38Sexual dimorphism like dioecy (separate male and female individuals) have evolved 39 in diverse multicellular eukaryotes while the molecular mechanisms underlying the 40 development of such a key biological trait remains elusive (1). The living fossil 41Ginkgo biloba represents an early diverged lineage of land plants with dioecy. 42However, its sex-determination system and molecular basis have long been 43 controversial or unknown. In the present research, we assembled the first and largest 44 to date chromosome-level genome of a non-model tree species using Hi-C data. With 45 this reference genome, we addressed both questions using genome resequencing data 46 gathered from 97 male and 265 female trees of ginkgo, as well as transcriptome data 47

Accurate species delimitation is the key to precise estimation of species diversity and is fundamental to most branches of biology. Unclear species boundaries within species complexes could lead to the underestimation of species diversity. However, species delimitation of species complexes remains challenging due to the continuum of phenotypic variations. To robustly examine species boundaries within a species complex, integrative approaches in phylogeny, ecology, and morphology were applied to the Stewartia sinensis complex (Theaceae) endemic to China. Multispecies coalescent‐based species delimitation using 572 nuclear ortholog sequences (anchored enrichment) supported reciprocal phylogenetic monophyly of the northern lineage (NL) and southern lineage (SL), which were not sister clades. Niche equivalency and similarity tests demonstrated significant climatic niche differentiation between NL and SL with observed Warren et al.'s I = 0.0073 and Schoener's D = 0.0021. Species distribution modeling also separated their potential distribution. Morphometric analyses suggested significant interlineage differentiation of multiple traits including the ratio of length and width, leaf width, and pedicel length, although overall similarity did not differ. Based on the integrative species concept, two distinct species were proposed with legitimate names of Stewartia gemmata for SL and S. sinensis for NL. Our empirical study of the S. sinensis complex highlights the importance of applying multiple species criteria, in particular the underappreciated niche differentiation, to species delimitation in species complexes pervasive in plants.

Although an increasing number of species-specific databases have been constructed to facilitate the global research community, comprehensive databases remain rare with multidimensional research resources rather than genomic data solely, in particular for non-model species. Here we introduced GinkgoDB, the ecological genome database for the world-renowned living fossil, Ginkgo biloba L., a tree species with extreme longevity, exceptional resistance, global landscape application and dioecy. The present version of GinkgoDB consists of four modules of genome, occurrence, quadrat and gallery. The genome module includes two versions of chromosome-level assembled genomes with high-quality annotation, expression profiles of each sex for different tissues and variants from 545 ginkgo genomes. A suite of tools were also deployed online for search, blast and further analyses. The occurrence module contains distribution information of over one million records across the world with an emphasis on 8926 individual-level tagged trees with phenotypic trait data in eastern Asia. The quadrat module provides monitoring data of both species and habitats from 27 plots of natural ginkgo forests and periodic data of functional traits measured for the entire plant communities. The gallery module collects field morphological photos and scanned phenotypic images of various ginkgo materials. GinkgoDB showcases a comprehensive and persistently growing database for a single non-model species. Database URL: https://ginkgo.zju.edu.cn/

Background Golden leaf in autumn is a prominent feature of deciduous tree species like Ginkgo biloba L., a landscape tree widely cultivated worldwide. However, little was known about the molecular mechanisms of leaf yellowing, especially its dynamic regulatory network. Here, we performed a suite of comparative physiological and dynamic transcriptional analyses on the golden-leaf cultivar and the wild type (WT) ginkgo to investigate the underlying mechanisms of leaf yellowing across different seasons. Results In the present study, we used the natural bud mutant cultivar with yellow leaves “Wannianjin” (YL) as materials. Physiological analysis revealed that higher ratios of chlorophyll a to chlorophyll b and carotenoid to chlorophyll b caused the leaf yellowing of YL. On the other hand, dynamic transcriptome analyses showed that genes related to chlorophyll metabolism played key a role in leaf coloration. Genes encoding non-yellow coloring 1 (NYC1), NYC1-like (NOL), and chlorophyllase (CLH) involved in the degradation of chlorophyll were up-regulated in spring. At the summer stage, down-regulated HEMA encoding glutamyl-tRNA reductase functioned in chlorophyll biosynthesis, while CLH involved in chlorophyll degradation was up-regulated, causing a lower chlorophyll accumulation. In carotenoid metabolism, genes encoding zeaxanthin epoxidase (ZEP) and 9-cis-epoxy carotenoid dioxygenase (NCED) showed significantly different expression levels in the WT and YL. Moreover, the weighted gene co-expression network analysis (WGCNA) suggested that the most associated transcriptional factor, which belongs to the AP2/ERF-ERF family, was engaged in regulating pigment metabolism. Furthermore, quantitative experiments validated the above results. Conclusions By comparing the golden-leaf cultivar and the wide type of ginkgo across three seasons, this study not only confirm the vital role of chlorophyll in leaf coloration of YL but also provided new insights into the seasonal transcriptome landscape and co-expression network. Our novel results pinpoint candidate genes for further wet-bench experiments in tree species.

Reproductive bud differentiation is one of the most critical events for the reproductive success of seed plants. Yet, our understanding of genetic basis remains limited for the development of the reproductive organ of gymnosperms, namely, unisexual strobilus or cone, leaving its regulatory network largely unknown for strobilus bud differentiation. In this study, we analyzed the temporal dynamic landscapes of genes, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) during the early differentiation of female strobilus buds in Ginkgo biloba based on the whole transcriptome sequencing. Results suggested that the functions of three genes, i.e., Gb_19790 (GbFT), Gb_13989 (GinNdly), and Gb_16301 (AG), were conserved in both angiosperms and gymnosperms at the initial differentiation stage. The expression of genes, lncRNAs, and miRNAs underwent substantial changes from the initial differentiation to the enlargement of ovule stalk primordia. Besides protein-coding genes, 364 lncRNAs and 15 miRNAs were determined to be functional. Moreover, a competing endogenous RNA (ceRNA) network comprising 10,248 lncRNA-miRNA-mRNA pairs was identified, which was highly correlated with the development of ovulate stalk primordia. Using the living fossil ginkgo as the study system, this study not only reveals the expression patterns of genes related to flowering but also provides novel insights into the regulatory networks of lncRNAs and miRNAs, especially the ceRNA network, paving the way for future studies concerning the underlying regulation mechanisms of strobilus bud differentiation.