JIPB
Journal of Integrative Plant Biology
Floral nectary, nectar production dynamics, and floral reproductive isolation among closely related species of Pedicularis Research Article
Ya-Nan Liu1,3, Yan Li2, Fu-Sheng Yang1* and Xiao-Quan Wang1* 1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, 2Institute of Alpine Economic Plant, Yunnan Academy of Agricultural Sciences, Lijiang 674100, China, 3University of Chinese Academy of Sciences, Beijing 100039, China. *Correspondences:
[email protected];
[email protected]
Abstract Floral nectar is thought to be one of the most important rewards that attract pollinators in Pedicularis; however, few studies have examined variation of nectary structure and/or nectar secretion in the genus, particularly among closely related species. Here we investigated nectary morphology, nectar quality, and nectar production dynamics in flowers of Pedicularis section Cyathophora. We found a conical floral nectary at the base of the ovary in species of the rex-thamnophila clade. Stomata were found on the surface of the nectary, and copious starch grains were detected in the nectary tissues. In contrast, a semi-annular nectary was found in flowers of the species of the superba clade. Only a few starch grains were observed in tissues of the semi-annular nectary, and the nectar sugar concentration in these flowers was much lower than that in the flowers of the rexthamnophila clade. Our results indicate that the floral nectary has experienced considerable morphological, structural, and
functional differentiation among closely related species of Pedicularis. This could have affected nectar production, leading to a shift of the pollination mode. Our results also imply that variation of the nectary morphology and nectar production may have played an important role in the speciation of sect. Cyathophora.
INTRODUCTION
species. Elucidating these key differences and pollination system shifts in driving speciation can fill a critical knowledge gap between microevolution and macroevolution in angiosperms (Van der Niet et al. 2014). For large numbers of wild plants, however, this kind of study was severely hampered by the lack of a robust species-level phylogeny, which can provide a framework for retrieving the floral evolutionary history in a specific lineage (Barraclough and Nee 2001; Bernardello 2007; Luo et al. 2015). Besides, the shape, size, and structure of a nectary and nectar production can be affected by different floral structures (Nilsson 1988; Galetto and Bernardello 2004; Galliot et al. 2006). Thus, the structural and functional alterations in nectaries, combined with different floral displays, may lead to reproductive isolation between closely related plant species by altering pollinator-mediated interactions. Pedicularis L. (lousewort) is one of the most species-rich genera of flowering plants. Its flowers vary greatly in corolla form, particularly in the shape of the galea (hood-like structure of the fused upper lips) and tube length (Tsoong 1963; Yang et al. 1998). Species of this genus are thought to have evolved by an adaptive radiation that diversified their corolla shapes to adapt to a diversity of pollinators (Li 1951; Tsoong 1955; Yang et al. 2003). However, increasing studies on plant-pollinator interactions have revealed that, with few exceptions, flowers of Pedicularis are pollinated exclusively by Bombus
It has long been recognized that the great diversity of angiosperms can be attributed to their floral adaptations to biotic pollination (Darwin 1877; Stebbins 1970; Whittall and Hodges 2007; Kay and Sargent 2009; van der Niet and Johnson 2012). Nectar is the most effective reward of flowers for their pollinators (De la Barrera and Nobel 2004; Brandenburg et al. 2009; Heil 2011). Despite the central role of the nectary and nectar in plant-pollinator interactions, nectar research has been overlooked by botanists for many years (Escalante-Perez and Heil 2012). Our limited knowledge about nectaries and nectar was obtained primarily from model plants (Stuurman 2004; Kram and Carter 2009; Kram et al. 2009; Liu et al. 2009) and a minority of species in the major angiosperm lineages (Bernardello 2007; Smith et al. 2008; van der Niet and Johnson 2012). However, nectaries are highly structurally and functionally diverse, and they might be easily lost or gained during evolution, associated with frequent pollination shifts and species/lineage divergence (Ree 2005; Crepet and Niklas 2008; Boberg et al. 2014; Liu et al. 2015). [Correction added on 21 January 2016, after first online publication: Liu et al. 2015 has been added to the list of references cited here and has been added to the reference list as well.] This process may be reflected in differences in nectar profile among closely related February 2016 | Volume 58 | Issue 2 | 178–187
Keywords: Pedicularis; nectary; nectar; pollination; reproductive isolation; speciation; section Cyathophora Citation: Liu YN, Li Y, Yang FS, Wang XQ (2016) Floral nectary, nectar production dynamics, and floral reproductive isolation among closely related species of Pedicularis. J Integr Plant Biol 58: 178–187 doi: 10.1111/jipb.12374 Edited by: Nicole van Dam, Department of Molecular Interaction, Germany Received Jan. 29, 2015; Accepted Jul. 6, 2015 Available online on Jul. 14, 2015 at www.wileyonlinelibrary.com/ journal/jipb © 2015 Institute of Botany, Chinese Academy of Sciences
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Variation of nectary and nectar production in Pedicularis (bumblebee) (Sprague 1962; Macior 1970, 1983, 1990, 1995; Macior and Tang 1997; Macior et al. 2001; Wang and Li 2005; Yang et al. 2007; Tang et al. 2007; Liao et al. 2011; Huang and Shi 2013; Armbruster et al. 2014; Huang et al. 2015). [Correction added on 21 January 2016, after first online publication: Huang et al. 2015 has been added to the list of references cited here and has been added to the reference list as well.] Moreover, there is not a species-specific relationship between plants and bumblebees. When bumblebees visit louseworts, they forage for pollen or nectar from flowers of different species and this leads to segregation in pollination behavior between nectarforaging and pollen-foraging pollinators. Although nectar is the most important reward for pollinators of Pedicularis, there have been no previous reports on variation of nectary structure or nectar production among closely related species. Moreover, field investigations on the lousewort flowers found that long curved beaks are often associated with a lack of nectar, whereas beakless galeas and galeas with short straight beaks are largely associated with nectar production (Macior and Tang 1997; Ree 2005). When species with long curved beaks were scored as nectarless, evolutionary analyses of morphological characters indicated that short corolla tubes are significantly associated with nectar production, and long corolla tubes with a lack of nectar (Ree 2005). Based on anatomical evidence, Huang and Fenster (2007) inferred that the loss of nectaries and nectar production in long-tubed species may be a key innovation. The results of these studies implied that the gain or loss of nectar, together with the morphological diversification of corolla, may have played a vital role in driving speciation of Pedicularis. Closely related species represent an early stage after their divergence from each other, and hence provide a good system for unraveling the mechanisms of reproduction isolation and speciation (Chen et al. 2014; Luo et al. 2015). However, few studies have examined either the variation of nectary morphology and nectar production or the pollination patterns among closely related Pedicularis species with different corolla types. As the only monophyletic section (Yang et al. 2003; Ree 2005) exhibiting all the basic corolla types present in Pedicularis, Cyathophora has become a model to elucidate floral evolution and speciation in the genus. Its low interspecific genetic variation indicates a recent and rapid species diversification, as also suggested by the results of
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molecular dating (Yu 2011). Notably, phylogenetic studies have provided increasing evidence to disentangle the evolutionary history of this section (Yang et al. 2003; Ree 2005; Yang and Wang 2007; Eaton and Ree 2013; Yu et al. 2013; Wang et al. 2015). In the present study, we investigate the variation of floral nectaries and nectar production among closely related species with different corolla types in Pedicularis sect. Cyathophora. Moreover, we discuss the degree of reproductive isolation among these species based on evidence from both pollination studies and molecular phylogenetic analyses.
RESULTS Visitation patterns of plant pollinators Our field observations showed that bumblebees are the primary and most effective pollinators of section Cyathophora, congruent with the results of previous pollination studies on species of Pedicularis (Macior and Tang 1997; Tang et al. 2007; Yang et al. 2007; Huang and Shi 2013). Bumblebees visited flowers of Pedicularis thamnophila by grasping the lower lips with its legs (Figure 1A). The lower lips formed a platform for pollinators to enter the corolla tube. When the bumblebee absorbed flower nectar in the upright position, the flower was pollinated by the tip of the beak pointing to the center surface of the dorsal thorax where pollen grains were deposited. Given that the folded lower lips of Pedicularis rex do not form a platform, the bumblebee had to grasp the corolla or the calyxes with its legs and entered the corolla tube from the left side of the flower (Figure 1B). The flower was pollinated by the tip of the beak contacting the left surface of the dorsal thorax. That is, the flowers of P. thamnophila and P. rex were pollinated nototribically. When a bumblebee visited the flower of Pedicularis superba, it landed on the large platform of the lower lips, entered the interspace between the galea and the lower corolla lip, and beat its wings (Figure 1C). The protruding stigma of the flower contacted the ventral side of the anterior thoracic region of the bumblebee, and then pollen grains fell onto the ventral surface of the bumblebee’s abdomen and the lower lip of flower (Figure 1C). When a bumblebee approached a flower of Pedicularis cyathophylla, it suspended itself from the galea by its mandibles and legs, and then turned its head
Figure 1. Bumblebee foraging behaviors on flowers of sect. Cyathophora (A, B) The bumblebee sucking nectar from Pedicularis thamnophila (A) and P. rex (B). (C, D) The bumblebee collecting pollen from P. superba (C) and P. cyathophylla (D). The arrow shows the stigma position. www.jipb.net
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to the throat of the corolla tube and gathered pollen by vibrating its wings (Figure 1D). The protruding stigma of the flower contacted the ventral side of the anterior thoracic region, while a shower of pollen fell on the ventral surface of the bumblebee’s abdomen. Pollen collection in flowers of P. cyathophylla was completed in about 2–3 s, which is shorter than that spent for nectar foraging (about 4–6 s). That is, the flowers of P. cyathophylla and P. superba were pollinated sternotribically. Given the small populations of Pedicularis cyathophylloides at high altitudes, we were not able to identify effective pollinators for this species. Characteristics of flower and nectary structure Pedicularis rex and P. thamnophila have typical short-tubed, beakless corolla, and a bidentate galea (Figure 2). Corolla tubes of the two species were 20.23 3.4 and 18.01 1.74 mm in length, and the widths of their upper corolla-tubes were 5.52 0.83 and 4.08 0.83 mm, respectively. Anthers of the four stamens were concealed in the galea, and the style extended dorsally within the galea and protruded slightly beyond the truncated opening. Plants of P. cyathophylloides were characterized by short-tubed corolla, and a toothless or obscurely dentate galea, with a tube length of 23.03 3.04 mm and a tube width of 2.21 0.69 mm. Pedicularis superba had a corolla tube of 25.47 2.87 mm long and 8.56 1.76 mm wide. The inflated galea was slightly skewed to right, with the elongated beak touching the right lower lip and extending left along the upper surface of the lower lips. The long-tubed
species, P. cyathophylla, had a greatly elongated corolla-tube (47.06 8.64 mm) and an elongated beak (approximately 7 mm) at the tip of the galea (Figure 2). Given that the corolla tube of P. cyathophylla was inverted forward at the throat position, the lower lips were vertical to the ground and the galea pointed downward. The long beak pointed to the throat of the corolla tube and then inverted to the left. Anthers of these three species were tightly packaged by the navicularshape galea, with the style extending through the short or elongated beak. We found a long conical structure at the abaxial side of the ovary in the two short-tubed, beakless species, P. rex and P. thamnophila (Figure 2). During stages 1 to 4 of the ovary development, we could not find any obvious changes at the base of the ovary (Figure 3). At stage 5, when the flower bud was about 4 mm long, an obvious protuberance was detected at the base of the ovary. The conical nectary elongated to approximately 1 mm at stage 8, with a flower bud of about 1 cm in length. At the same time, a semi-annular incrassated structure at the abaxial base of the ovary was detected. In the blooming flower, the nectary was as long as the ovary. The nectary-like conical structure was not detected in flowers of P. superba, P. cyathophylloides, and P. cyathophylla (Figure 2). In the beaked, short-tubed species P. superba, only a semiannular incrassated structure circling the abaxial base of the ovary was detected. In flowers of P. cyathophylloides and P. cyathophylla, the semi-annular incrassated structure surrounding the ovary was not as obvious as that in P. superba.
Figure 2. Floral morphology and nectary structure in sect. Cyathophora (A1, A2) Pedicularis thamnophila. (B1, B2) P. rex. (C1, C2) P. superba. (D1, D2) P. cyathophylloides. (E1, E2) P. cyathophylla. (A1–E1) Bar ¼ 1 cm. (A2–E2) Bar ¼ 2 mm. February 2016 | Volume 58 | Issue 2 | 178–187
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Figure 3. Nectary development of Pedicularis rex at the 12 stages of flower development Flower, bar ¼ 1 cm; Ovary, bar ¼ 1 mm.
Nectar production and sugar concentration The quantity of floral nectar varied among examined flowers of the five species, from zero in flowers of Pedicularis cyathophylloides to very small volumes in P. superba (on average 0.18 mL), and to moderate quantities in P. thamnophila (0.70– 1.20 mL), P. cyathophylla (on average 1.37 mL), and P. rex (0.23– 2.50 mL) (Table 1). With the elongation of corolla in P. rex, the quantity of nectar increased progressively from 0.33 mL to 2.50 mL (on average 1.13 0.68 mL). This increase of nectar production was also observed in flowers of P. thamnophila, with
a variation in quantity from 0.70 to 1.30 mL (on average 1.10 0.25 mL). The nectar sugar concentration (% NCW) varied from 6% to 41% among the four species that produced floral nectar (Table 1). With the exception of P. cyathophylloides, which does not produce nectar, the lowest sugar concentration was detected in P. superba, a short-tubed species with the largest lower lips (17–20 mm long, 27–33 mm wide) (Tsoong 1963). A strikingly low sugar concentration (7%) was also detected in P. cyathophylla with the longest corolla tube (35– 60 mm). In contrast, the highest sugar concentration (33% 5%)
Table 1. The volume and sugar content of nectar in flowers of different populations of Pedicularis Taxa
Population
No.
Stage
Mean volume (mL)
Mean sugar concentration (%)
Pedicularis rex
Kunming, YN
6 6 6 4 4 4 4 4 5 6 4 5 6
9 10 10 11 11 11 11 9 10 10 10 10 11
0.33 0.23 0.57 1.08 1.70 1.50 2.50 0.25 1.20 1.20 1.80 1.08 1.71 1.13 0.68a 0.70 1.30 1.30 1.20 1.00 1.10 0.25a 0.00 0.18 1.37
31 38 41 39 38 34 36 27 26 31 30 31 28 33 5a 25 22 25 20 18 22 3a 0 6 7
Lijiang, SC
Daocheng, SC
P. thamnophila
Daocheng, SC
6 6 3 5 3
9 10 10 11 11
P. cyathophylloides P. superba P. cyathophylla
Jiangda, T Lijiang, YN Yajiang, SC
40 20 20
9–11 9–11 9–11
Note: No., number of flowers analyzed; Stage, stage of flower development; aData are given as Mean SD. www.jipb.net
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was recorded in P. rex, a short-tubed species characterized by a folded and closed lower corolla lip. There was also a high sugar concentration (22% 3%) in P. thamnophila, a short-tubed species similar to P. rex in corolla width. Nectary microstructure and histochemistry The nectary of Pedicularis was located at the abaxial side of the ovary. Stomata were detected on the top surface of the nectary by scanning electronic microscopy (SEM) (Figure 4). We counted the number of modified stomata (secretory cells) on the median protuberance for three species of section Cyathophora. There were 244, 112 and 117 secretory cells per square millimeter of the nectary surface for P. rex, P. superba and P. cyathophylla, respectively. In longitudinal and cross sections near the base of the active nectary, several vascular bundles were found (Figure 5A, C). These vascular bundles were embedded in the tissue of nectariferous parenchyma, and bound to the connective tissues in the central nectary. The small isodiometric parenchyma cells in the tissue at the base of the nectary were covered with a continuous epidermis with stomata/secretory cells. These nectary-modified stomata may be responsible for nectar secretion. The purple grains in the nectariferous parenchyma indicated the accumulation of starch or amyloplast in the tissue. In the longitudinal section near the top of the active nectary (Figure 5B), dense starch grains were evident in the parenchyma cells. The starch grains in cells near the epidermis reduced in size, concomitant with a break in the cuticle. The longitudinal section through the whole nectary of P. rex indicated that the accumulation of starch grains occurred in nearly all tissues.
Figure 4. Scanning electron micrographs of the floral nectary (A and D) Pedicularis rex. (B and E) P. superba. (C and F) P. cyathophylla. The arrow shows stomata. OV, ovary; FN, floral nectary. Ovary, bar ¼ 1 mm; Nectary, bar ¼ 50 mm. February 2016 | Volume 58 | Issue 2 | 178–187
The different stages of nectary development for P. rex are shown in Figure 6. The cross sections of the nectary were stained with purple from the apex to the base, whereas all the sections of the ovary were blue in colour, indicating that large accumulations of starch grains only occurred in the nectary tissue. The gradual change in coloration of the stained sections indicated that starch grains reached the maximum quantity at stage 10, and then decreased gradually. In the sections near the base, the connective tissues at the central or abaxial position were stained blue. In the cross sections at the base of the ovary of P. rex, incrassated tissue surrounding the abaxial side of the ovary was detected (Figures 7, 8). That is, the conical nectary formed a semi-annular structure at the base of the ovary. The purple colour of the stained tissue indicated that starch grains accumulated at stages 9–10 and then decreased at stages 11–12, synchronous with the change in accumulation of starch grains in tissues of the conical nectary. In contrast, the nectary tissues of P. superba and P. cyathophylla were stained blue (Figures 7, 8). Plentiful purple starch grains were detected in the incrassated tissues of P. rex; however, only a few purple granules were detected in the cross sections of P. superba and P. cyathophylla.
DISCUSSION Nectary structure and nectar secretion in closely related species of Pedicularis Floral nectar of Pedicularis is one of the most important rewards for their pollinators; however, little is known about
Figure 5. Longitudinal sections (A, B) and cross section (C) of the nectary (stained with PAS) from flower of Pedicularis rex CT, connective tissue; EP, epidermis; NP, nectariferous parenchyma; ST, stomata; STA, starch/amyloplast; VB, vascular bundle. (A) bar ¼ 1 mm. (B and C) Bar ¼ 100 mm. www.jipb.net
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Figure 6. Cross sections from the top to the base of ovary and nectary in flowers of Pedicularis rex (A–E) Stage 9. (F–J) Stage 10. (K–O) Stage 11. (P–T) Stage 12. Bar ¼ 1 mm.
nectary formation and nectar secretion in the genus. In the present study, we found a single large conical nectary structure at the abaxial base of the ovary in flowers of Pedicularis rex and P. thamnophila (Figures 2–4). The conical nectary structure could be detected at stage 5 of flower development and it became mature at stage 11 (Figure 3). Stomata (secretory cells) were found on the surface of the nectary (Figure 4). Periodic acid–Schiff (PAS) reaction detected abundant starch grains in the nectary tissues of P. rex (Figures 5–8). Under the single layer of epidermis cells are 5–9 layers of parenchyma cells full of starch grains, with vascular bundles connecting to connective tissues. Taken together, morphological, anatomical, and histochemical evidence can confirm that this conical structure should be a nectary. Although there have been reports of annular gynoecial nectaries in Orobanchaceae, here conical nectaries are first found in this family. Field observations demonstrated that nectar secretion begins at stage 9, and the nectar volume of P. rex and P. thamnophila reaches peak at stage 11 when the level of starch www.jipb.net
accumulation begins to decline in the nectary tissues (Table 1). The period of the starch decline matches that of the nectar increase. Therefore, the floral nectary might rely on the accumulation of starch in the pre-flowering phase in order to reach the peak nectar secretion rate. The waxing and waning of starch accumulation and nectar secretion indicated that the starch in the nectary might be transformed into nectar during floral development. Previous molecular phylogenetic analyses revealed two sister clades, rex-thamnophila and superba, in sect. Cyathophora (Yang and Wang 2007; Eaton and Ree 2013; Yu et al. 2013; Wang et al. 2015). Interestingly, the conical nectary was detected only in flowers of the species of the rex-thamnophila clade (P. rex, P. thamnophila). Instead, we found a semi-annular nectary at the abaxial base of the ovary in flowers of the species of the superba clade (P. superba, P. cyathophylla, P. cyathophylloides) (Figures 2, 4, 7). Although SEM detected secretory cells on the nectary surface of P. superba and P. cyathophylla (Figure 4), the average number of secretory cells of the two species is about two times lower than that of P. rex February 2016 | Volume 58 | Issue 2 | 178–187
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Figure 7. Cross sections at the base of ovary at different floral development stages (A–D) Pedicularis rex. (E–H) P. superba. (I–L) P. cyathophylla. (A, E, and I) Stage 9. (B, F, and J) Stage 10. (C, G, and K) Stage 11. (D, H, and L) Stage 12. Bar ¼ 1 mm.
from the rex-thamnophila clade. Moreover, in the nectary tissues of P. superba and P. cyathophylla, we did not find any vascular bundles or connective tissues, which occur in the nectary tissues of P. rex (Figure 7). In contrast to the copious starch grains accumulated in the nectary tissue of P. rex, only a
few starch grains were detected in the stained tissues of P. superba and P. cyathophylla (Figure 8). Correspondingly, the mean sugar concentration in the flowers of the superba clade is much lower than that of the rex-thamnophila clade (Table 1). Gynoecial nectaries are common in Orobanchaceae, and
Figure 8. Cross sections of nectary at different floral development stages (A–D) Pedicularis rex. (E–H) P. superba. (I–L) P. cyathophylla. (A, E, and I) Stage 9. (B, F, and J) Stage 10. (C, G, and K) Stage 11. (D, H, and L) Stage 12. Bar ¼ 100 mm. February 2016 | Volume 58 | Issue 2 | 178–187
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Variation of nectary and nectar production in Pedicularis previous studies found that Pedicularis and some other genera in Rhinanthoideae can produce nectar to reward pollinators (Kwak 1979; Kampny 1995). Therefore, the loss of nectar in flowers of the superba clade should be a derived trait. Although the nectary structure still occurs in flowers of the superba clade, the small quantity of starch grains and nectar reveals an ongoing process of loss of nectar in this clade. Pollination shift and reproductive isolation between species of Cyathophora Flowers of Pedicularis were classified into two types, i.e., nectariferous and nectarless, in pollination studies (Macior and Tang 1997; Huang and Shi 2013; Armbruster et al. 2014). Our field investigations found that pollinators forage for nectar or pollen when they visit plants of sect. Cyathophora (Figure 1). In the two species of the rex-thamnophila clade that provide nectar to pollinators, the nectar sugar concentration is approximately 33% (P. rex) and 22% (P. thamnophila), respectively. This is comparable to the nectar sugar concentration previously reported in the species of Pedicularis from the HimalayasHengduan Mountains (Macior and Tang 1997; Tang et al. 2007). Although an obvious nectary structure was found in flowers of the superba clade, the sugar concentration (6% and 7%) is much lower than that reported in other Pedicularis species in this region (Table 1; Macior and Tang 1997; Tang et al. 2007). This is congruent with the results from the PAS analysis, which detected a few starch grains in the stained nectary tissues of P. superba and P. cyathophylla (Figures 7, 8). Our field investigations also indicate that, with a few exceptions, species in the rex-thamnophila clade are nototribically pollinated when visitors feed nectar from the base of the corolla (Figure 1; Tang et al. 2007), leading to the protrusive stigma contacting the residual pollen on the dorsal side of insects. In contrast, the species in the superba clade without sufficient nectar are sternotribically pollinated while bumblebees forage for pollen, resulting in a residual deposit and collection of pollen on the ventral side of insects. That is, two distinct modes of pollination occur in Pedicularis, nototribic for species with copious nectar and sternotribic for species with little or no nectar. The association between pollinator shifts and reward change has been found in different lineages of angiosperms (Fenster et al. 2004; Bernardello 2007; Boberg et al. 2014). For closely related species of Pedicularis, however, reward change of plants is associated with a shift of pollination modes rather than pollinators. According to the phylogenetic analyses of section Cyathophora that were conducted based on extensive sampling (Yu et al. 2013; Wang et al. 2015) and on RADseq (restriction-site associated DNA sequencing) data (Eaton and Ree 2013), no gene flow was detected between the two clades rexthamnophila and superba. The nototribic-sternotribic pollination shift leading to reproductive isolation was also detected in sympatric populations of two species of Pedicularis (Yang et al. 2007) and two species of Rhinanthus (Kwak 1978), a close relative of Pedicularis. In particular, Kwak (1978) found that bumblebees could mediate interspecific hybridization by visiting both plant species during one foraging trip as nototribic or as sternotribic pollinators. Conversely, reproductive isolation could also be achieved by bumblebees, which visit one species nototribically and the other sternotribically during one foraging trip. For species in the rex-thamnophila clade with a moderate www.jipb.net
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nectar production and nectar-foraging bumblebees as pollinators, the mechanism of reproductive isolation remains largely unknown. Although bumblebees can sporadically forage on flowers of P. rex for pollen in an inverted position (Tang et al. 2007), our observations indicate that the sternotribic bumblebees never visit flowers of P. thamnophila. For the two nectar-rewarding species P. rex and P. thamnophila, floral isolation might not be complete at all, as the two species have similar floral display and are pollinated by nototribic pollinators. Because the strong bumblebees can easily thrust into the pleated corolla tubes of the two species, the tiny difference between their corolla cannot prohibit them from sharing pollinators. Of course, we should keep in mind that this conclusion was drawn based on limited field observation. Nevertheless, a slight difference in pollination behavior is unlikely to result in effective floral reproductive isolation between species of Pedicularis, as the positions of pollen placement and stigma contact on the pollinator’s body are often not very precise (Huang and Shi 2013; Armbruster et al. 2014). Moreover, extensive gene flow between P. rex and P. thamnophila has been detected by phylogenetic analyses (Eaton and Ree 2013; Yu et al. 2013; Wang et al. 2015), which strongly supports that the subtle differences in pollination behavior are inadequate for promoting reproductive isolation between the two species. For species in the superba clade that produce no or little nectar and provide pollen as a reward for pollinators, no interspecific gene flow was detected, with the exception of an ancient hybridization/introgression event between Pedicularis superba and P. cyathophylla (Yu et al. 2013; Wang et al. 2015). Our extensive field investigations found that the two species never coexist in the same community; therefore, geographic isolation may have played a key role in driving speciation in the superba clade. Of course, a contribution of floral isolation to reproductive isolation could not be completely ruled out, considering that these close relatives exhibit striking differences in pollination syndrome. Despite a recent diversification of sect. Cyathophora (Wang et al. 2015), our results indicate that the floral nectary has experienced considerable morphological, structural, and functional differentiation between the two sister clades in this section, associated with a shift between two basic pollination modes. As reported in other angiosperm lineages (Van der Niet et al. 2014), this dichotomy between traits (nectariferous vs. nectarless) that mediate pollinator attraction is suggestive of pollinator-driven speciation. Because the loss of nectar has occurred repeatedly in different lineages of Pedicularis (Eaton et al. 2012), it is likely that the shift of pollination modes has played an important role in the floral diversification and speciation of the genus.
MATERIALS AND METHODS Section Cyathophora comprises five species, namely, Pedicularis rex, P. thamnophila, P. superba, P. cyathophylloides, and P. cyathophylla, which are distributed mainly in the Hengduan Mountains. This study was conducted in two communities where two species of the section co-existed, as well as in three communities where only one species of the section existed (Table 1). February 2016 | Volume 58 | Issue 2 | 178–187
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Flower characters and nectary structure We measured the length and width of the corolla tube which may determine the type of pollinators and pollination patterns, using a Vernier Caliper. The mean value and standard deviation were calculated based on 30 fully-blown flowers from 5–10 individuals of different populations. Flower development in Pedicularis was divided into 12 stages beginning with stage 1 (flower about 2 mm in diameter) to stage 12 (flower fully open) (Figure 2). Ovaries of mature flowers of P. rex have clear nectary structure, which was photographed in the field by a Nikon D90 digital camera. The flowers for anatomical studies were fixed in FAA (formaldehyde, acetic acid, 70% ethanol, 1:1:18 v/v/v) for at least 24 h. Fixed ovaries and potential nectaries of the five species were dissected and then were examined using a Nikon SMZ1000 stereomicroscope equipped with a digital camera. Furthermore, different stages of flower development of P. rex were examined to elucidate the general pattern of nectary development. Pollination observation Pollination ecology of the short-tubed P. rex has been studied extensively in recent years, as plants of this species often form large populations that are distributed mainly in relatively low altitude (1,800–3,600 m) habitats. The remaining species often grow in small fragmented populations at higher altitudes from 3,200–4,600 m; therefore, field work is quite difficult and reproductive biology of these species remains unknown. In the summers between 2009 and 2013, we recorded the foraging behavior of pollinators on species of section Cyathophora. Pollinator visits were monitored from flowers in a 5 5 m2 plot, and the visitor behavior was described based on field observations and/or analysis of photographs. Scanning electronic microscopy Fresh flowers of P. rex, P. superba, and P. cyathophylla were fixed in FAA. After fixation at 4 °C for 24 h, the dissected nectaries were further dehydrated through a graded ethanol series (from 70% to 100%) for 15 min, and then treated with a graded isoamyl acetate series (from 25% to 100%) in ethanol solution for 20 min. Finally, the materials were critical-point dried with CO2, coated with platinum, and examined with a HITACHI S-4800 Field Emission Scanning Electron Microscope. Nectar production and nectar sugar concentration To determine the pattern of nectar secretion throughout anthesis, we measured the nectar quantity in different developmental stages (Table 1). We did not find nectar in flowers earlier than stage 8. Thus, flowers from stages 9 to 11 were examined, and the fully open flowers (stage 12) were excluded to avoid influences of the environment and visitors. The nectar was collected from the base of the corolla tube by micropipette (2.0 and 10.0 mL), and was immediately expelled onto the prismatic surface of a hand refractometer (0%–80%; Taihua, Chengdu, China) to determine solute concentrations (percent nectar concentration by weight, %NCW). The refractometer readings were corrected according to the manufacturer’s instructions. We measured nectar volumes and concentrations for 12–40 flowers in each plot and a total of 167 flowers were examined, with the values expressed as mean standard deviation. February 2016 | Volume 58 | Issue 2 | 178–187
Nectary histochemistry Dissected ovaries and nectaries were investigated at four stages (8–11) for P. rex, P. superba, and P. cyathophylla, which represent beakless short-tubed, beaked short-tubed, and beaked long-tubed corolla types, respectively. Selected ovaries were dehydrated gradually in an ethanol series, and then were embedded in paraffin with ceresin (BR, 56–58 °C). Transverse sections were cut with a HM 340E microtome. Thin sections (3– 5 mm) were stained with periodic acid/Schiff’s reagent (PAS) to detect total insoluble polysaccharides, and then stained with fast green FCF to distinguish cell walls (blue) with polysaccharides (mauve). Stained sections were examined with a Leica DM4000B LED light microscope equipped with a digital camera.
ACKNOWLEDGEMENTS We thank Mr. Wei-Tao Li for his assistance in field work. This study was supported by the National Natural Science Foundation of China (31330008 and 31160047), the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-L07), and Science and Technology Basic Work (2013FY112100).
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