Morphological, cytological, and molecular evidences for natural hybridization between Roegneria stricta and Roegneria turczaninovii (Triticeae: Poaceae)

Abstract Some plants with low fertility are morphologically intermediate between Roegneria stricta and Roegneria turczaninovii, and were suspected to be natural hybrids between these species. In this study, karyotype analysis showed that natural hybrids and their putative parents were tetraploids (2n = 4x = 28). Meiotic pairing in natural hybrids is more irregular than its putative parents. Results of genomic in situ hybridization and fluorescence in situ hybridization indicate that natural hybrids contain the same genome as their putative parents. The nuclear gene DNA meiotic recombinase 1 (DMC1) and the chloroplast gene rps16 of natural hybrids and their putative parents were analyzed for evidence of hybridization. The results from molecular data supported by morphology and cytology demonstrated that the plants represent natural hybrids between R. stricta and R. turczaninovii. The study is important for understanding species evolution in the genus since it demonstrates for the first time the existence of populations of natural homoploid hybrids in Roegneria. The study also reports for the first time that the composition of the genomic formula of R. turczaninovii is StY, confirming that the current taxonomic status is correct.


| INTRODUC TI ON
Hybridization is the main driving force of plant evolution (Soltis & Soltis, 2009). It is estimated that about 25% of plant species are known to be involved in hybridization with other species (Mallet, 2005). These can provide source of genetic variation than on further evolution, through adaptation and selection leading to speciation (Arnold et al., 2012;Whitney et al., 2010). Hybridization can occur between species of the same ploidy level (homoploid hybridization) and between species of different ploidy levels (heteroploid hybridization). In plants, hybridization with an increase in ploidy (allopolyploidy) is associated with speciation much more commonly than homoploid hybridization, partly because of reproductive isolation between hybrids and parents with different ploidy (Soltis et al., 2014;Soltis & Soltis, 2009). So far, only about 20 cases of homoploid hybrids have been well documented in plants (Gross & Rieseberg, 2005;White et al., 2018).
The Triticeae (Poaceae) is an important economic gene pool for genetic improvement of cereal and forage crops, including about 450 diploid and polyploid species distributed in a wide range of ecological habitats over the temperate, subtropical, and tropical pine regions (Dewey, 1984). The majority of species are allopolyploids, and the ploidy levels range from diploid (2n = 2x) to dodecaploid (2n = 12x). With combining a wide variety of biological mechanisms and genetic systems, the tribe Triticeae is an excellent group for research in evolution, genetic diversity, and speciation in plant polyploids (von Bothmer & Salomon, 1994;Paštová et al., 2019).
Roegneria C. Koch is a relatively large perennial genus in Triticeae, and includes approximately 130 species, most of which are tetraploid with the StY genome, nearly 70 of which are found in China (Yang et al., 2008). Roegneria species not only provided genetic material for the improvement of forage crops but could also be used as potential contributors of genes for cereal crops (Keng, 1959), such as Roegneria stricta Keng and Roegneria turczaninovii (Drob.) Nevski. Predecessors have reported some studies on the hybrids of Roegneria, such as a hybrid of Roegneria and Hordeum (Zhou et al., 1995), a hybrid of R. ciliaris and Leymus multicaulis (Zhang et al., 2008). These hybrids were created by the artificial hybridization and could not replace the value of natural hybrids.
Early identification of hybridization is mainly based on morphological characteristics. However, the reliability of morphological markers is low, and morphological intermediacy is not always related to hybridization. It may also be caused by convergent evolution or environment (Rieseberg, 1995). Cytological markers have been used as important evidence for hybridization, including karyotype analysis, meiotic pairing analysis, Genomic in situ hybridization (GISH), and Fluorescence in situ hybridization (FISH) (Han et al., 2004;Mao et al., 2017). However, due to the high parental chromosome homology of interspecific hybrids, it is difficult to explore origin of hybrids by FISH and GISH (Soltis et al., 1992).
Single-or low-copy nuclear genes, which are less susceptible to concerted evolution, can serve as useful markers for studies of phylogenetic relationships (Lei et al., 2018;Sha et al., 2010). DNA meiotic recombinase 1 (DMC1) gene has been used to examine hybridization events (Tang et al., 2017). The chloroplast DNA (cp DNA) is maternally inherited in grasses (Smith et al., 2006), and ribosomal protein S16 (rps16) is used to identify the maternal donor of genera in Triticeae (Yan et al., 2014).
To cultivate new forage varieties, R. stricta and R. turczaninovii cv. Linxi were planted very close in Hong yuan Research Base of the Sichuan Academy of Grassland Science (SAGS), Sichuan Province, China (31.47°N,102.33°E). We harvested the seeds of the two species and planted them individually. In these plants, we found that some plants grew stronger and had lower seed setting rate than the surrounding plants (Figure 1a-c), and they had intermediate morphological characters of R. stricta and R. turczaninovii, such as pubescence of leaf, basal leaf sheath, and stem node (Figure 1d-o). We suspected that these plants are natural hybrids between R. stricta and R. turczaninovii. To determine if this is indeed the case, we conducted different methods including morphological analysis, cytological analysis, and phylogenetic analysis in these putative hybrids and their accompanying plants.

| Pollen fertility and seed set
The pollen grains from mature anthers were stained in an I 2 -KI solution for pollen fertility study. Seed set was estimated from a 10spike sample per plant.

| Karyotype and meiotic pairing analysis
Karyotype analysis was followed by Gill et al. (1991). The procedures of fixation, staining, and calculation of meiotic pairing followed Zhang and Zhou (2006).

| Chromosome preparation and in situ hybridization
Chromosomes were prepared for GISH analysis according to the method of Han et al. (2004). Total genomic DNA was extracted from fresh leaves by the CTAB method (Murray & Thompson, 1980). Plasmids (from positive clones that are St genome) and the StY genome were labeled with fluorescein-12-dUTP or Texas-red-5-dCTP using the nick translation method. Hybridization procedure, detection, and visualization were performed according to the method of Wang et al. (2017).

| Amplification and sequencing
The DMC1 and rps16 gene were amplified using the primers listed in Table S1 (Petersen & Seberg, 2002;Shaw et al., 2005). All PCRs were conducted in a 50μl reaction volume, with 1.5 U Ex Taq polymerase (TaKaRa, Shiga, Japan). The PCR amplification protocols for the DMC1 and rps16 gene are presented in Table S1. PCR products were cloned into the pMD19-T vector (TaKaRa). At least 15 random independent clones were selected for sequencing by Shanghai Sangon Biological Engineering and Technology Service Ltd. (Shanghai, China).

| Phylogenetic analysis
DNA sequences were confirmed through BLAST nucleotide alignment in the NCBI database, and sequence alignments were made    (Katoh & Standley, 2013). After preliminary phylogenetic analysis, the number of sequences is reduced. If there are more sequences of the same species form monophyletic groups, only one sequence is retained. ModelTest v3.06 (Posada & Crandall, 1998) was used to determine appropriate DNA substitution models and gamma rate heterogeneity using the Akaike information criterion (AIC).
The phylogenetic analyses of DMC1 and rps16 data were performed by using the maximum-likelihood (ML) method in PhyML 3.0 (Guindon et al., 2009). The best-fit evolutionary models determined were TPM1uf+G for DMC1 and TIM1+G for rps16. As a measurement of the robustness of tree clades, the bootstrap support (BS) values were calculated with 1000 replications and displayed in figure (above the branch) if the BS values were >50% (Felsenstein, 1985). Bayesian analyses were also performed using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003). The evolutionary model selected default settings.

| Morphological characteristics
The 57 natural hybrids were perennial grasses, which were similar in morphology and phenology to Roegneria species, such as one spikelet per node and palea equaling lemma. Most of hybrids were stronger than their surrounding plants (Figure 1a-c). These natural hybrids combined some unique characteristics of R. stricta and R. turczaninovii, such as leaf pubescence, stem node pubescence, and basal leaf sheath pubescence (Figure 1d-

| Evaluation of pollen fertility and seed set
The fertility, including pollen fertility and seed set, of R. stricta, R. turczaninovii, and putative hybrids, was shown in Figure 3. In R. stricta, the pollen fertilities were up to 92.05% and the seed sets were 90.02%. In R. turczaninovii, the pollen fertilities and seed set were high with 91.61% and 92.18%, respectively.
As for the hybrids of RH1, the pollen fertilities varied from 1.01% to 8.09%, and the seed sets were lower than those of their possible parents, varying from 0.41% to 4.50% ( Figure 3). As for the hybrids of RH2, the pollen fertilities varied from 0.83% to 13.63%, and seed set were lower, varying from 0.23% to 5.59% (Figure 3). It could be seen that the pollen fertilities and seed sets of putative hybrids were very low, indicating that they were hybrids and not stable species.
The meiotic configurations of the possible parent and the putative hybrids were listed in Table S2. Meiosis of R. stricta and R. turczaninovii were quite regular with 14 bivalents (Figure 5a-c, Table S2).  Table S2).

Meiotic pairing in
Except for hybrid RH2-31, all hybrids had univalents.

| FISH and GISH analysis
To further explore the genomic constitutions of natural hybrids, we selected some hybrids for in situ hybridization. Since the suspected parents of natural hybrids were R. turczaninovii and R. stricta (StY), and meiotic pairing in natural hybrids were comparatively high, we speculated that genomic constitution of natural hybrids was StY.

| Origin of natural hybrids
Natural hybrids are relatively common in flowering plants (Rieseberg & Ellstrand, 1993). Rieseberg (1997) reported that about 11% of plant species arose from interspecific hybridization. Artificial hybrids involving genus Roegeneria have been produced (Zhou et al., 1999), but there are no reports of natural hybrids. In this study, the low-fertility plants were suspected natural hybrids because of their morphologically intermediate between R. stricta and R. turczaninovii.
However, the natural hybrids had not been confirmed by cytological and molecular evidence. In this study, FISH and GISH analysis suggested that the genomic constitution of R. turczaninovii was StY.
This result was further confirmed by molecular data. Phylogenetic analyses based on DMC1 sequence suggested that R. turczaninovii has St and Y genomes. It is the first report that the composition of Additionally, meiotic pairing in 57 natural hybrids was comparatively high. This suggested that the genomes of their parents were homologous. This is consistent with our cytology and molecular data. Except for hybrid RH2-31, all hybrids had univalent. This also provides evidence for the low pollen fertility and seed setting rate of hybrids. Pairing and recombination among homologous chromosomes are common in nascent allopolyploids (Gaeta & Pires, 2010).
However, in the evolution of allopolyploids, homologous pairing is

| Formation process of natural hybrids
Triticeae is a young group; there is a large possibility of random hybridization among the relative genera in the Triticeae (Barkwoth & Bothmer, 2009

| Homoploid hybrid speciation
In the evolutionary history, many grasses from the Triticeae have undergone interspecific hybridization, resulting in allopolyploidy, which homoploid hybrid speciation (HHS) was found only in rye (Martis et al., 2013). Homoploid hybrid speciation is rare due to strongly reduced fitness of early generation hybrids and weak reproductive isolation with the progenitors (Mallet, 2007;Rieseberg & Willis, 2007 (Nolte & Tautz, 2010). Although such taxa may not eventually produce well-differentiated hybrid species, they can facilitate testing key predictions from models of hybridization and hybrid speciation (Barton, 2001;Buerkle et al., 2000). In this study, the natural homoploid hybrids are good research material for elucidating the first steps toward homoploid hybrids species. They can facilitate testing of key predictions from hybridization and hybrid speciation models. It can provide some references for the formation mechanism of natural hybrids of Triticeae.

| Utilization of natural hybrids
Hybridization among species can act as an additional, perhaps more abundant, source of adaptive genetic variation than mutation (Arnold & Martin, 2009;Kunte et al., 2011;Whitney et al., 2010). In this study, we found some natural hybrids with good forage traits in plant height, tillers, and leaf, but the fertility was very low. If these natural hybrids could be genetically improved to create new forage varieties, it would have good ecological and economic benefits. As a result of further reproduction, these hybrids could be a valid species because some highly sterile F 1 hybrids become species through adopting a vegetative mode of reproduction (Brysting et al., 2000).

ACK N OWLED G M ENTS
The authors are thankful to the National Natural Science Foundation of China (Grant Nos. 31870309 and 31670331) and the Science and Technology Bureau of Sichuan Province (2021NZZJ0010, R21YYJSYJ0014) for their financial supports.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.