Efficiency and Behavior of Visitors on Pollination of Agarwood (Aquilaria crassna Pierre ex Lec.) Flowers: a Comparison of Natural and Plantation Stands

W. Tasen1,2, K. Ogata1, S. Tangmitcharoen3, O. Tadauchi4 and M. Thakeaw5 1Kyushu University, Fukuoka, 2Kasetsart University, Bangkok, 3Royal Forest Department, Bangkok, Thailand 4Kyushu University, Fukuoka, Japan 5Department of National Parks, Wildlife and Plant Conservation Bangkok, Thailand

คลังความรู้ดิจิทัล มหาวิทยาลัยเกษตรศาสตร์ Abstract

The efficiency and behaviour of insect visitors to agarwood (Aquilaria crassna Pierre ex Lec.) was investigated in a natural forest at Khao Yai National Park and a plantation in Trat province, Thailand. This study investigated the diversity and abundance of flower visitors, their behaviour and the effectiveness of the major insect visitors on agarwood flowers. The data was collected during the flowering season in March, April and May from 2006 to 2008. A total of 103 insect species was found, comprising 34 families of 4 orders. Among this figure, (61 species) recorded the highest number of species found on the flowers, followed by Hymenoptera (26 species). Coleoptera and Diptera recorded only eight species each. In the natural forest, higher species diversity and species richness of the insect visitors were observed than in the plantation. The highest frequency of individual visits on the flowers was observed during the times of 20.00 to 22.00 hours, and 10.00 to 12.00 hours. In contrast, the lowest activity on the flowers was found during 16.00 to 18.00 hours.

The efficiency of the major insect visitors was estimated from the mean number of pollen grains deposited on the proboscis of each visiting insect. Parallelia rigidistria (Noctuidae, Lepidoptera) recorded the highest number of pollen grains on the proboscis with 25.57±8.61 (n=7). Endotricha species (Pyralidae, Lepidoptera) were the most common in terms of

Proceedings of the FORTROP II: Tropical Forestry Change in a Changing World, 17-20 November 2008, Kasetsart University, Bangkok, Thailand 134 FORTROP II: Tropical Forestry Change in a Changing World

frequency and abundance in both areas having pollen deposited on the proboscis at a rate of 16.29±5.36 (n=9) grains per individual.

Keywords: pollination, Aquilaria crassna, insect visitors, plantation, natural forest

Introduction

Agarwood (Aquilaria crassna Pierre ex Lec.) is one of the most important commercial, resinous, heartwood species. The raw material of the wood is คลังความรู้ดิจิทัลfamous for มหาวิทยาลัยเกษตรศาสตร์ producing perfume and incense in the Middle East. It is a member of the family Thymelaeaceae, which is native to continental Southeast Asia and found throughout tropical forests in many countries, including Cambodia, Laos, Thailand and Vietnam (Phengklai and Khamsai, 1985; Materials and Methods Nghia, 1998). Study Sites Traditionally, resin is mainly extracted from the genus Aquilaria including A. crassna, generally found in natural forests. It is considered to have a high quality and commands a high price due to its immune response to fungal attack (Nghia, 1998). In recent years, its demand has increased, leading to it being classified as ‘critically endangered’ as defined by the criteria of the IUCN red list and it is listed in Appendix II of the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora (Nghia, 1998). Therefore, without a good management system, A. crassna may become extinct in the near future.

To overcome this problem, the rate of establishment of agarwood plantation has been expanded rapidly in Thailand and neighbouring countries, in order to successfully produce high quality seed. However, the germination rate is one of the major problems because the rate of seed viability is low and has been found to decrease to 25% in the third week (Khangsap and Wachrinrat, 2005). Tissue culture could be an alternative means of coping with the germination rate problem. However, this technique requires high investment and is still in the developmental phase. Therefore, seed from plantations is probably still the best way to increase the quality and quantity of seed to meet farmers’ demands.

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Apart from an increase in the quantity of seed by increasing the planted area, it is also important to understand the factors affecting the pollination process. Some previous studies have focused on the reproduction of the genus Aquilaria (Soechartono et al., 2001) but studies on A. crassna, are still limited. Keywords: Moreover, there has been no report on the richness, abundance and efficiency of the species that act as insect pollinators of agarwood in natural forest and plantation. Introduction This study aimed to investigate and compare the role of pollinators in the canopies of natural forest and plantation during the agarwood flowering season. This work mainly focused on the: 1) diversity and abundance of คลังความรู้ดิจิทัลflower visitors;มหาวิทยาลัยเกษตรศาสตร์ 2) behaviour of major insect visitors; and (3) accessibility of flower visitors and their effectiveness as flower pollinators.

Materials and Methods

Study Sites

The study was conducted in two locations in Thailand: a natural forest at Kao Yai National Park; and a private plantation at Khao Saming district. Kao Yai National Park (101°22´E, 14°26´N), the second largest national park in Thailand, covers 216 000 ha in Nakhon Ratchasima, Saraburi, Prachinburi and Nakhon Nayok provinces. This area holds diverse and significant habitat in tropical rainforest ecosystems, containing hill evergreen forest, dry evergreen forest, dry deciduous forest, mixed deciduous forest, secondary forests and grassland (TDRI, 1995). The mean park elevation is 600 m, with the average annual rainfall being 1152.8 mm and the average annual temperature 26.09 °C. According to the official records, the maximum temperature of 33.85 °C and the minimum of 17.15 °C are observed in April and January, respectively (Sangtabtim, 2008). The park contains a wide variety of the generally threatened and endangered fauna and flora of Thailand. In 2005, the Park was designated an UNESCO World Heritage Site under the name ‘Dong Phaya Yen–Khao Yai Forest Complex’ (United Nations, 1992-2007; DNP, 2005).

The private plantation, in the Khao Saming district of Trat province in the eastern part of Thailand, covered 12.8 ha, with an average annual rainfall of 1753.0 mm and an average temperature of 28.15 °C. The maximum temperature is in May (33.40 °C), while the minimum is in December 136 FORTROP II: Tropical Forestry Change in a Changing World

(22.22 °C). At the time of the study, this plantation contained 15-year-old Nectar Measurement agarwood trees growing on a 2 m x 4 m spacing.

Flower Visitors

At each habitat, flower visitors were observed on the canopies of six trees of agarwood randomly selected based on their accessibility, flowering performance and isolation. Scaffolding was erected to a height of 4 to 8 m around each tree. A number and species of each visitor was collected with air-flight malaise traps. This method was chosen as this trap was considered effective at capturing Hymenoptera and Diptera (Grazoul, 1997). The flower คลังความรู้ดิจิทัลvisitors were มหาวิทยาลัยเกษตรศาสตร์ collected every week during the flowering period. In addition, Data Analyses visitor collection was supplemented by the use of a sweep net, which was rapidly swept back and forth through the canopy during a 24 hours period (collected for identification in two-hour periods from 16.00 to 16.00 hours).

A number of flower visitors and their behaviour were recorded for a 20-minute period each hour. The number of individuals and species, in ten inflorescences (n=10), were recorded in the first 10 minutes, while the visitor behaviour including foraging rate, visitation rate and frequency of flower Results and Discussions visits were then recorded in the second 10 minutes. This observation was performed in each of three years (2006-2008) during March to May, as this Flower Visitors was the dry season with abundant flower blooms. Collected specimens from the trapping and sweeping were labelled and recorded according to their time and location. The visitors were further identified to the species level at the forest entomology laboratory, Faculty of Forestry, Kasetsart University, Thailand and at the Institute of Tropical Agriculture, Kyushu University, Japan.

Efficiency Pollination Experiments

The efficiency of visitors was conducted on 10-15 inflorescences of each flowering tree species. Unopened flowers in each inflorescence were covered with a flower bag. The bags were then opened at 18:00, once blooming had commenced. The major insect visitors were collected to examine pollen deposits on their body after visiting the flowers. A proportion of the pollen grains deposited on each visitor specimen was counted using a light dissecting stereo microscope. A higher number of pollen grains represented a higher possibility of pollination and a resultant higher efficiency of fruit setting. Volume 7: Commercial Plantation Forestry 137

Nectar Measurement

Nectar volumes and concentrations were directly measured at the study site Flower Visitors in the evening period each hour (16:00 to 24:00). This measurement regime commenced as soon as the inflorescence started blooming. Nectar volumes were measured by inserting a 2 μl capillary tube down to the base of each flower (n=90 for sugar of nectar concentration, n=54 for nectar volume). Because of the small amount of nectar produced, several flowers were pooled for measuring the fresh nectar concentration using a hand-held refractometer (N.O.W., Tokyo, Japan).

คลังความรู้ดิจิทัลData A nมหาวิทยาลัยเกษตรศาสตร์alyses

The mean and standard error were calculated for all data. Means were compared by an F-test analysis of variance (ANOVA) of flower visitor by species, diversity and richness in the A. crassna flowering. Area variability in period of foraging and the behaviour of visitors were analyzed using Duncan’s new multiple range test.

Results and Discussions

Flower Visitors

The flower visitors recorded in both the diurnal and nocturnal periods (06:00 to 18:00 and 18:00 to 06:00) on the A. crassna flowers, represented 103 species and 34 families belonging to four orders within the Coleoptera, Diptera, Hymenoptera and Lepidoptera (Table 1). The most species visits on A. crassna flowers were by 61 Lepidoptera ( and butterflies) species, followed by 26 species of Hymenoptera and 8 species from the orders Efficiency Pollination Experiments Coleoptera and Diptera. There were 86 species from 33 families in the natural forest, while there were 38 species from 15 families in the plantation, with 21 species from 13 families found in both the native forest and plantation. Soehartono et al. (2001) found only 20 species visited Aquilaria flowers in plantation plots at Bogor, , of which half the species were nocturnal. The species diversity index, species richness and species evenness were analyzed by comparing the alpha (α) diversity or within-area diversity for variation between the areas (Whittaker, 1977). Shannon’s index, H', (Ludwig and Reynolds, 1988), was used as it has been recognized as a popular index of 138 FORTROP II: Tropical Forestry Change in a Changing World

diversity (Magurran, 2004; Trisurat, 2006). The results indicated that the Table 2 species diversity was higher in the forest than the plantation site, with values of 3.61 and 3.24, respectively. The most often found species in the forest was the pyralid (Xanthomelaena schematia), while Endotricha spp. was the most abundant in the plantation.

Species richness was analyzed by Margalef’s index (Magurran, 2004) and evenness by Pielou’s evenness index (Ludwig and Reynolds, 1988). The results showed that the species richness of the natural stand was higher than the plantation, with values of 32.23 and 19.42, respectively. However, the evenness index of the plantation was slightly higher than for the natural คลังความรู้ดิจิทัลstand. These มหาวิทยาลัยเกษตรศาสตร์ results indicated that the distribution of visitors at both places was moderate (Table 2). Behaviour and Foraging Periods Table 1 Number of insect species collected from agarwood flowering canopies of a natural and a plantation stand.

Habitat Number of Number of % of Order Natural/ Natural Plantation family species species plantation Lepidoptera 46 26 11 11 61 59.22 Hymenoptera 25 5 4 10 26 25.24 Diptera 8 5 5 7 8 7.77 Coleoptera 7 2 1 6 8 7.77 Mean±SE 21.5±9.15 9.5±5.54 5.25±2.10 8.5±1.19 25.75±12.49 Total 86 38 21 34 103 100.00

The proportion of each species on the flowers was analyzed by Whittaker’s measure (βw) (Whittaker, 1977) to determine the β diversity or differentiation diversity. The results of the ANOVA confirmed that the species richness of the natural stand was higher than in the plantation (F = 6.267, df= 1, 69; p = 0.015). The similarity index determined by the Morisita-Horn index (Wolda, 1981) was 0.36, which indicated that the similarity index was moderate to less for visitor species between the natural forest and plantation. Chey et al. (1997) found the species diversity was more towards moths in a study that considered plantation and natural forest, while Tangmitcharoen et al. (2006) reported that the diversity and abundance of in teak (Tectona grandis L.f.) canopies in a natural forest were higher than in plantation forests during the flowering season. Volume 7: Commercial Plantation Forestry 139

Table 2 Number of individuals and species diversity index of insect visitors collected from agarwood flowering canopies of natural forest and plantation.

Number of Diversity index Habitat individuals H' Evenness Richness β diversity Natural forest 1039 3.61 0.52 32.23 7.76 Plantation 377 3.24 0.55 19.42 4.26 Mean±SE 708±331.00 3.42±0.18 0.53±0.01 25.83±6.41 6.01±1.75 Total 1416 คลังความรู้ดิจิทัลNote: H'=Shannon’s มหาวิทยาลัยเกษตรศาสตร์ index

Behaviour and Foraging Periods Table 1 There was no significant difference between the foraging rates (one second per flower) at the two different sites, but the foraging period of visitors (number of visitors and anthesis on flowers) showed a highly significant difference (F = 36.996, df = 12; p < 0.01). Duncan’s new multiple range test was used in order to regroup data based on foraging period and individual visitors for the 12 periods of the day. The results indicated the day could be divided into six periods (Figure 1). The periods 20.00 to 22.00 and 10.00 to 12.00 hours were the peak periods for insect visits, while the number of insects was lowest during the period 16.00 to 18.00 hours.

The nectar volume and sugar concentration were measured in the agarwood flowers. The nectar volume and sugar concentration were 0.06±0.02 μl per flower and 12.54±1.22 % Brix, respectively. Both nectar volume and sugar concentration were highest at 19.00 hours (Figure 2). A period when the number of visiting insects was highest occurred after this specified time, with the nectar concentration increasing during the period of lowest visitation.

Moths might be the main group of first pollinators, as they tended to visit the flowers at the early blooming stage. Moreover, Sangtabtim (2008) reported the best period for pollination of agarwood was between 18.00 to 21.00 hours because that was the most receptive period for the stigma. Consequently, this period appeared to be important in achieving a high efficiency or success of fruit setting in this tree species.

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4040 f ff natural forest 35 e plantation 3030 e d 25 d c,d 2020 c,d cc visitors visitors 15 10 b 10 a,b a,b a,b b 5 % of individual of insect of insect % of individual a % of individual of insect a 00 16-18 18-20 20-22 22-24 00-02 02-04 04-06 06-08 08-10 10-12 12-14 14-16 Time of day (h EST) คลังความรู้ดิจิทัล มหาวิทยาลัยเกษตรศาสตร์Time of day (h EST)

Figure 1 Numbers of insect visitors in agarwood canopies during different flowering periods in both natural forest and plantation. Vertical error bars show the standard error; means of each variable with the same letter are not significantly different at p < 0.05 as determined by Duncan’s new multiple range test.

16 0.12 14 0.10 12 0.08 10 8 0.06 Efficiency of Insect Visitors 6 sugar concentration of nectar 0.04 4 an amount of nectar 0.02 2 An amount of nectar 0 0.00 Sugar concentration of nectar (%) 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00 24:00 Time of day

Figure 2 Relationship for sugar concentration of nectar (%) and an amount of nectar (μl) of agarwood flowers with each observation period during 16:00 to 24:00 h.

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During the daytime (06.00 to 18.00 hours), the highest period of diurnal visitors was during the period 10.00 to 12.00 hours, followed by 08.00 to 10.00 hours. The visiting insects during these periods were considered as the second pollinator for group that supplemented pollination by the nocturnal visitors. Research has shown that it required at least 12 hours of flowering for the stigma to become receptive, but it required at least 24 hours before pollination of agarwood flowers occurred (Sangtabtim, 2008). The diurnal visitation was considered the second pollination period because the flowers could use the insects during this period to help increase the fruit set.

The first pollination group or nocturnal visitors, which were mainly moths คลังความรู้ดิจิทัลshowed มหาวิทยาลัยเกษตรศาสตร์ that they were highly efficient in pollinating agarwood during the early period of flowering (night time) at both the forest and plantation sites. Figure 1 They were most often captured before midnight. Soehartono et al. (2001) also found that in Indonesia the moths visited Aquilaria spp. during the night time. Moreover, Makholela and Manning (2006) reported that moths were the first pollinators in Struthiola ciliate (Thymelaeaceae) in southern Africa. The present study indicated that diurnal insect groups were mostly present before noon in both of the natural and plantation stands. Diurnal insects were identified as the second pollinators of agarwood. Apoidea of the order Hymenoptera were more effective pollinators than other groups of diurnal visitors that visited and contacted with the stigma of agarwood trees. As a result, the second pollinator group could support or help pollination of any agarwood flowers that missed the first pollination.

Efficiency of Insect Visitors

The study of efficiency of insect visitors was mainly focused on nocturnal visitors as the priority was on the first pollination group. Efficiency was indicated by the number of pollen grains deposited on body of the visitor after visiting the flowers. For all ten different species of nocturnal flower visitors, most of the pollen grains were found on the proboscis (Figure 3).

Therefore, the results are limited to only reporting on the number of pollen Figure 2 grains on the proboscis. The highest number of pollen grain per individual deposit on the proboscis, 25.57±8.01 (n=7), was found on the noctuid moth (Parallelia rigidistria) belonging to Noctuidae family. This result also agreed with the findings of Makholela and Manning (2006), which indicated that the most pollen grains could be generally observed on the tip of the proboscis. The second and third highest numbers of pollen grains were found 142 FORTROP II: Tropical Forestry Change in a Changing World

on Salma sp. and cancellalis both of which are from the Pyralidae Table 3 family. Their number of pollen grains was 18.17±10.85 (n=6) and 17.67±2.60 (n=3), respectively. However, Endotricha species, which were the most frequent and abundant species in both the natural and plantation areas, had a pollen deposit of only 16.29±5.36 (n=9) grains per individual on the proboscis. In addition, Xanthomelaena schematias, which had the highest number of visits, showed the lowest number of pollen grains at 3.86±1.30 (n=17). The results imply that the possibility of pollination does not depend upon the number of visits. All data are presented in Table 3.

คลังความรู้ดิจิทัล มหาวิทยาลัยเกษตรศาสตร์

a) b)

c)

Conclusions

Figure 3 Pollen grains collected from a moth proboscis shown by arrows; a) proboscis of noctuid moth, scale bar = 0.5 mm., b) proboscis of pyralid moth, scale bar = 0.5 mm. and c) scanning electron micrographs (SEM) of agarwood pollen grain. Scale bar = 5 μm.

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Table 3 Mean number of pollen grains on the proboscis of ten moth visitors during 18:00 to 22:00 observation time and the number of individuals found in natural forest and plantation.

Mean of Number Number of visitors No. Family/Species pollen grains of Natural Plantation on moth proboscis visits forest Noctuidae 1 Parallelia rigidistria 25.57±8.01 7 14 10

Pyralidae

2 Salma sp. 18.17±10.85 6 - 7 คลังความรู้ดิจิทัล3 Sameodes มหาวิทยาลัยเกษตรศาสตร์ cancellalis 17.67±2.60 3 - 24 4 Endotricha spp. 16.29±5.36 9 69 45 Arctiidae 5 Cyana spp. 11.25±1.80 4 21 8 Geometridae 6 Cleora alienaria 10.67±2.19 3 - 7 Arctiidae 7 Ceryx imaon 9.67±4.91 3 - 3 Pyralidae 8 Gadessa nilusalis 8.25±3.87 4 30 15 9 Crypsiptya sp. 5.80±2.24 5 10 15 10 Xanthomelaena schematias 3.86±1.30 17 98 -

Conclusions

The results clearly show that moths are the major insect visitors playing an important role in the pollination of agarwood. The diversity and behavior of insect pollinators depend on several factors. One of the factors is the Figure 3 geographical and climatic conditions. It was clear that the efficiency and behavior of insect visitors was different between the natural forest and the plantation areas. Nevertheless, according to the study, various kinds of insect visitors are still found even in the plantation area. The finding implies that the probability of insect pollination is still high. This means that the pollination chance could be increased leading to an increase in fruit setting of agarwood. Therefore, plantation trees may be an alternative for high quality seed and seedling production. 144 FORTROP II: Tropical Forestry Change in a Changing World

Chemical use and habitat fragmentation are also important factors that have an impact on the distribution and abundance of insect visitors and which may affect changes in their activity. Thus, the greater the conservation of natural forest and plantation area, the higher the expected number of flower visitors. As a result, not only could fecundity be reduced, but fruit setting of agarwood could also be increased.

Acknowledgements

The authors acknowledge the support of the Kasetsart University Research and Development Institute (KURDI), Thailand and thank the managers and คลังความรู้ดิจิทัลMr Pravatsart มหาวิทยาลัยเกษตรศาสตร์ at Kao Yai National Park for providing the field facility. The authors are also grateful to Mr S. Pumpuang and Ms S. Sangtabtim who assisted with data collection during the field work. The authors wish to thank Mr Somporn and the family owners of the agarwood plantation at Trat province who kindly provided the plantation study area. This research would not have been possible without material and equipment from the Faculty of Forestry, Kasetsart University, Thailand and the Institute of Tropical Agriculture, Kyushu University, Japan.

References

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Makholela, T. and J.C. Manning. 2006. First of moth pollination in Struthiola ciliate (Thymelaeaceae) in Southern Africa. South African of Botany 72: 597-603. Nghia, N.H. 1998. Aquilaria crassna. In: 2007 IUCN Red List of Threatened Species. Available Source: www.iucnredlist.org, 31 October 2007. Phengklai, C. and S. Khamsai. 1985. Some non-timber species of Thailand. Thai Forest Bulletin (Botany) 1(15): 108-148. Acknowledgements Sangtabtim, S. 2008. Development of Flower and Fruit of Kritsana (Aquilaria crassna Pierre ex Lec.). MS. Thesis, Kasetsart University, Bangkok, Thailand. (in Thai). Soehartono, T. and A.C. Newton. 2001. Reproductive ecology of Aquilaria คลังความรู้ดิจิทัล มหาวิทยาลัยเกษตรศาสตร์spp. in Indonesia. Forest Ecology and Management 152: 59-71. Tangmitcharoen, S., T. Takaso, S. Siripatanadilox, W. Tasen and J.N. Owens. 2006. Diversity and abundance of Insects in the Teak (Tectona grandis L.f.) canopies of natural and plantation forests during flowering season. Forest Ecology and Management 222: 99-107. TDRI (Thailand Development Research Institute). 1995. Green finance: A case study of Khao Yai. Natural Resources and Environment Program, TDRI, Bangkok. Available Source: www.tdri.or.th/library/quarterly/ text/mingsarn.htm, 10 April 2008. References Trisurat, Y. 2006. Measuring of the Biodiversity. Department of Forest Biology, Faculty of Forestry, Kasetsart University. Bangkok, Thailand. United Nations. 1992-2007. UNESCO World Heritage Centre. Available Source: http://whc.unesco.org/en/list/590, 11 Dec 2007. Whittaker, R.H. 1977. Evolutionary of species diversity in land communities. In A.E. Magurran, ed. Ecological Diversity and Its Measurement. Princeton University Press, New Jersey. Wolda, H. 1981. Similarity indices, sample size and diversity. In A.E. Magurran. Ecological Diversity and Its Measurement. Princeton University Press, New Jersey.