The phytochrome gene family in legumes (Fabaceae) : evidence for a new locus and analysis of
evolutionary rates
by Elisa Jean Eshbaugh
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biological Sciences
Montana State University
© Copyright by Elisa Jean Eshbaugh (1998)
Abstract:
The phytochrome nuclear gene family has been investigated elsewhere to assess its potential
phylogenetic utility for plant systematics. Phylogenetic reconstruction of the phytochrome gene family
from sequences sampled throughout angiosperms detected four possible loci in legumes including
PHYA1, a potentially new and unique member of the multigene family. This study summarizes
attempts to further characterize the phytochrome gene family in legumes by addressing the following
questions: (1I) Does the phylogenetic pattern of the phytochrome sequences support designating the
PHYA1 sequences to locus status? (2) What does the evidence from analysis of nucleotide substitution
rates reveal about the PHYA1 sequences? The results from phylogenetic analysis and analysis of
nucleotide substitution rates support the hypothesis that the PHYA1 sequences are potentially a fourth
locus in legumes. In addition, the relative rate tests indicate that members of the gene family are
characterized as having heterogeneous rates of nucleotide substitution. Furthermore, it is concluded
that the Glycine max PHYA sequence is very likely to be unreliable for reconstructing organismal
relationships given its accelerated rate of evolution. Finally, the concrete designation of PHYA1 as a
functional gene awaits further evidence from the entire gene sequence. THE PHYTOCHROME GENE FAMILY IN LEGUMES (FABACEAE): EVIDENCE
FOR A NEW LOCUS AND ANALYSIS OF EVOLUTIONARY RATES
by
Elisa Jean Eshbaugh
A thesis submitted in partial fulfillment
o f the requirements for the degree
of
Master o f Science
in
Biological Sciences
M O NTANA STATE UNTVERSITY-BOZEMAN
Bozeman, Montana
October 1998
© COPYRIGHT
by
Elisa Jean Eshbaugh
. 1998
All Rights Reserved
11
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%
Dr. Matthew T. Lavin
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iv
ACKNOWLEDGMENTS
This work was supported by the Monts/EPScoR program and by funding provided
by the National Science Foundation Grant to Matt Lavin (B SR -9118785). The author
thanks Matt Lavin, Robert Sharrock and Rich Stout for their service as the graduate
committee, Sarah Mathews and Ted Clack for technical advice in the laboratory. In
addition the following individuals provided helpful comments and insights: Bonnie
McCaig, Elizabeth Friar, Micheal Clegg, Sudir Kumar, Dave Russel and Steve Eshbaugh.
V
TABLE OF CONTENTS
Page
IN TR O D U CTIO N .................................................................................................................. .1
MATERIALS AND M E T H O D S ...........................................................................................5
D N A Isolation ............................................................................................................... 5
D N A Am plification............................................................. .....................................5
C loning............................................................................................................................7
Sequencing................................................................................................................... 8
Data Management and Phylogenetic A nalysis...................................................... 8
Rates o f E volu tion ...................................................................................
10
RESULTS ....................................................................................................................................12
Amino Acid A lignm ent.............................................................................................. 12
Percent D ivergen ce...........................................
12
Within G roup s.............................................................................................. 12
Among G rou p s............................................................................................ 14
Phylogenetic R econstruction................................................................................. 15
Rates o f E volu tion ...................................................................................................... 21
Within PHYA T e s ts .................................................................................... 22
W ith in f# L 4 7 T e s ts ................................
24
Among PHYA-Xy1
PQ T e s ts ............................................................................24
Among PHYA-XyyQ and Within Taxa T e s ts ............................................25
D IS C U S S IO N ........................................
26
C O N C LU SIO N S.........................................................................................................................32
REFERENCES C IT E D ............................................................................................................. 34
APPENDICES
39
vi
LIST OF TABLES
Table
PaSe
1. Summary o f amino acid hallmarks and insertion/deletion ev en ts..............13
2. Summary o f percent divergence v a lu es.....................................................
3. Rate heterogeneity among and within PHY subfamilies......................... 23
14
vii
LIST OF FIGURES
Figure
Page
1. Idealized PHYA gene structure.......................................................................... 6
2. Neighbor-joining t r e e ................................................... ...................................... 17
3. Phylogenetic relationship depicted by parsimony analysis......................... 19
4. Neighbor-joining tree with non-papilionoideae sequences rem oved ........20
V lll
ABSTRACT
The phytochrome nuclear gene family has been investigated elsewhere to assess its
potential phylogenetic utility for plant systematics. Phylogenetic reconstruction o f the
phytochrome gene family from sequences sampled throughout angiosperms detected four
possible loci in legumes including PH YAl, a potentially new and unique member o f the
multigene family. This study summarizes attempts to further characterize the phytochrome
gene family in legumes by addressing the following questions: (I) D oes the phylogenetic
pattern o f the phytochrome sequences support designating the PHYAl sequences to locus
status? (2) What does the evidence from analysis o f nucleotide substitution rates reveal
about the PHYAl sequences? The results from phylogenetic analysis and analysis o f
nucleotide substitution rates support the hypothesis that the PHYAl sequences are
potentially a fourth locus in legumes. In addition, the relative rate tests indicate that
members o f the gene family are characterized as having heterogeneous rates o f nucleotide
substitution. Furthermore, it is concluded that the Glycine max PHYA sequence is very
likely to be unreliable for reconstructing organismal relationships given its accelerated rate
o f evolution. Finally, the concrete designation o f PHYAl as a functional gene awaits
further evidence from the entire gene sequence.
I
INTRODUCTION
Understanding molecular evolution requires detailed knowledge about different
evolutionary forces from a diversity o f sources (Wolfe et al., 1987). Taken together,
analysis o f a variety o f molecular data can advance areas o f research such as population
genetics, systematic biology and evolutionary ecology. Historically, much o f the alliance o f
molecular evolutionary analysis and other fields o f research has come from studies using
data from single-copy nuclear genes or from mitochondrial and chloroplast DNA. Lowcopy nuclear genes have been less thoroughly studied largely due to difficulties associated
with the very qualities that make them unique, like the potential for acquiring and losing
members o f gene families (Durbin et al., 1995; Gaut, 1997). Furthermore, the relative
paucity o f studies with multigene family data is particularly true o f data from plant sources
(Demmin et al., 1989; Sanderson and Doyle, 1992, Sang et al., 1997).
In their study o f the phytochrome gene family in grasses, Mathews and Sharrock
(1996) addressed one aspect o f molecular evolutionary analysis relevant to the application
o f gene family data to systematics, the analysis o f rates o f evolution. They found some
evidence for rate heterogeneity in duplicated members (paralogues) o f that gene family yet
the same was not observed for subfamilies acquired by speciation (orthologues). Their
study suggests that the phytochrome gene family is a potentially interesting group for
further study.
Phytochromes are one type o f photosensing pigment, or photoreceptor, that plants
use in the detection o f the radiant spectrum. They specifically function in the perception o f
a photon o f light in the red to far-red portion (ca. 600-800nm) o f the spectral region and
its subsequent conversion into a chemical signal (Quail, 1994; Pratt, 1995 ;Smith 1994).
The phytochrome molecule is composed o f tw o subunits that form a dimer between the
carboxyl termini o f the monomers (Sharrock et al., 1986). Each subunit has an open chain
tetrapyrrole chromophore linked to a polypeptide o f approximately 1100 to 1200 amino
acids (120- 127kD) by a covalent bond. This chromophore attachment site occurs at a
cysteine residue located in the N-terminal half o f the polypeptide (Quail, 1994) (Fig. I).
The structural features o f this molecule underlie its reversible photoconversion between
red light and far-red light absorbing forms that culminate in producing a molecular
switching mechanism. This regulatory switch alters the expression o f several nuclear genes
including those encoding the small unit o f ribulose bisphosphate carboxylase (rbcS) and
the chlorophyll a/b binding protein (cab) (Terzaghi and Cashmore, 1995). In this way
plants can coordinate growth and developmental events with information acquired about
relative amounts o f red/far-red light (Sharrock and Quail, 1989; Smith 1994).
The multigene family that encodes phytochromes (PHY) are from the low-copy
fraction o f the nuclear genome (Mathews, 1995). Sequence analysis o f the green alga,
Mesotaenium, revealed the presence o f an ancestral phytochrome sequence and
suggests that a phytochrome gene lineage existed well before the emergence o f land plants
(Mathews and Sharrock, 1997). Furthermore, the possibility o f still greater antiquity o f the
phytochrome gene lineage has not been discounted since sequences similar to
phytochrome have been identified in studies o f photoreceptors from cyanobacteria (Kehoe
and Grossman, 1996). In their review o f phytochrome gene family diversity, Mathews and
Sharrock (1997) state that PH Y subfamilies may have arisen from a repeated pattern o f
divergence from a common ancestor following gene duplication events. In phylogenetic
analysis o f full-length sequences from land plants and green algae, they also find evidence
to support the premise that these duplications most likely occurred around the time o f
major events in land plant evolution such as the emergence o f seed plants and flowering
plants.
The most extensively characterized phytochrome genes are known from
Arabidopsis where low stringency southern blot analysis and D N A sequence analysis have
revealed the presence o f five genes, PHYAy PHYB, PHYC, PHYD and PHYE (Sharrock
and Quail, 1989; Clack et al., 1994). Furthermore, there is evidence for functional
divergence among these subfamilies (Smith, 1995). Studies o f null mutants revealed that
PHYA in Arabidopsis controls plant response to far-red high irradiance while the PHYB
gene mediates a different group o f plant responses to red light and the red/far-red ratio.
Such diversification suggests unique functions might be assigned to other phytochrome
encoding genes as well.
The suite o f genes identified in Arabidopsis is considered representative o f
phytochrome diversity in flowering plants, although there may be some slight variation in
the composition o f subgroups among plant lineages. For example, there is some evidence
for additional gene duplication events within the PHYB/D lineage as there have been
several PHYBID homologues identified in tomato, carrpt, and Arabidopsis. Similarly,
there is evidence to suggest there are multiple PHYA homologues in carnation,
Ceratophyllum (an aquatic angiosperm) and legumes (Mathews and Sharrock, 1997). As
part o f a study to access the phylogenetic utility o f phytochrome sequence data, Mathews
et al. (1995) extensively surveyed angiosperms for PHY sequences. Polymerase chain
reaction using degenerate primers designed to amplify all possible PH Y homologues failed
to detect a homologue to PHYC and PHYD in legumes, however homologues o f
Arabidopsis PHYA, B and E were identified as well as unique PHYAYke, designated
PH YA l.
The work presented here was initiated as a satellite project to the investigation o f
the phylogeny within the legume tribe Millettieae by Lavin et al. (1998). That study used
the phytochrome gene family to elucidate problematic legume taxonomy. This paper
presents an extension o f the phylogenetic analysis o f the phytochrome gene family in
Fabaceae with emphasis on characterizing the PH YAl sequences. Additional evidence for
the hypothesis that PHYAl is a discrete locus from phylogenetic analysis, percent
divergence, conserved amino acid hallmarks and the analysis o f evolutionary rates is
discussed. Moreover, this paper diversifies the use o f this less studied fraction o f the
nuclear genome and attempts to diversify the use o f different types o f data used to explore
trends in molecular evolution.
MATERIALS AND METHODS
D N A Isolation
The sources for the plant tissues used in this study are given in Appendix I. Total
D N A was isolated from 0.1-2.0g o f fresh or dried material generally following the CTAB
method o f D oyle & D oyle (1987). Following this, the aqueous samples were subjected to
an additional extraction with phenol: chloroform (1:1 volume), purified over sepharose
CL-6B (Pharmacia, Piscataway, N I) columns and finally preserved in TE buffer for use as
templates for amplification.
D N A Amplification
Sequences obtained from the coding region o f exon I by Mathews et al. (1995)
were used to identify conserved peptide sequences. These conserved sequences were then
used to design degenerate PCR primers that would anneal fragments o f all possible
phytochrome loci (orthologs and paralogs) Mathews et al. (1995). The target region was a
portion o f exon I that encompasses the chromophore attachment site and a
phylogenetically informative region downstream from that site (Kolukisaoglu et al., 1995)
(Fig. I). Depending on the locus amplified, this target region was 600 to 630 bp and
increased the size o f the amplified fragment by approximately two-fold from that used for
phylogenetic analysis by Mathews et al. The conserved peptide sequences from which the
primers were designed are shown below in Figure I .
Figure I . Idealized PHYA gene structure illustrating the position o f exons, introns, the approximate position o f the
chromophore attachment site and the region of exon I sequenced tor this study. Exons (marked I-IV) are repiesented by open
boxes, introns appear as shaded boxes. The cross-hatched area represents the approximate location o f the 600-630 base pair
fragment sequenced and as described in this study. V ’ indicates the approximate chromophore attachment site. Fhe conserved
peptide sequences from which the primers were designed are also shown in bold followed by identifiers that proceed the
corresponding oligonucleotide sequence. Identifiers “Phy-S” and “Phy-N specify whether the sequence is identical to the sense
strand or nonsense strand of the phytochrome template DNA
YDRMAYKFHED: Phy-S: 5'-TAYGAYAGGGTIATGGCITAYAARTTYCAYGARGA-3'
NIMDLVKCDG: Phy-N: 5'-CCRTCRCAYTTIACTAGRTCCATDATRTT-3'
C-terminus
III
IV
IKb
(Modified from Mathews et al. 1995.)
Approximately 200 ng o f purified total D N A from each taxon was amplified in a
100 pi reaction volume containing 2.5 units o f Taq polymerase (Gibco BRL), 10 mM
Tris-HCl pH 8.4 reaction buffer, 2.5 mM MgCl2, 0.25 mM o f each dNTP andluM o f each
primer. Cycle parameters consisted o f an initial denaturing at 94°C for 5 minutes, five
cycles o f 94°C for I min, 48°C for I min, 3 min ramp to 12°C for I min and 10-30 cycles
o f 94°C for I min, 55°C for I min, I l 0C for I min. Upon completion o f the last cycle the
samples were incubated at I T C for 10 minutes. Additional amplifications with less
stringent annealing temperatures (e.g., 45-49° C) during the first five cycles were also
conducted representing a modification o f the protocols o f Erlich (1992) and Inms et al.
(1990). This approach, in concert with using degenerate primers, was designed to allow
for sampling o f all possible PH Y loci (Wagner et al., 1994). Amplifications products were
electrophoresed in 2% agarose gels to verify product size prior to cloning.
Cloning
Cloning was necessary to screen the pool o f PCR products amplified in each
individual PCR reaction. Over the course o f the study, multiple PCR reactions were
screened and cloned. Initially, the cloning procedure followed that o f Mathews et al.
(1995) wherein the amplifications products were treated with T4 ligase to generate bluntend products for ligating into bacteriophage M 13KRV8.2 (sensu Mathews et al.1995).
Alternatively, some sequences were obtained by cloning with the TA vector
(Invitrogen, San D iego, California) and subcloning into M 13m pl8 and m p l9 vectors.
Ligation products were then transformed into INVccF' or JM 109 competent cells and
8
grown overnight on LB plates. Blue/white screening was used to identify putative
phytochrome-containing recombinants. Eight colonies from each transformation were
selected and cultured according to standard cloning protocols o f Sambrook et al. (1989).
Single-stranded D N A for sequencing was prepared by alkalinelysis minipreparation o f
phage DNA. Following minipreparation, putative PtfFinserts were isolated from phage
D N A by digestion with restriction enzymes and screened on 1% agarose gels.
Sequencing
Single-stranded sequencing reactions followed protocols suggested by the Sequenase
version 2.0 Kit manufacturer (United States Biochemical, Cleveland, OH) using the -40
Primer. The sequences o f cloned PCR products were determined by obtaining both
orientations o f a sequence as facilitated by directional cloning into M 13m pl8 and m pl9
vectors, or from obtaining a consensus o f several clones. Following sequencing, the
reaction samples were electrophoresed in 6% acylamide gels, dried under vacuum at 80 C
and exposed to autoradiograph film for 24 hours. Sequences have been deposited in
GenBank and the accession numbers are given in Appendix I.
Data Mauapement and Phylogenetic Analysis
D N A sequences were read in manually and alignments obtained using the program
ALIGN (Scientific Education Software, State Line, Pennsylvania) with a gap penalty o f 10
and an extension penalty o f 0.5. Minor adjustments to the alignment were made by eye to
produce the best possible match upon comparison with previously sampled phytochrome
9
sequences. Additional sequences included in analyses that were taken from formerly
published sources are referenced in Appendix 2. The program MEGA (Kumar et al.,
1993) was used to generate an amino acid alignment from nucleotide data. Inspection o f
this alignment was the basis for initial classification o f sequences into a group or putative
loci based on the presence or absence o f identifying amino acid hallmarks and insertions or
deletions (indels) prior to analysis. The designation o f /W K4-type sequences as PHYA or
PHYAl was based on phylogenetic analysis with Arabidopsis PHYA (tree not shown).
Percent divergence for nucleotide sequences were calculated from the
nonsynonymous proportion o f the data with alignments generated in the program MEGA.
CLUSTAL X, available by anonymous FTP at “ftp-igbmc.u-strasbg.fr/pub/”, was used to
calculate percent divergence for amino acid sequences. Phylogenetic relationships among
sequences were inferred by both distance and parsimony methods. The nucleotides at the
5' and 3' primer sites were deleted from analyses. The Kimura two-parameter model o f
substitution was used to generate pairwise distances that were then subjected to cluster
analysis following the neighbor-joining method (Saitou and Nei, 1986) as implemented by
MEGA. Gaps and missing information were excluded. Strength o f support for the major
nodes within the trees were tested by bootstrap resampling o f the original data matrix
1000 times.
Phylogenetic analysis o f the data by maximum parsimony was obtained through
PAUP 3.1.1 (Swofford, 1993).,For this reconstruction, alignment gaps representing
insertions or deletions were treated as single characters. Heuristic searches were
conducted with all starting trees retained. For each search, the starting trees were
10
constructed with random stepwise addition. This search process was repeated 45 times so
as to reduce the probability that any single search converged on a local optima as an
artifact o f the initial addition sequence. Within this reconstruction framework, tree
bisection and reconnection (TBR) branch swapping algorithm was chosen to search for
optimal trees and all o f the most parsimonious trees were retained (MULPARS). PHYB
sequences were used to root the reconstructions when constructing the consensus tree.
Rates o f Evolution
Relative rate tests following the ID N method o f Tajima (1993) were applied to the
data to test the null hypothesis o f rate constancy among and within PHYA and PHYAltype loci. This method is applicable to data for which an alignment is possible, even when
phylogenetic reconstruction is inconclusive. Furthermore, the Tajima method is suitable
when the substitution rate varies among different sites and does not require knowledge o f
the pattern o f substitution rates. The neighbor-joining tree was used to guide the selection
o f pairs o f sequences for each test. Some sequences appeared to exhibit accelerated rates
based on branch length and were immediately included in the analysis. Other sequence
pairs were chosen after dividing the PHYAIPHYA l clade into smaller groups and devising
a scheme to sample from among those groups to achieve a representative cross section o f
the total comparisons possible. Initially all rate tests were done using Myrospermum
souscmum PHYB as the outgroup. Significant results were tested again by calculating the
relative evolutionary rate a second time using Arabidopsis PHYA. This measure was an
effort to reduce the chance that significant results were a result o f the long branch length
11
leading to the PHYB outgroup (Tourasse and Gouy, 1997). Within taxa and among loci
tests were conducted on all species from which A-, A l- and E-type sequences were
available. Primer sites were excluded from analysis o f evolutionary rates.
12
RESULTS
Amino Acid Alignment
The alignment o f 579 nucleotides from 43 phytochrome sequences (voucher
specimens listed in Appendix I) sampled from divergent species o f legumes is shown in
Appendix 2. Inspection o f the amino acid alignment revealed several amino acid hallmarks
as well as insertions and deletions useful for establishing the putative identity o f sequences
prior to further analysis (Appendix 3; Table I). Most o f the hallmarks are small features o f
a one to three amino acids, however the largest (not associated with an insertion or
deletion) is the twelve residue hallmark that spans from position 447 through 458 o f the
alignment (Table I). There are four amino acid hallmarks that are useful to distinguish
PH YAl from PHYA sequences; “VK” at alignment position 307-308, “T” at position 329,
“KKI” or “KKV” at position 354-357 and “NSC” or tcNSS55 at alignment position 383385.
Percent Divergence
Within Groups
A matrix o f percent nucleotide and amino acid divergence values were calculated
from pairwise alignment s to provide an initial estimate o f the evolutionary relatedness o f
sequences prior to phylogenetic analysis (Appendix 4). A summary o f the percent
nucleotide and amino acid divergence is provided in Table 2. The four groups or putative
S
Table I. Summary table o f amino acid hallmarks and insertion/ deletion events (indels) o f phytochrome sequences from legumes.
Dashes indicate indels and hallmarks that are unique PHYAl to are underlined._____________________________________________
PHYB
PHYE
VIA
VKKPG
L
TRFLFMK
V
KHVK
DKKIPFD/DKKVPFD
TLCG
A
S
L
NCS/NSS
VW
DNE/DND/DSD
WA
SKRPD
I
SRFLFKQ
C
SPVR
DEALVQP
CLVN
A
G
A
GST
vn
GND
WS
IRRSD
L
SRFLFKQ
V
KPVK
SEELRQP
CLVN
S
V/G
T
GST
——
—
—
GND
TT
EDGDSDA-VQPQKRK
V
NTTPRFV
LR
A
V
AIHVNKEIELEY
KNILRTQTL
M
APL
EDGDSDA-VQPQKRK
V
HTTPRFV
LR
A
V
AIHVNKEIELEY
KNILRTQTL
M
APL
EEGVCG------RSSM
V '
HTSARCI
LR
M
-A
GKQLYMEIQLAS
KRMLKTQTL
L
SPT
L
HTSPRW
VR/LR
M
A
GKQLYMEIQLAS
KRMLKTQTL
L
APF/APL
Length
Position
PHYA
3
5
I
7
I
4
7
4
I
I
I
3
3
3
2
16
I
7
2
I
I
12
9
I
3
303-305
307-311
316
329-335
342
347-350
354-360
363-365
370
373
376
383-385
392-394
396-398
399-400
399-415
421
425-431
435-436
443
445
447-458
463-471
477
480-483
VIA
ITKPG
L
ARFLRMK
V
KHVK
DEKLPFD
TLCG
A
S
L
DSI
VW
DNE/DND/DSD
PH YAl
vrwnv
14
gene subfamilies were found to have fairly consistent percent divergence values in all
pairwise comparisons made within groups at both the nucleotide and amino acid levels.
For most o f the within locus comparisons the percent nucleotide divergence values ranged
from very low divergence o f 0-1%, to values o f 11-12% for some comparisons. The
divergence values for the comparisons at amino acid level were higher overall with
percentages ranging from 0-19%. The highest percent divergence values reported for
these within group comparisons were from within PHYA and within PiTK4 7.
Table 2. Summary o f percent divergence values calculated from pairwise alignments.
Percent divergence o f amino acid sequences appears above diagonal and percent
divergence o f nucleotide sequences is shown below the diagonal. ________________
PH Y A
0-0.19
PH Y A
PHYAl
PH YE
PH Y B
0-0.11
0.03-0.15
0.24-0.32
0.23-0.29
PHYAl
0.01-0.25
0.02-0.17
0.01-0.12
0.26-0.34
0.25-0.31
PH Y E
0.29-0.42
0.27-0.40
0.02-0.13
0.01-0.07
0.13-0.18
PH YB
0.33-0.42
0.32-0.38
0.10-0.22
0.02-0.08
PHYA
PH YAl
PHYE
PHYB
0.01-0.03
Among Groups
As expected, the percent divergence values were higher for among group
comparisons, with values o f approximately 23-34% being fairly representative o f
nucleotide divergence, as compared to the values o f 12% or less from within group
divergence (Table 2). Notably, the percent divergence values from among E to B group
comparisons were found to be much lower overall than the range considered
representative; this is probably an artifact o f sampling since only three PHYB sequences
15
were obtained for inclusion in analysis. Yet, more relevant to the focus o f this study was
the range o f values calculated from pairwise comparisons among A to A l groups for both
nucleotide and amino acid sequences. Those values were found to be lower overall with
percent divergence values ranging from 3-15% and 1-25%, respectively.
Taken together, the high values for percent divergence for within presumptive
PHYA and PH YAl subfamilies combined with low percent divergence calculated for
among PHYA to PH YAl imply the tw o P/fK4-type sequences are consanguineous. High
divergence within genes and low divergence among them is often correlated with
sequences that are undergoing some type o f homogenizing process (Ohta, 1983), although
values calculated from percent divergence fail to indicate whether the relationship among
the sequences analyzed here are consistent with expectations to support a hypothesis on a
specific mechanism o f information transfer.
Phylogenetic Reconstruction
To understand the relationships among presumptive loci, both distance and
parsimony methods were used to infer the phytogeny o f all sequences. Neighbor-joining
methods and parsimony analysis resulted in trees with generally the same topology and
consistent relationships within clades. The phytogeny inferred from the phytochrome
sequences sampled in legumes support the position initially stated by Mathews et al.
(1995) that four loci appear to represent phytochrome gene diversity in that plant family,
three o f which are comparable to loci known from Arabidopsis (PHYA, PHYB, PHYE),
and one that is potentially unique in legumes, PH YA l.
Employing the neighbor-joining method using Kimura’s two-parameter model o f
nucleotide substitution resulted in a tree with the topology shown in Figure 2. Bootstrap
analysis o f the distance-based reconstruction showed high support (100%) for the internal
branches and distinguish the monophyly o f the following groups: all /WFZ? sequences, the
PHYE sequences, all PHYA-tyys sequences and finally the PHYB combined with the
PHYE sequences. Although many branches within the more terminal clades were poorly
supported, the same branching order and sequence relationships were consistently
reconstructed under a variety o f different conditions (e.g. reconstructed with the third
position removed; trees not shown). As stated above, parsimony analysis resulted in a tree
with similar topology to the distance tree. The strict consensus tree shown in Figure 3 was
produced from 18 minimal length trees, inferred from 1254 steps, with a consistency index
o f 0.508 and a retention index o f 0.805.
Resolution o f a better supported phytogeny within the entire PHYA group is
hindered by the inclusion o fta x a from the poorly represented M m osoideae and
Caesalpinioideae subfamilies (sensu Lavin et al. 1998). Using the distance approach again,
the phytogeny was inferred with the four taxa from those tw o legume subfamilies
removed. When Enterolobium cyclocarpum PHYA, Enterolobium cyclocarpum PH YAl,
Browneo sp. PHYA and Gymnoclcidus dioico PETYA are excluded from analysis the
PHYAl sequences formed a monophyletic group derived from within the PHYA subfamily
and bootstrap support was improved. Figure 4 shows PHYA-typQ sequences grouped by
presumptive loci rather than taxa. For example M illettia grandis PHYAl clusters with
other putative PHYAl subfamily sequences as opposed to clustering with M grandis
Figure 2. Neighbor-joining tree.
- Brownea sp. A
100 IGymnocladus dioica A
IEnterolobium cyclocarpum A
---------- Pisum sativum A
Cycloloblum nutans A
f:
o !------ Sophora affinis A
73^ l— Derris elliptica A
Dahlstedtia pinnata A
— Millettia dura A
------Pongamia pinnata A
— Mundulea sericea A
Glycine max A
- Austrosteensia blackil A
Dalberg iella n yassae A
5031t
64
Craibia brevicaudata A
MiIIettiagrandisA
- Myrospermum sousanum A
Millettia richardiana Al
Pongamiopsis amygdalina Al
Ostryocarpus stuhlmannii Al
„„ 68— ------ Lonchocarpus phaseolifolius Al
Millettia grandis Al
— Austrosteensia blackii Al
Dalbergiella n yassae Al
-------------- Carmichaelia sp. Al
Cycloloblum nutans Al
-------- Enteroloblum cyclocarpum Al
4—30
100
-
"jlW rp^
80
I
Figure 2. Continued.
--------- Enterolobium cyclocarpum E
Gleditsia triacanthos E
100
— Sophora affinis E
------ Poecilanthe falcata E
--------
Derris elliptica E
-------- Millettia dura E
Millettia richardiana E
----- Piscidia piscipula E
------- Tephrosia villosa E
100
- Millettia grandis E
48
Dalbergiella nyassae E
- Carmichaelia sp. E
Austrosteensia blackii E
98
100
------- Glycine max B
-------------- Mundulea sericea B
Myrospermum sousanum B
Figure 2. Phylogenetic relationship oiP H Y sequences sampled from legumes. Neighbor-joining tree constructed from genetic distances
using the Kimura two-parameter model o f nucleotide substitution. The number o f times out o f 1000 bootstrap replicates that a branch
was present is noted above the branch. Branch lengths are proportional to evolutionary distances.
19
Brownea sp . A
Gymnocladus dioica A
Enterolobium cyclocarpum A
Pisum sativum A
Cyclolobium nutans A
Sophora affinis A
D e n is elliptica A
Dahlstedtia pinnata A
Millettia dura A
Pongamia pinnata A
MunduIea sericea A
M illeltia grandis A
Glycine max A
Austrosteensia blackii A
Dalbergiella nyassae A
Craibia brevicaudata A
M yrospermum sousanum A
Milletlia richardiana A l
Pongam iopsis amygdalina A l
Ostryocarpus stuhlmannii A l
Lonchocarpus phaseolifolius A l
M ille ttia g ra n d isA l
■ AustrosteensiablackiiA l
■ Dalbergiella nyassae A l
■ Carmichaclia sp. A l
■
Cyclolobium nutans A l
■
Enterolobium cyclocarpum A l
-
Enterolobium cyclocarpum E
-
Sophora affinis E
- P oecilanthefalcataE
- Carmichaelia sp. E
-
D e n isellip tic a E
-
M ille ttia d u ra E
- M illettia richardiana E
-
P iscid ia p iscip u la E
- T ephrosiavillosaE
-
M illettia g ra n d isE
-
Austrosteensia blackii E
-
D albergiellanyassaeE
-
G leditsiatriacanthosE
-
G ly d n e m a x B
- M u n d uleasericeaB
- M yrospermum sousanum B
Figure 3. Phylogenetic relationship o f the PHY sequences from legumes reconstructed by
parsimony analysis o f D N A sequences. The strict consensus o f the eighteen most
parsimonious tees. Tree length, 1254; consistency index, 0.508; retention index, 0.805.
The tree is rooted with PHYB sequences.
O etryccarpa rti-h ln a n l I Al
Lonchoca-FUB f* h « o ] Ifol lue Al
Rngm loFeie aejgrfallna Al
M lllrttla rlch a-d lm a Al
— M lllrttla y m d le Al
--------- Auetroetemela black] I Al
B alberglrlla ngaeeae Al
------------- C m lch a-H a op- Al
Cycloloblui ru t m e Al
-------------- IkroeFrrnui mueanun A
Cycloloblui nutate A
-------- Sofhora a ffln le A
----------------------- Pleui eat Iuui A
I— Berrle r l I Ipt lea A
rP— B * le te d tla p lm ata A
L - M lllrttla dura A
— R ngm la p lm ata A
— Cralbla breuloaudata A
B alberglrlla nyaeear A
---- ----------------------------- GJyclnr nax A
— Auetroetemela black! I A
- M lllr ttla y a i d l e A
Murtulra e r r I ora A
to
O
Entcroloblui c y c l o c a m E
---- G ledltela tr la c a rther E
-------- Sofhora a ffln le E
------------ Rrecl la rth r fa lc a te E
Aueti ciutem ela black! I E
---------- C an lc h arlla op. E
B alberglrlla ryam ar E
M lllrttla w a t t l e E
----- Terhroela u lllc e a E
— Plecldla p lm lfu la E
M lllrttla r ic h a d la ra E
Berrle r l ll p t l c a E
M lllrttla d u e E
tty UOFr m u i a u e a r n I
----------- Glyclnr ihk J
----------------- Nurdulra w -Icra I
Figure 4. Neighbor-joining tree with all non-Papilionoideae PHY sequences removed. Relationships inferred using Kimura two
parameter model of nucleotide substitution. Branch lengths are proportional to evolutionary distances.
21
PHYA. This pattern o f reconstruction is more consistent with sequences representing
separate loci rather than allelic diversity and is also consistent with scenarios in which
sequence homogenization by gene conversion is low among paralogues (Waters, 1995).
Rates o f Evolution
Tests for heterogeneity in nucleotide substitution rates followed a relative rate
approach wherein the evolutionary rates o f two sequences were measured against a
reference sequence (Sarich and Wilson, 1967W u an^ Li, 1985). According to Tajima
(1993) a relative rate test is possible with nucleotide or amino acid sequences without
making assumptions about how the substitution rate varies from site to site, or that the
pattern o f substitution follows a specific substitution model. The basis o f the Tajima
method is that the equality o f constant rate o f substitution in two evolutionary lineages,
M l and M2, can be tested regardless o f the substitution model and despite substitution rate
variation among sites. Ifth e null expectation o f equality, M l = M2, fails the conclusion
drawn is that the substitution rate is not constant. The expectation that M l = M2 is tested
by calculating a chi-square. The value M l is the number o f changes in sequence I
compared to sequence 3 (a reference outgroup) and M2 is the number o f changes in
sequence 2 compared to sequence 3. Then, %2 = (M l - M2)2/ M l + M2. When transitional
and transversional nucleotide sites are calculated together, as done here, the chi-square
distribution has one degree o f freedom.
Thirty-seven tests were conducted to test the null hypothesis o f a constant substitution
rate within and among PHYA and PHYAl sequences using Myrospermum souscmum
22
PHYB as the reference outgroup. Significant chi-square values were tested again with
Arabidopsis PHYA as the outgroup. Similar results were obtained using the relative rate
test method o f Wu and Li (1985) on a subset o f data (results not shown).
Within PHYA Tests
Most o f the statistical tests comparing the relative rate o f nucleotide substitution o f
Glycine max PHYA to another PHYA sequence rejected the null hypothesis o f a constant
rate o f substitution (Table 3). Only two tests with Glycine max PHYA failed to reject the
null, one with the non-papilionoid legume, Enterolobium cyclocarpum PHYA, the other
twith the papilionoid legume, Brownea sp. PHYA. N o other tests for rate heterogeneity o f
PHYA sequences from within Papilionoideae rejected the null.
Two o f the within PHYA tests representing among legume subfamily comparisons
(Gymnocladus dioica PHYA to Millettia grcmdis PHYA and Enterolobium cyclocarpum
PHYA to Austrosteensia blackii PHYA) also rejected the null when Myrospermum
sousanum PHYB was the outgroup. However, they failed to reject the null when repeated
W ihArabidopsis PHYA. Results o f the within PHYA tests indicate that while
heterogenous rates were calculated for within PHYA lineages, only the Glycine max PHYA
sequence appeared to be evolving significantly faster.
23
Table 3. Rate heterogeneity among and within PH Y subfamilies calculated with Tajima’s
ID N method.
Reference
Within A Comparisons
Myrospermum PHYB Arabidopsis PHYA
2.7692
I. Brownea sp. -Myrospermum sousanum
1.0667
5.1200*
2. Gymnocladus dioica -Millettia grandis
15.2540***
8.9630***
3. Glycine max - Millettia grandis
0.2162
4. Enterlobium cyclocarpum - Glycine max
0.3913
5. Myrospermum sousanum - Dahlstedtia pinnata
0.5000
6. Pisum sativum - Millettia grandis
9.6800***
12.000***
I. Millettia dura - Glycine max
0.8571
8. Derris elliptica - Gymnocladus dioica
17.3333***
9.3820***
9. Glycine max -Austrosteensia. blackii
2.0864
10. Brownea sp. - Glycine max
2.0833
11. Gymnocladus dioica - Mundulea sericea
0.4902
5.3333*
12. Enterlobium cyclocarpum -Austrosteensia blackii.
3.7556
13. Gymnocladus dioica -Austrosteensia blackii
22.3448***
5.4528*
14. Glvcine max - Mundulea sericea
Within A l Comparisons
0.1176
15. Pongamiopsis amygdalina - Millettia grandis
0.3247
16. Enterlobium cyclocarpum -Austrosteensia blackii
0.2195
17. Cyclolobium nutans - Lonchocarpus phaseolifolius
0.2353
18. Enterlobium cyclocarpum - Ostryocarpusstuhlmanni
3.4286
19. Millettia richardiana - Carmichaelia sp.
3.5957
1.4545
20. Carmichaelia sp. - Millettia grandis
0.1268
21. Enterlobium cyclocarpum - Cyclolobium nutans
5.7692*
6.4286* ,
22. Carmichaelia sp. - Dalbergiella nyasse
0.3636
23. Millettia grandis - Austrosteensi 'a blackii
_______ Among A and A l______ __________ ____________________
24. Glycine max A-Cyclolobiumnutans A l
1.6575
25. Gymnocladus dioica A - Dalbergiella nyasse A l
2.5745
26. Gymnocladus dioica A - L phaseolifolius A l
0.0000
27. Glycinemax A-Ostryocarpusstuhlmannii A l
3.4595
11.6552***
28. Myrospermum sousanum A - Carmichaelia sp. A l
9.3830***
29. Glycine max A-M illettia grandis A l
2.5789
30. Sophora affinis A-Austrosteensia blackii A l
2.8800
31. Cyclolobium nutans A - Enterlobium cyclocarpum A l
1.2090
32. Mundulea sericea A - Cyclolobium nutans A l
1.6531
33. Derris elliptica A - Millettia grandis A l
2.7778
34. Derris elliptica A - Cyclolobium nutans A l
0.9697
35. Mundulea sericea A - Carmichaelia sp. A l
3.7692
5.1429*
36. Cyclolobium nutans A - Cyclolobium nutans A l
5.5385*
7.0784**
37. Crabia breicaudata A - Carmichaelia sp. A l_________ 5.8980*
*** Indicates significant at the 0.05% level. ** Indicates significant at the 1% level, indicates significant
at the 5% level.
24
Table 3. Continued.
Within Taxa and Among Loci Comparisons
Reference
PHYE
Myrospermum PHYB
Taxa Compared
0.1475
2.5224
Enterlobium
cyclocarpum
A-Al
I.
1.8837
3.4571
2. Millettia grandis A-Al
2.0000
0.2308
Austrosteensia
blackii
A-Al
3.
0.2571
0.2903
4. Dalbergiella nyasse A-Al
*** Indicates significant at the 0.05% level. ** Indicates significant at the 1% level. +Indicates significant
at the 5% level.
Within
7 Tests
N o observable pattern was recognized from the tests conducted within the group
, o f sequences, although the tests with Carmichaelia sp. PHYAl to Dalhergiella nyasse
PHYAl sequences rejected the H0 (x2 = 6.4286, P < 0.010) (Table 3). More tests with
Carmichaelia sp. PHYAl are necessary to make conclusive remarks about a larger trend.
Among PHYA-X v 1OQ Tests
A pattern is discernible with Carmichaelia sp. PHYAl in tests with PHYA
sequences (Table 3). All such comparisons are highly significant except Carmichaelia sp.
PHYAl to Mundulea sericea PHYA, however, the %2 from that test would reject the null
at the 10% level. The within species and among loci tests o f rates using Cyclolobium
nutans PHYA and PHYAI rejected H0 (%2 = 5.5385, P < 0.025). The absence o f* PHYE
or PHYB sequence from that species precluded testing for the effects o f phylogeny on
these tests results.
25
Among PHYA-tvyo, and Within Taxa Tests
Correction for phylogeny was possible in tests o f evolutionary rates among loci
and within a species with sequences from Enterolobium cyclocarpum, M illettia grandis,
Austrosteensia blackii and Dalbergiella nyasse (Table 3). All these PHYA to PHYAl tests
were conducted using the PHYE sequence from these taxa as the outgroup. N o significant
chi-square values were detected.
V
26
DISCUSSION
This paper describes the results o f phylogenetic analysis o f the phytochrome gene
family in Fabaceae and an analysis o f evolutionary rates among and within putative PHYAtype gene sequences. Although, additional data from the putative gene subfamily are
needed to explicitly remark on the identity o f PH YAl, the findings presented here are in
agreement with the suggestion o f Mathews et al. (1995) that the PHYA-Xike sequences,
PH YAl, most likely represent a forth locus in Fabaceae.
An initial survey o f the sequence alignments revealed that the chromophore
attachment site was conserved in the PHYAl sequences and no frameshift mutations,
misplaced start or stop codons were detected. Further screening o f the amino acid
alignments from four presumptive loci revealed four
-specific amino acid hallmarks
within the most conserved region o f the phytochrome gene. This was the first evidence to
suggest that PHYAl may be a new locus. The functional significance o f the changes are.
outside the scope o f this study, however there is evidence from other studies o f gene
families that the substitution o f even a single amino acid can have large effects in terms o f
protein function. For example, this was recently demonstrated in a study o f the a-esterase
multigene family evolution by Newcomb et al. (1998). Their investigation revealed that the
change o f a one amino acid from glycine to aspartic acid resulted in conversion o f the
enzyme from a phosphatase to a carboxylase and ultimately conferred insecticide
resistance in a blowfly species.
Calculations o f percent divergence from comparisons within gene families revealed
that the highest values come from comparisons within the PHYA and PH YAl lineages.
This implies that there is a higher rate o f substitution within these gene lineages. Higher
rates o f substitution are predicted as part o f the conceptual model o f multigene family
evolution wherein a gene is duplicated and the resulting copies begin to diverge in function
(Ohta, 1990; Ohta3 1988). Attempts to clarify the question o f substitution rates are
addressed further below.
In the calculations o f percent divergence among gene subfamilies, the percent
divergence values from nucleotide and amino acid data provided some evidence about the
identity o f the PHYAl sequences. PHYA and PHYAl sequences were found to be less
divergent from each other as compared to other loci (i.e. A to E 3 A l to B 3 B to E 3 etc.).
This was not surprising given their numerous shared amino acid hallmarks identified by
visual inspection o f the amino acid alignment. The levels o f divergence within and among
groups calculated from P7TK4-type sequences is concordant with several possible
explanations. One possibility is that tw o groups o fP # E 4 -ty p e sequences represent allelic
diversity. Alternatively, it is possible that the tw o P#K 4-type sequences are the products
o f recent gene duplication and have not had sufficient time to diverge or are experiencing
sequence homogenization or gene conversion. The percent divergence values from the
analysis presented here were inconclusive in supporting either hypothesis and so the
question was addressed further in the context o f phylogenetic analysis.
L ow divergence values were also found in the among loci comparisons o f PHYE to
PHYB, however the alignments clearly support the distinct identity o f PHYE and PHYB, as
does phylogenetic analysis. The low divergence values at the amino acid level reported in
the PHYE to PHYB comparisons are likely to be the result o f limited sampling o f those
loci or a high level o f synonymous substitutions (Demmin et al. 1989). Analysis to test the
latter are underway and will be discussed elsewhere.
As the estimation o f percent divergence did not allow for a rigorous statistical test
o f the values, phylogenetic analysis with bootstrap resampling was employed to further
address the question o f the identity o f the PHYAl sequences. The phylogenetic
reconstructions presented here are concordant with a picture o f P i / 7 gene family diversity
o f sequences sampled from Fabaceae first suggested by Mathews et al. (1995). This
picture includes four gene subfamilies represented by P i/7 4 , PHYB, PHYE and PHYA I,
the latter having no orthologue to PPTY sequences known from studies o f phytochrome
gene family diversity in Arcibidopsis. Given that the PHYAl sequences are present in
papilionoid, caesalpinoid and mimosoid legumes suggests that the gene duplication that
gave rise to the PHYAl predates the diversification o f legume subfamilies. Alternatively, it
is possible that there was insufficient data collected from non-papilionoid legumes to
reveal a larger trend o f multiple gene duplication events independently giving rise to
PH Y A l sequences in each legume subfamily. Therefore, the use o f PHYAl sequences for
systematic questions requires caution until further data collection and analysis permits
verification that PHYAl sequences are homologous.
Phylogenetic analysis resolves the PHYA/A1, PHYB and PHYE lineages with high
bootstrap support. Resolution within the PHYAIAI clade was contentious due to the
inclusion o f the poorly sampled non-papilionoid legumes. However, several lines o f
29
evidence are offered to support the assertion that the PHYAl sequences are most likely a
new locus. First, the removal o f caesalpinoid and mimosoid PHYA-type sequences
resolved all PH Y sequences by presumptive loci with the PHYAl sequences forming a
monophyletic group derived from within the PHYA clade. Secondly, all reconstruction
methods consistently inferred largely identical organismal relationships. Finally, the
strongest support for the designation o f PHYAl as a distinct loci was the observation that
all methods o f reconstruction consistently grouped sequences by presumptive gene
subfamily rather than by taxa. This pattern o f reconstruction is best explained under the
hypothesis that gene conversion is not prevalent among PHYA and PHYAl sequences
(Sitnikova and Nei, 1998; Sanderson and Doyle, 1992) from papilionoid legumes arid that
the PHYAl sequences represent a fourth locus in legumes.
In addition to phylogenetic analysis and percent divergence, a relative rate test
developed by Tajima (1993) was used to compare whether there was a pattern o f rate
heterogeneity among and within
-type loci and to assess whether the distribution o f
significant results were conserved across taxa. Motivation for this was twofold. Primarily,
it is thought that by testing for rate heterogeneity parallels might be drawn as to whether
the PHYAl sequences demonstrated patterns consistent with other PffK loci, or whether
there is a pattern indicative o f the expectation for sequences representing a pseudogene
wherein higher rates o f substitution are expected (Arctander, 1995; Ota and Nei, 1994).
Furthermore, while it is a common practice to test the molecular clock (Gaut et al., 1996;
Gaut et a l, 1997), it was decided to advance the efforts to do so with the less studied type
30
o f data represented here, ultimately advancing the efforts to formulate hypotheses
regarding larger trends in molecular evolution.
The Tajifna ID N method was used for testing rate heterogeneity with the
phytochrome data since it eliminated concern about selecting the appropriate outgroup
(Tajima, 1993). Also this method does not require knowledge o f the either the pattern o f
substitution rates or the rate variation among sites. The distance tree was used to identify
potential lineages exhibiting rate variation and to decide on pairs o f sequences to use for
rate tests. However, as suggested by M use and Gaut (1997), caution is applied in making
specific conclusions based on individual comparisons and instead broad patterns are
discussed.
Visual inspection o f the branch lengths indicated that some lineages may be
accelerated relative to others, although few significant patterns were revealed. Overall, the
lineages were found to have heterogeneous rates o f evolution consistent with the patterns
reported by Mathews and Sharrock (1996). There does appear to be some evidence for a
slightly more heterogeneous rate o f evolution among legume subfamilies (and among
orthologues), although too few data were available from Caesalpinoideae and
Mimosodeae to warrant conclusive remarks. Notably all tests with Glycine max PHYA
produced consistently high chi-square values that strongly support the assertion o f a
relative increase in the. evolutionary rate in that lineage. Strikingly,.there was no evidence
from rate heterogeneity to suggest the PHYAl sequences were behaving like pseudogenes.
Ifth e PHYAl locus was a pseudogene one would expect to see accelerated rate within the
31
PHYAl sequences as compared to other members o f the gene family, (Sitnikova and Nei,
1998) which was not observed with the analysis o f this data set.
Relative rate tests can be applied to test differences in substitution rates on a
number o f levels (Friar and Porter, 1998). The tests performed here were intended to
examine rate heterogeneity within a multigene family, however rate heterogeneity was also
tested within taxa and between loci to address the effects o f phylogeny on such
comparisons (Table 3). In all such tests no evidence was found for a pronounced change in
rates. Another consideration when undertaking a relative rate test is the possible effects o f
generation time on the lineages compared (Gaut et al., 1996). Without consideration o f
effects such as generation time, metabolic rates and D N A polymerase fidelity it is possible
to reduce the o f accuracy o f testing rate differences as a function o f solely time and
substitution rates (Muse and Weir, 1992). The effects o f generation time on these tests
were considered minimal because the taxa represented in this study are believed to have
comparable generation times (Matt Lavin, personal communication).
32
CONCLUSIONS
1. The analyses presented here suggest that the phytochrome gene family in
Fabaceae is represented by four loci: PHYA, PHYB, PHYE and PH YA l. The following
evidence is offered in support. The sampling procedure is believed to completely identify
all possible PHY-Vike sequences. Degenerate primers used in concert with variations in
stringency o f the annealing temperature during PCR failed to detect additional PHY-Mke
sequences, although this method was able to detect allelic diversity in a few taxa (Lavin et
al., 1998). Furthermore, the sampling strategy was so extensive that most sequences
amplified and cloned were redundant.
2. Phylogenetic analysis and relative rate tests o f the PH YAl sequences verify that
they conform to more to the expectations o f a fourth locus versus an allele or pseudogene.
While further evidence from the sequence o f the entire gene and verification o f its
expression is needed to be conclusive, the results presented here strongly suggest legumes
contain a locus that has no homologue to the loci known from Arabidopsis.
3. The analysis o f relative rates was based on partial sequence from the more
conserved proportion o f the gene. Similar analysis o f more complete data would be useful
to be more specific about patterns and process in this gene family. Nevertheless, the
limited information presented here on the level o f rate heterogeneity among PHY loci
suggest that phylogenetic reconstructions should be based on numerous terminal taxa to
control for heterogeneity.
33
4.
Additional analyses are underway to address differences in synonymous and
nonsynonymous rates o f evolution. These analyses will be used to assess questions about
possible selective constraints.
34
REFERENCES CITED
35
1. Arctander7P. 1995. Comparison o f a mitrochondrial gene and a corresponding
nuclear pseudogene. Proc. R. Soc. London. B. 262:13-19.
2. Clack, T., S. Mathews, R. A. Sharrock. 1994. The phytochrome apoprotein family in
Arabidopsis is encoded by five genes: the sequences and expression o f PHYD and
PHYE. PI. Mol. Biol. 25:413-427.
3. Demmin, D. S., E. J. Stockinger, Y. C. Chang, and L. L. Walling. 1989. Phylogenetic
relationships between the chlorophyll a/b binding protein (CAB) multigene family: an
intra- and interspecies study. J. Mol. Evol. 29:266-279.
4. Doyle, J. I. and J. L. Doyle. 1987. A rapid D N A isolation procedure for small
amounts o f fresh leaf tissue. Phytochem. Bull. 19:11-15.
5. Durbin, M. L., G. H. Learn, Jr., G. A. Huttley, and M. T. Clegg. 1995. Evolution o f
the chalcone synthase gene family in the genus Ipomoea. Proc. Natl. Acad. Sci. USA.
92:3338-3342.
6. Erlich, H. A. 1992. PCR Technology: principles and applications fo r DNA
amplification. W.H. Freeman, N ew York, NY.
7. Friar, E. A. and J. M. Porter. 1998. A tale o f three genomes I: rate heterogeneity o f
molecular evolution in noncoding regions in the nuclear, chloroplast and
mitrochondrial genomes. In Press.
8. Gaut, B. S., B. R. Morton, B. C. McCaig, and M. T. Clegg. 1996. Substitution rate
comparisons between grasses and palms: synonymous rate differences at the nuclear
gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl. Acad. Sci.
USA. 93:10274-10279.
9. Gaut, B. S., L. G Clark, J. F. Wendel, and S. V. Muse. 1997. Comparisons o f the
molecular evolutionsry process as rbcL and ndhFm the grass family (Poaceae). Mol.
Biol. Evol. 14:769-777.
10. Innis, M., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. PCRprotocols: a
guide to methods and applications. Academic Press, San D iego, CA.
11. Kehoe, D. M. and A. R. Grossman. 1996. Similarity o f a chromatic adaptation sensor
to phytochrome and ethylene receptors: Science. 274:1499-1412.
36
12. Kolukisaoglu, H. U., S. Marx, C. Wiegmann, S. Hanelt and H. A. W. Schneider. Poetsch 1995. Divergence o f the phytochrome gene family predates angiosperm
evolution and suggests that Selaginella and Equisetum arose prior to Psilotum. J.
Mol. Evol. 41:329-337.
13. Kumar S., K. Tamura, and M. Nei. 1993. MEGA: Molecular Evolutionary Analysis,
version 1.01. The Pennsylvania State University, University Park, Pennsylvania.
14. Lavin, M., E. Eshbaugh, J.-M. Hu, S. Mathews, and R. A. Sharrock. 1998.
Monophyletic subgroups o f the tribe Millettia (Leguminosae) as revealed by
phytochrome sequence data. Am. J. B o t 85:412-433.
15. Mathews, S. and R. A. Sharrock. 1996. Phytochrome gene family in grasses (Poacea)
a phylogeny and evidence that grasses have a subset o f the loci found in dicot
angiosperms. Mol. Biol. Evol. 13:1141-1150.
16. Mathews, S. and R. A. Sharrock. 1997. Phytochrome gene diversity. Plant, Cell, and
.Env. 20:666-671.
17. Mathews, S., M. Lavin, and R. A. Sharrock. 1995. Evolution o f the phytochrome
gene family and its utility for phylogenetic analyses o f angiosperms. Ann. Missouri
Bot. Gar. 82:296-321.
18. Muse, S. V. and B. S. Gaut. 1997. Comparing patterns o f nucleotide substitution
rates among chloroplast loci using the relative ratio test. Genetics. 146:393-399.
19. Muse, S. V. and B. S. Weir. 1992. Testing equality o f evolutionary rates. Genet.
132:269-276.
20. Newcomb, R., P. M Campbell, R. J. Russell, and J. G. Oakeshott. 1998. Insecticide
resistance in a blowfly conferred through evolution o f a catalytic mechanism. Society
for Molecular Biology and Evolution, 6th Annual International Meeting.
21. Ohta, T. 1983. O nthe evolution o f multigene families. Theor. Popl. Biol. 23:216240.
22. Ohta, T. 1988. Multigene and supergene families. In Harvey. P. H. and L. Partridge
(eds.) Oxford Survey in Evolutionary Biology. Oxford University Press, NY.
23. Ohta, T . 1990. H ow gene families evolve. Theoretical Population Biology. 37:213219.
■37
24. Ota, T. and M. Nei. 1994. Divergent evolution and evolution by the birth-and-death
process in the immunoglobulin V h gene family. Mol. Biol. Evol. 11:469-482.
25. Quail, P. H. 1994. Phytochrome genes and their expression. In Kendrick, R. E. and
G. H. M. Kroneberg (eds.) Photomorphogensis in Plants. 2nd ed. Kluwer Academic
Publishers, Dordrecht, Boston, London.
26. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for
reconstruction phylogenetic trees. Mol. Biol. Evol. 4:406-425.
27. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory
manual. Cold Spring Harbor, NY.
28. Sanderson M. I. and J. J. Doyle. 1992. Reconstruction o f organismal and gene
phylogenies from data on multigene families: concerted evolution, homoplasy, and
confidence. Syst. Biol. 41:4-17.
29. Sang, T., M. J. Donoghue, and D. Zhang. 1997. Evolution o f alcohol dehydrogenase
genes in peonies (Paeonia) : phylogenetic relationships o f putative nonhybrid species.
Mol. Biol. Evol. 14:994-1007.
30. Sarich, V. M. and A. C. Wilson. 1967. Immunological time scale for hominid
evolution. Science. 158:1200-1203.
31. Sharrock R. A. and P. H. Quail. 1989. N ovel phytochrome sequences in Arabidopsis
thaliana: structure, evolution, and differential expression o f a plant regulatory
photoreceptor family. Genes Dev. 3:1745-1757.
32. Sharrock, R. A., J. L. Lissemore and P. H. Quail. 1986. Nucleotide and amino acid
sequence o f a Curcurbia phytochrome cD N A clone: identification o f conserved
features by comparison with Avena phytochrome. Gene. 47:287-295.
33. Sitinikova, T. and M. Nei. 1998. Evolution o f immunoglobin kappa chain variable
region in genes in vertebrates. Mol. Biol. Evol. 15:50-60.
34. Smith, H. 1994. Sensing the light environment: the function o f the phytochrome
family. In Kendrick, R. E. and G. H. M. Kroneberg (eds.) Photomorphogensis in
Plants. 2nd ed. Kluwer Academic Publishers, Dordrecht, Boston, London.
35. Smith, H. 1995. Physiological and ecological function within the phytochrome family.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:289-315.
38
35. Swofford, D. L. 1993. PAUP: phylogenetic analysis using parsimony, version
3.1.1. Computer program distributed by the Illinois Natural History Survey,
Champaign, EL.
36. Tajima, F. 1993. Simple methods for testing the molecular evolutionary clock
hypothesis. Genet. 135:599-607.
37. Terzaghi, W. B. and A. R. Cashmore. 1995. Light-regulated transcription. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 46:445-474.
38. Tourasse, H J. and M. Gouy. 1997. Evolutionary distances between nucleotide
sequences based on the distribution o f substitution rates among sites as estimated by
parsimony. Mol. Biol. Evol. 14:287-298.
39. Wagner, A ., N. Blackstonej P. Cartwright, M. Dick, B. Mishof, P. Snow, G. P.
Wagner, J. Bartels, M. Murtha, and I Pendleton. 1994. Surveys o f gene families
using polymerase chain reaction: PCR selection and PCR Drift. Syst. Biol. 43:250261.
40. Waters, E. R. 1995. An evaluation o f the usefulness o f the small heat shock genes for
phylogenetic analysis in plants. Annl. Miss. Bot. Gard. 82:278-295.
41. W olfe, K. H., W.-H: Li, and P. M. Sharp. 1987. Rates o f nucleotide substitution vary
greatly among plant mitrochondrial, chloroplast, and nuclear DNAs. Proc. Nat. Acad.
S ci.U SA . 84:9054-9058.
42. Wu, C.-L, and W.-H. Li. 1985. Evidence for higher rates o f nucleotide substitution in
rodents than in man. Proc. Natl. Acad. Sci. USA. 82:1741-1745.
39
APPENDICES
40
APPENDIX A
41
Sources o f the plant tissues used in this study and GenBank accession numbers. Modified
from Lavin et al. (1998).
Species/ Source/ Voucher
GenBank Accession Number
1. Brownea sp. (Lavin 6225, BH)
2. Pisum sativum L. (GenBank accession)
3. Cyclolobium nutans Rizz. & Heringer (HG
Lima 3, RJ)
4. Denis elliptica (Roxb.) Benth. (no voucher,
specimen from Michigan State Univ.)
5. Gymnocladus dioica (L.) K.Koch (Lavin
6211, BH)
6. Enterolobium cyclocarpum (Jacquin)
Grisebach (Lavin 6226, BH)
7. Myrospermum sousanum (Delgado &
Johnston s.n., TEX)
8. Sophora affinis T. & G. (Escobar s.n.,
MONT)
9. Millettia dura (Lock 83/124, K)
10. Glycine max (L.) Merrill (GenBank
accessions)
11. Miilettia grandis (E. Meyer) Skeels (Lavin
& Lavin s.n., MONT)
12. Mundulea sericea (Willd.) A. Chev.
(Schrire 2529, K) PHYB: U78067
13. Dahlstedtia pinnata (Bentham) Malme (HG
Lima 4-1, RJ)
14. Austrosteensia (Kunstleria) blackii (F.
Muell.) Geesink (Pedley 5005, K)
15. Dalbergiella nyassae Baker f. (Muller
2686, K)
16. Pongamiapinnata (L ) Pierre (Gutierrez
s.n., K)
17. Craibia brevicaudata (Vatke) Dunn (Polhill
& Robertson 5296, K)
Millettia richardiana (Baillon) Du Puy &
Labat (Schrire 2555, K)
19. Carmichaelia sp. (Lavin 6170, MONT)
20. Enterolobium cyclocarpum (Jacquin)
Grisebach (Lavin 6226, BH)
21. Cyclolobium nutans Rizz. & Heringer (HG
Lima 3, RJ)
PHY± U78820
PHYA-. M37217
PHYA-. U78828
.
PHYA: U83270
PHYA\ U78822
PHYA-. U78825
PHYA-. U78830
PHYA\ U78835
PHYA-. AF004783
PHYA\ L34844
PHYA: AF004794
PHYA: U78064
PHYA\ AF004776
PHYA-. U78841
PHYA: U78068
PHYA: U83266
PHYA: U83269
PHYA1: AF004788
PHYA1: U78838
PHYA1: U78826
PHYAl: U78829
42
Appendix A. Continued.
22. Pongamiopsis amygdalina (Baillon) R.
Viguier (DuPuy M575, K)
23. Ostryocarpus iXerroderris') stuhlmannii
(Taubert) Harms (Corby 2162, K)
24. Millettia grandis (E. Meyer) Skeels (Lavin
& Lavin s.n., MONT)
25. Austrosteensia (Kunstleria) blackii (F.
Muell.) Geesink (Pedley 5005, K)
26. Dalbergiella nyassae Baker f. (Muller
2686, K)
27. Lonchocarpus phaseolifolius Bentham
(Hellin & Hughes 5, FHO)
28. Enterolobium cyclocarpum (Jacquin)
Grisebach (Lavin 6226, BH)
29. Sophora affinis T. & G (Escobar s.n.,
MONT)
30. Carmichaelia sp. (Lavin 6170, MONT)
31. Gleditsia triacanthos L. (Lavin s.n.,
MONT)
32. Derris elliptica (Roxb.) Benth. (no voucher
specimen from Michigan State Univ.)
33. Millettia dura (Lock 83/124, K)
34. Millettia richardiana (Baillon) Du Puy &
Labat (Schrire 2555, K)
35. Piscidiapiscipula (L ) Sarg. (Lavin &
Luckow 5793a, TEX)
36. Millettia grandis (E. Meyer) Skeels (Lavin
& Lavin s.n., MONT)
37. Austrosteensia (Kunstleria) blackii (F.
Muell.) Geesink (Pedley 5005, K)
38. Dalbergiella nyassae Baker f. (Muller
2686, K)
39. Tephrosia villosa (L.) Pers. (Lavin 6219,
BH)
40. Poecilanthe falcata (Veil.) Heringer (HG
Lima 2-1, RJ)
41. Glycine max (L.) Merrill (GenBank
accessions)
42. Myrospermum sousanum (Delgado &
Johnston s.n., TEX)
43. Mundulea sericea (Willd.) A. Chev.
(Schrire 2529, K)
44. Arabidopsis thaliana (L ) Heynh (GenBank
accession)
PHYAh AF004801
PHYAl \ AF004781.
PHYAh. AF004793
PHYAl: U78842
PHYAh U78069
PHYAh AF004800
PHYE-. U78827
PHYE: U78837
PHYE: U78839
PHYE: U78819
PHYE: U83272.
PHYE: AF004785
PHYE: AF004786
PHYE: AF004791
PHYE: AF004792
PHYE: U78843
PHYE: U78070
PHYE: AF004795
PHYE: U78848
PHYB: L34833
PHYB: U78832
PHYB: U78067
PHYA: L21154
43
APPENDIX B
44
Nucleic acid alignments o f PHY sequences showing positions o f insertion and deletion
events. Presumptive gene family is indicated by “A”, “B ”, “E” and “A l” following the
name o f each taxa. Primer sites (positions 1-36 and 616-642) are excluded from the
alignments.
37 40
B row n ea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a f f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k i i A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M il le t t ia r i ch ard i ana A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il l e t t i a d ura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a lc a t a E
G l y c i n e m ax B
M yrosp erm u m so u s a n u m B
M u n d u le a s e r i c e a B
50
SO
70
GATCACGGGG AAGTAATTGC TGAGATCACA AAGCCAGACA
GATCACGGGG AGGTGATTGC TGAGATAGCA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCACGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AAGTAATTGC TGAGATCACA AAGCCAGGTT
GATCATGGGG AAGTAATTGC TGAGATCACA AAGCCAGGTT
GATCATGGGG AGGTGGTCGC TGAGATCACA AAGTCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGAG AGGTGATTCG TGAGATAACA AAGCCCTGTC
GATCATGGGG AGGTGATTGC TGAGGTCAAA AGGCCTGGCC
GATCACGGGG AGGTGATTGC GGAGATAACA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCGGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGAC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AGGTGATTGC TGAGATAACA AAGCCAGGCC
GATCATGGGG AAGTGATTGC TGAGGTCAAA AAGCCAGGCC
GAACATGGAG AAGTGGTATC TGAGGTAAAA AAGCCAGGCT
GATCATGGGG AAGTGATTGC TGAGGTCACA AAGCCAGGCC
GATCATGGAG AAGTGATTGC TGAGGTCAAA AAGCCTGGCC
GATCATGGAG AAGTGATTGC TGAGGTCAAA AAGCCTGGCC
GATCATGGGG AGGTGATTGC TGAGCTCAAA AAGCCAGGCC
ATCATGGGGA AGGTGATTGC TGAGATAAAA AAGCCAGGCC
GATCATGGGG AAGTGGTTGC TGAGGTCAAA AAGCCAGGCC
GATCATGGAA GAGTGATTGC TGAGGTCAAA AAGCCTGGCC
GATCATGGGG AAGTTGTGTC TGAAATCAGA AGGTCAGATT
GATCATGGTG AGGTTGTTTC TGAGATTAGG AGGTCAGATT
GATCATGGTG AGGTTGTATC TGAGATTAGA AGGTCAGATT
GATCATGGTG AAGTTGTATC TGAAATCAGA AGGTCAGATT
GATCATGGTG AGATTGTATC TGAGATTAGA AGGTCGGATT
GATCATGGTG AGGTTGTATC CGAGATTAGA AGGTCGGATT
GATCATGGTG AGGTTGTATC TGAGATTAGG AGGTCAGATT
GATCATGGTG AGGTTGTATC CGAGATTAGG AGGTCAGATT
GATGATGGTG AGGTTGTATC TGAGATTAGG AGGTCGGATT
GATCATGGTG AGGTTGTATC TGAGATTAGG AGGTCAGATT
GAGCATGGTG AGGTTGTATC TGAGATTAGA AGGTCAGATT
GATCATGGTG AGGTTATAGC TGAGATTAGG AGGTCGGATT
GATCATGGTG AGGTTGTATC TGAGATTAGG AGGTCAGATT
GAGCATGGAG AGGTTGTTTC TGAGAGTAAG AGGCCTGATT
GAGCATGGTG AGGTTGTTGC TGAGAGTAAG AGGCCAGACT
? ? ? ? ? ? ? ? T G AGGTTGTTGC TGAGAGTAAG AGGCCTGATT
I 1111111112 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4
1234567890 1234567890 1234567890 1234567890
Numbering across the top o f the alignments indicates position relative to the alignments found in
Lavin et al. (1998; Appendix B). Numbering across the bottom marks the position within the target
region used for the analyses presented here.
45
Appendix B. Continued.
77 80
B r o w n ea s p . A
P isu m s a t i v u m A
C y c lo lo b i u m n u tarn s A
D e r r is e l l i p t i c a A
G y m n o cIa d u s d i o i c a A
E n t e r o lo b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
Sophora a f f i n i s A
M ille t t ia d ura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a lin a A l
O s tr y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e Al
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M ille t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s t e e n s ia b la c k i i E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
TGGAACCTTA
TAGAGCCATA
TAGAGCCATA
TTGAGCCATA
TAGAGCCATA
TAGAGCCATA
TAGAGCCATA
TAGAGCCTTA
TTGAGCCATA
TTGAGCCATA
TAGAGCCTTA
TTGAGCCGTA
TTGAGCCATA
TTGAGCCATA
TTGAGCCATA
TTGAGCCATA
TTGAGCAATA
TTGAGCCATA
TAGAGCCTTA
TAGAGTCATA
TAGAGCCATA
TAGAGCCTTA
TAGAGCCTTA
TAGAGCCATA
TAGAGCCATA
TAGAGCCATA
TAGAGCCTTA
TAGAACCCTT
TAGAGCCCTT
TAGAGCCTTA
TAGAGCCCTA
TGGAGCCATA
TGGAGCCATA
TGGAGCCATA
TGGAGCCATA
TGGAGCAGTG
TGGAGCCTTA
TGGAGCCTTA
TGGAGCCATA
TAGAGCCTTA
TGGAGCCTTA
TGGAGCCCTA
TGGAACCCTA
4444444445
1234567890
90
TCTG- --GGT
TCTA- --GGT
TTTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTT- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTA- --GGT
TCTG- --GGT
TATG- --GGT
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TATG- --GGC
TCTT- --GGT
TCTG- --GGT
TCTG- --GGT
TCTT- --GGC
TCTG- --GGT
TCTG- --GGT
TCTG- --GGT
TTTC- --GGT
ACTTCCGGTT
CTTG- --GGT
TTTG- --GGT
TTTG- --GGT
TTTG- --GGT
TTTG- --GGC
TTTG- --GGT
TTTG- --GGT
CTTG- --GGT
CTTG- --GGT
TTTG- --GGT
CTTG- --GGT
CATT- --GGA
CATC- --GGT
TATT- --GGT
5555555556
1234567890
100
TTGCATTATC
CTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCATTACC
TTGCATTACC
TTGCACTATC
TTACACTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCATTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTACACTATC
TTGCACTATC
TTGCATTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCACTATC
TTGCACTACC
TTGCACTACC
TTGCACTATC
TTGCATTACC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCATTATC
TTGCACTATC
TTGCATTATC
6666666667
1234567890
HO
CAGCCACTGA
CGGCGACAGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACCGA
CGGCCACTGA
CTGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CAGCCACTGA
CTGCCACTGA
CTGCCACGGA
CTTCCACAGA
CTGCCACTGA
CTGCCACAGA
CTGCCACAGA
CGGCCACAGA
CTGCCACAGA
CTGCCACTGA
CTGCCACAGA
CTGCCACTGA
CTGCCACAGA
CTGCCAAGGA
CTGCTACTGA
CTGCCACTGA
CTGCTACCGA
7777777778
1234567890
46
Appendix B Continued.
117 120
B row nea s p . A
P is u m s a t i v u m A
C y c X o lo b iu r a n u t a n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n te r o lo b iu m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a f f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s t e e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M i l l e t t i a dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosp erm u m so u s a n u m B
M u n d u le a s e r i c e a B
TATTCCACAG
TATTCCCCAG
TATTCCCCAG
TATTCCCCAG
CATTCCACAG
CATTCCACAG
TATTCCCCAG
TATCCCCCAG
TATTCCCCAG
CATTCCCCAG
TATTCCCCAG
TATTCCCCAG
TATTCCCCAG
TATTCCCCAG
TATTCCCCAG
TATTCCCCAG.
TATTCCTCAG
TATTCCCCAG
TGTTCCCCAG
TGTTCCACAG
TATTCCCCAG
TATTCCTCAG
TATTCCTCAG
TATCCCCCAG
TATTCCCCAG
TATTCCCCAG
TATTCCTCAG
TATCCCTCAA
TATTCCTCAA
TATCCCTCAA
TATCCCTCAA
TATCCCTCAA
TATCCCTCAG
TATCCCTCAA
TATTCCTCAA
TATCCCTCAA
TATCCCTCAG
TATCCCTCAA
TATCCCTCAA
TATCCCTCAA
TATTCCTCAG
TATTCCCCAG
TATTCCTCAA
130
GCTGCACGCT
GCTGCGCGGT
GCTGCACGGT
GCTGCACGCT
GCTGCACGTT
GCTGCACGTT
GCTGCACGTT
GCTGCAAGGT
GCTGCACGGT
GCTTCACGCT
GCTGCGCGCT
GCTTCACGCT
GCTGCACGGT
GCTGCACGTT
GCTGCACGCT
GCTGCACTGT
GCTGCACGCT
GCTGCACGCT
GCTGCACGCT
GCTACGAGAT
GCTACACGCT
GCTACGCGCT
GCTACGCGCT
GCTACACGCT
GCTACACGCT
GCTACACGCT
GCTACGCGCT
GCTGCTCGCT
GCTGCTCGTT
GCTGCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGCT
GCTTCTCGAT
GCTTCTCGCT
GCGGCTCGCT
GCTTCTAGGT
GCTTCAAGGT
GCTTCTAGGT
140
TTTTATTCAT
TTCTATTTAT
TTTTATTTAT
TTTTATTTAT
TTCTATTTAT
TTCTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTCAT
TTTTATTTAG
TTTTATTTAT
TCTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTTAT
TTTTATTCAT
TTTTATTAAT
TTTTATTTAT
TTTTATTTAT
TTTTGTTTAT
TTTTATTTAT
TTTCATTTAT
TTTTATTTAT
TCTTGTTCAA
TCTTGTTCCA
TCTTGTTCAA
TCTTATTTAA
TCTTGTTCAA
TCTTGTTCAA
TCTTGTTCAA
TCTTGTTCAG
TCTTGTTCAA
TTTTATTCAA
TATTGTTCAA
TCTTGTTCAA
TCTTGTTCAA
TTTTGTTTAA
TTTTGTTCAA
TTTTGTTTAA
I 1111111111
8888888889 9999999990 0000000001
1234567890 1234567890 1234567890
ISO
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAATAAG
GAAGAATAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAACAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAA
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GAAGAACAAG
GCAGAATAGA
GCAAAACCGG
GCAAAACCGG
GCAGAATCGG
GCAAAACCGT
GCAAAACCGT
GCAAAACCGT
GCAAAACCGC
GCAAAACCGT
GCAAAACCGG
GCAAAACCGG
GCAAAACCGC
GCAAAACCGG
GCAAAATAGA
GCAGAACCGT
GCAGAATAGG
1111111111
1111111112
1234567890
47
Appendix B Continued.
157 160
B row n ea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
S ophora a f f i n i s A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s t e e n s ia b la c k i i A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O str y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia d ura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k i i E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
170
180
190
GTCCGCATAA TTGTTGATTG TCGTGCAAAA CACGTGAAGG
GTCCGTATGA TAGTTGATTG TAATGCAAAA CATGTGAAGG
GTCCGTATGA TAGTTGATTG TCATGCAAAA CATGTGAAGG
GTCCGCATGA TAGTTGATTG TCATGCAAAA CACATGAAGG
GTGCGTATGA TAGTAGATTG TCATGCAAGG CATGTGAAGG
GTGCGTATGA TAGTAGATTG TCATGCAAGG CATGTGAAGG
GTCCGCATGA TAGTTGATTG TCATGCAAAA CATGTGAAGG
GTTCGTATGA TAGTTGACTG TCATGCAAAA CATGTGAAGG
GTCCGCATGA TAGTTGATTG TCATGCAAAA CATGTGAAGG
GTTCGTATGA TAGTTGACTG TCATGCAAAA CACGTGAGGG
GTCCGTATGA TAGTTGATTG TCATGCAAAA CACGTGAAGG
GTCCGTATGA TAGTTGATTG TCATGCAAAA CATGTGAAGG
GTCCGCATGA TAGTTGATTG TCATGCAAAA CACGTGAAAG
GTCCGTATGA TAGTTGATTG TCATGCAAAA CACGTGAAGG
GTCCGTATGA TAGTTGATTG TCATGCCAAA CACGTGAAGG
TACCGCATGA TAGTTGATTG TCATGCAAAA CATGTGAAGG
GTCCGTATGA TAGTTGATTG TCATGCAAAG CATGTGAAGG
GTTCGTATGA TAGTTGATTG TCGCGCAAAG CATGTGAATG
GTCCGTATAA TAGTTGATTG TAGTGCAAAG CGTGTAAAGG
GTTCGTATGA TAGTTGATTG TCGGGCAAAA CACGTGAAGG
GTCCGGTTAA TAGTTGATTG TTGCGCAAAG CATGTGAAGG
GTTCGTATGA TAGTTGATTG TCGCGCAAAG CATGTGAATG
GTTCGTATGA TAGTTGATTG TCGCGCAAAG CATGTGAATG
GTCCGTATGA TAGTTGATTG TCGCGCAAAG CATGTGAAGG
GTCCGTCAGA TAGTTGATTG CCGTGCAAAG CATGTGAAGG
GTCCGCATGA TAGTTGATTG TCATGCAAAG CATGTGAAGG
GTTCGTATGA TAGTTGATTG TCGCGCAAAG CATGTGAAGG
GTCAGGATGA TCTGTGATTG CCATGCAAAC CCAGTTAAGG
GTCAGGATGA TTTGTGATTG CCATGCAAAG CCTGTTAATG
GTAAGGATGA TTTGTGATTG CCATGCAAAG CCAGTTAAGG
GTCAGACTGA TCTGTGATTG CCATGCAAAT CCAGTAAGGG
GTCAGGATGA TTTGTGATTG CCATGCAAAG CCGGTTAAGG
GTCAGGATGA TTTGTGATTG TGGTGCAAAG CCGGTTAAGG
GTAAGGATGA TTTGTGATTG CCATGCAAAG CCGGTTAAGG
GTCAGGATGA TTTGCGATTG CCATGCAAAG CCAGTTAAGG
GTCAGGATGA TTTGTGATTG CCATGCAAAG CCAGTTAAGG
GTCAGGATGA TATGTGATTG CCATGCAAAG CCAGTTAAGG
GTCAGGATGA TTTGTGATTG CGATGCAAAG CCAGTTAAGG
GTCAGGATGA TTTGTGATTG CCATGCAAAG CCAGTTAAGG
GTCAGGATGA TTTTTGATTG CCATGCAAAG CCTGTGAATG
GTTAGGATGA TTGTGGATTG TCATGCTTCT GCTGTGAGGG
GTTAGGATGA TTGTGGATTG CCATGCTTCT CCGGTTGGGG
GTTAGAATGA TTGTGGATTG TCATGCTTCA CCAGTGAGGG
1111111111 1111111111 1111111111 1111111111
2222222223 3333333334 4444444445 5555555556
1234567890 1234567890 1234567890 1234567890
48
Appendix B Continued.
197 200
B row nea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m e y e lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a f f i n i s A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k i i E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a lc a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
210
220
230
TGCTTCAAGA TGAGAAACTT CCGTTTGATC TGACCTTGTG
TTCTTCAAGA CGAAAAACTC CCATTTGATT TGACTCTGTG
TTCTTCAAGA TGAAAAACTC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAAATC CCATTTGATT TGACTTTGTG
TGCTTCAAGA TGAAAAACTT CCATTTGAGC TAACCTTGTG
TGCTTCAAGA TGAAAAACTT CCATTTGAGC TAACCTTGTG
TTCTTCAAGA TGAAAAACTC CCATTTGATT TAACTCTGTG
TTCTTCAAGA TGAGAAACTC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAAATC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTC CAATTTGATT TGATTTTGTG
TTTTTCAAGA CGAAAAGCTC CCAATTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTG CCATTCGATT TGACATTGTG
TTCTTCAAGA TGAAAAAACC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTC CCACTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTC CCATTTGATT TGACTTTGTG
TTCTTCAAGA TGAAAAACTC CTGTTTGATT TGACTTTGTG
TTCTTCAAGA CAAAAAGGTT CCATTTGATT TAACTTTGTG
TGATTCAAGA CAAAAATATT CCATTTGATT TAACTTTGTG
TGCTTCAAGA TGAAAATCTT CCATATGATT TAACCTTCTG
TGCTTCAAGA CAAGAAGATT CCATTTGAGT TAACTTTGTG
TTCTTCAAGA CAAAAAGGTT CCATTTGATT TAACTTTGTG
TTCTTCAAGA CAAAAAGGTT CCATTTGATT TAACTTTGTG
TGCTTCAAGA CAAAAAAATC CCGTTTGATT TAACTCTATG
TGCTTCAAGA CAAAAACATT CCATTTGATT TAACTTTGTG
TGCTTCAAGA CAAAAAAATT CCATTTGATT TGACTTTGTG
TTCTTCAGGA CAAAAAGGTT CCATTTGATT TAACTTTGTG
TGATTCAAAG TGAAGAGTCA AGGCAGCCTC TTTGCTTGGT
TCATTCAGAG TGAAGAATTA AGGCAACCTC TTTGCTTAGT
TCATTCAGAG TGAAGAATTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAAGAATTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAGGAGTTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAGGAGTTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAGGAGTTA AGGCAACCTC TTTGCTTGGT
TAATTCAGAG TGAGGAGTTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAAGAGTTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAAGAATTA AGGCAACCTC TTTGCTTGGT
TCATTCAGAG TGAAGAATTA AGGCAACCTC TTTGCTTGGT
TCATCCAGAG CGAGGAGTTA AGGCAATCTC TTTGCTTGGT
TCATTCAGAG TGAAGAATTA AGACAACCTC TTTGCTTGGT
TGGTGCAGGA TGAGGCTCTT GTGCAGCCTT TGTGTTTGGT
TAATTCAGGA TGAAGGGCTT ATGCAGCCTT TGTGCTTGGT
TGGTTCAGGA TGAGGCTCTT GTGCAGCCTC TGTGTTTGGT
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1111111111 1111111112
6G666S6GG7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0
1234567890 1234567890 1234567890 1234567890
49
Appendix B Continued.
237 240
B r o w n ea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n te r o lo b iu m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
Sophora a E f in is A
M il le t t ia dura A
G l y c i n e m ax A
M ille t t ia g r a n d !s A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s t e e n s ia b la c k i i A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n te r o lo b iu m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s t e e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n te r o lo b iu m c y c lo c a r p u m E
Sophora a E f in is E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M ille t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n t h e E a lc a ta E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
250
CGGTTCAACC TTACGGGATC
CGGTTCGACC TTGAGAGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCGACC TTAAGGGCTC
CGGTTCAACC TTGAGGGCCC
CGGTTCAACC TTGAGGGCCC
TGGTTCAACC TTAAGGGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCGACC TTAAGGGCTC
TGGTTCCACC TTAAGAGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCGACC TTAAGGGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCAACC TTAAGGGCTC
TGGTTCCACG TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGTTCCACC TTGAGGGCCC
TGGTTCGACC TTACGGGCCC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
TGGATCAACC TTAAGGGCTC
CAATTCTACC CTTAGGTCAC
CAACTCAACC CTGAGGTCAC
TAACTCAACC CTTAGGTCAC
CATTTCAACC CTTCGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACC CTTAGGTCAC
GAACTCAACA CTTAGGTCAC
CAACTCAACC CTGAGGTCGC
TGGGTCCACC TTAGGGGCTC
TGGATCAACC CTGCGCGCAC
AGGGTCCACG CTT???GCTC
2222222222 2222222222
0000000001 1111111112
1234567890 1234567890
260
270
CTCATAGTTG CCATTTGCAA
CACATAGTTG CCATTTGCAG
CTCATAGTTG CCATTTGCAA
CCCATAGTTG CCATTTGCAA
CTCATACTTG CCATTTGCAA
CTCATACTTG CCATTTGCAA
CTCATAGCTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CCCATAGTTG CCATTTGCAA
CTCATAGTTG CCACGCGCAG
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CCCATAGTTG CCATTTGCAA
CTCATAGTTG CCACTTGCAA
CTCATAGTTG CCACTTGCAA
CCCATAGTTG CCATTTGCAA
CTCATAGTTG CCACTTGCAA
CTCATAGTTG CCATTTGCAA
CTCATAATTG CCATTTGCAA
CTCATAGTTG CCATGTGCAA
CTCATAGTTG CCATATACAA
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CTCATAGTTG CCATTTGCAA
CACATCAATG TCATGCACAG
CACTTGGTTG TCACACACAG
CACATGATTG TCACACACAG
CCCATGGATG TCACACACAG
CACATGTTTG TCACACACAG
CACATGTTTG TCACACACAG
CACATGTTTG TCACACACAG
CACATGTTTG TCACACACAG
CACATGTTTG TCACACACAG
CACATGGTTG TCACACACAG
CACATGGTTG TCACACACAG
CACATGTTTG TCACACACAG
CACATGGTTG TCACACACAA
CTCACGGTTG TCATGCTCAG
CACATGGTTG TCATGCCCAG
CGCACGGTTG TCATGCTCAG
2222222222 2222222222
2222222223 3333333334
1234567890 1234567890
50
Appendix B. Continued.
277 280
B row nea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a E f in is A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d !s A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a ta A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o lo b i u m c y c lo c a r p u m A l
C y c lo lo b i u m n u t a n s A l
P o n g a m io p s is a m y g d a l in a A l
O s tr y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
S ophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n t h e E a lc a ta E
G l y c i n e m ax B
M yrosperm um so u s a n u m B
M u n d u le a s e r i c e a B
290
TATATGGAAA ACATGAATTC
TACATGGCTA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCAA ACATGGATTC
TATATGGAGA ACATGAATTC
TATATGGAGA ACATGAATTC
TACATGGCGA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCTA ACATGGATTC
TACATGGCGA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCAA ACATGAATTC
TACATGGCGA ACATGGATTC
TACATGGCAA ACATGGATTC
TACATGGCGA ACATGGATTC
TACATGGAGA ACATGAATTG
TACATGGATA ACATGAAAGC
TATATGCAGA ACATGGATCT
TACATGTTGA ACATGAATTC
TACATGGAGA ACATGAATTG
TACATGGAGA ACATGAATTG
TACATGGAGA ACATGAATTC
TACATGAAGA ACATGAATTC
TACATGGAGA ACATGAATTC
TACATGGAGA ACATGAATTG
TACATGGAAA ACATGGGCTC
TACATGGCTA ACATGGGCTC
TATATGGCTA ATATGGGCTC
TACATGGCAA ACATGGGCTC
TATATGGCTA ACATGGGCTC
TATATGGCTA ACATGGGCTC
TATATGGCTA ACATGGGCTC
TATATGAGTA ATATGGGCTC
TATATGGCTA ACATGGGCTC
TACATGGCTA ACATGGGCTC
TACATGGCTA ACATGGGCTC
TATATGGCTA ATATGGGCTC
TACATGGCTA ACATGGGCTC
TATATGGCTA ACATGGGCTC
TATATGGCCA ATATGGGCTC
TACATGGCTA ATATGGGGTC
2222222222 2222222222
4444444445 5555555556
1234567890 1234567890
300
AGTTGCTTCC
AATTGCTTCG
AATTGCTTCC
AATTGCTTCT
CATTGCTTCC
CATTGCTTCC
AATTGCTTCC
AATTGCTTCC
AATTGCTTCT
AATTGCTTCC
AATTGCTTCT
AATTGCTTCT
AATTGCTTCT
AATTGCTTCC
CATTGCTTCC
AATTGCTTCT
AATTGCTTCC
TAGTGCTTCC
AAGTGCTTCC
AGTTGCATCC
AATTGCTTCC
TAGTGCTTCC
TAGTGCTTCC
TAGTGCTTCC
AAGTGCTTCC
TAGTGCTTCC
TAGTGCTTCC
AATTGCTTCT
AATTGCCTCT
AATTGCCTCT
AATTGCCTCT
AATTGCCTCT
AACTGCCTCT
CATTGCCTCT
AATTGCGTCT
AATTGCCTCT
AATTGCCTCT
AATTGCCTCT
AATTGCCTCT
AGTTGCCTCT
GATTGCGTCT
AATTGCTTCA
TATTGCGTCG
2222222222
6666666667
1234567890
310
TTGGTTATGG
TTGGTTATGG
CTGGTTATGG
CTGGTTATGG
CTGGTTATGG
CTGGTTATGG
CTGGTTCTGG
CTGGTCATGG
CTGGTTATGG
CTGGTTTTGG
CTGGTTATGG
CTGGTTATGG
CTGGTTATGG
CTGGTTCTGG
CTCGTTATGG
CTGGTTATGG
CTGGTTATGG
TTGGTTATGG
CTGGTTATGG
CTGGTTATGG
TTGGTTATGG
TTGGTTATGG
TTGGTTATGG
TTGGTTATGG
CTGGTAATGG
TTGGTTATGG
TTGGTTATGG
CTGGTTATGG
CTGGTGATGG
CTGGTGATGG
CTGGTGATGG
CTGGTTATGG
CTGGTTCTGG
CTGGTTATGG
CTGGTTATGG
CTGGTGATGG
CTGGTGATGG
CTGGTGATGG
CTGGTGATGG
CTGGTGATGG
TTGGTGATGG
TTGGTAATGG
TTGGTCATGG
2222222222
7777777778
1234567890
51
Appendix B Continued.
317 320
B row n ea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a f f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L on ch ocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia d ura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s t e e n s ia b la c k i i E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a lc a t a E
G l y c i n e m ax B
M yrosperm um so u s a n u m B
M u n d u le a s e r i c e a B
CTGTTGTGGT
CAGTTGTCGT
CAGTTGTTGT
CAGTTGTAGT
CAGTGGTGGT
CAGTGGTGGT
CAGTTGTGGT
CAGTTGTAGT
CAGTTGTAGT
CAGTTGTAGT
CGGTTGTAGT
CAGTTGTTGT
CAGTTGTAGT
CAGTTGTAGT
CAGTTGTAGT
CAGTTGTAGT
CAGTTGTAGT
CAGTTGTGGT
CAGTTGTGGT
CAGTTGTGAT
CAGTTGTGGT
CAGTTGTGGT
CAGTTGTGGT
CTGTTGTGGT
CAGTTGTAGT
CAGTCGTGGT
CAGTTGTGGT
CTGTTATTGT
CAGTTATAGT
CAGTTATAGT
CAGTTATAGT
CAATTATAGT
CAATTATAGT
CAATTATAGT
CAATTATAGT
CAATTATAGT
CAGTTACAGT
CAGTTATAAT
CAATTATAGT
CAGTTATAGT
CAGTTATTAT
CAGTCATAAT
CAGTTATTAT
2222222222
8888888889
1234567890
330
CAATGACAAC
CAATGACAGC
CAATGACAGT
TAATGACAAC
CAATGACAAT
CAATGACAAT
CAATGACAGT
CAATGACAGC
TAATGACAAC
CAATGACAAC
CAATGACAAC
CAATGACAAC
TAATGACAAC
CAATGACAAC
CAATGACAAC
TAATGACAAC
CAATGACAAT
CAATGACAAT
CAATGACAGC
CAATGACAGA
AAATGATAGT
CAATGACAAT
CAATGACAAT
CAATGACAAC
CAATGACAAT
CAATGACAAT
CAATGACAAT
CAATGGGAAT
CAATGGAAAT
CAATGGAAAT
CAATGGAACT
CAACGGAAAT
CAATGGAAAT
CAATGGAAAT
CAATGGAAAT
CAATGGAAAT
CAATGGAAAT
TAATGGAAAT
CAACGGAAAT
GAATGGAAAT
CAATGGGAAT
CAATGGGAAT
TAATGGGAAT
2222222223
9999999990
1234567890
340
GAT----------- G
GAT----------- G
GAT----------- G
GAA----------- G
GAT----------- G
GAT----------- G
GAT----------- G
GAT----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAA----------- G
GAT----------- G
AAT----------- G
GAC----------- G
GAT----------- G
GAT----------- G
GAT------------G
GAT----------- G
AAT----------- G
GAT----------- G
GAA----------- G
GATACCACAGATACAACAGATTCAACAGATACAACTGATACAACAGATACAACAGATAAAACAGATAAAAGAGATACAACAGATACAAGAGATACAACAGATACAACAGACACAACGGAC----------- G
GAT----------- G
GAT----------- G
3333333333
0000000001
1234567890
350
AAGATGGGGA
AAGATGGAGA
AAGATGGGAA
AAGATGGGGA
AAGATGTGGA
AAGATGTGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGGA
AAGATGGGAG
AAGATGGGGA
AAGATGGAGA
AAGATGGTGA
AAGATGGAGA
AAGATGGGGA
AAGATGGAGA
AAGATGGAGA
AAGATGGAGA
AAGATGGGGA
AAGATGGGGA
AAGATGGAGA
AGGAAGGCGT
AAGATACTGT
AGGAAGGTGT
3333333333
1111111112
1234567890
52
Appendix B. Continued.
357 360
B row nea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o lo b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
Sophora a f f i n i s A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a ta A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a l in a A l
O s tr y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il l e t t i a dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
CAGCTCTGAT
TAGCGCTGAC
TAGCTCTGAT
TAGCTCTGAT
CAGCTCTGAT
CAGCTCTGAT
TAGCTCTGAT
TAGCTCTGAT
TAGCTCTGAT
C-----ACTGAT
TAGTTCTGAT
TAGCTCTGAT
TAGCTCTGAT
TAGCTCTGAT
TAGCTCTGAT
TAGTTCTGAT
TAGCTCTGAT
TAGT-----GAT
TAGCTCTGAT
CAGCTCTGAT
TAGCTCTGAT
TAGT-----GAT
TAGT-----GAT
TAGCTCCGAT
CAGCTCGGAT
TAGCTCTGAT
TAGT-----GAT
370
TCT-----GTGC
GCA-----GTTC
GCT-----GTTC
GCT-----GTTC
TCT-----ATCC
TCT-----ATCC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT----- GTTC
TCT-----GTAC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----GTTC
GCT-----TTTC
GCT-----ATTC
GCT-----GTTC
380
AGCCACAAAA
TCCCACAAAA
AGCCACAAAA
AGCCGCAAAA
AGCCACAGAA
AGCCACAGAA
AACCTCAAAA
AGCCACAAAA
AGCCGCAAAA
AGCCACAAAA
AGCCACAAAA
AGCCTCAAAA
AGCCGCAAAA
AGCCACAAAA
AGCCACAAAA
AGCCGCAAAA
AGCCACAAAA
AGCCACAGAA
AACCACAAAA
AGCCACAGAA
AGCCACAAAA
AGCCACAGAA
AGCCACAGAA
AGCCACAAAA
AGCCACAGAA
AGCCACAAAA
AGCCACAGAA
CGCAG
TGGTGGT----- ---------------------CGTAG
TTGCAGT----- ---------------------AGGAG
TGGGGGA----- ---------------------3333333333 3333333333 3333333333
2222222223 3333333334 4444444445
1234567890 1234567890 1234567890
390
GAGGAAGAGA
GAAAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GACGGAGAGA
GAGAAAGAGA
GAGGAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAAAGG
GAGAAAGAGG
GAGAAAGAGG
GAGAAAGAGA
GAGAAAAAGA
GAGAAAAAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAGAGA
GAGAAAAAGA
............--AAG
------------- AGG
------------- AGG
------------- AAG
------------- AGA
------------- AGA
------------- AGA
------------- AGA
------------- AGG
------------- AGG
------------- AGG
------------- AGG
------------- AGG
TTCGATGAGG
CTCAATGAGG
TTCGATGCGG
3333333333
5555555556
1234567890
53
Appendix B Continued.
397 400
B r o w n ea s p . A
P is u m s a t i v u m A
C y c lo lo b i u m n u t a n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
Sophora a f f i n i s A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a ta A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o lo b i u m c y c lo c a r p u m A l
C y c lo lo b i u m n u t a n s A l
P o n g a m io p s is a m y g d a lin a A l
O s tr y o c a z p u s s t u b lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il l e t t i a dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um so u s a n u m B
M u n d u le a s e r i c e a B
CTCTGGGGTT
CTTTGGGGTT
CTCTGGGGTT
CTATGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTATGGGGTT
CTCTGGGGTT
CTATGGGGTT
CTTTGGGGTT
CTATGGGGTT
CTATGGGGTC
CTATGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTATGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTCTGGGGCT
CTCTGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTCTGGGGTT
CTTTGGGGTT
CTCTGGGGTT
CTATGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTCTGGGGTT
CTTTGGGGTT
CTTTGGGGTT
CTTTGGGGCT
CTGTGGGGGC
CTATGGGGCC
CTGTGGGGGC
3333333333
6666666667
1234567890
410
TAGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TGGTAGTTTG
TAGTAGTTTG
TAGTTGTTTG
TAGTAGTTTG
TGGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TAGTAGTTTG
TGCTTGTTTG
TGCTTGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTAGTTTG
TGCTGGTTTG
TGCTAGTTTG
TGCTAGTTTG
TTGTTGTCTG
TGGTTGTTTG
TTGTTGTTTG
3333333333
7777777778
1234567890
420
CCACAACACT
TCATAACACT
CCATAACACT
CCATAACACT
TCATAATACC
TCATAATACC
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACC
CCATAACACT
CCATAACACT
CCATAACACT
CCATAACACT
CCATCATATT
CCATAACTAT
CCATAACACT
CCATAACACT
CCATAACACT
CCATCACACT
CCATCACACT
CCATCACACT
CCATAACTCT
TCACCACACT
TCATCATACT
TCATCACTCT
CCATCACACT
TCATCACACT
TCATCACACT
TCATCACACT
TCATCACACT
TCATCACACT
TCATCATACT
TCATCACACT
TCATCACACT
TCATCACACT
CCACCATACC
CCATCACACT
CCATCATACT
3333333333
8888888889
1234567890
430
ACTCCCAGGT
ACTCCAAGGT
ACTCCCAGGT
ACTCCCAGGT
ACACCAAGGT
ACACCAAGGT
ACTCCTAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
AGTCCAAGGT
ACTCCCAGGT
ACTCCCAGAT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACCCCCAAGT
ACCCCTCGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ACTCCCAGGT
ATTCCCAGGT
TCGCCGCGTC
TCTCCCCGCT
TCACCTCGGT
TCACCACGCC
TCGCGC???T
TCACCACGCT
TCACCGCGCT
TCACTGCGCT
TCACCGCGCT
TCACCTCGCT
TCACCCCGTT
TCACCGCGCT
TCACCGCGCT
TCTGCCAGGT
TCTGCTCGAT
TCAGCTAGAT
3333333334
9999999990
1234567890
54
Appendix B Continued.
437 440
B ro w n ea s p . A
P isu m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o lo b i u m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sophora a f f i n i s A
M il le t t ia d ura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia d ura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k i i E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
450
460
470
TTGTTCCATT TCCTCTTAGG TATGCTTGTG AATTTCTGGT
TTGTTCCTTT TCCTCTAAGG TATGCTTGTG AGTTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCTTT TCCTCTTCGG TATGCTTGTG AATTTCTGGC
TTGTTCCTTT TCCTCTTCGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTTTGGC
TTGTTCCCTT TCCTCTAAGG TATGCAAGAG AATTTCTGCC
TTGTCCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCACTGAGG TATGC CTGTG AATTTCTCGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTACCCTT TCCTCTAAGG TATGCTTGTG AATTTCTAGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGTC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCTTT TCCACTTAGG TATGCCTGTG AATTCCTTGC
TTGTTCCCTT TCCTCTTAGA TATGCTTGTG AATTTCTGGC
TTGTTCCTTT TCCTCTTAGG TATGCATGTG AGTTCCTGGC
TTGTTCCCTT TCCTCTAAGG TATGCTTGTG AATTTCTGGC
TTGTTCCTTT TCCACTTAGG TATGCCTGTG AATTCCTTGC
TTGTTCCTTT TCCACTTAGG TATGCCTGTG AATTCCTTGC
TTGTTCCCTT TCCTCTGAGG TATGCCTGTG AATTCCTGGC
TTGTTCCTTT CCCTCTTAGG TATGCTTGTG AATTCCTGGC
TTGTTCCTTT CCCTCTTAGG TATGCTTGTG AATTCCTGGC
TTGTTCCCTT TCCTCTTAGG TATGCCTGTG AATTCCTTGC
ATGTCCCTTT TCCACTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT TCCTGTACGC TATGCTTGTG AGTTCCTTAT
ATGTGCCTTT CCCAGTTCGT TATGCTTGTG AGTTCCTAAT
ATGTACCTTT CCCCCTTCGC TATGCTTGTG AGTTCCTCAT
ATGTGCCTTT CCCAGTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCAGTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCAGTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCAGTTCGC TATGCCTGTG AGTTCCTAAT
ATGTGCCTTT CCCGGTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCAGTTCGC TATGCTTGTG AGTTCCTTAT
ATGTGCCTTT CCCAGTCTGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCGGTTCGC TATGCTTGTG AGTTCCTAAT
ATGTGCCTTT CCCAGTTCGC TATGCTTGTG AGTTCCTTAT
GTATTCCTTT TCCCTTGAGG TATGCTTGTG AGTTTCTGAT
GCATTCCTTT CCCTCTACGC TATGCTTGTG AGTTCCTAAT
GTATTCCTTT CCCATTGAGG TATGCCTGTG AGTTCTTGAT
4444444444 4444444444 4 4 4 4 4 4 4 4 4 4 4444444444
0000000001 1111111112 2222222223 3333333334
1234567890 1234567890 1234567890 1234567890
55
Appendix B. Continued.
477 480
B r o w n ea s p . A
P isu m s a t i v u m A
C y c lo lo b i u m n u t a n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o lo b i u m c y c lo c a r p u m A
M yrosperm um so u s a n u m A
Sop h ora a £ f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o lo b i u m c y c lo c a r p u m A l
C y c lo lo b i u m n u t a n s A l
P o n g a m io p s is a m y g d a lin a A l
O s tr y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s t e e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o lo b i u m c y c lo c a r p u m E
S ophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M i l l e t t i a dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a lc a t a E
G l y c i n e m ax B
M yrosperm um so u s a n u m B
M u n d u le a s e r i c e a B
490
500
510
TCAGGTATTT GCCATCCATG TCAACAAAGA ATTGGAATTA
TCAAGTGTTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATTCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCAATCCATG TGAACAAAGA AATAGAATTA
TCAAGTATTT GCCATCCATG TGAATAAAGA ATTAGAGTTA
TCAAGTATTT GCCATCCATG TGAATAAAGA ATTAGAGTTA
CCAGGTATTT GCCATCCATG TGAACAAAGA ATTTGAGCTA
TCAAGTGTTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCAATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCGACCATG TGCACAAAGA AATAGAGTTA
TCAGGTATTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA AATTGAGTTA
TCAAGTATTT GCAATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTGTTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCAATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCACG TGAACAAAGA ACTAGAGTTA
ACAAGTATTT GCCATCCATG TTAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TTAACAAAGA AATAGAGTTG
TCAAGTATTT GCCATTCATG TGAACAAGGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA AATAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA ACTAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA GCTAGAGTTG
TCAAGTATTT GCCTACCTAG TGAACAAAGA ACTAGAGATA
TCAAGTATTT GCCATCCATG TGAACAAAGA GCTAGAGTTA
TCAAGTATTT GCCATCCATG TGAACAAAGA ACTAGAGTTA
GCAGGCTTTT GGATTGCAGC TTTATATGGA GATGCAATTG
GCAGGCTTTT GGACTGCAGC TTTACATGGA GATTCAATTG
GCAGGCTTTT GGACTGCAGC TTTACATGGA GATTCAATTG
GCAGGCTTTT GGACTGCAGC TGTACTCGGA CATGCAACTA
GCAGGCTTTT GGAGTGCAGC TTTACATGGA GATTCAGATG
GCAGGCTTTT GGATTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGATTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGACTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGATTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGACTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGACTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGATTGCAGC TTTACATGGA GATTCAGTTG
GCAGGCTTTT GGACTGCAGC TGTATATGGA GATTCAATTG
GCAGGCGTTT GGGCTGCAGT TGAACATGGA GCTTCAGTTG
GCAGGCTTTT GGGCTCCAAC TGAATATGGA ACTTCAGCTG
GCAGGCGTTT GGGCTGCAGT TGAATATGGA GCTTCAGCTG
4444444444
4 4 4 4 4 4 4 4 4 4 4444444444 4 4 4 4 4 4 4 4 4 4
4444444445 5555555556 6666666667 7777777778
1234567890 1234567890 1234567890 1234567890
56
Appendix B Continued.
517 520
B r o w n ea s p . A
P isu ra s a t i v u m A
C y c lo lo b i u r a n u t a n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m e y e lo c a r p u m A
M yrosperm um s o u s a n u m A
S ophora a f f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s tu h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L on ch ocarpus p h a s e d i f o l i u s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
530
540
550
GAATACCAGA TTGTTGAGAA GAACATTCTG CGCACTCAGA
GAATATCAGA TTCTTGAGAA GAATATCCTG CGCACGCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACTCAGA
GAAAACCAAA TTGTTGAGAA AAAGATCCTG CGCACCCAGA
GAAAACCAAA TTGTTGAGAA AAAGATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGC TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CACCACCCAG
GAATATCAGA TTATTGAGAA GAATATCCTT CGCACCCAGA
GAAAATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATACCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAAA
GAATATCAGA TAATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA CAATATCCTG CGCACCCAGA
GAATATCAGA TTGTTGAGAA GAATATCCTG CGGACTCAAA
GAATATCAGG TTATTGAGAG GAATATCCTG CGCACTCAAA
GAATATCAGA TTATTGAGAA GAACATAATG CGAACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTATTGAGAA GAATATCCTG CGCACCCAGA
GAATATCAGA TTGTTGAGAA GAATATCCTG CGGACTCAAA
GAATATCAGA TTGTTGAGAA GAATATCCTG CGGACTCAAA
GCCTATCAAA TTGTTGAGAA GAATATCCTG CGCACTCAAA
GAATATCAGA TTGTTGAGAA GAATATCCTG CGCACTCAAA
GAATATCAGA TTGTTGAGAA GAATATCCTG CGGACTCAAA
GCATCACAGA TGGCAGAGAA GAGAACTCTT AAAACCCAAA
GCATCACAGA TGGCTGAGAA AAGACTTCTT AAAACACAAA
GCATCACAAA TGGCAGAGAA GAGAATTCTT AAAACACAAA
GCATCACAAA TGGCAGAGAA GAGAATTCTC AGAACTCAAA
GCATCACAGA TGGCAGAGAA GAGAATGCTT AAAACACAAA
GCATCACAGA AGGCAGAGAA GAGAATGCTT AAAACACAAA
GCATCGCAGA TGGCAGAGAA GAGAATGCTT AAAACACAAA
GCATCACAGA TGGCAGAGAA GAGAATGCTT AAAACACAAA
GCATCACAGA TGGCAGAGAA GAAAACTCTT AAAACACAAA
GCATCACAAA TGGCAGAGAA GAGGATTCTT AAAACACAAA
GCATCGCAAA TGGCAGAGAA GAGAATTCTT AAAACACAAA
GCTTCACAGA TGGCAGAGAA GAGAATTCTT AAAACACAAA
GCAACACAAT TGGCAGAGAA GAGAATTCTT AAAACACAAA
GCCGCGCAGT CGTTGGAGAA GCGGGTTTTG AGGACACAGA
GCATCGCAGT CATTGGAGAA ACGAGTTTTA AAGACCCAGA
GCAGCTCAGG CGTTGGACAA ACGAGTTTTG AGGACACAGA
4444444444
4444444445 5555555555 5555555555
8888888889 9999999990 0000000001 1111111112
1234567890 1234567890 1234567890 1234567890
57
Appendix B Continued.
557 560
B row nea s p . A
P isu m s a t i v u m A
C y c lo lo b i u m n u t a n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
S ophora a f f i n i s A
M i l l e t t i a dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k i i A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k i i A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia d ura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um s o u s a n u m B
M u n d u le a s e r i c e a B
CACTCTTGTG
CGCTGTTGTG
CCTTGTTGTG
CACTCTTGTG
CACTTCTATG
CACTTCTATG
CACTCTTGTG
CACTATTGTG
CACTCTTGTG
GGCACCTCTT
CACTATTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CTCTCTTGTG
CACTCTTGTG
CCCTCTTGTG
GTCTCCTGTG
CCTTGTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CACTCTTGTG
CCTTACTATG
CCCTACTGTG
CCTTACTGTG
CCTTACTATG
C C ????????
CCTTGCTGTG
CCTTGCTGTG
CCTTGCTGTG
CCTTGTTGTG
CCTTACTGTG
CCTTGCTGTG
CCTTGCTGTG
CCTTACTTTG
CTCTGTTGTG
CTCTCTTGTG
CGTTGTTGTG
5555555555
2222222223
1234567890
570
TGATATGTTG
TGATATGTTG
TGACATGCTA
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTA
TGATATGCTG
GTGTATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGTTG
TGATATGTTG
TGACATGCTA
TGATATGCTG
TGATATGCTG
TGATATGCTG
TGATATGCTG
CGATATGCTG
TGATATGCTG
TGACATGCTT
TGACATGCTC
TGACATGCTC
TGACATGCTC
??????????
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGACATGCTC
TGATATGCTT
TGACATGCTT
TGACATGCTT
5555555535
3333333334
1234567890
580
ATGCGAGATG
ATGCGAGATG
ATGCGAAATG
ATGCGAGATG
ATGCGAGA? ?
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGAGATG
ATGAGAGATG
ATGCGAGATG
ATGCGAGATG
ATGCGTGATG
ATGCGAAATG
ATGCGAGATG
ATGCGAGATG
ATGCGGGATG
ATGCGAGATG
ATGAGAGATG
ATGCGAGATG
CTCCGTGATG
CTTCGTGATG
CTACGTGATG
CTCCGTGATT
??????????
CTCCGTGATG
CTGCGTGATG
CTGCGTGATG
CTGCGTGATG
CTGCGTGATG
CTGCGTGATG
TTGCGTGATG
CTACGTGATG
CTTAGGGACT
CTTCGGGACT
CTTAGGGACT
5555555555
4444444445
1234567890
590
CACCTCTAGG
CACCCTTAGG
CACCCTTAGG
CACCGCTAGG
??????????
CACCCCTAGG
CACCCCTAGG
CACCCTTAGG
CACCACTAGG
CACCCCTAGG
CACCACTAGG
CACCACTAGG
CACCCCTAGG
CACCTCTAGG
CACCCCTAGG
CACCACTAGG
CACCCCTAGG
CGCCACTAGG
CACCCCTAGG
AACCCCTAGG
CACCCTTAGG
CACCACTAGG
CGCCACTTGG
CACCACTAGG
CGCCCCTAGG
CACCCCTGGG
CGCCACTAGG
CTCTTTTTAG
CACCATTTGG
CACCATTTGG
CCCCATTTGG
??????????
CGCCTCTTGG
CACCTCTTGG
CGCCTCTTGG
CGCCATTTGG
CACCATTTGG
CGCCATTTGG
CGCCTCTTGG
CACCTTTTGG
CGCCTACTGG
CTCCTATTGG
CTCCTACTGG
5555555555
5555555556
1234567890
58
Appendix B Continued.
597 600
B row nea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n t e r o l o b i u m c y c lo c a r p u m A
M yrosperm um s o u s a n u m A
Sophora a f f i n i s A
M il le t t ia dura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a t a A
A u s tr o s t e e n s ia b la c k i i A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o l o b i u m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m i o p s i s a m y g d a l in a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lif o liu s A l
E n t e r o l o b i u m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M il le t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n yassae E
T e p h r o s ia v i l l o s a E
P o e c ila m th e f a l c a t a E
G l y c i n e m ax B
M yrosperm um so u s a n u m B
M u n d u le a s e r i c e a B
TATTGTATCA
TATTGTATCA
TATTGTATCA
AATTGTATCA
??????????
TATTATATCA
CATTGTATCA
TATTGTATCA
AATTGTATCA
AATTGCATCA
AATAGTATCA
AATTGTATCA
AATTGTATCA
AATTATATCA
AATTGTATCC
AATTGTATCA
AATTGTATCA
CATTGTAACA
TATTATATCA
GATTATATCA
TATTGTATCA
AATTGTATCA
CATTGTAACA
CATTGTATCT
TATTGTATCA
TATTGTATCA
CATTGTAACA
TATTGTTACA
CATTGTGACT
AATTGTGACT
TATTGTTACT
??????????
TATTGTGACT
TATTGTGACT
TATTGTGACT
TATCGTGACT
CATTGTGAGT
CATTGTGAAT
TATTGTGACT
CATTGTGAAT
CATTGTTACT
CATTGTTACT
CATTGTTACT
5555555555
6666666667
1234567890
610
CAGGGCCCA
CAAAGCCCT
CAGAGTCCT
CAGAGCCCT
?????????
CAGAGCCCA
CAGAGCCCT
CAGAGTCCT
CAGAGCCCT
GAGAGTCCT
CAGAGCCCT
CAGAGCCCA
CAGAGCCCT
CAGAGCCCT
CAGAGCCCT
CAGAGCCCT
CAGAGCCCT
CAGAGGCCT
CAGAGCCCT
CAGAGCCCA
CAGAGTCCT
CAGAGCCCT
CAGAGGCCT
CAGAGGGCC
CAGAGTCCT
CAGAGCCCT
CAGAGGCCT
CAATCTCCT
CAATCTCCC
CAATCCCAA
CAATCTCCT
?????????
CAATCCCCA
CAATCCCCA
CAATCCCCA
CAATCCCCA
CAATCCCCA
CAATCCCCA
CAATCCCCA
CAATCTCCC
CAGAGTCCT
CAAAGCCCT
CAGAGTCCG
555555555
777777777
123456789
I b rsA
2 p isA
3 cynA
4 deeA
5 gydA
6 encA
7 m ysA
8 so aA
9 m idA
10 glm A
11 m igA
12 m u sA
13 d a p A
14 au b A
15 d a n A
16 p o p A
17 c rb A
18 m irl
19 c a s 1
20 e n d
21 cyn1
22 p o a l
23 o ss1
2 4 m ig l
2 5 au b 1
2 6 d an 1
2 7 Io p l
28 e n c E
29 so a E
30 c a s E
31 g ltE
32 d e e p
3 3 m idE
3 4 m irE
3 5 p ip E
36 m igE
37 au b E
38 d a n E
39 IevE
4 0 po fE
41 glm B
4 2 m y sB
43 m u s B
59
APPENDIX C
60
Amino acid alignments o f PHY sequences showing amino acid hallmarks o f the four
presumptive loci, the positions o f indels and the chromophore attachment site (marked
with “ U ” symbol). Presumptive gene family is indicated by “A”, “B”, “E” and “A l”
following the name o f each taxa. Primer sites are excluded.
B row nea s p . A
P is u m s a t i v u m A
C y c lo lo b iu m n u ta n s A
D e r r is e l l i p t i c a A
G y m n o c la d u s d i o i c a A
E n te r o lo b iu m c y c lo c a r p u m A
M y r o sp e r m u m s o u s a n u m A
Sophora a f f i n is A
M i l l e t t i a d ura A
G l y c i n e m ax A
M i l l e t t i a g r a n d is A
M u n d u le a s e r i c e a A
D a h ls t e d t ia p in n a ta A
A u s tr o s te e n s ia b la c k ii A
D a lb e r g ie lla n y a ssa e A
P o n g a m ia p i n n a t a A
C r a ib ia b r e v ic a u d a t a A
M i l l e t t i a r ic h a r d ia n a A l
C a r m ic h a e lia s p . A l
E n t e r o lo b iu m c y c lo c a r p u m A l
C y c lo lo b iu m n u ta n s A l
P o n g a m io p s is a m y g d a lin a A l
O s tr y o c a r p u s s t u h lm a n n ii A l
M i l l e t t i a g r a n d is A l
A u s tr o s te e n s ia b la c k ii A l
D a lb e r g ie lla n y a ssa e A l
L onchocarpus p h a s e o lifo liu s
E n t e r o lo b iu m c y c lo c a r p u m E
Sophora a f f i n i s E
C a r m ic h a e lia s p . E
G le d it s ia tr ia c a n th o s E
D e r r is e l l i p t i c a E
M ille t t ia dura E
M i l l e t t i a r ic h a r d ia n a E
P is c id ia p is c ip u la E
M i l l e t t i a g r a n d is E
A u s tr o s te e n s ia b la c k ii E
D a lb e r g ie lla n y a ssa e E
T e p h r o s ia v i l l o s a E
P o e c ila n th e f a lc a t a E
G ly c in e m ax B
M y r o sp e r m u m s o u s a n u m B
M u n d u le a s e r i c e a B
*
*
*
*
233333333333333333 333333333333333333333
900000000001111111 111222222222233333333
901234567890123456 789012345678901234567
DHGEVIAEITKPDMEPYL-GLHYPATDIPQAARFLFMKNK
DHGE VIAEIAKPGLEPYL - GLHYP ATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL - GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL- GLHYPATDI PQAARFLFMKNK
DHGEVIAEITKPGLEPYL - GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL- GLHYPATDIPQAARFLFMKNK
DHGEWAEITKSGLEPYL-GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL - GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL-GLHYPATDIPQAARFLFMKNK
DHGEVIREITKPCLEPYL-GLHYPATDIPQASRFLFRKNK
DHGEVIAEVKRPGLEPYL- GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLE PYL - GLHYPATDIPQASRFLFMKNK
DHGEVIAEITKPGLE PYL - GLHYPATDIPQAARFLFMKNK
DHGEVIAE ITKPGLE PY L- GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYL- GLHYPATDIPQAARFLFMKNK
DHGEVIAEITKPGLEPYM- GLHYPATDIPQAALFLFMKNK
DHGEVIAEITKPGLEQYL - GLHYPATDIPQAARFLFMNNK
DHGEVIAEITKPGLEPYL- GLHYPATDIPQAARFLFMKNK
DHGEVIAEVKKPGLEPYL - GLHYPATDVPQAARFLFMKNK
EHGEVVSEVKKPGLESYM-GLHYPATDVPQATRFLFMKNK
DHGEVIAEVTKPGLEPYL-GLHYPATDIPQATRFLLMKNK
DHGEVIAEVKKPGLEPYL-GLHYPATDIPQATRFLFMKNK
DHGEVIAEVKKPGLEPYL- GLHYPATDIPQATRFLFMKNK
DHGEVIAELKKPGLEPYL - GLHYPATDIPQATRFLFMKNK
IMGKVI AEI KKPGLE PYL- GLHYPATDIPQATRFLFMKNK
DHGEWAEVKKPGLEPYL-GLHYPATDIPQATRFSFMKNK
A l DHGRVIAEVKKPGLEPYL- GLHYPATDIPQATRFLFMKNK
DHGEVVSEIRRSDLEPFF- GLHYPATDIPQAARFLFKQNR
DHGEWSEIRRSDLEPLLPVLHYPATDIPQAARFLFQQNR
DHGEWSEIRRSDLEPYL-GLHYPSTDIPQAARFLFKQNR
DHGEVVSEIRRSDLEPYL-GLHYPATDIPQASRFLFKQNR
DHGEIVSEIRRSDLEPYL- GLHYPATDIPQASRFLFKQNR
DHGEWSEIRRSDLEPYL-GLHYPATDIPQASRFLFKQNR
DHGEVVSEIRRSDLEPYL- GLHYPATDIPQASRFLFKQNR
DHGEW SEIRRSDLEPYL - GLHYPATDIPQASRFLFRQNR
D DG EW SEIRRSDLEQ CL-GLHYPATDI PQASRFLFKQNR
D H G EW SEIRRSDLEPYL-GLHYPATDIPQASRFLFKQNR
EHGEW S EIRRSDLEPYL - GLHYPATDI PQASRLLFKQNR
DHGEVIAEIRRSDLEPYL- GLHYPATDIPQASRFLFKQNR
DHGEW SE IRRSDLEPYL - GLHYPAKDIPQAARFLFKQNR
EHGEW SESKRPDLEPYI - GLHYPATDI PQASRFLFKQNR
EHGEWAESKRPDLEPYI-GLHYPATDIPQASRFLFKQNR
? ? 7EW AESKRPDLEPYI -GLHYPATDIPQASRFLFKQNR
1111111111222222222233333333334
1234567890123456789012345678901234567890
Numbering of amino acid sites across the top of the alignments follows the system described by Lavin
et al. (1998; Appendix C) which includes the omission of sites 403, 404 and 481. Numbering across the
bottom marks codon position within the target region used for the analyses presented here.
61
Appendix C. Continued.
*
*
*
*
3333333333333333333333333333333333333333
334444444444555555555566G 66666G 677777777
8901234567890123456789012345678901234567
B row nea s p . A
VRIIVDCRAKHVKVLQDEKLPFDLTLCGSTLRDPHSCHLQ
VRMIVDCNAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
P is u m s a t i v u m A
VRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
C y c lo lo b iu m n u ta n s A
VRMIVDCHAKHMKVLQDEKIPFDLTLCGSTLRAPHSCHLQ
D e r r is e l l i p t i c a A
VRMIVDCHARHVKVLQDEKLPFELTLCGSTLRAPHTCHLQ
G y m n o c la d u s d i o i c a A
VRMIVDCHARHVKVLQDEKLPFELTLCGSTLRAPHTCHLQ
E n te r o lo b iu m c y c lo c a r p u m A
VRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
M y r o sp e r m u m s o u s a n u m A
VRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
Sophora a f f i n i s A
VRMIVDCHAKHVKVLQDEKIPFDLTLCGSTLRAPHSCHLQ
M il le t t ia d ura A
VRMIVDCHAKHVRVLQDEKLQFDLILCGSTLRAPHSCHAQ
G l y c i n e m ax A
VRMIVDCHAKHVKVFQDEKLPIDLTLCGSTLRAPHSCHLQ
M i l l e t t i a g r a n d is A
VRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
M u n d u le a s e r i c e a A
VRMIVDCHAKHVKVLQDEKTPFDLTLCGSTLRAPHSCHLQ
D a h ls t e d t ia p in n a ta A
VRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
A u s tr o s te e n s ia b la c k ii A
VRMIVDCHAKHVKVLQDEKLPLDLTLCGSTLRAPHSCHLQ
D a lb e r g ie lla n y a ssa e A
YRMIVDCHAKHVKVLQDEKLPFDLTLCGSTLRAPHSCHLQ
P o n g a m ia p i n n a t a A
VRMIVDCHAKHVKVLQDEKLLFDLTLCGSTLRAPHSCHLQ
C r a ib ia b r e v ic a u d a ta A
VRMIVDCRAKHVNVLQDKKVPFDLTLCGSTLRAPHSCHLQ
M i l l e t t i a r ic h a r d ia n a A l
VRIIVDCSAKRVKVIQDKNIPFDLTLCGSTLRAPHNCHLQ
C a r m ic h a e lia s p . A l
VRMIVDCRAKHV KVLQDENLPYDLTFCGSTLRAPHSCHVQ
E n te r o lo b iu m c y c lo c a r p u m A l
VRLIVDCCAKHVKVLQDKKIPFELTLCGSTLRAPHSCHIQ
C y c lo lo b iu m n u ta n s A l
VRMI VDCRAKHVNVLQDKKVP FDLTLCGSTLRAPH S CHLQ
P o n g a m io p s is a m y g d a lin a A l
VRMIVDCRAKHVNVLQDKKVPFDLTLCGSTLRAPHSCHLQ
O s tr y o c a r p u s s t u h lm a n n ii A l
VRMIVDCRAKHVKVLQDKKIPFDLTLCGSTLRAPHSCHLQ
M i l l e t t i a g r a n d is A l
VRQIVDCRAKHVKVLQDKNIPFDLTLCGSTLRAPHSCHLQ
A u s tr o s te e n s ia b la c k ii A l
VRMIVDCHAKHVKVLQDKKIPFDLTLCGSTLRAPHSCHLQ
D a lb e r g ie lla n y a ssa e A l
L o n c h o c a r p u s p h a s e o l i f o l i u s A l VRMIVDCRAKHVKVLQDKKVPFDLTLCGSTLRAPHSCHLQ
VRMICDCHANPVKVIQSEESRQPLCLVNSTLRSPHQCHAQ
E n te r o lo b iu m c y c lo c a r p u m E
VRMICDCHAKPVNVIQSEELRQPLCLVNSTLRSPLGCHTQ
Sophora a f f i n i s E
VRMICDCHAKPVKVIQSEELRQPLCLVNSTLRSPHDCHTQ
C a r m ic h a e lia s p . E
VRLICDCHANPVRVIQSEELRQPLCLVISTLRSPHGCHTQ
G le d it s ia tr ia c a n th o s E
VRMICDCHAKPVKVIQ S EELRQPLCLVNSTLRSPHVCHTQ
D e r r is e l l i p t i c a E
VRMICDCGAKPVKVIQSEELRQPLCLVNSTLRSPHVCHTQ
M ille t t ia dura E
VRMICDCHAKPVKVIQSEELRQPLCLVNSTLRSPHVCHTQ
M i l l e t t i a r ic h a r d ia n a E
VRMICDCHAKPVKVIQSEELRQPLCLVNSTLRSPHVCHTQ
P is c id ia p is c ip u la E
VRMICDCHAKPVKVIQSEELRQPLCLVNSTLRSPHVCHTQ
M i l l e t t i a g r a n d is E
VRMICDCHAKPVKVIQSEELRQPLCLVNSTLRSPHGCHTQ
A u s tr o s te e n s ia b la c k ii E
VRMICDCDAKPVKVIQSEELRQPLCLVNSTLRSPHGCHTQ
D a lb e r g ie lla n y a ssa e E
VRMICDCHAKPVKVIQSEELRQSLCLVNSTLRSPHVCHTQ
T e p h r o s ia v i l l o s a E
VRMIFDCHAKPVNVIQSEELRQPLCLVNSTLRSPHGCHTQ
P o e c ila n th e f a l c a t a E
VRMIVDCHASAVRWQDEALVQPLCLVGSTLGAPHGCHAQ
G l y c i n e m ax B
VRMIVDCHASPVGVIQDEGLMQPLCLVGSTLRAPHGCHAQ
M y r o sp e r m u m s o u s a n u m B
VRMIVDCHASPVRWQDEALVQPLCLVGSTL7APHGCHAQ
M u n d u le a s e r i c e a B
4444444445555555555666666666677777777778
1234567890123456789012345678901234567890
62
Appendix C. Continued.
333333333333333333333
3444444444444444
778888888888999999999
9000000000011111
890123456789012345678
9012567890123456
B row nea s p . A
YMENMNSVASLVMAVWNDND- -EDGDSSDSVQPQKRKR
P is u m s a t i v u m A
YMANMDSXASLVMAV W N D SD - -EDGDSADA VL PQKKKR
C y c lo lo b iu m n u ta n s A
YMANMDSIASLVMAW V N D S D --EDGNSSDAVQPQ KRKR
D e r r is e l l i p t i c a A
YMANMDSIA S LVMAVWNDNE- - EDGDS SDAVQPQ KRKR
G y m n o c la d u s d i o i c a A
YMENMNSIASLVMAVWNDND- -EDVDSSDSIQPQKRKR
E n te r o lo b iu m c y c lo c a r p u m A
YMENMNSIASLVMAWVNDND--EDVDSSDSIQPQKRKR
M y r o sp e r m u m s o u s a n u m A
YMANMDSIASLVLAVWNDSD- -EDGDSSDAVQPQKRKR
Sophora a f f i n i s A
YMANMDSIASLVMAW V N D S D --EDGDSSDAVQPQKRKR
YMANMDSIASLVMAW V N D N E --EDGDSSDAVQPQKRKR
M ille t t ia dura A
YMANMDSIASLVLAVWNDNE - -EDGD-TDAVQPQKTER
G l y c i n e m ax A
YMANMDSIASLVMAW VNDNE- - EDGDSSDAVQPQKRKR
M i l l e t t i a g r a n d is A
YMANMDSIASLVMAW VNDNE- - EDGDSSDAVQPQKRKR
M u n d u le a s e r i c e a A
YMANMDSIASLVMAW VNDNE- - EDGDSSDAVQPQKRKR
D a h ls t e d t ia p in n a ta A
YMANMNSIASLVLAW V N D N E -- EDGDSSDAVQPQKRKR
A u s tr o s te e n s ia b la c k ii A
YMANMDSIASLVMAWVNDNE- - EDGDSSDAVQPQKRKR
D a lb e r g ie lla n y a ssa e A
YMANMDSIASLVMAW VNDNE- - EDGSSSDAVQPQKRKR
P o n g a m ia p i n n a t a A
YMANMDSIASLVMAVWNDNE - - EDGDSSDAVQPQKRKR
C r a ib ia b r e v ic a u d a t a A
YMENMNCSASLVMAVWNDND- -EDGDS-DAVQPQKRKR
M i l l e t t i a r ic h a r d ia n a A l
YMDNMKASASLVMAVWNDSN- - EDGDSSDAVQPQKRKR
C a r m ic h a e lia s p . A l
YMQNMDLVASLVMAWINDRD- - EDGDS SDSVQPQKRKR
E n te r o lo b iu m c y c lo c a r p u m A l
YMLNMNSIASLVMAVWNDSD- - EDGDSSDAVQPQKRKR
C y c lo lo b iu m n u ta n s A l
YMENMNCSASLVMAWVNDND- -EDGDS-DAVQPQKRKR
P o n g a m io p s is a m y g d a lin a A l
YMENMNCSASLVMAWVNDND- -EDGDS-DAVQPQKRKR
O s tr y o c a r p u s s tu h lm a n n ii A l
YMENMNSSASLVMAWVNDND-- EDGDSSDAVQPQKRKR
M i l l e t t i a g r a n d is A l
YMKNMNSSASLVMAVWNDNN- -EDGDSSDAFQPQKRKR
A u s tr o s t e e n s ia b la c k ii A l
YMENMNSSASLVMAWVNDND--EDGDSSDAIQPQKRKR
D a lb e r g ie lla n y a ssa e A l
L o n c h o c a r p u s p h a s e d i f o l i u s A l YMENMNCSAS LVMAVWNDNE- - EDGDS - DAVQPQ KRKR
YMENMGSIAS LVMAVIVNGNDTT------------------------------- K
E n te r o lo b iu m c y c lo c a r p u m E
YMANMGSIASLVMAVIVNGNDTT----------R
Sophora a f f i n i s E
YMANMGSIASLVMAVIVNGNDST------------------------------- R
C a r m ic h a e lia s p . E
YMANMGSIASLVMAVIVNGTDTT--------------------------------K
G le d it s ia tr ia c a n th o s E
YMANMGSIASLVMAIIVNGNDTT--------------------------------R
D e r r is e l l i p t i c a E
YMANMGSTASLVLAIIVNGNDTT------------------------------- R
M il le t t ia dura E
YMANMGSIASLVMAIIVNGNDKT--------------------------------R
M i l l e t t i a r ic h a r d ia n a E
YMSNMGSIASLVMAIIVNGNDKR------------------------------- R
P is c id ia p is c ip u la E
YMANMGS IA S LVMAIIVNGNDTT--------------------------------R
M i l l e t t i a g r a n d is E
YMANMGS IASLVMAVTVNGNDTR--------------------------------R
A u s tr o s te e n s ia b la c k ii E
YMANMGS IASLVMAVIINGNDTT--------------------------------R
D a lb e r g ie lla n y a ssa e E
YMANMGS IASLVMAIIVNGNDTT------------------------------- R
T e p h r o s ia v i l l o s a E
YMANMGSVAS LVMAVI VNGNDTT------------------------------- R
P o e c ila n th e f a l c a t a E
YMANMGSIASLVMAVIINGND--EEGVGG----------RSSMR
G l y c i n e m ax B
YMANMGSIASLVMAVIINGND--ED TV CS----------RSSMR
M y r o sp e r m u m s o u s a n u m B
YMANMGSIASLVMAVIINGND--EEGVGG----------RSSMR
M u n d u le a s e r i c e a B
11111111111111111111
888888888999999999900000000001111111111
123456789012345678901234567890123456789
63
Appendix C. Continued.
4444444444444444444444444444444444444444
1112222222222333333333344444444445555555
7890123456789012345678901234567890123456
LWGLWCHNTTPRFVPFPLRYACEFLVQVFAIHVNKELEL
B row nea s p . A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
P is u m s a t i v u m A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
C y c lo lo b iu m n u ta n s A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIE L
D e r r is e l l i p t i c a A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
G y m n o c la d u s d i o i c a A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
E n te r o lo b iu m c y c lo c a r p u m A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEFEL
M y r o sp e r m u m s o u s a n u m A
LWGLWCHNTTPRFVPF PLR YACE FLAQVFAIHVNKEIE L
Sophora a f f i n i s A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
M ille t t ia dura A
LWGLWCHNTTPRFVPFPLRYAREFLPQVFADHVHKEIEL
G l y c i n e m ax A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
M i l l e t t i a g r a n d is A
LWGLWCHNTSPRFVPFPLRYACEFLAQVFAIHVNKEIEL
M u n d u le a s e r i c e a A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
D a h ls t e d t ia p in n a ta A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
A u s tr o s te e n s ia b la c k ii A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
D a lb e r g ie lla n y a ssa e A
LWGLWCHNTTPRFVPFPLRYACEFLSQVFAIHVNKEIEL
P o n g a m ia p i n n a t a A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
C r a ib ia b r e v ic a u d a t a A
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
M i l l e t t i a r ic h a r d ia n a A l
LWGLWCHHITPKFVPFPLRYACEFLAQVFAIHVNKEIEL
C a r m ic h a e lia s p . A l
LWGLWCHNYTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
E n te r o lo b iu m c y c lo c a r p u m A l
LWGLWCHNTTPRFVPFPLRYACE FLAQVFAIHVNKE IE L
C y c lo lo b iu m n u ta n s A l
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEIEL
P o n g a m io p s i s a m y g d a l i n a A l
LWGLWCHNTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
O s tr y o c a r p u s s tu h lm a n n ii A l
LWGLWCHHTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
M i l l e t t i a g r a n d is A l
LWGLWCHHTTPRFVPFPLRYACEFLAQVFAYL VNKELEI
A u s tr o s te e n s ia b la c k ii A l
LWGLWCHHTTPRFVPFPLRYACEFLAQVFAIHVNKELEL
D a lb e r g ie lla n y a ssa e A l
L o n c h o c a r p u s p h a s e o l i f o l i u s A l LWGLWCHNSIPRFVPFPLRYACEFLAQVFAIHVNKELEL
LWGLLVCHHTSPRHVPFPLRYACEFLMQAFGLQLYMEMQL
E n te r o lo b iu m c y c lo c a r p u m E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
Sophora a f f i n i s E
LWGLLVCHHSSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
C a r m ic h a e lia s p . E
LWGLLVCHHTSPRHVPFPLRYACEFLMQAFGLQLYSDMQL
G le d it s ia tr ia c a n th o s E
LWGLLVCHHTSR?YVPFPVRYACEFLMQAFGVQLYMEIQM
D e r r is e l l i p t i c a E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
M ille t t ia d ura E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
M i l l e t t i a r ic h a r d ia n a E
LWGLLVCHHTSLRYVPFPVRYACEFLMQAFGLQLYMEIQL
P is c id ia p is c ip u la E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
M i l l e t t i a g r a n d is E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
A u s tr o s te e n s ia b la c k ii E
LWGLLVCHHTSPRYVPFPVCYACE FLMQAFGLQLYMEIQL
D a lb e r g ie lla n y a ssa e E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
T e p h r o s ia v i l l o s a E
LWGLLVCHHTSPRYVPFPVRYACEFLMQAFGLQLYMEIQL
P o e c ila n th e f a lc a t a E
LWGLWCHHTSARCIPFPLRYACEFLMQAFGLQLNMELQL
G l y c i n e m ax B
LWGLWCHHTSARCIPFPLRYACEFLMQAFGLQ LNMELQL
M y r o sp e r m u m s o u s a n u m B
LWGLWCHHTSARCIPFPLRYACEFLMQAFGLQ LNMELQL
M u n d u le a s e r i c e a B
1111111111111111111111111111111111111111
2222222222333333333344444444445555555555
0123456789012345678901234567890123456780
64
Appendix C. Continued.
444444444444444444444444444444444
555666666666677777777778888888889
789012345678901234567890234567890
B row nea s p . A
EYQIVEKNILRTQTLLCDMLMRDAPLGXVSQGP
P is u m s a t i v u m A
EYQILEKNILRTQTLLCDMLMRDAPLGIVSQSP
C y c lo lo b iu m n u ta n s A
EYQIIEKNILRTQTLLCDMLMRNAPLGIVSQSP
E Y Q IIE KNlLRTQTLLCDMLMRDAPLGIVSQSP
D e r r is e l l i p t i c a A
ENQIVEKKILRTQTLLCDMLMR?? ? ? ? ? ? ? ? ? ?
G y m n o c la d u s d i o i c a A
E n te r o lo b iu m c y c lo c a r p u m A
ENQIVEKKILRTQTLLCDMLMRDAPLGIISQSP
EYQ IIEKNILRTQTLLCDMLMRDAPLGIVSQSP
M y r o sp e r m u m s o u s a n u m A
EYQLIEKNILRTQTLLCDMLMRDAPLGIVSQSP
Sophora a f f i n i s A
EYQIIEKNILRTQTLLCDMLMRDAPLGIVSQSP
M ille t t ia dura A
EYQIIBKNILHHPGHLLCMLMRDAPLGIASESP
G l y c i n e m ax A
EYQIIEKNILRTQTLLCDMLMRDAPLGIVSQSP
M i l l e t t i a g r a n d is A
ENQIIEKNILRTQTLLCDMLMRDAPLGIVSQSP
M u n d u le a s e r i c e a A
EYQIIEKNILRTQTLLCDMLMRDAPLGIVSQSP
D a h ls t e d t ia p in n a ta A
EYQIIEKNILRTQTLLCDMLMRDAPLGIIS Q S P
A u s tr o s te e n s ia b la c k ii A
E Y Q IIE K N ILRTQTLLCDMLMRDAPLGIVSQSP
D a lb e r g ie lla n y a ssa e A
E Y Q IIE K N ILRTQTLLCDMLMRDAPLGIVSQSP
P o n g a m ia p i n n a t a A
EYQI I E N N ILRTQTLLCDMLMRDAPLGIVSQSP
C r a ib ia b r e v ic a u d a ta A
EYQIVEKNILRTQTLLCDMLMRDAPLGIVTQRP
M i l l e t t i a r ic h a r d ia n a A l
EYQVIERNILRTQTLLCDMLMRDAPLGIISQSP
C a r m ic h a e lia s p . A l
E Y Q IIEKNIMRTQSLLCDMLMRDEPLGIIS Q S P
E n te r o lo b iu m c y c lo c a r p u m A l
EYQIIEKNILRTQTLLCDMLMRNAPLGIVSQSP
C y c lo lo b iu m n u ta n s A l
EYQIIEKNILRTQTLLCDMLMRDAPLGIVSQSP
P o n g a m io p s is a m y g d a lin a A l
EYQlVEKNTLRTQTLLCDMLMRDAPLGIVTQRP
O s tr y o c a r p u s s tu h lm a n n ii A l
EYQlVEKNTLRTQTLLCDMLMRDAPLGIVSQRA
M i l l e t t i a g r a n d is A l
AYQIVEKNILRTQTLLCDMLMRDAPLGIVSQSP
A u s tr o s te e n s ia b la c k ii A l
EYQIVEKNILRTQTLLCDMLMRDAPLGIVSQSP
D a lb e r g ie lla n y a ssa e A l
L o n c h o c a r p u s p h a s e o l i f o l i u s A l EYQIVEKNILRTQTLLCDMLMRDAPLGIVTQRP
ASQMAEKRTLKTQTLLCDMLLRDALFSIVTQSP
E n te r o lo b iu m c y c lo c a r p u m E
ASQMAEKRLLKTQTLLCDMLLRDAPFGIVTQSP
Sophora a f f i n i s E
ASQMAEKRILKTQTLLCDMLLRDAPFGIVTQSQ
C a r m ic h a e lia s p . E
ASQMAEKRILRTQTLLCDMLLRDSPFGIVTQSP
G le d it s ia tr ia c a n th o s E
ASQMAEKRMLKTQT?????? ? ? ? ? ? ? ? ? ? ? ? ? ?
D e r r is e l l i p t i c a E
ASQKAEKRMLKTQTLLCDMLLRDAPLGIVTQSP
M il le t t ia d ura E
ASQMAEKRMLKTQTLLCDMLLRDAPLGIVTQSP
M i l l e t t i a r ic h a r d ia n a E
ASQMAEKRMLKTQTLLCDMLLRDAPLGIVTQSP
P is c id ia p is c ip u la E
ASQMAEKKTLKTQTLLCDMLLRDAPFGIVTQSP
M i l l e t t i a g r a n d is E
ASQMAEKRILKTQTLLCDMLLRDAPFGIVSQSP
A u s tr o s t e e n s ia b la c k ii E
ASQMAEKRILKTQTLLCDMLLRDAPFGIVNQSP
D a lb e r g ie lla n y a ssa e E
ASQMAEKRILKTQTLLCDMLLRDAPLGIVTQSP
T e p h r o s ia v i l l o s a E
ATQLAEKRILKTQTLLCDMLLRDAPFGIVNQSP
P o e c ila n th e f a l c a t a E
AAQ SLE KRVLRTQTLLCDMLLRD S PTGI VTQS P
G l y c i n e m ax B
ASQSLEKRVLKTQTLLCDMLLRDSPIGIVTQSP
M y r o sp e r m u m s o u s a n u m B
AAQALEKRVLRTQTLLCDMLLRDSPTGIVTQSP
M u n d u le a s e r i c e a B
111111111111111111111111111111111
666666666777777777788888888889999
123456789012345678901234567890123
65
APPENDIX D
Percent divergence values.
I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
0
8
10
11
8
11
9
9
15
17
18
11
8
8
8
10
10
10
8
8
7
8
8
9
10
20
33
33
34
32
35
33
33
34
34
31
34
33
31
34
33
33
1
2
5
9
11
12
9
11
10
9
14
16
18
11
9
8
8
10
10
10
8
8
7
9
8
9
10
20
33
33
33
32
35
32
32
33
33
31
34
32
30
34
33
33
2
3
4
I
9
10
7
9
8
10
13
16
16
11
9
8
8
10
10
10
8
9
8
9
8
9
10
20
35
35
35
33
36
33
33
33
35
32
35
32
31
36
35
35
3
4
4
2
1
3
2
2
4
5
10
13
15
9
9
9
9
11
11
9
9
10
9
10
9
10
10
20
37
36
36
36
39
35
35
35
37
35
37
35
33
35
34
35
4
5
4
4
3
4
4
5
5
7
11
14
17
11
10
9
9
11
12
10
10
10
10
11
11
12
11
21
38
38
38
36
40
36
36
36
38
36
38
37
34
37
36
36
5
6
4
4
3
4
0
4
5
7
11
14
17
10
8
7
7
9
10
10
8
9
8
8
8
9
9
19
36
36
35
35
38
34
35
35
36
34
36
33
32
36
35
35
6
7
5
2
1
3
4
4
5
5
10
11
13
7
7
6
6
9
9
7
7
8
7
8
7
9
8
18
37
36
35
35
38
34
35
35
36
33
36
33
32
36
35
35
7
8
4
1
I
2
4
4
2
4
8
12
16
9
8
7
8
10
11
9
9
9
9
9
9
10
10
21
36
36
36
35
37
34
35
35
36
33
36
35
32
35
35
34
8
9
4
2
1
0
4
4
2
1
9
12
15
9
8
7
8
10
11
9
9
9
9
9
9
9
10
21
35
35
35
33
36
32
33
33
35
31
35
33
32
34
34
33
9
10 11
11 5
9 3
8 2
8 2
11 5
11 5
8 3
8 3
7 2
9
14
19 17
13 13
12 12
11 12
12 12
14 14
14 15
13 12
12 13
13 14
12 12
13 13
13 12
14 14
14 14
24 25
38 40
37 39
37 39
36 37
39 41
35 37
36 38
36 38
37 39
34 36
36 39
36 37
33 35
36 40
35 39
35 38
10 11
12 13
5 4
2 2
2 I
2 I
4 4
4 4
3 3
2 2
2 0
7 8
2 2
2
16
15 7
14 7
14 7
15 10
15 10
14 9
14 8
15 9
14 8
14 6
14 7
14 9
15 9
25 20
42 38
40 36
40 37
39 36
42 38
38 35
39 35
39 36
40 37
37 33
37 36
38 36
39 33
37 38
38 37
37 36
12 13
14 15
4 5
2 2
1
I
I
I
3 4
3 4
2 2
2 2
1 1
7 7
2 2
2 I
1
I
1
I
1
I
3 3
4 3
4 3
2 1
3 2
3 2
3 3
3 2
4 4
5 5
14 14
35 34
34 33
33 32
32 31
35 34
31 31
32 32
33 32
33 33
30 29
33 33
32 31
29 29
35 34
34 33
34 33
14 15
16 17
5 5
3 3
2 2
2 2
5 5
5 5
3 3
3 3
2 2
8 8
3 3
2 2
2 2
2 2
2 2
3
3
3 5
3 5
1 3
2 4
2 4
3 4
2 4
4 5
5 6
14 15
34 35
33 34
32 33
31 32
34 35
31 31
32 32
32 33
33 32
29 30
33 33
31 32
29 29
34 35
33 34
33 34
16 17
18 19 20
3 9 10
4 7 9
3 7 7
4 8 9
4 9 10
4 9 10
4 8 9
4 7 8
4 7 9
11 15 15
5 7 9
4 9 9
4 7 9
3 7 10
4 8 9
5 9 10
4 9 10
7 10
10
5
3 3
4 4 2
4 4 2
4 5 3
4 4 2
6 6 4
7 6 5
15 16 14
35 34 34
35 33 33
33 32 32
32 31 31
36 34 34
32 30 30
33 31 31
33 32 32
34 32 32
31 29 29
34 32 32
32 31 31
30 28 28
35 33 34
34 32 33
34 32 33
18 19 20
21 22
6 5
5 5
4 4
5 4
6 5
6 5
6 5
5 4
4 4
12 11
6 4
6 4
5 4
5 4
5 4
7 5
6 5
5 1
7 6
9 8
4
3
4 4
3 3
5 4
6 6
14 13
34 35
32 34
32 33
31 32
33 34
29 31
30 32
31 32
31 33
28 30
31 33
31 32
27 29
33 35
32 34
32 34
21 22
23 24
4 4
5 5
4 4
4 4
4 4
4 4
5 5
5 4
4 4
12 11
4 4
5 5
4 4
4 4
5 4
6 6
6 5
1 2
7 6
9 9
5 4
1 2
2
2
3 3
4 4
15 15
35 34
33 32
32 32
31 31
34 34
31 30
32 31
32 32
33 32
29 29
33 32
31 30
29 28
34 34
33 33
33 33
23 24
25 26
7 5
8 5
7 4
7 4
7 4
7 4
8 5
8 5
7 4
14 11
8 4
8 5
7 4
7 4
8 5
9 6
9 5
5 3
8 6
12 9
7 5
6 3
5 2
4 I
5
5
15 17
33 35
32 33
32 32
31 31
34 34
30 31
31 32
31 32
32 33
29 29
32 32
31 31
29 29
34 34
33 33
33 33
25 26
27
6
6
5
5
5
5
6
6
5
12
5
5
5
5
8
6
6
3
8
9
6
2
2
3
6
3
40
38
38
37
37
36
36
36
38
35
38
37
35
38
38
38
27
28
27
28
27
28
26
26
27
27
27
32
27
29
27
28
27
29
29
29
32
33
31
30
29
28
31
28
31
10
10
10
11
10
12
12
10
10
12
12
11
22
21
20
28
29
28
26
26
26
27
27
26
26
26
30
25
27
26
26
25
27
27
28
32
33
29
28
28
28
31
28
31
6
11
11
12
10
13
12
11
9
11
11
11
10
18
18
29
30
26
25
24
25
25
25
24
25
25
29
24
26
25
25
24
26
26
27
30
31
29
28
28
27
30
27
29
5
3
6
8
6
8
8
7
6
7
6
7
21
21
19
30
31
27
27
26
27
27
27
26
27
27
31
26
27
27
27
26
28
28
29
32
32
29
29
29
28
30
28
31
5
7
5
6
5
7
7
7
5
7
7
6
21
21
19
31
32
29
28
27
28
28
28
27
28
27
31
27
27
28
28
27
29
28
30
33
34
31
30
30
29
31
29
32
6
5
3
6
3
5
5
5
5
7
8
5
22
21
20
32
33
29
28
27
29
29
29
27
29
28
31
27
28
28
28
27
30
29
30
32
34
30
30
30
29
31
29
31
7
6
3
6
3
2
3
4
5
6
7
3
20
19
17
33
34
27
26
25
26
27
27
26
26
26
30
25
26
26
27
25
28
27
29
31
33
29
29
29
28
30
27
30
5
3
2
5
1
2
5
6
6
9
10
5
21
20
18
34
35
28
27
26
26
27
27
27
27
27
31
26
27
27
28
27
29
28
29
32
33
30
30
29
29
30
28
31
6
5
3
6
2
3
I
6
6
6
8
5
21
21
19
35
36
28
27
26
27
27
27
26
27
26
30
26
26
27
27
26
28
26
29
32
33
30
36
27
26
30
28
31
6
4
3
6
3
3
1
3
6
7
9
5
22
21
20
36
37
26
25
24
25
25
25
24
25
25
28
24
25
25
25
24
26
26
27
30
31
28
27
28
26
29
26
29
5
3
2
4
2
3
1
2
2
5
6
5
20
19
17
37
38
27
26
25
26
27
27
26
26
26
30
25
26
26
26
25
27
27
28
31
31
29
29
28
28
30
27
30
6
4
2
5
3
3
2
3
3
2
7
7
20
19
17
38
39
26
25
24
25
26
26
25
25
25
29
24
25
25
26
24
26
26
28
30
33
28
28
28
27
29
27
29
6
4
2
5
2
3
1
2
2
2
3
8
21
20
19
39
40
26
25
24
25
25
25
25
25
25
30
24
25
25
26
24
26
26
27
30
31
29
28
27
27
30
27
30
6
4
3
6
4
5
3
4
4
3
4
4
21
19
18
40
41
27
24
24
26
26
26
25
24
25
29
25
25
25
26
25
26
26
28
31
28
28
28
27
25
29
26
29
18
17
17
16
18
18
16
16
18
18
16
17
16
42
26
24
24
26
25
25
25
24
25
28
24
25
25
26
25
26
26
27
30
29
28
27
26
25
28
25
28
15
15
14
14
15
15
14
15
15
13
13
14
14
3
43
26 I brsA
24 2 pisA
24 3 cynA
25 4 deeA
25 5 gydA
25 6 encA
25 7 mysA
23 8 soaA
24 9 midA
29 10 glmA
24 11 migA
25 12 musA
25 13 dapA
25 14 aubA
25 15 danA
25 16 popA
25 17 crbA
28 18 mini
29 19 casl
29 20 e n d
28 21 cyn1
28 22 poa1
27 23 oss1
25 24 mig1
29 25 aubl
25 26 dan1
29 27lop1
17 28 encE
17 29 soaE
16 30 casE
16 31 gltE
17 32 deeE
17 33 midE
15 34 mirE
17 35 pipE
17 36 migE
15 37 aubE
15 38 danE
16 39 IevE
18 40 pofE
1 41 glmB
2
3 42 mysB
43 musB
8 7
41 42 43
Percent amino acid divergence is shown above the diagonal and percent nucleotide divergence appears below the diagonal. Taxa
numbered 1-43 are as shown in Appendix B
O'
o
- BOZEMAN
*