Unique Growth Paths of Heterospecific Pollen Tubes Result in Late
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Plant Biology ISSN 1435-8603 RESEARCH PAPER Unique growth paths of heterospecific pollen tubes result in late entry into ovules in the gynoecium of Sagittaria (Alismataceae) N. Lyu, W. Du & X.-F. Wang College of Life Sciences, Wuhan University, Wuhan, China Keywords ABSTRACT heterospecific pollen; interspecific pollination; pollen tube growth; pollen tube guidance; pollen-pistil interactions; Sagittaria. • Pollen-pistil interactions are a fundamental process in the reproductive biology of angiosperms and play a particularly important role in maintaining incipient species Correspondence that exist in sympatry. However, the majority of previous studies have focused on spe- X.-F. Wang, College of Life Sciences, Wuhan cies with syncarpous gynoecia (fused carpels) and not those with apocarpous gynoecia University, Wuhan, 430072, China. (unfused carpels). E-mail: [email protected] • In the present study, we investigated the growth of conspecific pollen tubes compared to heterospecific pollen tubes in Sagittaria species, which have apocarpous gynoecia. We Editor conducted controlled pollinations between S. pygmaea and S. trifolia and observed the G. Thiel growth of conspecific and heterospecific pollen tubes under a fluorescence microscope. • Heterospecific and conspecific pollen tubes arrived at locules within the ovaries near Received: 26 August 2015; Accepted: simultaneously. However, conspecific pollen tubes entered into the ovules directly, 24 September 2016 whereas heterospecific tubes passed through the carpel base and adjacent receptacle tissue, to ultimately fertilize other unfertilized ovules. This longer route taken by doi:10.1111/plb.12508 heterospecific pollen tubes therefore caused a delay in the time required to enter into the ovules. Furthermore, heterospecific pollen tubes displayed similar growth patterns at early and peak pollination. The growth pattern of heterospecific pollen tubes at late pollination was similar to that of conspecific pollen tubes at peak pollination. • Heterospecific and conspecific pollen tubes took different routes to fertilize ovules. A delayed entry of heterospecific pollen into ovules may be a novel mechanism of con- specific pollen advantage (CPA) for apocarpous species. 2010) and pistil-length mismatch (Lee et al. 2008). Environ- INTRODUCTION ment-dependent selection on pollen performance (Chapman Varying floral characters, such as corolla color and petal or pol- et al. 2005) may also play a role in pollen-pistil interactions. len scent (Adler & Irwin 2006), production of flower nectar These phenomena have been observed among taxa of Nicotiana (Mitchell & Waser 1992), as well as the structure of pollen (Lee et al. 2008), Iris (Emms et al. 1996), Mimulus (Fishman grain and stigma (Sannier et al. 2009) can determine outcomes et al. 2008), Helianthus (Rieseberg et al. 1995), Senecio (Chap- for plant pollination owing to their influence on complex man et al. 2005), Betula (Williams et al. 1999), Brassica (Hauser plant-pollinator interactions. For individuals of different spe- et al. 1997), Piriqueta (Wang & Cruzan 1998), Costus (Yost & cies that exist adjacent to one another, pollen exchange is pos- Kay 2009), and Chamerion angustifolium (Husband et al. 2002). sible via wind, and for those that exist in sympatry or Pollen-pistil interactions are more well characterized in syncar- parapatry, pollen exchange is possible via shared pollinators pous than apocarpous plants. (Arroyo & Dafni 1993; Brown & Mitchell 2001). Sagittaria is a genus of Alismataceae with conduplicate and Pollen-pistil interactions that lead mating barriers and prevent apocarpous carpels (Kaul 1976). There is incomplete fusion hybrid fertilizations are particularly important for the persistence of the mature carpel, which has an opening at the base due of incipient species that exist in sympatry. Hybrid fertilizations to the carpel margins being unfused (Huang 2014). In at reduce the fitness of one or both taxa as those ovules and sperm least four species of Sagittaria, pollen tubes from a carpel in are not used for conspecific matings (Levin 1975; Endress 1982; the gynoecium can either enter directly into an individual Armbruster et al. 2002; Husband et al. 2002; Ramsey et al. ovule or travel through the carpel basal opening and adjacent 2003). The pollen-pistil interactions of different taxonomic receptacle tissue to nearby unfertilized ovaries (Wang et al. groups employ varying strategies to maintain species integrity, 2002, 2006). The growth pattern of pollen tubes during such as inhibiting or retarding pollen germination on heterospecific pollination of these plants was unclear. There- heterospecific stigma (Hiscock & Dickinson 1993; Hodnett et al. fore, we used controlled pollination to examine the growth 2005), or unequal rates of pollen tube growth in the pistil (Car- of heterospecific pollen tubes in Sagittaria pygmaea gynoecia. ney et al. 1996; Klips 1999; Lankinene & Skogsmyr 2001). In We found that heterospecific and conspecific pollen tubes addition, several recent studies have reported mechanical mech- follow a different growth path in this species. This difference anisms of stigma closure and reopening (Sritongchuay et al. in pollen tube growth may represent a novel pollen-pistil Plant Biology © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands 1 Alien pollen tube growth in Sagittaria species Lyu, Du & Wang interaction in apocarpous species, which may extend our recounted carpels in which the pollen tubes entered into the understanding of these interactions and their importance in ovules and passed into the surface layer of the receptacle tissue plant evolution. simultaneously (Fig. 1, Type VI). One-way analysis of variance (ANOVA), t-test, and statistical graphics were performed using the STATISTICA software package for Windows (single user MATERIAL AND METHODS version 6.0) StatSoft Inc. (2002). Plant materials and study site The two monoecious species S. pygmaea and Sagittaria trifolia RESULTS are both widely distributed in marshes, wetlands, and rice fields Pollen tube growth route after conspecific and heterospecific in the south of China. Their flowers have three white petals in pollination in S. pygmaea a spiral arrangement on the scapes and the flowering period is from May to November (Chen 1989). In total, 60 plants of the Conspecific pollen tubes grew through the recipient styles and two species were collected in 2013 from a rice field in Lichuan, reached the vicinity of the ovary at approximately 1 h AP Hubei Province, China (30°390 N, 109°180 E), and transplanted (Fig. 2A). At roughly 1.5 h AP, one tube began to turn toward into an outdoor pond at Wuhan University. the ovule and smoothly penetrated the micropyle. By approxi- mately 2 h AP, a large proportion of the pollen tubes (44.74%) had entered the ovules. Redundant pollen tubes passed into Conspecific and heterospecific pollination and across the receptacle tissue to enter into other unfertilized ThefemaleflowersofbothS. pygmaea and S. trifolia were ovaries (Figs 2B and C, and 3A). bagged before flowering. The following day, flowers were polli- Heterospecific pollen tubes also reached the vicinity of the nated by applying pollen from either S. pygmaea or S. trifolia ovary at 1 h AP (Fig. 2D). Most heterospecific pollen tubes tan- directly onto the stigmata when the flowers opened naturally at gled together and displayed a crooked growth mode without any 0730 h which is when maximum pollen and stigma activity abnormal growth patterns. However, heterospecific growth tubes occurs in the two species (L Na, unpublished results) (herein were not seen to turn toward the ovules until 2.5 h AP (Fig. 3B). referred to as peak pollination). Pollinated flowers were then By 2.5 h AP, 5.13% of ovaries had received pollen tubes and harvested at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 74.23% of carpels contained pollen tubes at the receptacle (Type 6.0 h after pollination (AP). We collected 3–6 flowers pollinated by each species at each sampling time and fixed them in formalin–acetic acid–alcohol (FAA). In addition, using a stereomicroscope, pollen grains from S. pygmaea were applied to a single carpel in the gynoecium of a pistillate flower from S. pyg- maea, while the adjacent carpels were pollinated by S. trifolia. Pollinated flowers were collected 2 h AP and again fixed in FAA. Controlled pollination using pollen from S. trifolia on the gynoecia of S. pygmaea was also conducted at approximately 0930 h, meaning 2 h after the bagged flowers opened (herein referred to as late pollination). Similarly, we pollinated the gynoecia of S. pygmaea with S. trifolia at approximately 0630 h, meaning 1 h before peak pollination when the flow- ers were just opened (herein referred to as early pollination). Flowers were collected 2 h after early and late pollination and fixed in FAA. Observation of pollen tube growth in pistils Sampled flowers were rinsed with water and placed in NaOH (5%, w/v) at room temperature until most of the tissues had become translucent. The flowers were again rinsed in water and then stained with aniline blue (0.1% in 0.03 M K3PO4) for 4 h (Wang et al. 2002). Carpels were randomly picked using tweezers and placed on slides for observation under a fluores- cence microscope (Olympus BX-43, Tokyo, Japan). Fig. 1. Sagittaria pygmaea carpels classified into six types (I–VI) following Statistics and data analysis conspecific or heterospecific pollination, according to pollen tube front-end position. (I) Carpels with ungerminated pollen on the stigma. (II) Pollen ger- As shown in Fig. 1, the carpels were classified into six types mination and pollen tube beginning to emerge on the stigma. (III) Pollen (I–VI), according to pollen tube front-end position, with the tube navigating the style channel. (IV) Pollen tube arriving in the vicinity of exception of Type I that had carpels with ungerminated pollen. the ovary. (V) Pollen tube growing toward the ovule. (VI) Pollen tube passed At each time point from 0.5 to 6 h AP, we counted the carpels through the basal opening of the carpel toward the surface tissue layer of of each type using a fluorescence microscope.