Characterization of Wild Rice-Oryza Species Complexes in Sri Lanka

Rice is the staple food crop in Sri Lanka, which occupies 34% (0.77/million ha) of the total cultivated area. Sri Lanka currently produces 2.7 million tonnes of rough rice annually and satisfies around 95% of the domestic requirement. In Sri Lanka, genus Oryza consists of two species complexes, O. sativa (AA) and O. officinalis (CC). These two complexes are both pan tropical and have very similar overall distribution. Five wild rice species are reported in Sri Lanka, (O. nivara [AA], O. rufipogan (AA) O. eichengeri [CC], O. rhizomatis (CC) and O. granulate (GG). O. rhizomatis has been reported only in Sri Lanka and considered endemic to Sri Lanka. Recent studies demonstrated, the reliance on single source of information could mislead results in the phylogenetic inferences due to analytical inconsistency and biological processes. Therefore, exact number of wild rice species in Sri Lanka becomes uncertain and the necessity arises to assess Oryza species complexes in Sri Lanka using morphological, anatomical, and molecular information to enumerate number of species within each Oryza complex and characterization of species and species complexes. The study revealed, characterization of wild rice species, to a certain extent, can be made through morphological and anatomical characters, specially lamina anatomical characters. Molecular information is more reliable in delimitation of wild rice species complexes in Sri Lanka. O. rhizomatis and O. eichingeri (CC) are well separated from the rest of wild rice species (AA). Molecular data revealed, O. nivara and O. rufipogon have undergone independent evolution within Sri Lanka. Well separated five wild rice species are existing in Sri Lanka. Studies on ecological resilience of morphological, anatomical, and molecular studies are very useful for species enumeration of wild rice complexes in Sri Lanka. The findings led to conclude that wild rice species in Sri Lanka are “ecological swarms” and represents allopatric or sympatric populations. A comprehensive knowledge on genetic diversity and population structure of wild rice germplasm in Sri Lanka provides useful information to include these locally adapted and evolved wild rice species in rice crop improvement/breeding.


Introduction
Rice serves as the main staple food crop of nearly half of the world's population and it is obvious that genetic improvement of rice cultivars play an important role in the rice production for fulfilling ever increasing food demand. Rice is the staple food which occupies 34% (0.77/million ha) of the total cultivated area in Sri Lanka and currently produces 2.7 million tonnes of rough rice annually and satisfies around 95% of the domestic requirement [1].
The rice genus Oryza L. consists of ca. 21 wild and two cultivated species distributed in Asia, Africa, Australia, and the America [2,3] and these species have been categorized into ten different genome types, such as six diploids (AA, BB, CC, EE, FF, and GG) and four allotetraploid species (BBCC, CCDD, HHJJ, and HHKK) [4,5]. Wild rice spices are important in rice breeding programs because, these species comprise traits of agronomic interest, for example, the resistance and tolerance to biotic and abiotic stresses [2,[6][7][8]. However, due to the sterility barriers, most of the Oryza germplasm is of limited use in rice breeding programs [8,9]. Genetic resources of the AA-genome group also referred to as the Oryza complex, have long been a focal point of the rice breeders.
The Oryza sativa complex includes eight diploid species [2] and the Asian cultivated rice consists of main subspecies, O. sativa ssp. Indica and O. sativa ssp. Japonica [10][11][12] are of Asian origin and globally cultivated today. The two presumed wild progenitors; the perennial O. rufipogon (Figure 1) is distributed throughout tropical Asia and Oceania, whereas the annual O. nivara is distributed in tropical continental Asia (Figure 2). Another cultivated species in the genus, O. glaberrima, was parallelly domesticated in West Africa where it is endemic [2]. There are two additional wild species also endemic to Africa, O. barthii and O. longistaminata. The former is the annual wild progenitor of O. glaberrima, while the latter is a perennial, rhizomatous and partially self-incompatible grass species [13].
In Sri Lanka, the genus Oryza consists of two species complexes, the O. sativa complex that includes the AA genome species, the O. officinalis complex which includes the CC genome species [3,14] and a single species O. granulate (GG) [15,16] [17,18]. O. rhizomatis is one of the species of the O. officinalis complex (Figure 3). The taxonomy of O. officinalis complex in Sri Lankan has been puzzling due to insufficiency of satisfactory herbarium specimens and the living plant materials. As an attempt of resolving the problem of the morphological variation in the complex, Biswal and Sharma [19] retracted the name O. collina and considered this taxon to be synonymous with O. eichingeri. Thus, Biswal and Sharma [19] agreed with both Bor [20] and Tateoka [14] that O. eichingeri is the sole representative of O. officinalis complex in Sri Lanka (Figure 4). O. offocinalis in Sri Lanka grows in both shaded and open habitats, whereas O. eichingeri grows in the shade of forests in Uganda [21]. However, taxonomists were not able to give much weight to the habitat of this taxon since field notes are generally infrequent.
The new collections make known clear morphological and habitat differences in O. eichingeri and it is a larger taxon which occurs in the drier habitats in Sri Lanka [2]. This larger rhizomatous taxon has previously been called O. latifolia and O. officinalis. O. latifolia is a large non-rhizomatous tetraploid from South and Central America with broader leaves and whorled panicle branches. O. officinalis which usually has rhizomes, has smaller spikelets, shorter palea tip, more branches of approximately equal length from the lowest panicle node, and spikelets inserted away from the base of primary branches. O. officinalis is also genetically different from this Sri Lankan taxon with which it can form sterile hybrids. However, Sri Lankan taxon belongs to the same genome group as both O. officinalis and O. eichingeri, which is CC [22].
The nomenclature and the taxonomy of the elements of these complexes have been studied and nomenclatural changes have been suggested and certain de novo species was described to disentangle the problem within the complexes. Due to this reason, the exact number of wild rice species in Sri Lanka becomes uncertain and detailed studies specially, on morphological, anatomical, and molecular aspect of the Sri Lankan wild rice are needed for the delimitation of Oryza complexes in Sri Lanka.
Several recent studies demonstrated that the reliance on single source of information possibly misleading the results in the phylogenetic inferences due to analytical inconsistency and biological processes [27,28]. The inconsistencies among the  phylogenies have become one of the most common problems during the reconstructing molecular phylogenetics using different datasets, such as individual genes. Studies carried on the genome-wide markers have witnessed new phylogenetic reconstructions that use large quantities of genome-wide markers to illustrate former controversies on evolutionary relationships at all taxonomic levels [27][28][29][30][31]. In general, a gene tree does not necessarily reflect a species tree, even if the orthology of marker genes are clearly identified and employed. Therefore, many genetic markers, including unlinked loci with extensive functional representation as well as intergenic genomic regions, are needed to comprehensively track organismal history. Such a robust phylogeny will build a foundation for future insights into rice genome evolution.
Therefore, there is a need to delimit the Oryza species complexes in Sri Lanka using morphological, anatomical, and molecular information. The objectives of the present study are to enumerate the number of species within each Oryza complex (O. sativa complex and the O. officinalis complex) in Sri Lanka and characterization of species and species complexes with evidence generated from morphological, anatomical, and molecular studies.

Seed material
A total of four wild rice species; O. rufipogon, O. nivara, O. eichingeri and O. rhizomatis were collected from different localities of the Districts, Puttlam, Anuradhapura, Vavuniya, Trincomalee, Hambantota, Matara and Ampara of Sri Lanka. The botanical names and the acronyms used were given in Table 1. The collected samples were used for morphological, anatomical and molecular studies.

Morphological studies
The morphological characterization of each species collected was based on the Plant Genetics Resource Centre (PGRC), Sri Lanka Characterization Catalogue of Rice [32] ( Table 2). The leaf, culm, and rhizomes if available were collected and processed for micro sectioning. Temporary and permanent slides were prepared for cross sections of leaves, culm and rhizomes.

Anatomical studies
The free hand sections of the collected specimens were taken and observed under the light microscope. Measurements of anatomical characteristic features were made using standard methods.

Molecular studies
Total genomic DNA was extracted from 7-day old seedlings of wild rice species; O.rufipogon, O. nivara, O. eichingeri and O. rhizomatis respectively using Promega Plant DNA extraction kit. A total of twelve SSR primer pairs were used ( Table 3) for molecular study. SSR markers were obtained from Gramene (http://www.grame ne.org/). All SSR PCR amplification reactions were carried out in a total volume 30 μl of which consist 1 x PCR buffer, 1 mM dNTPs, 2 μM SSR primers, 2 mM  MgCl 2 , 50 ng of genomic DNA and 0.5 Units of Taq DNA polymerase. SSR alleles were resolved on Poly Acrylamide Gel. The SSR banding patterns were identified using Poly Acrylamide Gel Electrophoresis (PAGE).

Analysis of Data
Gathered data were analyzed with univariate, bivariate and multivariate statistical procedures. Suitable statistical software was employed in the analysis of data. In addition, data mining analysis were also attempted for the data gathered from the study to reduce the noise in the data set.
Molecular data were analyzed using Genemapper 4.1 software and SSR profiles were analyzed using PowerMarker 3.25.

Morphological studies
The mean values of the parametric morphological measurements of wild rice species are given in Table 4 Table 5, except ligule length, panicle length, the rest of the characters are significantly varying across the wild rice species.
The association of the non-parametric characters with wild rice species included in the study is shown in Table 6. The characters such as leaf blade pubescent, awn after full heading and intermodal color after full heading are not significantly differ across the species (p > 0.05). However, the rest of the characters are significantly associated with the wild rice species and are of potential characters in separating wild rice species.  Table 4.

Summary of the parametric morphological characters of the four wild rice species (Mean value and standard deviation below mean value).
A total of three clusters were resulted from the cluster analysis of morphological characters (

Anatomical studies
The variation of anatomical characters, especially the laminar anatomical features across the wild rice species are given in Table 7. Comparatively, the magnitude of mean values of bundle sheath cell width indicated a considerable variation between the wild rice species O. eichingeri (11.77 μm) and O. rufipogon (10.74 μm). The summary of the ANOVA (Table 8), indicated that the all the anatomical  characters except mesophyll height and bundle sheath height. The anatomy of the culm and leaf sheath of wild rice species indicated that the characteristic features of the structures reflect the habitat conditions (Figures 7 and 8).
The result of the cluster analysis of anatomical characters of wild rice species is shown in Figure 9. Comparatively, the Dendrogram resulted from the anatomical  features indicated that anatomical characters well-separate the samples of each wild rice species. The samples of O. rhizomatis formed a unique group at similarity level of 80%. The pattern of the sample grouping was similar to the results obtained from the cluster analysis of morphological characters. However, samples were homogenized representing each wild rice species by pure tree branch. The dendrogram resulted from the morphological and anatomical characters are shown in Figure 10. The grouping pattern of wild rice samples obtained from the analysis of morphological characters and anatomical characters reflect the same pattern observed in previously (Figures 5 and 9).

Molecular studies
A total of three clusters were resulted from the cluster analysis of molecular data (Figure 11) and species were grouped under each cluster with respect to

Discussion
The morphological and anatomical characters were investigated in relation to the species identification and delimitation of wild rice species complex in the country. The results of the morphological characters have indicated that they were useful in identification of wild rice species. However, the ecological resilience of the morphological characters is to be investigated before reaching a firm conclusion on the diagnostic value of the morphological characters. Compared to the morphological characters, the anatomical characters especially, lamina and culm anatomical characters are also indicted higher potential identification of species and delimitation of the wild rice species in each complex. Both morphological and anatomical characters can be used to separate the O. rhizomatis and O. eichingeri (CC) from the rest of wild rice species (AA). Further, based on both morphology and anatomy, O. rhizomatis can be distinguished from O. eichingeri. This finding suggests that species status of these two species deserved to maintain for further confirmation by molecular characterization. As far as the samples of two wild rice species of AA, O. nivara and O. rufipogon is concerned, there were considerable overleaps with respect to morphology and anatomy. However, the analysis of molecular data revealed that samples of O. nivara, O. rufipogon and O. rhizomatis have a distant relationship with each other and undergone independent evolution within Sri Lanka.
Finding of the study led to conclude that wild rice species in the island are "ecological swarms" and represents allopatric or sympatric populations. This finding is further supported by the connotations made by Nelson on the genus Oryza and its species in Sri Lanka [33].

Conclusions
The identification of wild rice species, to certain extent, can be made through the morphological and anatomical characters. The delimitation of the species complexes also achieved through the morphology and anatomy specially lamina anatomical characters. The nodal and culm anatomical characters are of limited value in the species identification and delimitation of wild rice species complexes.
However, molecular characterization is more reliable in characterization of wild rice species complexes in Sri Lanka.
The analysis of molecular data revealed that samples of O. nivara, O. rufipogon and O. rhizomatis have a distant relationship with each other and undergone independent evolution within Sri Lanka.
Therefore, studies on the ecological resilience of morphological characters in combination with anatomical and molecular studies are very useful for species enumeration of wild rice complexes in Sri Lanka. The finding led to conclude that wild rice species in Sri Lanka are "ecological swarms" and represents allopatric or sympatric populations.
A comprehensive knowledge on genetic diversity and population structure of wild rice germplasm in Sri Lanka provides useful information to include these locally adapled and evolved wild rice species in rice crop improvement and breeding programmes. rice species in Sri Lanka: some reproductive traits. In: Proceedings of the 7th YSF Symposium, Colombo, Sri Lanka; 2018. P. 7-10. https://www.resea rchgate.net/publication/326998769_ Diversity_of_wild_rice_species_of_Sri_ Lankasome_reproductive_traits#fullTe xtFileContent.