- Email: [email protected]
Biological study of Epicephala assamica (Lepidoptera: Gracilariidae) with notes on morphology of immature stages
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Biological study of Epicephala assamica (Lepidoptera: Gracilariidae) with notes on morphology of immature stages
MARK
Zhenguo Zhang, Houhun Li⁎ College of Life Sciences, Nankai University, Tianjin 300071, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Epicephala assamica Glochidion biology sex ratio immature stages morphology
The biology and immature stages of Epicephaha assamica Li (Lepidoptera: Gracilariidae) were investigated firstly based on the field observations and laboratory experiments, E. assamica pollinate for Glochidion assamicum (Muell. Arg.) Hook. (Phyllanthaceae) and then lay eggs in pollinated female flowers. In turn, hosts partly sacrifice seeds for larval growth and keeping some seeds for breeding, thus they form the most strictly integrated one-to-one nursery pollination mutualism. E. assamica have two generations correspondingly which are closely consistent with flowering phenology of G. assamicum, peak in March–April and September–October respectively. We investigated the benefits that each species obtains from partner. For G. assamicum, the ripening rate is 51.14%, the rate of consumed fruits is 79.52%, the average number of seeds consumed by each larva is 2.62, and the proportion of intact seeds is 68.03% that could keep the stabilization of mutualism in each population. The study first reports sex ratio in the genus Epicephala and suggests that the male-biased offspring in E. assamica can make sure that female moths have more mating opportunities. The eruciform larva bears three pairs of thoracic legs and four pairs of prolegs on abdominal segments III–V and X (caudal proleg), 11 crochets uniserial arranged in proleg. Antenne exceeds segment X in pupa. The study helps to further understand the evolutionary mechanism and driving force of coevolution in the obligate pollination mutualism between Epicephala and Glochidion.
Introduction The genus Epicephala Meyrick, 1880 (Gracillariidae: Ornixolinae) consists of 64 described species worldwide (Meyrick, 1880; Hu et al., 2011; Zhang et al., 2012a; Li and Yang, 2015; Li et al., 2015; De Prins and De Prins, 2016; Kawakita and Kato, 2016; Li and Zhang, 2016; Kawahara et al., 2017), and 19 species have been described from China in recent few years. Epicephala moths have recently become an important issue in ecology and evolutionary biology because of their mutualism with plants in the genera Glochidion, Breynia and Phyllanthus (Kato et al., 2003; Zhang et al., 2012b; Kawakita et al., 2015; Li et al., 2015). Pollinators and host plants have a variety of relationships. Given the described principle in most obligate pollination interactions, theoretical studies have predicted that cooperative interactions can keep evolutionarily stable only when both participants possess mechanism to prevent overexploitation by the other (Axelrod and Hamilton, 1981; Bull and Rice, 1991; Bronstein, 2001). It has been assumed that excessive exploitation of seeds by pollinators would confer a substantial cost to plants and would subsequently lead to a collapse of the
⁎
mutualistic relationship (Bull and Rice, 1991; Herre et al., 1999; Bronstein, 2001). The obligate pollination mutualisms between Epicephala moths and Phyllanthaceae plants (including Glochidion, Breynia and Phyllanthus) remain benefit balance by keeping some fruits and seeds (Kato et al., 2003; Kawakita and Kato, 2004, 2006; Zhang et al., 2012b; Zhang et al., 2016; Luo et al., 2017). However, few publications present the ripening rate of hosts and consumption of seeds by larvae in Glochidion-Epicephala mutualism. The study of how sexually reproducing organisms divide their resources between offspring of the two sexes (sex allocation) has proved one of the most successful areas in evolutionary biology (Charnov, 1982; Godfray, 1994; Herre et al., 1997; Peng et al., 2014). Several studies have reported the mating behaviours of Epicephala species (Okamoto et al., 2007; Zhang et al., 2012b; Zhang et al., 2016), however, no publications present the of sex ratio in the genus Epicephala. Immature characteristics is important for the phylogenetic classification and the morphological identification in the insect study (Di Giulio et al., 2003; Archangelsky, 2004; Mutanen et al., 2009), in particular larval characteristics. Larval characteristics are used widely in systematics, including for classification and phylogenetic
Corresponding author. E-mail address: [email protected] (H. Li).
http://dx.doi.org/10.1016/j.aspen.2017.05.003 Received 24 December 2016; Accepted 16 May 2017 Available online 19 May 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
is a monoecious plant which occurs in China and southeast Asia that flowers in bisexual axillary clusters, with many male and female flowers. The male flowers (Fig. 1B) grow at the base of branchlets, while the female flowers (Fig. 1C, D) tend to occur towards the apex. Fruits pedicels short (Fig. 1E); capsules depressed globose, usually 4locular, pericarp thinner (Li and Gilbert, 2008).
reconstruction (Archangelsky, 2004; Lee et al., 2007; Chenoweth et al., 2008; Mutanen et al., 2009; Regier et al., 2013), especially within Lepidoptera. In the mutualism between Glochidion and Epicephala, Epicephala species identification is performed based on male and female genitalia by rearing field-collected larvae until they become adults (Hu et al., 2011; Zhang et al., 2012a; Li and Yang, 2015; Li et al., 2015; Li and Zhang, 2016), however, few publications present the studies of immature stages on Epicephala moths. So, the characters of immature stages in Epicephala moths, including size, body color, chaetotaxy, proleg and crochets need to be studied. In this study, we investigated the biological characteristics between E. assamica and its obligate host-plant G. assamicum, including the life history and habits of E. assamica, the ripening rate of host-plant, consumption of seeds by pollinators and sex ratio in E. assamica, describing characteristics of some immature stages in order to understand the evolutionary mechanism in the obligate pollination mutualism between Epicephala moths and Glochidion plants and provide a scientific basis for the protection of the tropical rainforest ecosystem.
Biological observation We made tracking observations of the flower and fruit developmental stages of G. assamicum in XTBG for a total 46 individuals. We monitored 5 randomly selected branchlets each plant. We recorded the development of flowers and fruits, and counted the number of flowers and developing fruits of the selected plant individuals bi-week. We observed E. assamica during full anthesis and recorded their flowervisiting behaviours in detail, paying particular attention to their nocturnal activities, focused how they used their proboscis to collect pollen and pollinate flowers, and where they oviposited, photographs were taken with a Canon G11 digital camera in field. We recorded the times that the moths spent on pollination, oviposition and pollen collection. After the observations, moths collected from hosts were made dried specimens for species identification and sex determination. The genitalia of adult moths were dissected to identify the species follow the methods introduced by Li and Zheng (1996).
Materials and methods The study was performed from July 2013 to October 2014 in Xishuangbanna Tropical Botanical Garden (XTBG), Chinese Academy of Sciences (Fig. 1A), southwest Yunnan, China. The four seasons are not clear, but there are two obvious seasons, dry season between November and next April and the May to October rainy season. Dry season is divided into foggy-cool and dry-hot seasons, foggy-cool season lasts from November to February, heavy fog, wet in the morning and evening; dryhot season lasts from February to April, the fog gradually reduce, dry heat, less rainfall (Zhu, 1993). Epicephala assamica Li, 2016 is a new species for China (Li and Zhang, 2016), the specific name is derived from the name of the obligate host-plant Glochidion assamicum (Muell. Arg.) Hook. G. assamicum
The assessment of mutualism mechanisms To investigate ripening rate, we estimated based on total quantity of female flowers at peak anthesis, and retention of naturally matured fruits. To assess the seed production, we dissected mature fruits, recorded the number of intact seeds and consumed seeds. We collected developing and mature fruits and put them in a cylindrical plastic box (8.5 × 12 cm) group by group to rear Epicephala larvae for descriptive Fig. 1. The environment of researching site and the morphology of Glochidion assamicum. A. Landscape of XTBG; B. Male flowers; C. Female flowers; D. Pollinated female flowers during the flower-fruit interval season; E. Fruits.
919
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
The biological characteristics of Epicephala assamica
statistics. We then recorded how and when the larvae left fruits to cocoon and emerge as adults, the air temperature was monitored and maintained at 20 °C–26 °C, relative humidity 60%–80%. Moths reared from fruits were made dried specimens for species identification and sex determination.
Life history of Epicephala assamica Epicephala assamica have 2 generations correspondingly which are closely consistent with flowering phenology of G. assamicum, a complete life history lasts for about 6 months (Fig. 2). Overwintering eggs emerge into larvae in early February until fruits begin to develop, larvae that pupate from late February to early April could emerge into adults, pollinate for mature female flowers and lay eggs during middle March to late April. Oversummering eggs emerge into larvae in early August until fruits begin to develop, larvae that pupate in late August and begin to emerge as adults in middle September, pollinate for mature female flowers and lay eggs of the next generation during middle September to late October (Fig. 2). Pupal stage lasts for 13–17 days. Adults will survive 2–5 days by sucking nectar or dew for nutrition.
The description on morphological characteristics of immature stage Larvae were heated in 10% KOH at 100 °C for 5 min, subsequently dissected in 100% ethanol under Olympus SZ11 Stereo Microscope. Heads were detached by cutting along the edges, and then transferred into 100% glycerol where labiomaxillary complex, mandibles and labrum were detached from head capsules for line drawing. Body segments were cut open along the left side spiracular line, expanded and flattened, and transferred into 100% glycerol for line drawing. Chaetotary drawings were performed under Microscope CH30RF200, as well as pupa. The terminology of the larvae are based on Kumata (1978), pupa follows Davis (1987). All samples and voucher specimens, including the vouchered larvae and host plants are deposited in the Insect Collection of Nankai University, Tianjin, China (NKU).
Habits of Epicephala assamica Eggs that laid by last generational female moths start hatching larvae (Fig. 3A) until the fruits begin to develop, early instar larvae bore into seeds from testa by slashing mandibles. Early larva feeds inside only one seed, later larva continues to consume another seed inside the same locule or consumes other seeds in adjoining locule by drilling the ventricular when they consume away one seed except leaving seed coat. The hatching larvae need 3–5 weeks to develop into mature larvae, mature larvae drill from ripe fruits and fall in the litter of host-plant and surroundings to cocoon (Fig. 3B), mature larvae keep inactively after cocoon, pupate after 2–3 days and last for in the form of pupa (Fig. 3C). Consistent with flowering period, E. assamica will emerge as adult (Fig. 3D) all day, in particular at night, adults are nocturnal and insensitive to light. During the daytime, adults lie in the shadow of leaves, branchlets and circumjacent weeds, fly when be disturbed, flight ability is not strong, only a short flight. Adults become active at night, especially from 20:00 to 22:00. Epicephala moths usually fly or walk among trees or within a tree with antenna oscillating. The abdomen of male and female almost keep straight when mating (Fig. 4A), lasts for 3–7 h (n > 50). The females will creep or fly to the other branchlets with carrying males in case of interference, and mating will interrupt when
Results Phenology of Glochidion assamicum Glochidion assamicum bloom and yield twice per year (Fig. 2). The first flowering period lasts from late February to late April, especially from middle March to early April; the first fruiting period lasts from early August to late September. The second flowering period lasts from late August to late October, especially from middle September to middle October; the second fruiting period lasts from early February to late March. Female flowers don't develop after pollination during the flower-fruit interval season (Fig. 1D). The two interval seasons last for about 3 months which are high temperature in 1st generation and low temperature in 2nd generation (Zhu, 1993).
Fig. 2. The relevance between phenology of Glochidion assamicum, life history of Epicephala assamica and weather of habitat. Rainfall data from weather station of XTBG, 1st generation represented by green bars and 2nd generation by red bars in life history of pollinator. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
920
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 3. Morphology of Epicephala assamica at different developmental stages. A: Larvae; B: Cocoon; C: Pupa; D: Adult.
be interfered strongly. Mated females visit male flowers to collect pollen actively (Fig. 4B) by rubbing the proboscis against the anthers, the process lasts for 90–150 s and mated males have not been observed mating again and visiting female flowers. The females rub the proboscis to collect pollen back and forth, and store pollen to the base of proboscis via crimping proboscis efficiently, with a large number of pollen grains sticking to the base of proboscis (Fig. 4B, C). Pollen carriers don't pollinate for female flowers immediately instead just stay around male flowers or other places to “rest” with shaking their antennae, we consider that moths can locate hosts by discerning the odour of flowers via lots of receptors on antennae. One female collects pollen grains in only one male flower each time effectively, and visits approximately 10 female flowers sequentially, especially focusing on female flowers in the
Fig. 5. Comparison of time on pollination and oviposition between different times. Fig. 4. Behaviours of adult Epicephala assamica. A. Mating; B. Collecting pollen; C. Pollinating; D. Laying egg.
921
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Table 1 The ripening rate of Glochidion assamicum (September 2013–October 2014). Generations
Female flowers
Female flowers/cluster
Pollinated female flowers/cluster
Fruits
Fruits/cluster
Ripening rate
1st genenation 2nd genenation Sum Mean
2306 2311 4617 −
21.16 (n = 109) 18.34 (n = 126) − 19.65 (n = 235)
18.68 13.69 − 16.00
780 1581 2261 −
7.16 12.55 − 10.05
33.83% 68.42% − 51.14%
same cluster. Upon visiting female flowers (Fig. 4C), a female uncoils its proboscis and pushes the proboscis tip repeatedly into the stylar pit to pollinate the flowers by recoiling the proboscis several times to replenish pollen grains on the proboscis tip with moving the pollen from the base to the terminal of proboscis. After pollination, the female moth bends abdoman to basal calyx on ventral surface and inserts ovipositor through calyx lobes and ovary to lay eggs into ovary tissue (Fig. 4D). The pollination is both observed inside homophyletic plant and between different individuals. Epicephala moths are all pollen carriers which captured on female flowers examined under the Stereo Microscope. With the increasing frequency of pollination and oviposition, the homologous time consuming each time will increase (Fig. 5).
(Paired-samples Test, P (df = 4) = 0.02), the proportion of male moths is 0.64, 0.57 and 0.60, respectively, 0.62 as an average (Fig. 6D). The morphological characteristics of immature stage on Epicephala assamica Early instar larva (Fig. 3A): body eruciform, the newly-hatch larva is white translucent, the early instar larva has no crochets in proleg. Later instar larva has crochets in proleg, head capsule dark brown. Mature larva (Fig. 3A, 7A − J): body eruciform, with three pairs of well-developed thoracic legs and four pairs of fleshy ventral prolegs on abdominal segments III − V and X (caudal proleg). Head hypognathous. Length 5.52 ± 0.05 mm (n = 5) breadth of head capsule 0.65 ± 0.004 mm (n = 5). Head dark red, with dark brown patches in epicranial region and an ellipse of six ocelli. Prothoracic plate same as ground color, with an ivory triangle patch. Segment I dark red in anterior 1/3, ivory in posterior 2/3; segment II − VII ivory in anterior 1/5 and posterior 1/5, dark red in middle; segment VIII ivory in anterior 1/3 and dark red in posterior 2/3; segment IX − X dark red. Head chaetotaxy (Fig. 7A− E): coronal suture 1⁄ 3 length of frontal sclerite; frontal sclerite extended to almost 1 ⁄ 2 of head length, and nearly as broad at base as long (Figs. 7A). Distance between ocelli 1 and 6 slightly less than distance between ocelli 1 and 2, and distance between ocelli 5 and 6 nearly 1.5 × of distance between ocelli 1 and 2; ocelli 2 and 3 contiguous, and ocelli 3 and 4 close to each other; distance between ocelli 4 and 5, ocelli 5 and 6, equal in length (Fig. 7A, B). A-group trisetose with one puncture Aa, A1, A2 and A3, forming an acute triangle, A2 antero-dorsal ocelli 2, A3 antero-dorsal ocelli 1, A1 nearly 2⁄ 3 length of A3; A2 almost 4⁄ 7 length of A1; P-group bisetose with one puncture Pa, P1 and P2, P2 antero-ventral to P1, length of P1 almost 2.5 × of length of P2 or slightly less, Pa postero-dorsal to P 1; Lgroup unisetose with La. L1 antero-dorsal to A3, about 1/5 length of A3, La postero-dorsal to L1; V-group with three minute setae arranged in a vertical curve line, with one puncture Va, V1, V2 and V3 nearly equal in length; O-group with three setae O1, O2 and O3, with one puncture Oa, O1 postero-dorsal to ocelli 2, O2 postero-dorsal to ocelli 1, O3 between ocelli 1 and 6, O1 and O2 nearly equal in length and half length of O3; SO-group trisetose, SO1, SO2 and SO3, forming an obtuse triangle, with one puncture SOb, SO1 postero-ventral to ocelli 4, outside ocelli blotch, SO2 closer to ocellia 4 than to ocellia 5, SO3 posterior SO2, SO1 nearly 1/2 length of SO3, SO2 nearly 4/7 length of seta SO3; G-group unisetose with one puncture Ga, G1 postero-dorsal to SO3, Ga posterodorsal to G1, G1 and SO2 nearly equal in length; F-group with one seta F1 and one puncture Fa, Fa ventral to F1. C-group bisetose, C1 posteroventral to C2; C1 and C2 of nearly equal length, slightly more than F1, distance between C1 and C2 less than distance between F1 and C1. Mouthparts (Fig. 7C−E): labrum (Fig. 7C): three median setae M1, M2 and M3, M2 nearly equal to length of M3; M2 slightly more than
The ripening rate and consumption of seeds In 1st generation, ripening rate is only 33.83% (Table 1). Meanwhile, each fruit has 7.19 seeds (n = 426, in theory, 8 seeds each fruit, a few seeds without pollination abortive), 83.57% (n = 356) fruits are consumed by larvae. In the consumed fruits, 98.31% fruits consumed by only one larva (n = 350), 1.69% fruits consumed by two larvae and no fruit consumed by three or more larvae. Each larva consumes an average of 2.49 seeds (Table 2). In 2nd generation, ripening rate of 1st generation is 68.42% (Table 1). Meanwhile, each fruit has 6.75 seeds (n = 868, in theory, 8 seeds each fruit, a few seeds without pollination abortive), 77.53% (n = 673) fruits are consumed by larvae. In the consumed fruits, 92.27% (n = 621) fruits consumed by only one larva, 7.13% (n = 48) fruits consumed by two larvae, 0.59% (n = 4) fruits consumed by three larvae and no fruit consumed by four or more larvae. Each larva consumes an average of 2.67 seeds (Table 2). On average, the ripening rate is 51.14%, the rate of consumed fruits is 79.52%. In the consumed fruits, 94.36% fruits consumed by only one larva (n = 971), 5.25% (n = 54) fruits consumed by two larvae, 0.39% (n = 4) fruits consumed by three larvae and no fruit consumed by four or more larvae, each larva consumes 2.62 seeds to finish their larval stage. Overall, the proportion of intact seeds is 68.03% that can keep the stabilization of mutualism in each population (Table 2). Sex ratio of Epicephala assamica We collect mature fruits until larvae exit the fruits pupated and emerge as adults from 30 August 2013 to 17 September 2013 (Fig. 6A), from 4 March 2014 to 26 March 2014 (Fig. 6B) and from 12 September to 2 October 2014(Fig. 6C), respectively. Overall, we rear 236 male moths and 145 female moths. The study results of cumulative three different generations explained that male moths emerge as adults before than female moths and more male-biased offspring sex ratio Table 2 Fruits consumed by Epicephala assamica (September 2013–October 2014). Gen.
Fruits
Consumed fruits
Rate of consumed fruits
Seeds
Consumed seeds
Rate of consumed seeds
Larvae
Seeds consumed by one larva
1st gen 2nd gen Sum Mean
426 868 1294 −
356 673 1029 −
83.57% 77.53% − 79.52%
3064 5861 8925 −
903 1950 2853 −
29.47% 33.27% − 31.97%
362 729 1091 −
2.49 2.67 − 2.62
922
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 6. Sex Ratio of Epicephala assamica. A. The result of eclosion for the first time; B. The result of eclosion for the second time; C. The result of eclosion for the third time; D. The result of eclosion for the three times.
of L1; SV-group unisetose with SV, SV antero-dorsal to prolegs, SV1 slightly less than length of L2; V-group unisetose with V, close to ventrimeson; MV-group unisetose with MV, setae MV and V nearly equal in length. In segments A9, D-group bisetose, D1 and D2, D2 postero-dorsal to D1, nearly equal to length of D1; SD-group unisetose with SD1, D1 postero-dorsal to SD1 and slightly less than SD1; crochets of proleg (Fig. 7J): ventral prolegs on A3 to A5 and A10, 11 crochets uniserial, arranged in uniordinal crochet. Cocoon (Fig. 3B): white, 6.82 ± 0.07 mm (n = 12), oblong, transparent, with numbers of white spherical grains attached on surface which generally could be divided into six small rooms, there was a piece of silk under the grain went into the cocoon. Pupa (Figs. 8A, B): maximum length 5.0 mm (n = 13), breadth of head breadth 0.08 mm. Antenne exceeds segment X; yellowish green right after pupation, gradually turning yellowish brown, becoming dark brown right before emergence, slightly darker in ventral surface of thorax, caudal abdominal segments orangish. Vertex with cocoon cutter, all spiracles of nearly the same size. Forelegs extending to near posterior margin of segment III; midlegs extending to near posterior margin of segment IV; hindlegs have larger variation, extending near to end posterior margin of segments VI − VIII.
length of M1. Three epipharyngeal setae E1, E2 and E3, nearly equal length. Mandible (Fig. 7D): with four distinct pointed teeth along cutting margin (second and third one equal in size, the first and fourth tooth smaller); MD2 1⁄ 3 length of MD1. Maxillae and labium (Fig. 7E): spinneret pointed anteriorly, 1.5 × as long as breadth at base, 3× length of first segment of labial palpus; LB1 nearly equal to length of LB3, MX2 slightly less than length of MX1; MX3 slightly less than length of MX2; MX6 almost 1/2 length of MX1; MX3 and LB1 almost equal in length. Prothorax (Fig. 7F): XD-group unisetose, XD2 absent; D-group bisetose, D1 and D2, D1 anterior-dorsal D2, D1 postero-dorsal XD1, distance between D1 and D2 almost 2 × of distance between D1 and XD1; XD1 slightly > 3 × length of D1, D2 slightly > 1.5 × of length of D1; SD-group bisetose, SD1 postero-ventral to SD2, about twice length of SD2. L-group bisetose, L1 antero-dorsal to spiracle, nearly 4 × length of L2 or slightly less; SV-group bisetose, SV1 postero-ventral to SV2, SV1 almost 3× as long as SV2; V-group unisetose with V, postero-ventral to coxacava, close to ventrimeson; MV-group with three minute setae, MV1, MV2 and MV3; setae V1, MV1, MV2 and M3 nearly equal in length. Mesothorax and Metathorax (Fig. 7G): XD-group absent; D-group bisetose, D1 and D2, D1 antero-dorsal to D2, nearly 1/3 length D2; SDgroup bisetose, SD2 antero-dorsal to SD1, SD1 3 × length of SD2 or slightly more; L-group bisetose, L1 antero-ventral to L3 and slightly > 3 × length of seta L3; SV-group unisetose with SV1, SV1 antero-ventral to L3 and slightly less than length of L1. V-group unisetose with V, postero-ventral to coxacava, close to ventrimeson (imaginary line along the ventral surface of the body); MV-group with three minute setae, MV1, MV2 and MV3; setae V1, MV1, MV2 and M3 nearly equal in length. Abdomen (Figs. 7H, I): segments A1 to A8: D-group bisetose, D1 and D2, D1 postero-dorsal to D2 and slightly more than length of D2; SDgroup bisetose, SD1 postero-ventral to SD2, SD1 slightly > 4.5 × length of SD2; distance between SD1 and SD2 equal to distance between D1 and D2; distance between SD1 and D2 2.5 × of distance between D1 and D2; L-group bisetose, L1 antero-dorsal to L2, nearly equal to length
Discussion Epicephala assamica have 2 generations correspondingly which are closely consistent with flowering phenology of G. assamicum, peak in March–April and September–October, respectively. Flowering periods are in the border of the rainy and dry seasons in our study areas, the phenology of host-plant is closely related to climate of geographical distribution, we hypothesize that the period is favorable for pollinators to pollinate for host plants on account of the long-term co-adaptation between the local climate environment and host plants. Pollinated female flowers don't develop during the flower-fruit interval season, so it cannot provide food and living space for the E. assamica. The first interval season lasts from late May to late August is peak of rainy season, high temperature and humidity; the second interval season from late 923
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 7. Morphology of Epicephala assamica mature larva. A. Frontal view of head capsule; B. Lateral view of head capsule; C. Labrum; D. Mandible; E. Maxillae and labium; F. Prothorax; G. Mesothorax; H. Abdominal segment 3; I. Abdominal segment 9; J. Crochets of proleg. (Scales = 0.1 mm).
2016), fruit set of G. assamicum is almost same to other species. The reason may be that Glochidion plants abscise a fraction of their pollinated flowers as a result of resource limitation occurring after pollination (Goto et al., 2010), may also be aborted due to damage caused by enemy insects or related to the rainy season of high temperature, high humidity, composite can be concluded that the results of the above three aspects. The vast difference of ripening rate between 1st and 2nd generations is most likely due to the complex environment of rainy interval season rather than efficiency of pollination between two generations, because the number of pollinated female flowers/cluster is higher than 2nd generation. In conclusion, the offspring populations keep stability by means of efficient pollination mechanism. The above conclusions suggest that pollinators are very sensitive to the change of host-plants. Life history, habits of E. assamica and phenology of G. assamicum are closely related to climate of study sites. With the increasing frequency of pollination and oviposition, the homologous time each
November to next February is foggy-cool season, low temperature and foggy (Zhu, 1993). E. assamica in the form of egg exists in the ovary during those complex environments, the protection of plant tissue not only reduces the rate of the destroyed by natural enemies but also provides a relatively stable temperature and humidity for the preservation of dormant eggs, to reduce water evaporation from eggs to the dry atmosphere or keep appropriate humidity from eggs to the moist atmosphere. Most importantly, moths and seeds keep development synchronously. In the known obligate pollination mutualisms such as figs-fig wasp, yuccas-yucca moths and Glochidion-Epicephala, figs, yuccas and Glochidion plants depend exclusively on pollination of fig wasps, yucca moths and Epicephala respectively, while sacrificing some seeds for larvae and keeping some seeds for breeding (Janzen, 1979; Weiblen, 2002; Zhang et al., 2012b). Although the number of female flowers each cluster (n = 19.65) at peak anthesis (March) is higher than other species (Goto et al., 2010; Wang and Li, 2015; Zhang et al., 924
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 8. Pupa of Epicephala assamica. A. ventral view; B. lateral view.
influence of sex ratio on the population structure, mating patterns and the insect society (Charnov, 1979; Bulmer, 1986). So the results can help us understanding the pattern and mechanism of sex ratio allocation in natural condition, and the evolution mechanisms of mutualistic symbiosis between Glochidion and Epicephala as well. In insects, morphological studies of immature stages can play an important role in pest control and taxonomic and phylogenetic analyses (Van Emden, 1957; Huemer and Karsholt, 1999; Hebert et al., 2004; Piao, 2006; Kristensen et al., 2007;). The larval morphological study is important for the phylogenetic classification and the morphological identification in the larval stage. Early instar larvae of E. assamica feed seeds inside the fruit and the security of closed space is high, maybe this is the reason that the body color of early instar larvae is white. However, mature larvae need to drill out from ripe fruits and to cocoon in host-plant surroundings. Larvae creep slowly and exposed during the period, so the larval body color evolves into the red and white warning colouration (Fig. 3A) in evolutionary process to help to increase the survival probability of larvae. From a taxonomic perspective, because morphology generally differs among instars in insect larvae (e.g., chaetotaxy), the identification of larval instars is necessary for comparing homological characters among species (Kitching, 1984; Miller, 1991; Solodovnikov, 2007). Characters of larval chaetotaxy, i.e. the number and ordering of larval setae and pores, have particular potential in systematics (Kitching, 1984; Miller, 1991; Solodovnikov, 2007; Mutanen et al., 2009), in particular crochets in proleg within Lepidoptera. Epicephala moths are hardly distinguishable by superficial
time will increase (Fig. 5), we think this may be associated with fewer and fewer pollen grains in proboscis, worse and worse location that female flowers be oviposited in the same cluster. On the other hand, abortive seeds may be associated with the phenomenon. The study reports sex ratio of Epicephala moths firstly, males emerge before than females and more male-biased offspring sex ratio (P < 0.05) in E. assamica. In theory, in the random mating populations, natural selection support parental genetic investment between male and female offsprings is equal, only sex ratio 1:1 is evolutionary stable sex ratio (Fisher, 1930). In order to reproduce efficiently, more male-biased or more female-biased offspring sex ratio exists in some spider and insect groups (Hamilton, 1967; Charnov, 1982; Haidy, 2002). Sex ratio evolution is closely related to resource allocation, and some studies in the literature have dealt with how resource allocation affects sex allocation under different male and female function relationships (Zhang and Wang, 1994). In tightly coevolved mutualism between Glochidion and Epicephala, moth that no cilia found on the proboscis is considered to be non-pollinator (Kato et al., 2003; Li et al., 2015), which suggests that the males of Epicephala are unproductive for host-plants. We consider male-biased offspring sex ratio can make sure that female moths have more mating opportunities, because the Epicephala moths captured from Glochidion plants in the field are usually more females than males, and mated female Epicephala moths prefer the pollen-collecting behaviour than unmated moths (Okamoto et al., 2013). The study of sex ratio is important for understanding the causes and consequences of selective pressure, and understanding the 925
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Press, Cambridge, pp. 438. Hamilton, W.D., 1967. Extraordinary sex ratios. Science 156, 477–488. Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl. Acad. Sci. U. S. A. 101, 14812–14817. Herre, E.A., West, S.A., Cook, J.M., Compton, S.G. Kjellberg, F., 1997. Fig wasp mating system: pollinators and parasites, sex ratio adjustment and male polymorphism, population structure and its consequences. In: Social Competition and Cooperation in Insects and Arachnids. Vol. I: The Evolution of Mating Systems (Ed. by J. Choe and B. Crespi), Cambridge: Cambridge University Press. pp. 226 − 239. Herre, E.A., Knowlton, N., Mueller, G., Rehner, S.A., 1999. The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends Ecol. Evol. 14, 49–53. Hu, B.B., Wang, S.X., Zhang, J., Li, H.H., 2011. Taxonomy and biology of two seedparasitic gracillariid moths (Lepidoptera, Gracillariidae), with description of a new species. ZooKeys 83, 43–56. Huemer, P., Karsholt, O., 1999. Gelechiidae I (Gelechiinae: Teleiodini, Gelechini). Microlepidoptera of Europe. (ed. by Huemer, P., Karsholt, O., Lyneborg, L.), Vol. 3, pp. 1–356. Apollo Books, Stenstrup. Janzen, D.H., 1979. How many babies do figs pay for babies? Biotropica 11, 48–50. Kato, M., Takimura, A., Kawakita, A., 2003. An obligate pollination mutualism and reciprocal diversification in the tree genus Glochidion (Euphorbiaceae). Proc. Natl. Acad. Sci. 100 (9), 5264–5267. Kawahara, A., Plotkin, D., Ohshima, I., Lopen-Vaamonde, C., Houlihan, P., Breinholt, J.W., Kawakita, A., Xiao, L., Regier, J.C., Davis, D., Kumata, T., Sohn, J.C., De Prins, J., Miller, C., 2017. A molecular phylogeny and revised higher-level classification for the leaf-mining moth family Gracillariidae and its implications for larval host-use evolution. Syst. Entomol. 42, 60–81. Kawakita, A., Kato, M., 2004. Evolution of obligate pollination mutualism in new Caledonian Phyllanthus (Euphorbiaceae). Am. J. Bot. 91 (3), 410–415. Kawakita, A., Kato, M., 2006. Assessment of the diversity and species specificity of the mutualistic association between Epicephala moths and Glochidion trees. Mol. Ecol. 15 (12), 3567–3581. Kawakita, A., Kato, M., 2016. Revision of the Japanese species of Epicephala Meyrick with descriptions of seven new species (Lepidoptera, Gracillariidae). ZooKeys 568, 87–118. Kawakita, A., Mochizuki, K., Kato, M., 2015. Reversal of mutualism in a leafflower–leafflower moth association: the possible driving role of a third-party partner. Biol. J. Linn. Soc. 116 (3), 507–518. Kitching, I.J., 1984. The use of larval chaetotaxy in butterfly systematics, with special reference to the Danaini (Lepidoptera: Nymphalidae). Syst. Entomol. 9, 49–61. Kristensen, N.P., Scoble, M., Karsholt, O., 2007. Lepidoptera phylogeny and systematics: the state of inventorying moth and butterfly diversity. Zootaxa 1668, 699–747. Kumata, T., 1978. A new stem-miner of alder in Japan, with a review of the larval transformation in the Gracillariidae (Lepidoptera). Insecta Matsumurana 13, 1–27. Lee, C.F., Satô, M., Shepard, W.D., Jach, M.A., 2007. Phylogeny of Psephenidae (Coleoptera: Byrrhoidea) based on larval, pupal and adult characters. Syst. Entomol. 32, 502–538. Li, B.T., Gilbert, M.G., 2008. Flora of China. 11. Missouri Botanical Garden Press, USA, pp. 200. Li, H.H., Yang, X.F., 2015. Three new species of Epicephala Meyrick (Lepidoptera, Gracillariidae) associated with Phyllanthus microcarpus (Benth.) (Phyllanthaceae). ZooKeys 484, 71–81. Li, H.H., Zhang, Z.G., 2016. Five species of the genus Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) from China. Zootaxa 4084 (3), 391–415. Li, H.H., Zheng, Z.M., 1996. Methods and techniques of specimens of Microlepidoptera. J. Shaanxi Norm. Univ. (Natural Science Edition) 24 (3), 63–70 (in Chinese). Li, H.H., Wang, Z.B., Hu, B.B., 2015. Four new species of Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) associated with two Glochidion plants (Phyllanthaceae). ZooKeys 508, 53–67. Luo, S.X., Yao, G., Wang, Z.W., Zhang, D.X., Hembry, D., 2017. A novel, enigmatic basal leafflower moth lineage pollinating a derived leafflower host illustrates the dynamics of host shifts, partner replacement, and apparent coadaptation in intimate mutualisms. Am. Nat. http://dx.doi.org/10.1086/690623. Meyrick, E., 1880. Descriptions of New Zealand micro-lepidoptera III, Tineina. Proc. Linnean Soc. NSW 5 (1), 132–182. Miller, J.S., 1991. Cladistics and classification of the Notodontidae (Lepidoptera: Noctuoidea) based on larval and adult morphology. Bull. Am. Mus. Nat. Hist. 204, 1–230. Mutanen, M., Ruotsalainen, H., Kaila, L., 2009. Variation in larval chaetotaxy in Orthosia gothica (Lepidoptera: Noctuidae): effects of mother, sex and side, and implications for systematics. Syst. Entomol. 34, 712–723. Okamoto, T., Kawakita, A., Kato, M., 2007. Interspecific variation of floral scent composition in Glochidion and its association with host-specific pollinating seed parasite (Epicephala). J. Chem. Ecol. 33, 1065–1081. Okamoto, T., Kawakita, A., Goto, R., Svensson, G.P., Kato, M., 2013. Active pollination favours sexual dimorphism in floral scent. Proc. R. Soc. B Biol. Sci. 20132280. Peng, Y.Q., Zhang, Y., Compton, S.G., Yang, D.R., 2014. Fig wasps from the centre of figs have more chances to mate, more offspring and more female-biased offspring sex ratios. Anim. Behav. 98, 19–25. Piao, M.H., 2006. Morphological descriptions of three species larvae of Tortricidae (Lepidoptera) from Korea. Entomotaxonomia 28 (3), 201–208. Regier, J.C., Mitter, C., Zwick, A., Bazinet, A.L., Cummings, M.P., Kawahara, A.Y., Sohn, J.C., Zwickl, D.J., Cho, S., Davis, D.R., Baixeras, J., Brown, J., Parr, C., Weller, S., Lees, D.C., Mitter, K.T., 2013. A large-scale, higher-level, molecular phylogenetic study of the insect order Lepidoptera (moths and butterflies). PLoS One 8 (3), e58568.
characters of adults (Li and Zhang, 2016), as a result, larval characteristics can provide new additional implication for phylogenetic analysis, especially crochets of prolegs and chaetotaxy in Epicephala moths. On the other hand, pupal characterss are not species restricted that cannot be taken as the basis of interspecific classification. The larvae exhibit the characteristic adaptions associated with life mode that closely related to their host plants in the long-term natural selection, and showed species diversity (Thompson and Pellmyr, 1992), especially within interaction between insects and host plants. Therefore, the basic aspects of larval morphology, including intraspecific variation, effects of relatedness between pollinators and hosts, and the necessity of immature stages for the phylogenetic classification and the morphological identification in the insect study will be investigated in future to emphasize the importance of immature stages. Conclusion The study helps to further understand the evolutionary mechanism in the obligate pollination mutualism between Epicephala moths and Glochidion plants, and provides a scientific basis for the protection of the tropical rainforest ecosystem. One survival strategy of E. assamica is spending the flower-fruit interval season in G. assamicum in the form of eggs and synchronizing eclosion with the flowering phenology. Morphological and biological studies of E. assamica can contribute to understanding the evolutionary mechanisms in Glochidion-Epicephala mutualism. Acknowledgements The authors thank Professor D.R. Yang, Professor Y.Q. Peng and all members of Key Laboratory of Tropical Forest Ecology (XTBG) for their support and kind help in our field experiment. We also thank Dr. Z.B. Wang, Dr. X.F. Yang (NKU) for interesting discussion on the early draft of the manuscript. We specially thank Dr. Jurate De Prins (Belgium) for her review and comments on the manuscript. This research was supported by a grant from the National Natural Science Foundation of China (No. 31672372 & 30930014) and all the experiments comply with the current laws of China. References Archangelsky, M., 2004. Higher-level phylogeny of Hydrophilinae (Coleoptera: Hydrophilidae) based on larval, pupal and adult characters. Syst. Entomol. 29, 188–214. Axelrod, R., Hamilton, W.D., 1981. The evolution of co-operation. Science 211, 1390–1396. Bronstein, J.L., 2001. The exploitation of mutualisms. Ecol. Lett. 4, 277–287. Bull, J.J., Rice, W.R., 1991. Distinguishing mechanisms for the evolution of co-operation. J. Theor. Biol. 149, 63–74. Bulmer, M.G., 1986. Sex ratios theory in geographically structured populations. Heredity 56, 69–73. Charnov, E.L., 1979. The genetical evolution of patterns of sexuality: Darwinian fitness. Am. Nat. 113, 465–480. Charnov, E.L., 1982. The Theory of Sex Allocation. Princeton University Press, Princeton, pp. 355. Chenoweth, L.B., Fuller, S., Tierney, S.M., Park, Y.C., Schwarz, M.P., 2008. Hasinamelissa: a new genus of allodapine bee from Madagaskar revealed by larval morphology and DNA sequence data. Syst. Entomol. 33, 700–710. Davis, D.R., 1987. Gracillariidae. In: Stehr, F.W. (Ed.), Immature Insects. Vol. 1. Kendall/ Hunt Publ. Co., Dubuque, Iowa, pp. 372–374 376–378. De Prins, W., De Prins, J., 2016. Global taxonomic database of Gracillariidae (Lepidoptera). [2016 −9 − 18]. http://www.gracillariidae.net. Di Giulio, A., Fattorini, S., Kaupp, A., Taglianti, A.V., Nagel, P., 2003. Review of competing hypotheses of phylogenetic relationships of Paussinae (Coleoptera: Carabidae) based on larval characters. Syst. Entomol. 28, 509–537. Fisher, R.A., 1930. The Genetical Theory of Natural Selection. Oxford University Press, Oxford. Godfray, H.C.J., 1994. Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton, New Jersey. Goto, R., Okamoto, T., Kiers, E.T., Kawakita, A., Kato, M., 2010. Selective flower abortion maintains moth cooperation in a newly discovered pollination mutualism. Ecol. Lett. 13, 321–329. Haidy, L.C.W., 2002. Sex Ratio: Concepts and Research Methods. Cambridge University
926
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
44, 65–75. Zhang, J., Hu, B.B., Wang, S.X., Li, H.H., 2012a. Six new species of Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) associated with Phyllanthaceae plants. Zootaxa 3275, 43–54. Zhang, J., Wang, S.X., Li, H.H., Hu, B.B., Yang, X.F., Wang, Z.B., 2012b. Diffuse coevolution between two Epicephala species (Gracillariidae) and two Breynia species (Phyllanthaceae). PLoS One 7 (7), e41657. Zhang, Z.G., Teng, K.J., Li, H.H., 2016. Biological characteristics of Epicephala ancylopa (Lepidoptera: Gracillariidae) on host Glochidion sp. (Phyllanthaceae) and the compositional analysis of its floral scent. Acta Entomol. Sin. 59, 669–681. Zhu, H., 1993. The characteristics of the Xishuangbanna tropical rainforest flora. Trop. Geogr. 13 (4), 149–155.
Solodovnikov, A.Y., 2007. Larval chaetotaxy of Coleoptera (Insecta) as a tool for evolutionary research and systematics: less confusion, more clarity. J. Zool. Syst. Evol. Res. 45, 120–127. Thompson, J.N., Pellmyr, O., 1992. Mutualism with pollinating seed parasites amid copollinators: constraints on specialization. Ecology 73 (5), 1780–1791. Van Emden, F.J., 1957. The taxonomic significance of the characters of immature insects. Annu. Rev. Entomol. 2, 91–106. Wang, Z.B., Li, H.H., 2015. The biology and survival strategy of Epicephala sp. (Lepidoptera: Gracillariidae). Chin. J. Appl. Entomol. 52 (1), 162–172. Weiblen, G.D., 2002. How to be a fig wasp. Annu. Rev. Entomol. 47, 299–330. Zhang, D.Y., Wang, G., 1994. Evolutionarily stable reproductive strategies in sexual organisms: an integrated approach to life-history evolution and sex allocation. Am. Nat.
927
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Biological study of Epicephala assamica (Lepidoptera: Gracilariidae) with notes on morphology of immature stages
MARK
Zhenguo Zhang, Houhun Li⁎ College of Life Sciences, Nankai University, Tianjin 300071, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Epicephala assamica Glochidion biology sex ratio immature stages morphology
The biology and immature stages of Epicephaha assamica Li (Lepidoptera: Gracilariidae) were investigated firstly based on the field observations and laboratory experiments, E. assamica pollinate for Glochidion assamicum (Muell. Arg.) Hook. (Phyllanthaceae) and then lay eggs in pollinated female flowers. In turn, hosts partly sacrifice seeds for larval growth and keeping some seeds for breeding, thus they form the most strictly integrated one-to-one nursery pollination mutualism. E. assamica have two generations correspondingly which are closely consistent with flowering phenology of G. assamicum, peak in March–April and September–October respectively. We investigated the benefits that each species obtains from partner. For G. assamicum, the ripening rate is 51.14%, the rate of consumed fruits is 79.52%, the average number of seeds consumed by each larva is 2.62, and the proportion of intact seeds is 68.03% that could keep the stabilization of mutualism in each population. The study first reports sex ratio in the genus Epicephala and suggests that the male-biased offspring in E. assamica can make sure that female moths have more mating opportunities. The eruciform larva bears three pairs of thoracic legs and four pairs of prolegs on abdominal segments III–V and X (caudal proleg), 11 crochets uniserial arranged in proleg. Antenne exceeds segment X in pupa. The study helps to further understand the evolutionary mechanism and driving force of coevolution in the obligate pollination mutualism between Epicephala and Glochidion.
Introduction The genus Epicephala Meyrick, 1880 (Gracillariidae: Ornixolinae) consists of 64 described species worldwide (Meyrick, 1880; Hu et al., 2011; Zhang et al., 2012a; Li and Yang, 2015; Li et al., 2015; De Prins and De Prins, 2016; Kawakita and Kato, 2016; Li and Zhang, 2016; Kawahara et al., 2017), and 19 species have been described from China in recent few years. Epicephala moths have recently become an important issue in ecology and evolutionary biology because of their mutualism with plants in the genera Glochidion, Breynia and Phyllanthus (Kato et al., 2003; Zhang et al., 2012b; Kawakita et al., 2015; Li et al., 2015). Pollinators and host plants have a variety of relationships. Given the described principle in most obligate pollination interactions, theoretical studies have predicted that cooperative interactions can keep evolutionarily stable only when both participants possess mechanism to prevent overexploitation by the other (Axelrod and Hamilton, 1981; Bull and Rice, 1991; Bronstein, 2001). It has been assumed that excessive exploitation of seeds by pollinators would confer a substantial cost to plants and would subsequently lead to a collapse of the
⁎
mutualistic relationship (Bull and Rice, 1991; Herre et al., 1999; Bronstein, 2001). The obligate pollination mutualisms between Epicephala moths and Phyllanthaceae plants (including Glochidion, Breynia and Phyllanthus) remain benefit balance by keeping some fruits and seeds (Kato et al., 2003; Kawakita and Kato, 2004, 2006; Zhang et al., 2012b; Zhang et al., 2016; Luo et al., 2017). However, few publications present the ripening rate of hosts and consumption of seeds by larvae in Glochidion-Epicephala mutualism. The study of how sexually reproducing organisms divide their resources between offspring of the two sexes (sex allocation) has proved one of the most successful areas in evolutionary biology (Charnov, 1982; Godfray, 1994; Herre et al., 1997; Peng et al., 2014). Several studies have reported the mating behaviours of Epicephala species (Okamoto et al., 2007; Zhang et al., 2012b; Zhang et al., 2016), however, no publications present the of sex ratio in the genus Epicephala. Immature characteristics is important for the phylogenetic classification and the morphological identification in the insect study (Di Giulio et al., 2003; Archangelsky, 2004; Mutanen et al., 2009), in particular larval characteristics. Larval characteristics are used widely in systematics, including for classification and phylogenetic
Corresponding author. E-mail address: [email protected] (H. Li).
http://dx.doi.org/10.1016/j.aspen.2017.05.003 Received 24 December 2016; Accepted 16 May 2017 Available online 19 May 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
is a monoecious plant which occurs in China and southeast Asia that flowers in bisexual axillary clusters, with many male and female flowers. The male flowers (Fig. 1B) grow at the base of branchlets, while the female flowers (Fig. 1C, D) tend to occur towards the apex. Fruits pedicels short (Fig. 1E); capsules depressed globose, usually 4locular, pericarp thinner (Li and Gilbert, 2008).
reconstruction (Archangelsky, 2004; Lee et al., 2007; Chenoweth et al., 2008; Mutanen et al., 2009; Regier et al., 2013), especially within Lepidoptera. In the mutualism between Glochidion and Epicephala, Epicephala species identification is performed based on male and female genitalia by rearing field-collected larvae until they become adults (Hu et al., 2011; Zhang et al., 2012a; Li and Yang, 2015; Li et al., 2015; Li and Zhang, 2016), however, few publications present the studies of immature stages on Epicephala moths. So, the characters of immature stages in Epicephala moths, including size, body color, chaetotaxy, proleg and crochets need to be studied. In this study, we investigated the biological characteristics between E. assamica and its obligate host-plant G. assamicum, including the life history and habits of E. assamica, the ripening rate of host-plant, consumption of seeds by pollinators and sex ratio in E. assamica, describing characteristics of some immature stages in order to understand the evolutionary mechanism in the obligate pollination mutualism between Epicephala moths and Glochidion plants and provide a scientific basis for the protection of the tropical rainforest ecosystem.
Biological observation We made tracking observations of the flower and fruit developmental stages of G. assamicum in XTBG for a total 46 individuals. We monitored 5 randomly selected branchlets each plant. We recorded the development of flowers and fruits, and counted the number of flowers and developing fruits of the selected plant individuals bi-week. We observed E. assamica during full anthesis and recorded their flowervisiting behaviours in detail, paying particular attention to their nocturnal activities, focused how they used their proboscis to collect pollen and pollinate flowers, and where they oviposited, photographs were taken with a Canon G11 digital camera in field. We recorded the times that the moths spent on pollination, oviposition and pollen collection. After the observations, moths collected from hosts were made dried specimens for species identification and sex determination. The genitalia of adult moths were dissected to identify the species follow the methods introduced by Li and Zheng (1996).
Materials and methods The study was performed from July 2013 to October 2014 in Xishuangbanna Tropical Botanical Garden (XTBG), Chinese Academy of Sciences (Fig. 1A), southwest Yunnan, China. The four seasons are not clear, but there are two obvious seasons, dry season between November and next April and the May to October rainy season. Dry season is divided into foggy-cool and dry-hot seasons, foggy-cool season lasts from November to February, heavy fog, wet in the morning and evening; dryhot season lasts from February to April, the fog gradually reduce, dry heat, less rainfall (Zhu, 1993). Epicephala assamica Li, 2016 is a new species for China (Li and Zhang, 2016), the specific name is derived from the name of the obligate host-plant Glochidion assamicum (Muell. Arg.) Hook. G. assamicum
The assessment of mutualism mechanisms To investigate ripening rate, we estimated based on total quantity of female flowers at peak anthesis, and retention of naturally matured fruits. To assess the seed production, we dissected mature fruits, recorded the number of intact seeds and consumed seeds. We collected developing and mature fruits and put them in a cylindrical plastic box (8.5 × 12 cm) group by group to rear Epicephala larvae for descriptive Fig. 1. The environment of researching site and the morphology of Glochidion assamicum. A. Landscape of XTBG; B. Male flowers; C. Female flowers; D. Pollinated female flowers during the flower-fruit interval season; E. Fruits.
919
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
The biological characteristics of Epicephala assamica
statistics. We then recorded how and when the larvae left fruits to cocoon and emerge as adults, the air temperature was monitored and maintained at 20 °C–26 °C, relative humidity 60%–80%. Moths reared from fruits were made dried specimens for species identification and sex determination.
Life history of Epicephala assamica Epicephala assamica have 2 generations correspondingly which are closely consistent with flowering phenology of G. assamicum, a complete life history lasts for about 6 months (Fig. 2). Overwintering eggs emerge into larvae in early February until fruits begin to develop, larvae that pupate from late February to early April could emerge into adults, pollinate for mature female flowers and lay eggs during middle March to late April. Oversummering eggs emerge into larvae in early August until fruits begin to develop, larvae that pupate in late August and begin to emerge as adults in middle September, pollinate for mature female flowers and lay eggs of the next generation during middle September to late October (Fig. 2). Pupal stage lasts for 13–17 days. Adults will survive 2–5 days by sucking nectar or dew for nutrition.
The description on morphological characteristics of immature stage Larvae were heated in 10% KOH at 100 °C for 5 min, subsequently dissected in 100% ethanol under Olympus SZ11 Stereo Microscope. Heads were detached by cutting along the edges, and then transferred into 100% glycerol where labiomaxillary complex, mandibles and labrum were detached from head capsules for line drawing. Body segments were cut open along the left side spiracular line, expanded and flattened, and transferred into 100% glycerol for line drawing. Chaetotary drawings were performed under Microscope CH30RF200, as well as pupa. The terminology of the larvae are based on Kumata (1978), pupa follows Davis (1987). All samples and voucher specimens, including the vouchered larvae and host plants are deposited in the Insect Collection of Nankai University, Tianjin, China (NKU).
Habits of Epicephala assamica Eggs that laid by last generational female moths start hatching larvae (Fig. 3A) until the fruits begin to develop, early instar larvae bore into seeds from testa by slashing mandibles. Early larva feeds inside only one seed, later larva continues to consume another seed inside the same locule or consumes other seeds in adjoining locule by drilling the ventricular when they consume away one seed except leaving seed coat. The hatching larvae need 3–5 weeks to develop into mature larvae, mature larvae drill from ripe fruits and fall in the litter of host-plant and surroundings to cocoon (Fig. 3B), mature larvae keep inactively after cocoon, pupate after 2–3 days and last for in the form of pupa (Fig. 3C). Consistent with flowering period, E. assamica will emerge as adult (Fig. 3D) all day, in particular at night, adults are nocturnal and insensitive to light. During the daytime, adults lie in the shadow of leaves, branchlets and circumjacent weeds, fly when be disturbed, flight ability is not strong, only a short flight. Adults become active at night, especially from 20:00 to 22:00. Epicephala moths usually fly or walk among trees or within a tree with antenna oscillating. The abdomen of male and female almost keep straight when mating (Fig. 4A), lasts for 3–7 h (n > 50). The females will creep or fly to the other branchlets with carrying males in case of interference, and mating will interrupt when
Results Phenology of Glochidion assamicum Glochidion assamicum bloom and yield twice per year (Fig. 2). The first flowering period lasts from late February to late April, especially from middle March to early April; the first fruiting period lasts from early August to late September. The second flowering period lasts from late August to late October, especially from middle September to middle October; the second fruiting period lasts from early February to late March. Female flowers don't develop after pollination during the flower-fruit interval season (Fig. 1D). The two interval seasons last for about 3 months which are high temperature in 1st generation and low temperature in 2nd generation (Zhu, 1993).
Fig. 2. The relevance between phenology of Glochidion assamicum, life history of Epicephala assamica and weather of habitat. Rainfall data from weather station of XTBG, 1st generation represented by green bars and 2nd generation by red bars in life history of pollinator. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
920
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 3. Morphology of Epicephala assamica at different developmental stages. A: Larvae; B: Cocoon; C: Pupa; D: Adult.
be interfered strongly. Mated females visit male flowers to collect pollen actively (Fig. 4B) by rubbing the proboscis against the anthers, the process lasts for 90–150 s and mated males have not been observed mating again and visiting female flowers. The females rub the proboscis to collect pollen back and forth, and store pollen to the base of proboscis via crimping proboscis efficiently, with a large number of pollen grains sticking to the base of proboscis (Fig. 4B, C). Pollen carriers don't pollinate for female flowers immediately instead just stay around male flowers or other places to “rest” with shaking their antennae, we consider that moths can locate hosts by discerning the odour of flowers via lots of receptors on antennae. One female collects pollen grains in only one male flower each time effectively, and visits approximately 10 female flowers sequentially, especially focusing on female flowers in the
Fig. 5. Comparison of time on pollination and oviposition between different times. Fig. 4. Behaviours of adult Epicephala assamica. A. Mating; B. Collecting pollen; C. Pollinating; D. Laying egg.
921
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Table 1 The ripening rate of Glochidion assamicum (September 2013–October 2014). Generations
Female flowers
Female flowers/cluster
Pollinated female flowers/cluster
Fruits
Fruits/cluster
Ripening rate
1st genenation 2nd genenation Sum Mean
2306 2311 4617 −
21.16 (n = 109) 18.34 (n = 126) − 19.65 (n = 235)
18.68 13.69 − 16.00
780 1581 2261 −
7.16 12.55 − 10.05
33.83% 68.42% − 51.14%
same cluster. Upon visiting female flowers (Fig. 4C), a female uncoils its proboscis and pushes the proboscis tip repeatedly into the stylar pit to pollinate the flowers by recoiling the proboscis several times to replenish pollen grains on the proboscis tip with moving the pollen from the base to the terminal of proboscis. After pollination, the female moth bends abdoman to basal calyx on ventral surface and inserts ovipositor through calyx lobes and ovary to lay eggs into ovary tissue (Fig. 4D). The pollination is both observed inside homophyletic plant and between different individuals. Epicephala moths are all pollen carriers which captured on female flowers examined under the Stereo Microscope. With the increasing frequency of pollination and oviposition, the homologous time consuming each time will increase (Fig. 5).
(Paired-samples Test, P (df = 4) = 0.02), the proportion of male moths is 0.64, 0.57 and 0.60, respectively, 0.62 as an average (Fig. 6D). The morphological characteristics of immature stage on Epicephala assamica Early instar larva (Fig. 3A): body eruciform, the newly-hatch larva is white translucent, the early instar larva has no crochets in proleg. Later instar larva has crochets in proleg, head capsule dark brown. Mature larva (Fig. 3A, 7A − J): body eruciform, with three pairs of well-developed thoracic legs and four pairs of fleshy ventral prolegs on abdominal segments III − V and X (caudal proleg). Head hypognathous. Length 5.52 ± 0.05 mm (n = 5) breadth of head capsule 0.65 ± 0.004 mm (n = 5). Head dark red, with dark brown patches in epicranial region and an ellipse of six ocelli. Prothoracic plate same as ground color, with an ivory triangle patch. Segment I dark red in anterior 1/3, ivory in posterior 2/3; segment II − VII ivory in anterior 1/5 and posterior 1/5, dark red in middle; segment VIII ivory in anterior 1/3 and dark red in posterior 2/3; segment IX − X dark red. Head chaetotaxy (Fig. 7A− E): coronal suture 1⁄ 3 length of frontal sclerite; frontal sclerite extended to almost 1 ⁄ 2 of head length, and nearly as broad at base as long (Figs. 7A). Distance between ocelli 1 and 6 slightly less than distance between ocelli 1 and 2, and distance between ocelli 5 and 6 nearly 1.5 × of distance between ocelli 1 and 2; ocelli 2 and 3 contiguous, and ocelli 3 and 4 close to each other; distance between ocelli 4 and 5, ocelli 5 and 6, equal in length (Fig. 7A, B). A-group trisetose with one puncture Aa, A1, A2 and A3, forming an acute triangle, A2 antero-dorsal ocelli 2, A3 antero-dorsal ocelli 1, A1 nearly 2⁄ 3 length of A3; A2 almost 4⁄ 7 length of A1; P-group bisetose with one puncture Pa, P1 and P2, P2 antero-ventral to P1, length of P1 almost 2.5 × of length of P2 or slightly less, Pa postero-dorsal to P 1; Lgroup unisetose with La. L1 antero-dorsal to A3, about 1/5 length of A3, La postero-dorsal to L1; V-group with three minute setae arranged in a vertical curve line, with one puncture Va, V1, V2 and V3 nearly equal in length; O-group with three setae O1, O2 and O3, with one puncture Oa, O1 postero-dorsal to ocelli 2, O2 postero-dorsal to ocelli 1, O3 between ocelli 1 and 6, O1 and O2 nearly equal in length and half length of O3; SO-group trisetose, SO1, SO2 and SO3, forming an obtuse triangle, with one puncture SOb, SO1 postero-ventral to ocelli 4, outside ocelli blotch, SO2 closer to ocellia 4 than to ocellia 5, SO3 posterior SO2, SO1 nearly 1/2 length of SO3, SO2 nearly 4/7 length of seta SO3; G-group unisetose with one puncture Ga, G1 postero-dorsal to SO3, Ga posterodorsal to G1, G1 and SO2 nearly equal in length; F-group with one seta F1 and one puncture Fa, Fa ventral to F1. C-group bisetose, C1 posteroventral to C2; C1 and C2 of nearly equal length, slightly more than F1, distance between C1 and C2 less than distance between F1 and C1. Mouthparts (Fig. 7C−E): labrum (Fig. 7C): three median setae M1, M2 and M3, M2 nearly equal to length of M3; M2 slightly more than
The ripening rate and consumption of seeds In 1st generation, ripening rate is only 33.83% (Table 1). Meanwhile, each fruit has 7.19 seeds (n = 426, in theory, 8 seeds each fruit, a few seeds without pollination abortive), 83.57% (n = 356) fruits are consumed by larvae. In the consumed fruits, 98.31% fruits consumed by only one larva (n = 350), 1.69% fruits consumed by two larvae and no fruit consumed by three or more larvae. Each larva consumes an average of 2.49 seeds (Table 2). In 2nd generation, ripening rate of 1st generation is 68.42% (Table 1). Meanwhile, each fruit has 6.75 seeds (n = 868, in theory, 8 seeds each fruit, a few seeds without pollination abortive), 77.53% (n = 673) fruits are consumed by larvae. In the consumed fruits, 92.27% (n = 621) fruits consumed by only one larva, 7.13% (n = 48) fruits consumed by two larvae, 0.59% (n = 4) fruits consumed by three larvae and no fruit consumed by four or more larvae. Each larva consumes an average of 2.67 seeds (Table 2). On average, the ripening rate is 51.14%, the rate of consumed fruits is 79.52%. In the consumed fruits, 94.36% fruits consumed by only one larva (n = 971), 5.25% (n = 54) fruits consumed by two larvae, 0.39% (n = 4) fruits consumed by three larvae and no fruit consumed by four or more larvae, each larva consumes 2.62 seeds to finish their larval stage. Overall, the proportion of intact seeds is 68.03% that can keep the stabilization of mutualism in each population (Table 2). Sex ratio of Epicephala assamica We collect mature fruits until larvae exit the fruits pupated and emerge as adults from 30 August 2013 to 17 September 2013 (Fig. 6A), from 4 March 2014 to 26 March 2014 (Fig. 6B) and from 12 September to 2 October 2014(Fig. 6C), respectively. Overall, we rear 236 male moths and 145 female moths. The study results of cumulative three different generations explained that male moths emerge as adults before than female moths and more male-biased offspring sex ratio Table 2 Fruits consumed by Epicephala assamica (September 2013–October 2014). Gen.
Fruits
Consumed fruits
Rate of consumed fruits
Seeds
Consumed seeds
Rate of consumed seeds
Larvae
Seeds consumed by one larva
1st gen 2nd gen Sum Mean
426 868 1294 −
356 673 1029 −
83.57% 77.53% − 79.52%
3064 5861 8925 −
903 1950 2853 −
29.47% 33.27% − 31.97%
362 729 1091 −
2.49 2.67 − 2.62
922
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 6. Sex Ratio of Epicephala assamica. A. The result of eclosion for the first time; B. The result of eclosion for the second time; C. The result of eclosion for the third time; D. The result of eclosion for the three times.
of L1; SV-group unisetose with SV, SV antero-dorsal to prolegs, SV1 slightly less than length of L2; V-group unisetose with V, close to ventrimeson; MV-group unisetose with MV, setae MV and V nearly equal in length. In segments A9, D-group bisetose, D1 and D2, D2 postero-dorsal to D1, nearly equal to length of D1; SD-group unisetose with SD1, D1 postero-dorsal to SD1 and slightly less than SD1; crochets of proleg (Fig. 7J): ventral prolegs on A3 to A5 and A10, 11 crochets uniserial, arranged in uniordinal crochet. Cocoon (Fig. 3B): white, 6.82 ± 0.07 mm (n = 12), oblong, transparent, with numbers of white spherical grains attached on surface which generally could be divided into six small rooms, there was a piece of silk under the grain went into the cocoon. Pupa (Figs. 8A, B): maximum length 5.0 mm (n = 13), breadth of head breadth 0.08 mm. Antenne exceeds segment X; yellowish green right after pupation, gradually turning yellowish brown, becoming dark brown right before emergence, slightly darker in ventral surface of thorax, caudal abdominal segments orangish. Vertex with cocoon cutter, all spiracles of nearly the same size. Forelegs extending to near posterior margin of segment III; midlegs extending to near posterior margin of segment IV; hindlegs have larger variation, extending near to end posterior margin of segments VI − VIII.
length of M1. Three epipharyngeal setae E1, E2 and E3, nearly equal length. Mandible (Fig. 7D): with four distinct pointed teeth along cutting margin (second and third one equal in size, the first and fourth tooth smaller); MD2 1⁄ 3 length of MD1. Maxillae and labium (Fig. 7E): spinneret pointed anteriorly, 1.5 × as long as breadth at base, 3× length of first segment of labial palpus; LB1 nearly equal to length of LB3, MX2 slightly less than length of MX1; MX3 slightly less than length of MX2; MX6 almost 1/2 length of MX1; MX3 and LB1 almost equal in length. Prothorax (Fig. 7F): XD-group unisetose, XD2 absent; D-group bisetose, D1 and D2, D1 anterior-dorsal D2, D1 postero-dorsal XD1, distance between D1 and D2 almost 2 × of distance between D1 and XD1; XD1 slightly > 3 × length of D1, D2 slightly > 1.5 × of length of D1; SD-group bisetose, SD1 postero-ventral to SD2, about twice length of SD2. L-group bisetose, L1 antero-dorsal to spiracle, nearly 4 × length of L2 or slightly less; SV-group bisetose, SV1 postero-ventral to SV2, SV1 almost 3× as long as SV2; V-group unisetose with V, postero-ventral to coxacava, close to ventrimeson; MV-group with three minute setae, MV1, MV2 and MV3; setae V1, MV1, MV2 and M3 nearly equal in length. Mesothorax and Metathorax (Fig. 7G): XD-group absent; D-group bisetose, D1 and D2, D1 antero-dorsal to D2, nearly 1/3 length D2; SDgroup bisetose, SD2 antero-dorsal to SD1, SD1 3 × length of SD2 or slightly more; L-group bisetose, L1 antero-ventral to L3 and slightly > 3 × length of seta L3; SV-group unisetose with SV1, SV1 antero-ventral to L3 and slightly less than length of L1. V-group unisetose with V, postero-ventral to coxacava, close to ventrimeson (imaginary line along the ventral surface of the body); MV-group with three minute setae, MV1, MV2 and MV3; setae V1, MV1, MV2 and M3 nearly equal in length. Abdomen (Figs. 7H, I): segments A1 to A8: D-group bisetose, D1 and D2, D1 postero-dorsal to D2 and slightly more than length of D2; SDgroup bisetose, SD1 postero-ventral to SD2, SD1 slightly > 4.5 × length of SD2; distance between SD1 and SD2 equal to distance between D1 and D2; distance between SD1 and D2 2.5 × of distance between D1 and D2; L-group bisetose, L1 antero-dorsal to L2, nearly equal to length
Discussion Epicephala assamica have 2 generations correspondingly which are closely consistent with flowering phenology of G. assamicum, peak in March–April and September–October, respectively. Flowering periods are in the border of the rainy and dry seasons in our study areas, the phenology of host-plant is closely related to climate of geographical distribution, we hypothesize that the period is favorable for pollinators to pollinate for host plants on account of the long-term co-adaptation between the local climate environment and host plants. Pollinated female flowers don't develop during the flower-fruit interval season, so it cannot provide food and living space for the E. assamica. The first interval season lasts from late May to late August is peak of rainy season, high temperature and humidity; the second interval season from late 923
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 7. Morphology of Epicephala assamica mature larva. A. Frontal view of head capsule; B. Lateral view of head capsule; C. Labrum; D. Mandible; E. Maxillae and labium; F. Prothorax; G. Mesothorax; H. Abdominal segment 3; I. Abdominal segment 9; J. Crochets of proleg. (Scales = 0.1 mm).
2016), fruit set of G. assamicum is almost same to other species. The reason may be that Glochidion plants abscise a fraction of their pollinated flowers as a result of resource limitation occurring after pollination (Goto et al., 2010), may also be aborted due to damage caused by enemy insects or related to the rainy season of high temperature, high humidity, composite can be concluded that the results of the above three aspects. The vast difference of ripening rate between 1st and 2nd generations is most likely due to the complex environment of rainy interval season rather than efficiency of pollination between two generations, because the number of pollinated female flowers/cluster is higher than 2nd generation. In conclusion, the offspring populations keep stability by means of efficient pollination mechanism. The above conclusions suggest that pollinators are very sensitive to the change of host-plants. Life history, habits of E. assamica and phenology of G. assamicum are closely related to climate of study sites. With the increasing frequency of pollination and oviposition, the homologous time each
November to next February is foggy-cool season, low temperature and foggy (Zhu, 1993). E. assamica in the form of egg exists in the ovary during those complex environments, the protection of plant tissue not only reduces the rate of the destroyed by natural enemies but also provides a relatively stable temperature and humidity for the preservation of dormant eggs, to reduce water evaporation from eggs to the dry atmosphere or keep appropriate humidity from eggs to the moist atmosphere. Most importantly, moths and seeds keep development synchronously. In the known obligate pollination mutualisms such as figs-fig wasp, yuccas-yucca moths and Glochidion-Epicephala, figs, yuccas and Glochidion plants depend exclusively on pollination of fig wasps, yucca moths and Epicephala respectively, while sacrificing some seeds for larvae and keeping some seeds for breeding (Janzen, 1979; Weiblen, 2002; Zhang et al., 2012b). Although the number of female flowers each cluster (n = 19.65) at peak anthesis (March) is higher than other species (Goto et al., 2010; Wang and Li, 2015; Zhang et al., 924
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Fig. 8. Pupa of Epicephala assamica. A. ventral view; B. lateral view.
influence of sex ratio on the population structure, mating patterns and the insect society (Charnov, 1979; Bulmer, 1986). So the results can help us understanding the pattern and mechanism of sex ratio allocation in natural condition, and the evolution mechanisms of mutualistic symbiosis between Glochidion and Epicephala as well. In insects, morphological studies of immature stages can play an important role in pest control and taxonomic and phylogenetic analyses (Van Emden, 1957; Huemer and Karsholt, 1999; Hebert et al., 2004; Piao, 2006; Kristensen et al., 2007;). The larval morphological study is important for the phylogenetic classification and the morphological identification in the larval stage. Early instar larvae of E. assamica feed seeds inside the fruit and the security of closed space is high, maybe this is the reason that the body color of early instar larvae is white. However, mature larvae need to drill out from ripe fruits and to cocoon in host-plant surroundings. Larvae creep slowly and exposed during the period, so the larval body color evolves into the red and white warning colouration (Fig. 3A) in evolutionary process to help to increase the survival probability of larvae. From a taxonomic perspective, because morphology generally differs among instars in insect larvae (e.g., chaetotaxy), the identification of larval instars is necessary for comparing homological characters among species (Kitching, 1984; Miller, 1991; Solodovnikov, 2007). Characters of larval chaetotaxy, i.e. the number and ordering of larval setae and pores, have particular potential in systematics (Kitching, 1984; Miller, 1991; Solodovnikov, 2007; Mutanen et al., 2009), in particular crochets in proleg within Lepidoptera. Epicephala moths are hardly distinguishable by superficial
time will increase (Fig. 5), we think this may be associated with fewer and fewer pollen grains in proboscis, worse and worse location that female flowers be oviposited in the same cluster. On the other hand, abortive seeds may be associated with the phenomenon. The study reports sex ratio of Epicephala moths firstly, males emerge before than females and more male-biased offspring sex ratio (P < 0.05) in E. assamica. In theory, in the random mating populations, natural selection support parental genetic investment between male and female offsprings is equal, only sex ratio 1:1 is evolutionary stable sex ratio (Fisher, 1930). In order to reproduce efficiently, more male-biased or more female-biased offspring sex ratio exists in some spider and insect groups (Hamilton, 1967; Charnov, 1982; Haidy, 2002). Sex ratio evolution is closely related to resource allocation, and some studies in the literature have dealt with how resource allocation affects sex allocation under different male and female function relationships (Zhang and Wang, 1994). In tightly coevolved mutualism between Glochidion and Epicephala, moth that no cilia found on the proboscis is considered to be non-pollinator (Kato et al., 2003; Li et al., 2015), which suggests that the males of Epicephala are unproductive for host-plants. We consider male-biased offspring sex ratio can make sure that female moths have more mating opportunities, because the Epicephala moths captured from Glochidion plants in the field are usually more females than males, and mated female Epicephala moths prefer the pollen-collecting behaviour than unmated moths (Okamoto et al., 2013). The study of sex ratio is important for understanding the causes and consequences of selective pressure, and understanding the 925
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
Press, Cambridge, pp. 438. Hamilton, W.D., 1967. Extraordinary sex ratios. Science 156, 477–488. Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl. Acad. Sci. U. S. A. 101, 14812–14817. Herre, E.A., West, S.A., Cook, J.M., Compton, S.G. Kjellberg, F., 1997. Fig wasp mating system: pollinators and parasites, sex ratio adjustment and male polymorphism, population structure and its consequences. In: Social Competition and Cooperation in Insects and Arachnids. Vol. I: The Evolution of Mating Systems (Ed. by J. Choe and B. Crespi), Cambridge: Cambridge University Press. pp. 226 − 239. Herre, E.A., Knowlton, N., Mueller, G., Rehner, S.A., 1999. The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends Ecol. Evol. 14, 49–53. Hu, B.B., Wang, S.X., Zhang, J., Li, H.H., 2011. Taxonomy and biology of two seedparasitic gracillariid moths (Lepidoptera, Gracillariidae), with description of a new species. ZooKeys 83, 43–56. Huemer, P., Karsholt, O., 1999. Gelechiidae I (Gelechiinae: Teleiodini, Gelechini). Microlepidoptera of Europe. (ed. by Huemer, P., Karsholt, O., Lyneborg, L.), Vol. 3, pp. 1–356. Apollo Books, Stenstrup. Janzen, D.H., 1979. How many babies do figs pay for babies? Biotropica 11, 48–50. Kato, M., Takimura, A., Kawakita, A., 2003. An obligate pollination mutualism and reciprocal diversification in the tree genus Glochidion (Euphorbiaceae). Proc. Natl. Acad. Sci. 100 (9), 5264–5267. Kawahara, A., Plotkin, D., Ohshima, I., Lopen-Vaamonde, C., Houlihan, P., Breinholt, J.W., Kawakita, A., Xiao, L., Regier, J.C., Davis, D., Kumata, T., Sohn, J.C., De Prins, J., Miller, C., 2017. A molecular phylogeny and revised higher-level classification for the leaf-mining moth family Gracillariidae and its implications for larval host-use evolution. Syst. Entomol. 42, 60–81. Kawakita, A., Kato, M., 2004. Evolution of obligate pollination mutualism in new Caledonian Phyllanthus (Euphorbiaceae). Am. J. Bot. 91 (3), 410–415. Kawakita, A., Kato, M., 2006. Assessment of the diversity and species specificity of the mutualistic association between Epicephala moths and Glochidion trees. Mol. Ecol. 15 (12), 3567–3581. Kawakita, A., Kato, M., 2016. Revision of the Japanese species of Epicephala Meyrick with descriptions of seven new species (Lepidoptera, Gracillariidae). ZooKeys 568, 87–118. Kawakita, A., Mochizuki, K., Kato, M., 2015. Reversal of mutualism in a leafflower–leafflower moth association: the possible driving role of a third-party partner. Biol. J. Linn. Soc. 116 (3), 507–518. Kitching, I.J., 1984. The use of larval chaetotaxy in butterfly systematics, with special reference to the Danaini (Lepidoptera: Nymphalidae). Syst. Entomol. 9, 49–61. Kristensen, N.P., Scoble, M., Karsholt, O., 2007. Lepidoptera phylogeny and systematics: the state of inventorying moth and butterfly diversity. Zootaxa 1668, 699–747. Kumata, T., 1978. A new stem-miner of alder in Japan, with a review of the larval transformation in the Gracillariidae (Lepidoptera). Insecta Matsumurana 13, 1–27. Lee, C.F., Satô, M., Shepard, W.D., Jach, M.A., 2007. Phylogeny of Psephenidae (Coleoptera: Byrrhoidea) based on larval, pupal and adult characters. Syst. Entomol. 32, 502–538. Li, B.T., Gilbert, M.G., 2008. Flora of China. 11. Missouri Botanical Garden Press, USA, pp. 200. Li, H.H., Yang, X.F., 2015. Three new species of Epicephala Meyrick (Lepidoptera, Gracillariidae) associated with Phyllanthus microcarpus (Benth.) (Phyllanthaceae). ZooKeys 484, 71–81. Li, H.H., Zhang, Z.G., 2016. Five species of the genus Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) from China. Zootaxa 4084 (3), 391–415. Li, H.H., Zheng, Z.M., 1996. Methods and techniques of specimens of Microlepidoptera. J. Shaanxi Norm. Univ. (Natural Science Edition) 24 (3), 63–70 (in Chinese). Li, H.H., Wang, Z.B., Hu, B.B., 2015. Four new species of Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) associated with two Glochidion plants (Phyllanthaceae). ZooKeys 508, 53–67. Luo, S.X., Yao, G., Wang, Z.W., Zhang, D.X., Hembry, D., 2017. A novel, enigmatic basal leafflower moth lineage pollinating a derived leafflower host illustrates the dynamics of host shifts, partner replacement, and apparent coadaptation in intimate mutualisms. Am. Nat. http://dx.doi.org/10.1086/690623. Meyrick, E., 1880. Descriptions of New Zealand micro-lepidoptera III, Tineina. Proc. Linnean Soc. NSW 5 (1), 132–182. Miller, J.S., 1991. Cladistics and classification of the Notodontidae (Lepidoptera: Noctuoidea) based on larval and adult morphology. Bull. Am. Mus. Nat. Hist. 204, 1–230. Mutanen, M., Ruotsalainen, H., Kaila, L., 2009. Variation in larval chaetotaxy in Orthosia gothica (Lepidoptera: Noctuidae): effects of mother, sex and side, and implications for systematics. Syst. Entomol. 34, 712–723. Okamoto, T., Kawakita, A., Kato, M., 2007. Interspecific variation of floral scent composition in Glochidion and its association with host-specific pollinating seed parasite (Epicephala). J. Chem. Ecol. 33, 1065–1081. Okamoto, T., Kawakita, A., Goto, R., Svensson, G.P., Kato, M., 2013. Active pollination favours sexual dimorphism in floral scent. Proc. R. Soc. B Biol. Sci. 20132280. Peng, Y.Q., Zhang, Y., Compton, S.G., Yang, D.R., 2014. Fig wasps from the centre of figs have more chances to mate, more offspring and more female-biased offspring sex ratios. Anim. Behav. 98, 19–25. Piao, M.H., 2006. Morphological descriptions of three species larvae of Tortricidae (Lepidoptera) from Korea. Entomotaxonomia 28 (3), 201–208. Regier, J.C., Mitter, C., Zwick, A., Bazinet, A.L., Cummings, M.P., Kawahara, A.Y., Sohn, J.C., Zwickl, D.J., Cho, S., Davis, D.R., Baixeras, J., Brown, J., Parr, C., Weller, S., Lees, D.C., Mitter, K.T., 2013. A large-scale, higher-level, molecular phylogenetic study of the insect order Lepidoptera (moths and butterflies). PLoS One 8 (3), e58568.
characters of adults (Li and Zhang, 2016), as a result, larval characteristics can provide new additional implication for phylogenetic analysis, especially crochets of prolegs and chaetotaxy in Epicephala moths. On the other hand, pupal characterss are not species restricted that cannot be taken as the basis of interspecific classification. The larvae exhibit the characteristic adaptions associated with life mode that closely related to their host plants in the long-term natural selection, and showed species diversity (Thompson and Pellmyr, 1992), especially within interaction between insects and host plants. Therefore, the basic aspects of larval morphology, including intraspecific variation, effects of relatedness between pollinators and hosts, and the necessity of immature stages for the phylogenetic classification and the morphological identification in the insect study will be investigated in future to emphasize the importance of immature stages. Conclusion The study helps to further understand the evolutionary mechanism in the obligate pollination mutualism between Epicephala moths and Glochidion plants, and provides a scientific basis for the protection of the tropical rainforest ecosystem. One survival strategy of E. assamica is spending the flower-fruit interval season in G. assamicum in the form of eggs and synchronizing eclosion with the flowering phenology. Morphological and biological studies of E. assamica can contribute to understanding the evolutionary mechanisms in Glochidion-Epicephala mutualism. Acknowledgements The authors thank Professor D.R. Yang, Professor Y.Q. Peng and all members of Key Laboratory of Tropical Forest Ecology (XTBG) for their support and kind help in our field experiment. We also thank Dr. Z.B. Wang, Dr. X.F. Yang (NKU) for interesting discussion on the early draft of the manuscript. We specially thank Dr. Jurate De Prins (Belgium) for her review and comments on the manuscript. This research was supported by a grant from the National Natural Science Foundation of China (No. 31672372 & 30930014) and all the experiments comply with the current laws of China. References Archangelsky, M., 2004. Higher-level phylogeny of Hydrophilinae (Coleoptera: Hydrophilidae) based on larval, pupal and adult characters. Syst. Entomol. 29, 188–214. Axelrod, R., Hamilton, W.D., 1981. The evolution of co-operation. Science 211, 1390–1396. Bronstein, J.L., 2001. The exploitation of mutualisms. Ecol. Lett. 4, 277–287. Bull, J.J., Rice, W.R., 1991. Distinguishing mechanisms for the evolution of co-operation. J. Theor. Biol. 149, 63–74. Bulmer, M.G., 1986. Sex ratios theory in geographically structured populations. Heredity 56, 69–73. Charnov, E.L., 1979. The genetical evolution of patterns of sexuality: Darwinian fitness. Am. Nat. 113, 465–480. Charnov, E.L., 1982. The Theory of Sex Allocation. Princeton University Press, Princeton, pp. 355. Chenoweth, L.B., Fuller, S., Tierney, S.M., Park, Y.C., Schwarz, M.P., 2008. Hasinamelissa: a new genus of allodapine bee from Madagaskar revealed by larval morphology and DNA sequence data. Syst. Entomol. 33, 700–710. Davis, D.R., 1987. Gracillariidae. In: Stehr, F.W. (Ed.), Immature Insects. Vol. 1. Kendall/ Hunt Publ. Co., Dubuque, Iowa, pp. 372–374 376–378. De Prins, W., De Prins, J., 2016. Global taxonomic database of Gracillariidae (Lepidoptera). [2016 −9 − 18]. http://www.gracillariidae.net. Di Giulio, A., Fattorini, S., Kaupp, A., Taglianti, A.V., Nagel, P., 2003. Review of competing hypotheses of phylogenetic relationships of Paussinae (Coleoptera: Carabidae) based on larval characters. Syst. Entomol. 28, 509–537. Fisher, R.A., 1930. The Genetical Theory of Natural Selection. Oxford University Press, Oxford. Godfray, H.C.J., 1994. Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton, New Jersey. Goto, R., Okamoto, T., Kiers, E.T., Kawakita, A., Kato, M., 2010. Selective flower abortion maintains moth cooperation in a newly discovered pollination mutualism. Ecol. Lett. 13, 321–329. Haidy, L.C.W., 2002. Sex Ratio: Concepts and Research Methods. Cambridge University
926
Journal of Asia-Pacific Entomology 20 (2017) 918–927
Z. Zhang, H. Li
44, 65–75. Zhang, J., Hu, B.B., Wang, S.X., Li, H.H., 2012a. Six new species of Epicephala Meyrick, 1880 (Lepidoptera: Gracillariidae) associated with Phyllanthaceae plants. Zootaxa 3275, 43–54. Zhang, J., Wang, S.X., Li, H.H., Hu, B.B., Yang, X.F., Wang, Z.B., 2012b. Diffuse coevolution between two Epicephala species (Gracillariidae) and two Breynia species (Phyllanthaceae). PLoS One 7 (7), e41657. Zhang, Z.G., Teng, K.J., Li, H.H., 2016. Biological characteristics of Epicephala ancylopa (Lepidoptera: Gracillariidae) on host Glochidion sp. (Phyllanthaceae) and the compositional analysis of its floral scent. Acta Entomol. Sin. 59, 669–681. Zhu, H., 1993. The characteristics of the Xishuangbanna tropical rainforest flora. Trop. Geogr. 13 (4), 149–155.
Solodovnikov, A.Y., 2007. Larval chaetotaxy of Coleoptera (Insecta) as a tool for evolutionary research and systematics: less confusion, more clarity. J. Zool. Syst. Evol. Res. 45, 120–127. Thompson, J.N., Pellmyr, O., 1992. Mutualism with pollinating seed parasites amid copollinators: constraints on specialization. Ecology 73 (5), 1780–1791. Van Emden, F.J., 1957. The taxonomic significance of the characters of immature insects. Annu. Rev. Entomol. 2, 91–106. Wang, Z.B., Li, H.H., 2015. The biology and survival strategy of Epicephala sp. (Lepidoptera: Gracillariidae). Chin. J. Appl. Entomol. 52 (1), 162–172. Weiblen, G.D., 2002. How to be a fig wasp. Annu. Rev. Entomol. 47, 299–330. Zhang, D.Y., Wang, G., 1994. Evolutionarily stable reproductive strategies in sexual organisms: an integrated approach to life-history evolution and sex allocation. Am. Nat.
927