Functional trait diversity of wild rice species in Sri Lanka: implications for field identification and application

: The study assessed the variations in morphologically, physiologically and anatomically distinct traits of wild rice species in Sri Lanka; O . nivara , O . rufipogon , O . eichingeri , O. rhizomatis and O . granulata , which could be useful in rice breeding. The wild rice species were grown in a common garden, and the morphological traits were measured soon after heading. The results showed qualitative parameters such as the panicle type, awning, stigma colour, lemma and palea pubescence, seed coat colour, blade pubescence and ligule shape, are distinctive among the five species and are promising characters in their field identification. ANOVA revealed that the quantitative traits, such as flag leaf length, flag leaf width, culm length, culm diameter, panicle length, 100 grain weight and plant height are useful for further confirmation of species. The highest net photosynthetic rate (5.86 µmol m -2 s -1 ), high cluster width of the base (61.4 µm), and trichome density (184.33 per 25 mm 2 area) were observed in O . rufipogon compared to the rest, and such desirable traits are effective in rice breeding. Moreover, transpiration rates, stomatal conductance and sub-stomatal CO 2 concentration are ideal physiological traits to be considered in super rice breeding. Significant correlations were observed between transpiration and photosynthesis processes. Thus, our study provides a clear picture on habitat preferences, life cycle, distinctive morphologies and diverse functional traits to be effectively used in field identification and future utilisation of wild relatives of rice in the plant breeding programmes.


INTRODUCTION
The global rice (Oryza sativa) production is expected to increase in the next few decades, with special focus on productivity enhancement, owing to limited land and increase in demand (Lim et al., 2013;Tan & Norhaizan, 2020). Improving the yield potential of rice varieties has been the main breeding objective in many countries for several decades to meet this challenge. The ideotype breeding is a key approach for crop improvement. 'Crop ideotype' is an idealised plant type with a specific combination of characteristics favourable for photosynthesis, growth, and grain production based on knowledge of plant and crop physiology and morphology (Khan et al., 2015). In this context, wild species of rice provides a wide range of favourable characters and is a valuable reservoir of genetic resources (Khush, 1997). Moreover, improving rice varieties by incorporating desirable traits from wild relatives may lead to advances in rice breeding, as the wild species of rice seems to harbour significantly higher genetic and phenotypic diversity than the cultivated rice (Sarla et al., 2003). Consequently, the knowledge of functional trait diversity among wild relatives will largely enhance their efficient utilisation, in addition to effective conservation (Lu et al., 2002;Ren et al., 2003).

September 2021
Journal of the National Science Foundation of Sri Lanka 49 (3) Consequently, the knowledge of functional trait diversity among wild relatives will largely enhance their efficient utilisation, in addition to effective conservation (Lu et al., 2002;Ren et al., 2003). However, our understanding of functional trait diversity, particularly in wild Oryza species, is still limited (Duan et al., 2007;Rathore et al., 2016). Identifying traits and characterising their variation under different environmental factors is important to understand the functional trait diversity among species (Micol & Hake, 2003;Itoh et al., 2005;Kadioglu & Terzi, 2007;Alvarez et al., 2008;Tian et al., 2012). The variability of functional traits among species is indicative of their important physiological processes including photosynthesis. Of the functional traits of wild rice, net photosynthetic rate (net assimilation rate), transpiration, and stomatal conductance are significant parameters to regulate the plant growth and development (Rathore et al., 2016). Determination of diversity among the wild relatives of cultivated Oryza spp. based on functional traits will be needed in directing future efforts to discover desirable traits and thus facilitate effective germplasm conservation and utilisation in rice breeding (Zhu et al., 2014).
The genus Oryza contains approximately 24 species distributed in Asia, Africa, Australia, and America. Of the species, only two are cultivated species and the remaining 22 species are wild relatives of rice (Vaughan, 1989;Khush, 1997). Moreover, Sri Lanka is one of the secondary diversity centres for rice genetic resources (Ikeda & Vaughan, 1991). Five wild species of Oryza viz. O. nivara (AA genome), O. rufipogon (AA genome), O. eichingeri (CC genome), O. rhizomatis (CC genome) and O. granulata (GG genome) are known in Sri Lanka and O. rhizomatis is considered endemic to the country. Among these, O. nivara and O. rufipogon are closely related to Asian cultivated rice (Banaticla-Hilario, 2012). The pest and disease resistance in these wild rice species are well-documented (Liyanage & Senanayake, 2010;Madurangi et al., 2012). Their distribution, habitats, pollination and flowering patterns are also well described under Sri Lankan conditions Liyanage & Senanayake, 2010;Rajkumar et al., 2015). Such information directs rice breeders to identify genetically diverse parents to gain desired traits when developing new rice cultivars.
However, the understanding of morphological diversity, particularly in wild Oryza species is still limited and sometimes contradictory. Thus, identification of wild species in their natural habitat is difficult or misleading to draw proper conclusions. In this regard, distinct structures in different plants of the same or different species need to be examined in detail and compared (Sattler & Hall, 1994). The characters that are used in plant identification, classification and description should be diagnostic or key characters that can be either qualitative or quantitative or both qualitative and quantitative. Plant growth habitat, growth patterns, seedling characters, leaf characters, inflorescence and flowers, fruit characters and seed characters are the major traits considered in the proper identification process. However, plant descriptions are often limited, and the morphological, physiological and anatomical distinctions among these species are often vague and not clear enough for field identification and species differentiation. Thus, the field identification of these species is difficult and often confusing based on the available information. Therefore, this study aimed at characterisation of Sri Lankan wild rice species to identify morphologically distinct traits to support field identification, and physiologically and anatomically
Mature seeds or root stocks of ten individual plants of each wild rice species were collected from the naturally occurring populations keeping a minimum distance of 5 m between plants to prevent the collection of ramets from a single genet. Exact locations of samples collected were recorded by a Global Positioning System (GPS; Garmin Oregon 550). Thereafter, ten individuals from each wild rice population of the respective wild rice species were established in cement pots (40 cm length × 40 cm width × 45 cm height), using seeds or rootstocks having 10 pots per population, in a common garden at the Faculty of Agriculture of the University of Ruhuna (latitude 06.060337°N and longitude 80.5681455°E), Sri Lanka, from January to December 2016. Each pot per population was considered as a replicate. Pots were arranged in a completely randomised design and the morphological, physiological and leaf anatomical features were characterised. distinct traits that could be useful in rice breeding.
Journal of the National Science Foundation of Sri Lanka 49 (3) September 2021

Morphological characterisation
Morphological characters (qualitative and quantitative) from seedling to mature stage were measured as described in the list of descriptors for wild and cultivated rice (Oryza spp.) published by the Biodiversity International, International Rice Research Institute and West Africa Rice Development Association (BI-IRRI-WARDA, 2007). Morphological diversity was measured by 11 quantitative (Table 2) and 28 qualitative traits (Table 3). For each character, average measurements taken from three randomly selected tillers per plant, including the main culm was considered. As time of planting was same for all five species, measurements were taken soon after heading (except seedling height).

Functional trait characterisation
Photosynthetically active radiation (PAR), leaf transpiration rate (Evap), stomatal conductance (GS), leaf surface temperature (LT), net photosynthetic rate (PN) and sub-stomatal CO 2 concentration (C Int) were determined using TPS-2 (MA 01913, Portable Photosynthesis System, Amesbury, USA). The measurements were taken randomly from the fully expanded top five leaves of the main culm and matured tillers for each selected plant, and repeated in all replicates. The same leaves were sampled for anatomical investigations. The mid portion of each leaf blade was inserted in the leaf chamber for gas-exchange measurements. For all Oryza species except O. granulata, two leaf blades were used to fully cover the cuvette luminal surface area. The leaf width of O. granulata was higher than that of the cuvette diameter and thus, a single leaf blade was assembled. Data were obtained between 10 a.m. to 2 p.m. with an air temperature of around 30 °C. Measurements were taken after the plants were exposed to sunlight for approximately 1 h and the leaf functional traits that were given by the leaf gas exchange were recorded.

Fixing, staining and observation of leaf anatomy
A 3 cm long section of the first fully expanded leaf blade from each sampled plant was separated for the leaf structural studies. The leaf sections were cleared and fixed as described by Huckelhoven & Kogel (1998)

Statistical analysis
Statistical analysis was performed using SAS version 9.2 (SAS Institute) and Minitab version 17 (Minitab, 2014). First, ANOVA was carried out to describe the variability of each structural and functional leaf trait, based on the entire three (morphology, physiology and anatomy) datasets for five wild Oryza species. Quantitative traits of different wild rice species were statistically described using means and standard error of the means of particular traits to figure out the general information related to different species. Quantitative variables were subjected to Pearson's correlation analysis at p = 0.05. Pearson correlation matrices were calculated based on the mean values of each structural and functional trait of each leaf of each Oryza species, to evaluate the trait-to-trait associations.

Qualitative and quantitative traits for field identification of wild rice species
The results revealed that the quantitative traits evaluated showed a distinct variation among the species ( Table  2). The variation and unique morphological traits that could be useful in species identification in field level are illustrated in Figure 1.
Among the quantitative traits, flag leaf length (FLL) and flag leaf width (FLW), culm length (CL) and culm diameter (CD), panicle length (PL), 100-grain weight (SW) and plant height (PH) are distinctive parameters in identifying species in the field. Among the species, O. granulata showed the lowest (p < 0.0001) PH (60.3 cm), FLL (15.3 cm) and PL (8.9 cm) and a higher FLW (2.1 cm) indicating that this species is more appropriate for shade environments (Table 2). O. rufipogon recorded the highest CL (152.3 cm), CD (7.3 mm) and PH (184.8 cm) (p < 0.0001) compared to other species ( Table  2) indicating that the species has developed higher lodging resistance than the rest of the species. Further, the presence of such characteristics may help survival in permanently inundated habitats (Banaticla-Hilario et al., 2013). Previous studies have indicated that the culm-related traits such as a wider culm diameter and less number of tillers, are directly associated with crop physiology and yield due to increase in lodging resistance of the plant (Chuanren et al., 2004). O. nivara showed the highest 100-grain weight (p < 0.0001) among studied species. The 100-grain weight was also positively correlated (p < 0.001) with the LL (r = 0.929), FLL (r = 0.827), CL (r = 0.745) and PH (r = 0.696) (data not shown). The quantitative traits reported significant variations among species are distinctive indicators that could be used for field identification of wild rice species.  The values presented are the means ± standard error of means of 10 replicates in each of the five species. One way ANOVA was used to compare the mean values. Traits among species are significantly different at p < 0.0001. *LSD (least significant difference) at p = 0.05. The morphological differences among wild rice species are directly correlated to their natural habitats, life cycle and breeding system (Banaticla-Hilario et al., 2013). Ammiraju et al. (2010) reported that the genus Oryza has experienced a rapid diversification within a short evolutionary time period. In Sri Lanka, wild rice species are niched to diverse eco-geographic environments (Liyanage & Senanayake, 2010;Sandamal et al., 2018b).

Journal of the
Most of the morphological traits are influenced by the environmental factors and thus, we evaluated them in the common garden under same environmental conditions as reported by Abhayagunasekara et al. (2018 (Figure 1a). Comparatively long awns were detected in O. nivara and O. rufipogon. The floral morphology among species showed many characteristic differences in stamens. O. granulata had plumose type stigma with white colour stamens, which was clearly divided into two parts at the base of the stamen ( Figure  1c). O. eichingeri and O. rhizomatis had stamens of the same size (length and width) and shape but differed in colour, i.e. dark yellow and pale purple, respectively (field observation). Both O. nivara and O. rufipogon produced anthers of the same shape and colour (yellow) but O. rufipogon had longer anthers than O. nivara when compared to those reported by Banaticla-Hilario  (Table 3). Five wild rice species revealed large variations in seed coat colour, viz. red, light brown, brown and white (Figure 1b). Moreover, the dark green colour of lemma and palea was observed in the immature panicles of O. granulata. The size of the seeds (length and width) is one of the most stable characteristics (Table 2), which has a high heritability and therefore, can be used to distinguish species (Jackson, 1995).
Marginal differences were observed in the shapes and colour of auricle and ligule (Figure 1d) in wild rice species, which is one of the key characteristics to identify Oryza species from other species in the family Poaceae.  Generally, most rice varieties cultivated in Asia have pubescent leaves, and those in Africa and America are glabrous (Khush, 2001 (Table 3). Glabrous trait may be selectively neutral in rice. However, trichomes are thought to be vital for plant defence against biotic and abiotic stresses. Thus, breeding for pubescent rice varieties is mainly targeted at the practical advantages of paddy production. Except for these qualitative traits, others showed minor variations among the species (Table 3). In contrast, no variations were observed for six qualitative parameters, viz. blade colour, collar colour, panicle axis, sterile lemma colour, panicle shattering and panicle threshability.

Functional trait diversity of five wild rice species
The physiological functions of the five wild rice species used in this study varied from each other, indicating the potential of using such functional traits in rice breeding. Photosynthesis forms an essential aspect of plant metabolism and the balance sheet of growth and development, which is sensitive to different abiotic stresses (Gupta et al., 2002;Panda et al., 2008;Gauthami et al., 2014). Under the same environmental conditions, a remarkably high net photosynthetic rate was observed in O. rufipogon compared to the rest of the species (Table  4). The reduction in photosynthetic rates may be a result of the changes in stomatal and non-stomatal factors (Panda et al., 2008;Mathobo et al., 2017). Generally, a lower photosynthetic efficiency occurs due to the inhibition of photosynthetic enzymatic activity, and the decrease in chlorophyll and oxidative loads (Hayat et al., 2012). The five wild rice species had transpiration rates ranging from 0.7 to 2.  (Giuliani et al., 2013). Further, it helps cooling a plant and promote cell enlargement (Crawford et al., 2012). The highest stomatal conductance (p < 0.0001) in the present study was detected in O. rufipogon. The decrease of stomatal conductance was observed in species, except O. rufipogon under the existing environmental conditions, was may be due to the stomatal closure (Panda et al., 2008;Gauthami et al., 2014). The present study also reported a weak positive correlation between the photosynthetic rate and stomatal conductance. Siddique et al. (1999) reported that a strong relationship between net photosynthetic rate and stomatal conductance is an indication of the reduction in net photosynthetic rate, mostly due to stomatal closure, whereas a weak relationship indicates that the net photosynthetic rate is regulated by non-stomatal factors. An increase in sub stomatal CO 2 concentration (C int) suggests the predominance of non-stomatal limitation to photosynthesis, whereas a decrease in C int indicates the stomatal limitations dominated for the photosynthetic inhibition (Panda et al., 2008) One way ANOVA was used to compare the mean values. PAR -photosynthetically active radiation (µmol m -2 s -1 ); (p=0.1938) Evap -leaf evaporation rate (mmol m -2 s -1 ); (p<0.0001), GS -stomatal conductance (mmol m -2 s -1 ); (p<0.0001), LT -leaf temperature (°C); (p=0.0134), PN net photosynthetic rate (µmol m -2 s -1 ); (p=0.0622), C Int -sub-stomatal CO 2 concentration (µmol mol -1 ). (p=0.6212). *LSD (least significant difference) at p = 0.05. The present study showed that the vein density was not significantly different (p = 0.0612) among species (Table 5). However, the vascular bundle cell size and density are strongly correlated with the transpiration and photosynthetic rate of the species, which has a large culm (He & Zhang, 2003). Among the five wild rice species, O. rufipogon (Table 4) recorded the highest rate (p < 0.0001) of transpiration and stomatal conductance (p < 0.0001). Meanwhile, O. rufipogon showed a higher net photosynthesis rate among the tested species. O. rufipogon naturally grows and survives in environments such as deep-water habitats where water is not a limiting factor. Therefore, is an ideal species for super rice breeding when water is available at sufficient levels (Liu et al., 2015). As found in O. rufipogon,. cultivars or  species with a large culm has shown a higher apoplastic transport ability (Gong et al., 2006), which might help transfer water and nutrients more rapidly and efficiently thus, contributing to higher grain Bulliform cells are large, thin-walled and highly vacuolated cells that play a vital role in controlling leaf rolling in response to drought, salinity and high temperature (Itoh et al., 2005). The efflux of water from bulliform cells induce adaxial leaf curling (Liu et al., 2016). Expansion of the adaxial epidermal cells while an increase in bulliform cells, is closely related to abaxial rolling of the leaf. Furthermore, abaxial leaf rolling and their functions are bidirectional (Li et al., 2010). The present study showed that O. eichingeri and O. rufipogon had the highest number of bulliform cells (p=0.05) per cluster (Figure 3) whereas O. granulata showed the lowest number. The largest bulliform cell cluster and the highest width of middle bulliform cells were found in O. rhizomatis (Table 5). Moreover, the wider bulliform cell cluster and higher distance between the two clusters in the O. rufipogon indicated a systematic modification in morphology and anatomy involved in the development of rice in terms of drought resistance. study indicated that the sub stomatal CO 2 concentration was not significantly different (p = 0.6212) among species (Table 4).
The vascular tissues are the most important structural components in plant tissues, which are responsible for the transport of assimilates, minerals and water (Hose et al., 2001;Cholewa & Griffith, 2004).

September 2021
Journal of the National Science Foundation of Sri Lanka 49 (3) 2010), thus differ in O. nivara habitats. The typical natural habitats of O. rufipogon were stream banks, marshy lands, swamps, and deep-water lake edges (Sandamal et al., 2018a). It grows in water 10 cm -5 m deep. Perennial O. rufipogon is photoperiod sensitive plant with a bimodal flowering pattern and peak mature panicles were observed in April and October, separately (Ratnasekera et al., 2019).

CONCLUSION
This study has dissected the morphological, leaf anatomical and physiological traits of wild relatives of cultivated rice in Sri Lanka (O. nivara, O. rufipogon, O. eichingeri, O. rhizomatis and O. granulata ) and reports a significant morpho-physiological and anatomical diversity of the traits. Qualitative parameters such as the panicle type, awning, stigma colour, lemma and palea pubescence, seed coat colour, blade pubescence, and ligule shape showed vast differences among the five species and are useful and promising characters in field identification. Quantitative traits such as flag leaf length, flag leaf width, culm length, culm diameter, panicle length, 100-grain weight, and plant height are distinctive parameters among five species that could be used for further confirmation of species. The plant physiological characters such as net photosynthetic rate, transpiration rate, stomatal conductance and sub-stomatal CO 2 Further, the number of bulliform cells per cluster had a positive significant correlation indicating that a relatively higher cell number or cluster area in a species might play an important role in the adaptation to dry conditions (Giuliani et al., 2013). Stomata are microscopic apparatus in leaf epidermis enabling exchange of air and mainly contribute to the photosynthetic efficiency. The highest and the lowest stomatal density were recorded in O. granulata and O. nivara, respectively (Table 5). The stomatal density and stomatal size are the anatomical traits that contribute to gas diffusion (Giuliani et al., 2013). Leaf gas exchange was controlled by different stomatal traits such as stomata number, density and size (Panda et al., 2008). Stomatal density was influenced by the number of stomata per row, although on the abaxial surface, a greater number of rows across the leaf have also contributed to the stomatal density. The highest trichome density was observed in O. rufipogon (Table  5) indicating its enhancing antibiosis and antixenosis properties thus, reducing insect landing and feeding on leaf surface (Tian et al., 2012). This character could be exploited by breeders in the selection of superior genotypes in terms of phenotypic performance.

Specific habitat information for field collectors
Information on habitat preference, geographical distribution and life history traits are the most important facts that drive efficient sampling of wild genetic resources in their natural habitats. Some of these habitats are threatened due to various human activities (Sandamal et al., 2018a). Therefore, immediate actions are needed to conserve these valuable rice genetic resources (Abhayagunasekara et al., 2018).
O. nivara was mainly confined to the low country dry and intermediate zones. It was not found in the wet zone or upcountry dry/wet regions (Liyanage & Senanayake, 2010). O. nivara is distributed extensively in the dry zone and approximately more than 2 ha area in certain natural habitats can be seen. Swampy areas, at the edges of ponds and lakes, and beside streams are the major natural habitats of O. nivara. It generally begins seedling emergence with monsoon rain and grows in shallow water. However, continuous water logging condition is not required throughout the life cycle (life cycle observations). It is an annual plant; flowering occurs from January to May and peak mature panicles were recorded from April to May (Ratnasekera et al., 2019). Physiological functions and anatomical features of the five wild rice species vary from each other indicating the potential of using such functional traits in rice breeding programmes. There were significant correlations between several functional and structural traits, and physiological traits such as transpiration and photosynthesis. The findings of the present study help clear the way for field identification, conservation of the existing rice gene pool as well as provide useful information on important traits of the five rice genotypes for further utilisation. 100 grain weight; GL: grain length; GW: grain width; NCC: number of cells per cluster; DBC: distance between two cluster; CBW: cluster base width; SD: stomatal density; TD: trichome density; PAR: photosynthetically active radiation; Evap: transpiration rate from the leaf; GS: stomatal conductance; LT: leaf temperature; PN: net photosynthetic rate.