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

14

15

16

17

18

19

20

2 1

22

^'"TT 'yJS^

• ' ^

23

24

25

26

27

28

29

30

'^"/j^:^

3 1

32

^'?.d£fc.

33

34

35

36

37

38

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

56

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

58

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

60

'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

62

A. contortrix

A. b. taylori

A. b. bilineatus

A. piscivorus

Porthidium

A. blomhoffii

C. rhodostoma

D. acutus

Boa OUT

63

64

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

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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