Journal of Plant Research https://doi.org/10.1007/s10265-018-1016-y

REGULAR PAPER

Delayed selfing ensures reproductive assurance in Utricularia praeterita and Utricularia babui in Western Ghats Anjali Chaudhary1 · S. R. Yadav2 · Rajesh Tandon1 Received: 12 December 2017 / Accepted: 19 January 2018 © The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2018

Abstract Numerous bladderwort (Utricularia) species are distributed worldwide, but their reproductive biology is rarely investigated. Bladderworts are known to depend on tiny organisms to meet a significant proportion of their energy requirement by trapping them in bladders. However, information on the extent of their reliance on insects for pollination success is limited. We examined the reproductive strategy of two Utricularia species viz. Utricularia praeterita and U. babui, endemic to Western Ghats, India. The main aspects of the investigation involved floral biology, breeding system, pollination mechanism, and reproductive success. Flowers of both the species are structured for outbreeding through entomophilous floral suites, herkogamy, protandrous dichogamy and sensitive lobes of the stigma. With nearly 65% natural fruit-set, both the species appeared to be sufficiently open-pollinated. However, pollinators failed to show in plants of U. praeterita while in U. babui there was an apparent mismatch between the extent of fruit-set and pollinator visits. The study demonstrated that in the absence/insufficient visits of pollinators, the two species resort to autonomous selfing. In U. babui, denser patches of plants appeared to be crucial for attracting the pollinators. Both species are self-compatible, and reproductive success is predominantly achieved by delayed autonomous selfing. The sensitive stigma in the species fails to prevent selfing due to diminished herkogamy during the late anthetic stages. It is inferred that in the pollinator-limited environment, delayed selfing contributes to absolute natural fecundity in U. praeterita, while it produces a mixed progeny in U. babui. Keywords  Bladderworts · Herkogamy · Melittophily · Pollinator limitation · Protandry · Thigmosensitive stigma

Introduction Utricularia L. (Lentibulariaceae) species, popularly known as bladderworts, have captivated the attention of many biologists for their carnivorous trait. The subterranean/submerged bladder-like modified leaves of the plants trap various minute organisms such as invertebrates, protozoans, algae and digest them to obtain nutrition (Fineran 1985; Płachno et al. 2012). Utricularia has a cosmopolitan distribution except for some oceanic islands and arid regions (Taylor 1989), and in India the species has a wide range of distribution (Janarthanam and Henry 1992). Bladderworts prefer to grow in nutrient-poor soils of wet or marshy areas as floating aquatic * Rajesh Tandon [email protected] 1



Department of Botany, University of Delhi, 110007 New Delhi, Delhi, India



Department of Botany, Shivaji University, 416004 Kolhapur, Maharashtra, India

2

(15% species), terrestrial/lithophytic (60%) or as epiphytic (25%) plants (Janarthanam and Henry 1992; Taylor 1989). Out of 228 species of Utricularia known worldwide (Fleischmann 2012; Taylor 1989), 38 are believed to occur in India (Janarthanam and Henry 1992; Yadav et al. 2000, 2005). Despite their vast distribution and diversity in the world, information on the reproductive strategy of bladderworts is inadequate. So far, studies on Utricularia have been conducted on the mechanism, functioning (Llorens et al. 2012; Westermeier et al. 2017) and on the prey spectra of traps (Alkhalaf et  al. 2009; Koller-Peroutka et  al. 2015; Płachno et  al. 2012). Ultrastructural studies of corolla structure, female gametophyte development, post-fertilization events such as formation of endosperm haustoria and syncytium have also been done extensively ( Płachno and Świątek 2011, 2012; Płachno et al. 2013, 2017). Field ecological studies on bladderworts are fewer, perhaps because of their usual small population sizes in nature, and due to ephemerality of their habitats. A few species studied so far have shown

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that the plants are self-compatible and can self-pollinate (Araki and Kadono 2003; Jérémie 1989; Kausik and Raju 1955; Khosla et al. 1998; Yamamoto and Kadono 1990). There are only two reports on the outcrossing behaviour of bladderworts (Clivati et al. 2014; Hobbhahn et al. 2006). A study on U. reniformis is the only field study that focuses on some aspects of floral biology, and reproductive constraints (Clivati et al. 2014). Certain floral features of bladderworts such as bright colour, bi-lipped corolla with suitable landing space, concealed reproductive organs and well-developed spur with nectary glands are indicative of out-crossing. Touch sensitive stigma, which is usually considered to be an outbreeding device, has been reported from U. alpina (Jérémie 1989), U. vulgaris (Knuth 1899), U. reticulata and U. albocaerulea (personal observations) and U. reniformis (Clivati et al. 2014). Western Ghats, which is one of the biological hotspot with a high level of endemism (Myers et al. 2000; Nayar 1996), harbours approximately 50% of the Indian Utricularia species (Janarthanam and Henry 1992). We selected two of the terrestrial species viz. Utricularia praterita Taylor and Utricularia babui Yadav, Sardesai and Gaikwad, from the region to study their reproductive ecology. Floral biology, pollination mechanism and breeding system were the main aspects of the investigation. We demonstrate here for the first time that success in natural fruit-set among the bladderwort species is primarily achieved through delayed autonomous selfing. As both the taxa possessed a sensitive stigma, its role in reproductive success was evaluated. Further, we determined the relative contribution of pollinators and autonomous self-pollination in natural fruit-set of the two species.

Materials and methods Study sites and species Study was conducted during August–September for Utricularia praeterita P.Taylor and January–March for Utricularia babui S.R.Yadav, M.M. Sardesai & S.P.Gaikwad between 2015 and 2017. The peak flowering period of the two species is separated by almost 2 months and the fruiting phase is initiated during the middle of flowering period in both species. The site of U. praeterita is located at Tilhari, Kolhapur, Maharashtra (N 15°48′00.0″; E 074°10′32.0″) and that of U. babui is in Suleran village of Ajara district, Kolhapur, Maharashtra (N 16°04′58.2″ E 074°06′16.4″). Three populations each were identified at each study site. Both species of Utricularia (Lentibulariaceae) are annual carnivorous herbs. The plants generally occur along the margins of seasonal streamlets on the laterite plateaus, mostly with grasses (Fig. 2a, b). Both species are endemic

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to Western Ghats of India (Janarthanam and Henry 1992; Yadav et al. 2005). Utricularia praeterita has been categorised as ‘near-threatened’ by IUCN (Watve 2011), while the status of U. babui has not been evaluated. Utricularia praeterita is threatened due to urbanization, mining activities and increased tourism in Western Ghats. All of these factors are responsible for habitat decline (Watve 2011).

Floral biology Buds were randomly tagged (n = 100 plants) and monitored for the onset of anthesis and time of anther dehiscence. Floral lifespan was studied by randomly tagging another set of flowers (n = 50) 1 day prior to anthesis and monitoring them daily until senescence. The average amount of pollen and ovule produced in a flower (n = 25 flowers) was carried according to a method followed earlier (Khanduri et al. 2014). Pollen to ovule ratio was calculated to predict the possible breeding system (Cruden 1977). Pollen-viability (Fig. 1c, g) was ascertained by fluorochromatic reaction test (Heslop-Harrison and Heslop-Harrison 1970). Temporal details of the stigma receptivity were established by localising peroxidases (Fig. 1d, h) on the stigmatic surface at different stages of flowers recognized for the study (Dafni et al. 2005).

Breeding system To establish the breeding system, bagging experiments with the following floral manipulations (n = 50 plants, for each treatment) were performed, followed by monitoring the flowers for fruit-set: (i) Cross-pollination: flowers were emasculated and pollinated using fresh pollen obtained from a different plant, (ii) self-pollination: flowers were emasculated and pollinated using pollen from the same flower, (iii) Autopollination: floral buds were bagged, (iv) Apomixis: flowers were emasculated and bagged before anthesis, (v) Open-pollination: flowers (n = 250) were randomly tagged in the populations and monitored for fruit-set under natural conditions and considered as control, (vi) Pollen-removal: flowers were emasculated and kept unbagged to determine the amount of fruit-set achieved through pollinators. The data on fruit-set within each species were pooled, as there was no difference during the different flowering seasons (U. praeterita: t-test, t = − 0.391, P > 0.05; U. babui: t-test, t = 1.999, P > 0.05) and populations (U. praeterita: One-way ANOVA; F (2, 71) = 0.0001, P > 0.05,  U. babui: One-way ANOVA; F (2, 89) = 0.027, P > 0.05). Indices of self-fertility (SFI) and self-compatibility (SCI) were calculated for both the species for two flowering seasons in the three populations (Lloyd and Schoen 1992); the values were averaged.

Journal of Plant Research

Fig. 1  Details of floral biology of Utricularia babui (a–d) and U. praeterita (e–h). Scanning electron micrographs of inner surface of the spur with spherical glandular trichomes (arrow) (a, e) and conical papillae (b, f). Epifluorescence photomicrographs depicting pollen

viability; the brightly fluorescing pollen grains (arrow) are viable (c, g). Emergence of oxygen bubbles due to peroxidase activity on the stigmatic surface, indicating to its receptive condition (d, h). Scale bar = 50 µm (a, e, h); 10 µm (b, c, f, g); 100 µm (d)

Sensitivity of stigma

nectar. Nectar spot diameters were measured by Vernier calliper and nectar volume was calculated from a standardized curve of Nectar volume vs. spot diameter (Dafni et al. 2005; Punchihewa 1984). Total sugars and their concentration in the nectar were determined by hand-held refractometer (Brix 0–80%). Amino acid concentration was determined by comparing the sample with a histidine scale (Dafni et al. 2005).

The stigma is bi-lipped and touch-sensitive (thigmosensitive) in both species. A gentle touch with a finger was found to be sufficient to trigger the closure of the stigmatic lobes. Therefore, it was necessary to understand the pattern and process associated with the stigmatic movement. The observations in this regard were made during the peak period of flowering, and two sets of experiments were conducted for both species. First we determined the temporal details and extent of closure by touching the stigmatic lobes with ear buds. For this, 100 flowers at anthesis were touched and the time interval between closure and reopening of the stigmatic lobes was recorded. An angle of approx. 60° between the lobes was considered re-opened while that of between 30° and 60° as partially opened. Owing to the sensitive nature of stigma, the angle was measure from chemically fixed flowers (FAA, 4 h) at respective stages under the portable field stereo-microscope, and placing them over a plastic protractor. In the second experiment, we looked into the effect of pollen deposition (irrespective of the mating type) on closure/ reopening pattern. Pollen grains were gently dusted onto the opened stigmatic lobes (n = 50 flowers) directly from the dehisced anthers. Flowers (n = 50) dusted with sand particles were considered to be controls.

Floral rewards Floral nectar was extracted by carefully inserting the small triangular strips of Whatman filter paper no.1 to absorb

Pollination ecology To identify the pollinators, the floral visitors and their behaviour were observed for 10 consecutive days, at an interval of 30 min each between 0600 and 1800 h. The stigmatic pollen load, flower-handling time, frequency of visits and number of flowers visited/bout by the pollinators was recorded. Among the two species, only U. babui received pollinators. To record the pollinator frequency, insect visitation rate was calculated per day in a population of U. babui. One of the populations was demarcated into five patches (~ 150 plants ­patch−1) and the number of insects visiting the patch was counted; the values were then averaged for the population. As the display size of a solitary flower was small in U. babui, it was essential to ascertain if patch density had an effect on insect visits. To validate the correlation between the patch density and insect visits, an experiment was designed. Five quadrats (1 m2, each) were marked and each quadrat belonged to a category of plant patch density representing 25, 50, 75, 100, or 150 plants in a quadrat. The total number of insects visiting the marked patches was recorded. The observations were recorded for 10 days.

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Journal of Plant Research ◂Fig. 2  Flowering patches and the plants of selected Utricularia spe-

cies in bloom. Flower patches at natural habitats, U. praeterita (a) and U. babui (b). Flowering twig of U. praeterita (c) and that of U. babui (d). Floral visitors were noticed only on U. babui which was primarily pollinated by e Apis florea and f Apis mellifera. Bombylius major (g) and another Bomblyidae species (i) inserting the proboscis between the corolla lips. Episyrphus balteatus foraging the flower for floral reward (h). Scale bar = 10 mm (a, b); 2.5 mm (c, d, i); 5 mm (e–h)

Pollinator exclusion and delayed autonomous self‑pollination To ascertain the occurrence of delayed selfing, the period of self-pollen deposition vis-à-vis xenogamy had to be identified. Two treatments were performed at anthesis stage (n = 100 each treatment). In the first treatment (T1), randomly selected open-pollinated flowers were left exposed to pollinators while in the second treatment (T2), flowers were tagged and bagged to exclude the floral visitors. The amount of stigmatic pollen load was determined from open-pollinated flowers according to an earlier method (Tandon et al. 2001). In order to establish the mechanism of self-pollination, the temporal changes in the positioning of anthers with respect to stigma was recorded at different stages of flowers in both species. For U. praeterita, two stages—the day of anthesis (DOA) and 1 day after anthesis (1DAA), and for U. babui four stages—2 days before anthesis (2DBA), 1 day before anthesis (1DBA), day of anthesis (DOA) and the day of pollination (DOP) were selected. The flowers of both the species were fixed in 2.5% glutaraldehyde and dehydrated with a graded series of chilled acetone (10–100%, 30 min interval each). The samples were then critical point dried and mounted on aluminium stubs with a double-sided adhesive tape. The samples were coated with gold–palladium alloy (JFC 1600 Autofine Coater, Jeol, Japan). Observations were made by using a scanning electron microscope (JSM6610LV-Jeol, Japan). For measuring the length of filament in both species, pre- and post-pollination stages were selected. Measurement was done by using a digital Vernier calliper (n = 25, each stage). All data analyses were carried out with SPSS ver. 22 statistical software (IBM® SPSS® Amos™ 22; IBM Corp. Released 2013). Percentage data were root-square arcsinetransformed for homoscedasticity before analysis. The values are presented as mean ± standard error.

Results Floral biology The floral architecture is similar in both species. Flowers are zygomorphic and hermaphrodite with a bi-labiate corolla

(Fig. 2c, d). The larger ventral corolla lip is gibbous and descends into a nectariferous spur (Fig. 2c, d). Spherical glandular trichomes (Fig. 1a, e) intersperse with conical papillae that line the inner spur surface (Fig. 1b, f). Reproductive organs are attached to the base of upper corolla lip and androecium is ventrally aligned to the gynoecium. In U. praeterita, the flowers opened during crepuscular time (~ 1900 h) while those of U. babui opened during dawn (0500–0700 h). An inflorescence of U. babui produced an average of 2.6 ± 0.22 flowers, and that of U. praeterita produced an average of 1.98 ± 0.09 flowers (n = 50 plants, each species). Flowers were protandrous in both the species. Flowers of U. praeterita lasted for 20 h and those of U. babui for nearly 6 days, when not pollinated. The stigmatic lobes are unequal; the dorsal lobe being larger and receptive. The stigma receptivity in U. praeterita was attained 1DAA and was maximum between 1030 and 1330 h (Fig. 1h). The stigma of U. babui exhibited receptivity up to 4–6 DAA from the day of its inception on DOA (Fig. 1d). Pollen production is profuse, and a great majority of fresh pollen grains are viable (Fig. 1c, g), (U. praeterita: 79.08 ± 10.11%; U. babui: 80.14 ± 10.31%); the grains retained viability in U. babui up to 6 days after anther dehiscence. Pollen:ovule ratio was 19.71 ± 0.37 (n = 25) in U. praeterita, and 24.47 ± 1.21 (n = 25) in U. babui. Self-deposition of pollen is prevented in the freshly opened flowers because the stigma overarches the dehisced anther lobes (herkogamy). In U. praeterita, the herkogamous phase was maintained for ~ 20 h and by that time the stamens elongated to the level of the stigmatic lobes; the latter condition led to autopollination. On the other hand, the herkogamous condition was retained up to 4–6 DAA in U. babui and then the flowers get autopollinated.

Breeding system Both the species turned out to be self-compatible, as all the pollination treatments except those involving emasculated and unpollinated flowers (apomixis) led to fruit-set (Table 1), and the values of SFI (U. praeterita, 1.4; U. babui, 1.0) and SCI (U. praeterita, 1.7; U. babui, 1.06) were in accordance with the finding. Pollen:ovule ratios of both the species corresponded to obligate autogamous breeding system. Interestingly, manual cross-pollinations did not improve the extent of fruit-set (Table 1). In U. praeterita, autogamous fruit-set was significantly greater than that from xenogamous (t-test, t = 19.919, P = 0.001), and the pattern was the same during the different seasons of the study period. On other hand in U. babui, there was no significant difference between the autogamous and xenogamous fruit-set (t-test, t = − 1.267, P > 0.05). In pollenremoval treatment (unbagged and emasculated flowers), there was no fruit-set in U. praeterita, and it was also

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Table 1  Percentage fruit set of different pollination treatments in Utricularia praeterita and Utricularia babui 

Floral rewards

Treatments

Freshly opened flowers in both the species were brightly coloured with contrasting nectar guide marks. Interestingly, the flowers of U. praeterita neither produced nectar nor received any forager. In contrast, flowers of U. babui produced nectar in trace amounts which did not replenish; the nectar was produced as viscous and sugar rich (~ 60%) drop; nectar also contained phenolics and proteins. The amino concentration was 0.48 mg/mL. Nectar production was initiated a day before anthesis, but the volume reached its peak on the day of anthesis (0.25 ± 0.01 µL). Floral nectar was present in all tagged flowers that remained unvisited by the foragers. However, nectar volume in these flowers declined to less than 0.1 µL on 3DAA.

Cross-pollination (n = 50) Self-pollination (n = 50) Auto-Pollination (n = 50) Open-Pollination (n = 250) Pollen-removal (n = 50)

Per cent fruit-set Utricularia praeterita

Utricularia babui

67 87 65 63 –

80 78 68 65 16

significantly lower than the open-pollinated flowers in U. babui (t-test, t = 50.058, P = 0.001) (Table 1).

Stigmatic movement The lower of the two stigmatic lobes is larger and covers the upper smaller one in response to touch. In both species, the lobes closed within 5 s and there was no significant difference between the closure time in the two species (U. praeterita 4.5972 ± 0.04 s, U. babui 4.50 ± 0.05 s; t-test, t = − 0.943, P > 0.05). During the peak period of insect visitation in U. babui, 75% of the flowers of U. babui showed fully opened lobes (60°), and 25% flowers with partially opened lobes (30°). On the other hand, the stigmatic lobes were always seen in opened condition in U. praeterita. The time taken by the lobes to re-open was longer when stigmatic lobes were dusted with pollen grains (U. praeterita: 40.1 ± 0.56 min, U. babui: 100 ± 2.60 min) than with deposition of sand particles (U. praeterita: 21.56 ± 0.42 min, U. babui: 31 ± 0.53 min). The two species differed significantly in response when the lobes were dusted with pollen (t-test, t = − 22.961, P = 0.001) and sand (t-test, t = − 17.215, P = 0.001).

Pollination ecology A variety of insects belonging to different functional groups foraged the flowers of U. babui (Table 2). Among all the insects, Apis florea was the most frequent visitor. The other visitors included Apis mellifera, Episyrphus balteaus, Sceliphron caementarium, Ceratina sp., Nomia sp., Bombylius major (Table 2). In a population, there were blue, purple and pink flowers, whose corolla size did not vary significantly (One-way ANOVA; F (2, 59) = 1.490, P > 0.05). Visitors treated them as one, as the visiting frequency (One-way ANOVA; F(2,26) = 2.374, P > 0.05) and the flower-handling time of Apis florea was the same on all floral sizes (One-way ANOVA; F(2,29) = 0.031, P > 0.05). Foraging behaviour, flower handling, visiting frequency Foraging activity was confined between 1030 and 1630 h. The peak time of visitation was from 1130 to 1330 h. Bees and wasps were able to completely open the flower by pushing the lower lip downwards. Foraging led to opening of the two corolla lips thereby bringing the pollen laden body parts in contact with the receptive lobe of the stigma. Although

Table 2  Foraging characteristics of floral visitors of Utricularia babui  Floral visitor

Flower handling time (seconds)

Flowers visited per flight

Plants visited per flight

Stigmatic pollen load

Apis florea Apis mellifera Episyrphus balteatus Nomia sp Sceliphron caementarium Ceratina sp Bombylius major Bomblyidae Unidentified 1 Bomblyidae Unidentified 2

12 ± 0.37 (n = 20) 4.25 ± 0.22 (n = 20) 42 ± 3.26 (n = 10) 12.9 ± 1.04 (n = 7) 25.33 ± 2.90 (n = 3) 8.02 ± 0.95 (n = 8) 3.05 ± 0.18 (n = 10) 2.55 ± 0.16 (n = 10) 2.85 ± 0.20 (n = 10)

5.75 ± 0.36 (n = 8) 18 ± 1.65 (n = 5) 2.87 ± 0.22 (n = 10) 6 ± 0.5 (n = 2) 4.33 ± 0.33 (n = 3) 4.5 ± 0.5 (n = 2) 2.5 ± 0.26 (n = 10) 2.5 ± 0.18 (n = 10) 2.5 ± 0.18 (n = 10)

5.2 ± 0.2 (n = 10) 7.6 ± 0.67 (n = 5) 2.7 ± 0.21 (n = 10) 2.66 ± 0.18 (n = 3) 2.33 ± 0.18 (n = 3) 2.66 ± 0.18 (n = 3) 2.2 ± 0.13 (n = 10) 2.2 ± 0.13 (n = 10) 2.2 ± 0.13 (n = 10)

101.66 ± 18.76 (n = 4) 109 ± 15.42 (n = 8) 55 ± 2.88 (n = 3) 83 ± 19.88 (n = 6) 32 ± 3 (n = 2) 70.71 ± 14.36 (n = 7) – – –

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Apis mellifera (Fig. 2f) did not make frequent visits, the foraging behaviour of bees was effective in pollination (Table 2). All bee species recorded at the sites exhibited a short flight distance and foraged the denser patch before shifting to distantly scattered flowers in a bout. Visitation of bees led to pollination, and while leaving they collected pollen on their head and thoracic region of the visited flower (Fig. 2e, f). In U. babui, patch density influenced the frequency of visits (Fig. 3). A direct positive correlation was observed between the density and the insect visitation rate (R2 = 0.9904; Fig. 3). In the smallest patch investigated (~ 25 plants ­m−2), the visits were absent (Fig. 3). Thus, for visitation and pollination a threshold number of flowers were needed for presentation, which was approx.75 flowers ­m−2. On average, in a population of approximately 700 plants, 7.6 ± 0.65 visits ­day−1 were made by the pollinators, indicating to low pollinator frequency at the sites.

Pollinator exclusion and delayed autonomous self‑pollination The pollinator exclusion experiment yielded different results in the two species. In both the sets of flowers (T1 and T2) of U. praeterita, the stigma invariably collected pollen 1DAA (Fig. 4a, b). On the contrary, in the T1 flowers of U. babui, the pollen load was seen on the flowers from the onset to the end of anthesis, although the load also varied according to the availability, frequency and efficiency of pollinators (Fig. 4a). Although pollen load on pollinators varied, it was significantly lower than the quantity of pollen receipt through delayed selfing (t-test, t = − 2.667, P < 0.05). In T2 flowers of U. babui, the stigma lacked pollen deposition until 3DAA (Fig. 4b). The stigmatic pollen load was seen only when the flowers underwent autonomous self-pollination i.e. 4–6 DAA (Fig. 4b). These flowers also exhibited an appreciable amount of fruit-set (Table 1).

The mechanism of delayed autonomous self-pollination was similar in both species and consistently occurred at the end of anthetic phase. During the initial stages of anthesis, the flowers were distinctly herkogamous with anthers placed below the level of the dorsal stigmatic lip (Fig. 5a–d). In U. babui, this arrangement with undehisced anthers was maintained until bud stage (2DBA, 1DBA) (Fig. 5a, b) and at the time of anthesis (DOA) with dehisced anthers (Fig. 5c). In U. praeterita also, dehisced anthers were placed below the stigmatic lip on the day of anthesis (Fig. 5d). The filaments elongated up to the level of the stigma 1DAA in U. praeterita (Fig. 5f), and 4–6 DAA in U. babui (Fig. 5e). The extension of filaments and recurving of the dorsal stigmatic lip towards the dehisced anthers led to autonomous selfing (Fig. 5e, f). As the loss in herkogamous condition was necessary to pollinate the stigma, herkogamy in both the species is of ‘movement herkogamy’ type which belongs to category ‘Ordered herkogamy’ suggested by Webb and Lloyd (1986). A significant difference was seen between the filament length at pre- and post-pollination stages (pre: 0.28 ± 0.00 mm, post: 0.43 ± 0.01 mm; t-test, t = − 8.587, P = 0.001) in U. praeterita, as well as in U. babui (pre: 0.52 ± 0.03 mm, post: 0.92 ± 0.04 mm; t-test, t = − 11.658, P = 0.001).

Discussion Floral biology In flowering plants, a thigmosensitive stigma follows two patterns of responses to touch. In some species such as Oroxylum, the lobes close permanently in response to touch (Srithongchuay et al. 2008; Vikas et al. 2009) while in others species such as Mazus, the stigma reopens at regular intervals if not sufficiently pollinated (Jin et al. 2015).

Fig. 3  Relation between patch density and the total number of insects visited in a day on U. babui. Patch density had a significant influence on the number of visits

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Fig. 4  The outcome of pollinator exclusion experiment involving two treatments (T1 and T2). a Stigmatic pollen load on the two species in response to debagged flowers. Pollen grains were observed only on 1DAA in U. praeterita (white bar) while they were noticed at various stages form DOA to 4–6 DOP in U. babui (grey bars). b The amount of stigmatic pollen load in bagged flowers (pollinator exclusion) due to autonomous selfing. In U. praeterita (white bar), stigmatic pollen load was seen on the same day (1DAA), as in T1, while in U. babui (grey bar) pollen load was seen only on 4–6 DAA

These patterns can have ecological consequences on the reproductive success. Whereas reopening of the stigmatic lobes could be beneficial when pollen deposition is absent/ insufficient (Fetscher 1999), permanent closure of the lobes can be detrimental, if it is not pollination-induced (Vikas et al. 2009). In the selected species of Utricularia, the lobes showed the tendency to reopen irrespective of pollination. The insufficiently-pollinated lobes exhibited delayed (U. praeterita: 40.1 ± 0.56 min, U. babui: 100 ± 2.60 min) and partial (~ 30°) opening in comparison to the unpollinated stigmatic lobes, (U. praeterita: 21.56 ± 0.42 min, U. babui: 31 ± 0.53 min;~ 60°). Partial opening can be attributed to anchoring of the lobes due to pollen germination. Delayed reopening can help to reduce the chances of geitonogamy and thereby enhancing the possibility of out-crossing (Jin et al. 2015). Contrary to an earlier view (Jérémie 1989), the sensitive stigma also promotes outcrossing. Besides holding the pollen between the stigma lobes (Newcombe 1922), the mechanism works as a contrivance by minimizing the chances of

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self-pollen deposition (Clivati et al. 2014; Newcombe 1922; Vikas et al. 2009). The closure time of unpollinated lobes in both the species of Utricularia was more rapid (U. praeterita 4.59 ± 0.04 s, U. babui 4.50 ± 0.05 s) than that recorded in some taxa with thigmosensitive stigma such as Oroxylum indicum (~ 10 s., Srithongchuay et al. 2008; Vikas et al. 2009) and Mimulus guttatus (5–6 s, Friedman et al. 2017). A rapid closure movement of the stigmatic lobes is believed to prevent selfing in flowers that are foraged for a long duration by pollinators such as honeybees (Friedman et al. 2017).

Pollination and density‑dependent effects Floral features such as concealed location of nectar inside the spur, envelopment of essential organs within the corolla and highly concentrated nectar (~ 60% sugar) suggest the need of a suitable pollinator in the species. As the nectar is not replenished and produced only in a trace amount, the foraging period is likely to be restricted. Minute nectar quantity and diurnal foraging activity in the selected species

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Fig. 5  Scanning electron micrographs depicting the various stages of flower leading to autopollination in U. babui (a–c, e) and U. praeterita (d, f). The mechanism is similar in both the species. a–d Anthers are placed below the stigma before pollination (a-2DBA; b-1DBA;

c, d-DOA). e–f Elongation of anther filaments occurs and anthers approach the stigmatic lobes to shed the pollen grains on the larger and receptive stigmatic lip (1DAA in U. praeterita, 4DAA–6DAA in U. babui). Scale bar = 0.25 mm

is somewhat similar to U. reticulata and U. purpurescens (Hobbhahn et al. 2006). Nectarless flowers, as seen in U. praeterita, have also been reported from U. alpina (Jérémie 1989). Pollination guilds reported for the other Utricularia species are similar to that observed in U. babui (Clivati et al. 2014; Hobbhahn et al. 2006). Plant density can have a strong influence on pollination success (Feinsinger et al. 1991; Kunin 1993). Our findings on U. babui hold true to this notion, as plant density significantly correlated with insect visitation rate. Large-sized patches were favoured by the insects as they were able to attract legitimate pollinators. Thus, visual attraction which

increased with flowering patch density seemed necessary for insect visitation in U. babui. A longer floral lifespan further enhanced the floral visits by increasing the floral display size of the patch. Floral longevity is related to pollination rates and long-lived flowers are generally favoured when the pollinator visitation rate is low (Schoen and Ashman 1995). Bees visit the flowers of U. babui in denser patches before moving to sparser ones. Also the bees preferred denser patches, as the energy requirements will be accomplished by greater flower number with reduced average flight distance in a bout (de Jong et al. 1993; Mustajärvi et al. 2001).

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Plant density also appears to have a bearing on the mating pattern of the selected bladderwort species. Both the species in our work had lower density per patch (U. praeterita: 40–50 flowers ­m−2; U. babui: 70–100 flowers ­m−2) than the other Utricularia species studied so far, such as U. reticulata (500–1100 flowers m ­ −2), U. purpuracens (300–600 −2 flowers ­m ) and U. albocaerulea (300–700 flowers ­m−2) (Hobbhahn et al. 2006). Whereas these species with greater amount of floral display per unit area opt for xenogamy, U. babui perpetuates by mixed-mating and U. praeterita undergoes absolute selfing. Thus, there is a significant effect of density-dependent relationship between flowering (floral resource size) and mating pattern among these species.

Delayed selfing Timing of self-pollination at different stages of floral lifespan is a distinctive feature that defines the modes of autonomous self-pollination (Lloyd 1992). Prior selfing happens in the bud before anthesis, competing during anthesis when opportunity for cross-pollination also exists, and the delayed selfing occurs after the opportunity of crossing is over (Lloyd 1992). The outcome of pollinator exclusion experiments in the present study showed that self-pollination occurs late in floral life-span when the opportunity for outcrossing is lapsed (U. praeterita: 1DAA, U. babui: 4–6 DAA), and thus establishes the occurrence of autonomous delayed self-pollination in both species. This is in contrast to earlier reports which suggest that self-pollination occurs simultaneously with anthesis in U. reticulata (Kaushik and Raju 1955) and U. inflexa var. stellaris (Khosla et al. 1998). Pollen removal treatment in U. praeterita did not lead to fruit-set, indicating the non-availability of a suitable pollinator at the sites. And fruit-set in the control flowers of the treatment can be clearly attributed to delayed selfing. On other hand in U. babui, fruit formation occurs by both crosspollination and autonomous delayed selfing. Interestingly, fruit-set by delayed selfing was considerably greater than that caused by the pollinators. These results reiterate that natural fruit-set in both the Utricularia species is predominantly realized through selfing. Retention of pollen viability and stigma receptivity untill the later stage in open flowers of these taxa seems to contribute effectively to the success of delayed selfing. Delayed self-pollination in nature occurs by the movement of sexual organs or due to changes in spatial arrangement of male and female organs (Kalisz et al. 1999). Several mechanisms of delayed selfing are known but incidences with filament elongation as a necessity for pollination are few (Armbruster et al. 2002; Eckhert and Schaeffer 1998; Juncosa and Webster 1989; Mamut and Tan 2014). Delayed selfing in both Utricularia species is achieved by means of filament elongation (diminished herkogamy). Pollen

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deposition occurs when the recurving, lower stigmatic lobe brushes the pollen laden dehisced anthers mounted on an elongating filament. Thus, loss in herkogamous condition in the flowers results in autogamy. Besides the present study, filament elongation has been reported from various unrelated taxa such as Lupinus nanus (Juncosa and Webster 1989), Aquilegia canadensis (Eckhert and Schaeffer 1998) and Zygophyllum macropterum (Mamut and Tan 2014) and many species of Collinisia (Armbruster et al. 2002). Occurrence of delayed selfing in U. praeterita may also be due to the absence of suitable pollinators at the sites. Autonomous selfing is known to be favoured when the pollinators are unreliable in delivering the out-cross pollen (Kalisz and Vogler 2003). Unpredictable weather conditions such as daily thermal variation, prevailing winds, high rainfall with thunderstorms and fog may be possible reasons for the absence of pollinators at the site (Lekhak and Yadav 2012). Features such as herkogamy, dichogamy, sensitive stigma and fruit-set through manual cross-pollinations suggest conduciveness for xenogamy in bladderworts. However, consistent unpredictable and unsuitable weather conditions can give rise to an absolute selfing milieu (Husband and Barrett 1992; Schoen and Brown 1991), as observed in U. praeterita. In U. babui, the plants were distributed in small patches and low plant density was unable to attract legitimate pollinators. Scarcity of the pollinators forces plant to undertake delayed-selfing and therefore adopt a mixed-mating strategy (facultative xenogamy) for perpetuation. Pollinator activities are believed to be responsible for the evolution of mixed-mating strategy in plant species (Kalisz and Vogler 2003). It is suggested that when pollinator absence is contributing to the evolution of mixed-mating, autonomous self-pollination can be adaptive (Kalisz and Vogler 2003). Continuous autogamy in the absence of sufficient and suitable pollinators would favour the spread of selfing alleles in the population and maintenance of the trait (Fisher 1941). Thus, delayed selfing in Utricularia species appears to be favoured either when pollinators are absent (U. praeterita) or scarce (U. babui), and confers reproductive assurance in both species. The mechanism is considered as the most advantageous mode of self-pollination for combining “best of both worlds” (Lloyd 1992; Schoen et al. 1996). The present study demonstrates the prevalence of delayed selfing in Utricularia. The mechanism assures reproductive success in these self-compatible taxa. Our study also indicates that delayed-selfing could be a favoured mating strategy adopted by selected species in response to pollinator limitation. The sites were devoid of sufficient legitimate pollinator activity, and the outbreeding floral mechanisms appeared to remain unexploited. It is likely that the possibility of occurrence of delayed-selfing is overlooked or remains unnoticed in sympatric species occupying pollinator-limited landscapes, as it is manifested only during the later stages

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of floral development and due to failed xenogamy. Thus, there is a need to look into the reproductive biology of other bladderworts in this context. It would be pertinent to use a marker-assisted approach to measure the extent of mixedmating in species like U. babui. Acknowledgements  Financial assistance received from the Research and Development Grant (Grant no: RC/2015/9677) to RT is gratefully acknowledged. SRY thanks the University Grants Commission (UGC) for the BSR faculty fellowship awarded to him.

Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest.

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