The Influence of Altitude and Latitude on Breeding of Amomum tsaoko ( Zingiberaceae )

Objective: To explore the effect of altitude and latitude on breeding of Amomum tsaoko Crevost et Lemaire, a flexistylous ginger, which fruit is used as common materia medica and a food condiment. Methods: The 7 populations were selected randomly from the three floristic zones of Yunnan. Adult plants and infructescences were chosen randomly to gain flower number and fructification percentage per inflorescence, and seed number per fruit. All date was analyzed by SPSS (13.0 version). Results: As A. tsaoko was distributed (or transplanted) from a habitat at lower latitude and/or altitude to a site of higher latitude and/or altitude, the flower number per inflorescence increased, on the contrary, the fructification percentage per inflorescence decreased. The competition for reproductive resource was beneficial to increase flower number and seed production. Conclusions: The habitats in south of the tropic of cancer were favorable to the reproduction of A. tsaoko, which reproductive costs were lower and harvest was higher. Increasing flower number per inflorescence may be a strategy to promote the plant to distribute into alpine habitats for both female and male reproductive success.


Introduction
Amomum tsaoko Crevost et Lemaire is a flexistylous ginger cultivated in China for collection fruit as common materia medica [1] and a food condiment.The plant distributed in Yunnan province, Southwest China [2], but now its wild (or natural) population is almost extinct in Yunnan, and it is cultivated as an economic plant at many areas in the province.
In the course of cultivation of the plant, how did environmental factors influence on its reproduction?And how did the plant adapt to the environmental changes?Because flower, fruit and seed are reproductive organs of plant, so their quantitative change would reflect the reproduction conditions of a population, and reproductive resource status of the plant [3][4][5][6].Therefore, we studied the reproduction conditions of the 7 populations of A. tsaoko in different areas, and address the following questions: 1) how did latitude and altitude influence on the flower number and fructification percentage per inflorescence, seed number per fruit?2) How is the reproductive competition among the flower number and fructification percentage per inflorescence, seed number per fruit?3) How does the flowering strategy of the plant change to adapt to alpine habitats?4) How is the site chosen for cultivation of the plant?

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 [2].

Study Sites and Field Observation
Yunnan province was broken into five floristic zones [7].Our preliminary investigation showed that A. tsaoko was growing in the three floristic zones: 1) the south and southwest, 2) the southeast, 3) the west and northwest.
In each floristic zone, 2 or 3 populations were chosen to test the effect of altitude and latitude on breeding of the plant (Table 1).In each population, we selected adult plants at random, and ripe infructescences were sampled randomly from each individual in August-October.These infructescences were subjected to observations.We got both the number of mature fruits (excluding immature ones which maybe fell off early after anthesis) and scars of flowers in an infructescence (i.e. the number of flowers in a ripe inflorescence), and also got number of seeds in each fruit.

Statistical Analyses
Per infructescence, fructification percentage was the proportion of mature fruit number to total flower number.

Correlations among Flower Number per Inflorescence, Fruit and Seed Production in A. tsaoko
Previous studies on fruiting character of A. tsaoko showed that the fruit abortion occurred after initiation because of environmental factors (e.g.humility) [8].Therefore, in this study, the fructification percentage per inflorescence reflected the plant final reproductive success, which combined both fruit set and fruit abortion.By the optimal regression equations and ANOVA analysis, there was a trade-off between flower number and fructification percentage per inflorescence, and significantly positive correlation between flower number per inflorescence and seed number per fruit.The former relationship was a typical response found in the other plants because of limited reproduction resource [9][10][11].An inflorescence was more attractive to pollinator in comparison with a single flower because the integrated flowers were more striking to insects [12,13].Based on this, the latter relationship implied probably that the inflorescences with numerous flowers easily attracted pollinators for reproduction assurance.The results might be associated with the pollinators (i.e.bees) activity.Because bees didn't visit repeatedly those flowers with low reward, they were likely remembered by their pollinators [14].
Because of limited reproduction resource, a competition among reproductive organs for reproduction assurance was widespread in plants [9].Therefore, the correlations among flower number per inflorescence, fruit and seed production likely reflected that competition of reproduction resource in A. tsaoko.Results from our study showed the competition for reproductive resource was beneficial to increase flower number and seed production, in contrast, such competition wasn't partial to amplifying fruit number.

The Influence of Altitude and Latitude on Flower Number per Inflorescence, Fruit and Seed Production in A. tsaoko
Results from this study showed that there was a strong effect of both altitude and latitude on the flower number per inflorescence and the fruit production of A. tsaoko.When the latitude and/or altitude of those populations were elevated, the flower number per inflorescence increased significantly, on the contrary, the fructification percentage per inflorescence decreased significantly.On contrast to altitude, the influence of latitude on both flower number and fructification percentage per inflorescence was more significant according to the value of coefficients in their optimal regression equations.We suggested that A. tsaoko was distributed (or transplanted) from a habitat at lower latitude and/or altitude to a site of higher latitude and/or altitude, the flower number per inflorescence increased, on the contrary, the fructification percentage per inflorescence decreased.By its optimal regression equation, both altitude and longitude influenced significantly the seed number per fruit.Based on the comparison of their variation (altitude: 1425 -2070 m vs. longitude: 98˚46.501′-104˚49.333′) in the study, we considered that altitude was the main effect on the seed number per fruit, which increased when the altitude of population elevated.
It is known that change of day length is related to latitude, and yearly temperature averages will decrease 0.8˚C as latitude increase 1˚ from south to north in the Northern Hemisphere, in addition, temperature would decrease when altitude elevates (i.e.0.6˚C/100 m).The topography of Yunnan shows that the northwest is higher and the southeast lower, so from the east to the west the latitude is rising and the altitude also elevating continually.Therefore, the significant influence of both latitude and altitude on flower number per inflorescence likely reflected that lower temperature and higher temperature difference between day and night would be beneficial to increase the flower number per inflorescence.The fruit and seed production would partly depend on its pollinators.However, cooler temperatures could be a proximate cue for the ultimate factor of low pollinator availability [15].Therefore, low pollinator activity in habitat where latitude and/or altitude elevated would lead to less fruit and seed production.The tropic of cancer is the boundary between the north temperate zone and the tropic.The populations of A. tsaoko growing in south of the tropic of cancer had significantly less flower number per inflorescence, and significantly higher fruit and seed production than the population in north of the tropic of cancer.Such significant differences probably demonstrated that the habitats in south of the tropic of cancer were favorable to the reproduction of A. tsaoko, which reproductive costs were lower and harvest was higher.
These results would provide beneficial information for further study on the plant about the origin of cultivation, and for selection of cultivated site.

Flowering Strategy and Female Fitnesss
Floral longevity, the length of time a flower remains open and functional, was determined by a balance between female fitness accrual rates (pollen receipt), male fitness accrual rates (pollen dissemination), and the cost of flower maintenance [16][17][18].In plant reproductive ecology floral longevity played a central role, it determined plant reproductive assurance and overall fitness [19], because it affected the number of pollinator visits, the amount and quality of pollen received and disseminated, as well as floral display size [20,21].Floral longevity could ensure successful pollination in habitats where pollinators were few, unpredictable [17,18,20,22].For example, long floral duration could significantly increase successful pollination and fruit set in Kalmia latifolia [15].In addition, previous studies demonstrated that plants growing in alpine habitats, which typically had sparse or uncertain pollinators, had longer floral durations than plants at lower elevations with more abundant, predictable pollinators [23][24][25][26][27].
Floral longevity played a central role in the distribution of zingiberaceae from tropics to alpine habitats, in the course the duration of floral life had a tendency to increase for effective enhancement of both female and male fitness.Therefore, based on increasing of floral longevity, ginger was divorced from tropical habitat and distributed toward high elevation habitat [28].For A. tsaoko, as a flexistylous ginger, the longevity of a single flower was only 1 d, which opened at dawn and withered at dusk.Flexistyly is a unique and "active" floral mechanism to decrease inbreeding and promote outcrossing by changing position of style [29][30][31][32][33][34][35][36][37][38][39].Therefore, extension of floral longevity for a single A. tsaoko flower would not increase outcrossing.
Our study in Baoshan population suggested that floral longevity of A. tsaoko was only 1 d, and the flowers in inflorescence opened in proper order every day, i.e. 1 -6 flowers per 1 d for the most inflorescences and mean 3 -4 flowers per 1 d [40].Cui et al. suggested that floral longevity of A. tsaoko in Yuanjiang population was only 1 d, and 3 -5 flowers from an inflorescence opened every day [8].Based on this, the inflorescence with more flowers had longer anthesis, meaning that the floral longevity was prolonged at the inflorescence level.In addition, an inflorescence was more attractive to pollinator than a single flower [12,13].Therefore, increasing flower number per inflorescence may be a strategy to having greater attraction to pollinators for both female and male reproductive success.The strategy would promote the plant to distribute into those habitats with higher elevation, where its pollinator was unpredictable or sparse.
By the ANOVA analysis, both plant-intrinsic and environmental factors influenced significantly the flower number per inflorescence, fruit and seed production (P = 0.000 < 0.01).For the seed number per fruit, effect of the former was stronger than the latter.On the contrary, both flower number and fructification percentage per inflorescence were influenced by environmental factors more than plant-intrinsic factors.Therefore, the seed number per fruit possibly reflected that the certain plant-intrinsic trait, which might demonstrate that the plant individuals differed in reproductive resource status conditions.Such as different rate of nectar secretion among the plant individuals, and those flowers with higher rewards might receive more visits from pollinators (unpublished data), and possibly more seed number per fruit.A further study on the seed production will provide further insight into the fitness and the ecological adaptation of the plant.

Table 1 .
Information of the seven populations of A. tsaoko.

Table 2 .
Descriptives of the inflorescences from the seven populations.

Table 3 .
Tests of between-subjects effects on flower number and fructification percentage per inflorescence.

Table 4 .
Descriptives of seed number per fruit from the seven populations.

Table 5 .
Multiple comparisons of seed number per fruit among the seven populations (S-N-K, α = 0.05).

Table 6 .
Coefficients(a)for flower number per inflorescence.
a Dependent Variable: flower number per inflorescence.
a Dependent Variable: fructification percentage per inflorescence.

Table 8 .
Coefficients(a)for seed number per fruit.
a Dependent Variable: seed number per fruit.