T REPRO N DU The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.9-14 LA C P T I F V O E
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S H Functional floral morphology of two medicinal Mikania (Asteraceae) T species and their floral visitors
1* Marinês Eiterer , Adriano Valentin-Silva1*, Nelson S. Bittencourt Junior2 and Milene F. Vieira1
1Federal University of Viçosa, Department of Plant Biology, s/n P.H. Rolfs Ave., 36.570-900, Viçosa, MG, Brazil. 2UNESP – São Paulo State University, Institute of Biosciences, Letters and Exact Sciences, 2265 Cristóvão Colombo St., 15.054-000, São José do Rio Preto, SP, Brazil. *e-mail : [email protected] Received : 11. 09. 2018; Accepted and published online: 15.11.2018 ABSTRACT Mikania species, including the ones addressed in this study (M. glomerata and M. hirsutissima), are widely used as medicinal plants. In order to subsidize projects for the management and conservation of those two species, studies on their flower functionality, which involves several events that culminate in pollination, are highly necessary. We aimed to analyze the functional floral morphology of both species, including secondary pollen presentation (SPP), stigmatic receptivity and pollen grain viability. Additionally, we identified the species floral visitors and the potential pollinators among them. We studied natural populations from a seasonal semi-deciduous forest (Viçosa municipality, Minas Gerais state, southeastern Brazil) in 2003 and 2004. Flowers of both species are nectariferous, odoriferous, and open in the morning. SPP occurred in flower buds at pre- anthesis, with pollen (100% viable) being deposited on the dorsal portion of style branches, on sterile appendages, characterizing the beginning of the staminate phase. The beginning of the pistillate phase was characterized by gradual exposure of the four receptive stigmatic lines, on the third day of flower life; this phase could be long-lived, lasting up to 11 days. Stingless bees (meliponines) and Apis mellifera were the main visitors (> 50% of visitation frequency) and were therefore considered potential pollinators. Thus, the floral attributes of M. glomerata and M. hirsutissima, along with the main group of insect visitors (social bees), characterize melittophily. Nevertheless, the high diversity of insects observed on the species flowers indicates that the range of pollinators may be wider. Keywords : Mikania glomerata, Mikania hirsutissima, secondary pollen presentation, social bees, stigmatic areas. Mikania Willd. is large genus of the Asteraceae (430 several events that culminate in pollination, are necessary. An species; Cerana 1997) that occurs predominantly in the example of such studies is the one already performed by Hong Neotropical region (Robinson and King 1977). A total 201 et al. (2008) on M. micrantha Kunth. Those authors described species occur in Brazil (Flora do Brasil 2018), most of them the functional floral morphology of that species, including having a climbing habit and occurring in the Atlantic Forest secondary pollen presentation, style branch morphology, (Barroso 1958); only a few species inhabit other Brazilian stigmatic receptivity, and pollen grain viability. biomes, like the Amazon Forest (Holmes and Pruski 2000). On Other studies on species of the genus have focused on the other hand, representatives with erect herbaceous habit further reproductive aspects. Holmes (1991) reported the occur in the cerrado (Brazilian savanna) and in high altitude occurrence of gynodioecy in seven insular species. Cerana grasslands (Barroso 1958). (2004) reported autogamy and allogamy to M. urticifolia Some Mikania species are widely used as medicinal Hook. & Arn., a native species to Argentina, and emphasized plants. For instance, the species addressed in this study, M. that both systems coexist and are complementary; the species glomerata Spreng. and M. hirsutissima DC., have been is visited by several insects. Hong et al. (2007) reported self- officialized as phytopharmaceuticals as early as in the first incompatibility to M. micrantha, with the consequent edition of the Pharmacopeia of the United States of Brazil dependency on insect pollinators for the occurrence of (details in Lima et al. 2003). Mikania glomerata is widely fruiting. used in the treatment of respiratory diseases due to its high We aimed to analyze the functional floral morphology of contents of coumarin and kaurenoic acid, the major M. glomerata and M. hirsutissima, including secondary pollen metabolites with pharmacological activity found in the presentation (SPP), stigmatic receptivity and pollen grain species leaves (Czelusniak et al. 2012). Inflorescences of M. viability, as well as to identify floral visitors and the potential hirsutissima are used to relieve low back pain and to treat pollinators among them. kidney diseases (Brasileiro et al. 2006). MATERIAL AND METHODS Therefore, knowing the sexual reproduction of these plants is important to subsidize the development of Study area and studied species—We conducted the study in management and conservation plans for them. For that, 2003 and 2004, at the forest fragment “Station of Research, baseline studies on their flower functionality, which involves Environmental Training and Education Mata do Paraíso” 10 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.9-14
(hereafter, Mata do Paraíso). Mata do Paraíso is located at In 2004, we recorded the frequency of floral visitors in Viçosa municipality (20°48’07" S, 42°51’31" W), in the Zona five individuals of M. glomerata and four of M. hirsutissima. da Mata region of Minas Gerais state, southeastern Brazil, and On each plant, at regular 10-min intervals, we recorded the extends over 194 ha, having altitudes ranging between 690 presence of insects from 7:00 a.m. through 5:00 p.m., for 17 and 870 m. The site is the largest forest fragment in Viçosa days. These observations totalized 20 h equally distributed (Silva et al. 2014), and its vegetation belongs to the seasonal between the morning and afternoon periods, throughout the semideciduous montane forest formation (Veloso et al. 1991). entire flowering season. Mikania glomerata and M. hirsutissima both have wide We captured floral visitors and analyzed them to verify geographic distribution, occurring in midwestern, northeastern, southeastern and southern Brazil, as well as in the site of pollen deposition on their bodies. Then, we prepared Argentina and Paraguay (Barroso 1958, Ritter and Miotto them following Borror and Delong (1988) to enable their 2005). In the study area, these two species commonly occur in identification by specialists. forest gaps and edges, especially alongside water courses. RESULT AND DISCUSSIONS Mikania glomerata is a woody canopy-liana; the species capitula have four greenish flowers and the apex of each petal Functional floral morphology—In flower buds at pre- is wine-colored; fruits (cypselae) are crowned by the setose anthesis, on both species, we observed anther opening and pappus, which along with the seed characterize the diaspore pollen deposition on the dorsal region of style branches, on the (dispersal unit). Mikania hirsutissima is a twining herbaceous sterile appendages, thereby charactering the brushing-type vine that climbs low vegetation; the species capitula also have SSP (sensu Leins and Erbar 1990) and the beginning of the four flowers, yet white; diaspores of this species resemble staminate phase (Fig. 1); pollen viability was high (100%). those of M. glomerata. Further details on the flower Flower opening occurred in the morning, from 7:00 a.m. morphology of both species can be found in Barroso (1958). through 9:00 a.m.; during that period, the style elongated and In both species, the long style branches have four ventral its branches exserted above the anther tube and corolla (Fig. marginal stigmatic lines (two lines per branch), which are 1), exposing pollen grains. The juxtaposed branches therefore restricted to the lower half of the branches (Fig. 1). On the functioned as a false stamen, as the anthers remained within upper half, the sterile style appendages are densely papillose, the corolla tube. We observed flowers with available pollen being responsible for SPP (details in Bremer 1994). between 9:00 a.m. and 11:00 a.m., and the staminate phase In the study site, flowering of M. hirsutissima took place lasted up to two days (flowers with residual pollen). These from August through September, while the one of M. floral events are similar to the ones reported to other glomerata occurred from September through October. Asteraceae species, including in the Mikania genus (Cerana Flowering periods thus occurred during the dry season, and on 2004, Hong et al. 2008). Similarities include flower opening average lasted from 20 (M. glomerata) to 22 (M. hirsutissima) in the morning (Proctor and Yeo 1978) and SPP, the latter of days. We deposited voucher specimens in the VIC Herbarium which is universal in the family (Howell et al. 1993, Yeo 1993, (28,871 and 29,071). Lane 1996). The extension of the staminate phase during the Functional floral morphology—We recorded the times of first two days of anthesis resembled the one reported to M. flower opening and SPP as well as the duration of the micrantha (Hong et al. 2008). staminate and pistillate phases. SPP was identified following SPP is associated with protandry, a type of dichogamy in the classification by Leins and Erbar (1990). We assessed which pollen grains are released from anthers before stigmas stigmatic receptivity by immersing style branches in a neutral are receptive (Lloyd and Webb 1986); in the studied species, red solution (Bessa et al. 2010). We analyzed pollen grain pollen release occurred at the end of pre-anthesis, in flowers viability using carmine acetic (Radford et al. 1974). For that, that were still closed. Because of SPP, pollen was exposed to we used 25 flower buds at pre-anthesis per species. From each visitors in small “doses”, thereby favoring optimized flower bud, we counted 200 pollen grains per glass slide, pollination (Jeffrey 2009). Exposure of the receptive stigmatic separating the viable ones (red staining) from the non-viable lines (positive result in the test with neutral red) characterized (yellow staining). We assessed the presence of nectar in the beginning of the pistillate phase (Fig. 1), on the third day of previously bagged flowers using glucose strips (Alamar Tecno flower life. Initially, style branches separated by acropetally Científica Ltd.), which test for the presence of glucose, one of turning away from one another, and posteriorly by flexing the main sugars in nectar (Baker and Baker 1983). toward the flower base (Fig.1). Gradual exposure of stigmatic Additionally, we assessed odor production by isolating lines with acropetal separation of style branches differed from flowers in glass flasks (Kearns and Inouye 1993). what has been observed in M. micrantha (Hong et al. 2008) Floral visitors—We observed floral visitors throughout the and M. urticifolia (Cerana 2004). Style branches in those day for 215 h, from August through September 2003 and 2004. species initially separated at the apex and posteriorly they We recorded the resources (nectar and, or pollen) used by each flexed toward the flower base, and only then the stigmatic visitor. 2019 Functional floral morphology of two Mikania species 11
lines got exposed. These differences are probably associated floral visitors were attracted during a period in which they with reproductive particularities of each species, including the were able to remove the already exposed pollen and perform visiting behavior of their pollinators. pollination in flowers at the pistillate phase. The pistillate phase could be longer than the staminate Floral visitors—We recorded 32 insect taxons visiting one in both M. glomerata and M. hirsutissima, lasting from flowers of Mikania species (23 in M. hirsutissima and 18 in M. two to 11 days. Flowers that were visited and pollinated at glomerata) distributed across five orders (Table 1). All insects early pistillate phase tended to have lower longevity. Higher collected nectar, except for bees from family Halictidae, longevity of the pistillate phase, as observed in the studied which collected pollen. We recorded 249 visits in M. species, is a common trait of the Asteraceae (Dieringer and hirsutissima and 228 in M. glomerata. In both species, visits Cabrera 1989, Mani and Saravanan 1999, Fonseca et al. started in the morning, at around 7:00 a.m., and extended until 2013), and the duration of this floral event in both species the afternoon, at around 3:00 p.m. (Fig. 2). Pollen and nectar, seems to depend on pollination, as reported by Fonseca et al. as reported herein, are the floral resources of Mikania species (2013). Those authors observed that pollinated receptive (Cerana 2004), being available to visitors during the dry flowers senesced on the second day of flower life and that non- season (Morellato and Leitão-Filho 1996, Ferreira et al. pollinated flowers lasted up to four days. 2009). Indeed, flowering of the studied species took place at Mikania hirsutissima exhaled a strong sweet odor, while the end of that season. That may be why the floral resources of M. glomerata produced a discrete sweet odor. The presence of both species have been reported as an important food source odor has also been reported to M. micrantha (Hong et al. for native bees from the Atlantic Forest (Wilms et al. 1996, 2008). On the other hand, Cerana (2004) did not report odor Antonini et al. 2006), and our results reinforce such production in M. urticifolia. Verification of odor production as importance. assessed herein is subjective; in that sense, pollinators of Among the Hymenoptera, bees showed the highest apparently non-odoriferous species may detect the emission species richness (nine) and the highest visitation frequencies. of floral volatiles that are supposedly imperceptible to human Bees altogether performed more than half the visits in flowers nose, yet that attract them (Farré-Armengol et al. 2013). The of the two species (78.1% in M. glomerata and 51.8% in M. test for glucose confirmed the presence of nectar in flowers of hirsutissima; Table 1). These insects were not only frequent, the two studied species. Both odor emission and nectar but they also had pollen grains deposited on the head frontal secretion started at the end of the staminate phase. Thereby, region and on the body ventral region, thus indicating that they
Fig. 1 – Representation of flower anthesis of Mikania glomerata and M. hirsutissima in longitudinal section. From left to right, the first four drawings represent the staminate phase while the other four represent the pistillate phase. an = anther, co = corolla, fi = filament, sl = stigmatic line, ne = nectary, ov = ovary, sb = style branch. 12 The International Journal of Plant Reproductive Biology 11(1) Jan., 2019, pp.9-14
are the main pollinators. The other hymenopterans (ants and (other bees) wasps) were little frequent (Table 1) and rarely came into Megachilidae – Megachilini Megachile sp.* contact with the stigmatic lines of the flower. Halictidae Apidae was the most representative bee family, especially Morphospecies 1 meliponines (tribe Meliponini), which were represented by Morphospecies 2 ** five species, four of which were common visitors of the 5.3 1.6 studied plant species, as was the exotic Apis mellifera (tribe (ants and wasps) Apini). This exotic bee species visited predominantly M. Formicidae glomerata (51.3% of visits against 10.0% in M. hirsutissima), Morphospecies 1 while meliponines visited predominantly M. hirsutissima Morphospecies 2* Vespidae (40.2% of visits against 21.5% in M. glomerata) (Table 1). Brachygastra sp.** Brachymeria sp. ** Table 1 – Floral visitors of Mikania glomerata and M. hirsutissima and Eumeninae sp. ** their visitation frequencies in an Atlantic Forest fragment from Viçosa Polybia sp. ** municipality, southeastern Brazil. Morphospecies 1** ORDER (common name) Visitation frequency (%) 7.0 7.6 Family – Tribe M. glomerata M. hirsutissima LEPIDOPTERA (butterflies) Species1 Nymphalidae COLEOPTERA (beetles) Adelpha sp. ** Dircenna sp.** Chrysomelidae Prepona sp. ** Morphospecies 1 Morphospecies 1** 2.2 2.0 Morphospecies 2* DIPTERA (flies) Lycaenidae Arawacus sp. * Syrphidae Unidentified Morphospecies 1* Morphospecies 1* Tachinidae 2.6 2.8 Morphospecies 1* 1 Unidentified One asterisk: floral visitors exclusive to M. glomerata; two asterisks: Morphospecies 1* visitors exclusive to M. hirsutissima; no asterisk: visitors of both species. Morphospecies 2** Morphospecies 3** We considered social bees to be potential pollinators of Morphospecies 4** M. glomerata and M. hirsutissima due to their species 7.0 17.7 richness, visitation frequency and to the site of pollen HEMIPTERA (shield bugs) deposition on their bodies. Bees, especially solitary ones, are Reduviidae considered to be the main pollinators of the Asteraceae (Lane Apiomerus sp. 1996, Jeffrey 2009), which contrasts with our results. Social Morphospecies 1** bees were also reported as the main pollinators of Tilesia 3.1 18.1 baccata (L.f.) Pruski (Fonseca et al. 2013), an Asteraceae HYMENOPTERA species that has been analyzed at the same area of our study. (melliferous bee) These results may be related to the fact that those bees are Apidae – Apini eusocial (thereby being active throughout the year) and Apis mellifera Linnaeus, 1758 polyletic (Minussi and Alves-Santos 2007, Nogueira and 51.3 10.0 Augusto 2007). The latter feature renders them consumers of (stingless bees) flower resources of representatives from several families, Apidae – Meliponini including the species studied herein. Melipona quadrifasciata The visitation frequency of insects from the other orders Lepeletier, 1836* altogether represented 14.9% in M. glomerata and 40.6% in Scaptotrigona xanthotricha M. hirsutissima. Each species was visited by distinct insect Moure, 1950 Scaptotrigona sp. groups from orders Diptera and Lepidoptera (Table 1, Fig. 2). Schwarziana quadripunctata Those insects also had pollen on their bodies and may (Lepeletier, 1836) therefore have acted as pollinators of the studied plant species. Trigona spinipes (Fabricius, Insects from orders Coleoptera and Hemiptera, on the other 1793) hand, tended to remain on the flower or inflorescence for long 21.5 40.2 periods, thus disfavoring their role as pollinators. 2019 Functional floral morphology of two Mikania species 13
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