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This is to certify that the
thesis entitled

MONARDA SECTION CHEIL YCTIS: PATTERNS OF
SPECIATION AND ENDEMISM

presented by
JESSIE ANNE KEITH

has been accepted towards fulfillment
of the requirements for the

MS. degree in Plant Biology/ EEBB

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Date

 

 

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6/01 cJCIFiC/Dateouepes-pJ 5

MONARDA SECTION CHEILYCTIS: PATTERNS OF SPECIATION AND
ENDEMISM

By

Jessie Anne Keith

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Plant Biology

2003

 

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ABSTRACT
Monarda Section Cheilyctis: Patterns of Speciation and Endemism
By
Jessie Anne Keith
Monarda Section Cheilyctis (Lamiaceae) includes six species. Five are narrow endemics,
with four (M. viridissima, M. fruticulosa, M. maritima and M. stanfieldii) present in
unique edaphic conditions in Texas and the fifth (M. humilis) occurring in arid regions of
New Mexico. The sixth species, M. punctata, is widespread, occurring across much of the
United States. Phylogenies based on two Adh loci, Adh type 1 (Ath) and Adh type 2
(Ath), were used to investigate patterns of speciation, species relationships and endemic
status (neo- versus paleoendemic) of these Monarda species. It was hypothesized that
narrow endemic species were neoendemics that had speciated as peripheral isolates of M.
punctata. Both Adh] and A th phylogenies had three primary clades that depicted similar
relationships between taxa. Monarda punctata was the most ubiquitous taxon across both
phylogenies. Monardafruticulosa, M humilis, M. maritima and M viridissima sequences
exhibited polyphyly and patterns consistent with lineage sorting across both Ath and
Ath phylogenies. In both the Adhland Adh2 phylogeny sequences of M stanfieldii were
sister to a large clade including all samples of the other five species. Phylogenetic results
suggest that M. humilis, M fruticulosa, M. maritima and M. viridissima arose from their
widespread congener, M punctata, and that M. stanfieldii is a sister taxon to the other
five species. Likewise, it was concluded that M. fruticulosa, M. maritima and M.

viridissima and M. humilis are most likely neoendemics while M stanfieldii is likely a

paleoendemic species.

FOR THE TWO MEN THAT
INSPIRED ME TO PURSUE HIGHER EDUCATION

MY FATHER, DR. JAMES HILTON KEITH
AND
MY GRANDFATHER, DR. ARCHIE JUSTICE MACALPIN

iii

I”

 

 

ACKNOWLEDGMENTS

I thank the following people for all of their support in helping me complete this work.

Facultyt

Alan Prather, Tao Sang, J effery Conner and Richard Triemer

Fellow Graduate Students:

Anna Monfils, Nathan Sammons, Kristine Kern, Jay Sobel, Orlando Alvarez-Feuntes,

and Rachel Williams

Post-doctoral Associates:

Heather Hallen, Changbao Li and Eric Linton

I also acknowledge the following institutions for providing funding for this research:

The Herb Society of America ______________________________________ Research Grant
M SU Plant Biology Department __________________________________ Paul Taylor Funding
The MSU Graduate School __________________________________________ Travel Funding

The MSU EEBB ___________________________________________________________ Research Grant

TABLE OF CONTENTS

LIST OF TABLES ______________________________________________________________________________________________________ v iii
LIST OF FIGURES ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ix
CHAPTER l-Introduction
1.1 Project Summary ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 1
1.2 Peripatric Speciation ........................................................................... 1
1.3 Neoendemism versus Paleoendemism ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 4
1.4 Overview of Taxa _______________________________________________________________________________ 5
1.5 Objectives.m,,_mmnmwmmm"Hmumm; ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 7
CHAPTER 2-Materials and Methods
2.1 Study System _______________________________________________________________________________________ 8
i.Taxonomic Classification of Monarda Section Cheilyctis. ............. 8_
ii. Morphology and Life History Traits _______________________________________________ 9
iii. Chromosome Numbers .................................................................. 12
vi. Physiography and Vegetation ________________________________________________________ 12
v. Edaphic Constraints ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, l 4
vi. Species Recognitionmmw“WWW._W,m._m__._ ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 15
2.2 Sampling ............................................................................................. 16
2.3 Marker Choice ..................................................................................... 19
2.4 DNA Extraction ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 21
2.5 Primers 21

oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

CHAPTER 2—Materials and Methods continued

2.6 DNA Amplification ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 22
2.7 Cloning ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 3
2.8 Adh Clone Isolation ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 4
2.9 Restriction Digest Analysis of Adh _____________________________________________________ 2 S
2.10 Sequencing ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 6
2.11 DNA Alignment __________________________________________________________________________________ 2 6
2.12 Identification of Putative Adh and nchS Loci ................................... 2 7
2.13 Outgroup Selection and Rooting ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 7
2.14 Phylogenetic Analyses ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 2 8
2.1 5 Phylogenetic Interpretation ................................................................. 3 1
CHAPTER 3—Resnlts
3.1 Identification of Putative Loci ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 33
3.2 Adht] ___________________________________________________________________________________________________ 38
3.3 .4th and Adht3 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 4 2
3.4 .4th ___________________________________________________________________________________________________ 44
3.5 Adht3 ___________________________________________________________________________________________________ 4 9
3.6 Combined and Reduced Arflttl and AdhtZ __________________________________________ 51
3.7 nchS ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 53

CHAPTER 3—Discussion and Conclusions

4.1 General Overview of Results ______________________________________________________________ 55
4.2 Gene Trees and Species Trees ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 56
4.3 Gene Trees as Tools for Macroevolutionary Inference ,,,,,,,,,,,,,,,,,,,,,,, 57

vi

CHAPTER 3-Discussion and Conclusions continued

 

 

4.4 Peripatric Speciation ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 58
4.5 Neoendemic and Paleoendemic Species ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 59
4.6 Lineage Sorting and Noncoalescence ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 61
4.7 Species Protection and Conservation __________________________________________________ 62
4.8 Prospects for Future Studies of Monarda Section Cheilyctis ,,,,,,,,,,,,, 64
4.9 Conclusions _________________________________________________________________________________________ 66
APPENDICES
Aflil Sequence Alignment ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 6 8
Ath Sequence Alignment ____________________________________________ 81
Monarda Field Collection Data ______________________________________________________________________ 9 5
LITERATURE CITED 98

LIST OF TABLES

CHAPTER l-Introduction

Table 1.

Species in the Genus Monarda ..................................................

CHAPTER 2-Materials and Methods

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Taxonomic Ranks of Narrow Endemic Monarda

ooooooooooooooooooo

Morphology & Phenology of Monarda Section C heilyctism

DNA collection for Adh

DNA collection numbers for nchS .........................................

List of PCR Primers

PCR Thermocycler Programs ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, .

Maximum Likelihood Models

ooooooooooooooooooooooooooooooooooooooooooooooooooo

Phylogenetic Analyses Completed .......................................... .

viii

5

17

18

22

23

29

30

LIST OF FIGURES

CHAPTER l-Introduction

Figure 1.

Figure 2.

Phylogenetic Model of Peripatric Speciation ..........................

Geographic Distribution of Endemic Monarda ......................

CHAPTER 2-Materials and Methods

Figure 3. Collection Locations for Endemic Monarda ,,,,,,,,,,,,,,,,,,,,,,,,,,
Figure 4. Collection Locations for Monarda punctata ,,,,,,,,,,,,,,,,,,,,,,,,,,,,
Figure 5. EcoRI Restriction Digest Patterns of Adh Homologs ,,,,,,,,,,,,,
Figure 6. Base Frequencies in Adhl dataset ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
Figure 7. Base Frequencies in Adh2 dataset .............................................
CHAPTER 3-Results
Figure 8. Unrooted Phylogram of 99 Adh Ingroup Sequences ..............
Figure 9. Unrooted Phylogram of Ingroup and Outgroup Sequences___
Figure 10. Strict Consensus of Ingroup and Outgroup Sequences ___________
Figure 11. Rooted Parsimony Tree of Adhl ...............................................
Figure 12. Rooted Likelihood Tree of A0721 ...............................................
Figure 13. Strict Consensus Tree of Adhl .................................................
Figure 14. Strict Consensus Tree of Ath and Ari/73 .................................
Figure 15. Rooted Parsimony Tree of Ath ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
Figure 16. Rooted Likelihood Tree of Ath ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
Figure 17. Strict Consensus Tree of Ath ..................................................
Figure 18. Rooted Parsimony Tree of A dh3 ...............................................

ix

3

7

18

19

31

32

35

39

40

41

43

47

48

50

CHAPTER 3—Results continued
Figure 19. Combined and Reduced Adhl and Ari/22 Trees 52

Figure 20. Rooted Parsimony Tree of nchS ............................................. 54

INTRODUCTION

1.1 Project Summary

Many historical events, geographic and genetic, have been used to explain potential
modes of speciation. Allopatric and peripatric modes use geographic isolation of
populations to explain species divergence. Under these modes of speciation, geographic
isolation is expected to modify the genetic constitution of divided populations eventually

resulting in their morphological, ecological and genetic separation.

Phylogenetic studies of closely related species can be combined with geographic patterns
of distribution to infer mode of speciation (Harrison 1991). This study utilizes a small
cohesive group of North American mints (Lamiaceae) in the genus Monarda to test
peripatric speciation using phylogenies generated from Adh loci. These taxa appear well
suited for this purpose because the pattern of distribution found among its members

conesponded to those expected for peripatric speciation.

1.2 Peripatric Speciation
Speciation by peripheral isolation was first proposed by Mayr in 1942. Mayr believed
that most species diverged in allopatry but that geographic isolation of small peripheral
populations had the greatest potential for rapid speciation. He maintained that
marginalized populations would be subjected to a higher degree of reduced geneflow and
habitat dissimilarity resulting in founder effects (genetic drift in small populations),
increased selection pressures and thus rapid speciation and morphological change (Mayr

1942, 1 976, 1982). Founder populations are expected to have only a fraction of the

genetic composition found in parental populations (Mayr 1982). This genetic imbalance,
when coupled with a change in the abiotic environment, has the potential to lead to
heightened selection and ecological adaptation. This differs from the classic model of
allopatric speciation where larger allopatric populations are expected to require longer
periods of time to diverge phenotypically and genotypically due to larger more variable

gene pools and more homogeneous habitats

Harrison (1991) predicted that ancestral population structure between sister taxa could be
discerned using patterns of geographic dispersion and molecular phylogenic data. He
proposed a series of repeatable, testable patterns (geographic and phylogenetic) to model
allopatric, peripatric and sympatric speciation. In the model of divergence of peripheral
populations (peripatric speciation), he maintained that peripheral isolates would exist
along the distribution margins of a more widespread sister taxon while appearing

phylogenetically derived within a gene tree.

Narrowly endemic Monarda are located along the geographic periphery of their
widespread congener, M. punctata. This distribution pattern suggests that they may have
speciated as peripheral isolates from M. punctata. Likewise, ITS and chNA provided
little phylogenetic signal for these taxa which suggests they diversified recently (Prather
et a1 2002, Monfils and Prather unpubl. data). Harrison’s phylogenetic model for
peripheral isolation was used to test this hypothesis. In concordance with this model M.
punctata was expected to appear in a more basal position of the phylogeny and

paraphyletic to the endemic species. This pattern would indicate that M. punctata is an

 

 

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NEH...

 

ancestral species. For instance, in the hypothetical phylogenies below (Fig 1), M.
pwrctatahasamorebasalpositioninflretree,andisparaphyletictoMfiuticulosaand
M. virrHrZssima. Furthermore, M. fi'un'culosa and M. viridrlrsima are more closely related

to geographically proximate M. punctata samples in the phylogeny.

 

 

Figure 1: Geographic and hypothetical phylogenetic distributions given four samples of the widespread
taxon M puncrara and two samples representing M viridissima and M fi'utr'culosa.

1.3. Neoendemism versus Paleoendemism

Stebbins (1942 & 1942) defined two types of endemic species, paleoendemics and
neoendemics. Paleoendemics were described as relict species whose once large
distributions have been reduced to small disjunct populations that occur across a wide
area (Mayer & Soltis 1994, Qian 2001). Due to their once widespread distribution,
paleoendemics are expected to have highly heterozygous populations despite their small
size (Macnair & Gardner 1993). In contrast, neoendemics are newer species that are
expected to occur across very narrow range limits and are expected to have highly
homozygous populations. The, criteria used to distinguish paleoendemics from
neoendemics include geographic distribution, genetic variation and placement in the
phylogeny. The majority of North America’s neoendemics are restricted to the southwest.
This region is both a center for neoendemics in North America and the world (Qian,

2001).

Narrow endemic Monarda taxa in Section Cheilyctr's are hypothesized to have speciated
as peripheral isolates, therefore they are believed to be neoendemic species. Phylogentic
patterns that would support their neoendemic status would be consistent with those
mentioned for speciation by peripheral isolation. Neoendemics are expected to be derived

within M. punctata and paleoendemics are expected to exist on more basal branches

separate to M. punctata.

1.4 Overview of Tan

The genus Monarda contains 19 species that are split into two subgenera, one of which is
divided into two sections (Table 1). Section Cher'lyctis has five are narrow endemic
species. Monardafiwiculosa, M. maritima, M stanfieldii and M viridissima are endemic
to Texas, and M. humilrls', is endemic to New Mexico.

Table 1: Species in Monarda (Turner, 1994, Prather et al.2002 and Prather and Keith 2003) with scientific

and common names. Varieties and subspecies of Monarda punctata are included. Common names adapted
from the National Plants Database.

 

 

Species Common Names
SUBGENUS CHEIL YCTIS

SECTION ARISTA TAE

M. citriodora Cerv. ex Lag Lemon Beebalm

M. clincpodioides A. Gray Basil Beebalm

M pectinata Nutt. Pony Beebalm

SECTION CHEIL YCHS

M. fruticulosa Epling Shrubby Beebalm

M. humilis Prather & Keith Spreading Beebalm

M. maritima (Cory) B.L. Turner Seaside Beebalm

M. punctata L. Spotted Beebalm/Horsemint
- var. arkansana (McClintock & Epling) Shinners Arkansas Spotted Beebalm
- var. correllr’i B.L. Turner Correll’s Spotted Beebalm
- var. coryi (McClintock & Epling) Cory Cory’s Spotted Beebalm
- var. intermedia (McClintock & Epling) Waterfall Intermediate Spotted Beebalm
- var. Iasiodonta Gray Spotted Beebalm
- var. occidentalis (Epling) Palmer and Steyermar'k Western Spotted Beebalm
- var. villicaulr’s (Pennell) Shinners Villous Spotted Beebalm
- subw. punctata Epling Spotted Beebalm

M. stanfieldii Small Stanfield’s Beebalm

M viridissima Correll Green Beebalm
SUBGENUS MONARDA
M. didyma L. Scarlet Beebalm

M brarburiana Beck Eastern Beebalm

M clinopodia L. White Beebalm

M. eplingiana Stand]. Epling’s Beebalm

M. fistulosa L. Wild Beebalm or Bergamot
M. lindher'meri Engelm. & A. Gray Lindheirner’s Beebalm

M. media Willd. Purple Beebalm! Bergamot
M. pringlei Femald Pringle’s Beebalm
M russeliana Nutt. ex. Sims Redpurple Beebalm
M flimlandrdosa Waterf. Wild Beebalm

 

 

 

 

In contrast, M. punctata is widely distributed across the central and eastern United States,
southeast Canada, and northeast Mexico (Fig.2). These species also appear to have

distinct edaphic constraints in their distribution.

No formal field studies have addressed interspecific hybridization between these species,
but some field observations have been made. Monarda maritima is sympatric with M
puncata (Turner 1994, Prather pers comm, Keith pers. obs), Turner indicated that it was
found growing with other Monarda (unnamed taxa) and that no hybridization appeared to
occur between them. Monarda virrkiissima is also sympatric with M. punctata populations
(Turner 1994, Prather pers comm, Keith pets. Obs.). Turner found no evidence that these
two taxa overlapped in distribution but Prather and Keith observed sympatric populations
of M viridr’ssima and M punctata in Bastrop County Texas, although there was no
evidence that they bloomed at the same time. Monardafi-utr’culosa and M. prmctata also
grow together (Turner 1994, Prather pers comm., Keith pers. obs). However, Turner did
not find evidence that populations were confluent while Prather and Keith found evidence
of hybrid zones between them. Finally, M stanfieldii has not been found to intergrade
with M punctata (Turner 1994, Prather pers comm., Keith pers. obs.). Turner indicated
that M stanfieldr’i and M punctata do not intergrade, and Keith (pers. obs) found no
evidence that M stanfieldir' populations in Blanca, Bumet and Llano counties were
sympatric with M punctata. Lack of hybridization between these species suggests that

they maintain unique gene-pools and helps substantiate their species designation.

 

Figure 2: County distributions of Texas endemics, Monarda viridissima (v), M firdr'adosa (f), M
marinara (m), M stanfleldii (s), and the New Mexico endemic, M Inmilis (h). (based on Turner (1994),
Johnston and Correll (1979) and herbarium material from the University of New Mexico and New Mexico
State University)

1.5 Objectives

The purpose of this study was to test speciation by peripheral isolation and to determine
the neo- and paleoendemic status of the six species in Monarda Section Cheib’ctrlr. It was
hypothesized that the widespread taxon, M punctata, is the progenitor from which all
narrow endemic species have derived. Narrow endemic Monarda taxa were also
predicted to be neoendemic species. Geographic distributions and phylogenetic data were
used to test this. The low copy nuclear gene, Adh, was used as the genetic marker for
phylogenetic analyses. Maximum parsimony, maximum likelihood and Bayesian

methods of inference were used to generate phylogenic trees.

MATERIALS AND METHODS

2.1 Study System

i. Taxonomic Classification of Monarda Section Cheilyctis.

Three of the endemics have undergone conflicting taxonomic classifications (M
fi-utr'culosa, M maritima and M stanfieldii). The taxonomic ranks of these species have
been addressed in treatments by six primary authors (Table 2). Epling (1935), Epling and
McClintock (1942) and Turner (1994) recognized M fi-uticulosa as a species due to its
distinct morphology and apparent lack of hybridization with M punctata. Score (1967)
reduced its rank to a variety because he believed that did intergrade with M punctata var.
immaculata (a variety not recognized in this study). Similarly, both Cory (1936) and
Scora (1967) classified M maritima as a variety of M punctata because they believed it
shared characteristics with M fiwticulosa. In contrast, both Turner (1994) and Correll

(1968) treated it as a species due to its distinct morphology, habitat and distribution.

Monarda humilis was first described as a variety of M punctata (Torrey 1853) but was
recently recognized as a species by Prather and Keith (2003). This taxon was elevated to

specific status due to its distinct floral morphology and geographic distribution.

Monarda stanfieldir‘ was first described by Small (1903) and was later accepted as a
species by Turner ( 1994). Epling (1935) reduced it to a subspecies, and Cory (1936) and

Scora (1967) treated it as a variety of M punctata.

Jammie

 

 

 

Monarda viridissima was described as a species by Correll (1968) because of its unique
morphological characters and temporal reproductive isolation from M punctata. Correll
observed little evidence of hybridization with M punctata in the field. Turner (1994) and

Correll and Johnston (1979) maintained M viridissima at the species rank.

Turner’s recent treatment of these Monarda was used for this study. He recognized the
Texas endemics as species due to their distinct morphology, geographic distribution and
apparent lack of gene introgression with M punctata. He did not include the New

Mexican taxon (M humility) in his treatment.

ii. Morphology and Life History Traits
Several life history traits and morphological characters have been fundamental in
distinguishing these species. Key differences between them include life cycle and

phenology as well as floral and vegetative traits (Table 3).

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iii. Chromosome Numbers

All species in Monarda Section Cheilyctis have been found to be diploid. Scora (1967)
found that several species and varieties of M. punctata in Section Cheilyctis (M punctata
vars. arkansana, coryi and intermedia and M. fiuticulosa) had n=1 1 chromosomes, but
M. punctata var. villicaulis had n=12 and n=ll chromosomes. This result was further
confirmed in a study conducted by Bushnell (1936), which also showed the chromosome
number in M. punctata var. villicaulis was n=12. Monarda humilis was also found to

have n=12 chromosomes (Ward and Spellenberg 1984).

iv. Physiography and Vegetation
The Texas endemic Monarda seem to have distinct edaphic constraints on their

distributions. Less is known about the edaphic conditions that may limit M. humility.

Monardafiuticulosa grows in a region of South Texas called the Eolian Plain (Johnston
1963) that is divided into central and peripheral zones. The Central Eolian Plain has
loose, fine sandy soils that originally sustained copses of live oak (Quercus virginiana)
and some mesquite (Prosopis sp.). Many of the central plain soils are newer windblown
beach deposits, but also have older soils such as Galveston deep sands (Clover 1937).
The Peripheral Eolian Plain has loamy sand soils that once supported mesquite prairie.
Combinations of soil types may also exist in areas such as Brooks County where thin
layers of wind blown beach sands cover older soils. Suppression of natural fires, heavy
grazing and agriculture have altered the vegetation of the Eolian Plain, which is now

referred to as The Texas Brush Country.

12

Monarda humilis samples have been collected from the northwestern part of New Mexico
within the Rio Grand Basin. These plants most likely inhabit Penistaja soils, which are
deep, well drained, and comprised of alluvium fiom sandstone and shale. Penistaj a soils
are found primarily on upland regions such as plateaus and hills, and in drainageways

(USDA National Cooperative Soil Survey 2003).

Monarda maritima inhabits the deep sandy soils of the Coastal Belt (Turner 1994) that
extends across approximately a lOO-mile strip from Nueces to Galveston Bays. Its deep
coastal sands show little textural change with depth and are thought to be an old chain of
barrier islands from the late Pleistocene or Holocene called the Ingleside Terrace (Price
1933, Shideler 1986, Otvos 1991, Blum et al. 2002). Live oak (Quercus virginiana)

dominates these deep sands.

Monarda stanfieldii is primarily restricted to the middle course of the Colorado River
(Turner 1994) within the Central Texas Uplift. This region makes up the eastern
portion of The Texas Hill Country. Monarda stanfieldii only appears to inhabit
granitic sands (Turner 1994). Original vegetation in the Central Texas Uplift
consisted of grasslands and open savannah-like plains with small trees and brush

scrub growing along rocky slopes and stream bottoms (Bray 1901 and Buechner

1944).

Monarda viridissima is confined to fire Carrizo sand outcrop (Turner 1994) that runs

adjacent to The Blackland Prairies of East central Texas. Carrizo sands are unusually

13

homogeneous and harbor many endemic plant species (McBryde 1933). The Carrizo sand
belt originally harbored post oak (Quercus drummondii), blackjack oak (Q. marilandica)
and hickory (Carya texana) (Johnson 1963). Monarda viridissima grows in open post oak

or pine-oak forests

v. Edaphic Constraints

Vegetation and plant growth are largely reliant on the substrate in which a plant grows
(Jenny 1941). Most plant species are able to inhabit a wide range of soils but some have
adapted to become limited to specific soil types (Kruckeberg and Rabinowitz 1985).
These plants are referred to as edaphic endemics. Edaphic constraints can include
chemical, physical and biological properties. Edaphic endemics have been identified on
many substrates including soils composed of granite, limestone, Carrizo sand, oil shale,
gypsum and serpentines (McBryde 1933, Kruckeberg and Rabinowitz 1985). Edaphic
constraints may also be attributable to soil water holding capacity. Studies have found
that landscapes with sandy soils can have soil water environments that vary widely from

water permeated lowlands to arid dunes (Hoover and Parker 1991) and can sustain a

highly variable landscape flora (Ramaley 1939).

Several modes of speciation have been used to explain the evolution behind edaphically
restricted species including peripatric, parapatric and sympatric speciation (MacNair and
Gardner 1998). The most well documented and extreme examples of edaphic endemism
are in plants adapted to serpentine soils which are high in heavy metals and minerals such

as magnesium and calcium (Brooks 1987). Obligate edaphic endemics inhabiting

l4

serpentine soils are reported to have speciated very rapidly (Kruckeberg 1967) and often
are parapatric to sister taxa. All Texas endemic Monarda are reported as being edaphic

endemics (Turner 1994).

vi. Species Recognition

In this study it was assumed that the Monarda endemics were “good” species despite a
hypothesized phylogenetic outcome that did not render them reciprocally monophyletic
within the gene trees. This anticipated outcome conflicts with criteria set by some species
concepts, such as evolutionary, cladistic and phylogenetic species concepts, that delimit

species based on their phylogenetic relationships.

The lines that delimit species phenotypically, ecologically, genetically, and
phylogenetically do not always run parallel. If Monarda do not appear phylogenetically
distinct on the Adh gene trees it will not discredit their status as a biological species.
Based on their temporal and geographic separation they appear to be reproductively
isolated. Likewise, their morphological distinction suggests that they maintain their own
gene pools, and the putative edaphic constraints on their natural populations indicates that
they are ecologically distinct. These characteristics conform to several species concepts,

including Mayr’s (1942) biological species concept, and the phenetic species concept.
Other researchers have taken a comparable approach to recognizing species when faced
with marked differences in their ecological, morphological and phylogenetic distinction.

A phylogenetic study using ribosomal and chNA revealed that the widespread Mimulus

15

guttatus and several closely related serpentine endemic species were genetically indistinct
despite being highly dissimilar morphologically and ecologically (Macnair and Gardener
1998). Despite the lack of genetic distinction between these Mimulus, Macnair and
Gardener opted to recognize them as species because they maintained morphological and

ecological differences despite the fact they existed in sympatry.

2.2 Sampling

All Monarda species in Section Cheilyctis (Table 4) were included in the Acflr analyses.
Three individuals were sampled, each from a different population of M. fruticulosa, M.
maritima, M. stanfieldii and M. viridissima, one individual from a population of M.
humilis (Fig 3), and M. punctata from 19 separate geographic locations across the United

States (Table 4, Fig. 4).

Outgroup sequences included taxa of Monarda and Pycnanthemum (M. clinopodioides,
M. citriodora, P. Ioomisii, P. muticum and P. tenifolium). The Pycnanthemum sequences
were provided by Rachel Williams. Based on ITS phylogenies of Monarda and the
Lamiaceae subfamily Nepetoideae, Monarda Section Aristatae (M citriodora and M.
clinopodioides) is sister to Monarda Section Cheilyctis, and the genus Pycnanthemum is
closely related to Monarda (Prather et al. 2002 and Williams pers. com»). I chose these
outgroups based on this phylogenetic evidence.

In addition to Adh, some preliminary data was collected from a second molecular marker,
nchS. The nchS preliminary analysis included three endemic species (M fiuticulosa,

M maritima and M viridissima) and M punctata collected from four different

16

geographic locations (Table 5). Two Pycnanthemum species were used as orrtgroups.

Samples came from greenhouse, herbarium or silica dried plant material.

 

Table 4: DNA collection numbers and locations for all Monarda species in Acflrphxlogenetic analyses.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Specter DNA # State County Collector Collection #
M fiuliculosa 1920 Texas Kenedy Prather, LA 1858
2301 Texas Jim Hogg; Prather, LA 1860
2317 Texas Kenedy Keith, JA TX30
2420 Texas Zapata Prather, LA 2014
M hwm'lis 2176 New Mexico Socorro Tafoya, MA 94450 UNM
M mm'ifima 2181/2318 Texas Refugio Keith, JA TX36
2314 Texas Aransas Keith, JA TX23
2319 Texas Aransas Prather, LA 1940
M punctata 2060 Arkansas Calhoun Thomas, DR 141, 500
Amanson, C (7296) UTH
2303 Florida Bay Keith, JA FL4
2397 Florida Franklin Keith, JA FL9
2388 Florida Gadsden Keith, JA FM
2398 Oklahoma Marshall Barclay, A PI 326567
2305 Louisiana Iberville Thomas, DR 142, 284
Davies, M (7296) UTH
2401 S. Carolina Florence Churchill, JA 332325 MSU
2373 Texas Travis Keith, JA TX“
2387 Texas Zavala Ertter, B 527083 RMH
2393 Texas Aransas Keith, JA TX25
2396 Texas Bastrop Keith, JA TX14
2468 Texas Angelina Orzell, SL 18952 TAMU
Bridges, EL
M p. var. intermedia 2473 Texas Callahan Turner, BL, G 18900 TAMU
M p. var. villicaulis 1937 Delaware Sussex Keith, JA DE]
2048 Michigan Alleghan Keith, JA MI]
2172 Missouri Madison Ladd, D 4236830 MBG
Yatskievych G
1946/2189 New Jersey Burington Keith, JA NJ 1
M p. var. 2307 New Mexico Eddy Martin, WC 79100 UNM
occidentalis Schmidt, B (1215)
M p. subsp. monarda 2389 Florida Alachua Keith, JA FLll
M starfieldii 2339 Texas Llano Keith, JA TX7
2367 Texas Burnet Keith, JA TX2
2369 Texas Blanco Keith, JA TX9
M viridr'ssima 2055 Texas Milam Keith, JA 029007 Tamu
2441/2442 Texas Bastrop Keith, JA TX39
2444 Texas Milam Keith, JA TX40

 

 

in nchS phylogenetic analyses.

 

   

181 & 2318
2319
14

Figure 3: Collection locations of endemic species with their corresponding DNA numbers.
Texas samples include M fiwiculosa (1920, 2301, 2317 and 2420 (f)), M maritima (2314, 2319 and
2318/2181 (m)) M stanfieldii (2339, 2367 and 2369 (8)) and M viridissima (2055, 2441 and 2444 (V)).

The New Mexico sample is ofM humilis (2307 (11))

 

 

m, . 4 k
. 4
w v q~‘rmw

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4: Collection locations of the 16 M punctata samples with their corresponding DNA numbers.

2.3 Marker Choice

In this study, two homologs of the nuclear gene, Alcohol dehydrogenase (Arm), were
used for phylogenetic analysis. In addition to Adh data, some preliminary results were
also obtained for chloroplast expressed glutamine synthetase (nchS). Low copy nuclear
genes were chosen for this work because in previous studies ITS (Prather et al 2002) and
chNA (Prather and Monfils unpub. data) provided no phylogenetic information for
Monarda species in Section Cheilyctis.

Aflr has been widely used as a molecular marker in plant phylogenetics. Ath produced a
more resolved phylogeny in Paeom’a with sequence divergence one and one halftimes

higher than ITS (Sang 1997). Acflr and chNA sequences were also compared in

19

Gossypium and Adh was found to be far more informative (Small et al. 1998). Likewise,
in additional phylogenetic studies of Gossmium, .4th sequences were phylogenetically
informative and produced well-resolved trees (Small and Wendel 2000). Adh has also
been used to compare inter- and intraspecific relationships in DNA variation and codon
bias in the closely related brassicas Arabis and Arabidopsis (Miyashita et al. 1996), so it

can provide phylogenetic informative at the highest taxonomic levels.

Adh is referred to as “classical’ alcohol dehydrogenase. The alcohol dehydrogenase
produced by Adh is NAD+ dependent and oxidizes ethanol (reviewed in Chase 1999). In
most plant systems, there are two Adh loci. In Solanaceous plants, the loci differ in their
tissue-specific expression. One Adh enzyme is expressed when the plants are subjected to
anaerobic conditions (hypoxia) whereas the second is only expressed in anthers and
seeds. Moreover, two additional Adh genes have been identified and described in

Lycopersicon esculentum and Solanwn tuberoswn (Chase 1999).

Glutamine synthetase (nchS) encodes for an enzyme that functions in the assimilation
of ammonium into glutamine (reviewed in Ford & Cullimore 1989). In most plants, two
distinct classes of these genes exist. One class encodes for cytosolic enzymes and in most
plants four to six genes have been described. The other class encodes for a plastidic
protein and only one gene has been described in most plant species (Emshwiller & Doyle
1999). The choroplast-targeted glutamine synthetase was used to determine relationships
between eight Oxalis species and sequence divergence in intron regions was shown to be

higher (8.63%) than that of ITS sequences (7.75%; Emshwiller & Doyle 1999).

20

2.4 DNA Extraction

DNA was extracted using the 2xCTAB method (Doyle & Doyle 1987) as modified by
Zhang and Stewart (2000). DNA was extracted from living plants and herbarium
material. Extracted genomic DNA was cleaned using Schleicher & Schuell Elu-quick

DNA purification kit (Neese, NH).

2.5 Primers

Four sets of Adh primers, both general and specific, were used for PCR amplification of
Monarda. The general primers, Adh-R and Adh-F (Sang et al. 1997), amplified ~1400
basepairs of Adh sequence in Paeonia (Table 6). Adh-R and Adh-F primers were used to
amplify the first Adh sequences in these Monarda, but they provided poor amplification
results. Consequently, it was necessary to design Monarda specific general primers that
provided better Adh amplification in these plants.

Cloned Adh sequences were obtained from Monarda and Pycnanthemum using Arflr—F
and Adh-R Twenty of these sequences were aligned using Sequencher 3.0. (Gene Codes,
Ann Arbor, MI). General primers, labeled Adhm-F and Adhm-R (Adh-Monarda-forward
and reverse; Table 6), were designed at conserved regions near the 5’ and 3’ ends of this
alignment. The Adhm-F and Adhm-R primers amplified a ~1300 basepair region of Adh

in Monarda and Pycnanthemum.

An additional primer set, Adhmc—F and Adhmc-R (Adh-Monarda cut-forward and

reverse), was designed to differentially amplify a ~1200 basepair region believed to be

specific to a single locus, named Adh type 1 (Ad/21, see below). These primers were used

21

to test direct sequencing of the Adhl locus and allowed for more direct selection of these
clones for sequencing. PCR product amplified with Adhmc-F/Adhmc-R primers was
directly sequenced without cloning, but direct sequencing was problematic due to allelic
variation in Adhl sequences so as an alternative these primers were used to amplify Adhl
sequences for cloning. Finally, an additional primer Adhi—F (Adh-internal-forward) was
designed to sequence an internal region in all Adh homologs in Monarda. This was a
forward primer that amplified a region starting approximately 350 bases downstream
from the Adhm and Adhc forward primers that was needed for obtaining a complete

sequence of the entire amplified fragment.

One set of nchS primers, GScp687-f and GScp994-r (designed by Emshwiller & Doyle
1999) (Table 6), were used to amplify a 700 basepair region of the glutamine synthetase
gene in Monarda. This gene region was known to contain four intron regions in Oxalis

species (Emshwiller & Doyle 1999).

Table 6: Primer sequences used for the amplification of Acflr gene ree'ons, and nchS regions.
Adh-f: 5’-TAC TTCTGGGAAGCYAAG G-3’
Adh-r: 3’-CCTCGCATATTT GGTCA GAA G-5’
Adhm-f 5’-TATCTCAGGCGCAAGATTCA-3’
Adhm-r 3’-GCATCTTTGTGTGGAACTCCT-5’
Adhc-f 5’- GAACATCCCAGGCTCAAGAT-3’
Adhc-r 3’- TAGAGCAGAATGCAGCCACA-S’
Adhi-f 5’- CCCGTITACCAT'ITI‘CTT-T
GScp687-f 5’-GATGCTCACTACAAGGCTTG~3’
GScp994-r 5’-AATGTGCTC'I'I‘TGTGGCGAAG-3’

2.6 DNA Amplification
PCR was conducted on a PT C-1 00 thermocycler. Three separate thermocycler programs

were used (Table 7) depending on the case at which the DNA could be amplified. DNA

extracted fiom herbarium tissue generally required the use of either a stepdown program

or an annealing temperature of 55° C. Higher quality DNA could ofien be amplified at

60° C. Two Taq Polymerases were used for amplification. Platinum® Taq DNA

Polymerase (InvitrogenTM Life Technologies) was used to improve specificity and yield

of PCR product for DNA samples that were either difficult to amplify or showed a high

rate of PCR recombination. Standard Taq DNA Polymerase (New England Biolabs®,

Inc.) was used for all other PCR reactions. Amplification components follow those used

by Prather and Jansen (1998) with the addition of 5% DMSO to each reaction and a 5

minute hotstart at 94°C for each thermocycler run.

Table 7: Three thermocycler programs used for PCR amplification

55° and 60° C annealim'am

Stepdown program

 

1. 95° C for 5 minutes

2. 72° C for 2 minutes

3. 94° C for 1 minute

4. 55° or 60° C for 1 minute

5. 72° C for 3 minutes
6. 29 times to step 3

7. 72° c for 5 minutes
8. 15° C indefinitely

2.7 Cloning

1. 95° c for 5 minutes
2. 72° c for 2 minutes
3. 94° C for 1 minute
4. 56° C for 1 minute

5.72°C for3 minutes
6.2timestostep3
7. Repeatstep33t06butreducethe

annealing temperature by 3° C
each time until an annealing
temperature of 37° C is reached.
8. 72° C for 5 minutes
9. 15° C indefinitely

PCR products were gel purified from 2% agarose gels and cleaned using the Schleicher &

Schuell Elu-quick DNA purification kit (Neese, NH). Purified DNA was cloned into E.

23

coli using TOPOTM TA Sequence Cloning® Kit (Invitrogen, Carlesbad, CA). Plasmid
DNA was purified fiom bacteria by the use of either QIAGEN® QIAprep Spin Miniprep
Kits (Qiagenm) or the manual DNA extraction method for bacteria (Sambrook et al.

1989)

2.8 Adlr Clone Isolation

In several species it was necessary to pre-select sequences from a specific locus prior to
cloning. Potential loci were preselected by digesting the Adhm-F/Adhm-R PCR product
with two restriction enzymes. Ach was used for .4th selection, and EcoRI was used for
Ath and Adh3 selection (see below). Digested DNA was gel purified and undigested
bands were isolated. Ach-selected and purified PCR product was cleaned and directly
cloned. Ath and Adh3 selection (see below) involved additional steps due to the
presence of an EcoRI cutting site located 43 basepairs downstream from the 5’ end of the
amplified sequence. It was necessary to fill this EcoRI overhang and adjoin an adenine-
tail for insertion into the Topo® vector. Purified PCR products were cleaned and
precipitated. Precipitated product was then vacuum dried for 10 minutes in a Labnet
DyNAVap vacuum microfuge and rehydrated in 10 microliters of sterile distilled
deionized water. EcoRI overhangs were filled using Klenow DNA Polymerase I Large
Fragment (Tabor and Struhl, 1990) resulting in a blunt-ended product. Klenow reactions
were stopped by placement on a 75° Centigrade heating block for 10 minutes. For the
addition 3’ adenine overhangs, the heating block was lowered to 72° C, Taq polymerase

was added and, reactions were held at this temperature for 10 minutes. A-tailed DNA

24

samples were then precipitated, rehydrated in sterile distilled deionized water and directly

cloned using Topo® TA kits.

2.9 Restriction Digest Analysis of Ad]:

Prior to sequencing, Adh clones were digested with the restriction enzyme EcoRl .
Digested clones exhibited three digest patterns when gel purified: no digestion, digestion
at base 900 and digestion at base 400 and 900 (Fig 5). It was discovered that digested
clones (both those that were digested at bases 400 and at 900) had sequences that
represented one locus (Ad/11) while undigested clones had sequences that represented the
second and third locus (Ad/12 and Adh3). Restriction site cutting locations on sequences

were confirmed using Webcutter 2.0 (Heiman 1997).

Adhtl digest- Adhtl digest AdhtZ &.Adht3-
bp 400 & 900 bp 900 no digestion

 

Figure 5: Cloned samples of M punctata DNA (2048) digested from the Topo plasmid vector using EcoRl.
Digested bands show three digestion patterns that were found to correspond to the three putative Arfll loci:
Arflrl with single and double-digested bands and AM and AM with undigested bands.

2.10 Sequencing

Each Adh DNA insert was digested from the TOPOT“ plasmid vector using EcoRI and
quantified against a 100 basepair DNA ladder of known concentration (New England
Biolabs®, 1nc.). Sequence reactions were prepared using ABI PRISM® BigDyem
Terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase
(Applied Biosystems©) and thermocycler parameters were set to those specified in the
kit. Each Adh DNA insert was sequenced from the 5’ and 3’ ends and internally. All

sequences were obtained using an ABI-3 73 automated sequencer.
2.11 DNA Alignment

Sequences were aligned and edited manually using Sequencher 3.0. (Gene Codes, Ann

Arbor, MI). Aligned sequences were truncated so that they were all approximately 1200

26

basepairs in length. Complete datasets representing each marker were aligned first

followed by subsets that represented an individual locus.

2.12 Identification of Putative Adlr and nchS Loci

A tree containing the firll Adh dataset of 99 sequences was used to identify putative loci
(see results, Fig. 8) and served as a template to identify potential PCR recombinant
sequences. These sequences were manually aligned and analyzed by maximum
parsimony using Paup“ version 4.0bv (Swofford 2000) (see results, Fig. 6) and
parsimony settings were consistent with those used for all other analyses (see below).
Trees were viewed as unrooted phylograms. Phylogenetically divergent sequence sets
revealed three putative loci, labeled Adh type 1 (Ath), Adh type 2 (Ad/12) and Aflr type 3
(Ad/13). A second parsimony tree was used to identify suitable outgroup sequences for the
same three putative loci. An unrooted phylogram was generated using 22 outgroup
sequences (Pycnanthemum and Monarda spp.) and nine ingroup sequences representing
Ath, Ath and Adh3. The phylogeny was generated by maximum parsimony using the
same methods, and two equally parsimonious trees were created and tree one was used

for analysis (see results, Fig 9).

2.13 Outgroup Selection and Rooting

Outgroup sequences that appeared at the base of the ingroup sequences representing
Ath, Adh2 and Adh3 were used for analysis (see results, Fig 8). The Ath sequence set
was rooted with two sequences of M clinopodioides and sequences of P. muticum, P.
Ioomisii and P. tenuifolium. The Ath and Adh3 sequence set was rooted with P.

muticum, P. Ioomisii, three sequences of M citriodora, and three sequences of P.

27

tenuifolium. The Ath dataset was rooted with two sequences of M citriodora and two
sequences P. tenuifolium.Adh1 and Ath datasets were reduced to contain the same
samples. The reduced Adhl dataset was rooted with P. muticum, P. tenuifolium, and two
sequences of M clin0podioides and the reduced Ath dataset was rooted with the same

sequences used for the full Ath dataset.

nchS loci were identified using a phylogeny of nineteen ingroup sequences (twelve
taxa) and two outgroup sequences (Pycnanthemum). Phylogenetic analyses were the
same as those used for Adh. Results showed that there were two putative nchS loci that
were divided by 24 base changes (see results, Fig 20). These putative loci were labeled

GStI and GStZ.

2.14 Phylogenetic Analyses

For ease in interpreting the phylogenetic results, analyses completed for each dataset are
indexed in Table 9. Sequences were analyzed using Paup“ version 4.0bv (Swofford
2000). Maximum parsimony analyses were performed using Tree Bisection
Reconnection, Collapse, and Multrees options and Steepest Decent was inactive. Strict
consensus trees were obtained and bootstrap support was measured using 1000 bootstrap
replicates (F elsenstein 1985) with 100 addition-sequence replicates per bootstrap.

The sequences were also analyzed using maximum likelihood methods. Optimal
likelihood analyses were determined using Modeltest version 3.6 (Posada and Crandall
1998). The TrN+I+G model was selected for Ath and the TrNef+G model was selected

for Ath (Table 8). The Shimodaira-Hasegawa (S-H) test was used to determine whether

28

flat-.2, . .‘ .fifi All“

 

maximum parsimony trees were significantly worse than the maximum likelihood trees

(Goldman et al. 2000).

Table 8: Likelihood model parameters for TrN+I+G and Tmef+G

 

 

 

 

Likelihood Model TrN+I+G Tmef+G

Base Frequencies Freq A= 0.2766 Empirical frequencies
Freq C= 0.1752
Freq G= 0.2228
Freq T= 0.3254

Substitution Model [A-C]= 1.0000 [A-C]= 1.0000
[A-G]= 2.3130 [A-G]= 6.1615
[A-T]= 1.0000 [A-T]= 1.0000
[C-G]= 1.0000 [C-G]= 1.0000
[C-T]= 3.3248 [C-T]= 11.7499
[G-T]= 1.0000 [G-Tl= 1.0000

Proportion if =0.3488 = 0

invariable sites

Gamma distribution = 0.7773 = 0.1897

 

 

 

 

 

Maximum likelihood methods were used because data contained imbalanced base

frequencies (see below) and sites that have evolved quickly (intron regions) and slowly

(exon regions). When data trends show these types of anomalies, it is useful to include

likelihood-based methods of analysis (Collins et al. 1994).

The base frequencies for all Adh datasets were calculated on PAUP ‘ version 4.0bv

(Swofford 2000) and histograms were made using Microsoft Excel (Fig. 6 and 7). Results

were used to identify biases in the datasets that may potentially skew parsimony results.

The nucleotide frequencies among taxa showed a character state bias in the dataset.

29

 

 

1..

Table 9: List of all phylogenetic analyses completed for each Adh dataset. Check marks indicate a
'cular test was completed for that dataset.

 

 

 

 

 

 

 

 

Analysis Full Adh Outgroup .4th Ath & Adh3 Ari/12 Adh3 Reduced
Arflrl & .4er

Maximum
parsimony / I I I I \/ /
Parsimony strict
consensus I J I I I
Parsimony
13001811?!) I I I I
Maximum
likelihood / \/
Shimodaira-
Hasegawa test I I
Bayesian
analysis I J l /

 

 

 

 

 

 

 

 

Bayesian analyses were run using Mr. Bayes 3.0 (I-Iuelsenbeck, 2001). Bayesian
inference is a more recent tool for phylogenetic analysis that is just beginning to gain
popularity (Alfaro et al. 2003). This phylogenetic method uses Baye’s theorem of inverse
probability and likelihood parameters to measure tree topologies and branch lengths.
Bayesian methods apply probabilities that a given tree is correct given a particular dataset
and maximum likelihood parameters. In Bayesian analyses Bayesian confidence values
are also applied to the tree nodes. These tree node probabilities are determined using
Bayesian Markov Chain Monte Carlo (BMCMC) sampling method inference (Alfaro et
al. 2003). Probabilities are determined from multiple phylogenies generated by Bayesian
inference. The Markov chain samples the branch lengths and substitution parameters of a
given set of Bayesian trees to generate node probabilities. Node values above 90 are

considered to be well supported.

30

 

2.15 Phylogenetic Interpretation

Neoendemic species were distinguished from paleoendemic species based on their
phylogenetic position. Paleoendemics were expected to appear as the sister group to M
punctata and to have longer branch lengths, and neoendemics were expected to be
derived within M punctata and have shorter branches. In addition, species that speciated
as peripheral isolates of M punctata are expected to show tree topologies similar to those

shown in the hypothetical gene tree depicting peripatric speciation in Figure 1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Adm. Base Frequencies Among Sites (1096 sites)
035- - _ ;
>o.3———m__ ___~figgg___ ___§
c 0.25 “—' i 'r — — — —— 7 ~ * __
0 I
:r 0.2 —- ———_._ _ m___. 1
i “T
o 0.15 r— _____,__ ,__ ,.;_ _+__*, «mg.
3 1
1% 0.1 as __ _ a __ _ -—-—:
80.0513. A — __ _.__. __i
o . T . 3
Nucleotides Bases

 

 

 

Fig 6: Histogram of the base frequencies (Average frequency/ 100) in the Arflrl dataset. These results are
based on 1096 base sites.

31

 

Adh2 and Adh3 Base Frequencies Among Sites
(1 120 sites)

 

 

 

oouflouenbeig escrow

 

 

 

Nucleotide Bases

 

 

 

Fig 7: Histogram ofthe base frequencies (Average frequency/100) intheArflrZ andArflr3 datasets. These
resultsarebasedon 1120 base sites.

32

  

as}; dawn h?

 

RESULTS

3.1 Identification of Putative Loci

Ninety-nine Adh sequences, approximately 1,200 basepairs in length were obtained fi'orn
the six ingroup Monarda species. Two separate phylogenetic analyses were used to
delineate potential loci. The fast phylogeny was generated by maximum parsimony and
contained all 99 ingroup Monarda sequences. One hundred equally parsimonious trees
were generated for this dataset, tree length was 1663, the consistency index (CI) was
0.518 and the retention index (R1) was 0.870. In an unrooted phylogram (Fig. 8), Acflrl
and .4th were divided by 70 base changes and .4th and Add were divided by 20 base
changes. This phylogeny was used to determine how and where ingroup sequences

parsed, and allowed for better ingroup sequence selection for the second analysis.

The second analysis contained 23 potential outgroup sequences (seven species) and nine
ingroup sequences (five species) representing Ath , Adh2 and Adh3. The ingroup
sequences were selected from several points across the each putative locus. Data were
analyzed by maximum parsimony and two equally parsimonious trees were created with
a length of 797, a CI of 0.649 and a R1 of 0.806. In an unrooted phylogram (Fig. 9), Acflrl
and .4th were separated by 43 base changes and Adh2 and Adh3 were separated by 12
changes. A strict consensus tree was generated from the two most parsimonious trees
(Fig. 10), and compared to the unrooted phylogram. Branch length disparity between the
full Adh tree and outgroup tree was attributed to the increased sample number in the full

Aflr tree and the inclusion of outgroup samples. Pycnanthemum samples appear to have a

33

fourth putative locus adjacent to Ath that is not found in Monarda, and no
Pycnanthemum samples appear to branch within AflrZ.

The Ath dataset was rooted with two M clinopodioides samples and P. muticum, P.
Ioomisii and P. tenuifolium samples. The Adh2/Adh3 dataset was rooted with P. -muticum,
P. Ioomisii, three M citriodora samples, and three P. tenuifolium samples. The ArflrZ

dataset was rooted with two M citriodora and P. tenuifolium samples.

34

Locus MM ,1

70

20 \— Locus Adh3

Locus Adh2

— 5 changes
Figure 8: An unrooted phylogram of ninety-nine Adh sequences representing all ingroup Monarda samples.
The three putative loci, Adhl, Adh2 and Aflr3, are labeled. Seventy steps separate Acflrl and Adh2 and 20
steps separate Adh2 and Ad713. Several sequences branch between Adhl and Add but were found to fall in
the Acfl12 dataset when .4th and Adh3 were separated and outgroup sequences were incorporated.

35

Locus Adh1

M. stanfleldli TX 2367

P. loomlall 66-

P. tenulfollum 86-4
P. Ioomisli 66-

P. mufleum 52-4

P. Muflollom 85-4

P. tenulfollum 97-3
M. viridissima TX 2 . '

M. punctata NJ 2189
12

M. punctataLA2305
M. frutlculosa 1920

  
   

M. eltrlodora 2419

I. cm 2375
M. citriodora 2375

Locus Adh2

—-—-10diaiges

M. 11111in TX1920

M. citrlodora 2419 M. punctataNJ 2189

 
  
  

M. pmctata M0 2172

I. ellnopodloldea 2310
M. ellnopodloldea 2310
P. tenuifolium 55-4

P. tenuiican 975

P. mullcum 52-3
P. curvlpes 444

P. curvipes 446
P. virginianum M13

P. mutlcum 52-2
P. Ioornlall 66-1

P. vuginiaum Ml11
P. virg'nianum M17

P. tenultollum 80-8

   
  
 

M. punCtata SC 2401

Locus Adh3

Figure 9: Phylogeny of 23 outgroup sequences and nine ingroup sequences representing all three
putative loci identified (Add, AflrZ and Acflr3). The branch dividing Acflil and AcflrZ has 43
changes and the branch dividing Act/12 and Adh3 has 12 changes. Taxon labels include species and
DNA numbers. Samples in bold were chosen as outgroups for later analyses.

36

Strict

 

 

Adh1

 

M. fruticulosa TX 1920

 

 

 

 

P—-—— M. punctala MO 2172
M. stanfieldii TX 2367

 

 

 

 

 

 

 

 

M.cllnopodloldea 2310
——M.cllnopodloldea 2310

 

 

 

M. d’nopodioides 2310
P. Ioomlall 00-3

 

 

 

 

 

 

P. mutlcum 02-4

___: P.100misiiSS-2
P. tenultollum 004

 

 

 

 

 

 

 

 

 

P. swipes 444
r—-__‘[: R W 44.3
P. muticum 52-3
_: P. muticum 52-3

 

P. viginianum Ml3
r— P. tenuifolium 554

 

I—— P. tenuitolium 97-5

 

Adh2

 

 

P. mutlcum 02-2
r— P. tenulfollum 004

 

 

 

 

 

 

 

 

 

 

P. Muflollllm 97-3

____: M.dm2375
meltdown-2419

___E: .- cltrlodora 24".
I. citrlodm 2375

 

 

 

 

 

M. viridissima TX 2444
M. punctata NJ 2189

 

 

 

L—— MpuncldaLA2305

 

M. fruticulosaTX1920

 

Adh3

 

F——‘ M. pimctata SC 2401

 

 

 

 

 

M. punclala NJ 2189

 

P. tenulfollum 00-0

 

 

P. loomlall 00-1
P. virginianum 55-4

 

 

 

 

 

P. virghiamm 97a

 

Figure 10: Strict consensus tree of 2 equally parsimonious trees representing 23 potential outgroup
sequences and nine ingroup sequences from all three loci identified (Adhl , Adh2 and Adh3). The
Pycnanthemum clade between Adhl and Adh2 represents a fourth putative locus that is unique to
Pycnanthemum. Sequences that are in bold were chosen as outgroups. Taxon labels include species and

DNA numbers.

37

 

 

3.2 Adhl

The Adhl dataset had 35 ingroup sequences of all six species collected from 24
populations. Data were analyzed by maximum parsimony, parsimony bootstrap,
maximum likelihood, the S-H test and Bayesian analyses. Thirty-seven most-
parsirnonious trees were generated and each had a length of 480, a (C1) of 0.503 and a
(RI) of 0.746. All uninformative characters were removed from the datasets before CI
values were obtained. The S-H test was run on all 37 most parsimonious trees and one
maximum likelihood tree (TrNef+G). The best maximum parsimony tree identified (Fig
11) had several topological differences relative to the TrNef+G tree (Fig. 12) including
the placement of M stanfieldii, M maritima and M fi'uticulosa (2301 and 2317) samples.
The parsimony strict consensus tree (Fig. 13) and Bayesian consensus tree shared the
same topology, and bootstrap values and Bayesian node confidence values were applied

to this phylogeny.

The strict consensus tree had one major M punctata clade with M fiuticulosa, M
humilis, M maritima and M viridissima sequences derived within it (Fig 13). The M
punctata clade had a high Bayesian confidence value of 99 (NC99), but had bootstrap
support less than 50 (BS-). Most of the M punctata samples were together in two smaller
clades: One contained nine M punctata samples (2303, 2397, 2398, 2305, 2048, 2172,
2307 and 2396) and had strong support (BS81/N C100), while the second had four
samples (193 7, 2048, and 2189) and higher support (99BS/100NC). Monardafi'uticulosa
(2301 and 2317) and M maritima (2314, 2318 and 2319) samples shared two moderately

well-supported clades (-BS/99NC and 59BS/100NC) within the larger M punctata clade.

38

I. punctah PI. 2303
in. punctm no 2172
4 “ in. puma n. 2301
n. punctat- PL 2303
. 4 ii: In. punctnta our asee
Length. 480 16 re. w I I LA 2305
CI: 0.503 7 - 5 Iii. pence-ta m aces
RI: 0.743 I 9 I II. punctata rm 2301
6 1 I. punetata TX 2300

5 MfruticulosaTX1920
2 MfiuticulosaTX1920

 

 

 

4 2 M. maritime TX 2314
3 M. maritime TX 2319
1° M. maritime TX 2318
M. fiuticulosa TX 2301

 

 
  
 

M.fiuticuosaTX2317
I. punetata AR 2000
M.maritimaTx 2318
M.viiidissimaTX2441

1” M. staifielleX2239
fl 1° M. stanfleldiiTX2369
1‘ Msmaeiduszzas

I. prince-ta NJ 2100
3 I. pence-ta NJ 2100
I. punche- D! 1037
I. prince-ta m 2000

 

 

 

fl

 

 

 

 

   
   
 

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

M. viridiss'rna TX 2055
a. 5 l M. viridissima TX 2444
4 I. pallet-ta NI 2301
.9. 2| E a. par-cm em 2301
a 1‘ M. humilis NM 2176
‘° M. viridissima TX 2441
21 3° n. punctata n: ma
.7 1 M. stanfieldfi TX 2367
7 M. stmfieldil TX 2367
,1 M. clincpodloidesTX2310
L—‘ M. clinopodioides TX 2310
19 r—g— P. lOOMSll
‘1 P. whom
i P. tenuifolium
—— Schemes

Figure 11: The Adhl parsimony phylogram selected by the S-H test. Branches are labeled with steps.
Taxon labels include species, state of origin and DNA number. The larger M punctata clade is labeled by
the bar along the right margin. Tree length, CI and RI values are shown.

39

 

“Mon-Ma punchtaPL2303
HL—— Ionardapunctata I0 2172
I——-—M4marrla punctata PL 2307
Ji—‘E: Ion-m pence-ta Fl. 2303
. ummmmm
L—-————Ionar0a pom-2300
”—1 -—-- Menard- punctata Ill 2040
‘ Ila-adaption”!!! 2301

Inca-mum

[:— M. fl'uticulosa TX 1920
r M. frub'culosa TX 1920

__|———— M. fiuticulosa TX 2111

MmaritimaTX2318
—-—-— anilculosaTX2317

M. maitima TX 2318
— Monarch metat- 20001

__{-— M. maritime TX 2314
M. maitima TX 2319

m punctata 1031

 

 

 

 

 

 

 

 

 

 

 

 

 

Monarch parrot-ta 2100

_: We punctu- 2301
Menard. punctata 2307

 

 

 

———-————— M. humilisNM21763

r M.viridissimaTX2444

J M. staifieldilTX2367
'M.siarneidiirx2367

WWW

r—‘i MetanfleldliTX2239

MWITX2309

M.stanfleldiTX22$

__{ M.viritissimaTX2441

M.v'ridissimaTX2441

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

J M. clinopodbides 2310
L— M. clinopodioides 2310

r—~—-—— P. ioomisi 66-3

L- P. muticum 524

P. ionuiioium 86-4

—— 0.005 subsuuiionsisiia

 

 

Figure 12: The Acflil likelihood phylogram using the TrNef+G model of substitution. Branches are labeled
with values denoting substitutions per site. Taxon labels include species, state of origin and DNA number.

40

1001100

70/100

-/87 ‘

74/1 00

MCI. pallet-t- PI. 2303
M. planet-ta OK 2300

M. poi-cm PI. 2397
ll. punchh no 2172
u. puma La 2305
L———— re. punctlta PI. 2303

 

 

 

 

 

499

name-mama

r" re. pence-a NM 2307
1— M. punctu- TX 2300
,— M. tiuflculosaTX 1920

L— M. fruticulosa TX 1920
97190 M. man'tima TX 2314
M. maritime TX 2319
M. maritima TX 2318

 

 

 

M. fluticulosa TX 2317

M. fruticulosa TX 2301
553/100 M. punctata an aoeo
M. maitima TX 2318

 

 

 

 

 

 

 

 

70/100

100/100

M. viridissima TX 2441
M. stanfieldii TX 2239
l—I: M. stanfieldii TX 2369
M. stanfieldii TX 2239
u. punctata or: 1937

unmet-ta MI2040
npunceata M2100

99/100

 

 

 

 

 

I. panchr- NJ 2100
M. viridlssima TX 2055
M. viridissima TX 2444

I. puncture NI 2307
ifiCEu. puma m: 2307
M. humilis NM 2176

 

M. viiidisslma TX 2441

 

111N100

u. punchtl rx use
,— M. swoon 1x 236?
l—M. stanfieidii TX 2367
J—M. clinopodioides 2310
l—M. clinopodioides 2310
J— P. Ioomlsi 66-3

 

 

L— P. muticum 52-4

 

P. tenuifolium 86-4

Figure 13: Strict consensus of 37 maximum parsimony trees from the full Adhl dataset. Taxon labels
include species, state of origin and DNA number. Branches are labeled with bootstrap (left) and Bayesian

node confidence values (right); only values above 50 are shown and values below 50 are labeled with

dashes. The larger M punctata clade is labeled by the bar along the right margin.

41

This was the only narrow endemic species that was not within the M punctata clade.
Two M stanfieldii clones (2367) appeared on the most basal branch sister to the M
punctata clade and two additional M stanfieldii samples (2239 and 2369) were derived
within the larger M punctata clade but with no support. Monarda viridissima was more
widely dispersed in the phylogeny. Two samples (2055 and 2444) branched together with
M punctata and two clones of the third sample (244]) were not supported in the larger
M punctata clade. The single M humilis sample (2176) had good support (-BS/95 NC)

being in a clade with two M punctata from New Mexico (2307).

3.3 .4th and Adh3

These data were analyzed by maximum parsimony, parsimony bootstrap and Bayesian
analyses. The Adh2 and Adh3 dataset included 38 sequences and generated 6 most
parsimonious trees with a length of 571 steps, a CI of 0.434 and R1 of 0.743. The
parsimony and Bayesian consensus trees were identical and both parsimony bootstrap

values and Bayesian node confidence values were applied to this tree (Fig. 14).

Many sequences were replicated in both Adh2 and Adh3 trees. Outgroup sample, M
citriodora had two sequences at the base of each putative locus (Fig. 14). The branch that
delineated Adh2 and Adh3 had a high Bayesian node confidence value of 100 but no
bootstrap support. Nearly all the samples in Adh3 were of geographically separate M
punctata except for two M stanfieldii sequences (2369) in the clade with M citriodora
(2419). Clades in Adh3 had several high Bayesian node confidence values but little to no

bootstrap support.

42

Length: 786
CI: 0.0.434
RI: 0.743

56/100

100/100

84/100

73/92

86/100

—-1:

H. punctata NJ 2100
M. punctata NJ 2100

 

-/87

 

61/91

 

 

 

 

 

458

 

 

-/100

 

 

 

100/100

JZMLC:

 

M. punctata M1 2040
M. punctata TX 2300
M. punctata TX 2473
{—— N. 9006800. LA 2305

 

 

L—— M. punctata r1. ass-r

 

M. humilis NM 2176
M. viridlsslma TX 2444

 

71/89

92/100

MfruticulosaTX2317
MfiuliculosaTX2301

1’11:

 

 

57/99

 

 

 

 

fl. pence-ta TX 2303
M. fiuticulosa 2420

 

 

 

 

100/100

M. pence-ta PI. 2300
l"— M. fl'UllCUkBa TX 1920

 

60/ 100

100/100

L— M.fiuticulosaTX1920

69/75 M. vindlsslma TX 2055

 

 

 

 

 

100/100

 

M. punctata MI 2040
M. punctata OK 2300
——-—— M. maitima 2181
M. maritime 2181

 

 

~— flpunctataflm

1001—1“): M. 310111301011 TX 2387

 

62/99

 

499.3

 

-/100

—/99.2

84/100
-/99.9 —i:

 

M. cluiodora TX2419
M. citiiodora TX2375
M. puncture NJ 1040
ll. punches NJ 2100

M. 813111011111 TX 2367
100/1

 

 

4100

 

 

 

 

I. punctuta NJ 2109

 

I. punctata TX 2400

 

 

 

 

 

 

81/100

M. punctata TX 2307
r——-- M. punch“ TX 2303

 

 

51/999

L— 111. punctata so 2401
r—— ra. punctata TX 2300

 

L—-—- M. punctata rr. asae

 

4100

 

M. atmfieldii TX 2369
iS-S—LT.

M summon TX 2369
,-—— P. Ioomisii 66-1

 

M. cltriodora TX 2419
L— P. ionuiioium 86-8

 

 

P. muticum- 52-2
1“"" P. tenuitolium 55-4

 

L—— P.1onuiioium 97-3

Locus
Adh2

Locus
Adh3

Figure 14: Strict consensus of 6 maximum parsimony trees from the full .4er and Adh3 dataset. Taxon
labels include species, state of origin and DNA number. Branches are labeled with bootstrap (left) and
Bayesian node confidence values (right) and only values above 50 are shown. The Acflrl and Adh3 loci are
labeled by the bar along the right margin. Tree length, CI and RI values are shown.

43

3.4 Adh2

As with Adhl, these data were analyzed by maximum parsimony, parsimony bootstrap,
maximum likelihood, the S-H test and Bayesian analyses. The Adh2 dataset contained
only 24 ingroup samples. This was due in part to the presence of Adh3 and difficulties in
obtaining M maritima and M viridissima sequences for this locus. Many of the
sequences that were selected for the Adh2 dataset fell within Adh3 because both
duplicateS had the same restriction digest pattern and it was impossible to differentiate
between them prior to sequencing. Likewise, I had much difficulty in obtaining Adh2

sequences from M maritima and M viridissima.

Parsimony analysis of Adh2 data generated six trees with 446 steps, CI value of (0.588)
and RI value (0.830) than the Adh2 and Adh3 combined tree. The topology of the best
tree (Fig. 15) was identical to an additional tree generated by likelihood (Trn+I+G, Fig.
16) with the exception to its placement of M stanfieldii sequences relative to M

citriodora.

The parsimony strict consensus tree and the Bayesian tree shared the same tapology, and
parsimony bootstrap values and Bayesian node confidence values were applied to its
branches/nodes. This tree also contained one major clade of M punctata that had M
fi-uticulosa, M humilis, M maritima and M viridissima sequences nested within it (Fig.
14), but the clade had poor Bayesian and bootstrap support (55BS/64NC). Like the Adhl
tree, two clones of M stanfieldii (2367) were on a basal branch adjacent to the outgroup

and were not derived within M punctata.

44

Most of the M punctata samples were together on one supported branch (74BS/89NC)
that contained seven M punctata samples (2189, 2048, 2305, 2397, 2396 and 2473) with
one M humilis (2176) and one M viridissima (2444) sample nested within. All three M
fi'uticulosa samples were on one well-supported branch (68BS/93NC) with two M

punctata samples.

45

M. punctata NJ 2100

4 M. punctata NJ 21139

M. planet-ta MI 2040

M. punctata LA 2300

M. punctata PI. 2307

9 7 5 M. punctata rx 2300
__‘[-7—— M. punctata TX 2473

M. humilis NM 2176

M. viridissima TX 2444

  
 
  

Length: 446
CI: 0.588
RI: 0.830

19

 

 

 

25

  
 
  
  
 

M. fruticulosa TX 2317
M. truticulosa TX 2301
M. punctata TX 2393
M. truticulosa TX 2420
19 M. punctata PI. 2300
; I 12 [ M. truticulosa TX 1920
M. fruticulosa TX 1920

1 ‘ M. viridissima TX 2055
23 £111. punctata m 2048
1., '1 M. punctata OK 2300
16 M. maritima TX 2181
3 M. man'tlma TX 2181
‘3 u. punctata an 2on
11 M. stanfieldii TX 2367
1i- M. stantieldii TX 2367

 

 

 

 

   
   

 

 

[no

 

31

 

9 . .
33 {—— M. citriodora TX 2419
L—L— M. citriodora TX 2375

 

J— P. tenuitoiium 554

 

 

I P. tenuitoiium 97-3
— 5 changes

Figure 15: A phylogram of the full Acflrz tree. Taxon labels include species, state of origin, DNA number.

Branches are labeled with base change values. The larger M punctata clade is labeled with a bar along the
right margin.

46

r-- M. punctata NJ 2100

l“ u. punctata NJ ares
——-——— M. punctata M1 2040
~—-——— M. punctata TX 2300
r“ —~—-—— M. pence-ta TX 2413
M. punctat- L0 2300
M. punctata PL 2307
M. humilis NM 2176
M. viridissima TX 2444
r———-—-—- M. fruticulosa TX 2317

M. tmticulosa TX 2301
M. punctata TX 2303
M. huticulosa TX 2420
M. punctata PL 2300
___q M. fruticulosa TX 1920

—__[ M. fruticulosa 1x1920
M. viridlsslma TX 2055
M. punctata MI 2040
M. punctata OK 2300
r——— M. maritime TX 2181
M. maritima TX 2181
M. punch-ta All 2000

J—_ M. stanfieldii TX 2367
L— M. stanfieldii TX 2367
.___ M. citn'odora 2419
M. cttriodora 2375

 

 

 

 

 

 

 

 

 

 

 

~-—-1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P. muticum 52-2
P. tenuifolium 55-4

‘— P. tenuifotium 97-3
—--— 0.005 substitutions/site

 

 

 

 

Figure 16: The A4012 likelihood phylogram using the Tm+I+G model of substitution. Branches are labeled
with values denoting substitutions per site. Taxon labels include species, state of origin and DNA number.

47

M. punctata NJ 2100
”’95 M. patient. NJ 2100
56/79 M. pmctata in 204s
M. Metat- LA 2305
M. punctat- PI. 2307
100/100 95/100 r—— M. ptlim TX 2300
L—— M. punctat- TX 2473
I M. humilis NM 2176
M. viridissima TX 2444
M. fruticulosa TX 2317
M. fruticulosa TX 2301
M. pmetata TX 2303
M. initiculosa TX 2420
M. pretence PI. 2300
l——- M. truticulosa TX 1920
M. huticulosa TX 1920
88,78 M. viridissimaTX2055
100/100 M. punctat- M12040
55/- 861100 M. Metat- ON 2300
51/. M. maritime TX 2318
100/1 M. maritima TX 2318

1001100 I. W All 2000

100/100 J— M. stanfietdii TX 2367
l——— M. staniieidii TX 2367

 

74/89 96°00

 

 

 

88/100

 

 

 

 

55/100

 

 

 

 

 

55’“ 0 1001100

 

 

 

 

 

 

 

 

 

1001100 r—— M. citrlodoraTX2419
1— M. citnodora TX 2375

P. tenuitotium 55-4
P. tenuifotium 97-3

 

 

 

Figure 17: Strict consensus of the six most parsimonius trees of Adh2 with 20 ingroup sequences. Taxon
labels include species, state of origin, and DNA number. Branches are labeled with bootstrap (left) and
Bayesian node confidence values (right) and only values above 50 are shown. The larger M punctata clade
is labeled by the bar along the right margin.

48

3.5 Adh3

This dataset contained eight M punctata samples and two M stanfieldii samples and was
analyzed by parsimony analysis only. One maximum parsimony tree was generated and it
had a length of 358, a CI of 0.676 and a R] of 0.408. Monarda stanfieldii samples were at
the base of the tree with a M citriodora outgroup sequence. Monarda punctata samples
were in two clades. One had M punctata samples only and the second included the M

stanfieldii and M citriodora samples.

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

~——-11-—~-— M. punctata 14.1 2139
9
7 -——— 12 M. punctat- NJ 2100
a 23 M. panama NJ 21119
11 22 M. punctata TX use
26 M. punctata TX 23111
‘5 M. punctata 'nt 2393
8
——1°——— M. punctata cc 2401
6
2‘ M. punctata u use
9
15 2° M. prince-ta rt. 2333
———-—1-2—-—— M. stantieidii 2369
12
, 41 M. stantieldii 2369
7 I
3‘ M. citriodora 2419
23 P. ioomisii 66-1
1‘ P. tenuitoiium 86-8
10 changes

Figure 18: Adh3 parsimony tree with 11 ingroup sequences. Branches are labeled with steps. Taxon labels
include species, state of origin and DNA number. The M punctata clades are labeled by the bar along the
right margin.

50

3.6 Combined and Reduced Adhl and Adh2

As with full Adhl and Adh2 datasets, these data were analyzed by maximum parsimony,
parsimony bootstrap and Bayesian analyses. Adhl and Adh2 datasets were reduced so
they shared the same samples (Fig 19). Eighteen trees were generated in the maximum
parsimony analysis of the reduced Adhl dataset (length: 200, CI: 0.585, RI: 0.771). The
parsimony strict consensus tree and the Bayesian tree shared the same tree topology (Fig.
15). Two trees were generated in the maximum parsimony analysis of the reduced Adh2
dataset (length: 275, CI: 0.618, RI: 0.835) and the parsimony strict consensus tree and

Bayesian tree were also topologically the same.

Both trees placed M stanfieldii (2367) sequences at the most basal branch of the tree.
Monarda punctata was in two primary clades in both trees. Endemic species (M
fi-uticulosa, M humilis, M maritima and M viridilrsima) showed a higher degree of
variability regarding their placement in the trees, although each phylogeny had a clade
that consistently contained the majority of the M fruticulosa sequences included in the

study.

51

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983.52.

 

 

 

 

 

 

 

 

 

 

 

 

 

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52

3.7 nchS

Nineteen nchS sequences, approximately 600 basepairs in length were obtained from
four Monarda species (M. fruticulosa, M. maritima M. punctata and M. viridzlssima) with
two Pycnanthemum sequences set as outgroups. Results showed that there were two
distinct nchS loci (Fig.16) with 264 steps, a CI of 0.586 and a RI of 0.842. The two
putative loci were separated by 24 base changes. Due to the limited number of taxa in this

dataset, no further analyses were run on these trees.

53

 

15

Pycnanthemum 22

M. viridissima TX 1908

 

 

 

 

M. punctata TX 1907

 

 

__ ‘ M. viridissima TX 1989-1

 

 

M. fruticulosa TX 1910
[ M. punctata NJ 19461
M. punctata NJ 1946-2

M. fruticulosa TX 1920-1

 

 

 

 

“ENGI—

 

M. fruliculosa TX 1920-2

 

 

24

M. maritima TX 1977-1

I M. viridissima TX 1989-2
M. viridissima TX 1989-3

 

 

E M. punctataTX 1909-1
M. truficulosa TX 1920-3

M. frufiwlosa TX 1920-4

 

 

 

 

 

 

M. punctata Ml 1905-2

 

— M. punctataTX1909—2

 

 

 

 

M. fruficulosa TX 1920-5

 

 

 

- M. vin'dissimaTX 1908

 

---5Ch80995

Figure 20: Full nchS sequence data representing ten different taxa. Taxon labels include genus/species,

 

M. maritime TX 1977-2

 

 

 

Pycnanthemum 20

state of origin and DNA number. Locus l and 2 are listed along the right margin.

54

 

“Char.

DISCUSSION

4.1 General Overview of Results

The objectives of this project were to use the species in Monarda Section Cheilyctis to
test whether speciation by peripheral isolation had occurred among the species of the
section, and to determine whether the endemic species were paleo- or neoendemics. Data
showed a high degree of Adh allelic variability within and between Monarda species.
However, the phylogenetic results provided by Adh loci offered insight in answering

these questions.

In each dataset all of the methods employed, maximum parsimony, maximum likelihood,
and Bayesian analyses consistently produced trees with similar topologies. Consistency
in their phylogenetic results increased the chances that the best trees were found for
interpretation. The Adhl phylogeny had the most complete representation of samples. All
the endemic species were represented in the Adh2 phylogeny samples. Both phylogenies
were used for interpretation. The Adh3 phylogeny had only M. punctata and M.
stanfieldii samples. It is unknown whether this putative Adh locus is a pseudogene or
functional duplicate, but its distinction fiom Adh2 suggests that there was enough

variation in this duplicate to enable it to be distinguished as a separate type.
Phylogenetic data of nchS were not complete enough to be included in the analysis,
although Monarda to have two loci for the chloroplast-expressed nchS gene as opposed

a single locus, as documented in other plant systems (Emshwiller and Doyle 1999).

55

4.2 Gene Trees and Species Trees

There are limits that are inherent to the use of low copy nuclear genes for phylogenetic
analysis. Phylogenies are hypotheses that can be useful tools for the inference of species
relationships but there are caveats to those based on DNA markers. It is important to
recognize that gene trees, particularly those based on nuclear genes, may not clearly
reflect the same relationships as a true species tree (Doyle 1992, Schaal and Olsen 2000).
Coding and noncoding gene regions can accrue genetic variation fi'om point mutations,
insertions/deletions and meiotic recombination that can disseminate or be disseminated
within and among populations independent of speciation events. Genetic changes such as
these can convolute gene trees when species have not undergone complete lineage
sorting. Homoplasy can also be a source of incongruence between gene trees and species
trees (Doyle 1992). Because of these problems, it is prudent to obtain molecular
phylogenies from at least two different genetic markers. In this study only Aflr sequences
were used for analysis. Nonetheless, Adh has been widely used in phylogenetic studies
(Sang 2002) and has gene regions that are highly conserved between distant organisms

therefore greater confidence may be placed in its results.

Additional complications can arise when using low copy nuclear genes for phylogenetic
inference depending on whether gene homology is paralogous or orthologous.
Orthologous homologs precede speciation and are followed by divergence of an ancestral
sequence (Gogarten and Olendzenski 1999, Sang 2002). Therefore, orthologous
sequences can be used for phylogenetic reconstruction of species. Paralogous homologs

result from either a duplication or deletion event after that occurs after speciation.

56

__......__ _
‘ .. {.1121

 

P

~. v ‘ —~‘v.q.-V"

 

Paralogous sequences have been found to confound phylogenetic results, although some
studies have shown them to be phylogenetically useful for both rooting purposes and to
test comparative rates of divergence (Sang 2002). Ath and Adh2 are believed to be
orthologous because they duplicated prior to the divergence of monocotyledonous and
dicotyledonous plants (Sang pers. corn). These orthologs may be synonymous to Arflrl
and Adh2 loci identified in this study, but this cannot be confirmed because much
information about these genes is lacking. This conjecture was drawn from the fact that
AcflzI and Adh2 sequences were found in all six species while Adh3 sequences were only
found in M. punctata and M. stanfieldii. There is no evidence to suggest that A¢fl23 is

orthologous.

4.3 Gene Trees as Tools for Mecroevolutionary Inference

It is important to draw a clear distinction between population level research and studies
like this where species-level gene trees are used to infer population level processes.
Coalescent theory has supplied much of the theoretical foundation for predicted gene tree
topologies given different demographic histories of the populations they represent
(Maddison 1995, Avise 2000). In the absence of gene flow, inter-specific gene trees can
depict shared evolutionary histories and allelic variation that may coalesce to a common
ancestral gene (Harrison 1991). Population structure and genetic structure are key in
understanding how gene trees can be used to infer speciation events. If discontinuity in
population structure reflects historic barriers to geneflow then one might expect

comparable phylogenetic discontinuity in the genetic variation of neutral genetic markers.

57

 

 

 

1‘.--' :. 23min!

‘w-n “..‘..-)..nmu

This provides the underlying theory used by Harrison (1991) for his gene tree models of

speciation.

4.4 Peripatric Speciation

The hypothesis that narrow endemic Monarda taxa speciated as peripheral isolates of M.
punctata had phylogenetic support for some species but not all. In addition to being the
most morphologically variable and geographically widespread, M. punctata was the most
ubiquitous taxon throughout both Adhl and Adh2 phylogenies. All narrow endemic
species were derived within the large M. punctata clade with the exception of M.
stanfieldii. This suggests that M. punctata is the progenitor of M. fi-uticulosa, M. hwnilts,
M. maritima and M. viridissima and a sister taxon to M. stanfieldii. The Adh3 phylogeny
included few samples, but was consistent with the result that M. stanfieldii is the sister
group to M. punctata

It was unexpected that M. punctata samples did not tend to exist on longer branches
closer to outgroup samples. Factors that might explain why most M punctata appeared
on shallow terminal tree branches include recent divergence of species resulting in shared
ancestral polymorphisms, inter-specific gene introgression (Schaal et al. 1998, Templeton
et al. 1995) or inadequate samplinglf allopatric species are sexually compatible and their
populations expand resulting in a sympatric or parapatric distribution genetic distinction
between them can be blurred (Macnair and Gardener 1998). In addition, shared ancestral
polymorphisms that are maintained in sister species may also convolute gene tree results
and can be misinterpreted as evidence for recent gene flow (Schaal and Olsen 2000).

Based on what is known about the current geography, morphology, and life cycles of

58

these species these results are likely due to either low sampling or shared ancestral
polymorphisms. Species are either geographically or temporily separated and they
maintain unique morphological characteristics. It is likely that they speciated recently and
rapidly, and it would have been prudent to include more M. punctata samples from Texas

populations.

In the Adhl parsimony trees, M. fi-uticulosa and M. maritima sequences were consistently
on the same clades. Similarly, in the Adh2 parsimony trees all M. fi-uticulosa sequences
shared the same clade, although the only M. maritima sample in the phylogeny existed in
a different clade. Based on their placement in the Adhl phylogeny results suggest that M.
fiwiculosa and M. maritima share more allelic variation with each other than any of the
other narrow endemic species. This might suggest that there has been recent gene flow
between these species, or that they diverged recently from a common ancestor. However,
the results do not rule out the possibility that some of the endemics are sister taxa that

derived independently from M. punctata.

4.5 Neoendemic and Peleoendemic Species

Adh phylogenies provided useful information that could be used to infer the paleo- and
neoendemic status of these species. Most M. stanfieldii (2367, 2369) samples were not
derived within the larger M. punctata clade and had long branch lengths. This short-lived
herbaceous perennial is thought to be completely geographically isolated from M
punctata and inhabits a unique substrate (granitic sands) relative to the other narrow
endemic species. Overall, the phylogenies provided strong evidence that M. stanfieldii is

a paleoendemic

S9

 

 

w 3'. C (.4

 

 

In both .4th and Adh2 parsimony trees, one clade consisted almost entirely of M.
maritima and M. fruticulosa sequences as well as M. punctata samples from southeastern
Arkansas (2060), southeastern Texas (2393) and northcentral Florida (23 89) to name a
few. The close phylogenetic relationship between M. fi-uticulosa and M. maritima is not
surprising considering they share a woody habit, perennial life cycle and they are the
most geographically proximate of the narrow endemics. These results suggest these

species are neoendemics, but also may indicate they may share a common ancestor.

Monarda viridissima sequences are found in different positions across both phylogenies
which suggests they have a high degree of allelic variability. Regardless, most sample
sequences were on branches that were close to M. punctata samples, which suggests they
are closely related. Their close genetic relationships were most apparent in the Adhl tree
where M. viridissima sequences (2055 and 2444) clustered with M. punctata sequences.

Geographic and phylogenetic data support that M. viridissima is a neoendemic species.

Monarda humilis was not well represented in either dataset but some inferences can be
made regarding its placement in the phylogenies. This species is the most geographically
disjunct relative to the other southwestern narrow endemics, and like M. viridrlssima it
was found within the larger M. punctata clade. Even though this species was represented
by only one sample its apparently close genetic relationship suggests that it is also a

neoendemic that speciated from M. punctata.

4.6 Lineage Sorting and Noncoalescence

Several samples in the phylogeny appear to have allelic variation at given locus. In the
Adhl tree, two M punctata (2307 and 2048) and one M maritima (2318) had alleles that
consistently fell within the two separate clades. Likewise, two M viridissima sequences
(2055, 2444) were within one clade, while the two remaining samples (2441) were in two
separate locations in the phylogeny. Allelic variation in a phylogenetic marker leads to
incongruence between gene trees and species trees. Such allelic variation may be caused

by several phenomena such as lineage sorting, noncoalescence and gene flow between

species.

Lineage sorting refers to the fixation of different ancestral alleles for a given marker into
a daughter species at speciation (Small and Wendel 2000). In cases of lineage sorting
fixed alleles should be present throughout the species. If samples of a species from
multiple populations had the same patterns of allelic variation in a phylogeny it may
provide evidence for lineage sorting. In addition, shared ancestral polymorphisms caused
by lineage sorting should be divided by many steps in a phylogeny, while newer alleles
that have arisen afier speciation should have fewer steps dividing them. Lineage sorting is

a more common problem in species that have diverged recently (Sang 2002).

Noncoalescence refers to the introgression of alleles that are maintained as ancestral

polymorphisms in a multi-gene family (Small and Wendel 2000). The events that lead to

lineage sorting and noncoalescence both involve the maintenance of ancient allelic

61

polymorphisms and can produce phylogenetic results that show widely disparate taxa

closely related on a gene tree.

Introgression and hybridization both involve the transfer of genes from one species to
another (Small and Wendel 2000). Introgression results in the permanent fixation of
genes between species while species still maintain phenotypic distinction. In contrast,
hybridization results in phenotypic intermediates and progeny with novel gene
combinations which can either lead to a merging of species or create a platform for

speciation.

It is not certain which phenomena are the cause of allelic variation in the Adh
phylogenies. Phylogenetic data alone is not sufficient to distinguish between lineage
sorting, noncoalescence and gene flow between species. Further research is needed to

determine this.

4.7 Species Protection and Conservation

The Endangered Species Act does not use biological terms alone to define species.
Instead species are defined based on the most up to date knowledge of the genetics,
evolution and speciation of a given taxon. There are several considerations that may

warrant species protection for some of the Monarda taxa used in this study.

Monarda maritima has the most limited distribution of the Texas endemics. It is only

known to exist within the Ingleside Terrace sand body that extends approximately 100

62

miles along the Texas coast. The fact that it is a coastal endemic indicates that it has
likely suffered much habitat loss due to ranching and development associated with the oil
industry. This plant is challenging to find in the field and has not been extensively
collected, which suggests that it is not common in its range. All evidence indicates that

this species is a good candidate for conservation.

Monarda viridissima is limited to the Carrizo sands of the Blackland Prairies that extend
in a discrete belt from northeastern to southwestern Texas. Populations have been found
in only the central region of this belt. Monarda viridissima is not common in its range,
but I was able to find it in moderate sized populations (>25 individuals). It is also noted
as being rare in a list of southern U. S. endemics (Estill and Cruzan, 2001). Field
observations suggested that it inhabits more established plant communities rather than
newly disturbed sites, which concurs with its tendency to grow in established post-oak
and pine-oak forests. Like M maritima, it may be in need of protection due to its limited

range and propensity to exist in more established plant communities.

Monardafi-uticulosa is the most abundant of the Texas endemic Monarda species. It
occurs across the southern tip of the state in the coastal prairies and interior coastal
plains. Within its range it can commonly be found in large populations that are often
distributed along roadsides. Therefore, M fi-uticulosa is the least likely to be in

immediate need of conservation, despite its narrow endemic status.

63

Monarda stanfieldii has a narrow range across the Central Texas Uplift. Like M
viridzlssima, it only appears to grow in more established plant communities, and tends to
prefer open woodlands (pers. obs.). This species was also difficult to find in the field,
although one of the populations had a relatively good size (>50). Monarda stanfieldii is
also likely to be in need of protection particularly because of its apparent paleoendemic
status. It exists in more in established plant communities, its range is very small and it

grows in very specific soils.

4.8 Prospects for Future Studies of Monarda Section Cheilyctis

Several changes in our experimental design might have helped to better resolve
phylogenetic relationships between these species. An increase in sampling, particularly of
Texas M punctata, might have added useful information. Five varieties of M punctata
were not included in this study. A more complete sampling of these taxa may have
provided a better understanding of the origins of the Texas endemics. Additional samples

could also better resolve patterns due to incomplete lineage sorting and noncoalescence.

It would also would have been advantageous to include data fiom an additional low copy
nuclear gene. Molecular phylogenies from distinct genetic markers can be used to
compare and hopefirlly strengthen phylogenetic hypotheses, thus providing more tools for

distinguishing gene trees and species trees (Schaal and Olsen 2000)

A population-level study using Amplified Fragment Length Polymorphisms (AFLPs) for

analysis may have resolved complicated reticulating patterns between these taxa. In

several studies of Cichlid fishes, various genetic markers were used to determine the
relationships between species, but all showed a high level of DNA polymorphisms
among species (Klein et al. 1998, Moran and Kornfield 1995). Relationships between
these Cichlids were not fully resolved until AF LPs were used for analysis (Albertson et
al.1999). AF LPs are advantageous because thousands of genetic regions can be used to
generate an organism’sgenetic profile, and no preexisting knowledge of its genome is
required before use.

Some individual relationships between species may also spur additional investigation
regarding the history of these plants and their speciation. Manardafi-uticulosa and M
maritima share more allelic variation with each other than any of the other endemics.
This may suggest that there has been recent gene flow between them, or that they
diverged recently from either a common ancestor. Our current knowledge of these
species show that they do not overlap geographically or phenologically, which better
supports the latter, although they exist is such close proximity that their geographic
distributions may have overlapped recently. These species inhabit comparable substrates
(homogenous beach sands), they are the most geographically proximate and they are both
woody perennials. Geologic and physiographic history suggests that M maritima may
have been separated from mainland species when the Ingleside Terrace was a barrier
island. If M fi-uticulosa, or a shared M fi-uticulosa/M maritima ancestor had become

isolated on the barrier-island this may have given it a window of time to diverge.

Questions also arise regarding the speciation mechanism acting to maintain distinction

between M punctata and M fiuticulosa. These are the only two species in the study that

65

grow in syrnpatry, bloom at the same time and are thought to have hybrid zones (pers.
obs). They have been observed growing together along hilly sandy landscapes with M
fi-uticulosa growing at the sandy hilltops, M punctata at the base of the hills, and possible
hybrids along the cline between have been observed at one locality (Prather pers. comm).
This distribution better fits a parapatric species distribution with disruptive selection
acting to maintain genetic distinction between species. In a study by Leuth and Prather
(unpubl.), soils were collected from some of these sites and reciprocal seed plantings of
M punctata and M fiuticulosa were conducted. Results showed that M fi-uticulosa had
better survivorship on its own soil than it did on M punctata soil. Other factors that may
be involved include differences in water availability in high and low areas of the sandy

soils or subsoil component, such as high salt content.

4.9 Conclusions

Few studies have used phylogenies based on low copy nuclear genes to infer population-
level processes (Schaal 2000) such as speciation by peripheral isolation.

This research revealed a high degree of variation between alleles, loci and individuals in
both Adhl and Adh2 datasets. Monarda punctata sequences were widely distributed
across both phylogenies and M humilis, M fi-uticulosa, M maritima and M viridissima
sequences derived within this larger M punctata clade. These results support the
hypothesis that these narrow endemic Monarda speciated as peripheral isolates of M
punctata. In contrast, phylogenies suggest that M stanfieldii speciated as sister to of M
punctata. The data suggest that Monardafi-uticulosa, M humilis, M maritima and M

viridissima are neoendemics while M stanfieldii is a paleoendemic. Likewise, the high

degree of lineage sorting and noncoalescence in the dataset suggests that these species
diverged recently and rapidly. Despite the lack of phylogenetic distinction between these

species they have remained morphologically, geographically and phenologically distinct.

67

Adhl Sequence Alignment
~1200 nucleotides in length

68

B¢<GGU<H€Q¢Q¢H<<BUBHdddeZrB¢H<¢¢9Q10808a8406499
dewdudfidududfi<¢fiwhdedeZrB<H¢<<BQ<GBUBBB¢UQ<BB
deddUdeUGodadflewhafid¢0421B<H¢H<HQ¢GBUBBB<UQ<BH
addwéudaduo048449088414012IadadadawdwaUBBB‘UQ‘BB
BideUdeUdUdedawaadddUeradfidddawdwa0988406489
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deGdUdeUQU¢B<<HGBB<¢<UwZIB‘BflH4BQ‘UBUHBBdUQ449
H¢16<Q49400048449w88<d4042184909<8u468UBBBdUQddB
9446404840404844009H¢<<Q<2tB<H<H¢BO<GBUBBB<UG<IB
dewdudadumUdH<<HOBB<¢<U<Zrada<H<HQ<GBUBBB<UQ<HH
94464049400thddeuaedddu<zradsdfidaudwaosaaduwdaa
addeUdfideUdfiddfiuaadd<udzIH<H¢H<HUBG8UBBBdUQdBB
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B<4Q¢U<H<U¢U<B¢<HUBBdfldUUZIHdhdfiéawdwaoaaedumdda
H<<G<Q¢B<U<U¢H<<BUBB¢<¢U¢zradadfideudwaoaaaduudaa
B<¢Q<Q¢B<Q40484<808H<¢<Q<ZrBdfidadmudeUhaadomdhe
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844G40<B<U¢U<H<<BGB8444042184949486408089840Q498
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84<G<U<B<U¢0484¢809dewudzIH<H¢H<BQ<OBUBBB<UG<BB
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94<Q<Q<9404048449w99444042IB<H<B<BQ<UBUBBB<UQ<<H
deQ<Q<H¢U¢Q¢H<4909h<¢<U<zrB4B<B<BQ¢UHUHBB<UQ¢BB
addmdudadudodaddao884(4042rB4848<804090988406489
defidddfifiowudaddawaHdddUeradadedamdwauafiaduwdra
deddDdBdUdUdfiddBwaadddeZrBdadfidewdwaoeaaddm<<9
B<<w<Q<B<U<U<H¢40wH9(440421H4949¢9Q¢090989<UG<IB
addQ<U¢e¢U¢Q<H¢<HUBH¢<4Q<B¢H¢H<B¢HO4GBUBHBfiUQdBB
a¢¢Q<Q<e<U¢U<a<<BGB9444042rB<H<B<HO¢GBUBBB<UQ<HH
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addd<d¢a<o<0¢a<<awaa<<<u¢2r949494904680888406488
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B44Q<U<H<U¢Q<B<<BGBBd<<u<2IaUfldfidaudwauaafidu<<aa
addmdudfia0(048448G9841404219494949Q4680898400<98

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8(688Uhaddwekuahdwddua0989UB‘dUPdG‘QflOGBdHGOQflUH
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BdwaauaaddweauaadwddoaUBBBUdeUddeUdUGH‘H000408
8469909914699UBB<Q¢<UBUHBdUdeUHdeUdUGBdBOOQ‘UB
H¢GHHUBH¢4¢HBUHB¢G¢¢UB0989UdeUB<G<Q<UGB<HUOQ<UB
EdwaaoahdddaaUBBdedUB0898094409¢Q<Q<UOH<9000<UB
8459908844099UBH<G<<UB0888Dadduadwwuduoedswwwdoa
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BdGHHUBBd‘GBBUBBdedUBUhaiuadduadwduduwude00G<UH
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BdwaauaaidwkeUHH<Q¢<UH0888UBfidUHdGZrdUOHdHUGQdUB
adwaavaadddaaUBBde<UBUHHB08440940214008<BOUQ¢08
BdBHBBBB¢<GBBUHd<Qd<UB0989UBOdUBdUZrdUOhdHDUGdUG
949996HBfidGBBUH<<Q<<UBUBBBUB‘dUHdUZrdUOB<BGGQ<UO
B<w88899<4waaDaddddduduafiaUB<<UB<UZI¢U¢B¢HUOQ<U0

NrmmHN
HrmeN
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hmmu

bmmm

momn
wamw
mamm

NFHN
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wmvN

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NrvaN x9
cvvm x9
Hravvm x9
nmom x9
araomm x8
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79

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80

Adh2 Sequence Alignment
~1200 nucleotides in length

81

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89

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97

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