Gender plasticity uncovers multiple sexual morphs in natural populations of Cedrus deodara (Roxb.) G. Don

Background The expression of gender in gymnosperms is challenging because the extent of variability in gender segregations in tree crowns and selection pressure of nature can modify the gender through time. Methods An in-depth investigation on spatial segregation of genders in tree crowns and sex expression of a total of 500 trees in five different natural populations of Cedrus deodara was carried out and verified the occurrence of subdioecious (co-existence of male, female, and monoecious) genders in C. deodara. Results Five different sexual morphs were apparent among the 500 selected individuals as (1) pure male (M): bearing only male strobili in the whole crown with 22.2%; (2) pure female (F): bearing only female strobili in the whole crown with 12.4%; (3) mixed-monoecious (MM): bearing both male and female strobili with 13.6%; (4) predominantly female (PF) with 25.6%; and (5) predominantly male (PM) with 26.2%. The occurrence of multiple sexual morphs resulted from the complex selection pressure, which increased the stability of the populations. The segregation of genders in crown layers deemed to increase the fitness that may be a mechanism for accelerating outcrossing. The results of the study suggest that the subdioecious gender expression in C. deodara is evolved through the monoecy–paradioecy pathway. The production of male strobili revealed non-significant effect of tree sizes whereas a significant effect was observed for the production of female strobili. Our study established that the total pollen and seed outputs in C. deodara changed frequently according to gender expression. Conclusions The size of the tree, resource availability, sex allocation, plant architecture, gender segregation in crowns, and habitat conditions are the prominent causes for gender plasticity.

Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

Factors Affecting the Number of Pollen Grains per Male Strobilus in Japanese Cedar (Cryptomeria japonica)

Japanese cedar (Cryptomeria japonica) is the most important timber species in Japan; however, its pollen is the primary cause of pollinosis in Japan. The total number of pollen grains produced by a single tree is determined by the number of male strobili (male flowers) and the number of pollen grains per male strobilus. While the number of male strobili is a visible and well-investigated trait, little is known about the number of pollen grains per male strobilus. We hypothesized that genetic and environmental factors affect the pollen number per male strobilus and explored the factors that affect pollen production and genetic variation among clones. We counted pollen numbers of 523 male strobili from 26 clones using a cell counter method that we recently developed. Piecewise Structural Equation Modeling (pSEM) revealed that the pollen number is mostly affected by genetic variation, male strobilus weight, and pollen size. Although we collected samples from locations with different environmental conditions, statistical modeling succeeded in predicting pollen numbers for different clones sampled from branches facing different directions. Comparison of predicted pollen numbers revealed that they varied >3-fold among the 26 clones. The determination of the factors affecting pollen number and a precise evaluation of genetic variation will contribute to breeding strategies to counter pollinosis. Furthermore, the combination of our efficient counting method and statistical modeling will provide a powerful tool not only for Japanese cedar but also for other plant species.

Stem Branching of Cycad Plants Informs Horticulture and Conservation Decisions

The number of branches in male and female plants of Cycas micronesica K.D. Hill, Cycas edentata de Laub., Cycas wadei Merr., and Zamia encephalartoides D.W. Stev. were counted in Guam, Philippines, and Colombia, to confirm earlier reports that female plants develop fewer branches than males. Cycas plants produce determinate male strobili and indeterminate female strobili, but Zamia plants produce determinate strobili for both sexes. More than 80% of the female trees for each of the Cycas species were unbranched with a single stem, but more than 80% of the male trees exhibited two or more branches. The mean number of branches on male plants was more than double that of female plants. The number of branches of the Zamia male plants was almost triple that of female plants. Moreover, the Zamia plants produced 2.8-fold greater numbers of branches than the mean of the Cycas plants. Most of Guam’s unsexed C. micronesica trees in 2004 were unbranched, but after 15 years of damage from non-native insect herbivores, most of the remaining live trees in 2020 contained three or more branches. The results confirm that male Cycas and Zamia plants produce more branches than female plants and suggest cycad species with determinate female strobili produce more branches on female plants than species with indeterminate female strobili. Our results indicate that the years of plant mortality on Guam due to non-native insect herbivores have selectively killed more female C. micronesica trees. Horticulture and conservation decisions may be improved with this sexual dimorphism knowledge.

Fertility variation, seed collection and gene diversity in natural stands of Taurus cedar (Cedrus libani)

Background Seed stand is one of the important seed sources that is essential for global seed production programs. The primary objective of seed stands is to produce seeds as fast as possible. Characteristics of seed stand seeds depend mainly on stand quality, fertility variation and mating system. Individual fertility is an ability to produce progeny to next generation. Prediction of fertility variation is useful for seed production, genetic resources management and gene conservation. In this study, we report how to estimate fertility variation and gene diversity, and discuss its effect on the management of natural stands of Taurus cedar. Results Fertility variation and gene diversity were estimated based on the differences of strobilus production in female and male parents from three seed stands of Taurus cedar. A total of 50 trees were randomly chosen from each seed stand, and the female and male strobili were counted for three consecutive years. The coefficients of variation for female and male strobilus production were subjected to estimation female and male fertility variation. The total fertility variation (Ψ) was then estimated from the female and male fertility variation. The effective number of parents (Np) was calculated based on the Ψ. The mean of female strobili ranged from 31 to 150, and that of male strobili ranged between 77 and 828. The Ψ in a good crop year was smaller than in a poor year and the Np varied from 34.7 to 44.2. The Ψ was improved and the Np was increased when strobilus productions were pooled across three years or populations. The equal cone harvest could mitigate the fertility variation among individuals but caused loss of seed production. Conclusions The effective number of parents could estimate gene diversity of seeds from natural stands, which was based on the function of correlated fertility variation between female and male parents. Mixing seeds from different years could improve the fertility variation among individuals and increase the gene diversity of seeds. However, a balancing between the effective number of parents and the number of mixed years or populations should be carefully considered for maintaining the gene diversity.

Inspecting abundantly expressed genes in male strobili in sugi (Cryptomeria japonica D. Don) via a highly accurate cDNA assembly

Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. The field of functional genomics is rapidly developing. The genomics of this gymnosperm species is currently being studied. Although its genomic size is 11 Gbps, it is still too large to assemble well within a short period of time. Transcriptomics is the one another approach to address this. Moreover, it is a necessary step in obtaining the complete genomic data. Here we designed a three stages assembling workflow using the de novo transcriptome assembly tools, Oases and Trinity. The three stages in transcriptomics are independent assembly, automatic and semi-automatic integration, and refinement by filtering out potential contamination. We found a set of 49,795 cDNA and an equal number of translated proteins (CJ3006NRE). According to the benchmark of BUSCO, 87.01 % were complete genes, including very high “Complete and single-copy” genes–78.47%. Compared to other full-length cDNA resources, the extent of the coverage in CJ3006NRE suggests that it may be used as the standard for further studies. When two tissue-specific libraries were compared, principal component analysis (PCA) showed that there were significant differences between male strobili and leaf and bark sets. The highest three upregulated transcription factors stood out as orthologs to angiosperms. The identified signature-like domain of the transcription factors demonstrated the accuracy of the assembly. Based on the evaluation of different resources, we demonstrate that our transcriptome assembly output is valuable and useful for further studies in functional genomics and evolutionary biology.

Clonal variation in phenological synchronization and cone production in a Pinus patula seed orchard

Synchronization between female receptiveness and pollen dispersal, and fecundity of clones influence effective population size and genetic diversity of germplasm produced in seed orchards. Our objective was to determine clonal variation in phenological synchronization and in cone production in a Pinus patula seed orchard. Two-year phenology data of female and male strobili from a sample of 31 clones, and of male strobili in trees from neighboring natural stands of the same species were used. Synchronization indices between female receptiveness and pollen release of the same clone (O̅iP̅i), of other clones (O̅i P̅j), and of natural stands (O̅i P̅NS) were calculated for each clone and averaged per precocity group (early, intermediate, and late). Genetic parameters for cone production of clones during three consecutive cone harvests were estimated, as was their relationship to precocity and synchronization index O̅i P̅NS. Cone production showed a broad variation among clones and between years, with strong genetic control (H2c ≥ 0.80) and stable (rB ≥ 0.79) between yeas. There was wide clonal variation in synchronization indices, but no significant variation was found in most cases among precocity groups, neither were they consistent between years. Negative correlations [rp = -0.37 and -0.40 (P < 0.05)] in cone production per clone in two years with the O̅i P̅NS index in the first year, indicated that the most prolific clones had lower synchronization with pollen release in natural stands in the orchard’s vicinity, and thus less risk of genetic contamination.

Ephedra yangthangensis (Ephedraceae), a new species from Himachal Pradesh, India

A new species Ephedra yangthangensis Prabha Sharma & Rita Singh is described, and illustrated from Himachal Pradesh, India. This new species is most similar to E. intermedia Schr. & Meyer, from which it is distinguishable by its smaller male strobili, shorter length and curved synangiophore, yellowish orange fleshy bracts of bigger female strobili, fade orange scale leaves and light green robust stem as compared to the other flourishing species E. intermedia.Bangladesh J. Plant Taxon. 23(2): 195-198, 2016 (December)

Effect of giberellic acid (GA4/7) and girdling on induction of reproductive structures in Pinus patula

Aim of study: Seed production in forest tree species commonly takes a long time due to the length of the juvenile stage. Even though several treatments have been used to induce early flowering in conifer species, experience on their use in subtropical Pinus species is limited. This study aimed to evaluate the effect of GA4/7 dose (0, 1.27 and 2.54 mg cm-2) and application time (July-October), alone or in combination with partial stem girdling, on male and female strobili production in young Pinus patula Schiede ex Schltdl. et Cham. clones. Area of study: Nine clones with different flowering background of high-elevation Pinus patula growing in a six-year old seed orchard established in Central México (Aquixtla, Puebla) at 2,800 m elevation.Material and methods: Two independent flowering trials (FT1 and FT2) were carried out in the seed orchard during the 2009 and 2010 flowering cycles; similar factors were evaluated at both trials but time of application, clones tested, and experimental design used varied for each of them. Partial stem girdling was done at the base of the trunk and the GA4/7 solution was injected into the xylem above the point of girdling. The following spring, the percentage of trees with strobili and the number of strobili per tree were determined for both male and female structures.Main results: Significant differences (p ≤ 0.05) among clones in flowering capacity were found at both trials. None of the treatments applied in FT1 resulted in an increase of strobili formation, most probably because they were applied too late in the growing season. In FT2, however, application of GA4/7 combined with partial stem girdling increased the percentage of trees with strobili and the number of strobili of both sexes, particularly when applied in early July. Partial stem girdling was more effective on promoting male strobili than female ones in gibberellin-treated grafts.Research highlights: Timing of GA4/7 application and stem girdling was important, with a significant interaction with clones tested in FT2 on production of male strobili but not on female strobili. Application of 1.27 mg cm-2 GA4/7 and stem girdling in July promoted the highest percentage of trees with strobili, and increased 25-fold the number of female strobili and 5-fold the number of male strobili per tree as compared to the control treatment. Thus, operational use of this induction treatment would be valuable to increase and accelerate seed production in Pinus patula seed orchards in the region.Keywords: early flowering; gibberellins; juvenile stage; seed orchard; seed production.Abbreviations: GA: Gibberellic acid; GA4: Gibberellic acid 4; GA7: Gibberellic acid 7; GA4/7: Mix of gibberellic acids 4 and 7; FT1: Flowering trial carried out in the 2009 season; FT2: Flowering trial carried out in the 2010 season.