Temporal and Spatial Characteristics Endogenous Hormone Regulation During Male Cone Development of Dacrydium pectinatum

Background: Dacrydiumpectinatum de Laubenfels is a perennial gymnosperm dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been signicantly reduced. Furthermore, natural regeneration is poor. In order to better understand the reproductive process in D. pectinatum, we examined the morphological and anatomical changes during the development of male cone and analyzed the endogenous hormone dynamics. Results: Our study indicates that D. pectinatum male buds become distinguishable in April in tropical montane rain forests, while microspore sac forms in September and pollen mother cell forms and divides in December. Pollen grains mature and disperse in the following February. A mature male cone averages 8.5 mm in length. Level of GA, IAA, ABA and JA and their ratios uctuated during late August to late November when sporogenous tissues were actively differentiated. Conclusions: The differentiation of sporogenous tissues is accompanied by variations in levels of endogenous hormones (GA, ABA, IAA, and JA) and their balances. The new insights about the cone development in D. pectinatum lay the foundation for future cone induction with hormones and study of factors contributing to the species’ low rate of seed germination.

suspected to be long, since none of the trees with a DBH of 10 cm or smaller produced reproductive structures (Li et al. 2015). Among the 180,032 D. pectinatum seeds collected from a natural stand in Hainan province, China, the viability and germination rate was found to be 3.11% and 0.02%, respectively (Chen et al. 2016). These factors have seriously hindered arti cial cultivation and e cient use of resources.
Efforts have been undertaken to protect D. pectinatum and conserve its biodiversity. However, research on this endangered species is still in its infancy. Currently, most of the published studies are focused on the activity of medicinal ingredients, seedling growth, forest community structure, population genetic diversity, and origin of evolution (Huang et al. 2014;Su et al. 2010;Yang et al. 2008;Wu et al. 2018; Keppel et al. 2011). Limited information is available on reproduction. According to the descriptions in de Laubenfels (1988) and Flora of China (Fu et al. 1999), the reproductive structures of D. pectinatum occur on the terminals. Pollen cones (male, microsporangiate strobili) are 6-12 mm long and in clusters of 1-3. The seed-bearing structure (female cone, macrosporangiate strobili) is subtended by a short zone of small leaves which are ~ 2 mm long, while the cone bracts themselves may be up to 3 mm long. Seeds are ovoid and 4-4.5 mm long.
Reproductive structure formation is a signi cant process in a plant's life because of its important role in producing offspring and evolution. In this study, we investigated male cone development in D. pectinatum. We focused on the morphological and anatomical changes that occur during the development of the male cone, microsporangia and microsporangium wall, and analyzed the endogenous hormone changes. Our study provides further insights into male cone development of this important species.

Materials
Male reproductive structures of D. pectinatum were collected monthly from early April 2018 to February 2020 at Bawanglin Forest Reserve (Between 18°53'~19°30' north latitude, between 108°38'~109°17' east longitude) of Changjiang County, Hainan Province, China. Changjiang County is in a typical tropical monsoon climate zone. The annual average temperature is 24.3 ℃, with 39.8 ℃ as the highest and 0 ℃ as the lowest. There is no winter throughout the year, and the four seasons are like spring. The annual accumulated temperature is 8400 ~ 9100 ℃, while the total solar radiation is 135 kcal / cm 2 , Rainfall is abundant with average annual precipitation of 1676 mm. Male buds were collected from north, south, east and west sides of mature trees that are over 100 years old. Bracts were dissected and removed under a SMZ-168 stereomicroscope before buds were stored at 4℃ in 2.5% glutaraldehyde or FAA xative solution (formalin: acetic acid: 70% alcohol = 1:1:18). Photos were taken of male shoots at different stages of development with a Nikon digital camera.

Semi-thin Sectioning
Male buds xed in FAA were dehydrated and embedded in para n as described in Mazur et al (Mazur et al. 2016). Sections (~ 4 µm) were cut with a microtome (RM2016, Shanghai, China) and mounted on slides. After rehydration, specimens were stained with 1% saffron and 0.5% solid green and observed under a Nikon Eclipse Ci light microscope (Japan). Digital images were taken with a Nikon digital camera.

Observation of Morphological Development of Male Cones by the Scanning Electron Microscope (SEM)
Male buds originally xed in 2.5% glutaraldehyde were further treated with 1% osmium acid. After dehydration with a series of increasing concentration of ethanol and dipping into isoamyl acetate, dried samples were sputter-coated with gold prior to scanning electron microscopy examination (SEM, U8010, HITACHI, Japan), according to Kang (Kang et al. 2014).
Changes of Endogenous Hormones(IAA ABA GA, and JA) in Male Buds of Dacrydium pierrei

Sample preparation
Samples preserved at ultra-low temperature were ground (30 Hz, 1 min) to ne powders with a grinding machine. Ground samples of 50 mg was accurately weighed and dissolved in a 0.5 mL-extract solution, containing methanol, water, and formic acid (v:v:v = 15:4:1). After 10 minutes of extraction, supernatant was obtained by centrifugation for 5 min at 14 000 rpm. The extraction and centrifugation steps were repeated twice. All supernatants were combined and dried at 35℃ under nitrogen gas. The extracts were then resuspended with 100 µL of 80% methanol-water solution and sonicated for 1 min, followed by ltration through a 0.22 micron PTFE membrane.
Mass spectrometry conditions were as followed: electrospray ionization (ESI) temperature was 500℃, mass spectrometry voltage was 5500V, curtain gas was 35 psi, the collision-activated dissociation parameter was set to medium in the dissociation, and each ion pair was scanned according to the optimized cluster voltage and collision energy. The hormonal content obtained in the analysis was expressed as mg/g fresh weight.
Statistical analysis Data were analyzed by using the IMB® SPSS® version 22 and presented as mean values with their respective standard deviations (mean ± SD, n = 3). At 95% con dence level, Student's t-test and Fisher's Least Signi cant Difference (LSD) multiple comparison were used for statistical analyses. The statistical differences are mentioned in the text or considered as * p < 0.05 and ** p < 0.01.

Results
Arrangement and phenology of male cones As described by de Laubenfels (1988), D. pectinatum male reproductive structures appear on the top of current-year branches. Two or three microspore bulbs cluster together, showing a V-shaped distribution, subtended by decussate bud-scales (bracts) (Fig. 1a). In Bawanglin Forest Reserve, male reproductive structures were rst observed in early April, with a light green color and a diameter of 1-2 mm. From the beginning of May to the end of August, the cones gradually elongated and enlarged, and the green color deepened (Fig. 1b-e). The cones reached their maximum length of the year in September, while the width continued to growth (Fig. 2). By October, the outer scales of the cone appeared brown on the margin, and the scales became sharp (Fig. 1e). During November to the following January, the outer scales were greatly elongated and evenly thickened and became yellowish-brown ( Fig. 1f-g). Furthermore, the whole structure of the male cone became more compact. Cones enlarged rapidly in January and February. By late February, cones gradually expanded and became yellow-brown, with the outer scales cracked and mature microspore sacs dispersing pollens (Fig. 1g). Males cones were mainly found in the well sunlit parts of the outer crown, and there were approximately 45 male cones in each mature D. pectinatum tree.
The SEM images show that spirally-arranged microsporophylls were formed by April and underwent differentiation and enlargement through December (Fig. 4). Microsporophylls were tightly arranged around the main axis.
Microscopic anatomy of D. pectinatum male cone In April, microsporophyll primordia started to become visible, which were formed in the order of from the base of the bud to the top (Fig. 3a-b). The bud was wrapped by phylloclades, with the left and right phylloclades along the central axis forming a U shape close to the central axis. By September, sporogenous tissues in sac-like microsporangia became condense due to the further differentiation and accelerated cell division of the microsporophyll primordia (Fig. 3c-e). Microsporangia seemed to derive from a group of hypodermal cells of the microsporophyll. The formation and division of pollen mother cells were found in December. Mother cells were actively dividing, producing four microspores per mother cell through meiosis (Fig. 3e). Numerous pollen grains were formed in late January of the following year (Fig. 3f).
Male cone had a central axis on which 15 to 20 microsporophylls were spirally arranged. Each mcrosporophyll bored one or two microsporangia on the abaxial surface. A mature microsporangium consisted of one layer of epidermis, a multilayered endothecium, tapetum, and microspore mother cells ( Fig. 3e-f). Tapetum separated from endothecium as microsporangia developed.

Dynamic of Endogenous Hormones during Male cone Development
During male cone development, GA content decreased during September and October, then recovered by late November (Fig. 5a). IAA content did not change during August and September, while peaked in late October and then decreased to its lowest level in late November (Fig. 5b). ABA exhibited the most dynamic change, with signi cant difference among each time point (Fig. 5c). ABA content was lowest in early September and highest in late September, declining after the plateau. There was no signi cant change in JA content, with an exception in late October when a large increase occurred (Fig. 5d).
In terms of ratios among endogenous hormones, ABA/IAA was found the lowest in early September and highest in late September (Fig. 6a). ABA/GA peaked in late September and October, while no difference existed among the other timepoints (Fig. 6b). IAA/GA plateaued in late October and were the lowest in late August and late November (Fig. 6c). Mirroring the dramatic increase of JA content in late October, JA/IAA ratio in late October was much higher than the other periods (Fig. 6d), while ratios of ABA/JA and GA/JA were much lower ( Fig. 6e-f).

Discussion
There are 21 species in the genus Dacrydium (https://www.conifers.org/po/Dacrydium.php). Their natural distribution ranges from New Zealand, New Caledonia, Fiji and the Solomon Islands through New Guinea, Indonesia, Malaysia and the Philippines, to Thailand and southern China (de Laubenfels 1969; Quinn 1982). Currently no information is available about when reproductive cones start to initiate for Dacrydium species. In New Zealand, D. cupressinum cone initiation is suggested to occur in late summer or autumn with pollination occurring in spring (Norton et al. 1988). D. pectinatum male cones initiate before April in Hainan Island, China, because male buds are distinguishable by early April (Fig. 1a).
Different species and climates can be the contributing factors for the discrepancy observed. Buds will be collected in March and February for examination in order to determine the initiation period for D. pectinatum male strobili in the future. D. pectinatum male cones average 8.5 mm in length (Fig. 2), similar to de Laubenfels's report for the same species (6-12 mm) (de Laubenfels 1988), while longer than D.
Bidwillii's (2 to 6 mm) (Young 1907). When more information become available for other Dacrydium species, it will be interesting to compare their reproductive buds and cones' morphology and phenology. This is important for the diversity conservation.
Similar to other coniferous species such as Metasequoia glyptostroboides (also known as the dawn redwood, Chinese redwood and water r), D. pectinatum has microsporophylls spirally arranged around a main axis, and each microsporophyll consists of a phylloclade at the apex and one or two microsporangia at the base (Fig. 3). Like M. glyptostroboides (Jin et al. 2012), D. pectinatum male cones are mainly located around the outer and sunlit parts of crown. This is advantageous for pollen dispersal by wind, which is a common in conifers (Leslie 2011a, Leslie 2011b). Our study shows that the development of D. pectinatum microspore can be divided into four stages: initiation and differentiation of microsporophyll primordia, microspore sac formation, division of pollen mother cells, and pollen grain formation. This process lasts for about 12 months. A similar study will be conducted on female buds and cones. ). In our previous study with Metasequoia, higher levels of GA 1 + 3 and lower levels of IAA and ABA were bene cial to male primordium initiation, while higher levels of IAA and GA 1 + 3 and a lower level of ABA were favorable to female cone initiation (Liang and Yin 1994). In D. pectinatum, level of GA, IAA, ABA and JA and their ratios uctuated during late August to late November when sporogenous tissues were actively differentiated, suggesting their involvement in make cone development.
It is noteworthy that there was a dramatic increase of JA in male buds collected in late October after microspore sac was formed (Fig. 5d)  In summary, D. pectinatum male buds become distinguishable in April in tropical montane rain forests and continue to differentiate and develop until the following February. The dynamic change of endogenous hormones suggests their roles in cone development. Cone induction with hormones may provide an alternate approach to address the seed shortage issue due to the species' long juvenile phase. It is suggested that treatments for male cone induction should be applied no later than April before differentiation of vegetative and reproductive buds. This is the rst report on the anatomical and endogenous hormone changes that occur during the development of D. pectinatum the male cone.
Combining morphological analyses of reproductive development with transcriptome studies in the future may lead to the understanding of molecular mechanisms behind reproductive development in D. pectinatum.