Protogynous dichogamy, leaf morphology and leaf essential oil composition of selected Cinnamomum species in Sri Lanka

Wild relatives of cultivated cinnamon (Cinnamomum verum J. Presl) are underutilized and endangered in Sri Lanka. There is a lack of knowledge on wild relatives of cinnamon, hampering their utilization in breeding and industry. Morphology, floral behaviour and leaf essential oil composition of selected wild relatives were determined under ex-situ conservation in Dalpitiya, Sri Lanka. Floral cycles were determined in Cinnamomum dubium Nees (Cd) and Cinnamomum litsaeifolium Thwaites (Cl) along with Cinnamomum verum (Cv) variety Sri Gemunu (SG). Cl and SG belonged to type A, while Cd was type B of protogynous dichogamy. Partial overlapping of male and female phases in types A and B may lead to self-pollination. Leaf morphological characters varied among species. Gas Chromatography Mass Spectrometry revealed 34, 34, 12, 48, 8 and 18 chemical compounds from Cinnamomum capparu-coronde Blume (Cc), Cd, Cl (1), Cl (2), SG and variety Sri Wijaya (SW) respectively. The highest abundant chemical compound in leaf oil varied as Eugenol in Cc, SG and SW (33.11%, 82.11% and 90.80% respectively), Methyl eugenol in Cl (1) (59.27%), Eucaliptol in Cd (51.19%) and Linalool in Cl (2) (30.93%). The above variation of wild cinnamon provides insights on future cinnamon breeding and industry.

INTRODUCTION extinction risk level of wild cinnamon species in Sri Lanka. According to Kumarathilake (2009), Cinnamomum citriodorum, Cinnamomum rivulorum, Cinnamomum sinharajaense, Cc and Cl are critically endangered at the global level. Cinnamomum citriodorum and Cc are highly threatened at the national level. Cc is distributed in low country rain forests. Cl is a threatened species at the national level and is only found in the Knuckles forest of Sri Lanka. Cd with medicinal and timber value is not a threatened species and is grown in tropical rain forests and secondary forests in Sri Lanka. Habitat destruction for urbanization and intensive agricultural practices had been identified as the major threats to wild cinnamon species in Sri Lanka by Kumarathilake (2009) However, these ex-situ conservation sites consist of heterogenous plant collections as seedlings from exploration sites had been used for the establishment. During his study, Cc, Cinnamomum citriodorum and Cd were identified as potential candidates for Cv breeding. Among them, Cc was identified as the most suited species for domestication due to its desirable characters of the high amount of eugenol in leaf essential oil, the erect stem and medicinal and timber value. However, Kumarathilake (2009) reported that the above species were not successful in vegetative propagation. Prathibhani et al. (2020) reported the variation of shoot regeneration capacity of stem cuttings from several Cv genotypes. Geethakumary et al. (2007;2012)  In Family Lauraceae, protogynous dichogamy is observed: Flowers open in two phases, where the female phase is prior to the male phase. The first opening of flowers occurs during the morning in the type A plants, while it occurs during noon in type B plants. At the first phase only, the stigma appears as receptive. The first and third stamen whorls appear fused during this stage. First opening lasts for about five hours. The second phase begins after 24 hours of first opening. The third whorl of the stamens adheres to the pistil. The anthers become dehiscent after 1/2-1 hour of second opening. The stigma is shriveled and become non-receptive. Again, the flower is kept open for about five hours (Joseph 1981;Kubitzski and Kurz 1984;Sedgley and Griffin 1989). According to Azad et al. (2018), there is a variation in inflorescence as in panicle length, panicle type, flower colour, flower length, flower width, tepal length, tepal width, tepal number and tepal pubescence within Cv germplasm. Information on inflorescence type, size and flower colour of fifteen accessions from a field survey conducted in 15 locations of cultivated lands and wild habitats in Matara district, Sri Lanka has been reported by Azad et al. (2018). The same authors suggested the possible linkage between large flower size and type A flower behaviour.
There may be inter-species variation in the time of occurrence and active duration of the female and male phases among Cinnamomum species. Protogynous dichogamy, which leads to cross-pollination, contributes to allele richness in Cv germplasm (Azad et al. 2015). Bark, leaf, root and fruit essential oils of Cv bear each of unique chemical profiles has majored with cinnamaldehyde, eugenol, camphor and cadinene respectively (Senanayake et al. 1989;Paranagama et al. 2001).
The genetic diversity of wild cinnamon germplasm in Sri Lanka would be depicted through environment-independent leaf morphological variation. The wide chemical profiles of wild cinnamon species in Sri Lanka would be potential sources in food, pharmaceuticals and cosmetics. Revealing the extent of protogynous dichogamy among wild cinnamon would be useful in their utilization in breeding programmes.
One plant of Cc, one plant Cd and two plants of Cl through the exploration of Kumarathilake (2009) were maintained in the ex-situ conservation site at Mid Country Research Station. Seed setting had been observed at the above ex-situ conservation site in 2018 by the second author. As there were single plants of Cc, Cd and two plants of Cl (named as Cl-1 and Cl-2) at the site, we speculated that selfing within the single plant or cross-pollination with another Cinnamomum spp. had taken place. Therefore, this study was conducted in the following year (2019) to determine the morphological and chemical characters and flower type of each spp. as such information would be useful in identifying potential hybrids in the future. Further, the information on essential oil composition would be useful in industrial applications.

MATERIALS AND METHOD
The present study was based on the ex-situ wild cinnamon conservation site at Mid-Country Research Station, Department of Export Agriculture, Dalpitiya (WM2) (GPS: 7.1333031 N, 80.590026 E) established by Kumarathilake (2009). Observations on flowering of above Cinnamomum spp. were made. According to the availability of flowers during the study period of February to March 2019, the floral behaviour of two wild cinnamon species of Cd and Cl along with SG was determined. Floral cycles were determined through visual observation of flowers for two consecutive days from 8 am to 4 pm. Leaf morphology and leaf essential oil composition of wild cinnamon species of Cc, Cd and Cl along with SG and SW were determined. Mature leaves of one Cd, one Cc, two Cl (1), (2), SG and SW were collected randomly at 5 th to 6 th leaf from the tip of the branch for both morphological characterization and leaf essential oil analysis. Length, width and petiole length were measured as quantitative leaf morphological characters. Qualitative leaf morphological characters of shape, apex, base, texture, venation and margin were characterized using

RESULTS AND DISCUSSION
There was a variation in leaf morphological characters of leaf length, leaf width, leaf shape, leaf apex, leaf base, leaf texture, leaf venation, petiole length and leaf margin among Cinnamomum species (Table 1, Figure  1). Cc and Cd were lanceolate leaf-shaped while Cl (1) and Cl (2) were elliptic leafshaped. SG and SW were broadly ovate and ovate leaf-shaped respectively. Cl (1), (2), SG and SW had acute leaf apexes. Cc and Cd leaf apexes were acuminate with broad acumen and long acuminate respectively. Leaf base of Cd, SG and SW was round. Cc, Cd and Cl (1) leaf textures were thin to stiffly coriaceous. SG and SW had the same chartaceous leaf texture, while Cl (2) was of chartaceous to rigidly chartaceous leaf texture. Cl (1), (2), SG and SW had three-veined leaves while Cc and Cd had three-veined or five veined leaves. All Cc, Cd, Cl (1), (2) and SG had the entire leaf margin. SW was with the undulate leaf margin.
We observed wild cinnamon flowering from February to March 2019 in Dalpitiya. Sri Lanka. The flowering season of Cv begins in November and continues until early March. Cv fruits ripen from May to June (Joseph 1981;Kubitzki and Kurz 1984;Mohankumar et al. 1985). According to our observations in Southern Sri Lanka, Cv flowering starts from August to September and extends up to April in the following year. There are evidences that the floral behaviour of cinnamon might be affected by the environmental factors of day length, photon flux density and In SG and Cl, the inflorescence produced open flowers of two physiological stages during the morning. Flowers of one of the stages opened from 8.00 to 9.00 am with white colour stigma. They remained open around five hours till 1.00 pm and closed. They opened again on the next day around 11.00 am with brown colour stigma and tepals. After about five hours they permanently closed completing the floral cycle. According to the classification of flowers on dichogamy, variety SG and the studied Cl plant are included under type-A.
The studied Cd plant belonged to the type-B category. The flowers started opening in the afternoon around 1.00 pm with fresh white colour stigma and tepals. The stamen whorls appeared as fused. They remained opening about 3½ hours and closed around 4.00 pm. They opened again on the next day around 8.00 am. There were anthers dehiscent with pollens around 9.00-10.00 am. They were with brown colour stigma and brown colour tepals at this opening and remained open about 7 hours. According to the above observations, partial overlapping of functional male and functional female stages may lead to self-pollination in all three species.
Joseph (1981) reported that the female and male phases of cinnamon flowers separated by almost one day. According to our observations in Dalpitiya, Sri Lanka, the above observation was confirmed for the type A plants. The floral cycle of type B plants consisted of the female and male phases separated by twelve hours. In accordance with the report of Mohankumar et al. (1985), we also observed the maximum flower breath at the second phase of the floral cycle.
There are reports on the presence of type A and type B plants in Cv and C. camphora populations. Within a Cv population, 3/5 of the plants are type A and 2/5 of the plants are type B (Joseph 1981). In a natural population, A and B plant types are mixed for the availability of functional male and female flowers at any given time (Joseph 1981;Kubitzski and Kurz 1984). The majority of cultivars belong to type B among the studied ten cultivars of Cv for floral behaviour at the National Cinnamon Research and Training Center, Sri Lanka (Kumari et al. 2008). There is some unpublished information on floral behavior of two commercial varieties of Cv in Sri Lanka as SG is type A and SW is type B. Future work should be focused on the molecular basis of type A and type B plants of cinnamon, floral morphological markers for identifying the two types of plants and the environmental effects on floral behaviour of cinnamon.

CONCLUSION
There was a variation in leaf morphological characters in Cc, Cd, Cl, SG and SW. The observed Cl plant was of type A and Cd was of type B. Partial overlapping of functional male and female phases should be further investigated to determine the possibility of self-pollination. Eugenol was the major chemical compound of leaf essential oils from Cc (33.11%). Eucalyptol was the major chemical compound of Cd (51.19%). The highest chemical constituent in two Cl plants varied as methyl eugenol (59.27%) in one plant and linalool (30.93%) in the other. This information signifies the necessity of understanding the variation within a spp. and identification of chemotypes.

AUTHOR CONTRIBUTION
MRP, RAAKR and SG conceptualized and designed the study. MRP performed the experiments and analyzed the data. MRP, SAR and SG interpreted the data. MRP and SG drafted the manuscript. RAAKR and SG critically revised the manuscript.