Reproductive biology of the biofuel plant Jatropha curcas in its center of origin

Study site and biological material

The study was conducted within the living fences of a farm plot at Soconusco, Chiapas, Southern Mexico (14.5036 N, 92.1704 W, 58 m above sea level). In this zone the average annual temperature is 31 °C, the average annual humidity is 80%, and the average of rainfall is 2600 mm (Weather Station: 769043 MMTP of the Water National Commission, Mexico). The soil type was andosol with pH 5.7, 2.5% of organic matter and 0.2% of total nitrogen. Plants were selected based on their appearance (healthy and abundant foliage; approximately a 10% of the plants of the fence had foliar damage) and location (in a sunny area), on a 600 m transect. The living fence was 10 years old and underwent annual pruning. According to people living in the surrounding communities, the studied plants are considered toxic, with occasional cases of children intoxication caused by seed ingestion.

Floral phenology

Flowering and fruiting dynamics were studied in 10 plants every 14 days during one year. We determined the average number of inflorescences per primary branch. In five inflorescences per plant, randomly selected, male and female flowers were counted. To estimate the time of flower anthesis and stigma receptivity, in other 10 plants, an inflorescence in the stage of flower buds was marked and observed daily (30 d) at intervals of every 10 min from 0700–1200 h. To estimate the production of pollen and ovules, inflorescences were collected from other 10 plants with closed flowers, and 20 Female Flowers (FF) and 20 Male Flowers (FM) were randomly selected. Pollen grains were extracted from FM and mounted in glycerinated gelatin on a slide. Pollen was quantified using a stereomicroscope and the average number of pollen grains was estimated per anther and per flower. In FF the number of carpels and the number of ovules per carpel was counted. With these data the pollen to ovule ratio was estimated.

In order to comprehend the reproductive process, five pollination treatments were established: 1) geitonogamy or artificial pollination with pollen from the same inflorescence (GEI), 2) Xenogamy (XEN) or artificial pollination with pollen from another plant, 3) Apomixis (APO), which was performed by removing the male flowers and placing non-toxic white glue (Resistol®, Guadalajara, Mexico) on stigma, 4) Excluding Pollinators (ExP), and 5) Open Pollination (OpP). For each of the treatments 20 inflorescences were employed, one per plant (in total 20 plants for this experiment), which were covered, except in OpP, with tulle mesh bags of 1 mm mesh size. Fertilization was checked 14 days after pollination and the number of mature fruits per treatment was quantified at 55 days.

Insect flower visitors and pollinators

To determine insect pollinators and visitors, observations were made on ten inflorescences from independent plants, from 0800–1700 h at intervals of 10 min. For this, the time of arrival at the flower was taken into account, the time they stayed, the resource used (nectar or pollen), and movement among flowers of the same inflorescence and between inflorescences of the same or another plant.

In addition insects visiting the flowers of J. curcas were collected on another living fence, with similar characteristics, but distanced 500 m from the study site to avoid interference with the previous experiments. For this part of the study, we sampled the area of influence of 100 plants. The insect collection was performed with entomological nets from 0600–1800 h. All insects captured were examined under stereoscope, dissected into head and thorax, and identified using (Ayala, 1999; Michener, 2007) taxonomic keys. Moreover, the pollen adhered to the legs of the insects was identified by microscopy.

The visiting insects were classified as: a) efficient pollinators, b) occasional pollinators, c) accidental pollinators, or d) pillagers. We used the following criteria: 1) number of individuals collected during different times of the day, 2) recurrence and time of visit to the male and female flowers, 3) behavior observed on the flowers, and 4) presence of J. curcas pollen (pure or mixed) on different parts of the body.

Statistical analysis

To understand differences between visitor groups in the frequency and time of visit to male and female flowers, the Chi-square test was applied. The number of fruits, and the quality of these in the different treatments were compared by Analysis of Variance (ANOVA) and Tukey test (α = 0.05).

Flowering and anthesis time

The plants studied had three flowering peaks (80%) in the months of April, May and September. In the period from December to February the plants showed no flowering. Subsequent to that, there were three periods of peak fruiting in the months of May, June and October. There were no fruits in the months from December to March.

It was found that, under the study conditions, J. curcas produces an average of 1.25 inflorescences per branch, and the number of female flowers per inflorescence was 1–11, with an average of 2.2. Meanwhile, the number of male flowers ranged from 35–198 flowers per inflorescence (mean: 106.7). It was found that the proportion of female/male flowers was 1:60. Pollen production by another ranged from 266–647 pollen grains (mean: 475.1) and per flower was 3062–5016 pollen grains (mean: 4224.4). The proportion of pollen grains per ovule was 1408:1.

The male and female flowers begin to open at 0800 h, presenting the maximum aperture of female flowers (64.29%) and male (55.75%) at 0900 h. The stigma was receptive at the time of 1000–1200 h.

Pattern of flower opening

Flowers regularly opened over an average period of 15 days (Fig. 1). The pattern showed that the female flowers are the first to open and this process continues for eight days. Male flowers started opening two days after the female flowers, and lasted up to 13 days, with the highest peaks between days 8 and 10.

Diversity of insect visitors

The variety of insect visitors to the flowers of J. curcas came from 36 species, which were grouped into four orders, 12 families and 16 genera as recognized (Table 1). Hymenoptera were the most diverse (75% of the species) and dominant (72.6% of the relative abundance) group, followed by Diptera (diversity of 19.4%; dominance of 26.3%).

We found three types of pollinators: a) accidental, including the fly Thachinidae sp. 2, Vespidae sp. 1, Vespidae sp. 2 and one species of Cerambicidae; b) occasional, comprising 14 species that included bees, ants, and wasps; and c) efficient, composed nine species, of which most were bees and two species of flies. The remaining insects were considered pillagers or nectar robbers (Table 1). Some of the individuals were carrying pure pollen loads of J. curcas in different parts of the body, while others carried J. curcas pollen mixed with other pollen types [Ageratum aff. houstonianum (Mill.), Acacia aff. cornigera (L.) Willd., Tridax aff. procumbens (L.), Zea mays (L.)]. Pollen from other species represented less than 10% of the total loads (Table 2).

Frequency of visit

The activity of insects visiting the flowers of J. curcas was continuous, starting from 0600 h until shortly after 1800 h, showing a markedly bimodal behavior (Fig. 2). The main activity peaks coincided with increased secretion of female flower nectar during daylight 0700–0900 h. The greatest wealth of insects was recorded at 0900 and 1000 h (S = 8), registering the highest peak at 1400 h (S = 9) (Fig. 2).

We found differences in the frequency and time of insects visiting the female flowers (χ² = 21.78, p < 0.01) and male (χ² = 39.69, p < 0.01). Insects observed in the marked panicles were S. mexicana, T. (Trigona) fulviventris (Guérin), T. (Trigona) fuscipennis (Friese), Agapostemon nasutus (Smith), Augochlora (Augochlora) quiriguensis (Cockerell), Augochlora aurifera (Cockerell), Augochlora (Augochlora) smaragdina (Friese), Halictus (Seledonia) hesperus (Smith), Tachinidae sp. 1, Eristalis aff. (Williston), Camponotus (Mayr) and Vespidae sp. 1. Compared with other groups, the bees visited more flowers (43.2%) and stayed longer in resource foraging (38%). The second most important group was the Diptera with 39.3% (frequency) and 31% (time of visit). Specifically, bees visited a larger number of female flowers (55.6%) than male flowers (46.4%). Both bees and flies spent more time to visiting female flowers, while Vespidae sp. 1 had a preference for the male flowers. The Diptera foraged exclusively nectar, while bees collected nectar and pollen.

Jatropha curcas reproductive system

We found differences in the production and quality of fruits and seeds from the different reproductive systems of J. curcas (p < 0.001). The highest percentage of fruit set was recorded in free pollination treatments (OpP: 86.3 ± 2.2) and in xenogamy (XEN: 84.3 ± 6.3), being statistically equal between them; followed by the treatments with exclusion of pollinators (ExP: 18.1 ± 7.2) and geitonogamy (GEI: 16.2 ± 7.3). In the treatment of apomixis five fruits were formed, of which four were aborted and only one reached maturity (n = 55 female flowers). The only apomictic fruit had a high fresh weight, which contrasted with a low seed fresh weight, due to its thick endocarp.

Regarding the number of fruits that reached maturity, the OpP and XEN treatments were statistically superior to all other treatments (p < 0.001). The type of reproduction also influenced the quality of fruits and seeds, as the longest fruits were recorded in the OpP and XEN treatments (p < 0.001) and heaviest seeds occurred in OpP treatment (Table 3).