The size differs more than fluctuating asymmetry in Ficaria verna (Ranunculaceae) populations around

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The size differs more than fluctuating asymmetry in Ficaria verna (Ranunculaceae) populations around a woodland

Introduction Local community assemblages are ‘filtered’ by dispersal, abiotic, and competition factors (Houseman and Gross, 2006; Myers and Harms, 2009) which lead to the establishment of populations with traits or phenotypes, characteristics to that habitat (Pavoine et al., 2011; Kraft et al., 2015). Understanding the spatiotemporal variability of plant communities has relied on measuring functional traits (Mabry et al., 2000; Cornelissen et al., 2003) on indicator species (Verheyen and Hermy, 2001; Diekmann, 2003) which allow us to assess the quality of a certain habitat, area, landscape quantitatively (Dwyer and Laughlin, 2017).

Woodland habitats are of particular interest, as strong associations have been found between certain plant functional traits to environmental factors such as canopy cover (Jennings et al., 1999), soil pH (Hipps et al., 2005), soil water content (Leuschner and Lendzion, 2009), and competition with neighbouring species (Levine, 2000; Callaway, 2007). These variables interact can with each other, creating a vertically and horizontally heterogeneous habitat (Xiong et al., 2003; Niinemets and Valladares, 2004). Environmental limitation on clonal herbs can lead to ecological exclusion during different life stages (Verheyen and Hermy, 2001; Baeten et al., 2009). Clonal reproduction has numerous advantages (Whigham and Chapa, 1999) but plants need to compensate for their low genetic variability by phenotypic plasticity to survive in different environments (de Kroons and Hutchings, 1995; Dong, 1995; Kudoh et al., 1999). This plasticity can be used as an indicator of population fitness, stress, and genetic stability as it acts as a buffer against stress (Fazlioglu and Bonser, 2016; Liao et al., 2016). When a plant is exposed to long-term, extreme or multiple constraints during development, it can result in developmental instability, which has been quantified as fluctuating asymmetry (FA) (Rasmuson, 2002; Nikiforou and Manetas, 2017).

FA considers the random small deviations of bilateral characters, which depend on the same gene (Kozlov and Zvereva, 2015). Directional asymmetry (e.g. right, R side of the leaf is overall larger than the left side) and antisymmetrical sides (both right and left, L can be larger relative to each other) are defined based on the distribution of the absolute value of FA, which is calculated as FA = |R-L| (Palmer and Strobeck, 1986; Graham et al., 2010; Alves-Silva et al., 2018). The larger the FA absolute value, the more stressed the population is. The developmentally stable population is not constrained by environmental factors or its phenotypic plasticity is efficiently counteracting those and therefore the symmetrical features would be close to ‘perfect symmetry’ (Figure 1) (Velickovic, 2010). Despite the

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increasing numbers of studies using FA by measuring the ‘widest point on the leaf’, there are also increasing numbers of papers discussing the high error rate and hence unreliability of FA measurements (Heard et al., 1999; Kozlov et al., 2015; Kozlov et al., 2017; Alves-Silva et al., 2018). It is suggested that better-defined, anatomical points are needed when investigating FA (Klingenberg et al., 2002; Klingenberg et al., 2012).

Lesser celandine, Ficaria verna, formerly known as Ranunculus ficaria, is an ideal ephemeral, perennial species with a bilaterally symmetrical vein system (Figure 1). It mainly reproduces via vegetative growth, producing tubers and bulbils, creating connected ramets (Jung et al., 2008). Their morphological variability has been observed by Macdonell (1905) across Europe and was used to be grouped into ‘local races’ based upon their morphology, which has complicated their taxonomy (Heywood and Walker, 1961; Sell, 1994, Briggs and Walters, 2016). It is a stress-tolerant ruderal species (Grime et al., 2014; Packham and Cohn, 1990), however, Ficaria subsp. ficariiformis was found to be a stress-tolerant competitor (Huseyinoglu and Yalcin, 2017), while Ficaria subsp. bulbifer has been described as a poor competitor (Jung et al., 2008). Their variable colonisation capacity (Verheyen et al., 2003) has enabled this species to become invasive in different countries (Krings et al., 2005; Post et al., 2009; Axtell et al., 2010; Masters and Emery, 2015).

In this study, we used a new approach to measure leaf FA of F. verna, which is known for its phenotypic plasticity. Our first hypothesis was that F. verna populations from a river bank, a semi-natural grassland and a natural woodland will have different sizes and levels of FA. Secondly, we hypothesised that different veins will show different levels of relative FA, which would suggest the reliability of these measurements. Thirdly, we investigated the influence and interaction of canopy cover, population density, soil pH and water content on the size of the veins, leaves and flowers. We expected that the more distinct a site was, the more morphologically different the plants would be.

Figure 1. Plants emerge in January and flower from March to May when the temperature is between 15-20°C (Axtell et al., 2010). Prior to flowering, they require colder temperatures (4-6°C). The plants die back in June when the temperature is above 20°C. This study focused on the effect of abiotic and biotic stress factors on leaf and flower size difference and leaf vein asymmetry as opposed to ‘perfect’ bilateral symmetry.

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Methods 1. Site description and sampling methods Three different sites were used for this study within the 12-hectare Parc Natur Penglais Local Nature Reserve in Aberystwyth. Site A (N 52° 25' 8.706'', W 4° 3' 59.806'', 88.24 m altitude) was dominated by grasses, site B (N 52° 25' 7.835'', W 4° 3' 57.637'', 88.38 m altitude) was at a stream bank, and site C (N 52° 25' 8.452'', W 4° 4' 33.184'', 79.14 m altitude) was located near a footpath, in the woodland.

The distance between sites A-B was 56 m, A-C was 611.3 m, and B-C was 658.7 m. The reserve consisted of mature beech (Fagus sylvatica) woodland with an understory of holly (Ilex aquifolium) and hazel (Corylus avellana) with mature oaks (Quercus robur) and ash (Fraxinus excelsior).

At each location, three 0.5 m2 quadrats were randomly selected (Appendix 1). In each quadrat, species richness, number of plants, number of open flower and closed buds were counted. From these quadrats, five plants were chosen to measure peduncle length (0.1 cm), flower diameter (0.01 cm, with image analysis) and two leaves from each plant were measured. Nine measurements were recorded of each leaf with digital callipers to 0.1 mm accuracy (Figure 2a). All data was entered and processed in Excel (2016).

The soil of the nature reserve was Denbigh, relatively free draining soil with loamy topsoil texture and low soil fertility (National Soil Resources Institute, 2018). Four samples were taken from each site. The pH levels were measured by diluting 2 g of soil in 5 ml distilled water. After a 30-minute resting period, a probe (HI-98100 Checker Plus, Hanna) was used to record pH levels. For soil water content (WC), the ceramic pot weight with 0.01 g accuracy (W1) was measured, and then 5 g of soil was added to the pot (W2). After 12 hours of drying the soil samples at 105°C in an oven, the weight of soil with the pot was measured (W3). The soil moisture (%) was calculated as WC = (W2-W3/W3-W1) × 100.

2. Image analysis Pictures of flower diameters were taken in the field by placing a 2 cm diameter reference object covering half of the flower (Figure 2b). Measurements were recorded in ImageJ (Version 1.51w) with 0.01 mm accuracy.

Canopy cover percentage was calculated by dividing the number of pixels from the covering trees and shrubs by the number of pixels of the sky. We used the ‘Colour Threshold’ function in ImageJ, setting particle size circularity to 1,000 and by selecting the masks after the analysis, the number of pixels was measured (Figure 2c).

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b)

a)

c)

Figure 2. Measurements were taken from leaves (a) and flowers (b). The ‘Colour Threshold' function in ImageJ separating the trees and shrubs covering each site from the sky (c). Image analysis of the flowers and canopy cover combined with digital callipers used for leaf measurements led to precise measurements in this study.

3. Statistical methods

3.1. Size Natural log transformation for non-parametric distribution was used with Kruskal Wallis testing, crossvalidated by Dunn pairwise posthoc analyses with Bonferroni correction (Armstrong, 2014). To investigate a relationship between leaf vein lengths on opposite sides (R-L) and between life-size measurements (B, T), linear regressions were used. To investigate the effect of pH, canopy cover, and soil WC, linear and multiple General Linear Models (GLM) were used. The replicates of these measurements were multiplied to create a continuous variable while the mean, variance and standard error of the mean remained constant. The GLM treated the variance from factors as independent effects, and tested these interactive effects between each factor (Heikkinen et al. 2005). All statistical analysis was carried out in R 3.4.4 (R Core Team, 2018) software.

3.2. Fluctuating asymmetry Two calculations were used for expressing FA in between veins (R1-L1, R2-L2, R3-L3). Firstly, FA was calculated as the absolute value of the difference for each vein FA = |R-L|. The relative FA of both the veins and between B-T was calculated as FA = 100 Ă— |ln(L)-ln(R)| (Clarke, 1998; Pelabon et al., 2005).

To test if these veins are significantly different from each other and therefore are reliable measurements, Kruskal Wallis test with Dunn pairwise posthoc analysis were used.

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Results 1. Site differences Site A, the semi-natural grassland supported the densest population (nA=28) of Ficaria verna, consequently had the most number of flower buds (37) and open flowers (20) (Table 1). Site B and Site was very similar in terms of population count (nB=22, nC=23), the number of open flowers (12 and 11). Site B had the most canopy cover (79.54%), had the least acidic (pH=6) and soil water content (70.35%). Site C was moderately covered (39.18%), had the most acidic soil (pH=5.33) and the most soil water content (89.79%). Site A was similar to site C (34.71% cover, pH=5.58, 76.46% WC). The mean lengths of peduncles, flower diameters (Table 1) and leaf measurements (Appendix 2) showed that site C plants were larger than at other sites (Figure 3).

2. Size difference There was an overall significant difference between the sites (χ2 = 119.2, df = 2, P < 0.000) between site A-B and B-C (P < 0.000) but there was no difference between A-C (P = 1.00) (Table 2). Flower diameter measurements overall differed between sites (χ2 = 12.468, df = 2, P = 0.002) but only between sites B-C was this significant (P = 0.001). Peduncle lengths also varied between sites (χ2=12.468, df = 2, P = 0.002) and were significantly different for A-B (P = 0.007), A-C (P = 0.033) and B-C (P < 0.000).

2.1 Factor interaction at sites Linear GLMs between sites and abiotic factors (pH, canopy cover, WC) showed a highly significant response in vein lengths (F100,709 = 16.61, R2 = 0.451, P < 0.000) (Table 3). The measurements at site B were significantly different due to cover (P = 0.042), pH (P = 0.041), and soil WC (P = 0.042). Linear GLM between sides and sites showed that the right side veins were significantly different at site B (P = 0.043) and site C (P= 0.043). Multiple GLM, testing the interaction between several factors at different sites was overall significant (F33, 776 = 21.12, R2 = 0.451, P < 0.000) but there was only a significant interaction between pH and WC (P = 0.034) at site B.

Table 1. Biotic and abiotic factors collected at the three sample sites. For count data, we report the mean of the site means.

For length measurements (mm), we include standard error of the mean. The following mean values of canopy cover, pH and soil WC were used to test the response of the measurements and FA in the multiple GLMs. Site

Population

Species

Flower Buds

Open Flowers

Peduncle (cm)

Flower Diameter (cm) %Cover

pH

WC%

A

28

9

37

20

7.91 ± 0.432

2.95 ± 0.755

34.71%

5.58

76.46

B

22

7

24

12

12.99 ± 0.813

3.35 ± 1.213

79.54%

6

70.35

C

23

13

36

11

5.86 ± 0.234

2.69 ± 1.040

39.18%

5.33

89.79

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Figure 3. The leaf measurements at the three sites showing that leaves at site B were considerable larger compared to the other sites. The leaves at site A were relatively the smallest, while at site C they were intermediate between sites A-B.

Multiple GLMs sites, andsites population counts (F29, 780 =larger 51.65, R2 = 0.645, Figure 3. The leafbetween measurements at sides the three showing that leaveswas at sitesignificant B were considerable compared to the other sites. The leaves at site A were relatively the smallest, while at site C they were intermediate between sites A-B.

P < 0.000) (Table 3). They revealed that at site B both the left (P = 0.021) and right side (P = 0.004) responded to population density while at site C, only the right side (P = 0.038) responded. Linear regression between R-L veins (F1, 268 = 773.4, R2 = 0.86, P < 0.000) and B-T (F1, 88 = 214.3, R2 = 0.71, P < 0.000) showed high correlation (Appendix 3).

2. Fluctuating asymmetry The absolute value of FA was not statistically significantly different at the three sites (χ2 = 2.678, df = 2, P = 0.262) (Table 4) with Bonferroni correction, however, without the correction, the test was close to being statistically significant (P = 0.052) which suggests that the correction eliminated type I errors.

Relative FA between veins and B-T were different (χ2 = 56.896, df = 8, P< 0.000), however, the relative FA was not different between the first vein (FA1) and third (FA3) (P = 1.00).

Table 2. Kruskal Wallis test combined with Dunn pairwise analysis on leaf and flower measurements. χ2 Log(Lengths) A-B

Log(Lengths)

0.4507

119.2 χ2

2df

0.000 P

Linear

2

0.000

Site B : Cover

0.042

119.2

1.000

Site B : pH

0.041

0.000

Site B : WC

0.042

1.000

Site B : Right side

0.043

0.000

Site C : Right side

0.043

0.002

Multiple

0.000

0.000 12.468

2

B-C

0.002 0.094

A-B

12.468

2

A-C

0.504

A-B

0.094

B-C

0.001

A-C

0.504

Multiple with

0.000

sides

Peduncle

F100,709 = 16.61

30.629

2

0.001

B-C

F33,776 = 21.12

0.4507

< 0.000

< 0.000 0.034

Site B : pH : WC F29,780 = 51.65

0.6448

Site B: Left side : Population

0.021

0.000

0.033

Site B: Right side : Population

0.004

B-C

A-B

0.007 0.000

Site C : Right side : Population

0.038

A-C

0.033

B-C

0.000

Peduncle

A-C

30.629

2

Table 2. Kruskal Wallis test combined with Dunn pairwise

* Non-significant results are not reported.

* Non-significant results are not reported.

FA

χ2

df

P

2.678

2

0.262

Table 0.400 with Dunn A-B 4. Kruskal Wallis test combined pairwise analysis on FA and relative FA 1.000 calculations in between different sites and A-C different veins.

0.560

B-C Relative FA

129.120

3

0.000

FA1-FA2

0.000

FA2-FA3

0.000

FA3-FA1

1.000

FABT-FA1

0.000

FABT-FA2

0.000

FABT-FA3

0.000

< 0.000

0.007

A-B

Table 4. Kruskal Wallis test combined with Dunn pairwise analysis on FA and relative FA calculations in between different sites and veins.

P

GLM

A-B

Flower

R2

P

B-C Flower

F

df

A-C

A-C

Table 3. Significant responses* of the leaf veins to different abiotic factors compared to site A by linear and multiple GLMs.

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Discussion This study has shown that the size of the leaves, flowers and peduncles of Ficaria verna differed in contrasting habitats without significant changes in FA of the linearly correlated leaf veins. We also showed that two veins out of the three are reliable in terms of FA. At the most shaded and least acidic site (B), pH and soil WC factors interacted in differentiating the variances from sites A (semi-natural grassland) and C (woodland). This confirmed our hypothesis that the more distinct a location is in terms of environmental factors, the more diverse the plant morphology would be.

Site A had smaller leaves and flowers in comparison to the other sites, which could be explained by competitive exclusion with grasses and mowing (House et al., 2003; Gรถransson et al., 2008; Reisch and Scheitler, 2009; Cipollini and Schradin, 2011). Site C had the most number of species co-occurring with F. verna where competitive exclusion (Reisch and Scheitler, 2009) and allelopathy in F. verna (Cipollini and Schradin, 2011; Cipollini et al., 2012) can lead to more morphologically distinct genotypes within a habitat. The GLM showed that the right sides of the leaves interacted with population densities, separating these two sites. Hence, the statistically significant difference between the leaf and flower sizes between these two sites, are more likely to be caused by biotic interactions than abiotic ones (Turcotte and Levine, 2016). When the species composition, based on Ellenberg values, are compared (Table 5) (Hill et al., 1999), two distinct species (Fragaria vesca, Primula vulgaris) are indicators of a more or less infertile site while at site C, most species were indicators for intermediate soil fertility.

Non-significance of FA veins could be explained by the skewed data distribution (Figure 4) and small sample size (Gangestad and Thornhill, 1999; Alves-Silva et al., 2018). Alternatively, F. verna could be buffering the environmental stresses efficiently by adjusting the size of the leaves while maintaining its

Table 5. Species found at the different sample sites and their Ellenberg values*.

symmetry. Directional asymmetry or antisymmetry can impede the detection of FA and at sites A and C, while the distribution of FA at site B resembles platykurtic distribution, implying that it has more extreme deviations from the mean (Graham et al., 1994; Alves-Silva et al., 2018). The significant results from the GLM suggest that there were differences in between sites and sides. Despite the non-significant FA results, our measurements offer a well-defined, reproducible protocol, which could be used for other plant species.

Sites Species L F R N All Ficariafound vernaat the different sample 5 5sites5 and6 Table 5. Species Site Taraxacum 7 5 7 6 theirAEllenberg values*officinale Veronica persica 6 5 7 7 Fragaria vesca 6 5 6 4 Primula vulgaris 5 5 6 4 Site B Taraxacum officinale 7 5 7 6 Veronica hederifolia 6 5 7 6 Galium aparine 6 6 7 8 Hyacinthoides non-scripta 5 5 5 6 Ilex aquifolium 5 5 5 5 Hedera helix 4 5 7 6 Site C Geranium robertianum 8 4 7 6 Crataegus monogyna 6 5 7 6 Conopodium majus 6 5 5 5 Hyacinthoides non-scripta 5 5 5 6 Ilex aquifolium 5 5 5 5 Acer pseudoplatanus 4 5 6 6 Hedera helix 4 5 7 6 * L - Light, F - Moisture, R - Reaction, N - Nitrogen, S - Salt

S 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

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In comparison to our methods, the ‘widest point of the leaf’ results can lead to only 7.4% reproducibility (Kozlov, 2015). Even using a highly standardised protocol by Kozlov et al. (2017), found that 24% of total variation was explained by measurement error (Kozlov and Zvereva,

2015).

However,

the

differences in size reveal interesting patterns of abiotic effects and their interactions.

Canopy cover is known to increase clonal herb

growth

Silvertown,

rate 1998;

(Valverde

and

Hutching

and

Bradbury, 2000), leaf size, stolon and

Figure 4. The distribution of FA calculated on the third veins at different sites shows slight antisymmetry at sites A and C while site B is close to a platykurtic distribution with extreme values.

petiole length (Mitchel and Woodward, 1988) within woodlands. Taylor and Markham (1958) tested the effect irradiance Figure 4. The experimentally distribution of FA calculated on the thirdof veins at different sites shows slight antisymmetry at sites A and C while site B is close to a platykurtic distribution with extreme values.

on F. verna and found that in more shaded conditions, the specific leaf area and leaf weight ratio increased significantly confirming our findings that in a shaded habitat, the plants grow larger.

However, it is possible that our samples varied in their ploidy levels, contributing to the different morphologies (Ginzo and Lovell, 1973; Marchant and Brighton, 1974; Grime et al., 2014; Drenckhahn et al., 2017). Canopies, inter-canopies and their edges have also been shown to influence the precipitation reaching herbs on the forest floor, which creates a horizontally heterogeneous habitat in terms of soil water content (Bossuyt and Hermy, 2000; Breshears et al., 2009). At site C, dense Crataegus monogyna and Ilex aquifolium created an inter-canopy, which could have added to the different sizes of the leaves from site A. Soil pH also differed between our sample sites. The more acidic soils tend to be more nutrient deficient, affecting species composition (Härdtle et al., 2003; Goransson et al., 2008; Jeong et al., 2016).

Although F. verna can grow on a wide range of soil pH (4-8) they are mostly found on soils with pH 4.4 – 6.5 in the UK (Taylor and Markham, 1985; Grime et al., 2014). Soil chemistry also depends on soil WC (Özkan and Gökbulak, 2017), which affects plant distribution within a woodland (Leuschner and Lendzion, 2009; Grman et al., 2015; Wang et al., 2015) and ecosystem respiration (Xu et al., 2004).

Our WC measurements were larger compared to other woodland studies (Leuschner and Lendzion, 2009; De Keersmaeker, 2004) which could be caused by the short (12 h) drying time. Flower production (Marsden-Jones, 1935) and colonisation success (Swearingen, 2005) have been found to

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increase near riverbanks for F. verna. The flowers were larger and peduncles were longer at the stream bank, which is consistent with Taylor and Markham (1985). As initial tuber size is associated with flower production (Taylor and Markham, 1985; Kertabad et al., 2013) and colonisation capacity, therefore in future studies, long-term monitoring and more extensive sampling could enable us to gain in-depth knowledge about factors affecting F. verna and using it as a reliable indicator species.

This plant could be used to investigate the level of disturbance around footpaths within urban woodlands (Littlemore and Barker, 2001; Roovers et al., 2004). Geometric morphometrics (Silva et al 2012; Klingenberg et al., 2012) could also be a more reliable, image-based system to study FA (Klingenberg and McIntyre, 1998).

We conclude that F. verna shows phenotypic plasticity within a woodland, responding to canopy cover, soil characteristics and species competition. The size was a more reliable measurement than FA in the case of 45 plants due to unusual data distribution. It is suggested that two veins would be reliable for FA measurements, especially for image-based analysis.

Word count (excluding in-text references): 2487

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Appendix Appendix 1. Sampling design showing all the habitat characteristics and plant measurements and counts.

Appendix 1.

Appendix 2. Leaf measurements (mm) with standard error of the mean.

Site

R1

R2

R3

M

L1

L2

L3

B

T

1

2. ± 0.23 10.32 Appendix ± 0.271 9.68

11.17 ± 0.303

14.14 ± 0.374

11.22 ± 0.303

9.77 ± 0.245

9.89 ± 0.278

19.42 ± 0.48

15.75 ± 0.432

2

14.07 ± 0.285

13.38 ± 0.258

15.27 ± 0.352

19.00 ± 0.428

15.51 ± 0.326

13.83 ± 0.303

13.81 ± 0.264

27.60 ± 0.493

22.38 ± 0.48

3

10.42 ± 0.366

10.00 ± 0.308

11.44 ± 0.395

14.34 ± 0.527

11.9 ± 0.395

10.31 ± 0.342

10.53 ± 0.329

19.86 ± 0.625

15.66 ± 0.52

Appendix 3. Linear regression on the total right and left side veins showing high correlation.

Appendix 2.

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