Polyploid evolution in Oryza officinalis complex of the genus Oryza
Graduate University of the Chinese Academy of Sciences
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences
Background: Polyploidization is a prominent process in plant evolution, whereas the mechanism and tempo-spatial process remained poorly understood. Oryza officinalis complex, a polyploid complex in the genus Oryza, could exemplify the issues not only for it covering a variety of ploidy levels, but also for the pantropical geographic pattern of its polyploids in Asia, Africa, Australia and Americas, in which a pivotal genome, the C-genome, witnessed all the polyploidization process. Results: Tracing the C-genome evolutionary history in Oryza officinalis complex, this study revealed the genomic relationships, polyploid forming and diverging times, and diploidization process, based on phylogeny, molecular-clock analyses and fluorescent in situ hybridization using genome-specific probes. Results showed that C-genome split with B-genome at ca. 4.8 Mya, followed by a series of speciation of C-genome diploids (ca. 1.8-0.9 Mya), which then partook in successive polyploidization events, forming CCDD tetraploids in ca. 0.9 Mya, and stepwise forming BBCC tetraploids between ca. 0.3-0.6 Mya. Inter-genomic translocations between B- and Cgenomes were identified in BBCC tetraploid, O. punctata. Distinct FISH (fluorescent in situ hybridization) patterns among three CCDD species were visualized by C-genome-specific probes. B-genome was modified before forming the BBCC tetraploid, O. malampuzhaensis. Conclusion: C-genome, shared by all polyploid species in the complex, had experienced different evolutionary history particularly after polyploidization, e.g., inter-genomic exchange in BBCC and genomic invasion in CCDD tetraploids. It diverged from B-genome at 4.8 Mya, then participated in the tetraploid formation spanning from 0.9 to 0.3 Mya, and spread into tropics of the disjunct continents by transcontinentally long-distance dispersal, instead of vicariance, as proposed by this study, given that the continental splitting was much earlier than the C-genome species radiation. We also find reliable evidence indicated that an extinct BB diploid species in Asia was presumptively the direct genomic donor of their sympatric tetraploids.
Polyploidization is a prominent process in the evolution
of high plants. Between 50% and 70% of angiosperm
species were identified as polyploids by intensive screening,
while recent studies estimated that up to 100% of
angiosperms underwent genome duplication at least once
in their evolutionary history [1,2]. The commonity of
polyploidy suggests a potential advantage of polyploids to
survive better in harsh environments than diploids [3,4].
Thronged facts related to polyploidy were discovered, e.g.,
genomic divergence in allopolyploids by diploidization,
rapid genomic changes, and inter-genomic invasion [5-9].
However, more evidence is needed to reveal the
mechanism and tempo-spatial process of polyploidization.
Polyploid complex, a group of species with a variety of
ploidy levels, could be an ideal model to address the
problems. Oryza officinalis complex is an excellent example,
not only because it contains diploids and
heterochronically formed polyploids, but also it has a "pivotal
genome" , the C-genome, which participated in all
the polyploid formation, potentially as an inner criterion
to trace polyploid evolution. Moreover, geographic
patterns of the polyploids distributed pantropically to
isolated continents within a rather recent period, have
remained mysterious [11-15].
With agricultural importance, the genus Oryza comprises
23 species including cultivated rice, combined into four
species complexes [16-19]. In the last decades, molecular
methods have been used to reconstruct species phylogeny
and to trace evolution process in Oryza [14,15,20-26]. Ten
distinctive genomes were identified on the basis of DNA
sequences , or genomic in situ hybridization (GISH)
[27-29]. Particularly in O. officinalis complex, the largest
in Oryza, genomic relationships were found
extraordinarily complicated, e.g., the BBCC tetraploid species formed
independently with different parenthood by three
polyploidization events, in which O. eichingeri was the
maternal parent of tetraploid O. punctata while diploid O.
punctata was that of tetraploids O. malampuzhaensis and O.
minuta [14,22,30,31]. Furthermore, three tetraploid
species with CCDD genomes were assumed to be formed by
one polyploidization event, where the CC genome
progenitor served as the maternal parent [14,21,22,32,33].
Additionally, it seems much intriguing that the C-genome
diploids and tetraploids are distributed across Asian,
African and American tropics. And the CCDD tetraploids are
entirely endemic to Central and South Americas where no
extant diploid with C- or D-genome was found [11,12].
Therefore, the questions arose: (a) How was the
Cgenome, as the pivotal genome in all the tetraploids,
differentiated after polyploidizating? (b) When were the
tetraploids formed and how did they spread
transcontinentally? (c) Whether inter-genomic interaction, e.g.,
exchange or invasion, happened in the allopolyploids?
Focused on the questions, we reconstructed the
phylogenetic relationship, dated the divergence time among the
genomes in O. officinalis complex, and detected genomic
changes thereafter polyploidization by FISH methods
with genome-specific probes. The goal of this study is to
reveal the evolution history of the O. officinalis complex,
particularly the polyploidization and its genomic impact,
by tracing C-genome differentiating and dispersing
Thirty eight accessions representing eleven species of O.
officinalis complex were sampled, and one accession of O.
granulata, a species outside the complex, was used as
outgroup (Table 1). Of them, eight species with different
ploidy levels and geographic origins were used for
cytogenetic analysis. All the accessions used in this study, are
showed in Table 1, including their species names, genome
constitutions, original collection locations and GenBank
accession numbers. Total DNAs were extracted from fresh
leaves of individual plants by the CTAB method .
Primer design, PCR amplification and sequencing
Two genes, Starch debranching enzyme (SDBE) on
chromosome 4 and Os02 g0125000 (Os125) on chromosome 2 of
O. sativa, were chosen in the present study. SDBE is a
single copy gene , containing 25 introns, in which the
seventh was used in this study. Os125 is also identified as
a single copy gene by the criterion previously reported
, which had three introns and the second one was
selected. Primers used for PCR amplifying and sequencing
are listed in Table 2.
Amplification and purification of the PCR products were
performed by standard methods. Purified PCR products
were sequenced directly or after cloning into pGEM-T-easy
vectors (Promega, Madison, WI, USA). Sequencing was
performed by ABI 3730 automated sequencer (Applied
Biosystems, Foster City, CA, USA). All sequences obtained
in this study have been deposited to the GenBank
database under accession numbers FJ918688-FJ918822 (Table
Sequences were aligned with CLUSTAL_X version 1.81
. GC content, base frequency, pairwise divergence and
the percentage of phylogenetically informative characters
were calculated by MEGA4 .
Phylogenetic tree was built using maximum parsimony
(MP) and Bayesian inference (BI) methods. MP analyses
were performed using heuristic search with 1000
replicates of random stepwise addition and tree
bisectionreconnection (TBR) branch swapping in PAUP version
4.0b10 . Gaps were treated as missing data. Bootstrap
resampling  was conducted to assess topological
robustness with 1000 replicates. BI analyses were
performed in MrBayes version 3.1.2  by
Metropolis-coupled Markov Chain Monte Carlo algorithm. Sequences of
GenBank Accession No.
a The first three letters represent species name of an accession, followed by its origin country.
b All accessions were provided by the Genetic Resources Center of the International Rice Research Institute (IRRI), Los Banos, the Philippines.
c Accession selected to represent the species for divergence time analyses.
d Accession selected for fluorescent in situ hybridization.
e Heterozygous locus with more than one allele.
each gene were divided into three different partitions
(exon, intron and insertion), and the combined data have
six partitions. GTR+I+G model was applied for the exon of
Os125, GTR+G model for the insertion of SDBE, and HKY
model for the rest. Four Markov chains were conducted
for 1,000,000 generations, trees were sampled every 100
generations, and then the first 2500 trees were discarded
in the burn-in period. Optimal models and parameters
under the Akaike Information Criterion (AIC) were
determined by Modeltest 3.06  for Bayesian analyses.
When different alleles from heterozygotes were grouped
into one clade, one of them was excluded randomly in
phylogeny of the combined data, unless they were
otherwise grouped into different clades. Congruence between
SDBE and Os125 was evaluated using the partition
homogeneity test (PHT) , implemented in PAUP with 1000
replicates, random taxon addition (10 replicates), and
one tree saved per replicate. Results from the PHT
indiStarch Debranching Enzyme
cated that incongruence between these two genes was P =
0.01, ten folds higher than the suggested (P < 0.001) by
and rttmsd set to 1.36, rtrate and rtratesd to 0.04,
brownmean and brownsd to 0.7, according to preliminary dating
Divergence times were estimated by Bayesian dating
methods [44-46], using the programs Baseml ,
Estbranches  and Multidivtime . Splitting times of O.
officinalis complex from its affiliated genus Oryza and tribe
Oryzeae were determined through the plastid gene matK
of 11 representatives and two outgroups (Table 3). A
recent report suggested that origin of Oryzeae was about
34.5 6.8 Mya , based on newly discovered pollen
fossils [49,50] and phytoliths . These dates were used
as the maximum and the minimum constraints to the
crown node of Oryzeae, respectively. Other settings were
F84+G model  and 100,000 MCMC (markov chain
monte carlo) iterations, with rttm and rttmsd set at 6.0,
rtrate and rtratesd set at 0.02, brownmean and brownsd set at
0.16, and big time set at 100.
For divergence time estimation within the O. officinalis
complex, an MP tree with 19 sequences representing the
taxa of the complex (Table 1) was applied. Insert
sequences were excluded due to their considerably
variable lengths. Calculations were performed using the same
Bayesian relaxed clock methods stated above. Dating
constraints between the complex and its outgroup, and
between the first clades split within the complex, were set
as 13.6 3.6 Mya, and 8.0 2.9 Mya respectively, which
were determined by the dating of Oryzeae as described
above. Other specific parameters were set as follows: rttm
Preparation of genome specific sequences
C-genome-specific sequences (against B-genome) were
isolated by a modified subtractive hybridization methods
 as follows: genomic DNAs from O. officinalis (CC,
Accession 102460) and O. punctata (BB, Accession
105607) were digested with MseI (New England Biolabs,
Beverly, MA, USA) into 500 to 1000 bp fragments; and
then the fragments of C-genome were ligated with
adapter-C and those of B genome were ligated with
biotinylated adapter-B (Table 4). Ligation efficiency was
checked by PCR amplification using adapter specific
primers, C-adp1 and B-adp1. The C-genome ligation was
denatured and annealed together with excess B-genome
ligation in a single tube. The anneal temperature was
68C with 0.99 M sodium salt overnight, and then the
supernatant containing C-genome-specific sequences was
selectively recovered from the reaction mix with
streptavidin-coated magnetic beads (Dynabeads, Dynal, Lake
Success, NY, USA). A more round of subtracting process was
necessary to enrich the genome-specific sequences.
Finally, molecules containing the genome-specific
sequences were amplified with C-adp1 as primer, and
then used for the plasmid transformation.
The transformed plasmids were sequenced, and the
sequences were BLAST searched in GenBank. Then a series
of primers (Table 4) designed from the sequences were
* Outgroups used in the divergence-time analyses in Oryzeae.
* 5' end biotinylated
used to test whether the sequences were genome-specific
or not by PCR amplification onto the related accessions,
no correspond bands were seen in BB diploid species
(date not shown). Further, the genome-specific sequences
were labelled as probes and finally verified by fluorescent
in situ hybridization (FISH). The FISH images showed no
signal on the chromosomes and nuclei of BB diploid
species (data not showed). All these data confirmed that the
sequences were C-genome-specific.
Chromosome spreads were prepared by enzymatic
maceration/air-dry method [54,55]. Total genomic,
genomespecific and 45S rDNA probes were labelled by nick
translation with biotin-16-dUTP (Roche Diagnostics GmbH,
Mannheim, Germany) or DIG-11-dUTP (Roche
Diagnostics GmbH, Mannheim, Germany), respectively.
Multicolor fluorescence in situ hybridization (FISH) was
performed as described  with slight modification.
After overnight hybridization, the slides were given a
stringent wash in 20% (v/v) formamide in 0.1 SSC at
42C, resulting in 80%-85% stringency. The
biotinylatedprobes were detected by avidin-FITC (Roche Diagnostics
GmbH, Mannheim, Germany), and the
digoxigeninlabelled probes by anti-digoxigenin rhodamine conjugate
(Roche Diagnostics GmbH, Mannheim, Germany). The
chromosome spreads were mounted in Vectashield
mounting medium with DAPI (Vector Laboratories,
Burlingame, CA, USA), and examined under a Leica DMRBE
microscope (Leica, Wetzlar, Germany). Photographs were
captured by a SPOT cooled color digital camera system
(Diagnostic instruments Inc., MI, USA), then imported
into Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose,
CA, USA) for processing.
Two distinct sequences in both SDBE and Os125 genes
were identified from each accession of all tetraploids. One
was longer (740-2272 bp in SDBE and 871-1021 bp in
Os125) and the other was shorter (420-458 bp in SDBE
and 400-701 bp in Os125). Each longer sequence was
highly similar to and phylogenetically grouped with the
corresponding sequences of CC diploids (Figures 1, 2, 3),
and thereby was named as C-like copy. Each shorter one
was similar to that of BB or EE diploids, and thus was
named as B- or E- like copy accordingly. Aligned sequence
of SDBE (2337 bp) had 101 (4.3%) informative sites, and
that of the Os125 (1112 bp) contained 164 (14.7%)
informative sites. The combined sequence of these two
genes was aligned to be 3449 bp in length (Table 5).
A ~320 bp insertion in SDBE and a ~150 bp insertion in
Os125 were recognized in each of the C-genome-bearing
species. BLAST searches in the TIGR rice repeated database
http://www.tigr.org/tdb/e2k1/plant.repeats/ and then
mask against Repbase Update http://www.girinst.org
using CENSOR , identified the insertion of SDBE to
be MITE-MDM2 (miniature inverted transposable
element-MDM2), but no matching sequence of the Os125
insertion was found. The Os125 insertion was flanked by
a short direct repeat (sequence: TACATGGCTCTTTC), but
no terminal inverted-repeating sequence nor
tRNArelated region was found, suggesting that this fragment is
Aligned length (range)
GC (%) Mean sequence divergence (range)(%)
Variable sites (%) Informative sites (%)
Sequence divergence was estimated using the Jukes-Cantor distance
SoFtuirgsicuttrceoe1snsensus trees of SDBE gene from 72 most
parsimoniStrict consensus trees of SDBE gene from 72 most
parsimonious trees. Numbers above branches: bootstrap
values (only those > 50% showed), below: Posterior
probability (only those > 0.5 showed). a or b: alleles of a
heterozygous locus. 2 or 4: ploidy levels. Dash lines indicated the
nodes supported by Bayesian inference. Tree length = 184,
Consistency index (CI) = 0.8859, Retention index (RI) =
0.9694, Bayesian inference -ln L = -4517.46 (Gray Square: C
and C-like copy; White Square: B and B-like copy; Black
Square: E and E-like copy).
an unidentified retrotransposable element instead of a
SINE (short interspersed repetitive element) .
In addition, a partial fragment (~1.5 kb) of L1-type
retrotransposon family was found to insert into C-like SDBE
gene in some accessions of O. alta and O. grandiglumis.
Phylogeny reconstruction based on SDBE, Os125 and
Phylogenetic analyses of SDBE, Os125 and combined
dataset using maximum parsimony (MP) and Bayesian
inference (BI), all yielded similar topologies. Parsimony
analysis yielded 72, 500 and 126 equally most
parsimonious trees, from SDBE, Os125 and combined dataset,
respectively. The strict consensus trees of each dataset
were showed in Figures 1, 2 and 3 with general features as
follows: (a) The main clades were strongly supported by
bootstrap values and Bayesian posterior probability. (b)
B-, C- and E-like copies in tetraploid species formed three
monophyletic clades with the corresponding sequences of
BB, CC and EE diploid species, respectively. (c) In the
Cgenome clade, two monophyletic clades were formed, one
involving O. eichingeri and the tetraploid O. punctata, and
the other covering all the rest C-genome species. (d) The
B-like copies of BBCC tetraploid species were divided into
two clades, one including the Africa endemic O. punctata
(comprising BB diploid and BBCC tetraploid), and the
other including the Asian tetraploids only. We also put the
SDBE and Os125 sequences of O. sativa (A-genome,
GenBank Accession No. AB012915 and AP004885) into the
datasets, but the positions of B-, C- and E-like copies, and
the topology of the inferred cladegram, remained
unchanged (data not showed).
With more informative sites, the cladograms of the
complex constructed from Os125 and combined dataset were
more resolvable (Figures 2, 3). O. officinalis (CC) and
Clike copies of the BBCC tetraploids, i.e., O.
malampuzhaensis and O. minuta, were consistently united into one clade.
The clade was further grouped with C-like copies of all
CCDD tetraploid species. Apart from the C-like copies of
Os125 sequences, two E-like copies were isolated from
CCDD species, which formed two clades in parallel and
SFmtiorgincuitroecuos2ntsreenessus trees of Os125 genes from 500 most
parsiStrict consensus trees of Os125 genes from 500 most
parsimonious trees. Numbers above branches: bootstrap
values (only those > 50% showed), below: Posterior
probability (only those > 0.5 showed). a or b: alleles of a
heterozygous locus. 2 or 4: ploidy levels. Tree length = 319,
Consistency index (CI) = 0.8715, Retention index (RI) =
0.9807, Bayesian inference -ln L = -3585.67 (Gray Square: C
and C-like copy; White Square: B and B-like copy; Black
Square: E and E-like copy).
SpFtairgriscuitmrecoon3nisoeunsstursetersees of combined data set from 126 most
Strict consensus trees of combined data set from 126
most parsimonious trees. Numbers above branches:
bootstrap values (only those > 50% showed), below:
Posterior probability (only those > 0.5 showed). a or b: alleles of a
heterozygous locus. 2 or 4: ploidy levels. Tree length =
463, Consistency index (CI) = 0.8985, Retention index (RI) =
0.9771, Bayesian inference -ln L = -7735.44 (Gray Square: C
and C-like copy; White Square: B and B-like copy; Black
Square: E and E-like copy).
finally grouped with O. australiensis (EE) trichotomously
in both MP and BI trees (Figure 2).
It is noteworthy that two alleles of the heterozygous
accessions were grouped with each other, except eic_LAK1 and
eic_UGA2, in which one of the alleles was clustered with
that of different species (Figures 1, 2), suggesting that
interspecific hybridization and introgression in those
accessions occurred, as proposed by previous research
. For those heterozygous loci, the allele that clustered
into the O. eichingeri clade, was selected in the combined
As showed in Figure 4, the O. officinalis complex was
estimated to diverge from the rest of the genus Oryza at 7.9
1.6 Mya, and the separation between B- and C- genomes
took place at 4.8 1.3 Mya. The molecular dating
indicated that three C-genome diploid species radiated
between ca. 0.9-1.8 Mya during Pliocene. In BBCC
tetraploid species, C-like copy of O. punctata diverged from C
genome of O. eichingeri at 0.3 Mya, very close to the
divergent time (0.5 Mya) of the B-like copy from the B genome
of diploid O. punctata. In other two BBCC species (O.
malampuzhaensis and O. minuta), the C-like copies
diverged from their common paternal progenitor (O.
officinalis alike) at ca. 0.6 Mya, later than the divergence
time of their B-like copies from the B-genome of O.
punctata (BB) at ca. 1.8 Mya. Similarly, the divergence time
between C-like copies of CCDD tetraploids and their
Cgenome donor, was set at ca. 0.9 Mya, while the node to
separate their D-genomes from O. australiensis (EE) was
dated at ca. 2.8 Mya (Figure 4).
FDiigveurrgeen4ce times of main lineages in O. officinalis complex
Divergence times of main lineages in O. officinalis
complex. Calculated by Bayesian relaxed-clock methods
(details see Materials and Methods). Estimated Mya and the
standard deviation were noted above the branches. 2:
diploid; 4: tetraploid (Gray Square: C and C-like copy; White
Square: B and B-like copy; Black Square: E and E-like copy).
Figure 5 shows multicolor fluorescent in situ
hybridization images of O. officinalis complex, hybridized by
Cgenome-specific probes (red), together with B-genome
probes (green) or E-genome probes (green),
counterstained by DAPI (blue).
Figures 5a-d show that the C-genome-specific probes were
localized on all chromosomes of two diploid CC species,
O. officinalis and O. eichingeri. The C- genome-specific
sequences were scattered non-uniformly along each of the
chromosome as well as among twelve homologous pairs,
as the FISH patterns showed obviously (Figures 5a, c). The
total 24 chromosomes were karyotypically arranged into
twelve homologous pairs according to their FISH patterns,
relative length, centromere position and
heterochromatin, as showed in Figure 5b and Figure 5d.
Figures 5e and 5f show the FISH images of the tetraploid
O. punctata using digoxigenin-labelled C-genome-specific
probes and biotin-labelled total genomic DNA of diploid
O. punctata (BB). B- and C-genomes were clearly
discriminated in the same nucleus, where 24 chromosomes
showed strong bright green signals of the B-genome
probes, and the rest 24 chromosomes showed strong red
signals of the C-genome-specific probes (Figures 5e, f). It
is worth to notice that two pairs of B-genome
chromosomes were clearly involved in inter-genomic
translocations with the C-genome, one small and the other rather
Figure 5g shows that a prometaphase nucleus of O.
malampuzhaensis was hybridized with C- genome-specific
probes (red) together with B-genome probes (green). The
24 B-genome chromosomes exhibited strong green
signals, and the rest 24 chromosomes belonging to
Cgenome showed bright red signals. Two B-genome signals
were identified at short arm terminals of one pair of
Cgenome chromosomes. However, when O.
malampuzhaensis was hybridized with B-genome probes (green)
together with 45S rDNA probes (red), these two
Cgenome chromosomes with 45S rDNA signals were also
painted by B-genome signals on same areas (Additional
file 1). Therefore, in O. malampuzhaensis which was with
different origin from tetraploid O. punctata, B-genome
signals located on the two C-genome chromosomes may not
be inter-genomic translocation but homologous
sequences of 45S rDNAs.
Multicolor FISH was also used for three CCDD species,
where two probes were applied, one from O. australiensis
(EE) genome (labelled in green), and one from the
Cgenome-specific probe (labelled in red). Figure 5h shows
in O. latifolia, strong C-genome-specific signals (orange)
painted 24 chromosomes, while green signals (from the
E-genome probes) stained all chromosomes, in which 24
FMiuglutirceolo5r fluorescent in situ hybridization images of O. officinalis complex
Multicolor fluorescent in situ hybridization images of O. officinalis complex. Hybridized by C genome-specific probes
(red), together with B genome probes (green, e-g, n-s) or E genome probes (green, h-m, t-v), counterstained by DAPI (blue,
ad, k-m). Arrows indicated inter-genomic (B-C genomes) translocations, which were enlarged in the below box (e-g). (a) O.
officinalis (CC), and its karyotype (b). (c) O. eichingeri (CC), and its karyotype (d). (e-f) O. punctata (BBCC). (g) O. malampuzhaensis
(BBCC). (h, k) O. latifolia (CCDD). (i, l) O. grandiglumis (CCDD). (j, m) O. alta (CCDD). Parental genomes in the nuclei of
allotetraploids, separated spatially in O. punctata (n, interphase; o, prophase; p, anaphase), in O. malampuzhaensis (q and r,
interphase stages; s, anaphase), and in interphase nuclei of O. latifolia (t), O. grandiglumis (u), and O. alta (v), respectively. Bar, 5 m.
chromosomes with pure green signals should belong to
the D-genome. The FISH patterns of O. latifolia differed
remarkably from those of O. alta and O. grandiglumis. In
O. latifolia all chromosomes were painted by E-genome
signals (Figures 5h, K), whereas in O. alta and O.
grandiglumis all chromosomes were painted by C-genome signals
(red), in which merely some of the chromosomes showed
the E-genome signals (green) faintly or strongly near
centromere regions (Figures 5i-j; 5l-m). This difference could
also be seen in the interphase cells, as showed in Figures
5t-v, where nuclei of O. latifolia were dominantly painted
by E-genome probes while those of O. alta and O.
grandiglumis were strongly painted by C-genome-specific probes
with dot-like signals of E-genome probes.
Figure 5n-s shows each of the two parental genomes
separated spatially in BBCC tetraploid species in interphase,
prophase and anaphase nuclei. In O. malampuzhaensis,
about 10 chromocenters of B-genome were found at late
stage of interphase (Figure 5r); however, no similar
chromocenters were found in O. punctata.
The key to trace the complicated evolution process of
polyploid complex lies on a universal criterion. C-genome
in O. officinalis complex could play such a role. As the
pivotal genome, C-genome participated each of the
polyploid formation in the complex, and its evolution process
in genomic differentiation and geographical patterning
can therefore reflect the temporal and spatial history of
Genomic relationships in O. officinalis complex
In O. officinalis complex, four extant genomes, B, C, D or
E, were identified [14,21,27]. The present study showed
that each genome in the complex occurred only once
when rooted by the outgroup, O. granulata, where
Egenome sited at the basal position of the complex. The
clade of O. officinalis complex was first divided into two
clades, E-genome clade and the other clade involving
Band C-genomes. In E-genome clade, D-genome was
located as E's sister group. These results were consistent
with previous reports [14,21-23]. In the other clade,
Cgenomes in different diploid species had differentiated
apparently thereafter they partook in different polyploid
formation (Figures 1, 2, 3), in agreement with other
Although there is only one extant diploid with B-genome,
O. punctata, the B-genomes in tetraploids were
differentiated, as revealed by AFLP , RFLP , SSR  and
GISH . In this study, multicolor FISH (Figure 5g)
revealed that the B-genome of O. punctata (BB) was clearly
diverged from that of O. malampuzhaensis. Further
evidence of molecular phylogeny and dating showed that the
divergence happened even before polyploidization,
which formed O. malampuzhaensis and O. minuta (Figures
1, 2, 3, 4). Therefore, a diploid B-genome species extinct
nowadays in Asia was assumed to be the direct genomic
donor of Asian distributed BBCC tetraploids.
Since no diploid DD species has ever been found, the D
donor for the CCDD tetraploids has long been
controversial [14,27,28,32]. The Australian diploid, O. australiensis,
as the unique E-genome holder, was assumed to be
Dgenome donor by several authors [14,32]. Nevertheless,
genomic comparison by GISH and retrotransposon
analysis found obvious differences between D- and
Egenomes, and thus suspected E as the direct donor
[28,63]. Based on a universal criterion of C-genome
differentiation, our study in phylogeny and molecular dating
(Figures 1, 2, 3, 4) showed that D- and E-genome were
tied together as sister group, but they diverged much
earlier than CCDD tetraploid formation. Multicolor FISH
using E-genome probes for the CCDD tetraploids also
revealed obvious differentiation between D- and
Egenome, and this was even remarkable in D-genome
itself, as showed in Figure 5, where the D-genomes of O.
alta and O. grandiglumis exhibited sharply different from
that of O. latifolia.
C-genome variation and polyploid evolution in O.
To date six tetraploid species, three BBCC and three
CCDD, have been recorded in O. officinalis complex, and
all are C-genome carriers [16,64]. The relationship and
origin of the tetraploids have long been in debate
[14,21,22,32]. In this study, C-genome of diploid O.
eichingeri was localized at the basal of C-genomes, and it
subsequently diverged, resulting two C-genome diploids,
O. rhizomatis and O. officinalis. Later on, the three C
diploids participated separately in hybridization and
polyploidization, finally forming six tetraploids. For O.
eichingeri, it merely joined formation of O. punctata
(BBCC), while O. officinalis (CC) partook in formation of
O. malampuzhaensis and O. minuta. On the other hand, a
species closely related to present O. officinalis (or O.
rhizomatis) offered its C-genome to the three CCDD
tetraploids, O. alta, O. grandiglumis and O. latifolia (Figures 1,
C-genomes in different BBCC tetraploids confronted
variable fates, such as changes by inter-genomic translocation.
Multicolor FISH probing different genomes in an
allopolyploid can be a powerful indicator for identifying such
changes. As showed in multicolor FISH (Figure 5e-g),
inter-genomic translocations between C-and B-genomes
were visualized for the first time in two tetraploids of the
complex, which was speculated as the result of
diploidization impact [5,8,65,66]. In O. punctata two fragments of
C-chromosomes were translocated to different
B-chromosomes, while in O. malampuzhaensis no obvious
intergenomic translocation was found. Although C-genomes
experienced different history in various polyploid
formations, few fragments of C-genome-specific were detectably
lost after hybridization and polyploidization, as found in
multicolor FISH with genome-specific probes (Figures
The fate of C-genomes in CCDD tetraploids was different
even more. In O. alta and O. grandiglumis
C-genome-specific probes apparently dominated the nuclei, most
probably by inter-genomic invasion [5,8,67,68], as showed in
multicolor FISH images, while in O. latifolia C-genome
kept almost unchanged (Figures 5 5h-m, t-v). Considering
that O. alta and O. grandiglumis diverged from O. latifolia
(Figure 4), the inter-genomic invasion would have
happened during their speciation.
Temporal and spatial evolution of O. officinalis complex
Geographical pattern of intercontinental pantropics in O.
officinalis complex, framed by its relatively recent history,
makes its evolution process paradoxical for long time.
Based on molecular clock of matK and GAP1 sequences,
the origin of the complex was dated at late Miocene (ca.9
Mya) , and speciation of O. australiensis was set in ca.
8.5 Mya through Adh2 gene . However, re-dating the
origin and divergence times became necessary, because
(a) previous dating dealt mainly with diploids while the
polyploids evolution history remained unclear; (b) new
molecular timescales based on non-parametric rate
smoothing, penalized likelihood, and Bayesian-relaxed
clock methods have been recently developed for the
In this study, the estimated divergence time between O.
officinalis complex and its outgroup, O. granulata, was
13.6 3.6 Mya, earlier than the previous suggestion, and
the time of the first species divergence in the complex, was
7.9 1.6 Mya (Figure 4). C-genome was separated with
Bgenome at about 4.8 Mya, and then C-genome itself was
split into two clades in approximately 1.8-0.9 Mya, one
including O. eichingeri and the other including the rest
two CC diploid species. These times were earlier than
previously suggested , but closed to recent research
[24,25]. The time of polyploidization to synthesize
tetraploids was estimated to be ca. 0.3-0.9 Mya in Pleistocene,
in which the CCDD species (ca. 0.7-0.9 Mya) were formed
obviously earlier than BBCC species, also closed to that
recently reported .
If all C-genome species separated no earlier than two Mya,
the distribution of these species can be feasibly explained
by long-distance dispersal rather than vicariance, given
that the continental splitting was much earlier than the
species radiation. As suggest by Vaughan et al [11,12],
animal migration may play a role for this complex in seed
dispersal between Asia and Africa. Bird could be another
carrier, which could account for the disjunctive
distribution of some Oryza species, such as O. eichingeri [11,58].
For the CCDD tetraploids, this and previous studies 
both revealed that their putative parents were O. officinalis
and O. australiensis. The problem was that the putative
parents were confined to south Asia-Australia but the
CCDD tetraploids were nowadays endemic to the tropics
of Americas. Therefore, a new pathway to bridge these two
continents for long-distance dispersal was put forward
(Figure 6). The strong floristic affinities between the South
America and the antipodes were also confirmed by
biogeographical studies of other Poaceae species [70,71].
However, how the species in the complex could
transcontinentally spread across the oceans, remains
The genomic relationships, polyploid formations and
divergence times in Oryza officinalis complex of the genus
Oryza, were revealed based on DNA sequences and FISH
evidence. Focused on C-genome, the "pivotal genome" of
the polyploids, we found that the polyploids were formed
by stepwise polyploidizations in ca. 0.3-0.9 Mya, followed
by a series of inter-genomic translocations and invasions.
The pantropical distribution of the complex was suggested
to be formed by long-distance dispersal
transcontinenoBFfifogicguineraoelgisr6acpohmicpallesxcenario of species with C genome in O.
Biogeographical scenario of species with C genome
in O. officinalis complex. Above: divergence time
calculated using Bayesian relaxed-clock methods (the cladegram
narrowed from Figure 2). Below: Distribution areas (outline
and noted) and inferred migration procedure among
continents. CC diploids (eic = O. eichingeri, off = O. officinalis, rhi =
O. rhizomatis), BBCC tetraploids (pun = O. punctata, mal = O.
malampuzhaensis, min = O. minuta), and CCDD tetraploids
(lat = O. latifolia, alt = O. alta, gra = O. grandiglumis).
tally, instead of vicariance. This study offers a typical
example in tracing tempo-spatial process of
polyploidization, and for the first time it gives new stands for the
complex in dating the detailed times of polyploid formation,
visualizing inter-genomic changes, and viewing the
spatial evolution history of the polyploids.
BW carried out the molecular and cytogenetic studies,
wrote the manuscript and participated in the design of the
study. ZD, WL and JP made equal contributions in
chromosome preparation, data analyses, and phylogenetic
inference. CL provided partial DNA sequences for
phylogenetic analysis. SG identified all the Oryza materials and
modified the manuscript. DZ contributed to the design of
the study, supervised the experiment steps, and prepared
the manuscript. All authors read and approved the final
Additional file 1
Multicolour fluorescent in situ hybridization images of O.
malampuzhaensis. Prometaphase chromosomes of O. malampuzhaensis were
hybridized by 45S rDNA probes (red) together with B-genome probes
(green), and counterstained by DAPI (blue). Arrow indicated one pair of
C-genome chromosomes which painted by both 45S rDNA and B-genome
signals on same areas. (a) and (d) Two pairs of 45S rDNA loci (red). (b)
and (e) The B-genome chromosomes showing blue-green signals. (c) and
(f) The chromosomes counterstained by DAPI after hybridized with both
45S rDNA and B-genome probes. Bar, 5 m.
Click here for file
We thank Drs. Yuzhu Dong, Junxia Yuan and Zhukuan Cheng for their
technical assistance and helpful suggestions on the manuscript. We are also
grateful to the International Rice Research Institute (Los Banos, Philippines)
for providing seed samples. This work was supported by the National
Natural Science Foundation of China (30430030) and Program for Key
International S & T Cooperation project of P. R. China (2009CB119102).