PHYLOGENETIC ANALYSIS OF NORTH AMERICAN PLUMS (PRUNUS SECT. PRUNOCERASUS: ROSACEAE) BASED ON NUCLEAR LEAFY AND S6PDH SEQUENCES

2008·19.9 MB·English

by  Joseph R Rohrer

NORTH
AMERICAN PLUMS
PHYLOGENETIC OF
ANALYSIS
ON
PRUNOCERASUS: ROSACEAE) BASED
(PRUNUS
SECT.
AND
NUCLEAR SEQUENCES
LEAFY S6PDH
Megan
and Anderson
Joseph
Rohrer, 1 A. O'Brien, Julie A.
R.
Department of Biology
RESUMEN
INTRODUCTION
200 which most abundant
Prunus one of the larger genera of the Rosaceae with about species, are in the
is
North Temperate zone but also range into the tropics and Southern Hemisphere (Rehder 1940; Robertson
and
almonds,
1974). important commercially as the source of cherries, peaches, nectarines, apricots,
is
It
The
numerous grown
plums; species are ornamentally for their beautiful early-spring flowers. classifica-
tion of Prunus has been debated from Linnaeus to the present day, with some botanists arguing for a broad
McVaugh
mentioned above Robertson
concept of the genus that includes of the fruits 1951; 1974)
all (e.g.,
and others dividing Prunus into a variety of segregate genera including Amygdalus, Armeniaca, Cerasus,
and China Committee The
Iwatsuki 2001; Flora of Editorial 2003).
Laurocerasus, Padus, Persica
et
(e.g., al.
who
most widely used classification has been that of Rehder (1940) recognized Prunus in its broad sense
among
North American
At the species there has been considerable taxonomic uncertainty the
level
American
plums (Prunus Prunocerasus). In 1892 Bailey called Prunocerasus the "hardest puzzle in
sect.
Shaw more
pomology," memorable phrase quoted by and Small (2004) than century in reporting
a a later
cpDNA
Shaw
on enduring and Small provided an
the use of multiple sequences to bear this puzzle. (2004)
402 Journal of the Botanical Research Institute of Texas 2(1)
and taxonomic Prunoce
extensive review analysis of the history of
American plums
North
of the result of interspecifi
classification
is
probably hybridization.
interspecific
DNA
on
The using sequences examine relationships in Prunus focused a limited
earliest studies to
&
Uematsu 2001 two groups
number research
of cultivated species (Badenes Parfitt 1995; et 1991). In
al.
among subgenera and numerous sections of Prunus by ex-
sought phylogenetic relationships the
to clarify
&
Wen
amining on worldwide 2001; Lee 2001). Unfortunately relationships
species a scale (Bortiri et
al.
and found
among remained Both groups used ITS sequences
unresolved.
the species section Prunocerasus
of
among plums included Analyses by Bortiri (2001) using
slight variation the native in their studies. et al.
among North American
chloroplast trnL-trnF sequences also revealed only slight variation six species.
more
Rohrer (2004) used microsatellite analysis in an attempt to find molecular markers variable
et
al.
among species of Prunocerasus than ITS or trn sequences. However, all pairs of species in the study shared
among North American plums than
fewer than half of their alleles, indicating greater genetic divergence the
same
expected given their similar ITS and trn sequences. Accessions of two or three individuals of the spe-
did not suggesting that microsatellites might be too variable to be informative in
often cluster together,
cies
monophyly
resolving species relationships in Prunocerasus. Seeking to test the hypothesis of for Prunocerasus
and among Shaw and Small 2005) turned seven noncoding
resolve relationships the species, (2004, to
to
regions in the chloroplast genome shown to be highly variable across a wide array of taxa (Shaw et al. 2005).
Shaw
and
Given their taxonomically broad sampling of species both within the section across all of Prunus,
monophyly Within
and Small (2004, 2005) provided unequivocal support for the of sect. Prunocerasus. the
among They demonstrated strong support
section the analyzed sequences varied the species. for P.
little
An
was
unexpected the inclusion of the hairy-fruited
subcordata as sister to the rest of Prunocerasus. result
P and which formed
texana within Prunocerasus positioned between subcordata the other species, three
P.
Many more
have exemplars on than
primary clades with resolution within each clade. of the species
little
show with geography than
one and the chloroplast haplotypes greater correlation species limits.
clade,
among
Bortiri (2002) demonstrated that the nuclear gene s6pdh was more variable Prunus species
et
al.
than either ITS or trn and showed greater promise for resolving relationships in the genus. However, only
Oh
and
reported
three species of Prunocerasus were included in their analysis. In addition, Potter (2003)
LEAFY
more than
were informative
sequences from the second intron of the nuclear gene phylogenetically
we
cpDNA
either ITS or in the rosaceous genera Neillia and Stephanandra. In this study explore the use of
we
among
these low-copy nuclear genes resolving relationships the species of Prunocerasus. Specifically
for
expand the use of s6pdh commonly recognized species in the section and include sequences from the
to
all
North
Plant Material—-We were able to obtain at least one accession from each of the 15 (or so) species of
American plums. The exact number of species subject to the differences in opinion concerning species
is
plum and two
circumscriptions that animate taxonomic debate. Apricot armeniaca), cherry cerasifera),
(P.
(P.
com-
and were used outgroup
sand cherry pumila pumila pumila susquehanae) for
varieties of var. var.
P.
(P.
parison. In we analyzed 37 accessions of North American plums plus the four outgroup taxa (Table
1).
all,
from
Some specimens were collected from wild plant populations; others were collected cultivated plants of
USDA
Germplasm and
the University of California, Davis campus, the National Clonal Repository in Davis,
DNA
USDA documented
the Southeastern Fruit and Nut Research Station in Byron, GA. All sources of are
We
(UWEC).
Wisconsin-Eau herbarium
by voucher specimens deposited in the University of Claire verified
and herbarium
identifications against published descriptions authentic material.
DNA DNA
and Sequencing.— was by grinding approximately
Extraction, Amplification, Total isolated
mg
and DNeasy Mini
100 nitrogen using the Plant Kit (Qia-
(wet weight) of fresh or frozen leaves in liquid
We
PCR
gen) according the standard protocol. prepared reactions in 50-ul volumes containing -10 ng
to
Rohrer et Phylogenetk analysis of North American Prunus Prunocerasus
sect.
al.,
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Journal of the Botanical Research Institute of Texas 2(1)
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Rohrer Phylogenetic analysis of North American Prunus Prunocerasus 405
et sect.
al.,
DNA uM uM
PCR
DNA,
genomic ExTaq polymerase lx 200 dNTPs,
of 1.25 units of (Takara), buffer, 0.2 of
LEAFY
was
each primer, and water. Intron 2 of the nuclear gene amplified using primers modified
sterile
Oh 5»-GGCTTGTTGATGTAGCT-
5'-CAGAACATTGCCAAGGAGC-3',
from and LFY5: LFY4:
Potter (2003):
TGC-3'; and nuclear gene s6pdh from Exon 2 Exon 6 was amplified using primers modified from Bortiri
to
5'-CAAGAGTGAAGCAGACGTTGG-3',
new 5'-AGACCA-
primer
et (2002) plus a s6pdh-k: s6pdh-h:
al. (o):
ATGCTACGAACTAGGCCG-3', 5'-AGAATAAGGTGTTGGACATAGACG-3',
5'-AGAGTG-
s6pdh-o: s6pdh-p:
GTCCTGGATTTCTTATCTA-3'.
PCR, preceded by an melting 4
Thirty-five cycles of three-step step of
initial
min min
at 94°C and concluded by a final extension of 7 at 72°C, were carried out as follows: denaturation
94°C 30 primer annealing 52°C (LEAFY) or 54°C (s6pdK) min, and extension 72°C
at for sec, at for 1 at
2 min.
for
PCR
we from 1% and them
Following electrophoresis excised products agarose gels purified using a
We
PCR
QIAquick fragments sequencing using primers
Gel Extraction Kit (Qiagen). sent the for direct the
DNA
either Davis Sequencing (Davis, CA) or the Sequence Laboratory of the University of Wisconsin
to
PCR
number
Biotechnology Center (Madison, WI). products with unreadable sequences or a significant of
TOPO
TA
ambiguous base were cloned using the Cloning Kit Sequencing (Invitrogen) following
for
calls
DNA
the manufacturer's instructions. Sequencing of clones using T3 and T7 primers was done by the
all
UW
PCR
Sequence Laboratory of the Biotechnology Center. Following cloning, sequences with possible
artifacts were identified and removed from further analysis by comparing the cloned sequences to each other
When
more
and the original chromatogram obtained through direct sequencing. two or clones yielded
to
one was used
identical sequences, only in the analyses.
Chromas
Each sequence chromatogram was scrutinized for base determination errors using 2.22 (Tech-
&
GeneDoc
nelysium Pty and the sequences were aligned by eye in 2.6.002 (Nicholas Nicholas 1997).
Ltd),
among made
The Prunus manual alignment sequences For
close relationship species of the straightforward.
parsimony were coded binary Seven were
data informative indels as additional characters. indels
analysis,
LEAFY LEAFY
trimmed
included and sequences were Intron s6pdh sequences
for eight for s6pdh. to just
2;
Exon
were bp downstream from end primer and end bp
truncated to begin 18 the of k, located in 2, 14
upstream from primer Exon Sequences were submitted GenBank numbers
the of p in to (accession
start 6.
in Table
listed
1).
—
maximum
PAUP*
Phylogenetic Analyses. Both were analyzed using parsimony (MP) with
data
sets
&
and with MrBayes (Huelsenbeck Ronquist
4.0bl0 (Swofford 2002) Bayesian inference 3.1.2 2001;
(BI)
&
MP
TBR
Two
Ronquist Huelsebeck 2003). heuristic searches with branch swapping and the Multrees
and
option were performed: the with MaxTrees one million; in the second, the "nchuck" "chuck-
set to
first
score" options were employed so that 10,000 trees were saved and swapped for each of 30 random addition
replicates. The results of each search were summarized as strict consensus cladograms. Relative support
random
each branch was assessed by heuristic searches of 1000 bootstrap replications with 10 addition
for
TBR random
replicates each and branch swapping. For each addition replicate 100 trees were saved. For
was and ModelTest
BI analyses each data partitioned into nucleotide characters indel characters. 3.7
set
&
was model
(Posada Crandall 1998) used the substitution that best the nucleotide data using
to select
fit
TVMef LEAFY HKY+I+G
Akaike and Each two runs each
the information criterion: for for s6pdh. of BI for
We
drawn
gene consisted of three heated and one cold chain with samples every 100 generations. ran the
LEAFY we
500,000 whereas s6pdh ran
data 1,000,000 generations discarding the generations,
for first for
1,500,000 generations discarding the 750,000 generations. The runs for each data set were combined
first
67%
and a majority rule consensus tree constructed with a threshold.
—
Determination of chloroplast haplotypes. In order compare our phylogenetic results from nuclear
to
LEAFY Shaw we
and and sequenced
s6pdh with those of Small (2004, 2005), the trnL-trnF intergenic spacer
We
from the chloroplast genome for our accessions. amplified the spacer using the e-f primer pair of Taberlet
EU606152-
and used primer and sometimes primer sequencing (GenBank accessions
(1991) for
et
al. e,
f,
We
Shaw
EU606192). aligned our sequences with those from the 18 Prunocerasus exemplars used in and Small
406 Journal of the Botanical Research Institute of Texas 2(1)
plum
maximum
The parsimony from the combined dataset was used to place each of our native
(2004). tree
Shaw
and Small
accessions into one of the primary chloroplast haplotypes (A, B, C, S, or T) of (2005).
we by names on phy-
and sequences the
For clarity in the results discussion sections refer to specific their
name among from same by decimels
genus and clones the collection
lograms, omitting the differentiating
number.
following the three of the collection
last digits
LEAFY Alignment and Data Analysis.— PCR with primers LFY5 and LFY4 yielded fragments approxi-
250-bp Only 1000-bp fragments
mately 750 or 1000 nucleotides in length, the difference being a insertion.
PCR and two
were amplified Prunus hortulana and murrayana. All three individuals of rivularis of
for P.
P.
The
had both 750 and 1000-bp fragments other
munsoniana 676) (Fig
the four collections of (513,
1).
P.
Some
collections of munsoniana and the remainder of the species yielded only 750-bp fragments. of the
P.
cloned sequences appeared be recombinations of the 750 and 1000-bp sequences, similar to
rivularis to
P.
recombinant sequences
and murrayana respectively (Fig Putative
those of angustifolia hortulana/P. 2).
P. P.
LEAFY
Trimmed
ingroup sequences varied
were omitted from the phylogenetic analyses. to Intron 2, the
in length from 658 bp in texana to 925 bp in one clone of rivularis (528.22). The final alignment used
P.
P.
LEAFY 930 which 856 were 35 varied but
in the data analysis included nucleotide characters, of invariant,
were parsimony-uninformative, and 39 (4.2%) were parsimony informative for the ingroup taxa.
MP
The consensus cladograms from the two parsimony analyses are identical (not shown), each
strict
of length 194 with consistency index (CI) of 0.634 and retention index (RI) of 0.796. Their topologies are
by
similar to the Bayesian analysis phylogram (Fig. but several nodes (indicated the solid circles) are
3),
by
and
collapsed and there are minor rearrangements within Clades 3 4 (topology indicated dotted lines).
form monophyletic group three subcordata plus
Prunus Prunocerasus into lineages:
Species of a split P.
sect.
and composed and sequences from munsoniana
Clades Clade 3 of angustijolia-\ike
1 2. is P. angustifolia P.
from same
and The two species (names in bold on also have cloned sequences the
rivularis. latter Fig. 3)
P.
individuals on Clade Relationships among americana, mexicana, and nigra are unresolved; they
2. P. P. P.
mur-
from polytomy on Clade Sequences Clade including those of hortulana,
diverge a basal 2. of 4, P. P.
among
and have 250-bp insertion not found the other
rayana (except 525S), munsoniana, rivularis, a
P. P.
—
s6pdh Alignment and Data Analysis. The alignment the s6pdh analysis includes 1272 nucleotide
final for
and were
which 1197 were 46 varied but were parsimony-uninformative, 29 (2.3%)
characters, of invariant,
among
The ingroup
parsimony informative the Prunus Prunocerasus species. shortest sequences the
for sect.
TC
were those of hortulana maritima, and suhcordata at 1204 nucleotides, including 7 or 8
(628), P.
P. P.
had sequence 1256
nucleotides
repeats in Intron whereas one americana clone (505.2) the longest at
3, P.
TC
with
31
repeats.
MP
LEAFY
consensus cladograms from both parsimony analyses of
Just as in the analyses, the strict
and
s6pdh are identical (not shown) and have lengths of 153, consistency indices (CI) of 0.699, retention
MP
On
MP
phylogram
indices (RI) of 0.854. The cladograms are nearly identical to the BI (Fig. 4). the cla-
dograms, Clades 5 and 6 are groups such that Prunus sect. Prunocerasus forms a monophyletic group,
sister
by and
with bootstrap support than 50%, the branch indicated the black circle is collapsed,
albeit less
on by Clade
amencana.495.3/ameriama.505.23 Clade 5 (indicated dotted
americana.684.1 lines).
sister to
is
from americana and one
5 includes suhcordata plus subcordata-likz sequences five individuals of P. of P.
P.
on which
munsoniana. These accessions (names in bold on also have sequences Clade includes all
Fig. 4) 6,
among and
the other native plum taxa. There poor resolution these taxa. Prunus angustifolia, P. texana,
is
sequences from munsoniana and compose Clade Clade 8 consists solely of
angustifolia-like P. rivularis 7. P.
P.
which
americana and mexicana, plus similar sequence from one individual of umbellata (520.4), also
a P.
P.
among polytomy 520
has sequence the taxa of the Clade 6 basal (umbellata.
a
.3).
Fig. 1 Gel following electrophoresis of PCR products using LFY 5 and LFY 4 primers.
.
LEAFY
on sequence
Position alignment
51 4.24
r/Vt//a/fs.
GA
C
;
..
i
|
rivulari$.52$2
CTTCAC
GA
C
A
I |
-
CTTCAC
TG
A
A
! |
I
rivularis.5U.21
CTTCAC
TG
1*
A
A
rivularis.52725
I I
rivutaris.528.3
Fig. 2. Graphic representation of cloned LEAFY sequences from Prunus
rivularis accessions. Each bar represen
correspond
to close relationship with P. hortulana and P. murrayana sequences (Clade 4 on Fig.3) and light gray
ship with P. angustifolia sequences (Clade 3 on Fig. 3). Characters used to map sequence segr
(alignment
gap), stars indicate the presence of 6-bp and 9-bp and
deletions,
and
angustifolia-type sequences. The boundaries between sequence types are drawn to within
DISCUSSION
LEAFY
The
nuclear genes and weak
s6pdh provide among
resolution of relationships species Prunus
of
sect.
MP
The
Prunocerasus. majority of taxa diverge from polytomies on consensus and The
the
BI topology
trees.
cpDNA
of these from Shaw
trees is different the cladogram of and Small (2005) where and
subcordata then
P.
texana are groups remaining which
P. sister to the species, segregate primary American
into three
clades:
Journal of the Botanical Research Institute of Texas 2(1)
C
.513.2
C
munsoniana.676.Z
rivularts.527.24
A
mu/rayana.525$
C
w/arfs.514.24
C
.598.1
C
rfvu/ar/s.527,25
67%
LEAFY majorH
3.
Fig.
where
cladograms bla
differ
by epithet plus last 3 digits
(
Small (2005) as determined
Rohrer et Phyiogenetic analysis of North American Prunus sect. Prunocerasus
al.,
C
americana.495.2
A
amer/cana.505.2
C
amer/cana.508.4
C
americana.5Q7
C
amer/cana.684.2
—
C
82
rivularis.5U.22
C
rivularis.527.3
C
.528.4
T
texana.685
588
angustifblia.
C
munson/ana.592.21
-
jJITST C
1
nvularis.S27.2
92
C
munstv7/a/ia.513.1
'
A
n/gra,633
C
—alieghanien$i$AS7
alieghaniensisA68
B
geniculata.GQ9
C
gracMs.516
C
hortulana.598
A
1.628
B
.590
C
— .513.3
A
murrayana.524
A
murrayana.525
A
/Twrraya/73.526
-
cerasifera.541
M
Shaw
and
haplotypes of
Journal of the Botanical Research Institute of Texas 2(1)
410
LEAFY
Beach and Chickasaw Clade on the tree and s6pdh Clades 5 and 7 consist largely of
(A), (B), (C). 1
cpDNA
with haplotype otherwise taxa on the nuclear gene trees do not resolve into the A,
accessions C,
and C clades of Shaw and Small (2005). Incongruence between chloroplast and nuclear phylogenies is
B,
common long-branch
and such hybridization, lineage sorting, or attraction
typically attributed to factors as
&
Zhong The
Maddison Sang geographic distribution of
Doyle 2000).
(Albaladejo 2005; 1997; 1997;
et
al.
ABC which North
with
with capture given the ease
introgression chloroplast
the chloroplast clades suggests
&
known Shaw
American plums (Hedrick Small 2004).
hybridize 1911;
are to
Although Prunus known peachbush or Texas wild peach, has historically been placed in
texana, as
plum
Shaw and found has chloroplast
Amygdalus due hairy-skinned Small (2004) that a
sect. to its fruit, it
On
LEAFY
genome. Evidence from the nuclear genes and s6pdh confirms placement in sect. Prunocerasus.
its
we
cpDNA cladogram texana was the other plums of eastern North America, a position could
the sister to
P.
on
endemic Lake Wales Ridge in central Florida, our
not confirm. the rare of the
It is sister to P. geniculate,
weak
LEAFY phylogram. However, based on one character change and has bootstrap
relationship
this
is
On
and
support. the s6pdh phylogram texana part of Clade 7 with angustijolia the angustifolia-like
is P.
P.
and
sequences munsoniana
of rivularis.
P. P.
LEAFY
group on
and form paraphyletic
Prunus maritima, umbellata a
alleghaniensis, P. gracilis, P. P.
UPGMA
on dendrogram based on
similarity
Clade basal Clade These four species clustered the
to 3.
1,
These resemble each other
markers study Rohrer species closely
in the of (2004).
of microsatellite et
al.
On
American plums such Wight the s6pdh
morphologically and were by early students of as (1915).
allied
cladogram, they are part of the large Clade 6 basal polytomy.
when
working
Prunus americana and mexicana can be separate morphologically, especially
difficult to
P.
LEAFY
with herbarium specimens. Similarly their and s6pdh sequences were similar and without resolution
On LEAFY
and
phylogram americana
monophyletic americana and mexicana groups. the the
into P.
P. P.
and
from polytomy along with munsoniana.5921, Clade
mexicana sequences diverge a basal nigra, 4.
P.
P.
LEAFY and mexicana.545
americana.684 mexicana.518 (TX),
sequences americana.508 (WI), (PA), (cult.)
run and some heterozygous one or two
by length poly-A nucleotide characters for
differ trivially the of a
sequences but with only one of the two bases on the other sequences. Otherwise they are identical.
LEAFY
P
on
from mexicana sequences
Although do Prunus americana either
our data not separate
UPGMA
on
dendrogram, based
Boonprakob similarities
Clade 2 or s6pdh Clade (2001) calculated a
et
8, al.
RAPD and from
markers, on which 13 accessions of americana clustered together distinct
of their
of P.
all
more with wide geographic
distribution
a cluster of 11 accessions of mexicana. Clearly collections a
all P.
and more genes need be examined and correlated with the morphological characteristics of those
will to
complex
understood.
taxonomic americana-mexicana
accessions before the structure of the is
P.
two from Texas near
an Rohrer (2004) included collections east
In earlier microsatellite study, et
al.
morpho-
Marshall (designated (americana x mexicana?) 10517 and 10518 in that publication) that are
sp.
P.
between
The
and
between Prunus americana mexicana. lack of differentiation
intermediate typical
logically
P.
LEAFY ambiguous. However,
americana and mexicana and s6pdh sequences leaves their determination
P. P.
modern
sequence and because most exclude
because neither 517 nor 518 have subcordata-like s6pdh floras
a
americana from Texas, we have provisionally determined these accessions to be mexicana.
P.
P.
by
Two distinct sequences were isolated from five of the six accessions of Prunus americana cloning
s6pdh PCR products. One sequence from each pair on Clade 8 along with mexicana and the single
their is P.
sequence of americana 587; the other on Clade 5 with subcordata. Given that these P. americana se-
is P.
P.
and
quences form monophyletic group subcordata and that subcordata grows only in California
a sister to P.
P.
North America, seems unlikely that the americana
Oregon, far west of americana range in eastern it five P.
P.
s6pdh
collections with two s6pdh sequences are recent hybrids independently involving P. subcordata. If is
by and americana
concluded (2002)
a single copy gene in all diploid Prunus species as Bortiri et al. if P. is
&
being
be = Love Love then high degree of heterozygosity
diploid as reported (2n 1982), a is
to 16,
is
it
we two
heterologous
maintained However, cannot rule out the possibility that there are
a single locus.
at