Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Evolutionary Association of Stomatal Traits with Leaf Vein Density in Paphiopedilum, Orchidaceae

  • Shi-Bao Zhang ,

    Contributed equally to this work with: Shi-Bao Zhang, Zhi-Jie Guan

    Affiliations Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China, Key Laboratory of Tropical Plant Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, Yunnan, China

  • Zhi-Jie Guan ,

    Contributed equally to this work with: Shi-Bao Zhang, Zhi-Jie Guan

    Affiliations Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China, State Key Laboratory of Plant Physiology and Biochemistry and College of Agronomy and Biotechnology, China Agricultural University, Beijing, China

  • Mei Sun,

    Affiliation Key Laboratory of Tropical Plant Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, Yunnan, China

  • Juan-Juan Zhang,

    Affiliation Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China

  • Kun-Fang Cao,

    Affiliation Key Laboratory of Tropical Plant Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, Yunnan, China

  • Hong Hu

    huhong@mail.kib.ac.cn

    Affiliation Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China

Evolutionary Association of Stomatal Traits with Leaf Vein Density in Paphiopedilum, Orchidaceae

  • Shi-Bao Zhang, 
  • Zhi-Jie Guan, 
  • Mei Sun, 
  • Juan-Juan Zhang, 
  • Kun-Fang Cao, 
  • Hong Hu
PLOS
x

Abstract

Background

Both leaf attributes and stomatal traits are linked to water economy in land plants. However, it is unclear whether these two components are associated evolutionarily.

Methodology/Principal Findings

In characterizing the possible effect of phylogeny on leaf attributes and stomatal traits, we hypothesized that a correlated evolution exists between the two. Using a phylogenetic comparative method, we analyzed 14 leaf attributes and stomatal traits for 17 species in Paphiopedilum. Stomatal length (SL), stomatal area (SA), upper cuticular thickness (UCT), and total cuticular thickness (TCT) showed strong phylogenetic conservatism whereas stomatal density (SD) and stomatal index (SI) were significantly convergent. Leaf vein density was correlated with SL and SD whether or not phylogeny was considered. The lower epidermal thickness (LET) was correlated positively with SL, SA, and stomatal width but negatively with SD when phylogeny was not considered. When this phylogenetic influence was factored in, only the significant correlation between SL and LET remained.

Conclusion/Significance

Our results support the hypothesis for correlated evolution between stomatal traits and vein density in Paphiopedilum. However, they do not provide evidence for an evolutionary association between stomata and leaf thickness. These findings lend insight into the evolution of traits related to water economy for orchids under natural selection.

Introduction

Plants often exhibit considerable variations in their functional traits that affect the capture and utilization of resources and enable them to adapt to changing environments [1], [2]. The development of leaf cuticles and stomata might be linked to the success of terrestrial plants because they resolve two conflicting physiological requirements: increasing CO2 uptake vs. reducing water loss [3], [4]. Much of the evolutionary history of land plants involves leaf activities for obtaining water and preventing transpirational water losses, thereby improving their photosynthetic carbon gain and survival in dry habitats [5]. Both environment and evolutionary history are important to shape the hydraulic properties that determine how plants respond to water shortages [6]. Evolutionary pressures that drive such conservation strategies favor the coupling of the cuticle with the development of stomata [7]. Consequently, one might expect a correlated evolution between leaf attributes and stomatal traits [8]. However, little work has been done on such coordination within an evolutionary context even though one could gain valuable insights into ecological and evolutionary principles [8], [9].

Water is transpired from the leaf surface through either the outer epidermal cell walls or the stomata. Although cuticles can reduce water loss from the leaf to the atmosphere, they also slow the CO2 diffusion in the reverse direction [10]. Therefore, stomata can effectively regulate gas exchange where water vapor leaves the plant and CO2 enters. The potential transpirational demand is primarily determined by both stomatal aperture and density [11]. Over time, stomata have changed markedly in their size and numbers since first appearing on the leaf surface approximately 411 million years ago [12]. Stomatal density (SD) is negatively correlated with atmospheric CO2 concentration, while size is positively correlated [3], [13], [14]. Although the level of atmospheric CO2 is a main selective agent, SD is also related to water availability, light intensity, and temperature [13], [15], [16], [17]. Water deficits lead to more densely packed but smaller stomata [17], [18]. The efficiency with which CO2 is taken up and water loss restricted appears to be partially a function of stomatal size [19], [20]. Small stomata enable the leaf to attain high and rapid diffusive conductance under favourable conditions, and they afford greater water-use efficiency (WUE) in dry habitats because they can react more quickly to environmental stimuli [14]. By contrast, large stomata are slower to close. Although they are less able to prevent hydraulic dysfunction in dry habitats, this lag in response may be advantageous in cool, moist, or shaded environments [19], [20].

Leaf venation provides mechanical support and carboxylate transport, and aids in replacing the water transpired during photosynthesis [21], [22]. Vein density (VD) is correlated with SD, maximum hydraulic conductance, maximum photosynthetic rate, and WUE [11], . Vein patterns are highly diverse across species, and have a significant phylogenetic signal [5], [24],[25]. Historically, the evolution of VD resulted in high photosynthetic capacity during early angiosperm diversification, and promoted species diversity among angiosperms [5]. This feature can also serve as an environmental proxy [24]. For example, Dunbar-Co et al. have found that Hawaiian Plantago taxa in drier regions have higher VD values [9]. Loss of hydraulic conductance is accompanied by stomatal closure under water deficits [26]. The density of major veins plays a role in determining leaf drought tolerance [27].

Leaf structural traits determine how plants adapt to changes in water availability [15], [28]. For example, gametophyte morphology can influence water-holding capacity in ferns [29]. A leaf with a high mass per unit area is better able to store water and maintain more stable hydraulic functioning during droughty periods [30]. Consequently, leaf thickness tends to increase with site aridity [18], [28], [31]. The potential transpirational demand by plants is primarily determined by stomata. However, when water is severely limited and the stomata reach their minimum aperture, water loss from a leaf is mainly determined by epidermal conductance [32]. The cuticle is a hydrophobic and flexible membrane composed of cutin and associated solvent-soluble lipids. One of its functions is to protect against water loss from the leaf interior [33]. Cuticular property is often correlated with transpirational demand [33], [34]. Although a thick cuticle can help prevent water loss when moisture is limited [28], [35], thickness alone is not a good predictor of a species’ drought tolerance because it is not always correlated with cuticular water permeability [4], [36].

Leaf structure can also reflect the plant response to environmental stresses, such as a low supply of soil nutrients. Evolutionary pressures usually favour investment toward chemical and structural defences in stressed plants [31]. This drought response is often similar to that for nutrient limitations, i.e., the production of small leaves with thick cuticles [31], [37]. In fact, the thickened cuticles of sclerophylls can serve as a sink for excess photosynthate because those membranes do not require phosphorus or nitrogen to form cutin, suberin, and waxes [38]. Consequently, the sclerophyll protects against leaf herbivory and abiotic physical damage [37].

The well-known genus Paphiopedilum within Orchidaceae comprises 66 species, with plants usually occurring in limestone or mountainous forests of tropical and subtropical zones from Asia to the Pacific islands [39]. These species vary in their growing environments, developmental habit, and leaf morphology. The low capacity for water storage in the shallow soil layer of karst areas limits water supplies. Plants in this genus manifest three contrasting growth habits: terrestrial, facultative epiphytic or obligatory epiphytic. For epiphyte species, the amount of available moisture is a factor in determining the best sites for growth. Although periodic water deficit is a main environmental stressor that limits plant growth and survival within that genus [2], some species can adapt to relatively dry, calcareous regions [40]. Drought tolerance by Paphiopedilum is linked to leaf anatomy [2], which is evergreen and fleshy, with distinct epidermal cuticles, but no guard cell chloroplasts [2], [40]. This lack of guard cell chloroplasts slows the induction of photosynthesis, and is considered an ecophysiological adaptation to water shortage [41], [42]. Therefore, the wide range of morphological and ecological variations among Paphiopedilum species provides a valuable research system for understanding morphological evolution related to water-use traits [2], [42].

Plants adapt to challenging conditions through simultaneous configurations of multiple traits [9]. Their leaf vein network, stomatal design, leaf structure and cuticle are ordinately linked to water transport, regulation, storage and conservation, respectively. Here, we investigated the stomatal traits and leaf attributes of 17 species in Paphiopedilum when all plants were tested in the same growing environment. Our objectives were to assess the effect of phylogeny on leaf structure and stomatal traits, and to examine any correlated evolution between them. Because the responsiveness to environmental changes is generally more similar among closely related species than among those more distantly related, we expected that stomatal traits would manifest a correlated evolution with leaf attributes.

thumbnail
Table 1. Leaf carbon stable isotope ratios (δ13C) and stomatal traits of 17 Paphiopedilum species.

https://doi.org/10.1371/journal.pone.0040080.t001

thumbnail
Table 2. Leaf structural traits of 17 Paphiopedilum species. LMA, leaf mass per unit area (g m−2).

https://doi.org/10.1371/journal.pone.0040080.t002

thumbnail
Figure 1. Values for leaf traits and stomatal straits in Paphiopedilum species.

SL, stomatal length; SA, stomatal area; SD, stomatal density; LET, lower epidermal thickness; LT, leaf thickness; and VD, vein density. Names of subgenera are at left, and are based upon nuclear rDNA ITS trees from Cox et al. [46].

https://doi.org/10.1371/journal.pone.0040080.g001

thumbnail
Figure 2. Differences in stomatal traits and leaf thickness of Paphiopedilum due to growth habit.

SD, stomatal density; VD, vein density; SL, stomatal length; and LT, leaf thickness. Different letters above bars for each component indicate statistically different mean values (p≤0.05), as determined by LSD multiple comparison tests.

https://doi.org/10.1371/journal.pone.0040080.g002

Phylogenetic signals of SL, SA, UCT, and TCT were >1.0, demonstrating that these traits were phylogenetically conserved (Table 3). However, the K values for SD and SI were <0.5, indicating that these Paphiopedilum relatives resembled each other less than expected, under the Brownian model, along the phylogenetic tree. These results were confirmed by our phylogenetic distribution (Fig. 1).

Materials and Methods

Ethics Statement

None of these experimental materials was collected from national parks or other protected areas. No tested species are under first- or second-class state protection, and they are not listed in the Inventory of Rare and Endangered Plants of China (http://zrbhq.forestry.gov.cn/portal/zrbh/s/3053/content-457748.html), or the Key Protected Inventory of Wild Plants of China (http://zrbhq.forestry.gov.cn/uploadfile/zrbh/2010-10/file/2010-10-14-bb296addeaa047798d6b6c476aaa1da9.doc). These plants were used for only scientific research as permitted by the Wildlife Protection and Administration Office under the Forestry Department of Yunnan Province.

Plant Materials

Sample plants representing 17 species of Paphiopedilum were collected from their natural habitats and grown in a greenhouse at Kunming Institute of Botany, CAS (elev. 1990 m, E102°41′, N25°01′). Applying similar culturing practices largely helped to minimize any plastic differences among species in functional traits that might have resulted from environmental heterogeneity. Thus, any variations would likely reflect the role of a genetic component. Conditions included 30 to 40% of full sunlight controlled by shade nets and an ambient temperature of 20 to 25°C. Before the sample plants were analyzed, these plants were watered as needed, and were then cultivated for two to three years to ensure that their adaptation to a new environment was complete.

thumbnail
Table 3. Phylogenetic signal (K) of leaf attributes and stomatal traits in 17 Paphiopedilum species.

https://doi.org/10.1371/journal.pone.0040080.t003

Leaf Attributes

Six mature, undamaged leaves were evaluated from individual plants of each species. Leaf area (LA) was measured with a Li-Cor 3000A area meter (Li-Cor Inc., Lincoln, NE, USA). Each leaf was then divided along the midrib. One half was re-measured with the area meter, then oven-dried at 70°C for 48 h to obtain its dry weight. Specific leaf weight was expressed as leaf dry mass per unit area (LMA). The other half was cleaned for 1 h in a 5% NaOH aqueous solution. Three sections of leaf lamina were excised from the top, middle, and bottom portions, stained with 1% safranin, and mounted in glycerol to obtain the vein density (VD). Samples were photographed at 10× magnification with an Olympus U-CMAD3 light microscope (Olympus Inc., Tokyo, Japan). Vein lengths were determined from digital images via the IMAGEJ program (http://rsb.info.nih.gov/ij/). Values for VD were recorded as vein length per unit area (mm mm−2). Leaf stable carbon isotope ratio (δ13C) was analyzed using an IsoPrime100 isotope ratio mass spectrometer (Isoprime Ltd., Cheadle Hulme, UK).

thumbnail
Figure 3. Factor-loading for stomatal and leaf traits along 2 axes of principal component analysis (PCA).

SL, stomatal length; SW, stomatal width; SA, stomatal area; SD, stomatal density; UET, upper epidermal thickness; UCT, upper cuticular thickness; LET, lower epidermal thickness; LCT, lower cuticular thickness; LMA, leaf mass per unit area; LA, leaf area; VD, vein density; and MT, mesophyll thickness.

https://doi.org/10.1371/journal.pone.0040080.g003

thumbnail
Figure 4. Correlations vein density with stomatal traits or lower epidermal thickness.

Plate (a) to (e), Pearson’s regressions; and plate (f) to (j), phylogenetically independent contrast correlations. VD, leaf vein density; SL, stomatal length; SW, stomatal width; SA, stomatal area; SD, stomatal density; and LET, lower epidermal thickness.

https://doi.org/10.1371/journal.pone.0040080.g004

thumbnail
Figure 5. Correlations of lower epidermal thickness (LET) with stomatal traits.

Plate (a) to (d), Pearson’s regressions; and plate (e) to (h), phylogenetically independent contrast correlations. SL, stomatal length; SW, stomatal width; SA, stomatal area; and SD, stomatal density.

https://doi.org/10.1371/journal.pone.0040080.g005

Histological Observations

From samples of all 17 species, the middle portions of mature leaves were fixed in FAA (formalin, glacial acetic acid, ethanol, and distilled water; 10:5:50:35, v:v:v:v) for at least 24 h. They were then dehydrated in an ethanol series and embedded in paraffin for sectioning. Transverse sections, made on a Leica RM2126RT rotary microtome (Leica Inc., Bensheim, Germany), were mounted on glass slides. These tissues were examined and photographed under an Olympus U-CMAD3 light microscope. Thicknesses of the upper cuticle (UCT, µm), upper epidermis (UET, µm), palisade tissue (PTT, µm), spongy tissue (STT, µm), lower epidermis (LET, µm) and lower cuticle (LCT, µm) were measured at the midpoint of each transverse section with Adobe Photoshop 8.0 (Adobe Systems Inc., California, USA). For each species, six leaves were taken from different plants.

thumbnail
Figure 6. Correlation of upper epidermal thickness (UET) with stomatal density (SD).

(a) Pearson’s regression, and (b) phylogenetically independent contrast correlation.

https://doi.org/10.1371/journal.pone.0040080.g006

thumbnail
Figure 7. Correlation of stomatal length (SL) with stomatal density (SD)

. (a) Pearson’s regression, and (b) phylogenetically independent contrast correlation.

https://doi.org/10.1371/journal.pone.0040080.g007

Stomatal Observations

The adaxial and abaxial epidermises were peeled from the middle portions of fresh, mature leaves, and images were made under an Olympus U-CMAD3 light microscope. For each species, six leaves from different plants were used for stomatal observations. Their stomata were tallied in 30 randomly selected fields. Stomatal density (SD) was calculated as the number per unit leaf area. Stomatal size was represented as the guard cell length, possibly indicating the maximum potential opening of the pore [43]. Stomatal length (SL, µm) and stomatal width (SW, µm) were measured from 30 stomata selected randomly. Stomatal area (SA) was calculated as 1/4 × π × SL × SW [44]. Stomatal index (SI) was estimated as the ratio of stomatal numbers per given area divided by the total number of stomata and other epidermal cells within the same area.

Data Analysis

A phylogenetic signal (K) can be used to express the conservatism of traits. Cases where K<1 indicate convergent traits, K = 1 implies that closely related species have trait values that completely agree with a Brownian model, and K>1 represents traits more conserved than presumed from a Brownian expectation [45]. Our phylogenetic tree of Paphiopedilum, based on nuclear rDNA ITS sequences, was obtained from a previous report by Cox et al. [46]. The K value for each trait was calculated using ‘picante’, based on the R package 2.14 [47].

A principal component analysis (PCA) was performed with the ‘prcomp’ function of the R package ‘vegan’ to characterize the associations among leaf attributes and stomatal traits. Relationships among variables were analyzed using both Pearson regressions in R package 2.14 and phylogenetically independent contrasts (PICs). Possible evolutionary associations were assessed via PIC analysis, utilizing molecular phylogenetic trees [46]. This PIC analysis was evaluated with the “analysis of traits” (AOT) module in Phylocom, a program that calculates the internal node values for continuous traits [48], [49].

Results

None of the species tested within Paphiopedilum had pubescent leaves, and all were hypostomatic. Although leaf and stomatal traits varied considerably across species (Tables 1, 2, Fig. 1), the magnitudes of variation were generally smaller for the stomata. Among species, fluctuations in SL, SW, SI, LCT and TCT were less than 2.0-fold, while those in VD, LMA, LA, SA, SD, UET, UCT, PTT, STT, LET and MT differed by 2.1- to 5.7-fold. For stomatal traits, the magnitude of variation was largest for SD (3.9-fold) and smallest for SW (1.4-fold). For leaf attributes, UET exhibited the largest variation (5.7-fold) across species while LCT showed the smallest range. The stable carbon isotope ratio (δ13C) ranged from –27.24‰ to 23.32‰ (Table 1). Values for SD and VD differed significantly among growth habits, whereas the other traits showed no significant differences. Both SD and VD tended to increase from terrestrial to facultative and epiphytic orchids (Fig. 2).

All stomatal traits (SL, SW, SA and SD), plus VD, LET, and LA, loaded mainly on the first PCA axis, explaining 36.4% of the total variation (Fig. 3). By contrast, SD and VD loaded in the opposite direction on that axis. Leaf attributes, including LT, LMA, UET, MT, UCT, and LCT, loaded on the second axis, explaining 24.0% of the total.

Vein density was correlated with SL, SW, SA, SD, and LET; after phylogeny was considered, VD was still correlated with SL and SD (Fig. 4). Values for LET were correlated positively with SL, SW and SA, but negatively with SD (Fig. 5). After eliminating any phylogenetic effects via PICs, those correlations of LET with SW, SA, and SD became insignificant. Stomatal index was not correlated with any leaf structural straits.

The UET was not correlated with SD when phylogeny was not considered, but a significant correlation was found between them after phylogenetic correction (Fig. 6). Conversely, stomatal density was positively correlated with stomatal length when a Pearson regression was used, but that correlation became insignificant after correction (Fig. 7). Neither leaf size nor thickness was correlated with SD or VD under any circumstances.

Discussion

The evolutionary coordination of stomatal density with leaf thickness has been assessed in numerous species [8]. Here, we took a phylogenetically comparative approach to examine the correlated evolution between stomatal traits and leaf attributes from closely related species of Paphiopedilum grown under controlled conditions. Vein density had an evolutionary association with stomatal density and size, but traits for stomata and leaf thickness showed independent evolution.

Leaf Attributes and Stomatal Traits in Paphiopedilum

Leaves were fleshy and had cuticles on both sides. These characters are common among xeromorphic plants. Growth habit had no obvious influence on LMA, LT or cuticle thickness (Fig. 2). Samples from all species were hypostomatic, and their stomata were sunken into the leaf epidermis. This adaptive feature shields exerophytic plants from the effects of desiccating winds, and can help prevent excessive transpiration losses [50]. Compared with data reported from other angiosperms, Paphiopedilum members had relatively lower VD and SD, but larger stomata [9], [51]. In fact, previous study has suggested that the species in Orchidaceae have, relatively, the lowest SD values in the entire plant kingdom [40]. We noted that epiphytic Paphiopedilum had higher VD and SD than the terrestrial species (Fig. 2). Dunbar-Co et al. have also found that taxa in Plantago growing on drier sites have higher VD [9]. As a whole, these leaf attributes and stomatal traits reflect a general trend in how land plants adapt when water is limited.

Relationship of Leaf Attributes and Stomatal Traits to Phylogeny

Traits for both leaf anatomy and stomata varied significantly across species, although to a lesser extent for the latter (Table 1, Fig. 1). Several traits, such as SL, SA, UCT and TCT, showed strong phylogenetic signals while SD and SI exhibited a strong convergent evolution. This high level of conservatism demonstrates a distinct evolutionary shift among species [1]. Somewhat contradictory to our findings, Beaulieu et al. [43] did not report strong signals in SL (K = 0.685) or SD (K = 0.540) for 101 angiosperm species. However, Hodgson et al. [20] noted that stomatal size was related to both cytological status and phylogeny. The discrepancy between our observations and those of Beaulieu et al. are probably related to the choice of plant materials tested. In that earlier study, three growth forms were selected (herb, tree, and shrub), which led to large genetic differences. By contrast, our examination utilized tissues from the same genus, with all plants exposed to the same greenhouse conditions and, consequently, revealing only small genetic differences.

The strong signals for SL, SA, UCT, and TCT indicated that those traits are phylogenetically conserved. However, most traits had weak signals, possibly because of a departure from Brownian motion evolution, such as adaptive evolution, that would not have been correlated with phylogeny. Therefore, this reflected the outcome of selection in heterogeneous environments where species can best acclimate to their current growing conditions [1]. Caruso et al. [52] have suggested that any constraints on the development of stomatal traits in Lobelia cardinalis primarily arise from a lack of genetic variation. In our study, the correlation between LET and either SD or SA disappeared when the effect of phylogeny was considered, thus confirming that variations in stomatal traits and leaf attributes are related to that particular influence.

Evolutionary Associations of Stomatal Anatomy with Leaf Traits

Vein density in Paphiopedilum was positively correlated with stomatal density, whether or not phylogeny was considered. However, VD was negatively correlated with stomatal size (Fig. 4), indicating that leaf vein has an evolutionary association with stomatal anatomy. This result supports the notion that the development and function of leaf veins and stomata are coordinated [11], as the coordinated development of veins and stomata is important for optimizing photosynthetic yield relative to carbon investment in leaf venation [11]. Moreover, coordinated plasticity in veins and stomata is thought to be at least partially related to leaf size; the development of leaf-size plasticity can provide an efficient way for plants to acclimate their hydraulic and stomatal conductance to contrasting transpirational demands under different lighting conditions [11], [51]. However, we found that SD and VD for these 17 Paphiopedilum species were not affected by leaf size. This was because our experimental materials had been grown in the same environment, and had similar transpirational demands.

We found no evidence for correlated evolution between stomatal traits and leaf thickness or cuticle thickness, which suggests a lack of functional association. Although LET was correlated with stomatal traits when phylogeny was not considered, only two correlations (LET vs SL, UET vs SD) were significant after that correction. The discrepancy between our Pearson’s and PIC correlations can be explained in that PICs reflect the historical pattern of diversification among taxa, whereas traditional Pearson’s correlations describe present-day relations among taxa [1]. Similar to our results, Beerling and Kelly [8] have suggested that thicker leaves do not necessarily mean more stomata. Nevertheless, previous studies have also shown that species with thick leaves have moderately large stomata [20], and that leaf thickness is negatively correlated with SD along an acidity gradient [18].

The lack of evolutionary correlation of stomatal traits with leaf thickness or cuticle thickness may have several explanations. Selective pressure that drives their development can differ between the two. Evolutionary trends largely depend on the selective force endured in challenging environments [9]. Stomatal density can be influenced by atmospheric CO2 concentration, heat stress, water status, plant density and light intensity [13], [16], [17], whereas leaf thickness is affected by light intensity, UV-radiation, rainfall and the supply of soil nutrients [31], [35], [38]. This inconsistency in evolutionary correlations among functional traits suggests that fundamentally different selective pressures and constraints may be acting [53]. Consequently, for the genus studied here, periodic water shortages and low nutrient availability in karst regions would have contributed to the evolution of leaf anatomy.

The difference in function between leaf cuticle thickness and stomatal traits decreases the coordination between them. In fact, changes in leaf anatomy do not always reflect adaptations to water availability. For example, leaves of plants growing in habitats with reduced soil nutrients have thicker epidermises than do their relatives in high-nutrient soils [30] because those sclerophyllous tissues develop as a way to protect scarce nutrient investments in leaf material against herbivory and abiotic physical damage [37]. By contrast, in arid environments, a thick cuticle likely has other functions besides that of water barrier, such as preventing physical damage by herbivorous pests [54].

The structural investment toward different leaf traits is largely controlled by an evolutionary trade-off between the antagonistic demands to maximize both photosynthesis and WUE [19], [55]. Having a thicker cuticle implies a greater construction cost for the leaf protective structure [28]. If more biomass must be allocated to the same function, the investment is reduced toward other functions. This situation is not cost-efficient to plant survival and competitiveness. Therefore, a correlated evolution among those traits would limit such divergence and adaptive selection [1]. Although many leaf surface characters, e.g., crypts, wax and hairs, can modify the relationship between stomatal size and number, and stomatal function, an evolutionary association between leaf anatomical traits and stomatal traits does not always necessitate water conservation and ecological strategies.

Correlation between Stomatal Density and Size

Stomatal density was significantly correlated with SL, but that association disappeared when phylogeny was considered. The negative correlation found here between SD and SL has been described previously [43], [56]. Both stomatal aperture and density are linked to leaf conductance, photosynthetic carbon gain and transpiration [55]. The capacity of plants to fix carbon is constrained by their photosynthetic biochemistry and CO2 diffusion conductance. When the concentration of atmosphere CO2 decreases, stomata become denser while the rate of maximum Rubisco carboxylation (Vcmax) slows. This co-variation among SL, SD and the Vcmax rate reduces the impact that any change in atmospheric CO2 has on the assimilation of leaf CO2, resulting in minimum energy cost and reduced nitrogen requirements [3]. A negative correlation between SD and SL also increases plasticity in maximum stomatal conductance to water vapor and CO2, with minimal alterations in the balance of water loss and epidermal allocations to the stomata [14], [56].

In summary, phylogeny has a significant effect on leaf traits and stomatal traits in Paphiopedilum. Stomatal length and area and upper cuticle thickness are strongly conserved. We noted a correlated evolution between stomatal traits and vein density in Paphiopedilum, but not between stomatal traits and leaf thickness. These findings provide insight into the development of traits related to water economy by orchids under natural selection.

Author Contributions

Conceived and designed the experiments: HH SBZ. Performed the experiments: ZJG MS JJZ. Analyzed the data: ZJG SBZ. Contributed reagents/materials/analysis tools: ZJG JJZ SBZ. Wrote the paper: SBZ KFC HH.

References

  1. 1. Ackerly DD, Donoghue MJ (1998) Leaf size, sapling allometry, and Corner’s rules: Phylogeny and correlated evolution in maples (Acer). The American Naturalist 152: 767–791.DD AckerlyMJ Donoghue1998Leaf size, sapling allometry, and Corner’s rules: Phylogeny and correlated evolution in maples (Acer).The American Naturalist152767791
  2. 2. Guan ZJ, Zhang SB, Guan KY, Li SY, Hu H (2011) Leaf anatomical structures of Paphiopedilum and Cypripedium and their adaptive significance. Journal of Plant Research 124: 289–298.ZJ GuanSB ZhangKY GuanSY LiH. Hu2011Leaf anatomical structures of Paphiopedilum and Cypripedium and their adaptive significance.Journal of Plant Research124289298
  3. 3. Franks PJ, Beerling DJ (2009) CO2-forced evolution of plant gas exchange capacity and water-use efficiency over the Phanerozoic. Geobiology 7: 227–236.PJ FranksDJ Beerling2009CO2-forced evolution of plant gas exchange capacity and water-use efficiency over the Phanerozoic.Geobiology7227236
  4. 4. Pittermann J (2010) The evolution of water transport in plants: An integrated approach. Geobiology 8: 112–139.J. Pittermann2010The evolution of water transport in plants: An integrated approach.Geobiology8112139
  5. 5. Brodribb TJ, Feild TS (2010) Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. Ecology Letters 13: 175–183.TJ BrodribbTS Feild2010Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification.Ecology Letters13175183
  6. 6. Willson CJ, PS Manos, RB Jackson (2008) Hydraulic traits are influenced by phylogenetic history in the drought-resistant and invasive genus Juniperus (Cupressaceae). American Journal of Botany 95: 299–314.CJ WillsonManos PSJackson RB2008Hydraulic traits are influenced by phylogenetic history in the drought-resistant and invasive genus Juniperus (Cupressaceae).American Journal of Botany95299314
  7. 7. Raven JA (2002) Selection pressures on stomatal evolution. New Phytologist 153: 371–386.JA Raven2002Selection pressures on stomatal evolution.New Phytologist153371386
  8. 8. Beerling DJ, Kelly CK (1996) Evolutionary comparative analyses of the relationship between leaf structure and function. New Phytologist 134: 35–51.DJ BeerlingCK Kelly1996Evolutionary comparative analyses of the relationship between leaf structure and function.New Phytologist1343551
  9. 9. Dunbar-Co S, Sporck MJ, Sack L (2009) Leaf trait diversification and design in seven rare taxa of the Hawaiian Plantago radiation. International Journal of Plant Sciences 170: 61–75.S. Dunbar-CoMJ SporckL. Sack2009Leaf trait diversification and design in seven rare taxa of the Hawaiian Plantago radiation.International Journal of Plant Sciences1706175
  10. 10. Woodward FI (1998) Do plants really need stomata? Journal of Experimental Botany 49: 471–480.FI Woodward1998Do plants really need stomata?Journal of Experimental Botany49471480
  11. 11. Brodribb TJ, Jordan GJ (2011) Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees. New Phytologist 192: 437–448.TJ BrodribbGJ Jordan2011Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees.New Phytologist192437448
  12. 12. Edwards D, Kerp H, Hass H (1998) Stomata in early land plants: An anatomical and ecophysiological approach. Journal of Experimental Botany 49: 255–278.D. EdwardsH. KerpH. Hass1998Stomata in early land plants: An anatomical and ecophysiological approach.Journal of Experimental Botany49255278
  13. 13. Woodward FI (1987) Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels. Nature 327: 617–618.FI Woodward1987Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels.Nature327617618
  14. 14. Franks PJ, Drake PL, Beerling DJ (2009) Plasticity in maximum stomatal conductance constrained by negative correlation between stomatal size and density: An analysis using Eucalyptus globulus. Plant, Cell & Environment 32: 1737–1748.PJ FranksPL DrakeDJ Beerling2009Plasticity in maximum stomatal conductance constrained by negative correlation between stomatal size and density: An analysis using Eucalyptus globulus.Plant, Cell & Environment3217371748
  15. 15. Ashton PMS, Berlyn GP (1992) Leaf adaptations of some Shorea species to sun and shade. New Phytologist 121: 587–596.PMS AshtonGP Berlyn1992Leaf adaptations of some Shorea species to sun and shade.New Phytologist121587596
  16. 16. Schlüter U, Muschak M, Berger D, Altmann T (2003) Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1–1) under different light regimes. Journal of Experimental Botany 54: 867–874.U. SchlüterM. MuschakD. BergerT. Altmann2003Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1–1) under different light regimes.Journal of Experimental Botany54867874
  17. 17. Xu ZZ, Zhou GS (2008) Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. Journal of Experimental Botany 59: 3317–3325.ZZ XuGS Zhou2008Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass.Journal of Experimental Botany5933173325
  18. 18. Wang R, Huang W, Chen L, Ma L, Guo C, et al. (2011) Anatomical and physiological plasticity in Leymus chinensis (Poaceae) along large-scale longitudinal gradient in Northeast China. PLoS ONE 6: e26209.R. WangW. HuangL. ChenL. MaC. Guo2011Anatomical and physiological plasticity in Leymus chinensis (Poaceae) along large-scale longitudinal gradient in Northeast China.PLoS ONE6e26209
  19. 19. Aasamaa K, Sõber A, Rahi M (2001) Leaf anatomical characteristics associated with shoot hydraulic conductance, stomatal conductance and stomatal sensitivity to changes of leaf water status in temperate deciduous trees. Australian Journal of Plant Physiology 28: 765–774.K. AasamaaA. SõberM. Rahi2001Leaf anatomical characteristics associated with shoot hydraulic conductance, stomatal conductance and stomatal sensitivity to changes of leaf water status in temperate deciduous trees.Australian Journal of Plant Physiology28765774
  20. 20. Hodgson JG, Sharafi M, Jalili A, Díaz S, Montserrat-Martí G, et al. (2010) Stomatal vs. genome size in angiosperms: The somatic tail wagging the genomic dog? Annals of Botany 105: 573–584.JG HodgsonM. SharafiA. JaliliS. DíazG. Montserrat-Martí2010Stomatal vs. genome size in angiosperms: The somatic tail wagging the genomic dog?Annals of Botany105573584
  21. 21. Niklas KJ (1999) A mechanical perspective on foliage leaf form and function. New Phytologist 143: 19–31.KJ Niklas1999A mechanical perspective on foliage leaf form and function.New Phytologist1431931
  22. 22. Sack L, Frole K (2006) Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology 87: 483–491.L. SackK. Frole2006Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees.Ecology87483491
  23. 23. Brodribb TJ, Feild TS, Jordan GJ (2007) Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology 144: 1890–1898.TJ BrodribbTS FeildGJ Jordan2007Leaf maximum photosynthetic rate and venation are linked by hydraulics.Plant Physiology14418901898
  24. 24. Uhl D, Mosbrugger V (1999) Leaf venation density as a climate and environmental proxy: A critical review and new data. Palaeogeography, Palaeoclimatology, Palaeoecology 149: 15–26.D. UhlV. Mosbrugger1999Leaf venation density as a climate and environmental proxy: A critical review and new data.Palaeogeography, Palaeoclimatology, Palaeoecology1491526
  25. 25. Walls RL (2011) Angiosperm leaf vein patterns are linked to leaf functions in a global-scale data set. American Journal of Botany 98: 244–253.RL Walls2011Angiosperm leaf vein patterns are linked to leaf functions in a global-scale data set.American Journal of Botany98244253
  26. 26. Nardini A, Ramani M, Gortan E, Salleo S (2008) Vein recovery from embolism occurs under negative pressure in leaves of sunflower (Helianthus annuus). Physiologia Plantarum 133: 755–764.A. NardiniM. RamaniE. GortanS. Salleo2008Vein recovery from embolism occurs under negative pressure in leaves of sunflower (Helianthus annuus).Physiologia Plantarum133755764
  27. 27. Scoffoni C, Rawls M, McKown A, Cochard H, Sack L (2011) Decline of leaf hydraulic conductance with dehydration: Relationship to leaf size and venation architecture. Plant Physiology 156: 832–843.C. ScoffoniM. RawlsA. McKownH. CochardL. Sack2011Decline of leaf hydraulic conductance with dehydration: Relationship to leaf size and venation architecture.Plant Physiology156832843
  28. 28. Gratani L, Bombelli A (1999) Leaf anatomy, inclination and gas exchange relationships in evergreen sclerophyllous and drought semideciduous shrub species. Photosynthetica 37: 573–585.L. GrataniA. Bombelli1999Leaf anatomy, inclination and gas exchange relationships in evergreen sclerophyllous and drought semideciduous shrub species.Photosynthetica37573585
  29. 29. Watkins JE, Mack MC, Sinclair TR, Mulkey SS (2007) Ecological and evolutionary consequences of desiccation tolerance in tropical fern gametophytes. New Phytologist 176: 708–717.JE WatkinsMC MackTR SinclairSS Mulkey2007Ecological and evolutionary consequences of desiccation tolerance in tropical fern gametophytes.New Phytologist176708717
  30. 30. Bucci SJ, Goldstein G, Meinzer FC, Scholz FG, Franco AC, et al. (2004) Functional convergence in hydraulic architecture and water relations of tropical savanna trees: From leaf to whole plant. Tree Physiology 24: 891–899.SJ BucciG. GoldsteinFC MeinzerFG ScholzAC Franco2004Functional convergence in hydraulic architecture and water relations of tropical savanna trees: From leaf to whole plant.Tree Physiology24891899
  31. 31. Cunningham SA, Summerhayes B, Westoby M (1999) Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients. Ecological Monograph 69: 569–588.SA CunninghamB. SummerhayesM. Westoby1999Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients.Ecological Monograph69569588
  32. 32. Muchow RC, Sinclair TR (1989) Epidermal conductance, stomatal density and stomatal size among genotypes of Sorghum bicolour (L.) Moench. Plant, Cell & Environment 12: 425–431.RC MuchowTR Sinclair1989Epidermal conductance, stomatal density and stomatal size among genotypes of Sorghum bicolour (L.) Moench.Plant, Cell & Environment12425431
  33. 33. Helbsing S, Riederer M, Zotz G (2000) Cuticles of vascular epiphytes: Efficient barriers for water loss after stomatal closure? Annals of Botany 86: 765–769.S. HelbsingM. RiedererG. Zotz2000Cuticles of vascular epiphytes: Efficient barriers for water loss after stomatal closure?Annals of Botany86765769
  34. 34. Schreiber L, Riederer M (1996) Ecophysiology of cuticular transpiration: Comparative investigation of cuticular water permeability of plant species from different habitats. Oecologia 107: 426–432.L. SchreiberM. Riederer1996Ecophysiology of cuticular transpiration: Comparative investigation of cuticular water permeability of plant species from different habitats.Oecologia107426432
  35. 35. Manetas Y, Petropoulou Y, Stamatakis K, Nikolopoulos D, Levizou E, et al. (1997) Beneficial effects of enhanced UV-B radiation under field conditions: Improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer. Plant Ecology 128: 101–108.Y. ManetasY. PetropoulouK. StamatakisD. NikolopoulosE. Levizou1997Beneficial effects of enhanced UV-B radiation under field conditions: Improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer.Plant Ecology128101108
  36. 36. Riederer M, Schreiber L (2001) Protecting against water loss: Analysis of the barrier of plant cuticles. Journal of Experimental Botany 52: 2023–2032.M. RiedererL. Schreiber2001Protecting against water loss: Analysis of the barrier of plant cuticles.Journal of Experimental Botany5220232032
  37. 37. Turner IM (1994) Sclerophylly – primarily protective. Functional Ecology 8: 669–675.IM Turner1994Sclerophylly – primarily protective.Functional Ecology8669675
  38. 38. Kerstiens G (2006) Water transport in plant cuticles: An update. Journal of Experimental Botany 57: 2493–2499.G. Kerstiens2006Water transport in plant cuticles: An update.Journal of Experimental Botany5724932499
  39. 39. Cribb P (1998) The Genus Paphiopedilum (2nd Edition). Natural History Publications, Kota Kinabalu (Borneo) in association with Royal Botanic Gardens, Kew, UK. P. Cribb1998The Genus Paphiopedilum (2nd Edition).Natural History Publications, Kota Kinabalu (Borneo) in association with Royal Botanic Gardens, Kew, UK
  40. 40. Karasawa K, Saito K (1982) A revision of the genus Paphiopedilum (Orchidaceae). Bulletin of the Hiroshima Botanical Garden 5: 1–69.K. KarasawaK. Saito1982A revision of the genus Paphiopedilum (Orchidaceae).Bulletin of the Hiroshima Botanical Garden5169
  41. 41. Assmann SM, Zeiger E (1985) Stomatal responses to CO2 in Paphiopedilum and Phragmipedium – role of the guard cell chloroplast. Plant Physiology 77: 461–464.SM AssmannE. Zeiger1985Stomatal responses to CO2 in Paphiopedilum and Phragmipedium – role of the guard cell chloroplast.Plant Physiology77461464
  42. 42. Zhang S-B, Guan Z-J, Chang W, Hu H, Yin Q, et al. (2011) Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy. Physiologia Plantarum 142: 118–127.S-B ZhangZ-J GuanW. ChangH. HuQ. Yin2011Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy.Physiologia Plantarum142118127
  43. 43. Beaulieu JM, Leitch IJ, Patel S, Pendharkar A, Knight CA (2008) Genome size is a stronger predictor of cell size and stomatal density in angiosperms. New Phytologist 179: 975–986.JM BeaulieuIJ LeitchS. PatelA. PendharkarCA Knight2008Genome size is a stronger predictor of cell size and stomatal density in angiosperms.New Phytologist179975986
  44. 44. James SA, Bell DT (2001) Leaf morphological and anatomical characteristics of heteroblastic Eucalyptus globulus ssp. globulus (Myrtaceae). Australian Journal of Botany 49: 259–269.SA JamesDT Bell2001Leaf morphological and anatomical characteristics of heteroblastic Eucalyptus globulus ssp. globulus (Myrtaceae).Australian Journal of Botany49259269
  45. 45. Blomberg SP, Garland T Jr, Ives AR (2003) Testing for phylogenetic signal in comparative data: Behavioral traits are more labile. Evolution 57: 717–745.SP BlombergT. Garland JrAR Ives2003Testing for phylogenetic signal in comparative data: Behavioral traits are more labile.Evolution57717745
  46. 46. Cox AV, Pridgeon AM, Albert VA, Chase MW (1997) Phylogenetics of the slipper orchids (Cypripediodeae, Orchidaceae): Nuclear rDNA ITS sequences. Plant Systematics and Evolution 208: 197–223.AV CoxAM PridgeonVA AlbertMW Chase1997Phylogenetics of the slipper orchids (Cypripediodeae, Orchidaceae): Nuclear rDNA ITS sequences.Plant Systematics and Evolution208197223
  47. 47. R Development Core Team (2011) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. The R Project for Statistical Computing website. R Development Core Team2011R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. The R Project for Statistical Computing website. Available: http://www.R-project.org.Accessed 2011 Jul 10. Accessed 2011 Jul 10.
  48. 48. Webb CO, Ackerly DD, Kembel SW (2008) PHYLOCOM: Software for the analysis of phylogenetic community structure and trait evolution. Bioinformatics 24: 2098–2100.CO WebbDD AckerlySW Kembel2008PHYLOCOM: Software for the analysis of phylogenetic community structure and trait evolution.Bioinformatics2420982100
  49. 49. Felsenstein J (1985) Phylogenies and the comparative method. The American Naturalist 125: 1–15.J. Felsenstein1985Phylogenies and the comparative method.The American Naturalist125115
  50. 50. Jiménez S, Zellnig G, Stabentheiner E, Peters J, Morales D, et al. (2000) Structure and ultrastructure of Pinus canariensis needles. Flora 195: 228–235.S. JiménezG. ZellnigE. StabentheinerJ. PetersD. Morales2000Structure and ultrastructure of Pinus canariensis needles.Flora195228235
  51. 51. Murphy MRC, Jordan GJ, Brodribb TJ (2012) Differential leaf expansion can enable hydraulic acclimation to sun and shade. Plant, Cell & Environment. MRC MurphyGJ JordanTJ Brodribb2012Differential leaf expansion can enable hydraulic acclimation to sun and shade.Plant, Cell & Environment
  52. 52. Caruso CM, Maherall H, Mikulyuk A, Carlson K, Jackson RB (2005) Genetic variance and covariance for physiological traits in Lobelia: Are there constraints on adaptive evolution? Evolution 59: 826–832.CM CarusoH. MaherallA. MikulyukK. CarlsonRB Jackson2005Genetic variance and covariance for physiological traits in Lobelia: Are there constraints on adaptive evolution?Evolution59826832
  53. 53. Kembel SW, Cahill Jr JF (2011) Independent evolution of leaf and root traits within and among temperate grassland plant communities. PLoS ONE 6: e19992.SW KembelJF Cahill Jr2011Independent evolution of leaf and root traits within and among temperate grassland plant communities.PLoS ONE6e19992
  54. 54. Gentry G, Barbosa P (2006) Effects of leaf epicuticular wax on the movement, foraging behavior, and attack efficacy of Diaeretiella rapae. Entomologia Experimentalis et Applicata 121: 115–122.G. GentryP. Barbosa2006Effects of leaf epicuticular wax on the movement, foraging behavior, and attack efficacy of Diaeretiella rapae.Entomologia Experimentalis et Applicata121115122
  55. 55. Büssis D, von Groll U, Fisahn J, Altmann T (2006) Stomatal aperture can compensate altered stomatal density in Arabidopsis thaliana at growth light conditions. Functional Plant Biology 33: 1037–1043.D. BüssisU. von GrollJ. FisahnT. Altmann2006Stomatal aperture can compensate altered stomatal density in Arabidopsis thaliana at growth light conditions.Functional Plant Biology3310371043
  56. 56. Sack L, Grubb PJ, Marañón T (2003) The functional morphology of juvenile plants tolerant of strong summer drought in shaded forest understories in southern Spain. Plant Ecology 168: 139–163.L. SackPJ GrubbT. Marañón2003The functional morphology of juvenile plants tolerant of strong summer drought in shaded forest understories in southern Spain.Plant Ecology168139163