Floral traits and mating system of Hibiscus trionum (Malvaceae)

Acta Ecologica Sinica 37 (2017) 91–96

Contents lists available at ScienceDirect

Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes

Floral traits and mating system of Hibiscus trionum (Malvaceae) Qun Li a,b, Cheng-Jiang Ruan b,⁎, Jaime A. Teixeira da Silva c a b c

College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, Liaoning, China Institute of Plant Resources, Dalian Nationalities University, Dalian 116600, Liaoning, China Faculty of Agriculture, Graduate School of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

a r t i c l e

i n f o

Article history: Received 12 September 2016 Received in revised form 31 October 2016 Accepted 30 December 2016 Keywords: Hibiscus trionum Outcrossing rate ISSR markers Delayed selfing Reproductive assurance

a b s t r a c t Variations in floral traits and floral structures influence plant mating systems. Hibiscus trionum produces large, showy flowers typical of an outcrossing species, yet flowers are autonomously self-pollinated. In this study, we measured floral morphology, breeding system and outcrossing rate estimated by ISSR markers. Results indicate that two types of flowers were observed in H. trionum, and the type I with bigger petals appears to be much more visible to pollinators, demonstrated by than type II flowers with smaller petals. The flowers with hand pollination were closed 1 h earlier than intact flowers, whether they were type I or II. The relationship between the amount of pollen deposited on the stigma and the number of seeds per capsule was highly significant, and 80 or more pollens per flower can make the mean number of seeds (mean = 37) in H. trionum. Delayed selfing in H. trionum did not provide a large contribution to seed production, since reproductive assurance were only 0.025. However, successful reproduction of 72.5% flowers in the absence of pollinators suggested that selfing provides reproductive assurance during seasons, in which pollinators were limiting. The multilocus outcrossing rates in different populations varied from 0.982 to 1.200, with a mean of 1.116. Our data provide an empirical demonstration of a predominantly outcrossing species with potential delayed selfing when pollinators are absent or scarce. © 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Mating system determines the dynamics and genetic structure of populations with important consequences for plant ecology and evolution, especially influencing the levels of genetic diversity, effective population size, and population subdivision [1]. How species reproduce can occur at any point on a continuum between the two extremes of selffertilization and cross-fertilization [2]. Self-fertilization promotes local adaptation and the expression of recessive alleles. Conversely, outbreeding promotes gene flow, homogenizes populations, increases heterozygosity, and favors gametic linkage equilibrium [3]. In flowering plants breeding, one of the most frequent changes in evolutionary history is the transition from outbreeding to selfing [4,5]. Mixed mating systems, within the range between strict inbreeding and outbreeding, are characterized by a mixture of self- and cross-fertilization. This reproductive strategy presents a challenging problem for evolutionary biologists [2,6,7]. Despite natural selection against selfpollination, mixed mating is frequent in seed plants [7,8–10]. Recent works indicate mixed mating can become evolutionarily stable resulting from the selection of traits promoting outcrossing or assuring reproductive success when out breeding chances are limited [7,11,12]. Mixed mating often occurs as a result of selfing by pollinator-mediated ⁎ Corresponding author. E-mail address: [email protected] (C.-J. Ruan).

http://dx.doi.org/10.1016/j.chnaes.2016.12.011 1872-2032/© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

geitonogamy and autogamy in self-compatible plants. Another mechanism that can result in mixed mating is autonomous self-pollination [13–15]. Three modes of autonomous self-pollination (the prior, competing and delayed selfing) were recognized by Lloyd [16], in which, prior and competing selfing can lower the potential for outcrossing. On the other hand, delayed selfing allows outcrossing when pollinators are present, but provides reproductive assurance under unpredictable pollinator environments [13,16–18]. There is increasing evidence for the idea that delayed selfing has been considered the best-of-both-worlds response to pollinator unpredictability because it can provide reproductive assurance without decreasing outcrossing potential [17–21]. Mechanisms of delayed selfing frequently involve movements of floral parts that bring the anthers and stigmas into physical contact at the end of floral life. H. trionum is a native of Central Africa, exhibits delayed autonomous selfing, being introduced into China in 1406 (http://www.Chinaias.cn/). Here we extend previous work by Buttrose et al. [22], Ramsey et al. [23] and Seed et al. [24] on the breeding system of Hibiscus trionum (Malvaceae). Buttrose et al. [22] examined style curvature in H. trionum var. trionum, an introduced annual weed in Australia. Ramsey et al. [23] found high levels of autonomous selfing and low levels of inbreeding depression in H. trionum var. trionum. On the contrary, Seed et al. [24] estimated high inbreeding depression in H. trionum var. vesicarius, a native annual in Australia. In this study, we focus on H. trionum, an

92

Q. Li et al. / Acta Ecologica Sinica 37 (2017) 91–96

introduced taxa in China, probably of H. trionum var. trionum because of its narrow leaves. We investigate mechanisms and possible benefits of delayed autonomous selfing in this species. The objectives of this study were to: 1) describe the floral morphology of H. trionum; 2) test the breeding system, autofertility and reproductive assurance potentially provided by delayed selfing by different floral manipulations; and 3) estimate outcrossing rate using ISSR markers. 2. Methods 2.1. Species and site The flowers of Hibiscus trionum (Malvaceae) are large and have showy petals that are pale in color with a deep-purple centre. The numerous stamens are fused to form a tube that is united to the base of the corolla. The pistal has five stylar branches, each with a capitate stigma. The large and attractive flowers open in the morning, and close in the mid-afternoon [24]. If the stigma is not pollinated, the stylar branches will curve in the morning, until the stigmas contact the anthers in the mid-afternoon [22]. The floral visitors include beetles, flies, bees and larva of moths. Insects move among plants, potentially cross-pollinating flowers (Li pers. obs.). In September 2012, the seeds of H. trionum were collected from the Dongling Park in Shenyang city located in northern of Liaoning province, China. One fruit was collected from each of 32 plants that were growing at least 5 m apart. The collected seeds were stored in paper envelopes for at least 1 month before being used. To establish experimental populations, seeds were placed in Petri dishes in 2014. The seedlings with two or more leaves were planted in common garden in Shenyang Agricultural University (SYAU) of Shenyang city. Shenyang city (123°4′ E, 41°8′N) has a continental climate with an annual mean temperature of 8.3 °C and an annual mean rainfall of 500 mm. It has 183 frost-free days and a total annual radiation of 2539 h. There were approximately 500 individuals in the SYAU population in 2014 and planting interval is 50 cm. Plants were watered regularly and they can grow up to 1.0 m in height. We used this population to investigate floral morphology and breeding system. 2.2. Floral morphology We observed flower development during July to September 2014 in the population of SYAU. The average temperature in hottest July was 31.94 ± 3.43 °C and the average temperature in lowest September was 19.97 ± 2.06 °C in 2014. Previous observations found that the lengths of five petals in H. trionum flowers are not equal. We regarded the flowers as two types, based on whether the lengths of five petals are equal or not (type I and type II, respectively). The floral traits of types I and II flowers were measured: corolla width (diameter of the circle surrounding the five petal tips), the length and width of each petal, stamen column length. In addition, we randomly selected ten individuals to record the date of the first and the last flower, and the number of type I and II flowers produced by each plant (flowers were counted every day during anthesis). 2.3. Pollen-ovule ratio The pollen-ovule ratio (P/O) was measured by the method of Cruden [25]. For each flower, all stamens on the monadelphous column were collected before anthers dehisced, and the number of pollen grains per stamen was estimated while the number of ovules per flower was counted using a dissecting microscope (Olympus SZ2-ILST). For each day over 24 days, five flowers were randomly selected from five different individuals (one flower per individual) randomly selected from the garden of SYAU (n = 120). A total of 120 flowers were collected in 24 days. The breeding system was estimated by the data of Cruden [25,26] and Cruden & Hermann-Parker [27]: cleistogamous if the P/O

ratio ranged from 2.7 to 5.4, 18.1 to 39.0 for autogamous-obligate, 31.9 to 396.0 for autogamous-facultative, 244.7 to 2588.0 for xenogamous-facultative and 2108.0–195525.0 for xenogamousobligate. 2.4. Relationship between pollen deposition and seed set To determine the relationship between number of pollen loads on the number of seeds per capsule, different amounts of pollen were applied to one, two, three, four and five stigmas within a flower. Selected flowers were emasculated before anther dehiscence (04:00–04:30) by removing the anthers on the top of the monadelphous column using sharp, sterile scissors, randomly variable amounts of pollen grains from the unselected individuals were applied to each flower at 07:00, at which time all stigma lobes are just curving. The pollinated lobes of selected flowers were removed 4 h after pollination, and the number of pollen grains per stigma per flower was estimated using a dissecting microscope (Olympus SZ2-ILST). Mature capsules were harvested and the number of seeds in each capsule was counted. Ten individuals per treatment group were observed each day for 10 days, for a total of 100 flowers examined per treatment. Correlation analysis was used to test the relationship between pollen loads and seed set, using the software SPSS 11.5. 2.5. Breeding system, delayed selfing and reproductive assurance To assess the breeding system, we conducted an experiment in SYAU population during July to September 2014. Five treatments were designed, in which each treatment consisted of five individuals, and twenty flowers were randomly chosen from each individual. These included: (1) Io, intact open flowers; (2) As, autonomous self-pollination flowers; (3) Eo, emasculated flowers with open pollination, these flowers could only receive pollen delivered by pollinators; (4) Is, intact flowers with hand self-pollination; (5) Ec, emasculated flowers with hand cross-pollination. After 15 days, the fruits were collected and the number of mature seeds per fruit was counted. Differences in fruit set and the number of seeds per fruit among different treatments were analyzed using oneway ANOVA (SPSS 11.5) followed by a contrast test. The capacity for autonomous self-pollination, or autofertility (AF), was calculated as FAs/FIo, where FAs is the mean seed production of the As treatment, while FIo is the mean seed production of the Io treatment (including the flowers that did not produce any fruit at all) [13]. To test the contribution of AF to reproductive assurance (RA), a measure of RA was calculated as (FIo − FEo) / FIo, where FIo and FEo are the seed set of Io (which can produce seeds from either autonomous selfing or vector-assisted pollen movement) and Eo (which can only produce seeds via vector-assisted pollen movement) [28]. 2.6. Mating system We estimated outcrossing rates in three naturalized populations: Shenyang (SY), Anshan (AS) and Dalian city (DL) in Liaoning Province. They are located in centre and south of Liaoning province, where H. trionum grows widely. There were in average 90–100 individuals per ha in the SY, AS and DL populations. In each population, we randomly selected sixty individuals at least 10 m apart from each other, and all seeds per plant were collected from August to October 2011. Two months after collection, the seeds were germinated in growth chambers, with 45 μmol s−1 m−2 light, a 12 h photoperiod, 70–80% relative humidity, at 28 °C. Ten days after germination, at least nine seedlings per individual were randomly chosen, and between nine and 20 individuals were used for each population. A total of 505 seedlings from SY, AS and DL population were used for genomic DNA extraction. Total genomic DNA was extracted from fresh ten days old seedlings using the protocol of Doyle ﹠ Doyle [29]. A total number of 60 ISSR primers that were from the Biotechnology

Q. Li et al. / Acta Ecologica Sinica 37 (2017) 91–96

Laboratory, the University of British Columbia (UBC set No.9), were screened using four DNA samples. Based on the number and quality of polymorphic fragments, fourteen ISSR primers (Table. 1) were selected. PCR amplification was performed in 20 μl reaction volume containing 10 mM Tris-HCl pH 8.0, 50 mM KCl, 2.0 mM MgCl2, 0.5 mM of each dNTP, 0.5 μM of primer, 1 unit of Taq Polymerase and 30 ng of genomic DNA. Initial denaturation was for 3 min at 94 °C, followed by 45 cycles of 45 s at 94 °C, 45 s at 53 °C, 90 s at 72 °C, and a 7 min final extension step at 72 °C. PCR products were analyzed on 2% agarose gels at a constant voltage of 80 V for 1.5 h, then stained with ethidium bromide, visualized with ultraviolet light and photographed. ISSR markers are typically dominant. Bands were scored by considering only two possible alleles: band presence (1), band absence (2). The program MLTR version 3.2 [30] was used to estimate population multilocus (tm) and single-locus (ts), and the difference between the two (tm − ts), which is often used to characterize the level of biparental inbreeding, the inbreeding resulting from matings between closely related individuals. It also calculated the multilocus correlation of paternity within progeny arrays (rpm), which is the fraction of siblings that share the same father, and the inbreeding coefficient (F) of the maternal parents [30]. The expected inbreeding coefficient at equilibrium (Fe) was calculated using Fe = (1 − tm) / (1 + tm) [31,32]. By comparing parental F with Fe, it is possible to assess whether inbreeding is having a negative effect (inbreeding depression) on the survivorship of selfed progeny in the populations. Under significant inbreeding depression, survivorship of selfed progeny should be lower than survivorship of outcrossed progeny, and therefore parental F should be lower than Fe. Standard errors of these estimates were obtained based on 1000 bootstraps. 3. Results 3.1. Floral morphology and P/O ratio The life-time of a single flower was about 9 h. Flowers open about 4:30 in the morning, and close by mid-afternoon. In the SYAU population, the first flower of H. trionum opened on 12 July, 2014; the flowering period lasted 63 ± 4.32 days, with the peak blossoming dates (50% of flowering individuals) of 47.3 ± 3.65 days. Plants were 79.36 ± 12.93 cm tall, and each individual produced 23.76 ± 21.67 new flowers daily. The ratio of types I and II flowers was between 0.53 and 6.57 per plant (n = 10, Fig. 1). There were significant differences in petal length and petal basal width between types I and II flowers of H. trionum; and no significant differences were found between others traits (Table. 2). The time of floral closure was different when the Table 1 Sequences of the selected ISSR primers used for the analyses of outcrossing rate, annealing temperature and the number of bands they produced in three Hibiscus trionum populations (annealing temperature was 53 °C for all primers). Primers

Sequences (5′-3′)

UBC 807 UBC 809 UBC 810 UBC 811 UBC 812 UBC 813 UBC 814 UBC 822 UBC 823 UBC 834 UBC 835 UBC 836 UBC 881 UBC 887

(AG)8T (AG)8G (GA)8T (GA)8C (GA)8A (CT)8T (CT)8A (TC)8A (TC)8C (AG)8YT (AG)8YC (AG)8YA (GGGTG)3 DVD(TC)7

Y: C/T; D: A/G/T; V: A/C/G. Populations = SY: Shenyang; AS: Anshan; DL: Dalian.

Total number of bands SY

AS

DL

9 21 23 24 15 9 12 14 15 16 22 15 24 20

11 22 17 20 15 8 8 15 11 12 18 9 17 16

15 14 18 16 13 13 10 14 10 14 17 7 15 10

93

Fig. 1. The ratio values of the typeIto II flowers within one plant (n = 10).

flowers were pollinated by hand or not. The flowers with hand pollination were closed 1 h earlier than intact flowers, whether they were type I or II flowers. The P/O values of type I and II flowers were 42.15 ± 12.75 and 28.81 ± 13.10, respectively. There were significant difference between them (F = 0.263, t = 0.020). These values indicated that the breeding system of type I and II flowers of H. trionum belongs to facultative autogamy and obligate autogamy respectively, according to the criteria of Cruden [25]. 3.2. Effects of pollen loads on seed set The relationship between the number of pollinated stigma lobes and the number of seeds produced by flowers was first assessed, and the result indicated a significant positive correlation between them (F4,109 = 17.405, P b 0.000, Fig. 2). In order to account for the pollen number of the response variable, both linear and quadratic terms for seed set were included as independent variable [33]. The quadratic relationship between the amount of pollen grains and the number of seeds per capsule was obtained: y = 14.87 + 0.361x − 0.001x2, r2 = 0.32 (y, seeds per capsule; x, number of pollen per flower; n = 113 flowers) (Fig. 3). Furthermore, this quadratic relationship between them was extremely significant (regression, F1,112 = 29.506, P b 0.000; coefficients ± S.E., linear term: 23.976 ± 0.397, t = 5.432, P b 0.000). We calculated the minimum number of pollen grains needed per flower to obtain the mean observed number of seeds per fruit (mean = 37). We found that at least 80 pollen grains were required. 3.3. Breeding system, delayed selfing and reproductive assurance The H. trionum was capable of autonomous selfing, the AF of types I and II were 0.693 and 0.695, respectively. However, there were significant differences in seed set between autonomous selfing and other treatments (type I F4,132 = 6.815, P = 0.000; type II F4,90 = 2.694, P = 0.036; Fig. 4B). There were no significant difference in fruit and seed set between intact and hand-pollinated flowers with either self- or cross-pollen, indicating that H. trionum had a high level Table 2 Comparison of floral traits type I (n = 23) and type II (n = 32) in Hibiscus trionum. Floral trait (mm)

Type I

Type II

P (t-test)

Corolla diameter Petal length Petal basal width Stamen length Height of lowermost stamen Height of uppermost stamen Stamen column length Style length Herkogamy

26.40 ± 5.11 24.48 ± 2.19 16.15 ± 1.43 3.43 ± 0.30 4.34 ± 0.75 6.29 ± 1.33 7.99 ± 1.38 10.26 ± 1.11 3.45 ± 0.75

23.51 ± 3.97 21.05 ± 2.42 13.85 ± 1.66 3.59 ± 0.40 4.36 ± 0.70 6.67 ± 0.83 8.61 ± 1.53 10.60 ± 0.61 3.45 ± 0.48

0.024 0.000 0.000 0.132 0.953 0.200 0.132 0.203 0.982

94

Q. Li et al. / Acta Ecologica Sinica 37 (2017) 91–96

Fig. 2. The number of seed produced by flowers with different numbers of pollinated stigmas. Mean ± 1 SE, n = 10 different individuals.

of self-compatibility. Emasculation treatments affected the fruit set in the flowers of type II. There was significant differences in fruit set in the flowers of type II between emasculated open and intact flowers (t = 5.344, df = 4, P = 0.006), indicating that emasculation reduce the attractiveness of flowers to pollinators in the flowers of type II (Fig. 4A). To test for a significant contribution of flower size to fruit set, we compared the mean fruit set between types I and II flowers, and the difference was significant (t = 3.894, df = 4, P = 0.018). Delayed selfing in H. trionum did not provide a large contribution to seed production, since RA were only 0.025 (0.020 and 0.030 in the flowers of type I and II, respectively). 3.4. Mating system The multilocus outcrossing rate of H. rionum in three naturalized populations ranged from 0.982 to 1.200, with a mean of 1.116. This suggests that this species is predominantly outcrossing. The rates of biparental inbreeding (tm − ts) were ranging from 0.212 to 0.414, indicating that a considerable amounts of crossings occurred between closely related siblings in all three populations. The fraction of siblings that share the same father (rpm) were relatively low (b0.5) indicating a predominance of random mates in all three natural population. Interestingly, the maternal inbreeding coefficients (F) were higher than 0.5 in AS and DL population, indicating there is strong selection against selfed progeny in those populations. In addition, the values for Fe in the three populations were 0.009 (SY), 0.077 (AS) and, 0.091 (DL) respectively, were lower than the estimated F (Table 3). 4. Discussion Flower size is extremely important in attraction pollinators and larger targets are more attractive than smaller ones to most insects [34–36]. Our data showed that there were two type flowers in H. trionum, and the type I flower was predominant. The ratio values of type Ito II flower

Fig. 3. Quadratic relationship between the amount of pollen grains and the number of seeds per capsule in Hibiscus trionum. (y = 14.87 + 0.361x − 0.001x2, r2 = 0.32, F1,112 = 29.506, P b 0.000; n = 113 flowers).

Fig. 4. Effects of five pollination treatments on fruit set (A) and the number of seeds per fruit (excluding fruits with no seeds) (B) in the flowers of types I and II of Hibiscus trionum. Error bars are ±1 SE. Io: intact open flowers; As: autonomous self-pollination; Eo: emasculation with open pollination; Is: supplemental pollination with self-pollen on intact flowers; Ec: supplemental pollination with outcross-pollen on emasculated flowers.

were over 1.0 in most plants except one plant. There were significant differences in petal lengths and petal basal widths between type I and II flowers; and there was significant difference in fruit sets between type I and II flowers. These indicated that the larger sizes were much more visible to pollinators and should therefore be favored by selection. This is consistent with the results of Conner and Rush [35] who observed that increased flower size causes a weak increase in small-bee visitation in Raphanus raphanistrum. Furthermore, there was significant difference in fruit set in the flowers of type II between emasculated open and intact flowers. Those indicated that the petal may attracted more pollinators than pollen in H. trionum. Flower longevity in some species appears to be tightly coupled to the deposition or removal of pollen, although flowers may persist after pollination. This persistence may provide an adaptive advantage in attracting pollinators to un-pollinated flowers on the same plant [37]. Pollinated flowers closed much faster than control flowers in H. trionum, and this is a typical response that has been reported for different species [38–41]. However, there are significant difference in seed sets between un-pollinated flowers (autonomous selfing) and hand-pollinated flowers. This is consistent with the results of Holtsford [38] and Ashman and Schoen [39], who observed that greater floral longevity in Calochortus leichtlini and Clarkia tembloriensis caused a significant decrease in fruit and seed production. These indicated that pollination effects on floral longevity might change across plant resource status conditions and might represent an important source of variation in reproductive assurance and overall plant fitness. Our data show that H. trionum, regardless of flower size, are fully self-compatible and capable of delayed autonomous self-pollination. The curvature of style branches in H. trionum eventually brings some un-pollinated stigmas down to touches pollen, leading to successful reproduction of 72.5% of flowers in the absence of pollinators, indicating that the delayed selfing provides reproductive assurance when pollinators were scarce. This is consistent with the results of Seed et al. [24]

Q. Li et al. / Acta Ecologica Sinica 37 (2017) 91–96

95

Table 3 Mating system parameters in three populations of Hibiscus trionum. The parameters examined included the multilocus (tm) and average single-locus (ts) outcrossing rates, biparental inbreeding rate (tm − ts), multilocus correlation of paternity (rpm), and the inbreeding coefficient among maternal parents (F). Standard errors in parentheses. Population

tm

ts

tm − ts

rpm

F

SY AS DL

0.982(0.002) 1.166(0.079) 1.200(0.000)

0.770(0.006) 0.899(0.013) 0.786(0.028)

0.212(0.007) 0.268(0.067) 0.414(0.028)

0.379(0.009) 0.082(0.003) 0.110(0.014)

0.194(0.001) 0.561(0.011) 0.529(0.012)

Populations = SY: Shenyang; AS: Anshan; DL: Dalian.

who observed that the number of seeds per fruit following autonomous selfing was only 77% that following hand - selfing in H. trionum var. vesicarius. However, reproductive assurance from delayed autonomous self-pollination in H. trionum is low (RA = 0.025) in our populations where pollinators were abundant. The seed set was lower in autonomous selfing, indicating that pollinators are often required for maximum seed production. The curvature of style branches in H. trionum brings some un-pollinated stigmas down to touches unshed pollen, potentially resulting in delayed selfing. However, the herkogamy in H. trionum may promote outcrossing and reduce interference in intrafloral male-female interference [15]. H. trionum produce a mixture of selfed and crossed seeds, the frequency of each depending on pollinator abundance and visitation under natural conditions. Although there were significant correlations between the frequency of visits and the climatic factors for some plants [42–44]. There was no significant difference in seed set between self- or cross-pollen, indicating that the pollinators were abundantly available in our experimental population in 2014. Although the curvature of un-pollinated stigma lobes is independent of the amount of pollen load [24], increasing pollen load is helpful for seed set because the number of seeds per capsule is dependent on the pollen load. Stigma pollen load must be sufficiently high to produce a pollen population effect [26]. Our data show that 80 or more pollen grains per flower can make mean observed per flower seeds in H. trionum. In addition, a sufficiently number of pollen grains per ovule is needed to trigger germination and/or produce viable pollen tubes [45, 46]. The mean number of seeds was very similar between the handselfed flowers and the hand cross-pollination of emasculated flowers, indicating that self-pollen is efficient at fertilizing ovules. The flowers with five pollinated stigmas produced significantly more seeds than flowers with one stigma. This is consistent with the results of Seed et al. [24]who observed that the seed set of flowers with one stigma pollinated was only ~ 50% of that of flowers with five pollinated stigmas. Flowers with five stigmas have more chance for fertilization than the flowers with one to four stigmas [47]. The levels and distribution of genetic variability in populations may also be related to mating system [48]. Mating system analysis showed high levels of outcrossing in all populations studied in our word. On average, the outcrossing rates of three naturalized H. trionum populations were 1.116. When total outcrossing rates (tm) in our study were roughly estimated, there was significant difference between SY population and the other two populations. In SY population, only 0.8% of progeny would result from selfing. However, the values of tm were N1 in AS and DL population, suggesting that H. trionum is allogamous in those two studied area. However, the pollen-ovule ratios suggest an autogamous mating system in H. trionum, these strategies probably contribute to invasive success for this species. Self-fertilization rates can vary inversely with population density [49]. In the three studied H. trionum populations, the lower outcrossing rate appeared in the SY population, which has a relative low-density population compared with the other two populations. In spite of the high outcrossing rates detected in the three populations, the biparental inbreeding is inevitable, which might have been caused by mating among relatives. Biparental inbreeding may reflect (i) the spatial genetic substructure of the population caused by the limited dispersal of pollen and/or seeds [50,51], and (ii) the variation in flowering synchrony among individuals [52]. There was significant

difference in biparental inbreeding among the three populations. Biparental inbreeding in this species is possibly associated with plant density and the product of flower size and number [53]. In the three studied H. trionum populations, the highest biparental inbreeding appeared in the DL population, which has a relative small size, compared with the other two populations. The fraction of siblings that share the same father (rpm) was relatively high (0.379 in the SY population), indicating that this phenomenon contributes considerably to the inbreeding seen in this population. Herkogamy may encourage cross-fertilization, but it still allows pollination between flowers on the same individual (geitonogamous self-pollination) to occur [54,55]. Our field surveys have found that individual plants in fact display numerous open flowers at the same time, with large individuals displaying up to 34 flowers open concurrently. In the studied H. trionum population, the pollinators are beetles and bees, which often travel predominantly among neighbouring flowers or individuals. These observations indicate that geitonogamous selfing is indeed a very likely possibility in natural populations of H. trionum. However, there was no inbreeding depression observed in H. trionum, because the expected inbreeding coefficient Fe was lower than our estimated inbreeding coefficient F. The high maternal inbreeding coefficient in H. trionum can be explained by the high level of biparental inbreeding with many full sibs produced per fruit and a subsequent limited seed dispersal. In addition, the pollinators' activity can vary among environments either spatially or temporally [56]. In contrast, selfing in Kalmia latifolia is more prevalent within a flower than among flowers in the same plant [57]. Thus, herkogamy in H. trionum may act as a mechanism to reduce intrafloral male-female interference. The curvature of unpollinated styles towards the anther, which presumably facilitates self-pollination, is known to occur in several species of Malvaceae [58]. Our data showed that the floral traits in H. trionum contribute to out-crossing and reduce intrafloral male-female interference, but can still provide some reproductive assurance by autonomous selfing if pollination levels are low. In addition, the outcrossing rates are relatively high in this annual, self-compatible species. In conclusion, the growth and persistence of the invasive H. trionum populations is attributed to fitness homeostasis through a flexible reproductive strategy, the plants can reproduce without pollinators or conspecifics. Acknowledgments The authors wish to thank Mr P. Yang, D. Weng, and Mis H. Su for their field assistance. This work was funded by the National Natural Science Foundation of China (31100226). References [1] S. Pilar, V. Pablo, V. Marta, E.C. Maria, Mating system of Brassica napus and its relationship with morphological and ecological parameters in northwestern Spain, J. Hered. (2013) http://dx.doi.org/10.1093/jhered/est018. [2] D. Gottlieb, J.P. Holzman, Y. Lubin, A. Bouskila, S.T. Kelley, A.R. Harari, Mate availability contributes to maintain the mixed-mating system in a scolytid beetle, J. Evol. Biol. 22 (2009) 1526–1534. [3] R.F. Del Castillo, S. Trujillo, Effect of inbreeding depression on outcrossing rates among populations of a tropical pin, New Phytol. 178 (2008) 459. [4] G.L. Stebbins, Variation and Evolution in Plants, Columbia University Press, New York, 1950. [5] S.C. Barrett, The evolution of plant sexual diversity, Nat. Rev. Genet. 3 (2002) 274–284.

96

Q. Li et al. / Acta Ecologica Sinica 37 (2017) 91–96

[6] C.G. Oakley, Inbreeding Depression and Mating System Evolution in the Perennial Herb Viola septemloba; and the Evolutionary Maintenance of Cleistogamy(Master thesis) The Florida State University, 2004 4–7. [7] C. Goodwillie, S. Kalisz, C.G. Eckert, The evolutionary enigma of mixed mating systems in plants: occurrence, theoretical explanations, and empirical evidence, Annu. Rev. Ecol. Evol. Syst. 36 (2005) 47–79. [8] D.J. Schoen, A.H.D. Brown, Whole- and part-flower self-pollination in glycine clan destine and G. argyrea and the evolution of autogamy, Evolution 45 (1991) 1665–1674. [9] D.W. Vogler, S. Kalisz, Sex among the flowers: the distribution of plant mating systems, Evolution 55 (2001) 202–204. [10] C. Elena, G. Jaime, The role of a mixed mating system in the reproduction of a Mediterranean subshrub (Fumana hispidula, Cistaceae), J. Plant Res. 126 (2013) 33–40. [11] J.W. Busch, C.R. Herlihy, L. Gunn, W.J. Werner, Mixed mating in a recently derived self-compatible population of Leavenworthia alabamica (Brassicaceae), Am. J. Bot. 97 (2010) 1005–1013. [12] C.J. Ruan, J.A. Teixeira da Silva, Evolutionary assurance vs. Mixed mating, Crit. Rev. Plant Sci. 31 (4) (2012) http://dx.doi.org/10.1080/07352689.2011.645442. [13] D.G. Lloyd, D.J. Schoen, Self- and cross-fertilization in plants. I. Functional dimensions, Int. J. Plant Sci. 153 (1992) 358–369. [14] C.R. Herlihy, C.G. Eckert, Experimental dissection of inbreeding and its adaptive significance in a flowering plant, Aquilegia Canadensis (Ranunculaceae), Evolution 58 (2004) 2693–2703. [15] C.J. Ruan, J.A. Teixeira da Silva, P. Qin, Style curvature and its adaptive significance in the Malvaceae, Plant Syst. Evol. 288 (2010) 13–23. [16] D.G. Lloyd, Some reproductive factors affecting the selection of self-fertilization in plants, Am. Nat. 113 (1979) 67–79. [17] D.G. Lloyd, Self- and cross-fertilization in plants. II. The selection of self-fertilization, Int. J. Plant Sci. 153 (1992) 370–380. [18] C.J. Ruan, S. Mopper, J.A. Teixeira da Silva, P. Qin, X.Q. Zhang, Y. Shan, Contex-dependent style curvature in Kosteletzkya virginica (Malvaceae) offers reproductive assurance under unpredictable pollinator environments, Plant Syst. Evol. 277 (2009) 207–215. [19] S. Kalisz, D.W. Vogler, Benefits of autonomous selfing under unpredictable pollinator environments, Ecology 84 (2003) 2928–2942. [20] C.J. Ruan, Genetic relationships among sea buckthorn varieties from China, Russia and Mongolia using AFLP markers, J. Hortic. Sci. Biotechnol. 81 (3) (2006) 409–414. [21] F. Pérez, C. León, Muñoz, How variable is delayed selfing in a fluctuating pollinator environment? A comparison between a delayed selfing and a pollinator-dependent Schizanthus species of the high andes, Evol. Ecol. 27 (2013) 911–922. [22] M.S. Buttrose, W.J.R. Grant, J.N.A. Lott, Reversible curvature of style branches of Hibiscus trionum L., apollination mechanism, Aust. J. Bot. 25 (1977) 567–570. [23] M. Ramsey, L. Seed, G. Vaughton, Delayed selfing and low levels of inbreeding depression in Hibiscus trionum (Malvaceae), Aust. J. Bot. 51 (2003) 275–281. [24] L. Seed, G. Vaughton, M. Ramsey, Delayed autonomous selfing and inbreeding depression in the Australian annual Hibiscus trionum var. vesicarius (Malvaceae), Aust. J. Bot. 54 (2006) 27–34. [25] R.W. Cruden, Pollen-ovule ratios: a conservative indicator of breeding systems in flowering plants, Evolution 31 (1977) 32–36. [26] R.W. Cruden, Pollen grains: why so many? Plant Syst. Evol. 222 (2000) 143–165. [27] R.W. Cruden, S.M. Hermann-Parker, Temporal dioecism: An alternative to dioecism? Evolution 31 (1997) 863–866. [28] C.G. Eckert, K.E. Samis, S. Dart, Reproductive assurance and the evolution of uniparental reproduction in flowering plants, in: D.L. Harder, S.C.H. Barrett (Eds.), Ecology and Evolution of Flowers, Oxford University Press, Oxford, UK 2006, pp. 183–203. [29] J.J. Doyle, J.L. Doyle, A rapid DNA isolation procedure for small quantities of fresh leaf tissue, Phytochem. Bull. 19 (1987) 11–15. [30] K. Ritland, Extensions of models for the estimation of mating systems using n independent loci, Heredity 88 (2002) 221–228. [31] S. Wright, The interpretation of population structure by F-statistics with special regard to systems of mating, Evolution 19 (1965) 395–420. [32] P.W. Hedrick, Genetics of Populations, Jones and Bartlett, Boston, MA, 1985. [33] J. Neter, M.H. Kutner, C.J. Nachtsheim, W.W. Wasserman, Applied linear regression models, third ed. Irwin, Chicago, 1996.

[34] C. Galen, Measuring pollinator-mediated selection on morphometric floral traits: bumblebees and the alpine sky pilot, Polemonium viscosum, Evolution 43 (1989) 882–890. [35] J.K. Conner, S. Rush, Effects of flower size and number on pollinator visitation to wild radish, Raphanus raphanistrum, Oecologia 105 (1996) 509–516. [36] J. Spaethe, J. Tautz, L. Chittka, Visual constraints in foraging bumblebees: flower size and colour affect search time and flight behaviour, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 3898–3903. [37] C.E. Jones, M.B. Cruzan, Floral morphological changes and reproductive success in deer weed (Lotus scoparius, Fabaceae), Am. J. Bot. 86 (1999) 273–277. [38] T.P. Holtsford, Nonfruiting hermaphrodictic flowers of Calochortus leichtlini (Liliaceae): potential reproductive functions, Am. J. Bot. 72 (1985) 1687–1694. [39] T.L. Ashman, D.J. Schoen, The cost of floral longevity in Clarkia tembloriensis: an experimental investigation, Evol. Ecol. 11 (1) (1997) 289–300. [40] M. Stpiczynska, Flora longevity and nectar secretion of Platanthera chlorantha (Custer) Rchb (Orchidaceae), Ann. Bot. 92 (2003) 191–197. [41] E. Kozuharova, M. Anchev, Pollination ecology of Gentiana asclepiadea L. and G. pneumonanthe L. (Gentianaceae, sect. Pneumonanthe) in Bulgaria, Ann. Univ. Sofia, Book 2, Bot. 94 (2004) 39–58. [42] X.H. Wang, X. Liu, Y. Song, I. Chen, I. Jin, Correlations between environmental factors and wild bee behavior on alfalfa (Medicago sativa) in Northwestern China, Environ. Entomol. 38 (2009) 1480–1484. [43] R.J. Warren, I. Giladi, M.A. Bradford, Environmental heterogeneity and interspecific interactions influence nest occupancy by key seed dispersing ants, Environ. Entomol. 41 (2012) 463–468. [44] V. Daniele, F.V. Lyderson, P.L. Andrea, P. Fabio, Flower-visiting insects and phenology of Lippia alba (Lamiales: Verbenaceae): floral color changes and environmental conditions as cues for pollinators, Environ. Entomol. 45 (3) (2016) 685–693. [45] D.W. Schemske, C. Fenster, Pollen-grain interactions in a neotropical costus: effects of clump size and competitors, in: D.L. Mulcahy, E. Ottaviano (Eds.), Pollen: Biology and Implications for Plant Breeding, Elsevier Science, New York 1983, pp. 405–410. [46] M.B. Cruzan, Pollen tube distributions in Nicotiana glauca: evidence for density dependent growth, Am. J. Bot. 73 (1986) 902–907. [47] C.J. Ruan, P. Qin, J.A. Teixeira da Silva, Q.X. Zhang, Movement herkogamy in Kosteletzkya virginica: effect on reproductive success and contribution to pollen receipt and reproductive assurance, Acta Ecol. Sin. 29 (2009) 98–103. [48] J.C. Hamrick, J.W. Godt, Allozyme diversity in plant species, in: H.D. Brown, M.T. Clegg, A.L. Kahler, B.S. Weir (Eds.), Plant Population Genetics, Breeding, and Genetic Resources, Sinauer, Sunderland, Massachusetts, USA 1990, pp. 43–65. [49] M. Ward, C.W. Dick, R. Gribel, A.J. Lower, To self, or not to self… a review of outcrossing and pollen-mediated gene flow in neotropical trees, Heredity 95 (2005) 246–254. [50] R.A. Ennos, M.T. Clegg, Effect of population substructuring on estimates of outcrossing rate in plant population, Heredity 48 (1982) 283–292. [51] M.E. Turner, J.C. Stephens, W.W. Anderson, Homozygosity and patch structure in plant populations as a result of nearest neighbor pollination, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 203–207. [52] P. Hall, S. Walker, K.S. Bawa, Effect of forest fragmentation on genetic diversity and mating system in a tropical tree, Pithecellobium elegans, Conserv. Biol. 10 (1996) 757–768. [53] C. Goodwillie, R.E. Sargent, C.G. Eckert, E. Elle, M.A. Geber, M.O. Johnston, S. Kalisz, D.A. Moeller, R.H. Ree, M.V. Marin, A.A. Winn, Correlated evolution of mating system and floral display traits in flowering plants and its implications for the distribution of mating system variation, New Phytol. 185 (2010) 311–321. [54] D.G. Lloyd, C.J. Webb, The avoidance of interference between the presentation of pollen and stigmas in angiosperms: I. Dichogamy, New Zeal J. Bot. 24 (1986) 135–162. [55] R. Bertin, C.M. Newman, Dichogamy in angiosperms, Bot. Rev. 59 (1993) 112–152. [56] S. Bojana, O.C. Dierre, M. Sandrine, Does cleistogamy variation translate into outcrossing variation in the annual species Lamium amplexicaule (Lamiaceae)? Plant Syst. Evol. 300 (2014) 2105–2114. [57] M.A. Levri, A measure of the various modes of inbreeding in Kalmia latifolia, Ann. Bot. 80 (2000) 415–420. [58] C.J. Ruan, S.C. Chen, Q. Li, J.A. Teixeira da Silva, Adaptive evolution of context-dependent style curvature in some species of the Malvaceae: a molecular phylogenetic approach, Plant Syst. Evol. 297 (2011) 57–74.