1. Introduction
The
Oryza genus is comprised of 23 species with varying genome compositions and ploidy levels [
1]. The two cultivated species,
Oryza sativa and
Oryza glaberrima, belong to AA genome species, and their progenitors were wild
Oryza rufipogon and
Oryza barthii, respectively. The AA genome species are dispersed across the major continents and were once classified as a single species,
Oryza perennis, comprising Asian, American, African, and Oceanian forms [
2]. The Asian species,
O. rufipogon represents different life histories and varies from annual to perennial. Their life history is a continuum with annual, intermediate, and perennial forms [
3,
4]. The American species,
Oryza glumaepatula also varies from annual to perennial. African species,
O. barthii and
Oryza longistaminata, however, are exclusively annual and perennial types, respectively.
Oceanian species had been known as
O. perennis (later changed to the current species nomenclature) including annual and perennial types as a continuum within a single species [
3]. After rearrangement of the species classification, an annual type was defined as an Oceanian endemic species,
Oryza meridionalis and the perennial form as
O. rufipogon [
4]. Their distributions in Australia are well studied [
5,
6]. Speciation of these species has been confirmed using retrotransposon insertions [
7,
8] and crossing ability [
9,
10,
11].
In general, annual and perennial species have different adaptive strategies to allocate their energy resources [
3,
12]. Annual species tend to have higher seed productivity than perennial species.
O. meridionalis, the Australian annual species, produces plenty of seeds and disperses these seeds.
O. meridionalis inhabits ponds or the periphery of ponds, ditches, or lakes during the rainy season. Water levels in wild rice habitats recede and water in the peripheral areas of annual species disappears during the dry season [
13]. Annual species produce large amounts of seed for the next generation. In contrast, the life history of Australian perennial species is similar to Asian perennial species except for a unique taxon known as Jpn2 or taxon B [
6,
14]. In addition, Jpn2 type wild rice exhibits different morphological and genetic characteristics [
14]. Including the new wild rice type, Australian perennial and annual rice chloroplast (cp) genomes have been completely sequenced in order to understand the uniqueness in evolutionary relationships among other wild rice [
15,
16]. This showed that the cp genome of Australian
O. rufipogon, Jpn1 (taxon A) has a closer relationship to
O. meridionalis than to Asian
O. rufipogon, although its nuclear type tended to show higher similarity to Asian
O. rufipogon. Another perennial species, Jpn2 (taxon B), also shared similarity not only with the cp genome to
O. meridionalis but also the nuclear type [
14,
17,
18]. This analysis showed that all Australian wild rice shared some cp genetic similarity with
O. meridionalis. Nuclear genomes in Australia showed huge variation never seen in Asian wild rice. These findings with ecological observations confirmed that there were two types of perennial rice. Their distribution in northern Queensland and their unique morphological traits were also reported [
6,
14].
In this paper, we further characterized these two taxa at morphological and reproductive levels, which enabled us to determine how they have diverged at the species level. Cytoplasmic markers to distinguish them were developed and variation among natural populations was evaluated. These findings will help to distinguish these taxa in field research for further analysis and also give clues to their evolutionary origins. Retro-transposable elements were also used to screen the species examined in this study. Some of these provide clear evidence of phylogenetic relationships because of the unique mechanism of transposition insertion.
2. Results
2.1. Morphological Features
Two types of Australian perennial wild rice were collected (
Table 1). Based on our previous report [
14], identifying two types of perennials: Jpn1 (taxon A) and Jpn2 (taxon B), morphological traits were able to be discriminated between the Australian perennials. Bristle cells have a thorn-like architecture along the awns (
Figure 1). SEM enabled us to compare the density of these cells. They varied from 2.33 to 5.33 per 200 μm square among Asian wild rice (
Table 2). In
O. meridionalis, W1299 and W1300 had 12.67 and 14.67 per 200 μm
2, respectively. The density in Jpn1 was similar to that in Asian wild rice. That in Jpn2 was similar to
O. meridionalis. There were significant differences between the two groups, W1299/W1300/Jpn2 and W106/W0120/W0137/Jpn1. Other traits, such as anther length, suggested that Jpn2 shared short anthers with other annual accessions such as W0106 in
O. rufipogon, and W1299 and W1300 in
O. meridionalis.
2.2. Maternal Lineages
In order to trace maternal lineages, next-generation sequencing data obtained from Jpn1 and Jpn2 were used for re-sequencing and comparison with the Nipponbare complete cp genome sequence. More than 53 million reads were obtained from the two accessions. Two genome sequences of O. meridionalis, and O. rufipogon were added for comparison. In all cases, 100% coverage was achieved with 733 to 2002 mean depth. When the nuclear genome was used as a reference genome, 66%–88% coverage with 7.6 to 11.4 mean depth was obtained.
Simple sequence repeats were found at 20 loci in the cp genomes. Simple insertions or deletions (INDELs) were also found at 21 loci (
Table 3). Two loci were not amplified, and six loci were not confirmed because of difficulty of primer design for these fragments. One region ranging from nucleotide 17,336 to 17,392 of the Nipponbare cp genome was amplified as a single amplicon because of its short size. In total, 29 insertions/deletions (INDELs)/simple sequence repeats (SSRs) in the cp genome were polymorphic. Australian rice accessions including
O. meridionalis, Jpn1, and Jpn2 shared the same genotype at 26 out of the 29 loci developed by plastid INDELs and SSRs.
Five chloroplast markers, INDEL1, INDEL11, INDEL13, INDEL18, and INDEL19, represented polymorphisms among natural populations (
Table S2 (
Supplementary Materials)). Plastid types were defined as distinct combinations of each genotype. In total, nine plastid types (Type 1 to 9, r1, and r2) with r1 and r2 types in the control
O. rufipogon, were recognized. Asian
O. rufipogon and
O. sativa accessions, were obviously different from the Australian wild rices.
Three accessions in PNG O. rufipogon, W1235, W1238, and W1239, and W2109 in Australian O. rufipogon shared the Type 5 plastid type with O. meridionalis. W1230 in Papua New Guinea O. rufipogon shared the r2 plastid type with the Asian type. W1236 carried a unique plastid type. Jpn2 shared Type 1 with O. meridionalis. Other O. meridionalis in the core collection divided into three types, Types 1, 5, and 8. Only two types, Types 1 and 2, were detected in the Northern Territory and in Western Australia. Newly collected accessions from Queensland carried seven types. Five of them were newly detected.
2.3. Reproductive Isolation
Biological species can be detected by the pollen fertility of hybrids. Jpn1 and Jpn2 were crossed with Asian wild rice and O. meridionalis. Each F1 plant was grown in a greenhouse, and leaf samples were used to check whether they were hybrids originating from the cross. Anthers were taken to check pollen fertility by staining with I2–KI. Well-stained pollen grains were counted.
Seed fertility was also assessed but this may not reflect reproductive ability of the respective plants (
Table 4). W0106, W0120, and W1299 showed more than 95% pollen fertility. However, except for W0120, they showed lower seed fertility of 19.5% in W0106 and 22.3% in W1299. The panicles were bagged to prevent out-crossing and this might explain the low seed fertility. In combinations with Jpn1 and Asian
O. rufipogon, F
1 plants with W0106 and W0120 had more than 90% pollen fertility. However, seed fertility was relatively low, similar to self-pollination of W0106 and W1299. We relied on data from pollen fertility rather than seed fertility and concluded that by this criterion, Jpn1 is related to Asian
O. rufipogon, and that Jpn2 is not close to either
O. rufipogon or
O. meridionalis.
2.4. Unique Insertion of Retrotransposable Element in Jpn2
In total, six presumed insertions were confirmed only in the Jpn2 genome but not in Nipponbare (
Table 5). Two
pSINE1 insertions were shared among Jpn2 and 19
O. meridionalis accessions. Another insertion amplified with Chr3-10559212-r (w/L) and pSINE1-L showed an insertion shared among Jpn1, Jpn2, and 19
O. meridionalis accessions (
Figure 2). Chr1-4067055-f (w/L) and pSINE1-L amplified the same amplicons not only from Jpn2 and 19
O. meridionalis accessions but also with W0106, which originated in India, suggesting that some parts of the Jpn2 genome share the insertion with wild rice from India. No
O. rufipogon accessions in the core collection except for W2266 and W2267 were tested because of lack of DNA, and 19
O. meridionalis showed these insertions. Results suggested that the insertion was probably shared among
O. meridionalis and W0106. Chr3-10203820-f (w/L) can amplify with pSINE1-L only in Jpn2 and no other
O. meridionalis showed any amplicons. In screening for the insertion among 30
O. rufipogon accessions in the core collection, W0180 and W1921, both of which originated from Thailand, showed amplicons. The insertion sequence in Jpn2 was screened from the raw reads and 53 bp were recovered. When aligned with
pSINE1, 92.4% high similarity was retained. When
pSINE3 insertions were examined, three of the presumed insertions were amplified only among Jpn2 and 19
O. meridionalis accessions.