Sinocalycanthus chinensis Cheng et S. Y. Chang (Calycanthaceae), which has a unique systematic status, is listed as a national second-class protected plants of China . In this study, the genetic diversity, performance, and fitness of F1 progeny from crosses between the Damingshan (DMS) population of Sinocalycanthus chinensis and pollen parents from the Daleishan (DLS) and Longxushan (LXS) populations were examined. The DLS population has a relatively small population size, low genetic diversity, and considerable geographical and genetic distances from the DMS population relative to the LXS population. Compared with naturally occurring seeds, DLS-sired seeds had the highest thousand-seed weight, starch content, fat content, germination rate, germination index, and emergence rate, but the lowest protein content. Naturally occurring, open-pollinated seeds had the lowest thousand-seed weight, starch content, and fat content, but the highest protein content. Compared with natural F1 progeny, DMS × DLS seedlings had the highest genetic diversity, photosynthetic parameters, and growth characteristics, except for leaf mass ratio and stem mass ratio. Under strong light, DMS × DLS seedlings exhibited a Fv/Fm value of 0.75, while the other two seedling types exhibited Fv/Fm values of 0.65. DLS-sired seeds had the most vigorous growth characteristics except for leaf mass ratio and stem mass ratio. These results suggest that genetic rescue by transplanting seedlings from the DLS population or hand pollination with pollen from the DLS population would be effective methods to reduce inbreeding depression and obtain strong offspring with high genetic diversity and fitness in the DMS population.

Pollen sources

Plants from two S. chinensis populations located at two sites with different population sizes, levels of genetic diversity, and the relative geographical and genetic distances from the DMS seed parent were used as the pollen parents. One is located at Longxushan Mountain (LXS) in Anhui Province, China; this population is small with relatively high genetic diversity and with short geographical and low genetic distances from the DMS population (Table 1). This S. chinensis population is also within an evergreen broad-leaved forest. The main accompanying species are Litsea coreana var. sinensis, Symplocos setchuensi and Platycarya strobilacea. The other pollen parent population is located at Daleishan Mountain (DLS) in Tiantai County, Zhejiang Province, China, and it is a medium-sized population located a long geographical distance from the DMS population (Table 1). This S. chinensis population is located among shrubs within a valley. The main accompanying species are Camellia cuspidate, Spiraea salicifolia, Corylopsis sinensis, Rhododendron simsii, Actinidia chinensis, and Sargentodoxa cuneata. The DLS S. chinensis plants occur in the canopy and are intertwined with A. chinensis and S. cuneata.

Hand pollination and seed collection

Mature S. chinensis plants were selected as pollen donors. In May 2009, when the stigmas of S. chinensis flowers in DMS had matured, pollens were collected from flowers on 20 mature S. chinensis individuals in the LXS and DLS populations using Chinese brushes and stored in sterile plastic tubes. The pollen samples were maintained at 4°C and quickly transported to Damingshan Mountain, where the hand pollination was conducted with the DMS population. Pollen was transferred to DMS stigmas within 6 h. 30 mature individual plants in the DMS population were hand-pollinated with an average of 100 flowers per treatment. Each plant was randomly assigned to a crossing treatment and in total of 200 flowers were performed. All hand-pollinated stigmas were saturated with pollen. To exclude natural pollinators, plants were bagged with fine nylon mesh before flowering. Each hand-pollinated flower was also emasculated before the stigma became receptive to prevent selfing. Pollen was transferred directly from donor flower anthers onto receptive stigmas until the stigmas were saturated. The date was selected to ensure that each flower was encountered at the onset of its stigmatic surface receptivity. 30 naturally open pollinated DMS plants were selected as the control (Bossuyt, 2007; Holme, James & Hoffmann, 2008).

Fitness measurement

Fitness of the F1 progeny was defined as the relative reproductive success of a genotype as measured by survival, fecundity and other life history parameters (Molina-Montenegro et al., 2013) and was indicated by seed number per fruit, seed weight, seed size germination days, total germination rate, seedling emergence rate (Baker, Richards & Tremayne, 1994) and seedling biomass (Du, Yang, Guan & Li, 2016).

In October 2009, S. chinensis fruits were collected and air-dried, and seeds were collected from the dried fruits. Thousand-seed weights were measured using an electronic balance with an accuracy of 0.0001 g. The starch, lipid, and protein contents of seeds were measured using anthrone–sulfuric acid colorimetric, Soxhlet extractor, and UV-spectrophotometric methods, respectively (Song, Cheng, Jiang, Long & Huang, 2008).

In March, 2010, DMS × DLS (pollen) seeds (hereafter, DLSH), DMS × LXS (pollen) seeds (hereafter, LXSH), and control seeds were germinated in an illuminated incubator (Jiangnan Instrument Inc., Ningbo, China) with 30/15°C and 12 h/12 h light/dark cycles at 80% humidity. Seeds were immersed in H₂SO₄ for 3 min, rinsed with sterilized water, and surface sterilized with 70% ethanol. Seeds were immersed in sterilized water and incubated at 28ºC for 2 days. Fifty seeds were placed into 3-mm deep silicon sand. Seeds were covered with moist filter paper to prevent them from drying out. Three replicates were used with a total of 150 seeds for each treatment.

Seeds were considered to be germinated when their radicle length exceeded 2 mm (Hussain, Aljaloud, Alshammafy, Karimulla & Al-Aswad, 1997), and germination was recorded daily for 100 days. Germination-related indices were calculated as follows, according to previously described methods (Cai, 2008): (i) germination days were recorded as the number of days after sowing when the seeds begin germinating; (ii) total germination rate (%) = total number of germinated seeds on the 100^th day / total number of seeds used for germination experiment × 100 %.

In March 2010, S. chinensis seeds of F1 DLSH, F1 LXSH, and control progeny were immersed in H₂SO₄ for 3 min, rinsed with sterilized water, and surface sterilized with 70% ethanol. Seeds were immersed in sterilized water and incubated at 28ºC for 2 days. Seeds were planted 2-cm deep into soil-filled pots. Three seeds were planted per pot, and a total of 50 pots were used for each treatment. Sufficient tap water was added every day. One hundred days after planting, the seedling emergence rate was defined as the percentage of healthy seedlings that emerged, with a hypocotyl appearing on or above the soil surface (Demir & Mavi, 2004; Bolek, 2010).

After the performance measurement described below, plants were harvested and divided into leaves, stems, and roots. Plant material was oven-dried at 105°C for 1 h and then at 80°C until a constant weight was reached. The leaf, stem, and root biomasses were weighed with a balance to an accuracy of 0.1 mg, and the total biomass of seedlings was subsequently calculated.

Seedling photosynthetic physiological performance measurement

In May 2010, healthy F1 DLSH, FI LXSH, and control seedlings were transplanted into pots, with each pot containing one seedling. In August 2010, we conducted in-situ photosynthetic trait measurements on a sunny day using a mature middle leaflet at the same position across plants, using a GFS-3000 Portable Gas Exchange Fluorescence System (Heinz Walz GmbH, Effeltrich, Germany). The photosynthetically active radiation (PAR) was maintained at 800 μmol m^-2 s^-1 using a red-blue LED light source, and the temperature was maintained at 25°C with a relative humidity of 70% inside the leaf measurement chamber. The CO₂ concentration within the chamber was maintained at 400 µmol mol^-1. We recorded the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci) between 08:30 and 11:30. Three leaves per plant were chosen, and six consecutive measurements were performed (Li, Liao, Guan, Wang & Zhang, 2012).

To construct light response curves, we obtained all photosynthesis measurements between 09:30 and 11:00 (Beijing time) on a mature leaf from each plant with a leaf temperature of 25°C, a CO₂ concentration of 400 ppm, and a relative humidity of 70%. We used a red-blue LED light source attached to the system to produce steady photosynthetically active radiation (PAR). Prior to the measurements, we allowed the mature leaf to acclimate under a PAR of 2000 μmol m^-2 s^-1 for 30 min to avoid photo-inhibition. As soon as the value stabilized, we exposed the leaves to a series of PAR values for 20 min or so in the following order: 2000, 1500, 1200, 1000, 800, 600, 400, 200, 100, 50, 20 and 0 μmol m^-2 s^-1. The temporal interval between each concentration was 3 min. We fitted the entire photosynthetic light response curve in Origin 8.0 (OriginLab, Northampton, MA, USA) as a binary linear equation by calculating the maximum of the net photosynthetic rate (Pn) as Pmax. We utilized the following definitions. The light intensity at the maximum Pn value (Pmax) was defined as the light saturation point (LSP). The light intensity at a zero Pn value was defined as the light compensation point (LCP). The Pn at a maximum PAR of zero was defined as the dark respiration point (Rd).

Chlorophyll fluorescence parameters under strong light (1200 μmol photons m^-2 s^-1) were measured between 12:00 and 14:00 with a portable chlorophyll fluorometer (OS30P, Opti-Science Inc., Hudson, NH, USA). Measurements were performed on the third undamaged adult leaf from the top of each plant after 30 min of dark adaptation using light exclusion clips. For each measurement, three leaves per plant from three randomly selected plants per measurement were sampled. The data of three measurements of three leaves were averaged and used as the mean for each plant. The variable-to-maximum fluorescence ratio (Fv/Fm), which has been used to express the maximum PSII photochemical efficiency, was calculated.

Seedling morphological performance measurements

In September 2010, plant height, leaf length, and leaf width were measured using a ruler to an accuracy of 0.1 cm, and the leaf length-to-width ratio was then calculated. The number of leaves was also recorded. The basal diameter was measured using Vernier callipers to an accuracy of 0.02 cm. Furthermore, whole pots were immersed in water, and then entire root complexes were excavated and carefully washed with running water to remove fine soil particles. Intact root systems were spread in a Perspex tray (A3-size) to minimize overlap, scanned (resolution 300 dpi, Epson 1680, Seiko Epson Corporation, Japan), and analysed using the WinRhizo software package (Version 3.10, Regent Instruments Inc., Quebec City, Quebec, Canada) to obtain the root volume (RV), total root length (RL), root surface area (RSA) and the number of root tips (RT).

Original data for the figures and light response curve were provided.