Lindl. (Phalaenopsis pulcherrima
(Lindl.) J.J. Sm., voucher specimens, Hong Kong Kadoorie Program Team 5355, deposited in PE) is widespread in tropical Asia, including India, Myanmar, Vietnam, Laos, Thailand, Malaysia and China [20
]. The species mainly forms clones on marble outcrops at the borders between rocks and soil, among the bushes or under sparse forests at elevations of 200 to 500 m in the lowland tropical monsoon rainforests of the Hainan Island, China. Each stem produces 1–2 young shoots every year, forming dense clones with many shoots; the flowering period occurs from July to October. The flower colors vary from white and pale pink to darker pink, red and purple (Figure ). The lip attaches to the end of the column foot and is clawed and tri-lobed, containing pairs of pollen-like yellow calli on the claw and yellow appendages with blots in front of them (Figure ). The lateral lobes are erect, while the middle lobes are deflexed. Four yellowish pollinia are situated on a long and linear stipe. The pollinaria of Doritis pulcherrima
are long. Our previous field investigation indicated that they do not undergo reconfiguration when they are removed from the columns. No nectar has been reported in literatures to date [20
The variation of flower color and pseudopollen inDoritis pulcherrima.
This study was conducted using three populations from the southern and southwestern region of Hainan, China: the Yang-lin population (S1, elevation 386 m, 18°30’ N, 109°13’ E, approximately 2000 clones); the Wang-xia population (S2, elevation 469 m, 19°01’ N, 109°07’ E, approximately 320 clones); and the Ma-an-shi population (S3, elevation 200 m, 18°44’ N, 108°59’ E, approximately 140 clones). The climates of these three localities are tropical and strongly seasonal (for S1, the mean annual temperature is 25.8°C and the mean annual precipitation is 1392 mm and concentrated between June and October).
Pollinator observations were conducted in these three populations to reveal the pollinator specificities. The sizes of each of the D. pulcherrima clones were evaluated for S2, whereas the breeding system experiments were conducted using S1.
The behaviors of the flower visitors were observed during the day and night for the three populations between June and September of 2004 and July and August of 2005. The diurnal observation time was from 6:00 am to 20:00 pm (186 h in total), and the remaining observations were nocturnal (61 h in total). We recorded the behavior of the pollinators on D. pulcherrima, including the visitation behaviors, number of inflorescences and flowers visited per cluster, numbers of flowers visited per inflorescence, visitation period on each flower and inflorescence and numbers of pollinaria on each pollinator, were recorded. The visitors were collected from D. pulcherrima inflorescences and other co-blooming plants using nets and were killed with ether using a killing-bottle. The insects were identified by entomologists of the Entomology Department of Southwestern Forest University, Kunming, Yunnan, China. The insect voucher specimens (Jin X.H. P4, P7, P9, P15, and P20) were deposited in the Herbarium (PE) at the Institute of Botany, Chinese Academy of Sciences.
Measurements of the functional characteristics of flowers and pollinators
Ten full-blooming flowers were randomly selected from the Yang-lin population in 2010, and their functional characteristics were measured. The entrance and floral passage that were formed by the two lateral lobes of the lips (sides), columns (top) and lip claws (bottom) were considered to be the key functional characteristics of D. pulcherrima that would aid in the understanding of the morphological interactions between the plant and its pollinators. Four functional characteristics that were related to pollinia transfer and receipt were measured: the height (distance between the rostellum and the surface of the middle lobe), width (distance between the two lateral lobes of the lip at the entrance of the passage), depth (distance between the entrance and lip claw), and the distance between the stigma and surface of the lip. Six characteristics closely related to pollen transfer were measured: the width and height of the head, the height and width of the thorax, the length of the proboscis, and the total height of the pollinarium and position of attachment of the pollinarium to the pollinator. All of the above variables were measured to an accuracy of 0.1 mm using dial calipers (Zhengjiang Qiaosheng, HU 02270108).
To test whether the pollinaria of D. pulcherrima show PR, time-lapse photography (Nikon D700 digital camera on a tripod) was used in the natural habitat of Doritis pulcherrima (the S2 population ) during September of 2011. For ACR, ten pollinaria were carefully removed with toothpicks, and then photographs were taken every 5 seconds for 10 minutes of the anther caps that contained pollinaria. To observe PB and PS, photographs were taken every 5–10 seconds for 20 minutes of ten pollinaria that were positioned with toothpicks and had their anther caps removed with forceps.
Due to the densely branched, growing habitat of D. pulcherrima, we decided to use each clone as a minimum unit in our phenology census for the sake not to disturb the clones during the investigation. For the phenology census and natural fruit set assessments, 266 flowers from 21 clones in 2004 and 638 flowers from 44 clones in 2005 were randomly selected and marked as S1. Because the flowering season of D. pulcherrima extends into the monsoon season of Asia, only the following variables were recorded: (a) the numbers of flowers on each inflorescence and clone, which were checked once at the beginning of the investigation and counted as fertile bracts or flowers buds; (b) the numbers of open flowers on each inflorescence and clone, which were measured twice a week; and (c) the numbers of fruits (immature fruit) on each clone, which were obtained at the end of October in 2004 and August in 2005.
The floral rewards of Doritis pulcherrima were examined using S2 population in 2011. To measure the nectar concentrations and volumes, we randomly selected ten flowers and bagged them with nylon net bags before they opened. After anthesis, the flowers were removed to measure the nectar secretions. The presence/absence of nectar was examined visually and using an optical microscope and the 5 μL SIGMA micro-cap calibrated capillary tubes (Sigma-Aldrich, St. Louis, MI, USA). The volumes were determined by measuring the lengths of the filled tubes and converting the length measurements to microliters. To examine whether the pseudopollen on the lips was collected by pollinators during their visits, twenty S1 flowers whose pollinia had been removed by pollinators were examined using an optical microscope in 2005.
The presence/absence of nectar of the five co-blooming species, including two species of Lasianthus (Rubiaceae), one species of Leptodermis (Rubiaceae), one species Polygala (Polygalaceae), and one species of Helicteres (Sterculiaceae), visited by the pollinators were examined using an optical microscope in 2011.
The breeding system of Doritis pulcherrima was evaluated by controlled bagging in 2005 for S1. The treatments included artificial self-pollination (pollinia from the same clone), cross-pollination, and unmanipulated flowers (control). Pollinaria for the cross-pollination were obtained from plants that were situated over 20 m away. The numbers of clones, inflorescences and flowers that were used in each treatment are summarized in Table .
Percentage of fruiting success per treatment and natural fruit set in S1
All of the analyses were conducted using SPSS 16.0 for Windows. The data were analyzed using descriptive statistics. The variations in the natural fruit sets over the 2 years were analyzed using a one-way analysis of variance (ANOVA).