crucial assistance in PCR
• •
11
methodology. Dr. Randy Allen always kept his lab open to me,
allowed use of his computers, equipment, and provided helpful
discussions. I thank P. Scott White and Robert D. Bradley
for helpful discussions. Photographs for figures were
provided by Jonathan A. Caitpbell (Fig. li), Louis Porras
(Fig. 12) and Gordon Schuett (Fig. 3).
Lastly, I extend my gratitude to my father, Ralph A.
Knight, Sr., and to Margaret A. Knight, for the help they
provided in so many ways that enabled me to reach this goal,
so that I may now spend my future observing and describing
Nature. I thank my father for teaching me materialism, the
philosophy of science. This work is dedicated to the memory
of my mother, June Linder Knight.
This study was funded in part by grants from Sigma Xi
Grants-in-Aid of Research (RAK), Texas Tech University
Graduate School Stipend (RAK), NSF #BSR-8607420 (LDD) and
Texas Advanced Research Program Grant #003644-140 (LDD).
« • •
111
CONTENTS
ACKNOWLEDGMEITTS ii
ABSTRACT vi
LIST OF FIGURES viii
LIST OF ACROLJYMS X
I. INTRODUCTION 1
Purpose and Scope 1
New World Agkistrodon 3
Old World Agkistrodon 4
Allied Genera 6
Problems in Systematics of the Agkistrodon Complex 7
Objectives of this Study 10
Comparative Analysis of Mitochondrial DNA.... 11
The polymerase Chain Reaction 12
II. METHODS AND MATERIALS 40
Samples 4 0
DNA Isolation 41
Restriction Endonuclease Digestion 43
In Vitro DNA Amplification 44
Sequencing of DNAs 45
Data Analysis 4 6
III. RESULTS 49
Restriction Fragment Analysis 49
16S Sequence Analysis 52
IV
IV. DISCUSSION 66
Phylogeography of Agkistrodon contortrix 66
Phylogeography of Agkistrodon piscivorus 68
Evolution of New World Agkistrodon 69
Status of the Genus Gloydius 71
Allied Genera 7 2
Future Research 7 3
LITERATURE CITED 7 5
APPENDICES
A. RESTRICTION FRAGMENT DATA 80
B. 16S RIBOSOMAL GENE SEQUENCE DATA 93
V
ABSTRACT
This Study focused on the evolution and systematics of
the pitviper genus Agkistrodon and other genera traditionally
allied with Aakistrodon: Calloselasma. Deinagkistrodon and
Hypnale. One aim was to complement traditional work by
providing the first molecular systematic data sets for the
Aakistrodon complex, based on restriction fragment and
sequence analyses of mitochondrial DNAs.
Restriction fragment analysis showed low genetic
variation among . contortrix populations, suggesting a
recent radiation of the modern races. Geographic
distribution of mitochondrial DNA genotypes among A.
contortrix populations was discordant with distribution of
morphological variation. The divergence between eastern and
western A. piscivorus was of a degree greater than that
observed between some species, and suggested that populations
were isolated in refugia since the onset of the Pleistocene.
New World Aakistrodon is a monophyletic group.
Restriction fragment and 16S ribosomal gene sequence data
suggested an A. bilineatus-A. piscivorus association, with A.
contortrix the basal lineage of New World Agkistrodon. A
relatively large degree of independent evolution has occurred
along the A. contortrix lineage. Results of both analyses
showed that the inclusion of Asian and New World species
together forms a polyphyletic group, thereby supporting
vi
recognition of genus the Gloydius for the Asian species.
Sequence obtained for Hypnale was different from other
pitvipers and was not useful for phylogenetic purposes.
Calloselasma rhodostoma and Deinagkistrodon acutus appeared
to be remnants of an early pitviper radiation, and the
validity of their independent generic status was confirmed.
VI1
LIST OF FIGURES
FIGURE PAGE
1. Aakistrodon contortrix mokasen. the northern copperhead 14
2. Aakistrodon contortrix contortrix. the southern copperhead 16
3. Aakistrodon contortrix pictigaster. the Trans-Pecos copperhead 18
4. Aakistrodon piscivorus piscivorus. the eastern cottonmouth moccasin 20
5. Aakistrodon bilineatus taylori. the ornate cantil 22
6. Aakistrodon blomhoffii ussuriensis.the Ussuri mamushi 24
7. Aakistrodon halys halys. the Siberian pitviper... 26
8. Aakistrodon halys caraganus, the Karaganda pitviper 28
9. Agkistrodon intermedins saxatilis. the rock mamushi 30
10. Calloselasma rhodostoma. the Malaysian pitviper.. 32
11. Deinagkistrodon acutus. the hundred-pace snake... 34
12 . Hypnale hypnale. the hump-nosed pitviper 36
13. Proposed relationships among the three species of New World Agkistrodon 38
14. Autoradiograph of Hind III digested pitviper mtDNAs 56
15. UPGMA phenogram based on shared restriction fragments 58
16. PAUP (v. 2.4.1) strict consensus tree derived from analysis of 292 variable restriction fragments 60
Vlll
17. PAUP (v. 3.On) strict consensus of two shortest trees derived from an exhaustive search of aligned DNA sequences 62
18. PAUP (v. 3.On) maximum parsimony tree using C. rhodostoma and D. acutus as the outgroup 64
IX
LIST OF ACRONYMS
t>P base pairs
<^TP deoxyadenosine triphosphate
DNA deoxyribonucleic acid
EDTA ethylenediaminetetracetic acid
kb kilobase pairs
mtDNA mitochondrial DNA
Myr million years
PCR the polymerase chain reaction
RNA ribonucleic acid
rRNA ribosomal RNA
SDS sodium dodecyl sulfate
UPGMA unweighted pair group method of arithmetic averages
CHAPTER I
INTRODUCTION
Purpose and Scope
This study employs comparative analyses of mtDNAs in
order to better understand the evolution and systematics of
pitvipers of the genus Aakistrodon and three other genera
traditionally allied with Aakistrodon. The genera
Agkistrodon, CalloseXasma, Deinagkistrodon. and Hypnale. have
been collectively termed the "Aakistrodon complex." These
snakes are the subject of a recent monograph by Howard K.
Gloyd and Roger Conant (1990). The primary treatise is the
result of over a half-century of study, and reviews
morphology, zoogeography and phylogeny of the group. Also
included are ancillary papers by Carpenter and Gillingham,
Cole, Conant, Gloyd, Hardy, Kardong, Malnate, Minton, and Van
Devender and Conant, that address (respectively) behavior,
chromosomes, fossil history, palearctic species, venoms,
skull, bone and muscle variation, hemipenial structure,
immunological relationships, and historical biogeography of
these snakes (Gloyd and Conant, 1990).
One aim of my molecular study is to augment the work
presented in Gloyd and Conant's (1990) monograph by providing
the first data sets for this group derived from molecular
analyses. Snake phylogeny is particularly
difficult to assess by way of morphology. In general, snakes
possess fewer variable morphological characters suitable for
comparison than many other groups. Compounding this lack of
characters is convergent evolution and retention of primitive
character states. Considering these difficulties, molecular
data holds considerable promise to provide the most powerful
tool for achieving an understanding of the evolution of
snakes and taxonomy that reflects that evolutionary history.
The genus Aakistrodon was named by Palisot de Beauvois
(1799, p. 381). In accordance with naturalists of his day,
Palisot de Beauvois used rather general characters in his
description:
Agkistrodon. Large scales under the belly and tail. No rattles. The extremity of the upper jaw furnished with two hollow fangs or canine teeth. Venomous. In this division should be arranged the mokason.
Gloyd and Conant (1990) present a lengthy description of the
genus and include three New World species and eight Old World
species, including a new species, A- shedaoensis. described
shortly before publication (Zhao, 197 9). The three other
genera of the Agkistrodon complex, Calloselasma.
Deinagkistrodon. and Hypnale> are Asian.
It has been presumed that Agkistrodon or its progenitor
originated in Asia and spread to the New World, where it
gave rise to other New World pitviper genera (Brattstrom,
1964; Burger, 1971; Van Devender and Conant, 1990). Given 2
the present distribution of pitvipers in central and eastern
Asia and the New World, and their absence along the eastern
side of the Atlantic, a Beringean route seems probable. The
fossil record for pitvipers is poor, and the timing of this
colonization is speculative. Brattstrom (1964) hypothesized
a Cretaceous-Eocene crossing for pitvipers. Van Devender and
Conant (1990) speculated an early Miocene colonization. In
order to familiarize the reader with the Agkistrodon complex
species, a brief summary of taxa and their distribution
follows.
New World Agkistrodon
Agkistrodon contortrix. the copperhead
The type species of the genus, A- contortrix presently
includes five subspecies: the northern copperhead, h- Q-
mokasen (Fig. 1), in northeastern USA; the southern
copperhead, h. Q- contortrix (Fig. 2), along the Atlantic and
Gulf coastal plains; the Osage copperhead, A. Q. phaeogaster,
in the central USA; the broad-banded copperhead, h. £.
laticinctus. in the southwestern USA; and the Trans-Pecos
copperhead, A. Q- pictigaster (Fig. 3), in west Texas and
northeastern Mexico.
Agkistrodon piscivoniR. the cottonmouth moccasin
AgKistrodnn piscivorus presently includes three
subspecies. The Florida cottonmouth, A. p. conanti. occurs
in Florida, adjacent Georgia, and Alabama. To the north, the
eastern cottonmouth. A- p. piscivorus (Fig. 4), ranges
through much of the piedmont and coastal plain to
southeastern Virginia. The western cottonmouth, A. p.
leucostoma, is found in the southcentral and southwestern
USA.
Agkistrodon bilineatus. the cantil or Mexican moccasin
Aakistrodon bilineatus includes four subspecies. The
ornate cantil, A. h. taylori (Fig. 5), occurs in northeastern
Mexico. The Yucatecan cantil, A. h- russeolus. is known from
Yucatan and Belize. The common cantil, A. h. bilineatus.
ranges along the Pacific drainage of Mexico and Central
America from southern Sonora south to El Salvador. The
castellana, A. h- howardgloydi. occurs in Costa Rica,
Honduras, and Nicaragua. Agkistrodon h. russeolus and A. b.
howardgloydi are the only named forms of New World
Agkistrodon not included in this study.
Old World Agkistrodon
Monotypic species
Five of the eight Asian species of Aakistrodon are
monotypic. These forms (A. caliginosus. A. himalayanns^ .
monticola, A. shedaoensis, and A. strauchi) are rarely
imported into the western world. They were not included in
this analysis.
Agkistrodon blomhoffii, the mamushi
Aakistrodon blomhoffii ranges from southeastern China
northward to far eastern USSR, throughout the Korean
peninsula, and Japan. Agkistrodon blomhoffii includes the
subspecies blomhoffii, brevicaudus. dubitatus. sj.niticus> and
ussuriensis. It was represented in this study by the
subspecies A. h- ussuriensis. the Ussuri mamushi, from the
USSR (Fig. 6).
Agkistrodon halys. Pallas' viper
Agkistrodon halys occurs across central Asia. It
includes four subspecies. Figures 7 and 8 depict the two
subspecies represented in this study. The Siberian pitviper,
A. h. halys (Fig. 7), occurs in southern Siberia and
Mongolia. The Karaganda pitviper, A. h. caraganus (Fig. 8),
occurs from the Caspian Sea eastward across the central Asia
to western China. The Alashan pitviper, A. h. cognatus.
occurs in central China. Agkistrodon h. bohemei is a newly
described subspecies from Afghanistan.
Agkistrodon intermedins, the intermediate mamushi
Agkistrodon intermedins ranges across Asia from west and
south of the Caspian Sea in Azerbaydzhan and northern Iran
eastward to far eastern USSR and Korea. Four subspecies are
recognized. The central Asian pitviper, A. i. intermedius.
occurs across central Asia from eastern Kazakhastan through
northern China and Mongolia. The Caucasian pitviper, A. i.
caucasicus, occurs south and east of the Caspian Sea, chiefly
in Iran. The Gobi pitviper, A. i. stejnegeri. occurs in
central eastern China. The rock mamushi, A. i. saxatilis.
which represents the species for this study (Fig. 9), occurs
in the far east in China, the U.S.S.R., and Korea.
Allied Genera
During the history of pitviper taxonomy, three other
forms were often considered congeneric with Agkistrodon. but
all three have been relegated to distinct genera. These
Asian genera are considered closely allied to Aakistrodon by
traditional taxonomists (Gloyd and Conant, 1990) on the basis
of similarity due to shared, primitive traits. Two of these
genera, Calloselasma and Deinagkistrodon. are monotypic. The
other, Hypnale. is polytypic.
Calloselasma rhodostoma (Fig. 10) is known to occur in
Thailand, Laos, Cambodia, Vietnam, northern West Malaysia,
and Java. The hundred-pace viper or sharp-nosed viper,
Deinagkistrodon acutus (Fig. 11) occurs in extreme northern
Vietnam, southeastern China and Taiwan.
Genus Hypnale includes three species. The hump-nosed
viper, H. hypnale (Fig. 12), occurs in southwestern India and
Sri Lanka. The Sri Lankan hump-nosed viper, H. nepa. occurs
in southwestern Sri Lanka. Wall's hump-nosed viper, H-
wajli, is also known only from southwestern Sri Lanka.
Problems in Systematics of the Aakistrodon Complex
Phylogeography of A. contortrix and A. piscivorus
Subspecific divisions of A. contortrix and A- piscivorus
are based on exomorphology, that is, color pattern and, to a
lesser degree, scale counts (Gloyd and Conant, 1990). No
molecular assessment of genetic variation among the races of
these species existed. Comparative molecular data are
important for understanding the evolution of these various
populations and recovering their biogeographic history.
Also, within-species data provide a basis for evaluating
divergence among species.
Phylogenetic Systematics of New World Agkistrodon
There are three ways to arrange three species in a
dendrogram (not counting a trichotomy). In this case, all
three ways have been proposed (Fig. 13). Brattstrom (1964)
compared osteology and hypothesized that A. bilineatnc? and A.
piscivorus are closely related and that A. contortrix is
distant from them and more closely related to the Asian
forms. Immunological data presented by Minton (1990) also
supported a close relationship of A. bilineatus and A.
piscivorous. Electrophoretic protein profiles of venoms
(Jones, 197 6), and exomorphology (Conant, 1986; Van Devender
and Conant, 1990) supported an A. bilineatus-A. contortrix
clade, with A. piscivorus a more basal lineage. Biochemical
comparison of skin keratins in vipers (Campbell and Whitmore,
1989) suggested a sister species relationship for A-
contortrix and A. piscivorus. and more divergent A-
bilineatus. with some Asian forms in between.
A recurring question concerns origins of the copperheads
and moccasins. Are they descended from a single species
after it spread from Asia, or did more than one Asian
ancestor give rise to the three modern New World species?
Brattstrom (1964, p.64) argued that Agkistrodon is divisible
into two main groups:
One of these groups (A. acutus-bilineatus-piscivorus) may have differentiated in the New World into the present-day piscivorus and bilineatus...The other group of Agkistrodon survives today mainly in the Old World (hypnale. halys. etc.), with only contortrix in the New World.
Note that members of each of these groups have since been
removed from Agkistrodon. while Agkistrodon still retains
members in each group. The skin keratin analysis of Campbell
8
and Whitmore (1989) also suggests the possibility that more
than one Asian lineage gave rise to the three New World
species. Gloyd and Conant (1990) consider New World
Agkistrodon monophyletic. The question of the phylogenetic
position of the three New World species, in relation to each
other and to Old World forms, has not been resolved.
Monophyly of the Genus Aakistrodon
According to the concept of a natural group (Darwin,
1859) and to the principles of modern systematic zoology
(Hennig, 1979; Wiley, 1981; Mayr and Ashlock, 1991), a genus
must be composed of a common ancestor (known or hypothetical)
and all of its descendents. if Agkistrodon. as it now stands
as a group of eleven species spanning two continents, meets
this criterion, then this taxonomic designation is valid.
If, on the other hand, the common ancestor of all species
assigned to Agkistrodon also gave rise to one or more other
genera, then the group is not natural, but is an artificial
construct, either paraphyletic or polyphyletic.
Gloyd and Conant (1990) argue for retention of the Asian
species in Agkistrodon. based on morphological similarity
without consideration of character state polarity. Hoge and
Romano-Hoge (197 8) erected the genus Gloydius to include the
Asian Agkistrodon species, on the basis of slight
morphological differences between Old and New World forms,
also without attempting to determine whether the characters
considered were primitive or derived. The crux of the matter
is that it remains to be demonstrated that the genus
Agkistrodon/ as currently conceived, is a monophyletic group.
The phylogenetic positions of Calloselasma. Deinaakistrodon. and Hvpnale
For well over half a century, these taxa were assigned
to Agkistrodon/ based on similarity in retention of primitive
characteristics. Their elevations to generic status were
based on notable morphological differences. Using
Immunoelectrophoresis, Minton (1990) concluded that they may
be remnants of an early pitviper radiation. Gloyd and Conant
(1990) propose a close association between Calloselasma and
Hypnale, based on an aspect of dorsal color pattern and
considerations of biogeography. However, one may argue that
the turned up snout of Deinagkistrodon and Hypnale. as well
as the facial stripe through the eye, are synapomorphies
which unite these forms and exclude Calloselasma. The
phylogenetic positions among these taxa, and between these
taxa and Agkistrodon. have never been adequately resolved.
Objectives of this Study
Four objectives were set in order to address problems in
the systematics of the Agkistrodon complex. These were: (l)
to gain some measure of the intraspecific phylogeny and
geographic population structure, or phylogeography, among
populations of the two U.S. species of Aakistrodon: (2) to
10
determine the phylogenetic relationships among the three New
World species of Aakistrodon and their phylogenetic position
in relation to other "Aakistrodon complex" pitvipers; (3) to
test the hypothesis of monophyly for the genus Agkistrodon.
as it is presently recognized to include Asian and American
species; and (4) to determine the phylogenetic relationships
of Agkistrodon complex genera Calloselasma. Deinagkistrodon.
and Hypnaie, to each other and to Agkistrodon.
Comparative Analysis of Mitochondrial DNA
Animal mtDNA, with the exception of Hydra (Warrior and
Gall, 1985), is a closed-circular genome ranging from 16 to
over 40 kb. MtDNA has several properties that distinguish it
from nuclear DNA and make it ideally suited to address
certain questions in systematics and evolution. These are:
(1) small size, (2) ease of isolation, (3) maternal
inheritance, (4) variation in rate of evolution among
different regions of the genome, (5) its state as a haploid
genome composed of single copy genes, and (6) the genome has
been completely sequenced for a wide range of animals, which
makes possible the design of "universal" primers (Kocher et
al., 1989) for amplification by PCR and sequencing of primer-
defined regions. For reviews of animal mtDNA see Brown
(1985), Avise et al. (1987), Moritz et al. (1987), and
Spuhler (1988).
11
Restriction endonucleases recognize and cleave DNA at
specific sequences. DNA having the correct recognition
sequence is cleaved, resulting in a series of fragments which
may then be separated by electrophoresis. Fragment patterns
of these digests may then be compared. Or, by employing a
combination of single- and double-enzyme digests (Danna et
al., 1973), a restriction site map may be produced.
Depending on which method is employed, each variable
restriction site or fragment constitutes a character, and the
presence or absence of these characters yields discrete data
sets that lend themselves to phylogenetic analysis.
Restriction endonuclease analysis of mtDNA has been used
successfully for systematic studies of a large variety of
organisms (Brown, 1985; Avise et al., 1987; Moritz et al.,
1987; Spuhler, 1988).
The Polymerase Chain Reaction
Since the advent of automated in vitro enzymatic
amplification of nucleic acid sequences, applications of PCR
have expanded exponentially. This rapid method has
revolutionized many molecular studies, including molecular
systematics (Innis et al., 1990). For most applications, a
"target" region of unknown sequence of from a few hundred to
a few thousand base pairs (bp) is bracketed by
oligonucleotides (short synthetic single-stranded DNA
molecules) that are complementary to regions of known
12
sequence on either side of the target. Sample DNA is
introduced into a reaction mixture which also includes the
bracketing oligonucleotides which serve as primers, DNA
polymerase, and appropriate buffer. Following heat
denaturation the reaction mixture is cooled to annealing
temperature. One primer anneals to the heavy (H) strand and
the other anneals to the light (L) strand, one at one end of
the target sequence and the other at the other end. The 3'
end of each primer faces the target region. The temperature
is then adjusted for optimum polymerase activity.
Polymerases commonly used are isolated or cloned from the
thermophilic bacteria Thermus flavus or I. aquaticus.
allowing polymerization at elevated temperature, which limits
renaturation and secondary structure. Once a complementary
strand is synthesized for both original H- and L-strands the
first cycle is complete, with a result of a doubling of the
target DNA. The reaction mixture is immediately elevated to
denaturing temperature again, and the cycle is repeated. The
reaction mixture is usually subjected to 30 or more cycles,
with a doubling of the target sequence every cycle, and
resulting in a billion-fold amplification. Automation of the
PCR reaction with the development of the DNA thermal cycler
has made the process simple and effective in terms of time at
the lab bench.
13
CON BIL
PIS BIL CON
Brattstrom, 1964 Minton, 1990
BIL
Jones,1976 Conant, 1986 Van Devender
and Conant, 1990 Campbell & Whitmore, 1989
39
CHAPTER II
METHODS AND MATERIALS
Samples
Snakes were collected in the field, received as
donations, or obtained from animal dealers. Voucher
specimens were deposited in the collection of vertebrates.
The University of Texas at Arlington. The following named
forms were analyzed (number of individuals and general
locality where known are indicated for each): Agkistrodon
contortrix contortrix (6, SC: Jasper Co.; 2, MS); A. C
mokasen (1, NJ; 2, PA: Kempton); A. c. phaeogaster (2, KS:
Douglas Co.); A. Q. laticinctus (3, OK: Marshall Co.; 1, TX:
Cooke Co.; 2, TX: DeWitt Co.); A. C pictigaster (3, TX:
Brewster Co.); A. piscivorus piscivorus (1, SC: Jasper Co.);
A. p. conanti (1, FL: Collier Co.); A. p. leuCQStoma (1, TX:
Freestone Co.; 1, TX: Kimble Co.); A. bilineatus bilineatus
(1); A. b. taylori (1); A- halys halys (l/ Mongolia: North of
Ulan Bator); A. h. caraganus (1, USSR); A. intermedJUS
saxatilis (1, USSR); A. blomhoffii uSSUrJensJS (1/ USSR); Boa
oonstrictor imperator (1, Mexico: Nayarit: Cruz de
Huanacaxtl); Calloselasma rhodOStoma (1/ Malaysia); Crotalus
mniossus molossus (1/ TX: Kimble Co.); Deinagkistrodon
acutus (1); Hypnale hypnale (1); Porthidlum godmanj (1/
Honduras); Sistrurus catenatUS edwardsi (l/ TX: Yoakum Co.);
40
Trjmeresunip albolabris (1); T. mucrosquamatus (1); Vipera
ammodytes (i) .
Restriction fragment data were obtained for all of the
above individuals except Boa constrictor imperator and
Hypnale hypnale. In order to further address the second,
third, and fourth objectives of this study, sequence data
were obtained for a portion of the 16S gene for one
individual each of the following selected taxa: A.
contortrix mokasen. A. bilineatus taylori and A. b.
bilineatus. A. blomhoffii ussuriensis. A. piscivorus
leucostoma. Boa constrictor imperator. Calloselasma
rhodostoma, Deinagkistrodon acutus. Hypnale hypnale/ and
Porthjdium godmani.
DNA Isolation
Heart, liver, kidney, and muscle tissues were stored at
-7 0° C. Shed snake skins were found to be an excellent source
of DNA and were stored at -20° C. Hypnale tissues were
obtained from a formalin-fixed, alcohol-stored museum
specimen, and were lyophilized. For end-labeling, mtDNAs
were purified by cesium chloride density gradient
ultracentrifugation as described in Wright et al. (1983) and
modified in Densmore et al. (1985). For PCR amplification,
either purified mtDNAs or total DNAs isolated by one of the
following two methods, were used. The first method uses
proteinase K for protein degradation, and is a modification
41
of the method described in Hillis and Davis (1986). Tissues
were powdered in liquid nitrogen using a mortar and pestle.
Approximately 100 mg of the powdered tissue was placed into
500 jLil of STE (100 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5) .
25 jLil of a 20 mg/ml stock of proteinase K in STE was added
and mixed. Then 25 ixl of 20% SDS was added and the solution
mixed again. The solution was then incubated at 55° C for two
hours with occasional mixing, followed by two extractions
with PCI (25:24:1, phenol:chloroform:isoamyl alcohol) and
then two extractions with chloroform. DNA was precipitated
by adding I/IO volume 2 M NaCl, then 2 1/2 volumes ice cold
ethanol. DNA was pelleted in a microfuge at 11,000 X G for
ten minutes, air dried, and resuspended in 250 jul of water.
The second method uses SDS-urea for protein degradation,
and is a modification of the method described in Densmore and
White (in press). As in the first method, tissues were
powdered in liquid nitrogen using a mortar and pestle.
About 0.7 5 g powdered tissue was added to a 15 ml centrifuge
tube containing 2 ml SDS-urea (1% SDS in 8 M urea, 240 mM
Na2HP04, 1 mM EDTA, pH adjusted to 6.8 using phosphoric acid) .
This solution was vortexed, then incubated for 20 minutes at
room temperature, with occasional vortexing. This solution
volume was doubled with PCI and gently mixed, then
centrifuged in a Sorvall SS-34 fixed angle rotor, 14,000 RPM,
-10° C, for 20 minutes. The upper phase was removed using a
Pasteur pipette, extracted once with chloroform, and dialyzed
42
against l/io TE. All DNAs were stored in water or l/lO TE at
-20° C.
As I was unable to obtain a fresh tissue sample for
Hypnale, DNA was isolated from a formalin-fixed,
alcohol-stored museum specimen. Kidney tissue was first
lyophilized, then treated the same as fresh tissue. DNA was
isolated by the proteinase K method as described above.
Restriction Endonuclease Digestion
Restriction endonucleases were obtained from Bethesda
Research Laboratories, New England Biolabs, or Boehringer
Mannheim. The amount of enzyme, buffer, and other conditions
were those specified by the manufacturers.
MtDNAs purified from all individual A. contortrix
procured for this study were digested with the following 17
restriction endonucleases: ApaL I, Ava I, Bgl I, Bgl II,
EStE II, Cla I, Dra I, EQQR I, EQQR V, Hind III, i(pn I, ££t
I, Sma I, 2st 1/ Sst II, Stu I, and Xba I. MtDNAs from one
representative of each population of A. contortrix and A.
piscivorus. and from each other named form of viper studied
were digested with 27 different restriction endonucleases,
which included the above 17 as well as Apa I, Ase I, BamH I,
Ban 1/ B£l 1/ Hd£ 1/ Nru i, Sea i/ Sty i, and ^ho. i. These
enzymes recognize and cleave 6-base sequences (except BstE
II, which is a seven-base cutter). Of these 27 enzymes, 15
(ApaL I, Ava 1/ Ban i, B£l i, Bgl i, Bgl n, £la i, Dra i,
43
ECQR V, Hind III, Kpn I, M e I, Mm I, Sma i, and 2Q2a D were
found to have informative and clearly interpretable fragment
patterns for all taxa, and so were used in the final
analysis. MtDNA fragments were end-labeled using
a32p-deoxynucleotide triphosphates, separated by vertical
electrophoresis in 1.2% agarose, and visualized by
autoradiography (Wright et al., 1983; Densmore et al., 1985).
In Vitro DNA Amplification
One to 5 jul of purified DNA solution was placed in a PCR
reaction tube containing reagents supplied by Perkin Elmer
Cetus (Norwalk, CT) according to the manufacturer's
instructions. Primers used for the initial symmetrical
amplification were: (1) 5•-CGCCTGTTTATCAAAAACAT-3' and
(2) 5'-CCGGTCTGAACTCAGATCACG-3', which target a portion of
the 16S rRNA gene of animal mtDNA (Kessing et al., 1989).
The 3' end of primer #1 corresponds to chicken base 3214
(Desjardins and Morias, 1990) and initiates L-strand
synthesis. The 3' end of primer #2 corresponds to chicken
base 37 84 and initiates H-strand synthesis. Reactions were
carried out in a Perkin Elmer Cetus (Norwalk, CT) DNA Thermal
Cycler. Total reaction volume was 25 |ul. Reaction time
parameters were: 94° C/l minute, 50° C/l minute, 7 2° C/1.5
minutes for 35 cycles.
Double-stranded PCR product was gel purified using 1.4%
low melting point agarose (GIBCO BRL, Gaithersburg, MD). A
44
slice containing the amplified DNA was cut from the gel and
placed in a microfuge tube containing 750 |LI1 water. The tube
was heated for 5 minutes at 65° C and vortexed briefly to
liberate the DNA. Tubes were then stored at -20° C. Five jul
of this solution was used as template for asymmetrical PCR
(Gyllensten and Erlich, 1988) in a total reaction volume of
7 0 lul . Asymmetrical PCR reaction conditions were: 94° C/l
minute, 55° C/l minute, 72° C/l.5 minutes for 43 cycles.
Primers used in asymmetrical amplification were: (1)
5'-CGAGAAGACCCTATGGAGCTT-3' (L-Strand), the 3' end of which
corresponds to chicken base 3448 (Desjardins and Morals,
1990); (2) 5'-CCGTGCAAAGGTAGCGCA-3' (L-strand), the 3' end of
which corresponds to chicken base 3309 (Desjardins and
Morals, 1990); and (3) the H-strand primer was the same one
used in the double-stranded amplifications. The limiting
primer was diluted 100-fold. Single-stranded PCR product was
prepared for sequencing by microfiltration using Millipore
(Bedford, MA) filter units No. UFC3 TTK 00, centrifuged for
10 minutes, 3 times, at 4,000 RPM in a Sorvall SS-34 rotor,
and reconstituted to a final volume of 40 jul.
Sequencing of DNAs
Dideoxy sequencing (Sanger et al., 1977) was
accomplished using Sequenase v. 2 (United States Biochemical,
Cleveland, OH), according to the manufacturers' instructions.
DNAs were labeled with a^^s-dATP and run in 6.0 % acrylamide,
45
7.0 M urea, 50 x 21 cm gels at 1800 v. Autoradiographs were
produced using Kodak X-OMAT XAR 5 film exposed 24-36 hours.
Data Analysis
MtDNA restriction fragments were treated as
"presence/absence" characters. Phenetic analysis was
accomplished using the "FragCL" program (courtesy T.E.
Dowling) which uses the formula of Upholt (1977) to estimate
sequence divergence. This method derives percent sequence
divergence as a function of the fraction of conserved
fragments. It makes the assumption that changes in
restriction fragments are simple base substutions.
Observations by Upholt (1977) verified that, indeed, most
restriction site losses or gains are single point mutations.
He found that divergence estimated by this method is
consistant across enzymes, and that the method is reliable
below 15% divergence, which includes the taxa studied. The
UPGMA clustering method (Sokal and Sneath, 1963) was used to
produce a dendrogram.
The PAUP program, version 2.4.1 (Swofford, 1985) was
used for maximum parsimony analysis of restriction fragment
data, using global branch-swapping with MULPARS option. For
restriction site data, various methods of character weighting
may be used, as the loss of a specific site is more probable
than the gain of a site. However, the loss on gain of a
restriction site results in the loss and gain of restriction
46
fragments, and so the loss or gain of a restriction fragment
are equally probable events, and must be analyzed
accordingly. Therefore, maximum parsimony is appropriate for
restriction fragments. Global branch-swapping involves trial
of a very large number of trees. Each possible subtree is
removed from a tree and reinserted at all other positions on
the tree. The MULPARS option permits retention in memory of
each distinct tree that is equal in length to the shortest
yet found, and these are all input to the branch-swapping
procedure.
DNA sequences were aligned using the CLUSTAL program in
the PC Gene package (Intelligenetics, Mountainview, CA) , with
minor adjustments by hand. Aligned sequences were entered
into PAUP V. 3.On (Swofford, 1990), and analyzed using the
exhaustive search option. A problematical area that
contained deletions (Appendix B, bases 17 9-216) was not
phylogenetically informative and was omitted.
Equal weight was given to transitions and transversions.
Mindell (in press) observed that for the mitochondrial 12S
rRNA gene, which evolves at aproximately the same rate as the
16S gene (Brown, 1985; Mindell and Honeycutt, 1990), percent
transitions begins levelling off at about 50%. Transitions
then begin to saturate and become less phylogenetically
informative after about 20 million years divergence time.
Until saturation occurs, transitions are informative
character state changes, and should not be weighted (Mindell
47
and Honeycutt, 1990). The objectives for this study involve
resolution of relationships less than 20 Myr old (see Chapter
IV) and so all character state changes were assigned equal
weight.
48
CHAPTER III
RESULTS
Restriction Fragment Analysis
Of the taxa studied, intrapopulational variation was
assessed in only one species, A. contortrix. for which a
series was obtained for each population. Only one copperhead
population sampled exhibited within-population mtDNA
variation, this being A. Q. contortrix from Jasper Co., South
Carolina. Of six individuals, four were identical and two
exhibited size heteroplasmy, having one mitochondrial genome
of typical size for the species of about 17.3 kilobases (kb),
identical in restriction fragment patterns to the other
members of this population, and a second genome about 3.0 kb
larger. This larger genome was not taken into consideration
when comparing populations of copperheads. The two A. Q-
mokasen individuals from Pennsylvania and the single specimen
from New Jersey all shared identical restriction fragment
patterns for all enzymes. Another mtDNA genotype was shared
by the two A. £• phaeogaster from Kansas, another by the two
^. Q, contortrix from Mississippi, yet another among the six
^. Q. laticintus obtained from south Texas, north Texas, and
Oklahoma, and still another among the three A. Q. pictigaster
from the Davis Mountains in west Texas. Given the small
sample size, within-population variation was found to be, in
all cases except for the two heteroplasmic individuals, not
49
detectable in A. contortrix when using hexanucleotide
recognizing restriction enzymes (for example, see Fig. 14),
and was not assessed in other taxa.
Digestion of mtDNAs from single individuals of 24
viperid taxa or populations with 14 6- and one 7-base
recognizing restriction endonucleases yielded 292 variable
characters (Appendix A). From these data, a UPGMA phenogram
was produced (Fig. 15) . This analysis shows that A.
contortrix from across the species' range are all very
similar, differing overall by 1.26% sequence divergence.
Aakistrodon contortrix differs from other New World
Agkistrodon by 5.70%. Agkistrodon b. bilineatus and A. b.
taylori are 2.7 2% divergent. Agkistrodon bilineatus is most
similar to the species A. piscivorus. especially A. p.
leucostoma. differing by 3.10%. Agkistrodon p. leucostoma
from east Texas and west Texas were very similar, differing
by only 0.15%. Agkistrodon p. piscivorus from South Carolina
and A. p. conanti from south Florida also were very similar,
differing by 0.45%, but these two distinct eastern and
western lineages revealed a level of 3.70% divergence. The
New World pitviper Porthidium godmani was the most similar to
New World Agkistrodon. differing by 6.40%. The rattlesnake
Cr a l s molossus differed from the New World
figkj strodon/Porthidium godmani cluster by 7.64%. Old World
figki strodon differed from New World Agkistrodon by an average
of 8.02%, more than p. godmani or Q. molossus. The remaining
50
taxa revealed relatively high levels of divergence from other
taxa. Both Calloselasm; rhodostoma and Deinagkistrodon
acutus derived from near the base of the phenogram,
suggesting a lack of affinity with Agkistrodon. Old or New
World. Not surprisingly, the most divergent taxon in the
restriction fragment analysis was the viperine snake Vipera
ammodytes, which differed from pitvipers by 14.75%. All
divergence levels observed among these taxa were under 15%,
and so were in the range in which sequence divergence may be
estimated with accuracy (Upholt, 1977).
Using Vipera ammodytes as an outgroup, PAUP maximum
parsimony analysis of the 292 variable restriction fragments
derived for all viperid taxa studied yielded 25 equally
parsimonious trees from which a strict consensus tree was
produced (Fig. 16). New World Agkistrodon was resolved, and
formed a monophyletic group. Intraspecific A. contortrix and
A. piscivorus relationships were resolved with the same
topology as in the phenetic analysis. However, A. bilineatus
was paraphyletic. The subspecies A. b. taylori was sister
taxon to A. contortrix. and A. b. bilineatus was the next
most basally derived lineage. Agkistrodon intermedius
saxatilis and the two sampled subspecies of A. halys formed a
clade, and A. bjomhoffii ussuriensis was separate from its
congeners. Although p. godmani and £. rhodostoma formed a
clade, these two species shared less than 21% of their
restriction fragments according to phenetic analysis.
51
Relationships among other New and Old World pitvipers were
not resolved. Calloselasma rhodostoma and Deinagkistrodon
acutus were not related to Aakistrodon. Asian or American.
The two Trimeresums species included, albolabris and
mucrosquamatus, appear distinct from each other and other
pitvipers.
16S Sequence Analysis
Alignment of sequences (Appendix B) obtained for an
approximately 4 00 bp region of the 16S gene revealed regions
of strong conservation, moderate variation, and one region
where variation was extreme. Transitions only slightly
outnumbered transversions. Among fully consistent character
state changes, 34 were transversions and 35 were transitions.
These two types of point mutations occurred in approximately
equal frequency in the data set.
An exhaustive search, using Boa as outgroup taxon,
produced two equally parsimonious trees, from which a strict
consensus tree was produced (Fig. 17). The only difference
between the two shortest trees was equally parsimonious
placement of A. h- bilineatus as sister taxon to either A. b
taylori or A. piscivorus. Agkistrodon contortrix was the
basal lineage of New World Agkistrodon. Porthidium aodmani
was sister taxon to New World Aakistrodon. and Aakistrodon
blomhoffii was basal to the New World clade. Calloselasma
52
rhodostoina and then Deinagkistrodon acutus were the basal
lineages in the analysis.
All results of this study indicate a basal position for
Calloselasma rhodostoma and Deinagkistrodon acutus in
relation to the other pitvipers examined, and this is in
agreement with studies by Gloyd (197 8) and Minton (1990).
Therefore, an exhaustive analysis was performed using these
two taxa as outgroup and deleting Boa, in order to further
test the hypothesis of relationships for Agkistrodon by
designating an outgroup as close as possible to the ingroup.
Ingroup topology was identical to that derived using Boa as
outgroup. Again, two equally most parsimonious were found,
differing only in their placement of A. b. bilineatus. One
tree (not shown) had A. b. bilineatus as sister taxon to A.
p. leucostoma. united by one transition. The other tree
(Fig. 18) had the two subspecies of A. bilineatus as sister
taxa, united by one transversion. On the basis of
morphology, placement of the two A. bilineatus subspecies
together must be chosen over placement of A. b. bilineatus
with A. piscivorus.
As in the analysis using Boa as outgroup, A. contortrix
was the basal New World Agkistrodon. distinguished by four
autapomorphies (transversions) and two noncompatible
character states (homoplasies or reversals). The moccasins,
^. bilineatus and A. piscivorus. as a group were set apart by
only one compatible shared character state, a transition, and
53
four noncompatible character states. The moccasins, as a
group, showed little divergence from their common ancestor.
Aakistrodor) b. bilineatus was the most divergent moccasin,
distinguished by three autapomorphies (one transversion and
two transitions) and two noncompatible character states.
Aakistrodon piscivorus was set apart by one transition.
Porthidiimi aodmani was sister taxon to New World Agkistrodon.
but not a single compatible character state distinguished New
World Agkistrodon from Porthidium. Two noncompatible
character states set New World Agkistrodon apart from
Porthidium. Agkistrodon blomhoffii was the basal lineage of
the ingroup. No less than seven autapomorphies set this
Asian taxon apart (four transversions and three transitions)
from its common ancestor with the New World taxa. Two
compatible character states (transitions) set the New World
taxa apart from A. blomhoffii. Nine compatible and three
noncompatible unpolarized character state changes were
evident between the outgroup (£. rhodostoma and D. acutus)
and the ingroup. An even larger amount of autapomorphous
change was revealed along the independent lineages of Q.
rhodostoma and D. acutUS.
Although 300 bp of 16S sequence was obtained for Hypnale
hypnale. this taxon was not included in the analysis. The
Hypnale sequence was similar enough to align base by base
with the other snake sequences, but it was clearly so
54
different at so many sites that it differed from the other
pitvipers to a considerably greater extent than did Boa.
55
A. contortrix
(/) 5 o Q o g O O O 2 < C O = J 5 . 5 O I -J. U 2 . S X w A , A . Q O;. JPk Q. 5 H </)</) o oc
23.1 w
9.41 -^
6.56 ^
4.36 —"
t i ldyu
kb
2.32 2.03
0.56
+
57
rC A. c. laticinctus
4 A. c. pictigaster
A. c. ptiaeogaster
A. c. contortrix MS
A. c. contortrix sC
A. c. mokasen
— A. b. taylori
— A. b. bilineatus
[ A. p. leucostoma E.TX
A. p. leucostoma W.TX
CA. p. conanti S.FL
A. p. piscivorus SC
— P. godmani
— C. molossus
— A. h. halys
— A . h. caraganus
— A. I, saxatilis — A. b. ussuriensis
— 7. albolabris
— T. mucrosquamatus
— D. acutus
— S. catenatUS
— C. rhodostoma
— V. ammodytes
I I I I I I I I I I I I I I I I
1 5 1 4 1 3 1 2 1 1 10 9 8 7 6 5 4 3 2 1 0
Estimated Percent Sequence Divergence
59
'A. c. laticinctus
- A. c. pictigaster
-A. c. phaeogaster
- A. c. contortrix MS
- A. c. contortrix SC
- A. c. mokasen
•A. b. taylori
• A. b. bilineatus
•A. p. leucostoma E.TX
• A. p. leucostoma W.TX
'A. p. conanti S.FL
• A. p. piscivorus SC
•P. godmani
• C. rhodostoma
•A. i. saxatilis
-A. h. halys
.A. h. caraganus
• D. acutus
A. b. ussuriensis
S. catenatUS
• C. molossus
• T. albolabris
T. mucrosquamatus
V. ammodytes Outgroup
61
A. contortrix
A. b. taylori
A. b. bilineatus
A. piscivorus
Porthidium
A. blomhoffii
C. rhodostoma
D. acutus
Boa OUT
63
IIIIII
I I I I
I I I I A. contortrix
A. b. taylori
; I I I A. b. bilineatus
-^-A. piscivorus
P. godmani
I I i i >l. blomhoffii
Outgroup I I I r
I I I I I I I I I I I i I I C. rhodostoma
I I I I I I I D. acutus
65
CHAPTER IV
DISCUSSION
Phylogeography of Aakistrodon contortrix
Molecular variation in A. contortrix is low, suggesting
a recent radiation of the modern races. Based on restriction
fragment analysis, affinities of the mtDNA genotypes among A.
contortrix populations are discordant with subspecific status
based on morphology. The close relationship of A- contortrix
mtDNAs from the northern and southern Atlantic seaboard, and
the similarly close relationship of A. contortrix mtDNAs from
Kansas with those from Mississippi conflicts with
relationships based on morphology, which aligns the
Mississippi and South Carolina populations.
Three hypotheses might explain this conflict. One
hypothesis would be morphological convergence of copperheads
across the southeastern coastal plain. A second hypothesis
is nuclear DNA introgression. Nuclear DNAs may be spread by
male dispersion. Males moving through zones of
intergradation or hybridization may, over many generations,
saturate adjacent populations with their nuclear genome until
the original nuclear genome has been displaced. The original
mitochondrial genome would remain intact due to its maternal
inheritence. A third hypothesis is mtDNA introgression. It
may be that mtDNA genotypes have infiltrated adjacent
populations via hybridization and have become fixed, while
66
relatively little nuclear genetic introgession has occurred
across zones of intergradation or hybridization. It has been
demonstrated theoretically (Templeton, 1983) that low levels
of mtDNA introgression may lead to fixation of a
mitochondrial genome of different origin from the nuclear
genome. It has been shown experimentally that an introduced
mtDNA can approach fixation in three generations (Aubert and
Solinac, 1990).
In other words, these data suggest that the
phylogeography of the mtDNA haplotypes does not reflect the
phylogeography of the races of E. contortrix. Examples of
taxa in which mtDNA types have apparently spread via
hybridization and become fixed in populations which carry a
nuclear genome of different phylogenetic origin include
Drosophila (Powell, 1983), mice {Ferris et al., 1983), frogs
(Spolsky and Uzzell, 1984), deer (Carr et al., 1986), and a
salamander (Kraus and Miyamoto, 1990). In light of these
findings, it appears that hypothetical phylogenies based on
mtDNA data must be evaluated with horizontal transfer as a
viable alternative. This is particularly so among
populations which continue to interbreed, even at a low
level, or among closely related species in which
hybridization may have played a role in their evolutionary
history. In order to explain the discrepancy between mtDNA
and morphology in A. contortrix poplulations, an independent
nuclear genetic marker must be tested.
67
Phylogeography of Agkistrodon piscivorus
A large degree of independent evolution is apparent
along two lineages of A. piscivorus from the eastern and
western portions of the species' range. Possibly this
reflects long-term Pleistocene isolation in refugia. The
estimated sequence divergence between these eastern and
western populations is 3.7%. Brown (1985) shows that
overall, in vertebrates, mtDNA evolves at a rate of about
2.0%/Myr. Although this rate is not constant and has been
found to vary in some lineages, it provides a means of
estimating divergence times. At 2%/Myr, the timing of
divergence is 1.85 Myr, coinciding with the onset of
Pleistcene ice ages. It is well known that many species'
broad geographic ranges were reduced at this time, causing
fragmentation as ranges were reduced to refugia in Florida
and Texas (Blair, 1958). This seems a likely hypothesis for
the observed independent evolution of these two cottonmouth
lineages.
The divergence between eastern and western cottonmouth
moccasins is of a degree as great or greater than that
observed between some species, for example, between A.
bilineatus and A. piscivorus. It would be of interest to
obtain samples of cottonmouths along an east-west transect
from the east coast to Texas, and compare them using nuclear
markers such as venom proteins or nuclear DNAs, as well as
68
mtDNAs, to help determine if speciation has occurred between
these lineages.
Evolution of New World Agkistrodon
As yet, neither morphology, protein, or mtDNA data have
firmly established phylogeny of New World Agkistrodon.
Agkistrodon bilineatus is most similar in its mtDNA
restriction fragments to A. piscivorus. and yet in parsimony
analysis of restriction fragment data, character states
united it with A. contortrix. It is important to point out
here that restriction fragment characters were present which
supported an A. bilineatus-A. piscivorus clade, rather than
an A. bilineatus-A. contortrix clade. An . bilineatus-A.
contortrix clade was the most parsimonious given the
designated outgroup, but as the data are ambiguous, and the
phenetic analysis clusters A. bilineatus and A. piscivorus.
the parsimony analysis cannot be considered as having
resolved the issue. This parsimony analysis suggests A.
bilineatus may be paraphyletic, and A. b. taylori should be
recognized as a distinct species. Given the isolated
distribution of A. b- taylori in northeastern Mexico, and the
similarity of A. b. taylori in color pattern to A. Q.
pictigaster. this possibility merits consideration. The
sequence analysis supports an A- bilineatus-A. piscivorus
clade, with A- nontortrix the basal New World Aakistrodon
lineage. Taken as a whole, these data suggest, but do not
69
prove, an A. bilineatus-A. piscivorus clade. Both the
fragment and the sequence results are in agreement that a
relatively large degree of independent evolution has occurred
along the A. contortrix lineage following divergence from a
common New World Agkistrodon ancestor.
New World Agkistrodon is monophyletic. Copperheads and
the two species of moccasins are descended from one common
ancestor to the exclusion of other taxa. Restriction
fragment data reveal that this monophyletic group is more
similar, and likely more closely related, to certain other
New World genera (e.g., Porthidium) than to Old World taxa
presently included in Agkistrodon. The 16S sequence data
corroborate this conclusion. Also, the fragment analysis
showed that Old World Agkistrodon is more closely related to
the Asian genus Trimeresurus than to New World Agkistrodon.
The genus Trimeresurus contains highly divergent forms and
warrants further investigation.
The restriction fragment analysis reveals about 8.0%
divergence between Old and New World Agkistrodon. At 2%/Myr,
colonization of the New World by the common ancestor of the
copperheads and moccasins is dated at middle Pliocene. This
agrees with biogeographical data which documents biological
exchange across Beringea during this time (Wolfe and Hopkins,
1967). This estimate of time of entry into the New World is
much more recent than that of Van Devender and Conant
70
(1990) who estimated the time of entry at late Oligocene-
early Miocene.
Status of the Genus Gloydius
The genus Agkistrodon. as it now stands, is
polyphyletic. Serious consideration must be given to
recognition of the genus Gloydius Hoge and Romano-Hoge for
the Old World forms presently assigned to Agkistrodon. This
systematic issue will perhaps be settled as additional data
sets become available. Both data sets reveal that Porthidium
is very closely allied with Agkistrodon. Also, A. blomhoffii
is quite closely related to Porthidium and New World
Agkistrodon. On the basis of morphology and biogeography,
Gloyd and Conant (1990) considered A. blomhoffi to be the
most closely related of the Asian forms to New World
Agkistrodon. and so seemed the appropriate taxon to test
monophyly of Agkistrodon.
Although the data presented here suggest recognition of
the genus Gloydius. revision would not be justified as yet.
Conant (in Gloyd and Conant, 1990, p. 461) states:
Someone else will have to resolve the status of the genus Gloydius. Inasmuch as our monograph is based on morphology, it may be a student of molecular biology who will contribute toward eventually settling the matter. I would expect that person, however, to give full weight to the mass of morphological data we have presented.
Insofar as I am concerned, I would be delighted to see my colleague's name associated with a group of snakes to which he devoted so much time and
71
energy. Further, I think he would be immensely pleased if he could be aware of the signal honor conferred on him, despite his intense aversion to splitting the two groups.
Mayr (1989, p. 517) states that "no classification should be
abandoned until it is definitely falsified." I strongly
agree with this statement.
Data presented here fall just short of that goal. One
problem is the phenetic association of Trimeresurus
albolabris with A. blomhoffii (Fig. 15). It may be that
relationships among the eight Asian species of Agkistrodon
and among other Asian pitviper genera must be established
before generic status can be designated. A comprehensive
analysis of Asian pitvipers was beyond the scope of this
study. The best estimate of phylogeny determined by these
analyses is the topology depicted in Fig. 18, but revision of
Agkistrodon awaits incontrovertible resolution of
relationships. It is my contention that a large nuclear DNA
data set, obtained for New and Old World Agkistrodon and
selected representatives of Trimeresurus. would settle the
issue.
Allied Genera
The independent generic statuses of Calloselasma
rhodostoma and of DPI naakistrodon aciltus are supported by
72
the results of this study. In agreement with the
immunoelectrophoretic results of Minton (1990) , the
restriction fragment data and the 16S sequence data presented
here revealed an early divergence of these two distinct
lineages in the radiation of pitvipers. There is no alliance
between these relictual snakes and Agkistrodon. and so
"Agkistrodon complex" is really a misnomer from the viewpoint
of a natural group.
There are two possible explanations for the extremely
different sequence obtained from the formalin-fixed,
alcohol-stored Hypnale tissue. One possibility is that
Hypnale has undergone dramatic molecular evolution in this
region. Or, perhaps formaldehyde has chemically interacted
with, and modified many of the nucleotide bases, causing
incorperation of the wrong bases during the sequencing
reaction. In either case, this very different sequence is
not useful for phylogenetic purposes. Hopefully, this issue
will be settled when fresh Hypnale tissue is obtained for
analysis.
Future Research
This study has shed some light on the evolution of
pitvipers and has revealed biological questions which may be
addressed in future studies, primarily with analyses of
nuclear DNAs. These questions are: (1) What evolutionary
genetic mechanism has lead to the discrepancy between the
73
geographic distribution of mitochondrial DNA genotypes and
morphology in A. contortrix? (2) What are the evolutionary
genetic dynamics of the secondary contact zone between the
widely divergent eastern and western populations of A-
Piscivorus? (3) Is A. bilineatus the sister taxon to A-
contortrix or A. piscivorus? (4) Should A. bilineatus
taylori be elevated to separate specific status? (5) Should
the genus Gloydius be recognized? (6) What is the
phylogenetic position of Hypnale? (7) What are the
relationships of snakes assigned to the genus Trimeresurus?
74
LITERATURE CITED
Aubert, J. and M. Solinac. 1990. Experimental evidence for mitochondrial DNA introgression between Drosophila species. Evolution 44 (5):1272-1282.
Avise, J.C., J. Arnold, R.M. Ball, E. Bermingham, T. Lamb, J.E. Neigel, CA. Reeb, and N.C. Saunders. 1987. Intraspecific phylogeography: The mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18:489-522.
Blair, W.F. 1958. Distributional patterns of vertebrates in the southern United States in relation to past and present environments. In: C.L. Hubbs (Ed.), Zoogeography. Publication No. 51, Am. Assoc. Adv. Sci., Washington, D.C.
Brattstrom, B.H. 1964. Evolution of the pit vipers. Trans. San Diego Soc. Nat. Hist. 13 (11):185-268.
Brown, W.M. 1985. The mitochondrial genome of animals. Pp. 95-130. In: Maclntyre, R. J. (Ed.), Molecular Evolutionary Genetics. Plenum Press, New York.
Burger, Leslie W. 1971. Genera of pitvipers (Serpentes: Crotalidae). Ph.D. Dissertation, The University of Kansas, Lawrence.
Campbell, J.A. and D.H. Whitmore, Jr. 1989. A comparison of the skin keratin biochemistry in vipers with comments on its systematic value. Herpetologica 45 (2):242-249.
Carr, S.M., S.W. Ballinger, J.N. Derr, L.H. Blankenship, and J.W. Bickham. 1986. Mitochondrial DNA analysis of hybridization between sympatric white-tailed deer and mule deer in west Texas. Proc. Natl. Acad. Sci. U.S.A. 83:9576-9580.
Danna, K.J., G.H. Sack, and D. Nathans. 1973. Studies of Simian Virus 40 DNA. VII. A cleavage map of the SV40 genome. J. Mol. Biol. 78:363-376.
Darwin, C. 1859. On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. London: John Murray.
75
Densmore, L.D., J.W. Wright, and W.M. Brown. 1985. Length variation and heteroplasmy are frequent in mitochondrial DNA from parthenogenetic and bisexual lizards (Genus Cnemidophonis). Genetics 110:689-707.
Densmore, L.D. and P.S. White. 1991. The systematics and evolution of the Crocodilia as suggested by restriction endonuclease analysis of mitochondrial and ribosomal DNA. Copeia (in press).
Desjardins, P. and R. Morals. 1990. Sequence and gene organization of the chicken mitochondrial genome. A novel gene order in higher vertebrates. J. Mol. Biol. 212:599-634.
Dessauer, H.C., J.E. Cadle, and R. Lawson. 1987. Patterns of snake evolution suggested by their proteins. Fieldiana: Zoology 34:1-34.
Dowling, T.E. Department of Zoology, Arizona State University, Tempe AZ 85287.
Ferguson, A. 1980. Biochemical Systematics and Evolution. Wiley, New York.
Ferris, S.D., R.D. Sage, C.-M. Huang, J.T. Nielsen, U. Ritte, and A.C. Wilson. 1983. Flow of mitochondrial DNA across a species boundary. Proc. Natl. Acad. Sci. U.S.A. 80:2290-2294.
Gloyd, H.K. 1978. A new generic name for the hundred-pace viper. Proc. Biol. Soc. Wash. 91:963-964.
Gloyd, H.K. and R. Conant. 1990. Snakes of the Agkistrodon Complex: a Monographic Review. Society for the Study of Amphibians and Reptiles. Cornell University, Ithaca
Gyllensten, U.B. and H.A. Erlich. 1988. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. USA 85:7 652-7 656.
Hennig, W. 1979. Phylogenetic Systematics. University of Illinois Press, Urbana.
Hillis, D.M. and S.K. Davis. 1986. Evolution of ribosomal DNA: fifty million years of recorded history in the frog genus Eana. Evolution 40:127 5-1288.
76
Hoge, A.R. and S.A.R.W.L. Romano-Hoge. 1978. Poisonous snakes of the world. Part I. Check list of the pit vipers Viperoidea, Viperidae, Crotalinae. Mem. Inst. Butantan 42/43:179-310.
Innis, M.A., D.H. Gelfand, J.J. Sninsky, and T.J. White. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, Sna Diego.
Jones, J.M. 197 6. Variations of venom proteins in Aakistrodon snakes from North America. Copeia 1976(3):558-562.
Kessing, B., H. Croom, A. Martin, C. Mcintosh, W.O. McMillan, and S. Palumbi. 1989. The Simple Fool's Guide to PCR. Department of Zoology, University of Hawaii, Honolulu.
Kocher, T.D., W.K. Thomas, A. Meyer, S.V. Edwards, S, Paabo, F.X. Villablanca, and A.C. Wilson. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86:6196-6200.
Kraus, F., and M.M. Miyamoto. 1990. Mitochondrial genotype of a unisexual salamander of hybrid origin is unrelated to either of its nuclear haplotypes. Proc. Natl. Acad. Sci. USA 87:2235-2238.
Kurten, B. and E. Anderson. 1980. Pleistocene Mammals of North America. Columbia University Press, New York.
Mayr, E. 1989. A new classification of the living birds of the world. Auk 106:508-517.
Mayr, E. and P.D. Ashlock. 1991. Principles of Systematic Zoology. Second Edition. McGraw-Hill, New York.
Mindell, D.P. 1991. Aligning DNA sequences: homology and phylogenetic weighting. In: Miyamoto, M. and J. Cracraft (Eds.), Phylogenetic Analysis of DNA Sequences. Oxford University Press (in press).
Mindell, D.P and R.L. Honeycutt. 1990. Ribosomal RNA in vertebrates: evolution and phylogenetic applications. Annu. Rev. Ecol. Syst. 21:541-566.
Minton, S.A. 1990. Immunological relationships in Agkistrodon and related genera. In: Gloyd, H. K. and R. Conant, Snakes of the Aakistrodon Complex: a Monographic Review. Society for the Study of Amphibians and Reptiles. Cornell University, Ithaca.
77
Moritz, C , T.E. Dowling, and W.M. Brown. 1987. Evolution of animal mitochondrial DNA: Relevance for population biology and systematics. Ann. Rev. Ecol. Syst. 18:2 69-292.
Palisot de Beauvois, A.M.F.J. 1799. Memoir on Amphibia. No.XLII. Serpents. Trans. Amer. Philos. Soc. 4:362-381.
Powell, J.R. 1983. Interspecific cytoplasmic gene flow in the absence of nuclear gene flow: Evidence from Drosophila. Proc. Natl. Acad. Sci. USA 80:492-495.
Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain-terminating inhibitiors. Proc. Natl. Acad. Sci. USA 74:5463-5467.
Sokal, R.R. and P.H.A. Sneath. 1963. The Principles of Numerical Taxonomy. W.H. Freeman, San Francisco.
Spolsky, C. and T. Uzzell. 1984. Natural interspecies transfer of mitochondrial DNA in amphibians. Proc. Natl. Acad. Sci. USA 81:5802-5805.
Spuhler, J.N. 1988. Evolution of mitochondrial DNA in monkeys, apes, and humans. Yearbook of Physical Anthropology, Am. J. Phys. Anthropol. Suppl. 9:15-48.
Swofford, D.L. 1985. Phylogenetic Analysis Using Parsimony, V. 2.4.1. Illinois Natural History Survey, 607 East Peabody Drive, Champaign, IL 61820.
Swofford, D.L. 1990. Phylogenetic Analysis Using Parsimony, v. 3.On. Illinois Natural History Survey, 607 East Peabody Drive, Champaign, IL 61820.
Templeton, A.R. 1983. Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and apes. Evolution 37:221-244.
Upholt, W.B. 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acids Res. 4:1257-1265.
Van Devender, T.R. and R. Conant. 1990. Pleistocene forests and copperheads in the eastern United States, and the historical biogeography of New World Aakistrodon. m : Gloyd, H. K. and R. Conant, Snakes of the Aakistrodon Complex: a Monographic Review. Society for the Study of Amphibians and Reptiles. Cornell University, Ithaca.
78
Warrior, R. and J. Gall. 1985. The mitochondrial DNA of Hvdra attenuatta and Hydra littoralis consists of two linear molecules. Arch. Sc. Geneve. 3:339-45.
Wiley, E.O. 1981. Phylogenetics: the Theory and Practice of Phylogenetic Systematics. John Wiley and Sons, New York.
Wolfe, J.A. and D.M. Hopkins. 1967. Climatic changes recorded by Tertiary land floras in northwestern North America. In: Haitai, K. (Ed.), Tertiary correlations and climatic changes in the Pacific. 11th Pacific Sci. Congr., Symp. 25:67-76.
Wright, J.W. , CM. Spolsky and W.M. Brown. 1983. The origin of the parthenogenetic lizard Cnemidophorus laredoensis inferred from mitochondrial DNA analysis. Herpetologica 39:410-416.
Zhao, E. 1979. A new Agkistrodon from Shedao (Snake Island), Liaoning. [In Chinese with English summary.] Acta Herpetol. Sinica, old ser., 1:4-7.
79
APPENDIX A: RESTRICTION FRAGMENT DATA
Presence (l) or absence (0) of restriction fragments of mtDNA
Operational taxonomic units (OTUs) are: l, A.
contortrix. l tirjntlis; 2, A. C. mokasen: 3, A. c.
phaeogaster; 4, A. C. contortrix-south Carolina; 5, A. c.
COntOrtrix-Mississippi; 6, A. c. pictigaster: 7, A^
piscivorus conanti; 8, A. p. piscivorus: 9, A. p.
leucostoma-east Texas; 10, A. p. leucostoma-west Texas; 11,
A, bilineatus bilineatus; 12, A. b. taylori: 13, Porthidium
godmani; i4, calloselasma rhodostoma; 15, A. intermedins
saxatilis; I6, vipera ammodytes: 17, Deinagkistrodon acutus:
18, A. blomhoffii ussuriensis: 19, A. halys caraganus: 20,
A. h. halys; 21, sistrurus catenatus edwardsi: 22, Crotalus
molossus; 23, Trimeresurus albolabris: 24, TL.
mucrosquamatus. Fragment sizes are estimated to the nearest
100 base pairs (0.1 Kb). Certain small fragments which are
very close in size are distinguished by estimating size to
10 base pairs. Fragments smaller than 500 base pairs were
not included in the analysis. Genomes linearized by one
cleavage are designated L.
80
Fragment QTTT
1 1 1 1 1 1 1 1 1 1 2 2 2 2 2
Enzyme Kb i 2 ^ 4 R f ; 7 f l Q n i 2 i 4 s ^ 7 f t Q n i 2 i 4
ApaL I L 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 0 0 1 0 0 0
AcaL I 14.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
AcaL I 9.4 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
£j2aL I 7.9 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
ACaL I 3.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
Ava I 9.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
Ava I 8.8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Ava I 7.8 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0
A5Za I 7.2 0 0 1 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 1 0
Ava I 6.0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Ava I 5.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0
Ava I 5.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Ava I 5.3 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Ai a I 4.9 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Ava I 4.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0
Ava I 4.7 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Ava I 4.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0
Ava I 4.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0
Ava I 4.0 1 1 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Ava I 3.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
Ava I 3.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Ava I 3.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Ava I 3.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0
81
^^^ I 3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 1 0
A ^ I 3.1 1 1 0 1 0 0 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
^^^^ I 2.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0
Asta I 2.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0
Ava I 2.4 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Ava I 2.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ava I 2.2 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Ava I 2.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ava I 1.9 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0
Ava I 1.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
Ava I 1.7 0 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 1 1 1 0 1 1 1 0
Ava I 1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Ava I 1.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ava I 1.2 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 0
Ava I 0.9 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
Ava I 0.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0
Ava I 0.74 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Ava I 0.73 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Ava I 0.7 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Ava I 0.6 l l l l l i O O O O O l O O O l O l O l l l O O
Ban I 7.4 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0
Ban I 6.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Ban I 5.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Ban I 5.5 1 1 1 1 1 1 0 0 1 1 1 1 0 0 1 0 0 0 1 1 1 1 0 1
Ban I 5.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Ban I 4.6 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 0 1 1 0 0 0 1 1
82
2^^ I 4.4 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
^^^ I 4.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
^an I 4.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
San I 4.1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0
Ean I 3.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
Ean I 3.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ban I 3.5 0 0 0 0 1 0 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0
Ban I 2.8 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 0 1
Ban I 2.7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 0 1 1 1
Ban I 2.6 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Ban I 2.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Ban I 2.3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Ban I 2.2 0 0 0 0 0 0 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0
Ban I 1.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ban I 1.8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0
Ban I 1.6 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0
Ban I 1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ban I 1.4 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 1 1 1 0 0 0 1
Ban I 1.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ban I 1.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0
Ban I 1.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0
Ban I 1.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Ban I 0.95 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0
Ban I 0.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Ban I 0.8 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
T an I 0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0
83
Ban I
Ban I
Bnl I
Bel I
Bel I
BILL I
BILL I
E d I
Bel I
Bel I
Bri I
Bel I
Bel I
Bc.l 1
Bel I
Bel I
Bel I
Bel I
Bc.l I
BrA I
Bel I
Bel I
Bel I
Bel I
Bel I
Bel I
0.55 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
0.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
L
16.9
16.8
13.0
1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11.0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0
10.0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
9.5 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
8.8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0
8.4 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0
7.4 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
6.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
6.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
5.8 0 0 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0
5.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
4.4 0 0 0 0 0 0 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0
4.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
3.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
3.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
3.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
3.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
3.0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2.8 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 1
84
Bel I
Bel I
Bel I
Bel I
Bel I
Eel I
Bel I
Eal I
Bal I
Bgl I
Bgl I
Esl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Bgl I
Egl II
Bgl II
Bgl II
Bgl II
2.2
1.5
1.3
1.2
1.0
0.9
0.7
16.5
16.2
12.0
9.3
8.4
7.8
6.9
6.4
5.7
3.1
2.3
2.0
1.5
1.6
1.1
L
12.0
5.0
1.2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0
0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 1 1 1 1 0
0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0
1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 1 0 0 0 1 0 1 0 1 0 0 0 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0
0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 1 1 1 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
1 1 1 1 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 1 1 0 1 0 0 0 0
1 1 0 1 0 1 0 0 0 0 1 1 1 0 0 1 1 0 1 1 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
85
^^^ ^ L 0 1 1 1 1 0 0 0 0 0 0 1 0 0 0 1 0 1 1 0 0 1 1 0
^^^ I 14.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
^ ia I 3.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
^ la I 1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
I ra I L 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Era I 14.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Dra I 11.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Ura. I 9.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1
lira I 8.0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 0 0 1 0 0 1 0 1 1 1
Dra I 6.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Bra I 5.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Bra I 5.3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Dra I 4.7 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 0
Dra I 4.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
lira I 4.2 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Era I 3.7 0 0 0 0 0 0 1 1 1 1 1 0 1 0 0 0 1 1 0 1 0 1 1 0
Era I 3.5 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0 1 0 0
nra I 2.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Era I 2.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0
22ra I 1.3 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Era I 1.0 0 0 0 0 0 0 1 1 1 1 1 1 0 0 0 1 0 0 1 1 0 1 1 1
Era I 0.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
EceR V L 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
ECiiR V 14.0 0 1 0 1 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0
ECoR V 11.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
ECoK V 10.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
86
EeeR v 10.0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
ECQR V 9.2 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 8.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
ECOR V 8.3 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 7.3 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0
EeeR V 6.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0
EeeR V 5.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
EeeR V 5.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 0 0 0
EeeR V 5.2 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 5.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
BeeR V 4.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
EeeR V 4.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 0 0 0
EeeR V 4.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
EHQR V 4-2 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 4.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
EeeR V 4.0 0 1 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
ECQR V 3.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0
EeeR V 3.4 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
EixjR V 3.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0
EeeR V 3.05 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
ECQR V 3.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0
EeeR V 2.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
EceR V 2.8 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 2.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
EeeR V 2.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
87
E e e R v 2 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
E e e R V 1 .8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
E e e R v 1.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
EeeR V 1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
EeeR v 1.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
EeeR V 1.0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0
EeeR V 0.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
Bind III 10.5 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 1 1 0 0 1
Hind III 9.4 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Hind III 9.2 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Hind III 8.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
Hind III 8.2 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0
Hind III 6.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Hind III 6.6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 1 0 0
Hind III 6.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Hind III 6.0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Hind III 4.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0
Hind III 4.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1
Hind III 3.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
Hind III 3.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Hind III 3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0
Hind III 2.5 1 1 1 1 1 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0
Hind III 2.1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 1 1 0 1 1
Hind III 1-7 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
Hind III 1-45 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Hind III 1.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
88
Hind III 1.2 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 1 0 1 0 0 0 0
Hind III 0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0
Hind III 0.8 0 1 1 1 1 1 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0
Kpn I
Kim I
Kpn I
Kpn I
Kpn I
Kpn I
Kpn I
Kpn I
Ken I
Kpn I
Kpn I
Kpn I
Kim I
Kpn I
Kpn I
Nde I
Hde I
Hde I
mis. I
Nde I
Hd£ I
Nde I
Hde I
12.6 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 0 1 1 1
9.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
9.0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
8.3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
7.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
5.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
4.7 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 1 1 0 0 0 1 1
3.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
2.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0
2.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
1.8
1.3
0.7
0.5
17 .0
16.0
14.0
13.5
11.0
10.3
10.3
8.7
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
1.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
89
NdP I
lade I
Ndp I
Ndp I
NdS I
Nde I
Ndp I
M e I
Nde I
Hd£ I
Hde I
ude I
Hde I
Ndft I
Ndp I
Hd£ I
Hde I
Hde I
Hd£ I
Hde I
Nde I
Hde I
Nde I
Nru I
N m I
Urn I
8.2
8.1
7.6
7 .5
7.2
6.5
6.4
6.0
5.5
4.5
4.4
3.7
2.7
2.6
2.4
2.2
2.1
1.5
1.4
1.0
0.8
0.6
0.5
L
14.0
5.2
1 0 1 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1
1 0 1 0 1 1 1 1 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0
1 0 1 0 1 1 1 1 0 0 0 0 0 0 1 0 0 0 1 1 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
90
Sma
Sma
Sma
Sma
Sma
Sma
Sma
Sma
Sma
Sma
Sma
Xba
Xba
Xba
Xba
Xba
Xba
Xha
Xba
Xba
Xba
xba
Xba
Xba
Xba
Xba
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
L 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0
14.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
12.5 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 1 0 0 1 0
10.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
8.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
6.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
4.2 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 1 0 1 1 0 0 1 0
3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0
3.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
2.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0
1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0
L 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
16.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
14.5 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0
11.5 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
a.8 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0
8.4 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0
7,8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
7,4 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6.6
5.5
5.1
4.9
0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
4,6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
4.1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
91
Xl2a I 3.5 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Xha I 3.3 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0
Xba I 3.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0
Xba I 2.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0
Xba I 2.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0
Xba I 1.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Xba I 1.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
Xba I 1.1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Xba I 0.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0
92
APPENDIX B: 16S RIBOSOMAL
GENE SEQUENCE DATA
Aligned sequences of a portion of the mitochondrial 16S ribosomal gene
Sequence is L-strand, 5'-3'. Base No. 1 corresponds to
published chicken base No. 3350 and base No. 404 corresponds
to piiblished chicken base No. 37 83 (Desjardins and Morals,
1990). Taxa are: B.constr=BQa constrictor imperator.
A.contor=A. contortrix mokasen. A.b.tayl=A. bilineatus
taylori. A.b.bili=A. bilineatus bilineatus. A.pisciv=A.
piscivorus leucostoma. P.godman=Portllidium godmani.
A.blomho=^. blomhoffii ussuriensis. D.acutus=Deinagkistrodon
acutus. C.rhodos=Calloselasma rhodostoma. A period (.)
indicates identity with Boa. A dash (-) indicates a gap
inserted to maintain alignment.
B.constr A.contor A.b.tayl A.b.bill A.pisciv P.godman A.blomho D.acutus C.rhodos
1 2 3 4 1 1 1 1 1 ?????????a aactgtctct tataataaat caattaaact gatctcctag atgagagccc c atgagagtcc c atgagagtcc c -atgagagtcc c atgagagccc c atgagagccc c ? ? ? Pgggtct ggc atgaaagccc g c a
93
B.constr A.contor A.b.tayl A.b.bill A.pisciv P.godman A.blomho D.acutus C.rhodos
5 6 7 8 9 1 1 1 1 1 tacaaaagct agaataacta tataagacca gaagaccctg tgaagcttaa
t...ttat c t. tta. c t. tta. c t. tta. c t. eta. c t.
.c tta. c t. c.a. c t.
gtta. c t.
B.constr A.contor A.b.tayl A.b.bill A.pisciv P.godman A.blomho D.acutus C.rhodos
1 1 1 1 1 0 1 2 3 4 1 1 1 1 1 actaaactat taaaccaact aatagctact ttcggttggg gcgaccttgg
c c . t . t a c c
. . . . c .
. . . . c . aa..c. ,ta..c,
.a, ,ga.
..c.a,
..tea, t . . . a , t . . . a ,
.a. ,a. ,a. .a, t t t t
B.constr A.contor A.b.tayl A.b.bill A.pisciv P.godman A.blomho D.acutus C.rhodos
1 1 1 1 1 5 6 7 8 9 1 1 1 1 1 aacaaaacca aacttccaaa caaaatgagt tatacc-tat acctcatacc . . t . . ..aag - t - . t .
. . t t
.aag
.aag
.aag
.aag
.aag aag
t . . . .aag
t- . t . t- . t . t- . t . c-.t. t t . t . c- .c. c- .c.
B.constr A.contor A.b.tayl A.b.bill A.pisciv p.godman A.blomho D.acutus C.rhodos
2 2 2 2 2 0 1 2 3 4 1 1 1 1 I a tagg ccaacaagcc aacca-acga cccagtataa -cttcctcat aaa--ca... .a t-.tta -cttcctcat aaaatca... .a t-.tta -cttcctcat aaaacca... .g t tta, -cttcctcat aaa-tca... .g t-.tta. -ctccctcat aaaa-ca t-.cta. -cttc-tcat aaaat.a... .a --..eta. -ctccctcat aaaat.-... .a -c.cct. -c-tcctcat aaaat.a... .a....c... gcaa.t.t.
c. . .g . .a.. . .a.g
g
..c.egg
94
B.cons t r A.contor A . b . t a y l A . b . b i l i A .p i sc iv P.godman A.blomho D.acutus C.rhodos
2 2 2 2 2 5 6 7 8 9 1 1 1 1 1 c t g a t c a t t g aaceaagt ta etccagggat aacagegeta t e t t e t t e a a
a a t t . . a. . c . . . a t t . . a . . c . . . a t a . . e . . . a t t . . a a t t . . a . . c . . . a t t t . a . a . . . . a
• • • • y l — .L-.* •• o.C' • • • • • • • • • • • • • • • • • « • • • • • • • • • • • • • • • • • •
B.cons t r A.contor A . b . t a y l A . b . b i l i A .p i sc iv P.godman A.blomho D.acutus C.rhodos
3 3 3 3 3 0 1 2 3 4 1 1 1 1 1 gageccatat caaaaagaag g t t t acgace t ega tg t tgg atcaggaeae
t t t t t
B .cons t r A.contor A . b . t a y l A . b . b i l i A .p i sc iv P.godman A.blomho D.acutus C.rhodos
3 3 3 3 3 5 6 7 8 9 1 1 1 1 1 ccaaatggtg tagccgcta t t aacggt tcg t t t g t t c a a c gat taacagt . . c . g . a a . . c . a . . . t . . c . . . a . . c . g . a a . . c t . . c . . . a . . . . g . a a . . c . a . . . t . . c . . . a . . c . g . a a . . c t . . c . . . a . . c . g . a a . . c t . . c . . . a . . c . g . a a . . c . a . . . t . . c . . . a t . . . . . . . g . a a . . . . c . . . t . . c . . . a t . . . . . . . g . a a . . c t . . c . . . a t . . .
B.constr A.contor A.b.tayl A.b.bili A.pisciv p.godman A.blomho D.acutus C.rhodos
4 0 1 ccta
95
Recommended