Casuarina Species Research Articles

Differential responses of six Casuarina species to NaCl seed priming and salt stress during germination and early seedling growth

The degradation of salinized soils is imposing a steady decline in productivity, leading to significant socio-economic and environmental repercussions. As a promising solution for revitalizing depleted soil potential, the cultivation of salt-tolerant plants such as Casuarina emerges as a viable approach. This study delves into this prospect by evaluating six Casuarina species to discern their levels of tolerance to varying concentrations of NaCl, particularly in the initial stages of germination and seedling growth. The imposition of salt stress instigated a reduction in both germination capacity and speed. High variability in salt tolerance was observed between the tested species. Notably, the final germination percentages at 100 mM NaCl unveiled considerable disparities: C. torulosa, C. tenuissima, and C. stricta exhibited percentages of 33%, 19%, and 10%, respectively, while C. cunninghamiana, C. equisetifolia, and C. glauca showcased higher germination rates at 63%, 62%, and 55%, respectively. Furthermore, the increase of the recovery rate with high NaCl concentration confirmed that the salinity caused only an osmotic effect and not a toxic one on the studied species. Additionally, the adoption of seed priming showcased marked enhancements in germination rates across all NaCl concentrations, effectively expediting the germination process. In response to salinity stress, reductions were noted in root length (RL), and shoot length (SL). All germination parameters and the computation of the salt tolerance index (STI) facilitated the classification of the six Casuarina species in a descending order of salinity tolerance: C. cunninghamiana Miq. > C. equisetifolia L. > C. glauca Sieb. > C. torulosa Ait. > C. tenuissima Sieb. > C. stricta Ait.

Phytochemical analysis and evaluation of its antioxidant, antimicrobial, and cytotoxic activities for different extracts of Casuarina equisetifolia.

Casuarina equisetifolia belongs to the Casuarina species with the most extensive natural distribution, which contain various phytochemicals with potential health benefits. This study aimed to investigate the chemical composition and biological activities of different extracts of Casuarina equisetifolia. The n-hexane extract was analyzed for its unsaponifiable and fatty acid methyl esters fractions, while chloroform, ethyl acetate, and butanol extracts were studied for their phenolic components. Six different extracts of C. equisetifolia needles were evaluated for their total phenolic content, total flavonoid content, and their antioxidant, antimicrobial, and cytotoxic activities. The n-hexane extract contained mainly hydrocarbons and fatty acid methyl esters, while ten phenolic compounds were isolated and identified in the chloroform, ethyl acetate, and butanol extracts. The methanolic extract exhibited the highest total phenolic and flavonoid content, highest antioxidant activity, and most potent cytotoxic activity against HepG-2 and HCT-116 cancer cell lines. The ethyl acetate extract showed the most significant inhibition zone against Staphylococcus aureus and Bacillus subtilis. Casuarina equisetifolia extracts showed promising antioxidant, antimicrobial, and cytotoxic activities. Overall, Casuarina equisetifolia is a versatile tree with a variety of uses, and its plant material can be used for many different purposes.

Development of Volumetric Equation and Volume Table for Casuarina Species

Development of Volumetric Equation and Volume Table for Casuarina Species

Chromosome-scale de novo genome assembly and annotation of three representative Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana.

Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration. To characterize Casuarina genomic diversity, we sequenced and constructed de novo genome assemblies of the three most widely planted species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome-scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi-C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268,942,579 bp, 296,631,783 bp, and 293,483,606 bp, respectively, of which 25.91%, 27.15%, and 27.74% were annotated as repetitive sequences. We annotated 23,162, 24,673, and 24,674 protein-coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole-genome bisulfite sequencing (BS-seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA-seq) revealed differential expression of phytohormone-related genes between male and female plants. In summary, this study generated three chromosome-level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.

Floral biology, pollen viability and stigma receptivity in three species of Casuarina

Floral biology, pollen viability and stigma receptivity in three species of Casuarina

Casuarina species in Algeria: reviewing their identity, distribution and symbiotic status

Since their introduction at the beginning of the 19th century, Casuarina species have become important components of the Algerian landscape, yet the numbers of species present in Algeria and their identity, distribution, and symbiotic status are still not well understood. A survey conducted on 2,304 trees in 96 Casuarina stands across Algeria indicates that they are represented exclusively by five species: Casuarina cristata Miq., C. cunninghamiana Miq., C. equisetifolia L., C. glauca Sieber ex Spreng. and C. junghuhniana Miq. Two species, C. cunninghamiana and C. glauca, were the most common – the former occurring more often in the relatively humid and temperate areas of the country while the latter is most frequent in the drier and warmer areas. Production of seedlings of C. cunninghamiana and C. glauca in 12 nurseries resulted in low rates of spontaneous mycorrhizal infection frequency and nodulation, suggesting that inoculation of seedlings with selected mycorrhizal fungi and/or Frankia strains in the nursery would be necessary to improve their establishment in the field. This study is the first survey of the geographical distribution of Casuarina species in Algeria and should lead to more efficient utilization of this widely used tree genus in Algeria.

Hybridisation, pollen–pistil interactions and hybrid seed set among

Context Casuarina trees are extensively planted in the tropics for wood production and environmental services. The breeding program’s current focus is on developing hybrids that combine the specific characteristics of different species and are suitable for various planting environments and end uses. Aim Breeding programs aim to develop interspecific hybrids possessing desirable combinations of species-specific characters of Casuarina for increasing plantation productivity. Methods Controlled-pollination studies were conducted involving C. equisetifolia, C. junghuhniana and C. cristata to determine the possibility of hybridisation among them and to evaluate the hybridisation success rate in different cross-combinations. The pollen-tube growth in the pistil was studied to understand the role of pollen–pistil interactions in hybridisation success. Key result Fourteen cross-combinations of the three species resulted in 2–57% fruit set and 75.1–93.8% seed set. C. equisetifolia and C. junghuhniana hybridised reciprocally, whereas C. cristata hybridised with other species only as pollen parent. The larger flower size of C. cristata acted as a barrier preventing hybridisation with species having smaller-sized flowers. Variation in seed germination was large (3.6–91.3%), highest for C. junghuhniana and lowest for C. equisetifolia mothers. Fertilisation occurred 45 days after pollination in C. equisetifolia and C. junghuhniana and after 50 days in C. cristata. The ovary was immature at pollination and pollen tubes showed discontinuous growth in the pistil marked by periods of arrest for the ovules to develop. The stigma showed 3–5.7 pollen tubes, and invariably a single tube entered the ovary. The absence of major pre-fertilisation barriers but varying levels of hybridisation success indicates post-zygotic selection through embryo abortion. Conclusion Increasing the parental combinations of interspecific crosses where fruit set is limiting and pollinating more flowers per cross where germination is low may enhance hybridisation success. Implications The cross compatibility among different species of Casuarina provides scope to produce site and end-use specific hybrids to meet the commercial and livelihood needs.

Anthropogenic influences on the distribution of the Casuarina-Frankia symbiosis

Casuarina species are evergreen angiosperm trees native to Australia, South East Asia and the Pacific archipelagos. The genus comprises 14 confirmed species that are able to grow on a wide range of soils. Casuarina spp. are able to enter into an actinorhizal root nodule symbiosis with Frankia casuarinae, nitrogen-fixing soil actinobacteria; the symbiosis renders them independent of soil nitrogen sources. Casuarina spp. can also form symbiotic associations with ectomycorrhizal fungi (EMF) and/or arbuscular mycorrhizal fungi (AMF) for better phosphate supply. Under severe deficiency of phosphate and iron, several Casuarina spp. can form cluster roots. These trees are highly tolerant to salinity, drought, flooding and heavy metal pollution and are extensively used for the rehabilitation of degraded sites. Casuarina spp. plantations also play a significant role in prevention of soil erosion, coastal reclamation, dune stabilization, and as windbreaks in agricultural lands. Due to their multipurpose applications in agriculture and forestry, they have been widely introduced outside their native habitat especially in tropical, arid and semiarid countries across the globe. Therefore, the global distribution of Casuarina spp. has been significantly affected by anthropogenic influences. While in most of these countries, Casuarina spp. have become well-integrated into the regional farming systems, they have become invasive in other regions.

Feeding profitability is associated with Glossy Black-cockatoo (Calyptorhynchus lathami ssp. lathami) feed tree selection

ABSTRACT Habitat modification and destruction are known to be responsible for declines in avian taxa. Specialist species such as the vulnerable Glossy Black-cockatoo (Calyptorhynchus lathami ssp. lathami) are particularly at risk due to the limited number of Casuarina and Allocasuarina species on which they feed, which is compounded by their propensity for selecting only specific individual feed trees within patches. This study aims to address the lack of understanding surrounding Glossy Black-cockatoo feeding ecology in southeast Queensland, Australia, by examining determinants of tree selection for Allocasuarina littoralis and A. torulosa. We conducted surveys to identify feed and non-feed trees of both Allocasuarina species, and differences between the physical and chemical characteristics of trees, cones, and seeds were investigated. We found that larger trees were preferentially foraged, with nutritional profitability as the primary factor governing tree selection. Feed tree cones had higher seed number and seed mass, with seeds containing higher levels of fatty acids and nutrients than non-feed tree cones. We conclude that further research should aim to delineate the factors contributing to these differences, alongside determining appropriate management strategies to support viable feeding habitat, which is most likely comprised of mixed age patches, capable of maintaining mature trees offering sufficient nutrient reward.

Effect of Casuarina Plantations Inoculated with Arbuscular Mycorrhizal Fungi and Frankia on the Diversity of Herbaceous Vegetation in Saline Environments in Senegal

Land salinization is a major constraint for the practice of agriculture in the world. Considering the extent of this phenomenon, the rehabilitation of ecosystems degraded by salinization has become a priority to guarantee food security in semi-arid environments. The mechanical and chemical approaches for rehabilitating salt-affected soils being expensive, an alternative approach is to develop and utilize biological systems utilizing salt-tolerant plant species. Casuarina species are naturally halotolerant, but this tolerance has been shown to be improved when they are inoculated with arbuscular mycorrhizal fungi (AMF) and/or nitrogen-fixing bacteria (Frankia). Furthermore, Casuarina plantations have been proposed to promote the development of plant diversity. Thus, the aim of the current study was to evaluate the impact of a plantation comprising the species Casuarina inoculated with AMF and Frankia on the diversity of the sub-canopy and adjacent vegetation. Work was conducted on a plantation comprising Casurina equisetifolia and C. glauca variously inoculated with Frankia and Rhizophagus fasciculatus prior to field planting. The experimental area of 2500 m2 was divided into randomized blocks and vegetation sampling was conducted below and outside of the Casuarina canopy in 32 m2 plots. A total of 48 samples were taken annually over 3 years, with 24 taken from below the Casuarina canopy and 24 from outside the canopy. The results obtained show that co-inoculation with Frankia and Rhizophagus fasciculatus improves the height and survival rate of both species. After 4–5 years, there was greater species diversity and plant biomass in the sub-canopy environment compared with that of the adjacent environments. Our results suggest that inoculation of beneficial microbes can improve growth of Casuarina species and that planting of such species can improve the diversity of herbaceous vegetation in saline environments.

Effect of Casuarina Crushed Nodules, Rhizospheric Soil and Leaves Compost on Salt Tolerance of <i>Casuarina equisetifolia</i> L. and <i>Casuarina obesa</i> Miq.

Soil salinization is one of the major causes of land degradation. In Senegal, this phenomenon continues to grow, making soils unsuitable for agriculture. To rehabilitate salty lands, one of the recommended strategies is the use of salt-tolerant plants. Among them, plants of Casuarinaceae family form a relationship with symbiotic microorganisms such as arbuscular mycorrhizal fungi and nitrogen fixing bacteria. It has been shown that symbiotic microorganisms play an important role in the establishment of tolerant plants in saline conditions (Djighaly et al., 2018). They improve plant performance and reduce transplant shock under salt stress conditions (Diagne et al., 2014). These microorganisms can be used as biofertilizers. However, inocula containing symbiotic microorganisms are either too expensive or unavailable in many developing countries. The aim of this study is to test alternatively affordable and low-tech solutions to promote symbiotic interactions such as Casuarina crushed nodule, Casuarina rhizosphere soil and Casuarina leaves compost that may contain symbiotic microorganisms and also nutrients such as N and phosphorus. Two species of Casuarina (Casuarina equisetifolia L. and Casuarina obesa Miq.) were grown in the greenhouse on sterile soil to which an amendment was added (Casuarina crushed nodules, Casuarina Rhizospheric soil or Casuarina leaves compost). Plants were subjected to saline stress. After four months of cultivation, they were harvested and morphological and physiological parameters were determined. Results showed that inoculation with Casuarina crushed nodules, Casuarina rhizospheric soil and Casuarina leaves compost improved growth, total dry biomass, total chlorophyll and proline contents of C. equisetifolia and C. obesa plants in salt stress condition. These positive effects were more important in C. obesa plants amended with Casuarina leaves compost. This study shows that Casuarina leaves compost can play an important role in the rehabilitation of saline soils by improving Casuarina trees performance in saline conditions.

Infructescence and samara morphometrics and potential mechanism of samara release in Allocasuarina and Casuarina (Casuarinaceae)

In addition to samara aerodynamics, mechanisms of samara release will contribute to seed dispersal outcomes. Analysis of species descriptions of Australian members of the Casuarinaceae (sheoaks) indicated cone shrinkage to be a possible mechanism enhancing samara release. However, measurement of cones of nine species of Allocasuarina and Casuarina did not support this hypothesis, but outward movement of samaras both in the field and in oven-dried cones indicated that another mechanism was operating. Microscopic examination revealed that the proximal margins of the bracteoles are involute after drying, and it is postulated that incurving of the bracteoles during drying moves samaras outward against gravity enhancing samara release. This study also revealed inconsistencies in cone and samara metrics between field samples (1–3 specimens for 9 species) and the published descriptions. This could lead to identification uncertainties, and indicates the need to revise the descriptions to more fully encompass the variation in sheoaks across their natural and anthropogenic ranges.

Localization of typical and atypical Frankia isolates from Casuarina sp. in nodules formed on Casuarina equisetifolia

Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia. Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored. The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. qPCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only. These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia, but has likely been isolated as a surface contaminant from Casuarina nodules.

A novel set of 223 EST-SSR markers in Casuarina L. ex Adans.: polymorphisms, cross-species transferability, and utility for commercial clone genotyping

Simple sequence repeat (SSR) markers are very useful for genetic applications in plants, but SSR resource for the important tree genus Casuarina L. ex Adans. is still limited. In this study, we report a novel set of 223 SSR markers in Casuarina developed from expressed sequence tag (EST) resource of GenBank. The 223 EST-SSR markers were polymorphic among 10 unrelated individuals of C. equisetifolia L. Johnson, with the number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) averaging at 5.5, 0.72, 0.86, and 0.63, respectively. The rates of cross-species transferability ranged from 96.9% (C. glauca Sieber ex Sprengel) through 97.8% (C. cunninghamiana Miquel) to 99.1% (C. junghuhniana Miquel). Fifty-five C. equisetifolia clones widely planted in China were successfully genotyped with a subset of 20 EST-SSRs. These newly developed markers will have a great potential for genetic and breeding applications in Casuarina species and related taxa.

Development and validation of SSR markers based on transcriptome sequencing of Casuarina equisetifolia

Based on Casuarina equisetifolia transcriptome data, the present study developed and validated 15 transferable EST-SSR markers across Casuarina and Allocasuarina species. Casuarina species are valued as pioneer trees for coastal shelter forest construction. To accelerate Casuarina breeding for more robust, attractive, and cold-resistant cultivars, it is essential to introduce molecular marker-assisted breeding. Based on Casuarina equisetifolia transcriptome data, 10,291 EST-SSR loci with the types of 2–6 nucleotide repeats were identified from assembled 118,270 unigenes. An average of one EST-SSR was found every 13.54 kb, and the distribution frequency of SSRs was 8.7%. The most common repeat motifs were dinucleotide with AG/CT and trinucleotide with AAG/CTT. A total of 150 SSR markers were randomly selected for validation, among which 15 produced clear, reproducible and polymorphic bands, and thus were employed for genetic relationship analysis among 26 genotypes from four Casuarina and three Allocasuarina species. A total of 42 bands were identified, varying from 2 to 5 bands per locus with an average of 2.8. Polymorphism information content ranged from to 0.2624 to 0.6177 with a mean value of 0.4265, indicating a high level of polymorphism. The UPGMA dendrogram revealed a high genetic variation resided among species within two genera, Allocasuarina and Casuarina. Meanwhile, the 15 highly polymorphic Casuarina SSR markers were also proved to be transferable across Casuarina and Allocasuarina species. These valuable SSR markers can be applied further for population and evolutionary genetic studies in Casuarinaceae, and will be useful in selection and breeding of Casuarina species for improving cold tolerance.

Diversity of Pathogenic Fungi on Plantation Forests of North and North-West Ethiopia

Forest plantations in Ethiopia are mainly exotic genera of Eucalyptus, Cupressus, Casuarina, Pinus and native Juniperus species. Plantations species have suffered at varying degrees of attack by disease causing agents, particularly Amhara region is among regions with plantation forests that have in recent years been subjected to attack by diseases in Ethiopia. Plantation trees in commercial stands, farmlands and woodlots were surveyed for diseases symptoms in 20 selected areas of Amhara and Tigray from May to June 2016.Leaf blight, leaf spot, tip blight and stem canker were the most common symptoms appeared during the survey period with leaf spot and stem canker the most prevalent. Tree samples showing clear disease symptoms were collected, surface sterilized, cultured and morphologically characterized for pathogen identification. A total of 42 isolates of fungi colonies were identified from samples collected of 20 localities. Morphological characterization of fungal isolates reveals, six fungal genera belonging to Alternaria, Dioplodia, Pestalotiopsis, Curvularia, Phoma, and Penicillium were the cause of the symptoms of the symptoms of the observed disease. Among the isolates 14 (33.3%) were Alternaria species, 15(37.7%) were Phoma species, and the remaining 13 isolates were Diplodia3(7.2%), Pestalopsis7(16.7%), Curvularia2(4.7%) and Penicillium1 (2. 4%).Based on the findings of the study Phoma lingam, Phoma glomerata, Alternaria alternata, genera of Curvularia, Pestalotiopsis, Penicillium, and Diplodia were found to be the cause of diseases of the tree plantations. Phoma and Alternaria species were the most prevalent isolates, showing a majority of symptoms observed on plantations were due to their co-infection. The pathogenicity test result of the research also confirms fungal isolates were the cause of the symptoms of the disease observed. The findings of this research enable to study and design appropriate management options for the future prevention and control of the diseases especially when there is prolonged environmental stress in the country.

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees

BackgroundSoil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance.ResultsSalt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively.ConclusionGenetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.

Field Guide to Identify the Common Casuarina (Australian Pine) Species in Florida

This 6-page fact sheet that was submitted for sunset review provides practical information and photographs to anyone interested in field identification of Australian Pine. Written by William S. Castle and Michael Andreu, and originally published by the UF Department of Horticultural Sciences, May 2008. Reviewed July 2017. HS1140/HS394: Field Guide to Identify the Common Casuarina (Australian Pine) Species in Florida (ufl.edu)

Morpho-anatomical studies on the leaf reduction in Casuarina: the ecology of xeromorphy

The foliage characters found in Casuarina seedlings may represent the ancestral, scleromorphic ones found in the Casuarinaceae. In the adults studied, these are replaced by derived xeromorphic features. The ontogenetic changes in the foliage of two Casuarina species were investigated. While the cotyledons are flattened linear structures, all other leaf-types are strongly reduced. Except for the two primary leaves, all subsequent leaves are strongly fused to each other and also to the shoot axis, except for the leaf tips; the shoot axis is completely surrounded by photosynthetic leaf tissue and the branchlet is not made up of cladodes but of extended leaf sheaths which are a novel strategy for achieving reduced photosynthetic area. In seedlings there are four leaves per node, forming four shallow vertical furrows where light-exposed and non-encrypted stomata are developed. These features are also developed in the adult foliage within the strictly scleromorphic genus Gymnostoma, clearly the most mesic of the present day genera of Casuarinaceae and very likely to include the ancestral types. Thus, we assume that the Casuarina-seedling leaves reflect the ancestral scleromorphic condition. In the adult foliage, the number of leaves per node is strongly increased, which leads to the formation of several nearly closed vertical furrows on the shoot, where stomata are shaded and strongly encrypted. Thus, the adult foliage shows several xeromorphic features that are absent in the juvenile foliage. Our morpho-anatomical data mapped on ecological and palaeobotanical data show that within Casuarinaceae the foliage shifted from scleromorphic to xeromorphic. Thus, the adult xeromorphic foliage in Casuarina is the derived, advanced state.

Transcriptomic analysis of Casuarina equisetifolia L. in responses to cold stress

Casuarina species are valued as pioneer trees for coastal shelter forest construction. However, low temperature stress limits its cultivation expansion, fast growth, and high yield. To investigate the molecular mechanism by which Casuarina trees cope with cold stress and to provide a comprehensive sequence resource for further molecular genetic research on Casuarina species, we employed RNA-Seq technology to study the genome-wide expression profiles during cold stress in Casuarina equisetifolia L. Four cDNA libraries constructed from mRNAs of control and cold-treated seedlings (at −5 °C for 5 h) were sequenced by using the Illumina sequencing platform. A total of 118,270 unigenes were obtained by de novo assembly, creating an initial reference transcriptome dataset of C. equisetifolia. Among these unigenes, 4639 were identified as early cold response genes including 2331 cold up-regulated and 2308 down-regulated. Furthermore, the expression changes of 18 genes under cold stress from RNA-Seq analysis were further validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Functional network analysis indicate that modifications in the expression of genes involved in signal regulation by transcription factors, metabolic pathways including synthesis of carbohydrate, secondary metabolites, and antioxidant enzymes might play vital roles in responses to cold stress in C. equisetifolia. This study represents the first large-scale transcriptome annotation of C. equisetifolia and may be beneficial to elucidate the cold response mechanisms of Casuarina trees and will be useful in cold-tolerant Casuarina species breeding.