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Akiyama et al. BMC Evolutionary Biology 2011, 11:289
http://www.biomedcentral.com/1471-2148/11/289

RESEARCH ARTICLE

Open Access

Evolution of the apomixis transmitting
chromosome in Pennisetum
Yukio Akiyama1†, Shailendra Goel1†, Joann A Conner1, Wayne W Hanna2, Hitomi Yamada-Akiyama3 and
Peggy Ozias-Akins1*

Abstract
Background: Apomixis is an intriguing trait in plants that results in maternal clones through seed reproduction.
Apomixis is an elusive, but potentially revolutionary, trait for plant breeding and hybrid seed production. Recent
studies arguing that apomicts are not evolutionary dead ends have generated further interest in the evolution of
asexual flowering plants.
Results: In the present study, we investigate karyotypic variation in a single chromosome responsible for
transmitting apomixis, the Apospory-Specific Genomic Region carrier chromosome, in relation to species phylogeny
in the genera Pennisetum and Cenchrus. A 1 kb region from the 3’ end of the ndhF gene and a 900 bp region from
trnL-F were sequenced from 12 apomictic and eight sexual species in the genus Pennisetum and allied genus
Cenchrus. An 800 bp region from the Apospory-Specific Genomic Region also was sequenced from the 12
apomicts. Molecular cytological analysis was conducted in sixteen Pennisetum and two Cenchrus species. Our
results indicate that the Apospory-Specific Genomic Region is shared by all apomictic species while it is absent
from all sexual species or cytotypes. Contrary to our previous observations in Pennisetum squamulatum and
Cenchrus ciliaris, retrotransposon sequences of the Opie-2-like family were not closely associated with the
Apospory-Specific Genomic Region in all apomictic species, suggesting that they may have been accumulated after
the Apospory-Specific Genomic Region originated.
Conclusions: Given that phylogenetic analysis merged Cenchrus and newly investigated Pennisetum species into a
single clade containing a terminal cluster of Cenchrus apomicts, the presumed monophyletic origin of Cenchrus is
supported. The Apospory-Specific Genomic Region likely preceded speciation in Cenchrus and its lateral transfer
through hybridization and subsequent chromosome repatterning may have contributed to further speciation in the
two genera.

Background
Apomixis is an intriguing trait in plants that allows multiplication of maternal clones through seed reproduction
[1]. Besides the potential for apomixis to be a powerful
plant breeding tool due to the circumvention of genetic
segregation and maintenance of heterosis in hybrid progenies, the trait is also compelling in terms of evolutionary studies. Apomicts have long been regarded as
evolutionary dead ends [2] mainly because of their presumed lack of genetic variation in the absence of
* Correspondence: pozias@uga.edu
† Contributed equally
1
Department of Horticulture, The University of Georgia, 2360 Rainwater Rd.,
Tifton, GA 31793-5766, USA
Full list of author information is available at the end of the article

recombination and intermating, although various studies
have shown high levels of chromosomal and morphological variation within agamic complexes [3,4]. More
recently, levels of genetic diversity among asexual populations were found to be higher than expected when
compared to those in sexually reproducing populations
[5-9]. Apomicts can outcross when they produce viable
pollen, through the occasional reduced egg, or by fertilization of unreduced eggs, and can thus act as bridges
for introgressive hybridization between otherwise reproductively isolated taxa [10-14]. Hybrid lineages can be
stabilized by apomixis, allopolyploidy and recombinational speciation [15]. Apomixis does, however, reduce
the rate of chromosomal recombination in the female,
thereby diminishing the opportunity for unequal

© 2011 Akiyama et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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crossing over to reduce repetitive element copy number
[16], allowing instead an accumulation of transposons in
the genome and an increase in genome size, at least in
relatively recent lineages [17]. Recombination is further
constrained during male meiosis in apomicts in the
chromosomal region transmitting the trait to progeny
[18,19]. The fundamental importance of recombination
and the paradox of sex [20,21] have inspired interest in
deciphering the evolution of asexual organisms [14,22].
The Pennisetum/Cenchrus branch of the monophyletic
bristle clade of grasses [23] contains a major crop species, sexual pearl millet or Pennisetum glaucum (L.) R.
Br., and at least 17 aposporous species [18]. Relationships have been inferred among some of these species
using basic chromosome numbers, ITS (the internal
transcribed spacers of ribosomal RNA genes) DNA data
[24] and sequences from chloroplast genes such as ndhF
(F subunit of NADH dehydrogenase) [25], ndhF and
trnL-F [26], trnL-F and rpl16 [27]. Chemisquy [26] also
used a nuclear gene (knotted) to study the phylogeny in
Cenchrus, Pennisetum and related genera.
ITS sequences provide limited resolution to estimate
genetic similarities of hybrids and their parents due to
concerted evolution [28]. Though chloroplast DNA is
maternally inherited, and therefore can be criticized for
its inability to assess biparental contribution to the genome, it can provide sequences from specific genes or
intergenic regions that are phylogenetically informative.
The tobacco (Nicotiana tabacum) ndhF gene is 2223 bp
in length and has a nucleotide substitution rate [29]
which is, for example, two times greater than that of
rbcL, a second extensively studied chloroplast gene [30].
More recent studies have also demonstrated that the
3’end of ndhF is more variable than the 5’ region [31].
For the present study, we chose to sequence two chloroplast gene regions (a 1131-1155 bp fragment from the
3’end of ndhF and 811-872 bp region from trnL-F) and
a 792-799 bp segment from the ASGR-BBM-like gene,
also located within the p208 BAC used in fluorescence
in situ hybridization (FISH) analysis. We furthermore
report molecular cytogenetic analysis of the genomic
region associated with apomixis, the apospory-specific
genomic region (ASGR) that was previously identified in
P. squamulatum, C. ciliaris and now in 16 Pennisetum
and one additional Cenchrus species.
The ASGR is conserved between P. squamulatum and
C. ciliaris based on high sequence similarity between
putative orthologous genes within this region; syntenic
relationships between chromosomal sequences identified
by BAC probes; shared cytological features of hemizygosity, the heterochromatic nature of the ASGR, and a
region of low copy DNA flanked by high copy sequences
[32-37]. Nevertheless, there are distinct structural differences in the ASGR-carrier chromosomes of these two

Page 2 of 16

species. These previous observations suggested that a
conserved ASGR haplotype may occur in different chromosomal contexts among species. We now compare the
extent of conservation and variation in the ASGR and
ASGR-carrier chromosome in parallel with a Pennisetum
and Cenchrus species phylogeny constructed with
sequence data from chloroplast genes, ndhF and trnL-F.
Variability observed in chromosomal context should
enable a more precise delineation of the ASGR.

Results
Phylogenetic analysis based on ndhF and trnL-F
sequences

All species (Table 1) generated a 3’ ndhF sequence of
1134 bp except for P. hohenackeri (PS156) and P. polystachion (PS19). PS156 had an insertion of 21 bp while
PS19 showed a 3 bp deletion. For the trnL-F region, size
varied from 863 bp to 872 bp except in the case of P.
polystachion, P. pedicillatum and P. subangustum which
showed a length of 811 bp. ndhF and trnL-F produced
an aligned matrix of 1155 and 901 nucleotide positions
respectively thus giving a total aligned matrix of 2056
characters. The matrix had 1913 constant, 76 parsimony
uninformative and 67 parsimony informative characters.
A partition homogeneity test was done for 100 replicates, although the test was aborted during the 78 th
replicate due to time constraints (655:46 hr). The test
gave a P-value of 0.86 supporting the combination of
data sets for analysis.
A simple heuristic search of the aligned matrix using Phylogenetic Analysis Using Parsimony (PAUP) retained 28
trees. All trees were 166 steps in length and had a consistency index (CI) of 0.875, retention index (RI) of 0.895 and
rescaled consistency index (RC) of 0.775. The log likelihood
of all the trees ranged from 3974.04078 to 3971.98984. To
account for homoplasy generated by gaps, the gap creating
regions were ignored (accounting for ~97 characters of
aligned matrix). After exclusion, a heuristic search generated 9 trees each showing a length of 152 steps with CI of
0.875, RI of 0.914 and RC of 0.799. The log likelihood for
all the trees ranged from 3755.09184 to 3757.65531.
Phylogenetic trees with similar topologies were generated by Bayesian and maximum parsimony (MP) methods. Overall five groups emerged in the present
phylogenetic study (Figure 1A and 1B, Table 2). All
major groups showed good bootstrap support except
that the group of P. ramosum, P. nervosum and P.
mezianum showed low support in the Bayesian-based
analysis. These species also showed variation with
respect to their position in the two trees (Bayesian and
Maximum Parsimony). Subgroups I, II, and V contain
apomictic and obligately sexual species whereas subgroups III and IV contain apomictic species with sexual
cytotypes or facultative apomixis.

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Page 3 of 16

Table 1 Plant materials
Species

Primary ID

Secondary ID

Reported Chromosome No.

Ploidy

C. ciliaris

PS185

’LLANO’

36

4x

Reported MOR
A

C. ciliaris

PS186

’NUECES’

36

4x

A
A

C. setigerus

PS16

PI266185

36

4x

P. alopecuroides

PS938

9064-3

18

2x

S

P. basedowii

PS2

PI257782

54

6x

S

P. flaccidum

PS32

PI271601

18,36,45

2x,4x,5x

S,A

P. flaccidum

PS95

TIMOTHY C79I3

18,36,45

2x,4x,5x

S,A

P. glaucum
P. hohenackeri

23BE
PS156

ICRISAT

14
18

2x
2x

S
S

P. massaicum

PS680

IBPCR

36

4x

A

P. massaicum

PS953

WIPFF 87A11508

36

4x

A

P. mezianum

PS9

PI365021

P. nervosum

PS187

16, 32
#7-82

2x,4x

36,72

4x,8x

S,A
S

P. nervosum

PS38

PI316421

36,72

4x,8x

S

P. orientale

PS12

PI315867

18,27,36,45,54

2x-6x

S,A

P. orientale
P. pedicillatum

PS13
PS304

PI218097
HARLAN 682

18,27,36,45,54
36,54

2x-6x
4x,6x

S,A
A
A

P. polystachion

PS19

PI189347

36,54,63

4x,6x,7x

P. purpureum

N109

-

28

4x

S

P. purpureum

N168

-

28

4x

S

P. ramosum

PS29

PI331699

10

2x

S

P. ramosum

PS63

DEWET1641

10

2x

S

P. schweinfurthii

PS243

PI489685

14

2x

S

P. setaceum
P. setaceum

PS22
PS25

PI300087
PI364994

27,54
27,54

3x,6x
3x,6x

A
A

P. squamulatum

PS158

ICRISAT

54

6x

A

P. squamulatum

PS24

PI248534

54

6x

A

P. subangustum

PS163

IBADAN#2

P. villosum

PS249

TEL AVIV

Setaria viridis

GI:758770

-

36,54

4x,6x

A

18,27,36,45,54

2x-6x

S,A

18

2x

S

List of Cenchrus and Pennisetum species with corresponding identifiers, mode of reproduction and chromosome data. Reported chromosome numbers are from
Jauhar [57], Dujardin and Hanna [57] and the Kew C-value database (http://data.kew.org/cvalues/). MOR = Mode of Reproduction; A = apomictic; S = sexual.

A recent paper [26] also used ndhF and trnL-F
sequences to understand the relationship among Pennisetum and Cenchrus species. To compare their analysis
with that of the present study, the sequence alignment
was downloaded from TreeBase http://purl.org/phylo/
treebase/phylows/study/TB2:S10252. The resultant
matrix was too large to be analyzed by PAUP, hence it
was only analyzed by Mr. Bayes (Figure 2). Seven
sequences were removed from the analysis due to substantial amounts of missing data. The taxa used in the
present study are shown in blue while those shown in
red are from Chemisquy [26] whose grouping does not
entirely agree with that generated in the present study.
Phylogenetic analysis based on sequence from the ASGR
region

Eight primer pairs, previously identified as ASGR-linked
in F1 populations where P. squamulatum and C. ciliaris

were the apomictic parents, were tested on all species
used in this study (Additional File 1). Only the primer
pair p779/p780 which amplifies a portion of the ASGRBBM-like gene resulted in amplification of all the apomictic species but none of the sexual species. Primers
p779/p780 are located in the 4 th and 7 th exons of
ASGR-BBM-like2 (EU559277) and amplify a region
including 3 introns of 95 bp, 266 bp, and 154 bp. Based
on ASGR-linked BAC clone sequencing, P. squamulatum and C. ciliaris have duplicated ASGR-BBM-like
genes [38]. The p779/p780 primers amplify both copies,
although polymorphism between copies cannot be
detected in P. squamulatum while polymorphism is
detectable in C. ciliaris. The present analysis could differentiate two copies of the ASGR-BBM-like gene in C.
setigerus, P. orientale, P. mezianum and C. ciliaris. In P.
orientale, accession PS12 did not show two copies while
PS15 did. Among the two types of sequences obtained

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Bayesian

MP

S.viridis

S.viridis

P.villosum249
100

P.hohenackeri156
100
P.alopecuroides938

P.villosum249

II

1.00

P.hohenackeri156
1.00
P.alopecuroides938

P.basedowii2

P.basedowii2

P.mezianum9
P.nervosum38
96

P.mezianum9

V

P.nervosum38
1.00
P.nervosum187

0.50

0.50

P.nervosum187
1.00
P.massaicum953
70
76

C.ciliaris185

89

92

C.setigerus16

P.massaicum953

IV

C.setigerus16
1.00

P.polystachion19
100

1.00

C.ciliaris186
0.50
C.ciliaris185

1.00

P.pedicellatum304

0.50

61
P.subangustum163

1.00

P.pedicellatum304
1.00
P.subangustum163

P.orientale12

P.orientale12
1.00

P.orientale13

96

P.flaccidum32

1.00

III

P.flaccidum32
1.00
P.flaccidum95

P.flaccidum95
P.setaceum22
P.setaceum25
1.00

P.schweinfurthii243
56

P.squamulatum24
100

III

P.setaceum22
1.00
P.setaceum25

98

P.glaucum2

P.orientale13

0.50

90

80

IV

P.polystachion19

P.ramosum29
100
P.ramosum63
76

V

P.ramosum29
1.00
P.ramosum63

1.00

C.ciliaris186
88

II

P.schweinfurthii243
P.glaucum2

0.50

I

1.00

87
P.squamulatum158

P.squamulatum24
1.00
P.squamulatum158

I

0.50
P.purpureum168

P.purpureum168
1.00
P.purpureum109

57
P.pupureum109

A

B

Figure 1 Maximum Parsimony and Bayesian tree based on ndhF and trnL-F. Maximum parsimony (MP) and Bayesian trees based on the
ndhF+trnLF sequence alignments generated in the present study. Numbers at the nodes show bootstrap values obtained.

in C. ciliaris and C. setigerus, one showed similarity with
P. squamulatum while the other sequence grouped with
the other copy from Cenchrus (Figure 3).
The ASGR-BBM-like sequence ranged in size from 792799 bp. Alignment provided a matrix of 800 bp with
785 constant, 4 uninformative and 11 informative characters. Heuristic search retained only one tree. This tree
was only 15 steps in length with trichotomies and a log
likelihood score of 1206.32388 (Figure 3).
Detection of the ASGR-carrier chromosome in apomictic
Pennisetum species

The results of FISH with ASGR-linked BACs are summarized in Table 2 and Figs. 4 and 5. No sexual species showed discrete signals from hybridization of the
ASGR-linked BACs P001, P109 or P208. BAC P208
showed weak signal on the centromeres of not only
aposporous, but also sexual species. In aposporous species, the ASGR-linked BACs were detected as strong
signals on a single chromosome (Figure 4a, c-j) with
one exception (Figure 4b). In P. orientale (PS12), a 54-

chromosome accession, two ASGR-carrier chromosomes were observed (Figure 4b). The BACs sometimes showed strong and spatially distinct signals
within the ASGR indicating duplicated loci or repetitive sequences.
Localization of 25S rDNA on the ASGR-carrier
chromosome

The rDNA probe was used as a cytological marker to
test whether the ASGR in species other than C. ciliaris
[34,37] was associated with a 25S rDNA locus. Only two
other species, C. setigerus (PS16) and P. massaicum
(PS680) showed rDNA signals on the ASGR-carrier
chromosome. The ASGR-carrier chromosome in C. setigerus was indistinguishable from C. ciliaris in the position of the rDNA locus, i.e., terminal on the short arm
of the ASGR-carrier chromosome (Figure 4l), in addition to other characters (Figure 5). In P. massaicum, a
rDNA locus was distally located on the short arm of the
ASGR-carrier chromosome whereas the ASGR was
terminal on the long arm (Figure 4k).

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Page 5 of 16

Table 2 Species clusters and cytological characteristics
Group
No. in
MP Tree

Group No Species
in Bayesian Name
Tree

Primary
ID

MOR Number of
chromo-somes
observed

Number
of ASGR

25S rDNA on
ASGR-carrier
chromosome

Opie-2
around
ASGR

Opie-2 Enzyme
in
treatment (min)/
genome Denature (sec)

I

I

P. glaucum

23BE

S

14

0

-

-

low

80/70

I

I

P. purpureum N109

S

-

-

-

-

-

-

I
I

I
I

S
S

14

0

-

-

high

80/90

I

I

P. purpureum N168
P.
PS243
schweinfurthii
P. setaceum
PS22

A

-

-

-

-

-

-

I

I

P. setaceum

A

27

1

no

no

mid

80/90

I

I

P.
PS158
squamulatum

A

56

1

no

high

low

Ref. [50]

I

I

P.
PS24
squamulatum

A

56

1

no

high

low

120/90

II

II

P.
PS938
alopecuroides

S

18

0

-

-

low

80/90

II

II

P.
hohenackeri

PS156

S

-

-

-

-

-

-

II

II

P. villosum

PS249

A

45

1

no

no

mid

80/90

III

III

P. flaccidum

PS32

A

36

-

-

-

-

-

III

III

P. flaccidum

PS95

A

36

1

no

low

low

100/90

III

III

P. orientale

PS12

A

54

2

no

low

low

120/45

III

III

P. orientale

PS13

A

-

-

-

-

-

-

IV

IV

C. ciliaris

B12-9

A

36

1

yes (same arm)

high

high

Ref. [37]

IV
IV

IV
IV

C. ciliaris
C. ciliaris

Higgins
PS185

A
A

36
-

1
-

yes (same arm)
-

high
-

high
-

Ref. [37]
-

IV

IV

C. ciliaris

PS186

A

-

-

-

-

-

-

IV

IV

C. setigerus

PS16

A

36

1

yes (same arm)

high

high

80/90

IV

IV

P. massaicum PS680

A

35

1

yes (diff arm)

no

high (on 90/50
27/35
chr)

IV

IV

P. massaicum PS953

A

35

-

-

-

-

-

IV

IV

P.
pedicillatum

PS304

A

36

1

no

high

high

80/90

IV

IV

A

54

1

no

mid

mid

105/70

IV

IV

A

54

1

no

high

high

60/90

V

V

P.
PS19
polystachion
PS163
P.
subangustum
P. mezianum PS9

A

32

1

no

high

low

80/90

V

V

P. nervosum

PS187

S

54

0

-

-

high

100/90

V

V

P. nervosum

PS38

S

-

-

-

-

-

-

none

V

P. ramosum

PS29

S

-

-

-

-

-

-

none

V

P. ramosum

PS63

S

10

0

-

-

low

80/50

none

none

P. basedowii

PS2

S

54

0

-

-

low

80/50

PS25

Phylogenetic (ndhF) and cytological (ASGR and Opie-2 retrotransposon) analyses of Pennisetum and Cenchrus species.

Characteristics of the ASGR-carrier chromosome in
Pennisetum species

FISH experiments revealed that morphologies of the
mitotic ASGR-carrier chromosomes among the species
were different; therefore, image analysis was carried out
to quantify the differences (Table 2, Figure 5). Threshold
values of gray and black levels in the ideograms were
assigned to display differences in chromatin density
between chromosome regions. The lengths of ASGR-

carrier chromosomes ranged from 3.37 μm in P. orientale (PS12) to 7.20 μm in P. setaceum (PS25). The
ASGR position was estimated based on the mid-point of
the P208 signal and was always shown to be in or near
a moderately to highly condensed region, again with the
exception of P. massaicum.
Lengths and arm ratios of the two ASGR-carrier chromosomes in PS12 were compared to each other by a
paired t-test, which showed a significant difference for

1.00
1.00

70.000000
70.000000

0.50
0.50

1.00
1.00

93.000000
93.000000

1.00
1.00

1.00
1.00

1.00
1.00
1.00
1.00
1.00
1.00

0.50
0.50
1.00
1.00

0.50
0.50
1.00
1.00

0.50
0.50

65.000000
65.000000

1.00
1.00

0.50
0.50
1.00
1.00

C. ciliaris186A

chromosome length (t = 3.16 P < 0.01) but not for arm
ratio (t = 0.39, P = 0.70). The DNA distribution on the
two ASGR-carrier chromosomes of PS12 showed different patterns as measured by DAPI staining intensity
(Figure 5). The ASGR-carrier chromosome PS12a had a
0.50
0.50

C. ciliaris185A

1.00
1.00
1.00
1.00

C. setigres16A

0.50
0.50

1.00
1.00

c102 BAC

1.00
1.00

0.50
0.50

P. massaicum953

1.00
1.00

1.00
1.00

P. massaicum680

1.00
1.00

0.50
0.50

P. squamulatum24

1.00
1.00

1.00
1.00

C. ciliaris185B

0.50
0.50

0.50
0.50

C. ciliaris186B

0.50
0.50

P. squamulatum26

1.00
1.00

C100 BAC

1.00
1.00
1.00
1.00

P. squamulatum158

0.50
0.50

P. mezianum9B

1.00
1.00

1.00
1.00

P. mezianum9A

1.00
1.00

1.00
1.00

C. setigerus16B

1.00
1.00
1.00
1.00

p203 BAC

1.00
1.00
1.00
1.00

P. setaceum22

1.00
1.00

P. setaceum25

0.50
0.50
0.50
0.50

P. villosum249

1.00
1.00

P. orientale15B

1.00
1.00

P. flaccidum32

1.00
1.00

P. orientale12

1.00
1.00

P. flaccidum95

0.50
0.50

P. orientale15A

P. pedicellatum304

P. subangustum163

P. polystachion19

Cenchrus myosuroides

Cenchrus incertus

Cenchrus echinatus

Cenchrus pilosus

Cenchrus brownii

Pennisetum setaceum

Cenchrus setigerus

Cenchrus ciliaris

Pennisetum purpureum

Cenchrus setigerus16

Cenchrus ciliaris185

Cenchrus ciliaris186

Pennisetum massaicum

Pennisetum massaicum953

Pennisetum mezianum

Pennisetum mezianum9

Pennisetum subangustum163

Pennisetum pedicellatum304

Pennisetum polystachion19

Pennisetum polystachion polyst

Pennisetum polystachion atrich

Pennisetum pedicellatum

Pennisetum ramosum

Pennisetum ramosum29

Pennisetum ramosum63

Pennisetum nervosum

Pennisetum frutescens

Pennisetum nervosum187

Pennisetum nervosum38

Pennisetum basedowii2

Pennisetum flaccidum

Pennisetum flaccidum32

Pennisetum flaccidum95

Pennisetum orientale13

Pennisetum orientale12

Pennisetum violaceum

Pennisetum squamulatum

Pennisetum glaucum1

Pennisetum sieberianum

Pennisetum glaucum2

Pennisetum glaucum

Pennisetum squamulatum158

Pennisetum squamulatum24

Pennisetum purpureum109

Pennisetum purpureum168

Pennisetum schweinfurthii

Pennisetum schweinfurthii243

Pennisetum setaceum25

Pennisetum setaceum22

Pennisetum chilense

Pennisetum montanum

Pennisetum tristachyum

Pennisetum latifolium

Pennisetum unisetum

Pennisetum basedowii

Pennisetum foermeranum

Pennisetum alopecuroides938

Pennisetum hohenackeri156

Pennisetum alopecuroides

Pennisetum orientale

Pennisetum thunbergii

Pennisetum macrourum

Pennisetum clandestinum

Pennisetum villosum

Pennisetum villosum249

Odontelytrum abyssinicum

Pennisetum trachyphyllum

Setaria sphacelata

Setaria parviflora

Setaria palmifolia

Pennisetum lanatum

Ixophorus unisetus

Stenotaphrum secundatum

Paspalidium geminatum

Setaria viridis

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Page 6 of 16

1.00
1.00

1.00
1.00

1.00
1.00
0.50
0.50

1.00
1.00
0.50
0.50

0.50
0.50

0.50
0.50

1.00
1.00
1.00
1.00

0.50
0.50
0.50
0.50

0.50
0.50
0.50
0.50

0.50
0.50
0.50
0.50

0.50
0.50
1.00
1.00

1.00
1.00
0.50
0.50

Modified from con 50 majrule

Figure 2 Comparative Bayesian tree. Bayesian tree based on ndhF+trnLF sequence alignments generated from combined analysis of
sequence matrix generated in present study and Chemisquy et al. [26].

highly condensed heterochromatic region on the long
arm that was confined to the pericentromeric area of
PS12b. The ASGR itself was located on the distal end of
the short arm in both ASGR-carrier chromosomes.
Based on mitotic chromosome characteristics, the two

85.000000
85.000000

62.000000
62.000000

97.000000
97.000000

0
0

Modified from PAUP 1

Figure 3 ASGR-based Maximum Parsimony tree. Maximum parsimony tree based on the sequence alignments generated from the ASGR in
the present study. Numbers at the nodes show bootstrap values obtained.

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Page 7 of 16

Figure 4 Physical mapping with ASGR-linked BACs on chromosome spreads from various Pennisetum and Cenchrus species. a-j, Colormerged images of FISH signals and inverted DAPI-stained chromosomes; red and green arrows indicate P001 and P208, respectively; insets
show enlarged, pseudo-colored ASGR-carrier chromosome. k-m, Images of dual-labeled FISH on DAPI-stained chromosomes. a, m: P. mezianum
(PS9); b: P. orientale (PS12); c: C. setigerus (PS16); d: P. polystachion (PS19); e: P. setaceum (PS25); f: P. flaccidum (PS95); g: P. subangustum (PS163); h:
P. villosum (PS249); i: P. pedicillatum (PS304); j: P. massaicum; k: P. massaicum spread in panel j stripped and rehybridized with rDNA and P602
(red); green arrows and signals indicate rDNA; outlined chromosomes did not hybridize with P602. l: C. setigerus hybridized with rDNA and P208;
green signals are rDNA and red arrow indicates P208 signal. m: P. mezianum (PS9) hybridized with P208 (red arrow) and P602 (green signal). Bars
correspond to 10 μm.

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Cluster
I

Species

ASGR-Carrier Chromosome
Ideograms

Page 8 of 16

squamulatum and C. ciliaris are from Akiyama et al. (2004) and
Akiyama et al. (2005), respectively.

P. setaceum

ASGR-carrier chromosomes in PS12 were heteromorphic and suspected to be homeologous rather than
homologous chromosomes. However, physical mapping
of paired chromosomes at the pachytene stage of meiosis using BACs P001 and P208 showed that the two
ASGR-carrier chromosomes formed bivalents with one
another as would be expected of homologs (Figure 6). A
heterochromatic knob was observed in the ASGR.
Morphology of the ASGR-carrier chromosome in C.
setigerus was similar to that of C. ciliaris (Figure 5)
(data from [37]) and a t-test showed no significant difference in chromosome length (t = 0.18 P = 0.85), arm
ratio (t = 0.87, P = 0.38), or signal position of P208 (t =

P. squamulatum

II

P. villosum

III

P. flaccidum
P. orientale A
P. orientale B

IV

C. ciliaris
C. setigerus
P. massaicum
P. pedicillatum
P. polystachion
P. subangustum

V

P. mezianum

Chromatin density
Opie-2 signal intensity

BAC P208 signal

Gap in Opie-2 signal

45S rDNA signal

Figure 5 ASGR-carrier chromosome ideograms for apomictic
Pennisetum and Cenchrus species clustered according to the
phylogenetic analysis. In ideograms, dark, medium and light blue
indicate chromatin condensation pattern (regions of high, middle
and low condensation, respectively). Red and yellow circles indicate
BAC P208 and 25S rDNA, respectively. Opie-2 distribution, as
determined by P602 signal, is indicated as bars below each
ideogram and intensity of shading represents the approximate
intensity of P602 signal. Asterisks indicate the position of
discontinuous P602 signal. Bar corresponds to 1 μm. The data of P.

Figure 6 Physical mapping of BAC clones on pachytene
chromosomes of P. orientale (PS12). Upper image: Color-merged
images of DAPI and FISH signals. Red and green signals are P001
and P208, respectively. Lower image: inverted DAPI image. Arrow
indicates knob in the ASGR. Bar corresponds to 10 μm.

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1.08, P = 0.28). The signal position of C101, an ortholog
of P208 was used in C. ciliaris.
The ASGR-carrier chromosome in P. massaicum
(PS680) showed unique morphology among the species
with a highly condensed region in the middle of the
long arm (Figure 5). PS953, another P. massaicum
accession, showed the same ASGR-carrier chromosome
characteristics as PS680. The morphology of the ASGRcarrier chromosome was also sufficiently unique within
this species such that it sometimes could be distinguished under phase contrast without Giemsa staining
or FISH (Figure 4j, Figure 7). Comparison of the
Giemsa-stained chromosomes having rDNA indicated
that morphology was different among them (Figure 7).
Distribution of Opie-2 like retrotransposons

BAC P602 contains an Opie-2-like retrotransposon
abundant only in the ASGR of P. squamulatum, but
occurring throughout the genome of C. ciliaris [37].
The distribution of this repetitive sequence was clearly
different in two species that often are grouped as one, P.
mezianum (PS9) and P. massaicum (PS680) [26]. In P.
mezianum with 32 chromosomes, only the ASGR-carrier

Page 9 of 16

chromosome showed signal from hybridization of P602
(Figure 4m), whereas in P. massaicum, 27 out of 35
chromosomes, including the ASGR-carrier chromosome,
showed intense signal with this BAC (Figure 4k). This
retrotransposon family is present at varying copy numbers in different species within each cluster, regardless
of mode of reproduction (Table 2). For example, in
clade V it is abundant in P. nervosum but barely detectable in P. ramosum, both sexual species. Mainly two
patterns emerged in the apomicts, repeats detectable
flanking the ASGR plus either distributed throughout
the genome (buffelgrass pattern) or confined to the
ASGR-carrier chromosome (P. squamulatum pattern).
The latter pattern occurred in only two other species, P.
setaceum and P. mezianum. The Opie-2-like repeat was
of low abundance flanking the ASGR in P. setaceum, P.
villosum, and P. massaicum.
Phylogenetic reconstruction based on reproductive and
cytological features

Reconstruction of ancestral states was done by Mesquite. Four characters viz mode of reproduction, basic
chromosome number, distribution of Opie-2 on genomes, and distribution of Opie-2 on the ASGR (Table 2)
were used for reconstruction of ancestral states using
the tree generated by Bayesian method (Figure 8).

Discussion
Phylogenetic analysis

Figure 7 Ordered chromosomes based on Giemsa- and DAPIstained chromosomes of P. massaicum (PS680). These
chromosomes are from the spreads of Figure 1j and k and were
sorted according to their lengths. The bottom two rows of
chromosomes are the eight that did not hybridize with BAC P602.
Black and white arrow heads indicate ASGR and rDNA, respectively.

Earlier attempts to resolve phylogenetic relationships
among different species in the genus Pennisetum or
higher taxonomic levels including Pennisetum were
based on multiple approaches such as genome size variation [39], molecular markers [40,41], and DNA
sequence information from a) the internal transcribed
spacer region of ribosomal DNA [24], b) the nuclear
gene knotted [42,26], and c) the chloroplast genes ndhF
[25,26,42-44], rpoC2 [45], and rpl16/trnL-F [26,27]. The
present study, focused on apomixis, included twelve
apomicts and eight sexual species. The sequence data
obtained was carefully analyzed and resulted in a robust
tree with clear groupings which can be attributed to
quality sequence data. Our results show congruence
with earlier studies of these genera, but also emphasize
a few notable exceptions.
Chemisquy [26] recently used trnL-F and ndhF for
conducting a phylogenetic analysis. A combined analysis
of their sequences along with the sequences used in this
study generated a tree presented in Figure 2. Combined
analysis did not show any variation from the individual
analysis of the two datasets. The tree from the combined analysis did show discrepancies in the placement
of a few species from the present analysis as compared
to the one from Chemisquy [26]. These discrepancies

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Page 10 of 16

x=5
x=7
x=8
x=9

1A – Basic chromosome number

Low
High

1C – Presence of Opie2 in genome

Low
High

1B – Presence of Opie2 in the ASGR

Apomictic
Sexual

1D – Mode of reproduction

Figure 8 Ancestral states for different characters. Bayesian trees based on the ndhF+trnL-F sequence showing the ancestral state for different
characters. A. Chromosome number, B. Presence of Opie-2 at ASGR, C. Opie-2 distribution on genome, D. Mode of reproduction

could be due to errors in species identification or DNA
sequence.
Previous studies of nuclear and chloroplast gene
sequences suggested that, within the bristle clade of
grasses, the genus Cenchrus was monophyletic and
embedded in a polyphyletic Pennisetum [42]. Polyphyly
in Cenchrus was supported by additional chloroplast
sequence data and taxon sampling [27]. Our data support the placement of Cenchrus within Pennisetum, but
are incongruent with the result of Donadio [27] and
Chemisquy [26] that showed a close relationship
between one accession of P. purpureum, P. setaceum,
and Cenchrus spp. In the ITS-based phylogeny of Martel
[24], P. setaceum and P. villosum grouped with species
from section Brevivalvula in a clade that included Cenchrus ciliaris; therefore, their placement of P. setaceum,
but not P. villosum, was more consistent with Donadio
[27]. According to Chemisquy [26], P. villosum grouped
away from Cenchrus species and Brevivalvula section. In
contrast, results from the present study place P. setaceum as a member of a large clade that included the
cultivated species P. glaucum and excluded species from
section Brevivalvula while P. villosum grouped with P.
hohenackeri and P. alopecuroides. Doust and Kellogg
[23] also found P. setaceum to be closely related to P.

glaucum. Section Brevivalvula is considered more homogeneous compared with other sections in the genus Pennisetum [8] and morphologically is well differentiated
from the other sections [46]. The non-concordance
between relationships inferred from ITS and cpDNA
data sets could be due to a number of factors. First, the
ITS is part of a genomic region known to be affected by
the process of concerted evolution [47]. As with incomplete lineage sorting, concerted evolution can lead to
the phylogenetic association of lineages that did not
share a most recent common ancestor. In contrast to
the ITS, ndhF is a chloroplast gene and thus likely not
susceptible to the process of concerted evolution,
although nuclear capture of chloroplast DNA is possible
[48] but untested in this case.
The placement of P. setaceum in the P. glaucum clade
is supported by the current and prior [23] chloroplast
phylogeny and crossability studies [49]. This clade also
includes P. purpureum, P. squamulatum, and P.
schweinfurthii. P. squamulatum, recently proposed by
Akiyama et al. [50] to be a member of the secondary
gene pool (i.e., the group of biological species that will
cross with the crop species [51]), has strong support
from ndhF and cytological [50] data for a close relationship with P. glaucum (the primary gene pool) and P.

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purpureum (secondary gene pool). Since revision of the
basic chromosome number of P. squamulatum from x =
9 to x = 7 [50], all species in clade I, except for P. setaceum, have a basic chromosome number of x = 7 and
all (including P. setaceum) can be crossed with P. glaucum [52-55]. The accessions of P. purpureum we investigated were strongly supported as sister to P. glaucum,
confirming placement of this species in the secondary
gene pool of Pennisetum [56]. Natural hybrids have
been reported between P. glaucum and P. purpureum,
and one genome of P. purpureum has been suggested to
be homologous with pearl millet [57].
Based upon the ndhF data, the apomictic pentaploid P.
villosum is most closely related to the sexual diploids, P.
hohenackeri and P. alopecuroides. P. hohenackeri and P.
alopecuroides also grouped together in an ITS-based
phylogeny [24], while P. alopecuroides grouped with P.
villosum based on EST-microsatellites [41] and the
knotted-1 gene sequence [42], further supporting a close
phylogenetic relationship between all three species. In
contrast, P. alopecuroides was more closely related to P.
glaucum than P. villosum based on the chloroplast
sequence data of Donadio [27]. Significantly, meiotic
chromosome configurations in pentaploid P. villosum
are also consistent with its derivation through hybridization [57], and incongruence between studies could result
from multiple hybrid origins or ploidy level differences
among accessions.
A third group in the dendrogram consisted of two
apomictic species, P. orientale and P. flaccidum. P.
orientale and P. flaccidum were also closely related
based upon EST- microsatellite data, even though the P.
orientale cytotype examined was 2n = 2x = 18 and likely
sexual [41], as opposed to the 6x apomictic cytotypes
that we studied. The position of P. basedowii is uncertain. While Bayesian analysis places P. basedowii basal
to subgroups IV and V, MP analysis shows it as sister to
subgroups III, and IV/V. No previous phylogenetic studies have incorporated P. basedowii except for Chemisquy [26] where it grouped with P. glaucocladum, a
species not included in the present analysis, and to
which P. flaccidum was basal. Neither does our combined analysis (Figure 2) put the accession used by Chemisquy [26] with the one used in present study. In the
combined analysis, the position of the P. basedowii
accession used in this present study is consistent with
that of our Bayesian analysis.
Clade IV included 1) all three Pennisetum species
from section Brevivalvula (P. polystachion, P. pedicillatum, P. subangustum), 2) Pennisetum species from outside this section, P. massaicum, and 3) the Cenchrus
species. The close relationship between P. polystachion
and P. pedicillatum of section Brevivalvula was also
indicated by ITS and SSR data [24,41]. Furthermore,

Page 11 of 16

intermediate morphotypes and shared chloroplast haplotypes suggest considerable gene flow between species of
this section [58]. All species in clade IV contain apomictic cytotypes and have basic chromosome numbers of x
= 9, except for P. massaicum where the basic chromosome number is unclear. P. massaicum, with 35 chromosomes, 8 of which do not hybridize to the Opie-2like repeat, may be an aneuploid (4x-1) as the number
of rDNA loci suggests that it is tetraploid or it may
have been produced by interspecific hybridization
between species with basic chromosome numbers of x =
8 (low Opie-2 abundance, e.g., P. mezianum) and x = 9
(high Opie-2 abundance; e.g. Cenchrus). Another x = 8
species, P. montanum, falls outside of the Cenchrus
clade [27], but was not included in our study. All species
in clade IV contain apomictic cytotypes, predicting some
degree of asexual reproduction in all lineages. Hybridization between the largely asexual apomicts is possible
through rare fertilization of reduced or unreduced eggs
[59]. One accession of P. orientale used in the present
study uniquely showed two ASGR-carrier chromosomes,
each with a different morphology but able to pair at
meiosis, and may have resulted from fertilization of an
unreduced egg with reduced pollen, both carrying an
ASGR. In the case of the derivation of the apomictic P.
massaicum, such hybridization would have necessarily
involved at least one parental apomictic lineage that
contributed the ASGR-carrier chromosome. Pollen fertility in Pennisetum apomictic lineages is required for
pseudogamous apomixis, in which the endosperm develops only after fertilization of the central cell. Normal
male meiosis, giving rise to reduced pollen is typical,
but irregular segregation of genomes through aberrant
meioses also occurs. Either interspecific or intergeneric
hybridization may have precipitated speciation of P.
massaicum. Genomic in situ hybridization (GISH) studies could provide valuable insights regarding the putative hybrid origin of this species.
The fifth cluster contains sexual P. ramosum, P. nervosum and apomictic P. mezianum with divergent basic
chromosome numbers (x = 5, x = 8 and x = 9, respectively). Apomictic P. mezianum did show deviation in its
grouping among MP and Bayesian trees. The two sexual
species in this group differ in their distribution of Opie2-like repeats, abundant throughout the genome of P.
nervosum, but barely detectable by FISH in P. ramosum.
Even though P. ramosum has the smallest number of
chromosomes of any Pennisetum species (2n = 2x = 10),
it has one of the highest DNA contents estimated per
haploid genome (2.02 pg [39]) and the highest per chromosome (0.4 pg). Genome expansion through repetitive
DNA amplification is the most likely explanation,
although our results indicate that Opie-2-like repeats
probably played a minor role. These two species were

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positioned in separate clades based on the analysis of
Donadio [27]. Analysis based on trnL-F and ndhF did
put P. mezianum with P. ramosum but away from P.
nervosum [26].
Evolution of the ASGR-carrier Chromosome

Ancestral analysis shows x = 9 as the ancestral condition
and the other three basic chromosome numbers (x = 5,
x = 7, and x = 8) as derived states which originated
independently of each other (Additional File 1 Figure
S1A), which is in congruence with earlier studies
[26,27]. Ancestral analysis based on reproductive behavior (Additional File 1 Figure S1D) did not resolve sexuality or apomixis as being plesiomorphic. From the
sequence information generated from the ASGR and the
fact that the low copy BACs are shared by all the apomictic species, it is more likely that apomixis is the
result of a single event as suggested earlier [18] which
spread to other species through hybridization.
The three low-copy ASGR-linked BACs used in the
present study produced a discrete signal only in aposporous species confirming that these BAC sequences
have conserved homology in all aposporous species of
the genus Pennisetum and the closely related genus,
Cenchrus. By definition, therefore, all aposporous Pennisetum species/cytotypes possess an ASGR, while sexual
species/cytotypes of the genus lack an ASGR. These
results are consistent with our previous report for C.
ciliaris, where an ASGR, existing as a heterochromatic,
largely hemizygous chromosomal region on a heteromorphic chromosome, was observed in only aposporous
and not in sexual cytotypes [37]. Interestingly, the morphology of the ASGR-carrier chromosome and the position of the ASGR on the chromosome differ among the
species, indicating that the ASGR-carrier chromosome
has undergone rearrangement. Location of the ASGR in
a telomeric and condensed region of the chromosome
occurs in all clades containing apomictic cytotypes and
may thus be the ancestral state. In all Pennisetum species, the ASGR is located near the telomere of the chromosome, while it is interstitial in Cenchrus species and
inverted relative to P. squamulatum [34,35]. Given that
the intercalary position and linkage with rDNA on the
same chromosome arm are unique characters in Cenchrus, this is more likely to be a derived state. Additional data concerning the orientation of the ASGR, as
ascertained using FISH on the pachytene chromosomes
of all species are needed to test this hypothesis.
As explained earlier, an 800 bp region was amplified
from the ASGR and analyzed to further understand the
evolution of ASGR. This region was previously known
to be duplicated at the ASGR in P. squamulatum and C.
ciliaris [38]. The present investigation could detect two
copies in some species but not all the species

Page 12 of 16

investigated. Additional sequencing from the locus
could help to discover whether the locus is also duplicated in these species. This region of ASGR duplication
is recent and happened before the ASGR was transferred between Pennisetum and Cenchrus as one of the
copies from Cenchrus shows high similarity with Pennisetum. Among the species where duplication could not
be detected are the three species in section Brevivalvula,
which also group together based on the sequence from
the ASGR suggesting that they may contain the more
ancient form of the ASGR. Overall, variation assessed in
this region of the ASGR is very low. Although it is possible that there is an inconstant rate of evolution
between different regions of the ASGR, the level of variation detected suggests a recent origin of the ASGR.
Because of the low level of variation, the tree obtained
is of very low resolution (Figure 3). However, the tree
could still discriminate section Brevivalvula from other
species in the Pennisetum-Cenchrus complex.
Opie-2-like sequences were found to be abundant in
P. squamulatum only at the ASGR [36]. In contrast,
these sequences were associated with the centromeric
regions of all chromosomes in C. ciliaris [37]. In both
species, a repeat-poor portion of the ASGR is flanked by
Opie-2-rich regions [18]. Since this repeat now has been
detected as part of the ASGR in seven out of 12 aposporous species (exceptions include P. setaceum and P.
villosum of clades I and II, P. orientale and P. flaccidum
of clade III and P. massaicum of clade IV), we speculate
that the association of this repeat with the ASGR was
derived by either 1) translocation of the repeat-poor
portion of the ASGR into a repeat-rich region of the
genome or 2) transposition and accumulation of retrotransposons in proximity to the ASGR. Ancestral stage
analysis (Additional File 1 Figure S1C) did not show
either of the two (low and high) patterns of Opie-2 distribution on the genome as plesiomorphic, although
interestingly, low abundance of Opie-2 on the ASGR
(Additional File 1 Figure S1b) might be plesiomorphic.
Low abundance of this transposon repeat in the ASGR
species mentioned above could also be due to transposon elimination, although genome reduction seems less
likely than genome expansion for recently derived asexual taxa where recombination is suppressed [17]. Paradoxically, ancient, strictly asexual taxa such as bdelloid
rotifers are devoid of retrotransposons [60], and genome
expansion in strictly sexual Pennisetum species often has
exceeded that of the apomicts surveyed [39]. Transposable elements can spread more efficiently in sexually
reproducing populations, although sex also affords a
mechanism for purging the genome of deleterious mutations [61].
Deciphering the complex relationship between transposable element dynamics and mode of reproduction is

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further complicated by events of hybridization (intra- or
inter-specific) involving apomicts in diploid-polyploiddihaploid cycles [10]. Hybridization can elicit gene
expression, epigenome, and genome structural changes
[62] some of which have been documented in apomictic
Boechera [63]. One explanation for a pattern of Opie-2like repeats as identified in P. squamulatum and P.
mezianum, where repeats are clustered in the ASGR but
are of low abundance in the remainder of the genome,
is hybridization and introgression of the ASGR from an
Opie-2-rich genome to an Opie-2- poor genome. An
alternative explanation is local expansion of Opie-2-rich
repeats. The unique features of the ASGR-carrier chromosome in P. massaicum, the species most closely
related to Cenchrus, may reflect chromosome restructuring, perhaps as a consequence of hybridization, to distance the ASGR from either heterochromatin or an
Opie-2-rich region yet retain its linkage to rDNA as in
C. ciliaris and C. setigerus, albeit on the opposite chromosome arm compared with Cenchrus species.
The ASGR is present only in apomictic cytotypes of
Pennisetum species that either have diploid or higher
ploidy sexual cytotypes [57] or are closely related to
other species with only sexual cytotypes. The coexistence of sexual cytotypes, diversity of ASGR-carrier
chromosome structure, and yet phenotypic similarities
in the apomixis mechanism and conservation of the
ASGR argue for apomixis as a character that predates
speciation thus has been subject to repeated transfer via
introgressive hybridization.

Conclusions
The present investigation provides interesting insights
not only on the phylogeny of genus Pennisetum and
Cenchrus, but also on the origin and evolution of the
ASGR. FISH results reveal structural similarity within
the ASGR across Pennisetum and Cenchrus apomictic
species. This similarity is further supported by an ~800
base pair sequence generated from the ASGR. The fact
that apomictic species cluster with sexual species in the
chloroplast sequence-based phylogeny supports the view
that apomixis probably originated once and then spread
through repeated hybridization between the species. The
presence of different morphologies for the ASGR-carrier
chromosome in different species and variation in linkage
with rDNA infers that the ASGR can be translocated
within the genome and those genomes can support
gross chromosomal aberrations as a consequence. Polyploidy associated with apomixis likely increases tolerance of the genome to mutation and chromosomal
aberrations. It has often been speculated that apomicts
cannot be sustained in nature for a long period of time
due to their propensity for accumulating mutations in
the absence of sexual reproduction. Low rates of

Page 13 of 16

sequence variation at the ASGR, therefore, suggest that
the ASGR might be of recent origin.
Earlier studies from P. squamulatum and C. ciliaris
have shown association of high-copy Opie-2-like
sequences with the ASGR. Various species within the
present investigation have an ASGR containing fewer
copies of Opie-2-like sequences, from which we infer
that association of the Opie-2 sequence with the ASGR
is only a genome specific feature rather than a unique
feature associated with apomixis. Ancestral state analysis
suggests that the ASGR might have originated in a genome with low abundance of Opie-2 and was transferred
to a high Opie-2-copy genome while independent duplication of low-copy regions within the ASGR also
occurred. P. massaicum, which groups with Cenchrus
spp. and section Brevivalvula species, is a species where
genomes with low Opie-2 at the ASGR and high abundance of Opie-2 across the chromosome complement
coexist, and most likely were merged by hybridization.
Section Brevivalvula shows variation in distribution of
Opie-2 on the ASGR and throughout the genome with
P. polystachion displaying lower Opie-2 signal than the
other two species examined. These observations warrant
investigation into other species from the section. Additional sequence data from the ASGR also could resolve
the extent of gene duplication in this region.
In conclusion, the present investigations have provided
new insights into structure of the ASGR and its evolution based on experimental evidence. Further studies in
this direction can lead to information which can help in
deciphering the intrigues of apomixis.

Methods
Plant materials

Plant materials and their origins are listed in Table 1.
All species were grown in a greenhouse on the Tifton
Campus of the University of Georgia. For pachytene
chromosome preparation, immature panicles were collected and fixed in 3:1 ethanol:acetic acid for 2 days at
room temperature. The fixed material was stored at 4°C
and used within one year. For mitotic chromosome preparation, root tips of all species were collected and pretreated for 3 h by soaking in a saturated solution of abromonaphthalene on ice before fixation in 3:1 ethanol:
acetic acid.
Cytological analysis

Fluorescence in situ hybridization was carried out
according to Akiyama [36]. Enzymatic maceration of
root tips and anthers for chromosome spreads and
denaturation for probe hybridization were conducted
using the indicated times for each species (Table 2).
ASGR-linked bacterial artificial chromosome (BAC)
clones (P001 - containing SCAR A14M; P109 - SCAR

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
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Q8M; P208 - SCAR UGT197) have been described previously [35,38,64]. A BAC (P602) containing SCAR marker X18 and a large amount of repetitive DNA was
isolated by PCR screening of pooled BAC DNAs [36].
The BAC clones were labeled with Biotin-16-dUTP
(Roche, Indianapolis, IN) or digoxigenin-11-dUTP,
alkali-stable (Roche) by nick translation. Ribosomal loci
were detected using a 25S rDNA probe from rice [65]
cloned in pCR 2.1-TOPO (Invitrogen, Carlsbad, CA).
The pCR 2.1-TOPO insert was labeled using PCR
amplification with M13 primers. Denatured chromosomes were incubated with 10 μl denatured hybridization mixture consisting of approximately 5 ng/μl biotinor digoxigenin-labeled probe, 5% dextran sulfate, and
50% formamide in 2X SSC in a humidified chamber at
37°C overnight. After hybridization, the digoxigeninlabeled probes were detected with fluorescein using a
fluorescent antibody enhancer set (Roche). Biotinlabeled probes were detected with Texas-red streptavidin (Vector Laboratories, Burlingame, CA) and biotinylated anti-streptavidin (Vector Laboratories) for a
second layer of Texas-red streptavidin. After detection,
the slides were mounted in Vectashield (Vector Laboratories) containing 1.5 μg/ml DAPI and observed under a
fluorescence microscope, Olympus BX50. Images of
chromosomes were captured by a Sensys CCD camera
(Sensys Photometrics, Tucson, AZ) and Image Pro ver
4.1 software (Media Cybernetics, Silver Spring, MD).
Image analysis was performed with Object-Image 2.08
(http://simon.bio.uva.nl/object-image.html) and modified
CHIAS3 [66]. The statistical analysis was carried out by
Microsoft Excel 98 (Microsoft, Redmond, WA).
Amplification and sequencing of chloroplast NADH
dehydrogenase, ndhF, trnL-F and ASGR BBM-like genes

Total DNA was extracted from fresh leaves using the
procedure of Tai and Tanksley [67] or following the
protocol from the DNAeasy Plant Mini Kit (Qiagen,
Valencia, CA). All PCR amplifications were carried out
in a GeneAmp ® PCR system 9700 thermal cycler
(Applied Biosystems, Carlsbad, CA). The 3’ end of the
ndhF gene was amplified using the primers 972 and
2110R [29]. Amplification was carried out in 50 μl reactions containing 1X reaction buffer supplied by the
manufacturer plus 2.5 μM MgCl2, 0.2 mM dNTPs, 1.5
U Taq polymerase, and 0.2 μM of each primer. Cycling
conditions were denaturation at 94°C for 5 min followed
by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for
1.5 min, and a final extension for 7 min at 72°C. The
amplified products were purified using the Qiaquick
PCR purification kit (Qiagen, Valencia, CA). The purified products were sequenced using primers 972, 1603,
1603R and 2110R. In a few species, primers 1318,
1318R, 1955 and 1955R were used to complete the

Page 14 of 16

sequencing of amplified fragments. Sequencing was carried out on a Beckman CEQ8000 (Beckman Coulter,
Fullerton, CA) according to the manufacturer’s instructions. The trnL-F region was amplified using the primers trnL-F_c and trnL-F_f as per [68]. The ASGRBBM-like region was amplified using the primers p779/
p780. These primers amplify only apomicts from segregating F1 populations of both Pennisetum and Cenchrus
(unpublished results) and showed amplification only in
apomictic and not the sexual species in the present
study. Amplification was carried out in 50 μl reactions
containing 50-75 ng template DNA, 1X iProof GC buffer, 200 μm each dNTP, 0.5 μM each of trnL-F or
ASGR-BBM-like primers and 0.02 U/μl iProof DNA
polymerase (Bio-Rad Laboratories, Hercules, CA). The
cycling conditions were one cycle of 98°C for 30 s followed by 30 cycles of 98°C for 8 s, 60°C (trnL-F)/52°C
(ASGR-BBM like) for 20 s, 72°C for 25 s with a final 72°
C extension for 10 min. The amplified products were
ligated with the pCR®-BluntII-TOPO® vector (Invitrogen) and transformed into NEB E. coli DH5a following
the manufacturer’s instructions (New England BioLabs,
Ipswich, MA). For each trnL-F DNA template 3 clones
were fully sequenced. Sequencing was performed by the
Georgia Genomics Facility, (Athens, GA) using M13
Forward and Reverse primers. For each ASGR-BBM-like
DNA template 5 clones were sequenced using M13 Forward primer.
Phylogenetic Analysis

The nucleotide sequences generated were aligned by
Clustal X ver 1.81 [69]. The aligned sequences were
translated and compared with protein information for
ndhF available from the whole chloroplast genome
sequence in Nicotiana tabacum (Accn. No. Z00044
S54304). Sequence chromatograms were cross-checked
at positions where mutations were predicted. The corrected alignment was used for phylogenetic analysis.
The analysis was done by PAUP 4.0 beta 10 for windows [70] and Mr. Bayes 3.1.2 [71]. Mr. Model test V2
[72] was used to decide the best-fit model for parsimony
and Bayesian analysis. Parsimony analysis was done by
PAUP with 100 replicates being used for bootstrap analysis. For Bayesian analysis, Mr. Bayes was used and the
analysis continued until standard deviation of split frequencies became less than 0.01. For analysis of 29 taxa
analyzed in this study, analysis was run for 400,000 generations while for the combined analysis of sequences
with sequences from present investigation and Chemisquy et al. [26], analysis was run for 3,200,000 generations to achieve the desired standard deviation of split
frequencies. All trees were viewed by the program Tree
View ver 1.6.1 [73]. Mesquite ver 2.74 [74] was used to
alter the tree or to do phylogenetic reconstruction based

Akiyama et al. BMC Evolutionary Biology 2011, 11:289
http://www.biomedcentral.com/1471-2148/11/289

on a character to identify the ancestral state for that
character.

Page 15 of 16

9.

10.

Additional material
Additional file 1: Name and sequence of ASGR primers tested on
the species.

11.

12.
13.

Acknowledgements
We are grateful for the technical assistance of Anne Bell, Evelyn Perry,
Gunawati Gunawan and Jacolyn Merriman. We thank Dr. R. Geeta for the
help in carrying out sequence analysis. We thank Dr. Nobuko Ohmido for
providing rDNA plasmid, Prof. Kiichi Fukui and Mr. Seiji Kato for technical
advice for image analysis, and Dr. Michael Arnold for critical reading of the
manuscript. This work was supported by the National Science Foundation
(grant no. 0115911).
Author details
1
Department of Horticulture, The University of Georgia, 2360 Rainwater Rd.,
Tifton, GA 31793-5766, USA. 2Department of Crop and Soil Sciences, The
University of Georgia, 2360 Rainwater Rd., Tifton, GA 31793-5766, USA.
3
Graduate School of Agriculture, Iwate University, 3-18-8 Ueda, Morioka,
Iwate 020-8550, Japan.
Authors’ contributions
SG produced sequence for the ndhF chloroplast gene, produced and
analyzed all the evolutionary data and wrote those corresponding parts of
the paper. YA produced and did image analysis of the chromosomal and
FISH data and wrote those corresponding parts of the paper. JC produced
the sequence for the ASGR-BBM-like and trnL-F chloroplast genes and
participated in their analysis and writing of results. PO-A conceived of the
project, provided guidance, secured funding for the study, and coordinated
manuscript preparation. WWH provided plant research material and
contributed to cytological analysis. HY-A contributed with image analysis
and reconfirmed the PS680 rDNA results. All authors have read and
approved the manuscript.
Authors’ information
Current addresses:
YA: Livestock and Forage Research Division, Tohoku Agricultural Research
Center (TARC), National Agriculture and Food Research Organization (NARO),
Akahira 4, Shimokuriyagawa, Morioka, Iwate 020-0198, Japan
SG: Department of Botany, University of Delhi, Delhi 110007, India
Received: 29 June 2011 Accepted: 5 October 2011
Published: 5 October 2011
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Cite this article as: Akiyama et al.: Evolution of the apomixis
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11:289.

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