Differentiation in fructification percentage between two morphs of Amomum tsaoko (Zingiberaceae).

Amomum tsaoko is a flexistylous ginger. Flexistyly is a unique floral mechanism promoting outcrossing, which is known only in some species of Zingiberaceae till date. This is a pioneer report on flexistyly in A. tsaoko from the aspect of fructification percentage to clarify its influence on reproduction. We observed in 2007 and 2008 that the fructification percentage of the anaflexistyled and the cataflexistyled inflorescence were 14.89 ± 10.35% and 11.31 ± 7.91% respectively, with significant difference (d.f. = 141.920, t = 2.518, P = 0.013 < 0.05). The greatly significant difference between 2007 and 2008 were present in both the flower number (d.f. = 93, t = -2.819, P = 0.006 < 0.01) and the fructification percentage (d.f. = 93, t = -2.894, P = 0.005 < 0.01) of the cataflexistylous inflorescence. Although the two morphs were similar in morphological characteristics, there was some gender differentiation between them, showing a possibility that the anaflexistylous morph might function more as females and the cataflexistylous morph more as males. Reproduction of the cataflexistylous morph was significantly sensitive to change of environmental factors, in contrast to the anaflexistylous morph, thus the yield varied between the abundant year (2008) and the off year (2007).

Introduction

Plants are famously diverse in reproductive strategies (Barrett 2002), displaying a broad range of floral forms across species and presenting numerous examples of floral or reproductive polymorphisms maintained within a population (Pannell ). The sexual diversity in plants has been a main concern for evolutionary biologists (Barrett 2002).

Amomum tsaoko Crevost et Lemaire is a ginger with a flexistylous mechanism that is a unique and motile sexual dimorphism to promote outcrossing (Cui , 1995b, Li , 2001b, 2002, Takano , Wang , 2005b, Zhang ). There are two floral morphs in a flexistylous species, termed as anaflexistylous (protogynous) and cataflexistylous (protandrous) morphs according to the direction of stigma movement and time of pollen release during anthesis. Through active stylar curvature, each morph changes from one sexual phase to the other in the middle of the 1-d flowering period, and the two morphs form a reciprocal cooperation between stigmas and anthers to promote outcrossing (Li , Zhang and Li 2002). The sexual organs are separated temporarily and spatially by active stylar curvature, thus interference of male and female functions may reduce to small extent (Barrett 2002, Li , 2001b, Zhang 2004, Zhang , Zhang and Li 2002).

Fruit maturation indicates reproductive success of plants, which is the most basic purpose of flowering plants (Barrett and Harder 1996). In this context, fructification percentage is an important index of the reproductive success of plants, which also reflects the fitness of a plant. Other flexistylous species Alpinia nieuwenhuizii Val., a Bornean endemic, is known to be different in fruit set between the two morphs, and indicating some gender differentiation (Takano ). In this regard, it has not been reported in A. tsaoko yet (to distinguish Amomum from Alpinia, we abbreviate them as A. and Al., respectively thereafter).

With both flexistylous and non-flexistylous species in Amomum, which is an ideal model for discussing flexistyly phylogeny, flexistyly in this genus is worth to be studied in details (Ren ). To fully understand the evolution and maintenance of the sexual system in angiosperms, it is essential to investigate flexistyly in Amomum from various aspects, especially clarifying influence of this unique floral mechanism on reproduction and difference of reproductive trait between two morphs. Therefore, we reported the differentiation between the two morphs of A. tsaoko in the paper. The main goal of this study is to address the following questions: (1) is fructification percentage different between the two morphs of A. tsaoko? (2) Is there the same gender differentiation between the two morphs of A. tsaoko as those of Al. nieuwenhuizii? (3) Why does the yield of A. tsaoko vary between the abundant year and the off year?

Materials and Methods

Research species

A. tsaoko is a perennial herb of Zingiberaceae, usually 2–3 m in height. Inflorescence consists of a densely flowered spike that arises from rhizomes. Distinct characters of the plant from the other Amomum species are a showy yellow labellum with two red nectar guides, anther appendage, crimson and leathery ligule. Flowering occurs from April to June, and by September–December capsules are ripe (Wu and Raven 2000).

Study site

A. tsaoko distributes in Yunnan province, Southwest China (Wu and Raven 2000), but now its wild (or natural) population is almost extinct in Yunnan. Since its fruit is used as common materia medica (Chinese Pharmacopoeia Commission 2010), and a food condiment in China, it is cultivated as an economic plant in Yunnan.

The study site was at Biaopo (N 24°50.289′, E 98°46.501′, 2070 m in altitude), Gaoligong Mountain, Yunnan province, Southwest China. These plant individuals had grown for about 20 years in the humid valley covered by everygreen broadleaf forest, around which primary forest was kept as a part of natural reserve. These plant individuals grew naturally there, without artificial manipulation, and the villagers only entered for collection of the fruits in September–December.

Field observation and fructification percentage

The study was conducted from May 2007 to October 2008. Our preliminary observations suggested that the longevity of a single flower was only 1 d, both the nonfertilized and fertilized flowers withering at dusk. There were average 89.6 flowers (range from 48 to 129) and average 3–4 flowers opening for each day per inflorescence. Cui reported that the fertilized ovaries enlarged over the next five days, and grew rapidly from the next 20 d to 30 d, while some fertilized ovaries fell off under low humidity. On May 25, 2007, we selected at random 28 individuals in the population, including 12 cataflexistyled individuals and 16 anaflexistyled individuals for 2-year observation. In October 2007, six individuals of the 28 plants just had one infructescence respectively, so 1–4 infructescences were picked at random from each individual. Thus 26 anaflexistyled infructescences (352 fruits in total) and 31 cataflexistyled infructescences (201 fruits in total) were collected. In October 2008, 3–8 infructescences were picked at random from each individual, so 52 anaflexistyled infructescences (582 fruits in total) and 64 cataflexistyled infructescences (756 fruits in total) were collected. The infructescence of each morph that randomly selected was subjected to observations, and got both the number of mature fruits (excluding immature ones which fell off after anthesis) and scars of flowers in an infructescence, and the number of seeds in each fruit.

Statistical analyses

Fructification percentage was calculated by the formula (each infructescence: mature fruit number ÷ total flower number × 100%). Date on the fructification percentage, the flower number per inflorescence and the seed number per fruit were checked by Levene test, then compared between two morphs and between the two years by T Test in SPSS (13.0 version). Correlations were tested by Bivariate Correlation in SPSS (13.0 versions).

Results

Quantitative character of the inflorescence and fruit

As far as the population was concerned, 2008 was the abundant year and 2007 was the off year. The quantitative characters of the inflorescence also partly reflected the variation (Table 1).

Table 1

Quantitative character of the inflorescence and fruit of A. tsaoko in the study site

Quantitative Character	Year	Anaflexistylous morph	Cataflexistylous morph
Flower number per inflorescence	2007	81.00 ± 16.19 (46–106)*	80.77 ± 18.55 (52–129)
	2008	86.67 ± 21.24 (33–125)	94.23 ± 23.22 (21–135)
Fructification percentage per inflorescence (%)	2007	17.19 ± 8.31 (3.09–33.85)	8.05 ± 6.88 (0.78–30.12)
	2008	13.75 ± 11.12 (1.03–54.00)	12.88 ± 7.95 (1.57–37.78)
	Average**	14.89 ± 10.35 (1.03–54.00)	11.31 ± 7.91 (0.78–37.78)
Seed number per fruit	2007	27.13 ± 11.22 (5–67)	27.26 ± 11.40 (3–59)
	2008	29.31 ± 11.11 (5–64)	29.00 ± 11.05 (3–58)

Mean ± SD (range from 46 to 106).

The average of the two years.

Difference between the two morphs

Significant differences between the two morphs were only observed in the fructification percentage per inflorescence in 2007, and the average of the two years respectively (Table 2).

Table 2

Difference between the two morphs of A. tsaoko in the study site

Quantitative character	Year	d.f.	t	p
Flower number per inflorescence	2007	55	0.48	0.962
	2008	114	−1.812	0.073
Fructification percentage per inflorescence (%)	2007	55	4.545	0.000**
	2008	116	0.488	0.627
	Average***	141.920	2.518	0.013*
Seed number per fruit	2007	1711	−0.243	0.808
	2008	1289	0.506	0.613

The mean difference is significant at the 0.05 level.

The mean difference is greatly significant at the 0.01 level.

The average of the two years.

Difference between the two years

Of the anaflexistylous morph, there was greatly significant difference between the two years just in the seed number per fruit; of the cataflexistylous morph, greatly significant differences between the two years were detected in the flower number per inflorescence, the fructification percentage per inflorescence, and the seed number per fruit (Table 3).

Table 3

Difference between the two years in the study site

Quantitative character	Morph	d.f.	t	p
Flower number per inflorescence	Anaflexistylous	76	−1.198	0.235
	Cataflexistylous	93	−2.819	0.006**
Fructification percentage per inflorescence (%)	Anaflexistylous	76	1.396	0.167
	Cataflexistylous	93	−2.894	0.005**
Seed number per fruit	Anaflexistylous	1353	−3.517	0.000**
	Cataflexistylous	1647	−3.123	0.002**

The mean difference is greatly significant at the 0.01 level.

Correlation between flower number and fructification percentage

There were significantly negative correlations between flower number and fructification percentage per inflorescence (Table 4).

Table 4

Correlations between flower number and fructification percentage per inflorescence in the study site

D.f.	Pearson correlation (R)	Sig. (2-tailed) P	Note
172	−0.195	0.010*	In 2007 and 2008
115	−0.245	0.008**	In 2008
77	−0.324	0.004**	Among anaflexistylous morph

Correlation is significant at the 0.05 level (2-tailed).

Correlation is greatly significant at the 0.01 level (2-tailed).

Discussion

Gender (maleness or femaleness) was a quantitative phenomenon in plants; phenotypic gender was usually measured by the morphological feature of flowering, while functional gender was usually estimated by the relative success of female function (seed production) or male function (making female gametes to fertilize) (Lloyd 1979). Although the two morphs of A. tsaoko were similar in morphological characteristics, our study suggested that there was some gender differentiation between them. There was a possibility that the anaflexistylous morph might function more as females and the cataflexistylous morph more as males since higher fructification percentage and lower flower numbers in the anaflexistylous inflorescence in comparison to the cataflexistylous inflorescence supported this view, as reported previously flexistylous Al. nieuwenhuizii (Takano ). In addition, both the flower number and the fructification percentage in the cataflexistylous inflorescence were significantly different between the two years (2007 and 2008), in contrast to the anaflexistylous inflorescence. Our results indicated that both the flower number and the fructification percentage of the anaflexistylous morph was steady between the two years, while those of the cataflexistylous morph was inconstant. The result not only demonstrated that reproduction of the cataflexistylous morph could be easily influenced by environmental factors than the anaflexistylous morph, but also explained the main reason that the yield varied between the abundant year and the off year.

Because reproductive resources of a plant were limited, there would be tradeoff of two or more organs on account of competition in the same resource (Campbell 2000, Lloyd 1987). On the one hand, the tradeoff would show between male function and female function, such as between stamen and pistil, pollen and ovule, flower and fruit (Campbell 2000); on the other hand, the tradeoff would exhibit between number and size of organ, e.g. flower number and size, pollen number per flower and size, or seed number per fruit and size, etc. (Lloyd 1987, Thomson 1989). A significant negative relation was present between the flower number per inflorescence and the fructification percentage, indicating a tradeoff, which was more obvious or strong in the anaflexistylous inflorescences and in the abundant year (2008). The same tradeoff didn’t show in the cataflexistylous inflorescences with the lower fructification percentage, indicating a certain differentiation between the two morphs, and also presenting that the cataflexistylous flower would mainly provide pollen and function more as male. These averages of the flower number, the fructification percentage per inflorescence, the seed number per fruit were higher in 2008 than 2007, with exception of the fructification percentage of the anaflexistylous inflorescence. Likewise, the tradeoff was stronger in the abundant year (2008), thus the more abundant the yield, and the stronger the tradeoff.

The seed number per fruit was not significantly different between the two morphs, but that of each morph was greatly significant different between the two years, indicating that environmental factors would easily influence on the seed number per fruit of both morphs. It might also indicate that the condition of pollination was different between the two years.

A. tsaoko is a flexistylous ginger, its stylar curvature might be related to these differentiations. Firstly, style movement appeared to have asymmetrical effects, in contrast to male function, female function appeared to be affected weakly by stigma movement (Sun , 2011). Secondly, the anaflexistyled flower was proterogynous, which style curved upwards at around 15:00 in the afternoon to accomplish the gender transition from the female phase to the male phase (Cui ), so the female stage was longer than the male stage in one-day anthesis; the cataflexistyled flower was protandrous, which gender transition from the male phase to the female phase was accomplished at about 16:00 in the afternoon by the stylar downward curvature (Cui ), therefore the female stage was shorter than the male stage in one-day anthesis. Thirdly, the gender in the afternoon might be less efficient than the one in the morning (Takano ); the similar phenomenon might occur in A. tsaoko, and more observations would be needed. Further insight into reproductive trait would reveal better gender differentiation between the two morphs, such as an examination of the selfing rate in the two morphs using molecular markers.

Flexistyly was a special form of heterodichogamy promoting outcrossing (Ren ), and heterodichogamy was understood to come from synchronous dichogamy and was heading towards dioecy (Renner 2001). Since Darwin, most adaptive interpretations of sexual diversity had focused on mechanisms that functioned to promote outcrossing and reduced the likelihood of inbreeding depression (Takano ). However, reproductive assurance was a self-protecting mechanism or strategy (Baker 1955). Sex expression enhanced sexual reproduction through an optimal model of bisexual resource allocation (Charnov 1979), and plants invested more resources in male phasing than in female phasing (Zhang and Jiang 2001). Thus, the gender differentiation between the two morphs of A. tsaoko might exhibit its resource allocation strategy for enhancement of sexual reproduction or accomplishment of reproductive assurance. It might not only indicate that the influence of stylar movement on reproduction was different between the two morphs of A. tsaoko, and also the maintenance and evolutionary direction were different between the two morphs. As far as flexistyly in Amomum was concerned, further insight into which would be worth studying in details to fully understand its origin and evolution.