Thermogenic flowering of taro (Colocasia esculenta, Araceae)

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1 1557 Thermogenic flowering of taro (Colocasia esculenta, Araceae) Anton Ivancic, Vincent Lebot, Olivier Roupsard, José Quero Garcia, and Tom Okpul Abstract: Thermogenesis and its association with taro (Colocasia esculenta (L.) Schott) flowering was studied during the warmest period of the year (December 2002 February 2003) within a large collection of heterogeneous plant material on Espiritu Santo, Vanuatu. On each studied inflorescence, temperatures of the three main parts of the spadix and the ambient air were recorded during a period of 38 h. The investigation indicates that significant thermogenic activity of taro inflorescences takes place during two successive nights: (1) during the night when an inflorescence becomes odorous (the female phase) and (2) a night later, when microsporogenesis approaches its final phase (the male phase). The highest average difference between mean temperatures of the ambient air and inflorescences were documented during the female phase, at 0500 hours (the mean temperature of the sterile appendix was 29.1 ± 0.9 C (P = 0.05) and this was 6.8 C above the temperature of the ambient air). Thermogenic activity is synchronized with the protogynous nature of the species and insect pollination in the early morning hours. Its main putative functions are (1) to reduce the deviations of ambient air temperatures during the most critical periods of flowering, and (2) to promote crosspollination. It stops h after pollen has been released. Key words: taro, Colocasia esculenta, thermogenesis, inflorescence development, pollination. Résumé : La thermogénèse de la floraison du taro (Colocasia esculenta (L.) Schott) a été étudiée pendant la période la plus chaude de l anné (Décembre 2002 à Février 2003), au sein d une collection importante de germoplasmes présentant une forte diversité, sur l île de Espiritu Santo, au Vanuatu. Sur chacune des inflorescences étudiées, les températures des trois principales zones du spadice et de l air ambient ont été enregistrées pendant une période de 38 h. Ce travail indique qu il existe une thermogénèse significative des inflorescences de taro, et qu elle a lieu durant deux nuits successives : (1) durant la nuit où l inflorescence devient odorante (phase femelle); et (2) une nuit plus tard, lorsque la microsporogénèse approche sa phase finale (phase mâle). Les plus fortes différences entre les températures moyennes et la température de l air ont été enregistrées durant la phase femelle, à 0500 (la températue moyenne de l appendice stérile est alors de 29,1 C ± 0,9 C (P = 0,05) soit 6,8 C de plus que la température de l air ambiant). L activité de la thermogénèse est synchronisée avec la protogynie de l espèce et la pollinisation entomophile durant les premières heures du jour. Ses fonctions supposées sont : (1) de réduire les écarts de température de l air ambient durant les périodes critiques de la pollinisation; et (2) de promouvoir les pollinisations croisées. Cette activité cesse entre 1het1,5h après l émission de pollen. Mots clés : taro, Colocasia esculenta, thermogenèse, inflorescence développement, pollinisation. Ivancic et al Introduction Thermogenesis has been documented in several families of dicots (Annonaceae, Aristolochiaceae, Illiciaceae, Magnoliaceae, Nelumbonaceae, Nymphaeaceae, Rafflesiaceae) and monocots (Araceae, Arecaceae, Cyclanthaceae) (Knutson 1974; Meeuse 1975; Seymour and Schultze-Motel 1996, 1997; Patino et al. 2000; Thien et al. 2000; Lamprecht et al. 2002a, 2002b; Seymour et al. 2003). Among the Araceae, thermogenesis appears to be common. It has been studied in Received 26 February Published on the NRC Research Press Web site at on 16 November A. Ivancic. 1 Faculty of Agriculture, University of Maribor, Vrbanska 30, 2000 Maribor, Slovenia. V. Lebot. Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), P.O. Box 946, Port Vila, Vanuatu. O. Roupsard. Centre de coopération internationale en recherche agronomique pour le développement (CIRAD) Vanuatu Agricultural Research and Training Center (VARTC), P.O. Box 232, Espiritu Santo, Vanuatu. J. Quero Garcia. Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), Montpellier CEDEX 01, France. T. Okpul. 2 University of Vudal, PMBS, Rabaul, East New Britain, Papua, New Guinea. 1 Corresponding author ( anton.ivancic@uni-mb.si). 2 Present address: Sir Alkan Research Center, National Agricultural Research Institute (NARI), P.O. Box 1639, Lae, MP411, Papua New Guinea. Can. J. Bot. 82: (2004) doi: /B04-118

2 1558 Can. J. Bot. Vol. 82, 2004 Fig. 1. Development of a taro (Colocasia esculenta) inflorescence and fruit head over an 18-d period (the sketching took place at the beginning of 2003, in the morning hours between 0730 and 0800, at the experimental plot of the Vanuatu Agricultural Research and Training Center near Luganville, on the Island of Espiritu Santo, Vanuatu): 1, a 5-d-old inflorescence coming out of the membranous flag leaf (8 d before becoming odorous); 2, 7 d old; 3, 9 d old; 4, 11 d old; 5, 12 d old (1 d before becoming fragrant); 6, 13 d old (the release of odor; the spathe is loose, partly open, and odorous, expressing its final color, the stage when pollinating insects enter the floral chamber); 7, 14 d old (the release of pollen, the upper part of the spathe is fully open, whereas its constricted part is tight, preventing self-pollination); 8, 16 d old (3 d after becoming odorous); 9, 18 d old. several genera such as Alocasia, Amorphophallus, Anubias, Arum, Cercestis, Dieffenbachia, Dracunculus, Helicodiceros, Homalomena, Philodendron, Symplocarpus, and Sauromatum (Nagy et al. 1972; Meeuse 1975; Skubatz et al. 1991; Yafuso 1993; Bay 1995; Seymour 1999; Seymour and Blaylock 1999; Seymour and Schultze-Motel 1999; Barabé and Gibernau 2000; Gibernau and Barabé 2000; Seymour et al. 2003). Thermogenesis within the genus Colocasia has been known for about a century and was mentioned for the first time by Leick (1915). Taro (Colocasia esculenta (L.) Schott), which is one of the most important aroid root crops, has many typical aroid characteristics, one of them being thermogenic activity of its inflorescences. Many taro botanists and breeders are familiar with it, although it has not yet been systematically analyzed. There are several reasons for this: flowering is relatively rare, inflorescences (spadices) are small (especially the sterile appendix), and thermogenic activity is little expressed. Taro is an allogamous, monoecious, and protogynous species. Each floral unit includes an inflorescence placed on a floral stalk or peduncle (Figs. 1 and 2). Compared with other aroid species (e.g., Alocasia macrorrhizos (L.) G. Don, Amorphophallus campanulatus Bl. ex Decaisne), taro inflorescences are small, having a relatively thick spathe. They appear in groups or clusters, usually from two to five, depending on the genetic structure, plant age, and the environment; the latter is probably the most important. The highest number of inflorescences per cluster recorded is five (Young 1924; Ivancic and Lebot 2000). Each inflorescence consists of a spadix covered by a spathe. The spadix is divided into a female part (lower part), a sterile region, a male part, and a sterile tip (appendix). The female portion is generally shorter than the male portion. Female (pistillate) flowers are sessile and green, with welldeveloped stigmas and are usually mixed with pistoids (sterile female flowers), which can be distinguished by their light color. The male part of the spadix consists of sessile staminate (male) flowers. According to Backer and Bakhuizen van den Brink Jr. (1968) and Shaw (1975), male flowers consist of two to six sessile anther-cells, which are connate into more or less obpyramidal synandria. The apex of the synandria is mostly flat and hexagonal. Thecae are dehiscent by the terminal pore. The pollen grains are spherical and trinucleate.

3 Ivancic et al Fig. 2. A taro (Colocasia esculenta) inflorescence: 1, the structure of a spadix. a, peduncle; b, female portion with pistillate flowers; c, constricted sterile region between female and male portions; d, male portion with staminate flowers; e, sterile appendix; 2, transsection of an inflorescence in the morning when it became odorous; 3, the same inflorescence 24 h later. a, the length of the lower part of the spathe before elongation; b, the difference of the length after elongation. The spathe consists of two parts. The lower part is usually green or red, and forms the floral chamber in which female flowers are located. The upper part is predominantly yellow but sometimes can be purple, red, or blotched (associated with the pigmentation of the whole plant). The shape and size of the spathe are highly variable, depending on genotype, plant size, and environmental conditions (Ivancic 1995). During anthesis, the lower part of the spathe remains more or less closed and thus protecting the female portion, whereas the upper part opens and curves towards the opposite side of the opening and consequently exposing the upper part of the spadix (the male portion and the sterile appendix). In the tropical Pacific (e.g., in Papua New Guinea, Solomon Islands, Vanuatu), the most intense flowering takes place at the beginning and at the end of the rainy season. The beginning of taro flowering is usually associated with the emission of strong odor (mainly from the spathe). Its main purpose is to attract insect pollinators. The most important of these are small flies belonging to the Drosophilidae family (Carson and Okada 1980; Matthews 1990). Flies from this family, although different species, can also be found in other closely related aroids such as Alocasia odora (G. Lodd.) Spach (Yafuso 1993; Miyake and Yafuso 2003). An odorous inflorescence is erect, and its spathe is loose (Fig. 1, stage 6), enabling small pollinators (coming from a day older, pollinating inflorescences) to enter the floral chamber and distribute pollen on stigmas of the female flowers. Most of these insects will remain inside the inflorescence until the next morning, when the spathe will be fully open and pollen will be released. Insects are the most important agents of cross-pollination. Wind pollination can be significant only for openly flowering genotypes (with a fully exposed male portion) grown in dense populations. Rain mainly causes self-fertilization by washing pollen grains from the male part of the spadix to the pistillate region. Self-fertilization is possible, because the incompatibility system at the end of flowering becomes less efficient, and because there is an overlap between the period of stigma receptivity and the release of pollen. Stigmas (in humid conditions of Morobe Province in Papua New Guinea) can remain receptive for 10 days (Okpul and Ivancic 1995). Taro is a very sensitive species characterized by rare and erratic flowering (Taro Network for Southeast Asia and Oceania (TANSAO) 2002). Many of the genotypes never flower. For normal flowering and seed set, taro requires an optimal environment. In the situations of heavy rains or continuous rainy weather, or large deviations of ambient air temperatures, plants do not flower or produce sterile flowers (Ivancic and Lebot 2000). The hybridization technique is not complicated, and this is mainly because of monoecy and protogyny. The inflorescence has to be emasculated before the spathe becomes attractive for insects (in most cases this is 1 d before it becomes odorous). The emasculation procedure is very simple. With a small knife, one has to cut the inflorescence (the spadix and the spathe) in the constricted region (in the sterile zone between the male and female portions of the spadix). The female portion remains protected with the lower part of the spathe. Pollination is usually carried out immediately after emasculation. At first, the lower part of the spathe is carefully removed, then pollen is distributed on stigmas, and finally the pollinated female portion has to be protected by cotton or simply by the lower part of the spathe. One of the main problems associated with hybridization is the exactness of crosses, especially in areas with abundant insect pollinators. If taro generates temperature during anthesis, then thermogenesis should be considered as a factor that may influence hybridization results. From this point of view the data concerning thermogenic activity can be very useful for taro breeders. The paper represents the first systematic study of thermogenesis and its association with flowering of the true taro (Colocasia esculenta). The main objectives are to analyze: (1) the biology of inflorescences and its association with the prevention of self-pollination; (2) the dynamics of thermogenic activity; (3) its role in promoting cross-fertilization; and (4) its relationship with other processes associated with anthesis. The paper will also discuss the possible implications of the results on the crossing technique. Materials and methods The investigation took place from 5 December 2002 to 4 February 2003, at the Vanuatu Agricultural Research and Training Center (VARTC) near Luganville, on the Island of Espiritu Santo, Vanuatu ( S, E). The experimental plot was located on a deep and fertile soil, covering a

4 1560 Can. J. Bot. Vol. 82, 2004 coral limestone plateau, located 3 km from seashore at 80 m altitude. It was the end of the dry and the beginning of the wet season, which was characterized by relatively high temperatures and very intensive flowering. The plant materials used in this investigation consisted of hybrids between wild and cultivated germplasm, and flowering accessions from the national germplasm collection. These materials were used as parental components for hybridization within the existing recurrent selection programme. The main reason for using hybrids was their regular and natural flowering, relatively large inflorescences, and well-developed sterile appendices. Without hybrids, the sample would be too small, and flowering would have to be induced artificially (by treating plants with the gibberellic acid GA 3 ). The artificial induction of flowering is probably not the best solution for the investigation of the thermogenic activity. Treatments with gibberellic acid can cause serious deformations of inflorescences (Ivancic 1995). The study of the role of the spathe in the reproduction system (especially in preventing self-pollination) was based on the analysis of its growth and related changes. It included both parts of the spathe: the lower part (which enfolded the pistillate portion of the spadix and forms the floral chamber) and the upper part (which enfolded the male portion and the sterile appendix). Based on preliminary studies, we chose (for studying the developmental changes of spathes) the period of 39.5 h from 1800 (approximately 6 h before releasing odor) to 0930 (approximately 3 h after the first pollen had been released). The spathes were marked with two lines (the first at the bottom and the second in the middle of the constricted area), and distances (between the first and the second line, and between the second line and the tip) were recorded every 12 h. The investigation of thermogenesis involved 145 flowering plants, while the total number of studied inflorescences was 240 (small and deformed inflorescences were excluded). It was concentrated on two developmental periods: the female phase (which took place during the night when odor was released) and the male phase (which took place a night later and ended after the release of pollen). On each studied inflorescence, temperatures were recorded during the period of 38 h; starting at 2100 (in the night when an inflorescence became odorous) and ending at 1100 (approximately 3 4 h after pollen had been released). The determination of this period was based on a series of preliminary recordings, which took place in January 2002 and included the whole developmental period of taro inflorescences, lasting d. Temperatures were measured every 10 min, and later averages were computed for every 30 min. Before measuring, the spathe was gently loosened and partly unfolded, to determine the measuring points of the spadix. These points were marked on the outer surface of the spathe. Then the thin sensor of the thermometer (its end was slightly curved) was carefully inserted inside. Damaged inflorescences as well as those with very small sterile appendices were abandoned. The following temperatures were recorded: ambient air (under the shade of a fresh leaf, at the height of the base of the studied inflorescence) and spadix. The spadix temperatures represented the compromise between the temperature of the surface and the spongy tissue in the center and were measured in the outer zone of the spongy tissue of the female and male portion (the measuring points were at one half of their lengths) and the sterile appendix (the measuring point was at one third of its length, where normally developed appendices were on average the thickest). The temperatures were measured by the electronic thermometer Ebro TFN 1093 SK (sensor NiCr-Ni (thermocouple K), 175 mm 0.9 mm), having the resolution of 0.1 C and accuracy of ±0.4 C ± 1 digit ( C). Climatic parameters were measured continuously at the height of 22 m on a tower located 750 m away from the measurement site. They were monitored on Campbell Scientific 10 data logger every 30 s and averaged semi-hourly. Global radiation (Rg), silicon cell pyranometer SKS1110 (Skye Inst. Ltd., Powys, UK); air temperature and relative humidity, MP100a (Campbell Scientific, Shepshed, Leicestershire, UK); rainfall, ARG100 (Campbell Scientific). Results Climatic parameters The climate is tropical oceanic, averaging 2745 mm (±SD 710 mm) rainfall per year ( ). Rainfall peaks during the hot and rainy season (December-April), with an average of 335 mm month 1. A drier season usually occurs from July to September, with an average of 117 mm month 1.Average daily global radiation, maximum temperature, air humidity, maximum vapor pressure deficit, and daily potential evapotranspiration (Priestley Taylor) are, respectively, 20.0 MJ m 2 day 1, 30.3 C, 89.8%, 10.8 hpa, and 5.4 mm during the rainy season, and 14.5 MJ m 2 day 1, 27.6 C, 86.1%, 8.1 hpa, and 3.5 mm during the dry season. The period December 2002 March 2003 represented the warmest period of the year. The average air temperatures (at VARTC) were the following: 25.3 C in December, 25.6 C in January, and 26.0 C in February and March. The lowest temperatures (in most instances, they were recorded between 0300 and 0500) were as follows: 20.5 C in December, 20.4 C in January, and 21.7 C in February. December was relatively very dry (51.7 mm rain). January and February had some more rain (190 and 237 mm, respectively) but still not enough. To ensure an optimal environment, it was necessary to irrigate twice per week (using L of river water ha 1 ). Biology of inflorescences and prevention of selffertilization The developmental period of inflorescences, recorded from the moment when the upper part of the spathe came out of the membranous flag leaf, to the moment when pollen was released (Fig. 1, stages 1 7), varied from 14 to 17 d. Anthesis lasted 2 d, and its duration was very uniform. It was associated with several visible changes in an inflorescence. The approaching of the female phase was characterized by the elongation of the peduncle, spathe, and spadix (Fig. 1), and the change of color of the upper part of the spathe. The female phase began when the upper part of the spathe attained its final color (i.e., yellow, purple, red, or blotched) and started to unfold. This happened late in the afternoon, approximately h before the release of odor. The lower part remained green or nearly green. The first odor appeared between midnight and Its intensity was

5 Ivancic et al weak and gradually increased until (depending on genotype and weather conditions). The odorous spathes were relaxed, enabling small dipterous insects to enter the floral chamber. The mean length of spathes (measured on 4 February 2003) was ± 2.1 cm (P = 0.05). The spathes continued to elongate until the next morning (when pollen was ready to be released). The elongation of the spathe was reduced and stopped after pollen had been released. The average elongation determined for the period from 0830 to 1830 (on 4 February 2003), was 1.33 ± 0.1 cm, and this was mainly due to the growth of the lower part of the spathe. This part continued to grow during the following night, and its average elongation from 1830 to 0530 was 0.48 ± 0.1 cm. During these night hours, the spadix stopped elongating, and consequently the constricted part of the spathe moved upward toward the thick bottom of the male portion and closed the space between the male and the female portions (Fig. 2, illustrations 2 and 3). At the same time the lower part of the spathe (including the constriction) tightened. In this way, the female portion was completely isolated, and self-pollination was prevented. The upper part of the spathe, however, continued to loosen and unfold, and reached its maximum opening later in the morning, between 0745 and 0930, depending on weather conditions. The first pollen was observed at 0625 (on 5 February 2003), however, most of the inflorescences started releasing pollen 0.5 h later. On cool and wet days, the first pollen appeared after On rainy days, most inflorescences did not release pollen. There were obvious differences regarding the space available for the movement of insect pollinators inside odorous inflorescences, during the night and early morning hours. The spathe of some extreme genotypes loosened so much that even smaller honey bees could enter and move around the spadix. The other extremes were inflorescences that remained more or less completely closed with their spathes slightly opened after pollen had been released. There were also a few situations where the constricted part of the spathe did not close the space between the female and male portions (allowing self-pollination). Differences associated with the behavior of the dipterous pollinating insects were less obvious; however, it was possible to see some of them moving among inflorescences during the entire day and most of the night. Thermogenic cycles Significant thermogenic activity took place from midnight to 0930, during two successive nights: (i) the night when the inflorescence became fragrant (the female phase), and (ii) the night when microsporogenesis approached its final phase (the male phase). The female phase ended approximately 12 h after the first appearance of odor. After the end of this phase, there was and interval (8 10 h long) without thermogenic activity, which was followed by the male phase (Fig. 3). The female phase During this phase (Fig. 3), the generation of temperatures started before midnight, although temperature differences between the spadix tissues and the ambient air were relatively small. At 2200, the temperatures of these tissues were approximately 2 C higher than the temperature of the ambient air (the mean temperature of the ambient air was 24.2 ± 0.3 C). The mean temperature of the male portion was 26.2 ± 0.7 C. The second highest was the mean temperature of the sterile appendix (26.1 ± 0.9 C). The mean temperature of the female portion was 26.1 ± 1.1 C. The spadix tissues were warmer mainly because they were cooling slower than the ambient air. The thermogenic activity increased rapidly after 0330, and the most active was the sterile appendix, reaching its maximum at 0500 with the mean temperature 29.1 ± 0.9 C (6.8 C above the temperature of the ambient air). At the same time the mean temperature of the male portion was 28.8 ± 1.0 C (6.4 C above the temperature of the ambient air), whereas that of the female portion was 24.9 ± 0.6 C (only 2.6 C above the temperature of the ambient air). The female portion generated maximum temperatures between 0700 and 0900, reaching its peak at 0800, with the average temperature 32.2 ± 0.6 C (4.8 C above the temperature of the ambient air). After 0800, the thermogenic activity ceased, and the temperatures of the thermogenic tissues slowly approached the temperatures of the ambient air. During the warmest period of the day, no thermogenic activity was recorded. The female portion remained warmer than the male portion and the sterile appendix (Fig. 3). One of the reasons could be darker pigmentation of the lower part of the spathe (which absorbed more solar radiation). Another reason could be evaporation of odorous substances from the upper part of the spathe, which may act as a cooling system for the male portion and the sterile appendix. During the female phase, the generation of temperature did not appear to depend much on the weather conditions (dry weather, rain, wind), because temperature-generating organs were still well protected by the spathe. The male phase During this phase (which started when microsporogenesis approached its final stage and ended after the release of pollen), only the male portion of the spadix was thermogenically active. Its temperatures started to rise late in the evening, between 2000 and 2200 (Fig. 3), depending on genotype and weather conditions. At 2200, the mean temperature of the male portion was 27.0 ± 0.4 C (the mean temperature of the ambient air was 24.3 ± 0.4 C). It was gradually increasing until 0730 (the time when most of the pollen was released), reaching 30.6 ± 0.7 C (3.38 C above the temperature of the ambient air). The highest difference, however, was estimated much earlier, at 0400 (7.45 C above the temperature of the ambient air). This difference decreased rapidly once the spathe was more or less fully open (after 0730). The highest value (36.1 C) was recorded on January 28, at 0724 (when the ambient air temperature was 25.8 C). The thermogenic activity ended between 0830 and 0930 (1 1.5 h after pollen had been released). The mean temperatures of the sterile appendix were not much lower when compared with the male portion (in most situations 1 2 C), and this was probably due to the heat from the male portion. The male portion was also heating the female portion (in most situations, the mean temperature of the female portion was 1 5 C lower).

6 1562 Can. J. Bot. Vol. 82, 2004 Fig. 3. The course of temperature differences between the three main parts of the spadix of taro (Colocasia esculenta) and the ambient air during the period of 38 h, starting at 2100 (during the night when an inflorescence became odorous) and ending at 1100 (approximately 3 4 h after pollen had been released). The data were collected during the warmest period of the year (December 2002 February 2003) on the Island of Espiritu Santo, Vanuatu. During rainy days, the thermogenic activity of the male phase appeared to be less expressed. In many situations, especially after longer periods of heavy rains, the thermogenic activity stopped or was reduced to a very low level (because the spathes were already partly open and tiny spadices were exposed to rain), so that there were no significant differences between the temperatures of the male portion of the spadix and the ambient air. In such situations, most of the inflorescences did not release pollen. Discussion Biology inflorescences and prevention of selffertilization Size, shape, color, and odor of spathes, structure of spadices, and thermogenic activity indicate that taro is a cross-pollinating species adapted to insect pollination. For the evolution of this species, cross-pollination was probably crucial. In the case of self-pollination, combined with vegetative propagation, its evolution would be very slow. The mechanism that regulated cross-pollination appeared to be very efficient. The appearance of intensive odor and the release of pollen (on 24-h-old inflorescences) were perfectly synchronized. The investigation showed that the maximum intensity of odor was reached between 0800 and 0830 ( h after the release of pollen on 24-h-old inflorescences) and then it started to decrease. At 1900, it was already very weak and it was difficult to sense. When the weather was cool and cloudy, it could remain present until the release of pollen in the following morning. The presence of odor (although very weak) on pollinating inflorescences could have an important role in attracting insects that were not specialized for taro. The spathes of the pollinating inflorescences of most of the genotypes were widely open, so that insects could easily depart, carrying pollen on their bodies to the inflorescences that were 1 d younger and intensively odorous. The female flowers of these intensively odorous inflorescences were (because of protogyny) already fully receptive. Protogyny is probably not sufficient for preventing selfpollination, because stigmas in wet conditions may remain receptive for several (up to 10) days (Okpul and Ivancic 1995). The investigation indicated that the key role belonged to the constricted region of the spathe. Until the end of the female phase, the growth of the spathe and the spadix were synchronized, so that the constricted part of the spathe remained in the middle of the thin sterile region (between the female and the male part of the spadix), leaving enough space for the insects carrying pollen from other inflorescences to reach the floral chamber. As the moment of the release of pollen approached, the space between the female and the male portions closed. Another factor preventing self-fertilization is selfincompatibility. Although the incompatibility mechanisms of taro have not yet been systematically studied, practical experience indicates that they may not be as strong as those of some other species such as sweet potato (Ivancic and Lebot 2000). Thermogenic cycles The thermogenic activity of taro inflorescences probably has a long and complex evolution. It consists of two non-

7 Ivancic et al overlapping cycles and represents an important factor that helps to ensure successful fertilization and seed set in a wide range of environments. It might have two main functions: (i) to reduce deviations from optimal ambient air temperatures during the most critical periods of flowering, such as final stages of gametogenesis and early stages of embryo development, and (ii) to promote cross-pollination. The maximum increase of the inflorescence temperature due to thermogenic heating was approximately 7 C above the temperature of the ambient air and, on tropical Espiritu Santo, this was probably sufficient to maintain an optimal environment during the most critical stages of the floral development. The adaptation to a cooler climate would probably require the generation of higher temperatures. The examples of such an adaptation are inflorescences of the dead horse arum (Helicodiceros moscivorus), which can generate temperatures up to 15 C above those of the air (Seymour et al. 2003), and in that way maintain a more or less stable floral temperature at about 24 C. The thermogenic activity, which took place during two successive nights (i.e., during the female and the male phase), appeared to be well synchronized with the period of maximal receptivity of stigmas, the emission of intense odor, the release of pollen, and activities and movements of pollinating insects. Such a synchronization is very common in the Araceae family (Bay 1995; Seymour and Blaylock 1999; Gibernau and Barabé 2002; Miyake and Yafuso 2003). Compared with some thermogenic species such as Nelumbo nucifera Gaertn. (Seymour et al. 1998) and Alocasia odora (Yafuso 1993; Miyake and Yafuso 2003), the duration of thermogenic activity of taro inflorescences was found to be relatively short. However, this period is not shorter than that of Arum italicum Mill. (Barabé et al. 2003), Helicodicerus muscivorus Schott ex K. Koch (Seymour et al. 2003), Montrichardia arborescens L. (Gibernau et al. 2003), and Philodendron species (Gibernau and Barabé 2000). During the female phase, most of the heating was produced by the sterile appendix and the male portion. Their temperature peaks were reached very early in the morning (at 0500). Thermogenesis of these two portions probably interacted; however, it was difficult to demonstrate. The specific structure of taro inflorescences and the narrow space inside the spathe suggested that the male portion was heating the sterile appendix and vice versa. The exceptions were inflorescences with a very small appendix (smaller than 6 mm 2 mm), which probably had no significant influence on the temperature of the much larger male portion (having dimensions mm mm). The temperature course of the female portion differed significantly from that of the male portion and the sterile appendix. The peak was reached relatively late (at 0800), and this suggests that the female portion received at least some (if not all) heating from the upper portions of the spadix, which were thermogenically much more active (such heating was possible, because the constricted region between the female and the male portion was not yet closed). The course of the thermogenic activity of the male phase was synchronized with the final stages of microsporogenesis and the release of pollen. The final stages of the microsporogenesis took place when the air temperatures were relatively low and changeable, and thermogenesis was probably crucial to assure optimal and stable temperatures. Without it, microsporogenesis would probably last longer. Pollination Inflorescences in the female phase attracted pollinators in the early morning hours by odor and color of spathes, and perhaps infrared radiation. Odor was obviously the most important attractant. It has been demonstrated in aroids (Meeuse 1975) that odor is produced as a result of volatilization of various compounds caused by heating. Its intensity was gradually increasing with a current increase of temperature. Such a phenomenon is relatively common among thermogenic plants; e.g., Alocasia odora (Miyake and Yafuso 2003), Amorphophallus paeoniifolius (Dennst.) Nicolson, Sauromatum guttatum (Wall.) Schott, and Victoria cruziana Orb. (Lamprecht et al. 2002a). Infrared radiation was probably less important, because the majority of taro pollinators on Espiritu Santo started moving to odorous inflorescences when darkness was over (in January, it was after 0510) and the air temperature rose above 24 C. The spathe started to loosen late in the afternoon, between 1600 and 2000, depending on genotype and environmental conditions (e.g., weather conditions, presence of shade). The environmental conditions appeared to be much more important. After 0500, the upper part of the spathe was already partly unrolled (although its edges remain overlapped), enabling pollinating insects to enter and reach the male portion and the sterile appendix. Inflorescences with a sufficiently large opening on the lower part of the spathe (to enable insects to reach the pistillate flowers directly) were extremely rare. This characteristic, which is more frequent in Papua New Guinea (the example is the cv. Kowune from the Eastern Highlands), appears to be genetically controlled. In the morning, when odor is released, the lower part of the spathe (which protects the pistillate flowers) opens, whereas the upper part (which protects the staminate flowers and the sterile appendix) slightly unrolls itself. The lower part of the spathe then closes, whereas the upper part opens. Relatively high temperatures inside the upper part of the floral chamber increased the insects activity and movements, so that they reached also the female portion and distributed pollen. It was also possible that the temperature in the upper part of the spadix was too high to be comfortable, and therefore insects moved to a bit cooler female portion. Their movement was slow and most of them were crawling on the rough spadix surface, because the space inside the floral chamber was narrow and the spathe wall was slippery; in many ways similar to the situations described by Bay (1995) in the genus Arum. Most of the insects remained inside the inflorescence, being protected from direct insulation and extreme temperatures during the day and the following night. In most situations they were not trapped inside, because the spathe remained relaxed and (or) continued to unroll. The exceptions were spathes that gradually closed, as the air temperature approached 30 C, and started to reopen late in the afternoon. Among the exceptions were also a few genotypes

8 1564 Can. J. Bot. Vol. 82, 2004 with inflorescences that never opened (most of them were local cultivars with very small inflorescences). During the night, before the release of pollen, the heating was generated only by the male portion. The consequence was the movement of insects to the upper (warmer) part of the spadix. The lower part of the spathe elongated and became tight, and consequently closed the space between the male and female portions (to prevent self-pollination). In other related aroids such as Alocasia odora (Yafuso 1993), the situation appears to be very similar, although the role of the elongation of the lower part of the spathe has not been studied. In some aroids, e.g., Arum italicum (Barabé et al. 2003), the female and male portions separate because of the elongation of the inflorescence axis. After 0400, the upper part of the spathe started to unfold; at first slowly and later (after 0600) faster. When pollen was released (between 0730 and 0800), it was already open, exposing the upper part of the spadix (the male portion and the sterile appendix). The pollinating insects lost their shelter and were exposed to lower atmospheric temperature. Dusted with pollen, they had to search for new shelters, and these were a day younger (odorous) inflorescences. The results of this investigation indicate that thermogenesis is one of the key factors promoting crosspollination. Although its influence on artificial hybridization has not yet been studied, the breeders should take it into consideration when planning and carrying out crosses and self-pollinations. Thermogenically active (and intensively odorous) inflorescences, which are not protected from pollinating insects, should be avoided, because the insects with pollen from other inflorescences may already be inside the floral chamber. The most suitable time for hybridization (emasculation and pollination of inflorescences used as female components) is 2 d (two mornings) before pollen shed (23 30 h before they would have become thermogenically the most active and intensively odorous). This stage, which can be recognized by the change of the color of the upper part of the spathe, is also the most suitable time for the protection of the male components. The hybridization in such an early stage has been practiced by most of the breeders (Ivancic and Lebot 2000), although no systematic data about thermogenesis have been available. In further studies, it would be useful to determine the relationship between the expression of the thermogenic activity and spadix dimensions. It may also be useful to identify various volatile compounds responsible for odor. 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