Exposure to Water Increased Pollen Longevity of Pondweed

Evol Ecol (2010) 24:939–953 DOI 10.1007/s10682-010-9351-z

ORIGINAL PAPER

Exposure to water increased pollen longevity of pondweed (Potamogeton spp.) indicates different mechanisms ensuring pollination success of angiosperms in aquatic habitat

Xiao-lin Zhang • Robert W. Gituru • Chun-feng Yang • You-hao Guo

Received: 8 December 2008 / Accepted: 4 January 2010 / Published online: 22 January 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Pollen longevity in seven Potamogeton species representing different pollination systems (anemophily, epihydrophily and hydroautogamy) was assessed both under aerial condition and in contact with water to investigate how water impacts the sexual reproduction in these aquatic taxa. Stainability of pollen with MTT was considered as an indicator of pollen viability. The half-life of pollen longevity was calculated using exponential decay regression. Overall, pollen viability decreased relatively rapidly with time. Pollen grains of obligate anemophilic species had lower initial viability and shorter half- lives than those of facultative anemophilic species. Pollen in these latter species may take more time to reach the stigma. The pollen of Potamogeton may be categorized as partially hydrated pollen owing to its generally spherical shape and lack of furrows, rapid loss of viability, and fast pollen tube initiation. The half-life is positively correlated with pollen size. Smaller-sized grains are at greater risk of desiccation than larger grains. In contrast with the situation observed in most terrestrial angiosperms, contact with water increases pollen longevity in Potamogeton species. In our present study the half-lives of pollen longevity of Potamogeton species in which the pollen had come into contact with water (mean of 10.65 h) were markedly higher than those under aerial conditions (mean of 5.79 h, t = 2.622, P = 0.039). The results of our study contradict a widely held belief that water is detrimental to pollen viability in angiosperms and furthermore indicate that close

X. Zhang C. Yang (&) Y. Guo (&) College of Life Sciences, Wuhan University, 430072 Wuhan, China e-mail: [email protected] Y. Guo e-mail: [email protected]

X. Zhang Institute of Hydrobiology, Chinese Academy of Sciences, 430072 Wuhan, China

R. W. Gituru Botany Department, Jomo Kenyatta University of Agriculture and Technology, 62000-00200 Nairobi, Kenya

C. Yang Wuhan Botanical Garden, Chinese Academy of Sciences, 430074 Wuhan, China 123 940 Evol Ecol (2010) 24:939–953 proximity to water results in selection for wettability. The transition to a hydrated status together with its morphology, make Potamogeton pollen more adapted to the aquatic environment and thus serves to ensure reproductive process. Results of our present study may have direct implications for understanding the evolution of the sexual reproductive system in aquatic angiosperms.

Keywords Pollen longevity Half-life Selective pressure Pondweed Potamogeton

Introduction

Pollen loss during pollination process is a common phenomenon but the intensity of the loss is different in taxa with different pollination systems (Harder and Thomson 1989). Pollen loss is also considered to occur when a proportion of the pollen grains lose their male function e.g., when pollen grains lose viability before deposition on the stigma. Pollen viability is generally considered to indicate the ability of the pollen grain to perform its function following compatible pollination (Shivanna et al. 1991), and pollen longevity has been considered as an important ecological and evolutionary character of angiosperms (Dafni and Firmage 2000). Pollen viability commonly declines after release from the anthers. The term half-life is widely used in research because the data are easy to understand and they lend themselves easily to comparisons (Bryan et al. 1990). In addition, half-life provides a useful summary statistic for a population. However, only a few studies have attempted to quantify the analysis of pollen longevity (Khatun and Flowers 1995; Castellanos et al. 2006; Song et al. 2001). Many factors including humidity, temperature and the vagaries of the weather may reduce pollen viability and longevity (Bassani et al. 1994; Hedhly et al. 2005; Boavida and McCormick 2007; Song and Tachibana 2007). It has been demonstrated that contact with water and high levels of humidity markedly reduce pollen longevity in many terrestrial angiosperms (Gilissen 1977; Shivanna and Heslop-Harrison 1981; Huang et al. 2002; Mao and Huang 2009). Consequently, plants have evolved different mechanisms to avoid water related reduction in pollen longevity (Bynum and Smith 2001; Huang et al. 2002; Mao and Huang 2009) and ensure the accomplishment of male function even under unfavorable environments. Most aquatic angiosperms are thought to have evolved from their terrestrial ancestors on many independent occasions (Philbrick and Anderson 1987; Cook 1988, 1990; Philbrick and Les 1996) and hydrophily is considered to have been derived from anemophily (Arber 1920; Daumann 1963; Sculthorpe 1967; Philbrick 1988). In aquatic habitats, the pollination process is highly likely to be affected by the presence of water. Water must be an important source of selective pressure in the evolution of the pollination systems in plants living in these habitats. Considering that the preservation of pollen viability is of critical importance during the pollination process, it is crucial to investigate the response of the pollen of aquatic plants to contact with water in comparison to the pollen of terrestrial species. Such investigations may provide useful insights into the adaptation to aquatic environment instead of terrestrial environment of aquatic angiosperms since they are thought as a group derived from terrestrial ancestors. Potamogeton Linn. (Pondweed) is the largest angiosperm genus consisting entirely of aquatic species. Extensive morphological diversity and different pollination types have been found in this genus. This diversity is considered to have resulted from the intense selective 123 Evol Ecol (2010) 24:939–953 941 pressure in the aquatic habitat (Zhang et al. 2009). Although anemophily (wind pollination) is the main pollination type in this genus (Sculthorpe 1967; Cook 1988), epihydrophily (pollen grains are transported on water surface by water currents to the floating inflorescences) and hydroautogamy (self-pollination is achieved with the aid of bubbles when anthers dehisce Philbrick 1988; Guo and Cook 1990), also play a considerable role. Natural pollen transfer in almost all species of Potamogeton, may occur under two conditions: namely aerial condition and water condition. Under the aerial condition, pollen grains are transported entirely by air currents and they do not come into contact with water. In the water, pollen grains are transported by water currents or bubbles to the floating receptive inflorescences. Both of these two conditions of pollen transfer may occur in Potamogeton. Consequently, the pollen grains in these plants are likely to come into contact with water. There may exist a different pattern of pollen performance in aquatic plants com- pared to terrestrial angiosperms. It is therefore interesting to study how exposure to water affects the pollen grains in this genus. Our present study focused on the response of the pollen grains of Potamogeton under aerial and water conditions. Using information obtained partly from monitoring pollen viability under these two conditions and from calculation of the half-life of pollen longevity using the exponential decay regression, we proposed the likely mechanisms by which the pollen of Potamogeton maintains viability even after contact with water. The importance of hydration of pollen for Potamogeton species and its evolutionary implications are discussed. We also discuss the correlations among pollen traits, pollination types, and floral traits in Potamogeton.

Materials and methods

Study species

Potamogeton Linn., with more than 100 species, is the largest genus of ﬂowering plants in which all members are aquatic in habitat. The genus has a worldwide distribution but is concentrated in the Northern Hemisphere (Wiegleb and Kaplan 1998). Species in this genus display a range of morphological variation and habitat. Consequently three main pollination types are found in the genus namely anemophily, epihydrophily and hydroautogamy (Philbrick 1988; Zhang et al. 2009). For the present study we selected seven representative species representing all pollination types in the genus to investigate the effect of contact with water on pollen longevity. The experiments were performed during the summer of 2007 and 2008. Pollen was collected from several natural populations, and voucher specimens were preserved in Wuhan University Herbarium (WH), P. R. China. We also recorded the natural pollination process in these species and their overall morphology. Much of the detailed ﬂoral characteristics in our study populations, except for the natural seed set of the P. malaianus population which is located in Shanxi province, had been documented in an earlier study (Zhang et al. 2009).

Pollen longevity under different conditions

Pollen stainability is commonly used as an indicator of pollen viability (Dafni and Firmage 2000). In the present study we considered stainability of pollen with 2,5-diphenyl tetrazolium bromide (MTT) as an indication of pollen viability. MTT is a vital dye that detects the presence of dehydrogenase in viable pollen (Khatun and Flowers 1995; Rodriguez-Riano and Dafni 2000; Vizˇintin and Bohanec 2004). To assess the ability of the technique to discriminate 123 942 Evol Ecol (2010) 24:939–953 between viable and non-viable pollen, we also used the dye on heat-treated (boiled in water at 100°C for 2 h) pollen. We made attempts to germinate pollen grains (including the heat- treated pollen grains) in vitro. We placed the pollen grains in vials containing sucrose at -3 different concentrations (0, 2, 4, 6, 8, 10, 15 and 20%) and also in 1 9 10 MH3BO3.The vials were placed in room temperature (20°C) for 24 h and examined for evidence of germination. Pollen grains were considered to have germinated when pollen tube length was greater than or equal to pollen diameter (Rodriguez-Riano and Dafni 2000). We collected fresh pollen grains from just-dehisced anthers during the blooming period. A portion of the pollen from one plant was immediately stained with MTT to assess initial viability. The remaining pollen grains were divided into two equal sized groups. One group was put on a dry glass slide placed in the open air to simulate pollen waiting to be removed by airflow from the tepals. The other was dusted onto a glass slide which had drops of fresh water. The glass slides were placed at appropriate height in the field within the study populations in order to simulate as closely as possible the natural condition. Since the inflorescences of P. pectinatus are floating and constantly in contact with water during blooming, pollen viability for this species was tested only under the water condition. Samples of pollen were obtained periodically from each group at intervals of 2, 4, 6, and 8 etc., and tested for viability by staining for 20 min in MTT and examination under a light microscope fitted with a graticule. For each sample we studied about 200 grains each from five randomly selected equal portions of the microscope field of view. The percentage of viable (stained) pollen was calculated. The records of percentage of viable pollen were continued until it dropped to below 10% or until a drastic reduction was observed. The experiments were replicated five times for each species. In the present study we defined the half-life of pollen longevity as the time required for pollen viability to decline by one-half of its initial level. Longer pollen half-life was interpreted as an indicator of the ability of the pollen to withstand the effects of contact with water. Pollen grains of five Potamogeton species (P. natans, P. malaianus, P. perfoliatus, P. crispus and P. pectinatus) were collected from mature anthers and prepared for examination by scanning electron microscopy (SEM). Following dehydration in ethanol- acetone series and critical point drying with CO2, the samples were gold-coated in an evaporator and observed with an FEI scanning electron microscope at 30 kV.

Statistical analysis

We used non-linear estimation to describe the decline of pollen viability. We established that non-linear ﬁtted the data points well (Fig. 2). First-order exponential decay is described by the equation:

y ¼ c þ eðb0þb1xÞ ð1Þ where x is an index of time; e is the base of the natural logarithm; c, b0 and b1 are three parameters which can be obtained once the regression equation has been constructed from the data; y is 100 times of the measurement of pollen viability at time x. At 0 h, the b0 estimated initial viability is y^0 ¼ c þ e , at the half-life time x^1=2, the estimated viability 1 b0 should be y^1=2 ¼ 2ðc þ e Þ. By bringing the two variables into Eq. 1, we obtain: 1 ðc þ eb0 Þ¼c þ eðb0þb1x^1=2Þ ð2Þ 2

By rearranging the Eq. 2, experimental half-life time x^1=2 can be calculated as follows:

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b 1 e 0 c x^1=2 ¼ ln b ð3Þ b1 2e 0 Thus, Eq. 3 makes estimation of half-life possible once the three parameters of the regression Eq. 1 have been determined. We used correlation analysis to test the correlation between ﬂoral traits and pollen viability and the t-test for dependent samples to test the effect of contact with water on pollen half-life. STATISTICA for Windows statistical software (version 6.0) was used for all statistical analysis.

Results

MTT test and in vitro pollen germination

None of the heat-treated pollen grains were stained with MTT and neither did they germinate in vitro. In contrast viable pollen grains stained deep pink with MTT. This indicated that the dye successfully discriminated between the viable and non-viable pollen (Appendix 1). In vitro pollen germination was found to be minimally successful. The highest germination rate (12.06 ± 2.71%) was recorded from P. maackianus pollen after -3 24 h in distilled water with 1 9 10 MH3BO3. The germination rates for the other species were less than 10% (5.50, 3.21, 8.15, 9.55, 7.30 and 6.33% for P. distinctus, P. natans, P. malaianus, P. perfoliatus, P. crispus and P. pectinatus respectively).

Half-life of pollen longevity

The ﬁrst-order exponential decay suitably described the decline of pollen viability. In the aerial conditions, R ranged from 0.92661 (for P. malaianus) to 0.99939 (for P. distinctus). While, R ranged from 0.91723 (for P. perfoliatus) to 0.99857 (for P. pectinatus) in the water conditions (Fig. 1). The half-lives of pollen viability varied from 1.3 to 12.07 h in aerial conditions, and from 1.48 to 18.83 h in water conditions. The longest half-life recorded was 18.83 h for P. crispus in water condition, while the shortest was 1.3 h recorded for P. natans in aerial condition (Table 1).

Pollination types and pollen longevity

Two modes of pollination, obligate and facultative anemophily, were observed in the Potamogeton species studied. In the obligate anemophilic species, receptive stigmas receive pollen exclusively from the air, while the facultative anemophilic species, stigmas can receive pollen both from the air and from water currents. Our observation indicated that the three species (P. natans, P. distinctus and P. malaianus) displayed strictly obligate anemophily probably owing to their perpetually erect inﬂorescences. The other four species (P. pectinatus, P. crispus, P. maackianus and P. perfoliatus), whose inﬂorescences commonly undergo intermittent periods of submergence in turbulent water, displayed facultative anemophily (Table 1). The highest initial viability was 95.4 ± 1.63%, recorded for P. perfoliatus, while the lowest was 65.26 ± 2.56% recorded for P. malaianus (Table 1). The obligate anemophilous species had lower initial viability and half-lives of pollen longevity than the facultative anemophilous species (Fig. 2a, b). 123 944 Evol Ecol (2010) 24:939–953

Fig. 1 Exponential decay regression analysis for the reduction of pollen viability after anther dehiscence for each species in aerial and water conditions. Totally 5,000 pollen grains were counted for each point

Correlations between ﬂoral and pollen traits

Correlation analysis indicated that the half-life in aerial condition was significantly neg- atively correlated with P/O ratio (r =-0.869, P \ 0.05) and positively correlated with pollen diameter (r = 0.891, P \ 0.01; Table 2). Among the study species both the longest half-life of pollen viability and the largest pollen diameter were found in P. pectinatus. This is also the only species whose inflorescences float on the water surface during anthesis. The shortest half-life of pollen viability and the highest P/O ratio were found in P. natans.

Effect of water on pollen longevity

Pollen grains of Potamogeton species remain ﬂoating when placed on the surface of water. After about 6 h, some of the pollen begins to sink. Unexpectedly, most of the pollen remained viable in water for an extended period (Fig. 1), and no pollen grains were observed to burst. This indicated that the membranes maintained the integrity of the pollen grains in water. Analysis by t-test for dependent samples of the half-lives of pollen viability in aerial and in water conditions indicated signiﬁcant difference (t =-2.622, P = 0.039, df = 6). The mean half-life of longevity of the pollen grains in water (10.65 h) was considerably longer than that in aerial condition (5.79 h). This pattern was repeated for all the study species.

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Table 1 Floral traits (from Zhang et al. 2009, Table 2), pollination types, pollen viability and half-lives of the study Potamogeton species (the notation ane—stands for anemophily) Species P/O Ovule Pollen volume Pollen Seed set (%) Flowers per Pollination Initial viability Half-life (h) ratio number (lm3) diameter (lm) inﬂorescence type (%) Aerial Water

P. distinctus 25,563 2 7,547 24.20 66.21 26–54 Obligate ane- 88 ± 2.55 1.39 1.48 P. natans 38,637 4 5,233 21.33 51.50 28–60 Obligate ane- 74.38 ± 2.05 1.3 12.11 P. malaianus 24,839 4 7,892 24.43 48.93 33–46 Obligate ane- 65.26 ± 2.56 5.74 9.35 P. perfoliatus 19,466 4 11,151 27.58 74.13 12–25 Facultative ane- 95.4 ± 1.63 6.86 15.03 P. crispus 9,349 4 30,391 37.38 62.35 5–8 Facultative ane- 95.2 ± 3.65 8.21 18.83 P. maackianus 16,223 2 13,920 29.61 68.89 3–6 Facultative ane- 72.67 ± 1.85 4.94 5.68 P. pectinatus 6,752 4 17,567 39.55 73.65 6–12 Facultative ane- 81.01 ± 2.06 12.07 12.07 123 946 Evol Ecol (2010) 24:939–953

Fig. 2 Difference of pollen initial viability (a) and half-lives (b) between two pollination types. The bars are means, error bars present standard error, and sample sizes for obligate and facultative anemophily are 75 and 100

Table 2 Correlation analysis between pollen traits (sample size is 25 for each species, totally 7 species) P/O ratio Initial viability Half-life (aerial) Half-life (water)

Pollen diameter -0.9399* 0.3742 0.8902* 0.4714 P/O ratio -0.3792 -0.8686* -0.3179 Initial viability 0.2119 0.4107 Half-life (aerial) 0.5419

The number is r value, and the asterisk shows signiﬁcant correlation (P \ 0.05)

Discussion

Estimation of pollen viability and half-life of pollen longevity

Estimations of pollen viability obtained by staining with MTT have been found to be closely correlated with those obtained from actual pollen germination experiments. Staining with MTT is therefore considered to be one of the most reliable techniques of estimating pollen viability and is widely used (Khatun and Flowers 1995; Rodriguez-Riano and Dafni 2000; Vizˇintin and Bohanec 2004). Our results clearly demonstrate the capacity of MTT to discriminate between viable and non-viable pollen in the studied species of Potamogeton (Appendix 1). However, the pollen viability estimated by MTT was higher than the in vitro germination rate. Jin and Guo (2001) also reported low (less than 10%) in vitro germination rates for Potamogeton maackianus. However, examination for pollen tubes under fluorescence microscope after staining with aniline blue revealed in vivo germination rates of close to 90% for pollen of several Potamogeton species (R.-W. Gituru, Personal Observation). Several researchers have reported that the in vitro germination rate of pollen can be greatly influenced by different factors such as the genotype of the accession, the composition of the germination medium, temperature, and humidity (Vizˇintin and Bohanec 2004; Hedhly et al. 2005; Boavida and McCormick 2007; Ercisli 2007). It has been reported that in vitro germination tests are inappropriate for some systems and especially for the three-celled system (Shivanna et al. 1991). The results obtained in our experiments may indicate that the germination medium used may have 123 Evol Ecol (2010) 24:939–953 947 provided conditions that are different from those found on the receptive stigmas of Potamogeton plants. In view of the high rates of pollen germination observed in vivo (R.-W. Gituru, personal observation), we concluded that the rates of pollen viability estimated by staining with MTT were reliable. Up until now very few studies have focused on the half-life of pollen longevity. Fur- thermore, only a limited number of researchers have employed the regression analysis to describe the decline of pollen viability (Khatun and Flowers 1995; Song et al. 2001; Barabe´ et al. 2008), and they did not obtain the accurate half-lives by means of calculation. The model of exponential decay is widely used in biological research, especially in the fields of virology and bacteriology. Khatun and Flowers (1995) and Song et al. (2001) employed this model in their research on pollen viability in rice. Barabe´ et al. (2008) analyzed his results with the generalized linear regression, but the R2 was relatively low. In our present study using the first-order exponential decay regression analysis on the data points, we obtained remarkably high fitting degrees of regression equations for all the studied species (Fig. 1). We observed that pollen longevity in some species was greatly extended, and that the time it took for viability to cease was highly variable. The use of half-life of pollen viability provided a tool by which we could make the comparisons of pollen viability in different species and under different conditions more easily. This approach may also have wide significance in research on characters which vary regularly along a time gradient.

Pollen hydration status and the risk of desiccation

During the maturation process, pollen commonly undergoes dehydration and rehydration (Shivanna and Heslop-Harrison 1981; Heslop-Harrison 1987). On the basis of the water content of pollen, two types can be distinguished; namely partially dehydrated pollen (PD pollen) and partially hydrated pollen (PH pollen; Nepi et al. 2001; Franchi et al. 2002). PH pollen is ‘generally spherical in shape and lacks furrows’ (Nepi et al. 2001). Franchi et al. (2002) noted two more features of PH pollen namely: fast pollen tube initiation and rapid loss of viability, especially under conditions of low relative humidity. Results of our present study have revealed that pollen grains of the studied species of Potamogeton are all spherical (except for P. pectinatus) and lack furrows (Fig. 3). Pollen viability decreased more rapidly under aerial condition than in water condition, and pollen tube growth usually was initiated within 15 min after deposition on stigma (R.-W. Gituru, personal observation). Additionally, during preparation for SEM the pollen grains commonly crumpled into irregular shapes (Fig. 3), which is indicative of a rapid loss of water. On the basis of the foregoing observations and considering the aquatic habitat in the genus, it is reasonable to assume that the pollen in this genus is partially hydrated (PH). In the absence of any special mechanisms to retain moisture, PH pollen is more sensitive to desiccation than the PD pollen. In anemophilic Potamogeton species, PH pollen faces the distinct risk of desiccation during aerial transport. However, the level of risk is dependent on the size of the pollen grain. Our results revealed that larger pollen grains have longer half- life under aerial condition than smaller sized grains. This is consistent with the ﬁnding by Franchi et al. (2002) that large pollen grains survive longer than small sized ones due to their higher volume/surface area ratio which reduces the risk of desiccation.

Correlations of pollen longevity, pollination types and ﬂoral traits

In all the seven studied Potamogeton species, pollen viability decreased with time and the half-lives of pollen viability were all less than 20 h (Table 1). In comparison with the data 123 948 Evol Ecol (2010) 24:939–953

Fig. 3 S.E.M. photographs of pollen of five Potamogeton species. a P. natans; b P. malaianus; c P. perfoliatus; d P. crispus; e P. pectinatus for 33 species from different taxa [summarized by Dafni and Firmage (2000)] as well as the results of studies on 6 neotropical aroid species (Anaphyllopsis americana, Monstera adansonii, Montrichardia arborescens, Philodendron melinonii, Philodendron pedatum and Philodendron solimoesense; Barabe´ et al. 2008), the rate of decrease of pollen viability in Potamogeton may be considered to be relatively rapid. Although until now no work has been reported correlating pollen longevity with pollination type, entomophilic species are generally considered to have greater pollen longevity owing to the fact that the grains must wait for insect pollinators (Pacini et al. 1997). However, pollen longevity is also associated with features such as population density, weather, strategy of pollen presentation and the stage of development of the male gametophyte (Stanley and Linskens 1974; Dafni and Firmage 2000). Potamogeton is an entirely aquatic monocotyledonous genus, and the species usually develop crowded clonal populations. The high population density ensures that the distances traveled by the pollen during aerial transport are quite short. Under these circumstances any adaptation for longer half-life of pollen viability would be of minimal adaptive value to the plant (Dafni and Firmage 2000). In the studied species of Potamogeton the pollen of the obligate anemophilic species have relatively lower initial viability and shorter half-lives than the facultative anemophilic species (Fig. 2a, b). In the obligate anemophilic species there are numerous flowers per inflorescence and anthers dehisce in acropetal succession, while in the facultative anemophilic species there are fewer flowers per inflorescence and their anthers dehisce almost simultaneously. Additionally, the male stage in the obligate anemophilous species is much longer (Zhang et al. 2009). The results of the present study are in agreement with Lloyd and Yates (1982) who concluded that for species with relatively rapid pollen viability decline, selection might favor sequential anther dehiscence or to have numerous flowers open at the same time. Our results could also be explained by the fact that the pollen grains in obligate anemophilic species are smaller which places them at a greater risk of desiccation.

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The fate of pollen is different in obligate and facultative anemophily. In the obligate anemophilic species, pollen is mainly transported by airflow which provides a desic- cative environment while in the facultative anemophilic species; pollen has more likelihood of coming into contact with water. Our results bring to the fore two important questions namely: Why does the pollen of facultative anemophilic species have longer half-life of viability under aerial condition than that of obligate anemophilic species? And why does the pollen of obligate anemophilic species have higher half-life of viability in water than in the aerial condition? The first question could be explained mainly by the differences in the size of pollen grains. In an earlier study on the floral traits and pollination systems of 14 Potamogeton species (Zhang et al. 2009), the authors reported that species which have erect inflorescences and obligate anemophily have a much smaller pollen size. The smaller pollen size enhances the risk of desiccation, with a consequent loss of pollen viability (Franchi et al. 2002). However, considering that large numbers of small pollen grains are favorable to anemophily, and that the pollination process in the obligate anemophilic species is usually fast, the risk of desiccation can be considered as inconsequential. The second question could be explained by the hydrated status of pollen. The water condition prevents the loss of water from PH pollen and protects pollen viability. This finding contradicts a widely held belief that water is detrimental to pollen viability in many angiosperms (Gilissen 1977; Shivanna and Heslop-Harrison 1981; Dafni 1996; Huang et al. 2002; Mao and Huang 2009). Although the pollen of obligate anemophilic species is mainly transported by airflow, it will almost inevitably come into contact with water and the pollen grains that are not captured by stigmas eventually drop onto the water surface. In environments with minimal wind, pollen grains that fall down from inflorescences to the water surface gradually form a ‘‘pollen raft’’ (Appendix 2). The hydrated status confers on the pollen a longer half-life of viability in water. This feature reduces the loss of pollen viability and provides extra opportunity for pollination to occur when the viable drifting pollen meets a floating inflorescence with receptive stigmas (Appendix 2). Since Potamogeton species with different life forms are usually sympatric, and there are no obvious prezygotic reproductive barriers in this genus (X.-L. Zhang unpublished data), this pollination strategy also provides a potential opportunity for interspecific hybridization between obligate and facultative anemophilic species. The existence of several hybrids in the genus with parents of different morphological characters supports this viewpoint.

Evolutionary implications of hydrated pollen in aquatic angiosperms

Several studies have indicated that while pollen of most terrestrial species is killed by contact with water, pollen of some terrestrial species can withstand this exposure with various degrees of success. However, prior to our present study, contact with water has never before been known to prolong pollen longevity (Eisikowitch and Woodell 1974; Gilissen 1977; Shivanna and Heslop-Harrison 1981; Dafni 1996; Huang et al. 2002; Mao and Huang 2009). Our results show no detrimental effect on pollen of seven Potamogeton species when exposed to water and on the contrary, the half-lives of pollen longevity in these species are much longer in conditions of water (mean of 10.65 h) than in aerial conditions (mean of 5.79 h). To the best of our knowledge this is the ﬁrst report of prolonged half-life of pollen longevity due to exposure of water. In this case water represents a means of protection for partially hydrated pollen from the risk of desiccation. 123 950 Evol Ecol (2010) 24:939–953

It is generally accepted that most aquatic angiosperms have evolved from their terrestrial ancestors (Philbrick and Anderson 1987; Cook 1988, 1990; Philbrick and Les 1996). The evolutionary adaptations in reproductive characters are very important in this process. In Potamogeton, three pollination types are found namely anemophily, epihydrophily and hydroautogamy (Philbrick 1988). Hydroautogamy with aid of bubbles under water was considered as an intermediate stage between aerial systems and hypohydrophily (Philbrick 1988). Ackerman (1995) indicated an evolutionary path in seagrasses from spherical to ﬁlamentous pollen. Zhang et al. (2009) reported elliptical pollen in P. pectinatus, which is mainly pollinated by bubbles under water. Our study has shown that the partially hydrated (PH) pollen of facultative anemophilic Potamogeton species has higher half-life in water. The partially hydrated status of pollen is in line with the trend of evolution of pollen morphology in aquatic angiosperms. Franchi et al. (2002) suggested that the transition from PD to PH pollen have occurred in response to certain pollination and environmental constraints. We propose that the hydration status in pollen of aquatic angiosperms may have undergone an evolution process from PD pollen to PH pollen and that this transition was primarily due to the selective pressure of water. In the evolution of pollination system in aquatic angiosperms close proximity to water has resulted in selection for wettability.

Conclusion

Unlike in many terrestrial angiosperms, the pollen of Potamogeton species is not killed by contact with water. This is mainly due to its partially hydrated status. On the contrary, desiccation is the primary reason for the loss of pollen viability in this group because the pollen lacks mechanisms to retain water. Although smaller pollen grains have higher risk of desiccation, the copious numbers of pollen and the fast pollination process ameliorate the risk for obligate anemophilic species. The hydrated status of pollen has great evolutionary signiﬁcance in aquatic angiosperms. Along with the change in pollen morphology, partially hydrated pollen is better adapted to the aquatic environment. The longer half-life of viability in water and fast pollen tube initiation ensure the success of the reproductive process in unstable water environment. For many terrestrial plants, special characters have evolved to protect pollen viability in unfavorable environments (Bynum and Smith 2001; Huang et al. 2002; Mao and Huang 2009). In aquatic angiosperms, the pollen morphology and hydration status have evolved to adapt to the environment. In those plants that are characterized as entirely ‘‘aquatic’’ water no longer represents a threat to the pollination process but rather it serves to enhance it.

Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (No. 30430050) to Y.H.G. Many thanks go to Tao Wan and Zhi-yuan Du for their assistance in the ﬁeld work. We especially thank Prof. Z.P. Song for his helpful suggestions. We are grateful to Yun-yun Mao for her assistance in the laboratory and for her valuable comments on an earlier version of the manuscript. We would also like to thank the editor and the three anonymous reviewers for their comments and helpful suggestions.

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Appendix 1

See Fig. 4

Fig. 4 Viable (a) and unviable (b) pollen grains of P. perfoliatus stained by MTT. a viable pollen at 0 h; b unviable pollen at 20 h in aerial condition. The pollen grains were photographed at a magniﬁcation of 100

Appendix 2

See Fig. 5

Fig. 5 The ‘‘pollen raft’’ in natural populations. a an obligate anemophilous species P. distinctus; b facultative anemophilous species P. perfoliatus, in which pollen could reach the receptible ﬂoating inﬂorescence with the aid of pollen raft. The arrow points to a pollen raft

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