Examining phenotypic plasticity in Hydrocotyle bonariensis in response to variations in soil

conditions

Savannah D. Chiarello and Heather M. Joesting

Abstract

The ability of an organism to adjust its morphology and/or physiology in response to variations in abiotic

factors is known as phenotypic plasticity. Hydrocotyle bonariensis is a perennial herb found in both

coastal sand dune and inland coastal habitats of Georgia. The soil types of these two habitats differ in soil

characteristics (e.g., texture and pH) and chemistry (e.g., nutrient and organic matter content). The

purpose of this study was to explore potential phenotypic plasticity in H. bonariensis in response to

different soil types by comparing leaf and petiole morphology between individuals grown in soil from the

sand dune habitat and soil from an inland coastal habitat. Results showed significantly greater leaf area,

petiole fresh weight, petiole thickness, petiole length, and abaxial stomata density in individuals grown in

soil collected from the inland site compared to the sand dune soil, suggesting that the differences in soil

characteristics and chemistry between the two soil types, specifically those related to water-holding

capacity and nutrient availability, resulted in differences in leaf and petiole morphology. The ability of H.

bonariensis to respond plastically to changes in environmental conditions, such as variations in soil type,

likely plays an important role in its ability to successfully thrive and reproduce in multiple habitats.

Introduction

Phenotypic plasticity is defined as the ability of an organism to alter its morphology and/or physiology in

response to environmental variations. Plants have been shown to respond plastically in response to

herbivory, neighbor presence or absence, and variations in abiotic factors (Callaway et al. 2003). Of the

abiotic factors, sunlight exposure, water and nutrient availability, and soil type can strongly influence

morphological and physiological responses in plants.  Soil type is largely characterized by particle size

and shape (i.e., soil texture) and directly affects the amount of water and nutrients available for plant

uptake. Soil types that result in increased water and nutrient availability generally lead to increased

growth, reproduction, and overall success for plants growing in that environment.

Hydrocotyle bonariensis is a perennial clonal herb commonly found in both coastal sand dune

and inland coastal habitats of Georgia. Populations inhabiting coastal sand dune environments are

exposed to high growing season air and sand temperatures, high incident sunlight, salt spray, and periodic

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saltwater inundation, resulting in a patchy distribution of vegetation with little to no canopy cover.

Additionally, the soil consists of large, round sand particles that result in low water-holding capacity and

reduced nutrient retention (Banedjschafie and Durner 2015). In contrast, H. bonariensis populations in

inland coastal environments are exposed to more variable temperature and incident sunlight due to

increased canopy cover. Furthermore, the smaller grain size and soil chemistry properties of soil at inland

sites likely result in relatively greater water and nutrient retention compared to sandy soil.

The purpose of this study was to explore potential phenotypic plasticity in H. bonariensis in

response to variations in soil type.  Specifically, leaf and petiole morphological characteristics were

compared between plants grown in sand collected from the sand dune habitat and soil collected from an

inland coastal environment.  It was expected that the soil texture and chemistry would differ between soil

collected from the sand dune habitat and soil from the inland coastal habitat. Furthermore, it was

hypothesized that these differences would lead to differences in leaf and petiole morphology between

individuals grown in each soil type.

Materials and methods

Hydrocotyle bonariensis Comm ex. Lam (large-leaf pennywort) is a clonal perennial herb ranging from

Virginia, USA, to Chile in South America (Joesting et al. 2012). It consists of underground, horizontally

connected rhizomes and nodes consisting of a single leaf and root system (i.e., individual ramet). Each

node is also capable of producing inflorescences (Knight and Miller 2004). Resource foraging behavior

has been observed for this species and allows for both resource sharing (e.g., nutrients, water, and

photosynthates) between connected ramets and asexual reproduction via horizontal growth (Evans and

Whitney 1992; Evans and Cain 1995). Although sexual reproduction occurs, seedling survival rate is 1 -

3% for sand dune populations and therefore populations are predominantly established via clonal growth

(Evans 1992).

For this experiment, soil was collected on September 15, 2015, from Armstrong State University

(ASU), Savannah, GA, to represent soil from an inland site and sand was collected from a sand dune site

on North Tybee Beach on Tybee Island (TI), GA, on September 16, 2015. Large debris was removed

from the TI sand and ASU soil prior to sterilization in an autoclave, and samples of both TI sand and

ASU soil were collected for analysis of soil type, pH, organic matter content, ammonium content, nitrate,

and phosphorous at the University of Georgia Soil, Plant, and Water Laboratory.

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Twenty-eight rhizome fragments were haphazardly collected on September 16, 2015 from

portions of a H. bonariensis population interspersed within the sand dunes at North Tybee Beach by

digging out a portion of the rhizome and cutting each ramet approximately 2.5 cm from the node on each

side. Rhizome fragments were selected based on the presence of a mature leaf and lack of leaf

discoloration and injuries. Fragments were immediately stored on ice until planting. Rhizome fragments

were randomly assigned to one of two treatments: (1) TI sand and (2) ASU soil and planted the same day

as collection. A plant tray (5.5 cm in depth) was divided in half and two rhizome fragments were planted

on each side (N=2 treatments x 7 replicates x 2 rhizome fragments/replicate= 28 Total). Rhizome

fragments were grown and maintained under greenhouse conditions and watered from above weekly.

One leaf sample per replicate was collected weekly for seven weeks, from October 08, 2015 to

November 18, 2015. Leaf and petiole thickness was measured to the nearest 0.01 mm using calipers,

petiole length was measured to the nearest 0.1 cm, and fresh leaf and petiole weights were measured to

the nearest 0.001 g. Leaf samples were photographed and ImageJ (1.50d, Wayne Rasband National

Institute of Health, USA) was used to estimate leaf area. Leaf and petiole samples were dried at 65 °C for

at least 48 hours, after which dry weight was measured. Leaf and petiole water weight was determined by

subtracting dry weight from fresh weight. Stomata peels of both the abaxial and adaxial leaf surfaces were

created by applying a thin layer of clear nail polish on a portion of the leaf surface. Once dry, the peels

were mounted on a slide and photographed using an Olympus BX60 microscope and a Q Color 5 camera

(Olympus Corporation, Waltham, MA). Stomata densities were calculated using Adobe Photoshop (12.1

x64, Adobe Systems Incorporated) and ImageJ. A two-way ANOVA was conducted on each variable

independently (i.e., leaf area, leaf fresh weight, leaf dry weight, leaf water weight, leaf mass per area,

mean leaf thickness, petiole fresh weight, petiole dry weight, petiole water weight, petiole length, mean

petiole thickness, adaxial stomata density, and abaxial stomata density), with soil type and date as factors

and a significance level of P < 0.05.

Results

Soil collected from ASU was loamy sand, composed of 81.9% sand, 11.8% silt, and 6.3% clay, and was

more acidic with higher organic matter, potassium, nitrate and ammonium content compared to TI sand.

Sand from TI was 100% sand and had lower organic matter, potassium, nitrate, and ammonium but higher

phosphorus content compared to ASU soil (Table 1).

H. bonariensis individuals grown in ASU soil had significantly greater petiole length (df=1, 13,

F=7.1539; P=0.0096), petiole thickness (df=1,13, F=7.8486; P=0.0068), and petiole fresh weight

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(df=1,13, F=4.3657; P=0.041) (Fig. 1). Overall leaf area (df=1,13, F=4.3306; P= 0.0416) and abaxial

stomata density (df=1,13, F=7.7905; P=0.0071) was also significantly greater for individuals grown in

ASU soil (Fig. 2 and 3).

Discussion

H. bonariensis individuals grown in ASU soil had significantly greater leaf area, petiole fresh weight,

petiole thickness, petiole length, and abaxial stomata density compared to plants grown in TI sand. This is

likely a result of the greater organic matter content and the resulting greater potential water holding

capacity and nutrient availability in the ASU soil compared to the TI sand (Table 1). The increased

availability of nutrients may have led to greater photosynthesis, carbon gain, and growth in plants grown

in ASU soil, as inidcated by the significantly greater leaf area and petiole length. Furthermore, the

significantly higher abaxial stomata density of individuals grown in ASU soil suggests these plants may

have reached higher photosynthetic rates and thus experienced greater plant growth and carbon allocation

(Soares et al. 2007).

Soil texture between ASU soil and TI sand likely contributed to increased water holding capacity

and nutrient retention in ASU soil. Sand characteristically has relatively large particles (0.5-2 m) with

large pore spaces for water to flow through, and thus increased rate of water drainage and decreased water

available for plant uptake. In contrast, loamy sand contains a mixture of smaller particle sizes due to the

presence of silt (0.002-0.05mm) and clay (<0.002m), and the negative charge associated with clay

particles attracts and binds to positively charged ions. The results suggest that the potential increased

water availability in ASU soil led to greater water storage in tpetioles, as indicated by the significantly

greater petiole thickness and petiole fresh weight. The ability to obtain and store higher volumes of water

in plant tissues may contribute to the ability of H. bonariensis to adjust to and successfully inhabit various

environments, such as inland and coastal sand dune habitats.

The natural range and variety of environments inhabited by H. bonariensis suggests its aptitude

for surviving multiple climates and conditions, and the ability to alter morphological and physiological

responses to variations in the abiotic environment through phenotypic plasticity should contribute to this

ability. Phenotypic plasticity in response to variations in soil moisture has been shown to increase

survival and reproduction for Polygonum persicaria (Sultan and Bazzaz 1993) and likely plays a role in

the ability of H. bonariensis to successfully grow and reproduce in a variety of environments.

Furthermore, phenotypic plasticity, especially in response to soil environment, in H. bonariensis increases

the likelihood that this plant could be used in various management strategies, such as coastal sand dune

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restoration, long-term desert sand dune stabilization, control of sediment erosion, and waste removal from

polluted water bodies (Reddy and Tucker 1985; Williams 2007; Mahdavi and Bergmeier 2016).

To further investigate phenotypic plasticity in H. bonariensis, future studies should focus on

measuring additional morphological variables, such as belowground biomass, leaf longevity, and rhizome

length, in response to variations in soil type. In addition, studies should include irradiance and nutrient

availability as independent variables in combination with soil type to observe varying degrees of

phenotypic plastic responses to multiple abiotic factors (Evans 1991). Finally, a reciprocal transplant

study, in which individuals taken from the sand dune habitat are planted in either beach sand or soil from

an inland site and vice versa, would examine if observed morphological differences are due to phenotypic

plastic responses or local adaptation, in which a genetic component is responsible (Knight and Miller

2004).

Acknowledgements

The authors would like to thank John Counts and Esther Medrano for assistance in data collection and

data analysis.

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Table 1: Soil type, pH, organic matter (OM) content, ammonium (NH4-N), nitrate (NO3-N), phosphorus

(P), and potassium (K) content of soil collected from Tybee Island (TI) and Armstrong State University

(ASU).

TI ASU

Soil type Sand Loamy sand

pH 6.95 4.42

OM (%) 0.03 5.41

NH4-N (mg/kg) 3.51 5.27

NO3-N (mg/kg) 1.28 4.31

P (mg/kg) 139.0 49.4

K (mg/kg) 6.1 131.8

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Figure 1: Mean petiole length, petiole thickness, petiole dry weight, petiole fresh weight, and petiole

water weight for plants grown in soil collected from Tybee Island and Armstrong State University (ASU)

campus. Significant difference at P<0.05 is indicated by asterisk and error bars represent standard error.

1

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Figure 2: Mean leaf area, leaf thickness, leaf fresh weight, leaf dry weight, and leaf water weight for

plants grown in soil collected from Tybee Island and Armstrong State University (ASU) campus.

Significant difference at P<0.05 is indicated by asterisk and error bars represent standard error.

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Figure 3: Adaxial and abaxial stomata density for plants grown in soil collected from Tybee Island and

Armstrong State University (ASU) campus. Significant difference at P<0.05 is indicated by asterisk,

P<0.05, and error bars represent standard error.

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