Study species and sites
T. sinensis Hemsley (Linaceae) is a shrub or tree mainly distributed in Guangxi, Guizhou, Yunnan (southeast) of China at an elevation of 300–2000 m and usually grows in calcareous soil on mountain slopes or along trails. Plants can be 1–5 m tall with blade elliptic, obovate–elliptic or ovate leaf. The cymes inflorescence is generally 4 cm terminal or axillary at the top of stem or branch. Each flower consists of five green sepals and five white petals arranged into floral tubes, and the nectar is usually present and concealed at the base of the floral tube. Five stamens surround the central four pistils. Flowers are homogamy and usually last 3–4 days. Plants usually flower from May to August. Capsules contain three to eight seeds and mature 3 months after fertilisation . Our field investigation of pollination ecology revealed that T. sinensis is a typical distylous plant with L-morph (anthers are placed low in the corolla, and stigmas are located high) and S-morph (anthers are placed high, and stigmas are located low) (Figure 1A) in the field populations located in Laoshan Provincial Nature Reserve (104°49′ 62" E, 23°94′ 8" N, approximately 1700 m above sea level), Malipo country, Yunnan province, southwest China.
The plant materials in this research are obtained under the permission of Laoshan Nature Reserve Bureau, Yunnan province, China. The formal identification of the plant is undertook by Liu Changqiu, associate researcher, Guangxi Institute of Botany, Chinese Academy of Sciences. A voucher specimen photo of T. sinensis has been deposited in Plant Photo Bank of China (PPBC), the deposition number is xyc74220920100731.
Difference in traits between the L- and S-morphs of T. sinensis
To compare plant performance between the two morphs, we randomly selected 50 plants (each plant selected one flower) per morph and measured two vegetative and thirteen reproductive traits, including leaf length and width; sepal length and width; flower length, width and opening diameter; tube depth; petal length and width; stamen length; pistil length; and anther length, width and thickness to 0.01 mm using a caliper micrometre.
To compare pollen, ovule production and pollen size, we selected 30 flower buds from 30 L-morph individuals and 30 S-morph individuals respectively and stored them in a 1.5 ml centrifuge tube filled with 75% alcohol for fixation and preservation. The anther and ovary from one flower were separated using forceps in the laboratory, and the anthers were suspended in 500 ml of water. Three drops (each drop of 50 ml) of every pollen solution sample were counted under the Nikon E100 optical microscope. The mean of the three pollen drops was multiplied by 10 to estimate the pollen production of one flower. The ovules were counted under a stereomicroscope. The P/O ratio was equal to the number of pollen grains divided by the corresponding ovule number. For pollen size estimation, three pollen grains per flower were first photographed, and the length and width were then measured using Digimizer Version 4.6.0. To compare the single flower period between the two morphs, we marked one bud of the above selected 30 individuals each for L- and S-morph and recorded the first day of the opening state. Every 2 days, we recorded the flower opening state until the anthers and pistil lost function. These days were denoted as the single flower period.
Measurements of nectar volume, sugar concentration and properties of T. sinensis
To compare the nectar volume and concentration in T. sinensis during anthesis between day and night, we bagged and labelled 30 flowers before anthesis from 30 plants each from L-and S-morphs. During the male flowering phases, the nectar in the bagged flower was removed using glass microcapillary tube (0.3 mm in diameter) on the day before the measurement. Nectar was extracted from the flowers bagged from 18:30 to 06:30 (secreted during the night). After the treatments, the same flower was bagged again, and the nectar was extracted from 06:30 to 18:30 (secreted during the day) the next day. The length (L) of the microcapillary tube occupied by nectar was measured using a caliper micrometre. The volume (Vtotal) and length (Ltotal) of one standard microcapillary were calculated, and the volume of nectar (V) is equal to L/Ltotal * Vtotal. And the concentration of nectar was measured with a hand-held refractometer (Eclipse 0%–50%; Bellingham and Stanley Ltd., Basingstoke, United Kingdom; see .
To measure sugar components, we collected nectar from control-bagged flowers of L-morph (30 flowers from 30 individuals) and S-morph (27 flowers from 27 individuals) of T. sinensis by using microcapillary tubes. After the nectar length was measured using a caliper micrometre, the nectar was spotted onto filter paper and was air-dried at room temperature . The spotted filter papers were placed in a 1.5 ml centrifuge tube and stored in the refrigerator at −20 °C. The sugars were removed by elution with 100 µl of deionised water at room temperature for 24 h. Sugar type (glucose, fructose, sucrose and maltose) was identified, and the relative mass was quantified by High Performance Liquid Chromatography (HPLC, Waters Corporation, Milford, Massachusetts) with a refractive index detector and an Agilent Zorbax carbohydrate analysis column 843300-908 (Agilent Technologies, Santa Clara, California) under the column temperature of 35 °C. The mobile phase was 80% acetonitrile, the flow rate was 1 ml/min and the injection volume was 20 µl. Quantities of each sugar in nectar samples were determined by the standards (glucose, fructose, sucrose and maltose) using the regression equations (based on response peak areas to standard sugar mass) and were expressed as relative percentage by mass .
Pollinator species and abundance
To determine the pollinator species of T. sinensis, we observed all visits of different species over 2018 and 2019 in several populations with three or four individuals including hundreds of flowers. Visitor observation of L- and S-morphs lasted for 9 sunny days (July 15, 16, 17, 19, 21, 22, 23, 24, 26 and 29) in 2018 and 8 sunny days (June 22, 26, 27, 28, 29, 30 and July 1, 3) in 2019. Each session lasted for 30 minutes between 7:00 to 22:00, and the visitor’s observations of L- and S-morph were conducted simultaneously. We randomly selected 10 populations containing both L- and S-morph individuals and completed 40 and 44 sessions in 2018 and 2019, respectively. Visitor moves in one population were recorded to quantify visitation rates to L- and S-morphs. Visit number per foraging, visitor species and foraging behaviour were recorded, and the total opening flowers in each population were counted. The visit frequency of one visitor was expressed as the number of visits per flower per hour.
Pollen transfer efficiency of hawkmoths
To compare the pollination efficiency of hawkmoth between the L- and S-morphs, we estimated the pollen removal and receipt per morph. Male-phase inflorescence (previously unvisited) were bagged until anther dehiscence, and each inflorescence was allowed a single visit by a hawkmoth. To estimate pollen removal, we collected 48 visited flowers for L-morph and 46 visited flowers for S-morph from different plants with another 48 L-morph buds and 46 S-morph buds as the control. Each flower was stored in a 2 ml centrifuge tube with 75% alcohol. Pollen removal per flower was calculated from the mean number of pollen grains in unvisited flowers minus the mean number of pollen grains remaining after one visit. To estimate pollen receipt per visit, we removed undehisced anthers from the 48 male-phase flowers for L-morph and 46 male-phase flowers for S-morph and bagged these flowers with cotton mesh until they developed into the female phase. These female-phase inflorescences were then removed from the bag and allowed one visit by the hawkmoth. Stigmas of these visited emasculated flowers were collected and stored in a 1.5 ml centrifuge tube with alcohol. Pollen grains from the anthers and on the stigmas were counted under a light microscope (Nikon E100). The anthers were fully mashed with tweezers to form 0.5 ml of pollen suspension. Three drops (each drop of 50 μl) of every pollen solution sample were counted, and the mean was multiplied by 10 to estimate pollen production (for undehisced anthers) or pollen remaining per flower (one single visited anther) .
To determine whether T. sinensis is self- and intramorph incompatible, we conducted artificial pollination experiments as follows: (1) open pollination as control; (2) intramorph pollination (L-morph as pollen receptor and received L-morph pollen from other individuals and S-morph as pollen receptor and received S-morph pollen from other individuals); (3) intermorph pollination (L-morph as pollen receptor and received S-morph pollen from other individuals and S-morph as pollen receptor and received L-morph pollen from other individuals); (4) self-pollination (pollen from the flowers in the same individuals); (5) autogamy treatments (the flowers were bagged all the time without any treatments); and (6) emasculated treatments. In 30 individuals each for L- and S-morphs, six flowers were marked with a cotton thread of different colours. Four of the six flowers were emasculated and bagged until they developed into the female phase and then received intramorph, intermorph, self- and emasculated pollination treatments. The remaining two flowers were used as the control and autogamy pollination treatments. Three months after pollination, seeds per flower of six pollination treatments were collected and counted.
To assess the differences in plant performance between L- and S-morphs, we compared 15 plant vegetative and reproductive traits, single flowering days and P/O (pollen number/ ovule number) using a generalized linear model (GLM) with normal distribution and identity-link function. The pollen number and ovule number between L- and S-morphs were compared using Poisson distribution with loglinear-link function in GLM (all plant characters as dependent variable, and L- and S-morphs as factors). Nectar volume and sugar concentration were analysed using GLM with normal distribution and identity-link function (nectar volume and sugar concentration as dependent variables, and L- and S-morphs and day and night as factors) to compare the nectar traits of the two morphs between day and night. Glucose, fructose, sucrose and maltose contents in nectar were examined using GLM with normal distribution and identity-link function (sugar components as dependent variables, and L- and S-morphs as factors) to compare the sugar components between the two morphs. Data of visits were analysed using GLM with normal distribution and identity-link function (visitation rates as dependent variables, and flower morphs and visitor types as factors) to compare the visiting rates (visits/flower/hour) of all visitors between the two morphs. Pollen removal and receipt between the two morphs were compared using GLM with Poisson distribution with loglinear-link function (pollen number as dependent variable, and L- and S-morph as factors). Seed sets of all treatments were examined with binary logistic analysis in GLM (full seed number as event variable, total ovule number as trait variable, and pollination treatments and flower morph as factors) to compare the reproductive success of six pollination treatments between the two morphs.
All data were analysed in SPSS 20.0 (IBM Inc., New York, NY) software.