Cryptic Self-Incompatibility and Distyly in Hedyotis Acutangula Champ

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Cryptic self-incompatibility and distyly in Hedyotis acutangula Champ. (Rubiaceae)

ARTICLE in PLANT BIOLOGY · MAY 2010 Impact Factor: 2.63 · DOI: 10.1111/j.1438-8677.2009.00242.x · Source: PubMed

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RESEARCH PAPER Cryptic self-incompatibility and distyly in Hedyotis acutangula Champ. (Rubiaceae) X. Wu, A. Li & D. Zhang Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China

Keywords ABSTRACT Distyly; Hedyotis acutangula; pollination; Rubiaceae; self-incompatibility; thrum Distyly, floral polymorphism frequently associated with reciprocal herkogamy, self- flowers. and intramorph incompatibility and secondary dimorphism, constitutes an important sexual system in the Rubiaceae. Here we report an unusual kind of distyly Correspondence associated with self- and ⁄ or intramorph compatibility in a perennial herb, Hedyotis D. Zhang, South China Botanical Garden, acutangula. Floral morphology, ancillary dimorphisms and compatibility of the two Chinese Academy of Sciences, Guangzhou morphs were studied. H. acutangula did not exhibit precise reciprocal herkogamy, 510650, China. but this did not affect the equality of floral morphs in the population, as usually E-mail: [email protected] found in distylous plants. Both pin and thrum pollen retained relatively high viability for 8 h. The pollen to ovule ratio was 72.5 in pin flowers and 54.4 in thrum Editor flowers. Pistils of pin flowers remained receptive for longer than those of thrum F. Roux flowers. No apparent difference in the germination rate of pin and thrum pollen grains was observed when cultured in vitro, although growth of thrum pollen tubes Received: 23 January 2009; Accepted: 22 was much faster than that of pin pollen tubes. Artificial pollination revealed that June 2009 pollen tube growth in legitimate intermorph crosses was faster than in either intramorph crosses or self-pollination, suggesting the occurrence of cryptic self-incom- doi:10.1111/j.1438-8677.2009.00242.x patibility in this species. Cryptic self-incompatibility functioned differently in the two morphs, with pollen tube growth rates after legitimate and illegitimate pollination much more highly differentiated in pin flowers than in thrum flowers. No fruit was produced in emasculated netted flowers, suggesting the absence of apomixis. Our results indicate that H. acutangula is distylous, with a cryptic self-incompatibility breeding system.

Heterostyly is believed to have developed as a mechanism INTRODUCTION to promote out-crossing (Darwin 1877). Reciprocal herkoga- Heterostyly is a genetically controlled flower polymorphism, my between morphs compels pollinators to contact the floral characterised by a reversal in the position of stigmas and organs in a particular position with the same part of their anthers in different individuals within a single population body, thus promoting intermorph pollination (Barrett 2002; (Darwin 1877; Barrett 1990). It has been estimated that het- Sanchez et al. 2008). These convergences of morphological erostylous flowers occur in at least 28 animal-pollinated flow- characters, i.e. reciprocal herkogamy and physiological mech- ering plant families, particularly in species with tubular anisms favouring intermorph pollination presented in disty- flowers (Darwin 1877; Ganders 1979; Lloyd & Webb 1992; lous species act, as an efficient mechanism to avoid self- and Barrett & Shore 2008). Heterostyly is associated with two or intramorph pollination (Lloyd & Webb 1992). However, the three morphological forms, i.e. distyly and tristyly, with vari- effectiveness of the reciprocity has been doubted due to the ations in style and filament length; there can also be differ- occurrence of different degrees of reciprocal herkogamy in ences in pollen grain morphology, and in the size of the natural populations of some species considered to be disty- stigmatic papillae and corolla (Massinga et al. 2005). The lous (Faivre & McDade 2001; Castro & Araujo 2004). Fur- main way in which heterostyly is manifest is as distyly, which thermore, fruits are not only produced by intermorph is found in more than 25 families, with new cases continu- crosses, indicating that the incompatibility mechanism in ously being reported (Eckert & Barrett 2008). In distyly, the distylous species does not always function strictly as in typical flowers of one morph have a long style and short stamens S-locus gene-controlled self-incompatibility, which mostly (pin morph), while the flowers of the other morph have a prevents self-fertilisation (Washitani et al. 1994; Castro & short style and long stamens (thrum morph). Although het- Araujo 2004; Takayama & Isogai 2005). There may thus be erostyly has received considerable attention, the origin, devel- another mechanism involved in the competitive growth of opment and transition stages leading to this polymorphism self, intramorph and outcross pollen tubes once they have still remain unsolved. reached the style. Previous studies have revealed that, once

484 Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands Wu, Li & Zhang Distyly in Hedyotis acutangula pollen grains have been transferred to the style in cross-polli- olla and are arranged in cymes. The petals unfold synchro- nation, pollen tube elongation is more rapid than that of nously at anthesis. Flowers open at about 07:00–08:30, and self-pollination pollen tubes, which is defined as cryptic self- last about 24 h. The anthers dehisce about 2 h before anthe- incompatibility, a phenomenon first identified 50 years ago sis. (Bateman 1956; Weller & Ornduff 1977; Alonso & Company Newly open flowers were collected and preserved in forma- 2005). Unlike species with absolute self-incompatibility, self- lin ⁄ acetic acid ⁄ alcohol (FAA). For 20 flowers in each popula- pollen tubes can arrive at the ovule and achieve successful tion, tube length, height of stigma, anther and stigma lobe fertilisation when competing cross-pollen is absent (Riveros were measured. One flower from each plant was measured. et al. 1987, 1995; Castro & Araujo 2004). However, there After corolla tube length had been recorded, flowers were have been few comprehensive demonstrations of cryptic self- dissected to allow stigma height, anther height and stigma incompatibility in distylous species. The factors that might be lobe length to be measured. Sigma and anther heights were involved in cryptic self-incompatibility, including differential measured using calipers. Corolla tube length was measured pollen viability, pollen tube growth and differences in papillae from the base of the corolla tube to the sinus. Stigma height cells on the stigma between morphs, are yet to be elucidated. was measured from the base of the corolla tube to the tip of Rubiaceae is the fourth largest angiosperm family and the stigma lobe. Anther height was measured from the base contains the largest number of distylous genera (Ganders of the corolla tube to the tip of the anther, since the fila- 1979). Based on previous classification systems and phyloge- ments are thin and attached to the sides of corolla tubes. netic analyses of the Rubiaceae, the genus Hedyotis L. is Stigma lobe length was measured from the apex of the style included in the tribe Hedyotideae (Spermacoceae) and sub- to the tip of the stigma lobes. The pollen–ovule ratio (P ⁄ O) family Rubioideae (Robbrecht 1988; Bremer et al. 1995, was estimated using the method described by Dafni (1992) 1999; Bremer & Manen 2000; Robbrecht & Manen 2006). In from 20 pin and 20 thrum plants in each population. The a recent phylogenetic analysis, the monophyletic status of pollen grains in one anther from each bud were carefully dis- tribe Spermacoceae has been strongly supported, but the sected out, and transferred to calibrated tubes containing genus Hedyotis has been suggested only to include the Asian 0.5 ml distilled water. The suspension was stirred and 10–15 species in a clade that is sister to the clade that includes the separate samples of 1 ll each were transferred to a micro- genera Agathisanthemum, Lelya and three Oldenlandia species scope slide. Finally, the pollen grains were counted under the (one from North America, and two from Africa) (Ka˚rehed microscope. Ovaries were carefully dissected out of each et al. 2008). The genus Hedyotis has a broad spectrum of flower and placed in a drop of water on a microscope slide, breeding systems, including distyly, dioecy and herkogamy and all ovules counted. Nectar production was also compared (Robbrecht 1988; Wagner & Lorence 1998; Ko 1999). Previ- for each bagged flower from 10 pin and 10 thrum plants ous studies have demonstrated that pollen dimorphism with in each population. Nectar was collected with a 0.5-ll respect to size, shape and exine characteristics, occurs in microcapillary 4 h after anthesis. Sugar content was measured some distylous species of the Rubiaceae (Naiki & Nagamasu as sucrose equivalents using a portable refractometer (Brix 2003; Castro et al. 2004). However, several cases of distyly 0–32%; Atago). characterised by the absence of reciprocal herkogamy and ⁄ or For scanning electron microscopy (SEM), pollen grains incompatibility systems, have also been found in the Rubia- were collected from 10 individuals of each morph, brushed ceae, which contributes to a better understanding of the onto electron microscope stubs, then examined using an maintenance and breakdown of distyly (Barrett 1992; Rich- environmental SEM (Philips XL 30) to compare the exine ards & Koptur 1993; Riveros et al. 1995; Ree 1997). The sculpture of the two morphs, and to measure polar and equa- main goal of this work was to investigate the relevant mor- torial axes. Stigma morphology of five flowers was examined phological differences between the two floral morphs associ- under a SEM after fixation in glutaraldehyde and osmium ated with distyly in Hedyotis acutangula (Rubiaceae), and to tetroxide. To compare the transmitting tissues of styles in elucidate and illustrate the cryptic self-incompatibility system both morphs, samples were fixed in 3% buffered glutaralde- in this species. hyde and 1% buffered osmium tetroxide, then hydrated using an alcohol series and embedded in Spurr resin. Thin cross- sections (0.5 lm) were made with a Leica EM UC6 ultrami- MATERIALS AND METHODS crotome and stained with methylene blue. Plant species and study sites Pollen histochemistry, viability and longevity The occurrence of distyly in Hedyotis acutangula in the field was determined in Guangdong Province. Morph ratio was For histochemical observation, pollen grains from freshly evaluated by recording all individuals of H. acutangula in open flowers of the two morphs were sampled from 20 pin populations at Wutongshan, Shenzhen (11317¢–11418¢ E, and 20 thrum plants in each population. Detection of starch 2223¢–2243¢ N) and Dinghushan Nature Reserve (11230¢– with KI–I2, and lipids with Sudan IV followed the method 11233¢ E, 2309¢–2311¢ N), Zhaoqing, both in Guangdong, described in Wang et al. (2004). The nuclei of fresh pollen China. grains, just discharged from the anthers, were also examined ) by staining with 1 mgÆml 1 propidium iodide (PI) in phos- phate-buffered saline (PBS) (Fujie et al. 1994). Samples were Floral biology and ancillary polymorphisms examined using a microscope (BX41; Olympus) with fluores- Hedyosis acutangula is a herbaceous species, blooming from cence excitation for PI staining and bright-field illumination May to August. The bee-pollinated flowers have a white cor- for the other stains.

Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands 485 Distyly in Hedyotis acutangula Wu, Li & Zhang

To estimate pollen viability and longevity, fresh pollen RESULTS grains of H. acutangula were collected at intervals (0, 4, 8 Floral morphology and 24 h) after anthesis, then stained with 3-(4,5-dim- ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Both populations of Hedyotis acutangula included two dis- dissolved in 5% sucrose and incubated at 40 C for 15– tinct floral morphs: pin flowers have a long style with the 30 min. A total of 300–400 pollen grains were mounted on stigma positioned above the anthers, while thrum flowers each slide and the percentage stained was determined by light have a short style with the anthers raised above the stigma microscopy, with five replicates per estimation. To evaluate (Fig. 1A, B). The stigmas of pin flowers and anthers of thrum pollen viability more accurately, the pollen germination per- flowers are visible above the throats of the corolla. The pin centage in vitro was also determined in pollen grains cultured and thrum morphs were nearly equally abundant in the two in a medium containing 5% sucrose, 0.01% CaCl2, 0.01% populations. The population in Shenzhen had a pin:thrum 2 H3BO3, 0.02% MgCl2 and 0.01% KCl at 25 C with 90% morph ratio of 0.91:1 (k = 0.295, P > 0.05, n = 122), and in humidity. Pollen grains were considered to have germinated the Dinghu population, the ratio was 0.94:1 (k2 = 0.161, only if the tube length exceeded the diameter of the pollen P > 0.05, n = 155). The pin:thrum ratio did not differ from grain. More than 30 pollen tubes were measured in each of 1, therefore, the population of H. acutangula studied was five replicates to determine mean tube length, and the mean isopletic. was plotted against incubation time. The floral characteristics and sugar content of both morphs are presented in Table 1. It is noteworthy that anther height of thrum flowers was significantly lower than stigma height Pistil receptivity of pin flowers (t = 18.9, P < 0.01), while there was no signifi- To compare the receptivity of pistils of the two morphs, 20 cant difference between anther height of pin and stigma pistils each from pin and thrum flowers were tagged and col- height of thrum morphs (t = 1.28, P > 0.05). This uneven lected. Tests were conducted at four different times (0, 1, 12 height re Sudan IV staining duced the perfect reciprocal and 24 h; 0 h representing the time when the flower was still herkogamy. In addition, the volume of nectar produced by closed but an anther had just appeared) after flower opening. thrum morphs was considerably smaller than that produced Pistils were then stained with MTT dissolved in 5% sucrose by pin morphs (t = 10.26, P < 0.01). In contrast, the sugar and incubated at 40 C for 15–30 min. Finally, images were content in thrum flowers was higher than in pin flowers captured with an Olympus microscope. (t = 9.18, P < 0.01). Generally, the total mass of sugar in pin and thrum morphs was similar (t = 0.18, P > 0.05). Pollen production was not significantly different between morphs Breeding system (t = 0.68, P > 0.05), while the ovule number of thrum flow- We conducted artificial pollination experiments to determine ers exceeded that of pin morphs, leading to larger P ⁄ O ratio whether Hedyotis rectangula is self- and intramorph compati- in pin than thrum flowers (t = 2.5, P < 0.05). ble in Dinghushan Nature Reserve. The treatments included: (i) self-pollination, with pollen from the same individual Pollen morphology (pin, n = 45; thrum, n = 37); (ii) intramorph pollination with pollen from other individuals of the same morph (pin, The pollen size and shape measurements are presented in n = 45; thrum, n = 43); (iii) cross-pollination with pollen Table 1. Representative pollen grains of the two morphs are from the opposite morph (pin, n = 54; thrum, n = 46); (iv) illustrated in Fig. 2. Pollen grains of H. acutangula were tri- emasculation (pin, n = 24; thrum, n = 41); (v) netting with- colpate, with long colpi narrowed at both ends. On the basis out emasculation (pin, n = 62; thrum, n = 50); and (vi) open of the P ⁄ E (P, polar axis; E, equatorial diameter), the shape pollination as control (pin, n = 65; thrum, n = 51). To of the pollen could be classified as prolate spheroid. The exclude flower visitors, we covered pin and thrum inflores- exine sculpture of both pin and thrum pollen grains was cences (open flowers removed, buds left) with nylon mesh foveolate–perforate. However thrum pollen exhibited more before anthesis, except in the control treatment. For determi- perforation than pin pollen (Fig. 2A–F). In addition, the nation of pollen tube growth rate, each type of hand-pollina- P and E values indicate pollen dimorphism: thrum pollen tion was conducted on 40 flowers with 10 flowers per time was larger than pin pollen although the shape was similar interval (1, 2, 4 and 8 h). After rinsing with water, the fixed (Table 1). Accordingly, the average distance between the pistils were cleared in 1 m NaOH until the tissues became apices of colpi in thrum pollen was larger than in pin pollen transparent, washed with distilled water, and then stained grains (Table 1). with aniline blue according to Teng et al. (2006). Nylon mesh Both pin and thrum were classified as having wet papillose was removed once the corolla and pistil had wilted; fruit set stigmas and solid styles (Fig. 2I–P). The stigma was bilobed was counted 2 months after artificial pollination. and covered with sticky exudates at maturity. The styles were made up of four tissue elements: epidermis, cortex, transmitting tissue and two vascular bundles (Fig. 2O, P). However, Data analysis there were important differences in the stigma profiles of the Means (±SE) were calculated for all measurements using two morphs: the pin morph was spherical and the thrum Excel 2000. We tested for differences in each morphological conical (Fig. 2I–N). Furthermore, papillae cells covered the and biological attribute between floral morphs using t-tests. entire spherical surface of the pin stigma, whereas on thrum The frequency of the two morphs within each population stigma lobes, papillae cells were mainly confined to the was compared with Chi-square tests (Sokal & Rohlf 1981). two edges of the lobes and were sparse on the lateral surface.

486 Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands Wu, Li & Zhang Distyly in Hedyotis acutangula

A B

C D

E F

Fig. 1. Flowers of Hedyotis acutangula. Histochemical and ﬂuorescent staining of H. acutangula pollen, and pollen tube growth in vitro. Reciprocal herkogamy with a long style and short anthers in the pin morph (A) and long anthers and a short style in the thrum morph (B). St, stigma; An, anther; bar = 0.5 cm. C: I J KI–I2 staining of pin pollen grains, showing weak positive reaction (indicated by arrows); bar = 20 lm.

D: KI–I2 staining of thrum pollen grains; bar = 20 lm. E: Sudan IV staining of pin pollen grains, showing positive staining (indicated by arrow); bar = 20 lm. F: Sudan IV staining of thrum pollens, showing positive staining (indicated by arrow); bar = 20 lm. G: PI staining of pin pollen grains; bar = 20 lm. H: PI staining of thrum pollen grains; GC, generative cell; VC, vegetative cell; bar = 20 lm. Pollen tube growth of pin (I) and thrum (J) morphs after culture in medium for 1 h; bar = 100 lm.

In addition, hairs in the middle of the corolla tube of much more extensively (Fig. 1C, D). Pollen grains from both pin flowers were noticeably fewer than in thrum flowers morphs responded positively to Sudan IV staining (Fig. 1E, (Fig. 2G, H). F); however, we could only detect staining on the polar view of pollen grains. Histochemical staining indicated that thrum pollen is rich in starch, whereas pin pollen is lipid-rich but Pollen histochemistry, viability, longevity and pollen tube contains little starch. The nuclei of mature pollen grains were growth rate detected using PI, and synchronously maturing pollen from KI–I2 staining revealed that only a few particles stained black pin and thrum flowers contained a vegetative cell and two in pin pollen grains while thrum grains were stained black sperm cells (Fig. 1G, H).

Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands 487 Distyly in Hedyotis acutangula Wu, Li & Zhang

Table 1. Differences in ﬂoral and pollen attributes between pin and A B thrum ﬂowers of Hedyotis acutangula.

floral and pollen attributes pin (n = 40) thrum (n = 40) t-value tube height (cm) 2.69 ± 0.25 2.59 ± 0.21 1.94 5 µm 5 µm anther height (cm) 3.11 ± 0.12 4.18 ± 0.19 30.11a stigma height (cm) 5.42 ± 0.38 2.79 ± 0.33 33.05a stigma lobe height (cm) 0.59 ± 0.09 0.72 ± 0.12 5.48a CD nectar volume (ll) 10.9 ± 2.33 6.13 ± 1.79 10.26a sugar content (%) 11.88 ± 4.51 21.09 ± 4.46 9.18a total sugars in nectar (lg) 1.29 ± 0.18 1.23 ± 0.11 1.80 a polar axis (lm) 31.24 ± 2.77 35.09 ± 2.21 7.74 5 µm 10 µm equatorial axis (lm) 20.05 ± 1.11 21.81 ± 0.86 2.23b P ⁄ E 1.57 ± 0.15 1.64 ± 0.17 1.95 EF distance between apices 9.38 ± 1.45 10.22 ± 1.88 2.23a of two colpi (lm) no. of pollen grains per 2019 ± 534 1925 ± 688 0.68 flower no. of ovules per flower 30.1 ± 3.5 34.9 ± 5.8 4.48a P ⁄ O 66.7 ± 17.2 56.5 ± 20.6 2.40b 5 µm 5 µm P ⁄ E = the ratio of the length of the polar axis to the equatorial diameter; P ⁄ O = pollen–ovule ratio. GH aMorphs differ very significantly (P < 0.01) based t-test. bMorphs differ significantly (P < 0.05) based on t-test.

Different methods to assess pollen viability were applied (Fig. 3). The viabilities of pin and thrum pollen at the 500 µm 500 µm moment of anther dehiscence were 86.4 ± 5.8% and 93 ± 6.5%, respectively, when stained with MTT, but 80.7 ± 5.8% and 82.1 ± 6.9% when cultured in vitro. Further IJ statistical tests indicate no obvious difference in pollen viability between the two morphs through time (t0 = 0.84, t4h= 2.13, t8h= 1.36, t24 h = 1.9, P > 0.05). However, the difference between the two assessment methods was signiﬁ- cant (t = 5.46, P < 0.05). Staining with MTT and germina- 200 µm 100 µm tion in vitro indicated that pollen retains a high viability 4 h after anther dehiscence. After 8 h, viabilities of pin and thrum pollen declined to 50.6 ± 5.4% and 60.3 ± 4.6%, KL respectively, when cultured in medium. Twenty-four hours later, both pin and thrum pollen failed to germinate in vitro. Both methods demonstrate that the viability of both pollen types declined with time after anther dehiscence. Pollen started to germinate after 0.5 h in standard medium 200 µm 100 µm (Fig. 1I, J). The average growth rate of pin pollen tubes was ) 263.1 lmÆh 1, which was slower than thrum pollen tubes ) MN (435.6 lmÆh 1; t = 3.63, P < 0.01). Pollen tube growth rate of both morphs increased gradually during the ﬁrst 8 h of culture and reached a plateau after 12 h.

Fig. 2. Morphology of pollen grains and stigmas of Hedyotis acutangula 50 µm 50 µm under SEM and light microscopy. A, C: Surface view of pollen grains from a pin flower; E: polar view of pollen grains from a pin flower; B, D: surface O P view of pollen grains from a thrum flower; F: polar view of pollen grains from a thrum flower; G: sparse pubescence at the throat of the corolla tube of a pin flower; H: dense pubescence at the throat of a corolla tube of a thrum flower; I: spherical profile of a pin stigma; J: cone outline of a thrum stigma with fewer papillae cells; K: lateral side of a pin stigma; L: lateral side of a thrum stigma; M: secretion covering the papillae cells of a pin stigma; N: secretion covering the papillae cells of a thrum stigma; O: 100 µm 100 µm semi-ultrastructure of a pin style; P: semi-ultrastructure of a thrum style; Co, cortex cell; Ep, epidermis; TT, transmitting tissue; VB, vascular bundle.

488 Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands Wu, Li & Zhang Distyly in Hedyotis acutangula

100 thrum flowers resulted in the slowest pollen tube growth. Accordingly, the time taken for the pollen tube to arrive at the ovary varied with pollen tube growth rate and length of 80 style (Table 2). For illegitimate (i.e. self and intramorph) pollination, the pollen tube took longer to penetrate the pin style due to a slower pollen tube growth rate and a longer 60 style compared with pollen tubes in the thrum style. How- ever, for legitimate (i.e. intermorph) pollination, it took nearly the same time for pollen tubes to arrive at the ovary 40 in both pin and thrum styles. Moreover, with the same type Pin-MTT staining of pollination, pollen tube growth in pin flowers was faster Pollen viability (%) Pollen Thrum-MTT staining Pin-pollen germination in vitro than in thrum flowers. Furthermore, the difference in pollen 20 Thrum-pollen germination in vitro tube growth rate between legitimate and illegitimate pollination was greater in pin flowers than in thrum flowers (difference between intermorph and self-pollination: t = 16.9; 0 difference between intermorph and intramorph pollination: 04824 Time (h) t = 36.8, P < 0.01 for both). When comparing pollen tube growth rate in vitro and legitimate pollination in vivo, the lat- Fig. 3. Pollen variability of Hedyotis acutangula determined by MTT stain- ter was significantly faster than the former (t = 29.7, ing and germination in vitro. Data shown are means, vertical bars repre- P < 0.01). Under florescence microscopy, callose plugs were sent standard deviation of the mean. present at regular intervals in both the illegitimate and legitimate pollen tubes, except in those growing in self-pollinated Following artificial pollination, pollen germinated on the thrum styles. Upon self-pollination, pollen tubes in thrum stigma within 1 h and, irrespective of the type of pollination, styles exhibited abnormal callose plugs of irregular length. the tubes emerged and grew down into the style (Figs 4 and Further, a slightly swollen tube was also observed after stain- 5). Tube growth rate differed significantly between the differ- ing with aniline blue (Fig. 5B). ent kinds of pollination. In pin flowers, pollen tubes were observed at the base of the style 8 h after self-pollination and Pistil receptivity and fecundity intramorph pollination, but after only 4 h in intermorph pollination (Fig. 5A, C and E). In thrum flowers 8 h after self- The MTT test showed that the pistil of pin and thrum flowers pollination, pollen tubes had nearly reached the base of the became receptive shortly before anther dehiscence and lasted style (Fig. 5B). However, 8 h after intramorph pollination for 12 h after anthesis (Fig. 6). Stigmatic receptivity then grad- and 4 h after intermorph pollination, some pollen tubes had ually decreased. When flowers wilted 24 h after anthesis, the already entered the ovary (Fig. 5D, F). Average pollen tube pistil completely lost receptivity (Fig. 6). It was noticeable that ) growth rates were: 256.3 ± 27.4 lmÆh 1 (pin) and MTT staining of pin stigmas was stronger than in thrum stig- ) 164.9 ± 9.8 lmÆh 1 (thrum) for self-pollination; mas 12 h after anthesis, indicating a higher level of receptivity ) ) 436.7 ± 144.9 lmÆh 1 (pin) and 373.8 ± 32.2 lmÆh 1 (thrum) of pin pistils in comparison with thrum pistils (Fig. 6E, F). ) for intramorph pollination; and 925 ± 119.8 lmÆh 1 (pin) Fruit set in artificially pollinated flowers ranged from 40% ) and 500.85 ± 88.1 lmÆh 1 (thrum) for intermorph pollina- to 88% (Table 3). Fruit set did not differ significantly tion (Fig. 4). Thus, pollen tube growth was fastest after inter- between morphs following legitimate pollination (pin versus morph pollination in pin flowers, while self-pollination in thrum: 80.0% versus 78.1%), emasculation and netting (pin versus thrum: 0% versus 0%), and open-pollination (pin versus thrum: 82.8% versus. 81.7%). When self-pollinated (pin 8000 versus thrum: 47.0% versus 40.8%) or pollinated with pollen Pin: self-pollination Pin: intramorph pollination from individuals of the same floral morph (pin versus thrum: Pin: intermorph pollination 55.1% versus 49.8%), fruit set of pin flowers was higher than 6000 Thrum: self-pollination that of thrum flowers (self-pollination: t = 2.87; intramorph Thrum: intramorph pollination Thrum: intermorph pollination pollination: t = 2.59, P < 0.01 for both). Upon netting without emasculation, a significant difference was found between 4000 pin and thrum flowers (pin versus thrum: 0% versus 40.1%, t = 31.1, P < 0.01). Both pin and thrum flowers that had been legitimately pollinated exhibited higher fruit set (intermorph pollination versus self-pollination, t = 34.5, Pollen tube length (µm) Pollen 2000 pin tthrum = 49.6; intermorph pollination versus intramorph pollination, tpin = 15.3, tthrum = 22.5, P < 0.01 for all). Apomixis did not occur in either morph. 0 124 8 Time (h) DISCUSSION Fig. 4. Pollen tube length of Hedyotis acutangula after artificial pollination in vivo. Data are means, vertical bars represent standard deviation of Populations of Hedyotis acutangula contain two flower mor- the mean. PSH, pin style height; TSH, thrum style height. phs, characterised by reciprocal herkogamy, strongly asyn-

Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands 489 Distyly in Hedyotis acutangula Wu, Li & Zhang

A B

C D

E F

Fig. 5. Relative extent of pollen tube growth in stigma lobes and style of Hedyotis acutangula following self-, intramorph and intermorph pollination. A: Pin morph stigma lobes and style 8 h after self-pollination; B: thrum morph stigma lobes and style 8 h after self-pollination; red ellipse showing abnormal callose plug with slightly swollen tube compared with normal callose plugs marked with white ellipses; C: pin morph stigma lobes and style 8 h after intramorph pollination (D) thrum morph stigma lobes and style 8 h after intramorph pollination; E: pin morph stigma lobes and style 4 h after intermorph pollination; F: thrum morph stigma lobes and style 4 h after intermorph pollination. Arrows indicate arrival at the tip of pollen tubes. Bar = 500 lm.

Table 2. The time taken for pollen tubes to arrive at the ovary in pin and anthers was lower in thrum flowers, it did not correspond thrum flowers of Hedyotis acutangula. well with stigmas of the pin morph; this kind of imprecise reciprocal herkogamy is similar to that found in most Rubia- time (h, mean ± SE) ceae species (Faivre & McDade 2001; Castro & Araujo 2004). treatment pin (n = 10) thrum (n = 10) t value It could be expected that the dissimilar heights of the stigma and anthers in the two morphs might lead to imprecise self-pollination 21.5 ± 1.3 17.1 ± 1.8 6.29a cross-morph pollen transfer and increased potential for self- intramorph pollination 12.6 ± 0.9 7.7 ± 0.8 12.9a pollination in thrum flowers. However, the approximate 1:1 intermorph pollination 5.9 ± 0.4 5.8 ± 0.5 0.5 morph ratio and equivalent fruit set after artificial inter- aMorphs differ very significantly (P < 0.01) based on t-test. morph pollination and open-pollination provided strong evi- dence that significant disassortative (intermorph) mating prevailed in populations of H. acutangula, as is common in chronous pollen tube growth and a wide range of ancillary distylous species of the Rubiaceae (Pailler & Thompson 1997; polymorphism. Reciprocal herkogamy is believed to be one Massinga et al. 2005; Garcıá-Robledo & Mora 2007) that have of the most important factors affecting breeding strategies of a typical self-incompatibility system. Because flowers of both a species because it can effectively reduce both self- and morphs are protandrous, and stigmas are receptive before intramorph pollination while promoting intermorph pollen anthesis, reciprocal herkogamy in this species is an effective flow by pollen segregation on pollinator bodies (Lloyd & mechanism favouring high rates of intermorph pollination. Webb 1992; Massinga et al. 2005). However, considerable dis- The similar fruit set of morphs with legitimate pollination parities in the precise positions of anthers and stigmas in and open-pollination indicated that pollination is by insects distylous plants and, thus, imprecise reciprocal herkogamy and that there is no pollen limitation, despite the imperfect occur widely, including in the Rubiaceae (Lloyd & Webb reciprocal herkogamy. In addition, upon netting without 1992; Arroyo & Barrett 2000; Faivre & McDade 2001; Deng emasculation, no fruit was produced in pin morphs, com- 2007). In H. acutangula, although the reciprocal placement of pared with 40% fruit set in thrum morphs, suggesting that in

490 Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands Wu, Li & Zhang Distyly in Hedyotis acutangula

A B However, fruit set was never zero in artificial self-pollination, suggesting reproductive assurance in the absence of pollinators. The majority of distylous species investigated have larger floral parts in thrum than pin morphs. It is generally accepted that such asymmetry between morphs is a mechanism to compensate for the bias towards pollen reaching pin stigmas, compelling thrum flowers to attract more pollinators than pin morphs (Dulberger 1992; Castro et al. 2004). How- ever, such asymmetry was not absolute in H. acutangula on the basis of the characters assessed (Table 1). In our study, thrum stigma lobes were significantly larger than those of the C D pin morph. Most data indicate that pin stigmas are generally larger than those in thrum morphs (Dulberger 1992); however, exceptions are found in Hedyotis (Ornduff 1980), Psychotria (Castro et al. 2004) and Guettarda (Richards & Koptur 1993). Our results corresponded well with those for Hedyotis caerulea (Ornduff 1980), which seems to compensate for asymmetric pollen flow to the pin stigma. Interest- ingly, papillae cells are concentrated on the edge of thrum stigma lobes, which would facilitate outcross pollen grains carried by pollinators in comparison with self-pollen falling from the anther to the pistil. This is a feature that has not E F been previously described in heterostylous species, and might be a mechanism to avoid self-pollen disturbance during insect pollination in thrum flowers. Pollen dimorphism, including size, surface sculpture and stigmatic papillae, has always been associated with distyly (Darwin 1877; Ganders 1979; Naiki & Nagamasu 2003; Quinet et al. 2004). It is generally considered that differences in pollen size could be attributed to selection for increased numbers of pollen grains (Lloyd 1979). Previous investiga- tions revealed that small pin grains can pollinate the less- exposed thrum stigma most efficiently, while large thrum pollen is necessary to provide energy reserves or facilitate G H faster growth of the pollen tube down the longer pin style (Cruden & Lyon 1985; Richards 1997). As expected, pollen grains from thrum flowers of H. acutangula are larger, contain more starch, and grow faster in vitro and in vivo than those from pin flowers. The same dichotomy is found in other distylous species of the Rubiaceae (Naiki & Nagamasu 2003; Massinga et al. 2005). The number of pin pollen grains is higher than thrum pollen grains, consistent with the hypothesis that there is a trade-off between size and number of pollen grains (Lloyd 1979). Furthermore, smaller pin pollen grains are most efficient for pollinating thrum flowers with fewer papillae cells on their stigmas, thus also promot- Fig. 6. Variation in receptivity of pin and thrum pistils detected by MTT ing legitimate pollination between morphs. Interestingly, this staining. A: Pistil of a pin flower during pre-anthesis with a broken anther; is the first report of starch content dimorphism, which is B: pistil of thrum flower during pre-anthesis with a broken anther; C: pistil known to play an important role in pollen germination and of a pin flower 1 h after anthesis; D: pistil of thrum flower 1 h after pollen tube growth (Baker & Baker 1979). anthesis; E: pistil of pin flower 12 h after anthesis; F: pistil of thrum flower In most distylous species, there is no opportunity for direct 12 h after anthesis; G: pistil of pin flower 24 h after anthesis; H: pistil of thrum flower 24 h after anthesis. Bar = 100 lm. competition between pin and thrum pollen because a system of heteromorphic incompatibility precludes self- and intramorph cross-fertilization and allows only intermorph cross- the case of pollinator limitation, self-pollinated fruits from fertilization (Ree 1997; Castro & Oliveira 2002; Ornelas et al. thrum flowers will prevail in the studied populations. The 2004). However, there are other heterostylous species, e.g. similarity of fruit set after artificial self-pollination in pin and Decodon verticillatus, Hedyotis nigricans and Amsinckia gran- thrum flowers, and autonomous selfing in thrum flowers sig- diflora, which also have a self- or intramorph compatible sys- nificantly lower than in open- and legitimate pollination tem (Bahadur 1968; Weller & Ornduff 1977; Eckert & Allen treatments, indicates that there is some self-incompatibility. 1997). Furthermore, the degree of morph compatibility can

Plant Biology 12 (2010) 484–494 ª 2009 German Botanical Society and The Royal Botanical Society of the Netherlands 491 Distyly in Hedyotis acutangula Wu, Li & Zhang

Table 3. Fruit set of Hedyotis acutangula under different treatments.

fruit set (%, mean ± SE) treatment pin thrum t value self-pollination 47.0 ± 7.6 (n = 45) 40.8 ± 11.9 (n = 37) 2.87a intramorph pollination 55.1 ± 10.8 (n = 45) 49.8 ± 8.1 (n = 43) 2.59a intermorph pollination 80 ± 4.7 (n = 54) 78.1 ± 2.7 (n = 46) 1.89 emasculation and netting 0 (n = 24) 0 (n = 41) ⁄ netting without emasculation 0 (n = 62) 40.1 ± 10.2 (n = 50) 7.91a open-pollination 82.8 ± 5.9 (n = 65) 81.7 ± 3.8 (n = 51) 1.16 aMorphs differ very significantly (P < 0.01) based on t-test. vary at both species and population levels, particularly in the (Athanasiou et al. 2003). However, further studies on the Rubiaceae (Sobrevila et al. 1983). Pollen tube competition formation of callose plugs are required to reveal how experiments between pin and thrum morphs of A. grandiflora classical self-incompatibility acts in the thrum morph of revealed that pollen growth after intramorph pollination was H. acutangula. slower than after intermorph pollination, leading to a cryptic In conclusion, the results of the present study indicate that self-incompatibility system (Weller & Ornduff 1977). Conse- H. acutangula is a distylous plant with floral dimorphism quently, intermorph fertilisation predominated during natural with respect to several characters (e.g. styles, stigma outline, reproduction. Here, we observed the apparent competitive stamens, ovule number and pollen grain size). The breeding advantage of thrum pollen over pin pollen and faster pollen system in H. acutangula is cryptic self-incompatible and tube growth following legitimate compared to illegitimate dominated by legitimate (intermorph) mating. In a recent pollination within each morph. Therefore, we conclude that reconstruction of phylogeny in the tribe Spermacoceae (Ka˚r- H. acutangula possesses a cryptic self-incompatibility system. ehed et al. 2008), species with apparent affinities to H. acut- The fact that thrum pollen grains grow faster in vivo as well angula, Hedyotis effusa Hance and Hedyotis consanguinea as in vitro may indicate that differential pollen tube growth Hance, appear in clade C. This Agathisanthemum–Hedyotis in cryptic self-incompatibility is largely determined by mater- clade also includes Oldenlandia uniflora L. (=Hedyotis salzma- nal resource levels. nii), a Chilean species that has self- and intramorph compati- Incompatible pollen tubes usually have abnormal plugs, bility (Riveros et al. 1995). However, one of the species and unstable and variable callose deposition in the walls known to have a typical self- and intramorph incompatibility compared with compatible pollen tubes (Vithanage et al. system, i.e. Arcytophyllum lavarum (Garcıá-Robledo & Mora 1980; Murfett et al. 1994; Lush & Clarke 1997). Castro et al. 2007), appears in clade D, sister to clade C. One of the out- (2004) reported that distylous Psychotria brotula exhibited groups used in their analysis, Pentanisia, also consists of spe- self- and intramorph incompatibility reactions based on the cies with typical self- and intramorph incompatibility occurrence of abnormal callose plugs. Interestingly, upon (Massinga et al. 2005). Since the sexual systems of most spe- self-pollination, pollen tubes in short-styled plants do not cies in the tribe are still unknown, it is difficult to judge if produce callose plugs, while all other pollinations result in the occurrence of cryptic self-incompatibility in H. acutangu- callose plug formation in Turnera species (Tamari et al. la and O. uniflora definitely represents a breakdown of typical 2001). In our study, callose plugs occurred not only in the self-incompatibility found in most heterostylous species. The illegitimate pollen tubes, but also in the legitimate pollen abnormal callose deposition in thrum self-pollen may indi- tubes in both pin and thrum styles. However, in H. acutan- cate that a remnant of the pollen tube arresting mechanism gula, the abnormal callose plugs were only observed in self- is still functioning in H. acutangula, which would support a pollinated thrum styles, similar to those reported in other breakdown of heteromorphic self-incompatibility. self-incompatible Rubiaceous species (Bawa & Beach 1983; Scribailo & Barrett 1991), indicating the classical pollen tube ACKNOWLEDGEMENTS arresting mechanism may play a role in self-pollination in the thrum morph. However, most pollen tubes penetrate tis- This work was supported by the National Science Fund of sues of the stigma and even result in normal fruits, which is China (Grant Nos. 30700040 and 30570314), and by the significantly different from cases previous reported in Rubia- Knowledge Innovation Programme of the Chinese Academy ceae, where short-style plants always had stigmatic inhibition of Sciences (KSCX2-YW-Z-027 and KZCX2-YW-414). The of pollen tubes following self- and intramorph pollination authors are indebted to Drs Shijin Li, Pu Zou, Xiaofang (Bawa & Beach 1983; Dulberger 1992). Thus, we suggest that Deng, and Shixiao Luo for field assistance on observation of there might be a difference in the mechanisms of delayed the species in Dinghushan Nature Reserve. pollen tube growth between morphs. In the thrum morph, cryptic self-incompatibility might be partly induced by a clas- REFERENCES sical tube-arresting mechanism characterised by abnormal callose plug formation, which was not observed in pin mor- Alonso J.M., Company R.S. (2005) Differential pollen tube growth phs in the present study, in accordance with a putative in inbred self-compatible almond genotypes. Euphytica, 144, incompatibility protein identified only in the thrum morph 207–213.

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